AU2003291839A1 - Nicotinamide-based kinase inhibitors - Google Patents

Description

WO 2004/054977 PCT/AU2003/001666 I Nicotinamide-based Kinase Inhibitors FIELD OF THE INVENTION The present invention involves compounds represented by Formula (I) herein below, pharmaceutical compositions comprising such compounds and methods of suppressing 5 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 10 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 15 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 20 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 cytokinos 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, 25 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 PCT/AU2003/001666 2 over-expression or inappropriate activation of the enzyme; or by over- Or under production of cytokines or growth factors also participating in the transduction of signals upstream or downstream of the kinase. In all of these instances, selective inhibition of the action of the kinase might be expected to have a beneficial effect. Diseases where aberrant 5 kinase activity-has been implicated include: diabetes; restenosis; atherosclerosis; fibrosis of the liver and kidney; ocular diseases; myelo- and lymphoproliferative disorders; cancer such as prostate cancer, colon cancer, breast cancer, head and neck cancer, leukemia and lymphoma; and, auto-immune diseases such as Atopic Dermatitis, Asthma, rheumatoid arthritis, Crohn's disease, psoriasis, Crouzon syndrome, achondroplasia, and 10 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;YES and YRK); the BRK Family 15 (including: BRK; FRK, SAD; and SRM); the CSK family (including: CSK and CTK); the BTK family, (including BTK;1TK; T; MKK2 and TXK), the Janus kinase family, (including: JAKI, JAK2, JAK3 and Tyk2), the FAK family (including, FAK and PYK2); the Fes family (including FES and FER), the ZAP70 family (including ZAP70 and SYK); the ACK family (including ACKI and ACK2); and the Abl family (including ABL and ARG). The RTK 20 family includes the EGF-Receptor family (including, EGFR, HER2, HER3 and HER4); the Insulin Receptor family (including INS-R and IGFI-R); the PDGF-Receptor family (including PDGFRx, PDGFR, CSF1R, KIT, FLK2); the VEGF-Receptor family (including; FLT FLK1 and FLT4); the FGF-Receptor family (including FCFRL FGFR2, FGFR3 and FCFR4 ); the CCK4 family (induding CCK4); the MET family (including MET and RON); 25 the TRK family (including TRKA, TRKB, and TRKC ); the AXL family (including AXL, MER, and SKY); the TIE/TEK family (including TIE and TIE2/TEK); the EPH family (including EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPI-B2, EPH33, EPHB4, EPHB5, EPHB6); the RYK family (including RYK); the MCK family (including MCK and TYRO10); the ROS family (including ROS); the RET family 30 (including RET); the LTK family (including LTK and ALK); the ROR family (including RORI and ROR2); The Musk family (including Musk); the LMR family including LMRL, LMR2 and LMR3); and the SuRTK106 family (including SuRTKi06).

WO 2004/054977 PCT/AU2003/001666 s Similarly, the serino /threonine specific kinases (STKs) comprise a number of distinct sub families, including; the extracellular signal regulated kinases, (p42/ERK2 and p44/ERKJ); c-Jun NH2-terminal kinase (JNK); cAMP-responsive element-binding protein kinases (CREBK); cAMF-dependent kinase (CAFK); mitogen-activated protein kinase-activated 5 protein kinase (MAPK and its relatives); stress-activated protein kinase p38/SAPK2; mitogen-and stress-activated kinase (MSK); protein kinases, PA, 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. 10 In one embodiment the method of the invention is used in the treatment of sarcomas, carcinomas ana/ or leukemias. 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, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, 15 mesothelioma, Ewing's tumor, leiomyosarcorna, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous ccll 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, 20 choriocarcinoma, seminona, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangoblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. 25 In certain embodiments, the method of the invention is be 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 cel tumors, e.g., lipomas, fibrolipomas, lipoblastomas, lipomatosis, hibemomas, hemangiomas and/or liposarcomas. 30 WO 2004/054977 PCT/AU2003/001666 4 SUMMARY OF THE INVENTION 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 genera 5 formula W 0 Q A B N or pharmaceutically acceptable prodrugs, salts, hydrates, solvates, crystal forms or 10 diastereomers thereof, wherein: A is selected from 0, S, NRI, where R1 is selected from H, C, 4 alkyl; B is aryl hetaryl optionally substituted with 0-3 substituents independently chosen from halogen, C 14 alkyl, CF,, CN, aryl, hetaryl, OH, OCF 3 , OC 1 4 alkyl,

OC

2 ,alkylNR2R3, Oaryl, Ohetaryl, CO2R2, CONR2R3, NR2R3, C,. alkylNR2R3, 15 NR4C4alkyINR2R3, NR2COR3, OC(0)NR2R3, NR4CONR2R3, NR2S0 2 R3; and R2, R are each independently H, C 1 4 alkyl, C14 alkyl heterocyclyl, aryl, hetaryl,

C

1 4 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 0, 5, NR5; and R4 is selected from H, C14 alkyl; and R5 is selected from H, Cp- alkyl; 20 Q is a bond, or C alkyl; W is selected from H, Cbalkyl, C 2 6 alkenyl; where C 1 4 alkyl or C 2 6 alkenyl may be optionally substitv ted with C 1 4alkyl, OH, OCI-alkyl, N1RC(O)R7, CONR6R7, OR6, NR6R7; and R6, and 37 are each independently H, C,. alkyl, C 1 4 alkyl cycloalkyl,

C.

4 alkyl heterocyclyl, aryl, hetaryl, or may be joined to form an optionally WO 2004/054977 PCT/AU2003/001666 5 substituted 3-8 membered ring optionally containing an atom selected from 0, S, NRS and R8 is selected from H, C- 4 alIkyl; Y is H, aryl or hetaryl optionally substituted with 0-3 substituents independently chosen from halogen, C 1 , alkyl, CF3, aryl, hetaryl, OH, 0CF CN, C 2

.

4 alkynyl, 0C, 4 5 alkyl, QC2.salkylNR9RI0, Oaryl, Ohetaryl, CO2R9, CONR9R1O, NR9R1O, C 1 4 alkyNR9R1O, NR1C, 4 alkylNR9R10, NR9COR1O, NR11CONR9RIO, NR95O 2 RI0; and R9, R10 are each independently H, CI. alkyl, C 14 alkyl heterocyclyl, aryl, hetaryl, C 14 alkyl aryl, C 1 - alkyl hetaryl. or may be joined to form an optionally substituted 3-8 membered ring optionally containing an atom selected from 0, S, 10 NR12; and RI is selected from H, C 1 4 alkyl; and R12 is selected from H, C,., alkyl. 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 kinase-associated disease state in a subject, the method comprising administering a 15 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 20 W o Y' A B N or pharmaceutically acceptable prodrugs, salts, hydrates, solvates, crystal forms or diastereomers thereof, wherein: 25 A is selected from 0, S, NR1, where RI is selected from K, C, 4 alkyl; WO 2004/054977 PCT/AU2003/001666 B is aryl hetaryl optionally substituted with 0-3 substituents independently chosen from halogen, C 1 4 alkyl, CF, CN, aryl, hetary, OH, OCF, QC 1 -alkyl, 0C 2 .salkylNR2R3, Oaryl, Ohetaryl, CO2R2, CONE2R3, NR2R3, Ci4 alkylNR2RS, NR4C 1 .alkylNR2R3, NR2COR3, OC(O)NR2R3, NR4CONR2R3, NR250 2 R3; and R2, 5 R3 are each independently H, C 14 alkyl C1 alkyl heterocyclyl, aryl, hetaryl,

C

14 alkyl aryl, C,, alkyl hetaryl, or may be joined to form an optionally substituted 3-8 membered ring optionally containing an atom selected from 0, S, NR5; and R4 is selected from H, C.

4 alkyl; and R5 is selected from H, C, alkyl; Q is a bond, or C, alkyl; 10 W is selected from H; Calkyl, C 2 alkenyl; where C 1 4 alkyl or C 24 -alkenyl may be optionally substituted with C 1 .alkyl, OH, OC.

4 alkyl, NRIC(0)R7, CONR6R7, OR6, NR6R7; and R6, and R7 are each independently H, C 1 4 alkyl, C 14 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 0, S, 15 NR8 and R8 is selected from H, C,., alkyl; Y is H, aryl or hetaryl optionally substituted with 0-3 substituents independently chosen from halogen, C,., alkyl, CF3, aryl, hetaryl OH, OCF', CN, C 2 4 alkynyl, OC. alkyl OQC2.alkylNR9R1O, Caryl, Ohetaryl, C0 2 R9, CONR9R1O, NR9R10, C 1

-

4 alkylNR9R1O, NR11C.

4 alkylNR9R10, NR9COR10, NRI ICONR9R1O, NR9SO 2 R1O; 20 and R9, R10 are each independently H, C,, alkyl, C 1

.

4 aIkyl heterocyclyl, aryl, hetaryl, C,.

4 alkyl aryl, C 1 4 alkyl hetaryl, or may be joined to form an optionally substituted 3-8 membered ring optionally containing an atom selected from 0, S, NIR12; and R11 is selected from H, C 1 _ alkyl; and R12 is selected from H, C, 4 alkyl. In the above description it will be appreciated that: 25 C 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 0, N, S.

WO 2004/054977 PCT/AU2003/001666 7 Cycloalkyl means a 3-8 membered saturated ring Heterocyclyl means a 3-8 membered saturated ring containing 1-3 heteroatoms selected from 0, S, NR13, where R13 is -L C1 4 alkyl, aryl, hetaryl. Preferably, the compound is selected from the compounds of Table 1 and Table 2. 5 In a further preferred embodiment the compound is selected from compounds of the general formula IL W 0 Q N 1 B N II or pharmaceutically acceptable prodrugs, salts, hydrates, solvates, crystal forms or 10 diastereomers thereof, wherein: RI is selected from H, C, alkyl; B is aryl, hetaryl optionally substituted with 0-3 substituents independently chosen from halogen, C1 alkyl, CF_, aryl, hetaryl, OH, OCF, OC 1 4 alkyl, OCalky]NR2R3, Oaryl, Ohetaryl, CO 2 R2, CONR2R3, NR2R3, C,., alky1NR2R3, NR4C, 4 alkylNR2R3, 15 NR2COR3, NR4CONR2R3, NR2S 2 R3; and R2, F3 are each independently H, C 1 alkyl, C 1 , alkyl heterocydyl, aryl, hetaryl, C 1 4 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 C, S, NR5; and R4 is selected from H, C- 4 alkyl; and R5 is selected from H, C,., alkyl; 20 Q is a bond, or C,. alkyl; W is selected from H, C 1

.

4 alkyl, C26alkenyl; where C 1 4 alkyl or Cualkenyl may be optionally substituted with Calkyl, OH, OC1.

4 alkyl, NR6R7; and R6, and R7 are each independently H, C-, alkyl, C1.

4 alkyl cycloalkyl, C.

4 alkyl heterocyclyl, aryl, hetaryL or may be joined to form an optionally substituted 3-8 membered ring WO 2004/054977 PCT/AU2003/001666 S optionally containing an atom selected from 0,5, NR8 and R8 is selected from H,

C

1 4 alkyl; Y is H, aryl or hetaryl optionally substituted with 0-3 substituents independently chosen from halogen, C 1 4 alkyl, CF, aryl, hetary1, OH, OCF, OC 1 4 alkyl, 5 CQalkylNR9RiO, Oaryl, Ohtaryl, CO 2 R9, CONR9RIO, NR9R10, CIA alkylNR9R1O, NR11C 34 alkyINR9R1O, NR9COR10, NR11CONR9R1O, NR9SO 2 R1O; and R9, R10 are each independently H, C 1

,

1 alkyl, C 1 4 alkyl heterocyclyl, ary, hetaryl, C 1 4 alkyl aryl, C 14 alkyl hetaryl, or may be joined to form an optionally substituted 3-8 membered ring optionally containing an atom selected from 0, S, 10 NR12; and R11 is selected from H, C- 4 alkyl; and R12 is selected from H, C 1 4 alkyl. 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. 15 Hetaryl means an unsubstituted or optionally substituted 5- or 6-membered heteroaroma tic ring containing one or more heteroatoms selected from 0, N, S. Cycloalkyl means a 3-8 membered saturated ring Heterocyclyl means a 3-6 membered saturated ring containing 1-3 heteroatoms selected from 0, S, NR13, where R13 is H, C 1 4 alkyl, aryl, hetaryl. 20 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 25 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 WO 2004/054977 PCT/AU2003/001666 9 preventing disorders that can be treated or prevented by the inhibition of protein kinases comprising administering prodrugs of compounds of the formula L 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 5 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 . The amino acid residues include the 20 naturally occurring amino acids commonly designated by three letter symbols and also include, 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvaline, 10 beta-alanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine and methinine sulfone. Prodrugs also include compounds wherein carbonates, carbamates, aides 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 acids, salts of acids, or esters) 15 joined through a phosphorus-oxygen bond to a free hydroxyl of compounds of formula L 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 20 N-(acyloxyalkyl)- or N-(phosphonooxyalkyl)-pyridinium salt. This invention also encompasses pharmaceutical compositions containing prodrugs of compounds of the formula L 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. 25 In a third aspect the present invention provides a method of treating a tyrosine kinase-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 30 group consisting of Atopy, such as Allergic Astuna, Atopic Dermatitis (Eczema), and Allergic Rhinitis; Cell Mediated Hypersensitivity, such as Allergic Contact Derma titis and Hypersensitivity Pneumoni tis; Rheumatic Diseases, such as Systemic Lupus WO 2004/054977 PCT/AU2003/001666 10 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, S 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, leiornyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, 10 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, choriocarcinona, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung 15 carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, 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 20 as adipose cell tumors, e.g., lipomas, fibrolipomas, lipoblastomas, lipomatosis, hibemomas, hemangiornas 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. 25 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 30 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/AU2003/001666 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 (e.g., as sterile injectable aqueous or non 5 aqueous 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 10 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 15 not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine, canine, feline, rodent or marine species can be treated. However, the method can also be practiced in other species, such as avian species (e.g., 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 20 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 limi ted to, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine, canine, feline, rodent or murine species, 25 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 30 - 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/AU2003/001666 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 5 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. All methods include the step of bringing the active ingredient into association with the carrier, which constitutes one or 10 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 15 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 20 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, 25 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; 30 granulating and disintegrating agents, for example, com starch, or alginic add; 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/AU2003/001666 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. 5 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 10 the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodiurn alginate, polyvinyl-pyrrolidon, gurn 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 15 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 bexitol 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 20 aqueous suspensions may also contain one or more preservatives, for example ethyl, or n propyl, p-hydroxybenzoate, 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 25 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 30 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/AU2003/001666 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 tical compositions of the invention may also be in the form of oil-in-water 5 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, naturally occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty adds and hexitol anhydrides, for example sorbitan monooleate, and 10 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 elxdrs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a 15 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 20 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 25 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 30 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/AU2003/001666 15 por 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. 5 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, 10 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 15 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 20 therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects. Examples of other therapeutic agents include the following: cyclosporins (e.g., cyclosporin A), 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, 25 agents blocking the interaction between CD40 and gp39, such as antibodies specific for CD40 and/or gp3 9 (i.e., 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 30 (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 methotrexate, PK506 (tacrolimus, Prograf), mycophenolate mofetil, antineoplastic agents such as azathioprine, VP 6, etoposide, fludarabine, cispla tin, doxorubicin, adriamycin, amsacrine, camptothecin, cytarabine, gemcitabine, vinblastine, vincristine, fluorodeoxyuridine, melphalan and cyclophosphamide, TNF-a inhibitors such 5 as tenidap, anti-TNF antibodies or soluble TNF receptor, and rapamycin (sirolimus or Rapamune) 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. 10 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, multidrug resistance-associated protein, lung resistance protein and glutathione $-transferase isoenzymes alpha, mu, pi, sigma, 15 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 20 these systems include; verapamil, probenecid, dipyridamole, ethacrynic acid, indomethacin, sulfasalazine, buthionine sulfoximine, cyclosporin A and tamoxifen. 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 mul tiple doses. Preferably, 25 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 S to 50 mg/kg per day. For oral administration, the compositions are preferably provided in the form of tablets containing 1.0 to 1000 30 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, 500.0, 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/AU2003/001666

-

27 treated. The compounds may be administered on a regimen of 1 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 5 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 10 "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 15 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. 20 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 25 Compounds are generally prepared in a 2-step process starting from a protected 5 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 wi th 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 5 Stille, J.IC, Angew. Chem , Int Ed. RngL, 1986, 25, 508) (Scheme 1). 0 0 Br R1--M2k Pd catalyst base N N Scheme I The Suzuki coupling is the preferred coupling method and is typically performed in a solvent such as DME, THF, DMF, ethanol, propanol, toluene, or I4-dioxane in the presence 10 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 Pld(PPh,) 4 , Pd(OAc) 2 , [PdCl2(dpp)], Pd 2 (dba) 3 /P(t-Bu), palladium on carbon. Methods to protect 5-bromonicotinic acid are known to those skilled in the art and may 15 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 t-Butyl 5-broniorcotinate employed in the first step can be readily prepared from commercial 5-bromonicotinic acid using conventional methods well known to those skilled 20 in the art. These methods include the coupling of 5-bromonicotinic acid with t-butanol using coupling reagents such as dicyclohexylcarbodiimide or 1-( 3 -dimethylaminopropyl) 3-ethylcarbodiimide in a solvent such as dichloromethane, or treatment of 5 bromonicotinic acid with di-t-butyldicarbonate in the presence of a base such as triethylamine In a solvent such as tetrahydrofuran. 25 The second step of the synthesis is amide, ester or thioester fonmation by coupling the 5 arylnicotinic acid with a primary or secondary amine, an alcohol or phenol, or an alkyl or aryl mercaptan (Scheme 2).

WO 2004/054977 PCT/AU2003/001666 19 0 Ho N., Ar RI-XH FN ., Ar N X=0,S,NH,NR2 N Scheme 2 Initially the protected ester forms of the 5-arylicotinate derivatives prepared as in Scheme 1 arc cleaved to the corresponding acids using methods well known to those skilled in the 5 art. The 5-arylnicotinic acid derivatives are typically coupled with amines, alcohols, phenols, thiols or thiophenols using coupling reagents such as dicyclohexylcarbodiimnide, 1-(3 dimethylaminopropyl)-3-ethylearbodiimide, diisopropylkarbodiimide or carbonyldiirmidazole in solvents such as dichloromethane and tetrahydrofuran. 10 Alternatively, the deprotected 5-arylnicotinic 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, t-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 15 thiophenol in the presence of a base such as triethylamine, diisopropylethylanine or solid phase equivalent in a solvent such as dichIoromethane, tetrahydrofuran, dioxane or ethyl acetate at ambient or elevated temperatures, to generate the desired 5-aryl nicotinic add derivatives. As a further alternative, the deprotected 5-aryInicotinic acid derivatives prepared as in 20 Scheme I can be converted to the corresponding active ester intermediates, such as the succini midyl, pentafluorophenyl or p-nitrophenyl esters, This can be achieved by coupling the 5-arylnicotinic add derivatives with A-hydroxysuccinimide, pentafluorophenol or p nitrophenol using coupling reagents such as dicyclohexylcarbodiimide, 1-(3 dimethylaminopropyl)-3-ethylcarbodiimide, diisopropylcarbodiimide or 25 carbonyldiirnidazole in solvents such as dichloromethane and tetrahydrofuran. Active acyl intermediates can also be formed directly by reaction of the 5-arylnicolinic add derivatives with reagents such as diphenylphosphoryl azide, pentafluorophenyl acetate, WO 2004/054977 PCT/AU2003/001666 20 pentafluoropheny diphenylphosphinate or cyanuric chloride using methods well known to those skilled in the art. The products formed from this reaction sequence may be further derivatised using techniques well known to those skilled in the art. 5 Example 1 Tettayl5-romonicoinate To a mixture of 5-bromonicotinic acid (0.30g, 1.49mmol) and di- tert-butyldicarbonate (0.45g, 2.06nmmol) in THF (15mL) was added triethylamine (0.25mL, 1.79mmnol) followed by 4-(pyrrolidino)pyridine (30mg, 0.20mmol). The resultant solution was stirred at 10 ambient temperature for 48 hr. The volatiles were then removed under vacuum and the residue was purified by column chromatography on silica (gradient CH 2

C

2 to 3% MeOH/CH 2 C2) to provide the product as a white solid (0.35g, 91%) IH-n.m.r. (CDCI ) SL60 (s, 91-, t-butyl), 8.35 (m, iH, Ar), 8.79 (d, J25 Hz, 1H, Ar), 9.06 (d,J 1.4 Hz,, 1H, Ar). 15 Example 2 Tert-Butyl5-(3,4-methylenedioxypheny)nacodnate In a flask was placed tert-butyl 5-bromonicotinate (1.00g 3.87mmol), 10% w/w palladium on carbon (210mg, -0.2nmol Pd), potassium carbonate (110g, 7.96mmol), 3,4 methylenedioxy-phenyl boronic acid (0.

9 6g, 5.79nmmol), dimethylformanide (50mL) and 20 water (250L). The flask was purged with nitrogen and then heated, with stirring, to 90*C for 15hr. The reaction mixture was allowed to cool, diluted with water (350mL) and extracted with CH- 2

C

2 (4X). The combined extracts was washed with water, dried (MgSO 4 ) and concentrated in vacuo. The crude residue was purified by flash chromatography on silica (gradient CH 2 C1 2 to 50%ether/ CH 2 C1 2 ) to provide an off-white solid (1.

0 2g, 88%). 25 'I-n.m.r. (CDCla) 51.63 (s, 911, t-bu tyl), 6.04 (s, 211, CH2), 6.90 (m, 1H, Ar), 7.08 (m, 21, Ar), 8.34 (m, 1I, Ar), 8.89 (d, J1.9 H-z, 115, Ar), 9.09 (d, J2.0 Hz, 1I, Ar). Example 3 WO 2004/054977 PCT/AU2003/001666 21 5-(3,4-methylenedioyphenyl)nkcoainoyl choide A solution of kart-butyI 5-(3,4-methylenedioxyphenyl)nicotinate (0.80g, 2.67mmol) in trifluoroacetic acid (10mL) was stirred at roorn temperature for 2hr. The trifluoroacetic acid was removed in vacuo and the residue treated twice with toluene followed by 5 removal under vacuum (to remove residual TFA). The yellow/green residue was then treated with thionyl chloride (10mL) and dimethylformamide (iOL) and the mixture was heated to reflux for 15hir. 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 vacuo to leave a yellow solid. To the 10 crude add 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 (70mg, 15 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 (0-48mL, 80mol). The plate was sealed with a webseal mat and the plate was sonicated for 1hr in a sonicator bath. Amberlite IRA-67 (30mg, 0.17mmol) was added to each well, the plate was re-sealed and sonication continued for a further 30min. The contents of the 96-well plate was transferred 20 v/a pipettor to a 96-well filter plate and filtered into a second 96-well deep well plate. 1,4 Dioxane (0.40nL) was placed in each well of the original 96-well plate (to rinse). This was aspirated with the pipettor 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 25 CI-I 2 C1 2 (0.50mL). These CH 2

CI

2 solutions were transferred to a second filter plate loaded with silica (200mg/well) and filtered into a third 96-well deep well plate. A 10% methanol/CH 2 C1 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. 30 Further examples of 5-arylnicotinanides are shown in Table 1 along with their experimental m/z values.

WO 2004/054977 PCT/AU2003/001666 Table I. CHEMISTRY' M/Z (El) 0 T~o346.6 N H 0 1 H C20H19N302 HO ~ 284.4 017H2ON202 1100 N -~. N.846.6 H 0 "C OH37. Ci 8H20N204 WO 2004/054977 PCT/AU2003/001666 23 Table 1 (cont) CHEMISTRY m/z (EI) F l ~ 361.2 H 020H15FN2O3 404.2 0H C23H21NS04 07 K347.1 H 0211H18N203 309.9 C1H16N2OS 0 274.3 C14H17NSOS WO 2004/054977 PCT/AU2003/001666 24 Table 1 (conL) CHEMISTRY- mlz (EI) 0~ 0 "LIN 310,1 Cl 81-I N20S _____ N~ 331.2 C21 H21 N30 HH CISH13N302_______ _____ WO 2004/054977 PCT/AU2003/001666 25 Table 1 (cont.) CHEMISTRY m/z (E) 0 N N290.0 405.4 NN 024H25FN40 FN H H 302.0 H C17H19FN2O2 F NN IJ~ N'391.4 F . N N 0A . 023H23FN40 F FNF 325.2 C19H14F2N20 _____ WO 2004/054977 PCT/AU2003/001666 26 Table 1 (cont.) CHEMI$TRY m/z (El) HON 378.4 N C22H22N204 0 'N.334.2 HDH C20H1N203 ci, N. cz N 385.3 C20H16C12N202 0 N.. 366-0 C21 H20N204 _____ WO 2004/054977 PCT/AU2003/001666 27 SCREENING JAK Tyrosine Kinase Domain Production JAK kinase domains were produced in the following manner: JAK1 5 The kinase domain of human JAIC1 was amplified from U937mRNA using the polymerase chain reaction with the following primers: XHoI-p 5'-CCG CTC GAG ACT GAA GTG GAC CCC ACA CAT-3' J1-KPNI 5'-CGG GGT ACC TTA TT TAA AAG TGC TTC AAA-3' JAKT PCR products were cloned into the pFastBac Hib expression vector (Gibco) via the 10 Xho I and Kpn I sites, The JAK1 plasmid was then transformed into competent DHIOBac 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 15 chain reaction with the following primers: 5ALI-jk2 5'-ACG CGT CGA CGG TG CTT TGA AGA CCG GGA T-3' jk2-NOT 5'-ATA G'r TAG CGG CCG CTC AGA ATG AAG GTC ATT T-3' JAK2 PCR products were cloned into the pFastBac Hic expression vector (Gibco) via the Sal I and Not I sites. The JAK2 plasmid was then transformed into competent DH1OBac 20 cells (Gibco), and the recombinant baculovirus produced prepared for transfection into Sf9 insect cells. JAK3 The kinase domain of humanjAK3 was amplified fromU937mnRNA using the polymerase chain reaction with the following primers: WO 2004/054977 PCT/AU2003/001666 28 XHOI-Ja 5'-CCG CTC GAG TAT GCC TGC CAA GAC CCC ACC-3' J3-KPNI 5'-CGG OCT 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 DH1OBac 5 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: 10 T2EK 5'-GGA GCA CTC GAG ATG GTA GCA CAC AAC CAG GTG-3' ITY2.2R 5'-GGA GCA GGA ATT CCG GCG CTG CCC GTC AAA TCT GG-3' TYK2 PCR products were cloned into pBlueBacHis2A (Invitrogen) via the EcoRI site. The recombinant TYK2 baculovirus produced was prepared for transfected into Sf9 insect cells. Large Scale Production Of Kinase Domains 15 Baculovirus preparations from each of the JAK family members were infected into five Iitres 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-S.0. Cells were harvested and lysed. JAK kiriase domains were purified by affinity chromatography on a Probond (Invitrogen) nickel chelate affinity column. 20 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 50mM HEFES, pH 7.5, 10mM MgCI, 150mM NaCI and 10pM-1mM ATP. The biotinylated 25 substrate biotin-EGPWLEEEEEAYGWMF-NH2 (final concentration 5M) was used as substrate. In the ELISA assay tyrosine phosphorylation was quantitated following transfer to an avidin coated ELISA plate using peroxidase-linked anti-phospho-tyrosine anybody WO 2004/054977 PCT/AU2003/001666 29 PY20. 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 Alpa. Inhibitors were added to the assays fifteen minutes prior to the addition of ATP. Inhibitors were 5 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 sM (measured under standard conditions, see Methods), are designated as "+". Compounds not tested are 10 designated "NT"; while compounds that did not inhibit enzyme activity by 50% at 10 gM are designated "-".

WO 2004/054977 PCT/AU2003/001666 30 Table 2 CHEMISTRY .ioIQ JaWc obf f-~ fis hoic Zo~ -:IxI~. 0NT NT 02;3H24N2OB 00 H NT - - - + N, 01 71-12N205 __ H C17H19NO4

N

WO 2004/054977 PCT/AU2003/001666 31 Table 2 (conL) CHEMISTRY Jak2 Jak3 abi fes fns lick Za7o NN 0OH~-14C2N2O2 - 4 NT O18H14N2O2 NNT C21HF-20N204 0

HOH

WO 2004/054977 PCT/AU2003/001666 32 Table 2 (cont.) CHEMISTRY Jak2l Jak3l abi fog I c3 NN H NT T . C20HIBN202 NTN 020H418N202 HO KC~F - - NT C0215FN202 NQNT NY C20HI8N202 WO 2004/054977 PCT/AU2003/001666 33 Table 2 (sCont) CHEMISTRY Jak2 Jak3 abi les fins hok ZWDO - - NT NT - H -1 - - NT - + + 019H1 6N202 NN C2oH1 7IN202 H - - NT - NT C22H22N202 HH C22H22N202______ WO 2004/054977 PCT/AU2003/001666 34 TabIl 2 (c~ont.) CHEMISTRY _Aj k Jak3 _abi FOS fi hck Zap7O HO r~ 0 rIZ - - - NT + C2DH18N2 HO NN. 0 0 - - NT + 023H24N204 HOH C A1NO + H~j:: N- - - - -H WO 2004/054977 PCT/AU2003/001666 3-5 Table 2 (cont) CHEMISTRY jok2 JakS aM fes fs hck Zp0 NT C22H22N203 - - - - NT N 022H22N202 __ HO 0 - - + + + NT 022H22N202 S- - NT NT ++ 021 HIOFN202 N :i Z- + - + + NT H 021H1l9FN202 _______ WO 2004/054977 PCT/AU2003/001666 Table 2 (cont) CHEMISTRiY Jak1l Jak3 abi fe I _G _ ZmP70 Qk: l- - NT N C24H26N202 HOo NI I - - - -NT NT C22H22N204 Ho, 0 CM1H2ON202 00 ~- ~ ~- - NT NT HH C20H25CINP-05 WO 2004/054977 PCT/AU2003/001666 37 Table 2 (conIL) CHEMISTRiV ____ .Jac abl fes tmns ick ZE 70 00 1e, L I - - NT 00 H 021 H20CIN304 __ H - N - NT 021 H2OCIFN2O4 - - - -NNT i~aH ClSH20I1N204 - 0 NT NT 0H2CIN204

J_____

WO 2004/054977 PCT/AU20031001666 38 Table 2 (cent) CHEMISTRY Jaki2 Jpk3 obi lOS hIMS hct ZanTO)

-C

~-y'~k~r~ .x~- - T - NT C231-2301N204 NT + + N Ci N 023201IN204 WO 2004/054977 PCT/AU2003/001666 39 Table 2 (cont. CHEMISTRY Jak2 Jak3 obi fes fins hok Zap7O 0 Y1 Or - - - NT N 02BH-270IN204 r - - - - - NT CM323CIN206 101 0 + 022H21 CIN204 -~ I - - NT C-24H25CIN20:7 NNT 'N H 024H23N304 WO 2004/054977 PCT/AU2003/001666 40 Table 2 (cont.) CHEMitSTRY ___2 JpR ____ h < NT - 021ON203 <G~i ~ J1 *~, - - NT C2IHl 8N203 - -NT - NT C20H-14CM2203 H N T - C18H20N204 KNA- -NT NT C15H14N204 WO 2004/054977 PCT/AU2003/001666 41 Table 2 (cornt.) CHEMISTRY JaN2 JakS abi fee fins hzek p7 0 - - - - - NT C1OH14N203 - NT -NT 022H20N205 0- ~ 0N C2OH1 6FN203 NNT H 021H18N204 WO 2004/054977 PCT/AU2003/001666 42 Table 2 (cont.) CHEMISTRY ___ J~k3ab fes fI Is hok a7 0 0 jH C2HI 5N203 0I ; 1 F F -0- - N T -

-

C20HI5F'N203 ~-~- - - NT - - NT 02MHUN905 020H1 6N203 0 - NT - NT H C21H21N302S - - - WO 2004/054977 PCT/AU2003/001666 43 Table 2 (c~nnL) CHEMISTRIY _______ Ja aI es fms ho aD0 NN W-11 N20 O - NT - N C17i-I12CN20S 0 H NT HF -H- NT HH C16H12N20$S <NNT

H

WO 2004/054977 PCT/AU2003/001666 44 Table 2 (Cont.) CHEISTY ____ Jak2 Jak3 obi fes fins lick Za 7o 01 F NY~ 0I7HiSFN2OS C13Hi4N2OS Il ~NT - + 018H16N208______ - ____ H NT CU HI 6208 02OH19NS02S __ WO 2004/054977 PCT/AU2003/001666 45 Table 2 (cont.) CHEMISTRY Jak2 Jak3 abi fes frs hok Zaw7 H +- NT C18H16N208 H F _ - + + 017H13FN20S oF NT - - - - NT C17H18FN20S C18H1N2OS C2aH20N20S WO 2004/054977 PCT/AU2003/001666 46 Table 2 (cont.) CHEMSTRYJuI2 IJak$ abi -!a- fins I hk Z75 -L NT - N . NTN C18H14FN20S CISH15PN208S H 019H17N30 WO 2004/054977 PCT/AU2003/001666 Table 2 (Cont.) CHEMISTRY Jak2 JakS abi feS nS hiek Lam~ H NT - H' 017H13N30 N ,, 0 - - - NT C19H17N303 'NT 0 ~ m C18H15N$O WO 2004/054977 PCT/AU2003/001666 48 Table 2 (conQ. CHEMISTRY JA2Q Jok3 abI e fins lik Zqp7o - - NT - - NT H 02OHI 7FN20 Fr.~ -- - - NT H N C17H19FN202 __ C16H-IBFNSO __ WO 2004/054977 PCT/AU2003/001666 49 Table 2 (conQ. CHEMISTRY .1aR JAk3 abi fee 1mc hok ao INIT 018H13FN20 F 0 + - - - - NT 0 -~ - - - NT Cl SHI4FNf3O F '-0 - NT C20Hl FN20 WO 2004/054977 PCT/AU2003/001666 50 Table 2 (cont. _________ CHEMISTRY_____________ JaF3 abi lea fns lck zu 70 K - - - NT - NT 02SH23FN40 ~ N H C2IH19FN202 - - NT C20HI7FN20 FF NT - T C2oH17FN2O _____ WO 2004/054977 PCT/AU2003/001666 51 Table 2 (cont.) CHEMISTRY JaIQ JaI3 abi fez fins hoI & - - NT -NT 02OH17FN203 F 0 ;[ 0

-

-N C21H 7FN2O3 __ F0 0 - - -NTN NT 09-1N01 WO 2004/054977 PCT/AU2003/001666 52 Table 2 (cont.) OHEMISTRY Jak2 J0Pc3 abi fes tins hck-Zao C0? ~ 2 - - NT - - NT C1IHIN302 F C) - NT - - NT H N NN 2IH20CN2 Q C21 H20N202 WO 2004/054977 PCT/AU2003/001666 Table 2 (cont.) CHEMISTRY Jak2 Jak3 I t esa fmas lik ZHP70 I- - - NT - - - + NN Cl 9H16N202L 0 0 022H22N204 __ a+ 018H1614203 __ r021 H20N203 __ WO 2004/054977 PCT/AU2003/001666 194 Table 2 (cont.) CHEMISTRY Jak JokS obi fog iriF ho N 021 H20N202 C21 H20N202 022H2.2N203- - - - - r~ m - - - - - + + C2H2QN202

N.

0 A ~ ,- - -NT NT + H C20H17FN202 WO 2004/054977 PCT/AU2003/001666 Table 2 (cont.) CHEMISTRIY "0k Jak3 abi foo fins lick ZW F 0 ;, - --+ NT' C20H17FW'202

C

0

NT-

02SH24N202 NN T 021 H20N204 020HI 8N202___ - - NT - - 022H22N204 WO 2004/054977 PCT/AU2003/001666 56 Table 2 (cont.) CHEMISTRY .Jnc2_ Jak3 abi tes fins hCk a7 u HOH 00 HOH ~~+ + NT N F H C20H17FN203 HOH 0 N I1-'N - - T -T i 020H1 8N203 NI C19H16FN30 WO 2004/054977 PCT/AU2003/001666 57 Table 2 (cont) CHEMISTRY Jak2 Jak3 abi Tinsf hoft Za7 NT + 0Hi NT NT -+ Ci BH14FN30 N H INT NT - - - ClBH14PNSO to H NT NT-- - C18H14FN3O H ~NT NT - - - CISH14FN30 - -- -_ WO 2004/054977 PCT/AU2003/001666 59 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 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 5 restrictive.

Claims (2)

1. 3-8 membered ring optionally containing an atom selected from 0, S, NRS; and R4 15 is selected from 14, C., alkyl; and R5 is selected from H, C, alkyl; o is a bond, or Cj4 alkyl; W is selected from H, C 1 4 alkyl, C 2 alkenyl; where Cualkyl or C 2 4 alkenyl may be optionally substituted with C 14 alkyl, OH, OC 1 4 alkyl, NRC(O)R7, CONR6R7, OR6, NR6R7; and R6, and R7 are each independently H, CIA alkyl, C, alkyl cycloalkyl, 20 C 14 alkyl heterocyclyl, aryl, hetary, or may be joined to form an optionally substituted 3-8 membered ring optionally containing an atom selected from 0, S, NR8 and R8 is selected from H, C- alkyl; Y is H, aryl or hetaryl optionally substituted with 0-3 substituents independently chosen from halogen, CI4 alkyl, CF, aryl, hetaryl, OH, OCFO, CN, C2 alkynyl, 0C. 4 alkyl, 25 OC 4 alkyJNR9R1O, Oaryl, Ohetaryl, C0 2 R9, CONR9RI10, NR9R1O, C 14 alkylNR9R1O, WO 2004/054977 PCT/AU2003/001666 60 NR11C 1 .alkyINR9R1Q, NR9COR1O, NR1CONR9R10, NR9SOR10; and R9, RIO are each independently -1, C, 4 alkyl, C14 alkyl heterocyclyL aryl, hetaryl, Cl 4 alkyl aryL C 14 alkyl betaryl, or may be joined to form an optionally substituted 3-8 membered ring optionally containing an atom selected from 0, S, NR12; and R11 is selected from H, C 1 4 alkyI; and 5 R12 Is selected from H, C,, alkyl. 2. A compound according to claim 1 of the general formula II: W 0 Y N R1I. N II or pharmaceutically acceptable prodrugs, salts, hydrates, solvates, crystal forms or 10 diastereomers thereof, wherein: R1 is selected from H, C 4 aky; B is aryl, hetaryl optionally substituted with 0-3 substituents independently chosen from halogen, C- alkyl, CF, aryL, hetaryl, OH, OCFq, OC 1 4 alkyl, OC 2 .alkylNR2R3, Oaryl, Ohetaryl, CO 2 R2, CONR2R3, NR2R3, C 1 , alkylNR2R3, NR4C 1 4 alkylNR2R3, 15 NR2COR3, NR4CONR2R3, NR2SOR3; and R2, R3 are each independently H, C alkyl, C 14 alkyl heterocyclyl, aryl, hetaryL Calkyl aryl, C 1 4 alkyl hetaryl, or may be joined to form an optionally substituted 3-8 membered ring optionally containing an atom selected from 0, S, NR5; and R4 is selected from H, C 14 alkyl; and R5 is selected from H, C 14 alkyl; 20 Q is a bond, or C,- alkyl; W is selected from H, C 14 alkyl, qalkenyl; where Calkyl or C 2 . 6 alkenyl may be optionally substituted with C 1 4 alkyl, OH, OC. 4 alkyl, NR6R7; and R6, and R7 are each independently H, C.4 alkyl, C, 4 alkyl cycloalkyl, Ca 1 alkyl heterocyclyl, aryl, hetaryl, or may be joined to form an optionally substituted 3-8 membered ring 25 optionally containing an atom selected from 0, S, NRS and R8 is selected from H, C. 4 alkyl; WO 2004/054977 PCT/AU2003/001666 61 Y is H, aryl or hetaryl optionally substituted with 0-3 substituents independently chosen from halogen, C- alkyl, CF3, aryl, hetaryl, 01-, OCF, OCalkyl, Oq 5 aIkyNR9RIO, Oaryl, Ohetaryl, COzR9, CONR9R10, NR9R10, C. . alkyINR9R1O, NR11C 4 alkylNR9RI0, NR9COR10, NR11CONR9R1O, NR9S0 2 R10; 5 and R9, RIO are each independently H, C 1 . 4 alkyl, C1, alkyl heterocydyl, aryl, hetaryl, C 4 alkyI aryl, C,- alkyl hetaryl, or may be joined to form an optionally substituted 3-8 membered ring optionally containing an atom selected from 0, S, NR12; and R11 is selected from H, C,., alkyl; and R12 is selected from L C, 4 alkyl. 3. A compound according to claim 1 wherein the compound is selected from the 10 group consisting of: o 0 N N C22H22N204 C23H23CIN204 15 N 01 NN 020H18N202 C22H2CCIFN204 20 N Fal I H N rl-19HSFPJ2O2 C22H2001FN204 WO 2004/054977 PCT/AU2003/001666 62 H H C19H16N202 C22H21 CIN204 HO H C19H184N2O4 013H14N2OS 15 C N2 K- H IIV 022H24N204 Cl GH122O WO 2004/054977 PCT/AU2003/001666 63 HO F 0 021 H19FN202 C20H19N202 5 HO NN F H C21H19FN202 C18Hi6N2OS 10 HO-. 0 > C21H20N202 C17H1SFN20S 15 aF C17H13FN20$ 021 H20N202 200 1 H1 N 20 NN CISH1SN2Q$ 021 H2ON2Qz2 WO 2004/054977 PCT/AU20031001666 64 NN 020H20N20S C22H22N2Q3 50 H CIT1H14NPOS C2i H20N20)2 100 N Cl OHISFN2O)S 020H17FN202 15 10 H 0 N H CISHl7N3oS 020H17FN202 20 WO 2004/054977 PCT/AU2003/001666 65 C19H7NS2 C20H1ON202 5 NN' H C19H167N202 C21H20N2O3 10 N HONF
2. 022-H22N2O4 C2OH17FN203 N 0 15 H 0F HH 018H16N203 020H17FN203 20K HO N 21 0 2 N' o22HaW2O4 020-IS7N203 WO 2004/054977 PCT/AU2003/001666 66 4. A composition comprising a carrier and at least one compound of any one of claims I to 3. 5. A composition comprising a carrier and at least one compound of any one of claims I to 4. 5 6. A method of treating a tyrosine kinase-associated disease state in a subject, the method comprising administering a therapeutically effective amount of at least one compound of any one of claims 1 to 4 or a therapeutically effective amount of a composition of claim 5. 7. A method according to claim 6 wherein the disease state is selected from the group 10 consisting of Atopy, such as Allergic Asthma, Atopic Dermatitis (Eczema), and Allergic Rhinitis; Cell Mediated Hypersensitivity, such as Allergic Contact Dermatitis and Hypersensitivity Pneumonitis; Rheumatic Diseases, such as Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis, Juvenile Arthritis, Sjbgren's Syndrome, Scleroderma, Polymyositis, Ankylosing Spondylitis, Psoriatic 15 Arthritis; Other autoimmune diseases such as Type I diabetes, autoimmujne 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, 20 endothciosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, Ieiomyosarcoma, 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 25 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, 30 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 WO 2004/054977 PCT/AU2003/001666 67 disorders arising in adipose tissue, such as adipose cell tumors, e.g., lipomas, fibrolipomas, lipoblastomas, lipomatogis, hibemomas, hemanglomas and/or liposarconas. 8. The use of at least one of the compounds of any one of claims 1 to 4 in the 5 preparation of a medicament for the treatment of a tyrosine kinase-associated disease state.