AU2003291839B2 - Nicotinamide-based kinase inhibitors - Google Patents

Description

WO 2004/054977 PCT/AU2003/001666 Nicotinamide-based Kinase Inhibitors FIELD OF THE INVENTION The present invention involves compounds represented by Formula herein below, pharmaceutical compositions comprising such compounds and methods of suppressing the growth of cancers and other proliferative diseases.

BACKGROUND OF THE INVENTION Normal cellular proliferation is a well-controlled balance between the rate of cell cycle progression and programmed cell death (apoptosis). This balance is maintained by the appropriate transmission of extracellular signals by intracellular signal transduction circuitry. In tumours this equilibrium becomes disturbed by either unrestrained completion of the cell cycle, or loss of normal apoptotic cell death. In many cases this deregulation comes about by the autonomous activation of the intracellular signal transduction circuitry that controls the cell cycle and apoptosis pathways. Central to the regulation of these pathways are members of the protein kinase family, and a.promising avenue to the generation of treatments for hyperproliferative diseases such as cancer, are compounds that target those kinases involved in this regulation.

Protein kinases are a family of enzymes that catalyse the phosphorylation of specific residues in proteins. In general protein kinases fall into several groups; those which preferentially phosphorylate serine and/or threonine residues, those which preferentially phosphorylate tyrosine residues and those which phosphorylate both tyrosine and Ser/ Thr residues. Protein kinases are therefore key elements in signal transduction pathways responsible for transducing extracellular signals, including the action of cytokines on their receptors, to the nuclei, triggering various biological events. The many roles of protein kinases in normal cell physiology include cell cycle control and cell growth, differentiation, apoptosis, cell mobility and mitogenesis.

Inappropriately high protein kinase activity has been implicated in many diseases resulting from abnormal cellular function. This might arise either directly or indirectly, for example by failure of the proper control mechanisms for a kinase, related for example to mutation, WO 2004/054977 PCTIAU2003!001666 2 over-expression or inappropriate activation of the enzyme; or by over- Or underproduction of cytokines or growth factors also participating in the transduction of signals upstream or downstream of the kinese. In all of these instances, selective inhibition of the action of the. kinase might be expected to have a beneficial effect. Diseases where aberrant kinase activity-has been implicated include: diabetes; restenosis; atherosclerosis; fibrosis of the liver and kidney; ocular diseases; myelo- and lymphoproliferative disorders; cancer such as prostae cancer, colon cancer, breast cancer, head and neck cancer, leukemia and lymphoma; and, auto-immune diseases such as Atopic Dermnnatitis, Asthma, rheumatoid arthritis, Crohn's disease, psoriasis, Crouzon syndrome, achondroplasia, and thanatophoric dysplasia.

Protein kinases include, for example, but are not limited to, members of the Protein Tyrosine Kinase family (PTKs), which in turn can be divided into the cytoplasmic PTKs (CTKs) and the receptor PTKs (RTKs). The cytoplasmic PTKS include the SRC family, (including: BLK; FGR; FYN; HCK; LCK; LYN; SRC;Y]S and YRK); the BRK Family (inclu.ding BRK; FRK, SAD; and SRM); the CSK family (including: CSK and CTK); the BTK family, (including BTFK; m; 'TC; MKK2 and TXK), the Janus kinase family, (including: JAKI, JAK2, JAK3 and Tyk2), the FAK family (including, FAK and PYK2); the Fes family (including PBS and FER), the ZAP70 family (including ZAP70 and SYK); the ACK family (including ACKI and ACK2); and the Abl family (including ABL and ARC). The RTK family includes the EGF-Receptor family (including, EGFR, HER2, HER3 and HER4); the Insulin Receptor family (including INS-R and IGPI-R); the PDGF-Receptor family (including PDCFRa, PDGFRI, CSFiR, KIT, FLK2); the VEGF-Receptor family (including; FLTI, FLKi and PLT4); the FGF-Receptor family (including FGFRI, FGFR2, FGFRS and FGFR4); the CCK4 family (including CCK4); the MET family (including MET and RON); the TRK family (including TRKA, TRKB, and TKC the AXL family (including AXL, MER, and SKY); the TIE/TEEK family (including TIE and TE2/TEK); the EPH family (including EPHA1, EPHA, EPA, EPHA4, EPHA5, EPHA6, EPHA EP-A8, EPHB1, EPI-B2, EPHB3, EPHB4, EPI-B5, EPHB6); the RYK family (including RYK); the MCK family (including MCK and TYRO10); the ROS family (including ROS); the RET family (including RET); the LTK family (including LTK and ALK); the ROR family (including ROR1 and ROR2); The Musk family (including Musk); the LMR family including LMR1, LMRZ and LMR3); and the SuRTK106 family (including SuRTX1K06).

WO 2004/054977 PCTIAU2003!001666 3 Similarly, the serine /threonine specific kinases (STKs) comprise a number of distinct subfamilies, including; the extracellular signal regulated kinases, (p42/ERK2 and p44/ERQ); c-Jun NH2-termial kinase (JNK); cAMP-responsive element-binding protein kinases (CREBC); cAMP-dependent kinase (CAPK); mitogen-activated protein kinase-activated protein kinase (MAPK and its relatives); stress-activated protein kidnase p38/SAP2; mitogen-and stress-activated kinase (MSK); protein kinases, PIKA, PKB and PKC inter alia.

Additionally, the genomes of a number of pathogenic organisms possess genes encoding protein kinases. For example, the malarial parasite Plasmodium falciparum and viruses such as HPV and Hepatitis viruses appear to bear kinase related genes.

In one embodiment, the method of the invention is used in the treatment of sarcomas, carcinomas and/or lcukenias. Exemplary disorders for which the subject method can be used alone or as part of a treatment regimen include: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, Qndotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcorna, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cull carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma;Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastomrna.

In certain embodiments, the method of the invention isbe used to treat disorders such as carcinomas forming from tissue of the breast, prostate, kidney, bladder or colon.

In other embodiments, the method of the invention is used to treat hyperplastic or neoplastic disorders arising in adipose tissue, such as adipose cell tumors, lipomas, fibrolipomas, lipablastomas, lipomatosis, hibemomas, hemangiomas and/or liposarcomas.

-4- O SUMMARY OF THE INVENTION ct The present inventors have found that a group of compounds based upon a disubstituted pyridine scaffold are inhibitors of the growth and proliferation of cancer cells.

Accordingly, in a first aspect the present invention provides a compound of the general formula 00 W OO ci Y/Q A B

N

or pharmaceutically acceptable prodrugs, salts, hydrates, solvates, crystal forms or diastereomers thereof, wherein: A is selected from O, S, NRI, where R1 is selected from H, CM4 alkyl; B is aryl or hetaryl optionally substituted with 0-3 substituents independently is chosen from halogen, C 1 4 alkyl, CF 3 CN, aryl, hetaryl, OH, OCF 3 OCi-4alkyl,

OC

2 .5alkylNR2R3, Oaryl, Ohetaryl, C0 2 R2, CONR2R3, NR2R3, NR4CI.

4 alkylNR2R3, NR2COR3, OC(O)NR2R3, NR4CONR2R3, NR2SO 2 R3; and R2, R3 are each independently H, C14 alkyl, C 1 -4 alkyl heterocyclyl, aryl, hetaryl, C 1 4 alkyl aryl, CI-4 alkyl hetaryl, or may be joined to form an optionally substituted 3-8 membered ring optionally containing an atom selected from O, S, NR5; and R4 is selected from H, CI4 alkyl; and R5 is selected from H, C 14 alkyl; Q is absent or C1-4 alkyl; W is selected from H, Ci-4alkyl, C 2 6 alkenyl; where C 14 alkyl or C 2 -6alkenyl may be optionally substituted with C 14 alkyl, OH, OC14alkyl, NR 6 C(O)R7, CONR6R7, OR6, NR6R7; and R6, and R7 are each independently H, C-4 alkyl, CI 4 alkyl cycloalkyl, C-4 alkyl heterocyclyl, aryl, hetaryl, or may be joined to form an optionally substituted 3-8 membered ring optionally containing an atom selected from O, S, NR8 and R8 is selected from H, C.4 alkyl; Y is H, aryl or 5- or 6- membered heteroaromatic ring containing one or more heteroatoms selected from O, N, S, optionally substituted with 0-3 substituents independently chosen from halogen, C-4 alkyl, CF 3 aryl, hetaryl, OH, OCF 3 CN, C 2 4 alkynyl, OC 1 4 alkyl, OC2.

5 alkylNR9R10, Oaryl, Ohetaryl, C0 2 R9, N:\OIIboumONCasas\Pant\7200072999%P725IAU.2SpecrsP72516.AU.2 FirtAmendment.doc CONR9RIO, NR9RIO, Ci-4 alkylNR9RIO, NRI JIC 14 alkylNR9R1O, NR9CORIO, NRI ICONR9RIO, NR9SO 2 R1O; and R9, RIO are each ct independently H, CI4 alkyl, CI4 alkyl heterocyclyl, aryl, hetaryl, C-4alkyl aryl, CIA4 alkyl hetaryl, or may be joined to form an optionally substituted 3-8 membered ring optionally containing an atom selected from O, S, NRI2; and RI 1 is selected from H, CI-4 alkyl; and R12 is selected from H, C-4 alkyl, with the proviso that when RI is C-4 alkyl and Q and W are absent, then Y is r not aryl substituted at the ortho position with CO 2 R9, CN, NH 2 or hetaryl.

C 1to In a second aspect the present invention provides a composition comprising a carrier and at least one compound of the first aspect of the invention.

In a third aspect the present invention provides a method of treating a tyrosine kinaseassociated disease state in a subject, the method comprising administering a therapeutically effective amount of at least one compound of the first aspect of the invention or a therapeutically effective amount of a composition of the second aspect of the invention.

DETAILED DESCRIPTION OF THE INVENTION In a first aspect the present invention provides a compound of the general formula W 0 1

N

or pharmaceutically acceptable prodrugs, salts, hydrates, solvates, crystal forms or diastereomers thereof, wherein: A is selected from O, S, NRI, where RI is selected from H, CA4 alkyl; B is aryl or hetaryl optionally substituted with 0-3 substituents independently chosen from halogen, CI4 alkyl, CF 3 CN, aryl, hetaryl, OH, OCF 3 OCi4alkyl,

OC

2 5 alkylNR2R3, Oaryl, Ohetaryl, CO 2 R2, CONR2R3, NR2R3, NR4CJ.

4 alkylNR2R3, NR2COR3, OC(O)NR2R3, NR4CONR2R3, NR2SO 2 R3; and R2, R3 are each independently H, C14 alkyl, C-4 alkyl heterocyclyl, aryl, hetaryl, Cl4alkyl aryl, C14 alkyl hetaryl, or may be joined to form an optionally substituted 3-8 membered ring optionally containing an atom N:\Meboume\CasesPatent72OO-72999\P725 16AU.2SpeczsP72516.AU 2 First Amendrentsdoc -6selected from O, S, NR5; and R4 is selected from H, CM4 alkyl; and R5 is selected from H, C-4 alkyl; ¢c Q is absent or CM alkyl; W is selected from H, C 14 alkyl, C2-6alkenyl; where C.4alkyl or C26alkenyl may be optionally substituted with C 14 alkyl, OH, OC.4alkyl, NR 6 C(O)R7, CONR6R7, OR6, NR6R7; and R6, and R7 are each independently H, C-4 0\ alkyl, C 1 -4 alkyl cycloalkyl, C 14 alkyl heterocyclyl, aryl, hetaryl, or may be O joined to form an optionally substituted 3-8 membered ring optionally containing an atom selected from O, S, NR8 and R8 is selected from H, C 1 -4 NC1 10 alkyl; Y is H, aryl or 5- or 6- membered heteroaromatic ring containing one or more Sheteroatoms selected from O, N, S optionally substituted with 0-3 substituents independently chosen from halogen, C14 alkyl, CF 3 aryl, hetaryl, OH, OCF 3 CN, C 24 alkynyl, OC1-4alkyl, OC2-5alkylNR9RI0, Oaryl, Ohetaryl, CO 2 R9, CONR9R10, NR9RIO, C-4 alkylNR9RO0, NRI lC 14 NR9COR10, NRI ICONR9RIO, NR9SO 2 RI0; and R9, RIO are each independently H, C-4 alkyl, C 1 4 alkyl heterocyclyl, aryl, hetaryl, C 14 alkyl aryl, C-4 alkyl hetaryl, or may be joined to form an optionally substituted 3-8 membered ring optionally containing an atom selected from O, S, NRI2; and RI 1 is selected from H, C-4 alkyl; and R12 is selected from H, C-4 alkyl, with the proviso that when RI is C 1 4 alkyl and Q and W are absent, then Y is not aryl substituted at the ortho position with CO 2 R9, CN, NH 2 or hetaryl.

In the above description it will be appreciated that:

C

1 4 alkyl means an unsubstituted or optionally substituted straight or branched alkyl chain.

Aryl means unsubstituted or optionally substituted phenyl or naphthyl.

Hetaryl means an unsubstituted or optionally substituted 5- or 6-membered heteroaromatic ring containing one or more heteroatoms selected from O, N, S.

Cycloalkyl means a 3-8 membered saturated ring.

Heterocyclyl means a 3-8 membered saturated ring containing 1-3 heteroatoms selected from O, S, NRI3, where R13 is H, C 1 -4 alkyl, aryl, hetaryl.

N:\MelboumeNCases\Patent\72000-72999lP725 6 AU 2\Specis\P72516 AU 2 Firt Amendments doc Preferably, the compound is selected from the compounds of Table 1 and Table 2.

In a further preferred embodiment the compound of the general formula I is selected from the compounds of the general formula II.

w 0 QN B I

I

Sor pharmaceutically acceptable prodrugs, salts, hydrates, solvates, crystal forms or diastereomers thereof, wherein: RI is selected from H, C 1 4 alkyl; B is aryl or hetaryl optionally substituted with 0-3 substituents independently chosen from halogen, C.4 alkyl, CF 3 aryl, hetaryl, OH, OCF 3 OC.4alkyl,

OC

2 .5alkylNR2R3, Oaryl, Ohetaryl, C0 2 R2, CONR2R3, NR2R3, NR4CI.

4 alkylNR2R3, NR2COR3, NR4CONR2R3, NR2SO 2 R3; and R2, R3 are each independently H, C-4 alkyl, C14 alkyl heterocyclyl, aryl, hetaryl, C.4alkyl aryl, C-4 alkyl hetaryl, or may be joined to form a.n optionally substituted 3-8 membered ring optionally containing an atom selected from O, S, NR5; and R4 is selected from H, C-4 alkyl; and R5 is selected from H, C 1 4 alkyl; Q is absent or C-4 alkyl; W is selected from H, C14alkyl, C2-6alkenyl; where Ci 4 alkyl or C 2 6 alkenyl may be optionally substituted with C 1 4alkyl, OH, OC.4alkyl, NR6R7; and R6, and R7 are each independently H, C-4 alkyl, CI 4 alkyl cycloalkyl, C-4 alkyl heterocyclyl, aryl, hetaryl, or may be joined to fonn an optionally substituted 3-8 membered ring optionally containing an atom selected from O, S, NR8 and R8 is selected from H, C 14 alkyl; Y is H, aryl or 5- or 6- membered heteroaromatic ring containing one or more heteroatoms selected from O, N, S optionally substituted with 0-3 substituents independently chosen from halogen, C 14 alkyl, CF 3 aryl, hetaryl, OH, OCF 3

OC

14 alkyl, OC 2 5 alkylNR9RIO, Oaryl, Ohetaryl, CO0 2 R9, CONR9R10, NR9RIO, C-4 alkylNR9RO0, NRI ICi 4 alkylNR9R.10, NR9CORIO, NR ICONR9RIO, NR9SO 2 R10; and R9, RIO are each independently H, C.4 alkyl, C-4 alkyl heterocyclyl, aryl, hetaryl, C 1 i 4 alkyl aryl, C 14 alkyl hetaryl, or may be joined to form an optionally substituted 3-4 membered ring optionally N:WMelboume\Case Patent\72000-72999\P7251,AU.2\Speci3P72516 AU 2 First Amendment.doc containing an atom selected from O, S, NRI2; and RI 1 is selected from H, C-4 alkyl; and R12 is selected from H, C14 alkyl.

In the above description it will be appreciated that: CI4 alkyl means an unsubstituted or optionally substituted straight or branched alkyl chain.

00 Aryl means unsubstituted or optionally substituted phenyl or naphthyl.

(N Hetaryl means an unsubstituted or optionally substituted 5- or 6-membered Sheteroaromatic ring containing one or more heteroatoms selected from O, N, S.

Cycloalkyl means a 3-8 membered saturated ring.

Heterocyclyl means a 3-8 membered saturated ring containing 1-3 heteroatoms selected from O, S, NRI3, where R13 is H, C-4 alkyl, aryl, hetaryl.

The compounds of this invention include all conformational isomers (eg. cis and trans isomers). The compounds of the present invention may have asymmetric centers and therefore may exist in different enantiomeric and diastereomeric forms. This invention relates to the use of all optical isomers and stereoisomers of the compounds of the present invention, and mixtures thereof and to all pharmaceutical compositions and methods of treatment that may employ or contain them. The compounds of formula I may also exist as tautomers. This invention relates to the use of all such tautomers and mixtures thereof.

This invention also encompasses pharmaceutical compositions containing prodrugs of compounds of the formula I. This invention also encompasses methods of treating or N:\MelboumeCesersPate\720-72999\P725IO.AU.2Zpecis P72516.AU.2 First Amendmnts.doc WO 2004/054977 PCT/AU20031001666 9 preventing disorders that can be treated or prevented by the inhibition of protein kinases comprising administering prodrugs of compounds of the formula I. Compounds of formula I having free amino, amido, hydroxy or carboxylic groups can be converted into prodrugs. Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (eg, two, three or four) amino add residues which are covalently joined through peptide bonds to free amino, hydroxy and carboxylic acid groups of compounds of formula I. The amino acid residues include the 20 naturally occurring amino acids commonly designated by three letter symbols and also include, 4-hydroxyprolinc, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvaline, beta-alanine, gamnia-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine and methioine sulfone. Prodrugs also include compounds wherein carbonates, carbamates, amides and alkyl esters which are covalently bonded to the above substituents of formula I through the carbonyl carbon prodrug sidechain. Prodrugs also include phosphate derivatives of compounds of formula I (such as adds, salts of acds, or esters) joined through a phosphorus-oxygen bond to a free hydroxyl of compounds of formula I.

Prodrugs also include compounds wherein acyloxyalkyl or phosphonooxyalkyl moieties are covalently attached to compounds of formula I possessing a free hydroxyl group.

Acyloxyalkyl or phosphonooxyalkyl moieties may also be covalently attached to compounds of formula I possessing a pyridyl ring through formation of a N-(acyloxyalkyl)- or N-(phosphonooxyalkyl)-pyridinium salt This invention also encompasses pharmaceutical compositions containing prodrugs of compounds of the formula I.

In a second aspect the present invention provides a composition comprising a carrier and at least one compound of the first aspect of the Invention.

In a third aspect the present invention provides a method of treating a tyrosine Idnase-associated disease state, the method comprising administering a therapeutically effective amount of at least one compound of the first aspect of the invention or a therapeutically effective amount of a composition of the second aspect of the invention.

In a preferred embodiment of the present invention the disease state is selected from the group consisting of Atopy, such as Allergic Asthma, Atopic Dermatitis (Eczema), and Allergic Rhinitis; Cell Mediated Hypersensitivity, such as Allergic Contact Derma titis and Hypersensitivity Pneumoni is; Rheumatic Diseases, such as Systemic Lupus WO 2004/054977 PCT/AU20031001666 Erythematosus (SLE), Rheumatoid Arthritis, Juvenile Arthritis, Sj8gren's Syndrome, Scleroderma, Polymyositis, Ankylosing Spondylitis, Psoriatic Arthritis; Other autoimmune diseases such as Type I diabetes, autoimmune thyroid disorders, and Alzheimer's disease; Viral Diseases, such as Epstein Barr Virus (EBV), Hepatitis B, Hepatitis C, HIV, HTLV 1, Varicella-Zoster Virus (VZV), Human Papilloma Virus (IIPV); Cancer, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealona, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoina, neuroblastoma, and retinoblastoma, and carcinomas forming from tissue of the breast, prostate, kidney, bladder or colon, and neoplastic disorders arising in adipose tissue, such as adipose cell tumors, lipomas, fibrolipomas, lipoblastomas, lipomatosis, hibemomas, hemangiomas and/or liposarcomas.

As used herein the term "tyrosine kinase-associated disease state" refers to those disorders which result from aberrant tyrosine kinase activity and/or which are alleviated by inhibition of one or more of these enzymes.

The present invention provides pharmaceutical compositions comprising at least one of the compounds of the formula I or II capable of treating a kinase associated disorder in an amount effective therefore, and a pharmaceutically acceptable vehicle or diluent. The compositions of the present invention may contain other therapeutic agents as described below, and may be formulated, for example, by employing conventional solid or liquid vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the mode of desired administration (for example, excipients, binders, preservatives, stabilizers, flavours, etc.) according to techniques such as those well known in the art of pharmaceutical formulation.

WO 2004/054977 PCT/AU20031001666 11 The compounds of the formula I or II may be administered by any suitable means, for example, orally, such as in the form of tablets, capsules, granules or powders; sublingually; buccally; parenterally, such as by subcutaneous, intravenous, intramuscular, or intracisternal injection or infusion techniques as sterile injectable aqueous or nonaqueous solutions or suspensions); nasally such as by inhalation spray; topically, such as in the form of a cream or ointment; or rectally such as in the form of suppositories; in dosage unit formulations containing non-toxic, pharmaceutically acceptable vehicles or diluents.

The compounds may, for example, be administered in a form suitable for immediate release or extended release. Immediate release or extended release may be achieved by the use of suitable pharmaceutical compositions comprising the present compounds, or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps.

In addition to primates, such as humans, a variety of other mammals can be treated according to the method of the present invention. For instance, mammals including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine, canine, feline, rodent or murine species can be treated. However, the method can also be practiced in other species, such as avian species chickens).

Diseases and conditions associated with inflammation and infection can be treated using the method of the present invention. In a preferred embodiment, the disease or condition is one in which the actions of eosinophils and or lymphocytes are to be inhibited or promoted, in order to modulate the inflammatory response.

The subjects treated in the above methods, in whom which cell growth inhibition is desired, are mammals, including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine, canine, feline, rodent or murine species, and preferably a human being, male or female.

The term "therapeutically effective amount" means the amount of the subject composition that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.

The term "composition" as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified WO 2004/054977 PCT/AU20031001666 12 amounts. By "pharmaceutically acceptable" it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The terms "administration of" and or "administering a" compound should be understood to mean providing a compound of the invention to the individual in need of treatment.

The pharmaceutical compositions for the administration of the compounds of this invention may conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the art of pharmacy. Al methods include the step of bringing the active ingredient into association with the carrier, which constitutes one or more accessory ingredients. In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the pharmaceutical composition the active object compound is included in an amount sufficient to produce the desired effect upon the process or condition of diseases. As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.

Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients, which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acd; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be WO 2004/054977 PCT/AU20031001666 13 coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed, They may also be coated to form osmotic therapeutic tablets for control release.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum. acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate, The aqueous suspensions may also contain one or more preservatives, for example ethyl, or npropyl, p-hydroxybcnzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic add.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or WO 2004/054977 PCT/AU20031001666 14 wetting agents and suspending agents are exemplified by those already mentioned above.

Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

The pharmaceu tcal compositions of the invention may also be in the Form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally- occurring gums, for example gum acacia or gum tragacanth, naturallyoccurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acds and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that maybe employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The compounds of the present invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.

WO 2004/054977 PCT/AU20031001666 For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the compounds of the present invention are employed. (For purposes of this application, topical application shall include mouthwashes and gargles.) The compounds of the present invention can also be administered in the form of liposomes.

As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound of the present invention, stabilisers, preservatives, excipients and the like. The preferred lipids are the phospholipids and phosphatidyl cholines, both natural and synthetic. Methods to form liposomes are known in the art.

The pharmaceutical composition and method of the present invention may further comprise other therapeutically active compounds as noted herein which are usually applied in the treatment of the above mentioned pathological conditions. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

Examples of other therapeutic agents include the following: cydosporns cyclosporin CTLA4-Ig, antibodies such as ICAM-3, anti-IL-2 receptor (Anti-Tac), anti-CD45RB, anti-CD2, anti-CD3 (OKT-3), anti-CD4, anti-CD80, anti-CD86, agents blocking the interaction between CD40 and gp39, such as antibodies specific for and/or gp 3 9 CD154), fusion proteins constructed from CD40 and gp39 (CD401g and CD8gp39), inhibitors, such as nuclear translocation inhibitors, of NF-kappa B function, such as deoxyspergualin (DSG), cholesterol biosynthesis inhibitors such as HMG CoA reductase inhibitors (lovastatin and simvastatin), non-steroidal antiinflammatory drugs (NSAIDs) such as ibuprofen, aspirin, acetaminophen and cyclooxygenase inhibitors such as rofecoxib, steroids such as prednisolone or dexamethasone, gold compounds, WO 2004/054977 PCT/AU2003/001666 16 antiproliferative agents such as mcthotrexate, PK506 (tacrolimus, Prograf), mycophenolate mofetil, antineoplastic agents such as azathioprine, VP-16, etoposide, fludarabine, cisplatin, doxorubidn, adriamycin, amsacrine, camptothecin, cytarabine, gemcitabine, vinblastine, vincristine, fluorodeoxyuridine, melphalan and cyclophosphamide, TNF-c inhibitors such as tenidap, anti-TNF antibodies or soluble TNF receptor, and rapamycin (sirolimus or Rapamunc) or derivatives thereof.

When other therapeutic agents are employed in combination with the compounds of the present invention they may be used for example in amounts as noted in the Physician Desk Reference (PDR) or as otherwise determined by one of ordinary skill in the art.

The pharmaceutical composition and method of the present invention may further comprise other therapeutically active compounds as noted herein which are known inhibitors or substrates of drug efflux systems or drug detoxification and excretory systems. Such systems include P-glycoprotein, nultidrug resistance-associated protein, lung resistance protein and glutathione S-transferase isoenzymes alpha, mu, pi, sigma, theta, zeta and kappa. Co-administration of drugs known to inhibit or reduce the activity of these systems may increase the efficacy of the compounds described in the present invention through increasing the amount of therapeutic agent in the cell. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages, thus reducing the potential for adverse side effects. Examples of inhibitors or substrates for these systems include; verapamil, probenecid, dipyridamole, ethacrynic acd, indomethacin, sulfasalazine, butlhioninc sulfoximine, cyclosporin A and tamoxifan.

In the treatment or prevention of conditions which require protein tyrosine kinase inhibition an appropriate dosage level will generally be about 0.01 to 500 mg per kg patient body weight per day which can be administered in single or multiple doses. Preferably, the dosage level will be about 0.1 to about 250 mg/kg per day; more preferably about 0.5 to about 100 mg/kg per day. A suitable dosage level may be about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg kg per day. Within this range the dosage maybe 0.05 to 0.5, 0..5 to 5 or 5 to 50 mg/kg per day. For oral administration, the compositions are preferably provided in the form of tablets containing 1.0 to 1000 milligrams of the active ingredient, particularly 1.0, 5.0, 10.0, 15.0. 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0,400.0, 500.(, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be WO 2004/054977 PCT/AU20031001666 17 treated. The compounds may be administered on a regimen of I to 4 times per day, preferably once or twice per day.

It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

All publications mentioned in this specification are herein incorporated by reference.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

In order that the nature of the present invention may be more clearly understood preferred forms thereof will now be described by reference to the following non-limiting Examples.

EXAMPLES

MATERIALS AND METHODS: Compound Synthesis Compounds are generally prepared in a 2-step process starting from a protected bromonicotinic acid.

WO 2004/054977 PCT/AU2003/001666 18 The first step of the synthesis typically involves a palladium mediated cross-coupling of the protected 5-bromonicotinic acid with a suitably functionalised coupling partner.

Typical coupling partners are boronic acids (Suzuki coupling: see for example Miyaura, N.

and Suzuki, Chem Rev. 1995, 952457) or organostannanes (Stille coupling: see for example Stille, J.K, Angew. Chem, Int. Ed. Eng., 1986, 25, 508) (Scheme 1).

0 0 B r P R1 R-

M

1 a Pd catalyst base Scheme 1 The Suzuki coupling is the preferred coupling method and is typically performed in a solvent such as DME, TIF, DMF, ethanol, propanol, toluene, or ,4-dioxane in the presence of a base such as potassium carbonate, sodium carbonate, lithium hydroxide, caesium carbonate, sodium hydroxide, potassium fluoride or potassium phosphate. The reaction may be carried out at elevated temperatures and the palladium catalyst employed may be selected from Pd(PPh,), Pd(OAc) 2 [PdCl(dppf)], Pd(dba)/P(t-Bu) 3 palladium on carbon.

Methods to protect 5-bromonicotinic acid are known to those skilled in the art and may include the formation of a 2-(trimethylsilyl)ethyl ester, 2 -(trimethylsilyl)ethoxymethyl ester, tetrahydrofuranyl ester or t-butyl ester. The t-butyl ester is the preferred protecting group.

The -Butyl 5-bromonicotinate employed in the first step can be readily prepared from commercial 5-bromonicotinic acd using conventional methods well known to those skilled in the art. These methods indude the coupling of 5-bromonicotinic acid with t-butanol using coupling reagents such as dicyclohexylearbodiimide or l-(3-dimethylaminopropyl)- 3-ethylcarbodiimide in a solvent such as dichloromethane, or treatment of bromonicotlnic acid with di- -butyldicarbonate in the presence of a base such as triethylamine in a solvent such as tetrahydrofuran.

The second step of the synthesis is amide, ester or thioester formation by coupling the arylnicotinic acid with a primary or secondary amine, an alcohol or phenol, or an alkyl or aryl mercaptan (Scheme 2).

WO 2004/054977 PCT/AU20031001666 19 0 Ar RI-X P Ar HO XA A NX O,S,NH,NR2 N Scheme 2 Initially the protected ester forms of the 5-arylnicotinate derivatives prepared as in Scheme 1 are cleaved to the corresponding acids using methods well known to those skilled in the art.

The 5-arylnicotinic acid derivatives are typically coupled with amines, alcohols, phenols, thiols or thiophenols using coupling reagents such as dicyclohexylcarbodiiide, 1-(3dimethylamninopropyl)-3-ethylearbodiimide, diisopropylcarbodiimide or carbonydiimidazole in solvents such as dichloromethane and tetrahydrofuran.

Alternatively, the deprotected 5-aryInicotinic acid derivatives prepared as in Scheme 1 can be converted to the respective acid chloride derivatives using thionyl chloride or oxalyl chloride, or to the mixed anhydride species using, for example, -butyl chloroformate, using procedures well known to those skilled in the art. The acid chloride or mixed anhydride derivatives can then be reacted with the desired amine, alcohol, phenol, thiol or thiophenol in the presence of a base such as triethylamine, diisopropylethylamine or solid phase equivalent in a solvent sudch-i as dichloromethane, tetrahydrofuran, dioxane or ethyl acetate at ambient or elevated temperatures, to generate the desired 5-aryl nicotinic aidd derivatives.

As a further alternative, the deprotected 5-arylnicotinic acid derivatives prepared as in Scheme 1 can be converted to the corresponding active ester intermediates, such as the succinimidyl, pentafluorophenyl or p-nitrophenyl esters, This can be achieved by coupling the 5-arylnicotinic acid derivatives with Athydroxysuccinimide, pentafluorophenol or pnitrophenol using coupling reagents such as dicyclohexylcarbodiimide, 1-(3dimethylandnopropyl)-3-ethylcarbodiimide, diisopropylcarbodiiide or carbonyldiimrnidazole in solvents such as dichloromethane and tetrahydrofuran. Active acyl intermediates can also be formed directly by reaction of the 5-aryInicofinic acid derivatives with reagents such as diphenylphlosphoryl azide, pentafluorophenyl acetate, WO 2004/054977 WO 204/04977PCT1A1J20031001666 pentafluorophenyl diphenyiphosphinate or cyanuric chloride using methods well known to those skilled in the art.

The products formed from this reaction sequence may be fufther derivatised using techniques well known to those skilled in the art.' S Example I Tert-0uafyl To a mixture of 5-bromonicotinkc adid (0.30g, 1L49mmol) and di- tert-butyldicarbonate (0.45g, 2.06n-tmol) in TI-F (l1rnL) was added triethylaminie (0.2SinL, 1.79mnol) followed by 4-Cpyrroidino)pyridinc (30mg, 0.Zoinmol). T'he resultant solution was stirred at ambient temperature for 48 hr. The volatiles were then removed under vacutum and the residue was purified by column chromatography on silica (gradient CR2C 2 to 3% MeOH/Cl 2

C

2 to provide the product as a white solid (0.35g, 91%) 1 1±n~m.r. (C~D 3 I) 61.60 91-I, t-but-yl), 8.35 (in, 11-, Ar), 8.79 (di, J2.5 Hy-, IN, Ar), 9.06 (d,J Example 2 Tert-2tly1 -F-04-me-Myilen ed/oyph enyyaivcodtn af In a flask was placed tert-butyl 5-bronwnicotinate (1.00g. 3.B7mmol), 101/ wlw palladium on carbon (210mg, -0.2rnmol Id), potassium carbonate (1-10g, 7.96mnmol), 3,4methylcnedioxy-phenyl boronric acid 55'Orrunol), dimethylformamicle (5OinL and water (25Gm). The flask was plvlfed with nitrogen and then heated, with stirring, to 901C for l5hr. The reaction mrixture was allowed to cool, diluted with water (350mL) and extracted with CHl-I (4XY The combined. extracts was washed with water, dried (MgSO 4 and concentrated in vacua. The crude residue was purified by flash chromatography on silica (gradient, CH 2 C 2 to 50%oether/ CH 2

CI

2 to provide an off-wbite solid (1.

0 2g, 88%).

(CDCba) 31.63 911I, t-bu tyl), 6.04 ZH, CH-I, 6.90 (in, IH, Ar), 7.0$ (ni, 21-1, Ar), 8.34 (iii, i1-, Ar), 8-S9 (di, Jt19 I1-K, Ar), 9.09 (di, J2.0 Hiz, in, Ar).

ExampleS3 WO 2004/054977 PCT/AU20031001666 21 S-c3,4-mnethyldioenedioxyphenyl)Vicotylcode A solution of tert-butyl 5-(3,4-meAtylenedioxyphenyl)nicotinate (0.80g, 2.67mmol) in trilluoroacetic acid (10mL) was stirred at room temperature for 2hr. The trifluoroacetic acid was removed in vacuo and the residue treated twice with toluene followed by removal under vacuum (to remove residual TFA). The yellow/green residue was then treated with thionyl chloride (10tL) and dimethylformamide (100L) and the mixture was heated to reflux for 15hr. The reaction mixture was allowed to cool to ambient temperature, then the Volatiles were removed on a water aspirator. The residue was treated twice with toluene followed by removal in vacua to leave a yellow solid. To the crude acd chloride was added 1,4-dioxane (16mL) to make a 0.167M suspension, which was used without further treatment in the next step.

Example 4 Formation of nicotinamides in 96-well format To each well of a 96-well deep well plate was added Amberlyst A-21 resin 0.33mmol). To each well was then added a 0.19M 1,4-dioxane solution of amine (0.30mL, 57mol) followed by a 0.167M suspension of 5-arylnicotinoyl chloride (0A48mL, 80mol). The plate was sealed with a webseal mat and the plate was sonicated for Thr in a sonicator bath.

Amberlite IRA-67 (30mg, 0.17rmmol) was added to each well, the plate was re-sealed and sonication continued for a further 30mmn. The contents of the 96-well plate was transferred via pipettor to a 96-well filter plate and filtered into a second 96-well deep well plate. 1,4- Dioxane (0.40mL) was placed in each well of the original 96-well plate (to rinse). This was aspirated with the pipcttor and then transferred to the filter plate and filtered into the second 96-well plate. The second 96-well plate was stripped of all volatiles on a Christ rotary vacuum concentrator, and then the residues in each well were re-dissolved in CI-HC1, (0.50mL). These CH.C solutions were transferred to a second filter plate loaded with silica (200mg/well) and filtered into a third 96-well deep well plate. A methanol/CH 2

CI

2 solution (0.50mL) was added to each well of the second filter plate and filtered into the third deep well plate. The contents of the third plate was analysed by LC- MS and then concentrated under vacuum using the Christ rotary vacuum concentrator.

Further examples of 5-arylnicotinamides are shown in Table 1 along with their experimental m z values.

WO 2004/054977 WO 204/04977PCT/A1J20031001666 Table I CHEMISTRY' miz2 (El) 346.6

H

C22H2-2N202

H

H

02OH19N302

HO

28.

C171J20N202

HO,.

HH

rco 327.8 Cl1I-120N204 WO 2004/054977 WO 204/04977PCT1A1J20031001666 23 Table 1 (cont) CHEMISTRY mlz (El)

F

N J1~) 51.2 0 F- 71 0~N K- N 274.3 C1HNS WO 2004/054977 WO 204/04977PCT/A1J20031001666 24 Table 1 (COWL CHEMISTRiY miz (El 0W 0 N S36.1 CI5H17N203 0 H 225~1.2(M1) 1H1N302 WO 2004/054977 PCT1A1J20031001666 Table 1 (cont.) WO 2004/054977 WO 204104977PCTiAU2003!001666 Table 1 (cant) CHEMISTRY mt(ED) 00 378.4

N

022H22N204 Ho

H

020N18N203 305.3 I,

P

-A

*C2HC2N202 WO 2004/054977 PCTIAU2003!001666 27

SCREENING

JAK Tyrosine Kinase Domain Production JAK kinase domains were produced in the following manner: JAK1 The kinase domain of human JAI was amplified from U937mRNA using the polymerase chain reaction with the following primers: XHOI-1 5'-CCG CTC GAG ACT GAA GTG GAC CCC ACA CAT-3' J1-KPNI 5'-CGG GGT ACC TT'A TIT TAA AAG TGC TTC AAA-3' JAKI PCR products were cloned into the pFastBac HTb expression vector (Gibco) via the Xho I and Kpn I sites. The JAK1 plasmid was then transformed into competent DH1OBac cells (Gibco), and the recombinant baculovirus produced prepared for transfection into Sf9 insect cells.

JAK2 The kinase domain of humanJAK2 was amplified from U937mRNA using the polymerase chain reaction with the following primers: SALI-jk2 5'-ACG CGT CGA CGG TGC CTT TGA AGA CCG GGA T-3' jk2-NOTJ 5'-ATA GTI' TAG CGG CCG (ZTC AGA ATG AAG GTC ATT T-3' JAK2 PCR products were cloned into the pFastBac ftc expression vector (Gibco) via the Sal I and Not I sites. The JAK2 plasmid was then transformed into competent DHIOBac cells (Gibco), and the recombinant baculovirus produced prepared for transfection into Sf9 insect cells.

JAK3 The kinase domain of human)AKS was amplified from U937mRNA using the polymerase chain reaction with the following primers: WO 2004/054977 PCT/AU20031001666 28 XHOI-JP 5'-CCG CTC GAG TAT GCC TGC CAA GAC CCC ACG-3' JS-KPNI 5'-CGG GG'' ACC CTA TGA AAA GGA CAG GGA GTG-3' JAK3 PCR products were cloned into the pFastBac HTb expression vector (Gibco) via the Xho I and Kpn I sites. The JAK3 plasmid was then transformed into competent DHIOBac cells (Gibco), and the recombinant baculovirus produced prepared for transfection into Sf9 insect cells.

TYK2 The kinase domain of humanTYK2 was amplified from A549 mRNA using the polymerase chain reaction with the following primers: HT2EK 5'-GGA GCA CTC GAG ATG GTA GCA CAC AAC CAG GTG-3' TlY2.2R 5'-GGA GCA GGA ATT CCG GCG CTG CCG GTC AAA TCT GG-3' TYK2 PCR products were cloned into pl3ueBacHis2A (Invitrogen) via the EcoRI site. The recombinant TYK2 baculovirus produced was prepared for transfected into Sf9 insect cells.

Large Scale Production Of Kinase Domains Baculovirus preparations from each of theJAK family members were infected into five litres of High Five cells (Invitrogen) grown in High Five serum free medium (Invitrogen) to a cell density of approximately 1-2 X 10' cells/ml. Cells are infected with virus at a MOI of 0.8-3.0. Cells were harvested and lysed. JAK kinase domains were purified by affinity chromatography on a Probond (Invitrogen) nickel chela te affinity column.

Assay Protocols Kinase assays were performed either in a 96 well capture-based ELISA assay or in 384 well Optiplates (Packard) using an Alphascreen Protein Tyrosine Kinase kit In either casse using approximately 1.5 gg of affinity purified PTK domain in the presence of HS'ES, pH 7.5, 10mM MgCI, 150mM NaCI and 10pM-1mM ATP. The biotinylated substrate biolin-EGPWLEEEEEAyGWMDP)-NI-1 2 (final concentration 5gM) was used as substrate. In the ELISA assay tyrosine phosphorylation was quantitated following transfer to an avidin coated EISA plate using peroxidase-linked anti-phospho-tyrosine antibody WO 2004/054977 PCTIAU2003/001666 29 In the Alphascreen assay, Alphascreen phosphotyrosine acceptor beads followed by streptavidin donor beads were added under subdued light. The ELISA plates were read on a BMG Fluorostar, the Alphascreen plates were read on a Packard Fusion Alpha Inhibitors were added to the assays fifteen minutes prior to the addition of ATP. Inhibitors were added in aqueous DMSO, with DMSO concentrations never exceeding 1%.

Results The activity of a range of compounds is shown in Table 2. Compounds that exhibited a capacity to inhibit 50% or greater of enzyme activity at a concentration of 10 RM (measured under standard conditions, see Methods), are designated as Compounds not tested are designated while compounds that did not inhibit enzyme activity by 50% at 10 xM are designated WO 2004/054977 PCT/A1J20031001666 Table 2 0 NT NT G23H24N206 0 NT NT NT

N,

01 7H20N205

H

C17H19NOQ4 020HIBN204 WO 2004/054977 WO 204104977PCTiAU2003!001666 Table 2 (cant) H00

H

WO 2004/054977 PCT1A1J20031001666 32 Table 2 (couL) WO 2004/054977 PCT/A1J20031001666 33 Table 2 (aCont) WO 2004/054977 WO 204104977PCTiAU2003!001666 TabL 2 (C~ont.) CHEMISTRY- JabI Jak 2t- ZR7O NT C20Hla1N202 0 NT- ro2H24N204 0 0 -1 N Clr~jJQ H0IH WO 2004/054977 PCTiAU2003!001666 TAble 2 (cont) WO 2004/054977 PCT/A1J20031001666 Table 2 (cont) WO 2004/054977 WO 204/04977PCT/A1J20031001666 Table 2 kcont) CHEMISTRY Jak JaW ai~ f ff lick

ITNT

-0 0 ~NT 021 H2OCIN304 KL NTT

H

CISH210IN204 J 0 NT NT C1 I 9-; 02G230N204 WO 2004/054977 PCT/A1J20031001666 38 Table 2 (conL) WO 2004/054977 PCT/A1J20031001666 Tabk 2 (cont.

WO 2004/054977 PCT/A1J20031001666 Table 2 (cont.) WO 2004/054977 WO 204104977PCTiAU2003!001666 Table 2 (cont.) CHEMISTRY Jak3 abi 7ie- fIns rck p NT ClOH14N2OS NT -NT C22H12ON205

F

<OIJK)- NT Q2QHI 5FN203 -o

NN

021H18N204 co

NT

021H18N20S WO 2004/054977 WO 204104977PCTiAU2003!001666 Table 2 (comL) CHEMISTRY Jah2 JaI abi fes fI Is Ihak Zap7O I

NT

C21BN203 020HI 5IN203

SF

NT NT 020H16FN203 -NT NT N C21H21N325_____ WO 2004/054977 WO 204/04977PCT/A1J20031001666 Table 2 (tcont-) CHEMISTY Jsk2 ~Jak -Ij hoq -a~

NN

FP0

NT

N

H -NT- C1lH18N202S 0 H NT C12H12M202S Q 0w- o 016H-12N2c'$ 0 NT

H

C1GH13MsOS WO 2004/054977 WO 204104977PCTiAU2003!001666 Table 2 (colnt.) CHEMISTRY- Jak2 Jak3 ObW fes; fins lck ?ZD7-

'NT

NN

Irv 017Hi4FN208 Il NT CISH16N2OS______

NN

C2ON3O2S WO 2004/054977 WO 204104977PCTiAU2003!001666 Table 2 (cont.) CHEMISTRY Jakc2 Jnk3 abi tea fins hok IZapf H NT O1I-i16N208 H- Oi07H1SFN2OSI >yF NT NT M09 C2Of-1WN208 WO 2004/054977 WO 204/04977PCT/A1J20031001666 Table 2 (corkt.) CHEMISTRY Jui2 AMi~ bi- fp fins lick

H

CIOHIGN203S 0Q H NT C17H14N20S

F+

N

CISH15FN2O8

NT

019HU1 N2048 N NT IYI

H

N

019H17N30 WO 2004/054977 PCT/A1J20031001666 Table 2 (cont.) WO 2004/054977 PCT/A1J20031001666 Table 2 (conQL

IH

WO 2004/054977 PCT/A1J20031001666 49 Table 2 (conQ.

WO 2004/054977 PCTiAU2003!001666 Table 2 (cont.

WO 2004/054977 PCTiAU2003!001666 51 Table 2 (cont.) WO 2004/054977 PCT/A1J20031001666 52 Table 2 (cont.) WO 2004/054977 PCTiAU2003!001666 Table 2 (cont.) CIHEMISTRY Jak2 JaIQ abi fe-s- fma- haok NT

N

NN

Cl 9H17N202 '0 Cl 9H22N204 0 rO21H20N2O4 WO 2004/054977 PCT/A1J20031001666 54 Table 2 (cont.) WO 2004/054977 WO 204/04977PCT/A1J20031001666 Table 2 (cont.) CHEISRYJdk2 Jgk3 albi foo fins hek ZaiO

NT'

C~oH17F!W2O2 CL NT-- C23H-24N202

TN

021H20N204-

NNT

H

N202

I__

NT

NNI

0;22H22N204 WO 2004/054977 WO 204104977PCTiAU2003!001666 Table 2 (coot.) CHIEMISTRY Jakl2 JAS~ abi fes tns- lck

HO

NT 021H42ON203 ~I +i NT

NN

H

020HIS7N203

NT-

Cl WO 2004/054977 PCTiAU2003!001666 Table 2 (cont) CHEMISTRY Jak2 iJU abi- fes Tins ho i; 7 NT ClSH17NSO HNT NT- Cl BH-14PNSO to H NT Cl5H14IFN30 0 K>Le NT NT-- Q18H14FN30I -58- It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments Mc without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

In the claims which follow and in the preceding description of the invention, except 00 where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an NC 10 inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

N:\MboumEasesa\Paent7200D7299P72 51.AU.2\SpoCCsP72518 AU.2 First Amendments doc

Claims (3)

1. 2. A compound according to claim I of the general formula 11: w 0 B Ri N Cc) II or pharmaceutically acceptable prodrugs, salts, hydrates, solvates, crystal forms or diastereomers thereof, wherein: RI is selected from H, CI4 alkyl; B is aryl or hetaryl optionally substituted with 0-3 substituents independently chosen from halogen, Cl4 alkyl, CF 3 aryl, hetaryl, OH, OCF 3 OCi4alkyl, OC2-5.salkylNR2R3, Oaryl, Ohetaryl, CO 2 R2, CONR2R3, NR2R3, NR4C 1 4 alkylNR2R3, NR2COR3, NR4CONR2R3, NR2SO 2 R3; and R2, R3 are each independently H, CI4 alkyl, CI-4 alkyl heterocyclyl, aryl, hetaryl, C14alkyl aryl, CI4 alkyl hetaryl, or may be joined to form an optionally substituted 3-8 membered ring optionally containing an atom selected from O, S, NR5; and R4 is selected from H, C 1 4 alkyl; and R5 is selected from H, CI4 alkyl; Q is absent or CIA alkyl; W is selected from H, Cl4alkyl, C2-6alkenyl; where Cl-4alkyl or C 2 6 alkenyl may be optionally substituted with CI4alkyl, OH,. OCI-4alkyl, NR6R7; and R6, and R7 are each independently H, CI4 alkyl, CI4 alkyl cycloalkyl, CI4 alkyl heterocyclyl, aryl, hetaryl, or may be joined to form an optionally substituted
2. 3-8 membered ring optionally containing an atom selected from O, S, NR8 and R8 is selected from H, CI4 alkyl; Y is H, aryl or 5- or 6- membered heteroaromatic ring containing one or more heteroatoms selected from O, N, S optionally substituted with 0-3 substituents independently chosen from halogen, C14 alkyl, CF 3 aryl, hetaryl, OH, OCF 3 OC-4alkyl, OC2-salkylNR9RIO, Oaryl, Ohetaryl, CO 2 R9, CONR9RIO, NR9RIO, C-4 alkylNR9RIO, NRI ICI4akylNR9RIO, NR9CORIO, NRI ICONR9RIO, NR9SO 2 R IO; and R9, RIO are each independently H, CI-4 alkyl, CI-4 alkyl heterocyclyl, aryl, hetaryl, CI-4alkyl aryl, C14 alkyl hetaryl, or may bejoined to form an optionally substituted 3-8 membered ring optionally N Welbo oumCoS303'atcnt\7200-72999P7:516AU2Speci3 P72516AU2 First Amendmens.doc -61 containing an atom selected from 0, S, NR12; and RI 1 is selected from H, C 1 4 alkyl; and R12 is selected from H, CI-alkyl. 3. A compound according to claim I wherein the compound is selected from the group consisting of:. 0 0 00 N 0 0 N0 N N I II c i N N C22H22N204 C23H23CIN204 18N202 C22H20CIFN2O4 C19HI5FN202 C22H20CIFN204 N.\eltoum\Caes alet\70007299T7!il.AU2\Sed3P7218 U.2First Amendmentsdoc WO 2004/054977 WO 204/04977PCT1A1J20031001666 62 N.N 019H16N202 C22 C22HION2O4 H i N C2OHi 9N802 C2OH1QlN2O3 HOH NN 02PH22N202 CI 6HI2N208 WO 2004/054977 WO 204/04977PCT1A1J20031001666 63 H 021 H19FN202 HO 'N 021H19FN202 HO.0 HO-- l H> 021H2M202 017'HlarN2oS IF1i C2oHisNso02s C18SHION20S Cl 7HlSFN2Q$ 021 H20N202 CISH1GN2Q$02H0N0 C21HgONP-02 WO 2004/054977 WO 204/04977PCT1A1J20031001666 O2OH2ONQOS N 0 C22H22N203 0 CMi 20N202 CITH14NPOS CIBHISFN2CS 020H17FN202 019H17N303 C2Cl7FN202 WO 2004/054977 WO 204/04977PCT1A1J20031001666 N 0 N 1o C19Hl17N302 00 N'N 010H16DN202 001,1 12 0 HO 'N 0 HO 01 BH1 N203 0 H O2OH16NZOP 021H20N203 C2OJ-17FN208 G2O0N 7FN903 021 HPONP03 2H1N0 020HISN20a
3. 66- O 4. A composition comprising a carrier and at least one compound of any one of claims 1 to 3. A composition according to claim 4 which further comprises another therapeutically active compound. 6. A method of treating a tyrosine kinase-associated disease state in a subject, the 00 method comprising administering a therapeutically effective amount of at least one compound of any one of claims I to 3 or a therapeutically effective amount of a 10 composition of claim 4 or 7. A method according to claim 6 wherein the disease state is selected from the group consisting of Atopy, such as Allergic Asthma, Atopic Dermatitis (Eczema), and Allergic Rhinitis; Cell Mediated Hypersensitivity, such as Allergic Contact Dermatitis and Hypersensitivity Pheumonitis; Rheumatic Diseases, such as Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis, Juvenile Arthritis, Sjogren's Syndrome, Scleroderma, Polymyositis, Ankylosing Spondylitis, Psoriatic Arthritis; Other autoimmune diseases such as Type I diabetes, autoimmune thyroid disorders, and Alzheimer's disease; Viral Diseases, such as Epstein Barr Virus (EBV), Hepatitis B, Hepatitis C, HIV, HTLV 1, Varicella-Zoster Virus (VZV), Human Papilloma Virus (HPV); Cancer, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma, and carcinomas forming from tissue of the breast, prostate, kidney, bladder or colon, and neoplastic disorders arising in adipose tissue, such as adipose cell tumors, lipomas, fibrolipomas, lipoblastomas, lipomatosis, hibemomas, hemangiomas and/or liposarcomas. N:\Melboume\Case\Patent\72000-72999\P72516.AU.2\SpecsP72518.AU.2 Fit Amendment.doc -67- ct 8. Use of at least one of the compounds of any one of claims 1 to 3 in the preparation of a medicament for the treatment of a tyrosine kinase-associated disease state. 9. Use of at least one of the compounds of any one of claims 1 to 3 for the treatment of a tyrosine kinase-associated disease state. Q and A process for the preparation of a compound of any one of claims 1 to 3 when W are absent which comprises the steps of: coupling a compound of formula III wherein is a protecting group with B-M wherein B is as defined in claim 1 or 2 and M is a metal to prepare a compound of formula IV wherein 0 and B are as defined above; and (ii) deprotecting the compound of formula IV; and (iii) coupling the compound of formula IV with Y-AH in which Y and A are as defined in claim I or 2. I1. Compounds of the general formulae I or II, processes for their preparation, compositions containing them or methods or uses involving them, substantially as herein described with reference to the accompanying examples. N:WslboumeCases\Patent\7000-72999\P7516.AU.2SpeP72516AU.2 First Amendments.doc