US007338957B2

(12) United States Patent (10) Patent No.: US 7,338,957 B2 Ding et al. (45) Date of Patent: Mar. 4, 2008

(54) COMPOUNDS AND COMPOSITIONS AS (58) Field of Classification Search ...... 544/280; KNASE INHIBITORS 51.4/265.1 See application file for complete search history. (75) Inventors: Qiang Ding, San Diego, CA (US); (56) References Cited Nathanael Schiander Gray, San Diego, CA (US); Bing Li, San Diego, OTHER PUBLICATIONS CA (US); Yi Liu, San Diego, CA (US); West, Anthony R., Solid State Chemistry and its Applications, Tetsuo Uno, San Diego, CA (US) Wiley, New York, 1988, pp. 358 & 365.* Vippagunta et al., Advanced Drug Delivery Reviews 48: 3-26, (73) Assignee: IRM LLC, Hamilton (BM) 2001.* Cecil Textbook of Medicine, edited by Bennet, J.C., and Plum F. *) Notice: Subject to anyy disclaimer, the term of this 20th edition,vol. 1, 1004-1010, 1996.* Mass, R. D., Int. J. Radiation Oncology Bio. Phys.vol. 58(3): patent is extended or adjusted under 35 932-940, 2004.* U.S.C. 154(b) by 393 days. Fabbro et al. Pharmacology & therapeutics 93, 79-98, 2002.* Vlahovic G. et al. Activation of Tyrosine Kinases in Cancer, (21) Appl. No.: 10/927,992 Oncologist. 2003, vol. 8, No. 6, pp. 531-538. * cited by examiner (22) Filed: Aug. 26, 2004 Primary Examiner Venkataraman Balasubram (74) Attorney, Agent, or Firm Emily Tongco Wu: Institute of the Novartis Research Foundation (65) Prior Publication Data US 2005/O159391 A1 Jul. 21, 2005 (57) ABSTRACT The invention provides a novel class of compounds, phar Related U.S. Application Data maceutical compositions comprising Such compounds and (60) Provisional application No. 60/498.532, filed on Aug. methods of using such compounds to treat or prevent 28, 2003. diseases or disorders associated with abnormal or deregu lated kinase activity, particularly diseases or disorders that (51) Int. Cl. involve abnormal activation of the Abl, BCR-Abl, Bmx, CO7D 487/04 (2006.01) CSK, TrkB, FGFR3, Fes, , B-RAF C-RAF, MKK6, A 6LX 3/59 (2006.01) SAPK2C. and SAPK2|B kinases. A6IP35/00 (2006.01) (52) U.S. Cl...... 514/265.1: 544/280 8 Claims, No Drawings US 7,338,957 B2 1. 2 COMPOUNDS AND COMPOSITIONS AS hydrogen and Calkyl, R, is selected from Coaryl, PROTEIN KNASE INHIBITORS Csoheteroaryl, C-cycloalkyl and C-sheterocycloalkyl, or R and R, together with the nitrogen to which both R and CROSS-REFERENCE TO RELATED R, are attached form C-sheterocycloalkyl or Csohet APPLICATIONS eroaryl; wherein any aryl, heteroaryl, cycloalkyl or hetero cycloalkyl of R, or of the combination of R and R, can be This application claims the benefit of priority under 35 optionally substituted with 1 to 3 radicals selected from halo, U.S.C. S 119(e) to U.S. Provisional Patent Application No. cyano, nitro, Calkyl, C2-alkenyl, C2-galkynyl, halo-Sub 60/498.532, filed Aug. 28, 2003. The disclosure of the stituted-Calkyl, Calkoxy, halo-substituted-Calkoxy, priority application is incorporated herein by reference in its 10 —XNRRs, XC(O)NR'Rs, XC(O)NRXORs, XS entirety and for all purposes. (O)NRRs —XS(O)o-Rs, —XNRS(O)o-Rs. —XNRC(O)Rs, XNRSRs, XP(O)NR'Rs, XCR BACKGROUND OF THE INVENTION (ORs)Rs —XOC(O)Rs —XORs and —XOR; wherein X is a bond or C-alkylene, Rs is independently selected from 1. Field of the Invention 15 hydrogen and Calkyl, R is selected from Coaryl and The invention provides a novel class of compounds, Cs-oheteroaryl; pharmaceutical compositions comprising such compounds and methods of using Such compounds to treat or prevent R is selected from hydrogen and Calkyl; diseases or disorders associated with abnormal or deregu R is selected from hydrogen, hydroxy, Calkyl, Calk lated kinase activity, particularly diseases or disorders that enyl, C2-alkynyl, Calkoxy, mercapto, halo, nitro and involve abnormal activation of the Abl, BCR-Abl, Bmx, cyano; CSK, TrkB, FGFR3, Fes, Lck, B-RAF, C-RAF, MKK6, n is 0, 1 or 2: SAPK2C. and SAPK2B kinases. R is selected from hydrogen, halo, hydroxy, cyano, nitro, 2. Background Calkyl, halo-substituted-Calkyl, Calkoxy, halo-Sub The protein kinases represent a large family of , 25 stituted-Calkoxy and —XRo, wherein X is a bond or which play a central role in the regulation of a wide variety Coalkylene, Rio is selected from Co-oaryl, Csohet of cellular processes and maintaining control over cellular eroaryl, C-acycloalkyl and C-sheterocycloalkyl, wherein function. A partial, non-limiting, list of these kinases any aryl, heteroaryl, cycloalkyl or heterocycloalkyl of Rio is include: receptor tyrosine kinases Such as the nerve growth optionally substituted with a radical selected from halo, factor receptor, trkB, and the fibroblast growth factor recep 30 hydroxy, cyano, nitro, Calkyl, halo-substituted-Calkyl, tor, FGFR3; non-receptor tyrosine kinases such Abl and the Calkoxy and halo-substituted-Calkoxy; fusion kinase BCR-Abl, Lck, Csk, Fes, Bmx and c-src; and m is 1, 2 or 3; and wherein the phenyl rings A and B can serine/threonine kinases such as c-RAF, Sgk, MAP kinases independently have up to four —C= groups replaced by (e.g., MKK4, MKK6, etc.), SAPK2C. and SAPK2B. Aber —N=; and the N-oxide derivatives, prodrug derivatives, rant kinase activity has been observed in many disease states 35 protected derivatives, individual isomers and mixture of including benign and malignant proliferative disorders as isomers thereof, and the pharmaceutically acceptable salts well as diseases resulting from inappropriate activation of and Solvates (e.g. hydrates) of Such compounds. the immune and nervous systems. In a second aspect, the present invention provides a The novel compounds of this invention inhibit the activity pharmaceutical composition which contains a compound of of one or more protein kinases and are, therefore, expected 40 Formula I or a N-oxide derivative, individual isomers and to be useful in the treatment of kinase-associated diseases. mixture of isomers thereof, or a pharmaceutically acceptable salt thereof, in admixture with one or more suitable excipi SUMMARY OF THE INVENTION entS. In a third aspect, the present invention provides a method In one aspect, the present invention provides compounds 45 of treating a disease in an animal, including human, in which of Formula I: inhibition of kinase activity, particularly the Abl, BCR-Abl, Bmx, CSK, TrkB, FGFR3, Fes, Lck, B-RAF, C-RAF, MKK6. SAPK2C. and/or SAPK2B activity, can prevent, R inhibit or ameliorate the pathology and/or symptomology of 50 the diseases, which method comprises administering to the C N (R3)n animal a therapeutically effective amount of a compound of e Formula I or a N-oxide derivative, individual isomers and mixture of isomers thereof, or a pharmaceutically acceptable S.-->{ X), N salt thereof. Y, 55 /n 1s B --R) In a fourth aspect, the present invention provides the use O 21 of a compound of Formula I in the manufacture of a R medicament for treating a disease in an animal in which kinase activity, particularly the Abl, BCR-Abl, Bmx, CSK, in which: 60 TrkB, FGFR3, Fes, Lck, B-RAF, C-RAF, MKK6, SAPK2O. W is selected from CH and N: and/or SAPK2B activity, contributes to the pathology and/or Y is selected from C, S and S(O); symptomology of the disease. Z is a divalent radical selected from Y(O)NRs and In a fifth aspect, the present invention provides a process - NRY(O) ; wherein Y is selected from C, S and S(O); for preparing compounds of Formula I and the N-oxide and Rs is selected from hydrogen and C-alkyl: 65 derivatives, prodrug derivatives, protected derivatives, indi R is selected from hydrogen, halo, hydroxy Calkyl, vidual isomers and mixture of isomers thereof, and the Calkoxy and —NRR, wherein R is selected from pharmaceutically acceptable salts thereof. US 7,338,957 B2 3 4 DETAILED DESCRIPTION OF THE Coheteroaryl; or R and R, together with the nitrogen to INVENTION which both R and R, are attached form C-sheterocy cloalkyl; wherein any aryl, heteroaryl or heterocycloalkyl of Definitions R, or of the combination of R and R, can be optionally “Alkyl as a group and as a structural element of other substituted with 1 to 3 radicals selected from halo, cyano, groups, for example halo-substituted-alkyl and alkoxy, can Calkyl, Calkenyl, halo-substituted-Calkyl, halo-Sub be either straight-chained or branched. C-alkoxy includes, stituted-Calkoxy, XNRRs, XC(O)NR'Rs, XC(O) methoxy, ethoxy, and the like. Halo-substituted alkyl NRXORs —XS(O)NRRs —XSRs —XNRS(O)Rs. includes trifluoromethyl, pentafluoroethyl, and the like. —XNRC(O)Rs —XOC(O)Rs, XORs and XORs: Unless otherwise defined, “alkyl can be optionally inter 10 wherein X is a bond or Calkylene, Rs is hydrogen or rupted by —O— or optionally substituted by —OH. C-calkyl, Ro is Co-oaryl; "Aryl means a monocyclic or fused bicyclic aromatic In a further embodiment, R is selected from hydrogen ring assembly containing six to ten ring carbon atoms. For and Calkyl; R is Calkyl; n is 1; and R is selected from example, aryl may be phenyl or naphthyl, preferably phenyl. halo, halo-substituted-Calkyl and —XR, wherein X is a 15 bond or Calkylene, Ro is selected from Csoheteroaryl "Arylene' means a divalent radical derived from an aryl and C-sheterocycloalkyl, wherein any heteroaryl or hetero group. “Heteroaryl is as defined for aryl where one or more cycloalkyl of Rio is optionally Substituted with Calkyl; of the ring members are a heteroatom. For example het and m is 1, 2 or 3. eroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl, In another embodiment, Z is a divalent radical selected quinolinyl, benzofuranyl, benzopyranyl, benzothiopyranyl. from –C(O)NH- and - NHC(O)-. benzo. 1.3dioxole, imidazolyl, benzo-imidazolyl, pyrimidi In another embodiment, R is selected from hydrogen, nyl, furanyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl pyra halo, hydroxy and —NHR,; wherein R, is selected from Zolyl, thienyl, etc. phenyl and pyridinyl: or R and R, together with the nitro “Cycloalkyl means a saturated or partially unsaturated, gen to which both R and R7 are attached form morpholino; monocyclic, fused bicyclic or bridged polycyclic ring 25 wherein any aryl, heteroaryl or heterocycloalkyl of R, or of assembly containing the number of ring atoms indicated. For the combination of R and R, can be optionally substituted example, Cocycloalkyl includes cyclopropyl, cyclobutyl, with 1 to 3 radicals selected from halo, cyano, trifluorom cyclopentyl, cyclohexyl, etc. “Heterocycloalkyl means ethyl, trifluoromethoxy, dimethylamino, amino, aminocar cycloalkyl, as defined in this application, provided that one bonyl, methyl-aminocarbonyl, aminosulfonyl, 1-hydroxy or more of the ring carbons indicated, are replaced by a 30 ethyl, hydroxymethyl, acetoxy-methyl, methyl-sulfanyl. moiety selected from —O— —N=, —NR , —C(O)—, phenoxy, methyl-carboxy-methyl, butyl-amino-Sulfonyl, —S —S(O)— or —S(O) , wherein R is hydrogen, methyl, isopropenyl, methoxy-ethyl-amino-carbonyl, 1-hy Calkyl or a nitrogen protecting group. For example, droxy-1-methyl-ethyl, methyl-Sulfonyl-amino and methyl C-sheterocycloalkyl as used in this application to describe carbonyl-amino; and R is hydrogen or methyl. compounds of the invention includes morpholino, pyrrolidi 35 In a further embodiment, R is selected from halo, trif nyl, piperazinyl, piperidinyl, piperidinylone, 1,4-dioxa-8- luoromethyl and —XRo: wherein X is a bond or methylene: aza-spiro4.5dec-8-yl, etc. Ro is selected from imidazolyl, piperazinyl and mor "Halogen' (or halo) preferably represents chloro or pholino; wherein any heteroaryl or heterocycloalkyl is fluoro, but may also be bromo or iodo. optionally substituted with methyl. “Treat”, “treating and “treatment” refer to a method of 40 Preferred compounds of the invention are selected from: alleviating or abating a disease and/or its attendant symp 4-(3-Trifluoromethyl-phenylamino)-pyrrolo2,3-dipyrimi tOmS. dine-7-carboxylic acid 3-(4-morpholin-4-yl-phenylcarbam oyl)-phenyl-amide: 4-(4-Trifluoromethyl-1-phenylamino)- DESCRIPTION OF THE PREFERRED pyrrolo[2,3-dipyrimidine-7-carboxylic acid {2-methyl-5-4- EMBODIMENTS 45 (4-methyl-piperazin-1-ylmethyl)-benzoylaminol-phenyl amide, 4-(3-Dimethylamino-phenylamino)-pyrrolo2,3-d The fusion protein BCR-Abl is a result of a reciprocal pyrimidine-7-carboxylic acid (2-methyl-5-3-(4-methyl translocation that fuses the Abl proto-oncogene with the Bcr imidazol-1-yl)-5-trifluoromethyl-benzoylaminol-phenyl . BCR-Abl is then capable of transforming B-cells amide; 4-(3-Carbamoyl-phenylamino)-pyrrolo2,3-d through the increase of mitogenic activity. This increase 50 pyrimidine-7-carboxylic acid (2-methyl-5-4-(4-methyl results in a reduction of sensitivity to apoptosis, as well as piperazin-1-ylmethyl)-benzoylaminol-phenyl-amide: 4-(3- altering the adhesion and homing of CML progenitor cells. Sulfamoyl-phenylamino)-pyrrolo2,3-dipyrimidine-7- The present invention provides compounds, compositions carboxylic acid (2-methyl-5-4-(4-methyl-piperazin-1- and methods for the treatment of kinase related disease, ylmethyl)-benzoylaminol-phenyl-amide: 4-3-(1- particularly the Abl, BCR-Abl, Bmx, CSK, TrkB, FGFR3, 55 Hydroxy-ethyl)-phenylamino-pyrrolo2,3-dipyrimidine-7- Fes, Lck, B-RAF, C-RAF, MKK6. SAPK2C. and/or SAPK2 B carboxylic acid (2-methyl-5-3-(4-methyl-imidazol-1-yl)-5- kinase related diseases. For example, leukemia and other trifluoromethyl-benzoylaminol-phenyl-amide: 4-(3- proliferation disorders related to BCR-Abl can be treated Carbamoyl-phenylamino)-pyrrolo2,3-dipyrimidine-7- through the inhibition of wild type and mutant forms of carboxylic acid (2-methyl-5-3-(4-methyl-imidazol-1-yl)-5- BCR-Ab1. 60 trifluoromethyl-benzoylaminol-phenyl-amide: 4-(3- In one embodiment, with reference to compounds of Sulfamoyl-phenylamino)-pyrrolo2,3-dipyrimidine-7- Formula I, W is N. Y is C; Z is a divalent radical selected carboxylic acid (2-methyl-5-3-(4-methyl-imidazol-1-yl)-5- from —C(O)NRs and NRC(O)—; wherein Rs is trifluoromethyl-benzoylaminol-phenyl-amide: 4-3-(1- selected from hydrogen and Calkyl; Hydroxy-ethyl)-phenylamino-pyrrolo2,3-dipyrimidine-7- In another embodiment, is selected from hydrogen, halo, 65 carboxylic acid 5-(3-fluoro-5-trifluoromethyl hydroxy and —NRR7; wherein R is selected from hydro benzoylamino)-2-methyl-phenyl-amide; 4-Hydroxy gen and Calkyl; R, is selected from Coaryl and pyrrolo[2,3-dipyrimidine-7-carboxylic acid {2-methyl-5-3-

US 7,338,957 B2 7 8 Abelson (i.e. Abl, c-Abl) is involved in the JNKs, along with other MAPKs, have been implicated in regulation of the cell cycle, in the cellular response to having a role in mediating cellular response to cancer, genotoxic stress, and in the transmission of information thrombin-induced platelet aggregation, immunodeficiency about the cellular environment through integrin signaling. disorders, autoimmune diseases, cell death, allergies, Overall, it appears that the Abl protein serves a complex role osteoporosis and heart disease. The therapeutic targets as a cellular module that integrates signals from various related to activation of the JNK pathway include chronic extracellular and intracellular sources and that influences myelogenous leukemia (CML), rheumatoid arthritis, decisions in regard to cell cycle and apoptosis. Abelson asthma, osteoarthritis, ischemia, cancer and neurodegenera tyrosine kinase includes Sub-types derivatives such as the tive diseases. As a result of the importance of JNK activation chimeric fusion (oncoprotein) BCR-Abl with deregulated 10 associated with liver disease or episodes of hepatic tyrosine kinase activity or the V-Abl, BCR-Abl is critical in ischemia, compounds of the invention may also be useful to the pathogenesis of 95% of chronic myelogenous leukemia treat various hepatic disorders. A role for JNK in cardiovas (CML) and 10% of acute lymphocytic leukemia. STI-571 cular disease such as myocardial infarction or congestive (Gleevec) is an inhibitor of the oncogenic BCR-Abl tyrosine heart failure has also been reported as it has been shown kinase and is used for the treatment of chronic myeloid 15 JNK mediates hypertrophic responses to various forms of leukemia (CML). However, some patients in the blast crisis cardiac stress. It has been demonstrated that the JNK cas stage of CML are resistant to STI-571 due to mutations in cade also plays a role in T-cell activation, including activa the BCR-Abl kinase. Over 22 mutations have been reported tion of the IL-2 promoter. Thus, inhibitors of JNK may have to date with the most common being G250E, E255V, T315I, therapeutic value in altering pathologic immune responses. F317L and M351.T. A role for JNK activation in various cancers has also been Compounds of the present invention inhibit kinase, established, suggesting the potential use of JNK inhibitors in especially V-abl kinase. The compounds of the present cancer. For example, constitutively activated JNK is asso invention also inhibit wild-type BCR-Abl kinase and muta ciated with HTLV-1 mediated tumorigenesis Oncogene tions of BCR-Abl kinase and are thus suitable for the 13:135-42 (1996). JNK may play a role in Kaposi's sar treatment of Bcr-abl-positive cancer and tumor diseases, 25 coma (KS). Other proliferative effects of other cytokines Such as leukemias (especially chronic myeloid leukemia and implicated in KS proliferation, such as vascular endothelial acute lymphoblastic leukemia, where especially apoptotic growth factor (VEGF), IL-6 and TNFC, may also be medi mechanisms of action are found), and also shows effects on ated by JNK. In addition, regulation of the c-jun gene in the Subgroup of leukemic stem cells as well as potential for p210 BCR-ABL transformed cells corresponds with activity the purification of these cells in vitro after removal of said 30 of JNK, suggesting a role for JNK inhibitors in the treatment cells (for example, bone marrow removal) and reimplanta for chronic myelogenous leukemia (CML) Blood 92:2450 tion of the cells once they have been cleared of cancer cells 60 (1998). (for example, reimplantation of purified bone marrow cells). Certain abnormal proliferative conditions are believed to The trk family of neurotrophin receptors (trkA, trkB, be associated with raf expression and are, therefore, believed trkC) promotes the survival, growth and differentiation of 35 to be responsive to inhibition of raf expression. Abnormally the neuronal and non-neuronal tissues. The TrkB protein is high levels of expression of the raf protein are also impli expressed in neuroendocrine-type cells in the Small intestine cated in transformation and abnormal cell proliferation. and colon, in the alpha cells of the pancreas, in the mono These abnormal proliferative conditions are also believed to cytes and macrophages of the lymph nodes and of the be responsive to inhibition of raf expression. For example, spleen, and in the granular layers of the epidermis (Shiba 40 expression of the c-raf protein is believed to play a role in yama and Koizumi, 1996). Expression of the TrkB protein abnormal cell proliferation since it has been reported that has been associated with an unfavorable progression of 60% of all lung carcinoma cell lines express unusually high Wilms tumors and of neuroblastomas. TkrB is, moreover, levels of c-raf mRNA and protein. Further examples of expressed in cancerous prostate cells but not in normal cells. abnormal proliferative conditions are hyper-proliferative The signaling pathway downstream of the trk receptors 45 disorders such as cancers, tumors, hyperplasia, pulmonary fibrosis, angiogenesis, psoriasis, atherosclerosis and Smooth involves the cascade of MAPK activation through the Shc, muscle cell proliferation in the blood vessels, such as activated Ras, ERK-1 and ERK-2 , and the PLC Stenosis or restenosis following angioplasty. The cellular gammal transduction pathway (Sugimoto et al., 2001). signaling pathway of which raf is a part has also been The Tec family kinase, Bmx, a non-receptor protein 50 implicated in inflammatory disorders characterized by T-cell tyrosine kinase, controls the proliferation of mammary epi proliferation (T-cell activation and growth). Such as tissue thelial cancer cells. graft rejection, endotoxin shock, and glomerular nephritis, Fibroblast 3 (FGFR3) was shown for example. to exert a negative regulatory effect on bone growth and an The stress activated protein kinases (SAPKs) area family inhibition of chondrocyte proliferation. Thanatophoric dys 55 of protein kinases that represent the penultimate step in plasia is caused by different mutations in fibroblast growth signal transduction pathways that result in activation of the factor receptor 3, and one mutation, TDII FGFR3, has a c-jun transcription factor and expression of genes regulated constitutive tyrosine kinase activity which activates the by c-jun. In particular, c-jun is involved in the transcription transcription factor Stat1, leading to expression of a cell of genes that encode proteins involved in the repair of DNA cycle inhibitor, growth arrest and abnormal bone develop 60 that is damaged due to genotoxic insults. Therefore, agents ment (Suet al., Nature, 1997, 386, 288-292). FGFR3 is also that inhibit SAPK activity in a cell prevent DNA repair and often expressed in multiple myeloma-type cancers. sensitize the cell to agents that induce DNA damage or Lck plays a role in T-cell signaling. Mice that lack the Lck inhibit DNA synthesis and induce apoptosis of a cell or that gene have a poor ability to develop thymocytes. The func inhibit cell proliferation. tion of Lck as a positive activator of T-cell signaling 65 Mitogen-activated protein kinases (MAPKs) are members Suggests that Lck inhibitors may be useful for treating of conserved signal transduction pathways that activate autoimmune disease such as rheumatoid arthritis. transcription factors, translation factors and other target US 7,338,957 B2 10 molecules in response to a variety of extracellular signals. calcium salt and/or polyethyleneglycol, for tablets also c) MAPKs are activated by phosphorylation at a dual phos binders, e.g., magnesium aluminum silicate, starch paste, phorylation motif having the sequence Thr-X-Tyr by mito gelatin, tragacanth, methylcellulose, Sodium carboxymeth gen-activated kinases (MKKS). In higher ylcellulose and or polyvinylpyrrolidone; if desired d) disin eukaryotes, the physiological role of MAPK signaling has tegrants, e.g., starches, agar, alginic acid or its sodium salt, been correlated with cellular events such as proliferation, or effervescent mixtures; and/or e) absorbents, colorants, oncogenesis, development and differentiation. Accordingly, flavors and Sweeteners. Injectable compositions can be the ability to regulate signal transduction via these pathways aqueous isotonic solutions or Suspensions, and Suppositories (particularly via MKK4 and MKK6) could lead to the can be prepared from fatty emulsions or Suspensions. The development of treatments and preventive therapies for 10 compositions may be sterilized and/or contain adjuvants, human diseases associated with MAPK signaling, such as Such as preserving, stabilizing, wetting or emulsifying inflammatory diseases, autoimmune diseases and cancer. agents, solution promoters, salts for regulating the osmotic Csk down-regulates kinase activity by phosphorylating a pressure and/or buffers. In addition, they may also contain single tyrosine residue in the C-terminus of the Src other therapeutically valuable substances. Suitable formula (Okada et al., J. Biol. Chem. 266:24249-24252, 1991; and 15 tions for transdermal applications include an effective Bergman et al., EMBO J. 11:2919-2924, 1992). Owing to amount of a compound of the present invention with a this premier regulatory function, Csk has direct effects on carrier. A carrier can include absorbable pharmacologically many biological functions including T cell activation, neu acceptable solvents to assist passage through the skin of the ronal development, cytoskeletal organization, and cell cycle host. For example, transdermal devices are in the form of a control. bandage comprising a backing member, a reservoir contain Examples of the diseases associated with activity of the ing the compound optionally with carriers, optionally a rate non- from the Fes family include, controlling barrier to deliver the compound to the skin of the but are not limited to, tumors of mesenchymal origin and host at a controlled and predetermined rate over a prolonged tumors of hematopoietic origin. period of time, and means to secure the device to the skin. In accordance with the foregoing, the present invention 25 Matrix transdermal formulations may also be used. Suitable further provides a method for preventing or treating any of formulations for topical application, e.g., to the skin and the diseases or disorders described above in a subject in need eyes, are preferably aqueous solutions, ointments, creams or of such treatment, which method comprises administering to gels well-known in the art. Such may contain solubilizers, said subject a therapeutically effective amount (See, stabilizers, tonicity enhancing agents, buffers and preserva “Administration and Pharmaceutical Compositions, infra) 30 tives. of a compound of Formula I or a pharmaceutically accept Compounds of the invention can be administered in able salt thereof. For any of the above uses, the required therapeutically effective amounts in combination with one or dosage will vary depending on the mode of administration, more therapeutic agents (pharmaceutical combinations). For the particular condition to be treated and the effect desired. example, synergistic effects can occur with other immuno 35 modulatory or anti-inflammatory Substances, for example Administration and Pharmaceutical Compositions when used in combination with cyclosporin, rapamycin, or In general, compounds of the invention will be adminis ascomycin, or immunosuppressant analogues thereof, for tered in therapeutically effective amounts via any of the example cyclosporin A (CSA), cyclosporin G, FK-506, rapa usual and acceptable modes known in the art, either singly mycin, or comparable compounds, corticosteroids, cyclo or in combination with one or more therapeutic agents. A 40 phosphamide, azathioprine, methotrexate, brequinar, therapeutically effective amount may vary widely depending leflunomide, mizoribine, mycophenolic acid, mycopheno on the severity of the disease, the age and relative health of late mofetil, 15-deoxyspergualin, immunosuppressant anti the Subject, the potency of the compound used and other bodies, especially monoclonal antibodies for leukocyte factors. In general, satisfactory results are indicated to be receptors, for example MHC, CD2, CD3, CD4, CD7. CD25, obtained systemically at daily dosages of from about 0.03 to 45 CD28, B7, CD45, CD58 or their ligands, or other immuno 2.5 mg/kg per body weight. An indicated daily dosage in the modulatory compounds, such as CTLA41g. Where the com larger mammal, e.g. humans, is in the range from about 0.5 pounds of the invention are administered in conjunction with mg to about 100 mg, conveniently administered, e.g. in other therapies, dosages of the co-administered compounds divided doses up to four times a day or in retard form. will of course vary depending on the type of co-drug Suitable unit dosage forms for oral administration comprise 50 employed, on the specific drug employed, on the condition from ca. 1 to 50 mg active ingredient. being treated and so forth. Compounds of the invention can be administered as The invention also provides for a pharmaceutical combi pharmaceutical compositions by any conventional route, in nations, e.g. a , comprising a) a first agent which is a particular enterally, e.g., orally, e.g., in the form of tablets or compound of the invention as disclosed herein, in free form capsules, or parenterally, e.g., in the form of injectable 55 or in pharmaceutically acceptable salt form, and b) at least Solutions or Suspensions, topically, e.g., in the form of one co-agent. The kit can comprise instructions for its lotions, gels, ointments or creams, or in a nasal or Supposi administration. tory form. Pharmaceutical compositions comprising a com The terms “co-administration' or “combined administra pound of the present invention in free form or in a pharma tion' or the like as utilized herein are meant to encompass ceutically acceptable Salt form in association with at least 60 administration of the selected therapeutic agents to a single one pharmaceutically acceptable carrier or diluent can be patient, and are intended to include treatment regimens in manufactured in a conventional manner by mixing, granu which the agents are not necessarily administered by the lating or coating methods. For example, oral compositions same route of administration or at the same time. can be tablets or gelatin capsules comprising the active The term “pharmaceutical combination” as used herein ingredient together with a) diluents, e.g., lactose, dextrose, 65 means a product that results from the mixing or combining Sucrose, mannitol, Sorbitol, cellulose and/or glycine; b) of more than one active ingredient and includes both fixed lubricants, e.g., silica, talcum, Stearic acid, its magnesium or and non-fixed combinations of the active ingredients. The US 7,338,957 B2 11 12 term “fixed combination” means that the active ingredients, Compounds of Formula I, in which Y is carbon, can be e.g. a compound of Formula I and a co-agent, are both prepared by proceeding as in the following Reaction Scheme administered to a patient simultaneously in the form of a II: single entity or dosage. The term “non-fixed combination' means that the active ingredients, e.g. a compound of 5 Formula I and a co-agent, are both administered to a patient Reactions Scheme II as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein Such (R3)n administration provides therapeutically effective levels of the 2 compounds in the body of the patient. The latter also 10 --Z N applies to cocktail therapy, e.g. the administration of 3 or I CN H-R). more active ingredients. R2 21 PROCESSES FOR MAKING COMPOUNDS OF (4) THE INVENTION 15 The present invention also includes processes for the R preparation of compounds of the invention. In the reactions described, it can be necessary to protect reactive functional N21 N groups, for example hydroxy, amino, imino, thio or carboxy groups, where these are desired in the final product, to avoid their unwanted participation in the reactions. Conventional ls-- protecting groups can be used in accordance with standard Y. practice, for example, see T. W. Greene and P. G. M. Wuts / N in “Protective Groups in Organic Chemistry”, John Wiley (5) and Sons, 1999, 3" Edition. 25 Y Compounds of Formula I, in which R is —NRR, can be R prepared by proceeding as in the following Reaction Scheme I: N21 30

Reaction Scheme I V

N21 N (R3) 35

sus Pry in which R. R. R. R. W. Z. m and n are as defined for Y. N Formula I in the Summary of the Invention and L is a leaving / N H-R)n 40 group. R 21 A compound of formula I can be prepared by reacting a compound of formula 4 with a compound of formula 5 in the (2) presence of an appropriate solvent (e.g., THF, and the like), NHRR, using triphosgene orphosgene and an appropriate base (e.g., (3) 45 DIEA, and the like), at a temperature range of 0°C. to about 90° C. and can take up to 24 hours to complete. R Reactions Schemes I and II are exemplified in the refer ences and examples, infra.

50 Additional Processes for Making Compounds of the Inven C N (R3)n tion s N 2) A compound of the invention can be prepared as a Y. N i-Z N pharmaceutically acceptable acid addition salt by reacting / NN H-R). the free base form of the compound with a pharmaceutically 2 55 acceptable inorganic or organic acid. Alternatively, a phar R maceutically acceptable base addition salt of a compound of the invention can be prepared by reacting the free acid form of the compound with a pharmaceutically acceptable inor in which R. R. R. R. W. Y. Z. m and n are as defined ganic or organic base. Alternatively, the salt forms of the for Formula I in the Summary of the Invention and V 60 compounds of the invention can be prepared using salts of represents a methylsulfonyl group, or a halo group, for the starting materials or intermediates. example iodo or chloro, preferably chloro. The free acid or free base forms of the compounds of the A compound of Formula I can be prepared by reacting a invention can be prepared from the corresponding base compound of formula 2 with a compound of formula 3 in the addition salt or acid addition salt from, respectively. For presence of a suitable solvent (e.g., n-butanol, acetic acid, or 65 example a compound of the invention in an acid addition salt the like), at a temperature range of 60 to 80° C. and can take form can be converted to the corresponding free base by up to 36 hours to complete. treating with a Suitable base (e.g., ammonium hydroxide US 7,338,957 B2 13 14 Solution, Sodium hydroxide, and the like). A compound of (e) optionally converting an N-oxide form of a compound the invention in a base addition salt form can be converted of the invention to its unoxidized form; to the corresponding free acid by treating with a suitable acid (f) optionally resolving an individual isomer of a com (e.g., hydrochloric acid, etc.) pound of the invention from a mixture of isomers; Compounds of the invention in unoxidized form can be 5 (g) optionally converting a non-derivatized compound of prepared from N-oxides of compounds of the invention by the invention into a pharmaceutically acceptable prodrug treating with a reducing agent (e.g., Sulfur, Sulfur dioxide, derivative; and triphenyl phosphine, lithium borohydride, sodium borohy (h) optionally converting a prodrug derivative of a com dride, or the like) in a suitable inert organic solvent (e.g. pound of the invention to its non-derivatized form. acetonitrile, ethanol, aqueous dioxane, or the like) at 0 to 80° 10 Insofar as the production of the starting materials is not C. particularly described, the compounds are known or can be Prodrug derivatives of the compounds of the invention prepared analogously to methods known in the art or as can be prepared by methods known to those of ordinary skill disclosed in the Examples hereinafter. in the art (e.g., for further details see Saulnier et al., (1994), One of skill in the art will appreciate that the above Bioorganic and Medicinal Chemistry Letters, Vol. 4, p. 15 transformations are only representative of methods for 1985). For example, appropriate prodrugs can be prepared preparation of the compounds of the present invention, and by reacting a non-derivatized compound of the invention that other well known methods can similarly be used. with a Suitable carbamylating agent (e.g., 1,1-acyloxyalky lcarbanochloridate, para-nitrophenyl carbonate, or the like). EXAMPLES Protected derivatives of the compounds of the invention can be made by means known to those of ordinary skill in The following examples provide detailed descriptions of the art. A detailed description of techniques applicable to the the preparation of intermediates (References) and represen creation of protecting groups and their removal can be found tative compounds of the invention (Examples) and are in T. W. Greene, “Protecting Groups in Organic Chemistry’. offered to illustrate, but not to limit the present invention. 3" edition, John Wiley and Sons, Inc., 1999. 25 Compounds of the present invention can be conveniently Reference 1 prepared, or formed during the process of the invention, as Solvates (e.g., hydrates). Hydrates of compounds of the 4-Chloro-pyrrolo2,3-dipyrimidine-7-carboxylic present invention can be conveniently prepared by recrys acid 4-(4-morpholin-4-yl-phenylcarbamoyl)-phe tallization from an aqueous/organic solvent mixture, using 30 nyl-amide resin organic solvents such as dioxin, tetrahydrofuran or metha nol. Compounds of the invention can be prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to 35 form a pair of diastereoisomeric compounds, separating the diastereomers and recovering the optically pure enanti omers. Additionally enantiomers can be separated by a C chiral preparative HPLC or using enantiomerically pure reagents. While resolution of enantiomers can be carried out 40 using covalent diastereomeric derivatives of the compounds of the invention, dissociable complexes are preferred (e.g., crystalline diastereomeric salts). Diastereomers have dis tinct physical properties (e.g., melting points, boiling points, solubilities, reactivity, etc.) and can be readily separated by 45 taking advantage of these dissimilarities. The diastereomers can be separated by chromatography, or preferably, by PAL-resin (Midwest Bio-Tech) bearing 4-morpholino separation/resolution techniques based upon differences in aniline (1 g, 1 mmol), DIEA (1.04 mL, 6 mmol), and solubility. The optically pure enantiomer is then recovered, nitrobenzoyl chloride (3 mmol) are mixed in CHCl (10 50 mL) and the Solution is shaken at room temperature for 16 along with the resolving agent, by any practical means that hours. The resultant mixture is filtered and the resin is would not result in racemization. A more detailed descrip washed with DMF (3x10 mL), CHCl (3x10 mL), MeOH tion of the techniques applicable to the resolution of stere (3x10 mL) and dried under vacuum. The resin (10 mgs) is oisomers of compounds from their racemic mixture can be treated with TFA/CH.Cl/HO (45/50/5) (200 uL) for 30 found in Jean Jacques, Andre Collet, Samuel H. Wilen, 55 minutes. LC-MS revealed only one peak: observed MS "Enantiomers, Racemates and Resolutions”, John Wiley (M+H") is 328.1; calculated MS (M+H") is 328.12. And Sons, Inc., 1981. The resulting resin is mixed with SnCl2.H2O (2.26 g. 10 In Summary, the compounds of Formula I can be made by mmol) in NMP (10 mL), and is shaken for 48 hours. The a process, which involves: solution is filtered and the resin is washed with NMP (3x10 (a) those of reaction schemes I and II; and 60 mL). CHCl (3x10 mL), MeOH (3x10 mL), and dried (b) optionally converting a compound of the invention under vacuum to give Solid-supported 4-amino-N-(4-mor into a pharmaceutically acceptable salt; pholin-4-yl-phenyl)-benzamide. This resin (10 mgs) is (c) optionally converting a salt form of a compound of the treated with TFA/CHC1/HO (45/50/5) (200 L) for 30 invention to a non-salt form; minutes. LC-MS revealed only one peak: observed MS (d) optionally converting an unoxidized form of a com 65 (M+H") is 298.1; calculated MS (M+H") is 298.15. pound of the invention into a pharmaceutically acceptable 6-Chloro-7-deazapurine (300 mg, 2 mmol), triphosgene N-oxide; (290 mg, 1 mmol), and DIEA (680 uL, 3.9 mmol) are stirred US 7,338,957 B2 15 16 in THF (10 mL) at 0° C. for 2 hours. THF is removed by 6-Chloro-7-deazapurine (1.50 g, 10 mmol), triphosgene evaporation and the residue is dissolved in THF (10 mL). (1.45 g, 5 mmol), and DIEA (3.4 mL, 19.5 mmol) are stirred The solution is mixed with 4-amino-N-(4-morpholin-4-yl in THF (50 mL) at 0°C. for 3 hours. After removing THF phenyl)-benzamide resin and shaken for 18 hours. The by evaporation, the residue is dissolved in THF (50 mL) and solution is filtered and the resin is washed with DMF (3x10 5 the solution is mixed with N-(3-amino-4-methyl-phenyl)-N- mL). CHCl (3x10 mL), MeOH (3x10 mL), and dried methyl-4-(4-methyl-piperazin-1-ylmethyl)-benzamide resin under vacuum to give 4-chloro-pyrrolo2,3-dipyrimidine-7- and shaken for 18 hours. The solution is filtered and the resin carboxylic acid-4-(4-morpholin-4-yl-phenylcarbamoyl)- is washed with DMF (3x50 mL), CHC1 (3x50 mL), MeOH phenyl-amide resin. 10 mgs of the resin is further treated (3x50 mL), and dried under vacuum to give 4-chloro with TFA/CHC1/HO (45/50/5) (200 uL) for 30 minutes. 10 pyrrolo[2,3-dipyrimidine-7-carboxylic acid {2-methyl-5-4- LC-MS showed only one peak: observed MS (M--H) is 477.10; calculated MS (M+H") is 477.14. 4-Chloro-pyrrolo (4-methyl-piperazin-1-ylmethyl)-benzoylaminol-phenyl 2,3-dipyrimidine-7-carboxylic acid methyl-4-(3-morpho amide resin. This resin (10 mgs) is treated with TFA/ lin-4-yl-phenylcarbamoyl)-phenyl-amide resin and CHC1/HO (45/50/5) (200 uL) for 30 minutes. LC-MS: 4-chloro-pyrrolo2,3-dipyrimidine-7-carboxylic acid 3-(4- 15 observed MS (M+H") is 518.20; calculated MS (M+H") is morpholin-4-yl-phenylcarbamoyl)-phenyl-amide are pre 518.2. pared in a similar manner using appropriate starting mate rials. Reference 3 Reference 2 4-Chloro-pyrrolo2,3-dipyrimidine-7-carboxylic acid (2-methyl-5-3-(4-methyl-imidazol-1-yl)-5- 4-Chloro-pyrrolo2,3-dipyrimidine-7-carboxylic trifluoromethyl-benzoylaminol-phenyl-amide resin acid {2-methyl-5-4-(4-methyl-piperazin-1-ylm ethyl)-benzoylaminol-phenyl)-amide resin 25 FC /s N N N N \ / 30 CO21 --> ---,s N \, C CJ21 s Cl N O era. 35 PAL-resin 4-methyl-3-nitro-aniline (5 g, 5 mmol), bis(2- oxo-3-oxazolidinyl)phosphinic chloride (5.09 g, 20 mmol), DIEA (3.5 mL, 20 mmol), and 3-(4-methyl-imidazol-1-yl)- 4-Methyl-3-nitro-analine PAL-resin (5 g, 5 mmol), DIEA 5-trifluoromethyl-benzoic acid (2.71 g, 10.0 mmol) are (5.34 mL. 20 mmol), and 4-(chloromethyl)-benzoyl chloride 40 mixed in DMF (50 mL) with shaking at room temperature (1.89 g, 10 mmol) are mixed in CHCl (50 mL) and shaken for 18 hours. The resultant mixture is filtered and the resin at room temperature for 18 hours. The solution is filtered and is washed with DMF (3x50 mL), CHC1 (3x50 mL), MeOH the resin is washed with DMF (3x50 mL), CHCl (3x50 (3x50 mL), and dried under vacuum. 10 mgs of this resin is mL), MeOH (3x50 mL) before drying under vacuum. This treated with TFA/CH.Cl/HO (45/50/5) (200 uL) for 30 resin (10 mgs) is treated with TFA/CHC1/HO (45/50/5) 45 minutes. LC-MS shows only one peak: observed MS (200 uL) for 30 minutes. LC-MS shows one major peak: (M+H") is 405.1; calculated MS (M+H") is 405.11. observed MS (M+H") is 305.0; calculated MS (M+H") is The resulting resin is mixed with SnCl2.2H2O (11.3 g, 50 3O4.06. mmol) in NMP (50 mL), and shaken at room temperature for The resin is mixed with 1-methylpiperazine (5.56 mL, 48 hours. The solution is filtered and the resin is washed with 49.2 mmol) in DMSO (50 mL) and shaken overnight before 50 NMP (3x50 ml), CHCl (3x50 ml), MeOH (3x50 mL), and filtering, washing with DMF (3x50 mL), CHCl (3x50 dried under vacuum to give N-(3-Amino-4-methyl-phenyl)- mL), MeOH (3x50 mL) and drying under vacuum. This 3-(4-methyl-imidazol-1-yl)-5-trifluoromethyl-benzamide resin (10 mgs) is treated with TFA/CHC1/HO (45/50/5) resin. This resin (10 mgs) is treated with TFA/CHCl2/H2O (200 uL) for 30 minutes. LC-MS shows only one peak: (45/50/5) (200 uL) for 30 minutes. LC-MS shows only one observed MS (M+H") is 369.2: calculated MS (M+H") is 55 peak: observed MS (M+H") is 375.1; calculated MS 369.18. (M+H") is 375.14. The resulting resin is mixed with SnCl2.H2O (11.3 g, 50 6-Chloro-7-deazapurine (1.50 g, 10.0 mmol), triphosgene mmol) in NMP (50 mL), and is shaken for 48 hours. The (1.45 g, 5.00 mmol), and DIEA (3.4 mL, 20 mmol) are solution is filtered and the resin is washed with NMP (3x50 60 stirred in THF (50 mL) at 0° C. for 3 hours. The THF is mL), CHCl (3x50 mL), MeOH (3x50 mL), and dried removed by evaporation, the residue is dissolved in THF (50 under vacuum to give solid-supported N-(3-amino-4-me mL) and the solution is mixed with Pal N-(3-amino-4- thyl-phenyl)-N-methyl-4-(4-methyl-piperazin-1-ylmethyl)- methyl-phenyl)-3-(4-methyl-imidazol-1-yl)-5-trifluorom benzamide. This resin (10 mgs) is treated with TFA/CH.Cl/ ethyl-benzamide resin (5 g, 5 mmol) with shaking at room HO(45/50/5) (200L) for 30 minutes. LC-MS shows only 65 temperature for 18 hours. The solution is filtered and the one peak: observed MS (M+H") is 339.2; calculated MS resin is washed with DMF (3x50 ml), CHCl (3x50 ml), (M+H") is 339.21. MeOH (3x50 mL) and dried under vacuum to give US 7,338,957 B2 17 18 4-Chloro-pyrrolo2,3-dipyrimidine-7-carboxylic acid Example 1 {2-methyl-5-3-(4-methyl-imidazol-1-yl)-5-trifluorom ethyl-benzoylaminol-phenyl-amide resin. This resin (10 4-(3-Trifluoromethyl-phenylamino)-pyrrolo2,3-d mgs) is treated with TFA/CHC1/HO (45/50/5) (200 uL) pyrimidine-7-carboxylic acid 3-(4-morpholin-4-yl for 30 minutes. LC-MS shows only one peak: observed MS phenylcarbamoyl)-phenyl-amide (M+H") is 554.1; calculates MS (M+H") is 554.12.

Reference 4 10 A solution phase synthesis of Reference 3 is achieved by O mixing 3-nitro-4-methylaniline (3.00 g, 11.1 mmol), O-(7- FC NTylSN H aZabenzotriazol-1-yl)-N.N.N',N'-tetramethyluronium y \N, N N hexafluorophosphate (5.49 g, 14.4 mmol), DIEA (5.00 mL. 15 28.8 mmol), and 3-(4-methyl-imidazol-1-yl)-5-trifluoro-me V- " ?o N thyl-benzoic acid (2.02 g, 13.3 mmol) in DMF (15 mL) with stirring at room temperature for 12 hours. Water (150 mL) is added into the solution and the light yellow precipitate is filtered, washed with water and dried under vacuum to give 4.3 g of product (yield 97%). LC-MS showed only one peak: observed MS (M+H") is 405.1; calculated MS (M+H") is 4-Chloro-pyrrolo2,3-dipyrimidine-7-carboxylic acid 3 405.11. (4-morpholin-4-yl-phenylcarbamoyl)-phenyl-amide resin The light yellow solid is stirred with 10-wt % Pd/C (600 25 (50 mg, 0.05 mmol), prepared as in reference 1, in n-butanol mg) in MeOH (50 mL) under H. After 24 hours, the (2 mL) is mixed with acetyl chloride (17.8 uL, 0.25 mmol) suspended solution is filtered and the Pd/C is washed with and 3-trifluoromethyl-aniline (31 uL, 0.25 mmol), or only MeOH (3x50 mL). The combined MeOH solution is con mixed with HCl salt of 3-trifluoromethyl-aniline (50 mg. centrated and dried under vacuum to give N-(3-amino-4- 0.25 mmol), and shaken at 80°C. for 24 hours. The resulting methyl-phenyl)-3-(4-methyl-imidazol-1-yl)-5-trifluorom 30 mixture is filtered and the resin is treated with TFA/CH.Cl/ ethyl-benzamide (3.5g yield 84%). LC-MS shows only one HO(45/50/5) (600 uL) for 30 minutes before filtering and peak: observed MS (M+H") is 375.1; calculated MS washing with CHCl (3x2 mL), MeOH (3x2 mL). The (M+H") is 375.14. washes are then combined together and concentrated. The residue is purified by preparative HPLC to give 4-(3- 6-Chloro-7-deazapurine (3.9 g, 25.5 mmol), triphosgene 35 trifluoromethyl-phenylamino)-pyrrolo2,3-dipyrimidine-7- (3.80 g, 12.8 mmol), and DIEA (8.9 mL, 51 mmol) are carboxylic acid 3-(4-morpholin-4-yl-phenylcarbamoyl)- stirred in THF (100 mL) at 0°C. for 3 hours. The THF is removed by evaporation and the residue is dissolved in THF phenyl-amide (19.0 mg, yield 64%). "H NMR (400 MHz, (100 mL). The solution is mixed with N-(3-amino-4-methyl DMSO-d) & 11.75 (s, 1H), 10.16 (s, 1H), 10.09 (s, 1H), 8.69 phenyl)-3-(4-methyl-imidazol-1-yl)-5-trifluoromethyl-ben 40 (s, 1H), 8.31 (s, 1H), 8.22 (d. J=8.3 Hz, 1H), 8.15 (s, 1H), Zamide (3.5 g., 9.3 mmol) and shaken at room temperature 7.94 (d. J=8.2 Hz, 1H), 7.92 (d. J=3.9 Hz, 1H), 7.77 (d. J=7.7 for 18 hours. The solvent is evaporated and the residue is HZ, 1H), 7.64 (d. J=8.8 Hz, 2H), 7.63 (d. J=6.9 Hz, 1H), 7.58 purified by silica gel column chromatography (2% MeOH in (t, J=8.0 Hz, 1H), 7.44 (d. J=7.7 Hz, 1H), 7.13 (d. J=4.0 Hz, CHCl) to obtain 4-chloro-pyrrolo2,3-dipyrimidine-7-car 45 1H), 6.95 (d. J=9.0 Hz, 2H), 3.74 (t, J=4.6 Hz, 4H), 3.08 (t, boxylic acid (2-methyl-5-3-(4-methyl-imidazol-1-yl)-5-tri J=4.7 Hz, 4H); ESIMS m/z 602.20 (M"+1). fluoromethyl-benzoylaminol-phenyl-amide (3.4 g, yield 65%). "H NMR (400 MHz, DMSO-d) & 11.07 (s, 1H), Example 2 10.71 (s, 1H), 9.62 (s, 1H), 8.99 (s, 1H), 8.62 (s, 1H), 8.55 50 4-(4-Trifluoromethyl-1-phenylamino)-pyrrolo2,3-d (s, 1H), 8.46 (d. J=4.0 Hz, 1H), 8.24 (d. J=4.0 Hz, 1H), 8.18 pyrimidine-7-carboxylic acid (2-methyl-5-4-(4- (s, 1H), 7.62 (d. J=8.0 Hz, 1H), 7.35 (d. J=8.0 Hz, 1H), 6.96 methyl-piperazin-1-ylmethyl)-benzoylamino-phe (d. J=4.0 Hz, 1H), 2.47 (s, 3H), 2.37 (s, 3H); ESIMS m/z 554.1 (M+1). nyl-amide

US 7,338,957 B2 31 32

TABLE 1-continued

Physical Data Compound H NMR 400 MHz (DMSO-d) Number Structure and/or MS (m/z)

28 F ESIMS m/z 593.20 (M* + 1).

29 ESIMS m/z 625.20 (M" + 1).

30 ESIMS m/z 636.20 (M* + 1).

31 ESIMS m/z 554.10 (M* + 1).

32 ESIMS m/z 518.10 (M" + 1).

33 ESIMS m/z 646.30 (M" + 1).

US 7,338,957 B2 39 40

TABLE 1-continued Physical Data Compound H NMR 400 MHz (DMSO-d) Number Structure and/or MS (m/z)

50 O CF ESIMS m/z 704.2 (M + 1).

51 FC ESIMS m/z 605.2 (M + 1).

52 FC ESIMS m/z 543.20 (M* + 1). O er

53 ESIMS m/z 654.20 (M* + 1).

ESIMS m/z 654.20 (M" + 1). US 7,338,957 B2 41 42 ASSAYS NP-40) containing protease and phosphatase inhibitors. 50 uL of cell lysate is added to 96 well optiplates previously Compounds of the present invention are assayed to mea coated with anti-abl specific antibody and blocked. The sure their capacity to selectively inhibit cell proliferation of plates are incubated for 4 hours at 4°C. After washing with 32D cells expressing BCR-Abl (32D-p210) compared with 5 TBS-Tween 20 buffer, 50 uL of alkaline-phosphatase con parental 32D cells. Compounds selectively inhibiting the jugated anti-phosphotyrosine antibody is added and the plate proliferation of these BCR-Abl transformed cells are tested is further incubated overnight at 4°C. After washing with for anti-proliferative activity on Ba/F3 cells expressing TBS-Tween 20 buffer, 90 uL of a luminescent substrate are either wild type or the mutant forms of BCR-Abl. In added and the luminescence is quantified using the addition, compounds are assayed to measure their capacity 10 AcquestTM system (Molecular Devices). Test compounds of to inhibit B-RAF. the invention that inhibit the proliferation of the BCR-Abl Inhibition of Cellular BCR-Abl Dependent Proliferation expressing cells, inhibit the cellular BCR-Ablautophospho (High Throughput Method) rylation in a dose-dependent manner. The murine cell line used is the 32D hemopoietic pro Effect on Proliferation of Cells Expressing Mutant Forms of genitor cell line transformed with BCR-Abl cDNA (32D 15 BCR-Ab1 p210). These cells are maintained in RPMI/10% fetal calf Compounds of the invention are tested for their antipro serum (RPMI/FCS) supplemented with penicillin 50 ug/mL, liferative effect on Ba/F3 cells expressing either wild type or streptomycin 50 g/mL and L-glutamine 200 mM. Untrans the mutant forms of BCR-Abl (G250E, E255V, T315I, formed 32D cells are similarly maintained with the addition F317L, M351T) that confers resistance or diminished sen of 15% of WEHI conditioned medium as a source of IL3. sitivity to STI571. The antiproliferative effect of these 50 ul of a 32D or 32D-p210 cells suspension are plated in compounds on the mutant-BCR-Abl expressing cells and on Greiner 384 well microplates (black) at a density of 5000 the nontransformed cells were tested at 10, 3.3, 1.1 and 0.37 cells per well. 50 mL of test compound (1 mM in DMSO uM as described above (in media lacking IL3). The ICso stock solution) is added to each well (STI571 is included as values of the compounds lacking toxicity on the untrans a positive control). The cells are incubated for 72 hours at 25 formed cells were determined from the dose response curves 37° C., 5% CO. 10 uL of a 60% Alamar Blue solution (Tek obtained as describe above. diagnostics) is added to each well and the cells are incubated for an additional 24 hours. The fluorescence intensity (Exci B-RAF tation at 530 nm, Emission at 580 nm) is quantified using the Compounds of the invention are tested for their ability to AcquestTM system (Molecular Devices). 30 inhibit the activity of B-RAF. The assay is carried out in 384-well MaxiSorp plates (NUNC) with black walls and Inhibition of Cellular BCR-Abl Dependent Proliferation clear bottom. The substrate, IKBO. is diluted in DPBS 32D-p210 cells are plated into 96 well TC plates at a (1:750) and 15 uL is added to each well. The plates are density of 15,000 cells per well. 50 uL of two fold serial incubated at 4°C. overnight and washed 3 times with TBST dilutions of the test compound (C is 40 uM) are added to 35 (25 mM Tris, pH 8.0, 150 mM. NaCl and 0.05% Tween-20) each well (STI571 is included as a positive control). After using the EMBLA plate washer. Plates are blocked by incubating the cells for 48 hours at 37° C., 5% CO, 15 LL Superblock (15 uL/well) for 3 hours at room temperature, of MTT (Promega) is added to each well and the cells are washed 3 times with TBST and pat-dried. Assay buffer incubated for an additional 5 hours. The optical density at containing 20 uM ATP (10 uL) is added to each well 570 nm is quantified spectrophotometrically and ICs val 40 followed by 100 mL or 500 mL of compound. B-RAF is ues, the concentration of compound required for 50% inhi diluted in the assay buffer (1 uL into 25 ul) and 10 uL of bition, determined from a dose response curve. diluted B-RAF is added to each well (0.4 ug/well). The Effect on Cell Cycle Distribution plates are incubated at room temperature for 2.5 hours. The 32D and 32D-p210 cells are plated into 6 well TC plates kinase reaction is stopped by washing the plates 6 times with at 2.5x10° cells per well in 5 mL of medium and test 45 TBST. Phosph-IKBC. (Ser32/36) antibody is diluted in compound at 1 or 10 uM is added (STI571 is included as a Superblock (1:10,000) and 15 uL is added to each well. The control). The cells are then incubated for 24 or 48 hours at plates are incubated at 4°C. overnight and washed 6 times 37° C., 5% CO. 2 ml of cell suspension is washed with with TBST. AP-conjugated goat-anti-mouse IgG is diluted in PBS, fixed in 70% EtOH for 1 hour and treated with Superblock (1:1,500) and 15uL is added to each well. Plates PBS/EDTA/RNase A for 30 minutes. Propidium iodide 50 are incubated at room temperature for 1 hour and washed 6 (Cf-10 ug/ml) is added and the fluorescence intensity is times with TBST. 15 uL of Attophos AP substrate is added quantified by flow cytometry on the FACScaliburTM system to each well and plates are incubated at room temperature for (BD Biosciences). Test compounds of the present invention 15 minutes. Plates are read on Acquest or Analyst GT using demonstrate an apoptotic effect on the 32D-p210 cells but do a Fluorescence Intensity Nanxin BBT anion (505 dichroic not induce apoptosis in the 32D parental cells. 55 mirror). Effect on Cellular BCR-Abl Autophosphorylation Upstate KinaseProfilerTM Radio-enzymatic filter binding BCR-Abl autophosphorylation is quantified with capture assay Elisa using a c-abl specific capture antibody and an Compounds of the invention are assessed for their ability antiphosphotyrosine antibody. 32D-p210 cells are plated in 60 to inhibit individual members of a panel of kinases (a partial, 96 well TC plates at 2x10 cells per well in 50 uL of non-limiting list of kinases includes: BMX, CSK, TrkB, medium. 50 uL of two fold serial dilutions of test com FGFR3, Fes, Lck, B-RAF, C-RAF, MKK6, SAPK2O. and pounds (C is 10 uM) are added to each well (STI571 is SAPK2B). The compounds are tested in duplicates at a final included as a positive control). The cells are incubated for 90 concentration of 10 LM following this generic protocol. minutes at 37° C., 5% CO. The cells are then treated for 1 65 Note that the kinase buffer composition and the substrates hour on ice with 150 uL of lysis buffer (50 mM Tris-HCl, pH vary for the different kinases included in the “Upstate 7.4, 150 mM. NaCl, 5 mM EDTA, 1 mM EGTA and 1% KinaseProfilerTM panel. The compounds are tested in dupli US 7,338,957 B2 43 44 cates at a final concentration of 10 LM following this generic We claim: protocol. Note that the kinase buffer composition and the 1. A compound of formula I: substrates vary for the different kinases included in the “Upstate KinaseProfilerTM panel. Kinase buffer (2.5 uL. 10x—containing MnCl when required), active kinase (I) (0.001-0.01 Units; 2.5 LL), specific or Poly(Glu-4-Tyr) pep R tide (5-500 uM or 0.01 mg/ml) in kinase buffer and kinase 2 buffer (50 uM; 5 uL) are mixed in an eppendorf on ice. A N N "R- Mg/ATP mix (10 uL: 67.5 (or 33.75) mM MgCl, 450 (or 10 ls N x^ 225) uM ATP and 1 uCi/uly-'P-ATP (3000 Ci/mmol)) is W V s! N added and the reaction is incubated at about 30° C. for about YN B - (R) 10 minutes. The reaction mixture is spotted (20 uL) onto a %2 N\ 2 2 cmx2 cm P81 (phosphocellulose, for positively charged R2 peptide substrates) or Whatman No. 1 (for Poly (Glu-4-Tyr) peptide Substrate) paper square. The assay squares are 15 washed 4 times, for 5 minutes each, with 0.75% phosphoric in which: acid and washed once with acetone for 5 minutes. The assay W is N: squares are transferred to a scintillation vial, 5 ml Scintilla Y is selected from C, S and S(O); tion cocktail are added and P incorporation (cpm) to the Z is a divalent radical selected from Y(O)NRs and peptide Substrate is quantified with a Beckman Scintillation —NRY(O)—; wherein Y is selected from C, S and counter. Percentage inhibition is calculated for each reac S(O); and Rs is selected from hydrogen and C-alkyl, tion. R is selected from hydrogen, halo, hydroxy Calkyl, Calkoxy and —NRR, wherein R is selected from Compounds of Formula I, in free form or in pharmaceu hydrogen and Calkyl, R is selected from Coaryl, tically acceptable salt form, exhibit valuable pharmacologi 25 Csoheteroaryl, C-cycloalkyl and C-sheterocy cal properties, for example, as indicated by the in vitro tests cloalkyl, or R and R, together with the nitrogen to described in this application. For example, compounds of which both R and R, are attached form C-sheterocy Formula I preferably show an ICso in the range of 1x10' cloalkyl or Csoheteroaryl; wherein any aryl, het to 1x10 M, preferably less than 100 nM for wild type 30 eroaryl, cycloalkyl or heterocycloalkyl of R, or of the BCR-Abl and G250E, E255V, T315I, F317L and M351T combination of R and R, can be optionally substituted BCR-Abl mutants. with 1 to 3 radicals selected from halo, cyano, nitro, For example: 4-(3-carbamoyl-phenylamino)-pyrrolo2.3- Calkyl, Calkenyl, C-alkynyl, halo-substituted dpyrimidine-7-carboxylic acid {2-methyl-5-3-(4-methyl Calkyl, Calkoxy, halo-substituted-Calkoxy, imidazol-1-yl)-5-trifluoromethyl-benzoylaminol-phenyl 35 - XNRRs, XC(O)NR'Rs, XC(O)NRXORs. amide (Example 7) has an ICs of 5 nM, 47 nM, 31 nM. 115 —XS(O)NRRs —XS(O)o-Rs —XNRS(O)o-Rs. nM 9 nM and 5 nM for wild type, G250E, E255V, T315I, - XNRC(O)Rs - XNRSRs, XP(O)NR'Rs, F317L and M351T BCR-Abl, respectively. At a concentra —XCR(ORs)Rs, XOC(O)Rs. XORs and tion of 10 LM, the compound inhibits the activity of Abl, —XOR, wherein X is a bond or C-alkylene, Rs is BCR-Abl, Bmx, CSK, TrkB, FGFR3, Fes, Lck, B-RAF, 40 independently selected from hydrogen and Calkyl, C-RAF, MKK6. SAPK2C. and SAPK2 B kinases by greater Ro is selected from Coaryl and Csoheteroaryl; than 50%, preferably greater than 75%. For example, 4-(3- R is selected from hydrogen and Calkyl; carbamoyl-phenylamino)-pyrrolo2,3-dipyrimidine-7-car R is selected from hydrogen, hydroxy, Calkyl, Calk boxylic acid (2-methyl-5-3-(4-methyl-imidazol-1-yl)-5-tri enyl, C2-galkynyl, Calkoxy, mercapto, halo, nitro fluoromethyl-benzoylaminol-phenyl-amide (Example 7) 45 and cyano; shows a percentage inhibition of Bmx (100%), c-Raf n is 0, 1 or 2: (99%), CSK (100%), Fes (98%), FGFR3 (95%), Lck (96%), R is selected from hydrogen, halo, hydroxy, cyano, nitro, MKK6 (98%), SAPK2O (100%), SAPK2|B (100%) and Calkyl, halo-substituted-Calkyl, Calkoxy, TrkB (98%); halo-substituted-Calkoxy and —XRo, wherein X 4-(3-Dimethylamino-phenylamino)-pyrrolo2,3-dipyri 50 is a bond or Calkylene, Rio is selected from midine-7-carboxylic acid {2-methyl-5-3-(4-methyl-imida Coaryl, Csoheteroaryl, C-cycloalkyl and Zol-1-yl)-5-trifluoromethyl-benzoylaminol-phenyl-amide C-sheterocycloalkyl, wherein any aryl, heteroaryl, (Example 3) has an ICs of 22 nM, 170 nM, 220 nM, 370 cycloalkyl or heterocycloalkyl of Rio is optionally nM, 50 nM and 29 nM for wild type, G250E, E255V, T315I, substituted with a radical selected from halo, hydroxy, F317L and M351T BCR-Abl, respectively. The compound 55 cyano, nitro, C-alkyl, halo-substituted-Calkyl, has an ICs of 32 nM for B-Raf. At a concentration of 10 LM, Calkoxy and halo-substituted-Calkoxy; the compound shows a percentage inhibition of Bmx m is 1, 2 or 3; and wherein the phenyl rings A and B can (100%), c-Raf (83%), CSK (90%), Fes (82%), Lck (95%), independently have up to four —C= groups replaced SAPK2|B(95%) and TrkB (100%). by N=; It is understood that the examples and embodiments 60 or the pharmaceutically acceptable salts thereof. described herein are for illustrative purposes only and that various modifications or changes in light thereof will be 2. The compound of claim 1 in which: Suggested to persons skilled in the art and are to be included W is N: within the spirit and purview of this application and scope of Y is C; the appended claims. All publications, patents, and patent 65 Z is a divalent radical selected from —C(O)NRs and applications cited herein are hereby incorporated by refer —NRC(O)—; Rs is selected from hydrogen and ence for all purposes. Calkyl,