Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
3-METHYLIDENYL-2-INDOLINONE MODULATORS OF PROTEIN KINASE
INTRODUCTION
The present invention relates generally to organic
chemistry, biochemistry, pharmacology and medicine. More
particularly, it relates to 3-methylidenyl-2-indolinone
derivatives, and their physiologically acceptable salts and
prodrugs, which modulate the activity of protein kinases
("PKs") and are expected to exhibit a salutary effect
against disorders related to abnormal PK activity.
BACKGROUND OF THE INVENTION
The following is offered as background information
only and is not admitted to be prior art to the present
invention.
PKs are enzymes that catalyze the phosphorylation of
hydroxy groups on tyrosine, serine and threonine residues
of proteins. The consequences of this seemingly simple
activity are staggering; cell growth, differentiation and
proliferation, i.e., virtually all aspects of cell life in
one way or another depend on PK activity. Furthermore,
abnormal PK activity has been related to a host of
disorders, ranging from relatively non-life threatening
diseases such as psoriasis to extremely virulent diseases
such as glioblastoma (brain cancer).
The PKs can be conveniently be broken down into two
classes, the protein tyrosine kinases (PTKs) and the
serine-threonine kinases (STKs).
One of the prime aspects of PTK activity is their
involvement with growth factor receptors. Growth factor
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receptors are cell-surface proteins. When bound by
growth factor ligand, growth factor receptors ale converted
to an active form which interacts with proteins on tai
inner surface of a cell membrane. This leads to
phosphorylation on tyrosine residues of the receptor and
other proteins and to the formation inside the cell of
complexes with a variety of cytoplasmic signaling molecules
that, in turn, effect numerous cellular responses such as
cell division (proliferation), cell differentiation, cell
growth, expression of cellular metabolic processes to the
extracellular microenvironment, etc. For a more complete
discussion, see Schlessinger and Ullrich, Neuron, 9:303-391
(1992) which is incorporated by reference, including any
drawings, as if fully set forth herein.
Growth factor receptors with PTK activity are known as
receptor tyrosine kinases ("RTKs"). They comprise a large
family of transmembrane receptors with diverse biological
activity. At present, at least nineteen (19) distinct
subfamilies of RTKs have been identified. An example of
these is the subfamily designated the "HER" RTKs, which
include EGFR (epithelial growth factor receptor), HER2,
HERS and HER4. These RTKs consist of an extracellular
glycosylated ligand binding domain, a transmembrane domain
and an intracellular cytoplasmic catalytic domain that can
phosphorylate tyrosine residues on proteins.
Another RTK subfamily consists of insulin receptor
(IR), insulin-like growth factor I receptor (IGF-1R) and
insulin receptor related receptor (IRR). IR and IGF-1R
interact with insulin, IGF-I and IGF-II to form a
heterotetramer of two entirely extracellular glycosylated a
subunits and two (3 subunits which cross the cell membrane
and which contain the tyrosine kinase domain.
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A third RTK subfamily is referred to as the platelet
derived growth factor receptor ("PDGFR"' group, which
includes PDGFRa, PDGFR~3, CSFIR, c-kit and c-fms. These
receptors consist of glycosylated extracellular domains
composed of variable numbers of immunoglobin-like loops and
an intracellular domain wherein the tyrosine kinase domain
is interrupted by unrelated amino acid sequences.
Another group which, because of its similarity to the
PDGFR subfamily, is sometimes subsumed into the later group
is the fetus liver kinase ("flk") receptor subfamily. This
group is believed to be made of up of kinase insert domain-
receptor fetal liver kinase-1 (KDR/FLK-1), flk-1R, flk-4
and fms-like tyrosine kinase 1 (flt-1).
A further member of the tyrosine kinase growth factor
receptor family is the fibroblast growth factor ("FGF")
receptor subgroup. This group consists of four receptors,
FGFRl-4, and seven ligands, FGF1-7. While not yet well
defined, it appears that, like the PDGF receptors, the FGF
receptors consist of a glycosylated extracellular domain
containing a variable number of immunoglobin-like loops and
an intracellular domain in which the tyrosine kinase
sequence is interrupted by regions of unrelated amino acid
sequences.
Still another member of the tyrosine kinase growth
factor receptor family is the vascular endothelial growth
factor (VEGF") receptor subgroup. VEGF is a dimeric
glycoprotein similar to PDGF but has different biological
functions and target cell specificity in vivo. In
particular, VEGF is presently thought to play an essential
role is vasculogenesis and angiogenesis.
A more complete listing of the known RTK subfamilies
is described in Plowman et al., DN&P, 7(6):334-339 (1994)
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which is incorporated by reference, including any drawings,
as if fully set forth herein.
In addition to the RTKs, there also exists a family of
entirely intracellular PTKs called "non-receptor tyrosine
kinases" or "cellular tyrosine kinases." This latter
designation, abbreviated "CTK," will be used herein. CTKs
do not contain extracellular and transmembrane domains. At
present, over 24 CTKs in 11 subfamilies (Src, Frk, Btk,
Csk, Abl, Zap70, Fes, Fps, Fak, Jak and Ack) have been
identified. The Src subfamily appear so far to be the
largest group of CTKs and includes Src, Yes, Fyn, Lyn, Lck,
Blk, Hck, Fgr and Yrk. For a more detailed discussion of
CTKs, see Bolen, Oncoaene, 8:2025-2031 (1993), which is
incorporated by reference, including any drawings, as if
fully set forth herein.
The serine/threonine kinases, STKs, like the CTKs, are
predominantly intracellular although there are a few
receptor kinases of the STK type. STKs are the most common
of the cytosolic kinases; i.e., kinases that perform their
function in that part of the cytoplasm other than the
cytoplasmic organelles and cytoskelton. The cytosol is the
region within the cell where much of the cell's
intermediary metabolic and biosynthetic activity occurs;
e.g., it is in the cytosol that proteins are synthesized on
ribosomes.
RTKs, CTKs and STKs have all been implicated in a host
of pathogenic conditions including, significantly, cancer.
Other pathogenic conditions which have been associated
with PTKs include, without limitation, psoriasis, hepatic
cirrhosis, diabetes, angiogenesis, restenosis, ocular
diseases, rheumatoid arthritis and other inflammatory
disorders, immunological disorders such as autoimmune
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disease, cardiovascular disease such as atherosclerosis and
a variety of renal disorders.
With regard to cancer, two of the major hypotheses
advanced to explain the excessive cellular proliferation
5 that drives tumor development relate to functions known to
be PK regulated. That is, it has been suggested that
malignant cell growth results from a breakdown in the
mechanisms that control cell division and/or
differentiation. It has been shown that the protein
products of a number of proto-oncogenes are involved in the
signal transduction pathways that regulate cell growth and
differentiation. These protein products of proto-oncogenes
include the extracellular growth factors, transmembrane
growth factor PTK receptors (RTKs), cytoplasmic PTKs (CTKs)
and cytosolic STKs, discussed above.
In view of the apparent link between PK-related
cellular activities and wide variety of human disorders, it
is no surprise that a great deal of effort is being
expended in an attempt to identify ways to modulate PK
activity. Some of these have involved biomimetic
approaches using large molecules patterned on those
involved in the actual cellular processes (e. g., mutant
ligands (U.S. App. No. 4,966,849); soluble receptors and
antibodies (App. No. WO 94/10202, Kendall and Thomas, Proc.
Nat'1 Acad. Sci., 90:10705-09 (1994), Kim, et al., Nature,
362:841-844 (1993)); RNA ligands (Jelinek, et al.,
Biochemistry, 33:10450-56); Takano, et al., Mol. Bio. Cell
4:358A (1993); Kinsella, et al., Exp. Cell Res. 199:56-62
(1992); Wright, et al., J. Cellular Phys., 152:448-57) and
tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; WO
91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani, et
al., Proc. Am. Assoc. Cancer Res., 35:2268 (1994)).
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In addition to the above, attempts have been made to
identify small molecules which act as PK inhibitors. For
example, bis-monocylic, bicyclic and heterocyclic aryl
compounds (PCT WO 92/20642), vinylene-azaindole derivatives
(PCT WO 94/14808) and 1-cyclopropyl-4-pyridylquinolones
(U. S. Pat. No. 5,330,992) have been described as tyrosine
kinase inhibitors. Styryl compounds (U.S. Pat. No.
5,217,999), styryl-substituted pyridyl compounds (U. S. Pat.
No. 5,302,606), quinazoline derivatives (EP App. No. 0 566
266 A1), selenaindoles and selenides (PCT WO 94/03427),
tricyclic polyhydroxylic compounds (PCT WO 92/21660) and
benzylphosphonic acid compounds (PCT WO 91/15495) have all
been described as PTK inhibitors useful in the treatment of
cancer.
SUMMARY OF THE INVENTION
Our own efforts to identify small organic molecules
which modulate PK activity and which, therefore, are
expected to be useful in the treatment and prevention of
disorders involving abnormal PK activity, has led us to the
discovery of a family of 3-methylidenyl-2-indolinone
derivatives and their prodrugs and physiologically
acceptable salts, which exhibit PK modulating ability and
are thereby expected to have a salutary effect against
disorders related to abnormal PK activity; it is these
compounds which is the subject of this invention.
Thus, the present invention relates generally to 3-
methylidenyl-2-indolinones and their prodrugs and
physiologically acceptable salts, which modulate the
activity of receptor tyrosine kinases (RTKs), non-receptor
protein tyrosine kinases (CTKs) and serine/threonine
protein kinases (STKs). In addition, the present invention
relates to the preparation and use of pharmaceutical
compositions of the disclosed compounds and their
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physiologically acceptable salts and prodrugs in the
treatment or prevention of PK driven disorders such as, by
way of example and not limitation, cancer, diabetes,
hepatic cirrhosis, cardiovasacular disease such as
atherosclerosis, angiogenesis, immunological disease such
as autoimmune disease and renal disease.
The terms "2-indolinone," indolin-2-one and "2-
oxindole" are used interchangeably herein to refer to a
molecule having the chemical structure:
Ri.~ ~ ~O
N
A
R
where A is carbon or nitrogen.
A "3-methylidenyl-2-indolinone" refers to a molecule
having the chemical structure:
H
R~_4
A N O
R
where, again, A can be carbon or nitrogen.
A "pharmaceutical composition" refers to a mixture of
one or more of the compounds described herein, or
physiologically acceptable salts or prodrugs thereof, with
other chemical components, such as physiologically
acceptable carriers and excipients. The purpose of a
pharmaceutical composition is to facilitate administration
of a compound to an organism.
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A "prodrug" refers to an agent which is converted into
the parent drug in vivo. Prodrugs are often useful
because, in some situations, they may be easier to
administer than the parent drug. They may, for instance,
be bioavailable by oral administration whereas the parent
drug is not. The prodrug may also have improved solubility
in pharmaceutical compositions over the parent drug. An
example, without limitation, of a prodrug would be a
compound of the present invention which is administered as
an ester (the "prodrug") to facilitate transmittal across a
cell membrane where water solubility is detrimental to
mobility but then is metabolically hydrolyzed to the
carboxylic acid, the active entity, once inside the cell
where water solubility is beneficial.
A further example of a prodrug might be a short
polypeptide, for example, without limitation, a 2 - 10
amino acid polypeptide, bonded through a terminal amino
group to a carboxy group of a compound of this invention
wherein the polypeptide is hydrolyzed or metabolized in
vivo to release the active molecule.
As used herein, a "physiologically acceptable carrier"
or a "pharmaceutically acceptable carrier" refers to a
carrier or diluent that does not cause significant
irritation to an organism and does not abrogate the
biological activity and properties of the administered
compound.
A "carrier" refers to a chemical compound which
faciliates the incorporation of a compound of interest into
cells and tissues. An example, without limitation, of a
carrier is dimethyl sulfoxide (DMSO).
A "diluent" refers to a chemical compound, usually a
liquid, which dissolves or disperses a compound of interest
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thereby reducing the concentration of the compound to less
than that of the compound alone.
An "excipient" refers to an inert substance added to a
pharmaceutical composition to further facilitate
administration of a compound. Examples, without
limitation, of excipients include calcium carbonate,
calcium phosphate, various sugars and types of starch,
cellulose derivatives, gelatin, vegetable oils and
polyethylene glycols.
1. CHEMISTRY
A. General Structural Features.
In one aspect, the the present invention relate to 2-
indolinones having chemical structure 1:
R~ H
R2 / / Q
R3 A N O
R4 Ro
wherein:
A is selected from the group consisting of carbon and
nitrogen;
Q is selected from the group consisting of
7
Rs R Rs R12 R11 H ~H~r R14
R16
R9 R5 ~ N~ 1o N~R13
' H R and R1~ ~H~~ H '
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bonds a and b may be either single or double bonds as
indicated by the dotted lines with the proviso that a and b
are both single bonds or both double bonds in any one
compound of this invention;
5
r is 1 when a and b are double bonds;
r is 2 when a and b are single bonds;
10 R° is selected from the group consisting of hydrogen, alkyl,
-C (O) OR19 and -C (O) R19;
R' is selected from the group consisting of hydrogen, alkyl,
aryl, alkoxy, halo, -C (O) OR19, - (CHz) nOC (O) R19, -C (O) NR19 and
(CHz)nR2°, wherein:
Rl9 is selected from the group consisting of hydrogen,
alkyl, alkenyl, alkynyl, cycloalkyl and aryl;
n is 1, 2, 3 or 4;
R2° is selected from the group consisting of hydroxy,
halo, -OC (O) NRzlRzz; _0C (S) NR21Ra2; _0C (O) NHSO2Rzl, -C (0) OR19,
-NRZIRzz and a heteroalicylic group containing at least one
nitrogen atom in the ring, the ring being bonded to the
adjacent CHz group through the nitrogen atom;
R2 is selected from the group consisting of hydrogen, alkyl,
trihalomethyl, aryl, heteroaryl, heteroalicyclic, alkoxy,
halo, - (CH2) nR2°, -SOZNRz1R22, -C (O) OR19, -C (O) R19, -NHC (O)
OR19,
NHC (O) R19, -C (O) (CHz) nRz°, -NRzlRzz arid -N=CR23 wherein
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Rzl and R22 are independently selected from the group
consisting of hydrogen, alkyl, alkenyl, alkynyl,
cycloalkyl, aryl, heteroaryl and heteroalicyclic;
Rz3 is selected from the group consisting of alkyl,
alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl;
R3 is selected from the group consisting of hydrogen, alkyl,
trihalomethyl, alkoxy, aryl, aryloxy, heteroaryl,
heteroalicyclic, hydroxy, halo, -S02NR21R22, -NHSOZRls, -
C (O) ORl9, -NRZIRzz and -NHC (O) R24, wherein
R24 is selected from the group consisting of alkyl,
alkenyl, alkynyl, aryl, heteroaryl and
heteroalicyclic;
R' is selected from the group consisting of hydrogen, alkyl,
alkoxy and halo;
RS is selected from the group consisting of hydrogen, alkyl,
cycloalkyl, halo, aryl and heteroaryl;
R6 is selected from the group consisting of hydrogen, alkyl,
alkoxy, halo, cycloalkyl, aryl and, combined with R18, a
heteroalicyclic group having the structure
Y Y
O O
Rs Rs
\ or I \
R9 / R5 Rs / R5
wherein
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y and y' are independently selected from the group
consisting of hydrogen, alkyl and aryl;
R' is OR18, wherein R18 is selected from the group consisting
of alkyl, - (CH) nR2° and, combined with R6 or Re, a
heteroalicyclic group having the structure
Y Y Y Y
O O O O
Ra Rs Rs Rs
\ or ( \ or I \ or I \
R9 / R5 R9 ~ R5 R9 ~ R5 R9 ~ Rs
RB is selected from the group consisting of hydrogen, alkyl,
cycloalkyl, alkoxy, halo, aryl, heteroaryl and, combined
with R18, a heteroalicyclic ring having the structure
Y Y
O O
\ Rs \ Rs
or
R9 ~ R5 Rs ( ~ Rs
R9 is selected from the group consisting of hydrogen, alkyl,
alkoxy, halo and -NRzlRaz;
Rl° is selected from the group consisting of alkyl and
-C (O) OR19;
Rll is selected from the group consisting of hydrogen, alkyl
and -C (O) OR19;
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Rlz iS _ (CHz) nRz°;
R13 is either a bond through which Q is bonded to the rest
of the molecule or, when it is not, a group selected from
the group consisting of hydrogen and alkyl;
R14 is either a bond through which Q is bonded to the rest
of the molecule or, when it is not, a group selected from
the group consisting of hydrogen, alkyl and - (CHz) nRz°;
RlSis either a bond through which Q is bonded to the rest
of the molecule or, when it is not, hydrogen;
with the proviso that only one of R13, R14 or R15 is the
bond linking Q to the rest of the molecule in any one
compound herein;
R16 and R1', when a and b are single bonds, are independently
selected from the group consisting of hydrogen, alkyl and
aryl and, when a and b are double bonds, R16 is hydrogen and
R1' does not exist; and,
physiologically acceptable salts and prodrugs thereof.
As used herein, the term "alkyl" refers to a saturated
aliphatic hydrocarbon including straight chain and branched
chain groups. Preferably, the alkyl group has 1 to 20
carbon atoms (whenever a numerical range; e.g. "1-20", is
stated herein, it means that the group, in this case the
alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3
carbon atoms, etc. up to and including 20 carbon atoms).
More preferably, it is a medium size alkyl having 1 to 10
carbon atoms. Most preferably, it is a lower alkyl having
1 to 4 carbon atoms. The alkyl group may be substituted or
unsubstituted. When substituted, the substituent groups)
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is preferably one or more independently selected from the
group consisting of halo hydroxy, unsubstituted lower
alkoxy, aryl optionally substituted with one or more halo,
hydroxy, unsubstituted lower alkyl or unsubstituted lower
alkoxy groups, aryloxy optionally substituted with one or
more halo, hydroxy, unsubstituted lower alkyl or
unsubstituted lower alkoxy groups, 6-member heteroaryl
having from 1 to 3 nitrogen atoms in the ring, the carbons
in the ring being optionally substituted with one or more
halo, hydroxy, unsubstituted lower alkyl or unsubstituted
lower alkoxy groups, 5-member heteroaryl having from 1 to 3
heteroatoms selected from the group consisting of nitrogen,
oxygen and sulfur, the carbon atoms of the group being
optionally substituted with one or more halo, hydroxy,
unsubstituted lower alkyl or unsubstituted lower alkoxy
groups, 5- or 6-member heteroalicyclic group having from 1
to 3 heteroatoms selected from the group consisting of
nitrogen, oxygen and sulfur, the carbon and nitrogen (if
present) atoms in the group being optionally substituted
with one or more halo, hydroxy, unsubstituted lower alkyl
or unsubstituted lower alkoxy groups, mercapto,
(unsubstituted lower alkyl)thio, arylthio optionally
substituted with one or more halo, hydroxy, unsubstituted
lower alkyl or unsubstituted lower alkoxy groups, cyano,
acyl, thioacyl, 0-carbamyl, N-carbamyl, 0-thiocarbamyl, N-
thiocarbamyl, C-amido, N-amido, vitro, N-sulfonamido, S-
sulfonamido, RZSS (0) -, R25S (0) 2-, -C (0) OR25, R2sC (0) 0-, and
-NR25RZS, wherein R25 and R26 are independently selected from
the group consisting of hydrogen, unsubstituted lower
alkyl, trihalomethyl, unsubstituted (C3-C6)cycloalkyl,
unsubstituted lower alkenyl, unsubstituted lower alkynyl
and aryl optionally optionally substituted with one or more
halo, hydroxy, unsubstituted lower alkyl or unsubstituted
lower alkoxy groups.
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A "cycloalkyl" group refers to a 3 to 8 member all-
carbon monocyclic ring, an all-carbon 5-member/6-member or
6-member/6-member fused bicyclic ring or a multicyclic
fused ring(a "fused" ring system means that each ring in
5 the system shares an adjacent pair of carbon atoms with
each other ring in the system)group wherein one or more of
the rings may contain one or more double bonds but none of
the rings has a completely conjugated pi-electron system.
Examples, without limitation, of cycloalkyl groups are
10 cyclopropane, cyclobutane, cyclopentane, cyclopentene,
cyclohexane, cyclohexadiene, adamantane, cycloheptane and,
cycloheptatriene. A cycloalkyl group may be substituted or
unsubstituted. When substituted, the substituent groups)
is preferably one or more independently selected from the
15 group consisting of unsubstituted lower alkyl,
trihaloalkyl, halo, hydroxy, unsubstituted lower alkoxy,
aryl optionally substituted with one or more halo, hydroxy,
unsubstituted lower alkyl or unsubstituted lower alkoxy
groups, aryloxy optionally substituted with one or more
halo, hydroxy, unsubstituted lower alkyl or unsubstituted
lower alkoxy groups, 6-member heteroaryl having from 1 to 3
nitrogen atoms in the ring, the carbons in the ring being
optionally substituted with one or more halo, hydroxy,
unsubstituted lower alkyl or unsubstituted lower alkoxy
groups, 5-member heteroaryl having from 1 to 3 heteroatoms
selected from the group consisting of nitrogen, oxygen and
sulfur, the carbon atoms of the group being optionally
substituted with one or more halo, hydroxy, unsubstituted
lower alkyl or unsubstituted lower alkoxy groups, 5- or 6-
member heteroalicyclic group having from 1 to 3 heteroatoms
selected from the group consisting of nitrogen, oxygen and
sulfur, the carbon and nitogen (if present)atoms in the
group being optionally substituted with one or more halo,
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hydroxy, unsubstituted lower alkyl or unsubstituted lower
alkoxy groups, mercapto,(unsubstituted lower alkyl)thio,
arylthio optionally substituted with one or more halo,
hydroxy, unsubstituted lower alkyl or unsubstituted lower
alkoxy groups, cyano, acyl, thioacyl, O-carbamyl, N-
carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido,
nitro, N-sulfonamido, S-sulfonamido, RzSS (O) -, RZSS (O) 2-,
-C (0) ORzS, RzsC (0) O-, and -NRzSRzs are as defined above.
An "alkenyl" group refers to an alkyl group, as
defined herein, consisting of at least two carbon atoms and
at least one carbon-carbon double bond.
An "alkynyl" group refers to an alkyl group, as
defined herein, consisting of at least two carbon atoms and
at least one carbon-carbon triple bond.
An "aryl" group refers to an all-carbon monocyclic or
fused-ring polycyclic (i.e., rings which share adjacent
pairs of carbon atoms) groups having a completely
conjugated pi-electron system. Examples, without
limitation, of aryl groups are phenyl, naphthalenyl and
anthracenyl. The aryl group may be substituted or
unsubstituted. When substituted, the substituted groups)
is preferably one or more independently selected from the
group consisting of unsubstituted lower alkyl,
trihaloalkyl, halo, hydroxy, unsubstituted lower alkoxy,
mercapto,(unsu~stituted lower alkyl)thio, cyano, acyl,
thioacyl, 0-carbamyl, N-carbamyl, 0-thiocarbamyl, N-
thiocarbamyl, C-amido, N-amido, nitro, N-sulfonamido, S-
sulfonamido, RZSS (O) -, R25S (0) 2-, -C (0) OR25, RzsC (0) 0-, and
-NR25R2s, with R25 and R26 as defined above.
As used herein, a "heteroaryl" group refers to a
monocyclic or fused ring (i.e., rings which share an
adjacent pair of atoms) group having in the rings) one or
more atoms selected from the group consisting of nitrogen,
oxygen and sulfur and, in addition, having a completely
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conjugated pi-electron system. Examples, without
limitation, of heteroaryl groups are pyrrole, furan,
thiophene, imidazole, oxazole, thiazole, pyrazole,
pyridine, pyrimidine, quinoline, isoquinoline, purine and
carbazole. The heteroaryl group may be substituted or
unsubstituted. When substituted, the substituted groups)
is preferably one or more independently selected from the
group consisting of unsubstituted lower alkyl,
trihaloalkyl, halo, hydroxy, unsubstituted lower alkoxy,
mercapto,(unsubstituted lower alkyl)thio, cyano, acyl,
thioacyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-
thiocarbamyl, C-amido, N-amido, vitro, N-sulfonamido, S-
sulfonamido, RZSS (0) -, R25S (0) 2-, -C (0) ORzs, R25C (0) 0-, and
-NR25Rz6, with R25 and R26 as defined above.
A "heteroalicyclic" group refers to a monocyclic or
fused ring group having in the rings) one or more atoms
selected from the group consisting of nitrogen, oxygen and
sulfur. The rings may also have one or more double bonds.
However, the rings do not have a completely conjugated pi-
electron system. The heteroalicyclic ring may be
substituted or unsubstituted. When substituted, the
substituted groups) is preferably one or more
independently selected from the group consisting of
unsubstituted lower alkyl, trihaloalkyl, halo, hydroxy,
unsubstituted lower alkoxy, mercapto,(unsubstituted lower
alkyl)thio, cyano, acyl, thioacyl, 0-carbamyl, N-carbamyl,
0-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, vitro, N-
sulfonamido, S-sulfonamido, R25S (0) -, R25S (0) 2-, -C (0) OR25,
RZ5C (0) O-, and -NR25R2s, with R25 and Rz6 as defined above.
A "hydroxy" group refers to an -OH group.
An "alkoxy" group refers to both an -O-(unsubstituted
alkyl) and an -O-(unsubstituted cycloalkyl) group.
An "aryloxy" group refers to both an -O-aryl and an -
O-heteroaryl group, as defined herein.
A "mercapto" group refers to an -SH group.
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A "alkylthio" group refers to both an S-(unsubstituted
alkyl) and an -S-(unsubstituted cycloalkyl) group.
A "arylthio" group refers to both an -S-aryl and an
-S-heteroaryl group, as defined herein.
An "acyl" group refers to a -C(O)-R" group, where R"
is selected from the group consisting of hydrogen,
unsubstituted lower alkyl, trihalomethyl, unsubstituted
cycloalkyl, aryl optionally substituted with one or more
unsubstituted lower alkyl, trihalomethyl, unsubstituted
lower alkoxy, halo and
-NR25Rzs groups, heteroaryl (bonded through a ring carbon)
optionally substituted with one or more unsubstituted lower
alkyl, trihaloalkyl, unsubstituted lower alkoxy, halo and
-NRZSR2s groups and heteroalicyclic (bonded through a ring
carbon) optionally substituted with one or more
unsubstituted lower alkyl, trihaloalkyl, unsubstituted
lower alkoxy, halo and -NR25R26 groups .
An "aldehyde" group refers to an acyl group in which
R" is hydrogen.
A "thioacyl" group refers to a -C(S)-R" group, with R"
as defined herein.
An "ester" group refers to a -C(O)O-R" group with R"
as defined herein except that R" cannot be hydrogen.
An "acetyl" group refers to a -C(O)CH3 group.
A "halo" group refers to fluorine, chlorine, bromine
or iodine.
A "trihalomethyl" group refers to a -CX3 group wherein
X is a halo group as defined herein.
A "trihalomethanesulfonyl" group refers to a X3CS(=O)z-
groups with X as defined above.
A "cyano" group refers to a -C---N group.
SUBSTITUTE SHEET (RULE 26)
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A "methylenedioxy" group refers to a -OCHzO- group
where the two oxygen atoms are bonded to adjacent carbon
atoms.
An "ethylenedioxy" group refers to a -OCH2CH20- where
the two oxygen atoms are bonded to adjacent carbon atoms.
An "O-carbamyl" group refers to a -OC (O) NRz5Ra6 group
with R25 and Rz6 as defined herein.
An "N-carbamyl" group refers to an Rz50C (O) NR26- group,
with R25 and Rz6 as defined herein.
An "O-thiocarbamyl" group refers to a -OC(S)NR25Rzs
group with R25 and R26 as defined herein.
An "N-thiocarbamyl" group refers to a Rz50C (S) NR2s-
group, with Rzs and R26 as defined herein.
An "amino" group refers to an -NRz5R26 group, wherein Rzs
and Rz6 are both hydrogen.
A "C-amido" group refers to a -C (O) NR25R26 group with RZs
and R26 as defined herein.
An "N-amido" group refers to a RzSC (O) NR26- group, with
R25 and R26 as defined herein.
A "nitro" group refers to a -N02 group.
B. Preferred Structural Features.
A presently preferred aspect of this invention is a
compound in which Q is
R~
R8 R6
Rs ~ R5
Another presently preferred aspect of this invention
is a compound in which Q is
SUBSTITUTE SHEET (RULE 26)
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R~
R$ R6
Rs ~ R5
and
R1 is selected from the group consisting of hydrogen, lower
alkyl and -C(O)OH;
5 R2 is selected from the group consisting of hydrogen, lower
alkyl, halo, -C (O) R19, -C (O) OR19, lower alkoxy, -NR~1R22,
- ( CHZ ) nRz° and -NHC ( O ) ORl9 ;
R3 is selected from the group consisting of hydrogen,
hydroxy, halo, -NHC (O) O (lower alkyl) , -NHSO2R19, -NHC (O) R24,
10 trihalomethyl, and aryl optionally substituted with one or
more lower alkoxy groups;
R4 is hydrogen;
R19 is selected from the group consisting of hydrogen
and lower alkyl;
15 n is 2 or 3;
Rz° is selected from the group consisting of hydroxy,
-C(O)OH, morpholin-4-yl, piperidin-1-yl, piperazin-1-
yl , pyrrol idin-1-yl and -NRZIRzz ;
R21 and R22 are independently selected from the group
20 consisting of hydrogen and lower alkyl; and,
R24 is selected from the group consisting of hydrogen
and lower alkyl.
It is likewise a presently preferred embodiment of
this invention that Q is
SUBSTITUTE SHEET (RULE 26)
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21
R~
R$ R6
Rs ~ Rs
R1 is selected from the group consisting of hydrogen, lower
alkyl and -C (O) OH;
R2 is selected from the group consisting of hydrogen, lower
alkyl, halo, -C (O) R19, -C (O) OR19, lower alkoxy, -NRz1R22,
- ( CH2 ) nR2 ° and -NHC ( O ) OR19 ;
R3 is selected from the group consisting of hydrogen,
hydroxy, halo, -NHC (O) O (lower alkyl) , -NHSOZR19, -NHC (O) R24,
trihalomethyl, and aryl optionally substituted with one or
more lower alkoxy groups;
R4 is hydrogen;
Rl9 is selected from the group consisting of hydrogen
and lower alkyl;
n is 2 or 3;
RZ° is selected from the group consisting of hydroxy,
-C(O)OH, morpholin-4-yl, piperidin-1-yl, piperazin-1-
yl , pyrrol idin-1-yl and -NR21R22 ;
R21 and Rz2 are independently selected from the group
consisting of hydrogen and lower alkyl;
Rz4 is selected from the group consisting of hydrogen
and lower alkyl;
RS is selected from the group consisting of hydrogen, lower
alkyl, halo, five-member ring heteroaryl and aryl
optionally substituted with one or more groups selected
from the group consisting of lower alkyl, halo, hydroxy,
-NRzlRzz and lower alkoxy;
R6 is selected from the group consisting of hydrogen, lower
alkyl, 3 to 7-member cycloalkyl, lower alkoxy, halo, aryl
SUBSTITUTE SHEET (RULE 26)
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optionally substituted with one or more groups
independently selected from the group consisting of lower
alkyl, halo, hydroxy, -NRZIRzz and lower alkoxy and, combined
with Ria, a group having the structure
Y Y
O O
Rs Rs
\ or I \
Rs / Rs Rs / Rs
wherein y and y' are either both hydrogen or both lower
alkyl;
R' is -OR18 wherein R18 is selected from the group consisting
of lower alkyl, - (CHz) nR2° and, combined with R6 or Re, a
group having the structure
Y Y Y Y
O O O O
Rs Rs Rs Rs
\ or I \ or I \ or I \
Rs / Rs Rs / Rs Rs / R5 Rs / Rs
R8 is selected from the group consisting of hydrogen, lower
alkyl, 3 to 6-member ring cycloalkyl, lower alkoxy, halo,
aryl optionally substituted with one or more groups
selected from the group consisting of lower alkyl, lower
alkoxy, halo, -NR21Rz2 and -NHC (O) (lower alkyl) , five-member
heteroaryl having from 1 to 3 heteroatoms in the ring and
6-member heteroaryl having from 1 to 3 heteroatoms in the
ring and, combined with R18 a group having the structure
SUBSTITUTE SHEET (RULE 26)
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23
Y Y
O O
Rs Rs
or
Rs ~ R5 Rs ~ ~ Rs
R9 is selected from the group consisting of hydrogen, lower
alkyl ; hydroxy, lower alkoxy, halo and -NRzlRzz
A further presently preferred aspect of this invention
is a compound in which Q is
R~
R8 Rs
Rs ~ Rs
R1 is selected from the group consisting of hydrogen, lower
alkyl and -C(O)OH;
Rz is selected from the group consisting of hydrogen, lower
alkyl, halo, -C (O) R19, -C (O) OR19, lower alkoxy, -NR2lRzz,
- ( CHz ) nR2° and -NHC ( O ) ORl9 ;
R3 is selected from the group consisting of hydrogen,
hydroxy, halo, -NHC (O) O (lower alkyl) , -NHSOzRI9, -NHC (O) Rz°,
trihalomethyl, and aryl optionally substituted with one or
more lower alkoxy groups;
R4 is hydrogen;
R19 is selected from the group consisting of hydrogen
and lower alkyl;
n is 2 or 3;
R2° is selected from the group consisting of hydroxy,
-C(O)OH, morpholin-4-yl, piperidin-1-yl, piperazin-1-
yl , pyrrol idin-1-yl and -NRzlRa2 ;
SUBSTITUTE SHEET (RULE 26)
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24
R21 and Rzz are independently selected from the group
consisting of hydrogen and lower alkyl;
R24 is selected from the group consisting of hydrogen
and lower alkyl;
RS is selected from the group consisting of hydrogen, lower
alkyl, thien-2-yl, thien-3-yl and aryl optionally
substituted with one or more lower alkoxy groups;
R6 is selected from the group consisting of hydrogen, lower
alkyl, 5 or 6-member cycloalkyl, aryl optionally
substituted with one or more lower alkoxy groups and,
combined with R18, a group having the structure
Y Y
O O
Rs Rs
or
Rs / Rs Rs / Rs
wherein y and y' are either both hydrogen or both
lower alkyl;
R' is -OR18, wherein R18 is selected from the group
consisting of lower alkyl, -(CH2)nRzo and, combined with R6
or Re, a group having the structure
Y Y Y Y
O O O O
R$ $ Rs s
R R
or I ~ or I ~ or
Rs / Rs Rs / Rs Rs / R5 Rs / Rs
n is 2 or 3;
SUBSTITUTE SHEET (RULE 26)
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R2° is selected from the group consisting of hydroxy,
-C(O)OH, morpholin-4-yl, piperidin-1-yl, piperazin-1-yl
pyrrolidin-1-yl and -NRzlRaz;
R8 is selected from the group consisting of hydrogen, lower
5 alkyl, 5 or 6-member ring cycloalkyl, lower alkoxy, aryl
optionally substituted with a -NHC(O)(lower alkyl) group,
thien-2-yl, thien-3-yl, pyridin-2-yl, pyridin-3-yl and,
combined with R18, a group having the structure
Y Y
O O
Rs Rs
.
or I , and,
Rs / R5 Rs / Rs
10 R9 is selected from the group consisting of hydrogen and
lower alkyl.
Still another presently preferred aspect of this
invention is a compound in which Q is:
R~2 R»
Rio
N
H
15 It is a presently preferred aspect of this compound
that, in a compound in which Q is
R~ 2 R~ ~
Rio
N
H
R1 is selected from the group consisting of hydrogen, lower
alkyl and - (CH2) "R2°;
n is 2 or 3;
SUBSTITUTE SHEET (RULE 26)
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26
Rz° is selected from the group consisting of hydroxy
and -C (O) OH;
Rz is selected from the group consisting of hydrogen, halo,
lower alkyl, lower alkoxy, -SOzNRzlRzz and -C (O) OH;
R3 is selected from the group consisting of hydrogen, halo,
lower alkyl, lower alkoxy and aryl optionally substituted
with one or more lower alkoxy groups; and,
R' i s hydrogen .
Likewise, in a compound in which Q is
R12 R11
Rio
N
H
it is a presently preferred aspect of this invention that
R1 is selected from the group consisting of hydrogen, lower
alkyl and - ( CHz ) nRzo ;
n is 2 or 3 ;
Rz° is selected from the group consisting of hydroxy
and -C (O) OH;
Rz is selected from the group consisting of hydrogen, halo,
lower alkyl, lower alkoxy, -SOZNRzIRzz and -C (O) OH;
R3 is selected from the group consisting of hydrogen, halo,
lower alkyl, lower alkoxy and aryl optionally substituted
with one or more lower alkoxy groups;
R4 is hydrogen;
Rl° is selected from the group consisting of lower alkyl and
-C (O) OR19;
Rll is selected from the group consisting of~hydrogen, lower
alkyl and -C (O) OR19;
Rlz i S _ ( CH2 ) nRzo ;
SUBSTITUTE SHEET (RULE 26)
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27
R19 is selected from the group consisting of hydrogen
and lower alkyl;
n is 2 or 3; and,
Rz° is selected from the group consisting of hydroxy,
-C(O)OH, morpholin-4-yl, piperidin-1-yl, piperazin-1-yl,
pyrrolidin-1-yl and -NRzlRzz.
It is yet another presently preferred aspect of this
invention that, in a compound in which Q is
R12 R11
Rio
N
H
R1 is selected from the group consisting of hydrogen, lower
alkyl and - (CHz) nRzo;
n is 2 or 3;
Rz° is selected from the group consisting of hydroxy
and -C (O) OH;
Rz is selected from the group consisting of hydrogen, halo,
lower alkyl , lower alkoxy, -S02NRz1Rzz and -C (O) OH;
R3 is selected from the group consisting of hydrogen, halo,
lower alkyl, lower alkoxy and aryl optionally substituted
with one or more lower alkoxy groups;
R4 is hydrogen;
Rl° is selected from the group consisting of lower alkyl and
-C (O) O (lower alkyl) ;
Rll is selected from the group consisting of hydrogen, lower
alkyl and -C (O) OR19;
Rlz is - ( CHz ) nRz° ;
R19 is selected from the group consisting of hydrogen
and lower alkyl;
n is 2; and,
SUBSTITUTE SHEET (RULE 26)
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28
R2° is selected from the group consisting of -C(0)OH,
morpholin-1-yl, piperidin-1-yl, piperazin-1-yl,
pyrrolidin-1-yl and -N(lower alkyl)z.
It is also a presently preferred aspect of this
invention that Q is
[H]r
H R~4
R~5 a.
..
R~6
R17 '. y R~3
[H]r
R1 is selected from the group consisting of hydrogen and
lower alkyl;
Rz is selected from the group consisting of hydrogen, lower
alkyl, halo, -C (O) OR19, -C (O) R19, -NR2IRZZ, -SOzNRzlRaz and
-N-C-RZa ;
R3 is selected from the group consisting of hydrogen, halo,
lower alkyl, lower alkoxy, morpholin-4-yl, piperidin-1-yl,
piperazin-1-yl, pyrrolidin-1-yl and aryl optionally
substituted with one or more lower alkoxy groups,; and,
R4 i s hydrogen .
It is a presently preferred aspect of this invention
that, in a compound in which Q is
[H] r
H R~4
R~s
~a
R~s
R~~ ''' H R~s
[H]r
a and b are both single bonds;
r is 2;
SUBSTITUTE SHEET (RULE 26)
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29
R1 is selected from the group consisting of hydrogen and
lower alkyl;
Rz is selected from the group consisting of hydrogen, lower
alkyl, halo, -C (O) OR19, -C (O) R19, -NR21Rz2, -602NR21Raz arid
-N=C-R23 ;
R3 is selected from the group consisting of hydrogen, halo,
lower alkyl, lower alkoxy, morpholin-4-yl, piperidin-1-yl,
piperazin-1-yl, pyrrolidin-1-yl and aryl optionally
substituted with one or more lower alkoxy groups;
R9 i s hydrogen ;
R13 is a covalent bond through which Q is bonded to the rest
of the molecule;
R14 is selected from the group consisting of hydrogen, lower
alkyl and - ( CHz ) nRzo ;
Rls is hydrogen; and,
R16 and Rl' are independently selected from the group
consisting of hydrogen and lower alkyl.
It is also a presently preferred aspect of this
invention that, in a compound having the substituents
indicated in the paragraph immediately above, R2° is -NRZIRa2
wherein Rzl and R22 are independently selected from the group
consisting of hydrogen and lower alkyl.
It is yet another presently preferred aspect of this
invention that, in a compound in which Q is
~H~r
H R~4
R~5
R~6
R» ~ H~R~3
[H]r
a and b are both double bonds;
r is 1;
SUBSTITUTE SHEET (RULE 26)
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R1 is selected from the group consisting of hydrogen and
lower alkyl;
Rz is selected from the group consisting of hydrogen, lower
alkyl, halo, -C (O) OR19, -C (O) R19, -NRZIRzz, -SOzNRzlRzz arid
5 -N=C-Rza ;
R3 is selected from the group consisting of hydrogen, halo,
lower alkyl, lower alkoxy, morpholin-4-yl, piperidin-1-yl,
piperazin-1-yl, pyrrolidin-1-yl and aryl optionally
substituted with one or more lower alkoxy groups;
10 R4 is hydrogen;
R13 is a covalent bond through which Q is bonded to the rest
of the molecule;
R14 is selected from the group consisting of hydrogen, lower
alkyl and - ( CHz ) nRzo ;
15 Rls and R16 are hydrogen; and,
Rl' does not exist .
A compound in which Q is
14
15
R
R1s
X13
I
(H)r
a and b are both double bonds;
20 r is 1;
R1 is selected from the group consisting of hydrogen and
lower alkyl;
Rz is selected from the group consisting of hydrogen, lower
alkyl, halo, -C (O) OR19, -C (O) R19, -NR2lRzz, -SOzNRzlRzz arid
25 -N=C-Rz3;
R3 is selected from t'~e group consisting of hydrogen, halo,
lower alkyl, lower alkoxy, morpholin-4-yl, piperidin-1-yl,
piperazin-1-yl, pyrrolidin-1-yl and aryl optionally
SUBSTITUTE SHEET (RULE 26)
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31
substituted with one or more lower alkoxy groups;
R4 is hydrogen;
Rlj is selected from the group consisting of hydrogen and
lower alkyl;
R1' is a covalent bond through which Q is bonded to the rest
of the molecule;
R15 and R16 are hydrogen; and,
Rl' does not exist is another presently preferred aspect of
this invention.
It is a presently preferred aspect of this invention
that, in a compound where Q is
r
H R' 4
R15 . a.
R~s
Ri~~~H R~s
[H]r
a and b are double bonds;
r is 1;
R1 is selected from the group consisting of hydrogen and
lower alkyl;
Rz is selected from the group consisting of hydrogen, lower
alkyl, halo, -C (O) OR19, -C (O) R19, -NRzlRzz, -SOzNRz''Rzz and
-N-C-Rza ;
R3 is selected from the group consisting of hydrogen, halo,
lower alkyl, lower alkoxy, morpholin-4-yl, piperidin-1-yl,
piperazin-1-yl, pyrrolidin-1-yl and aril optionally
substituted with one or more lower alkoxy groups;
R4 is hydrogen;
R13 is selected from the group consisting of hydrogen and
lower alkyl;
R14 is selected from the group consisting of hydrogen, lower
alkyl and - (CHz) nRzo;
SUBSTITUTE SHEET (RULE 26)
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32
R15 is a covalent bond through which Q is bonded to the rest
of the molecule;
R16 are hydrogen; and,
Rl' does not exist .
A compound selected from the group consisting of:
3-(3,5-Diisopropyl-4-methoxybenzylidene)-1,3-dihydroindol-2-
one
5-Chloro-3-(3,5-diisopropyl-4-methoxybenzylidene)-1,3-
dihydroindol-2-one
N-[3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-
dihydro-1H-indol-6-yl]-acetamide
3-(3,5-Diisopropyl-4-methoxybenzylidene)-6-hydroxy-1,3-
dihydroindol-2-one
5-Acetyl-3-(3,5-diisopropyl-4-methoxybenzylidene)-1,3-
dihydroindol-2-one
3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-dihydro-
1H-indole-5-carboxylic acid methyl ester
3-(3-Isopropyl-4-methoxybenzylidene)-1,3-dihydroindol-2-one
3-(5-Isopropyl-4-methoxy-2-methylbenzylidene)-1,3-
dihydroindol-2-one
5-Chloro-3-(5-isopropyl-4-methoxy-2-methylbenzylidene)-1,3-
dihydroindol-2-one
3-(3-Cyclopentyl-4-methoxybenzylidene)-1,3-dihydroindol-2-
one
3-(3-Cyclopentyl-4-methoxybenzylidene)-5-fluoro-1,3-
dihydroindol-2-one
3-(3-Cyclohexyl-4-methoxybenzylidene)-1,3-dihydroindol-2-one
5-Bromo-3-(6-methoxybiphenyl-3-ylmethylene)-1,3-
dihydroindol-2-one
5-Chloro-3-(2,3-dihydrobenzofuran-5-ylmethylene)-1,3-
dihydroindol-2-one
5-Chloro-3-(2,2-dimethylchroman-6-ylmethylene)-1,3-
dihydroindol-2-one
SUBSTITUTE SHEET (RULE 26)
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33
N-~3-[3-Cyclohexyl-4-(2-morpholin-4-ylethoxy)-benzylidene]-
2-oxo-2,3-dihydro-1H-indol-6-yl}-acetamide
3-(3,5-Diisopropyl-4-methoxybenzylidene)-5-methoxy-1,3-
dihydroindol-2-one
N-[3-(4-Methoxy-3-thiophen-3-ylbenzylidene)-2-oxo-2,3-
dihydro-1H-indol-6-yl]-acetamide
3-(3,5-Diisopropyl-4-methoxybenzylidene)-5-methyl-1,3-
dihydroindol-2-one
5-Amino-3-(3,5-diisopropyl-4-methoxybenzylidene)-1,3-
dihydroindol-2-one
5-Chloro-3-(4-methoxy-3,5-dimethylbenzylidene)-1,3-
dihydroindol-2-one
3-(3,5-Diisopropyl-4-methoxybenzylidene)-6-fluoro-1,3-
dihydroindol-2-one
3-(2,2-Dimethylchroman-6-ylmethylene)-5-fluoro-1,3-
dihydroindol-2-one
5-Chloro-3-[3,5-diisopropyl-4-(2-morpholin-4-ylethoxy)-
benzylidene]-1,3-dihydroindol-2-one
3-(3,5-Diisopropyl-4-methoxybenzylidene)-7-fluoro-1,3-
dihydroindol-2-one
3-(4-Methoxy-3-thiophen-3-ylbenzylidene)-5-(2-morpholin-4-
ylethyl)-1,3-dihydroindol-2-one
N-[3-(5-Isopropyl-4-methoxy-2-methylbenzylidene)-2-oxo-2,3-
dihydro-1H-indol-6-yl]-acetamide
3-(3,5-Diisopropyl-4-methoxybenzylidene)-5-ethyl-1,3-
dihydroindol-2-one
N-[2'-Methoxy-5'-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-
biphenyl-3-yl]-acetamide
5-Fluoro-3-(5-isopropyl-4-methoxy-2-methylbenzylidene)-1,3-
dihydroindol-2-one
N-[3-(4-Methoxy-3-thiophen-2-ylbenzylidene)-2-oxo-2,3-
dihydro-1H-indol-6-yl]-acetamide
6-Amino-3-(3,5-diisopropyl-4-methoxybenzylidene)-1,3-
SUBSTITUTE SHEET (RULE 26)
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34
dihydroindol-2-one
N-[3-(2,2-Dimethylchroman-6-ylmethylene)-2-oxo-2,3-dihydro-
1H-indol-6-yl]-acetamide
5-Bromo-3-(2,2-dimethylchroman-6-ylmethylene)-1,3-
dihydroindol-2-one
3-(4-Methoxy-3-thiophen-3-ylbenzylidene)-1,3-dihydroindol-2-
one
5-Bromo-3-(5-isopropyl-4-methoxy-2-methylbenzylidene)-1,3-
dihydroindol-2-one
5-Fluoro-3-(6-methoxybiphenyl-3-ylmethylene)-1,3-
dihydroindol-2-one
3-(3-Isopropyl-4-methoxybenzylidene)-4-methyl-1,3-
dihydroindol-2-one
3-(4,5-Dimethoxy-2-thiophen-2-ylbenzylidene)-1,3-
dihydroindol-2-one
N-{3-[4-(2-Morpholin-4-ylethoxy)-3-thiophen-2-
ylbenzylidene]-2-oxo-2,3-dihydro-1H-indol-6-yl}-acetamide
3-(2,2-Dimethylchroman-6-ylmethylene)-4-methyl-1,3-
dihydroindol-2-one
3-(2,3-Dihydrobenzofuran-5-ylmethylene)-5-fluoro-1,3-
dihydroindol-2-one
3-(3-Cyclohexyl-4-methoxybenzylidene)-5-fluoro-1,3-
dihydroindol-2-one
5-Fluoro-3-(3-isopropyl-4-methoxybenzylidene)-1,3-
dihydroindol-2-one
3-(5-Isopropyl-4-methoxy-2-methylbenzylidene)-1,3-
dihydropyrrolo[2,3-b]pyridin-2-one
3-(3'-Ethoxy-6-methoxybiphenyl-3-ylmethylene)-1,3-
dihydroindol-2-one
3-(3-Cyclopentyl-4-methoxybenzylidene)-1,3-
dihydropyrrolo[2,3-b]pyridin-2-one
3-(3-Cyclopentyl-4-methoxybenzylidene)-4-methyl-1,3-
dihydroindol-2-one
SUBSTITUTE SHEET (RULE 26)
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3-(4,5,2'-Trimethoxybiphenyl-2-ylmethylene)-1,3-
dihydroindol-2-one
N-~3-[4-(2-Morpholin-4-ylethoxy)-3-thiophen-3-
ylbenzylidene]-2-oxo-2,3-dihydro-1H-indol-6-yl~-acetamide
5-Chloro-3-(3-cyclohexyl-4-methoxybenzylidene)-1,3-
dihydroindol-2-one
[3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-dihydro-
1H-indol-6-yl]-carbamic acid tert-butyl ester
3-(3,5-Diisopropyl-4-methoxybenzylidene)-4-methyl-1,3-
dihydroindol-2-one
5-Bromo-3-(3,5-diisopropyl-4-methoxybenzylidene)-1,3-
dihydroindol-2-one
N-f3-[3-tert-Butyl-4-(2-morpholin-4-ylethoxy)-benzylidene]-
2-oxo-2,3-dihydro-1H-indol-6-yl}-acetamide
3-(4-Methoxy-3,5-dimethylbenzylidene)-1,3-
dihydropyrrolo[2,3-b]pyridin-2-one
5-Bromo-3-[3,5-diisopropyl-4-(2-morpholin-4-ylethoxy)-
benzylidene]-1,3-dihydroindol-2-one
3-(3'-Ethoxy-4,5-dimethoxybiphenyl-2-ylmethylene)-1,3-
dihydroindol-2-one
5-Chloro-3-(4-methoxy-3-thiophen-2-ylbenzylidene)-1,3-
dihydroindol-2-one
5-Chloro-3-(4-methoxy-3-pyridin-3-ylbenzylidene)-1,3-
dihydroindol-2-one
5-Chloro-3-(4,5,3'-trimethoxybiphenyl-2-ylmethylene)-1,3-
dihydroindol-2-one
3-(4,5-Dimethoxy-2-naphthalen-2-ylbenzylidene)-1,3-
dihydroindol-2-one
N-[3-(3'-Acetylamino-6-methoxybiphenyl-3-ylmethylene)-2-oxo-
2,3-dihydro-1H-indol-6-yl)-acetamide
6-Methoxy-3-(4-methoxy-3-thiophen-3-ylbenzylidene)-1,3-
dihydroindol-2-one
3-(6-Methoxybiphenyl-3-ylmethylene)-1,3-dihydroindol-2-one
SUBSTITUTE SHEET (RULE 26)
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3-(2,3-Dihydrobenzofuran-5-ylmethylene)-1,3-dihydroindol-2-
one
5-Chloro-3-(6-methoxybiphenyl-3-ylmethylene)-1,3-
dihydroindol-2-one
3-(3-Cyclohexyl-4-methoxybenzylidene)-4-methyl-1,3-
dihydroindol-2-one
3-(2,3-dihydrobenzofuran-5-ylmethylene)-4-methyl-1,3-
dihydroindol-2-one
3-(3-Isopropyl-4-methoxybenzylidene)-1,3-dihydropyrrolo[2,3-
b]pyridin-2-one
3-(6-Methoxybiphenyl-3-ylmethylene)-1,3-dihydropyrrolo[2,3-
b]pyridin-2-one
3-(3-Cyclohexyl-4-methoxybenzylidene)-1,3-
dihydropyrrolo[2,3-b]pyridin-2-one
3-(2,3-Dihydrobenzofuran-5-ylmethylene)-1,3-
dihydropyrrolo[2,3-b]pyridin-2-one
3-(3,5-Diisopropyl-4-methoxybenzylidene)-1,3-
dihydropyrrolo[2,3-b]pyridin-2-one
5-Bromo-3-(3-isopropyl-4-methoxybenzylidene)-1,3-
dihydroindol-2-one
5-Bromo-3-(3-cyclopentyl-4-methoxybenzylidene)-1,3-
dihydroindol-2-one
5-Chloro-3-(3-cyclopentyl-4-methoxybenzylidene)-4-methyl-
1,3-dihydroindol-2-one
5-Chloro-3-(6-methoxybiphenyl-3-ylmethylene)-4-methyl-1,3-
dihydroindol-2-one
5-Chloro-3-(5-isopropyl-4-methoxy-2-methylbenzylidene)-4-
methyl-1,3-dihydroindol-2-one
5-Chloro-3-(4-methoxy-3,5-dimethylbenzylidene)-4-methyl-1,3-
dihydroindol-2-one
3-(3,5-Diisopropyl-4-methoxybenzylidene)-6-trifluoromethyl-
1,3-dihydroindol-2-one
6-Chloro-3-(3,5-diisopropyl-4-methoxybenzylidene)-1,3-
SUBSTITUTE SHEET (RULE 26)
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dihydroindol-2-one
3-[3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-
dihydro-1H-indol-5-yl]-propionic acid
3-(3,5-Diisopropyl-4-methoxybenzylidene)-6-methoxy-1,3-
dihydroindol-2-one
5-Butyl-3-(3,5-diisopropyl-4-methoxybenzylidene)-1,3-
dihydroindol-2-one
3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-dihydro-
1H-indole-4-carboxylic acid
3-(3,5-Diisopropyl-4-methoxybenzylidene)-6-(3-
methoxyphenyl)-1,3-dihydroindol-2-one
7-Chloro-3-(3,5-diisopropyl-4-methoxybenzylidene)-5-methyl-
1,3-dihydroindol-2-one
[3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-dihydro-
1H-indol-5-yl]-carbamic acid tent-butyl ester
5-Chloro-3-(3-isopropyl-4-methoxybenzylidene)-1,3-
dihydroindol-2-one
5-Chloro-3-(3-cyclopentyl-4-methoxybenzylidene)-1,3-
dihydroindol-2-one
3-(6-Methoxybiphenyl-3-ylmethylene)-4-methyl-1,3-
dihydroindol-2-one
3-(5-Isopropyl-4-methoxy-2-methylbenzylidene)-4-methyl-1,3-
dihydroindol-2-one
5-Bromo-3-(2,3-dihydrobenzofuran-5-ylmethylene)-1,3-
dihydroindol-2-one
5-Chloro-3-(3-isopropyl-4-methoxybenzylidene)-4-methyl-1,3-
dihydroindol-2-one
5-Chloro-3-(3,5-diisopropyl-4-methoxybenzylidene)-4-methyl-
1,3-dihydroindol-2-one
5-Chloro-3-(2,2-dimethylchroman-6-ylmethylene)-4-methyl-1,3-
dihydroindol-2-one
3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-dihydro-
1H-indole-5-carboxylic acid
SUBSTITUTE SHEET (RULE 26)
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3-(3,5-Diisopropyl-4-methoxybenzylidene)-5,6-dimethoxy-1,3-
dihydroindol-2-one
N-[3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-
dihydro-1H-indol-6-yl]-methanesulfonamide
N-[3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-
dihydro-1H-indol-6-yl]-benzamide
3-(3,5-Diisopropyl-4-methoxybenzylidene)-6-(3-ethoxyphenyl)-
1,3-dihydroindol-2-one
3-(3,5-Diisopropyl-4-methoxybenzylidene)-6-phenyl-1,3-
dihydroindol-2-one
3-(3,5-Diisopropyl-4-methoxybenzylidene)-5-fluoro-1,3-
dihydroindol-2-one
5-Fluoro-3-(4-methoxy-3,5-dimethylbenzylidene)-1,3-
dihydroindol-2-one
3-(2,2-dimethylchroman-6-ylmethylene)-1,3-dihydroindol-2-one
is also a preferred embodiment of this invention.
Likewise, a compound selected from the group consisting of:
5-methyl-3-(3-methyl-1H-indol-2-ylmethylene)-1,3-
dihydroindol-2-one,
3-(3-methyl-1H-indol-2-ylmethylene)-2-oxo-2,3-dihydro-1H-
indol-5-sulfonic acid amide,
3(3-methyl-1H-indol-2-ylmethylene)-2-oxo-2,3-dihydro-1H-
indole-5-sulfonic acid methylamide,
3-(3-methyl-1H-indole-2-ylmethylene)-2-oxo-2,3-dihydro-1H-
indole-5-sulfonic acid dimethylamide,
3-(3-methyl-1H-indol-2-ylmethylene)-2-oxo-2,3-dihydro-1H-
indole-5-carboxylic acid,
5-acetyl-3-(3-methyl-1H-indol-2-ylmethylene)-1,3-
dihydroindol-2-one,
5-acetyl-3-(1H-indol-2-ylmethylene)-1,3-dihydroindol-2-
one,
3-(1H-indol-2-ylmethylene)-2-oxo-2,3-dihydro-1H-indol-5-
sulfonic acid amide,
SUBSTITUTE SHEET (RULE 26)
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5-amino-3-(1H-indol-2-ylmethylene)-1,3-dihydroindol-2-one,
3-(1H-indol-2-ylmethylene)-2-oxo-2,3-dihydro-1H-indole-5-
carboxylic acid,
6-chloro-3-(1H-indol-2-ylmethylene)-1,3-dihydroindol-2-
one,
3-(1H-indol-2-ylmethylene)-1,3-dihydroindol-2-one,
5-chloro-3-(1H-indol-2-ylmethylene)-1,3-dihydroindol-2-
one,
5-bromo-3-(1H-indol-2-ylmethylene)-1,3-dihydroindol-2-one,
3-(1H-indol-2-ylmethylene)-4-methyl-1,3-dihydroindol-2-
one,
3-(3-methyl-1H-indol-2-ylmethylene)-1,3-dihydroindol-2-
one,
5-chloro-3-(3-methyl-1H-indol-2-ylmethylene)-1,3-
dihydroindol-2-one,
5-bromo-3-(3-methyl-1H-indol-2-ylmethylene)-1,3-
dihydroindol-2-one,
4-methyl-3-(3-methyl-1H-indol-2-ylmethylene)-1,3-
dihydroindol-2-one, and
3-(1H-indol-2-ylmethylene)-5[(1H-indol-2-
ylmethylene)amino]-1,3-dihydroindol-2-one
is also a presently preferred embodiment of this invention.
Finally, with regard to chemical compounds of this
invention, a presently preferred embodiment comprises
compounds selected from the group consisting of:
3-[2-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-4,5,6,7-
tetrahydro-1H-indol-3-yl]-propionic acid,
3-[2-(5-chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-
4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic acid,
3-2-(5-bromo-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-
4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic acid,
3-[2-(4-methyl-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-
4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic acid,
SUBSTTTUTE SHEET (RULE 26)
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3-[2-(5-methyl-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-
4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic acid,
3-[2-(6-chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-
4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic acid,
3-[2-(6-methoxy-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-
4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic acid,
N,N-dimethyl-3-[2-(2-oxo-1,2-dihydro-indol-3-
ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-
propionamide,
3-[3-(3-dimethylamino-propyl)-4,5,6,7-tetrahydro-1H-indol-
2-ylmethylene]-1,3-dihydro-indol-2-one,
3-[2-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-4,5,6,7-
tetrahydro-1H-indol-3-yl]-propionamide,
3-[3-(3-morpholin-4-yl-3-oxo-propyl)-4,5,6,7-tetrahydro-
1H-indol-2-ylmethylene]-1,3-dihydro-indol-2-one,
N-methyl-3-[2-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-
4,5,6,7-tetrahydro-1H-indol-3-yl]-propionamide,
N-(2-morpholin-4-yl-ethyl)-3-[2-(2-oxo-1,2-dihydro-indol-
3-ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-
propionamide, 3-[2-(2-oxo-1,2-dihydro-pyrrolo[2,3-
b]pyridin-3-ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-
yl]-propionic acid,
3-{2-[6-(3-methoxy-phenyl)-2-oxo-1,2-dihydro-indol-3-
ylidenemethyl]-4,5,6,7-tetrahydro-1H-indol-3-yl}-propionic
acid,
3-{2-[6-(4-methoxy-phenyl)-2-oxo-1,2-dihydro-indol-3-
ylidenemethyl]-4,5,6,7-tetrahydro-1H-indol-3-yl}-propionic
acid,
3-(2-(2-oxo-6-phenyl-1,2-dihydro-indol-3-ylidenemethyl)-
4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic acid,
3-{2-[6-(2-methoxy-phenyl)-2-oxo-1,2-dihydro-indol-3-
ylidenemethyl]-4,5,6,7-tetrahydro-1H-indol-3-yl}-propionic
acid,
SUBSTITUTE SHEET (RULE 26)
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3-[2-(5-isopropylsulfamoyl-2-oxo-1,2-dihydro-indol-3-
ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic
acid,
3-[2-(6-morpholin-4-yl-2-oxo-1,2-dihydro-indol-3-
ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic
acid,
3-[2-(5-chloro-4-methyl-2-oxo-1,2-dihydro-indol-3-
ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic
acid,
3-[2-(5-bromo-4-methyl-2-oxo-1,2-dihydro-indol-3-
ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic
acid,
3-[2-(5-bromo-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-
4,5,6,7-tetrahydro-1H-indol-3-yl]-N-(2-morpholin-4-yl-
ethyl)-propionamide,
3-[2-(5-chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-
4,5,6,7-tetrahydro-1H-indol-3-yl]-N-(2-morpholin-4-yl-
ethyl)-propionamide, and
3-[2-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-phenyl]-
propionic acid.
2. SYNTHESIS/COMBINATORIAL LIBRARIES
An additional aspect of this invention is a
combinatorial library of at least ten 3-methylidenyl-2-
indolinone compounds that can be formed by reacting
oxindoles of structure 5 with aldehydes of structure 6, 7
or 8:
R~ R~ R~2 Ro
R2 R8 R6
3
R R4 Ro O R9 ~ R5 OHC N~R~o
CHO H
5 6 7
SUBSTITUTE SHEET (RULE 26)
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[H]r
f ~HO, R~4
R~5, OHC
R~s
CHO, R~3
[H] r
8
wherein R1, RZ, R3, R4, R5, R6, R', Re, R9, Rlo, R11, R12, R13,
R14~ R15, Ris and R1~ have the meanings set forth above.
As used herein, a "combinatorial library" refers to all
the compounds formed by the reaction of each compound of one
dimension with a compound in each of the other dimensions in
a mufti-dimensional array of compounds. In the context of
the present invention, the array is two dimensional and one
dimension represents all the oxindoles of the invention and
the second dimension represents all the aldehydes of the
invention. Each oxindole may be reacted with each and every
aldehyde in order to form a substituted 3-methylidenyl-2-
indolinone compound. All substituted 3-methylidenyl-2-
indolinone compounds formed in this way are within the scope
of the present invention. Also within the scope of the
present invention are smaller combinatorial libraries formed
by the reaction of some of the oxindoles with all of the
aldehydes, all of the oxindoles with some of the aldehydes,
or some of the oxindoles with some of the aldehydes.
The oxindole in the above combinatorial library is
preferably selected from the group consisting of oxindole
itself and substituted oxindoles such as, without
limitation, 6-bromooxindole, 5-hydroxyoxindole, 5-
methoxyoxindole, 6-methoxyoxindole, 5-phenylaminosulfonyl-
oxindole, 4-[2-(2-isopropylphenoxy)ethyl]oxindole, 4-[2-(3-
isopropylphenoxy)-ethyl]oxindole, 4-[2-(4-isopropylphenoxy)-
SUBSTITUTE SHEET (RULE 26)
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ethyl]oxindole, 5-fluorooxindole, 6-fluorooxindole, 7-
fluorooxindole, 6-trifluoromethyloxindole, 5-chlorooxindole,
6-chlorooxindole, 4-carboxyindole, 5-bromooxindole, 5-bromo-
4-methyloxindole, 6-(N-acetamido)oxindole, 4-methyloxindole,
5-methyloxindole, 4-methyl-5-chlorooxindole, 5-
ethyloxindole, 6-hydroxyoxindole, 5-acetyloxindole, 5-
carboxyindole, 5-aminooxindole, 6-aminooxindole, 4-(2-N-
morpholinoethyl)oxindole, 7-azaoxindole, oxindole-4-
carabamic acid t-butyl ester, oxindole-6-carbamic acid t-
butyl ester,4-(2-carboxyethyl)oxindole, 5-n-butyloxindole,
5,6-dimethoxyoxindole, 6-(methanesulfonamido)-oxindole, 6-
(benzamido)oxindole, 5-ethoxyoxindole, 6-phenyloxindole, 6-
(2-methoxyphenyl)oxindole, 6-(3-methoxyphenyl)oxindole, 6-
(4-methoxyphenyl)oxindole, 5-aminosulfonyloxindole, 5-
isopropyl-aminosulfonyloxindole, 5-dimethylaminosulfonyl-
oxindole, 5-(N-morpholinosulfonyl)oxindole, 4-(2-
hydroxyethyl)oxindole, 6-(3-ethoxyphenyl)oxindole, 6-
(morpholin-4-yl)oxindole, 5-(2-(N-morpholino)ethyl)oxindole,
5-(methanesulfonamido)oxindole, 5-methoxycarbonyloxindole
and 5-carboxyethyloxindole.
The aldehyde in the above combinatorial library is
preferably selected from the group consisting of 3-(1-
Benzyl-5-formyl-2,4-dimethyl-1H-pyrrol-3-yl)propionic acid,
3-(5-Formyl-1-methoxycarbonylmethyl-2,4-dimethyl-1H-pyrrol-
3-yl)propionic acid, 3-(5-Formyl-1,2,4-trimethyl-1H-pyrrol-
3-yl)propionic acid, 3-[5-Formyl-1-(3-methoxybenzyl)-2,4-
dimethyl-1H-pyrrol-3-yl]propionic acid methyl ester, 3-(1-
Cyclohexylmethyl-5-formyl-2,4-dimethyl-1H-pyrrol-3-yl)-
propionic acid methyl ester, 3-[1-(2,2-Dimethyl-propyl)-5-
formyl-2,4-dimethyl-1H-pyrrol-3-yl]propionic acid methyl
ester, 1,3,5-Trimethyl-4-(3-morpholin-4-yl-3-oxo-propyl)1H-
pyrrole-2-carbaldehyde, 3-(5-Formyl-1,2,4-trimethyl-1H-
pyrrol-3-yl)-N-(2-morpholin-4-ylethyl)propionamide, 3-(5-
Formyl-1,2,4-trimethyl-1H-pyrrol-3-yl)-1,~-phenylpropionamide,
1,3,5-trimethyl-4-(3-oxo-3-piperidin-1-ylpropyl)-1H-pyrrole-
SUBSTITUTE SHEET (RULE 26)
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2-carbaldehyde, 1,3,5-Trimethyl-4-(3-oxo-3-pyrrolidin-1-
ylpropyl)-1H-pyrrole-2-carbaldehyde, 3-(5-formyl-1,2,4-
trimethyl-1H-pyrrol-3-yl)-N-(4-methoxyphenyl)propionamide,
3-(5-Formyl-1,2,4-trimethyl-1H-pyrrol-3-yl)-N-(4-
methoxyphenyl)propionamide, N-(4-fluorophenyl)-3-(5-formyl-
1,2,4-trimethyl-1H-pyrrol-3-yl)-propionamide, 3-(5-Formyl-
1,2,4-trimethyl-1H-pyrrol-3-yl)-N-(4-trifluoromethylphenyl)-
propionamide, 3-[5-Formyl-1-(3-methoxy-benzyl)-2,4-dimethyl-
1H-pyrrol-3-yl]propionic acid, 3-(1-cyclohexylmethyl-5-
formyl-2,4-dimethyl-1H-pyrrol-3-yl)propionic acid, 3-[1-(3-
fluorobenzyl)-5-formyl-2,4-dimethyl-1H-pyrrol-3-yl]propionic
acid methyl ester, 3-(1-Benzyl-5-formyl-2,4-dimethyl-1H-
pyrrol-3-yl)propionic acid, 3-[1-(4-fluorobenzyl)-5-formyl-
2,4-dimethyl-1H-pyrrol-3-yl]propionic acid methyl ester, 3-
[1-(4-fluorobenzyl)-5-formyl-2,4-dimethyl-1H-pyrrol-3-yl]-
propionic acid, 3-[1-(3-fluorobenzyl)-5-formyl-2,4-dimethyl-
1H-pyrrol-3-yl]propionic acid, 3,5-dimethyl-4-(3-morpholin-
4-yl-propyl)-1H-pyrrole-2-carbaldehyde, 4-(3-
dimethylaminopropyl)-3,5-dimethyl-1H-pyrrole-2-carbaldehyde,
5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid, 3,5-
dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrole-2-
carbaldehyde, 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic
acid (2-dimethylaminoethyl)amide, 3-(2-formyl-4,5,6,7-
tetrahydro-1H-indol-3-yl)propionic acid, 3-(3-
dimethylaminopropyl)-2-formyl-4,5,6,7-tetrahydro-1H-indole,
indole-2-carbaldehyde and 3-methylindole-2-carbaldehyde.
Another aspect of this invention provides a method for
the synthesis of a 3-methylidenyl-2-indolinone of formula 1
comprising reacting an oxindole of formula 5 with an
aldehyde of formula 6, 7 or 8 in a solvent, preferably in
the presence of a base.
Examples of the oxindoles of formula 5 which may be
reacted with an aldehyde of formula 6, 7 or 8 to give the
substituted 3-methylidenyl-2-indolinones of formula 1 are
oxindole itself and substituted oxindoles such as, without
SUBSTTTUTE SHEET (RULE 26)
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limitation, 6-bromooxindole, 5-hydroxyoxindole, 5-
methoxyoxindole, 6-methoxyoxindole, 5-phenylaminosulfonyl-
oxindole, 4-[2-(2-isopropylphenoxy)ethyl]oxindole, 4-[2-(3-
isopropylphenoxy)-ethyl]oxindole, 4-[2-(4-isopropylphenoxy)-
5 ethyl]oxindole, 5-fluorooxindole, 6-fluorooxindole, 7-
fluorooxindole, 6-trifluoromethyloxindole, 5-chlorooxindole,
6-chlorooxindole, 4-carboxyindole, 5-bromooxindole, 5-bromo-
4-methyloxindole, 6-(N-acetamido)oxindole, 4-methyloxindole,
5-methyloxindole, 4-methyl-5-chlorooxindole, 5-ethyl-
10 oxindole, 6-hydroxyoxindole, 5-acetyloxindole, 5-
carboxyindole, 5-aminooxindole, 6-aminooxindole, 4-(2-N-
morpholinoethyl)oxindole, 7-azaoxindole, oxindole-4-
carabamic acid t-butyl ester, oxindole-6-carbamic acid t-
butyl ester,4-(2-carboxyethyl)oxindole, 5-n-butyloxindole,
15 5,6-dimethoxyoxindole, 6-(methanesulfonamido)-oxindole, 6-
(benzamido)oxindole, 5-ethoxyoxindole, 6-phenyloxindole, 6-
(2-methoxyphenyl)oxindole, 6-(3-methoxyphenyl)oxindole, 6-
(4-methoxyphenyl)oxindole, 5-aminosulfonyloxindole, 5-
isopropyl-aminosulfonyloxindole, 5-dimethylaminosulfonyl-
20 oxindole, 5-(N-morpholinosulfonyl)oxindole, 4-(2-
hydroxyethyl)oxindole, 6-(3-ethoxyphenyl)oxindole, 6-
(morpholin-4-yl)oxindole, 5-(2-(N-morpholino)ethyl)oxindole,
5-(methanesulfonamido)oxindole, 5-methoxycarbonyloxindole
and 5-carboxyethyloxindole.
25 Examples of aldehydes of structure 6, 7 or 8 which may
be reacted with oxindoles of structure 2 are, without
limitation, 3-(1-benzyl-5-formyl-2,4-dimethyl-1H-pyrrol-3-
yl)propionic acid, 3-(5-formyl-1-methoxycarbonylmethyl-2,4-
dimethyl-1H-pyrrol-3-yl)-propionic acid, 3-(5-formyl-1,2,4-
30 trimethyl-1H-pyrrol-3-yl)-propionic acid, 3-[5-formyl-1-(3-
methoxybenzyl)-2,4-dimethyl-1H-pyrrol-3-yl]propionic acid
methyl ester, 3-(1-cyclohexylmethyl-5-formyl-2,4-dimethyl-
1H-pyrrol-3-yl)propionic acid methyl ester, 3-[1-(2,2-
dimethylpropyl)-5-formyl-2,4-dimethyl-1H-pyrrol-3-yl]-
35 propionic acid methyl ester, 1,3,5-trimethyl-4-(3-morpholin-
SUBSTITUTE SHEET (RULE 26)
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46
4-yl-3-oxopropyl)-1H-pyrrole-2-carbaldehyde, 3-(5-formyl-
1,2,4-trimethyl-1H-pyrrol-3-yl)-N-(2-morpholin-4-
ylethyl)propionamide, 3-(5-formyl-1,2,4-trimethyl-1H-pyrrol-
3-yl)-N-phenylpropionamide, 1,3,5-trimethyl-4-(3-oxo-3-
piperidin-1-ylpropyl)-1H-pyrrole-2-carbaldehyde, 1,3,5-
trimethyl-4-(3-oxo-3-pyrrolidin-1-yl-propyl)-1H-pyrrole-2-
carbaldehyde, 3-(5-formyl-1,2,4-trimethyl-1H-pyrrol-3-yl)-N-
(4-methoxyphenyl)propionamide, 3-(5-formyl-1,2,4-trimethyl-
1H-pyrrol-3-yl)-N-(4-methoxyphenyl)propionamide, N-(4-
fluorophenyl)-3-(5-formyl-1,2,4-trimethyl-1H-pyrrol-3-yl)-
propionamide, 3-(5-formyl-1,2,4-trimethyl-1H-pyrrol-3-yl)-N-
(4-trifluoromethylphenyl)propionamide, 3-[5-formyl-1-(3-
methoxy-benzyl)-2,4-dimethyl-1H-pyrrol-3-yl]propionic acid,
3-(1-cyclohexylmethyl-5-formyl-2,4-dimethyl-1H-pyrrol-3-
yl)propionic acid, 3-[1-(3-fluorobenzyl)-5-formyl-2,4-
dimethyl-1H-pyrrol-3-yl]propionic acid methyl ester, 3-(1-
benzyl-5-formyl-2,4-dimethyl-1H-pyrrol-3-yl)propionic acid,
3-[1-(4-fluorobenzyl)-5-formyl-2,4-dimethyl-1H-pyrrol-3-
yl]propionic acid methyl ester, 3-[1-(4-fluorobenzyl)-5-
formyl-2,4-dimethyl-1H-pyrrol-3-yl]-propionic acid, 3-[1-(3-
fluorobenzyl)-5-formyl-2,4-dimethyl-1H-pyrrol-3-yl]propionic
acid, 3,5-dimethyl-4-(3-morpholin-4-yl-propyl)-1H-pyrrole-2-
carbaldehyde, 4-(3-dimethylaminopropyl)-3,5-dimethyl-1H-
pyrrole-2-carbaldehyde, 5-formyl-2,4-dimethyl-1H-pyrrole-3-
carboxylic acid, 3,5-dimethyl-4-(4-methylpiperazine-1-
carbonyl)-1H-pyrrole-2-carbaldehyde, 5-formyl-2,4-dimethyl-
1H-pyrrole-3-carboxylic acid (2-dimethylaminoethyl)amide, 3-
(2-formyl-4,5,6,7-tetrahydro-1H-indol-3-yl)propionic acid,
3-(3-dimethylaminopropyl)-2-formyl-4,5,6,7-tetrahydro-1H-
indole, indole-2-carbaldehyde and 3-methylindole-2-
carbaldehyde.
The synthesis of an aminopropyl-4,5,6,7-tetrahydro-1H-
indole, structure 9, below, which is an intermediate to some
of the compounds of this invention, is another aspect of
this invention:
SUBSTITUTE SHEET (RULE 26)
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47
OH NXY
0 0 O O O
base, Solve~n~t 0 _Amine,~ O
0 NHz~HCI O R~ 0 / ~ R~ CHZCi2, rt / ~ R~
Rz N Rz N Rz
step 1 step 2
reducing agent
O
solvent
step 3
NXY NXY
NXY
R=, N O POCI3~ DMF
R~ t
~ N~ RZ base, solvent, / ~ R~ 5 ~C ~ / ~ R~
0 O R
R--~~O H N Rz step 4 N 2
step 5
In Step 1, the base can be an inorganic or an organic
base. Examples of inorganic and organic bases are
presented elsewhere herein. Preferrably the base is sodium
acetate. The "solvent" may be any solvent in which base and
the other reactants are sufficiently soluble for reaction
to take place. Preferred solvents are polar erotic solvents
(defined elsewhere herein) such as water, methanol and
ethanol. The presently preferred solvent is water. The
reaction is carried out at temperatures of from about 60° C
to about 180° C. Preferably the temperature is between about
60° C and 180° C, more preferably between 80° C and
150° C,
most preferably between 100 and 120° C. The reaction is
allowed to run for from 1 to 30 hours, preferably from 2 to
20 hours, most preferably from 4 to 15 hours.
In step 2, a carboxylic acid group is converted to an
amide group. Procedures for accomplishing this conversion
are well-known in the art. The reaction of an amine with
the carboxylic acid in the presence of 1,1'-carbonyl
diimidazole is the presently preferred method. Any aprotic
solvent may be used; in a presently preferred embodiment
the solvent is a non-polar aprotic solvent, in particular
SUBSTITUTE SHEET (RULE 26)
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48
dichoromethane. The reaction may be carried out at room
temperature or at an elevated temperature up to about 100°
C; a presently preferred embodiment is to run the reaction
at about room temperature.
Step 3 is the simultaneous reduction of the keto and
the amido group using a reducing agent such as, for example
and without limitation, lithium aluminum hydride. The
reaction is carried out in an aprotic solvent (defined
elsewhere herein). A presently preferred solvent is
tetrahydrofuran. The reaction is carried out at
temperatures from about room temperature to about 80° C,
preferrably from about 50° C to about 70° C, most
preferably, the reaction is carried out in tetrahydrofuran
at reflux.
Step 4 is the well-known formylation of the aromatic
pyrrole ring using phosphorus oxychloride and N,N-dimethyl
formamide.
Step 5, the condensation of an aldehyde with an
oxindole to form a 3-methylidenyl-2-indolinone of this
invention is carried out in a solvent which may contain a
base. The base may be an organic or an inorganic base. If
an organic base is used, preferably it is a nitrogen base.
Examples of organic nitrogen bases include, but are not
limited to, diisopropylamine, trimethylamine, triethylamine,
aniline, pyridine, 1,8-diazabicyclo-[5.4.1]undec-7-ene,
pyrrolidine and piperidine.
Examples of inorganic bases are, without limitation,
ammonia, alkali metal or alkaline earth hydroxides,
phosphates, carbonates, bicarbonates, acetates, bisulfates
and amides. The alkali metals include, lithium, sodium and
potassium while the alkaline earths include calcium,
magnesium and barium.
In a presently preferred embodiment of this invention,
when the solvent is a erotic solvent, such as water or
SUBSTITUTE SHEET (RULE 26)
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49
alcohol, the base is an alkali metal or an alkaline earth
inorganic base, preferably, a alkali metal or an alkaline
earth hydroxide.
It will be clear to those skilled in the art, based
both on known general principles of organic synthesis and on
the disclosures herein which base would be most appropriate
for the reaction contemplated.
The solvent in which the reaction is carried out may be
a erotic or an aprotic solvent, preferably it is a erotic
solvent. A "erotic solvent" is a solvent which has hydrogen
atoms) covalently bonded to oxygen or nitrogen atoms which
renders the hydrogen atoms appreciably acidic and thus
capable of being "shared" with a solute through hydrogen
bonding. Examples of erotic solvents include, without
limitation, water and alcohols.
An "aprotic solvent" may be polar or non-polar but, in
either case, does not contain acidic hydrogens and therefore
is not capable of hydrogen bonding with solutes. Examples,
without limitation, of non-polar aprotic solvents, are
pentane, hexane, benzene, toluene, methylene chloride and
carbon tetrachloride. Examples of polar aprotic solvents
are chloroform, tetrahydro-furan, dimethylsulfoxide and
dimethylformamide.
In a presently preferred embodiment of this invention,
the solvent is a erotic solvent, preferably water or an
alcohol such as ethanol.
The reaction is carried out at temperatures greater
than room temperature. The temperature is generally from
about 30° C to about 150° C, preferably about 80°C to
about
100° C, most preferable about 75° C to about 85° C, which
is
about the boiling point of ethanol.
By "about" is meant that a temperature range described
herein is preferably within 10 degrees Celcius of the
indicated temperature, more preferably within 5 degrees
SUBSTTTLTTE SHEET (RITLE 26)
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Celsius of the indicated temperature and, most preferably,
within 2 degrees Celsius of the indicated temperature.
Thus, for example, by "about 75° C" is meant 75° C ~
10° C,
preferably 75° C ~ 5° C and most preferably, 75° C ~
2° C.
5 Some representative compounds of this invention are
shown in Table 1. The compounds shown are examples only and
are not to be construed as limiting the scope of this
invention in any manner whatsoever.
SUBSTITUTE SHEET (RULE 26)
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S1
TABLE 7
CompoundStructures Names
~
/
o
1 ', 3-(3,5-Diisopropyl-4-methoxybenzylidene)-1,3-dihydroindol-2-
one
0
N
H
O
5-Chloro-3-(3,5-diisopropyl-4-methoxybenzylidene)-1,3-
dihydroindol-2-one
0
', N
H
/
O
N-[3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-dihydro-
0 1 H-indol-6-yl]-acetamide
~O
~N
H H
/
O
3-(3,5-Diisopropyl-4-methoxybenzylidene)-6-hydroxy-1,3-
dihydroindol-2-one
0
I H H
i /
I O
5-Acetyl-3-(3,5-diisopropyl-4-methoxybenzylidene)-1,3-
dihydroindol-2-one
o i
H
O
I
6 0 ~ 3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-
dihydro-1
H-
0 indole-5-carboxylic acid methyl ester
0
N
i H
.O
7 ' 3-(3-Isopropyl-4-methoxybenzylidene)-1,3-dihydroindol-2-one
0
N
H
O
3-(5-Isopropyl-4-methoxy-2-methylbenzylidene)-1,3-dihydroindol-
0 2-one
N
H
O
5-Chloro-3-(5-isopropyl-4-methoxy-2-methylbenzylidene)-1,3-
~~ ~ o dihydroindol-2-one
N
H
O
3-(3-Cyclopentyl-4-methoxybenzylidene)-1,3-dihydroindol-2-one
0
N
H
SUBSTITUTE SHEET (RULE 26)
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52
0
11 3-(3-Cyclopentyl-4-methoxybenzylidene)-5-fluoro-1,3-
dihydroindol
F 2-one
0
N
H
O
12 3-(3-Cyclohexyl-4-methoxybenzylidene)-1,3-dihydroindol-2-
one
o
N
H
/
O I
13 I 5-Bromo-3-(6-methoxybiphenyl-3-ylmethylene)-1,3-
dihydroindol-2
O
I one
i
N I
H
O
14 G 5-Chloro-3-(2,3-dihydrobenzofuran-5-ylmethylene)-1,3-
o ~ dihydroindol-2-one
N
H
O
15 5-Chloro-3-(2,2-dimethylchroman-6-ylmethylene)-1,3-
dihydroindol
~
~, ,
2-one
I O
N I
H
i JN'~o I
16 ! N-{3-[3-Cyclohexyl-4-(2-morpholin-4-ylethoxy)-
benzylidene]-2-oxo
o I 2,3-dihydro-1 H-indol-6-yl}-acetamide
~N N ~
H H
O
17 3-(3,5-Diisopropyl-4-methoxybenzylidene)-5-methoxy-1,3-
,o~ dihydroindol-2-one
~ r0
N
H i
~I .O I
i
S ! N-[3-(4-Methoxy-3-thiophen-3-ylbenzylidene)-2-oxo-2,3-dihydro-
i ~ 0 1 H-indol-6-yl]-acetamide
i
I
i o
I
19 3-(3,5-Diisopropyl-4-methoxybenzylidene)-5-methyl-1,3-
dihydroindol-2-one
0
N i
H
O
~
20 ' 5-Amino-3-(3,5-diisopropyl-4-methoxybenzylidene)-1,3-
H,N ; dihydroindol-2-one
0
N
H
SUBSTITUTE SHEET (RULE 26)
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53
o
21 ~ ! 5-Chloro-3-(4-methoxy-3,5-dimethylbenzylidene)-1,3-dihydroindol
I
2-one
I o
i N i
H
I
O
3-(3,5-Diisopropyl-4-methoxybenzylidene)-6-fluoro-1,3-
22 ~
o dihydroindol-2-one
F H
I
I
O I
23 I' I 3-(2,2-Dimethylchroman-6-ylmethylene)-5-fluoro-1,3-dihydroindol-
2-one
o I
!
I I
I
n
~,. vN~p ''..
24 5-Chloro-3-[3,5-diisopropyl-4-(2-morpholin-4-ylethoxy)-
benzylidene]-1,3-dihydroindol-2-one
0
j H
I i
I p i
I I 3-(3,5-Diisopropyl-4-methoxybenzylidene)-7-fluoro-1,3-
25 i ~o ~ dihydroindol-2-one
N I
F H
I
.O
01 s ~ 3-(4-Methoxy-3-thiophen-3-ylbenzylidene)-5-(2-morpholin-4-
26 i ~N~o ; ylethyl)-1,3-dihydroindol-2-one
I - ~ H~- I
I
~O
27 ~ ~ N-[3-(5-Isopropyl-4-methoxy-2-methylbenzylidene)-2-oxo-2,3
0 o dihydro-1 H-indol-6-yl]-acetamide
Ii ~ H H i
i / I.
i O
2$ ',, 3-(3,5-Diisopropyl-4-methoxybenzylidene)-5-ethyl-1,3-
~~ dihydroindol-2-one
i ~o
I H I
.O
29 I N-[2'-Methoxy-5'-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-
o I biphenyl-3-yl]-acetamide
I N I
I H
i
I
O
30 ' . 5-Fluoro-3-(5-isopropyl-4-methoxy-2-methylbenzylidene)-1,3-
F dihydroindol-2-one
0
N I
H
SUBSTITUTE SKEET (RULE 26)
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54
_o
31 s N-[3-(4-Methoxy-3-thiophen-2-ylbenzylidene)-2-oxo-2,3-
dihydro-
o ' 1 H-indol-6-ylj-acetamide
~
N N
H H
O
32
6-Amino-3-(3,5-diisopropyl-4-methoxybenzylidene)-1,3-
dihydroindol-2-one
o
ii
H
i O I
I~
33 I N-[3-(2,2-Dimethylchroman-6-ylmethylene)-2-oxo-2,3-dihydro-
1
o H-
o ' ~ NH ,, indol-6-ylj-acetamide
~
I ~H i
I
I
O
34 5-Bromo-3-(2,2-dimethylchroman-6-ylmethylene)-1,3-
dihydroindol
2-one
o
N
H
_O
i
I
35 s 3-(4-Methoxy-3-thiophen-3-ylbenzylidene)-1,3-dihydroindol-
2-one
o .
i H I.
O
I I
36 ' ; 5-Bromo-3-(5-isopropyl-4-methoxy-2-methylbenzylidene)-
1,3-
0
I dihydroindol-2-one
N
I H
I I
37 i, 5-Fluoro-3-(6-methoxybiphenyl-3-ylmethylene)-1,3-
dihydroindol-2
0
one
N
H
I
' 3-(3-Isopropyl-4-methoxybenzylidene)-4-methyl-1
3-dihydroindol-
38 ,
' 2-one
I
I H
i
~ _O
j _ i
s ~~ 3-(4,5-Dimethoxy-2-thiophen-2-ylbenzylidene)-1,3-dihydroindol-2-
39
' o one
N
H
JNZO
40 s I N-{3-[4-(2-Morpholin-4-ylethoxy)-3-thiophen-2-
ylbenzylidenej-2-
o ~o ' oxo-2,3-dihydro-1 H-indol-6-yl}-acetamide
~ N-~'~ ~H
I H
41 0 3-(2,2-Dimethylchroman-6-ylmethylene)-4-methyl-1,3-
dihydroindol
N
2-one
H
SUBSTITUTE SHEET (RULE 26)
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WO 00/08202 PCT/US99/17845
I o j
3-(2,3-Dihydrobenzofuran-5-ylmethylene)-5-fluoro-1,3-
42 F o , dihydroindol-2-one
I N
H
i ~ I
I
43 3-(3-Cyclohexyl-4-methoxybenzylidene)-5-fluoro-1,3-dihydroindol-
F !, 2-one
0
' H
j I
j ~O I
i
I F ! 5-Fluoro-3-(3-isopropyl-4-methoxybenzylidene)-1,3-dihydroindol-2
' o ' one
I p f
II H I
j i
/
i °
45 3-(5-Isopropyl-4-methoxy-2-methylbenzylidene)-1,3-
I dihydropyrrolo[2,3-b]pyridin-2-one
0
N N
H
O' I
_O I
46 ~ j 3-(3'-Ethoxy-6-methoxybiphenyl-3-ylmethylene)-1,3-dihydroindol-
2-one
~~o
N
I H
i
j I
i O
I 3-(3-Cyclopentyl-4-methoxybenzylidene)-1,3-dihydropyrrolo[2,3-
47 ~ ~ I b]pyridin-2-one
o I
N N
I H I
O
4$ ~ 3-(3-Cyclopentyl-4-methoxybenzylidene)-4-methyl-1,3-
N o dihydroindol-2-one
I H I
.0
j ' 3- 4 5,2'-Trimethox bi hen I-2- Imeth lene -1,3-dih droindol-2-
49 ~ o I ( , Y P Y Y Y ) Y
' ' i, one
I ° I
I H I
JN..'O I
50 ~ s ~ N-(3-[4-(2-Morpholin-4-ylethoxy)-3-thiophen-3-ylbenzylidene]-2-
0 ~0 oxo-2,3-dihydro-1 H-indol-6-yl}-acetamide
~N~ I
H ,
i
i
O
51 ~ 'I 5-Chloro-3-(3-cyclohexyl-4-methoxybenzylidene)-1,3-dihydroindol
i 2-one
o
N
H
SUBSTTTUTE SHEET (RULE 26)
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WO 00/08202 PCT/US99/17845
56
o i
52 ~ [3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-
dihydro-1
H-
indol-6-yl]-carbamic acid tert-butyl
O ester
O~ I
i H I
I
i i
. 3-(3,5-Diisopropyl-4-methoxybenzylidene)-4-methyl-1,3-
53
j dihydroindol-2-one
N
i H
I
I
O
5-Bromo-3-(3,5-diisopropyl-4-methoxybenzylidene)-1,3-
54
~~ dihydroindol-2-one
0
i N
I H
I
55 ' ' N-{3-(3-tert-Butyl-4-(2-morpholin-4-ylethoxy)-
benzylidene]-2-oxo-
0 2,3-dihydro-1 H-indol-6-yl}-acetamide
~N N O
H H
p I
I
56 I~ ' 3-(4-Methoxy-3,5-dimethylbenzylidene)-1,3-
dihydropyrrolo(2,3-
i, b]pyridin-2-one
0
i N H i
n
uN~ I
57 ! ~ 5-Bromo-3-[3,5-diisopropyl-4-(2-morpholin-4-ylethoxy)-
benzylidene]-1,3-dihydroindol-2-one
0
N
H
58 of 3-(3'-Ethoxy-4,5-dimethoxybiphenyl-2-ylmethylene)-1,3-
0
dihydroindol-2-one
I N
H
~O
59 ' S~V~ 5-Chloro-3-(4-methoxy-3-thiophen-2-ylbenzylidene)-1,3-
G dihydroindol-2-one
N
H
'O
I N
60 I , 5-Chloro-3-(4-methoxy-3-pyridin-3-ylbenzylidene)-1,3-
'
! dihydroindol-2-one
i N
I H
I .O
o- ~ 5-Chloro-3-(4,5,3'-trimethoxybiphenyl-2-ylmethylene)-1,3-
61 ~ '
~I c~~ , dihydroindol-2-one
~
~
.o
N
' i
~'~
~
H
SUBSTITUTE SHEET (RULE 26)
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57
.o
62 ~ ~ ~ 3-(4,5-Dimethoxy-2-naphthaten-2-ylbenzylidene)-1,3-dihydroindol
o ; 2-one
H ,
o H I
-N .O
63 N-[3-(3'-Acetylamino-6-methoxybiphenyl-3-ylmethylene)-2-oxo
o I 2,3-dihydro-1 H-indol-6-yl]-acetamide
~ I N o
I N H
H
O
i
', s 6-Methoxy-3-(4-methoxy-3-thiophen-3-ylbenzylidene)-1,3-
o ' dihydroindol-2-one
~ H
O
I
65 ; 3-(6-Methoxybiphenyl-3-ylmethylene)-1,3-dihydroindol-2-one
0
N
H
O
66 ( 3-(2,3-Dihydrobenzofuran-5-ylmethylene)-1,3-dihydroindol-2-one
i o I
H
O
67 ' S-Chloro-3-(6-methoxybiphenyl-3-ylmethylene)-1,3-dihydroindol-2
' G one
0
N
H
i o ! 3-(3-Cyclohexyl-4-methoxybenzylidene)-4-methyl-1,3-
dihydroindol-2-one
I H
69 ! o ' 3-(2,3-dihydrobenzofuran-5-ylmethylene)-4-methyl-1,3-
I ~ o dihydroindol-2-one
N
H
I ~O i
I ~ 3-(3-Isopropyl-4-methoxybenzylidene)-1,3-dihydropyrrolo[2,3-
o ~ b]pyridin-2-one
I N H
O
71 ~ i 3-(6-Methoxybiphenyl-3-ylmethylene)-1,3-dihydropyrrolo[2,3-
b]pyridin-2-one
i ~'~. o ;
i N H I
I O
72 j 3-(3-Cyclohexyl-4-methoxybenzylidene)-1,3-dihydropyrrolo[2,3-
j b]pyridin-2-one
0
N N
H
SUBSTITUTE SHEET (RULE 26)
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58
0
73 I ' 3-(2,3-Dihydrobenzofuran-5-ylmethylene)-1,3-dihydropyrrolo(2,3-
o , bJpyridin-2-one
N H '
i I
O
i
74 I 3-(3,5-Diisopropyl-4-methoxybenzylidene)-1,3-dihydropyrrolo[2,3-
b]pyridin-2-one
H
I
i
I I
i ' S-Bromo-3-(3-isopropyl-4-methoxybenzylidene)-1,3-dihydroindol-
75 I e,~o 2-one
i N~ i
H
I
76 i 5-Bromo-3-(3-cyclopentyl-4-methoxybenzylidene)-1,3-
i e~ a i dihydroindol-2-one
N i
I H
77 ' o, !, 5-Chloro-3-(3-cyclopentyl-4-methoxybenzylidene)-4-methyl-1,3
o ~ dihydroindol-2-one
i N
H i
i i
o ~' 5-Chloro-3-(6-methoxybiphenyl-3-ylmethylene)-4-methyl-1,3-
I ci
78 I I dihydroindol-2-one
N
I H
I O I
79 ' ci ~ ; 5-Chloro-3-(5-isopropyl-4-methoxy-2-methylbenzylidene)-4-
o i methyl-1,3-dihydroindol-2-one
N
H
80 i c~ ~ 5-Chloro-3-(4-methoxy-3,5-dimethylbenzylidene)-4-methyl-1,3-
N o dihydroindol-2-one
H
i I
O
8~ _ 3-(3,5-Diisopropyl-4-methoxybenzylidene)-6-trifluoromethyl-1,3-
dihydroindol-2-one
I FF N O I
i F H
i i
O
/ I.
82 i i 6-Chloro-3-(3,5-diisopropyl-4-methoxybenzylidene)-1,3-
I , dihydroindol-2-one
i o I.
j G H i
i O
3-[3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-dihydro-
83 x
Ho 1 H-indol-5-yl]-propionic acid
0
N
H
SUBSTITUTE SHEET (RULE 26)
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59
I
84 ' ' 3-(3,5-Diisopropyl-4-methoxybenzylidene)-6-methoxy-1,3-
dihydroindol-2-one
H i.
I
O
85 i 5-Butyl-3-(3,5-diisopropyl-4-methoxybenzylidene)-1,3-
~ dihydroindol-2-one
i
N
H I
i
I
0 off o, i 3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-dihydro-1
86 H-
I o ! indole-4-carboxylic acid
N
H
O
8~ 3-(3,5-Diisopropyl-4-methoxybenzylidene)-6-(3-
methoxyphenyl}-
1,3-dihydroindol-2-one
I H
I
O
88 j 7-Chloro-3-(3,5-diisopropyl-4-methoxybenzylidene)-5-methyl-
1,3-
dihydroindol-2-one
0
H
G
O
89 [3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-
dihydro-1
H-
~o N indol-5-yl]-carbamic acid tert-butyl
Y ester
o
o
I H
j ~ i
j . 5-Chloro-3-(3-isopropyl-4-methoxybenzylidene)-1,3-dihydroindol-
90
~ c.
i o ' 2-one
N
I H i
I !
O
91 5-Chloro-3-(3-cyclopentyl-4-methoxybenzylidene)-1,3-
dihydroindol-2-one
0
N
H
O I
~ 3-(6-Methoxybiphenyl-3-ylmethylene)-4-methyl-1
3-dihydroindol-2
92 ; ,
~ ~
j one
H
!
' 3-(5-Isopropyl-4-methoxy-2-methylbenzylidene)-4-methyl-1,3-
93 ~
i dihydroindol-2-one
I H
!
0
94 ~ ~ 5-Bromo-3-(2,3-dihydrobenzofuran-5-ylmethylene)-1,3-
& dihydroindol-2-one
0
N
H
SUBSTITUTE SHEET (RULE 26)
CA 02383623 2001-11-29
WO 00/08202 PCT/US99/17845
° 5-Chloro-3-(3-isopropyl-4-methoxybenzylidene)-4-methyl-1,3-
95 c~
° dihydroindol-2-one
N
H
0 5-Chloro-3-(3,5-diisopropyl-4-methoxybenzylidene)-4-methyl-1,3-
96 ~, dihydroindol-2-one
N
H
° 5-Chloro-3-(2,2-dimethylchroman-6-ylmethylene)-4-methyl-1,3-
97 ci
° dihydroindol-2-one
N
H i
i
~i
0
98 I 3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-dihydro-1 H-
I ° ! indole-5-carboxylic acid
HO I N O
H
O
99 ' 3-(3,5-Diisopropyl-4-methoxybenzylidene)-5,6-dimethoxy-1,3-
.o dihydroindol-2-one
o
I .o
Ov /
N-(3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-dihydro-
100 I cH;N_° / 1H-indol-6-yl]-methanesulfonamide
N
H. IS H
O
1
O
101 N-[3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-dihydro
~ op ° 1 H-indol-6-yl]-benzamide
l!/' N N
H H
p
102 I I 3-(3,5-Diisopropyl-4-methoxybenzylidene)-6-(3-ethoxyphenyl)-1,3
° dihydroindol-2-one
VO N
H
i I
i o
103 I ', 3-(3,5-Diisopropyl-4-methoxybenzylidene)-6-phenyl-1,3-
° dihydroindol-2-one
N
H
O
104 3-(3,5-Diisopropyl-4-methoxybenzylidene)-5-fluoro-1,3-
dihydroindol-2-one
0
N
H
SUBSTITUTE SHEET (RULE 26)
CA 02383623 2001-11-29
WO 00/08202 PCT/US99/17845
61
0
105 5-Fluoro-3-(4-methoxy-3,5-dimethylbenzylidene)-1,3-
dihydroindol-
2-one
0
N
H
O
106 3-(2,2-dimethylchroman-6-ylmethylene)-1,3-dihydroindol-2-
one
0
N
H
OH
O
107 ~ 3-{2-[6-(4-Fluoro-phenyl)-2-oxo-1,2-dihydroindol-3-
ylidenemethyl}-
~
H 5-methyl-1 H-pyrrol-3-yl}-propionic
~ acid
i ~ H
H
I ~ / H ..
F /
q,
O
O
O
108 ~ ~~~ ~ 4-(2-Carboxy-ethyl)-5-[6-(4-fluoro-phenyl)-2-oxo-1,2-
dihydroindo1-
H 3-ylidenemethyl}-2-methyl-1 H-pyrrole-3-carboxylic
i H acid ethyl ester
i F /
I
OH
O
109 ~ 3-{2-[6-(2-Methoxy-phenyl)-2-oxo-1,2-dihydroindol-3-
I ~ ~
'
~ ylidenemethyl}-5-methyl-1 H-pyrrol-3-yl}-propionic
i acid
I ~ / H
/ O I
'
OH
O
O
4-(2-Carboxy-ethyl)-5-[6-(2-methoxy-phenyl)-2-oxo-1,2-
~
110 , ~ ~ dihydroindol-3-ylidenemethyl}-2-methyl-1
H H-pyrrole-3-carboxylic
H acid ethyl ester
i/ H
0
111 3-[2-(5-Chloro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-5-
methyl-
~ \ 1 H-pyrrol-3-yl]-propionic acid
I o
~ N i
H i
OH
O
O
112 ~ 1 ~ 4-(2-Carboxy-ethyl)-5-(5-chloro-2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-2-methyl-1 H-pyrrole-3-carboxylic
I o acid ethyl ester
~ N
H
O
O
113 ~ ~ ~ 4-(2-Carboxy-ethyl)-2-methyl-5-(2-oxo-1,2-dihydroindol-3-
~ N~ ylidenemethyl)-1H-pyrrole-3-carboxylic
H acid ethyl ester
O
N
H
SUBSTITUTE SHEET (RULE 26)
CA 02383623 2001-11-29
WO 00/08202 PCT/US99/17845
62
OH
O
O
114 ~ ~ ~ 5-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4-(2-
carboxy
i ~N'~ ethyl)-2-methyl-1 H-pyrrole-3-carboxylic
\ H acid ethyl ester
O
/ N
H
OH
O
115 ~ ~ 3-[5-Methyl-2-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-
pyrrol-
\ i ~H~ 3-yl]-propionic acid
/ H
H
OH
O
116 ~ ~ 3-[2-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-5-
methyl-
i N'~ 1 H-pyrrol-3-yl]-propionic acid
\ N
O
/ N
H
O
~ O~ 4-(2-Carboxy-ethyl)-3-methyl-5-(2-oxo-1,2-dihydroindol-3-
117 i
N
ylidenemethyl)-1 H-pyrrole-2-carboxylic
off acid ethyl ester
\ o
N
H
N
2-Methyl-5-(5-methylsulfamoyl-2-oxo-1,2-dihydroindol-3-
118 ~ ~ ~ ylidenemethyl)-4-(3-morpholin-4-yl-propyl)-1
H-pyrrole-3-
H~-9 \ i "~ ! carboxylic acid ethyl ester
o I
~
I
/ N
H
l
N
2-Meth I-4- 3- 4-meth t- i erazin-1-
o I - ro I -5- 5
Y [ ( Y PP Y)P PY] ( -
119 ~ methylsulfamoyl-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-
HH.S , ~ ~ ', pyrrole-3-carboxylic acid ethyl ester
O= \
~ , O"
H
\N-
O
4-(3-Dimethylamino-propyl)-5-(5-methoxy-2-oxo-1,2-dihydroindol-
120
~
i I3-ylidenemethyl)-2-methyl-1H-pyrrole-3-carboxylic
v acid ethyl ester
N
i
O \ H
O
/ N
H
N
O
5-(5-Methoxy-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-methyl-4
121
~
' i (3-morpholin-4-yl-propyl)-1H-pyrrole-3-carboxylic
~ acid ethyl ester
-
N
r
O \ H
O
/ N
N
5-(5-Methoxy-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-methyl-4
122 ~ [3-(4-methyl-piperazin-1-yl)-propyl]-1H-pyrrole-3-
carboxylic
~ acid
o , ethyl ester
~~
!
SUBSTITUTE SHEET (RULE 26)
CA 02383623 2001-11-29
WO 00/08202 PCT/US99/17845
63
~~
N
2-Methyl-4-(3-morpholin-4-yl-propyl)-5-(2-oxo-5-sulfamoyl-1,2-
123 , ' ~ dihydroindol-3-ylidenemethyl)-1 H-pyrrole-3-carboxylic
acid ethyl
:a r " ester
O
H
~~
2-Methyl-4-[3-(4-methyl-piperazin-1-yl)-propyl}-5-(2-oxo-5-
124 ~ sulfamoyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-
r
~
~-a r carboxylic acid ethyl ester
~
I
I ~~
3-(4-Ethoxycarbonyl-5-methyl-3-(3-morpholin-4-yl-propyl)-1H-
~
125 r \ ~ pyrrol-2-ylmethylene]-2-oxo-2,3-dihydro-1
~ H-indole-5-carboxylic
N acid
HO \ r H
I O
N
N
,- 2-Methyl-5-(5-methylsulfamoyl-2-oxo-1,2-dihydroindol-3-
126 ~ \ ylidenemethyl)-4-(3-pyrrolidin-1-yl-propyl)-1H-pyrrole-3-
carboxylic
~
\ r " acid ethyl ester
i
~ H I
H
N
~ ' 5-(5-Methoxy-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2-
methyl-4
127
(3-pyrrolidin-1-yl-propyl)-1 H-pyrrole-3-carboxylic
acid ethyl ester
\ I N
H
O
~ N
H
~ 3-[4-Ethoxycarbonyl-5-methyl-3-(3-pyrrolidin-1-yl-propyl)-
1H-
128 ~ \ pyrrol-2-ylmethylene}-2-oxo-2,3-dihydro-1H-indole-5-
carboxylic
~
i H acid
' HO
\
~
~ N
H
3-(4-Ethoxycarbonyl-5-methyl-3-[3-(4-methyl-piperazin-1-yl)-
-
129 ' propyl)-1H-pyrrol-2-ylmethylene}-2-oxo-2,3-dihydro-1H-
indole-5-
r r~~ carboxylic acid
\
y O
N
~ 2-Methyl-5-(2-oxo-5-sulfamoyl-1,2-dihydroindol-3-
ylidenemethyl)-
130
4-(3-pyrrolidin-1-yl-propyl)-1 H-pyrrole-3-carboxylic
acid ethyl este
H~-.,$ r ,N,
O ~ ~ O
N
H
SUBSTITUTE SHEET (RULE 26)
CA 02383623 2001-11-29
WO 00/08202 PCT/US99/17845
64
N
5-[4-(2-Hydroxy-ethyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-2-
~
131 ~ r ~ methyl-4-(3-morpholin-4-yl-propyl)-1
H-pyrrole-3-carboxylic acid
r ~~~ ~ ethyl ester
/ N
N
~ 5-[4-(2-Hydroxy-ethyl)-2-oxo-1,2-dihydroindol-3-
ylidenemethyl]-2-
132 ~ ~ ~ methyl-4-(3-pyrrolidin-1-yl-propyl)-1
'~ H-pyrrole-3-carboxylic acid
i ~ ethyl ester
i
/ N
H
\ N-
~- 4-(3-Dimethylamino-propyl)-2-methyl-5-(2-oxo-5-sulfamoyl-
1,2-
133 r ~ dihydroindol-3-ylidenemethyl)-1 H-pyrrole-3-carboxylic
acid ethyl
9 i \ r H ester
/ H
H
\N_
-- 3-[3-(3-Dimethylamino-propyl)-4-ethoxycarbonyl-5-methyl-
1H-
134 r v pyrrol-2-ylmethylene]-2-oxo-2,3-dihydro-1H-indole-5-
carboxylic
r
H
Hp I \ acid
/ N
H
N-
4-(3-Dimethylamino-propyl)-5-[4-(2-hydroxy-ethyl)-2-oxo-1,2-
~
135 H r ~ dihydroindol-3-ylidenemethyl]-2-methyl-1
H-pyrrole-3-carboxylic
i \ r " ' acid ethyl ester
/ N
H
5-[4-(2-Hydroxy-ethyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-2-
136 ~ methyl-4-[3-(4-methyl-piperazin-1-yl)-propyl]-1
H-pyrrole-3-
~
~ carboxylic acid ethyl ester
N
I N~
I
4-(3-Dimethylamino-propyl)-2-methyl-5-(5-methylsulfamoyl-2-oxo-
137 r v ~ 1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylic
acid
.s r
" ethyl ester
/ N
N-
i
O
138 ~ 4-(3-Dimethylamino-propyl)-2-methyl-5-(5-methylsulfamoyl-
2-oxo-
,5 r rN~ 1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylic
o i H acid
N
H
O
HO
139 r ~ 4-(2-Carboxy-ethyl)-3-methyl-5-(4-methyl-2-oxo-1,2-
dihydroindol-
\ i H~('~ 3-ylidenemethyl)-iH-pyrrole-2-carboxylic
/ N acid ethyl ester
H
SUBSTITUTE SHEET (RULE 26)
CA 02383623 2001-11-29
WO 00/08202 PCT/US99/17845
HO
r ~ 4-(2-Carboxy-ethyl)-5-(5-methoxy-2-oxo-1,2-dihydroindol-3-
140
~ ylidenemethyl)-3-methyl-1H-pyrrole-2-carboxylic
\ r H~~ acid ethyl ester
;i
N
H
O
HO
141 r i 4-(2-Carboxy-ethyl)-5-(6-methoxy-2-oxo-1,2-dihydroindol-3-
'
'~
r ~ ylidenemethyl)-3-methyl-1 H-pyrrole-2-carboxylic
1f acid ethyl ester
O
~O ~ N
O
HO
5-(6-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4-(2-carboxy
142
~
~
\ r ethyl)-3-methyl-1 H-pyrrole-2-carboxylic
H acid ethyl ester
~
i
N
Br H
O
HO
143 5-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4-(2-
carboxy
ethyl)-3-methyl-1 H-pyrrole-2-carboxylic
N acid ethyl ester
H
O ,
HD
i
r i ~ i 4-(2-Carboxy-ethyl)-3-methyl-5-(2-oxo-6-phenyl-1,2-
dihydroindol-
i ~ r ~~ 3-ylidenemethyl)-1 H-pyrrole-2-carboxylic
acid ethyl ester
I
H
"
O
HO
4-(2-Carboxy-ethyl)-3-methyl-5-(2-oxo-5-sulfamoyl-1,2-
s r rN t ~ dihydroindol-3-ylidenemethyl)-1 H-pyrrole-2-carboxylic
145 acid ethyl
. ester
".N , ~ H
N
O
HD
4-(2-Carboxy-ethyl)-3-methyl-5-(5-methylsulfamoyl-2-oxo-1,2-
146 ~ dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic
5_ r rH~J acid ethyl
, ester
O"
N
O
HO
4-(2-Carboxy-ethyl)-5-(5-dimethylsulfamoyl-2-oxo-1,2-
147 0 r i ~ dihydroindol-3-ylidenemethyl)-3-methyl-1
H-pyrrole-2-carboxylic
' acid ethyl ester
~ O"
H
O
Hp
-(2-Carboxy-ethyl)-5-(5-isopropylsulfamoyl-2-oxo-1,2-
148 , rN~ ~ dihydroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2-
carboxylic
~
"' , " acid ethyl ester
SUBSTITUTE SHEET (RULE 26)
CA 02383623 2001-11-29
WO 00/08202 PCT/US99/17845
66
/N
149 ~ ~ 3-[3-(3-Dimethylamino-propyl)-1H-indol-2-ylmethylene)-2-
oxo-2,3
~
N dihydro-1 H-indole-5-sulfonic acid
0; . methylamide
~ H
-N
H ~ N
H
N-
150 ~ 3-[3-(3-Dimethylamino-propyl)-1 H-indol-2-ylmethylene]-4-
(2-
H
~ 1 hydroxy-ethyl)-1,3-dihydroindol-2-one
NJ~
H
I O
N
H
\N
151 ~ 3-[3-(3-Dimethylamino-propyl)-1 H-indol-2-ylmethylene]-5-
methoxy
~N ~ ~ 1,3-dihydroindol-2-one
~ ~ H
I / O
N
H
H
CHI H
152 H / /N 1 ~ 5-methyl-3-(3-methyl-1H-indol-2-ylmethylene)-1,3-
dihydroindol-2-
"
~H
,
H one
I
O "
~
H N
H
H
H
H
H ~ ~ ~ H 3-(3-methyl-1 H-indol-2-ylmethylene)-2-oxo-2,3-dihydro-1
153 5 i 'N"~ H-indol-5
HsN- sulfonic acid amide
o I ~ O" "
i
N
H
H
H i
H
H H
154 H r ~N 1 ' 3(3-methyl-1 H-indol-2-ylmethylene)-2-oxo-2,3-dihydro-1
" H-indole-
H~
NS 5-sulfonic acid methylamide
~H, o I ~ "
H
H / N
H
H
H
CHI H
155 3-(3-methyl-1 H-indole-2-ylmethylene)-2-oxo-2,3-dihydro-1
~N. H-
CH' H ~ ~
' "
S
~H; o I ~ N indole-5-sulfonic acid dimethylamide
"
H H
H
H
CHI H
3(3-methyl-1 H-indol-2-ylmethylene)-2-oxo-2,3-dihydro-1
156 Hooc H-indole-
~
H
H
I ~ 5-carboxylic acid
O
H
" H
H
H
(~h H
\
" ~ ~ 5-acetyl-3-(3-methyl-1H-indol-2-ylmethylene)-1,3-dihydroindol-2-
157 "
/ ,N,~
",c-II
~ one
I o
H N
H
H
H
H H
II " I I /
H
158 "'o-o ~ H' 5-acetyl-3-(1H-indol-2-ylmethylene)-1,3-dihydroindol-2-
one
H
I o
" N
H
H
SUBSTITUTE SHEET (RULE 26)
CA 02383623 2001-11-29
WO 00/08202 PCT/US99/17845
67
H
H H
" ~ ~ ~ H 3-(1 H-indol-2-ylmethylene)-2-oxo-2,3-dihydro-1
159 s H-indol-5-sulfonic
r '
"
HzN- acid amide
~
p
o "
H p
H
H
H H
" I 1 / H
160 N"~ 5-amino-3-(1H-indol-2-ylmethylene)-1,3-dihydroindol-2-one
~ ~
p~ H
~
H / N
H
H
H
H H -
1 /J~ 3-(1 H-indol-2-ylmethylene)-2-oxo-2,3-dihydro-1
161 Hooc ~ ~ H~H H-indole-5-
o " carboxylic acid
H N
H
H
H
H H
H I ~ / H
162 " 6-chloro-3-(1H-indol-2-ylmethylene)-1,3-dihydroindol-2-
one
~ I
p~ H
~
a / p
H
H
H H
H I ' / H
"
r
~ H
163 ~ ~ 3-(1H-indol-2-ylmethylene)-1,3-dihydroindol-2-one
a
H / N
H
H
H
H H
I H I ~ / H
164 i a S-chloro-3-(1H-indol-2-ylmethylene)-1,3-dihydroindol-2-
one
I o ~ H
~ /
F1 p
H
I
H
H H
165 ~ 5-bromo-3-(1H-indol-2-ylmethylene)-1,3-dihydroindol-2-one
~ ~
p~"
~
H / p
H
H
H H
I ~ / H
~
~
166 " 3-(1 H-indol-2-ylmethylene)-4-methyl-1,3-dihydroindol-2-
one
~
p
H
~
H / N
H
H
H
Ha H
H I ' / H
N
~
~
167 " ~ ~ 3-(3-methyl-1H-indol-2-ylmethylene)-1,3-dihydroindol-2-
one
o
H p
H
I
H
i ~~ H
168 I a H / Ip~" 5-chloro-3-(3-methyl-1H-indol-2-ylmethylene)-1,3-
dihydroindol-2-
H ~ , N~ one
H
H
H
CHI H
169
/ " 5-bromo-3-(3-methyl-1 H-indol-2-ylmethylene)-1,3-dihydroindol-2-
v
~ " I I v
I ~ o " one
H p
H
SUBSTITUTE SHEET (RULE 26)
CA 02383623 2001-11-29
WO 00/08202 PCT/US99/17845
68
H
q-p H
170 " ~~ ~ /N , 4-methyl-3-(3-methyl-1H-indol-2-ylmethylene)-1,3-
dihydroindol-2-
~ "
ff
o one
"
H
H
~
171 ~ 3-(1H-indol-2-ylmethylene)-5[(1H-indol-2-ylmethylene)-
amino]-1,3
~" " dih
i
l
d
d
2
y
ro
n
-
o
-one
i\
172 ~ "''' 3-(1H-Indol-3-ylmethylene)-2-oxo-2,3-dihydro-1H-indole-5-
"zN~s
\ sulfonic acid amide
o
~ I
~
N
H
CH3
~ ~
3-(1H-Indol-2-ylmethylene)-2-oxo-2,3-dihydro-1H-indole-5-
173 '~
'
N
~
'~
s
, sulfonic acid methylamide
,
'
;
o
N
H
/ \
174 " ~ \ NH 3-(1H-Indol-3-ylmethylene)-2-oxo-2,3-dihydro-1H-indole-5-
i ' carboxylic acid
N
H
' S ~ ~N , ~ 3-(3-Methyl-1H-indol-2-ylmethylene)-2-oxo-2,3-dihydro-1H-indole
175
HzN ~ I % o" 5-sulfonic acid amide
N
i H
I
~l
;".S , ~ ~ 3-(1H-Indol-5-ylmethylene)-2-oxo-2,3-dihydro-1H-indole-5-
176 cH
, o i ~ sulfonic acid methylamide
o
N
H
-1
NH
177 oH 3-(1H-Indol-5-ylmethylene)-2-oxo-2,3-dihydro-1H-indole-5-
~ \ ~
\ carboxylic acid
o
o
N
H
CH3
178 HN-O ~ NH 3-(1H-Indol-3-ylmethylene)-2-oxo-2,3-dihydro-1H-indole-5-
~
~~ i . sulfonic acid methylamide
o
O ~ N
H
1
_
NH
179 "~N I \ ~ 0 5-Amino-3-(1 H-indol-5-ylmethylene)-1,3-dihydroindol-2-
one
N
H
CH _
180 ~ ~ 5-Amino-3-(3-methyl-1 H-indol-2-ylmethylene)-1,3-
dihydroindol-2-
~
o one
,.,2N I \
N
H
SUBSTITUTE SHEET (RULE 26)
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WO 00/08202 PCT/US99/17845
69
CH3 /_\
181 "N.~ , N" 3-(2-Methyl-1H-indol-3-ylmethylene)-2-oxo-2,3-dihydro-1H-
indole
o/ , ~ o cH3 5-sulfonic acid methylamide
N
H
off ~ N" 3-(2-Methyl-1H-indol-3-ylmethylene)-2-oxo-2,3-dihydro-1H-indole
182 ,~
0 5-carboxylic acid
i o c"s
N
H
N"
C"3
183 ~N~g ~ ~ ~ 3-(1H-Indol-5-ylmethylene)-2-oxo-2,3-dihydro-1H-indole-5-
c"3 0, ~ , sulfonic acid dimethylamide
o
'
N
H
CH3
184 NH 3-(2-Methyl-1H-indol-3-ylmethylene)-2-oxo-2,3-dihydro-1H-
indole
N.~
~
\ 5-sulfonic acid dimethylamide
c"3 as ~ /
C CH3
N
H
.,
185 "ZN~S / ~ 3-(1H-Indol-5-ylmethylene)-2-oxo-2,3-dihydro-1H-indole-5-
~
o ~ , N o sulfonic acid amide
H
i CHy O
~
;N,11 ~ N 3-(1H-Indol-2-ylmethylene)-2-oxo-2,3-dihydro-1H-indole-5-
186 ~
s
cH,
.,
i ~ d sulfonic acid dimethylamide
o
N
H
/ \
187 , N" 5-Amino-3-(2-methyl-1 H-indol-3-ylmethylene)-1,3-
dihydroindol-2-
" N one
o ~c"3
i N
H
CHI O
188 N," ~ ,NH 3-(1H-Indol-3-ylmethylene)-2-oxo-2,3-dihydro-1H-indole-5-
cH3' Ds ., sulfonic acid dimethylamide
N o
H
i v
Q
189 "2N ;s ~ 'NH 3-(2-Methyl-1H-indol-3-ylmethylene)-2-oxo-2,3-dihydro-1H-
indole
Q ~ ~ N oc"3 5-sulfonic acid amide
H
COOH
190 " ~ ~ 3-[2-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-
tetrahydro-
~
~ ~
" i 1 H-indol-3-yl]-propionic acid
o"
N
H
H
H
COOH
" r \ 3-[2-(5-chloro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-
191
0' o" tetrahydro-1 H-indol-3-yl]-propionic
acid
" H
H
SUBSTITUTE SHEET (RULE 26)
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coo"
192 " ~ 3-[2-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-
4,5,6,7-
B'~" tetrahydro-1 H-indol-3-yl]-propionic
' acid
''
H
~
H
H
COOH
193 H CHI ~N~ 3-[2-(4-methyl-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-
4,5,6,7-
"
i o tetrahydro-1 H-indol-3-yl]-propionic
acid
H N
H
H
COON
194 H 3-[2-(5-methyl-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-
4,5,6,7-
H tetrahydro-1 H-indol-3-yl]-propionic
~~ acid
i o
H N
H
H
COOH
195 H r v 3-[2-(6-chloro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-
4,5,6,7-
H i -H'" tetrahydro-1 H-indol-3-yl]-propionic
i o acid
p
H
COOH
196 H 3-[2-(6-methoxy-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-
4,5,6,7-
" i o ; tetrahydro-1 H-indol-3-yl]-propionic
acid
"' b
H
C(O)N(Cti~)~
197 " ~ ~ N,N-dimethyl-3-[2-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-
~
~ ~
" 4,5,6,7-tetrahydro-1 H-indol-3-yl]-propionamide
~
I o
" N
N
H
CH:-N(CH~
198 H i v 3-[3-(3-dimethylamino-propyl)-4,5,6,7-tetrahydro-1
- v H-indol-2-
H i ~ oH ylmethylene]-1,3-dihydroindol-2-one
H H
H
C(D)NHz
199 " ~ ~ 3-(2-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-
tetrahydro-
~
~ v
" i 1 H-indol-3-yl]-propionamide
o
H H
H
O
~ I I
O N-C
200 H ~ ( 3-(3-(3-morpholin-4-yl-3-oxo-propyl)-4,5,6,7-tetrahydro-1
H-indol-2
ylmethylene]-1,3-dihydroindol-2-one
H H
H
C(OyJH(CH~
201 " N N-methyl-3-[2-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-
4,5,6,7-
" i o" tetrahydro-1 H-indol-3-yl]-propionamide
" N
H H
SUBSTITUTE SHEET (RULE 26)
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71
_
'..,\ ~~ ,o
202 '" ' N-(2-morpholin-4-yl-ethyl)-3-[2-(2-oxo-1,2-dihydroindol-3-
o' ylidenemethyl)-4,5,6,7-tetrahydro-1
" ., H-indol-3-yl]-propionamide
Hooc
203 i ~ 3-[2-(2-oxo-1,2-dihydro-pyrrolo[2,3-b]pyridin-3-
ylidenemethyl)-
i p 4,5,6,7-tetrahydro-1 H-indol-3-yl]-propionic
acid
N N
H
204 H H N 3-{2-[6-(3-methoxyphenyl)-2-oxo-1,2-dihydroindol-3-
ylidenemethyl]-4,5,6,7-tetrahydro-1
H H-indol-3-yl}-propionic acid
COOFi
205 " ~ . ~, 3-{2-[6-(4-methoxyphenyl)-2-oxo-1,2-dihydroindol-3-
ylidenemethyl]-4,5,6,7-tetrahydro-1
H H-indol-3-yl}-propionic acid
CFi~O
COOH
H ~ N~ 3-[2-(2-oxo-6-phenyl-1,2-dihydroindol-3-ylidenemethyl)-
4,5,6,7-
206
H
~
\ tetrahydro-1 H-indol-3-yl]-propionic
i ~ N ~ acid
I ~ H H
cooH -
207 H H ~ H 3-{2-[6-(2-methoxyphenyl)-2-oxo-1,2-dihydroindol-3-
I N o ylidenemethyl]-4,5,6,7-tetrahydro-1
H-indol-3-yl}-propionic acid
H H
COOH
208 H~ H N 3-[2-(5-isopropylaminosulfonyl-2-oxo-1,2-dihydroindol-3-
~NSCOh ~, ylidenemethyl)-4,5,6,7-tetrahydro-1
H-indol-3-yl]-propionic acid
CHI-C
H N
H
C
a H
H ~ ]J 3-(2-(6-morpholin-4-yl-2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-
209
H i ~ 0 4,5,6,7-tetrah dro-1 H-indol-3- I
y y ]-propionic acid
II N N
O~ H H
COOH
~~ ~ _ _ 3-[2-(5-chloro-4-methyl-2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-
210
c 4,5,6,7-tetrahydro-1 H-indol-3-yl]-propionic
0 acid
H H
H
COON
Nil 3-[2-(5-bromo-4-methyl-2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-
211 ~
I o 4,5,6,7-tetrahydro-1 H-indol-3-yl]-propionic
acid
H H
H
SUBSTITUTE SHEET (RULE 26)
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72
%
~
212 ~ ~"~.' 3-[2-(5-bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-
4,5,6,7-
"' '
tetrahydro-1 H-indol-3-yl]-N-(2-morpholin-4-yl-ethyl)-propionamide
L~
~. ~"~
213 '" ' 3-[2-(5-chloro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-
4,5,6,7-
N, o"' tetrahydro-1 H-indol-3-yl]-N-(2-morpholin-4-yl-ethyl)-
propionamide
COOH
/
214 3-[2-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-phenyl]-
propionic
I o acid
N
H
COOH
i
CH3
" N ~ 3-[4-Methyl-2-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1
215 H H-pyrrol-
/
' I ' 3-yl]-propionic acid
off
H w N
H
H
COOH
CHI
216 ci , / H1 3-[2-(5-Chloro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4-
methyl-
I o 1 H-pyrrol-3-yl]-propionic acid
H ~ N
H
H
i COOH
CH3
217 I H , / H~ ~3-[2-(6-Methoxy-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4-
methyl
o 1 H-pyrrol-3-yl]-propionic acid
I
~
N
H3C0
H
H
COOH
CHI
218 H ,H3 / H~ 3-[2-(4-Methyl-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4-
methyl-
I ko 1 H-pyrrol-3-yl]-propionic acid
H ~ N
H
H
COOH j
CH3
219 H , / H1 3-[2-(6-Chloro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4-
methyl-
i
I o ,
' 1 H-pyrrol-3-yl]-propionic acid
CI ~ N
H
H
COOH
CH
220 a~ , / H' 3-[2-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4-
methyl-
I ko 1 H-pyrrol-3-yl]-propionic acid
H w N
H
H
CHI
3- 2- 5-Meth I-2-oxo-1,2-dih droindol-3-
221 H3c H / N~ lidenemeth I -4-meth 1-
[ ( Y Y Y Y ) Y
I o 1 H-pyrrol-3-yl]-propionic acid
H ~ N
H
H
SUBSTITUTE SHEET (RULE 26)
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73
HOOC
CH,
222 H ~ ~ 3-[2-(5-Methoxy-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4-methyl
HaCO ~ ~ H
I o 1 H-pyrrol-3-yl}-propionic acid
H ~ N ,
H H
C",
223 I " ~ ~ H 3-{2-[6-(3-Methoxyphenyl)-2-oxo-1,2-dihydroindol-3-
cH,o ~ I N~° ylidenemethyl}-4-methyl-1 H-pyrrol-3- I
" y }-propionic acid
"ooc
CH,
224 H ~ , N~ 3-{2-[6-(3-Ethoxyphenyl)-2-oxo-1,2-dihydroindol-3-yl
ct,,cHzo , i N ~ 4-methyl-1 H-pyrrol-3-yl}-propionic acid
H
H
SUBSTITUTE SHEET (RULE 26)
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74
3. BIOCHEMISTRY/PHARMACOTHERAPY
Another aspect of this invention relates to a method for
the modulation of the catalytic activity of a PK by contacting
a PK with a compound of this invention or a physiologically
acceptable salt or prodrug thereof.
As used herein, "PK" refers to receptor protein tyrosine
kinase (RTKs), non-receptor or "cellular" tyrosine kinase
(CTKs) and serine-threonine kinases (STKs).
The term "method" refers to manners, means, techniques
and procedures for accomplishing a given task including, but
not limited to, those manners, means, techniques and
procedures either known to, or readily developed from known
manners, means, techniques and procedures by, practitioners cf
the chemical, pharmaceutical, biological, biochemical and
medical arts.
As used herein, the term "modulation" or "modulating"
refers to the alteration of the catalytic activity of RTKs,
CTKs and STKs. In particular, modulating refers to the
activation of the catalytic activity of RTKs, CTKs and STKs,
preferably the activation or inhibition of the catalytic
activity of RTKs, CTKs and STKs, depending on the
concentration of the compound or salt to which the RTK, CTK cr
STK is exposed or, more preferably, the inhibition of the
catalytic activity of RTKs, CTKs and STKs.
The term "catalytic activity" as used herein refers to
the rate of phosphorylation of tyrosine under the influence,
direct or indirect, of RTKs and/or CTKs or the phosphorylaticn
of serine and threonine under the influence, direct or
indirect, of STKs.
The term "contacting" as used herein refers to bringing a
compound of this invention and a target PK together in such a
manner that the compound can affect the catalytic activity of
the PK, either directly, i.e., by interacting with the kinase
SUBSTITUTE SHEET (RULE 26)
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itself, or indirectly, i.e., by interacting with another
molecule on which the catalytic activity of the kinase is
dependent. Such "contacting" can be accomplished "in vitro,"
i.e., in a test tube, a petri dish or the like. In a test
5 tube, contacting may involve only a compound and a PK of
interest or it may involve whole cells. Cells may also be
maintained or grown in cell culture dishes and contacted with
a compound in that environment. In this context, the ability
of a particular compound to affect a PK related disorder,
10 i.e., the ICso of the compound, defined below, can be
determined before use of the compounds in vivo with more
complex living organisms is attempted. For cells outside the
organism, multiple methods exist, and are well-known to those
skilled in the art, to get the PKs in contact with the
15 compounds including, but not limited to, direct cell
microinjection and numerous transmembrane carrier techniques.
A further aspect of this invention is that the
modulation of the catalytic activity of PKs using a
compound of this invention may be carried out in vitro or
20 in vivo.
"In vitro" refers to procedures performed in an
artificial environment such as, e.g., without limitation,
in a test tube or culture medium.
As used herein, "in vivo" refers to procedures
25 performed within a living organism such as, without
limitation, a mouse, rat, rabbit of human being.
A still further aspect of this invention is that the
protein kinase whose catalytic activity is being modulated
by a compound of this invention is selected from the group
30 consisting of receptor protein tyrosine kinases, a cellular
(or non-receptor) tyrosine kinases and serine-threonine
kinases.
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It is an aspect of this invention that the receptor
protein kinase whose catalytic activity is modulated by a
compound of this invention is selected from the group
consisting of EGF, HER2, HERS, HERO, IR, IGF-1R, IRR,
PDGFRa, PDGFR~i, CSFIR, C-Kit, C-fms, Flk-1R, Flk4,
KDR/Flk-1, Flt-1, FGFR-1R, FGFR-2R, FGFR-3R and FGFR-4R.
In addition, it is an aspect of this invention that
the cellular tyrosine kinase whose catalytic activity is
modulated by a compound of this invention is selected
from the group consisting of Src, Frk, Btk, Csk, Abl,
ZAP70, Fes/Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk,
Hck, Fgr and Yrk.
Another aspect of this invention is that the serine-
threonine protein kinase whose catalytic activity is
modulated by a compound of this invention is selected from
the group consisting of CDK2 and Raf.
A pharmaceutical composition of a compound of this
invention with a pharmaceutically acceptable carrier or
excipient is yet another aspect of this invention. Such
pharmaceutical composition may contain both carriers and
excipients as well as other components generally known to
those skilled in the formulation of pharmaceutical
compositions.
A method for treating or preventing a protein kinase
related disorder in an organism comprising administering a
therapeutically effective amount of a compound, salt or
prodrug of this invention to an organism in need of such
treatment is another aspect of this invention.
As used herein, "PK related disorder," "PK driven
disorder," and "abnormal PK activity" all refer to a
condition characterized by inappropriate, i.e., under or,
more commonly, over, PK catalytic activity, where the
particular PK can be an RTK, a CTK or an STK. Inappropriate
SUBSTTTUTE SHEET (RULE 26)
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catalytic activity can arise as the result of either: (1) PK
expression in cells which normally do not express PKs, (2)
increased PK expression leading to unwanted cell
proliferation, differentiation and/or growth, or, (3)
decreased PK expression leading to unwanted reductions in
cell proliferation, differentiation and/or growth. Over-
activity of a PK refers to either amplification of the gene
encoding a particular PK or production of a level of PK
activity which can correlate with a cell proliferation,
differentiation and/or growth disorder (that is, as the level
of the PK increases, the severity of one or more of the
symptoms of the cellular disorder increases). Under-activity
is, of course, the converse, wherein the severity of one or
more symptoms of a cellular disorder increase as the level of
the PK activity decreases.
As used herein, the terms "prevent", "preventing" and
"prevention" refer to a method for barring an organism from
acquiring a PK related disorder in the first place.
As used herein, the terms "treat", "treating" and
"treatment" refer to a method of alleviating or abrogating
a PK mediated cellular disorder and/or its attendant
symptoms. With regard particularly to cancer, these terms
simply mean that the life expectancy of an individual
affected with a cancer will be increased or that one or
more of the symptoms of the disease will be reduced.
The term "organism" refers to any living entity
comprised of at least one cell. A living organism can be
as simple as, for example, a single eukariotic cell or as
complex as a mammal, including a human being.
The term "therapeutically effective amount" as used
herein refers to that amount of the compound being
administered which will relieve to some extent one or more
of the symptoms of the disorder being treated. In
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reference to the treatment of cancer, a therapeutically
effective amount refers to that amount which has the effect
of (1) reducing the size of the tumor, (2) inhibiting (that
is, slowing to some extent, preferably stopping) tumor
metastasis, (3) inhibiting to some extent (that is, slowing
to some extent, preferably stopping) tumor growth, and/or,
(4) relieving to some extent (or, preferably, eliminating)
one or more symptoms associated with the cancer.
It is an aspect of this invention that the above-
referenced protein kinase related disorder is selected from
the group consisting of a receptor protein tyrosine kinase
related disorder, a cellular tyrosine kinase disorder and a
serine-threonine kinase related disorder.
In yet another aspect of this invention, the above
referenced protein kinase related disorder is selected from
the group consisting of an EGFR related disorder, a PDGFR
related disorder, an IGFR related disorder and a flk
related disorder.
The above referenced protein kinase related disorder
is a cancer selected from the group consisting of squamous
cell carcinoma, sarcomas such as Kaposi's sarcoma,
astrocytoma, glioblastoma, lung cancer, bladder cancer,
colorectal cancer, gastrointestinal cancer, head and neck
cancer, melanoma, ovarian cancer, prostate cancer, breast
cancer, small-cell lung cancer and glioma in a further
aspect of this invention.
The above referenced protein kinase related disorder
is selected from the group consisting of diabetes, an
autoimmune disorder, a hyperproliferation disorder, von
Hippel-Lindau disease, restenosis, fibrosis, psoriasis,
osteoarthritis, rheumatoid arthritis, an inflammatory
disorder and angiogenesis in yet another aspect of this
invention.
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Additional disorders which may be treated or prevented
using the compounds of this invention are immunological
disorders such as autoimmune disease (AIDS) and
cardiovasular disorders such as atherosclerosis.
It is as aspect of this invention that a chemical
compound that modulates the catalytic activity of a protein
kinase may be identified by contacting cells expressing
said protein kinase with a compound, salt or prodrug of the
present invention and then monitoring said cells for an
effect.
By "monitoring" is meant observing or detecting the
effect of contacting a compound with a cell expressing a
particular PK. The observed or detected effect can be a
change in cell phenotype, in the catalytic activity of a PK
or a change in the interaction of a PK with a natural
binding partner. Techniques for observing or detecting
such effects are well-known in the art.
The above-referenced effect is selected from a change
or an absence of change in a cell phenotype, a change or
absence of change in the catalytic activity of said protein
kinase or a change or absence of change in the interaction
of said protein kinase with a natural binding partner in a
final aspect of this invention.
"Cell phenotype" refers to the outward appearance of a
cell or tissue or the biological function of the cell or
tissue. Examples, without limitation, of a cell phenotype
are cell size, cell growth, cell proliferation, cell
differentiation, cell survival, apoptosis, and nutrient
uptake and use. Such phenotypic characteristics are
measurable by techniques well-known in the art.
A "natural binding partner" refers to a polypeptide
that binds to a particular PK in a cell. Natural binding
partners can play a role in propagating a signal in a PK-
SUBSTITUTE SHEET (RULE 26)
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mediated signal transduction process. A change in the
interaction of the natural binding partner with the PK can
manifest itself as an increased or decreased concentration
of the PK/natural binding partner complex and, as a result,
5 in an observable change in the ability of the PK to mediate
signal transduction.
It is also an aspect of this invention that a compound
described herein, or its salt or prodrug, might be combined
with other chemotherapeutic agents for the treatment of the
10 diseases and disorders discussed above. For instance, a
compound, salt or prodrug of this invention might be
combined with alkylating agents such as fluorouracil (5-FU)
alone or in further combination with leukovorin; or other
alkylating agents such as, without limitation, other
15 pyrimidine analogs such as UFT, capecitabine, gemcitabine
and cytarabine, the alkyl sulfonates, e.g., busulfan (used
in the treatment of chronic granulocytic leukemia),
improsulfan and piposulfan; aziridines, e.g., benzodepa,
carboquone, meturedepa and uredepa; ethyleneimines and
20 methylmelamines, e.g., altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide and
trimethylolmelamine; and the nitrogen mustards, e.g.,
chlorambucil (used in the treatment of chronic lymphocytic
leukemia, primary macroglobulinemia and non-Hodgkin's
25 lymphoma), cyclophosphamide (used in the treatment of
Hodgkin's disease, multiple myeloma, neuroblastoma, breast
cancer, ovarian cancer, lung cancer, Wilm's tumor and
rhabdomyosarcoma), estramustine, ifosfamide, novembrichin,
prednimustine and uracil mustard (used in the treatment of
30 primary thrombocytosis, non-Hodgkin's lymphoma, Hodgkin's
disease and ovarian cancer); and triazines, e.g.,
dacarbazine (used in the treatment of soft tissue sarcoma).
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Likewise a compound, salt or prodrug of this invention
might be expected to have a beneficial effect in
combination with other antimetabolite chemotherapeutic
agents such as, without limitation, folic acid analogs,
e.g. methotrexate (used in the treatment of acute
lymphocytic leukemia, choriocarcinoma, mycosis fungiodes
breast cancer, head and neck cancer and osteogenic sarcoma)
and pteropterin; and the purine analogs such as
mercaptopurine and thioguanine which find use in the
treatment of acute granulocytic, acute lymphocytic and
chronic granulocytic leukemias.
A compound, salt or prodrug of this invention might
also be expected to prove efficacious in combination with
natural product based chemotherapeutic agents such as,
without limitation, the vinca alkaloids, e.g., vinblastin
(used in the treatment of breast and testicular cancer),
vincristine and vindesine; the epipodophylotoxins, e.g.,
etoposide and teniposide, both of which are useful in the
treatment of testicular cancer and Kaposi's sarcoma; the
antibiotic chemotherapeutic agents, e.g., daunorubicin,
doxorubicin, epirubicin, mitomycin (used to treat stomach,
cervix, colon, breast, bladder and pancreatic cancer),
dactinomycin, temozolomide, plicamycin, bleomycin (used in
the treatment of skin, esophagus and genitourinary tract
cancer); and the enzymatic chemotherapeutic agents such as
L-asparaginase.
In addition to the above, a compound, salt or prodrug
of this invention might be expected to have a beneficial
effect used in combination with the platinum coordination
complexes (cisplatin, etc.); substituted ureas such as
hydroxyurea; methylhydrazine derivatives, e.g.,
procarbazine; adrenocortical suppressants, e.g., mitotane,
aminoglutethimide; and hormone and hormone antagonists such
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as the adrenocorticosteriods (e. g., prednisone), progestins
(e. g., hydroxyprogesterone caproate); estrogens (e. g.,
diethylstilbesterol); antiestrogens such as tamoxifen;
androgens, e.g., testosterone propionate; and aromatase
inhibitors (such as anastrozole.
Finally, the combination of a compound of this
invention might be expected to be particularly effective in
combination with mitoxantrone or paclitaxel for the
treatment of solid tumor cancers or leukemias such as,
without limitation, acute myelogenous (non-lymphocytic)
leukemia.
DETAILED DESCRIPTION OF THE INVENTION
1. BRIEF DESCRIPTION OF THE TABLES
TABLE 1 shows the chemical structures of some exemplary
compounds of this invention. The compound numbers correspond
to the Example numbers in the Examples section. That is, the
synthesis of Compound 1 in Table 1 is described in Example 1.
The compounds presented in Table 1 are exemplary only and are
not to be construed as limiting the scope of this invention in
any manner.
TABLE 2 shows the results of biological testing of some
exemplary compounds of this invention. The results are
reported in terms of ICso, the micromolar (~tM) concentration
of the compound being tested which causes a 50% change i~ the
activity of the target PKT compared to the activity of the PTK
in a control to which no test compound has been added.
Specifically, the results shown indicate the concentration of
a test compound needed to cause a 50% reduction of the
activity of the target PTK. The bioassays used are described
in detail below.
2. INDICATIONS/TARGET DISEASES
The PKs whose catalytic activity is modulated by the
compounds of this invention include protein tyrosine
SUBSTITUTE SHEET (RULE 26)
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83
kinases of which there are two types, receptor tyrosine
kinases (RTKs) and cellular tyrosine kinases (CTKs), and
serine-threonine kinases (STKs). RTK mediated signal
transduction, is initiated by extracellular interaction
with a specific growth factor (ligand), followed by
receptor dimerization, transient stimulation of the
intrinsic protein tyrosine kinase activity and
phosphorylation. Binding sites are thereby created for
intracellular signal transduction molecules and lead to the
formation of complexes with a spectrum of cytoplasmic
signaling molecules that facilitate the appropriate
cellular response (e. g., cell division, metabolic effects
on the extracellular microenvironment, etc.). See,
Schlessinger and Ullrich, 1992, Neuron 9:303-391.
It has been shown that tyrosine phosphorylation sites
on growth factor receptors function as high-affinity
binding sites for SH2 (src homology) domains of signaling
molecules. Fantl et al., 1992, Cell 69:413-423, Songyang
et al., 1994, Mol. Cell. Biol. 14:2777-2785), Songyang et
al., 1993, Cell 72:767-778, and Koch et al., 1991, Science
252:668-678. Several intracellular substrate proteins that
associate with RTKs have been identified. They may be
divided into two principal groups: (1) substrates that have
a catalytic domain, and (2) substrates which lack such
domain but which serve as adapters and associate with
catalytically active molecules. Songyang et al., 1993,
Cell 72:767-778. The specificity of the interactions
between receptors and SH2 domains of their substrates is
determined by the amino acid residues immediately
surrounding the phosphorylated tyrosine residue.
Differences in the binding affinities between SH2 domains
and the amino acid sequences surrounding the
phosphotyrosine residues on particular receptors are
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consistent with the observed differences in their substrate
phosphorylation profiles. Songyang et al., 1993, Cell
72:767-778. These observations suggest that the function
of each RTK is determined not only by its pattern of
expression and ligand availability but also by the array of
downstream signal transduction pathways that are activated
by a particular receptor. Thus, phosphorylation provides
an important regulatory step which determines the
selectivity of signaling pathways recruited by specific
growth factor receptors, as well as differentiation factor
receptors.
STKs, being primarily cytosolic, affect the internal
biochemistry of the cell, often as a down-line response to
a PTK event. STKs have been implicated in the signaling
process which initiates DNA synthesis and subsequent
mitosis leading to cell proliferation.
Thus, PK signal transduction results in, among other
responses, cell proliferation, differentiation, growth and
metabolism. Abnormal cell proliferation may result in a
wide array of disorders and diseases, including the
development of neoplasia such as carcinoma, sarcoma,
glioblastoma and hemangioma, disorders such as leukemia,
psoriasis, arteriosclerosis, arthritis and diabetic
retinopathy and other disorders related to uncontrolled
angiogenesis and/or vasculogenesis.
A precise understanding of the mechanism by which the
compounds of this invention inhibit PKs is not required in
order to practice the present invention. However, while
not hereby being bound to any particular mechanism or
theory, it is believed that the compounds interact with the
amino acids in the catalytic region of PKs. PKs typically
possess a bi-lobate structure wherein ATP appears to bind
in the cleft between the two lobes in a region where the
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amino acids are conserved among PKs. Inhibitors~of PKs are
believed to bind by non-covalent interactions such as
hydrogen bonding, van der Waals forces and ionic
interactions in the same general region where the aforesaid
5 ATP binds to the PKs. More specifically, it is thought
that the 2-indolinone component of the compounds of this
invention binds in the general space normally occupied by
the adenine ring of ATP. Specificity of a particular
molecule for a particular PK may then arise as the result
10 of additional interactions between the various substituents
on the 2-indolinone core and the amino acid domains
specific to particular PKs. Thus, different indolinone
substituents may contribute to preferential binding to
particular PKs. The ability to select compounds active at
15 different ATP (or other nucleotide) binding sites makes the
compounds of this invention useful for targeting any
protein with such a site. The compounds disclosed herein
may thus have utility as in vitro assays for such proteins
as well as exhibiting in vivo therapeutic effects through
20 interaction with such proteins.
In another aspect, the protein kinase, the catalytic
activity of which is modulated by contact with a compound
of this invention, is a protein tyrosine kinase, more
particularly, a receptor protein tyrosine kinase. Among the
25 receptor protein tyrosine kinases whose catalytic activity
can be modulated with a compound of this invention, or salt
thereof, are, without limitation, EGF, HER2, HER3, HER4,
IR, IGF-1R, IRR, PDGFRa, PDGFR~3, CSFIR, C-Kit, C-fms, Flk-
1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R, FGFR-2R, FGFR-3R and
30 FGFR-4R.
The protein tyrosine kinase whose catalytic activity
is modulated by contact with a compound of this invention,
or a salt or a prodrug thereof, can also be a non-receptor
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or cellular protein tyrosine kinase (CTK). Thus, the
catalytic activity of CTKs such as, without limitation,
Src, Frk, Btk, Csk, Abl, ZAP70, Fes, Fps, Fak, Jak, Ack,
Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk, may be modulated
by contact with a compound or salt of this invention.
Still another group of PKs which may have their
catalytic activity modulated by contact with a compound of
this invention are the serine-threonine protein kinases
such as, without limitation, CDK2 and Raf.
In another aspect, this invention relates to a method
for treating or preventing a PK related disorder by
administering a therapeutically effective amount of a
compound of this invention, or a salt or a prodrug thereof,
to an organism.
It is also an aspect of this invention that a
pharmaceutical composition containing a compound of this
invention or a salt or prodrug thereof is administered to
an organism for the purpose of preventing or treating a PK
related disorder.
This invention is therefore directed to compounds that
modulate PK signal transduction by affecting the enzymatic
activity of RTKs, CTKs and/or STKs, thereby interfering
with the signals transduced by such proteins. More
particularly, the present invention is directed to
compounds which modulate RTK, CTK and/or STK mediated
signal transduction pathways as a therapeutic approach to
the treatment of many kinds of solid tumors, including but
not limited to carcinomas, sarcomas including Kaposi's
sarcoma, erythroblastoma, glioblastoma, meningioma,
astrocytoma, melanoma and myoblastoma. Treatment or
prevention of non-solid tumor cancers such as leukemia are
also contemplated by this invention. Indications may
include, but are not limited to brain cancers, bladder
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cancers, ovarian cancers, gastric cancers, pancreas
cancers, colon cancers, blood cancers, lung cancers and
bone cancers.
Further examples, without limitation, of the types of
disorders related to inappropriate PK activity that the
compounds described herein may be useful in preventing,
treating and studying, are cell proliferative disorders,
fibrotic disorders and metabolic disorders.
Cell proliferative disorders, which may be prevented,
treated or further studied by the present invention include
cancer, blood vessel proliferative disorders and mesangial
cell proliferative disorders.
Blood vessel proliferative disorders refer to
disorders related to abnormal vasculogenesis (blood vessel
formation) and angiogenesis (spreading of blood vessels).
While vasculogenesis and angiogenesis play important roles
in a variety of normal physiological processes such as
embryonic development, corpus luteum formation, wound
healing and organ regeneration, they also play a pivotal
role in cancer development where they result in the
formation of new capillaries needed to keep a tumor alive.
Other examples of blood vessel proliferation disorders
include arthritis, where new capillary blood vessels invade
the joint and destroy cartilage, and ocular diseases, like
diabetic retinopathy, where new capillaries in the retina
invade the vitreous, bleed and cause blindness.
Two structurally related RTKs have been identified to
bind VEGF with high affinity: the fms-like tyrosine 1 (fit-
1) receptor (Shibuya et al., 1990, Onco~ne,5:519-524; De
Vries et al., 1992, Science, 255:989-991) and the KDR/FLK-1
receptor, also known as VEGF-R2. Vascular endothelial
growth factor (VEGF) has been reported to be an endothelial
cell specific mitogen with in vitro endothelial cell growth
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promoting activity. Ferrara & Henzel, 1989, Biochein.
Biophys. Res. Comm., 161:851-858; Vaisman et al., 1990, J.
Biol. Chem., 265:19461-19566. Information set forth in U.S.
application Ser. Nos. 08/193,829, 08/038,596 and
07/975,750, strongly suggest that VEGF is not only
responsible for endothelial cell proliferation, but also is
the prime regulator of normal and pathological
angiogenesis. See generally, Klagsburn & Soker, 1993,
Current Bioloay, 3(10)699-702; Houck, et al., 1992, J.
Biol. Chem., 267:26031-26037.
Normal vasculogenesis and angiogenesis play important
roles in a variety of physiological processes such as
embryonic development, wound healing, organ regeneration
and female reproductive processes such as follicle
development in the corpus luteum during ovulation and
placental growth after pregnancy. Folkman & Shing, 1992, J.
Bioloaical Chem., 267(16):10931-34. Uncontrolled
vasculogenesis and/or angiogenesis has been associated with
diseases such as diabetes as well as with malignant solid
tumors that rely on vascularization for growth. Klagsburn &
Soker, 1993, Current Bioloay, 3(10):699-702; Folkham, 1991,
J. Natl. Cancer Inst., 82:4-6; Weidner, et al., 1991, New
Enal. J. Med., 324:1-5.
The surmised role of VEGF in endothelial cell
proliferation and migration during angiogenesis and
vasculogenesis indicates an important role for the KDR/FLK-
1 receptor in these processes. Diseases such as diabetes
mellitus (Folkman, 198, in XIth Congress of Thrombosis and
Haemostasis (Verstraeta, et al., eds.), pp. 583-596, Leuven
University Press, Leuven) and arthritis, as well as
malignant tumor growth may result from uncontrolled
angiogenesis. See e.g., Folkman, 1971, N. Engl. J. Med.,
285:1182-1186. The receptors to which VEGF specifically
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binds are an important and powerful therapeutic target for
the regulation and modulation of vasculogenesis and/or
angiogenesis and a variety of severe diseases which involve
abnormal cellular growth caused by such processes. Plowman,
et al., 1994, DN&P, 7(6):334-339. More particularly, the
KDR/FLK-1 receptor's highly specific role in
neovascularization make it a choice target for therapeutic
approaches to the treatment of cancer and other diseases
which involve the uncontrolled formation of blood vessels.
Thus, one aspect of the present invention relates to
compounds capable of regulating and/or modulating tyrosine
kinase signal transduction including KDR/FLK-1 receptor
signal transduction in order to inhibit or promote
angiogenesis and/or vasculogenesis, that is, compounds that
inhibit, prevent, or interfere with the signal transduced
by KDR/FLK-1 when activated by ligands such as VEGF.
Although it is believed that the compounds of the present
invention act on a receptor or other component along the
tyrosine kinase signal transduction pathway, they may also
act directly on the tumor cells that result from
uncontrolled angiogenesis.
Although the nomenclature of the human and murine
counterparts of the generic "flk-I" receptor differ, they
are, in many respects, interchangeable. The murine
receptor, Flk-1, and its human counterpart, KDR, share a
sequence homology of 93.4% within the intracellular domain.
Likewise, murine FLK-I binds human VEGF with the same
affinity as mouse VEGF, and accordingly, is activated by
the ligand derived from either species. Millauer et al.,
1993, Cell, 72:835-846; Quinn et al., 1993, Proc. Natl.
Acad. Sci. USA, 90:7533-7537. FLK-1 also associates with
and subsequently tyrosine phosphorylates human RTK
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substrates (e.g., PLC-y or p85) when co-expressed in 293
cells (human embryonal kidney fibroblasts).
Models which rely upon the FLK-1 receptor therefore
are directly applicable to understanding the KDR receptor.
5 For example, use of the murine FLK-1 receptor in methods
which identify compounds that regulate the murine signal
transduction pathway are directly applicable to the
identification of compounds which may be used to regulate
the human signal transduction pathway, that is, which
10 regulate activity related to the KDR receptor. Thus,
chemical compounds identified as inhibitors of KDR/FLK-1 in
vitro, can be confirmed in suitable in vivo models. Both in
vivo mouse and rat animal models have been demonstrated to
be of excellent value for the examination of the clinical
15 potential of agents acting on the KDR/FLK-1 induced signal
transduction pathway.
Thus, in one aspect, this invention is directed to
compounds that regulate, modulate and/or inhibit
vasculogenesis and/or angiogenesis by affecting the
20 enzymatic activity of the KDR/FLK-1 receptor and
interfering with the signal transduced by KDR/FLK-1. In
another aspect, the present invention is directed to
compounds which regulate, modulate and/or inhibit the
KDR/FLK-1 mediated signal transduction pathway as a
25 therapeutic approach to the treatment of many kinds of
solid tumors including, but not limited to, glioblastoma,
melanoma and Kaposi's sarcoma, and ovarian, lung, mammary,
prostate, pancreatic, colon and epidermoid carcinoma. In
addition, data suggest the administration of compounds
30 which inhibit the KDR/Flk-1 mediated signal transduction
pathway may also be used in the treatment of hemangioma,
restenois and diabetic retinopathy.
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A further aspect of this invention relates to the
inhibition of vasculogenesis and angiogenesis by other
receptor-mediated pathways, including the pathway
comprising the flt-1 receptor.
' Receptor tyrosine kinase mediated signal transduction
is initiated by extracellular interaction with a specific
growth factor (ligand), followed by receptor dimerization,
transient stimulation of the intrinsic protein tyrosine
kinase activity and autophosphorylation. Binding sites are
thereby created for intracellular signal transduction
molecules which leads to the formation of complexes with a
spectrum of cytoplasmic signalling molecules that
facilitate the appropriate cellular response, e.g., cell
division and metabolic effects to the extracellular
microenvironment. See, Schlessinger and Ullrich, 1992,
Neuron, 9:1-20.
The close homology of the intracellular regions of
KDR/FLK-1 with that of the PDGF-~3 receptor (50.3% homology)
and/or the related flt-1 receptor indicates the induction
of overlapping signal transduction pathways. For example,
for the PDGF-(3 receptor, members of the src family (Twamley
et al., 1993, Proc. Natl. Acad. Sci. USA, 90:7696-7700),
phosphatidylinositol-3'-kinase (Hu et al., 1992, Mol. Cell.
Biol., 12:981-990), phospholipase cy (Kashishian & Cooper,
1993, Mol. Cell. Biol., 4:49-51), ras-GTPase-activating
protein, (Kashishian et al., 1992, EMBO J., 11:1373-1382),
PTP-ID/syp (Kazlauskas et al., 1993, Proc. Natl. Acad. Sci.
USA, 10 90:6939-6943), Grb2 (Arvidsson et al., 1994, Mol.
Cell. Biol., 14:6715-6726), and the adapter molecules Shc
and Nck (Nishimura et al., 1993, Mol. Cell. Biol., 13:6889-
6896), have been shown to bind to regions involving
different autophosphorylation sites. See generally,
Claesson-Welsh, 1994, Prog. Growth Factor Res., 5:37-54.
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Thus, it is likely that signal transduction pathways
activated by KDR/FLK-1 include the ras pathway (Rozakis et
al., 1992, Nature, 360:689-692), the PI-3'-kinase, the src-
mediated and the plcy-mediated pathways. Each of these
pathways may play a critical role in the angiogenic and/or
vasculogenic effect of KDR/FLK-1 in endothelial cells.
Consequently, a still further aspect of this invention
relates to the use of the organic compounds described
herein to modulate angiogenesis and vasculogenesis as such
processes are controlled by these pathways.
Conversely, disorders related to the shrinkage,
contraction or closing of blood vessels, such as
restenosis, are also implicated and may be treated or
prevented by the methods of this invention.
Fibrotic disorders refer to the abnormal formation of
extracellular matrices. Examples of fibrotic disorders
include hepatic cirrhosis and mesangial cell proliferative
disorders. Hepatic cirrhosis is characterized by the
increase in extracellular matrix constituents resulting in
the formation of a hepatic scar. An increased extracellular
matrix resulting in a hepatic scar can also be caused by a
viral infection such as hepatitis. Lipocytes appear to
play a major role in hepatic cirrhosis. Other fibrotic
disorders implicated include atherosclerosis.
Mesangial cell proliferative disorders refer to
disorders brought about by abnormal proliferation of
mesangial cells. Mesangial proliferative disorders include
various human renal diseases such as glomerulonephritis,
diabetic nephropathy and malignant nephrosclerosis as well
as such disorders as thrombotic microangiopathy syndromes,
transplant rejection, and glomerulopathies. The RTK PDGFR
has been implicated in the maintenance of mesangial cell
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proliferation. Floege et al., 1993, Kidney International
43:47S-54S.
Many cancers are cell proliferative disorders and, as
noted previously, PKs have been associated with cell
proliferative disorders. Thus, it is not surprising that
PKs such as, for example, members of the RTK family have
been associated with the development of cancer. Some of
these receptors, like EGFR (Tuzi et al., 1991, Br. J.
Cancer 63:227-233, Torp et al., 1992, APMIS 100:713-719)
HER2/neu (Slamon et al., 1989, Science 244:707-712) and
PDGF-R (Kumabe et al., 1992, Onco_ene, 7:627-633) are over-
expressed in many tumors and/or persistently activated by
autocrine loops. In fact, in the most common and severe
cancers these receptor over-expressions (Akbasak and Suner-
Akbasak et al., 1992, J. Neurol. Sci., 111:119-133, Dickson
et al., 1992, Cancer Treatment Res. 61:249-273, Korc et
al., 1992, J. Clin. Invest. 90:1352-1360) and autocrine
loops (Lee and Donoghue, 1992, J. Cell. Biol., 118:1057-
1070, Korc et al., supra, Akbasak and Suner-Akbasak et al.,
su ra) have been demonstrated. For example, EGFR has been
associated with squamous cell carcinoma, astrocytoma,
glioblastoma, head and neck cancer, lung cancer and bladder
cancer. HER2 has been associated with breast, ovarian,
gastric, lung, pancreas and bladder cancer. PDGFR has been
associated with glioblastoma and melanoma as well as lung,
ovarian and prostate cancer. The RTK c-met has also been
associated with malignant tumor formation. For example, c-
met has been associated with, among other cancers,
colorectal, thyroid, pancreatic, gastric and hepatocellular
carcinomas and lymphomas. Additionally c-met has been
linked to leukemia. Over-expression of the c-met gene has
also been detected in patients with Hodgkins disease and
Burkitts disease.
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IGF-IR, in addition to being implicated in nutritional
support and in type-II diabetes, has also been associated with
several types of cancers. For example, IGF-I has been
implicated as an autocrine growth stimulator for several tumor
types, e.g. human breast cancer carcinoma cells (Arteaga et
al., 1989, J. Clin. Invest. 84:1418-1423) and small lung tumor
cells (Macauley et al., 1990, Cancer Res., 50:2511-2517). In
addition, IGF-I, while integrally involved in the normal
growth and differentiation of the nervous system, also appears
to be an autocrine stimulator of human gliomas. Sandberg-
Nordqvist et al., 1993, Cancer Res. 53:2475-2478. The
importance of IGF-IR and its ligands in cell proliferation is
further supported by the fact that many cell types in culture
(fibroblasts, epithelial cells, smooth muscle cells, T-
lymphocytes, myeloid cells, chondrocytes and osteoblasts (the
stem cells of the bone marrow)) are stimulated to grow by IGF-
I. Goldring and Goldring, 1991, Eukaryotic Gene
Expression,1:301-326. In a series of recent publications,
Baserga suggests that IGF-IR plays a central role in the
mechanism of transformation and, as such, could be a preferred
target for therapeutic interventions for a broad spectrum of
human malignancies. Baserga, 1995, Cancer Res., 55:249-252,
Baserga, 1994, Cell 79:927-930, Coppola et al., 1994, Mol.
Cell. Biol., 14:4588-4595.
STKs have been implicated in many types of cancer
including, notably, breast cancer (Cance, et al., Int. J.
Cancer, 54:571-77 (1993)).
The association between abnormal PK activity and
disease is not restricted to cancer. For example, RTKs
have been associated with diseases such as psoriasis,
diabetes mellitus, endometriosis, angiogenesis,
atheromatous plaque development, Alzheimer's disease, von
Hippel-Lindau disease, epidermal hyperproliferation,
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neurodegenerative diseases, age-related macular
degeneration and hemangiomas. For example, EGFR has been
indicated in corneal and dermal wound healing. Defects in
Insulin-R and IGF-1R are indicated in type-II diabetes
5 mellitus. A more complete correlation between specific RTKs
and their therapeutic indications is set forth in Plowman
et al., 1994, DN&P 7:334-339.
As noted previously, not only RTKs but CTKs including,
but not limited to, src, abl, fps, yes, fyn, lyn, lck, blk,
10 hck, fgr and yrk (reviewed by Bolen et al., 1992, FASEB J.,
6:3403-3409) are involved in the proliferative and metabolic
signal transduction pathway and thus could be expected, and
have been shown, to be involved in many PTK-mediated disorders
to which the present invention is directed. For example,
15 mutated src (v-src) has been shown to be an oncoprotein (pp60"-
gr°) in chicken. Moreover, its cellular homolog, the proto-
oncogene pp60°-er° transmits oncogenic signals of many
receptors. Over-expression of EGFR or HER2/neu in tumors
leads to the constitutive activation of pp60~sr°
which is
20 characteristic of malignant cells but absent in normal cells.
On the other hand, mice deficient in the expression of c-src
exhibit an osteopetrotic phenotype, indicating a key
participation of c-src in osteoclast function and a possible
involvement in related disorders.
25 Similarly, Zap70 has been implicated in T-cell
signaling which may relate to autoimmune disorders.
STKs have been associated with inflamation, autoimmune
disease, immunoresponses, and hyperproliferation disorders such
as restenosis, fibrosis, psoriasis, osteoarthritis and
30 rheumatoid arthritis.
PKs have also been implicated in embryo implantation.
Thus, the compounds of this invention may provide an effective
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method of preventing such embryo implantation and thereby be
useful as birth control agents.
Finally, both RTKs and CTKs are currently suspected as
being involved in hyperimmune disorders.
A method for identifying a chemical compound that
modulates the catalytic activity of one or more of the
above discussed protein kinases is another aspect of this
invention. The method involves contacting cells expressing
the desired protein kinase with a compound of this
invention (or its salt or prodrug) and monitoring the cells
for any effect that the compound has on them. The effect
may be any observable, either to the naked eye or through
the use of instrumentation, change or absence of change in
a cell phenotype. The change or absence of change in the
cell phenotype monitored may be, for example, without
limitation, a change or absence of change in the catalytic
activity of the protein kinase in the cells or a change or
absence of change in the interaction of the protein kinase
with a natural binding partner.
Examples of the effect of a number of exemplary
compounds of this invention on several PTKs are shown in
Table 2. The compounds and data presented are not to be
construed as limiting the scope of this invention in any
manner whatsoever.
3. PHARMACEUTICAL COMPOSITIONS AND USE
A compound of the present invention, a prodrug thereof
or a physiologically acceptable salt of either the compound
or its prodrug, can be administered as such to a human
patient or can be administered in pharmaceutical
compositions in which the foregoing materials are mixed
with suitable carriers or excipient(s). Techniques for
formulation and administration of drugs may be found in
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"Remington's Pharmacological Sciences," Mack Publishing
Co., Easton, PA, latest edition.
Routes of Administration.
As used herein, "administer" or "administration"
refers to the delivery of a compound, salt or prodrug of
the present invention or of a pharmaceutical composition
containing a compound, salt or prodrug of this invention to
an organism for the purpose of prevention or treatment of a
PK-related disorder.
Suitable routes of administration may include, without
limitation, oral, rectal, transmucosal or intestinal
administration or intramuscular, subcutaneous,
intramedullary, intrathecal, direct intraventricular,
intravenous, intravitreal, intraperitoneal, intranasal, or
intraocular injections. The preferred routes of
administration ary oral and parenteral.
Alternatively, one may administer the compound in a
local rather than systemic manner, for example, via
injection of the compound directly into a solid tumor,
often in a depot or sustained release formulation.
Furthermore, one may administer the drug in a targeted
drug delivery system, for example, in a liposome coated
with tumor-specific antibody. The liposomes will be
targeted to and taken up selectively by the tumor.
Composition/Formulation.
Pharmaceutical compositions of the present invention
may be manufactured by processes well known in the art,
e.g., by means of conventional mixing, dissolving,
granulating, dragee-making, levigating, emulsifying,
encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with
the present invention may be formulated in conventional
manner using one or more physiologically acceptable
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carriers comprising excipients and auxiliaries which
facilitate processing of the active compounds into
preparations which can be used pharmaceutically. Proper
formulation is dependent upon the route of administration
chosen.
For injection, the compounds of the invention may be
formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiological saline buffer. For
transmucosal administration, penetrants appropriate to the
barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art.
For oral administration, the compounds can be
formulated by combining the active compounds with
pharmaceutically acceptable carriers well known in the art.
Such carriers enable the compounds of the invention to be
formulated as tablets, pills, lozenges, dragees, capsules,
liquids, gels, syrups, slurries, suspensions and the like,
for oral ingestion by a patient. Pharmaceutical
preparations for oral use can be made using a solid
excipient, optionally grinding the resulting mixture, and
processing the mixture of granules, after adding other
suitable auxiliaries if desired; to obtain tablets or
dragee cores. Useful excipients are, in particular,
fillers such as sugars, including lactose, sucrose,
mannitol, or sorbitol, cellulose preparations such as, for
example, maize starch, wheat starch, rice starch and potato
starch and other materials such as gelatin, gum tragacanth,
methyl cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinyl-pyrrolidone (PVP).
If desired, disintegrating agents may be added, such as
cross-linked polyvinyl pyrrolidone, agar, or alginic acid.
A salt such as sodium alginate may also be used.
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Dragee cores are provided with suitable coatings. For
this purpose, concentrated sugar solutions may be used
which may optionally contain gum arabic, talc, polyvinyl
pyrrolidone, carbopol gel, polyethylene glycol, and/or
titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures. Dyestuffs or pigments may be
added to the tablets or dragee coatings for identification
or to characterize different combinations of active
compound doses.
Pharmaceutical compositions which can be used orally
include push-fit capsules made of gelatin, as well as soft,
sealed capsules made of gelatin and a plasticizer, such as
glycerol or sorbitol. The push-fit capsules can contain
the active ingredients in admixture with a filler such as
lactose, a binder such as starch, and/or a lubricant such
as talc or magnesium stearate and, optionally, stabilizers.
In soft capsules, the active compounds may be dissolved or
suspended in suitable liquids, such as fatty oils, liquid
paraffin, or liquid polyethylene glycols. Stabilizers may
be added in these formulations, also.
For administration by inhalation, the compounds for
use according to the present invention are conveniently
delivered in the form of an aerosol spray using a
pressurized pack or a nebulizer and a suitable propellant,
e.g., without limitation, dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetra-fluoroethane or
carbon dioxide. In the case of a pressurized aerosol, the
dosage unit may be controlled by providing a valve to
deliver a metered amount. Capsules and cartridges of, for
example, gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
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The compounds may also be formulated for parenteral
administration, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in
unit dosage form, e.g., in ampoules or in mufti-dose
containers, with an added preservative. The compositions
may take such forms as suspensions, solutions or emulsions
in oily or aqueous vehicles, and may contain formulating
materials such as suspending, stabilizing and/or dispersing
agents.
Pharmaceutical compositions for parenteral
administration include aqueous solutions of a water soluble
form, such as, without limitation, a salt, of the active
compound. Additionally, suspensions of the active compounds
may be prepared in a lipophilic vehicle. Suitable
lipophilic vehicles include fatty oils such as sesame oil,
synthetic fatty acid esters such as ethyl oleate and
triglycerides, or materials such as liposomes. Aqueous
injection suspensions may contain substances which increase
the viscosity of the suspension, such as sodium
carboxymethyl cellulose, sorbitol, or dextran. Optionally,
the suspension may also contain suitable stabilizers and/or
agents that increase the solubility of the compounds to
allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in powder
form for constitution with a suitable vehicle, e.g.,
sterile, pyrogen-free water, before use.
The compounds may also be formulated in rectal
compositions such as suppositories or retention enemas,
using, e.g., conventional suppository bases such as cocoa
butter or other glycerides.
In addition to the fomulations described previously,
the compounds may also be formulated as depot preparations.
Such long acting formulations may be administered by
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implantation (for example, subcutaneousiy or
intramuscularly) or by intramuscular injection. A compound
of this invention may be formulated for this route of
administration with suitable polymeric or hydrophobic
materials (for instance, in an emulsion with a
pharamcologically acceptable oil), with ion exchange
resins, or as a sparingly soluble derivative such as,
without limitation, a sparingly soluble salt.
A non-limiting example of a pharmaceutical carrier for
the hydrophobic compounds of the invention is a cosolvent
system comprising benzyl alcohol, a nonpolar surfactant, a
water-miscible organic polymer and an aqueous phase such as
the VPD co-solvent system. VPD is a solution of 3% w/v
benzyl alcohol, 8% w/v of the nonpolar surfactant
Polysorbate 80TM, and 65% w/v polyethylene glycol 300, made
up to volume in absolute ethanol. The VPD co-solvent
system (VPD:DSW) consists of VPD diluted 1:1 with a 5%
dextrose in water solution. This co-solvent system
dissolves hydrophobic compounds well, and itself produces
low toxicity upon systemic administration. Naturally, the
proportions of such a co-solvent system may be varied
considerably without destroying its solubility and toxicity
characteristics. Furthermore, the identity of the co-
solvent components may be varied: for example, other low-
toxicity nonpolar surfactants may be used instead of
Polysorbate 80TM, the fraction size of polyethylene glycol
may be varied, other biocompatible polymers may replace
polyethylene glycol, e.g., polyvinyl pyrrolidone, and other
sugars or polysaccharides may substitute for dextrose.
Alternatively, other delivery systems for hydrophobic
pharmaceutical compounds may be employed. Liposomes and
emulsions are well known examples of delivery vehicles or
carriers for hydrophobic drugs. In addtion, certain
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organic solvents such as dimethylsulfoxide also may be
employed, although often at the cost of greater toxicity.
Additionally, the compounds may be delivered using a
sustained-release system, such as semipermeable matrices of
solid hydrophobic polymers containing the therapeutic
agent. Various sustained-release materials have been
established and are well known by those skilled in the art.
Sustained-release capsules may, depending on their
chemical nature, release the compounds for a few weeks up
to over 100 days. Depending on the chemical nature and the
biological stability of the therapeutic reagent, additional
strategies for protein stabilization may be employed.
The pharmaceutical compositions herein also may
comprise suitable solid or gel phase carriers or
excipients. Examples of such carriers or excipients
include, but are not limited to, calcium carbonate, calcium
phosphate, various sugars, starches, cellulose derivatives,
gelatin, and polymers such as polyethylene glycols.
Many of the PK modulating compounds of the invention may
be provided as physiologically acceptable salts wherein the
claimed compound may form the negatively or the positively
charged species. Examples of salts in which the compound
forms the positively charged moiety include, without
limitation, quaternary ammonium (defined elsewhere herein),
salts such as the hydrochloride, sulfate, carbonate, lactate,
tartrate, maleate, succinate wherein the nitrogen atom of the
quaternary ammonium group is a nitrogen of the selected
compound of this invention which has reacted with the
appropriate acid. Salts in which a compound of this invention
forms the negatively charged species include, without
limitation, the sodium, potassium, calcium and magnesium salts
formed by the reaction of a carboxylic acid group in the
compound with an appropriate base (e. g. sodium hydroxide
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(NaOH), potassium hydroxide (KOH), Calcium hydroxide
(Ca (OH) z) , etc. ) .
Dosa~.
Pharmaceutical compositions suitable for use in the
present invention include compositions wherein the active
ingredients are contained in an amount sufficient to
achieve the intended purpose, i.e., the modulation of PK
activity or the treatment or prevention of a PK-related
disorder.
More specifically, a therapeutically effective amount
means an amount of compound effective to prevent, alleviate
or ameliorate symptoms of disease or prolong the survival
of the subject being treated.
Determination of a therapeutically effective amount is
well within the capability of those skilled in the art,
especially in light of the detailed disclosure provided
herein.
For any compound used in the methods of the invention,
the therapeutically effective amount or dose can be
estimated initially from cell culture assays. Then, the
dosage can be formulated for use in animal models so as to
achieve a circulating concentration range that includes the
ICso as determined in cell culture (i.e., the concentration
of the test compound which achieves a half-maximal
inhibition of the PK activity). Such information can then
be used to more accurately determine useful doses in
humans.
Toxicity and therapeutic efficacy of the compounds
described herein can be determined by standard
pharmaceutical procedures in cell cultures or experimental
animals, e.g., by determining the ICso and the LDSO (both of
which are discussed elsewhere herein) for a subject
compound. The data obtained from these cell culture assays
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and animal studies can be used in formulating a range of
dosage for use in humans. The dosage may vary depending
upon the dosage form employed and the route of
administration utilized. The exact formulation, route of
administration and dosage can be chosen by the individual
physician in view of the patient's condition. (See e.g.,
Fingl, et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1 p.1).
Dosage amount and interval may be adjusted
individually to provide plasma levels of the active species
which are sufficient to maintain the kinase modulating
effects. These plasma levels are referred to as minimal
effective concentrations (MECs). The MEC will vary for
each compound but can be estimated from in vitro data,
e.g., the concentration necessary to achieve 50-90%
inhibition of a kinase may be ascertained using the assays
described herein. Dosages necessary to achieve the MEC
will depend on individual characteristics and route of
administration. HPLC assays or bioassays can be used to
determine plasma concentrations.
Dosage intervals can also be determined using MEC
value. Compounds should be administered using a regimen
that maintains plasma levels above the MEC for 10-90% of
the time, preferably between 30-90% and most preferably
between 50-90%.
In cases of local administration or selective uptake,
the effective local concentration of the drug may not be
related to plasma concentration and other procedures known
in the art may be employed to determine the correct dosage
amount and interval.
The amount of a composition administered will, of
course, be dependent on the subject being treated, the
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severity of the affliction, the manner of administration,
the judgment of the prescribing physician, etc.
Packag~ing .
The compositions may, if desired, be presented in a
pack or dispenser device, such as an FDA approved kit,
which may contain one or more unit dosage forms containing
the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration. The pack or dispenser may also be
accompanied by a notice associated with the container in a
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals, which notice
is reflective of approval by the agency of the form of the
compositions or of human or veterinary administration.
Such notice, for example, may be of the labeling approved
by the U.S. Food and Drug Administration for prescription
drugs or of an approved product insert. Compositions
comprising a compound of the invention formulated in a
compatible pharmaceutical carrier may also be prepared,
placed in an appropriate container, and labeled for
treatment of an indicated condition. Suitable conditions
indicated on the label may include treatment of a tumor,
inhibition of angiogenesis, treatment of fibrosis,
diabetes, and the like.
4. SYNTHESIS
The compounds of this invention, as well as the
precursor oxindoles and aldehydes, may be readily
synthesized using techniques well known in the chemical
arts. It will be appreciated by those skilled in the art
that other synthetic pathways for forming the compounds of
the invention are available and that the following is
offered by way of example and not limitation.
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General synthetic procedures.
The following general methodologies may be used to
prepare intermediates to and compounds of this invention.
Preparation of substituted oxindoles
5-Amino-2-oxindole
5-Nitro-2-oxindole (6.3 g) was hydrogenated in
methanol over 10% palladium on carbon to give 3.0 g (60%
yield) of the title compound as a white solid.
5-Bromo-2-oxindole
2-Oxindole (1.3 g) in 20 mL acetonitrile was cooled to
-10 °C and 2.0 g N-bromosuccinimide was slowly added with
stirring. The reaction was stirred for 1 hour at -10 °C and
2 hours at 0 °C. The precipitate was collected, washed with
water and dried to give 1.9 g (90 % yield) of the title
compound.
4-Methyl-2-oxindole
Diethyl oxalate (30 mL) in 20 mL of dry ether was
added with stirring to 19 g of potassium ethoxide suspended
in 50 mL of dry ether. The mixture was cooled in an ice
bath and 20 mL of 3-nitro-o-xylene in 20 mL of dry ether
was slowly added. The thick dark red mixture was heated to
reflux for 0.5 hr, concentrated to a dark red solid, and
treated with 10% sodium hydroxide until almost all of the
solid dissolved. The dark red mixture was treated with 30%
hydrogen peroxide until the red color changed to yellow.
The mixture was treated alternately with 10% sodium
hydroxide and 30% hydrogen peroxide until the dark red
color was no longer present. The solid was filtered off and
the filtrate acidified with 6N hydrochloric acid. The
resulting precipitate was collected by vacuum filtration,
washed with water, and dried under vacuum to give 9.8 a
(45% yield) of 2-methyl-6-nitrophenylacetic acid as an off-
white solid. The solid was hydrogenated in methanol over 10
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% palladium on carbon to give 9.04 g of the title compound
as a white solid.
7-Bromo-5-chloro-2-oxindole
5-Chloro-2-oxindole (16.8 g) and 19.6 g of
N-bromosuccinimide were suspended in 140 mL of acetonitrile
and refluxed for 3 hours. Thin layer chromatography
(silica, ethyl acetate) at 2 hours of reflux showed
5-chloro-2-oxindole or N-bromosuccinimide (Rf 0.8), product
(Rf 0.85) and a second product (Rf 0.9) whose proportions
did not change after another hour of reflux. The mixture
was cooled to 10 °C, the precipitate was collected by vacuum
filtration, washed with 25 mL of ethanol and sucked dry for
minutes in the funnel to give 14.1 g of wet product (56
yield). The solid was suspended in 200 mL of denatured
15 ethanol and slurry-washed by stirring and refluxing for 10
minutes. The mixture was cooled in an ice bath to 10 °C. The
solid product was collected by vacuum filtration, washed
with 25 mL of ethanol and dried under vacuum at 40 °C to
give 12.7 g (51% yield) of 7-bromo-5-chloro-2-oxindole.
20 5-Fluoro-2-oxindole
5-Fluoroisatin (8.2 g) was dissolved in 50 mL of
hydrazine hydrate and refluxed for 1.0 hr. The reaction
mixtures were then poured in ice water. The precipitate was
then filtered, washed with water and dried in a vacuum oven
to afford the title compound.
5-Nitro-2-oxindole
2-Oxindole (6.5 g) was dissolved in 25 mL concentrated
sulfuric acid and the mixture maintained at -10 to -15 °C
while 2.1 mL of fuming nitric acid was added dropwise.
After the addition of the nitric acid the reaction mixture
was stirred at 0 °C for 0.5 hr and poured into ice-water.
The precipitate was collected by filtration, washed with
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water and crystallized from 50% acetic acid. The
crystalline product was then filtered, washed with water
and dried under vacuum to give 6.3 g (70%) of 5-vitro-2-
oxindole.
5-Aminosulfonyl-2-oxindole
To a 100 mL flask charged with 27 mL of chlorosulfonic
acid was added slowly 13.3 g of 2-oxindole. The reaction
temperature was maintained below 30 °C during the addition.
After the addition, the reaction mixture was stirred at
room temperature for 1.5 hr, heated to 68 °C for 1 hr,
cooled, and poured into water. The precipitate was washed
with water and dried in a vacuum oven to give 11.0 g of
5-chlorosulfonyl-2-oxindole (50% yield) which was used
without further purification.
5-Chlorosulfonyl-2-oxindole (2.1 g) was added to 10 mL
of ammonium hydroxide in 10 mL of ethanol and stirred at
room temperature overnight. The mixture was concentrated
and the solid collected by vacuum filtration to give 0.4 g
(20% yield) of the title compound as an off-white solid.
5-Methylaminosulfonyl-2-oxindole
A suspension of 3.38 g of 5-chlorosulfonyl-2-oxindole
in 10 mL 2M methylamine in tetrahydrofuran was stirred at
room temperature for 4 hours during which time a white
solid formed. The precipitate was collected by vacuum
filtration, washed twice with 5 mL of water and dried under
vacuum at 40 °C overnight to give 3.0 g (88 % yield) of
5-methylaminosulfonyl-2-oxindole.
5-(4-Trifluoromethylphenylaminosulfonyl)-2-oxindole
A suspension of 2.1 g of 5-chlorosulfonyl-2-oxindole,
1.6 g of 4-trifluoromethylaniline and 1.4 g of pyridine in
20 mL of dichloromethane was stirred at room temperature
for 4 hours. The precipitate which formed was collected by
vacuum filtration, washed twice with 5 mL of water and
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dried under vacuum at 40 °C overnight to give 2.4 g of crude
product containing some impurities by thin layer
chromatography. The crude product was chromatographed on
silica gel eluting with ethyl acetate: hexane (1:2) to give
1.2 g (37 % yield) of 5-(4-trifluoromethylphenyl-amino
sulfonyl)-2-oxindole.
5-(Morpholinosulfonyl)-2-oxindole
A suspension of 2.3 g of 5-chlorosulfonyl-2-oxindole
and 2.2 g of morpholine in 50 mL of dichloromethane was
stirred at room temperature for 3 hours. The white
precipitate was collected by vacuum filtration, washed with
ethyl acetate and hexane and dried under vacuum at 40 °C
overnight to give 2.1 g (74 % yield) of 5-(morpholino
sulfonyl)-2-oxindole.
6-Trifluoromethyl-2-oxindole
Dimethylsulfoxide (330 mL) was added to 7.9 g of
sodium hydride followed by dropwise addition of 43.6 g
diethyloxalate. The mixture was heated to 100 °C for 1.0
hour and cooled to room temperature. 2-Nitro-4-trifluoro
methyltoluene (31.3 g) was added, the reaction stirred for
minutes at room temperature and then heated to 100 °C for
1 hour. The reaction was cooled and poured into a mixture
of saturated aqueous ammonium chloride, ethyl acetate and
hexane. The organic layer was washed with saturated
25 ammonium chloride, water and brine, dried, and concentrated
to give dimethyl 2-(2-nitro-4-trifluoromethylphenyl)
malonate.
The diester was dissolved in a mixture of 6.4 g of
lithium chloride and 2.7 mL of water in 100 mL of
30 dimethylsulfoxide and heated to 100 °C for 3 hours. The
reaction was cooled and poured into a mixture of ethyl
acetate and brine. The organic phase was washed with brine,
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dried with sodium sulfate, concentrated and chromatographed
on silica gel (10 % ethyl acetate in hexane). The fractions
containing product were evaporated to give 25.7 g of methyl
2-nitro-4-trifluoromethylphenylacetate.
Methyl 2-nitro-4-trifluoromethylphenylacetate (26 mg)
was hydrogenated over 10 o palladium on carbon and then
heated at 100 °C for 3 hours. The catalyst was removed by
filtration and the solvent evaporated to give the title
compound.
4-Carboxy-2-oxindole
A solution of trimethylsilyldiazomethane in hexane (2M)
was added dropwise to a solution of 2.01 g of 2-chloro-3-
carboxy-nitrobenzene in 20 mL methanol at room temperature
until no further gas evolution occurred. The excess
trimethylsilyldiazo-methane was quenched with acetic acid. The
reaction mixture was dried by rotary pump and the residue was
further dried in a vacuum oven overnight. The product (2-
chloro-3-methoxycarbonyl-nitrobenzene) was pure enough for the
following reaction.
Dimethyl malonate (6.0 mL) was added to an ice-cold
suspension of 2.1 g of sodium hydride in 15 mL of DMSO. The
reaction mixture was then stirred at 100 °C for 1.0 h and
then cooled to room temperature. 2-Chloro-3-methoxy
carbonyl-nitrobenzene (2.15 g) was added to the above
mixture in one portion and the mixture was heated to 100 °C
for 1.5 h. The reaction mixture was then cooled to room
temperature and poured into ice water, acidified to pH 5,
and extracted with ethyl acetate. The organic layer was
washed with brine, dried over anhydrous sodium sulfate and
concentrated to give 3.0 g of the dimethyl 2-methoxy
carbonyl-6-nitrophenylmalonate.
Dimethyl 2-methoxycarbonyl-6-nitrophenylmalonate (3.0
g) was refluxed in 50 mL of 6 N hydrochloric acid
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overnight. The mixture was concentrated to dryness and
refluxed for 2 hours with 1.1 g of tin(II) chloride in 20
mL of ethanol. The mixture was filtered through Celite,
concentrated and chromatographed on silica gel (ethyl
acetate:hexane:acetic acid) to give 0.65 g (37% yield) of
4-carboxy-2-oxindole as a white solid.
5-Carboxyethyl-2-oxindole
5-Cyanoethyl-2-oxindole (4.02 g) in 10 mL of water
containing 25 mL of concentrated hydrochloric acid was
refluxed for 4 hours. The mixture was cooled, water added
and the resulting solid collected by vacuum filtration,
washed with water and dried to give 1.9 g (44% yield) of
the title compound as a yellow solid.
5-(Morpholin-4-ethyl)-2-oxindole
5-Chloroethyl-2-oxindole (2.3 g), 1.2 mL of morpholine
and 1.2 mL of diisopropylethylamine were heated overnight
at 100 °C in 10 mL of dimethylsulfoxide. The mixture ws
cooled, poured into water and extacted with ethyl acetate.
The organic layer was washed with brine, dried and
evaporated. The residue was chromatographed on silica gel
(5 % methanol in chloroform) to give 0.9 g (31%) of the
title compound as a white solid.
5-(4-Methoxycarbonylbenzamido)-2-oxindole
A mixture of 82.0 mg 5-amino-2-oxindole and 131.0 mg
4methoxycarbonylbenzoyl chloride in pyridine was stirred at
room temperature for 3 hr and poured into ice water. The
precipitate was filtered, washed with water and dried in a
vacuum oven to give 138.0 mg of 5-(4-methoxycarbonyl
benzamido)-2-oxindole (81% yield).
5-Methoxy-2-oxindole
Chloral hydrate (9.6 g) was dissolved in 200 mL of
water containing 83 g of sodium sulfate. The solution was
warmed to 60 °C, a solution of 11.4 g of hydroxylamine
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hydrochloride in 50 mL of water was added and the mixture
was held at 60 °C. In a separate flask, 6.4 g of 4-anisidine
and 4.3 mL of concentrated hydrochloric acid in 80 mL of
water was warmed to 80 °C. The first solution was added to
the second and the mixture refluxed for 2 minutes after
which it was cooled slowly to room temperature and then
cooled in an ice bath. The tan precipitate was collected by
vacuum filtration, washed with water and dried under vacuum
to give 8.6 g ( 85% yield) of N-(2-hydroximinoacetyl)
anisidine.
Concentrated sulfuric acid (45 mL) containing 5 mL of
water was warmed to 60 °C and 8.6 g of N-(2-hydroximino
acetyl)anisidine was added in one portion. The stirred
mixture was heated to 93 °C for 10 minutes and then allowed
to cool to room temperature. The mixture was poured into
500 g of ice and extracted 3 times with ethyl acetate. The
combined extracts were dried over anhydrous sodium sulfate
and concentrated to give 5.1 g (65% yield) of 5-methoxy-
isatin as a dark red solid. 5-Methoxyisatin (5.0 g) and 30
mL of hydrazine hydrate were heated to reflux for 15
minutes. The reaction mixture was cooled to room
temperature and 50 mL of water was added. The mixture was
extracted 3 times with 25 mL of ethyl acetate each time,
the organic layers combined, dried over anhydrous sodium
sulfate and concentrated to give a yellow solid. The solid
was stirred in ethyl acetate and 1.1 g of insoluble
material was removed by vacuum filtration and saved. This
material proved to be 2-hydrazinocarbonylmethyl-4-
anisidine. The filtrate was concentrated and chromatographed
on silica gel eluting with ethyl acetate: hexane (1:1) to give
0.7 g of 5-methoxy-2-oxindole as a yellow solid. The 1. 1 g
of 2-hydrazino-carbonylmethyl-4-anisidine was refluxed for 1
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hour in 20 mL of 1N sodium hydroxide. The mixture was cooled,
acidified to pH 2 with concentrated hydrochloric acid and
extracted 3 times with 25 mL of ethyl acetate each time. The
organic extracts were combined, washed with brine, dried over
anhydrous sodium sulfate and concentrated to give 0.8 g of
5-methoxy-2-oxindole as a yellow solid. The combined yield
was 1.5 g or 33%.
7-Azaoxindole
3,3-Dibromo-7-azaoxindole (2.9 g) was dissolved in a
mixture of 20 mL of acetic acid and 30 mL of acetonitrile.
To the solution was added 6.5 g of zinc dust. The mixture
was stirred for 2 hrs at room temperature. The solid was
filtered from the mixture and the solvent evaporated. The
residue was slurried with ethyl acetate. The ethyl acetate
solution containing insoluble solid was passed through a
short column of silica gel. The collected ethyl acetate
solution was evaporated and the residue dried under vacuum
to give 1.8 g (yield 91%) of 7-azaoxindole acetic acid
salt.
5-Dimethylaminosulfonyl-2-oxindole
A suspension of 2.3 g 5-chlorosulfonyl-2-oxindole in
10 mL 2M dimethylamine in methanol was stirred at room
temperature for 4 hours at which time a white solid formed.
The precipitate was collected by vacuum filtration, washed
with 5 mL of 1N sodium hydroxide and 5 mL of 1N
hydrochloric acid and dried under vacuum at 40 °C overnight
to give 1.9 g (79% yield) of 5-dimethylaminosulfonyl-2-
oxindole.
6-Phenyl-2-oxindole
Dimethyl malonate (10 mL) in 25 mL of dimethyl-
sulfoxide was added dropwise to 3.5 g sodium hydride
suspended in 25 mL dimethylsulfoxide and the mixture heated
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at 100 °C for 10 minutes. The mixture was cooled to room
temperature and 4.7 g of 4-fluoro-3-nitrobiphenyl in 25 mL
dimethylsulfoxide was added. The mixture was heated at 100
°C for 2 hours, cooled and quenched with 300 mL of saturated
ammonium chloride solution. The mixture was extracted three
times with ethyl acetate and the combined organic layers
washed with water and brine and evaporated to give, as a
yellow oil, crude dimethyl-3-nitrobiphenyl-4-malonate.
Crude dimethyl-3-nitrobiphenyl-4-malonate was refluxed
in 30 mL of 6 N hydrochloric acid for 24 hours. The
precipitate was collected by filtration, washed with water
and dried to give 4.5 g of 3-nitrobiphenyl-4-acetic acid as
a cream colored solid.
Iron powder (2.6 g) was added all at once to 4.5 g of
3-nitrobiphenyl-4-acetic acid in 40 mL of acetic acid. The
mixture was refluxed for 2 hours, concentrated to dryness
and taken up in ethyl acetate. The solids were removed by
filtration and the filtrate washed twice with 1N
hydrochloric acid and brine and dried over anhydrous sodium
sulfate. The filtrate was concentrated to give 3.4 g (93%
yield) of 6-phenyl-2-oxindole as a light brown solid.
6-(3-Methoxyphenyl)-2-oxindole
Tetrakis(triphenylphosphine)palladium (0.8 g) was
added to a mixture of 5 g 3-methoxyphenylboronic acid, 5 g
5-bromo-2-fluoro-nitrobenzene and 11 mL of 2 M sodium
carbonate solution in 100 mL of toluene. The mixture was
refluxed for 2 hours, diluted with water and extracted with
ethyl acetate. The ethyl acetate was washed with saturated
sodium bicarbonate and brine and then dried and
concentrated to give an oily solid. The solid was
chromatographed on silica gel (ethyl acetate: hexane (1:6))
to give 4.3 g (77% yield) of 4-fluoro-3'-methoxy-3-
nitrobiphenyl.
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Dimethyl malonate (9.7 mL) was added dropwise to 2.0 g
sodium hydride suspended in 50 mL dimethylsulfoxide. The
mixture was heated to 100 °C for 35 minutes and cooled to
room temperature. 4-Fluoro-2'-methoxy-3-nitrobiphenyl (4.2
g) in 50 mL dimethylsulfoxide was added and the mixture was
heated at 100 °C for 1 hour. The reaction mixture was cooled
and quenched with 300 mL of saturated ammonium chloride
solution and extracted twice with ethyl acetate. The
extracts were combined, washed with brine, dried over
anhydrous sodium sulfate and concentrated to give crude
dimethyl 3'-methoxy-3-nitrobiphenyl-4-malonate as a pale
yellow solid.
Crude dimethyl 3'-methoxy-3-nitrobiphenyl-4-malonate
was heated at 110 °C in 45 mL 6N hydrochloric acid for 4
days and then cooled. The precipitate was collected by
filtration, washed with water and hexane, and dried to give
5.3 g of 3'-methoxy-2-nitrobiphenyl-4-acetic acid as a
light tan solid.
3'-Methoxy-3-nitrobiphenyl-4-acetic acid (5.2 g) was
dissolved in methanol and hydrogenated over 0.8 g of 10%
palladium on carbon for 3 hours at room temperature. The
catalyst was removed by filtration, washed with methanol
and the filtrates combined and concentrated to give a brown
solid. The solid was chromatographed on silica gel in ethyl
acetate:hexane:acetic acid (33:66:1) to give 3.0 g of 6-(3-
methoxypheny)-2-oxindole as a pink solid.
5-Acetyl-2-oxindole
2-Oxindole (3 g) was suspended in 1,2-dichloroethane
and 3.2 mL acetyl chloride were slowly added. The resulting
suspension was heated to 50 °C for 5 hours, cooled, and
poured into water. The resulting precipitate was collected
by vacuum filtration, washed copiously with water and dried
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under vacuum to give 2.9 g (73% yield) of the title
compound as a brown solid.
5-Cyanoethyl-2-oxindole
Potassium cyanide (2.0 g) was added to 15 mL of
dimethyl-sulfoxide and heated to 90 °C. 5-Chloroethyl-2-
oxindole (3.0 g) dissolved in 5 mL dimethyl sulfoxide was
added slowly with stirring, and the reaction heated to 150
°C for 2 hours. The mixture was cooled, poured into ice
water and the precipitate collected by vacuum filtration,
washed with water, dried and then chromatographed on silica
gel (5% methanol in chloroform) to give 1 .2 g (42 % yield)
of the title compound.
6-(Morpholin-4-yl)-2-oxindole
6-Amino-2-oxindole (2.2 g), 4.0 g 2, 2'-dibromoethyl
ether and 7.9 g sodium carbonate were refluxed in 20 ml
ethanol overnight, concentrated and diluted with 50 ml of
water. The mixture was extracted three times with 50 ml of
ethyl acetate and the organic extracts combined, washed
with 20 ml of brine, dried over anhydrous sodium sulfate
and concentrated to dryness. The solid was chromatographed
on a column of silica gel (ethyl acetate: hexane (1:1)
containing 0.7% acetic acid) to give 1.2 g (37% yield) of
the title compound as a beige solid.
Methvlation of phenols
An appropriate phenol (1 equivalent) is stirred
overnight at room temperature with an equal volume of
dimethylformamide containing 1.3 equivalents of methyl
iodide and 1.3 equivalents of potassium carbonate. The
mixture is diluted with water and extracted with ethyl
acetate. The ethyl acetate layer is washed with water and
brine. The organic layer is separated, dried over
anhydrous sodium sulfate and evaporated to dryness to give
the anisole derivative.
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Formulation of anisoles
The anisole (1 equivalent), dimethylformamide (1-3
equivalents) and phosphorus oxychloride (1-3 equivalents)
are heated to 100° C for 1-3 hours. The reaction mixture is
cooled to room temperature and dichloromethane is added.
The mixture is cooled in ice and water is then added,
followed by 4-12 equivalents of concentrated sodium
hydroxide until a pH of 9-10 is reached. The organic layer
is separated and washed with water and then with brine,
dried over anhydrous sodium sulfate and evaporated to give
the crude aldehyde. The crude aledehyde is dissolved in
boiling hexane containing activated carbon and the hexane
solution is decanted and filtered hot through a layer of
silica gel. The filtrate is evaporated to dryness to give
the benzaldhyde derivative.
Formulation of pvrroles
POC13 (1.1 equiv.) is added dropwise to
dimethylformamide (3 equiv.) in dichloromethane at -10°C
followed by the appropriate pyrrole. After stirring for two
hours, the reaction mixture is diluted with Hz0 and basified
to pH 11 with 10 N KOH. The precipitate which forms is
collected by filtration, washed with HZO and dried in a
vacuum oven to give the desired aldehyde.
Saponification of pvrrolecarboxvlic acid esters
A mixture of a pyrrolecarboxylic acid ester and KOH (2
- 4 equiv.) in EtOH is refluxed until reaction completion
is indicated by thin layer chromatography (TLC). The cooled
reaction mixtrue is acidified to pH 3 with 1 N HC1. The
precipitate which forms is collected by filtration, washed
with Hz0 and dried in a vacuum oven to give the desired
pyrrolecarboxylic acid.
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Amidation of pyrrolecarboxylic acids
To a stirred solution of a pyrrolecarboxylic acid
dissolved in dimethylformamide(0.3M) is added 1-ethyl-3-(3-
dimethylaminopropyl)carboiimide (1.2 equiv.), 1-
hydroxybenzotriazole (1.2 equiv.), and triethylamine (2
equiv.). The appropriate amine is added (1 equiv.) and the
reaction stirred until completion is indicated by TLC. The
reaction is diluted with saturated NaHC03 and brine (with
extra salt), dried over anhydrous MgS04and concentrated to
afford the desired amide.
Preparation of pyrrole and indole carbaldehydes
3-(2-Formyl-5-methyl-1H-pyrrol-3-yl)propionic acid
4-(2-Carboxyethyl)-3-ethoxycarbonyl-2-methylpyrrole
(Bulter, A.R., and George, S.D. (1993) Tetrahedron 49:
7017-7026) was hydrolyzed using potassium hydroxide (KOH)
in ethanol (EtOH) to give 4-(2-carboxyethyl)-2-methyl-1H-
pyrrole-3-carboxylic acid (64%).
1HNMR (DMSO-d6) 8 11.63 (s, 1H, COOH), 10.81 (s, 1H,
NH) , 6.36 (s, 1H, CH) , 2.78, 2.45 (2 x t, 4H, CHZCHz) ,
2 .34 (s, 3H, CH3) .
MS m/z 22 5 [M+2 ] .
4-(2-Carboxyethyl)-2-methyl-1H-pyrrole-3-carboxylic
acid was decarboxylated by heating for one hour at 200°C to
give 3-(5-methyl-1H-pyrrol-3-yl)propionic acid.
1HNMR (DMSO-ds) b 10.07 (s, 1H, NH) , 9.60 (br s, 1H,
COOH), 6.30 (s, 1H, CH), 5.56 (s, 1H, CH), 2.51, 2.36 (2 x
t, 4H, CH2CHz) , 2. 10 (s, 3H, CH3) .
MS m/z 154 [M+1] .
3-(5-Methyl-1H-pyrrol-3-yl)propionic acid was
formylated using phosphorus oxycloride (POC13) and N,N-
dimethylformamide (DMF) to give 3-(2-formyl-5-methyl-1H-
pyrrol-3-yl)propionic acid.
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1HNMR (300 MHz, DMSO-d6) 8 12. 09 (br s, 1H, COOH) ,
11.54 (br s, 1H, NH), 9.44 (s, 1H, CHO), 5.86 (d, J = 1.8
Hz, 1H) , 2.87 (t, J = 7.5 Hz, 2H, CHZ) , 2.47 (t, J = 7.5 Hz,
2H, CHz) , 2.16 (s, 3H, CH3) .
MS-EI m/z 181 [M+] .
4-(2-Carboxyethyl)-5-formyl-3-methyl-1H-pyrrole-2-
carboxylic acid ethyl ester
To a suspension of 10 g (38.9 mmol) ethyl 2,4-
dimethyl-5-(ethoxycarbonyl)-3-pyrrolepropionate
(commercially available) in a mixture of water (250 mL)
and methanol (MeOH)(50 mL) was added 3.0 g of KOH in 50
mL of water. The mixture was stirred at 70°C for 4 hr.
The reaction mixture was cooled and the methanol was
stripped from the mixture under vacuum. The remaining
solution was filtered and the filtrate was then acidified
with 6N hydrochloric acid to pH 3. The precipitate which
formed was collected by filtration, washed with a 2:1
mixture of water and ethanol and dried under vacuum to
give 7.5 g of 4-(2-carboxy-ethyl)-3,5-dimethyl-1H-
pyrrole-2-carboxylic acid ethyl ester as a white solid.
1HNMR (360 MHz, DMSO-d6) 8 12.03 (br s, 1H, COOH) ,
11.02 (br s, 1H, NH) , 4.16 (q, J = 7.2 Hz, 2H, OCH2CH3) ,
2.53 (t, J = 7.8 Hz, 2H, CHZCHZ) , 2.25 (t, J = 7.8 Hz, 2H,
CHzCHz) , 2.16 (s, 3H, CH3) , 2. 11 (s, 3H, CH3) , 1 .25 (t, J =
7.2 Hz, 3H, OCH2CH3) .
MS-EI m/z 239 [M+] .
To a mixture of 7 g 4-(2-carboxyethyl)-3,5-dimethyl-
1H-pyrrole-2-carboxylic acid ethyl ester in 35 mL each of
tetrahydrofuran (THF), acetic acid (AcOH) and water at -10°
C was added 70 g of ceric ammonium nitrate in portion over
20 minutes, the reaction temperature being maintained at
about 5°C. The resulting mixture was them cooled to 0°C,
stirred for another 2 hr and then diluted with 250 mL of
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brine. The mixture was then extracted with 2 x 300 mL 10%
MeOH in dichloromethane (DCM). The organic extracts were
combined and dried over anhydrous magnesium sulfate (MgS04),
filtered and concentrated. The residue was purified on
silica gel column with hexanes(hex)/ethyl acetate
(EtOAc)/AcOH (6:4:0.05) followed by crystallization in
EtOAc to give 1.5 g of 4-(2-carboxyethyl)-5-formyl-3-
methyl-1H-pyrrole-2-carboxylic acid ethyl ester as a white
solid.
1HNMR (360 MHz, DMSO-d6) 8 12.36 (br s, 1H, COOH),
12.01 (br s, 1H, NH) , 9.77 (s, 1H, CHO) , 4.27 (q, J = 7.1
Hz, 2H, OCHZCH3) , 2 . 89 (t, J = 7. 6 Hz, 2H, CH2CHZ) , 2.37 (t,
J = 7.6 Hz, 2H, CHzCHz) , 2.21 (s, 3H, CH3) , 1.30 (t, J = 7.1
Hz, 3H, OCHZCH3) .
MS-EI m/z 253 [M+] .
4-(3-Dimethylaminopropyl)-5-formyl-2-methyl-1H-pyrrole-3-
carboxylic acid ethyl ester
To a suspension of 4-(2-carboxyethyl)-3-
ethoxycarbonyl-2-methylpyrrole (2 g, 8.88 mmol) (Bulter,
A.R., and George, S.D. (1993) Tetrahedron 49: 7017-7026) in
18 mL of DMF was added 1.73 g (10.65 mmol) 1,1'-
carbonyldiimidazole (CDI) followed by the dropwise addition
of 8.9 mL (17.76 mmol) of 2M dimethylamine in THF. After
stirring for 2 hr, the reaction was diluted with water (200
mL) and cooled. The precipitate which formed was collected
by filtration, washed with water and dried to give 1.0 g of
the product as a white crystalline solid. The filtrate was
extracted with EtOAc, the organic layer was washed with
brine, dried and concentrated to obtain an additional 0.9 g
of the product for a total of 1.9 g of 4-(2-dimethyl-
carbamoyl-ethyl)-2-methyl-1H-pyrrole-3-carboxylic acid
ethyl ester (85%).
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1HNMR (360 MHz, DMSO-d6) 8 10.89 (br s, 1H, NH), 6.40
(d, J = 2.5 Hz, 1H) , 4. 13 (q, J = 7.1 Hz, 2H, OCHzCH3) , 2.92
(s, 3H, NCH3) , 2.79 (s, 3H, NCH3) , 2.75 (m, 2H, CH2CH2) , 2.45
(m, 2H, CHZCH2) , 2.35 (s, 3H, CH3) , 1.23 (t, J = 7.1 Hz, 3H,
OCHzCH3 ) .
MS-EI m/z 252 [M'] .
To a heterogeneous mixture of 4-(2-dimethylcarbamoyl-
ethyl)-2-methyl-1H-pyrrole-3-carboxylic acid ethyl ester
(1.01 g, 4 mmol) in THF (9 mL) was added dropwise 8 mL of
borane-tetrahydrofuran complex (1M in THF). The mixture was
refluxed overnight. Then 9.0 mL of MeOH was added slowly to
the reaction and refluxing was continued for another 2 hr.
The reaction mixture was cooled, quenched with 1N HC1 and
extracted with EtOAc. The aqueous layer was basified with
aqueous KOH and extracted with EtOAc. The EtOAc extract
was washed with brine, dried and concentrated to give 616
mg (65%) of 4-(3-dimethylaminopropyl)-2-methyl-1H-pyrrole-
3-carboxylic acid ethyl ester as a faint orange oil.
1HNMR (360 MHz, DMSO-ds) 8 10.84 (br s, 1H, NH) , 6.36
(d, J = 2.5 Hz, 1H) , 4. 12 (q, J = 7. 1 Hz, 2H, OCHZCH3) , 2.52
(m, 2H, CH2) , 2 .34 (s, 3H, CH3) , 2 . 17 (m, 2H, CHz) , 2.08 (s,
6H, N (CH3) 2) , 1 . 57 (m, 2H, CH2CH2 CH2) , 1 .23 (t, J = 7. 1 Hz,
3H, OCHzCH3) .
MS-EI m/z 238 [M+] .
4-(3-Dimethylaminopropyl)-2-methyl-1H-pyrrole-3-
carboxylic acid ethyl ester (600 mg, 2.5 mmol) was
formylated using POC13 and DMF to give 645 mg (96%) of 4-(3-
dimethylamino-propyl)-5-formyl-2-methyl-1H-pyrrole-3-
carboxylic acid ethyl ester.
'HNMR (360 MHz, DMSO-d6) 8 12.15 (br s, 1H, NH) , 9.59
(s, 1H; CHO) , 4.19 (q, J = 7.2 Hz, 2H, OCHZCH3) , 2.93 (m,
2H, CHZ) , 2.41 (s, 3H, CH3) , 2 . 18 (m, 2H, CHz) , 2 . 09 (s, 6H,
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N (CH3) 2) , 1 . 63 (m, 2H, CH2CH2 CHz) , 1 .27 (t, J = 7.2 Hz, 3H,
OCHZCH3 ) .
MS-EI m/z 266 [M'] .
3-(3-Dimethylaminopropyl)-1H-indole-2-carbaldehyde
Ethyl indole-2-carboxylate (10 g, 52.8 mmol) was
formylated using POC13 (1.3 equiv.) and DMF (1.3 equiv.) as
above to give 3-formyl-1H-indole-2-carboxylic acid ethyl
ester as a white solid.
1HNMR (360 MHz, DMSO-ds) 8 12. 0 (br s, 1H, NH) , 10.6
(s, 1H, CHO), 8.24 (dd, J = 0.7 & 8.0 Hz, 1H), 7.57 (d,
1H), 7.38 (m, 1H), 7.26 (m, 1H), 4.44 (q, J = 7.12 Hz, 2H,
OCHZCH3 ) , 1 . 3 9 ( t , J = 7 . 12 Hz , 3H, OCHZCH3 ) .
MS-EI m/z 217 [M'] .
Butyllithium (1.1 equiv.) was added to a suspension of
(2-dimethylaminoethyl)triphenylphosphonium bromide in THF
(0.2 M) at 0°C. After stirring for 30 minutes, lithium
diethylamine (1.1 equiv.) was added dropwise followed by
the cold suspension of 3-formyl-1H-indole-2-carboxylic acid
ethyl ester (3.96 g, 18.2 mmol) in THF. The resulting
orange suspension was stirred for 18 hours. The reaction
was then quenched with saturated ammonium chloride and
extracted with 10% MeOH in DCM. The organic layer was
washed with saturated sodium bicarbonate, dried and
concentrated to give 3-(3-dimethylaminopropenyl)-1H-indole-
2-carboxylic acid ethyl ester as a brownish-red waxy solid.
1HNMR (360 MHz, DMSO-d6) b 11. 8 (br s, 1H, NH) , 7.93
(d, 1H) , 7.49 (d, 1H) , 7.41 (d, 1H) , 7.3 (t, 1H) , 7.14 (t,
1H) , 6.33-6.42 (m, 1H) , 4 .36 (q, 2H, OCHZCH3) , 3 .38 (d, 2H,
CHz) , 2 .44 (s, 6H, 2xCH3) , 1 .36 (t, 3H, OCHZCH3) .
MS-EI m/z 272 [M+] .
3-(3-Dimethylaminopropenyl)-1H-indole-2-carboxylic
acid ethyl ester was hydrogenated using 10% palladium over
carbon to reduce the double bond followed by a lithium
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aluminum hydride (LAH) reduction of the ester group to an
alcohol resulting in the formation of [3-(3-dimethylamino-
propyl)-1H-indol-2-yl]methanol as an orange oil.
Manganese dioxide (10 equiv.) was added to a solution
of [3- (3-dimethylaminopropyl) -1H-indol-2-yl] methanol (2.2
g) in DCM. The mixture was stirred at room temperature for
18 hr. Insoluble materials were removed by filtrationand
the filtrate was concentrated. The residue was column
chromatographed to give 3-(3-dimethylaminopropyl)-1H-
indole-2-carbaldehyde as a cream-colored solid.
1HNMR (300 MHz, DMSO-d6) 8 11.58 (br s, 1H, NH) , 10.0
(s, 1H, CHO), 7.72 (d, 1H), 7.39 (d, 1H), 7.29 (t, 1H),
7.07 (t, 1H) , 3.08 (t, 2H, CHZ) , 2.20 (t, 2H) , 2.12 (s, 6H,
2xCH3) , 1 .73 (t, 2H, CHz) .
MS-EI m/z 230 [M+] .
Preparation of aminotetrahydoindolecarbaldehydes
A mixture of 5-aminolevulinic acid hydrochloride (1
equiv.), 1,3-cyclohexanedione (1 equiv.) and sodium acetate
(2 equiv. ) in water (1M) is stirred at 110° C for 4 - 12 hr
and then cooled. The precipitate which forms is collected
by vacuum filtration, washed with 30% ethanol in water and
dried under vacuum to give the amido-keto-tetrahydroindole
in 50-70% yield.
Lithium aluminum hydride (LAH, 4 equiv.)is added
dropwise to a suspension of the appropriate amido-keto-
tetrahydroindole (1 equiv.) in THF (0.5 M). The mixture is
refluxed overnight. The mixture is then cooled and water is
added carefully until no more gas is generated, then a few
drops of 15% NaOH/water is added. The mixture is then
stirred at room temperature for 0.5 hr and filtered to
remove insoluble materials. The filtrate is concentrated to
give the amino-tetrahydroindole.
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Examples of syntheses of aminotetrahydroindolecarb-
aldehydes
The following syntheses of aminotetrahydroindole-
carbaldehydes are shown by way of example only and are not
to be construed as limiting the scope of this invention in
any manner whatsoever.
3-(3-(4-methylpiperazin-1-yl)propyl]-4,5,6,7-tetrahydro-1H-
indole-2-carbaldehyde
Step l: A mixture of 5-aminolevulinic acid
hydrochloride (1.68 g, 10 mmol), 1,3-cyclohexanedione (1.12
g, 10 mmol) and sodium acetate (1.64 g, 20 mmol) in water
(10 mL) was stirred at 110°C for 4 hr and then cooled. The
precipitate which formed was collected by vacuum
filtration, washed with 30% of ethanol in water and dried
under vacuum to give 1.7 g (82%) of 3-(4-oxo-4,5,6,7-
tetrahydro-1H-indol-3-yl)-propionic acid.
1HNMR (360 MHz, DMSO-d6) ~ 11.91 (br s, 1H, COOH),
10.99 (br s, 1H, NH), 6.45 (d, J = 1.4 Hz, 1H), 2.76 (t,
2H, CHz) , 2.69 (t, 2H, CHZ) , 2.44 (t, 2H, CH2) , 2.26 (t, 2H,
CH2) , 1.96 (m, 2H, CH2) .
MS-EI m/z 207 [M+] .
Step 2: To a suspension of 10 g of the product of step
1 (48 mmol) in dichloromethane(60 mL) was added 9.3 g (57.6
mmol) of 1,1'-carbonyldiimidazole. The mixture was stirred
at room temperature for 2 hours and then 5.3 mL (48 mmol)
1-methylpiperazine and 8.4 mL (48 mmol) N,N-diisopropyl-
ethylamine was added. The dark red reaction mixture was
then stirred at room temperature overnight. The reaction
was then poured into water, the organic layer separated and
washed with brine, dried and concentrated to give 8 g (57%)
of 3-[3-(4-methylpiperazin-1-yl)-3-oxo-propyl]-1,5,6,7-
tetrahydroindol-4-one.
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1HNMR (360 MHz, DMSO-d6) 8 10.97 (br s, 1H, NH), 6.47
(d, J = 2.0 Hz, 1H), 3.43 (m, 4H, 2xCH2), 2.67-2.75 (m, 4H,
2xCH2) , 2.51 (m, 2H, CHZ) , 2 .27 (m, 2H, CHZ) , 2.20 (m, 4H,
2xCH2) , 2 .15 (s, 3H, CH3) , 1. 97 (m, 2H, CH2) .
MS-EI m/z 289 [M'] .
Step 3: LAH (2.6 g, 68 mmol) was added dropwise to a
suspension of 3-[3-(4-methylpiperazin-1-yl)-3-oxo-propyl]-
1,5,6,7-tetrahydroindol-4-one (5g, 17 mmol) in THF (300
mL). The mixture was then refluxed overnight. The mixture
was then cooled and 2.6 mL each of water followed by a few
drops of 15% NaOH was added. The reaction was stirred at
room temperature for 30 min and then filtered to remove
insolubles. The filtrate was concentrated to give 4.5 g
(100%) of 3-[3-(4-methylpiperazin-1-yl)propyl]-4,5,6,7-
tetrahydro-1H-indole.
1HNMR (360 MHz, DMSO-d6) 8 9.79 (br s, 1H, NH), 6.22
(d, J = 2.0 Hz, 1H), 2.44 (m, 2H, CHZ), 2.21-2.3 (m, 14H,
7xCHz) , 2.12 (s, 3H, CH3) , 1.65 (m, 4H, 2xCH2) , 1.53 (m, 2H,
CH2 ) .
MS-EI m/z 261 [M+] .
Step 4: POC13 (1.8 mL, 18.9 mmol) was added dropwise to
N,N-dimethylformamide(DMF, 3.8 mL, 51.6 mmol) at -5° C. The
mixture was then allowed to come to room temperature and
then stirred for 30 minutes after which it was again cooled
to -5°C. A solution of 3-[3-(4-methylpiperazin-1-yl)-
propyl]-4,5,6,7-tetrahydro-1H-indo1e (4.5 g, 17.2 mmol) in
DMF (9 mL) was added dropwise. The mixture was again
allowed to come to room temperature and then stirred at
that temperature overnight. The reaction was then quenched
with ice, followed by 10 N NaOH to adjust the pH to 10-11.
After stirring at room temperature for 1 hr, the reaction
was extracted with ethyl acetate (EtOAc), the organic layer
separated, washed with brine, dried and concentrated to
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give 3 . 1 g (62%) of 3- [3- (4-methylpiperazin-1-yl) -propyl] -
4,5,6,7-tetrahydro-1H-indole-2-carbaldehyde.
1HNMR (360 MHz, DMSO-d6) 8 11.24 (br, s, 1H, NH), 9.42
(s, 1H, CHO), 2.60 (t, 2H, CHz), 2.51 (m, 2H, CHz), 2.35 (m,
2H, CHz) , 2.28 (m, 8H, 4xCH2) , 2.21 (m, 2H, CH2) , 2.12 (s,
3H, CH3) , 1.57-1.68 (m, 6H, 3xCH2) .
MS-EI m/z 289 [M+] .
3-(3-dimethylaminopropyl)-4,5,6,7-tetrahydro-1H-indole-2-
carbaldehyde
The procedure was the same as that employed in Example A
except that the amine in Step 2 was dimethylamine (2. OM
solution in tetrahydrofuran).
Step 2: N,N-Dimethyl-3-(4-oxo-4,5,6,7-tetrahydro-1H-
indol-3-yl)-propionamide:
1HNMR (300 MHz, DMSO-d6) 8 11.0 (br s, 1H, NH), 6.48
(d, J = 1 .6 Hz, 1H) , 2 .95 (s, 3H, CH3) , 2 .79 (s, 3H, CH3) ,
2.71 (m, 4H, 2xCHz) , 2.47 (m, 2H, CH2) , 2 .27 (t, 2H, CHz) ,
1.96 (m, 2H, CHz) .
MS-EI m/z 234 [M+] .
Step 3: Dimethyl-[3-(4,5,6 ,7-tetrahydro-1H-indol-3-
yl) propyll-amine:
1HNMR (300 MHz, DMSO-d6) 8 9.83 (br s,
1H, NH),
6.22
(d, J = 2.3 Hz, 1H) , 2 .43 (m, (m, 2H, CHz)
2H, CHZ) , 2 .28 ,
2 . 4-2 . 25 (m, 4H, CHZ) , 2 6H, N (CH3) 1 . 64 (m,
1 . 08 (s, 2) , 4H,
2xC H2) , 1 .53 (m, 2H, CHZ) .
MS-EI m/z 206 [M'] .
Step 4: 3-(3-Dimethylamino progyl)-4,5,6,7-tetrahydro-
1H- indole-2-carbaldehyde:
1HNMR (300 MHz, DMSO-d6) 8 11.29 (br, 1H, NH), 9.40
s,
(s, 1H, CHO) , 2.59 (t, 2H, CHZ) 2.5 (m, 2H, CHz) , 2 .34
, (m,
2H, CHZ) , 2 . 16 (m, 2H, CH2) 8 (s, 6H,
, 2 . 0 N (CH3) 2)
, 1 . 67
(m,
4H, 2xCHz) , 1.56 (m, 2H, CHz)
.
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MS-EI m/z 234 [M'] .
3-(3-pyrrolidin-1-ylpropyl)-4,5,6,7-tetrahydro-1H-indole-2-
carbaldehyde
The procedure employed was that same as that in
Example A except that the amine used in Step 2 was
pyrrolidine.
Step 2: 3-(3-Oxo-3-pyrrolidin-l~lpropyl)-1 5 6 7-
tetrahydroindol-4-one:
1HNMR (300 MHz, DMSO-d6) 8 11.05 (br s, 1H, NH), 6.46
(d, J = 1.5 Hz, 1H) , 3.35 (m, 2H, CHz) , 2 .24 (m, 2H, CHZ) ,
2.66-2.73 (m, 4H, 2xCH2) , 2.44 (m, 2H, CHz) , 2.26 (m, 2H,
CHz) , 1.96 (m, 2H, CHZ) , 1. 82 (m, 2H, CH2) , 1.73 (m, 2H,
CH2 ) .
MS-EI m/z 260 [M+] .
Step 3: 3-(3-Pvrrolidin-1 ylproQvl)-4 5 6 7-
tetrahydro-1H-indole:
1HNMR (300 MHz, DMSO-d6) 8 9.82 (br s, 1H, NH), 6.22
(s, 1H), 2.2-2.5 (m, 12H, 6xCHz), 1.5-1.64(m, 10H, 5xCH2).
MS-EI m/z 232 [M'] .
Step 4: 3-(3-Pyrrolidin-1-yl~ropyl)-4 5 6 7-
tetrahydro-1H-indole-2-carbaldehyde:
1HNMR (300 MHz, DMSO-d6) 8 11.28 (br, s, 1H, NH), 9.38
(s, 1H, CHO), 2.59 (t, 2H, CH2), 2.46 (m, 2H, CHz), 2.3-2.44
(m, 8H, 4xCH2), 1.55-1.65 (m, 10H, 5xCH2).
MS-EI m/z 260 [M+] .
3-(3-diethylamino-propyl)-4,5,6,7-tetrahydro-1H-indole-2-
carbaldehyde
The procedure used was the same as that in Example A
except that the amine used was diethylamine.
Step 2: N,N-Diethyl-3-(4-oxo-4,5,6,7-tetrahydro-1H-
indol-3-yl)-propionamide:
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iHNMR (300 MHz, DMSO-d6) 8 11.0 (br s, 1H, NH), 6.47
(d, J = 2 . 0 Hz, 1H) , 3 .24 (m, 4H, N (CH2CH3) 2) , 2 . 69 (m, 4H,
2xCH2) , 2.46 (m, 2H, CHZ) , 2.27 (m, 2H, CHz) , 1. 96 (m, 2H,
CHz) , 1. 04 (t, J = 7. 0 Hz, 3H, NCHzCH3) , 0. 98 (t, J = 7.0
Hz, 3H, NCHzCH3) .
Step 3: Diethyl-(3-(4,5,6,7-tetrahydro-1H-indol-3-yl)-
prowl l -amine
1HNMR (300 MHz, DMSO-d6) 8 9.83 (br s, 1H, NH), 6.22
(d, J = 2. 0 Hz, 1H) , 2 . 19-2 .44 (m, 12H, 6xCHz) , 1.64 (m, 4H,
2xCH2) , 1.51 (m, 2H, CHZ) , 0. 90 (t, J = 7. 0 Hz, 6H,
N ( CH2CH3 ) 2 ) .
MS-EI m/z 234 [M+] .
Step 4: 3-(3-Diethylaminopropyl)-4,5,6,7-tetrahydro-
1H-indole-2-carbaldehyde:
1HNMR (360 MHz, DMSO-d6) 8 11.24 (br, s, 1H, NH), 9.42
(s, 1H, CHO) , 2.59 (t, 2H, CHz) , 2.34-2 .53 (m, 10H, 5xCHz) ,
1.67 (m, 4H, 2xCH2) , 1.57 (m, 2H, CHz) , 0.91 (t, J = 7.0 Hz,
6H, N (CHZCH3) 2) .
MS-EI m/z 262 [M+] .
3-(3-Diethylaminopropyl)-6,6-dimethyl-4,5,6,7-tetrahydro-
1H-indole-2-carbaldehyde
Step l: A mixture of 5-aminolevulinic acid
hydrochloride (1.68 g, 10 mmol) , 5,5-dimethyl-1,3-
cyclohexandione (1.4 g, 10 mmol) and sodium acetate (1.64
g, 20 mmol) in water (10 mL) was stirred at 110°C for 4 hr
and then cooled. The precipitate which formed was collected
by vacuum filtration, washed with 30% of ethanol (EtOH) in
water and dried under vacuum to give 1.6 g (68%) of 3-(4-
oxo-6-dimethyl-4,5,6,7-tetrahydro-1H-indol-3-yl)propionic
acid.
1HNMR (360 MHz, DMSO-d6) 8 11.89 (br s, 1H, COOH),
10.94 (br s, 1H, NH) , 6.45 (d, J = 1.4 Hz, 1H) , 2.76 (t,
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2H, CHz) , 2.57 (s, 2H, CHZ) , 2.44 (t, 2H, CHZ) , 2.16 (s, 2H,
CHz) , 0.99 (s, 6H, 2xCH3) .
MS-EI m/z 235 [M+] .
Step 2: To a suspension of 1.18 g (5 mmol) of the
product of step 1 in dichloromethane (25 mL) was added 0.97
g (6 mmol) of CDI. After stirring at room temperature for 2
hr, 2.1 mL (20 mmol) diethylamine was added. The mixture
was stirred at room temperature overnight. The reaction was
concentrate and the residue was dissolved in
dichloromethane, washed with brine, dried and concentrated
to give 1.2 g (83%) of 3-(6,6-dimethyl-4-oxo-4,5,6,7-
tetrahydro-1H-indol-3-yl)-N,N-diethylpropionamide as a
white solid.
1HNMR (360 MHz, DMSO-d6) ~ 10.91 (br s, 1H, NH), 6.46
(s, 1H), 3.20-3.29 (m, 4H, 2xCH2), 2.72-2.76 (m, 2H, CHZ),
2.57 (s, 2H, CHZ), 2.45 (m, 2H, CH2), 2.17 (s, 2H, CHz),
0.96-1.06 (m, 12H, 4xCH3).
MS-EI m/z 290 [M+] .
Step 3: LAH (0.57 g, 15.1 mmol) was added dropwise to
a suspension of 3-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-
1H-indol-3-yl)-N,N-diethylpropionamide (1.1 g, 3.8 mmol) in
THF (80 mL). The mixture was refluxed overnight. The
reaction was cooled and ice was added until no more gas was
generated. A few drops of 15% NaOH in water was then
added. The mixture was stirred at room temperature for 30
minutes and then filtered to remove insolubles. The
filtrate was concentrated to give 0.9 g of [3-(6,6-
dimethyl-4,5,6,7-tetrahydro-1H-indol-3-yl)-propyl]-diethyl-
amine as a light yellow oil.
1HNMR (360 MHz, DMSO-d6) 8 9.75 (br s, 1H, NH), 6.24
(s, 1H), 2.19-2.44 (m, 14H, 7xCHz), 1.53 (m, 2H, CHz), 1.40
(m, 2H, CHz) , 0.88-0.92 (m, 12H, 4xCHj) .
MS-EI m/z 262 [M+] .
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Step 4: POClj (0.35 mL, 3.74 mmol) was added dropwise
to DMF (0.8 mL, 10.3 mmol) at -5° C. After stirring at room
temperature for 30 min, the mixture was cooled to -5°C. A
solution of [3-(6,6-dimethyl-4,5,6,7-tetrahydro-1H-indol-3-
yl)-propyl]-diethylamine (0.9 g, 3.4 mmol) in DMF (2 mL)
was then added dropwise. The mixture was stirred at room
temperature for 3 hours and then quenched with ice,
followed by 10 N KOH to adjust pH to 10-11. After stirring
at room temperature for 1 hour, the mixture was extracted
with EtOAc, washed with brine, dried and concentrated to
give 0.55 g 3-(3-diethylaminopropyl)-6,6-dimethyl-4,5,6,7-
tetrahydro-1H-indole-2-carbaldehyde.
1HNMR (360 MHz, DMSO-d6) 8 11.23 (br, s, 1H, NH), 9.41
(s, 1H, CHO) , 2.61 (t, 2H, CHz) , 2.30-2 .43 (m, 10 H, 5x
CHz) , 1.58 (m, 2H, CHz) , 1.45 (t, 2H, CHz) , 0.93 (s, 6H,
2xCH3) , 0.89 (t, 6H, N(CHzCH3)z) .
MS-EI m/z 290 [M'] .
6,6-Dimethyl-3-(3-pyrrolidin-1-ylpropyl)-4,5,6,7-
tetrahydro-1H-indole-2-carbaldehyde
The procedure employed was the same as that in Example
E except that the amine in Step 2 was pyrrolidine.
Step 2: 6,6-Dimethyl-3-(3-oxo-3-pyrrolidin-1-
ylpropyl)-1,5,6,7-tetrahydroindol-4-one
1HNMR (360 MHz, DMSO-d6) 8 10.90 (br s, 1H, NH), 6.46
(s, 1H) , 3 .34 (t, 2H, CHz) , 3.25 (t, 2H, CHz) , 2.76 (m, 2H,
CHz) , 2.58 (s, 2H, CHz) , 2 .43 (m, 2H, CHz) , 2.17 (s, 2H,
CHz) , 1.74-1-1.74 (m, 4H, 2xCHz) , 1.00 (s, 6H, 2xCH3) .
MS-EI m/z 288 [M'] .
Step 3: 6~6-Dimethyl-3-(3-pyrrolidin-1-ylpropyl)-
4,5,6,7-tetrahydro-1H-indole
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1HNMR (360 MHz, DMSO-d6) b 9.75 (br s, 1H, NH), 6.23
(d, 1H), 2.22-2.37 (m, 12H, 6xCHz), 1.55-1.66 (m, 6H,
3xCH2) , 1.40 (m, 2H, CHz) , 0. 92 (s, 6H, 2xCH3) .
MS-EI m/z 260 [M+) .
Step 4: 6,6-Dimethyl-3-(3=pyrrolidin-1 ylpropyl)-
4,5,6,7-tetrahydro-1H-indole-2-carbaldehyde
1HNMR (360 MHz, DMSO-d6) 8 11.25 (br, s, 1H, NH), 9.41
(s, 1H, CHO) , 2.64 (t, 2H, CHz) , 2.31-2.38 (m, 10 H, 5x
CH2) , 1.59-1. 67 (m, 6H, 3xCH2) , 1.46 (t, 2H, CHZ) , 0. 93 (s,
6H, 2xCH3 ) .
MS-EI m/z 288 [M+] .
6,6-Dimethyl-3-[3-(4-methylpiperazin-1-yl)-propyl]-4,5,6,7-
tetrahydro-1H-indole-2-carbaldehyde
The procedure used was that same as that in Example E
except that the amine in Step 2 was 1-methylpiperazine.
Step 2: 6.6-Dimethyl-3-[3-(4-methylpi~erazin-1-yl)-3-
oxo-bropvll-1,5,6,7-tetrahvdroindol-4-one
1HNMR (360 MHz, DMSO-d6) 8 10.93 (br s, 1H, NH), 6.48
(s, 1H), 3.42 (m, 4H, 2xCH2), 3.73 (m, 2H, CH2), 2.57 (s,
2H, CH2) , 2.48 (m, 2H, CHZ) , 2.21 (m, 4H, 2xCH2) , 2. 17 (s,
2H, CHz) , 2. 15 (s, 3H, CH3) , 1 . 00 (s, 6H, 2xCH3) .
MS-EI m/z 317 [M+] .
Step 3: 6,6-Dimethyl-3-f3-(4-met~lpiperazin-1-yl)-
propyll-4,5,6,7-tetrahydro-1H-indole
1HNMR (360 MHz, DMSO-d6) 8 9.74 (br s, 1H, NH), 6.24
(s, 1H), 2.21-2.30 (m, 16H, 8xCH2), 2.12 (s, 3H, CH3), 1.56
(m, 2H, CHz) , 1.40 (m, 2H, CHZ) , 0.92 (s, 6H, 2xCH3) .
MS-EI m/z 289 [M'] .
Step 4: 6,6-Dimethyl-3-f3-(4-methylpiperazin-1-yl)-
propel]-4,5,6,7-tetrahydro-1H-indole-2-carbaldehyde
1HNMR (360 MHz, DMSO-d6) b 11.21 (br, s, 1H, NH), 9.42
(s, 1H, CHO), 2.62 (t, 2H, CH2), 2.18-2.37 (m, 14 H, 7x
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CHZ) , 2.12 (s, 3H, CH3) , 1.61 (m, 2H, CHz) , 1.45 (t, 2H,
CHz) , 0.93 (s, 6H, 2xCH3) .
MS-EI m/z 317 [M~] .
6,6-Dimethyl-3-(3-morpholin-4-ylpropyl)-4,5,6,7-tetrahydro-
1H-indole-2-carbaldehyde
The procedure used was the same as that in Example E
except that the amine used was morpholine.
Step 2: 6,6-Dimethyl-3-(3-morpholin-4-yl-3-oxopropvl)-
1,5,6,7-tetrahydroindol-4-one
1HNMR (360 MHz, DMSO-d6) b 10.94 (br s, 1H, NH), 6.49
(s, 1H), 3.42-3.50 (m, 8H, 4xCH2), 2.74 (m, 2H, CHz), 2.57
(s, 2H, CHZ) , 2 .48 (m, 2H, CH2) , 2 . 17 (s, 2H, CHz) , 1.00 (s,
6H, 2xCH3) .
MS-EI m/z 304 [M+] .
Step 3: 6,6-Dimethyl-3-(3-morpholin-4-ylpropyl)-
4,5,6,7-tetrahydro-1H-indole
1HNMR (360 MHz, DMSO-d6) 8 9.75 (br s, 1H, NH), 6.24
(s, 1H), 3.54 (m, 4H, 2xCH2), 2.23-2.31 (m, 12H, 6xCH2),
1.58 (m, 2H, CHZ) , 1.40 (m, 2H, CH2) , 0. 92 (s, 6H, 2xCH3) .
MS-EI m/z 276 [M+] .
Step 4: 6,6-Dimethyl-3-(3-mor~holin-4 ylpropyl)-
4,5,6,7-tetrahydro-1H-indole-2-carbaldehyde
1HNMR (360 MHz, DMSO-d6) 8 11.25 (br, s, 1H, NH), 9.43
(s, 1H, CHO) , 3.54 (m, 4H, 2xCHz) , 2 .63 (t, 2H, CH2) , 2.20
2.37 (m, 10 H, 5xCHz) , 1.62 (m, 2H, CHZ) , 1 .45 (t, 2H, CHz) ,
0.93 (s, 6H, 2xCH3) .
MS-EI m/z 304 [M+] .
3-(3-Dimethylaminopropyl)-6,6-dimethyl-4,5,6,7-tetrahydro-
1H-indole-2-carbaldehyde
The procedure was the same as that in Example E except
that the amine used was dimethylamine.
Step 2: 3-(6,6-Dimethyl-4-oxo-4,5 6 7-tetrahydro-1H-
indol-3-vl) -N,N-dimethylpropionamide
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1HNMR (360 MHz, DMSO-d6) 8 10.93 (br s, 1H, NH), 6.48
(s, 1H) , 2 .94 (s, 3H, CH3) , 2.79 (s, 3H, CH3) , 2.72 (m, 2H,
CHz) , 2.57 (s, 2H, CHz) , 2.48 (m, 2H, CHz) , 2.17 (s, 2H,
CHz) , 1.00 (m, 6H, 2xCH3) .
MS-EI m/z 262 [M+] .
Step 3: [3- (6, 6-Dimeth~rl-4 5 6 7-tetra~dro-1H-indol-
3-vl)-propylldimethylamine
1HNMR (360 MHz, DMSO-d6) 8 9.75 (br s, 1H, NH), 6.24
(s, 1H), 3.36 (m, 2H, CHz), 2.07-2.3 (m, 14H, 7xCH2), 1.53
(m, 2H, CH2) , 1.40 (m, 2H, CHZ) , 0. 92 (s, 6H, 2xCH3) .
MS-EI m/z 234 [M+] .
Step 4: 3-(3-Dimethylaminopropyl)-6 6-dimethyl-
4~5,6,7-tetrahydro-1H-indole-2-carbaldehyde
1HNMR (360 MHz, DMSO-d6) 8 11.25 (br s, 1H, NH), 9.41
(s, 1H, CHO) , 2.62 (t, 2H, CHZ) , 2 .36 (t, 2H, CHZ) , 2.30 (s,
2H, CHz) , 2.21 (t, 2H, CHz) , 2.12 (s, 6H, 2xCH3) , 1.60 (m,
2H, CHz) , 1 .46 (t, 2H, CH2) , 0. 93 (s, 6H, 2xCH3) .
MS-EI m/z 262 [M+] .
Condensation of aldehydes and oxindoles containing
carboxylic acid substituents
A mixture of the appropriate oxindole (1 equivalent),
1 equivalent of the appropriate aldehyde and 1 - 3
equivalents of piperidine (or pyrrolidine) in ethanol (0.4
M) is stirred at 90-100°C until the reaction complete as
indicated by thin layer chromatography. The mixture is
then concentrated and the residue is triturated with dilute
hydrochloric acid. The resulting precipitate is collected
by vacuum filtration, washed with water and ethanol and
dried to give the product.
Condensation of aldehvde and oxindole not containing
carboxylic acid substituents
A mixture of the appropriate oxindole (1 equivalent),
1 equivalent of the appropriate aldehyde and 1 - 3
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equivalents of piperidine (or pyrrolidine) in ethanol (0.4
M) is stirred at 90-100°C until the reaction complete as
indicated by thin layer chromatography. The mixture is
cooled to room temperature and the precipitate which forms
is collected by vacuum filtration, washed with ethanol and
dried to give the product. Where a precipitate does not
form, the mixture is concentrated and the product isolated
by column chromatography.
Condensation of aldehyde and oxindole containing amino
substituents
The aminooxindole is first protected with a BOC-
group. After the condensation with the appropriate
aldehyde, the solid which forms is deprotected using
trifluoroacetic acid to yield the desired product.
C. Examples - Synthesis of 3-methylidenyl-2-
indolinones of this invention.
The following syntheses of representative compounds of
this invention are shown by way of example only and are not
to be construed as limiting the scope of this invention in
any manner whatsoever.
Example l: 3- (3,5-Diisopropyl-4-methoxybenzylidene)-
1,3-dihydroindol-2-one
3,5-Diisopropyl-4-hydroxybenzaldehyde was methylated
to give 3,5-diisopropyl-4-methoxybenzaldehyde.
1HNMR (ds-DMSO) b: 10.1 (s, 1H, CHO), 6.9 (s, 2H,
aromatic), 3.9 (s, 3H, OCH3), 3.2 (m, 2H, 2xCH), 1.2 (d,
12H, 4xCH3 ) .
3,5-Diisopropyl-4-methoxybenzaldehyde was then
condensed with 2-oxindole to give 0.25 g of 3-(3,5-
Diisopropyl-4-methoxybenzylidene)-1,3-dihydroindol-2-one as
a yellow-orange solid.
1HNMR (ds-DMSO) 8: 10.54 (s, 1H, CONH), 7.6, 7.59,
7.49, 7.21, 6.81-6.88 (multiplets, 7H, =CH-, aromatic),
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3.73 (s, 3H, OCH3) , 3.26-3.33 (m, 2H, 2x-CH) , 1.2 (d, J =
7.23Hz, 12H, 4xCH3).
MS m/z 336.
Example 2: 5-Chloro-3-(3,5-diisopropyl-4-
methoxybenzylidene)-1,3-dihydroindol-2-one
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 5-chloro-2-oxindole to give 0.3 g of 5-chloro-3-(3,5-
diisopropyl-4-methoxybenzylidene)-1,3-dihydroindol-2-one as
a yellow-orange solid.
1HNMR (d6-DMSO) 8: 10.69 (s, 1H, CONH), 7.67 (s, 1H,
=CH-), 7.6 (d, J = 1.85Hz, 1H, H-4), 7.51 (s, 2H,
aromatic), 7.26 (dd, J = 1.85, 8.3Hz, 1H, H-6), 6.89 (d, J
- 8.3Hz, 1H, H-7), 3.74 (s, 3H, OCH3), 3.27-3.34 (m, 2H, 2x-
CH) , 1.22 (d, J = 6. 7Hz, 12H, 4xCH3) .
Example 3: N-(3-(3,5-Diisopro~yl-4-methoxybenzylidene)-
2-oxo-2,3-dihydro-1H-indol-6-yl]-acetamide
Tin chloride dehydrate (225 g) was added to a solution
of 2,4-dinitrophenylacetic acid (22.6 g) in ethanol (450
ml). The mixture was heated at 90° C for 10 hours. The
reaction mixture was cooled and adjusted to pH 11 with 12M
sodium hydroxide. The solids were removed by filtration and
the filtrate was concentrated. The residue was treated with
ethanol (300 ml). Insoluble materials were removed by
filtration and washed with ethanol (5 x 60 ml). The
combined ethanol solutions were evaporated and the solid
obtained was dried under vacuum to give 15g of 6-amino-2-
oxindole as a brown powder.
1HNMR (360 MHz, DMSO-d6) ~: 10.03 (s, br, NH), 6.78 (d,
J = 8.55Hz, 1H, H-4), 6.09-6.11 (m, 2H), 4.95 (s, br, 2H,
NH2), 3.22 (s, 2H, H-3). MS (+ APCI) m/z (relative
intensity, %)
MS m/z 147 ( [M-1]+, 100) .
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To a mixture of 6-amino-2-oxindole (1 g) and acetyl
chloride (0.79 g) in 10 ml of dichloromethane at 0° C was
added triethylamine (1 g). The mixture was brought to room
temperature and stirred for 4 hours protected from
atmospheric moisture with a dry-tube. The solvent was then
evaporated and the residue was recrystallized from methanol
to give 0.98 g (77%) of 6-acetylamino-2-oxindole as a beige
solid.
iHNMR (360 MHz, DMSO-d6) 8: 10.28 (s, br, NH), 9.83 (s,
br, NH), 7.34 (d, J = l.4Hz, 1H, H-7), 7.06 (d, J = 7.9Hz,
1H, H-4), 6.97 (dd, J = 1.4, 7.9Hz, 1H, H-5), 3.37 (s, 2H,
H-3) , 2.01 (s, 3H, CH3) .
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 6-acetamido-2-oxindole to give 0.3 g of N-[3-(3,5-
diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-dihydro-1H-
indol-6-yl]-acetamide as a yellow-orange solid.
1HNMR (d6-DMSO) 8: 10.48 (s, 1H, CONH) , 9. 99 (s, 1H,
CONH), 7.51 (d, J = 8Hz, 1H, H-4), 7.46 (s, 2H, aromatic),
7.44 (s, 1H, =CH-), 7.41 (d, J = 2Hz, 1H, H-7), 6.91 (dd, J
- 2,8Hz, 1H, H-5), 3.73 (s, 3H, OCH3), 3.26-3.34 (m, 2H),
2.03 (s, 3H, CH3) , 1.21 (d, J = 7Hz, 12H, 4xCH3) .
MS m/z 392.
Example 4: 3-(3,5-Diisopropvl-4-methoxybenzylidene)-6-
hydroxy-1,3-dihydroindol-2-one
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 6-hydroxy-2-oxindole to give 0.3 g of 3-(3,5-
Diisopropyl-4-methoxybenzylidene)-6-hydroxy-1,3-
dihydroindol-2-one as a yellow-orange solid.
1HNMR (d6-DMSO) 8: 10.34 (s, 1H, CONH), 9.79 (s, 1H,
OH), 7.42 (d, J = 8Hz, 1H, H-4), 7.43 (s, 2H, aromatic),
7.33 (s, 1H, =CH-), 6.32 (d, J = 2Hz, 1H, H-7), 6.21 (dd, J
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- 2,8Hz, 1H, H-5), 3.72 (s, 3H, OCH3), 3.25-3.33 (m, 2H),
1.2 (d, J = 7Hz, 12H, 4xCH3) .
MS m/z 351.
Example 5: 5-Acetyl-3-(3,5-diisopropvl-4-
methoxybenzylidene)-1,3-dihvdroindol-2-one
2-Oxindole (3 g) was suspended in 1,2-dichloroethane
and slowly treated with 3.2 ml of acetyl chloride. The
resulting suspension was heated at 50° C for 5 hours,
cooled, and poured into water. The resulting precipitate
was collected by vacuum filtration, washed copiously with
water and dried under vacuum to give 2.9 g (73% yield) of
5-acetyl-2-oxindole as a brown solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.75 (s, br, NH), 7.83 (d,
J = 8.23Hz, 1H), 7.78 (s, 1H, H-4), 6.88 (d, J = 8.23Hz,
1H), 3.53 (s, 2H, CH2), 2.49 (s, 3H, CH3).
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 5-acetyl-2-oxindole to give 0.3 g of 5-Acetyl-3-(3,5-
diisopropyl-4-methoxybenzylidene)-1,3-dihydroindol-2-one as
a yellow-orange solid.
1HNMR (ds-DMSO) 8: 10.95 (s, 1H, CONH) , 8.22 (d, J =
2Hz, 1H, H-4), 7.93 (dd, J = 2,8Hz, 1H, H-6), 7.69 (s, 1H,
=CH-), 7.53 (s, 2H, aromatic), 6.97 (d, J = 8Hz, 1H, H-7),
3.75 (s, 3H, OCH3), 3.24-3.34 (m, 2H), 2.48 (s, 3H, CH3),
1.24 (d, J = 7Hz, 12H, 4xCH3) .
MS m/z 377.
Example 6: 3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-
oxo-2,3-dihydro-1H-indole-5-carboxylic acid methyl ester
2-Oxindole (82.9 g) was suspended in 630 ml of acetic
acid in a reaction vessel equipped with mechanical stirring
and the mixture cooled to 10° C in an ice water bath. Solid
N-iodosuccinimide (175 g) was added in portions over 10
minutes. .After the addition was complete the mixture was
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stirred for 1 hour at 10° C. The suspended solid which was
always present became very thick at this time. The solid
was collected by vacuum filtration, washed with 100 ml of
50% acetic acid in water and then with 200 ml of water and
subjected to vacuum filtration for 20 minutes to partially
air dry it. The product was then dried under vacuum to give
93.5 g (36%) of 5-iodo-2-oxindole containing about 5% 2-
oxindole by proton NMR.
1HNMR (360 MHz, DMSO-d6) 8: 10.45 (s, 1H, NH-1), 7.49
(s, 1H, H-4) , 7.48 (d, J = 8.10 Hz, 1H, H-6) , 6.64 (d, J =
8.10 Hz, 1H, H-7) , and 3.46 (s, 2H, CH2-3) .
MS (m/z (relative intensity, %)) 258 ([M-1]+, 13).
5-Iodo-2-oxindole (17 g) was refluxed with 2 g of
palladium diacetate, 18.2 g of triethylamine, 150 ml of
methanol, 15 ml of dimethylsulfoxide and 2.6 g of DPPP in
an atmosphere saturated with carbon monoxide. After 24
hours, the reaction was filtered to remove the catalyst and
the filtrate was concentrated. The concentrate was
chromatographed on silica gel using 30% ethyl
acetate/hexane as the eluent. The fractions containing
product were concentrated and allowed to stand. The
precipitated product was collected by vacuum filtration to
give 0.8 g (7%) of 5-methoxycarbonyl-2-oxindole as an off-
white solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.70 (s, br, 1H, NH-1),
7.83 (dd, J = 1.77, 8.29 Hz, 1H, H-6), 7.77 (s, br, 1H, H-
4), 6.89 (d, J = 8.29 (Hz, 1H, H-7), 3.80 (s, 3H, COOCH3-5),
3.51 (s, 2H, CHz-3) .
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 5-methoxycarbonyl-2-oxindole to give 0.25 g of 3-(3,5-
Diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-dihydro-1H-
indole-5-carboxylic acid methyl ester as a yellow-orange
solid.
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1HNMR (ds-DMSO) 8: 10.96 (s, 1H, CONH) , 8.29 (d, J =
lHz, 1H, H-4), 7.88 (dd, J = l,8Hz, 1H, H-6), 7.7 (s, 1H,
=CH-), 7.55 (s, 2H, aromatic), 7.0 (d, J = 8Hz, 1H, H-7),
3 .76 (s, 3H, OCH3) , 3 . 73 (s, 3H, OCH3) , 3 .26-3 .35 (m, 2H) ,
1.23 (d, J = 7Hz, 12H, 4xCH3) .
MS m/z 393.
Example 7: 3-(3-Isopropyl-4-methoxybenzylidene)-1,3-
dihvdroindol-2-one
2-Isopropylphenol was methylated and then formylated
to give 3-isopropyl-4-methoxybenzaldehyde. 3-Isopropyl-4-
methoxybenzaldehyde was condensed with 2-oxindole to give
0.3 g of 3-(3-isopropyl-4-methoxybenzylidene)-1,3-
dihydroindol-2-one as a yellow-orange solid.
1HNMR (ds-DMSO) 8: 10.5 (s, 1H, CONH), 7.7, 7.6, 7.2,
7.1, 6.8 (multiplets, 8H, aromatic, =CH-), 3.9 (s, 3H,
OCH3) , 3.2 (m, 1H, CH) , 1.2 (d, 6H, 2xCH3) .
Example 8: 3-(5-Isopropyl-4-methoxy-2-
methvlbenzylidene)-1,3-dihydroindol-2-one
5-Isopropyl-4-methoxy-2-methylbenzaldehyde was
condensed with 2-oxindole to give 0.25 g of 3-(5-Isopropyl-
4-methoxy-2-methylbenzylidene)-1,3-dihydroindol-2-one as a
yellow-orange solid.
1HNMR (d6-DMSO) 8: 10.5 (s, 1H, CONH), 7.6, 7.4, 7.3,
7.2, 6.9, 6.8, 6.8 (multiplets, 7H, aromatic, =CH-), 3.9
(s, 3H, OCH3) , 3 .2 (m, 1H, CH) , 2.3 (s, 3H, CH3) , 1.2 (d,
6H, 2xCH3 ) .
Example 9: 5-Chloro-3-(5-isopropyl-4-methoxy-2-
methvlbenzylidene)-1,3-dihydroindol-2-one
2-Isopropyl-5-methylphenol was methylated and then
formylated to give 5-isopropyl-4-methoxy-2-
methylbenzaldehyde.
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1HNMR (d6-DMSO) 8: 10.1 (s, 1H, CHO), 7.6, 6.9 (2xs,
2H, aromatic), 3.9 (s, 3H, OCH3), 3.2 (m, 1H, CH), 2.6 (s,
3H, CH3) , 1.2 (d, 6H, 2xCH3) .
5-Isopropyl-4-methoxy-2-methylbenzaldehyde was
condensed with 5-chloro-2-oxindole to give 0.3 g of 5-
Chloro-3-(5-isopropyl-4-methoxy-2-methylbenzylidene)-1,3-
dihydroindol-2-one as a yellow-orange solid.
1HNMR (d6-DMSO) 8: 10.65 (s, 1H, CONH), 7.73 (s, 1H,),
7.49 (s, 1H,), 7.4 (d, J = 2Hz, 1H, H-4), 7.23 (dd, J =
2,8Hz, 1H, H-6), 6.98 (s, 1H), 6.86 (d, J = 8Hz, 1H, H-7),
3.86 (s, 3H, OCH3) , 3.23-3.3 (m, 2H) , 2.33 (s, 3H, CH3) ,
1.15 (d, J = 7Hz, 6H, 2xCH3) .
MS m/z 341.
Example 10: 3-(3-Cyclopentyl-4-methoxybenzylidene)-1,3-
dihydroindol-2-one
3-Cyclopentyl-4-methoxybenzaldehyde was condensed with
2-oxindole to give 0.25 g of 3-(3-Cyclopentyl-4-
methoxybenzylidene)-1,3-dihydro-indol-2-one as a yellow-
orange solid.
1HNMR (d6-DMSO) b: 10.5 (s, 1H, CONH), 7.6, 7.5, 7.2,
7.1, 6.8 (multiplets, 8H, aromatic, =CH-), 3.9 (s, 3H,
OCH3) , 3.2 (m, 1H, CH) , 1.8 (multiplets, 8H, 4xCH2) .
Example 11: 3-(3-Cyclopentyl-4-methoxybenzylidene)-5-
fluoro-1,3-dihvdroindol-2-one
2-Cyclopentylphenol was methylated and then formylated
to give 3-cyclopentyl-4-methoxybenzaldehyde.
1HNMR (d6-DMSO) 8: 9.9 (s, 1H, CHO), 7.8, 7.7, 7.2
(multiplets, 3H, aromatic), 3.9 (s, 3H, OCH3), 3.2 (m, 1H,
CH) , 1.2 (multiplets, 8H, 4xCHz) .
3-Cyclopentyl-4-methoxybenzaldehyde was condensed with
5-fluoro-2-oxindole to give 0.25 g of 3-(3-cyclopentyl-4-
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methoxybenzylidene)-5-fluoro-1,3-dihydroindol-2-one as a
yellow-orange solid.
1HNMR (d6-DMSO) b: 10.5 (s, 1H, CONH), 7.6, 7.6, 7.6,
7.4, 7.1, 7.0, 6.8 (multiplets, 7H, aromatic, vinyl CH),
3.9 (s, 3H, CH3), 3.3 (m, 1H, CH), 2.0-1.5 (multiplets, 8H,
4xCHz ) .
MS m/z 337.
Example 12: 3-(3-Cyclohexyl-4-methoxybenzylidene)-1,3-
dihydroindol-2-one
2-Cyclohexylphenol was methylated and then formylated.
The aldehyde was condensed with oxindole to give 0.3 g
of 3-(3-Cyclohexyl-4-methoxybenzylidene)-1,3-dihydroindol-
2-one as a yellow-orange solid.
1HNMR (d6-DMSO) b: 10.5 (s, 1H, CONH), 7.7, 7.6, 7.6,
7.2, 7.1, 6.8, 6.8 (multiplets, 7H, aromatic, =CH-), 3.9
(s, 3H, OCH3), 3.0 (m, 1H, CH), 1.8, 1.4 (multiplets, 10H,
5xCHz ) .
Example 13: 5-Bromo-3-(6-methoxybiphenyl-3-vlmethylene)-
1,3-dihydroindol-2-one
2-Phenylanisole was formylated to give 4-methoxy-3-
phenylbenzaldehyde.
1HNMR (d6-DMSO) 8: 9.9 (s, 1H, CHO), 7.9, 7.8, 7.5,
7.4, 7.4, 7.3 (multiplets, 8H, aromatic), 3.9 (s, 3H, OCH3.
MS m/z 213.
4-Methoxy-3-phenylbenzaldehyde was condensed with 5-
bromo-2-oxindole to give 0.3 g of 5-Bromo-3-(6-methoxy-
biphenyl-3-ylmethylene)-1,3-dihydroindol-2-one as a yellow-
orange solid.
1HNMR (d6-DMSO) 8: 10.6 (s, 1H, CONH), 7.8-6.8
(multiplets, 12H, aromatic, =CH-), 3.9 (s, 3H, OCH3).
Example 14: 5-Chloro-3-(2,3-dihydro-benzofuran-5-
ylmethylene)-1,3-dihydroindol-2-one
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2,3-Dihydro-5-formylbenzofuran was condensed with 5-
chloro-2-oxindole to give 0.25 g of 5-Chloro-3-(2,3-
dihydro-benzofuran-5-ylmethylene)-1,3-dihydroindol-2-one as
a yellow-orange solid.
1HNMR (d6-DMSO) 8: 10.6 (s, 1H, CONH), 8.6, 8.2, 7.7,
7.6, 7.2, 6.9, 6.8 (multiplets, 7H, aromatic, =CH-), 4.6
(t, 2H, OCHZ) , 3.2 (t, 2H, OCHZ) .
Example 15: 5-Chloro-3-(2,2-dimethyl-chroman-6-
Ylmethylene)-1,3-dihydroindol-2-one
2,2-Dimethyl-6-formylchromane was condensed with 5-
chloro-2-oxindole to give 0.3 g of 5-Chloro-3-(2,2-
dimethylchroman-6-ylmethylene)-1,3-dihydroindol-2-one as a
yellow-orange solid.
iHNMR (d6-DMSO) 8: 10.61 (s, 1H, CONH), 8.31, 8.27,
7.76, 7.17, 6.79 (multiplets, 6H, aromatic), 7.8 (s, 1H,
=CH-) , 2.78 (t, 2H, CH2) , 1.81 (t, 2H, CHz) , 1.31 (s, 6H,
2xCH3 ) .
MS m/z 340.5 (M+1).
Example 16: N-~3-f3-Cyclohexyl-4-(2-morpholin-4-
ylethoxy)-benzylidenel-2-oxo-2,3-dihydro-1H-indol-6-yl~-
acetamide
Triphenylphosphine (7.47 g, 19.58 mmol) was added to a
solution of 2-cyclohexyl-4-chlorophenol (6 g, 28.48 mmol)
and 2-hydroxyethylmorpholine (3.5 g, 28.48 mmol) in
tetrahydrofuran (50 ml), followed by the dropwise addition
of diethylazodicarboxylate (4.5 ml, 28.48 mmol). The
mixture stirred at room temperature for 12 hours. The
reaction mixture was concentrated under reduced pressure
and triturated with dichloromethane/ hexanes and then
washed with more hexanes. Chromatography (silica, 10-20-
30% ethyl acetate/hexanes) afforded 8.4 g (95%) of 3-
cyclohexyl-4-morpholinoethoxy-chlorobenzene as a yellowish
oil.
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1HNMR (360 MHz, DMSO-d6) 8: 7.12 (m, 2H, 2xAr-H), 6.93
(d, J = 9Hz, 1H, Ar-H), 4.04 (m, 2H, O-CH2CH2), 3.55 (m, 4H,
2x ring0-CH2CHz), 2.84 (m, 1H, CHcyclohexyl), 2.68 (m, 2H,
N-CH2CHz) , 2.47 (m, 4H, 2x ringN-CHZCHz) , 1.7 (m, 5H,
cyclohexyl), 1.3 (m, 5H, cyclohexyl).
MS m/z 324.4 and 325.4, [M+1]' and [M+3)'.
Naphthalene (3.48 g, 27.2 mmol) was added to a
suspension of 30% lithium dispersion (3.2 g, 138 mmol, pre-
washed with tetrahydrofuran) in tetrahydrofuran (80 ml).
The suspension was stirred until a green color appeared
(approximately 1 hour). The reaction was cooled to -78°C,
and a solution of the 3-cyclohexyl-4-morpholinoethoxy-
chlorobenzene (8 g, 24.7 mmol) in tetrahydrofuran (20m1)
was added. After 1 hour, the reaction was warmed to 0°C and
stirred for an additional hour. Dimethylformamide (9.6 ml,
123.5 mmol) was added, the reaction was stirred at 0°C for
one more hour and then warmed to room temperature and
stirred for another hour. The reaction was quenched with
methanol (30 ml), added to 1N hydrochloric acid (300 ml)
and extracted with ethyl acetate (400 ml). The organic
layer was washed with water (300 ml), saturated sodium
bicarbonate solution (300 ml) and brine (300 ml), dried
over magnesium sulfate and concentrated. Chromatography
(silica, 40-60% ethyl acetate/hexanes) afforded 3 g (34%)
of 3-cyclohexyl-4-morpholinoethoxybenzaldehyde as a light
yellow oil.
1HNMR (360 MHz, DMSO-d6) 8: 9.83 (s, 1H, CHO), 7.12 (m,
2H, 2xAr-H), 7.13 (d, J = 8Hz, 1H, Ar-H), 4.19 (m, 2H, 0-
CHZCHZ) , 3.55 (m, 4H, 2x ring0-CHzCH2) , 2.88 (m, 1H,
CHcyclohexyl), 2.74 (m, 2H, N-CHZCHZ), 2.49 (m, 4H, 2x
ringN-CHZCHZ), 1.7 (m, 5H, cyclohexyl), 1.3 (m, 5H,
cyclohexyl).
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MS m/z 318.4 [M+1]'.
A mixture of 3-cyclohexyl-4-
morpholinoethoxybenzaldehyde (3 g, 9.45 mmol), 6-acetamido-
2-oxindole (3.5g, 18.9 mmol) and piperidine (5 ml, 50 mmol)
in ethanol (35 ml) was held in a sealed tube at 100°C for 6
hours. The reaction mixture was cooled, diluted with ethyl
acetate (500 ml) and filtered to remove excess 6-acetamido-
2-oxindole. The filtrate was extracted with 0.5N
hydrochloric acid (200 ml) and brine (2 X 200 ml). The
acid wash was basified with solid sodium bicarbonate to pH
9 and extracted with ethyl acetate (2 x 200 ml). The
combined organic layers were washed with brine (200 ml),
dried over magnesium sulfate and concentrated. The
combined solids were dissolved in dichloromethane (25 ml)
and precipitated with diethyl ether (500 ml) to afford 1.6g
of a mustard yellow solid. The solid was dissolved in
methanol (100 ml) and filtered, affording a yellow solid
(which was set aside) and then precipitated with water (400
ml) and filtered. The solid residue was redissolved in
methanol and concentrated to a solid. This process was
repeated twice to afford -.82% pure isomer 1 by HPLC. The
filtered yellow solid was 77% isomer 2 by HPLC.
1HNMR (360 MHz, DMSO-d6) b: 10.45 (s, 1H, NH), 10.0 (s,
1H, NHAc), 7.55 (m, 3H, Ar-H), 7.44 (m, 2H, Ar-H and Ar-
CH=C), 7.07 (d, 1H, J = 8Hz, Ar-H), 6.89 (dd, 1H, J =2 and
BHz, Ar-H) , 4. 16 (m, 2H, Ar-OCHz) , 3 .57 (m, 4H, OCHzCH2N) ,
2.92 (m, 1H, chexCH), 2.75 (m, 2H, ArOCH2-CHzN), 2.51 (m,
4H, NCHZCH20) , 2. 02 (s, 3H, NHCOCH3) , 1. 8 (m, 5H, chexCHz) ,
1.38 (m, 5H, chexCH2). 20% isomer 1; 77% isomer 2
1HNMR (360 MHz, DMSO-d6) 8: 10.39 (s, 1H, NH), 9.92 (s,
1H, NHAc), 8.37 (d, 1H, J = 2Hz, Ar-H), 8.24 (m, 1H, Ar-H),
7.55 (s, 1H, Ar-CH=C), 7.52 (d, 1H, J = 8Hz, Ar-H), 7.36
(m, 1H, Ar-H), 7.03 (m, 2H, Ar-H), 4.16 (m, 2H, Ar-OCHz),
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3.57 (m, 4H, OCH2CHZN), 2.88 (m, 1H, chexCH), 2.74 (m, 2H,
ArOCH2-CHIN) , 2.51 (m, 4H, NCHzCH20) , 2. 03 (s, 3H, NHCOCH3) ,
1.8 (m, 5H, chexCHz), 1.38 (m, 5H, chexCH2).
Example 17: 3-(3,5-Diisopropyl-4-methoxybenzvlidene)-5-
methoxy-1,3-dihydroindol-2-one
3,5-Diisopropyl-4-hydroxybenzaldehyde was methylated
to give 3,5-diisopropyl-4-methoxybenzaldehyde.
1HNMR (360 MHz, DMSO-d6) 8: 10.1 (s, 1H, CHO), 6.9 (s,
2H, aromatic), 3.9 (s, 3H, OCH3), 3.2 (m, 2H, 2xCH), 1.2 (d,
12H, 4xCH3 ) .
A solution of 11.4 g of hydroxylamine hydrochloride in
water (50 ml) was added to a solution of chloral hydrate
(9.6 g) and sodium sulfate (83 g) in water (200 ml) at 60°C.
The mixture was held at 60°C while, in a separate flask, a
solution of 4-anisidine (6.4 g) and concentrated
hydrochloric acid (4.3 ml) in water (80 ml) was warmed to
80°C. The first solution was then added to the second and
the reaction was refluxed for 2 minutes, cooled slowly to
room temperature and then cooled in an ice bath. The tan
precipitate which formed was collected by vacuum
filtration, washed with water and dried under vacuum to
give 8.6 g (85 % yield) of N-(2-hydroximinoacetyl)-
anisidine.
Concentrated sulfuric acid (45 ml) containing water (5
ml) was warmed to 60°C and 8.6 g of N-(2-hydroximinoacetyl)
anisidine was added in one portion. The mixture was
stirred at 93°C for 10 minutes and then allowed to cool to
room temperature. The mixture was poured into 500 g of ice
and extracted 3 times with ethyl acetate. The combined
extracts were dried over anhydrous sodium sulfate and
concentrated to give 5.1 g (65 % yield) of 5-methoxyisatin
as a dark red solid.
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A mixture of 5-methoxyisatin (5.0 g) and 30 ml of
hydrazine hydrate was refluxed for 15 minutes. The
reaction mixture was cooled to room temperature and 50 ml
of water was added. The mixture was extracted 3 times with
25 ml of ethyl acetate, the organic layers combined, dried
over anhydrous sodium sulfate and concentrated to give a
yellow solid. The solid was stirred in ethyl acetate and
1.1 g of insoluble material removed by vacuum filtration
and saved. This material proved to be 2-hydrazinocarbonyl-
methyl-4-anisidine. The filtrate was concentrated and
chromatographed on silica gel eluting with ethyl
acetate:hexane 1:1 to give 0.7 g of 5-methoxy-2-oxindole as
a dirty yellow solid. The 1.1 g of 2-hydrazinocarbonyl-
methyl-4-anisidine was refluxed for 1 hour in 20 ml of 1 N
sodium hydroxide. The mixture was cooled, acidified to pH
2 with concentrated hydrochloric acid and extracted 3 times
with 25 ml of ethyl acetate. The organic extracts were
combined, washed with brine, dried over anhydrous sodium
sulfate and concentrated to give 0.8 g of 5-methoxy-2-
oxindole as a dirty yellow solid. The combined yield was
1.5 g or 33 %.
1HNMR (360 MHz, DMSO-d6) 8: 10.13 (s, 1H, NH-1), 6.84
(s, 1H, H-4), 6.72 (d, J = 9Hz, 1H, H-6), 6.69 (d, J = 9Hz,
1H, H-7) , 3.68 (s, 3H, OCH3-5) , 3.41 (s, 2H, CHz-3) .
MS m/z 163 [M+1] +.
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 5-methoxy-2-oxindole to give 0.3 g of 3-(3,5-
Diisopropyl-4-methoxybenzylidene)-5-methoxy-1,3-
dihydroindol-2-one as a yellow-orange solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.34 (s, 1H, NH), 7.59 (s,
2H), 7.47 (s, 2H), 7.15 (d, J = 2Hz, 1H), 6.85 (dd, J = 2,
8Hz, 1H), 6.58 (d, J = 8Hz, 1H), 3.73 (s, 3H, OCH3), 3.63
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(s, 3H, OCH3) , 3 .25-3 .39 (m, 2H, 2 x (CH3) 2CH) , 1 .2 (d, 12H,
2 x (CH3) ZCH) .
MS APCI m/z 366 . 1 [M+1] +.
Example 18: N-f3-(4-Methoxy-3-thiophene-3-
ylbenzylidene)-2-oxo-2,3-dihydro-1H-indol-6-yll-acetamide
Tetrakis(triphenylphosphine)palladium(0) (1.35 g) was
added to a solution of 4-methoxy-3-bromobenzaldehyde (6.72
g) in toluene (45 ml) and ethanol (45 ml), followed by
addition of 2M aqueous sodium carbonate (80 ml). To this
mixture was added thiophene-3-boronic acid (5 g). The
mixture was then refluxed for 12 hours. The reaction
mixture was poured into water (200 ml) and extracted into
ethyl acetate (2 x 150 ml). The organic layer was washed
with saturated aqueous sodium bicarbonate (150 ml) and
brine (150 ml), dried over magnesium sulfate and
concentrated. Chromatography (silica, 20% ethyl-
acetate/hexanes) afforded 6 g (88%) of 3 (3-thiophene) -4-
methoxybenzaldehyde as a yellow oil.
1HNMR (360 MHz, DMSO-d6) 8: 9.92 (s, 1H, CHO), 8.04 (d,
1H, J = 2Hz, lxAr-H), 7.8 (m, 2H, Ar-H), 7.58 (dd, 1H, J =
3 and 5Hz, thiophene and Ar-H), 7.50 (dd, 1H, J = 2 and
5Hz, Ar-H), 7.28 (d, 1H, J = 9Hz, thiophene), 3.94 (s, 3H,
OCH3 ) .
MS m/z 219.2 [M+1]'.
A mixture of 3(3-thiophene)-4-methoxybenzaldehyde
(1.72 g), 6-acetamido-2-oxindole (1.5g) and piperidine (4
ml) in ethanol (26 ml) was held in a sealed tube for 12
hours at 100°C. The reaction was cooled and poured into
diethyl ether. The precipitate which formed was removed by
filtration, washed with diethyl ether and then hexanes and
dried to afford 0.9 g (29%) of N-[3-(4-methoxy-3-thiophene-
3-ylbenzylidene)-2-oxo-2,3-dihydro-1H-indol-6-yl]-acetamide
as a tan solid.
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1HNMR 360 MHz, DMSO-d6) 8: 10.52 (s, 1H, NH), 10.22 (s,
1H, NHAc), 7.85 (m, 2H, 2xAr-H), 7.7 (dd, J = 2 and 9Hz,
1H, Ar-H), 7.6 (m, 2H, 2xAr-H), 7.5 (dd, 1H, J = 1 and SHz,
Ar-H), 7.47 (s, 2H, Ar-H and Ar-CH=C), 7.25 (d, 1H, J =9Hz,
Ar-H), 6.96 (dd, 1H, J =2 and 9Hz, Ar-H), 3.92 (s, 3H,
OCH3) , 2. 04 (s, 3H, NHCOCHj) .
MS m/z 391.2 [M+1]'.
Example 19: 3-(3,5-Diisopropyl-4-methoxybenzylidene)-5-
methvl-1,3-dihydroindol-2-one
A mixture of 5-methylisatin (15.0 g) and 60 ml of
hydrazine hydrate was stirred at 140 - 160°C for 4 hours.
The reaction mixture was cooled to room temperature, poured
into 300 ml of ice water and acidified to pH 2 with 6 N
hydrochloric acid. After standing at room temperature for
2 days, a precipitate formed which was collected by vacuum
filtration, washed with water and dried under vacuum to
give 6.5 g (47 % yield) of 5-methyl-2-oxindole.
1HNMR (360 MHz, DMSO-d6) 8: 10.20 (s, br, 1H, NH-1),
6.99 (s, 1H, H-4) , 6.94 (d, J = 8 Hz, 1H, H-6) , 6.68 (d, J
- 8Hz, 1H, H-7), 3.39 (s, 2H, CH2-3), and 2.22 (s, 3H, CHj-
5) .
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 5-methyl-2-oxindole to give 0.3 g of 3-(3,5-
diisopropyl-4-methoxybenzylidene)-5-methyl-1,3-
dihydroindol-2-one as a yellow-orange solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.41 (s, br, 1H, NH), 7.56
(s, 1H), 7.51 (s, 2H), 7.50 (s, br, 1H), 7.03 (d, br, J =
8Hz, 1H), 6.76 (d, J = 8Hz, 1H), 3.74 (s, 3H, OCH3), 3.26-
3 .35 (m, 2H, 2 x (CH3) ZCH) , 2 . 15 (s, 3H, CH3) , 1 .22 (d, J =
7Hz, 12H, 2 x (CH3) zCH) .
MS APCI m/z 350.2 [M+1]'.
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Example 20: 5-Amino-3-(3,5-diisopropyl-4-methoxy-
benzvlidene)-1,3-dihydroindol-2-one
2-Oxindole (6.5 g) was dissolved in concentrated
sulfuric acid (25 ml) and the mixture maintained at -10 to
15°C while fuming nitric acid (2.1 ml) was added dropwise.
After the addition of the nitric acid the reaction mixture
was stirred at 0°C for 0.5 hour and poured into ice-water.
The precipitate which formed was collected by filtration,
washed with water and crystallized from 50% acetic acid.
The final crystalline product was then filtered, washed
with water and dried under vacuum to give 6.3 g (70%) of 5-
nitro-2-oxindole.
5-Nitro-2-oxindole (6.3 g) was hydrogenated in
methanol over 10% palladium on carbon to give 3.0 g (60%
yield) of 5-amino-2-oxindole as a white solid.
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 5-amino-2-oxindole to give 0.3 g of 5-Amino-3-(3,5-
diisopropyl-4-methoxybenzylidene)-1,3-dihydroindol-2-one as
a yellow-orange solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.62 (br s, 1H, NH), 8.31
(s, 2H), 7.8 (s, 1H), 7.65 (s, 1H), 7.55 (d, 1H), 7.47 (s,
1H), 7.09 (dd, J = 2, 8Hz, 1H), 6.88 (d, J = 8Hz, 1H), 3.72
(3H, OCH3) , 3 .24-3 . 32 (m, 2H, 2 x (CH3) 2CH) , 1 .23 (d, J =
6. 5Hz, 12H, 2 x (CH3) ZCH) .
Example 21: 5-Chloro-3-(4-methoxy-3,5-
dimethylbenzylidene)-1,3-dihydroindol-2-one
2,6-Dimethylphenol was methylated and then formylated.
1HNMR (d6-DMSO) 8: 10.1(s, 1H, CHO), 6.9(s, 2H,
aromatic) , 3 . 9 (s, 3H, OCH3) , 2 . 6 (s, 6H, 2xCH3) .
3,5-Dimethyl-4-methoxybenzaldehyde was condensed with
5-chloro-2-oxindole to give 0.3 g of 5-chloro-3-(4-methoxy-
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3,5-dimethylbenzylidene)-1,3-dihydroindol-2-one as a
yellow-orange solid.
Example 22: 3-(3,5-Diisopropyl-4-methoxybenzylidene)-6-
fluoro-1,3-dihydroindol-2-one
A suspension of sodium hydride (2.6 g) and
dimethylmalonate (14.5 g) in dimethylsulfoxide (160 ml) was
stirred at 100°C for 1 hour. The mixture was cooled to room
temperature and 7.95 g of 2,5-difluoronitrobenzene was
added and stirring was continued for 30 minutes. The
mixture was then heated to 100°C and stirred for 1
additional hour, then cooled to room temperature and poured
into 400 ml of saturated ammonium chloride. The mixture
was extracted with ethyl acetate (200 ml) and the organic
layer washed with brine, dried over anhydrous sodium
sulfate and concentrated under vacuum. The residue was
crystallized from methanol to give 24.4 g (80% yield) of
dimethyl 4-fluoro-2-nitrophenylmalonate as a white solid.
The filtrate was concentrated and chromatographed on a
column of silica gel (ethyl acetate: hexane 1:8) to give
5.03 g of dimethyl 4-fluoro-2-nitrophenylmalonate, for a
total of 29.5 g (96 % yield).
Dimethyl 4-fluoro-2-nitrophenylmalonate (5.0 g) was
refluxed in 20 ml of 6 N hydrochloric acid for 24 hours.
The reaction was cooled and the white solid which formed
was collected by vacuum filtration, washed with water and
dried to give 3.3 g (87% yield) of 4-fluoro-2-
nitrophenylacetic acid.
4-Fluoro-2-nitrophenylacetatic acid (3.3 g) dissolved
in acetic acid (15 ml) was hydrogenated over 0.45 g of 10%
palladium on carbon under 60 psi for 2 hours. The catalyst
was removed by filtration and washed with 15 ml of
methanol. The combined filtrates were concentrated and
diluted with water. The precipitate was collected by
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vacuum filtration, washed with water and dried to give 1.6
g (70% yield) of 6-fluoro-2-oxindole. The filtrate was
concentrated to give a purple solid with an NMR spectrum
similar to the first crop. Chromatography of the purple
solid (ethyl acetate: hexane 1:2, silica gel) gave a second
crop of 6-fluoro-2-oxindole as a white solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.43 (s, 1H, NH-1), 7.17
(t, J = 8Hz, H-4), 6.69 (ddd, J = 2, 8, lOHz, 1H, H-5), 6.6
(dd, J = 2, 9Hz, 1H, H-7), and 3.42 (s, 2H, CHZ-3).
MS m/z 152.8 [M+1]+.
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 6-fluoro-2-oxindole to give 0.3 g of 3-(3,5-
diisopropyl-4-methoxybenzylidene)-6-fluoro-1,3-
dihydroindol-2-one as a yellow-orange solid.
1HNMR (360 MHz, DMSO-d6) b: 10.69 (s, br, 1H, NH), 7.58
(s, 1H, vinyl), 7.55-7.59 (m, 1H), 7.47 (s, 2H), 6.68 (m,
2H) , 3 . 73 (s, 3H, OCH3) , 3 .25-3 .34 (m, 2H, 2 x (CH3) 2CH) ,
1 .2 (d, J = 7Hz, 12H, 2 x (CH3) zCH) .
MS m/z 353.
Example 23: 3-(2,2-Dimethylchroman-6-ylmethylene)-5-
fluoro-1,3-dihydroindol-2-one
2,2-Dimethyl-6-formylchromane (commercially available)
was condensed with 5-fluoro-2-oxindole to give 0.3 g of 3-
(2,2-Dimethylchroman-6-ylmethylene)-5-fluoro-1,3-
dihydroindol-2-one as a yellow-orange solid.
Example 24: 5-Chloro-3-[3,5-diisopropyl-4-(2-morpholin-
4-ylethoxy)-benzylidenel-1,3-dihydroindol-2-one
Triphenylphosphine (5.14 g, 19.58 mmol) was added to a
solution of 3,5-diisopropyl-4-hydroxybenzaldehyde (4 g,
19.58 mmol) in tetrahydrofuran (40 ml) followed by addition
of 2-hydroxyethylmorpholine (2.57 g, 19.58 mmol) and then
the dropwise addition of diethylazodicarboxylate (3.41 g,
19.58 mmol). The mixture was stirred at room temperature
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for 12 hours. The reaction was concentrated under reduced
pressure and partitioned between 2N hydrochloric acid (200
ml) and ethyl acetate (150 ml). The aqueous layer was
extracted with ethyl acetate (2 x 150m1), basified to pH 9
with solid sodium bicarbonate and extracted with ethyl
acetate (3 x 150 ml). The organic layers were combined,
dried over magnesium sulfate and concentrated to afford 900
mg (14%) of 3,5-diisopropyl-4-(2-morpholin-4-ylethoxy)-
benzaldehyde as a yellowish oil.
1HNMR (360 MHz, DMSO-d6) 8: 9.91 (s, 1H, CHO), 9.37 (s,
2H, 2xAr-H) , 3 .85 (t, J = 5Hz, 2H, O-CHzCH2) , 3 .59 (m, J = 5
and 4Hz, 4H, 2x ring0-CHzCH2), 3.39 (sept, J = 7Hz, 2H,
2xCHCH3) , 2 .72 (t, J = 5Hz, 2H, N-CHZCHZ) , 2 .4 (m on DMSO,
4H, 2x ringN-CH2CHz) , 1.2 (d, J = 7Hz, 12H, 4xCHj) .
A mixture of 3,5-diisopropyl-4-(2-morpholin-4-
ylethoxy)-benzaldehyde (0.4 g, 1.26 mmol), 5-chloro-2-
oxindole (0.218, 1.26 mmol) and pyrrolidine (0.5 ml, 6.3
mmol) in ethanol (2 ml) was held in sealed tube at 100°C for
12 hours. The mixture was then poured into 1N hydrochloric
acid (100 ml) and the solid which remained were filtered
and washed with more water (50 ml). The solid was then
dissolved in ethyl acetate (200 ml), the solution dried
over magnesium sulfate, fiiltered and then concentrated.
The solid which remained was chromatographed (silica, 4/4/1
dichloromethane/ hexanes/ methanol) to give 60 mg (10%) of
5-chloro-3-[3,5-diisopropyl-4-(2-morpholin-4-ylethoxy)-
benzylidene]-1,3-dihydroindol-2-one as a brownish-orange
solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.54 (s, 1H, NH), 9.43 (d,
J = lOHz, 1H, Ar-H), 7.58 (d, J = 9Hz, 1H, Ar-H), 7.46 (d,
J = SHz, 2H, Ar-H), 7.3 (m, 1H, Ar-H), 7.19 (m, J = 9 and
lOHz, 1H, Ar-H), 6.99 (m, J = 9 and 9Hz, 1H, Ar-H), 6.83
(m, J = 9 and lHz, 1H, Ar-H), 3.61 (dd, J = 9 and 22 HZ 1H,
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lxCH2CH) , 3 .43 (m, 1H, CHCH3) , 2 .86 (dd, J = 4 and 22Hz, 1H,
lxCH2CH) , 1.35 (d, J = 9Hz, 3H, CH3) .
MS m/z 262.0 [M+1]+.
Example 25: 3-(3,5-Diisopropvl-4-methoxybenzvlidene)-7-
fluoro-1,3-dihydroindol-2-one
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 7-fluoro-2-oxindole to give 0.25 g of 3-(3,5-
diisopropyl-4-methoxybenzylidene)-7-fluoro-1,3-
dihydroindol-2-one as a yellow-orange solid.
Example 26: 3-(4-Methoxy-3-thiophen-3-ylbenzylidene)-5-
(2-morpholin-4-yl-ethyl)-1,3-dihydroindol-2-one
A solution of 5-chloroacetyl-2-oxindole (4.18 g) in
trifluoroacetic acid (30 ml) was cooled in an ice bath and
4.65 g of triethylsilane added. The mixture was then
stirred at room temperature for 3 hours. The mixture was
poured into 150 ml of water and the precipitate which
formed was collected by vacuum filtration, washed with 50
ml of water and dried to give 2.53 g (65 % yield) of 5-(2-
chloroethyl)-2-oxindole as a reddish-brown solid.
1HNMR (360 MHz, DMSO-d6) b: 10.26 (s, br, NH), 7.11 (s,
1H, H-4), 7.05 (d, J = 8Hz, 1H), 6.73 (d, J = 8Hz, 1H),
3.77 (t, J = 7Hz, 2H, CHZ) , 3.42 (s, 2H, CH2) , 2.94 (t, J =
7Hz, 2H, CHz) .
A solution of 5-(2-chloroethyl)-2-oxindole (2.3 g),
morpholine (1.2 ml) and diisopropylethylamine (1.2 ml) in
dimethylsulfoxide (10 ml) was heated overnight at 100°C.
The mixture was cooled, poured into water and extracted
with ethyl acetate. The organic layer was washed with
brine, dried and evaporated. The residue was
chromatographed (silica gel, 5 % methanol in chloroform) to
give 0.9 g (31 %) of 5-(2-morpholin-4-yl-ethyl)-2-oxindole
as a white solid.
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1HNMR (360 MHz, DMSO-d6) 8: 10.21 (s, br, 1H, NH), 7.06
(s, br, 1H, H-4) , 7.0 (d, J = 8Hz, 1H, H-6) , 6.71 (d, J =
8Hz, 1H, H-7), 3.55-3.58 (m, 4H), 3.41 (s, 2H, H-3), 2.63-
2.68 (m, 2H), 2.39-2.47 (m, 6H).
A mixture of 3(3-thiophene)-4-methoxybenzaldehyde
(0.27 g, 1.22 mmol), 5-(2-morpholin-4-yl-ethyl)-2-oxindole
(0.3g, 1.22 mmol) and piperidine (0.6 ml, 6.1 mmol) in
ethanol (4 ml) was held in a sealed tube at 100°C for 12
hours. The reaction was cooled and poured into diethyl
ether (150 ml) and hexanes (150 ml). The solid which
remained was removed by filtration, washed with diethyl
ether and then hexanes and dried to afford 0.27 g (50%) of
3-(4-methoxy-3-thiophen-3-ylbenzylidene)-5-(2-morpholin-4-
yl-ethyl)-1,3-dihydroindol-2-one as an orange-yellow solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.41 (s, 1H, NH), 8.86 (d,
J = 2.15Hz, 1H, Ar-H), 8.39 (dd, J = 2 and 9Hz, 1H, Ar-H),
7.8 (dd, 1H, J = 1 and 3Hz, Ar-H), 7.7 (s, 1H, Ar-CH=C),
7.6 (m, 1H, ArH), 7.5 (m, 2H, 2xArH), 7.21 (d, J = 9Hz, 1H,
Ar-H), 7.02 (dd, 1H, J = 1 and 8Hz, Ar-H), 6.73 (d, 1H, J =
8Hz, Ar-H) , 3 .93 (s, 3H, OCH3) , 3.58 (m, 4H, 2x OCHZCHZN) ,
2.7 (m, 2H, ArCH2CH2) , 2 .5 (m, 2H, ArCH2CH2N) , 2 .4 (m, 4H,
2xOCH2CH2N) .
MS m/z 447.2 [M+1]+.
Example 27: N-[3-(5-Isogropyl-4-methoxy-2-methyl-
benzvlidene)-2-oxo-2,3-dihydro-1H-indol-6-yll-acetamide
A mixture of 5-isopropyl-4-methoxy-2-
methylbenzaldehyde (0.25 g, 1.3 mmol), 6-acetamido-2-
oxindole (0.25g, 1.3 mmol) and pyrrolidine (0.54 ml, 6.5
mmol) in ethanol (4 ml) was held in a sealed tube at 100°C
for 12 hours. The reaction was cooled and then poured into
water (100 ml). The solid which formed was removed by
filtration, washed with water and then dissolved in ethyl
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acetate (200 ml). The ethyl acetate solution was dried
over magnesium sulfate and concentrated. The resulting
solid was triturated with dichloromethane/ hexanes to
afford 0.33 g (70%) of N-[3-(5-isopropyl-4-methoxy-2-
methylbenzylidene)-2-oxo-2,3-dihydro-1H-indol-6-yl]-
acetamide as a brownish yellow solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.46 (s, 1H, NH), 7.49 (s,
1H, Ar-H), 7.46 (s, 1H, Ar-H), 7.41 (s, 1H, Ar-H), 6.93 (m,
1H, Ar-CH=C), 6.83 (dd, 1H, J =2 and 8Hz, Ar-H), 3.83 (s,
3H, OCH3) , 2.30 (s, 3H, Ar-CHj) , 2. 02 (s, 3H, NHCOCH3) , 1.2
(m, 1H, CH (CH3) 2) , 1 . 1 (d, J = 7Hz, 6H, CH (CH3) z) .
MS m/z 365.2 [M+1] +.
Example 28: 3-(3,5-Diisopropyl-4-methoxybenzylidene)-5-
ethyl-1,3-dihydroindol-2-one
2-Oxindole (3 g) suspended in 1,2-dichloroethane was
slowly treated with 3.2 ml of acetyl chloride. The
resulting suspension was stirred at 50°C for 5 hours,
cooled, and poured into water. The resulting precipitate
was collected by vacuum filtration, washed copiously with
water and dried under vacuum to give 2.9 g (73 % yield) of
5-acetyl-2-oxindole as a brown solid.
1HNMR (360 MHz, DMSO-d6) 8 . 10.75 (s, br, NH), 7.83
(d, J = 8Hz, 1H), 7.78 (s, 1H, H-4), 6.88 (d, J = 8Hz, 1H),
3.53 (s, 2H, CH2) , 2.49 (s, 3H, CH3) .
5-Acetyl-2-oxindole (2 g) and 15 ml of trifluoroacetic
acid in an ice bath was slowly treated with 1.8 g of
triethylsilane and then stirred at room temperature for 5
hours. One ml of triethylsilane was added and stirring
continued overnight. The reaction mixture was poured into
ice water and the resulting precipitate collected by vacuum
filtration, washed copiously with water and dried under
vacuum to give 1.3 g (71 % yield) of 5-ethyl-2-oxindole as
a yellow solid.
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1HNMR (360 MHz, DMSO-d6) 8: 10.25 (s, br, NH-1), 7.03
(s, 1H, H-4), 6.97 (d, J = 8Hz, 1H, H-6), 6.69 (d, J = 8Hz,
1H, H-7) , 3.40 (s, 2H, CHZ-3) , 2.51 (q, J = 8Hz, 2H, CHzCH3-
5) , and 1.12 (t, J = 7Hz, 3H, CHzCH3-5) .
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 5-ethyl-2-oxindole to give 0.3 g of 3-(3,5-
diisopropyl-4-methoxybenzylidene)-5-ethyl-1,3-dihydroindol-
2-one as a yellow-orange solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.42 (s, 1H, NH), 7.56 (s,
1H), 7.51 (s, 2H), 7.49 (s, br, 1H), 7.08 (dd, J = 8Hz,
1H), 6.79 (d, J = 8Hz, 1H), 3.74 (s, 3H, OCH3), 3.25-3.55
(m, 2H, 2 x (CH3) 2CH) , 2 .44 (q, J = 8Hz, 2H, CH3CHz) , 1 .22
(d, J = 7Hz, 12H, 2 x (CH3)ZCH), 1.08 (t, J = 8Hz, 3H,
CH3CH2 ) .
MS APCI m/z 364 . 3 [M+1] +.
Example 29: N-[2'-Methoxy-5'-(2-oxo-1,2-di~droindol-3-
ylidenemethyl)-biphenyl-3-yll-acetamide
A mixture of 3-bromo-p-anisaldehyde (1 g, 4.65 mmol),
pyrrolidine (1.9 ml, 23.2 mmol) and oxindole (0.62 g, 4.65
mmol) in dimethylformamide (7 ml) was held in a sealed tube
at 100'C for 12 hours. The mixture was cooled to room
temperature and poured into 1N hydrochloric acid (100 ml).
The precipitate which formed was filtered and washed with
water. The precipitate was then dissolved in ethyl acetate
(200 ml), the solution washed with brine, dried over
magnesium sulfate and then concentrated. The solid obtained
was chromatographed (silica, 30-40% ethyl acetate/ hexanes)
to give 0.63 g (42%) of 3-(3-bromo-4-methoxybenzylidene)-
1;3-dihydroindol-2-one as a yellow solid.
1HNMR (360 MHz, DMSO-d6) b: 10.5 (m, 1H, NH), 7.7 (m,
1H, Ar-H), 7.6 (m, 1H, Ar-CH=C), 7.5 (m, 1H, Ar-H), 7.2 (m,
2H, 2xAr-H), 6.9 (m, 1H, Ar-H), 6.8 (m, 1H, Ar-H), 3.9 (s,
3H, OCH3). MS m/z 330.0 and 332Ø
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[M]' and [M+2]'.
Tetrakis(triphenylphosphine)palladium(0) (0.02 g, 0.02
mmol) was added to a solution of 3-(3-Bromo-4-methoxy-
benzylidene)-1,3-dihydroindol-2-one (0.2 g, 0.61 mmol) in
toluene (1 ml) and ethanol (2 ml), followed by addition of
2M aqueous sodium carbonate (1.2 ml, 2.4 mmol). To this
mixture was added 3-acetamidophenylboronic acid (0.12 g,
0.67 mmol), and the mixture was heated to 100°C in a sealed
tube and held there for 12 hours. The reaction was then
poured into water (50 ml) and extracted with ethyl acetate
(2x100 ml). The combined organic layers were washed with
saturated aqueous sodium bicarbonate (50 ml) and brine (50
ml). The organic layer was dried over magnesium sulfate and
concentrated. Chromatography (silica, 30-40% ethyl
acetate/hexanes) afforded a waxy solid that was triturated
with diethyl ether/hexanes and then dried to afford 0.05 g
(22%) of N-[2'-methoxy-5'-(2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-biphenyl-3-yl]-acetamide as a reddish orange
solid.
1HNMR (360 MHz, DMSO-d6) b: 10.6 (m, 1H, NH), 9.88 (s,
1H, NHAc), 7.7 (m, 1H, Ar-H), 7.5 (m, 3H, 2xAr-H and Ar-
CH=C), 7.2 (m, 4H, Ar-H), 7.0 (m, 2H, Ar-H), 6.8 (m, 2H,
Ar-H) , 3.9 (two s, 3H, OCH3) , 2.02 (2, 3H, COCH3) .
MS m/z 385.2 [M+1]+.
Example 30: 5-Fluoro-3-(5-isopropyl-4-methoxy-2-methyl-
benzvlidene)-1,3-dihvdroindol-2-one
5-Isopropyl-4-methoxy-2-methylbenzaldehyde was
condensed with 5-fluoro-2-oxindole to give 0.25 g of 5-
fluoro-3-(5-isopropyl-4-methoxy-2-methylbenzylidene)-1,3-
dihydroindol-2-one as a yellow-orange solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.54 (s, 1H, NH), 7.72 (s,
1H), 7.47 (s, 1H), 7.1 (dd, 1H), 7.04 (ddd, 1H), 6.98 (s,
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1H), 6.82-6.86 (m, 1H), 3.86 (s, 3H, OCH3), 3.25-3.28 (m,
1H, (CH3) 2CH) , 2 .32 (s, 3H, CH3) , 1 . 14 (d, 6H, (CH3) 2CH) .
EI MS m/z 325 [M]'.
Example 31: N-[3-(4-Methoxy-3-thiophen-2-ylbenzylidene)-
2-oxo-2,3-dihydro-1H-indol-6-yl]-acetamide
Tetrakis(triphenylphosphine)palladium(0) (0.24 g) was
added to a solution of 4-methoxy-3-bromobenzaldehyde (1.5
g) in toluene (15 ml) and ethanol (15 ml), followed by
addition of 2M aqueous sodium carbonate (14 ml). To this
mixture was added thiophene-2-boronic acid (0.98 g), and
the mixture was heated to reflux. After 3 hours, the
reaction was partitioned between water (100 ml) and ethyl
acetate (250 ml). The organic layer was washed with
saturated aqueous sodium bicarbonate (75 ml) and brine (75
ml), dried over magnesium sulfate and concentrated.
Chromatography (silica, 20% ethyl acetate/hexanes) afforded
1.3 g (87%) of 3-(2-thiophene)-4-methoxybenzaldehyde as a
yellow oil.
1HNMR (360 MHz, DMSO-d6) 8: 9.92 (s, 1H, CHO), 8.2 (d,
1H, J = 3Hz, lxAr-H), 7.8 (dd, 1H, J = 10 and lOHz,
SCHCHCH), 7.65 (m, 1H, Ar-H), 7.60 (dd, 1H, J = 2 and 6Hz,
Ar-H), 7.32 (d, 1H, J = lOHz, SCHCHCH), 7.13 (d, 1H, J = 10
and 6Hz, SCHCHCH), 3.99 (s, 3H, OCH3).
A mixture of 3-(2-thiophene)-4-methoxybenzaldehyde
(0.25 g, 1.15 mmol), 6-acetamido-2-oxindole (0.228, 1.15
mmol) and pyrrolidine (0.48 ml, 5.75 mmol) in ethanol (4
ml) was held in a sealed tube at 100°C for 12 hours. The
reaction was cooled and the precipitate which formed was
removed by filtration, washed with ethanol and then hexanes
and dried to afford 0 . 15 g (33%) of N- [3- (4-methoxy-3-
thiophen-2-ylbenzylidene)-2-oxo-2,3-dihydro-1H-indol-6-yl]-
acetamide as a yellow solid.
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'HNMR (360 MHz, DMSO-d6) d: 10.49 (s, 1H, NH), 9.95 (s,
1H, NHAc), 8.99 (d, J = 2Hz, 1H, Ar-H), 8.28 (dd, J = 2 and
9Hz, 1H, Ar-H), 7.6 (m, 2H, 2xAr-H), 7.5 (m, 2H, Ar-H and
Ar-CH=C), 7.38 (d, 1H, J =lHz, Ar-H), 7.22 (d, 1H, J = 9Hz,
Ar-H), 7.14 (dd, 1H, J = 4 and 5Hz, Ar-H), 7.05 (dd, 1H, J
=2 and 9Hz, Ar-H), 3.97 (s, 3H, OCH3), 2.04 (s, 3H,
NHCOCH3 ) .
MS m/ z 3 91 . 3 [M] ' and [M+2 ] ' .
Example 32: 6-Amino-3-(3,5-diisoprop~l-4-methoxy-
benzvlidene)-1,3-dihydroindol-2-one
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 6-amino-2-oxindole to give 0.3 g of 6-amino-3-(3,5-
diisopropyl-4-methoxybenzylidene)-1,3-dihydroindol-2-one as
a yellow-orange solid.
Example 33: N-f3-(2,2-Dimethylchroman-6-ylmethylene)-2-
oxo-2,3-dihvdro-1H-indol-6=yll-acetamide
A mixture of 6-acetamido-2-oxindole (285 mg), 2,2-
dimethylchroman-6-carboxaldehyde (285 mg) and piperidine
(0.2 ml) was stirred at 90°C overnight. The reaction mixture
was cooled and diluted with 20 ml of water. The sticky
solid which formed was filtered and chromatographed (2%
ethanol in ethyl acetate) to give 240 mg (44%) of N-[3-
(2,2-dimethylchroman-6-ylmethylene)-2-oxo-2,3-dihydro-1H-
indol-6-yl]-acetamide as an orange red solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.44 (s, 1H, NH), 10.0 (s,
1H, NH), 7.6, 7.46, 7.42, 7.37, 6.93, 6.82 (m, 7H, 6 Ar-H
and 1 vinyl-H), 2.8 (t, J = 6Hz, 2H, CHZ), 2.03 (s, 3H,
CH3) , 1.83 (t, J = 6Hz, 2H, CHz) , 1.32 (s, 6H, 2x CH3) .
MS m/z 363.1 [M+1]'.
Example 34: 5-Bromo-3-(2,2-dimethylchroman-6-
ylmethylene)-1,3-dihydroindol-2-one
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2-Oxindole (1.3 g) in 20 ml of acetonitrile was cooled
to -10°C and 2.0 g of N-bromosuccinimide was slowly added
with stirring. The reaction was stirred for 1 hour at -10°C
and then 2 hours at 0°C. The precipitate which formed was
collected, washed with water and dried to give 1.9 g (90%
yield) of 5-bromo-2-oxindole.
1HNMR (360 MHz, DMSO-d6) 8: 10.44 (s, br, 1H, NH-1),
7.32-7.36 (m, 2H), 6.76 (d, J = 8.5 Hz, 1H, H-7), 3.5 (s,
2H, CHz ) . MS m/ z 212 . 1 and 214 . 1 , [M] ' and [M+2 ] ' .
2,2-Dimethyl-6-formylchromane (commercially available)
was condensed with 5-bromo-2-oxindole to give 0.3 g of 5-
bromo-3-(2,2-dimethylchroman-6-ylmethylene)-1,3-
dihydroindol-2-one as a yellow-orange solid.
Isomer 1 (67%) : 1HNMR (360 MHz, DMSO-d6) 8: 10.65 (s,
br, 1H, NH), 7.73 (d, 1H), 7.6 (s, 1H, vinyl), 7.5 (s, br,
1H), 7,47 (dd, 1H), 7.37 (dd, 1H), 6.86 (d, 1H), 6.82 (d,
1H), 2.77-2.81 (m, 4H, 2 x CH2), 1.81-1.84 (m, 4H, 2 x CHz),
1.32 (s, 6H, 2 x CH3) .
Isomer 2 (33%): 1HNMR (360 MHz, DMSO-d6) 8: 10.63 (s,
br, 1H, NH) , 8.32 (dd, 1H) , 8.27 (s, br, 1H) , 7. 89 (d, 1H) ,
7, 81 (s, 1H, vinyl) , 7.3 (dd, 1H) , 6. 79 (d, 1H) , 6.76 (d,
1H), 2.77-2.81 (m, 4H, 2 x CHZ), 1.81-1.84 (m, 4H, 2 x CHZ),
1.31 (s, 6H, 2 x CHj) .
MS APCI m/z 384.1 and 385.9, [M]' and [M+2]+.
Example 35: 3-(4-Methoxy-3-thiophen-3-ylbenzZrlidene)-
1,3-dihydroindol-2-one
Tetrakis(triphenylphosphine)palladium(0) (0.02 g, 0.02
mmol) was added to a solution of 3-(3-bromo-4-methoxy-
benzylidene)-1,3-dihydroindol-2-one (0.2 g, 0.61 mmol) in
toluene (1 ml) and ethanol (1 ml), followed by addition of
2M aqueous sodium carbonate (1.2 ml, 2.4 mmol). To this
mixture was added thiophene-3-boronic acid (0.09 g, 0.67
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mmol) and the mixture was held at 100°C in a sealed tube for
12 hours. The reaction was then poured into water (50 ml)
and extracted with ethyl acetate (2x100 ml). The combined
organic layers were washed with saturated aqueous sodium
bicarbonate (50 ml) and brine (50 ml), dried over magnesium
sulfate and concentrated. Chromatography (silica,
methylene chloride then 30-40% ethyl acetate/hexanes)
afforded 0.1 g (50%) of 3-(4-methoxy-3-thiophen-3-
ylbenzylidene)-1,3-dihydroindol-2-one as a yellow solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.54 (s, 1H, NH), 7.8 (m,
2H, 2xAr-H), 7.6 (m, 5H, 4xAr-H and Ar-CH=C), 7.2 (m, 2H,
Ar-H), 6.8 (m, 2H, Ar-H), 3.92 (s, 3H, OCH3).
MS m/z 334 . 3 [M+1] '.
Example 36: 5-Bromo-3-(5-isopropyl-4-methoxy-2-methyl-
benzylidene)-1,3-dihydroindol-2-one
5-Isopropyl-4-methoxy-2-methylbenzaldehyde was
condensed with 5-bromo-2-oxindole to give 0.3 g of 5-bromo-
3-(5-isopropyl-4-methoxy-2-methylbenzylidene)-1,3-
dihydroindol-2-one as a yellow-orange solid.
1HNMR (d6-DMSO) 8: 10.5 (s, 1H, CONH), 7.7, 7.6, 7.5,
7.4, 7.0, 6.8 (m, 6H, aromatic, =CH-), 3.9 (s, 3H, OCH3),
3.3 (m, 1H, CH) , 2.3 (s, 3H, CHj) , 1.2 (d, 6H, 2xCH3) .
Example 37: 5-Fluoro-3-(6-methoxybiphenyl-3-
ylmethylene)-1,3-dihydroindol-2-one
4-Methoxy-3-phenylbenzaldehyde was condensed with 5-
fluoro-2-oxindole to give 0.3 g of 5-fluoro-3-(6-
methoxybiphenyl-3-ylmethylene)-1,3-dihydroindol-2-one as a
yellow-orange solid.
Example 38: 3-(3-Isopropyl-4-methoxybenzylidene)-4-
methyl-1,3-dihydroindol-2-one
Diethyl oxalate (30 ml) in 20 ml of dry ether was
added with stirring to 19 g of potassium ethoxide suspended
in 50 ml of dry ether. The mixture was cooled in an ice
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bath and 20 ml of 3-nitro-o-xylene in 20 ml of dry ether
was slowly added. The thick dark red mixture which formed
was refluxed for 0.5 hour, concentrated to a dark red
solid, and treated with 10 % sodium hydroxide until almost
all of the solid dissolved. The dark red mixture was
treated with 30% hydrogen peroxide until the red color
changed to yellow. The mixture was then treated
alternately with 10% sodium hydroxide and 30% hydrogen
peroxide until the dark red color no longer formed on
addition of sodium hydroxide. The remaining solid was
filtered and the filtrate acidified with 6 N hydrochloric
acid. The resulting precipitate was collected by vacuum
filtration, washed with water, and dried under vacuum to
give 9.8 g (45% yield) of 1-methyl-6-nitrophenylacetic acid
as an off-white solid. The solid was hydrogenated in
methanol over 10% palladium on carbon to give 9.04 g of 4-
methyl-2-oxindole as a white solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.27 (s, br, 1H, NH), 7.06
(t, J = 8Hz, 1H, H-6) , 6.74 (d, J = 8Hz, H-5) , 6.63 (d, J
- 8Hz, 1H, H-7), 3.36 (s, 2H, CHz), 2.18 (s, 3H, CH3).
3-Isopropyl-4-methoxybenzaldehyde was condensed with
4-methyl-2-oxindole to give 0.25 g of 3-(3-isopropyl-4-
methoxybenzylidene)-4-methyl-1,3-dihydroindol-2-one as a
yellow-orange solid.
1HNMR (ds-DMSO) 8: 10.4(s, 1H, NH), 8.2, 8.1, 7.6, 7.2,
7.1, 6.8, 6.7 (m, 7H, aromatic, =CH-), 3.9 (s, 3H, OCH3),
3.2 (m, 1H, CH), 2.6 (m, 1H, CH), 1.2 (d, 6H, 2xCH3).
Example 39: 3-(4,5-Dimethoxy-2-thiophen-2-
Ylbenzylidene)-1,3-dihydroindol-2-one
A mixture of 6-bromoveratraldehyde (1 g, 4.08 mmol),
oxindole (0.54 g, 4.08 mmol) and pyrrolidine (1.7 ml, 20.4
mmol) in dimethylformamide (6 ml) was held in a sealed tube
at 100°C for 12 hours. The reaction mixture was cooled and
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added to 1N hydrochloric acid (100 ml). The precipitate
which formed was filtered and washed with water. The
precipitate was then dissolved in ethyl acetate (200 ml),
the solution washed with brine, dried over magnesium
sulfate and concentrated. The solid obtained was
chromatographed (silica, 30-4-% ethyl acetate/ hexanes) to
give 1.2 g (81%) of 3-(3-bromo-4,5-dimethoxybenzylidene)-
1,3-dihydroindol-2-one as a yellow solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.5 (s, 1H, NH), 7.4 (s,
1H, Ar-CH=C), 7.3 (m, 2H, 2xAr-H), 7.2 m, 1H, Ar-H), 6.8
(m, 2H, 2xAr-H), 3.8 (s, 3H, OCH3), 3.7 (s, 3H, OCH3).
MS m/z 360.5 and 362.5, [M]' and [M+2]'.
Tetrakis(triphenylphosphine)palladium(0) (0.03 g, 0.03
mmol) was added to a solution of 3-(3-bromo-4,5-
dimethoxybenzylidene)-1,3-dihydroindol-2-one (0.32 g, 0.89
mmol) in toluene (2 ml) and ethanol (2 ml), followed by
additiona of 2M aqueous sodium carbonate (1.8 ml, 3.6
mmol). To this mixture was added thiophene-2-boronic acid
(0.13 g, 0.98 mmol), and the mixture was held at 100°C in a
sealed tube for 12 hours. The reaction mixture was poured
into water (50 ml) and extracted with ethyl acetate (2x100
ml). The combined organic layers were washed with
saturated aqueous sodium bicarbonate (50 ml) and brine (50
ml), dried over magnesium sulfate and concentrated.
Chromatography (silica, 30-40% ethyl acetate/hexanes)
afforded an orange solid that was triturated with methylene
chloride and dried to afford 0.19 g (59%) Of 3-(4,5-
dimethoxy-2-thiophen-2-ylbenzylidene)-1,3-dihydroindol-2-
one as an orange solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.5 (m, 1H, NH), 7.6 (m,
1H, Ar-H), 7.5 (m, 2H, Ar-H), 7.3 (m, 1H, Ar-CH=C), 7.2 (m,
4H, Ar-H), 6.8 (m, 2H, Ar-H), 6.8 (m, 2H, Ar-H), 3.9 (two
s, 3H, OCH3) , 3.7 (two s, 3H, COCH3) .
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MS m/z 364.0 [M+1]'.
Example 40: N-~3-L4-(2-Morpholin-4-ylethoxy)-3-thiophen
2-ylbenzylidene]-2-oxo-2,3-dihydro-1H-indol-6-yl~-acetamide
Triphenylphosphine (6.85 g) was added to a solution of
3-bromo-4-hydroxybenzaldehye (5 g) in tetrahydrofuran (40
ml), followed by addition of 2-hydroxyethylmorpholine (3.01
ml) and then the dropwise addition of
diethylazodicarboxylate (4.11 ml). After 12 hours, the
reaction was concentrated under reduced pressure, poured
into 2N hydrochloric acid (200 ml) and extracted with ethyl
acetate (2x 150 ml). The aqueous layer was basified to pH
9 with solid sodium bicarbonate and extracted with ethvl
acetate (2 x 150 ml). The organic layers were dried over
magnesium sulfate and concentrated to afford 4.4 g (56%) of
4-morpholinoethoxy-3-bromobenzaldehyde as a reddish oil.
1HNMR (360 MHz, DMSO-d6) 8: 9.84 (s, 1H, NH), 8.07 (d,
1H, J = 2Hz, Ar-H), 7.90 (dd, 1H, J = 2 and 8Hz, Ar-H),
7.32 (d, 1H, J = 8Hz, Ar-H), 4.29 (t, 2H, J = 6Hz, Ar-OCHz),
3 .56 (m, 4H, OCH2CHZN) , 2 .76 (t, 2H, J = 6Hz, ArOCH2-CH2N) ,
2.50 (m, 4H, NCHzCH20) .
Tetrakis(triphenylphosphine)palladium(0) (0.23) was
added to a solution of 4-morpholinoethoxy-3-bromobenz-
aldehyde (2.13 g) in toluene (10 ml) and ethanol (10 ml),
followed by addition of 2M aqueous sodium carbonate (13 ml).
To this mixture was added thiophene-2-boronic acid (1.13 g),
and the mixture was refluxed for 12 hours. The reaction
mixture was then poured into water (100 ml) and extracted
into ethyl acetate (3 x 100 ml). The product was extracted
into 1N hydrochloric acid (2 x 100 ml), the aqueous layers
basified to pH 9 with solid sodium bicarbonate and extracted
with ethyl acetate (2 x 100m1). The organic layers were
washed with brine (75 ml), dried over magnesium sulfate and
concentrated to afford 1.7 g (75%) of 3-(2-thiophene)-4-
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morpholinoethoxybenzaldehyde as a greenish/yellow oil.
1HNMR (360 MHz, DMSO-d6) 8: 9.93 (s, 1H, CHO), 8.2 (d,
1H, J = 2Hz, Ar-H), 7.82 (dd, 1H, J = 2 and 8Hz, Ar-H),
7.76 (dd, 1H, J = 1 and 4Hz, Ar-H), 7.59 (dd, 1H, J = 1 and
5Hz, Ar-H), 7.35 (d, 1H, J = 9Hz, Ar-H), 7.14 (dd, 1H, J =
4 and 5Hz, Ar-H) , 4.34 (t, J = 6Hz, 2H, 0-CHzCH~) , 3.56 (m,
4H, 2x ring0-CHZCH2) , 2 . 82 (t, J = 6Hz, 2H, N-CHzCHz) , 2.5 (m
on DMSO, 4H, 2x ringN-CHZCHz) .
MS m/ z 318 . 2 [M+1 ] ' .
A mixture of 3(2-thiophene)-4-morpholinoethoxy-
benzaldehyde (1.74 g), 6-acetamido-2-oxindole(lg) and
piperidine (2.6 ml) in ethanol (11 ml) was held in sealed
tube at 100°C for 12 hours. The reaction was cooled and
added to diethyl ether and the ether decanted. The oily
residue was dissolved in methylene chloride (10 ml) and
precipitated in ether (200 ml) and hexanes (100 ml). The
precipitate was removed by filtration, washed with ether
and then hexanes and dried to afford a brownish solid.
Chromatography (silica, 5% methanol/dichloromethane)
afforded N-(3-[4-(2-morpholin-4-ylethoxy)-3-thiophen-2-
ylbenzylidene]-2-oxo-2,3-dihydro-1H-indol-6-yl~-acetamide
as a dark yellow solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.49 (s, 1H, NH), 10.01
(s, 1H, NHAc), 8.05 (d, J = 2Hz, 1H, Ar-H), 7.72 (m, 1H,
Ar-H), 7.65 (m, 1H, Ar-H), 7.48 (m, 2H, 2xAr-H), 7.47 (s,
1H, Ar-CH=C), 7.45 (d, 1H, J =2Hz, Ar-H), 7.29 (d, 1H, J =
9Hz, Ar-H), 7.13 (m, 1H, Ar-H), 6.90 (dd, 1H, J =2 and 9Hz,
Ar-H) , 4 .32 (m, 2H, ArOCHzCH2N) , 3 . 58 (m, 4H, 2xOCH2CH2N) ,
2 . 84 (m, 2H, ArOCH2CH2N) , 2 . 5 (m under DMSO, 4H, 2xOCH2CH2N) ,
2.03 (s, 3H, NHCOCH3) .
MS m/z 490.0 [M+1]'.
Example 41: 3-(2,2-Dimethylchroman-6-ylmethylene)-4-
methyl-1,3-dihydroindol-2-one
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2,2-Dimethyl-6-formylchromane (commercially available)
was condensed with 4-methyl-2-oxindole to give 0.25 g of 3-
(2,2-dimethylchroman-6-ylmethylene)-4-methyl-1,3-
dihydroindol-2-one as a yellow-orange solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.45 (s, br, 1H, N~i), 8.17
(dd, J = 2, 8HZ, 1H) , 8.02 (d, J = 2Hz, 1H) , 7.6 (s, 1H,
vinyl), 7.05 (t, J = BHz, 1H), 6.75 (t, 2H), 6.67 (d, J =
8Hz, 1H), 2.77 (t, J = 7Hz, 2H, CHz), 2.55 (s, 3H, CH3), 1.8
(t, J = 7Hz, 2H, CH2) , 1.31 (s, 6H, 2 x CH3) .
1O MS APCI m/z 320.2 [M+1]'.
Example 42: 3-(2,3-Dihydrobenzofuran-5-ylmeth~lene)-5-
fluoro-1,3-dihydroindol-2-one
2,3-Dihydro-5-formylbenzofuran (commercially
available) was condensed with 5-fluoro-2-oxindole to give
0.25 g of 3-(2,3-dihydrobenzofuran-5-ylmethylene)-5-fluoro-
1,3-dihydroindol-2-one as a yellow-orange solid.
Example 43: 3-(3-Cyclohexyl-4-methoxybenzylidene)-5-
fluoro-1,3-dihydroindol-2-one
2-Cyclohexylphenol was methylated then formylated.
The aldehyde was condensed with 5-fluoro-2-oxindole to give
0.3 g of 3-(3-cyclohexyl-4-methoxybenzylidene)-5-fluoro-
1,3-dihydroindol-2-one as a yellow-orange solid.
Example 44: 5-Fluoro-3-(3-isopropyl-4-
methoxvbenzylidene)-1,3-dihydroindol-2-one
3-Isopropyl-4-methoxybenzaldehyde was condensed with
5-fluoro-2-oxindole to give 0.25 g of 5-fluoro-3-(3-
isopropyl-4-methoxybenzylidene)-1,3-dihydroindol-2-one as a
yellow-orange solid.
Example 45: 3-(5-Isopropyl-4-methoxy-2-
methylbenzylidene)-1,3-dihydro-pyrrolo[2 3-b]pyridin-2-one
Zinc dust (6.5 g) was added to a solution of 3,3-
dibromo-7-azaoxindole (2.9 g) in acetic acid (20 ml) and
acetonitrile (30 ml). The mixture was stirred for 2 hours
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at room temperature. The solids were removed by filtration
and the solvent evaporated. The residue was treated with
ethyl acetate. The ethyl acetate solution containing
insoluble solid was passed through a short column of silica
gel. The collected ethyl acetate solution was evaporated
and the residue dried under vacuum to give 1.8 g (91%
yield) of 7-aza-2-oxindole acetic acid salt.
1HNMR (360 MHz, DMSO-d6) b: 10.90 (s, br, 1H, NH),
8.01, 7.52, 6.91 (m, 3H), 3.52 (s, 2H, CH2).
MS APCI 135 (M+1).
5-Isopropyl-4-methoxy-2-methylbenzaldehyde condensed
with 7-aza-2-oxindole to give 0.25 g of 3-(5-isopropyl-4-
methoxy-2-methylbenzylidene)-1,3-dihydro-pyrrolo[2,3-
b]pyridin-2-one as a yellow-orange solid.
1HNMR (d6-DMSO) 8: 10.5(s, 1H, CONH), 8.3-6.8(m, 6H,
aromatic, =CH-), 3.9(s, 3H, OCH3), 3.3(m, 1H, CH), 2.3(s,
3H, CH3) , 1.2 (d, 6H, 2xCH3) .
Example 46: 3-(3'-Ethoxy-6-methoxvbiphenvl-3-
ylmethylene)-1,3-dihydroindol-2-one
Tetrakis(triphenylphosphine)palladium(0) (0.02 g, 0.02
mmol) was added to a solution of 3-(bromo-4-methoxy-
benzylidene)-1,3-dihydroindol-2-one (0.2 g, 0.61 mmol) in
toluene (1 ml) and ethanol (1 ml), followed by addition of
2M aqueous sodium carbonate (1.2 ml, 2.4 mmol). To this
mixture was added 3-ethoxyphenylboronic acid (0.11 g, 0.67
mmol), and the mixture was held at100°C in a sealed tube for
12 hours. The reaction mixture was added to water (40 ml)
and extracted with ethyl acetate (75 ml). The combined
organic layers were washed with saturated aqueous sodium
bicarbonate (50 ml) and brine (50 ml). The organic layer
was dried over magnesium sulfate and concentrated. The
resulting solid was triturated with dichloromethane/
hexanes to afford 0.9 g (39%) of 3-(3'-ethoxy-6-methoxy-
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biphenyl-3-ylmethylene)-1,3-dihydroindol-2-one as a ye-~iow
solid.
'HNMR (360 MHz, DMSO-d6) 8: 10.57 (s, 1H, NH), 7.6~ (m,
5H, 4xAr-H and Ar-CH=C), 7.3 (m, 3H, 4xAr-H), 7.15 (m, 2H,
Ar-H), 6.9 (m, 2H, Ar-H), 4.05 (q, 2H, OCHZCH3), 3.85 (s,
3H, OCH3) , 1.35 (t, 3H, OCHzCH3) .
MS m/z 372.5 [M+1]'.
Example 47: 3-(3-Cyclopentyl-4-methoxybenzylidene)-1,3-
dihvdropyrrolo(2,3-blpyridin-2-one
3-Cyclopentyl-4-methoxybenzaldehyde was condensed with
7-aza-2-oxindole to give 0.25 g of 3-(3-cyclopentyl-4-
methoxybenzylidene)-1,3-dihydropyrrolo[2,3-b]pyridin-2-one
as a yellow-orange solid.
1HNMR (d6-DMSO) b: 1.1 (s, 1H, CONH), 8.3-6.8 (m, 7H,
aromatic, =CH-), 3.9(s, 3H, OCH3), 3.2(m, 1H, CH), 1.8 (m,
8H, 4xCHz ) .
Example 48: 3-(3-Cyclopentyl-4-methoxybenzvlidene)-4-
methyl-1,3-dihvdroindol-2-one
3-Cyclopentyl-4-methoxybenzaldehyde was condensed with
4-methyl-2-oxindole to give 0.25 g of 3-(3-cyclopentyl-4-
methoxybenzylidene)-4-methyl-1,3-dihydroindol-2- as a
yellow-orange solid.
1HNMR (d6-DMSO) 8: 10.4 (s, 1H, CONH), 8.3, 8.1, 7.6,
7.0, 7.0, 6.8, 6.6 (m, 7H, aromatic, =CH-), 3.9 (s, 3H,
OCH3) , 3.2 (m, 1H, CH) , 1.8 (m, 8H, 4xCH2) .
Example 49: 3-(4,5,2~-Trimethoxybiphenyl-2-ylmethylene)-
1,3-dihvdroindol-2-one
Tetrakis(triphenylphosphine)palladium(0) (0.02 g, 0.02
mmol) was added to a solution of 3-(3-bromo-4,5-
dimethoxybenzylidene)-1,3-dihydroindol-2-one (0.20 g, 0.56
mmol) in toluene (1m1) and ethanol (1 ml), followed by
addition of 2M aqueous sodium carbonate (1.1 ml, 2.2 mmol).
To this mixture was added 2-methoxyphenylboronic acid 10.09
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g, 0.62 mmol), and the mixture was held at 100°C in a sealed
tube for 12 hours. The reaction mixture was added to water
(50 ml) and extracted with ethyl acetate (2x100 ml). The
combined organic layers were washed with saturated aqueous
sodium bicarbonate (50 ml) and brine (50 ml). The organic
layer was dried over magnesium sulfate and concentrated.
Chromatography (silica, 30-40% ethyl acetate / hexanes)
afforded 0.14 g (640) of 3-(4,5,2'-trimethoxy-biphenyl-2-
ylmethylene)-1,3-dihydroindol-2-one as an orange solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.4 (m, 1H, NH), 7.5 (m,
1H, Ar-H), 7.3 (m, 2H, Ar-H and Ar-CH=C), 7.2 (m, 4H, Ar-
H), 6.9 (m, 4H, Ar-H), 3.8 (two s, 3H, COCH3), 3.7 (two s,
3H, OCH3) , 3 .5 (two s, 3H, COCH3) .
MS m/z 388.2 [M+1]'.
Example 50: N-~3-(4-(2-Morpholin-4-ylethoxy)-3-thiophen-
3-ylbenzylidene]-2-oxo-2,3-dihydro-1H-indol-6-yl~-acetamide
Tetrakis(triphenylphosphine)palladium(0) (0.21 g) was
added to a solution of 4-morpholinoethoxy-3-bromobenz-
aldehyde (1.92 g) in toluene (10 ml) and ethanol (10 ml),
followed by addition of 2M aqueous sodium carbonate (12
ml). To this mixture was added thiophene-3-boronic acid
(1.02 g), and the mixture was heated to reflux. After 12
hours, the reaction mixture was added to water (100 ml) and
extracted into ethyl acetate (3 x 100 ml). The product was
then extracted into 1N hydrochloric acid (2 x 100 ml), the
aqueous layers basified to pH 9 with solid sodium
bicarbonate and extracted with ethyl acetate (2 x 100 ml).
The organic layers were washed with brine (75 ml), dried
over magnesium sulfate and concentrated to afford 1.7 g
(83%) of 3-(3-thiophene)-4-morpholinoethoxy benzaldehyde a
reddish oil.
1HNMR (360 MHz, DMSO-d6) b: 9.91 (s, 1H, CHO), 8.08 (m,
2H, Ar-H), 7.83 (dd, 1H, J = 2 and 9Hz, Ar-H), 7.76 (dd,
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1H, J = 1 and 4Hz, Ar-H), 7.59 (dd, 1H, J = 1 and 5Hz, Ar-
H), 7.35 (d, 1H, J = 9Hz, Ar-H), 7.64 (dd, 1H, J = 1 and
5Hz, Ar-H), 7.58 (dd, 1H, J = 3 and 5Hz, Ar-H), 7.31 (d,
1H, J = 9Hz, Ar-H) , 4.27 (t, J = 5Hz, 2H, O-CHzCH2) , 3.56
(m, 4H, 2x ring0-CHZCHZ) , 2 .75 (t, J = 6Hz, 2H, N-CH2CH2) ,
2.48 (m on DMSO, 4H, 2x ringN-CH2CH2).
MS m/z 318.2 [M+1]'.
A mixture of 3-(3-thiophene)-4-morpholinoethoxy-
benzaldehyde (1.74 g), 6-acetamido-2-oxindole (1 g)
(prepared as described in example 1) and piperidine (2.6
ml) in ethanol (11 ml) was held in a sealed tube at 100°C
for 12 hours. The reaction was cooled and added to diethyl
ether (200 ml). The solvent was decanted, and the oily
solid which remained was dissolved in dichloromethane (15
ml) and precipitated in diethyl ether (200 ml) and hexanes
(200 ml), filtered, washed with diethyl ether and then
hexanes and dried to afford a mustard yellow solid.
Chromatography (silica, 5% methanol/ dichloromethane)
afforded N-f3-[4-(2-morpholin-4-ylethoxy)-3-thiophen-3-
ylbenzylidene]-2-oxo-2,3-dihydro-1H-indol-6-yl)-acetamide
as a yellow foam.
1HNMR (360 MHz, DMSO-d6) S: 10.47 (s, 1H, NH), 10.00
(s, 1H, NHAc), 8.07 (m, 1H, Ar-H), 7.91 (d, J = 2Hz, 1H,
Ar-H), 7.63 (m, 2H, 2xAr-H), 7.58 (m, 2H, 2xAr-H), 7.48 (s,
1H, Ar-CH=C), 7.45 (d, 1H, J = 2Hz, Ar-H), 7.26 (d, 1H, J =
9Hz, Ar-H), 6.90 (dd, 1H, J =2 and 9Hz, Ar-H), 4.26 (m, 2H,
ArOCH2CH2N), 3.58 (m, 4H, 2xOCH2CH2N), 2.76 (m, 2H,
ArOCH2CH2N), 2.48 (m under DMSO, 4H, 2xOCH2CH2N), 2.03 (s,
3H, NHCOCH3) .
MS m/z 490.0 [M+1]'.
Example 51: 5-Chloro-3-(3-cyclohexyl-4-
methoxybenzylidene)-1,3-dihvdroindol-2-one
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2-Cyclohexylphenol was methylated and then formylated.
The aldehyde was condensed with 5-chloro-2-oxindole to give
0.3 g of 5-chloro-3-(3-cyclohexyl-4-methoxy-benzylidene)-
1,3-dihydroindol-2-one as a yellow-orange solid.
1HNMR (d6-DMSO) ~: 10.6(s, 1H, CONH), 7.7-6.8(m, 7H,
aromatic, =CH-), 3.9(s, 3H, OCH3), 3.0 (m, 1H, CH), 1.8, 1.4
(m, 10H, 5xCH2) .
Example 52: [3-(3,5-Diisonrorwl-4-methoxybenz~lidene)-2-
oxo-2,3-dihydro-1H-indol-6-yl~-carbamic acid tert-butyl
ester
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 6-tert-butyloxycarbonylamino-2-oxindole to give 0.35 g
of [3-(3,5-diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-
dihydro-1H-indol-6-yl]-carbamic acid tert-butyl ester as a
yellow-orange solid.
Example 53: 3-(3,5-Diisopropyl-4-methoxybenzylidene)-4-
methvl-1,3-dihvdroindol-2-one
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 4-methyl-2-oxindole to give 0.3 g of 3-(3,5-
diisopropyl-4-methoxybenzylidene)-4-methyl-1,3
dihydroindol-2-one as a yellow-orange solid.
1HNMR (d6-DMSO) 8: 10.41 (s, 1H, CONH) , 8 . 0 (s, 2H) ,
7.66 (s, 1H), 7.07 (t, 1H), 6.76 (d, 1H), 6.67 (d, 1H), 3.7
(s, 3H, OCH3), 3.21-3.3 (m, 1H), 1.21 (d, 12H, 4 x CH3).
Example 54: 5-Bromo-3-(3,5-diisopropyl-4-methoxy-
benzylidene)-1,3-dihydroindol-2-one
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 5-bromo-2-oxindole to give 0.3 g of 5-bromo-3-(3,5-
diisopropyl-4-methoxybenzylidene)-1,3-dihydroindol-2-one as
a yellow-orange solid.
Example 55: N-~3-[3-tert-Butyl-4-(2-morpholin-4-
~lethoxy)-benzylidenel-2-oxo-2,3-dihvdro-1H-indol-6-vl~-
acetamide
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Triphenylphosphine (5.89 g, 22.44 mmol) was added to a
solution of 3-tert-butyl-4-hydroxybenzaldehyde (4 g, 22.44
mmol) in tetrahydrofuran (40 ml), followed by addition of
2-hydroxyethylmorpholine (2.94 g, 22.44 mmol) and then the
dropwise addition of diethylazodicarboxylate (3.91 g, 22.44
mmol). The mixture was allowed to stir at room temperature
for 3 days. The reaction was concentrated under reduced
pressure and partitioned between 2N hydrochloric acid (200
ml) and ethyl acetate (150 ml). The aqueous layer was
extracted with ethyl acetate (2 x 150 ml), basified to pH 9
with solid sodium bicarbonate, saturated with solid sodium
chloride and extracted with ethyl acetate (3 x 150 ml). The
combined organic layers were dried over magnesium sulfate
and concentrated to afford 2.8 g (44o) of 3-tert-butyl-4-
(2-morpholin-4-ylethoxy)benzaldehyde as a yellowish oil.
1HNMR (360 MHz, DMSO-d6) 8: 9.8 (s, 1H, CHO), 7.75
(m, 2H, 2xAr-H), 7.16 (d, J = lOHz, 1H, Ar-H), 4.19 (t, J =
7Hz, 2H, O-CHzCH2) , 3 .55 (m, 4H, 2x ring0-CHzCH2) , 2 .75 (t, J
- 7Hz, 2H, N-CHZCHz) , 2.4 (m on DMSO, 4H, 2x ringN-CH2CH2) ,
1 .36 (s, 9H, C (CH3) 3) .
MS m/z 292.0 [M+1]'.
A mixture of 3-tert-butyl-4-(2-morpholin-4-ylethoxy)-
benzaldehyde (0.25 g), 6-acetamido-2-oxindole (0.16g) and
piperidine (0.43 ml) in ethanol (3 ml) was held in a sealed
tube at 100°C for 12 hours. The reaction was cooled, added
to ethyl acetate (250 ml), washed with 1N hydrochloric acid
(100 ml) and brine (100 ml), dried with magnesium sulfate
and concentrated. Some product went into the acid wash, so
this was basified with solid sodium bicarbonate to pH 9 and
extracted with ethyl acetate (2 x 150 ml). The organic
layers were dried over magnesium sulfate, combined with the
oil from the first crop of product and concentrated.
Chromatography (silica, 40% ethyl acetate followed by 5-10%
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methanol / methylene chloride) afforded 100 mg (27%) of N-
{3-[3-tert-butyl-4-(2-morpholin-4-ylethoxy)benzylidene]-2-
oxo-2,3-dihydro-1H-indol-6-yl}-acetamide as a yellow solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.46 (s, 1H, NH), 9.99 (s,
1H, NHAc), 7.58 (m, 3H, Ar-H), 7.41 (m, 2H, Ar-H and Ar-
CH=C), 7.12 (d, 1H, Ar-H), 6.92 (dd, 1H, J =2 and 8Hz, Ar-
H) , 4. 18 (m, 2H, Ar-OCHZ) , 3 .57 (m, 4H, OCH2CHZN) , 2.77 (m,
2H, ArOCH2-CHzN) , 2.49 (m, 4H, NCHZCHzO) , 2. 02 (s, 3H,
NHCOCH3), 1.38 (s, 9H, Ar-tBu).
MS m/z 464.0 [M+1]'.
Example 56: 3-(4-Methoxy-3,5-dimethylbenzylidene)-1,3-
dihvdropyrrolo[2,3-blpvridin-2-one
2,6-Dimethylphenol was methylated and then formylated.
1HNMR (d6-DMSO) 8: 10.1 (s, 1H, CHO), 6.9 (s, 2H,
aromatic), 3.9 (s, 3H, OCH3), 2.6 (s, 6H, 2xCH3).
The aldehyde was condensed with 7-aza-2-oxindole to
give 0.2 g of 3-(4-methoxy-3,5-dimethylbenzylidene)-1,3-
dihydropyrrolo[2,3-b]pyridin-2-one as a yellow-orange
solid.
1HNMR (ds-DMSO) b: 1.5(s, 1H, CONH), 8.2-6.8(m, 6H,
aromatic, =CH-) , 3 .8 (s, 3H, OCH3) , 2 .5 (s, 6H, 2xCH3) , 2.4 (s,
3H, CH3) .
Example 57: 5-Bromo-3-(3,5-diisopropyl-4-(2-morpholin-4-
ylethoxy)benzylidene]-1,3-dihydroindol-2-one
A mixture of 3,5-diisopropyl-4-(2-morpholin-4-
ylethoxy)-benzaldehyde (0.4 g), 5-bromo-2-oxindole (0.27g)
and pyrrolidine (0.5 ml) in ethanol (2 ml) was held in a
sealed tube at 100°C for 12 hours. The mixture was then
added to 1N hydrochloric acid (100 ml) and the solids
removed by filtration and washed with more water (50 ml).
The solids were then dissolved in ethyl acetate (200 ml),
the solution washed with 1N hydrochloric acid (75 ml) and
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brine (75 ml), dried with magnesium sulfate and
concentrated. The crude solid was chromatographed (silica,
4:4:1 dichloromethane: hexanes: methanol) to give 80 mg
(13%) of 5-bromo-3-[3,5-diisopropyl-4-(2-morpholin-4-
ylethoxy)-benzylidene]-1,3-dihydroindol-2-one as a reddish-
brown solid.
'HNMR (360 MHz, DMSO-d6) 8: 10.54 (s, 1H, NH), 9.43 (d,
J = lOHz, 1H, Ar-H), 7.58 (d, J = 9Hz, 1H, Ar-H), 7.46 (d,
J = SHz, 2H, Ar-H), 7.3 (m, 1H, Ar-H), 7.19 (m, J = 9 and
lOHz, 1H, Ar-H), 6.99 (m, J = 9 and 9Hz, 1H, Ar-H), 6.83
(m, J = 9 and lHz, 1H, Ar-H), 3.61 (dd, J = 9 and 22Hz 1H,
lxCHzCH) , 3.43 (m, 1H, CHCH3) , 2.86 (dd, J = 4 and 22Hz, 1H,
lxCH~CH) , 1.35 (d, J = 9 Hz, 3H, CH3) .
MS m/z 262.0 [M+1]'.
Example 58: 3-(3'-Ethoxy-4,5-dimethoxybiphenyl-2-
ylmethylene)-1,3-dihydroindol-2-one
Tetrakis(triphenylphosphine)palladium(0) (0.02 g, 0.02
mmol) was added to a solution of 3-(3-bromo-4,5-
dimethoxybenzylidene)-1,3-dihydroindol-2-one (0.2 g, 0.56
mmol) in toluene (1 ml) and ethanol (1 ml), followed by
addition of 2M aqueous sodium carbonate (1 ml, 2mmo1). To
this mixture was added 3-ethoxyphenylboronic acid (0.1 g,
0.62 mmol) and the mixture was held at 100°C in a sealed
tube for 12 hours. The reaction mixture was then added to
water (40 ml) and extracted with ethyl acetate (2 x 75 ml).
The combined organic layers were washed with saturated
aqueous sodium bicarbonate (50 ml) and brine (50 ml), dried
over magnesium sulfate and concentrated. The resulting
solid was triturated with methylene chloride / hexanes to
afford 0.14 g (64%) of 3-(3'-ethoxy-4,5-dimethoxybiphenyl-
2-ylmethylene)-1,3-dihydroindol-2-one as a yellow/orange
solid.
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'HNMR (360 MHz, DMSO-d6) d: 10.5 (s, 1H, NH), 7.6 (d, J
- 9Hz, 1H, Ar-H), 7.2 (m, 4H, 3xAr-H and Ar-CH=C), 7.08 (s,
2H, Ar-H), 6.8 (m, 5H, 5xAr-H), 4.0 (q, J = 8Hz, 2H,
OCHZCH3) , 3.89 (s, 3H, OCH3) , 3.77 (s, 3H, OCH3) , 1.29 (t, J
- 8Hz, 3H, OCHZCH3) .
MS m/z 402.5 [M+1]'.
Example 59: 5-chloro-3-(4-Methoxy-3-thiophen-2-
ylbenzylidene)-1,3-dihydroindol-2-one
A mixture of 3-(2-thiophene)-4-methoxybenzaldehyde
(0.21 g), 5-chloro-2-oxindole (0.16g) and pyrrolidine (0.4
ml) in ethanol (1 ml) was placed in a sealed tube and
heated to 100°C. A precipitate formed within 10 minutes so
the reaction was diluted with another 10 ml of ethanol and
heating resumed. After 4 hours, the reaction was cooled and
the solid which formed was removed by filtration and washed
with ethanol and then hexanes to afford 0.21 g (60%) of 5-
chloro-3-(4-methoxy-3-thiophen-2-ylbenzylidene) -1,3-
dihydroindol-2-one as a yellow-orange solid.
1HNMR (360 MHz, DMSO-d6) b: 10.7 (s, 1H, NH), 7.8-9.1
(m, 3H, Ar-H), 7.6 (m, 3H, 2xAr-H and Ar-CH=C), 7.2 (m, 3H,
Ar-H), 6.9 (m, 1H, Ar-H), 3.9 (m, 3H, OCHj).
MS m/z 368.1 and 369.9 [M]' and [M+2]'.
Example 60: 5-Chloro-3-4-methoxy-3-pryidin-3-
ylbenzylidene)-1,3-dihydroindol-2-one
Tetrakis(triphenylphosphine)palladium(0) (0.2 g) was
added to a solution of 3-bromo-4-methoxybenzldehyde (1.25
g) in toluene (10 ml) and ethanol (10 ml), followed by
addition of 2M aqueous sodium carbonate (11 ml). To this
mixture was added pyridine-3-boronic acid propane diol (1
g), and the mixture was heated to reflux. After 2 hours,
the reaction was added to water (75 ml) and extracted with
ethyl acetate (2x 75 ml). The combined organic layers were
washed with saturated aqueous sodium bicarbonate (100 ml)
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and brine (100 ml), dried over magnesium sulfate and
concentrated. Chromatography (silica, 20-50% ethyl
acetate/hexanes) afforded 1.08 g (87%) of 3-(3-pyridyl)-4-
methoxybenzaldehyde as a white solid.
1HNMR (360 MHz, DMSO-d6) 8: 9.93 (s, 1H, CHO), 8.7 (d,
1H, J = 3Hz, Ar-H), 8.56 (dd, 1H, J = 1 and 6Hz, Ar-H), 7.9
(m, 3H, 3xAr-H), 7.46 (dd, 1H, J = 6 and 9Hz, Ar-H), 7.35
(d, 1H, J = lOHz, Ar-H) , 3.89 (s, 3H, OCH3) .
A mixture of 3-(3-pyridyl)-4-methoxybenzaldehyde (0.25
g), 5-chloro-2-oxindole (0.2g) and pyrrolidine (0.5 ml) in
ethanol (2 ml) was placed in a sealed tube and heated to
100°C. A precipitate formed within 10 minutes so the
reaction was diluted with another 10 ml of ethanol and
heating resumed. After 4 hours, the reaction was cooled and
the solid was removed by filtration and washed with ethanol
and then hexanes to afford 0.27 g (61%) of 5-chloro-3-(3-
(3-pyridyl)-4-methoxybenzylidene)-1,3-dihydroindol-2-one as
a yellow-orange solid.
1HNMR (360 MHz, DMSO-d6) 8: 0.67 (s, 1H, NH), 8.6 (m,
3H, 2xAr-H and Ar-CH=C), 7.8 (m, 4H, Ar-H), 7.4 (m, 1H, Ar-
H), 7.35 (m, 2H, Ar-H), 6.85 (m, 1H, Ar-H), 3.88 (s, 3H,
OCH3 ) .
MS m/z 363.0 and 365.1, [M]' and [M+2]+.
Example 61: 5-Chloro-3-(4,5-3'-Trimethoxybiphenyl-2-
ylmethylene)-1,3-dihydroindol-2-one
Tetrakis(triphenylphosphine)palladium(0) (0.36 g) was
added to a solution of 4,5-dimethoxy-2-bromobenzaldehyde
(2.5 g) in toluene (20 ml) and ethanol (20 ml), followed by
addition of 2M aqueous sodium carbonate (20 ml). To this
mixture was added 3-methoxyphenylboronic acid (1.7 g), and
the mixture was heated to reflux. After 1 hour, the
reaction was partitioned between water (150 ml) and ethyl
acetate (300 ml). The organic layer was washed with
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saturated aqueous sodium bicarbonate (150 ml) and brine
(150 ml), dried over magnesium sulfate and concentrated.
Chromatography (silica, 20-30-40% ethyl acetate / hexanes)
afforded 2.7 g (98%) of 2- (3-methoxyphenyl) -4, 5-
dimethoxybenzaldehyde as a yellow oil which solidified upon
standing.
'HNMR (360 MHz, DMSO-d6) b: 9.71 (s, 1H, CHO), 7.38 (m,
2H, 2xAr-H), 7.0 (m, 4H, 4xAr-H), 3.9 (s, 3H, OCH3), 3.84
(s, 3H, OCH3) , 3.8 (s, 3H, OCH3) .
MS m/z 273.5 [M+1]'.
A mixture of 2-(3-methoxyphenyl)-4,5-dimethoxy-
benzaldehyde (0.25 g), 5-chloro-2-oxindole (0.15g) and
pyrrolidine (0.4 ml) in ethanol (1 ml) was placed in a
sealed tube and heated to 100°C. A precipitate formed
within 10 minutes so the reaction was diluted with another
10 ml of ethanol and heating resumed. After 2.5 hours, the
reaction was cooled, the solid was removed by filtration
and washed with ethanol and then hexanes to afford 0.36 g
(92%) of 3-(4,5-3'-trimethoxybiphenyl-2-ylmethylene)-1,3-
dihydroindol-2-one as a yellow solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.67 (s, 1H, NH), 7.6 (d,
J = 2.5Hz, 1H, Ar-H), 7.3 (m, 4H, Ar-H), 7.12 (s, 1H, Ar-
CH=C), 6.9 (m, 4H, Ar-H), 3.9 (s, 3H, OCH3), 3.79 (s, 3H,
OCH3) , 3 .75 (s, 3H, OCH3) .
MS m/z 422.0 and 424.1 [M]' and [M+2]'.
Example 62: 3-(4,5-Dimethoxy-2-naphthalen-2-
~rlbenzylidene)-1,3-dihydroindol-2-one
Tetrakis(triphenylphosphine)palladium(0) (0.02 g) was
added to a solution of 3-(3-bromo-4,5-
dimethoxybenzylidene)-1,3-dihydroindol-2-one (0.20 g) in
toluene (1 ml) and ethanol (1 ml) followed by addition of
2M aqueous sodium carbonate (1.1 ml). To this mixture was
added naphthalene-2-boronic acid (0.11 g), and the mixture
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was held at 100~C in a sealed tube for 12 hours. The
precipitate which formed was filtered, washed with ethanol
and then dissolved in 20% methanol / toluene, filtered and
concentrated. The resulting solid was triturated with
hexanes and dried to afford 0.07 g (29%) of 3-(4,5-
dimethoxy-2-naphthalen-2-ylbenzylidene)-1,3-dihydroindol-2-
one as an orange solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.5 (m, 1H, NH), 7.9 (m,
4H, Ar-H), 7.7 (m, 1H, Ar-H), 7.5 (m, 3H, Ar-H), 7.4 (s,
1H, Ar-CH=C), 7.2 (m, 3H, Ar-H), 6.9 (m, 2H, Ar-H), 3.9
(two s, 3H, COCH3) , 3.8 (two s, 3H, OCHj) .
MS m/z 408.0 [M+1]'.
Example 63: N-f3-(3~-Acetylamino-6-methoxvbiphenyl-3-
ylmethylene)-2-oxo-2,3-dihydro-1H-indol-6-yll-acetamide
3-Bromo-4-methoxybenzaldehyde was condensed with 6-
acetamido-2-oxindole to give N-[3-(3-bromo-4-methoxy-
benzylidene)-2-oxo-2,3-dihydro-1H-indol-6-yl]-acetamide.
Tetrakis(triphenylphosphine)palladium(0) (0.01 g) was
added to a solution of N-[3-(3-bromo-4-methoxybenzylidene)-
2-oxo-2,3-dihydro-1H-indol-6-yl]-acetamide (0.14 g) in
toluene (1 ml) and ethanol (1 ml) followed by addition of
2M aqueous sodium carbonate (0.72 ml). To this mixture was
3-acetylamino-phenylboronic acid (0.07 g), and the mixture
was held at 100° C in a sealed tube for 12 hours. The
reaction mixture was added to water (50 ml) and extracted
with ethyl acetate (2x100 ml).
The combined organic layers were washed with saturated
aqueous sodium bicarbonate (50 ml) and brine (50 ml), dried
over magnesium sulfate and concentrated. Chromatography
(silica, dichloromethane then 5-10% methanol/
dichloromethane) afforded 0. 07 g (43%) of N- [3- (3' -
acetylamino-6-methoxybiphenyl-3-ylmethylene)-2-oxo-2,3-
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dihydro-1H-indol-6-yl)-acetamide as a yellow solid. About
26% isomer by 'HNMR.
yHNMR (360 MHz, DMSO-d6) 8: 10.49 (s, 1H, NH), 10.42
(s, 1H, NHAc), 9.95 (s, 1H, NHAc), 7.76 (m, 2H, 2xAr-H),
7.58 (m, 3H, 3xAr-H), 7.4 (m, 2H, Ar-H and Ar-CH=C), 7.3
(m, 2H, Ar-H), 7.18 (m, 1H, Ar-H), 6.9 (dd, 2H, J = 2 and
8.5Hz, Ar-H), 3.84 (s, 3H, OCH3), 2.05 (s, 3H, NHCOCH3),
2.03 (s, 3H, NHCOCH3) . MS m/z 442.2 [M+1]'.
Example 64: 6-Methoxy-3-(4-methoxy-3-thiophen-3-
ylbenzylidene)-1;3-dihydroindol-2-one
A mixture of 3(3-thiophene)-4-methoxybenzaldehyde
(0.53 g, 2.45 mmol), 6-methoxy-2-oxindole (0.4g, 2.45 mmol)
and piperidine (1.2 ml, 12.25 mmol) in 5 ml of ethanol was
held in a sealed tube at 100°C for 12 hours. The reaction
was cooled and added to diethyl ether (150 ml) and hexanes
(150 ml). The precipitate which formed was removed by
filtration, washed with diethyl ether and then hexanes and
dried to afford 0.57 g (64%) of 6-methoxy-3-(4-methoxy-3-
thiophen-3-ylbenzylidene)-1,3-dihydroindol-2-one as a
mustard yellow solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.45 (s, 1H, NH), 8.78 (d,
J = 2Hz, 1H, Ar-H), 8.30 (dd, J = 2 and 9Hz, 1H, Ar-H), 7.8
(dd, 1H, J = 1.5 and 3Hz, Ar-H), 7.5 (m, 3H, 2xAr-H and Ar
CH=C), 7.18 (d, J = 9Hz, 1H, Ar-H), 6.55 (dd, 1H, J = 2 and
8.5Hz, Ar-H), 6.39 (d, 1H, J = 2Hz, Ar-H), 3.91 (s, 3H,
OCH3) , 3 .76 (s, 3H, OCH3) , 2.04 (s, 3H, NHCOCH3) .
MS m/z 364.1 [M+1]'.
Example 65: 3-(6-Methoxybiphenvl-3-ylmethylene)-1,3-
dihydroindol-2-one
4-Methoxy-3-phenylbenzaldehyde was condensed with 2-
oxindole to give 0.3 g of 3-(6-methoxybiphenyl-3-
ylmethylene)-1,3-dihydroindol-2-one as a yellow-orange
solid.
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iHNMR (d6-DMSO) 8: 10.5(s, 1H, CONH), 8.5, 8.5, 7.8,
7.7, 7.5, 7.5, 7.4, 7.4, 7.3, 7.2, 7.2, 7.0, 6.8(m, 13H,
aromatic, =CH-), 3.9(s, 3H, OCH3).
Example 66: 3-(2,3-Dihydrobenzofuran-5-ylmethylene)-1,3-
dihydroindol-2-one
2,3-Dihydro-5-formylbenzofuran (commercially
available) was condensed with 2-oxindole to give 0.25 g cT
3-(2,3-dihydrobenzofuran-5-ylmethylene)-1,3-dihydroindol-2-
one as a yellow-orange solid.
1HNMR (d6-DMSO) 8: 10.4 (s, 1H, CONH), 8.6, 8.2, 7.7,
7.6, 7.2, 7.0, 6.9, 6.8 (m, 8H, aromatic, =CH-), 4.6(t, 2H,
OCH2 ) , 3 . 2 ( t , 2H, OCH2 ) .
Example 67: 5-Chloro-3-(6-methoxybi~phenyl-3-
ylmethylene)-1,3-dihydroindol-2-one
4-Methoxy-3-phenylbenzaldehyde was condensed with 5-
chloro-2-oxindole to give 0.3 g of 5-chloro-3-(6-
methoxybiphenyl-3-ylmethylene)-1,3-dihydroindol-2-one as a
yellow-orange solid.
1HNMR (d6-DMSO) 8: 11.5 (s, 1H, CONH), 7.8-6.8 (m, 12H,
aromatic, =CH-), 3.9 (s, 3H, OCH3).
Example 68: 3-(3-Cyclohexyl-4-methoxybenzylidene)-4-
methyl-1,3-dihydroindol-2-one
3-Cyclohexyl4-methoxybenzaldehyde was condensed with
4-methyl-2-oxindole to give 0.3 g of 3-(3-cyclohexyl-4-
methoxybenzylidene)-4-methyl-1,3-dihydroindol-2-one as a
yellow-orange solid.
1HNMR (ds-DMSO) 8: 10.6 (s, 1H, CONH) , 7.7-6.8 (m, 7'r_',
aromatic, =CH-), 3.9 (s, 3H, OCH3), 3.0 (m, 1H, CH), 2.5 ;s,
3H, CH3) , 1.8-1.4 (m, 10H, SxCHz) .
Example 69: 3-(2,3-Dihydrobenzofuran-5-ylmethylene)-4-
methyl-1,3-dihydroindol-2-one
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2,3-Dihydro-5-formylbenzofuran (commercially
available) was condensed with 4-methyl-2-oxindole to give
0.25 g of 3-(2,3-dihydrobenzofuran-5-ylmethylene)-4-methyl-
1,3-dihydroindol-2-one as a yellow-orange solid.
1HNMR (dE-DMSO) 8: 10.5(s, 1H, CONH), 8.4-6.6 (m, 7H,
aromatic, =CH-), 4.6 (t, 2H, OCH2), 3.2 (t, 2H, OCHZ), 2.5
(s, 3H, CH3) .
Example 70: 3-(3-Isopropyl-4-methoxybenzylidene)-1,3-
dihvdropyrrolof2,3-b]pyridin-2-one
3-Isopropyl-4-methoxybenzaldehyde was condensed with
7-aza-2-oxindole to give 0.25 g of 3-(3-isopropyl-4-
methoxybenzylidene)-1,3-dihydropyrrolo[2,3-b]pyridin-2-one
as a yellow-orange solid.
1HNMR (d6-DMSO) 8: 11.5(s, 1H, CONH), 8.0-6.8 (m, 7H,
aromatic, =CH-), 3.7 (s, 3H, OCH3), 3.2 (m, 1H, CH), 1.2 (d,
6H, 2xCH3 ) .
Example 71: 3-(6-Methoxybiphenyl-3-ylmethylene)-1,3-
dihydropyrrolof2,3-blpyridin-2-one
4-Methoxy-3-phenylbenzaldehyde was condensed with 7-
aza-2-oxindole to give 0.25 g of 3-(6-methoxybiphenyl-3-
ylmethylene)-1,3-dihydropyrrolo[2,3-b]pyridin-2-one as a
yellow-orange solid.
1HNMR (d6-DMSO) 8: 10.5 (s, 1H, CONH), 8.5-6.8 (m, 12H,
aromatic, =CH-), 3.9 (s, 3H, OCH3).
Example 72: 3-(3-Cyclohexyl-4-methoxybenzylidene)-1,3-
dihvdropyrrolo(2,3-b]pyridin-2-one
3-Cyclohexyl-4-methoxybenzaldehyde was condensed with
7-aza-2-oxindole to give 0.25 g of 3-(3-Cyclohexyl-4-
methoxybenzylidene)-1,3-dihydropyrrolo[2,3-b]pyridin-2-one
as a yellow-orange solid.
Example 73: 3-(2,3-Dihydrobenzofuran-5-ylmethylene)-1,3-
dihydropyrrolof2,3-bLpyridin-2-one
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2,3-Dihydro-5-formylbenzofuran (commercially
available) was condensed with 7-aza-2-oxindole to give 0.2
g of 3-(2,3-dihydrobenzofuran-5-ylmethylene)-1,3-
dihydropyrrolo[2,3-b]pyridin-2-one as a yellow-orange
solid.
1HNMR (d6-DMSO) 8: 10.4 (s, 1H, CONH), 8.3-6.8 (m, 7H,
aromatic, =CH-) , 4.6 (t, 2H, OCHz) , 3.2 (t, 2H, OCHz) .
Example 74: 3-(3,5-Diisopropyl-4-methoxybenzylidene)-
1,3-dihydropyrrolo(2,3-bl~yridin-2-one
3,5-Diisopropyl-4-methoxybenzaldehyde was methylated
and then formylated. The aldehyde was condensed with 7-
aza-2-oxindole to give 0.2 g of 3-(3,5-diisopropyl-4-
methoxybenzylidene)-1,3-dihydropyrrolo[2,3-b]pyridin-2-one
as a yellow-orange solid.
1HNMR (d6-DMSO) 8: 11.5 (s, 1H, CONH), 8.0-6.8 (m, 7H,
aromatic, =CH-), 3.7 (s, 3H, OCH3), 3.2 (m, 2H, 2xCH), 1.2
(m, 12H, 4xCH3) .
Example 75: 5-Bromo-3-(3-isopropyl-4-
methoxybenzvlidene)-1,3-dihydroindol-2-one
3-Isopropyl-4-methoxybenzaldehyde was condensed with
5-bromo-2-oxindole to give 0.3 g of 5-bromo-3-(3-isopropyl-
4-methoxybenzylidene)-1,3-dihydroindol-2-one as a yellow-
orange solid.
1HNMR (d6-DMSO) 8: 10.6 (s, 1H, CONH), 8.5, 8.4, 7.9,
7.9, 7.3, 7.1, 6.8 (m, 7H, aromatic, =CH-), 3.9 (m, 3H,
OCH3) , 3 .3 (m, 1H, CH) , 1 .2 (d, 6H, 2xCH3) .
Example 76: 5-Bromo-3-(3-cyclopentyl-4
methoxybenzylidene)-1,3-dihydroindol-2-one
3-Cyclopentyl-4-methoxybenzaldehyde was condensed with
5-bromo-2-oxindole to give 0.3 g of 5-bromo-3-(3-
cyclopentyl-4-methoxybenzylidene)-1,3-dihydroindol-2-one as
a yellow-orange solid.
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'HNMR (d6-DMSO) d: 10.6 (s, 1H, CONH), 8.6-6.7 (m, 7H,
aromatic, =CH-) , 3 .9 (s, 3H, OCH3) , 3.2 (m, 1H, CH) , 1.8 (m,
8H, 4xCH~) .
Example 77: 5-Chloro-3-(3-cyclopentyl-4-methoxy-
benzvlidene)-4-methyl-1,3-dihydroindol-2-one
A suspension of 3.0 g of 4-methyl-2-oxindole was
stirred in 50 ml of acetonitrile at room temperature while
3.3 g of N-chlorosuccinimide was added in portions.
Trifluoroacetic acid (1 ml) was then added. The suspension
was stirred at room temperature for 3 days during which
time solids were always present. The solids were collected
by vacuum filtration, washed with a small amount of cold
acetone and dried overnight in a vacuum oven at 40°C to give
2.5 g (68 %) of 5-chloro-4-methyl-2-oxindole.
3-Cyclopentyl-4-methoxybenzaldehyde was condensed with
4-methyl-5-chloro-2-oxindole to give 0.3 g of 5-Chloro-3-
(3-cyclopentyl-4-methoxybenzylidene)-4-methyl-1,3-
dihydroindol-2-one as a yellow-orange solid.
Example 78: 5-Chloro-3-(6-methoxybiphenyl-3-
ylmethvlene)-4-methyl-1,3-dihydroindol-2-one
4-Methoxy-3-phenylbenzaldehyde was condensed with 4-
methyl-5-chloro-2-oxindole to give 0.3 g of 5-chloro-3-(6-
methoxybiphenyl-3-ylmethylene)-4-methyl-1,3-dihydroindol-2-
one as a yellow-orange solid.
Example 79: 5-Chloro-3-(5-isopropyl-4-methoxv-2-methyl-
benzylidene)-4-methyl-1 3-dihydroindol-2-one
5-Isopropyl-4-methoxy-2-methylbenzaldehyde was
condensed with 4-methyl-5-chloro-2-oxindole to give 0.3 g
of 5-chloro-3-(5-isopropyl-4-methoxy-2-methylbenzylidene)-
4-methyl-1,3-dihydroindol-2-one as a yellow-orange solid.
Example 80: 5-Chloro-3-(4-methoxy-3,5-
dimethylbenzylidene)-4-methyl-1,3-dihydroindol-2-one
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2,6-Dimethylphenol was methylated and then formylated.
The aldehyde was condensed with 4-methyl-5-chloro-2-
oxindole to give 0.3 g of 5-chloro-3-(4-methoxy-3,5-
dimethylbenzylidene)-4-methyl-1,3-dihydroindol-2-one as a
yellow-orange solid.
Example 81: 3-(3,5-DiisopropYl-4-methoxybenzylidene)-6-
trifluoromethyl-1,3-dihydroindol-2-one
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 6-trifluoromethyl-2-oxindole to give 0.35 g of 3-(3,5-
diisopropyl-4-methoxybenzylidene)-6-trifluoromethyl-1,3-
dihydroindol-2-one as a yellow-orange solid.
Example 82: 6-Chloro-3-(3,5-diisopropyl-4-
methoxybenzylidene)-1,3-dihydroindol-2-one
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 6-chloro-2-oxindole to give 0.3 g of 6-chloro-3-(3,5-
diisopropyl-4-methoxybenzylidene)-1,3-dihydroindol-2-one as
a yellow-orange solid.
Example 83: 3-f3-(3,5-Diisopropyl-4-methoxybenzylidene)-
2-oxo-2,3-dihydro-1H-indol-5-yl]-propionic acid
Potassium cyanide (2.0 g) was added to 15 ml of
dimethylsulfoxide and heated to 90°C. 5-chloroethyl-2-
oxindole (3.0 g) dissolved in 5 ml of dimethylsulfoxide was
added slowly with stirring, and the reaction was heated to
150°C and stirred for 2 hours. The mixture was cooled,
poured into ice water and the precipitate which formed was
collected by vacuum filtration, washed with water, and
dried to give crude product. The crude material was
chromatographed on silica gel (5% methanol in chloroform)
to give 1.2 g (42%) of 5-cyanoethyl-2-oxindole.
1HNMR (360 MHz, DMSO-d6) 8: 10.28 (s, br, NH), 7.12 (s,
1H, H-4), 7.07 (d, J = 8Hz, 1H), 6.74 (d, J = 8Hz, 1H),
3.43 (s, 2H, CHZ) , 2.71-2.82 (m, 4H, 2xCH2) .
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5-Cyanoethyl-2-oxindole (4.02 g) in 10 ml of water
containing 25 ml of concentrated hydrochloric acid was
refluxed for 4 hours. The mixture was cooled, water added
and the resulting solid collected by vacuum filtration,
washed with water and dried to give 1.9 g (44 % yield) of
5-carboxyethyl-2-oxindole as a yellow solid.
1HNMR (360 MHz, DMSO-d6) 8: 12.00 (s, br, 1H, 5-
CHZCHzCOOH) , 10.21 (s, 1H, NH-1) , 7.05 (s, 1H, H-4) , 6.99
(d, J = 9Hz, 1H, H-6), 6.69 (d, J = 9Hz, 1H, H-7), 3.40 (s,
2H, CHz-3) , 2.74 (t, J = 7Hz, 2H, 5-CH2CHZCOOH) and 2.46 (t,
J = 7Hz, 2H, 5-CHzCH2CO0H) .
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 5-(2-carboxyethyl)-2-oxindole to give 0.35 g of 3-[3-
(3,5-diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-dihydro-
1H-indol-5-yl]-propionic acid as a yellow-orange solid.
Example 84: 3-(3,5-Diisopropyl-4-methoxybenzylidene)-6-
methoxv-1,3-dihydroindol-2-one
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 6-methoxy-2-oxindole to give 0.3 g of 3-(3,5-
diisopropyl-4-methoxybenzylidene)-6-methoxy-1,3
dihydroindol-2-one as a yellow-orange solid.
Example 85: 5-Butyl-3-(3,5-diisoprotwl-4-methoxy-
benzylidene)-1,3-dihydroindol-2-one
To 15 g of aluminum chloride suspended in 30 ml of
1,2-dichloroethane in an ice bath was added 7.5 g of 2-
oxindole and then 12 g of butyryl chloride. The resulting
suspension was stirred at 50 °C overnight. The mixture was
poured into ice water and extracted 3 times with ethyl
acetate. The combined ethyl acetate layers were washed
with brine, dried over sodium sulfate, and concentrated to
dryness to give a brown solid. The solid was
chromatographed on silica gel (50 % ethyl acetate in
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hexane) to give 4.3 g (38%) of 5-butanoyl-2-oxindole as a
yellow solid.
'HNMR (360 MHz, DMSO-d6) d: 10.67 (s, br, NH), 7.84 (d,
J = 8.39Hz, 1H), s, 1H, H-4), 6.88 (d, J = 8.39Hz, 1H),
3.53 (s, 2H, CH2), 2.88 (t, J = 7.16Hz, 2H, CH2), 1.58-1.64
(m, 2H) , 0.9 (t, J = 7.58Hz, 3H, CH3) .
Triethylsilane (2.3 g) was added to 2 g of 5-butanoyl-
2-oxindole in 20 ml of trifluoroacetic acid at room
temperature and the solution stirred for 3 hours. The
reaction was poured into ice water to give a red oil which
solidified after standing. The solid was collected by
vacuum filtration, washed with water and hexane and dried
to give 1.7 g (91% yield) of 5-butyl-2-oxindole as an off-
white solid.
1HNMR (360 MHz, DMSO-d6) 8: 10.19 (s, br, NH), 7.01 (s,
1H, H-4), 6.95 (d, J = 8Hz, 1H), 6.69 (d, J = 8Hz, 1H), 3.4
(s, 2H, CHZ) , 2.49 (t, J = 8Hz, 2H, CHZ) , 1.48-1 .52 (m, 2H) ,
1.25-1.31 (m, 2H), 0.88 (t, J = 7Hz, 3H).
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 5-butyl-2-oxindole to give 0.3 g of 5-butyl-3-(3,5-
diisopropyl-4-methoxybenzylidene)-1,3-dihydroindol-2-one as
a yellow-orange solid.
Example 86: 3-(3,5-Diisopropvl-4-methoxybenzylidene)-2-
oxo-2,3-dihydro-1H-indole-4-carboxylic acid
Trimethylsilyldiazomethane in hexane (2 M) was added
dropwise to the solution of 2-chloro-3-carboxynitrobenzene
(2.01 g) in methanol (20 ml) at room temperature until no
further gas evolution occurred. The excess trimethylsilyl-
diazomethane was quenched with acetic acid. The reaction
mixture was dried by rotary pump and the residue was
further dried in oven for overnight. The product (2-
chloro-3-methoxycarbonyl-nitrobenzene) was pure enough for
the following reaction.
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Dimethyl malonate (6.0 mi) was added to an ice-cold
suspension of sodium hydride (2.1 g) in dimethylsulfoxide
(15 ml). The reaction mixture was heated to 100°C, stirred
for 1 hour and then cooled to room temperature. 2-Chloro-
3-methoxycarbonylnitrobenzene (2.15 g) was added in one
portion and the mixture was stirred at 100°C for 1.5 hour.
The reaction mixture was then cooled to room temperature
and poured into ice water, acidified to pH 5, and extracted
with ethyl acetate. The organic layer was washed with
brine, dried over anhydrous sodium sulfate and concentrated
to give 3.0 g of the dimethyl 2-methoxycarbonyl-6-
nitrophenylmalonate.
Dimethyl 2-methoxycarbonyl-6-nitrophenylmalonate (3.0
g) was refluxed in 50 ml of 6 N hydrochloric acid
overnight. The mixture was concentrated to dryness and
refluxed for 2 hours with 1.1 g of tin(II) chloride in 20
ml of ethanol. The mixture was filtered through Celite,
concentrated and chromatographed on silica gel (ethyl
acetate:hexane:acetic acid) to give 0.65 g (37 % yield) of
4-carboxy-2-oxindole as a white solid.
1HNMR (360 MHz, DMSO-d6) 8: 12.96 (s, br, 1H, COOH),
10.74 (s, br, 1H, NH) , 7.53 (d, J = 8Hz, 1H) , 7.39 (t, J =
8Hz, 1H, H-6), 7.12 (d, J = 8Hz, 1H, H-7), 3.67 (s, 2H, H-
3) .
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 4-carboxy-2-oxindole to give 0.3 g of 3-(3,5-
diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-dihydro-1H-
indole-4-carboxylic acid as a yellow-orange solid.
Example 87: 3-(3,5-Diisopropyl-4-methoxybenzylidene)-6-
(3-methoxyphenyl)-1,3-dih~droindol-2-one
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 6-(3-methoxyphenyl)-2-oxindole to give 0.4 g of 3-
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(3,5-diisopropyl-4-methoxybenzylidene)-6-(3-methoxyphenyl)-
1,3-dihydroindol-2-one as a yellow-orange solid.
Example 88: 7-Chloro-3-(3,5-diisopro~yl-4-
methoxvbenzylidene)-5-methyl-1,3-dihydroindol-2-one
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 5-methyl-7-chloro-2-oxindole to give 0.35 g of 7
chloro-3-(3,5-diisopropyl-4-methoxybenzylidene)-5-methyl-
1,3-dihydroindol-2-one as a yellow-orange solid.
Example 89: (3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-
oxo-2,3-dihydro-1H-indol-5-yll-carbamic acid tert-butyl
ester
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 5-tert-butyloxy-carbonylamino-2-oxindole to give 0.4 g
of [3-(3,5-diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-
dihydro-1H-indol-5-yl]-carbamic acid tert-butyl ester as a
yellow-orange solid.
Example 90: 5-Chloro-3-(3-isopropyl-4-
methoxvbenzylidene)-1,3-dihydroindol-2-one
3-Isopropyl-4-methoxybenzaldehyde was condensed with
5-chloro-2-oxindole to give 0.3 g of 5-chloro-3-(3-
isopropyl-4-methoxybenzylidene)-1,3-dihydroindol-2-one as a
yellow-orange solid.
1HNMR (d6-DMSO) 8: 10.6 (s, 1H, CONH), 8.5, 8.4, 7.9,
7.8, 7.2, 7.1, 6.8 (m, 7H, aromatic, =CH-), 3.9 (s, 3H,
OCH3) , 3 .3 (m, 1H, CH) , 1.2 (d, 6H, 2xCH3) .
Example 91: 5-Chloro-3-(3-cycloQentyl-4-methoxy-
benzylidene)-1,3-dihydroindol-2-one
3-Cyclopentyl-4-methoxybenzaldehyde was condensed with
5-chloro-2-oxindole to give 0.3 g of 5-chloro-3-(3-
cyclopentyl-4-methoxybenzylidene)-1,3-dihydroindol-2-one as
a yellow-orange solid.
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'HNMR (d6-DMSO) 8: 10.6 (s, 1H, CONH), 8.5, 8.3, 7.9,
7.8, 7.2, 7.1, 6.8 (m, 7H, aromatic, =CH-), 3.9 (s, 3H,
OCH3) , 3 .3 (m, 1H, CH) , 1.8 (m, 8H, 4xCH2) .
Example 92: 3-(6-Methoxybiphenyl-3-ylmethylene)-4-
methyl-1,3-dihydroindol-2-one
4-Methoxy-3-phenylbenzaldehyde was condensed with 4-
methyl-2-oxindole to give 0.3 g of 3-(6-methoxybiphenyl-3-
ylmethylene)-4-methyl-1,3-dihydroindol-2-one as a yellow-
orange solid.
1HNMR (d6-DMSO) 8: 10.5 (s, 1H, CONH), 8.3-6.7 (m, 12H,
aromatic, =CH-), 3.9 (s, 3H, OCH3), 2.6 (s, 3H, CH3).
Example 93: 3-(5-Isopropyl-4-methoxy-2-
methylbenzylidene)-4-methyl-1,3-dihydroindol-2-one
5-Isopropyl-4-methoxy-2-methylbenzaldehyde was
condensed with 4-methyl-2-oxindole to give 0.3 g of 3-(5-
isopropyl-4-methoxy-2-methylbenzylidene)-4-methyl-1,3-
dihydroindol-2-one as a yellow-orange solid.
1HNMR (ds-DMSO) 8: 10.3 (s, 1H, CONH), 8.2, 7.8, 7.1,
6.8, 6.8, 6.7 (m, 6H, aromatic, =CH-), 3.8 (s, 3H, OCH3),
3.2 (m, 1H, CH) , 2.5 (s, 3H, CH3) , 2.4 (s, 3H, CH3) , 1.2 (d,
6H, 2xCH3 ) .
Example 94: 5-Bromo-3-(2,3-dihydrobenzofuran-5-
ylmethylene)-1,3-dihydroindol-2-one
2,3-Dihydro-5-formylbenzofuran (commercially
available) was condensed with 5-bromo-2-oxindole to give
0.3 g of 5-bromo-3-(2,3-dihydrobenzofuran-5-ylmethylene)-
1,3-dihydroindol-2-one as a yellow-orange solid.
1HNMR (d6-DMSO) 8: 10.64 (s, 1H, CONH) , 7.71 (d, 1H) ,
7.62(vinyl, 1H), 7.61 (m, 1H), 7.52 (m, 1H), 7.37 (dd, 1H),
6.93 (d, 1H) , 6.984 (d, 1H) , 4.64 (t, 2H, CHZ) , 3.22 (t, 2H,
CHz ) .
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Example 95: 5-Chloro-3-(3-isopropyl-4-
methoxybenzylidene)-4-methyl-1,3-dihydroindol-2-one
3-Isopropyl-4-methoxybenzaldehyde was condensed with
4-methyl-5-chloro-2-oxindole to give 0.3 g of 5-chloro-3
(3-isopropyl-4-methoxybenzylidene)-4-methyl-1,3
dihydroindol-2-one as a yellow-orange solid.
Example 96: 5-Chloro-3-(3,5-diisopro~yl-4-
methoxybenzylidene)-4-methyl-1 3-dihydroindol-2-one
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 4-methyl-5-chloro-2-oxindole to give 0.4 g of 5-
chloro-3-(3,5-diisopropyl-4-methoxybenzylidene)-4-methyl-
1,3-dihydroindol-2-one as a yellow-orange solid.
Example 97: 5-Chloro-3-(2,2-dimethylchroman-6-
ylmethylene)-4-methyl-1,3-dihydroindol-2-one
2,2-Dimethyl-6-formylchromane (commercially available)
was condensed with 4-methyl-5-chloro-2-oxindole to give 0.3
g of 5-chloro-3-(2,2-dimethyl-chroman-6-ylmethylene)-4-
methyl-1,3-dihydroindol-2-one as a yellow-orange solid.
Example 98: 3-(3,5-Diisopropyl-4-methoxybenzylidene)-2-
oxo-2,3-dihydro-1H-indole-5-carboxylic acid
2-Oxindole (6.7 g) was added to a suspension of
aluminum chloride (23 g) in dichloroethane (30 ml) cooled
in an ice bath. Chloroacetyl chloride (11.3 g) was slowly
added and hydrogen chloride gas was evolved. After ten
minutes of stirring, the reaction was warmed to 40 - 50°C
for 1.5 hours. The mixture was cooled to room temperature
and poured into ice water. The precipitate was collected
by vacuum filtration, washed with water and dried under
vacuum to give 10.3 g (98%) of 5-chloroacetyl-2-oxindole as
an off-white solid.
A suspension of 9.3 g of 5-chloroacetyl-2-oxindole was
stirred in 90 ml pyridine at 80 - 90°C for 3 hours then
cooled to room temperature. The precipitate was collected
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by vacuum filtration and washed with 20 ml of ethanol. The
solid was dissolved in 90 ml of 2.5 N sodium hydroxide and
stirred at 70 to 80°C for 3 hours. The mixture was cooled
to room temperature and acidified to pH 2 with 0.5 N
hydrochloric acid. The precipitate was collected by
vacuum filtration and washed thoroughly with water to give
crude 5-carboxy-2-oxindole as a dark brown solid. After
standing overnight the filtrate yielded 2 g of 5-carboxy-2-
oxindole as a yellow solid. The crude dark brown product
was dissolved in hot methanol, the insoluble material
removed by filtration and the filtrate concentrated to give
5.6 g of 5-carboxy-2-oxindole as a brown solid. The
combined yield was 97%.
1HNMR (360 MHz, DMSO-d6) 8: 2.56 (s, br, 1H, COOH-5),
10.70 (s, 1H, NH-1), 7.82 (dd, J = 2, 8Hz, 1H, H-6), 7.74
(s, br, 1H, H-4), 6.87 (d, J = 8Hz, 1H, H-7), and 3.53 (s,
2H, CH2-3 ) .
MS m/z 178 [M+1]'.
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 5-carboxy-2-oxindole to give 0.4 g of 3-(3,5-
diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-dihydro-1H-
indole-5-carboxylic acid as a yellow-orange solid.
Example 99: 3-(3,5-Diisopropyl-4-methoxybenzylidene)-
5,6-dimethoxy-1,3-dihydroindol-2-one
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 5,6-dimethoxy-2-oxindole to give 0.4 g of 3-(3,5-
diisopropyl-4-methoxybenzylidene)-5,6-dimethoxy-1,3-
dihydroindol-2-one as a yellow-orange solid.
Example 100: N-f3-(3,5-Diisopropyl-4-methoxybenzvlidene)-
2-oxo-2,3-dihydro-1H-indol-6-vl]-methanesulfonamide
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 6-methylsulfonylamino-2-oxindole to give 0.4 g of N-
[3-(3,5-diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-
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dihydro-1H-indol-6-yl]-methanesulfonamide as a yellow-
orange solid.
Example 101: N-f3-(3,5-Diisonrot~yl-4-methoxybenzylidene)-
2-oxo-2,3-dihydro-1H-indol-6-yl]-benzamide
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 6-benzamido-2-oxindole to give 0.4 g of N-[3-(3,5-
diisopropyl-4-methoxybenzylidene)-2-oxo-2,3-dihydro-1H-
indol-6-yl]-benzamide as a yellow-orange solid.
Example 102: 3-(3,5-Diisopropyl-4-methoxybenzylidene)-6-
(3-ethoxyphenyl)-1,3-dihydroindol-2-one
Tetrakis(triphenylphosphine)palladium (0.8 q) was
added to a mixture of 4.2 g of 3-ethoxyphenylboronic acid,
5.0 g of 5-bromo-2-fluoronitrobenzene and 22 ml of 2 M
sodium carbonate solution in 50 ml of toluene and 50 ml of
ethanol. The mixture was heated to reflux for 2 hours. The
cooled mixture was concentrated, water was added and the
mixture was extracted twice with ethyl acetate. The ethyl
acetate layer was washed with water, brine, dried, and
concentrated. The residue was chromatographed on silica
gel (5 % ethyl acetate in hexane) to give 5.3 g (90 %
yield) of crude 4-fluoro-3'-ethoxy-3-nitrobiphenyl as a
yellow oil.
Dimethyl malonate (11.4 ml) was added dropwise to 4.0
g of sodium hydride suspended in 20 ml of
dimethylsulfoxide. The mixture was heated to 100°C for 10
minutes and cooled to room temperature. Crude 4-fluoro-3'-
ethoxy-3-nitrobiphenyl (5.3 g) in 25 ml of
dimethylsulfoxide was added and the mixture was stirrede at
100°C for 2 hours. The reaction mixture was cooled and
quenched with 300 ml of saturated ammonium chloride
solution and extracted three times with ethyl acetate. The
extracts were combined, washed with water and brine, dried
over anhydrous sodium sulfate and concentrated to give
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crude dimethyl 3'-ethoxy-3-nitrobiphenyl-4-malonate as a
yellow oil.
Crude dimethyl 3'-ethoxy-3-nitrobiphenyl-4-malonate in
60 ml of 6 N hydrochloric acid was stirred at 100°C for 4
days and and then cooled. The precipitate which formed was
collected by filtration, washed with water and hexane, and
dried to give 4.7 g (77% based on 5-bromo-2-
fluoronitrobenzene) of crude 3'-ethoxy-3-nitrobiphenyl-4-
acetic acid as a light tan solid.
Iron chips (2.4 g) were added in one portion to 4.6 g
of 3'-ethoxy-3-nitrobiphenyl-4-acetic acid in glacial
acetic acid (40 ml). The mixture was refluxed for 2 hours.
The reaction mixture was cooled and concentrated to
dryness. The residue was treated repeatedly with ethyl
acetate and filtered to remove insolubles. The filtrate
was washed twice with 1 N hydrochloric acid and brine,
dried over anhydrous sodium sulfate and concentrated to
give 3.5 g (91%) of 6-(3-ethoxyphenyl)-2-oxindole as a
light brown solid.
1HNMR (360 MHz, DMSO-d6) b: 10.4 (s, br, 1H, NH), 7.33
(t, J = 8Hz, 1H, H-3'), 7.35 (d, J = 8Hz, 1H), 7.19 (dd, J
- 1, 8Hz, 1H), 7.13 (d, J = 8Hz, 1H), 7.07-7.08 (m, 1H),
7.0 (s, br, 1H) , 6.9 (dd, J = 3, 8Hz, 1H) , 4.08 (q, J =
7Hz, 2H, O CH~CH3) , 3.49 (s, 2H, CHz) , 1.34 (t, J = 7Hz, 3H,
OCHzCH3) .
MS m/z 254.2 [M+1]'.
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 6-(3-ethoxyphenyl)-2-oxindole to give 0.4 g of 3-(3,5-
diisopropyl-4-methoxybenzylidene)-6-(3-ethoxyphenyl)-1,3-
dihydroindol-2-one as a yellow-orange solid.
Example 103: 3-(3,5-Diisopropyl-4-methoxybenzylidene)-6-
phenvl-1,3-dihydroindol-2-one
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Tetrakis(triphenylphosphine)palladium (0.8 g) was
added to a mixture of 3.1 g of benzeneboronic acid, 5 g of
5-bromo-2-fluoronitrobenzene and 22 ml of 2M aqueous sodium
carbonate in 50 ml of toluene and 50 ml of ethanol. The
mixture was refluxed for 2 hours, concentrated, and the
residue extracted twice with ethyl acetate. The ethyl
acetate layer was washed with water and brine, dried, and
concentrated to give a yellow oil. The oil was
chromatographed on silica gel (5% ethyl acetate in hexane)
to give 4.75 g (96%) of 4-fluoro-3-nitrobiphenyl as a
yellow oil.
Dimethyl malonate (10 ml) in 25 ml of
dimethylsulfoxide was added dropwise to 3.5 g of sodium
hydride suspended in 25 ml of dimethylsulfoxide and the
mixture stirred at 100°C for 10 minutes. The mixture was
cooled to room temperature and 4.7 g of 4-fluoro-3-
nitrobiphenyl in 25 ml of dimethylsulfoxide was added. The
mixture was stirred at 100°C for 2 hours, cooled and
quenched with 300 ml of saturated ammonium chloride
solution. The mixture was extracted three times with ethyl
acetate and the combined organic layers washed with water
and brine and evaporated to give a yellow oil, crude
dimethyl-3-nitrobiphenyl-4-malonate.
Crude dimethyl-3-nitrobiphenyl-4-malonate was refluxed
in 30 ml of 6 N hydrochloric acid for 24 hours. The
precipitate was collected by filtration, washed with water
and dried to give 4.5 g (80% based on 4-fluoro-3-
nitrobiphenyl) of 3-nitrobiphenyl-4-acetic acid as a cream
colored solid.
Iron chips (2.6 g) were added all at once to 4.5 g of
3-nitrobiphenyl-4-acetic acid in 40 ml of acetic acid. The
mixture was refluxed for 2 hours, concentrated to dryness
and the residue taken up in ethyl acetate. The solids were
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removed by filtration and the filtrate washed twice with 1
N hydrochloric acid and brine and dried over anhydrous
sodium sulfate. The filtrate was concentrated to give 3.4
g (93 % yield) of 6-phenyl-2-oxindole as a light brown
solid.
1HNMR (360 MHz, DMSO-d6) b: 10.4 (s, br, 1H, NH-1),
7.57-7.6 (m, 2H), 7.42-7.46 (m, 2H), 7.32-7.37 (m, 1H),
7.27 (d, J = 8, 1H, H-4), 7.19 (dd, J = 2 and 8Hz, 1H, H-
5) , 7.01 (d, J = 2Hz, 1H, H-7) , 3.49 (s, 2H, CHZ) .
MS m/z 210 [M+1]'.
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 6-phenyl-2-oxindole to give 0.4 g of 3-(3,5-
diisopropyl-4-methoxybenzylidene)-6-phenyl-1,3-
dihydroindol-2-one as a yellow-orange solid.
Example 104: 3-(3,5-Diisopropyl-4-methoxvbenzylidene)-5-
fluoro-1,3-dihydroindol-2-one
3,5-Diisopropyl-4-methoxybenzaldehyde was condensed
with 5-fluoro-2-oxindole to give 0.3 g of 3-(3,5-
diisopropyl-4-methoxybenzylidene)-5-fluoro-1,3-
dihydroindol-2-one as a yellow-orange solid.
Example 105: 5-Fluoro-3-(4-methoxy-3,5-
dimethylbenzylidene)-1,3-dihydroindol-2-one
3,5-Dimethyl-4-methoxybenzaldehyde was condensed with
5-fluoro-2-oxindole to give 0.3 g of 5-fluoro-3-(4-methoxy-
3,5-dimethylbenzylidene)-1,3-dihydroindol-2-one as a
yellow-orange solid.
Example 106: 3-(2,2-Dimeth~lchroman-6-ylmethylene)-1,3-
dihydroindol-2-one
2,2-Dimethyl-6-formylchromane (commercially available)
was condensed with 2-oxindole to give 0.3 g of 3-(2,2-
dimethylchroman-6-ylmethylene)-1,3-dihydroindol-2-one as a
yellow-orange solid.
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Example 107: 3-~~2-[6-(4-Fluorophenyl)-2-oxo-1,2-
dihvdroindol-3-vlidenemethyll-5-methyl-1H-pyrrol-3-
yl~propionic acid
6-(4-Fluorophenyl)-1,3-dihydroindol-2-one (70 mg, 0.31
mmol) was condensed with 3-(2-formyl-5-methyl-1H-pyrrol-3-
yl)-propionic acid (56 mg) to give the title compound.
1HNMR (360 MHz, DMSO-d6) 8 13.38 (s, br, 1H, COOH),
12.08 (s, br, 1H, NH), 10.87 (s, br, 1H, NH), 7.79 (d, J =
7.9 Hz, 1H), 7.63-7.69 (m, 3H), 7.23-7.29 (m, 3H), 7.07 (d,
J = 1.4 Hz, 1H), 6.06 (d, J = 2.2 Hz, 1H), 2.96 (t, J= 7.2
Hz, 2H, CHZ) , 2 .52 (t, J = 7.2 Hz, 2H, CHZ) , 2 .32 (s, 3H,
CH3 ) .
MS-EI m/z 390 [M'] .
Example 108: 4-(2-Carboxvethyl)-5-[6-(4-fluorophenyl)-2-
oxo-1,2-dihydro-indol-3-ylidenemethyl~-2-methyl-1H-pyrrole-
3-carboxylic acid ethyl ester
6-(4-Fluorophenyl)-1,3-dihydroindol-2-one (60 mg, 0.26
mmol) was condensed with 4-(2-carboxyethyl)-5-formyl-2-
methyl-1H-pyrrole-3-carboxylic acid ethyl ester (65 mg),
prepared by formylation of 4-(2-carboxyethyl)-3-
ethoxycarbonyl-2-methylpyrrole (Butter, A.R., and George,
S.D. (1993) Tetrahedron 49: 7017-7026), to give the title
compound.
1HNMR (360 br, 1H, NH), 12.03
MHz, DMSO-d6)
b 13.85 (s,
(s, br, 1H, COOH) 11.05 (s, br, 1H, NH) 7.83 (d, J = 7.9
, ,
Hz, 1H), 7.76 (s, 1H, H-vinyl), 7.66-7.70(m, 2H), 7.25-
7.30 (m, 3H) , 7.08(d, J = 1.4 Hz, 1H) .22 (q, J = 7.0
, 4
Hz, 2H, OCHzCH3) .20 (t, J = 7.6 Hz, CHZ) , 2.54 (s,
, 3 2H,
3H, CH3) , 2.45 2H, CH2) , 4.22 (t, 7.0 Hz, 2H,
(m, J =
3 0 OCHZ
CH3
) ,
MS-EI m/z 462 [M'] .
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Example 109: 3-~2-(6-(2-Methoxyphenyl)-2-oxo-1,2-
dihvdroindol-3-vlidenemethyll-5-methyl-1H-pvrrol-3-yl~-
progionic acid
6-(2-Methoxyphenyl)-1,3-dihydroindol-2-one (71 mg,
0.3 mmol) was condensed with 3-(2-formyl-5-methyl-1H-
pyrrol-3-yl)-propionic acid (54 mg) to give the title
compound.
iHNMR (300 MHz, DMSO-d6) 8 13.33 (s, br, 1H, NH) , 12.07
(s, br, 1H, COOH), 10.74 (s, br, 1H, NH), 7.68 (d, J = 7.8
Hz, 1H), 7.56 (s, 1H, H-vinyl), 7.25-7.28 (m, 2H), 6.94-
7.05 (m, 4H), 5.99 (br s, 1H), 3.70 (s, 3H, OCH3), 2.89 (m,
2H, CH2) , 2.45 (m, 2H, CHz) , 2.26 (s, 3H, CH3) .
MS-EI m/z 402 [M'] .
Example 110: 4-(2-Carboxyethyl)-5-(6-(2-methox henyl)-2-
oxo-1,2-dihydro-indol-3-ylidenemethyll-2-methyl-1H-pvrrole-
3-carboxylic acid ethyl ester
1HNMR (300 MHz, DMSO-d6) 8 13.88 (s, br, 1H, NH), 12.07
(s, br, 1H, COOH), 10.99 (s, br, 1H, NH), 7.77 (d, J = 7.8
Hz, 1H), 7.74 (s, 1H, H-vinyl), 7.28-7.36 (m, 2H), 7.07-7.1
(m, 2H), 6.99-7.04 (m, 2H), 4.21 (q, J = 7.0 Hz, 2H,
OCHzCH3) , 3.76 (s, 3H, OCH3) , 3.19 (m, 2H, CH2) , 2.54 (s, 3H,
CH3) , 2.50 (m, 2H, CH2) , 1.04 (t, J = 7.0 Hz, 3H, OCHZCH3) .
MS-EI m/z 474 [M'] .
Example 111: 3-(2-(5-Chloro-2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-5-methyl-1H-pvrrol-3-yllprogionic acid
5-Chloro-1,3-dihydroindol-2-one (61 mg, 0.36 mmol) was
condensed with 3-(2-formyl-5-methyl-1H-pyrrol-3-yl)-
propionic acid (66 mg) to give the title compound.
MS-EI m/z 300 and 302 [M-1 and M+1] .
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Example 112: 4-(2-Carboxyethyl)-5-(5-chloro-2-oxo-1,2-
dihvdroindol-3-ylidenemethyl)-2-methyl-1H-pyrrole-3-
carboxvlic acid ethyl ester
1HNMR (300 MHz, DMSO-d6) b 13.88 (s, br, 1H, NH), 12.04
(s, br, 1H, COOH) , 11.08 (s, br, 1H, NH) , 7.93 (d, J = 2.0
Hz, 1H), 7.83 (s, 1H, H-vinyl), 7.15 (dd, J = 2.0, 8.3 Hz,
1H), 6.87 (d, J = 8.3 Hz, 1H), 4.20 (q, J = 7.1 Hz, 2H,
OCHZCH3) , 3.21 (t, J = 7.6 Hz, 2H, CH2) , 2.53 (s, 3H, CH3) ,
2.45 (t, J = 7.6 Hz, 2H, CH2) , 1.28 (t, J = 7. 1 Hz, 3H,
OCHZCH3 ) .
MS-EI m/z 402 and 404 [M-1 and Ma-1) .
Example 117: 4-(2-Carboxyethyl)-3-methyl-5-(2-oxo-1,2-
dihvdroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid
ethyl ester
Oxindole (0.258, 1.88 mmol) was condensed with 4-(2-
carboxyethyl)-5-formyl-3-methyl-1H-pyrrole-2-carboxylic
acid ethyl ester (0.5 g, 1.97 mmol) to give 0.45 g (65%) of
the title compound.
1HNMR (300 MHz, DMSO-d6) 8 13.85 (s, br, 1H, NH), 12.12
(s, br, 1H, COOH) , 10.99 (s, br, 1H, NH) , 7. 78 (d, J = 7.6
Hz, 1H), 7.70 (s, 1H, H-vinyl), 7.17 (m 1H), 6.99 (m, 1H),
4.25 (q, J = 7.1 Hz, 2H, OCHZCH3) , 2.93 (t, J = 7.4 Hz, 2H,
CHZ) , 2.38 (t, J = 7.4 Hz, 2H, CH2) , 2.24 (s, 3H, CH3) , 1.29
(t, J = 7.1 Hz, 3H, OCH2CH3) .
MS-EI m/z 368 [M') .
Example 118: 2-Methyl-5-(5-methylsulfamoyl-2-oxo-1,2-
dihydroindol-3-ylidenemethyl)-4-(3-morpholin-4-ylpropyl)-
1H-twrrole-3-carboxylic acid ethyl ester
2-Oxo-2,3-dihydro-1H-indole-5-sulfonic acid
methylamide was stirred with 5-formyl-2-methyl-4-(3-
morpholin-4-ylpropyl)-1H-pyrrole-3-carboxylic acid ethyl
ester overnight at room temperature to give the title
compound.
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1HNMR (300 MHz, DMSO-d6) b 13.86 (s, br, 1H, NH), 11.39
(s, br, 1H, NH) , 8.16 (d, J = 1.6 Hz, 1H, H-4) , 7.82 (s,
1H, H-vinyl), 7.57 (dd, J = 1.6 & 6.9 Hz, 1H, H-6), 7.21
(m, 1H, CH3NHSOz) , 7.07 (d, J = 6.9 Hz, 1H, H-7) , 4.21 (q, J
- 7.0 Hz, 2H, OCH2CH3) , 3.52 (m, 4H, 2xCH2) , 3. 03 (m, 2H,
CHz) , 2.55 (s, 3H, CH3) , 2.40 (d, J = 5.0 Hz, 3H, CH3NHS02) ,
2.25 (m, 6H, 3xCH2) , 1.69 (m, 2H, CHz) , 1.29 (t, J = 7.0 Hz,
3H, OCH2CH3 ) .
MS-EI m/z 516 [M'] .
Example 119: 2-Methyl-4-[3-(4-methylpiperazin-1-yl)-
propyl]-5-(5-methylsulfamoyl-2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-1H-pyrrole-3-carboxylic acid ethyl ester
1HNMR (300 MHz, DMSO-d6) 8 13.88 (s, br, 1H, NH), 11.38
(s, br, 1H, NH), 8.16 (d, J = 1.6 Hz, 1H, H-4), 7.79 (s,
1H, H-vinyl), 7.57 (dd, J = 1.6 & 6.6 Hz, 1H, H-6), 7.21
(m, 1H, CH3NHS0z) , 7.05 (d, J = 6.6 Hz, 1H, H-7) , 4.21 (q, J
- 7. 0 Hz, 2H, OCH2CH3) , 3.01 (m, 2H, CH2) , 2.55 (s, 3H, CH3) ,
2.40 (d, J = 5.4 Hz, 3H, CH3NHS02) , 2.57 (m, 10H, 5xCH2) ,
2.09 (s, 3H, CH3N) , 1. 67 (m, 2H, CHz) , 1.29 (t, J = 7. 0 Hz,
3H, OCH2CH3) .
MS-EI m/z 529 [M'] .
Example 120: 4-(3-Dimethvlaminopropyl)-5-(5-methoxy-2-oxo-
1,2-dihydroindol-3-ylidenemethyl)-2-methyl-1H-pyrrole-3-
carboxylic acid ethyl ester
1HNMR (360 MHz, DMSO-d6) b 13.95 (s, br, 1H, NH), 10.76
(s, br, 1H, NH), 7.70 (s, 1H, H-vinyl), 7.31 (d, J = 2.2
Hz, 1H, H-4), 6.78 (d, J = 8.3 Hz, 1H, H-7), 6.73 (dd, J =
2.2 & 8.3 Hz, 1H, H-6), 4.21 (q, J = 7.0 Hz, 2H, OCH2CH3),
3 .76 (s, 3H, OCH3) , 2. 98 (m, 2H, CH2) , 2.53 (s, 3H, CH3) ,
2. 19 (m, 2H, CHz) , 2 . 11 (s, 6H, N (CH3) 2) , 1 . 64 (m, 2H, CH2) ,
1.30 (t, J = 7.0 Hz, 3H, OCH2CH3) .
MS-EI m/z 411 [M'] .
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Example 121: 5-(5-Methoxy-2-oxo-1,2-dihydroindol-3-
Ylidenemethyl)-2-methyl-4-(3-morpholin-4-ylpropyl)-1H-
pyrrole-3-carboxylic acid eth~rl ester
1HNMR (300 MHz, DMSO-d6) 8 14.0 (s, br, 1H, NH), 10.77
(s, br, 1H, NH), 7.65 (s, 1H, H-vinyl), 7.40 (m, 1H), 6.76
(m, 2H) , 4.2 (q, J = 7. 0 Hz, 2H, OCHzCH3) , 3 .76 (s, 3H,
OCH3) , 3 .54 (m, 4H, 2xCHz) , 2.99 (m, 2H, CH2) , 2.52 (s, 3H,
CH3) , 2 .25 (m, 6H, 3xCH2) , 1 .67 (m, 2H, CHz) , 1 .29 (t, J =
7.0 Hz, 3H, OCHzCH3) .
MS-EI m/z 453 [M'] .
Example 122: 5-(5-Methoxy-2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-2-methyl-4-[3-(4-methylpiperazin-1-yl)-
propvl]-1H-pyrrole-3-carboxylic acid ethyl ester
1HNMR (300 MHz, DMSO-d6)8 13.99 (s, br, 1H, NH), 10.78
(s, br, 1H, NH), 7.63 (s, 1H, H-vinyl), 7.39 (d, J = 1.8
Hz, 1H, H-4), 6.76 (m, 2H), 4.2 (q, J = 7.1 Hz, 2H,
OCHzCH3) , 3 .77 (s, 3H, OCH3) , 2. 97 (m, 2H, CH2) , 2.52 (s, 3H,
CH3), 2.25 (m, 10H, 5xCH2), 2.08 (s, 3H, NCH3), 1.66 (m, 2H,
CHz) , 1.29 (t, J = 7. 1 Hz, 3H, OCHzCH3) .
MS-EI m/z 466 [M+] .
Example 123: 2-Methyl-4-(3-morpholin-4-vlpropyl)-5-(2-oxo-
5-sulfamoyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pvrrole-3-
carboxylic acid ethyl ester
1HNMR (300 MHz, DMSO-d6) 8 13.85 (s, br, 1H, NH), 11.33
(s, br, 1H, NH), 8.19 (d, J = 1.8 Hz, 1H, H-4), 7.75 (s,
1H, H-vinyl), 7.63 (dd, J = 1.8 & 8.3 Hz, 1H, H-6), 7.16
(s, 2H, H2NS02) , 7.02 (d, J = 8.3 Hz, 1H, H-7) , 4.21 (q, J =
7.3 Hz, 2H, OCHZCH3) , 3.53 (m, 4H, 2xCH2) , 3 . O1 (m, 2H, CHz) ,
2.55 (s, 3H, CH3), 2.28 (m, 6H, 3xCH2), 1.68 (m, 2H, CHz),
1.29 (t, J = 7.3 Hz, 3H, OCHzCH3) .
MS-EI m/z 502 [M'] .
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Example 124: 2-Methyl-4-[3-(4-methylpiperazin-1=yl)-
propyl]-5-(2-oxo-5-sulfamoyl-1,2-dihydroindol-3-
ylidenemethyl)-1H-pyrrole-3-carboxylic acid ethyl ester
'HNMR (300 MHz, DMSO-ds) b 13.85 (s, br, 1H, NH), 11.31
(s, br, 1H, NH) , 8.18 (d, J = 1.5 Hz, 1H, H-4) , 7.74 (s,
1H, H-vinyl), 7.63 (dd, J = 1.5 & 8.1 Hz, 1H, H-6), 7.15
(s, 2H, HzNSOz) , 7.01 (d, J = 8. 1 Hz, 1H, H-7) , 4.21 (q, J =
7.1 Hz, 2H, OCHZCH3) , 3.0 (m, 2H, CHZ) , 2.54 (s, 3H, CH3) ,
2.26 (m, 10H, 5xCH2) , 2. 09 (s, 3H, NCH3) , 1 . 67 (m, 2H, CHz) ,
1.29 (t, J = 7.1 Hz, 3H, OCHZCH3) .
MS-EI m/z 515 [M+] .
Example 125: 3-[4-Ethoxvcarbonyl-5-methyl-3-(3-morpholin-
4-ylpropyl)-1H-pyrrol-2-ylmethylenel-2-oxo-2,3-dihydro-1H-
indole-5-carboxylic acid
1HNMR (300 MHz, DMSO-d6) 8 13.88 (s, br, 1H, NH), 11.03
(s, br, 1H, COOH), 8.17 (s, 1H, H-4), 7.76 (d, J = 8.4 Hz,
1H, H-6), 7.59 (s, 1H, H-vinyl), 6.80 (d, J = 8.4 Hz, 1H,
H-7) , 4.21 (q, J = 7.2 Hz, 2H, OCHzCH3) , 3.54 (m, 4H,
2xCHz), 2.98 (m, 2H, CHZ), 2.49 (s, 3H, CH3), 2.29 (m, 6H,
3xCH2) , 1.64 (m, 2H, CH2) , 1 .29 (t, J = 7.2 Hz, 3H, OCHzCH3) .
MS-EI m/z 467 [M'] .
Example 126: 2-Methyl-5-(5-methylsulfamoyl-2-oxo-1,2-
dihydroindol-3-ylidenemethyl)-4-(3 wrrolidin-l~rlpropyl)-
1H-pyrrole-3-carboxylic acid ethyl ester
1HNMR (360 MHz, DMSO-d6) b 13.84 (s, 1H, NH) , 11.28 (br
s, 1H, NH) , 8.06 (d, J = 2.1 Hz, 1H) , 7.83 (s, 1H, H-
vinyl), 7.57 (dd, J = 2.1 & 8.3 Hz, 1H), 7.20 (m, 1H,
HNCH3) , 7.06 (d, J = 8.3 Hz, 1H) , 4.22 (q, J = 7.0 Hz, 2H,
OCH2CH3) , 3.04 (m, 2H, CHz) , 2.55 (s, 3H, CH3) , 2.40 (d, J =
4.8 Hz, 3H, HNCH3), 2.3-2.36 (m, 6H, 3xCHz), 1.66-1.71 (m,
6H, 3xCHz) , 1.30 (t, J = 7.0 Hz, 3H, OCHzCH3) .
MS-EI m/z 500 [M'] .
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Example 127: 5-(5-Methoxy-2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-2-methyl-4-(3-pyrrolidin-1-ylpropyl)-1H-
pvrrole-3-carboxylic acid ethyl ester
1HNMR (300 MHz, DMSO-d6) 8 13.96 (s, 1H, NH) , 10.75 (br
s, 1H, NH), 7.65 (s, 1H, H-vinyl), 7.28 (d, J = 2.2 Hz,
1H) , 6. 71-6. 79 (m, 2H) , 4.20 (q, J = 7. 1 Hz, 2H, OCHzCH3) ,
3.75 (s, 3H, OCH3) , 3 .0 (m, 2H, CHz) , 2.52 (s, 3H, CH3) ,
2.26-2.35 (m, 6H, 3xCHz), 1.65-1.71 (m, 6H, 3xCH2), 1.29 (t,
J = 7.1 Hz, 3H, OCH2CH3) .
MS-EI m/z 437 [M+] .
Example 128: 3-(4-Ethoxycarbonyl-5-methyl-3-(3-pyrrolidin-
1-vlpropyl)-1H-pyrrol-2-ylmethylenel-2-oxo-2,3-dihydro-1H-
indole-5-carboxylic acid
iHNMR (300 MHz, DMSO-d6) 8 13.80 (s, 1H, NH), 11.26 (br
s, 1H, NH), 8.2 (s, 1H), 7.77 (m, 2H), 6.92 (d, J = 8.1 Hz,
1H) , 4.23 (q, J = 6.9 Hz, 2H, OCHZCH3) , 3. 03 (m, 2H, CHZ) ,
2.54 (s, 3H, CH3), 2.40 (m, 6H, 3xCH2), 1.68 (m, 6H,
3xCH2) , 1.29 (t, J = 6. 9 Hz, 3H, OCHZCHj) .
MS-EI m/z 451 [M'] .
Example 130: 2-Methyl-5-(2-oxo-5-sulfamoyl-1,2-
dihvdroindol-3-ylidenemethyl)-4-(3-pyrrolidin-1-ylpropyl)-
1H-pyrrole-3-carboxylic acid ethyl ester
1HNMR (300 MHz, DMSO-ds) b 13.98 (s, 1H, NH) , 11.30 (br
s, 1H, NH), 8.10 (d, J = 1.9 Hz, 1H), 7.79 (s, 1H, H-
vinyl), 7.63 (dd, J = 1.9 & 8.2 Hz, 1H), 7.16 (br s, 2H,
HZNSOz) , 7. 02 (d, J = 8.2 Hz, 1H) , 4.21 (q, J = 6. 9 Hz, 2H,
OCHZCH3) , 3.03 (m, 2H, CHZ) , 2.54 (s, 3H, CH3) , 2.33 (m, 6H,
3xCH2) , 1.68 (m, 6H, 3xCH2) , 1.29 (t, J = 6. 9 Hz, 3H,
OCHZCH3 ) .
MS-EI m/z 486 [M+] .
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Example 131: 5-L4-(2-Hydroxvethyl)-2-oxo-1,2-dihydroindol-
3-ylidenemethyll-2-methyl-4-(3-morpholin-4-ylpronyl)-1H-
pvrrole-3-carboxylic acid ethyl ester
1HNMR (360 MHz, DMSO-d6) 8 13.93 (s, 1H, NH) , 10.95 (br
s, 1H, NH), 7.65 (s, 1H, H-vinyl), 7.06 (t, J = 7.6 Hz,
1H) , 6. 83 (d, J = 7.6 Hz, 1H) , 6.77 (d, J = 7. 6 Hz, 1H) ,
4.85 (br s, 1H, OH) , 4.21 (q, J = 7. 0 Hz, 2H, OCH2CH3) , 3.73
(m, 2H, CHZ) , 3.53 (m, 4H, 2xCH2) , 3.09 (m, 2H, CH2) , 2.92
(m, 2H, CHz), 2.53 (s, 3H, CH3), 2.31 (m, 6H, 3xCH2), 1.68
(m, 2H, CHz) , 1.29 (t, J = 7.0 Hz, 3H, OCHzCH3) .
MS-EI m/z 467 [M'] .
Example 132: 5-f4-(2-Hydroxyethyl)-2-oxo-1,2-di ~droindol-
3-vlidenemethyll-2-methyl-4-(3-pvrrolidin-1-ylpropyl)-1H-
pyrrole-3-carboxylic acid ethyl ester
1HNMR (360 MHz, DMSO-d6) 8 13.93 (s, 1H, NH) , 10.94 (br
s, 1H, NH), 7.68 (s, 1H, H-vinyl), 7.06 (t, J = 7.3 Hz,
1H), 6.83 (d, J = 7.3 Hz, 1H), 6.77 (d, J = 7.3 Hz, 1H),
4.87 (br s, 1H, OH) , 4.21 (q, J = 7.2 Hz, 2H, OCH2CH3) , 3.72
(m, 2H, CH2) , 3. 09 (m, 2H, CHz) , 2.94 (m, 2H, CHZ) , 2.53
(s, 3H, CH3) , 2.38 (m, 6H, 3xCH2) , 1.67 (m, 6H, 3xCH2) , 1.29
(t, J = 7.2 Hz, 3H, OCHZCH3) .
MS-EI m/z 451 [M'] .
Example 133: 4-(3-Dimethvlaminopropyl)-2-methyl-5-(2-oxo-
5-sulfamovl-1,2-dihvdroindol-3-vlidenemethvl)-1H-pYrrole-3-
carboxylic acid ethyl ester
1HNMR (300 MHz, DMSO-d6) 8 13.73 (s, 1H, NH) , 11.25 (br
s, 1H, NH), 8.09 (d, J = 1.6 Hz, 1H), 7.90 (s, 1H, H-
vinyl), 7.62 (dd, J = 1.6 & 8.1 Hz, 1H), 7.20 (br s, 2H,
HZNSOZ) , 7. 02 (d, J = 8.1 Hz, 1H) , 4.21 (q, J = 7. 1 Hz, 2H,
OCHZCH3) , 3 . O1 (m, 2H, CHz) , 2.54 (s, 3H, CH3) , 2 . 16 (m, 2H,
CH2) , 2 . 11 (s, 6H, N (CH3) z) , 1 . 66 (m, 2H, CHZ) , 1 .29 (t, J =
7.1 Hz, 3H, OCHzCH3) .
MS-EI m/z 460 [M+] .
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Example 134: 3-L3-(3-Dimethylaminopropyl)-4-
ethoxvcarbonvl-5-methyl-1H-pyrrol-2-vlmethylenel-2-oxo-2,3-
dihydro-1H-indole-5-carboxylic acid
1HNMR (300 MHz, DMSO-d6) b 13 .77 (s, 1H, NH) , 11.25 (br
s, 1H, NH), 8.24 (s, 1H), 7.85 (s, 1H, H-vinyl), 7.76 (d,
1H) , 6. 82 (d, 1H) , 4 .21 (q, J = 6. 9 Hz, 2H, OCHzCH3) , 3 . 0
(m, 2H, CH2) , 2.54 (s, 3H, CH3) , 2.20 (m, 2H, CHZ) , 2. 14 (s,
6H, N (CH3) 2) , 1 . 64 (m, 2H, CHZ) , 1 .29 (t, J = 6 . 9 Hz, 3H,
OCHzCH3 ) .
MS m/z 426.2 [M+H].
Example 135: 4-(3-Dimethylaminopropyl)-5- 4-(2-
hvdroxvethvl)-2-oxo-1,2-dihydroindol-3-ylidenemethvl]-2-
methvl-1H-pyrrole-3-carboxylic acid ethyl ester
1HNMR (300 MHz, DMSO-d6) b 13.95 (s, 1H, NH) , 10.97 (br
s, 1H, NH), 7.69 (s, 1H, H-vinyl), 7.05 (t, J = 7.7 Hz,
1H), 6.82 (d, J = 7.7 Hz, 1H), 6.75 (d, J = 7.7 Hz, 1H),
4. 90 (br s, 1H, OH) , 4 .20 (q, J = 7.2 Hz, 2H, OCHZCH3) , 3 .72
(m, 2H, CHz) , 3.09 (m, 2H, CHZ) , 2. 90 (m, 2H, CH2) , 2.52 (s,
3H, CH3) , 2.20 (m, 2H, CHz) , 2.07 (s, 6H, N(CH3) 2) , 1.63 (m,
2H, CHz) , 1.28 (t, J = 7.2 Hz, 3H, OCHZCH3) .
MS-EI m/z 425 [M'] .
Example 136: 5-f4-(2-Hydroxyethyl)-2-oxo-1,2-dihydroindol-
3-ylidenemethyl]-2-methyl-4-[3-(4-methylpiperazin-1-yl)-
propvll-1H-pvrrole-3-carboxylic acid ethyl ester
1HNMR (360 MHz, DMSO-d6) 8 14.0 (s, 1H, NH) , 10. 94 (br
s, 1H, NH), 7.70 (s, 1H, H-vinyl), 7.06 (t, J = 7.4 Hz,
1H), 6.83 (d, J = 7.4 Hz, 1H), 6.76 (d, J = 7.4 Hz, 1H),
4.77 (br s, 1H, OH) , 4.22 (q, J = 7.21 Hz, 2H, OCHZCH3) ,
3.72 (m, 2H, CHz) , 3.36 (m, 2H, CH2) , 3 . 13 (m, 4H, 2xCH2) ,
2.56 (m, 2H, CHZ) , 2.53 (s, 3H, CH3) , 2.09 (m, 8H, 4xCH2) ,
1.28 (t, J = 7.1 Hz, 3H, OCHzCH3) .
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Example 137: 4-(3-Dimethvlaminopropyl)-2-methyl-5-(5-
methvlsulfamoyl-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-
pyrrole-3-carboxylic acid ethyl ester
2-Oxo-2,3-dihydro-1H-indole-5-sulfonic acid
methylamide (113 mg, 0.5 mmol) was condensed with 4-(3-
dimethylamino-propyl)-5-formyl-2-methyl-1H-pyrrole-3-
carboxylic acid ethyl ester (133 mg) to give 96 mg (40%) of
the title compound.
1HNMR (360 MHz, DMSO-d6) b 13.73 (s, br, 1H, NH), 11.32
(s, br, 1H, NH) , 8.05 (d, J = 1.8 Hz, 1H, H-4) , 7.92 (s;
1H, H-vinyl), 7.57 (dd, J = 1.8 & 8.1 Hz, 1H, H-6), 7.22
(m, 1H, CHjNHSOZ) , 7. 05 (d, J = 8.1 Hz, 1H, H-7) , 4.21 (q, J
- 7.1 Hz, 2H, OCH2CH3) , 3.01 (m, 2H, CHz) , 2.54 (s, 3H, CH3) ,
2.41 (d, J = 4.0 Hz, 3H, CH3NHS02) , 2.14 (s, 8H, CHZ &
N(CH3)2) , 1.66 (m, 2H, CH2) , 1.29 (t, J = 7.1 Hz, 3H,
OCHZCH3 ) .
MS-EI m/z 474 [M'] .
Example 138: 4-(3-Dimethvlaminopropyl)-2-methyl-5-(5-
methvlsulfamoyl-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-
pyrrole-3-carboxylic acid
A mixture of 4-(3-dimethylaminopropyl)-5-formyl-2-
methyl-1H-pyrrole-3-carboxylic acid ethyl ester (300 mg,
1.13 mmol) and sodium hydroxide in MeOH (4 mL)/water (2 mL)
was stirred at 80°C for 19 hr. The reaction was then
diluted with EtOAc. The aqueous layer was separated,
neutralized and concentrated. The residue was dissolved in
MeOH, filtered and the filtrate was concentrated to give
251 mg (94%) of 4-(3-dimethylaminopropyl)-5-formyl-2-
methyl-1H-pyrrole-3-carboxylic acid.
1HNMR (300 MHz, DMSO-ds) 8 11.88 (br s, 1H, NH), 9.50
(s, 1H, CHO) , 2.96 (m, 2H, CHZ) , 2.39 (s, 3H, CH3) , 2.22 (m,
2H, CH2) , 2.14 (s, 6H, N(CH3)z) , 1.68 (m, 2H, CHZCHZ CH2) .
MS-EI m/z 238 [M+] .
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2-Oxo-2,3-dihydro-1H-indole-5-sulfonic acid
methylamide (113 mg) was condensed with 4-(3-
dimethylaminopropyl)-5-formyl-2-methyl-1H-pyrrole-3-
carboxylic acid (119 mg) to give 50 mg 4-(3-
dimethylaminopropyl)-2-methyl-5-(5-methylsulfamoyl-2-oxo-
1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carboxylic
acid.
1HNMR (360 MHz, DMSO-d6) 8 13.52 (s, br, 1H, NH), 11.25
(s, br, 1H, COOH), 8.01 (d, J = 1.7 Hz, 1H, H-4), 7.84 (s,
1H, H-vinyl), 7.52 (dd, J = 1.7 & 8.3 Hz, 1H, H-6), 7.22
(m, 1H, CH3NHS0z) , 7.05 (d, J = 8.3 Hz, 1H, H-7) , 3.08 (m,
2H, CH2) , 2.56 (s, 3H, CH3) , 2.40 (m, 3H, CH3NHS02) , 2.17 (m,
8H, CHz & N (CH3) 2) , 1 . 72 (m, 2H, CHz) .
MS-EI m/z 446 [M'] .
Example 139: 4-(2-Carboxyethyl)-3-methyl-5-(4-methyl-2-
oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-
carboxylic acid ethyl ester
1HNMR (300 MHz, DMSO-d6) 8 13.97 (s, br, 1H, NH), 12.15
( br s, 1H, COOH ), 11.03 (s, 1H, NH), 7.69 (s, 1H, H-
vinyl), 7.10 (t, J = 7.5 Hz, 1H), 6.83 (d, J = 7.5 Hz, 1H),
6.76 (d, J = 7.5 Hz, 1H) , 4.28 (q, J = 7. 0 Hz, 2H, OCHzCH3) ,
2.88 (t, 2H, CHz) , 2.62 (s, 3H, CH3) , 2.43 (t, 2H, CHZ) ,
2.26 (s, 3H, CH3) , 1.31 (t, J = 7. 0 Hz, 3H, OCH2CH3) .
MS-EI m/z 382 [M'] .
Example 140: 4-(2-Carboxyethyl)-5-(5-methoxy-2-oxo-1,2-
dihvdroindol-3-ylidenemethyl)-3-methyl-1H-pyrrole-2-
carboxylic acid ethyl ester
1HNMR (300 MHz, DMSO-ds) F 13.97 (s, br, 1H, NH) , 12.14
(br s, 1H, COOH ), 10.81 (s, 1H, NH), 7.71 (s, 1H, H-
vinyl), 7.46 (m, 1H), 6.78 (m, 2H), 4.28 (q, J = 7.0 Hz,
2H, OCHZCH3) , 3.76 (s, 3H, OCH3) , 2.97 (t, 2H, CHZ) , 2.40 (t,
2H, CH2) , 2.26 (s, 3H, CH3) , 1.31 (t, J = 7. 0 Hz, 3H,
OCHzCH3 ) .
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MS-EI m/z 398 [M'] .
Example 141: 4-(2-Carboxyethyl)-5-(6-methoxy-2-oxo-1,2-
dihydroindol-3-ylidenemethyl)-3-methyl-1H-t~yrrole-2-
carboxvlic acid ethyl ester
1HNMR (300 MHz, DMSO-d6) 8 13.70 (s, br, 1H, NH), 12.11
( br s, 1H, COOH ), 10.95 (s, 1H, NH), 7.0 (d, J = 8.4 Hz,
1H, H-4), 7.55 (s, 1H, H-vinyl), 6.59 (dd, J = 2.7 & 8.4
Hz, 1H, H-5), 6.45 (d, J = 2.7 Hz, 1H, H-7), 4.27 (q, J =
7.0 Hz, 2H, OCHzCH3) , 3.76 (s, 3H, OCH3) , 2.92 (t, 2H, CH2) ,
2.39 (t, 2H, CHZ) , 2.25 (s, 3H, CH3) , 1.31 (t, J = 7.0 Hz,
3H, OCHzCH3 ) .
MS-EI m/z 398 [M+] .
Example 142: 5-(6-Hromo-2-oxo-1,2-dihydroindol-3-
Srlidenemethyl)-4-(2-carboxy-ethyl)-3-methyl-1H-pyrrole-2-
carboxylic acid ethyl ester
1HNMR (300 MHz, DMSO-d6) 8 13.77 (s, br, 1H, NH), 12.13
( br s, 1H, COOH ) , 11. 14 (s, 1H, NH) , 7.77 (br s, 2H) , .12
(m, 2H) , 4.28 (m, 2H, OCH2CH3) , 2 .95 (m, 2H, CHZ) , 2 .40 (m,
2H, CHZ) , 2.26 (s, 3H, CH3) , 1.29 (m, 3H, OCHzCH3) .
MS-EI m/z 446 & 448 [M-1 & M+1] .
Example 143: 5-(5-Bromo-2-oxo-1,2-dihydroindol-3-
Ylidenemethyl)-4-(2-carboxy-ethyl)-3-methyl-1H-pyrrole-2-
carboxvlic acid ethyl ester
1HNMR (300 MHz, DMSO-d6) 8 13.86 (s, br, 1H, NH), 12.2
( br s, 1H, COOH), 11.12 (s, 1H, NH), 8.11 (d, J = 2.0 Hz,
1H, H-4), 7.83 (s, 1H, H-vinyl), 7.33 (dd, J = 2.0 & 8.3
Hz, 1H, H-6), 6.83 (d, J = 8.3 Hz, 1H, H-7), 4.28 (q, J =
7. 0 Hz, 2H, OCH2CH3) , 2. 98 (t, 2H, CH2) , 2.39 (m, 2H, CH2) ,
2.25 (s, 3H, CH3) , 1.31 (t, J = 7.0 Hz, 3H, OCH2CH3) .
MS-EI m/z 446 & 448 [M-1 & M+1] .
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Example 144: 4-(2-Carboxyethyl)-3-methyl-5-(2-oxo-6-
phenyl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-
carboxylic acid ethyl ester
1HNMR (300 MHz, DMSO-d6) b 13.86 (s, br, 1H, NH), 12.2
( br s, 1H, COOH ), 11.11 (s, 1H, NH), 7.89 (d, J = 7.7 Hz,
1H), 7.77 (s, 1H, H-vinyl), 7.65 (m, 2H), 7.46 (t, 2H),
7.32-7.39 (m, 2H), 7.12 (d, J = 1.6 Hz, 1H), 4.29 (q, J =
7. 0 Hz, 2H, OCHzCH3) , 2.97 (m, 2H, CH2) , 2.42 (m, 2H, CH2) ,
2.27 (s, 3H, CH3) , 1.31 (t, J = 7.0 Hz, 3H, OCHZCH3) .
MS-EI m/z 444 [M'] .
Example 145: 4-(2-Carboxyethyl)-3-methyl-5-(2-oxo-5-
sulfamoyl-1,2-dihvdro-indol-3-ylidenemethyl)-1H-pyrrole-2-
carboxylic acid ethyl ester
1HNMR (300 MHz, DMSO-d6) b 13.84 (s, br, 1H, NH), 11.0
( br s, 2H), 8.26 (d, J = 1.6 Hz, 1H, H-4), 7.88 (s, 1H, H-
vinyl), 7.67 (dd, J = 1.6 & 8.4 Hz, 1H, H-6), 7.22 (br m,
2H, HzNSOz) , 7.03 (d, J = 8.4 Hz, 1H, H-7) , 4.29 (q, J = 7.1
Hz, 2H, OCHzCH3) , 2.97 (t, 2H, CHz) , 2.37 (m, 2H, CHZ) , 2.26
(s, 3H, CH3) , 1.31 (t, J = 7.1 Hz, 3H, OCH2CH3) .
MS-EI m/z 447 [M'] .
Example 146: 4-(2-Carboxyethyl)-3-methyl-5-(5-
methylsulfamoyl-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-
pyrrole-2-carboxylic acid ethyl ester
1HNMR (300 MHz, DMSO-d6) b 13.89 (s, br, 1H, NH), 12.2
( br s, 1H, COOH ) , 11.43 (s, 1H, NH) , 8.25 (d, J = 1.8 Hz,
1H, H-4), 7.94 (s, 1H, H-vinyl), 7.62 (dd, J = 1.8 & 8.4
Hz, 1H, H-6) , 7.27 (m, 1H, F~1S02) , 7. 07 (d, J = 8 .4 Hz, 1H,
H-7) , 4.29 (q, J = 7. 1 Hz, 2H, OCH2CH3) , 3.01 (t, 2H, CHZ) ,
2.45 (m, 2H, CH2) , 2.42 (d, J = 5.3 Hz, 3H, NCH3) , 2.27 (s,
3H, CH3) , 1.32 (t, J = 7.1 Hz, 3H, OCH2CH3) .
MS-EI m/z 461 [M'] .
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Example 147: 4-(2-Carboxyethyl)-5-(5-dimethvlsulfamoyl-2-
oxo-1,2-dihydro-indol-3-ylidenemethyl)-3-methyl-1H-pyrrole-
2-carboxylic acid ethyl ester
1HNMR (300 MHz, DMSO-d6) 8 13.88 (s, br, 1H, NH), 12.14
( br s, 1H, COOH ) , 11.47 (s, 1H, NH) , 8.26 (d, J = 1.8 Hz,
1H, H-4), 8.0 (s, 1H, H-vinyl), 7.58 (dd, J = 1.8 & 7.9 Hz,
1H, H-6), 7.10 (d, J = 7.9 Hz, 1H, H-7), 4.29 (q, J = 7.2
Hz, 2H, OCHzCH3) , 3.03 (t, 2H, CH2) , 2.61 (s, 6H, N(CH3)z) ,
2.42 (t, 2H, CHz) , 2.26 (s, 3H, CH3) , 1.32 (t, J = 7.2 Hz,
3H, OCHzCH3) .
MS-EI m/z 489 [M+] .
Example 148: 4-(2-Carboxyethyl)-5-(5-isopropylsulfamoyl-2-
oxo-1,2-dihydro-indol-3-ylidenemethyl)-3-methyl-1H-pyrrole-
2-carboxylic acid ethyl ester
1HNMR (300 MHz, DMSO-d6) 8 13.84 (s, br, 1H, NH), 11.0
( br s, 2H, COOH & NH), 8.23 (d, J = 1.5 Hz, 1H, H-4), 7.92
(s, 1H, H-vinyl), 7.64 (dd, J = 1.5 & 8.4 Hz, 1H, H-6),
7.43 (br m, 1H, HI~TC (CHj) 2) , 7. 05 (d, J = 8 .4 Hz, 1H, H-7) ,
4.29 (q, J = 7.1 Hz, 2H, OCHZCH3) , 2.96 (t, 2H, CHz) , 2.34
(t, 2H, CHz) , 2.25 (s, 3H, CH3) , 1.31 (t, J = 7. 1 Hz, 3H,
OCHzCH3) , 0 . 95 (d, J = 6 . 6 Hz, 6H, HNC (CH3) 2) .
MS-EI m/z 489 [M'] .
Example 149: 3-f3-(3-Dimethylaminopropyl)-1H-indol-2-
ylmethylene~-2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid
methyl amide
2-Oxo-2,3-dihydro-1H-indole-5-sulfonic acid
methylamide (98 mg, 0.43 mmol) was condensed with 3-(3-
dimethylaminopropyl)-1H-indole-2-carbaldehyde (100 mg) to
give the title compound as an orange solid.
1HNMR (300 MHz, DMSO-d6) 8 12.90 (s, 1H, NH), 11.40 (br
s, 1H, NH), 8.17 (d, J = 1.5 Hz, 1H), 8.15 (s, 1H, H-
vinyl), 7.68 (d, J = 8.4 Hz, 1H), 7.63 (dd, J = 1.5 & 8.4
Hz, 1H), 7.54 (d, J = 8.7 Hz, 1H), 7.30 (m, 2H), 7.08 (m,
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2H) , 3.15 (t, J = 6.6 Hz, 2H, CHZ) , 2 .43 (d, J = 4.2 Hz, 3H,
NCH3) , 2.17 (t, J = 6.6 Hz, 2H, CHz) , 2.14 (s, 6H, N(CH3)Z) ,
1.78 (m, 2H, CHz) .
MS-EI m/z 438 [M'] .
Example 150: 3-f3-(3-Dimethylaminopropyl)-1H-indol-2
ylmethylene]-4-(2-hydroxyethyl)-1,3-dihydroindol-2-one
1HNMR (300 MHz, DMSO-d6) 8 13. 12 (s, 1H, NH) , 11.01 (br
s, 1H, NH) , 7.95 (s, 1H, H-vinyl) , 7.66 (d, J = 7.9 Hz,
1H), 7.52 (d, J = 7.9 Hz, 1H), 7.26 (t, J = 7.54 Hz, 1H),
7.11 (t, J = 7.9 Hz, 1H), 7.06 (t, J = 7.4 Hz, 1H), 6.87
(d, J = 7.4 Hz, 1H), 6.78 (d, J = 7.4 Hz, 1H), 4.91 (br s,
1H, OH), 3.79 (m, 2H, CH2), 3.16 (m, 2H, CH2), 3.04 (m, 2H,
CHz) , 2 .21 (m, 2H, CHz) , 2 . 09 (s, 6H, N (CH3) 2) , 1 . 77 (m, 2H,
CHz ) .
MS m/z 389 [M'] .
Example 151: 3-(3-(3-DimethylaminopropYl)-1H-indol-2-
ylmethylene]-5-methoxy-1,3-dihydroindol-2-one
1HNMR (300 MHz, DMSO-d6) b 13.11 (s, 1H, NH) , 10.84 (br
s, 1H, NH) , 7.92 (s, 1H, H-vinyl) , 7.65 (d, J = 8.1 Hz,
1H), 7.50 (d, J = 8.1 Hz, 1H), 7.44 (s, 1H), 7.26 (t, J =
7.5 Hz, 1H), 7.05 (t, J = 7.5 Hz, 1H), 6.79 (s, 2H), 3.79
(s, 3H, OCH3), 3.11 (m, 2H, CHZ), 2.20 (m, 2H, CHz), 2.12
(s, 6H, N (CH3) Z) , 1 . 76 (m, 2H, CHz) .
MS m/z 375 [M'] .
Example 152: 5-Methyl-3-(3-methyl-1H-indol-2=ylmethylene)-
1,3-dihydro-indol-2-one
3-Methylindole-2-carbaldehyde was prepared as
described in the literature: 1) David W.M. Benzies, Pilar
Matinez Fresneda and R. Alan Jones, Synthetic
Communications, 16 (14), 1799-1807 (1986) and
2) Chatterjee, A. and Biswas, K.M., J. Org. Chem., 1973,
38, 4002.
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A mixture of 5-methyl-2-oxindole (59 mg), 3-
methylindole-2-carbaldehyde (56 mg) and piperidine (30 mg)
in ethanol(1 mL).-was held in a sealed tube at 90°C
overnight. The mixture was cooled to room temperature. The
solid was collected by vacuum filtration, washed with cold
ethanol and dried in a vacuum oven to give 72 mg (74%) of
5-methyl-3-(3-methyl-1H-indol-2-ylmethylene)-1,3-
dihydroindol-2-one.
1H NMR (360 MHz, DMSO-d6) 13.03 (s, br, 1H, NH), 10.88
(s, br, 1H, NH), 7.84 (s, 1H, vinyl), 7.71 (s, br, 1H),
6.62 (d, J = 8Hz, 1H), 7.48 (d, J = 8Hz, 1H), 7.26 (t, J =
7Hz, 1H), 7.06 (t, J = 7Hz, 1H), 7.0 (d, J = 8Hz, 1H), 6.78
(d, J = 8Hz, 1H) , 2.59 (s, 3H, CH3) , 2.32 (s, 3H, CH3) .
MS m/z 289 [M+1]'.
Example 153: 3-(3-Methyl-1H-indol-2-ylmethylene)-2-oxo-
2,3-dihydro-1H-indole-5-sulfonic acid amide
A mixture of 5-aminosulfonyl-2-oxindole (84 mg), 3-
methylindole-2-carbaldehyde (56 mg) (prepared according to
Synthetic Communications, 1986, 16, 1799) and piperidine
(30 mg) in ethanol (1 mL) was held in a sealed tube at 90°C
overnight. The mixture was cooled to room temperature.
The solid was collected by vacuum filtration, washed with
cold ethanol and dried in a vacuum oven to give 88 mg (71%
yield) of 3-(3-methyl-1H-indol-2-ylmethylene)-2-oxo-2,3-
dihydro-1H-indole-5-sulfonic acid amide.
1H NMR (360 MHz, DMSO-d6) 12.86 (s, br, 1H, NH), 11.3
(s, br, 1H, NH), 8.3 (d, J = 2Hz, 1H), 7.97 (s, 1H, vinyl),
7.63 (dd, J = 2 and 8Hz, 1H), 7.6 (d, J = 8Hz, 1H), 7.47
(d, J = 8Hz, 1H), 7.24 (t, J = 7.5Hz, 1H), 7.12 (s, br, 2H,
NHz), 7.02 (t, J = 7.5Hz, 1H), 6.98 (d, J = 8Hz, 1H), 2.57
(s, 3H, CH3) .
MS m/z 354.1 (M+1]'
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Example 154: 3-(3-Methyl-1H-indol-2-ylmethylene)-2-oxo-
2,3-dihydro-1H-indole-5-sulfonic acid methvlamide
A suspension of 5-chlorosulfonyl-2-oxindole (3.38 g)
in 2M methylamine in tetrahydrofuran (10 mL) was stirred at
room temperature for 4 hours at which time a white solid
had formed. The solid was collected by vacuum filtration,
washed twice with 5 mL of water and dried under vacuum at
40°C to give 3.0 g (88% yield) of 5-methylaminosulfonyl-2-
oxindole.
1H NMR (300 MHz, DMSO-d6) 8 10.87 (s, br, 1H, NH-1),
7.86 (s, br, 1H, 5-SOZNHCH3),7.61 (d, J = 8Hz 1H, H-6), 7.32
(d, J = 5Hz, 1H, H-4), 6.97 (d, J = 8Hz, 1H, H-7), 2.53 (s,
2H, CHz-3) , and 2.36 (s, 3H, 5-SO2NHCH3) .
MS m/z 226.
A mixture of 5-methylaminosulfonyl-2-oxindole (90 mg),
3-methylindole-2-carbaldehyde (56 mg) (prepared according
to Synthetic Communications, 1986, 16, 1799) and piperidine
(30 mg) in ethanol (1 mL) was held in a sealed tube at 90°C
overnight. The mixture was cooled to room temperature.
The solid was collected by vacuum filtration, washed with
cold ethanol and dried in a vacuum oven to give 90 mg (70%
yield) of 3-(3-methyl-1H-indol-2-ylmethylene)-2-oxo-2,3-
dihydro-1H-indole-5-sulfonic acid methylamide (mixture of
isomers).
1H NMR (360 MHz, DMSO-d6) 11.13 (s, br, 1H, NH), 10.94
(s, br, 1H, NH), 8.84 (d, J = l6Hz, 1H), 7.98 (s, 1H),
6.97-7.66 (m, 6H, Ar-H), 2.6 (s, 3H, CH3), 2.4 (d, J = 5Hz,
3H, CH3N.
Example 155: 3-(3-Methyl-1H-indol-2-ylmethylene)-2-oxo-
2,3-dihydro-1H-indole-5-sulfonic acid dimethvlamide
A mixture of 5-dimethylaminosulfonyl-2-oxindole (96
mg), 3-methylindole-2-carbaldehyde (56 mg) (prepared
according to Synthetic Communications, 1986, 16, 1799) and
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piperidine (30 mg) in ethanol (1 mL) was held in a sealed
tube at 90°C overnight. The mixture was cooled to room
temperature. The solid was collected by vacuum filtration,
washed with cold ethanol and dried in a vacuum oven to give
76 mg (57% yield) of 3-(3-methyl-1H-indol-2-ylmethylene)-2-
oxo-2,3-dihydro-1H-indole-5-sulfonic acid dimethylamide
(mixture of isomers).
1H NMR (360 MHz, DMSO-d6) 11.09 (s, br, 1H, NH), 10.95
(s, br, 1H, NH), 8.78 (d, J = l6Hz, 1H), 7.8 (s, 1H,
vinyl), 7.45-7.54 (m, 2H), 7.32 (d, J = 8Hz, 1H), 7.1 (t,
1H), 6.98 (d, J = 8Hz, 1H), 6.93 (t, 1H), 2.56 (s, 6H, 2 x
CHj) , 2.39 (s, 3H, CH3)
Example 156: 3-(3-Methyl-1H-indol-2-ylmethylene)-2-oxo-
2,3-dihydro-1H-indole-5-carboxZrlic acid (piperidine salt)
A mixture of 5-carboxy-2-oxindole (113 mg), 3-
methylindole-2-carbaldehyde (56 mg) (prepared according to
Synthetic Communications, 1986, 16, 1799) and piperidine
(30 mg) in ethanol (1 mL) was held in a sealed tube at 90°C
overnight. The mixture was cooled to room temperature.
The solid was collected by vacuum filtration, washed with
cold ethanol and dried in a vacuum oven to give 75 mg (58%
yield) of 3-(3-methyl-1H-indol-2-ylmethylene)-2-oxo-2,3-
dihydro-1H-indole-5-carboxylic acid (piperidine salt).
1H NMR (360 MHz, DMSO-d6) 13.0 (s, br, 1H, NH), 8.35
(d, J = l.5Hz, 1H, H-4), 7.91 (s, 1H, vinyl), 7.82 (dd, J =
1.5 and 8Hz, 1H, H-6), 7.63 (d, J = 8Hz, 1H), 7.49 (d, J =
8Hz, 1H), 7.26 (t, J = 7.5Hz, 1H), 7.06 (t, J = 7.5Hz, 1H),
6.87 (d, J = 8Hz, 1H), 2.91 (t, 4H, piperidine), 2.6 (s,
3H, CH3) , 1.6 (m, 4H, piperidine) , 1.54 (m, 2H, piperidine) .
Example 157: 5-Acetyl-3-(3-methyl-1H-indol-2-ylmethylene)-
1~3-dihydroindol-2-one
2-Oxindole (3 g) was suspended in 1,2-dichloroethane
and 3.2 mL of acetyl chloride slowly added. The resulting
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suspension was stirred at 50°C for 5 hours, cooled, and
poured into water. The resulting precipitate was collected
by vacuum filtration, washed copiously with water and dried
under vacuum to give 2.9 g (73% yield) of 5-acetyl-2-
oxindole as a brown solid.
'H NMR (360 MHz, DMSO-d6) 8 10.75 (s, br, NH), 7.83 (d,
J = BHz, 1H) , 7.78 (s, 1H, H-4) , 6.88 (d, J = BHz, 1H) ,
3 .53 (s, 2H, CH2) , 2.49 (s, 3H, CH3) .
A mixture of 5-acetyl-2-oxindole (70 mg), 3-
methylindole-2-carbaldehyde (56 mg) (prepared according to
Synthetic Communications, 1986, 16, 1799) and piperidine
(30 mg) in ethanol (1 mL) was held in a sealed tube at 90°C
overnight. The mixture was cooled to room temperature.
The solid was collected by vacuum filtration, washed with
cold ethanol and dried in a vacuum to give 83 mg (75%
yield) of 5-acetyl-3-(3-methyl-1H-indol-2-ylmethylene)-1,3-
dihydro-indol-2-one.
1H NMR (360 MHz, DMSO-d6) 12.86 (s, br, 1H, NH), 11.29
(s, br, 1H, NH), 8.43 (d, J = l.SHz, 1H, H-4), 8.01 (s, 1H,
vinyl), 7.78 (dd, J = 1.5 and 8Hz, 1H, H-6), 7.58 (d, J =
8Hz, 1H), 7.45 (d, J = 8Hz, 1H), 7.23 (t, 1H), 7.01 (t,
1H), 6.92 (d, J = 8Hz, 1H), 2.58 (s, 3H, CH3), 2.54 (s, 3H,
CH3 ) .
MS m/z 317.2 [M+1]'.
Example 158: 5-Acetyl-3-(1H-indol-2-ylmethylene)-1,3-
dihydro-indol-2-one
Indole-2-carbaldehyde was prepared as described in the
literature: Michel Barbier, Michel Devys, Christiane
Tempete, Albert Kollmann and Jean-Fran~ois Bousquet,
Synthetic Communications, 23(22), 3109-3117 (1993).
A mixture of 5-acetyl-2-oxindole (88 mg), indole-2-
carbaldehyde (87 mg) and piperidine (4 mg) in ethanol (2
mL) was held in a sealed tube at 90°C for 3 hours. The
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mixture was cooled to room temperature. The solid which
formed was collected by vacuum filtration, washed with cold
ethanol and dried in a vacuum oven to give 133 mg (88%
yield) of 5-acetyl-3-(1H-indol-2-ylmethylene)-1,3-dihydro-
indol-2-one as an orange solid.
1H NMR (360 MHz, DMSO-d6) 12.81 (s, br, 1H, NH), 11.38
(s, br, 1H, NH), 8.38 (d, J = lHz, H-4), 8.17 (s, 1H,
vinyl), 7.88 (dd, J = 1 and BHz, 1H, H-6), 7.69 (d, J =
8Hz, 1H), 7.58 (d, J = 8Hz, 1H), 7.29 (t, 1H), 7.21 (s,
1H), 7.09 (t, 1H), 7.0 (d, J = 8Hz, 1H), 2.59 (s, 3H, CH3).
MS m/z 303.1 [M+1]'.
Example 159: 3-(1H-Indol-2-ylmethylene)-2-oxo-2,3-dihydro-
1H-indole-5-sulfonic acid amide
A mixture of 5-aminosulfonyl-2-oxindole (106 mg),
indole-2-carbaldehyde (87 mg) (prepared according to
Synthetic Communications, 1993, 23, 3109) and piperidine
(4 mg) in ethanol (2 mL) was held in a sealed tube at 90°C
for 3 hours. The mixture was cooled to room temperature.
The solid was collected by vacuum filtration, washed with
cold ethanol and dried in a vacuum oven to give 140 mg (83%
yield) of 3-(1H-indol-2-ylmethylene)-2-oxo-2,3-dihydro-1H-
indole-5-sulfonic acid amide as an orange solid.
1H NMR (360 MHz, DMSO-d6) 12.81 (s, br, 1H, NH), 11.38
(s, br, 1H, NH), 8.18 (d, J = lHz, H-4), 8.12 (s, 1H,
vinyl), 7.7 (m, 2H), 7.6 (d, J = 8Hz, 1H), 7.26-7.29 (m,
2H), 7.2 (s, 2H, NH2), 7.05-7.11 (m, 2H).
MS m/z 340 [M+1]'.
Example 160: 5-Amino-3-(1H-indol-2-ylmethylene)-1,3-
dihydro-indol-2-one
A mixture of 5-tert-butoxycarbonylamino-2-oxindole
(124 mg), indole-2-carbaldehyde (87 mg) (prepared according
to Synthetic Communications, 1993, 23, 3109) and piperidine
(4 mg) in ethanol (2 mL was held in a sealed tube at 90°C
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for 3 hours. The mixture was cooled to room temperature.
The solid was collected by vacuum filtration, washed with
cold ethanol and dried in a vacuum overn to give 196 mg of
5-tert-butoxycarbonylamino-3-(1H-indol-2-ylmethylene)-1,3-
dihydro-indol-2-one as a bright orange solid. The prodcuct
was then dissolved in trifluoroacetic acid/dichloromethane
(2 mL each) and stirred at room temperature for 1 hour.
The reaction mixture was then concentrated. The residue
was dissolved in water and basified with saturated sodium
bicarbonate solution. The solid was collected by vacuum
filtration, washed with water and dried in a vacuum oven to
give 129 mg of 5-amino-3-(1H-indol-2-ylmethylene)-1,3-
dihydroindol-2-one as a brown solid.
1H NMR (360 MHz, DMSO-d6) 13.1 (s, br, 1H, NH), 10.57
(s, br, 1H, NH), 7.65 (s, 1H, vinyl), 7.64 (d, J = 8Hz,
1H), 7.54 (d, J = 8Hz, 1H), 7.24 (t, 1H), 7.1 (s, 1H), 7.06
(t, 1H), 6.93 (d, J = 2Hz, 1H), 6.61 (d, J = 8Hz, 1H), 6.5
(dd, J = 2 and 8Hz, 1H) , 4.8 (s, br, 2H, NHz) .
MS m/z 276.1 [M+1J'.
Example 161: 3-(1H-Indol-2-ylmethylene)-2-oxo-2,3-dihydro-
1H-indole-5-carboxylic acid
A mixture of 5-carboxy-2-oxindole (88.5 mg), indole-2-
carbaldehyde (87 mg) (prepared according to Synthetic
Communications, 1993, 23, 3109) and piperidine (1 drop) in
ethanol (3 mL) was held in a sealed tube at 95°C for 4
hours. The mixture was cooled to room temperature. The
solid was filtered and acidified with 2N hydrochloric acid.
The resulting solid was collected by vacuum filtration,
washed with cold ethanol and dried in vacuum oven to give
60 mg (40% yield) of 3-(1H-indol-2-ylmethylene)-2-oxo-2,3-
dihydro-1H-indole-5-carboxylic acid as a mustard-colored
solid.
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'H NMR (360 MHz, DMSO-d6) 12.8 (s, br, 1H, NHI, 12.69
(s, 1H, COOH), 11.35 (s, br, 1H, NH), 8.33 (s, 1H), 8.16
(s, 1H), 7.86 (dd, J = 1 and 8Hz, 1H), 7.68 (d, J = 8Hz,
1H) , 7.59 (d, J = 8Hz, 1H) , 7.28 (t, 1H) , 7.21 (s, 1H) ,
7.08 (t, 1H) , 6.99 (d, J = 8Hz, 1H) .
MS m/z 304 [M]'.
Example 162: 6-Chloro-3-(1H-indol-2-ylmethylene)-1,3-
dihydro-indol-2-one
A mixture of 6-chloro-2-oxindole (41 mg) (commercially
available), indole-2-carbaldehyde (42 mg) (prepared
according to Synthetic Communications, 1993, 23, 3109) and
piperidine (1 drop) in ethanol (3 mL) was held in a sealed
tube at 95°C for 3 hours. The mixture was cooled to room
temperature. The solid was collected by vacuum filtration,
washed with cold ethanol and dried in vacuum oven to give
62 mg (87% yield) of 6-chloro-3-(1H-indol-2-ylmethylene)-
1,3-dihydroindol-2-one as a shiny orange solid.
1H NMR (360 MHz, DMSO-d6) 12.83 (s, br, 1H, NH), 11.17
(s, br, 1H, NH), 7.98 (s, 1H), 7.74 (d, J = 8Hz, 1H), 7.67
(d, J = 8Hz, 1H), 7.58 (d, J = 8Hz, 1H), 7.28 (m, 1H), 7.15
(s, br, 1H) , 7.08 (m, 2H) , 6.91 (d, J = 2Hz, 1H) .
MS m/z 293 and 295.
Example 163: 3-(1H-Indol-2-ylmethylene)-1,3-dihydro-
indol-2-one
A mixture of 2-oxindole (133 mg), indole-2-
carbaldehyde (174 mg) (prepared according to Synthetic
Communications, 1993, 23, 3109) and piperidine (10 mg) in
ethanol was held in a sealed tube at 95°C for 4 hours. The
mixture was cooled to room temperature. The solid was
collected by vacuum filtration, washed with cold ethanol
and dried in a vacuum oven to give 3-(1H-indol-2-
ylmethylene)-1,3-dihydro-indol-2-one.
1H NMR (360 MHz, DMSO-d6) 8 12.97 (s, br, 1H, NH),
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11.01 (s, br, 1H, NH), 7.92 (s, 1H, H-vinyl), 7.72 (d, J =
7Hz, 1H), 7.66 (d, J = 7Hz, 1H), 7.57 (dd, J = 1, 8Hz, 1H),
7.27 (dt, J = 1, 7Hz, 1H), 7.21 (dt, J = 1, BHz, 1H), 7.13
(s, 1H), 7.01-7.1 (m, 2H), 6.91 (d, J = 8Hz, 1H).
MS E1 260.
Example 164: 5-Chloro-3-(1H-indol-2-ylmethylene)-1,3-dihydro-
indol-2-one
A mixture of 5-chloro-2-oxindole (167 mg), indole-2-
carbaldehyde (174 mg) (prepared according to Synthetic
Communications, 1993, 23, 3109) and piperidine (10 mg) in
ethanol was held in a sealed tube at 95°C for 4 hours. The
mixture was cooled to room temperature. The solid was
collected by vacuum filtration, washed with cold ethanol
and dried in a vacuum oven to give 5-chloro-3-(1H-indol-2-
ylmethylene)-1,3-dihydroindol-2-one.
1H NMR (360 MHz, DMSO-d6) b 12.88 (s, br, 1H, NH),
11.12 (s, br, 1H, NH), 8.07 (s, 1H, H-vinyl), 7.86 (d, J =
2Hz, 1H), 7.68 (d, J = 8Hz, 1H), 7.59 (dd, J = 1, 8Hz, 1H),
7.28 (m, 1H), 7.24 (dd, J = 2, BHz, 1H), 7.14 (s, 1H), 7.09
(dt, J = 1, 8Hz, 1H), 6.90 (d, J = 8Hz, 1H).
MS E1 294 and 296.
Example 165: 5-Bromo-3-(1H-indol-2-ylmethylene)-1,3-
dihvdro-indol-2-one
A mixture of 5-bromo-2-oxindole (212 mg), indole-2-
carbaldehyde (174 mg) (prepared according to Synthetic
Communications, 1993, 23, 3109) and piperidine (10 mg) in
ethanol was held in a sealed tube at 95°C for 4 hours. The
mixture was cooled to room temperature. The solid was
collected by vacuum filtration, washed with cold ethanol
and dried in a vacuum oven to give 5-bromo-3-(1H-indol-2-
ylmethylene)-1,3-dihydroindol-2-one.
1HNMR (360 MHz, DMSO-d6) 8 12.87 (s, br, 1H, NH), 11.12
(s, br, 1H, NH), 8.07 (s, 1H, H-vinyl), 7.98 (d, J = 2Hz,
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1H), 7.68 (d, J = 8Hz, 1H), 7.59 (d, J = 8Hz, 1H), 7.36
(dd, J = 2, 8Hz, 1H) , 7.28 (dt, J = 1, 8Hz, 1H) , 7.14 (s,
1H), 7.08 (dt, J = l, 8Hz, 1H), 6.86 (d, J = 8Hz, 1H).
MS E1 338 and 340.
Example 166: 3-(1H-indol-2-ylmethylene)-4-methyl-1,3-
dihydro-indol-2-one
A mixture of 4-methyl-2-oxindole (147 mg), indole-2-
carbaldehyde (174 mg) (prepared according to Synthetic
Communications, 1993, 23, 3109) and piperidine (10 mg) in
ethanol was held in a sealed tube at 95°C for 4 hours. The
mixture was cooled to room temperature. The solid was
collected by vacuum filtration, washed with cold ethanol
and dried in a vacuum oven to give 3-(1H-indol-2-
ylmethylene)-4-methyl-1,3-dihydroindol-2-one.
1H NMR (360 MHz, DMSO-d6) 8 13.12 (s, br, 1H, NH),
11.02 (s, br, 1H, NH), 7.79 (s, 1H, H-vinyl), 7.64 (d, J =
8Hz, 1H), 7.57 (d, J = 8Hz, 1H), 7.27 (d, J = 7Hz, 1H),
7.23 (s, 1H), 7.05 - 7.14 (m, 2H), 6.84 (d, J = 8Hz, 1H),
6.78 (d, J = 8Hz, 1H) , 2.61 (s, 3H, CH3) .
MS E1 274.
Example 167: 3-(3-Methyl-1H-indol-2 ylmethylene)-1,3-
dihydro-indol-2-one
A mixture of 2-oxindole (133 mg), 3-methyiindole-2-
carbaldehyde (190 mg) (prepared according to Synthetic
Communications, 1986, 16, 1799) and piperidine (10 mg) in
ethanol was held in a sealed tube at 95°C for 4 hours. The
mixture was cooled to room temperature. The solid was
collected by vacuum filtration, washed with cold ethanol
and dried in a vacuum oven to give 3-(3-methyl-1H-indol-2-
ylmethylene)-1,3-dihydroindole-2-one.
1H NMR (360 MHz, DMSO-d6) b 13.01 (s, br, 1H, NH),
10.99 (s, br, 1H, NH), 7.89 (s, 1H, H-vinyl), 7.88 (d, J =
7Hz, 1H) , 7.63 (d, J = 8Hz, 1H) , 7.49 (d, J = BHz, 1H) ,
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7.27 (dt, J = 1, 8Hz, 1H) , 7.2 (dt, J = 1, 8Hz, 1H) , 7.01-
7.08 (m, 2H) , 6.9 (d, J = 8Hz, 1H) , 2 .59 (s, 3H, CH3) .
MS El 274.
Example 168: 5-Chloro-3-(3-methyl-1H-indol-2-ylmethylene)-
1,3-dihydro-indol-2-one
A mixture of 5-chloro-2-oxindole (167 mg), 3-
methylindole-2-carbaldehyde (190 mg) (prepared according to
Synthetic Communications, 1986, 16, 1799) and piperidine
(10 mg) in ethanol was held in a sealed tube at 95°C for 4
hours. The mixture was cooled to room temperature. The
solid was collected by vacuum filtration, washed with cold
ethanol and dried in a vacuum oven to give 5-chloro-3-(3-
methyl-1H-indol-2-ylmethylene)-1,3-dihydroindol-2-one.
1H NMR (360 MHz, DMSO-d6) 8 12.96 (s, br, 1H, NH),
11.09 (s, br, 1H, NH) , 8.09 (d, J = 2Hz, 1H) , 8.02 (s, 1H,
H-vinyl), 7.63 (d, J = 8Hz, 1H), 7.5 (d, J = 8Hz, 1H), 7.29
(dt, J = 1, 7Hz, 1H) , 7.21 (dd, J = 2, 8Hz, 1H) , 7.07 (dt,
J = 1, 7Hz, 1H), 6.89 (d, J = 8Hz, 1H), 2.61 (s, 3H, CH3).
MS E1 308.
Example 169: 5-Bromo-3-(3-methyl-1H-indol-2-ylmethylene)-
1,3-dihydro-indol-2-one
A mixture of 5-bromo-2-oxindole (212 mg), 3-methyl-
indole-2-carbaldehyde (190 mg) (prepared according to
Synthetic Communications, 1986, 16, 1799) and piperidine
(10 mg) in ethanol was held in a sealed tube at 95°C for 4
hours. The mixture was cooled to room temperature. The
solid was collected by vacuum filtration, washed with cold
ethanol and dried in a vacuum oven to give 5-bromo-3-(3-
methyl-1H-indol-2-ylmethylene)-1,3-dihydro-indol-2-one.
1H NMR (360 MHz, DMSO-d6) 8 12.96 (s, br, 1H, NH),
11.09 (s, br, 1H, NH) , 8.21 (d, J = 2Hz, 1H) , 8.02 (s, 1H,
H-vinyl), 7.63 (d, J = 8Hz, 1H), 7.5 (d, J = 8Hz, 1H), 7.33
(dd, J = 2, SHz, 1H) , 7.29 (dt, J = 1, 7Hz, 1H) , 7.07 (dt,
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J = 1, 7Hz, 1H) , 6.84 (d, J = 8Hz, 1H) , 2.62 (s, 3H, CH3) .
MS El 352 and 354.
Example 170: 4-Methyl-3-(3-methyl-1H-indol-2-ylmethylene)-
1,3-dihydroindol-2-one
A mixture of 4-methyl-2-oxindole (147 mg), 3-methyl-
indole-2-carbaldehyde (190 mg) (prepared according to
Synthetic Communications, 1986, 16, 1799) and piperidine
(10 mg) in ethanol was held in a sealed tube at 95°C for 4
hours. The mixture was cooled to room temperature. The
solid was collected by vacuum filtration, washed with cold
ethanol and dried in a vacuum oven to give 4-methyl-3-(3-
methyl-1H-indol-2-ylmethylene)-1,3-dihydro-indol-2-one.
1H NMR (360 MHz, DMSO-d6) 13.07 (s, br, 1H, NH), 11.0
8
(s, br, 1H, NH),7.78 (s, 1H, H-vinyl),
7.64 (d, J =
8Hz,
1H) 7.51 (d, = 8Hz, 1H) , 7.27(dt, J = 1, 8Hz, 1H) ,
, J
7.04 - 7.12 (m, 2H), 6.85 (d, = 8Hz, 1H), 6.78 (d, J =
J
8Hz, 1H) , 2.64 s, 3H, CH3) , (s, 3H, CH3) .
( 2.52
MS E1 288.
Example 171: 3-(1H-indol-2-ylmethylene)-5-f(1H-indol-2-
ylmethylene)-aminol-1,3-dihydroindol-2-one
A mixture of 5-amino-2-oxindole (74 mg), indole-2-
carbaldehyde (87 mg) (prepared according to Synthetic
Communications, 1983, 23, 3109) and piperidine (4 mg) in
ethanol (2 mL) was held in a sealed tube at 95 °C for 3
hours. The mixture was cooled to room temperature. The
solid was collected by vacuum filtration, washed with cold
ethanol and dried in a vacuum oven. The crude solid was
then chromatographed on silica gel to give 50 mg (25%
yield) of 3-(1H-indol-2-ylmethylene)-5-[(1H-indol-2-
ylmethylene)-amino]-1,3-dihydro-indol-2-one
Example 172: 3-(1H-Indol-3-ylmethylene)-2-oxo-2.3-dihydro-
1H-indole-5-sulfonic acid amide
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1HNMR (300 MHz, DMSO-d6) 8 12.03 (br, s, 1H, NH), 10.80
(br, s, 1H, NH), 9.37 (s, 1H), 8.09-8.24 (m, 3H), 7.51 (m,
1H) , 7.41 (m, 1H) , 7.13 (m, 2H) , 7.03 (s, 2H, NHZ) , 6.85 (d,
J = 7.8 Hz, 1H).
MS-EI m/z 339 [M]'.
Example 173: 3-(1H-Indol-2-ylmethylene)-2-oxo-2,3-dihydro-
1H-indole-5-sulfonic acid methvlamide
1HNMR (300 MHz, DMSO-d6) 8 12.75 (br, s, 1H, NH), 11.40
(br, s, 1H, NH), 8.15 (s, 1H, H-vinyl), 8.10 (d, J = 1.5
Hz, 1H), 7.54-7.65 (m, 3H), 7.20-7.27 (m, 3H), 7.02-7.06
(m, 2H), 2.38 (d, J = 4.8 Hz, 3H, CH3).
MS-EI m/z 353 [M]'.
Example 174: 3-(1H-Indol-3-ylmethylene)-2-oxo-2,3-dihydro-
1H-indole-5-carboxylic acid
1HNMR (300 MHz, DMSO-d6) 8 12.13 (br, s, 1H, NH), 10.88
(br, s, 1H, NH), 8.22-8.50 (m, 2H), 7.66-7.94 (m, 2H), 7.52
(m, 1H) , 7.23 (m, 2H) , 6. 94 (m, 1H) .
MS-EI m/z 304 [M]'.
Example 175: 3-(3-Methyl-1H-indol-2-ylmethylene)-2-oxo-
2,3-dihydro-1H-indole-5-sulfonic acid amide
1HNMR (360 MHz, DMSO-d6) 8 12.92 (br, s, 1H, NH) , 11.35
(br, s, 1H, NH), 8.36 (d, J = 2.2 Hz, 1H), 8.03 (s, 1H, H-
vinyl), 7.67 (m, 2H), 7.53 (d, J = 7.6 Hz, 1H), 7.30 (m,
1H), 7.16 (s, 2H, NHz), 7.08 (m, 1H), 7.03 (d, J = 8.6 Hz,
1H) , 2.63 (s, 3H, CH3) .
MS-EI m/z 353 [M] ' .
Example 176: 3-(1H-Indol-5-vlmethylene)-2-oxo-2,3-dihvdro-
1H-indole-5-sulfonic acid methylamide
1HNMR (300 MHz, DMSO-d6) 8 11.38 (br, s, 1H, NH), 10.85
(br, s, 1H, NH), 8.11 (m, 1H), 8.01 (m, 1H), 7.90 (m, 1H),
7.78 (m, 1H), 7.37-7.53 (m, 3H), 7.10 (m, 1H), 6.95 (m,
1H) , 6.4 (m, 1H) , 2 .28 (s, 3H, CH3) .
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MS-EI m/z 353 [M]'.
Example 177: 3-(1H-Indol-5-ylmethylene)-2-oxo-2,3-dihydro-
1H-indole-5-carboxylic acid
1HNMR (300 MHz, DMSO-d6) 8 12.54 (br, s, 1H, NH), 10.86
(br, s, 1H, NH), 8.56, 7.76, 7.46, 6.91 (m, 9H).
Example 178: 3-(1H-Indol-3 ylmethylene)-2-oxo-2,3-dihydro-
1H-indole-5-sulfonic acid methylamide
1HNMR (300 MHz, DMSO-d6) 8 12.03 (br, s, 1H, NH), 10.84
(br, s, 1H, NH), 9.39 (s, 1H), 8.25 (s, 1H, H-vinyl), 8.22
(d, J = 1.5 Hz, 1H), 8.16 (m, 1H), 7.40-7.46 (m, 2H), 7.06-
7.15 (m, 2H) , 7. 04 (m, 1H, CH3NH) , 6.88 (d, J = 7 . 8 Hz, 1H) ,
2.32 (d, J = 5.1 Hz, 3H, CH3) .
MS-EI m/z 355 [M]'.
Example 179: 5-Amino-3-(1H-indol-5 ylmethylene)-1,3-
dihydro-indol-2-one
MS-EI m/z 275 [M]'.
Example 180: 5-Amino-3-(3-methyl-1H-indol-2-vlmethylene)-
1~3-dih~rdroindol-2-one
1HNMR (300 MHz, DMSO-d6) 8 13.13 (br, s, 1H, NH), 10.57
(br, s, 1H, NH), 7.63 (s, 1H, H-vinyl), 7.61 (d, J = 7.6
Hz, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.25 (t, J = 7.6 Hz,
1H), 7.03-7.06 (m, 2H), 6.61 (d, J = 8.3 Hz, 1H), 6.49 (dd,
J = 1.4 & 8.3 Hz, 1H) , 4.70 (br s, 2H, NHZ) , 2.55 (s, 3H,
CH3 ) .
MS-EI m/z 289 [M]'.
Example 182: 3-(2-Methyl-1H-indol-3-ylmethylene)-2-oxo-
2,3-dihydro-1H-indole-5-carboxylic acid
1HNMR (300 MHz, DMSO-d6) b 12.0 (br, s, 1H, NH), 10.85
(br, s, 1H, NH), 7.90 (s, 1H, H-vinyl), 7.79 (dd, 1H),
7.41-7.44 (m, 2H), 7.02-7.18 (m, 3H), 6.91 (d, 1H), 2.45
(s, 3H, CH3) .
MS-EI m/z 318 [M]'.
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Example 183: 3-(1H-Indol-5=ylmethylene)-2-oxo-2,3-dihydro-
1H-indole-5-sulfonic acid dimethylamide
MS-EI m/z 367 [M]'.
Example 184: 3-(2-Methyl-1H-indol-3-ylmethylene)-2-oxo-
2,3-dihydro-1H-indole-5-sulfonic acid dimethvlamide
1HNMR (300 MHz, DMSO-d6) b 11.95 (br, s, 1H, NH), 10.86
(br, s, 1H, NH), 7.84 (s, 1H, H-vinyl), 7.41 (dd, J = 1.8 &
8.4 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.04 (m, 1H), 6.85-
6.95 (m, 4H) .
MS-EI m/z 3 81 [M] ' .
Example 185: 3-(1H-Indol-5-vlmethylene)-2-oxo-2,3-dihydro-
1H-indole-5-sulfonic acid amide
MS-EI m/z 339 [M]'.
Example 186: 3-(1H-Indol-2-vlmethvlene)-2-oxo-2,3-dihvdro-
1H-indole-5-sulfonic acid dimethylamide
1HNMR (360 MHz, DMSO-d6) ~ 12.80 (br, s, 1H, NH), 11.44
(br, s, 1H, NH), 8.30 (s, 1H, H-vinyl), 8.15 (d, J = 2.2
Hz, 1H), 7.70 (d, J = 7.2 Hz, 1H), 7.60 (m, 2H), 7.30 (m,
1H), 7.23 (s, 1H), 7.07-7.12 (m, 2H), 2.63 (s, 6H, 2xCH3).
MS-EI m/z 367 [M]'.
Example 187: 5-Amino-3-(2-methyl-1H-indol-3-ylmethylene)-
1 3-dihydroindol-2-one
MS-EI m/z 289 [M]'.
Example 188: 3-(1H-Indol-3-ylmethylene)-2-oxo-2,3-dihydro-
1H-indole-5-sulfonic acid dimethylamide
1HNMR (360 MHz, DMSO-d6) b 12.11 (br, s, 1H, NH), 10.94
(br, s, 1H, NH) , 9.50 (s, 1H) , 8.44 (s, 1H) , 8.33 (m, 2H) ,
7.51-7.53 (m, 2H), 7.25 (m, 2H), 7.03 (d, J = 7.9 Hz, 1H),
2.63 (s, 6H, 2xCH3) .
MS-EI m/z 367 [M]'.
Example 189: 3-(2-Methyl-1H-indol-3-ylmethylene)-2-oxo-
2 3-dihydro-1H-indole-5-sulfonic acid amide
MS-EI m/z 353 [M] ' .
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Example 190: 3-f2-(2-Oxo-1,2-dihydro-indol-3-
ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-t~ropionic
acid
1-(Morpholin-4-yl)cyclohexene (300 g), 214 g of
triethylamine and 1400 mL of dichloromethane were refluxed
for 15 minutes and then cooled in a water bath to 15-20 °C.
Ethyl succinyl chloride (266 g) dissolved in 500 mL of
dichloromethane was added over 30 minutes. The mixture was
refluxed for 30 minutes and cooled to ambient temperature
in a water bath. The solid was collected by vacuum
filtration, washed with 100 mL of dichloromethane and
discarded. The filtrate was returned to the flask and the
solvent removed by distillation to give 454 g of crude 4-
(2-morpholin-4-yl-cyclohex-1-enyl)-4-oxo-butyric acid ethyl
ester as an oil.
Crude 4-(2-morpholin-4-ylcyclohex-1-enyl)-4-oxo-
butyric acid ethyl ester (454 g), 398 g of diethyl
aminomalonate hydrochloride, 162 g of sodium acetate and
350 mL of glacial acetic acid were heated to 108°C over 30
minutes. The mixture was held at 100-108 °C for 2 hours
and then cooled to about 50°C in a water bath. Water (2500
mL) and 700 mL of ethyl acetate were added. The ethyl
acetate layer was separated and washed three times with
brine, twice with saturated sodium bicarbonate solutiona
and once with brine. The solution was dried over anhydrous
sodium sulfate, and the ethyl acetate was removed using a
rotary evaporator to give 494 g (105% yield) of crude 3-(2-
ethoxycarbonylethyl)-4,5,6,7-tetrahydro-1H-indole-2-
carboxylic acid ethyl ester as an oil. The crude mixture
was chromatographed on a silica gel column using a 1:10
mixture of ethyl acetate: hexane as the eluent to give 122.1
g of pure 3-(2-ethoxycarbonylethyl)-4,5,6,7-tetrahydro-1H-
indole-2-carboxylic acid ethyl ester as a low melting
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solid.
1H NMR (ds-DMSO): ~ 11.0 (s, 1H, pyrrole NH), 4.2, 4.0
( t , each 4H, COCHZ ) , 2 . 8 , 2 . 4 ( t , each 4H, -CHzCH2C0- ) , 2 . 4
(m, 4H, -CH2- , -CHZ- ) , 1 . 7 (m, 4H, -CHzCHz- ) .
Purified 3-(2-ethoxycarbonylethyl)-4,5,6,7-tetrahydro-
1H-indole-2-carboxylic acid ethyl ester (122.1 g) and 328
mL of 5N sodium hydroxide were refluxed for 80 minutes.
The heat was turned off and 165 mL of 10 N hydrochloric
acid was carefully added to the vigorously stirred mixture
using an addition funnel over the reflux condenser.
Hydrochloric acid was added until the pH was 2-3. The
mixture was then cooled in an ice bath at which time the
oil in the mixture solidified. The solid was collected by
vacuum filtration, washed 3 times with water and dried
under vacuum at 50-60°C to give 54.9 g (71% yield) of 3-
(4,5,6,7-tetrahydro-1H-indol-3-yl)-propionic acid as a dark
brown solid.
1H NMR (d6-DMSO) : b 13.1 (s, 1H, pyrrole NH) , 11.8 (br
s, 1H, COOH), 9.8 (s, 1H, CH), 2.5, 2.3 (t, 4H, -CHzCH2C0-),
2.4 (m, 4H, -CHZ-, -CH2-) , 1.7 (m, 4H, -CHZCHz-) .
A mixture of 24 g of dimethylformamide and 300 mL of
dichloromethane was cooled to -9°C. Phosphorus oxychloride
(50 g) was rapidly added via an addition funnel. 3-
(4,5,6,7-tetrahydro-1H-indol-3-yl)-propionic acid (54.9 g)
was added in portions with vigorous stirring. The mixture
was warmed to room temperature and then refluxed for 10
minutes. It was then cooled to 5 °C and diluted with 300
mL of water. The pH was adjusted to 10 using lON sodium
hydroxide. The layers were separated. The aqueous layer
was cooled to 10°C and the pH adjusted to 2-3 with about
130 mL of 10 N hydrochloric acid. The oil which formed
solidified and was collected by vacuum filtration, washed
three times with water and dried under vacuum at 50°C to
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give 52.8 g (93% yield) of 3-(2-formyl-4,5,6,7-tetrahydrc-
1H-indol-3-yl)-propionic acid as a dark brown solid.
1H NMR (d6-DMSO): b 13.1 (s, 1H, pyrrole NH), 11.7 (br
s, 1H, COOH), 9.4 (s, 1H, CHO), 2.8, 2.5(t, 4H, -CHzCH~CO-),
2.4 (m, 4H, -CH2-, -CHz-) , 1.7 (m, 4H, -CH2CH~-) .
3-(2-Formyl-4,5,6,7-tetrahydro-1H-indol-3-yl)-propicnic
acid (5.4 g), 3.6 g of 2-oxindole and 2.7 g of piperidine (or
2.2 g of pyrrolidine) in 25 mL of ethanol were refluxed for 4
hours. Upon slow addition of acetic acid (8 mL), a
precipitate formed. The mixture was refluxed for 5 minutes
and cooled to ambient temperature. The precipitate was
collected by vacuum filtration and washed with 20 mL of
ethanol. The solids were slurry-washed refluxing in 30 r~.L of
ethanol, cooled, collected by vacuum filtration, washed with
30 mL of ethanol and dried under vacuum to give 5.5 g (68%
yield) of 3-[2-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-
4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic acid as an orange
solid: mp 263-265 °C.
1H NMR (d6-DMSO): 8 13.1 (s, 1H, pyrrole NH), 12.0 (br s,
1H, COOH), 10.7 (s, 1H, CONH), 7.7, 7.1, 6.9, 6.8 (m, each
4H, aromatic), 7.6 (s, 1H, -CH=), 2.9, 2.7 (t, each 4H, -
CH2CH2C0-) , 2.4 (m, 4H, -CH2-, -CHZ-) , 1.7 (m, 4H, -CH~CH2-
Example 191: 3- 2-(5-Chloro-2-oxo-1,2-dihydroindol-3-
ylidenemethvl)-4,5,6,7-tetrahydro-1H-indol-3-yll-propionic
acid
3-(2-Formyl-4,5,6,7-tetrahydro-1H-indol-3-yl)-
propionic acid (5.4 g), 3.7 g of 5-chloro-2-oxindole and
2.7 g of piperidine (or 2.2 g of pyrrolidine) in 25 mL cf
ethanol was refluxed for 4 hours. Slow addition of acetic
acid (8 mL) resulted in a precipitate. The mixture was
refluxed for 5 minutes and cooled to ambient temperature.
The precipitate was collected by vacuum filtration and
washed with 20 mL of ethanol. The solids were slurry-
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washed by refluxing in 30 mL of ethanol, cooled, collected
by vacuum filtration, washed with 30 mL of ethanol and
dried under high vacuum to give 6.5 g (80% yield) 3-[2-(5-
chloro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-
tetrahydro-1-H-indol-3-yl]-propionic acid as an orange
solid: mp 370-384 °C.
1H NMR (d6-DMSO) : b 13.3 (s, 1H, pyrrole NH) , 12.0 (br
s, 1H, COOH), 10.7 (s, 1H, CONH), 7.8, 7.1, 6.8 (m, each
3H, aromatic), 7.7 (s, 1H, -CH=), 2.9, 2.7 (t, each 4H,
CH2CH2C0) , 2.5 (m, 4H, -CHZ-, -CH2-) , 1.7 (m, 4H, -CHzCHz-) .
Example 192: 3-f2-(5-Bromo-2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic
acid
A mixture of 3-(2-Formyl-4,5,6,7-tetrahydro-1H-indol-
3-yl)-propionic acid (3.4 g), 2.7 g of 5-bromo-2-oxindole
and 1.4 g of pyrrolidine in 25 mL of ethanol was refluxed
for 4 hours. Upon slow addition of acetic acid (8 mL), a
precipitate formed. The mixture was refluxed for 5 minutes
and cooled to ambient temperature. The precipitate was
collected by vacuum filtration and washed with 20 mL of
ethanol. The solids were slurry-washed by refluxing in 30
mL of ethanol, cooled, collected by vacuum filtration,
washed with 30 mL of ethanol and dried under vacuum to give
5.2 g (98% yield) of 3-[2-(5-bromo-2-oxo-1,2-dihydroindol-
3-ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-
propionic acid as a red-orange solid, mp 286-289 °C.
1H NMR (d6-DMSO): 8 13.3 (s, 1H, pyrrole NH), 12.0 (br
s, 1H, COOH), 10.7 (s, 1H, CONH), 7.9, 7.1, 6.8 (m, each
3H, aromatic), 7.7 (s, 1H, -CH=), 2.9, 2.7 (t, each 4H, -
CHZCHzCO-) , 2.5 (m, 4H, -CHz-, -CHz-) , 1.7 (m, 4H, -CH2CH2-) .
Example 193: 3-[2-(4-Methyl-2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-4,5,6 7-tetrahydro-1H-indol-3-yl~-propionic
acid
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Diethyl oxalate (30 mL) in 20 mL of dry ether was
added with stirring to 19 g of potassium ethoxide suspended
in 50 mL of dry ether. The mixture was cooled in an ice
bath and 20 mL of 3-nitro-o-xylene in 20 mL of dry ether
was slowly added. The thick dark red mixture was refluxed
for 0.5 hr, concentrated, and treated with 10% sodium
hydroxide until almost all of the solid dissolved.
Hydrogen peroxide (30%) was then added to the dark red
mixture until the red color changed to yellow. The mixture
was treated alternately with 10% sodium hydroxide and 30%
hydrogen peroxide until the dark red color was no longer
present. The solid was filtered off and the filtrate
acidified with 6 N hydrochloric acid. The resulting
precipitate was collected by vacuum filtration, washed with
water, and dried under vacuum to give 9.8 g (45% yield) of
1-methyl-6-nitrophenylacetic acid as an off-white solid.
The solid was hydrogenated in methanol over 10% palladium
on carbon to give 9.04 g of 4-methyl-2-oxindole as a white
solid.
1H NMR (d6-DMSO, 360 MHz) b 10.27 (s, br, 1H, NH-1) ,
7.06 (t, 7.71 Hz, 1H, H-6), 6.74 (d, 7.73 Hz, H-5), 6.63
(d, 7.73 Hz, 1H, H-7), 3.36 (s, 2H, CH2), 2.18 (s, 3H, CH3).
A mixture of 3-(2-formyl-4,5,6,7-tetrahydro-1H-indol
3-yl)-propionic acid (5.4 g), 3.2 g of 4-methyl-2-oxindole
and 2.7 g of piperidine in 25 mL of ethanol was refluxed
for 4 hours. Upon slow addition of acetic acid (8 mL), a
precipitate formed. The mixture was refluxed for 5 minutes
and cooled to ambient temperature. The precipitate was
collected by vacuum filtration and washed with 20 mL of
ethanol. The solids were slurry-washed in 30 mL of
refluxing ethanol, cooled, collected by vacuum filtration,
washed with 30 mL of ethanol and dried under vacuum to give
6.2 g (80% yield) of 3-[2-(4-methyl-2-oxo-1,2-dihydroindol-
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3-ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-
propionic acid as an orange solid.
1H NMR (d6-DMSO): 8 13.3 (s, 1H, pyrrole NH), 12.0 (br
s, 1H, COOH), 10.7 (s, 1H, CONH), 7.7 (s, 1H, -CH=), 7.0,
6.8 (m, each 2H, aromatic), 2.8, 2.7 (t, each 4H, -CH2CHzC0-
2.6 (s, 1H, CH3) , 2 .5 (m, 4H, -CHZ-, -CH2-) , 1 .7 (m, 4H,
-CH2CH2-).
Example 194: 3-[2-(5-Methyl-2-oxo-1,2-dihydroindol-3-
xlidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yll-propionic
acid
A mixture of 3-(2-formyl-4,5,6,7-tetrahydro-1H-indol-
3-yl)-propionic acid (5.4 g), 3.2 g of 5-methyl-2-oxindole
and 2.7 g of piperidine in 25 mL of ethanol was refluxed
for 4 hours. Upon addition of acetic acid (8 mL), a
precipitate formed. The mixture was refluxed for 5 minutes
and cooled to ambient temperature. The precipitate was
collected by vacuum filtration and washed with 20 mL of
ethanol. The solids were slurry-washed with 30 mL of
refluxing ethanol, cooled, collected by vacuum filtration,
washed with 30 mL of ethanol and vacuum dried to give 6.2 g
(80% yield) of 3-[2-(5-methyl-2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic
acid as an orange solid.
1H NMR (d6-DMSO): 8 13.3 (s, 1H, pyrrole NH), 12.0 (br s,
1H, COOH), 10.7 (s, 1H, CONH), 7.7 (s, 1H, -CH=), 7.0, 6.8
(m, each 3H aromatic), 2.8, 2.7 (t, each 4H, -CHzCH2C0-), 2.6
(s, 1H, CH3) , 2.5 (m, 4H, -CH2-, -CH2-) , 1.7 (m, 4H, -CHZCH2-) .
MS: m/z 349.
Example 195: 3 [2-(6-Chloro-2-oxo-1,2-dihydroindol-3-
ylidenemethyl) 4,5 6,7-tetrahydro-1H-indol-3-yl]-propionic
acid
A mixture of 3-(2-formyl-4,5,6,7-tetrahydro-1H-indol-
3-yl)-propionic acid (5.4 g), 3.7 g of 6-chloro-2-oxindole
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and 2.7 g of piperidine in 25 mL of ethanol was refluxed
for 4 hours. Upon slow addition of acetic acid (8 mL), a
precipitate formed. The mixture was refluxed for 5 minutes
and cooled to ambient temperature. The precipitate was
collected by vacuum filtration and washed with 20 mL of
ethanol. The solids were slurry-washed in 30 mL of
reflusing ethanol, cooled, collected by vacuum filtration,
washed with 30 mL of ethanol and vacuum dried to give 6.5 g
(80%) of 3-[2-(6-chloro-2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic
acid as an orange solid.
1H NMR (d6-DMSO) : 8 13.3 (s, 1H, pyrrole NH) , 12.0 (br
s, 1H, COOH), 10.7 (s, 1H, CONH), 7.7, 7.0, 6.9 (m, each
3H, aromatic), 7.6 (s, 1H, -CH=), 2.9, 2.7 (t, each 4H, -
CHzCH2C0- ) , 2 . 4 (m, 4H, -CH2- , -CH2- ) , 1 . 7 (m, 4H, -CH2CH2- ) .
MS: m/z 371.
Example 196: 3-[2-(6-Methox~r-2-oxo-1,2-dihydroindol-3-
ylidenemethyl) 4,5,6,7-tetrahydro-1H-indol-3-yl~-propionic
acid
A mixture of 3-(2-formyl-4,5,6,7-tetrahydro-1H-indol-
3-yl)-propionic acid (5.4 g), 3.6 g of 6-methoxy-2-oxindole
and 2.7 g of piperidine in 25 mL of ethanol was refluxed
for 4 hours. Upon slow addition of acetic acid (8 mL), a
precipitate formed. The mixture was refluxed for 5 minutes
and cooled to ambient temperature. The precipitate was
collected by vacuum filtration and washed with 20 mL of
ethanol. The solids were slurry-washed in 30 mL of
reflusing ethanol, cooled, collected by vacuum filtration,
washed with 30 mL of ethanol and vacuum dried to give 6.4 g
(80% yield) of 3-[2-(6-methoxy-2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic
acid as an orange solid, mp 263-266 °C.
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1H NMR (ds-DMSO) : 8 13 . 0 (s, 1 H, pyrrole NH) , 12 . 0 (s,
1H, COOH), 10.7 (s, 1H, CONH), 7.6, 6.5, 6.4 (m, each 3H,
aromatic), 7.4 (s, 1H, -CH=), 3.7 (s, 3H, CH3), 2.9, 2.7 (t,
each 4H, -CHzCH2C0- ) , 2 . 5 (m, 4H -CHZ- , -CH2- ) , 1 . 7 (m, 4H,
-CHzCH2- ) .
MS: m/z 365.
Example 197: N,N-Dimethyl-3-f2-(2-oxo-1,2-dihydroindol-3-
~lidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yll-
propionamide
3-[2-(2-Oxo-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-
tetrahydro-1H-indol-3-yl]-propionic acid (10 g ) was
dissolved in 100 mL of dimethylformamide.
Carbonyldiimidazole (6.3 g) was added and the mixture
stirred at ambient temperature for 1 hour. Dimethylamine
(2.7 g) and 30 mL of dimethylformamide were added and the
stirring continued overnight at room temperature. Fifty mL
of water was added to the mixture and stirring was
continued for 10 minutes. The precipitate was collected by
vacuum filtration, washed with 20 mL of water and then 20
mL of ethanol. The solid was slurry-washed in 30 mL of
refluxing ethanol for 5 minutes and cooled to room
temperature. The solid was collected by vacuum filtration,
washed with 20 mL of ethanol and vacuum dried to give 8.9 g
(83% yield) of N,N-dimethyl-3-[2-(2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-
propionamide.
1H NMR (d6-DMSO): 8 13.1 (s, 1H, pyrrole NH), 10.7 (s,
1H, CONH), 7.7, 7.1, 6.9, 6.8 (m, each 4H, aromatic), 7.6
(s, 1H, -CH=), 3.3 (s, 6H, CHj), 2.9, 2.7 (t, each 4H, -
CH2CHZC0-) , 2.5 (m, 4H -CH2-, -CHz-) , 1.7 (m, 4H, -CH2CHz-) .
MS: m/z 364.
Example 198: 3-(3-(3-Dimethylaminopropyl)-4,5,6,7-
tetrahydro-1H-indol-2 ylmethvlene~-1,3-dihvdroindol-2-one
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3-(4,5,6,7-tetrahydro-1H-indol-3-yl)-propionic acid
(9.7 g) in 100 mL of tetrahydrofuran was stirred with 8.1 g
of carbonyl diimidazole for 1 hour. Dimethylamine (2.5 g)
was added and the mixture stirred for 2 hours. The solvent
was evaporated and the residue taken up in ethyl acetate,
washed with water, 0.1 N hydrochloric acid, water, and
brine, dried over sodium sulfate and evaporated to give 7.7
g (70% yield) of 3-(4,5,6,7-tetrahydro-1H-indol-3-yl)-
propionic acid dimethyl amide.
3-(4,5,6,7-Tetrahydro-1H-indol-3-yl)-propionic acid
dimethyl amide (7.7 g) and 13 g of borane-tetrahydrofuran
complex in 50 mL of tetrahydrofuran was refluxed for 3
hours. The reaction was quenched with acetone and then
water and evaporated to dryness. The residue was
chromatographed on silica gel to give 2 g (20% yield) of
3-(3-dimethylaminopropyl)-4,5,6,7-tetrahydro-1H-indole as a
yellow oil.
Dimethylformamide (0.8 g) and 13 mL of dichloromethane
was cooled to -9 °C. Phosphorus oxychloride (1.7 g) was
rapidly added via a dropping funnel. 3-(3-dimethylamino-
propyl)-4,5,6,7-tetrahydro-1H-indole (2 g) was added in
portions with vigorous stirring. The mixture was warmed to
room temperature and then refluxed for 10 minutes. It was
then cooled to 5°C, and diluted with 20 mL of water. The
pH was adjusted to 10 with lON sodium hydroxide. The
layers were separated. The organic layer was washed with
water and brine, dried over anhydrous sodium sulfate and
evaporated to give 1.8 g (80%) of 3-(3-
dimethylaminopropyl)-2-formyl-4,5,6,7-tetrahydro-1H-indole
as a dark oil which solidified.
3-(3-Dimethylaminopropyl)-2-formyl-4,5,6,7-tetrahydro-
1H-indole (1.8 g), 1 g of 2-oxindole and 0.1 g of
piperidine in 10 mL of ethanol was refluxed for 4 hours and
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then cooled to room temperature. The precipitate which
formed was collected by vacuum filtration and washed with 4
mL of ethanol. The solids were slurry-washed with 8 mL of
refluxing ethanol, cooled, collected by vacuum filtration,
washed with 3 mL of ethanol and vacuum dried to give 1.8 g
(70% yield) of 3-[3-(3-dimethylaminopropyl)-4,5,6,7-
tetrahydro-1H-indol-2-ylmethylene]-1,3-dihydroindol-2-one
as an orange solid.
1H NMR (d6-DMSO): 8 13.1 (s, 1H, pyrrole NH), 10.7 (s,
1H, CONH), 7.6, 7.1, 7.0, 6.8 (m, each 4H, aromatic), 7.6
(s, 1H, -CH=) , 3 .3 (s, 6H, CH3) , 2.7 (m, 4H, -CH2-, -CHZ-) ,
2 . 5 , 2 . 3 , 1 . 4 ( t , each 6H, -CH2CHZCHzN- ) , 1 . 7 (m, 4H, -
CHZCH2-) .
MS: m/z 350.
Example 199: 3-(2-(2-Oxo-1,2-dihydroindol-3-
ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-vl]-
propionamide
3-[2-(2-Oxo-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-
tetrahydro-1H-indol-3-yl]-propionic acid (10 g) was
dissolved in 100 mL of dimethylformamide.
Carbonyldiimidazole (6.3 g) was added and the mixture
stirred at ambient temperature for 1 hour. Ammonia (1 g)
in 30 mL of dimethylformamide was added and the stirring
continued overnight. Fifty mL of water was added to the
mixture and stirring was continued for 10 minutes. The
precipitate that formed was collected by vacuum filtration,
washed with 20 mL of water and then 20 mL of ethanol. The
solid was slurry-washed with 30 mL of refluxing ethanol for
5 minutes and cooled to room temperature. The solid was
collected by vacuum filtration, washed with 20 mL of
ethanol and vacuum dried to give 11 g (83% yield) of 3-[2-
(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-
tetrahydro-1H-indol-3-yl]-propionamide.
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'H NMR (d6-DMSO) : 8 13.3 (s, 1H, pyrrole NH) , 12.0 (br
s, 1H, COOH), 10.7 (s, 1H, CONH), 7.6, 7.1, 6.9, 6.8 (m,
each 4H, aromatic), 7.6 (s, 1H, -CH=), 7.2, 6.7 (s, each
2H, NHZ) , 2.9, 2.7 (t, 4H, -CH~CHZCO-) , 2.4 (m, 4H, -CHz-,
-CH2-) , 1.7 (m, 4H, -CH~CH2-) .
MS: m/z 336.
Example 200: 3-f3-(3-Morpholin-4-vl-3-oxopropyl)-4,5,6,7-
tetrahydro-1H-indol-2-ylmethylene~-1,3-dihydroindol-2-one
3-[2-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-
tetrahydro-1H-indol-3-yl]-propionic acid (10 g) was
dissolved in 100 mL of dimethylformamide.
Carbonyldiimidazole (6.3 g) was added and the mixture
stirred at ambient temperature for 1 hour. Morpholine (5.2
g) in 30 mL of dimethylformamide was added and the stirring
continued overnight. Fifty mL of water was added to the
mixture and stirring was continued for 10 minutes. The
precipitate was collected by vacuum filtration, washed with
mL of water and then 20 mL of ethanol. The solid that
formed was slurry-washed with 30 mL of refluxing ethanol
20 for 5 minutes and cooled to room temperature. The solid
was collected by vacuum filtration, washed with 20 mL of
ethanol and vacuum dried to give 9.6 g (80%) of 3-[3-(3-
morpholin-4-yl-3-oxopropyl)-4,5,6,7-tetrahydro-1H-indol-2-
ylmethylene]-1,3-dihydroindol-2-one as an orange solid.
1H NMR (d6-DMSO): 8 13.3 (s, 1H, pyrrole NH), 12.0 (br
s, 1H, COOH), 10.7 (s, 1H, CONH), 7.6, 7.1, 6.9, 6.8 (m,
each 4H, aromatic), 7.6 (s, 1H, -CH=), 3.3 (multipets, 8H,
morpholine CH2) , 2.9, 2.7 (t, each 4H, -CHzCH2C0-) , 2.5 (m,
4H, CH2, CHZ) , 1.7 (m, 4H, -CHZCH2-) .
MS: m/z 406.
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Example 201: N-Methyl-3-(2-(2-oxo-1,2-dihydroindol-3-
~lidenemeth~l)-4,5,6,7-tetrahydro-1H-indol-3-yll-
propionamide
3-[2-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-
tetrahydro-1H-indol-3-yl]-propionic acid (10 g) was
dissolved in 100 mL of dimethylformamide.
Carbonyldiimidazole (6.3 g) was added and the mixture
stirred at ambient temperature for 1 hour. Methyl amine
(1.8 g) in 30 mL of dimethylformamide was added and the
stirring continued overnight. Fifty mL of water was added
to the mixture and stirring was continued for 10 minutes.
The precipitate was collected by vacuum filtration, washed
with 20 mL of water and then 20 mL of ethanol. The solid
was slurry-washed in 30 mL of refluxing ethanol for 5
minutes and cooled to room temperature. The solid was
collected by vacuum filtration, washed with 20 mL of
ethanol and vacuum dried to give 8.3 g (80% yield) of N-
methyl-3-[2-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-
4,5,6,7-tetrahydro-1H-indol-3-yl]-propionamide as an orange
solid.
MS: m/z 350.
Example 202: N (2 Morpholin-4-ylethvl)-3-(2-(2-oxo-1,2-
dihydroindol 3 ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-
3-yl]-propionamide
3-[2-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-
tetrahydro-1H-indol-3-yl]-propionic acid (10 g) was
dissolved in 100 mL of dimethylformamide.
Carbonyldiimidazole (6.3 g) was added and the mixture
stirred at ambient temperature for 1 hour. 4-(2-
Aminoethyl)morpholine (7.7 g) and 30 mL of
dimethylformamide were added and the stirring continued
overnight. Fifty mL of water was added to the mixture and
stirring was continued for 10 minutes. The precipitate was
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collected by vacuum filtration, washed with 20 mL of water
and then 20 mL of ethanol. The solid was slurry-washed in
30 mL of refluxing ethanol for 5 minutes and cooled to room
temperature. The solid was collected by vacuum filtration,
washed with 20 mL of ethanol and vacuum dried to give 1 g
(83%) of N-(2-morpholin-4-ylethyl)-3-[2-(2-oxo-1,2-dihydro-
indol-3-ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-
propionamide, mp 256-258 °C.
1H NMR (d6-DMSO) : 8 13 .1 (s, 1H, pyrrole NH) , 10.7 (s,
1H, -CONH-), 7.7 (t, 1H, -CONHCHz-), 7.6, 7.1, 6.9, 6.8 (m,
each 4H, aromatic), 7.5 (s, 1H, -CH=), 3.5 (t, each 4H,
-CHzCH2-) , 3 .1, 2.8 (m, each 2H, -CHzNCH2-) , 2.7, 2.5 (t,
each 4H, -CH2CH2C0- ) , 2 . 3 , 2 . 2 (m, each 4H, -NHCH~CHzN- ) , 2 . 2
(m, 4H, -CHz- , -CHz- ) , 1 . 7 (m, 4H, -CHzCH2- ) .
Example 203: 3-f2-(2-Oxo-1,2-dihydropyrrolof2,3-
blQyridin-3-ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-
yll-propionic acid
A mixture of 7-azaoxindole (99 mg), 110 mg of 3-(2-
formyl-4,5,6,7-tetrahydro-1H-indol-3-yl)-propionic acid and
2 drops of piperidine in 2 mL of ethanol was refluxed for 5
h. The reaction mixture was cooled and concentrated. The
residue was acidified with acetic acid to pH 6. The
resulting precipitate was collected by vacuum filtration,
washed with water and dried in a vacuum oven at 40 °C to
give 25.4 mg (13% yield) of 3-[2-2-oxo-1,2-
dihydropyrrolo[2,3-b]pyridin-3-ylidenemethyl)-4,5,6,7-
tetrahydro-1H-indol-3-yl]-propionic acid.
MS: m/z 338.
Example 204: 3-,~2-f6-(3-Methoxynhenvl)-2-oxo-1,2-dihydro-
indol 3-ylidenemethyl]-4,5,6,7-tetrahydro-1H-indol-3-yl~-
propionic acid
A mixture of 103 mg 6-(3-methoxyphenyl)-2-oxindole, 95
mg 3-(2-formyl-4,5,6,7-tetrahydro-1H-indol-3-yl)-propionic
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acid and piperidine (3 drops) in ethanol (2 mL) was heated
in sealed tube to 90°C and held there overnight. The
reaction mixture was concentrated and acidified with 6 N
hydrochloric acid. The precipitate that formed was
collected by filtration and washed with water and hexane to
give 156 mg of 3-{2-[6-(3-methoxyphenyl)-2-oxo-1,2-
dihydroindol-3-ylidenemethyl]-4,5,6,7-tetrahydro-1H-indol-
3-yl~-propionic acid as a brown solid (82%).
1H NMR (d6-DMSO, 360 MHz) : 8 13.26 (s, br, 1H, NH-1)
1.78 (s, br, 1H, NH-1), 7.72 (d, 8.1 Hz, 1H, H-4), 7.65 (s,
1H, H-vinyl), 7.35 (d, 7.9 Hz, 1H), 7.26 (dd, 1.3 Hz, 8.1
Hz, 1H, H-5), 7.18 (d, 7.9 Hz 1H), 7.13 (t, 2.0 Hz, 1H ),
7.09 (d, 1.3 Hz, 1H, H-7) , 6.90 (dd" 2.0 Hz, 1H) , 3.82 (s,
3H, OCH3) , 2. 91 (t, 7.4 Hz, 2H, CHZCH2COOH) , 2 .66 (t, 5.9
Hz, 2H, H-7'), 2.38-2.46 (m, 4H, CH2CHzCOOH and H-4'), 1.69-
1.76 (m, 4H, H-5',6'). MS: m/z 443.2.
Example 205: 3-~2-f6-(4-Methoxynhenyl)-2-oxo-1,2-dihvdro-
indol-3-ylidenemethyll-4,5,6,7-tetrahvdro-1H-indol-3-yl~-
propionic acid
A mixture of 103 mg 6-(4-methoxyphenyl)-2-oxindole, 95
mg 3-(2-formyl-4,5,6,7-tetrahydro-1H-indol-3-yl)-propionic
acid and piperidine (3 drops) in ethanol (2 mL) was heated
to 90°C in a sealed tube and held there for 4 hrs. The
reaction mixture was concentrated and acidified with 6 N
hydrochloric acid. Ethyl acetate was added upon which a
solid precipitated from the aqueous layer. The precipitate
was collected by filtration, and washed with water and
hexane to give 57 mg of 3-(2-[6-(4-Methoxyphenyl)-2-oxo-
1,2-dihydroindol-3-ylidenemethyl]-4,5,6,7-tetrahydro-1H-
indol-3-yl}-propionic acid as a brown solid (30%).
1H NMR (d6-DMSO, 360 MHz): 8 13.24 (s, br, 1H, NH-1),
11.61 (s, br, 1H, COOH), 10.76 (s, br, 1H, NH-1), 7. (d,
8.1 Hz, 1H, H-4), 7.61 (s, 1H, H-vinyl), 7.56 (d, 8.8 Hz,
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2H, H-3, 5), 7.21 (dd, 1.5 Hz, 8.1 Hz, 1H, H-5), 7.04 (d,
J = 1.5 Hz, 1H, H-7), 7.01 (d, 8.8 Hz, 2H, H-2",6" ), 3.79
(s, 3H, OCHj) , 2.91 (t, 7.4 Hz, 2H, CH2CH2COOH ) , 2.67 (t,
5.9 Hz, 2H, H-7'), 2.40-2.46 (m, 4H, CH2CHzCOOH and H-4'),
1.72-1.78 (m, 4H, H-5',6').
MS: m/z 441.2.
Example 206: 3-(2-(2-Oxo-6-phenyl-1,2-dihydroindol-3-
ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic
acid
A mixture of 90 mg 6-phenyl-2-oxindole, 95 mg 3-(2-
formyl-4,5,6,7-tetrahydro-1H-indol-3-yl)-propionic acid and
piperidine (3 drops) in ethanol (2 mL) was held in a sealed
tube at 90 °C for 4 hrs. The reaction mixture was
concentrated and acidified with 6 N hydrochloric acid. The
precipitate was collected by filtration and washed with
water and hexane to give 59 mg of 3-[2-(2-oxo-6-phenyl-1,2-
dihydro-indol-3-ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-
3-yl]-propionic acid as a brown solid (31% yield).
1H NMR (d6-DMSO, 360 MHz): 8 13.27 (s, br, 1H, NH-1'),
12.06 (s, v br, 1H, COOH), 10.80 (s, br, 1H, NH-1), 7.74
(d, 7.9 Hz, 1H, H-4), 7.64 (s, 1H, H-vinyl), 7.62 (d, 7.7
Hz, 2H), 7.44 (t, 7.7 Hz, 2H), 7.32 (dd, 7.7 Hz, 1H), 7.27
(dd, 1.1, 7.9 Hz 1H, H-5), 7.10 (d, 1.1 Hz, 1H, H-7), 2.92
(t, 7.3 Hz, 2H, CHZCH2COOH), 2.67 (t, 5.5 Hz, 2H, H-7'),
2.41-2.46 (m, 4H, CH2CHZCOOH and H-4'), 1.73-1.76 (m, 4H, H-
5' , 6' ) .
MS: m/z 411.2.
Example 207: 3-~2-f6-(2-Methoxyphenyl)-2-oxo-1,2-dihydro-
indol-3-ylidenemethyll-4,5,6,7-tetrahydro-1H-indol-3-yl~-
propionic acid
Tetrakis(triphenylphosphine)palladium (1 g) was added
to a mixture of 5 g of 2-methoxyphenylboronic acid, 6.6 g
of 5-bromo-2-fluoronitrobenzene and 30 mL of 2 M sodium
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carbonate solution in 50 mL of toluene and 50 mL of
ethanol. The mixture was refluxed for 2 hours,
concentrated, and the residue extracted twice with ethyl
acetate. The ethyl acetate layer was washed with water and
brine, dried, and concentrated to give a dark green oil
which solidified on standing to give crude 4-fluoro-2'-
methoxy-3-nitrobiphenyl.
Dimethyl malonate (14 mL) was added dropwise to 2.9 g
of sodium hydride suspended in 50 mL of dimethylsulfoxide.
The mixture was stirred at 100°C for 15 minutes and cooled
to room temperature. Crude 4-fluoro-2'-methoxy-3-
nitrobiphenyl in 60 mL of dimethylsulfoxide was added and
the mixture stirred at 100 °C for 2 hours. The reaction
mixture was cooled, quenched with 300 mL of saturated
ammonium chloride solution and extracted twice with ethyl
acetate. The extracts were combined, washed with saturated
ammonium chloride, water, and brine, dried over anhydrous
sodium sulfate and concentrated to give crude dimethyl 2'-
methoxy-3-nitrobiphenyl-4-malonate as a yellow oil.
Crude 2'-methoxy-3-nitrobiphenyl-4-malonate was
stirred at 100 °C in 50 mL of 6 N hydrochloric acid for 24
hours and cooled. The precipitate that formed was
collected by filtration, washed with water and hexane, and
dried to give 9.8 of 2'-methoxy-2-nitrobiphenyl-4-acetic
acid as a light tan solid.
Iron chips (5 g) were added in one portion to 9.8 g of
2'-methoxy-3-nitrobiphenyl-4-acetic acid in 50 mL of
glacial acetic acid and the mixture was stirred at 100 °C
for 3 hours. The reaction mixture was concentrated to
dryness, sonicated in ethyl acetate and filtered to remove
insolubles. The filtrate was washed twice with 1 N
hydrochloric acid, then with water and brine, dried over
anhydrous sodium sulfate and concentrated. The residue was
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chromatographed on silica gel using ethyl acetate: hexane
1:2 as the eluent to give 5.4 g (69% based on 5-bromo-2-
fluoronitrobenzene) of 6-(2-methoxyphenyl)-2-oxindole as a
rose colored solid.
A mixture of 103 mg of 6-(2-methoxyphenyl)-2-oxindole,
95 mg of 3-(2-formyl-4,5,6,7-tetrahydro-1H-indol-3-yl)-
propionic acid and piperidine (3 drops) in ethanol (2 mL)
was held in a sealed tube at 90 °C for 4 hrs. The reaction
mixture was concentrated and acidified with 6 N
hydrochloric acid. The precipitate was collected by
filtration and washed with water and hexane to give 67 mg
of 3-(2-[6-(2-methoxyphenyl)-2-oxo-1,2-dihydroindol-3-
ylidenemethyl]-4,5,6,7-tetrahydro-1H-indol-3-yl~-propionic
acid as a brown solid (35% yield).
1H NMR (d6-DMSO, 360 MHz): 8 13.26 (s, br, 1H, H1, 20,
yr H OH) 10.71 (s, br, 1H, NH-1), 7.67 (d, 7.7 Hz, 1H, H-
4), 7.61 (s, 1H, H-vinyl), 7.27-7.34 (m, 2H,), 7.01-7.10
(m, 2H), 7.05 (dd, 1.2, 7.7 Hz, 1H, H-5), 6.99 (d, 1.2 Hz,
1H, H-7), 3.75 (s, 3H, OCH3), 2.91 (t, 7.5 Hz, 2H,
CH2CH2COOH), 2.68 (t, 7 Hz, 2H, H-7'), 2.40-2.46 (m, 4H,
CHzCHzC00H and H-4' ) , 1 . 71-1 . 78 (m, 4H, .H-5' , 6' ) .
MS m/z 441.2.
Example 208: 3-f2-(5-Isopropylaminosulfonyl-2-oxo-1,2-
dihydroindol-3-vlidenemethvl)-4,5,6,7-tetrahydro-1H-indol-
3-yl]-propionic acid
To a 100 mL flask charged with 27 mL of chlorosulfonic
acid was slowly added 13.3 g of 2-oxindole. The reaction
temperature was maintained below 30 °C during the addition.
After the addition, the reaction mixture was stirred at
room temperature for 1.5 hr, heated to 68°C and stirred an
additional 1 hr, cooled, and poured into water. The
precipitate was washed with water and dried in a vacuum
oven to give 11.0 g of 5-chlorosulfonyl-2-oxindole (50%
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yield) which was used without further purification.
A suspension of 3 g of 5-chlorosulfonyl-2-oxindole,
1.15 g of isopropylamine and 1.2 mL of pyridine in 50 mL of
dichloromethane was stirred at room temperature for 4 hours
at which time a white solid was present. The solid was
collected by vacuum filtration, slurry-washed with hot
ethanol, cooled, collected by vacuum filtration and vacuum
dried at 40°C to give 1.5 g (45%) of 5-isopropylamino-
sulfonyl-2-oxindole.
1H NMR (d6-DMSO, 300 MHz) 8 10.69 (s, br, 1H, NH), 7.63
(dd, 1.8 Hz, 1H, H-6), 7.59 (d, 1 Hz, 1H, H-4), 7.32 (d, 7
Hz, 1H, NH_-SOz-), 6.93 (d, 8 Hz, 1H, H-7), 3.57 (s, 2H, H-
3) _, 3. 14-3 .23 (m, 1H, CH- (CH3) 2) , 0. 94 (d, 7 Hz, 6H, CH3) .
A mixture of (2-Formyl-4,5,6,7-tetrahydro-1H-indol-3-
yl)-propionic acid (5.4 g), 5.7 g of 5-isopropylamino-
sulfonyl-2-oxindole and 2.7 g of piperidine in 25 mL of
ethanol was refluxed for 4 hours. Upon addition of acetic
acid (8 mL), a precipitate formed. The mixture was
refluxed for 5 minutes and cooled to ambient temperature.
The precipitate was collected by vacuum filtration and
washed with 20 mL of ethanol. The solids were slurry-
washed in 30 mL of reflusing ethanol, cooled, collected by
vacuum filtration, washed with 30 mL of ethanol and vacuum
dried to give 8.1 g (80% yield) of 3-[2-(5-isopropylamino-
sulfonyl-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-
tetrahydro-1H-indol-3-yl]-propionic acid as an orange
solid.
1H NMR (d6-DMSO): b 13.3 (s, 1H, pyrrole NH), 12.0 (br
s, 1H, COOH), 10.7 (s, 1H, CONH), 8.1, 7.5, 7.3, 7.0 (m,
each 4H, aromatic), 7.7 (s, 1H, -CH=), 2.3 (m, 1H, -CH-),
2.9, 2.7 (t, each 4H, -CHZCH2C0-) , 2.4 (m, 4H, -CHz-, -CHz-) ,
1.7 (m, 4H, -CHZCHZ-) , 0.9 (d, 6H, CH3) .
MS m/z 458.
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Example 209: 3-I2-(6-Morpholin-4-yl-2-oxo-1,2-
dihvdroindol-3-ylidenemethyl)-4,5,6,7-tetrahvdro-1H-indol-
3-yl]-propionic acid
A mixture of 4 g of 6-(morpholin-4-yl)-2-oxindole,
3.75 g of 3-(2-formyl-4,5,6,7-tetrahydro-1H-indol-3-
yl)propionic acid, and 1.8 mL of piperidine in ethanol (60
mL) was refluxed for 6 hrs. The reaction mixture was
concentrated and acidified with 6 N hydrochloric acid to pH
6. The precipitate was collected by filtration, washed
once with water, twice with ethyl acetate and twice with
methanol to give 2.6 g of 3-[2-(6-Morpholin-4-yl-2-oxo-1,2-
dihydroindol-3-ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-
3-yl]propionic acid as an orange solid (34% yield).
'H NMR (d6-DMSO, 360 MHz): b 13.04 (s, br, 1H, NH-1'),
12.05 (s, vbr, 1H, COOH), 10.60 (s, br, 1H, NH-1), 7.50 (d,
8.0 Hz, 1H, H-4), 7.39 (s, 1H, H-vinyl), 6.60 (d, 8.0 Hz,
1H, H-5) , 6.43 (s, 1H, H-7) , 3 .73 (d, 4 .7, 4H, H-2" , 6" ) ,
3.09 (s, 4H, H-3" , 5" ) , 2.86 (t, 7. 1 Hz, 2H, CHZCHZCOOH) ,
2.64 (s, br, 2H, H-7' ) , 2 .37-2.43 (m, 4H, CHzCH2COOH and H-
4'), 1.71-1.75 (m, 4H, H-5',6').
MS m/z 422.3.
Example 210: 3-[2-(5-Chloro-4-methyl-2-oxo-1,2-dihydro-
indol-3-ylidenemethyl)-4,5,6,7-tetrahvdro-1H-indol-3-yll-
propionic acid
A suspension of 3.0 g of 4-methyl-2-oxindole was
stirred in 50 mL of acetonitrile at room temperature while
3.3 g of N-chlorosuccinimide was added in portions.
Trifluoroacetic acid (1 mL) was then added. A white
precipitate formed. The suspension was stirred at room
temperature for 3 days. The white solid was collected by
vacuum filtration, washed with a small amount of cold
acetone and dried overnight in a vacuum oven at 40°C to
give 2.5 g (68%) of 5-chloro-4-methyl-2-oxindole.
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A mixture of (2-Formyl-4,5,6,7-tetrahydro-1H-indol-3-
yl)-propionic acid (5.4 g), 4.0 g of 5-chloro-4-methyl-2-
oxindole and 2.7 g of piperidine in 25 mL of ethanol was
refluxed for 4 hours. Addition of acetic acid (8 mL)
resulted in the formation of a precipitate. The mixture
was refluxed for 5 minutes and cooled to ambient
temperature. The precipitate was collected by vacuum
filtration and washed with 20 mL of ethanol. The solids
were slurry-washed with 30 mL of refluxing ethanol, cooled,
collected by vacuum filtration, washed with 30 mL of
ethanol and vacuum dried to give 6.8 g (80%) of3-[2-(5-
Chloro-4-methyl-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-
4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic acid as an
orange solid.
1H NMR (d6-DMSO): 8 13.1 (s, 1H, pyrrole NH), 12.0 (br
s, 1H, COOH), 10.7 (s, 1H, CONH), 7.6 (s, 1H, -CH=), 7.3,
6.7(m, each 2H, aromatic), 2.9, 2.7 (t, each 4H, -CHzCH2
CO-) , 2.7 (s, 3H, CH3) , 2.4 (m, 4H, -CH2-, -CHz-) , 1.7 (m,
4H, -CHzCH2- ) .
Example 211: 3-(2-(5-Bromo-4-methyl-2-oxo-1,2-
dihydroindol-3-ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-
3-vll-propionic acid
A mixture of (2-Formyl-4,5,6,7-tetrahydro-1H-indol-3-
yl)-propionic acid (5.4 g), 5.0 g of 5-bromo-4-methyl-2-
oxindole and 2.7 g of piperidine in 25 mL of ethanol was
refluxed for 4 hours. Acetic acid (8 mL) was slowly added
and a precipitate formed. The mixture was refluxed for 5
minutes and cooled to ambient temperature. The precipitate
was collected by vacuum filtration and washed with 20 mL of
ethanol. The solids were slurry-washed in 30 mL of
refluxing ethanol, cooled, collected by vacuum filtration,
washed with 30 mL of ethanol and vacuum dried to give 7.6 g
(80%) of 3-[2-(5-bromo-4-methyl-2-oxo-1,2-dihydroindol-3-
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ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic
acid as an red-orange solid.
1H NMR (d6-DMSO): 8 13.1 (s, 1H, pyrrole NH), 12.0 (br
s, 1H, COOH), 10.7 (s, 1H, CONH), 7.8 (s, 1H, -CH=), 7.3,
6.7, (m, each 2H, aromatic), 2.9, 2.7 (t, each 4H,
-CHZCHZCO-) , 2.7 (s, 3H, CH3) , 2.4 (m, 4H -CH2-, -CHz-) , 1.7
(m, 4H, -CH2CHz- ) .
MS: m/z 427,429.
Example 212: 3-(2-(5-Bromo-2-oxo-1,2-dihydroindol-3
y_lidenemethvl)-4,5,6,7-tetrahydro-1H-indol-3-yll-N-(2
morpholin-4 ~lethyl)-propionamide
3-[2-(5-Bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-
4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic acid(12.3 g )
was dissolved in 150 mL of dimethylformamide.
Carbonyldiimidazole (6.3 g) was added and the mixture
stirred at ambient temperature for 1 hour. 4-(2-
Aminoethyl)morpholine (7.7 g) and 30 mL of
dimethylformamide were added and the stirring continued
overnight at room temperature. Fifty mL of water was added
to the mixture and stirring was continued for 10 minutes.
The precipitate was collected by vacuum filtration, washed
with 20 mL of water and then 20 mL of ethanol. The solid
was slurry-washed in 30 mL of refluxing ethanol for 5
minutes and cooled to room temperature. The solid was
collected by vacuum filtration, washed with 20 mL of
ethanol and vacuum dried to give 12.5 g (80%) of 3-[2-(5-
bromo-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-
tetrahydro-1H-indol-3-yl]-N-(2-morpholin-4-ylethyl)-
propionamide.
Example 213: 3-[2-(5-Chloro-2-oxo-1,2-dihydroindol-3-
ylidenemethvl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-N-(2-
morpholin-4-vlethvl)-propionamide
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3-[2-(5-Chloro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)
-4,5,6,7-tetrahydro-1H-indol-3-yl]-propionic acid (11 g )
was dissolved in 150 mL of dimethylformamide.
Carbonyldiimidazole (6.3 g) was added and the mixture
stirred at ambient temperature for 1 hour. 4-(2-Aminoethyl)
morpholine (7.7 g) and 30 mL of dimethylformamide were
added and the stirring continued overnight at room
temperature. Fifty mL of water was added to the mixture
and stirring was continued for 10 minutes. The precipitate
was collected by vacuum filtration, washed with 20 mL of
water and then 20 mL of ethanol. The solid was slurry-
washed in 30 mL of refluxing ethanol for 5 minutes and
cooled to room temperature. The solid was collected by
vacuum filtration, washed with 20 mL of ethanol and vacuum
dried to give 11.5 g (80%) of 3-[2-(5-chloro-2-oxo-1,2-
dihydroindol-3-ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-
3-yl]-N-(2-morpholin-4-ylethyl)-propionamide.
Example 214: 3-[2-(2-Oxo-1,2-dihydroindol-3-
ylidenemethyl)-phenyl]-propionic acid
To a solution of 13.4 g of phthalic dicarboxaldehye in
100 mL of dichloromethane was added in portions over 5
minutes 35 g of ethyl (triphenylphosphoranylidene)acetate.
The reaction mixture was stirred at room temperature
overnight and concentrated. The residue was stirred in 500
mL of a 6:1 mixture of hexanes:ethyl acetate for 1 hr. The
solids were removed by fitration and the filtrates
concentrated. The product was chromatographed on a silica
gel column to give 10 g of ethyl 3-(2-formylphenyl)
propenate as a mixture of E and Z isomers.
The above mixture was dissolved in 100 mL of ethyl
acetate containing 100 mg of 5% palladium on carbon and
stirred under hydrogen (balloon pressure) for 10 hrs. The
mixture was filtered through a bed of celite which was
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washed with ethyl acetate. The combined filtrates were
concentrated to give 8 g of ethyl 3-(2-formylphenyl)
propionate.
A mixture of 1 g of ethyl 3-(2-formylphenyl)propenate,
500 mg oxindole and 0.1 mL piperidine in 5 mL of ethanol
was stirred at 90 °C overnight. The mixture was evaporated
and purified on a silica gel column to give 450 mg of ethyl
(E)-3-[(2-oxo-1,2-dihydroindol-3-ylidenemethyl)phenyl]-
propionate (M+1 = 322).
To 300 mg of ethyl (E)-3-[(2-oxo-1,2-dihydroindol-3-
ylidenemethyl)phenyl]-propionate in 3 mL of ethanol was
added 2 mL of 2N sodium hydroxide. The mixture was stirred
at 90°C for 2 hrs, cooled and acidified with 6 N
hydrochloric acid to pH 3. The solid was collected by
filtration and washed with cold ethanol to give 30 mg of 3-
[2-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-phenyl]-
propionic acid as a yellow solid.
1H NMR (d6-DMSO) : 8 12 . 1 (s, 1H, COOH) , 10 . 6 (S, 1H,
CONH), 7.7 (s, 1H, =CH) 7.6, 7.4, 7.3, 7.2, 7.0, 6.8 ,6.7
(m, 8H, aromatic) , 2 .9, 2.5 (t, each 4H, CH2CH2) .
MS: m/z 294.
Example 215: 3-f4-Methvl-2-(2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-1H-pyrrol-3-yll-propionic acid
3,5-Dimethyl-4-(2-methoxycarbonylethyl)-1H-pyrrole-2-
carboxylic acid ethyl ester (127 g) was dissolved in acetic
acid (1900 mL), water (1900 mL) and tetrahydrofuran (1900
mL) and cooled to -30 °C. Cerric ammonium nitrate (1097 g)
was added in portions with stirring to give a reddish-
orange suspension. The suspension was stirred at 0 °C for
2 hours, neutralized to pH 7 with sodium bicarbonate and
extracted with ethyl acetate (2000 mL). The ethyl acetate
layer was separated, washed with brine (200 mL) and dried
over anhydrous sodium sulfate (20 g). The solvent was
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removed to give 80.2 g (60%) of 5-formyl-4-(2-methoxy-
carbonylethyl)-3-methyl-1H-pyrrole-2-carboxylic acid ethyl
ester as an oil.
5-Formyl-4-(2-methoxycarbonylethyl)-3-methyl-1H-
pyrrole-2-carboxylic acid ethyl ester (80.2 g), 2-oxindole
(37.9 g) and ethanol (300 mL) were warmed to 70 °C in a 500
mL, 3-neck round bottom flask equipped with mechanical
stirring and a reflux condenser. Piperidine (1.3 g) was
added and the mixture was refluxed for 4 hours. The
mixture was cooled to 10°C and the orange precipitate
collected by vacuum filtration and washed with 30 mL of
ethanol. The solid was slurry-washed in 150 mL of
refluxing ethanol, cooled, collected by vacuum filtration,
washed with 30 mL of ethanol and vacuum dried to give 81.7
g (75%) of 4-(2-Methoxycarbonylethyl)-3-methyl-5-(2-oxo-
1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic
acid ethyl ester as an orange solid.
4-(2-Methoxycarbonylethyl)-3-methyl-5-(2-oxo-1,2-
dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid
ethyl ester (81.7 g) ), 56.5 g of potassium hydroxide, 200
mL of ethanol and 200 mL of water were charged to a 1 L, 3
neck round bottom flask equipped with mechanical stirring
and a thermometer. The mixture was stirred at 90°C for 90
minutes, cooled to room temperature, and acidified with
acetic acid until a precipitate formed. The precipitate
was collected by vacuum filtration, washed with 50 mL of
water and vacuum dried to give 69.1 g (85 %) of 4-(2-
carboxyethyl)-3-methyl-5-(2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-1H-pyrrole-2-carboxylic acid as a red solid.
4-(2-carboxyethyl)-3-methyl-5-(2-oxo-1,2-dihydroindol-
3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid (10 g)
suspended in 50 mL of ethylene glycol (b.p. 198 °C) was
sealed in a 1 L pressure reactor, the reactor was
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pressurized to 120 psi with nitrogen and then heated to 150
°C for 3 hours. The reaction mixture was cooled to room
temperature and then diluted with 50 mL of water. The
resulting precipitate was collected by vacuum filtration
and was washed twice with 100 mL of water to give a mixture
of 3-[4-methyl-2-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-
1H-pyrrol-3-yl]-propionic acid and 3-[4-methyl-2-(2-oxo-
1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrol-3-yl]-propionic
acid 2-hydroxyethyl ester as a dark orange solid. The
solid was used in the next step without drying.
1H NMR (d6-DMSO) 8 13.3 (s, br, 1H, NH), 10.77 (s, 1 H,
NH), 7.6 (s, 1 H, H-vinyl), 7.67, 7.08, 6.97, 6.85 (m, 4 H,
Ar-H), 4.73 (t, J = 6Hz, 1 H, OH), 3.97-4.0 (m, 2H, CHz),
3.5-3.55 (m, 2 H, CHz) , 2.98 (t, J = 7.5 Hz, 2 H, CHz) , 2.51
(t, J = 7.5 Hz, 2 H, CHZ) , 2.04 (s, 3 H, CH3) .
MS m/z 341 (M+1).
3-[4-methyl-2-(2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-1H-pyrrol-3-yl]-propionic acid and 3-[4-
methyl-2-(2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-
pyrrol-3-yl]-propionic acid 2-hydroxyethyl ester, 1.9 g
of potassium hydroxide, 50 mL of water, and 50 mL of
ethanol in a 500 mL 3 neck round bottom flask were
stirred at 70°C for 1 hour. The mixture was cooled to
room temperature and acidified with 2 N hydrochloric acid
until a precipitate formed. The precipitate was
collected by vacuum filtration and washed with
ethanol:water mixture (1:1, 100mL). The solid was
slurry-washed with ethyl acetate: ethanol mixture (1:1,
100 mL) at 70°C for 30 minutes and then cooled to room
temperature. The product was collected by vacuum
filtration and vacuum dried at 40°C overnight to give 7.8
g (90% overall yield) of 3-[4-methyl-2-(2-oxo-1,2-
dihydroindol-3-ylidenemethyl)-1H-pyrrol-3-yl]-propionic
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acid as a dark orange solid. (R. B. Woodward et al.,
Tetrahedron, 1990, 46 (22), 7599-7659)
1HNMR (d6-DMSO) b 13.28 (s, br, 1 H, NH), 12.05 (s, 1
H, COOH), 10.78 (s, 1 H, NH), 7.68 (d, J = 7 Hz, 1 H, Ar-
H), 7.64 (s, 1 H, H-vinyl), 7.11 (t, J = 7 Hz, 1 H, Ar-
H), 7.11 (s, 1 H), 6.97 (t, J = 7 Hz, 1 H, Ar-H), 6.86
(d, J = 7 Hz, 1 H, Ar-H), 2.94 (t, J = 7.5 Hz, 2 H, CHz),
2.41 (t, J = 7.5 Hz, 2 H, CH2) , 2.04 (s, 3H, CH3) .
Example 216: 3-(2-(5-Chloro-2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-4-methyl-1H-pyrrol-3-yl)-propionic acid
5-Formyl-4-(2-ethoxycarbonylethyl)-3-methyl-1H-
pyrrole-2-carboxylic acid ethyl ester (281 mg), 5-chloro-
2-oxindole (168 mg), and piperidine (2 drops) in ethanol
(2 mL) were refluxed for 2 hours. The reaction mixture
was cooled and the precipitate that formed was filtered,
washed with ethanol and hexanes, and dried to give 369 mg
(86%) of 4-(2-ethoxycarbonylethyl)-3-methyl-5-(5-chloro-
2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-
carboxylic acid ethyl ester as a light yellow needle
crystals.
A mixture of 4-(2-ethoxycarbonylethyl)-3-methyl-5-
(5-chloro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-
pyrrole-2-carboxylic acid ethyl ester (346 mg) and
potassium hydroxide (560 mg) in ethanol (5 mL) was heated
to 95°C and then cooled, upon which red crystals formed.
The crystals were dissolved in water and acidified with 2
N hydrochloric acid to pH 2. The precipitate was
filtered, washed with water, and dried in a vacuum oven
overnight to give 299 mg (100%) of 4-(2-carboxyethyl)-3-
methyl-5-(5-chloro-2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-1H-pyrrole-2-carboxylic acid as a brown
solid.
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4-(2-carboxyethyl)-3-methyl-5-(5-chloro-2-oxo-1,2-
dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic
acid suspended in ethylene glycol (5 mL) was heated in a
sealed tube in a pre-heated oil bath at 200 °C for 2
hours. The reaction mixture was cooled to 90 °C and
potassium hydroxide (2 pellets) were added. The mixture
was then heated at 90°C for 30 minutes, after which time
it was cooled, poured into water, and acidified with 2 N
hydrochloric acid to pH 2. The precipitate that formed
was filtered, washed with water, and dried in a vacuum
oven overnight to give 77 mg (29%) of 3-[2-(5-Chloro-2-
oxo-1,2-dihydroindol-3-ylidenemethyl)-4-methyl-1H-pyrrol-
3-yl]-propionic acid.
1HNMR (d6-DMSO) 8 13.31 (s, br. 1 H, NH), 12.05 (s, 1
H, COOH), 10.89(s, br, 1 H, NH), 7.85 (d, J = 2 Hz, 1 H,
H-4), 7.75 (s, 1 H, H-vinyl), 7.16 (d, J = 3 Hz, 1 H),
7.11 (dd, J = 2.8 Hz, 1 H, H-6), 6.84 (d, J = 8 Hz, 1 H,
H-7) , 2.97 (t, J = 7.5 Hz, 2 H, CH2) , 2.41 (t, J = 7.5 Hz,
2 H, CHZ) , 2 .04 (s, 3 H, CH3) .
MS m/z 331 (M+1).
Example 217: 3-[2-(6-Methoxy-2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-4-methyl-1H-pyrrol-3-yl]-propionic acid
5-Formyl-4-(2-ethoxycarbonylethyl)-3-methyl-1H-
pyrrole-2-carboxylic acid ethyl ester (591 mg), 6-
methoxy-2-oxindole (333 mg) and piperidine (0.1 mL) in
ethanol (4 mL) were stirred at 90°C for 2 hours.
Potassium hydroxide (537 mg) was added to the mixture and
it was then stirred at 95°C for and additional hour. The
reaction mixture. was cooled and concentrated, dissolved
in water, and acidified with 2 N hydrochloric acid to pH
2. The precipitate was filtered, washed with water, and
dried in a vacuum oven overnight to give 730 mg (99%) of
4-(2-carboxyethyl)-3-methyl-5-(6-methoxy-2-oxo-1,2-
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dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic
acid.
4-(2-carboxyethyl)-3-methyl-5-(6-methoxy-2-oxo-1,2-
dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic
acid (501 mg) suspended in ethylene glycol (10 mL) was
heated in a sealed tube in a pre-heated oil bath at 200°C
for 2 hours. The reaction mixture was cooled to 90 °C and
potassium hydroxide (2 pellets) was added. The mixture
was then stirred at 90°C for 30 minutes, after which time
it was cooled, poured into water, and acidified with 2 N
hydrochloric acid to pH 2. The precipitate was filtered,
washed with water, and dried in a vacuum oven overnight
to give 221 mg (48%) of 3-[2-(6-methoxy-2-oxo-1,2-
dihydro-indol-3-ylidenemethyl)-4-methyl-1H-pyrrol-3-yl]-
propionic acid.
1HNMR (d6-DMSO) 8 13.09 (s, 1 H, NH) , 12.05 (s, 1 H,
COOH), 10.74 (s, 1 H, NH), 7.58 (d, J = 8 Hz, 1 H, H-4),
7.48 (s, 1 H, H-vinyl), 7.04 (d, J = 2 Hz, 1 H), 6.56
(dd, J = 2.8 Hz, 1 H, H-5), 6.43 (d, J = 2 Hz, 1 H), 3.75
(s, 3 H, OCH3) , 2. 91 (t, J = 7.5 Hz, 2 H, CHZ) , 2 .4 (t, J
- 7.5 Hz, 2 H, CHz) , 2.03 (s, 3 H, CH3) .
MS m/z 325 (M-1).
Example 218: 3-(2-(4-Methyl-2-oxo-1,2-dih~rdroindol-3-
~rlidenemethyl)-4-methyl-1H-pyrrol-3-yl]-propionic acid
A mixture of 5-formyl-4-(2-ethoxycarbonylethyl)-3-
methyl-1H-pyrrole-2-carboxylic acid ethyl ester (281 mg),
4-methyl-2-oxindole (147 mg) and piperidine (2 drops) in
ethanol (2 mL) was stirred at 90°C for 2 hours.
Potassium hydroxide (213 mg) was added, the temperature
was increased to 95°C and held there for 1 hour. The
reaction mixture was cooled and concentrated. The
residue was dissolved into water and acidified with 2 N
hydrochloric acid to pH 2. The precipitate was filtered,
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washed with water and dried in a vacuum oven overnight to
give 337 mg (95%) of 4-(2-carboxyethyl)-3-methyl-5-(4-
methyl-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-
pyrrole-2-carboxylic acid.
4-(2-carboxyethyl)-3-methyl-5-(4-methyl-2-oxo-1,2-
dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid
(300 mg) suspended in ethylene glycol (5 mL) was heated in
a sealed tube in a pre-heated oil bath at 200 °C for 2
hours. The reaction mixture was cooled to 90°C and
potassium hydroxide (1 pellet) was added. It was then
stirred at 90°C for 30 minutes. The reaction mixture was
cooled, poured into water, and acidified with 2 N
hydrochloric acid to pH 2. The precipitate was filtered,
washed with water, and purified on a silica gel column to
give 115 mg (44% yield) of 3- [2- (4-methyl-2-oxo-1,2-
dihydroindol-3-ylidenemethyl)-4-methyl-1H-pyrrol-3-yl]-
propionic acid.
1HNMR (d6-DMSO) 8 13.37 (s, 1 H, NH) , 12.04 (s, 1 H,
COOH), 10.81 (s, 1 H, NH), 7.69 (s, 1 H, H-vinyl), 7.09 (d,
J = 2.5 Hz, 1 H), 7.01 (t, J = 7.5 HZ, 1 H, Ar-H), 6.79 (d,
J = 7.5 Hz, 1 H, Ar-H), 6.74 (d, J = 7.5 Hz, 1 H, Ar-H),
2.88 (t, J = 7.2 Hz, 2 H, CHz), 2.61 (s, 3 H, CH3-4), 2.44
(t, J = 7.2 Hz, 2 H, CHZ) , 2 . 04 (s, 3 H, CH3) .
MS m/z 309 (M-1) .
Example 219: 3-f2-(6-Chloro-2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-4-methyl-1H-pyrrol-3-yl]-propionic acid
5-Formyl-4-(2-ethoxycarbonylethyl)-3-methyl-1H-
pyrrole-2-carboxylic acid ethyl ester (281 mg), 6-chloro-
2-oxindole (168 mg) and piperidine (2 drops) in ethanol
(2 mL) were stirred at 90 °C for 2 hours. Potassium
hydroxide (537 mg) was added to the mixture and it was
then stirred at 95°C for and additional hour. The
reaction mixture was cooled and concentrated. The
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residue was dissolved into water and acidified with 2 N
hydrochloric acid to pH 2. The precipitate was filtered,
washed with water, and dried in a vacuum oven overnight
to give 270 mg (72%) of 4-(2-carboxy-ethyl)-3-methyl-5-
(6-chloro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-
pyrrole-2-carboxylic acid.
4-(2-carboxyethyl)-3-methyl-5-(6-chloro-2-oxo-1,2-
dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid
(240 mg) suspended in ethylene glycol (5 mL) was held in a
sealed tube in a pre-heated oil bath at 200 °C for 2 hours.
The reaction mixture was cooled to 90°C and potassium
hydroxide (2 pellets) was added. It was then stirred at
90°C for 30 minutes. The reaction mixture was cooled,
poured into water, and acidified with 2 N hydrochloric acid
to pH 2. The precipitate was filtered, washed with water,
and purified on a silica gel column using ethyl
acetate:hexanes:glacial acetic acid 50:50:10 as eluent to
give 45 mg (21%) of 3-[2-(6-chloro-2-oxo-1,2-dihydroindol-
3-ylidenemethyl)-4-methyl-1H-pyrrol-3-yl)-propionic acid.
1HNMR (d6-DMSO) 8 13.2 (s, 1H, NH) , 12.04 (s, 1 H,
COOH), 10.92 (s, 1 H, NH), 7.72 (d, J = 8 Hz, 1 H, H-4),
7.68 (s, 1 H, H-vinyl), 7.15 (d, J = 2.4 HZ, 1 H), 7.01
(dd, J = 2.8 Hz, 1 H, H-6), 6.86 (d, J = 2 Hz, 1 H, H-7),
2.94 (t, J = 7.5 Hz, 2 H, CH2) , 2.4 (t, J = 7.5 Hz, 2 H,
CHz) , 2.03 (s, 3 H, CH3) . MS m/z 329 (M-1) .
Example 220: 3-f2-(5-Bromo-2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-4-methyl-1H-pyrrol-3-yll-propionic acid
A mixture of 5-formyl-4-(2-ethoxycarbonylethyl)-3-
methyl-1H-pyrrole-2-carboxylic acid ethyl ester (281 mg),
5-bromo-2-oxindole (220 mg), and piperidine (2 drops) in
ethanol (2 mL) was stirred at 90 °C for 2 hours.
Potassium hydroxide (537 mg) was added and the
temperature was increased to 95° C for 1 hour. The
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reaction mixture was cooled and concentrated. The
residue was dissolved into water and acidified with 2 N
hydrochloric acid to pH 2. The precipitate that formed
was filtered, washed with water and dried in a vacuum
oven overnight to give 411 mg (98%) of 4-(2-
carboxyethyl)-3-methyl-5-(5-bromo-2-oxo-1,2-dihydro-
indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid.
4-(2-carboxyethyl)-3-methyl-5-(5-bromo-2-oxo-1,2-
dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid
(380 mg) suspended in ethylene glycol (5 mL) was held in a
sealed tube in a pre-heated oil bath at 200°C for 2 hours.
The reaction mixture was cooled to 90°C and potassium
hydroxide (1 pellet) was added. It was then stirred at
90°C for 30 minutes. The reaction mixture was cooled,
poured into water, and acidified with 2 N hydrochloric acid
to pH 2. The precipitate was filtered, washed with water,
and purified using silica gel column chromatography to give
168 mg (49% yield) of 3-[2-(5-bromo -2-oxo-1,2-
dihydroindol-3-ylidenemethyl)-4-methyl-1H-pyrrol-3-yl]-
propionic acid.
1HNMR (d6-DMSO) 8 13.32 (s, 1 H, NH) , 12.0 (s, 1 H,
COOH), 10.9 (s, 1 H, NH), 7.97 (d, J = 2 HZ, 1 H, h-4),
7.75 (s, 1 H, H-vinyl), 7.23 (dd, v = 2.8 Hz, 1 H, H-6),
7.16 (d, v = 2.6 Hz, 1 H), 6.8 (d, J = 8 Hz, 1 H, H-7),
2.97 (t, J = 7.7 Hz, 2 H, CHZ) , 2.41 (t, J = 7.7 Hz, 2 H,
CHz ) , 2 . 0 4 ( s , 3 H , CH3 ) .
MS m/z 375/377.
Example 221: 3-[2-(5-Methyl-2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-4-methyl-1H-pyrrol-3-yl]-propionic acid
5-Methylisatin (15.0 g) and 60 mL of hydrazine hydrate
were heated to 140-160°C for 4 hours. Thin layer
chromatography (ethyl acetate: hexane 1:2, silica gel)
showed no starting material remaining. The reaction
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mixture was cooled to room temperature, poured into 300 mL
of ice water, and acidified to pH 2 with 6 N hydrochloric
acid. After standing at room temperature for 2 days, the
precipitate was collected by vacuum filtration, washed with
water, and dried under vacuum to give 6.5 g (47%) of 5-
methyl-2-oxindole.
1HNMR (360 MHz, DMSO-d6) b 10.20 (s, br, 1 H, NH-1),
6.99 (s, 1 H, H-4), 6.94 (d, J = 8.11 Hz, 1 H, H-6), 6.68
(d, J = 8.11 Hz, 1 H, H-7), 3.39 (s, 2 H, CHz-3), and 2.22
(s, 3 H, CH3-5) .
5-Formyl-4-(2-ethoxycarbonylethyl)-3-methyl-1H-
pyrrole-2-carboxylic acid ethyl ester (560 mg), 5-methyl-
2-oxindole (300 mg), and piperidine (4 drops) in ethanol
(4 mL) were stirred at 90°C for 2 hours. Potassium
hydroxide (537 mg) was added to the mixture and it was
then stirred at 95°C for an additional hour. The
reaction mixture was cooled and concentrated. The
residue was dissolved into water and acidified with 2 N
hydrochloric acid to pH 2. The precipitate was filtered,
washed with water, and dried in a vacuum oven overnight
to give 496 mg of 4-(2-carboxyethyl)-3-methyl-5-(5-methyl
-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-
carboxylic acid.
4-(2-carboxyethyl)-3-methyl-5-(5-methyl-2-oxo-1,2-
dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid
(496 mg) suspended in ethylene glycol (2 mL) was held in a
sealed tube in a pre-heated oil bath at 200°C for 2 hours.
The reaction mixture was cooled to 90°C and potassium
hydroxide (157 mg) was added. It was then stirred at 90°C
for 30 minutes. The reaction mixture was cooled, poured
into water, and acidified with 2 N hydrochloric acid to pH
2. The precipitate was filtered, washed with water, and
dried in a vacuum oven overnight to give 128 mg (29%) of 3-
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[2-(5-methyl-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-4-
methyl-1H-pyrrol-3-yl)-propionic acid.
1HNMR (d6-DMSO) 8 13.31 (s, 1 H, NH) , 12.07 (s, 1 H,
COOH), 10.68 (s, 1 H, NH), 7.59 (s, 1 H), 7.51 (br s, 1 H,
H-4), 7.09 (d, J = 2.7 Hz, 1H), 6.91 (br d, J = 8 HZ, 1 H,
H-6), 6.73 (d, J = 8 Hz, 1 H, H-7), 2.93 (t, J = 7.5 Hz, 2
H, CHz) , 2.41 (t, J = 7.5 Hz, 2 H, CH2) , 2.3 (s, CH3-5) ,
2.04 (s, 3 H, CH3) .
MS m/z 311 (M+1) .
Example 222: 3-[2-(5-Methoxy-2-oxo-1,2-dihydroindol-3-
ylidenemethyl)-4-methyl-1H-pyrrol-3=yl]-propionic acid
A mixture of 5-formyl-4-(2-ethoxycarbonylethyl)-3-
methyl-1H-pyrrole-2-carboxylic acid ethyl ester (562 mg),
5-methoxy-2-oxindole (326 mg), and piperidine (2 drops)
in ethanol (2 mL) were stirred at 90° C for 2 hours.
Potassium hydroxide (537 mg) was added, the temperature
was increased to 95°C and held there for 1 hour. The
reaction mixture was cooled and concentrated. The
residue was dissolved into water and acidified with 2 N
hydrochloric acid to pH 2. The precipitate was filtered,
washed with water, and dried in a vacuum oven overnight
to give 240 mg (65%) of 4-(2-carboxy-ethyl)-3-methyl-5-
(5-methoxy-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-1H-
pyrrole-2-carboxylic acid.
4-(2-carboxyethyl)-3-methyl-5-(5-methoxy-2-oxo-1,2-
dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid
(240 mg) suspended in ethylene glycol (2 mL) was held in a
sealed tube in a pre-heated oil bath at 200°C for 2 hours.
The reaction mixture was cooled to 90°C and potassium
hydroxide (1 pellet) was added. It was then stirred at
90°C for 30 minutes. The reaction mixture was cooled,
poured into water and acidified with 2 N hydrochloric acid
to pH 2. The precipitate was filtered, washed with water,
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and dried in a vacuum oven overnight to give 30.5 mg (14%)
of 3-[2-(5-methoxy-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-
4-methyl-1H-pyrrol-3-yl]-propionic acid.
1HNMR (d6-DMSO) 8 13.38 (s, 1 H, NH) , 12.07 (s, 1 H,
COOH), 10.59 (s, 1 H, NH), 7.63 (s, 1 H, H-vinyl), 7.1 (d,
J = 2.1 Hz, 1 H), 6.75 (d, J = 8 Hz, 1 H, H-7), 6.69 (dd, J
- 2.8 Hz, 1 H, H-6) , 3.76 (s, 3 H, OCH3) , 2.96 (t, J = 7.4
Hz, 2 H, CHZ) , 2.41 (t, J = 7.4 Hz, 2 H, CHZ) , 2.04 (s, 3 H,
CH3 ) .
MS m/z 327 (M+1).
Example 223: 3-~2-[6-(3-Methoxyphenyl)-2-oxo-1,2-dihydro-
indol-3-vlidenemethyl]-4-methyl-1H-pyrrol-3-yl~~-propionic
acid
Tetrakis(triphenylphosphine)palladium (0.7 g) was
added to a mixture of 5 g of 3-methoxyphenylboronic acid,
3.8 g of 5-bromo-2-fluoronitrobenzene, and 11 mL of 2 M
sodium carbonate solution in 100 mL of toluene. The
mixture was refluxed for 2 hours, diluted with water and
extracted with ethyl acetate. The ethyl acetate was washed
with saturated sodium bicarbonate and brine, dried, and
concentrated to give an oily solid. The solid was purified
on a silica gel column using a 1:6 mixture of ethyl
acetate: hexane as eluent, to give 4.3 g (77%) of 4-fluoro-
3'-methoxy-3-nitrobiphenyl.
Dimethyl malonate (9.7 mL) was added dropwise to 2.0 g
of sodium hydride suspended in 50 mL of dimethylsulfoxide.
The mixture was stirred at 100°C for 35 minutes and cooled
to room temperature. 4-Fluoro-2'-methoxy-3-nitrobiphenyl
(4.2 g) in 50 mL of dimethylsulfoxide was added and the
mixture was stirred at 100°C for 1 hour. The reaction
mixture was cooled, quenched with 300 mL of saturated
ammonium chloride solution and extracted twice with ethyl
acetate. The extracts were combined, washed with brine,
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dried over anhydrous sodium sulfate and concentrated to
give crude dimethyl 3'-methoxy-3-nitrobiphenyl-4-malonate
as a pale yellow solid.
Crude 3'-methoxy-3-nitro-biphenyl4-malonate was
stirred at 110°C in 45 mL of 6 N hydrochloric acid for 4
days and then cooled. The precipitate which formed was
collected by filtration, washed with water and hexane, and
dried to give 5.3 g of 3'-methoxy-2-nitrobiphenyl-4-acetic
acid as a light tan solid.
3'-Methoxy-3-nitrobiphenyl-4-acetic acid (5.2 g) was
dissolved in methanol and hydrogenated over 0.8 g of 10%
palladium on carbon for 3 hours at room temperature. The
catalyst was removed by filtration, washed with methanol,
and the filtrates combined and concentrated to give a
brown solid. The solid was purified on a silica gel
column, using a 33:66:1 mixture of ethyl
acetate:hexane:acetic acid as eluent, to give 3.0 g (75
based on 4-fluoro-3'-methoxy-3-nitrobiphenyl) of 6-(3-
methoxypheny)-2-oxindole as a pink solid.
1HNMR (360 MHz, DMSO-d6) 8 10.39 (s, br, 1 H, NH),
7.35 (t, J = 7.85 Hz, 1 H), 7.26 (d, J = 7.78 Hz, 1 H),
7.19 (dd, J = 1.22, 7.8 Hz, 1 H), 7.13-7.16 (m, 1 H),
7.09-7.1 (m, 1 H), 7.01 (d, J'= 1.48 Hz, 1 H), 6.90-6.93
(m, 1 H), 3.8 (s, 3 H, OCH3), 3.49 (s, 2H, CHZ).
MS m/z (relative intensity, %) 240.0 ([M+1]', 100).
5-Formyl-4-(2-ethoxycarbonylethyl)-3-methyl-1H-
pyrrole-2-carboxylic acid ethyl ester (281 mg), 6-(3-
methoxy-phenyl)-2-oxindole (287 mg), and piperidine (2
drops) in ethanol (5 mL) were stirred at 90°C overnight.
The precipitate was filtered and washed with ethanol.
The yellow orange solid and potassium hydroxide (4
pellets) were stirred in ethanol (3 mL) at 90°C for 2.5
hours. The reaction mixture was cooled and concentrated.
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The residue was dissolved into water and acidified with 2
N hydrochloric acid to pH 2. The precipitate was
filtered, washed with water, and dried in a vacuum oven
overnight to give 413 mg of 4-(2-Carboxyethyl)-5-[6-(3-
methoxyphenyl)-2-oxo-1,2-dihydroindol-3-ylidenemethyl]-3-
methyl-1H-pyrrole-2-carboxylic acid.
4-(2-Carboxyethyl)-5-[6-(3-methoxyphenyl)-2-oxo-1,2-
dihydroindol-3-ylidenemethyl]-3-methyl-1H-pyrrole-2-
carboxylic acid (413 mg) suspended in ethylene glycol (5
mL) was held in Baled tube in a pre-heated oil bath at
200°C for 2 ~ hours. The reaction mixture was cooled to
90°C and potassium hydroxide (4 pellets) was added. It was
then stirred at 100°C for 2 more hours. The reaction
mixture was cooled, poured into water and acidified with 2
N hydrochloric acid to pH 2. The precipitate was filtered,
washed with water and dried in a vacuum oven overnight. The
crude solid was purified on a silica gel column using
33:66:1 ethyl acetate:hexanes:glacial acetic acid as eluent
to give 75 mg (20%) of 3-~2- [6- (3-methoxyphenyl) -2-oxo-l, 2-
dihydroindol-3-ylidenemethyl]-4-methyl-1H-pyrrol-3-yl)-
propionic acid as an orange-red solid.
1HNMR (d6-DMSO) 8 13.27 (s, 1 H, NH), 12.06 (s, 1
H, COOH), 10.86 (s, 1 H, NH), 7.76 (d, J = 8 Hz, 1 H, H-4),
7.68 (s, 1 H, H-vinyl), 7.36 (t, J = 8 Hz, 1 H), 7.29 (dd,
J = 1.5, 8 Hz, 1 H), 6.92, 7.09, 7.13,7.2 (m, 5 H, Ar-H),
3 .82 (s, 3 H, OCH3) , 2 . 96 (t, J = 7.4 Hz, 2 H, CH2) , 2 .42
(t, J = 7.4 Hz, 2 H, CHz) , 1.97 (s, 3H, CH3) .
Example 224: 3-~2-[6-(3-Ethoxyphenyl)-2-oxo-1,2-dihydro-
indol-3-vlidenemethvl]-4-methyl-1H-pvrrol-3-yl~,-propionic
acid
A mixture of 5-formyl-4-(2-ethoxycarbonylethyl)-3-
methyl-1H-pyrrole-2-carboxylic acid ethyl ester (281 mg),
6-(3-ethoxyphenyl)-2-oxindole (304 mg), and piperidine (2
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drops) in ethanol (5 mL) were held at 90 °C overnight.
The precipitate that formed was filtered, washed with
ethanol. The precipitate, an orange solid, was stirred
with potassium hydroxide (4 pellets) in ethanol (3 mL) at
90°C for 2.5 hours. The reaction mixture was cooled and
concentrated. The residue was dissolved into water and
acidified with 2N hydrochloric acid to pH 2. The
precipitate was filtered, washed with water, and dried in
a vacuum oven overnight to give 370 mg of 4-(2-
Carboxyethyl)-5-[6-(3-ethoxyphenyl)-2-oxo-1,2-
dihydroindol-3-ylidenemethyl]-3-methyl-1H-pyrrole-2-
carboxylic acid.
4-(2-Carboxyethyl)-5-[6-(3-ethoxyphenyl)-2-oxo-1,2-
dihydroindol-3-ylidenemethyl]-3-methyl-1H-pyrrole-2-
carboxylic acid (350 mg) suspended in ethylene glycol (5
mL) was held in a sealed tube in a pre-heated oil bath at
200°C for 2.5 hours. The reaction mixture was cooled to
100°C and potassium hydroxide (4 pellets) was added. It
was then stirred at 100°C for 2 hours. The reaction
mixture was cooled, poured into water, and acidified with 2
N hydrochloric acid to pH 2. The precipitate was filtered,
washed with water, and dried in a vacuum oven overnight.
The crude solid was purified on a silica gel column using
33:66:1 ethyl acetate:hexanes:glacial acetic acid to give
140 mg (44%) of 3-~2- [6- (3-ethoxyphenyl) -2-oxo-1,2-
dihydroindol-3-ylidenemethyl]-4-methyl-1H-pyrrol-3-yl~-
propionic acid as a brown solid.
1HNMR (ds-DMSO) 8 13 .28 (s, 1 H, NH) , 12 . 04 (s, 1 H,
COOH), 10.86 (s, 1 H, NH), 7.76 (d, J = 8 Hz, 1 H, H-4),
7.68 (s, 1 H, H-vinyl), 7.34 (t, J = 8 Hz, 1 H), 7.28 (dd,
J = 2, 8 Hz, 1 H, H-5), 7.08 (d, J = 2 Hz, 1 H, H-7), 7.18,
7.13, 6.9 (m, 4 H, Ar-H), 4.1 (q, J = 7 Hz, 2 H, OCHZCH3),
SUBSTITUTE SKEET (RULE 26)
CA 02383623 2001-11-29
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262
2.96 (t, J = 7.5 Hz, 2 H, CHZ) , 2.43 (t, J = 7.5 Hz, 2 H,
CHz) , 2.05 (s, 3 H, CH3) , 1.35 (t, J = 7 Hz, 3 H, OCHZCH3) .
5. BIOLOGICAL EVALUATION
It will be appreciated that, in any given series of
compounds, a spectrum of biological activity will be
afforded. In its presently preferred embodiments, this
invention relates to 3-methylidenyl-2-indolinones which
demonstrate the ability to modulate RTK, CTK, and STK
activity. The assays employed to select those compounds
demonstrating of the desired activity are set forth below.
Table 2 shows the results of testing of compounds of this
invention using the described assays. The results shown are
not to be construed as limiting the scope of this invention
in any manner whatsoever.
SUBSTITUTE SHEET (RULE 26)
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263
TABLE 2
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SUBSTITUTE SHEET (RULE 26)
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269
A. Assay Procedures.
The following in vitro assays may be used to
determine the level of activity and effect of the
different compounds of the present invention on one or
more of the PKs. Similar assays can be designed along
the same lines for any PK using techniques well known in
the art.
The cellular/catalytic assays described herein are
performed in an ELISA format. The general procedure is
as follows: a compound is introduced to cells expressing
the test kinase, either naturally or recombinantly, for a
selected period of time after which, if the test kinase
is a receptor, a ligand known to activate the receptor is
added. The cells are lysed and the lysate is transferred
to the wells of an ELISA plate previously coated with a
specific antibody recognizing the substrate of the
enzymatic phosphorylation reaction. Non-substrate
components of the cell .lysate are washed away and the
amount of phosphorylation on the substrate is detected
with an antibody specifically recognizing phosphotyrosine
compared with control cells that were not contacted with
a test compound. The cellular/biologic assays
described herein measure the amount of DNA made in
response to activation of a test kinase, which is a
general measure of a proliferative response. The general
procedure for this assay is as follows: a compound is
introduced to cells expressing the test kinase, either
naturally or recombinantly, for a selected period of time
after which, if the test kinase is a receptor, a ligand
known to activate the receptor is added. After
incubation at least overnight, a DNA labeling reagent
such as Bromodeoxyuridine (BrdU) or 3H-thymidine is
added. The amount of labeled DNA is detected with either
SUBSTITUTE SHEET (RULE 26)
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an anti-BrdU antibody or by measuring radioactivity and
is compared to control cells not contacted with a test
compound.
Cellular/Catalvtic Assays
Enzyme linked immunosorbent assays (ELISA) may be used
to detect and measure the presence of PK activity. The
ELISA may be conducted according to known protocols which
are described in, for example, Voller, et al., 1980,
"Enzyme-Linked Immunosorbent Assay," In: Manual of Clinical
Immunology, 2d ed., edited by Rose and Friedman, pp 359-
371 Am. Soc. Of Microbiology, Washington, D.C.
The disclosed protocol may be adapted for determining
activity with respect to a specific PK. That is, the
preferred protocols for conducting the ELISA experiments
for specific PKs is provided below. However, adaptation of
these protocols for determining a compound's activity for
other members of the RTK family, as well as for CTKs and
STKs, is well within the scope of knowledge of those
skilled in the art.
FLK-1 Assa
An ELISA assay is conducted to measure the kinase
activity of the FLK-1 receptor and more specifically, the
inhibition or activation of TK activity on the FLK-1
receptor. Specifically, the following assay can be
conducted to measure kinase activity of the FLK-1 receptor
in cells genetically engineered to express Flk-1.
Materials and Reagents.
a. Corning 96-well ELISA plates (Corning Catalog No.
25805-96),
b. Cappel goat anti-rabbit IgG (catalog no. 55641),
c. PBS (Gibco Catalog No. 450-1300EB),
d. TBSW Buffer (50 mM Tris (pH 7.2), 150 mM NaCl and
0.1% Tween-20),
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e. Ethanolamine stock (10% ethanolamine (pH 7.0),
stored at 4°C),
f. HNTG buffer (20mM HEPES buffer (pH 7.5), 150mM
NaCl, 0.2% Triton X-100, and 10% glycerol),
g. EDTA (0.5 M (pH 7.0) as a 100X stock),
h. Sodium orthovanadate (0.5 M as a 100X stock),
i. Sodium pyrophosphate (0.2 M as a 100X stock),
j. NUNC 96 well V bottom polypropylene plates
(Applied Scientific Catalog No. AS-72092),
k. NIH3T3 C7#3 Cells (FLK-1 expressing cells),
1. DMEM with 1X high glucose L-Glutamine (catalog
No. 11965-050),
m. FBS, Gibco (catalog no. 16000-028),
n. L-glutamine, Gibco (catalog no. 25030-016),
0. VEGF, PeproTech, Inc. (catalog no. 100-20)(kept
as 1 ~g/100 ~,1 stock in Milli-Q dH20 and stored at -20° C,
p. Affinity purified anti-FLK-1 antiserum,
q. UB40 monoclonal antibody specific for
phosphotyrosine (see, Fendley, et al., 1990, Cancer
Research 50:1550-1558),
r. EIA grade Goat anti-mouse IgG-POD (BioRad catalog
no. 172-1011),
s. 2,2-azino-bis(3-ethylbenz-thiazoline-6-sulfonic
acid (ABTS) solution (100mM citric acid (anhydrous), 250 mM
Na2HP04 (pH 4.0), 0.5 mg/ml ABTS (Sigma catalog no. A-
1888)), solution should be stored in dark at 4° C until
ready for use,
t. HZOz (30% solution)(Fisher catalog no. H325),
u. ABTS/ Hz02 (15m1 ABTS solution, 2 ~1 H20z) prepared
5 minutes before use and left at room temperature,
v. 0.2 M HC1 stock in HzO,
w. dimethylsulfoxide (100%)(Sigma Catalog No. D-
8418), and
SUBSTITUTE SHEET (RULE 26)
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272
y. Trypsin-EDTA (Gibco BRL Catalog No. 25200-049).
Protocol.
1. Coat Corning 96-well ELISA plates with 1.0 ~g per
well Cappel Anti-rabbit IgG antibody in O.1M Na2C03 pH 9.6.
Bring final volume to 150 ~1 per well. Coat plates
overnight at 4°C. Plates can be kept up to two weeks when
stored at 4°C.
2. Grow cells in Growth media (DMEM, supplemented
with 2.0 mM L-Glutamine, 10% FBS) in suitable culture
dishes until confluent at 37°C, 5% COz.
3. Harvest cells by trypsinization and seed in
Corning 25850 polystyrene 96-well round bottom cell plates,
25,000 cells/well in 200 ~,1 of growth media.
4. Grow cells at least one day at 37°C, 5% C02.
5. Wash cells with D-PBS 1X.
6. Add 200 ~1/well of starvation media (DMEM, 2.OmM
1-Glutamine, 0.1% FBS). Incubate overnight at 37°C, 5% C02.
7. Dilute Compounds 1:20 in polypropylene 96 well
plates using starvation media. Dilute dimethylsulfoxide
1:20 for use in control wells.
8. Remove starvation media from 96 well cell culture
plates and add 162 ~1 of fresh starvation media to each
well.
9. Add 18 ~1 of 1:20 diluted compound dilution (from
step 7) to each well plus the 1:20 dimethylsulfoxide
dilution to the control wells (+/- VEGF), for a final
dilution of 1:200 after cell stimulation. Final
dimethylsulfoxide is 0.5%. Incubate the plate at 37°C, 5%
COz f or two hours .
10. Remove unbound antibody from ELISA plates by
inverting plate to remove liquid. Wash 3 times with TBSW +
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0.5% ethanolamine, pH 7Ø Pat the plate on a paper towel
to remove excess liquid and bubbles.
11. Block plates with TBSW + 0.5% ethanolamine, pH
7.0, 150 ~1 per well. Incubate plate thirty minutes while
shaking on a microtiter plate shaker.
12. Wash plate 3 times as described in step 10.
13. Add 0.5 ~g/well affinity purified anti-FLU-1
polyclonal rabbit antiserum. Bring final volume to 150
~1/well with TBSW + 0.5% ethanolamine pH 7Ø Incubate
plate for thirty minutes while shaking.
14. Add 180 ~l starvation medium to the cells and
stimulate cells with 20 ~,1/well 10.0 mM sodium
orthovanadate and 500 ng/ml VEGF (resulting in a final
concentration of 1.0 mM sodium orthovanadate and 50 ng/ml
VEGF per well) for eight minutes at 37°C, 5% CO~. Negative
control wells receive only starvation medium.
15. After eight minutes, media should be removed from
the cells and washed one time with 200 ~1/well PBS.
16. Lyse cells in 150 ~.1/well HNTG while shaking at
room temperature for five minutes. HNTG formulation
includes sodium ortho vanadate, sodium pyrophosphate and
EDTA.
17. Wash ELISA plate three times as described in step
10.
18. Transfer cell lysates from the cell plate to
ELISA plate and incubate while shaking for two hours. To
transfer cell lysate pipette up and down while scrapping
the wells.
19. Wash plate three times as described in step 10.
20. Incubate ELISA plate with 0.02 Etg/well UB40 in
TBSW + 05% ethanolamine. Bring final volume to 150
~1/well. Incubate while shaking for 30 minutes.
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21. Wash plate three times as described in step 10.
22. Incubate ELISA plate with 1:10,000 diluted EIA
grade goat anti-mouse IgG conjugated horseradish peroxidase
in TBSW plus 0.5% ethanolamine, pH 7Ø Bring final volume
to 150 ~,1/well. Incubate while shaking for thirty minutes.
23. Wash plate as described in step 10.
24. Add 100 ~1 of ABTS/H202 solution to well.
Incubate ten minutes while shaking.
25. Add 100 ~l of 0.2 M HC1 for 0.1 M HC1 final
concentration to stop the color development reaction.
Shake 1 minute at room temperature. Remove bubbles with
slow stream of air and read the ELISA plate in an ELISA
plate reader at 410 nm.
EGF Receptor-HER2 Chimeric Receptor Assay In Whole
Cells.
HER2 kinase activity in whole EGFR-NIH3T3 cells
are measured as described below:
Materials and Reagents.
a. EGF: stock concentration: 16.5 ILM, EGF 201,
TOYOBO, Co., Ltd. Japan.
b. 05-101 (UBI) (a monoclonal antibody
recognizing an EGFR extracellular domain).
c. Anti-phosphotyrosine antibody (anti-Ptyr)
(polyclonal)(see, Fendley, et al., supra).
d. Detection antibody: Goat anti-rabbit 1gG
horseradish peroxidase conjugate, TAGO, Inc., Burlingame,
CA.
e. THST buffer:
Tris-HCl, pH 7.2 50 mM
NaCl 150 mM
Triton X-100 0.1
f. HNTG 5X stock:
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HEPES 0.1 M
NaCl 0.75 M
Glycerol 50%
Triton X-100 1.0%
g. ABTS stock:
Citric Acid 100 mM
Na2HP04 2 5 0 mM
HCl, conc. 0.5 mM
ABTS* 0.5mg/ml
* (2,2'-azinobis(3-
ethylbenzthiazolinesulfonic acid)). Keep solution in dark
at 4°C until use.
h. Stock reagents of:
EDTA 100 mM pH 7.0
Na3V04 0.5 M
Na4 ( P20~ ) 0 . 2 M
Procedure.
Pre-coat ELISA Plate
1. Coat ELISA plates (Corning, 96 well, Cat.
#25805-96) with 05-101 antibody at 0.5 ~.g per well in PBS,
100 ~1 final volume/well, and store overnight at 4°C.
Coated plates are good for up to 10 days when stored at
4°C.
2. On day of use, remove coating buffer and
replace with 100 ~1 blocking buffer (5% Carnation Instant
Non-Fat Dry Milk in PBS). Incubate the plate, shaking, at
room temperature (about 23°C to 25°C) for 30 minutes. Just
prior to use, remove blocking buffer and wash plate 4 times
with TBST buffer.
Seeding Cells
1. An NIH3T3 cell line overexpressing a
chimeric receptor containing the EGFR extracellular domain
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and intracellular HER2 kinase domain can be used for this
assay.
2. Choose dishes having 80-90% confluence for
the experiment. Trypsinize cells and stop reaction by
adding 10% fetal bovine serum. Suspend cells in DMEM medium
(10% CS DMEM medium) and centrifuge once at 1500 rpm, at
room temperature for 5 minutes.
3. Resuspend cells in seeding medium (DMEM,
0.5% bovine serum), and count the cells using trypan blue.
Viability above 90% is acceptable. Seed cells in DMEM
medium (0.5% bovine serum) at a density of 10,000 cells per
well, 100 ~1 per well, in a 96 well microtiter plate.
Incubate seeded cells in 5% C02 at 37°C for about 40 hours.
Assay Procedures
1. Check seeded cells for contamination using
an inverted microscope. Dilute drug stock (10 mg/ml in
DMSO) 1:10 in DMEM medium, then transfer 5 ~l to a TBST
well for a final drug dilution of 1:200 and a final DMSO
concentration of 1%. Control wells receive DMSO alone.
Incubate in 5% COZ at 37°C for two hours.
2. Prepare EGF ligand: dilute stock EGF in DMEM
so that upon transfer of 10 ~tl dilute EGF (1:12 dilution;,
100 nM final concentration is attained.
3. Prepare fresh HNTG* sufficient for 100 u1
per well, and place on ice.
HNTG* ( 10 ml )
HNTG stock 2.0 ml
milli-Q H20 7.3 ml
EDTA, 100 mM, pH 7.0 0.5 ml
Na3V04 (0.5 M) 0.1 ml
Na9 ( P20., ) ( 0 . 2 M ) 0 . 1 ml
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4. After 120 minutes incubation with drug, add
prepared SGF ligand to cells, 10 ~1 per well, to a final
concentration of 100 nM. Control wells receive DMEM alone.
Incubate with shaking, at room temperature, for 5 minutes.
5. Remove drug, EGF, and DMEM. Wash cells
twice with PBS. Transfer HNTG* to cells, 100 ~1 per well.
Place on ice for 5 minutes. Meanwhile, remove blocking
buffer from other ELISA plate and wash with TBST as
described above.
6. With a pipette tip securely fitted to a
micropipettor, scrape cells from plate and homogenize cell
material by repeatedly aspirating and dispensing the HNTG*
lysis buffer. Transfer lysate to a coated, blocked, and
washed ELISA plate. Incubate shaking at room temperature
for one hour.
7. Remove lysate and wash 4 times with TBST.
Transfer freshly diluted anti-Ptyr antibody to ELISA plate
at 100 ~1 per well. Incubate shaking at room temperature
for 30 minutes in the presence of the anti-Ptyr antiserum
(1:3000 dilution in TBST).
8. Remove the anti-Ptyr antibody and wash 4
times with TBST. Transfer the freshly diluted TAGO anti-
rabbit IgG antibody to the ELISA plate at 100 ~l per well.
Incubate shaking at room temperature for 30 minutes (anti
rabbit IgG antibody: 1:3000 dilution in TBST).
9. Remove TAGO detection antibody and wash 4
times with TBST. Transfer freshly prepared ABTS/H202
solution to ELISA plate, 100 ~l per well. Incubate shaking
at room temperature for 20 minutes. (ABTS/HzOz solution: 1.0
~tl 30% H202 in 10 ml ABTS stock) .
10. Stop reaction by adding 50 ~1 5N HzS04
(optional), and determine O.D. at 410 nm.
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11. The maximal phosphotyrosine signal is
determined by subtracting the value of the negative
controls from the positive controls. The percent inhibition
of phosphotyrosine content for extract-containing wells is
then calculated, after subtraction of the negative
controls.
PDGF-R Assay
All cell culture media, glutamine, and fetal bovine
serum can be purchased from Gibco Life Technologies
(Grand Island, NY) unless otherwise specified. All cells
are grown in a humid atmosphere of 90-95o air and 5-l00
COZ at 37°C. All cell lines are routinely subcultured
twice a week and are negative for myco~lasma as
determined by the Mycotect method (Gibco).
For ELISA assays, cells (U1242, obtained from Joseph
Schlessinger, NYU) are grown to 80-90% confluency in
growth medium (MEM with 10% FBS, NEAA, 1 mM NaPyr and 2
mM GLN) and seeded in 96-well tissue culture plates in
0.5% serum at 25,000 to 30,000 cells per well. After
overnight incubation in 0.5% serum-containing medium,
cells are changed to serum-free medium and treated with
test compound for 2 hr in a 5% CO2, 37°C incubator. Cells
are then stimulated with ligand for 5-10 minute followed
by lysis with HNTG (20 mM Hepes, 150 mM NaCl, 10%
glycerol, 5 mM EDTA, 5 mM Na3V04, 0.2% Triton X-100, and 2
mM NaPyr). Cell lysates (0.5 mg/well in PBS) are
transferred to ELISA plates previously coated with
receptor-specific antibody and which had been blocked
with 5% milk in TBST (50 mM Tris-HC1 pH 7.2, 150 mM NaCl
and 0.1% Triton X-100) at room temperature for 30 min.
Lysates are incubated with shaking for 1 hour at room
temperature. The plates are washed with TBST four times
and then incubated with polyclonal anti-phosphotyrosine
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antibody at room temperature for 30 minutes. Excess
anti-phosphotyrosine antibody is removed by rinsing the
plate with TBST four times. Goat anti-rabbit IgG
antibody is added to the ELISA plate for 30 min at room
temperature followed by rinsing with TBST four more
times. ABTS (100 mM citric acid, 250 mM NazHPO~ and 0.5
mg/mL 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic
acid) ) plus H202 (1.2 mL 30% H20z to 10 ml ABTS) is added
to the ELISA plates to start color development.
Absorbance at 410 nm with a reference wavelength of 630
nm is recorded about 15 to 30 min after ABTS addition.
IGF-1 RECEPTOR Assay
The following protocol may be used to measure
phosphotyrosine level on IGF-1 receptor, which indicates
IGF-1 receptor tyrosine kinase activity.
Materials and Reagents.
a. The cell line used in this assay is 3T3/IGF-1R, a
cell line genetically engineered to overexpresses IGF-1
receptor.
b. NIH3T3/IGF-1R is grown in an incubator with 5% C02
at 37°C. The growth media is DMEM + 10% FBS (heat
inactivated)+ 2mM L-glutamine.
c. Affinity purified anti-IGF-1R antibody 17-69.
d. D-PBS:
KH2P04 0.20 g/1
KHZPOQ 2.16 g/1
KC1 0.20 g/1
NaCl 8.00 g/1 (pH 7.2)
e. Blocking Buffer: TBST plus 5% Milk (Carnation
Instant Non-Fat Dry Milk).
f. TBST buffer:
Tris-HC1 50 mM
NaCl 150mM (pH 7.2/HCl 10N)
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Triton X-100 0.1%
Stock solution of TBS (10X) is prepared, and
Triton X-100 is added to the buffer during
dilution.
g. HNTG buffer:
HEPES 20 mM
NaCl 150 mM (pH 7.2/HC1 1N)
Glycerol 10%
Triton X-100 0.2%
Stock solution (5X) is prepared and kept at 4°C.
h. EDTA/HC1: 0.5 M pH 7.0 (NaOH) as 100X stock.
i. Na3V04: 0.5 M as 100X stock and aliquots are kept
at 80°C.
j . Na4Pz0,: 0.2 M as 100X stock.
k. Insulin-like growth factor-1 from Promega (Cat#
65111 ) .
1. Rabbit polyclonal anti-phosphotyrosine antiserum.
m. Goat anti-rabbit IgG, POD conjugate (detection
antibody), Tago (Cat. No. 4520, Lot No. 1802): Tago, Inc.,
Burlingame, CA.
n. ABTS (2,2'-azinobis(3-ethylbenzthiazolinesulfonic
acid)) solution:
Citric acid 100 mM
NazHP04 2 5 0 mM ( pH 4 . 0 / 1 N HC1 )
ABTS 0.5 mg/ml
ABTS solution should be kept in dark and 4°C. The
solution should be discarded when it turns green.
o. Hydrogen Peroxide: 30% solution is kept in the
dark and at 4°C.
Procedure.
All the following steps are conducted at room
temperature unless specifically indicated otherwise. All
ELISA plate washings are performed by rinsing the plate
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with tap water three times, followed by one TBST rinse.
Pat plate dry with paper towels.
Cell Seeding:
1. The cells, grown in tissue culture dish
(Corning 25020-100) to 80-90% confluence, are harvested
with Trypsin-EDTA (0.25%, 0.5 ml/D-100, GIBCO).
2. Resuspend the cells in fresh DMEM + 10% FBS
+ 2mM L-Glutamine, and transfer to 96-well tissue culture
plate (Corning, 25806-96) at 20,000 cells/well (100
f.tl/well). Incubate for 1 day then replace medium to serum-
free medium (90/1) and incubate in 5% CO2 and 37°C
overnight.
ELISA Plate Coating and Blocking:
1. Coat the ELISA plate (Corning 25805-96) with
Anti-IGF-1R Antibody at 0.5 ~.g/well in 100 ~1 PBS at least
2 hours.
2. Remove the coating solution, and replace
with 100 ~1 Blocking Buffer, and shake for 30 minutes.
Remove the blocking buffer and wash the plate just before
adding lysate.
Assay Procedures:
1. The drugs are tested under serum-free condition.
2. Dilute drug stock (in 100% DMSO) 1:10 with DMEM
in 96-well poly-propylene plate, and transfer 10 ~,1/well of
this solution to the cells to achieve final drug dilution
1:100, and final DMSO concentration of 1.0%. Incubate the
cells in 5% COZ at 37°C for 2 hours.
3. Prepare fresh cell lysis buffer (HNTG*)
HNTG 2 ml
EDTA 0.1 ml
Na3V04 0.1 ml
Na4 ( P20, ) 0 . 1 ml
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Hz 0 7 . 3 ml
4. After drug incubation for two hours, transfer 10
~tl/well of 200nM IGF-1 Ligand in PBS to the cells (Final
Conc. is 20 nM), and incubate at 5% COz at 37°C for 10
minutes.
5. Remove media and add 100 ~1/well HNTG* and shake
for 10 minutes. Look at cells under microscope to see if
they are adequately lysed.
6. Use a 12-channel pipette to scrape the cells from
the plate, and homogenize the lysate by repeated aspiration
and dispensing. Transfer all the lysate to the antibody
coated ELISA plate, and shake for 1 hour.
7. Remove the lysate, wash the plate, transfer anti-
pTyr (1:3,000 with TBST) 100 ~l/well, and shake for 30
minutes.
8. Remove anti-pTyr, wash the plate, transfer TAGO
(1:3,000 with TBST) 100 ~1/well, and shake for 30 minutes.
9. Remove detection antibody, wash the plate, and
transfer fresh ABTS/H20z (1.2 ~1 Hz02 to 10 ml ABTS) 100
~.1/well to the plate to start color development.
Measure OD at 410 nm with a reference wavelength of
630 nm in Dynatec MR5000.
EGFR Assay
EGF Receptor kinase activity in cells genetically
engineered to express human EGF-R can be measured as
described below:
Materials and Reagents.
a. EGF Ligand: stock concentration = 16.5 ~M, EGF
201, TOYOBO, Co., Ltd. Japan.
b. 05-101 (UBI) (a monoclonal antibody recognizing
an EGFR extracellular domain).
c. Anti-phosphotyosine antibody (anti-Ptyr)
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(polyclonal).
d. Detection antibody: Goat anti-rabbit 1gG horse
radish peroxidase conjugate, TAGO, Inc., Burlingame,
CA.
e. TBST buffer:
Tris-HC1, pH 7 50 mM
NaCl 150 mM
Triton X-100 0.1
f. HNTG 5X stock:
HEPES 0.1 M
NaCl 0.75 M
Glycerol 50
Triton X-100 1.0%
g. ABTS stock:
Citric Acid 100 mM
Na3V04 250 mM
HCl, conc. 4.0 pH
ABTS* 0.5 mg/ml
Keep solution in dark
at 4C until used.
h. Stock reagents of:
EDTA 100 mM pH 7.0
Na3V0q 0.5 M
Na4 ( Pz 0, ) 0 . 2
M
Procedure.
Pre-coat ELISA Plate
1. Coat ELISA plates (Corning, 96 well, Cat. #25805-
96) with 05-101 antibody at 0.5 ~g per well in PBS, 150 ~,l
final volume/well, and store overnight at 4°C. Coated
plates are good for up to 10 days when stored at 4°C.
2. On day of use, remove coating buffer and replace
with blocking buffer (5% Carnation Instant NonFat Dry Milk
in PBS). Incubate the plate, shaking, at room temperature
(about 23°C to 25°C) for 30 minutes. Just prior to use,
remove blocking buffer and wash plate 4 times with TBST
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buffer.
Seeding Cells
1. NIH 3T3/C7 cell line (Honegger, et al., Cell
51:199-209, 1987) can be use for this assay.
2. Choose dishes having 80-90% confluence for the
experiment. Trypsinize cells and stop reaction by adding
10% CS DMEM medium. Suspend cells in DMEM medium (10% CS
DMEM medium) and centrifuge once at 1000 rpm at room
temperature for 5 minutes.
3. Resuspend cells in seeding medium (DMEM, 0.5%
bovine serum), and count the cells using trypan blue.
Viability above 90% is acceptable. Seed cells in DMEM
medium (0.5% bovine serum) at a density of 10,000 cells per
well, 100 ~l per well, in a 96 well microtiter plate.
Incubate seeded cells in 5% COz at 37°C for about 40 hours.
Assay Procedures.
1. Check seeded cells for contamination using an
inverted microscope. Dilute test compounds stock (10 mg/ml
in DMSO) 1:10 in DMEM medium, then transfer 5 ~l to a test
well for a test compounds drug dilution of 1:200 and a
final DMSO concentration of 1%. Control wells receive DMSO
alone. Incubate in 5% C02 at 37°C for one hour.
2. Prepare EGF ligand: dilute stock EGF in DMEM so
that upon transfer of 10 ~tl dilute EGF (1:12 dilution), 25
nM final concentration is attained.
3. Prepare fresh 10 ml HNTG* sufficient for 100 ~1
per well wherein HNTG* comprises: HNTG stock (2.0 ml),
milli-Q HZO (7.3 ml) , EDTA, 100 mM, pH 7.0 (0.5 ml) , Na3V0;
0 . 5 M ( 0 . 1 ml ) and Na4 ( PzO, ) , 0 . 2 M ( 0 . 1 ml ) .
4. Place on ice.
5. After two hours incubation with drug, add
prepared EGF ligand to cells, 10 ~1 per well, to yield a
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final concentration of 25 nM. Control wells receive DMEM
alone. Incubate, shaking, at room temperature, for 5
minutes.
6. Remove test compound, EGF, and DMEM. Wash cells
twice with PBS. Transfer HNTG* to cells, 100 ~1 per well.
Place on ice for 5 minutes. Meanwhile, remove blocking
buffer from other ELISA plate and wash with TBST as
described above.
7. With a pipette tip securely fitted to a
micropipettor, scrape cells from plate and homogenize cell
material by repeatedly aspirating and dispensing the HNTG*
lysis buffer. Transfer lysate to a coated, blocked, and
washed ELISA plate. Incubate shaking at room temperature
for one hour.
8. Remove lysate and wash 4 times with TBST.
Transfer freshly diluted anti-Ptyr antibody to ELISA plate
at 100 ~l per well. Incubate shaking, at room temperature
for 30 minutes in the presence of the anti-Ptyr antiserum
(1:3000 dilution in TBST).
9. Remove the anti-Ptyr antibody and wash 4 times
with TBST. Transfer the freshly diluted TAGO 30 anti-rabbit
IgG antibody to the ELISA plate at 100 ~1 per well.
Incubate shaking at room temperature for 30 minutes (anti-
rabbit IgG antibody: 1:3000 dilution in TBST).
10. Remove detection antibody and wash 4 times with
TBST. Transfer freshly prepared ABTS/H202 solution to ELISA
plate, 100 ~l per well. Incubate at room temperature for
20 minutes. ABTS/HzOz solution: 1.2 ~1 30% HZOZ in 10 ml
ABTS stock.
11. Stop reaction by adding 50 ~1 5N HzS04 (optional),
and determine O.D. at 410 nm.
12. The maximal phosphotyrosine signal is determined
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by subtracting the value of the r_egative controls from the
positive controls. The percent inhibition of
phosphotyrosine content for extract-containing wells is
then calculated, after subtraction of the negative
controls.
Met Autophosphorylation Assay
This assay determines Met tyrosine kinase activity by
analyzing Met protein tyrosine kinase levels on the Met
receptor.
Reagents
a. HNTG (5X stock solution): Dissolve 23.83 g HEPES
and 43.83 g NaCl in about 350 ml dHzO. Adjust pH to 7.2
with HC1 or NaOH, add 500 ml glycerol and 10 ml Triton X-
100, mix, add dH20 to 1 L total volume. To make 1 L of 1X
working solution add 200 ml 5X stock solution to 800 ml
dHZO, check and adjust pH as necessary, store at 4°C.
b. PBS (Dulbecco's Phosphate-Buffered Saline), Gibco
Cat. # 450-1300EB (1X solution).
c. Blocking Buffer: in 500 ml dH20 place 100 g BSA,
12.1 g Tris-pH7.5, 58.44 g NaCl and 10 ml Tween-20, dilute
to 1 L total volume.
d. Kinase Buffer: To 500 ml dH~O add 12.1 g TRIS (pH
7.2), 58.4 g NaCl, 40.7 g MgClz and 1.9 g EGTA, bring to 1 L
total volume with dHzO.
e. PMSF (Phenylmethylsulfonyl fluoride), Sigma Cat.
# P-7626, to 435.5 mg, add 100% ethanol to 25 ml total
volume, vortex.
f. ATP (Bacterial Source), Sigma Cat. # A-7699,
store powder at -20°C, to make up solution for use,
dissolve 3.31 mg in 1 ml dH20.
g. RC-20H HRPO Conjugated Anti-Phosphotyrosine,
Transduction Laboratories Cat. # E120H.
h. Pierce 1-Step T"' Turbo TMB-ELISA (3, 3' , 5, 5' -
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tetramethylbenzidine, Pierce Cat. # 34022.
i . HzS04, add 1 ml conc . ( 18 N) to 35 ml dH20.
j. TRIS HCL, Fischer Cat. # BP152-5, to 121.14 g of
material, add 600 ml MilliQ H20, adjust pH to 7.5 (or 7.2)
with HC1, bring volume to 1 L with MilliQ H20.
k. NaCl, Fischer Cat. # S271-10, make up 5M
solution.
1. Tween-20, Fischer Cat. # S337-500.
m. Na3V04, Fischer Cat. # 5454-50, to 1.8 g material
add 80 ml MilliQ H20, adjust pH to 10.0 with HCl or NaOH,
boil in microwave, cool, check pH, repeat procedure until
pH stable at 10.0, add MilliQ H20 to 100 ml total volume,
make 1 ml aliquots and store at -80°C.
n. MgCl2, Fischer Cat. # M33-500, make up 1M
solution.
o. HEPES, Fischer Cat. # BP310-500, to 200 ml MilliQ
HzO, add 59.6 g material, adjust pH to 7.5, bring volume to
250 ml total, sterile filter.
p. Albumin, Bovine (BSA), Sigma Cat. # A-4503, to 30
grams material add sterile distilled water to make total
volume of 300 ml, store at 4°C.
q. TBST Buffer: to approx. 900 ml dHZO in a 1 L
graduated cylinder add 6.057 g TRIS and 8.766 g NaCl, when
dissolved, adjust pH to 7.2 with HCl, add 1.0 ml Triton X-
100 and bring to 1 L total volume with dH20.
r. Goat Affinity purified antibody Rabbit IgG (whole
molecule), Cappel Cat. # 55641.
s. Anti h-Met (C-28) rabbit polyclonal IgG antibody,
Santa Cruz Chemical Cat. # SC-161.
t. Transiently Transfected EGFR/Met chimeric cells
(EMR) (Komada, et al., Oncogene, 8:2381-2390 (1993).
u. Sodium Carbonate Buffer, (Na2C04, Fischer Cat. #
S495): to 10.6 g material add 800 ml MilliQ HzO, when
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dissolved adjust pH to 9.6 with NaOH, bring up to 1 L tc-al
volume with MilliQ H20, filter, store at 4°C.
Procedure
All of the following steps are conducted at room
temperature unless it is specifically indicated otherwise.
All ELISA plate washing is by rinsing 4X with TBST.
EMR Lysis
This procedure can be performed the night before or
immediately prior to the start of receptor capture.
1. Quick thaw lysates in a 37° C waterbath with a
swirling motion until the last crystals disappear.
2. Lyse cell pellet with 1X HNTG containing 1 mM
PMSF. Use 3 ml of HNTG per 15 cm dish of cells. Add 1/2
the calculated HNTG volume, vortex the tube for 1 min., add
the remaining amount of HNTG, vortex for another min.
3. Balance tubes, centrifuge at 10,000 x g for 10
min at 4°C.
4. Pool supernatants, remove an aliquot for protein
determination.
5. Quick freeze pooled sample in dry ice/ethanol
bath. This step is performed regardless of whether lysa~e
will be stored overnight or used immediately following
protein determination.
6. Perform protein determination using standard
bicinchoninic acid (BCA) method (BCA Assay Reagent Kit from
Pierce Chemical Cat. # 23225).
ELISA Procedure
1. Coat Corning 96 well ELISA plates with 5 ~g per
well Goat anti-Rabbit antibody in Carbonate Buffer for a
total well volume of 50 ~tl. Store overnight at 4°C.
2. Remove unbound Goat anti-rabbit antibody by
inverting plate to remove liquid.
3. Add 150 ~1 of Blocking Buffer to each well.
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Incubate for 30 min. with shaking.
4. Wash 4X with TBST. Pat plate on a paper towel to
remove excess liquid and bubbles.
5. Add leg per well of Rabbit anti-Met antibody
diluted in TBST for a total well volume of 100 ~tl.
6. Dilute lysate in HNTG (90 ~tg lysate/100~1)
7. Add 100 ~1 of diluted lysate to each well. Shake
for 60 min.
8. Wash 4X with TBST. Pat on paper towel to remove
excess liquid and bubbles.
9. Add 50 ~1 of 1X lysate buffer per well.
10. Dilute compounds/extracts 1:10 in 1X Kinase
Buffer in a polypropylene 96 well plate.
11. Transfer 5.5 ~l of diluted compound to ELISA
plate wells. Incubate at room temperature with shaking for
min.
12. Add 5.5 ~1 of 60 ~M ATP solution per well.
Negative controls do not receive any ATP. Incubate for 90
min., with shaking.
20 13. Wash 4X with TBST. Pat plate on paper towel to
remove excess liquid and bubbles.
14. Add 100 ~l per well of RC20 (1:3000 dilution in
Blocking Buffer). Incubate 30 min. with shaking.
15. Wash 4X with TBST. Pat plate on paper towel to
remove excess liquid and bubbles.
16. Add 100 ~1 per well of Turbo-TMB. Incubate with
shaking for 30-60 min.
17. Add 100 ~l per well of 1M HZSOQ to stop reaction.
18. Read assay on Dynatech MR7000 ELISA reader. Test
Filter = 450 nm, reference filter = 410 nm.
Biochemical src assay
This assay is used to determine src protein kinase
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activity measuring phosphorylation of a biotinylated
peptide as the readout.
Materials and Reagents:
a. Yeast transformed with src (Sugen, Inc., Redwood
City, California).
b. Cell lysates: Yeast cells expressing src are
pelleted, washed once with water, re-pelleted and stored at
-80°C until use.
c. N-terminus biotinylated EEEYEEYEEEYEEEYEEEY is
prepared by standard procedures well known to those skilled
in the art.
d. DMSO: Sigma, St. Louis, M0.
e. 96 Well ELISA Plate: Corning 96 Well Easy Wash,
Modified flat Bottom Plate, Corning Cat. #25805-96.
f. NUNC 96-well V-bottom polypropylene plates for
dilution of compounds: Applied Scientific Cat. # A-72092.
g. Vecastain ELITE ABC reagent: Vector, Burlingame,
CA.
h. Anti-src (327) mab: Schizosaccharomyces Pombe is
used to express recombinant Src (Superti-Furga, et al.,
EMBO J., 12:2625-2634, Superti-Furga, et al., Nature
Biochem., 14:600-605). S. Pombe strain SP200 (h-s leul.32
ura4 ade210) is grown as described and transformations are
pRSP expression plasmids are done by the lithium acetate
method (Superti-Furga, supra). Cells are grown in the
presence of 1 ~,M thiamine to repress expression from the
nmtl promoter or in the absence of thiamine to induce
expression.
i. Monoclonal anti-phosphotyrosine, UBI 05-321 (UB40
may be used instead).
j. Turbo TMB-ELISA peroxidase substrate: Pierce
Chemical.
Buffer Solutions
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a. PBS (Dulbecco's Phosphate-Buffered Saline): GIBCO
PBS, GIBCO Cat. # 450-1300EB.
b. Blocking Buffer: 5% Non-fat milk (Carnation) in
PBS.
c. Carbonate Buffer: Na2C04 from Fischer, Cat. #
5495, make up 100 mM stock solution.
d. Kinase Buffer: 1.0 ml (from 1M stock solution)
MgCl2, 0.2 ml (from a 1M stock solution) MnCl2, 0.2 ml
(from a 1M stock solution) DTT, 5.0 ml (from a 1M stock
solution) HEPES, 0.1 ml TX-100, bring to 10 ml total volume
with MilliQ H20.
e. Lysis Buffer: 5.0 HEPES (from 1M stock
solution.), 2.74 ml NaCl (from 5M stock solution), 10 ml
glycerol, 1.0 ml TX-100, 0.4 ml EDTA (from a 100 mM stock
solution), 1.0 ml PMSF (from a 100 mM stock solution), 0.1
ml Na3V09 (from a 0.1 M stock solution), bring to 100 ml
total volume with MilliQ H20.
f. ATP: Sigma Cat. # A-7699, make up 10 mM stock
solution (5.51 mg/ml).
g TRIS-HCl: Fischer Cat. # BP 152-5, to 600 ml
MilliQ H20 add 121.14 g material, adjust pH to 7.5 with HCl,
bring to 1 L total volume with MilliQ H20.
h. NaCl: Fischer Cat. # S271-10, Make up 5M stock
solution with MilliQ H20.
i. Na3V04: Fischer Cat. # 5454-50, to 80 ml MilliQ
HzO, add 1.8 g material, adjust pH to 10.0 with HC1 or NaOH,
boil in a microwave, cool, check pH, repeat pH adjustment
until pH remains stable after heating/cooling cycle, bring
to 100 ml total volume with MilliQ H20, make 1 ml aliquots
and store at -80°C.
j. MgCl2: Fischer Cat. # M33-500, make up 1M stock
solution with MilliQ H20.
k. HEPES: Fischer Cat. # BP 310-500, to 200 ml
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MilliQ H,O, add 59.6 g material, adjust pH to 7.5, bring ~.o
250 ml total volume with MilliQ H20, sterile filter (1M
stock solution) .
1. TBST Buffer: TBST Buffer: To 900 ml dHzO add
6.057 g TRIS and 8.766 g NaCl, adjust pH to 7.2 with HC1,
add 1.0 ml Triton-X100, bring to 1 L total volume with dH20.
m. MnCl2: Fischer Cat. # M87-100, make up 1M stock
solution with MilliQ H20.
n. DTT: Fischer Cat. # BP172-5.
0. TBS (TRIS Buffered Saline): to 900 ml MilliQ H,0
add 6.057 g TRIS and 8.777 g NaCl, bring to 1 L total
volume with MilliQ HzO.
p. Kinase Reaction Mixture: Amount per assay plate
(100 wells): 1.0 ml Kinase Buffer, 200 ~g GST-' , bring to
final volume of 8.0 ml with MilliQ HzO.
g. Biotin labeled EEEYEEYEEEYEEEYEEEY: Make peptide
stock solution (lmM, 2.98 mg/ml) in water fresh just before
use.
r. Vectastain ELITE ABC reagent: To prepare 14 ml of
working reagent, add 1 drop of reagent A to 15 ml TBST and
invert tube several times to mix. Then add 1 drop of
reagent B. Put tube on orbital shaker at room temperature
and mix for 30 minutes.
Procedures:
Preparation of src coated ELISA plate.
1. Coat ELISA plate with 0.5 ~.g/well anti-src mab in
100 ~l of pH 9.6 sodium carbonate buffer, hold at 4°C
overnight.
2. Wash wells once with PBS.
3. Block plate with 0.15 ml 5% milk in PBS for 30
min. at room temperature.
4. Wash plate 5X with PBS.
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5. Add 10 ~g/well of src transformed yeast lysates
diluted in Lysis Buffer (0.1 ml total volume per well).
(Amount of lysate may vary between batches.) Shake plate
for 20 minutes at room temperature.
Preparation of phosphotyrosine antibody-coated ELISA
plate.
1. 4610 plate: coat 0.5 ~g/well 4610 in 100 ~l PBS
overnight at 4°C and block with 150 ~l of 5% milk in PBS
for 30 minutes at room temperature.
Kinase assay procedure.
1. Remove unbound proteins from plates and wash
plates 5X with PBS.
2. Add 0.08 ml Kinase Reaction Mixture per well
(containing 10 ~1 of lOX Kinase Buffer and 10 ~M (final
concentration) biotin-EEEYEEYEEEYEEEYEEEY per well diluted
in water.
3. Add 10 ~tl of compound diluted in water containing
10% DMSO and pre-incubate for 15 minutes at room
temperature.
4. Start kinase reaction by adding 10 ul/well of
0.05 mM ATP in water (5 ~M ATP final).
5. Shake ELISA plate for 15 min. at room
temperature.
6. Stop kinase reaction by adding 10 ~,l of 0.5 M
EDTA per well.
7. Transfer 90 ~tl supernatant to a blocked 4610
coated ELISA plate.
8. Incubate for 30 min. while shaking at room
temperature.
9. Wash plate 5X with TBST.
10. Incubate with Vectastain ELITE ABC reagent (100
~.1/well) for 30 min. at room temperature.
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11. Wash the wells 5X with TBST.
12. Develop with Turbo TMB.
Biochemical lck Assay
This assay is used to determine lck protein kinase
activities measuring phosphorylation of GST-~ as the
readout.
Materials and Reagents:
a. Yeast transformed with lck. Schizosaccharomyces
Pombe is used to express recombinant Lck (Superti-Furga, et
al., EMBO J, 12:2625-2634, Superti-Furga, et al., Nature
Biotech., 14:600-605). S. Pombe strain SP200 (h-s leul.32
ura4 ade210) is grown as described and transformations with
pRSP expression plasmids are done by the lithium acetate
method (Superti-Furga, supra). Cells are grown in the
presence of 1 ~M thiamine to induce expression.
b. Cell lysates: Yeast cells expressing lck are
pelleted, washed once in water, re-pelleted and stored
frozen at -80°C until use.
c. GST-~: DNA encoding for GST-~ fusion protein for
expression in bacteria obtained from Arthur Weiss of the
Howard Hughes Medical Institute at the University of
California, San Francisco. Transformed bacteria are grown
overnight while shaking at 25°C. GST-~ is purified by
glutathione affinity chromatography, Pharmacia, Alameda,
CA.
d. DMSO: Sigma, St. Louis, MO.
e. 96-Well ELISA plate: Corning 96 Well Easy Wash,
Modified Flat Bottom Plate, Corning Cat. #25805-96.
f. NUNC 96-well V-bottom polypropylene plates for
dilution of compounds: Applied Scientific Cat: # AS-72092.
g. Purified Rabbit anti-GST antiserum: Amrad
Corporation (Australia) Cat. #90001605.
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h. Goat anti-Rabbit-IgG-HRP: Amersham Cat. #
V010301.
i. Sheep ant-mouse IgG (H+L): Jackson Labs Cat. #
5215-005-003.
j. Anti-Lck (3A5) mab: Santa Cruz Biotechnology Cat
# sc-433.
k. Monoclonal anti-phosphotyrosine UBI 05-321 (UB40
may be used instead).
Huffer solutions:
a. PBS (Dulbecco's Phosphate-Buffered Saline) 1X
solution: GIBCO PBS, GIBCO Cat. # 450-1300EB.
b. Blocking Buffer: 100 g. BSA, 12.1 g. TRIS
(pH7.5), 58.44 g NaCl, 10 ml Tween-20, bring up to 1 L
total volume with MilliQ H20.
c. Carbonate Buffer: Na2C04 from Fischer, Cat. #
5495, make up 100 mM solution with MilliQ H20.
d. Kinase Buffer: 1.0 ml (from 1M stock solution)
MgCl2, 0.2 ml (from a 1M stock solution) MnCl2, 0.2 ml (from
a 1M stock solution) DTT, 5.0 ml (from a 1M stock solution)
HEPES, 0.1 ml TX-100, bring to 10 ml total volume with
MilliQ H20.
e. Lysis Buffer: 5.0 HEPES (from 1M stock
solution.), 2.74 ml NaCl (from 5M stock solution), 10 ml
glycerol, 1.0 ml TX-100, 0.4 ml EDTA (from a 100 mM stock
solution), 1.0 ml PMSF (from a 100 mM stock solution), 0.1
ml Na3V04 (from a 0.1 M stock solution), bring to 100 ml
total volume with MilliQ HzO.
f. ATP: Sigma Cat. # A-7699, make up 10 mM stock
solution (5.51 mg/ml).
g TRIS-HC1: Fischer Cat. # BP 152-5, to 600 ml
MilliQ Hz0 add 121.14 g material, adjust pH to 7.5 with HC1,
bring to 1 L total volume with MilliQ HzO.
h. NaCl: Fischer Cat. # S271-10, Make up 5M stock
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solution with MilliQ H~O.
i Na3V04: Fischer Cat. # 5454-50, to 80 ml MilliQ
HZO, add 1.8 g material, adjust pH to 10.0 with HC1 or NaOH,
boil in a microwave, cool, check pH, repeat pH adjustment
until pH remains stable after heating/cooling cycle, bring
to 100 ml total volume with MilliQ HzO, make 1 ml aliquots
and store at -80°C.
j. MgCl2: Fischer Cat. # M33-500, make up 1M stock
solution with MilliQ H20.
k. HEPES: Fischer Cat. # BP 310-500, to 200 ml
MilliQ H20, add 59.6 g material, adjust pH to 7.5, bring to
250 ml total volume with MilliQ H20, sterile filter (1M
stock solution).
1. Albumin, Bovine (BSA), Sigma Cat. # A4503, to 150
ml MilliQ H20 add 30 g material, bring 300 ml total volume
with MilliQ HzO, filter through 0.22 ~m filter, store at
4°C.
m. TBST Buffer: To 900 ml dHzO add 6.057 g TRIS and
8.766 g NaCl, adjust pH to 7.2 with HC1, add 1.0 ml Triton-
X100, bring to 1 L total volume with dHzO.
n. MnCl2: Fischer Cat. # M87-100, make up 1M stock
solution with MilliQ HZO.
o. DTT: Fischer Cat. # BP172-5.
p. TBS (TRIS Buffered Saline): to 900 ml MilliQ Hz0
add 6.057 g TRIS and 8.777 g NaCl, bring to 1 L total
volume with MilliQ HzO.
q Kinase Reaction Mixture: Amount per assay plate
(100 wells): 1.0 ml Kinase Buffer, 200 ~g GST-~, bring to
final volume of 8.0 ml with MilliQ HZO.
Procedures:
Preparation of Lck coated ELISA plate.
1. Coat 2.0 ~g/well Sheep anti-mouse IgG in 100 ~1
of pH 9.6 sodium carbonate buffer at 4°C overnight.
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2. Wash well once with PBS.
3. Block plate with 0.15 ml of blocking Buffer for
30 min. at room temp.
4. Wash plate 5X with PBS.
5. Add 0.5 ~g/well of anti-lck (mab 3A5) in 0.1 ml
PBS at room temperature for 1-2 hours.
6. Wash plate 5X with PBS.
7. Add 20 ~g/well of lck transformed yeast lysates
diluted in Lysis Buffer (0.1 ml total volume per well).
Shake plate at 4°C overnight to prevent loss of activity.
Preparation of phosphotyrosine antibody-coated ELISA
plate.
1. UB40 plate: 1.0 ~g/well UB40 in 100 ~1 of PBS
overnight at 4°C and block with 150 ~1 of Blocking Buffer
for at least 1 hour.
Kinase assay procedure.
1. Remove unbound proteins from plates and wash
plates 5X with PBS.
2. Add 0.08 ml Kinase Reaction Mixture per well
(containing 10 ~l of lOX Kinase Buffer and 2 ~,g GST-~ per
well diluted with water).
3. Add 10 ~1 of compound diluted in water containing
10% DMSO and pre-incubate for 15 minutes at room
temperature.
4. Start kinase reaction by adding 10~1/well of 0.1
mM ATP in water (10 ~tM ATP final).
5. Shake ELISA plate for 60 min. at room
temperature.
6. Stop kinase reaction by adding 10 ~,1 of 0.5 M
EDTA per well.
7. Transfer 90 ~1 supernatant to a blocked 4610
coated ELISA plate from section B, above.
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8. Incubate while shakir~g for 30 min. at room
temperature.
9. Wash plate 5X with TBST.
10. Incubate with Rabbit anti-GST antibody at 1:5000
dilution in 100 ~1 TBST for 30 min. at room temperature.
11. Wash the wells 5X with TBST.
12. Incubate with Goat anti-Rabbit-IgG-HRP at
1:20,000 dilution in 100 u1 of TBST for 30 min. at room
temperature.
13. Wash the wells 5X with TBST.
14. Develop with Turbo TMB.
Assay measuring phosphorylating function of RAF.
The following assay reports the amount of RAF-
catalyzed phosphorylation of its target protein MEK as well
as MEK's target MAPK. The RAF gene sequence is described
in Bonner et al., 1985, Molec. Cell. Biol., 5:1400-1407,
and is readily accessible in multiple gene sequence data
banks. Construction of the nucleic acid vector and cell
lines utilized for this portion of the invention are fully
described in Morrison et al., 1988, Proc. Natl. Acad. Sci.
USA, 85:8855-8859.
Materials and Reagents
1. Sf9 (Spodoptera frugiperda) cells, GIBCO-BRL,
Gaithersburg, MD.
2. RIPA buffer: 20 mM Tris/HC1 pH 7.4, 137 mM NaCl,
10% glycerol, 1 mM PMSF, 5 mg/L Aprotenin, 0.5 % Triton X-
100,
3. Thioredoxin-MEK fusion protein (T-MEK): T-MEK
expression and purification by affinity chromatography are
performed according to the manufacturer's procedures.
Catalog# K 350-O1 and R 350-40, Invitrogen Corp., San
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Diego, CA
4. His-MAPK (ERK 2), His-tagged MAPK is expressed in
XL1 Blue cells transformed with pUCl8 vector encoding His-
MAPK. His-MAPK is purified by Ni-affinity chromatography.
Cat# 27-4949-O1, Pharmacia, Alameda, CA, as described
herein.
5. Sheep anti mouse IgG: Jackson laboratories, West
Grove, PA. Catalog, # 515-006-008, Lot# 28563
6. RAF-1 protein kinase specific antibody: URP2653
from UBI.
7. Coating buffer: PBS, phosphate buffered saline,
GIBCO-BRL, Gaithersburg, MD.
8. Wash buffer: TBST (50 mM Tris/HCL pH 7.2, 150 mM
NaCl, 0.1 % Triton X-100).
9. Block buffer: TBST, 0.1 % ethanolamine pH 7.4
10. DMSO, Sigma, St. Louis, MO
11. Kinase buffer (KB): 20 mM HEPES/HCl pH 7.2, 150
mM NaCl, 0.1 % Triton X-100, 1 mM PMSF, 5 mg/L Aprotenin,
75 mM sodium orthovanadate, 0.5 MM DTT and 10 mM MgCl2.
12. ATP mix: 100 mM MgClz, 300 mM ATP, 10 mCi y33P ATP
(Dupont-NEN)/mL.
13 Stop solution: 1% phosphoric acid, Fisher,
Pittsburgh, PA.
14. Wallac Cellulose Phosphate Filter mats, Wallac,
Turku, Finnland.
15. Filter wash solution: 1% phosphoric acid, Fisher,
Pittsburgh, PA.
16. Tomtec plate harvester, Wallac, Turku, Finnland.
17. Wallac beta plate reader # 1205, Wallac, Turku,
Finnland.
18. NUNC 96-well V bottom polypropylene plates for
compounds Applied Scientific Catalog # AS-72092.
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Procedure
All of the following steps are conducted at room
temperature unless specifically indicated otherwise.
1. ELISA plate coating: ELISA wells are coated with
100 ml of Sheep anti mouse affinity purified antiserum (1
mg/100 mL coating buffer) over night at 4° C. ELISA plates
can be used for two weeks when stored at 4° C.
2. Invert the plate and remove liquid. Add 100 mL
of blocking solution and incubate for 30 min.
3. Remove blocking solution and wash four times with
wash buffer. Pat the plate on a paper towel to remove
excess liquid.
4. Add 1 mg of antibody specific for RAF-1 to each
well and incubate for 1 hour. Wash as described in step 3.
5. Thaw lysates from RAS/RAF infected Sf9 cells and
dilute with TBST to 10 mg/100 mL. Add 10 mg of diluted
lysate to the wells and incubate for 1 hour. Shake the
plate during incubation. Negative controls receive no
lysate. Lysates from RAS/RAF infected Sf9 insect cells are
prepared after cells are infected with recombinant
baculoviruses at a MOI of 5 for each virus, and harvested
48 hours later. The cells are washed once with PBS and
lysed in RIPA buffer. Insoluble material is removed by
centrifugation (5 min at 10,000 x g). Aliquots of lysates
are frozen in dry ice/ethanol and stored at -80 °C until
use.
6. Remove non-bound material and wash as outlined
above (step 3).
7. Add 2 mg of T-MEK and 2 mg of His-MAEPK per well
and adjust the volume to 40 ml with kinase buffer. Methods
for purifying T-MEK and MAPK from cell extracts are
provided herein by example.
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8. Pre-dilute compounds (stock solution 10 mg/ml
DMSO) or extracts 20 fold in TBST plus 1% DMSO. Add 5 ml
of the pre-diluted compounds/extracts to the wells
described in step 6. Incubate for 20 min. Controls receive
no drug.
9. Start the kinase reaction by addition of 5 ml ATP
mix, Shake the plates on an ELISA plate shaker during
incubation.
10. Stop the kinase reaction after 60 min by addition
of 30 mL stop solution to each well.
11. Place the phosphocellulose mat and the ELISA
plate in the Tomtec plate harvester. Harvest and wash the
filter with the filter wash solution according to the
manufacturer's recommendation. Dry the filter mats. Seal
the filter mats and place them in the holder. Insert the
holder into radioactive detection apparatus and quantify
the radioactive phosphorous on the filter mats.
Alternatively, 40 mL aliquots from individual wells of
the assay plate can be transferred to the corresponding
positions on the phosphocellulose filter mat. After air
drying the filters, put the filters in a tray. Gently rock
the tray, changing the wash solution at 15 min intervals
for 1 hour. Air-dry the filter mats. Seal the filter mats
and place them in a holder suitable for measuring the
radioactive phosphorous in the samples. Insert the holder
into a detection device and quantify the radioactive
phosphorous on the filter mats.
CDK2/CYClin A - Inhibition Assay
This assay analyzes the protein kinase activity of
CDK2 in exogenous substrate.
Reagents:
A. Buffer A: (80 mM Tris ( pH 7.2), 40 mM MgCl2),
4.84 g. Tris (F. W. =121.1 g/mol), 4.07 g. MgClZ (F. W.=203.31
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g/mol) dissolved in 500 ml H~O. Adjust pH to 7.2 with HC1.
B. Histone H1 solution (0.45 mg/ml Histone H1 and 20
mM HEPES pH 7.2: 5 mg Histone H1 (Boehinger Mannheim) in
11.111 ml 20 mM HEPES pH 7.2 (477 mg HEPES (F. W.= 238.3
g/mol) dissolved in 100 ml ddH20, stored in 1 ml aliquots at
-80° C.
C. ATP solution (60 ~M ATP, 300 ~g/ml BSA, 3 mM
DTT): 120 ~1 10 mM ATP, 600 ~tl 10 mg/ml BSA to 20 ml,
stored in 1 ml aliquots at -80° C.
D. CDK2 solution: cdk2/cyclin A in 10 mM HEPES pH
7.2, 25 mM NaCl, 0.5 mM DTT, 10% glycerol, stored in 9 ~1
aliquots at
-80° C.
Protocol
1. Prepare solutions of inhibitors at three times
the desired final assay concentration in ddH20/15% DMSO by
volume.
2. Dispense 20 ~tl of inhibitors to wells of
polypropylene 96-well plates (or 20 ~1 15% DMSO for
positive and negative controls).
3. Thaw Histone H1 solution (1 ml/plate), ATP
solution (1 ml/plate plus 1 aliquot for negative control),
and CDK2 solution (9 ~tl/plate). Keep CDK2 on ice until
use. Aliquot CDK2 solution appropriately to avoid repeated
freeze-thaw cycles.
4. Dilute 9 ~,l CDK2 solution into 2.1 ml Buffer A
(per plate). Mix. Dispense 20 ~1 into each well.
5. Mix 1 ml Histone H1 solution with 1 ml ATP
solution (per plate) into a 10 ml screw cap tube. Add y33P
ATP to a concentration of 0.15 ~Ci/20~1 (0.15 ~Ci/well in
assay). Mix carefully to avoid BSA frothing. Add 20 ~1 to
appropriate wells. Mix plates on plate shaker. For
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negative control, mix ATP solution with an equal amount of
20 mM HEPES pH 7.2 and add y33P ATP to a concentration of
0.15 ~Ci/20~1 solution. Add 20 ~1 to appropriate wells.
6. Let reactions proceed for 60 minutes.
7. Add 35 ~1 10% TCA to each well. Mix plates on
plate shaker.
8. Spot 40 ~tl of each sample onto P30 filter mat
squares. Allow mats to dry (approx. 10-20 minutes).
9 Wash filter mats 4 X 10 minutes with 250 ml 1%
phosphoric acid (10 ml phosphoric acid per liter ddH20).
10. Count filter mats with beta plate reader.
Cellular/Biologic Assays
PDGF-Induced BrdU Incorporation Assay
Materials and Reagents:
(1) PDGF: human PDGF B/B, 1276-956, Boehringer
Mannheim, Germany.
(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4),
Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(3) FixDenat: fixation solution (ready to use), Cat.
No. 1 647 229, Boehringer Mannheim, Germany.
(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated
with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim,
Germany.
(5) TMB Substrate Solution: tetramethylbenzidine
(TMB), ready to use, Cat. No. 1 647 229, Boehringer
Mannheim, Germany.
(6) PBS Washing Solution . 1X PBS, pH 7.4 (Sugen,
Inc., Redwood City, California).
(7) Albumin, Bovine (BSA): fraction V powder, A-8551,
Sigma Chemical Co., USA.
(8) 3T3 cell line genetically engineered to express
human PDGF-R.
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Protocol
(1) Cells are seeded at 8000 cells/well in DMEM, 10%
CS, 2mM Gln in a 96 well plate. Cells are incubated
overnight at 37°C in 5% COz.
(2) After 24 hours, the cells are washed with PBS,
and then are serum starved in serum free medium (0%CS DMEM
with 0.1% BSA) for 24 hours.
(3) On day 3, ligand (PDGF, 3.8 nM, prepared in DMEM
with 0.1% BSA) and test compounds are added to the cells
simultaneously. The negative control wells receive serum
free DMEM with 0.1% BSA only, the positive control cells
receive the ligand (PDGF) but no test compound. Test
compounds are prepared in serum free DMEM with ligand in a
96 well plate, and serially diluted for 7 test
concentrations.
(4) After 20 hours of ligand activation, diluted BrdU
labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the
cells are incubated with BrdU (final concentration=10 ~.M)
for 1.5 hours.
(5) After incubation with labeling reagent, the
medium is removed by decanting and tapping the inverted
plate on a paper towel. FixDenat solution is added (50
~1/well) and the plates are incubated at room temperature
for 45 minutes on a plate shaker.
(6) The FixDenat solution is thoroughly removed by
decanting and tapping the inverted plate on a paper towel.
Milk is added (5% dehydrated milk in PBS, 200 ~tl/well) as
a blocking solution and the plate is incubated for 30
minutes at room temperature on a plate shaker.
(7) The blocking solution is removed by decanting and
the wells are washed once with PBS. Anti-BrdU-POD solution
(1:100 dilution in PBS, 1% BSA) is added (100 ~l/well) and
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the plate is incubated for 90 minutes at room temperature
on a plate shaker.
(8) The antibody conjugate is thoroughly removed by
decanting and rinsing the wells 5 times with PBS, and the
plate is dried by inverting and tapping on a paper towel.
(9) TMB substrate solution is added (100 ~1/well) and
incubated for 20 minutes at room temperature on a plate
shaker until color development is sufficient for
photometric detection.
(10) The absorbance of the samples are measured at 410
nm (in "dual wavelength" mode with a filter reading at 490
nm, as a reference wavelength) on a Dynatech ELISA plate
reader.
EGF-Induced BrdU Incorporation Assay
Materials and Reagents
(1) EGF: mouse EGF, 201, Toyobo, Co., Ltd. Japan.
(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4),
Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(3) FixDenat: fixation solution (ready to use), Cat.
No. 1 647 229, Boehringer Mannheim, Germany.
(4) Anti-BrdU-POD: mouse monoclonal antibody
conjugated with peroxidase, Cat. No. 1 647 229, Boehringer
Mannheim, Germany.
(5) TMB Substrate Solution: tetramethylbenzidine
(TMB), ready to use, Cat. No. 1 647 229, Boehringer
Mannheim, Germany.
(6) PBS Washing Solution . 1X PBS, pH 7.4 (Sugen,
Inc., Redwood City, California).
(7) Albumin, Bovine (BSA): fraction V powder, A-8551,
Sigma Chemical Co., USA.
(8) 3T3 cell line genetically engineered to express
human EGF-R.
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Protocol
(1) Cells are seeded at 8000 cells/well in 10% CS,
2mM Gln in DMEM, in a 96 well plate. Cells are incubated
overnight at 37°C in 5% CO2.
(2) After 24 hours, the cells are washed with PBS,
and then are serum starved in serum free medium (0% CS DMEM
with 0.1% BSA) for 24 hours.
(3) On day 3, ligand (EGF, 2 nM, prepared in DMEM
with 0.1% BSA) and test compounds are added to the cells
simultaneously. The negative control wells receive serum
free DMEM with 0.1% BSA only, the positive control cells
receive the ligand (EGF) but no test compound. Test
compounds are prepared in serum free DMEM with ligand in a
96 well plate, and serially diluted for 7 test
concentrations.
(4) After 20 hours of ligand activation, diluted BrdU
labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the
cells are incubated with BrdU (final concentration = 10 ~M)
for 1.5 hours.
(5) After incubation with labeling reagent, the
medium is removed by decanting and tapping the inverted
plate on a paper towel. FixDenat solution is added (50
~tl/well) and the plates are incubated at room temperature
for 45 minutes on a plate shaker.
(6) The FixDenat solution is thoroughly removed by
decanting and tapping the inverted plate on a paper towel.
Milk is added (5% dehydrated milk in PBS, 200 ~,1/well) as a
blocking solution and the plate is incubated for 30 minutes
at room temperature on a plate shaker.
(7) The blocking solution is removed by decanting and
the wells are washed once with PBS. Anti-BrdU-POD solution
(1:100 dilution in PBS, 1% BSA) is added (100 ~1/well) and
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the plate is incubated for 90 minutes at room temperature
on a plate shaker.
(8) The antibody conjugate is thoroughly removed by
decanting and rinsing the wells 5 times with PBS, and the
plate is dried by inverting and tapping on a paper towel.
(9) TMB substrate solution is added (100 ~1/well) and
incubated for 20 minutes at room temperature on a plate
shaker until color development is sufficient for
photometric detection.
(10) The absorbance of the samples are measured at 410
nm (in "dual wavelength" mode with a filter reading at 490
nm, as a reference wavelength) on a Dynatech ELISA plate
reader.
EGF-Induced Her2-Driven HrdU Incorporation
Materials and Reagents
(1) EGF: mouse EGF, 201, Toyobo, Co., Ltd. Japan
(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4),
Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(3) FixDenat: fixation solution (ready to use), Cat.
No. 1 647 229, Boehringer Mannheim, Germany.
(4) Anti-BrdU-POD: mouse monoclonal antibody
conjugated with peroxidase, Cat. No. 1 647 229, Boehringer
Mannheim, Germany.
(5) TMB Substrate Solution: tetramethylbenzidine
(TMB), ready to use, Cat. No. 1 647 229, Boehringer
Mannheim, Germany.
(6) PBS Washing Solution: 1X PBS, pH 7.4, made in
house.
(7) Albumin, Bovine (BSA): fraction V powder, A-8551,
Sigma Chemical Co., USA.
(8) 3T3 cell line engineered to express a chimeric
receptor having the extra-cellular domain of EGF-R and the
intra-cellular domain of Her2.
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Protocol
(1) Cells are seeded at 8000 cells/well in DMEM, 10%
CS, 2mM Gln in a 96- well plate. Cells are incubated
overnight at 37° C in 5% COz.
(2) After 24 hours, the cells are washed with PBS,
and then are serum starved in serum free medium (0% CS DMEM
with 0.1% BSA) for 24 hours.
(3) On day 3, ligand (EGF = 2 nM, prepared in DMEM
with 0.1% BSA) and test compounds are added to the cells
simultaneously. The negative control wells receive serum
free DMEM with 0.1% BSA only, the positive control cells
receive the ligand (EGF) but no test compound. Test
compounds are prepared in serum free DMEM with ligand in a
96 well plate, and serially diluted for 7 test
concentrations.
(4) After 20 hours of ligand activation, diluted BrdU
labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the
cells are incubated with BrdU (final concentration = 10 ~M)
for 1.5 hours.
(5) After incubation with labeling reagent, the
medium is removed by decanting and tapping the inverted
plate on a paper towel. FixDenat solution is added (50
~l/well) and the plates are incubated at room temperature
for 45 minutes on a plate shaker.
(6) The FixDenat solution is thoroughly removed by
decanting and tapping the inverted plate on a paper towel.
Milk is added (5% dehydrated milk in PBS, 200 ~1/well) as a
blocking solution and the plate is incubated for 30 minutes
at room temperature on a plate shaker.
(7) The blocking solution is removed by decanting and
the wells are washed once with PBS. Anti-BrdU-POD solution
(1:100 dilution in PBS, 1% BSA) is added (100 ~1/well) and
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the plate is incubated for 90 minutes at room temperature
on a plate shaker.
(8) The antibody conjugate is thoroughly removed by
decanting and rinsing the wells 5 times with PBS, and the
plate is dried by inverting and tapping on a paper towel.
(9) TMB substrate solution is added (100 ~1/well) and
incubated for 20 minutes at room temperature on a plate
shaker until color development is sufficient for
photometric detection.
(10) The absorbance of the samples are measured at 410
nm (in "dual wavelength" mode with a filter reading at 490
nm, as a reference wavelength) on a Dynatech ELISA plate
reader.
IGF1-Induced BrdU Incorporation Assay
Materials and Reagents
(1) IGF1 Ligand: human, recombinant, 6511, Promega Corp,
USA.
(2) BrdU Labeling Reagent: 10 mM, in PBS (pH7.4),
Cat. No. 1 647 229, Boehringer Mannheim, Germany.
(3) FixDenat: fixation solution (ready to use), Cat.
No. 1 647 229, Boehringer Mannheim, Germany.
(4) Anti-BrdU-POD: mouse monoclonal antibody conjugated
with peroxidase, Cat. No. 1 647 229, Boehringer Mannheim,
Germany.
(5) TMB Substrate Solution: tetramethvlhen~ir3inP
(TMB), ready to use, Cat. No. 1 647 229, Boehringer
Mannheim, Germany.
(6) PBS Washing Solution: 1X PBS, pH 7.4 (Sugen,
Inc., Redwood City, California).
(7) Albumin, Bovine (BSA): fraction V powder, A-8551,
Sigma Chemical Co., USA.
(8) 3T3 cell line genetically engineered to express
human IGF-1 receptor.
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Protocol
(1) Cells are seeded at 8000 cells/well in DMEM, 10%
CS, 2mM Gln in a 96- well plate. Cells are incubated
overnight at 37°C in 5% CO2.
(2) After 24 hours, the cells are washed with PBS,
and then are serum starved in serum free medium (0%CS DMEM
with 0.1% BSA) for 24 hours.
(3) On day 3, ligand (IGF1 - 3.3 nM, prepared in DMEM
with 0.1% BSA) and test compounds are added to the cells
simultaneously. The negative control wells receive serum
free DMEM with 0.1% BSA only, the positive control cells
receive the ligand (IGFl) but no test compound. Test
compounds are prepared in serum free DMEM with ligand in a
96 well plate, and serially diluted for 7 test
concentrations.
(4) After 16 hours of ligand activation, diluted BrdU
labeling reagent (1:100 in DMEM, 0.1% BSA) is added and the
cells are incubated with BrdU (final concentration=10 ~.M)
for 1.5 hours.
(5) After incubation with labeling reagent, the
medium is removed by decanting and tapping the inverted
plate on a paper towel. FixDenat solution is added (50
~1/well) and the plates are incubated at room temperature
for 45 minutes on a plate shaker.
(6) The FixDenat solution is thoroughly removed by
decanting and tapping the inverted plate on a paper towel.
Milk is added (5% dehydrated milk in PBS, 200 ~l/well) as
a blocking solution and the plate is incubated for 30
minutes at room temperature on a plate shaker.
(7) The blocking solution is removed by decanting and
the wells are washed once with PBS. Anti-BrdU-POD solution
(1:100 dilution in PBS, 1% BSA) is added (100 ~l/well) and
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the plate is incubated for 90 minutes at room temperature
on a plate shaker.
(8) The antibody conjugate is thoroughly removed by
decanting and rinsing the wells 5 times with PBS, and the
plate is dried by inverting and tapping on a paper towel.
(9) TMB substrate solution is added (100 ul/well) and
incubated for 20 minutes at room temperature on a plate
shaker until color development is sufficient for
photometric detection.
(10) The absorbance of the samples are measured at 410
nm (in "dual wavelength" mode with a filter reading at 490
nm, as a reference wavelength) on a Dynatech ELISA plate
reader.
FGF-Induced BrdU incorporation Assay
This assay measures FGF-induced DNA synthesis in
3Tc7/EGFr cells that express endogenous FGF receptors.
Materials and Reagents:
1. FGF: human FGF2/bFGF (Gibco BRL, No. 13256-029).
2. BrdU Labeling reagent, (10 mM PBS (pH 7.4),
Boehringer Mannheim Cat No. 1 647 229).
3. Fixdenat fixation solution (Boehringer Mannheim
Cat No. 1 647 229).
4. Anti-BrdU-POD (mouse monoclonal antibody
conjugated with peroxidase, Boehringer Mannheim Cat. No. 1
647 229).
5. TMB (tetramethylbenzidine, Boehringer Mannheim
Cat. No. 1 647 229).
6. PBS washing solution, pH 7.4 (Sugen, Inc.).
7. Albumin, bovine (BSA), fraction V powder (Sigma
Chemical Co., Cat. No. A-8551)
Procedure
1. 3T3 engineered cell line: 3T3c7/EGFr.
2. Cells are seeded at 8,000 cells/well in DMEM, 10%
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CS and 2 mM Gln in a 96-well plate. Incubate 24 hours at
37° C in 5% CO2.
3. After 24 hours, wash cells with PBS then serum
starve in serum free medium (0% DMEM, 0.1% BSA) for 24
hours.
4. Add ligand (FGF2 (1.5 nM in DMEM with 0.1% BSA)
and test compound simultaneously. Negative control wells
receive serum free DMEM with 0.1% BSA only, positive
control wells receive FGF2 ligand but no test compound.
Test compounds are prepared in serum-free DMEM with ligand
in a 96-well plate and serially diluted to make seven (7)
test concentrations.
5. After 20 hours, add diluted BrdU labeling reagent
(1:100 BrdU:DMEM, 0.1% BSA, final concentration is 10 ~M)
to the cells and incubate for 1.5 hours.
6. Decant medium. Remove traces of material with
paper towel. Add FixDenat (50 ~.1/well) and incubate at
room temperature for 45 minutes on a plate shaker.
7. Remove Fixdenat solution. Add blocking solution
(5% dehydrated milk in PBS (200 ~.1/well)) and incubate for
minutes at room temperature on a plate shaker.
8. Decant blocking solution, wash wells once with
PBS. Add anti-BrdU-POD solution (1:100 dilution in PBS,
0.1% BSA), incubate for 90 minutes at room temperature on a
25 plate shaker.
9. Decant antibody conjugate, rinse wells 5 times
with PBS. Dry plate by inverting on paper towel and
tapping.
10. Add TMB solution (100 ~tl/well), incubate 20
30 minutes at room temperature on a plate shaker until color
development is sufficient for photometric detection.
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11. Measure absorbance at 410 nM on a Dynatech ELISA
plate reader using "Dual wavelength" mode with a filter at 490
nM.
Biochemical EGFR Assay
This assay measures the in vitro kinase activity
of EGFR using ELISA.
Materials and Reagents
1. Corning 96-well Elisa plates (Corning Catalog No.
25805-96).
2. SUMO1 monoclonal anti-EGFR antibody (Biochemistry
Lab, SUGEN, Inc.).
3. PBS (Dulbecco's Phosphate-Buffered Saline, Gibco
Catalog No. 450-1300EB).
4. TBST Buffer
Reagent M.W. Working Amount
Concentration per L
Tris 121.14 50 mM 6.057 g
NaCl 58.44 150 mM 8.766 g
Triton X-100 NA 0.1% 1.0 ml
5. Blocking Buffer:
Reagent M.W. Working Amount per
Concentration 100 ml
Carnation Instant 5% 5.0 g
Non-Fat Milk
PBS NA NA 100 ml
6. A431 cell lysate (Screening Lab, UGEN, Inc.)
S
7. TBS Buffer:
Reagent M.W. Working Amount
Concentration per L
Tris 121.14 50 mM 6.057 g
NaCl 58.44 150 mM 8.766 g
8. TBS + 10% DMSO
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Reagent M.W. Working Amount.
Concentration per L
Tris 121.14 50 mM 1.514 g
NaCl 58.44 150 mM 2.192 g
DMSO NA 10% 25 ml
9. Adenosine-5'-triphosphate (ATP, from Equine
muscle, Sigma Cat. No. A-5394).
Prepare a 1.0 mM solution in dH20. This reagent
should be made up immediately prior to use and kept on ice.
10 . MnClz .
Prepare a 1.0 M stock solution in dH20.
11. ATP/MnCl2 phosphorylation mix
Reagent Stock Amount Working
solution per 10 ml Concentration
ATP 1.0 mM 300 ~.1 30 ~M
MnClz 1.0 M 500 ~l 50 mM
dH20 9.2 ml
This reagent should be prepared immediately
before use and kept on ice.
12. NUNC 96-well V bottom polypropylene plates
(Applied Scientific Cat. No. AS-72092).
13. Ethylenediaminetetraacetic acid (EDTA)
Prepare 200 mM working solution in dHZO. Adjust to
pH 8.0 with 10 N NaOH.
14. Rabbit polyclonal anti-phosphotyrosine serum
(Biochemistry Lab, SUGEN, Inc.)
15. Goat anti-rabbit IgG peroxidase conjugate
(Biosource Cat. No. ALI0404)
16. ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-
sulfonic acid), Sigma Cat. No. A-1888).
Reagent M.W. Working Amount
Concentration per L
Citric Acid 192.12 100 mM 19.21 g
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Na2HP04 141.96 250 mM 35.49 g
ABTS NA 0.5 mg/ml 500 mg
Mix first two ingredients in about 900 ml dH20, adjust
pH to 4.0 with phosphoric acid. Add ABTS, cover, let sit
about 0.5 hr., filter. The solution should be kept in the
dark at 4° C until ready to use.
17. Hydrogen peroxide 30°s solution (Fisher Cat. No.
H325)
18. ABTS/H20z
Mix 15 ml ABTS solution and 2.0 ~1 Hz02. Prepare 5
minutes before use.
19. 0.2 M HC1
Procedure
1. Coat Corning 96 well ELISA plates with 0.5 ~.g
SUMOl in 100 ~1 PBS per well, store overnight at 4° C.
2. Remove unbound SUMO1 from wells by inverting
plate to remove liquid. Wash lx with dHzO. Pat the plate on
a paper towel to remove excess liquid.
3. Add 150 ~,1 of Blocking Buffer to each well.
Incubate for 30 min. at room temperature with shaking.
4. Wash plate 3x with deionized water, then once
with TBST. Pat plate on a paper towel to remove excess
liquid and bubbles.
5. Dilute lysate in PBS (7 ~g lysate/100 ~l PBS).
6. Add 100 ~l of diluted lysate to each well. Shake
at room temperature for 60 min.
7. Wash plates as described in 4, above.
8. Add 120 ~1 TBS to ELISA plate containing captured
EGFR.
9. Dilute test compound 1:10 in TBS in 96-well
polypropylene plates (ie. 10 ~1 compound + 90 ~.1 TBS).
10. Add 13.5 ~1 diluted test compound to ELISA plate.
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To control wells (wells which do not receive any test
compound), add 13.5 ~tl TBS + 10% DMSO.
11. Incubate for 30 minutes while shaking at room
temperature.
12. Add 15 ~1 phosphorylation mix directly to all
wells except negative control well which does not receive
ATP/MnCl2 (final well volume should be approximately 150 ~.1
with 3 ~M ATP/5 mM MnClz final concentration in each well.)
Incubate 5 minutes while shaking.
13. After 5 minutes, stop reaction by adding 16.5 ~tl
of 200 mM EDTA (pH 8.0) to each well, shaking continuously.
After the EDTA has been added, shake for 1 min.
14. Wash 4x with deionized water, twice with TBST.
15. Add 100 ~1 anti-phosphotyrosine (1:3000 dilution
in TBST) per well. Incubate 30-45 min. at room
temperature, with shaking.
16. Wash as described in 4, above.
17. Add 100 ~tl Biosource Goat anti-rabbit IgG
peroxidase conjugate (1:2000 dilution in TBST) to each
well. Incubate 30 min. at room temperature, with shaking.
18. Wash as described in 4, above.
19. Add 100 ~1 of ABTS/H202 solution to each well.
20. Incubate 5 to 10 minutes with shaking. Remove any
bubbles.
21. If necessary stop reaction with the addition of
100 ~1 0.2 M HC1 per well.
22. Read assay on Dynatech MR7000 ELISA reader. Test
Filter: 410 nM Reference Filter: 630 Nm.
Biochemical PDGFR Assay
This assay measures the in vitro kinase activity of
PDGFR using ELISA.
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Materials and Reagents
Unless otherwise noted, the preparation of working
solution of the following reagents is the same as that for
the Biochemical EGFR assay, above.
1. Corning 96-well Elisa plates (Corning Catalog No.
25805-96) .
2. 28D4C10 monoclonal anti-PDGFR antibody
(Biochemistry Lab, SUGEN, Inc.).
3. PBS (Dulbecco's Phosphate-Buffered Saline, Gibco
Catalog No. 450-1300EB)
4. TBST Buffer.
5. Blocking Buffer.
6. PDGFR-(3 expressing NIH 3T3 cell lysate (Screening
Lab, SUGEN, Inc.).
7. TBS Buffer.
8. TBS + 10% DMSO.
9. Adenosine-5'-triphosphate (ATP, from Equine
muscle, Sigma Cat. No. A-5394).
10 . MnCl2 .
11. Kinase buffer phosphorylation mix.
Reagent Stock Amount Working
solution per 10 ml Concentration
Tris 1 M 250 ~l 25 mM
NaCl 5 M 200 ~1 100 mM
MnCl2 1 M 100 ~,1 10 mM
TX-100 100 mM 50 ~tl 0.5 mM
12. NUNC 96-well V bottom polypropylene plates
(Applied Scientific Cat. No. AS-72092).
13. Ethylenediaminetetraacetic acid (EDTA).
14. Rabbit polyclonal anti-phosphotyrosine serum
(Biochemistry Lab, SUGEN, Inc.).
15. Goat anti-rabbit IgG peroxidase conjugate
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(Biosource Cat. No. ALI0404).
16. 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic
acid) (ABTS, Sigma Cat. No. A-1888).
17. Hydrogen peroxide 30% solution (Fisher Cat. No.
H325).
18. ABTS/H202.
19. 0.2 M HC1.
Procedure
1. Coat Corning 96 well ELISA plates with 0.5 ~g
28D4C10 in 100 ~1 PBS per well, store overnight at 4° C.
2. Remove unbound 28D4C10 from wells by inverting
plate to remove liquid. Wash lx with dH20. Pat the plate on
a paper towel to remove excess liquid.
3. Add 150 ~1 of Blocking Buffer to each well.
Incubate for 30 min. at room temperature with shaking.
4. Wash plate 3x with deionized water, then once
with TBST. Pat plate on a paper towel to remove excess
liquid and bubbles.
5. Dilute lysate in HNTG (10 ~g lysate/100 ~,l HNTG)
6. Add 100 ~l of diluted lysate to each well. Shake
at room temperature for 60 min.
7. Wash plates as described in 4, above.
8. Add 80 ~l working kinase buffer mix to ELISA
plate containing captured PDGFR.
9. Dilute test compound 1:10 in TBS in 96-well
polypropylene plates (i.e., 10 ~1 compound + 90 ~1 TBS).
10. Add 10 ~1 diluted test compound to ELISA plate.
To control wells (wells which do not receive any test
compound), add 10 ~1 TBS + 10% DMSO.
11. Incubate for 30 minutes while shaking at room
temperature.
12. Add 10 ~,1 ATP directly to all wells except
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negative control well (final well volume should be
approximately 100 ~l with 20 ~tM ATP in each well.) Incubate
30 minutes while shaking.
13. After 30 minutes, stop reaction by adding 10 ~1
of 200 mM EDTA (pH 8.0) to each well.
14. Wash 4x with deionized water, twice with TBST.
15. Add 100 ~1 anti-phosphotyrosine (1:3000 dilution
in TBST) per well. Incubate 30-45 min. at room
temperature, with shaking.
16. Wash as described in 4, above.
17. Add 100 ~,1 Biosource Goat anti-rabbit IgG
peroxidase conjugate (1:2000 dilution in TBST) to each
well. Incubate 30 min. at room temperature, with shaking.
18. Wash as described in 4, above.
19. Add 100 ~1 of ABTS/H202 solution to each well.
20. Incubate 10 to 30 minutes with shaking. Remove
any bubbles.
21. If necessary stop reaction with the addition of
100 ~1 0.2 M HC1 per well.
22. Read assay on Dynatech MR7000 ELISA reader: test
filter: 410 nM, reference filter: 630 nM.
Biochemical FGFR Assay
This assay measures in vitro kinase activity of the
Myc-GyrB-FGFR fusion protein using ELISA.
Materials and Reagents
1. HNTG
Reagent M.W. 5x Stock Amount lx Working
Concentration per L Concentration
HEPES 238.3 100 mM 23.83 g 20 mM
NaCl 58.44 750 mM 43.83 g 150 mM
Glycerol NA 50% 500 ml 10%
Triton X- 100 NA 5% 10 ml 1.0%
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To make a liter of 5x stock solution, dissolve HEPES
and NaCl in about 350 ml dH20, adjust pH to 7.2 with HCl or
NaOH (depending on the HEPES that is used), add glycerol,
Triton X-100 and then dH20 to volume.
2. PBS (Dulbecco's Phosphate-Buffered Saline, Gibco
Catalog # 450-1300EB).
3. Blocking Buffer.
4. Kinase Buffer.
Reagent M.W. lOx Stock lx Working
Concentration Concentration
HEPES (pH 7.2) 238.3 500 mM 50
MnClz 20 mM 2 mM
MgCl2 203.32 200 mM 10 mM
Triton-X-100 1 % 0.1%
DTT 380.35 5 mM 0.5 mM
5. Phenylmethylsulfonyl fluoride (PMSF, Sigma, Cat.
No. P-7626):
Working solution: 100 mM in ethanol.
6. ATP (Bacterial source, Sigma Cat. No. A-7699)
Use 3.31 mg per ml MilliQ H20 for a stock
concentration of 6 mM.
7. Biotin conjugated anti-phosphotyrosine mab
(clone 4610, Upstate Biotechnology Inc. Cat. No. 16-103,
Ser. No. 14495).
8. Vectastain Elite ABC reagent (Avidin peroxidase
conjugate, Vector Laboratories Cat. No. PK-6 100).
9. ABTS Solution.
10. Hydrogen peroxide 30% solution ( Fisher Catalog
# H325) .
3 0 11 . ARTS /H202 .
12. 0.2 M HC1.
13. TRIS HC1 (Fischer Cat. No. BP 152-5).
Prepare 1.0 mM solution in MilliQ H20, adjust pH
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to 7.2 with HC1.
14. NaCl (Fisher Cat. No. S271-10).
Prepare 5 M solution in MilliQ H20.
15. MgCl~ (Fisher Cat. No. M33-500).
Prepare 1 M solution in MilliQ HZO.
16. HEPES (Fisher Cat. No. BP310-500).
Prepare 1 M solution in MilliQ H20, adjust pH to
7.5, sterile filter.
17. TBST Buffer.
18. Sodium Carbonate Buffer (Fisher Cat. No. S495):
Prepare 0.1 M solution in MilliQ H20, adjust pH to
9.6 with NaOH, filter.
19. Dithiothreitol (DTT, Fisher Cat. No. BP172-25).
Prepare 0.5 mM working solution in MilliQ H20 just
prior to use. Store at -20° C until used, discard any
leftover.
20. MnClz.
21. Triton X-100.
22. Goat a-Rabbit IgG (Cappel).
23. Affinity purified Rabbit a GST GyrB (Biochemistry
Lab. SUGEN, Inc.).
Procedure
All of the following steps are conducted at room
temperature unless otherwise indicated.
1. Coat Corning 96-well ELISA plates with 2 ~g Goat
a-Rabbit antibody per well in Carbonate Buffer such that
total well volume is 100 ~1. Store overnight at 4° C.
2. Remove unbound Goat a-Rabbit antibody by
inverting plate to remove liquid. Pat plate on a paper
towel to remove excess liquid and bubbles
3. Add 150 ~1 Blocking Buffer (5% Low Fat Milk in
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PBS) to each well. Incubate while shaking on a micro-titer
plate shaker for 30 min.
4. Wash 4x with TBST. Pat plate on a paper towel to
remove excess liquid and bubbles.
5. Add 0.5 ~tg Rabbit a-GyrB antibody per well.
Dilute antibody in DPBS to a final volume of 100 ~l per
well. Incubate with shaking on a micro-titer plate shaker
at room temperature for 1 hour.
6. Wash 4x with TBST as described in step 4.
7. Add 2 ~g COS/FGFR cell lysate (Myc-GyrB-FGFR
source) in HNTG to each well to give a final volume of 100
~1 per well. Incubate with shaking on a micro-titer plate
shaker for 1 hour.
8. Wash 4X with TBST as described in step 4.
9. Add 80 ~1 of lx kinase buffer per well.
10. Dilute test compound 1:10 in lx kinase buffer +
1% DMSO in a polypropylene 96 well plate.
11. Transfer 10 ~l of diluted test compound solution
and control wells from polypropylene plate wells to the
corresponding ELISA plate wells, incubate with shaking on a
micro-titer plate shaker for 20 minutes.
12. Add 10 ~l of 70 ~M ATP diluted in kinase buffer
to positive control and test wells (Final ATP concentration
is 7 ~M/well). Add 10 ~1 lx kinase buffer to negative
control wells. Incubate with shaking on a micro-titer plate
shaker for 15 min.
13. Stop kinase reaction by adding 5 ~.1 0.5 M EDTA to
all wells.
14. Wash 4x with TBST as described in step 4.
15. Add 100 ~l biotin conjugated a-phosphotyrosine
mab (b4G10) diluted in TBST to each well. Incubate with
shaking on a micro-titer plate shaker for 30 minutes.
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16. Prepare Vectastain ABC reagent. Add 1 drop
reagent A to 15 ml TBST. Mix by inverting tube several
times. Add 1 drop reagent B and mix again.
17. Wash 4x with TBST as described in step 4.
18. Add 100 ~1 ABC HRP reagent to each well. Incubate
with shaking on a micro-titer plate shaker for 30 minutes.
19. Wash 4x with TBST as described in step 4.
20. Add 100 ~1 of ABTS/H202 solution to each well.
22. Incubate 5 to 15 minutes with shaking. Remove any
bubbles.
23. If necessary stop reaction by adding 1 00 ~l of
0.2M HC1/well.
24. Read assay on Dynatech MR7000 ELISA Plate Reader,
test filter: 410 nM, reference filter: 630 nM.
Biochemical FLK-1 Assay
This assay evaluates flk-1 autophosphorylation
activity in vitro using ELISA.
Materials And Reagents
1. 15 cm tissue culture dishes
2. Flk-1/NIH cells: NIB fibroblast line over-
expressing human flk-1 clone 3 (SUGEN, Inc., obtained from
MPI, Martinsried, Germany).
3. Growth medium: DMEM plus heat inactivated loo FBS
and 2 mM Glutamine (Gibco-BRL).
4. Starvation medium: DMEM plus 0.5% heat
inactivated FBS, 2 mM Glutamine (Gibco-BRL).
5. Corning 96-well ELISA plates (Corning Cat. No.
25805-96) .
6. L4 or E38 monoclonal antibody specific for flk-1,
Purified by Protein-A agarose affinity chromatography
(SUGEN, Inc.).
7. PBS (Dulbecco's Phosphate-Buffered Saline) Gibco
Cat. No. 450-1300EB).
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8. HNTG (see BIOCHEMICAL FGFR for preparation).
9. Pierce BCA protein determination kit.
10. Blocking buffer
11. TBST (pH 7.0)
12. Kinase Buffer
13. Kinase Stop Solution: 200 mM EDTA.
14. Biotinylated 4610, specific for phosphotyrosine
(UBI, Cat. No. No. 16-103).
15. AB kit (Vector Laboratories Cat. No. PK 4000).
16. DMSO
17. NUNC 96-well V bottom polypropylene plates
(Applied Scientific Cat. No. AS-72092).
18. Turbo-TMB (Pierce).
19. Turbo-TMB stop solution: 1 M HZS04.
20. ATP (Sigma Cat. No. A-7699).
21. 20% DMSO in TBS (pH 7.0).
Procedure
Cell Growth and Lysate Preparation.
1. Seed cell into growth medium and grow for 2-3
days to 90-100% confluency at 37° C and 5% C02. Do not
exceed passage #20.
2. Remove the medium and wash the cells twice with
PBS. Lyse with HNTG lysis buffer. Collect all lysates and
vortex mix them for 20-30 seconds.
3. Remove insoluble material by centrifugation (5-10
min at about 10,000 xg).
4. Determine the protein concentration using BCA
kit.
5. Partition lysate into 1 mg aliquots, store at -80°
C.
Assay Procedure
1. Coat Corning 96-well ELISA plates with 2 ~g/well
purified L4 (or E 38) in 100 ~1 of PBS. Store overnight at
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4° C.
2. Remove unbound proteins from wells by inverting
the plate to remove the liquid. Wash one time with dH20, pat
plate on paper towel to remove excess liquid.
3. Block plates with 150 ~tl blocking buffer per
well. Incubate for 45-60 minutes with shaking at 4° C.
4. Remove the blocking buffer and wash the ELISA
plate three times with dHzO and one time with TBST. Pat
plate on paper towel to remove excess liquid.
5. Dilute lysate in PBS to give final concentraticn
of 50 ~g/100 ~1. Add 100 ~l of diluted lysate to each
well. Incubate with shaking at 4° C overnight.
6. Remove unbound proteins from wells by invertinc
the plate. Wash as in step 4.
7. Add 80 ~1 of kinase buffer to wells (90 ~l to
negative control wells).
8. Dilute test compounds (normally 10-fold) into
wells of a polypropylene plate containing 20% DMSO in TBS.
9. Add 10 ~1 of the diluted compounds to the ELISA
wells containing immobilized flk-1 and shake. Control webs
receive no compounds.
10. From stock 1 mM ATP, prepare 0.3 mM ATP solutic~
in dH20 (alternatively, kinase buffer may be used).
11. Add 10 ~1 of 0.3 mM ATP to all wells except the
negative controls. Incubate for 60 min. at room temperature
with shaking.
12. After 1 hr stop the kinase reaction by adding 11
~.1 200 mM EDTA. Shake for 1-2 min.
13. Wash the ELISA plate 4 times with dH20 and twice
with TBST.
14. Add 100 ~1 of 1:5000 biotinylated 4G10:TBST to
all wells. Incubate 45 min with shaking at room
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temperature.
15. While the above is incubating, add 50 ~1 of
solutions A & B from the ABC kit to 10 ml of TBST. These
solutions must be combined approximately 30 min prior to
use.
16. Wash plates as in step 4.
17. Add 100 ~1 of the preformed A & B complex to all
wells. Incubate 30 min with shaking at room temperature.
18. Wash plates as in step 4.
19. Add 100 ~tl turbo-TMB. Shake at room temperature
for 10- 15 min.
20. When the color in the positive control wells
reaches an absorbance of about 0.35 - 0.4, stop the
reaction with 100 ~tl of turbo-TMB stop solution.
21. Read plates on Dynatech MR7000 ELISA reader, test
filter: 450 nM, reference filter: 410 nM.
HUV-EC-C Assay
The following protocol may also be used to measure a
compound's activity against PDGF-R, FGF-R, VEGF, aFGF or
Flk-1/KDR, all of which are naturally expressed by HUV-EC
cells.
DAY 0
1. Wash and trypsinize HUV-EC-C cells (human
umbilical vein endothelial cells, (American Type Culture
Collection, catalogue no. 1730 CRL). Wash with Dulbecco's
phosphate-buffered saline (D-PBS, obtained from Gibco BRL,
catalogue no. 14190-029) 2 times at about 1 m1/10 cm2 of
tissue culture flask. Trypsinize with 0.05% trypsin-EDTA
in non-enzymatic cell dissociation solution (Sigma Chemical
Company, catalogue no. C-1544). The 0.05% trypsin is made
by diluting 0.25% trypsin/1 mM EDTA (Gibco, catalogue no.
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25200-049) in the cell dissociation solution. Trypsinize
with about 1 m1/25-30 cm2 of tissue culture flask for about
minutes at 37°C. After cells have detached from the
flask, add an equal volume of assay medium and transfer to
5 a 50 ml sterile centrifuge tube (Fisher Scientific,
catalogue no. 05-539-6).
2. Wash the cells with about 35 ml assay medium in
the 50 ml sterile centrifuge tube by adding the assay
medium, centrifuge for 10 minutes at approximately 200x g,
aspirate the supernatant, and resuspend with 35 ml D-PBS:
Repeat the wash two more times with D-PBS, resuspend the
cells in about 1 ml assay medium/15 cmz of tissue culture
flask. Assay medium consists of F12K medium (Gibco BRL,
catalogue no. 21127-014) and 0.5% heat-inactivated fetal
bovine serum. Count the cells with a Coulter Counter~
(Coulter Electronics, Inc.) and add assay medium to the
cells to obtain a concentration of 0.8-1.0 x 105 cells/ml.
3. Add cells to 96-well flat-bottom plates at 100
~.1/well or 0.8-1.0 x 104 cells/well, incubate -.24h at 37°C,
2 0 5 % COZ .
DAY 1
1. Make up two-fold test compound titrations in
separate 96-well plates, generally 50 ~M on down to 0 ~M.
Use the same assay medium as mentioned in day 0, step 2
above. Titrations are made by adding 90 ~l/well of test
compound at 200 uM (4X the final well concentration) to the
top well of a particular plate column. Since the stock
test compound is usually 20 mM in DMSO, the 200 ~M drug
concentration contains 2% DMSO.
A diluent made up to 2% DMSO in assay medium (F12K +
0.5% fetal bovine serum) is used as diluent for the test
compound titrations in order to dilute the test compound
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but keep the DMSO concentration constant. Add this diluent
to the remaining wells in the column at 60 ~tl/well. Take
60 ~l from the 120 ~1 of 200 ~M test compound dilution in
the top well of the column and mix with the 60 u1 in the
second well of the column. Take 60 ~1 from this well and
mix with the 60 ~tl in the third well of the column, and so
on until two-fold titrations are completed. When the next-
to-the-last well is mixed, take 60 ~1 of the 120 ~tl in this
well and discard it. Leave the last well with 60 ~tl of
DMSO/media diluent as a non-test compound-containing
control. Make 9 columns of titrated test compound, enough
for triplicate wells each for: (1) VEGF (obtained from
Pepro Tech Inc., catalogue no. 100-200, (2) endothelial
cell growth factor (ECGF) (also known as acidic fibroblast
growth factor, or aFGF) (obtained from Boehringer Mannheim
Biochemica, catalogue no. 1439 600), or, (3) human PDGF B/B
(1276-956, Boehringer Mannheim, Germany) and assay media
control. ECGF comes as a preparation with sodium heparin.
2. Transfer 50 ~,1/well of the test compound
dilutions to the 96-well assay plates containing the 0.8-
1.0x104 cells/100 ~1/well of the HUV-EC-C cells from day 0
and incubate ~2 h at 37° C, 5% CO2.
3. In triplicate, add 50 ~tl/well of 80 ~.g/ml VEGF,
20 ng/ml ECGF, or media control to each test compound
condition. As with the test compounds, the growth factor
concentrations are 4X the desired final concentration. Use
the assay media from day 0 step 2 to make the
concentrations of growth factors. Incubate approximately
24 hours at 37°C, 5% COZ. Each well will have 50 ~1 test
compound dilution, 50 ~1 growth factor or media, and 100 ~1
cells, which calculates to 200 ~1/well total. Thus the 4X
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concentrations of test compound and growth factors become
1X once everything has been added to the wells.
DAY 2
1. Add 3H-thymidine (Amersham, catalogue no. TRK-686)
at 1 ~Ci/well (10 ~tl/well of 100 ~tCi/ml solution made up in
RPMI media + 10% heat-inactivated fetal bovine serum) and
incubate ~24 h at 37°C, 5% COz. RPMI is obtained from Gibco
BRL, catalogue no. 11875-051.
DAY 3
1. Freeze plates overnight at -20°C.
DAY 4
Thaw plates and harvest with a 96-well plate harvester
(Tomtec Harvester 96~) onto filter mats (Wallac, catalogue
no. 1205-401), read counts on a Wallac BetaplateT"" liquid
scintillation counter.
In Vivo Animal Models
XenoQraft Animal Models
The ability of human tumors to grow as xenografts in
athymic mice (e. g., Balb/c, nu/nu) provides a useful in vivo
model for studying the biological response to therapies for
human tumors. Since the first successful
xenotransplantation of human tumors into athymic mice,
(Rygaard and Povlsen, 1969, Acta Pathol. Microbial. Scand.
77:758-760), many different human tumor cell lines (e. g.,
mammary, lung, genitourinary, gastro-intestinal, head and
neck, glioblastoma, bone, and malignant melanomas) have been
transplanted and successfully grown in nude mice. The
following assays may be used to determine the level of
activity, specificity and effect of the different compounds
of the present invention. Three general types of assays are
useful for evaluating compounds: cellular/catalytic,
cellular/biological and in vivo. The object of the
cellular/catalytic assays is to determine the effect of a
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compound on the ability of a TK to phosphorylate tyrosines
on a known substrate in a cell. The object of the
cellular/biological assays is to determine the effect of a
compound on the biological response stimulated by a TK in a
cell. The object of the in vivo assays is to determine the
effect of a compound in an animal model of a particular
disorder such as cancer.
Suitable cell lines for subcutaneous xenograft
experiments include C6 cells (glioma, ATCC # CCL 107), A375
cells (melanoma, ATCC # CRL 1619), A431 cells (epidermoid
carcinoma, ATCC # CRL 1555), Calu 6 cells (lung, ATCC # HTB
56), PC3 cells (prostate, ATCC # CRL 1435), SKOV3TP5 cells
and NIH 3T3 fibroblasts genetically engineered to
overexpress EGFR, PDGFR, IGF-1R or any other test kinase.
The following protocol can be used to perform xenograft
experiments:
Female athymic mice (BALB/c, nu/nu) are obtained from
Simonsen Laboratories (Gilroy, CA). All animals are
maintained under clean-room conditions in Micro-isolator
cages with Alpha-dri bedding. They receive sterile rodent
chow and water ad libitum.
Cell lines are grown in appropriate medium (for
example, MEM, DMEM, Ham's F10, or Ham's F12 plus 5% - 10%
fetal bovine serum (FBS) and 2 mM glutamine (GLN)). All
cell culture media, glutamine, and fetal bovine serum are
purchased from Gibco Life Technologies (Grand Island, NY)
unless otherwise specified. All cells are grown in a humid
atmosphere of 90-95% air and 5-10% CO2 at 37°C. All cell
lines are routinely subcultured twice a week and are
negative for mycoplasma as determined by the Mycotect
method (Gibco).
Cells are harvested at or near confluency with 0.05%
Trypsin-EDTA and pelleted at 450 x g for 10 min. Pellets
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are resuspended in sterile PBS cr media (without FBS) to a
particular concentration and the cells are implanted into
the hindflank of the mice (8 - 10 mice per group, 2 - 10 x
106 cells/animal). Tumor growth is measured over 3 to 6
weeks using venier calipers. Tumor volumes are calculated
as a product of length x width x height unless otherwise
indicated. P values are calculated using the Students t-
test. Test compounds in 50 - 100 uL excipient (DMSO, or
VPD:D5W) can be delivered by IP injection at different
concentrations generally starting at day one after
implantation.
Tumor Invasion Model
The following tumor invasion model has been
developed and may be used for the evaluation of
therapeutic value and efficacy of the compounds
identified to selectively inhibit KDR/FLK-1 receptor.
Procedure
8 week old nude mice (female) (Simonsen Inc.) are
used as experimental animals. Implantation of tumor
cells can be performed in a laminar flow hood. For
anesthesia, Xylazine/Ketamine Cocktail (100 mg/kg
ketamine and 5 mg/kg Xylazine) are administered
intraperitoneally. A midline incision is done to expose
the abdominal cavity (approximately 1.5 cm in length) to
inject 10' tumor cells in a volume of 100 ~tl medium. The
cells are injected either into the duodenal lobe of the
pancreas or under the serosa of the colon. The
peritoneum and muscles are closed with a 6-0 silk
continuous suture and the skin is closed by using wound
clips. Animals are observed daily.
Analysis
After 2-6 weeks, depending on gross observations of
the animals, the mice are sacrificed, and the local tumor
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metastases to various organs (lung, liver, brain,
stomach, spleen, heart, muscle) are excised and analyzed
(measurement of tumor size, grade of invasion,
immunochemistry, in situ hybridization determination,
etc . ) .
Measurement Of Cell Toxicity
Therapeutic compounds should be more potent in inhibiting
receptor tyrosine kinase activity than in exerting a cytotoxic
effect. A measure of the effectiveness and cell toxicity of a
compound can be obtained by determining the therapeutic index,
i . a . , ICso/LDso . ICSO, the dose required to achieve 50 0
inhibition, can be measured using standard techniques such as
those described herein. LDso,the dosage which results in 50%
toxicity, can also be measured by standard techniques as
well(Mossman, 1983, J. Immunol. Methods, 65:55-63), by
measuring the amount of LDH released (Korzeniewski and
Callewaert, 1983, J. Immunol. Methods, 64:313, Decker and
Lohmann-Matthes, 1988, J. Immunol. Methods, 115:61), or by
measuring the lethal dose in animal models. Compounds with a
large therapeutic index are preferred. The therapeutic index
should be greater than 2, preferably at least 10, more
preferably at least 50.
B. Examples - Biological Activity.
Examples of the in vitro potency of compounds of this
invention are shown in Table 2. The examples shown are not
to be construed as limiting the scope of this invention in
any manner whatsoever.
CONCLUSION
It will be appreciated that the compounds, methods
and pharmaceutical compositions of the present invention
are effective in modulating PK activity and therefore are
expected to be effective as therapeutic agents against
RTK, CTK-, and STK-related disorders.
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One skilled in the art would also readily appreciate
that the present invention is well adapted to carry out the
objects and obtain the ends and advantages mentioned, as
well as those inherent herein. The molecular complexes and
the methods, procedures, treatments, molecules, specific
compounds described herein are presently representative of
preferred embodiments, are exemplary, and are not intended
as limitations on the scope of the invention. Changes
therein and other uses will occur to those skilled in the
art which are encompassed within the spirit of the
invention are defined by the scope of the claims.
It will be readily apparent to one skilled in the art
that varying substitutions and modifications may be made to
the invention disclosed herein without departing from the
scope and spirit of the invention.
All patents and publications mentioned in the
specification are indicative of the levels of those skilled
in the art to which the invention pertains. All patents
and publications are herein incorporated by reference to
the same extent as if each individual publication was
specifically and individually indicated to be incorporated
by reference.
The invention illustratively described herein suitably
may be practiced in the absence of any element or elements,
limitation or limitations which is not specifically
disclosed herein. Thus, for example, in each instance
herein any of the terms "comprising", "consisting
essentially of" and "consisting of" may be replaced with
either of the other two terms. The terms and expressions
which have been employed are used as terms of description
and not of limitation, and there is no intention that in
the use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
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thereof, but it is recognized that various modifications
are possible within the scope of the invention claimed.
Thus, it should be understood that although the present
invention has been specifically disclosed by preferred
embodiments and optional features, modification and
variation of the concepts herein disclosed may be resorted
to by those skilled in the art, and that such modifications
and variations are considered to be within the scope of
this invention as defined by the appended claims.
In addition, where features or aspects of the
invention are described in terms of Markush groups, those
skilled in the art will recognize that the invention is
also thereby described in terms of any individual member or
subgroup of members of the Markush group. For example, if
X is described as selected from the group consisting of
bromine, chlorine, and iodine, claims for X being bromine
and claims for X being bromine and chlorine are fully
described.
Other embodiments are within the following claims.
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