Patent application title: KINASE INHIBITORS USEFUL FOR THE TREATMENT OF MYLEOPROLIFIC DISEASES AND OTHER PROLIFERATIVE DISEASES
Inventors:
Daniel L. Flynn (Lawrence, KS, US)
Peter A. Petillo (Lawrence, KS, US)
Peter A. Petillo (Lawrence, KS, US)
Michael D. Kaufman (Lawrence, KS, US)
Michael D. Kaufman (Lawrence, KS, US)
Assignees:
Deciphera Pharmaceuticals, LLC
IPC8 Class: AA61K314439FI
USPC Class:
5142278
Class name: Hetero ring is six-membered and includes at least nitrogen and sulfur as ring members 1,4-thiazines additional hetero ring attached directly or indirectly to the 1,4-thiazine by nonionic bonding
Publication date: 2008-10-23
Patent application number: 20080261961
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Patent application title: KINASE INHIBITORS USEFUL FOR THE TREATMENT OF MYLEOPROLIFIC DISEASES AND OTHER PROLIFERATIVE DISEASES
Inventors:
Daniel L. Flynn
Peter A. Petillo
Michael D. Kaufman
Agents:
HOVEY WILLIAMS LLP
Assignees:
Deciphera Pharmaceuticals, LLC
Origin: OVERLAND PARK, KS US
IPC8 Class: AA61K314439FI
USPC Class:
5142278
Abstract:
Compounds of the present invention find utility in the treatment of
mammalian cancers and especially human cancers including but not limited
to malignant, melanomas, glioblastomas, ovarian cancer, pancreatic
cancer, prostate cancer, lung cancers, breast cancers, kidney cancers,
cervical carcinomas, metastasis of primary tumor sites,
myeloproliferative diseases, leukemias, papillary thyroid carcinoma, non
small cell lung cancer, mesothelioma, hypereosinophilic syndrome,
gastrointestinal stromal tumors, colonic cancers, ocular diseases
characterized by hyperproliferation leading to blindness including
various retinopathies, rheumatoid arthritis, asthma, chronic obstructive
pulmonary disease, mastocyctosis, mast cell leukemia, a disease caused by
c-Abl kinase, oncogenic forms thereof, aberrant fusion proteins thereof
and polymorphs thereof, or a disease caused by c-Kit kinase, oncogenic
forms thereof, aberrant fusion proteins thereof and polymorphs thereof.Claims:
1. Compounds of the formula Iaand wherein the pyridine ring may be
optionally substituted with one or more R20 moieties;each D is
individually taken from the group consisting of C, CH, C--R20, N-Z3, N, O
and S, such that the resultant ring is taken from the group consisting of
triazolyl, isoxazolyl, isothiazolyl, oxazolyl, and thiadiazolyl;wherein E
is selected from the group consisting of phenyl, pyridyl, and
pyrimidinyl;E may be optionally substituted with one or two R16
moieties;wherein A is a ring system selected from the group consisting of
phenyl, naphthyl, cyclopentyl, cyclohexyl, G1, G2, and G3;G1 is a
heteroaryl taken from the group consisting of pyrrolyl, furyl, thienyl,
oxazolyl, thiazolyl, isoxazol-4-yl, isoxazol-5-yl, isothiazolyl,
imidazolyl, pyrazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl,
pyrazinyl, pyridazinyl, triazinyl, pyridinyl, and pyrimidinyl;G2 is a
fused bicyclic heteroaryl taken from the group consisting of indolyl,
indolinyl, isoindolyl, isoindolinyl, indazolyl, benzofuranyl,
benzothienyl, benzothiazolyl, benzothiazolonyl, benzoxazolyl,
benzoxazolonyl, benzisoxazolyl, benzisothiazolyl, benzimidazolyl,
benzimidazolonyl, benztriazolyl, imidazopyridinyl, pyrazolopyridinyl,
imidazolonopyridinyl, thiazolopyridinyl, thiazolonopyridinyl,
oxazolopyridinyl, oxazolonopyridinyl, isoxazolopyridinyl,
isothiazolopyridinyl, triazolopyridinyl, imidazopyrimidinyl,
pyrazolopyrimidinyl, imidazolonopyrmidinyl, thiazolopyridiminyl,
thiazolonopyrimidinyl, oxazolopyridiminyl, oxazolonopyrimidinyl,
isoxazolopyrimidinyl, isothiazolopyrimidinyl, triazolopyrimidinyl,
dihydropurinonyl, pyrrolopyrimidinyl, purinyl, pyrazolopyrimidinyl,
phthalimidyl, phthalimidinyl, pyrazinylpyridinyl, pyridinopyrimidinyl,
pyrimidinopyrimidinyl, cinnolinyl, quinoxalinyl, quinazolinyl,
quinolinyl, isoquinolinyl, phthalazinyl, benzodioxyl,
benzisothiazoline-1,1,3-trionyl, dihydroquinolinyl, tetrahydroquinolinyl,
dihydroisoquinolyl, tetrahydroisoquinolinyl, benzoazepinyl,
benzodiazepinyl, benzoxapinyl, and benzoxazepinyl;G3 is a heterocyclyl
taken from the group consisting of oxetanyl, azetadinyl,
tetrahydrofuranyl, pyrrolidinyl, oxazolinyl, oxazolidinyl, imidazolonyl,
pyranyl, thiopyranyl, tetrahydropyranyl, dioxalinyl, piperidinyl,
morpholinyl, thiomorpholinyl, thiomorpholinyl S-oxide, thiomorpholinyl
S-dioxide, piperazinyl, azepinyl, oxepinyl, diazepinyl, tropanyl, and
homotropanyl;the A ring may be optionally substituted with one or two R2
moieties;X is selected from the group consisting of --O--,
--S(CH2)n--, --N(R3)(CH2)n--, --(CH2)p--,
and wherein the carbon atoms of --(CH2)n--,
--(CH2)p--, of X may be further substituted by oxo or one or
more C1-C6alkyl moieties;when A, G1, G2 or G3 has one or more
substitutable sp2-hybridized carbon atoms, each respective sp2 hybridized
carbon atom may be optionally substituted with a Z1 substituent;when A,
G1, G2 or G3 has one or more substitutable sp3-hybridized carbon atoms,
each respective sp3 hybridized carbon atom may be optionally substituted
with a Z2 substituent;when A, G1, G2 or G3 has one or more substitutable
nitrogen atoms, each respective nitrogen atom may be optionally
substituted with a Z4 substituent;each Z1 is independently and
individually selected from the group consisting of C1-6alkyl, branched
C3-C7alkyl, C3-C8cycloalkyl, halogen, fluoroC1-C6alkyl wherein the alkyl
moiety can be partially or fully fluorinated, cyano, C1-C6alkoxy,
fluoroC1-C6alkoxy wherein the alkyl moiety can be partially or fully
fluorinated, --(CH2)nOH, oxo, C1-C6alkoxyC1-C6alkyl,
(R4)2N(CH2)n--, (R3)2N(CH2)n--,
(R4)2N(CH2)qN(R4)(CH2)n--,
(R4)2N(CH2)qO(CH2)n--, (R3)2NC(O)--,
(R4)2NC(O)--, (R4)2NC(O)C1-C6alkyl-, --(R4)NC(O)R8,
C1-C6alkoxycarbonyl-, -carboxyC1-C6alkyl, C1-C6alkoxycarbonylC1-C6alkyl-,
(R3)2NSO2--, --SOR3, (R4)2NSO2--, --N(R4)SO2R8,
--O(CH2)qOC1-C6alkyl, --SO2R3, --SOR4, --C(O)R8, --C(O)R6,
--C(═NOH)R6, --C(═NOR3)R6, --(CH2)nN(R4)C(O)R8,
--N(R3)(CH2)qO-alkyl, --N(R3)(CH2)qN(R4)2,
nitro, --CH(OH)CH(OH)R4, --C(═NH)N(R4)2,
--C(═NOR3)N(R4)2, --NHC(═NH)R8, R17 substituted G3, R17
substituted pyrazolyl and R17 substituted imidazolyl;in the event that Z1
contains an alkyl or alkylene moiety, such moieties may be further
substituted with one or more C1-C6alkyls;each Z2 is independently and
individually selected from the group consisting of aryl, C1-C6alkyl,
C3-C8cycloalkyl, branched C3-C7alkyl, hydroxyl, hydroxyC1-C6alkyl-,
cyano, (R3)2N--, (R4)2N--, (R4)2NC1-C6alkyl-,
(R4)2NC2-C6alkylN(R4)(CH2)n--,
(R4)2NC2-C6alkylO(CH2)n--, (R3)2NC(O)--,
(R4)2NC(O)--, (R4)2NC(O)--C1-C6alkyl-, carboxyl,
-carboxyC1-C6alkyl, C1-C6alkoxycarbonyl-, C1-C6alkoxycarbonylC1-C6alkyl-,
(R3)2NSO2--, (R4)2NSO2--, --SO2R8,
--(CH2)nN(R4)C(O)R8, --C(O)R8, ═O, ═NOH, and
═N(OR6);in the event that Z2 contains an alkyl or alkylene moiety,
such moieties may be further substituted with one or more
C1-C6alkyls;each Z3 is independently and individually selected from the
group consisting of H, C1-C6alkyl, branched C3-C7alkyl, C3-C8cycloalkyl,
fluoroC1-C6alkyl wherein the alkyl moiety can be partially or fully
fluorinated, hydroxyC2-C6alkyl-, C1-C6alkoxycarbonyl-, --C(O)R8,
R5C(O)(CH2)n--, (R4)2NC(O)--, (R4)2NC(O)C1-C6alkyl-,
R8C(O)N(R4)(CH2)q--, (R3)2NSO2--,
(R4)2NSO2--, --(CH2)qN(R3)2, and
--(CH2)qN(R4)2;each Z4 is independently and individually
selected from the group consisting of C1-C6alkyl, branched C3-7alkyl,
hydroxyC2-C6alkyl-, C1-C6alkoxyC2-C6alkyl-. (R4)2N--C2-C6alkyl-,
(R4)2N--C2-C6alkylN(R4)-C2-C6alkyl-,
(R4)2N--C2-C6alkyl-O--C2-C6alkyl-(R4)2NC(O)C1-C6alkyl-,
carboxyC1-C6alkyl, C1-C6alkoxycarbonylC1-C6alkyl-,
--C2-C6alkylN(R4)C(O)R8, R8-C(═NR3)--, --SO2R8, and --COR8;in
the event that Z4 contains an alkyl or alkylene moiety, such moieties may
be further substituted with one or more C1-C6alkyls;each R2 is selected
from the group consisting of H, C1-C6alkyl, branched C3-C8alkyl, R19
substituted C3-C8cycloalkyl-, fluoroC1-C6alkyl- wherein the alkyl is
fully or partially fluorinated, halogen, cyano, C1-C6alkoxy-, and
fluoroC1-C6alkoxy- wherein the alkyl group is fully or partially
fluorinated, hydroxyl substituted C1-C6alkyl-, hydroxyl substituted
branched C3-C8alkyl-, cyano substituted C1-C6alkyl-, cyano substituted
branched C3-C8alkyl-, (R3)2NC(O)C1-C6alkyl-, and
(R3)2NC(O)C3-C8 branched alkyl-;wherein each R3 is independently and
individually selected from the group consisting of H, C1-C6alkyl,
branched C3-C7alkyl, and C3-C8cycloalkyl;each R4 is independently and
individually selected from the group consisting of H, C1-C6alkyl,
hydroxyC1-C6alkyl-, dihydroxyC1-C6alkyl-, C1-C6alkoxyC1-C6alkyl-,
branched C3-C7alkyl, branched hydroxyC1-C6alkyl-, branched
C1-C6alkoxyC1-C6alkyl-, branched dihydroxyC1-C6alkyl-,
--(CH2)pN(R7)2, --(CH2)pC(O)N(R7)2,
--(CH2)nC(O)OR3, and R19 substituted C3-C8cycloalkyl-;each R5
is independently and individually selected from the group consisting
ofand wherein the symbol (##) is the point of attachment to Z3;each R6 is
independently and individually selected from the group consisting of
C1-C6alkyl, branched C3-C7alkyl, and R19 substituted
C3-C8cycloalkyl-;each R7 is independently and individually selected from
the group consisting of H, C1-C6alkyl, hydroxyC2-C6alkyl-,
dihydroxyC2-C6alkyl-, C1-C6alkoxyC2-C6alkyl-, branched C3-C7alkyl,
branched hydroxyC2-C6alkyl-, branched C1-C6alkoxyC2-C6alkyl-, branched
dihydroxyC2-C6alkyl-, --(CH2)nC(O)OR3, R19 substituted
C3-C8cycloalkyl- and --(CH2)nR17;each R8 is independently and
individually selected from the group consisting of C1-C6alkyl, branched
C3-C7alkyl, fluoroC1-C6alkyl- wherein the alkyl moiety is partially or
fully fluorinated, R19 substituted C3-C8cycloalkyl-, --OH, C1-C6alkoxy,
--N(R3)2, and --N(R4)2;each R10 is independently and
individually selected from the group consisting of --CO2H,
--CO2C1-C6alkyl, --C(O)N(R4)2, OH, C1-C6alkoxy and
--N(R4)2;each R16 is independently and individually selected from
the group consisting of H, C1-C6alkyl, branched C3-C7alkyl, R19
substituted C3-C8cycloalkyl-, halogen, fluoroC1-C6alkyl- wherein the
alkyl moiety can be partially or fully fluorinated, cyano, hydroxyl,
C1-C6alkoxy, fluoroC1-C6alkoxy- wherein the alkyl moiety can be partially
or fully fluorinated, --N(R3)2, --N(R4)2, R3 substituted
C2-C3alkynyl- and nitro;each R17 is independently and individually
selected from the group consisting of H, C1-C6alkyl, branched C3-C7alkyl,
R19 substituted C3-C8cycloalkyl-, halogen, fluoroC1-C6alkyl- wherein the
alkyl moiety can be partially or fully fluorinated, cyano, hydroxyl,
C1-C6alkoxy, fluoroC1-C6alkoxy- wherein the alkyl moiety can be partially
or fully fluorinated, --N(R3)2, --N(R4)2, and nitro;each R19 is
independently and individually selected from the group consisting of H,
OH and C1-C6alkyl;each R20 is independently and individually selected
from the group consisting of C1-C6alkyl, branched C3-C7alkyl, R19
substituted C3-C8cycloalkyl-, halogen, fluoroC1-C6alkyl- wherein the
alkyl moiety can be partially or fully fluorinated, cyano, hydroxyl,
C1-C6alkoxy, fluoroC1-C6alkoxy- wherein the alkyl moiety can be partially
or fully fluorinated, --N(R3)2, --N(R4)2, --N(R3)C(O)R3,
--C(O)N(R3)2 and nitro and wherein two R4 moieties independently and
individually taken from the group consisting of C1-C6alkyl, branched
C3-C6alkyl, hydroxyalkyl-, and alkoxyalkyl and attached to the same
nitrogen heteroatom may cyclize to form a C3-C7 heterocyclyl ring; k is 0
or 1; n is 0-6; p is 1-4; q is 2-6; r is 0 or 1; t is 1-3; v is 1 or 2; m
is 0-2;and stereo-, regioisomers and tautomers of such compounds.
2. Compounds of claim 1 whereinis selected from the group consisting ofwherein the symbol (**) indicates the point of attachment to the pyridine ring.
3. Compounds of claim 2 having the formula Ibwherein A is any possible isomer of pyrazole.
4. Compounds of claim 3 having formula Ic
5. Compounds of claim 3 having formula Id
6. Compounds of claim 3 having formula Ie
7. Compounds of claim 2 having the formula If
8. Compounds of claim 7 having formula Ig
9. Compounds of claim 2 having the formula Ihwherein A is selected from the group consisting of any possible isomer of phenyl and pyridine.
10. Compounds of claim 9 having formula Ii
11. Compounds of claim 9 having formula Ij
12. Compounds of claim 2 having the formula Ik
13. Compounds of claim 12 having formula Il
14. A method of treating mammalian disease wherein the disease etiology or progression is at least partially mediated by the kinase activity of c-Abl kinase, bcr-Abl kinase, Flt-3 kinase, VEGFR-2 kinase mutants, c-Met, PDGFR-alpha kinase, PDGFR-beta kinase, HER-1, HER-2, HER-3, HER-4, FGFR, c-Kit, oncogenic forms thereof, aberrant fusion proteins thereof and polymorphs of any of the foregoing, comprising the step of administering to the mammal a compound of claim 1.
15. A method of claim 14 wherein said kinase is selected from the group consisting of bcr-Abl fusion protein kinases p210, bcr-Abl fusion protein kinases p190, bcr-Abl fusion protein kinases bearing the T315I gatekeeper mutant in the Abl kinase domain of p210, bcr-Abl fusion protein kinases bearing the T3315I gatekeeper mutant in the Abl kinase domain of p190, and other bcr-Abl polymorphs of any of the foregoing kinases.
16. The method of claim 15, wherein said bcr-Abl fusion protein kinases p210 having SEQ ID NO:3 & SEQ ID NO:4, wherein said bcr-Abl fusion protein kinase p190 has SEQ ID NO:5, wherein said bcr-Abl fusion protein kinases p210 bearing the T315I mutation in the Abl kinase domain has SEQ ID NO:6 & SEQ ID NO:7, and wherein said bcr-Abl fusion protein kinase p190 bearing the T315I mutation in the Abl kinase domain has SEQ ID NO:8.
17. A method of claim 14 wherein said kinase is selected from the group consisting of ckit protein kinase, PDGFR-alpha kinase, and any fusion protein, mutation and polymorphs of any of the foregoing.
18. A method of claim 14 wherein said kinase is selected from the group consisting of c-Met protein kinase, and any fusion protein, mutation and polymorphs of any of the foregoing.
19. A pharmaceutical composition comprising a compound of claim 1, together with a pharmaceutically acceptable carrier, optionally containing an additive selected from the group including adjuvants, excipients, diluents, and stabilizers.
20. A method of treating an individual suffering from a condition selected from the group consisting of cancer, hyperproliferative diseases, metabolic diseases, neurodegenerative diseases, or diseases characterized by angiogenesis, such as solid tumors, melanomas, glioblastomas, ovarian cancer, pancreatic cancer, prostate cancer, lung cancers, breast cancers, renal cancers, hepatic cancers, cervical carcinomas, metastasis of primary tumor sites, myeloproliferative diseases, chronic myelogenous leukemia, leukemias, papillary thyroid carcinoma, non-small cell lung cancer, mesothelioma, hypereosinophilic syndrome, gastrointestinal stromal tumors, colonic cancers, ocular diseases characterized by hyperproliferation leading to blindness including retinopathies, diabetic retinopathy, age-related macular degeneration and hypereosinophilic syndrome, rheumatoid arthritis, asthma, chronic obstructive pulmonary, mastocytosis, mast cell leukemia, or disease a disease caused by c-Kit kinase, oncogenic forms thereof, aberrant fusion proteins thereof and polymorphs thereof, comprising the step of administering to such individual a compound of claim 1.
21. The method of claim 20, said compound being administered by a method selected from the group consisting of oral, parenteral, inhalation, and subcutaneous.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001]This application claims the benefit of Provisional Application 60/913,216 filed Apr. 20, 2007. This provisional application is incorporated by reference herein in its entirety.
SEQUENCE LISTING
[0002]This application contains a Sequence Listing in both paper and computer readable format in accordance with 37 C.F.R. 1.821 (c) and (e), the contents of which are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0003]The present invention relates to novel kinase inhibitors and modulator compounds useful for the treatment of various diseases. More particularly, the invention is concerned with such compounds, kinase/compound adducts, methods of treating diseases, and methods of synthesis of the compounds. Preferably, the compounds are useful for the modulation of kinase activity of C-Abl, c-Kit, VEGFR, PDGFR kinases, Flt-3, c-Met, FGFR, the HER family and disease polymorphs thereof.
BACKGROUND OF THE INVENTION
[0004]Several members of the protein kinase family have been clearly implicated in the pathogenesis of various proliferative and myleoproliferative diseases and thus represent important targets for treatment of these diseases. Some of the proliferative diseases relevant to this invention include cancer, rheumatoid arthritis, atherosclerosis, and retinopathies. Important examples of kinases which have been shown to cause or contribute to the pathogensis of these diseases include C-Abl kinase and the oncogenic fusion protein bcr-Abl kinase; c-Kit kinase, PDGF receptor kinase; VEGF receptor kinases; and Flt-3 kinase.
[0005]C-Abl kinase is an important non-receptor tyrosine kinase involved in cell signal transduction. This ubiquitously expressed kinase--upon activation by upstream signaling factors including growth factors, oxidative stress, integrin stimulation, and ionizing radiation--localizes to the cell plasma membrane, the cell nucleus, and other cellular compartments including the actin cytoskeleton (Van Etten, Trends Cell Biol. (1999) 9: 179). There are two normal isoforms of Abl kinase: Abl-1A and Abl-1B. The N-terminal half of c-Abl kinase is important for autoinhibition of the kinase domain catalytic activity (Pluk et al, Cell (2002) 108: 247). Details of the mechanistic aspects of this autoinhibition have recently been disclosed (Nagar et al, Cell (2003) 112: 859). The N-terminal myristolyl amino acid residue of Abl-1B has been shown to intramolecularly occupy a hydrophobic pocket formed from alpha-helices in the C-lobe of the kinase domain. Such intramolecular binding induces a novel binding area for intramolecular docking of the SH2 domain and the SH3 domain onto the kinase domain, thereby distorting and inhibiting the catalytic activity of the kinase. Thus, an intricate intramolecular negative regulation of the kinase activity is brought about by these N-terminal regions of c-Abl kinase. An aberrant dysregulated form of c-Abl is formed from a chromosomal translocation event, referred to as the Philadelphia chromosome (P. C. Nowell et al, Science (1960) 132: 1497; J. D. Rowley, Nature (1973) 243: 290). This abnormal chromosomal translocation leads aberrant gene fusion between the Abl kinase gene and the breakpoint cluster region (BCR) gene, thus encoding an aberrant protein called bcr-Abl (G. Q. Daley et al, Science (1990) 247: 824; M. L. Gishizky et al, Proc. Natl. Acad. Sci. USA (1993) 90: 3755; S. Li et al, J. Exp. Med. (1999) 189: 1399). The bcr-Abl fusion protein does not include the regulatory myristolylation site (B. Nagar et al, Cell (2003) 112: 859) and as a result functions as an oncoprotein which causes chronic myeloid leukemia (CML). CML is a malignancy of pluripotent hematopoietic stem cells. The p210 form of bcr-Abl is seen in 95% of patients with CML, and in 20% of patients with acute lymphocytic leukemia and is exemplified by sequences such as e14a2 and e13a2. The corresponding p190 form, exemplified by the sequence e1a2 has also been identified. A p185 form has also been disclosed and has been linked to being causative of up to 10% of patients with acute lymphocytic leukemia. It will be appreciated by one skilled in the art that "p210 form", "p190 form" and "p185 form" each describe a closely related group of fusion proteins, and that Sequence ID's used herein are merely representative of each form and are not meant to restrict the scope solely to those sequences.
[0006]C-KIT (Kit, CD117, stem cell factor receptor) is a 145 kDa transmembrane tyrosine kinase protein that acts as a type-III receptor (Pereira et al. J Carcin. (2005), 4: 19). The c-KIT proto-oncocgene, located on chromosome 4q11-21, encodes the c-KIT receptor, whose ligand is the stem cell factor (SCF, steel factor, kit ligand, mast cell growth factor, Morstyn G, et al. Oncology (1994) 51(2):205. Yarden Y, et al. Embo J (1987) 6(11):3341). The receptor has tyrosine-protein kinase activity and binding of the ligands leads to the autophosphorylation of KIT and its association with substrates such as phosphatidylinositol 3-kinase (Pi3K). Tyrosine phosphorylation by protein tyrosine kinases is of particular importance in cellular signalling and can mediate signals for major cellular processes, such as proliferation, differentiation, apoptosis, attachment, and migration. Defects in KIT are a cause of piebaldism, an autosomal dominant genetic developmental abnormality of pigmentation characterized by congenital patches of white skin and hair that lack melanocytes. Gain-of-function mutations of the c-KIT gene and the expression of phosphorylated KIT are found in most gastrointestinal stromal tumors and mastocytosis. Further, almost all gonadal seminomas/dysgerminomas exhibit KIT membranous staining, and several reports have clarified that some (10-25%) have a c-KIT gene mutation (Sakuma, Y. et al. Cancer Sci (2004) 95:9, 716). KIT defects have also been associated with testicular tumors including germ cell tumors (GCT) and testicular germ cell tumors (TGCT).
[0007]The role of c-kit expression has been studied in hematologic and solid tumours, such as acute leukemias (Cortes J. et al. Cancer (2003) 97(11):2760) and gastrointestinal stromal tumors (GIST, Fletcher C. D. et al. Hum Pathol (2002) 33(5):459). The clinical importance of c-kit expression in malignant tumors relies on studies with Gleevec® (imatinib mesylate, STI571, Novartis Pharma AG Basel, Switzerland) that specifically inhibits tyrosine kinase receptors (Lefevre G. et al. J Biol Chem (2004) 279(30):31769). Moreover, a clinically relevant breakthrough has been the finding of anti-tumor effects of this compound in GIST, a group of tumors regarded as being generally resistant to conventional chemotherapy (de Silva C M, Reid R: Pathol Oncol Res (2003) 9(1):13-19). GIST most often become Gleevec resistant and molecularly targeted small therapies that target c-KIT mutations remain elusive.
[0008]c-MET is a unique receptor tyrosine kinase (RTK) located on chromosome 7p and activated via its natural ligand hepatocyte growth factor. c-MET is found mutated in a variety of solid tumors (Ma P. C. et al. Cancer Metastasis (2003) 22:309). Mutations in the tyrosine kinase domain are associated with hereditary papillary renal cell carcinomas (Schmidt L et al. Nat. Genet. (1997)16:68; Schmidt L, et al. Oncogene (1999) 18:2343), whereas mutations in the sema and juxtamembrane domains are often found in small cell lung cancers (SCLC; Ma P. C. et al. Cancer Res (2003) 63:6272). Many activating mutations are also found in breast cancers (Nakopoulou et al. Histopath (2000) 36(4): 313). The panoply of tumor types for which c-Met mediated growth has been implicated suggests this is a target ideally suited for modulation by specific c-MET small molecule inhibitors.
[0009]The TPR-MET oncogene is a transforming variant of the c-MET RTK and was initially identified after treatment of a human osteogenic sarcoma cell line transformed by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (Park M. et al. Cell (1986) 45:895). The TPR-MET fusion oncoprotein is the result of a chromosomal translocation, placing the TPR3 locus on chromosome 1 upstream of a portion of the c-MET gene on chromosome 7 encoding only for the cytoplasmic region. Studies suggest that TPR-MET is detectable in experimental cancers (e.g. Yu J. et al. Cancer (2000) 88:1801). Dimerization of the M, 65,000 TPR-MET oncoprotein through a leucine zipper motif encoded by TPR leads to constitutive activation of the c-MET kinase (Zhen Z. et al. Oncogene (1994) 9:1691). TPR-MET acts to activated wild-type c-MET RTK and can activate crucial cellular growth pathways, including the Ras pathway (Aklilu F. et al. Am J Physiol (1996) 271:E277) and the phosphatidylinositol 3-kinase (PI3K)/AKT pathway (Ponzetto C. et al. Mol Cell Biol (1993) 13:4600). Conversely, in contrast to c-MET RTK, TPR-MET is ligand independent, lacks the CBL binding site in the juxtamembrane region in c-MET, and is mainly cytoplasmic. c-Met immunohistochemical expression seems to be associated with abnormal β-catenin expression, and provides good prognostic and predictive factors in breast cancer patients.
[0010]The majority of small molecule kinase inhibitors that have been reported have been shown to bind in one of three ways. Most of the reported inhibitors interact with the ATP binding domain of the active site and exert their effects by competing with ATP for occupancy. Other inhibitors have been shown to bind to a separate hydrophobic region of the protein known as the "DFG-in-conformation" pocket wherein such a binding mode by the inhibitor causes the kinase to adopt the "DFG-out" conformation, and still others have been shown to bind to both the ATP domain and the "DFG-in-conformation" pocket again causing the kinase to adopt the "DGF-out" conformation. Examples specific to inhibitors of Raf kinases can be found in Lowinger et al, Current Pharmaceutical Design (2002) 8; 2269; Dumas, J. et al., Current Opinion in Drug Discovery & Development (2004) 7: 600; Dumas, J. et al, WO 2003068223 A1 (2003); Dumas, J., et al, WO 9932455 A1 (1999), and Wan, P. T. C., et al, Cell (2004) 116: 855.
[0011]Physiologically, kinases are regulated by a common activation/deactivation mechanism wherein a specific activation loop sequence of the kinase protein binds into a specific pocket on the same protein which is referred to as the switch control pocket. Such binding occurs when specific amino acid residues of the activation loop are modified for example by phosphorylation, oxidation, or nitrosylation. The binding of the activation loop into the switch pocket results in a conformational chance of the protein into its active form (Huse, M. and Kuriyan, J. Cell (109) 275)
SUMMARY OF THE INVENTION
[0012]Compounds of the present invention find utility in the treatment of mammalian cancers and especially human cancers including but not limited to malignant, melanomas, glioblastomas, ovarian cancer, pancreatic cancer, prostate cancer, lung cancers, breast cancers, kidney cancers, cervical carcinomas, metastasis of primary tumor sites, myeloproliferative diseases, leukemias, papillary thyroid carcinoma, non small cell lung cancer, mesothelioma, hypereosinophilic syndrome, gastrointestinal stromal tumors, colonic cancers, ocular diseases characterized by hyperproliferation leading to blindness including various retinopathies, rheumatoid arthritis, asthma, chronic obstructive pulmonary disorder, a disease caused by c-Abl kinase, oncogenic forms thereof, aberrant fusion proteins thereof and polymorphs thereof, or a disease caused by c-Kit, oncogenic forms thereof, aberrant fusion proteins thereof and polymorphs thereof.
SECTION 1--DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0013]The following descriptions refer to various compounds, stereo-, regioisomers and tautomers of such compounds and individual moieties of the compounds thereof.
[0014]Cycloalkyl refers to monocyclic saturated carbon rings taken from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptanyl and cyclooctanyl;
[0015]Aryl refers to monocyclic or fused bicyclic ring systems characterized by delocalized π electrons (aromaticity) shared among the ring carbon atoms of at least one carbocyclic ring; preferred aryl rings are taken from phenyl, naphlthyl, tetrahydronaphthyl, indenyl, and indanyl;
[0016]Heteroaryl refers to monocyclic or fused bicyclic ring systems characterized by delocalized π electrons (aromaticity) shared among the ring carbon or heteroatoms including nitrogen, oxygen, or sulfur of at least one carbocyclic or heterocyclic ring; heteroaryl rings are taken from, but not limited to, pyrrolyl, furyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyrazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, indolyl, indolinyl, isoindolyl, isoindolinyl, indazolyl, benzofuranyl, benzothienyl, benzothiazolyl, benzothiazolonyl, benzoxazolyl, benzoxazolonyl, benzisoxazolyl, benzisothiazolyl, benzimidazolyl, benzimidazolonyl, benztriazolyl, imidazopyridinyl, pyrazolopyridinyl, imidazolonopyridinyl, thiazolopyridinyl, thiazolonopyridinyl, oxazolopyridinyl, oxazolonopyridinyl, isoxazolopyridinyl, isothiazolopyridinyl, triazolopyridinyl, imidazopyrimidinyl, pyrazolopyrimidinyl, imidazolonopyrimidinyl, thiazolopyridiminyl, thiazolonopyrimidinyl, oxazolopyridiminyl, oxazolonopyrimidinyl, isoxazolopyrimidinyl, isothiazolopyrimidinyl, triazolopyrimidinyl, dihydropurinonyl, pyrrolopyrimidinyl, purinyl, pyrazolopyrimidinyl, phthalimidyl, phthalimidinyl, pyrazinylpyridinyl, pyridinopyrimidinyl, pyrimidinopyrimidinyl, cinnolinyl, quinoxalinyl, quinazolinyl, quinolinyl, isoquinolinyl, phthalazinyl, benzodioxyl, benzisothiazoline-1,1,3-trionyl, dihydroquiniolinyl, tetrahydroquinolinyl, dihydroisoquinolyl, tetrahydroisoquinolinyl, benzoazepinyl, benzodiazepinyl, benzoxapinyl, and benzoxazepinyl;
[0017]Heterocyclyl refers to monocyclic rings containing carbon and heteroatoms taken from oxygen, nitrogen, or sulfur and wherein there is not delocalized π electrons (aromaticity) shared among the ring carbon or heteroatoms; heterocyclyl rings include, but are not limited to, oxetanyl, azetadinyl, tetrahydrofuranyl, pyrrolidinyl, oxazolinyl, oxazolidinyl, thiazolinyl, thiazolidinyl, pyranyl, thiopyranyl, tetrahydropyranyl, dioxalinyl, piperidinyl, morpholinyl, thiomorpholinyl, thiomorpholinyl S-oxide, thiomorpholinyl S-dioxide, piperazinyl, azepinyl, oxepinyl, diazepinyl, tropanyl, and homotropanyl;
[0018]Poly-aryl refers to two or more monocyclic or fused aryl bicyclic ring systems characterized by delocalized π electrons (aromaticity) shared among the ring carbon atoms of at least one carbocyclic ring wherein the rings contained therein are optionally linked together;
[0019]Poly-heteroaryl refers to two or more monocyclic or fused bicyclic systems characterized by delocalized π electrons (aromaticity) shared among the ring carbon or heteroatoms including nitrogen, oxygen, or sulfur of at least one carbocyclic or heterocyclic ring wherein the rings contained therein are optionally linked together, wherein at least one of the monocyclic or fused bicyclic rings of the poly-heteroaryl system is taken from heteroaryl as defined broadly above and the other rings are taken from either aryl, heteroaryl, or heterocyclyl as defined broadly above;
[0020]Poly-heterocyclyl refers to two or more monocyclic or fused bicyclic ring systems containing carbon and heteroatoms taken from oxygen, nitrogen, or sulfur and wherein there is not delocalized π electrons (aromaticity) shared among the ring carbon or heteroatoms wherein the rings contained therein are optionally linked, wherein at least one of the monocyclic or fused bicyclic rings of the poly-heteroaryl system is taken from heterocyclyl as defined broadly above and the other rings are taken from either aryl, heteroaryl, or heterocyclyl as defined broadly above;
[0021]Alkyl refers to straight or branched chain C1-C6alkyls;
[0022]Halogen refers to fluorine, chlorine, bromine, and iodine;
[0023]Alkoxy refers to --O-(alkyl) wherein alkyl is defined as above;
[0024]Alkoxylalkyl refers to -(alkyl)-O-(alkyl) wherein alkyl is defined as above;
[0025]Alkoxylcarbonyl refers to --C(O)O-(alkyl) wherein alkyl is defined as above;
[0026]CarboxylC1-C6alkyl refers to --C(O)-alkyl wherein alkyl is defined as above;
[0027]Substituted in connection with a moiety refers to the fact that a further substituent may be attached to the moiety to any acceptable location on the moiety.
[0028]The term salts embraces pharmaceutically acceptable salts commonly used to form alkali metal salts of free acids and to form addition salts of free bases. The nature of the salt is not critical, provided that it is pharmaceutically-acceptable. Suitable pharmaceutically-acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, and heterocyclyl containing carboxylic acids and sulfonic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, stearic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, toluenesulfonic, 2-hydroxyethanesulfonic, sulfanilic, cyclohexylaminosulfonic, algenic, 3-hydroxybutyric, galactaric and galacturonic acid. Suitable pharmaceutically-acceptable salts of free acid-containing compounds of Formula I include metallic salts and organic salts. More preferred metallic salts include, but are not limited to appropriate alkali metal (group Ia) salts, alkaline earth metal (group IIa) salts and other physiological acceptable metals. Such salts can be made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc. Preferred organic salts can be made from primary amines, secondary amines, tertiary amines and quaternary ammonium salts, including in part, tromethamine, diethylamine, tetra-N-methylammonium, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine(N-methylglucamine) and procaine.
[0029]The term prodrug refers to derivatives of active compounds which revert in vivo into the active form. For example, a carboxylic acid form of an active drug may be esterified to create a prodrug, and the ester is subsequently converted in vivo to revert to the carboxylic acid form. See Ettmayer et. al, J. Med. Chem (2004) 47: 2393 and Lorenzi et. al, J. Pharm. Exp. Therapeutic (2005) 883 for reviews.
[0030]Structural, chemical and stereochemical definitions are broadly taken from IUPAC recommendations, and more specifically from Glossary of Terms used in Physical Organic Chemistry (IUPAC Recommendations 1994) as summarized by P. Muller, Pure Appl. Chem., 66, 1077-1184 (1994) and Basic Terminology of Stereochemistry (IUPAC Recommendations 1996) as summarized by G. P. Moss Pure and Applied Chemistry, 68, 2193-2222 (1996). Specific definitions are as follows:
[0031]Atropisomers are defined as a subclass of conformers which can be isolated as separate chemical species and which arise from restricted rotation about a single bond.
[0032]Regioisomers or structural isomers are defined as isomers involving the same atoms in different arrangements.
[0033]Enatiomers are defined as one of a pair of molecular entities which are mirror images of each other and non-superimposable.
[0034]Diastereomers or diastereoisomers are defined as stereoisomers other than enantiomers. Diastereomers or diastereoisomers are stereoisomers not related as mirror images.
[0035]Diastereoisomers are characterized by differences in physical properties, and by some differences in chemical behavior towards achiral as well as chiral reagents.
[0036]Tautomerism is defined as isomerism of the general form
[0037]where the isomers (called tautomers) are readily interconvertible; the atoms connecting the groups X,Y,Z are typically any of C, H, O, or S, and G is a group which becomes an electrofuge or nucleofuge during isomerization. The commonest case, when the electrofuge is H.sup.+, is also known as "prototropy".
[0038]Tautomers are defined as isomers that arise from tautomerism, independent of whether the isomers are isolable.
[0039]1. First Aspect of the Invention--Compounds, Methods, Preparations and Adducts
[0040]and wherein the pyridine ring may be optionally substituted with one or more R20 moieties;
[0041]each D is individually taken from the group consisting of C, CH, C--R20, N-Z3, N, O and S, such that the resultant ring is taken from the group consisting of triazolyl, isoxazolyl, isothiazolyl, oxazolyl, and thiadiazolyl;
[0042]wherein E is selected from the group consisting of phenyl, pyridyl, and pyrimidinyl;
[0043]E may be optionally substituted with one or two R16 moieties;
[0044]wherein A is a ring system selected from the group consisting of phenyl, naphthyl, cyclopentyl, cyclohexyl, G1, G2, and G3;
[0045]G1 is a heteroaryl taken from the group consisting of pyrrolyl, furyl, thienyl, oxazolyl, thiazolyl, isoxazol-4-yl, isoxazol-5-yl, isothiazolyl, imidazolyl, pyrazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyrazinyl, pyridazinyl, triazinyl, pyridinyl, and pyrimidinyl;
[0046]G2 is a fused bicyclic heteroaryl taken from the group consisting of indolyl, indolinyl, isoindolyl, isoindolinyl, indazolyl, benzofuranyl, benzothienyl, benzothiazolyl, benzothiazolonyl, benzoxazolyl, benzoxazolonyl, benzisoxazolyl, benzisothiazolyl, benzimidazolyl, benzimidazolonyl, benztriazolyl, imidazopyridinyl, pyrazolopyridinyl, imidazolonopyridinyl, thiazolopyridinyl, thiazolonopyridinyl, oxazolopyridinyl, oxazolonopyridinyl, isoxazolopyridinyl, isothiazolopyridinyl, triazolopyridinyl, imidazopyrimidinyl, pyrazolopyrimidinyl, imidazolonopyrimidinyl, thiazolopyridiminyl, thiazolonopyrimidinyl, oxazolopyridiminyl, oxazolonopyrimidinyl, isoxazolopyrimidinyl, isothiazolopyrimidinyl, triazolopyrimidinyl, dihydropurinonyl, pyrrolopyrimidinyl, purinyl, pyrazolopyrimidinyl, phthalimidyl, phthalimidinyl, pyrazinylpyridinyl, pyridinopyrimidinyl, pyrimidinopyrimidinyl, cinnolinyl, quinoxalinyl, quinazolinyl, quinolinyl, isoquinolinyl, phthalazinyl, benzodioxyl, benzisothiazoline-1,1,3-trionyl, dihydroquinolinyl, tetrahydroquinolinyl, dihydroisoquinolyl, tetrahydroisoquinolinyl, benzoazepinyl, benzodiazepinyl, benzoxapinyl, and benzoxazepinyl;
[0047]G3 is a heterocyclyl taken from the group consisting of oxetanyl, azetadinyl, tetrahydrofuranyl, pyrrolidinyl, oxazolinyl, oxazolidinyl, imidazolonyl, pyranyl, thiopyranyl, tetrahydropyranyl, dioxalinyl, piperidinyl, morpholinyl, thiomorpholinyl, thiomorpholinyl S-oxide, thiomorpholinyl S-dioxide, piperazinyl, azepinyl, oxepinyl, diazepinyl, tropanyl, and homotropanyl;
[0048]the A ring may be optionally substituted with one or two R2 moieties;
[0049]X is selected from the group consisting of --O--, --S(CH2)n--, --N(R3)(CH2)n--, --(CH2)p--, and wherein the carbon atoms of --(CH2)n--, --(CH2)p--, of X may be further substituted by oxo or one or more C1-C6alkyl moieties;
[0050]when A, G1, G2 or G3 has one or more substitutable sp2-hybridized carbon atoms, each respective sp2 hybridized carbon atom may be optionally substituted with a Z1 substituent;
[0051]when A, G1, C2 or G3 has one or more substitutable sp3-hybridized carbon atoms, each respective sp3 hybridized carbon atom may be optionally substituted with a Z2 substituent;
[0052]when A, G1, G2 or G3 has one or more substitutable nitrogen atoms, each respective nitrogen atom may be optionally substituted with a Z4 substituent;
[0053]each Z1 is independently and individually selected from the group consisting of C1-6alkyl, branched C3-C7alkyl, C3-C8cycloalkyl, halogen, fluoroC1-C6alkyl wherein the alkyl moiety can be partially or fully fluorinated, cyano, C1-C6alkoxy, fluoroC1-C6alkoxy wherein the alkyl moiety can be partially or fully fluorinated, --(CH2)nOH, oxo, C1-C6alkoxyC1-C6alkyl, (R4)2N(CH2)n--, (R3)2N(CH2)n--, (R4)2N(CH2)qN(R4)(CH2)n--, (R4)2N(CH2)qO(CH2)n--, (R3)2NC(O)--, (R4)2NC(O)--, (R4)2NC(O)C1-C6alkyl-, --(R4)NC(O)R8, C1-C6alkoxycarbonyl-, -carboxyC1-C6alkyl, C1-C6alkoxycarbonylC1-C6alkyl-, (R3)2NSO2--, --SOR3, (R4)2NSO2--, --N(R4)SO2R8, --O(CH2)qOC1-C6alkyl, --SO2R3, --SOR4, --C((O)R8, --C(O)R6, --C(═NOH)R6, --C(═NOR3)R6, --(CH2)nN(R4)C(O)R8, --N(R3)(CH2)qO-alkyl, --N(R3)(CH2)qN(R4)2, nitro, --CH(OH)CH(OH)R4, --C(═NH)N(R4)2, --C(═NOR3)N(R4)2, --NHC(═NH)R8, R17 substituted G3, R17 substituted pyrazolyl and R17 substituted imidazolyl;
[0054]in the event that Z1 contains an alkyl or alkylene moiety, such moieties may be further substituted with one or more C1-C6alkyls;
[0055]each Z2 is independently and individually selected from the group consisting of aryl, C1-C6alkyl, C3-C8cycloalkyl, branched C3-C7alkyl, hydroxyl, hydroxyC1-C6alkyl-, cyano, (R3)2N--, (R4)2N--, (R4)2NC1-C6alkyl-, (R4)2NC2-C6alkylN(R4)(CH2)n--, (R4)2NC2-C6alkylO(CH2)n--, (R3)2NC(O)--, (R4)2NC(O)--, (R4)2NC(O)--C1-C6alkyl-, carboxyl, -carboxyC1-C6alkyl, C1-C6alkoxycarbonyl-, C1-C6alkoxycarbonylC1-C6alkyl-, (R3)2NSO2--, (R4)2NSO2--, --SO2R8, --(CH2)nN(R4)C(O)R8, --C(O)R8, ═O, ═NOH, and ═N(OR6);
[0056]in the event that Z2 contains an alkyl or alkylene moiety, such moieties may be further substituted with one or more C1-C6alkyls;
[0057]each Z3 is independently and individually selected from the group consisting of H, C1-C6alkyl, branched C3-C7alkyl, C3-C8cycloalkyl, fluoroC1-C6alkyl wherein the alkyl moiety can be partially or fully fluorinated, hydroxyC2-C6alkyl-, C1-C6alkoxycarbonyl-, --C(O)R8, R5C(O)(CH2)n--, (R4)2NC(O)--, (R4)2NC(O)C1-C6alkyl-, R8C(O)N(R4)(CH2)q--, (R3)2NSO2--, (R4)2NSO2--, --(CH2)qN(R3)2, and --(CH2)qN(R4)2;
[0058]each Z4 is independently and individually selected from the group consisting of C1-C6alkyl, branched C3-7alkyl, hydroxyC2-C6alkyl-, C1-C6alkoxyC2-C6alkyl-, (R4)2N--C2-C6alkyl-, (R4)2N--C2-C6alkylN(R4)--C2-C6alkyl-, (R4)2N--C2-C6alkyl-O--C2-C6alkyl-(R4)2NC(O)C1-C6alkyl-, carboxyC1-C6alkyl, C1-C6alkoxycarbonylC1-C6alkyl-, --C2-C6alkylN(R4)C(O)R8, R8-C(═NR3)--, --SO2R8, and --COR8;
[0059]in the event that Z4 contains an alkyl or alkylene moiety, such moieties may be further substituted with one or more C1-C6alkyls;
[0060]each R2 is selected from the group consisting of H, C1-C6alkyl, branched C3-C8alkyl, R19 substituted C3-C8cycloalkyl-, fluoroC1-C6alkyl- wherein the alkyl is fully or partially fluorinated, halogen, cyano, C1-C6alkoxy-, and fluoroC1-C6alkoxy- wherein the alkyl group is fully or partially fluorinated, hydroxyl substituted C1-C6alkyl-, hydroxyl substituted branched C3-C8alkyl-, cyano substituted C1-C6alkyl-, cyano substituted branched C3-C8alkyl-, (R3)2NC(O)C1-C6alkyl-, and (R3)2NC(O)C3-C8 branched alkyl-;
[0061]wherein each R3 is independently and individually selected from the group consisting of H, C1-C6alkyl, branched C3-C7alkyl, and C3-C8cycloalkyl;
[0062]each R4 is independently and individually selected from the group consisting of H, C1-C6alkyl, hydroxyC1-C6alkyl-, dihydroxyC1-C6alkyl-, C1-C6alkoxyC1-C6alkyl-, branched C3-C7alkyl, branched hydroxyC1-C6alkyl-, branched C1-C6alkoxyC1-C6alkyl-, branched dihydroxyC1-C6alkyl-, --(CH2)pN(R7)2, --(CH2)pC(O)N(R7)2, --(CH2)nC(O)OR3, and R19 substituted C3-C8cycloalkyl-;
[0063]each R5 is independently and individually selected from the group consisting of
[0064]and wherein the symbol (##) is the point of attachment to Z3;
[0065]each R6 is independently and individually selected from the group consisting of C1-C6alkyl, branched C3-C7alkyl, and R19 substituted C3-C8cycloalkyl-;
[0066]each R7 is independently and individually selected from the group consisting of H, C1-C6alkyl, hydroxyC2-C6alkyl-, dihydroxyC2-C6alkyl-, C1-C6alkoxyC2-C6alkyl-, branched C3-C7alkyl, branched hydroxyC2-C6alkyl-, branched C1-C6alkoxyC2-C6alkyl-, branched dihydroxyC2-C6alkyl-, --(CH2)nC(O)OR3, R19 substituted C3-C8cycloalkyl- and --(CH2)nR17;
[0067]each R8 is independently and individually selected from the group consisting of C1-C6alkyl, branched C3-C7alkyl, fluoroC1-C6alkyl- wherein the alkyl moiety is partially or fully fluorinated, R19 substituted C3-C8cycloalkyl-, --OH, C1-C6alkoxy, --N(R3)2, and --N(R4)2;
[0068]each R10 is independently and individually selected from the croup consisting of --CO2H, --CO2C1-C6alkyl, --C(O)N(R4)2, OH, C1-C6alkoxy, and --N(R4)2;
[0069]each R16 is independently and individually selected from the group consisting of H, C1-C6alkyl, branched C3-C7alkyl, R19 substituted C3-C8cycloalkyl-, halogen, fluoroC1-C6alkyl- wherein the alkyl moiety can be partially or fully fluorinated, cyano, hydroxyl, C1-C6alkoxy, fluoroC1-C6alkoxy- wherein the alkyl moiety can be partially or fully fluorinated, --N(R3)2, --N(R4)2, R3 substituted C2-C3alkynyl- and nitro;
[0070]each R17 is independently and individually selected from the group consisting of H, C1-C6alkyl, branched C3-C7alkyl, R19 substituted C3-C8cycloalkyl-, halogen, fluoroC1-C6alkyl- wherein the alkyl moiety can be partially or Filly fluorinated, cyano, hydroxyl, C1-C6alkoxy, fluoroC1-C6alkoxy- wherein the alkyl moiety can be partially or fully fluorinated, --N(R3)2, --N(R4)2, and nitro;
[0071]each R19 is independently and individually selected from the group consisting of H, OH and C1-C6alkyl;
[0072]each R20 is independently and individually selected from the group consisting of C1-C6alkyl, branched C3-C7alkyl, R19 substituted C3-C8cycloalkyl-, halogen, fluoroC1-C6alkyl- wherein the alkyl moiety can be partially or fully fluorinated, cyano, hydroxyl, C1-C6alkoxy, fluoroC1-C6alkoxy- wherein the alkyl moiety can be partially or fully fluorinated, --N(R3)2, --N(R4)2, --N(R3)C(O)R3, --C(O)N(R3)2 and nitro and wherein two R4 moieties independently and individually taken from the group consisting of C1-C6alkyl, branched C3-C6alkyl, hydroxyalkyl-, and alkoxyalkyl and attached to the same nitrogen heteroatom may cyclize to form a C3-C7 heterocyclyl ring;
[0073]k is 0 or 1; n is 0-6; p is 1-4; q is 2-6; r is 0 or 1; t is 1-3; v is 1 or 2; m is 0-2;
[0074]and stereo-, regioisomers and tautomers of such compounds.
[0075]1.1 Compounds of Formula Ia which Exemplify Referred D Moieties
[0076]In a preferred embodiment of compounds of formula Ia, said compounds have preferred
[0077]moieties of the formula:
[0078]wherein the symbol (**) indicates the point of attachment to the pyridine ring.
[0079]1.1.1 Compounds of Formula Ia which Exemplify Preferred A Moieties
[0080]In a preferred embodiment of compounds of formula Ia, said compounds have structures of formula Ib
[0081]wherein A is any possible isomer of pyrazole.
[0082]1.1.2 Compounds of Formula Ia which Exemplify Preferred A and R16 Moieties
[0083]In a more preferred embodiment of compounds of formula Ib, said compounds have structures of formula Ic
[0084]1.1.3 Compounds of Formula Ia which Exemplify Preferred A and R16 Moieties
[0085]In a more preferred embodiment of compounds of formula Ib, said compounds have structures of formula Id
[0086]1.1.4 Compounds of Formula Ia which Exemplify Preferred A and R16 Moieties
[0087]In a more preferred embodiment of compounds of formula Ib, said compounds have structures of formula Ie
[0088]1.1.5 Compounds of Formula Ia which Exemplify Preferred A Moieties
[0089]In a more preferred embodiment of compounds of formula Ia, said compounds have structures of formula If
[0090]1.1.6 Compounds of Formula Ia which Exemplify Preferred A and R16 Moieties
[0091]In a more preferred embodiment of compounds of formula Ia, said compounds have structures of formula Ig
[0092]1.1.7 Compounds of Formula Ia which Exemplify Preferred A Moieties
[0093]In a preferred embodiment of compounds of formula Ia, said compounds have structures of formula Ih
[0094]wherein A is selected from the group consisting of any possible isomer of phenyl and pyridine.
[0095]1.1.8 Compounds of Formula Ia which Exemplify Preferred A and R16 Moieties
[0096]In a more preferred embodiment of compounds of formula Ih, said compounds have structures of formula Ii
[0097]1.1.9 Compounds of Formula Ia which Exemplify Preferred A and R16 Moieties
[0098]In a more preferred embodiment of compounds of formula Ih, said compounds have structures of formula Ij
[0099]1.1.10 Compounds of Formula Ia which Exemplify Preferred A Moieties
[0100]In a more preferred embodiment of compounds of formula Ia, said compounds have structures of formula Ik
[0101]1.1.11 Compounds of Formula Ia which Exemplify Preferred A and R16 Moieties
[0102]In a more preferred embodiment of compounds of formula Ik, said compounds have structures of formula Il
[0103]1.1.12 Most Preferred Compounds of Formula Ia
[0104]1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(oxazol-5- -yl)pyridin-4-yloxy)phenyl)urea, 1-(4-(2-(1H-1,2,3-triazol-4-yl)pyridin-4-yloxy)-2-fluorophenyl)-3-(3-tert- -butyl-1-methyl-1H-pyrazol-5-yl)urea, 1-(4-(2-(1H-1,2,4-triazol-1-yl)pyridin-4-yloxy)-2-fluorophenyl)-3-(3-tert- -butylisoxazol-5-yl)urea and 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(oxazol-2-yl)p- yridin-4-yloxy)phenyl)urea.
[0105]1.2 Methods
[0106]1.2a Methods of Protein Modulation
[0107]The invention includes methods of modulating kinase activity of a variety of kinases, e.g. C-Abl kinase, bcr-Abl Kinase, Flt-3, VEGFR-2 kinase mutants, c-Met, c-Kit, PDGFR and the HER family of kinases. The kinases may be wildtype kinases, oncogenic forms thereof, aberrant fusion proteins thereof or polymorphs of any of the foregoing. The method comprises the step of contacting the kinase species with compounds of the invention and especially those set forth in sections section 1. The kinase species may be activated or unactivated, and the species may be modulated by phosphorylations, sulfation, fatty acid acylations glycosylations, nitrosylation, cystinylation (i.e. proximal cysteine residues in the kinase react with each other to form a disulfide bond) or oxidation. The kinase activity may be selected from the group consisting of catalysis of phospho transfer reactions, inhibition of phosphorylation, oxidation or nitrosylation of said kinase by another enzyme, enhancement of dephosphorylation, reduction or denitrosylation of said kinase by another enzyme, kinase cellular localization, and recruitment of other proteins into signaling complexes through modulation of kinase conformation.
[0108]1.2b Treatment Methods
[0109]The methods of the invention also include treating individuals suffering from a condition selected from the group consisting of cancer and hyperproliferative diseases. These methods comprise administering to such individuals compounds of the invention, and especially those of section 1, said diseases including, but not limited to, malignant melanomas, glioblastomas, ovarian cancer, pancreatic cancer, prostate cancer, lung cancers, breast cancers, kidney cancers, cervical carcinomas, metastasis of primary tumor secondary sites, myeloproliferative diseases, leukemias, papillary thyroid carcinoma, non small cell lung cancer, mesothelioma, hypereosinophilic syndrome, gastrointestinal stromal tumors, colonic cancers, ocular diseases characterized by hyperproliferation leading to blindness including various retinopathies including diabetic retinopathy and age-related macular degeneration, rheumatoid arthritis, asthma, chronic obstructive pulmonary disorder, mastocytosis, mast cell leukemia, a disease caused by c-Abl kinase, oncogenic forms thereof, aberrant fusion proteins thereof and polymorphs thereof, or a disease caused by a c-Kit kinase, oncogenic forms thereof, aberrant fusion proteins thereof and polymorphs thereof. The administration method is not critical, and may be from the group consisting of oral, parenteral, inhalation, and subcutaneous.
[0110]1.3 Pharmaceutical Preparations
[0111]The compounds of the invention, especially those of section 1 may form a part of a pharmaceutical composition by combining one or more such compounds with a pharamaceutically acceptable carrier. Additionally, the compositions may include an additive selected from the group consisting of adjuvants, excipients, diluents, and stabilizers.
SECTION 2. SYNTHESIS OF COMPOUNDS OF THE PRESENT INVENTION
[0112]The compounds of the invention are available by the procedures and teachings of WO 2006/071940, incorporated by reference, and by the general synthetic methods illustrated in the Schemes below and the accompanying examples.
[0113]As indicated in Scheme 1, ureas of general formula 1 can be readily prepared by the union of amines of general formula 2 with isocyanates 3 or isocyanate surrogates, for example trichloroethyl carbamates (4) or isopropenyl carbamates (5). Preferred conditions for the preparation of compounds of general formula 1 involve heating a solution of 4 or 5 with 2 in the presence of a tertiary base such as diisopropylethylamine, triethylamine or N-methylpyrrolidine in a solvent such as dimethylformamide, dimethylsulfoxide, tetrahydrofuran or 1,4-dioxane at a temperature between 50 and 100° C. for a period of time ranging from 1 hour to 2 days.
[0114]As shown in Scheme 2, isocyanates 3 can be prepared from amines A-NH2 6 with phosgene, or a phosgene equivalent such as diphosgene, triphosgene, or N,N-dicarbonylimidazole. Trichloroethyl carbamates 4 and isopropenyl carbamates 5 are readily prepared from amines A-NH2, (6) by acylation with trichloroethyl chloroformate or isopropenyl chloroformate by standard conditions familiar to those skilled in the art. Preferred conditions for the preparation of 4 and 5 include include treatment of compound 6 with the appropriate chloroformate in the presence of pyridine in an aprotic solvent such as dichloromethane or in the presence of aqueous hydroxide or carbonate in a biphasic aqueous/ethyl acetate solvent system.
[0115]Additionally, compounds of formula 1 can also be prepared from carboxylic acids 7 by the intermediacy of in-situ generated acyl azides (Curtius rearrangement) as indicated in Scheme 3. Preferred conditions for Scheme 3 include the mixing of acid 7 with amine 2 and diphenylphosphoryl azide in a solvent such as 1,4-dioxane or dimethylformamide in the presence of base, such as triethylamine and raising the temperature of the reaction to about 80-120° C. to affect the Curtius rearrangement.
[0116]By analogy to Schemes 1 and 3 above, it will be recognized by those skilled in the art that the compounds of formula 1 can also be prepared by the union of amines A-NH2 6 with isocyanates 8 (Scheme 4). Isocyanates 8 can be prepared from general amines 2 by standard synthetic methods. Suitable methods for example, include reaction of 2 with phosgene, or a phosgene equivalent such as diphosgene, triphosgene, or N,N-dicarbonylimidazole. In addition to the methods above for converting amines 2 into isocynates 8, the isocyanates 8 can also be prepared in situ by the Curtius rearrangement and variants thereof. Those skilled in the art will further recognize that isocycanates 8 need not be isolated, but may be simply generated in situ. Accordingly, acid 9 can be converted to compounds of formula 1 either with or without isolation of 8. Preferred conditions for the direct conversion of acid 9 to compounds of formula 1 involve the mixing of acid 9, amine A-NH2 6, diphenylphosphoryl azide and a suitable base, for example triethylamine, in an aprotic solvent, for example dioxane. Heating said mixture to a temperature of between 80 and 120° C. provides the compounds of formula 1.
[0117]Additionally, compounds of formula 1 can also be prepared from amines 2 by first preparing stable isocyanate equivalents, such as carbamates (Scheme 5). Especially preferred carbamates include trichloroethyl carbamates (10) and isopropenyl carbamates (11) which are readily prepared from amine 2 by reaction with trichloroethyl chloroformate or isopropenyl chloroformate respectively using standard conditions familiar to those skilled in the art. Further reaction of carbamates 10 or 11 with amine A-NH2 6 provides compounds of formula 1. Those skilled in the art will further recognize that certain carbamates can also be prepared from acid 9 by Curtius rearrangement and trapping with an alcoholic co-solvent. For example, treatment of acid 9 (Scheme 5) with diphenylphosphoryl azide and trichloroethanol at elevated temperature provides trichloroethyl carbamate 10.
[0118]Many methods exist for the preparation of amines A-NH2 6 and acids A-CO2H 7, depending on the nature of the A-moiety. Indeed, many such amines (6) and acids (7) useful for the preparation of compounds of formula 1 are available from commercial vendors. Some non-limiting preferred synthetic methods for the preparation of amines 6 and acids 7 are outlined in the following schemes and accompanying examples.
[0119]As illustrated in Scheme 6, Z4-substituted pyrazol-5-yl amines 14 (a preferred aspect of A-NH2 6, Scheme 2) are available by the condensation of hydrazines 12 and beta-keto nitrites 13 in the presence of a strong acid. Preferred conditions for this transformation are by heating in ethanolic HCl. Many such hydrazines 12 are commercially available. Others can be prepared by conditions familiar to those skilled in the art, for example by the diazotization of amines followed by reduction or, alternately from the reduction of hydrazones prepared from carbonyl precursors.
[0120]Another preferred method for constructing Z4-substituted pyrazoles is illustrated by the general preparation of pyrazole acids 19 and 20. (Scheme 7), aspects of of general acid A-CO2H 7 (Scheme 3). As indicated in Scheme 7, pyrazole 5-carboxylic esters 17 and 18 can be prepared by the alkylation of pyrazole ester 16 with Z4-X 15, wherein X represents a leaving group on a Z4 moiety such as a halide, triflate, or other sulfonate. Preferred conditions for the alkylation of pyrazole 16 include the use of strong bases such as sodium hydride, potassium tert-butoxide and the like in polar aprotic solovents such as dimethylsulfoxide, dimethylformamide or tetrahydrofuran. Z4-substituted pyrazoles 17 and 18 are isomers of one another and can both be prepared in the same reactions vessel and separated by purification methods familiar to those skilled in the art. The esters 17 and 18 in turn can be converted to acids 19 and 20 using conditions familiar to those skilled in the art, for example saponification in the case of ethyl esters, hydrogenation in the case of benzyl esters or acidic hydrolysis in the case of tert-butyl esters.
[0121]Scheme 8 illustrates the preparation of pyrazole amine 25, a further example of general amine A-NH2 6. Acid-catalyzed condensation of R2-substituted hydrazine 21 with 1,1,3,3-tetramethoxypropane 22 provides R2-substituted pyrazole 23. Those skilled in the art will further recognize that R2-substituted pyrazole 23 can also be prepared by direct alkylation of pyrazole. Pyrazole 23 can be regioselectively nitrated to provide nitro-pyrazole 24 by standard conditions familiar to those skilled in the art. Finally, hydrogenation of nitro-pyrazole 24 employing a hydrogenation catalyst, such as palladium or nickel provides pyrazole amine 25, an example of general amine A-NH2 6.
[0122]Additional pyrazoles useful for the synthesis of compounds of formula 1 can be prepared as described in Scheme 9. Thus, keto-ester 26 can be reacted with N,N-dimethylformamide dimethyl acetal to provide 27. Reaction of 27 with either 21 or 28 (wherein P is an acid-labile protecting group) in the presence of acid provides 29 or 30. In practice, both 29 and 30 can be obtained from the same reaction and can be separated by standard chromatographic conditions. In turn, esters 29 and 30 can be converted to acids 31 and 32 respectively as previously described in Scheme 7.
[0123]In a manner similar to Scheme 9, NH-pyrazole 34 can be prepared by reaction of acrylate 33 with hydrazine (Scheme 10). Alkylation of 34 with R2-X 35 as described above for Scheme 7 provides mixtures of pyrazole esters 36 and 37 which are separable by standard chromatographic techniques. Further conversion of esters 36 and 37 to acids 38 and 39 can be accomplished as described above in Scheme 7.
[0124]General amines 6 containing an isoxazole ring can be prepared as described in Scheme 11. Thus, by analogy to Scheme 6, reaction of keto-nitrile 9 with hydroxylamine can provide both the 5-aminoisoxazole 40 and 3-aminoisoxazole 41. Preferred conditions for the formation of 5-aminoisoxazole 40 include the treatment of 9 with hydroxylamine in the presence of aqueous sodium hydroxide, optionally in the presence of an alcoholic co-solvent at a temperature between 15 and 100° C. Preferred conditions for the formation of 3-aminoisoxazole 41 include the treatment of 9 with hydroxylamine hydrochloride in a polar solvent such as water, an alcohol, dioxane or a mixture thereof at a temperature between 15 and 100° C.
[0125]Amines 2 useful for the invention can be synthesized according to methods commonly known to those skilled in the art. Amines of general formula 2 contain three rings and can be prepared by the stepwise union of three monocyclic subunits as illustrated in the following non-limiting Schemes. Scheme 12 illustrates one mode of assembly in which an E-containing subunit 42 is combined with the central pyridine ring 43 to provide the bicyclic intermediate 44. In one aspect this general Scheme, the "M" moiety of 42 represents a hydrogen atom of a heteroatom on the X linker that participates in a nucleophilic aromatic substitution reaction with monocycle 43. Such reactions may be facilitated by the presence of bases (for example, potassium tert-butoxide), thus M may also represent a suitable counterion (for example potassium, sodium, lithium, or cesium) within an alkoxide, sulfide or amide moiety. Alternately, the "M" group can represent a metallic species (for example, copper, boron, tin, zirconium, aluminum, magnesium, lithium, silicon, etc.) on a carbon atom of the X2 moiety that can undergo a transition-metal-mediated coupling with monocycle 43.
[0126]The "Y" group of monocyclic species 42 is an amine or an amine surrogate, such as an amine masked by a protecting group ("P" in formula 45), a nitro group, or a carboxy acid or ester that can be used to prepare an amine via known rearrangement. Examples of suitable protecting groups "P" include but are not limited to tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), and acetamide. In the instances wherein the "Y"-group of intermediate 42 is not an amine, the products of Scheme 11 will be amine surrogates such as 45 or 46 that can be converted to amine 2 by a deprotection, reduction or rearrangement (for example, Curtius rearrangement) familiar to those skilled in the all.
[0127]In these instances, the "LG" of monocycle 43 represents a moiety that can either be directly displaced in a nucleophilic substitution reaction (with or without additional activation) or can participate in a transition-mediated union with fragment 42. The W group of monocycle 43 or bicycle 44 represents a moiety that allows the attachment of the pyrazole. In one aspect, the "W" group represents a halogen atom that will participate in a transition-metal-mediated coupling with a pre-formed heterocyclic reagent (for example a boronic acid or ester, or heteroaryl stannane) to give rise to amine 2. In another aspect, the "W" group of 43 and 44 represents a functional group that can be converted to a five-membered heterocycle by an annulation reaction. Non-limiting examples of such processes would include the conversion of a cyano, formyl, carboxy, acetyl, or alkynyl moiety into a pyrazole moiety. It will be understood by those skilled in the art that such annulations may in fact be reaction sequences and that the reaction arrows in Scheme 11 may represent either a single reaction or a reaction sequence. Additionally, the "W" group of 44 may represent a leaving group (halogen or triflate) that can be displaced by a nucleophilic nitrogen atom of a pyrazole ring.
[0128]Some non-limiting examples of general Scheme 12 are illustrated in the Schemes below. Scheme 13 illustrates the preparation of pyrazole 51, an example of general amine 2. In Scheme 13, commercially available 3-fluoro-4-aminophenol (47) is reacted with potassium tert-butoxide and 2,4-dichloropyridine 48 to provide chloropyridine 49. The preferred solvent for this transformation is dimethylacetamide at a temperature between 80 and 100° C. Subsequent union of chloropyridine 49 with the commercially available oxazole-4-boronic acid pinacol ester 50 in the presence of a palladium catalyst, preferably tetrakis(triphenylphosphine)palladium, provides oxazole amine 51.
[0129]Scheme 14 illustrates a non-limiting example of Scheme 12 wherein the "W" group is a leaving group for nucleophilic aromatic substitution. Thus, amine 53, an example of general amine 2, can be prepared from general intermediate 49 by reaction with 1,2,4-triazole (52). Preferred conditions include the use of polar aprotic solvents such as 1-methyl-2-pyrrolidinone, dimethylacetamide, or dimethylsulfoxide in the presence of non-nucleophilic bases such as potassium carbonate, sodium hydride, 1,8-diaza-bicyclo[5.4.0]undec-7-ene (DBU), and the like. Preferred temperatures are from ambient temperature up to about 250° C. and may optionally include the use of microwave irradiation or sonication. Those skilled in the art will recognize that the general methods of scheme 14 can be used to prepare additional triazole isomers by employing either 1,2,4-triazole 52, or alternatively, by employing 1,2,3-triazole in place of 52.
[0130]Scheme 15 illustrates the preparation of amine 55 and 56, non-limiting examples of general amine of formula 2, by way of an annulation sequence according to general Scheme 12. Conversion of chloropyridine 49 into alkyne 53 can be accomplished by Sonogashira cross-coupling with trimethylsilylacetylene, followed by aqueous hydrolysis of the trimethylsilyl group, conditions familiar to those skilled in the art. Further reaction of alkyne 53 with azidomethyl pivalate (54) in the presence of copper sulfate and sodium ascorbate provides the N-pivaloylymethyl triazole amine 55. (see Loren, et. al. Synlett, (2005), 2847). Deprotection under standard conditions, preferably dilute aqueous sodium hydroxide, provides 56. Alternatively, the amine 55 can be used directly to produce ureas of formula 1 prior to the removal of the N-pivaloylmethyl protecting group.
[0131]Additional examples of general amines of formula 2 can be prepared as illustrated in Scheme 16. Thus, by analogy to Scheme 12, the general intermediate 40 can be converted by palladium-mediated Stille-coupling into oxazoles 57 or 59 by reaction with the tributylstannanes 58 (see: Cheng et al., Biorg. Med. Chem. Lett., 2006, 2076) or 60 (Aldrich Chemical). Preferred palladium catalysts for the Stille reactions include dichlorobis(triplhenylphosphine)palladium, dichloro[11'-bis(diphenylphosphino)ferrocene]palladium and tetrakis(triphenylphosphine)palladium. Similarly, isoxazoles 61 and 63 can be obtained by the palladium-catalyzed reaction of 40 with 4-isoxazoleboronic acid pinacol ester 62 (Frontier Scientific) or tributylstannane 64 (see: Sakamoto, et al. Tetrahedron, 1991, 5111).
[0132]As an extension of Schemes 12, 13 and 16, amines of general formula 2 containing an isothiazole ring can also be prepared by the methods described above. Scheme 17 shows a non-limiting example wherein a palladium-catalyzed Stille reaction of trimethylstannane 65 (see: Wentland, et al. J. Med. Chem., 1993, 1580) with 40 can provide isothiazole 67. In a similar fashion, palladium-catalyzed Suzuki-cross coupling between 40 and the boronate ester 66 (see: Blackaby, et al., U.S. Pat. No. 7,030,128) gives rise to isothiazole amine 68.
[0133]Additional preferred synthetic methods for the preparation of compounds of formula 1 are found in the following examples.
SECTION 3. EXAMPLES
[0134]General Method A: To a stirring solution of the carboxylic acid (0.24 mmol) and TEA (1.2 mmol) in 1,4-dioxane (4.5 mL) at RT was added DPPA (0.29 mmol). After stirring for 0.5 h at RT, the appropriate amine (0.71 mmol) was added and the reaction was stirred with heating at 100° C. for 2 h. The reaction was cooled to RT, diluted with brine (15 mL) and extracted with EtOAc (3×30 mL). The combined organic layers were dried (MgSO4) and concentrated. The residue was purified by chromatography to afford the target compound.
[0135]General Method B: To a solution of the starting pyrazole amine (1 eq) in EtOAc were added 2,2,2-trichloroethylchloroformate (1.1 eq) and saturated NaHCO3 (2-3 eq) at 0° C. After stirring for 3 h at RT, the layers were separated and the aqueous layer extracted with EtOAc. The combined organic extracts were washed with brine, dried (Na2SO4) and concentrated under vacuum to yield the crude TROC carbamate of the pyrazole amine.
[0136]To the TROC carbamate (1 eq) in DMSO were added diisopropylethylamine (2 eq), the appropriate amine (2 eq) and the mixture was stirred at 60° C. for 16 h or until all the starting carbamate was consumed. Water was added to the mixture and the product was extracted with EtOAc (2×25 mL). The combined organic extracts were washed with brine solution, dried (Na2SO4) and concentrated to yield crude product, which was purified by column chromatography to yield the target compound.
Example A1
[0137]A suspension of 3-fluoro-4-aminophenol (8.0 g, 63.0 mmol) in dimethylacetamide (80 mL) was de-gassed in vacuo and treated with potassium tert-butoxide (7.3 g, 65 mmol). The resultant mixture was stirred at RT for 30 min. 2,4-Dichloropyridine (8 g, 54 mmol) was added and the mixture was heated to 80° C. for 12 h. The solvent was removed under reduced pressure to give a residue which was partitioned between water and EtOAc (3×100 mL). The organic layers were washed with saturated brine, dried (MgSO4), concentrated in vacuo and purified by silica gel column chromatography to give 4-(2-chloro-pyridin-4-yloxy)-2-fluoro-phenylamine (11 g, 86% yield). 1H NMR (300 MHz, DMSO-d6), δ 8.24 (d, J=5.7 Hz, 1 H), 7.00 (dd, J=9.0, 2.7 Hz, 1 H), 6.89-6.73 (m, 4 H), 5.21 (br s, 2 H); MS (ESI) m/z: 239.2 (M+H+).
[0138]To a degassed solution of 4-(2-chloropyridin-4-yloxy)-2-fluorobenzenamine (0.801 g, 3.36 mmol) in DMF (2 mL) and TEA (2 mL) was added ethynyltrimethylsilane (0.929 ml, 6.71 mmol), trans-dichloro-bis(triphenyl phosphine)palladium(0) (0.236 g, 0.336 mmol) and copper (I) iodide (0.064 g, 0.336 mmol) and the mixture was stirred at 90° C. for 16 h. Water (60 ml) was added to the mixture, product was extracted with EtOAc (2×45 ml) and the combined organics were washed with brine, dried (Na2SO4) concentrated to afford crude product. The product was dissolved in methanol (10 ml), K2CO3 (0.5 g) was added and the mixture was stirred at RT for 2 h. Solvent was removed, to the crude residue was added water (60 mL) and EtOAc (40 ml), the layers were separated and the aqueous layer was extracted with EtOAc (1×30 mL). The combined organic layer was washed with brine, dried (Na2SO4) and concentrated to afford crude product which was purified by column chromatography (ethylacetate/hexane) to afford 4-(2-ethynylpyridin-4-yloxy)-2-fluorobenzenamine as a thick residue (0.56 g, 73% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.37 (d, J=6.0 Hz, 1H), 6.98 (dd, J=8.0 Hz, 2.4 Hz, 1H), 6.95 (d, J=6.0 Hz, 1H), 6.87 (dd, J=6.0 Hz, 2.4 Hz, 1H), 6.81-6.73 (m, 2H), 5.20 (brs, 2H), 4.03 (s, 1H); MS (ESI) m/z: 229.1 (M+H.sup.+).
[0139]Sodium azide (1.942 g, 29.9 mmol) was added to a suspension of chloromethyl pivalate (3.00 g, 19.92 mmol) in water (5 mL) and stirred vigorously at 90° C. for 16 h. The reaction mixture was diluted with water (20 mL) and EtOAc (20 ml). The organic layer was washed with brine, dried (Na2SO4) and concentrated to afford azidomethyl pivalate as a liquid (2 g, 64% yield). 1H NMR (400 MHz, Acetone-d6): δ 5.23 (s, 2H), 1.22 (s, 9H).
[0140]To a suspension of azidomethyl pivalate (0.075 g, 0.477 mmol), 4-(2-ethynylpyridin-4-yloxy)-2-fluorobenzenamine (0.109 g, 0.477 mmol) in t-butanol (0.6 mL) and water (0.6 mL) was added sodium ascorbate (0.021 g, 0.095 mmol). Copper(II)sulfate in water (0.048 ml, 0.048 mmol) was added to the above suspension and the dark red mixture was stirred for 3 h at RT. It was diluted with water (30 mL) and EtOAc (20 mL), the layers were separated and the aqueous layer was extracted with EtOAc (2×15 mL). The combined organics were washed with brine, dried (Na2SO4) and concentrated to afford (4-(4-(4-amino-3-fluorophenoxy)pyridin-2-yl)-1H-1,2,3-triazol-1-yl)methyl pivalate as a red solid. (0.165 g, 90% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.54 (s, 1H), 8.46 (brs, 1H), 7.60 (s, 1H), 6.98 (d, J=8.8 Hz, 1H), 6.94 (d, J=3.6 Hz, 1H), 6.83-6.81 (m, 2H), 6.42 (s, 2H), 4.78 (s, 2H), 1.17 (s, 9H); MS (ESI) m/z: 386.1 (M+H.sup.+).
Example A2
[0141]To a solution of 4-(2-chloropyridin-4-yloxy)-2-fluorobenzenamine from Example A1 (1.0 g, 4.2 mmol) in NMP (10 ml) was added DBU (0.94 mL, 6.3 mmol) and 1,2,4-triazol sodium salt (0.57 g, 6.3 mmol) and the mixture was heated overnight under argon atmosphere at 160° C. The reaction mixture cooled to RT, diluted with water (100 mL) and the solution was extracted with EtOAc (3×). The organics were combined and washed with LiCl solution and brine (2×), dried (Na2SO4) and concentrated in vacuo. The residue was slurried in EtOAc (5 mL), the solid was filtered and washed with EtOAc to obtain a mixture of product and SM. The filtrate was concentrated in vacuo, the residue was slurried in CH2Cl2, filtered and washed with CH2Cl2 to obtain 4-(2-(1H-1,2,4-triazol-1-yl)pyridin-4-yloxy)-2-fluorobenzenamine (0.35 g). MS (ESI) m/z: 272.2 (M+H.sup.+).
Example A3
[0142]4-(2-Chloropyridin-4-yloxy)-2-fluorobenzenamine from Example A1 (150 mg, 0.629 mmol), 2-(tri-n-butylstannyl)oxazole (0.132 ml, 0.629 mmol) and PdCl2(dppf)-CH2Cl2 (51.3 mg, 0.063 mmol) were combined in DMF (3 ml) under Ar and stirred with heating at 90° C. After 3 h, the completed reaction was cooled to RT and treated with satd. aq. KF (5 ml; prepared from equal portions of KF.H2O and H2O) and stirred at RT for 1 h. The suspension was diluted with EtOAc and filtered through Celite®, rinsing forward with EtOAc. The filtrate was diluted with H2O and the layers were separated. The aqueous was extracted with EtOAc (2×). The combined organics were washed with brine (2×), dried (MgSO4), concentrated in vacuo and purified by flash column chromatography (EtOAc/hexanes) to afford 80 mg of 2-fluoro-4-(2-(oxazol-2-yl)pyridin-4-yloxy)benzenamine (0.295 mmol, 47% yield) as an oil that solidified on standing. 1H NMR (400 MHz, DMSO-d6): δ 8.52 (d, J=5.8 Hz, 1H), 8.26 (s, 1H), 7.40 (d, J=2.3 Hz, 1H), 7.38 (s, 1H), 7.05-7.02 (m, 2H), 6.88-6.78 (m, 2H), 5.23 (s, 2H); ); MS (ESI) m/z: 272.1 (M+H.sup.+).
Example A4
[0143]In a 3:1 mix of DMF:H20 (6 mL) was placed 4-(2-chloropyridin-4-yloxy)-2-fluorobenzenamine from example A1 (245 mg, 1.026 mmol), cesium carbonate (1.337 g, 4.10 mmol), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)oxazole (300 mg, 1.538 mmol) and tetrakistriphenylphosphine Pd(0) (178 mg, 0.154 mmol). The mix was degassed, placed under Ar, warmed to 80° C. and stirred overnight. The reaction was cooled to RT, diluted with water (20 mL) and extracted with ethyl acetate (3×20 mL). The combined organic phases were washed with brine, dried (Na2SO4) and concentrated in vacuo to give 2-fluoro-4-(2-(oxazol-5-yl)pyridin-4-yloxy)benzenamine (415 mg, 149% yield) as a dark oil. LC and LCMS shows mostly desired product as well as triphenylphosphine oxide as a by product. Used as is. MS (ESI) m/z: 272.1 (M+H.sup.+).
Example A5
[0144]In a microwave reaction vial, 4-(2-ethynylpyridin-4-yloxy)-2-fluorobenzenamine from Example A1 (0.201 g, 0.881 mmol) was dissolved in THF (4 mL). Acetaldoxime (0.078 g, 1.321 mmol), triethylamine (0.246 ml, 1.761 mmol), and 1-chloropyrrolidine-2,5-dione (0.176 g, 1.321 mmol) were added and the mixture was stirred at 130° C. for 45 min under microwave irradiation. An additional 1.5 eq each of acetaldoxime and 1-chloropyrrolidine-2,5-dione were added and microwave heating was heated for an additional 45 min at 130° C. This process repeated one more time. The mixture was poured into a biphasic solution of water (40 mL) and EtOAc (30 mL). The organic layer was separated and the aqueous layer was extracted with EtOAc (2×20 mL). The combined organics were washed with brine, dried (Na2SO4) and concentrated. The residue was purified by silica gel chromatography to afford 2-fluoro-4-(2-(3-methylisoxazol-5-yl)pyridin-4-yloxy)benzenamine (58 mg, 23% yield) as light red colored residue. MS (ESI) m/z: 286.1 (M+H.sup.+).
Example B1
[0145]To an aqueous solution of sodium hydroxide solution (40.00 g, 1 mol, in 200 ml of water) was added hydroxylamine hydrochloride (24.00 g, 346 mmol) and pivaloylacetonitrile (40.00 g, 320 mmol). The resulting solution was stirred at 50° C. for 3 hrs. The reaction mixture cooled and the resultant white crystalline solid filtered, washed with water and dried to provide 3-t-butylisoxazol-5-amine as a white crystalline solid (34 g, yield 76% yield). 1H NMR (DMSO-d6) δ 6.41 (brs, 2H), 4.85 (s, 1H), 1.18(s, 9H): LC-MS (ES, m/z, M+H) 141.3.
Example 1
[0146]Using General Method A, 3-tert-butyl-1-methyl-1H-pyrazole-5-carboxylic acid (0.054 g, 0.3 mmol), Example A1 (0.1 g, 0.25 mmol), triethylamine (0.76 g, 0.75 mmol) and DPPA (0.137 g, 0.5 mmol) were combined and purified by column chromatography (ethylacetate/hexanes) to afford (4-(4-(4-(3-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)ureido)-3-fluorophenox- y)pyridin-2-yl)-1H-1,2,3-triazol-1-yl)methyl pivalate as a white solid (0.115 g, 82% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.89 (s, 1H), 8.84 (s, 1H), 8.68 (s, 1H), 8.50 (d, J=5.6 Hz, 1H), 8.19 (t, J=8.8 Hz, 1H), 7.46 (d, J=2.4 Hz, 1H), 7.33 (dd, J=12.0 Hz, 2.8 Hz, 1H), 7.06 (dd, J=8.8 Hz, 1.6 Hz, 1H), 6.97 (dd, J=5.6 Hz, 2.4 Hz, 1H), 6.35 (s, 2H), 6.07 (s, 1H), 3.60 (s, 3H), 1.19 (s, 9H), 1.10 (s, 9H); MS (ESI) m/z: 565.2 (M+H.sup.+).
[0147]To a solution of (4-(4-(4-(3-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)ureido)-3-fluorophenox- y)pyridin-2-yl)-1H-1,2,3-triazol-1-yl)methyl pivalate (0.11 g, 0.195 mmol) in MeOH (1 mL) was added 2M NaOH (0.4 mL) and the mixture was stirred for 30 min at RT. Solvent was removed, crude residue was diluted with water (5 mL) and neutralized with 50% aqueous acetic acid (1 ml). The resultant solid was filtered and dried to afford 1-(4-(2-(1H-1,2,3-triazol-4-yl)pyridin-4-yloxy)-2-fluorophenyl)-3-(3-tert- -butyl-1-methyl-1H-pyrazol-5-yl)urea as a white solid (75 mg, 85% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.89 (s, 1H), 8.84 (s, 1H), 8.68 (s, 1H), 8.49 (d, J=6.0 Hz, 1H), 8.36 (brs, 1H), 8.19 (t, J=9.2 Hz, 1H), 7.42 (s, 1H), 7.33 (dd, J=11.6 Hz, 2.4 Hz, 1H), 7.06-7.04 (m, 1H), 6.94 (dd, J=5.6 Hz, 2.0 Hz, 1H), 6.07 (s, 1H), 3.60 (s, 3H), 1.19 (s, 9H); MS (ESI) m/z: 451.1 (M+H.sup.+).
Example 2
[0148]To a solution of 2,2,2-trichloroethyl 3-tert-butylisoxazol-5-ylcarbamate (0.080 g, 0.25 mmol), formed via General method B from Example B1, in dioxane (3 mL) was added Example A2 (70 mg, 0.25 mmol) and 1-methylpyrrolidine (22 mg, 0.25 mmol). The reaction mixture was heated overnight at 65° C. The reaction mixture cooled to RT, concentrated in vacuo, DCM (2 mL) was added and the slurry was stirred for 1 hour. The solid was filtered and air dried to obtain 1-(4-(2-(1H-1,2,4-triazol-1-yl)pyridin-4-yloxy)-2-fluorophenyl)-3-- (3-tert-butylisoxazol-5-yl)urea. 1H NMR (400 MHz, DMSO-d6): δ 10.3 (s, 1H), 9.56 (s, 1H), 8.70 (s, 1H), 8.34 (s, 1H), 8.29 (d, J=6.0 Hz, 1H), 8.03 (t, J=9.2 Hz, 1H), 7.68 (dd, J=2.0, and 5.6 Hz, 1H), 7.57 (d, J=1.2 Hz, 1H), 7.26 (dd, J=2.8, and 12.0 Hz, 1H), 7.03 (m, 1H), 6.05 (s, 1H), 1.24 (s, 9H); MS (ESI) m/z: 438.1 (M+H.sup.+).
Example 3
[0149]To a solution of 3-(t-butyl)-1-methyl-1H-pyrazole-5-carboxylic acid (0.054 g, 0.295 mmol) in dioxane (3 ml) was added TEA (0.123 ml, 0.885 mmol) followed by DPPA (0.095 ml, 0.442 mmol). The mixture was stirred at RT for 30 min and then treated with a solution of Example A3 (0.080 g, 0.295 mmol) in dioxane (3.00 ml). The reaction was then placed in an oil bath preheated to 100° C. and stirred with heating overnight. The completed reaction was cooled to RT. Without aqueous workup, the reaction mixture was purified directly by reverse phase chromatography (MeCN (w/0.1% TFA)/H2O (w/0.1% TFA)) to afford 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(oxazol-2-yl)p- yridin-4-yloxy)phenyl)urea of 96.8% purity. MS (ESI) m/z: 451.1 (M+H.sup.+).
Example 4
[0150]Using General Method A, 3-tert-butyl-1-methyl-1H-pyrazole-5-carboxylic acid (205 mg, 1.127 mmol), triethylamine (415 mg, 4.10 mmol), Example A4 [max theoretical yield from previous reaction] (278 mg, 1.025 mmol) and DPPA (338 mg, 1.23 mmol) were combined and purified by reverse phase chromatography (acetonitrile/water/-0.1% TFA) to give a residue which was treated with 10% potassium carbonate (10 mL) and extracted with ethyl acetate (3×25 mL). The combined organic phases were washed with brine (25 mL), dried (Na2SO4) and concentrated in vacuo to give 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(oxazol-5-yl)p- yridin-4-yloxy)phenyl)urea (131 mg, 28% yield) as a film. 1H NMR (400 MHz, DMSO-d6): δ 1.15 (s, 9 H), 3.60 (s, 3 H), 6.07 (s, 1 H), 6.91-6.93 (m, 1 H), 7.04-7.07 (m, 1 H), 7.23 (d, 1 H), 7.30-7.34 (m, 1 H), 7.78 (s, 1 H), 8.18 (t, 1 H), 8.48-8.51 (m, 2 H), 8.85 (d, 1 H), 8.90 (s, 1 H); MS (ESI) m/z: 451.1 (M+H.sup.+).
Example 5
[0151]Using general method A, 3-tert-butyl-1-methyl-1H-pyrazole-5-carboxylic acid (0.042 g, 0.23 mmol), Example A5 (0.51 g, 0.18 mmol), triethylamine (0.54 g, 0.53 mmol) and DPPA (0.1 g, 0.35 mmol) were combined and purified by column chromatography using (EtOAc/hexanes) to afford 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(3-methylisoxa- zol-5-yl)pyridin-4-yloxy)phenyl)urea as white solid (0.015 g, 18% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.89 (s, 1H), 8.85 (s, 1H), 8.56 (d, J=6.0 Hz, 1H), 8.20 (t, J=8.8 Hz, 1H), 7.38 (d, J=2.4 Hz, 1H), 7.34 (dd, J=12.0 Hz, 2.4 Hz, 1H), 7.08-7.05 (m, 1H), 7.03-7.00 (m, 1H), 6.97 (s, 1H), 6.07 (s, 1H), 3.60 (s, 3H), 2.82 (s, 3H), 1.19 (s, 9H); MS (ESI) m/z: 465.1 (M+H.sup.+).
[0152]Using the synthetic procedures and methods described herein and methods known to those skilled in the art, the following compounds are made: 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(4-methy- loxazol-2-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(3-methyl-1,2,- 4-thiadiazol-5-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(isoxazol-5-yl- )pyridin-4-yloxy)phenyl)urea, 1-(4-(2-(4H-1,2,4-triazol-4-yl)pyridin-4-yloxy)-2-fluorophenyl)-3-(3-tert- -butyl-1-methyl-1H-pyrazol-5-yl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(isothiazol-4-- yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(3-methyl-1H-1- ,2,4-triazol-1-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl-3-(2-fluoro-4-(2-(isothiazol-3-y- l)pyridin-4-yloxy)phenyl)urea 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(isothiazol-5-- yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(4-methyl-2H-1- ,2,3-triazol-2-yl)pyridin-4-yloxy)phenyl)urea, 1-(5-tert-butylisoxazol-3-yl)-3-(2-fluoro-4-(2-(4-methyl-4H-1,2,4-triazol- -3-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(2-methyl-2H-1- ,2,3-triazol-4-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(5-methyl-1,2,- 4-thiadiazol-3-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(4-methyl-4H-1- ,2,4-triazol-3-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(3-methyl-3H-1- ,2,3-triazol-4-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(isoxazol-4-yl- )pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(isoxazol-3-yl- )pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butylisoxazol-5-yl)-3-(2-fluoro-4-(2-(2-methyl-2H-1,2,3-triazol- -4-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butylisoxazol-5-yl)-3-(2-fluoro-4-(2-(5-methyl-1,2,4-thiadiazol- -3-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butylisoxazol-5-yl)-3-(2-fluoro-4-(2-(4-methyl-4H-1,2,4-triazol- -3-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butylisoxazol-5-yl)-3-(2-fluoro-4-(2-(3-methyl-3H-1,2,3-triazol- -4-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butylisoxazol-5-yl)-3-(2-fluoro-4-(2-(isoxazol-4-yl)pyridin-4-y- loxy)phenyl)urea, 1-(3-tert-butylisoxazol-5-yl)-3-(2-fluoro-4-(2-(isoxazol-3-yl)pyridin-4-y- loxy)phenyl)urea, 1-(1-tert-butyl-1H-pyrazol-4-yl)-3-(2-fluoro-4-(2-(isoxazol-4-yl)pyridin-- 4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(4-methyloxazo- l-2-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(3-methyl-1,2,- 4-thiadiazol-5-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(isoxazol-5-yl- )pyridin-4-yloxy)phenyl)urea, 1-(4-(2-(4H-1,2,4-triazol-4-yl)pyridin-4-yloxy)-2-fluorophenyl)-3-(3-tert- -butyl-1-methyl-1H-pyrazol-5-yl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(isothiazol-4-- yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(3-methyl-1H-1- ,2,4-triazol-1-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(isothiazol-3-- yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(isothiazol-5-- yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(4-methyl-2H-1- ,2,3-triazol-2-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(2-methyl-2H-1- ,2,3-triazol-4-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(5-methyl-1,2,- 4-thiadiazol-3-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(4-methyl-4H-1- ,2,4-triazol-3-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(3-methyl-3H-1- ,2,3-triazol-4-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(isoxazol-4-yl- )pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(isoxazol-3-yl- )pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butylisoxazol-5-yl)-3-(2-fluoro-4-(2-(2-methyl-2H-1,2,3-triazol- -4-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butylisoxazol-5-yl)-3-(2-fluoro-4-(2-(5-methyl-1,2,4-thiadiazol- -3-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butylisoxazol-5-yl)-3-(2-fluoro-4-(2-(4-methyl-4H-1,2,4-triazol- -3-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butylisoxazol-5-yl)-3-(2-fluoro-4-(2-(3-methyl-3H-1,2,3-triazol- -4-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butylisoxazol-5-yl)-3-(2-fluoro-4-(2-(isoxazol-4-yl)pyridin-4-y- loxy)phenyl)urea, 1-(3-tert-butylisoxazol-5-yl)-3-(2-fluoro-4-(2-(isoxazol-3-yl)pyridin-4-y- loxy)phenyl)urea, 1-(1-tert-butyl-1H-pyrazol-4-yl)-3-(2-fluoro-4-(2-(isoxazol-4-yl(pyridin-- 4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(1-methyl-1H-1- ,2,3-triazol-4-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(1-methyl-1H-1- ,2,4-triazol-3-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butylisoxazol-5-yl)-3-(2-fluoro-4-(2-(1-methyl-1H-1,2,3-triazol- -4-yl)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butylisoxazol-5-yl)-3-(2-fluoro-4-(2-(1-methyl-1H-1,2,4-triazol- -3-yl)pyridin-4-yloxy)phenyl)urea, 1-(1-tert-butyl-1H-pyrazol-4-yl)-3-(2-fluoro-4-(2-(1-methyl-1H-1,2,3-tria- zol-4-yl)pyridin-4-yloxy)phenyl)urea, 1-(1-tert-butyl-1H-pyrazol-4-yl)-3-(2-fluoro-4-(2-(1-methyl-1H-1,2,4-tria- zol-3-yl)pyridin-4-yloxy)phenyl)urea, 1-(2-fluoro-4-(2-(1-methyl-1H-1,2,3-triazol-4-yl)pyridin-4-yloxy)phenyl)-- 3-(5-(trifluoromethyl)pyridin-3-yl)urea, 1-(2-fluoro-4-(2-(1-methyl-1H-1,2,3-triazol-4-yl)pyridin-4-yloxy)phenyl)-- 3-(5-isopropylpyridin-3-yl)urea, 1-(2-fluoro-4-(2-(1-methyl-1H-1,2,4-triazol-3-yl)pyridin-4-yloxy)phenyl)-- 3-(5-(trifluoromethyl)pyridin-3-yl)urea, 1-(2-fluoro-4-(2-(1-methyl-1H-1,2,4-triazol-3-yl)pyridin-4-yloxy)phenyl)-- 3-(5-isopropylpyridin-3-yl)urea, 1-(2-fluoro-4-(2-(3-methyl-1,2,4-thiadiazol-5-yl)pyridin-4-yloxy)phenyl)-- 3-(5-(trifluoromethyl)pyridin-3-yl)urea, 1-(2-fluoro-4-(2-(3-methyl-1 2,4-thiadiazol-5-yl)pyridin-4-yloxy)phenyl)-3-(5-isopropylpyridin-3-yl)ur- ea, 1-(2-fluoro-4-(2-(3-methyl-1H-1,2,4-triazol-1-yl)pyridin-4-yloxy)pheny- l)-3-(5-(trifluoromethyl)pyridin-3-yl)urea, 1-(2-fluoro-4-(2-(3-methyl-1H-1,2,4-triazol-1-yl)pyridin-4-yloxy)phenyl)-- 3-(5-isopropylpyridin-3-yl)urea, 1-(4-(2-(4H-1,2,4-triazol-4-yl)pyridin-4-yloxy)-2-fluorophenyl)-3-(5-isop- ropylpyridin-3-yl)urea, 1-(4-(2-(4H-1,2,4-triazol-4-yl)pyridin-4-yloxy)-2-fluorophenyl)-3-(5-(tri- fluoromethyl)pyridin-3-yl)urea, 1-(2-fluoro-4-(2-(3-methyl-1H-1,2,4-triazol-1-yl)pyridin-4-yloxy)phenyl)-- 3-(5-isopropylpyridin-3-yl)urea, 1-(2-fluoro-4-(2-(2-methyl-2H-1,2,3-triazol-4-yl)pyridin-4-yloxy)phenyl)-- 3-(5-(trifluoromethyl)pyridin-3-yl)urea, 1-(2-fluoro-4-(2-(2-methyl-2H-1,2,3-triazol-4-yl)pyridin-4-yloxy)phenyl)-- 3-(5-isopropylpyridin-3-yl)urea, 1-(2-fluoro-4-(2-(5-methyl-1,2,4-thiadiazol-3-yl)pyridin-4-yloxy)phenyl)-- 3-(5-(trifluoromethyl)pyridin-3-yl)urea, 1-(2-fluoro-4-(2-(5-methyl-1,2,4-thiadiazol-3-yl)pyridin-4-yloxy)phenyl)-- 3-(5-isopropylpyridin-3-yl)urea, 1-(2-fluoro-4-(2-(2-methyl-2H-1,2,3-triazol-4-yl)pyridin-4-yloxy)phenyl)-- 3-(5-(trifluoromethyl)pyridin-3-yl)urea, 1-(2-fluoro-4-(2-(isoxazol-4-yl)pyridin-4-yloxy)phenyl)-3-(5-isopropylpyr- idin-3-yl)urea, 1-(2-fluoro-4-(2-(isoxazol-4-yl)pyridin-4-yloxy)phenyl)-3-(5-(trifluorome- thyl)pyridin-3-yl)urea, 1-(2-fluoro-4-(2-(2-methyl-2H-1,2,3-triazol-4-yl)pyridin-4-yloxy)phenyl)-- 3-(5-isopropylpyridin-3-yl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-5-(2-(1-methyl-1H-p- yrazol-4-yl)pyridin-4-yloxy)phenyl)urea, 1-(5-(2-(1H-pyrazol-4-yl)pyridin-4-yloxy)-2-fluorophenyl)-3-(3-tert-butyl- -1-methyl-1H-pyrazol-5-yl)urea, 1-(5-(2-(1H-pyrazol-3-yl)pyridin-4-yloxy)-2-fluorophenyl)-3-(3-tert-butyl- -1-methyl-1H-pyrazol-5-yl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-5-(2-(oxazol-4-yl)p- yridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-5-(2-(oxazol-2-yl)p- yridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-5-(2-(oxazol-5-yl)p- yridin-4-yloxy)phenyl)urea, 1-(1-tert-butyl-1H-pyrazol-4-yl)-3-(2-fluoro-5-(2-(3-methylisoxazol-5-yl)- pyridin-4-yloxy)phenyl)urea, 1-(1-tert-butyl-1H-pyrazol-4-yl)-3-(2-fluoro-5-(2-(isoxazol-4-yl)pyridin-- 4-yloxy)phenyl)urea, 1-(1-tert-butyl-1H-pyrazol-4-yl)-3-(2-fluoro-5-(2-(isothiazol-4-yl)pyridi- n-4-yloxy)phenyl)urea, 1-(1-tert-butyl-1H-pyrazol-4-yl)-3-(2-fluoro-5-(2-(3-methylisothiazol-5-y- l)pyridin-4-yloxy)phenyl)urea, 1-(5-(2-(1H-1,2,4-triazol-1-yl)pyridin-4-yloxy)-2-fluorophenyl)-3-(1-tert- -butyl-1H-pyrazol-4-yl)urea, 1-(5-(2-(4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-2-fluorophenyl)-3-(1-tert- -butyl-1H-pyrazol-4-yl)urea, 1-(5-(2-(1H-1,2,3-triazol-1-yl)pyridin-4-yloxy)-2-fluorophenyl)-3-(1-tert- -butyl-1H-pyrazol-4-yl)urea, 1-(5-(2-(1,3,4-thiadiazol-2-yl)pyridin-4-yloxy)-2-fluorophenyl)-3-(1-tert- -butyl-1H-pyrazol-4-yl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(oxazol-4-yl)p- yridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(oxazol-5-yl)p- yridin-4-yloxy)phenyl)urea, 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(3-methylisoxa- zol-5-yl)pyridin-4-yloxy)phenyl)urea, 1-(1-tert-butyl-1H-pyrazol-4-yl)-3-(2-fluoro-4-(2-(3-methylisothiazol-5-y- l)pyridin-4-yloxy)phenyl)urea, 1-(3-tert-butylisoxazol-5-yl)-3-(2-fluoro-4-(2-(1-methyl-1H-imidazol-4-yl- )pyridin-4-yloxy)phenyl)urea, 1-(4-(2-(1H-1,2,4-triazol-1-yl)pyridin-4-yloxy)-2-fluorophenyl)-3-(3-tert- -butyl-1-methyl-1H-pyrazol-5-yl)urea, 1-(4-(2-(4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-2-fluorophenyl)-3-(1-tert- -butyl-1H-pyrazol-4-yl)urea, 1-(4-(2-(1H-1,2,3-triazol-1-yl)pyridin-4-yloxy)-2-fluorophenyl)-3-(1-tert- -butyl-1H-pyrazol-4-yl)urea, 1-(4-(2-(1,3,4-thiadiazol-2-yl)pyridin-4-yloxy)-2-fluorophenyl)-3-(1-tert- -butyl-1H-pyrazol-4-yl)urea, 1-(4-(2-(1H-1,2,4-triazol-1-yl)pyridin-4-yloxy)-2-fluorophenyl)-3-(3-tert- -butylisoxazol-5-yl)urea, and 1-(3-tert-butyl-1-methyl-1H-pyrazol-5-yl)-3-(2-fluoro-4-(2-(oxazol-2-yl)p- yridin-4-yloxy)phenyl)urea.
SECTION 4. BIOLOGICAL DATA
[0153]Abl Kinase (SEQ ID NO:1) Assay
[0154]Activity of Abl kinase (SEQ ID NO:1) was determined by following the production of ADP from the kinase reaction through coupling with the pyruvate kinase/lactate dehydrogenase system (e.g., Schindler, et al. Science (2000) 289, 1938-1942). In this assay, the oxidation of NADH (thus the decrease at A340nm was continuously monitored spectrophometrically. The reaction mixture (100 μl) contained Abl kinase (1 nM. Abl from deCode Genetics), peptide substrate (EAIYAAPFAKKK, 0.2 mM), MgCl2 (10 mM), pyruvate kinase (4 units), lactate dehydrogenase (0.7 units), phosphoenol pyruvate (1 mM), and NADH (0.298 mM) in 90 mM Tris buffer containing 0.2% octyl-glucoside and 3.5% DMSO, pH 7.5. Test compounds were incubated with Abl (SEQ ID NO:1) and other reaction reagents at 30° C. for 2 h before ATP (500 μM) was added to start the reaction. The absorption at 340 nm was monitored continuously for 2 hours at 30° C. on Polarstar Optima plate reader (BMG). The reaction rate was calculated using the 1.0 to 2.0 h time frame. Percent inhibition was obtained by comparison of reaction rate with that of a control (i.e. with no test compound). IC50 values were calculated from a series of percent inhibition values determined at a range of inhibitor concentrations using software routines as implemented in the GraphPad Prism software package.
TABLE-US-00001 Abl kinase (SEQ ID NO: 1) GTSMDPSSPNYDKWEMERTDITMKHKLGGGQYGEVYEGVWKKYSLTVAVK TLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVCTREPPFYIITEFMTYG NLLDYLRECNRQEVNAVVLLYMATQISSAMEYLEKKNFIHRDLAARNCLV GENHLVKVADFGLSRLMTGDTYTAHAGAKFPIKWTAPESLAYNKFSIKSD VWAFGVLLWEIATYGMSPYPGIDLSQVYELLEKDYRMERPEGCPEKVYEL MRACWQWNPSDRPSFAEIHQAFETMFQE
[0155]Abl Kinase (SEQ ID NO:2) Assay
[0156]Activity of T3 15I Abl kinase (SEQ ID NO:2) was determined by following the production of ADP from the kinase reaction through coupling with the pyruvate kinase/lactate dehydrogenase system (e.g., Schindler, et al. Science (2000) 289, 1938-1942). In this assay, the oxidation of NADH (thus the decrease at A340nm) was continuously monitored spectrophometrically. The reaction mixture (100 μl) contained Abl kinase (4.4 nM. M315I Abl from deCode Genetics), peptide substrate (EAIYAAPFAKKK, 0.2 mM), MgCl2 (10 mM), pyruvate kinase (4 units), lactate dehydrogenase (0.7 units), phosphoenol pyruvate (1 mM), and NADH (0.28 mM) in 90 mM Tris buffer containing 0.2% octyl-glucoside and 1% DMSO, pH 7.5. Test compounds were incubated with T315I Abl (SEQ ID NO:2) and other reaction reagents at 30° C. for 1 h before ATP (500 μM) was added to start the reaction. The absorption at 340 nm was monitored continuously for 2 hours at 30° C. on Polarstar Optima plate reader (BMG). The reaction rate was calculated using the 1.0 to 2.0 h time frame. Percent inhibition was obtained by comparison of reaction rate with that of a control (i.e. with no test compound). IC50 values were calculated from a series of percent inhibition values determined at a range of inhibitor concentrations using software routines as implemented in the GraphPad Prism software package.
TABLE-US-00002 Abl T3151 kinase (SEQ ID NO: 2) GTSMDPSSPNYDKWEMERTDITMKHKLGGGQYGEVYEGVWKKYSLTVAVK TLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVCTREPPFYIIIEFMTYG NLLDYLRECNRQEVNAVVLLYMATQISSAMEYLEKKNFIHRDLAARNCLV GENHLVKVADFGLSRLMTGDTYTAHAGAKFPIKWTAPESLAYNKFSIKSD VWAFGVLLWEIATYGMSPYFGIDLSQVYELLEKDYRMERPEGCPEKVYEL MRACWQWNPSDRPSFAEIHQAFETMFQE BCR-Abl p210-e14a2 (SEQ ID NO: 3) MVDPVGFAEAWKAQFPDSEPPRMELRSVGDIEQELERCKASIRRLEQEVN QERFRMIYLQTLLAKEKKSYDRQRWGFRRAAQAFDGASEPRASASRPQPA PADGADPPPAEEPEARPDGEGSPGKARPGTARRPGAAASGERDDRGPPAS VAALRSNFERIRKGHGQPGADAEKPFYVNVEFHHERGLVKVNDKEVSDRI SSLGSQAMQMERKKSQHGAGSSVGDASRPPYRGRSSESSCGVDGDYEDAE LNPRFLKDNLIDANGGSRPPWPPLEYQPYQSIYVGGIMEGEGKGPLLRSQ STSEQEKRLTWPRRSYSPRSFEDCGGGYTPDCSSNENLTSSEEDFSSGQS SRVSPSPTTYRMFRDKSRSPSQNSQQSFDSSSPPTPQCHKRHRHCPVVVS EATIVGVRKTGQIWPNDDEGAFHGDADGSFGTPPGYGCAADRAEEQRRHQ DGLPYIDDSPSSSPHLSSKGRGSRDALVSGALKSTKASELDLEKGLEMRK WVLSGILASEETYLSHLEALLLPMKPLKAAATTSQPVLTSQQIETIFFKV PELYEIHKESYDGLFPRVQQWSHQQRVGDLFQKLASQLGVYRAFVDNYGV AMEMAEKCCQANAQFAEISENLRARSNKDAKDPTTKNSLETLLYKPVDRV TRSTLVLHDLLKHTPASHPDHPLLQDALRISQNFLSSINEEITPRRQSMT VKKGEHRQLLKDSFMVELVEGARKLRHVFLFTDLLLCTKLKKQSGGKTQQ YDCKWYIPLTDLSFQMVDELEAVPNIPLVPDEELDALKIKISQIKSDIQR EKRANKGSKATERLKKKLSEQESLLLLMSPSMAFRVHSRNGKSYTFLISS DYERAEWRENIREQQKKCFRSFSLTSVELQMLTNSCVKLQTVHSIPLTIN KEDDESPGLYGFLNVIVHSATGFKQSSKALQRPVASDFEPQGLSEAARWN SKENLLAGPSENDPNLFVALYDFVASGDNTLSITKGEKLRVLGYNHNGEW CEAQTKNGQGWVPSNYITPVNSLEKHSWYHGPVSRNAAEYPLSSGINGSF LVRESESSPSQRSISLRYEGRVYHYRINTASDGKLYVSSESRFNTLAELV HHHSTVADGLITTLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLG GGQYGEVYEGVWKKYSLTVAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLV QLLGVCTREPPFYIITEFMTYGNLLDYLRECNRQEVNAVVLLYMATQISS AMEYLEKKNFIHRDLAARNCLVGENHLVKVADFGLSRLMTGDTYTAHAGA KFPIKWTAPESLAYNKFSIKSDVWAFGVLLWEIATYGMSPYPGIDRSQVY ELLEKDYRMKRPEGCPEKVYELMRACWQWNPSDRPSFAEIHQAFETMFQE SSISDEVEKELGKQGVRGAVTTLLQAPELPTKTRTSRRAAEHRDTTDVPE MPHSKGQGESDPLDHEPAVSPLLPRKERGPPEGGLNEDERLLPKDKKTNL FSALIKKKKKTAPTPPKRSSSFREMDGQPERRGAGEEEGRDISNGALAFT PLDTADPAKSPKPSNGAGVPNGALRESGGSGFRSPHLWKKSSTLTSSRLA TGEEEGGGSSSKRFLRSCSVSCVPHGAKDTEWRSVTLPRDLQSTGRQFDS STFGGHKSEKPALPRKRAGENRSDQVTRGTVTPPPRLVKKNEEAADEVFK DIMESSPGSSPPNLTPKPLRRQVTVAPASGLPHKEEAWKGSALGTPAAAE PVTPTSKAGSGAPRGTSKGPAEESRVRRHKHSSESPGRDKGKLSKLKPAP PPPPAASAGKAGGKPSQRPGQEAAGEAVLGAKTKATSLVDAVNSDAAKPS QPAEGLKKPVLPATFKPHPAKPSGTPISPAPVPLSTLPSASSALAGDQPS STAFIPLISTRVSLRKTRQPPERASGAITKGVVLDSTEALCLAISGNSEQ MASHSAVLEAGKNLYTFCVSYVDSIQQMRNKFAFREAINKLENNLRELQI CPASAGSGPAATQDFSKLLSSVKEISDIVQR BCR-Abl p210-e13a2 (SEQ ID NO: 4) MVDPVGFAEAWKAQFPDSEPPRMELRSVGDIEQELERCKASIRRLEQEVN QERFRMIYLQTLLAKEKKSYDRQRWGFRRAAQAPDGASEPRASASRPQPA PADGADPPPAEEPEARPDGEGSPGKARPGTARRPGAAASGERDDRGPPAS VAALRSNFERIRKGHGQPGADAEKPFYVNVEFHHERGLVKVNDKEVSDRI SSLGSQAMQMERKKSQHGAGSSVGDASRPPYRGRSSESSCGVDGDYEDAE LNPRFLKDNLIDANGGSRPPWPPLEYQPYQSIYVGGIMEGEGKGPLLRSQ STSEQEKRLTWPRRSYSPRSFEDCGGGYTPDCSSNENLTSSEEDFSSGQS SRVSPSPTTYRMFRDKSRSPSQNSQQSFDSSSPPTPQCHKRHRHCPVVVS EATIVGVRKTGQIWPNDDEGAFHGDADGSFGTPPGYGCAADRAEEQRPHQ DGLPYIDDSPSSSPHLSSKGRGSRDALVSGALKSTKASELDLEKGLEMRK WVLSGILASEETYLSHLEALLLPMKPLKAAATTSQPVLTSQQIETIFFKV PELYEIHKESYDGLFPRVQQWSHQQRVGDLFQKLASQLGVYRAFVDNYGV AMEMAEKCCQANAQFAEISENLRARSNKDAKDPTTKNSLETLLYKPVDRV TRSTLVLHDLLKHTPASHPDHPLLQDALRISQNFLSSINEEITPRRQSMT VKKGEHRQLLKDSFMVELVEGARKLRHVFLFTDLLLCTKLKKQSGGKTQQ YDCKWYIPLTDLSFQMVDELEAVPNIPLVPDEELDALKIKISQIKSDIQR EKRANKGSKATERLKKKLSEQESLLLLMSPSMAFRVHSRNGKSYTFLISS DYERAEWRENIREQQKKCFRSFSLTSVELQMLTNSCVKLQTVHSIPETIN KEEALQRPVASDFEPQGLSEAARWNSKENLLAGFSENDPNLFVALYDFVA SGDNTLSITKGEKLRVLGYNHNGEWCEAQTKNGQGWVPSNYITPVNSLEK HSWYHGPVSRNAAEYPLSSGINGSFLVRESESSPSQRSISLRYEGRVYHY RINTASDGKLYVSSESRFNTLAELVHHHSTVADGLITTLHYPAPKRNKPT VYGVSPNYDKWEMERTDITMKHKLGGGQYGEVYEGVWKKYSLTVAVKTLK EDTMEVEEFLKEAAVMKEIKHPNLVQLLGVCTREPPFYIITEFMTYGNLL DYLRECNRQEVNAVVLLYMATQISSAMEYLEKKNFIHRDLAARNCLVGEN HLVKVADFGLSRLMTGDTYTAHAGAKFPIKWTAPESLAYNKFSIKSDVWA FGVLLWEIATYGMSPYPGIDRSQVYELLEKDYRMKRPEGCPEKVYELMRA CWQWNPSDRPSFAEIHQAFETMFQESSISDEVEKELGKQGVRGAVTTLLQ APELPTKTRTSRRAAEHRDTTDVPEMPHSKGQGESDPLDHEPAVSPLLPR KERGPPEGGLNEDERLLPKDKKTNLFSALIKKKKKTAPTPPKRSSSFREM DGQPERRGAGEEEGRDISNGALAFTPLDTADPAKSPKPSNGAGVPNGALR ESGGSGFRSPHLWKKSSTLTSSRLATGEEEGGGSSSKRFLRSCSVSCVPH GAKDTEWRSVTLPRDLQSTGRQFDSSTFGGHKSEKPALPRKRAGENRSDQ VTRGTVTPPPRLVKKNEEAADEVFKDIMESSPGSSPPNLTPKPLRRQVTV APASGLPHKEEAWKGSALGTPAAAEPVTPTSKAGSGAPRGTSKGPAEESR VRRHKHSSESPGRDKGKLSKLKPAPPPPPAASAGKAGGKPSQRPGQEAAG EAVLGAKTKATSLVDAVNSDAAKPSQPAEGLKKPVLPATPKPHPAKPSGT PISPAPVPLSTLPSASSALAGDQPSSTAFIPLISTRVSLRKTRQPPERAS GAITKGVVLDSTEALCLAISGNSEQMASHSAVLEAGKNLYTFCVSYVDSI QQMRNKFAFREAINKLENNLRELQICPASAGSGPAATQDFSKLLSSVKEI SDIVQR BCR-Abl p190-e1a2 (SEQ ID NO: 5) MVDPVGFAEAWKAQFPDSEPPRMELRSVGDIEQELERCKASIRRLEQEVN QERFRMIYLQTLLAKEKKSYDRQRWGFRRAAQAPDGASEPRASASRPQPA PADGADPPPAEEPEARPDGEGSPGKARPGTARRPGAAASGERDDRGPPAS VAALRSNFERIRKGHGQPGADAEKPFYVNVEFHHERGLVKVNDKEVSDRI SSLGSQAMQMERKKSQHGAGSSVGDASRPPYRGRSSESSCGVDGDYEDAE LNPRFLKDNLIDANGGSRPPWPPLEYQPYQSIYVGGIMEGEGKGPLLRSQ STSEQEKRLTWPRRSYSPRSFEDCGGGYTPDCSSNENLTSSEEDFSSGQS SRVSPSPTTYRMFRDKSRSPSQNSQQSFDSSSPPTPQCHKRHRHCPVVVS EATIVGVRKTGQIWPNDDEGAFHGDAEALQRPVASDFEPQGLSEAARWNS KENLLAGPSENDPNLFVALYDFVASGDNTLSITKGEKLRVLGYNHNGEWC EAQTKNGQGWVPSNYITPVNSLEKHSWYHGPVSRNAAEYPLSSGINGSFL VRESESSPSQRSISLRYEGRVYHYRINTASDGKLYVSSESRFNTLAELVH HHSTVADGLITTLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLGG GQYGEVYEGVWKKYSLTVAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQ LLGVCTREPPFYIITEFMTYGNLLDYLRECNRQEVNAVVLLYMATQISSA MEYLEKKNFIHRDLAARNCLVGENHLVKVADFGLSRLMTGDTYTAHAGAK FPIKWTAPESLAYNKFSIKSDVWAFGVLLWEIATYGMSPYPGIDRSQVYE LLEKDYRMKRPEGCPEKVYELMRACWQWNPSDRPSFAEIHQAFETMFQES SISDEVEKELGKQGVRCAVTTLLQAPELPTKTRTSRRAAEHRDTTDVPEM PHSKGQGESDPLDHEPAVSPLLPRKERGPPEGGLNEDERLLPKDKKTNLF SALIKKKKKTAPTPPKRSSSFREMDGQPERRGAGEEEGRDISNGALAFTP LDTADPAKSPKPSNGAGVPNGALRESGGSGFRSPHLWKKSSTLTSSRLAT GEEEGGGSSSKRFLRSCSVSCVPHGAKDTEWRSVTLPRDLQSTGRQFDSS TFGGHKSEKPALFRKRAGENRSDQVTRGTVTPPPRLVKKNEEAADEVFKD IMESSPGSSPPNLTPKPLRRQVTVAPASGLPHKREAWKGSALGTPAAAEP VTPTSKAGSGAPRGTSKGPAEESRVRRHKHSSESPGRDRGKLSKLKPAPP PPPAASAGKAGGKPSQRPGQEAAGEAVLGAKTKATSLVDAVNSDAAKPSQ PAEGLKKPVLPATPKPHPAKPSGTPISPAPVPLSTLPSASSALAGDQPSS TAFIPLISTRVSLRKTRQPPERASGAITKGVVLDSTEALCLAISGNSEQM ASHSAVLEAGKNLYTFCVSYVDSIQQMRNKFAFREAINKLENNLRELQIC PASAGSGPAATQDFSKLLSSVKEISDIVQR BCR-Abl p210-e14a2 T315I (SEQ ID NO: 6) MVDPVGFAEAWKAQFPDSEPPRMELRSVGDIEQELERCKASIRRLEQEVN QERFRMIYLQTLLAKEKKSYDRQRWGFRRAAQAPDGASEPRASASRPQPA PADGADPPPAEEPEARPDGEGSPGKARPGTARRPGAAASGERDDRGPPAS VAALRSNFERIRKGHGQPGADAEKPFYVNVEFHHERGLVKVNDKEVSDRI
SSLGSQAMQMERKKSQHGAGSSVGDASRPPYRGRSSESSCGVDGDYEDAE LNPRFLKDNLIDANGGSRPPWPPLEYQPYQSIYVGGIMEGEGKGPLLRSQ STSEQEKRLTWPRRSYSPRSFEDCGGGYTPDCSSNENLTSSEEDFSSGQS SRVSPSPTTYRMFRDKSRSPSQNSQQSFDSSSPPTPQCHKRHRHCPVVVS EATIVGVRKTGQIWPNDDEGAFHGDADGSFGTPPGYGCAADRAEEQRRHQ DGLPYIDDSPSSSPHLSSKGRGSRDALVSGALKSTKASELDLEKGLEMRK WVLSGILASEETYLSHLEALLLPMKPLKAAATTSQPVLTSQQIETIFFKV PELYEIHKESYDGLFPRVQQWSHQQRVGDLPQKLASQLGVYRAFVDNYGV AMEMAEKCCQANAQFAEISENLRARSNKDAKDPTTKNSLETLLYKPVDRV TRSTLVLHDLLKHTPASHPDHPLLQDALRISQNFLSSINEEITPRRQSMT VKKGEHRQLLKDSFMVELVEGARKLRHVFLFTDLLLCTKLKKQSGGKTQQ YDCKWYIPLTDLSFQMVDELEAVPNIPLVPDEELDALKIKISQIKSDIQR EKRANKGSKATERLKKKLSEQESLLLLMSPSMAFRVHSRNGKSYTFLISS DYERAEWRENIREQQKKCFRSFSLTSVELQMLTNSCVKLQTVHSIPLTIN KEDDESPGLYGFLNVIVHSATGFKQSSKALQRPVASDFEPQGLSEAARWN SKENLLAGPSENDPNLFVALYDFVASGDNTLSITKGEKLRVLGYNHNGEW CEAQTKNGQGWVPSNYITPVNSLEKHSWYHGPVSRNAAEYPLSSGINGSF LVRESESSPSQRSISLRYEGRVYHYRINTASDGKLYVSSESRFNTLAELV HHHSTVADGLITTLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLG GGQYGEVYEGVWKKYSLTVAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLV QLLGVCTREPPFYIIIEFMTYGNLLDYLRECNRQEVNAVVLLYMATQISS AMEYLEKKNFIHRDLAARNCLVGENHLVKVADFGLSRLMTGDTYTAHAGA KFPIKWTAPESLAYNKFSIKSDVWAFGVLLWEIATYGMSPYPGIDRSQVY ELLEKDYRMKRPEGCPEKVYELMRACWQWNPSDRPSFAEIHQAFETMFQE SSISDEVEKELGKQGVRGAVTTLLQAPELPTKTRTSRRAAEHRDTTDVPE MPHSKGQGESDPLDHEPAVSPLLPRKERGPPEGGLNEDERLLPKDKKTNL FSALIKKKKKTAPTPPKRSSSFREMDGQPERRGAGEEEGRDISNGALAFT PLDTADPAKSPKPSNGAGVPNGALRESGGSGFRSPHLWKKSSTLTSSRLA TGEEEGGGSSSKRFLRSCSVSCVPHGAKDTEWRSVTLPRDLQSTGRQFDS STFGGHKSEKPALPRKRAGENRSDQVTRGTVTPPPRLVKKNEEAADEVFK DIMESSPGSSPPNLTPKPLRRQVTVAPASGLPHKEEAWKGSALGTPAAAE PVTPTSKAGSGAPRGTSKGPAEESRVRRHKHSSESPGRDKGKLSKLKPAP PPPPAASAGKAGGKPSQRPGQEAAGEAVLGAKTKATSLVDAVNSDAAKPS QPAEGLKKPVLPATPKPHPAKPSGTPISPAPVPLSTLPSASSALAGDQPS STAFIPLISTRVSLRKTRQPPERASGAITKGVVLDSTEALCLAISGNSEQ MASHSAVLEAGKNLYTFCVSYVDSIQQMRNKFAFREAINKLENNLRELQI CPASAGSGFAATQDFSKLLSSVKEISDIVQR BCR-Abl p210-e13a2 T315I (SEQ ID NO: 7) MVDPVGFAEAWKAQFPDSEPPRMELRSVGDIEQELERCKASIRRLEQEVN QERFRMIYLQTLLAKEKKSYDRQRWGFRRAAQAPDGASEPRASASRPQPA PADGADPPPAEEPEARPDGEGSPGKARPGTARRPGAAASGERDDRGPPAS VAALRSNFERIRKGHGQPGADAEKPFYVNVEFHHERGLVKVNDKEVSDRI SSLGSQAMQMERKKSQHGAGSSVGDASRPPYRGRSSESSCGVDGDYEDAE LNPRFLKDNLIDANGGSRPPWPPLEYQPYQSIYVGGIMEGEGKGPLLRSQ STSEQEKRLTWPRRSYSPRSFEDCGGGYTPDCSSNENLTSSEEDFSSGQS SRVSPSPTTYRMFRDKSRSPSQNSQQSFDSSSPPTPQCHKPHRHCPVVVS EATIVGVRKTGQIWPNDDEGAFHGDADGSFGTPPGYGCAADRAEEQRRHQ DGLPYIDDSPSSSPHLSSKGRGSRDALVSGALKSTKASELDLEKGLEMRK WVLSGILASEETYLSHLEALLLPMKPLKAAATTSQPVLTSQQIETIFFKV PELYEIHKESYDGLFPRVQQWSHQQRVGDLFQKLASQLGVYRAFVDNYGV AMEMAEKCCQANAQFAEISENLRARSNKDAKDPTTKNSLETLLYKPVDRV TRSTLVLHDLLKHTPASHPDHPLLQDALRISQNFLSSINEEITPRRQSMT VKKGEHRQLLKDSFMVELVEGARKLRHVFLFTDLLLCTKLKKQSGGKTQQ YDCKWYIPLTDESFQMVDELEAVPNIPLVPDEELDALKIKISQIKSDIQR EKRANKGSKATERLKKKLSEQESLLLLMSPSMAFRVHSRNGKSYTFLISS DYERAEWRENIREQQKKCFRSPSLTSVELQMLTNSCVKLQTVHSIPLTIN KEEALQRPVASDFEPQGLSEAAPWNSKENLLAGPSENDPNLFVALYDFVA SQDNTLSITKGEKLRVLGYNHNGEWCEAQTKNGQGWVPSNYITPVNSLEK HSWYHGPVSRNAAEYPLSSGINGSFLVRESESSPSQRSISLRYEGRVYHY RINTASDGKLYVSSESRFNTLAELVHHHSTVADGLITTLHYPAPKRNKPT VYGVSPNYDKWEMERTDITMKHKLGGGQYGEVYEGVWKKYSLTVAVKTLK EDTMEVEEFLKEAAVMKEIKHPNLVQLLGVCTREPPFYIIIEFMTYGNLL DYLRECNRQEVNAVVLLYMATQISSAMEYLEKKNFIHRDLAARNCLVGEN HLVKVADFGLSRLMTGDTYTAHAGAKFPIKWTAFESLAYNKFSIKSDVWA FGVLLWEIATYGMSPYPGIDRSQVYELLEKDYRMKRPEGCPEKVYELMRA CWQWNPSDRPSFAEIHQAFETMFQESSISDEVEKELGKQGVRGAVTTLLQ APELPTKTRTSRRAAEHRDTTDVPEMPHSKGQGESDPLDHEPAVSPLLPR KERGPFEGGLNEDERLLPKDKKTNLFSALIKKKKKTAPTPPKRSSSFREM DGQFERRGAGEEEGRDISNGALAFTPLDTADPAKSFKPSNGAGVPNGALR ESGGSGFRSPHLWKKSSTLTSSRLATGEEEGGGSSSKRFLRSCSVSCVPH GAKDTEWRSVTLPRDLQSTGRQFDSSTFGGHKSEKPALPRKRAGENRSDQ VTRGTVTPPPRLVKKNEEAADEVFKDIMESSPGSSPPNLTPKPLRRQVTV APASGLPHKEEAWKGSALGTPAAAEPVTPTSKAGSGAPRGTSKGPAEESR VRRHKHSSESPGRDKGKLSKLKPAPPPPPAASAGKAGGKPSQRPGQEAAG EAVLGAKTKATSLVDAVNSDAAKPSQPAEGLKKPVLPATPKPHPAKPSGT PISPAPVPLSTLPSASSALAGDQPSSTAFIPLISTRVSLRKTRQPPERAS GAITKGVVLDSTEALCLAISGNSEQMASHSAVLEAGKNLYTFCVSYVDSI QQMRNKFAFREAINKLENNLRELQICPASAGSGPAATQDFSKLLSSVKEI SDIVQR BCR-Abl p190-e1a2 (SEQ ID NO: 8) MVDPVGFAEAWKAQFPDSEPPRMELRSVGDIEQELERCKASIRRLEQEVN QERFRMIYLQTLLAKEKKSYDRQRWGFRRAAQAFDGASEPRASASRPQPA PADGADPPPAEEPEARPDGEGSPGKARPGTARRPQAAASGERDDRGPPAS VAALRSNFERIRKGHGQPGADAEKPFYVNVEFHHERGLVKVNDKEVSDRI SSLGSQAMQMERKKSQHGAGSSVGDASRPPYRGRSSESSCGVDGDYEDAE LNPRFLKDNLIDANGGSRPPWFPLEYQPYQSIYVGGIMEGEGKGPLLRSQ STSEQEKRLTWPRRSYSPRSFEDCGGGYTPDCSSNENLTSSEEDFSSGQS SRVSPSPTTYRMFRDKSRSPSQNSQQSFDSSSPPTPQCHKRHRHCPVVVS EATIVGVRKTGQIWPNDDEGAFHGDAEALQRPVASDFEPQGLSEAARWNS KENLLAGPSENDPNLFVALYDFVASGDNTLSITKGEKLRVLGYNHNGEWC EAQTKNGQGWVPSNYITPVNSLEKHSWYHGPVSRNAAEYPLSSGINGSFL VRESESSPSQRSISLRYEGRVYHYRINTASDGKLYVSSESRFNTLAELVH HHSTVADGLITTLHYPAPKRNRPTVYGVSPNYDKWEMERTDITMKHKLGG GQYGEVYEGVWKKYSLTVAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQ LLGVCTREPPFYIIIEFMTYGNLLDYLRECNRQEVNAVVLLYMATQISSA MEYLEKKNFIHRDLAARNCLVGENHLVKVADFGLSRLMTGDTYTAHAGAK FPIKWTAPESLAYNKFSIKSDVWAFGVLLWEIATYGMSPYPGIDRSQVYE LLEKDYRMKRPEGCPEKVYELMPACWQWNPSDRPSFAEIHQAFETMFQES SISDEVEKELGKQGVRGAVTTLLQAPELPTKTRTSRRAAEHRDTTDVPEM PHSKGQGESDPLDHEPAVSFLLPRKERGPPEGGLNEDERLLPKDKKTNLF SALIKKKKKTAPTPPKRSSSFREMDGQPERRGAGEEEGRDISNGALAFTP LDTADPAKSPKPSNGAGVPNGALRESGGSGFRSPHLWKKSSTLTSSRLAT GEEEGGGSSSKRFLRSCSVSCVPHGAKDTEWRSVTLPRDLQSTGRQFDSS TFGGHKSEKPALPRKRAGENRSDQVTRGTVTPPPRLVKKNEEAADEVFKD IMESSPGSSPPNLTPKPLRRQVTVAPASGLPHKEEAWKGSALGTPAAAEP VTPTSKAGSGAPRGTSKGPAEESRVRRHKHSSESPGRDKGKLSKLKPAPP PPPAASAGKAGGKPSQRPGQEAAGEAVLGAKTKATSLVDAVNSDAAKPSQ PAEGLKKPVLPATPKPHPAKPSGTPISPAPVPLSTLPSASSALAGDQPSS TAFIPLISTRVSLRKTRQPPERASGAITKGVVLDSTEALCLAISGNSEQM ASHSAVLEAGKNLYTFCVSYVDSIQQMRNKFAFREAINKLENNLRELQIC PASAGSGPAATQDFSKLLSSVKEISDIVQR C-Kit kinase (SEQ ID NO: 9) assay
[0157]Activity of c-Kit kinase (SEQ ID NO:9) was determined by following the production of ADP from the kinase reaction through coupling with the pyruvate kinase/lactate dehydrogenase system (e.g.. Schindler, et al. Science (2000) 289, 1938-1942). In this assay, the oxidation of NADH (thus the decrease at A340 nm) was continuously monitored spectrophometrically. The reaction mixture (100 μl) contained c-Kit (cKIT residues T544-V976, from ProQinase, 5.4 nM), polyE4Y (1 mg/ml), MgCl2 (10 mM), pyruvate kinase (4 units), lactate dehydrogenase (0.7 units), phosphoenol pyruvate (1 mM), and NADH (0.28 mM) in 90 mM Tris buffer containing 0.2% octyl-glucoside and 1% DMSO, pH 7.5. Test compounds were incubated with C-Met (SEQ ID NO:9) and other reaction reagents at 22° C. for <2 min before ATP (200 μM) was added to start the reaction. The absorption at 340 nm was monitored continuously for 0.5 hours at 30° C. on Polarstar Optima plate reader (BMG). The reaction rate was calculated using the 0 to 0.5 h time frame. Percent inhibition was obtained by comparison of reaction rate with that of a control (i.e. with no test compound). IC50 values were calculated from a series of percent inhibition values determined at a range of inhibitor concentrations using software routines as implemented in the GraphPad Prism software package.
TABLE-US-00003 c-Kit with N-terminal GST fusion (SEQ ID NO: 9) LGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPN LPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVDIRYG VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFML YDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIWPLQGW QATFGGGDHPPKSDLVPRHNQTSLYKKAGSAAAVLEENLYFQGTYKYLQK PMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPRNRLSFGKTLGAGAF GKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNH MNIVNLLGACTTGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEA ALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRIGSYIER DVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILL THGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFES DVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSFEHAPAEMY DIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPV VDHSVRINSVGSTASSSQPLLVHDDV
[0158]C-Met Kinase (SEQ ID NO:10) Assay
[0159]Activity of C-Met kinase (SEQ ID NO:10) was determined by following the production of ADP from the kinase reaction through coupling with the pyruvate kinase/lactate dehydrogenase system (e.g., Schindler, et al. Science (2000) 289, 1938-1942). In this assay, the oxidation of NADH (thus the decrease at A340 nm) was continuously monitored spectrophometrically. The reaction mixture (100 μl) contained C-Met (c-Met residues: 956-1390, from Invitrogen, catalogue #PV3143, 6 nM), polyE4Y (1 mg/ml), MgCl2 (10 mM), pyruvate kinase (4 units), lactate dehydrogenase (0.7 units), phosphoenol pyruvate (1 mM), and NADH (0.28 mM) in 90 mM Tris buffer containing 0.25 mM DTT, 0.2% octyl-glucoside and 1% DMSO, pH 7.5. Test compounds were incubated with C-Met (SEQ ID NO:10) and other reaction reagents at 22° C. for 0.5 h before ATP (100 μM) was added to start the reaction. The absorption at 340 nm was monitored continuously for 2 hours at 30° C. on Polarstar Optima plate reader (BMG). The reaction rate was calculated using the 1.0 to 2.0 h time frame. Percent inhibition was obtained by comparison of reaction rate with that of a control (i.e. with no test compound), IC50 values were calculated from a series of percent inhibition values determined at a range of inhibitor concentrations using software routines as implemented in the GraphPad Prism software package.
TABLE-US-00004 cMet Kinase (SEQ ID NO: 10) MSYYHHHHHHDYDIPTTENLYFQGAMLVPRGSPWIPFTMKKRKQIKDLGS ELVRYDARVHTPHLDRLVSARSVSPTTEMVSNESVDYRATFPEDQFPNSS QNGSCRQVQYPLTDMSPILTSGDSDISSPLLQNTVHIDLSALNPELVQAV QHVVIGPSSLIVHFNEVIGRGHFGCVYHGTLLDNDGKKIHCAVKSLNRIT DIGEVSQFLTEGIIMKDFSHPNVLSLLGICLRSECSPLVVLPYMKHGDLR NFIRNETHNPTVKDLIGFGLQVAKGMKYLASKKFVHRDLAARNCMLDEKF TVKVADFGLARDMYDKEYYSVHNKTGAKLPVKWMALESLQTQKFTTKSDV WSFGVLLWELMTRGAPPYPDVNTFDITVYLLQGRRLLQPEYCPDPLYEVM LKCWHPKAEMRPSFSELVSRISAIFSTFIGEHYVHVNATYVNVKCVAPYP SLLSSEDNADDEVDTRPASFWETS
[0160]The biochemical IC50 Values of other compounds disclosed herein are at least 10 μM against Abl enzyme.
Cell Culture
[0161]BaF3 cells (parental or transfected with the following: wild type p210 BCR-Abl and T315I p210 BCR-Abl was obtained from Professor Richard Van Etten (New England Medical Center, Boston, Mass.). Briefly, cells were grown in RPMI 1640 supplemented with 10% characterized fetal bovine serum (HyClone, Logan, Utah) at 37 degrees Celsius, 5% CO2, 95% humidity. Cells were allowed to expand until reaching 80% saturation at which point they were subcultured or harvested for assay use.
Cell Proliferation Assay
[0162]A serial dilution of test compound was dispensed into a 96 well black clear bottom plate (Coming, Coming, N.Y.). For each cell line, three thousand cells were added per well in complete growth medium. Plates were incubated for 72 hours at 37 degrees Celsius, 5% CO2, 95% humidity. At the end of the incubation period Cell Titer Blue (Promega, Madison, Wis.) was added to each well and an additional 4.5 hour incubation at 37 degrees Celsius, 5% CO2, 95% humidity was performed. Plates were then read on a BMG Fluostar Optima (BMG, Durham, N.C.) using an excitation of 544 nM and an emission of 612 nM. Data was analyzed using Prism software (Graphpad, San Diego. Calif.) to calculate IC50's.
Sequence CWU
1
101278PRTHomo sapiens 1Gly Thr Ser Met Asp Pro Ser Ser Pro Asn Tyr Asp Lys
Trp Glu Met1 5 10 15Glu
Arg Thr Asp Ile Thr Met Lys His Lys Leu Gly Gly Gly Gln Tyr 20
25 30Gly Glu Val Tyr Glu Gly Val Trp
Lys Lys Tyr Ser Leu Thr Val Ala 35 40
45Val Lys Thr Leu Lys Glu Asp Thr Met Glu Val Glu Glu Phe Leu Lys
50 55 60Glu Ala Ala Val Met Lys Glu Ile
Lys His Pro Asn Leu Val Gln Leu65 70 75
80Leu Gly Val Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile
Thr Glu Phe 85 90 95Met
Thr Tyr Gly Asn Leu Leu Asp Tyr Leu Arg Glu Cys Asn Arg Gln
100 105 110Glu Val Asn Ala Val Val Leu
Leu Tyr Met Ala Thr Gln Ile Ser Ser 115 120
125Ala Met Glu Tyr Leu Glu Lys Lys Asn Phe Ile His Arg Asp Leu
Ala 130 135 140Ala Arg Asn Cys Leu Val
Gly Glu Asn His Leu Val Lys Val Ala Asp145 150
155 160Phe Gly Leu Ser Arg Leu Met Thr Gly Asp Thr
Tyr Thr Ala His Ala 165 170
175Gly Ala Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu Ser Leu Ala Tyr
180 185 190Asn Lys Phe Ser Ile Lys
Ser Asp Val Trp Ala Phe Gly Val Leu Leu 195 200
205Trp Glu Ile Ala Thr Tyr Gly Met Ser Pro Tyr Pro Gly Ile
Asp Leu 210 215 220Ser Gln Val Tyr Glu
Leu Leu Glu Lys Asp Tyr Arg Met Glu Arg Pro225 230
235 240Glu Gly Cys Pro Glu Lys Val Tyr Glu Leu
Met Arg Ala Cys Trp Gln 245 250
255Trp Asn Pro Ser Asp Arg Pro Ser Phe Ala Glu Ile His Gln Ala Phe
260 265 270Glu Thr Met Phe Gln
Glu 2752278PRTHomo sapiens 2Gly Thr Ser Met Asp Pro Ser Ser Pro
Asn Tyr Asp Lys Trp Glu Met1 5 10
15Glu Arg Thr Asp Ile Thr Met Lys His Lys Leu Gly Gly Gly Gln
Tyr 20 25 30Gly Glu Val Tyr
Glu Gly Val Trp Lys Lys Tyr Ser Leu Thr Val Ala 35
40 45Val Lys Thr Leu Lys Glu Asp Thr Met Glu Val Glu
Glu Phe Leu Lys 50 55 60Glu Ala Ala
Val Met Lys Glu Ile Lys His Pro Asn Leu Val Gln Leu65 70
75 80Leu Gly Val Cys Thr Arg Glu Pro
Pro Phe Tyr Ile Ile Ile Glu Phe 85 90
95Met Thr Tyr Gly Asn Leu Leu Asp Tyr Leu Arg Glu Cys Asn
Arg Gln 100 105 110Glu Val Asn
Ala Val Val Leu Leu Tyr Met Ala Thr Gln Ile Ser Ser 115
120 125Ala Met Glu Tyr Leu Glu Lys Lys Asn Phe Ile
His Arg Asp Leu Ala 130 135 140Ala Arg
Asn Cys Leu Val Gly Glu Asn His Leu Val Lys Val Ala Asp145
150 155 160Phe Gly Leu Ser Arg Leu Met
Thr Gly Asp Thr Tyr Thr Ala His Ala 165
170 175Gly Ala Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu
Ser Leu Ala Tyr 180 185 190Asn
Lys Phe Ser Ile Lys Ser Asp Val Trp Ala Phe Gly Val Leu Leu 195
200 205Trp Glu Ile Ala Thr Tyr Gly Met Ser
Pro Tyr Pro Gly Ile Asp Leu 210 215
220Ser Gln Val Tyr Glu Leu Leu Glu Lys Asp Tyr Arg Met Glu Arg Pro225
230 235 240Glu Gly Cys Pro
Glu Lys Val Tyr Glu Leu Met Arg Ala Cys Trp Gln 245
250 255Trp Asn Pro Ser Asp Arg Pro Ser Phe Ala
Glu Ile His Gln Ala Phe 260 265
270Glu Thr Met Phe Gln Glu 27532031PRTHomo sapiens 3Met Val Asp
Pro Val Gly Phe Ala Glu Ala Trp Lys Ala Gln Phe Pro1 5
10 15Asp Ser Glu Pro Pro Arg Met Glu Leu
Arg Ser Val Gly Asp Ile Glu 20 25
30Gln Glu Leu Glu Arg Cys Lys Ala Ser Ile Arg Arg Leu Glu Gln Glu
35 40 45Val Asn Gln Glu Arg Phe Arg
Met Ile Tyr Leu Gln Thr Leu Leu Ala 50 55
60Lys Glu Lys Lys Ser Tyr Asp Arg Gln Arg Trp Gly Phe Arg Arg Ala65
70 75 80Ala Gln Ala Pro
Asp Gly Ala Ser Glu Pro Arg Ala Ser Ala Ser Arg 85
90 95Pro Gln Pro Ala Pro Ala Asp Gly Ala Asp
Pro Pro Pro Ala Glu Glu 100 105
110Pro Glu Ala Arg Pro Asp Gly Glu Gly Ser Pro Gly Lys Ala Arg Pro
115 120 125Gly Thr Ala Arg Arg Pro Gly
Ala Ala Ala Ser Gly Glu Arg Asp Asp 130 135
140Arg Gly Pro Pro Ala Ser Val Ala Ala Leu Arg Ser Asn Phe Glu
Arg145 150 155 160Ile Arg
Lys Gly His Gly Gln Pro Gly Ala Asp Ala Glu Lys Pro Phe
165 170 175Tyr Val Asn Val Glu Phe His
His Glu Arg Gly Leu Val Lys Val Asn 180 185
190Asp Lys Glu Val Ser Asp Arg Ile Ser Ser Leu Gly Ser Gln
Ala Met 195 200 205Gln Met Glu Arg
Lys Lys Ser Gln His Gly Ala Gly Ser Ser Val Gly 210
215 220Asp Ala Ser Arg Pro Pro Tyr Arg Gly Arg Ser Ser
Glu Ser Ser Cys225 230 235
240Gly Val Asp Gly Asp Tyr Glu Asp Ala Glu Leu Asn Pro Arg Phe Leu
245 250 255Lys Asp Asn Leu Ile
Asp Ala Asn Gly Gly Ser Arg Pro Pro Trp Pro 260
265 270Pro Leu Glu Tyr Gln Pro Tyr Gln Ser Ile Tyr Val
Gly Gly Ile Met 275 280 285Glu Gly
Glu Gly Lys Gly Pro Leu Leu Arg Ser Gln Ser Thr Ser Glu 290
295 300Gln Glu Lys Arg Leu Thr Trp Pro Arg Arg Ser
Tyr Ser Pro Arg Ser305 310 315
320Phe Glu Asp Cys Gly Gly Gly Tyr Thr Pro Asp Cys Ser Ser Asn Glu
325 330 335Asn Leu Thr Ser
Ser Glu Glu Asp Phe Ser Ser Gly Gln Ser Ser Arg 340
345 350Val Ser Pro Ser Pro Thr Thr Tyr Arg Met Phe
Arg Asp Lys Ser Arg 355 360 365Ser
Pro Ser Gln Asn Ser Gln Gln Ser Phe Asp Ser Ser Ser Pro Pro 370
375 380Thr Pro Gln Cys His Lys Arg His Arg His
Cys Pro Val Val Val Ser385 390 395
400Glu Ala Thr Ile Val Gly Val Arg Lys Thr Gly Gln Ile Trp Pro
Asn 405 410 415Asp Asp Glu
Gly Ala Phe His Gly Asp Ala Asp Gly Ser Phe Gly Thr 420
425 430Pro Pro Gly Tyr Gly Cys Ala Ala Asp Arg
Ala Glu Glu Gln Arg Arg 435 440
445His Gln Asp Gly Leu Pro Tyr Ile Asp Asp Ser Pro Ser Ser Ser Pro 450
455 460His Leu Ser Ser Lys Gly Arg Gly
Ser Arg Asp Ala Leu Val Ser Gly465 470
475 480Ala Leu Lys Ser Thr Lys Ala Ser Glu Leu Asp Leu
Glu Lys Gly Leu 485 490
495Glu Met Arg Lys Trp Val Leu Ser Gly Ile Leu Ala Ser Glu Glu Thr
500 505 510Tyr Leu Ser His Leu Glu
Ala Leu Leu Leu Pro Met Lys Pro Leu Lys 515 520
525Ala Ala Ala Thr Thr Ser Gln Pro Val Leu Thr Ser Gln Gln
Ile Glu 530 535 540Thr Ile Phe Phe Lys
Val Pro Glu Leu Tyr Glu Ile His Lys Glu Ser545 550
555 560Tyr Asp Gly Leu Phe Pro Arg Val Gln Gln
Trp Ser His Gln Gln Arg 565 570
575Val Gly Asp Leu Phe Gln Lys Leu Ala Ser Gln Leu Gly Val Tyr Arg
580 585 590Ala Phe Val Asp Asn
Tyr Gly Val Ala Met Glu Met Ala Glu Lys Cys 595
600 605Cys Gln Ala Asn Ala Gln Phe Ala Glu Ile Ser Glu
Asn Leu Arg Ala 610 615 620Arg Ser Asn
Lys Asp Ala Lys Asp Pro Thr Thr Lys Asn Ser Leu Glu625
630 635 640Thr Leu Leu Tyr Lys Pro Val
Asp Arg Val Thr Arg Ser Thr Leu Val 645
650 655Leu His Asp Leu Leu Lys His Thr Pro Ala Ser His
Pro Asp His Pro 660 665 670Leu
Leu Gln Asp Ala Leu Arg Ile Ser Gln Asn Phe Leu Ser Ser Ile 675
680 685Asn Glu Glu Ile Thr Pro Arg Arg Gln
Ser Met Thr Val Lys Lys Gly 690 695
700Glu His Arg Gln Leu Leu Lys Asp Ser Phe Met Val Glu Leu Val Glu705
710 715 720Gly Ala Arg Lys
Leu Arg His Val Phe Leu Phe Thr Asp Leu Leu Leu 725
730 735Cys Thr Lys Leu Lys Lys Gln Ser Gly Gly
Lys Thr Gln Gln Tyr Asp 740 745
750Cys Lys Trp Tyr Ile Pro Leu Thr Asp Leu Ser Phe Gln Met Val Asp
755 760 765Glu Leu Glu Ala Val Pro Asn
Ile Pro Leu Val Pro Asp Glu Glu Leu 770 775
780Asp Ala Leu Lys Ile Lys Ile Ser Gln Ile Lys Ser Asp Ile Gln
Arg785 790 795 800Glu Lys
Arg Ala Asn Lys Gly Ser Lys Ala Thr Glu Arg Leu Lys Lys
805 810 815Lys Leu Ser Glu Gln Glu Ser
Leu Leu Leu Leu Met Ser Pro Ser Met 820 825
830Ala Phe Arg Val His Ser Arg Asn Gly Lys Ser Tyr Thr Phe
Leu Ile 835 840 845Ser Ser Asp Tyr
Glu Arg Ala Glu Trp Arg Glu Asn Ile Arg Glu Gln 850
855 860Gln Lys Lys Cys Phe Arg Ser Phe Ser Leu Thr Ser
Val Glu Leu Gln865 870 875
880Met Leu Thr Asn Ser Cys Val Lys Leu Gln Thr Val His Ser Ile Pro
885 890 895Leu Thr Ile Asn Lys
Glu Asp Asp Glu Ser Pro Gly Leu Tyr Gly Phe 900
905 910Leu Asn Val Ile Val His Ser Ala Thr Gly Phe Lys
Gln Ser Ser Lys 915 920 925Ala Leu
Gln Arg Pro Val Ala Ser Asp Phe Glu Pro Gln Gly Leu Ser 930
935 940Glu Ala Ala Arg Trp Asn Ser Lys Glu Asn Leu
Leu Ala Gly Pro Ser945 950 955
960Glu Asn Asp Pro Asn Leu Phe Val Ala Leu Tyr Asp Phe Val Ala Ser
965 970 975Gly Asp Asn Thr
Leu Ser Ile Thr Lys Gly Glu Lys Leu Arg Val Leu 980
985 990Gly Tyr Asn His Asn Gly Glu Trp Cys Glu Ala
Gln Thr Lys Asn Gly 995 1000
1005Gln Gly Trp Val Pro Ser Asn Tyr Ile Thr Pro Val Asn Ser Leu
1010 1015 1020Glu Lys His Ser Trp Tyr
His Gly Pro Val Ser Arg Asn Ala Ala 1025 1030
1035Glu Tyr Pro Leu Ser Ser Gly Ile Asn Gly Ser Phe Leu Val
Arg 1040 1045 1050Glu Ser Glu Ser Ser
Pro Ser Gln Arg Ser Ile Ser Leu Arg Tyr 1055 1060
1065Glu Gly Arg Val Tyr His Tyr Arg Ile Asn Thr Ala Ser
Asp Gly 1070 1075 1080Lys Leu Tyr Val
Ser Ser Glu Ser Arg Phe Asn Thr Leu Ala Glu 1085
1090 1095Leu Val His His His Ser Thr Val Ala Asp Gly
Leu Ile Thr Thr 1100 1105 1110Leu His
Tyr Pro Ala Pro Lys Arg Asn Lys Pro Thr Val Tyr Gly 1115
1120 1125Val Ser Pro Asn Tyr Asp Lys Trp Glu Met
Glu Arg Thr Asp Ile 1130 1135 1140Thr
Met Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Glu Val Tyr 1145
1150 1155Glu Gly Val Trp Lys Lys Tyr Ser Leu
Thr Val Ala Val Lys Thr 1160 1165
1170Leu Lys Glu Asp Thr Met Glu Val Glu Glu Phe Leu Lys Glu Ala
1175 1180 1185Ala Val Met Lys Glu Ile
Lys His Pro Asn Leu Val Gln Leu Leu 1190 1195
1200Gly Val Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Thr Glu
Phe 1205 1210 1215Met Thr Tyr Gly Asn
Leu Leu Asp Tyr Leu Arg Glu Cys Asn Arg 1220 1225
1230Gln Glu Val Asn Ala Val Val Leu Leu Tyr Met Ala Thr
Gln Ile 1235 1240 1245Ser Ser Ala Met
Glu Tyr Leu Glu Lys Lys Asn Phe Ile His Arg 1250
1255 1260Asp Leu Ala Ala Arg Asn Cys Leu Val Gly Glu
Asn His Leu Val 1265 1270 1275Lys Val
Ala Asp Phe Gly Leu Ser Arg Leu Met Thr Gly Asp Thr 1280
1285 1290Tyr Thr Ala His Ala Gly Ala Lys Phe Pro
Ile Lys Trp Thr Ala 1295 1300 1305Pro
Glu Ser Leu Ala Tyr Asn Lys Phe Ser Ile Lys Ser Asp Val 1310
1315 1320Trp Ala Phe Gly Val Leu Leu Trp Glu
Ile Ala Thr Tyr Gly Met 1325 1330
1335Ser Pro Tyr Pro Gly Ile Asp Arg Ser Gln Val Tyr Glu Leu Leu
1340 1345 1350Glu Lys Asp Tyr Arg Met
Lys Arg Pro Glu Gly Cys Pro Glu Lys 1355 1360
1365Val Tyr Glu Leu Met Arg Ala Cys Trp Gln Trp Asn Pro Ser
Asp 1370 1375 1380Arg Pro Ser Phe Ala
Glu Ile His Gln Ala Phe Glu Thr Met Phe 1385 1390
1395Gln Glu Ser Ser Ile Ser Asp Glu Val Glu Lys Glu Leu
Gly Lys 1400 1405 1410Gln Gly Val Arg
Gly Ala Val Thr Thr Leu Leu Gln Ala Pro Glu 1415
1420 1425Leu Pro Thr Lys Thr Arg Thr Ser Arg Arg Ala
Ala Glu His Arg 1430 1435 1440Asp Thr
Thr Asp Val Pro Glu Met Pro His Ser Lys Gly Gln Gly 1445
1450 1455Glu Ser Asp Pro Leu Asp His Glu Pro Ala
Val Ser Pro Leu Leu 1460 1465 1470Pro
Arg Lys Glu Arg Gly Pro Pro Glu Gly Gly Leu Asn Glu Asp 1475
1480 1485Glu Arg Leu Leu Pro Lys Asp Lys Lys
Thr Asn Leu Phe Ser Ala 1490 1495
1500Leu Ile Lys Lys Lys Lys Lys Thr Ala Pro Thr Pro Pro Lys Arg
1505 1510 1515Ser Ser Ser Phe Arg Glu
Met Asp Gly Gln Pro Glu Arg Arg Gly 1520 1525
1530Ala Gly Glu Glu Glu Gly Arg Asp Ile Ser Asn Gly Ala Leu
Ala 1535 1540 1545Phe Thr Pro Leu Asp
Thr Ala Asp Pro Ala Lys Ser Pro Lys Pro 1550 1555
1560Ser Asn Gly Ala Gly Val Pro Asn Gly Ala Leu Arg Glu
Ser Gly 1565 1570 1575Gly Ser Gly Phe
Arg Ser Pro His Leu Trp Lys Lys Ser Ser Thr 1580
1585 1590Leu Thr Ser Ser Arg Leu Ala Thr Gly Glu Glu
Glu Gly Gly Gly 1595 1600 1605Ser Ser
Ser Lys Arg Phe Leu Arg Ser Cys Ser Val Ser Cys Val 1610
1615 1620Pro His Gly Ala Lys Asp Thr Glu Trp Arg
Ser Val Thr Leu Pro 1625 1630 1635Arg
Asp Leu Gln Ser Thr Gly Arg Gln Phe Asp Ser Ser Thr Phe 1640
1645 1650Gly Gly His Lys Ser Glu Lys Pro Ala
Leu Pro Arg Lys Arg Ala 1655 1660
1665Gly Glu Asn Arg Ser Asp Gln Val Thr Arg Gly Thr Val Thr Pro
1670 1675 1680Pro Pro Arg Leu Val Lys
Lys Asn Glu Glu Ala Ala Asp Glu Val 1685 1690
1695Phe Lys Asp Ile Met Glu Ser Ser Pro Gly Ser Ser Pro Pro
Asn 1700 1705 1710Leu Thr Pro Lys Pro
Leu Arg Arg Gln Val Thr Val Ala Pro Ala 1715 1720
1725Ser Gly Leu Pro His Lys Glu Glu Ala Trp Lys Gly Ser
Ala Leu 1730 1735 1740Gly Thr Pro Ala
Ala Ala Glu Pro Val Thr Pro Thr Ser Lys Ala 1745
1750 1755Gly Ser Gly Ala Pro Arg Gly Thr Ser Lys Gly
Pro Ala Glu Glu 1760 1765 1770Ser Arg
Val Arg Arg His Lys His Ser Ser Glu Ser Pro Gly Arg 1775
1780 1785Asp Lys Gly Lys Leu Ser Lys Leu Lys Pro
Ala Pro Pro Pro Pro 1790 1795 1800Pro
Ala Ala Ser Ala Gly Lys Ala Gly Gly Lys Pro Ser Gln Arg 1805
1810 1815Pro Gly Gln Glu Ala Ala Gly Glu Ala
Val Leu Gly Ala Lys Thr 1820 1825
1830Lys Ala Thr Ser Leu Val Asp Ala Val Asn Ser Asp Ala Ala Lys
1835 1840 1845Pro Ser Gln Pro Ala Glu
Gly Leu Lys Lys Pro Val Leu Pro Ala 1850 1855
1860Thr Pro Lys Pro His Pro Ala Lys Pro Ser Gly Thr Pro Ile
Ser 1865 1870 1875Pro Ala Pro Val Pro
Leu Ser Thr Leu Pro Ser Ala Ser Ser Ala 1880 1885
1890Leu Ala Gly Asp Gln Pro Ser Ser Thr Ala Phe Ile Pro
Leu Ile 1895 1900 1905Ser Thr Arg Val
Ser Leu Arg Lys Thr Arg Gln Pro Pro Glu Arg 1910
1915 1920Ala Ser Gly Ala Ile Thr Lys Gly Val Val Leu
Asp Ser Thr Glu 1925 1930 1935Ala Leu
Cys Leu Ala Ile Ser Gly Asn Ser Glu Gln Met Ala Ser 1940
1945 1950His Ser Ala Val Leu Glu Ala Gly Lys Asn
Leu Tyr Thr Phe Cys 1955 1960 1965Val
Ser Tyr Val Asp Ser Ile Gln Gln Met Arg Asn Lys Phe Ala 1970
1975 1980Phe Arg Glu Ala Ile Asn Lys Leu Glu
Asn Asn Leu Arg Glu Leu 1985 1990
1995Gln Ile Cys Pro Ala Ser Ala Gly Ser Gly Pro Ala Ala Thr Gln
2000 2005 2010Asp Phe Ser Lys Leu Leu
Ser Ser Val Lys Glu Ile Ser Asp Ile 2015 2020
2025Val Gln Arg 203042006PRTHomo sapiens 4Met Val Asp Pro
Val Gly Phe Ala Glu Ala Trp Lys Ala Gln Phe Pro1 5
10 15Asp Ser Glu Pro Pro Arg Met Glu Leu Arg
Ser Val Gly Asp Ile Glu 20 25
30Gln Glu Leu Glu Arg Cys Lys Ala Ser Ile Arg Arg Leu Glu Gln Glu
35 40 45Val Asn Gln Glu Arg Phe Arg Met
Ile Tyr Leu Gln Thr Leu Leu Ala 50 55
60Lys Glu Lys Lys Ser Tyr Asp Arg Gln Arg Trp Gly Phe Arg Arg Ala65
70 75 80Ala Gln Ala Pro Asp
Gly Ala Ser Glu Pro Arg Ala Ser Ala Ser Arg 85
90 95Pro Gln Pro Ala Pro Ala Asp Gly Ala Asp Pro
Pro Pro Ala Glu Glu 100 105
110Pro Glu Ala Arg Pro Asp Gly Glu Gly Ser Pro Gly Lys Ala Arg Pro
115 120 125Gly Thr Ala Arg Arg Pro Gly
Ala Ala Ala Ser Gly Glu Arg Asp Asp 130 135
140Arg Gly Pro Pro Ala Ser Val Ala Ala Leu Arg Ser Asn Phe Glu
Arg145 150 155 160Ile Arg
Lys Gly His Gly Gln Pro Gly Ala Asp Ala Glu Lys Pro Phe
165 170 175Tyr Val Asn Val Glu Phe His
His Glu Arg Gly Leu Val Lys Val Asn 180 185
190Asp Lys Glu Val Ser Asp Arg Ile Ser Ser Leu Gly Ser Gln
Ala Met 195 200 205Gln Met Glu Arg
Lys Lys Ser Gln His Gly Ala Gly Ser Ser Val Gly 210
215 220Asp Ala Ser Arg Pro Pro Tyr Arg Gly Arg Ser Ser
Glu Ser Ser Cys225 230 235
240Gly Val Asp Gly Asp Tyr Glu Asp Ala Glu Leu Asn Pro Arg Phe Leu
245 250 255Lys Asp Asn Leu Ile
Asp Ala Asn Gly Gly Ser Arg Pro Pro Trp Pro 260
265 270Pro Leu Glu Tyr Gln Pro Tyr Gln Ser Ile Tyr Val
Gly Gly Ile Met 275 280 285Glu Gly
Glu Gly Lys Gly Pro Leu Leu Arg Ser Gln Ser Thr Ser Glu 290
295 300Gln Glu Lys Arg Leu Thr Trp Pro Arg Arg Ser
Tyr Ser Pro Arg Ser305 310 315
320Phe Glu Asp Cys Gly Gly Gly Tyr Thr Pro Asp Cys Ser Ser Asn Glu
325 330 335Asn Leu Thr Ser
Ser Glu Glu Asp Phe Ser Ser Gly Gln Ser Ser Arg 340
345 350Val Ser Pro Ser Pro Thr Thr Tyr Arg Met Phe
Arg Asp Lys Ser Arg 355 360 365Ser
Pro Ser Gln Asn Ser Gln Gln Ser Phe Asp Ser Ser Ser Pro Pro 370
375 380Thr Pro Gln Cys His Lys Arg His Arg His
Cys Pro Val Val Val Ser385 390 395
400Glu Ala Thr Ile Val Gly Val Arg Lys Thr Gly Gln Ile Trp Pro
Asn 405 410 415Asp Asp Glu
Gly Ala Phe His Gly Asp Ala Asp Gly Ser Phe Gly Thr 420
425 430Pro Pro Gly Tyr Gly Cys Ala Ala Asp Arg
Ala Glu Glu Gln Arg Arg 435 440
445His Gln Asp Gly Leu Pro Tyr Ile Asp Asp Ser Pro Ser Ser Ser Pro 450
455 460His Leu Ser Ser Lys Gly Arg Gly
Ser Arg Asp Ala Leu Val Ser Gly465 470
475 480Ala Leu Lys Ser Thr Lys Ala Ser Glu Leu Asp Leu
Glu Lys Gly Leu 485 490
495Glu Met Arg Lys Trp Val Leu Ser Gly Ile Leu Ala Ser Glu Glu Thr
500 505 510Tyr Leu Ser His Leu Glu
Ala Leu Leu Leu Pro Met Lys Pro Leu Lys 515 520
525Ala Ala Ala Thr Thr Ser Gln Pro Val Leu Thr Ser Gln Gln
Ile Glu 530 535 540Thr Ile Phe Phe Lys
Val Pro Glu Leu Tyr Glu Ile His Lys Glu Ser545 550
555 560Tyr Asp Gly Leu Phe Pro Arg Val Gln Gln
Trp Ser His Gln Gln Arg 565 570
575Val Gly Asp Leu Phe Gln Lys Leu Ala Ser Gln Leu Gly Val Tyr Arg
580 585 590Ala Phe Val Asp Asn
Tyr Gly Val Ala Met Glu Met Ala Glu Lys Cys 595
600 605Cys Gln Ala Asn Ala Gln Phe Ala Glu Ile Ser Glu
Asn Leu Arg Ala 610 615 620Arg Ser Asn
Lys Asp Ala Lys Asp Pro Thr Thr Lys Asn Ser Leu Glu625
630 635 640Thr Leu Leu Tyr Lys Pro Val
Asp Arg Val Thr Arg Ser Thr Leu Val 645
650 655Leu His Asp Leu Leu Lys His Thr Pro Ala Ser His
Pro Asp His Pro 660 665 670Leu
Leu Gln Asp Ala Leu Arg Ile Ser Gln Asn Phe Leu Ser Ser Ile 675
680 685Asn Glu Glu Ile Thr Pro Arg Arg Gln
Ser Met Thr Val Lys Lys Gly 690 695
700Glu His Arg Gln Leu Leu Lys Asp Ser Phe Met Val Glu Leu Val Glu705
710 715 720Gly Ala Arg Lys
Leu Arg His Val Phe Leu Phe Thr Asp Leu Leu Leu 725
730 735Cys Thr Lys Leu Lys Lys Gln Ser Gly Gly
Lys Thr Gln Gln Tyr Asp 740 745
750Cys Lys Trp Tyr Ile Pro Leu Thr Asp Leu Ser Phe Gln Met Val Asp
755 760 765Glu Leu Glu Ala Val Pro Asn
Ile Pro Leu Val Pro Asp Glu Glu Leu 770 775
780Asp Ala Leu Lys Ile Lys Ile Ser Gln Ile Lys Ser Asp Ile Gln
Arg785 790 795 800Glu Lys
Arg Ala Asn Lys Gly Ser Lys Ala Thr Glu Arg Leu Lys Lys
805 810 815Lys Leu Ser Glu Gln Glu Ser
Leu Leu Leu Leu Met Ser Pro Ser Met 820 825
830Ala Phe Arg Val His Ser Arg Asn Gly Lys Ser Tyr Thr Phe
Leu Ile 835 840 845Ser Ser Asp Tyr
Glu Arg Ala Glu Trp Arg Glu Asn Ile Arg Glu Gln 850
855 860Gln Lys Lys Cys Phe Arg Ser Phe Ser Leu Thr Ser
Val Glu Leu Gln865 870 875
880Met Leu Thr Asn Ser Cys Val Lys Leu Gln Thr Val His Ser Ile Pro
885 890 895Leu Thr Ile Asn Lys
Glu Glu Ala Leu Gln Arg Pro Val Ala Ser Asp 900
905 910Phe Glu Pro Gln Gly Leu Ser Glu Ala Ala Arg Trp
Asn Ser Lys Glu 915 920 925Asn Leu
Leu Ala Gly Pro Ser Glu Asn Asp Pro Asn Leu Phe Val Ala 930
935 940Leu Tyr Asp Phe Val Ala Ser Gly Asp Asn Thr
Leu Ser Ile Thr Lys945 950 955
960Gly Glu Lys Leu Arg Val Leu Gly Tyr Asn His Asn Gly Glu Trp Cys
965 970 975Glu Ala Gln Thr
Lys Asn Gly Gln Gly Trp Val Pro Ser Asn Tyr Ile 980
985 990Thr Pro Val Asn Ser Leu Glu Lys His Ser Trp
Tyr His Gly Pro Val 995 1000
1005Ser Arg Asn Ala Ala Glu Tyr Pro Leu Ser Ser Gly Ile Asn Gly
1010 1015 1020Ser Phe Leu Val Arg Glu
Ser Glu Ser Ser Pro Ser Gln Arg Ser 1025 1030
1035Ile Ser Leu Arg Tyr Glu Gly Arg Val Tyr His Tyr Arg Ile
Asn 1040 1045 1050Thr Ala Ser Asp Gly
Lys Leu Tyr Val Ser Ser Glu Ser Arg Phe 1055 1060
1065Asn Thr Leu Ala Glu Leu Val His His His Ser Thr Val
Ala Asp 1070 1075 1080Gly Leu Ile Thr
Thr Leu His Tyr Pro Ala Pro Lys Arg Asn Lys 1085
1090 1095Pro Thr Val Tyr Gly Val Ser Pro Asn Tyr Asp
Lys Trp Glu Met 1100 1105 1110Glu Arg
Thr Asp Ile Thr Met Lys His Lys Leu Gly Gly Gly Gln 1115
1120 1125Tyr Gly Glu Val Tyr Glu Gly Val Trp Lys
Lys Tyr Ser Leu Thr 1130 1135 1140Val
Ala Val Lys Thr Leu Lys Glu Asp Thr Met Glu Val Glu Glu 1145
1150 1155Phe Leu Lys Glu Ala Ala Val Met Lys
Glu Ile Lys His Pro Asn 1160 1165
1170Leu Val Gln Leu Leu Gly Val Cys Thr Arg Glu Pro Pro Phe Tyr
1175 1180 1185Ile Ile Thr Glu Phe Met
Thr Tyr Gly Asn Leu Leu Asp Tyr Leu 1190 1195
1200Arg Glu Cys Asn Arg Gln Glu Val Asn Ala Val Val Leu Leu
Tyr 1205 1210 1215Met Ala Thr Gln Ile
Ser Ser Ala Met Glu Tyr Leu Glu Lys Lys 1220 1225
1230Asn Phe Ile His Arg Asp Leu Ala Ala Arg Asn Cys Leu
Val Gly 1235 1240 1245Glu Asn His Leu
Val Lys Val Ala Asp Phe Gly Leu Ser Arg Leu 1250
1255 1260Met Thr Gly Asp Thr Tyr Thr Ala His Ala Gly
Ala Lys Phe Pro 1265 1270 1275Ile Lys
Trp Thr Ala Pro Glu Ser Leu Ala Tyr Asn Lys Phe Ser 1280
1285 1290Ile Lys Ser Asp Val Trp Ala Phe Gly Val
Leu Leu Trp Glu Ile 1295 1300 1305Ala
Thr Tyr Gly Met Ser Pro Tyr Pro Gly Ile Asp Arg Ser Gln 1310
1315 1320Val Tyr Glu Leu Leu Glu Lys Asp Tyr
Arg Met Lys Arg Pro Glu 1325 1330
1335Gly Cys Pro Glu Lys Val Tyr Glu Leu Met Arg Ala Cys Trp Gln
1340 1345 1350Trp Asn Pro Ser Asp Arg
Pro Ser Phe Ala Glu Ile His Gln Ala 1355 1360
1365Phe Glu Thr Met Phe Gln Glu Ser Ser Ile Ser Asp Glu Val
Glu 1370 1375 1380Lys Glu Leu Gly Lys
Gln Gly Val Arg Gly Ala Val Thr Thr Leu 1385 1390
1395Leu Gln Ala Pro Glu Leu Pro Thr Lys Thr Arg Thr Ser
Arg Arg 1400 1405 1410Ala Ala Glu His
Arg Asp Thr Thr Asp Val Pro Glu Met Pro His 1415
1420 1425Ser Lys Gly Gln Gly Glu Ser Asp Pro Leu Asp
His Glu Pro Ala 1430 1435 1440Val Ser
Pro Leu Leu Pro Arg Lys Glu Arg Gly Pro Pro Glu Gly 1445
1450 1455Gly Leu Asn Glu Asp Glu Arg Leu Leu Pro
Lys Asp Lys Lys Thr 1460 1465 1470Asn
Leu Phe Ser Ala Leu Ile Lys Lys Lys Lys Lys Thr Ala Pro 1475
1480 1485Thr Pro Pro Lys Arg Ser Ser Ser Phe
Arg Glu Met Asp Gly Gln 1490 1495
1500Pro Glu Arg Arg Gly Ala Gly Glu Glu Glu Gly Arg Asp Ile Ser
1505 1510 1515Asn Gly Ala Leu Ala Phe
Thr Pro Leu Asp Thr Ala Asp Pro Ala 1520 1525
1530Lys Ser Pro Lys Pro Ser Asn Gly Ala Gly Val Pro Asn Gly
Ala 1535 1540 1545Leu Arg Glu Ser Gly
Gly Ser Gly Phe Arg Ser Pro His Leu Trp 1550 1555
1560Lys Lys Ser Ser Thr Leu Thr Ser Ser Arg Leu Ala Thr
Gly Glu 1565 1570 1575Glu Glu Gly Gly
Gly Ser Ser Ser Lys Arg Phe Leu Arg Ser Cys 1580
1585 1590Ser Val Ser Cys Val Pro His Gly Ala Lys Asp
Thr Glu Trp Arg 1595 1600 1605Ser Val
Thr Leu Pro Arg Asp Leu Gln Ser Thr Gly Arg Gln Phe 1610
1615 1620Asp Ser Ser Thr Phe Gly Gly His Lys Ser
Glu Lys Pro Ala Leu 1625 1630 1635Pro
Arg Lys Arg Ala Gly Glu Asn Arg Ser Asp Gln Val Thr Arg 1640
1645 1650Gly Thr Val Thr Pro Pro Pro Arg Leu
Val Lys Lys Asn Glu Glu 1655 1660
1665Ala Ala Asp Glu Val Phe Lys Asp Ile Met Glu Ser Ser Pro Gly
1670 1675 1680Ser Ser Pro Pro Asn Leu
Thr Pro Lys Pro Leu Arg Arg Gln Val 1685 1690
1695Thr Val Ala Pro Ala Ser Gly Leu Pro His Lys Glu Glu Ala
Trp 1700 1705 1710Lys Gly Ser Ala Leu
Gly Thr Pro Ala Ala Ala Glu Pro Val Thr 1715 1720
1725Pro Thr Ser Lys Ala Gly Ser Gly Ala Pro Arg Gly Thr
Ser Lys 1730 1735 1740Gly Pro Ala Glu
Glu Ser Arg Val Arg Arg His Lys His Ser Ser 1745
1750 1755Glu Ser Pro Gly Arg Asp Lys Gly Lys Leu Ser
Lys Leu Lys Pro 1760 1765 1770Ala Pro
Pro Pro Pro Pro Ala Ala Ser Ala Gly Lys Ala Gly Gly 1775
1780 1785Lys Pro Ser Gln Arg Pro Gly Gln Glu Ala
Ala Gly Glu Ala Val 1790 1795 1800Leu
Gly Ala Lys Thr Lys Ala Thr Ser Leu Val Asp Ala Val Asn 1805
1810 1815Ser Asp Ala Ala Lys Pro Ser Gln Pro
Ala Glu Gly Leu Lys Lys 1820 1825
1830Pro Val Leu Pro Ala Thr Pro Lys Pro His Pro Ala Lys Pro Ser
1835 1840 1845Gly Thr Pro Ile Ser Pro
Ala Pro Val Pro Leu Ser Thr Leu Pro 1850 1855
1860Ser Ala Ser Ser Ala Leu Ala Gly Asp Gln Pro Ser Ser Thr
Ala 1865 1870 1875Phe Ile Pro Leu Ile
Ser Thr Arg Val Ser Leu Arg Lys Thr Arg 1880 1885
1890Gln Pro Pro Glu Arg Ala Ser Gly Ala Ile Thr Lys Gly
Val Val 1895 1900 1905Leu Asp Ser Thr
Glu Ala Leu Cys Leu Ala Ile Ser Gly Asn Ser 1910
1915 1920Glu Gln Met Ala Ser His Ser Ala Val Leu Glu
Ala Gly Lys Asn 1925 1930 1935Leu Tyr
Thr Phe Cys Val Ser Tyr Val Asp Ser Ile Gln Gln Met 1940
1945 1950Arg Asn Lys Phe Ala Phe Arg Glu Ala Ile
Asn Lys Leu Glu Asn 1955 1960 1965Asn
Leu Arg Glu Leu Gln Ile Cys Pro Ala Ser Ala Gly Ser Gly 1970
1975 1980Pro Ala Ala Thr Gln Asp Phe Ser Lys
Leu Leu Ser Ser Val Lys 1985 1990
1995Glu Ile Ser Asp Ile Val Gln Arg 2000
200551530PRTHomo sapiens 5Met Val Asp Pro Val Gly Phe Ala Glu Ala Trp Lys
Ala Gln Phe Pro1 5 10
15Asp Ser Glu Pro Pro Arg Met Glu Leu Arg Ser Val Gly Asp Ile Glu
20 25 30Gln Glu Leu Glu Arg Cys Lys
Ala Ser Ile Arg Arg Leu Glu Gln Glu 35 40
45Val Asn Gln Glu Arg Phe Arg Met Ile Tyr Leu Gln Thr Leu Leu
Ala 50 55 60Lys Glu Lys Lys Ser Tyr
Asp Arg Gln Arg Trp Gly Phe Arg Arg Ala65 70
75 80Ala Gln Ala Pro Asp Gly Ala Ser Glu Pro Arg
Ala Ser Ala Ser Arg 85 90
95Pro Gln Pro Ala Pro Ala Asp Gly Ala Asp Pro Pro Pro Ala Glu Glu
100 105 110Pro Glu Ala Arg Pro Asp
Gly Glu Gly Ser Pro Gly Lys Ala Arg Pro 115 120
125Gly Thr Ala Arg Arg Pro Gly Ala Ala Ala Ser Gly Glu Arg
Asp Asp 130 135 140Arg Gly Pro Pro Ala
Ser Val Ala Ala Leu Arg Ser Asn Phe Glu Arg145 150
155 160Ile Arg Lys Gly His Gly Gln Pro Gly Ala
Asp Ala Glu Lys Pro Phe 165 170
175Tyr Val Asn Val Glu Phe His His Glu Arg Gly Leu Val Lys Val Asn
180 185 190Asp Lys Glu Val Ser
Asp Arg Ile Ser Ser Leu Gly Ser Gln Ala Met 195
200 205Gln Met Glu Arg Lys Lys Ser Gln His Gly Ala Gly
Ser Ser Val Gly 210 215 220Asp Ala Ser
Arg Pro Pro Tyr Arg Gly Arg Ser Ser Glu Ser Ser Cys225
230 235 240Gly Val Asp Gly Asp Tyr Glu
Asp Ala Glu Leu Asn Pro Arg Phe Leu 245
250 255Lys Asp Asn Leu Ile Asp Ala Asn Gly Gly Ser Arg
Pro Pro Trp Pro 260 265 270Pro
Leu Glu Tyr Gln Pro Tyr Gln Ser Ile Tyr Val Gly Gly Ile Met 275
280 285Glu Gly Glu Gly Lys Gly Pro Leu Leu
Arg Ser Gln Ser Thr Ser Glu 290 295
300Gln Glu Lys Arg Leu Thr Trp Pro Arg Arg Ser Tyr Ser Pro Arg Ser305
310 315 320Phe Glu Asp Cys
Gly Gly Gly Tyr Thr Pro Asp Cys Ser Ser Asn Glu 325
330 335Asn Leu Thr Ser Ser Glu Glu Asp Phe Ser
Ser Gly Gln Ser Ser Arg 340 345
350Val Ser Pro Ser Pro Thr Thr Tyr Arg Met Phe Arg Asp Lys Ser Arg
355 360 365Ser Pro Ser Gln Asn Ser Gln
Gln Ser Phe Asp Ser Ser Ser Pro Pro 370 375
380Thr Pro Gln Cys His Lys Arg His Arg His Cys Pro Val Val Val
Ser385 390 395 400Glu Ala
Thr Ile Val Gly Val Arg Lys Thr Gly Gln Ile Trp Pro Asn
405 410 415Asp Asp Glu Gly Ala Phe His
Gly Asp Ala Glu Ala Leu Gln Arg Pro 420 425
430Val Ala Ser Asp Phe Glu Pro Gln Gly Leu Ser Glu Ala Ala
Arg Trp 435 440 445Asn Ser Lys Glu
Asn Leu Leu Ala Gly Pro Ser Glu Asn Asp Pro Asn 450
455 460Leu Phe Val Ala Leu Tyr Asp Phe Val Ala Ser Gly
Asp Asn Thr Leu465 470 475
480Ser Ile Thr Lys Gly Glu Lys Leu Arg Val Leu Gly Tyr Asn His Asn
485 490 495Gly Glu Trp Cys Glu
Ala Gln Thr Lys Asn Gly Gln Gly Trp Val Pro 500
505 510Ser Asn Tyr Ile Thr Pro Val Asn Ser Leu Glu Lys
His Ser Trp Tyr 515 520 525His Gly
Pro Val Ser Arg Asn Ala Ala Glu Tyr Pro Leu Ser Ser Gly 530
535 540Ile Asn Gly Ser Phe Leu Val Arg Glu Ser Glu
Ser Ser Pro Ser Gln545 550 555
560Arg Ser Ile Ser Leu Arg Tyr Glu Gly Arg Val Tyr His Tyr Arg Ile
565 570 575Asn Thr Ala Ser
Asp Gly Lys Leu Tyr Val Ser Ser Glu Ser Arg Phe 580
585 590Asn Thr Leu Ala Glu Leu Val His His His Ser
Thr Val Ala Asp Gly 595 600 605Leu
Ile Thr Thr Leu His Tyr Pro Ala Pro Lys Arg Asn Lys Pro Thr 610
615 620Val Tyr Gly Val Ser Pro Asn Tyr Asp Lys
Trp Glu Met Glu Arg Thr625 630 635
640Asp Ile Thr Met Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Glu
Val 645 650 655Tyr Glu Gly
Val Trp Lys Lys Tyr Ser Leu Thr Val Ala Val Lys Thr 660
665 670Leu Lys Glu Asp Thr Met Glu Val Glu Glu
Phe Leu Lys Glu Ala Ala 675 680
685Val Met Lys Glu Ile Lys His Pro Asn Leu Val Gln Leu Leu Gly Val 690
695 700Cys Thr Arg Glu Pro Pro Phe Tyr
Ile Ile Thr Glu Phe Met Thr Tyr705 710
715 720Gly Asn Leu Leu Asp Tyr Leu Arg Glu Cys Asn Arg
Gln Glu Val Asn 725 730
735Ala Val Val Leu Leu Tyr Met Ala Thr Gln Ile Ser Ser Ala Met Glu
740 745 750Tyr Leu Glu Lys Lys Asn
Phe Ile His Arg Asp Leu Ala Ala Arg Asn 755 760
765Cys Leu Val Gly Glu Asn His Leu Val Lys Val Ala Asp Phe
Gly Leu 770 775 780Ser Arg Leu Met Thr
Gly Asp Thr Tyr Thr Ala His Ala Gly Ala Lys785 790
795 800Phe Pro Ile Lys Trp Thr Ala Pro Glu Ser
Leu Ala Tyr Asn Lys Phe 805 810
815Ser Ile Lys Ser Asp Val Trp Ala Phe Gly Val Leu Leu Trp Glu Ile
820 825 830Ala Thr Tyr Gly Met
Ser Pro Tyr Pro Gly Ile Asp Arg Ser Gln Val 835
840 845Tyr Glu Leu Leu Glu Lys Asp Tyr Arg Met Lys Arg
Pro Glu Gly Cys 850 855 860Pro Glu Lys
Val Tyr Glu Leu Met Arg Ala Cys Trp Gln Trp Asn Pro865
870 875 880Ser Asp Arg Pro Ser Phe Ala
Glu Ile His Gln Ala Phe Glu Thr Met 885
890 895Phe Gln Glu Ser Ser Ile Ser Asp Glu Val Glu Lys
Glu Leu Gly Lys 900 905 910Gln
Gly Val Arg Gly Ala Val Thr Thr Leu Leu Gln Ala Pro Glu Leu 915
920 925Pro Thr Lys Thr Arg Thr Ser Arg Arg
Ala Ala Glu His Arg Asp Thr 930 935
940Thr Asp Val Pro Glu Met Pro His Ser Lys Gly Gln Gly Glu Ser Asp945
950 955 960Pro Leu Asp His
Glu Pro Ala Val Ser Pro Leu Leu Pro Arg Lys Glu 965
970 975Arg Gly Pro Pro Glu Gly Gly Leu Asn Glu
Asp Glu Arg Leu Leu Pro 980 985
990Lys Asp Lys Lys Thr Asn Leu Phe Ser Ala Leu Ile Lys Lys Lys Lys
995 1000 1005Lys Thr Ala Pro Thr Pro
Pro Lys Arg Ser Ser Ser Phe Arg Glu 1010 1015
1020Met Asp Gly Gln Pro Glu Arg Arg Gly Ala Gly Glu Glu Glu
Gly 1025 1030 1035Arg Asp Ile Ser Asn
Gly Ala Leu Ala Phe Thr Pro Leu Asp Thr 1040 1045
1050Ala Asp Pro Ala Lys Ser Pro Lys Pro Ser Asn Gly Ala
Gly Val 1055 1060 1065Pro Asn Gly Ala
Leu Arg Glu Ser Gly Gly Ser Gly Phe Arg Ser 1070
1075 1080Pro His Leu Trp Lys Lys Ser Ser Thr Leu Thr
Ser Ser Arg Leu 1085 1090 1095Ala Thr
Gly Glu Glu Glu Gly Gly Gly Ser Ser Ser Lys Arg Phe 1100
1105 1110Leu Arg Ser Cys Ser Val Ser Cys Val Pro
His Gly Ala Lys Asp 1115 1120 1125Thr
Glu Trp Arg Ser Val Thr Leu Pro Arg Asp Leu Gln Ser Thr 1130
1135 1140Gly Arg Gln Phe Asp Ser Ser Thr Phe
Gly Gly His Lys Ser Glu 1145 1150
1155Lys Pro Ala Leu Pro Arg Lys Arg Ala Gly Glu Asn Arg Ser Asp
1160 1165 1170Gln Val Thr Arg Gly Thr
Val Thr Pro Pro Pro Arg Leu Val Lys 1175 1180
1185Lys Asn Glu Glu Ala Ala Asp Glu Val Phe Lys Asp Ile Met
Glu 1190 1195 1200Ser Ser Pro Gly Ser
Ser Pro Pro Asn Leu Thr Pro Lys Pro Leu 1205 1210
1215Arg Arg Gln Val Thr Val Ala Pro Ala Ser Gly Leu Pro
His Lys 1220 1225 1230Glu Glu Ala Trp
Lys Gly Ser Ala Leu Gly Thr Pro Ala Ala Ala 1235
1240 1245Glu Pro Val Thr Pro Thr Ser Lys Ala Gly Ser
Gly Ala Pro Arg 1250 1255 1260Gly Thr
Ser Lys Gly Pro Ala Glu Glu Ser Arg Val Arg Arg His 1265
1270 1275Lys His Ser Ser Glu Ser Pro Gly Arg Asp
Lys Gly Lys Leu Ser 1280 1285 1290Lys
Leu Lys Pro Ala Pro Pro Pro Pro Pro Ala Ala Ser Ala Gly 1295
1300 1305Lys Ala Gly Gly Lys Pro Ser Gln Arg
Pro Gly Gln Glu Ala Ala 1310 1315
1320Gly Glu Ala Val Leu Gly Ala Lys Thr Lys Ala Thr Ser Leu Val
1325 1330 1335Asp Ala Val Asn Ser Asp
Ala Ala Lys Pro Ser Gln Pro Ala Glu 1340 1345
1350Gly Leu Lys Lys Pro Val Leu Pro Ala Thr Pro Lys Pro His
Pro 1355 1360 1365Ala Lys Pro Ser Gly
Thr Pro Ile Ser Pro Ala Pro Val Pro Leu 1370 1375
1380Ser Thr Leu Pro Ser Ala Ser Ser Ala Leu Ala Gly Asp
Gln Pro 1385 1390 1395Ser Ser Thr Ala
Phe Ile Pro Leu Ile Ser Thr Arg Val Ser Leu 1400
1405 1410Arg Lys Thr Arg Gln Pro Pro Glu Arg Ala Ser
Gly Ala Ile Thr 1415 1420 1425Lys Gly
Val Val Leu Asp Ser Thr Glu Ala Leu Cys Leu Ala Ile 1430
1435 1440Ser Gly Asn Ser Glu Gln Met Ala Ser His
Ser Ala Val Leu Glu 1445 1450 1455Ala
Gly Lys Asn Leu Tyr Thr Phe Cys Val Ser Tyr Val Asp Ser 1460
1465 1470Ile Gln Gln Met Arg Asn Lys Phe Ala
Phe Arg Glu Ala Ile Asn 1475 1480
1485Lys Leu Glu Asn Asn Leu Arg Glu Leu Gln Ile Cys Pro Ala Ser
1490 1495 1500Ala Gly Ser Gly Pro Ala
Ala Thr Gln Asp Phe Ser Lys Leu Leu 1505 1510
1515Ser Ser Val Lys Glu Ile Ser Asp Ile Val Gln Arg 1520
1525 153062031PRTHomo sapiens 6Met Val Asp
Pro Val Gly Phe Ala Glu Ala Trp Lys Ala Gln Phe Pro1 5
10 15Asp Ser Glu Pro Pro Arg Met Glu Leu
Arg Ser Val Gly Asp Ile Glu 20 25
30Gln Glu Leu Glu Arg Cys Lys Ala Ser Ile Arg Arg Leu Glu Gln Glu
35 40 45Val Asn Gln Glu Arg Phe Arg
Met Ile Tyr Leu Gln Thr Leu Leu Ala 50 55
60Lys Glu Lys Lys Ser Tyr Asp Arg Gln Arg Trp Gly Phe Arg Arg Ala65
70 75 80Ala Gln Ala Pro
Asp Gly Ala Ser Glu Pro Arg Ala Ser Ala Ser Arg 85
90 95Pro Gln Pro Ala Pro Ala Asp Gly Ala Asp
Pro Pro Pro Ala Glu Glu 100 105
110Pro Glu Ala Arg Pro Asp Gly Glu Gly Ser Pro Gly Lys Ala Arg Pro
115 120 125Gly Thr Ala Arg Arg Pro Gly
Ala Ala Ala Ser Gly Glu Arg Asp Asp 130 135
140Arg Gly Pro Pro Ala Ser Val Ala Ala Leu Arg Ser Asn Phe Glu
Arg145 150 155 160Ile Arg
Lys Gly His Gly Gln Pro Gly Ala Asp Ala Glu Lys Pro Phe
165 170 175Tyr Val Asn Val Glu Phe His
His Glu Arg Gly Leu Val Lys Val Asn 180 185
190Asp Lys Glu Val Ser Asp Arg Ile Ser Ser Leu Gly Ser Gln
Ala Met 195 200 205Gln Met Glu Arg
Lys Lys Ser Gln His Gly Ala Gly Ser Ser Val Gly 210
215 220Asp Ala Ser Arg Pro Pro Tyr Arg Gly Arg Ser Ser
Glu Ser Ser Cys225 230 235
240Gly Val Asp Gly Asp Tyr Glu Asp Ala Glu Leu Asn Pro Arg Phe Leu
245 250 255Lys Asp Asn Leu Ile
Asp Ala Asn Gly Gly Ser Arg Pro Pro Trp Pro 260
265 270Pro Leu Glu Tyr Gln Pro Tyr Gln Ser Ile Tyr Val
Gly Gly Ile Met 275 280 285Glu Gly
Glu Gly Lys Gly Pro Leu Leu Arg Ser Gln Ser Thr Ser Glu 290
295 300Gln Glu Lys Arg Leu Thr Trp Pro Arg Arg Ser
Tyr Ser Pro Arg Ser305 310 315
320Phe Glu Asp Cys Gly Gly Gly Tyr Thr Pro Asp Cys Ser Ser Asn Glu
325 330 335Asn Leu Thr Ser
Ser Glu Glu Asp Phe Ser Ser Gly Gln Ser Ser Arg 340
345 350Val Ser Pro Ser Pro Thr Thr Tyr Arg Met Phe
Arg Asp Lys Ser Arg 355 360 365Ser
Pro Ser Gln Asn Ser Gln Gln Ser Phe Asp Ser Ser Ser Pro Pro 370
375 380Thr Pro Gln Cys His Lys Arg His Arg His
Cys Pro Val Val Val Ser385 390 395
400Glu Ala Thr Ile Val Gly Val Arg Lys Thr Gly Gln Ile Trp Pro
Asn 405 410 415Asp Asp Glu
Gly Ala Phe His Gly Asp Ala Asp Gly Ser Phe Gly Thr 420
425 430Pro Pro Gly Tyr Gly Cys Ala Ala Asp Arg
Ala Glu Glu Gln Arg Arg 435 440
445His Gln Asp Gly Leu Pro Tyr Ile Asp Asp Ser Pro Ser Ser Ser Pro 450
455 460His Leu Ser Ser Lys Gly Arg Gly
Ser Arg Asp Ala Leu Val Ser Gly465 470
475 480Ala Leu Lys Ser Thr Lys Ala Ser Glu Leu Asp Leu
Glu Lys Gly Leu 485 490
495Glu Met Arg Lys Trp Val Leu Ser Gly Ile Leu Ala Ser Glu Glu Thr
500 505 510Tyr Leu Ser His Leu Glu
Ala Leu Leu Leu Pro Met Lys Pro Leu Lys 515 520
525Ala Ala Ala Thr Thr Ser Gln Pro Val Leu Thr Ser Gln Gln
Ile Glu 530 535 540Thr Ile Phe Phe Lys
Val Pro Glu Leu Tyr Glu Ile His Lys Glu Ser545 550
555 560Tyr Asp Gly Leu Phe Pro Arg Val Gln Gln
Trp Ser His Gln Gln Arg 565 570
575Val Gly Asp Leu Phe Gln Lys Leu Ala Ser Gln Leu Gly Val Tyr Arg
580 585 590Ala Phe Val Asp Asn
Tyr Gly Val Ala Met Glu Met Ala Glu Lys Cys 595
600 605Cys Gln Ala Asn Ala Gln Phe Ala Glu Ile Ser Glu
Asn Leu Arg Ala 610 615 620Arg Ser Asn
Lys Asp Ala Lys Asp Pro Thr Thr Lys Asn Ser Leu Glu625
630 635 640Thr Leu Leu Tyr Lys Pro Val
Asp Arg Val Thr Arg Ser Thr Leu Val 645
650 655Leu His Asp Leu Leu Lys His Thr Pro Ala Ser His
Pro Asp His Pro 660 665 670Leu
Leu Gln Asp Ala Leu Arg Ile Ser Gln Asn Phe Leu Ser Ser Ile 675
680 685Asn Glu Glu Ile Thr Pro Arg Arg Gln
Ser Met Thr Val Lys Lys Gly 690 695
700Glu His Arg Gln Leu Leu Lys Asp Ser Phe Met Val Glu Leu Val Glu705
710 715 720Gly Ala Arg Lys
Leu Arg His Val Phe Leu Phe Thr Asp Leu Leu Leu 725
730 735Cys Thr Lys Leu Lys Lys Gln Ser Gly Gly
Lys Thr Gln Gln Tyr Asp 740 745
750Cys Lys Trp Tyr Ile Pro Leu Thr Asp Leu Ser Phe Gln Met Val Asp
755 760 765Glu Leu Glu Ala Val Pro Asn
Ile Pro Leu Val Pro Asp Glu Glu Leu 770 775
780Asp Ala Leu Lys Ile Lys Ile Ser Gln Ile Lys Ser Asp Ile Gln
Arg785 790 795 800Glu Lys
Arg Ala Asn Lys Gly Ser Lys Ala Thr Glu Arg Leu Lys Lys
805 810 815Lys Leu Ser Glu Gln Glu Ser
Leu Leu Leu Leu Met Ser Pro Ser Met 820 825
830Ala Phe Arg Val His Ser Arg Asn Gly Lys Ser Tyr Thr Phe
Leu Ile 835 840 845Ser Ser Asp Tyr
Glu Arg Ala Glu Trp Arg Glu Asn Ile Arg Glu Gln 850
855 860Gln Lys Lys Cys Phe Arg Ser Phe Ser Leu Thr Ser
Val Glu Leu Gln865 870 875
880Met Leu Thr Asn Ser Cys Val Lys Leu Gln Thr Val His Ser Ile Pro
885 890 895Leu Thr Ile Asn Lys
Glu Asp Asp Glu Ser Pro Gly Leu Tyr Gly Phe 900
905 910Leu Asn Val Ile Val His Ser Ala Thr Gly Phe Lys
Gln Ser Ser Lys 915 920 925Ala Leu
Gln Arg Pro Val Ala Ser Asp Phe Glu Pro Gln Gly Leu Ser 930
935 940Glu Ala Ala Arg Trp Asn Ser Lys Glu Asn Leu
Leu Ala Gly Pro Ser945 950 955
960Glu Asn Asp Pro Asn Leu Phe Val Ala Leu Tyr Asp Phe Val Ala Ser
965 970 975Gly Asp Asn Thr
Leu Ser Ile Thr Lys Gly Glu Lys Leu Arg Val Leu 980
985 990Gly Tyr Asn His Asn Gly Glu Trp Cys Glu Ala
Gln Thr Lys Asn Gly 995 1000
1005Gln Gly Trp Val Pro Ser Asn Tyr Ile Thr Pro Val Asn Ser Leu
1010 1015 1020Glu Lys His Ser Trp Tyr
His Gly Pro Val Ser Arg Asn Ala Ala 1025 1030
1035Glu Tyr Pro Leu Ser Ser Gly Ile Asn Gly Ser Phe Leu Val
Arg 1040 1045 1050Glu Ser Glu Ser Ser
Pro Ser Gln Arg Ser Ile Ser Leu Arg Tyr 1055 1060
1065Glu Gly Arg Val Tyr His Tyr Arg Ile Asn Thr Ala Ser
Asp Gly 1070 1075 1080Lys Leu Tyr Val
Ser Ser Glu Ser Arg Phe Asn Thr Leu Ala Glu 1085
1090 1095Leu Val His His His Ser Thr Val Ala Asp Gly
Leu Ile Thr Thr 1100 1105 1110Leu His
Tyr Pro Ala Pro Lys Arg Asn Lys Pro Thr Val Tyr Gly 1115
1120 1125Val Ser Pro Asn Tyr Asp Lys Trp Glu Met
Glu Arg Thr Asp Ile 1130 1135 1140Thr
Met Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Glu Val Tyr 1145
1150 1155Glu Gly Val Trp Lys Lys Tyr Ser Leu
Thr Val Ala Val Lys Thr 1160 1165
1170Leu Lys Glu Asp Thr Met Glu Val Glu Glu Phe Leu Lys Glu Ala
1175 1180 1185Ala Val Met Lys Glu Ile
Lys His Pro Asn Leu Val Gln Leu Leu 1190 1195
1200Gly Val Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Ile Glu
Phe 1205 1210 1215Met Thr Tyr Gly Asn
Leu Leu Asp Tyr Leu Arg Glu Cys Asn Arg 1220 1225
1230Gln Glu Val Asn Ala Val Val Leu Leu Tyr Met Ala Thr
Gln Ile 1235 1240 1245Ser Ser Ala Met
Glu Tyr Leu Glu Lys Lys Asn Phe Ile His Arg 1250
1255 1260Asp Leu Ala Ala Arg Asn Cys Leu Val Gly Glu
Asn His Leu Val 1265 1270 1275Lys Val
Ala Asp Phe Gly Leu Ser Arg Leu Met Thr Gly Asp Thr 1280
1285 1290Tyr Thr Ala His Ala Gly Ala Lys Phe Pro
Ile Lys Trp Thr Ala 1295 1300 1305Pro
Glu Ser Leu Ala Tyr Asn Lys Phe Ser Ile Lys Ser Asp Val 1310
1315 1320Trp Ala Phe Gly Val Leu Leu Trp Glu
Ile Ala Thr Tyr Gly Met 1325 1330
1335Ser Pro Tyr Pro Gly Ile Asp Arg Ser Gln Val Tyr Glu Leu Leu
1340 1345 1350Glu Lys Asp Tyr Arg Met
Lys Arg Pro Glu Gly Cys Pro Glu Lys 1355 1360
1365Val Tyr Glu Leu Met Arg Ala Cys Trp Gln Trp Asn Pro Ser
Asp 1370 1375 1380Arg Pro Ser Phe Ala
Glu Ile His Gln Ala Phe Glu Thr Met Phe 1385 1390
1395Gln Glu Ser Ser Ile Ser Asp Glu Val Glu Lys Glu Leu
Gly Lys 1400 1405 1410Gln Gly Val Arg
Gly Ala Val Thr Thr Leu Leu Gln Ala Pro Glu 1415
1420 1425Leu Pro Thr Lys Thr Arg Thr Ser Arg Arg Ala
Ala Glu His Arg 1430 1435 1440Asp Thr
Thr Asp Val Pro Glu Met Pro His Ser Lys Gly Gln Gly 1445
1450 1455Glu Ser Asp Pro Leu Asp His Glu Pro Ala
Val Ser Pro Leu Leu 1460 1465 1470Pro
Arg Lys Glu Arg Gly Pro Pro Glu Gly Gly Leu Asn Glu Asp 1475
1480 1485Glu Arg Leu Leu Pro Lys Asp Lys Lys
Thr Asn Leu Phe Ser Ala 1490 1495
1500Leu Ile Lys Lys Lys Lys Lys Thr Ala Pro Thr Pro Pro Lys Arg
1505 1510 1515Ser Ser Ser Phe Arg Glu
Met Asp Gly Gln Pro Glu Arg Arg Gly 1520 1525
1530Ala Gly Glu Glu Glu Gly Arg Asp Ile Ser Asn Gly Ala Leu
Ala 1535 1540 1545Phe Thr Pro Leu Asp
Thr Ala Asp Pro Ala Lys Ser Pro Lys Pro 1550 1555
1560Ser Asn Gly Ala Gly Val Pro Asn Gly Ala Leu Arg Glu
Ser Gly 1565 1570 1575Gly Ser Gly Phe
Arg Ser Pro His Leu Trp Lys Lys Ser Ser Thr 1580
1585 1590Leu Thr Ser Ser Arg Leu Ala Thr Gly Glu Glu
Glu Gly Gly Gly 1595 1600 1605Ser Ser
Ser Lys Arg Phe Leu Arg Ser Cys Ser Val Ser Cys Val 1610
1615 1620Pro His Gly Ala Lys Asp Thr Glu Trp Arg
Ser Val Thr Leu Pro 1625 1630 1635Arg
Asp Leu Gln Ser Thr Gly Arg Gln Phe Asp Ser Ser Thr Phe 1640
1645 1650Gly Gly His Lys Ser Glu Lys Pro Ala
Leu Pro Arg Lys Arg Ala 1655 1660
1665Gly Glu Asn Arg Ser Asp Gln Val Thr Arg Gly Thr Val Thr Pro
1670 1675 1680Pro Pro Arg Leu Val Lys
Lys Asn Glu Glu Ala Ala Asp Glu Val 1685 1690
1695Phe Lys Asp Ile Met Glu Ser Ser Pro Gly Ser Ser Pro Pro
Asn 1700 1705 1710Leu Thr Pro Lys Pro
Leu Arg Arg Gln Val Thr Val Ala Pro Ala 1715 1720
1725Ser Gly Leu Pro His Lys Glu Glu Ala Trp Lys Gly Ser
Ala Leu 1730 1735 1740Gly Thr Pro Ala
Ala Ala Glu Pro Val Thr Pro Thr Ser Lys Ala 1745
1750 1755Gly Ser Gly Ala Pro Arg Gly Thr Ser Lys Gly
Pro Ala Glu Glu 1760 1765 1770Ser Arg
Val Arg Arg His Lys His Ser Ser Glu Ser Pro Gly Arg 1775
1780 1785Asp Lys Gly Lys Leu Ser Lys Leu Lys Pro
Ala Pro Pro Pro Pro 1790 1795 1800Pro
Ala Ala Ser Ala Gly Lys Ala Gly Gly Lys Pro Ser Gln Arg 1805
1810 1815Pro Gly Gln Glu Ala Ala Gly Glu Ala
Val Leu Gly Ala Lys Thr 1820 1825
1830Lys Ala Thr Ser Leu Val Asp Ala Val Asn Ser Asp Ala Ala Lys
1835 1840 1845Pro Ser Gln Pro Ala Glu
Gly Leu Lys Lys Pro Val Leu Pro Ala 1850 1855
1860Thr Pro Lys Pro His Pro Ala Lys Pro Ser Gly Thr Pro Ile
Ser 1865 1870 1875Pro Ala Pro Val Pro
Leu Ser Thr Leu Pro Ser Ala Ser Ser Ala 1880 1885
1890Leu Ala Gly Asp Gln Pro Ser Ser Thr Ala Phe Ile Pro
Leu Ile 1895 1900 1905Ser Thr Arg Val
Ser Leu Arg Lys Thr Arg Gln Pro Pro Glu Arg 1910
1915 1920Ala Ser Gly Ala Ile Thr Lys Gly Val Val Leu
Asp Ser Thr Glu 1925 1930 1935Ala Leu
Cys Leu Ala Ile Ser Gly Asn Ser Glu Gln Met Ala Ser 1940
1945 1950His Ser Ala Val Leu Glu Ala Gly Lys Asn
Leu Tyr Thr Phe Cys 1955 1960 1965Val
Ser Tyr Val Asp Ser Ile Gln Gln Met Arg Asn Lys Phe Ala 1970
1975 1980Phe Arg Glu Ala Ile Asn Lys Leu Glu
Asn Asn Leu Arg Glu Leu 1985 1990
1995Gln Ile Cys Pro Ala Ser Ala Gly Ser Gly Pro Ala Ala Thr Gln
2000 2005 2010Asp Phe Ser Lys Leu Leu
Ser Ser Val Lys Glu Ile Ser Asp Ile 2015 2020
2025Val Gln Arg 203072006PRTHomo sapiens 7Met Val Asp Pro
Val Gly Phe Ala Glu Ala Trp Lys Ala Gln Phe Pro1 5
10 15Asp Ser Glu Pro Pro Arg Met Glu Leu Arg
Ser Val Gly Asp Ile Glu 20 25
30Gln Glu Leu Glu Arg Cys Lys Ala Ser Ile Arg Arg Leu Glu Gln Glu
35 40 45Val Asn Gln Glu Arg Phe Arg Met
Ile Tyr Leu Gln Thr Leu Leu Ala 50 55
60Lys Glu Lys Lys Ser Tyr Asp Arg Gln Arg Trp Gly Phe Arg Arg Ala65
70 75 80Ala Gln Ala Pro Asp
Gly Ala Ser Glu Pro Arg Ala Ser Ala Ser Arg 85
90 95Pro Gln Pro Ala Pro Ala Asp Gly Ala Asp Pro
Pro Pro Ala Glu Glu 100 105
110Pro Glu Ala Arg Pro Asp Gly Glu Gly Ser Pro Gly Lys Ala Arg Pro
115 120 125Gly Thr Ala Arg Arg Pro Gly
Ala Ala Ala Ser Gly Glu Arg Asp Asp 130 135
140Arg Gly Pro Pro Ala Ser Val Ala Ala Leu Arg Ser Asn Phe Glu
Arg145 150 155 160Ile Arg
Lys Gly His Gly Gln Pro Gly Ala Asp Ala Glu Lys Pro Phe
165 170 175Tyr Val Asn Val Glu Phe His
His Glu Arg Gly Leu Val Lys Val Asn 180 185
190Asp Lys Glu Val Ser Asp Arg Ile Ser Ser Leu Gly Ser Gln
Ala Met 195 200 205Gln Met Glu Arg
Lys Lys Ser Gln His Gly Ala Gly Ser Ser Val Gly 210
215 220Asp Ala Ser Arg Pro Pro Tyr Arg Gly Arg Ser Ser
Glu Ser Ser Cys225 230 235
240Gly Val Asp Gly Asp Tyr Glu Asp Ala Glu Leu Asn Pro Arg Phe Leu
245 250 255Lys Asp Asn Leu Ile
Asp Ala Asn Gly Gly Ser Arg Pro Pro Trp Pro 260
265 270Pro Leu Glu Tyr Gln Pro Tyr Gln Ser Ile Tyr Val
Gly Gly Ile Met 275 280 285Glu Gly
Glu Gly Lys Gly Pro Leu Leu Arg Ser Gln Ser Thr Ser Glu 290
295 300Gln Glu Lys Arg Leu Thr Trp Pro Arg Arg Ser
Tyr Ser Pro Arg Ser305 310 315
320Phe Glu Asp Cys Gly Gly Gly Tyr Thr Pro Asp Cys Ser Ser Asn Glu
325 330 335Asn Leu Thr Ser
Ser Glu Glu Asp Phe Ser Ser Gly Gln Ser Ser Arg 340
345 350Val Ser Pro Ser Pro Thr Thr Tyr Arg Met Phe
Arg Asp Lys Ser Arg 355 360 365Ser
Pro Ser Gln Asn Ser Gln Gln Ser Phe Asp Ser Ser Ser Pro Pro 370
375 380Thr Pro Gln Cys His Lys Arg His Arg His
Cys Pro Val Val Val Ser385 390 395
400Glu Ala Thr Ile Val Gly Val Arg Lys Thr Gly Gln Ile Trp Pro
Asn 405 410 415Asp Asp Glu
Gly Ala Phe His Gly Asp Ala Asp Gly Ser Phe Gly Thr 420
425 430Pro Pro Gly Tyr Gly Cys Ala Ala Asp Arg
Ala Glu Glu Gln Arg Arg 435 440
445His Gln Asp Gly Leu Pro Tyr Ile Asp Asp Ser Pro Ser Ser Ser Pro 450
455 460His Leu Ser Ser Lys Gly Arg Gly
Ser Arg Asp Ala Leu Val Ser Gly465 470
475 480Ala Leu Lys Ser Thr Lys Ala Ser Glu Leu Asp Leu
Glu Lys Gly Leu 485 490
495Glu Met Arg Lys Trp Val Leu Ser Gly Ile Leu Ala Ser Glu Glu Thr
500 505 510Tyr Leu Ser His Leu Glu
Ala Leu Leu Leu Pro Met Lys Pro Leu Lys 515 520
525Ala Ala Ala Thr Thr Ser Gln Pro Val Leu Thr Ser Gln Gln
Ile Glu 530 535 540Thr Ile Phe Phe Lys
Val Pro Glu Leu Tyr Glu Ile His Lys Glu Ser545 550
555 560Tyr Asp Gly Leu Phe Pro Arg Val Gln Gln
Trp Ser His Gln Gln Arg 565 570
575Val Gly Asp Leu Phe Gln Lys Leu Ala Ser Gln Leu Gly Val Tyr Arg
580 585 590Ala Phe Val Asp Asn
Tyr Gly Val Ala Met Glu Met Ala Glu Lys Cys 595
600 605Cys Gln Ala Asn Ala Gln Phe Ala Glu Ile Ser Glu
Asn Leu Arg Ala 610 615 620Arg Ser Asn
Lys Asp Ala Lys Asp Pro Thr Thr Lys Asn Ser Leu Glu625
630 635 640Thr Leu Leu Tyr Lys Pro Val
Asp Arg Val Thr Arg Ser Thr Leu Val 645
650 655Leu His Asp Leu Leu Lys His Thr Pro Ala Ser His
Pro Asp His Pro 660 665 670Leu
Leu Gln Asp Ala Leu Arg Ile Ser Gln Asn Phe Leu Ser Ser Ile 675
680 685Asn Glu Glu Ile Thr Pro Arg Arg Gln
Ser Met Thr Val Lys Lys Gly 690 695
700Glu His Arg Gln Leu Leu Lys Asp Ser Phe Met Val Glu Leu Val Glu705
710 715 720Gly Ala Arg Lys
Leu Arg His Val Phe Leu Phe Thr Asp Leu Leu Leu 725
730 735Cys Thr Lys Leu Lys Lys Gln Ser Gly Gly
Lys Thr Gln Gln Tyr Asp 740 745
750Cys Lys Trp Tyr Ile Pro Leu Thr Asp Leu Ser Phe Gln Met Val Asp
755 760 765Glu Leu Glu Ala Val Pro Asn
Ile Pro Leu Val Pro Asp Glu Glu Leu 770 775
780Asp Ala Leu Lys Ile Lys Ile Ser Gln Ile Lys Ser Asp Ile Gln
Arg785 790 795 800Glu Lys
Arg Ala Asn Lys Gly Ser Lys Ala Thr Glu Arg Leu Lys Lys
805 810 815Lys Leu Ser Glu Gln Glu Ser
Leu Leu Leu Leu Met Ser Pro Ser Met 820 825
830Ala Phe Arg Val His Ser Arg Asn Gly Lys Ser Tyr Thr Phe
Leu Ile 835 840 845Ser Ser Asp Tyr
Glu Arg Ala Glu Trp Arg Glu Asn Ile Arg Glu Gln 850
855 860Gln Lys Lys Cys Phe Arg Ser Phe Ser Leu Thr Ser
Val Glu Leu Gln865 870 875
880Met Leu Thr Asn Ser Cys Val Lys Leu Gln Thr Val His Ser Ile Pro
885 890 895Leu Thr Ile Asn Lys
Glu Glu Ala Leu Gln Arg Pro Val Ala Ser Asp 900
905 910Phe Glu Pro Gln Gly Leu Ser Glu Ala Ala Arg Trp
Asn Ser Lys Glu 915 920 925Asn Leu
Leu Ala Gly Pro Ser Glu Asn Asp Pro Asn Leu Phe Val Ala 930
935 940Leu Tyr Asp Phe Val Ala Ser Gly Asp Asn Thr
Leu Ser Ile Thr Lys945 950 955
960Gly Glu Lys Leu Arg Val Leu Gly Tyr Asn His Asn Gly Glu Trp Cys
965 970 975Glu Ala Gln Thr
Lys Asn Gly Gln Gly Trp Val Pro Ser Asn Tyr Ile 980
985 990Thr Pro Val Asn Ser Leu Glu Lys His Ser Trp
Tyr His Gly Pro Val 995 1000
1005Ser Arg Asn Ala Ala Glu Tyr Pro Leu Ser Ser Gly Ile Asn Gly
1010 1015 1020Ser Phe Leu Val Arg Glu
Ser Glu Ser Ser Pro Ser Gln Arg Ser 1025 1030
1035Ile Ser Leu Arg Tyr Glu Gly Arg Val Tyr His Tyr Arg Ile
Asn 1040 1045 1050Thr Ala Ser Asp Gly
Lys Leu Tyr Val Ser Ser Glu Ser Arg Phe 1055 1060
1065Asn Thr Leu Ala Glu Leu Val His His His Ser Thr Val
Ala Asp 1070 1075 1080Gly Leu Ile Thr
Thr Leu His Tyr Pro Ala Pro Lys Arg Asn Lys 1085
1090 1095Pro Thr Val Tyr Gly Val Ser Pro Asn Tyr Asp
Lys Trp Glu Met 1100 1105 1110Glu Arg
Thr Asp Ile Thr Met Lys His Lys Leu Gly Gly Gly Gln 1115
1120 1125Tyr Gly Glu Val Tyr Glu Gly Val Trp Lys
Lys Tyr Ser Leu Thr 1130 1135 1140Val
Ala Val Lys Thr Leu Lys Glu Asp Thr Met Glu Val Glu Glu 1145
1150 1155Phe Leu Lys Glu Ala Ala Val Met Lys
Glu Ile Lys His Pro Asn 1160 1165
1170Leu Val Gln Leu Leu Gly Val Cys Thr Arg Glu Pro Pro Phe Tyr
1175 1180 1185Ile Ile Ile Glu Phe Met
Thr Tyr Gly Asn Leu Leu Asp Tyr Leu 1190 1195
1200Arg Glu Cys Asn Arg Gln Glu Val Asn Ala Val Val Leu Leu
Tyr 1205 1210 1215Met Ala Thr Gln Ile
Ser Ser Ala Met Glu Tyr Leu Glu Lys Lys 1220 1225
1230Asn Phe Ile His Arg Asp Leu Ala Ala Arg Asn Cys Leu
Val Gly 1235 1240 1245Glu Asn His Leu
Val Lys Val Ala Asp Phe Gly Leu Ser Arg Leu 1250
1255 1260Met Thr Gly Asp Thr Tyr Thr Ala His Ala Gly
Ala Lys Phe Pro 1265 1270 1275Ile Lys
Trp Thr Ala Pro Glu Ser Leu Ala Tyr Asn Lys Phe Ser 1280
1285 1290Ile Lys Ser Asp Val Trp Ala Phe Gly Val
Leu Leu Trp Glu Ile 1295 1300 1305Ala
Thr Tyr Gly Met Ser Pro Tyr Pro Gly Ile Asp Arg Ser Gln 1310
1315 1320Val Tyr Glu Leu Leu Glu Lys Asp Tyr
Arg Met Lys Arg Pro Glu 1325 1330
1335Gly Cys Pro Glu Lys Val Tyr Glu Leu Met Arg Ala Cys Trp Gln
1340 1345 1350Trp Asn Pro Ser Asp Arg
Pro Ser Phe Ala Glu Ile His Gln Ala 1355 1360
1365Phe Glu Thr Met Phe Gln Glu Ser Ser Ile Ser Asp Glu Val
Glu 1370 1375 1380Lys Glu Leu Gly Lys
Gln Gly Val Arg Gly Ala Val Thr Thr Leu 1385 1390
1395Leu Gln Ala Pro Glu Leu Pro Thr Lys Thr Arg Thr Ser
Arg Arg 1400 1405 1410Ala Ala Glu His
Arg Asp Thr Thr Asp Val Pro Glu Met Pro His 1415
1420 1425Ser Lys Gly Gln Gly Glu Ser Asp Pro Leu Asp
His Glu Pro Ala 1430 1435 1440Val Ser
Pro Leu Leu Pro Arg Lys Glu Arg Gly Pro Pro Glu Gly 1445
1450 1455Gly Leu Asn Glu Asp Glu Arg Leu Leu Pro
Lys Asp Lys Lys Thr 1460 1465 1470Asn
Leu Phe Ser Ala Leu Ile Lys Lys Lys Lys Lys Thr Ala Pro 1475
1480 1485Thr Pro Pro Lys Arg Ser Ser Ser Phe
Arg Glu Met Asp Gly Gln 1490 1495
1500Pro Glu Arg Arg Gly Ala Gly Glu Glu Glu Gly Arg Asp Ile Ser
1505 1510 1515Asn Gly Ala Leu Ala Phe
Thr Pro Leu Asp Thr Ala Asp Pro Ala 1520 1525
1530Lys Ser Pro Lys Pro Ser Asn Gly Ala Gly Val Pro Asn Gly
Ala 1535 1540 1545Leu Arg Glu Ser Gly
Gly Ser Gly Phe Arg Ser Pro His Leu Trp 1550 1555
1560Lys Lys Ser Ser Thr Leu Thr Ser Ser Arg Leu Ala Thr
Gly Glu 1565 1570 1575Glu Glu Gly Gly
Gly Ser Ser Ser Lys Arg Phe Leu Arg Ser Cys 1580
1585 1590Ser Val Ser Cys Val Pro His Gly Ala Lys Asp
Thr Glu Trp Arg 1595 1600 1605Ser Val
Thr Leu Pro Arg Asp Leu Gln Ser Thr Gly Arg Gln Phe 1610
1615 1620Asp Ser Ser Thr Phe Gly Gly His Lys Ser
Glu Lys Pro Ala Leu 1625 1630 1635Pro
Arg Lys Arg Ala Gly Glu Asn Arg Ser Asp Gln Val Thr Arg 1640
1645 1650Gly Thr Val Thr Pro Pro Pro Arg Leu
Val Lys Lys Asn Glu Glu 1655 1660
1665Ala Ala Asp Glu Val Phe Lys Asp Ile Met Glu Ser Ser Pro Gly
1670 1675 1680Ser Ser Pro Pro Asn Leu
Thr Pro Lys Pro Leu Arg Arg Gln Val 1685 1690
1695Thr Val Ala Pro Ala Ser Gly Leu Pro His Lys Glu Glu Ala
Trp 1700 1705 1710Lys Gly Ser Ala Leu
Gly Thr Pro Ala Ala Ala Glu Pro Val Thr 1715 1720
1725Pro Thr Ser Lys Ala Gly Ser Gly Ala Pro Arg Gly Thr
Ser Lys 1730 1735 1740Gly Pro Ala Glu
Glu Ser Arg Val Arg Arg His Lys His Ser Ser 1745
1750 1755Glu Ser Pro Gly Arg Asp Lys Gly Lys Leu Ser
Lys Leu Lys Pro 1760 1765 1770Ala Pro
Pro Pro Pro Pro Ala Ala Ser Ala Gly Lys Ala Gly Gly 1775
1780 1785Lys Pro Ser Gln Arg Pro Gly Gln Glu Ala
Ala Gly Glu Ala Val 1790 1795 1800Leu
Gly Ala Lys Thr Lys Ala Thr Ser Leu Val Asp Ala Val Asn 1805
1810 1815Ser Asp Ala Ala Lys Pro Ser Gln Pro
Ala Glu Gly Leu Lys Lys 1820 1825
1830Pro Val Leu Pro Ala Thr Pro Lys Pro His Pro Ala Lys Pro Ser
1835 1840 1845Gly Thr Pro Ile Ser Pro
Ala Pro Val Pro Leu Ser Thr Leu Pro 1850 1855
1860Ser Ala Ser Ser Ala Leu Ala Gly Asp Gln Pro Ser Ser Thr
Ala 1865 1870 1875Phe Ile Pro Leu Ile
Ser Thr Arg Val Ser Leu Arg Lys Thr Arg 1880 1885
1890Gln Pro Pro Glu Arg Ala Ser Gly Ala Ile Thr Lys Gly
Val Val 1895 1900 1905Leu Asp Ser Thr
Glu Ala Leu Cys Leu Ala Ile Ser Gly Asn Ser 1910
1915 1920Glu Gln Met Ala Ser His Ser Ala Val Leu Glu
Ala Gly Lys Asn 1925 1930 1935Leu Tyr
Thr Phe Cys Val Ser Tyr Val Asp Ser Ile Gln Gln Met 1940
1945 1950Arg Asn Lys Phe Ala Phe Arg Glu Ala Ile
Asn Lys Leu Glu Asn 1955 1960 1965Asn
Leu Arg Glu Leu Gln Ile Cys Pro Ala Ser Ala Gly Ser Gly 1970
1975 1980Pro Ala Ala Thr Gln Asp Phe Ser Lys
Leu Leu Ser Ser Val Lys 1985 1990
1995Glu Ile Ser Asp Ile Val Gln Arg 2000
200581530PRTHomo sapiens 8Met Val Asp Pro Val Gly Phe Ala Glu Ala Trp Lys
Ala Gln Phe Pro1 5 10
15Asp Ser Glu Pro Pro Arg Met Glu Leu Arg Ser Val Gly Asp Ile Glu
20 25 30Gln Glu Leu Glu Arg Cys Lys
Ala Ser Ile Arg Arg Leu Glu Gln Glu 35 40
45Val Asn Gln Glu Arg Phe Arg Met Ile Tyr Leu Gln Thr Leu Leu
Ala 50 55 60Lys Glu Lys Lys Ser Tyr
Asp Arg Gln Arg Trp Gly Phe Arg Arg Ala65 70
75 80Ala Gln Ala Pro Asp Gly Ala Ser Glu Pro Arg
Ala Ser Ala Ser Arg 85 90
95Pro Gln Pro Ala Pro Ala Asp Gly Ala Asp Pro Pro Pro Ala Glu Glu
100 105 110Pro Glu Ala Arg Pro Asp
Gly Glu Gly Ser Pro Gly Lys Ala Arg Pro 115 120
125Gly Thr Ala Arg Arg Pro Gly Ala Ala Ala Ser Gly Glu Arg
Asp Asp 130 135 140Arg Gly Pro Pro Ala
Ser Val Ala Ala Leu Arg Ser Asn Phe Glu Arg145 150
155 160Ile Arg Lys Gly His Gly Gln Pro Gly Ala
Asp Ala Glu Lys Pro Phe 165 170
175Tyr Val Asn Val Glu Phe His His Glu Arg Gly Leu Val Lys Val Asn
180 185 190Asp Lys Glu Val Ser
Asp Arg Ile Ser Ser Leu Gly Ser Gln Ala Met 195
200 205Gln Met Glu Arg Lys Lys Ser Gln His Gly Ala Gly
Ser Ser Val Gly 210 215 220Asp Ala Ser
Arg Pro Pro Tyr Arg Gly Arg Ser Ser Glu Ser Ser Cys225
230 235 240Gly Val Asp Gly Asp Tyr Glu
Asp Ala Glu Leu Asn Pro Arg Phe Leu 245
250 255Lys Asp Asn Leu Ile Asp Ala Asn Gly Gly Ser Arg
Pro Pro Trp Pro 260 265 270Pro
Leu Glu Tyr Gln Pro Tyr Gln Ser Ile Tyr Val Gly Gly Ile Met 275
280 285Glu Gly Glu Gly Lys Gly Pro Leu Leu
Arg Ser Gln Ser Thr Ser Glu 290 295
300Gln Glu Lys Arg Leu Thr Trp Pro Arg Arg Ser Tyr Ser Pro Arg Ser305
310 315 320Phe Glu Asp Cys
Gly Gly Gly Tyr Thr Pro Asp Cys Ser Ser Asn Glu 325
330 335Asn Leu Thr Ser Ser Glu Glu Asp Phe Ser
Ser Gly Gln Ser Ser Arg 340 345
350Val Ser Pro Ser Pro Thr Thr Tyr Arg Met Phe Arg Asp Lys Ser Arg
355 360 365Ser Pro Ser Gln Asn Ser Gln
Gln Ser Phe Asp Ser Ser Ser Pro Pro 370 375
380Thr Pro Gln Cys His Lys Arg His Arg His Cys Pro Val Val Val
Ser385 390 395 400Glu Ala
Thr Ile Val Gly Val Arg Lys Thr Gly Gln Ile Trp Pro Asn
405 410 415Asp Asp Glu Gly Ala Phe His
Gly Asp Ala Glu Ala Leu Gln Arg Pro 420 425
430Val Ala Ser Asp Phe Glu Pro Gln Gly Leu Ser Glu Ala Ala
Arg Trp 435 440 445Asn Ser Lys Glu
Asn Leu Leu Ala Gly Pro Ser Glu Asn Asp Pro Asn 450
455 460Leu Phe Val Ala Leu Tyr Asp Phe Val Ala Ser Gly
Asp Asn Thr Leu465 470 475
480Ser Ile Thr Lys Gly Glu Lys Leu Arg Val Leu Gly Tyr Asn His Asn
485 490 495Gly Glu Trp Cys Glu
Ala Gln Thr Lys Asn Gly Gln Gly Trp Val Pro 500
505 510Ser Asn Tyr Ile Thr Pro Val Asn Ser Leu Glu Lys
His Ser Trp Tyr 515 520 525His Gly
Pro Val Ser Arg Asn Ala Ala Glu Tyr Pro Leu Ser Ser Gly 530
535 540Ile Asn Gly Ser Phe Leu Val Arg Glu Ser Glu
Ser Ser Pro Ser Gln545 550 555
560Arg Ser Ile Ser Leu Arg Tyr Glu Gly Arg Val Tyr His Tyr Arg Ile
565 570 575Asn Thr Ala Ser
Asp Gly Lys Leu Tyr Val Ser Ser Glu Ser Arg Phe 580
585 590Asn Thr Leu Ala Glu Leu Val His His His Ser
Thr Val Ala Asp Gly 595 600 605Leu
Ile Thr Thr Leu His Tyr Pro Ala Pro Lys Arg Asn Lys Pro Thr 610
615 620Val Tyr Gly Val Ser Pro Asn Tyr Asp Lys
Trp Glu Met Glu Arg Thr625 630 635
640Asp Ile Thr Met Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Glu
Val 645 650 655Tyr Glu Gly
Val Trp Lys Lys Tyr Ser Leu Thr Val Ala Val Lys Thr 660
665 670Leu Lys Glu Asp Thr Met Glu Val Glu Glu
Phe Leu Lys Glu Ala Ala 675 680
685Val Met Lys Glu Ile Lys His Pro Asn Leu Val Gln Leu Leu Gly Val 690
695 700Cys Thr Arg Glu Pro Pro Phe Tyr
Ile Ile Ile Glu Phe Met Thr Tyr705 710
715 720Gly Asn Leu Leu Asp Tyr Leu Arg Glu Cys Asn Arg
Gln Glu Val Asn 725 730
735Ala Val Val Leu Leu Tyr Met Ala Thr Gln Ile Ser Ser Ala Met Glu
740 745 750Tyr Leu Glu Lys Lys Asn
Phe Ile His Arg Asp Leu Ala Ala Arg Asn 755 760
765Cys Leu Val Gly Glu Asn His Leu Val Lys Val Ala Asp Phe
Gly Leu 770 775 780Ser Arg Leu Met Thr
Gly Asp Thr Tyr Thr Ala His Ala Gly Ala Lys785 790
795 800Phe Pro Ile Lys Trp Thr Ala Pro Glu Ser
Leu Ala Tyr Asn Lys Phe 805 810
815Ser Ile Lys Ser Asp Val Trp Ala Phe Gly Val Leu Leu Trp Glu Ile
820 825 830Ala Thr Tyr Gly Met
Ser Pro Tyr Pro Gly Ile Asp Arg Ser Gln Val 835
840 845Tyr Glu Leu Leu Glu Lys Asp Tyr Arg Met Lys Arg
Pro Glu Gly Cys 850 855 860Pro Glu Lys
Val Tyr Glu Leu Met Arg Ala Cys Trp Gln Trp Asn Pro865
870 875 880Ser Asp Arg Pro Ser Phe Ala
Glu Ile His Gln Ala Phe Glu Thr Met 885
890 895Phe Gln Glu Ser Ser Ile Ser Asp Glu Val Glu Lys
Glu Leu Gly Lys 900 905 910Gln
Gly Val Arg Gly Ala Val Thr Thr Leu Leu Gln Ala Pro Glu Leu 915
920 925Pro Thr Lys Thr Arg Thr Ser Arg Arg
Ala Ala Glu His Arg Asp Thr 930 935
940Thr Asp Val Pro Glu Met Pro His Ser Lys Gly Gln Gly Glu Ser Asp945
950 955 960Pro Leu Asp His
Glu Pro Ala Val Ser Pro Leu Leu Pro Arg Lys Glu 965
970 975Arg Gly Pro Pro Glu Gly Gly Leu Asn Glu
Asp Glu Arg Leu Leu Pro 980 985
990Lys Asp Lys Lys Thr Asn Leu Phe Ser Ala Leu Ile Lys Lys Lys Lys
995 1000 1005Lys Thr Ala Pro Thr Pro
Pro Lys Arg Ser Ser Ser Phe Arg Glu 1010 1015
1020Met Asp Gly Gln Pro Glu Arg Arg Gly Ala Gly Glu Glu Glu
Gly 1025 1030 1035Arg Asp Ile Ser Asn
Gly Ala Leu Ala Phe Thr Pro Leu Asp Thr 1040 1045
1050Ala Asp Pro Ala Lys Ser Pro Lys Pro Ser Asn Gly Ala
Gly Val 1055 1060 1065Pro Asn Gly Ala
Leu Arg Glu Ser Gly Gly Ser Gly Phe Arg Ser 1070
1075 1080Pro His Leu Trp Lys Lys Ser Ser Thr Leu Thr
Ser Ser Arg Leu 1085 1090 1095Ala Thr
Gly Glu Glu Glu Gly Gly Gly Ser Ser Ser Lys Arg Phe 1100
1105 1110Leu Arg Ser Cys Ser Val Ser Cys Val Pro
His Gly Ala Lys Asp 1115 1120 1125Thr
Glu Trp Arg Ser Val Thr Leu Pro Arg Asp Leu Gln Ser Thr 1130
1135 1140Gly Arg Gln Phe Asp Ser Ser Thr Phe
Gly Gly His Lys Ser Glu 1145 1150
1155Lys Pro Ala Leu Pro Arg Lys Arg Ala Gly Glu Asn Arg Ser Asp
1160 1165 1170Gln Val Thr Arg Gly Thr
Val Thr Pro Pro Pro Arg Leu Val Lys 1175 1180
1185Lys Asn Glu Glu Ala Ala Asp Glu Val Phe Lys Asp Ile Met
Glu 1190 1195 1200Ser Ser Pro Gly Ser
Ser Pro Pro Asn Leu Thr Pro Lys Pro Leu 1205 1210
1215Arg Arg Gln Val Thr Val Ala Pro Ala Ser Gly Leu Pro
His Lys 1220 1225 1230Glu Glu Ala Trp
Lys Gly Ser Ala Leu Gly Thr Pro Ala Ala Ala 1235
1240 1245Glu Pro Val Thr Pro Thr Ser Lys Ala Gly Ser
Gly Ala Pro Arg 1250 1255 1260Gly Thr
Ser Lys Gly Pro Ala Glu Glu Ser Arg Val Arg Arg His 1265
1270 1275Lys His Ser Ser Glu Ser Pro Gly Arg Asp
Lys Gly Lys Leu Ser 1280 1285 1290Lys
Leu Lys Pro Ala Pro Pro Pro Pro Pro Ala Ala Ser Ala Gly 1295
1300 1305Lys Ala Gly Gly Lys Pro Ser Gln Arg
Pro Gly Gln Glu Ala Ala 1310 1315
1320Gly Glu Ala Val Leu Gly Ala Lys Thr Lys Ala Thr Ser Leu Val
1325 1330 1335Asp Ala Val Asn Ser Asp
Ala Ala Lys Pro Ser Gln Pro Ala Glu 1340 1345
1350Gly Leu Lys Lys Pro Val Leu Pro Ala Thr Pro Lys Pro His
Pro 1355 1360 1365Ala Lys Pro Ser Gly
Thr Pro Ile Ser Pro Ala Pro Val Pro Leu 1370 1375
1380Ser Thr Leu Pro Ser Ala Ser Ser Ala Leu Ala Gly Asp
Gln Pro 1385 1390 1395Ser Ser Thr Ala
Phe Ile Pro Leu Ile Ser Thr Arg Val Ser Leu 1400
1405 1410Arg Lys Thr Arg Gln Pro Pro Glu Arg Ala Ser
Gly Ala Ile Thr 1415 1420 1425Lys Gly
Val Val Leu Asp Ser Thr Glu Ala Leu Cys Leu Ala Ile 1430
1435 1440Ser Gly Asn Ser Glu Gln Met Ala Ser His
Ser Ala Val Leu Glu 1445 1450 1455Ala
Gly Lys Asn Leu Tyr Thr Phe Cys Val Ser Tyr Val Asp Ser 1460
1465 1470Ile Gln Gln Met Arg Asn Lys Phe Ala
Phe Arg Glu Ala Ile Asn 1475 1480
1485Lys Leu Glu Asn Asn Leu Arg Glu Leu Gln Ile Cys Pro Ala Ser
1490 1495 1500Ala Gly Ser Gly Pro Ala
Ala Thr Gln Asp Phe Ser Lys Leu Leu 1505 1510
1515Ser Ser Val Lys Glu Ile Ser Asp Ile Val Gln Arg 1520
1525 15309676PRTHomo sapiens 9Leu Gly Tyr
Trp Lys Ile Lys Gly Leu Val Gln Pro Thr Arg Leu Leu1 5
10 15Leu Glu Tyr Leu Glu Glu Lys Tyr Glu
Glu His Leu Tyr Glu Arg Asp 20 25
30Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu Phe
35 40 45Pro Asn Leu Pro Tyr Tyr Ile
Asp Gly Asp Val Lys Leu Thr Gln Ser 50 55
60Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn Met Leu Gly Gly65
70 75 80Cys Pro Lys Glu
Arg Ala Glu Ile Ser Met Leu Glu Gly Ala Val Asp 85
90 95Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr
Ser Lys Asp Phe Glu Thr 100 105
110Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe
115 120 125Glu Asp Arg Leu Cys His Lys
Thr Tyr Leu Asn Gly Asp His Val Thr 130 135
140His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr
Met145 150 155 160Asp Pro
Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys Lys
165 170 175Arg Ile Glu Ala Ile Pro Gln
Ile Asp Lys Tyr Leu Lys Ser Ser Lys 180 185
190Tyr Ile Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly Gly
Gly Asp 195 200 205His Pro Pro Lys
Ser Asp Leu Val Pro Arg His Asn Gln Thr Ser Leu 210
215 220Tyr Lys Lys Ala Gly Ser Ala Ala Ala Val Leu Glu
Glu Asn Leu Tyr225 230 235
240Phe Gln Gly Thr Tyr Lys Tyr Leu Gln Lys Pro Met Tyr Glu Val Gln
245 250 255Trp Lys Val Val Glu
Glu Ile Asn Gly Asn Asn Tyr Val Tyr Ile Asp 260
265 270Pro Thr Gln Leu Pro Tyr Asp His Lys Trp Glu Phe
Pro Arg Asn Arg 275 280 285Leu Ser
Phe Gly Lys Thr Leu Gly Ala Gly Ala Phe Gly Lys Val Val 290
295 300Glu Ala Thr Ala Tyr Gly Leu Ile Lys Ser Asp
Ala Ala Met Thr Val305 310 315
320Ala Val Lys Met Leu Lys Pro Ser Ala His Leu Thr Glu Arg Glu Ala
325 330 335Leu Met Ser Glu
Leu Lys Val Leu Ser Tyr Leu Gly Asn His Met Asn 340
345 350Ile Val Asn Leu Leu Gly Ala Cys Thr Ile Gly
Gly Pro Thr Leu Val 355 360 365Ile
Thr Glu Tyr Cys Cys Tyr Gly Asp Leu Leu Asn Phe Leu Arg Arg 370
375 380Lys Arg Asp Ser Phe Ile Cys Ser Lys Gln
Glu Asp His Ala Glu Ala385 390 395
400Ala Leu Tyr Lys Asn Leu Leu His Ser Lys Glu Ser Ser Cys Ser
Asp 405 410 415Ser Thr Asn
Glu Tyr Met Asp Met Lys Pro Gly Val Ser Tyr Val Val 420
425 430Pro Thr Lys Ala Asp Lys Arg Arg Ser Val
Arg Ile Gly Ser Tyr Ile 435 440
445Glu Arg Asp Val Thr Pro Ala Ile Met Glu Asp Asp Glu Leu Ala Leu 450
455 460Asp Leu Glu Asp Leu Leu Ser Phe
Ser Tyr Gln Val Ala Lys Gly Met465 470
475 480Ala Phe Leu Ala Ser Lys Asn Cys Ile His Arg Asp
Leu Ala Ala Arg 485 490
495Asn Ile Leu Leu Thr His Gly Arg Ile Thr Lys Ile Cys Asp Phe Gly
500 505 510Leu Ala Arg Asp Ile Lys
Asn Asp Ser Asn Tyr Val Val Lys Gly Asn 515 520
525Ala Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ser Ile Phe
Asn Cys 530 535 540Val Tyr Thr Phe Glu
Ser Asp Val Trp Ser Tyr Gly Ile Phe Leu Trp545 550
555 560Glu Leu Phe Ser Leu Gly Ser Ser Pro Tyr
Pro Gly Met Pro Val Asp 565 570
575Ser Lys Phe Tyr Lys Met Ile Lys Glu Gly Phe Arg Met Leu Ser Pro
580 585 590Glu His Ala Pro Ala
Glu Met Tyr Asp Ile Met Lys Thr Cys Trp Asp 595
600 605Ala Asp Pro Leu Lys Arg Pro Thr Phe Lys Gln Ile
Val Gln Leu Ile 610 615 620Glu Lys Gln
Ile Ser Glu Ser Thr Asn His Ile Tyr Ser Asn Leu Ala625
630 635 640Asn Cys Ser Pro Asn Arg Gln
Lys Pro Val Val Asp His Ser Val Arg 645
650 655Ile Asn Ser Val Gly Ser Thr Ala Ser Ser Ser Gln
Pro Leu Leu Val 660 665 670His
Asp Asp Val 67510474PRTHomo sapiens 10Met Ser Tyr Tyr His His His
His His His Asp Tyr Asp Ile Pro Thr1 5 10
15Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Leu Val Pro
Arg Gly Ser 20 25 30Pro Trp
Ile Pro Phe Thr Met Lys Lys Arg Lys Gln Ile Lys Asp Leu 35
40 45Gly Ser Glu Leu Val Arg Tyr Asp Ala Arg
Val His Thr Pro His Leu 50 55 60Asp
Arg Leu Val Ser Ala Arg Ser Val Ser Pro Thr Thr Glu Met Val65
70 75 80Ser Asn Glu Ser Val Asp
Tyr Arg Ala Thr Phe Pro Glu Asp Gln Phe 85
90 95Pro Asn Ser Ser Gln Asn Gly Ser Cys Arg Gln Val
Gln Tyr Pro Leu 100 105 110Thr
Asp Met Ser Pro Ile Leu Thr Ser Gly Asp Ser Asp Ile Ser Ser 115
120 125Pro Leu Leu Gln Asn Thr Val His Ile
Asp Leu Ser Ala Leu Asn Pro 130 135
140Glu Leu Val Gln Ala Val Gln His Val Val Ile Gly Pro Ser Ser Leu145
150 155 160Ile Val His Phe
Asn Glu Val Ile Gly Arg Gly His Phe Gly Cys Val 165
170 175Tyr His Gly Thr Leu Leu Asp Asn Asp Gly
Lys Lys Ile His Cys Ala 180 185
190Val Lys Ser Leu Asn Arg Ile Thr Asp Ile Gly Glu Val Ser Gln Phe
195 200 205Leu Thr Glu Gly Ile Ile Met
Lys Asp Phe Ser His Pro Asn Val Leu 210 215
220Ser Leu Leu Gly Ile Cys Leu Arg Ser Glu Gly Ser Pro Leu Val
Val225 230 235 240Leu Pro
Tyr Met Lys His Gly Asp Leu Arg Asn Phe Ile Arg Asn Glu
245 250 255Thr His Asn Pro Thr Val Lys
Asp Leu Ile Gly Phe Gly Leu Gln Val 260 265
270Ala Lys Gly Met Lys Tyr Leu Ala Ser Lys Lys Phe Val His
Arg Asp 275 280 285Leu Ala Ala Arg
Asn Cys Met Leu Asp Glu Lys Phe Thr Val Lys Val 290
295 300Ala Asp Phe Gly Leu Ala Arg Asp Met Tyr Asp Lys
Glu Tyr Tyr Ser305 310 315
320Val His Asn Lys Thr Gly Ala Lys Leu Pro Val Lys Trp Met Ala Leu
325 330 335Glu Ser Leu Gln Thr
Gln Lys Phe Thr Thr Lys Ser Asp Val Trp Ser 340
345 350Phe Gly Val Leu Leu Trp Glu Leu Met Thr Arg Gly
Ala Pro Pro Tyr 355 360 365Pro Asp
Val Asn Thr Phe Asp Ile Thr Val Tyr Leu Leu Gln Gly Arg 370
375 380Arg Leu Leu Gln Pro Glu Tyr Cys Pro Asp Pro
Leu Tyr Glu Val Met385 390 395
400Leu Lys Cys Trp His Pro Lys Ala Glu Met Arg Pro Ser Phe Ser Glu
405 410 415Leu Val Ser Arg
Ile Ser Ala Ile Phe Ser Thr Phe Ile Gly Glu His 420
425 430Tyr Val His Val Asn Ala Thr Tyr Val Asn Val
Lys Cys Val Ala Pro 435 440 445Tyr
Pro Ser Leu Leu Ser Ser Glu Asp Asn Ala Asp Asp Glu Val Asp 450
455 460Thr Arg Pro Ala Ser Phe Trp Glu Thr
Ser465 470
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