AU2002226197B2 - Methods of inhibiting kinases - Google Patents

Description

WO 02/060492 PCT/AU02/00089 Methods ofInhibiting Kinases FIELD OF THE INVENTION The present invention relates generally to the field of non-peptidyl inhibitors of protein tyrosine kinases. More particularly, the present invention concerns methods of inhibiting specific protein tyrosine kinases, including members of the JAK family of protein tyrosine kinases.

BACKGROUND OF THE INVENTION Since the immune system is central to the protection of an individual from an external biological threat, diseases of the immune system are therefore a consequence of one or a combination of three problems with the immune system.

Underproduction or suppression of the immune system AIDS or

SIDS);

Overproduction of cells of the immune system (e.g Leukemia or Lymphoma); Overproduction of the effects of the immune system Inflammation); Inappropriate activation of the effects of the immune system (e.g.

allergy).

Treatments of diseases of the immune system are therefore aimed at either the augmentation of immune response or the suppression of inappropriate responses. Since cytokines play a pivotal role in the regulation of the immune system, they are appropriately considered to be key targets for therapeutic intervention in immune pathologies. Similarly, the intracellular signal transduction pathways that are regulated by cytokines are potential points of therapeutic intervention in diseases that involve overproduction of cytokine signalling.

There are many different types of protein kinases. Each type has the ability to add a phosphate group to an amino acid in a target protein. The phosphate is provided by hydrolyzing ATP to ADP. Typically, a protein kinase has an ATP-binding site and a catalytic domain that can bind to the target protein molecule. The JAK family of protein tyrosine kinases (PTKs) play a central role in the cytokine dependent regulation of the proliferation and end function of several important cell types of the immune system.

SUBSTITUTE SHEET (RULE 26) WO 02/060492 PCT/AU02/00089 2 play a central role in the cytokine dependent regulation of the proliferation and end function of several important cell types of the immune system.

A direct comparison of the four currently known mammalian JAK family members reveals the presence of seven highly conserved domains (Harpur et al, 1992). In seeking a nomenclature for the highly conserved domains characteristic of this family of PTKs, the classification used was guided by the approach of Pawson and co-workers (Sadovski et al, 1986) in their treatment of the .SRC homology (SH) domains. The domains have been enumerated accordingly with most C-terminal homology domain designated JAK Homology domain 1 (JH1). The next domain N-terminal to JH1 is the kinase-related domain, designated here as the JH2 domain. Each domain is then enumerated up to the JH7 located at the N-terminus. The high degree of conservation of these JAK homology (JH) domains suggests that they are each likely to play an important role in the cellular processes in which these proteins operate. However, the boundaries of the JAK homology domains are arbitrary, and may or may not define functional domains. Nonetheless, their delineation is a useful device to aid the consideration of the overall structural similarity of this class of proteins.

The feature most characteristic of the JAK family of PTKs is the possession of two kinase-related domains (JH1 and JH2) (Wilks et al, 1991).

The putative PTK domain of JAK1 (JH1) contains highly conserved motifs typical of PTK domains, including the presence of a tyrosine residue at position 1022 located 11 residues C-terminal to sub-domain VII that is considered diagnostic of membership of the tyrosine-specific class of protein kinases. Alignment of the human JAK1 PTK domain (255 amino acids), with other members of the PTK class of proteins revealed homology with other functional PTKs (for example, 28% identity with c-fes (Wilks and Kurban, 1988) and 37% homology to TRK (Kozma et al, 1988). The JH1 domains of each of the JAK family members possess a interesting idiosyncrasy within the highly conserved sub-domain VIII motif (residues 1015 to 1027 in JAK2) that is believed to lie close to the active site, and define substrate specificity. The phenylalanine and tyrosine residues flanking the conserved tryptophan in this motif are unique to the JAK family of PTKs. Aside from this element, the JH1 domains of each of the members of the JAK family are typical PTK domains.

WO 02/060492 PCT/AU02/00089 3 The central role played by the JAK family of protein tyrosine kinases in the cytokine dependent regulation of the proliferation and end function of several important cell types means that agents which inhibit JAK are useful in the prevention and chemotherapy of disease states dependent on these enzymes. Potent and specific inhibitors of each of the currently known four JAK family members will provide a means of inhibiting the action of those cytokines that drive immune pathologies, such as asthma IL-13; JAK1, JAK2), and leukemia/lymphoma IL-2: JAK1 and JAK3).

Furthermore, certain types of cancer such as prostate cancer develop autocrine production of certain cytokines as a selectable mechanism of developing growth and/or metastatic potential. An example of this is cancer of the prostate, where IL-6 is produced by and stimulates the growth of prostate cancer cell lines such as TSU and TC3 (Spiotto MT, and Chung TD, 2000). Interestingly, levels of IL-6 are elevated in sera of patients with metastatic prostate cancer, A great deal of literature covers the area of cytokine signalling. The present inventors have focussed on the JAK/STAT pathway that is involved in the direct connection of cytokine receptor to target genes (such as cell cycle regulators p21) and anti-apoptosis genes (such as Bcl-XL)).

The JAK/STAT Pathway The delineation of a particularly elegant signal transduction pathway downstream of the non-protein tyrosine kinase cytokine receptors has recently been achieved. In this pathway the key components are: A cytokine receptor chain (or chains) such as the Interleukin-4 receptor or the Interferon y receptor; (ii) a member (or members) of the JAK family of PTKs; (iii) a member(s) of the STAT family of transcription factors, and (iv) a sequence specific DNA element to which the activated STAT will bind.

A review of the JAK/STAT literature offers strong support to the notion that this pathway is important for the recruitment and marshalling of the host immune response to environmental insults, such as viral and bacterial infection. This is well exemplified in Table 1 and Table 2. Information accumulated from gene knock-out experiments have underlined the importance of members of the JAK family to the intracellular signalling triggered by a number of important immune regulatory cytokines. The therapeutic possibilities stemming from inhibiting (or enhancing) the WO 02/060492 PCT/AU02/00089 4 JAK/STAT pathway are thus largely in the sphere of immune modulation, and as such are likely to be promising drugs for the treatment of a range of pathologies in this area. In addition to the diseases listed in Tables 1 and 2, inhibitors of JAKs could be used as immunosuppresive agents for organ transplants and autoimmune diseases such as lupus, multiple sclerosis, rheumatoid arthritis, Type I diabetes, autoimmune thyroid disorders, Alzheimer's disease and other autoimmune diseases. Additionally, treatment of cancers such as prostate cancer by JAK inhibitors is indicated.

Table 1 Disease Type Cell Types Involved Characteristics Atopy Allergic Asthma (Mast Cells T-cell activation of B-cells Atopic Dermatitis (Eczema) (Eosinophils followed by IgE mediated activation of resident Mast Allergic Rhinitis (T-Cells actan of esiden t as Scells and Eosinophils (B-Cells Cell Mediated Hypersensitivity (T-cells Allergic Contact Dermatitis (B-cells T-cell hypersensitivity Hypersensitivity Pneumonitis Rheumatic Diseases Systemic Lupus Erythematosus (SLE) Rheumatoid Arthritis (Monocytes Cytokine Production Juvenile Arthritis (Macrophages (e.g.TNF, IL-1, CSF-1, GM-

CSF)

Sjbgren's Syndrome (Neutrophils S T-cell Activation Scleroderma (Mast Cells JAK/STAT activation Polymyositis (Eosinophils J STAT activation Ankylosing Spondylitis (T-Cells Psoriatic Arthritis (B-Cells Viral Diseases Epstein Barr Virus (EBV) Lymphocytes JAK/STAT Activation Hepatitis B Hepatocytes JAK/STAT Activation Hepatitis C Hepatocytes JAK/STAT Inhibition HIV Lymphocytes JAK/STAT Activation HTLV 1 Lymphocytes JAK/STAT Activation Varicella-Zoster Virus (VZV) Fibroblasts JAK/STAT Inhibition Human Papilloma Virus (HPV) Epithelial cells JAK/STAT Inhibition Cancer Leukemia Lymphoma Leucocytes Lymphocytes (Cytokine production (JAK/STAT Activation WO 02/060492 WO 02/60492PCT/AU02/00089 Target Disease Cytoine JAK family Strength of member Association Asthma IL-4 IL-9 JAKI &JAK3 IL-13 JAIK1 &JAK2...

_IL-5 JAK2 Eczema IL-4 JAK1 JAK3...

IFN-L JAK1 &JAK2...

Food Allergy IL-4 JAK1 JAK3 Inflammatory Bowel IL-4 JAK1 JAK3...

Disease Crobn's Disease Leukaemia And (IL-2) JAK3, JAK1 Lymphoma JAK2 Cutaneous GM-CSF IL- JAIKi JAK2 Inflammation 6 Immune Suppression IL-10 JAK1 TYK2 By Solid Tumour Prostate Cancer IL-6 JAK1, JAK2 Table 2: Diseases Potentially Treatable ByJAK-Based Drug Therapies SUMMARY OF THE E\TVENTION The present inventors have found that a group of compounds based either upon a 2-amino-6-carba-disubstituted pyrazine scaffold or a 2-amino-6carba-disubstituted pyridine scaffold are JAK inhibitors.

Accordingly, in a first aspect the present invention consists in a method of inhibiting JAK in a cell, the method comprising administering to the cell an effective amount of a composition comprising a carrier and a compound of the general formula I: R1N N R2 or pharmaceutically acceptable salts, hydrates, solvates, crystal forms or diastereomers thereof, wherein X is either carbon or nitrogen R1 is C-o 1 0 Alkyl, C2- 1 0 Alkenyl, C2- 1 0 Alkynyl, C2- 10 Alkylaryl, Aryl, or Heterocyclyl, or R1 with N may form a substituted or unsubstituted heterocyclyl, wherein the Alkyl, Alkenyl, Alkynyl, Alkylaryl, Aryl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 membered ring and in which the hetero atom is O, N or S; R2 is selected from Cl-lo Alkyl, C2- 1 0 Alkenyl, C2- 1 0 Alkynyl, C2-10 Alkylaryl, Aryl, Halo, OH, or 6-7 membered Heterocyclyl, wherein the Alkyl, Alkenyl, Alkynyl, Alkylaryl, Aryl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 membered ring and in which the hetero atom is O, N or S; with the proviso that when X is CH, R2 is not C 1 i 6 alkyl or piperazinyl.

In a second aspect the present invention consists in a method of inhibiting JAK in a cell, the method comprising administering to the cell an effective amount of a composition comprising a carrier and a compound of the general formula II: 200527036 1 HN/R6

HN

R7 N or pharmaceutically acceptable salts, hydrates, solvates, crystal forms or diastereomers thereof, wherein R6 is CIlo Alkyl, C2- 10 Alkenyl, C2- 1 0 Alkynyl, C2- 1 0 Alkylaryl, Aryl, or Heterocyclyl, or RI with N may form a substituted or unsubstituted heterocyclyl, wherein the Alkyl, Alkenyl, Alkynyl, Alkylaryl, Aryl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 membered ring and in which the hetero atom is O, N or S; R7 is Cl-o 1 Alkyl, C2- 10 Alkenyl, C2- 10 Alkynyl, C2- 10 Alkylaryl, Aryl, OH, or Heterocyclyl, wherein the Alkyl, Alkenyl, Alkynyl, Alkylaryl, Aryl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 membered ring and in which the hetero atom is O, N or S.

The present invention also includes the use of compounds of formula I or II is the prophylaxis and/or treatment of JAK-associated disease states.

DETAILED DESCRIPTION OF THE INVENTION In a first aspect the present invention consists in a method of inhibiting JAK in a cell, the method comprising administering to the cell an effective amount of a composition comprising a carrier and a compound of the general formula I: 200527036 1 N R2 R1

X

or pharmaceutically acceptable salts, hydrates, solvates, crystal forms or diastereomers thereof, wherein X is either carbon or nitrogen RI is C-o 1 0 Alkyl, C2- 10 Alkenyl, C 2 -1o Alkynyl, C2- 1 0 Alkylaryl, Aryl, or Heterocyclyl, or RI with N may form a substituted or unsubstituted heterocyclyl, wherein the Alkyl, Alkenyl, Alkynyl, Alkylaryl, Aryl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 membered ring and in which the hetero atom is O, N or S; R2 is selected from Ci-lo Alkyl, C 2 -1 0 Alkenyl, C2- 1 0 Alkynyl, C 2 1 0 Alkylaryl, Aryl, Halo, OH, or 6-7 membered Heterocyclyl, wherein the Alkyl, Alkenyl, Alkynyl, Alkylaryl, Aryl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 membered ring and in which the hetero atom is O, N or S; with the proviso that when X is CH, R2 is not C-6alkyl or piperazinyl.

In a preferred embodiment of the present invention X is N.

In yet a further preferred embodiment of the present invention the compound is of the general formula: z.x Y N N R3 200527036 1 WO 02/060492 PCT/AU02/00089 9 wherein one of X,Y and Z is nitrogen and the other two are carbon, or all three are carbon; R3, R4 and R5 are the same or different and are H, halo, OH, hydroxyamide, amino, hydroxyalkyl, aminoalkylamide, alkylamide, arylamide or alkoxy.

In a more preferred embodiment when R1 with N forms a heterocycle it is preferred that the heterocycle includes two heteroatoms, preferably two nitrogen atoms.

In a more preferred embodiment of the present invention the compound is of the general formula: x H R3 R1 R5 R4

N

wherein X is nitrogen or carbon; R1 is C2., Alkylphenyl, Phenyl, or Heterocyclyl, wherein the Alkyl, Phenyl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 member ring and in which the hetero atom is O, N or S; R3, R4 and R5 are the same or different and are H, halo, OH, hydroxyamide, amino, hydroxyalkyl, aminoalkylamide, alkylamide, arylamide or alkoxy.

In a still further preferred embodiment the compound is selected from the compounds set out in Table 4.

In a second aspect the present invention consists in a method of inhibiting JAK in a cell, the method comprising administering to the cell an effective amount of a composition comprising a carrier and a compound of the general formula II: H R6

HN/

R7) N or pharmaceutically acceptable salts, hydrates, solvates, crystal forms or diastereomers thereof, wherein R6 is Cl-lo Alkyl, C2- 1 o Alkenyl, C2- 1 0 Alkynyl, C2- 10 Alkylaryl, Aryl, or Heterocyclyl, or RI with N may form a substituted or unsubstituted heterocyclyl, wherein the Alkyl, Alkenyl, Alkynyl, Alkylaryl, Aryl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 member ring and in which the hetero atom is O, N or S; R7 is C.

0 lo Alkyl, C2- 1 0 Alkenyl, C2- 1 0 Alkynyl, C2- 10 Alkylaryl, Aryl, OH, or Heterocyclyl, wherein the Alkyl, Alkenyl, Alkynyl, Alkylaryl, Aryl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 member ring and in which the hetero atom is O, N or S.

In a preferred embodiment of the present invention the compound is of the general formula: 200527036_1 WO 02/060492 PCT/AU02/00089 11 ,R6

HN

R8 R9 wherein one of X,Y or Z is nitrogen and the other two are carbon, or all three are carbon R8, R9 and R10 are the same or different and are H, halo, OH, hydroxyamide, amino, hydroxyalkyl, aminoalkylamide, alkylamide, arylamide or alkoxy.

In a more preferred embodiment when R1 with N forms a heterocycle it is preferred that the heterocycle includes two heteroatoms, preferably two nitrogen atoms.

In a more preferred embodiment of the present invention the compound is of the general formula: H R6

HN

R8 R9 in which: R6 is C 2 1 0 Alkylphenyl, Phenyl, or Heterocyclyl, wherein the Alkyl, Phenyl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 membered ring and in which the hetero atom is O, N or

S;

WO 02/060492 PCT/AU02/00089 12 R3, R4 and R5 are the same or different and are H, halo, OH, hydroxyamide, amino, hydroxyalkyl, aminoalkylamide, alkylamide, arylamide or alkoxy.

In a still further preferred embodiment the compound is selected from the compounds set out in Tables 6 and 7.

In a further preferred embodiment the method is conducted in vivo. It is also preferred that the JAK is JAK1, JAK2, JAK3 or TYK2.

In a third aspect the present invention consists in a method of treating an individual suffering from a JAK-associated disease state, the method comprising administering to the individual a composition comprising a pharmaceutically acceptable carrier and a compound of the general formula: RN N R2 or pharmaceutically acceptable salts, hydrates, solvates, crystal forms or diastereomers thereof, wherein X is either carbon or nitrogen R1 is CI.

1 o Alkyl, Co 10 Alkenyl, C2- 1 Alkynyl, C2- 1 o Alkylaryl, Aryl, or Heterocyclyl, or R1 with N may form a substituted or unsubstituted heterocyclyl, wherein the Alkyl, Alkenyl, Alkynyl, Alkylaryl, Aryl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 membered ring and in which the hetero atom is O, N or S; R2 is selected from C.

10 Alkyl, C 2 1 Alkenyl, C2 1 -o Alkynyl, C 21 Alkylaryl, Aryl, Halo, OH, or 6-7 membered Heterocyclyl, wherein the Alkyl, Alkenyl, Alkynyl, Alkylaryl, Aryl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, WO 02/060492 PCT/AU02/00089 13 hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 member ring and in which the hetero atom is O, N or S.

In a preferred embodiment of the present invention the compound is of the general formula: z^Y H R3 R ,N N R5 R4 RIN .R5 R4

N

wherein one of X,Y and Z is nitrogen and the other two are carbon, or all three are carbon; R3, R4 and R5 are the same or different and are H, halo, OH, hydroxyamide, amino, hydroxyalkyl, aminoalkylamide, alkylamide, arylamide or alkoxy.

In a more preferred embodiment when R1 with N forms a heterocycle it is preferred that the heterocycle includes two heteroatoms, preferably two nitrogen atoms.

In a more preferred embodiment of the present invention the compound is of the general formula:

X

H R3 RI I R5 R4

N

wherein X is nitrogen or carbon; WO 02/060492 PCT/AU02/00089 14 R1 is C 2 Alkylphenyl, Phenyl, or Heterocyclyl, wherein the Alkyl, Phenyl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of chloro, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 membered ring and in which the hetero atom is O, N or

S;

R3, R4 and R5 are the same or different and are H, halo, OH, hydroxyamide, amino, hydroxyalkyl, aminoalkylamide, alkylamide, arylamide or alkoxy.

In a still further preferred embodiment the compound is selected from the compounds set out in Table 4.

In a fourth aspect the present invention consists in a method of treating an individual suffering from a JAK-associated disease state, the method comprising administering to the individual a composition comprising a pharmaceutically acceptable carrier and a compound of the general formula: ,R6

HN

N- N R7)N or pharmaceutically acceptable salts, hydrates, solvates, crystal forms or diastereomers thereof, wherein R6 is CI.

1 o Alkyl, C2-10 Alkenyl, C2.o Alkynyl, C2.

1 Alkylaryl, Aryl, or Heterocyclyl, or R1 with N may form a substituted or unsubstituted heterocyclyl, wherein the Alkyl, Alkenyl, Alkynyl, Alkylaryl, Aryl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy [in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 member ring and in which the hetero atom is O, N or S; WO 02/060492 PCT/AU02/00089 R7 is Alkyl, C 2 10 Alkenyl, C,.o Alkynyl, C, 2 Alkylaryl, Aryl, Halo, OH, or Heterocyclyl, wherein the Alkyl, Alkenyl, Alkynyl, Alkylaryl, Aryl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 membered ring and in which the hetero atom is O, N or S.

In a preferred embodiment of the present invention the compound is of the general formula: H R6

HN

Sz N/

R

10 R8 R9 wherein one of X,Y or Z is nitrogen and the other two are carbon, or all three are carbon R8, R9 and R10 are the same or different and are H, halo, OH, hydroxyamide, amino, hydroxyalkyl, aminoalkylamide, alkylamide, arylamide or alkoxy.

In a more preferred embodiment when R1 with N forms a heterocycle it is preferred that the heterocycle includes two heteroatoms, preferably two nitrogen atoms.

In a more preferred embodiment of the present invention the compound is of the general formula: WO 02/060492 PCT/AU02/00089 16 ,R6

HN

R8 R9 in which: R6 is Co,, Alkylphenyl, Phenyl, or Heterocyclyl, wherein the Alkyl, Phenyl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 membered ring and in which the hetero atom is O, N or

S;

R3, R4 and R5 are the same or different and are H, halo, OH, hydroxyamide, amino, hydroxyalkyl, aminoalkylamide, alkylamide, arylamide or alkoxy.

In a still further preferred embodiment the compound is selected from the compounds set out in Tables 6 and 7.

In a further preferred embodiment the disease state involves JAK1, JAK2, JAK3 or TYK2.

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 Dermatitis and Hypersensitivity Pneumonitis; Rheumatic Diseases, such as Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis, Juvenile Arthritis, Sj6gren'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 WO 02/060492 PCT/AU02/00089 17 Papilloma Virus (HPV), Cancer, such as Leukemia, Lymphoma and Prostate Cancer.

In further aspects the present invention provides the use of the compounds described in the preparation of medicaments for the treatment of JAK-associated disease states.

As used herein the term "JAK", "JAK kinase" or "JAK family" refers to protein tyrosine kinases which possess the characterizing features of JAK1, JAK2, JAK3 and TYK as described herein.

As used herein the term "JAK-associated disease state" refers to those disorders which result from aberrant JAK 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 I capable of treating a JAK-associated disorder in an amount effective therefor, 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, flavors, etc.) according to techniques such as those well known in the art of pharmaceutical formulation.

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 non-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 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. The compounds may also be administered liposomally.

WO 02/060492 PCT/AU02/00089 18 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 JAK 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 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. All 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 WO 02/060492 PCT/AU02/00089 19 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 acid; 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 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.

WO 02/060492 PCT/AU02/00089 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 n-propyl, phydroxybenzoate, 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 acid.

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 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 pharmaceutical 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- WO 02/060492 PCT/AU02/00089 21 occurring gums, for example gum acacia or gum tragacanth, naturallyoccurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids 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 may be 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 nonirritating 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.

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 pharmaceutical composition and method of the present invention may further comprise other therapeutically active compounds as noted WO 02/060492 PCT/AU02/00089 22 herein which are usually applied in the treatment of the above mentioned pathological conditions.

Examples of other therapeutic agents include the following: cyclosporins 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 and gp39, such as antibodies specific for CD40 and/or gp39 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 and cyclooxygenase inhibitors such as rofecoxib, steroids such as prednisone or dexamethasone, gold compounds, antiproliferative agents such as methotrexate, FK506 (tacrolimus, Prograf), mycophenolate mofetil, cytotoxic drugs such as azathioprine and cyclophosphamide, TNF-ao inhibitors such 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.

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, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient for the symptomatic adjustment of the WO 02/060492 PCT/AU02/00089 23 dosage to the patient to be 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 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.

WO 02/060492 PCT/AU02/00089 24 MATERIALS AND METHODS: Compound Synthesis All compounds may be prepared in a 2-step process starting from a dihalogenated heterocycle. The dihalogenated heterocyclic starting materials 2,6-dichloropyrazine and 2,6-dibromopyridine are obtained commercially.

6,8-Dibromo-imidazo-[1,2-a]-pyrazine can be prepared following the literature route (see for example, Sablayrolles, C. et al, J. Med. Chem., 1984, 27, 206).

The first step is a nucleophilic aromatic substitution to generate a monoamino-monohalo intermediate. (Scheme 1).

CI N Cl R1-NH, R N Cl RIN K base

H

Br N Br Br1 N

I

W R1-NH, R1 N N base Scheme 1 The nucleophilic aromatic substitution is typically carried out by addition of a primary amine to the di-halogenated heterocycle in a solvent such as ethanol, isopropanol, tert-butanol, dioxane, THF, DMF, toluene or xylene. The reaction is typically performed at elevated temperature in the presence of excess amine or a non-nucleophilic base such as triethylamine or diisopropylethylamine, or an inorganic base such as potassium carbonate or sodium carbonate.

The second step of the synthesis typically involves a palladium mediated cross-coupling of the monoamino-monohalo intermediate with a WO 02/060492 PCT/AU02/00089 suitably functionalised coupling partner. Typical coupling partners are boronic acids (Suzuki coupling: see for example Miyaura, N. and Suzuki, Chem Rev. 1995, 95 2457] or stannanes (Stille coupling: see for example Stille, Angew. Chem., Int. Ed. Engl., 1986, 25, 508) (Scheme 2).

H H N Cl R2-M Ny N R2 RIR1 R2-R-M Pd catalyst N N Pd catalyst

N

_N base Scheme 2 The Suzuki coupling is the preferred coupling method and is typically performed in a solvent such as DME, THF, DMF, ethanol, toluene, or 1,4dioxane in the presence of a base such as potassium 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)J, Pd(OAc) [PdCl,(dppf)], Pd,(dba),/Pt-Bu),.

Representative syntheses are reported below.

WO 02/060492 WO 02/60492PCT/AU02/00089 26 Example I CI- N CI N _N CI N~r

NY

A solution of R-c-methylbenzylamnine (3.64g, 30.Ommol) and 2,6dichioropyrazine (1.50g, 1O.Ommol) in dioxane (5 m1L) was heated at reflux under N 2 for 48 hours. The solvent was removed and the product crystallised from toluene-hexane to give 2-(R-ca-methylbenzylamino) -6-chioro-pyrazine.

(GDCl 3 81.59 3H, J 6.9Hz, GCHJ, 4.88 1H-, J=6.6Hz, CHI), 5.13 (br s, 1H, NIT), 7.27-7,36 (in, 5H, ArHl), 7.64 1Hl, pyraz-H), 7.79 1H, pyraz-H).

mlz (El) 235 233 WO 02/060492 WO 02/60492PCT/AU02/00089 27 Example 2

N

N, N CIN N

OYN

N~r

N

To a solution of 2-(S-cc-methylbenzylamino)-6-chloro-pyrazine (120mg, 0.51 mmol) (prepared via an analogous procedure to that outlined in Example 1), -tetramethyl-i, 3,2-dioxaborolan-2-yl) -pyridine (144mg, 0,61 mmcl) and Pd(PPh,) 4 (64mg, 0.05 mmol) in toluene (3mL) was added an aqueous solution of NaCO, (0.3 lmL, 2M). The resulting mixture was heated at reflux under N 2 for 16 hours. Upon cooling the mixture was diluted with water and the product extracted with EtOAc (2 x 15m-L). The combined organic layers were dried (NaSO,) and the solvent removed under reduced pressure to furnish the crude product. Column chromatography using EtOAc-hexane 1) as eluant furnished the purified product as a colorless oil (146mg, (CDCl 3 51.63 3H, J=6.6Hz, 3.90 3H, OCH,), 5.04 (in, 1H, CH), 5.14 (mn, 1H, NH), 7.23-7.71 (mn, 6H, Ar-H), 7.82 (mn, 2H, pyraz-H), 8.28 1H, Ar-H1), 8.3 3 1H, Ar-H), 8. 72 1H1, Ar-H), m/z (ES) 307 WO 02/060492 WO 02/60492PCT/AU02/00089 28 Example 3 HHN 0 N C1 N N NT

N

A mixture of 2-(S-xc-methylbenzylamino)-6-chloro-pyrazine (100mg, 0.43 mmol), 2-methoxypyridyl-5-boronic acid (78mg, 0.52 mmol), and Pd(PPhj 4 (53mg, 0.05 mmol) in toluene (3mL) and aqueous NaCO, (0.26mL, 2M) was treated as for Example 2 to furnish the product as a colorless oil (12 3mg, (CDGl 3 51.62 3H, I 6.3Hz, CH 3 3.99 3H, OGH 3 5.01-5.06 (in, 2H, CH and NIH), 6.81 lIH, J 8.7 Hz, Ar-H], 7.23-7.42 (in, 5H, Ar-H), 7.72 111, pyraz-H), 8.09 (dd, 11H, J= 8.7, 2.4 Hz, Ar-H), 8.20 1H, pyraz- 8.73 1H, J 2.4 Hz, Ar-H).

m/lz (ES) 307 (M-iH).

WO 02/060492 WO 02/60492PCT/AU02/00089 29 Example 4

H

N NC I N N 0 N

N

A mixture of 2-(R-o-methylbenzylamino) -6-chioro-pyrazine (66mg, 0.28 mmol), N-1j4-(4,4,5 ,5 -tetramethyl-1, 3, 2-dioxaborolan-2-yl)] acetamide (88mg, 0.34 mmol) and Pd(PPh,), (16mg, 0.02 mmol) in toluene (3m1i) and aqueous NaCO, (0.I5iL, 2MJ was treated as for Example 2 to furnish the product as a colorless oil (123mg, 949,).

(CDGl 3 51.65 3H, J 6.9 Hz, Gil 3 2.21 3H1, NIICH 3 4.99 (in, 111T, CHi), 6.39 (hr s, 1H1, 7.24-7.43 (in, 5H, Ar-H), 7.65-7.78 (mn, 4H1, Ar-H), 7.87 (AA'XX', 2H, Ar-H).

m/z (ES) 333 WO 02/060492 WO 02/60492PCT/AU02/00089 Example 0 0 "N N CI N N K 0 N:

N

A mixture of 2-(R-c-methylbenzylamino)-6-chloro-pyrazine (500mg, 2.2 mmol), 3,4,5-trimethoxybeuzeneboronic acid (547mg, 2.6 mmol) and Pd(PPh,), (124 mg, 0.11 nnolj in toluene (10 ml) and aqueous Na 2

CGO,

(1.3mL, 2M) was treated as for Example 2 to furnish the product as a colorless oil (5 74mg, The product was obtained as pale yellow crystals by recrystallisation from methanol 132-1330).

(GDGl 3 51.61 3H, J= 6.2 Hz, 3.88 3H, 0C11 3 3.90 (s, 611, OCHT,), 5.02 (in, 111, CH), 5. 09 111, I 5.9 Iz, NE., 7. 10 2H1, Ar-H), 7.24-7.42 (in, 5H1, Ar-H), 7.74 111, Ar-H), 8.22 111, Ar-H).

m/z (El) 366 WO 02/060492 WO 02/60492PCT/AU02/00089 31 Example 6 CI N CI H NN ,N -CI N:

N

A solution of ortho-toluidine (320mg, 3.0 mruol), 2,6-dichioropyrazine (150mg, 1.0 mmol), bis (tributylphosphine) palladium (26mg, O.O5mmol) and sodium tert-butoxide (144mg, 1.5 mmol) in toluene (2mL) was heated at overnight. Upon cooling to room temperature, the solution was

(CDCJ

3 62.29 3H, GH 3 6.35 (br s, 1H, NHl), 7.03-7.06 (in, 2H, Ar-H), 7.15-7.31 (mn, 1H, Ar-H), 7.43 1H, J=7.8 Hz, Ar-H), 7.88 1H, Ar- 7.96 1H, Ar-H).

WO 02/060492 WO 02/60492PCT/AU02/00089 32 Example 7 H/ 0 N, N ci H N~ N A mixture of 2-(2-methylphenyl)-6-chloro-pyrazine (110mg, 0.5 rnm-ol), 3,4,5trimethoxybenzeneboronic acid (127 mg, 0.6 mrnol) and Fd(PPh,) 4 (6 2 mg, 0.05 mmol) in toluene (3 mL) and aqueous NaCO, (0.3mL, 2M) was treated as for Example 2 to furnish the product as a white solid (147mg, 84%).

(GDC1 3 52.27 3H, CH 3 3.84 3H, OCHJ, 3.89 611, OCH,), 6.40 (br s, 1H1, NH), 7.07 111, J= 7,6 Hz, Ar-H), 7.15-7.32 (in, 4H1, Ar-H), 7.55 1H, I 7.T8 Hz, Ar-H), 7.93 (br s, 1H, Ar-H), 8.33 (br s, 1H, Ar-H).

m/z (ES) 352 WO 02/060492 PCT/AU02/00089 33 Compound Dilution For screening purposes, compounds were diluted in 96 well plates at a concentration of 50p[M or 20 jM. Plates were warmed at 37 0 C for 30 minutes before assay.

JAK Tyrosine Kinase Domain Production JAK kinase domains were produced in the following manner: JAK1 The kinase domain of humanJAK1 was amplified from U937mRNA using the polymerase chain reaction with the following primers: XHOI-J1 5'-CCG CTC GAG ACT GAA GTG GAC CCC ACA CAT-3' J1-KPNI 5'-CGG GGT ACC TTA TTT TAA AAG TGC TTC AAA-3' JAK1 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-NOTI 5'-ATA GTT TAG CGG CCG CTC AGA ATG AAG GTC ATT T-3' JAK2 PCR products were cloned into the pFastBac HTc expression vector (Gibco) via the Sal I and Not I sites. The JAK2 plasmid was then transformed into competent DH1OBac cells (Gibco), and the recombinant baculovirus produced prepared for transfection into Sf9 insect cells.

JAK3 The kinase domain of humanJAK3 was amplified from U937mRNA using the polymerase chain reaction with the following primers: XHOI-J3 5'-CCG CTC GAG TAT GCC TGC CAA GAC CCC ACG-3' J3-KPNI 5'-CGG GGT ACC CTA TGA AAA GGA CAG GGA GTG-3' WO 02/060492 PCT/AU02/00089 34 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 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' ITY2.2R 5'-GGA GCA GGA ATT CCG GCG CTG CCG 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 Baculovirus preparations from each of the JAK 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 chelate affinity column.

Assay Protocols Kinase assays were performed in a 96 well capture-based ELISA assay, using approximately 1.5 pg of affinity purified PTK domain in the presence of HEPES, pH 7.5, 10mM MgC1 2 150mM NaCl and 10-20 tM ATP. The biotinylated substrate biotin-EGPWLEEEEEAYGWMDF-NH 2 (final concentration 5 iM) was used as substrate, and tyrosine phosphorylation was quantitated following transfer to an avidin coated ELISA plate using peroxidase-linked anti-phospho-tyrosine antibody 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%.

WO 02/060492 PCT/AU02/00089 Cellular assays were performed as follows: Cell suspensions were prepared by harvesting cells from culture. Cell used in this test should be in later log phase growth and high viability. Cells were diluted in correct growth medium to 1.lx final concentration (from 50000 cell/ml to 200,000 cell/ml, depending on cell line). 90pL was added to samples, diluted in PBS to 10x final concentration in flat-bottom 96-well plates (10iL). After incubation for 40 hr in 37 °C 5% CO 2 incubator, MTT (in PBS, filter sterile) 20 gl per well was added. The plates were returned to incubator for another 6 hours. Lysis Buffer (10% SDS, 0.01N HC1) 100 Rl per well was added and the plate put back in incubator overnight. The plate was then read at 590 nm.

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 restrictive.

WO 02/060492 PCT/AU02/00089 36

TABLES

Table 4: Selected 2-amino-6-carba-disubstituted pyrazines and 2-amino- 6-carba-disubstituted pyridines possessing JAK inhibitory activity Compounds that exhibited a capacity to inhibit 50% of JAK activity at a concentration of 50 M (measured under standard conditions, see Methods), are shown.

Table 5: Selected 2-amino-6-carba-disubstituted pyrazines possessing inhibitory activity on certain cells Compounds that exhibited a capacity to inhibit 50% of cellular growth (measured under standard conditions, see Methods), are shown.

Table 6 and 7: 6-carba-8-amino-disubstituted imidazo-[1,2-a]-pyrazine possessing JAK inhibitory activity Compounds that exhibited a capacity to inhibit 50% of JAK activity at a concentration of 50iM (measured under standard conditions, see Methods), are shown.

WO 02/060492 PCT/AU02/00089 37 References Spiotto MT, and Chung TD. (2000) STAT3 mediates IL-6-induced growth inhibition in the human prostate cancer cell line LNCaP. Prostate 42 88-98 Sadowski HB, Stone JC, Pawson T. (1986) A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P1308-",* Mol Cell Biol 6 4396-408.

Harpur AG, Andres AC, Ziemiecki A, Aston R.R. and Wilks, (1992) JAK2, a third member of the JAK family of protein tyrosine kinases.

Oncogene; 7 1347-53.

Kozma SC, Redmond SMS, Xiao-Chang F, et al. (1988] Activation of the receptor kinase domain of the trk oncogene by recombination with two different cellular sequences. EMBO 7 147-54.

Wilks AF, Harpur AG, Kurban RR, Ralph SJ, Zurcher G, Ziemiecki (1991] Two novel protein-tyrosine kinases, each with a second phosphotransferaserelated catalytic domain, define a new class of protein kinase. Mol Cell Biol.

11 2057-65.

Wilks AF, Kurban RR. (1988) Isolation and structural analysis of murine c-fes cDNA clones, Oncogene 3 289-94.

WO 02/060492 PCT/AU02/00089 38 TABLE 4 CHEMISTRY CYT ID JAKI JAK2 JAK3 TYK2 Ab Hck 0 CYT13809 13809 CYT14607 c 14607 CYT14804

H

14804 HNl N 0 CYT20406 20406 HN CYT20307 20307 CYT20303 20303 H N& CYT20510 100 20510 0 N~CYT20508 20508 WO 02/060492 PCT/AU02/00089 CHEMISTRY CYT ID JAM JAK2 JAK3 TYK2 Abi Hck H4 CYT20504

"N

20504 H4N_16 CYT20506 20506 1 0l 0 CYT20310 20310 N l N 0CYT21404

N

21404 HO N -c-CYT21103

N-

21103 N 0 CYT21507

N

HN N_ P CYT4108 24108

HN

HNCCYT24510 24510____ WO 02/060492 PCT/AU02/00089 CHEMISTRY CYT ID JAKI JAK2 JAK3 TYK2 Abi Hck C YT24605

N

24605 N N -CYT24803 24803 0, 0 wt CYT25210

OH

25210

N

iN N'b CYT35210 2210 NCYT21607 32107 WO 02/060492 WO 02/60492PCT/AU02/00089 CHEMISTRY CYT ID JAKI JAK2 JAK3 TYK2 Abi Hck 0 O 4rCYT32302 32302 jCYT32602 32602 o N u CYT34503 34503 ii 2~N)CYT34205 S N NCYT34702 34702 WO 02/060492 PCT/AU02/00089 42 Table CHE ISRYT-cell activation Jurkat ICSO TSU 060TY CG (UM) (um) (UM)

N)

JIN N all 30 50 24108 allN N 15 50 24510

H

N N N)10 20

N

32107 0-~ HN N 15 20 25209 0- NH 12 35 20510

HH

Hl 1 20 20 20504 40 21103 C 0 6 30 40 20310 WO 02/060492 PCT/AU02/00089 43 ____Table 6 CHEMISTRY JAKI JAK2 JAK3 TYK2 Abi

N

No

-N

_N

Chemistry N N+

N-

Chemistry 23_ WO 02/060492 PCT/AU02/00089 CHEMISTRY JAKI JAK2 JAK3 TYK2 AbN

-N

No 0 Chemistry 4 0

-N

0 +1

N

Chemistry 5

-N

F

6N H Chemistry 6 0- -N o Chemistry 7 WO 02/060492 PCT/AU02/00089 CHEMISTRY JAKI JAK2 -JAK3 TYK2 Abi N-j Chemistry 8 N 14

-N

N-

Chemistry 9 N O N+ H 0 Chemistry

NN

2 Chemistry 11 WO 02/060492 PCT/AU02/00089 CHEMISTRY JAKI JAK2 JAK3 TYK2 ANi A n 0 Chemistry 12

NPV

N +1 Chemistr 13 0

~NH

Nr

H

HI -U Chemistry 14 N+ 00 Chemistry

XN\\

-N

H

Chemistry WO 02/060492 PCT/AU02/00089 CHEMISTRY JAKI JAK2 JAK3 TYK2 AbN

N

I-N

0- Chemistry 17

NN

N

N

NN

lChemistry 20 WO 02/060492 PCT/AU02/00089 CHEMISTRY JAKI JAK2 JAK3 TYK2 Abi

N

Chemistry 21 No 0 Chemistry 22 X N Br

N

HO

Chemistry 23

H

N

N+ +I

N--

Chemistry 24

N

N N Chemistry 25 WO 02/060492 WO 02/60492PCT/AU02/00089 Table 7 CHEMISTRY Jaki Jak2 Jak3 Tyk2 Chemistry 0 Chmitr 0 Chemistry

F

OF

F

Fr+ Chemistry 12 Chemistry 123____ Chemistry 13 WO 02/060492 WO 02/60492PCT/AU02/00089 CHEMISTRY Jaki Jak2 Jak3 Ty k2 Chemistry Chemistry 16 Chemistry 17 Xr+ Chemistry 18 Chemistry 19 Chemistry 2 0+ Chemistry 20 WO 02/060492 WO 02/60492PCT/AU02/00089 CHEMISTRY Jaki Jak2 Jak3 Tyk2 0 Chemistry 21 ChmitrH2 Chemistry 22 Chemistry 23 0 Chemistry 245____ Chemistry 25 Chemistry 26 WO 02/060492 PCT/AU02/00089 CHEMISTRY Jaki Jak2 Jak3 Tyk2 ~H

OH

Chemistry 28

N

Chemistry 29 Chemistry 3 C+

OH

Chemistry 30

N,

OH-

Chmsr 32 HO+ Chemistry 323____ WO 02/060492 WO 02/60492PCT/AU02/00089 CHEMISTRY Jaki Jak2 Jak3 Ty k2 Chemistry 34

CH

Chemistry OH Chemistry Chemistry 38 WO 02/060492 WO 02/60492PCT/AU02/00089 CHEMISTRY Jaki Jak2 Jak3 Tyk2 Chemistry 40 Chemistry 41 Chemistry 421 Chemistry 43 Chemistry 44 Chemistry IChemistry 46 WO 02/060492 WO 02/60492PCT/AU02/00089 CHEMISTRY Jaki Jak2 Jak3 Tyk2 Chemistry 47 Chemistry 48 Chemistry 49 Chemistry 5 Chemistry Chemistry 51 WO 02/060492 PCT/AU02/00089 CHEMISTRY Jaki Jak2 Jak3 Tyk2 0 Chemistry 52 0 02 Chemistry 53

HN

0 Chemistry 54 Chemistry

NF

Chemistry 57 WO 02/060492 WO 02/60492PCT/AU02/00089 CHEMISTRY Jaki Jak2 Jak3 Ty k2

SN

~ON+ Chemistry 58 Chemistry 59 Chemistry 6 Chemistry 60 Chemistry 61 Chemistry 62 Chemistry 63 WO 02/060492 WO 02/60492PCT/AU02/00089 CHEMISTRY Jaki IJak2 Jak3 Tyk2

OH

Chemistry 645____ Chemistry 656____ 0", Chemistry 667____ AnAI No 0 Chemistry 68 r\ Chemistry 697___ Chemistry 70 WO 02/060492 PCT/AU02/00089 CHEMISTRY Jaki Jak2 Jak3 Tyk2

N

Chemitry 7 0 Chemistry 72

N

Chemistry Chemistry 74 Chemistry AAI.+ Chemistry 77 e'N \I0,, N 0+ Chemistry 77 WO 02/060492 PCT/AU02/00089 WO 02/060492 WO 02/60492PCT/AU02/00089 CHEMISTRY Jaki Jak2 Jak3 Tyk2 Chemistry 85 0 FN+ Chemistry 86 Chemistry 87 0 S+ Chemistry 9

Claims (30)

1. A method of inhibiting JAK in a cell, the method comprising administering to the cell an effective amount of a composition comprising a carrier and a compound of the general formula I: RIN R2 x or pharmaceutically acceptable salts, hydrates, solvates, crystal forms or diastereomers thereof, wherein X is either carbon or nitrogen R1 is C-o 1 0 Alkyl, C2- 10 Alkenyl, C2- 10 Alkynyl, C2- 10 Alkylaryl, Aryl, or Heterocyclyl, or R1 with N may form a substituted or unsubstituted heterocyclyl, wherein the Alkyl, Alkenyl, Alkynyl, Alkylaryl, Aryl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 member ring and in which the hetero atom is O, N orS; R2 is selected from Ci-lo Alkyl, C2- 1 0 Alkenyl, C 2 10 Alkynyl, C 210 Alkylaryl, Aryl, Halo, OH, or 6-7 membered Heterocyclyl, wherein the Alkyl, Alkenyl, Alkynyl, Alkylaryl, Aryl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 membered ring and in which the hetero atom is O, N or S; 200527036 1 with the proviso that when X is CH, R2 is not Cl. 6 alkyl or piperazinyl.

2. A method as claimed in claim 1 wherein X is N.

3. A method as claimed in claim 1 in which the compound is of the general formula: wherein one of X,Y and Z is nitrogen and the other two are carbon, or all three are carbon; R3, R4 and R5 are the same or different and are H, halo, OH, hydroxyamide, amino, hydroxyalkyl, aminoalkylamide, alkylamide, arylamide or alkoxy.

4. A method as claimed in any one of claims 1 to 3 in which RI with N forms a heterocycle. A method as claimed in claim 4 in which the heterocycle includes two heteroatoms, preferably two nitrogen atoms.

6. A method as claimed in any one of claims 1 to 5 in which the compound is of the general formula: SR H R3 wherein X is nitrogen or carbon; R1 is C 2 -o 1 Alkylphenyl, Phenyl, or Heterocyclyl, wherein the Alkyl, Phenyl, and 6-7 membered Heterocyclyl, is optionally substituted with one to three members 200527036 1 selected from the group consisting of chloro, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 membered ring and in which the hetero atom is O, N or S; R3, R4 and R5 are the same or different and are H, halo, OH, hydroxyamide, amino, hydroxyalkyl, aminoalkylamide, alkylamide, arylamide or alkoxy.

7. A method as claimed in any one of claims 1 to 6 in which the compound is selected from the group consisting of 200527036_1

8. A method of inhibiting JAK in a cell, the method comprising administering to the cell an effective amount of a composition comprising a carrier and a compound of the general formula II: 200527036_1 H R6 HN R7)N or pharmaceutically acceptable salts, hydrates, solvates, crystal forms or diastereomers thereof, wherein R6 is Cl-1o Alkyl, C2- 1 0 Alkenyl, C2-10 Alkynyl, C2-1 0 Alkylaryl, Aryl, or Heterocyclyl, or R1 with N may form a substituted or unsubstituted heterocyclyl, wherein the Alkyl, Alkenyl, Alkynyl, Alkylaryl, Aryl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 membered ring and in which the hetero atom is O, N or S; R7 is C-o 1 0 Alkyl, C2- 1 0 Alkenyl, C2- 1 0 Alkynyl, C2- 1 0 Alkylaryl, Aryl, OH, or Heterocyclyl, wherein the Alkyl, Alkenyl, Alkynyl, Alkylaryl, Aryl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 member ring and in which the hetero atom is O, N or S.

9. A method as claimed in claim 8 in which the compound is of the general formula: 200527036 1 H R6 N 1 N1/ II R8 R9 wherein one of X,Y or Z is nitrogen and the other two are carbon, or all three are carbon R8, R9 and R10 are the same or different and are H, halo, OH, hydroxyamide, amino, hydroxyalkyl, aminoalkylamide, alkylamide, arylamide or alkoxy. A method as claimed in claim 8 or 9 in which Ri with N forms a heterocycle.

11. A method as claimed in claim 9 in which the heterocycle includes two heteroatoms, preferably two nitrogen atoms.

12. A method as claimed in any one of claims 8 to 11 in which the compound is of the general formula: HN/R x N II 31 Rio R8 R9 in which: R6 is C 2 10 Alkylphenyl, Phenyl, or Heterocyclyl, wherein the Alkyl, Phenyl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of chloro, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 member ring and in which the hetero atom is O, N or S; 200527036_1 67a. R3, R4 and R5 are the same or different and are H, halo, OH, hydroxyamide, amino, hydroxyalkyl, aminoalkylamide, alkylamide, arylamide or alkoxy.

13. A method as claimed in any one of claims 8 to 12 in which the compound is selected from the group consisting of HO-\-H HN HN N N N" N/ N N r HN HNHN 0 0 0 200527036 1 WO 02/060492 PCT/AU02/00089 0 HN (N 'N /OH HN 0- N NH N\ HNH -N HN 0 N N HN 0 HN QN HN 0 SUBSTITUTE SHEET (RULE 26) WO 02/060492 WO 02/60492PCT/AU02/00089 F F- OH _F NH 0\N ol N /,F N N NN N o H N I- N N Br F N~ N HN N H H H 0 Br NH~ H N N( N N N N N N N N HN HN HO 0N N N0 0N N HN N 0 NNH 0N (NN -N HN N OH 0 SUBSTITUTE SHEET (RULE 26) WO 02/060492 WO 02/60492PCT/AU02/00089 N INNN HNH N 0 N\ IH HN N 0N HNN N N 0 OH -NN NH H NHN NNN N N HN N N- HNN -N-NH N OH 0 j HO N NN HN _N N N p HN H 0 Nj No HO HO SUBSTITUTE SHEET (RULE 26) WO 02/060492 WO 02/60492PCT/AU02/00089 N F rN cj HN HO 0 r~N\ 'I OH 0 HN H 0 NH rI N H 0 ~N OH -N NI N HN O ON 0 0 HNH N H N= F HN N4~ NN H 11),-0 SUBSTITUTE SHEET (RULE 26) WO 02/060492 WO 02/60492PCT/AU02/00089 7N\ \N 0 N HNN 0 H\ NH HN 0- N 0 N 0 HN OH 0 -N HN Na F xF F o N 0 N F HN 0/- 0 0 N NN N N SUBSTITUTE SHEET (RULE 26) WO 02/060492 PCT/AU02/00089 HN HN N N HN No HN OH N1 HO -N HN N00 H N N HN No 0 SUBSTITUTE SHEET (RULE 26) WO 02/060492 PCT/AU02/00089 N N, N\ NN-/ fj 0 HO N- 1 0 N H,~N OH HN N\ H HN Nz N N-S 0 NI O OH 0 HN HN N \N &~N 0- H OH OH HH NH, HN SUBSTITUTE SHEET (RULE 26)

14. A method as claimed in any one of claims 1 to 13 in which the method is conducted in vivo. A method as claimed in any one of claims 1 to 14 in which the JAK is JAK1.

16. A method as claimed in any one of claims 1 to 14 in which the JAK is JAK2.

17. A method as claimed in any one of claims 1 to 14 in which the JAK is JAK3.

18. A method as claimed in any one of claims 1 to 14 in which the JAK is TYK2.

19. A method of treating an individual suffering from a JAK-associated disease state, the method comprising administering to the individual a composition comprising a pharmaceutically acceptable carrier and a compound of the general formula: N N .N R2 R1 I NX x or pharmaceutically acceptable salts, hydrates, solvates, crystal forms or diastereomers thereof, wherein X is either carbon or nitrogen R1 is C- 0 lo Alkyl, C 2 10 Alkenyl, C2- 10 Alkynyl, C2- 1 0 Alkylaryl, Aryl, or 6-7 membered Heterocyclyl, or R1 with N may form a substituted or unsubstituted heterocyclyl, wherein the Alkyl, Alkenyl, Alkynyl, Alkylaryl, Aryl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 membered ring and in which the hetero atom is O, N or S; R2 is selected from C 1 -lo Alkyl, C2- 1 0 Alkenyl, C2-1 0 Alkynyl, C2- 1 0 Alkylaryl, Aryl, Halo, OH, or 6-7 membered Heterocyclyl, wherein the Alkyl, Alkenyl, 200527036 1 Alkynyl, Alkylaryl, Aryl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 member ring and in which the hetero atom is O, N or S with the proviso that when X is CH, R2 is not Ci- 6 alkyl or piperazinyl. A method as claimed in claim 19 wherein X is N.

21. A method as claimed in claim 19 in which the compound is of the general formula: z Y HR3 N N R1 R5 R4 N wherein one of X,Y and Z is nitrogen and the other two are carbon, or all three are carbon; R3, R4 and R5 are the same or different and are H, halo, OH, hydroxyamide, amino, hydroxyalkyl, aminoalkylamide, alkylamide, arylamide or alkoxy.

22. A method as claimed in any one of claims 19 to 21 in which R1 with N forms a heterocycle.

23. A method as claimed in claim 22 in which the heterocycle includes two heteroatoms, preferably two nitrogen atoms.

24. A method as claimed in any one of claims 19 to 23 in which the compound is of the general formula: 200527036 1 H R3 /N N R1 R5 R4 N wherein X is nitrogen or carbon; R1 is C2- 1 0 Alkylphenyl, Phenyl, or Heterocyclyl, wherein the Alkyl, Phenyl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of chloro, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 member ring and in which the hetero atom is O, N or S; R3, R4 and R5 are the same or different and are H, halo, OH, hydroxyamide, amino, hydroxyalkyl, aminoalkylamide, alkylamide, arylamide or alkoxy. A method as claimed in any one of claims 19 to 24 in which the compound is selected from the group consisting of HN N, N HN NN N- H N Hr 200527036 1 I9OLMOZ 0- 0 N~ N 0 H

26. A method of treating an individual suffering from a JAK-associated disease state, the method comprising administering to the individual a composition comprising a pharmaceutically acceptable carrier and a compound of the general formula: H R6 NN R7 or pharmaceutically acceptable salts, hydrates, solvates, crystal forms or diastereomers thereof, wherein R6 is Cl-lo Alkyl, C2- 1 0 Alkenyl, C2- 1 0 Alkynyl, C2- 1 0 Alkylaryl, Aryl, or Heterocyclyl, or R1 with N may form a substituted or unsubstituted heterocyclyl, wherein the Alkyl, Alkenyl, Alkynyl, Alkylaryl, Aryl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 membered ring and in which the hetero atom is O, NorS; R7 is Cl-lo Alkyl, C2- 10 Alkenyl, C2- 1 0 Alkynyl, C2- 10 Alkylaryl, Aryl, OH, or Heterocyclyl, wherein the Alkyl, Alkenyl, Alkynyl, Alkylaryl, Aryl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular methoxy), heterocyclic alkyl in which heterocycle is a 5-7 membered ring and in which the hetero atom is O, N or S.

27. A method as claimed in claim 26 in which the compound is of the general formula: 200527036 1 HNR6 HN z N/ R8 R9 wherein one of X,Y or Z is nitrogen and the other two are carbon, or all three are carbon R8, R9 and R10 are the same or different and are H, halo, OH, hydroxyamide, amino, hydroxyalkyl, aminoalkylamide, alkylamide, arylamide or alkoxy.

28. A method as claimed in claim 26 or 27 in which R1 with N forms a heterocycle it is preferred that the heterocycle.

29. A method as claimed in claim 28 in which the heterocycle includes two heteroatoms, preferably two nitrogen atoms.

30. A method as claimed in any one of claims 26 to 29 in which the compound is of the general formula: H R6 HN x II R10 Y R IO R8 R9 in which: R6 is C2-10 Alkylphenyl, Phenyl, or Heterocyclyl, wherein the Alkyl, Phenyl, and Heterocyclyl, is optionally substituted with one to three members selected from the group consisting of halo, amino, hydroxy, hydroxyalkyl, alkylamide, arylamide, hydroxyalkylamide, nitrilo, aminoalkylamide, nitriloaryl, alkoxy (in particular 200527036 I methoxy), heterocyclic alkyl in which heterocycle is a 5-7 membered ring and in which the hetero atom is O, N or S; R3, R4 and R5 are the same or different and are H, halo, OH, hydroxyamide, amino, hydroxyalkyl, aminoalkylamide, alkylamide, arylamide or alkoxy.

31. A method as claimed in any one of claims 26 to 30 in which the compound is selected from the group consisting of HO\ H N N HN HN N o H N N Q 200527036 1 WO 02/060492 WO 02/60492PCT/AU02/00089 SUBSTITUTE SHEET (RULE 26) WO 02/060492 WO 02/60492PCT/AU02/00089 SUBSTITUTE SHEET (RULE 26) WO 02/060492 WO 02/60492PCT/AU02/00089 SUBSTITUTE SHEET (RULE 26) WO 02/060492 WO 02/60492PCT/AU02/00089 SUBSTITUTE SHEET (RULE 26) WO 02/060492 WO 02/60492PCT/AU02/00089 NNH HNJ _NI) N N Y'N\ N _-N QNH 0H OH NNO HN N HN 0 N K-HN HO X NH F0 N0 -N 0N -6 N HN/ HN HN OH- N N N N HN F N HN 0N N r NO HN NN0 N N N N.s HH ofH0 OH SUBSTITUTE SHEET (RULE 26) WO 02/060492 PCT/AU02/00089 N -N No HNO OH N Q HO N H HN HN /N i H N o 0N F F (N N F N F N HN H HNNF N0 N -N HH OH HH HN N Q-NO0 OH N N NH HN (N\ No N N- NN HH SUBSTITUTE SHEET (RULE 26) WO 02/060492 PCT/AU02/00089 I N IN< OH'IN HN 1 HN N= NN N N -NN 0N N N 0 HO 00 N INI NC 2 NOH HN HN N H N~ N N-N NH HIN H H (N HI N-oN HN HN SUBSTITUTE SHEET (RULE 26)

32. A method as claimed in any one of claims 19 to 31 in which the JAK-associated disease state involves JAK1.

33. A method as claimed in any one of claims 19 to 31 in which the JAK-associated disease state involves JAK2.

34. A method as claimed in any one of claims 19 to 31 in which the JAK-associated disease state involves JAK3. A method as claimed in any one of claims 19 to 31 in which the JAK-associated disease state involves TYK2.

36. A method as claimed in any one of claims 19 to 30 in which the JAK-associated 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 Pneumonitis; 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 Leukemia, Lymphoma and Prostate Cancer. Dated: 30 January 2006 Cytopia Pty Ltd Patent Attorneys for the Applicant BLAKE DAWSON WALDRON PATENT SERVICES 200527036 1