Note: Descriptions are shown in the official language in which they were submitted.
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PYRROLOPYRIMIDINES AS PROTEIN ICINASE INHIBITORS
This invention is directed to substituted pyrrolopyrimidines, their
preparation, pharmaceutical
compositions containing these compounds, and their pharmaceutical use in the
treatment of disease
states capable of being modulated by the inhibition of the protein kinases.
Protein kinases participate in the signalling events which control the
activation, growth and
differentiation of cells in response to extracellular mediators and to changes
in the environment. In
general, these kinases fall into several groups; those which preferentially
phosphorylate serine andlor
threonine residues and those which preferentially phosphorylate tyrosine
residues [S.K.Hanks and
T.Hunter, FASEB. J., 1995, 9, pages 576-596]. The serine/threonine kinases
include for example,
protein kinase C isoforms [A.C.Newton, J. Biol. Chem., 1995, 270, pages 28495-
28498] and a group of
cyclin-dependent kinases such as cdc2 [J.Pines, Trends in
Biochemical Sciences, 1995, 18, pages 195-197]. The tyrosine kinases include
membrane-spanning
growth factor receptors such as the epidermal growth factor receptor
[S.Iwashita and M.Kobayashi,
Cellular Signalling, 1992, 4, pages 123-132], and cytosolic non-receptor
kinases such as p56tck,
p59fYn, ZAP-70 and csk kinases [C.Chan et. al., Ann. Rev. Immunol., 1994,12,
pages 555-592].
Inappropriately high protein kinase activity has been implicated in many
diseases resulting from
abnormal cellular function. This might arise either directly or indirectly,
for example by failure of the
proper control mechanisms for the kinase, related for example to mutation,
over-expression or
inappropriate activation of the enzyme; or by over- or underproduction of
cytokines or growth factors
also participating in the transduction of signals upstream or downstream of
the kinase. In all of these
instances, selective inhibition of the action of the kinase might be expected
to have a beneficial effect.
Syk is a 72-kDa cytoplasmic protein tyrosine kinase that is expressed in a
variety of hematopoietic
cells and is an essential element in several cascades that couple antigen
receptors to cellular responses.
Thus, Syk plays a pivotal role in signalling of the high affinity IgE
receptor, FcsRl, in mast cells and
in receptor antigen signalling in T and B lymphocytes. The signal transduction
pathways present in
mast, T and B cells have common features. The ligand binding domain of the
receptor lacks intrinsic
tyrosine kinase activity. However, they interact with transducing subunits
that contain
immunoreceptor tyrosine based activation motifs (ITAMs) [M.Reth, Nature, 1989,
338, pages 383-
384]. These motifs are present in both the (3 and y subunits of the FcERl, in
the ~-subunit the of T cell
receptor (TCR) and in the IgGoc and IgG (3 subunits of the B cell receptor
(BCR). [N.S.van Oers and
A.Weiss, Seminars in Immunology, 1995, 7, pages 227-236] Upon binding of
antigen and
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multimerization, the ITAM residues are phosphorylated by protein tyrosine
kinases of the Src family.
Syk belongs to a unique class of tyrosine kinases that have two tandem Src
homology 2 (SH2) domains
and a C terminal catalytic domain. These SH2 domains bind with high affinity
to ITAMs and this SH2
-mediated association of Syk with an activated receptor stimulates Syk kinase
activity and localises
Syk to the plasma membrane.
In Syk deficient mice, mast cell degranulation is inhibited, suggesting that
this is an important target
for the development of mast cell stabilising agents [P.S.Costello, Oncogene,
1996, 13, pages 2595-
2605]. Similar studies have demonstrated a critical role for Syk in BCR and
TCR signalling
[A.M.Cheng, Nature, 1995, 378, pages 303-306, (1995) and D.H.Chu et al.,
Immunological Reviews,
1998, 165, pages 167-180]. Syk also appears to be involved in eosinophil
survival in response to IL-5
and GM-CSF [S.Youse~ et al., J. Exp. Med., 1996, 183, pages 1407-1414].
Despite the key role of
Syk in mast cell, BCR and T cell signalling, little is known about the
mechanism by which Syk
transmits downstream effectors. Two adaptor proteins, BLNK (B cell Linker
protein, SLP-65) and
SLP-76 have been shown to be substrates of Syk in B cells and mast cells
respectively and have been
postulated to interface Syk with downstream effectors [M.Ishiai et al.,
Immunity, 1999, 10, pages 117-
125 and L.R.Hendricks-Taylor et al., J.Biol. Chem, 1997, 272, pages 1363-
1367]. In addition Syk
appears to play an important role in the CD40 signalling pathway, which plays
an important role in B
cell proliferation [M.Faris et al., J.Exp. Med., 1994, 179, pages 1923-I93I].
Syk is further involved in the activation of platelets stimulated via the low-
affinity IgG receptor (Fc
gamma-RIIA) or stimulated by collagen [F.Yanaga et al., Biochem. J., 1995,
311, (Pt. 2) pages 471-
478].
Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase involved in
integrin-mediated signal
transduction pathways. FAK colocalizes with integrins in focal contact sites
and FAK activation and
its tyrosine phosphorylation have been shown in many cell types to be
dependent on integrins binding
to their extracellular ligands. Results from several studies support the
hypothesis that FAK inhibitors
could be useful in cancer treatment. For example, FAK-deficient cells migrate
poorly in response to
chemotactic signals and overexpression of C-terminal domain of FAK blocks cell
spreading as well as
chemotactic migration (Sieg et al, J. Cell Science,1999, 112, 2677-2691;
Richardson A, and Parsons
T., Cell, 1997, 97, 221-231) ; in addition, tumor cells treated with FAK
antisense oligonucleotides lost
their attachment and underwent apoptosis (Xu et al, Cell Growth Differ. 1996,
4, 413-418). FAK has
been reported to be overexpressed in prostate, breast, thyroid, colon and lung
cancers. The level of
expression of FAK is directly correlated with tumors demonstrating the most
aggressive phenotype.
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Angiogenesis or the formation of new blood vessels by sprouting from the
preexisting vasculature is of
central importance for embryonic development and organogenesis. Abnormal
enhanced
neovascularization is observed in rheumatoid arthritis, diabetic retinopathy
and during tumor
development (Folkman, Nat. Med., 1995, 1, 27-31.). Angiogenesis is a complex
multistage process
which includes activation, migration, proliferation and survival of
endothelial cells. Extensive studies
in the field of tumor angiogenesis in the past two decades have identified a
number of therapeutic
targets including kinases, proteases and integrins resulting in the discovery
of many new anti-
angiogenic agents, including KDR inhibitors some of which are currently under
clinical evaluation
(Jekunen, et al Cancer Treatment Rev. 1997 , 23, 263-286.). Angiogenesis
inhibitors may be used in
frontline, adjuvant and even preventive settings for the emergence or regrowth
of malignancies.
Several proteins involved in chromosome segregation and spindle assembly have
been identified in
yeast and drosophila. Disruption of these proteins results in chromosome
missegregation and
monopolar or disrupted spindles. Among these kinases are the Ipl l and aurora
kinases from
IS S.cerevisiae and drosophila respectively, which are required for centrosome
separation and
chromosome segregation. One human homologue of yeast Ipl l was recently cloned
and characterized
by different laboratories. This kinase termed Aurora2, STKIS or BTAK belongs
to the
serine/threonine kinase family. Bischoff et al showed that Aurora2 is
oncogenic and is amplified in
human colorectal cancers (EMBO J, 1998, 17, 3052-3065). It has also been
exemplified in cancers
involving epithelial tumors such as breast cancer.
30
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This invention concerns substituted pyrrolopyrimidines of formula (I), which
have the ability to inhibit
one or more protein kinases, more particularly, FAIL, KDR , Syk kinase or
Aurora2, especially Syk
kinase.
(I)
wherein
R 1 represents hydrogen, -C(=O)-NY 1 Y~, -C(=O)-ORS, -S02-NY 1 Y2 , -S02-R~ , -
C(=O)R~, or R 1
may be alkenyl, alkenyloxy, alkyl, alkynyl, aryl, heteroaryl,
heterocycloalkyl, cycloalkyl or
cycloalkylalkyl, each optionally substituted by one or more groups selected
from aryl, cycloalkyl,
cyano, halo, heteroaryl, heterocycloalkyl, -CHO (or a 5-, 6- or 7-membered
cyclic acetal derivative
thereof), -C(=O)-NYlY2, -C(=O)-ORS, -NYIY~, -N(R6)-C(=O)-R~, -N(R6)-C(=O)-
NY3Y4,
-N(R6)-S02-R~, -N(R~)-S02-NY3y4, _OR~, -C(=O)-R~, hydroxy, alkoxy and carboxy;
R~ represents one or more groups selected from hydrogen, acyl, alkylenedioxy,
alkenyl, alkenyloxy,
alkynyl, aryl, cyano, halo, hydroxy, heteroaryl, heterocycloalkyl, nitro, R4, -
C(=O)-NYlY2,
-C(=O)-ORS, -NY 1 Y2, -N(R6)-C(=O)-R~, -N(R6)-C(=O)-NY3Y4, -N(R6)-C(=O)-ORS,
-N(R6)-S02-R~, -N(R6)-S02-NY3Y4, -S02-NYly2 and -ZR4;
R3 represents H, cyano, halo, hydroxy, nitro, R4, NYlY~, -ZR4, -C(=O)-ORS, -
C(=0)-R~,
-C(=O)-NYlY2, -N(Rg)-C(=O)-R4, -N(R8)-C(=O)-NYlY2, -N(Rg)-C(=O)-ORS, -S02-
NY3Y4, or
-N(R8)-S02-R~, or R3 represents aryl, heteroaryl, alkenyl or alkynyl, each
optionally substituted by
one or more groups selected from aryl, cyano, halo, hydroxy, heteroaryl,
heterocycloalkyl, nitro,
-C(°O)-~'1y2~ -C('-O)-ORS, -NYIy~, -N(R6)-C(°O)-R~° -
N(R6)-C(°O)-1'IZ'3y'l,
-N(R6)-C(=O)-ORS, -N(R6)-S02-R~, -N(R6)-SO?-NY3Y4, -S02-NYIY~ or -ZR4;
R4 represents alkyl, cycloalkyl or cycloallylalkyl each optionally substituted
by one or more groups
selected from aryl, cycloalkyl, cyano, halo, heteroaryl, heterocycloalkyl,
hydroxy, -CHO (or a 5-, 6- or
7-membered cyclic acetal derivative thereof), -C(=O)-NYIY~, -C(=O)-ORS, -
NYly~,
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-N(R6)-C(=O)-R~, -N(R6)-C(=O)-NY3Y4, -N(R6)-S02-R~, -N(R6)-S02-NY3Y4, -ORS and
-C(=O)-R~; R4 can also be optionally interspersed with a group selected from
O, S(O)n, NR6;
RS represents hydrogen, alkyl, alkenyl, aryl, arylalkyl, heteroaryl or
heteroarylalkyl;
R6 represents hydrogen or lower alkyl;
R~ represents alkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl,
heteroarylalkyl,
heterocycloalkyl or heterocycloalkylalkyl;
Rg represents hydrogen or lower alkyl;
Y 1 and Y2 are independently hydrogen, alkenyl, aryl, cycloalkyl, heteroaryl
or alkyl optionally
substituted by one or more groups selected from aryl, halo, heteroaryl,
hydroxy, -C(=O)-NY3Y4,
-C(=O)-ORS, -NY3Y4, -N(R6)-C(=O)-R~, -N(R6)-C(=O)-NY3Y4, -N(R6)-S02-R~,
-N(R6)-SO~,-NY3Y4 and -ORS; or the group -NYlY2 may form a cyclic amine;
Y3 and Y4 are independently hydrogen, alkenyl, alkyl, aryl, arylalkyl,
cycloalkyl, heteroaryl or
heteroarylalkyl; or the group -NY3Y4 may form a cyclic amine;
Z represents O or S(O)n;
n is zero or an integer 1 or 2;
and their corresponding N-oxides, and their prodrugs, and their acid
bioisosteres; and pharmaceutically
acceptable salts and solvates (e.g. hydrates) of such compounds and their N-
oxides and their prodrugs,
and their acid bioisosteres; together with one or more pharmaceutically
acceptable carriers or
excipients.
In the present specification, the term "compounds of the invention", and
equivalent expressions, are
meant to embrace compounds of general formula (I) as hereinbefore described,
which expression
includes the prodrugs, the pharmaceutically acceptable salts, and the
solvates, e.g. hydrates, where the
context so permits. Similarly, reference to intermediates, whether or not they
themselves are claimed,
is meant to embrace their salts, and solvates, where the context so permits.
For the sake of clarity,
particular instances when the context so permits are sometimes indicated in
the text, but these instances
are purely illustrative and it is not intended to exclude other instances when
the context so permits.
As used above, and throughout the description of the invention, the following
terms, unless otherwise
indicated, shall be understood to have the following meanings:-
"Patient" includes both human and other mammals.
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"Acid bioisostere" means a group which has chemical and physical similarities
producing broadly
similar biological properties to a carboxy group (see Lipinski, Annual Reports
in Medicinal Chemistry,
1986,21,p283 "Bioisosterism In Drug Design"; Yun, Hwahak Sekye, 1993, 33,
pages 576-579
"Application Of Bioisosterism To New Drug Design"; Zhao, Huaxue Tongbao, 1995,
pages 34-38
"Bioisosteric Replacement And Development Of Lead Compounds In Drug Design";
Graham,
Theochem, 1995, 343, pages 105-109 "Theoretical Studies Applied To Drug
Design:ab initio
Electronic Distributions In Bioisosteres"). Examples of suitable acid
bioisosteres include:
-C(=O)-NHOH, -C(=O)-CH20H, -C(=O)-CH2SH, -C(=O)-NH-CN, sulfo, phosphono,
alkylsulfonylcarbamoyl, tetrazolyl, arylsulfonylcarbamoyl,
heteroarylsulfonylcarbamoyl,
N-methoxycarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-
oxadiazolidinyl or
heterocyclic phenols such as 3-hydroxyisoxazolyl and 3-hydoxy-I-
methylpyrazolyl.
"Acyl" means an H-CO- or alkyl-CO- group in which the alkyl group is as
described herein.
"Acylamino" is an acyl-NH- group wherein acyl is as defined herein.
"Alkenyl" means an aliphatic hydrocarbon group containing a carbon-carbon
double bond and which
may be straight or branched having about 2 to about 15 carbon atoms in the
chain. Preferred alkenyl
groups have 2 to about 12 carbon atoms in the chain; and more preferably 2 to
about 6 carbon atoms
(e.g. 2 to 4 carbon atoms) in the chain. "Branched," as used herein and
throughout the text, means that
one or more lower alkyl groups such as methyl, ethyl or propyl are attached to
a linear chain; here a
linear alkenyl chain. "Lower alkenyl" means about 2 to about 4 carbon atoms in
the chain, which may
be straight or branched. Exemplary alkenyl groups include ethenyl, propenyl, n-
butenyl, i-butenyl,
3-methylbut-2-enyl, n-pentenyl, heptenyl, octenyl, cyclohexylbutenyl and
decenyi.
"Alkenyloxy" is an alkenyl-O- group wherein alkenyl is as defined above.
Exemplary alkenyloxy
groups include allyloxy.
"Alkoxy" means an alkyl-O- group in which the alkyl group is as described
herein. Exemplary alkoxy
groups include difluoromethoxy, methoxy, trifluoromethoxy, ethoxy, n-propoxy,
i-propoxy, n-butoxy
and heptoxy.
"Alkoxycarbonyl" means an alkyl-O-CO- group in which the alkyl group is as
described herein.
Exemplary alkoxycarbonyl groups include methoxy- and ethoxycarbonyl.
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"Alkyl" means, unless otherwise specified, an aliphatic hydrocarbon group
which may be straight or
branched chain having about 1 to about 15 carbon atoms in the chain,
optionally substituted by one or
more halogen atoms. Particular alkyl groups have from 1 to about 6 carbon
atoms. "Lower alkyl" as a
group or part of a lower alkoxy, lower alkylthio, lower alkylsulfinyl or lower
alkylsulfonyl group
means unless otherwise specified, an aliphatic hydrocarbon group which may be
a straight or branched
chain having 1 to about 4 carbon atoms in the chain. Exemplary alkyl groups
include methyl, ethyl,
n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, 3-pentyl, heptyl,
octyl, nonyl, decyl and dodecyl.
Exemplary alkyl groups substituted by one or more halogen atoms include
trifluoromethyl.
"Alkylene" means an aliphatic bivalent radical derived from a straight or
branched alkyl group, in
which the alkyl group is as described herein. Exemplary alkylene radicals
include methylene, ethylene
and trimethylene.
"Alkylenedioxy" means an -O-alkylene-O- group in which alkylene is as defined
above. Exemplary
alkylenedioxy groups include methylenedioxy and ethylenedioxy.
"Alkylsulfinyl" means an alkyl-SO- group in which the alkyl group is as
previously described.
Preferred alkylsulfinyl groups are those in which the alkyl group is Cl-
q.alkyl.
"Alkylsulfonyl" means an alkyl-S02- group in which the alkyl group is as
previously described.
Preferred alkylsulfonyl groups are those in which the alkyl group is
Cl_q.alkyl.
"Alkylsulfonylcarbamoyl" means an alkyl-S02-NH-C(=O)- group in which the alkyl
group is as
previously described. Preferred alkylsulfonylcarbamoyl groups are those in
which the alkyl group is
C 1 _q.alkyl.
"Alkylthio" means an alkyl-S- group in which the alkyl group is as previously
described. Exemplary
alkylthio groups include methylthio, ethylthio, isopropylthio and heptylthio.
"Alkynyl" means an aliphatic hydrocarbon group containing a carbon-carbon
triple bond and which
group may be a straight or branched chain having about 2 to about 15 carbon
atoms in the chain.
Preferred alkynyl groups have 2 to about 12 carbon atoms in the chain; and
more preferably 2 to about
6 carbon atoms (e.g. 2 to 4 carbon atoms) in the chain. Exemplary alkynyl
groups include ethynyl,
propynyl, n-butynyl, i-butynyl, 3-methylbut-2-ynyl, and n-pentynyl.
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"Aroyl" means an aryl-CO- group in which the aryl group is as described
herein. Exemplary aroyl
groups include benzoyl and 1- and 2-naphthoyl.
"Aroylamino" is an aroyl-NH- group wherein amyl is as previously defined.
"Aryl" as a group or part of a group denotes: (i) an optionally substituted
monocyclic or multicyclic
aromatic carbocyclic moiety of about 6 to about 14 carbon atoms, such as
phenyl or naphthyl; or (ii) an
optionally substituted partially saturated multicyclic aromatic carbocyclic
moiety in which an aryl and
a cycloalkyl or cycloalkenyl group are fused together to form a cyclic
structure, such as a
tetrahydronaphthyl, indenyl or indanyl ring. Except where otherwise defined,
aryl groups may be
substituted with one or more aryl group substituents, which may be the same or
different, where "aryl
group substituent" includes, for example, acyl, acylamino, alkoxy,
alkoxycarbonyl, alkylenedioxy,
alkylsulfinyl, alkylsulfonyl, alkylthio, aroyl, aroylamino, aryl,
arylalkyloxy, arylalkyloxycarbonyl,
arylalkylthio, aryloxy, aryloxycarbonyl, arylsulfinyl, arylsulfonyl, arylthio,
carboxy (or an acid
bioisostere), cyano, halo, heteroaroyl, heteroaryl, heteroarylalkyloxy,
heteroaroylamino, heteroaryloxy,
hydroxy, nitro, trifluoromethyl, -NY3Y4, -CONY3Y4, _S02Ny3y4, -Ny3_C(-O)alkyl,
-NY3S02alkyl or alkyl optionally substituted with aryl, heteroaryl, hydroxy,
or -NY3Y4.
"Arylalkyl" means an aryl-alkyl- group in which the aryl and alkyl moieties
are as previously
described. Preferred arylalkyl groups contain a C I _4alkyl moiety. Exemplary
arylalkyl groups
include benzyl, 2-phenethyl and riaphthlenemethyl.
"Arylalkyloxy" means an arylalkyl-O- group in which the arylalkyi groups is as
previously described.
Exemplary arylalkyloxy groups include benzyloxy and 1- or 2-
naphthalenemethoxy.
"Arylalkyloxycarbonyl" means an arylalkyl-O-CO- group in which the arylalkyl
groups is as previously
described. An exemplary arylalkyloxycarbonyl group is benzyloxycarbonyl.
"Arylalkylthio" means an arylalkyl-S- group in which the arylalkyl group is as
previously described.
An exemplary arylalkylthio group is benzylthio.
"Aryloxy" means an aryl-O- group in which the aryl group is as previously
described. Exemplary
aryloxy groups include phenoxy and naphthoxy, each optionally substituted.
"Aryloxycarbonyl" means an aryl-O-C(=O)- group in which the aryl group is as
previously described.
Exemplary aryloxycarbonyl groups include phenoxycarbonyl and
naphthoxycarbonyl.
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"Arylsulfmyl" means an aryl-SO- group in which the aryl group is as previously
described.
"Arylsulfonyl" means an aryl-S02- group in which the aryl group is as
previously described.
"Arylsulfonylcarbamoyl" means an aryl-S02-NH-C(=O)- group in which the aryl
group is as
previously described.
"Arylthio" means an aryl-S- group in which the aryl group is as previously
described. Exemplary
arylthio groups include phenylthio and naphthylthio.
"Azaheteroaryl" means an aromatic carbocyclic moiety of about 5 to about 10
ring members in which
one of the ring members is nitrogen and the other ring members are selected
from carbon, oxygen,
sulfur, and nitrogen. Examples of azaheteroaryl groups include benzimidazolyl,
imidazolyl,
indazolinyl, indolyl, isoquinolinyl, pyridyl, pyrimidinyl, pyrrolyl,
quinolinyl, quinazolinyl and
tetrahydroindolizinyl.
"Cyclic amine" means a 3 to 8 membered monocyclic cycloalkyl ring system
wherein one of the ring
carbon atoms is replaced by nitrogen and which (i) may also contain a further
heteroatom-containing
group selected from O, S, 502, or NYS (where YS is hydrogen, alkyl, aryl,
arylalkyl, -C(=O)-R~,
-C(=O)-ORS or -S02R~); and (ii) may be fused to additional aryl (e.g. phenyl),
heteroaryl (e.g.
pyridyl), heterocycloalkyl or cycloalkyl rings to form a bicyclic or tricyclic
ring system. Exemplary
cyclic amines include pyrrolidine, piperidine, morpholine, piperazine,
indoline, pyrindoline,
tetrahydroquinoline and the like groups.
"Cycloalkenyl" means a non-aromatic monocyclic or multicyclic ring system
containing at least one
carbon-carbon double bond and having about 3 to about 10 carbon atoms.
Exemplary monocyclic
cycloalkenyl rings include cyclopentenyl, cyclohexenyl and cycloheptenyl.
"Cycloalkyl" means a saturated monocyclic or bicyclic ring system of about 3
to about 10 carbon
atoms, optionally substituted by oxo. Exemplary monocyclic cycloalkyl rings
include C3_gcycloalkyl
rings such as cyclopropyl, cyclopentyl, cyclohexyl and cycloheptyl.
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"Cycloalkylalkyl" means a cycloalkyl-alkyl- group in which the cycloalkyl and
alkyl moieties are as
previously described. Exemplary monocyclic cycloalkylalkyl groups include
cyclopropylmethyl,
cyclopentylmethyl, cyclohexylmethyl and cycloheptylrnethyl.
"Halo" or "halogen" means fluoro, chloro, bromo, or iodo. Preferred are fluoro
and chloro.
"Heteroaroyl" means a heteroaryl-C(=O)- group in which the heteroaryl group is
as described herein.
Exemplary heteroaryl groups include pyridylcarbonyl.
"Heteroaroylamino" means a heteroaroyl-NH- group in which the heteroaryl
moiety is as previously
described.
"Heteroaryl" as a group or part of a group denotes: (l) an optionally
substituted aromatic monocyclic or
multicyclic organic moiety of about 5 to about 10 ring members in which one or
more of the ring
members is/are elements) other than carbon, for example nitrogen, oxygen or
sulfur (examples of such
groups include benzimidazolyl, benzthiazolyl, furyl, imidazolyl, indolyl,
indolizinyl, isoxazolyl,
isoquinolinyl, isothiazolyl, oxadiazolyl, pyrazinyl, pyridazinyl, pyrazolyl,
pyridyl, pyrimidinyl,
pyrrolyl, quinazolinyl, quinolinyl, 1,3,4-thiadiazolyl, thiazolyl, thienyl and
triazolyl groups, optionally
substituted by one or more aryl group substituents as defined above except
where otherwise deEned);
(ii) an optionally substituted partially saturated multicyclic
heterocarbocyclic moiety in which a
heteroaryl and a cycloalkyl or cycloalkenyl group are fused together to form a
cyclic structure
(examples of such groups include pyrindanyl groups, optionally substituted by
one or more "aryl group
substituents" as defined above, except where otherwise defined). Optional
substituents include one or
more "aryl group substituents" as deEned above, except where otherwise
defined.
"Heteroarylalkyl" means a heteroaryl-alkyl- group in which the heteroaryl and
alkyl moieties are as
previously described. Preferred heteroarylalkyl groups contain a C1_4alkyl
moiety. Exemplary
heteroarylalkyl groups include pyridylmethyl.
Heteroarylalkyloxy" means an heteroarylalkyl-O- group in which the
heteroarylalkyl group is as
previously described. Exemplary heteroaryloxy groups include optionally
substituted pyridylmethoxy.
"Heteroaryloxy" means an heteroaryl-O- group in which the heteroaryl group is
as previously
described. Exemplary heteroaryloxy groups include optionally substituted
pyridyloxy.
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"Heteroarylsulfonylcarbamoyl" means a heteroaryl-S02-NH-C(=O)- group in which
the heteroaryl
group is as previously described.
"Heterocycloalkyl" means: (i) a cycloalkyl group of about 3 to 7 ring members
which contains one or
more heteroatoms or heteroatom-containing groups selected from O, S and NYS
and mat be optionally
substituted by oxo; (ii) a partially saturated multicyclic heterocarbocyclic
moiety in which an aryl (or
heteroaryl) ring, each optionally substituted by one or more "aryl group
substituents," and a
heterocycloalkyl group are fused together to form a cyclic structure.
(Examples of such groups include
chromanyl, dihydrobenzofuranyl, indolinyl and pyrindolinyl groups).
"Heterocycloalkylalkyl" means a heterocycloalkyl-alkyl- group in which the
heterocycloalkyl and alkyl
moieties are as previously described.
"Prodrug" means a compound which is convertible in vivo by metabolic means
(e.g. by hydrolysis) to a
compound of formula (I), including N-oxides thereof. For example an ester of a
compound of formula
(I) containing a hydroxy group may be convertible by hydrolysis in vivo to the
parent molecule.
Alternatively, an ester of a compound of formula (I) containing a carboxy
group may be convertible by
hydrolysis in vivo to the parent molecule.
Suitable esters of compounds of formula (I) containing a hydroxy group axe,
for example acetates,
citrates, lactates, tartrates, malonates, oxalates, salicylates, propionates,
succinates, fumarates,
maleates, methylene-bis-(3-hydroxynaphthoates, gentisates, isethionates, di-p-
toluoyltartrates,
methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates,
cyclohexylsulfamates
and quinates.
Suitable esters of compounds of formula (I) containing a carboxy group are,
for example, those
described by F.J.Leinweber, Drug Metab. Res., 1987, 18, page 379.
Suitable esters of compounds of formula (I) containing both a carboxy group
and a hydroxy group
within the moiety -L1-Y include lactones formed by loss of water between said
carboxy and hydroxy
groups. Examples of such lactones include caprolactones and butyrolactones.
An especially useful class of esters of compounds of formula (I), containing a
hydroxy group, may be
formed from acid moieties selected from those described by Bundgaard et. al.,
J. Med. Chem., 1989, 32
, page 2503-2507, and include substituted (aminomethyl)-benzoates, for example
dialkylamino-methylbenzoates in which the two alkyl groups may be joined
together and/or interrupted
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by an oxygen atom or by an optionally substituted nitrogen atom, e.g. an
alkylated nitrogen atom, more
especially (morpholino-methyl)benzoates, e.g. 3- or 4-(morpholinomethyl)-
benzoates, and
(4-alkylpiperazin-1-yl)benzoates, e.g. 3- or 4-(4-alkylpiperazin-1-
yl)benzoates.
Where the compound of the invention contains a carboxy group, or a
sufficiently acidic bioisostere,
base addition salts may be formed and are simply a more convenient form for
use; in practice, use of
the salt form inherently amounts to use of the free acid form. The bases which
can be used to prepare
the base addition salts include preferably those which produce, when combined
with the free acid,
pharmaceutically acceptable salts, that is, salts whose rations are non-toxic
to the patient in
pharmaceutical doses of the salts, so that the beneficial inhibitory effects
inherent in the free base are
not vitiated by side effects ascribable to the rations. Pharmaceutically
acceptable salts, including those
derived from alkali and alkaline earth metal salts, within the scope of the
invention include those
derived from the following bases: sodium hydride, sodium hydroxide, potassium
hydroxide, calcium
hydroxide, aluminium hydroxide, lithium hydroxide, magnesium hydroxide, zinc
hydroxide, ammonia,
ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline,
N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-
benzylphenethylamine,
diethylamine, piperazine, tris(hydroxymethyl)aminomethane, tetramethylammonium
hydroxide, and
the like.
Some of the compounds of the present invention are basic, and such compounds
are useful in the form
of the free base or in the form of a pharmaceutically acceptable acid addition
salt thereof.
Acid addition salts are a more convenient form for use; and in practice, use
of the salt form inherently
amounts to use of the free base form. The acids which can be used to prepare
the acid addition salts
include preferably those which produce, when combined with the free base,
pharmaceutically
acceptable salts, that is, salts whose anions are non-toxic to the patient in
pharmaceutical doses of the
salts, so that the beneficial inhibitory effects inherent in the free base are
not vitiated by side effects
ascribable to the anions. Although pharmaceutically acceptable salts of said
basic compounds are
preferred, alt acid addition salts are useful as sources of the free base form
even if the particular salt,
per se, is desired only as an intermediate product as, for example, when the
salt is formed only for
purposes of purification, and identification, or when it is used as
intermediate in preparing a
pharmaceutically acceptable salt by ion exchange procedures. Pharmaceutically
acceptable salts within
the scope of the invention include those derived from mineral acids and
organic acids, and include
hydrohalides, e.g. hydrochlorides and hydrobromides, sulfates, phosphates,
nitrates, sulfamates,
acetates, citrates, lactates, tartrates, malonates, oxalates, salicylates,
propionates, succinates, fumarates,
maleates, methylene-bis-beta-hydroxynaphthoates, gentisates, isethionates, di-
p-toluoyltartrates,
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methane-sulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates,
cyclohexylsulfamates
and quinates.
As well as being useful in themselves as active compounds, salts of compounds
of the invention are
useful for the purposes of purification of the compounds, for example by
exploitation of the solubility
differences between the salts and the parent compounds, side products and/or
starting materials by
techniques well known to those skilled in the art.
With reference to formula (I) above, the following are particular and
preferred groupings:
RI may particularly represent:
(i) hydrogen
(ii) CI_q.alkyl [e.g.-CH3 or-CH2CH3];
(iii) C1_4alkyl substituted by halo [e.g. -CH2CF3];
(iv) C I-q.alkyl substituted by hydroxy [e.g. -CH20H , -CH2CHZOH or -
CH2CHzCH20H ];
(v) CI-q.alkyl substituted by -N(R6)C(=O)-R~ [e.g. -CHaCH2CHZNHC (=O) CH3 ];
(vi) CI_q.alkyl substituted by -C(=O)-NYIY2 [e.g. -CH2 C (=O) -N~ ]; or
H2C-CH2
(vii) cycloalkylalkyl substituted by hydroxy (e.g. -C-CHI ].
CH~OH
Compounds of formula (I) in which RI represents hydrogen, -CH3, -CH2CH3, -
CH2CF3 or
-CH2 C (=O) -NN O are especially preferred. RI more especially represents
hydrogen.
R2 may particularly represent:
N~NH
(i) carboxy or an acid bioisostere (e.g. --<~ ~N );
N~
(ii) hydroxy;
(iii) alkyl substituted by carboxy [e.g. -CH2CH2C02H ];
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N CH3
w
(iv) heteroaryl [e.g. ---<~ ~ or pyridyl];
NCO
(v) -OR4 in which R4 is alkyl [e.g. -OCH3 ];
(vi) -OR4 in which R4 is alkyl or cycloalkylalkyl substituted by one or more
hydroxy groups
[e.g. -OCH2CH20H , -OCH2CH2CH~OH , -OCH ( CH3 ) CH20H ,
H2 I ~ H2
-OCH2CH (OH) CH3 , -O- ~ -CH2 or -OCH2CH (OH) CH20H ];
CH20H
(vii) -OR4 in which R'l is alkyl substituted by one or more alkoxy groups
[e.g.
-OCH (CH3) CHZOCH3 ];
(viii) -OR4 in which R'l is alkyl or cycloalkyl substituted by one or more
carboxy groups [e.g.
H2C-CHI
-OCH2C02H, -OCH (CH3) CO2H or -O- ~ -CH2 ];
C02H
H2C-CHI
(ix) -OR4 in which R4 is cycloalkyl substituted by -C(=O)-NYlY~ [e.g. -O-C-CH2
or
CONHZ
HZC ~ H2
-O- ~ -CH2 ];
CONHCH3
(x) -C(=O)-R in which R is alkyl (e.g. -C (=O) -CH3 ];
(xi) -C(=O)-NYlY~ [e.g. -CONH2 , -CONHCH3 , -CONHCH (CH20H) 2 ,
-CONHCH2CH20H , -CONHC ( CH3 ) ~CH20H , -CONHCH2CH20CH3 ,
N
-CONHCH2CH2CONH2 , -CONHCH2C (CH3) 20H or -CONH--C~ ]; or
N~NH
(xii) -N(R6)-C(=O)-R~ [e.g. -NHC (=O) CH3 ]
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Compounds of formula (I) in which R2 represents -OCH3 or -CONHC (CH3) 2CH~OH
are especially
preferred. R2 more especially represents -OCH3
R3 may particularly represent:
(i) hydrogen;
(ii) cyano;
(iii) optionally substituted aryl (e.g. phenyl);
(iv) optionally substituted heteroaryl (e.g. optionally substituted pyridyl or
optionally
Me0
substituted indolyl, especially ~ N > or \ N );
~H
(v) alkyl (e.g. methyl or ethyl);
(vi) alkyl substituted by one or more halogen atoms (e.g. trifluoromethyl);
(vii) alkyl substituted by -C(=O)-NYlY2, especially -CH2-CH2-C(=O)NHCH3;
(viii) alkyl substituted by -ORS (e.g. -CH2-CH2-OCH3);
(ix) -ZR4, especially -OCH3 , -OCH2CH3, -OCF2H or -OCH2-CH2-OCH3;
(x) -C(=O)-ORS, especially -C(=O)-OH;
(xi) -C(=O)-NYlY2, especially -C(=O)NHCH3 or -C(=O)-NH-C(CH3)2-CH20H; and
(xii) -NYlY2, especially -N O .
Compounds of formula (I) in which R3 represents hydrogen, cyano, pyridyl,
trifluoromethyl, -CH2-
CH2-C(=O)NHCH3, -OCF2H, -C(=O)-NH-C(CH3)2-CH20H or -N~ are especially
preferred. R3 more especially represents -OCH3.
R2 is preferably attached to position S of the indole ring.
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s
The group is preferably attached to the 3 position of the indole ring
H
It is to be understood that this invention covers all appropriate combinations
of the particular and
preferred groupings referred to herein.
Particular preferred compounds of the invention are:-
Me
~Me
~CH20H
H ...I3
; o
(II) (III)
> ;
(N) (V)
H
N
~O CF3 .
; ,
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(VI) (VII)
~,CH20H
H
0
(VIII) (IX)
3
H H
> > >
(X) (XI) (XII)
a
de
H
i
to (xIII) (xIV)
Me0 (CH,
Y
H H H
i i i
(XV) (XVI) (XVII)
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_ I8-
H
H H H
> ;
(XVIII) (XIX) (XX)
~CH20H
i
H H
(XXI) (XXII) (XXIII)
'CH20H
i
H n
0 0
(XXVI)
(XXIV) (XXV)
v
IO H ;
(XXVII)
and the corresponding N-oxides, and their prodrugs; and pharmaceutically
acceptable salts and solvates
(e.g. hydrates) of such compounds and their N-oxides and prodrugs.
Especially preferred compounds of the invention are:-
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(XII)
4-methoxy-6-(5-methoxy-1 H-indol-3-yl)-7H-pyrrolo[2,3-d]pyrimidine;
and the corresponding N-oxides, and their prodrugs; and pharmaceutically
acceptable salts and solvates
(e.g. hydrates) of such compounds and their N-oxides and prodrugs.
The compounds of the invention exhibit useful pharmacological activity and
accordingly are
incorporated into pharmaceutical compositions and used in the treatment of
patients suffering from
certain medical disorders. The present invention thus provides, according to a
further aspect,
compounds of the invention and compositions containing compounds of the
invention for use in
therapy.
Compounds within the scope of the present invention block kinase catalytic
activity according to tests
described in the literature and in vitro procedures described hereinafter, and
which tests results are
1S believed to correlate to pharmacological activity in humans and other
mammals. Thus, in a further
embodiment, the present invention provides compounds of the invention and
compositions containing
compounds of the invention for use in the treatment of a patient suffering
from, or subj ect to,
conditions which can be ameliorated by the administration of protein kinase
(e.g. Syk, FAK, KDR or
Aurora2) inhibitors, in particular a Syk kinase inhibitor. For example,
compounds of the present
invention are useful in the treatment of inflammatory diseases, for example
asthma: inflammatory
dermatoses (e.g. psoriasis, dematitis herpetiformis, eczema, necrotizing and
cutaneous vasculitis,
bullous disease); allergic rhinitis and allergic conjunctivitis; joint
inflammation, including arthritis,
rheumatoid arthritis and other arthritic conditions such as rheumatoid
spondylitis, gouty arthritis,
traumatic arthritis, rubella arthritis, psoriatic arthritis and
osteoarthritis. The compounds are also
2S useful in the treatment of Chronic Obstructive Pulmonary Disease (COPD),
acute synovitis,
autoimmune diabetes, autoimmune encephalomyelitis, collitis, atherosclerosis,
peripheral vascular
disease, cardiovascular disease, multiple sclerosis, restenosis, myocarditis,
B cell lymphomas,
systemic lupus erythematosus, graft v host disease and other transplant
associated rejection events,
cancers and tumours (such as colorectal, prostate, breast, thyroid, colon and
lung cancers) and
inflammatory bowel disease. Additionally, the compounds are useful as tumor
anti-angiogenic agents.
A special embodiment of the therapeutic methods of the present invention is
the treating of asthma.
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Another special embodiment of the therapeutic methods of the present invention
is the treating of
psoriasis.
Another special embodiment of the therapeutic methods of the present invention
is the treating of joint
inflammation.
Another special embodiment of the therapeutic methods of the present invention
is the treating of
inflammatory bowel disease.
Another special embodiment of the therapeutic methods of the present invention
is the treating of
cancers and tumours.
According to a further feature of the invention there is provided a method for
the treatment of a human
or animal patient suffering from, or subject to, conditions which can be
ameliorated by the
administration of a protein kinase (e.g. Syk, FAK, KDR or Aurora2) inhibitor
for example conditions
as hereinbefore described, which comprises the administration to the patient
of an effective amount of
a compound of the invention or a composition containing a compound of the
invention. "Effective
amount" is meant to describe an amount of compound of the present invention
effective in inhibiting
the catalytic activity a protein kinase, such as Syk, FAK, KDR or Aurora2, and
thus producing the
ZO desired therapeutic effect.
References herein to treatment should be understood to include prophylactic
therapy as well as
treatment of established conditions.
The present invention also includes within its scope pharmaceutical
compositions comprising at least
one of the compounds of the invention in association with a pharmaceutically
acceptable carrier or
excipient.
Compounds of the invention may be administered by any suitable means. In
practice, compounds of
the present invention may be administered parenterally, topically, rectally,
orally or by inhalation,
especially by the oral route.
Compositions according to the invention may be prepared according to the
customary methods, using
one or more pharmaceutically acceptable adjuvants or excipients. The adjuvants
comprise, inter olio,
diluents, sterile aqueous media and the various non-toxic organic solvents.
The compositions may be
presented in the form of tablets, pills, granules, powders, aqueous solutions
or suspensions, injectable
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solutions, elixirs or syrups, and can contain one or more agents chosen from
the group comprising
sweeteners, flavourings, colourings, or stabilisers in order to obtain
pharmaceutically acceptable
preparations. The choice of vehicle and the content of active substance in the
vehicle are generally
determined in accordance with the solubility and chemical properties of the
active compound, the
particular mode of administration and the provisions to be observed in
pharmaceutical practice. For
example, excipients such as lactose, sodium citrate, calcium carbonate,
dicalcium phosphate and
disintegrating agents such as starch, alginic acids and certain complex
silicates combined with
lubricants such as magnesium stearate, sodium lauryl sulfate and talc may be
used for preparing tablets.
To prepare a capsule, it is advantageous to use lactose and high molecular
weight polyethylene glycols.
When aqueous suspensions are used they can contain emulsifying agents or
agents which facilitate
suspension. I~iluents such as sucrose, ethanol, polyethylene glycol, propylene
glycol, glycerol and
chloroform or mixtures thereof may also be used.
For parenteral administration, emulsions, suspensions or solutions of the
products according to the
invention in vegetable oil, for example sesame oil, groundnut oil or olive
oil, or aqueous-organic
solutions such as water and propylene glycol, injectable organic esters such
as ethyl oleate, as well as
sterile aqueous solutions of the pharmaceutically acceptable salts, are used.
The solutions of the salts
of the products according to the invention are especially useful for
administration by intramuscular or
subcutaneous injection. The aqueous solutions, also comprising solutions of
the salts in pure distilled
water, may be used for intravenous administration with the proviso that their
pH is suitably adjusted,
that they are judiciously buffered and rendered isotonic with a sufficient
quantity of glucose or sodium
chloride and that they are sterilised by heating, irradiation or
microfiltration.
For topical administration, gels (water or alcohol based), creams or ointments
containing compounds of
the invention may be used. Compounds of the invention may also be incorporated
in a gel or matrix
base for application in a patch, which would allow a controlled release of
compound through the
transdermal barrier.
For administration by inhalation compounds of the invention may be dissolved
or suspended in a
suitable carrier for use in a nebuliser or a suspension or solution aerosol,
or may be absorbed or
adsorbed onto a suitable solid carrier for use in a dry powder inhaler.
Solid compositions for rectal administration include suppositories formulated
in accordance with
known methods and containing at least one compound of the invention.
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The percentage of active ingredient in the compositions of the invention may
be varied, it being
necessary that it should constitute a proportion such that a suitable dosage
shall be obtained.
Obviously, several unit dosage forms may be administered at about the same
time. The dose employed
will be determined by the physician, and depends upon the desired therapeutic
effect, the route of
administration and the duration of the treatment, and the condition of the
patient.
In the adult, the doses are generally from about 0.001 to about 50, preferably
about 0.001 to about 5,
mg/kg body weight per day by inhalation, from about 0.01 to about 100,
preferably 0.1 to 70, more
especially 0.5 to 10, mg/kg body weight per day by oral administration, and
from about 0.001 to about
10, preferably 0.01 to l, mg/kg body weight per day by intravenous
administration. In each particular
case, the doses will be determined in accordance with the factors distinctive
to the subject to be treated,
such as age, weight, general state of health and other characteristics which
can influence the efficacy of
the medicinal product.
The compounds according to the invention may be administered as frequently as
necessary in order to
obtain the desired therapeutic effect. Some patients may respond rapidly to a
higher or lower dose and
may find much weaker maintenance doses adequate. For other patients, it may be
necessary to have
long-term treatments at the rate of 1 to 4 doses per day, in accordance with
the physiological
requirements of each particular patient. Generally, the active product may be
administered orally 1 to
4 times per day. Of course, for some patients, it will be necessary to
prescribe not more than one or
two doses per day.
Compounds of the invention may be prepared by the application or adaptation of
known methods, by
which is meant methods used heretofore or described in the literature, for
example those described by
R.C.Larock in Comprehensive Organic Transformations, VCH publishers, 1989.
In the reactions described hereinafter it may be necessary to protect reactive
functional groups, for
example hydroxy, amino, imino, thio or carboxy groups, where these are desired
in the final product, to
avoid their unwanted participation in the reactions. Conventional protecting
groups may be used in
accordance with standard practice, for examples see T.W. Greene and P.G.M.Wuts
in "Protective
Groups in Organic Chemistry" John Wiley and Sons, 1991.
Compounds of formula (I), wherein R1, R2 and R3 are as hereinbefore defined,
are prepared by
reaction of compounds of formula (XXVIII):-
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X1
(XXVIII)
wherein R3 is as hereinbefore defined and X1 is a halogen, preferably iodine,
atom or a triflate group,
with compounds of formula (XXIX):-
4 5
(HO)2B
R1
(XXIX)
wherein R1 and R2 are as defined hereinbefore. The coupling reaction may
conveniently be carried
out, for example, in the presence of a complex metal catalyst such as
tetrakis(triphenylphosphine)palladium(0) and sodium bicarbonate, in aqueous
dimethylformamide at a
temperature up to reflux temperature. This reaction is conveniently carried
out with the pyrrole NH in
compound (XXVIII) protected with for example a tosyl group and the indole NH
in compound (XXIX)
protected with, for example, a tent-butyloxycarbonyl group.
Compounds of formula (I) wherein R2 and R3 are as hereinbefore defined and R1
is optionally
substituted alkyl are prepared by reaction of the corresponding compounds of
formula (I) wherein R2
and R3 are as hereinbefore defined and R1 is hydrogen with the appropriate
alkyl halide R2-X2 in
which R2 is optionally substituted alkyl and X2 is halo. This reaction is
particularly suitable for the
preparation of compounds of formula (I) wherein Rl is morpholinoacetyl.
Compounds of the invention may also be prepared by interconversion of other
compounds of the
invention.
Thus, for example, compounds of formula (I) containing a carboxy group may be
prepared by
hydrolysis of the corresponding esters. The hydrolysis may conveniently be
carried out by alkaline
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hydrolysis using a base, such as an alkali metal hydroxide, e.g. lithium
hydroxide, or an alkali metal
carbonate, e.g. potassium carbonate, in the presence of an aqueous/organic
solvent mixture, using
organic solvents such as dioxan, tetrahydrofuran or methanol, at a temperature
from about ambient to
about reflux. The hydrolysis of the esters may also be carried out by acid
hydrolysis using an inorganic
acid, such as hydrochloric acid, in the presence of an aqueous/inert organic
solvent mixture, using
organic solvents such as dioxan or tetrahydrofuran, at a temperature from
about 50°C to about 80°C.
As another example compounds of formula (I) containing a carboxy group may be
prepared by acid
catalysed removal of the test-butyl group of the corresponding te~~t-butyl
esters using standard reaction
conditions, for example reaction with trifluoroacetic acid at a temperature at
about room temperature.
As another example compounds of formula (I) containing a carboxy group may be
prepared by
hydrogenation of the corresponding benzyl esters. The reaction may be carried
out in the presence of
ammonium formate and a suitable metal catalyst, e.g. palladium, supported on
an inert carrier such as
carbon, preferably in a solvent such as methanol or ethanol and at a
temperature at about reflux
temperature. The reaction may alternatively be carried out in the presence of
a suitable metal catalyst,
e.g. platinum or palladium optionally supported on an inert carrier such as
carbon, preferably in a
solvent such as methanol or ethanol.
As another example of the interconversion process, compounds of formula (I)
containing a
-C(=O)-NYlY2 group may be prepared by coupling compounds of formula (I)
containing a carboxy
group with an amine of formula HNYlY2 to give an amide bond using standard
peptide coupling
procedures, for example coupling in the presence of O-(7-azabenzotriazol-1-yl)-
1,1,3,3-
tetramethyluronium hexafluorophosphate and triethylamine (or
diisopropylethylamine) in
tetrahydrofuran (or dimethylformamide) at room temperature. This procedure is
particularly useful for
the preparation of (i) compounds of formula (I) wherein R3 represents -C(=O)-
NYlY2 or (ii)
compounds of formula (I) wherein R2 represents -C(=O)-NYlY2. The coupling may
also be brought
about by reaction of compounds of formula (I) containing a carboxy group with
N-{(dimethylamino)( 1 H-1,2,3-triazaolo[4,5-b]pyridin-1-yI)methylene}-N-
methylmethanaminium
hexafluorophosphate N-oxide in the presence of a suitable base, such as
diisopropylethylamine, in an
inert solvent, such as dimethylformamide, and at a temperature at about room
temperature, followed by
reaction with an amine of formula HNYlY2 (ammonium chloride can be used for
the preparation of
compounds of formula (I) containing a -C(=O)-NH2 group). The coupling may also
be brought about
by reaction of compounds of formula (I) containing a carboxy group with 2-(1H-
benzotriazole-1-
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yl)1,1,3,3-tetramethyluronium hexafluorophosphate, in dry dimethyIformamide,
followed by reaction
with an amine of formula HNYlY2 in the presence of diisopropylethylamine.
As another example of the interconversion process, compounds of formula (I)
containing a -CH20H
group may be prepared by the reduction of corresponding compounds of formula
(I) containing a -CHO
or -C02R~ (in which R~ is lower alkyl) group. For example, the reduction may
conveniently be
carried out by means of reaction with lithium aluminium hydride, in an inert
solvent, such as
tetrahydrofuran, and at a temperature from about room temperature to about
reflex temperature.
As another example of the interconversion process, compounds of formula (I) in
which R2 is hydroxy
may be prepared by reaction of the corresponding compounds of formula (I) in
which R1 is methoxy
with a Lewis acid, such as boron tribromide, in an inert solvent, such as
dichloromethane and at a
temperature from about 0°C to about room temperature.
As another example of the interconversion process, compounds of formula (I) in
which R2 is -OR4 (in
which R4 is optionally substituted alkyl, cycloalkyl, cycloalkylalkyl,
heterocycloalkyl or
heterocycloalkylalkyl) may be prepared by alkylation the corresponding
compounds of formula (I) in
which R2 is hydroxy, with compounds of formula (XXX):-
R4-X3 (XXX)
wherein R4 is as just hereinbefore defined and X3 is a halogen, preferably
bromo, atom, or a tosyl
group, using standard alkylation conditions. The alkylation may for example be
carried out in the
presence of a base, such as an alkali metal carbonate (e.g. potassium
carbonate or cesium carbonate),
an alkali metal alkoxide (e.g. potassium tertiary butoxide) or alkali metal
hydride (e.g. sodium
hydride), in dimethylformamide, or dimethyl sulfoxide, at a temperature from
about 0°C to about
100°C.
As another example of the interconversion process, compounds of formula (I) in
which R1 is alkyl,
alkenyl, cycloalkyl, heterocycloalkyl, or alkyl substituted by -C(=O)NYlY2, -
ORS, -C(=O)-ORS,
-NYIY~ may be prepared by alkylation of the corresponding compounds of formula
(Ia) in which R1 is
hydrogen, with the appropriate halide of formula (XXXI):-
R1 _X4
(XXXI)
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wherein Rl is alkyl, alkenyl, cycloalkyl, heterocycloalkyl, or alkyl
substituted by -C(=O)NYlY2,
-ORS, -C(=O)-ORS, -NYlY2 and X4 is a halogen, preferably bromine, atom, using
standard alkylation
conditions for example those described hereinbefore.
As another example of the interconversion process, compounds of formula (I)
containing sulfoxide
linkages may be prepared by the oxidation of corresponding compounds
containing -S- linkages. For
example, the oxidation may conveniently be carried out by means of reaction
with a peroxyacid, e.g.
3-chloroperbenzoic acid, preferably in an inert solvent, e.g. dichloromethane,
preferably at or near
room temperature, or alternatively by means of potassium hydrogen
peroxomonosulfate in a medium
such as aqueous methanol, buffered to about pHS, at temperatures between about
0°C and room
temperature. This latter method is preferred for compounds containing an acid-
labile group.
As another example of the interconversion process, compounds of formula (I)
containing sulfone
linkages may be prepared by the oxidation of corresponding compounds
containing -S- or sulfoxide
linkages. For example, the oxidation may conveniently be carried out by means
of reaction with a
peroxyacid, e.g. 3-chloroperbenzoic acid, preferably in an inert solvent, e.g.
dichloromethane,
preferably at or near room temperature.
As another example of the interconversion process, compounds of formula (I)
containing a cyano group
may be prepared by reaction of the corresponding compounds of formula (I)
containing a -C(=O)-NHZ
group with phosphorus pentachloride in the presence of triethylamine. The
reaction may conveniently
be carried out in an inert solvent, such as tetrahydrofuran, and at a
temperature at about reflux
temperature.
As another example of the interconversion process, compounds of formula (I)
containing a
-C(=O)-NH2 group may be prepared by reaction of the corresponding compounds of
formula (I)
containing a cyano group with hydrogen peroxide in the presence of sodium
hydroxide. The reaction
may conveniently be carried out in methanol at a temperature at about room
temperature.
As another example of the interconversion process, compounds of formula (I) in
which R3 is -NYlY2
(wherein Y1 and Y2 are as hereinbefore defined), may be prepared by reaction
of the corresponding
compounds of formula (I) in which R3 is halo (e.g. chloro) with an amine of
formula HNYlY2
(wherein Y 1 and Y2 are as immediately hereinbefore defined).
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As another example of the interconversion process, compounds of formula (I) in
which R3 is cyano
may be prepared by reaction of compounds of formula (I) in which X 1 is halo,
preferably chloro, with
zinc cyanide in the presence of zinc powder, [1'1-
bis(diphenylphosphino)ferrocene]
dichloropalladium(II) complex and dichloromethane (catalytic amount) and N,N-
dimethylacetamide at
a temperature up to about 150°C.
As another example of the interconversion process, compounds of formula (I)
containing a
-C(=O)-ORS group (in which RS is as hereinbefore defined) may be prepared by
reaction of the
corresponding compounds of formula (I) containing a -C(=O)-OH group with
alcohols of formula
RS-OH. For example when RS is tent-butyl the reaction may conveniently be
carried out in the
presence of 1-1'-carbonyldiimidazole and 1,8-diazabicyclo[5.4.0]undec-7-ene at
a temperature at about
room temperature.
It will be appreciated that compounds of the present invention may contain
asymmetric centres. These
asymmetric centres may independently be in either the R or S configuration. It
will be apparent to
those skilled in the art that certain compounds of the invention may also
exhibit geometrical
isomerism. It is to be understood that the present invention includes
individual geometrical isomers
and stereoisomers and mixtures thereof, including racemic mixtures, of
compounds of formula (I)
hereinabove. Such isomers can be separated from their mixtures, by the
application or adaptation of
known methods, for example chromatographic techniques and recrystallisation
techniques, or they are
separately prepared from the appropriate isomers of their intermediates.
According to a further feature of the invention, acid addition salts of the
compounds of this invention
may be prepared by reaction of the free base with the appropriate acid, by the
application or adaptation
of known methods. For example, the acid addition salts of the compounds of
this invention may be
prepared either by dissolving the free base in water or aqueous alcohol
solution or other suitable
solvents containing the appropriate acid and isolating the salt by evaporating
the solution, or by
reacting the free base and acid in an organic solvent, in which case the salt
separates directly or can be
obtained by concentration of the solution.
The acid addition salts of the compounds of this invention can be regenerated
from the salts by the
application or adaptation of known methods. For example, parent compounds of
the invention can be
regenerated from their acid addition salts by treatment with an alkali, e.g.
aqueous sodium bicarbonate
solution or aqueous ammonia solution.
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Compounds of this invention can be regenerated from their base addition salts
by the application or
adaptation of known methods. For example, parent compounds of the invention
can be regenerated
from their base addition salts by treatment with an acid, e.g. hydrochloric
acid.
Compounds of the present invention may be conveniently prepared, or formed
during the process of the
invention, as solvates (e.g. hydrates). Hydrates of compounds of the present
invention may be
conveniently prepared by recrystallisation from an aqueous/organic solvent
mixture, using organic
solvents such as dioxan, tetrahydrofuran or methanol.
According to a further feature of the invention, base addition salts of the
compounds of this invention
may be prepared by reaction of the free acid with the appropriate base, by the
application or adaptation
of known methods. For example, the base addition salts of the compounds of
this invention may be
prepared either by dissolving the free acid in water or aqueous alcohol
solution or other suitable
solvents containing the appropriate base and isolating the salt by evaporating
the solution, or by
reacting the free acid and base in an organic solvent, in which case the salt
separates directly or can be
obtained by concentration of the solution.
The starting materials and intermediates may be prepared by the application or
adaptation of known
methods, for example methods as described in the Reference Examples or their
obvious chemical
equivalents.
Intermediates of formula (XXVIII) wherein R3 is as hereinbefore defined, Xl is
iodo and the pyrrole
NH is protected with a tosyl group may be prepared as shown in scheme 1.
SCHEME I
R3 R3
N/ I ~ N/ ~ ~ I
N N N N
g Tosyl Tosyl
(XXXII)
(XXXIII) (XXXIV)
Thus for example compounds of formula (XXXIV) may be prepared by:
(i) reaction of compounds of formula (XXXII) with papa-toluenesulfonyl
chloride in the presence of
aqueous sodium hydroxide and tetrabutyl ammonium sulfate in an inert solvent,
such as toluene, and at
room temperature;
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(ii) subsequent treatment of the resulting compound of formula (XXXIII) with
butyl lithium in
tetrahydrofuran, at a temperature at about -78°C;
(iii) reaction of the resulting anion with iodine.
Intermediates of formula (XXXIII) wherein R3 is heteroaryl may be prepared by
reaction of
compounds of formula (XXXIII) wherein R3 is halo, e.g. chloro, with a borane
of formula R3BEt2
wherein R3 is heteroaryl. The reaction may conveniently be carried out in the
presence of
tetrakis(triphenylphosphine)palladium(0) and potassium carbonate, in
tetrahydrofuran at a temperature
up to reflux temperature. This reaction is particularly suitable for the
preparation of compounds of
formula (XXXIII) wherein R3 is pyridyl.
Intermediates of formula (XXXIII) wherein R3 is heteroaryl may also be
prepared by reaction of
compounds of formula (XXXIII) wherein R3 is halo, e.g. chloro, with heteroaryl-
boronic acids of
formula R3B(OH)2 in the presence of tetrakis(triphenylphosphine)palladium(0)
and aqueous sodium
bicarbonate, in dimethylformamide at a temperature up to reflux temperature.
This reaction is
particularly suitable for the preparation of compounds of formula (XXXIII)
wherein R3 is optionally
substituted indolyl.
Intermediates of formula (XXXIII) wherein R3 is OR4, in which R4 is as
hereinbefore defined, may be
prepared by reaction of compounds of formula (XXXIII) wherein R3 is halo, e.g.
chloro, with
compounds of formula R40Na (prepared by reacting alcohols of formula R40H with
sodium) at a
temperature up to about 65°C. This reaction is particularly suitable
for the preparation of compounds
of formula (XXXIII) wherein R3 is OMe.
The present invention is further exemplified but not limited by the following
illustrative Examples and
Reference Examples.
High Pressure Liquid Chromatography - Mass Spectrometry (LC-MS) conditions for
determination of
retention times (RT) were as follows:-
Method A: Hypersil BDS C-18 column (4.6 mm x 50 mm) reverse phase operated
under gradient
elution conditions with mixtures of (A) water containing 0.05% trifluoroacetic
acid and (B) acetonitrile
containing O.OS% trifluoroacetic acid as the mobile phase gradient : (0.00
minutes 100%A:0%B; linear
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gradient to 100% B at 2 minutes; then hold until 3.S minutes); flow rate 1
mL/minute with
approximately 0.25mL/minute split to the Mass Spectrometer; injection volume
10 pL; Hewlett
Packard Model HPl 100 Series UV detector wavelength 200nm; Evaporative light
scattering (ELS)
detection - temperature 46°C, nitrogen pressure 4bar.
Method B: Gilson 21 S injector model using a Hypersil HyPURITY C-18 -S p
column (4.6 mm x SO
mm) operated under gradient elution conditions with mixtures of (A) water
containing 0.05%
trifluoroacetic acid and (B) acetonitrile containing 0.05% trifluoroacetic
acid as the mobile phase
gradient : (0.00 minutes 9S%A:S%B; linear gradient to 95% B at 4 minutes; then
to S% B at 4.S
minutes, then hold until 6 minutes); injection volume S p,L and flow rate
ImLlminute to UV (DAD)
detector followed by approximately O.I OOmL/minute split to the Mass
Spectrometer (positive
electrospray) with remainder to ELS detector.
METHOD C: Micromass instrument model LCT linked to an HP 1100 model
instrument. Compound
abundance were detected using an HP model G1315A photodiode array detector in
the 200-600 nm
wavelength range and a Sedex model 6S evaporative light scattering detector.
Mass spectra were
acquired in the 180 to 800 range. Data were analysed using the Micromass
MassLynx software.
Separation were carried out on a Hypersil BDS C18, 3 ~.m particle size column
(SO x 4.6 mm) eluted
by a linear gradient of 5 to 90% acetonitrile containing 0.05% (v/v)
trifluoroacetic acid in water
containing 0.05% (v/v) trifluoroacetic acid in 3.5 minutes at a flow rate of 1
ml/minute. The total
runtime including column reequilibration was 7 minutes.
EXAMPLE I
2-f 5-Methoxy-3-(4-trifluorometh~pyrrolo~2,3-b]pyrimidin-6-yl~-indol-1-~]-I-
morpholin-4-yl-
ethanone
O
I . _~H ~N 2 . 3 .
The compound of formula (I), wherein R is 2 ~ , R is -OMe, R is -CF3 the
~O
group is attached to the 3 position of the indole ring and the group R2 is
H
attached to the 5 position of the indole ring, represented by formula (II):
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Me0
CF3
0
~~N~
N H N
~O
(II)
is prepared as shown in the following scheme:
Br
N \ \ mCPBA N \ \ POBr3 \ TosCl, phase
N
transfer
'N H~ ~ N+
~_
(1) O
(2) (3)
Br CF CF3
3
NI \ ~ KF, Cul N \ (i) LDA N \ ~ Pd(0)
N/ ref 1 ~ ~ ~ \~ (ii) Iz ~ ' / N~I
N \ N/ ~ N \
Tos Tos Tos
(5) (6) 'eo~
5N KOH NaH
0
CI~N
~1IO
(7) (8)
(II)
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(i) treatment of 7H-pyrrolo[2,3-b]pyrimidine (1) with 3-chloroperbenzoic acid
in
dichloromethane at about 0°C to give 7H-pyrrolo[2,3-b]pyrimidine-N-
oxide (2);
(ii) reaction of (2) with phosphorous oxybromide at about 50°C to give
4-bromo-7H-
pyrrolo[2,3-b]pyrimidine (3);
(iii) reaction of (3) with 4-toluene sulfonyl chloride in the presence of
tetrabutylammonium
sulfate and aqueous sodium hydroxide in toluene, to give 4-bromo-7H-
pyrrolo[2,3-
b]pyrimidine (4);
(iv) reaction of (4) with trifluoromethyltrimethylsilane in the presence of
potassium fluoride
and copper(I) iodide in dimethylformamide at about 60°C, to give 7-
(toluene-4-sulfonyl)-4-
trifluoromethyl-7H-pyrrolo[2,3-b]pyrimidine (5);
(v) treatment of (5) with lithium diisopropylamide in tetrahydrofuran, at
about -78°C,
followed by reaction of the resulting anion with iodine to give 6-iodo-7-
(toluene-4-sulfonyl)-4-
trifluoromethyl-7H-pyrrolo[2,3-b]pyrimidine (6).
(vi) coupling of (6) with 1-tent-butyloxycarbonyl-5-methoxy-1H-indole-3-
boronic acid in the
presence of tetrakis(triphenylphosphine)palladium(0) and sodium bicarbonate,
in aqueous
dimethylformamide at about reflux temperature and removal of the tart-
butyloxycarbonyl
protecting group followed by treatment with methyl iodide in the presence of
sodium hydride,
in tetrahydrofuran, to give 6-(5-methoxy-1H-indol-3-yl)-7-(toluene-4-sulfonyl)-
4-
trifluoromethyl-7H-pyrrolo[2,3-b]pyrimidine (7);
(viii) removal of the tosyl protecting group in (7) by treatment with
potassium hydroxide in
methanol to give 6-(5-methoxy-1H-indol-3-yl)-4-trifluoromethyl-7H-pyrrolo[2,3-
b]pyrimidine
(8); and
(ix) alkylation of (8) with 4-(2-chloroacetyl)morpholine in the presence of
sodium hydride, in
dimethylformamide to give 2-[5-methoxy-3-(4-trifluoromethyl-7H-pyrrolo[2,3-
b]pyrimidin-6-
yl)-indol-1-yl]-1-morpholin-4-yl-ethanone (II).
EXAMPLE 2
1-Methyl-3-(7H-pyrrolo[2,3-b]pyrimidine-6-~)-1H-indole-5-carbox lic acid 2-
hydrox~-1,1-dimethyl-
ethyll-amide
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O Me Me
The compound of formula (I), wherein Rl is -CH3, R2 is ~N~CH OH ° R3 is
-H the group
H
N~
\>--- is attached to the 3 position of the indole ring and the group RZ is
attached to
N
N
H
the 5 position of the indole ring, represented by formula (III):
H Me
Me
CH20H
H ~.,~i3
(III)
is prepared as shown in the following scheme:
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i N ~ N~N N ~N N I
N H is \ts
(9) (10) (11)
OH
HO-B°
\ COZCH3
N I
bac
(12)
O~H COzCH3
N \ ~ \ I N \ ~~ \
Ni N N 'N N~N\
CH3 H
(14) (13)
(III)
(i) reaction of (9) with 4-toluene sulfonyl chloride in the presence of
tetrabutylammonium
sulfate and aqueous sodium hydroxide in toluene, to give ( 10);
(ii) treatment of (10) with lithium diisopropylamide in tetrahydrofuran, at
about -78°C,
followed by reaction of the resulting anion with iodine to give ( 11 );
(iii) coupling of (11) with 1-tent-butyloxycarbonyl-5-methoxy-1H-indole-3-
boronic acid (12) in
the presence of tetrakis(triphenylphosphine)palladium(0) and sodium
bicarbonate, in aqueous
dimethylformamide at about reflux temperature and removal of the tert-
butyloxycarbonyl
protecting group followed by treatment with methyl iodide in the presence of
sodium hydride,
in tetrahydrofuran, to give 6-[(1-methyl-5-carbomethoxyindole)3-yl]-7H-
pyrrolo[2,3-
b]pyrimidine (13);
(iv) treatment of (13) with aqueous methanolic potassium hydroxide at reflux
to give 6-[(1-
methyl-5-carboxyindole)3-yl]-7H-pyrrolo[2,3-b]pyrimidine (14); and
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(v) coupling of (14) with 2-hydroxy-1,1-dimethylethylamine in the presence of
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and
diisopropylethylamine in dimethylformamide to give 1-methyl-3-(7H-pyrrolo[2,3-
b]pyrimidine-6-yl)-1H-indole-5-carboxylic acid (2-hydroxy-1,1-dimethyl-ethyl)-
amide (III).
EXAMPLE 3
2-I f 5-Methoxy-3-(7H-p.~rrolo [2,3-b]pyrimidine-6-yl)-indol-1-yl]-1-morphol
in-4-X11-ethanone
O
1 . _0H ~N 2 . 3 .
The compound of formula (I), wherein R is 2 ~ , R ~s -OMe, R is -H, the group
~O
H is attached to the 3 position of the indole ring and the group R2 is
attached to
the 5 position of the indole ring, represented by formula (IV):
OMe
r ~~
~N N~N
H
O
N
~O
(
is prepared as shown in the following scheme:
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- 36-
OH
HO-g'
\ \ OMe
N I /
is boc
(11 ) (15) (16)
OMe
O
I ~.gr
N \ \ N
'N N
H
(17)
O N
O
(IV)
(i) coupling of 6-iodo-7-(toluene-4-sulfonyl)-7H-pyrrolo[2,3-b]pyrimidine (11)
1-tert-
butyloxycarbonyl-5-methoxyindole-3-boronic acid (15) in the presence of
tetrakis(triphenylphosphine)palladium(0) and sodium bicarbonate, in aqueous
dimethylformamide at about reflux temperature and removal of the tent-
butyloxycarbonyl
protecting group, to give 6-[(5-rnethoxyindole)-3-yl]-7-(toluene-4-sulfonyl)-
7H-pyrrolo[2,3-
b]pyrimidine ( 16);
(ii) ) treatment of ( I6) with aqueous methanolic potassium hydroxide at
reflux to give 6-[(5-
methoxyindole)3-yl]-7H-pyrrolo[2,3-b]pyrimidine (17); and
(iii) reaction of (17) with sodium hydride in dimethylformamide followed by
reaction with 2-
bromoacetic acid morpholineamide to give 2-{[5-methoxy-3-(7H-pyrrolo[2,3-
b]pyrimidine-6-
yl)-indol-1-yl]-1-morpholin-4-yl}-ethanone (IV).
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EXAMPLE 4
The compound of formula (I), wherein Rl is -CH2CF3, R2 is -OMe, R3 is-CN, the
group
is attached to the 3 position of the indole ring arid the group R2 is attached
to
H
the 5 position of the indole ring, represented by formula (VII):
H
CF3
(VII)
is prepared as shown in the following scheme:
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NaOEt HO OEt
OEt OEt CN EfOH
~ ' + NC C02Et --- ~ ~
Et0- vBr Nal Et0_ v 'COzEt S N~
(18) (1g) K (20) H2N"NHZ HS N NHZ
(21)
Br Br Br O
N~ \ E FDA, THF ~ ~ TosCl ~ ~ E POBr3 H
I
I ~ I NaH, THF
~N~ N \ N H
Tos Tos
(25) (24) (23) (22)
Me Me Me0
Br Zn(CN)2
CF3CH21 Br / Pd N /
_ ~ DMA, 140 C
N i ~ ~ ~ N, \ w ---~ N i I ~ w
I ~ NH NaH, THF I ~ N
N ~ N N
t N \
Tos Tos Tos
F3 ~ Fa
(26) (27) (28)
KOH 5N, MeOH
Me0
N
i
\N
N
F3
(VII)
(i) reaction of (18) and (19) in the presence of potassium carbonate and
sodium iodide to give
(20);
(ii) reaction of (20) with thiourea in the presence of sodium ethoxide in
ethanol to give (21 );
(iii) cyclisation of (21) by heating in toluene at about reflux to give (22);
(iv) reaction of (22) with phosphorus oxybromide to give 4-bromo-7H-
pyrrolo[2,3-
b]pyrimidine (23);
(v) reaction of (23) with 4-toluenesulfonyl chloride in the presence of
tetrabutylammonium
sulfate and aqueous sodium hydroxide in toluene to give (24);
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(vi) treatment of (24) with lithium diisopropylamide in tetrahydrofuran, at
about -78°C,
followed by reaction of the resulting anion with iodine to give (25);
(vii) coupling of (25) with 1-tert-butyloxycarbonyl-S-methoxyindole-3-boronic
acid in the
presence of tetrakis(triphenylphosphine)palladium(0) and sodium bicarbonate,
in aqueous
dimethylformamide at about reflux temperature and removal of the tent-
butyloxycarbonyl
protecting group, to give 4-bromo-6-[(5-methoxyindole)3-yl]-7-(toluene-4-
sulfonyl)-7H-
pyrrolo[2,3-b~pyrimidine (26);
(viii) reaction of (26) with sodium hydride in tetrahydrofuran followed by
reaction with 2-
trifluoro-iodoethane to give (27);
(ix) reaction of (27) with zinc cyanide in the presence of palladium in N'N-
dimethylaniline at
about 140°C to give (28); and
(x) treatment of (28) with aqueous methanolic potassium hydroxide at reflux to
give (VII).
EXAMPLE 5
O Me Me
The compound of formula (I), wherein Rl is -CH3, R2 is -OMe, R3 is ~N.~CH OH ~
the
H 2
group is attached to the 3 position of the indole ring and the group R2 is
H
attached to the 5 position of the indole ring, represented by formula (IX):
CH2OH
w
N~~N
N H \
(IX)
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is prepared as shown in the following scheme;
Me0 Me0 Me
Br / CO, MeOH COzMe /
/ 1 ~ Pd, reflux
N~ \ ~ Mel,~ N~ \ ~ ~ Ni I \ w
\N I N ~ NH \N I ~ ~ N\ \N N ~ N\
Tos Tos Tos
(29) (30)
(26)
KOH 5N, MeOH
Me0
HzN~ CO~H /
OIH Ni ~
I ~ N\
HATU ~N H
DIPEA
DMF
(3~)
(!x)
(ii) reaction of (26) with sodium hydride in tetrahydrofuran followed by
reaction with methyl
iodide to give (29);
(ii) reaction of (29) with carbon monoxide in the presence of palladium in
methanol at reflux to
give (30);
(iii) treatment of (30) with aqueous methanolic potassium hydroxide at reflux
to give (31); and
(iv) coupling of (31) with 2-hydroxy-I, I-dimethylethylamine in the presence
of
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and
diisopropylethylamine in dimethylformamide to give (IX).
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EXAMPLE 6
O
1 -CH ~N
The compound of formula (I), wherein R ><s 2 ~ , R is -OMe, R is
~O
O
/CH2~CH~NHMe . . .
the group H is attached to the 3 pos>tton of the mdole
ring and the group R~ is attached to the 5 position of the indole ring,
represented by formula (V):
H
O N
~O
(V)
is prepared as shown in the following scheme:
Ni --~ NI~ ~z Ni
N I N ~ NH Heck ~N~\ ~ N ' \ ~ N'
~H N'~
1
Tos Tos Tos H
(32) (33)
(25)
KOH 5N, MeOH
CI- y 0 CONHMe Me0 COzH Me
~N
/ ~ /
0 N~ \ ~ N,
w ~ I ~N
NaH, THF ~N \ NCH HATUz N H
DIPEA
DMF
(35) (34)
(V)
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(i) reaction of (26) with methyl acrylate in the presence of palladium
acetate, triphenyl
phosphine and triethylamine at about 110°C to give (32);
S (ii) hydrogenation of (32) in the presence of palladium on carbon to give
(33);
(iii) treatment of (33) with aqueous methanolic potassium hydroxide at reflux
to give the acid
(34);
(iv) coupling of (34) with methylamine in the presence of O-(7-azabenzotriazol-
1-yl)-I,1,3,3-
tetramethyluronium hexafluorophosphate and diisopropylethylamine in
dimethylformamide to
give (35); and
(v) alkylation of (35) with 4-(2-chloroacetyl)morpholine in the presence of
sodium hydride, in
1S dimethylformamide to give (V).
EXAMPLE 7
O
1 . _CH ~N 2 . 3 .
The compound of formula (I), wherein R is 2 ~ , R is -OMe, R is
~O
the group is attached to the 3 position of the indole ring and
N
H
the group R2 is attached to the 5 position of the indole ring, represented by
formula (VI):
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~ N Me0
N~
N~~N
N H
' \N~
~O
(VI)
is prepared as shown in the following scheme:
Me0 ~ ~ N ~ ~ N Me0
Br ,/ / /
N~ I ~ ~ I HO'B~OH N,~ I
N NH N ~NH
Tos DMF reflux Tos
aq. Na2C03
Pd(PPh3)4 (36)
(26)
' ICON 5N, MeOH
O
N
~N Me0
'/
I
NaH, THF Ni
N ~ ~ NH
H
(VI)
(37)
(i) coupling of (26) with pyridine-3-boronic acid in the presence of
tetrakis(triphenylphosphine)palladium(0) and sodium bicarbonate, in aqueous
IO dimethylformarnide at about reflux temperature to give 4-(pyridin-3-yl)-6-
[(5-
methoxyindole)3-yl]-7-(toluene-4-sulfonyl)-7H-pyrrolo[2,3-b]pyrimidine (36);
(ii) treatment of (36) with aqueous methanolic potassium hydroxide at reflux
to give (37,
Example 9); and
IS
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(iii) alkylation of (37, Example 9) with 4-(2-chloroacetyl)morpholine in the
presence of sodium
hydride, in dimethylformamide to give 2-[5-methoxy-3-(4-(pyridin-3-yl)-7H-
pyrrolo[2,3-
b]pyrimidin-6-yl)-indol-1-yl]-1-morpholin-4-yl-ethanone (VI).
EXAMPLE 8
The compound of formula (I), wherein R1 is -CH~CH3, R~ is -OMe, R3 is - 0 ,
the group
is attached to the 3 position of the indole ring and the group R~ is attached
to
H
the 5 position of the indole ring, represented by formula (VIII):
(VIII)
is prepared as shown in the following scheme:
Me0 Me0
Br
Br
Etl, NaH Ni
~N I \ ~ NH ~ N ~ , \ N
Tos Tos
(26) (38)
C~~
CO, Me0 CO, Me0
NJ / I NJ / I
N~ ~ KOH 5N, MeOH N~
~N ~ \ ~ N N ~ ~ N
Tos
(vup
(39)
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(i) alkylation of (26) with ethyl iodide in the presence of sodium hydride, in
dimethylformamide to give (3 8);
(ii) reaction of (38) with morpholine in a microwave oven at about
200°C in a,a,a-
trifluorotoluene to give (39); and
(iii) treatment of (39) with aqueous methanolic potassium hydroxide at reflux
to give (VIII).
EXAMPLE 9
6-(S-Methoxy-1 H-indol-3-yl)-4-pin-3-yl-7H-pyrrolo [2,3-d]~yrimidine
EI
a
A solution of 6-iodo-7-[(4-methylphenyl)sulfonyl]-4-pyridin-3-yl-7H-
pyrrolo[2,3-d]pyrimidine
[260mg, Reference Example 1] and I-tee~t-butyl-carboxyl-5-methoxy-1H indole-3-
boronic acid
[178mg, Reference Example 12] in dimethylformamide (I OmL) was treated with
palladium tetrakis
triphenyl phosphine (l3mg) and sodium hydrogen carbonate (8mg). The reaction
mixture was stirred at
reflux for 2 hours and allowed to cool to room temperature. The solution was
evaporated under
reduced pressure and the residue partitioned between water and ethyl acetate.
The organic phase was
separated, then dried over magnesium sulfate and then evaporated under reduced
pressure. The residue
was subjected to flash column chromatography on silica eluting with a mixture
of ethyl acetate and
methanol (95:5, v/v) to give 6-(5-Methoxy-1H-indol-3~1~4-pyridin-3 yl-7H-
pyrrolo[2,3-d]'pyrimidine
(20mg) as an amorphous solid. MS: 342 [MH]+, LCMS (Method A) RT = 2.57
minutes.
EXAMPLE 10
4-Methoxy-6-(5-methoxy-I-methyl-1H-indol-3-~)-7H-pyrrolo[2,3-d]pyrimidine
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Me0
pie
H
A solution of 4-methoxy-6-(5-methoxy-1-methyl-1H-indol-3-yl)-7-[(4-
methylphenyl)sulfonyl]-7H-
pyrrolo[2,3-d]pyrimidine [361mg, Reference Example 4] in methanol (20mL) was
treated with
potassium hydroxide (1.53g). The reaction mixture was stirred for 16 hours at
room temperature and
refluxed 1 hour. The solution was evaporated under reduced pressure and the
residue partitioned
between water and ethyl acetate. The organic phase was separated, then dried
over magnesium sulfate
and evaporated under reduced pressure. The residue was triturated with diethyl
ether to give
4-methoxy-6-(5-methoxy-1-methyl-1H-indol-3-~~7H-p r~olo[2,3-d]ipyrimidine
(155mg) as a solid
m.p. = 184°C. MS: 309 [MH]+.
EXAMPLE 11
4-Methoxy-6-(5-methoxy-1 H-indol-3-~~7H-pyrrolo[2,3-d]pyrimidine
Me0
H
A solution of 4-methoxy-6-(5-methoxy-1H-indol-3-yl)-7-[(4-
methylphenyl)sulfonyl]-7H-pyrrolo[2,3-
d]pyrimidine [448mg, Reference Example 5] in methanol (lSmL) was treated with
potassium
hydroxide (1.96g). The reaction mixture was stirred for 2 hours at room
temperature and the solvent
was evaporated under reduced pressure. The residue was partitioned between
water and ethyl acetate.
The organic phase was separated, then dried over magnesium sulfate and then
evaporated under
reduced pressure. The residue was subjected to flash column chromatography on
silica eluting with a
mixture of ethyl acetate and cyclohexane (80:20, v/v) to give 4-methoxy-~5-
methoxy-1 H-indol-3-yl~-
7H-p~rrolo[2,3-d]pyrimidine (320mg) as a yellow solid m.p. > 260°C. MS:
295 [MH]~.
EXAMPLE 12
4-(5-Methoxy-1H indol-3-yl)-6-(5-methoxy-1-methyl-1H indol-3- ly_)-7H p
rrolo[2,3-d]~pyrimidine
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a
de
H
A solution of 4-(5-methoxy-I-[(4-methylphenyl)sulfonyl]-1H indol-3-yl)-6-(5-
methoxy-I-methyl-IH
indol-3-yl)-7-[(4-methylphenyl)sulfonyl]-7H pyrrolo[2,3-d]pyrimidine [93mg,
Reference Example 9]
in methanol (SmL) was treated with potassium hydroxide (249mg). The reaction
mixture was stirred
for I6 hours at room temperature. The solution was evaporated under reduced
pressure and the residue
partitioned between ethyl acetate and water. The organic phase was separated,
then dried over
magnesium sulfate and then evaporated under reduced pressure. The residue was
purified by HPLC to
give 4-(5-methoxy-1H indol-3-~)-~5-methoxy-1-methyl-IH indol-3-~)-7H
p~roloj2,3-d~pyrimidine
(9mg) as a gum. MS: 424 [MH]+. LCMS (Method B) RT = 3.15 minutes.
REFERENCE EXAMPLE 1
6-Iodo-7-[(4-meth~phenyl)sulfon~]-4-pyridin-3 yl-7H-pyrroloL,3-d~pyrimidine
To a solution of 7-[(4-methylphenyl)sulfonyl]-4-pyridin-3-yl-7H-pyrrolo[2,3-
d]pyrimidine [1g,
Reference Example 2] in tetrahydrofuran (20mL) at -78°C was added drop
wise a solution of butyl
lithium in hexane (2mL, 1.6M) under inert atmosphere. The solution was stirred
at that temperature for
1.5 hour and iodine (796mg) was added. The reaction mixture was stirred at -
78°C for another 1 hour
and allowed to reach room temperature. The reaction mixture was partitioned
between ethyl acetate
and aqueous sodium sulfite solution. The organic phase was separated, then
dried over magnesium
sulfate and then evaporated under reduced pressure. The residue was subjected
to flash column
chromatography on silica eluting with a gradient of ethyl acetate and
cyclohexane (50:50, to 100, v/v)
to give the title compound (260mg) as an amorphous solid. MS: 477 [MH]+. LCMS
(Method B) RT =
3.26 minutes.
REFERENCE EXAMPLE 2
7-~ (4-Methylphenyl)sulfon~]-4-pyridin-3-~H-pyrrolo[2,3-d]pyrimidine
A solution of 4-chloro-7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo[2,3-
d]pyrimidine [4g, Reference
Example 3] and diethyl-3-pyridyl-borane (2.Ig) in tetrahydrofuran (180mL) was
treated with palladium
tetrakis triphenylphosphine (0.65g) and potassium carbonate (3.59g). The
solution was stirred at reflux
for 24 hours and evaporated under reduced pressure. The residue was
partitioned between ethyl acetate
and brine. The organic phase was separated, than dried over magnesium sulfate
and then evaporated
under reduced pressure. The residue was subjected twice to flash column
chromatography on silica
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eluting with a mixture of ethyl acetate and methanol (90:10, v/v) and a
mixture of ethyl acetate and
cyclohexane (50:50, v/v) to give the title compound (2.5g) as an amorphous
solid. MS: 351 [MH]+.
LCMS (Method B) RT = 3.05 minutes.
REFERENCE EXAMPLE 3
4-Chloro-7-[(4-methylphen~)sulfon~)-7H-p rrolo[2,3-d)pyrimidine
A solution of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (Reference: Gerster, John
F. ; Hinshaw, Barbara
C.; Robins, Roland I~.; Townsend, Leroy B. Study of electrophylic substitution
in the pyrrolo[2,3-
d]pyrimidine ring. J. Heterocycl. Chem. (1969), -(2), 207-213) (20g) andpa~~a-
toluene sulfonylchioride
(28.6g) in toluene (1L) was treated with a solution of sodium hydroxide (50g)
in water (800mL), and
tetrabutyl ammonium sulfate (462mg). The solution was stirred vigorously at
room temperature for 2
hours and partitioned between ethyl acetate and brine. The organic phase was
separated, then dried
over magnesium sulfate and then evaporated under reduced pressure. The residue
was subjected to
flash column chromatography on silica eluting with a gradient of ethyl acetate
and cyclohexane (50:50
to 80:20, v/v) to give the title compound (2.5g) as a solid m.p. =
143°C. LCMS (Method B) RT = 2.78
minutes.
REFERENCE EXAMPLE 4
4-Methoxy_-6-(5-methoxy-1-methyl-1 H-indol-3-yl)-7-[(4-methyl~henyl)sulfonyl]-
7H-
p rroloj2,3d]pyrimidine
To a solution of 4-methoxy-6-(5-methoxy-1H-indol-3-yl)-7-[(4-
methylphenyl)sulfonyl]-7H-
pyrrolo[2,3-d]pyrimidine [448mg, Reference Example 5] in dimethylformamide
(20mL) was added the
sodium hydride (44mg, 60% dispersion in oil) and methyl iodide-(156mg) under
inert atmosphere. The
solution was stirred for 1 hour at room temperature and the solvent was
evaporated under reduced
pressure. The residue was partitioned between water and ethyl acetate. The
organic phase was
separated, then dried over magnesium sulfate and then evaporated under reduced
pressure. The residue
was subjected to flash column chromatography on silica eluting with a mixture
of ethyl acetate and
cyclohexane (30:70, v/v) to give the title com ound (260mg) as an amorphous
solid. MS: 464 [MH]+.
LCMS (Method B) R-r = 4.39 minutes.
REFERENCE EXAMPLE 5
4-Methoxy-6-(5-methox~ 1 H-indol-3-vl)-7-f (4-methvlphenvl)sulfonvll-7H-
pvrrolof2,3-dlnvrimidine
A solution of 6-iodo-4-methoxy-7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo[2,3-
d]pyrimidine [1.98g,
Reference Example 6] and 1-test-butyl-carboxyl-5-methoxy-1H indole-3-boronic
acid [1.26g,
Reference Example 12] in dimethylformarnide (40mL) was treated successively
with a saturated
aqueous solution of sodium bicarbonate (IOmL) and palladium tetrakis
triphenylphosphine (165mg)
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The reaction mixture was stirred at reflux for 3 hours and the solvent was
evaporated under reduced
pressure. The residue was partitioned between ethyl acetate and water. The
organic phase was
separated, then dried over magnesium sulfate and then evaporated under reduced
pressure. The residue
was subjected to flash column chromatography on silica eluting with a mixture
of ethyl acetate and
cyclohexane (50:50, v/v) to give the title compound (1.8g) as a grey solid.
m.p.= 131°C. MS: 450
[MH]+.
REFERENCE EXAMPLE 6
6-Iodo-4-methoxy-7-[(4-meth~phen~)sulfonyl]-7H-p r~[2,3-d]pyrimidine
To a solution of 4-methoxy-7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo[2,3-
d]pyrimidine [2.23g,
Reference Example 7] in tetrahydrofuran (35mL) at -78°C was added drop
wise a solution of butyl
lithium in hexane (SmL, 1.6M) under inert atmosphere. The solution was stirred
at -70°C for 1 hour
and iodine (2.05g) was added. The reaction mixture was stirred at -70°C
for another 1 hour, allowed to
reach room temperature and partitioned between ethyl acetate and aqueous
sodium sulfite solution. The
1S organic phase was separated, then dried over magnesium sulfate and then
evaporated under reduced
pressure to give the title compound (2.64g) as an amorphous solid. MS: 430
[MH]+. LCMS (Method
B) RT = 4.15 minutes.
REFERENCE EXAMPLE 7
4-Methoxy-7-[(4-methxlphenysulfonyl]-7H-pyrrolo~2,3-d]p~imidine
A solution of 4-rnethoxy-7H-pyrrolo[2,3-d]pyrimidine [1.2g, Reference Example
8] and para-toluene
sulfonylchloride (1.77g) in toluene (60mL) was treated with a solution of
sodium hydroxide (3.2g) in
water (30mL), and tetrabutyl ammonium sulfate (27mg). The solution was stirred
vigorously at room
temperature for 4 hours and partitioned between ethyl acetate and brine. The
organic phase was
2S separated, then dried over magnesium sulfate and then evaporated under
reduced pressure. The residue
was subjected to flash column chromatography on silica eluting with a gradient
of ethyl acetate and
cyclohexane (50:50 to 80:20, v/v) to give the title compound (2.23g) as an
amorphous solid. MS: 304
[MH]+. LCMS (Method B) RT = 3.88 minutes.
REFERENCE EXAMPLE 8
4-Methox -~p ry rolo[2,3-d]pyrimidine
To a solution of sodium methoxide prepared by adding portion wise the sodium
(2g) in methanol
(100mL) under an inert atmosphere, was added 4-chloro-7H-pyrrolo[2,3-
d]pyrimidine (Reference:
Gerster, John F. ; Hinshaw, Barbara C.; Robins, Roland K.; Townsend, Leroy B.
Study of electrophylic
3S substitution in the pyrrolo[2,3-d]pyrimidine ring. J. Heterocycl. Chem.
(1969), -(2), 207-13.) (3.5g).
The solution was stirred at 65°C for 16 hours and then partitioned
between ethyl acetate and brine.
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The organic phase was separated, then dried over magnesium sulfate and then
evaporated under
reduced pressure. The residue was subjected to flash column chromatography on
silica eluting with a
mixture of ethyl acetate and cyclohexane (50:50, v/v) to give the title
compound (1.2g) as an
amorphous solid. MS: 1 SO [MH]+. LCMS (Method B) RT = 2.39 minutes.
REFERENCE EXAMPLE 9
4-(S-Methoxy _1-'[(4-methXlphenyl)sulfonyl]-1H indol-3-yl)-6-(S-methoxy-I-
methyl-IH indol-3- Ice)-7-
[ 4-meth Iy_phen,Kl)sulfon~]-7H p r~rolo[2,3-d]pyrimidine
To a solution of 4-(5-methoxy-1-[(4-methylphenyl)sulfonyl]-1H indol-3-yl)-6-(S-
methoxy-1H indol-3-
yl)-7-[(4-methylphenyl)sulfonyl]-7H pyrrolo[2,3-d]pyrimidine [270mg, Reference
Example 10] in
dimethylformamide (IOmL) was added the sodium hydride (lOmg, 60% dispersion in
oil) and methyl
iodide (0.02SmL) under inert atmosphere. The solution was stirred for 16 hours
at room temperature
and the solvent was evaporated under reduced pressure. The residue was
partitioned between water and
ethyl acetate The organic phase was separated, then dried over magnesium
sulfate and then evaporated
under reduced pressure. The residue was subjected to flash column
chromatography on silica eluting
with a mixture of ethyl acetate and cyclohexane (SO:SO, v/v) to give the title
compound (93mg) as an
amorphous solid. MS: 732 [MH]+. LCMS (Method B) RT = 4.68 minutes.
REFERENCE EXAMPLE 10
4~5-Methoxy-1-f(4-methylphe~l)sulfonyl]-1H indol-3-yl)-6-(S-methoxy-IH indol-3-
yl)-7-[(4-
methylphen~)sulfony~-7H p~rrolo[2,3-d]p~imidine
A solution of 4-chloro-6-iodo-7-[(4-methylphenyl)sulfonyl]-7H pyrrolo[2,3-
d]pyrimidine [1.72g,
Reference Example 11] and 1-test-butyl-carboxyl-5-methoxy-1H indole-3-boronic
acid [1.26g,
Reference Example 12] in dimethylformamide (36.SmL) was treated successively
with a saturated
aqueous solution of sodium bicarbonate (9.lmL) and palladium tetrakis
triphenylphosphine (0.3g). The
reaction mixture was stirred at reflux for 2 hours and the solvent was
evaporated under reduced
pressure. The residue was partitioned between ethyl acetate and water. The
organic phase was
separated, then dried over magnesium sulfate and then evaporated under reduced
pressure. The residue
was subjected to flash column chromatography on silica eluting with a mixture
of ethyl acetate and
cyclohexane (30:70, v/v) to give the title compound (270mg) as a gum. MS: 718
[MH]+. LCMS
(Method B) RT = 4.44 minutes.
REFERENCE EXAMPLE 11
4-Chloro-6-iodo-7-[(4-meths 1p Zenyl)sulfon~rl -1 7H uyrrolo[2,3-d]pyrimidine
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To a solution of 4-chloro-7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo[2,3-
d]pyrimidine [5.4g, Reference
Example 3], in tetrahydrofuran (96mL) at-78°C was added drop wise a
solution of butyl lithium in
hexane (12.1mL, 1.6M) under inert atmosphere. The solution was stirred at-
78°C for 3 hours and
iodine (8.9g) was added. The reaction mixture was stirred at -78°C for
2 hours, and allowed to reach
room temperature. The reaction mixture was partitioned between ethyl acetate
and aqueous sodium
sulfite solution, dried over magnesium sulfate and the solvent was evaporated
under reduced pressure.
The residue was subjected to flash column chromatography on silica eluting
with a gradient of ethyl
acetate and cyclohexane (50:50, to 100, v/v) to give the title compound
(1.52g) as an amorphous solid.
MS: 434 [MH]+. LCMS (Method B) RT = 4.26 minutes.
REFERENCE EXAMPLE 12
1-tent-butyl-carbox,~-5-methoxy-1H indole-3-boronic acid
A stirred solution of 3-bromo-5-methoxy-indole-1-carboxylic acid, tart-butyl
ester [50g, Reference
Example 13)] in tetrahydrofuran (800 mL), under nitrogen, was treated with
tributylborate (49.5 mL)
then cooled to -100°C and then treated with a solution of n-
butyllithium in hexanes (94 mL, 2.5M)
whilst keeping the temperature below -90°C. Once the addition was
complete the mixture was allowed
to warm slowly to room temperature over 1 hour and quenched by the addition of
ice (1 Og). The
organics were removed under reduced pressure and the residue was partitioned
between ethyl acetate
(500 mL) and water (400 mL). The organic layer was dried over magnesium
sulfate and then
evaporated to afford the title compound as a cream coloured solid (28g). MS:
314 [M+Na]+. LCMS
(Method C) RT = 4.07 minutes.
REFERENCE EXAMPLE 13
3-Bromo-5-methoxy-indole-I-carboxylic acid, tent-butyl ester
A solution of 5-methoxyindole (10g) in dry dimethylformamide (150 mL) at
ambient temperature was
treated with bromine (4 mL) dropwise ensuring the temperature did not rise
above 30°C. The mixture
was treated immediately with triethylamine (28 mL) and 4-dimethylaminopyridine
(0.5g) followed by a
solution of di-tart-butyldicarbonate (I8g) in dry dimethylformamide (80 mL)
and stirring was
continued for a further 4 hours. The reaction mixture was evaporated and the
residue was partitioned
between ethyl acetate (250 mL) and water (200 mL). The aqueous layer was
extracted with ethyl
acetate (100 mL). The combined organic phases were washed with water (100 mL),
then with brine
(100 mL), then dried over magnesium sulfate and then evaporated. The residue
was subjected to flash
column chromatography on silica eluting with a mixture of pentane and ethyl
acetate (19/I, v/v) to give
the title compound (23.4g) as a colourless solid, m.p. 111-112°C.
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IN VITRO TEST PROCEDURES FOR SYK
1. Inhibitory effects of compounds on Syk kinase
Inhibitory effects of compounds on Syk kinase were determined using a time-
resolved fluorescent
assay.
The catalytic domain of Syk kinase (residues A340-N63S ) was expressed as a
fusion protein in yeast
cells and purified to homogeneity. Kinase activity was determined in SOmM Tris-
HCl buffer pH 7.0
containing SOmM NaCI, SmM MgCl2, SmM MnCl2, lp,M adenosine triphosphate and
lOp,M synthetic
peptide Biotin-( ~i-Alanine)3-DEEDYEIPP-NH2. Enzyme reactions were terminated
by the addition of
buffer containing 0.4M KF, 133mM EDTA, pH 7.0, containing a streptavidin-XL66S
conjugate and a
monoclonal phosphospecfic antibody conjugated to a europium cryptate (Eu-K).
Features of the two
fluorophores, XL-665 and Eu-K are given in G.Mathis et al., Anticancer
Research, 1997, 17, pages
3011-3014. The specific long time signal of XL-665, produced only when the
synthetic peptide is
phosphorylated by Syk, was measured on an LrL Biosystems Analyst AD microplate
reader. Inhibition
of syk activity with compounds of the invention was expressed as percentage
inhibition of control
activity exhibited in the absence of test compounds. Particular preferred
compounds of the invention
inhibit syk activity with ICSOs in the range 100 micromolar to 100 nanomolar.
Especially preferred
compounds of the invention inhibit syk activity with ICSOs in the range 1
micromolar to 100
nanomolar.
2. Antigen-induced dee~,ranulation of Rat Basophilic leukemia (RBL cells
2.1 Cell culture, labelling of RBL-2H3 cells and performance of assay.
RBL-2H3 cells are maintained in T7S flasks at 37°C and S%C02, and
passaged every 3-4 days. To
harvest cells, S ml trypsin-EDTA is used to rinse the flask once, then S ml
trypsin is added to each
flask, and incubated at room temperature for 2 minutes. Cells are transferred
to a tube with 14m1
medium, spun down at 1100 rpm RT for S minutes and resuspended at 2x10S/ml.
Cells are sensitized
by adding 1 p.1 of DNP-specific IgE to every I 0 ml of cells. 200p.I of cells
are added to each well of a
flat-bottom 96 well plate (40,000 cells/well), and the plate incubated
overnight at 37°C and S%C02.
The next day compounds are prepared in 100% DMSO at l OmM. Each compound is
then diluted
1:100 in assay buffer and then diluted further in 1 % DMSO-assay buffer to
obtain final concentrations
of 0.03-30p,M. 80p,1 assay buffer is added to each well, followed by l Op.l of
diluted compound.
Incubation follows for 5 minutes. lOp,l of DNP-HSA (I OOng/ml) is added to
each well and incubated
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at 37°C (no C02) for 30 minutes. As one control, 1% DMSO alone (no
compound) is added to a set of
wells to determine total release. As another control, add buffer instead of
DNP-HSA to another set of
wells to determine the assay background. After the 30 minutes incubation, the
supernatants are
transferred to a new 96-well plate. Add SOp,I supernatant to each well of an
assay plate. Add I OOp,I of
substrate solution to each well and incubate at 37°C for 90 minutes.
Add SOp.I of 0.4 M glycine
solution to stop the reaction and the plate is read at 405 nm on a Molecular
Devices SpectraMax 250
plate reader.
2.2 Calculation of results
(i) The mean ~ SD of each set of triplicate wells was calculated.
(ii) Maximum response was the positive control wells containing antigen
(100ng/mL) but no
compound.
(iii) Minimum response was the control wells containing buffer (no antigen)
and no compound.
(iv) Using these values as the maximum (100%) and minimum (0%) values
respectively, the
experimental data was calculated to yield a percentage of the maximum response
(designated
control).
(v) A dose response curve was plotted and the IC50 of the compound was
calculated using Prism
GraphPad software and nonlinear least squares regression analysis.
Compounds of the invention inhibit antigen-induced degranulation of Rat
Basophilic leukemia (RBL)
cells with ECSOs in the range 100 micromolar to 1 micromolar.
