Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
' NOVEL HETEROARYL COMPOUNDS FOR THE MODULATION OF PROTEIN
TYROSINE ENZYME RELATED CELLULAR SIGNAL TRANSDUCTION
RELATED APPLICATIONS
The present application is related to and claims priority from provisional
application
serial no. 60/049,560, dated June 13, 1997, which application is incorporated
as if fully set
forth herein.
INTRODUCTION
The present invention relates generally to organic chemistry, biochemistry,
pharmacology and medicine. More particularly, it relates to novel heteroaryl
compounds and
their physiologically acceptable salts and prodrugs, which modulate the
activity of protein
tyrosine enzymes related to cellular signal transduction and, therefore, are
expected to exhibit
a salutary effect against disorders associated with abnormal protein tyrosine
enzyme related
cellular signal transduction.
BACKGROUND OF THE INVENTION
Cellular signal transduction is a fundamental mechanism whereby external
stimuli that
regulate diverse cellular processes are relayed to the interior of cells. The
biochemical
pathways through which signals are transmitted within cells comprise a
circuitry of directly
or functionally connected interactive proteins. One of the key biochemical
mechanisms of
signal transduction involves the reversible phosphorylation of tyrosine
residues on proteins.
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The phosphorylation state of a protein may affect its conformation and/or
enzymic activity as
well as its cellular location. The phosphorylation state of a protein is
modified through the
reciprocal actions of protein tyrosine kinases (PTKS) and protein tyrosine
phosphatases
(PTPS) at various specific tyrosine residues.
A common mechanism by which receptors regulate cell function is through an
inducible tyrosine kinase activity which is either endogenous to the receptor
or is imparted by
other proteins that become associated with the receptor. (Darnell et al.,
1994, Science,
264:1415-1421; Heldin, 1995, Cell, 80:213-223; Pawson, 1995, Nature, 373:573-
580).
Protein tyrosine kinases comprise a large family of transmembrane receptor and
intracellular enzymes with multiple functional domains (Taylor et al., 1992,
Ann. Rev. Cell
Biol. 8:429-62). The binding of Iigand allosterically transducer a signal
across the cell
membrane where the cytoplasmic portion of the PTKs initiates a cascade of
molecular
interactions that disseminate the signal throughout the cell and into the
nucleus. Many
receptor protein tyrosine kinase (RPTKs), such as epidermal growth factor
receptor (EGFR)
and platelet-derived growth factor receptor (PDGFR) undergo oligomerization
upon ligand
binding, and the receptors self phosphorylate (via autophosphorylation or
transphosphorylation) on specific tyrosine residues in the cytoplasmic
portions of the receptor
(Schlessinger and Ullrich, 1992, Neuron, 9:383-91, Heldin, 1995, Cell,
80:213223).
Cytoplasmic protein tyrosine kinases (CPTKs), such as Janus kinases (e.g.,
JAKl, JAK2,
TYK2) and Src kinases (e.g., src, lck, fyn) are associated with receptors for
cytokines (e.g.,
IL-2, IL-3, IL-6, erythropoietin), interferons and antigens. These receptors
also undergo
oligomerization, and have tyrosine residues that become phosphorylated during
activation,
but the receptor polypeptides themselves do not possess kinase activity.
Like the PTKS, the protein tyrosine phosphatases (PTPS) comprise a family of
transmernbrane and cytoplasmic enzymes, possessing at least an approximately
230 amino
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acid catalytic domain containing a highly conserved active site with the
consensus motif
[I/V]IHCXAGXXR[S/T]G. The substrates of PTPs may be PTKs which possess
phosphotyrosine residues or the substrates of PTKS. (Hunter, 1989, Cell,
58:1013-16;
Fischer et aL, 1991, cience, 253:401-6; Saito & Streuli, 1991, Cell Growth and
S Differentiation, 2:59-6S; Pot and Dixon, 1992, Biochem. Biophvs. Acta, I
136:35-43).
Transmembrane or receptor-like PTPs (RPTPS) possess an extracellular domain, a
single transmembrane domain, and one or two catalytic domains followed by a
short
cytoplasmic tail. The extracellular domains of these RPTPs are highly
divergent, with small
glycosylated segments (e.g., RPTPa, RPTPE), tandem repeats of immunoglobulin-
like and/or
I 0 fibronectin type III domains (e.g., LAR) or carbonic anhydrase like
domains (e.g., RPTPa,
RPTP(3). These extracellular features might suggest that these RPTPs function
as a receptor
on the cell surface, and their enzymatic activity might be modulated by
ligands. Intracellular
or cytoplasmic PTPs (CPTPs), such as PTP 1 C and PTP 1 D, typically contain a
single catalytic
domain flanked by several types of modular conserved domains. For example, PTP
I C a
1 S hemopoietic cell CPTP is characterized by two Src homology 2 (SH2) domains
that recognize
short peptide motifs bearing phosphotyrosine (pTyr).
In general, these modular conserved domains influence the intracellular
localization of
the protein. SH2-containing proteins are able to bind pTyr sites in activated
receptors and
cytoplasmic phosphoproteins. Another conserved domain known as SH3 binds to
proteins
20 with proline-rich regions. A third type known as the pleckstrin-homology
(PH) domain has
also been identified. These modular domains have been found in both CPTKs and
CPTPs as
well as in noncatalytic adapter molecules, such as Grbs (Growth factor
Receptor Bound),
which mediate protein-protein interactions between components of the signal
transduction
pathway (Skolnik et al., 1991, Cell, 65:83-90; Pawson, 1995, Nature, 373:573-
580).
2S
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Multiprotein signaling complexes comprising receptor subunits, kinases,
phosphatases
and adapter molecules are assembled in subcellular compartments through the
specific and
dynamic interactions between these domains with their binding motifs. Such
signaling
complexes integrate the extracellular signal with the ligand-bound receptor
and relay the
signal to other downstream signaling proteins or complexes in other locations
inside the cell
or in the nucleus (Koch et al., 1991, Science, 252:668-674; Pawson, 1994,
Nature,
373:573-580; Mauro et al., 1994, Trends Biochem. Sci., 19:151-155; Cohen et
al., 1995, Cell,
80:237-248).
The levels of tyrosine phosphorylation required for normal cell growth and
differentiation at any time are achieved through the coordinated action of
PTKs and PTPS.
Depending on the cellular context, these two types of enzymes may either
antagonize or
cooperate with each other during signal transduction. An imbalance between
these enzymes
may impair normal cell functions leading to metabolic disorders and cellular
transformation.
For example, insulin binding to the insulin receptor, which is a PTK, triggers
a variety
of metabolic and growth promoting effects such as glucose transport,
biosynthesis of
glycogen and fats, DNA synthesis, cell division and differentiation. Diabetes
mellitus, which
is characterized by insufficient or a lack of insulin signal transduction, can
be caused by any
abnormality at any step along the insulin signaling pathway. (Olefsky, 1988,
"Cecil
Textbook of Medicine," 18th Ed., 2:1360-81).
It is also well known, for example, that the overexpression of PTKS, such as
HER2, can
play a decisive role in the development of cancer {Slamon et al., 1987,
cience, 235:77-82) and
that antibodies capable of blocking the activity of this enzyme can abrogate
tumor growth
(Drebin et al., 1988, Onco ene, 2:387-394). Blocking the signal transduction
capability of
tyrosine kinases such as Flk-1 and the PDGF receptor have been shown to block
tumor growth
in animal models (Millauer et al., 1994, Nature, 367:577; Ueno et al.,
Science, 252:844-848).
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Relatively less is known with respect to the direct role of tyrosine
phosphatases in
signal transduction; PTPs may play a role in human diseases. For example,
ectopic
expression of RPTPa produces a transformed phenotype in embryonic fibroblasts
(Zheng et
al., Nature, 359:336-339), and overexpression of RPTPa in embryonal carcinoma
cells
5 causes the cells to differentiate into a cell type with a neuronal phenotype
(den Hertog, et al.,
EMBO Journal, 12:3789-3798). The gene for human RPTPy has been localized to
chromosome 3p21 which is a segment frequently altered in renal and small lung
carcinoma.
Mutations may occur in the extracellular segment of RPTPy which renders the
RPTP no
longer responsive to external signals (LaForgia et al., Wary et aL, 1993,
Cancer Res.,
52:478-482). Mutations in the gene encoding PTP 1 C (also known as HCP or SHP)
are the
cause of the moth-eaten phenotype in mice which suffer from severe
immunodeficiency, and
systemic autoimmune disease accompanied by hyperproliferation of macrophages
(Schultz et
al., 1993, Cell, 73:1445-1454). PTP1D (also known as Syp or PTP2C) has been
shown to
bind through SH2 domains to sites of phosphorylation in PDGFR, EGFR and
insulin
receptor substrate I (IRS-1). Reducing the activity of PTP1D by microinjection
of
anti-PTP1D antibody has been shown to block insulin or EGF-induced mitogenesis
(Xiao et
al., 1994, J. Biol. Chem., 269:21244-21248).
It has been reported that some of the biological effects of insulin can be
mimicked by
vanadium salts such as vanadates and pervanadates. Vanadates and pervanadates
are known
to be non-specific phosphatase inhibitors. However, this class of compounds is
toxic because
each compound contains a heavy metal (U.S. Patent No. 5,155,031; Fantus et
al., 1989,
Biochem., 28:8864-71; Swarup et aL, 1982, Biochem. Biophys. Res. Commun.,
107:1104-9).
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SUMMARY OF THE INVENTION
The present invention relates generally to novel heteroaryl compounds which
modulate the activity of protein tyrosine enzymes which are related to
cellular signal
transduction; namely, protein tyrosine kinases (PTKs) and protein tyrosine
phosphatases
(PTPs). In particular, the compounds of this invention are expected to
modulate protein
tyrosine phosphatase activity. In addition, the present invention relates to
the preparation
and use of pharmaceutical compositions of the disclosed compounds and their
physiologically
acceptable salts and prodrugs for the treatment or prevention of disorders
associated with
abnormal protein tyrosine enzyme related cellular signal tranduction
including, but not
limited to, cancer and diabetes.
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.
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 wherein it is administered as an ester (the
"prodrug") to
facilitate transmittal across a cell membrane where water solubility is not
beneficial, but then
it is metabolically hydrolyzed to the carboxylic acid once inside the cell
where water
solubility is beneficial.
As used herein, an "ester" is a C-carboxy group, as defined herein, wherein R"
is any
of the listed groups other than hydrogen.
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As used herein, a "physiologically 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.
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.
THE COMPOUNDS
General structural features.
In one aspect this invention relates to heteroaryl compounds having the
general
chemical structure shown in Formula 1:
R1 Rio
R2~ ~ A Q M ~ R9
L
R3/D\fE~/F'R5R6/G'Wls K\RS
14 i7
In Formula 1, r and s are independently 0 or 1.
When r or s is 1 then A, B, D, E, F, G, J, K, L, and M are independently
selected
from the group consisting of carbon and nitrogen, it being understood that the
six-member
nitrogen heteroaryl rings so formed are those known in the chemical arts; it
being further
understood that when A, B, D, E, F, G, J, K, L or M is nitrogen, R', R2, R3,
R', R5, R6, R', R8,
R9 or R'°, respectively, do not exist.
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At least one of A, B, D, E or F and at least one of G, J, K, L and M must be
nitrogen.
When r or s is 0 then A, B, D, and F or G, K, L and M, respectively, are
independently
selected from the group consisting of carbon, nitrogen, oxygen and sulfur, it
being understood
that the five-member heteroaryl rings so formed are those known in the
chemical arts; it being
fizrther understood that when A, B, D, F, G, K, L or M is oxygen or sulfur or
A, B, D, F, G,
K, L or M is nitrogen and that nitrogen is participating in a heteroaryl ring
double bond, R',
R2, R3, R5, R6, Ra, R9 or R'° do not exist.
R', R2, R', R', R5, R6, R', R8, R9 and R'° are independently selected
from the group
consisiting of hydrogen, alkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl,
aryl, heteroaryl,
heteroalicyclic, alkoxy, aryloxy, thioalkoxy, thioaryloxy, heteroaryloxy,
heteroalicycloxy,
sulfinyl, sulfonyl, S-sulfonamido, N-Sulfonamido, trihalomethanecarbonyl,
trihalomethanesulfonyl, carbonyl, C-carboxy, O-carboxy, C-amido, C-thioamido,
N-amido,
hydrazino, cyano, vitro, halo, O-carbamyl, N-carbamyl, O-thiocarbamyl,
phosphonyl, N-
thiocarbamyl, guanyl, guanidino, ureido, amino, trihalomethane sulfonamido,
and -NR"R'Z.
R" and R'2 are independently selected from the group consisting of hydrogen,
alkyl,
cycloalkyl, alkenyl, alkynyl, aryl, carbonyl, C-carboxy, sulfonyl,
trihalomethanesulfonyl,
trihalomethanecarbonyl and, combined, a five- or six-member heteroalicyclic
ring.
When r or s is 0 and A, B, D, or F; or G, K, L or M, respectively, is a
nitrogen atom
which is not participating in a heteroaryl ring double bond, then R', R2, R3,
R5, R6, R8, R9 and
R'° are independently selected from the group consisting of hydrogen,
alkyl, cycloalkyl,
alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,
trihalomethanecarbonyl,
sulfonyl, trihaiomethane- sulfonyl, cyano, C-carboxy, O-carboxy, C-amido, C-
thioamido and
guanyl;
Q is selected from the group consisting of oxygen, sulfur, sulfinyl, sulfonyl
and -NR'3.
R'3 is selected from the group consisting of hydrogen, alkyl, cycloalkyl,
alkenyl,
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alkynyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, cyano,
trihalomethanecarbonyl,
sulfonyl, trihalomethanesulfonyl, C-carboxy, O-carboxy, C-amido, C-thioamido
and guanyl.
Any two adjacent R groups may combine to form an additional aryl, cycloalkyl,
heteroaryl or heteroalicyclic ring fused to the ring initially bearing those R
groups.
When r is 0, A is sulfur, F is nitrogen, Q is sulfur and Rz is vitro, then G,
J, K, L, M,
R6, R', Re, R9 and R'° are selected so as to afford the compounds of
Table 1.
Physiologically acceptable salts and prodrugs of the compounds disclosed
herein are
within the scope of this invention.
As used herein, the phrase "participating in a heteroaryl ring double bond",
with
regard to A, B, D, F, G, K, L and M when they are nitrogen atoms, refers to
the formal double
bonds in the two tautomeric structures which comprise five-member ring
heteroaryl groups:
~>
An "alkyl" group 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
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) is
preferably one or
20 more individually selected from trihaloalkyl, cycloalkyl, aryl, heteroaryl,
heteroalicyclic,
hydroxy, alkoxy, aryloxy, heteroaryloxy, heteroalicycloxy, thiohydroxy,
thioalkoxy,
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thioaryloxy, thioheteroaryloxy, thioheteroalicycloxy, cyano, halo, vitro,
carbonyl,
thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amide,
C-
thioamido, N-amide, C-carboxy, O-carboxy, sulfinyl, sulfonyl, sulfonamide,
trihalomethane-
sulfonamide, trihalomethanesulfonyl, silyl, guanyl, guanidine, ureido,
phosphonyl, amino
S and -NR"R'2 wherein R" and R'z are independently selected from the group
consisting of
hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, carbonyl, C-carboxy,
sulfonyl,
trihalomethanesulfonyl, trihalomethanecarbonyl, and, combined, a five- or six-
member
heteroalicyclic ring.
A "cycloalkyl" group refers to an all-carbon monocyclic or fused ring (i.e.,
rings
10 which share an adjacent pair of carbon atoms) group wherein one of more of
the rings does
not have a completely conjugated pi-electron system. Examples, without
limitation, of
cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene,
cyclohexane,
cyclohexadiene, cycloheptane, cycloheptatriene and adamantane. A cycloalkyl
group may be
substituted or unsubstituted. When substituted, the substituent groups) is
preferably one or
more individually selected from alkyl, aryl, heteroaryl, heteroalicyclic,
hydroxy, alkoxy,
aryloxy, heteroaryloxy, heteroalicycloxy, thiohydroxy, thioalkoxy,
thioaryloxy,
thioheteroaryloxy, thioheteroalicycloxy, cyano, halo, vitro, carbonyl,
thiocarbonyl, O-
carbamyl, N-carbamyl, O-thiocarbamyi, N-thiocarbamyl, C-amide, C-thioamido, N-
amide,
C-carboxy, O-carboxy, sulfinyl, sulfonyl, sulfonamide, trihalo-
methanesulfonamido,
trihalomethanesulfonyl, silyl, guanyl, guanidine, ureido, phosphonyl, amino
and -NR"R'z
with R" and R'2 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.
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An "aryl" group refers to an all-carbon monocyclic or fused-ring polycyclic
(i.e., rings
which share adjacent pairs ofcarbon 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
S substituted groups) is preferably one or more selected from alkyl,
cycloalkyl, aryl,
heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, heteroaryloxy,
heteroalicycloxy,
thiohydroxy, thioalkoxy, thioaryloxy, thioheteroaryloxy, thioheteroalicycloxy,
cyano, halo,
nitro, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-
thiocarbamyl, C-
amido, C-thioamido, N-amido, C-carboxy, O-carboxy, sulfinyl, sulfonyl,
sulfonamido,
trihalomethanesulfonamido, trihalomethane- sulfonyl, silyl, guanyl, guanidino,
ureido,
phosphonyl, amino and -NR"R'2 with R" and R'2 as defined above.
As used herein, a "heteroaryl" group refers to a monocyclic or fused ring
(i.e., rings
which share an adj acent 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
1 S completely 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 selected from alkyl, cycloallcyl, aryl, heteroaryl, heteroalicyclic,
hydroxy, alkoxy,
aryloxy, heteroaryloxy, heteroalicycloxy, thiohydroxy, thioalkoxy,
thioaryloxy,
thioheteroaryloxy, thioheteroalicycloxy, cyano, halo, carbonyl, thiocarbonyl,
O-carbamyl, N-
carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, C-thioamido, N-amido, C-
carboxy, 0-
carboxy, sulfinyl, sulfonyl, sulfonamido, vitro, trihalomethanesulfonamido,
trihalomethanesulfonyl, silyl, guanyl, guanidino, ureido, phosphonyl, amino
and -NR"R'2
with R" and R'2 as defined above.
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A "heteroalicycIic" 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 selected
from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy,
aryloxy,
heteroaryloxy, heteroalicycloxy, thiohydroxy, thioalkoxy, thioaryloxy,
thioheteroaryloxy,
thioheteroalicycloxy, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-
carbamyl, O-
thiocarbamyl, N-thiocarbamyl, C-amido, C-thioamido, N-amido, C-carboxy, O-
carboxy,
sulfinyl, sulfonyl, sulfonamido, vitro, trihalomethanesulfonamido,
trihalomethanesulfonyl,
silyl, guanyl, guanidino, ureido, phosphonyl, amino and -NR"R'z with R" and
R'2 as defined
above.
A "hydroxy" group refers to an -OH group.
An "alkoxy" group refers to both an -O-alkyl and an -O-cycloalkyl group, as
defined
herein.
An "aryloxy" group refers to both an -O-aryl and an -O-heteroaryl group, as
defined herein
A "heteroaryloxy" group refers to a heteroaryl-O- group with heteroaryl as
defined herein.
A "heteroalicycloxy" group refers to a heteroalicyclic-O- group with
heteroalicyclic as
defined herein.
A "thiohydroxy" group refers to an -SH group.
A "thioalkoxy" group refers to both an S-alkyl and an -S-cycloalkyl group, as
defined
herein.
A "thioaryloxy" group refers to both an -S-aryl and an -S-heteroaryl group, as
defined
herein.
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A "thioheteroaryloxy" group refers to a heteroaryl-S- group with heteroaryl as
defined
herein.
A "thioheteroalicycloxy" group refers to a heteroalicyclic-S- group with
heteroalicyclic as defined herein.
A "carbonyl" group refers to a -C(=O)-R" group, where R" is selected from the
group
consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl
(bonded through a
ring carbon) and heteroalicyclic (bonded through a ring carbon), as each is
defined herein.
An "aldehyde" group refers to a carbonyl group where R" is hydrogen.
A "thiocarbonyl" group refers to a -C(=S)-R" group, with R" as defined herein.
A "keto" group refers to a -CC(=O)C- group wherein the carbon on either or
both sides
of the C=O may be alkyl, cycloalkyl, aryl or a carbon of a heteroaryl or
heteroaliacyclic group.
A "trihalomethanecarbonyl" group refers to a X3CC(=O)- group with X as defined
herein.
A "C-carboxy" group refers to a -C(=O)O-R" groups, with R" as defined herein.
1 S An "O-carboxy" group refers to a R"C(=O)O- group, with R" as defined
herein.
A "carboxylic acid" group refers to a C-carboxy group in which R" is hydrogen.
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 "trihalomethanecarbonyl" group refers to an X3CC(=O)- group with X as
defined
above.
A "trihalomethanesulfonyl" group refers to an X3CS(=O)z- groups with X as
defined
above.
A "trihalomethanesulfonamido" group refers to a X3CS(=O)ZNR'3- group with X
and
R" as defined herein.
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A "sulfmyl" group refers to a -S(=O)-R" group, with R" as defined herein and,
in
addition, as a bond only; i.e., -S(O)-.
A "sulfonyl" group refers to a -S(=O)ZR" group, with R" as defined herein and,
in
addition as a bond only; i.e., -S(O)2-.
An "S-sulfonamido" group refers to a -S(=O)ZNR"R'2, with R" and R'2 as defined
herein.
An "N-Sulfonamido" group refers to a R"S(=O)ZNR'Z- group, with R" and R'2 as
defined herein.
An "O-carbamyl" group refers to a -OC(=O)NR"R''- group with R" and R'2 as
defined
herein.
An "N-carbamyl" group refers to a R"OC(=O)NR'2 group, with R" and R'2 as
defined herein.
An "O-thiocarbamyl" group refers to a -OC{=S}NR"R'z group with R" and R''- as
defined herein.
An "N-thiocarbamyl" group refers to a R"OC(=S)NR''- group, with R" and R'z as
defined herein.
An "amino" group refers to an -NHZ group.
A "C-amido" group refers to a -C(=O}NR"R'2 group with R" and R'~ as defined
herein.
A "C-thioamido" group refers to a -C(=S}NR"R'Z group, with R" and R'z as
defined
herein.
An "N-amido" group refers to a R"C(=O)NR'Z- group, with R" and R'2 as defined
herein.
A "ureido" group refers to a -NR"C(=O)NR'zR'4 group, with R" and R'2 as
defined
herein and R'° defined the same as R" and R'2
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A "guanidine" group refers to a -R"NC(=N)NR'2R'4 group, with R", R'2 and R'4
as
defined herein.
A "guanyl" group refers to a R"R'ZNC(=N)- group, with R" and R'Z as defined
herein.
A "cyano" group refers to a -C----N group.
A "silyl" group refers to a -Si(R")3, with R" as defined herein.
A "phosphonyl" group refers to a P(=O)(OR")z with R" as defined herein.
A "hydrazine" group refers to a -NR"NR'ZR'4 group with R", R'2 and R'4 as
defined
herein.
Preferred Structural Features.
Preferred structural features of this invention are those in which r and s are
0 and Q is
sulfur.
Further preferred structural features are those in which r is 1 and s is 0.
Other preferred structural features are those in which r is 0, A is sulfur, F
is nitrogen,
1 S Q is sulfur and RZ is nitre.
The compounds in Table 1 provide additional preferred structural features of
this
invention.
THE BIOCHEMISTRY
The present invention is directed to the use of compounds capable of
modulating or
regulating signal transduction in normal or diseased cells. The present
invention is also
directed to the use of compounds capable of inhibiting the activity of protein
tyrosine
enzymes, in particular protein tyrosine kinases (PTKs) and protein tyrosine
phosphatases
(PTPs), to modulate or trigger signal transduction. The invention is fiuther
directed to the
regulation of cellular processes that are controlled by signal transduction
through the
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16
inhibition of the activity of PTKs and PTPs by the compounds. The invention
further
provides for the use of such compounds in the treatment of a subject having a
disorder caused
by dysfunctional signal transduction.
In one embodiment of the invention, the compounds of the invention are capable
of
inhibiting the activity of protein tyrosine phosphatases, that are
transmembrane or
intracellular, and that may have one or more characteristic catalytic domains.
The amino acid
sequences of the PTPs in the catalytic domains may include but are not limited
to
[I/V]HCXAGXXR(S/T]G (single-letter amino acid code; X is any amino acid). In
addition,
the PTPs may possess one or more modular conserved domains, which include but
are not
limited to, SH2, SH3 and PH domains. In a specific embodiment of the
invention, the
compounds of the invention can be used to inhibit the phosphatase activity of
PTP1B
(Chaxbonneau, et al., 1989, Proc. Natl. Acad. Sci., USA, 86: 5252-5256), T-
cell PTP (Cool,
et al., 1989, Proc. Natl. Acad. Sci., USA, 86: 52575261, PTP1C (Shen, et al.,
1991, Nature,
352: 736-739), PTP1D (Vogel, et al., 1993, Science, 259: 1611-1614), RPTPa,
RPTP[i,
i 5 RPTPy (Kaplan, et al., 1990, Proc. Natl. Acad. Sci., USA, 87: 70007004),
RPTPa (Yan, et
al., 1993, J. Biol. Chem., 268: 24880-24886), RPTPx (Jiang , et al., 1993,
Mol. Cell Biol.,
13: 2942-2951 ) and CD45 (Charbonneau, et al., 1988, Proc. Natl. Acad. Sci.,
USA, 85:
7182-7186). The PTKs and PTPs preferred in the invention are of human origin.
Inhibition
of phosphatase activity that is substantially specific to a PTP or a set of
PTPs in a signaling
pathway is preferred. While the inhibition of phosphatase activity is believed
to be the
mechanism of action of the compounds of the present invention with respect to
their ability to
modulate and/or regulate signal transduction, additional mechanisms have not
been ruled out.
The term "signal transduction" as used herein is not limited to transmembrane
signaling, and includes the multiple pathways that branch off throughout the
cell and into the
nucleus. Such signaling pathways may include but are not limited to the Ras
pathway
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(Schlessinger, 1994, Curr. Opin. Genet. Dev., 4:25-30), the JAK/STAT pathways
(Sadowski, et al., 1994, cience, 261:1739-1744), the phosphoinositide 3-kinase
pathway and
the phospholipase C-y pathway. As used herein, the term "modulation" or
"modulating" shall
mean upregulation or downregulation of a signaling pathway. Cellular processes
under the
control of signal transduction may include, but are not limited to,
transcription of specific
genes; normal cellular functions, such as metabolism, proliferation,
differentiation, adhesion,
apoptosis and survival; as well as abnormal processes, such as transformation,
blocking of
differentiation and metastasis.
A signal may be triggered by the binding of a ligand to its receptor on the
cell surface,
and the signal is transduced and propagated by the phosphorylation or
dephosphorylation of
specific tyrosine residues on various substrates inside the cell. The specific
interactions
between the PTKs, PTPs and their substrates may involve the formation of a
transient or
stable multimolecular complex on the inner face of the plasma membrane or in
other
subcellular compartments including the nucleus. A substrate may contain one or
more
tyrosine residues that are phosphorylated or dephosphorylated by PTKs or PTPs
in the
signaling pathway. Such substrates may include the receptor and its subunits,
molecules
associated with or recruited to the receptor such as cytoplasmic kinases,
cytoplasmic
phosphatases, adapter molecules, cytoskeletal proteins and transcription
factors. The term
receptor as used herein may include, but is not limited to, insulin receptor,
members of the
insulin-like growth factor receptor family, epidermal growth factor receptor
family, fibroblast
growth factor receptor family, hepatocyte growth factor receptor family,
vascular endothelial
growth factor receptor family, neurotrophin receptor (trk) family, the T-cell
receptor, the B
cell receptor and members of the Type I-IV cytokine receptor families (Heldin,
1995, Cell,
80: 213-223; Taniguchi, 1995, cience, 268: 251-255). Adapter molecules that
are substrates
may include the Grb proteins, IRS-1, Zap-70 and Shc (Pawson, et al., 1995,
Nature, 373:
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573-580). Cytoskeletal proteins such as actin and transcription factors such
as the STAT
proteins (Ihle, et al., Trends Biochem. Sci., 19:222-227) may also serve as
substrates. As
used herein, the term ligand is synonymous with extracellular signaling
molecules, and
includes but is not limited to growth factors such as insulin, EGF, PDGF,
fibroblast growth
factors, vascular endothelial growth factor, and neurotrophins; and cytokines
such as growth
hormone, erythropoietin, tumor necrosis factor, interleukins and interferons.
The term ligand
is not limited to soluble molecules, and includes, for example, extracellular
matrix proteins,
cell adhesion molecules as well as antigenic peptides associated with the
major
histocompatibility complex proteins on the surface of an antigen-presenting
cell.
In one embodiment of the invention, the compounds of the invention can be used
to
trigger or upregulate signal transduction in cells so that the effect of
ligand binding to a
receptor is enhanced, or mimicked if the ligand is not present. The compounds
exert the
effect by inhibiting or diminishing the activity of a phosphatase in the
signaling pathway
which normally acts negatively toward signaling. One mechanism by which PTPs
normally
downregulate signal transduction involves the dephosphorylation of specific
phosphotyrosine
residues (pTyr) on PTKs and their substrates since many PTKs require
phosphorylation of
some of its own tyrosine residues in order to become optimally active in the
signaling
pathway. The compounds of the invention can be used to prevent the
dephosphorylation of
pTyr residues on receptors or their subunits which normally becomes
phosphorylated upon
ligand binding, thereby enhancing the extent and duration of PTK
phosphorylation. The
compounds of the invention can also be used to prevent the dephosphorylation
of PTKs in
which the tyrosine residues become autophosphorylated or transphosphorylated
due to its
basal activity. In these PTKS, a signal may be triggered by the compounds of
the invention
in the absence of ligand binding since the basal activity of PTKs is
sufficient to promote a
signal if constitutive PTP activity is inhibited or diminished by the
compounds.
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A preferred embodiment of the invention is directed to a method of triggering,
enhancing or sustaining insulin receptor signal transduction by inhibiting the
constitutive,
dephosphorylation of the pTyr sites on the activated insulin receptor. This
would allow the
insulin receptor to remain phosphorylated, thus enhancing or sustaining the
insulin signal.
Furthermore, since it has been shown that insulin receptor is phosphorylated
at a low level
even in the absence of insulin (Goldstein, 1992, J. Cell Biol., 48:33-42), the
compounds of
the invention can be used to trigger a signal, even in the absence of insulin,
by allowing the
tyrosine residues on the receptor to become self phosphorylated.
Another mechanism by which PTPs may exert a negative effect on signaling is
through the dephosphorylation of specific pTyr sites to which SH2-containing
molecules bind
during signaling. The absence of such pTyr sites would prevent the recruitment
of
SH2-containing molecules to specific subcellular compartments to form
multiprotein
signaling complexes, thereby, preventing the further propagation of the
signal. Thus, the
compounds of the invention can be used to upregulate or prolong signal
transduction by
preventing the dephosphorylation of pTyr sites on substrate proteins that
normally serve as
binding sites for SH2-containing proteins which promote signaling. In another
embodiment
of the invention, the compounds of the invention may be used to prevent the
dephosphorylation of specific pTyr residues on any substrate, which pTyr
residues are
essential to the transmissions or propagation of the signal. Furthermore, the
compounds of
the invention may be used to prevent the dephosphorylation of specific pTyr
residues on any
substrate, which pTyr residues are inhibitory to signal transduction.
The compounds of the invention can also be used to suppress or downregulate
signal
transduction in cells so that the effect of ligand binding to a receptor is
abolished or
attenuated. The compounds can inhibit a phosphatase in a signaling pathway
which normally
acts positively toward signaling. For example, PTPs promote signaling through
the activation
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of members of the Src family of PTKs. Src family PTKs have an inhibitory site
of
phosphorylation in their carboxy termini which by dephosphorylation activates
kinase
activity. Thus the compounds of the invention can be used to prevent the
dephosphorylation
of the inhibitory pTyr in the carboxy termini of kinases which function
normally to promote
5 signal transductions. Src family PTKs may include Src, Fyn, Lck, Lyn, Blk,
Hck, Fgr and
Yrk. Other kinases which may be similarly regulated by a phosphatase may
include Fak and
Csk (Taniguchi, 1995, Science, 268: 251-255).
PHARMACOLOGICAL COMPOSITIONS AND THERAPEUTIC APPLICATIONS
10 As used herein, "pharmaceutically acceptable salt" refers to those salts
which retain
the biological effectiveness and properties of the compound and which are
obtained by
reaction with acids such as hydrochloric acid, hydrobromic acid, sulfuric
acid, nitric acid,
phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic
acid, salicylic
acid and the like.
1 S In addition to the above compounds and their pharmaceutically acceptable
salts, the
present invention is further directed, where applicable, to solvated as well
as unsolvated
forms of the compounds (e.g., hydrated forms) having the ability to regulate
and/or modulate
phosphatase activity.
The compounds described above may be prepared by any process known to be
20 applicable to the preparation of chemically-related compounds. Suitable
processes are
illustrated by the representative examples provided, infra. Necessary starting
materials may
be obtained by standard procedures of organic chemistry.
Pharmaceutical Formulations And Routes Of Administration
A compound of this invention can be administered to a human patient as such or
in
pharmaceutical compositions in which a therapeutically effective dose is mixed
with suitable
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carriers or excipient(s) at doses to treat or ameliorate a variety of
disorders, including solid
cell tumor growth, including Kaposi's sarcoma, glioblastoma, and melanoma and
ovarian,
lung, mammary, prostate, pancreatic, colon and epidermoid carcinoma, diabetes,
diabetic
retinopathy, hemangioma and rheumatoid arthritis. A therapeutically effective
dose further
refers to that amount of the compound sufficient to result in amelioration of
symptoms of
uncontrolled vasculogenesis and angiogenesis. Techniques for formulation and
administration of the compounds such as those of this invention may be found
in
"Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest
edition.
The formulations of the present invention normally will consist of at least
one
compound of formula I mixed with a Garner, or diluted by a carrier, or
enclosed or
encapsulated by an ingestible carrier in the form of a capsule, sachet,
cachet, paper or other
container or by a disposable container such as an ampoule. A carrier or
diluent may be a
solid, semi-solid or liquid material, which serves as a vehicle, excipient or
medium for the
active therapeutic substance.
Some examples of the diluents or Garners which may be employed in the
pharmaceutical compositions of the present invention are lactose, dextrose,
sucrose, sorbitol,
mannitol, propylene glycol, liquid paraffin, white soft paraffin, kaolin,
microcrystalline
cellulose, calcium silicate, silica polyvinylpyrrolidone, cetostearyl alcohol,
starch, gum
acacia, calcium phosphate, cocoa butter, oil of theobroma, arachis oil,
alginates, tragacanth,
gelatin, syrup B.P., methyl cellulose, polyoxyethylene sorbitan monolaurate,
ethyl lactate and
propylhydroxybenzoate, sorbitan trioleate, sorbitan sesquioleate and oleyl
alcohol.
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 pharamacological composition
containing a
compound, salt or prodrug of this invention to an organism for the purpose of
prevention or
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treatment of a disorder associated with an abnormal protein tyrosine enzyme
related cellular
signal transduction.
As used herein, a "disorder associated with an abnormal protein tyrosine
enzyme
related cellular signal transduction" refers to a condition characterized by
inappropriate; i.e.,
under or, more commonly, over, catalytic activity on the part of a protein
tyrosine enzyme.
Inappropriate catalytic activity can arise as the result of either: (1}
protein tyrosine enzyme
expression in cells which normally do not express protein tyrosine enzymes;
(2) increased
protein tyrosine enzyme expression leading to unwanted cell proliferation,
differentiation
andlor growth; or, (3) decreased protein tyrosine enzyme expression leading to
unwanted
reductions in cell proliferation, differentiation and/or growth. Over-activity
of protein
tyrosine enzymes refers to either amplification of the gene encoding a
particular protein
tyrosine enzyme or production of a level of protein tyrosine enzyme activity
which can
correlate with a cell proliferation, differentiation and/or growth disorder
(that is, as the level
of the protein tyrosine enzyme increases, the severity of one or more of the
symptoms of the
I S cellular disorder increases). Underactivity is, of course, the converse,
wherein the severity of
one or more symptoms of a cellular disorder increase as the level of the
protein tyrosine
enzyme decreases.
As used herein, the terms "prevent", "preventing" and "prevention" refer to a
method
for barring an organism from in the first place acquiring a disorder
associated with abnormal
protein tyrosine enzyme related cellular signal transduction.
As used herein, the terms "treat", "treating" and "treatment" refer to a
method of
alleviating or abrogating the abnormal protein tyrosine enzyme related
cellular signal
transduction 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.
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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.
Suitable routes of administration include, without limitation, oral, rectal,
transmucosal, or intestinal administration; intramuscular, subcutaneous,
intramedullary,
intrathecal, direct intraventricular, intravenous, intraperitoneal,
intranasal, or intraocular
injections; transdermal, topical and vaginal application, and the like. Dosage
forms include
but are not limited to tablets, troches, dispersions, suspensions,
suppositories, solutions,
capsules, creams, patches, minipumps and the like.
Alternately, 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.
CompositionlFormulation
The pharmaceutical compositions of the present invention may be manufactured
by
processes well known in the art, for example and without limitation 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
thus
may be formulated in conventional manner using one or more physiologically
acceptable
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.
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For injection, the agents of the invention may be formulated in aqueous
solutions,
preferably in physiologically compatible buffers such as Hanks's 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 readily by combining
the
active compounds with pharmaceutically acceptable carriers well known in the
art. Such
Garners enable the compounds of the invention to be formulated as tablets,
pills, dragees,
capsules, liquids, gels, syrups, slurnes, suspensions and the like, for oral
ingestion by a
patient to be treated. Pharmaceutical preparations for oral use can, for
instance, be prepared
by adding a compound of this invention to a solid excipient, optionally
grinding the resulting
mixture and processing the mixture of granules, after adding suitable
auxiliaries, if desired, to
obtain tablets or dragee cores. Suitable 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, potato starch, gelatin, gum
tragacanth, methyl
cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or
polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added,
such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof
such as sodium
alginate.
Dragee cores are provided with suitable coatings. For this purpose,
concentrated
sugar solutions may be used, which may optionally contain gum arabic, talc,
polyvinyl
pyrroiidone, 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.
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Pharmaceutical preparations 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 filler
S such as lactose, binders such as starches, and/or lubricants 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. In addition, stabilizers may be added. All formulations for oral
administration
should be in dosages suitable for such administration.
10 For buccal administration, the compositions may take the form of tablets or
lozenges
formulated in conventional manner. For administration by inhalation, the
compounds for use
according to the present invention are conveniently delivered in the form of
an aerosol spray
presentation from pressurized packs or a nebulizer, with the use of a suitable
propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane,
carbon dioxide
15 or other suitable gas. In the case of a pressurized aerosol the dosage unit
may be determined
by providing a valve to deliver a metered amount. Capsules and cartridges of
e.g., 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.
The compounds of this invention may be formulated for parenteral
administration by
20 injection, e.g., by bolus injection or continuous infusion. Formulations
for injection may be
presented in unit dosage form, e.g., in ampoules or in multidose containers,
with an added
preservative. The compositions may take such forms as suspensions, solutions
or emulsions
in oily or aqueous vehicles, and may contain formulatory agents such as
suspending,
stabilizing and/or dispersing agents.
25 Pharmaceutical formulations for parenteral administration include aqueous
solutions
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of the active compounds in water-soluble form. Additionally, suspensions of
the active
compounds may be prepared as appropriate oily injection suspensions. Suitable
lipophilic
solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty
acid esters, such as
ethyl oleate or triglycerides, or 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 or agents which 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, e.g., containing conventional suppository bases such as
cocoa butter or
other glycerides.
In addition to the formulations described previously, the compounds may also
be
formulated as a depot preparation. Such long acting formulations may be
administered by
implantation (for example subcutaneously or intramuscularly) or by
intramuscular injection.
Thus, for example, the compounds may be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable oil) or ion
exchange
resins, or as sparingly soluble derivatives such as, for example, a sparingly
soluble salt.
A pharmaceutical carrier for the hydrophobic compounds is a cosolvent system
comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic
polymer, and an
aqueous phase. The cosolvent system may be the VPD co-solvent system. VPD is a
solution
of 3% wlv benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80,
and b5% w/v
polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-
solvent
system (VPD:OSV~ consists of VPD diluted 1:1 with a S% dextrose in water
solution. This
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co-solvent system dissolves hydrophobic compounds well, and itself produces
low toxicity
upon systemic administration. Naturally, the proportions of 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 80~; 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. Certain organic solvents such as
dimethylsulfoxide also
may be employed, although usually 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.
Numerous
sustained release products 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 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.
In addition to the common dosage forms set out above, the compounds of the
present
invention may also be administered by controlled release means and/or delivery
devices
including Alzet~ osmotic pumps which are available from Alza Corporation.
Suitable
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28
delivery devices are described in U.S. Patent Nos. 3,845,770; 3,916,899;
3,536,809;
3,598,123; 3,944,064 and 4,008,719, the disclosures of which are incorporated
in their
entirety by reference herein.
Many of the phosphatase modulating compounds of the invention may be provided
as
salts with pharmaceutically compatible counterions. Pharmaceutically
compatible salts may
be formed with many acids, including but not limited to hydrochloric,
sulfuric, acetic, lactic,
tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or
other protonic
solvents that are the corresponding free base forms.
Do_ saEe
Pharmaceutical compositions suitable for use in the present invention include
compositions wherein the active ingredients are contained in an amount
effective to achieve
its intended purpose. 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 reference to the treatment of
cancer, a
1 S 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; andJor, (4) relieving to some extent (or preferably eliminating)
one or more
symptoms associated with the cancer. Determination of the therapeutically
effective amount
of a compound of this invention is well within the capability of those skilled
in the art,
especially in light of the detailed disclosure provided herein.
The therapeutically effective dose can be estimated initially from cell
culture assays.
For example, a dose can be formulated in animal models to achieve a
circulating
concentration range that includes the IC50 as determined in cell culture
(i.e., the
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29
concentration of the test compound which achieves a half maximal inhibition of
the PTP
activity). Such information can be used to more accurately determine useful
doses in
humans.
Thus, a therapeutically effective dose refers to that amount of the compound
that
results in amelioration of symptoms in or a prolonged survival of a patient.
Toxicity and
therapeutic efficacy of such compounds can be determined by standard
pharmaceutical
procedures in cell cultures or experimental animals, e.g., for determining the
LD50 (the dose
lethal to SO% of the population) and the ED50 (the dose therapeutically
effective in 50% of
the population). The ratio of toxic does to therapeutic effective, LD50/ ED50,
is the
therapeutic index. Compounds which exhibit high therapeutic indices are
preferred. The data
obtained from cell culture assays and animal studies can be used in
formulating a range of
dosages for use in humans. A dosage preferably lies within a range of
circulating
concentrations that include the ED50 and exhibits little or no toxicity. The
dosage may vary
within this range depending upon the dosage form employed and the route of
administration
1 S 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.l).
Dosage amount and interval may be adjusted individually to provide plasma
levels of
the active moiety which are sufficient to maintain the tyrosine enzyme
modulating effects,
known as the minimal effective concentration (MEC). The MEC will vary for each
compound but can be estimated from in vitro data; for example, without
limitation, the
concentration necessary to achieve a 50-90% inhibition of the tyrosine enzyme
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.
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Dosage intervals can also be determined using the MEC value. Compounds should
be
administered using a regimen which maintains plasma levels above the MEC for
10-90%,
preferably between 30-90% and most preferably between 50-90% of the time.
5 Usual patient dosages for systemic administration range from 1 to 2000
mg/day,
commonly from 1 to 250 mg/day, and typically from 10 to I 50 mg/day. Stated in
terms of
patient body weight, usual dosages range from 0.02 to 25 mg/kg/day, commonly
from 0.02 to
3 mglkg/day, typically from 0.2 to 1.5 mg/kg/day. Stated in terms of patient
body surface
areas, usual dosages range from 0.5 to 1200 mg/mz/day, commonly from 0.5 to
150
10 Mg/m2/day, typically from 5 to 100 Mgl m2/day. Usual average plasma levels
should be
maintained within 50 to 5000 ~g/ml, commonly 50 to 1000 pg/ml, and typically
100 to 500
~eg/ml.
In cases of local administration or selective uptake, the effective local
concentration of
the drug may not be related to plasma concentration.
15 The amount of a particular composition administered will, of course, be
dependent on
the subject being treated, on the subject's weight, the severity of the
affliction, the manner of
administration and the judgment of the prescribing physician.
Desirable blood levels may be maintained by a continuous infusion of the
compound;
plasma level can be monitored by HPLC. It should be noted that the attending
physician
20 would know how and when to terminate, interrupt or adjust therapy to lower
dosage due to
toxicity, or bone marrow, liver or kidney dysfunctions. Conversely, the
attending physician
would also know to adjust treatment to higher levels if the clinical response
is not adequate
and toxicity os not a problem.
The size of a prophylactic or therapeutic dose of a compound in the acute or
chronic
25 management of disease will vary with the severity of the condition to be
treated and the route
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of administration. Again, it should be noted that the clinician or physician
would know when
to interrupt and/or adjust the treatment dose due to toxicity or bone marrow,
liver or kidney
dysfunctions. The dose, and perhaps the dosage frequency, will also vary
according to the
age, body weight, and response of the individual patient. In general, as
discussed above, the
total daily dose ranges for the compounds for the majority of the disorders
described herein,
is from about 0.02 to about 25 mg/kg patient. Preferably, a daily dose range
should be
between about 0.02 to about 3 mg/kg, while most preferably a daily dose range
should be
between about 0.2 to about 1.5 mglkg per day. It is further recommended that
infants,
children, and patients over 65 years, and those with impaired renal, or
hepatic function,
initially receive low doses and that they be titrated based on individual
clinical responses)
and blood level(s). It may be necessary to use dosages outside the above
ranges in some
cases; situations requiring such a decision will be apparent to those of
ordinary skill in the art.
Packagin>r
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 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
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on the label may include treatment of a tumor, inhibition of angiogenesis,
treatment of
fibrosis, diabetes, and the like.
Methods Of Treatment
Any compound of the invention which inhibits or diminishes protein tyrosine
enzyme
activity in a signaling pathway may be used in the therapeutic methods of the
invention. In a
preferred embodiment, the activity of the compound is sufficiently specific
for the paticular
protein tyrosine enzyme pathway so that the compound does not interfere with
the function of
other enzymatic activity, including other tyrosine enzyme activity, in the
cell.
The compounds and pharmaceutical compositions of the invention can be used for
treating diabetes mellitus. The pathogenesis of diabetes generally involves
insufficient or a
total lack of insulin signal transduction. The paucity or absence of the
insulin signal may be
caused by a variety of factors such as a lack of insulin, loss of binding
affinity, defective
receptor or under expression of receptor. Insulin receptor activity can be
modulated by
inhibiting tyrosine phosphatases in the signaling using the compounds of the
invention.
Unlike currently available treatment modalities that are based on the insulin
receptor, the
insulin signal may be restored or stimulated in cells through the inhibition
of
dephosphorylating activity, even in the absence of insulin. The example of
diabetes mellitus
illustrates the principles of therapeutic applications of the compounds of
this invention which
may be applied to other disorders that implicate signal transduction by
tyrosine enzymes, in
particular, phosphotyrosine phosphatases. The compounds and pharmaceutical
compositions
of the invention may be used to treat immune disorders in which cytokine
signal transduction
is deficient. Cytokines play a crucial role in hemopoiesis as well as
coordinating immune and
inflammatory responses. The compounds may be used to replace or enhance the
activity of a
cytokine in signaling the differentiation and proliferation of hemopoietic
cells, as well as B
and T cells in response to antigenic stimulation, and thus be usefizl for
treating anemia and
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immunodeficiency. The compounds may also be used as an antiinflammatory agent
to treat
disorders such as rheumatoid arthritis. The compounds may also be
therapeutically useful in
treating neurodegenerative diseases by stimulating the growth and
differentiation of neuronal
cells which is regulated by neurotrophin-mediated signal transduction.
In another embodiment of the invention, the compounds and pharmaceutical
compositions
of the invention may be used to treat cancer, such as glioma, melanoma,
Kaposi's sarcoma,
hemangioma and ovarian, breast, lung, pancreatic, liver, prostate, colon and
epidermoid cancer,
in which the malignant cells proliferate and/or metastasize as a result of
uncontrolled signal
transduction mediated by growth factors. For example, over expression of a
PTK, such as HER2
has been shown to correlate with the aberrant growth characteristics of tumor
cells.
Vasculogenesis and/or angiogenesis that facilitates tumor growth may also be
inhibited by the
compounds of this invention. The compounds may modulate signal transduction in
these tumor
cells so that normal growth characteristics are restored. The compounds may
also be useful in
treating psoriasis which is caused by excessive epidermal growth factor
mediated signal
transduction.
SYNTHESIS
The compounds of the present invention as well as the starting materials 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 this
invention are available and that the following examples are in no way to be
considered
limiting in any manner whatsoever with regard to preparation of the compounds
of this
invention.
Example 1. 3-(5-nitrothiazol-2-yl)mercaptol-5-phenyl-1,2,4-triazole (Compound
1)
The starting material 2-brono-5- nitrothiazol was prepared by treating 2-amino-
5-
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nitrothiazol (Aldrich) with sodium nitrite and hydrogen bromide {Fr. Demande
2,015,4 34,
1970). 3-Phenyl-1,2,4-triazole-5-thione (E. Hogarth, J. Chem. Soc. (1949)
1163) was
prepared by first reacting benzoyl chloride with thiosemicarbazide in pyridine
at 0 °C to give
benzoyl thiosemicarbazide. Benzoyl thiosemicarbazide was treated with
potassium
S hydroxide in ethanol to give 3phenyl-1,2,4-triazole-5-thione. 3-Phenyl-1,2,4-
triazole-Sthione
(1.77 g) was then dissolved in 50 mL of methanol and treated with 0.57 g of
95% sodium
methoxide, and then with 2-bromo-S-nitrothiazole (2.09 g). The mixture was
stirred at room
temperature for 2 hours and the precipitated sodium bromide was removed by
filtration. The
methanol was evaporated and the product crystallized from ethanol and watre to
give 1.5 g of
3-[(5-nitrothiazol-2-yl)mercapto]-S-phenyl 1,2,4-triazole, a white solid, MP
I55-157°C.
Example 2. 2-[(5-vitro-thiazol-2-yl)mercapto]-5-t-butyl-1,2,4-triazole
The title compound was prepared in the manner described in Example 1.
Substituting
pivaloyl chloride for the benzoyl chloride in Example 1 gave pivaloyl
thiosemicarbazide and
then 3-t-butyl-1,2,4-triazole-S-thione. Reaction of 1.79 g of the sodium salt
of the thione
with 2.09 g of 2-bromo-S-nitrothiazole as in Example I yielded 1 g of 2-[(5-
nitro-
thiazol-2-yl)- mercapto)-5-t-butyl-1,2,4-triazole, a yellow solid, MP 219-
221° C.
Example 3. 3-[(S-nitrothiazol-2-yl)mercapto]-5-(thien-2-yl)-1,2,4-triazole
The title compound was prepared in the manner described in Example 1.
Substituting
the acid chloride of thiophene-2-carboxylic acid (prepared from the acid and
oxalyl chloride)
for the benzoyl chloride in Example 1 gave the thiosemicarbazide of thiophene-
2-carboxylic
acid and then 3-(thien-2-yl)-1,2,4-triazole-S-thione. Reaction of 1.73 g of
the sodium salt of
the thione with 2.09 g of 2-bromo-5-nitrothiazole as in Example 1 yielded 1 g
of 3-[(S-
nitrothiazol- 2-yl)mercapto]-5-(thien-2-yl)-1,2,4-triazole, an orange solid,
MP 179-181° C.
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Example 4. 3-(4-chlorophenyl)-5-((5-nitrothiazol-2y1)mercapto]-1,2,4-triazole
The title compound was prepared in the manner described in Example 1.
Substituting
4-chlorobenzoyl chloride for the benzoyl chloride in Example 1 gave 4-
chlorobenzoyl
5 thiosemicarbazide and then 3-{4-chlorophenyl)-1,2,4-triazole-5-thione.
Reaction of 2.34 g of
the sodium salt of 3-(4-chlorophenyl)-1,2,4-triazole-5-thione with 2.09 g of 2-
bromo--
S-nitrothiazole as in Example 1 yielded 1.5 g of 3-(4--
chlorophenyl)-5-[(5-nitrothiazol-2-yl)mercapto[-1,2,4triazole, a light brown
solid, MP
181-184°C.
Example 5. 3-hydroxy-5-[(5-nitrothiazol-2 yl)mercapto]-4-phenyl-1,2,4-triazole
The title compound was prepared by the general method described by Potts,
K.T.,
1961, Chem. Rev., 61:87. 4-Phenyl-3-thiosemicarbazide {4.18g) (Aldrich) was
dissolved in
50 mL of pyridine and treated with 2.71 g of ethyl chloroformate at
O°C. The reaction was
I 5 stirred for 2 hours and then refluxed for 18 hours. Evaporation of the
solvent and trituration
with water gave 2.5 g of 3-hydroxy-5-mercapto-4-phenyl-1,2,4-triazole.
3-Hydroxy-5-mercapto-4-phenyl- 1,2,4-triazole (1.93 g) was stirred in 10 mL of
ethanol with
1.1 equivalent of potassium carbonate in I O mL ethanol for one hour and then
reacted with
2.09 g of 2-bromo-5-nitrothiazole as in Example 1. Crystallization from
ethanol and water
gave 0.6 g of 3-hydroxy-5-[(5-vitro- thiazol-2-yl)mercapto]-4-phenyl-1,2,4-
triazole, a dark
yellow solid, M.P. 188-190°C.
Example 6. 4-Cyclohexyl-3-hydroxy-5-[(5-nitrothiazol-2-yl)mercapto]-1,2,4-
triazole
The title compound was prepared in a manner similar to that described in
Example 5.
Cyclohexyl isothiocyanate (3.53 g) in 10 mL of acetonitrile was added to
hydrazine (0.8 g) in
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20 mL of acetonitrile over a period of 30 minutes. The reaction was stirred
for two additional
hours and evaporated to dryness to give 4.4 g of 4-cyclohexylcarbonyl-3-
thiosemicarbazide.
4-Cyclohexylcarbonyl-3-thiosemicarbazide (2.02 g) was treated as in Example 5
with ethyl
chloroformate (1.08 g). The reaction product 3-hydroxy-5-mercaptol-4-
cyclohexyl-1,2,4-triazole (1.0 g) was reacted with 1.05 g of 2-bromo-5-
nitrothiazole as in
example S. Crystallization from ethanol and water gave 0.3 g of 3-hydroxy-5-
[(5-
nitrothiazoi-2-yl)mercapto] -4-cyclohexyl- 1,2,4-triazale, a yellow solid, MP
237-239°C.
Example 7. 4-benzyl-3-hydroxy-5-[(5-nitrothien-2-yl)mercapto[-1,2,4-triazole
The title compound was prepared in a manner similar to that described in
Example 5
starting with benzyl isothiocyanate. The intermediate 4-benzyl-3-
thiosemicarbazide (1.81 g)
was treated with ethyl chloroformate (1.09 g) as in Example 5. The reaction
product,
4-benzyl-3-hydroxy-5-mercapto-1,2,4-triazole (1.04 g), was reacted with 1.05 g
of
2-bromo-S-nitrothiazole as in Example 5. Crystallization from ethanol and
water gave 0.3 g
of 4benzyl-3-hydroxy-5-[(5-nitrothien-2-yl)mercapto]-1,2,4-triazole, a yellow
solid, MP
221-224° C.
Example 8. 3-hydroxy-5-[(5-nitrothiazol-2-yl)mercapto]-4-[2-{trifluoromethyl)
phenyl]-1,2,4-triazole
The title compound was prepared in a manner similar to that described in
Example 5
starting with 2-(trifluoromethyl) phenyl isothiocyanate. The intermediate 4-[2-
(trifluoromethyl)- phenyl]-3-thiosemicarbazide (2.04 g) was treated with ethyl
chloroformate
(1.09 g) as in example 5. The reaction product, 3-hydroxy-S-mercapto-4-[2-
(trifluoromethyl)phenyl]-1,2,4-triazole (0.78 g) was reacted with 0.63 g of
2-bromo-5-nitrothiazole as in Example 5. Crystallization from ethanol and
water gave 0.3 g
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of 3hydroxy-5-[(5-nitrothiazol-2-yl)mercapto]-4-[2(trifluoro- methyl)phenylJ-
1,2,4-triazole, a
yellow solid, MP 183-185° C.
Example 9.
3-(l-ethyl-3-methylpyrazol-5-yl)-4-(3-methoxy-n-propyl)-5-[5-(nitrothiazol-2
yl)mercapto]-1,2,4-triazole
The title compound was synthesized in a manner similar to that described in
Example
1. 3-Methoxy-n-propyl isothiocyanate was prepared from 3-methoxy-n-propylamine
and
thiophosgene at high temperature and then reacted with hydrazine in pyridine
to give the
intermediate 4-(3-methoxy-n-propyl)-3-thiosemicarbazide. 4-(3-Methoxy-n-
propyl)
3-thiosemicarbazide (1.64 g) was reacted with 1-ethyl-3-methylpyrazole-5-
carboxylic acid
chloride (I.73 g, prepared from the acid and oxalyl chloride) to give 2 g of 1-
(I-ethyl-
3-methylpyrazole-5-carbonyl)-4-(3-methoxy-n-propyl)-3-thiosemicarbazide.
Treatment of
1-(I-ethyl-3-methylpyrazole-5-carbonyl)-4-(3-methoxy-n-propyl)-3-
thiosemicarbazide with
potassium hydroxide in ethanol gave 3-(1-ethyl-3ethylpyrazol-5-yl)-5-mercapto-
4-(3-methoxy-n-propyl)-1,2,4-triazole. Reaction of the sodium salt of 3-(1-
ethyl-3-
methylpyrazol-5-yl)-5-mercapto-4-(3-methoxy-n-propyl)-1,2,4-triazole(0.73 g)
with
2-bromo-5-nitrothiazole (0.52 g) yielded crude 3-(1
-ethyl-3-methylpyrazol-5-yl)-4-(3-methoxy-
n-propyl)-5-[5-(nitrothiazol-2-yl)mercaptoJ-1,2,4-triazole as in Example 1.
Crystallization
from ethanol and water gave 0.3 g of 3-(1-ethyl-3-methylpyrazol-5-yl)-4-(3-
methoxy-n-
propyl)-5-[5-(nitrothiazol-2-yl)mercaptoJ-1,2,4-triazole, a light brown solid,
MP 117-118° C.
Example 10. 3-(4-chlorophenyl)-5-[(S-nitrothiazol-2-yl)amino]-1,2,4-triazole
The title compound was prepared in a similar manner to that described in
Example 1
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by heating 3-amino-5-(4-chlorophenyl)-1,2,4-triazole with 2-bromo-5-
nitrothiazole in
refluxing tetrahydrofuran followed by silica gel column chromatograph using a
mixture of
dichloromethane and methanol as the eluent to yield 3-(4-chlorophenyl)-5-((5-
nitrothiazo-
2-yl)amino]-1,2,4-triazole.
Example 11. 4-Allyl-3-hydroxy-5-[5-nitrothien-2-yl)mercapto]-1,2,4-triazole
The title compound was prepared in a similar manner to that described in
Example 5
starting with allyl isothiocyanate. 4-Allyl-3-hydroxy-5-mercapto-1,2,4-
triazole was reacted
with 2-bromo-5-nitrothiazole as in Example 5. Crystallization from ethanol and
water gave
4-allyl-3-hydroxy-5-[(5nitrothien-2-yl)mercapto]-1,2,4-triazole as a yellow
solid.
Example 12. 3-[(5-nitrothiazol-2-yl)mercapto]-4-(4
methoxyphenyl)-5-(thien-2-yl)-1,2,4- triazole
2-Bromo-5-nitrothiazole
To 72.5 g of 2 -amino-5-nitrothiazole in 300 mL of 48 % hydrobromic acid and
200
mL of water stirred and cooled to about -10° C was slowly added, in
portions, S I .8 g of
sodium nitrite dissolved in 80 mL of water from one addition funnel and 250 mL
of n-amyl
alcohol from a second addition funnel. The addition of both solutions required
about 3 hours.
The cooling bath was removed and the mixture allowed to warm to about I
S° C overnight
and then stirred at room temperature for 2 hours. The solid was collected by
vacuum
filtration and steam distilled to give 67 g of crude product. The crude
product was
recrystallized from hot ethanol to give 6I g (60 k yield) of the 2-bromo-5-
nitrothiazole as a
yellow solid.
N1-(4-Methoxyphenylaminothiocarbonyll-N2-(thien-2carbonvllhjrdrazine
Thien-2-carboxyhydrazide (2 g) and 2.3 g of 4methoxyphenylisothiocyanate in 25
mL
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of tetrahydrofuran are stirred overnight at 25° C. The reaction is
concentrated to give a
precipitate which is collected by vacuum filtration and dried to give 3.5 g of
the title
compound as an off white solid.
4-l4-Meth oxvp h enyll-5-(th ien-2-vl)-1.2,4-triazol-3-th lone
Nl-(4-Methoxyphenylaminothiocarbonyl)-N2-(thien-2carbonyl)hydrazine (1 g) and
0.2 g of sodium ethoxide in 10 mL of ethanol are refluxed for 6 hours. The
mixture is cooled
and the precipitate collected by vacuum filtration and dried to give 0.6 g of
the title
compound as an off white solid. 4-{4-Methoxyphenyl)-5-(thien-2-yl)-1,2,4-
triazol-3-thione
(0.5 g} and 0.4 g of 2-bromo-S-nitrothiazole in 50 mL of acetonitrile are
refluxed for 6 hours.
The solvent is concentrated to give crude product which is collected by vacuum
filtration.
The crude product is crystallized from ethanol and dried to give 0.6 g of the
desired triazole
compound as an off white solid.
Example I3. 3-[(5-Nitrothiazole -2-yl)mercapto]-4-methyl-1,2,4-triazol
Methylaminothicarbonyl-hydrazine
By substituting 1 g of formic acid hydrazide for the 2 g of thien-2-
carboxyhdrazide and 1 g of
methylisothiocyanate for the 4-methoxyphenylisothiocyanate in the method of
Example 12, 1
g of the title compound is obtained as an off white solid.
Example 14. 3-[(5-nitrothiazol-2-yl)niercapto]-4-butyl-1,2,4-triazole
Butanoyl hvdrazide
Butanoyl hydrazide is prepared according to the general method of A. I. Vogel,
Practical Organic Chemistry, 3rd edition, 1956 (Longman Group, London) p 395.
Ten grams
of ethyl butanoate is refluxed in 10 mL of hydrazine hydrate for 15 minutes.
Absolute
ethanol is added, reflux is continued for 3 hours, and the ethanol distilled.
The solution is
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cooled and the crystalline hydrazide isolated by vacuum filtration and dried
to give 8 g of
butanoyl hydrazide. By substituting 1.7 g of butanoyl hydrazide for the formic
acid
hydrazide in Example 14, 0.6 g of the desired triazole compound is obtained as
an off white
solid.
Example 15. 5-[(5-Nitrothiazol-2-yl)mercapto]-1-(4-methoxyphenyl)tetrazole
2-Bromo-5-nitrothiazole
To 72.5 g of 2-amino-5-nitrothiazole in 300 mL of 48% hydrobromic acid and 200
mL of water stirred and cooled to about -10° C was slowly added, in
portions, 51.8 g of
sodium nitrite dissolved in 80 mL of water from one addition funnel and 250 mL
of n-amyl
alcohol from a second addition funnel. The addition of both solutions required
about 3
hours. The cooling bath was removed and the mixture allowed to warm to about
15° C
overnight and then stirred at room temperature for 2 hours. The solid was
collected by
vacuum filtration and steam distilled to give 67 g of crude product. The crude
product was
recrystallized from hot ethanol to give 61 g (60 k yield) of the 2-bromo-5-
nitrothiazole as a
yellow solid.
1-f4-Methoxyphenyl)tetrazol-5-thione
4-Methoxyphenylisothiocyanate (2 g) and 1 g of sodium azide in 50 mL of
ethanol is
refluxed for 5 hours, cooled and concentrated (see, for instance, Canadian
Journal of
Chemistrv 1959, p 101). The precipitate is collected by vacuum filtration and
dried to give
1.5 g of 1-(4methoxyphenyl)tetrazol-S-thione as an off white solid.
2-Bromo-5-nitrothiazole (1.1 g) and 1 g of 1-{4-methoxyphenyl)tetrazol-5-
thione in
50 mL of acetonitrile are refluxed for 5 hours and the acetonitrile
evaporated. The residue is
crystallized from ethanol to give 1.2 g of the desired tetrazole compound as
an off white
solid.
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Example 16. 2-[(5-Nitrothiazol-2-yl)mercapto]-5-methyl-1,3,4-thiadiazole
2-Bromo-5-nitrothiazole
To 72.5 g of 2-amino-5-nitrothiazole in 300 mL of 48 % hydrobromic acid and
200
mL of water stirred and cooled to about -10° C was slowly added, in
portions, S 1.8 g of
sodium
nitrite dissolved in 80 mL of water from one addition funnel and 250 mL of n-
amyl alcohol
from a second addition funnel. The addition of both solutions required about 3
hours. The
cooling bath was removed and the mixture allowed to warm to about 15° C
overnight and
then stirred at room temperature for 2 hours. The solid was collected by
vacuum filtration
and steam distilled to give 67 g of crude product. The crude product was
recrystallized from
hot ethanol to give 61 g (60 % yield) of 2-bromo-5-nitrothiazole as a yellow
solid.
Z-Mercapto-5-methyl-1.3,4-thiadiazoie
2-Mercapto-5-methyl-I,3,4-thiadiazole is prepared according to the general
method
1 S described by S. G. Boots and C. C. Cheng, 1967, J. Hetercyclic Chemistry,
4: 272-283.
Acetic acid hydrazide (7.4 g) 160 mL of methanol, 5.0 g of 85 % potassium
hydroxide
pellets, and 10 mL of carbon disulfide is stirred at room temperature for 4
hours. Ether (400
mL) is added and and the mixture is cooled in an ice bath to give 10 g of
solid potassium
acetyldithiocabazate, which is collected by vacuum filtration, dried, and used
immediately.
The crude potassium acetyldithiocarbazate in a mixture of 300 mL of
dichloromethane and 54
mL of boron trifluoride etherate is stirred under nitrogen for 18 hours. The
orange solution is
poured onto ice and extracted with ether. The ether extract is washed with 10
% potassium
hydroxide solution and the aqueous phase acidified to pH 2 with cold 10 %
hydrochloric acid.
The precipitate is collected by vacuum filtration and dried to give 2.5 g of
the title compound
as a beige solid.
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2-Bromo-5-nitrothiazole (2.09 g) and 1.32 g of 2-mercapto-5-methyl-1,3,4-
thiadiazole
in 10 mL of tetrahydrofuran are stirred at room temperature overnight,
triethylamine is added
to neutrality, and stirring is continued. The precipitated solid is dissolved
in
dichloromethane, washed with water, and the organic layer evaporated to give
crude product.
The crude product is crystallized from ethyl acetate/hexane to give 1 g of
desired
thiazole-thiadiazole compound as an off white solid.
Alternatively, 2-[(5-nitrothiazol-2-yl)mercapto]-5-methy-1,3,4-thiadiazole is
prepared
by the general method of J. Bourdais, et al., 1981, Eur. J. Chem. Chim. Ther.,
16: 233-240.
2Bromo-5-nitrothiazole (2.09 g) and 1.32 g of 2-mercapto-Smethyl-1,3,4-
thiadiazole in 10
mL of ethanol and 10 mL of 1 N potassium hydroxide are stirred at room
temperature
overnight. The precipitated solid is collected by vacuum filtration, washed
with water and
crystallized from ethyl acetate/hexane to give 1 g of the title compound as an
off white solid.
Alternatively, 2-[(5-nitrothiazol-2-yl)mercapto]-S-methyl-1,3,4-thiadiazole is
prepared by forming and isolating the sodium salt of 2-mercapto-5-methyl-1,3,4-
thiadiazole,
reacting it with 2-bromo-5-nitrothiazole in a suitable inert solvent at room
temperature and
isolating the product as described above. For an example of this method see M.
F. Abdel-
Lateef and Z. Eckstein, 1972, Rocz. Chem., 46: 1647- 1658.
Example 17. 2-[(5-Nitrothiazol-2-yl}mercapto-5-propyl-1,3,4-thiadiazole
Butanoyl hydrazide is prepared according to the general method of A. I. Vogel,
Practical Organic Chemistry, 3rd edition, 1956 (Longman Group, London} p 395.
Ten grams
of ethyl butanoate is refluxed in 10 mL of hydrazine hydrate for 15 minutes.
Absolute
ethanol is added, reflux is continued for 3 hours, and the ethanol distilled.
The solution is
cooled and the crystalline hydrazide isolated by vacuum filtration and dried
to give 8 g of
CA 02293400 1999-12-10
WO 98156376 PCT/US98/12333
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propanoyl hydrazide.
2-Mercapto-5-propyl-1,3,4-thiadiazole is prepared as for 2--
mercapto-5-methyl-1,3,4-thia.diazole by substituting propanoyl hydrazide for
acetic hydrazide
in Example 16.
By substituting 2-mercapto-5-propyl-1,3,4-thiadiazole for 2 -mercapto-5-methyl-
1,3,4-thiadiazole in Example 17, 2-[(5-nitrothiazol-2-yl)mercapto]-5-propyl-
1,3,4-thiadiazole
is isolated as an off white solid.
BRIEF DESCRIPTION OF THE TABLES.
Table 1 shows preferred chemical structures which are within the scope of this
invention. The compounds shown are in no way to be construed as limiting the
scope of this
invention.
BIOLOGICAL EVALUATION
It will be appreciated that, in any given series of compounds, a spectrum of
biological
activity will be observed. In a preferred embodiment, the present invention
relates to novel
heteroaryl compounds demonstrating the ability to modulate protein tyrosine
enzymes related
to cellular signal transduction, most preferrably, protein tyrosine
phosphatase. The assays
described below are employed to select those compounds demonstrating the
optimal degree
of the desired activity .
As used herein, the phrase "optimal degree of desired activity" refers to the
highest
therapeutic index, defined above, against a protein tyrosine enzyme which
mediates cellular
signal transduction and which is related to a particular disorder so as to
provide a patient,
preferably a human, suffering from such disorder with a therapeutically
effective amount of a
compound of this invention at the lowest possible dosage.
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TABLE 1
\ l
s
N
S S
NOy~~ N-N
O.
S
/N S
O N ~ O O~ ~=O
N
~ v
S ~ N
~ i
\N \N"S~N
O-
S
Qi ~ S N N_N
N N S ~ N
S N'1r
CI N O
~S
'S~ F F
N~ .
S S N ' CI O/~ p ~ F
i ~N ~-~ O_
I
O~N S Nw S iNv
~I ~~ S ~~~~N
S' \Si "NH CHI ?-N
oN' NH
N~S ~ ~ O~NH I \. NON%N O O_
O
O N-O_ O / ~N
S
/ N N- \ ~S~S
N \ I \ S
-S N
O~N' S N ~ O N O CH
3
0- H3C~N\N/N O NO N~N
~s O
S \N S N S S
~N _ ~S N ~ F N
O~ ~S S / N O N O. \ / F ~Na.O_
N~ F
\ Br O
O CH3
N
S ~
S N H3 ~.O~N~~S~S N
~S N ~ O p- ~ N
N~ O-N~ H3C O O S
O. O '--CH _
3
O/
SUBSTITUTE SHEET (RULE 26)
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WO 98/56376 PCT/US98/12333
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TABLE I (cont'd)
S N
HO N_ / N
O ~ ~ O
N'
~S O O:N ~O_ O
S' \\
S
OH N~
N S N
N' /S' /N
O
HO--~N~ i0 O N 0 S NH SO--O
N n
S S O' ~ O:N ~
r ~ O'
H N S NH
2
0
S N~ Q~ S S NH
N~SJ~ ~O- O ~ ~ CI
~N
O._N ~O
~-~. HOC
Br ~ NH // S ~ ~ ~ N >--S
~ i
H C ~ N S N~S~N p~ ~ NH ~=N
3 ('.H3
N / Nip- HsC CHs
'O O ~N ~O
_ ,O F F
O ~N NH S NH
S~S~NH N ~ ~S ~ ~ ~ F ~~ ~ / \ F F
0 \\N N~S~N O~N ~O CI - F
0 H~C~CHs
N\ ~~S~NH
C ~~''~ '~I
O°N'~S~ ~ O--N ~ ~ S N ~ ~ 0
O. N~ H ~//S NI~ ~ ~ O-Nv CHs
i \N%\S/ 'N O.
~CH3
HsC
NIN / I S I N
HO~ S NH
N N O=N. ~ ~ C s
S~S~N,:O O'
O-
SUBSTITUTE SHEET (RULE 26)
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WO 98/56376 PCT/US98/12333
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TABLE 1 (cont'd)
N N-N
F F N~ ~S~ S O S~ JI
S NH
O_N \ \ O ~~N ~ N
/ O. \ S \ OwCH3
N CHs
ON w0_
O
+~0
N_ S~N ~ NH~S'<~N+\O_ CI ~ I ON
CH N O\ ~N'N NH S S \
3
p CHs ~ ~N
O N'\O_ N~N~S
O-
,N S F O_
'+ S CHs F
N\ S N O ~~ ~ ~~'' ~~ I F ~ ~~N ~O
< N J ~N~S
O_ +~--S ~/ N
N /~_
O_ S ~ ~ O
CHs + S N S a
N ~~ ~,' N+ S~ ~
N O ~ N N N ~ I O ~~S~N
S ~ NJ
~N NH~
O_N+ N' ~~N
O N
HzC~
O_ S\,iS\ 'N' S NYS ~N
N S p' ~ ~N~ Nr~ ~ ~ I IS
_-N N ,N+
N
O_N \ ~N.N
O CHs //
S
~ S S N~S~N~N
N~S~N/ N/\S <\ S S
~S ~NH ..""SCI O-N \r ''~S
p-N \ - ONE ~O_
O_ HsC
O
N~N
N S \
N- NH ~ N S S
\ O~CHg ~S O~ \
O
O:N ~O_ ON ~O_ /
SUBSTITUTE SHEET (RULE 26)
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WO 98/56376 PCT/US98/12333
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TABLE 1 (cont'd)
N
~NH CH3
0-
S / _ S S N O
S S H C \ / O S N ~ N~ pN~~~ ~ /
N ~--O
N a0 OH
O_, N~N / _ O~CHs
N~S~ ~ O_ O . S fI S I N \ V
v
\ S N ~ S S N O~CH3 N ~ I
ON'~~ I O~ N N ~
O_.N~O \ O
N~
O~CH3 O- S S N H3C
N
N_ _ .,N ~N N ~ I ~S~
S-
N=
I, S /
O_.NWO
O-
ON
O_ S S N~ O~CH3 O_ O O- S S
N N~ S S i ~ N''W il
p, ~N N / 0~ N y CH3 ~ ~~N N~N
OH
N
CH3 O-
S
-- N
NOz S~S N S Oo ~~S
~N ~ / ~ I Ct \ I
N ~F
-/F
F
/ \N
S N
S
~S~NH
N ~l'
O O N\\ N
O
I~
N, '
1
SUBSTITUTE SHEET (RULE 26)
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WO 98/56376 PCT/US98/12333
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TABLE 1 (cont'd)
cl o- o_
f , ,N
S~ N ~ O,N N yOH O.Nr N S NH S
O p ''N CI ~ ~S~N/CHa ~ /
/ N~ N S NH S
_ ~N
O S S 1\ / ~ 1 O S S N ~ /
N. ~ N-~ O.
~N N ~ ~--OH
N~N
HpN
_ N~S~N/
O ~ ,/~N N_N
N I
O CHa
O_
,, S
N S N H3C
O ~N N~ S NH S
N\ OH \ I ~ N \
NHp
S N F
HZN~N~ ~~1=O F N S NH S
O H3C~0 I ~ N N N
O
S
O S S ~N
S N
N F
O,IV'_ ~N N N F F
H3C N"5\ 'NH S
S I \~' '~ / \ ~ F F
N N.
~~S~N ~ N F S N
O ~ 'N'Y IN ~ ~ v ~CHa NHZ I ~ N N~~,j
S O ~ S NH \ J
HaC~O N~ S NH S
HaCCH~ ~ ~ I ~ ~ N \ ~ F
-~'~N N~=O HpN N N
CHa ~ Ha
O
SUBSTITUTE SHEET (RULE 26)
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WO 98/56376 PCTIUS98/12333
49
TABLE 1 (cont'd)
S NH S S
i ~ / \ ~ i0 S 11 N/ \ I
N N N~ ~ N_N
N
p-
p.N \ N~ S NH S
S
/ N ~ N \ ~ ~ O~S~N~ \ I
N N_N
~O~ N S NH S
H3C ~ ~ / \ ~ S
N\ /N N~N p S\ /NH \ I
,O ~ ~~ ~N-
H3C
NH N S NH S S
\ I p S' /S N
N ~ N N~N ~' ~ / \
H2N~N N_N
F /
F
S
H ~~O N'\ /S\ 'NH S I p S S NH~
N ,\~N' 'N~~N~ / \ N
[ /
F
N~N~ S~NH S
p~S~NHNH~ / N.
O
SUBSTITUTE SHEET (RULE 26)
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WO 98/56376 PCT/US98/12333
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TABLE 1 (cont'd)
N CH3
S S
S
~~ ON+-~~
O " N / ~~ ~N
N / CH3 O
S N H
O S~
ONa \ ~ ~ ~ N~-~~ I I S
O ~N \ ~ ~N
OEt
_N=O O
O
CH3 O
1\ .
_ N,O_
O S S~N O ,
N. ~ N~ /~~
O ~N N
D,S~O
SYN
O N. \ ~ ~NI ~
~N
~N~--~~S~S~S>--N CH3
0 VN N~N
S S O
ON. S I! ~ / vN=O_
~N N~N
O ~ \ H3C CH3
a
N_=O
O
S
O N. \ ~ N
Q ~N
C1
O . S S S H
% N ~ ~ /~-N C-~\H3
~N N
N\ w . H3C
/ CHI
SUBSTITUTE SHEET (RULE 26)
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WO 98156376 PCT/US98/12333
51
TABLE 1 (cont'd)
O
ii.
/ N~O_
O
~ ~ S
S ~N~
~N
~N'--~~S~S~ / \ I O
~N N-N
H
/ ~H
/
N S
N S ~N~ S~S~ / \ I
~N~ S~S~ / \ I p ~N N N
O ~N N-N
H
N\ 'CH3
/ OO
~N S
~N~--~~S~S~ / \ I
0 ~N N_N
SUBSTITUTE SHEET (RULE 26)
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TABLE 1 (cont'd)
OH
p\N. S~S~S~--N CHI
O ~N ~N / \ / \
O- S S N~
C! N ~ ~ N
~' ~N N~~
O
F
\ o- ,
N ,
Y N
ON~~S~N~N O
N N
O~NHEt ON~
S N/ O
ON. \ ~ N
O
NH
0 N=O . O~S~O
i
CHI _
S ~! O S II NN
~N~ ~S~N N N~~ N~~~
O ~N ~ \ O
CF3
p- S 5 N O
O tJ' \ ~ ~ ON~ S~S
OEt
OEt O ~N N
O
SUBSTITUTE SHEET (RULE 26)
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TABLE 1 (cont'd)
H
O S S N~ N~OH
/ NN
O
OH O
N
~ OH p
CHI
ONy S~S''~N,N S S S
~N N N ~ / NCH
S S w \
CH
ON'--~~ ~ ~ / s
O ~N
- S N\ NHz
O'N~~ ~ / OEt
O ~ N
O
0
~N
.- O
O O_
oN~-~~ S II S II N~N S N.., CH3
~~ ~N N~N N'--~~S~ I
~~ ~N /
0 /
., /
ON~ ~
O
SUBSTTTUTE SHEET (RULE 26)
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54
TABLE 1 (cont'd)
OEt 0 S
ON, S~S~Nw N
~~ ~N N / O CI
0
CI CI
S N S 1
O. S ~ / \
O- S S ~;~~~ N-N
O
O N
O OEt
O O I \
\ S N S
S N S ~ ~N~ S ~ / \
S~ ~ / \ ~, ~~ N-N
~N~~ N-N O
O
OMe
\ \ ~ ~ \ O
Me0 ~ N S 1
S O . SY S \1 / \
~N, S~ ~ N \ 0;~~~ N-N
_ S N\
~N
H~C CI
/
N_
ON~~S~ / S
O ~N~CH3
N~
N
CFA
SUBSTITUTE SHEET (RULE 26)
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SS
TABLE 1 {cont'd)
Ci
s
O_ S N~. , N~O ~ ~ / / I
v . S ~ S
N O
N N / Ct
O
HzN
N S ,
/ ON~ S~S~-/ \
~N '' N
N_ v
O S~/
O,N~~S~ /
N
S
C
O
HzN
NJ
O ~N S
N. S~S~ / \
VN N-N
O N S ,
N S~S~-/ \ H
0 ~N N a
N
O,N~~S
O N
CFA /
S N
O~ ~S~ ~OH
~~ ~TN 'N\ //N
O
O' S N
S~ ~ / \
O ~IN' N-N
SUBSTITUTE SHEET (RULE 26)
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TABLE 1 (cont'd)
OMe Me0
CH3 O- S S N~ OMe
--~~ O
O . S S ~ N/ I ~N ON~~ ~ /
N N N~~ OMe
O Et
OMe
N \
- S N~
O
N ~~ ~ / /
N O- S S N
N=-~jl' ~ I
O N
S S N \ ~ ~ \
O
\ I N-N N=O
HsC N~O Me0 O_
l_
O
H S
H S \ / ~ S S~N /
O S S N \ ' O S \ I 1N1-N
I I ~ p s.0
~S \ I N-N N,
O N~O
O-.N' OI_
O
O'
_ N S N
w ~ O , S~
O ~ N-N N
OEt ~ \ N
SU6STITUTE SHEET (RULE 26)
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TABLE I (cont'd)
S N S ,
NEW ~ / \
HZN ~ 1 I~ I / N-N
O ~H
S
S 11 /
H S N ~ I N-N
N N
N' ~ S~ /
H3C / ' , / N-N Et0 O
w N
H
S S N
i
N ~ I N-N
N~O
O-N\
O
SUBSTITUTE SHEET (RULE 26)
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Assays For Determining Inhibitory Activity
Various procedures known in the art may be used for identifying, evaluating or
assaying the inhibition of activity of protein tyrosine enzymes, in particular
protein tyrosine
phosphatases, by the compounds of the invention. For example but without
limitation, with
regard to phosphatases such assays involve exposing target cells in culture to
the compounds
and a) biochemically analyzing cell lysates to assess the level and/or
identity of tyrosine
phosphorylated proteins; or (b) scoring phenotypic or functional changes in
treated cells as
compared to control cells that were not exposed to the test substance.
Where mimics of the natural ligand for a signal transducing receptor are to be
identified or evaluated, the cells are exposed to the compound of the
invention and compared
to positive controls which are exposed only to the natural ligand, and to
negative controls
which were not exposed to either the compound or the natural ligand. For
receptors that are
known to be phosphorylated at a basal level in the absence of the natural
ligand, such as the
insulin receptor, the assay may be carried out in the absence of the ligand.
Where inhibitors
or enhancers of ligand induced signal transduction are to be identified or
evaluated, the cells
are exposed to the compound of the invention in the presence of the natural
Iigand and
compared to controls which are not exposed to the compound of the invention.
The assays described below may be used as a primary screen to evaluate the
ability of
the compounds of this invention to inhibit phosphatase activity of the
compounds of the
invention. The assays may also be used to assess the relative potency of a
compound by
testing a range of concentrations, in a range from 100 ~,M to I pM, for
example, and
computing the concentration at which the amount of phosphorylation or signal
transduction is
reduced or increased by SO% (IC50) compared to controls.
Biochemical Assays
Target cells having a substrate molecule that is phosphorylated or
dephosphorylated
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on a tyrosine residue during signal transduction are exposed to the compounds
of the
invention and radiolabelled phosphate, and thereafter, lysed to release
cellular contents,
including the substrate of interest. The substrate may be analyzed by
separating the protein
components of the cell lysate using a sodium dodecyl sulphate-polyacrylamide
gel
electrophoresis (SDS-PAGE) technique, in either one or two dimensions, and
detecting the
presence of phosphorylated proteins by exposing to X-ray film. In a similar
technique, but
without radioactive labeling, the protein components separated by SDS-PAGE are
transferred
to a nitrocellulose membrane, the presence of pTyr is detected using an
antiphosphotyrosine
(anti-pTyr) antibody. Alternatively, it is preferred that the substrate of
interest be first
isolated by incubating the cell lysate with a substrate-specific anchoring
antibody bound to a
solid support, and thereafter, washing away non-bound cellular components, and
assessing
the presence or absence of pTyr on the solid support by an anti-pTyr antibody.
This preferred
method can readily be performed in a microtiter plate format by an automated
robotic system,
allowing for testing of large numbers of samples within a reasonably short
time frame.
The anti-pTyr antibody can be detected by labeling it with a radioactive
substance
which facilitates its detection by autoradiography. Alternatively, the anti-
pTyr antibody can
be conjugated with an enzyme, such as horseradish peroxidase, and detected by
subsequent
addition of an appropriate substrate for the enzyme, the choice of which would
be clear to one
skilled in the art. A further alternative involves detecting the anti-pTyr
antibody by reacting
with a second antibody which recognizes the anti-pTyr antibody, this second
antibody being
labelled with either a radioactive substance or an enzyme as previously
described. Any other
methods for the detection of an antibody known in the art may be used.
The above methods may also be used in a cell-free system wherein cell lysate
containing the signal-transducing substrate molecule and phosphatase is mixed
with a
compound of the invention and a kinase. The substrate is phosphorylated by
initiating the
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kinase reaction by the addition of adenosine triphosphate (ATP). To assess the
activity of the
compound, the reaction mixture may be analyzed by the SDS-PAGE technique or it
may be
added to a substrate-specific anchoring antibody hound to a solid support, and
a detection
procedure as described above is performed on the separated or captured
substrate to assess the
presence or absence of pTyr. The results are compared to those obtained with
reaction
mixtures to which the compound is not added. The cell-free system does not
require the
natural ligand or knowledge of its identity. For example, Posner et al. {U.S.
Patent No.
5,155,031) describes the use of insulin receptor as a substrate and rat
adipocytes as target
cells to demonstrate the ability of pervanadate to inhibit PTP activity. Burke
et al., Biochem.
Biophys. Res. Comm., 1994, 204:129-134) describes the use of
autophosphorylated insulin
receptor and recombinant PTP 1B in assessing the inhibitory activity of a
phosphotyrosyl
mlmetlc.
In addition to measuring phosphorylation or dephosphorylation of substrate
proteins,
activation or modulation of second messenger production, changes in cellular
ion levels,
I 5 association, dissociation or translocation of signaling molecules, gene
induction or
transcription or translation of specific genes may also be monitored. These
biochemical
assays may be performed using conventional techniques developed for these
purposes.
Biological Assays
The ability of the compounds of this invention to modulate the activity of
PTPs, which
control signal transduction, may also be measured by scoring for morphological
or functional
changes associated with ligand binding. Any qualitative or quantitative
techniques known in
the art may be applied for observing and measuring cellular processes which
come under the
control of phosphatases in a signaling pathway. Such cellular processes may
include, but are
not limited to, anabolic and catabolic processes, cell proliferation, cell
differentiation, cell
adhesion, cell migration and cell death.
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The techniques that have been used for investigating the various biological
effects of
vanadate as a phosphatase inhibitor may be adapted for use with the compounds
of the
invention. For example, vanadate has been shown to activate an insulin-
sensitive facilitated
transport system for glucose and glucose analogs in rat adipocytes (Dubyak, et
al., 1980, J
Biol. Chem., 256:5306-5312). The activity of the compounds of the invention
may be
assessed by measuring the increase in the rate of transport of glucose analog
such as
2-deoxy-3Hglucose in rat adipocytes that have been exposed to the compounds.
Vanadate
also mimics the effect of insulin on glucose oxidation in rat adipocytes
(Shechter, et al., 1980,
Nature, 284:556-558). The compounds of this invention may be tested for
stimulation of
glucose oxidation by measuring the conversion of '4C-glucose to '°COz.
Moreover, the effect
of sodium orthovanadate on erythropoietin-mediated cell proliferation has been
measured by
cell cycle analysis based on DNA content as estimated by incorporation of
tritiated thymidine
during DNA synthesis (Spivak, et al., 1992, Exp. Hematol., 20:500-504).
Likewise, the
activity of the compounds of this invention toward phosphatases that play a
role in cell
proliferation may be assessed by cell cycle analysis.
The activity of the compounds of this invention can also be assessed in
animals using
experimental models of disorders caused by or related to dysfunctional signal
transduction.
For example, the activity of a compound of this invention may be tested for
its effect on
insulin receptor signal transduction in non-obese diabetic mice (Lund et al.,
1990, Nature,
345:727-729), B B Wistar rats and streptozotocin-induced diabetic rats
(Solomon et al., 1989,
Am. J. Med. Sci., 297:372-376). The activity of the compounds may also be
assessed in
animal carcinogenesis experiments since phosphatases can play an important
role in
dysfunctional signal transduction leading to cellular transformation. For
example, okadaic
acid, a phosphatase inhibitor, has been shown to promote tumor formation on
mouse skin
(Suganuma et al., 1988, Proc. Natl. Acad. Sci., 85:1768-1771).
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The data obtained from these cell culture assays and animal studies can be
used in
formulating a range of dosages for use in humans. The dosage of the compounds
of the
invention should lie within a range of circulating concentrations with little
or no toxicity.
The dosage may vary within this range depending on the dosage form employed
and the route
of administration.
The above-described assays are exemplary and not intended to limit the scope
of the
invention in any manner. Other assays known to those skilled in the art may be
employed to
ascertain the ability of the compounds of this Those of skill in the art would
appreciate that
modifications can be made to the assays to develop equivalent assays that
obtain the same
result.
Phosphatase Inhibitors
The present invention encompasses compounds capable of regulating and/or
modulating signal transduction by, including but not limited to, inhibiting
the activity of
protein tyrosine phosphatases. More specifically, the present invention
encompasses
compounds capable of inhibiting protein tyrosine phosphatase activity. These
compounds
will be referred to herein generically as "phosphatase inhibitors", even
though these
compounds either upregulate or downregulate cellular processes that are
controlled by signal
transduction.
Phosphotvrosine Enzyme Linked Immunosorbent Assav_
This assay may be used to test the ability of the compounds of the invention
to inhibit
dephosphorylation of phosphotyrosine (ptyr) residues on insulin receptor (IR).
Those skilled
in the art will recognize that other substrate molecules, such as platelet
derived growth factor
receptor, may be used in the assay by using a different target cell and
anchoring antibody. By
using different substrate molecules in the assay, the activities of the
compounds of this
invention toward different protein tyrosine enzymes may be assessed. In the
case of IR, an
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endogenous kinase activity is active at low level even in the absence of
insulin binding.
Thus, no insulin is needed to stimulate phosphorylation of 1R. That is, after
exposure to a
compound, cell lysates can be prepared and added to microtiter plates coated
with anti-insulin
receptor antibody. The level of phosphorylation of the captured insulin
receptor is detected
S using an anti-pTyr antibody and an enzyme-linked secondary antibody.
Materials And Methods
1. The cell line used for the IR assay is NIH3T3 cells (ATCC# CRL 1658)
engineered to over-express the human IR (H2S cells). Growth media for these
cells is
DMEM (Gibco) containing 10% fetal bovine serum, 1 % L-glutamine, and 20 mM
Hepes.
2. The anchoring antibody used was BBE which recognizes the extracellular
domain of human IR (Enzymology Laboratories, Sugen Inc.).
3. PBS (Gibco): KHZPO, (0.20 gll), KZHP04 (2.16 gll), KC1 (0.20 g/1), NaCI
(8.0
g/1), pH 7.2.
4. Rabbit polyclonal antiphosphotyrosine antibody (anti-pTyr, Enzymology
1 S Laboratories, Sugen, Inc.).
S. Goat anti-rabbit IgG POD conjugate (Tago, Burlingame, CA, Cat.No. 6430) is
used as the secondary antibody.
6. TBST buffer: SO mM Tris-HCI, 1S0 mM NaCI, 0.1% Triton X-100, adjusted to
pH 7.2 with lON HC1.
7. Blocking buffer: PBS plus S% milk (Carnation instant non-fat dry milk).
8. SX HNTG buffer: 100 mM HEPES, 750 mM NaCI, SO% glycerol, 0.5% Triton
X-100, pH 7.5.
9. ABTS solution: 100 mM citric acid, 2S0 mM NazHP04, O.S mg/ml ABTS
(2,2'-azinobis(3-ethylbenzthiazlinesulfonic acid), adjusted to pH 4.0 with 1N
HC1.
10. Cell iysis buffer: HNTG containing 1mM NajV04, (O.SM solution kept as a
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100X stock at -80°C in aliquots), SmM NaP20, and SmM EDTA prepared
fresh and
keep on ice until ready for use.
1 I. Hydrogen peroxide: 30% solution.
Preparation Of Assa, P
Microtiter plates (96-well, Easy Wash ELISA plate, Corning 25805-96) are
coated with the anchoring antibody at 0.5 pg per well, in 100 ~1 PBS for at
least two hours at
room temperature or overnight at 4° C. Before use, the coating buffer
is replaced with 100 lZl
blocking buffer, and the precoated assay plate was shaken at room temperature
for 30
minutes. The wells are then washed 3 times with water and once with TBST
buffer before
adding lysate.
Seedins Cells
Cells are grown in a l5cm culture dish (Corning 25020-100) in DMEM media
containing 10% fetal bovine serum (FES) until 80-90% confluent. The cells are
harvested
with trypsin-EDTA (0.25%, O.SmI, Gibco), resuspended in fresh medium
containing 10%
FBS, 1% L-glutamine and Hepes, and transferred to round bottom 96-well tissue
culture
plates (Corning 25806-96) at 25,000 cells/well, 100 wl/well. The cells are
incubated at 37° C
at 5% COz for 24 hours. The media is then changed by inverting the plate, and
adding
DMEM medium containing 0.5% FBS and Hepes. The cells are further incubated
overnight
at 37°C, 5% COz.
Assay Procedure
The assay is set up in the 96-well tissue culture plate. Before adding the
compounds
to the cells, media in the wells is replaced by serum free DMEM medium, 90 p.l
per well.
Positive control wells receive 80 ~Zl DMEM. Negative controls received 90 pl
DMEM. The
test compound is diluted 1:10 with DMEM and 10 p,llwell of the diluted test
substances are
transferred to the cells in the wells to achieve a final dilution of 1:100.
Positive and negative
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control wells both receive 10p1/well of dimethyl sulfoxide (DMSO) to achieve a
final
concentration of 1 %. Positive control wells additionally received 10 pl/welll
of O.1M
Na3V04 so that that the final concentration is 10 mM. The tissue culture plate
is shaken for 1
minute before incubation at 37°C, 5% C02. After 90 minutes of
incubation, the media is
S removed by inversion of the plate, and 100pUwell of lysis buffer is added.
The tissue culture
plate is shaken for 5 minutes and then placed on ice for 10 minutes. The cells
are
homogenized by repeated aspirating and dispensing, and the lysate is then
transferred to the
corresponding wells of a precoated assay plate.
The substrate in the cell lysates is allowed to bind to the anchoring antibody
for 1 hour
with shaking at room temperature. The lysate is then removed, and the assay
plate is washed.
All ELISA plate washings are done by rinsing in water 3 times followed by one
rinse with
TBST. The plate is dried by tapping it on paper towels. Phosphotyrosine is
detected by
adding 100 pllwell anti-pTyr antiserum diluted 1:3000 with TBST to the wells
and incubating
for 30 minutes shaking at room temperature. The unbound excess anti-pTyr
antiserum is then
removed, and the assay plate is washed as described above. A secondary
antibody diluted
1:3000 with TBST, is added to the wells and incubated for 30 minutes with
shaking at room
temperature. The secondary antibody is then removed, the plate washed, and
fresh
ABTS/HZOz (I.2 p.l 30% HZOz to 10 ml 0.5 mg/ml 2,2'-azinobis(3-
ethylbenzethiazline)sulfonic
acid in 100 rnM citric acid, 250 mM Na2HP04, pH 4.0) is added to start color
development.
The reaction is stopped after 10 minutes by adding 100 pl/well of 0.2M HCl,
and incubating
with shaking for 1 minute. Absorbance values at 410 nm were measured using an
ELISA
plate reader (Dynatec MR5000).
Glucose Transuort Assav
This assay is used to assess the ability of the compounds of the invention to
inhibit
phosphatase activity that is involved in the signaling pathway that regulates
the
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insulin-induced transport of glucose into adipocytes. It has been shown that
incubation of
isolated adipocytes with vanadate resulted in a dose-dependent increase in the
rate of glucose
uptake.
Materials And Methods
The cell line used for the glucose transport assay is 3T3-Ll, a preadipocyte
cell line
(American Type Culture Collection CCL92.1 ) which overexpress the insulin
receptor. The
3T3-L1 cells are first differentiated by treating the cells under confluent
growth conditions in
DMEM containing 10% fetal bovine serum (FBS) with 0.5 mM 3-isobutyl-1-
methylxanthine,
5 p.l/ml porcine insulin and 250 mM dexamethasone for 2 days. The cells are
then grown in
DMEM containing 10% FBS and 5 p,llml porcine insulin for two days, after which
the cells
are cultured in DMEM containing only 10% FBS.
Cells for use in the assay are first grown overnight in DMEM media and 1% FBS
at
37°C at S% COz. Two hours before use, the overnight media is replaced
with serum free
DMEM containing SmM glucose. After washing the cells twice with phosphate
buffered
saline (PBS), serial dilutions of the test compound 1:100 into DMEM are added
to the wells
for a final concentration range of 0.1 p,M to 500 p,M. Negative control wells
received
DMEM only. The cells are incubated with the test compound for 1-4 hours at
37°C at S%
C02. Fifteen minutes before the end of the selected time, 2-deoxy-3H-glucose
is added to a
final concentration of 50 pM and 0.5 ~Ci/ml. At the end, the compound is
removed and the
wells are washed twice with PBS containing 10 mM glucose. The cells are lysed
with SOpI
O.SN NaOH and the cell lysates are transferred to a scintillation vial and
mixed with 5.2 pl of
glacial acetic acid. The wells are each washed with 200 pl PBS which is then
transferred to
the corresponding scintillation vial. 3H radioactivity is then counted with a
Beclanan
counter.
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Primary Adipocyte Glucose Uptake Assay
This assay is used to assess the effect of the compounds of the invention on
insulin-mediated signal transduction of primary adipocytes as determined by
glucose uptake
by the cells.
MATERIALS AND METHODS
Reagents
The following buffers and solutions are used in the primary adipocyte glucose
uptake
assay:
Mixed Salts
76.74g NaCI
3.Slg KCl
3.06g MgS04~7H20
3.638 CaCh ~2H20 (2.748 CaClz)
The volume was brought up to 1 liter with distilled HZO.
HEPES Buffer
23.8g HEPES
3.42g NaHZP04~Hz0 {3.87g NaHzP04~2HZ0)
were dissolved in approximately 600m1 of H20. The pH of the solution was
adjusted to 7.6
and the volume was brought up to 1 liter with distilled H20.
Albumin Collagenase Buffer
44.8mi distilled H20
10.0 ml HEPES Buffer
10.0 ml Mixed Salts
35.0 ml 10% BSA
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0.2m1 Glucose (300mM)
100mg Collagenase
Transport buffer
48m1 distilled HZO
8m1 Mixed salts
8m1 HEPES Buffer
16m1 10% Bovine Serum Albumin
Excision of epididymal fat pads
Primary adipocytes used in the assay are obtained from euthanized male rats
(Sprague-Dawley or other appropriate strains) with a body weight of 200-250
grams. Old and
heavier rats are not used as these rats may be resistant to insulin and do not
provide a good
response. Each animal is expected to yield 1-1.5 g of fat. Approximately 2.5 g
of fat is
required to run 40 reactions, with 20 samples in the glucose uptake assay and
a matching set
of 20 LDH samples.
Using sterile techniques, a midline abdominal incision is made through the
skin
followed by a 4-6 cm incision through the peritoneum. The fat body adjoining
the testes is
identified by tracing the vas deferens to the testes. The fat pads are
carefully cut away from
the epididymis and testes, and the innervating blood vessels. The excised fat
pads are
weighed, finely chopped, and digested with 5 ml of collagenase buffer at
37°C for 1 hour.
The digested material was then strained through a 250-micron nylon mesh sieve.
The cells
float to the top, and are collected and washed three times with transport
buffer. The cell
concentration is determined by one of the following methods:
(1) Cells as percentage of solution: The cells in suspension are centrifuged
at SOOx g
for S-10 minutes in a hematocrit tube. The total length of the column of
liquid and the length
of column of "white" cellular material at the bottom of the tube is measured
in millimeters.
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The cell concentration is estimated as a percentage of the length of cell
column to the total
length. For the glucose uptake assay, approximately 2-3 % of the cells in the
final reaction
volume are used. For a S00 pl reaction volume, the fat cell stock solution is
diluted to 2S%
cells with transport buffer, and SO pl aliquots are added to each sample.
(2) Cell Number: Cells are first fixed with osmium tetroxide in collidine
buffer so that
the adipocytes sink in suspension. The fixed cells are centrifuged to remove
the osmium
tetroxide, and then counted with a Coulter counter. Once the cell number is
determined, the
cell concentration is adjusted and SO p,l aliquots of the cells are added to
each sample.
Assav
Adipocytes collected from rats are exposed to a test compound in the absence
or
presence of saturation levels of insulin. '4C-labelled glucose, which would
normally be taken
up by the cells via an insulin-induced mechanism, are added to the cells. The
amount of
radioactive glucose retained by the treated cells is determined and compared
to that of
untreated cells to assess the activity of the test compounds.
1 S A typical assay can be set up as follows:
Sample buffer Compound DMSO cells '4C-Glucose
blank 447.5 2.S SO
basal 397.5 2.5 SO SO
insulin 347.5 2.S SO SO
sample 397.5 2.S 50 50
duplicate 397.5 2.S 50 50
Reaction vials (polyethylene scintillation vials l7mm) containing the
appropriate buffer and
compounds are set up while the cells are being prepared. SO p,l of insulin at
80 nM, which
represent saturation levels, is added to the appropriate samples just prior to
addition of the
adipocytes. A lower concentration of insulin may also be used in the assay.
Dimethyl
sulfoxide (DMSO; below O.S%) is used as a vehicle for the compounds of the
invention. Test
compounds at 200x, depending on solubility in DMSO (approximately SO pM), is
used. The
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adipocytes in 50 Ml aliquots is added to the reaction vials and incubated at
37° C for 30
minutes with gentle shaking. "C-Glucose is then added to each sample which was
incubated
for a further 60 minutes at 37°C with shaking.
The cells are separated from the reaction buffer by centrifugation. The amount
of
S glucose taken up by the cells is determined by standard scintillation
counting. Thus the cells
(in duplicates) can be separated by centrifugation in narrow bore
microcentrifuge tubes (5.8 x
47.5 mm, 0.4 ml volume) containing 100 pl of dimethyl silicon fluid SF96/50).
Each
reaction sample is then stirred to ensure even distribution of cells and 200
pl is transferred to
a narrow bore tube. The mixture is centrifuged at 13000 rpm for 10 minutes.
After
centrifugation, the cells float to the top and are separated at the silicon
fluid interface. The
top layer is then transferred to a borosilicate vial with 7-10 ml
scintillation fluid and counted
for approximately 10 minutes. 50 pl of '4C was used a control for the total
amount of
radioactivity in each reaction.
Cellular Insulin Receptor Activation Assax
This assay is used to provide a consistent method for detenmination of
catalytic insulin
receptor activity in intact cells in a 96 well ELISA format. A EY-20mer
peptide is used as
the IR substrate in vitro for determination of the activation state of IR in
an enzyme-linked-
immunosorbent-assay (ELISA).
Reagent and su~nliers:
1 ) Corning 96-well ELISA plates (Corning cat.# 25905-96)
2) PBS (Phosphate Buffered Saline)
Formulation: 2.7mM KCL
1.1 mM KHZP04
1.5 mM MgCl2 (anhydrous)
138 mM NaCI
8.1 mM Na2HP04
3) HNTG lysis Buffer
Formulation: 20 mM HEPES buffer pH 7.5
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1 SO mM NaCI
0.2% Triton X-100
10% Glycerol
S 4) HNTG* Formulation:
1 X HNTG
SmM Na304
2mM NaPPi
SmM EDTA
1S
S) DMEM (Dulbecco's Modified Eagle Medium) with IX high glucose,
L-Glutamine (GibCco Cat# 11965 -OSO}
6) FBS (Fetal Bovine Serum) (GibCo Cat# 16000-044)
7) L-Glutamine (200mM stock) (GibCo Cat# 11965-OS0)
8} Growth media: DMEM 10% (heat activated) FBS+ 2mM L-glutamine
9) Starve media: DMEM
2S
10) H2S cells (NIH 3T3c7 cells transfected with a plasmid expressing the human
insulin receptor) grown in growth media containing S% COz at 37°
C.
11 ) Anti insulin receptor antibody (Santa Cruz Biotech) (SC-710 or SC-09).
I2) EY-Tide: Biotin linked peptide (sequence biotin
EYEYEYEYEYEYEYEYEYEY, MW=3280.4) (Protein chemistry lab
SUGEN, Inc.).
12) ABTS solution:
formulation: 100 mM Citric Acid (anhydrous)
2SOmM Na2HP04, pH 4.0, O.S mg/ml ABTS (2,2-azino-
3S bis (3-ethylbenzthiszoline-6-sulfonic acid) (Sigma Cat#
A4-888), keep solution in dark at 4° C until ready to
use.
14) Hydrogen peroxide 30% : Fisher (cat#H32S).
1S) ABTSIH202:
formulation: 1 S mL ABTS solution
2 ~L Hz02
4S 16) Kinase Buffer
formulation: 2S mM HEPES/CL, pH 7.0
1S0 mM NaCI
0.1 % Triton X-100
10 mM MnCl2
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17) TBST Buffer (Tris-buffered Saline with Triton X0199)
formulation: 50 mM Tris, pH 7.2
150mM NaCI
S 0.1 % Triton X-100
18) Polypropylene 96-well V bottom plates. (Applied Scientific Cat. # AS-
72092)
19) Blocking buffer: 5% powdered non-fat milk in PBS (w/v).
20) ABC kit : Avidin-HRP Developing reagents : Vector Labs (Cat. # PK-4000)
21 ) DMSO (Dimethylsulfoxide): Sigma (Cat. # D-8418)
i5 22) ATP : Sigma (Cat. # D-8418)
25
23) 4610: Phosphotyrosine specific monoclonal antibody (UBI)
24) Insulin, Crystalline, Bovine, Zinc: GibCo cat# 13007-018
Assay Procedure
1 ) Seed H25 cells in 96 well Tissue culture plate at 15,000 cells well in
Growth
media.
2) Coat Corning ELISA plate with 0.5 pg/well of anti-insulin receptor antibody
in PBS. Final volume per well is 100 p,l. Keep plate over night at 4°
C.
3) Coat another Corning ELISA plate with 0.5 p,g/well of 4610 antibody in PBS.
Final volume per well is 100 ~1. Keep plate over night at 4° C.
4) Starve cells 2 hours before assay by removing growth media, washing with
PBS and adding 90 ~1 of starve media.
5) Remove unbound antibody from wells of anti-insulin receptor antibody coated
plate by inverting plate to remove liquid. Wash plate 3x with TBST.
6) Block plate with 150 pl blocking buffer per well. Incubate while shaking on
a
microtiter plate shaker at room temperature for 30 minutes.
7) Dilute compounds/extracts and DMSO controls 1:10 in DMEM on a
polypropylene plate.
8) Add diluted compounds/extracts and DMSO controls 1:10 to 96 well cell
plate. Incubate plate for 20 minutes in incubator at 37°C and 5% COz.
9) After 20 minutes of incubation add 100 ~,1 of 0.2 p,M insulin to positive
control wells. This results in a final insulin concentration of 0.1 pM per
well.
Incubate plate for 10 minutes in incubator at 37°C and 5% CO2.
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10) Remove media from cell plate by inverting plate to remove liquid. Wash lx
with PBS, then add 100 p.l/well HNTG*. Let plate sit on ice for 5 minutes.
S 11 ) Remove blocking buffer and wash the anti-insulin receptor antibody
coated
ELISA plate 3x times with TBST. Pat the plate on a paper towel to remove
excess liquid and bubbles.
12) Use a 12 channel pipetter to scrape the cells from the plate, and
homogenize
the lysate by repeating aspiration and dispensing. Transfer the lysate to the
corresponding wells of the anti-insulin receptor antibody coated ELISA plate.
Incubate while shaking on a microtiter plate shaker at room temperature for 1
hour.
13) Remove unbound protein from wells by inverting anti-insulin receptor
antibody coated ELISA plate. Wash plate 3x with TBST. Pat the plate on a
paper towel to remove excess liquid and bubbles.
14) Prepare kinase buffer solution containing 6 ~M peptide and 2 ~M ATP. Add
50 ul of solution to each well. Cover ELISA plate with Parafilm and incubate
while shaking on a microtiter plate shaker at 4°C overnight
(approximately 15-
17 hrs.).
15) The next morning block a 4610-coated plate with 100 pl blocking buffer to
each well. Incubate while shaking on a microtiter plate shaker at room
temperature for 30 min.
16) Remove anti-insulin receptor antibody coated plate from 4°C cooling
medium
and add 50 p.l of kinase buffer to each well.
17) Wash UB40 coated plate 3X with TBST. Pat the plate on a paper towel to
remove excess liquid and bubbles.
18) Transfer 80 pl/well of kinase buffer (containing EY Tide peptide and ATP)
from anti-insulin receptor antibody coated plate to the corresponding wells of
the 4610 coated plate. Incubate for 30 minutes while shaking on a microtiter
plate shaker at room temperature. Discard the anti insulin receptor antibody
coated plate.
19) Right after above step mix up ABC kit reagent in 15 ml of TBST. Vortex and
incubate on bench top for 30 minutes.
20) Wash UB40 coated plate 3x times with TBST. Pat the plate on a paper towel
to remove excess liquid and bubbles.
21 ) Add 100 ~cl/well of pre-mixed ABC reagents to UB40 plate. Incubate for 30
minutes while shaking on a microtiter plate shaker at room temperature.
22) Wash 4610-coated plate 3x times with TBST. Pat the plate on a paper towel
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to remove excess liquid and bubbles.
23) Remove liquid from plate and wash 3x with TBST.
24) Add 100 ~1/ well of ABST/H202 solution. Incubate for S minutes while
shaking on a microtiter plate shaker. Place ELISA plate in ELISA plate reader
and determine absorption at 410 nm, reference at 630 nm.
Results are calculated as an ICSO.
CONCLUSION
Thus, it will be appreciated that the compounds, methods and pharmaceutical
compositions of the present invention are expected to modulate the activity of
protein
tyrosine enzymes which mediate cellular signal transduction, in particular,
protein tyrosine
phosphatase, and therefore are expected to be effective as therapeutic agents
against disorders
associated with protein tyrosine enzyme related cellular signal transduction.
Although certain embodiments and examples have been used to describe the
present
invention, it will be apparent to those skilled in the art that changes to the
embodiments and
examples shown may be made without departing from the scope and spirit of this
invention.
Other embodiments are within the following claims.
