Enantiomerically pure aminoheteroaryl compounds as protein kinase inhibitors

Description

Field of the Invention

[0001] The invention relates generally to novel chemicals and methods. More particularly, the invention relates to enantiomerically pure aminoheteroaryl compounds, particularly aminopyridines and aminopyrazines, which exhibit protein tyrosine kinase activity, and methods for the synthesis and use of such compounds. Preferred compounds are c-MET inhibitors useful in the treatment of abnormal cell growth such as cancer cells.

Background of the invention

[0002] It has been shown that in many human cancers the receptor (cMET or HGFR) of the hepatocyte growth factor (HGF) of the receptor tyrosine kinase (RTK) family is involved in the formation of tumors, the development of tumors with increased cell motility and tumor invasion, as well as in metastasis formation (see e.g. Ma, PC, Maulik, G., Christensen, J. & Salgia, R. (2003b). Cancer Metastasis Rev, 22, 309-25; Maulik, G., Shrikhande, A., Kijima , T., Ma, PC, Morrizon, PT & Salgia, R. (2002b). Growth Factor Rev. 13, 41-59). c-MET (HGFR) can be activated by overexpression or mutations in a variety of human cancers including small cell lung cancer (SCLC) (Ma, PC, Kijima, T., Maulik, G., Fox, EA, Sattler, M., Griffin, JD, Johnson, BE & Salgia, R. (2003a). Cancer Res, 63, 6272-6281).

[0003] c-MET is a receptor tyrosine kinase that is encoded by the Met proto-oncogene and transduces the biological effects of hepatocyte growth factor (HGF), also referred to as scattering factor (SF). Jiang et al., Crit. Rev. Oncol. Hematol. 29: 209-248 (1999). c-MET and HGF are expressed in numerous tissues, although their expression is typically largely restricted to cells of epithelial and mesenchymal origin, respectively. c-MET and HGF are necessary for proper mammalian development and have been shown to be important in cell migration, cell proliferation and survival, in morphogenic differentiation, and in organizing 3-dimensional tubular structures (e.g., cylindrical kidney cells, in gland formation, e.t.c.). In addition to its effect on epithelial cells, HGF / SF has been reported to be an angiogenic factor, and c-MET signaling in endothelial cells can induce many of the cellular responses necessary for angiogenesis (proliferation, mobilization, invasion).

[0004] The c-MET receptor has been shown to be expressed in many human carcinomas. It has also been shown that c-MET and its ligand, HGF, are collectively expressed on elevated levels in a variety of human carcinomas (particularly in sarcoma). However, since the receptor and ligand are typically expressed by different cell types, c-MET signaling is most commonly regulated by tumor-stroma (tumor-host) interactions. In addition, enhancement, mutation, and reorganization of the c-MET gene were observed in a subset of human cancers. Families with germline mutations that activate c-MET kinase are susceptible to many kidney tumors as well as tumors in other tissues. Numerous studies have correlated the expression of c-MET and / or HGF / SF with disease progression of various types of cancer (including cancers of the lung, colon, breast, prostate, liver, pancreas, brain, kidney, ovary, stomach, skin, and bone). Moreover, it has been shown that overexpression of c-MET or HGF is correlated with poor prognosis and leads to the development of many of the major human cancers including lung, liver, stomach, and breast cancer. c-MET is also directly involved in cancers without an effective treatment regimen, such as pancreatic cancer, glioma, and hepatocellular carcinoma.

[0005] Examples of c-MET (HGFR) inhibitors, their synthesis and use can be found in US patent application US No. US 10/786610 entitled "Aminoheteroaryl Compounds as Protein Kinase Inhibitors, filed February 26, 2004, and in the corresponding international patent application PCT PCT / US2004 / 005495 ​​of the same title, filed February 26)

2004 years, the disclosures of which are herein incorporated by reference in their entirety.

[0006] It is desirable to develop new c-MET inhibitors (HGFRs) and methods of using such inhibitors to treat abnormal cell growth such as in cancer.

The essence of the invention

[0007] In one embodiment, the invention relates to an enantiomerically pure compound of Formula 1,

R1

<img file="PL1786785T3_D0001.tif" />

wherein:

Y, Rl and r2 are as defined in claim 1 1, or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0008] In another particular aspect of this embodiment, Y is N.

[0009] In another particular aspect of this embodiment, Y is CR³³ .

[0010] In another particular aspect of this embodiment, Y is CR³³ and r12 is H.

[0011] In another particular aspect of this embodiment, and in conjunction with any other non-contradictory aspect, R.³is selected from furan, thiophene, pyrrole, pyrroline, pyrrolidine, dioxolane, oxazole, thiazole, imidazole, imidazoline, imidazolidine, pyrazole, pyrazoline, pyrazolidine, isoxazole, isothiazole, oxadiazole, triazole, thiadiazole, pyroxaneridine, pyridine, pyridine, , morpholine, dithian, thiomorpholine, pyridazine, pyrimidine, pyrazine, piperazine, triazine, trithian, or phenyl, and each hydrogen in R³· Is optionally substituted with one or more R³ .

[0012] In another particular aspect of this embodiment, and in combination with any other non-contradictory aspect, R.⁷is fused ring heteroaryl, and each hydrogen atom is wr! is optionally substituted with one or more substituents.

[0013] In another particular aspect of this embodiment, and in combination with any other non-contradictory aspect, R.⁷is hydrogen.

[0014] In another particular aspect of this embodiment, and in combination with any other non-contradictory aspect, R.⁷is halogen.

[0015] In another embodiment, the invention provides an enantiomerically pure compound of Formula Ia.

<img file="PL1786785T3_D0002.tif" />

wherein:

Y is CH;

is selected from furan, thiophene, pyrrole, pyrroline, pyrrolidine, dioxolane, oxazole, thiazole, imidazole, imidazoline, imidazolidine, pyrazole, pyrazoline, pyrazolidine, isoxazole, isothiazole, oxadiazole, triazole, thiadiazole, pyroxaneridine, pyridine, pyridine, , morpholine, dithian, thiomorpholine, pyridazine, pyrimidine, pyrazine, piperazine, triazine, trithian, azitidine or phenyl; and each hydrogen in w is optionally substituted with R5;

each is independently halogen, Cy_yg alkyl, C2-12 alkenyl, C2-12 alkynyl, 03-22 cycloalkyl, C8-12 aryl, 3-12 membered heteroalicycle, 5-12 membered heteroaryl, -S (O)_mR⁴, -SO2NR⁴R⁵, -S (O) 2OR⁴, -NO2, -NR⁴R⁵, - (CR⁶R⁷) nOR⁴, -CN, -C (O) R⁴, -OC (O) R⁴, -O (CR⁶R⁷) nR⁴, -NR⁴C (O) R⁵,

- (CR⁶R⁷) nC (O) OR⁴, - (CR⁶R⁷) nOR⁴, - (CR⁵R⁷)_nC (O) NO⁴R⁵,

- (CR⁵R⁷) nNCR⁴R⁵, —C (= NO⁶) NO⁴R⁵, -NR⁴C (O) NO⁵R⁶, -NR⁴S (O) pR⁵ or -C (O) No. 4R5, each hydrogen in R⁸ is optionally substituted with R⁸, and R⁸ adjacent atoms can combine to form a group such as 05-32 aryl, 5-12 membered heteroaryl, 03-32 cycloalkyl or 3-12 membered heteroalicyclic;

each R ^, R⁸, R⁸ and r⁸ is independently hydrogen, halogen, C12 alkyl, C2-32 alkenyl, C2-32 alkynyl, 03-12 cycloalkyl, C1-12 aryl, 3-12 membered heteroalicyclic, 5-12 membered heteroaryl; or any two of R ^, R⁸, R⁸ and R7 attached to the same nitrogen atom may, together with the nitrogen atom they are attached to, form a 3 to 12 membered heteroalicycle or a 5-12 membered heteroaryl optionally containing 1 to 3 additional heteroatoms selected from N, O, and S; or any two of R ^, R⁸, R⁸ and R7 attached to the same carbon atom can combine to form a ^ 3-12 cycloalkyl, ^ 6-12 aryl, 3-12 membered heteroalicycle, or 5-12 membered heteroaryl; and each hydrogen in R ^, R⁸,⁸ and r7 is optionally substituted with R⁸;

each R⁸ is independently halogen, C-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, 03-12 cycloalkyl, 05.32 aryl, 3-12 membered heteroalicyclic, 5-12 membered heteroaryl, -NH2, -one, -OH, - 0-03.32 alkyl, -O- (CH2) ηΟ3_12 cycloalkyl, -O- (CH2) nC8] _2 aryl, -O- (CH2) n (3-12 membered heteroalicycle) or -O- (CH2) n (5- 12 membered heteroaryl); and each hydrogen in R⁸ is optionally substituted with R5; each R⁸⁸is independently halogen, C1-12 alkyl, C1-12 alkoxy, 03-32 cycloalkyl, 05.32 aryl, 3-12 membered heteroalicyclic, 5-12 membered heteroaryl, -0-03-32 alkyl, -O- (CH2)_nC3-12 cycloalkyl, -O- (CH₂)_n^c6-12 aryl, -O- (CH₂)_n(3-12 membered heteroalicycle), -O- (CH2)_n (5-12 membered heteroaryl) or -CN, and each hydrogen in rH is optionally substituted with halogen, -OH, -CN, -Ci-12 alkyl which may be partially or fully halogenated, -0-03-32 alkyl, which may be partially or fully halogenated, -CO, -SO or -SO2;

each R⁷is independently halogen, C-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, 03-12 cycloalkyl, C8-18 aryl, 3-12 membered heteroalicycle, 5-12 membered heteroaryl, -S (O) mR⁴, -SO2NR⁴R⁵, -S (O) 2OR⁴, -no2, -no⁴r⁵, - (CR⁶R⁷) nOR⁴, -CN, -C (O) R⁴, -OC (O) R⁴, -O (CR⁶R⁷) nR⁴, -NR⁴C (O) R⁵,

- (CR⁶R⁷) nC (O) OR⁴, - (CR⁶R⁷) nOR⁴, - (CR⁶R⁷)_nC (O) NO⁴R⁵,

- (CR⁶R⁷) nNCR⁴R⁵, —C (= NO⁶) NO⁴R⁵, -NR⁴C (O) NO⁵R⁶, -NR⁴S (O) pR⁵, -C (O) NO⁴r5, - (CR $ R⁷)_n (3-12 membered heteroalicycle),

- (CR $ R⁷) n (03-12 cycloalkyl), - (CR $ R⁷) n (C8 -12 aryl), - (CR $ R⁷) n (5-12 membered heteroaryl), - (CR5R⁷) nC (O) NO⁴R ^, or - (CR $ R⁷)_nC (O) R⁴, R13 on adjacent atoms can combine to form a C8-12 aryl, 5-12 membered heteroaryl, 03-12 cycloalkyl or 3-12 membered heteroalicycle, and each hydrogen atom in R^77s is optionally substituted with R⁷⁵;

each m is independently 0, 1 or 2;

each n is independently 0, 1, 2, 3 or 4;

each p is independently 1 or 2;

or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0016] In another embodiment, the invention provides an enantiomerically pure compound selected from the group consisting of 5-bromo-3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyrazine-2 -ylamine; 5-iodo-3 - [(R) 1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-2-ylamine; 5-bromo-3- [1 (R) (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-2-ylamine; 4- {5-amino-6 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyrazin-2-yl} benzoic acid; (4- {5-amino-6 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyrazin-2-yl} phenyl) piperazin-1-ylmethanone; 4- (4- {5-amino-6 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyrazin-2-yl} benzoyl) -piperazine-1-carboxylic acid tert-butyl ester; 3 - [(IR) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- [4- (piperazin-1-ylcarbonyl) phenyl] pyridine-2amine; 4- {6-amino-5 - [(IR) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} -N- [2- (dimethylamino) ethyl] -N-methylbenzamide ; (4- {6-amino-5 - [(IR) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} phenyl) methanol; 4- {6-amino-5 - [(IR) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} -N- [3- (dimethylamino) propyl] -N-methylbenzamide; Tert-butyl 4- (4- {6-amino-5 - [(IR) -1 (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} benzoyl) piperazine-1-carboxylate; 3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- [1- (1-methylpiperidin-4-yl) -1H-pyrazol-4-yl] pyridin-3-ylamine; 1- [4- (4- {6-amino-5 - [(R) -1 (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} pyrazol-1-yl) piperidin-1-yl] -2-hydroxyethanone; 3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- (1-piperidin-4-yl-1H-pyrazol-4-yl) pyridin-2-ylamine; 3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- (1-piperidin-4-yl-1H-pyrazol-4-yl) pyridin-2-ylamine; 3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- (1-piperidin-4-yl-1H-pyrazol-4-yl) pyrazin-2-ylamine; 3 - [(R) 1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- (1H-pyrazol-4-yl) pyrazin-2-ylamine; 1- [4- (4- {5-amino-6 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyrazin-2-yl} pyrazol-1-yl) piperidinl-yl] - 2-hydroxyethanone; 3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- [1- (1-methylpiperidin-4-yl) -1H-pyrazol-4-yl] pyrazin-2- ylamine; 1- [4- (4- {5-amino-6 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyrazin-2-yl} pyrazol-1-yl) piperidin-yl] - 2-dimethylaminoethanone; 3 - [(R) -1- (2-chloro-3,6-difluorophenyl) ethoxy] -5- (1-piperidin-4-yl-1H-pyrazol-4-yl) pyridin-2-ylamine; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0017] In another embodiment, the invention provides a pharmaceutical composition comprising any of the compounds of the invention and a pharmaceutically acceptable carrier. Examples of such compositions are described below.

[0018] Preferred compounds of the invention include those having c-MET inhibitory activity as defined by any one or more of IC50, Ki, or percent inhibition (% I). One skilled in the art can easily determine whether a compound exhibits such activity by performing a suitable test, and descriptions of such tests are provided in the Examples section of this description. In one embodiment, particularly preferred compounds have a c-MET Ki of less than 5 µΜ or less than 2 µΜ, or less than 1 µΜ, or less than 500 nM, or less than 200 nM or less than 100 nM. In another embodiment, particularly preferred compounds exhibit c-MET inhibition at 1 μΜ of at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60% or at least 70%. % or at least 80% or at least 90%. Methods for measuring activity against c-MET / HGFR are described in the Examples herein.

In another embodiment, the invention relates to a compound of Formula 1 or Ia for treating abnormal cell growth in a mammal, in another embodiment, the invention relates to the use of such a compound for the manufacture of medicaments for treating abnormal cell growth in a mammal.

In a specific embodiment of any of the methods of the invention described herein, the abnormal cell growth relates to cancer such as, but not limited to, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, cancer ovarian cancer, rectal cancer, rectal cancer, stomach cancer, colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid gland cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, kidney cell cancer, renal pelvic cancer, CNS neoplasms, primary CNS lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, or combinations of one or more of the above cancers. In another embodiment of said method, the abnormal cell growth relates to a benign proliferative disease, including, but not limited to, psoriasis, benign prostatic hyperplasia, or restenosis.

[0021] In further specific embodiments of the invention described herein, the compounds of the invention are administered in combination with an amount of one or more substances selected from anti-neoplastic agents, angiogenesis inhibitors, signal transduction inhibitors, and anti-proliferative agents that are together in amounts. effective in treating said abnormal cell growth. Such substances include those disclosed in international PCT patent applications WO 00/38715, WO 00/38716, WO 00/38717, WO 00/38718, WO 00/38719, WO 00/38730, WO 00/38665, WO 00 / 37107 and WO 00/38786, the disclosures of which are herein incorporated by reference in their entirety.

[0022] Examples of anti-cancer agents include mitosis inhibitors, e.g. vinca alkaloids such as vinblastine, vinorelbine, vindescine and vincristine; colchicine derivatives, allocolchicine, halichondrin, N-benzoyltrimethylcolchicinic acid methyl ether, dolastatin 10, maytansine, rhizoxine, taxanes such as taxol (paclitaxel), docetaxel (taxoter), 2'-N- [3- (dimethylamino) propyl (derivative] glutatin) taxol), thiocolchicine, tritylcysteine, teniposide, methotrexate, azathioprine, fluorouracil, cytosine arabinoside, 2'2'-difluorodeoxycytidine (gemcitabine), adriamycin and mitamycin. Alkylating agents, e.g. such as cis-platinum, carboplatin, oxyplatin, iproplatin, N-acetyl-DL-sarcosyl-L-leucine ethyl ester (Asatey or Asalex), 2,5-bis (1-aziridinyl) -3,8-dioxo-1 acid diethyl ester , 4-cyclohexadieno-1,4-dicarbamic (diazinone), 1,4-bis (methanesulfonyloxy) butane (bisulfane or leucosulfan), chlormorozotocin, clomesone (2-chloroethyl- (methylsulfonyl) methanesulfonate), cyanomorpholinodoxorhydroganlycin, cyclomorpholate, hematroganilicin, cyclomorpholate, hematroganilicine , mitomycin C, hycantheonemitomycin C, mitozolamide, 1- (2-chloroethyl) -4- (3-chloropropyl) piperazine dihydrochloride, piperazidione, pipobroman, portiromycin, spirohydantoin mustard, teroxirone, tetraplatin, thiotepa, triethylenemelamine, uracil derivative of nitrogen mustard, bis (3-chloropropyl) amine hydrochloride such as cyclohexylchloroethylnitrosourea, methylcyclohexylchloroethylnitrosourea, 1- (2-chloroethyl) -3- (2,6-dioxo-3-piperidyl) -1-nitrosourea, bis (2-chloroethyl) nitroso urea, procarbazine, dacarbazine, nitrogen mustard derivatives such as mechlorethamine, cyclophosphamide, ifosamide, melphalan, chlorambucil, estramustine sodium phosphate, streptozocin, and temozolamide. DNA antimetabolites, e.g. such as 5-fluorouracil, cytosine arabinoside, hydroxyurea, 2 [(3-hydroxy-2-pyrinodinyl) methylene] hydrazinecarbothioamide, deoxyfluorouridine, 5-hydroxy-2-formylpyridine thiosemicarbazone, alpha-2'-deoxy-6-glycyanidine , 5-azadeoxycytidine, betathioguanine deoxyriboside, cyclocytidine, guanazole, inosine glycodialdehyde, macbecin II, pyrazolimidazole, cladribine, pentostatin, thioguanine, mercaptopurine, bleomycin, 2-chlorodeoxyadenosine, thymidylate synthase inhibitors such as raltitrexed and disodium pemetrexed, clofarabine, floxuridine and fludarabine. DNA / RNA antimetabolites, e.g. L-alanosine, 5-azacytidine, acivicin, aminopterin and derivatives thereof such as N- [2-chloro-5 - [[(2,4-diamino-5-methyl-6-quinazolinyl) methyl] amino] benzoyl] -L -aspartic acid, N- [4 - [[(2,4-diamino-5-ethyl-6-quinazolinyl) methyl] amino] benzoyl] -Lasparaginic acid, N- [2-chloro-4 - [[(2, 4-diaminopteridinyl) methyl] amino] benzoyl] -L-aspartic, Baker antifol solution, dichloroallyl lawsone, brechinar, phtoraph, dihydro-5-azacytidine, methotrexate, tetrasodium N (phosphonoacetyl) -L-aspartic acid addition salt, pyrazofuran, trimetrexate, plicamycin, actinomycin D, cryptophycin, and analogs such as cryptophycin-52 or, e.g. one of the preferred antimetabolites disclosed in European Patent Application EP 239362 such as EP 239362 N- (5- [N- (3,4-Dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl) -N-methylamino] -2tenoyl) -L-glutamic acid; growth factor inhibitors; cell cycle inhibitors; intercalation antibiotics, e.g. adriamycin and bleomycin; proteins, e.g. interferon; and antihormones, e.g. antiestrogens such as Nolvadex ™ (tamoxifen) or, e.g. antiandrogens such as Casodex ™ (4 'cyano-3- (4-fluorophenylsulfonyl) -2-hydroxy-2-methyl-3' (trifluoromethyl) propionanilide ). Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment.

[0023] Agents that inhibit angiogenesis include MMP-2 (Matrix Metalloproteinase 2) inhibitors, MMP-9 (Matrix Metalloproteinase-9) inhibitors and COX-II (cyclooxygenase II) inhibitors. Examples of useful COX-II inhibitors include CELEBREX ™ (alecoxib), valdecoxib, and rofecoxib. Examples of useful extracellular matrix metalloproteinase inhibitors are described in WO 96/33172 (published October 24, 1996) and WO 96/27583 (published March 7, 1996), in European patent applications No. EP 97304971.1 (filed July 8, 1997) and EP 99308617.2 (filed October 29, 1999), in international patent applications WO 98/07697 (published February 26, 1998), WO 98/03516 (published January 29, 1998), WO 98/34918 (published August 13, 1998), WO 98/34915 (published August 13, 1998), WO 98/33768 (published August 6, 1998), WO 98 / 30566 (published July 16, 1998), in European patents EP 606,046 (published July 13, 1994), EP 931,788 (published July 28, 1999), in international patent applications WO 90/05719 (published May 31, 1990), WO 99/52910 (published October 21, 1999), WO 99/52889 (published October 21, 1999), WO 99/29667 (published June 17, 1999), in POT International Patent Application No. PCT / IB98 / 01113 ( filed July 21, 1998), European patent application EP 99302232.1 (filed March 25, 1999), British patent application GB 9912961.1 (filed June 3, 1999), US provisional patent application US US North America No. 60/148464 (filed August 12, 1999), in U.S. Pat. US US 5,863,949 (published January 26, 1999), US 5,861,510 (published January 19, 1999), and European Patent EP 780,386 (published June 25, 1997), the disclosures of which are herein incorporated by reference in their entirety. Preferred MMP-2 and MMP-9 inhibitors are those that show little or no MMP-1 inhibitory activity. More preferred are those inhibitors that selectively inhibit MMP-2 and / or MMP-9 relative to other matrix metalloproteinases (i.e. MMP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP -7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13).

[0024] Examples of MMP inhibitors include AG-3340, RO 32-3555, RS 13-0830, and the following compounds: 3- [[4- (4-fluorophenoxy) benzenesulfonyl] - (1-hydroxycarbamoylcyclopentyl) amino] propionic acid; 3-exo-3- [4- (4-fluorophenoxy) benzenesulfonylamino] -8-oxabicyclo [3.2.1] octane-3-carboxylic acid hydroxyamide; (2R, 3R) -1- [4- (2-chloro-4-fluorobenzyloxy) benzenesulfonyl] -3-hydroxy-3-methylpiperidine-2-carboxylic acid hydroxyamide; 4- [4 (4-fluorophenoxy) benzenesulfonylamino] tetrahydropyran-4-carboxylic acid hydroxyamide; 3- [[4- (4-fluorophenoxy) benzenesulfonyl] - (1-hydroxycarbamoylcyclobutyl) amino] propionic acid;

4- [4- (4-chlorophenoxy) benzenesulfonylamino] tetrahydropyran-4-carboxylic acid hydroxyamide; 3 [4- (4-chlorophenoxy) -benzenesulfonylamino] tetrahydropyran-3-carboxylic acid hydroxyamide; (2R, 3R) -1- [4- (4-fluoro-2-methylbenzyloxy) benzenesulfonyl] -3-hydroxy-3-methyl-piperidine-2-carboxylic acid hydroxyamide; 3 - [[4- (4-fluorophenoxy) benzenesulfonyl] - (1-hydroxycarbamoyl-1-methylethyl) amino] propionic acid; 3 - [[4- (4-fluorophenoxy) benzenesulfonyl] - (4-hydroxycarbamoyltetrahydropyran-4-yl) amino] propionic acid;

3-exo-3- [4- (4-chlorophenoxy) benzenesulfonylamino] -8-oxabicyclo [3.2.1] octane-3-carboxylic acid hydroxyamide; 3-endo-3- [4- (4-fluorophenoxy) benzenesulfonylamino] -8-oxabicyclo [3.2.1] octane-3-carboxylic acid hydroxyamide; 3- [4- (4-fluorophenoxy) benzenesulfonylamino] tetrahydrofuran-3-carboxylic acid hydroxyamide; and the pharmaceutically acceptable salts, solvates and hydrates thereof.

[0025] Examples of signal transduction inhibitors include agents that can inhibit EGFR (epidermal growth factor receptor) responses such as EGFR antibodies, EGF antibodies, and molecules that are inhibitors of EGFR; VEGF (vascular endothelial growth factor) inhibitors; and erbB2 receptor inhibitors such as organic molecules or antibodies that bind to the erbB2 receptor, e.g. HERCEPTIN35 ™ (Genentech, Inc. of South San Francisco, California, USA). EGFR inhibitors are described e.g. in international patent applications WO 95/19970 (published July 27, 1995), WO 98/14451 (published April 9, 1998), WO 98/02434 (published January 22, 1998), and in U.S. Patent No. . US No. 5,747,498 (issued May 5, 1998). EGFR inhibitors include, but are not limited to, monoclonal antibodies C225 and antiEGFR 22Mab (ImClone Systems Incorporated of New York, New York, USA), compounds ZD-1839 (AstraZeneca), ΒΙΒΧ-1382 (Boehringer Ingelheim), MDX-447 (Medarex Inc., Annandale , New Jersey, USA), and OLX-103 (Merck &amp; Co., Whitehouse Station, NJ, USA), VRCTC-310 (Ventech Research), and an EGF fusion toxin (Seragen Inc., Hopkinton, Massachusetts).

[0026] VEGF inhibitors, e.g., SU-5416 and SU-6668 (Sugen Inc., South San Francisco, California, USA), may also be combined or administered simultaneously with the composition. VEGF inhibitors are described e.g. in international patent application WO 99/24440 (published May 20, 1999), in international patent application PCT PCT / IB99 / 00797 (filed May 3, 1999), in international patent applications WO 95/21613 (published August 17, 1995) ), WO 99/61422 (published December 2, 1999), in US Patent No. US No. 5,834,504 (published November 10, 1998), in WO 98/50356 (published November 12, 1998), in US Pat. US Nos. 5,883,113 (published March 16, 1999), 5,886,020 (published March 23, 1999), US 5,792,783 (published August 11, 1998), in international patent applications WO 99/10349 (published March 4, 1999), WO 97 / 32856 (published September 1997), WO 97/22596 (published June 26, 1997), WO 98/54093 (published December 31, 998), WO 98/02438 (published January 22, 1998), WO 99/16755 (published April 8, 1999), and WO 98/02437 (published January 22, 1998), the disclosures of which are herein incorporated by reference in their entirety. Other examples of some specific VEGF inhibitors include IM862 (Cytran Inc., Kirkland, Washington, USA); anti-VEGF monoclonal antibody bevacizumab (Genentech, Inc., South San Francisco, California); and angiozyme, a synthetic ribozyme from Ribozyme (Boulder, Colorado) and Chiron (Emeryville, California).

[0027] ErbB2 receptor inhibitors such as GW-282974 (Glaxo Wellcome pic), and monoclonal antibodies AR-209 (Aronex Pharamceuticals Inc., The Woodlands, Texas, USA) and 2B-551 (Chiron) may be administered in combination with composition. Such erbB2 inhibitors include those described in WO 98/02434 (published January 22, 1998), WO 99/35146 (published July 15, 1999), WO 99/35132 (published July 15, 1999), WO 98/02437 (published January 22, 1998), WO 97/13760 (published April 17, 1997), WO 95/19970 (published July 27, 1995), in US Pat. US US 5,587,458 (published December 24, 1996), and US 5,877,305 (published March 2, 1999), the disclosures of which are hereby incorporated by reference in their entirety. ErbB2 receptor inhibitors useful in the present invention are also described in US provisional patent applications US 60/117341, filed January 27, 1999, and 60/117346, filed January 27, 1999, the disclosures of which are hereby incorporated by reference in their entirety.

[0028] Other anti-proliferative agents that can be used include inhibitors of the enzyme farnesyl protein transferase and inhibitors of the receptor tyrosine kinase PDGFr, including the compounds disclosed and claimed in the following US patent applications. US America:

09/221946 (filed December 28, 1998); 09/454058 (filed December 2, 1999); 09/501163 (filed February 9, 2000); 09/539930 (filed March 31, 2000); 09/202796 (filed May 22, 1997); 09/384339 (filed August 26, 1999); and 09/383755 (filed August 26, 1999); and the compounds disclosed and claimed in the following US provisional patent applications US Americas: US 60/168207 (filed November 30, 1999); 60/170119 (filed December 10, 1999); 60/177718 (filed January 21, 2000); 60/168217 (filed November 30, 1999), and 60/200834 (filed May 1, 2000). Each of the foregoing patent applications and provisional patent applications are herein incorporated by reference in their entirety.

The compositions of the invention can also be used with other agents useful in the treatment of abnormal cell growth or cancer, including, but not limited to, agents capable of enhancing anti-tumor immune responses such as CTLA4 (cytotoxic lymphocyte antigen 4) antibodies, and other agents capable of increasing anti-tumor immune responses. for blocking CTLA4; and anti-proliferative agents such as other farnesyltransferase inhibitors. Specific CTLA4 antibodies that can be used in the present invention include those described in US Provisional Patent Application No. US 60/113647 (filed December 23, 1998), the entire contents of which are hereby incorporated by reference.

Definition of e

[0030] Unless otherwise indicated, the following terms used in the specification and claims have the meanings discussed below. The variables described in this section, such as R, X, n, etc., are included in this section for reference only, and used outside of this section may have different meanings. In addition, many of the groups described herein can be optionally substituted. The list of typical substituents provided throughout the definition is exemplary and is not intended to limit the selection of substituents described elsewhere in the specification and claims.

[0031] The term "alkyl" refers to saturated aliphatic hydrocarbon substituents including straight and branched chain groups of 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, more preferably 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 up to 4 carbon atoms. The term "lower alkyl" refers to an alkyl of 1 to 4 carbon atoms. Examples of such alkyls include methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, etc. Alkyl can be substituted or unsubstituted. Typical substituents include cycloalkyl, aryl, heteroaryl, heteroalicycle, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halocarbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl , N-amido, C-carboxy, O-carboxy, nitro, silyl, amino, and -NR^XRY, with R.^x and RY are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, acetyl, sulfonyl, trifluoromethanesulfonyl, and a linked, five or six membered teteroalicyclic ring.

[0032] The term "cycloalkyl refers to a 3 to 8 membered monocyclic ring containing only carbon atoms, a 5/6 membered or 6/6 membered fused bicyclic ring containing only carbon atoms, or a multicyclic fused ring (" fused ring system means that each ring in the system shares an adjacent pair of carbon atoms with any other ring in the system), wherein one or more of the rings may contain one or more double bonds but none of the rings has a fully conjugated pi electron system. Non-limiting examples of cycloalkyl! include cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, adamantane, cycloheptane, cycloheptatriene, etc. The cycloalkyl may be substituted or unsubstituted. Typical substituents include such as alkyl, aryl, heteroaryl, heteroalicycle, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halocarbonyl, thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl, C-amido, N -amido, nitro, amino and -NR^XRY, with R.^x and RY are as defined above. Illustrative examples of cycloalkyls include, but are not limited to, the following:

<img file="PL1786785T3_D0003.tif" />

<img file="PL1786785T3_D0004.tif" />

[0033] The term "alkenyl" refers to an alkyl, as defined herein, containing at least two carbon atoms and at least one carbon-carbon double bond. Representative examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl, etc.

[0034] The term "alkynyl refers to an alkyl, as defined herein, containing at least two carbon atoms and at least one carbon-carbon triple bond. Representative examples include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-, 2-, or 3-butynyl, etc.

[0035] The term "aryl" refers to a monocyclic or fused, cyclic, polycyclic, fully conjugated pi-electron system of 6 to 12 carbon atoms. Non-limiting examples of aryls include phenyl, naphthalenyl, and anthracenyl. The aryl can be substituted or unsubstituted. Typical substituents include halogen, trihalomethyl, alkyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl, sulfonyl, amino and -NR^XRY, with R.^x and RY are as defined above.

[0036] The term "heteroaryl" refers to a monocyclic or fused ring of 5 to 12 atoms containing one, two, three or four heteroatoms selected from N, O, and S, the remaining ring atoms are carbon atoms, furthermore, it is a completely coupled pi-electron system. Non-limiting examples of unsubstituted heteroaryls are pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, purine, tetrazole, triazine, and carbazole. The heteroaryl can be substituted or unsubstituted. Typical substituents include alkyl, cycloalkyl, halogen, trihalomethyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, nitro, carbonyl, thiocarbonyl, sulfonoamido, C-carboxy, O-carboxy, sulfinyl, sulfonyl, O- carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, amino and -NR^XRY, with R.^x and RY are as defined above.

[0037] A pharmaceutically acceptable heteroaryl is a heteroaryl that is stable enough to be attached to a compound of the invention, incorporated into a pharmaceutical composition, and then administered to a patient in need thereof.

[0038] Non-limiting examples of typical monocyclic heteroaryls include:

^about θ ά furan thiophene pyrrole, r: -i \ pyrazole (furanyl) (thiophenyl) (pyrrolyl) (pyrazolyl)

HO OS

N ^N u /) V qw Ww isoxazole oxazole isothiazole imidazole (isoxazolyl) (oxazolyl) (isothiazolyl) (imidazolyl)

<img file="PL1786785T3_D0005.tif" />

l-oxa-2,3-diazole (l-oxa-2,3-diazolyl) l-thia-2,3-diazole (l-thia-2,3-diazolyl) ύ

thiazolyl (thiazolyl)

<img file="PL1786785T3_D0006.tif" />

l-oxa-2,4-diazole (l-oxa-2,4-diazolyl)

<img file="PL1786785T3_D0007.tif" />

l-thia-2,4-diazole (l-thia-2,4-diazolyl)

<img file="PL1786785T3_D0008.tif" />

pyridine (pyridinyl)

[0039] condensed

HNZ ^XN

1,2,3-triazole (1,2,3-triazolyl)

O l-oxa-2,5-diazole (l-oxa-2,5-diazolyl)

S Ν N 1-thia-2,5-diazole (1-thia-2,5-diazolyl)

U pyridazine (pyridazinyl)

H.

N

ABOUT

1,3,4-triazole (1,3,4-triazolyl) ύ

1-oxa-3,4-diazole (1-oxa-3,4-diazolyl) ύ

l-thia-3,4-diazole (l-thia-3,4-diazolyl)

<img file="PL1786785T3_D0009.tif" />

pyrimidine

Non-limiting annular

<img file="PL1786785T3_D0010.tif" />

benzofuran benzothiophene (benzofuranyl) (benzothiophenyl)

<img file="PL1786785T3_D0011.tif" />

tetrazole (tetrazolyl)

N ί dr pyrazine (pyrimidinyl) (pyrazinyl) examples of suitable heteroaryls include:

<img file="PL1786785T3_D0012.tif" />

111) indole benzimidazole (indolyl) (benzimidazo21

<img file="PL1786785T3_D0013.tif" />

<img file="PL1786785T3_D0014.tif" />

Η

<img file="PL1786785T3_D0015.tif" />

Η

<img file="PL1786785T3_D0016.tif" />

H indazole (indazolyl) benzotriazole (benzotriazolyl) pyrrolo [2,3b] pyridine (pyrrolo [2,3-b] pyridinyl) pyrrolo [2,3c] pyridine (pyrrolo [2,3-c] pyridinyl)

<img file="PL1786785T3_D0017.tif" />

<img file="PL1786785T3_D0018.tif" />

H.

<img file="PL1786785T3_D0019.tif" />

H.

<img file="PL1786785T3_D0020.tif" />

H pyrrolo [3,2-c] - pyrrolo [3,2-b] - imidazo [4,5-b pyridine pyridine] pyridine (pyrrolo [3,2-c] - (pyrrolo [3,2-b] - (imidazo [4,5-pyridinyl) pyridinyl) b] pyridinyl) imidazo [4,5-c] pyridine (imidazo [4,5c] pyridinyl)

<img file="PL1786785T3_D0021.tif" />

H.

<img file="PL1786785T3_D0022.tif" />

pyrazolo [4,3-d] pyridine (pyrazolo [4,3-d] pyridinyl)

<img file="PL1786785T3_D0023.tif" />

isoindole (isoindolyl) pyrazolo [4,3c] pyridine (pyrazolo [4,3-c] pyridinyl)

<img file="PL1786785T3_D0024.tif" />

indazole (indazolyl) pyrazolo [3,4c] pyridine (pyrazolo [3,4 - c] pyridinyl)

<img file="PL1786785T3_D0025.tif" />

H purine (purinyl) pyrazolo [3,4b] pyridine (pyrazolo [3,4-b] pyridinyl)

<img file="PL1786785T3_D0026.tif" />

indolizine (indolininyl)

<img file="PL1786785T3_D0027.tif" />

<img file="PL1786785T3_D0028.tif" />

imidazo [1,2a] pyridine (imidazo [1,2a] pyridinyl) imidazo [1.5a] pyridine (imidazo [1,5- a] pyridinyl) pyrazolo [1.5a] pyridine (pyrazolo [1,5-a] pyridinyl) pyrrolo [1,2b] pyridazine (pyrrolo [1,2b] pyridazinyl

<img file="PL1786785T3_D0029.tif" />

imidazo [1,2c] pyrimidine (imidazo [1,2c] pyrimidinyl)

<img file="PL1786785T3_D0030.tif" />

<img file="PL1786785T3_D0031.tif" />

<img file="PL1786785T3_D0032.tif" />

<img file="PL1786785T3_D0033.tif" />

quinoline (quinolinyl) isoquinoline (isoquinolinyl cinnoline (cinnolinyl) quinazoline (azachinazoline)

<img file="PL1786785T3_D0034.tif" />

<img file="PL1786785T3_D0035.tif" />

<img file="PL1786785T3_D0036.tif" />

<img file="PL1786785T3_D0037.tif" />

quinoxaline (quinoxalinyl phthalazine (phthalazinyl)

1,6naphthyridine (1,6naphthyridinyl)

1,7-naphthyridine (1,7-naphthyridinyl)

<img file="PL1786785T3_D0038.tif" />

<img file="PL1786785T3_D0039.tif" />

<img file="PL1786785T3_D0040.tif" />

1,8-naphthyridine (1,8-naphthyridinyl)

1,5-naphthyridine (1,5-naphthyridinyl)

2,6-naphthyridine (2,6-naphthyridinyl)

2,7-naphthyridine (2,7-naphthyridinyl)

<img file="PL1786785T3_D0041.tif" />

<img file="PL1786785T3_D0042.tif" />

<img file="PL1786785T3_D0043.tif" />

<img file="PL1786785T3_D0044.tif" />

pyrido [3,2d] pyrimidine (pyrido [3,2—

d] pyrimidinyl) pyrido [4,3d] pyrimidine (pyrido [4,3d] pyrimidinyl) pyrido [3,4-pyrido [2,3d] pyrimidine d] pyrimidine (pyrido [3,4- (pyrido [2,3d]) pyrimidinyl)

d] pyrimidinyl)

<img file="PL1786785T3_D0045.tif" />

<img file="PL1786785T3_D0046.tif" />

<img file="PL1786785T3_D0047.tif" />

<img file="PL1786785T3_D0048.tif" />

pyrido [2,3-b] pyrazine (pyrido [2,3-b] pyrazinyl) pyrido [3,4-b] pyrazine (pyrido [3,4-b] pyrazinyl) pyrimido [5,4d] pyrimidine (pyrimido [ 5,4d] pyrimidinyl) pyrazino [2,3b] pyrazine (pyrazine [2,3 b] pyrazinyl)

<img file="PL1786785T3_D0049.tif" />

pyrimido [4.5d] pyrimidine (pyrimido [4.5d] pyrimidinyl)

[0040] The term "heteroalicyclic or" heterocycle refers to a monocyclic or fused ring of 3 to 12 atoms in which one or two atoms are heteroatoms selected from N, O, and S (O)_n (where n is 0, 1 or 2), with the remaining ring atoms being carbon. The rings may also contain one or more double bonds. However, the rings do not have a completely conjugated pi-electron system.

Non-limiting examples of suitable saturated heteroalicycles include:

<img file="PL1786785T3_D0050.tif" />

<img file="PL1786785T3_D0051.tif" />

<img file="PL1786785T3_D0052.tif" />

oxirane (oxiranyl) tirane (tiranyl) aziridine (aziridinyl)

<img file="PL1786785T3_D0053.tif" />

thietane (tietanyl) azetidine (azetidinyl) tetrahydrofuran (tetrahydrofuranyl)

H.

<img file="PL1786785T3_D0054.tif" />

<img file="PL1786785T3_D0055.tif" />

<img file="PL1786785T3_D0056.tif" />

pyrrolidine (pyrrolidinyl) tetrahydropyran

<img file="PL1786785T3_D0057.tif" />

(tetrahydropyranyl) tetrahydrothiopyran (tetrahydrothiopyranyl) HN oxetane (oxetanyl) -tetrahydrothiophene (tetrahydrothiophenyl) HN piperidine (piperidinyl)

<img file="PL1786785T3_D0058.tif" />

<img file="PL1786785T3_D0059.tif" />

<img file="PL1786785T3_D0060.tif" />

1,4-dioxane (1,4-dioxanyl)

1,4-oxathate (1,4-oxathanyl) morpholine (morpholinyl)

1,4-dithian (1,4-dithianyl)

<img file="PL1786785T3_D0061.tif" />

<img file="PL1786785T3_D0062.tif" />

1,4-azathate (1,4-azathanyl)

<img file="PL1786785T3_D0063.tif" />

<img file="PL1786785T3_D0064.tif" />

oxepane (oxepanyl)

<img file="PL1786785T3_D0065.tif" />

<img file="PL1786785T3_D0066.tif" />

tiepan (tiepanyl)

<img file="PL1786785T3_D0067.tif" />

H azepan (azepanyl)

1,4-dioxepane (1,4-dioxepanyl)

1,4-oxatiepanyl (1,4-oxatiepanyl)

1,4-oxazepane (1,4-oxazepanyl)

<img file="PL1786785T3_D0068.tif" />

1,4-ditiepan (1,4-ditiepanyl)

<img file="PL1786785T3_D0069.tif" />

1,4-thiazepane (1,4-thiazepanyl)

H.

<img file="PL1786785T3_D0070.tif" />

H.

1,4-diazepane (1,4-diazepa [0041] nyl)

Non-limiting examples of suitable partially unsaturated heteroalicycles include:

<img file="PL1786785T3_D0071.tif" />

<img file="PL1786785T3_D0072.tif" />

^0

<img file="PL1786785T3_D0073.tif" />

3,4-dihydro2H-pyran (3,4-dihydro2H-pyranyl)

5,6-dihydro2H-pyran (5,6-dihydro2H-pyranyl)

2H-pyran (2H-pyranyl)

Η Η

Ο ο

1,2,3,4-tetra- 1,2,5,6-tetrahydropyridine hydropyridine (1,2,3,4-tetra- (1,2,5,6-tetrahydropyridinyl) hydropyridinyl)

[0042] The heterocycle is optionally substituted with one or two substituents independently selected from halogen, lower alkyl, lower alkyl substituted with carboxy, hydroxyester, or mono or dialkylamino.

[0043] The term "hydroxy refers to the group -OH.

[0044] The term "alkoxy" refers to both an -O- (alkyl) and an -O- (unsubstituted cycloalkyl) moiety. Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, etc.

[0045] The term "haloalkoxy" refers to an -O- (haloalkyl) group. Representative examples include, but are not limited to, trifluoromethoxy, tribromomethoxy, etc.

[0046] The term "aryloxy" refers to -O-aryl or -O-heteroaryl as defined herein. Representative examples include, but are not limited to, phenoxy, pyridinyloxy, furanyloxy, thienyloxy, pyrimidinyloxy, pyrazinyloxy, and the like, and derivatives thereof.

[0047] The term "mercapto" refers to a -SH moiety.

[0048] The term "alkylthio refers to an -S- (alkyl) or -S- (unsubstituted cycloalkyl) moiety.

Representative examples include, but are not limited to, methylthio, ethylthio, propylthio, butylthio, cyclopropylthio, cyclobutylthio, cyclopentylthio, cyclohexylthio, etc.

[0049] The term "arylthio refers to -S-aryl or -S-heteroaryl as defined herein. Representative examples include, but are not limited to, phenylthio, pyridinylthio, furanylthio, thienylthio, pyrimidinylthio, and the like and derivatives thereof.

The term "acyl or" carbonyl refers to the group -C (O) R, where R is selected from the group consisting of hydrogen, lower alkyl, trihalomethyl, unsubstituted cycloalkyl, aryl optionally substituted with one or more, preferably one , two or three substituents selected from the group consisting of lower alkyl, trihalomethyl, lower alkoxy, halogen and -NR^XRV, heteroaryl (linked via a ring carbon) optionally substituted with one or more, preferably one, two, or three substituents selected from the group consisting of lower alkyl, trihaloalkyl, lower alkoxy, halogen and -NR^XRV and heteroalicyclic (linked via a ring carbon) optionally substituted with one or more, preferably one, two or three substituents selected from the group consisting of lower alkyl, trihaloalkyl, lower alkoxy, halogen and -NR^XRV. Representative acyls include, but are not limited to, acetyl, trifluoroacetyl, benzoyl, etc.

[0051] The term "aldehyde" refers to an acyl where R is hydrogen.

[0052] The term "thioacyl or" thiocarbonyl refers to the group -C (S) R, where R is as defined above.

[0053] The term "thiocarbonyl refers to a -C (S) R moiety, where R is as defined above.

[0054] The term "C-carboxy" refers to the moiety

-C (O) OR, where R is as defined above.

[0055] The term "O-carboxy" refers to the moiety

-OC (O) R, where R is as defined above.

[0056] The term "ester" refers to the -C (O) OR moiety, wherein R is as defined herein, except that R cannot be hydrogen.

[0057] The term "acetyl refers to the group -C (O) CH₃.

[0058] The term "halo" refers to a fluorine, chlorine, bromine or iodine atom, preferably a fluorine or chlorine atom.

[0059] The term "trihalomethyl" refers to methyl having three halo substituents, such as trifluoromethyl.

[0060] The term "cyano refers to the group -C = N.

[0061] The term "sulfinyl" refers to the group -S (O) R where, in addition to the meaning defined above, R may also be hydroxy.

[0062] The term "sulfonyl" refers to the moiety -S (O) gR where, in addition to the meaning defined above, R may also be hydroxy.

[0063] The term "S-sulfonamido" refers to the group -S (O) 2NR^xRY, where R.^x and RY are as defined above.

[0064] The term "N-sulfonamido" refers to an -NR moiety^XS (O) 2 ^ ' ⁱⁿ which R.^x and RY are as defined above.

[0065] The term "O-carbamyl refers to the moiety -OC (O) NR^XRY, where R.^x and RY are as defined above.

[0066] The term "N-carbamyl" refers to the RYOC (O) NR moiety^X- where R.^x and RY are as defined above.

[0067] The term "O-thiocarbamyl refers to the group -OC (S) NR^XRY, where R.^x and RY are as defined above.

[0068] The term "N-thiocarbamyl" refers to the RYOC (S) NR moiety^X- where RY and R^x have the meaning defined above.

[0069] The term "amino refers to an -NR moiety^XRY, where R.^x and RY are hydrogen.

[0070] The term "C-amido" refers to the group -C (O) NR^XRY, where R.^x and RY are as defined above.

[0071] The term "N-amido refers to the R group^xC (O) NRy-, where R.^x and RY are as defined above.

[0072] The term "nitro refers to an -NO moiety₂·

[0073] The term "haloalkyl means alkyl, preferably lower alkyl, i.e. substituted with one or more, same or different halogens, e.g. -CH₂C1, -CF₃, -CH₂CF₃, -CH₂CC1₃, etc.

[0074] The term "hydroxyalkyl means alkyl, preferably lower alkyl, i.e. substituted with one, two, or three hydroxy substituents; e.g., hydroxymethyl, 1 or 2-hydroxyethyl, 1,2-, 1,3-, or 2,3-dihydroxypropyl, etc.

[0075] The term "aralkyl" means alkyl, preferably lower alkyl, i.e. substituted with aryl as defined above; e.g. -CH₂-phenyl, - (CH₂)₂-phenyl, - (CH₂)₃phenyl, CH₃CH (CH₃) CH₂-phenyl, etc. and their derivatives.

[0076] The term "heteroarylalkyl means alkyl, preferably lower alkyl, i.e. substituted with heteroaryl; e.g. -CH₂-pyridinyl, - (CH₂)₂-pyrimidinyl, - (CH₂)₃-imidazolyl, etc., and derivatives thereof.

[0077] The term "monoalkylamino" denotes an -NHR substituent where R is alkyl or unsubstituted cycloalkyl; e.g. methylamino, (1-methylethyl) amino, cyclohexylamino, etc.

[0078] The term "dialkylamino" means -NRR where each R is independently alkyl or unsubstituted cycloalkyl; such as dimethylamino, diethylamino, (1-methylethyl) ethylamino, cyclohexylmethylamino, cyclopentylmethylamino, etc. The term "optional or" optionally means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and cases in which it does not. By way of example, the term "heterocycle optionally substituted with alkyl means that alkyl may or may not be present, and the description includes situations where the heterocycle is alkyl substituted and situations where the heterocycle is not alkyl substituted.

[0079] The term "pharmaceutical composition" refers to a mixture of one or more compounds described herein, or physiologically / pharmaceutically acceptable salts, solvates, hydrates or prodrugs thereof, with other chemical ingredients such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration of the compound to the body.

[0080] As used herein, the term "physiologically / pharmaceutically acceptable carrier refers to a carrier or diluent that does not cause significant irritation to the body and does not cancel out the biological activity and properties of the administered compound.

[0081] The term "pharmaceutically acceptable excipient refers to an inert substance added to a pharmaceutical composition to facilitate administration of the compound. Non-limiting examples of the excipients include calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0082] The term "pharmaceutically acceptable salt as used herein" refers to a salt that retains the biological effectiveness and properties of the parent compound. Such salts include:

(1) acid addition salts obtainable by reaction of the free base of the parent compound with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, perchloric acid and the like, or with organic acids such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid or malonic acid etc .; or (2) salts formed by replacing an acidic proton present in the parent compound with a metal ion, e.g., an alkali metal ion, an alkaline earth ion, an aluminum ion; or by coordination bonding with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucosamine, etc.

[0083] As used herein, the term "PK refers to receptor protein tyrosine kinases (RTKs), non-receptor or" cellular tyrosine kinases (CTKs), and serine / threonine kinases (STKs)).

[0084] As used herein, the terms "modulation or modulation" refer to altering the catalytic activity of RTKs, CTKs and STKs. In particular, modulation refers to the activation of the catalytic activity of RTKs, CTKs and STKs, preferably to the activation or inhibition of the catalytic activity of RTKs, CTKs and STKs, depending on the concentration of the compound or salt to be treated with RTK, CTK or STK or, more preferably, to inhibition of the catalytic activity of RTKs, CTKs and STKs.

[0085] As used herein, the term "catalytic activity refers to the rate of tyrosine phosphorylation under the influence, directly or indirectly, of RTKs and / or CTKs, or the phosphorylation of serine and threonine under the influence, directly or indirectly, of STKs.

[0086] As used herein, the term "contacting refers to bringing a compound of the invention into contact with a target PK in such a way that the compound can affect the catalytic activity of the PK, either directly, i.e. by interacting with the kinase itself, or indirectly, i.e. , by interaction with another molecule on which the catalytic activity of the kinase depends. Such "contacting" can be carried out in vitro, ie in a test tube, a petri dish or the like. In a tube, contacting may include only the compound and the PK of interest, or may include whole cells. Cells can also be maintained or grown in cell culture vessels and contacted with a compound in that environment. In this context, the ability of a particular compound to influence a PK-related disorder is tested, ie, the IC50 value of a compound, as defined below, can be determined prior to using the compounds in vivo on more complex living organisms. For cells outside the body, there are a number of methods that are well known to those skilled in the art for bringing PK kinases into contact with compounds including, but not limited to, direct cell microinjection and numerous transmembrane transport techniques.

[0087] As used herein, the term "in vitro" refers to methods performed in an artificial environment such as, but not limited to, a test tube or culture medium.

[0088] The term "in vivo" as used herein refers to methods carried out inside a living organism such as, but not limited to, a mouse, rat, or rabbit.

[0089] As used herein, the terms "PK related disorder," PK mediated disorder, and "abnormal PK activity refer to conditions characterized by inappropriate, i.e., too low or, more commonly, too high, catalytic PK activity, where a particular PK it can be RTK, CTK or STK. Inappropriate catalytic activity can occur as a result of either: (1) PK expression in cells that normally do not express PKs, (2) increased PK expression leading to undesirable cell proliferation, differentiation and / or growth, or (3) decreased PK expression leading to an undesirable reduction in cell proliferation, differentiation and / or growth. Excessive PK activity refers to either enhancing a gene encoding a particular PK kinase or producing a level of PK activity that may correlate with abnormal cell proliferation, differentiation, and / or growth (i.e., as PK levels increase, one or more symptoms increase in severity). cellular disorders). Too low activity is, of course, the opposite, in which the severity of one or more symptoms of a cellular disorder increases as the level of PK activity decreases.

[0090] As used herein, the terms "treat," treatment, and "therapy" refer to a method of ameliorating or abolishing a cellular disorder mediated by PK and / or its attendant symptoms. When referring specifically to cancer, these terms simply mean that the average length of a cancer sufferer will be increased, or that one or more symptoms of the disease will be reduced.

[0091] The term "organism" as used herein refers to any living entity comprised of at least one cell. A living organism can be as simple as, for example, a single eukaryotic cell, or as complex as a mammal, including a human.

[0092] The term "therapeutically effective amount as used herein refers to that amount of a compound the administration of which will alleviate to some extent one or more symptoms of the disorder treated. With regard to the treatment of cancer, a therapeutically effective amount refers to that amount that produces at least one of the following effects:

(1) reducing tumor size;

(2) inhibiting (ie, slowing to some extent, preferably stopping) tumor metastasis;

(3) inhibiting to some extent (ie, slowing to some extent, preferably arresting) tumor growth, and (4) relieving to some extent (or, preferably, eliminating) one or more symptoms associated with cancer.

[0093] As used herein, the term "monitoring refers to observing or detecting the effect of contacting a compound with a cell on the expression of a particular PK kinase. The observed or detected effect may be a change in the cell phenotype, in the catalytic activity of the PK, or a change in the interaction of the PK with its natural binding partner. Techniques for observing or detecting such influences are well known in the art. The effect is selected from a change or no change in the phenotype of a cell, a change or no change in the catalytic activity of said protein kinase, or a change or no change in the interaction of said protein kinase with a natural binding partner, in a final aspect of the invention.

[0094] As used herein, the term "phenotype of a cell" refers to the outward appearance of a cell or tissue or the biological function of a cell or tissue. Examples, but not limited to, of a cell phenotype include cell size, cell growth, cell proliferation, cell differentiation, cell survival, apoptosis, and nutrient uptake and use. Such phenotypic properties are measured using techniques well known in the art.

[0095] As used herein, the term "natural binding partner" refers to a polypeptide that binds to a particular PK in a cell. Natural binding partners may be important in propagating a signal in a PK-mediated signal transduction process. A change in the interaction of a natural binding partner with a PK may manifest itself as an increased or decreased concentration of the PK complex / natural binding partner and consequently as an observable change in the ability of a PK kinase to mediate signal transduction.

[0096] As used herein, the terms "optically pure," enantiomerically pure, "pure enantiomer," and "optically pure enantiomer" refer to a composition that contains one enantiomer of a compound and substantially free of the opposite enantiomer of that compound. A typical optically pure compound comprises greater than about 80% by weight of one enantiomer of the compound and less than about 20% by weight of the opposite enantiomer of the compound, more preferably greater than about 90% by weight of one enantiomer of the compound, and less than about 10% by weight of the opposite enantiomer of the compound, even more preferably greater than about 95% by weight of one compound. the enantiomer of the compound and less than about 5% by weight of the opposite enantiomer of the compound. and most preferably greater than about 97% by weight of one enantiomer of the compound and less than about 3% by weight of the opposite enantiomer of the compound.

Detailed description

[0097] General schemes for the synthesis of compounds of the invention are provided in the examples.

[0098] Some of the general methods are shown for the synthesis of compounds where the 1- (2,6-dichloro-3-fluorophenyl) ethoxy moiety is the pure (R) isomer and some are shown for compounds where said moiety it is a racemic mixture.

It should be understood that by selecting the appropriate racemic or enantiomerically pure starting materials, these methods can be used to synthesize racemic compounds or enantiomerically pure (R) isomers.

[0099] The methods set out herein can be used to synthesize a wide variety of enantiomerically pure compounds by selecting the appropriate enantiomerically pure starting materials. Furthermore, the invention also relates to enantiomerically pure compounds corresponding to compounds such as 3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-2-ylamine and 3 [1- (2,6-dichloro 3-fluorophenyl) ethoxy] pyrazin-2-ylamine described in US Pat. US North America No. US 10/786610 (PCT / US2004 / 005495); in U.S. Patent No. US Of America assigned application number PC 32546, filed on August 26, 2004 and entitled "PyrazoloSubstituted Aminoheteroaryl Compounds as Protein Kinase Inhibitors; and in U.S. Patent No. US Of America assigned application number PC 32548, filed August 26, 2004 and entitled "Aminoheteroaryl Compounds as Protein Kinase Inhibitors. The full disclosures of these documents are hereby incorporated by reference.

[0100] Unless otherwise indicated, all references for compounds of the invention include references to their salts, solvates, hydrates, and complexes, and to solvates, hydrates, and salt complexes thereof, including their polymorphs, stereoisomers, and isotope-labeled analogs thereof.

[0101] Pharmaceutically acceptable salts include acid addition and base salts (including disalts). Suitable acid addition salts are formed from acids which form non-toxic salts. Examples are acetate, aspartate, benzoate, benzenesulfonate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptan, gluconate, glucuronate, hexafluorophosphate, hibenzatebenzoate (4- (4) ), hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malesate, maleate, malonate, mesylate, methylsulfate, naphthate, 2napsylate, nicotinate, nitrate, orotate (1,2,3,6-tetrahydro-2,6-dioxopyrimidine-4-carboxylate), oxalate, palmitate, pamamate, phosphate / dibasic / dihydrogen phosphate, sucrose, stearate, succinate, tartrate, tosylate and trifluoroacetate.

[0102] Suitable base salts are formed from bases which form non-toxic salts. Examples are aluminum, calcium, arginine, benzathine, choline, diethylamine, diolamine, glycine, lysine, magnesium salts, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts.

[0103] For a review of suitable salts, see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002), the disclosure of which is herein incorporated by reference in its entirety.

[0104] A pharmaceutically acceptable salt of a compound of the invention can be readily prepared by mixing appropriate solutions of the compound and the desired acid or base. The salt can crash out of solution and can be collected by filtration or can be recovered by evaporating the solvent. The degree of ionization in the resulting salt can vary from completely ionized to almost non-ionized.

[0105] The compounds of the invention may exist in both solvated and unsolvated forms. The term "solvate" is used to describe a molecular complex comprising a compound of the invention and one or more pharmaceutically acceptable solvent molecules, e.g., ethanol. The term "hydrate" is used when that solvent is water. Pharmaceutically acceptable solvates according to the invention include hydrates and solvates, and the crystallization solvent may be isotopic substituted, e.g., with DgO, dg-acetone, dg-DMSO.

Also included within the scope of the invention are complexes such as clathrates, drug-host inclusion complexes, in contrast to the aforementioned solvates, the drug and host are present in stoichiometric or non-stoichiometric amounts. The invention also includes drug complexes containing two or more organic and / or inorganic components, which may be present in stoichiometric or non-stoichiometric amounts. The resulting complexes can be ionized, partially ionized or non-ionized. Such complexes are reviewed in J Pharm Sci, 64 (8), 1269-1288 published by Haleblian (August 1975), the disclosure of which is herein incorporated by reference in its entirety.

[0107] Also included within the scope of the invention are polymorphs, prodrugs, and isomers (including optical, geometric, and tautomeric isomers) of the compounds of the invention.

[0108] Derivatives of the compounds of the invention showing little or no pharmacological activity may, when administered to a patient, be converted into the compounds of the invention, e.g. by hydrolytic cleavage. Such derivatives are referred to as "prodrugs." For more information on the use of prodrugs, see "Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (T. Higuchi and W. Stella) and in "Bioreversible Carriers in Drug Design, Pergamon Press, 1987 (Ed. EB Roche, American Pharmaceutical Association), the disclosures of which are herein incorporated by reference in their entirety.

[0109] Prodrugs of the invention can, for example, be prepared by replacing the corresponding functional groups present in the compounds of the invention with certain groups known to those skilled in the art as "pro-groups as described, e.g., by H. Bundgaard in" Design of Prodrugs (Elsevier, 1985). ), the disclosure of which is herein incorporated by reference in its entirety.

[0110] Some examples of prodrugs of the invention include:

(i) an ester thereof, when the compound contains a carboxyl function (-COOH), e.g. by replacing a hydrogen atom with (C 1 -C₈) alkyl;

(ii) its ether, when the compound contains an alcohol function (-OH), e.g. by replacing the hydrogen atom with (C₃-C6) alkanoyloxymethyl; and (iii) an amide thereof, when the compound contains a primary or secondary amine functionality (-NH6 or -NHR where RH), e.g. by replacement of one or both of the hydrogen atoms with (C1-C6) alkanoyl.

[0111] Further examples of group replacement according to the above examples and examples of other types of prodrugs can be found in the aforementioned references.

[0112] Finally, certain compounds of the invention may themselves act as prodrugs of other compounds of the invention.

[0113] Compounds of the invention containing one or more asymmetric carbon atoms can exist as two or more stereoisomers. When a compound of the invention contains an alkenyl or alkenylene group, geometric cis / trans (or Z / E) isomers are also possible. When a compound contains, for example, a ketone, oxime, or aromatic group, tautomeric isomerism ("tautomerism") may occur. A single compound may exhibit more than one type of isomerism.

[0114] Included within the scope of the invention are all stereoisomers, geometric isomers and tautomeric forms of the compounds of the invention, including compounds exhibiting more than one type of isomerism, and mixtures thereof. Also included within the scope of the invention are acid addition salts or base salts wherein the counterion is optically active, e.g. D-lactate or L-lysine, or racemates, e.g. DL-tartrate or DL-arginine.

[0115] The cis / trans isomers may be separated using conventional techniques well known to those skilled in the art, such as, for example, chromatography and fractional crystallization.

[0116] Conventional techniques for the preparation / separation of individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of a racemate (or a racemate of salts or derivatives) using, e.g., chiral high pressure liquid chromatography (HPLC).

[0117] Alternatively, the racemate (or racemic precursor) may be reacted with a suitable optically active compound, e.g. an alcohol, or, where the compound contains an acidic or basic group, with an acid or base such as tartaric acid or 1-phenylethylamine. The resulting diastereomeric mixture can be separated by chromatography and / or fractional crystallization and one or both of the diastereoisomers can be converted to the corresponding pure enantiomer (s) by methods well known to those skilled in the art.

[0118] The chiral compounds of the invention (and their chiral precursors) can be obtained in enantiomerically enriched form using chromatography, typically HPLC, on an asymmetric resin with a hydrocarbon, typically heptane or hexane mobile phase, containing from 0 to 50% isopropanol, typically 2 to 20%, and 0 to 5% alkylamine, typically 0.1% diethylamine. After concentrating the eluate, an enriched mixture is obtained.

[0119] Stereoisomeric conglomerates may be separated using conventional techniques known to those skilled in the art; see, for example, "Stereochemistry of Organic Compounds EL Eliel (Wiley, New York, 1994), the disclosure of which is herein incorporated by reference in its entirety.

[0120] The invention also includes isotopically labeled compounds of the invention in which one or more atoms are replaced by an atom having the same atomic number but an atomic weight or mass number other than the predominant atomic mass or mass number normally predominant in nature. Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen such as carbon such as HC, 1¾ and 14q, chlorine such as fluorine such as ΙθΕ, iodine such as 123j and 125j, nitrogen such as 1¾ and 15 ^^ oxygen, such as ^ - ^ 0, and Ιθο, phosphorus, such as 22p, and sulfur, such as 25g. Certain isotopically labeled compounds of the invention, for example, those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. Due to their ease of incorporation and easy means of detecting them, the radioactive isotopes of tritium and carbon-14 are particularly useful for this purpose. Substitution with heavier isotopes such as deuterium may provide some therapeutic benefits resulting from greater metabolic stability, e.g., increased half-life. in vivo or reducing the required doses and therefore may be beneficial in some circumstances. Substitution with positron emitting isotopes such as HC, ΙθΡ, 1 ^ 0, and 1¾ may be useful in positron emission tomography (PET) studies of the degree of receptor occupancy by a substrate.

[0121] Isotopic labeled compounds of the invention can generally be prepared by conventional techniques known to those skilled in the art or by methods analogous to those described herein, using appropriate isotopically labeled reagents in place of the previously used unlabeled reagent.

[0122] Pharmaceutically acceptable solvates of the invention include those in which the crystallization solvent may be isotopic substituted, e.g., DgO, dgacetone, dg-DMSO.

[0123] Compounds of the invention intended for pharmaceutical use may be administered as crystalline or amorphous products, or mixtures thereof. They can be obtained, for example, in the form of solid plugs, powders or films, by methods such as precipitation, crystallization, freeze drying, spray drying or evaporative drying. For this purpose, microwave or radiofrequency drying can be used.

[0124] The compounds may be administered alone or in combination with one or more other compounds of the invention, or in combination with one or more other drugs (or any combination thereof). Generally, they will be administered as a formulation with one or more pharmaceutically acceptable excipients. The term "excipient" is used to describe any ingredient other than the compound (s) of the invention. The choice of excipient will largely depend on factors such as the particular route of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[0125] Pharmaceutical compositions suitable for the delivery of compounds of the invention and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in "Remington's Pharmaceutical Sciences, 19 th edition (Mack Publishing Company, 1995), the disclosure of which is hereby incorporated by reference in its entirety.

Oral administration

[0126] The compounds of the invention may be administered orally. Oral administration can include swallowing so as to introduce the compound into the gastrointestinal tract, or buccal or sublingual administration can be used, by which the compound is introduced into the bloodstream directly from the oral cavity.

[0127] Formulations suitable for oral administration include solid preparations such as tablets, capsules containing particles, liquids or powders, lozenges (including filled lozenges), chewing preparations, multi- and nanoparticles, gels, solid solutions, liposomes, films (including mucoadhesive films), suppositories, sprays and liquid preparations.

[0128] Liquid preparations include suspensions, solutions, syrups and elixirs. Such preparations can be used as fillings in soft or hard capsules and typically include a carrier, e.g., such as water, ethanol, polyethylene glycol, propylene glycol, methyl cellulose, or a suitable oil, and one or more emulsifying and / or suspending agents. Liquid preparations can also be made by reconstituting a solid preparation, e.g. from a bag.

[0129] The compounds of the invention may also be used in rapidly dissolving, rapidly disintegrating dosage forms, such as those described in Expert Opinion in Therapeutic Patents, 11 (6), 981-986 by Liang and Chen (2001), the disclosure of which is appended by here it is entirely on a cross-reference basis.

[0130] For tablet dosage forms, depending on dose, the drug may make up from 1 wt% to 80 wt% of the dosage form, more typically from 5 wt% to 60 wt% of the dosage form. In addition to the drug, tablets generally contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, sodium croscarmellose, crospovidone, polyvinyl pyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl substituted hydroxypropyl cellulose, and sodium starch, pregelated starch. Generally, the disintegrant will make up from 1 wt% to 25 wt%, preferably from 5 wt% to 20 wt% of the dosage form.

[0131] Binders are generally used to achieve the desired cohesive properties in a tablet formulation. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose, and hydroxypropyl methylcellulose. Tablets may also contain diluents such as lactose (as mono hydrate, spray dried mono hydrate, anhydrous etc.), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch, and dibasic calcium phosphate dihydrate.

[0132] Tablets may also optionally contain a surfactant such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc. If present, the surfactant typically comprises 0.2 wt.% To 5 wt.% Of the tablet, and lubricants typically comprises 0.2 wt.% To 1 wt.% Of the tablet.

[0133] Tablets also generally contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulfate. Lubricants are generally present in an amount of from 0.25 wt% to 10 wt%, preferably from 0.5 wt% to 3 wt% of the tablet.

[0134] Other typical ingredients include antioxidants, dyes, flavors, preservatives, and taste-masking agents.

[0135] Exemplary tablets contain up to about 80 wt.% Drug, about 10 wt.% To about 90 wt.% Binder, about 0 wt.% To about 85 wt.% Diluent, about 2 wt.% To about 10 wt.% Disintegrant. , and from about 0.25 wt.% to about 10 wt.% of a lubricant.

[0136] Tablet blends may be compressed directly or on a roller into tablets. Tablet blends or portions of blends may alternatively be wet, dry or melt granulated, melt solidified, or extruded prior to tableting. The final formulation may contain one or more layers, and may be coated or uncoated; or encapsulated.

[0137] Tablet formulation is discussed in detail in Pharmaceutical Dosage Forms: Tablets, Vol. 1, H. Lieberman and L. Lachman, Marcel Dekker, NY, NY, 1980 (ISBN 0-82476918-Χ), the disclosure of which is hereby incorporated by reference in its entirety. on a cross-reference basis.

[0138] Solid formulations for oral administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release formulations.

[0139] Suitable modified release formulations are described in US Pat. US No. 6,106,864. Details on other suitable release techniques such as high energy dispersions and osmotic and coated particles can be found in: Verma et al., Pharmaceutical Technology On-line, 25 (2), 1-14 (2001). The use of chewing gum to achieve controlled release is described in the international patent application WO 00/35298. The disclosures of these references are hereby incorporated by reference in their entirety.

Parenteral administration

[0140] The compounds of the invention may also be administered directly into the bloodstream, intramuscularly, or into an internal organ. Suitable routes for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and infusion techniques.

Preparations for parenteral administration typically are aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers (preferably at a pH of 3 to 9), but, for some applications, these preparations may be more suitably formulated as sterile. a non-aqueous solution or in dry form for use in association with a suitable vehicle such as sterile pyrogen-free water.

[0142] Preparation of formulations for parenteral administration under sterile conditions, e.g. by lyophilization, can be readily accomplished using standard pharmaceutical techniques well known to those skilled in the art.

[0143] The solubility of the compounds of the invention used in the preparation of solutions for parenteral administration may be increased by the use of appropriate formulation techniques, such as the incorporation of solubilizers.

[0144] Formulations for parenteral administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release formulations. Thus, the compounds of the invention may be formulated as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot providing modified release of the active ingredient. Examples of such formulations include drug coated stents and PGLA microspheres.

Topical administration

[0145] The compounds of the invention may also be administered topically to the skin or mucosa, ie, dermally or transdermally. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin dressings, wafers, implants, sponges, fibers, bandages, and microemulsions. Liposomes can also be used. Typical carriers include alcohol, water, mineral oil, liquid paraffin, white petroleum, glycerin, polyethylene glycol, and propylene glycol. Penetration enhancers may also be added: see, e.g., J Pharm Sci, 88 (10). 955-958, Finnin and Morgan (October 1999). Other methods of topical administration include delivery by electroporation, iontophoresis, phonophoresis, sonophoresis, and microneedle or needle-free injection (eg Powderject ™, Bioject ™, etc.). The disclosures of these references are hereby incorporated by reference in their entirety.

[0146] Formulations for topical administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release formulations.

Inhalation / nasal administration

[0147] The compounds of the invention may also be administered intranasally or by inhalation, typically in the form of a dry powder (alone or as a mixture, e.g. in dry admixture with lactose, or as mixed particles of ingredients, e.g. mixed with phospholipids such as phosphatidylcholine) using a powder inhaler or as an aerosol spray using a pressurized container, pump, atomizer, atomizer (preferably an atomizer using electrohydrodynamic atomization to produce a fine mist), or a nebulizer, with or without a suitable propellant such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. For nasal use, the powder may contain a bioadhesive agent, e.g. chitosan or cyclodextrin.

[0148] The pressurized container, pump, atomiser, atomizer, or nebulizer contains a solution or suspension of a compound (s) of the invention containing, e.g., ethanol, an aqueous ethanol solution, or a suitable alternative agent to disperse, dissolve, or sustain release of the active ingredient. a propellant (s) as a solvent and an optional surfactant such as sorbitan trioleate, oleic acid, or oligolactic acid.

[0149] Before use in a powder or suspension formulation, the drug is micronized to a size suitable for inhalation administration (typically less than 5 microns). This may be achieved by using any suitable comminution method, such as spiral jet milling, fluid bed milling, supercritical fluid treatment to form nanoparticles, high pressure homogenization, or spray drying.

Capsules (made from e.g. gelatin or HPMC), blisters and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention, a suitable powder base such as lactose or starch, and an action modifier. such as l-leucine, mannitol, or magnesium stearate. Lactose may be anhydrous or in the form of the monohydrate, preferably in the latter form. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.

[0151] A suitable formulation solution for use in an atomizer using electrohydrodynamic spraying to produce a fine mist may contain from 1 µg to 20 mg of the compound of the invention per spray and the volume of one spray may vary from 1 µΐ to 100 µΐ. A typical formulation includes a compound of the invention, propylene glycol, sterile water, ethanol, and sodium chloride. Alternative solvents that can be used in place of propylene glycol include glycerol and polyethylene glycol.

[0152] Suitable flavoring agents, such as menthol and levomenthol, or sweetening agents, such as saccharin or sodium saccharin, may be added to those formulations of the invention to be administered by inhalation / nasal administration.

[0153] Formulations for inhalation / intranasal administration may be formulated to be immediate and / or modified release using, eg, poly (DL-lactoglycolic) acid (PGLA). Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release formulations.

[0154] In the case of powder inhalers and aerosols, the dosage unit form is determined by a valve that delivers a measured amount. The units of the invention are typically arranged to deliver a medicament in a metered dose or puff containing a desired amount of a compound of the invention. Generally, the daily dose can be administered in a single dose or, more typically, as divided doses throughout the day.

Rectal / vaginal administration

[0155] The compounds of the invention may be administered rectally or vaginally, eg, in the form of a suppository, pessary, or enema. Cocoa butter is a conventional suppository base, but various alternatives may also be used as appropriate.

[0156] Formulations for rectal / vaginal administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, target and programmed release formulations.

Ocular administration

[0157] The compounds of the invention may also be administered directly to the eye or ear, typically as drops of a micronized suspension or solution in isotonic, pH-adjusted, sterile saline. Other formulations suitable for ocular or ear administration include ointments, biodegradable (e.g., absorbable gel sponges, collagen) and non-biodegradable (e.g., silicone) implants, wafers, lenses, and partial or vesicular systems such as niosomes or liposomes. A polymer such as cross-linked polyacrylic acid, polyvinyl alcohol, hyaluronic acid, a cellulose polymer, e.g. hydroxypropylmethyl cellulose, hydroxyethyl cellulose, or methyl cellulose, or a heteropolysaccharide polymer, e.g. gellan gum, may be introduced together with a preservative such as benzalkonium chloride. Such preparations can also be delivered by iontophoresis.

[0158] Formulations for ocular / aural administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, target and programmed release formulations.

Other technologies

[0159] The compounds of the invention can be combined with soluble macromolecular units such as cyclodextrin and its corresponding derivatives or polyethylene glycol containing polymers to improve their solubility, dissolution rate, taste masking, bioavailability and / or stability for use in any of the following. before the types of administration.

[0160] For example, drug cyclodextrin complexes have been found to be generally useful for most dosage forms and administration routes. Both inclusion complexes and non-inclusion complexes can be used. As an alternative to direct complexation with the drug, cyclodextrin can be used as an auxiliary additive, i.e.

as a carrier, diluent, or solubilizer. For these purposes, alpha-, beta-, and gamma-cyclodextrins are most commonly used, examples of which can be found in PCT International Patent Applications Nos. WO 91/11172, WO 94/02518 and WO 98/55148, the disclosures of which are hereby incorporated by reference in their entirety on a cross-reference basis.

Dosage

[0161] The amount of active ingredient administered will depend on the treated patient, the severity of the disease or condition, the rate of administration, the disposition of the compound and the judgment of the prescribing physician. However, the effective dose is typically from about 0.001 to about 100 mg per kg of body weight per day, preferably from about 0.01 to about 35 mg / kg / day, in a single dose or in divided doses. For a 70 kg human, the dose may be from about 0.07 to about 7000 mg / day, preferably from about 0.7 to about 2500 mg / day. In some cases, lower dosage levels than the lower end of the aforementioned range may be more appropriate, while in other cases even higher doses may be used without causing any deleterious side effects, such higher doses typically being split into several smaller doses for administration. during the day.

Kit parts

[0162] Since it may be desirable to use a medicament as a combination of active ingredients, e.g., to treat a particular disease or condition, two or more pharmaceutical compositions of which at least one comprises a compound of the invention, which may be suitably combined with in the form of a kit suitable for the simultaneous administration of the composition. Thus, the kit of the invention comprises two or more separate pharmaceutical compositions, at least one of which contains a compound of the invention, and means for separately retaining said compositions, such as a container, divided bottle, or divided foil pouch.

An example of such a kit is the known blister pack used for the packaging of tablets, capsules etc.

[0163] The kit of the invention is particularly suitable for the administration of different dosage forms, e.g. oral and parenteral, for the administration of separate compositions at different dosing intervals, or for dispensing separate compositions against each other. To assist in adhering to proper dosing, the kit typically includes administration directions and may be accompanied by an abridged manual.

Examples

[0164] In the following examples, "Et is ethyl," Ac is acetyl, "Me is methyl," Ms is methanesulfonyl (CHgSO4), "iPr is isopropyl," HATU is 2- (7-aza-1H-benzotriazole hexafluorophosphate). 1-yl) 1,1,3,3-tetramethyluronium, "Ph is phenyl," Boc is tert-butoxycarbonyl, "EtOAc is ethyl acetate," HOAc is acetic acid, "NEtg or" EtgN is triethylamine, "THF is tetrahydrofuran , "DIC is diisopropylcarbodiimide," HOBt is hydroxybenzotriazole, "MeOH means methanol," 1-ProAc means isopropyl acetate, "KOAc means potassium acetate," DMSO means dimethyl sulfoxide, "AcCl ™ means acetyl chloride," CDClg means deuterated chloroform, "MTBE means t-butyl methyl ether," DMF means dimethylformamide , "AcgO is acetic anhydride," MegSOI is trimethylsulfoxonium iodide, "DMAP is 4-dimethylaminopyridine," dppf is diphenylphosphine ferrocene, "DME is ethylene glycol dimethyl ether, HOBT is 1-hydroxybenzotriazole, EDO is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.

[0165] The following examples are provided to illustrate the invention. It should be understood, however, that the invention is not limited to the specific conditions or details described in these examples.

[0166] The reagents can be synthesized by the methods set forth herein, and can be obtained from commercially available sources (e.g., Aldrich, Milwaukee, WI; Acros, Morris Plains, NJ; Biosynth International, Naperville, It; Frontier Scientific, Logan, UT; TCI America) , Portland, OR; Combi-Blocks, San Diego, CA; Matrix Scientific, Columbia, SC; Acros, Morris Plains, NJ; Alfa Aesar, Ward Hill, MA; Apollo Scientific, UK; etc.) or can be synthesized by known methods in the field.

[0167] A method to synthesize several specific reagents is described in US Patent No. US America's Pin. US 10/786610 entitled "Aminoheteroaryl Compounds as Protein Kinase Inhibitors, filed February 26, 2004, and international application PCT / US2004 / 005495 ​​of the same title, filed February 26, 2004, respectively. Other reagents can be synthesized by suitably adapting the methods set forth herein. One skilled in the art can readily adapt these methods to produce the desired compounds. In addition, these references contain general methods and specific examples for the preparation of many heteroarylamine compounds, and one skilled in the art can readily adapt such methods and examples to prepare the compounds of the present invention. The disclosures of these references are hereby incorporated by reference in their entirety.

[0168] Based on the general or exemplary synthetic procedure described, one skilled in the art can readily determine suitable reagents, if not indicated. Some of the general methods are given as examples for the preparation of specific compounds. One skilled in the art can readily adapt such methods to synthesize other compounds. It should be understood that the R groups described in the general methods are conventional and non-limiting and do not correspond to the definitions of the R groups given elsewhere in this document. Each such R group represents one or more semenemic moieties which may be the same or different from other chemical moieties also represented by the same R symbol. One skilled in the art can readily recognize a suitable range of R groups in the exemplary synthetic methods.

Furthermore, depiction of unsubstituted positions in the formulas depicted, or in the formulas referenced by the general methods, is provided for convenience, and does not exclude substitution described elsewhere in the description. Specific groups that may appear either as R groups in general methods or as optional substituents not shown refer to the descriptions elsewhere in this document, including the claims, the spirit and the detailed description.

[0169] Some of the general methods are shown for the synthesis of compounds where the 1- (2,6-dichloro-3-fluorophenyl) ethoxy group is the pure (R) isomer and some are shown for compounds where the above group is a mixture racemic. It should be understood that these methods can be used to generate racemic compounds or enantiomerically pure (R) isomers by selecting the appropriate racemic or enantiomerically pure starting materials.

[0170] By selecting the appropriate enantiomerically pure starting materials, the methods described herein can be used to produce a wide variety of enantiomerically pure compounds. Furthermore, the invention also relates to enantiomerically pure compounds corresponding to compounds such as 3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-2-ylamine and 3- [1- (2,6-dichloro-3- -fluorophenyl) ethoxy] pyrazine-2-ylamine described in US Patent No. US America's Pin. No. US 10/786610 (PCT / US2004 / 005495); in U.S. Patent No. US America assigned application number PC 32546, filed August 26, 2004 and entitled "Pyrazolo-Substituted Aminoheteroaryl Compounds as Protein Kinase Inhibitors; and in U.S. Patent No. US Of America assigned application number PC 32548, filed August 26, 2004 and entitled "Aminoheteroaryl Compounds as Protein Kinase Inhibitors. The disclosures of these documents are incorporated herein by reference in their entirety.

Selection of starting substances

5-bromo-3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-2-ylamine (racemate):

[0171]

Br

<img file="PL1786785T3_D0074.tif" />

1. 2,6-dichloro-3-fluoroacetophenone (15 g, 0.072 mol) was stirred in THF (150 mL, 0.5M) at 0 ° C using an ice bath for 10 minutes. Lithium aluminum hydride (2.75 g, 0.072 mol) was slowly added and the reaction mixture was stirred at ambient temperature for 3 hours. The mixture was cooled in an ice bath, water (3 mL) was slowly added dropwise, then 15% NaOH solution (3 mL) was slowly added. The reaction mixture was stirred at ambient temperature for 30 minutes. A 15% NaOH solution (9 mL) and MgSO4 were added and the mixture was filtered to remove solids. The solid was washed with THF (50 ml) and the filtrate concentrated to give 1- (2,6-dichloro-3-fluorophenyl) ethanol (14.8 g, 95% yield) as a yellow oil.³1 H NMR (400 MHz, DMSO-d₆) δ 1.45 (d, 3H), 5.42 (m, 2H), 7.32 (m, 1H), 7.42 (m, 1H).

2. To a mixed solution of triphenylphosphine (8.2 g, 0.03 mol) and DEAD (13.65 ml of a 40% solution in toluene) in THF (200 ml) at 0 ° C was added a solution of 1- (2,6-dichloro 3-fluorophenyl) ethanol (4.55 g, 0.021 mol) and 3-hydroxynitropyridine (3.35 g, 0.023 mol) in THF (200 ml). The resulting light orange solution was stirred under nitrogen at ambient temperature for 4 hours as a result of which all starting materials were consumed. The solvent was removed, the crude material was directly loaded onto silica gel and eluted with ethyl acetate-hexanes (20:80) to give 3- (2,6-dichloro-3-fluorobenzyloxy) -2-nitropyridine (6.21 g, 0.021 mol, 98%) as a pink solid. ⁸H NMR (CDCl₃, 300 MHz) δ 1.8-1.85 (d, 3H), 6.0-6.15 (q, 1H), 7.0-7.1 (t, 1H), 7.2-7, 21 (d, 1H), 7.25-7.5 (m, 2H), 8.0-8.05 (d, 1H).

3. 3- (2,6-Dichloro-3-fluorobenzyloxy) -2-nitropyridine (9.43 g, 0.028 mol) and iron turnings (15.7 g, <RTI ID = 0.0>) were suspended in a stirred mixture of AcOH (650 mL) and EtOH (500 mL). 0.28 mol). The reaction mixture was slowly warmed to reflux and allowed to stir for 1 hour. The reaction mixture was cooled to room temperature, then diethyl ether (500 ml) and water (500 ml) were added. The solution was carefully neutralized by adding sodium carbonate. The combined organic extracts were washed with a saturated solution of NaHCO3 (2 x 100 mL), HgO (2 x 100 mL), and brine (1 x 100 mL), then dried (Na2SO4), filtered and concentrated to dryness in vacuo to give 3- (2,6-dichloro-3-fluorobenzyloxy) pyridin-2-ylamine (9.04 g, 0.027 mol, 99%) as a light pink solid. ⁸H NMR (CDCl3, 300 MHz) δ 1.8-1.85 (d, 3H), 4.9-5.2 (br s, 2H), 6.7-6.84 (q, 1H), 7, 07.1 (m, 1H), 7.2-7.3 (m, 1H), 7.6-7.7 (m, 1H).

4. A mixed solution of 3- (2,6-dichloro-3-fluorobenzyloxy) pyridin-2-ylamine (9.07 g, 0.03 mol) in acetonitrile was cooled to 0 ° C with an ice bath. To this solution was added N-bromosuccinimide (NBS) (5.33 g, 0.03 mol) in portions. The reaction mixture was stirred at 0 ° C for 15 minutes then concentrated to dryness under reduced pressure. The resulting dark oil was dissolved in EtOAc (500 mL) and purified by silica gel chromatography. The solvents were then removed under reduced pressure to give 5-bromo-3- (2,6-dichloro-3-fluorobenzyloxy) pyridin-2-ylamine (5.8 g, 0.015 mol, 51%) as a white crystalline solid. ⁸H NMR (CDCl3, 300 MHz) δ 1.85-1.95 (d, 3H), 4.7-5.0 (br s, 2H), 5.9-6.01 (q, 1H), 6, 8-6.95 (d, 1H), 7.017.2 (t, 1H), 7.4-7.45 (m, 1H), 7.8-7.85 (d, 1H).

5-iodo-3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-2-ylamine (racemate):

[0172]

AND

<img file="PL1786785T3_D0075.tif" />

[0173] To a solution of 3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-2-ylamine (10.0 g, 33.2 mmol) in acetonitrile (600 mL) and acetic acid (120 ml), N-iodosuccinimide (11.2 g, 49.8 mmol) was added. The reaction mixture was stirred at room temperature for 4 hours and a cold NagSgOg solution was added quickly. After evaporation, the residue was partitioned between ethyl acetate and water. The organic layer was washed with 2N NaOH, brine and dried over Na2SO4. The crude product was purified on a silica gel column to give 5-iodo-3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-2-ylamine (7.1 g, 50% yield). MS m / z 427 [M + 1]. 1 h NMR (400 MHz, DMSO-D6) δ ppm 1.74 (d, J = 6.57 Hz, 3H) 5.91-5.99 (m, 3H) 6.82 (d, J = 1.10 26 Hz, 1H) 7.46 (t, J = 8.72 Hz, 1H) 7.56 (dd, J = 8.97, 4.93 Hz, 1H) 7.62 (d, J = 1.52 Hz, 1H).

5-bromo-3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyrazin-2-ylamine (racemate):

[0174]

Br

<img file="PL1786785T3_D0076.tif" />

1. 2,6-dichloro-3-fluoroacetophenone (15 g, 0.072 mol) was stirred in THF (150 mL, 0.5M) at 0 ° C using an ice bath for 10 minutes. Lithium aluminum hydride (from Aldrich, 2.75 g, 0.072 mol) was slowly added and the reaction mixture was stirred at ambient temperature for 3 hours. The mixture was cooled in an ice bath, water (3 mL) was added dropwise, then 15% NaOH solution (3 mL) was slowly added. The reaction mixture was stirred at ambient temperature for 30 minutes, 15% NaOH solution (9 mL), MgSO4 was added and the mixture was filtered to remove solids. The solid was washed with THF (50 ml) and the filtrate concentrated to give 1- (2,6-dichloro-3-fluorophenyl) ethanol (14.8 g, 95% yield) as a yellow oil. 1 H NMR (400 MHz, DMSO-d 6) δ 1.45 (d, 3H), 5.42 (m, 2H), 7.32 (m, 1H), 7.42 (m, 1H).

2. 5-bromo-3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyrazin-2-ylamine was prepared following procedure 2 given below, starting from 1- (2,6-dichloro-3-fluorophenyl) ) ethanol and 3,5-dibromopyrazin-2-ylamine. 1 H NMR (400 MHz, DMSO-d₆) δ 1.74 (d, 3H), 6.40 (m, 1H), 6.52 (brs, 2H), 7.30 (m, 1H), 7.48 (m, 1H), 7.56 (s. 1H); MS m / z 382 (M + 1).

Enantiomerically pure starting materials

[0175] PLE is an enzyme manufactured by Roche and marketed by Biocatalitics Inc. in the form of crude porcine liver esterase, commonly known as PLE-AS (purchased from Biocatalitics as ICR-123, sold as an ammonium sulfate slurry). The enzyme is classified in the CAS registry as a "carboxylic-ester hydrolase, CAS No. 9016-18-6." This enzyme has an EC number of 3.1.1.1. The enzyme is known to be broadly specific to the hydrolysis of a wide range of esters. Lipase activity is determined from the hydrolysis of ethyl butyrate in a pH titrator. 1 LU (lipase unit) is the amount of enzyme which liberates 1 pmol of titratable butyric acid per minute at 22 ° C and pH 8.2. The preparation described herein (PLE-50AS, as a suspension) is typically provided as a cloudy brown-green liquid with a declared activity> 45 LU / mg (protein content around 40 mg / ml).

(1S) -1- (2,6-dichloro-3-fluorophenyl) ethanol

[0176] (1S) -1- (2,6-Dichloro-3-fluorophenyl) ethanol, shown as compound (S-1) in the schemes below, was prepared by the combined enzymatic hydrolysis of racemic 1- (2,6-dichloro) acetate -3-fluorophenyl) ethyl, esterification and inversion chemical hydrolysis according to scheme B. Racemic 1- (2,6-dichloro-3-fluorophenyl) ethyl acetate (compound A2) was prepared according to scheme A. Scheme A

<img file="PL1786785T3_D0077.tif" />

1- (2,6-Dichloro-3-fluorophenyl) ethanol (Al): For a solution of 2 ', 6'-dichloro-3'-fluoroacetophenone (Aldrich, cat no. 52, 294-5) (207 mg , 1 mmol) in 2 mL anhydrous CHgOH was added sodium borohydride (90 mg, 2.4 mmol). After stirring at room temperature for 1 hour, the reaction mixture was concentrated to a colorless oily residue. The residue was purified by flash chromatography (eluting with 0 to 10% EtOAc in hexanes) to give compound A1 as a colorless oil (180 mg; 0.88 mmol; 86.5% yield); MS (APCI) (MH) "208; 1H NMR (400 MHz, chloroform-D) δ ppm 1.64 (d, J = 6.82 Hz, 3H) 3.02 (d, J = 9.85 Hz, 1H) 6.977.07 (m, 1H) 7.19-7.33 (m, 1H).

1- (2,6-Dichloro-3-fluorophenyl) ethyl acetate (A2): To a solution of compound Al (2.2 g, 10.5 mmol) in 20 mL of CHgCl3 was added acetic anhydride (1.42 mL , 15 mmol) and pyridine (1.7 mL, 21 mmol). The reaction mixture was stirred at room temperature for 12 hours, then the solvent was evaporated to leave a yellowish oily residue. The residue was purified by flash chromatography (eluting with 7 to 9% EtOAc in hexanes) to give Compound A2 as a colorless oil (2.26 g;

9.0 mmol; 85.6% yield); NMR (400 MHz, chloroform-D) δ ppm 1.88 (d, J = 6.82 Hz, 3H) 2.31 (s, 3H) 6.62 (q, J = 6.62 Hz, 1H) 7 , 25 (t, J = 6.46 Hz, 1H) 7.49 (dd, J = 8.84, 5.05 Hz, 1H).

Scheme B

<img file="PL1786785T3_D0078.tif" />

A2 s-2 Rl

<img file="PL1786785T3_D0079.tif" />

[0179] In a 50 mL jacketed flask equipped with a pH electrode, mechanical stirrer, and base addition channel (IM NaOH), 12 mL of 100 mM phosphate buffer (potassium phosphate) pH 7.0 and 0.13 mL of PLE AS suspension were placed. . Then, compound A2 (0.13 g, 0.5 mmol, 1.00 eq.) Was added dropwise and the resulting reaction mixture was stirred at room temperature for 20 hours, keeping the pH of the reaction mixture constant at 7.0 using 1M NaOH solution. Conversion and enantiomeric excess were monitored by RP-HPLC, and the reaction was stopped after 50% consumption of the starting material (approximately 17 hours under these conditions). The mixture was then extracted three times with 10 mL of ethyl acetate to recover the ester and alcohol as a mixture of R1 and S-2.

Methanesulfonyl chloride (0.06 mL, 0.6 mmol) was added to a mixture of R1 and S-2 (0.48 mmol) in 4 mL of pyridine under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 3 hours, then the solvent was evaporated to an oil. Water (20 mL) was added to the mixture followed by EtOAc (20 mL x 2) to extract the aqueous solution. The organic layers were combined, dried, filtered and concentrated to give a mixture of compounds R-3 and S-2. This mixture was used in the next reaction step without further purification. 1H NMR (400 MHz, chloroform-D) δ ppm 1.66 (d, J = 7.1 Hz, 3H) 1.84 (d, J = 7.1 Hz, 3H) 2.09 (s, 3H) 2.92 (s, 3H) 6.39 (q, J = 7.0 Hz, 1H) 6.46 (q, J = 6.6 Hz, 1H) 6.98-7.07 (m, 1H) 7.07-7.17 (m, 1H) 7.23-7.30 (m, 1H) 7.34 (dd, J = 8.8, 4.80 Hz, 1H).

[0181] Potassium acetate (0.027 g, 0.28 mmol) was added to a mixture of compounds R-3 and S-2 (0.48 mmol) in 4 mL of DMF under a nitrogen atmosphere. The reaction mixture was heated at 100 ° C for 12 hours. Water (20 mL) and EtOAc (20 mL × 2) were added to the reaction mixture to extract the aqueous solution. The combined organic layers were dried, filtered and concentrated to give an oil of S-2 (72 mg, 61% yield over two steps). Enantiomeric excess: 97.6%. 1 H NMR (400 MHz, chloroform-D) δ ppm 1.66 (d, J = 7.1 Hz, 3H) 2.09 (s, 3H) 8.39 (q, J = 8.8 Hz, 1H ) 7.02 (t, J = 8.5 Hz, 1H) 7.22-7.30 (m, 1H).

[0182] Sodium methoxide (19 mmol; 0.5M methanol solution) was slowly added to compound S-2 (4.64 g, 18.8 mmol) under nitrogen at 0 ° C. The resulting reaction mixture was stirred at room temperature for 4 hours. The solvent was evaporated and HgO (100 ml) was added. The cooled reaction mixture was neutralized with sodium acetate-acetic acid buffer to pH 7. Ethyl acetate (100 mL × 2) was added to extract the aqueous solution. The combined organic layers were dried over NagSO 4, filtered, and concentrated to a white solid (4.36 g, 94.9% yield); SFC-MS: enantiomeric excess: 97%. ^ H NMR (400 MHz, chloroform-D) δ ppm 1.65 (d, J = 6.6 Hz, 3H) 5.58 (q, J = 6.6 Hz, 1H) 8.96-7.10 (m, 1H) 7.22-7.36 (m, 1H).

3 - [(IR) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -2-nitropyridine

[0183]

<img file="PL1786785T3_D0080.tif" />

C ^ N ^^ N ^

[0184] To a stirred solution of (1S) -1- (2,6-dichloro-3-fluoro-phenyl) ethanol (229.8 mg, 1.1 mmol) in THF (10 ml), under nitrogen atmosphere, 3-hydroxy -2-nitropyridine (175 mg, 1.21 mmol) and triphenylphosphine (440 mg, 1.85 mmol). The reaction mixture was kept at room temperature for 1 hour and then diisopropyl azodicarboxylate (0.34 mL, 1.65 mmol) was added at 0 ° C. The reaction mixture was stirred for an additional 12 hours, then the solvent was evaporated in vacuo to yield an oil. The residue was purified by flash chromatography (eluting with 20 to 25% EtOAc in hexanes) to give the title compound as a white solid (321.5 mg; 0.97 mmol; 88.3% yield); MS (ARC1) (M + H)⁺331; SFC-MS: enantiomeric excess: 99.5%. ^ H NMR (400 MHz, chloroform-D) δ ppm 1.85 (d, J = 6.6 Hz, 3H) 6.10 (q, J = 6.6 Hz, 1H) 7.04-7.13 (m, 1H) 7.21 (dd, J = 8.5, 1.14 Hz, 1H) 7.30 (dd, J = 9.0, 4.9 Hz, 1H) 7.37 (dd, J = 8.8, 4.6 Hz, 1H) 8.04 (dd, J = 4.8, 1.3 Hz, 1H).

3 - [(IR) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-2-amine

[0185]

<img file="PL1786785T3_D0081.tif" />

To a stirred solution of 3 - [(IR) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -2-nitropyridine (321 mg, 0.97 mmol) in a mixture of EtOH (2 mL) and 2M HCl ( 0.2 ml), at 0 ° C, iron (385 mg) was added. The resulting solution was heated to 85 ° C for 2 hours. Celite (0.5 g) was added to the cooled reaction mixture. This mixture was filtered through a bed of celite and the solvent was evaporated to give the title compound as a dark oil. MS (APCI) (M + H)⁺301.

5-bromo-3- [1 (R) - (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-2-ylamine:

[0187]

Br

<img file="PL1786785T3_D0082.tif" />

[0188] The enantiomerically pure R isomer was prepared by the method described above for the racemate but using the enantiomerically pure starting materials described above. ⁷H NMR (400 MHz, DMSO-d6) δ 1.74 (d, 3H), 6.40 (m, 1H), 6.52 (br s, 2H), 7.30 (m, 1H), 7.48 (m, 1H), 7.56 (s, 1H); MS m / z 382 (M + 1).

5-iodo-3 - [(R) 1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-2-ylamine:

[0189]

AND

<img file="PL1786785T3_D0083.tif" />

[0190] To a mixed solution of 3 - [(IR) -1- (2,6-dichloro-3-fluorophenyl) ethoxypyridin-2-amine (0.97 mmol) in a mixture of acetic acid (3 ml) and HgO (0.5 ml ) periodic acid (60 mg, 0.24 mmol), iodine (130 mg, 0.5 mmol) and sulfuric acid (0.03 ml) were successively added. The resulting solution was heated at 80 ° C for 5 hours. Cold NagSO4 (80 mg) was quickly added to the cooled reaction mixture and the mixture was neutralized with a saturated solution of NagCOg (2 x 100 ml) to pH 7. CHgCl3 (2 x 50 mL) was added to extract the aqueous solution. The combined organic layers were dried over NagSO 4 then filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with 35 to 40% EtOAc in hexanes) to give the title compound as a yellow oil (254 mg; 0.6 mmol; 61.6% yield); MS (APCI) (M + H)⁺ 426.

NMR (400 MHz, chloroform-D) δ ppm 1.81 (d, J = 6.8 Hz, 3H) 4.86 (s, 2H) 5.98 (q, J = 6.57 Hz, 1H) 6 .96 (d, J = 1.5 Hz, 1H) 7.08 (dd, J = 9.0, 8.0 Hz, 1H) 7.31 (dd, J = 8.8, 4.8 Hz, 1H) 7.78 (d, J = 1.8Hz, 1H).

5-bromo-3 - [(R) -1- (2,6-dicinloro-3-fluorophenyl) ethoxy] pyrazin-2-ylamine:

[0191]

Br

<img file="PL1786785T3_D0084.tif" />

[0192] The title compound was prepared following procedure 2 starting from (1S) -1- (2,6-dichloro-3-fluorophenyl) ethanol. 1H NMR (400MHz, DMSO-d₆) δ 7.53 (s, 1H), 7.48 (m, 1H), 7.39 (t, 1H), 6.48 (s, 2H), 6.41 (q, 1H), 1.74 (d, 3H); LCMS: 381 [M + 1], c-MET Ki: 0.796 µΜ.

General scheme I for the synthesis of 5-aryl-3- (substituted benzyloxy) pyridin-2-ylamine (6):

[0193]

<img file="PL1786785T3_D0085.tif" />

benzyloxy) pyridin-2-ylamine (5):

[0194]

1. Preparation of 3- (substituted benzyloxy) -2-nitropyridine (3): to a mixed solution of CsgCOg (1.0 molar eq.) In DMF (0.2 M) containing 3-hydroxy-4-nitropyridine (Aldrich, 1.0 molar eq. ) under Ng atmosphere, substituted benzyl bromide (1.0 molar equivalent) was added. The reaction mixture was stirred for 6 hours at ambient temperature, then diluted with EtOAc and partitioned with HgO. The aqueous layer was extracted twice with EtOAc. The organic layers were then combined, washed with HgO and brine, dried over NagSO 4, filtered, and concentrated to dryness under reduced pressure to give 3- (substituted benzyloxy) -2-nitropyridine (3) as a solid.

2. Preparation of 3- (substituted benzyloxy) pyridin-2-ylamine (4): in a mixture of AcOH and EtCH (1.3: 1), 3- (substituted benzyloxy) -2-nitropyridine (1.0 molar equivalent, 1 M) was suspended with stirring. ) and iron turnings (1.0 molar equivalent). The reaction mixture was slowly warmed to reflux and allowed to stir for 1 hour. The mixture was then cooled to room temperature and filtered through a celite pad. The resulting filtrate was neutralized with concentrated NH 4 OH solution and then extracted three times with EtOAc. The combined organic extracts were washed with saturated NaHCO 3, HgO, and brine, dried over NagSO 4, filtered, and concentrated to dryness in vacuo to afford 3- (substituted benzyloxy) pyridin-2-ylamine (4) as a solid.

3. Preparation of 5-bromo-3- (substituted benzyloxy) pyridin-2-ylamine (5): a mixed solution of 3- (substituted benzyloxy) pyridin-2-ylamine (4) (1.0 molar eq) in acetonitrile cooled to 0 ° C, using an ice bath. To this solution was added N-bromosuccinimide (Aldrich, 1.0 molar equivalent) in portions. The reaction mixture was stirred at 0 ° C for 15 minutes then concentrated under reduced pressure to dryness. The resulting dark oil was dissolved in EtOAc and partitioned using HgO. The organic layer was then washed twice with saturated NaHCOg solution and once with brine. Activated charcoal was added to the organic layer, and it was heated to reflux temperature. The solution was then cooled to room temperature and filtered through a pad of celite. The organic layer was then concentrated in vacuo to dryness to give one third of the original volume. The solid was then filtered off to give 5 bromo-3- (substituted benzyloxy) pyridin-2-ylamine (5) as a solid.

General scheme II for the synthesis of 5-aryl-3- (substituted benzyloxy) pyrazin-2-ylamine:

[0195]

<img file="PL1786785T3_D0086.tif" />

Pd (PPh₃) ₂C1₂DME / Na₂WHAT₃/ H₂About 80 ° C

ArB (OH)₂

<img file="PL1786785T3_D0087.tif" />

General Procedure 2 for the synthesis of 5-bromo-3- (substituted benzyloxy) pyrazin-2-ylamine:

[0196]

Br

<img file="PL1786785T3_D0088.tif" />

NH₂

[0197] Sodium hydride (1.0 molar equiv.) And anhydrous tetrahydrofuran (0.14 M) was slowly added to an ice-cooled solution of substituted benzyl alcohol (1.0 molar equivalent) under a nitrogen atmosphere. After stirring for 30 minutes, 3,5-dibromopyrazin-2-ylamine (1.0 molar eq) in tetrahydrofuran (0.56 M) was added quickly dropwise via an addition funnel. After the addition was complete, the ice bath was removed and the reaction mixture was refluxed under a nitrogen atmosphere and the progress of the reaction was monitored by reverse phase HPLC. After 18 hours, HPLC analysis showed that most of the starting 3,5-dibromopyrazin-2-ylamine had been consumed and the reaction was allowed to cool to room temperature. The reaction mixture was then concentrated, diluted with ethyl acetate, and washed with brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated under high vacuum. The crude product was purified over silica gel eluting with 1: 1 ethyl acetate / dichloromethane to give 5-bromo-3- (substituted benzyloxy) pyrazin-2-ylamine as a white solid. Yield: 60-90%.

General Procedure 3 for the synthesis of 5-aryl-3- (substituted benzyloxy) pyridin-2-ylamine and 5-aryl-3- (substituted benzyloxy) pyrazin-2-ylamine: [0198]

<img file="PL1786785T3_D0089.tif" />

Pd (PPh₃) ₂C1₂

DME / Na₂WHAT₃/ H₂ABOUT

Y: CH or N

80 ° C -------- ► arylboronic acid

<img file="PL1786785T3_D0090.tif" />

Ar

<img file="PL1786785T3_D0091.tif" />

[0199] A mixture of 5-bromo-3- (substituted benzyloxy) pyridin-2-ylamine or 5-bromo-3- (substituted benzyloxy) pyrazin-2-ylamine (1 molar eq ), aryl boronic acid or its ester (1 2 mole equiv.), Bis (triphenylphosphine) palladium II chloride (0.03 mole equiv.) And sodium carbonate (3.0 mole equiv.) In ethylene glycol dimethyl ether and water (10: 0.5, 0.03 M) were degassed purged with nitrogen three times and then heated to reflux, under nitrogen overnight. The reaction mixture was cooled to ambient temperature and diluted with ethyl acetate. The mixture was washed with water, brine, dried over Na2SO4 and purified on a silica gel column to afford 5-aryl-3 (substituted benzyloxy) pyridin-2-ylamine, or 5-aryl-3 (substituted benzyloxy) pyrazin-2-ylamine.

General Procedure 4 for the amidation reaction of 6-amino-5- (substituted benzyloxy) pyridin-3-yl] benzoic acid: [0200]

<img file="PL1786785T3_D0092.tif" />

[0201] To a solution of 6-amino-5- (substituted benzyloxy) pyridin-3-yl] benzoic acid (1 molar equivalent), 1-hydroxybenzotriazole hydrate (HOBT, 1.2 molar equivalents) and 1- (3-dimethylaminopropyl hydrochloride) ) -3-ethylcarbodiimide (EDC, 1.2 molar equivalents) in DMF (0.2 M) was added the amine (1.2 molar equivalents). The solution was stirred at room temperature overnight, then diluted with EtOAc, and partitioned using HgO. The organic layer was separated and the aqueous layer was extracted with EtOAc. The organic layers were combined, washed with saturated NaHCO 3, and concentrated under reduced pressure to dry. The material was purified by column chromatography (silica gel, 99: 1 to 95: 5 CHgCl 3 / MeOH). The product containing fractions were concentrated under reduced pressure to yield the amide product.

General procedure 5 for the preparation of 3- (substituted benzyloxy) -5- (3-dialkylaminomethyl-1H-indol-5-yl) pyridin-2-ylamine: [0202]

<img file="PL1786785T3_D0093.tif" />

[0203] To a solution of the benzotriazole (1.0 molar equivalent) in dichloromethane (0.2 M) was added the amine (1.0 molar equivalent). After the reaction mixture was stirred for 5 minutes at room temperature, formaldehyde (37 wt%, 1.0 molar equivalent) was added, the mixture was covered and stirred at room temperature for 3 hours. When TLC analysis (10% ethyl acetate: dichloromethane) indicated consumption of the starting benzotriazole, the mixture was dried with anhydrous magnesium sulfate (10 g), filtered and concentrated under reduced pressure. The crude product was purified on a silica gel column eluting with 1: 1 ethyl acetate: dichloromethane to afford the desired product as a white solid.

Aluminum chloride (2.0 molar equivalent) was added to an intermediate solution of aminomethylbenzotriazole (1.0 molar equivalent) in dichloromethane (0.43M) followed by 3 (2,6-dichlorobenyloxy) -5- (1H-indole). -5-yl) pyridin-2-ylamine (1.1 molar equivalents). The mixture was covered and heated with stirring at 40 ° C for 3-4 hours.

The reaction mixture was then removed from the bath and allowed to cool to room temperature. The mixture was diluted with sodium hydroxide (0.2 M) and chloroform, covered again, and stirred vigorously at room temperature to dissolve any residue in the vial. The chloroform layer was separated from the water layer, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified on a silica gel column first eluting with 1: 1 ethyl acetate: dichloromethane to elute less polar impurities and then the product was eluted with 90: 9: 1 chloroform: methanol: ammonium hydroxide. (Yield 10-67%).

General procedure 6 for the synthesis of 3- (substituted benzyloxy) -5-phenyl-pyridin-2-ylamine using 3- (3-methoxybenzyloxy) -5-phenyl-pyridin-2-ylamine:

[0205]

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<img file="PL1786785T3_D0095.tif" />

[0206] To a solution of 3-benzyloxy-5-phenylpyridin-2-ylamine (example 1-87, 3.27 g, 11.8 mmol) in methanol (30 ml) was added Pd (OH) 2 (2.5 g, 2.37 mmol). The mixture was degassed and flushed three times with hydrogen, then stirred under a hydrogen balloon for 5 hours. The reaction mixture was filtered through celite, washed with methanol and concentrated. After drying in vacuo, it was obtained

2-amino-5-phenylpyridin-3-ol (2.04 g, 93% yield). MS m / z 187 [M + 1].

NaH (1.31 g, 32.85 mmol) was slowly added to a solution of 2-amino-5-phenylpyridin-3-ol (2.04 g, 10.95 mmol) in THF (anhydrous, 30 mL). The reaction mixture was stirred under nitrogen for 20 minutes and then trityl chloride (3.66 g, 13.14 mmol) was added. The reaction mixture was stirred under nitrogen at room temperature overnight. The solvent was evaporated, the residue was dissolved in dichloromethane, washed with water and dried over Na2SO4. After filtration and condensation, the crude product was purified on a silica gel column, eluting with EtOAc-hexane (1:10) to give 5-phenyl-2- (tritylamino) pyridin-3-ol (1.09 g, 23% yield). MS m / z 427 [M + 1].

[0208] To a solution of 5-phenyl-2- (tritylamino) pyridin-3-ol (100 mg, 0.24 mmol) in THF (3 mL) was added CsCOg (79 mg, 0.24 mmol). The reaction mixture was stirred at room temperature for 20 minutes, then 3-methoxybenzyl bromide (0.037 mL, 0.26 mmol) was added. The reaction mixture was stirred at room temperature overnight, diluted with dicloromethane (5 mL), and filtered to remove salt. The solvents were evaporated and the residue was dissolved in a 10% solution of trifluoroacetic acid in dicinloromethane (2 ml). The reaction mixture was stirred for 2 hours and evaporated, the residue was dissolved in dicloromethane, washed with saturated NaHCO 3 solution and dried over Na 2 SO 4. After filtration and concentration, the crude product was purified on a silica gel column, eluting with methanol: dichloromethane (gradient from 3% to 15% ) to give 3 (3-methoxybenzyloxy) -5-phenylpyridin-2-ylamine as a white solid (43.5 mg, 60% yield).

General Procedure 7 for the Synthesis of 3- (substituted benzyloxy) -5arylpyridin-2-ylamine using 5- [4- (2-morpholin-4-ylethoxy) phenyl] -3- (3-nitrobenzyloxy) pyridin-2-ylamine: [0209]

<img file="PL1786785T3_D0096.tif" />

WELL₂

[0210] To a solution of 2-amino-5- [4- (2-morpholin-4-ylethoxy) phenyl] pyridin-3-ol (prepared according to the preparation method of 2-amino-5-phenylpyridin-3-ol described in Example 1) In U.S. Patent No. 10/786610 (PCT / US2004 / 005495) (45.5 mg, 0.14 mmol) in DMF (3 ml), NaH (60% in oil) (5.6 mg, 0.14 mmol) and the reaction mixture was stirred at 0 ° C for 20 minutes. Then 1-bromomethyl-3-nitrobenzene was added and the reaction mixture was stirred at 0 ° C for 1 hour and at room temperature for 2 hours. Cold 1N aqueous HCl (0.1 mL) was added and the solvent was removed under reduced pressure. The residue was purified by chromatography on silica gel (CHgCl3: MeOH: NH4OH = 100: 3: 0.3) to obtain 5 [4 - (2-morpholin-4-ylethoxy) phenyl] -3- (3-nitrobenzyloxy) pyridin-2 -ylamine as a yellow solid (44 mg, 68%).

General Procedure 8 for the synthesis of {4- [6-amino-5- (substituted benzyloxy) pyridin-3-yl] phenyl} - [(2R) -2-pyrrolidin-1-ylmethylpyrrolidin-1-yl] methanone using {4- [6-amino-5- (4-fluoro-2-trifluoromethylbenzyloxy) pyridin-3-yl] phenyl} - [(2R) -2-pyrrolidin-1-ylmethyl-pyrrolidin-1-yl] methanone:

<img file="PL1786785T3_D0097.tif" />

by following procedure 3 with 3-benzyloxy-5-bromopyridin-2-ylamine and 4- (4,4,5,5-tetramethyl- [1,3,2] dioxaborolan-2-yl) benzoic acid. MS m / z 321 (M + 1).

2. [4- (6-Amino-5-benzyloxypyridin-3-yl) phenyl] - [(2R) 2-pyrrolidin-1-ylmethylpyrrolidin-1-yl] methanone was prepared by following procedure 4 using 6-amino-5-benzyloxynicotinic acid and (2R) -pyrrolidin-1-ylmethylpyrrolidine (prepared according to example 1-39 of US Patent No. 10/786610 (PCT / US2004 / 005495)).

MS m / z 457 (M + 1).

3. To a solution of [4- (6-amino-5-benzyloxypyridin-3-yl) phenyl] - [(2R) -pyrrolidin-1-ylmethylpyrrolidin-1-yl] methanone (2.28 g, 5.00 mmol) in methanol (25 ml) 10% Pd / C (100 mg) was added. The mixture was degassed, flushed three times with hydrogen, then stirred under a hydrogen balloon overnight. The reaction mixture was filtered through celite, washed with methanol and concentrated. After drying in vacuo, [4- (6-amino-5-hydroxypyridin-3-yl) phenyi] - [(2R) -2-pyrrolidin-1-ylmethyl-pyrrolidin-1-yl) methanone (1.74 g, yield 95%). NMR (400 MHz, DMSO-d6) δ 7.79 (s, 1H), 7.54 (m, 3H), 7.46 (m, 2H), 7.14 (s, 1H), 5.68 ( s, 2H), 4.22 (m, 1H), 3.45 (m, 2H), 2.66 (m, 1H), 2.52 (m, 4H), 1.96 (m, 2H), 1.84 (m, 3H), 1.64 (m, 4H); MS m / z 367 (M + 1).

4. To a stirred solution of [4- (6-amino-5-hydroxypyridin-3-yl) phenyl] - [(2R) -2-pyrrolidin-1-ylmethyl-pyrrolidin-1-yl] methanone (100 mg, 0.27 mmol) in anhydrous DMF (15 mL), under nitrogen atmosphere, sodium hydride (60% dispersion in mineral oil, 11 mg, 0.49 mmol) was added at 0 ° C. The reaction mixture was stirred at 0 ° C for 30 minutes then 1- (bromomethyl) -4-fluoro-2- (trifluoromethyl) benzene (0.046 mL, 0.27 mmol) was added. After stirring at room temperature for 2 hours, the reaction mixture was diluted with EtOAc and partitioned with HgO. Extraction with EtOAc (2 x 25 mL) was performed. The organic layers were combined, washed with HgO (1 x 15 mL), brine (1 x 15 mL), dried over MgSO4, filtered, concentrated, and purified on a silica gel column to afford {4- [6-amino-5- (4-fluoro -2-trifluoromethylbenzyloxy) pyridin-3-yl] phenyl} [(2R) -2-pyrrolidin-1-ylmethylpyrrolidin-1-yl] methanone as off-white crystals.

General Procedure 9 for the synthesis of 2-dialkylaminoethanesulfonic acid [6-amino-5- (substituted benzyloxy) pyridin-3-yl] phenylamide using {4- [6-amino-5- (2-chloro-3,6-difluorobenzyloxy) pyridin-3 2-diethylaminoethanesulfonic acid-yl] phenyl} amide. [0212]

<img file="PL1786785T3_D0098.tif" />

about

<img file="PL1786785T3_D0099.tif" />

1. To a solution of 4- (4,4,5,5-tetramethyl- [1,3,2] dioxaborolan-2-yl) phenylamine (5 g, 22.8 mmol) in dichloromethane (120 mL) was added N-methylmorpholine (7 , 5 mL, 68.4 mmol). The mixture was cooled to 0 ° C under nitrogen atmosphere then 2-chloroethanesulfonyl chloride (2.5 mL, 23.9 mmol) in dichloromethane (60 mL) was added dropwise while stirring. After the addition was complete, the mixture was stirred at 0 ° C for 1 hour and then at room temperature, monitoring the reaction by TLC (1: 1 ethyl acetate: hexanes) and staining with ninhydrin. After stirring for 4 hours, there was still some remaining starting boronic ester and an additional 0.2 equivalents (0.5 ml) of 2-chloroethanesulfonyl chloride in dichloromethane (25 ml) was added dropwise at room temperature. After 1 hour, TLC analysis showed consumption of the boronic ester and the total volume of the reaction mixture was reduced to one half by rotary evaporation. The contents of the flask were diluted with ethyl acetate (200 mL), washed with 50% brine (2 x 100 mL), dried over anhydrous sodium sulfate, and concentrated under high vacuum. The crude product was purified using silica gel (120g) and eluting with 10% ethyl acetate, dichloromethane to give [4- (4,4,5,5-tetramethyl- [1,3,2] dioxaborolan-2-yl) ethenesulfonic acid phenyl] amide as a white solid (6.2 g, 20.2 mmol, 89% yield). ⁸H NMR (CDCl 3, 300 MHz), δ 7.76 (d, J = 8.4, 2H), 7.12 (d, J = 8.45, 2H), 6.65 (s, 1H), 6 . 55 (dd, J = 9.77, 6.7, 1H), 6.31 (d, J = 16.54, 1H), 5.96 (d, J = 9.8, 1H), 1. 33 (s, 12H).

2. To a solution of ethenesulfonic acid [4- (4,4,5,5-tetramethyl- [1,3,2] dioxaborolan-2-yl) phenyl] amide (0.500 g, 1.6 mmol) in methanol (5 mL) was added diethylamine (0.707 g, 4.0 mmol) in methanol (5 ml) was stirred at room temperature and the reaction was monitored by TLC (1: 1 ethyl acetate: hexanes). After 2 hours, the reaction mixture was concentrated in vacuo and the residue was partitioned between ethyl acetate (50 ml) and water (50 ml). The ethyl acetate portion was then washed with 50% brine (1 x 50 ml), dried over anhydrous sodium sulfate, filtered and concentrated under high vacuum. The crude product was purified on a 10 g pre-packed silica gel column eluting with 1: 1 ethyl acetate: dichloromethane to give [4- (4,4,5,5-tetramethyl- [1,3,2] dioxaborolan-2-yl) phenyl 2-diethylaminoethanesulfonic acid amide as a white solid (0.346 g, 0.90 mmol, 56%).⁸H NMR (CDCl3, 300 MHz) δ 7.78 (d, J = 6.65, 2H) 7.15 (d, J = 6.66, 2H), 3.20 (m, 2H), 3.0 (m, 2H), 2.55 (q, J = 7.15, 7.16 4H), 1.34 (s, 12H), 1.05 (t, J = 7.19, 6H).

3. 2-Diethylaminoethanesulfonic acid {4- [6-amino-5- (2-chloro-3,6-difluorobenzyloxy) pyridin-3-yl] phenyl} amide was prepared following General Suzuki Coupling Procedure 3 starting from 5-bromo 3- (2-chloro-3,6-difluorobenzyloxy) pyridin-2-ylamine and prepared in part 2 [4- (4,4,5,5 tetramethyl- [1,3,2] dioxaborolan-2-yl) phenyl ] 2-diethylaminoethanesulfonic acid amide as a white solid, yield 60%.

General Procedure 10: [0213]

Dissolve 1: 4- (4,4,5,6-tetramethyl-1,2-dioxaboralan-2-yl) aniline (3 g, 0.013 mol) in dichloromethane (350 ml) and then add pyridine (1 0.2 g, 0.013 mol) and 4-nitrophenyl chloroformate. The reaction mixture was stirred for 13 hours at which time TLC analysis showed all starting materials were consumed. It was washed with saturated NaHCO 3 solution (3 x 50 ml), water (3 x 50 ml) and brine (3 x ml). The organic layer was dried over Na2SO4 and the solvent removed to give [4- (4,4,5,5-tetramethyl- [1,3,2] dioxaborolan-2-yl) phenyl] carbamic acid phenyl ester, 4.45 g, 91% , in the form of a white crystalline solid. ⁷Η NMR (CDCl 8 300 MHz) δ 1.4 (s, 12H), 7.1 (brs, 1H), 7.3 (d, 2H), 7.5 (d, 2H), 7.8 (d, 2H), 8.3 (d, 2H).

<img file="PL1786785T3_D0100.tif" />

2: [4- (4,4,5,5-Tetramethyl- [1,3,2] dioxaborolan-2-yl) phenyl] carbamic acid phenyl ester (500 mg, 1.3 mmol) was dissolved in anhydrous dichloromethane (0 5 mL) and triethylamine (0.187 mL, 1.3 mmol). To the stirred solution was added 1-methylpiperazine (or any other amine) (0.144 mL, 1.3 mmol). The solution turned yellow immediately and TLC analysis showed all starting material consumed. The mixture was washed with water (3 x 500 ml), saturated sodium bicarbonate (2 x 200 ml) and dried and the solvents were removed in vacuo. The boronic esters were used without purification.

3: To a mixture of 2.1 ml DME and 2.8 ml of 2N NagCOg solution was added 100 mg of bromide, 1 equivalent of boronic acid and 5 mol% PdiPPh6). The reaction mixture was stirred and heated at 80 ° C overnight in a 2-drachma vial. The crude mixture was filtered through celite and extracted with EtOAc (2 x 100 mL). The combined extracts were washed with NaHCO 3 (1 x 100 mL), water (1 x 100 mL), and then saturated brine (1 x 100 mL). The resulting mixture was concentrated under high vacuum. The residue was dissolved in hexane and purified by column chromatography.

General Procedure 11: [0214]

<img file="PL1786785T3_D0101.tif" />

1: To a solution of 3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-2-ylamine (10.0 g, 33.2 mmol) in acetonitrile (600 ml) and acetic acid (120 ml ) N-iodosuccinimide (11.2 g, 49.8 mmol) was added. The reaction mixture was stirred at room temperature for 4 hours and a cold NagSgOg solution was added quickly. After evaporation, the residue was partitioned between ethyl acetate and water. The organic layer was washed with 2N NaOH, brine and dried over NagSO 4. The crude product was purified on a silica gel column to give 3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5-iodopyridin-2-ylamine ( 7.1 g, 50% yield). MS m / z427 [M + 1]

2: To a solution of 3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5-iodopyridin-2-ylamine (7.1 g, 16.6 mmol), prop-2 acid tert-butyl ester -ynylcarbamic acid (3.1 g, 20.0 mmol) in THF (60 ml) and EtgN (60 ml) were added CuI (63 mg, 0.3 mmol) and Pd (PPh3) 4 (384 mg, 0.3 mmol) ). The reaction mixture was stirred under a nitrogen atmosphere and the course of the reaction was monitored by TLC until completion. The mixture was extracted with EtOAc and washed with water. The crude product was purified on a silica gel column eluting with 20-40% EtOAc in cexanacine to give (3- {6-amino-5- [1- (2,6-dicinloro-3-fluorophenyl) ethoxy] pyridin- acid tert-butyl ester. 3-yl} prop-2-ynyl) carbamic acid (2.2 g, 29% yield).

3: Solution of (3- {6-amino-5- [1 (2,6-dicinloro-3-fluorophenyl) ethoxy] pyridin-3-yl} prop-2-ynyl) carbamic acid tert-butyl ester in 25% TFA in dichloromethane was stirred for 2 hours, then washed with 2N NaOH, twice with water, brine and dried over NagSO 4. Filtration and evaporation gave 5- (3-aminoprop-1-ynyl) -3- [1- (2,6-dichloro -3-fluorophenyl) ethoxy] pyridin-2-ylamine, 93% yield.

4: To a solution of 5- (3-aminoprop-1-ynyl) -3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-2-ylamine (0.282 mmol, 1 eq.) And 4- chloroformate nitrophenyl (1 eq.) in dry dichloromethane (10 mL) was added pyridine (1 eq.). The reaction mixture was stirred for 4 hours under nitrogen atmosphere, then the selected amine (1 eq.) And triethylamine (1 eq.) Were added. The mixture was heated to reflux for 5 minutes and cooled to room temperature. The mixture was then washed with water. The organic layer was evaporated and purified on a silica gel column eluting with 0-20% methanol in dichloromethane on pre-packed silica columns. The final yield was between 24% and 71%.

General Procedure 12: [0215]

<img file="PL1786785T3_D0102.tif" />

1: To a solution of 5- (3-aminoprop-1-ynyl) -3- [1- (2,6-dicinloro-3-fluorophenyl) ethoxy] pyridin-2-ylamine (prepared according to procedure 11) (400 mg, 1 1 mmol) in dicloromethane (17 mL) was added chloroacetyl chloride (153 mg, 1.4 mmol).

The reaction mixture was stirred at room temperature while monitoring the progress of the reaction by TLC. After completion of the reaction, the solvent was evaporated to yield a crude product.

2: To a solution of N- (3- {6-amino-5- [1- (2,6-dicinloro-3-fluorophenyl) ethoxy] pyridin-3-yl} prop-2-ynyl) -2-chloroacetamide ( 1 eq.) In acetonitrile (5 eq.) Was added the amine (5 eq.). A mixture of refluxed under nitrogen overnight. After evaporation of the solvent, the residue was purified on a silica gel column eluting with 1-10% methanol in dicloromethane to afford the product, the yield was between 47% and 97%.

General Procedure 13: [0216]

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<img file="PL1786785T3_D0104.tif" />

1. To a stirred solution of 2-amino-3-benzyloxypyridine (42.0 g, 0.21 mol) in CH 3 CN (600 ml) at 0 ° C, N-bromosuccinimide (37.1 g, 0.21 g, 0.21 g) was added over 30 minutes. mole). After the reaction mixture was stirred for 0.5 hours, the mixture was diluted with EtOAc (900 mL) and partitioned using HgO (900 mL). The organic layer was washed with brine, dried (NagSO 4, filtered, and concentrated to dryness in vacuo to give 3-benzyloxy-5-bromopyridin-2-ylamine (31.0 g, 0.11 mol, 53%). NMR (CDCl 3, 300 MHz) δ 4.63-4.78 (brs, 2H), 5.04 (s, 2H), 7.07 (d, 1H, J, 1.8Hz), 7.33-7.42 (m , 5H), 7.73 (d, 1H, J,

1.8 Hz).

2. To a stirred mixture of 3-benzyloxy-5-bromopyridin-2-ylamine (31.0 g, 0.11 mol) in a mixture of DME (600 ml) and HgO (600 ml) was added 4-carboxymethylboronic acid (29.9 g, 0.11 mol), Pd (PPhg) 4 (6.4 g, 5.55 mmol) and NagCOg (82.0 g, 0.78 mol). The reaction mixture was slowly warmed to reflux temperature and allowed to stir for 3 hours. The mixture was cooled to room temperature, then diluted with CHgCl3 (1.5 L) and partitioned using HgO (700 mL). The organic layer was washed with a saturated NaHCO 3 solution (700 mL), dried (NagSO 4), filtered, and concentrated in vacuo. The crude material was purified by column chromatography (silica gel, 1: 1 to 4: 1 EtOAc: hexanes), product containing fractions were combined and concentrated under reduced pressure to give 4- (6-amino-5-benzyloxypyridin-3-yl) acid methyl ester ) benzoic acid (29.4 g, 0.086 mol, 79%). NMR (CDCl 3, 300 MHz) δ 3.92 (s, 3H), 4.82-4.94 (br s, 2H), 5.15 (s, 2H), 7.22 (d, 1H, J, 1 , 8Hz), 7.33-7.42 (m, 5H), 7.54 (d, 2H, J, 8.6), 7.98 (d, 1H, J, 1.8Hz), 8 .06 (d, 2H, J, 8.6Hz).

3. To a stirred solution of 4- (6-amino-5-benzyloxypyridin-3-yl) benzoic acid methyl ester (10.0 g, 0.03 mol) in EtOH: HgO (95: 5, 600 mL) was added Pd / C ( 15.9 g, 0.015 mol) (the mixture was degassed under reduced pressure). The solution was stirred under an atmosphere of hydrogen for 22 hours. The solution was filtered through damp celite and the celite was washed with EtOH. The filtrate was concentrated under reduced pressure to give 4- (6-amino-5-hydroxypyridin-3-yl) benzoic acid methyl ester (2.3 g, 9.3 mmol, 31%). ^ H NMR (MeOD, 300 MHz) δ 3.90 (s, 3H), 7.21 (d, 1H, J, 1.9 Hz), 7.62 (d, 2H, J, 8.5 Hz) , 7.76 (d, 1H, J, 1.9Hz), 8.04 (d, 2H, J, 8.5Hz).

4. N, N-Diisopropylethylamine (3.2 ml) was added to a stirred solution of 4- (6-amino-5-hydroxypyridin-3-yl) benzoic acid methyl ester (2.3 g, 9.3 mmol) in CH 2 Cl 3 (180 ml). 0.019 mol), 4-methylbenzenesulfonyl chloride (2.66 g, 0.014 mol) and PS-DMAP (catalytic amount). The reaction mixture was stirred at ambient temperature for 6 hours and then filtered to remove the gum. The resin was washed with CHgCl3 (3 x 20 mL), the combined fractions were washed with 10% citric acid (100 mL), saturated NaCl (100 mL), dried (NagSO 4), filtered and concentrated in vacuo. The resulting crude material was purified by column chromatography (silica gel, 100% CH 6 Cl 3 to 95: 5 CH 6 Cl 3: MeOH), the fractions containing the desired product were combined and concentrated under reduced pressure to give 4- [6- amino-5- (toluene-toluene) acid methyl ester. 4-sulfonyloxy) pyridin-3-yl] benzoic acid (3.3 g, 8.2 mmol, 88%). 1 H NMR (CDCl 3, 300 MHz) δ 2.47 (s, 3H), 3.93 (s, 3H), 4.81-4.88 (br s, 2H), 7.36-7.44 (m, 5H),

7.81 (d, 2H, J, 8.3Hz), 8.05 (d, 2H, J, 8.4Hz), 8.19-8.27 (br s, 1H).

5. To a stirred solution of 1- (3-fluoro-2-trifluoromethylphenyl) ethanol (2.0 g, 9.6 mmol) in dry DMF (500 mL) at 0 ° C, NaH (0.38 g, 9.6 mmol). The reaction mixture was stirred for 0.5 hours. A solution of 4- [6-amino-5- (toluene-4-sulfonyloxy) pyridin-3-yl] benzoic acid methyl ester (3.8 g, 9.6 mmol) in dry DMF (30 mL) was added to the reaction mixture. it was allowed to warm to ambient temperature and stirred at this temperature for 21 hours. The reaction mixture was diluted with EtOAc (500 mL) and HgO (100 mL). The organic layer was separated and the aqueous layer was then extracted with EtOAc (1 x 200 mL). The organic layers were combined and washed with brine (1 x 100 mL), dried with NagSO4, and concentrated to dry in vacuo. The crude mixture was purified by column chromatography (silica gel, 40:60 to 70:30 EtOAc: hexanes) and the product containing fractions were combined and concentrated under reduced pressure to give 4- {6-amino-5- [1] acid methyl ester (3 -fluoro-2-trifluoromethylphenyl) ethoxypyridin-3-yl} benzoic acid (1.4 g, 3.2 mmol, 34%).³H NMR (CDCl 3, 300 MHz) δ 1.73 (d, 3H, J, 6.2Hz), 3.91 (s, 3H), 4.87-4.64 (brs, 2H), 5.81 (q, 1H, J, 6.1, 6.3Hz), 6.92 (d, 1H, J, 1.8Hz), 7.38 (d, 2H, J, 8.5Hz), 7 , 46-7.66 (m, 3H), 7.93 (d, 1H, J, 1.8Hz), 8.02 (d, 2H, J, 8.5Hz).

6. To a stirred solution of 4— {6-amino-5- [1- (3-fluoro-2-trifluoromethylphenyl) ethoxy] pyridin-3-yl] -benzoic acid methyl ester (1.4 g, 3.2 mmol) in warm IPA (72 mL ) HgO (38 ml) containing LiOH (0.68 g, 16.2 mmol) was added. The reaction mixture was heated to reflux for 3.5 hours. The mixture was neutralized, diluted with EtOAc (200 mL), and extracted after cooling. The organic layer was washed with brine (50 ml), dried over NagSO4 and concentrated in vacuo to give 4- {6-amino-5- [1- (3-fluoro-2-trifluoromethylphenyl) ethoxy] pyridin-3-yl} benzoic acid (1 , 2 g, 2.8 mmol, 88%). ³H NMR (MeOD, 300 MHz) δ

1.75 (d, 3H, J, 6.2Hz), 4.88-4.93 (m, 1H), 7.01 (d, 1H, J, 1.8Hz), 7.39 (d , 2H, J, 8.3Hz), 7.52-7.67 (m, 3H), 7.80 (d, 1H, J, 1.8Hz), 7.97 (d, 2H, J, 8.3 Hz).

7. Preparation of amide compounds: A mixed solution of 4- {6-amino-5- [1- (3-fluoro-2-trifluoromethylphenyl) ethoxy] pyridin-3- 3 was added to a 2 drachma vial containing NHR] R 2 (0.12 mmol). ylJbenzoic acid (50 mg, 0.12 mmol), EDC (27.0 mg, 0.13 mmol), and HOBt (18.0 mg, 0.13 mmol) in DMF (2 mL). After stirring at room temperature for 18 hours, the reaction mixture was diluted with CHgCl3 (3 mL) and partitioned using HgO. The organic layer was separated, washed with saturated NaCl solution (1x2 ml) and saturated NaHCOg solution (1x2 ml). The organic layer was concentrated to dry under reduced pressure. The material was purified by column chromatography (silica gel, 99: 1 to 95: 5 CHgCl 3 / MeOH). The product containing fractions were concentrated under reduced pressure to afford the amide compounds.

General Procedure 14: [0217]

<img file="PL1786785T3_D0105.tif" />

1: To a mixture of 1- (2-chloroethyl) pyrrolidine hydrochloride (200 mg, 1.18 mmol) and 4- [4- (4,4,5,5-tetramethyl [1,3,2] dioxaborolan-2-yl) ) phenyl] -IH-pyrazole (229 mg, 1.19 mmol) in DMF (6 ml) was added CsOCOg. The reaction mixture was stirred at room temperature overnight. Water (10 ml) was then added to the mixture. The product was extracted with EtOAc (3 x 10 mL). The combined extracts were washed with brine (5 x 10 mL) to remove DMF then dried over NagSO 4 and concentrated (142 mg, 41% yield).

2: A mixture of 3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5-iodopyridin-2-ylamine (200 mg, 0.468 mmol), boronic acid pinacol ester (1) was placed in a reaction vessel for use in a microwave reactor. 2 eq.), NagCOg (149 mg, 1.41 mmol) in water (1.25 ml) and dimethylethyl glycol (3.75 ml, 0.1M) and PdiPPhgJgCl6 (16 mg, 0.020 mmol) was added. The system was degassed and flushed with nitrogen. The reaction mixture was stirred at 160 ° C in a microwave reactor for 15 minutes. The mixture was then cooled to room temperature and water (10 ml) was added. The product was extracted with EtOAc (3 x 20 mL), dried over NagSO 4, and concentrated. The crude product was purified by reverse phase HPLC using 0.1% TFA in water and acetonitrile.

General Procedure 15: [0218]

Br

<img file="PL1786785T3_D0106.tif" />

1: To a solution of 3H-oxazolo [4,5-b] pyridin-2-one (13.6 g, 100 mmol) in acetonitrile (600 ml) and acetic acid (120 ml) was added N-bromosuccinimide (21.4 g , 120 mmol). The reaction mixture was stirred at room temperature for 4 hours and a cold NagSgOg solution was added quickly. After concentration, the residue was partitioned between ethyl acetate and water. The organic layer was washed with 2N NaOH, brine and dried over Na2SO4. The crude product was purified on a silica gel column to give 6-bromo-3H-oxazolo [4,5-b] pyridin-2-one (11.5 g, yield 55 %).

2: 6-bromo-3H-oxazolo [4,5-b] pyridin-2-one (21.5 g, 100 mmol) was suspended in NaOH solution (2N, 250 mL, 500 mmol). The mixture was heated at reflux overnight to give a clear solution. After cooling to room temperature, the solution was neutralized to pH 7. A large amount of COg was released and a precipitate formed.

The product was filtered off, washed with water and dried in vacuo to give 2-amino-5-bromopyridin-3-ol as an off-white solid (17.8 g, 98% yield).

3: To a solution of 2-amino-5-bromopyridin-3-ol (358 mg, 1.89 mmol) in DMF (8 mL) was added Cs 6 CO 3 (620 mg, 1.89 mmol). The reaction mixture was stirred at room temperature under nitrogen for 1 hour then bromine compound (0.9 eq) in DMF (5 mL) was slowly added. The solution was stirred under nitrogen for five hours and then partitioned between water and ethyl acetate. The organic layer was washed with brine three times and dried over MgSO4. The crude product was purified on a silica gel column eluting with hexane: ethyl acetate (4: 1) to give the product, 70% -80% yield.

General Procedure 16 using Example 1-488 of US Pat. US America's Pin. No. US 10/786610 (PCT / US2004 / 005495): [0219]

<img file="PL1786785T3_D0107.tif" />

1. To a solution of 3-benzyloxy-5-bromopyridin-2-ylamine (1 g, 3.58 mmol) in dimethyl sulfoxide (7 ml) was added bis (pinacolane) diborane (1.0 g, 3.94 mmol), potassium acetate (1.05 g, 10.7 mmol) and [1,1'-bis (diphenylphosphino) ferrocene] dichloropalladium (II) dichloromethane (1: 1) complex (146 mg, 0.18 mmol). The mixture was heated at 80 ° C for 16 hours and then cooled to room temperature. The mixture was diluted with ethyl acetate (50 ml) and filtered. The filtrate was washed with water (2 X 50 mL) and dried over magnesium sulfate. After concentration under reduced pressure, the crude boronate was obtained as a brown solid (1.13 g, 97%). iH NMR (CDCl8) δ 1.32 (s, 12H), 5.08 (s, 2H), 5.44 (brs, 2H), 7.33-7.42 (m, 6H), 8.03 ( s, 1H).

2. An 18 mL reaction vessel was charged with crude 3-benzyloxy-5- (4,4,5,5-tetramethyl- [1,3,2] dioxaborolan-2-yl) pyridin-2-ylamine (161 mg, 0.49 mmol), dimethoxyethane (3 mL), and 2-bromopyridine (117 mg, 0.74 mmol). To this solution was added [1,1'-bis (diphenylphosphino) ferrocene] dichloropalladium (II) dichloromethane (1: 1) complex (20mg, 0.05mmol) and a 2M solution of cesium carbonate in water (0.75ml, 1.5 mmol). The mixture was heated at 80 ° C under nitrogen for 66 hours and then cooled to room temperature. The reaction mixture was partitioned between ethyl acetate (5 ml) and water (5 ml). The organic layer was washed with additional water (5 ml) and diluted with dimethylformamide (5 ml). Polymer bound sulfonic acid (0.5 g, 2.1 mmol) was added and the resulting mixture was gently stirred for 2 hours. The resin was filtered off and washed with dimethylformamide, methanol, and methylene chloride (3 x 5 mL of each solvent). The polymer was then treated with a 2M solution of ammonia in methanol for 1 hour. The resin was filtered off and washed with an additional 2M ammonia in methanol solution (2 x 5 ml) and the combined filtrates were concentrated under reduced pressure. Purification of the crude product by flash column chromatography gave 52.2 mg of the product as a tan solid (38% yield).

General Procedure 17: [0220]

<img file="PL1786785T3_D0108.tif" />

1. To the solution of 3- (2-chloro-3,6-difluorobenzyloxy) -5 (4,4,5,5-tetramethyl- [1,3,2] dioxaborolan-2-yl) pyridin-2-ylamine (procedure 16) (10 0 g, 24.3 mmol) in t-butyl alcohol (50 ml) was added boc anhydride (5.83 g, 26.7 mmol) and the reaction mixture was stirred at room temperature overnight. Additional boc anhydride (2.25 g, 10.3 mmol) was added and the reaction mixture was stirred overnight again. The mixture was concentrated to a sticky black oil and used as such.

2. To a solution of sodium bicarbonate (16.3 g, 194 mmol) in water (150 ml) and acetone (23 ml) was added the crude boronic ester (theoretically 24.3 mmol) in THF (150 ml). The mixture was cooled to 2 ° C and oxone (13.5 g, 21.9 mmol) was slowly added while keeping the temperature below 8 ° C. After the addition was complete, the reaction mixture was stirred for 5 minutes and then a cold solution of sodium bisulfite (14.2 g) in water (28 mL) was quickly added. Ethyl acetate (200 ml) was added and the layers were separated. The aqueous layer was neutralized with 6N HCl and extracted with ethyl acetate (2 x 200 ml). The combined organic layers were washed with water (250 mL) and brine (250 mL), dried (NagSO 4), and concentrated to a crude black oil. Chromatography on silica gel (ethyl acetate / hexane) gave the product as a light brown foam (4.78 g, 49.0%). iH NMR (CDCl6) δ 1.48 (s, 9H), 1.74 (d, 3H), 5.75 (q, 1H), 6.61 (d, 1H), 76.89 (dt, 1H) , 6, 94-7.04 (m, 2H), 7.26 (d, 1H), 8.19 (bs, 1H). MS m / z 401 (M + H)⁺.

3. [3- (2-Chloro-3,6-difluorobenzyloxy) -5-hydroxypyridin-2-yl] -carbamic acid tert-butyl ester (100 mg, 0.25 mmol) in dry DMF (1) was added to cesium carbonate in a 2 drachma vial. mL) followed by benzyl bromide (89.2 µΐ, 0.75 mmol). The vial was capped and stirred at 90 ° C overnight. The reaction mixture was filtered through a 5 ml Chem-Elut column previously wetted with water (3.5 ml) and eluted with 1: 1 ethyl acetate: methylene chloride. After partial concentration, 4N HCl in dioxane (1-2 mL) was added and the solution was concentrated. Reverse phase chromatography (water: acetonitrile, 0.05% TFA) followed by lyophilization afforded the desired product as an off-white amorphous solid (25.3 mg, 20.0%) and a brown bis-addition product an amorphous solid (35.2 mg, 23.7%).

General Procedure 18: [0221]

<img file="PL1786785T3_D0109.tif" />

Well₂

[0222] Sodium borohydride (1.5 molar equivalents) was added to a solution of the ketone (3.89 mmol) in 10 mL of ethanol under nitrogen atmosphere. After the resulting reaction mixture was stirred at room temperature for 12 hours, it was placed in an ice bath and diluted aqueous HCl was added quickly. The ethanol was evaporated and EtOAc was added to extract the aqueous solution. The EtOAc layer was dried over Na2SO4. NagSO4 filtered and the filtrate evaporated to give an oily residue, Compound A5. The residue was used without further purification.

[0223] To a solution of compound A5 (1.1 mmol) in 10 mL of THF, 3-hydroxy-2-nitropyridine (1.1 molar eq.) And triphenylphosphine (1.5 molar eq.) Were added. The reaction mixture was then placed in an ice bath and diisopropyl azodicarboxylate (1.5 molar equivalents) was added. The ice bath was removed and the reaction mixture was stirred at room temperature for 12 hours. The solvent was evaporated to leave a yellow oily residue. The residue was purified by chromatography on silica gel (eluting with EtOAc in hexanes) to provide compound Al.

[0224] 2M HCl (0.2 ml) was added to a solution of compound A1 (0.97 mmol) in 2 mL of ethanol. The mixture was then placed in an ice bath and iron powder (365 mg) was slowly added. The reaction mixture was heated at 85 ° C for 1 hour then cooled to room temperature. Celite (0.5 g) was added to the mixture, it was filtered through celite and rinsed with ethanol. The filtrate was evaporated to give a brown oily residue, Compound A2. The residue was used without further purification.

[0225] Periodic acid (0.25 molar equiv.), Iodine (0.5 molar equiv.), HgO (0.5 mL) and concentrated sulfuric acid (0.03 mL) were added to a solution of compound A2 in 3 mL acetic acid. The reaction mixture was heated at 85 ° C for 5 hours, then cooled in an ice bath and basified with saturated aqueous NagCOg solution to pH 3-4. Ethyl acetate was added to extract the aqueous solution. The EtOAc layer was dried over NagSO4. NagSO4, filtered and the filtrate was evaporated to leave a brown oily residue. The residue was purified by chromatography on silica gel (eluting with EtOAc and hexanes) to give the desired product, Compound A3.

General Procedure 19:

[0226]

IR

<img file="PL1786785T3_D0110.tif" />

A3 A4

[0227] Boronic ester or boronic acid (1.3 molar equivalents) was added to a solution of compound A3 (0.47 mmol) in 5 mL of DME. The mixture was purged with nitrogen several times and then dichlorobis (triphenylphosphine) palladium (II) (0.05 molar equivalent) was added. Sodium carbonate (3 molar equivalents) in 1 mL of HgO was added to the reaction mixture and the resulting solution was heated at 85 ° C for 12 hours. Water was added to terminate the reaction. Then, EtOAc was added to extract the aqueous solution. The EtOAc layer was dried over Na2SO4. NagSO4 was filtered off and the filtrate was evaporated to a dark brown oily residue. The residue was purified by chromatography on silica gel (eluting with CH3O3OH, CH6Cl3, EtOAc, and hexanes) to give the desired product, Compound A4.

General Procedure 20: [0228]

R

COOH R

<img file="PL1786785T3_D0111.tif" />

[0229] Compound A6 was prepared according to general procedure 19. To a solution of compound A6 (0.17 mmol) in 3 mL of DMF under a nitrogen atmosphere was added O- (7-azabenzotriazol-1-yl) -Ν, Ν, Ν ', Ν'- hexafluorophosphate. tetramethyluronium (HATU) (1.1 molar equivalents), diisopropylethylamine (5 molar equivalents) and amine (1.3 molar equivalents). The solution was allowed to stir at room temperature for 12 hours. A saturated solution of NaHCOg was added to terminate the reaction. Then, EtOAc was added to extract the aqueous solution. The EtOAc layer was dried over NagSO4. NagSO4 was filtered and the filtrate evaporated to leave a brown oily residue. The residue was purified by chromatography on silica gel (eluting with EtOAc and hexanes) to afford the desired amide, Compound A7, as a yellow oil.

General Procedure 21: [0230]

<img file="PL1786785T3_D0112.tif" />

[0231] Acid (16 molar equivalents or less) was added to compound A7 (0.13 mmol) at room temperature.

The resulting solution was stirred at room temperature or 60 ° C for 12 hours. The reaction mixture was concentrated and the residue was purified by silica gel chromatography (eluting with CHgOH, EtOAc, and CHgCl3) to afford the desired amide, A8, as a yellowish to white solid.

General Procedure 22: [0232]

<img file="PL1786785T3_D0113.tif" />

[0233] Compound A9 was prepared according to general procedure 19. To a solution of compound A9 (3 mmol) in 20 mL of DMF was added di-tert-butyl dicarbonate (3 molar equivalent) and 4- (dimethylamino) pyridine (0.14 molar equivalent). The reaction mixture was stirred at room temperature for 12 hours. Water was added to terminate the reaction. EtOAc was then added to extract the aqueous solution. The EtOAc layer was dried over NagSO 4, NgSO 4 was filtered off and the filtrate was evaporated to give a brown yellow oily residue.

The residue was purified by silica gel chromatography (eluting with 25 to 30% EtOAc in hexanes) to afford the desired product, Compound A10 as a yellowish oil (87.8% yield). Ozone was bubbled through a solution of A10 in 50 mL CHgCl3 at -78 ° C and dimethyl sulfide was added to terminate the reaction. A saturated sodium chloride solution was added to the reaction mixture, and EtOAc was added to extract the aqueous solution. The combined EtOAc layers were dried over NagSO4. NagSO4 filtered and the filtrate evaporated to leave a yellow oily residue. The residue was purified by silica gel chromatography (eluting with 35 to 40% EtOAc in hexanes) to give the desired product, Compound Ali, as a yellowish oil (58.4% yield).

General Procedure 23: Reductive Amination [0234]

<img file="PL1786785T3_D0114.tif" />

Ali Al2

[0235] To a solution of Compound Ali (0.45 mmol) in 4 mL of CHgOH under a nitrogen atmosphere, amine hydrochloride (1.2 molar eq.) And sodium acetate (2 molar eq. Versus amine hydrochloride) were added. Molecular sieves (0.5 g) were added to the reaction mixture, followed by sodium cyanoborohydride (2 molar equivalents). The resulting reaction mixture was stirred at room temperature under nitrogen for 12 hours. The mixture was then filtered through celite, the filtrate was evaporated and purified by chromatography on silica gel (eluting with CH3OH, EtOAc, and CHgCl3) to afford the desired product, Compound A12 as an oil (52.6% yield). Acid (16 molar equivalents or less) was added to compound A12 (0.17 mmol) at room temperature. The resulting solution was stirred at room temperature or 60 ° C for 12 hours. The solvent was evaporated and the residue was purified by chromatography on silica gel (eluting with CHgOH, EtOAc and CHgCl3) to give the desired product, Compound A13.

General Procedure 24: [0236]

<img file="PL1786785T3_D0115.tif" />

Ali A14 A15

[0237] O-Phenyldiamine (1.2 molar equivalents) and sodium bisulfite (2.1 molar equivalents) were added to a solution of compound Ali (0.41 mmol) in 5 mL of DMA. The resulting solution was heated at 110 ° C for 12 hours. Water was added to terminate the reaction. Then, EtOAc was added to extract the aqueous solution. The EtOAc layer was dried over NagSO4. NagSO4 was filtered and the filtrate was evaporated to leave a brown-yellow oily residue. The residue was purified by chromatography on silica gel (eluting with EtOAc in hexanes) to give the desired product, Compound A14. Acid (16 molar equivalents or less) was added to compound A14 (0.16 mmol) at room temperature. The resulting solution was stirred at room temperature or heated to 60 ° C for 12 hours. The reaction mixture was concentrated and the residue was purified by silica gel chromatography (eluting with CH3OH, EtOAc, and CHgCl3) to afford the desired amide, Compound A15.

General Procedure 25: [0238]

<img file="PL1786785T3_D0116.tif" />

[0239] Di-tert-butyl dicarbonate (3 molar equivalents), 4- (dimethylamino) pyridine (0.14 molar equivalents) was added to a solution of compound A3b (2 mmol) in 10 mL of DMF. The reaction mixture was stirred at room temperature for 12 hours. Water was added to terminate the reaction. Then, EtOAc was added to extract the aqueous solution. The EtOAc layer was dried over Na2SO4. Na2SO4 was filtered off and the filtrate was evaporated to give a brown-yellow oily residue (Compound A16). The residue was used without further purification.

[0240] To a solution of compound A16 in 4 mL of DMSO, bis (pinacolane) diboron (1.2 molar equivalents) and potassium acetate (3.4 molar equivalents) were added. The mixture was purged with nitrogen several times and then dicinlorobis (triphenylphosphine) palladium (II) (0.05 molar equivalent) was added. The resulting solution was heated at 80 ° C for 12 hours. Water was added to terminate the reaction. Then, EtOAc was added to extract the aqueous solution. The EtOAc layer was dried over Na2SO4. Na2SO4 was filtered off and the filtrate was evaporated to leave a dark brown oily residue. The residue was purified by silica gel chromatography (eluting with 30% EtOAc in hexanes) to afford the desired product, Compound A17 (76% yield). HCl (5 molar equiv.) Was added to a solution of compound A17 (0.43 mmol) in 4 mL of CHgClg. The resulting mixture was heated at 50 ° C for 12 hours. To neutralize the reaction mixture, a saturated solution of NaHCOg was added. EtOAc was then added to extract the aqueous solution. Dry the EtOAc layer over NagSO 4. NagSO4 was filtered off and the filtrate was evaporated to afford the desired product (Compound A18) as a yellow solid (75% yield).

General Procedure 26: [0241]

<img file="PL1786785T3_D0117.tif" />

Al 7

Compound A17 (1.3 molar eq) was added to a solution of the aryl halide (0.36 mmol) in 3 mL of DME. The mixture was purged with nitrogen several times and then dichlorobis (triphenylphosphine) palladium (II) (0.05 molar equivalent) was added. Sodium carbonate (3 molar equivalents) in 0.8 mL of HgO was added to the reaction mixture and the resulting solution was heated at 85 ° C for 12 hours. Water was added to terminate the reaction. Then, EtOAc was added to extract the aqueous solution. The EtOAc layer was dried over Na2SO4. Na2SO4 was filtered off and the filtrate was evaporated to leave a dark brown oily residue. The residue was purified by chromatography on silica gel (eluting with EtOAc in hexanes) to give the desired product, Compound A19 (74.4% yield). HCl (5 molar equiv.) Was added to a solution of compound A19 (0.26 mmol) in 10 mL IPA. The resulting mixture was heated at 50 ° C for 12 hours. The solvent was evaporated to yield the desired product, compound A20.

<img file="PL1786785T3_D0118.tif" />

General Procedure 27: [0243]

<img file="PL1786785T3_D0119.tif" />

A21

A18

[0244] Compound A18 (1.3 molar equiv.) Was added to a solution of the aryl halide (0.21 mmol) in 3 mL of DME.

The mixture was purged with nitrogen several times and then dichlorobis (triphenylphosphine) palladium (II) (0.05 molar equivalent) was added. Sodium carbonate (3 molar equivalents) in 0.6 mL of HgO was added to the reaction mixture and the resulting solution was heated at 85 ° C for 12 hours. Water was added to terminate the reaction. Then, EtOAc was added to extract the aqueous solution. The EtOAc layer was dried over Na2SO4. Na2SO4 was filtered off and the filtrate was evaporated to leave a dark brown oily residue. The residue was purified by chromatography on silica gel (eluting with CHgOH, CHgCl3, EtOAc, and hexanes) to afford the desired product, Compound A21.

General Procedure 28:

[0245]

<img file="PL1786785T3_D0120.tif" />

A2 2

Al 7

X

I, Br, Cl

[0246] To a solution of 4-fluorobenzyl halide (1.0 molar equivalent) in 2 ml of toluene was added the amine (1.5 molar equivalent) and KgCOg (1.5 molar equivalent). The resulting mixture was microwaved using a Smithsynthesizer (150 ° C, 1 hour). Water was added to terminate the reaction. Then, EtOAc was added to extract the aqueous solution. The EtOAc layer was dried over NagSO4. NagSO4 was filtered off and the filtrate was evaporated to give the desired product, Compound A23. For the synthesis of A22, the residue was used according to procedure 11 without further purification.

General Procedure 29:

[0247]

<img file="PL1786785T3_D0121.tif" />

X = I, Br, Cl A24

[0248] The amine (1.2 molar equivalents) and diisopropylamine (5 molar equivalents) were added to a solution of 4-bromobenzenesulfonyl chloride (0.77 mmol) in 5 ml CHCl 3 under nitrogen atmosphere. The resulting reaction mixture was stirred at room temperature for 4 hours. Water was added to terminate the reaction. EtOAc was then added to extract the aqueous solution. The EtOAc layer was dried over Na2SO4. NagSO4 was filtered off and the filtrate was evaporated to give the desired product, Compound A25. For the synthesis of A24, the residue was used according to procedure 11 without further purification.

General Procedure 30: [0249]

<img file="PL1786785T3_D0122.tif" />

[0250] Boronic ester or boronic acid (1.2 molar equivalents) was added to a solution of 1-chloro-4-iodobenzene (0.84 mmol) in 10 ml DME under nitrogen atmosphere. The mixture was purged with nitrogen several times and then dichlorobis (triphenylphosphine) palladium (II) (0.05 molar equivalent) was added. Sodium carbonate (3 molar equivalents) in 1.8 mL of HgO was added to the reaction mixture and the resulting solution was heated at 85 ° C for 12 hours. Water was added to terminate the reaction. Then, EtOAc was added to extract the aqueous solution. Dry the EtOAc layer over NagSO 4. NagSO4 was filtered off and the filtrate was evaporated to leave a dark brown oily residue. The residue was purified by chromatography on silica gel (eluting with CHgOH, CHgCl3, EtOAc, and hexanes) to afford the desired product, Compound A27. Compound A27 was used in procedure 11 to synthesize compound A26.

General procedure 31 for chiral resolution of racemates:

[0251] The racemic sample was purified by SFC-MS supercritical liquid chromatography. Exemplary purification conditions: chiralpak AD-H column, 250 x 21mm, 5 microns, 100A column (column #: ADHOCJ-C1003); column temperature 35 ° C; mobile phase: 35% methanol solution (with 0.1% isopropylamine) modified with COg; flow rate 52 ml / minute; isobaric conditions at a pressure of 120 bar.

General procedure 32: using (4- {6-amino-5- [1 (3-trifluoromethylphenyl) ethoxy] pyridin-3-yl} phenyl) - (3,5-dimethylpiperazin-1-yl) methanone [0252]

<img file="PL1786785T3_D0123.tif" />

To a mixture of 4 - [4 - (6-amino-5-hydroxypyridin-3-yl) benzoyl] -2,6-dimethylpiperazine-1-carboxylic acid tert-butyl ester (100mg, 0.23mmol) and 1- (1-Bromoethyl) -3-trifluoromethylbenzene (64 mg, 0.25 mmol) in DMF (2 mL) was added NaH (12 mg, 0.47 mmol) at 0 ° C.

The reaction mixture was stirred overnight. LCMS analysis showed complete reaction, DMF and water removed. TFA (2 ml) was added to the residue, and the mixture was stirred at room temperature for 3 hours. TFA was removed and then methanol was added. The residue was purified by preparative HPLC to obtain (4- {6-amino-5- [1- (3-trifluoromethylphenyl) ethoxy] pyridin-3-yl} phenyl) - (3,5-dimethylpiperazin-1-yl) methanone ( 30 mg, 25.7% yield).

General procedure 33: using (4- {6-amino-5- [l (2-trifluoromethylphenyl) ethoxy] pyridin-3-yl} phenyl) - (3,5-

<td>dimethylpiperazin-1-yl) methanone [0254] H.₃C 0 H. / ^ N ^ ot-Bu r CA N d 0. N ^ ch₃1 cf₃ ch₃ . 1 ό y — y yy A NH₂ NH₂in situ</td><td>₃C 0 H.₃C. ^^ N ^^ Ot-Bu .L from 1. tfa Γ i cf₃ ch₃JL JL JL AND[ ⁰ τ IL Λ nh₂</td>

100

To a mixture of 4 - [4 - (6-amino-5-hydroxypyridin-3-yl) benzoyl] -2,6-dimethylpiperazine-1-carboxylic acid tert-butyl ester (50mg, 0.12mmol) and 1- (1-bromoethyl) -2-trifluoromethylbenzene (32 mg, 0.12 mmol) in DMF (2 ml) was added a 2M solution of CsgCOg (0.18 ml, 0.35 mmol) followed by water (0.5 ml) , the reaction mixture was stirred overnight and then heated at 70 ° C for 8 hours. LCMS analysis showed the reaction was complete. DMF and water were removed. TFA (2 ml) was added to the residue, and the mixture was stirred at room temperature for 3 hours. TFA was removed and then methanol was added. The residue was purified by preparative HPLC to obtain (4- {6-amino-5- [1- (2-trifluoromethylphenyl) ethoxy] pyridin-3-yl} phenyl) - (3,5-dimethylpiperazin-1-yl) methanone (20 mg, yield 34.2%).

General procedure 34: using {4- [6-amino-5- (2-methylbenzyloxy) pyridin-3-yl] phenyl} - (3,5-dimethylpiperazin-1-yl) methanone [0256]

<img file="PL1786785T3_D0124.tif" />

[0257] To a mixture of (2R, 6S) -4- [4- (6-amino-5-hydroxypyridin-3-yl) benzoyl] -2,6-dimethylpiperazine-1-carboxylic acid tert-butyl ester (100 mg, 0.05 23mmol) and 1-bromomethyl-2-methylbenzene (47mg, 0.25mmol) in DMF (2ml) were added a 2M solution of C5CO3 (0.35ml, 0.7mmol) followed by water (0.5ml). The reaction mixture was stirred at room temperature overnight. LCMS analysis showed complete reaction, DMF was removed, then 4N HCl in dioxane (2 mL) was added and the reaction mixture was stirred at room temperature for 3 hours. Volatiles were removed and then methanol was added. This solution was purified

101 by preparative HPLC to obtain {4- [6-amino-5- (2-methylbenzyloxy) pyridin-3-yl] phenyl} - (3,5-dimethylpiperazin-1-yl) methanone (47 mg, 46.6% yield).

General procedure 35: using (6-amino-3-azabicyclo [3.1.0] hex-3-yl) - (4 - {6-amino-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} phenyl) methanone [0258]

<img file="PL1786785T3_D0125.tif" />

To a mixture of [3- (4-iodobenzoyl) -3-aza-bicyclo [3.1.0] hex-6-yl] carbamic acid tert-butyl ester (100 mg, 0.234 mmol) and 3- [ 1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- (4,4,5,5-tetramethyl- [1,3,2] dioxaborolan-2-yl) pyridin-2-ylamine (100 mg, 0.234 Pd (dppf) gCl8 · CHgCl6 (10 mg, 0.012 mmol) and CsgCOg (351 mg, 0.702 mmol) were added) in DME (2 mL). The mixture was bubbled with nitrogen for 10 minutes, followed by microwave heating at 150 ° C for 30 minutes. LCMS analysis showed the reaction was complete. The crude reaction mixture was diluted with ethyl acetate and then washed with water and brine. The solution was dried over MgSO4. Purification by preparative HPLC gave a solid. The solid was stirred at room temperature with a 4N HCl solution in dioxane (3 mL) for 3 hours. Removal of a portion of the airports left a residue which was purified by preparative HPLC to obtain (6-amino-3-aza-bicyclo [3.1.0] x-3-yl) - (4- {6 amino-5- [1- ( 2,6-dicinloro-3-fluorophenyl) ethoxy] pyridin-3-yl} phenyl) methanone (30 mg, 26% yield).

102

General procedure 36: using 5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] -6 '- (2-morpholin-4-ylethoxy) - [3,3'] bipyridinyl-6-ylamine [0260]

<img file="PL1786785T3_D0126.tif" />

To a mixture of α-amino-α-t1-yl-dichloro-Sfluorophenyl) ethoxy] - [3,3 '] bipyridinyl-6-ol (78 mg,

0.20 mmol), triphenylphosphine (63 mg, 0.24 mmol) and 2-morpholin-4-ylethanol (0.026 ml, 0.22 mmol) were added DEAD (0.034 ml, 0.22 mmol). After stirring overnight, additional PPhg (63 mg, 0.24 mmol) and DEAD (0.034 mL, 0.22 mmol) were added. After a few hours, more alcohol (0.026 mL, 0.22 mmol) was added. After another few hours, another portion of PPhg (63 mg, 0.24 mmol) and more DEAD (0.034 mL, 0.22 mmol) was added. After stirring overnight, the mixture was partitioned between dichloromethane and semi-saturated brine. The phases were separated and the aqueous phase was extracted with dichloromethane. The combined organic phases were dried over NagSO4 and concentrated on the rotary evaporator. The residue was purified by chromatography on silica gel, eluting with a gradient of dichloromethane, methanol to give 5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] -6 '- (2-morpholin-4-ylethoxy) - [3, 3 '] bipyridinyl-6-ylamine (53 mg, 53%).

103

General procedure 37: using the compound of Example 1-650 of US Pat. US Of North America No. US 10/786610 (PCT / US2004 / 005495) [0262]

<img file="PL1786785T3_D0127.tif" />

<img file="PL1786785T3_D0128.tif" />

3- (2,6-Dichloro-3-fluorobenzyloxy) -5-thiazol-2-yl-pyridin-2-ylamine: a microwave reactor tube with stirrer was charged with iodopyridyl starting material (300 mg, 0.702 mmol), tetrakis (triphenylphosphine) palladium (0) (40 mg, 5 mol) and tetrahydrofuran (anhydrous, 6 ml). The vial was capped and purged with nitrogen for 5 minutes. 2-thiazolylzinc bromide (0.5M in THF, 1.4 mmol, 2.8 mL) was then added via syringe. The vial was heated at 120 ° C in a microwave reactor for 10 minutes. TLC analysis (1: 1 ethyl acetate: methylene chloride) indicated that a large amount of starting material remained. Additional 2-thiazolylzinc bromide (0.5M in THF, 500 µL) was added and the vial was heated at 120 ° C in a microwave reactor for 20 minutes. TLC analysis (1: 1 ethyl acetate: methylene chloride) indicated that a large amount of starting material still remained. Additional 2-thiazolylzinc bromide (0.5M in THF, 500 µ 500) was added and the vial was heated to 120 ° C in a microwave reactor for 60 minutes. TLC analysis (1: 1 ethyl acetate: methylene chloride) still showed high starting material and impurity. The vial contents were poured into a saturated NH4Cl solution (10 mL) and this solution was extracted with ethyl acetate (2 x 30 mL). The combined ethyl acetate layers were dried over NagSO4, filtered, and concentrated under reduced pressure. The crude product was loaded onto a 10 g prepacked silica gel column and desired

104 the product was eluted with 1: 1 ethyl acetate: methylene chloride. (40 mg, 15%).

General procedure 38: using the compound of Example 1-652 of US Pat. US Of North America No. US 10/786610 (PCT / US2004 / 005495) [0264]

<img file="PL1786785T3_D0129.tif" />

3- [1- (2,6-Dichloro-3-fluorophenyl) ethoxy] -5- (1-methyl-1H-imidazol-2-yl) pyridin-2-ylamine: N-methylimidazole was placed in a 50 mL round bottom flask (92 mg, 1.1 mmol) and dissolved in tetrahydrofuran (anhydrous, 4 mL). The flask contents were cooled with a dry ice / acetone bath under nitrogen. N-Butyllithium (2.5M, 562 µ,, 1.4 mmol) was added in 100 µ dodano aliquots by syringe over 5 minutes.

The reaction mixture was stirred at -70 ° C for 30 minutes. Solid zinc chloride (anhydrous, 383 mg, 2.8 mmol) was added and the reaction mixture was stirred for 15 minutes. The ice bath was then removed and the mixture was allowed to warm to room temperature. When all the zinc chloride was in solution and the mixture was at room temperature, starting iodide (400 mg, 0.936 mmol) in tetrahydrofuran (anhydrous, 4 ml) was added followed by tetrakis (triphenylphosphine) palladium (0) (108 mg, 10 mol%). ) and the reaction mixture was heated to reflux. The course of the reaction was monitored by LC / MS until all the starting iodide was consumed. The reaction mixture was allowed to cool and then diluted with saturated NH 4 Cl solution (20 mL). This solution was extracted with ethyl acetate (2 x 50 ml). The combined ethyl acetate layers were dried over NagSO4, filtered, and concentrated under reduced pressure. The crude product was loaded onto a 10 g pre-packed column

105 filled with silica gel and the desired product was eluted with 10% methanol: ethyl acetate (25mg, 7%).

General Procedure 39: Using the compound of Example 1-657 of US Pat. US Of North America No. US 10/786610 (PCT / US2004 / 005495) [0266]

FFF

<img file="PL1786785T3_D0130.tif" />

[0267] Gas was bubbled through a solution of 6-amino-5- [1- (2,8-dichloro-3-fluorophenyl) ethoxy] nicotinonitrile (400mg, 1.23mmol) in 70ml of dry methanol at 0 ° C for 3 minutes. HCl. The mixture was stirred overnight at 3 ° C. The volatiles were removed and the solid was washed with diethyl ether to yield quantitatively the imidate. Down

200 mg of imidate in 4 ml of methanol at 0 ° C, a 2N solution of methylamine in THF (837 µΐ) was added. It was allowed to stir on the ARC for about 1 hour and then allowed to warm to room temperature overnight. The volatiles were removed and the residue was chromatographed eluting with 10-20% methanol / dichloromethane to give 70 mg of product.

General Procedure 40: [0268]

<img file="PL1786785T3_D0131.tif" />

B3

<img file="PL1786785T3_D0132.tif" />

5-hydroxynicotinic acid (BI) (7.0 g, 50 mmol) w

106 with concentrated H 2 SO 4, 9 mL of fuming HNO 3 (90%) (9 mL) was added. The reaction mixture was stirred at 55-60 ° C in a sealed vial for four days. The mixture was then poured onto ice and the pH was adjusted to 3 with 50% NaOH. MgSO4 was added to saturate the aqueous mixture, followed by extraction with isopropyl alcohol (4 x 45 mL). After removal of the isopropyl alcohol under reduced pressure, 5.93 g (64% yield) of B2 was obtained as a yellow solid. MS (APCI), (M + H)⁺ 185. ⁷H NMR (DMSO-d₆) δ 8.01 (d, 1H, Ar-H), 8.41 (d, 1H, Ar-H).

2. 2,6-dichlorobenzyl 6-nitro-5 - [(2,6-dichlorobenzyl) oxy] nicotinate (B3): Place 6-nitro-5-hydroxynicotinic acid (B2) in a 250 mL round bottom flask (B2) (3.4 g, 18.5 mmol), 2,8-dichlorobenzyl bromide (8.88 g, 37 mmol), DIPEA (5.5 g, 42.5 mmol), this was dissolved in DMF (25 ml), the reaction mixture was stirred at room temperature for 4 5 hours and then concentrated under reduced pressure. The resulting mixture was poured onto ice and filtered. The collected solid was dried in vacuo to provide 4.25 g (46% yield) of B3. MS (APCI) (M + H)⁺503. 1 h NMR (DMSO-d8) δ 5.47 (s, 2H, ArCH₂O), 5.71 (s, 2H, ArCH₂O), 7.24-7.43 (m, 6H, Ar-H), 8.26 (d, 1H, Ar-H), 8.66 (d, 1H, Ar-H).

3. 2,6-dichlorobenzyl 6-amino-5 - [(2,6-dichlorobenzyl) oxy] nicotinate (B4): a mixture of 2,6-dichlorobenzyl 6-nitro-5 - [(2,6-dichlorobenzyl) oxy] nicotinate (B3) (5.5 g, 10.96 mmol), iron powder (0.92 g, 16.43 mmol), glacial acetic acid (20 mL) and methanol (17 mL) were stirred at 85 ° C for three hours. The reaction mixture was concentrated to near dryness, and ammonium hydroxide (30%) was added to neutralize the mixture. A minimal amount of DM was added to dissolve the mixture and the mixture was purified by flash column chromatography (eluent: EtOAc-EtOH 9: 1) to give 4.5 g (87%) of B4 as a light yellow solid. MS (APCI) (M + H)⁺ 473.

4. 6-amino-5 - [(2,6-dichlorobenzyl) oxy] nicotinic acid (B5): 6-amino-5 - [(2,8-dichlorobenzyl) oxy] nicotine mixture

107 2,6-dichlorobenzylnate (B4) (3.5 g, 7.4 mmol), lithium hydroxide (0.41 g, 17 mmol), water (22 ml), and methanol (30 ml) were stirred and refluxed , 85 ° C for 5 hours. The mixture was concentrated to dryness under reduced pressure. The resulting residue was dissolved in water, extracted with Et₂O / hexane (1: 1, 4 x 25 mL), neutralized with 1N HCl, afforded a white precipitate, which was filtered off and dried in vacuo to afford 1.83 g (79%) of B5 as a white solid. MS (APCI) (M + H) + 313.1 H NMR (DMSO-d8) δ 5.26 (s, 2H, ArCH₂ABOUT) . 6.37 (s, 2H, NH₂), 7.43-7.48 (t, 1H, Ar-H), 7.54 (s, 2H, ArH), 7.58 (s, 1H, Ar-H), 8.18 (s, 1H , Ar-H).

<img file="PL1786785T3_D0133.tif" />

[0269] 400 μ (0.2M in DMF) of 4- [6-amino-5- (2,8-dichloro-3-) acid was added to 400 μ aliquots of 0.2M solutions of various amines in DMF on a 96-well plate. fluorobenzyloxy) pyridin-3-yl] benzoic acid, 80 µL of triethylamine (IM in DMF), and 160 µL of HATU (0.5 M in DMF) and the solutions were stirred at 70 ° C for 2 hours. The solvent was removed using a SpeedVac, the crude reaction mixtures were redissolved in DMSO and transferred using a liquid dispenser to a 1 ml 96-well plate to give a final theoretical concentration of 10 mM. The mixtures were analyzed by LC / MS and the product was positively identified. The stock stock was diluted to 50 nM and tested for percent c-MET inhibition at 50 nM.

108

General Procedure 41: [0270]

<img file="PL1786785T3_D0134.tif" />

[0271] Up to 400 μΐ aliquots of 0.2M solutions of various amines in

DMF in a 96-well plate was added 400 μΐ (0.2M in DMF) 6-amino-5 [(2,6-dicinlorobenzyl) oxy] nicotinic acid, 80 μΐ of triethylamine (IM in DMF) and 160 μΐ of HATU (0, 5 M in DMF) and the solutions were stirred at 70 ° C for 2 hours. The solvent was removed using a SpeedVac, the crude reaction mixtures were redissolved in DMSO and transferred using a liquid dispenser to a 1 ml 98-well plate to give a final theoretical concentration of 10 mM. The mixtures were analyzed by LC / MS and the product was positively identified. The stock stock was diluted to 1 µΜ and tested.

109

General Procedure 42 using 2- {4- (6-amino-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxypyridin-3-yl} pyrazol-1-yl) -N- (3-dimethylaminopropyl) isobutyramide [ 0272]

<img file="PL1786785T3_D0135.tif" />

<img file="PL1786785T3_D0136.tif" />

[0273] To a solution of 4- (4,4,5,5-tetramethyl- [1,3,2] dioxaborolan-2-yl) -IH-pyrazole (5 g, 25.77 mmol) and 2-methyl ester bromo-2-methylpropionic acid (12.6 g, 27.06 mmol) in DMF (85 mL) was added C 8 CO 6 (12.6 g, 38.65 mmol). The reaction mixture was heated to 90 ° C in an oil bath overnight. The solution was cooled to room temperature and partitioned between water and ethyl acetate. The combined organic layers were washed five times with water, dried over NagSO4 and concentrated to give the product, methyl 2- [4- (4,4,5,5-tetramethyl- [1,3,2] dioxaborolan-2-acid methyl ester). -yl) pyrazol-1-yl] propionic acid (4.776 g, 63% yield).

110

[0274] To a solution of 3- [1- (2,6-dicinloro-3-fluoro-phenyl) ethoxy] -5-iodopyridin-2-ylamine (6.363 g, 14.901 mmol) and 2-methyl-2- [4] acid methyl ester - (4,4,5,5-tetramethyl [1,3,2] dioxaborolan-2-yl) pyrazol-1-yl] propionic acid (4.6 g, 15.64 mmol) in DME (27 ml) was added a solution CsF (6.79 g, 44.7 mmol) in water (9.3 mL). The reaction mixture was degassed 3 times with nitrogen. Pd (dppf) CHgCl8 was added and the mixture was degassed 3 times with nitrogen. The reaction mixture was heated in the microwave to 120 ° C (thereafter Pd was added at 30 minute intervals until the reaction was complete). Water was added, the mixture was extracted with EtOAc, dried over NagSO 4 and concentrated to give 2- (4- {6-amino-5- [1- (2,6-dicinloro-3-fluoro-phenyl) ethoxy] -pyridin-3- 3-acid methyl ester. yl} pyrazol-1-yl) -2-methylpropionic acid. The crude product was purified by column chromatography on silica eluting with a gradient of 25% -50% EtOAc / hexanes to give 2- (4- {6-amino-5- [1- (2,6-dichloro-3-fluoro-phenyl) -ethoxy) methyl ester ] pyridin-3-yl} pyrazol-1-yl) 2-methylpropionic acid (1.46 g, 21% yield) from R 0.11 (50% EtOAc / hexanes).

[0275] To a solution of the methyl ester (2.92 g, 6.25 mmol) in MeOH (31 mL) was added a solution of LiOH (450 mg, 18.76 mmol) in water (6.25 mL). The reaction mixture was heated at 60 ° C until LCMS analysis showed complete hydrolysis (approximately 45 minutes). MeOH was removed under reduced pressure and MeOH (2.5 mL) and water (1 mL) were added. The pH was adjusted to 5 with 1N HCl, after which the product precipitated. Filtration gave the product 2- (4 - {6-amino-5- [1- (2,6-dicinloro-3-fluorophenyl) ethoxy] pyridin-3-yl} pyrazol-1-yl) -2-methylpropionic acid (2.825 g, quantitatively).

[0276] To a solution of 2- (4- {6-amino-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} pyrazol-1-yl) 2-methyl-propionic acid (1, 00 g, 2.20 mmol) in DME (5.5 ml) added HOBT (300 mg, 2.20 mmol), EDC (633 mg, 3.30 mmol) and N, N-dimethylpropane-1,3-diamine (225 mg, 2.20 mmol). The reaction mixture was stirred overnight at room temperature, then purified by preparative HPLC in

111 reversed phase system, C-18, eluting with acetonitrile / water with 0.1% acetic acid to give 2- (4- {6 amino-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin- 3-yl} pyrazol-1-yl) -N- (3-dimethylaminopropyl) isobutyramide (170 mg, 14% yield).

General Procedure 43 using 3- [1- (2,6-Dicinloro-3-fluorophenyl) ethoxy] -5- (3-methylpyrazol-1-yl) pyridin-2-ylamine [0277]

CH₃

<img file="PL1786785T3_D0137.tif" />

To a stirred solution of 3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5-iodopyridin-2-ylamine (100 mg,

0.23 mmol) and 3-methyl-1H-pyrazole (59 mg, 0.70 mmol) in DMSO (1 mL) added K3PO4 (101 mg, 0.47 mmol), dodecane (0.015 mL, 0.05 mmol) , cyclohexanediamine (0.009 mL, 0.07 mmol) and copper iodide (CuI) (14 mg, 0.07 mmol). Nitrogen was bubbled through the solution for 5 minutes, followed by exposure to microwaves for 2 hours at 150 ° C. LCMS confirmed the completion of the reaction and the mixture was purified by preparative HPLC to give 3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- (3-methylpyrazol-1-yl) pyridin-2-ylamine (30 mg), 34.2% yield

112

General Procedure 44 [0279]

<img file="PL1786785T3_D0138.tif" />

²⁵

[0280] 2.5-dibromopyridine (1 molar equivalent) was dissolved in anhydrous toluene (0.085M) and cooled to -78 ° C. Over 5 minutes, n-BuLi (1.2 molar eq.) Was slowly added and then the resulting mixture was allowed to stir at -78 ° C. After 2 hours, R 2 -COR 3 (1.3 molar eq.) Was added and the solution was kept at -78 ° C. After 1 hour, saturated aqueous NH4Cl was added and warmed to room temperature. The product was extracted with EtOAc (3X), the organic extracts were combined, dried (Na2SO4), concentrated, and purified by column chromatography (10% EtOAc / hexanes to 100% EtOAc) to give the crude product that was used directly for procedure 27 to afford Compound 25.

General procedure 45

[0281]

NN

<img file="PL1786785T3_D0139.tif" />

To a solution of 3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-2-ylamine (1.8 98% (2.07 g, 12.07 mmol) ferrocene, 97% ( 0.4 g, 0.712 g, 6.04 mmol), zinc cyanide, and 1,1'-bis (diphenylphosphino) mmol) in DMF (48 ml) were added

113 complex of [1,1'-bis (diphenylphosphino) ferrocene] dichloropalladium (II) with dichloromethane (1: 1) (0.25 g, 0.30 mmol). The reaction mixture was heated under nitrogen at 150 ° C overnight. The reaction mixture was diluted with EtOAc (50 ml), washed with 4: 1: 4 saturated NH4Cl / 28% NH4OH / H2O (2 x 28 ml) and dried over Na2SO4. The crude mixture was purified on a silica gel column. eluting with a linear 25% -50% gradient (EtOAc / hexanes) to afford 2- [1- (2-aminopyridin-3-yloxy) ethyl] -3-chloro-4-dimethylaminobenzonitrile as a yellow solid (37% yield) and 2- [1- (2-Aminopyridin-3-yloxy) ethyl] -4-dimethylaminoisophthalonitrile as a dark brown solid (33% yield).

General Procedure 46 [0283]

ABOUT

<img file="PL1786785T3_D0140.tif" />

To a mixture of 4-bromoimidazole (995mg, 6.77mmol), potassium hydroxide (380mg, 6.77mmol), potassium carbonate (936mg, 6.77mmol) and tetra-n-butylammonium bromide (109mg, 0.339 mmol) in dichloromethane (7 ml) was added tert-butyl bromoacetate (0.50 ml, 3.4 mmol). After stirring overnight, the reaction mixture was filtered. The filtrate was dried over sodium sulfate, filtered and concentrated on a rotary evaporator. The residue was purified by chromatography on silica gel, eluting with a gradient of dichloromethane, ethyl acetate to give (4-bromo-imidazol-1-yl) acetic acid tert-butyl ester (696 mg, 79%).

114

General Procedure 47 [0285]

<img file="PL1786785T3_D0141.tif" />

[0286] To a solution of (4- {6-amino-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} imidazol-1-yl) acetic acid tert-butyl ester (86 mg, 0 , 18 mmol) in dichloromethane (2 mL) was added a 4M solution of hydrochloric acid in dioxane (0.22 mL, 0.89 mmol). After stirring for two days, the reaction mixture was concentrated on a rotary evaporator and the residue was dissolved in the minimum amount of methanol. This solution was added dropwise to ether and the resulting mixture was allowed to stand overnight. The mixture was then filtered, washed with ether and air dried to give (4- {6-amino-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} imidazol-1-yl) acetic acid. (83 mg, 93%).

General procedure 48

[0287]

<img file="PL1786785T3_D0142.tif" />

[0288] A mixture of 4-bromoimidazole (217 mg, 1.48 mmol) and cesium carbonate (875 mg, 2.69 mmol) in dimethylformamide (5 mL) was stirred for 30 minutes. 4- (2-chloroethyl) morpholine hydrochloride (250 mg, 1.34 mmol) was added and the mixture was warmed to 50 ° C. After heating overnight, the reaction mixture was concentrated on the rotary evaporator.

115

The residue was suspended in a mixture of dichloromethane and methanol and filtered. The filtrate was concentrated on a rotary evaporator. The residue was purified by chromatography on silica gel, eluting with a gradient of dichloromethane, methanol to give 4- [2- (4-bromoimidazol-1-yl) ethyl] morpholine (148 mg, 42%).

General Procedure 49 [0289]

<img file="PL1786785T3_D0143.tif" />

AND

[0290] To a solution of N-iodosuccinimide (2.3 g, 10 mmol) in trifluoroacetic acid (20 mL) was added isoxazole (0.64 mL, 10 mmol). After stirring overnight, water (50 mL), hexanes (50 mL), and sodium bisulfite were added to the reaction mixture. The phases were separated and the organic phase was dried over NagSO 4, filtered and concentrated by rotary evaporation to give 4-iodoisoxazole (218 mg, 11%).

General procedure 50

[0291]

<img file="PL1786785T3_D0144.tif" />

<img file="PL1786785T3_D0145.tif" />

[0292] To a solution of 6'-bromo-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] - [3,3 '] bipyridinyl-6-yl-bis- (tert-butoxy carbonyl) amine (1.3 g, 2.0 mmol) in dichloromethane (15 mL) was added trifluoroacetic acid (5 mL). After 3 hours, aliquots of water and a saturated aqueous solution of sodium bicarbonate were added. The phases were separated and the aqueous phase was extracted with dichloromethane. The combined organic phases were dried over

116

NagSO 4 and concentrated by rotary evaporation to give 6'-bromo-5 [1- (2,6-dichloro-3-fluorophenyl) ethoxy] - [3.3 '] bipyridinyl-6-ylamine (968 mg, 106%).

[0293] A tube was charged with 6'-bromo-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] - [3.3 '] bipyridinyl-6-ylamine (92mg, 0.20mmol), 4- pyrrolidin-1-ylpiperidine (0.62 g, 4.0 mmol) and N-methyl pyrrolidinone (0.8 ml). The tube was then sealed and the mixture was heated at 80 ° C overnight. The temperature was increased to 100 ° C for 5.5 hours, and then heating was terminated. The reaction mixture was partitioned between ethyl acetate and water. The phases were separated and the aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over MgSO4 and concentrated on the rotary evaporator. The residue was purified by chromatography on silica gel, eluting with a gradient of dichloromethane, methanol, ammonium hydroxide, yielding 5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] -4-pyrrolidin-1-yl-3,4,5, 6-tetrahydro-2H- [1,2 '; 5', 3] terpyridin-6-ylamine (53 mg, 50%).

General procedure 51

[0294]

<img file="PL1786785T3_D0146.tif" />

[0295] Sodium hydride (56 mg, 2.3 mmol) was added to a solution of piperidin-4-ol (214 mg, 2.11 mmol) in DMSO (8 mL). After stirring for 30 minutes, 2,5-dibromopyridine was added. After stirring for 24 hours, sodium hydride (56 mg, 2.3 mmol) was added. After stirring for another 24 hours, the reaction mixture was partitioned between ethyl acetate and water. The phases were separated and the aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over MgSO4 and concentrated on the rotary evaporator. The residue was purified by a method

117 chromatography on silica gel, eluting with a gradient of dichloromethane, methanol, ammonium hydroxide, to give 5-bromo-2- (piperidin-4-yloxy) pyridine (316 mg, 58%).

General procedure 52

[0296]

Boc

<img file="PL1786785T3_D0147.tif" />

[0297] 2,5-Dibromopyridine (0.24 g, 1.0 mmol), 4-aminopiperidine-1-carboxylic acid tert-butyl ester (0.22 g, 1.1 mmol), diisopropylethylamine (0 , 19 mL, 1.1 mmol) and N-methylpyrrolidinone (1.0 mL). The tube was sealed and the mixture was heated at 80 ° C overnight. The temperature was increased to 120 ° C and heated again overnight. The reaction mixture was partitioned between ethyl acetate and water. The phases were separated and the aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over MgSO4 and concentrated on the rotary evaporator. The residue was purified by silica gel chromatography eluting with a gradient of ethyl acetate and hexanes to afford 4- (5-bromopyridin-2-ylamino) piperidine-1-carboxylic acid tert-butyl ester (36mg, 10%).

General Procedure 53 [0298]

<img file="PL1786785T3_D0148.tif" />

4- (4- {6-Amino-5- [1- (2,6-dichloro-3-ethoxyphenyl) ethoxy] pyridin-3-yl} benzoyl) -piperazine-1-carboxylic acid tert-butyl ester: to 4 ml of DMSO was added 0.124 ml

118 ethanol followed by 32 mg of NaH. After stirring for 30 minutes, 250 mg of 4- (4- {6-amino-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl] benzoyl) -piperazine-1- tert-butyl ester were added. carboxylic acid and the reaction mixture was heated to 40 ° C. After three hours, the mixture was cooled and poured into water to precipitate. After neutralization to pH 6, 200 mg of a brown solid, 77%, was isolated.

General Procedure 54 [0300]

<img file="PL1786785T3_D0149.tif" />

[03 01] (4- {6-amino-5- [1- (2,6-dichloro-3-hydroxyphenyl) ethoxy] pyridin-3-yl} phenyl) piperazin-1-ylmethanone: up to 140 mg tert- 4- [4- (6-amino-5- {1- [2,6-dichloro-3- (2,4,6-trimethoxybenzyloxy) phenyl] ethoxy} pyridin-3-yl) benzoyl] piperazine-1-carboxylic acid butyl (from general procedure 53) 1 mL of TFA was added, the solution turned red, then, 3 seconds later, 100 µL of triethylsilane was added. The solution turned yellow. After stirring for four hours, 5 ml of toluene was added and the solvent was removed under reduced pressure. Chromatography eluting with 10% MeOH / CHgCl3 to 0.5% to 1% NH4OH / 9.5 to 9% MeOH / 90% CHgCl3 provided 65 mg of a white solid, 62% yield.

119

OH Cl

General Procedure 55 [0302]

<img file="PL1786785T3_D0150.tif" />

8a 8b

<img file="PL1786785T3_D0151.tif" />

2- (4-bromo-2-methoxyphenoxy) ethanol (8a): to a solution of ethylene carbonate (1.8 g, 20 mmol) and 4-bromo-2-methoxyphenol (1.05 g, 5 mmol) in 5 ml of toluene, under an inert atmosphere, potassium carbonate (1.4 g, 10 mmol) was added. The mixture was heated at 115 ° C for 12 hours, then water (50 ml) and ethyl acetate (2 x 100 ml) were added with stirring. The organic layers were combined, dried, filtered and the solvent was evaporated to yield a yellow oily residue. The residue was purified by flash chromatography (eluting with 40 to 45% EtOAc in hexanes) to give 8a as a light brown / yellow oil (1 g; 4.13 mmol; 82.6% yield); MS (APCI) (M + H)⁺248. ⁸H NMR (400 MHz, chloroform-D) δ ppm 2.83 (t, J = 6.3 Hz, 1H) 3.84 (s, 3H) 3.89-4.01 (m, 2H) 4.03 -4.13 (m, 2H) 6.78 (d, J = 8.3 Hz, 1H) 6.99 (d, 1H) 7.02 (d, 1H).

4-bromo-1- (2-chloroethoxy) -2-methoxybenzene (8b): to a solution of compound 1 in 1 ml of pyridine in an ice bath, thionyl chloride (0.3 ml) was added. The reaction mixture was stirred in the ice bath for 10 minutes and then heated at 100 ° C for 2 hours. The mixture was cooled to room temperature and neutralized with dilute HCl (IM). To extract an aqueous solution

120 CHgCl6 (2 x 100 ml) was added. The combined organic layers were dried over NagSO4 then concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with 10 to 15% EtOAc in hexanes) to give 8b as a colorless oil (485 mg; 1.84 mmol; 50.3% yield); MS (APCI) (M + H)⁺ 264.1 H NMR (400 MHz, chloroform-D) δ ppm 3.81 (t, J = 6.2 Hz, 2H) 3.85 (s, 3H) 4.23 (t, J = 6.2 Hz, 2H) 6.78 (d, J = 8.6 Hz, 1H).

Compound 9: Compounds of formula 9 can be prepared following an exemplary procedure: Compound A18 (1.3 molar equivalent) is added to a solution of the aryl halide (0.51 mmol) in 7 ml of DME. The mixture is purged with nitrogen several times, then dichlorobis (triphenylphsophino) palladium (II) (0.05 molar equivalent) is added. Sodium carbonate (3 molar equivalents) in 1.5 mL of HgO is added to the reaction mixture and the resulting solution is heated at 85 ° C for 12 hours. Water (20 mL) was added to terminate the reaction. Then, EtOAc (50 mL × 2) is added to extract the aqueous solution. The EtOAc layer is dried over Na2SO4. Na2SO4 is filtered off and the filtrate is concentrated to a dark brown oily residue. The residue was purified by silica gel chromatography (eluting with CHgOH, CHgCl3, EtOAc, and hexanes) to give the desired product, compound 9.

Compound 10: Compounds of formula 10 can be prepared following an exemplary procedure: amine (7 molar equivalents) is added to a solution of compound 9 (0.17 mmol) in 3 ml of 2-methoxyethanol. The resulting solution is heated to 85 ° C for 12 hours. Water (20 mL) was added to terminate the reaction. Then, EtOAc (50 mL × 2) is added to extract the aqueous solution. The EtOAc layer is dried over Na2SO4. NagSO4 is filtered off and the filtrate is concentrated to leave a light brown oily residue. The residue was purified by silica gel chromatography (eluting with CH3OH, CHgCl3, EtOAc, and hexanes) to give the desired product, compound 10.

121

General Procedure 56 [0307]

<img file="PL1786785T3_D0152.tif" />

X = C1, OR

RO

<img file="PL1786785T3_D0153.tif" />

Compound 14: Compounds of formula 14 can be prepared by following an exemplary procedure: lithium hexamethyldisilazide (1.2 molar equivalents; IM in THF) is added to a solution of alcohol (1 mmol) in 2 mL THF. The reaction mixture is stirred at room temperature under nitrogen for 30 minutes and then 5 bromo-2-chloropyrimidine (1 molar equivalent) is added. The resulting solution is heated to 75 ° C for 12 hours. Water (20 mL) was added to terminate the reaction. Then, EtOAc (50 mL × 2) is added to extract the aqueous solution. The EtOAc layer was dried over NaSO4 NgSO4 filtered and the filtrate was concentrated to an oily residue. The residue was purified by chromatography on silica gel (eluting with EtOAc in the exhaust) to give the desired product, compound 14.

Compound 11: Compound A18 (1.3 molar equivalent) was added to a solution of 5-bromo-2-chloropyrimidine or compound 14 (1 mmol) in 24 mL of DME. The mixture was purged with nitrogen several times and then dichlorobis was added

122 (triphenylphosphine) palladium (II) (0.05 molar equivalent). Sodium carbonate (3 molar equivalents) in 3 mL of HgO was added to the reaction mixture and the resulting solution was heated at 85 ° C for 12 hours. Water (50 mL) was added to terminate the reaction. Then, EtOAc (100 mL × 2) was added to extract the aqueous solution. The EtOAc layer was dried over Na2SO4. Na2SO4 was filtered off and the filtrate was evaporated to leave a dark brown oily residue. The residue was purified by flash chromatography (eluting with 40 to 55% EtOAc in hexanes) to provide compound 11.

[0310] Compounds 12: amine (2 molar equivalents) was added to a solution of compound 11 in 3 mL n-butanol. The reaction mixture was exposed to microwave radiation at 120 ° C for 30 minutes. The resulting mixture was poured into HgO and EtOAc (100 ml; v: v: 1: 1). The organic layer was dried, filtered and the solvent was evaporated to leave a light brown oily residue. The residue was purified by chromatography on silica gel (eluting with CHgOH, CHgCl3, EtOAc, and hexanes) to give the desired product, compound 12.

Compound 13: acid (16 molar equivalents or less) was added to compound 12 (0.14 mmol) at room temperature. The resulting solution was stirred at room temperature or 60 ° C for 12 hours. The solvent was evaporated and the residue was purified by silica gel chromatography (eluting with CH3OH, EtOAc, and CHgCl3) to afford the desired amide product, Compound 13, as a yellowish to white solid.

123

General Procedure 57 [0312]

<img file="PL1786785T3_D0154.tif" />

A18 16 17

Compound 15: Sodium hydride (1.3 molar equivalent) and RX (1.1 molar equivalent) were added to a solution of 2-amino-5-bromopyridine (0.84 mmol) in 3 mL of DMF. The reaction mixture was irradiated with microwave radiation at 100 ° C for 20 minutes. It was poured into a mixture of HgO and EtOAc (100 ml; v: v: 1: 1). The organic layer was dried, filtered, and concentrated to a light brown oily residue. The residue was purified by chromatography on silica gel (eluting with CHgOH, CHgCl3, EtOAc, and hexanes) to give the desired product, compound 15.

Compound 16: Compound A18 (1.3 molar equivalent) was added to a solution of compound 15 (0.25 mmol) in 5 mL of DME. The mixture was purged with nitrogen several times and then dichlorobis (triphenylphosphine) palladium (II) (0.05 molar equivalent) was added. Sodium carbonate (3 molar equivalents) in 0.8 mL of HgO was added to the reaction mixture and the resulting solution was heated at 85 ° C for 12 hours. Water (50 mL) was added to terminate the reaction. Then, EtOAc (100 mL × 2) was added to extract the aqueous solution. The EtOAc layer was dried over NaSO4 NagSO4 filtered and the filtrate evaporated to leave a dark brown oily residue. The residue was purified by flash chromatography (eluting with CH3OH, CHgCl3, EtOAc, and hexanes) to give the desired product, compound 16.

Compound 17: acid (16 molar equivalents or less) was added to compound 16 (0.114 mmol) at room temperature. The resulting solution was stirred at temperature

124 room or heated to 60 ° C for 12 hours. The solvent was evaporated and the residue was purified by silica gel chromatography (eluting with CHgOH, EtOAc and CHgCl3) to give the desired amide product, compound

17, as a yellowish to white solid.

General procedure 58

[0316]

<img file="PL1786785T3_D0155.tif" />

1-1 1-2 1-3 1-4

<img file="PL1786785T3_D0156.tif" />

1-6

<img file="PL1786785T3_D0157.tif" />

1-9 1-10 1-11

1- (t-Butoxycarbonyl) azetidine-3-carboxylic acid (1-1) (AXL016917, 1000 mg, 4.97 mmol) was dissolved in MeOH (5 ml) / toluene (20 ml) and then cooled down. to 0 ° C. TMSCHNN (trimethylsilyl diazomethane) (7.45 mmol) was then added dropwise over 15 minutes while observing a slight bubbling. The solution turned clear and slowly turned yellow. The solution was stirred for 10 minutes at 0 ° C and then warmed to room temperature for 30 minutes. The solution was then concentrated and pumped to remove the toluene to give 1.055 g of 1-t-butyl 3-methyl azetidine-1,3-dicarboxylate (1-2) which was used directly without purification in the next step (99% yield, crude).

[0318] 1-t-Butyl 3-methyl azetidine-1,3-dicarboxylate (1055 mg, 4.90 mmol) was dissolved in THF (17 mL) and then cooled to 0 ° C. MeOH was added sequentially

125 (0.397 mL, 9.80 mmol) and L1BH4 (14.7 mmol). The reaction mixture was warmed to room temperature for 3 hours. Then 10% aqueous potassium sodium tartrate tetrahydrate (Rochelle's salt) (30 ml) and EtOAc (30 ml) were added and the solution was stirred at room temperature for 30 minutes. The organic layer was separated, dried (NagSO 4) and concentrated to give 674 mg of t-butyl 3- (hydroxymethyl) azetidine-1-carboxylate (1-2) as a crude product (clear oil). The product was used directly without purification in the next step.

[0319] T-butyl 3- (hydroxymethyl) azetidine-1-carboxylate (674mg, 3.60mmol) was dissolved in CH₂C1₂ (13 mL, 0.25M) and then Et was added sequentially at 0 ° C by slowly adding MsCl₃N (1.0 mL, 7.20 mmol), DMAP (44 mg, 0.360 mmol), and methanesulfonyl chloride (0.31 mL, 3.96 mmol). The solution was warmed to room temperature for 1 hour. After 15 hours, saturated aqueous NaHCO 3 solution (50 mL) was added and then the product was extracted with CH₂C1₂ (2 x 50 mL), the combined organic extracts were washed with brine (50 mL), dried (Na₂SO4), concentrated, and purified by flash chromatography (Horizon biotage-10% EtOAc / hexanes-100% EtOAc) to give 962 mg of compound (1-4) as an oil (quantitative).

Combine NaH (95%, 96mg, 3.99mmol) under nitrogen with DMF (10ml) at room temperature. Then 4-bromopyrazole (533 mg, 3.63 mmol) was added and the reaction mixture was stirred at room temperature. After 30 minutes, compound (1-4) was added and the solution was heated to 95 ° C. After 2 hours, saturated aqueous NH4Cl (50 mL) and EtOAc (50 mL) were added. The organic layer was dried (Na₂SO4), concentrated and then passed through a thin pad of silica gel eluting with 50% EtOAc / hexanes to give 846 mg of crude compound (1-5) which was used directly in the next step (74% yield, crude).

Compound (1-5) (846 mg, 2.68 mmol), Compound (1-6) (815 mg, 3.21 mmol), [1,1'-bis (diphenylphosphino) ferrocene) dichloropalladium (108 mg, 0.133 mmol) and KOAc (893 mg,

126

9.10 mmol) was combined in DMSO (10 mL, Ng purged for 10 min) and then the solution was heated to 80 ° C. After 16 hours, the solution was filtered through celite and HgO (50 mL) and EtOAc (50 mL) were added. The organic phase was extracted, dried (NagSO 4, concentrated then passed through a silica plug eluting with 50% EtOAc / hexane. The solvent was evaporated to yield 1.22 g of crude compound (1-7) which was used directly in the next step.

[0322] Boronic ester (1-7) (4144 mg, 11.4 mmol), compound (1-8) (2890 mg, 7.60 mmol), dichlorobis (triphenylphosphine) palladium (II) (534 mg, 0.760 mmol) ), DME (40 mL, degassed 30 min Ng) and IN NagCOg (40 mL, degassed 30 min Ng) were combined and heated to 80 ° C. After 16 hours, the mixture was cooled to room temperature and EtOAc (80 mL) was added. The solution was filtered through celite and then water (80 ml) was added. The organic layer was separated, dried (NagSO 4 and concentrated. The product was purified by flash chromatography to give 1486 mg of compound (1-9) as a brown solid (36%).

[0323] 1 g of DOWEX 50WX2-400 ion exchange resin was prepared by washing it with HgO (500 mL), 1: 1 HgO / MeOH, MeOH (5 x 250 mL), CHgCl3 (500 mL), and hexanes (500 mL). The DOWEX was then dried in a vacuum oven at 40 ° C for 1 day. Compound (1-9) was dissolved in MeOH and then DOWEX (588 mg, 1.096 mmol) was added. The solution was stirred at room temperature for 2 hours, then filtered, the resin washed with MeOH (3 x 200 mL) and the filtrate discarded. The resin was then washed with 3.5M NHg / MeOH and collected. The solution was then concentrated to give 374 mg of compound (1-10) as a gummy solid (78%).

[0324] To prepare compounds of formula (1-11), the following exemplary procedure can be followed. 1 molar equivalent of compound (1-10) was dissolved in DMF or CHgCl 3 and then base (3 molar equivalents) and / or coupling reagent (1.5 molar equivalents) was added. XR (1.1 molar eq.) Was added to the solution where X

127 is e.g. Cl, Br, I, OMs, COC1, CO, COOH, ethylene or carbonate, and R is a desired group such as shown in the examples or the like. The resulting solution was stirred at room temperature for 4 hours. HgO and EtOAc were added, the organic phase was extracted, dried (NagSO 4 and concentrated. The crude product can be purified by preparative HPLC or other method well known in the art to afford the product (1-11).

General Procedure 59 [0325]

OH

<img file="PL1786785T3_D0158.tif" />

2-1

<img file="PL1786785T3_D0159.tif" />

2-6(98%) 2-8(97%)

<img file="PL1786785T3_D0160.tif" />

<img file="PL1786785T3_D0161.tif" />

2-10(63%) 2-11(85%) 2-12

3-azetidinol (2-2): mixture of N-benzohydrylazetidin-3-ol hydrochloride (2.76 g, 10.0 mmol) with palladium hydroxide, 20% Pd (dry basis) on C (400 mg) in 50 ml MeOH was hydrogenated at 55 psi for 48 hours. The mixture was filtered through celite and washed with MeOH. The filtrate was concentrated under reduced pressure in a water bath at room temperature. The residue was treated with ether (3 x 30 ml) and the solvent decanted. The solid was air dried to obtain 571 mg of the hydrochloride (2-2) w

128 white solid form (52% yield), 1 H NMR (400 MHz, DMSO-D 6) δ ppm 3.33 (s, 1H) 3.63-3.80 (m, 2H) 3.934.09 (m, 2H) 4.40-4.58 (m, 1H) 6.18 (d, J = 6.32 Hz, 1H).

3-Hydroxyazetidine-1-carboxylic acid tert-butyl ester (3-3): to a cold (0 ° C bath) mixed solution of compound (2-2) (570 mg, 5.20 mmol) in 10 mL of EtOH was added EtGN (1.8 mL, 13.0 mmol) and di-tert-butyl dicarbonate (1.702 g, 7.38 mmol). The resulting clear solution was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure. The residue was partitioned between EtOAc (200 ml), 0.5N citric acid (30 ml) and brine (30 ml). The organic layer was dried (NagSO 4) then concentrated under reduced pressure to give 899 mg of compound (2-3) as a clear oil (52%). 1 H NMR (400 MHz, chloroform-D) δ ppm 1.42 (s, 9H) 3.78 (dd, J = 9.47, 4.42 Hz, 2H) 4.13 (dd, J = 9, 35.6, 57Hz, 2H) 4.49-4.63 (m, 1H).

3-Methanesulfonyloxyazetidine-1-carboxylic acid tert-butyl ester (2-4): to a solution of compound (2-3) (466 mg; 2.69 mmol) with Et 8 N (0.75 mL; 5.38 mmol) ) and 4- (dimethylamino) pyridine (33 mg, 0.269 mmol) in 10 mL CH 6 Cl 3 at 0 ° C, methanesulfonyl chloride (0.25 mL 3.23 mmol) was added. The resulting brown solution was stirred at 0 ° C to room temperature overnight. Cold NaHCOg was added quickly then partitioned between CHgCl3 (200 mL) and saturated NaHCOg solution (50 mL). The organic layer was dried (NagSO 4), filtered through silica gel, eluted with 1: 1 hexane: EtOAc and the filtrate was concentrated under reduced pressure to give 614 mg of compound (2-4) as a yellow oil (91% yield). 1 H NMR (400 MHz, chloroform-D) δ ppm 1.43 (s, 9H) 3.05 (s, 3H) 4.08 (dd, J = 10.36, 4.29 Hz, 2H) 4. 26 (dd, J = 10.36.6.82Hz, 2H) 5.11-5.26 (m, 1H).

4-Bromopyrazole-3-azetidine-1-carboxylic acid tert-butyl ester (2-6): charge (2-4) (304mg, 1.21mmol) to a 5ml tube for microwave use; 4-bromopyrazole (2-5, 178 mg,

129

1.21 mmol) and NaH 60% in mineral oil (73 mg, 1.82 mmol) with 2 mL of DMF. The resulting mixture was irradiated with microwaves at 110 ° C for 30 minutes. The mixture was partitioned between EtOAc (200 mL) and saturated NaHCO3 solution (2 x 50 mL) and brine (50 mL). The organic layer was dried (NagSO4) and concentrated under reduced pressure to give 360 ​​mg of compound (2-6) as a yellow oil (98%). NMR (400 MHz, DMSO-D6) δ ppm 1.36-1.43 (m, 9H) 4.08 (s, 2H) 4.18-4.31 (m, 2H) 5.12-5.22 (m, 1H) 7.67 (s, 1H) 8.14 (s, 1H).

Tert-Butyl 3- [4- (4,4,5,5-tetramethyl-1,3-dioxoborolan-2-yl) -IH-pyrazol-1-yl] azetidine-1-carboxylate (2-8): a mixture of compound (2-6) (225 mg, 0.74 mmol) and bis (pinacolane) diboron (2-7, 227 mg, 0.89 mmol) with KOAc (247 mg, 2.52 mmol) in 3 ml DMSO purged with Ng for 15 minutes, then PdClg (dppf) gCHgCl6 (30 mg, 2.52 mmol) was added. The resulting reaction mixture was stirred at 80 ° C under nitrogen overnight. It was then cooled to room temperature, filtered through celite and washed with EtOAc. The filtrate was extracted with HgO (2 x 50 ml) and brine (50 ml). The organic layer was dried (Na2SO4) then concentrated under reduced pressure. The residue was filtered through silica gel, eluted with 3: 2 hexane: EtOAc. The filtrate was concentrated under reduced pressure to give 250 mg of compound (2-8) as a clear oil (97% yield). 1 H NMR (400 MHz, chloroform-D) δ ppm 1.181.27 (m, 9H) 1.28-1.34 (m, 6H) 1.41-1.49 (m, 6H) 4.22-4 . 33 (m, 2H) 4.36 (t, J = 8.59 Hz, 2H) 4.98-5.13 (m, 1H) 7.83 (s, 2H).

Tert-butyl 3- (4- {6-amino-5- [1- (2,6-dichloro-3-fluorophenylethoxy] pyridin-3-yl} -IH-pyrazol-1-yl) azetidinol-carboxylate (2 -10): mixture of compound (2-6) (459 mg; 1.31 mmol) and 3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5-iodopyridin-2-amine (2- 9) (374 mg; 0.88 mmol) in ml of anhydrous ethylene glycol dimethyl ether (DME) was washed with Ng for 15 minutes, then Pd (II) (PPhgJgClg (46 mg, 0.07 mmol) was added and the whole was washed with Ng for another 15 minutes. Another 1.0N NagCOg solution (3.9 mL; 3.9 mmol) was added

130 rinsed with Ng for 15 minutes. The resulting reaction mixture was stirred at 85 ° C under a nitrogen atmosphere overnight, then filtered through celite and washed with MeOH. The filtrate was concentrated under reduced pressure. The residue was partitioned between EtOAc (200 mL), saturated NaHCO3 solution (2 x 50 mL), and brine (50 mL). The organic layer was dried (NagSO 4 and concentrated in vacuo. The residue was purified using the Biotage system (25 M, 100% CHgCl6; 100% CHgCl6 to 90% CHgCl3 with 10% MeOH), collecting the desired fraction, 421 mg of compound (2-10) as a brown lubricant (92% yield). ⁸H NMR (400 MHz, chloroform-D) δ ppm 1.17 -1.26 (m, 9H) 1.80-1.87 (m, 3H) 4.04-4.18 (m, 2H) 4. 20-4.33 (m, 2H) 4.344.41 (m, 1H) 4.79 (s, 2H) 5.02 (d, J = 7.58 Hz, 1H) 7.04 (t, J = 8 , 46Hz, 1H) 7.33-7.41 (m, 1H) 7.44-7.52 (m, 1H) 7.53-7.58 (m, 1H) 7.59-7.65 ( m, 1H) 7.72-7.78 (m, 1H); LCMS calcd for C24H25Cl2FN5O3 (M + H) 523, found 523.

5- (1-Azetidin-3-yl-1H-pyrazol-4-yl) -3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-2-amine (2-11): a mixture of compound (2-10) (421 mg; 0.81 mmol) with a 4.0 M solution of HCl in dioxane (2.0 ml; 8.1 mmol) in 5 ml of CHgCl3 was stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure. The residue was treated with EtOAc. The precipitate was filtered off, washed with EtOAc and hexane, then dried in vacuo to provide 275 mg of compound (2-11) as a hydrochloride salt, a sandy solid (81% yield). ⁸H NMR (400 MHz, DMSO-D6) δ ppm 1.79-1.89 (m, 3H) 3.56 (s, 1H) 4.35 (s, 4H) 5.40 (s, 1H) 6, 23 (d, J = 6.57 Hz, 2H) 7.09 (s, 1H) 7.40-7.54 (m, 1H) 7.59 (dd, J = 8.84, 5.05 Hz, 1H) 7.73-7.83 (m, 1H) 7.86 (s, 1H) 8.12 (s, 1H) 9.20 (s, 1H). LCMS calcd for C] x gH] x gCl8 FN5O (M + H) 423, found 423.

Compounds of formula 2-12 can be prepared by the following exemplary procedure: to a mixture of compound (2-11) (1.0 eq.) With EtgN (2.0 eq.) In 2.0 mL DMF at room temperature is added alkyl bromide (1.1 eq.) is obtained. The resulting reaction mixture was stirred under nitrogen atmosphere at room temperature overnight.

131

The mixture is partitioned between EtOAc (200 mL), saturated NaHCO3 solution (2 x 50 mL), and brine (50 mL). The organic layer is dried (NagSO 4 and concentrated in vacuo. The residue is purified using Dionex (5% to 95% MeCN: H₂0 in 0.1% HOAc buffer), collecting the desired fraction of compound (2-12).

[0334] Alternatively, compounds of formula 2-12 can be prepared by following an exemplary procedure: to an alkylamine solution (1.0 eq.) With! Pr₂EtN (diisopropylethylamine) (3.0 eq.) In 2.0 mL DMF is added HATU (1.5 eq.). After stirring for 30 minutes, compound (2-11) (1.0 eq.) Is added.

The resulting reaction mixture was stirred at room temperature overnight then partitioned between EtOAc (200 mL), saturated NaHCO 3 g (2 x 50 mL) and brine (50 mL). The organic layer is dried (NagSO 4 and concentrated in vacuo. The residue is purified using a Dionex system (5% to 95% McCN: H₂0 in 0.1% HOAc), collecting the desired product (2-12).

General procedure 60:

[0335]

<img file="PL1786785T3_D0162.tif" />

3-6(68%) 3-7(63%)

[0336] tert-butyl 1-oxa-6-azaspiro [2,5] octane-6-carboxylate (3-2): a solution of dimethylsulfoxonium methylide was prepared under a nitrogen atmosphere with NaH (60% dispersion in mineral oil) (440mg; 11. 0 mmol) and trimethyl sulfoxonium iodide (2.421 g; 11.0 mmol) in 5 ml dry DMSO.

132

Another solution of tert-butyl 4-oxo-1-piperidinecarboxylate (3-1, 1.993 g; 10.0 mmol) in 5 mL of DMSO was added dropwise. The resulting reaction mixture was stirred at 55 ° C for 6 hours. The cooled mixture was poured into ice-HgO and extracted with EtOAc (2 x 200 ml). The combined organic layers were washed with HgO (50 ml), brine (50 ml), dried (NagSO 4, then concentrated in vacuo to give 1.4791 g of compound (3-2) as a yellow oil (69% yield).⁷H NMR (400 MHz, chloroform-D) δ ppm 1.37-1.52 (m, 11H) 1.71-1.84 (m, 2H) 2.63-2.72 (m, 2H) 3, 35-3.49 (m, 2H), 3.623.78 (m, 2H).

Tert- 4-hydroxy-4 - {[4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) -IH-pyrazol-1-yl] methyl} piperidine-1-carboxylate butyl (3-4): mixture of compound (3-2) (214 mg; 1.0 mmol) and 4- (4,4,5,5-tetramethyl-1,2,2-dioxaborolan-2-yl) - IH-pyrazole (3-3, 194 mg; 1.0 mmol) with NaH (60% dispersion in mineral oil) (60 mg; 1.5 mmol) in 3 ml of DME was stirred at 90 ° C for 3 hours. The mixture was partitioned between EtOAc (200 mL), saturated NaHCO3 solution (50 mL), and brine (50 mL). The organic layer was dried (NagSO 4 and concentrated under reduced pressure to give 361 mg of the compound (3-4) as a yellow lubricant (89% yield). ⁷H NMR (400 MHz, chloroform-D) δ ppm 1.21-1.34 (m, 12H) 1.391.50 (m, 9H) 1.56-1.78 (m, 4H) 3.14 (s, 2H) 3.72-3.91 (m, J = 32.34 Hz, 2H) 4.05 (s, 2H) 7.65 (s, 1H) 7.80 (s, 1H) 8.00 (s , 1H). LCMS calcd for C20H34BN3O5 (M + H) 408, found 408. HPLC purity 85%.

[0338] 4 - [(4 - {6-amino-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} -IH-pyrazol-1-yl) methyl] - Tert-Butyl 4-hydroxypiperidine-1-carboxylate (3-6): mixture of compound (3-4) (361 mg; 0.89 mmol) and 3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] - 5-iodopyridin-2-amine (3-5) (378 mg;

0.89 mmol) in 9.0 mL anhydrous ethylene glycol dimethyl ether (DME) was purged with Ng for 15 minutes, then Pd (II) (PPhgJgClg (32 mg, 0.05 mmol) was added and the whole was purged again with Ng for another 15 minutes. Another 1.0N NagCOg solution (3.9 mL; 3.9 mmol) was added and rinsed with Ng for 15 minutes.

133

The resulting reaction mixture was stirred at 85 ° C under a nitrogen atmosphere overnight, then filtered through celite and washed with MeOH. The filtrate was concentrated under reduced pressure. The residue was partitioned between EtOAc (200 mL), saturated NaHCO3 solution (2 x 50 mL), and brine (50 mL). The organic layer was dried (NagSO 4 and concentrated in vacuo. The residue was purified using the Dionex system (25% to 95% MeCNiHgO in 0.1% HOAc buffer), collecting the desired fraction to give 147 mg of compound (3-6) as a white solid (28% yield). 1H NMR (400 MHz,

DMSO-D6) δ ppm 1.34-1.39 (m, 9H) 1.70-1.77 (m, 2H) 1.79 (d, J = 6.57 Hz, 3H) 3.06 (d , J = 12.63 Hz, 2H) 3.62 (s, 2H) 4.03 (s, 2H) 4.79 (s, 1H) 5.66 (s, 2H) 6.08 (d, J = 6.82 Hz, 1H) 6.86 (d, J = 1.52 Hz, 1H) 7.44 (t, J = 8.72 Hz, 1H) 7.51-7.58 (m, 2H) 7 , 58-7.65 (m, 2H) 7.73 (d, J = 1.52Hz, 1H) 7.78 (s, 1H). LCMS calcd for C27H32Cl2FN5O4 (M + H) 581, found 581.

HPLC purity 87%.

4 - [(4 - {6-amino-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} -IH-pyrazol-1-yl) methyl] piperidin -4ol (3-7): a mixture of compound (3-6) (145 mg; 0.25 mmol) with

4, OM HCl in dioxane (2.0 mL; 8.1 mmol) in 5 mL CHgCl3 was stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure. The residue was purified using the Dionex system (5% to 95% MeCNiHgO in 0.1% HOAc buffer), collecting the desired fraction to give 76 mg of compound (3-7) as a yellow lubricant (63% yield). 1 h NMR (400 MHz, DMSO-D6) δ ppm 1.41-1.55 (m, 2H) 1.59-1.71 (m, 2H) 1.81 (d, J = 6.57 Hz, 3H) 2.88-3.00 (m, 2H) 3.02-3.14 (m, 2H) 4.08 (s, 2H) 5.17 (s, 2H) 6.14-6.27 ( m, J = 6.57 Hz, 1H) 7.05 (s, 1H) 7.40-7.49 (m, J = 8.72, 8.72 Hz, 1H) 7.51-7.60 ( m, J = 9.09.4, 80Hz, 1H) 7.63 (s, 1H) 7.76 (s, 1H) 7.91 (s, 1H) 8.51 (s, 1H) 8.81 (s, 1H). LCMS calculated for^C.22^H.24^C12^FN5 ° 2 (M + H) 481, found 481. HPLC purity 98%. Analysis (C22^H.24^C12^FN5°2 ^x 2.2 HOAc x 2.3H₂O) C, Η, N.

134

General Procedure 61:

[0340]

<img file="PL1786785T3_D0163.tif" />

Ethyl 2 - [(4-bromo-1H-pyrazol-1-yl) methyl] cyclopropanecarboxylate (4-3): to a solution of ethyl 2- (hydroxymethyl) cyclopropanecarboxylate (4-145) (577 mg; 4.0 mmol) in EtGN (1.1 mL; 8.0 mmol) and DMAP (49 mg; 0.4 mmol) in 12M CH 3 Cl 3 at 0 ° C was added methanesulfonyl chloride (0.4 ml; 4.8 mmol). The resulting brown suspension was stirred at 0 ° C to room temperature under nitrogen overnight. Cold NaHCO3 was added quickly, then partitioned between CHgCl3 (200 mL), saturated NaHCO3 solution (50 mL), and brine (50 mL). The organic layer was dried (NagSO4) and filtered through silica gel eluting with 1: 1 hexane: EtOAc. The filtrate was concentrated under reduced pressure to obtain 880 mg of ethyl 2 - {[(methylsulfonyl) oxy] methyl} cyclopropanecarboxylate as a yellow oil (99% yield).³H NMR (400 MHz, chloroform-D) δ ppm

0.91-1.02 (m, 1H) 1.26 (q, J = 6.99Hz, 3H) 1.29-1.36 (m, 1H)

1. 63-1.74 (m, 1H) 1.79-1.92 (m, 1H) 3.02 (s, 3H) 3.99-4.24 (m, 4H).

135

Ethyl 2 - {[(methylsulfonyl) oxy] methyl} cyclopropanecarboxylate (880mg; 4.0mmol), 4-bromopyrazole (4-2.588mg, 4.0mmol) and NaH (60% in mineral oil ) (240 mg, 6.0 mmol) was mixed together with 3.0 mL of DMF. The resulting reaction mixture was stirred at 90 ° C under nitrogen for four hours. The mixture was partitioned between EtOAc (200 mL), saturated NaHCO3 solution (2 x 50 mL), and brine (50 mL). The organic layer was dried (NagSO 4) then concentrated under reduced pressure to give 812 mg of compound (4-3) as a yellow oil (74%). NMR (400 MHz, chloroform-D) δ ppm 0.85 (dd, J = 7.96, 3.16 Hz, 1H) 0.88-0.98 (m, 1H) 1.18-1.29 ( m, 3H) 1.56-1.71 (m, 1H) 1.79-1.94 (m, 1H) 3.96-4.08 (m, 2H) 4.07-4.17 (m, 2H) 7.45 (d, J = 3.79Hz, 2H). LCMS calcd for CgoHggBrNgOg (M + H) 274, found 274. HPLC purity 95%.

Ethyl 2 - {[4- (4,4,5,5-tetramethyl-1,3-dioxoborolan-2-yl) -IH-pyrazol-1-yl] methyl} cyclopropanecarboxylate (4-4): a mixture of the compound ( 4-3) (812 mg, 2.97 mmol) and diboron bis (pinacolane) (906 mg, 3.57 mmol) with KOAc (991 mg, 10.10 mmol) in 10.0 mL DMSO was rinsed with Ng for 15 minutes followed by the addition of PdClg (dppf) gCHgCl6 (122 mg, 0.15 mmol). The resulting reaction mixture was stirred at 80 ° C under nitrogen overnight. After cooling to room temperature, the mixture was filtered through celite and washed with EtOAc. The filtrate was extracted with HgO (2 x 50 ml) and brine (50 ml). The organic layer was dried (NagSO 4) then concentrated under reduced pressure. The residue was filtered through silica gel and eluted with 3: 1 hexane: EtOAc. The filtrate was concentrated under reduced pressure to give 945 mg of compound (4-4) as a yellow oil (98% yield). 1 H NMR (400 MHz, chloroform-D) δ ppm 0.85 (dd, J = 7.83, 3.03 Hz, 1H) 0.90-0.96 (m, 1H) 1.20-1, 24 (m, 3H) 1.29-1.34 (m, 12H) 1.62-1.71 (m, 1H) 1.84-1.97 (m, 1H) 3.964.07 (m, 1H) 4.06-4.14 (m, 2H) 4.15-4.23 (m, J = 14.27,

6.44 Hz, 1H) 7.73 (s, 1H) 7.77 (s, 1H).

[0344] 2 - [(4 - {6-amino-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} -IH-pyrazol-1-yl) methyl] cyclo

136 Ethyl propane carboxylate (4-6): a mixture of compound (4-4) (643 mg; 2.01 mmol) and 3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5-iodopyridine-2- amine (4-5) (572 mg; 1.34 mmol) w

20.0 mL of anhydrous ethylene glycol dimethyl ether (DME) was rinsed with Ng for 15 minutes, then Pd (II) (PPhgJgClg (71 W / 0/1 mmol) was added and the whole was purged with Ng for another 15 minutes. Another 1.0N was added. NagCOg solution (6.0 mL; 6.0 mmol) was purged with Ng for 15 minutes The resulting reaction mixture was stirred at 85 ° C under nitrogen overnight then filtered through Celite and washed with MeOH The filtrate was concentrated under reduced pressure. The residue was partitioned between EtOAc (200 mL), saturated NaHCO3 solution (2 x 50 mL), and brine (50 mL). The organic layer was dried (NagSO4) and concentrated under reduced pressure. The residue was purified by Biotage (25M CHgClg 100%; CHgClg 100% to 90% CHgCl6: 10% MeOH) collecting the desired fraction to give 600 mg of compound (4-6) as a brown lubricant (91% yield). 1h NMR (400 MHz, DMSO-D6) δ ppm 0.96-1.10 (m, 2H) 1.15 (t, J = 7.07 Hz, 2H) 1.74 (s, 3H) 1.79 (d, J = 6.57 Hz, 3H) 3.95-4.14 (m, 4H) 5.66 (s, 2H) 6.08 (d, J = 6.57 Hz, 1H) 6.88 (s, 1H) 7.43 (t, J = 8.72 Hz, 1H) 7.49-7.62 (m, 2H) 7.73 (s, 1H) 7.88 (s, 1H). LCMS calcd for C 8 H 8 8 Cl 6 F 2 O 6 (M + H) 494, found 494. HPLC purity 95%.

[0345] 2- [(4- {6-Amino-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} -IH-pyrazol-1-yl) methyl acid] cyclopropanecarboxylic acid (4-7): to a solution of compound (4-6) (377 mg, 0.76 mmol) in 5.0 mL of MeOH at room temperature under nitrogen atmosphere was added 2.0 N NaOH solution (2) (1.5 mL ,

3.04 mmol). The resulting reaction mixture was stirred at 80 ° C for 3 hours, then concentrated under reduced pressure to remove most of the MeOH and acidified with 2M HCl to pH 4.0. The mixture was extracted with CHgCl6 (2 x 200 mL); the organic layers were washed with brine (50 mL), dried (NagSO4) and concentrated under reduced pressure to give 324 mg of compound (4-7) as a yellow solid. (92% efficiency). 1 H NMR (400 MHz, DMSO-D6) δ ppm

137

0.92-1.04 (m, 2H) 1.57-1.72 (m, 2H) 1.76-1.90 (m, 3H) 3.984.18 (m, 2H) 6.46 (s, 2H) 6.89-7.02 (m, 1H) 7.29-7.52 (m, 2H) 7.52-7.63 (m, 2H) 7.73 (d, J = 1.52Hz , 1H) 7.94 (s, 1H) 12.19 (s, 1H). LCMS calculated for Ο21Η₁₉Ο12ΕΝ₄Ο₃ (MH) 463, found 463. HPLC purity 87%.

[0346] 2 - [(4 - {6-amino-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} -IH-pyrazol-1-yl) methyl] - N-methylcyclopropanecarboxamide (4-8) (R = Me, R '= H): to a solution of compound (4-7) (1.0 eq.) With iPrgEtN (2.0 eq.) In 1.0 ml DMF was added HATU ( 1.5 eq). After stirring for 30 minutes, alkylamine (1.1 eq.) Was added. The resulting reaction mixture was stirred at room temperature overnight then partitioned between EtOAc (200 mL), saturated NaHCO3 solution (2 x 50 mL), and brine (50 mL). The organic layer was dried (Na₉SO₄) and concentrated under reduced pressure. The sample was made base free by partitioning between EtOAc (200 mL), saturated NaHCO3 solution (50 mL), and brine (50 mL). The organic layer was dried (Na₉SO₄) and concentrated under reduced pressure. The residue was treated with 1.0 mL of HgO and lyophilized to afford compound (4-8).

138

General Procedure 62: [0347]

Br Br

<img file="PL1786785T3_D0164.tif" />

<img file="PL1786785T3_D0165.tif" />

Boc

<img file="PL1786785T3_D0166.tif" />

<img file="PL1786785T3_D0167.tif" />

[0348] To a solution of 5-bromo-3 - [(R) -1- (2,6-dichloro-fluorophenyl) ethoxy] pyridin-2-ylamine (12.83 g, 33.76 mmol) in anhydrous DMF ( 100 ml), di-tert-butyl dicarbonate (21.25 g, 97.35 mmol) and 4-dimethylaminopyridine (0.793 g, 6.49 mmol) were added. The reaction mixture was stirred at ambient temperature under nitrogen for 18 hours. Saturated NaHCOg (300 mL) was added to the mixture, and the mixture was extracted with EtOAc (3 x 250 mL). The combined extracts were washed with water (5 x 100 mL), saturated NaHCO 3 solution, and brine then dried over NagSO 4. After filtration, evaporation and drying in vacuo, the di-boc protected 5-bromo-3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-2-ylamine was obtained as an off-white a foamy solid (19.59 g, 100% yield).⁷H NMR (DMSO-dg, 400 MHz) δ 8.18 (d, 1H), 7.83 (d, 1H), 7.59 (dd, 1H), 7.48

139 (t, 1H), 6.25 (q, 1H), 1.75 (d, 3H), 1.39 (s, 9H), 1.19 (s, 9H).

[0349] To a di-boc solution of 5-bromo-3 - [(R) 1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-2-ylamine (19.58 g, 33.76 mmol) in DMSO (68 ml), potassium acetate (11.26 g, 114.78 mmol) and bis (pinacolane) diboron (10.29 g, 40.51 mmol) were added. The mixture was degassed and purged with nitrogen three times, then Pd (dppf) Clg · CHgCl6 (1.38 g, 1.69 mmol) was added. The mixture was degassed and purged with nitrogen three times, then stirred at 80 ° C in an oil bath under nitrogen for 12 hours. The mixture was cooled to ambient temperature, diluted with ethyl acetate (100 ml) and filtered through celite which was then washed with ethyl acetate. The combined ethyl acetate solutions (700 ml) were washed with water (5 x 100 ml), brine (100 ml) and dried over Na2SO4. After filtration and concentration, the residue was purified on a silica gel column, eluting with EtOAc / cexane (0% -50 %) to give di-boc protected 3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] 5- (4,4,5,5-tetramethyl- [1,3,2] dioxaborolan-2-yl) pyridin-2-ylamine as a foamy solid (20.59 g, 97% yield). 1 H NMR (DMSO-d₆, 400 MHz) δ 8.20 (d, 1H), 7.70 (d, 1H), 7.63 (dd, 1H), 7.47 (t, 1H), 6.20 (q, 1H), 1.73 (d, 3H), 1.50-1.13 (m, 30H).

[0350] To a di-boc solution of 3- [(R) -1- (2,6-dichloro-3-fluoraphenyl) ethoxy] -5- (4,4,5,5-tetramethyl [1,3,2] - dioxaborolan-2-yl) pyridin-2-ylamine (20.34 g,

32.42 mmol) in CHgCl3 (80 ml) was added a solution of dry HCl in dioxane (4N, 40.5 ml, 162 mmol). The solution was stirred at 40 ° C in an oil bath under nitrogen for 12 hours. The mixture was cooled to ambient temperature, diluted with EtOAc (400 mL) then washed carefully but quickly with saturated NaHCO3 solution until the aqueous layer was basic (pH> 8). The organic layer was washed with brine and dried over Na2SO4. After filtration, evaporation and drying under high vacuum, 3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) was obtained

140 ethoxy] -5- (4,4,5,5-tetramethyl- [1,3,2] dioxaborolan-2-yl) pyridin-2-ylamine as an off-white foamy solid (13.48 g, 97% yield) . 1 H NMR (DMSO-d 6, 400 MHz) δ 8.01 (d, 1H), 7.27 (dd, 1H), 7.17 (d, 1H), 7.03 (t, 1H), 6 , 12 (q, 1H), 5.08 (bs, 2H), 1.81 (d, 3H), 1.30 (s, 6H), 1.28 (s, 6H).

[0351] To a stirred solution of 3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- (4,4,5,5-tetramethyl- [1,3,2] 2-dioxaborolan) -yl) pyridin-2-ylamine (4.2711 g, 10.0 mmol) and 4- (4-bromopyrazol-1-yl) piperidine-1-carboxylic acid tert-butyl ester (see Procedure 11) (3.9628 g, 12.0 mmol) in DME (40 ml) was added a solution of Na₂WHAT₃ (3.1787 g, 30.0 mmol) in water (10 mL). The solution was degassed and purged with nitrogen three times. Pd (PPh₃)₂Cl₂ (351 mg, 0.50 mmol), then the solution was degassed and purged with nitrogen again three times. The solution was stirred at 87 ° C in an oil bath for about 16 hours (or until the pinacol ester of the corona acid was consumed), cooled to ambient temperature and diluted with EtOAc (200 mL). The mixture was filtered through a pad of celite and washed with EtOAc. The EtOAc solution was washed with brine, dried over Na₂SO4 and concentrated. The crude product was purified on a silica gel column eluting with EtOAc / cexane (0% EtOAc to 100% EtOAc) to give 4- (4- {6-amino-5- [(R) -1- ( 2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} pyrazol-1-yl) piperidine-1-carboxylic acid (3.4167 g, 65% yield, 95% purity) with an Rf of 0.15 (50%

EtOAc / hexanes). MS m / e 550 (M + 1)⁺.

[0352] In a solution of 4- (4— {6-amino-5 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} pyrazol-1- [0352] acid tert-butyl ester, yl) piperidine-1-carboxylic acid (566.7 mg, 1.03 mmol) in methanol (5 mL) or dichloromethane (30 mL) was added 4N HCl / dioxane (15 mL). The solution was stirred for about 1 hour or until the deprotection reaction was complete. The solvents were evaporated, the residue was dissolved in methanol and purified by preparative reverse phase C-18 HPLC, eluting with acetonitrile / water with

141

0.1% acetic acid from 5% to 30%, linear gradient. After lyophilization, 3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- (1-piperidin-4-yl-1H-pyrazol-4-yl) pyridin-2- acetate was obtained. ylamine as a white solid (410 mg, 78% yield, 100% HPLC purity, 96.4% enantiomeric excess). 1 H NMR (DMSO-d 6, 400 MHz) δ 7.84 (s, 1H), 7.68 (d, 1H), 7.50 (dd, 1H), 7.46 (s, 1H), 7, 37 (t, 1H), 6.83 (d, 1H), 6.02 (q, 1H), 5.57 (bs, 2H), 4.09 (m, 1H), 2.98 (m,

2H), 2.53 (m, 2H), 1.88 (m, 2H), 1.82 (s, 3H), 1.73 (d, 3H),

1.70 (m, 2H). MS m / e 450 (M + 1)⁺.

General Procedure 63: [0353]

<img file="PL1786785T3_D0168.tif" />

[0354] To a suspension of 3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- (1-piperidin-4-yl-1H-pyrazol-4-yl) pyridin-2-ylamine hydrochloride (procedure 6 ) (150 mg, 0.288 mmol) w

CHgCl6 (2 mL) was added NEtg (0.121 mL, 0.863 mmol) and the mixture was stirred for 30 minutes at room temperature. The mixture was cooled to 0 ° C, acetic acid chlorocarbonylmethyl ester was added and the mixture was stirred for 1 hour at room temperature. The course of the reaction was monitored by LC-MS and after complete conversion to the desired product, water (2 ml) was added. The mixture was extracted with EtOAc (4 x 10 mL), dried over NagSO4 and concentrated to give the 2- [4- (4- {6-amino-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin- ester. Acetic acid 3-yl} pyrazol-1-yl) piperidin-1-yl] -2-oxoethyl (164 mg, quantitative yield).

[0355] To a solution of 2- [4- (4- {6-amino-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} pyrazol-1-yl) ester solution

142 piperidin-1-yl] -2-oxoethyl acetic acid (164 mg, 0.298 mmol) in MeOH (4 mL) was added LiOH (7 mg, 0.298 mmol) dissolved in 1 mL of water. The reaction mixture was stirred for 30 minutes at room temperature. At this time, LCMS analysis showed complete conversion to 1- [4- (4- {6-amino-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} pyrazol-1-yl) piperidines -1-yl] -2-hydroxyethanone. The product was purified by preparative reverse phase C-18 HPLC, eluting with acetonitrile / water with 0.1% acetic acid from 10% to 40%.

General Procedure 64: [0356]

<img file="PL1786785T3_D0169.tif" />

[0357] A 100 mL flask equipped with a stirrer was oven dried and cooled under a dry nitrogen atmosphere. The flask was fitted with a rubber injection stopper. The flask was immersed in an ice-water bath under nitrogen atmosphere and 1.6 mL (1.6 mmol) of 1.0 M borane in THF was introduced. Then 2- (4- {5-amino-6- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyrazin-2-yl} pyrazol-1-yl) 2-methylpropionic acid was introduced (procedure 5) (0 , 1 g, 0.221 mmol) in anhydrous THF (1.0 mL). The resulting reaction mixture was stirred at ambient temperature for 5 hours under nitrogen atmosphere, 6N HCl (1.1 mL) was slowly added and then HCl was introduced.₂O (1.1 ml) and MeOH (7.4 ml). The reaction mixture was continuously stirred overnight. Most of the solvents were evaporated under reduced pressure and then the pH was adjusted to 11 with 1N NaOH. Water was added, the solution was extracted with EtOAc (3 x 30 mL) and dried over Na₂SO4 · After filtration and concentration, the crude product

143 purified by preparative reverse-phase HPLC eluting with acetonitrile / water containing 0.1% acetic acid from 10% to 60%. After lyophilization of the pure fractions, 2- (4- {6-amino5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} pyrazol-1-yl) -2-methylpropane acetate was obtained -1-ol as a white solid (21 mg, 22% yield).

General Procedure 65: [0358]

<img file="PL1786785T3_D0170.tif" />

<img file="PL1786785T3_D0171.tif" />

[0359] To a stirred 0 ° C solution of 4-hydroxypiperidine-1-carboxylic acid tert-butyl ester (7.94 g, 39.45 mmol) in CHgCl3 (100 mL), NEtg (5.54 mL) was slowly added. , 39.45 mmol) followed by methanesulfonyl chloride (3.06 mL, 39.45 mmol) and DMAP (48 mg, 0.39 mmol). The reaction mixture was stirred at room temperature overnight then water (30 ml) was added. Extraction with CH 8 Cl 3 (3 x 30 ml), drying (NagSO 4) and removal of the solvent in vacuo afforded 4-methanesulfonyloxypiperidine-1-carboxylic acid tert-butyl ester as a white solid (11.00 g, 99% yield). 1 H NMR (CDCl 3, 400 MHz) δ 4.89 (m, 1H), 3.69 (m, 2H), 3.31 (m, 2H), 3.04 (s, 3H), 1.95 ( m, 2H), 1.83 (m, 2H), 1.46 (s, 9H).

144

To a stirred 0 ° C solution of 4-bromopyrazole (10.44 g, 71.03 mmol) in dry DMF (96 mL) was slowly added NaH (60% in mineral oil) (3.13 g, 78.133 mmol) . The solution was stirred for 1 hour at 0 ° C. 4-Methanesulfonyloxypiperidine-1-carboxylic acid tert-butyl ester (19.82 g, 71.03 mmol) was added slowly and the reaction mixture was heated at 100 ° C overnight or until pyrazole was consumed as shown by NMR. The reaction mixture was cooled to room temperature, water (20 mL) was added, followed by extraction with EtOAc. The combined extracts were washed with saturated aqueous NaCl (4 x 20 mL), dried with NagSO4, and concentrated to give 4- (4-bromopyrazol-1-yl) piperidine-1-carboxylic acid tert-butyl ester as an orange oil. The oil was purified by c-chromatography on silica eluting with 10% EtOAc / hexanes to 25% EtOAc / hexanes to afford 4- (4-bromopyrazol-1-yl) piperidine-1-carboxylic acid tert-butyl ester as a white solid (10 55 g, 45% efficiency) with Rf = 0.4 (25%

EtOAc / hexanes, developed in iodine). ⁷H NMR (CDCl 3, 400 MHz) δ 7.46 (s, 1H), 7.43 (s, 1H), 4.23 (m, 3H), 2.88 (m, 2H), 2.10 (m , 2H), 1.88 (m, 2H), 1.47 (s, 9H).

[0361] TFA (3 mL) was added to a solution of 4- (4-bromopyrazol-1-yl) -piperidine-1-carboxylic acid tert-butyl ester (500 mg, 1.515 mmol) in CH 6 Cl 3 (3 mL). The reaction mixture was stirred at room temperature until LCMS analysis indicated complete reaction. The solvents were removed under reduced pressure and the residue was dissolved in MeOH (15 mL). The pH of the solution was adjusted to 9 with resin hydroxide to give 4- (4-bromopyrazol-1-yl) piperidine.

[0362] To a solution of 4- (4-bromopyrazol-1-yl) piperidine (375 mg, 1.63 mmol) in DMF (3.26 mL) was added NEtg (230 µΐ, 1.63 mmol) and stirred for 5 minutes. Methyl iodide (MeI) (1.63 ml, 1M Mel in DMF, freshly prepared) was added and the reaction mixture was stirred overnight at room temperature. Water was added and the solution was extracted with EtOAc (4 x

145 ml). The organic layer was washed with brine, dried with NagSO4, concentrated and dried in vacuo to give 4- (4-bromopyrazol-1-yl) -1-methylpiperidine (251 mg, 63% yield).

General Procedure 66: [0363]

Boc

<img file="PL1786785T3_D0172.tif" />

[0364] To a solution of 3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- (1H-pyrazol-4-yl) pyrazin-2-ylamine (295 mg, 0.80 mmol ) in anhydrous DMF (4 ml), NaH (60% in mineral oil, 30.7 mg, 0.80 mmol) was added. The reaction mixture was stirred at ambient temperature under nitrogen for 0.5 h and then 4-methanesulfonyloxypiperidine-1-carboxylic acid tert-butyl ester (223.5 mg, 0.80 mmol) was introduced. The reaction mixture was heated to 90 &amp; lt; 0 &amp; gt; C oil bath for 0.5 h under nitrogen, then cooled to ambient temperature. Water was slowly added to the mixture, then it was extracted with EtOAc, washed with brine and dried over Na2SO4. The crude product was purified on a silica gel column to give 4- (4- {5-amino-6 - [(R) - acid tert-butyl ester) L (2,6-dichloro-3-fluorophenyl) ethoxy] pyrazin-2-yl} pyrazol-1-yl) piperidine-l-carboxylic acid as a white solid (265 mg, 59% yield).

[0365] To a solution of 4- (4- {5 - amino-6- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyrazin-2-yl} pyrazol-1-yl) piperidine acid tert-butyl ester 1-carboxylate (265 mg, 0.48 mmol) in CH 6 Cl 3 was added 4N HCl / dioxane (4 ml). The reaction mixture was stirred at ambient temperature for one hour. After evaporation, the residue was dissolved in methanol

146 (2.5 mL), purified by preparative reverse phase C-18 HPLC eluting with acetonitrile / water containing 0.1% acetic acid, linear gradient 10% -40%. After lyophilization, 3 - [(R) -1 (2,6-dichloro-3-fluorophenyl) ethoxy] -5- (1-piperidin-4-yl-1H-pyrazol-4-yl) pyrazin-2-ylamine acetate was obtained. as a white solid (125 mg, 51% yield).

General Procedure 67: [0366]

<img file="PL1786785T3_D0173.tif" />

[0367] To a solution of 2- (4- {6-amino-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} pyrazol-1-yl) propionic acid (69 mg 0.16 mmol), triethylamine (0.024 mL, 0.17 mmol), and 3-dimethylaminopropylamine (0.022 mL,

0.17 mmol) in 1.6 mL of DMF was added O- (7-azabenzotriazol-1-yl) -Ν, Ν, Ν ', Ν'-tetramethyluronium hexafluorophosphate (HATU) (66 mg, 0.17 mmol). After stirring for 3 hours, the reaction mixture was concentrated on the rotary evaporator. The residue was purified by chromatography on silica gel, eluting with a gradient of dichloromethane, methanol, ammonium hydroxide to give 2- (4- {6-amino-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} pyrazol-1-yl) N- (3-dimethylaminopropyl) propionamide. (41 mg, 50%).

147 '—Boc

General Procedure 68: [0368]

H.₃C.

<img file="PL1786785T3_D0174.tif" />

[0369] Diethyl azodicarboxylate (0.48 mL, 3.1 mmol) was added to a solution of triphenylphosphine (0.80 g, 3.1 mmol) in THF (20 mL) cooled to 0 ° C. After stirring for 5 minutes, 4-bromopyrazole (0.30 mg, 2.0 mmol) was added. After stirring for another 5 minutes, (2-hydroxyethyl) methylcarbamic acid tert-butyl ester (0.45 g, 2.6 mmol) was added. The reaction mixture was allowed to warm to room temperature and stirred overnight. The mixture was cooled to 0 ° C and filtered. The filtrate was concentrated on a rotary evaporator. The residue was purified by chromatography on silica gel, eluting with a gradient of dichloromethane, ethyl acetate to give [2- (4-bromopyrazol-1-yl) ethyl] methylcarbamic acid tert-butyl ester

<td>(541 mg, 87%). General Procedure 69: [0370] N-NH n // \ + ^C.K JL / Br H₃[0371] To solution 4.1 mmol) in DMF (10 ml) 4.9 mmol). After mixing</td><td>h₃c Ό⁰⁼\ ch₃Ν — N NaH h \ CH₃ ------- ► &lt; DMF Br 4-bromo-4H-pyrazole (0.60 g, sodium hydride (0.12 g, over 10 minutes, solution added</td>

148 2-chloropropionic acid methyl ester in DMF (4 ml). After stirring for 4 hours, the reaction mixture was partitioned between ethyl acetate and water. The phases were separated and the aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over MgSO4 and concentrated on the rotary evaporator. The residue was purified by silica gel chromatography eluting with a gradient of ethyl acetate and hexanes to give 2- (4-bromopyrazol-1-yl) propionic acid methyl ester (733mg, 77%).

General Procedure 70: [0372]

<img file="PL1786785T3_D0175.tif" />

<img file="PL1786785T3_D0176.tif" />

LiOH, H.₂ABOUT

<img file="PL1786785T3_D0177.tif" />

[0373] To a solution of 2- (4- {6-amino5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} pyrazol-1-yl) propionic acid methyl ester (70 mg 0.15 mmol) in a mixture of THF (1.5 mL) and MeOH (0.4 mL) was added a solution of LiOH (34 mg, 1.4 mmol) in water (0.4 mL). After stirring overnight, the reaction mixture was partitioned between dichloromethane and semi-saturated brine. A small amount of ethanol was added and the pH was adjusted to 7 with 1M HCl. The phases were separated and the aqueous phase was extracted with dichloromethane. The combined organic phases were dried over NagSO 4, filtered, and concentrated by rotary evaporation to give 2- (4- {6-amino-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} pyrazole acid. -1-yl) propionic (69 mg, 100%).

149

General Procedure 71: [0374]

<img file="PL1786785T3_D0178.tif" />

<img file="PL1786785T3_D0179.tif" />

[0375] To a stirred solution of 4 (3- {6-amino-5- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} pyrazol-1-yl) pyrrolidine-2-acid methyl ester carboxylate (105 mg, 0.21 mmol) in THF (5 mL) was added a 2M solution of CH3NH6 in THF (1.06 mL, 2.12 mmol), the reaction mixture was stirred and heated at 55 ° C for 18 hours. LCMS analysis showed the reaction was complete, THF was removed and the residue was purified by preparative HPLC to give 4- (4- {6-amino-5- [1- (2,6-dicinloro-3-fluoro-phenyl) ethoxy] acid methylamide) pyridin-3-yl} pyrazol-1-yl) pyrrolidine-2-carboxylic acid (30 mg), yield 28.6%.

General Procedure 72:

[0376]

Boc ✓

<img file="PL1786785T3_D0180.tif" />

21-2 21-3 21-4

Tert-Butyl 4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) -IH-pyrazole-1-carboxylate (21-1): to a solution of 4,4,5 , 5-tetramethyl-2- (1H-pyrazol-4-yl) -1,3,2-dioxaborolane (6 mmol) in 40 ml of DMF was added di-tert-butyl dicarbonate (7.2 mol eq) and 4- (dimethylamino)

150 pyridine (0.84 molar equivalents). The reaction mixture was stirred at room temperature for 12 hours. Water was added to terminate the reaction. EtOAc was then added to extract the aqueous solution. The EtOAc layer was dried over NagSO4. NagSO4 was filtered and the filtrate evaporated to give a brown-yellow oily residue, compound 211 (1.32 g; 4.56 mmol; 76%).⁷H NMR (400 MHz, chloroform-D) δ ppm 1.32 (s, 12H) 1.63 (s, 9H) 7.91 (s, 1H) 8.37 (s, 1H). The residue was used in the next step reaction without further purification.

Compound 21-3, provided as a specific example - 3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- (1H-pyrazol-4-yl) pyridin-2-amine (21-3a ):

N-NH

<img file="PL1786785T3_D0181.tif" />

[0379] To a solution of compound 21-2a (compound 21-2, with R substituents to give 2,6-dichloro-3-fluorophenyl) (1.92 mmol) in 20 mL of DME was added compound 21-1 (1.0 molar equiv. ). The reaction mixture was stirred at room temperature under nitrogen for 30 minutes and then dichlorobis (triphenylphosphine) palladium (II) (0.05 molar equivalent) was added. Sodium carbonate (3 molar equivalents) in 4 mL of HgO was added to the reaction mixture and the resulting solution was heated at 85 ° C for 12 hours. Alternatively, the base of CsF and CsGCOg can be used in 1 or 2 equivalents of the boronic ester, and the reaction carried out at room temperature (CsF) or 80 ° C (all). Water was added to terminate the reaction. Then, EtOAc (150 mL × 2) was added to extract the aqueous solution. The EtOAc layer was dried over NagSO4 NagSO4 filtered and the filtrate evaporated to a dark brown oily

151 residues. The residue was purified by chromatography on silica gel (eluting with 0 to 10% MeOH in ethyl acetate) to give the desired product, compound 21-3a (2.05 g, 53.6% yield). NMR (400 MHz, chloroform-D) δ ppm 1.60 (s, 1H) 1.84 (d, J = 6.57 Hz, 3H) 5.07 (s, 2H) 6.06 (q, J = 6.57 Hz, 1H) 6.89 (d, J = 1.77 Hz, 1H) 6.96-7.06 (m, 1H) 7.22-7.33 (m, 1H) 7.67 ( s, 2H) 7.80 (d, J = 1.52Hz, 1H).

[0380] To prepare compounds of formula 21-4, the following exemplary procedure can be used: sodium hydride (1.2 molar equivalent) is added to a solution of compound 21-3 (0.87 mmol) in 10 mL of DMF. The reaction mixture is stirred at room temperature under nitrogen for 30 minutes and then compound 21-6 (1 molar equivalent) is added. The resulting solution is heated to 85-90 ° C for 12 hours. Water (20 mL) was added to terminate the reaction. Then, EtOAc (50 mL × 2) is added to extract the aqueous solution. The EtOAc layer was dried over NgSO 4 NgSO 4 filtered and the filtrate was concentrated. The residue is purified by chromatography on silica gel (eluting with EtOAc in exanation) to afford the desired product, compound 21-4 (20-50% yield).

General Procedure 73:

[0381]

<img file="PL1786785T3_D0182.tif" />

L = Br, Cl, COOH, GOCI, OMs, ethylene carbonate, aldenide

Compounds of formula 22-3 can be prepared by the following exemplary procedure: to a solution of compound 22-1 (0.24 mmol) and base (3-5 molar equivalents) and / or coupling reagent (1 molar equivalent) in 5 ml of DMF is added to compound 22-2 (1.2 eq

152 molar). The reaction mixture was stirred under nitrogen for 12 hours. Water (20 mL) was added to terminate the reaction. Then, EtOAc (50 mL × 2) is added to extract the aqueous solution. The EtOAc layer was dried over NgSO 4 NgSO 4 filtered and the filtrate was concentrated. The residue is purified by c-chromatography on silica gel (eluting with CHgOH, CHgCl3, EtOAc, and Hexanes) to afford the desired product, compound 22-3.

General Procedure 74:

[0383] The following procedure can be used to prepare the monohydrate of piperidinopyrazole-2-aminopyridine.

.Boc Joc> Boc

<img file="PL1786785T3_D0183.tif" />

23lb

153

<img file="PL1786785T3_D0184.tif" />

<img file="PL1786785T3_D0185.tif" />

Tert-Butyl 4- (4-iodo-1H-pyrazol-1-yl) piperidine-1-carboxylate (23-Ia)

To a stirred solution of 4-iodopyrazole (0.57 mmol) in DMF (2 L) at 4 ° C was added NaH (1.2 eq, 0.68 mmol) portionwise. The resulting reaction mixture was stirred for 1 hour at 4 ° C, then compound 23-4 (1.1 eq, 0.63 mmol) was added. The resulting mixture was heated to 100 ° C for 12 hours. Cold H was added quickly₂It was extracted with EtOAc several times. The combined organic layers were dried, filtered, and concentrated to an orange oil. The residue was purified by silica gel chromatography (eluting with 5% EtOAc in pentane) to afford 23-Ia as a white solid (140 g, 66%).

Tert-Butyl 4- [4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) -1H-pyrazol-1-yl] piperidine-1-carboxylate (23-1b)

To a solution of compound 23-Ia (140 g, 0.37 mol) in 1.5 L DMSO, bis (pinacolane) diboron (1.4 eq, 134 g, 0.52 mol) and potassium acetate (4 equivalents, 145 g, 1.48 mol). The mixture is several times

154 flush with nitrogen then dichlorobis (triphenylphosphine) palladium (II) (0.05 equiv, 12.9 g, 0.018 mol) was added. The resulting mixture was heated at 80 ° C for 2 h, then cooled to room temperature, filtered through celite, and washed with EtOAc. The filtrate was washed with saturated NaCl solution (500 mL x 2), dried over NagSO 4, filtered, and concentrated. The residue was purified by silica gel chromatography (eluting with 5% EtOAc in hexanes) to give compound 23-1b as a white solid (55 g, 40%).

Compound 23-2 (1.0 molar equivalent) was added to a solution of compound 23-1b (1.3 molar equivalent) in 15 mL of DME. The mixture was purged with nitrogen several times and then dichlorobis (triphenylphosphine) palladium (II) (0.05 molar equivalent) was added. Cesium carbonate (3 molar equivalents) in 4 mL of HgO was added to the reaction mixture and the resulting solution was heated to 85 ° C for 12 hours. Water (10 mL) was added to terminate the reaction. Then, EtOAc (150 mL × 2) was added to extract the aqueous solution. Dry the EtOAc layer over NagSO 4. NagSO4 was filtered off and the filtrate was evaporated to leave a dark brown oily residue. The residue was purified by silica gel chromatography (eluting with 75 to 100% EtOAc in hexanes) to afford compound 23-3a (61% yield).

[0387] To a solution of compound 23-3a (0.63 mmol) in MeOH (4 mL) was added the hydrochloride salt (19 equiv, 12 mmol). The reaction mixture was stirred at room temperature for 12 hours. The solvent was evaporated and HgO (10 ml) was added. To neutralize the solution to pH 7, saturated NaHCOg solution was added. To extract the aqueous solution, ethyl acetate (100 mL × 2) was added. The combined organic layers were dried over NagSO4, filtered, and concentrated to give compound 23-5a as a solid (0.6 mmol, 95% yield).

Compounds of formula 23-7 can be prepared according to the following general procedure: to a solution of compound 23-5a (0.24 mmol) and base (3-5 molar equivalents) and / or coupling reagent (1 molar equivalent) in 5 ml

155

Compound 23-8 (1.2 molar equivalents) is added with DMF.

The reaction mixture was stirred under nitrogen for 12 hours. Water (20 mL) was added to terminate the reaction. Then, EtOAc (50 mL × 2) is added to extract the aqueous solution. The EtOAc layer is dried over NagSO 4

The NagSO4 is filtered off and the filtrate is concentrated to an oily residue. The residue is purified by c-chromatography on silica gel (eluting with CHgOH, CHgCl3, EtOAc, and nexanes) to afford the desired product, compound 23-7a.

156

General Procedure 75:

[03891

<img file="PL1786785T3_D0186.tif" />

157 TFA (2 mL) was added and the reaction mixture was stirred and warmed to room temperature. It was then heated to 50 ° C for 5 hours. LCMS analysis indicated the reaction was complete and THF was removed and the residue was purified by preparative HPLC to give 3- (4- {6-amino-5- [1- (2,6-dicinloro-3-fluorophenyl) ethoxy] pyridin-3-yl} pyrazol-1-yl) propane-1,2-diol (102 mg), 74.2% yield.

General Procedure 77: [0393]

ABOUT

H.

N 0 N

N^x '' br \ NaH N^X '' b

M. ⁺ L> ° dm_F. - M.

V ⁰ \_r

[0394] Sodium hydride was added to a stirred solution of 4-bromo-1H-pyrazole in DMF at room temperature. The reaction mixture was stirred for 30 minutes, [1,3] dioxolan-2-one was added, the mixture was stirred and slowly warmed to room temperature. The course of the reaction was monitored by TLC. After completion of the reaction, EtOAc was added, it was washed with saturated NaHCO 3 solution, water and brine, dried with Na₂SO4, filtered and concentrated. The residue was purified on silica gel, eluting with EtOAc and DCM 10% to give 2- (4-bromopyrazol-1-yl) ethanol, 0.22 g, 34% yield. 1 H NMR (400 MHz, chloroform-D) δ ppm 7.49 (s, 1H) 7.46 (s, 1H) 4.184.23 (m, 2H) 3, 93-3.98 (m, 2H) 3 . 09 (s, 1H).

Example 1: 5-bromo-3 - [(R) -1- (2,6-dicinloro-3-fluorophenyl) ethoxy] pyrazin-2-ylamine [0395]

<img file="PL1786785T3_D0187.tif" />

AcOH

158

[0396] The title compound was prepared following procedure 2 starting from (1S) -1- (2,6-dichloro-3-fluorophenyl) ethanol. 1H NMR (400MHz, DMSO-d6) δ 7.53 (s, 1H), 7.48 (m, 1H), 7.39 (t, 1H), 6.48 (s, 2H), 6 , 41 (q, 1H), 1.74 (d, 3H); LCMS: 381 [M + 1]; c-MET Ki: 0.796 μΜ.

Example 2: 4- {5-amino-6 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyrazin-2-yl} benzoic acid [0397]

<img file="PL1786785T3_D0188.tif" />

[0398] The title compound was prepared following procedure 3. NMR (400 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.84 (d, 2H), 7.77 (d, 2H), 7 . 53 (m, 1H), 7.37 (t, 1H), 6.64 (s, 2H), 6.53 (q, 1H), 1.78 (d, 3H); LCMS: 422 [M + 1]; c-MET Ki: 0.154 μΜ.

Example 3: (4- {5-amino-6 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyrazin-2-yl} phenyl) piperazin-1-ylmethanone [0399]

<img file="PL1786785T3_D0189.tif" />

[0400] The title compound was prepared following procedure 4. NMR (400 MHz, DMSO-d6) δ 8.11 (s, 1H), 7.73 (d, 2H), 7.53 (m, 1H), 7 . 37 (t, 1H), 7.31 (d, 2H), 6.55 (m,

159

3Η), 3.51 (br, 2H), 3.32 (br, 2H), 2.67 (br, 4H), 1.77 (d, 3H); LCMS: 490 [M + 1]; c-MET Ki: 0.027 μΜ.

Example 425: 4- (4- {5-amino-6 [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyrazin-2-yl} benzoyl) -piperazine-1 acid tert-butyl ester -carboxylic acid [0401]

<img file="PL1786785T3_D0190.tif" />

[0402] The title compound was prepared following procedure 16 followed by 20. NMR (400 MHz, DMSO-d 6) δ 8.12 (s, 1H), 7.72 (d, 2H), 7.50 (m, 1H), 7.33 (t, 3H), 6.55 (m, 3H), 3.51 (br, 2H), 3.39 (m, 3H), 3.32 (br, 3H), 1. 77 (d, 3H), 1.40 (s, 9H); LCMS: 590 [M + 1]; c-MET Ki: 0.335 μΜ.

Example 5: 3 - [(IR) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- [4- (piperazin-1-ylcarbonyl) phenyl] pyridin-2-amine [0403]

<img file="PL1786785T3_D0191.tif" />

[0404] The title compound was prepared according to procedure 20 followed by 21 as a racemic mixture with the appropriate S-enantiomer according to Example 119 followed by separation by chiral chromatography. The title compound was also prepared enantiomerically

160 pure compound starting from a chiral starting material. 1 h NMR (400 MHz, DMSO-D6) δ ppm 1.83 (d, J = 6.57 Hz, 3H) 3.35 (s, 4H) 3.69 (s, 4H) 6.24 (q, J = 6.57 Hz, 1H) 6.91-7.08 (m, 2H) 7.10 (d, J = 1.26 Hz, 1H) 7.46 (t, J = 8.72 Hz, 1H ) 7.50 (s, 4H) 7.58 (dd, J = 8.97, 4.93 Hz, 1H) 7.91 (d, J = 1.77 Hz, 1H) 9.35 (s, 2H ); LCMS: 490 [M + 1]; c-MET Ki: 0.01 μΜ.

Example 6: 4- {6-amino-5 - [(IR) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} -N- [2- (dimethylamino) ethyl] - N-methylbenzamide [0405]

<img file="PL1786785T3_D0192.tif" />

The title compound was prepared following the procedure 20. &amp; lt; 1 &amp; gt; H NMR (400 MHz, DMSO-D6) δ ppm 1.80 (d,

J = 6.82 Hz, 3H) 1.97 (s, 3H) 2.19 (s, 3H) 2.30-2.42 (m,

J = 1.77 Hz, 2H) 2.93 (s, 3H) 3.22-3.29 (m, 1H) 3.44-3.61 (m,

1H) 5.95 (s, 2H) 6.14 (q, J = 6.57 Hz, 1H) 6.98 (d, J = 1.01 Hz, 1H) 7.30-7.39 (m, 2H) 7.40-7.47 (m, 3H) 7.51-7.62 (m, 1H)

7.87 (d, J = 1.77Hz, 1H); LCMS: 506 [M + 1]; c-MET Ki: 0.01 μΜ.

Example 7: (4- {6-amino-5 - [(IR) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} phenyl) methanol [0407]

<img file="PL1786785T3_D0193.tif" />

oh

161

[0408] The title compound was prepared following the procedure 27. NMR (400 MHz, DMSO-D6) δ ppm 1.84 (d,

J = 6.57 Hz, 3H) 4.49 (d, J = 5.81 Hz, 2H) 5.20 (t, J = 5.81 Hz, 1H) 6.25 (q, J = 6.57 Hz, 1H) 6.46-6.88 (m, 2H) 7.04 (d, J = 1.52 Hz, 1H) 7.34 (s, 4H) 7.46 (t, J = 8.72 Hz, 1H) 7.59 (dd, J = 8.97, 4.93 Hz, 1H) 7.76 (d, J = 1.52 Hz, 1H); LCMS: 408

[M + 1]; c-MET Ki: 0.051 μΜ.

Example 8: 4- {6-amino-S - [(IR) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} -N- [3- (dimethylamino) propyl] - N-methylbenzamide

[0409]

<img file="PL1786785T3_D0194.tif" />

The title compound was prepared following the procedure 27. H NMR (400 MHz, DMSO-D6) δ ppm 1.60-1.73 (m, 2H) 1.80 (d, J = 6.57 Hz, 3H) 1.94 (s, 3H) 2.13 (s, 3H) 2.202.29 (m, 2H) 2.92 (s, 3H) 3.36-3.50 (m, 2H) 5.96 ( s, 2H) 6.14 (q, J = 6.57 Hz, 1H) 6.98 (s, 1H) 7.37 (s, 2H) 7.40-7.51 (m, 3H) 7.55 (dd, J = 8.84.4, 80Hz, 1H) 7.86 (d, J = 1.77Hz, 1H); LCMS: 520 [M + 1]; c-MET Ki: 0.01 μΜ.

162

Example 9: tert-butyl 4- (4- {6-amino-5 - [(IR) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl] benzoyl) -piperazine-1 carboxylate

[0411]

<img file="PL1786785T3_D0195.tif" />

[0412] The title compound was prepared following procedure 20. NMR (400 MHz, chloroform-D) δ ppm 1.46 (s, 9H) 1.86 (d, J = 6.82 Hz, 3H) 3.30-3 . 89 (m, 8H) 4.90 (s, 2H) 6.11 (q, J = 6.57 Hz, 1H) 6.98 (d, J = 1.52 Hz, 1H) 7.01-7 . 10 (m, 1H) 7.30 (dd, J = 8.97, 4.93 Hz, 1H) 7.35-7.43 (m, 4H) 7.88 (d, J = 1.77 Hz , 1H); LCMS: 590 [M + 1]; c-MET Ki: 0.03 μΜ.

Example 10: 3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- [1- (1-methylpiperidin-4-yl) -1H-pyrazol-4-yl] pyridines -2-ylamine [0413]

<img file="PL1786785T3_D0196.tif" />

[0414] The title compound was prepared following procedure 62 using 3 - [(R) -1- (2,6-dichloro-3-fluoro

163 phenyl) ethoxy] -5- (4,4,5,5-tetramethyl- [1,3,2] dioxaborolan-2-yl) pyridin-2-ylamine and 4- (4-bromopyrazol-1-yl) -1-methylpiperidine (prepared according to general procedure 11). ⁷H NMR (400 MHz, CDCl 3) δ 7.65 (s, 1H), 7.55 (s, 1H), 7.50 (s, 1H),

7.31 (m, 1H), 7.06 (m, 1H), 6.87 (s, 1H), 6.08 (m, 1H), 5.50 (bs, 2H), 4.18 (m , 1H), 3.11 (m, 2H), 2.40 (s, 3H), 2.30 (m,

2H), 2.20 (m, 4H), 2.07 (s, 3H), 1.86 (d, J = 8Hz, 3H); LCMS:

464 [M + 1]; c-MET Ki: 0.01 μΜ.

Example 11: 1- [4- (4- {6-amino-5 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-3-yl} pyrazol-1-yl) piperidin- 1-yl] -2-hydroxyethanone [0415]

<img file="PL1786785T3_D0197.tif" />

[0416] The title compound was prepared following procedure 63. ⁷H NMR (400 MHz, CDCl 3) δ 7.72 (s, 1H), 7.57 (s, 1H), 7.47 (s, 1H), 7.31 (m, 1H), 7.06 (m , 1H), 6.86 (s, 1H), 6.08 (m, 1H), 5.00 (bs, 2H), 4.70 (m, 1H), 4.36 (m, 1H), 4 , 21 (s, 1H), 3.70 (m, 1H), 3.18 (m, 1H), 3.00 (m, 1H), 2.223 (m, 2H), 2.01 (m, 2H) , 1.86 (d, J = 8Hz, 3H); LCMS: 508 [M + 1]; c-MET Ki: 0.004 μΜ.

164

Example 12: 3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- (1-piperidin-4-yl-1H-pyrazol-4-yl) pyridin-2-ylamine [0417]

<img file="PL1786785T3_D0198.tif" />

[0418] The title compound was prepared following procedure 62 using 3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- (4,4,5,5-tetramethyl- [ 1,3,2] dioxaborolan-2-yl) pyridin-2-ylamine and 4- (4-bromopyrazol-1-yl) -1-cyclopentylpiperidine (prepared according to general procedure 11 using bromocyclopentane as alkylating agent). 1 H NMR (400 MHz, CDCl 3) δ 7.73 (s, 1H), 7.55 (s, 1H), 7.48 (s, 1H), 7.31 (m, 1H), 7.07 ( m, 1H), 6.88 (s, 1H), 6.08 (m, 1H), 4.64 (m, 1H), 2.04 (m, 2H), 1.98 (m, 2H), 1.86 (d, J = 8 Hz, 3H), 1.73 (m, 2H); LCMS: 435 [M + 1]; c-MET Ki: 0.02 μΜ.

Example 13: 3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- (1-piperidin-4-yl-1H-pyrazol-4-yl) pyridin-2-ylamine [0419]

<img file="PL1786785T3_D0199.tif" />

165

[0420] The title compound was prepared following the procedure 62. NMR (400 MHz, CDCl 3) δ 7.69 (s, 1H), 7.56 (s, 1H), 7.50 (s, 1H), 7.32 (m, 1H), 7.07 (m, 1H), 6.87 (m, 1H), 6.07 (m, 1H), 5.25 (bs, 2H), 4.30 (m, 1H) , 3.41 (m, 2H), 2.96 (m, 2H), 2.26 (m, 2H), 2.12 (m, 2H), 1.86 (d, J = 8Hz, 3H) ; LCMS: 450 [M + 1]; c-MET Ki: 0.003 μΜ.

Example 14: 3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- (1-piperidin-4-yl-1H-pyrazol-4-yl) pyrazin-2-ylamine [0421]

<img file="PL1786785T3_D0200.tif" />

<img file="PL1786785T3_D0201.tif" />

F.

AcOH

The title compound was prepared following the procedure 66. 1 H NMR (400 MHz, DMSO-d 6) δ 7.86 (s, 1H), 7.76 (s, 1H), 7.63 (m, 2H) , 7.54 (m, 1H), 7.37 (t, 1H), 6.46 (q, 1H), 6.15 (s, 1H), 4.10 (m, 1H), 3.01 ( m, 2H), 1.95 (m, 2H), 1.85 (s, 2H), 1.75 (d, 3H), 1.67 (dd, 1H); LCMS: 451 [M + 1]; c-MET Ki: 0.010 μΜ.

Example 15: 3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- (1H-pyrazol-4-yl) pyrazin-2-ylamine

[0423]

N-NH

<img file="PL1786785T3_D0202.tif" />

166

[0424] The title compound was prepared following procedure 3 using 5-bromo-3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyrazin-2-ylamine and 4-acid tert-butyl ester. - (4,4,5,5-tetramethyl- [1,3,2] dioxaborolan-2-yl) pyrazole-1-carboxylic acid. NMR (400 MHz, DMSO-d6) δ 12.81 (s, 1H), 7.79 (s, 1H), 7.48 (m, 1H), 7.36 (t, 1H), 6.48 ( q, 1H), 6.12 (s, 2H), 1.75 (d, 3H); LCMS: 368 [M + 1]; c-MET Ki: 0.065 μΜ.

Example 16: 1- [4- (4- {5-amino-6 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyrazin-2-yl} pyrazol-1-yl) piperidin- 1-yl] -2-hydroxyethanone [0425]

<img file="PL1786785T3_D0203.tif" />

[0426] The title compound was prepared according to methods 62 and 63, using 5-bromo-3- [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyrazin-2-ylamine as starting material. 1 H NMR (400 MHz, DMSO-d₆) δ 7.91 (s, 1H), 7.76 (s, 1H), 7.64 (s, 1H), 7.49 (m, 1H), 7.36 (t, 1H), 6.46 (q, 1H), 6.15 (s, 2H), 4.57 (br, 1H), 4.40 (m, 2H), 4.12 (br, 2H), 3.77 (m, 1H) , 3.35 (m, 2H), 3.43 (m, 1H), 3.16 (m, 2H), 1.75 (d, 3H); LCMS: 509 [M + 1]; c-MET Ki: 0.015 μΜ.

167

Example 17: 3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- [1- (1-methylpiperidin-4-yl) -1H-pyrazol-4-yl] pyrazine -ylamine [0427]

<img file="PL1786785T3_D0204.tif" />

[0428] The title compound was prepared following procedure 62 using 5-bromo-3 - [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyrazin-2-ylamine and 4- (4-bromopyrazole- lyl) -1-methylpiperidine (prepared according to general procedure 11). 1H NMR (400MHz, DMSO-d6) δ 7.88 (s, 1H), 7.76 (s, 1H), 7.64 (s, 1H), 7.49 (m, 1H), 7 . 36 (t, 1H), 6.46 (q, 1H), 6.15 (s, 2H), 4.02 (m, 1H), 2.84 (m, 2H), 2.19 (s, 3H), 2.00 (m, 4H), 1.85 (m, 3H), 1.75 (d, 3H); LCMS: 465 [M + 1]; c-MET Ki: 0.03 μΜ.

Example 18: 1- [4- (4- {5-amino-6- [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyrazin-2-yl} pyrazol-1-yl) piperidin- 1-yl] -2-dimethylaminoethanone

[0429]

<img file="PL1786785T3_D0205.tif" />

168

[0430] The title compound was prepared following procedure 63 using 3- [(R) -1- (2,6-dichloro-3-fluorophenyl) ethoxy] -5- (1-piperidin-4-yl-1H-pyrazole -4-yl) pyrazin-2-ylamine conjugated with dimethylaminoacetic acid in the presence of HOBt / EDC / triethylamine in DMF as described in procedure 5 using 5-bromo-3 - [(R) -1- (2,6-dichloro 3-fluorophenyl) ethoxy] pyrazin-2-ylamine as starting material. i H NMR (400 MHz, DMSO-d₆) δ 7.90 (s, 1H), 7.76 (s, 1H), 7.65 (s, 1H), 7.49 (m, 1H), 7.36 (t, 1H), 6.47 (q, 1H), 6.15 (s,

2H), 4.39 (m, 1H), 4.16 (m, 1H), 3.16 (m, 2H), 3.02 (m, 1H),

2.75 (m, 1H), 2.19 (s, 6H), 2.01 (m, 2H), 1.88 (s, 1H), 1.75 (d, 3H); LCMS: 536 [M + 1]; c-MET Ki: 0.015 μΜ.

Example 19: 3 - [(R) -1- (2-chloro-3,6-difluorophenyl) ethoxy] -5- (1-piperidin-4-yl-1H-pyrazol-4-yl) pyridin-2-ylamine [0431]

<img file="PL1786785T3_D0206.tif" />

[0432] The title compound was prepared following procedure 62 using 5-bromo-3 - [(R) -1- (2-chloro-3,6-difluorophenyl) ethoxy] pyridin-2-ylamine as the starting material (according to by the synthesis of 5-bromo-3- [1- (2,6-dichloro-3-fluorophenyl) ethoxy] pyridin-2-ylamine from (S) -1- (2-chloro-3,6-difluorophenyl) ethanol, obtained from SynChem, Inc .). 1 H NMR (400 MHz, DMSO-d₆) δ 7.88 (s, 1H), 7.70 (s, 1H), 7.50 (s,

1H), 7.38 (m, 1H), 7.25 (m, 1H), 6.99 (s, 1H), 5.88 (m, 1H),

5.48 (bs, 2H), 4.08 (m, 1H), 2.96 (m, 2H), 2.53 (m, 1H), 2.45 (m, 1H), 1.89 (m , 1H), 1.80 (m, 4H), 1.67 (m, 4H); LCMS: 434

[M + 1]; c-MET Ki: 0.09 μΜ.

169

Biological examples

[0433] It is understood that in any given series of compounds, a range of biological activity will be observed. In its presently preferred aspects, the present invention relates to novel compounds capable of modulating, regulating and / or inhibiting protein kinase activity. The following assays can be used to select those compounds that exhibit the optimal degree of activity desired.

Testing methods

[0434] The following in vitro assay can be used to determine the level of activity and the effect of various compounds of the invention on one or more PKs. Similar assays can be designed following the same basics for any PK kinase using techniques well known in the art. Literature reference: Technikova-Dobrova Z, Sardanelli AM, Papa S FEBS Lett. November 4, 1991; 292: 69-72).

[0435] The general procedure is as follows: compounds and kinase test reagents are introduced into test wells. The test begins by adding the enzyme kinase. Enzyme inhibitors reduce the measured enzyme activity.

[0436] In a continuous-coupled spectrophotometric assay, the time-dependent production of ADP by the kinase is determined by analyzing the NADH consumption rate by measuring the decrease in absorbance at 340 nm. Since PK produces ADP, it is converted back to ATP by reaction with phosphoenolpyruvate and pyruvate kinase. Pyruvate is also produced in this reaction. Pyruvate is then converted to lactate by reaction with lactate dehydrogenase, which also converts NADH to NAD. NADH has a measurable absorbance at 340 nm while NAD does not.

[0437] The currently preferred protocol to perform a continuous-coupled spectrophotometric experiment for specific PKs kinases is outlined below. However, it is within the knowledge of those skilled in the art to adapt this protocol to determine the activity of the compounds against other RTKs as well as against CTKs and STKs.

170

Continuous-coupled HGFR spectrophotometric assay

[0438] This assay analyzes HGFR tyrosine kinase activity on a Met-2 substrate peptide, a peptide from activation of the HGFR loop.

Materials and Reagents: [0439]

1. HGFR enzyme from Upstate (Met, active) Cat. # 14-526

2. Met-2 peptide (HGFR activation loop) AcARDMYDKEYYSVHNK (MW = 1960). It is dissolved in 200 mM HEPES, pH 7.5 at 10 mM starting concentration.

3. IM PEP (phosphoenolpyruvate) in 200 mM HEPES, pH 7.5

4. 100 mM NADH (adenine B-nicotinamide, dinucleotide, reduced form) in 200 mM HEPES, pH 7.5

5. 4M MgCl₂ (magnesium chloride) in double distilled H.₂ABOUT

6. IM DTT (dithiothreitol) in 200 mM HEPES, pH 7.5

7. 15 units / ml LDH (lactic acid dehydrogenase)

8. 15 units / ml PK (pyruvate kinase)

9. 5M NaCl dissolved in double distilled H.₂ABOUT

10. Tween-20 (for proteins) 10% solution

11. IM HEPES buffer: (N- [2-hydroxyethyl] piperazine-N- [2-ethanesulfonic acid] sodium salt). It dissolves in double distilled H.₂0, the pH is adjusted to 7.5, the volume is adjusted to 1 L. Filter at 0.1 µm.

12. Water for HPLC; Burdick and Jackson # 365-4, 1X4 liters (or equivalent amount)

13. 100% DMSO (SIGMA)

14. Costar # 3880 - Black plates with a transparent flat bottom half of the plate for determining Ki and% inhibition

15. Costar # 3359 - 96-well polypropylene plates with round bottom for serial dilution

16. Costar # 3635 - UV plates with a transparent flat bottom for determining the% inhibition

17. Beckman DU-650 microcuvette holders w /

18. Beckman microcuvette item 4

171

Procedure:

[0440] Preparation of dilution buffer (DB) for the enzyme (for preparation of 30mL)

1. The final DB solution contains 2 mM DTT, 25 mM NaCl₂, 5 mM MgCl₂, 0.01% Tween-20, and 50 mM HEPES buffer, pH 7.5.

2. Dilute 50 mM HEPES by adding 1.5 ml of IM HEPES to 28.1 ml double distilled H₂O. The remaining reagents are added. To a 50 mL conical vial, add 60 µM of IM DTT, 150 µL of 5M NaCl₂, 150 μΐ IM MgCl₂, and 30 μΐ 10% Tween-20 to make a total volume of 30 ml.

3. It is vortexed for 5-10 seconds.

4. 1 ml / tube of DB is dispensed and the tubes are labeled DB HGFR

5. Note: The buffer can be prepared in advance and stored.

6. Freeze unused samples in microcentrifuge tubes in a freezer at -20 ° C.

Preparation of compounds [0441]

1. On the compound dilution plate, 4 µL of a 10 mM stock is added to column 1 of the plate, and made up to 100 µL with 100% DMSO.

2. The Precision 2000 dilution method is adjusted. Final concentration is 200 µL of compound in 50% DMSO, 100 mM HEPES (1: 2 serial dilution).

Preparation of the conjugated enzyme buffer: [0442]

1. Final concentration in test:

Reagent (concentration of stock solution), final concentration in the assay

a. PEP (IM) 1 mM

b. NADH (100 mM) 300 µΜ

c. MgCl₂ (4M) 20mM

d. DTT (IM) 2 mM

e. ATP (500 mM) 300 µΜ

f. HEPES 200 mM (pH 7.5) 100 mM

g. Pyruvate kinase (PK) 15 units / ml

172

h. Lactic acid dehydrogenase (LDH) 15 units / ml

i. Met-2 peptide (10 mM) 0.500 mM

j. HGFR 50 nM

2. 10 μΐ IM PER, 33 μΐ 100 mM NADH, 50 μΐ 4M MgCl are added to 10 ml of the reaction buffer₂, 20 µM DTT, 6 µM 500 mM ATP, and 500 µM 10 mM Met-2 peptide to 100 mM HEPES buffer, pH 7.5 and vortexed / mixed.

3. The coupling enzymes LDH and PK are added to the reaction mixture. It mixes by gentle inversion.

Carrying out measurements

[0443]

1. Spectrophotometer settings:

i. Absorbance at wavelength (λ): 340 nm ii. Incubation time: 10 minutes iii. Measurement duration: 10 minutes iv. Temperature: 37 ° C

2. 85 µΐ CE Reaction Mix is ​​added to each well of the assay plate.

3. Add 5 µΐ of diluted compound to the well of the assay plate.

4. Add 5 µΐ 50% DMSO as negative control to the last column of the assay plate.

5. It is mixed using a multichannel pipette or circular shaker.

6. It is pre-incubated for 10 minutes at 37 ° C.

7. Add 10 µΐ 500 nM HGFR to each well of the assay plate; final HGFR concentration is 50 nM in a final total volume of 100 µM.

8. The activity is measured for 10 minutes at λ = 340 nm and at 37 ° C.

[0444] The following in vitro assays can be used to determine the level of activity and the effect of various compounds of the invention on one or more PK. Similar assays can be designed following the same guidelines for any PK kinase using techniques well known in the art.

173

[0445] Several of the assays described herein are performed in ELISA (Double Binding Enzyme Immunoassay) format (Voller, et al., 1980, Enzyme-Linked Immunosorbent Assay, Manual of Clinical Immunology, 2nd Edition, Rose and Friedman, Am. Soc. . Of Microbiology, Washington, DC, pp. 359-371). The general procedure is as follows: the compound is introduced into cells expressing the test kinase, either naturally or recombinantly, for a selected period of time, after which, if the test kinase is a receptor, a ligand known to activate the receptor is added. The cells are lysed and the lysate is transferred to the wells of an ELISA plate previously coated with a specific antibody recognizing the substrate of the enzymatic phosphorylation reaction. Non-substrate components of the cell lysate are washed away and the degree of substrate phosphorylation is determined using an antibody that specifically recognizes phosphotyrosine, as compared to untreated control cells.

[0446] The currently preferred protocols for running an ELISA experiment for specific PK kinases are shown below. However, adapting these protocols to determine the activity of compounds against other RTKs, as well as for CTK and STK, is within the knowledge of those skilled in the art.

[0447] Other assays described herein measure the amount of DNA produced in response to activation of a test kinase, which is an overall measure of the proliferative response. The general procedure for this assay is as follows: the compound is introduced into cells expressing the test kinase, either naturally or recombinantly, for a selected period of time, after which, if the test kinase is a receptor, a ligand known to activate that receptor is added. . After incubation at least overnight, a DNA labeling reagent such as 5-bromodeoxyuridine (BrdU) or H 6 -thymidine is added. The amount of labeled DNA is detected using an anti-BrdU antibody or by measuring radioactivity, and

174 are compared with untreated control cells.

MET transphosphorylation test

[0448] This assay is used to measure phosphotyrosine levels on a poly (glutamic acid: tyrosine, 4: 1) substrate as a means to identify agonists / antagonists of the meth transphosphorylation substrate.

Materials and reagents:

[0449]

1. Corning 96-well ELISA plates, Catalog # 25805-96.

2. Poly (glu-tyr), 4: 1, Sigma, Cat #; P 0275.

3. PBS, Gibco, catalog # 450-1300EB

4. 50 mM HEPES

5. Blocking buffer: 25 g of Bovine Serum Albumin, Sigma, Cat # A-7888, are dissolved in 500 ml of PBS, filtered through a 4 µm filter.

6. Purified GST fusion protein containing the Met kinase domain, SUGEN, Inc.

7. TBST buffer.

8. 10% Aqueous (MilliQue HgO) DMSO.

9. 10mM aqueous (dHgO) adenosine-5'-triphosphate solution, Sigma, catalog number A-5394.

10. 2X Kinase Dilution Buffer: per 100 ml, mix 10 ml of IM HEPES pH 7.5 with 0.4 ml of 5% BSA / PBS, 0.2 ml of 0.1M sodium orthovanadate and 1 ml of 5M sodium chloride at 88, 4 ml distilled HgO.

11. 4X ATP reaction mixture: per 10 mL, mix 0.4 mL of 1M manganese chloride and 0.02 mL of 0.1M ATP in 9.56 mL of distilled HgO.

12. 4X mixture of negative controls: for 10 mL, mix 0.4 mL of 1M manganese chloride in 9.6 mL of distilled HgO.

13. NUNC 96-Well V-Bottom Polypropylene Plates, Applied Scientific Catalog # S-72092

14. 500 mM EDTA.

175

15. Antibody dilution buffer: per 100 ml, mix 10 ml 5% BSA / PBS, 0.5 ml 5% Camation® Instant Milk in PBS and 0.1 ml 0.1M sodium orthovanadate in 88.4 ml TBST.

16. Rabbit anti-phosphotyrosine polyclonal antibody, SUGEN, Inc.

17. Goat anti-rabbit charzanic peroxidase conjugated antibody, Biosource, Inc.

18. ABTS solution: for 1 L, 19.21 g of citric acid, 35.49 g of Na are mixed₂HPO4 and 500 mg ABTS with sufficient distilled H₂O to receive 1 1.

19. ABTS / H₂ABOUT₂: mix 15 ml of ABST solution with 2 μΐH₂ABOUT₂ five minutes before use.

20. 0.2M HCl

Procedure:

[0450]

1. ELISA plates are coated with 2 µg poly (Glu-Tyr) in 100 µl PBS, and kept at 4 ° C overnight.

2. The plate is blocked with 150 µL of 5% BSA / PBS for 60 minutes.

3. The plate is washed twice with PBS and then once with 50 mM HEPES buffer, pH 7.4.

4. Add 50 µΐ of diluted kinase to all wells. (Purified kinase is diluted with kinase dilution buffer. Final concentration should be 10 ng / well.)

5. 25 μΐ test compound (in 4% DMSO) or DMSO alone (4% in distilled H₂O) to the control wells on the plate.

6. The kinase / compound mixture is incubated for 15 minutes.

7. 25 µΐ 40 mM MnCl is added₂ into negative control wells.

8. 25 µL of ATP / MnCl mixture is added₂ to all other wells (except negative controls). It is incubated for 5 minutes.

9. 25 μΐ 500 mM EDTA is added to stop the reaction.

176

10. The plate is washed 3x with TBST.

11. 100 µL of rabbit anti-Ptyr polyclonal antibody diluted 1: 10,000 in antibody dilution buffer is added to each well. Incubate with shaking at room temperature for one hour.

12. The plate is washed 3x with TBST.

13. The HRP-conjugated anti-rabbit antibody of Biosource is diluted 1: 6000 in antibody dilution buffer. 100 µL per well is added and incubated at room temperature with shaking for one hour.

14. The plate is washed with 1X PBS.

15. 100 µΐ of ABTS / HgOg solution is added to each well.

16. If necessary, the progress of the reaction is stopped by the addition of 100 µM 0.2M HCl per well.

17. The plate is read on a Dynatecń MR7000 ELISA reader, using the assay filter at 410 nM and the reference filter at 630 nM.

BrdU input tests

[0451] The following assays use cells genetically engineered to express the selected receptor to evaluate the effect of a compound of interest on ligand-induced DNA synthesis activity by determining BrdU incorporation into DNA. Generally, the following materials, reagents, and procedure are used for each of the following BrdU incorporation tests. Changes to specific tests are given.

General Materials and Reagents: [0452] 1. Appropriate ligand.

2. Relevant genetically modified cells.

3. BrdU labeling reagent: 10 mM, in PBS, pH 7.4 (Roche Molecular Biochemicals, Indianapolis, IN).

4. FxDenat: fixative solution (Roche Molecular Biochemicals, Indianapolis, IN).

177

5. Anti-BrdU-POD: peroxidase-conjugated murine monoclonal antibody (Chemicon, Temecula, CA).

6. TMB Substrate Solution: Tetramethylbenzidine (TMB, ready to use, Roche Molecular Biochemicals, Indianapolis, IN).

7. PBS washing solution: IX PBS, pH 7.4.

8. Bovine albumin (BSA) fraction V powder (Sigma Chemical Co., USA).

General procedure:

[0453]

1. Cells are seeded at 8,000 cells / well in 10% CS, 2 mM Gln in DMEM, in a 96-well plate. Cells are incubated overnight at 37 ° C in 5% COg.

2. After 24 hours, cells are washed with PBS, and then serum starved in serum free medium (0% CS DMEM with 0.1% BSA) for 24 hours.

3. On day 3, the appropriate ligand and test compound are added to the cells simultaneously. Only serum-free DMEM with 0.1% BSA is added to the negative control wells; the ligand is added to the positive control cells, but without the test compound. Test compounds are made up into serum-free DMEM with ligand in a 96-well plate, and serially diluted to 7 test concentrations.

4. After 18 hours of ligand activation, diluted BrdU labeling reagent (1: 100 in DMEM, 0.1% BSA) is added and cells are incubated with BrdU (final concentration is 10 µΜ) for 1.5 hours.

5. After incubation with the labeling reagent, the medium is removed by decanting and tapping the inverted plate on a paper towel. FixDenat solution is then added (50 µΐ / well) and the plates are incubated at room temperature for 45 minutes on a plate shaker.

6. The FixDenat solution is removed by decanting and tapping the inverted plate on a paper towel. Milk (5% dehydrated milk in PBS, 200 μΐ / well) is then added as a blocking solution and the plate incubated

178 for 30 minutes at room temperature on a plate shaker.

7. Blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution is then added (1: 200 dilution in PBS, 1% BSA, 50 µΐ / well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.

8. The conjugated antibody is removed by decanting and washing the wells 5 times with PBS, and the plate is dried by inverting and tapping on a paper towel.

9. TMB substrate solution is added (100 µΐ / well) and incubated for 20 minutes at room temperature on a plate shaker until a color develops sufficient for photometric detection.

10. The absorbance of the samples is measured at 410 nm (in dual wavelength mode with filter reading at 490 nm as reference wavelength) on a Dynatecń ELISA plate reader.

HGF test induced by BrdU incorporation

Materials and reagents:

[0454]

1. Recombinant human HGF (Cat. No. 249-HG, R&amp;D Systems, Inc. USA).

2. BxPC-3 cells (ATCC CRL-1687).

Other materials and reagents, as above.

Procedure:

[0455]

1. Cells are seeded at 9,000 cells / well in RPMI with 10% FBS in a 96-well plate. Cells are incubated overnight at 37 ° C in 5% COg.

2. After 24 hours, cells are washed with PBS, and then serum depleted in 100 µl serum free medium (RPMI with 0.1% BSA) for 24 hours.

3. On day 3, 25 µl of a ligand-containing solution (prepared at 1 µg / ml in RPMI with 0.1% BSA; final HGF concentration is 200 ng / ml) and test compounds are added to the cells. Into negative control wells

179 25 μΐ serum free RPMI with 0.1% BSA only is added; the ligand (HGF) is added to the positive control cells, but no test compound. Test compounds are prepared at a 5-fold final concentration in serum-free RPMI medium with ligand in a 96-well plate, and serially diluted to 7 test concentrations. Typically, the highest final concentration of test compound is 100 μΜ, and a 1: 3 dilution (i.e. the final concentration of the test compound ranges from 0.137 to 100 μΜ).

4. After 18 hours of ligand activation, 12.5 µL of diluted BrdU labeling reagent (1: 100 in RPMI, 0.1% BSA) is added to each well and cells are incubated with BrdU (final concentration 10 µM) for 1 hour.

5. Same as in the general procedure.

6. Same as in the general procedure.

7. Blocking solution is removed by decanting and the wells are washed once with PBS. Anti-BrdU-POD solution (diluted 1: 100 in PBS, 1% BSA) is then added (100 µΐ / well) and the plate is incubated for 90 minutes at room temperature on a plate shaker.

8. Same as in the general procedure.

9. Same as in the general procedure.

10. Same as in the general procedure.

Cellular HGFR autophosphorylation test

[0456] A549 cells (ATCC) are used in this assay. Cells are plated in growth medium (RPMI + 10% FBS) in 96-well plates and grown overnight at 37 ° C until attachment. Cells are treated with starvation media (RPMI + 0.05% BSA). Diluted inibitor solutions are added to the plates and incubated at 37 ° C for 1 hour. Cells are then stimulated by adding 40 ng / ml HGF for 15 minutes. Cells are washed once with 1 mM NagVO4 in HBSS and then lysed. Lysates are diluted with 1 mM NagVO4 in HBSS and transferred to a 96-well plate coated with goat anti-rabbit antibody (Pierce) that has been pre-coated with anti-HGFR antibody (Zymed

180

Laboratories). Plates are incubated overnight at 4 ° C and washed with 1% Tween 20 in PBS for seven times. HRP-PY20 (Santa Cruz) is diluted and added to plates and incubated for 30 minutes. The plates are washed again, TMB peroxidase substrate (Kirkegaard &amp; Perry) is added and incubated for 10 minutes. The reaction is then stopped by adding 0.09N H2SO4. Plates are analyzed at OD-450 nm using a spectrophotometer. IC50 values ​​are calculated by curve fitting using a four-parameter analysis.

[0457] Compounds of the invention were measured for HGFR inhibitory activity; the data is presented in each example. Ki data was obtained using the continuous coupled HGFR spectrophotometric assay, and IC50 data was obtained using the HGFR cellular autophosphorylation assay described above.

[0458] While the invention has been illustrated by reference to specific and preferred embodiments, those skilled in the art will recognize that changes and modifications can be made through routine experimentation and during the practice of the invention. Thus, it is intended that the invention is not limited by the foregoing description, but is defined by the appended claims and their equivalents.

[0459] All references cited herein, including any priority documents, are herein incorporated by reference in their entirety.

181