U.S. patent application number 14/076457 was filed with the patent office on 2014-03-20 for intracellular kinase inhibitors.
This patent application is currently assigned to MannKind Corporation. The applicant listed for this patent is MannKind Corporation. Invention is credited to David W. Brandt, Subrata Chakravarty, Mary Faris, Gary A. Flynn, Sandra A. Lee.
Application Number | 20140080833 14/076457 |
Document ID | / |
Family ID | 38723876 |
Filed Date | 2014-03-20 |
United States Patent
Application |
20140080833 |
Kind Code |
A1 |
Flynn; Gary A. ; et
al. |
March 20, 2014 |
INTRACELLULAR KINASE INHIBITORS
Abstract
Intracellular kinase inhibitors and their therapeutic uses for
patients with T cell malignancies, B cell malignancies, autoimmune
disorders, and transplanted organs.
Inventors: |
Flynn; Gary A.; (Tucson,
AZ) ; Lee; Sandra A.; (Sherman Oaks, CA) ;
Faris; Mary; (Los Angeles, CA) ; Brandt; David
W.; (Moorpark, CA) ; Chakravarty; Subrata;
(Valencia, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MannKind Corporation |
Valencia |
CA |
US |
|
|
Assignee: |
MannKind Corporation
Valencia
CA
|
Family ID: |
38723876 |
Appl. No.: |
14/076457 |
Filed: |
November 11, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11750866 |
May 18, 2007 |
8604031 |
|
|
14076457 |
|
|
|
|
60869664 |
Dec 12, 2006 |
|
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60801074 |
May 18, 2006 |
|
|
|
Current U.S.
Class: |
514/238.8 ;
435/184; 435/188; 514/426; 514/428; 544/175; 548/557; 548/571 |
Current CPC
Class: |
A61P 7/06 20180101; C07D
265/36 20130101; A61K 31/40 20130101; A61P 37/00 20180101; A61K
31/538 20130101; C07D 207/14 20130101; C07D 217/04 20130101; C07D
295/185 20130101; A61P 19/02 20180101; C07D 211/26 20130101; A61P
7/04 20180101; C07D 211/10 20130101; C07D 211/30 20130101; C07D
333/56 20130101; A61P 35/00 20180101; C07D 295/215 20130101; A61K
31/4035 20130101; A61K 31/137 20130101; C07D 307/52 20130101; A61P
1/04 20180101; A61P 1/16 20180101; A61P 13/12 20180101; A61K 31/33
20130101; C07D 319/18 20130101; A61P 17/00 20180101; A61K 31/5375
20130101; A61P 25/00 20180101; A61P 25/02 20180101; A61P 21/00
20180101; C07D 207/08 20130101; C07D 209/44 20130101; A61K 31/5377
20130101; A61P 29/00 20180101; A61P 37/02 20180101; A61K 31/445
20130101; C07D 401/06 20130101; A61K 31/47 20130101; A61K 31/495
20130101; A61P 7/00 20180101; C07D 317/54 20130101; C07D 307/80
20130101; A61P 3/10 20180101; A61P 43/00 20180101; A61P 1/02
20180101; A61P 37/06 20180101; A61P 7/02 20180101; A61P 11/06
20180101; C07D 295/192 20130101; C07D 401/04 20130101; A61P 35/02
20180101; C07D 295/108 20130101 |
Class at
Publication: |
514/238.8 ;
548/571; 514/428; 544/175; 548/557; 514/426; 435/188; 435/184 |
International
Class: |
C07D 295/108 20060101
C07D295/108; C07D 207/14 20060101 C07D207/14; C07D 207/08 20060101
C07D207/08 |
Claims
1. A protein kinase inhibitor which has a structural formula
selected from the group consisting of: (a) ##STR00261## wherein
R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are independently hydrogen
or optionally substituted C.sub.1-C.sub.6 alkyl, wherein R.sup.22
is selected from hydrogen, C.sub.1-C.sub.4 alkyl, --NR'R'', --COH,
--COOH, --CNR'R'', and --CONHR', and wherein R' and R'' are
independently selected from hydrogen and C.sub.1-C.sub.4 alkyl; (b)
##STR00262## wherein R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are as
defined above and wherein R.sup.24 is ##STR00263## (c) ##STR00264##
wherein L is ##STR00265## and wherein R.sup.3, R.sup.4, R.sup.5,
and R.sup.6 are as defined above; (d) ##STR00266## wherein T, U, V,
and W independently are selected from hydrogen; halogen; --O;
C.sub.1-C.sub.3 alkyl; and C.sub.1-C.sub.3 alkyloxy; and wherein
R.sup.25 is hydrogen or C.sub.1-C.sub.3 alkyl; (e) ##STR00267##
wherein T, U, V, and W independently are selected from hydrogen;
halogen; --O; C.sub.1-C.sub.3 alkyl; and C.sub.1-C.sub.3 alkyloxy;
and wherein R.sup.8 is hydrogen or C.sub.1-C.sub.3 alkyl; (f)
##STR00268## wherein T, U, V, and W independently are selected from
hydrogen; halogen; --O; C.sub.1-C.sub.3 alkyl; and C.sub.1-C.sub.3
alkyloxy; and wherein R.sup.8 is hydrogen or C.sub.1-C.sub.3 alkyl;
(g) ##STR00269## wherein D is S, O, or NH; (h) ##STR00270## wherein
D is as defined above; and (i) ##STR00271## wherein G' is NH or CH,
or a pharmaceutically acceptable salt thereof.
2. The protein kinase inhibitor of claim 1, which is a compound of
formula (X).
3. The protein kinase inhibitor of claim 1, which is a compound of
formula (XII).
4. The protein kinase inhibitor of claim 1, which is a compound of
formula (XIII).
5. The protein kinase inhibitor of claim 1, which is a compound of
formula (XIV).
6. The protein kinase inhibitor of claim 1, which is a compound of
formula (XV).
7. The protein kinase inhibitor of claim 1, which is a compound of
formula (XVI).
8. The protein kinase inhibitor of claim 1, which is a compound of
formula (XVII).
9. The protein kinase inhibitor of claim 1, which is a compound of
formula (XVIII).
10. The protein kinase inhibitor of claim 1, which is a compound of
formula (XIX).
11. A composition, comprising: (a) a pharmaceutically acceptable
vehicle; and (b) the protein kinase inhibitor of claim 1.
12. An adduct formed between (a) the protein kinase inhibitor of
claim 1; and (b) a DKC triad kinase domain.
13. The adduct of claim 12, wherein the DKC triad kinase comprises
ITK.
14. A complex, comprising the protein kinase inhibitor of claim 1
which is bound to a DKC triad kinase.
15. The complex of claim 14, wherein the DKC triad kinase comprises
ITK.
16. A method of inhibiting kinase activity, comprising contacting a
DKC triad kinase with the protein kinase inhibitor of claim 1 or a
pharmaceutically acceptable salt thereof, whereby kinase activity
of the DKC triad kinase is inhibited.
17. The method of claim 16, wherein the DKC triad kinase comprises
ITK.
18. The method of claim 16, wherein the contacting occurs in a
cell-free system.
19. The method of claim 16, wherein the contacting occurs in a
cell.
20. The method of claim 19, wherein the cell is in vitro.
21. The method of claim 19, wherein the cell is in a patient.
22. The method of claim 21, wherein patient has an organ
transplant, an autoimmune disease, or a blood cell malignancy.
23. The method of claim 22, wherein the patient has a blood cell
malignancy and the blood cell malignancy is selected from the group
consisting of a B cell malignancy and a T cell malignancy.
24. The method of claim 16, wherein the DKC triad kinase comprises
ITK.
Description
[0001] This application is a division of Ser. No. 11/750,866 filed
on May 18, 2007, which claims the benefit of provisional
application Ser. No. 60/801,074 filed May 18, 2006 and Ser. No.
60/869,664 filed Dec. 12, 2006.
[0002] This application incorporates by reference the contents of a
25.1 kb text file created on Nov. 6, 2013 and named
"sequencelisting.txt," which is the sequence listing for this
application.
FIELD OF THE INVENTION
[0003] The invention relates to intracellular kinase inhibitors and
their therapeutic uses.
BACKGROUND OF THE INVENTION
[0004] Intracellular kinases play important functions in cells of
the immune system. For example, interleukin-2 inducible tyrosine
kinase (ITK) plays a key role in T cell development and
differentiation; it regulates IL-2 production via phospholipase C
.gamma.1 (PLC.gamma.1) and nuclear factor of activated T cells
(NFAT); it mediates Th2 cell differentiation; and it regulates T
cell migration and recruitment to lymphatic organs. Bruton's
tyrosine kinase (BTK) is involved in signal transduction pathways
which regulate growth and differentiation of B lymphoid cells. BTK
also is involved in platelet physiology by regulating the
glycoprotein VI/Fc receptor .gamma. chain (GPVI-FcR.gamma.)-coupled
collagen receptor signaling pathway. For these reasons, inhibitors
of intracellular kinases are useful for treating blood cell
malignancies, solid tumors and for suppressing the immune system,
for example in patients with autoimmune disorders or organ
transplants. Intracellular kinase inhibitors also are useful for
preventing or reducing the risk of thromboembolism.
BRIEF DESCRIPTION OF THE DRAWING
[0005] FIG. 1. Results of a BIACORE.RTM. experiment in which the
ITK kinase domain was immobilized on a biosensor and evaluated for
its ability to bind and dissociate from a small molecule.
[0006] FIG. 2. Alignment of human ITK (SEQ ID NO:1) and BTK (SEQ ID
NO:2).
[0007] FIG. 3. Alignment of kinase domains. Bolded amino acids,
hinge; bolded and underlined amino acids, gatekeeper; italicized
and bolded amino acids, Cys442 equivalents.
DETAILED DESCRIPTION OF THE INVENTION
[0008] The invention provides compounds which inhibit intracellular
kinases, particularly ITK and BTK, with an IC.sub.50 of 1 .mu.M or
below in an in vitro kinase assay as disclosed herein. The
invention also provides pharmaceutical compositions and methods of
using the compounds therapeutically. Patients who can be treated
include those with blood cell malignancies, solid tumors,
autoimmune disorders, and transplanted organs.
[0009] A review of the literature and patent database revealed the
existence of compounds that inhibit ITK or BTK kinases. However,
these compounds differ significantly from the compounds disclosed
herein. In several instances, the compounds are pyrrolopyridines
(e.g., US 2005/0215582). In other instances, the compounds are
methyl dimethylbenzoates that belong thiazolyl family of compounds
(e.g., US 2004/0077695). In all cases, these published compounds
differ from the compounds disclosed herein based on the following
parameters: the compounds do not correspond to the general
structure shown in this application, do not require the amino acid
triad DKC found in the kinase binding site and necessary for
optimal compound inhibitory capability described herein, do not
undergo elimination, and do not bind covalently to the kinase
binding pocket.
[0010] Compounds of the invention which inhibit ITK can be used,
e.g., to treat T cell malignancies. Preferred compounds of the
invention inhibit both ITK and BTK with an IC.sub.50 of 1 .mu.M or
below for each enzyme. Such compounds can be used, e.g., to treat
both T and B cell malignancies, as well as EGFR or HER positive
tumors.
[0011] The Tec family of kinases share a common subunit structure
composed of a Src homology domain 2 (SH.sub.2), an SH.sub.3 and a
catalytic kinase domain. Further, they are uniquely identified by
the presence of a Tec homology region (TH) and a pleckstrin
homology (PH) domain. There are four known crystallographic
structures described for the Tec family of kinases. These include
(a) two structures representing the phosphorylated and
unphosphorylated staurosporine-bound ITK (PDB codes 1 SM2, 1 SNU);
(b) one structure of the unphosphorylated apo-form of ITK (PDB code
1 SNX), and (c) one structure for the unphosphorylated apo-form of
BTK (Mao et al. J. Biol. Chem. 2001, 276, 41435-41443). For the
purpose of clarity of explanation, this disclosure will represent
these kinase structures with those of the nearly identical ITK
structures in (a) and (b) incorporated herein by reference (Brown
et al. J. Biol. Chem. 2004, 279, 18727-18732) focusing attention on
the ATP binding site. For the sake of uniformity, the residue
numbering in these kinase structures as represented in the Protein
Data Bank have been incorporated throughout this document to
describe the kinase domain. The amino acid sequence of human ITK is
shown in SEQ ID NO:1. The amino acid sequence of human BTK is shown
in SEQ ID NO:2. Homologous residues in the other kinases and
sequences from other sources may be numbered differently.
[0012] Referring to FIG. 2, The ITK kinase domain (residues
357-620) can be broken down into two components: the N-terminal
lobe (residues 357-437) and the C-terminal lobe (residues 438-620).
Like most kinases, the connecting region between the two lobes is a
flexible hinge region described below, that forms part of the
catalytic active site. The ordered nature of the C-helix places the
catalytically important residues of Glu406, Lys391 and Asp500 in an
orientation typical of the active form of a protein kinase. The
Gly-rich loop (residues 362-378), commonly observed in kinases,
assumes an extended and open conformation typical of an active
kinase.
[0013] The boundaries of the ATP binding site are demarcated by the
following residues: (a) the glycine-rich loop (Gly370, Ser371,
Gly372, Gln373, Phe374 and Gly375); (b) the hinge region (Phe435,
Glu436, Phe437, Met438, Glu439, His440, Gly441 and Cys442); and (c)
the catalytic residues Lys391 and Asp500. Additionally, the active
site also comprises several other hydrophobic residues including
Ala389, Ile369, Val377, Val419, and Leu 489 as well as the
hydrophilic residue Ser499.
[0014] Similar to other kinases, the hinge region of ITK contains
two backbone carbonyls and one backbone amino group as potential
hydrogen bond acceptor and donor sites respectively. Similar
backbone interactions have been observed in the interaction of
kinases with the adenine base of ATP and several competitive
inhibitors have been designed pursuing this concept. At the
N-terminal end of the hinge region lies the "gatekeeper" residue,
Phe435. This residue blocks access to an internal hydrophobic
pocket, and, at the same time, provides a potential site of
interaction for aromatic or hydrophobic groups. This "gatekeeper"
residue is a significant difference between ITK and BTK. Despite
the strong overall sequence identity between BTK and ITK, the
presence of the smaller threonine residue as a gatekeeper in the
active site of BTK justifies a key similarity of the latter to the
active site of several kinases such as Src/Abl/EGFR. The absence of
the bulkier Phe gatekeeper allows access to an internal hydrophobic
pocket for these kinases, a fact that has been exploited for the
design of allosteric inhibitors, and to improve the affinity of
ATP-competitive inhibitors through the addition of a hydrophobic
pharmacophore.
DEFINITIONS
[0015] "Alkyl" is a monovalent linear or branched saturated
hydrocarbon radical and can be substituted or unsubstituted. Linear
or branched alkyls typically have between 1 and 12 carbon atoms
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). Lower alkyls, or
"C.sub.1-C.sub.6 alkyls," have between 1 and 6 carbon atoms (e.g.,
1, 2, 3, 4, 5, or 6). Optional substitutents include halogen,
hydroxyl, alkoxy, aryloxy, amino, N-alkylamino, N,N-dialkylamino,
alkylcarbamoyl, arylcarbamoyl, aminocarbamoyl,
N-alkylaminocarbamoyl, N,N-dialkylaminocarbamoyl,
alkylsulfonylamino, arylsulfonylamino, carboxy, carboxyalkyl,
N-alkylcarboxamido, N,N-dialkylcarboxamido, alkylthio,
alkylsulfinyl, alkylsulfonyl, trihaloalkylsulfonylamino (e.g.,
trifluoromethylsulfonylamino), arylthio, arylsulfinyl,
arylsulfonyl, and heterocyclyl. Examples of linear or branched
C.sub.1-C.sub.6 alkyl are methyl, ethyl, propyl, isopropyl,
sec-butyl, tert-butyl, n-butyl, n-pentyl, sec-pentyl, tert-pentyl,
n-hexyl, isopentyl, fluoromethyl, trifluoromethyl, hydroxybutyl,
dimethylcarboxyalkyl, aminoalkyl, and benzylpropyl.
[0016] "Acyl" (or "alkylcarbonyl") is the radical --C(O)R.sup.8,
wherein R.sup.8 is an optionally substituted lower alkyl. Examples
of acyl include, but are not limited to, acetyl, propionyl,
n-butyryl, sec-butyryl, t-butyryl, iodoacetyl, and
benzylacetyl.
[0017] "Acyloxy" is the radical --OC(O)R.sup.8, wherein R.sup.8 is
an optionally substituted lower alkyl. Examples of acyloxy include,
but are not limited to, acetoxy, propionyloxy, butyryloxy,
trifluoroacetoxy, and diiodobutyryloxy.
[0018] "Alkoxy" is the radical --OR.sup.8, wherein R.sup.8 is an
optionally substituted lower alkyl. Examples of alkoxy include
methoxy, ethoxy, propoxy, 2-propoxy, butoxy, sec-butoxy,
tert-butoxy, pentyloxy, hexyloxy, fluoromethoxy, and
iodoethoxy.
[0019] "Alkylamino" is the radical --NR.sup.7R.sup.8, wherein
R.sup.7 is hydrogen or an optionally substituted lower alkyl and
R.sup.8 is an optionally substituted lower alkyl. Examples of
alkylamino groups are methylamino, ethylamino, isopropylamino,
dimethylamino, diethylamino, and trifluoromethylamino.
[0020] "Alkylaminocarbonyl" (or "alkylcarbamoyl") is the radical
--C(O)NR.sup.7R.sup.8, wherein R.sup.7 is hydrogen or an optionally
substituted lower alkyl and R.sup.8 is an optionally substituted
lower alkyl. Examples of alkylaminocarbonyl include, but are not
limited to, methylaminocarbonyl, dimethylaminocarbonyl,
t-butylaminocarbonyl, n-butylaminocarbonyl,
iso-propylaminocarbonyl, and trifluoromethylaminocarbonyl.
[0021] "Alkylaminosulfonyl" is the radical
--S(O).sub.2NR.sup.7R.sup.8, wherein R.sup.7 is hydrogen or an
optionally substituted lower alkyl and R.sup.8 is an optionally
substituted lower alkyl. Examples of alkylaminosulfonyl include,
but are not limited to, methylaminosulfonyl, dimethylaminosulfonyl,
and triiodomethylaminosulfonyl.
[0022] "Alkoxycarbonyl" or "alkyl ester" is the radical
--C(O)OR.sup.8, wherein R.sup.8 is an optionally substituted lower
alkyl. Examples of alkoxycarbonyl radicals include, but are not
limited to, methoxycarbonyl, ethoxycarbonyl, sec-butoxycarbonyl,
isopropyloxycarbonyl, and difluoromethoxycarbonyl.
[0023] "Alkylcarbonylamino" is the radical --NR.sup.7C(O)R.sup.8,
wherein R.sup.7 is hydrogen or an optionally substituted lower
alkyl and R.sup.8 is an optionally substituted lower alkyl.
Examples of alkylcarbonylamino include, but are not limited to,
methylcarbonylamino, iso-propylcarbonylamino, and
t-butylcarbonylamino.
[0024] "Alkylcarboxamido" is the radical --C(O)NR.sup.7R.sup.8,
wherein R.sup.7 is hydrogen or an optionally substituted lower
alkyl and R.sup.8 is an optionally substituted lower alkyl.
Examples of alkylcarboxamidos are methylcarboxamido,
ethylcarboxamido, isopropylcarboxamido, and
n-propylcarboxamido.
[0025] "Alkylsulfonyl" is the radical --S(O).sub.2R.sup.8, wherein
R.sup.8 is an optionally substituted lower alkyl. Examples of
alkylsulfonyl include, but are not limited to, methylsulfonyl,
trifluoromethylsulfonyl, and propylsulfonyl.
[0026] "Alkylsulfonylamino" is the radical
--NR.sup.7S(O).sub.2R.sup.8, wherein R.sup.7 is hydrogen or an
optionally substituted lower alkyl and R.sup.8 is an optionally
substituted lower alkyl. Examples of alkylsulfonylamino include,
but are not limited to, methylsulfonylamino, propylsulfonylamino,
and trifluoromethylsulfonylamino.
[0027] "Aryl" is the monovalent aromatic carbocyclic radical of one
individual aromatic ring or two or three fused rings in which at
least one of the fused rings is aromatic. Aryls can be optionally
substituted on one or more rings with one or more of halogen,
hydroxyl, alkoxy, aryloxy, amino, N-alkylamino, N,N-dialkylamino,
alkylcarbamoyl, arylcarbamoyl, aminocarbamoyl,
N-alkylaminocarbamoyl, N,N-dialkylaminocarbamoyl,
alkylsulfonylamino, arylsulfonylamino, carboxy, carboxyalkyl,
N-alkylcarboxamido, N,N-dialkylcarboxamido, alkylthio,
alkylsulfinyl, alkylsulfonyl, trifluoromethylsulfonylamino,
arylthio, arylsulfinyl, arylsulfonyl, hydroxyalkyl, alkoxyalkyl,
aryloxalkyl, aminoalkyl, N-alkylaminoalkyl, N,N-dialkylaminoalkyl,
alkylcarbamoylalkyl, arylcarbamoylalkyl, aminocarbamoylalkyl,
N-alkylaminocarbamoylalkyl N,N-dialkylaminocarbamoylalkyl,
alkylsulfonylaminoalkyl, arylsulfonylaminoalkyl, alkylcarboxy,
alkylcarboxyalkyl, N-alkylcarboxamindoalkyl,
N,N-dialkylcarboxamindoalkyl, alkylthioalkyl, alkylsulfinylalkyl,
alkylsulfonylalkyl, trifluoromethylsulfonylaminoalkyl,
arylthioalkyl, arylsulfinylalkyl, and arylsulfonylalkyl. Examples
of aryls are phenyl, naphthyl, tetrahydronaphthyl, indanyl,
indanonyl, tetralinyl, tetralonyl, fluorenonyl, phenanthryl,
anthryl, and acenaphthyl.
[0028] "Arylalkoxycarbonyl" or "arylalkyl ester" is the radical
--C(O)OR.sup.8X, wherein R.sup.8 is an optionally substituted lower
alkyl and X is an optionally substituted aryl. Examples of
aryloxycarbonyl radicals include, but are not limited to, benzyl
ester, phenyl ethyl ester, and dimethylphenyl ester.
[0029] "Arylalkylcarbamoyl" is the radical --C(O)NHR.sup.8X,
wherein R.sup.8 is an optionally substituted lower alkyl and X is
an optionally substituted aryl. Examples of arylalkylcarbamoyl
include, but are not limited to, benzylcarbamoyl,
phenylethylcarbamoyl, and cyanophenylcarbamoyl.
[0030] "Arylalkylcarbonyl" (or "aralkylcarbonyl") is the radical
--C(O)R.sup.8X, wherein R.sup.8 is an optionally substituted lower
alkyl and X is an optionally substituted aryl. Examples of
arylalkylcarbonyl radicals include, but are not limited to,
phenylacetyl and fluorophenylacetyl.
[0031] "Arylaminocarbonyl" (or "arylcarbamoyl") is the radical
--C(O)NXX', wherein X is an optionally substituted aryl and X' is
hydrogen or an optionally substituted aryl. Examples of
arylaminocarbonyl include, but are not limited to,
phenylaminocarbonyl, methoxyphenylaminocarbonyl,
diphenylaminocarbonyl, and dimethoxyphenyl-aminocarbonyl.
[0032] "Arylaminosulfonyl" is the radical --S(O).sub.2NXX', wherein
X is an optionally substituted aryl and X' is hydrogen or an
optionally substituted aryl. Examples of arylaminosulfonyl include,
but are not limited to, phenylaminosulfonyl,
methoxyphenylaminosulfonyl, and triiodomethylaminosulfonyl.
[0033] "Arylcarbonyl" is the radical --C(O)X, wherein X is an
optionally substituted aryl. Examples of arylcarbonyl radicals
include, but are not limited to, benzoyl, naphthoyl, and
difluorophenylcarbonyl.
[0034] "Arylcarbonylamino" is the radical --NHC(O)X, wherein X is
an optionally substituted aryl. Examples of arylcarbonylamino
include, but are not limited to, phenylcarbonylamino,
tosylcarbonylamino, and cyanophenylcarbonylamino.
[0035] "Aryloxy" is --OX, wherein X is an optionally substituted
aryl. Examples of aryloxys include phenyloxy, naphthyloxy,
tetrahydronaphthyloxy, indanyloxy, indanonyloxy, biphenyloxy,
tetralinyloxy, tetralonyloxy, fluorenonyloxy, phenanthryloxy,
anthryloxy, and acenaphthyloxy.
[0036] "Aryloxycarbonyl" or "aryl ester" is the radical --C(O)OX,
wherein X is an optionally substituted aryl. Examples of
aryloxycarbonyl radicals include, but are not limited to, phenyl
ester, naphthyl ester, dimethylphenyl ester, and trifluorophenyl
ester.
[0037] "Arylsulfonyl" is the radical --S(O).sub.2X, wherein X is an
optionally substituted aryl. Examples of arylsulfonyl include, but
are not limited to, phenylsulfonyl, nitrophenylsulfonyl,
methoxyphenylsulfonyl, and 3,4,5-trimethoxyphenylsulfonyl.
[0038] "Arylsulfonylamino" is the radical --NS(O).sub.2X, wherein X
is an optionally substituted aryl. Examples of arylsulfonylamino
include, but are not limited to, phenylsulfonylamino,
naphthylsulfonylamino, 2-butoxyphenylsulfonylamino,
4-chlorophenylsulfonylamino, 2,5-diethoxysulfonylamino,
4-hexyloxyphenylsulfonylamino, 4-methylphenylsulfonylamino,
naphtylsulfonylamino, 4-methoxyphenylsulfonylamino,
N-methylphenylsulfonylamino, and 4-cyanophenylsulfonylamino,
phenylsulfonylamino, 4-methylphenylsulfonylamino,
naphtylsulfonylamino. phenylsulfonylamino, and
4-methylphenylsulfonylamino.
[0039] "Arylsulfonyloxy" is the radical --OS(O).sub.2X, wherein X
is an optionally substituted aryl. Examples of arylsulfonyloxy
include, but are not limited to, benzenesulfonyloxy and
4-chloro-benzenesulfonyloxy.
[0040] "Cycloalkyl" is a monovalent saturated carbocyclic radical
consisting of one or more rings, preferably one, of three to seven
carbons per ring and can be optionally substituted with one or more
of hydroxyl, alkoxy, aryloxy, amino, N-alkylamino,
N,N-dialkylamino, alkylcarbamoyl, arylcarbamoyl, aminocarbamoyl,
N-alkylaminocarbamoyl, N,N-dialkylaminocarbamoyl,
alkylsulfonylamino, arylsulfonylamino, carboxy, carboxyalkyl,
N-alkylcarboxamido, N,N-dialkylcarboxamido, alkylthio,
alkylsulfinyl, alkylsulfonyl, trifluoromethylsulfonylamino,
arylthio, arylsulfinyl, arylsulfonyl, hydroxyalkyl, alkoxyalkyl,
aryloxalkyl, aminoalkyl, N-alkylaminoalkyl, N,N-dialkylaminoalkyl,
alkylcarbamoylalkyl, arylcarbamoylalkyl, aminocarbamoylalkyl,
N-alkylaminocarbamoylalkyl N,N-dialkylaminocarbamoylalkyl,
alkylsulfonylaminoalkyl, arylsulfonylaminoalkyl, alkylcarboxy,
alkylcarboxyalkyl, N-alkylcarboxamindoalkyl,
N,N-dialkylcarboxamindoalkyl, alkylthioalkyl, alkylsulfinylalkyl,
alkylsulfonylalkyl, trifluoromethylsulfonylaminoalkyl,
arylthioalkyl, arylsulfinylalkyl, and arylsulfonylalkyl. Examples
of cycloalkyls are cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, adamantyl, cyclooctyl, cycloheptyl,
tetrahydro-naphthalene, methylenecylohexyl, indanyl, indenyl, and
fluorenyl.
[0041] "Cycloalkylcarbonyl" is the radical --C(O)R, wherein R is an
optionally substituted cycloalkyl radical. Examples of
cycloalkylcarbonyl radicals include, but are not limited to,
cyclobutanoyl, cyclopentanoyl, cyclohexanoyl, and
trifluorocyclopentanoyl.
[0042] "Halogen" includes fluorine, chlorine, bromine, and
iodine.
[0043] "Heteroaryl" is a monovalent aromatic cyclic radical having
one or more rings, preferably one to three rings, of four to eight
atoms per ring, incorporating one or more heteroatoms selected
independently from nitrogen, oxygen, silicon, and sulfur.
Heteroaryls can be optionally substituted on one or more rings with
one or more of halogen, hydroxyl, alkoxy, aryloxy, amino,
N-alkylamino, N,N-dialkylamino, alkylcarbamoyl, arylcarbamoyl,
aminocarbamoyl, N-alkylaminocarbamoyl, N,N-dialkylaminocarbamoyl,
alkylsulfonylamino, arylsulfonylamino, carboxy, carboxyalkyl,
N-alkylcarboxamido, N,N-dialkylcarboxamido, alkylthio,
alkylsulfinyl, alkylsulfonyl, trifluoromethylsulfonylamino,
arylthio, arylsulfinyl, arylsulfonyl, hydroxyalkyl, alkoxyalkyl,
aryloxalkyl, aminoalkyl, N-alkylaminoalkyl, N,N-dialkylaminoalkyl,
alkylcarbamoylalkyl, arylcarbamoylalkyl, aminocarbamoylalkyl,
N-alkylaminocarbamoylalkyl N,N-dialkylaminocarbamoylalkyl,
alkylsulfonylaminoalkyl, arylsulfonylaminoalkyl, alkylcarboxy,
alkylcarboxyalkyl, N-alkylcarboxamindoalkyl,
N,N-dialkylcarboxamindoalkyl, alkylthioalkyl, alkylsulfinylalkyl,
alkylsulfonylalkyl, trifluoromethylsulfonylaminoalkyl,
arylthioalkyl, arylsulfinylalkyl, and arylsulfonylalkyl.
[0044] Representative examples of monocyclic ring system
heteroaryls include, but are not limited to, azetidinyl, azepinyl,
aziridinyl, diazepinyl, 1,3-dioxolanyl, dioxanyl, dithianyl, furyl,
imidazolyl, imidazolinyl, imidazolidinyl, isothiazolyl,
isothiazolinyl, isothiazolidinyl, isoxazolyl, isoxazolinyl,
isoxazolidinyl, morpholinyl, oxadiazolyl, oxadiazolinyl,
oxadiazolidinyl, oxazolyl, oxazolinyl, oxazolidinyl, piperazinyl,
piperidinyl, pyranyl, pyrazinyl, pyrazolyl, pyrazolinyl,
pyrazolidinyl, pyridyl, pyrimidinyl, pyridazinyl, pyrrolyl,
pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl,
tetrazinyl, tetrazolyl, thiadiazolyl, thiadiazolinyl,
thiadiazolidinyl, thiazolyl, thiazolinyl, thiazolidinyl,
thiophenyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl,
thiopyranyl, triazinyl, triazolyl, and trithianyl.
[0045] Bicyclic ring systems include any of the above monocyclic
ring systems fused to an aryl group, a cycloalkyl group, or another
heteroaryl monocyclic ring system. Representative examples of
bicyclic ring systems include but are not limited to,
benzimidazolyl, benzothiazolyl, benzothiophenyl, benzoxazolyl,
benzofuranyl, benzopyranyl, benzothiopyranyl, benzodioxinyl,
1,3-benzodioxolyl, cinnolinyl, indazolyl, indolyl, indolinyl,
indolizinyl, naphthyridinyl, isobenzofuranyl, isobenzothiophenyl,
isoindolyl, isoindolinyl, isoquinolyl, phthalazinyl, pyranopyridyl,
quinolyl, quinolizinyl, quinoxalinyl, quinazolinyl,
tetrahydroisoquinolyl, tetrahydroquinolyl, and
thiopyranopyridyl.
[0046] Tricyclic rings systems include any of the above bicyclic
ring systems fused to an aryl group, a cycloalkyl group, or a
heteroaryl monocyclic ring system. Representative examples of
tricyclic ring systems include, but are not limited to, acridinyl,
carbazolyl, carbolinyl, dibenzofuranyl, dibenzothiophenyl,
naphthofuranyl, naphthothiophenyl, oxanthrenyl, phenazinyl,
phenoxathiinyl, phenoxazinyl, phenothiazinyl, thianthrenyl,
thioxanthenyl, and xanthenyl.
[0047] "Heteroarylaminocarbonyl" is the radical --C(O)NZZ', wherein
Z is an optionally substituted heteroaryl and Z' is hydrogen or an
optionally substituted heteroaryl. Examples of
heteroarylaminocarbonyl include, but are not limited to,
pyridinylaminocarbonyl, and thienylaminocarbonyl,
furanylaminocarbonyl.
[0048] "Heteroarylaminosulfonyl" is the radical --S(O).sub.2N ZZ',
wherein Z is an optionally substituted heteroaryl and Z' is
hydrogen or an optionally substituted heteroaryl. Examples of
heteroarylaminosulfonyl include, but are not limited to,
thienylaminosulfonyl, piperidinylamino sulfonyl,
furanylaminosulfonyl, and imidazolylaminosulfonyl.
[0049] "Heteroarylcarbonyl" is the radical --C(O)Z, wherein Z is an
optionally substituted heteroaryl. Examples of heteroarylcarbonyl
radicals include, but are not limited to, pyridinoyl,
3-methylisoxazoloyl, isoxazoloyl, thienyl, and furoyl.
[0050] "Heteroarylsulfonyl" is the radical --S(O).sub.2Z, wherein Z
is an optionally substituted heteroaryl. Examples of
heteroarylsulfonyl include, but are not limited to,
thienylsulfonyl, furanylsulfonyl, imidazolylsulfonyl, and
N-methylimidazolylsulfonyl.
[0051] "Heteroarylsulfonyloxy" is the radical --OS(O).sub.2Z,
wherein Z is an optionally substituted heteroaryl. An examples of
hetroarylsulfonyloxy is thienylsulfonyloxy.
[0052] "Heterocycle" is a saturated or partially unsaturated
carbocyclic radical having one, two, or three rings each containing
one or more heteroatoms selected independently from nitrogen,
oxygen, silicon, and sulfur. A heterocycle can be unsubstituted or
substituted on any or all of the rings with one or more of halogen,
aryl, heteroaryl, hydroxy, alkoxy, aryloxy, amino, N-alkylamino,
N,N-dialkylamino, alkylsulfonylamino, arylsulfonylamino,
alkylcarbamoyl, arylcarbamoyl, aminocarbamoyl,
N-alkylaminocarbamoyl, N,N-dialkylaminocarbamoyl, carboxy,
alkylcarboxy, N-alkylcarboxamido, N,N-dialkylcarboxamido,
alkylthio, alkylsulfinyl, alkylsulfonyl,
trifluoromethylsulfonylamino, arylthio, arylsulfinyl, arylsulfonyl,
carboxyalkyl, hydroxyalkyl, alkoxyalkyl, aryloxalkyl, aminoalkyl,
N-alkylaminoalkyl, N,N-dialkylaminoalkyl, alkylcarbamoylalkyl,
arylcarbamoylalkyl, aminocarbamoylalkyl, N-alkylaminocarbamoylalkyl
N,N-dialkylaminocarbamoylalkyl, alkylsulfonylaminoalkyl,
arylsulfonylaminoalkyl, alkylcarboxyalkyl,
N-alkylcarboxamindoalkyl, N,N-dialkylcarboxamindoalkyl,
alkylthioalkyl, alkylsulfinylalkyl, alkylsulfonylalkyl,
trihaloalkylsulfonylaminoalkyl, arylthioalkyl, arylsulfinylalkyl,
and arylsulfonylalkyl. Examples of heterocycles include
piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl,
thiamorpholinyl, pyrrolyl, phthalamide, succinamide, and
maleimide.
[0053] "Heterocyclylcarbonyl" (or "heterocyclocarbonyl") is the
radical --C(O)M', wherein M' is an optionally substituted
heterocycle. Examples of heterocyclylcarbonyl include, but are not
limited to, piperazinoyl, morpholinoyl, and pyrrolindinoyl.
[0054] "Heterocyclylsulfonyl" is the radical --S(O).sub.2Z',
wherein M' is an optionally substituted heterocycle. Examples of
heterocyclylsulfonyl include, but are not limited to,
piperidinylsulfonyl and piperazinylsulfonyl.
[0055] "Heterocyclylsulfonyloxy" is the radical --OS(O).sub.2M',
wherein M' is an optionally substituted heterocycle. Examples of
heterocyclylsulfohyloxy include, but are not limited to,
3,5,dimethyl-isoxazolesulfonyloxy and pyrrolidinylsulfonyloxy.
Compounds
[0056] This invention provides compounds which inhibit tyrosine
kinases, particularly Tec (e.g., ITK, BTK), Src (Src, Lck, etc.)
and EGFR kinases (e.g., EGFR1, Her 2, Her 4), and Jak kinase (e.g.,
Jak3), having structures that exploit a discrete mechanistic
rationale described herein. This mechanism provides for the
utilization of the kinase catalytic machinery, described in the ITK
crystallographic structures as the acid-base pair residues Lys391
and Asp500 (herein referred to as the "catalytic dyad"), to trigger
a transformation that activates the proposed inhibitory compounds
within the enzyme active site. This transformation involves the
elimination of a leaving group, resulting in the in situ formation
of an electrophilic intermediate capable of forming a covalent
adduct with an active site cysteine residue thereby irreversibly
inhibiting the function of the target enzyme. This cysteine residue
is identifiable as Cys442 in the ITK crystallographic structure.
The group of kinases with the above described triad, including ITK,
BTK, BMX, Tec, TXK, BLK, EGFr, Her 2, Her 4 and JAK3, will be
referred to as the DKC triad kinases. Various embodiments of the
invention relate to this group, its possible sub-groupings, and to
its individual members.
[0057] It is known that several compounds, typically containing
electrophilic Michael acceptors, form covalent adducts with
enzymatic nucleophiles present in the active site to irreversibly
inhibit the target enzyme (Slichenmeyer, W. J.; Elliott, W. C.;
Fry, D. W. Semin. Oncol. 2001, 28, 80-85; Shimamura, T.; Ji, H.;
Minami, Y.; Thomas, R. K.; Lowell, A. M.; Sha, K.; Greulich, H.;
Glatt, K. A.; Meyerson, M.; Shapiro, I.; Wong, K.-K. Cancer Res.
2006, 66, 6487-6491). However, the compounds described in this
invention are unique in that the transformation that forms the
electrophilic intermediate takes place preferentially in situ, i.e.
within the enzyme active site. Outside of an appropriate active
site, these compounds are much less likely to undergo
beta-elimination and form adducts with other proteins. The
compounds described within must first bind in the active site of
the target kinase and achieve a specific conformational geometry
with respect to the relevant catalytic residues in order to
effectively trigger elimination of the leaving group, thereby
unmasking the adduct-forming intermediate. This intermediate forms
a covalent, irreversible adduct with the proximal active site
cysteine residue. In some embodiments the reaction proceeds
stepwise; in other embodiments it is concerted. In preferred
embodiments additional portions of the inhibitor molecule interact
with other portions of the kinase, particularly in the active site,
to promote favorable binding affinity and positioning. Such
interactions contribute to the specificity of various inhibitors so
that some inhibitors are inhibit a single kinase whereas others
inhibit multiple kinases with similar or different IC.sub.50s. To
our knowledge, this is the first example of an in situ formation of
an active inhibitor in a kinase active site.
Compound Interaction with the Kinase Domain
[0058] Without specifying the kinetics of the reaction, the
inhibition of the target kinase goes through the following sequence
of steps to form the adduct with the inhibitory compounds: [0059]
(1) The catalytic lysine N--H is positioned within hydrogen bonding
distance (approximately 1.8-4.0 Angstroms) of a hydrogen bond
acceptor Y in the compound that exists in the form of a C.dbd.Y
(Y.dbd.O, S, NOR) functionality. Polarization of the C.dbd.Y bond
results in increasing the acidity of the proton (H.sub.A) at a
carbon atom alpha to the C.dbd.Y group. [0060] (2) Acting as a
base, the aspartate of the catalytic dyad extracts the acidic
proton H.sub.A, leaving behind a conjugated carbanion that forms
for Y.dbd.O, an enol, H-bonded enolate through standard electronic
rearrangement. For Y.dbd.S, it would form a thioenol or H-bonded
thioenolate, and for Y.dbd.NOR, it would form an alkoxy (R=alkyl),
aryloxy (R=aryl) or hydroxy (R.dbd.H) enamine. [0061] (3) The
formation of the enol/thioenol/enamine facilitates the elimination
of the leaving group attached at a carbon beta to C.dbd.Y, through
a process known as ".beta.-elimination." The leaving group,
attached to the compound through protonatable heteroatom Z, may
optionally be additionally tethered to the rest of the
compound.
[0062] (4) Being a strong nucleophilic species, the sulfhydryl
group of the neighboring cysteine residue reacts with the newly
formed electrophilic elimination product. This addition reaction
(thioalkylation) forms the covalent adduct to the kinase resulting
in its irreversible inhibition and abrogation of activity.
[0063] The inhibitory activity of this class of compounds toward
select kinases is dependent on their ability to bind effectively in
proximity to the appropriate calalytic environment, the existence
of a polarizable C.dbd.Y group (C.dbd.O in formula (I), below) with
appropriate reactivity and an adjacent alpha proton to allow
elimination of the beta leaving group.
[0064] In turn, the elimination process that generates a reactive
electrophilic species requires removal of the abstractable alpha
proton that is facilitated by adequate positioning of the C.dbd.Y
group in the catalytic environment. The generated electrophilic
Michael acceptor, in turn is required to be positioned within
reactive distance of the key cysteine residue. The appropriate
positioning of the abstractable proton in the kinase binding site
is achieved through pharmacophoric elements that include: [0065]
(i) a C.dbd.Y moiety that serves the dual purpose of polarizing the
proximal C--H bond of the abstractable proton, and hydrogen bonding
to the lysine residue of the catalytic pair; [0066] (ii) a
hydrophobic aryl or heteroaryl group that interacts with specific
hydrophobic residues in the binding site at an approximate distance
of 3-5 .ANG. from Y, [0067] (iii) several (one to 3) hydrophilic
pharmacophores that interact with the backbone in the hinge region,
[0068] (vi) a carbon atom in the beta position from the C.dbd.Y
carbon atom, that is positioned within reactive distance of the
sulfhydryl group of the relevant cysteine as explained below.
[0069] The effective "reactive distance" to the cysteine sulfhydryl
group as stated above is observed in the range of about 3-10 .ANG.
using computational design methods that test the binding of
inhibitors to the ITK ATP binding site, wherein the enzyme is
maintained in a fixed conformation. While a distance of 10 .ANG. in
a rigid system would be too far to effect a chemical reaction, the
enzymatic nucleophilic moiety and the inhibitor's electrophilic
moiety can readily be brought together through a series of low
energy barrier rotations around the flexible inhibitor bonds as
well as the cysteinyl side chain. Overall global conformational
changes, common to kinase systems, cannot be ruled out either but
are not readily measurable. Such conformational changes, which can
be envisioned by computational predictions, are adequate in
bringing the two reactive pieces in close enough proximity to
effect covalent bond formation.
[0070] Compounds according to the invention have the structural
formula:
##STR00001##
wherein: Ar is optionally substituted aryl or optionally
substituted heteroaryl; R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are
independently hydrogen or optionally substituted C.sub.1-C.sub.6
alkyl; and R.sup.1 and R.sup.2 (a) are independently hydrogen,
optionally substituted C.sub.1-C.sub.6 alkyl, piperidine, or
furanyl; or (b) are taken together with the nitrogen atom to which
they are attached to form (i) a 5- to 7-membered optionally
substituted aryl, (ii) a 5- to 7-membered optionally substituted
heteroaryl, or (iii) a 5- to 7-membered optionally substituted
heterocycle which may be unfused or fused to an optionally
substituted aryl.
[0071] In some embodiments Ar is selected from the group consisting
of:
##STR00002##
wherein A, B, E, and Q are independently CH, O, or N; and D and D'
are independently CH.sub.2, NH, O, or S.
[0072] In other embodiments Ar is selected from the group
consisting of:
##STR00003##
[0073] Examples of 5- to 7-membered heterocycles include:
##STR00004##
wherein:
G is N, CH, or S;
G' is NH, CH, or S;
[0074] n=0-2; R.sup.1 and R.sup.2 are as defined above; and R.sup.7
is hydrogen, optionally substituted C.sub.1-C.sub.6 alkyl,
optionally substituted aryl, or optionally substituted
heteroaryl.
[0075] Preferred 5- to 7-membered heterocycles are piperazinyl,
piperidinyl, pyrrolidinyl, and morpholinyl. Preferred substituents
for piperazinyl are C.sub.1-C.sub.6 alkyl, dialkyl C.sub.1-C.sub.6
aminoalkyl, aryl, aralkyl, cycloalkyl, and cycloalkyl-alkyl.
Preferred substituents for piperidinyl are C.sub.1-C.sub.6 alkyl
and aralkyl. In some embodiments piperidinyl is benzofused to form
isoquinolinyl. Preferred substituents for pyrrolidinyl are
C.sub.1-C.sub.6 alkyl, aryl, and aralkyl. In some embodiments
pyrrolidinyl is benzofused to form isoindolyl. Preferred
substituents for morpholinyl are C.sub.1-C.sub.6 alkyl and
arylalkyl.
[0076] Some compounds have the structural formula:
##STR00005##
wherein: [0077] R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are as
defined above; R.sup.9 is selected from,
##STR00006## ##STR00007## ##STR00008## ##STR00009##
##STR00010##
[0077] and [0078] R.sup.10 is hydrogen, --OH, --COOH, --CONH.sub.2,
or --NCO, [0079] wherein if R.sup.9 is naphthyl, then R.sup.5 and
R.sup.6 are not both methyl.
[0080] Examples of these compounds include:
##STR00011## ##STR00012## ##STR00013## ##STR00014## ##STR00015##
##STR00016##
[0081] Other compounds have the structural formula:
##STR00017##
wherein: [0082] R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are as
defined above; [0083] R.sup.11 and R.sup.12 are independently
selected from hydrogen, --OCH.sub.3, halogen, --NO.sub.2, --CN,
--CF.sub.3, --NCOR' (wherein R' is hydrogen or C.sub.1-C.sub.4
alkyl), phenyloxy, --OCF.sub.3, --NR'R'' (wherein R' and R'' are
independently hydrogen or C.sub.1-C.sub.4 alkyl), C.sub.1-C.sub.4
alkyl, C.sub.r C.sub.4 alkoxy, and --SO.sub.2R' (wherein R' is
hydrogen or C.sub.1-C.sub.4 alkyl); and [0084] R.sup.13 is
hydrogen, C.sub.1-C.sub.4 alkyl,
##STR00018##
[0084] with the proviso that formula (III) does not include the
following compounds:
##STR00019## ##STR00020## ##STR00021##
One example of a compound of formula (III) is
##STR00022##
[0085] Some compounds of the invention have the structural
formula:
##STR00023##
wherein R.sup.1 and R.sup.2 are as defined above, with the
exception of
##STR00024##
[0086] Examples of such compounds include those of with the
following structural formulae:
##STR00025##
in which: [0087] GG is hydrogen, dimethylaminoalkyl, aryl,
C.sub.1-C.sub.6 alkyl, cyclohexylalkyl, pyridine, --COCF.sub.3;
--CONR'R'', or
[0087] ##STR00026## [0088] J is hydrogen, aralkyl, C.sub.1-C.sub.6
alkyl, --CNHCOOR', or NR'R''; [0089] K is hydrogen, pyridine, aryl,
--COOH, --CONR'R'', --COH, or --CNR'R''; [0090] L is hydrogen or
alkyloxy; and [0091] R.sup.2 is as defined above.
[0092] Other compounds of formula (IV) include:
##STR00027## ##STR00028## ##STR00029##
[0093] Other compounds have the structural formula:
##STR00030##
wherein R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.10 are as
defined above.
[0094] Other compounds have the structural formula:
##STR00031##
wherein: [0095] R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and
R.sup.6 are as defined above; and [0096] R.sup.14 is hydrogen or
.dbd.O; [0097] and D is CH or NH, with the exception of:
##STR00032##
[0098] Other compounds have the structural formula:
##STR00033##
wherein: [0099] R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are as
defined above; and [0100] R.sup.1 and R.sup.2 are independently
hydrogen, C.sub.1-C.sub.4 alkyl,
##STR00034##
[0100] (wherein R.sup.15 is halogen or C.sub.1-C.sub.4 alkyl and
R.sup.16 is C.sub.1-C.sub.4 alkyl), or R.sup.1 and R.sup.2 together
with the nitrogen to which they are attached form an aryl group
selected from
##STR00035##
(wherein R.sup.17 and R.sup.18 are independently hydrogen or
--OCH.sub.3),
##STR00036##
(wherein R.sup.1 and R.sup.2 are independently hydrogen or
C.sub.1-C.sub.4 alkyl),
##STR00037##
phenyl-C.sub.1-C.sub.4 alkyl (optionally substituted with halogen),
with the exception of
##STR00038##
[0101] Other compounds have the structural formula:
##STR00039##
wherein R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are as defined
above.
[0102] Other compounds have the structural formula:
##STR00040##
wherein R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are as defined above
and wherein R.sup.1 is hydrogen and R.sup.2 is
##STR00041##
wherein R.sup.19 is selected from hydrogen and
##STR00042##
or R.sup.1 and R.sup.2 together with the nitrogen to which they are
attached are
##STR00043##
A is N or O;
[0103] R.sup.20 is phenyl-C.sub.1-C.sub.4 alkyl optionally
substituted with one or more halogens, hydrogen, C.sub.1-C.sub.4
alkyl, amino-C.sub.1-C.sub.4 alkyl,
##STR00044##
R.sup.17 and R.sup.18 are independently hydrogen or
--OCH.sub.3;
R.sup.21 is --CONR'R'', --COR',
##STR00045##
[0104] and R' and R'' are independently selected from hydrogen and
C.sub.1-C.sub.4 alkyl.
[0105] In other embodiments compounds have the structural
formula:
##STR00046##
wherein R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are as defined above
and wherein R.sup.22 is selected from hydrogen, C.sub.1-C.sub.4
alkyl, --NR'R'', --COH, --COOH, --CNR'R'', and --CONHR', wherein R'
and R'' are as defined above.
[0106] In other embodiments compounds have the structural
formula:
##STR00047##
wherein R.sup.3, R.sup.4, R.sup.5, R.sup.6, G, and G' are as
defined above; and R.sup.23 is hydrogen, --NR'R'' C.sub.1-C.sub.4
linear alkyl, C.sub.1-C.sub.4 alkyl, phenyl-C.sub.1-C.sub.4 alkyl,
--CONH.sub.2, and --COR'R'', wherein R' and R'' are as defined
above.
[0107] In other embodiments compounds have the structural
formula:
##STR00048##
R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are as defined above and
wherein R.sup.24 is
##STR00049##
[0108] In other embodiments compounds have the structural
formula:
##STR00050##
wherein L is
##STR00051##
and wherein R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are as defined
above.
[0109] Some compounds have the structural formula:
##STR00052##
wherein T, U, V, and W independently are selected from hydrogen;
halogen; --O; C.sub.1-C.sub.3 alkyl; and C.sub.1-C.sub.3 alkyloxy;
and wherein R.sup.25 is hydrogen or C.sub.1-C.sub.3 alkyl.
Representative compounds include:
##STR00053##
[0110] Other compounds have the structural formula:
##STR00054##
wherein T, U, V, and W independently are selected from hydrogen;
halogen; --O; C.sub.1-C.sub.3 alkyl; and C.sub.1-C.sub.3 alkyloxy;
and wherein R.sup.8 is hydrogen or C.sub.1-C.sub.3 alkyl.
[0111] Still other compounds have the structural formula:
##STR00055##
wherein T, U, V, and W independently are selected from hydrogen;
halogen; --O; C.sub.1-C.sub.3 alkyl; and C.sub.1-C.sub.3 alkyloxy;
and wherein R.sup.8 is hydrogen or C.sub.1-C.sub.3 alkyl.
[0112] Other compounds have the structural formula:
##STR00056##
wherein D is S, O, or NH; i.e.,
##STR00057##
[0113] Other compounds of the invention include those with the
structural formula:
##STR00058##
wherein D is defined above; i.e.,
##STR00059##
[0114] Other compounds of the invention include those with the
structural formula:
##STR00060##
wherein G' is NH or CH; i.e.,
##STR00061##
[0115] The invention also includes the compounds identified in
Examples 15 and 16.
[0116] The compounds of the present invention may have asymmetric
centers and may occur as racemates, stereoisomers, and tautomers.
The invention includes all possible racemates, tautomers,
stereoisomers, and mixtures thereof
[0117] Suitable methods of preparing compounds of the invention are
illustrated by the representative examples provided below. Starting
materials are known compounds and can be obtained by standard
procedures of organic chemistry.
[0118] Provisos for Compound Claims
[0119] Compounds of the invention preferably do not have one or
more of the following activities: vasodilator, hypotensive,
bradycardiac, anti-depressant, anti-arrhythmic,
anti-arteriosclerotic, serum cholesterol lowering, triglyceride
level lowering, neuroleptic, anti-inflammatory, tranquilizing,
anti-convulsant, anesthetic, muscle relaxing, anti-fungal,
anti-bacterial, insecticidal, fumigant, anti-parasitic, central
nervous system depressant, antagonization of sedation,
antipollakiurea, antihistamine, anti-allergy, bronchodilating,
analgesic, spasmolytic, muscarinic antagonist, preventing or
decreasing production of abnormally phosphorylated paired helical
filament epitopes associated with Alzheimer's Disease,
hypolipidemic, male anti-fertility, anti-sporicidal, inhibition of
nitric oxide production, or central nervous system stimulant
activities.
[0120] To the extent any of the following compounds are not novel,
Applicants reserve the right to present compound and/or composition
claims which include a proviso excluding the compounds and/or their
pharmaceutically acceptable salts from the scope of the claims:
[0121] a. compounds having the structural formula:
[0121] ##STR00062## [0122] wherein n is 0, 1, 2, or 3 and R.sup.1
and R.sup.2 together with the nitrogen atom to [0123] which they
are attached are
##STR00063##
[0123] and Y is alkyl, halogen, halogenoalkyl, alkyoxy, alkylthio,
halogenoalkyloxy, halogenoalkylthio, cycloalkyl, or a cyano
radical; [0124] b. compounds of formula (I) in which Ar is phenyl,
if R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are each hydrogen, and
R.sup.1 and R.sup.2 together form a ring with the nitrogen atom to
which they are attached
[0124] ##STR00064## [0125] c. compounds having the structural
formula:
[0125] ##STR00065## [0126] in which Ph is an optionally substituted
monocyclic carbocyclic aryl radical, Alk is C.sub.1-C.sub.3 lower
alkyl, and Am is a tertiary amino group, salts, N-oxides, or
quaternary ammonium derivatives thereof; [0127] d. compounds having
the structural formula:
[0127] ##STR00066## [0128] in which Ph.sub.1 and Ph.sub.2 are
monocyclic carboxylic aryl radicals and the acid addition salts
thereof; [0129] e. compounds having the structural formula:
[0129] ##STR00067## [0130] in which RR is selected from the group
consisting of aliphatic, aromatic, and araliphatic radicals;
RR.sup.1 is selected from the group consisting of hydrogen,
aliphatic, aromatic, and araliphatic radicals; --N(XX) is the
residue of a secondary amine selected from the group consisting of
dialkylamine and dialkylamines; [0131] f. compounds having the
structural formula:
[0131] ##STR00068## [0132] wherein R.sup.1 and R.sup.2 are as
defined in formula (I), including the compound
[0132] ##STR00069## [0133] g. compounds having the structural
formula:
[0133] ##STR00070## [0134] wherein R.sup.30 is an ethyl-, propyl-,
isopropyl-, butyl-, or isobutyl group or a cycloalkyl group having
5-7 carbon atoms; [0135] h. compounds having the structural
formula:
[0135] ##STR00071## [0136] in which M.sup.2 is hydrogen, halogen,
or C.sub.1-C.sub.12 alkoxy, M.sup.1 is hydrogen or halogen, and
M.sup.3 and M.sup.4 are lower alkyl or, taken together with the
nitrogen atom to which they are attached, (a) are a heterocyclic
amino group or an N-lower alkyl quaternary heterocyclic ammonium
group or (b) a tri-lower alkyl-ammonium; [0137] i. compounds having
the structural formula:
[0137] ##STR00072## [0138] or a picrate salt thereof, wherein
M.sup.5 is a simple or substituted aryl group and M.sup.6 is a
simple or substituted amino group; [0139] compounds having the
structural formula:
[0139] ##STR00073## [0140] in which M.sup.7 is thienyl, phenyl or
substituted phenyl, [0141] k. compounds having the structural
formula:
[0141] ##STR00074## [0142] in which each of X.sup.1, X.sup.2, and
X.sup.3 are independently hydrogen or an alkyl group, and each of
X.sup.5 and X.sup.4 are independently hydrogen or an alkyl group
or, together with the nitrogen atom to which they are attached,
form a heterocyclic group with 5, 6, or 7 ring atoms, optionally,
containing, in addition to N, a further heteroatom selected from N,
S, and O; [0143] l. compounds of formula (II) in which R.sup.9 is
phenyl and R.sup.3, R.sup.4, and R.sup.6 are each hydrogen; [0144]
m. compounds having the structural formula:
[0144] ##STR00075## [0145] in which. X.sup.6 forms with the
nitrogen atom pyrrolidine, piperidine, morpholine,
hexamethyleneimine, 3-azabicyclo-3,2,2 nonane, including the
compound
[0145] ##STR00076## [0146] n. compounds having the structural
formula:
[0146] ##STR00077## [0147] in which X.sup.7 is hydrogen or
fluorine; X.sup.8 is N(X.sup.9)phenyl (wherein the phenyl is
optionally monosubstituted with C.sub.1-C.sub.8 alkoxy,
C.sub.1-C.sub.8 alkyl, trifluoromethyl, or halogen),
--C(OH)(X.sup.9) phenyl (wherein the phenyl is optionally
monosubstituted with C.sub.1-C.sub.8 alkoxy, C.sub.1-C.sub.8 alkyl,
trifluoromethyl, or halogen), or phenyl (Wherein the phenyl is
optionally monosubstituted with C.sub.1-C.sub.8 alkoxy,
C.sub.1-C.sub.8 alkyl, trifluoromethyl, or halogen); and X.sup.9 is
hydrogen, C.sub.1-C.sub.8 alkyl, or lower alkanoyl; [0148] o.
compounds having the structural formula:
[0148] ##STR00078## [0149] wherein X.sup.9 and X.sup.10 each
designate a saturated or unsaturated aliphatic hydrocarbon having 1
to 4 carbon atoms or, together with the nitrogen to which they are
attached, form a heterocyclic radical selected from pyrrolidino,
piperidine, perhydroazepino, and morpholino; [0150] compounds
having the structural formula:
[0150] ##STR00079## [0151] in which X.sup.11 is C.sub.2-C.sub.3
alkyl; [0152] q. compounds having the structural formula:
[0152] ##STR00080## [0153] in which X.sup.11 is hydrogen, halogen,
C.sub.1-C.sub.4 alkoxy, nitro, or C.sub.1-C.sub.4 secondary amine;
X.sup.12 (CH.sub.2).sub.n--OX.sup.13; n is 2 or 3; and X.sup.13 is
C.sub.1-C.sub.4 alkoxyphenyl, nitrophenyl, trifluoromethylphenyl,
or phenyl disubstituted with two halogens, two C.sub.1-C.sub.4
alkyls, halogen and nitro, halogen and C.sub.1-C.sub.4 alkyl,
halogen and C.sub.1-C.sub.4 alkoxy, or C.sub.1-C.sub.4 alkoxy and
C.sub.1-C.sub.4 alkoyl; [0154] r. compounds having the structural
formula:
[0154] ##STR00081## [0155] in which X.sup.14, X.sup.15, and
X.sup.16 are independently hydrogen, halogen, C.sub.1-C.sub.4
alkyl, halogeno-C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, or a
cycloalkyl group having 3-8 carbon atoms and two of X.sup.14,
X.sup.15, and X.sup.16 may combine to form methylenedioxy or
ethyleneoxy; X.sup.18 is hydrogen or C.sub.1-C.sub.4 alkyl; and
X.sup.17 is pyrrolidinyl-, morpholinyl-, or azepinyl; [0156] s.
compounds having the structural formula:
[0156] ##STR00082## [0157] Ar denotes an aryl radical; and X.sup.19
and X.sup.20 (a) are both C.sub.1-C.sub.6 alkyl or (b) together
with the N atom form the remaining members of a saturated
heterocyclic radical and X.sup.21 is --OH, C.sub.1-C.sub.6 alkyl,
or aryl; [0158] t. compounds having the structural formula:
[0158] ##STR00083## [0159] wherein R.sup.1 and R.sup.2
independently represent an alkyl radical; or [0160] R.sup.1 and
R.sup.2, together with the nitrogen atom to which they are bonded
complete an optionally substituted heterocyclic radical of the
formula
[0160] ##STR00084## [0161] R.sup.3 is hydrogen or C.sub.1-C.sub.4
alkyl; and X.sup.22, X.sup.23, and X.sup.24 are independently
C.sub.1-C.sub.4 alkyl; halogen, or a halogeno C.sub.1-C.sub.4
alkyl, C.sub.1-C.sub.4 alkoxy, alkylthio, halogeno-C.sub.1-C.sub.4
alkoxy, halogeno-C.sub.1-C.sub.4 alkylthio, cycloalkyl 3 to 7
carbon atoms, or cyano; [0162] u. compounds having the structural
formula:
[0162] ##STR00085## [0163] wherein Ar is non-substituted aryl or
aryl substituted with a hydroxyl group, lower alkoxy group or
halogen, or non-substituted benzo[b]thienyl group or
benzo[b]thienyl group substituted by hydroxyl group, lower alkyl
group, lower alkoxy group, aryl group or halogen; R.sup.5 is
hydrogen or C.sub.1-C.sub.4 alkyl; and X.sup.25 is a group other
than piperidine; [0164] v. compounds having the structural
formula:
[0164] ##STR00086## [0165] wherein L.sup.1 and L.sup.2 are
independently halogen or alkyl; L.sup.6 and L.sup.7 are
independently hydrogen or alkyl; and L.sup.3 and L.sup.4 are
independently hydrogen or an aliphatic group or combine together
with the nitrogen to which they are attached to form a ring; [0166]
w. compounds of formula, (I), (IV), (VI), (VII), (IX), and (XI) in
which if R.sup.3 and R.sup.4 are hydrogen, then
[0166] ##STR00087## is not
##STR00088## wherein L.sup.8 is a carbonyl, sulfonyl, methylene, or
methylene substituted with optionally substituted phenyl; and Ar is
an aryl group; [0167] x. compounds having the structural
formula:
[0167] ##STR00089## [0168] in which T.sup.1 is O, S, or NT.sup.7;
T.sup.7 is hydrogen, C.sub.1-C.sub.4 alkyl, and
CH.sub.2CH.sub.2COAr.sub.1; T.sup.6 is hydrogen, C.sub.1-C.sub.6
alkyl, or T.sup.6 and a substituent the an group together represent
CH.sub.2, CH.sub.2CH.sub.2, CH.sub.2O, or CH.sub.2S to form a five
or six membered ring where the ring is optionally substituted with
C.sub.1-C.sub.6 alkyl or phenyl; T.sup.5 is hydrogen,
C.sub.1-C.sub.6 alkyl, or optionally substituted phenyl; T.sup.2,
T.sup.3, and T.sup.4 are independently hydrogen or C.sub.1-C.sub.6
alkyl; and Ar and Ar.sub.1 are aryl or optionally substituted
phenyl; and [0169] y. the following compounds:
##STR00090##
[0169] Pharmaceutical Preparations
[0170] Compounds of the invention can be formulated as
pharmaceuticals using methods well known in the art. Pharmaceutical
formulations of the invention typically comprise at least one
compound of the invention mixed with a carrier, diluted with a
diluent, and/or enclosed or encapsulated by an ingestible carrier
in the form of a capsule, sachet, cachet, paper or other container
or by a disposable container such as an ampoule.
[0171] A carrier or diluent can be a solid, semi-solid or liquid
material. Some examples of diluents or carriers which may be
employed in the pharmaceutical compositions of the present
invention are lactose, dextrose, sucrose, sorbitol, mannitol,
propylene glycol, liquid paraffin, white soft paraffin, kaolin,
microcrystalline cellulose, calcium silicate, silica
polyvinylpyrrolidone, cetostearyl alcohol, starch, gum acacia,
calcium phosphate, cocoa butter, oil of theobroma, arachis oil,
alginates, tragacanth, gelatin, methyl cellulose, polyoxyethylene
sorbitan monolaurate, ethyl lactate, propylhydroxybenzoate,
sorbitan trioleate, sorbitan sesquioleate and oleyl alcohol.
[0172] Pharmaceutical compositions of the invention can be
manufactured by methods well known in the art, including
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping, or lyophilizing
processes.
[0173] For injection, the agents of the invention may be formulated
in aqueous solutions, preferably in physiologically compatible
buffers such as acetate, Hanks's solution, Ringer's solution, or
physiological saline buffer. Preferably the solutions are sterile
and non-pyrogenic. For transmucosal administration, penetrants
appropriate to the barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.
[0174] For oral administration, the active compound(s) can be
combined with pharmaceutically acceptable carriers which enable the
compound(s) to be formulated as tablets, pills, dragees, capsules,
liquids, gels, syrups, slurries, suspensions and the like. Fillers
can be used, such as gelatin, sugars (e.g., lactose, sucrose,
mannitol, or sorbitol); cellulose preparations (e.g., maize starch,
wheat starch, rice starch, potato starch, gum tragacanth, methyl
cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose); and/or polyvinylpyrrolidone (PVP). If
desired, disintegrating agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0175] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0176] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compound(s)
may be dissolved or suspended in suitable liquids, such as fatty
oils, liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
preferably are in dosages suitable for such administration.
[0177] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0178] For administration by inhalation, pharmaceutical
preparations of the invention can be delivered in the form of an
aerosol sprays from pressurized packs or a nebulizer, with the use
of a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide,
or other suitable gas. If desired, a valve can be used to deliver a
metered amount. Capsules and cartridges of e.g., gelatin for use in
an inhaler or insufflator, may be formulated containing a powder
mix of a compound and a suitable powder base such as lactose or
starch.
[0179] Compounds of the invention can be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection can be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions can take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0180] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds. Additionally,
suspensions of the active compounds may be prepared as appropriate
oily injection suspensions. Suitable lipophilic solvents or
vehicles include fatty oils such as sesame oil, or synthetic fatty
acid esters, such as ethyl oleate or triglycerides, or liposomes.
Aqueous injection suspensions may contain substances which increase
the viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Optionally, the suspension may
also contain suitable stabilizers or agents which increase the
solubility of the compounds to allow for the preparation of highly
concentrated solutions.
[0181] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
[0182] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0183] Compounds of the invention typically are soluble and stable
in 50 mM acetate at a concentration of 10 mg/ml or above, and can
be delivered intraperitoneally and orally in this buffer. Some
compounds are soluble in hydroxypropyl-b-cyclodextrin (HBPCD,
3-5%), and can be delivered intraperitoneally and orally in this
solvent. For intravenous delivery, compounds can be suspended or
dissolved in 5% mannitol.
[0184] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0185] The pharmaceutical compositions also may comprise suitable
solid or gel phase carriers or excipients. Examples of such
carriers or excipients include but are not limited to, calcium
carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin, and polymers such as polyethylene
glycols.
[0186] In addition to the common dosage forms set out above, the
compounds of the present invention may also be administered by
controlled release means and/or delivery devices including
ALZET.RTM. osmotic pumps which are available from Alza Corporation.
Suitable delivery devices are described in U.S. Pat. Nos.
3,845,770; 3,916,899; 3,536,809; 3,598,123; 3,944,064 and
4,008,719.
Therapeutic Methods
[0187] The identified compounds can be administered to a human
patient, either alone or in pharmaceutical compositions where they
are mixed with suitable carriers or excipient(s) at doses to treat
or ameliorate blood-related cancers (e.g., lymphomas and leukemias)
and autoimmune disorders. Reduction of intracellular kinase
activity also is useful to suppress the immune system of transplant
patients prior to, during, and/or after transplant.
[0188] Lymphomas are malignant growths of B or T cells in the
lymphatic system, including Hodgkin's lymphoma and non-Hodgkin's
lymphoma. Non-Hodgkin's lymphomas include cutaneous T cell
lymphomas (e.g., Sezary syndrome and Mycosis fungoides), diffuse
large cell lymphoma, HTLV-1 associated T cell lymphoma, nodal
peripheral T cell lymphoma, extranodal peripheral T cell lymphoma,
central nervous system lymphoma, and AIDS-related lymphoma.
[0189] Leukemias include acute and chronic types of both
lymphocytic and myelogenous leukemia (e.g, acute lymphocytic or
lymphoblastic leukemia, acute myelogenous leukemia, acute myeloid
leukemia, chronic myelogenous leukemia, chronic lymphocytic
leukemia, T cell prolymphocytic leukemia, adult T cell leukemia,
and hairy cell leukemia).
[0190] Autoimmune disorders include systemic lupus erythematosus,
anti-phospholipid antibody syndrome, multiple sclerosis, ulcerative
colitis, Crohn's disease, rheumatoid arthritis, asthma, Hashimoto's
thyroiditis, Reiter's syndrome, Sjogren's syndrome, Guillain-Barre
syndrome, myasthenia gravis, large vessel vasculitis, medium vessel
vasculitis, polyarteritis nodosa, pemphigus vulgaris, scleroderma,
Goodpasture's syndrome, glomerulonephritis, primary biliary
cirrhosis, Grave's disease, membranous nephropathy, autoimmune
hepatitis, celiac sprue, Addison's disease, polymyositis,
dermatomyositis, monoclonal gammopathy, Factor VIII deficiency,
cryoglobulinemia, peripheral neuropathy, IgM polyneuropathy,
chronic neuropathy, autoimmune hemolytic anemia, autoimmune
thrombocytopenic purpura, pernicious anemia, ankylosing
spondylitis, vasculitis, inflammatory bowel disease, and type I
diabetes mellitus. The autoimmune disease may involve a secretory
cell, such as a T lymphocyte, B lymphocyte, Mast cell, or dendritic
cell. Compounds of the invention also can be used to treat patients
who undergo protein replacement therapies and who develop
antibodies to the replacement.
[0191] Routes of Administration
[0192] Pharmaceutical preparations of the invention can be
administered locally or systemically. Suitable routes of
administration include oral, pulmonary, rectal, transmucosal,
intestinal, parenteral (including intramuscular, subcutaneous,
intramedullary routes), intranodal, intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal,
intraocular, transdermal, topical, and vaginal routes. As described
in more detail above, dosage forms include, but are not limited to,
tablets, troches, dispersions, suspensions, suppositories,
solutions, capsules, creams, patches, minipumps and the like.
Targeted delivery systems also can be used (for example, a liposome
coated with target-specific antibody).
[0193] Dosage
[0194] A pharmaceutical composition of the invention comprises at
least one active ingredient in a therapeutically effective amount.
A "therapeutically effective dose" is the amount of an active agent
which, when administered to a patient, results in a measurable
improvement in a characteristic of the disease being treated (e.g.,
improved laboratory values, retarded development of a symptom,
reduced severity of a symptom, improved levels of a biological
marker such as CD25a or IL2). The improvement can be evident after
a single administration of the therapeutically effective dose. More
usually multiple administrations are utilized in order to achieve
or maintain optimal effect. In preferred embodiments frequency of
administration can range from twice a month to once a week to
several times a day, for example 1-4 times a day. In alternative
embodiments administration can be by time-release formulations, or
extended or continuous infusions. The frequency of administration
can be selected to achieve a systemic or local concentration at or
above some predetermined level for a period of time. The period of
time can be all or a substantial portion of the interval between
administrations or comprise the period of time-release or infusion.
In some embodiments, the treatment schedule can require that a
concentration of the compound be maintained for a period of time
(e.g., several days or a week) and then allowed to decay by ceasing
administration for a period of time (e.g., 1, 2, 3, or 4
weeks).
[0195] Determination of therapeutically effective amounts is well
within the capability of those skilled in the art. A
therapeutically effective dose initially can be estimated from in
vitro enzyme assays, cell culture assays and/or animal models. For
example, a dose can be formulated in an animal model to achieve a
circulating concentration range that includes the IC.sub.50 as
determined in an in vitro enzyme assay or in a cell culture (i.e.,
the concentration of the test compound which achieves a
half-maximal inhibition of ITK or BTK activity). Such information
can be used to more accurately determine useful doses in
humans.
[0196] Appropriate animal models for the relevant diseases are
known in the art. See, e.g., Exp Hematol. 34, 284-88, 2006
(aggressive systemic mastocytosis and mast cell leukemia); Leuk.
Lymphoma. 47, 521-29, 2006 (acute myeloid leukemia); Leuk.
Lymphoma. 7, 79-86, 1992 (disseminated human B-lineage acute
lymphoblastic leukemia and non-Hodgkins lymphoma); J. Virol. 79,
9449-57, 2006 (adult T-cell leukemia); Neoplasia 7, 984-91, 2005
(lymphoma); Oligonucleotides 15, 85-93, 005 (lymphoma); Transfus.
Apher. Sci. 32, 197-203, 2005 (cutaneous T cell lymphoma); Nature
17, 254-56, 1991 (follicular lymphoma and diffuse large cell
lymphoma); Cell. Mol. Immunol. 2, 461-65, 2005 (myasthenia gravis);
Proc. Natl. Acad. Sci. USA 102, 11823-28, 2005 (type I diabetes);
Arthritis Rheum. 50, 3250-59, 2004 (lupus erythymatosus); Clin.
Exp. Immunol. 99, 294-302, 1995 (Grave's disease); J. Clin. Invest.
116, 905-15, 2006 (multiple sclerosis); Pharmacol Res. e-published
Feb. 1, 2006 (ulcerative colitis); J. Pathol. e-published Mar. 21,
2006 (Crohn's disease); J. Clin. Invest. 116, 961-973, 2006
(rheumatoid arthritis); Endocrinol. 147, 754-61, 2006 (asthma); Exp
Mol Pathol. 77, 161-67, 2004 (Hashimoto's thyroiditis); J.
Rheumatol. Suppi. 11, 114-17, 1983 (Reiter's syndrome); Rheumatol.
32, 1071-75, 2005 (Sjogren's syndrome); Brain Pathol. 12, 420-29,
2002 (Guillain-Barre syndrome); J. Clin. Invest. 110, 955-63, 2002
(vessel vasculitis); Vet. Pathol. 32, 337-45, 1995 (polyarteritis
nodosa); Immunol. Invest. 3, 47-61, 2006 (pemphigus vulgaris);
Arch. Dermatol. Res. 297, 333-44, 2006 (scleroderma); J. Exp. Med.
191, 899-906, 2000 (Goodpasture's syndrome); J. Vet. Med. Sci. 68,
65-68, 2006 (glomerulonephritis); Liver Int. 25, 595-603, 2005
(primary biliary cirrhosis); Clin. Exp. Immunol. 99, 294-302, 1995
(Grave's disease); J. Clin. Invest. 91, 1507-15, 1993 (membranous
nephropathy); J. Immunol. 169, 4889-96, 2002 (autoimmune
hepatitis); Isr. J. Med. Sci. 15, 348-55, 1979 (celiac sprue);
Surgery 128, 999-1006, 2000 (Addison's disease); J. Neuroimmunol.
98, 130-35, 1999 (polymyositis); Am. J. Pathol. 120, 323-25, 1985
(dermatomyositis); Bone 20, 515-20, 1997 (monoclonal gammopathy);
Haemophilia 11, 227-32, 2005 (Factor VIII deficiency); Proc. Natl.
Acad. Sci. USA 94, 233-36, 1997 (cryoglobulinemia); Pain 110,
56-63, 2004 (peripheral neuropathy); Ann. Neurol. 49, 712-20, 2001
(IgM polyneuropathy); J. Neurosci. Res. 44, 58-65, 1996 (chronic
neuropathy); Eur. J. Immunol. 32, 1147-56, 2002 (autoimmune
hemolytic anemia); Haematologica 88, 679-87, 2003 (autoimmune
thrombocytopenic purpura); Curr. Top. Microbiol. Immunol. 293,
153-77, 2005 (pernicious anemia); J. Immunol. 175, 2475-83, 2005
(ankylosing spondylitis); Inflamm. Res. 53, 72-77, 2004
(vasculitis); Vet. Pathol. 43, 2-14, 2006 (inflammatory bowel
disease); and J. Biol. Chem. 276, -13821, 2001 (anti-phospholipid
antibody syndrome).
[0197] LD.sub.50 (the dose lethal to 50% of the population) and the
ED.sub.50 (the dose therapeutically effective in 50% of the
population) can be determined by standard pharmaceutical procedures
in cell cultures and/or experimental animals. Data obtained from
cell culture assays or animal studies can be used to determine
initial human doses. As is known in the art, the dosage may vary
depending upon the dosage form and route of administration
used.
[0198] As is well known, the FDA guidance document "Guidance for
Industry and Reviewers Estimating the Safe Starting Dose in
Clinical Trials for Therapeutics in Adult Healthy Volunteers"
(HFA-305) provides an equation for use in calculating a human
equivalent dose (HED) based on in vivo animal studies. Based on the
studies described in Example 16, below, the human equivalent dose
ranges between 1.5 mg/kg and 8 mg/kg, with some compounds showing
considerable efficacy at lower or higher doses than those estimated
by the HED. Thus, human dosages for systemic administration can
range from, e.g., 1.5 mg/kg to 3 mg/kg; 2 mg/kg to 4 mg/kg; 5 mg/kg
to 7 mg/kg; and 4 mg/kg to 8 mg/kg. The amount of composition
administered will, of course, be dependent on the subject being
treated, on the subject's weight, the severity of the disorder, the
manner of administration and the judgment of the prescribing
physician.
[0199] All patents, patent applications, and references cited in
this disclosure are expressly incorporated herein by reference. The
above disclosure generally describes the present invention. A more
complete understanding can be obtained by reference to the
following specific examples, which are provided for purposes of
illustration only and are not intended to limit the scope of the
invention.
Example 1
Preparation of 1-naphthalen-2-yl-prop-2-en-1-ol
##STR00091##
[0201] Naphthaldehyde (5.0 g, 32.0 mmole) was dissolved in
anhydrous tetrohydrofuran and stirred at -78.degree. C. under
N.sub.2 (g) atmosphere. To the mixture was added vinyl magnesium
bromide (50 ml, 1 M solution in THF) and the reaction was warmed to
room temperature and stirred overnight. The reaction was quenched
with water and partitioned between EtOAc and water. The organic
layer was washed with brine, dried over sodium sulfate, filtered,
and concentrated under vacuum to give the desired product as yellow
oil (5.0 g, 85%). ESI-MS m/z 185 (M+H).sup.+.
Example 2
Preparation of 1-naphthalen-2-yl-propenone
##STR00092##
[0203] To a solution of 1-naphthalen-2-yl-prop-2-en-1-ol (1.3 g,
7.0 mmole) in 30 ml of dichloromethane was added pyridinium
chlorochromate (1.5 g, 7.0 mmole). The mixture was stirred at room
temperature until oxidation was complete. The solution was filtered
through celite and the solvent was concentrated under vacuum. The
residue was redissolved in EtOAc and washed with water and brine,
dried over sodium sulfate, filtered, and concentrated under vacuum.
The residue was purified by HPLC using a 0-100% EtOAc-Hx gradient
to give the desired product as yellow oil (280 mg, 22%). ESI-MS m/z
183 (M+H).sup.+.
Example 3
Preparation of 1-naphthalen-2-yl-3-piperidin-1-yl-propan-1-one
##STR00093##
[0205] 1-Naphthalen-2-yl-propenone (10 mg, 0.05 mmole) was
dissolved in 100 .mu.l of DMSO in one well of a 96 well
polypropylene plate. To the mixture was added piperidine (12 .mu.l,
0.10 mmole) and diisopropylethyl amine (17 .mu.l, 0.1 mmole). After
completion, the product was purified using HPLC to give the desired
product (50 mm.times.10 mm Phenomenex GEMINI.TM. column using a
30-100% acetonitrile-water gradient). ESI-MS m/z 268
(M+H).sup.+.
Example 4
Preparation of 1H-Pyrrolo[2,3-b]pyridine 7-oxide
##STR00094##
[0207] 7-Azaindole (10 g, 84.7 mmol) was dissolved in ether (300
mL) at room temperature. M-CPBA (29.1 g, 1.5 eq.) was added in
portions and stirred by manual agitation. After all oxidant was
added, the mixture was stirred at room temperature for a further 3
hours. LC/MS showed complete conversion. The mixture was filtered,
and the solid was washed with ether (40 mL.times.3) and air-dried.
NMR analysis of this solid in d6-DMSO obtained showed the product
as mostly the meta-Chloro benzoic acid salt of
1H-Pyrrolo[2,3-b]pyridine 7-oxide (off white, 17.9 g); MS: m/z
135.3 [MH.sup.+].
Example 5
Preparation of 4-Chloro-1H-pyrrolo[2,3-b]pyridine
##STR00095##
[0209] The m-CBA salt of 1H-Pyrrolo[2,3-b]pyridine 7-oxide (9 g)
was taken into POCl.sub.3 (46 mL, 7.5 eq.). The mixture was heated
at 90.degree. C. for 15 hours and to 106.degree. C. for another 4
hours. The mixture was cooled to room temperature, and most of the
POCl.sub.3 was distilled off under high vacuum. The residue was
dissolved in CH.sub.3CN (10 mL). Water (20 mL) was added slowly to
quench the reaction. The resulted mixture was adjusted to
pH.about.9 using 10 N NaOH. The solid was filtered. The crude solid
was redissolved in several ml of THF and combiflashed using 0-10%
MeOH in DCM to give 4-Chloro-1H-pyrrolo[2,3-b]pyridine as a
slightly yellowish solid. (4 g). MS: m/z 154.9 [MH.sup.+]
Example 6
Preparation of
1-[4-(1H-Pyrrolo[2,3-b]pyridin-4-yl)-phenyl]ethanone
##STR00096##
[0211] 4-Chloro-1H-pyrrolo[2,3-b]pyridine (500 mg, 3.27 mmol) was
dissolved in dioxane (11 mL). 4-Acetyl phenylboronic acid (802 mg,
4.9 mmol, 1.5 eq), dppfPdCl.sub.2 (41 mg, 0.03 mmol, 0.01 eq) and
Na.sub.2CO.sub.3 (2 N aq., 8.6 mL) were charged. The mixture was
vacuumed and flushed with N.sub.2 and microwaved at 160.degree. C.
for 15 minutes. Six batches of this same reaction were carried out.
The crude mixture was pooled and partitioned between DCM (40 mL)
and water (20 mL). Combi-flash of the residue using hexane/EtOAc
(0% to 100%) gave the free base azaindole derivative as a slightly
yellowish solid. The solid was redissolved in DCM (20 mL) and
stirred in an ice bath. A 2M HCl solution in ether (10 mL) was
added dropwise. The precipitate was filtered and dried to give
1-[4-(1H-Pyrrolo[2,3-b]pyridin-4-yl)-phenyl]-ethanone. (2.5 g,
48%). MS: m/z 237.3 [MH.sup.+].
Example 7
Preparation of 1-[3-(2-Chloro-pyridin-4-yl)-phenyl]-ethanone
##STR00097##
[0213] 2-Chloropyridine-4-boronic acid (11.0 g, 69.9 mmol),
3-Bromoacetophenone (11.2 mL, 83.9 mmol, 1.2 eq.), Na.sub.2CO.sub.3
(35 mL, 244.65 mmol, 3.5 eq.) and dppfPdCl.sub.2 (572 mg, 0.07
mmol, 0.01 eq.) were mixed in THF (200 mL). The mixture was heated
to reflux and continued at this temperature for 6 hours. It was
then cooled and concentrated in vacuo. The residue was partitioned
between DCM and water (100 mL/40 mL). The layers were separated and
the aqueous layer was washed further with DCM (2.times.40 mL). The
combined organic layer was dried (Na.sub.2SO.sub.4) and filtered.
The filtrate was concentrated, and the residue was chromatographed
using 1/1 hexane/EtOAc to give
1-[3-(2-Chloro-pyridin-4-yl)-phenyl]-ethanone as a white solid (9.5
g, 58%). MS: m/z 232.1 [MH.sup.+].
Example 8
Preparation of N-[4-(3-Acetyl-phenyl)-pyridin-2-yl]-benzamide
##STR00098##
[0215] A degassed mixture of
1-[3-(2-Chloro-pyridin-4-yl)-phenyl]-ethanone (500 mg, 2.16 mmol),
benzamide (523 mg, 4.32 mmol, 2 eq.), Xantphos (120 mg, 0.21 mmol,
0.1 eq.), Pd(OAc).sub.2 (24 mg, 0.10 mmol, 0.05 eq.),
K.sub.2CO.sub.3 (448 mg, 3.24 mmol, 1.5 eq.) in dioxane (12 mL) was
irradiated with microwaves at 150.degree. C. for 1 hour. LC/MS
control. Conversion was mostly 100% based on disappearance of
starting material. Dimer (M+: 392) being the major by-product. If
any starting material is unreacted at this point, another portion
of Xantphos and Pd(OAc).sub.2 may be added and the mixture
microwaved for another 30 minutes. The mixture was then partitioned
between DCM and water (20 mL/10 mL). The layers were separated and
the aqueous layer was washed further with DCM (2.times.20 mL). The
combined organic layer was dried (Na.sub.2SO.sub.4) and filtered.
The filtrate was concentrated and the residue was chromatographed
using 1/1 Hexane/EtOAc to give
N-[4-(3-Acetyl-phenyl)-pyridin-2-yl]-benzamide as a white solid
(375 mg, 55%). MS: m/z 317.1 [MH.sup.+].
Example 9
Preparation of
N-{4-[3-(3-Morpholin-4-yl-propionyl)-phenyl]-pyridin-2-yl}-benzamide
##STR00099##
[0217] N-[4-(3-Acetyl-phenyl)-pyridin-2-yl]-benzamide (200 mg,
0.632 mmol), morpholine HCl salt (78 mg, 0.632 mmol, 1 eq.) and
paraformaldehyde (19 mg, 0.632 mmol, 1 eq.) were mixed with dioxane
(2 mL) in a microwave tube. It was irradiated at 180.degree. C. for
15 minutes. The mixture was partitioned between DCM/water (10 mL/5
mL). The aqueous layer was washed further with DCM (2.times.10 mL).
The combined organic layer was dried (Na.sub.2SO.sub.4) and
filtered. The filtrate was concentrated and the residue was
chromatographed using 20/1 DCM/MeOH to give
N-{4-[3-(3-Morpholin-4-yl-propionyl)-phenyl]-pyridin-2-yl}-benzamide
as a slightly yellow solid (100 mg, 38%). MS: m/z 416.3
[MH.sup.+].
Example 10
Preparation of
1-[3-(2-Amino-pyridin-4-yl)-phenyl]-3-morpholin-4-yl-propan-1-one
##STR00100##
[0219]
N-{4-[3-(3-Morpholin-4-yl-propionyl)-phenyl]-pyridin-2-yl}-benzamid-
e (100 mg, 0.32 mmol) was dissolved in HCl (2 mL, 6 N). The mixture
was irradiated with microwaves at 140.degree. C. for 30 minutes.
The mixture was diluted with DCM (20 mL) and neutralized with NaOH
to pH.about.9. The layers were separated and the aqueous layer was
washed further with DCM (2.times.15 mL). The combined organic layer
was dried (Na.sub.2SO.sub.4) and filtered. The filtrate was
concentrated and the residue was purified to give
1-[3-(2-Amino-pyridin-4-yl)-phenyl]-3-morpholin-4-yl-propan-1-one
(TFA salt) as a white solid (84 mg, 78%). MS: m/z 312.3
[MH.sup.+].
Example 11
Preparation of
N-[4-(3-Acetyl-phenyl)-pyridin-2-yl]-4-tert-butyl-benzamide
##STR00101##
[0221] According the same procedure for the preparation of
N-[4-(3-Acetyl-phenyl)-pyridin-2-yl]-benzamide,
N-[4-(3-Acetyl-phenyl)-pyridin-2-yl]-4-tert-butyl-benzamide (130
mg, 81%, slight impurity) was obtained from
1-[3-(2-Chloro-pyridin-4-yl)-phenyl]-ethanone (100 mg, 0.43 mmol)
and 4-tert-butylbenzamide (153 mg, 0.86 mmol). MS: m/z 373.1
[MH.sup.+].
Example 12
Preparation of
4-tert-Butyl-N-{4-3-(3-morpholin-4-yl-propionyl)-phenyl]-pyridin-2-yl}-be-
nzamide
##STR00102##
[0223] According to the same procedure for the preparation of
1-[3-(2-Amino-pyridin-4-yl)-phenyl]-3-morpholin-4-yl-propan-1-one,
4-tert-Butyl-N-{4-3-(3-morpholin-4-yl-propionyl)-phenyl]-pyridin-2-yl}-be-
nzamide (12 mg, 30%) was obtained from
N-[4-(3-Acetyl-phenyl)-pyridin-2-yl]-4-tert-butyl-benzamide (36 mg,
0.1 mmol). MS: m/z 472.3 [MH.sup.+].
Example 13
Preparation of N-[4-(3-Acetyl-phenyl)-pyridin-2-yl]-acetamide
##STR00103##
[0225] According the same procedure for the preparation of
N-[4-(3-Acetyl-phenyl)-pyridin-2-yl]-benzamide,
N-[4-(3-Acetyl-phenyl)-pyridin-2-yl]-acetamide (50 mg, 50%, slight
impurity) was obtained from
1-[3-(2-Chloro-pyridin-4-yl)-phenyl]-ethanone (100 mg, 0.43 mmol)
and acetamide (26 mg, 0.86 mmol). MS: m/z 255.1 [MH.sup.+].
Example 14
Preparation of
N-{4-[3-(3-Morpholin-4-yl-propionyl)-phenyl]-pyridin-2-yl}-acetamide
##STR00104##
[0227] According to the same procedure for the preparation of
1-[3-(2-Amino-pyridin-4-yl)-phenyl]-3-morpholin-4-yl-propan-1-one,
N-{4-[3-(3-Morpholin-4-yl-propionyl)-phenyl]-pyridin-2-yl}-acetamide
(10 mg, 20%) was obtained from
N-[4-(3-Acetyl-phenyl)-pyridin-2-yl]-acetamide (50 mg, 0.2 mmol).
MS: m/z 354.3 [MH.sup.+].
Example 15
In Vitro Assays
[0228] Measurement of IL-2 Production
[0229] Human T cell lines were plated in 96 well plates pre-coated
with anti-CD3 monoclonal antibodies. Wells were either left
untreated or treated with anti-CD28 for 2 days. The supernatant was
collected and tested for IL-2 production in the presence or absence
of a test compound using a human IL-2 ELISA assay.
[0230] T Cell Proliferation Assay
[0231] Human T cell lines were plated in 96 well plates pre-coated
with anti-CD3 monoclonal antibodies. Wells were either left
untreated or treated with anti-CD28 for 2 days. Cell proliferation
was measured in the presence or absence of a test compound using a
commercially available CELLTITER-GLO.TM. assay (Promega).
[0232] In Vitro Kinase Assays
[0233] Compounds were screened using the HITHUNTER.TM. enzyme
fragment complementation method (Discoverx). Briefly, a
recombinantly produced, N-terminally His-tagged ITK kinase domain
(amino acids 352-617) was incubated with various concentrations of
individual compounds. ATP and substrate were added, and the kinase
reaction was allowed to proceed for 2-16 hours. Commercially
available detection reagents were added and allowed to react for
2-4 hours. The reaction was evaluated by luminescence. Initial
results were confirmed using full-length recombinant ITK
protein.
[0234] Similarly, commercially available reagents such as
HITHUNTERT.TM. were used to evaluate the effect of compounds on the
activity of additional kinases. The kinase domains of BTK, LCK and
ERK were expressed as recombinant purified proteins were used for
these studies.
[0235] The compounds in Table 1 were tested and shown to inhibit
IL-2 production, to inhibit T cell proliferation, and to inhibit
ITK with an IC.sub.50 of less than 1 .mu.M.
TABLE-US-00001 TABLE 1 Compound IC.sub.50 (.mu.M) ##STR00105##
0.01807 ##STR00106## 0.00954 ##STR00107## 0.01355 ##STR00108##
0.02851 ##STR00109## 0.00533 ##STR00110## 0.00426 ##STR00111##
0.05043 ##STR00112## 0.0114 ##STR00113## 0.01327 ##STR00114##
0.00686 ##STR00115## 0.02855 ##STR00116## 0.01825 ##STR00117##
0.00085 ##STR00118## 0.07194 ##STR00119## 0.01964
[0236] The compounds in Tables 2-5 were tested in in vitro kinase
assays:
TABLE-US-00002 TABLE 2 IC.sub.50 ITK IC.sub.50 BTK IC.sub.50 LCK
Compound (.mu.M) (.mu.M) (.mu.M) ##STR00120## 0.005 0.42482
12.55299 ##STR00121## 0.040 0.27584 2.89341 ##STR00122## 0.04022
0.0369 8.03843 ##STR00123## 0.013 1.41274 27.7419 ##STR00124##
0.013 0.10223 34.05941 ##STR00125## 0.014 1.83528 23.89837
##STR00126## 0.020 ##STR00127## 0.025 0.36501 NO IC50 ##STR00128##
0.029 0.64341 413.06105 ##STR00129## 0.035 0.94241 16.4214
##STR00130## 0.036 0.039 19.969 ##STR00131## 0.043 0.6561 27.11277
##STR00132## 0.056 0.86517 NO IC50 ##STR00133## 0.065 1.24489
18.43928 ##STR00134## 0.067 0.12463 28.09552 ##STR00135## 0.072
0.7368 NO IC50 ##STR00136## 0.065 0.39763 23.16665 ##STR00137##
0.091 0.09415 18.46087 ##STR00138## 0.077 0.77538 47.61179
##STR00139## 0.096 1.53948 20.8277 ##STR00140## 0.104 0.23242 NO
IC50 ##STR00141## 0.148 0.77352 28.01341 ##STR00142## 0.180 1.52018
163.63704 ##STR00143## 0.186 3.67569 20.64831 ##STR00144## 0.199
0.4735 NO IC50 ##STR00145## 0.207 0.09415 18.46087 ##STR00146##
0.208 2.89272 33.1157 ##STR00147## 0.207 0.08071 NO IC50
##STR00148## 0.219 1.30729 NO IC50 ##STR00149## 0.223 1.47599
21.15799 ##STR00150## 0.241 0.81405 NO IC50 ##STR00151## 0.290
0.68214 25.86619 ##STR00152## 0.305 0.74064 NO IC50 ##STR00153##
0.345 3.1355 21.10834 ##STR00154## 0.381 3.03351 32.57859
##STR00155## 0.385 1.47531 25.34326 ##STR00156## 0.385 3.92321
23.25 ##STR00157## 0.385 0.75252 23.94596 ##STR00158## 0.468
1.21899 NO IC50 ##STR00159## 0.560 3.06627 24.36134 ##STR00160##
0.569 1.01979 NO IC50 ##STR00161## 0.611 2.31114 NO IC50
##STR00162## 0.797 3.62429 NO IC50 ##STR00163## 0.935 0.99267
29.52378 ##STR00164## 0.874 2.57662 NO IC50 ##STR00165## 1.279
0.55617 704.77096 ##STR00166## 1.406 4.1378 27.08267
TABLE-US-00003 TABLE 3 IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 ITK
BTK ITK BTK Compound (.mu.M) (.mu.M) naphthyl analog (.mu.M)
(.mu.M) ##STR00167## 0.0059 0.7810 ##STR00168## 0.020 0.03947
##STR00169## 0.030 0.276 ##STR00170## 0.007 0.01276 ##STR00171##
0.072 1.894 ##STR00172## 0.007 0.00215 ##STR00173## 0.010 0.300
##STR00174## 0.006 0.00338 ##STR00175## 0.018 0.481 ##STR00176##
0.008 0.00152 ##STR00177## 0.013 0.150 ##STR00178## 0.009 0.00654
##STR00179## 0.023 0.544 ##STR00180## 0.009 0.00072
TABLE-US-00004 TABLE 4 Compound IC.sub.50 ITK (.mu.M) IC.sub.50 BTK
(.mu.M) ##STR00181## 0.05666 ##STR00182## 0.03034 ##STR00183##
0.09281 ##STR00184## 0.02285 ##STR00185## 0.07489 ##STR00186##
0.020 0.039 ##STR00187## 0.048 5.001 ##STR00188## 1.684 no
IC.sub.50 ##STR00189## 0.189 0.100 ##STR00190## 0.176 8.174
##STR00191## 0.049 0.296 ##STR00192## 0.134 0.611 ##STR00193##
0.075 2.038 ##STR00194## 0.060 0.334 ##STR00195## 56.392 no
IC.sub.50 ##STR00196## 0.010 0.017 ##STR00197## 0.030 0.006
##STR00198## 3.339 2.364 ##STR00199## 0.005 0.001 ##STR00200##
0.042 0.002
TABLE-US-00005 TABLE 5 Compound IC.sub.50 ITK (.mu.M) IC.sub.50 BTK
(.mu.M) ##STR00201## 0.0205 0.0395 ##STR00202## 0.1369 8.21849
##STR00203## 0.0080 0.06706 ##STR00204## 0.0169 ##STR00205## 0.0602
0.12799 ##STR00206## 0.0148 ##STR00207## 0.5375 ##STR00208## 0.8516
##STR00209## 0.0309 ##STR00210## 0.0212 ##STR00211## 0.1609
##STR00212## 0.0242 ##STR00213## 0.0072 0.11532 ##STR00214## 0.3096
##STR00215## 0.0069 0.0492 ##STR00216## 0.0187 ##STR00217## 0.0095
##STR00218## 0.0162 ##STR00219## 0.0359 ##STR00220## 0.0147
##STR00221## 0.0092 ##STR00222## 0.0062 0.16054 ##STR00223## 0.0163
##STR00224## 0.117 0.410 ##STR00225## 0.023 0.153 ##STR00226##
0.056 0.452 ##STR00227## 0.060 0.242 ##STR00228## 0.066 0.089
##STR00229## 0.064 0.360 ##STR00230## 0.054 0.018 ##STR00231##
0.087 0.051 ##STR00232## 0.031 0.071 ##STR00233## 0.066 0.117
##STR00234## 0.049 0.123 ##STR00235## 0.086 0.084 ##STR00236##
0.284 0.486 ##STR00237## 0.217 0.266 ##STR00238## 0.163 0.100
##STR00239## 0.036 0.004 ##STR00240## 0.737 0.373
Example 16
[0237] In Vivo Studies
[0238] Several representative compounds were evaluated for efficacy
in mouse in vivo tumor models. NOD/SCID mice were implanted
intraperitoneally with T cell leukemia/lymphoma cells. One group
was treated with vehicle alone (mock treatment) while the other
groups were treated with several small molecule inhibitors via
intraperitoneal route. Tumor growth was evaluated by peritoneal
lavage and FACS analysis. Table 6 summarizes percent inhibition of
tumor growth relative to a mock group treated with vehicle alone.
Doses of compounds evaluated in this study were below the maximal
tolerated dose, and showed minimal toxicity.
[0239] The compounds in Table 6 were tested and inhibited tumor
growth by at least 50% at the concentrations shown.
TABLE-US-00006 TABLE 6 Compound mg/kg % inhibition tumor growth
##STR00241## 100 70-90 ##STR00242## 100 85-98 ##STR00243## 80 92-99
##STR00244## 80 50-80 ##STR00245## 80 90-99 ##STR00246## 80 99
##STR00247## 80 67-81 ##STR00248## 80 92-99 ##STR00249## 100 99
##STR00250## 80 97-99 ##STR00251## 30 99 ##STR00252## 100 99
##STR00253## 80 81-95 ##STR00254## 20 40 vehicle -- 0
Example 17
[0240] Compound Activity Mechanism
[0241] The compound class interacts selectively with kinase domains
of such kinase families as Tec and EGFR, as well as a few
additional kinases. There is evidence indicating that this class of
compounds reacts irreversibly at the ATP binding site of the kinase
binding domain, through a mechanism that involves the exposure of a
reactive aminoethyl C.dbd.Y warhead through the in situ elimination
of a leaving group. The compounds contain an abstractable proton
adjacent to the C.dbd.Y group, which upon exposure to an
appropriate catalytic environment in the active site of a kinase of
interest will promote elimination of the beta-amino functionality.
This elimination thus generates a reactive electrophilic species
(commonly termed a Michael acceptor moiety) which, due to the
existence of a proximal cysteine residue in the kinase active site,
rapidly forms a covalent adduct between this cysteine residue and
the in situ generated electrophilic species. The combination of a
kinase with the catalytic environment in close proximity to a
nucleophilic cysteine, is a vital and unique requirement that
describes this mechanism of action. The data below support that in
situ elimination promotes the inhibitory activity of compounds in
depicted in this invention. When a compound is modified in a manner
that prevents elimination, the compound fails to exhibit inhibitory
activity.
TABLE-US-00007 TABLE 7 compound IC.sub.50 ITK ##STR00255## 0.0446
##STR00256## no IC.sub.50 ##STR00257## no IC.sub.50
Example 18
[0242] Covalent Binding to Select Kinases
[0243] As a result of elimination in proximity of a relevant
cysteine, a covalent adduct is formed between the compound and the
kinase domain. The irreversible binding that ensues can be
demonstrated by several methods, including surface plasmon
resonance (SPR) and co-precipitation of the compound with the
kinase.
[0244] BIACORE.RTM. is a SPR-based protein interaction approach,
whereby the kinase is immobilized on the sensor chip, and a small
molecule solution allowed to interact with the kinase. Detection of
small molecule/kinase interaction occurs in real time, and is
detected as a difference in SPR response. FIG. 1 shows a
BIACORE.RTM. experiment in which the ITK kinase domain was
immobilized on a biosensor, and evaluated for its ability to bind
and dissociate form a small molecule. The data indicates that
compounds depicted in this application bind to the ITK kinase
domain irreversibly.
[0245] In the co-precipitation assay, 1-10 mM labeled compound is
incubated with cell lysates from either kinase expressing or kinase
lacking cells. The label is then used to precipitate the compound
and any bound proteins. The mixture is separated by SDS-PAGE and
proteins are identified by western blotting and/or Mass
spectrophotometry.
Example 19
[0246] Contribution of Cysteine 442 to Adduct Formation
[0247] In order to confirm the mechanism by which compounds
depicted herein interact with the kinase domain of Tec and EGFR
kinases, we created a point mutant of the ITK kinase domain,
whereby the key amino acid, namely C442 was mutated to alanine. The
protein was expressed in a commercial baculovirus expression system
using the manufacturer's general protocol (Invitrogen, pBlueBac).
Protein was expressed and purified using standard techniques. Both
wild type (WT) ITK kinase domain and C442A kinase domain exhibited
kinase activity. While the activity of WT-ITK was inhibited by
compounds depicted in this application, the same compounds had no
activity towards the C442A mutant kinase domain.
TABLE-US-00008 TABLE8 IC.sub.50 (.mu.M) compound wild-type ITK
C442A-ITK control (BMS-488516) 0.0392 0.0532 ##STR00258## 0.011
>10 ##STR00259## 0.0496 >10 ##STR00260## 0.0111 >10
Sequence CWU 1
1
421620PRTHomo sapiens 1Met Asn Asn Phe Ile Leu Leu Glu Glu Gln Leu
Ile Lys Lys Ser Gln1 5 10 15 Gln Lys Arg Arg Thr Ser Pro Ser Asn
Phe Lys Val Arg Phe Phe Val 20 25 30 Leu Thr Lys Ala Ser Leu Ala
Tyr Phe Glu Asp Arg His Gly Lys Lys 35 40 45 Arg Thr Leu Lys Gly
Ser Ile Glu Leu Ser Arg Ile Lys Cys Val Glu 50 55 60 Ile Val Lys
Ser Asp Ile Ser Ile Pro Cys His Tyr Lys Tyr Pro Phe65 70 75 80 Gln
Val Val His Asp Asn Tyr Leu Leu Tyr Val Phe Ala Pro Asp Arg 85 90
95 Glu Ser Arg Gln Arg Trp Val Leu Ala Leu Lys Glu Glu Thr Arg Asn
100 105 110 Asn Asn Ser Leu Val Pro Lys Tyr His Pro Asn Phe Trp Met
Asp Gly 115 120 125 Lys Trp Arg Cys Cys Ser Gln Leu Glu Lys Leu Ala
Thr Gly Cys Ala 130 135 140 Gln Tyr Asp Pro Thr Lys Asn Ala Ser Lys
Lys Pro Leu Pro Pro Thr145 150 155 160 Pro Glu Asp Asn Arg Arg Pro
Leu Trp Glu Pro Glu Glu Thr Val Val 165 170 175 Ile Ala Leu Tyr Asp
Tyr Gln Thr Asn Asp Pro Gln Glu Leu Ala Leu 180 185 190 Arg Arg Asn
Glu Glu Tyr Cys Leu Leu Asp Ser Ser Glu Ile His Trp 195 200 205 Trp
Arg Val Gln Asp Arg Asn Gly His Glu Gly Tyr Val Pro Ser Ser 210 215
220 Tyr Leu Val Glu Lys Ser Pro Asn Asn Leu Glu Thr Tyr Glu Trp
Tyr225 230 235 240 Asn Lys Ser Ile Ser Arg Asp Lys Ala Glu Lys Leu
Leu Leu Asp Thr 245 250 255 Gly Lys Glu Gly Ala Phe Met Val Arg Asp
Ser Arg Thr Ala Gly Thr 260 265 270 Tyr Thr Val Ser Val Phe Thr Lys
Ala Val Val Ser Glu Asn Asn Pro 275 280 285 Cys Ile Lys His Tyr His
Ile Lys Glu Thr Asn Asp Asn Pro Lys Arg 290 295 300 Tyr Tyr Val Ala
Glu Lys Tyr Val Phe Asp Ser Ile Pro Leu Leu Ile305 310 315 320 Asn
Tyr His Gln His Asn Gly Gly Gly Leu Val Thr Arg Leu Arg Tyr 325 330
335 Pro Val Cys Phe Gly Arg Gln Lys Ala Pro Val Thr Ala Gly Leu Arg
340 345 350 Tyr Gly Lys Trp Val Ile Asp Pro Ser Glu Leu Thr Phe Val
Gln Glu 355 360 365 Ile Gly Ser Gly Gln Phe Gly Leu Val His Leu Gly
Tyr Trp Leu Asn 370 375 380 Lys Asp Lys Val Ala Ile Lys Thr Ile Arg
Glu Gly Ala Met Ser Glu385 390 395 400 Glu Asp Phe Ile Glu Glu Ala
Glu Val Met Met Lys Leu Ser His Pro 405 410 415 Lys Leu Val Gln Leu
Tyr Gly Val Cys Leu Glu Gln Ala Pro Ile Cys 420 425 430 Leu Val Phe
Glu Phe Met Glu His Gly Cys Leu Ser Asp Tyr Leu Arg 435 440 445 Thr
Gln Arg Gly Leu Phe Ala Ala Glu Thr Leu Leu Gly Met Cys Leu 450 455
460 Asp Val Cys Glu Gly Met Ala Tyr Leu Glu Glu Ala Cys Val Ile
His465 470 475 480 Arg Asp Leu Ala Ala Arg Asn Cys Leu Val Gly Glu
Asn Gln Val Ile 485 490 495 Lys Val Ser Asp Phe Gly Met Thr Arg Phe
Val Leu Asp Asp Gln Tyr 500 505 510 Thr Ser Ser Thr Gly Thr Lys Phe
Pro Val Lys Trp Ala Ser Pro Glu 515 520 525 Val Phe Ser Phe Ser Arg
Tyr Ser Ser Lys Ser Asp Val Trp Ser Phe 530 535 540 Gly Val Leu Met
Trp Glu Val Phe Ser Glu Gly Lys Ile Pro Tyr Glu545 550 555 560 Asn
Arg Ser Asn Ser Glu Val Val Glu Asp Ile Ser Thr Gly Phe Arg 565 570
575 Leu Tyr Lys Pro Arg Leu Ala Ser Thr His Val Tyr Gln Ile Met Asn
580 585 590 His Cys Trp Lys Glu Arg Pro Glu Asp Arg Pro Ala Phe Ser
Arg Leu 595 600 605 Leu Arg Gln Leu Ala Glu Ile Ala Glu Ser Gly Leu
610 615 620 2659PRTHomo sapiens 2Met Ala Ala Val Ile Leu Glu Ser
Ile Phe Leu Lys Arg Ser Gln Gln1 5 10 15 Lys Lys Lys Thr Ser Pro
Leu Asn Phe Lys Lys Arg Leu Phe Leu Leu 20 25 30 Thr Val His Lys
Leu Ser Tyr Tyr Glu Tyr Asp Phe Glu Arg Gly Arg 35 40 45 Arg Gly
Ser Lys Lys Gly Ser Ile Asp Val Glu Lys Ile Thr Cys Val 50 55 60
Glu Thr Val Val Pro Glu Lys Asn Pro Pro Pro Glu Arg Gln Ile Pro65
70 75 80 Arg Arg Gly Glu Glu Ser Ser Glu Met Glu Gln Ile Ser Ile
Ile Glu 85 90 95 Arg Phe Pro Tyr Pro Phe Gln Val Val Tyr Asp Glu
Gly Pro Leu Tyr 100 105 110 Val Phe Ser Pro Thr Glu Glu Leu Arg Lys
Arg Trp Ile His Gln Leu 115 120 125 Lys Asn Val Ile Arg Tyr Asn Ser
Asp Leu Val Gln Lys Tyr His Pro 130 135 140 Cys Phe Trp Ile Asp Gly
Gln Tyr Leu Cys Cys Ser Gln Thr Ala Lys145 150 155 160 Asn Ala Met
Gly Cys Gln Ile Leu Glu Asn Arg Asn Gly Ser Leu Lys 165 170 175 Pro
Gly Ser Ser His Arg Lys Thr Lys Lys Pro Leu Pro Pro Thr Pro 180 185
190 Glu Glu Asp Gln Ile Leu Lys Lys Pro Leu Pro Pro Glu Pro Ala Ala
195 200 205 Ala Pro Val Ser Thr Ser Glu Leu Lys Lys Val Val Ala Leu
Tyr Asp 210 215 220 Tyr Met Pro Met Asn Ala Asn Asp Leu Gln Leu Arg
Lys Gly Asp Glu225 230 235 240 Tyr Phe Ile Leu Glu Glu Ser Asn Leu
Pro Trp Trp Arg Ala Arg Asp 245 250 255 Lys Asn Gly Gln Glu Gly Tyr
Ile Pro Ser Asn Tyr Val Thr Glu Ala 260 265 270 Glu Asp Ser Ile Glu
Met Tyr Glu Trp Tyr Ser Lys His Met Thr Arg 275 280 285 Ser Gln Ala
Glu Gln Leu Leu Lys Gln Glu Gly Lys Glu Gly Gly Phe 290 295 300 Ile
Val Arg Asp Ser Ser Lys Ala Gly Lys Tyr Thr Val Ser Val Phe305 310
315 320 Ala Lys Ser Thr Gly Asp Pro Gln Gly Val Ile Arg His Tyr Val
Val 325 330 335 Cys Ser Thr Pro Gln Ser Gln Tyr Tyr Leu Ala Glu Lys
His Leu Phe 340 345 350 Ser Thr Ile Pro Glu Leu Ile Asn Tyr His Gln
His Asn Ser Ala Gly 355 360 365 Leu Ile Ser Arg Leu Lys Tyr Pro Val
Ser Gln Gln Asn Lys Asn Ala 370 375 380 Pro Ser Thr Ala Gly Leu Gly
Tyr Gly Ser Trp Glu Ile Asp Pro Lys385 390 395 400 Asp Leu Thr Phe
Leu Lys Glu Leu Gly Thr Gly Gln Phe Gly Val Val 405 410 415 Lys Tyr
Gly Lys Trp Arg Gly Gln Tyr Asp Val Ala Ile Lys Met Ile 420 425 430
Lys Glu Gly Ser Met Ser Glu Asp Glu Phe Ile Glu Glu Ala Lys Val 435
440 445 Met Met Asn Leu Ser His Glu Lys Leu Val Gln Leu Tyr Gly Val
Cys 450 455 460 Thr Lys Gln Arg Pro Ile Phe Ile Ile Thr Glu Tyr Met
Ala Asn Gly465 470 475 480 Cys Leu Leu Asn Tyr Leu Arg Glu Met Arg
His Arg Phe Gln Thr Gln 485 490 495 Gln Leu Leu Glu Met Cys Lys Asp
Val Cys Glu Ala Met Glu Tyr Leu 500 505 510 Glu Ser Lys Gln Phe Leu
His Arg Asp Leu Ala Ala Arg Asn Cys Leu 515 520 525 Val Asn Asp Gln
Gly Val Val Lys Val Ser Asp Phe Gly Leu Ser Arg 530 535 540 Tyr Val
Leu Asp Asp Glu Tyr Thr Ser Ser Val Gly Ser Lys Phe Pro545 550 555
560 Val Arg Trp Ser Pro Pro Glu Val Leu Met Tyr Ser Lys Phe Ser Ser
565 570 575 Lys Ser Asp Ile Trp Ala Phe Gly Val Leu Met Trp Glu Ile
Tyr Ser 580 585 590 Leu Gly Lys Met Pro Tyr Glu Arg Phe Thr Asn Ser
Glu Thr Ala Glu 595 600 605 His Ile Ala Gln Gly Leu Arg Leu Tyr Arg
Pro His Leu Ala Ser Glu 610 615 620 Lys Val Tyr Thr Ile Met Tyr Ser
Cys Trp His Glu Lys Ala Asp Glu625 630 635 640 Arg Pro Thr Phe Lys
Ile Leu Leu Ser Asn Ile Leu Asp Val Met Asp 645 650 655 Glu Glu
Ser335PRTHomo sapiens 3Leu Thr Phe Leu Lys Glu Leu Gly Thr Gly Gln
Phe Gly Val Val Lys1 5 10 15 Tyr Gly Lys Trp Arg Gly Gln Tyr Asp
Val Ala Ile Lys Met Ile Lys 20 25 30 Glu Gly Ser 35 435PRTHomo
sapiens 4Ile Thr Leu Leu Lys Glu Leu Gly Ser Gly Gln Phe Gly Val
Val Gln1 5 10 15 Leu Gly Lys Trp Lys Gly Gln Tyr Asp Val Ala Val
Lys Met Ile Lys 20 25 30 Glu Gly Ser 35 535PRTHomo sapiens 5Leu Thr
Phe Met Arg Glu Leu Gly Ser Gly Leu Phe Gly Val Val Arg1 5 10 15
Leu Gly Lys Trp Arg Ala Gln Tyr Lys Val Ala Ile Lys Ala Ile Arg 20
25 30 Glu Gly Ala 35 635PRTHomo sapiens 6Leu Ala Phe Ile Lys Glu
Ile Gly Ser Gly Gln Phe Gly Val Val His1 5 10 15 Leu Gly Glu Trp
Arg Ser His Ile Gln Val Ala Ile Lys Ala Ile Asn 20 25 30 Glu Gly
Ser 35 735PRTHomo sapiens 7Leu Thr Phe Val Gln Glu Ile Gly Ser Gly
Gln Phe Gly Leu Val His1 5 10 15 Leu Gly Tyr Trp Leu Asn Lys Asp
Lys Val Ala Ile Lys Thr Ile Arg 20 25 30 Glu Gly Ala 35 835PRTHomo
sapiens 8Leu Arg Leu Val Arg Lys Leu Gly Ser Gly Gln Phe Gly Glu
Val Trp1 5 10 15 Met Gly Tyr Tyr Lys Asn Asn Met Lys Val Ala Ile
Lys Thr Leu Lys 20 25 30 Glu Gly Thr 35 940PRTHomo sapiens 9Phe Lys
Lys Ile Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr1 5 10 15
Lys Gly Leu Trp Ile Pro Glu Gly Glu Lys Val Lys Ile Pro Val Ala 20
25 30 Ile Lys Glu Leu Arg Glu Ala Thr 35 40 1040PRTHomo sapiens
10Leu Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr1
5 10 15 Lys Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val
Ala 20 25 30 Ile Lys Val Leu Arg Glu Asn Thr 35 40 1140PRTHomo
sapiens 11Leu Lys Arg Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr
Val Tyr1 5 10 15 Lys Gly Ile Trp Val Pro Glu Gly Glu Thr Val Lys
Ile Pro Val Ala 20 25 30 Ile Lys Ile Leu Asn Glu Thr Thr 35 40
1240PRTHomo sapiens 12Leu Lys Tyr Ile Ser Gln Leu Gly Lys Gly Asn
Phe Gly Ser Val Glu1 5 10 15 Leu Cys Arg Tyr Asp Pro Leu Ala His
Asn Thr Gly Ala Leu Val Ala 20 25 30 Val Lys Gln Leu Gln His Ser
Gly 35 40 1336PRTHomo sapiens 13Met Ser Glu Asp Glu Phe Ile Glu Glu
Ala Lys Val Met Met Asn Leu1 5 10 15 Ser His Glu Lys Leu Val Gln
Leu Tyr Gly Val Cys Thr Lys Gln Arg 20 25 30 Pro Ile Phe Ile 35
1436PRTHomo sapiens 14Met Ser Glu Asp Glu Phe Phe Gln Glu Ala Gln
Thr Met Met Lys Leu1 5 10 15 Ser His Pro Lys Leu Val Lys Phe Tyr
Gly Val Cys Ser Lys Glu Tyr 20 25 30 Pro Ile Tyr Ile 35 1536PRTHomo
sapiens 15Met Cys Glu Glu Asp Phe Ile Glu Glu Ala Lys Val Met Met
Lys Leu1 5 10 15 Thr His Pro Lys Leu Val Gln Leu Tyr Gly Val Cys
Thr Gln Gln Lys 20 25 30 Pro Ile Tyr Ile 35 1636PRTHomo sapiens
16Met Ser Glu Glu Asp Phe Ile Glu Glu Ala Lys Val Met Met Lys Leu1
5 10 15 Ser His Ser Lys Leu Val Gln Leu Tyr Gly Val Cys Ile Gln Arg
Lys 20 25 30 Pro Leu Tyr Ile 35 1736PRTHomo sapiens 17Met Ser Glu
Glu Asp Phe Ile Glu Glu Ala Glu Val Met Met Lys Leu1 5 10 15 Ser
His Pro Lys Leu Val Gln Leu Tyr Gly Val Cys Leu Glu Gln Ala 20 25
30 Pro Ile Cys Leu 35 1835PRTHomo sapiens 18Met Ser Pro Glu Ala Phe
Leu Gly Glu Ala Asn Val Met Lys Ala Leu1 5 10 15 Gln His Glu Arg
Leu Val Arg Leu Tyr Ala Val Val Thr Lys Glu Pro 20 25 30 Ile Tyr
Ile 35 1937PRTHomo sapiens 19Ser Pro Lys Ala Asn Lys Glu Ile Leu
Asp Glu Ala Tyr Val Met Ala1 5 10 15 Ser Val Asp Asn Pro His Val
Cys Arg Leu Leu Gly Ile Cys Leu Thr 20 25 30 Ser Thr Val Gln Leu 35
2037PRTHomo sapiens 20Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu
Ala Tyr Val Met Ala1 5 10 15 Gly Val Gly Ser Pro Tyr Val Ser Arg
Leu Leu Gly Ile Cys Leu Thr 20 25 30 Ser Thr Val Gln Leu 35
2137PRTHomo sapiens 21Gly Pro Lys Ala Asn Val Glu Phe Met Asp Glu
Ala Leu Ile Met Ala1 5 10 15 Ser Met Asp His Pro His Leu Val Arg
Leu Leu Gly Val Cys Leu Ser 20 25 30 Pro Thr Ile Gln Leu 35
2239PRTHomo sapiens 22Pro Asp Gln Gln Arg Asp Phe Gln Arg Glu Ile
Gln Ile Leu Lys Ala1 5 10 15 Leu His Ser Asp Phe Ile Val Lys Tyr
Arg Gly Val Ser Tyr Gly Pro 20 25 30 Gly Arg Pro Glu Leu Arg Leu 35
2333PRTHomo sapiens 23Ile Thr Glu Tyr Met Ala Asn Gly Cys Leu Leu
Asn Tyr Leu Arg Glu1 5 10 15 Met Arg His Arg Phe Gln Thr Gln Gln
Leu Leu Glu Met Cys Lys Asp 20 25 30 Val 2433PRTHomo sapiens 24Val
Thr Glu Tyr Ile Ser Asn Gly Cys Leu Leu Asn Tyr Leu Arg Ser1 5 10
15 His Gly Lys Gly Leu Glu Pro Ser Gln Leu Leu Glu Met Cys Tyr Asp
20 25 30 Val 2533PRTHomo sapiens 25Val Thr Glu Phe Met Glu Arg Gly
Cys Leu Leu Asn Phe Leu Arg Gln1 5 10 15 Arg Gln Gly His Phe Ser
Arg Asp Val Leu Leu Ser Met Cys Gln Asp 20 25 30 Val 2633PRTHomo
sapiens 26Val Thr Glu Phe Met Glu Asn Gly Cys Leu Leu Asn Tyr Leu
Arg Glu1 5 10 15 Asn Lys Gly Lys Leu Arg Lys Glu Met Leu Leu Ser
Val Cys Gln Asp 20 25 30 Ile 2733PRTHomo sapiens 27Val Phe Glu Phe
Met Glu His Gly Cys Leu Ser Asp Tyr Leu Arg Thr1 5 10 15 Gln Arg
Gly Leu Phe Ala Ala Glu Thr Leu Leu Gly Met Cys Leu Asp 20 25 30
Val 2840PRTHomo sapiens 28Val Thr Glu Tyr Met Ala Arg Gly Cys Leu
Leu Asp Phe Leu Lys Thr1 5 10 15 Asp Glu Gly Ser Arg Leu Ser Leu
Pro Arg Leu Ile Asp Met Ser Ala 20 25 30 Gln Ile Ala Glu Gly Met
Ala Tyr 35 40 2939PRTHomo sapiens 29Ile Thr Gln Leu Met Pro Phe Gly
Cys Leu Leu Asp Tyr Val Arg Glu1 5 10 15 His Lys Asp Asn Ile Gly
Ser Gln Tyr Leu Leu
Asn Trp Cys Val Gln 20 25 30 Ile Ala Lys Gly Met Asn Tyr 35
3039PRTHomo sapiens 30Val Thr Gln Leu Met Pro Tyr Gly Cys Leu Leu
Asp His Val Arg Glu1 5 10 15 Asn Arg Gly Arg Leu Gly Ser Gln Asp
Leu Leu Asn Trp Cys Met Gln 20 25 30 Ile Ala Lys Gly Met Ser Tyr 35
3139PRTHomo sapiens 31Val Thr Gln Leu Met Pro His Gly Cys Leu Leu
Glu Tyr Val His Glu1 5 10 15 His Lys Asp Asn Ile Gly Ser Gln Leu
Leu Leu Asn Trp Cys Val Gln 20 25 30 Ile Ala Lys Gly Met Met Tyr 35
3239PRTHomo sapiens 32Val Met Glu Tyr Leu Pro Ser Gly Cys Leu Arg
Asp Phe Leu Gln Arg1 5 10 15 His Arg Ala Arg Leu Asp Ala Ser Arg
Leu Leu Leu Tyr Ser Ser Gln 20 25 30 Ile Cys Lys Gly Met Glu Tyr 35
3335PRTHomo sapiens 33Phe Leu His Arg Asp Leu Ala Ala Arg Asn Cys
Leu Val Asn Asp Gln1 5 10 15 Gly Val Val Lys Val Ser Asp Phe Gly
Leu Ser Arg Tyr Val Leu Asp 20 25 30 Asp Glu Tyr 35 3435PRTHomo
sapiens 34Phe Ile His Arg Asp Leu Ala Ala Arg Asn Cys Leu Val Asp
Arg Asp1 5 10 15 Leu Cys Val Lys Val Ser Asp Phe Gly Met Thr Arg
Tyr Val Leu Asp 20 25 30 Asp Gln Tyr 35 3535PRTHomo sapiens 35Phe
Ile His Arg Asp Leu Ala Ala Arg Asn Cys Leu Val Ser Glu Ala1 5 10
15 Gly Val Val Lys Val Ser Asp Phe Gly Met Ala Arg Tyr Phe Leu Asp
20 25 30 Asp Gln Tyr 35 3635PRTHomo sapiens 36Tyr Ile His Arg Asp
Leu Ala Ala Arg Asn Cys Leu Val Ser Ser Thr1 5 10 15 Cys Ile Val
Lys Ile Ser Asp Phe Gly Met Thr Arg Tyr Val Leu Asp 20 25 30 Asp
Glu Tyr 35 3735PRTHomo sapiens 37Val Ile His Arg Asp Leu Ala Ala
Arg Asn Cys Leu Val Gly Glu Asn1 5 10 15 Gln Val Ile Lys Val Ser
Asp Phe Gly Met Thr Arg Phe Val Leu Asp 20 25 30 Asp Gln Tyr 35
3839PRTHomo sapiens 38Ile Glu Arg Met Asn Ser Ile His Arg Asp Leu
Arg Ala Ala Asn Ile1 5 10 15 Leu Val Ser Glu Ala Leu Cys Cys Lys
Ile Ala Asp Phe Gly Leu Ala 20 25 30 Arg Ile Ile Asp Ser Glu Tyr 35
3940PRTHomo sapiens 39Leu Glu Asp Arg Arg Leu Val His Arg Asp Leu
Ala Ala Arg Asn Val1 5 10 15 Leu Val Lys Thr Pro Gln His Val Lys
Ile Thr Asp Phe Gly Leu Ala 20 25 30 Lys Leu Leu Gly Ala Glu Glu
Lys 35 40 4040PRTHomo sapiens 40Leu Glu Asp Val Arg Leu Val His Arg
Asp Leu Ala Ala Arg Asn Val1 5 10 15 Leu Val Lys Ser Pro Asn His
Val Lys Ile Thr Asp Phe Gly Leu Ala 20 25 30 Arg Leu Leu Asp Ile
Asp Glu Thr 35 40 4140PRTHomo sapiens 41Leu Glu Glu Arg Arg Leu Val
His Arg Asp Leu Ala Ala Arg Asn Val1 5 10 15 Leu Val Lys Ser Pro
Asn His Val Lys Ile Thr Asp Phe Gly Leu Ala 20 25 30 Arg Leu Leu
Glu Gly Asp Glu Lys 35 40 4240PRTHomo sapiens 42Leu Gly Ser Arg Arg
Cys Val His Arg Asp Leu Ala Ala Arg Asn Ile1 5 10 15 Leu Val Glu
Ser Glu Ala His Val Lys Ile Ala Asp Phe Gly Leu Ala 20 25 30 Lys
Leu Leu Pro Leu Asp Lys Asp 35 40
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