U.S. patent application number 15/782214 was filed with the patent office on 2018-02-01 for heteroaromatic compounds as btk inhibitors.
The applicant listed for this patent is Boehringer Ingelheim International GmbH. Invention is credited to Joerg BENTZIEN, Todd BOSANAC, Michael Jason BURKE, Ryan Michael FRYER, Eric Thomas LARSON, Wang MAO, Bryan Patrick MCKIBBEN, Yue SHEN, Fariba SOLEYMANZADEH, Matt Aaron TSCHANTZ.
Application Number | 20180030037 15/782214 |
Document ID | / |
Family ID | 58737849 |
Filed Date | 2018-02-01 |
United States Patent
Application |
20180030037 |
Kind Code |
A1 |
BOSANAC; Todd ; et
al. |
February 1, 2018 |
HETEROAROMATIC COMPOUNDS AS BTK INHIBITORS
Abstract
The present invention encompasses compounds of the formula (I)
##STR00001## wherein the groups R.sub.1, Cy and Y are defined
herein, which are suitable for the treatment of diseases related to
BTK, and processes for making these compounds, pharmaceutical
preparations containing these compounds, and their methods of
use.
Inventors: |
BOSANAC; Todd; (New Milford,
CT) ; BENTZIEN; Joerg; (White Plains, NY) ;
BURKE; Michael Jason; (Newtown, CT) ; FRYER; Ryan
Michael; (Brewster, NY) ; LARSON; Eric Thomas;
(Bethel, CT) ; MAO; Wang; (Milford, CT) ;
MCKIBBEN; Bryan Patrick; (New Milford, CT) ; SHEN;
Yue; (Ridgefield, CT) ; SOLEYMANZADEH; Fariba;
(Danbury, CT) ; TSCHANTZ; Matt Aaron; (Newtown,
CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Boehringer Ingelheim International GmbH |
Ingelheim am Rhein |
|
DE |
|
|
Family ID: |
58737849 |
Appl. No.: |
15/782214 |
Filed: |
October 12, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15379745 |
Dec 15, 2016 |
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15782214 |
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62431008 |
Dec 7, 2016 |
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62268278 |
Dec 16, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 27/02 20180101; A61P 9/00 20180101; A61P 11/02 20180101; A61P
17/04 20180101; C07D 403/14 20130101; A61P 1/04 20180101; A61P
19/02 20180101; A61P 13/12 20180101; A61P 17/00 20180101; A61P
29/00 20180101; C07D 401/14 20130101; A61P 11/06 20180101; A61P
7/00 20180101; A61P 37/02 20180101; A61P 37/08 20180101; A61P 35/02
20180101; A61P 37/06 20180101; A61P 37/00 20180101 |
International
Class: |
C07D 403/14 20060101
C07D403/14; C07D 401/14 20060101 C07D401/14 |
Claims
1-23. (canceled)
24. A compound of the formula (I) ##STR00263## wherein: Cy is
chosen from ##STR00264## each R.sub.1 is independently chosen from
hydrogen or methyl; R.sub.2 is L--Ar, wherein Ar is phenyl or
pyridinyl and each is optionally substituted by one or more of
halogen, halo C.sub.1-4 alkyl, C.sub.1-4 alkyl, C.sub.1-4 alkoxy,
--CN, halo C.sub.1-4 alkoxy, or cycloalkyl; L is --(CH.sub.2)-- or
--(CHCH.sub.3)--; Y is C.sub.6-C.sub.8 spirocycle containing 1 ring
nitrogen atom, and is substituted by one R.sub.3; R.sub.3 is chosen
from ##STR00265## and each R.sub.4 is independently chosen from
hydrogen, C.sub.1-4 alkyl, or C.sub.3-4 cycloalkyl; each group
defined above for R.sub.1-R.sub.4 and Y can be where possible
partially or fully halogenated; or a pharmaceutically acceptable
salt or hydrate thereof.
25. The compound according to claim 24, wherein Y is chosen from
##STR00266## or a pharmaceutically acceptable salt or hydrate
thereof.
26. The compound according to claim 1, wherein Cy is ##STR00267##
or a pharmaceutically acceptable salt or hydrate thereof.
27. The compound according to claim 24 and wherein Cy is
##STR00268## or a pharmaceutically acceptable salt or hydrate
thereof.
28. The compound according to claim 24 chosen from: ##STR00269##
##STR00270## ##STR00271## ##STR00272## ##STR00273## ##STR00274##
##STR00275## ##STR00276## ##STR00277## ##STR00278## ##STR00279##
##STR00280## ##STR00281## ##STR00282## ##STR00283## ##STR00284##
##STR00285## or the pharmaceutically acceptable salts or hydrates
thereof.
29. The compound according to claim 28 of the formula: ##STR00286##
or the pharmaceutically acceptable salt or hydrate thereof.
30. The compound according to claim 28 of the formula: ##STR00287##
or the pharmaceutically acceptable salt or hydrate thereof.
31. The compound according to claim 28 of the formula: ##STR00288##
or the pharmaceutically acceptable salt or hydrate thereof.
32. The compound according to claim 28 of the formula: ##STR00289##
or the pharmaceutically acceptable salt or hydrate thereof.
33. The compound according to claim 28 of the formula: ##STR00290##
or the pharmaceutically acceptable salt or hydrate thereof.
34. The compound according to claim 28 of the formula: ##STR00291##
or the pharmaceutically acceptable salt or hydrate thereof.
35. The compound according to claim 28 of the formula: ##STR00292##
or the pharmaceutically acceptable salt or hydrate thereof.
36. The compound according to claim 28 of the formula: ##STR00293##
or the pharmaceutically acceptable salt or hydrate thereof.
37. The compound according to claim 28 of the formula: ##STR00294##
or the pharmaceutically acceptable salt or hydrate thereof.
38. The compound according to claim 28 of the formula: ##STR00295##
or the pharmaceutically acceptable salt or hydrate thereof.
39. The compound according to claim 28 of the formula: ##STR00296##
or the pharmaceutically acceptable salt or hydrate thereof.
40. The compound according to claim 28 of the formula: ##STR00297##
or the pharmaceutically acceptable salt or hydrate thereof.
41. The compound according to claim 28 of the formula: ##STR00298##
or the pharmaceutically acceptable salt or hydrate thereof.
42. A pharmaceutical composition comprising a therapeutically
effective amount of a compound according to claim 24 or a
pharmaceutically acceptable salt or hydrate thereof.
43. A method of treating a disease chosen from rheumatoid
arthritis, systemic lupus erythromatosis, lupus nephritis,
Sjogren's disease, vasculitis, scleroderma, asthma, allergic
rhinitis, allergic eczema, B cell lymphoma, multiple sclerosis,
juvenile rheumatoid arthritis, juvenile idiopathic arthritis,
inflammatory bowel disease, graft versus host disease, psoriatic
arthritis, ankylosing spondylitis and uveitis, comprising
administering to a patient a therapeutically effective amount of a
compound according to claim 24 or a pharmaceutically acceptable
salt or hydrate thereof.
Description
TECHNICAL FIELD
[0001] The present invention relates to novel compounds which
inhibit BTK and their use as medicaments.
BACKGROUND INFORMATION
[0002] Members of the protein kinase family of human enzymes play
important regulatory roles in a multitude of distinct signal
transduction processes due to their post-translational modification
of specific proteins via the addition of a phosphate group (Hunter,
Cell 1987, 50, 823-829). Bruton's tyrosine kinase (BTK) is a member
of the Tec family of tyrosine kinases and plays a critical role in
B cell development, activation and antibody production.
[0003] The contribution of BTK to B cell biology is exemplified in
the X-linked agammaglobulinemia (XLA) immunodeficiency in humans
(reviewed in Lindvall, Immunol. Rev. 2005, 203, 200-215) that
display attenuated calcium signaling upon B cell receptor (BCR)
engagement, lack mature B cells in the periphery due to block
between pro- and pre-B cell stage and have lower levels of
circulating antibodies than normal healthy subjects. The outcome of
recent clinical trials with B cell depleting anti-CD20 molecules in
diseases such as rheumatoid arthritis (RA) and multiple sclerosis
(MS) support the hypothesis that B cells offer an important
intervention node for controlling autoimmune disorders (Townsend,
Immunol. Rev. 2010, 237, 264-283). As such, attenuation of B cell
activation and proliferation via inhibition of BTK may offer
similar therapeutic benefit and is consistent with the demonstrated
resistance of BTK-deficient mice to collagen induced arthritis
(Jansson, Clin. Exp. Immunol. 1993, 94, 459-465) and experimental
autoimmune encephalitis (Svensson, Eur. J. Immunol. 2002, 32,
1939-1946 and Mangla, Blood 2004, 104, 1191-1197). Similarly, the
clinical efficacy observed with a neutralizing antibody to the B
cell stimulating factor BlyS supports a role for B cells in the
pathophysiology of systemic lupus erythematosus (SLE) (La Cava,
Expert Opin. Biol. Ther. 2010, 10, 1555-1561). Given the necessity
for BTK for the production of autoantibodies, including anti-DNA
antibodies, in murine models of SLE (Steinberg, J. Clin. Invest.
1982, 70, 587-597; Golding, J. Immunol. 1983, 130, 1043-1046;
Scribner, J. Immunol. 1987, 138, 3611-3617; Seldin, J. Exp. Med.
1987, 166, 1585-1590; Takeshita, Int. Immunol. 1998, 10,
435-4444;Whyburn, J. Immunol. 2003, 171, 1850-1858), BTK inhibitors
may offer therapeutic benefit to SLE patients.
[0004] Within myeloid cells, BTK signal transduction is necessary
for the stimulated release of inflammatory cytokines such as
TNF.alpha. from stimulated monocytes (Horwood, J. Exp. Med. 2003,
197, 1603-1611) and for optimal actin cytoskeletal organization and
lacunar bone resorption in isolated osteoclasts (Danks, J. Bone
Miner. Res. 2010, 26, 182-192). Bone marrow derived mast cells
lacking BTK exhibit impaired activation-induced degranulation and
cytokine release. Given the role of BTK in signal transduction
processes across multiple cell types implicated in the pathogenesis
of autoimmune and allergic disorders, inhibition of BTK activity
may provide clinical benefit in diseases such as RA, MS, SLE, lupus
nephritis, Sjogren's disease, vasculitis, asthma and allergic
disorders.
SUMMARY OF THE INVENTION
[0005] Currently, compounds such as A and C (discussed below), and
those depicted in, for example, PCT publication number WO2014025976
are known as BTK inhibitors. However, as provided herein below,
these compounds cross-react with and inhibit other kinases. Hence,
these representatives are not selective for BTK over other targets.
The lack of selective BTK inhibition increases the likelihood of
adverse effects in a clinical setting.
[0006] Beside efficacy and selectivity, a therapeutic compound must
have a favorable safety profile such as cardiovascular safety. One
parameter for assessing the cardiovascular (CV) safety of a
compound is the mean arterial pressure (MAP). A statistically
significant change in MAP in a pre-clinical rat CV safety study is
indicative of adverse cardiovascular events in human. As provided
herein below, comparative compounds A, B, and C show statistically
significant increases in MAP in a rat CV study. The data suggests
that these compounds may not have a favorable cardiovascular safety
profile in human.
[0007] In view of the above-mentioned safety concerns with the
other known BTK inhibitors, there still remains a need for
additional compounds that are highly selective for BTK inhibition
and do not have an adverse impact on relevant cardiovascular
parameters such as MAP.
[0008] The compounds of the present invention maintain the
requisite potent inhibitory activity against BTK to treat the
aforementioned BTK-related diseases, and solve the selectivity and
cardiovascular safety problems associated with other known BTK
inhibitors such as those represented by comparative compounds A, B,
and C (discussed below). The BTK selectivity and the favorable
cardiovascular safety profile that are demonstrated by the
compounds of the instant invention represent unexpected and
surprising improvements over other known BTK inhibitors.
[0009] In particular, the compounds of the present invention solve
the efficacy and safety problems associated with other known BTK
inhibitors by maintaining a high level of potency and selectivity
in inhibiting the BTK activity without having any statistically
significant effects on MAP.
[0010] Accordingly, this invention comprises a novel class of
heteroaromatic compounds and methods for making and using the same.
These compounds are useful for the treatment of autoimmune and
allergic disorders in that they exhibit excellent inhibitory effect
upon BTK.
[0011] In a first generic embodiment, there is provided a compound
of the formula (I)
##STR00002##
[0012] in which:
[0013] Cy is chosen from
##STR00003##
[0014] each R.sub.1 is independently chosen from hydrogen or
methyl;
[0015] R.sub.2 is L--Ar, wherein Ar is phenyl or pyridinyl and each
is optionally substituted by one or more of halogen, halo C.sub.1-4
alkyl, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, --CN, halo C.sub.1-4
alkoxy, or cycloalkyl;
[0016] L is --(CH.sub.2)-- or --(CHCH.sub.3)--;
[0017] Y is C.sub.6-C.sub.8 spirocycle containing 1 ring nitrogen
atom, and is substituted by one R.sub.3;
[0018] R.sub.3 is chosen from
##STR00004##
[0019] each R.sub.4 is independently chosen from hydrogen, C
.sub.1-4 alkyl, or C.sub.3-4 cycloalkyl;
[0020] each group defined above for R.sub.1-R.sub.4 and Y can be
where possible partially or fully halogenated; or a
pharmaceutically acceptable salt or hydrate thereof.
[0021] In a further embodiment, there is provided a compound of the
formula (I) according to the embodiment herein-above and in
which:
[0022] Y is chosen from
##STR00005##
[0023] or a pharmaceutically acceptable salt or hydrate
thereof.
[0024] In a further embodiment, there is provided a compound of the
formula (I) according to the embodiments herein-above and in
which:
[0025] Cy is
##STR00006##
[0026] Y is chosen from
##STR00007##
[0027] R.sub.3 is
##STR00008##
[0028] in which each R.sub.4 is independently chosen from hydrogen,
C.sub.1-4 alkyl, or C.sub.3-4 cycloalkyl; or a pharmaceutically
acceptable salt or hydrate thereof.
[0029] In a further embodiment, there is provided a compound of the
formula (I) according to the embodiment herein-above and in
which:
##STR00009##
[0030] Y is chosen from
##STR00010##
[0031] R.sub.3 is
##STR00011##
[0032] in which R.sub.4 is chosen from hydrogen, C.sub.1-4 alkyl,
or C.sub.3-4 cycloalkyl; or a pharmaceutically acceptable salt or
hydrate thereof.
[0033] In a further embodiment, there is provided a compound of the
formula (I) according to the embodiment herein-above and in
which:
[0034] Cy is
##STR00012##
[0035] Y is chosen from
##STR00013##
[0036] R.sub.3 is
##STR00014##
[0037] in which R.sub.4 is chosen from hydrogen, C.sub.1-4 alkyl,
or C.sub.3-4 cycloalkyl; or a pharmaceutically acceptable salt or
hydrate thereof.
[0038] In a further embodiment, there is provided a compound of the
formula (I) according to the embodiment herein-above and in
which:
[0039] Cy is
##STR00015##
[0040] Y is chosen from
##STR00016##
[0041] R.sub.3is
##STR00017##
[0042] in which each R.sub.4 is independently chosen from hydrogen,
C.sub.1-4 alkyl, or C.sub.3-4 cycloalkyl; or a pharmaceutically
acceptable salt or hydrate thereof.
[0043] In a further embodiment, there is provided a compound of the
formula (I) according to the embodiment herein-above and in
which:
[0044] Cy is
##STR00018##
[0045] Y is chosen from
##STR00019##
[0046] R.sub.3is
##STR00020##
[0047] in which each R.sub.4 is independently chosen from hydrogen,
C.sub.1-4 alkyl, or C.sub.3-4 cycloalkyl; or a pharmaceutically
acceptable salt or hydrate thereof.
[0048] In a further embodiment, there is provided a compound of the
formula (I) according to any of the embodiments herein-above and in
which:
[0049] each R.sub.4 is independently chosen from hydrogen, methyl,
or cyclopropyl; or a pharmaceutically acceptable salt or hydrate
thereof.
[0050] In a further embodiment, there is provided a compound of the
formula (I) according to any of the embodiments herein-above and in
which:
[0051] R.sub.2 is L--Ar, wherein Ar is phenyl or pyridinyl and each
is optionally substituted by one or more of halogen, halomethyl,
methyl, methoxy, --CN, halomethoxy, or cyclopropyl;
[0052] L is --(CH.sub.2)-- or --(CHCH.sub.3)--
[0053] or a pharmaceutically acceptable salt or hydrate
thereof.
[0054] In another embodiment, the invention provides made compounds
in Table I which can be made in view of the general schemes,
examples and methods described herein and those known in the
art.
TABLE-US-00001 TABLE I Biological and physical properties of
representatives of the present invention BTK IC.sub.50 HPLC RT m/z
Example Structure (nM) Method (min) [M + H].sup.+ 1 ##STR00021## 36
A 0.65 446.4 2 ##STR00022## 0.3 B 0.596 491.1 3 ##STR00023## 0.4 B
0.797 559.1 4 ##STR00024## 1.8 B 0.795 540.3 5 ##STR00025## 0.4 B
0.808 540.3 6 ##STR00026## 0.4 B 0.813 540.3 7 ##STR00027## 3.3 B
0.778 555.2 8 ##STR00028## 0.9 B 0.80 559.1 9 ##STR00029## 0.6 A
0.80 542.3 10 ##STR00030## 0.5 B 0.751 551.3 11 ##STR00031## 0.7 B
0.647 483.3 12 ##STR00032## 0.5 B 0.770 526.4 13 ##STR00033## 9.1 B
0.665 527.2 14 ##STR00034## 0.5 B 0.7 527.2 15 ##STR00035## 1.1 B
0.814 540.3 16 ##STR00036## 1.5 B 0.595 472.5 17 ##STR00037## 1.7 B
0.798 539.2 18 ##STR00038## 0.6 B 0.798 539.2 19 ##STR00039## 0.9 B
0.777 540.3 20 ##STR00040## 22 B 0.741 528.7 21 ##STR00041## 5.8 B
0.786 554.3 22 ##STR00042## 0.3 B 0.760 526.3 23 ##STR00043## 1.3 B
0.796 540.3 24 ##STR00044## 0.8 B 0.801 512.4 25 ##STR00045## 2.6 B
0.843 526.3 26 ##STR00046## 2.6 C 1.78 532.4 27 ##STR00047## 1.7 B
0.769 506.3 28 ##STR00048## 0.3 B 0.692 476.4 29 ##STR00049## 0.8 B
0.69 476.4 30 ##STR00050## 0.6 B 0.786 544.4 31 ##STR00051## 0.8 B
0.779 544.3 32 ##STR00052## 0.5 B 0.772 544.4 33 ##STR00053## 0.4 B
0.78 544.2 34 ##STR00054## 0.7 B 0.753 506.2 35 ##STR00055## 0.9 B
0.724 522.3 36 ##STR00056## 0.2 B 0.716 508.3 37 ##STR00057## 0.8 B
0.788 512.3 38 ##STR00058## 3.0 A 0.87 540.2 39 ##STR00059## 0.9 B
0.758 560.3 40 ##STR00060## 4.2 B 0.722 472.4 41 ##STR00061## 0.3 B
0.734 484.3 42 ##STR00062## 78 B 0.716 514.4 43 ##STR00063## 3.0 B
0.722 486.4 44 ##STR00064## 0.3 B 0.672 458.3 45 ##STR00065## 6.0 A
0.64 444.3 46 ##STR00066## 7.9 B 0.761 526.3 47 ##STR00067## 0.5 A
0.78 512.3 48 ##STR00068## 16 A 0.59 432.3 49 ##STR00069## 45 B
0.802 580.3 50 ##STR00070## 63 B 0.728 511.1 51 ##STR00071## 0.3 B
0.766 498.4 52 ##STR00072## 1.2 B 0.7345 486.4 53 ##STR00073## 0.3
B 0.743 510.3 54 ##STR00074## 0.5 B 0.73 492.2 55 ##STR00075## 0.7
B 0.84 510.3 56 ##STR00076## 0.6 B 0.775 544.3 57 ##STR00077## 0.9
B 0.778 544.3 58 ##STR00078## 0.4 B 0.809 559.1 59 ##STR00079## 0.3
B 0.707 494.2 60 ##STR00080## 0.5 B 0.688 476.3
[0055] In a second generic embodiment, there is provided a
pharmaceutical composition comprising a therapeutically effective
amount of a compound according to the first embodiment or any of
its related embodiments or a pharmaceutically acceptable salt
thereof.
[0056] In a third generic embodiment, there is provided a method of
treating a disease chosen from rheumatoid arthritis, systemic lupus
erythromatosis, lupus nephritis, Sjogren' s disease, vasculitis,
scleroderma, asthma, allergic rhinitis, allergic eczema, B cell
lymphoma, multiple sclerosis, juvenile rheumatoid arthritis,
juvenile idiopathic arthritis, inflammatory bowel disease, graft
versus host disease, psoriatic arthritis, ankylosing spondylitis
and uveitis, comprising administering to a patient a
therapeutically effective amount of a compound according to the
first embodiment or any of its related embodiments or a
pharmaceutically acceptable salt thereof.
[0057] In a forth generic embodiment, there is provided a process
for preparation of a compound according to the first embodiment or
any of its related embodiments by:
[0058] (i) coupling a compound of formula A
##STR00081##
[0059] with a compound of formula E
##STR00082##
[0060] to form a compound of formula G
##STR00083##
[0061] wherein each R.sub.1 is independently chosen from hydrogen
or methyl; X is a halogen (i.e. chloro, bromo, or iodo); LG is a
leaving group; and Y' is C.sub.6-C.sub.8 spirocycle containing 1
ring nitrogen capped by a protecting group;
[0062] (ii) coupling the compound of formula (I-1) with a
heterocyclic boronic ester or acid of formula C
##STR00084##
[0063] in presence of a suitable base and palladium catalyst
followed by hydrolysis of the nitrile to carboxamide to form a
compound of formula (II-1)
##STR00085##
[0064] wherein each R group of the compound of formula C is H,
alkyl, or both R groups are connected to form a ring;
[0065] Cy in the compound of formula (II-1) is chosen from
##STR00086##
[0066] R.sub.2 is L--Ar, wherein Ar is phenyl or pyridinyl and each
is optionally substituted by one or more of halogen, halo C.sub.1-4
alkyl, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, --CN, halo C.sub.1-4
alkoxy, or cycloalkyl;
[0067] L is --(CH.sub.2)-- or --(CHCH.sub.3)--; and
[0068] (iii) Deprotecting the capped nitrogen of the compound of
formula (II-1) under an acidic condition and coupling the
deprotected compound of formula (II-1) with a compound chosen
from
##STR00087##
[0069] to form a compound of formula (I)
##STR00088##
[0070] wherein Y is C.sub.6-C.sub.8 spirocycle containing 1 ring
nitrogen bonded or covalently linked to R.sub.3, wherein
[0071] R.sub.3 is
##STR00089##
[0072] each R.sub.4 is independently chosen from hydrogen,
C.sub.1-4 alkyl, or C.sub.3-4 cycloalkyl; or a pharmaceutically
acceptable salt thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0073] The accompanying drawing, which is included to provide
further understanding of the subject technology and is incorporated
in and constitute a part of this specification, illustrates aspects
of the subject technology and together with the description serves
to explain the principles of the subject technology.
[0074] FIG. 1 shows that the compounds of the present invention,
e.g., Examples 12 and 22, elicit no effect on mean arterial
pressure (MAP) in-vivo in comparison to the comparative compounds
A--C (described in the example section).
DETAILED DESCRIPTION OF THE INVENTION
[0075] Definitions
[0076] Terms that are not specifically defined here have the
meanings that are apparent to the skilled man in the light of the
overall disclosure and the context as a whole.
[0077] As used herein, the following definitions apply, unless
stated otherwise:
[0078] The use of the prefix C.sub.x-y, wherein x and y each
represent a natural number, indicates that the chain or ring
structure or combination of chain and ring structure as a whole,
specified and mentioned in direct association, may consist of a
maximum of y and a minimum of x carbon atoms.
[0079] Alkyl denotes monovalent, saturated hydrocarbon chains,
which may be present in both straight-chain (unbranched) and
branched form. If an alkyl is substituted, the substitution may
take place independently of one another, by mono- or
polysubstitution in each case, on all the hydrogen-carrying carbon
atoms.
[0080] For example, the term "C.sub.1-5 alkyl" includes for example
H.sub.3C--, H.sub.3C--CH.sub.2--, H.sub.3C--CH.sub.2--CH.sub.2--,
H.sub.3C--CH(CH.sub.3)--, H.sub.3C--CH.sub.2--CH.sub.2--CH.sub.2--,
H.sub.3C--CH.sub.2--CH(CH.sub.3)--,
H.sub.3C--CH(CH.sub.3)--CH.sub.2--, H.sub.3C--C(CH.sub.3).sub.2--,
H.sub.3C--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--,
H.sub.3C--CH.sub.2--CH.sub.2--CH(CH.sub.3)--,
H.sub.3C--CH.sub.2--CH(CH.sub.3)--CH.sub.2--,
H.sub.3C--CH(CH.sub.3)--CH.sub.2--CH.sub.2--,
H.sub.3C--CH.sub.2--C(CH.sub.3).sub.2--,
H.sub.3C--C(CH.sub.3).sub.2--CH.sub.2--,
H.sub.3C--CH(CH.sub.3)--CH(CH.sub.3)-- and
H.sub.3C--CH.sub.2--CH(CH.sub.2CH.sub.3)--.
[0081] Further examples of alkyl are methyl (Me; --CH.sub.3), ethyl
(Et; 13 CH.sub.2CH.sub.3), 1-propyl (n-propyl; n-Pr;
--CH.sub.2CH.sub.2CH.sub.3), 2-propyl (i-Pr; iso-propyl;
--CH(CH.sub.3).sub.2), 1-butyl (n-butyl; n-Bu;
--CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-methyl-1-propyl (iso-butyl;
i-Bu; --CH.sub.2CH(CH.sub.3).sub.2), 2-butyl (sec-butyl; sec-Bu;
--CH(CH.sub.3)CH.sub.2CH.sub.3), 2-methyl-2-propyl (tert-butyl;
t-Bu; --C(CH.sub.3).sub.3), 1-pentyl (n-pentyl;
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-pentyl
(--CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.3), 3-pentyl
(--CH(CH.sub.2CH.sub.3).sub.2), 3-methyl-1-butyl (iso-pentyl;
--CH.sub.2CH.sub.2CH(CH.sub.3).sub.2), 2-methyl-2-butyl
(--C(CH.sub.3).sub.2CH.sub.2CH.sub.3), 3-methyl-2-butyl
(--CH(CH.sub.3)CH(CH.sub.3).sub.2), 2,2-dimethyl-1-propyl
(neo-pentyl; --CH.sub.2C(CH.sub.3).sub.3), 2-methyl-1-butyl
(--CH.sub.2CH(CH.sub.3)CH.sub.2CH.sub.3), 1-hexyl (n-hexyl;
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-hexyl
(--CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 3-hexyl
(--CH(CH.sub.2CH.sub.3)(CH.sub.2CH.sub.2CH.sub.3)),
2-methyl-2-pentyl (--C(CH.sub.3).sub.2CH.sub.2CH.sub.2CH.sub.3),
3-methyl-2-pentyl (--CH(CH.sub.3)CH(CH.sub.3)CH.sub.2CH.sub.3),
4-methyl-2-pentyl (--CH(CH.sub.3)CH.sub.2CH(CH.sub.3).sub.2),
3-methyl-3-pentyl (--C(CH.sub.3)(CH.sub.2CH.sub.3).sub.2),
2-methyl-3-pentyl (--CH(CH.sub.2CH.sub.3)CH(CH.sub.3).sub.2),
2,3-dimethyl-2-butyl (--C(CH.sub.3).sub.2CH(CH.sub.3).sub.2),
3,3-dimethyl-2-butyl (--CH(CH.sub.3)C(CH.sub.3).sub.3),
2,3-dimethyl-1-butyl (--CH.sub.2CH(CH.sub.3)CH(CH.sub.3)CH.sub.3),
2,2-dimethyl-1-butyl (--CH.sub.2C(CH.sub.3).sub.2CH.sub.2CH.sub.3),
3,3-dimethyl-1-butyl (--CH.sub.2CH.sub.2C(CH.sub.3).sub.3),
2-methyl-1-pentyl (--CH.sub.2CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.3),
3-methyl-1-pentyl (--CH.sub.2CH.sub.2CH(CH.sub.3)CH.sub.2CH.sub.3),
1-heptyl (n-heptyl), 2-methyl-1-hexyl, 3-methyl-1-hexyl,
2,2-dimethyl-1-pentyl, 2,3-dimethyl-1-pentyl,
2,4-dimethyl-1-pentyl, 3,3-dimethyl-1-pentyl,
2,2,3-trimethyl-1-butyl, 3-ethyl-1-pentyl, 1-octyl (n-octyl),
1-nonyl (n-nonyl); 1-decyl (n-decyl) etc.
[0082] By the terms propyl, butyl, pentyl, hexyl, heptyl, octyl,
nonyl, decyl etc. without any further definition are meant
saturated hydrocarbon groups with the corresponding number of
carbon atoms, wherein all isomeric forms are included.
[0083] The above definition for alkyl also applies if alkyl is a
part of another (combined) group such as for example C.sub.x-y
alkylamino or C.sub.x-y alkoxy.
[0084] Unlike alkyl, alkenyl consists of at least two carbon atoms,
wherein at least two adjacent carbon atoms are joined together by a
C--C double bond and a carbon atom can only be part of one C--C
double bond. If in an alkyl as hereinbefore defined having at least
two carbon atoms, two hydrogen atoms on adjacent carbon atoms are
formally removed and the free valencies are saturated to form a
second bond, the corresponding alkenyl is formed.
[0085] Alkenyl may optionally be present in the cis or trans or E
or Z orientation with regard to the double bond(s).
[0086] Unlike alkyl, alkynyl consists of at least two carbon atoms,
wherein at least two adjacent carbon atoms are joined together by a
C--C triple bond. If in an alkyl as hereinbefore defined having at
least two carbon atoms, two hydrogen atoms in each case at adjacent
carbon atoms are formally removed and the free valencies are
saturated to form two further bonds, the corresponding alkynyl is
formed.
[0087] Haloalkyl (haloalkenyl, haloalkynyl) is derived from the
previously defined alkyl (alkenyl, alkynyl) by replacing one or
more hydrogen atoms of the hydrocarbon chain independently of one
another by halogen atoms, which may be identical or different. If a
haloalkyl (haloalkenyl, haloalkynyl) is to be further substituted,
the substitutions may take place independently of one another, in
the form of mono- or polysubstitutions in each case, on all the
hydrogen-carrying carbon atoms.
[0088] Examples of haloalkyl (haloalkenyl, haloalkynyl) are
--CF.sub.3, --CHF.sub.2, --CH.sub.2F, --CF.sub.2CF.sub.3,
--CHFCF.sub.3, --CH.sub.2CF.sub.3, --CF.sub.2CH.sub.3,
--CHFCH.sub.3, --CF.sub.2CF.sub.2CF.sub.3,
--CF.sub.2CH.sub.2CH.sub.3, --CF.dbd.CF.sub.2, --CCl.dbd.CH.sub.2,
--CBr.dbd.CH.sub.2, --C.ident.C--CF.sub.3, --CHFCH.sub.2CH.sub.3,
--CHFCH.sub.2CF.sub.3 etc.
[0089] Halogen relates to fluorine, chlorine, bromine and/or iodine
atoms.
[0090] Cycloalkyl is made up of the subgroups monocyclic
hydrocarbon rings, bicyclic hydrocarbon rings and spiro-hydrocarbon
rings. The systems are saturated. In bicyclic hydrocarbon rings two
rings are joined together so that they have at least two carbon
atoms together.
[0091] If a cycloalkyl is to be substituted, the substitutions may
take place independently of one another, in the form of mono- or
polysubstitutions in each case, on all the hydrogen-carrying carbon
atoms. Cycloalkyl itself may be linked as a substituent to the
molecule via every suitable position of the ring system.
[0092] Examples of cycloalkyl are cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl.
[0093] Corresponding groups are an example:
[0094] cyclohexyl
##STR00090##
[0095] Spirocycle is a spiro-hydrocarbon ring one carbon atom
(spiroatom) belongs to two rings together.
[0096] Aryl denotes mono-, bi- or tricyclic carbocycles with at
least one aromatic carbocycle. Preferably, it denotes a monocyclic
group with six carbon atoms (phenyl) or a bicyclic group with nine
or ten carbon atoms (two six-membered rings or one six-membered
ring with a five-membered ring), wherein the second ring may also
be aromatic or, however, may also be saturated or partially
saturated.
[0097] If an aryl is to be substituted, the substitutions may take
place independently of one another, in the form of mono- or
polysubstitutions in each case, on all the hydrogen-carrying carbon
atoms. Aryl itself may be linked as a substituent to the molecule
via every suitable position of the ring system.
[0098] Examples of aryl are phenyl and naphthyl.
[0099] The above definition of aryl also applies if aryl is part of
another (combined) group as for example in arylamino, aryloxy or
arylalkyl.
[0100] Heterocyclyl denotes ring systems, which are derived from
the previously defined cycloalkyl or spirocycle by replacing one or
more of the groups --CH.sub.2-- independently of one another in the
hydrocarbon rings by the groups --O--, --S-- or --NH--, wherein a
total of not more than five heteroatoms may be present, at least
one carbon atom may be present between two oxygen atoms and between
two sulphur atoms or between one oxygen and one sulphur atom and
the ring as a whole must have chemical stability. Heteroatoms may
optionally be present in all the possible oxidation stages
(sulphur.fwdarw.sulphoxide --SO--, sulphone --SO.sub.2--;
nitrogen.fwdarw.N-oxide).
[0101] If a heterocyclyl is substituted, the substitutions may take
place independently of one another, in the form of mono- or
polysubstitutions in each case, on all the hydrogen-carrying carbon
and/or nitrogen atoms. Heterocyclyl itself may be linked as a
substituent to the molecule via every suitable position of the ring
system.
[0102] Examples of heterocyclyl are tetrahydrofuranyl,
tetrahydropyranyl, piperidinyl, piperazinyl, pyrrolidinyl,
morpholinyl,
[0103] or the following heterocyclic spirocycles
##STR00091##
[0104] Heteroaryl denotes monocyclic heteroaromatic rings or
polycyclic rings with at least one heteroaromatic ring, which
compared with the corresponding aryl or cycloalkyl, instead of one
or more carbon atoms, one or more identical or different
heteroatoms, selected independently of one another from among
nitrogen, sulphur and oxygen, wherein the resulting group must be
chemically stable. The prerequisite for the presence of heteroaryl
is a heteroatom and a heteroaromatic system.
[0105] If a heteroaryl is to be substituted, the substitutions may
take place independently of one another, in the form of mono- or
polysubstitutions in each case, on all the hydrogen-carrying carbon
and/or nitrogen atoms. Heteroaryl itself may be linked as a
substituent to the molecule via every suitable position of the ring
system, both carbon and nitrogen.
[0106] Examples of heteroaryl are pyridinyl, pyridazinyl,
pyrimidinyl, pyrazinyl, benzoxazolyl, indolyl, isoindolyl,
benzofuranyl, benzimidazolyl, benzothiazolyl, and the like.
[0107] Heteroatoms may optionally be present in all the possible
oxidation stages (sulphur.fwdarw.sulphoxide --SO--, sulphone
--SO.sub.2--; nitrogen.fwdarw.N-oxide).
[0108] Carbocycles include hydrocarbon rings containing from three
to twelve carbon atoms. These carbocycles may be either aromatic
either aromatic or non-aromatic ring systems. The non-aromatic ring
systems may be mono- or polyunsaturated. Preferred carbocycles
include but are not limited to cyclopropyl, cyclobutyl,
cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl,
cycloheptanyl, cycloheptenyl, phenyl, indanyl, indenyl,
benzocyclobutanyl, dihydronaphthyl, tetrahydronaphthyl, naphthyl,
decahydronaphthyl, benzocycloheptanyl and benzocycloheptenyl.
[0109] All cyclic and acyclic systems defined in this section
hereinabove shall be understood to be optionally partially or fully
halogenated where possible and unless otherwise indicated.
[0110] Stereochemistry/solvates/hydrates: Unless specifically
indicated, throughout the specification and appended claims, a
given chemical formula or name shall encompass tautomers and all
stereo, optical and geometrical isomers (e.g. enantiomers,
diastereomers, E/Z isomers, etc.) and racemates thereof as well as
mixtures in different proportions of the separate enantiomers,
mixtures of diastereomers, or mixtures of any of the foregoing
forms where such isomers and enantiomers exist, as well as salts,
including pharmaceutically acceptable salts thereof. The compounds
and salts of the invention can exist in unsolvated as well as
solvated forms with pharmaceutically acceptable solvents such as
water, ethanol and the like. In general, the solvated forms such as
hydrates are considered equivalent to the unsolvated forms for the
purposes of the invention.
[0111] Compounds of the invention also include their
isotopically-labelled forms. An isotopically-labelled form of an
active agent of a combination of the present invention is identical
to said active agent but for the fact that one or more atoms of
said active agent have been replaced by an atom or atoms having an
atomic mass or mass number different from the atomic mass or mass
number of said atom which is usually found in nature. Examples of
isotopes which are readily available commercially and which can be
incorporated into an active agent of a combination of the present
invention in accordance with well established procedures, include
isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous,
fluorine and chlorine, e.g., .sup.2H, .sup.3H, .sup.13C, .sup.14C,
.sup.15N, .sup.18O, .sup.17O, .sup.31P, .sup.32P, .sup.35S,
.sup.18F, and .sup.36Cl, respectively. An active agent of a
combination of the present invention, a prodrug thereof, or a
pharmaceutically acceptable salt of either which contains one or
more of the above-mentioned isotopes and/or other isotopes of other
atoms is contemplated to be within the scope of the present
invention.
[0112] Salts: The phrase "pharmaceutically acceptable" is employed
herein to refer to those compounds, materials, compositions, and/or
dosage forms which are, within the scope of sound medical
judgement, suitable for use in contact with the tissues of human
beings and animals without excessive toxicity, irritation, allergic
response, or other problem or complication, and commensurate with a
reasonable benefit/risk ratio.
[0113] As used herein "pharmaceutically acceptable salts" refers to
derivatives of the disclosed compounds wherein the parent compound
is modified by making acid or base salts thereof. Examples of
pharmaceutically acceptable salts include, but are not limited to,
mineral or organic acid salts of basic residues such as amines;
alkali or organic salts of acidic residues such as carboxylic
acids; and the like.
[0114] For example, such salts include acetates, ascorbates,
benzenesulphonates, benzoates, besylates, bicarbonates,
bitartrates, bromides/hydrobromides, Ca-edetates/edetates,
camsylates, carbonates, chlorides/hydrochlorides, citrates,
edisylates, ethane disulphonates, estolates esylates, fumarates,
gluceptates, gluconates, glutamates, glycolates,
glycollylarsnilates, hexylresorcinates, hydrabamines,
hydroxymaleates, hydroxynaphthoates, iodides, isothionates,
lactates, lactobionates, malates, maleates, mandelates,
methanesulphonates, mesylates, methylbromides, methylnitrates,
methylsulphates, mucates, napsylates, nitrates, oxalates, pamoates,
pantothenates, phenyl acetates, phosphates/diphosphates,
polygalacturonates, propionates, salicylates, stearates,
subacetates, succinates, sulphamides, sulphates, tannates,
tartrates, teoclates, toluenesulphonates, triethiodides, ammonium,
benzathines, chloroprocaines, cholines, diethanolamines,
ethylenediamines, meglumines and procaines.
[0115] Further pharmaceutically acceptable salts can be formed with
cations from metals like aluminium, calcium, lithium, magnesium,
potassium, sodium, zinc and the like (also see Pharmaceutical
salts, Birge, S. M. et al., J. Pharm. Sci., (1977), 66, 1-19).
[0116] The pharmaceutically acceptable salts of the present
invention can be synthesised from the parent compound which
contains a basic or acidic moiety by conventional chemical methods.
Generally, such salts can be prepared by reacting the free acid or
base form of these compounds with a sufficient amount of the
appropriate base or acid in water or in an organic diluent like
ether, ethyl acetate, ethanol, isopropanol, or acetonitrile, or a
mixture thereof.
[0117] Salts of other acids than those mentioned above which for
example are useful for purifying or isolating the compounds of the
present invention (e.g. trifluoroacetates), also comprise a part of
the invention.
[0118] Some abbreviated notations and their structure
correspondences are listed below:
[0119] In a representation such as for example
##STR00092##
[0120] the solid line means that the ring system may be attached to
the molecule via the carbon atom 1, 2 or 3, and is thus equivalent
to the following representation
##STR00093##
[0121] By a therapeutically effective amount for the purposes of
this invention is meant a quantity of substance that is capable of
obviating symptoms of illness or alleviating these symptoms, or
which prolong the survival of a treated patient.
TABLE-US-00002 List of abbreviations Ac Acetyl ACN Acetonitrile aq
Aqueous Ar Argon ATP adenosine triphosphate Bn Benzyl Bu Butyl Boc
tert-butyloxycarbonyl cat Catalyst conc concentrated d day(s) DCM
Dichloromethane DIPEA N,N-diisopropylethylamine DMAP
4-N,N-dimethylaminopyridine DMA Dimethylacetamide DME
1,2-dimethoxyethane DMF N,N-dimethylformamide DMSO
Dimethylsulphoxide dppf 1.1'-bis(diphenylphosphino)ferrocene EDC
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide ESI electron spray
ionization Et Ethyl Et.sub.2O diethyl ether EtOAc ethyl acetate
EtOH Ethanol h hour(s) HATU
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate Hep Heptane HPLC high performance liquid
chromatography i Iso IPAc Isopropyl acetate LC liquid
chromatography LiHMDS lithium bis(trimethylsilyl)amide sln.
Solution mCPBA 3-Chloroperoxbenzoic acid Me Methyl MeOH Methanol
min Minutes MPLC medium pressure liquid chromatography MS mass
spectrometry m/z mass-to-charge ratio NBS N-bromo-succinimide NIS
N-iodo-succinimide NMM N-methylmorpholine NMP N-methylpyrrolidone
NP normal phase n.a. not available PBS phosphate-buffered saline Ph
Phenyl Pr Propyl Pyr Pyridine rac Racemic Rf (R.sub.f) retention
factor RP reversed phase RT Retention time (HPLC) rt ambient
temperature TBAF tetrabutylammonium fluoride TBDMS
tert-butyldimethylsilyl TBME tert-butylmethylether TBTU
O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyl-uronium
tetrafluoroborate tBu tert-butyl TEA Triethylamine temp.
Temperature tert Tertiary Tf Triflate TFA trifluoroacetic acid THF
Tetrahydrofuran TMS Trimethylsilyl TRIS
tris(hydroxymethyl)-aminomethane Ts p-Tosyl TsOH p-toluenesulphonic
acid UV Ultraviolet
[0122] Features and advantages of the present invention will become
apparent from the following detailed examples which illustrate the
fundamentals of the invention by way of example without restricting
its scope:
[0123] Preparation of the Compounds According to the Invention
[0124] General Synthetic Methods
[0125] Optimum reaction conditions and reaction times may vary
depending on the particular reactants used. Unless otherwise
specified, solvents, temperatures, pressures and other reaction
conditions may be readily selected by one of ordinary skill in the
art. Specific procedures are provided in the Synthetic Examples
section. Intermediates and products may be purified by
chromatography on silica gel, recrystallization and/or reverse
phase HPLC (RHPLC). Discrete enantiomers may be obtained by
resolution of racemic products using chiral HPLC. RHPLC
purification methods used anywhere from 0-100% acetonitrile in
water containing 0.1% formic acid, 0.1% TFA, or 2.5 mM ammonium
bicarbonate and used one of the following columns:
[0126] a) Waters Sunfire OBD C18 5 .mu.m 30.times.150 mm column
[0127] b) Waters XBridge OBD C18 5 .mu.m 30.times.150 mm column
[0128] c) Waters ODB C8 5 .mu.m 19.times.150 mm column
[0129] d) Waters Atlantis ODB C18 5 .mu.m 19.times.50 mm column
[0130] e) Waters Atlantis T3 OBD 5 .mu.m 30.times.100 mm column
[0131] f) Phenomenex Gemini Axia C18 5 .mu.m 30.times.100 mm
column
[0132] HPLC Methods:
TABLE-US-00003 TABLE 1 Analytical HPLC Method A Gradient Mobile
Mobile Time Flow Method Phase A Phase B (min) % A % B (mL/min.)
Column A 0.05% Formic 0.05% Formic 0 90.0 10.0 0.8 CSH C18 Acid in
95% Acid in ACN 1.19 0 100 2.1 .times. 50 mm, water/5% ACN 1.70 0
100 1.7 .mu.m particle diameter
TABLE-US-00004 TABLE 2 Analytical HPLC Method B Gradient Mobile
Mobile Time Flow Method Phase A Phase B (min) % A % B (mL/min.)
Column A 0.1% Formic 0.1% Formic 0 95.0 5.0 0.8 BEH 2.5 .times. 50
mm Acid in Water Acid in ACN 1.0 5.0 95.0 C18, 1.7 .mu.m 1.3 5.0
95.0 particle diameter 1.4 95.0 5.0 1.7 95.0 5.0
TABLE-US-00005 TABLE 3 Analytical HPLC Method C Gradient Mobile
Mobile Time Flow Method Phase A Phase B (min) % A % B (mL/min.)
Column A 0.05% Formic 0.05% Formic 0 90.0 10.0 0.8 CSH C18 Acid in
95% Acid in ACN 4.45 0 100 2.1 .times. 50 mm, water/5% ACN 4.58 0
100 1.7 .mu.m particle diameter
[0133] The compounds according to the invention are prepared by the
methods of synthesis described hereinafter in which the
substituents of the general formulae have the meanings given
hereinbefore. These methods are intended as an illustration of the
invention without restricting its subject matter and the scope of
the compounds claimed to these examples. Where the preparation of
starting compounds is not described, they are commercially
obtainable or may be prepared analogously to known compounds or
methods described herein. Substances described in the literature
are prepared according to the published methods of synthesis.
[0134] Amide bond formations may be carried out by standard
coupling conditions well-known in the art (e.g., Bodanszky, M. The
Practice of Peptide Synthesis, Springer-Verlag, 1984, which is
herein incorporated by reference in its entirety), such as reacting
a carboxylic acid and an amine in the presence of a coupling
reagents such as
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HATU). Use of protective groups (i.e.,
protection or de-protection of a functional group) may be carried
out by standard conditions well-known in the art (e.g., Greene, T.
W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 3rd Ed.
New York, Wiley, 1999, which is herein incorporated by reference in
its entirety).
[0135] Compounds of formula I may be prepared as shown in Scheme I
or II below.
##STR00094##
[0136] In Scheme I, a pyrazole of formula A, in which X may be
bromo, chloro, or iodo, is reacted with a suitable boronic acid of
formula B (R.dbd.H), a suitable boronic ester of formula B
(R=methyl), or a suitable boronic ester of formula C under a
palladium catalysed cross-coupling condition such as the presence
of a suitable base (e.g., aqueous Cs.sub.2CO.sub.3, NaH), a
suitable catalyst [e.g., tetrakis(triphenylphosphine)palladium(0)],
in a suitable solvent (e.g., DME) and at a suitable temperature to
provide a compound of formula D. The heterocycle D is reacted with
a compound of formula E, wherein LG is a suitable leaving group
(e.g., O-Ts), in a suitable solvent (e.g., DMA), in the presence of
a suitable base (e.g., NaH) and at a suitable temperature to afford
a compound of formula F. The nitrile F is hydrolysed to the
corresponding carboxamide under a suitable condition such as in a
suitable solvent or a mixture of solvents (e.g., a mixture of water
and ethanol), in the presence of a suitable reagent such as
(hydrido(dimethylphosphinous acid-KP) [hydrogen
bis(dimethylphosphinito-KP)]platinum(II) and at a suitable
temperature. The subsequent deprotection and amide coupling using
conditions well-known in the art such as those described above
provide a compound of formula (I).
[0137] Additionally, compounds of formula I may be prepared
according to Scheme II.
##STR00095##
[0138] According to Scheme II, a pyrazole of formula A, in which X
may be bromo, chloro, or iodo, may be reacted with a compound of
formula E, wherein LG is a leaving group (e.g., O-Ts), in a
suitable solvent (e.g., acetone), in the presence of a suitable
base such (e.g., Cs.sub.2CO.sub.3, NaH) and at a suitable
temperature to afford a heterocycle of formula G. The
amino-pyrazole G may be reacted with a suitable boronic acid of
formula B (R.dbd.H), a suitable boronic ester of formula B
(R=methyl) or a suitable boronic ester of formula C under a
palladium catalysed cross-coupling condition such as the presence
of a suitable base (e.g., aqueous K.sub.2CO.sub.3), a suitable
catalyst [e.g., tetrakis(triphenylphosphine)palladium(0)], in a
suitable solvent (e.g., DME) and at a suitable temperature to
generate a compound of formula F. The nitrile F may be converted to
a compound of formula (I) according to the method described in
Scheme I.
SYNTHETIC EXAMPLES
[0139] Method A
[0140] Synthesis of Intermediate I-1
##STR00096##
[0141] Cs.sub.2CO.sub.3 is added to a solution of the R-1 (22.0 g,
118 mmol) and R-2 (47.6 g, 129 mmol) in acetone (250 mL). The
mixture is heated at 80.degree. C. for 2 days. The mixture is
diluted with water (200 mL) and extracted with CH.sub.2Cl.sub.2
(100 mL.times.2). The organics are then collected and concentrated
to give I-1 (25 g), m/z=382.1 [M+H].
[0142] Method B
[0143] Synthesis of Intermediate I-2
##STR00097##
[0144] Sodium hydride (14.3 g; 372.2 mmol) is added to a solution
of the R-1 (58 g; 310.2 mmol) in DMA (460 mL). After 30 min, R-3
(130.2 g; 341.2 mmol) is added and heated at 80.degree. C. for 18
h. The reaction is cooled to room temperature and diluted with MeOH
(250 mL) and water (35 mL). The reaction is then stirred vigorously
overnight. The heterogeneous mixture is vacuum filtered to yield,
after drying, 96 g of a solid as a 1:1 mixture of pyrazole isomers.
The solid is combined with 240 mL of CH.sub.2Cl.sub.2 and stirred
vigorously overnight. The heterogeneous mixture is vacuum filtered
and yielded 40 g of an off white solid. The solid is combined with
58 mL of CH.sub.2Cl.sub.2 and stirred vigorously. After 2 h, the
heterogeneous solution is sonicated for 5 minutes and then cooled
to 5.degree. C. and stirred for 1 h. The heterogeneous solution is
vacuum filtered and the solid is washed with cold CH.sub.2Cl.sub.2
(2.times.), collected and dried to yield I-2 (27.7 g). The combined
filtrates are diluted with 180 mL of i-PrOH and stirred vigorously
for 3 h. The heterogeneous solution is filtered and the solid is
washed with a small amount of i-PrOH (2.times.). The filtrate is
concentrated in vacuo to give a residue that is combined with 32 mL
of CH.sub.2Cl.sub.2 and sonicated for 5 minutes. After an
additional 1 h of stirring, the solution is cooled to 0.degree. C.
and stirred 1 h. The heterogeneous solution is filtered and solid
collected and dried to yield additional amounts of I-2(5.6 g).
Total amount of I-2 isolated is 33.3 g, m/z 394.0/396.0 [M+H].
[0145] Method C
[0146] Synthesis of Intermediate I-3
##STR00098##
[0147] I-I (1.1 g, 2.9 mmol), R-4 (1.71 g, 3.2 mmol), 2M aqueous
potassium carbonate (2.9 ml, 5.8 mmol),
tetrakis(triphenylphosphine)palladium(0) (333 mg, 0.3 mmol) and DME
(6 mL) are combined and sealed in a microwave tube and heated to
120.degree. C. thermally overnight. The mixture is filtered, then
diluted with water (100 mL) and extracted with EtOAc (4.times.200
mL). The combined EtOAc layers are dried over sodium sulfate and
concentrated. The crude residue is purified by flash chromatography
(SiO.sub.2, 0-60% EtOAc/Heptane) to yield 1.2 g of I-3, m/z=500.5
[M+H].
[0148] The following intermediate is prepared in similar
fashion:
TABLE-US-00006 Structure Intermediate m/z ##STR00099## I-4 460.7 [M
+ H]
[0149] Method D
[0150] Synthesis of Intermediate I-5
##STR00100##
[0151] R-1 (2.0 g, 10.7 mmol), R-4 (6.4 g, 60%, 11.8 mmol), 2M
aqueous Cs.sub.2CO.sub.3 (10.7 ml; 21 mmol),
tetrakis(triphenylphosphine)palladium(0) (1.2 g; 1.1 mmol), and DME
(6 mL) are combined in a microwave tube and heated to 135.degree.
C. in a microwave for 2 hours. The mixture is filtered, then
diluted with water and extracted with EtOAc. The combined extracts
are dried over sodium sulfate and concentrated to provide a crude
residue that is purified by flash chromatography (0-100% EtOAc in
heptane) to yield 3.2 g of I-5, m/z=382.1 [M+H].
[0152] The following intermediates are prepared in similar
fashion:
TABLE-US-00007 Structure Intermediate m/z ##STR00101## I-6 319.1 [M
+ H] ##STR00102## I-7 265.2 [M + H]
[0153] Method E
[0154] Synthesis of Intermediate I-8
##STR00103##
[0155] Sodium hydride (250 mg, 6.5 mmol) is added to a solution of
I-5 (1.64 g, 5.4 mmol) in DMA (10 mL). After 5 min, R-3 (2.26 g,
5.9 mmol) is added and heated at 70.degree. C. for 18 h. The
mixture is diluted with water (20 mL) and extracted with EtOAc
(4.times.10 mL). The combined EtOAc extracts are dried over sodium
sulfate, filtrate and then concentrated in vacuo. The crude residue
is purified by flash chromatography (SiO.sub.2, 0-50% EtOAc in
heptane) to provide 1.1 g of I-8, m/z =514.5 [M+H].
[0156] The following intermediate is prepared in similar
fashion:
TABLE-US-00008 Structure Intermediate m/z ##STR00104## I-9 528.3 [M
+ H]
[0157] Method F
[0158] Synthesis of Intermediate I-10
##STR00105##
[0159] I-3 (845 mg, 1.7 mmol) is dissolved in THF (15 mL). A 1M
solution of R-5 in THF (5.1 ml, 5.1 mmol) is added to the solution.
The mixture is stirred at 70.degree. C. overnight. The reaction
solution is partitioned between saturated NH.sub.4Cl (aq. solution)
and EtOAc. The layers are separated and the organic layer is
concentrated in vacuo. A small amount of CH.sub.2Cl.sub.2 is added
to the residue and the resulting solid is filtered to yield 900 mg
of I-10, m/z=370.3 [M+H].
[0160] The following intermediates are prepared in similar
fashion:
TABLE-US-00009 Structure Intermediate m/z ##STR00106## I-11 384.3
[M + H] ##STR00107## I-12 398.2 [M + H]
[0161] Method G
[0162] Synthesis of Intermediate I-13
##STR00108##
[0163] Potassium carbonate (270 mg, 1.94 mmol) is added to a
solution of I-10 (143 mg, 0.39 mmol) in DMA (5 mL). After 5 min,
R-6 (110 mg, 0.47 mmol) is added and the solution is heated to
70.degree. C. for 18 h. The crude solution is loaded directly onto
a silica column and purified (Gradient: 0-60% EtOAc in heptane) to
yield 71 mg of I-13, m/z=528.4 [M+H].
[0164] The following intermediates are prepared in similar
fashion:
TABLE-US-00010 Structure Intermediate m/z ##STR00109## I-14 328.4
[M + H] ##STR00110## I-15 542.4 [M + H] ##STR00111## I-16 596.4 [M
+ H] ##STR00112## I-17 542.4 [M + H] ##STR00113## I-18 576.3 [M +
H] ##STR00114## I-19 556.4 [M + H] ##STR00115## I-20 556.3 [M +
H]
[0165] Method H
[0166] Synthesis of Intermediate I-21
##STR00116##
[0167] To a stirred solution of R-7 (19.2 g, 99.1 mmol) in DMA (54
mL) is added potassium carbonate (27.4 g, 198.1 mmol). R-8 (23.0g,
109 mmol) is then added slowly. The reaction is stirred at room
temperature for 6 h. The reaction is then quenched with water and
extracted with EtOAc. The EtOAc is concentrated in vacuo and
residue is purified by flash chromatography (SiO.sub.2, 10% EtOAc
in hexanes) to yield 18 g of I-21, m/z=324.4 [M+H].
[0168] The following intermediates are prepared in similar
fashion:
TABLE-US-00011 Structure Intermediate m/z ##STR00117## I-22 367.2
[M + H] ##STR00118## I-23 313.6 [M + H] ##STR00119## I-24
337.1/339.2 [M + H] ##STR00120## I-25 319.2/321.1 [M + H]
##STR00121## I-26 370.1/371.9 [M + H] ##STR00122## I-27 370.3 [M +
H] ##STR00123## I-28 321.4 [M + H] ##STR00124## I-29 303.4 [M + H]
##STR00125## I-30 286.0 [M + H]
[0169] Method I
[0170] Synthesis of Intermediate I-31
##STR00126##
[0171] In a 1 L flask is placed R-7 (25 g, 128.8 mmol) and
potassium carbonate (35.6 g, 257.7 mmol) in 100 ml of DMF. To this
mixture is added R-9 (33.9 g, 141.7 mmol) and the reaction allowed
to stir overnight. The reaction is then filtered and concentrated.
The residue is dissolved in CH.sub.2Cl.sub.2 and filtered through
Celite. The filtrate is concentrated to provide 45.4 g of I-31,
m/z=353.4 [M+H]. Intermediate I-31 is used in subsequent steps
without further purification.
[0172] The following intermediates are prepared in similar
fashion:
TABLE-US-00012 Structure Intermediate m/z ##STR00127## I-32 367.1
[M + H] ##STR00128## I-33 ##STR00129## I-34 ##STR00130## I-35 367.2
[M + H] ##STR00131## I-36 383.1 [M + H] ##STR00132## I-37 387.1 [M
+ H] ##STR00133## I-38 319.2 [M + H] ##STR00134## I-39 431.1 [M +
H] ##STR00135## I-40 369.2 [M + H] ##STR00136## I-41 310.1 [M + H]
##STR00137## I-42 377.7 [M + H] ##STR00138## I-43 300.5 [M + H]
##STR00139## I-44 353.9 [M + H] ##STR00140## I-45 367.3 [M + H]
##STR00141## I-46 354.3 [M + H] ##STR00142## I-47 330.0 [M + H]
##STR00143## I-48 349.4 [M + H] ##STR00144## I-49 353.5 [M + H]
##STR00145## I-50 313.1 [M + H] ##STR00146## I-51 336.1/338.1 [M +
H]
[0173] Method J
[0174] Synthesis of Intermediate I-52
##STR00147##
[0175] In a 1 L flask is placed R-7 (75 g, 386.5 mmol) and
K.sub.2CO.sub.3 (106.7 g, 773 mmol) in 100 mL DMF. To this is added
R-10 (101.6 g, 425.2 mmol) and the reaction allowed to stir
overnight. The reaction is filtered and concentrated. The residue
is dissolved in CH.sub.2Cl.sub.2 and filtered through Celite. The
filtrate is concentrated to provide 136 g of I-52, m/z=353.0 [M+H].
Intermediate I-52 is used in subsequent steps without further
purification.
[0176] Method K
[0177] Synthesis of mixture of Intermediates I-53
##STR00148##
[0178] To a mixture of R-7 (5.0 g, 25.8 mmol), acetonitrile (29 mL)
and potassium carbonate (7.1 g, 51.5 mmol) is added R-11 (3.9 mL,
25.6 mmol). The mixture is stirred for 18 h under Ar. The reaction
is then concentrated and the residue is partitioned between EtOAc
and water. The layers are separated and the aqueous layer is
extracted with EtOAc (2.times.). The combined organic layers are
washed with brine, dried over MgSO.sub.4, filtered and concentrated
to yield 8.75 g of I-53, m/z=271.0 [M+H]. Intermediate I-53 mixture
is used in subsequent steps without further purification.
[0179] The following intermediates are prepared in similar
fashion:
TABLE-US-00013 Inter- me- Structure diate m/z ##STR00149## I-54 289
[M + H] ##STR00150## I-55 289 [M + H] ##STR00151## I-56 289 [M + H]
##STR00152## I-57 289 [M + H] ##STR00153## I-58 221 [M + H]
##STR00154## I-59 221 [M + H]
[0180] Method L
[0181] Synthesis of Intermediate I-60
##STR00155##
[0182] I-2 (1.1 g, 2.8 mmol), I-53 (1.12 g, 4.16 mmol), cesium
carbonate (1.81 g, 5.5 mmol) are combined in a microwave tube and
the vessel is flushed with Ar. DME (6.6 mL) and Pd(PPh.sub.3).sub.4
(320 mg, 0.28 mmol) are added and the vessel is degassed and
thermally heated to 125.degree. C. overnight. The mixture is
filtered through Celite and the Celite is washed with EtOAc and
water. The layers are separated and the aqueous is extracted with
EtOAc (2.times.). The combined organic layers are washed with
brine, dried over MgSO.sub.4, filtered and concentrated. The
residue is purified by flash chromatography (SiO.sub.2, 10-80%
EtOAc in heptane) to give 407 mg of I-60, m/z=542.2 [M+H].
[0183] Method M
[0184] Synthesis of Intermediate I-61
##STR00156##
[0185] I-2 (1.0 g, 1.31 mmol), I-30 (790 mg, 2.8 mmol), cesium
carbonate (1.6 g, 5.1 mmol), Pd(PPh.sub.3).sub.4 (0.29 g, 0.25
mmol) and DME (6 mL) are combined in a microwave tube and heated
thermally to 125.degree. C. overnight. The mixture is filtered,
then diluted with water (30 mL) and extracted with EtOAc
(4.times.30 mL). The combined organic extracts are dried over
sodium sulfate, filtered and concentrated to provide the crude
residue. The crude material is purified via flash chromatography
(SiO.sub.2, 0-100% EtOAc in heptane) to yield 1.1 g of I-61,
m/z=474.3 [M+H].
[0186] The following intermediates are prepared in similar
fashion:
TABLE-US-00014 Inter- me- Structure diate m/z ##STR00157## I-62
488.5 [M + H] ##STR00158## I-63 508.2/510.2 [M + H] ##STR00159##
I-64 558.4 [M + H] ##STR00160## I-65 488.4 [M + H] ##STR00161##
I-66 492.0 [M + H] ##STR00162## I-67 492.0 [M + H] ##STR00163##
I-68 524.3 [M + H] ##STR00164## I-69 542.5 [M + H] ##STR00165##
I-70 508.2/510.2 [M + H] ##STR00166## I-71 576.3 [M + H]
##STR00167## I-72 576.3 [M + H] ##STR00168## I-73 560.0 [M + H]
##STR00169## I-74 560.0 [M + H] ##STR00170## I-75 560.0 [M + H]
##STR00171## I-76 560.0 [M + H] ##STR00172## I-77 526.2/528.2 [M +
H] ##STR00173## I-78 526.3/528.2 [M + H] ##STR00174## I-79 556.4 [M
+ H] ##STR00175## I-80 543.3 [M + H] ##STR00176## I-81 543.3 [M +
H] ##STR00177## I-82 520.2/522.2 [M + H] ##STR00178## I-83 502.3 [M
+ H] ##STR00179## I-84 528.4 [M + H] ##STR00180## I-85 556.3 [M +
H] ##STR00181## I-86 538.4 [M + H]
[0187] Method N
[0188] Synthesis of Intermediate I-87
##STR00182##
[0189] I-2 (0.7 g, 1.8 mmol), I-39 (1.5 g, 3.5 mmol), cesium
carbonate (1.15 g, 3.5 mmol), Pd(PPh.sub.3).sub.4 (0.2 g, 0.21
mmol), are combined in a microwave tube. Degassed dioxane (8 mL)
and water (2 mL) are added. The reaction vessel is sealed under Ar
and heated in a microwave for 60 min at 125.degree. C. The reaction
is transferred to a separatory funnel, diluted with EtOAc and
rinsed with water and brine. The organics are dried, filtered, and
evaporated in vacuo. The residue is then purified via flash
chromatography (SiO.sub.2, 0-55% EtOAc/heptane) to yield 710 mg of
I-87, m/z=622.2 [M+H].
[0190] The following intermediates are prepared in similar
fashion:
TABLE-US-00015 Inter- Structure mediate m/z ##STR00183## I-88 558.4
[M + H] ##STR00184## I-89 ##STR00185## I-90 556.3 [M + H]
##STR00186## I-91 556.3 [M + H] ##STR00187## I-92 556.4 [M + H]
##STR00188## I-93 572.4 [M + H] ##STR00189## I-94 576.3 [M + H]
[0191] Method O
[0192] Synthesis of Intermediate I-95
##STR00190##
[0193] I-2 (310 mg, 0.78 mmol), I-21 (380 mg, 1.17 mmol),
tricyclohexylphosphine (175 mg, 0.63 mmol) and potassium phosphate
(500 mg, 2.3 mmol) are combined in 20 mL microwave vial in 8 ml of
dioxane and 2 mL of water. Ar is bubbled through the solution for
10 minutes. Tris(dibenzylideneacetone)dipalladium (0) is then added
and Ar is bubbled through the reaction for another 5 minutes. The
reaction is sealed and heated in a microwave for 60 min at
120.degree. C. After cooling to rt, the reaction solution is
diluted with water and extracted with EtOAc (2.times.).
[0194] The combined organic extracts are dried over MgSO.sub.4,
filtered, and concentrated in vacuo. The crude reside is purified
by flash chromatography (SiO.sub.2, 10-90% EtOAc in heptane) and
yields 340 mg of I-95, m/z=514.3 [M+H].
[0195] The following intermediates are prepared in similar
fashion:
TABLE-US-00016 Inter- Structure mediate m/z ##STR00191## I-96 492.3
[M + H] ##STR00192## I-97 560.2 [M + H] ##STR00193## I-98 560.0 [M
+ H] ##STR00194## I-99 510.3 [M + H] ##STR00195## I-100 571.7 [M +
H]
[0196] Method P
[0197] Synthesis of Intermediate I-101
##STR00196##
[0198] In a 1 L flask is placed I-2 (32.0 g, 80.8 mmol), I-31 (56.9
g, 161.5 mmol), cesium carbonate (52.6 g, 161.5 mmol) and
Pd(PPh.sub.3).sub.4 in 225 ml of Ar degassed DMA and 75 ml of
water. This is equipped with a condenser under argon and then
heated to 140.degree. C. on a preheated reaction block. After 45
min, the reaction is cooled to rt and then filtered. The solids are
rinsed with minimal EtOAc. The combined filtrates are transferred
to a 2 L separatory funnel, diluted with approximately 750 mL of
water and extracted with EtOAc (750 mL). The EtOAc is then rinsed
with another 750 mL of water and then 750 mL of brine. The organics
are then combined, dried over sodium sulfate, filtered and
concentrated in vacuo. Flash chromatography (SiO.sub.2, 0-75%
EtOAc/heptane) yields 25 g of I-101. The impure fractions are
isolated and re-purified by flash chromatography (SiO.sub.2, 0-75%
EtOAc/heptane) to yield 7.5 g of I-101. Total 33 g of I-101 (75%),
m/z=560.4 [M+H].
[0199] The following intermediate is prepared in similar
fashion:
TABLE-US-00017 Inter- Structure mediate m/z ##STR00197## I-102
542.3/543.3 [M + H]
[0200] Method Q
[0201] Synthesis of Intermediate I-103
##STR00198##
[0202] Hydrido(dimethylphosphinous acid-KP)[hydrogen
bis(dimethylphosphinito-KP)]platinum(II) (79 mg, 0.19 mmol) is
added to I-95 (1.0 g, 1.9 mmol) in water (3.0 mL) and ethanol (15
mL). The heterogeneous reaction is heated to 80.degree. C. After 18
h, the reaction is cooled to rt. The reaction is concentrated in
vacuo. The residue is combined with EtOAc and filtered. The
filtrate is concentrated in vacuo to yield 500 mg of I-103,
m/z=532.3 [M+H]. The product is used in subsequent steps without
further purification.
[0203] The following intermediates are prepared in similar
fashion:
TABLE-US-00018 Structure Intermediate m/z ##STR00199## I-104 560.4
[M + H] ##STR00200## I-105 546.4 [M + H] ##STR00201## I-106 614.4
[M + H] ##STR00202## I-107 614.4 [M + H] ##STR00203## I-108 560.4
[M + H] ##STR00204## I-109 ##STR00205## I-110 492.3 [M + H]
##STR00206## I-111 ##STR00207## I-112 506.4 [M + H] ##STR00208##
I-113 542.3 [M + H] ##STR00209## I-114 560.2 [M + H] ##STR00210##
I-115 594.3 [M + H] ##STR00211## I-116 594.3 [M + H] ##STR00212##
I-117 574.3 [M + H] ##STR00213## I-118 574.4 [M + H] ##STR00214##
I-119 574.4 [M + H] ##STR00215## I-120 594.3 [M + H] ##STR00216##
I-121 683.3/640.3 [M + H] ##STR00217## I-122 578 [M + H]
##STR00218## I-123 578 [M + H] ##STR00219## I-124 578 [M + H]
##STR00220## I-125 578 [M + H] ##STR00221## I-126 574.2 [M + H]
##STR00222## I-127 592.2/594.4 [M + H] ##STR00223## I-128 588.3 [M
+ H] ##STR00224## I-129 520.4 [M + H]
[0204] Method R
[0205] Synthesis of Intermediate I-130
##STR00225##
[0206] I-102 (61.5 g; 113.6 mmol) is dissolved in ethanol (200 mL)
and water (40 mL). Hydrido(dimethylphosphinous acid-KP)[hydrogen
bis(dimethylphosphinito-KP)]platinum(II) (2.91 g; 6.8 mmol) is
added and the reaction allowed to stir at 80.degree. C. for 16 h.
The reaction solution is diluted with water, extracted with 5%
MeOH/CH.sub.2Cl.sub.2 and the organic layer is collected, dried
over MgSO.sub.4, filtered and concentrated in vacuo. The residue is
purified by flash chromatography (SiO.sub.2, 0-100% EtOAc in Hep
then 0-20% MeOH in CH.sub.2Cl.sub.2) to yield 57.2 g of I-130,
m/z=560.3 [M+H].
[0207] Method S
[0208] Synthesis of Intermediate I-131
##STR00226##
[0209] Hydrido(dimethylphosphinous acid-KP)[hydrogen
bis(dimethylphosphinito-KP)]platinum(II) (2.91 g; 6.8 mmol) (863 mg
2.0 mmol) is added to the solution of I-101 (11.4 g, 20.2 mmol) in
water (30 mL) and ethanol (100 mL) in a sealable vessel. The vessel
is sealed and heated to 95.degree. C. overnight. The reaction is
concentrated in vacuo, diluted with EtOAc and filtered through
Celite. The filtrate is concentrated in vacuo to yield 12g of
I-131, m/z=560.4 [M+H]. The material (I-131) is used without
further purification.
[0210] Method T
[0211] Synthesis of Intermediate I-132
##STR00227##
[0212] I-109 (1.04 g, 2.5 mmol), I-41 (1.5 g, 5.0 mmol), cesium
carbonate (1.64 g, 5.0 mmol), Pd(PPh.sub.3).sub.4 (0.29 g, 0.25
mmol), are combined in a microwave tube. Degassed dioxane (8 mL)
and water (2 mL) are added. The reaction vessel is sealed under Ar
and heated in a microwave for 60 min at 125.degree. C. The reaction
is transferred to a separatory funnel, diluted with EtOAc and
rinsed with water and brine. The organics are dried, filtered, and
concentrated in vacuo. The residue is then purified via flash
chromatography (SiO.sub.2, 0-20% MeOH in DCM) to yield 1000 mg of
I-132, m/z=517.4 [M+H].
[0213] Method U
[0214] Synthesis of Intermediate I-133
##STR00228##
[0215] I-95 (1.34 g, 2.6 mmol) is heated to 140.degree. C. in
trimethylorthoformate (R-12) (17.4 mL). After 18 h the excess
trimethylorthoformate is removed in vacuo. The yellow residue is
diluted with absolute ethanol (15 mL), sodium borohydride (R-13)
(118 mg, 3.1 mmol) is added and the mixture stirred at rt. After 3
h, the solvent is removed in vacuo. The residue is diluted with
water, extracted with EtOAc, dried over MgSO.sub.4, filtered and
concentrated in vacuo. The crude residue is purified by flash
chromatography (SiO.sub.2, 10-80% EtOAc in heptane) to yield 920 mg
of I-133, m/z=528.3 [M+H].
[0216] The following intermediates are prepared in similar
fashion:
TABLE-US-00019 Inter- Structure mediate m/z ##STR00229## I-134
##STR00230## I-135 556.5 [M + H]
[0217] Method V
[0218] Synthesis of Intermediate I-136
##STR00231##
[0219] Hydrido(dimethylphosphinous acid-KP)[hydrogen
bis(dimethylphosphinito-KP)]platinum(II) (70 mg, 0.16 mmol) is
added to I-133 (890 mg, 1.7 mmol) in water (0.8 mL) and ethanol
(2.4 mL). The heterogeneous reaction is heated to 80.degree. C.
After 18 h, the reaction is cooled to rt. Additional
hydrido(dimethylphosphinous acid-KP)[hydrogen
bis(dimethylphosphinito-KP)]platinum(II) (80 mg, 0.19 mmol) is
added and the reaction is heated to 80.degree. C. for 96 h. The
reaction is concentrated in vacuo and partitioned between EtOAc and
water. The layers are separated and the aqueous layer is extracted
with EtOAc (2.times.). The combined organic layers are washed with
brine, dried over MgSO.sub.4, filtered and concentrated to give a
residue that is purified by flash chromatography (SiO.sub.2,
30-100% EtOAc in heptane) yielding 500 mg of I-136, m/z=546.4
[M+H].
[0220] The following intermediates are prepared in similar
fashion:
TABLE-US-00020 Structure Intermediate m/z ##STR00232## I-137 478.7
[M + H] ##STR00233## I-138 560.3 [M + H] ##STR00234## I-139 526.2
[M + H] ##STR00235## I-140 576.4 [M + H] ##STR00236## I-141 510 [M
+ H] ##STR00237## I-142 510 [M + H] ##STR00238## I-143 526.2 [M +
H] ##STR00239## I-144 576.4 [M + H] ##STR00240## I-145 544.2/546.2
[M + H] ##STR00241## I-146 544.2/546.2 [M + H] ##STR00242## I-147
544.2/546.1 [M + H] ##STR00243## I-148 528.3 [M + H] ##STR00244##
I-149 590.4 [M + H] ##STR00245## I-150 578.2 [M + H] ##STR00246##
I-151 578.3 [M + H] ##STR00247## I-152 574.3 [M + H] ##STR00248##
I-153 556.3 [M + H] ##STR00249## I-154 546.4 [M + H] ##STR00250##
I-155 538.3/540.3 [M + H] ##STR00251## I-156 574.2 [M + H]
##STR00252## I-157 540 [M + H]
[0221] Method W
[0222] Synthesis of Example 1
##STR00253##
[0223] I-110 (84 mg, 0.17 mmol) is treated with a 4.0M HCl solution
in dioxane (0.427 ml, 1.7 mmol) and stirred at rt for 0.5 h. The
reaction is concentrated in vacuo to afford 120 mg of I-158. To a
solution of acryloyl chloride (0.03 ml 0.37 mmol) in
CH.sub.2Cl.sub.2 (5 mL) is added I-158 and DIEA (0.15 mL, 0.84
mmol). After stirring at rt overnight, saturated aqueous ammonium
chloride (4 mL) is added and the mixture is extracted with EtOAc
(4.times.20 mL). The combined organic extracts are dried over
sodium sulfate, filtered and concentrated in vacuo. The residue is
purified by RHPLC (Column: Luna PFP(2) Prep; Gradient: 25% to
30%ACN in Water (0.1% TFA)) to give 5 mg of Example 1.
[0224] The following compound is made in similar fashion: Example
26.
[0225] Method X
[0226] Synthesis of Example 2
##STR00254##
[0227] To a solution of I-139 (220 mg, 0.42 mmol) in
CH.sub.2Cl.sub.2 (5 mL) is added a 4.0M HCl solution in dioxane
(2.0 ml; 8.0 mmol) and the reaction is stirred at rt for 16 h. The
solution is concentrated in vacuo to afford 175 mg of I-159.
[0228] To a solution of 2-butynoic acid (35 mg; 0.41 mmol) in THF
(5 ml) is added isobutyl chloroformate (62 mg; 0.45 mmol) and
N-methylmorpholine (166 mg; 1.6 mmol). The reaction is stirred at
rt for 15 min then is transferred to a solution of I-159 (175 mg;
0.41 mmol) in THF (10 mL) and stirred for 1 h at rt. The mixture is
then portioned between 10% MeOH in CH.sub.2Cl.sub.2 and water and
filtered through a phase separator and filtrate is concentrated.
The residue is purified by flash chromatography (SiO.sub.2, Ethyl
acetate in heptane 0-100%, then MeOH in CH.sub.2Cl.sub.2 0-20%) to
yield, after concentrating in-vacuo, 127 mg of Example 2.
[0229] The following compounds are made in similar fashion:
Examples 3-9, 13, 14, 19, 24, 27-29, 34-37, 44, 52-60.
[0230] Method Y
[0231] Synthesis of Example 12
##STR00255##
[0232] To a solution of I-130 (57 g, 102 mmol) in CH.sub.2Cl.sub.2
(250 mL) is added a 4.0M HCl solution in dioxane (101.9 mL, 407.4
mmol). This reaction solution is allowed to stir at rt for 16 h
then concentrated in vacuo to afford 57.5g of I-160 that is used
without further purification. A solution of 2-butynoic acid (11.6
g, 138 mmol) in IPAc (228 mL) is cooled to 0.degree. C. and
isobutyl chloroformate (18 mL, 138 mmol) followed by
N-methylmorpholine (50.5 mL, 460 mmol) are added sequentially
dropwise . The solution is allowed to stir at 0.degree. C. for 15
min then is transferred to a solution of I-160 (57 g, 115 mmol) in
IPAc (200 mL). The reaction mixture is stirred for 1 h then diluted
with 300 mL of water and warmed to 50.degree. C. for 3 h, then
stirred overnight at rt. The heterogeneous mixture is vacuum
filtered and the solid is washed with water, collected and dried to
yield 39 g of Example 12. The filtrate is collected and layers are
separated. The IPAc layer is concentrated and the residue suspended
in EtOAc and heated until a homogeneous solution is observed. The
solution is cooled to rt and the resulting precipitate is filtered,
collected and dried to yield an additional 8.2 g of Example 12.
[0233] Method Z
[0234] Synthesis of Example 22
##STR00256##
[0235] To a solution of I-131 (77.4 g, 138.3 mmol) in
CH.sub.2Cl.sub.2 (250 mL) is added MeOH (50 mL) followed by a 4M
HCl solution in dioxane (138.3 mL, 553.3 mmol). This reaction
solution is allowed to stir at rt for 4 h and then concentrated in
vacuo to yield 69.6 g of I-161 that is used without further
purification.
[0236] A solution of 2-butynoic acid (14.3 g, 168.4 mmol) in IPAc
(350 mL) is cooled to 0.degree. C. and isobutyl chloroformate (25.4
g, 182. 4 mmol) followed by N-methylmorpholine (57.3 g, 561 mmol)
are added sequentially dropwise. The solution is allowed to stir at
0.degree. C. for 30 min then is transferred to a solution of I-161
(69.6 g, 140.3 mmol) in IPAc (350 mL). The solution is warmed to rt
and stirred for 1 h then diluted with 800 ml of water and warmed to
50.degree. C. for 45 minutes. The mixture is then cooled to rt and
stirred for 30 min and then filtered. The solid is collected and
dried to yield 55 g of Example 22.
[0237] Method AA
[0238] Synthesis of Example 25
##STR00257##
[0239] To a solution of I-139 (624 mg, 1.15 mmol) in
CH.sub.2Cl.sub.2 (10 mL) is added a solution of HCl in dioxane (4M,
2.8 mL, 11.5 mmol) dropwise. The solution is decanted and the
residue is dried in vacuo to yield 571 mg of I-162. The crude
material (I-162) is used without further purification. A solution
of I-162 (571 mg, 1.51 mmol) in DMF (10 mL) and DIEA (0.60 mL, 3.4
mmol) is stirred for 15 minutes then 2-butynoic acid (97 mg, 1.51
mmol) and HATU (440 mg, 1.1 mmol) are added. After 30 minutes,
saturated aqueous NH.sub.4Cl (50 mL) is added, and the mixture is
extracted with EtOAc. The organic extract is washed with water and
brine, dried over sodium sulfate, filtered and concentrated in
vacuo to give a crude residue that is purified by flash
chromatography (SiO.sub.2, 0-10% MeOH in EtOAc) yielding 55 mg of
Example 25.
[0240] The following compounds are made in similar fashion:
Examples 15-18, 21, 23, 30-33, 38, 39, 40, 41, 51.
[0241] Method AB
[0242] Synthesis of Example 43
##STR00258##
[0243] To a solution of I-103 (1.2g, 2.3 mmol) in CH.sub.2Cl.sub.2
(15 mL) is added a HCl solution in dioxane (4M, 5mL, 20 mmol). The
mixture is stirred at rt for 1 h then concentrated in vacuo and the
residue is triturated with CH.sub.2Cl.sub.2. The solid is filtered,
collected and dried to yield 1.09 g of I-163 that is used without
further purification.
[0244] To a solution of the acrylic acid (50 mg, 0.69 mmol) and
HATU (264 mg, 0.69 mmol) in DMA (2.5 mL) is added I-163 (250 mg,
0.53 mmol) and DIEA (0.47 mL, 2.7 mmol). After stirring at rt
overnight, the reaction is concentrated in vacuo to afford a
residue that is purified by flash chromatography (SiO.sub.2, 0-10%
MeOH in CH.sub.2Cl.sub.2) giving 106 mg of Example 43.
[0245] The following compounds are made in similar fashion:
Examples 20, 42, 48.
[0246] Method AC
[0247] Synthesis of Example 45
##STR00259##
[0248] To a solution of I-137 (100 mg, 0.21 mmol) in
CH.sub.2Cl.sub.2 (5 mL) is added TFA (1.5 mL) and the mixture is
stirred at rt overnight. The reaction is concentrated in vacuo to
yield I-164 that is used without further purification.
[0249] To a solution of the 2-butynoic acid (20 mg, 0.24 mmol) and
EDC (78 mg, 0.41 mmol) in DMF (1 mL) is added DIEA (0.12 mL, 0.80
mmol). After 15 min, I-164 (100 mg, 0.27 mmol) is added. After
stirring at rt overnight, the reaction is concentrated in vacuo.
Purification by RHPLC (10-90%: ACN/H.sub.2O with 0.1% TFA) yielded
9 mg of Example 45.
[0250] Method AD
[0251] Synthesis of Example 47
##STR00260##
[0252] I-106 (87 mg, 0.159 mmol) is dissolved in 5 mL of
CH.sub.2Cl.sub.2. TFA (1 mL) is added and the mixture is stirred at
room temperature for 1 hour. The solution is concentrated in vacuo
and the residue is dissolved in MeOH and filtered through a 500 mg
Agilent StratoSpheres SPE column (MP PL-HCO.sub.3). The filtrate is
concentrated in vacuo to yield I-165 that is used without further
purification.
[0253] To a solution of the 2-butynoic acid (17 mg, 0.207 mmol) and
HATU (79 mg 0.21 mmol) in DMA (1 mL), is added I-165 (71 mg, 0.159
mmol) and DIEA (0.083 mL, 0.48 mmol). After stirring at rt
overnight, saturated aqueous NH.sub.4Cl (4 mL) is added and the
mixture is extracted with EtOAc (4.times.20 mL). The combined
organic extracts are dried over sodium sulfate, filtered and
concentrated in vacuo to afford a residue that is purified by flash
chromatography (SiO.sub.2, 1-6% MeOH in CH.sub.2Cl.sub.2) to give
21 mg of Example 47.
[0254] The following compounds are made in similar fashion:
Examples 46, 49, 50.
[0255] Method AE
[0256] Synthesis of Example 48
##STR00261##
[0257] In a vial is placed I-164 (100 mg, 0.27 mmol), acrylic acid
(28 mg, 0.4 mmol), TBTU (127 mg, 0.4 mmol) and triethylamine (40
mg, 0.4 mmol) in 1 mL of DMF. After stirring at rt overnight, the
solvent is removed in vacuo to provide a residue that is purified
by RHPLC (10-80% MeCN/water+0.1% TFA) to yield 20 mg of Example
48.
[0258] Method AF
[0259] Synthesis of Example 11
##STR00262##
[0260] To a solution of I-132 (1.0 g, 1.94 mmol) in
CH.sub.2Cl.sub.2 (5 mL) is added TFA (3 mL) dropwise. After 3 h at
rt, the solvent is removed to provide a residue that is dissolved
in MeOH and passed through multiple 500 mg Agilent StratoSpheres
SPE columns (MP PL-HCO.sub.3). The cartridges are washed with MeOH.
The filtrate is concentrated in vacuo to provide 806 mg of I-167
that is used without further purification.
[0261] To a solution of 2-butynoic acid (197 mg, 2.3 mmol) in EtOAc
(10 mL) is added isobutyl chloroformate (350 mg, 2.5 mmol) followed
by N-methylmorpholine (0.79 g, 7.7 mmol). The mixture is stirred
for 10 min then is added to a solution of I-167 (806 mg, 1.9 mmol)
in THF (10 mL) and stirred for 30 min at rt. The reaction is
diluted with water and extracted with EtOAc, dried over MgSO.sub.4,
filtered, and concentrated. The crude residue is purified by flash
chromatography (SiO.sub.2, 0-10% MeOH in CH.sub.2Cl.sub.2) to yield
370 mg of Example 11.
[0262] The following compounds are made in similar fashion: Example
10
[0263] Therapeutic Use
[0264] On the basis of their biological properties the compounds of
formula (I) according to the invention, or their tautomers,
racemates, enantiomers, diastereomers, mixtures thereof and the
salts of all the above-mentioned forms are suitable for treating
autoimmune and allergic disorders in that they exhibit good
inhibitory effect upon BTK.
[0265] Such diseases include for example: rheumatoid arthritis,
systemic lupus erythromatosis, lupus nephritis, Sjorgen's disease,
vasculitis, scleroderma, asthma, allergic rhinitis, allergic
eczema, B cell lymphoma, multiple sclerosis, juvenile rheumatoid
arthritis, juvenile idiopathic arthritis, inflammatory bowel
disease, graft versus host disease, psoriatic arthritis, ankylosing
spondylitis and uveitis.
[0266] The compounds of formula (I) may be used on their own or in
combination with at least one other active substance according to
the invention, and/or optionally also in combination with at least
one other pharmacologically active substance. The other
pharmacologically active substance may be an immunomodulatory
agent, anti-inflammatory agent, or a chemotherapeutic agent.
Examples of such agents include but are not limited to
cyclophosphamide, mycophenolate (MMF), hydroxychloroquine,
glucocorticoids, corticosteroids, immunosuppressants, NSAIDs,
non-specific and COX-2 specific cyclooxygenase enzyme inhibitors,
tumour necrosis factor receptor (TNF) receptors antagonists and
methotrexate.
[0267] Suitable preparations include for example tablets, capsules,
suppositories, solutions--particularly solutions for injection
(s.c., i.v., i.m.) and infusion--elixirs, emulsions or dispersible
powders. The content of the pharmaceutically active compound(s)
should be in the range from 0.1 to 90 wt.-%, preferably 0.5 to 50
wt.-% of the composition as a whole, i.e. in amounts which are
sufficient to achieve the dosage range specified below. The doses
specified may, if necessary, be given several times a day.
[0268] Suitable tablets may be obtained, for example, by mixing the
active substance(s) with known excipients, for example inert
diluents such as calcium carbonate, calcium phosphate or lactose,
disintegrants such as corn starch or alginic acid, binders such as
starch or gelatine, lubricants such as magnesium stearate or talc
and/or agents for delaying release, such as carboxymethyl
cellulose, cellulose acetate phthalate, or polyvinyl acetate. The
tablets may also comprise several layers.
[0269] Coated tablets may be prepared accordingly by coating cores
produced analogously to the tablets with substances normally used
for tablet coatings, for example collidone or shellac, gum arabic,
talc, titanium dioxide or sugar. To achieve delayed release or
prevent incompatibilities the core may also consist of a number of
layers. Similarly the tablet coating may consist of a number of
layers to achieve delayed release, possibly using the excipients
mentioned above for the tablets.
[0270] Syrups or elixirs containing the active substances or
combinations thereof according to the invention may additionally
contain a sweetener such as saccharine, cyclamate, glycerol or
sugar and a flavour enhancer, e.g. a flavouring such as vanillin or
orange extract. They may also contain suspension adjuvants or
thickeners such as sodium carboxymethyl cellulose, wetting agents
such as, for example, condensation products of fatty alcohols with
ethylene oxide, or preservatives such as p-hydroxybenzoates.
[0271] Solutions for injection and infusion are prepared in the
usual way, e.g. with the addition of isotonic agents, preservatives
such as p-hydroxybenzoates, or stabilisers such as alkali metal
salts of ethylenediamine tetraacetic acid, optionally using
emulsifiers and/or dispersants, whilst if water is used as the
diluent, for example, organic solvents may optionally be used as
solvating agents or dissolving aids, and transferred into injection
vials or ampoules or infusion bottles.
[0272] Capsules containing one or more active substances or
combinations of active substances may for example be prepared by
mixing the active substances with inert carriers such as lactose or
sorbitol and packing them into gelatine capsules.
[0273] Suitable suppositories may be made for example by mixing
with carriers provided for this purpose such as neutral fats or
polyethyleneglycol or the derivatives thereof.
[0274] Excipients which may be used include, for example, water,
pharmaceutically acceptable organic solvents such as paraffins
(e.g. petroleum fractions), vegetable oils (e.g. groundnut or
sesame oil), mono- or polyfunctional alcohols (e.g. ethanol or
glycerol), carriers such as e.g. natural mineral powders (e.g.
kaolins, clays, talc, chalk), synthetic mineral powders (e.g.
highly dispersed silicic acid and silicates), sugars (e.g. cane
sugar, lactose and glucose), emulsifiers (e.g. lignin, spent
sulphite liquors, methylcellulose, starch and polyvinylpyrrolidone)
and lubricants (e.g. magnesium stearate, talc, stearic acid and
sodium lauryl sulphate).
[0275] The preparations are administered by the usual methods,
preferably by oral or transdermal route, most preferably by oral
route. For oral administration the tablets may of course contain,
apart from the above-mentioned carriers, additives such as sodium
citrate, calcium carbonate and dicalcium phosphate together with
various additives such as starch, preferably potato starch,
gelatine and the like. Moreover, lubricants such as magnesium
stearate, sodium lauryl sulphate and talc may be used at the same
time for the tabletting process. In the case of aqueous suspensions
the active substances may be combined with various flavour
enhancers or colourings in addition to the excipients mentioned
above.
[0276] For parenteral use, solutions of the active substances with
suitable liquid carriers may be used.
[0277] The dosage for intravenous use is from 1-1000 mg per hour,
preferably between 5 and 500 mg per hour.
[0278] However, it may sometimes be necessary to depart from the
amounts specified, depending on the body weight, the route of
administration, the individual response to the drug, the nature of
its formulation and the time or interval over which the drug is
administered. Thus, in some cases it may be sufficient to use less
than the minimum dose given above, whereas in other cases the upper
limit may have to be exceeded. When administering large amounts it
may be advisable to divide them up into a number of smaller doses
spread over the day.
[0279] Description of Biological Properties
[0280] BTK v. EGFR Inhibition Assay
[0281] BTK Lanthscreen.RTM. Eu Kinase Binding Assay:
[0282] A Lanthscreen.RTM. Eu Kinase Binding assay (Life
Technologies) is performed to quantitate the ability of test
compounds to bind to BTK. The assay is based on the binding and
displacement of Alexa Fluor647-labeled Kinase Tracer # 236 to the
ATP-binding site of human full length His-tagged BTK (Life
Technologies cat #PV3587) with TR-FRET detection using a
europium-labeled anti-His antibody. The assay is assembled in
384-well low volume NBS black plates (Corning) where 2 nM BTK and
test compound in DMSO at varying concentrations are pre-incubated
for 30 min at 28.degree. C. in assay buffer consisting of 50 mM
HEPES, pH 7.4, 10 mM MgCl.sub.2, 1 mM EGTA. 100 .mu.M
Na.sub.3VO.sub.4 and 0.01% Brij 35. Then, 2 nM of Eu-anti His
antibody and 30 nM Kinase Tracer are added and incubated for 60 min
at 28.degree. C. Following incubation, TR-FRET signal is read on an
Envision plate reader (Excitation: 340 nm; Emissions:615 and 665
nm). The 665:615 nm emission ratio is calculated and converted to
POC compared to control and blank wells.
[0283] Inhibition of IL-6 Production in B cells Co-Stimulated with
ODN 2006 and Anti-hlgD
[0284] Primary CD19+ B cells (AllCells # PB010F) are thawed and
plated in RPMI containing 10% HI FBS in a 384-well tissue cultured
plate at 20,000 cells/well. The cells are treated with test
compound (0.5% DMSO final concentration) and incubated for 1 hour
at 37.degree. C., 5% CO2. Cells are then stimulated with 5 ug/mL
Goat F(ab')2 anti-human IgD (SouthernBiotech # 2032) and 2 uM ODN
2006 (InvivoGen # tlrl-2006) and incubated for 18-24 hours at
37.degree. C., 5% CO.sub.2. IL-6 in the supernatant is measured
using Meso Scale Discovery kit # K211AKB-6.
[0285] Inhibition of EGFR Autophosphorylation in A431 Human
Epithelial Cells Stimulated with Epithelial Growth Factor
[0286] A431cells (ATCC # CRL-1555 FZ) are thawed and plated in DMEM
containing 10% FBS in a 384-well tissue culture treated plate at
15,000 cells/well. After incubating for 24 hours at 37.degree. C.,
5% CO.sub.2, the cells are treated with test compound (1% DMSO
final concentration) and incubated for 16 hours at 37.degree. C.,
5% CO.sub.2. EGF (Millipore, 01-107) is added at a final
concentration of 60 ng/mL and incubated for 10 minutes. The medium
is removed, the cells are lysed, and phospho EGFR is measured (Meso
Scale Diagnostics, N31CB-1).
[0287] Representative compounds of the present invention are tested
and show BTK inhibition (Table I). Thus, they have the ability to
demonstrate clinical benefit for the treatment of autoimmune
disorders. Additionally, compounds of the present invention, as
represented by examples in Table II, are selective for BTK
inhibition over other related kinases. For example, the data
presented in Table II demonstrates that the compounds of the
present invention possess high degree of BTK selectivity over EGFR.
In this table the BTK activity is measured by IL-6 production in
primary CD19.sup.+ B cells, and the EGFR activity is measured by
EGFR phosphorylation in A431 cells.
TABLE-US-00021 TABLE II EGFR selectivity data for representative
compounds of the present invention Example B-cell IL-6 IC.sub.50
(nM) A431 p-EGFR IC.sub.50 (nM) 2 0.3 >10000 3 1.2 >10000 6
1.0 >10000 7 72 >10000 8 2.5 >10000 10 1.1 >10000 12
0.5 >10000 14 1.1 >10000 16 2.0 >10000 18 8.0 >10000 19
2.3 >10000 21 9.2 >10000 22 0.8 >10000 23 4.5 >10000 25
6.1 >10000 26 4.0 >10000 27 3.4 >10000 30 2.4 >10000 32
1.3 >10000 33 1.2 >10000 36 0.5 >10000 44 0.7 >10000 47
1.1 >10000 51 1.9 >10000 52 0.5 >10000 53 0.4 >10000 54
0.4 >10000 55 1.5 >10000 56 0.7 >10000 58 3.4 >10000 59
0.4 >10000 60 0.6 >10000
[0288] Therefore, as can be appreciated by a person skilled in the
art, the compounds of the present invention have a lower potential
for adverse effects due to off-target activity, as demonstrated by
their high selectivity against EGFR in cellular assays.
[0289] BTK v. BMX, TEC and TXK Inhibition Assays
[0290] Preferred compounds of the present invention display a range
of selective inhibition of BTK over other related kinases BMX, TEC,
and TXK relative to known BTK inhibitors. The following are used as
test compounds: compounds of the present invention and 1-[(3R)-3
[4-amino-3-(4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]-1-piperidyl]pr-
op-2-en-1-one (comparative compound A, ibrutinib),
5-amino-1-(7-but-2-ynoyl-7-azaspiro[3.4]octan-2-yl)-3-(4-isopropoxyphenyl-
) pyrazole-4-carboxamide (comparative compound B, Example 168
WO2014/025976),
N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)
phenyl)acrylamide (comparative compound C, Journal of Pharmacology
and Experimental Therapeutics 2013, 346:219-228) which are known
BTK inhibitors.
[0291] BTK, BMX, and TXK Assays
[0292] Z'-LYTE.TM. assay (Life Technologies):
[0293] The Z'-Lyte assay employs a FRET-based, coupled-enzyme
format and is based on the differential sensitivity of
phosphorylated and non-phosphorylated peptides to proteolytic
Cleavage. The activity of human recombinant BTK (full length,
His-tagged), BMX (full length, His-tagged) or Txk (full length,
GST-tagged) is estimated by measuring the phosphorylation of a
synthetic FRET peptide substrate labeled with Coumarin and
Fluorescein. The 10 .mu.L assay mixtures contain 50 mM HEPES (pH
7.5), 0.01% Brij-35, 10 mM MgCl2, 1 mM EGTA, 2 .mu.M FRET peptide
substrate (Z'-LYTE.TM. Tyr 1 Peptide for BTK and BMX, and Tyr 06
peptide for TXK), and kinase (1.3-9.3 ng BTK; 2.8-45.0 ng BMX;
2.3-93.6 ng TXK). Incubations are carried out at 22.degree. C. in
black polypropylene 384-well plates (Corning). Prior to the assay,
kinase, FRET peptide substrate and serially diluted test compounds
are pre-incubated together in assay buffer (7.5 .mu.L) for 10 min,
and the assay is initiated by the addition of 2.5 .mu.L assay
buffer containing 4.times.ATP (25 .mu.M for BTK; 100 .mu.M for both
BMX and TXK). Following the 60 min incubation, the assay mixtures
are quenched by the addition of 5 .mu.L of Z'-LYTE.TM. development
reagent, and 1 hour later the emissions of Coumarin (445 nm) and
Fluorescein (520 nm) are determined after excitation at 400 nm
using an Envision plate reader. An emission ratio (445 nm/520 nm)
is determined to quantify the degree of substrate
phosphorylation.
[0294] TEC Assay
[0295] Lanthscreen.RTM. Eu Kinase Binding Assay (Life
Technologies):
[0296] Lanthscreen.RTM. Eu Kinase Binding assay for BMX is
performed as described above for BTK except that 1 nM human
recombinant full length TEC (His-tagged) kinase and 1 nM Alexa
Fluor647-labeled Kinase Tracer #178 were used instead.
[0297] Representative compounds of the present invention are
assessed for inhibition of BTK, BMX, and TXK measuring
phosphorylation of a substrate (Z'-LYTE.TM. assay, Life
Technologies) and TEC measuring displacement of a "tracer"
(Lanthscreen.RTM. Eu Kinase Binding assay, Life Technologies).
TABLE-US-00022 TABLE III BMX, TEC and TXK selectivity for compounds
of the present invention BTK IC.sub.50 BMX IC.sub.50 Example (nM)
(nM) TEC IC.sub.50 (nM) TXK IC.sub.50 (nM) Compound A 1.4 0.8 12
2.3 Compound B 0.9 2.2 44 2.3 Compound C 6.1 3.2 6.8 22 2 0.8 16 14
25 3 1.2 17 45 27 6 1.5 33 65 43 7 29 870 1200 630 8 4.4 85 130 120
10 1.4 50 120 100 11 1.8 37 73 150 12 1.7 21 92 130 14 1.7 150 92
180 16 13 160 220 430 18 3.1 120 110 270 19 5.0 290 160 220 21 40
3000 1100 1900 22 0.7 29 21 46 23 8.3 280 130 600 25 19 300 160 850
26 18 1200 430 3000 27 6.6 120 66 120 28 1.2 56 39 95 29 3.6 78 79
92 30 3.0 120 64 160 32 1.9 64 49 55 33 1.3 25 48 59 36 1.0 37 22
50 44 1.4 24 20 78 47 2.7 500 34 310 51 0.8 8.0 18 12 52 7.0 160
100 260 53 1.3 34 47 84 54 1.5 27 35 110 55 2.5 35 67 79 56 2.2 54
120 150 58 2.6 46 38 67 59 0.8 38 36 68 60 1.7 27 35 100
[0298] These results show that the compounds of the present
invention are selective for BTK inhibition as compared to other
kinases by at least about 10 folds. See Table III
[0299] In-Vivo Assay--Comparison Between the Compounds of the
Present Invention and Comparative Compounds A, B and C
[0300] In a side-by-side in-vivo study, select compounds of the
present invention and comparative compounds A-C are evaluated in
telemetry-instrumented conscious rats to determine their effects on
mean arterial pressure (MAP) at doses at or above therapeutically
relevant concentrations. The following compounds are evaluated at
10 mg/kg po qd and 30 mg/kg po qd over the course of five days:
Examples 12 and 22 of the present invention and comparative
compounds A-C, i.e.,
1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)pyrazolo[3
,4-d]pyrimidin-1-yl]-1-piperidyl]prop-2-en-1-one (comparative
compound A, ibrutinib),
5-amino-1-(7-but-2-ynoyl-7-azaspiro[3.4]octan-2-yl)-3-(4-isopropoxyphenyl-
) pyrazole-4-carboxamide (comparative compound B, Example 168
WO2014/025976) and
N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)
phenyl)acrylamide (comparative compound C, Journal of Pharmacology
and Experimental Therapeutics 2013, 346:219-228).
[0301] Experimental Protocol
[0302] All animals (telemetry-instrumented) are single housed in
metabolic cages. Rats are acclimated to the metabolic cage for at
least 3 days and then dosed with vehicle for up to 4 days. Blood
pressure, heart rate, and bodyweight are collected during the
baseline period and animals are randomized into 3 groups based on
these parameters (n=8-9/group). Treatment groups are: vehicle and
test compound (10 mg/kg po and 30 mg/kg po qd); animals are treated
with compound for 5 days. The following day, rats are dosed again
with the test compound and plasma samples are collected via tail
bleed for compound exposures at multiple timepoints post-dose to
capture the T.sub.max (n=3-9/group). Mean arterial pressure (MAP)
and heart rate (HR) are collected continuously throughout the
study. Statistical analyses is performed using GraphPad Prism based
on the average 24-hr mean value during five days of compound
administration (one-way ANOVA with Dunnett's post-test vs. Vehicle;
p<0.05 is considered statistically significant).
TABLE-US-00023 TABLE IV Dose C.sub.max (nM) 5-Day 24-h MAP (mmHg)
Example (mg/kg) Day 6 Change versus Control Example 12 10 111 .+-.
37 No statistically significant effect on MAP Example 12 30 344
.+-. 106 No statistically significant effect on MAP Compound A 10
319 .+-. 36 3 .+-. 1 mmHg Compound A 30 561 .+-. 183 4 .+-. 1 mmHg
Compound B 10 182 .+-. 17 3 .+-. 1 mmHg Compound B 30 480 .+-. 53 2
.+-. 1 mmHg Compound C 10 644 .+-. 96 4 .+-. 1 mmHg Compound C 30
1731 .+-. 434 5 .+-. 1 mmHg Example 22 10 170 .+-. 29 No
statistically significant effect on MAP Example 22 30 720 .+-. 262
No statistically significant effect on MAP
[0303] The results show that the compounds of the present
invention, e.g., Examples 12 and 22, elicit no effect on MAP in
rats as compared to the comparative compounds A, B and C. As can be
appreciated by a person skilled in the art, significant changes in
the mean arterial pressure in rats could be indicative of higher
risk of adverse cardiovascular events in a clinical setting.
Therefore, the fact that the compounds of the present invention do
not display statistically significant effects on MAP is surprising
and unexpected. See Table IV and FIG. 1.
[0304] All patent and non-patent documents or literature cited in
this application are herein incorporated by reference in their
entirety.
* * * * *