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Title:
INHIBITORS OF NECROPTOSIS
Document Type and Number:
WIPO Patent Application WO/2021/168521
Kind Code:
A1
Abstract:
This invention relates to compounds of Formula (I) and salts, solvates, prodrugs, tautomers, N-oxides, stereoisomers, polymorphs and physiologically functional derivatives thereof. Also disclosed are methods of use of Formula (I), including in the inhibition of necroptosis and treatment of associated diseases, conditions and/or disorders.

Inventors:
LESSENE GUILLAUME LAURENT (AU)
GARNIER JEAN-MARC DANIEL (AU)
FEUTRILL JOHN THOMAS (AU)
CUZZUPE ANTHONY NICHOLAS (AU)
CZABOTAR PETER EDWARD (AU)
Application Number:
PCT/AU2021/050176
Publication Date:
September 02, 2021
Filing Date:
March 01, 2021
Export Citation:
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Assignee:
ANAXIS PHARMA PTY LTD (AU)
International Classes:
C07D471/04; A61P39/00
Domestic Patent References:
WO2016127213A12016-08-18
WO2015172203A12015-11-19
WO2019089442A12019-05-09
Other References:
XIE YANGCHUN; ZHU SHAN; ZHONG MEIZUO; YANG MANHUA; SUN XIAOFAN; LIU JINBAO; KROEMER GUIDO; LOTZE MICHAEL; ZEH HERBERT J.; KANG RUI: "Inhibition of Aurora Kinase A Induces Necroptosis in Pancreatic Carcinoma", GASTROENTEROLOGY, ELSEVIER INC., US, vol. 153, no. 5, 1 January 1900 (1900-01-01), US, pages 1429, XP085279222, ISSN: 0016-5085, DOI: 10.1053/j.gastro.2017.07.036
Attorney, Agent or Firm:
FPA PATENT ATTORNEYS PTY LTD (AU)
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Claims:
CLAIMS:

1. A compound of Formula (I):

Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, polymorph and/or physiologically functional derivative thereof, wherein:

W is N or C-R, wherein R is selected from the group consisting of hydrogen, flouro, chloro, C1-C2 alkyl, C1-C2 haloalkyl, methoxy, halomethoxy or cyano; and Ri is selected from the group consisting of hydrogen, C1-C4 alkyl, deuterated C1-C4 alkyl, and C1-C4 hydroxyalkyl, and 3-7 membered heterocyclyl;

J is selected from the group consisting of hydrogen, C1-C4 alkyl, C1-C4 hydroxyalkyl, and 3-7 membered heterocyclyl;

L is-(CHRa)n- wherein n is 0 or 1 and Ra is selected from the group consisting of hydrogen, Ci- C4 alkyl, C1-C4 hydroxyalkyl, C3-C7 cycloalkyl, and 3-7 membered heterocyclyl; and

Xi, X2, X3, X4 and X5 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C3-C7 cycloalkyl, 3-7 membered heterocyclyl, aryl, heteroaryl, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy, aryloxy, aralkoxy, halo, -CN, -NR'R', N(FI)C(0)R", N(H)C(0)0R", N(H)C(0)NR'R', N(H)S(0) R", OR", 0C(0)RR", C(0)R", SR", -S(0)R"',

S(0) R'", and S(0)2NR'R', wherein R' is selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, 3-7 membered heterocyclyl, -OR1, -SR1, -S(0)2R1, -S(0)R1, and C(0)R1; and

R" is selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, 3-7 membered heterocyclyl, -OR1, -NR3R4, -S(0)2R1, -S(0)R1 and C(0)R1; and

R'" is selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C3-C7 heterocyclyl, -OR1 and -NR3R4;

A is selected from the group consisting of 5-6 membered heteroaryl and C& aryl;

Q is hydrogen or is one or more substituents, wherein one substituent of the one or more substituents is selected from a group consisting of:

(i) -(Co-C2alkyl)3-7 membered heterocyclyl, and

(ii) the group defined by -(Z)m-(Z1)n-(Z2), wherein

Z is CH2 and m is 0, 1 , 2, or 3; and Z1 is C(O), and n is 0 or 1 ; and

Z2 is C1 -C4 alkyl, C1 -C4 alkoxy, C3-C7 cycloalkyl, 3-7 membered heterocyclyl, NR3R4, aryl, arylamino, aralkyl, aralkoxy, cyano or heteroaryl; wherein R3 and R4 are each independently selected from hydrogen, and C1-C6 alkyl; and optionally, the one or more substituents comprise a further substituent or substituents independently selected from the group consisting of halogen, C1-C2 alkoxy, and C1-C2 haloalkoxy; Ci -C3 alkyl, CrCshaloalkyl, and -OR1.

2. A compound according to claim 1 , or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, wherein Ri is methyl.

3. A compound according to claim 1 , or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, wherein Ri is 3-7 membered heterocyclyl wherein the heteroatom is oxygen.

4. A compound according to claim 3, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, wherein Ri is oxetane.

5. A compound according to any one of claims 1 to 4, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, wherein X2 and X4 are each independently selected from -F, -Cl, -OCF3 and -CF3 and Xi, X3 and X5 are each hydrogen.

6. A compound according to any one of claims 1 to 5, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, wherein X3 is other than hydrogen and Xi, X2, X4 and X5 are hydrogen.

7. A compound according to claim 6, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, wherein X3 is -OCF3.

8. A compound according to any one of claims 1 to 7, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, wherein J is methyl.

9. A compound according to any one of claims 1 to 8, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, wherein W is CR and R is hydrogen, methyl, chloro, fluoro or trifluoromethyl.

10. A compound according to claim 9, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, wherein R is hydrogen.

11. A compound according to any one of claims 1 to 10, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, wherein A is 5 membered heteroaryl.

12. A compound according to claim 11 , or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, wherein A is selected from the group consisting of pyrazole, imidazole, isoxazole or oxazole.

13. A compound according to claim 12, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, wherein A is pyrazole.

14. A compound according to any one of claims 1 to 13, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, wherein A is pyrazole.

15. A compound according to any one of claims 1 to 14, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, wherein Q is (C0-C2 alkyl)3-7 membered heterocyclyl.

16. A compound according to claim 15, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, wherein Q is a 4 membered heterocyclyl wherein the heteroatom is N.

17. A compound according to claim 16, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, wherein Q is: or

18. A compound according to claim 15, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, wherein Q is (Cialkyl)C6heterocyclyl comprising two O heteroatoms. 19. A compound according to claim 18, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, wherein Q is

20. A compound according to any one of claims 1 to 19, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, wherein L is absent, ie n is zero.

21. A compound according to any one of claims 1 to 19, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, wherein L is -CH2-.

22. A compound according to claim 1 selected from the group consisting of: salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof.

23. A composition comprising a compound according to any one of claims 1 to 22, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof, and a pharmaceutically acceptable excipient.

24. A method for inhibiting necroptosis in a subject in need thereof, the method comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 22, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof; or a composition according to claim 23.

25. The method of claim 24, wherein the subject has a disease selected from the group consisting of diseases of the bones, joints, connective tissue and cartilage, muscular diseases, skin diseases, cardiovascular diseases, circulatory diseases, hematological and vascular diseases, diseases of the lung, diseases of the gastro-intestinal tract, diseases of the liver, diseases of the pancreas, metabolic diseases, diseases of the kidneys, viral and bacterial infections, severe intoxications, degenerative diseases associated with the Acquired Immune Deficiency Syndrome (AIDS), disorders associated with aging, inflammatory diseases, auto immune diseases, dental disorders, ophthalmic diseases or disorders, diseases of the audition tracts, diseases associated with mitochondria, neuronal loss, ischemic reperfusion injury, cancer and metastatic cancer.

26. Use of a compound according to any one of claims 1 to 22, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof; or a composition according to claim 23, in the preparation of a medicament for the inhibition of necroptosis in a subject. 27. Use of a compound according to any one of claims 1 to 22, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof; or a composition according to claim 23, for inhibiting necroptosis.

28. A compound according to any one of claims 1 to 22, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof; or a composition according to claim 23, for use in inhibiting necroptosis.

29. A compound according to any one of claims 1 to 22, or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, and/or polymorph thereof; or a composition according to claim 23, when used for inhibiting necroptosis.

Description:
INHIBITORS OF NECROPTOSIS

This application claims priority to Australian provisional patent application no. 2020900562 (filed on 27 February 2020), the entire contents of which is hereby incorporated by reference.

FIELD OF THE INVENTION

The present disclosure relates to novel heterocyclic compounds which inhibit necroptosis and methods for their use.

BACKGROUND OF THE INVENTION

In many diseases, cell death is mediated through apoptotic and/or necrotic pathways. While much is known about the mechanisms of action that control apoptosis, control of necrosis is not as well understood. Understanding the mechanisms in respect of both necrosis and apoptosis in cells is essential to being able to treat conditions, such as neurodegenerative diseases, stroke, coronary heart disease, kidney disease, liver disease, AIDS and the conditions associated with AIDS.

Cell death has traditionally been categorized as either apoptotic or necrotic based on morphological characteristics (Wyllie et al., Int. Rev. Cytol. 68: 251 (1980)). These two modes of cell death were also initially thought to occur via regulated (caspase-dependent) and non- regulated processes, respectively. More recent studies, however, demonstrate that the underlying cell death mechanisms resulting in these two phenotypes are much more complicated and under some circumstances interrelated. Furthermore, conditions that lead to necrosis can occur by either regulated caspase-independent or non-regulated processes.

One regulated caspase-independent cell death pathway with morphological features resembling necrosis, called necroptosis, has been described (Degterev eta!., Nat. Chem. Biol. 1 :112, 2005). This manner of cell death can be initiated with various stimuli (e.g., TNF-[alpha] and Fas ligand) and in an array of cell types (e.g., monocytes, fibroblasts, lymphocytes, macrophages, epithelial cells and neurons). Necroptosis may represent a significant contributor to and in some cases predominant mode of cellular demise under pathological conditions involving excessive cell stress, rapid energy loss and massive oxidative species generation, where the highly energy- dependent apoptosis process is not operative.

In WO2015/172203, we reported that particular compounds described in US2005/0085637 have been found to be suitable for inhibiting necroptosis. We also discussed particularly suitable compounds for inhibiting necroptosis in WO2016/127213. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification 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 disclosure as it existed before the priority date of each claim of this application. SUMMARY OF THE INVENTION

As discussed above, certain compounds described in WO2016/127213, US2005/0085637 and WO2015/172203 have been found to be suitable for inhibiting necroptosis. Surprisingly, the inventors of this invention have now discovered that other types of compounds are also suitable for inhibiting necroptosis. Further, and equally surprising, the compounds described in this invention target two key effectors of the necroptotic pathway, namely RIP1 and MLKL. RIP1 is the switch by which cell death is either directed towards an apoptotic or necroptotic phenotype. In our case, apoptosis being disengaged, the cell death process is mainly necroptotic. MLKL is the last known effector of the necroptotic cascade. Thus, inhition of these two proteins may represent an added benefit over compounds that inhibit only one of these proteins. In one aspect, the present invention provides a compound of Formula (I):

Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, polymorph and/or physiologically functional derivative thereof, wherein:

W is N or C-R, wherein R is selected from the group consisting of hydrogen, flouro, chloro, Ci- C2 alkyl, C1-C2 haloalkyl, methoxy, halomethoxy or cyano; and Ri is selected from the group consisting of hydrogen, C1 -C4 alkyl, deuterated C1 -C4 alkyl, and C1-C4 hydroxyalkyl, and 3-7 membered heterocyclyl;

J is selected selected from the group consisting of hydrogen, C1 -C4 alkyl, C1 -C4 hydroxyalkyl, and 3-7 membered heterocyclyl;

L is the linker -(CHR a ) n - wherein n is 0 or 1 and R a is selected from the group consisting of hydrogen, C1 -C4 alkyl, C1 -C4 hydroxyalkyl, C3-C7 cycloalkyl, and 3-7 membered heterocyclyl;

Xi, X 2 , X3, X4 and X 5 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C3-C7 cycloalkyl, 3-7 membered heterocyclyl, aryl, heteroaryl, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy, aryloxy, aralkoxy, halo, -CN, -NR'R', N(H)C(0)R", N(H)C(0)0R", N(H)C(0)NR'R', N(H)S(0) 2 R", OR", 0C(0)RR", C(0)R", SR", -S(0)R"', S(0) 2 R"', and S(0) 2 NR'R', wherein

R' is selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, 3-7 membered heterocyclyl, -OR 1 , -SR 1 , -S(0) 2 R 1 , -S(0)R 1 , and C(0)R 1 ; and

R" is selected from the group consisting of hydrogen, C1 -C6 alkyl, C3-C7 cycloalkyl, 3-7 membered heterocyclyl, -OR 1 , -NR 3 R 4 , -S(0) 2 R 1 , -S(0)R 1 and C(0)R 1 ; and

R'" is selected from the group consisting of hydrogen, C1 -C6 alkyl, C3-C7 cycloalkyl, 3-7 membered heterocyclyl, -OR 1 and -NR 3 R 4 ;

A is selected from the group consisting of 5-6 membered heteroaryl and C& aryl;

Q is hydrogen or is one or more substituents, wherein one substituent is selected from a group consisting of:

(i) -(Co-C 2 alkyl)3-7 membered heterocyclyl, and

(ii) the group defined by -(Z) m -(Z 1 ) n -(Z 2 ), wherein

Z is CH 2 and m is 0, 1 , 2, or 3; and

Z 1 is C(O), and n is 0 or 1 ; and

Z 2 is C1 -C4 alkyl, C1 -C4 alkoxy, C3-C7 cycloalkyl, 3-7 membered heterocyclyl, NR 3 R 4 , aryl, arylamino, aralkyl, aralkoxy, or heteroaryl; wherein R 3 and R 4 are each independently selected from hydrogen, and C1-C6 alkyl; and optionally, the one or more substituents comprise a further substituent or substituents independently selected from the group consisting of halogen, C 1 -C 2 alkoxy, and C 1 -C 2 haloalkoxy; C 1 -C 3 alkyl, CrCs haloalkyl, and -OR 1 .

In one aspect, there is provided a composition comprising a compound according to Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof, and a pharmaceutically acceptable excipient.

In another aspect, there is provided a method for inhibiting necroptosis in a subject in need thereof, the method comprising administering a therapeutically effective amount of a compound or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof of Formula (I) to a subject.

In another aspect, there is provided a method for inhibiting necroptosis in a subject in need thereof, the method comprising administering a therapeutically effective amount of a composition containing a compound or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof of Formula (I) to a subject.

In another aspect, there is provided a method for inhibiting necroptosis in a subject in need thereof, the method comprising administering a therapeutically effective amount of a compound according to Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof, that binds to the ATP-binding site of the pseudokinase domain of Mixed Lineage Kinase Domain-like (MLKL) protein.

In another aspect, there is provided a method for inhibiting necroptosis in a subject in need thereof, the method comprising administering a therapeutically effective amount of a composition comprising a compound according to Formula (I) a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof, that binds to the ATP-binding site of the pseudokinase domain of Mixed Lineage Kinase Domain-like (MLKL) protein.

In another aspect, there is provided use of a compound of Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof, in the preparation of a medicament for the inhibition of necroptosis in a subject.

In another aspect, there is provided use of a composition comprising a compound of Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof, in the preparation of a medicament for the inhibition of necroptosis in a subject. In another aspect, there is provided use of a compound according to Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof, for inhibiting necroptosis.

In another aspect, there is provided use of a composition comprising a compound according to Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof, for inhibiting necroptosis.

In yet another aspect, there is provided a compound according to Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof, for use in inhibiting necroptosis.

In another aspect, there is provided a composition comprising a compound according to Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof, for use in inhibiting necroptosis.

In yet another aspect, there is provided a compound according to Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof, when used for inhibiting necroptosis.

In yet another aspect, there is provided a composition comprising a compound according to Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof, when used for inhibiting necroptosis.

Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides a compound of Formula (I): Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer, polymorph and/or physiologically functional derivative thereof, wherein:

W is N or C-R, wherein R is selected from the group consisting of hydrogen, flouro, chloro, C1-C2 alkyl, C1-C2 haloalkyl, methoxy, halomethoxy or cyano; and

Ri is selected from the group consisting of hydrogen, C1-C4 alkyl, deuterated C1-C4 alkyl, and C1-C4 hydroxyalkyl, and 3-7 membered heterocyclyl; J is selected from the group consisting of hydrogen, C1-C4 alkyl, C1-C4 hydroxyalkyl, and 3-7 membered heterocyclyl;

L is the linker -(CHR a ) n - wherein n is 0 or 1 and R a is selected from the group consisting of hydrogen, C1-C4 alkyl, C1-C4 hydroxyalkyl, C3-C7 cycloalkyl, and 3-7 membered heterocyclyl; and

Xi, X 2 , X3, X4 and X 5 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C3-C7 cycloalkyl, 3-7 membered heterocyclyl, aryl, heteroaryl, Ci-C 6 alkoxy, Ci-C 6 haloalkoxy, hydroxy, aryloxy, aralkoxy, halo, -CN, -NR'R', N(H)C(0)R", N(H)C(0)0R", N(H)C(0)NR'R', N(H)S(0) R", OR", 0C(0)RR", C(0)R", SR", -S(0)R"', S(0) R'", and S(0) 2 NR'R', wherein

R' is selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, 3-7 membered heterocyclyl, -OR 1 , -SR 1 , -S(0) 2 R 1 , -S(0)R 1 , and C(0)R 1 ; and R" is selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, 3-7 membered heterocyclyl, -OR 1 , -NR 3 R 4 , -S(0) 2 R 1 , -S(0)R 1 and C(0)R 1 ; and

R'" is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, C 3 -C 7 cycloalkyl, 3-7 membered heterocyclyl, -OR 1 and -NR 3 R 4 ;

A is selected from the group consisting of 5-6 membered heteroaryl and C& aryl;

Q is hydrogen or is one or more substituents, wherein one substituent of the one or more substituents is selected from a group consisting of:

(i) -(Co-C 2 alkyl)3-7 membered heterocyclyl, and

(ii) the group defined by -(Z) m -(Z 1 ) n -(Z 2 ), wherein

Z is CH 2 and m is 0, 1 , 2, or 3; and Z 1 is C(O), and n is 0 or 1 ; and

Z 2 is C 1 -C 4 alkyl, C 1 -C 4 alkoxy, C 3 -C 7 cycloalkyl, 3-7 membered heterocyclyl, NR 3 R 4 , aryl, arylamino, aralkyl, aralkoxy, or heteroaryl; wherein R 3 , and R 4 are each independently selected from hydrogen, and C1-C6 alkyl; and optionally, the one or more substituents comprise a further substituent or substituents independently selected from the group consisting of hydrogen, halogen, C1-C2 alkoxy, and C1-C2 haloalkoxy; Ci -C 3 alkyl, CrCshaloalkyl, and -OR 1 .

In one embodiment, Ri is methyl.

In another embodiment, Ri is 3-7 membered heterocyclyl wherein the heteroatom is oxygen. Preferably, R1 is oxetane.

In one embodiment, X 2 and X 4 are each independently selected from -F, -Cl, -OCF 3 and -CF 3 and Xi, X 3 and X 5 are each hydrogen.

In another preferred embodiment, X 3 is other than hydrogen and Xi, X 2 , X 4 and X 5 are hydrogen. Preferably, X 3 is -OCF 3 .

In one embodiment, J is methyl.

In one embodiment, W is CR and R is hydrogen, methyl, chloro, fluoro or trifluoromethyl. In a preferred embodiment, R is hydrogen.

In one embodiment, A is 5 membered heteroaryl. Preferably, A is selected from the group consisting of pyrazole, imidazole, isoxazole or oxazole. More preferably, pyrazole.

In one embodiment, Q is (Co-C 2 alkyl)3-7 membered heterocyclyl.

In a preferred embodiment, Q is a 4 membered heterocyclyl wherein the heteroatom is N. More preferably Q is: or

In another preferred embodiment, Q is (Cialkyl)C 6 heterocyclyl comprising two O heteroatoms.

In one embodiment, Q is

In one embodiment, L is absent, ie n is zero.

In another embodiment, L is -CH2-. In one embodiment, the compound of formula (I) is selected from the group consisting of:

In yet another aspect, there is provided a composition comprising a compound according to Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof, and a pharmaceutically acceptable excipient.

As used herein the term "alkyl" refers to a straight or branched chain hydrocarbon radical having from one to twelve carbon atoms, or any range between, i.e. it contains 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. The alkyl group may be unsubstituted or optionally substituted with one or more substituent(s), multiple degrees of substitution being allowed, for example 1 -6 substitutents. Examples of "alkyl" as used herein include, but are not limited to, methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, and the like.

As used herein, the terms "C1-C3 alkyl", "C1-C4 alkyl" and "C1-C6 alkyl" refer to an alkyl group, as defined above, containing at least 1 , and at most 3, 4 or 6 carbon atoms respectively, or any range in between (e.g. alkyl groups containing 2-5 carbon atoms are also within the range of Ci- Ce). Where the term “C0-C2 alkyl” is used, there may be no alkyl group, or an alkyl group containing 1 or 2 carbon atoms.

As used herein, the term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) and the term "halo" refers to the halogen radicals fluoro (-F), chloro (-CI), bromo (-Br), and iodo (- I). Preferably, ‘halo’ is fluoro or chloro.

As used herein, the term "cycloalkyl" refers to a non-aromatic cyclic hydrocarbon ring. In a like manner the term "C3-C 7 cycloalkyl" refers to a non-aromatic cyclic hydrocarbon ring having from three to seven carbon atoms, or any range in between. For example, the C3-C 7 cycloalkyl group would also include cycloalkyl groups containing 4 to 6 carbon atoms. The alkyl group is as defined above, and may be substituted. Exemplary "C3-C 7 cycloalkyl" groups useful in the present invention include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

As used herein, the terms "heterocyclic" or "heterocyclyl" refer to a nonaromatic heterocyclic ring, being saturated or having one or more degrees of unsaturation, containing one or more heteroatom substitution selected from S, S(O), S(0) 2 , O, or N. The term "C3-C7 heterocyclyl" refers to a non-aromatic cyclic hydrocarbon ring having from three to seven ring atoms containing one or more heteroatom substitutions as referred to herein. Accordingly, the term “C3-C 7 heterocyclyl” may alternatively be referred to as a 3-7 membered heterocyclyl. The heterocyclic moiety may be unsubstituted or optionally substituted, multiple degrees of substitution being allowed. The term "C3-C 7 heterocyclyl" also includes heterocyclyl groups containing C4-C5, C5-C 7 , C6-C 7 , C4-C 7 , C4-C6 and C5-C6 carbon atoms. Similarly, the term "3-7 membered heterocyclyl " also includes heterocyclyl groups containing 4-5 membered rings, 5-7 membered rings, 6-7 membered rings, 4-7 membered rings, 4-6 membered rings and 5-6 membered rings. Preferably, the heterocyclic ring contains four to six carbon atoms and one or two heteroatoms. More preferably, the heterocyclic ring contains five carbon atoms and one heteroatom, or four carbon atoms and two heteroatom substitutions, or five carbon atoms and one heteroatom. Such a ring may be optionally fused to one or more other "heterocyclic" ring(s) or cycloalkyl ring(s). Examples of "heterocyclic" moieties include, but are not limited to, tetrahydrofuran, pyran, oxetane, 1 ,4- dioxane, 1 ,3-dioxane, piperidine, piperazine, /V-methylpiperazinyl, 2,4-piperazinedione, pyrrolidine, imidazolidine, pyrazolidine, morpholine, thiomorpholine, tetrahydrothiopyran, tetrahydrothiophene, and the like.

As an example of substituted heterocyclic groups, the term “(C 0 -C 2 alkyl)3-7 membered heterocyclyl” includes heterocyclyl groups containing either no alkyl group as a linker between the compound and the heterocycle, or an alkyl group containing 1 or 2 carbon atoms as a linker between the compound and the heterocycle (i.e. heterocycle, -CH 2 -heterocycle or -CH 2 CH 2 - heterocycle). These heterocycles may be further substituted.

Substituted cycloalkyl and heterocyclyl groups may be substituted with any suitable substituent as described below. They may be substituted at any of the carbons on the ring with another cycloalkyl or heterocyclic moiety to form a spiro compound.

As used herein, the term "aryl" refers to an optionally substituted benzene ring or to an optionally substituted benzene ring system fused to one or more optionally substituted benzene rings to form, for example, anthracene, phenanthrene, or napthalene ring systems. The aryl group may be unsubstituted or optionally substituted with one or more substituent(s), multiple degrees of substitution being allowed, Examples of "aryl" groups include, but are not limited to, phenyl, 2- naphthyl, 1 -naphthyl, biphenyl, as well as substituted derivatives thereof. Preferred aryl groups include arylamino, aralkyl, aralkoxy, heteroaryl groups.

As used herein, the term "heteroaryl" refers to a monocyclic five, six or seven membered aromatic ring, or to a fused bicyclic or tricyclic aromatic ring system comprising at least one monocyclic five, six or seven membered aromatic ring. These heteroaryl rings contain one or more nitrogen, sulfur, and/or oxygen heteroatoms, where N-oxides and sulfur oxides and dioxides are permissible heteroatom substitutions and may be optionally substituted with up to three members. The heteroaryl moiety may be unsubstituted or optionally substituted, multiple degrees of substitution being allowed. The terms “C 5 -C 6 heteroaryl” or “5-6 membered heteroaryl” refers to an aromatic ring having 5 to 6 ring atoms, of which one or more of the ring atoms are heteroatom substitutions as referred to herein. Examples of "heteroaryl" groups used herein include furanyl, thiophenyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, oxo-pyridyl, thiadiazolyl, isothiazolyl, pyridyl, pyridazyl, pyrazinyl, pyrimidyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothiophenyl, indolyl, indazolyl, benzimidazolyl, and substituted versions thereof.

As used herein, “haloalkyl” refers to refers to an alkyl group as defined above substituted with one or more halogens as defined above.

As used herein, “hydroxyalkyl” refers to refers to an alkyl group as defined above bound to a hydroxyl group.

As used herein, “alkoxy” refers to an alkyl group as defined above covalently bound via an O linkage.

As used herein, “haloakoxy” refers to an alkyl group as defined above substituted with one or more halogens as defined above, wherein the alkyl group is covalently bound via an O linkage.

As used herein, “aryloxy” refers to an aryl group as defined above covalently bound via an O linkage.

As used herein, “aralkoxy” refers to refers to an aryl group as defined above covalently bound via an alkyl linkage as defined above, wherein the alkyl linkage is covalently bound via an O linkage.

As used herein, “arylamino” refers to an aryl group as defined above covalently bound via an amino linkage.

A "substituent" as used herein, refers to a molecular moiety that is covalently bonded to an atom within a molecule of interest. For example, a "ring substituent" may be a moiety such as a halogen, alkyl group, or other substituent described herein that is covalently bonded to an atom, preferably a carbon or nitrogen atom, that is a ring member. The term "substituted," as used herein, means that any one or more hydrogens on the designated atom is replaced with a selection from the indicated substituents, provided that the designated atom's normal valence is not exceeded, and that the substitution results in a stable compound, i.e., a compound that can be isolated, characterized and tested for biological activity.

The terms "optionally substituted" or “may be substituted” and the like, as used throughout the specification, denotes that the group may or may not be further substituted or fused (so as to form a polycyclic system), with one or more non-hydrogen substituent groups. Suitable chemically viable substituents for a particular functional group will be apparent to those skilled in the art.

Examples of substituents include but are not limited to:

C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 haloalkoxy, C 1 -C 6 hydroxyalkyl, 3-7 membered heterocyclyl, C 3 -C 7 cycloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylsulfanyl, C 1 -C 6 alkylsulfenyl, C 1 -C 6 alkylsulfonyl, C 1 -C 6 alkylsulfonylamino, arylsulfonoamino, alkylcarboxy, alkylcarboxyamide, oxo, hydroxy, mercapto, amino, acyl, carboxy, carbamoyl, aryl, aryloxy, heteroaryl, aminosulfonyl, aroyl, aroylamino, heteroaroyl, acyloxy, aroyloxy, heteroaroyloxy, alkoxycarbonyl, nitro, cyano, halogen, ureido or C 1 -C 6 perfluoroalkyl. Any of these substituents may be further substituted by any of the above- mentioned substituents, where appropriate. For example, alkylamino, or dialkylamino, C 1 -C 6 alkoxy, etc. In one embodiment, cyclic or heterocyclic substituents may form a spiro substituent with a carbon in the moiety from which the cyclic or heterocyclic group is substituted.

As used herein, denotes a bond forming a point of attachment to the compound.

In one aspect of the present invention, there is provided a method for inhibiting necroptosis in a subject in need thereof, the method comprising administering a therapeutically effective amount of a compound or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof of Formula (I), Formula (II), Formula (III) and/or Formula (IV) to a subject.

In another aspect, there is provided a method for inhibiting necroptosis in a subject in need thereof, the method comprising administering a therapeutically effective amount of a composition containing a compound or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof of Formula (I) to a subject.

In another aspect of the present disclosure, there is provided a method for inhibiting necroptosis in a subject in need thereof, the method comprising administering a therapeutically effective amount of a compound according to Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof, that binds to the ATP-binding site of the pseudokinase domain of Mixed Lineage Kinase Domain-like (MLKL) protein.

In another aspect of the present disclosure, there is provided a method for inhibiting necroptosis in a subject in need thereof, the method comprising administering a therapeutically effective amount of a composition comprising a compound according to Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof, that binds to the ATP-binding site of the pseudokinase domain of Mixed Lineage Kinase Domain-like (MLKL) protein.

As used herein, the term "effective amount" means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function.

In one embodiment of the present disclosure, administration of a compound according to Formula (I) inhibits a conformational change of MLKL. In another embodiment, the conformational change of MLKL involves release of the four-helix bundle (4HB) domain of MLKL. In another embodiment, administration of the compound inhibits oligomerisation of MLKL. In yet another embodiment, administration of the compound inhibits translocation of MLKL to the cell membrane. In a further embodiment, administration of the compound inhibits a conformational change of MLKL, inhibits oligomerisation of MLKL and inhibits translocation of MLKL to the cell membrane.

It is envisaged that some compounds of the present disclosure can bind to MLKL in various species and inhibit necroptosis.

As used herein, the term “pseudokinase domain” as understood by a person skilled in the art, means a protein containing a catalytically-inactive or catalytically-defective kinase domain. “Pseudokinase domains” are often referred to as “protein kinase-like domains” as these domains lack conserved residues known to catalyse phosphoryl transfer. It would be understood by a person skilled in the art that although pseudokinase domains are predicted to function principally as catalysis independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions. Accordingly, in the present disclosure the term “pseudokinase domain” includes “pseudokinase domains” which lack kinase activity and “pseudokinase domains” which possess weak kinase activity.

As used herein, the term “ATP-binding site” as understood by a person skilled in the art, means a specific sequence of protein subunits that promotes the attachment of ATP to a target protein. An ATP binding site is a protein micro-environment where ATP is captured and hydrolyzed to ADP, thereby releasing energy that is utilized by the protein to work by changing the protein shape and/or making the enzyme catalytically active. In pseudokinase domains, the “ATP-binding site” is often referred to as the “pseudoactive site”. The term “ATP-binding site” may also be referred to as a “nucleotide-binding site” as binding at this site includes the binding of nucleotides other than ATP. It would be understood by a person skilled in the art that the term “nucleotide” includes any nucleotide. Exemplary nucleotides include, but are not limited to, AMP, ADP, ATP, AMPPNP, GTP, CTP and UTP.

As described herein, inhibition of necroptosis includes both complete and partial inhibition of necroptosis. In one embodiment, inhibition of necroptosis is complete inhibition. In another embodiment, inhibition of necroptosis is partial inhibition.

Binding of the compound to the ATP-binding site of the pseudokinase domain of MLKL may inhibit phosphorylation of MLKL by an effector kinase or binding of the compound to the ATP-binding site of the pseudokinase domain of MLKL may not inhibit phosphorylation of MLKL by an effector kinase. The present disclosure demonstrates that compounds that bind to the ATP-binding site of the pseudokinase domain of the MLKL protein, as described herein, can inhibit necroptosis without inhibiting phosphorylation of MLKL by an effector kinase. In one embodiment, binding of the compound to the ATP-binding site of the pseudokinase domain of MLKL does not inhibit phosphorylation of MLKL by an effector kinase. In another embodiment, binding of the compound to the ATP-binding site of the pseudokinase domain of MLKL inhibits phosphorylation of MLKL by an effector kinase.

Some compounds of this invention may bind to RIP1 . RIP1 kinase is the switch between apoptosis and necroptosis. It has already been shown that, in cases where apoptosis is inhibited (for example using a pan caspase inhibitor such as QVD-OPh), RIP1 mediates a necroptotic response. Moreover, it has also been shown that small molecule inhibitors of RIP1 can potently inhibit necroptosis (see for example Degterev et al, Nat Chem Biol, pp 112 - 119, 2005 and Harris, J Med Chem, 2016). Therefore compounds in this invention may inhibit necroptosis by inhibiting RIP1 .

In addition to their inhibitory activity on RIP1 , compounds of this invention may also bind to MLKL and block its ability to mediate necroptosis. MLKL is the last known effector of necroptosis (Murphy, Immunity, 39, pp 443 - 453, 2013). Without it, necroptosis cannot unfold properly. MLKL acts downstream of RIP1 : upon necroptotic stimulus (e.g. using the combination of TNF, SMAC mimetic and QVD-OPh on suitable cell lines), RIP1 is auto-phosphorylated leading to association with RIP3, which in turn auto-phosphorylates itself. Finally activated RIP3 phosphorylates MLKL leading to a putative conformational change that triggers its necroptotic activity (Murphy, Immunity, 39, pp 443 -453, 2013). Compounds of this invention may bind to MLKL and block this conformational change or any other key event in its activation. Compounds that can simultaneously inhibit RIP1 auto-phosphorylation and MLKL activation may represent very powerful inhibitors of necroptosis due to the fact that they interfere with two key components of the pathway.

In another aspect, there is provided use of a compound of Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof in the preparation of a medicament for the inhibition of necroptosis in a subject.

In another aspect, there is provided use of a composition comprising a compound of Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof in the preparation of a medicament for the inhibition of necroptosis in a subject.

In another aspect, there is provided use of a compound of Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof for inhibiting necroptosis.

In another aspect, there is provided use of a composition comprising a compound of Formula (I) or a salt, solvate, prodrug tautomer, N-oxide, stereoisomer and/or polymorph thereof for inhibiting necroptosis.

In yet another aspect, there is provided a compound according to Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof for use in inhibiting necroptosis.

In yet another aspect, there is provided a composition comprising a compound according to Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof for use in inhibiting necroptosis.

In yet another aspect, there is provided a compound according to Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof when used for inhibiting necroptosis.

In yet another aspect, there is provided a composition comprising a compound according to Formula (I) or a salt, solvate, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof when used for inhibiting necroptosis.

The salts of the compounds of Formula (I) are preferably pharmaceutically acceptable, but it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the present disclosure, since these are useful as intermediates in the preparation of pharmaceutically acceptable salts. The term “pharmaceutically acceptable” may be used to describe any pharmaceutically acceptable salt, hydrate or prodrug, or any other compound which upon administration to a subject, is capable of providing (directly or indirectly) a compound of Formula (I) or an active metabolite or residue thereof.

Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, malic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.

Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, zinc, ammonium, alkylammonium such as salts formed from triethylamine, alkoxyammonium such as those formed with ethanolamine and salts formed from ethylenediamine, choline or amino acids such as arginine, lysine or histidine. General information on types of pharmaceutically acceptable salts and their formation is known to those skilled in the art and is as described in general texts such as “Handbook of Pharmaceutical saltd P.H. Stahl, C.G.Wermuth, 1st edition, 2002, Wiley-VCH.

In the case of compounds that are solids, it will be understood by those skilled in the art that the inventive compounds, agents and salts may exist in different crystalline forms, all of which are intended to be within the scope of the present invention and specified formulae. Amorphous solid forms of the compounds are also intended to be within the scope of the present invention and specified formulae.

The compound of Formula (I) or salts, tautomers, N-oxides, stereoisomers, solvates and/or prodrugs thereof that form crystalline solids may demonstrate polymorphism. All polymorphic forms of the compounds, salts, tautomers, N-oxides, stereoisomers, solvates and/or prodrugs are within the scope of this invention and may be used in the methods of the invention.

Formula (I) is intended to cover, where applicable, solvated as well as unsolvated forms of the compounds. Thus, Formula (I) includes compounds having the indicated structures, including the hydrated or solvated forms, as well as the non-hydrated and non-solvated forms.

As used herein, the term "solvate" refers to a complex of variable stoichiometry formed by a solute (in this invention, a compound of Formula (I) or a salt, prodrug, tautomer, N-oxide, stereoisomer and/or polymorph thereof) and a solvent. Such solvents for the purpose of the invention may not interfere with the biological activity of the solute. Examples of suitable solvents include, but are not limited to, water, methanol, ethanol and acetic acid. Preferably the solvent used is a pharmaceutically acceptable solvent. Examples of suitable pharmaceutically acceptable solvents include, without limitation, water, ethanol and acetic acid. Most preferably the solvent used is water.

Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.

Nitrogen containing groups within a compound of the invention may also be oxidised to form an N-oxide.

The compound of the invention may demonstrate tautomerism. Tautomers are two interchangeable forms of a molecule that typically exist within an equilibrium. Any tautomers of the compounds described herein are to be understood as being within the scope of the invention and may be used in the methods of the invention.

The compound of the invention may contain one or more stereocentres. All stereoisomers of the compounds described herein are within the scope of the invention. Stereoisomers include enantiomers, diastereomers, geometric isomers (E and Z olephinic forms and cis and trans substitution patterns) and atropisomers. In some embodiments, the compound is a stereoisomerically enriched at any stereocentre. The compound may be enriched in one stereoisomer over another by about 60, 70, 80, 90, 95, 98 or 99%.

The compound of the invention or its salts, tautomers, solvates, N-oxides, polymorphs and/or stereoisomers, may be isotopically enriched with one or more of the isotopes of the atoms present in the compound. For example, the compound may be enriched with one or more of the following minor isotopes: 2 H, 3 H, 13 C, 14 C, 15 N and/or 17 0. An isotope may be considered enriched when its abundance is greater than its natural abundance.

A "prodrug" is a compound that may not fully satisfy the structural requirements of the compounds provided herein, but is modified in vivo, following administration to a subject or patient, to produce a compound of formula (I) provided herein. For example, a prodrug may be an acylated derivative of a compound as provided herein. Prodrugs include compounds wherein hydroxy, carboxy, amine or sulfhydryl groups are bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxy, carboxy, amino, or sulfhydryl group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, phosphate and benzoate derivatives of alcohol and amine functional groups within the compounds provided herein. Prodrugs of the compounds provided herein may be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved in vivo to generate the parent compounds.

Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (eg, two, three or four) amino acid residues which are covalently joined to free amino, and amido groups of compounds of Formula (I). The amino acid residues include the 20 naturally occurring amino acids commonly designated by three letter symbols and also include, 4- hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvlin, beta-alanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine and methionine sulfone. Prodrugs also include compounds wherein carbonates, carbamates, amides and alkyl esters which are covalently bonded to the above substituents of Formula (I) through the carbonyl carbon prodrug sidechain.

The compounds of Formula (I) and prodrugs thereof may be covalent irreversible or covalent reversible inhibitors of the active site of a protein.

Pharmaceutical compositions may be formulated from compounds according to Formula (I) for any appropriate route of administration including, for example, topical (for example, transdermal or ocular), oral, buccal, nasal, vaginal, rectal or parenteral administration. The term parenteral as used herein includes subcutaneous, intradermal, intravascular (for example, intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and intraperitoneal injection, as well as any similar injection or infusion technique. In certain embodiments, compositions in a form suitable for oral use or parenteral use are preferred. Suitable oral forms include, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. For intravenous, intramuscular, subcutaneous, or intraperitoneal administration, one or more compounds may be combined with a sterile aqueous solution which is preferably isotonic with the blood of the recipient. Such formulations may be prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride or glycine, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering said solution sterile. The formulations may be present in unit or multi-dose containers such as sealed ampoules or vials. Examples of components are described in Martindale - The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.), Remington's Pharmaceutical Sciences. In the context of this specification the term “administering” and variations of that term including “administer” and “administration”, includes contacting, applying, delivering or providing a compound or composition of the invention to an organism, or a surface by any appropriate means.

For the inhibition of necroptosis, the dose of the biologically active compound according to the invention may vary within wide limits and may be adjusted to individual requirements. Active compounds according to the present invention are generally administered in a therapeutically effective amount. Preferred doses range 5 from about 0.1 mg to about 140 mg per kilogram of body weight per day (e.g. about 0.5 mg to about 7 g per patient per day). The daily dose may be administered as a single dose or in a plurality of doses. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the subject treated and the particular mode of administration. Dosage unit forms will generally contain between about 1 mg to about 500 mg of an active ingredient.

It will be understood, however, that the specific dose level for any particular subject and will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination (i.e. other drugs being used to treat the subject), and the severity of the particular disorder undergoing therapy. The dosage will generally be lower if the compounds are administered locally rather than systemically, and for prevention rather than for treatment. Such treatments may be administered as often as necessary and for the period of time judged necessary by the treating physician. A person skilled in the art will appreciate that the dosage regime or therapeutically effective amount of the compound of formula (I) to be administered may need to be optimized for each individual. The pharmaceutical compositions may contain active ingredient in the range of about 0.1 to 2000 mg, preferably in the range of about 0.5 to 500 mg and most preferably between about 1 and 200 mg. A daily dose of about 0.01 to 100 mg/kg body weight, preferably between about 0.1 and about 50 mg/kg body weight, may be appropriate. The daily dose can be administered in one to four doses per day.

It will also be appreciated that different dosages may be required for treating different disorders. An effective amount of an agent is that amount which causes a statistically significant decrease in necroptosis.

For in vitro analysis, the necroptosis inhibition may be determined by assays used to measure TSQ-induced necroptosis, as described in the biological tests defined herein. The terms “therapeutically effective amount” or “effective amount” refer to an amount of the compound of formula (I) that results in an improvement or remediation of the symptoms of necroptosis and/or associated diseases or their symptoms.

The terms “treating”, “treatment” and “therapy” are used herein to refer to curative therapy, prophylactic therapy and preventative therapy. Thus, in the context of the present disclosure the term “treating” encompasses curing, ameliorating or tempering the severity of necroptosis and/or associated diseases or their symptoms.

“Preventing” or "prevention" means preventing the occurrence of the necroptosis or tempering the severity of the necroptosis if it develops subsequent to the administration of the compounds or pharmaceutical compositions of the present invention.

“Subject” includes any human or non-human animal. Thus, in addition to being useful for human treatment, the compounds of the present invention may also be useful for veterinary treatment of mammals, including companion animals and farm animals, such as, but not limited to dogs, cats, horses, cows, sheep, and pigs.

The term “inhibit” is used to describe any form of inhibition that results in prevention, reduction or otherwise amelioration of necroptosis, including complete and partial inhibition.

The compounds of the present invention may be administered along with a pharmaceutical carrier, diluent or excipient as described above.

The methods of the present disclosure can be used to prevent or treat the following disease in a subject:

• diseases of the bones, joints, connective tissue and of cartilage, such as osteoporosis, osteomyelitis, arthritises including for example osteoarthritis, rheumatoid arthritis and psoriatic arthritis, avascular necrosis, progressive fibrodysplasia ossificans, rickets, Cushing's syndrome;

• muscular diseases such as muscular dystrophy, such as for example Duchenne's muscular dystrophy, myotonic dystrophies, myopathies and myasthenias;

• diseases of the skin, such as dermatitis, eczema, psoriasis, aging or even alterations of scarring; • cardiovascular diseases such as cardiac and/or vascular ischemia, myocardium infarction, ischemic cardiopathy, chronic or acute congestive heart failure, cardiac dysrythmia, atrial fibrillation, ventricular fibrillation, paroxystic tachycardia, congestive heart failure, hypertrophic cardiopathy, anoxia, hypoxia, secondary effects due to therapies with anti-cancer agents;

• circulatory diseases such as atherosclerosis, arterial scleroses and peripheral vascular diseases, cerebrovascular strokes, aneurisms;

• haematological and vascular diseases such as: anemia, vascular amyloidosis, haemorrhages, drepanocytosis, red cell fragmentation syndrome, neutropenia, leukopenia, medullar aplasia, pantocytopenia, thrombocytopenia, haemophilia;

• lung diseases including pneumonia, asthma; obstructive chronic diseases of the lungs such as for example chronic bronchitis and emphysema;

• diseases of the gastro-intestinal tract, such as ulcers;

• diseases of the liver such as for example hepatitis particularly hepatitis of viral origin or having as causative agent other infectious agents, auto-immune hepatitis, fulminating hepatitis, certain hereditary metabolic disorders, Wilson's disease, cirrhoses, non alcoholic hepatic steatosis, diseases of the liver due to toxins and to drugs such as drug- induced liver injury;

• diseases of the pancreas such as for example acute or chronic pancreatitis;

• metabolic diseases such as diabetes mellitus and insipid diabetes, thyroiditis;

• diseases of the kidneys such as for example acute renal disorders or glomerulonephritis;

• viral and bacterial infections such as septicemia;

• severe intoxications by chemicals, toxins or drugs;

• degenerative diseases associated with the Acquired Immune Deficiency Syndrome (AIDS);

• disorders associated with aging such as the syndrome of accelerated aging; • inflammatory diseases such as Terminal ileitis including Crohn's disease, rheumatoid polyarthritis, TNF-induced systemic inflammatory syndrome;

• auto-immune diseases such as erythematous lupus;

• dental disorders such as those resulting in degradation of tissues such as for example periodontitis;

• ophthalmic diseases or disorders including diabetic retinopathies, glaucoma, macular degenerations, retinal degeneration, retinitis pigmentosa, retinal holes or tears, retinal detachment, retinal ischemia, acute retinopathies associated with trauma, inflammatory degenerations, post-surgical complications, medicinal retinopathies, cataract, cone cell degeneration;

• disorders of the audition tracts, such as otosclerosis and deafness induced by antibiotics;

• Ischemic reperfusion injury;

• Neuronal loss;

• diseases associated with mitochondria (mitochondrial pathologies), such as Friedrich's ataxia, congenital muscular dystrophy with structural mitochondrial abnormality, certain myopathies (MELAS syndrome, MERFF syndrome, Pearson's syndrome), MIDD (mitochondrial diabetes and deafness) syndrome, Wolfram's syndrome, dystonia; and

• cancer and metastasis including but not limited to cancers of the lung and bronchus, including non-small cell lung cancer (NSCLC), squamous lung cancer, brochioloalveolar carcinoma (BAC), adenocarcinoma of the lung, and small cell lung cancer (SCLC); prostate cancer, including androgen-dependent and androgen-independent prostate cancer; breast cancer, including metastatic breast cancer; pancreatic cancer; cancers of the colon and rectum; thyroid cancer; cancers of the liver and intrahepatic bile duct; hepatocellular cancer; gastric cancer; endometrial cancer; melanoma; cancers of the kidney, renal pelvis, urinary bladder, uterine corpus and uterine cervix; ovarian cancer, including progressive epithelial or primary peritoneal cancer; multiple myeloma; oesophageal cancer, including squamous cell carcinoma and adenocarcinoma of the oesophagus; acute myelogenous leukemia (AML); chronic myelogenous leukemia (CML), including accelerated CML and CML blast phase (CML-BP); lymphocytic leukemia; myeloid leukemia; acute lymphoblastic leukemia (ALL); chronic lymphocytic leukemia (CLL); Hodgkin's disease (HD); non- Hodgkin's lymphoma (NHL), including follicular lymphoma and mantle cell lymphoma; B-cell lymphoma, including diffuse large B-cell lymphoma (DLBCL); T-cell lymphoma; multiple myeloma (MM); amyloidosis; Waldenstrom's macroglobulinemia; myelodysplastic syndromes (MDS), including refractory anemia (RA), refractory anemia with ringed siderblasts (RARS), (refractory anemia with excess blasts (RAEB), and RAEB in transformation (RAEB-T); and myeloproliferative syndromes; cancers of the brain, including glioma/glioblastoma, anaplastic oligodendroglioma, and adult anaplastic astrocytoma; neuroendocrine cancers, including metastatic neuroendocrine tumors; cancers of the head and neck, including , e.g., squamous cell carcinoma of the head and neck, and nasopharyngeal cancer; cancers of the oral cavity, pharynx and small intestine; bone cancer; soft tissue sarcoma; and villous colon adenoma.

The methods can also be used for protecting cells, tissues and/or transplanted organs, whether before, during (removal, transport and/or re-implantation) or after transplantation.

The methods and compounds described herein are described by the following illustrative and non-limiting examples.

EXAMPLES

1.1 Materials and Methods Compounds.

All temperatures referred to are in °C.

The names of the following compounds have been obtained using ChemDraw Ultra 12.0.

Abbreviations

AcOH acetic acid

BINAP 2,2'-Bis(diphenylphosphino)-1,T-binaphthyl (BOC)20 di-tert-butyl dicarbonate CDCI 3 deuterochloroform

CDI 1,T-Carbonyldiimidazole CS2CO3 cesium carbonate DMSO-d 6 deuterated dimethylsulfoxide DCC dicyclohexylcarbodiimide DCM dichloromethane DIPEA diisopropylethylamine DMF A/,A/-dimethylformamide DMSO dimethylsulfoxide TEA triethylamine EtOAc ethylacetate EtOH ethanol hr hour(s)

HATU 1-[Bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b] pyridinium 3-oxid hexafluorophosphate

HCI hydrochloric acid/hydrogen chloride HPLC high performance liquid chromatography

K 2 CO 3 potassium carbonate

LCMS liquid chromatography-mass spectrometry

M molar (concentration)

MeOH methanol min minute(s)

M/Z mass/charge ratio (mass spectrometry) Na2CC>3 sodium carbonate NaH sodium hydride NaHC03 sodium bicarbonate NaOH sodium hydroxide Na 2 SC> 4 sodium sulphate NH4CI ammonium chloride NMP N-methyl-2-pyrrolidinone NMR nuclear magnetic resonance Pd/C palladium on activated charcoal Pd2(dba)3 tris(dibenzylideneacetone)dipalladium(0)

Rt retention time rt room temperature

SEM-CI 2-(Trimethylsilyl)ethoxymethyl chloride SOCI2 thionyl chloride TFA trifluoroacetic acid THF tetrahydrofuran TsOH tosyl chloride LCMS methodology

Electrospray mass spectroscopy (MS) was carried out using the following method; Method B (5 min method): LC model: Agilent 1200 (Pump type: Binary Pump, Detector type: DAD) MS model: Agilent G6110A Quadrupole. Column: Xbridge-C18, 2.5 pm, 2.1x30 mm. Column temperature: 30°C. Acquisition of wavelength: 214 nm, 254 nm. Mobile phase: A: 0.07% HCOOH aqueous solution, B: MeOH. Run time: 5 min. MS: Ion source: ES+ (or ES-). MS range: 50-900 m/z. Fragmentor: 60. Drying gas flow: 10 L/min. Nebulizer pressure: 35 psi. Drying gas temperature: 350°C. Vcap: 3.5 kV.

Preparative HPLC

Preparative HPLC

Instrument type:

VARIAN 940 LC. Pump type: Binary Pump. Detector type: PDA LC conditions:

Column: Waters SunFire prep C18 OBD, 5 pm, 19x100 mm. Acquisition wavelength: 214 nm, 254 nm. Mobile Phase: A: 0.07% TFA aqueous solution, B: MeOH

NMR

Nuclear magnetic resonance ( 1 H NMR, 400 MHz) spectra were obtained at 300 K with the CDCI3 as the solvent, unless otherwise indicated. Chemical shifts are reported in ppm on the <5 scale and referenced to the appropriate solvent peak.

Synthesis of intermediate A

Step 1 : A/-methyl-3,5-dinitropyridin-2-amine

To a solution of 2-chloro-3,5-dinitropyridine (5 g, 24.6 mmol) in MeOH (30 ml_) was added methanamine hydrochloride (1.6 g, 24.6 mmol) and the mixture was stirred at RT under nitrogen atmosphere overnight. A precipitate formed and the solid was collected by filtration and dried under reduced pressure to give the title compound (4 g, 83%) as a white solid. LCMS (acidic 5 min): 1.53 min [MH] + =199.1.

Step 2: N2-methyl-5-nitropyridine-2, 3-diamine

To a solution of N-methyl-3,5-dinitropyridin-2-amine (4 g, 20 mmol) in EtOH (20 ml_) were added (NH ) S (9.8 g, 120 mmoL). The reaction mixture was stirred at 100 °C under nitrogen overnight. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with ethyl acetate (20 ml_), and the mixture was washed with H O (20 ml_). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAc 5:1) to give N2-methyl- 5-nitropyridine-2, 3-diamine (2.8 g, 84%) as a yellow solid. LCMS (acidic 5 min): 0.46 min [MH] + =169.1.

Step 3: 3-methyl-6-nitro-N-(4-(trifluoromethoxy)phenyl)-3H-imidazo[4 ,5-b]pyridin-2- amine

To a solution of N2-methyl-5-nitropyridine-2, 3-diamine (7.2 g, 43 mmol) in MeOH (120 mL) was added 1-isocyanato-4-(trifluoromethoxy)benzene (9.4 g, 43 mmol). The mixture was stirred at room temperature under nitrogen atmosphere overnight and then concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate 20:1) to give 3-methyl-6-nitro-N-(4-(trifluoromethoxy)phenyl)-3H-imidazo[4 ,5-b]pyridin-2- amine (6.5 g, 43%) as a yellow solid. LCMS (acidic 5 min): 3.09 min [MH] + =354.1.

Step 4: 3-methyl-6-nitro-N-(4-(trifluoromethoxy)phenyl)-N-((2-(trime thylsilyl)ethoxy) methyl)-3H- imidazo[4,5-b]pyridin-2-amine To a solution of 3-methyl-6-nitro-N-(4-(trifluoromethoxy)phenyl)-3H-imidazo[4 ,5-b]pyridin-2-amine (1.5 g, 4.3 mmol) in THF (20 ml_) was added NaH (310 mg, 15 mmol) at 0 °C for 30 min, followed by SEMCI (852 mg, 5.1 mmol). The reaction mixture was stirred at rt for 3 h. The reaction mixture was then concentrated and the residue was diluted with ethyl acetate (25 ml_) and washed with H2O (25 ml_). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAc 20:1) to give 3-methyl-6-nitro-N-(4-(trifluoromethoxy)phenyl)-N-((2- (trimethylsilyl)ethoxy)methyl)- 3H-imidazo[4,5-b]pyridin-2-amine (800 mg, 40%) as a yellow solid. LCMS (acidic 5 min): 3.86min [MH] + =484.2.

Step 5: 3-methyl-/\/2-(4-(trifluoromethoxy)phenyl)-N2-((2-(trimethyl silyl)ethoxy) methyl)-3H- imidazo[4,5-b]pyridine-2, 6-diamine

To a solution of 3-methyl-6-nitro-/\/-(4-(trifluoromethoxy)phenyl)-N-((2-

(trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridin-2 -amine (300 mg, 0.62 mmol) in MeOH (10 ml_) was added zinc (323 mg, 5 mmol) and NH4CI (aq, 10 ml_). The reaction mixture was allowed to stir at 60 °C under nitrogen atmosphere overnight. The reaction mixture was then concentrated and the residue was diluted with ethyl acetate (10 ml_), and the mixture was washed with H2O (10 ml_). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate 20:1) to give 3-methyl-/\/2-(4-(trifluoromethoxy)phenyl)-N2-((2- (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine (280 mg, 100%) as a yellow solid. LCMS (acidic 5 min): 3.28 min [MH] + =454.2.

Step 6: A/6-(2-chloropyrimidin-4-yl)-3-methyl-N2-(4-(trifluoromethox y)phenyl)-N2- ((2-

(trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine- 2, 6-diamine

To a solution of 3-methyl-/\/2-(4-(trifluoromethoxy)phenyl)-N2-((2-(trimethyl silyl)ethoxy)methyl)- 3H-imidazo[4,5-b]pyridine-2, 6-diamine (280 mg, 0.62 m mol) in DMSO (10 mL) were added 2,4- dichloropyrimidine (184 mg, 1.2 mmol) and Cs 2 CC> 3 (606 g, 1.86 mmol). The mixture was heated to 100 °C under nitrogen atmosphere overnight. The mixture was partitioned between water and EtOAc and the organics were extracted with ethyl acetate (3x20 ml_). The combined organics were dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAC 10:1) to give A/6-(2-chloropyrimidin- 4-yl)-3-methyl-N2-(4-(trifluoromethoxy)phenyl)-N2-((2-(trime thylsilyl)ethoxy)methyl)-3H- imidazo[4,5-b]pyridine-2, 6-diamine (150 mg, 42%) as a yellow solid. LCMS (acidic 5 min): 3.70 min [MH] + =566.2.

Step 7: (Intermediate A) A/6-(2-chloropyrimidin-4-yl)-N6,3-dimethyl-N2-(4-

(trifluoromethoxy)phenyl)-N2-((2-(trimethylsilyl)ethoxy)m ethyl)-3H-imidazo[4,5-b] pyridine-2, 6- diamine

To a solution of A/6-(2-chloropyrimidin-4-yl)-3-methyl-N2-(4-(trifluoromethox y)phenyl)-N2-((2- (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine (150 mg, 0.26 mmol) in DMSO (10 mL) were added CH 3 I (56 mg, 0.4 mmol) and Cs 2 CC> 3 (254 g, 0.78 mmol). The resulting mixture was heated to 30 °C for 3 h. and partitioned between water and EtOAc. The organic layer was separated, dried over sodium sulfate and concentrated to give a residue which was purified by column chromatography on silica gel (PE/EtOAC 20:1) to give N6-(2-chloropyrimidin-4-yl)- N6,3-dimethyl-N2-(4-(trifluoromethoxy)phenyl)-N2-((2-(trimet hylsilyl)ethoxy)methyl)-3H- imidazo[4,5-b]pyridine-2, 6-diamine (130 mg, 86%) as a yellow solid. LCMS (acidic 5 min): 3.74 min [MH] + =580.1.

Synthesis of intermediate B Step 1 : N-methyl-3,5-dinitropyridin-2-amine

A solution of 2-chloro-3,5-dinitropyridine (10 g, 49 mmol), methanamine in methanol solution (20 ml_, 200 mmol,) in MeOH (40 ml.) was stirred for 5 hours at room temperature and then concentrated under reduced pressure to give the desired product (9.7 g, 99%) as a yellow solid. LCMS (acidic 5 min): 1 .51 min [MH] + =199.1 .

Step 2: N2-methyl-5-nitropyridine-2, 3-diamine

To a solution of N-methyl-3,5-dinitropyridin-2-amine (9.0 g, 45 mmol) in EtOH (45 ml.) and H2O (10 ml.) was added ammonium sulfide (13.6 g, 200 mmol). The mixture was stirred for 5 hours at room temperature and the solvent was evaporated under reduced pressure. The residue was diluted with ethyl acetate (100 ml.) and saturated NH CI solution (50 ml_). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure to give the desired product (6.2 g, 80%) as a brown solid. LCMS (acidic 5 min): 0.39 min [MH] + =169.1.

Step 3: 3-methyl-6-nitro-1 ,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one

To a solution of N2-methyl-5-nitropyridine-2, 3-diamine (4.8 g, 28.57 mmol) in THF (20 mL) and was added triphosgene (17 g,57.2 mmol). The mixture was stirred for 12 hours at room temperature. A saturated NaHCC>3 solution (30 mL) was added and the resulting mixture was diluted with ethyl acetate (45 mL) and washed with H 2 0 (15 mL). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure to give the desired product (3.8 g, 70%) as a yellow solid. LCMS (acidic 5 min):0.76 min [MH] + =195.0.

Step 4: 2-chloro-3-methyl-6-nitro-3H-imidazo[4,5-b]pyridine (Intermediate B) A solution of 3-methyl-6-nitro-1 H-imidazo[4,5-b]pyridin-2(3H)-one (2.0 g, 10.3 mmol) in POCI3 (30 ml.) was stirred at 120 °C for 8 hours, and then concentrated under reduced pressure. The residue was diluted with saturated NaHCC>3 solution (30 ml.) and ethyl acetate (50 ml_). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate=2:1 ) to give the desired product (600 mg, 27%) as a yellow solid. LCMS (acidic 5 min): 1.24 min [MH] + =213.0.

Used directly without further purification.

Example 1

Step 1 : 2-methyl-5-nitrobenzoyl chloride

To a solution of 2-methyl-5-nitrobenzoic acid (3.0 g, 16.6 mmol) in thionyl chloride (15 ml.) kept under nitrogen atmosphere, was added /V,/V-dimethylformamide (1 drop). The mixture was heated to reflux for 4 h. The mixture was diluted with dichloromethane (20 ml.) and concentrated to dryness to give a white solid (3.3 g, quantitative).

Step 2: N,2-dimethyl-5-nitrobenzamide

To a solution of 2-methyl-5-nitrobenzoyl chloride (1.1 g, 5.5 mmol) in dichloromethane (20 ml.) at 0 °C were added methylamine (411 mg, 6.08 mmol) and triethylamine (1.67 g, 16.6 mmol). The mixture was stirred at room temperature overnight and then washed with water (30 ml.) and brine (20 ml_), dried over sodium sulfate and concentrated to give a white solid (800 mg, 75%). LCMS (acidic, 5 min): 2.58 min [MH]+= 195.0.

Step 3: 5-amino-/V,2-dimethylbenzamide

To a solution of A/,2-dimethyl-5-nitrobenzamide (350 mg, 1.80 mmol) in methanol (20 ml.) were added zinc powder (1.5 g) and saturated ammonium chloride aqueous solution (20 ml_). The mixture was stirred at room temperature overnight. The solid was filtered off and the solvent methanol was removed under reduced pressure to give a residue which was partitioned between water and ethylacetate. The organic phase was separated and washed with water, brine, dried over sodium sulfate and concentrated under reduced pressure to give 5-amino-/V,2- dimethylbenzamide (250 mg, 85%) as a light yellow solid. LCMS (acidic 5 min): 2.96 min [MH] + = 165.2.

Step 4: A/,2-dimethyl-5-((4-(methyl(3-methyl-2-((4-(trifluoromethoxy )phenyl) ((2-

(trimethylsilyl)ethoxy)methyl)amino)-3H-imidazo[4,5-b]pyr idin-6-yl)amino)pyrimidn-2-yl) amino)benzamide

To a solution of intermediate A (130 mg, 0.22 mmol) in 2-propanol (10 mL) were added 5-amino- N,2-dimethylbenzamide (48 mg, 0.22 mmol) and HCI aq (2M, 2 drops). The resulting mixture was heated to 90 °C under nitrogen atmosphere overnight. Upon cooling a precipitate formed and the solid was collected by filtration, washed with diethyl ether and dried under reduced pressure to give the title compound (150 mg, 96%) as a yellow solid. LCMS (acidic 5 min): 2.56 min Step 5: A/,2-dimethyl-5-((4-(methyl(3-methyl-2-((4-(trifluoromethoxy )phenyl)amino) -3H- imidazo[4,5-b]pyridin-6-yl)amino)pyrimidin-2-yl)amino)benzam ide

To a solution of A/,2-dimethyl-5-((4-(methyl(3-methyl-2-((4-(trifluoromethoxy )phenyl)((2- (trimethylsilyl)ethoxy)methyl)amino)-3H-imidazo[4,5-b]pyridi n-6-yl)amino)pyrimidin-2-yl)amino) benzamide (150 mg, 0.21 mmol) in MeOH (10 ml_) was added HCI (6 M,1 ml_). The resulting mixture was heated to 90 °C for 4 h. The solid was collected by filtration, washed with ether and dried to give the title compound (50 mg, 41%) as a yellow solid.

LCMS (acidic 5 min): 2.48 min [MH] + =578.3.

1 HNMR (400 MHz, DMSO-d 6 ): <5 ppm 2.36 (s, 3H), 2.82 (d, J = 4.0 Hz, 3H), 3.63 (s, 3H), 3.88 (s, 3H), 6.00 (br s, 1 H), 7.19-7.40 (m, 1 H), 7.47 (d, J = 8.8 Hz, 2H), 7.50-7.80 (m, 2H), 7.89 (d, J =

1.6 Hz, 1 H), 7.92-8.05 (m, 1 H), 8.12 (d, J = 8.8Hz, 2H), 8.20 (d, J = 2.0 Hz, 1 H), 8.25-8.35 (m, 1 H), 9.80 (s, 1H), 10.70 (s, 1 H).

Example 2

Step 1 : 2-(4-nitro-1 /-/-pyrazol-1-yl)acetamide

A solution of ethyl 2-(4-nitro-1 H-pyrazol-1 -yl)acetate (from example 3, step 1 , 0.3 g, 1 .5mmol) and ammonium hydroxide (15 ml.) in ethanol (10 ml.) was heated to 75 °C over 24 h in a sealed tube. After cooling, the mixture was concentrated under reduce pressure and then diluted with ethyl acetate (20 ml.) and washed with H 2 0 (10 ml_). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to give the desired product (160 mg, 62%) as a white solid. LCMS (acidic 5 min): 0.27 min [MH] + =171 .1 . Step 2: 2-(4-amino-1 H-pyrazol-1-yl)acetamide

To a solution of 2-(4-nitro-1 H-pyrazol-1 -yl)acetamide (160 mg, 0.94 mmol) in MeOH (3 ml.) was added 10% Pd/C (5 mg) and the mixture was stirred under H 2 at RT for 12 h. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was washed with Et 2 0 (2 ml.) to afford the desired product (92.4 mg, 70%) as a yellow solid. LCMS (acidic 5 min): 0.26 min [MH] + =141 .1

Step 2: 2-(4-((4-(methyl(3-methyl-2-((4-(trifluoromethoxy)phenyl)ami no)-3H- imidazo[4,5- b]pyridin-6-yl)amino)pyrimidin-2-yl)amino)-1 H-pyrazol-1 -yl) acetamide

To a solution of intermediate A (50 mg, 0.086 mmol) and 2-(4-amino-1 H-pyrazol-1 -yl) acetamide (12 mg, 0.086 mmol) in iso-propanol (5 ml.) was added concentrated HCI (1 drop). The mixture was stirred at 85 °C overnight. The mixture was concentrated under reduced pressure and the residue purified by prep TLC (DCM/MeOH/ammonia 9:1 :0.1) to give the desired product (12 mg, 25%) as a white solid.

LCMS (acidic 5 min): 2.38 min [MH] + =554.2, [MNa] + =576.1 .

1 H NMR (400 MHz, DMSO-d 6 ) d 9.52 (s, 1 H), 9.36 (s, 1H), 8.09-7.98 (m, 3H), 7.80 (d, J= 6.2 Hz, 1 H), 7.73 (d, J = 1 .8 Hz, 1 H), 7.55-7.11 (m, 6H), 5.72 (s, 1 H), 4.60 (s, 2H), 3.78 (s, 3H), 3.48 (s, 3H).

Example 3 Step 1 : ethyl 2-(4-nitro-1 H-pyrazol-1 -yl)acetate

A mixture of 4-nitro-1 H-pyrazole (11.3 g, 0.1 mol), ethyl 2-bromoacetate (20.0 g, 0.12 mol) and K 2 CC>3 (20.7 g, 0.15 mol) in acetone (100 ml.) was stirred for 3 h at 65 °C. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (PE/EA 2:1) to give the desired product (18.5 g, 93%) as a yellow solid LCMS (acidic 5 min): 0.86 min [MH] + =200.0

Step 2: A/-methyl-2-(4-nitro-1 H-pyrazol-1 -yl)acetamide

Ethyl 2-(4-nitro-1 H-pyrazol-1-yl)acetate (199.0 mg, 1.0 mmol) and CH 3 NH 2 in EtOH (33%, 8 mL) were stirred for 10 h at 75 °C. The mixture was concentrated under reduced pressure and the residue was partitioned between H 2 0 (5 mL) and EtOAc (10 mL). The organic layer was dried over Na 2 S0 4 and concentrated under reduced pressure to give the titled compound (160 mg, 87%) as a yellow solid. LCMS (acidic 5 min): 0.33 min [MNa] + =207.0.

Step 3: 2-(4-amino-1 H-pyrazol-1-yl)-/V-methylacetamide

To a solution of A/-methyl-2-(4-nitro-1 /-/-pyrazol-1 -yl)acetamide (184.0 mg, 1.0 mmol) in methanol (5mL) were added zinc (650.0 mg, 10 mmol) and an aqueous solution of ammonium chloride (5 ml_). The mixture was stirred for 10 h at 65 °C. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was partitioned between H 2 0 (5 ml.) and EtOAc (10 ml_). The organic layer was dried over Na 2 S0 4 and concentrated under reduced pressure and the residue was purified by column chromatography on silica gel (DCM/MeOH 10:1) to afford the titled compound (100 mg, 65%) as a yellow solid. LCMS (acidic 5 min): 0.26 min [MH] + =155.0 Step 4: N-methyl-2-(4-((4-(methyl(3-methyl-2-((4-(trifluoromethoxy)p henyl) amino)-3H- imidazo[4,5-b]pyridin-6-yl)amino)pyrimidin-2-yl)amino)-1 H-pyrazol-1-yl)acetamide

To a solution of Intermediate A (84 mg, 0.14 mmol) and 2-(4-amino-1 H-pyrazol-1-yl)-N- methylacetamide (22.3 mg, 0.14 mmol) in iso-propanol (5 ml.) was added concentrated HCI (1 drop). The mixture was stirred at 85 °C overnight. The mixture was concentrated under reduced pressure and the residue was washed with diethylether to give the desired product (42 mg, 51%) as a white solid.

LCMS (acidic 5 min): 2.32 min [MH] + = 568.2, [MNa] + = 590.2. 1 H NMR (400 MHz, DMSO-de) d 9.47 (s, 1H), 9.02 (s, 1H), 8.03 (m, 3H), 7.91-7.63 (m, 4H), 7.44 (s, 1 H), 7.37 (d, J= 8.7 Hz, 2H), 5.67 (s, 1 H), 4.56 (s, 2H), 3.77 (s, 3H), 3.45 (s, 3H), 2.57 (d, J =

4.3 Hz, 3H).

Example 4

Step 1 : tert-butyl 3-(4-nitro-1 H-pyrazol-1-yl)azetidine-1-carboxylate

To a solution of 4-nitro-1 H-pyrazole (2.26 g, 20 mmol) and tert-butyl 3-hydroxyazetidine-1- carboxylate (3.56 g, 20 mmol) in THF (35 ml.) were added triphenylphosphine (6.28 g, 24 mmol) and DIAD (5.3 g, 26 mmol) dropwise at 0 °C. The mixture was stirred under nitrogen at room temperature overnight, then partitioned between water and EtOAc. The organic was separated and the aqueous layer was extracted with EtOAc. The combined organics were washed with brine, dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (PE/EA 10:1) to give the desired product (3.2 g, 60%) as the white solid. LCMS (acidic 5 min): 2.42 min [M-55] + =213.1 , [MNa] + =291 .1 .

Step 2: tert-butyl 3-(4-amino-1 H-pyrazol-1 -yl)azetidine-1 -carboxylate

To a solution of tert-butyl 3-(4-nitro-1 H-pyrazol-1 -yl)azetidine-1 -carboxylate (200 mg, 0.74 mmol) in methanol (5 mL) was added Pd/C (20 mg) and the mixture was stirred under H 2 at room temperature overnight. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give the desired product (177.6 mg, 100%) as a yellow oil. LCMS (acidic 5 min): 0.49 min [M-55] + =183.1 .

Step 3: tert-butyl 3-(4-((4-(methyl(3-methyl-2-((4-(trifluoromethoxy)phenyl) ((2- (trimethylsilyl)ethoxy)methyl)amino)-3H-imidazo[4,5-b]pyridi n-6-yl)amino) pyrimidin-2-yl)amino)- 1 H-pyrazol-1 -yl)azetidine-1 -carboxylate

To a solution of tert-butyl 3-(4-amino-1 H-pyrazol-1 -yl)azetidine-1 -carboxylate (41 mg, 0.17 mmol) and Intermediate A (100 mg, 0.17 mmol) in 1 ,4-dioxane (10 ml.) were added cesium carbonate (168.1 mg, 0.516 mmol), Xantphos (20 mg, 0.0344 mmol) and Pd 2 (dba) 2 (16 mg, 0.017 mmol). The mixture was stirred at 110 °C under ISh overnight. The mixture was filtered and the filtrate was concentrated under reduced pressure. The crude residue was purified by prep TLC (DCM/MeOH 8:1) to give the desired product (28 mg, 21%) as a yellow oil. LCMS (acidic 5 min): 3.39 min [MH] + =782.3.

Step 4: N6-(2-((1-(azetidin-3-yl)-1 H-pyrazol-4-yl)amino)pyrimidin-4-yl)- N6,3-dimethyl-N2-(4- (trifluoromethoxy)phenyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine A solution of tert-butyl 3-(4-(4-(methyl(3-methyl-2-((4-(trifluoromethoxy)phenyl) ((2- (trimethylsilyl)ethoxy)methyl)amino)-3H-imidazo[4,5-b]pyridi n-6-yl)amino) pyrimidin-2-ylamino)- 1 H-pyrazol-1-yl)azetidine-1 -carboxylate (28 mg, 0.036 mmol) in HCI in EtOH solution (3 M, 5 ml.) was stirred at RT overnight. The mixture was partitioned between water (10 ml.) and ethyl acetate (10 ml_). The organic phase was separated, washed with brine, dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified by prep TLC (DCM/MeOH/NH 3 8:1 :0.1) to give the desired product (3 mg, 15%) as a brown solid.

LCMS (acidic 5 min): 2.18 min [MH] + =552.1 .

1 H NMR (400 MHz, MeOD) d 8.10 (d, J= 2.0 Hz, 1 H), 7.96 (s, 1 H), 7.82 (m, 3H), 7.66 (d, J= 2.0 Hz, 2H), 7.31 (d, J = 8.6 Hz, 2H), 5.99 (br s, 1 H), 5.40 (br s, 1 H), 4.55 (d, J = 7.0 Hz, 4H), 3.85 (s, 3H), 3.66 (s, 3H).

Example 5

Step 1 : tert-butyl 2-(4-nitro-1 H-pyrazol-1 -yl)ethylcarbamate

To a solution of 4-nitro-1 H-pyrazole (2.3 g, 20 mmol) and tert-butyl 2-hydroxyethylcarbamate (3.2 g, 20 mmol) in THF (30 ml.) were added triphenylphosphine (6.28 g, 24 mmol) followed by DIAD (5.3 g, 26 mmol) dropwise at 0 °C. The mixture was stirred under nitrogen at room temperature overnight. Water was added and the solution was extracted with ethyl acetate (3 x). The combined organics were washed with brine, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EA 10:1) to give the desired product (5 g, 98%) as a white solid. LCMS (acidic 5 min): 2.15 min [M-55]+=201 .1 , [MNa]+=279.1 . Step 2: tert-butyl (2-(4-amino-1 H-pyrazol-1 -yl)ethyl)carbamate

To a solution of tert-butyl 2-(4-nitro-1 H-pyrazol-1 -yl)ethylcarbamate (2.1 g, 8.2 mmol) in methanol (20 ml.) was added Pd/C (200 mg) and stirred under H 2 at room temperature overnight. The reaction mixture was filtered and the filtrate was concentrated to give the desired product (1.85 g, 100%) as a black oil. LCMS (acidic 5 min): 0.40 min [MH]+=227.1 , [MNa]+=249.1 .

Step 3: tert-butyl (2-(4-((4-(methyl(3-methyl-2-(1-(4-(trifluoromethoxy)phenyl) - 2-(2- (trimethylsilyl)ethoxy)ethyl)-3H-imidazo[4,5-b]pyridin-6-yl) amino) pyrimidin-2-yl)amino)-1 H- pyrazol-1 -yl)ethyl)carbamate

To a solution of tert-butyl 2-(4-amino-1 H-pyrazol-1-yl)ethylcarbamate (39 mg, 0.17 mmol) and intermediate A (100 mg, 0.17 mmol) in 1 ,4-dioxane (10 ml.) were added cesium carbonate (168.1 mg, 0.516 mmol), Xantphos (20 mg, 0.0344 mmol) and Pd 2 (dba) 3 (16 mg, 0.017 mmol). The mixture was stirred at 110 °C under nitrogen overnight. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by prep TLC (DCM/MeOH/ammonia 10:1 :0.1 ) to give the desired product (36 mg, 27%) as a yellow oil. LCMS (acidic 5 min): 3.24 min [MH] + =770.3.

Step 4: N6-(2-((1-(2-aminoethyl)-1 H-pyrazol-4-yl)amino)pyrimidin-4-yl)-N6,3- dimethyl-N2-(4- (trifluoromethoxy)phenyl)-3H-imidazo[4,5-b]pyridine-2,6- diamine

A solution of tert-butyl 2-(4-(4-(methyl(3-methyl-2-((4-(trifluoromethoxy)phenyl) ((2- (trimethylsilyl)ethoxy)methyl)amino)-3H-imidazo[4,5-b]pyridi n-6-yl)amino) pyrimidin-2-ylamino)- 1 H-pyrazol-1-yl)ethylcarbamate (36 mg, 0.047 mmol) in a solution of HCI in EtOH (3 M, 5 ml.) was stirred at room temperature overnight. The mixture was partitioned between water and ethyl acetate. The organic phase was separated, washed with brine, dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified by pre-HPLC to give the desired product (5.5 mg, 22%) as a brown solid as a TFA salt.

LCMS (acidic 5 min): 2.15 min [MH] + =540.2.

1 H NMR (400 MHz, MeOD) d 8.07 (d, J = 2.0 Hz, 1 H), 7.79 (m, 3H), 7.63 (m, 2H), 7.30 (m, 3H), 5.94 (s, 1 H), 4.37 (br s, 2H), 3.83 (s, 3H), 3.60 (s, 3H). 3.43 (t, J= 5.2 Hz, 2H).

Example 6

Step 1 : A/,/V-dimethyl-2-(4-nitro-1 H-pyrazol-1 -yl)acetamide

To a solution of ethyl 2-(4-nitro-1 H-pyrazol-1-yl)acetate (from example 3, step 1 , 530 mg, 2.66 mmol) in EtOH (10 ml.) was added dimethylamine in ethanol (30%, 800 mg, 5.32 mmol). The mixture was stirred at 100 °C for 20 h in a sealed tube. The mixture was concentrated under reduced pressure and the residue was purified by column chromatography on silica gel (PE/EtOAc 1 :1) to afford the title compound (470 mg, 89%) as a yellow solid. LCMS (acidic 5 min): 0.34 min [MH] + =199.0.

Step 2: 2-(4-amino-1 H-pyrazol-1-yl)-/V,/V-dimethylacetamide

To a solution of A/,/V-dimethyl-2-(4-nitro-1 H-pyrazol-1 -yl)acetamide (100 mg, 0.5 mmol) in THF (5 mL) was added Pd/C (10 mg). The mixture was stirred at rt for 20 h under H 2 atmosphere. Pd/C was filtered off and the filtrate was concentrated under reduced pressure to give the title compound (81 mg, 96%) as a yellow solid. LCMS (acidic 5 min): 0.25 min [MH] + =169.0.

Step 3: /V,/V-dimethyl-2-(4-((4-(methyl(3-methyl-2-((4-(trifluoromet hoxy)phenyl) amino)-3H- imidazo[4,5-b]pyridin-6-yl)amino)pyrimidin-2-yl)amino)-1 H-pyrazol-1 -yl) acetamide

To a solution of intermediate A (100 mg, 0.17 mmol) and 2-(4-amino-1 H-pyrazol-1 -yl)-N,N- dimethylacetamide (29 mg, 0.17 mmol) in iso-propanol (5 ml.) was added concentrated HCI (2 drops). The mixture was stirred at 85 °C overnight. The mixture was concentrated under reduced pressure and the residue was washed with diethyl ether to give the desired product (66 mg, 66%) as a white solid.

LCMS (acidic 5 min): 2.32 min [MH] + =582.2.

1 H NMR (400 MHz, DMSO-d 6 ) d 9.47 (s, 1 H), 9.00 (s, 1H), 8.11-7.96 (m, 4H), 7.82 (d, J= 5.9 Hz, 1 H), 7.71 (d, J = 2.1 Hz, 1 H), 7.50-7.33 (m, 3H), 5.66 (br s, 1 H), 4.90 (s, 2H), 3.78 (s, 3H), 3.45 (s, 3H), 2.97 (s, 3H), 2.80 (s, 3H).

Example 7

Step 1 : 1-(azetidin-3-yl)-4-nitro-1 H-pyrazole

A mixture of tert-butyl 3-(4-nitro-1 H-pyrazol-1 -yl)azetidine-1 -carboxylate (from example 4, step 1 , 500 mg, 1.86 mmol) in HCI in EtOH solution (30%, 10 mL) was stirred at room temperature overnight. The mixture was partitioned between water (10 mL) and ethyl acetate (10 mL). The organic phase was removed, washed with brine, dried over sodium sulfate and concentrated under reduced pressure to give the desired product (313 mg, 100%) as a yellow oil. LCMS (acidic 5 min): 0.27 min [MH] + =169.1

Step 2: 1-(1-methylazetidin-3-yl)-4-nitro-1 H-pyrazole

To a solution of 1 -(azetidin-3-yl)-4-nitro-1 H-pyrazole (313 mg, 1.86 mmol) in methanol (15 ml.) were added paraformaldehyde (223.6 mg, 7.45 mmol), sodium cyanoborohydride (234 mg, 3.73 mmol) and acetic acid (1 drop). The mixture was stirred at room temperature overnight. The mixture was partitioned between water (10 ml.) and ethyl acetate (10 ml_). The organic phase was removed, washed with brine, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (DCM/MeOH 40:1 ) to give the desired product (150 mg, 44%) as yellow oil. LCMS (acidic 5 min): 0.27 min [MH] + =183.0.

Step 3: 1-(1-methylazetidin-3-yl)-1 H-pyrazol-4-amine

To a solution of 1-(1-methylazetidin-3-yl)-4-nitro-1 H-pyrazole (150 mg, 0.82 mmol) in methanol (10 mL) was added Pd/C (20 mg) and the mixture was stirred under H 2 at room temperature for 2 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give the desired product (125.3 mg, 100%) as a yellow oil. LCMS (acidic 5 min): 0.42 min [MH] + =153.1 .

Step 4: N6,3-dimethyl-N6-(2-((1-(1-methylazetidin-3-yl)-1 H-pyrazol-4-yl) amino)pyrimidin-4-yl)- N2-(4-(trifluoromethoxy)phenyl)-3H-imidazo[4,5-b] pyridine-2, 6-diamine

To a solution of 1 -(1-methylazetidin-3-yl)-1 H-pyrazol-4-amine (52 mg, 0.34 mmol) and intermediate A (200 mg, 0.34 mmol) in iso-propanol (10 ml.) was added concentrated HCI (2 drops). The mixture was stirred at 85 °C overnight. The mixture was concentrated under reduced pressure and the residue was purified by prep HPLC to give the desired product (4 mg, 2%) as a brown solid.

LCMS (acidic 5 min): 2.14 min [MH] + =566.2

1 H NMR (400 MHz, MeOD) d 8.10 (d, J= 2.1 Hz, 1 H), 7.94-7.63 (m, 6H), 7.30 (d, J= 8.7 Hz, 2H), 6.01 (br s, 1 H), 5.38 (br s, 1 H), 4.55 (br s, 4H), 3.86 (s, 3H), 3.66 (s, 3H), 3.10 (s, 3H).

Example 8

Step 1 : 1 H-pyrazol-4-amine

To a solution of 4-nitro-1 H-pyrazole (2 g, 17.7 mmol) in methanol (20 mL) was added Pd/C (200 mg) and the resulting mixture was stirred under H 2 at room temperature overnight. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to afford the desired product (1 .8 g, 100%) as a black oil. LCMS (acidic 5 min): 0.64 min [MH] + =84.0

Step 2: N6-(2-((1 H-pyrazol-4-yl)amino)pyrimidin-4-yl)-N6,3-dimethyl-N2-(4- (trifluoromethoxy)phenyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine

A mixture of 1 H-pyrazol-4-amine (41 mg, 0.17 mmol) and intermediate A (200 mg, 0.34 mmol) in iso-propanol (5 mL) was stirred at 85 °C overnight. The mixture was concentrated under reduced pressure and the residue was purified by prep TLC (DCM/MeOH 10:1 ) to give the desired product (28 mg, 21%) as a white solid.

LCMS (acidic 5 min): 2.38 min [MH] + =497.1

1 H NMR (400 MHz, DMSO-d 6 ) d 12.51 (s, 1 H), 9.57 (s, 1H), 8.05 (m, 3H), 7.86-7.51 (m, 4H), 7.37 (d, J= 8.7 Hz, 2H), 5.75 (br s, 1 H), 3.79 (s, 3H), 3.51 (s, 3H). Example 9

Step 1 : 3,5-dinitro-N-(oxetan-3-yl)pyridin-2-amine To a solution of 2-chloro-3,5-dinitropyridine (4 g, 20 mmol) in MeOH (30 ml_) was added oxetan- 3-amine (1.7 g, 20 mmol), and the mixture was stirred at room temperature under nitrogen overnight. A precipitated solid formed and was collected by filtration to give the desired product (3.9 g, 81%) as a white solid. LCMS (acidic 5 min): 1.47 min [MH] + =241.1.

Step 2: 5-nitro-N2-(oxetan-3-yl)pyridine-2, 3-diamine

To a solution of 3,5-dinitro-N-(oxetan-3-yl)pyridin-2-amine (2 g, 8.5 mmol) in MeOH (20 ml_) was added (NH^S (2.4 g, 43 mmol). The reaction mixture was stirred at 60 °C under nitrogen for 5 h. The reaction mixture was then concentrated under reduced pressure. The residue was diluted with ethyl acetate (20 ml_), and washed with H2O (20 ml_). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAc 5:1) to give the desired product (600 mg, 33%) as a yellow solid. LCMS (acidic 5 min): 0.79 min [MH] + =211.1.

Step 3: 6-nitro-3-(oxetan-3-yl)-N-(4-(trifluoromethoxy)phenyl)-3H-im idazo [4,5-b]pyridin-2-amine

To a solution of 5-nitro-N2-(oxetan-3-yl)pyridine-2, 3-diamine (600 mg, 2.8 mmol) in CH3CN (15 ml_) were added 1-isocyanato-4-(trifluoromethoxy) benzene (810 mg, 3.7 mmol) and TEA (862 mg, 8.5 mmol). The resulting mixture was stirred at RT under nitrogen for 2 h. DBU (864 mg, 5.7 mmol) and HATU (1.6 g, 4.2 mmol) were added. The mixture was stirred at 65 °C overnight. The mixture was concentrated under reduced pressure and the residue was partitioned between EtOAc and H2O. The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAc 20:1) to afford the title product (400 mg, 36%) as a yellow solid. LCMS (acidic 5 min): 3.17 min [MH] + =396.0.

Step 4: 6-nitro-3-(oxetan-3-yl)-N-(4-(trifluoromethoxy)phenyl)-N-((2 - (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridin-2-am ine

To a solution of 6-nitro-3-(oxetan-3-yl)-N-(4-(trifluoromethoxy)phenyl) -3H-imidazo[4,5-b]pyridin- 2-amine (400 mg, 0.8 mmol) in THF (10 ml_) was added NaH (96 mg, 2.4 mmol) at 0 °C and the solution was stirred for 30 min, and then SEMCI (201 mg, 1.2 mmol) was added. The reaction mixture was stirred at RT for 3 h. The reaction mixture was then concentrated under reduced pressure and the residue was diluted with ethyl acetate (25 ml_), and washed with H2O (25 ml_). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAc 20:1) to give the desired product (400 mg, 75%) as a yellow solid. LCMS (acidic 5 min): 3.71 min [MH] + =526.1. Step 5: 3-(oxetan-3-yl)-N2-(4-(trifluoromethoxy)phenyl)-N2-((2-(trim ethylsilyl) ethoxy)methyl)-3H- imidazo[4,5-b]pyridine-2, 6-diamine

To a solution of 6-nitro-3-(oxetan-3-yl)-N-(4-(trifluoro ethoxy)phenyl)- N-((2- (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridin-2-am ine (400 g, 0.76 mmol) in MeOH (10 ml_) was added zinc (297 mg, 4.6 mmol) and NhUCI aq (10 ml_). The reaction mixture was stirred at 60 °C under nitrogen overnight. The reaction mixture was concentrated under reduced pressure and the residue was diluted with ethyl acetate (10 ml_), and washed with H2O (10 ml_). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAc 5:1) to give the desired product (300 mg, 79%) as a yellow solid. LCMS (acidic 5 min): 3.05 min [MH] + =496.2.

Step 6: N6-(2-chloropyrimidin-4-yl)-3-(oxetan-3-yl)-N2-(4-(trifluoro methoxy) phenyl)-N2-((2- (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2,6 - diamine

To a solution of 3-(oxetan-3-yl)-N2-(4-(trifluoromethoxy)phenyl)-N2- ((2- (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine (300 mg, 0.6 mmol) in 2- propanol (10 ml_) were added 2,4-dichloropyrimidine (134 mg, 0.9 mmol) and DIEA (230 mg, 1.8 mmol). The resulting mixture was heated to 80 °C under nitrogen overnight, cooled to RT and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAC 10:1) to give the desired product (300 mg, 82%) as a yellow solid. LCMS (acidic 5 min): 3.67 min [MH] + =608.2. Step 7: N6-(2-chloropyrimidin-4-yl)-N6-methyl-3-(oxetan-3-yl)-N2-(4- (trifluoromethoxy)phenyl)- N2-((2-(trimethylsilyl)ethoxy)methyl)-3H-imidazo [4, 5-b]pyridine-2, 6-diamine

To a solution of N6-(2-chloropyrimidin-4-yl)-3-(oxetan-3-yl)-N2-(4- (trifluoromethoxy)phenyl)-N2- ((2-(trimethylsilyl)ethoxy)methyl)-3H-imidazo [4, 5-b]pyridine-2, 6-diamine (300 g, 0.5 mmol) in DMSO (10 ml_) was added CH 3 I (85 mg, 0.6 mmol) and Cs 2 CC> 3 (478 mg, 1.5 mmol). The mixture was warmed to 30 °C for 3 h. The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAC 20:1) to give the desired product (180 mg, 59%) as a yellow solid. LCMS (acidic 5 min): 3.72 min [MH] + =622.2.

Step 8: 2-(4-((4-(methyl(3-(oxetan-3-yl)-2-((4-(trifluoromethoxy)phe nyl) ((2- (trimethylsilyl)ethoxy)methyl)amino)-3H-imidazo[4,5-b]pyridi n-6-yl)amino) pyrimidin-2-yl)amino)- 1 H-pyrazol-1 -yl)acetamide

To a solution of N6-(2-chloropyrimidin-4-yl)-N6-methyl-3-(oxetan-3-yl)- N2-(4- (trifluoromethoxy)phenyl)-N2-((2-(trimethylsilyl)ethoxy)meth yl)-3H- imidazo[4,5-b]pyridine-2,6- diamine (180 mg, 0.29 mmol) in 2-propanol (10 ml_) were added 2-(4-amino-1 H-pyrazol-1 - yl)acetamide (49 mg, 0.35 mmol) and cone HCI (2 drops). The resulting mixture was heated to 80 °C under nitrogen overnight. The solids were collected by filtration, washed with diethylether and dried under reduced pressure to give the desired product (110 mg, 58%) as a yellow solid. LCMS (acidic 5 min): 2.91 min [MH] + =726.2. Step 9: 2-(4-((4-(methyl(3-(oxetan-3-yl)-2-((4-(trifluoromethoxy)phe nyl) amino)-3H-imidazo[4,5- b]pyridin-6-yl)amino)pyrimidin-2-yl)amino)-1 H-pyrazol-1-yl)acetamide

To a solution of 2-(4-(4-(methyl(3-(oxetan-3-yl)-2-((4-(trifluoromethoxy) phenyl)((2- (trimethylsilyl)ethoxy)methyl)amino)-3H-imidazo[4,5-b]pyridi n-6-yl) amino)pyrimidin-2-ylamino)- 1 H-pyrazol-1-yl)acetamide (65 mg, 0.1 mmol) in MeOH (10 ml_) was added aqueous HCI (5 M, 1 ml_). The resulting mixture was heated to 90 °C overnight. The reaction mixture was concentrated under reduced pressure and the residue was purified by prep TLC (PE/EtOAc 2:1) to give 2-(4- ((4-((3-(1-chloro-3-hydroxypropan-2-yl)-2-((4-(trifluorometh oxy)phenyl)amino)-3H-imidazo[4,5- b]pyridin-6-yl)(methyl)amino) pyrimidin-2-yl) amino)-1 H-pyrazol-1 -yl) acetamide (22 mg, 37%) as a yellow solid. LCMS (acidic 5 min): 2.56 min [MH] + = 632.2.

To a solution of 2-(4-(4-((3-(1-chloro-3-hydroxypropan-2-yl)-2- (4-(trifluoromethoxy)phenylamino)- 3H-imidazo[4,5-b]pyridin-6-yl)(methyl) amino)pyrimidin-2-ylamino)-1H-pyrazol-1-yl)acetamide (22 mg, 0.034 mmol) in DMSO (5 ml_) was added TEA (14 mg, 0.14 mmol). The resulting mixture was heated to 90 °C for overnight. The reaction mixture was concentrated under reduced pressure and the residue was purified by prep TLC (PE/EtOAc 1 :1) to give the desired product (2.5 mg, 12%) as a white solid.

LCMS (acidic 5 min): 2.31 min [MH] + =596.2.

1 H NMR (400 MHz, MeOD) d 8.03 (d, J = 2.0 Hz, 1 H), 7.94-7.91 (m, 3H), 7.72 (d, J= 2.0 Hz, 1 H), 7.70 (br s, 1 H), 7.48 (br s, 1 H), 7.42 (d, J = 8.4 Hz, 2H), 6.00 (br s, 1 H), 5.09-5.01 (m, 2H), 4.80 (s, 2H), 4.62-4.56 (m, 1 H), 4.38-4.31 (m, 1 H), 4.00-3.94 (m, 1H), 3.56 (s, 3H).

Example 10

Step 1: 2-(1,3-dihydroxypropan-2-yl)isoindoline-1,3-dione 2-aminopropane-1,3-diol (5.0 g, 54.88 mml) and isobenzofuran-1,3-dione (8.13 g, 54.88 mmol) were dissolved in DMF (50 ml_). The resulting mixture was stirred at 95 °C for 5 h under N2. After cooling to RT, the mixture was diluted with H2O and extracted with EtOAc (2 x). The combined organic layers were washed with H2O, brine, dried over Na2SC>4 and concentrated under reduced pressure. The residue was washed with PE/EtOAc (2/1 v/v) to afford the title compound (4.2 g, 35%) as a white solid. LCMS (acidic 5 min): 0.43 min [MH]+= 222.1.

Step 2: 2-(2-(bromomethyl)-1 ,3-dioxan-5-yl)isoindoline-1 ,3-dione

A 500 mL flask were charged with 2-(1,3-dihydroxypropan-2-yl)isoindoline-1,3-dione (4.0 g, 18.08 mmol), 2-bromo-1,1-diethoxyethane (4.3 g, 21.7 mmol), p-Toluenesulfonic acid monohydrate (0.69 g, 3.6 mmol) and toluene (120 mL). The mixture was heated to 110 °C and stirred overnight under N2. After cooling, the reaction mixture was diluted with saturated aq. NaHCCh (50 mL) and brine (50 mL). The organic layer was dried over Na2SC>4, filtered and evaporated to dryness. The residue was re-crystallized from ethyl acetate to give 2-(2-(bromomethyl)-1,3-dioxan-5- yl)isoindoline-1,3-dione (2.8 g, 47%) as a white solid. LCMS (acidic 5 min): 2.59 min [MH]+= 326.1/328.1. 1 H NMR (400 MHz, DMSO) d 7.85 (m, 4H), 4.83 (t, J= 4.0 Hz, 1 H), 4.35-4.24 (m, 3H), 4.15-4.04 (m, 2H), 3.50 (d, J = 4.0 Hz, 2H).

Step 3: 2-((2-((4-nitro-1 H-pyrazol-1-yl)methyl)-1,3-dioxan-5-yl)carbamoyl) benzoic acid

2-(2-(bromomethyl)-1,3-dioxan-5-yl)isoindoline-1,3-dione (450 mg, 1.38 mmol), 4-nitro-1 H- pyrazole (164 mg, 1.45 mmol) and CS2CO3 (674 mg, 2.07 mmol) were suspended in CH3CN (15 ml_). The resulting mixture was stirred at 88 °C under N2 overnight. After cooling to RT, the mixture was diluted with H2O, and washed with EtOAc. The aqueous layer was adjusted to pH 2~3 with an HCI solution (3 M). The precipitate was collected by filtration, and dried to afford the title compound (370 mg, 71%) as a grey solid. LCMS (acidic 5 min): 1.18 min [MH]+= 377.1.

Step 4: 2-(2-((4-nitro-1 H-pyrazol-1-yl)methyl)-1,3-dioxan-5-yl)isoindoline -1,3-dione

2-((2-((4-nitro-1 H-pyrazol-1-yl)methyl)-1,3-dioxan-5-yl)carbamoyl)benzoic acid (150 mg, 0.39 mol) was dissolved in EtOH (10 ml_). The solution was refluxed at 88 °C for 20 h under N2. After cooling, the mixture was concentrated under reduced pressure and the residue was purified by column chromatography on silica gel (DCM/PE 3:1 to C^Ch/MeOH 100:1) to afford the titled compound (41 mg, 29 %) as a white solid. LCMS (acidic 5 min): 2.46 min [MH]+= 358.9.

1 H NMR (400 MHz, DMSO) d 8.86 (s, 1 H), 8.29 (s, 1 H), 7.93-7.79 (m, 4H), 5.07 (t, J = 4.7 Hz, 1 H), 4.39 (d, J = 4.7 Hz, 2H), 4.33-4.20 (m, 3H), 4.14-4.03 (m, 2H).

Step 5: 2-(2-((4-amino-1 H-pyrazol-1-yl)methyl)-1 ,3-dioxan-5-yl)isoindoline -1 ,3-dione

To a solution of 2-(2-((4-nitro-1H-pyrazol-1-yl)methyl)-1,3-dioxan-5-yl) isoindoline-1 ,3-dione (40 mg, 0.11 mmol) in MeOH (5 ml_) was added Pd/C (10% wet, 15 mg). The resulting mixture was stirred at 38 °C under H2 overnight. After cooling to RT, Pd/C was filtered out and the filtrate was concentrated under reduced pressure to afford the title compound (39 g, 100 %) as a yellow oil, which was used in the next step without further purification. LCMS (acidic 5 min): 0.65 min [MH]+= 329.0.

Step 6: 2-(2-((4-((4-(methyl(3-methyl-2-((4-(trifluoromethoxy)phenyl )amino)- 3H-imidazo[4,5- b]pyridin-6-yl)amino)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl) methyl)-1 ,3-dioxan-5-yl)isoindoline- 1 ,3-dione

To a mixture of 2-(2-((4-amino-1 H-pyrazol-1-yl)methyl)-1,3-dioxan-5-yl)isoindoline-1 ,3-dione (53 mg, 0.16 mmol) and intermediate A (94 mg, 0.16 mmol) in isopropanol (5 ml_) was added cone. HCI (2 drops). The resulting mixture was stirred at 88 °C under N2 for 16 h. After cooling to RT, the solvent was removed under reduced pressure, and the residue was purified by prep TLC (DCM/MeOH 10:1) to give the title compound (30 mg, 25%) as a light-yellow solid. LCMS (acidic 5 min): 2.64 min [MH] + = 742.2.

Step 7: N6-(2-((1-((5-amino-1 ,3-dioxan-2-yl)methyl)-1 H-pyrazol-4-yl)amino) pyrimidin-4-yl)-N6,3- dimethyl-N2-(4-(trifluoromethoxy)phenyl)-3H-imidazo [4, 5-b]pyridine-2, 6-diamine

2-(2-((4-((4-(methyl(3-methyl-2-((4-(trifluoromethoxy)phe nyl)amino)-3H- imidazo[4,5-b]pyridin-6- yl)amino)pyrimidin-2-yl)amino)-1 H-pyrazol-1-yl)methyl) -1 ,3-dioxan-5-yl)isoindoline-1,3-dione (30 mg, 0.04 mmol) was suspended in CH 3 NH 2 (30 % in H2O, 7 mL), and the resulting mixture was stirred at 65 °C in for 1 h. After cooling to RT, the reaction mixture was evaporated under reduced pressure, and the residue was purified by prep HPLC to give the title compound (5 mg, 20 %) as a yellow oil as a mono TFA salt.

LCMS (acidic 5 min): 2.19 min [MH] + = 612.2.

1 H NMR (400 MHz, DMSO-d 6 ) d 10.45 (s, 1H), 9.59 (s, 1 H), 8.11 (d, J = 1.8 Hz, 1 H), 8.06-8.00 (m, 4H), 7.82 (s, 1H), 7.38 (d, J= 8.7 Hz, 2H), 4.96-4.67 (m, 1H), 4.47-3.95 (m, 4H), 3.79 (s, 3H), 3.57 (s, 3H), 2.02-1.05 (m, 2H), 1.47-1.43 (m, 1 H). Other 5 protons were covered.

Example 11 „ Boc

Step 1 : tert-butyl 2-(4-nitro-1 H-pyrazol-1-yl)ethylcarbamate

To a solution of 4-nitro-1 H-pyrazole (2.3 g, 20 mmol) and tert-butyl 2-hydroxyethylcarbamate (3.2 g, 20 mmol) in THF (30 ml.) were added triphenylphosphine (6.28 g, 24 mmol) followed by DIAD (5.3 g, 26 mmol) dropwise at 0 °C. The mixture was stirred under nitrogen at room temperature overnight. Water was added and the solution was extracted with ethyl acetate (3 x). The combined organics were washed with brine, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EA 10:1) to give the desired product (5 g, 98%) as a white solid. LCMS (acidic 5 min): 2.15 min [M-55] + =201 .1 , [MNa] + =279.1.

Step 2: tert-butyl methyl(2-(4-nitro-1 H-pyrazol-1-yl)ethyl)carbamate

To a solution of tert-butyl 2-(4-nitro-1 H-pyrazol-1-yl)ethylcarbamate (2 g, 7.8 mmol) in N,N- dimethylformamide (20 ml.) were added sodium tert-butoxide (2.25 g, 23.4 mmol) and methyl iodide (2.21 g, 15.6 mmol). The mixture was stirred at 75 °C for 5 hours. Water (20 ml.) was added and the reaction mixture was extracted with ethyl acetate (3 x). The combined organic phases were washed with brine, dried over sodium sulfate and concentrated under reduced pressure to give the desired product (2.1 g, 100%) as a yellow oil. LCMS (acidic 5 min): 2.31 min [MNa] + =293.1

Step 3: tert-butyl (2-(4-amino-1 H-pyrazol-1-yl)ethyl)(methyl)carbamate

To a solution of tert-butyl methyl(2-(4-nitro-1 H-pyrazol-1-yl)ethyl)carbamate (2.1 g, 7.8 mmol) in methanol (20 mL) was added Pd/C (200 mg) and the mixture was stirred under H 2 at room temperature overnight. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give the desired product (1.12 g, 60%) as the black oil. LCMS (acidic 5 min): 0.41 min [MH] + =241.1 , [MNa] + =263.1 .

Step 4: tert-butyl methyl(2-(4-((4-(methyl(3-methyl-2-((4-(trifluoromethoxy) phenyl)((2- (trimethylsilyl)ethoxy)methyl)amino)-3H-imidazo[4,5-b]pyridi n-6-yl) amino)pyrimidin-2-yl)amino)- 1 H-pyrazol-1-yl)ethyl)carbamate

To a solution of tert-butyl 2-(4-amino-1 H-pyrazol-1-yl)ethyl(methyl)carbamate (124.3 mg, 0.52 mmol) and Intermediate A (300 mg, 0.52 mmol) in 1 ,4-dioxane (20 mL) were added cesium carbonate (505.6 mg, 1.55 mmol), Xantphos (60 mg, 0.1 mmol) and Pd 2 (dba)3 (47.4 mg, 0.052 mmol). The mixture was stirred under nitrogen at 110 °C overnight. The mixture was filtered and the filtrate was concentrated under reduced pressure. The crude product was purified by prep HPLC to give the desired product (20 mg, 5%) as a white solid. LCMS (acidic 5 min): 3.30 min [MH] + =784.3, [MNa] + =806.3. Step 5: N6,3-dimethyl-N6-(2-((1-(2-(methylamino)ethyl)-1 H-pyrazol-4-yl)amino) pyrimidin-4-yl)- N2-(4-(trifluoromethoxy)phenyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine

To a solution of tert-butyl methyl(2-(4-(4-(methyl(3-methyl-2- ((4-(trifluoromethoxy)phenyl)((2- (trimethylsilyl)ethoxy)methyl)amino)-3H- imidazo[4,5-b]pyridin-6-yl)amino)pyrimidin-2-ylamino)- 1 H-pyrazol-1 -yl)ethyl) carbamate (20 mg, 0.026 mmol) in hydrochloric acid ethanol solution (3 M, 5 ml.) was stirred at room temperature overnight. The mixture was concentrated under reduced pressure and washed with diethylether to give the desired product (4 mg, 28%) as a brown solid as HCI salt.

LCMS (acidic 5 min): 2.09 min [MH] + =554.3.

1 H NMR (400 MHz, MeOD) d 8.39 (s, 1H), 8.08 (s, 1H), 7.81 (m, 5H), 7.51 (s, 2H), 5.96 (br s, 1 H), 4.57 (br s, 2H), 3.95 (s, 3H), 3.68 (s, 3H), 3.56 (br s, 2H), 2.76 (s, 3H).

Example 12

Step 1 : N,N-dimethyl-2-(4-nitro-1 H-pyrazol-1 -yl)ethan-1-amineTo a solution of 4-nitro-1 H- pyrazole (3 g, 26.5 mmol) and 2-(dimethylamino)ethanol (2.36 g, 26.5 mmol) in THF (40 ml.) was added triphenylphosphine (10.4 g, 39.8 mmol) followed by DIAD (7.8 ml_, 39.8 mmol) dropwise under nitrogen at 0 °C. The mixture was stirred at room temperature overnight, and partitioned between water and ethyl acetate. The organic phase was removed, washed with brine, dried and concentrated under reduced pressure to give the desired product (3 g, 62%) as a yellow solid. LCMS (acidic 5 min): 0.26 min [MH] + =185.1 . Step 2: 1-(2-(dimethylamino)ethyl)-1 H-pyrazol-4-amine

To a solution of N,N-dimethyl-2-(4-nitro-1 H-pyrazol-1 -yl)ethan-1 -amine (2 g, 10.86 mmol) in MeOH (20 ml.) was added Pd/C (200 mg) and the resulting mixture was stirred under H 2 at room temperature for 16h. The reaction mixture was filtered, rinsed with MeOH and the combined filtrates were concentrated to give the desired product (1 .8 g, 100%) as a yellow oil. LCMS (acidic 5 min): 0.25 min [MH] + =155.1. Step 3: N6-(2-((1 -(2-(dimethylamino)ethyl)-1 H-pyrazol-4-yl)amino)pyrimidin- 4-yl)-N6,3-dimethyl- N2-(4-(trifluoromethoxy)phenyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine

To a solution of 1-(2-(dimethylamino)ethyl)-1 H-pyrazol-4-amine (26.6 mg, 0.17 mmol) and Intermediate A (100 mg, 0.17 mmol) in isopropyl alcohol (5 ml.) was added concentrated HCI (1 drop). The mixture was stirred at 85°C overnight. The mixture was concentrated under reduced pressure, and purified by prep TLC (DCM/MeOH=10/1 ) to give the desired product (15 mg, 15%) as a white solid.

LCMS (acidic 5 min): 2.0 min [MH] + =568.3.

1 H NMR (400 MHz, d 6 -DMSO) d 9.79 (s, 1 H), 8.97 (s, 1H), 8.06 - 7.97 (m, 4H), 7.83 (d, J = 5.7 Hz, 1 H), 7.68 (s, 1 H), 7.34 (m, 3H), 5.72 (s, 1 H), 3.93 (s, 2H), 3.79 (s, 3H), 3.44 (s, 3H), 2.07 (s, 6H). 2 H are covered. Example 13

Step 1 : N-methyl-3,5-dinitropyridin-2-amine A solution of 2-chloro-3,5-dinitropyridine (5 g, 24.6 mmol) in MeOH (30 ml.) was added methylamine hydrochloride (1.6 g, 24.6 mmol) and the mixture was stirred at RT under nitrogen overnight. The precipitated solid was collected by filtration to give the desired product (4 g, 83%) as a yellow solid. LCMS (acidic 5 min): 1.48 min [MH] + =199.1 .

Step 2: N2-methyl-5-nitropyridine-2, 3-diamine

To a solution of N-methyl-3,5-dinitropyridin-2-amine (4 g, 20 mmol) in EtOH (20 ml.) was slowly added an aqueous solution of (NhU^S (6.4 g in 20 ml. of H 2 0). The reaction mixture was stirred at 60°C under nitrogen for 3h and then concentrated under reduced pressure. The residue was diluted with ethyl acetate (20 ml_), and washed with H 2 0 (20 ml_). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAc = 20/1 ) to afford the title product (2.8 g, 84%) as a yellow solid. LCMS (acidic 5 min): 0.36 min [MH] + =169.1. Step 3: 3-methyl-6-nitro-N-phenyl-3H-imidazo[4,5-b]pyridin-2-amine

To a solution of N2-methyl-5-nitropyridine-2, 3-diamine (1.0 g, 6 mmol) in CH 3 CN (15 mL) was added isothiocyanatobenzene (973 mg, 7.2 mmol) and TEA (1.8 g, 18 mmol). The mixture was stirred at rt under nitrogen for 2h before the addition of DBU (1 .8 g, 12 mmol) and HATU (3.4 g, 9 mmol). The mixture was stirred at 65°C overnight and then partitioned between EtOAc and H 2 0. The organics were separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAc = 20/1) to afford the title product (1.3 g, 81%) as a yellow solid. LCMS (acidic 5 min): 3.07 min [MH] + =270.0

Step 4: 3-methyl-6-nitro-N-phenyl-N-((2-(trimethylsilyl)ethoxy)methy l)-3H- imidazo[4,5-b]pyridin- 2-amine

To a solution of 3-methyl-6-nitro-N-phenyl-3H-imidazo[4,5-b]pyridin-2-amine (1.3 g, 4.8 mmol) in THF (20 mL) cooled to 0°C was slowly added NaH (576 mg, 14.4 mmol), followed by SEMCI (1.2 g,7.2 mmol). The reaction mixture was stirred at rt for 3h and partitioned between water (25 mL) and EtOAc (25 mL). The organic layer was separated, dried under sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAc = 20/1) to afford the title product (1 .5 g, 78%) as a yellow oil. LCMS (acidic 5 min): 3.86min [MH] + =400.1.

Step 5: 3-methyl-N2-phenyl-N2-((2-(trimethylsilyl)ethoxy)methyl)-3H- imidazo [4,5-b]pyridine-2,6- diamine To a solution of 3-methyl-6-nitro-N-phenyl-N-((2-(trimethylsilyl)ethoxy)methy l)-3H-imidazo[4,5- b]pyridin-2-amine (1.5 g, 3.8 mmol) in MeOH (10 ml.) were added zinc (1.5 g, 22.5mmol) and aqueous NhUCI (10 ml_). The reaction mixture was stirred at 60°C for 16 hours. The mixture was concentrated under reduced pressure and the residue was diluted with ethyl acetate (10 ml_), and washed with H 2 0 (10 ml_). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The crude residue was purified by column chromatography on silica gel (PE/EtOAc = 20/1) to give the desired product (1.1 g, 79%) as yellow oil. LCMS (acidic 5 min): 2.53 min [MH]+=370.0.

Step 6: N6-(2-chloropyrimidin-4-yl)-3-methyl-N2-phenyl-N2-((2-(trime thylsilyl) ethoxy)methyl)- 3H-imidazo[4,5-b]pyridine-2, 6-diamine

To a solution of 3-methyl-N2-phenyl-N2-((2-(trimethylsilyl)ethoxy)methyl)- 3H-imidazo[4,5- b]pyridine-2, 6-diamine (1.1 g, 3 m mol) in 2-propanol (15 ml.) were added 2,4-dichloropyrimidine (670 mg, 4.5 mmol) and DIEA (774mg, 6mmol). The mixture was stirred at 80°C under an inert atmosphere. The mixture was partitioned between water and EtOAc. The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAC=10/1) to give the desired product (1 .1 g, 78%) as yellow oil. LCMS (acidic 5 min): 2.84min [MH] + =482.1 .

Step 7: N6-(2-chloropyrimidin-4-yl)-N6,3-dimethyl-N2-phenyl-N2-((2-

(trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine- 2, 6-diamine

To a solution of N6-(2-chloropyrimidin-4-yl)-3-methyl-N2-phenyl- N2-((2- (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine (1.1 g, 2.3 m mol) in acetone (15 ml.) were added CH 3 I (487 mg, 3.4 mmol) and Cs2CC>3 (2.2 g, 6.9 mmol). The mixture was stirred at RT for 3h and then concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAC= 20/1 ) to give the title product (1 g, 88%) as yellow oil. LCMS (acidic 5 min): 3.54min [MH]+=496.2.

Step 8: 2-(4-((4-(methyl(3-methyl-2-(phenyl((2-(trimethylsilyl)ethox y)methyl) amino)-3H- imidazo[4,5-b]pyridin-6-yl)amino)pyrimidin-2-yl)amino)-1 H- pyrazol-1 -yl)acetamide

To a solution of N6-(2-chloropyrimidin-4-yl)-N6,3-dimethyl-N2-phenyl- N2-((2- (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine (200mg, 0.4 mmol) in 2- propanol (10 ml.) was added 2-(4-amino-1 H- pyrazol-1 -yl)acetamide (68 mg, 0.48 mmol) and HCI (2M, 2 drops). The resulting mixture was heated to 90°C under an inert atmosphere overnight. The solid was collected by filtration, washed with diethyl ether and dried under reduced pressure to afford the title product (200 mg, 83%) as yellow oil. LCMS (acidic 5 min): 2.845min [MH]+=600.3.

Step 9: 2-(4-((4-(methyl(3-methyl-2-(phenylamino)-3H-imidazo[4,5-b]p yridin- 6-yl) amino)pyrimidin-2-yl)amino)-1 H-pyrazol-1 -yl)acetamide

A mixture of 2-(4-((4-(methyl(3-methyl-2-(phenyl((2-(trimethylsilyl)ethox y) methyl)amino)-3H- imidazo[4,5-b]pyridin-6-yl)amino)pyrimidin-2-yl)amino)-1 H- pyrazol-1 -yl)acetamide (150 mg, 0.25 mmol) in 2-propanol (10 mL) was added concentrated HCI (2 drops). The mixture was heated to 115 °C under nitrogen overnight. The solid was collected by filtration, washed with diethyl ether and dried under reduced pressure to give the title product (60 mg, 83%) as a brown solid.

LCMS (acidic 5 min): 3.02 min [MH] + =470.2. 1 H NMR (400 MHz, DMSO-d 6 ) d 9.34 (s, 1 H), 9.00 (br s, 1 H), 8.0 (s, 1 H), 7.94-7.87 (m, 2H), 7.85- 7.78 (m, 1 H), 7.68 (s, 2H), 7.46 (s, 1 H), 7.40-7.26 (m, 3H), 7.20 (s, 1 H), 7.06-6.95 (m, 1 H), 5.65 (br s, 1 H), 4.60 (s, 2H), 3.77 (s, 3H), 3.45 (s, 3H). Example 14 and Example 15

Step 1 : N,N-dimethyl-2-(5-methyl-4-nitro-1 H-pyrazol-1-yl)ethan-1 -amine and N,N-dimethyl-2-(3- methyl-4-nitro-1 H-pyrazol-1 -yl)ethan-1 -amine To a solution of 5-methyl-4-nitro-1 H-pyrazole (3 g, 23.6 mmol) and 2-(dimethylamino)ethanol (2.1 g, 23.6 mmol) in THF (40 mL) was added PPh 3 (9.3 g, 35.4 mmol), followed by DIAD (7.2 ml_, 35.4 mmol) under nitrogen at 0°C. The mixture was then stirred at room temperature overnight. The mixture was partitioned between water and ethyl acetate. The organic phase was washed with brine, dried (MgSC ) and concentrated under reduced pressure to give a mixture of desired products (2 g, 43%) as a yellow solid. LCMS (acidic 5 min): 0.28 min [MH] + =199.1 .

Step 2: 1 -(2-(dimethylamino)ethyl)-5-methyl-1 H-pyrazol-4-amine and 1-(2-(dimethylamino)ethyl)- 3-methyl-1 H-pyrazol-4-amine To a solution of N,N-dimethyl-2-(5-methyl-4-nitro-1 H-pyrazol-1 -yl)ethan-1 -amineand N,N- dimethyl-2-(3-methyl-4-nitro-1 H-pyrazol-1 -yl)ethan-1 -amine (2 g, 10.86 mmol) in methanol (20 mL) was added Pd/C (200 mg) and the mixture was stirred under H 2 at room temperature overnight. The reaction mixture was filtered and filtrate was concentrated to give the mixture of desired products (1.8 g, 100%) as yellow oil. LCMS (acidic 5 min): 0.25 min [MH] + =169.1.

Step 3: N6-(2-((1 -(2-(dimethylamino)ethyl)-5-methyl-1 H-pyrazol-4-yl)amino) pyrimidin-4-yl)-N6,3- dimethyl-N2-(4-(trifluoromethoxy)phenyl)-3H-imidazo[4,5-b]py ridine-2, 6-diamine and N6-(2-((1 - (2-(dimethylamino)ethyl)-3-methyl-1 H-pyrazol-4-yl)amino)pyrimidin-4-yl)-N6,3-dimethyl-N2-(4- (trifluoromethoxy) phenyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine

To a solution of 1 -(2-(dimethylamino)ethyl)-5-methyl-1 H-pyrazol-4-amine and 1-(2- (dimethylamino)ethyl)-3-methyl-1 H-pyrazol-4-amine (58 mg, 0.34 mmol) and intermediate A (200 mg, 0.34 mmol) in isopropanol (5 ml.) was added concentrated hydrochloric acid (1 drop). The mixture was stirred at 85°C overnight. The mixture was concentrated, and purified by prep-TLC (DCM/MeOH=10/1 ) to give N6-(2-((1-(2-(dimethylamino)ethyl)-5-methyl-1 H-pyrazol-4- yl)amino)pyrimidin-4-yl)-N6,3-dimethyl-N2-(4-(trifluorometho xy) phenyl)-3H-imidazo[4,5- b]pyridine-2, 6-diamine (4 mg, 2%) as a brown solid LCMS (acidic 5 min): 2.09 min [MH] + =582.3.

1 H NMR (400 MHz, d 4 -methanol) d 8.07 (d, J= 2.1 Hz, 1 H), 7.84 (d, J = 9.0 Hz, 2H), 7.77 (d, J = 6.2 Hz, 1 H), 7.72 (s, 1 H), 7.65 (d, J = 2.0 Hz, 1 H), 7.33 (d, J = 8.5 Hz, 2H), 5.85 (d, J = 5.9 Hz, 1 H), 4.26 (s, 2H), 3.86 (s, 3H), 3.54 (s, 3H), 3.15 (s, 2H), 2.61 (s, 6H), 2.24 (s, 3H); and N6-(2-((1 -(2- (dimethylamino)ethyl)-3-methyl-1 H-pyrazol-4-yl)amino) pyrimidin-4-yl)-N6,3- dimethyl-N2-(4-(trifluoromethoxy)phenyl)-3H-imidazo[4,5-b]py ridine-2, 6-diamine (5 mg, 3%) as a brown solid.

LCMS (acidic 5 min): 2.12 min [MH] + =582.3.

1 H NMR (400 MHz, MeOD) d 8.01 (d, J = 2.1 Hz, 1 H), 7.80 (d, J = 9.0 Hz, 2H), 7.70 (d, J = 6.1 Hz, 1 H), 7.59 (d, J = 2.0 Hz, 1 H), 7.50 (s, 1 H), 7.30 (d, J = 8.9 Hz, 2H), 5.73 (d, J = 6.1 Hz, 1 H), 4.17 (t, J = 7.2 Hz, 2H), 3.83 (s, 3H), 3.46 (s, 3H), 2.74 (t, J = 7.2 Hz, 2H), 2.31 (s, 6H), 2.23 (s, 3H).

Example 16 Step 1 :

To a solution of 2,4,5-trichloropyrimidine (160 mg, 0.88 mmol) in THF (8 ml.) and ethanol (24 ml.) were added NaHC03 (148 mg, 1.76 mmol) and 3-methyl-N2-(4-(trifluoromethoxy)phenyl)-N2-((2- (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine (360 mg, 0.8 mmol) at room temperature. The resulting mixture was refluxed for 6hr. The mixture was filtered, evaporated, then diluted with ethyl acetate (25 ml_). The resulting mixture was washed with H 2 0 (15 ml_), and the organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by prep HPLC to give the desired product (110 mg, 23%) as a yellow oil. LCMS (acidic 5 min): 3.71 min [MH] + =600.1.

Step 2: N6-(2,5-dichloropyrimidin-4-yl)-N6,3-dimethyl-N2-(4-(trifluo romethoxy) phenyl)-N2-((2- (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2,6 - diamine

A mixture of N6-(2,5-dichloropyrimidin-4-yl)-3-methyl-N2-(4-(trifluoromet hoxy) phenyl)-N2-((2- (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2,6 - diamine (110 mg, 0.18 mmol), CH 3 I (26 mg, 0.18 mmol), CS2CO3 (117 mg, 0.36 mmol) in acetone (5 ml.) was stirred at room temperature under nitrogen overnight, and then diluted with ethyl acetate (25 ml.) and washed with H2O (15 ml_). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by Prep TLC (PE/ EtOAc =4:1) to give the desired product (50 mg, 45%) as a yellow oil. LCMS (acidic 5 min): 4.03 min [MH] + =614.1 . Step 3: 2-(4-((5-chloro-4-(methyl(3-methyl-2-((4-(trifluoromethoxy)p henyl) amino)-3H- imidazo[4,5-b]pyridin-6-yl)amino)pyrimidin-2-yl)amino)-1 H-pyrazol-1 -yl)acetamide

The mixture of N6-(2-chloro-5-fluoropyrimidin-4-yl)-N6,3-dimethyl-N2 -(4- (trifluoromethoxy)phenyl)-N2-((2-(trimethylsilyl)ethoxy)meth yl)-3H-imidazo[4,5-b]pyridine-2,6- diamine (40 mg, 0.064 mmol), 2-(4-amino-1 H-pyrazol-1 -yl)acetamide (20 mg, 0.128 mmol), HCI cc (1 drop) in IPA (5 ml.) was stirred at 80°C under nitrogen for 48 hours, and concentrated under reduced pressure. The residue was purified by Prep TLC (PE/ EtOAc =2:1 ) to give the desired product (7 mg, 15%) as a white solid.

LCMS (acidic 5 min): 2.76 min [MH] + =588.1 .

1 H NMR (400 MHz, DMSO-d 6 ) d ppm 9.48 (s, 1H), 9.35 (s, 1 H), 8.03 (d, J = 8.8 Hz, 2H), 7.94 (s, 1 H), 7.91 (d, J = 2.4 Hz, 1 H), 7.86 (s, 1 H),7.65 (d, J = 2.0 Hz, 1 H), 7.54 (s, 1 H), 7.37 (d, J = 8.8

Hz, 2H), 7.35 (s, 1 H), 7.20 (s, 1 H),4.69 (s, 2H), 3.76 (s, 3H), 3.49 (s, 3H).

Example 17 Step 1 : N-methyl-3,5-dinitropyridin-2-amine

To a solution of 2-chloro-3,5-dinitropyridine (5 g, 24.6m mol) in MeOH (30 ml.) was added methylamine hydrochloride (1.6 g, 24.6 mmol) and the resultant mixture was stirred at room temperature under nitrogen overnight. The precipitated solid was collected by filtration to give the desired product (4 g, 83%) as a yellow solid. LCMS (acidic 5 min): 1 .53 min [MH] + =199.1 .

Step 2: N2-methyl-5-nitropyridine-2, 3-diamine

To a solution of N-methyl-3,5-dinitropyridin-2-amine (4 g, 20 mmol) in EtOH (20 ml.) was added (NH ) S (6.4 g, 100 mmol). The reaction mixture was stirred at 60°C under nitrogen for 5h. The reaction mixture was concentrated under reduced pressure. The residue was diluted with ethyl acetate (20 ml_), and washed with H 2 0 (20 ml_). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAc = 20/1 ) to afford the title product (2.8 g, 84%) as a yellow solid. LCMS (acidic 5 min): 0.46 min [MH] + =169.1 .

Step 3: N-cyclopentyl-3-methyl-6-nitro-3H-imidazo[4,5-b]pyridin-2-am ine

To a solution of N2-methyl-5-nitropyridine-2, 3-diamine (1 g, 6 mmol) in MeOH (120 mL) was added isothiocyanatocyclopentane (1.1 g, 9 mmol), TEA (1.8 g, 18 mmol), sulfur (230 mg, 7.2 mmol) and HgO (1.6 g,7.2 mmol). The mixture was stirred at 80°C under nitrogen for 16 hours. The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate=20/1 ) to give the title product (800 mg, 51 %) as a yellow solid. LCMS (acidic 5 min): 3.09 min [MH] + =262.1 .

Step 4: N-cyclopentyl-3-methyl-6-nitro-N-((2-(trimethylsilyl)ethoxy) methyl)-3H- imidazo[4,5- b]pyridin-2-amine

To a solution of N-cyclopentyl-3-methyl-6-nitro-3H-imidazo[4,5-b] pyridin-2-amine (800 mg, 3 mmol) in THF (20 ml.) cooled to 0 °C was added NaH (360 mg, 9 mmol) followed by SEMCI (750 mg, 4.5 mmol). The reaction mixture was stirred at rt for 3h. The mixture was then concentrated under reduced pressure and the residue was diluted with ethyl acetate (25 ml_), and washed with H 2 0 (25 ml_). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAc = 20/1) to give the title product (800 mg, 68%) as a yellow oil. LCMS (acidic 5 min): 3.86min [MH] + =392.2.

Step 5: N2-cyclopentyl-3-methyl-N2-((2-(trimethylsilyl)ethoxy)methyl )-3H-imidazo[4,5-b]pyridine- 2,6-diamine

To a solution of N-cyclopentyl-3-methyl-6-nitro-N-((2-(trimethylsilyl)ethoxy) methyl)-3H- imidazo[4,5-b]pyridin-2-amine (800 mg, 2 mmol) in MeOH (10 ml.) were added zinc (780 mg, 12 mmol) and aqueous saturated NH CI solution (10 ml_). The reaction mixture was stirred at 60°C for 16 hours. The reaction mixture was concentrated and the residue was diluted with ethyl acetate (10 ml_), and washed with H 2 0 (10 ml_). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAc = 20/1) to give the desired product (720 mg, 100%) as a yellow oil. LCMS (acidic 5 min): 3.28 min [MH] + =362.2. Step 6: N6-(2-chloropyrimidin-4-yl)-N2-cyclopentyl-3-methyl-N2-((2-( trimethylsilyl) ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine

To a solution of N2-cyclopentyl-3-methyl-N2-((2-(trimethylsilyl)ethoxy)methyl )-3H-imidazo[4,5- b]pyridine-2, 6-diamine (720 mg, 2 mmol) in 2-propanol (10 ml.) was added 2,4-dichloropyrimidine (357 mg, 2.4 mmol) and DIEA (774 mg, 6 mmol). The mixture was stirred at 80°C overnight. The mixture was partitioned between water and EtOAc and the organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAC=10/1 ) to give the title product (340 mg, 35%) as a yellow oil. LCMS (acidic 5 min): 3.70min [MH] + =474.2.

Step 7: N6-(2-chloropyrimidin-4-yl)-N2-cyclopentyl-N6,3-dimethyl-N2- ((2- (trimethylsilyl) ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine

A solution of N6-(2-chloropyrimidin-4-yl)-N2-cyclopentyl-3-methyl-N2-((2-( trimethylsilyl) ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine (400 mg, 0.60 mmol) in acetone (10 ml.) were added CH 3 I (130 mg, 0.90. mmol) and Cs 2 CC> 3 (678 mg, 1.2 mmol). The resulting mixture was warmed to 30°C and stirred for 3h. The mixture was partitioned between ethyl acetate and water. The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAC=20/1) to give the desired product (300 mg, 99%) as a yellow oil. LCMS (acidic 5 min): 3.74min [MH] + =488.1.

Step 8: 2-(4-((4-((2-(cyclopentylamino)-3-methyl-3H-imidazo[4,5-b]py ridin-6-yl) (methyl) amino)pyrimidin-2-yl)amino)-1 H-pyrazol-1 -yl)acetamide To a solution of N6-(2-chloropyrimidin-4-yl)-N2-cyclopentyl-N6,3-dimethyl-N2- ((2- (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine (300 mg, 0.46 mmol) in 2- propanol (10 ml.) was added 2-(4-amino-1 H-pyrazol-1-yl)acetamide (129 mg, 0.92 mmol) and concentrated HCI (2 drops). The mixture was stirred at 90°C under nitrogen overnight. The solid was collected by filtration, washed with ether and dried under reduced pressure to afford the title product (20 mg, 5%) as a white solid.

LCMS (acidic 5 min): 2.66 min [MH] + =462.2.

1 H NMR (400 MHz, DMSO-d 6 ) d 9.00 (s, 1H), 7.90-7.65 (m, 3H), 7.50-7.35 (m, 2H), 7.35-7.10 (m, 2H), 7.05-6.85 (m, 1 H), 5.60 (br s, 1 H), 4.60 (s, 2H), 4.25 (br s, 1 H), 3.54 (s, 3H), 3.41 (s, 3H), 2.10-1.95 (m, 2H), 1.80-1.77 (m, 2H), 1.77-1.52 (m, 4H).

Example 18 Step 1 : 2-(3-nitro-1 H-pyrazol-1 -yl)acetamide

T o the solution of 3-nitro-1 H-pyrazole (1 g, 8.85 mmol) and 2-bromoacetamide (1 .46 g, 10.6 mmol) in DMF(10 ml.) was added NaH (60% in oil, 425 mg, 17.7 mmol) at 0°C and the mixture was stirred at room temperature for 1 hour. The mixture was partitioned between water and ethyl acetate. The organics were separated, washed with brine, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (DCM/MeOH = 30:1 ) to give the title product (900 mg, 60%) as a white solid. LCMS (acidic 5 min): 0.360 min [M-H] =169.1 . Step 2: 2-(3-amino-1 H-pyrazol-1-yl)acetamide

A mixture of 2-(3-nitro-1 H-pyrazol-1-yl)acetamide (50 mg, 0.294 mmol) and Pd/C (10 mg) in MeOH (10 ml.) was stirred at room temperature under H2 atmosphere overnight. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give the desired amine (30 mg, 73%) as a white solid. LCMS (acidic 5 min): 0.343 min [MH] + =141 .1 .

Step 3: 2-(3-((4-(methyl(3-methyl-2-((4-(trifluoromethoxy)phenyl)ami no)-3H- imidazo[4,5- b]pyridin-6-yl)amino)pyrimidin-2-yl)amino)-1 H-pyrazol-1 -yl) acetamide

To the mixture of 2-(3-amino-1 H-pyrazol-1 -yl)acetamide (30 mg, 0.214 mmol) and intermediate A (124 mg, 0.214 mmol) in isopropanol (10 ml.) was added concentrated HCI (1 drop) and the mixture was stirred at 80°C overnight. The mixture was concentrated under reduced pressure, purified by prep-TLC (DCM/MeOH=/20/1 ) to give the desired target (5 mg, 2.0%) as a brown solid. LCMS (acidic 5 min): 2.39 min [MH] + =554.2.

1 H NMR (400 MHz, CD 3 OD) d ppm 8.23 (d, J = 6.0 Hz, 1 H), 8.00-7.60 (m, 5H), 7.39 (d, J = 8.4 Hz, 2H), 6.19-6.17 (m, 1 H), 6.04-6.01 (m, 1 H), 4.90 (s, 2H), 3.90 (s, 3H), 3.65 (s, 3H).

Example 19 Step 1 : N6-(2-chloro-5-methylpyrimidin-4-yl)-3-methyl-N2-(4-(trifluo romethoxy) phenyl)-N2-((2- (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine

To a solution of 2,4-dichloro-5-methylpyrimidine (68 mg, 0.4 mmol) in n-BuOH (20 ml.) were added DIEA (104 mg, 0.8 mmol) and 3-methyl-N2-(4-(trifluoromethoxy)phenyl)-N2-((2- (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine (180 mg, 0.4 mmol) at room temperature. The resulting mixture was refluxed for 8hr. The mixture was evaporated, then diluted with ethyl acetate (25 ml.) and washed with H 2 0 (15 ml_). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by prep HPLC to give the desired product (40 mg, 17%) as a yellow solid. LCMS (acidic 5 min): 3.48 min [MH] + =580.1 .

Step 2: N6-(2-chloro-5-methylpyrimidin-4-yl)-N6,3-dimethyl-N2-(4- (trifluoromethoxy) phenyl)-N2- ((2-(trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine -2, 6-diamine

N6-(2-chloro-5-methylpyrimidin-4-yl)-3-methyl-N2-(4-(trif luoromethoxy)phenyl)-N2-((2- (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine (40 mg, 0.068 mmol), CH 3 I (10 mg, 0.068 mmol), CS2CO3 (44 mg, 0.136 mmol) were dissolved in acetone (5 ml.) and the mixture was stirred at 60°C for 3 days under nitrogen, and then diluted with ethyl acetate (25 ml.) and washed with H 2 0 (15 ml_). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by Prep TLC (DCM:MeOH =20:1 ) to give the desired product (18 mg, 44%) as yellow oil. LCMS (acidic 5 min): 3.85 min [MH] + =594.1 . Step 3: 2-(4-((5-methyl-4-(methyl(3-methyl-2-((4-(trifluoromethoxy)p henyl) amino)-3H- imidazo[4,5-b]pyridin-6-yl)amino)pyrimidin-2-yl)amino)-1 H-pyrazol-1 -yl)acetamide

A mixture of N6-(2-chloro-5-methylpyrimidin-4-yl)-N6,3-dimethyl-N2-(4-(tr ifluoromethoxy) phenyl)-N2-((2-(trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5 -b]pyridine-2, 6-diamine (18 mg, 0.03 mmol), 2-(4-amino-1 H-pyrazol-1-yl)acetamide (8.5 mg, 0.06 mmol) and concentrated HCI (1 drop) in IPA (5 ml.) was stirred at 80°C under nitrogen for 8 hours, and then concentrated under reduced pressure. The residue was purified by prep TLC (DCM:MeOH =10:1 ) to give the desired product (3 mg, 14%) as a white solid. LCMS (acidic 5 min): 2.50 min [MH] + =568.2.

1 H NMR (400 MHz, CD 3 OD) d ppm: 8.07 (d, J = 2.4 Hz, 1 H), 7.97 (s, 1 H), 7.83 (d, J = 9.2 Hz, 2H), 7.68-7.67 (m, 2H),7.52 (s, 1 H),7.32 (d, J = 8.8 Hz, 2H), 4.93 (s, 2H), 3.83 (s, 3H), 3.65 (s, 3H), 1.40 (s, 3H). Example 20

Step 1 : N6-(2-chloro-5-(trifluoromethyl)pyrimidin-4-yl)-3-methyl-N2- (4- (trifluoromethoxy) phenyl)- N2-((2-(trimethylsilyl)ethoxy)methyl)-3H-imidazo [4, 5-b]pyridine-2, 6-diamine A mixture of 3-methyl-N2-(4-(trifluoromethoxy)phenyl)-N2-((2-(trimethylsi lyl) ethoxy)methyl)-3H- imidazo[4,5-b]pyridine-2, 6-diamine (400 mg, 0.88 mmol), 2,4-dichloro-5- (trifluoromethyl)pyrimidine (208 mg, 0.96 mmol), DIEA (248 mg, 1.72 mmol) in isopropanol (10 ml.) was stirred at room temperature for 48 hours, then concentrated under reduced pressure. The residue was diluted with ethyl acetate (15 ml_), washed with H 2 0 (10 ml_). The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by Prep TLC (PE:EtOAc = 3:1) to give the desired product (400 mg, 72%) as a white solid. LCMS (acidic 5 min): 2.76 min [MH] + =634.2

Step 2: N6-(2-chloro-5-(trifluoromethyl)pyrimidin-4-yl)-N6,3-dimethy l-N2-(4-(trifluoro methoxy)phenyl)-N2-((2-(trimethylsilyl)ethoxy)methyl)-3H-imi dazo[4,5-b]pyridine-2, 6-diamine

To a solution of 6-(2-chloro-5-(trifluoromethyl)pyrimidin-4-yl)-3-methyl-N2-( 4- (trifluoromethoxy)phenyl)-N2-((2-(trimethylsilyl)ethoxy)meth yl)-3H-imidazo[4,5-b] pyridine-2, 6- diamine (300 mg, 0.6 mmol) in DMSO (10 ml.) was added methyl iodide (100 mg, 0.7 mmol) and CS2CO3 (391 mg, 1.2 mmol). The resulting mixture was stirred at RT for 3h and partitioned between water and EtOAc. The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAC= 2/1) to give the title product (300 mg, 67%) as a yellow oil. LCMS (acidic 5 min): 2.56 min [MH] + =648.1.

Step 3: 2-(4-((4-(methyl(3-methyl-2-((4-(trifluoromethoxy)phenyl)ami no)-3H- imidazo[4,5- b]pyridin-6-yl)amino)-5-(trifluoromethyl)pyrimidin-2-yl)amin o)-1 H- pyrazol-1 -yl)acetamide

To a solution of N6-(2-chloro-5-(trifluoromethyl)pyrimidin-4-yl)-N6, 3-dimethyl- N2-(4- (trifluoromethoxy)phenyl)-N2-((2-(trimethylsilyl)ethoxy)meth yl)-3H-imidazo[4,5-b]pyridine -2,6- diamine (300 mg, 0.46 mmol) in 2-propanol (10 ml.) was added 2-(4-amino-1 H-pyrazol-1- yl)acetamide (129 mg, 0.92 mmol) and concentrated HCI (2 drops). The mixture was heated to 90°C under nitrogen overnight. The solid was collected by filtration, washed with ether and dried to give the desired product (20 mg, 5%) as a grey solid. LCMS (acidic 5 min): 3.04 min [MH] + =622.2.

1 H NMR (400 MHz, MeOH-d 4 ) d 8.32 (d, J = 2.4 Hz, 1 H), 8.22 (s, 1 H), 7.76-7.73 (m, 3H), 7.52- 7.45 (m, 4H), 4.58 (s, 2H), 3.91 (s, 3H), 3.61 (s, 3H).

Example 21

Step 1 : 4-methyl-3-nitro-1 H-pyrazole

To a stirred solution of 4-methyl-1 H-pyrazole (1.0 g, 12.19 mmol) in H 2 SC> 4 (15 ml.) at 30-40°C was added fuming nitric acid (0.64 g, 15.23 mmol). The reaction mixture was stirring at 105°C for 2h. Upon completion, the reaction mixture was diluted with water and purged with air for 30 min, and then the mixture was extracted with ethyl acetate. The organic layer was washed with water and brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude residue was purified by column chromatography (DCM/MeOH=40/1) to afford the title compound (0.4 g, 26%). LCMS (acidic 5 min): 0.63 min [MH] + =128.1. 1 H NMR (400 MHz, DMSO- d 6 ) d 13.69 (br s, 1 H), 7.85 (s, 1 H), 2.25 (s, 3H). Step 2: 2-(4-methyl-3-nitro-1 H-pyrazol-1 -yl)acetamide

A mixture of 2-(4-methyl-3-nitro-1 H-pyrazol-1 -yl)acetamide (100 mg, 0.79 mmol), 2- bromoacetamide (131 mg, 0.95 mmol), K 2 CO 3 (163 mg, 1 .18 mmol) in acetone (20 ml.) was stirred at 80°C for 2 hours. The reaction mixture was concentrated under reduced pressure and the residue was diluted with ethyl acetate (50 ml_), washed with H 2 0 and brine. The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (DCM: MeOH=20:1 ) to give the desired product (80 mg, 55%) as a white solid. LCMS (acidic 5 min): 0.48 min [MH] + =185.1. 1 H NMR (400 MHz, DMSO-de) d 7.81 (s, 1 H), 7.67 (s, 1 H), 7.39 (s, 1H), 4.87 (s, 2H), 2.26 (s, 3H).

Step 3: 2-(3-amino-4-methyl-1 H-pyrazol-1-yl)acetamide

To a solution of 2-(3-amino-4-methyl-1 H-pyrazol-1-yl)acetamide (100 mg, 0.54 mmol) in methanol (2 ml.) was added Pd/C (17 mg, 0.16 mmol)under H2, and the mixture was stirred at RT for 8 hours. The reaction mixture was filtered and the filtering cake was rinsed with methanol. The solvent was removed under reduced pressure to give the desired product (50 mg, 60 %) as a grey solid. LCMS (acidic 5 min): 0.34 min [MH]+=155.1 . 1 H NMR (400 MHz, DMSO-de) d 7.18 (s, 1 H), 7.12 (t , 1 H), 7.09 (s, 1 H), 4.43 (s, 2H), 4.36 (s, 2H), 1 .81 (s, 3H).

Step 4: 2-(4-methyl-3-((4-(methyl(3-methyl-2-((4-(trifluoromethoxy)p henyl) amino)-3H- imidazo[4,5-b]pyridin-6-yl)amino)pyrimidin-2-yl)amino)-1 H- pyrazol-1 -yl)acetamide

To a solution of N6-(2-chloropyrimidin-4-yl)-N6,3-dimethyl-N2-(4- (trifluoromethoxy)phenyl)-N2- ((2-(trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b] pyridine-2, 6-diamine (100 mg, 0.17 mmol) and 2-(3-amino-4-methyl-1 H-pyrazol-1-yl)acetamide (27 mg, 0.17 mmol) in isopropanol (5 mL) was added concentrated HCI (1 drop), and the mixture was stirred at 85°C overnight. The mixture was concentrated under reduced pressure and purified by prep-TLC (DCM/MeOH=10/1) to give the title product (10 mg, 10%) as a yellow solid.

LCMS (acidic 5 min):2.46 min [MH] + =568.2.

1 H NMR (400 MHz, d 6 -DMSO) d 9.57 (s, 1 H), 9.11 (br s, 1 H), 8.04 (m, 3H), 7.71 (d, J = 6.4 Hz, 2H), 7.47 (s, 1 H), 7.36 (m, 3H), 7.23 (s, 1 H), 5.68 (s, 1 H), 4.63 (s, 2H), 3.78 (s, 3H), 3.42 (s, 3H),

1.92 (s, 3H).

Example 22

Step 1 : 2-(3,5-dimethyl-4-nitro-1 H-pyrazol-1 -yl)acetamide

To the solution of 3,5-dimethyl-4-nitro-1 H-pyrazole (1 g, 7.09 mmol) and 2-bromoacetamide (1.17 g, 8.51 mmol) in acetone (10 ml.) was added potassium carbonate (1.96 g, 14.2 mmol). The mixture was stirred at 60°C overnight and then partitioned between water and ethyl acetate. The organics were separated, dried over sodium sulfate and concentrated under reduced pressure to give the desired product (1.4 g, 100%) as a white sold. LCMS (acidic 5 min): 0.48 min [MH] + =199.1 . Step 2: 2-(4-amino-3,5-dimethyl-1 H-pyrazol-1 -yl)acetamide A mixture of 2-(3,5-dimethyl-4-nitro-1 H-pyrazol-1 -yl)acetamide (100 mg, 0.505 mmol) and Pd/C (10 mg) in CH 3 OH (10 ml.) was stirred at room temperature under hydrogen atmosphere overnight. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give the desired compound (70 mg, 82%) as a brown solid. LCMS (acidic 5 min): 0.30 min [MH] + =169.1 .

Step 3: 2-(3,5-dimethyl-4-((4-(methyl(3-methyl-2-((4-(trifluorometho xy)phenyl) amino)-3H- imidazo[4,5-b]pyridin-6-yl)amino)pyrimidin-2-yl)amino)-1 H-pyrazol-1 -yl)acetamide To the mixture of 2-(4-amino-3,5-dimethyl-1 H-pyrazol-1-yl)acetamide (70 mg, 0.41 mmol) and intermediate A (242 mg, 0.417 mmol) in isopropanol (10 ml.) was added concentrated HCI (1 drop), and the mixture was heated to 80°C and stirred overnight. The mixture was concentrated under reduced pressure, and purified by preparative TLC (DCM/MeOH = 20/1 ) to give the title compound (5 mg, 2%) as a brown solid. LCMS (acidic 5 min): 2.36 min [MH] + =582.2.

1 H NMR (400 MHz, CD 3 OD) d ppm 8.02 (d, J= 2.0 Hz, 1 H), 7.79 (d, J = 9.2 Hz, 2H), 7.60 (m, 2H), 7.28 (d, J = 8.8 Hz, 2H), 5.74 (d, J = 6.4 Hz, 1 H), 4.72 (s, 2H), 3.81 (s, 3H), 3.50 (s, 3H), 2.14 (s, 3H), 2.11 (s, 3H). Example 23 Step 1 : N6-(2-((1 H-pyrazol-4-yl)amino)pyrimidin-4-yl)-N6,3-dimethyl-N2-(4- (trifluoro methoxy)phenyl)-N2-((2-(trimethylsilyl)ethoxy)methyl)-3H-imi dazo[4,5-b]pyridine-2, 6-diamine

A solution of 1 H-pyrazol-4-amine (40 mg, 0.48 mmol) and N6-(2-chloropyrimidin-4-yl)-N6,3- dimethyl-N2-(4-(trifluoromethoxy)phenyl)-N2-((2-(trimethylsi lyl)ethoxy)methyl)-3H-imidazo[4,5- b]pyridine-2, 6-diamine (286 mg, 0.48 mmol) in isopropanol (5 ml.) was stirred at 85°C overnight. The mixture was concentrated under reduced pressure and the crude residue was purified by prep-TLC (DCM/MeOH=10/1 ) to give the title product (110 mg, 37%) as a yellow solid. LCMS (acidic 5 min):3.30 min [MH] + =627.3.

Step 2: 2-(4-((4-(methyl(3-methyl-2-((4-(trifluoromethoxy)phenyl)((2 - (trimethylsilyl) ethoxy)methyl)amino)-3H-imidazo[4,5-b]pyridin-6-yl)amino)pyr imidin-2-yl)amino)-1 H-pyrazol-1- yl)acetonitrile

To a solution of N6-(2-(1 H-pyrazol-4-ylamino)pyrimidin-4-yl)-N6,3-dimethyl-N2-(4- (trifluoromethoxy)phenyl)-N2-((2-(trimethylsilyl)ethoxy)meth yl)-3H-imidazo[4,5-b] pyridine-2, 6- diamine (110 mg, 0.18 mmol) in THF (5 ml.) cooled to 0°C was added NaH (7 mg, 0.18 mmol). The mixture was stirred at room temperature for 48 hours. The mixture was partitioned between water and ethyl acetate. The organic phase was washed with brine, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by prep-TLC (DCM/MeOH = 10/1 ) to give the title product (40 mg, 34%) as a yellow oil. LCMS (acidic 5 min): 3.12 min [MH] + =666.3, min [MNa] + =688.3. Step 3: 2-(4-((4-(methyl(3-methyl-2-((4-(trifluoromethoxy)phenyl)ami no)-3H- imidazo[4,5- b]pyridin-6-yl)amino)pyrimidin-2-yl)amino)-1 H-pyrazol-1 -yl) acetonitrile

A solution of 2-(4-(4-(methyl(3-methyl-2-((4-(trifluoromethoxy)phenyl)((2-

(trimethylsilyl)ethoxy)methyl)amino)-3H-imidazo[4,5-b]pyr idin-6-yl)amino)pyrimidin-2-ylamino)- 1 H-pyrazol-1 -yl)acetonitrile (40 mg, 0.06 mmol) in isopropanol (5 ml.) was added concentrated

HCI (1 drop) and the mixture was stirred at 85°C overnight. The reaction mixture was concentrated under reduce pressure and the residue was purified by prep-TLC (DCM/MeOH = 10/1) to give the title product (10 mg, 31%) as a yellow solid.

LCMS (acidic 5 min):2.42 min [MH] + =536.2. 1 H NMR (400 MHz, d 4 -methanol) d 8.08 (d, J = 2.1 Hz, 1 H), 7.79 (m, 4H), 7.67 - 7.58 (m, 2H), 7.29 (d, J= 8.6 Hz, 2H), 6.04 (s, 1 H), 4.79 (s, 2H), 3.84 (s, 3H), 3.61 (s, 3H).

Example 24

Step 1 : N6-(2-chloro-5-fluoropyrimidin-4-yl)-3-methyl-N2-(4-(trifluo romethoxy) phenyl)-N2-((2- (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine

To a solution of 2,4-dichloro-5-fluoropyrimidine (0.33 g, 2.0 mmol) in i-PrOH (10 ml.) was added DIEA (0.52 g, 4.0 mmol) and 3-methyl-N2-(4-(trifluoromethoxy)phenyl)-N2-((2- (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine (0.9 g, 2.0 mmol) at room temperature. The mixture was refluxed for 6hr. The mixture was evaporated, then diluted with ethyl acetate (25 ml.) and washed with H 2 0 (15 ml_). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by prep HPLC to give the desired product (0.35 g, 29%) as a white solid. LCMS (acidic 5 min): 3.64 min [MH] + =584.1 .

Step 2: N6-(2-chloro-5-fluoropyrimidin-4-yl)-N6,3-dimethyl-N2-(4- (trifluoromethoxy) phenyl)-N2- ((2-(trimethylsilyl)ethoxy)methyl)-3H-imidazo [4, 5-b]pyridine-2, 6-diamine

A mixture of N6-(2-chloro-5-fluoropyrimidin-4-yl)-3-methyl-N2-(4- (trifluoromethoxy) phenyl)-N2- ((2-(trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine -2, 6-diamine (0.35 g, 0.6 mmol), CH 3 I (0.085 g, 0.6 mmol) and CS2CO3 (0.39 g, 1.2 mmol) in acetone (5 mL) was stirred at room temperature under nitrogen overnight, and then diluted with ethyl acetate (25 mL) and washed with H2O (15 mL). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (DCM/MeOH=50:1 ) to give the desired product (0.15 g, 42%) as a yellow solid. LCMS (acidic 5 min): 3.95 min [MH] + =598.1.

Step 3: 2-(4-((5-fluoro-4-(methyl(3-methyl-2-((4-(trifluoromethoxy)p henyl) amino)-3H- imidazo[4,5-b]pyridin-6-yl)amino)pyrimidin-2-yl)amino)-1 H-pyrazol-1 -yl)acetamide

A mixture of N6-(2-chloro-5-fluoropyrimidin-4-yl)-N6,3-dimethyl-N2-(4-(tr ifluoromethoxy) phenyl)- N2-((2-(trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyrid ine-2,6- diamine (150 mg, 0.25 mmol), 2-(4-amino-1 H-pyrazol-1 -yl)acetamide (70 mg, 0.50mmol), concentrated HCI (1 drop) in IPA (15 ml.) was stirred at 80°C under nitrogen for 4 hours, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (DCM/MeOH=50:1) to give the desired product (15 mg, 9%) as a white solid.

LCMS (acidic 5 min): 2.74 min [MH] + =572.1 . 1 H NMR (400 MHz, DMSO-d 6 ) d ppm 9.44 (s, 1 H), 9.09 (s, 1 H), 8.04 (m, 3H), 7.88 (d, J = 6.0 Hz, 1 H), 7.74 (d, J = 2.4 Hz, 2H), 7.47 (s, 1 H), 7.38 (d, J = 8.8 Hz, 2H), 7.31 (s, 1 H), 7.19 (s, 1 H), 4.62 (s, 2H), 3.76 (s, 3H), 3.49 (s, 3H).

Example 25

Step 1 : N-(methyl-d3)-3,5-dinitropyridin-2-amine

A solution of 2-chloro-3,5-dinitropyridine (3 g, 14.7 mmol) in MeOH (30 ml.) was added CD3NH2.HCI (1 g, 14.7 mmol) and the mixture was stirred at room temperature under nitrogen overnight. The precipitated solid was collected by filtration to give the desired product (2.8 g, 99%) as a yellow solid. LCMS (acidic 5 min): 1.53 min [MH] + =202.1 .

Step 2: N2-(methyl-d3)-5-nitropyridine-2, 3-diamine

To a solution of N-(methyl-d3)-3,5-dinitropyridin-2-amine (2.6 g, 13 mmol) in EtOH (20 ml.) was added (NH^S (30 ml, 69 mmol). The reaction mixture was stirred at 60°C under nitrogen for 5hrs and concentrated under reduced pressure. The residue was diluted with ethyl acetate (20 ml_), and washed with H 2 0 (20 ml_). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAc=20/1) to give the desired product (2 g, 90%) as a yellow solid. LCMS (acidic 5 min): 0.46 min [MH] + =172.1.

Step 3: 3-(methyl-d3)-6-nitro-N-(4-(trifluoromethoxy)phenyl)-3H-imid azo[4,5-b] pyridin-2-amine

To a solution of N2-(methyl-d3)-5-nitropyridine-2, 3-diamine (1 .71 g, 10 mmol) in MeOH (120 ml.) was added 1-isothiocyanato-4-(trifluoromethoxy)benzene (2.42 g, 11 mmol). The mixture was stirred at room temperature under nitrogen overnight. The mixture was partitioned between EtOAc and water. The organics were separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAc = 20/1) to give the desired product (2.8 g, 80%) as a yellow solid. LCMS (acidic 5 min): 3.09 min [MH] + =357.1 .

Step 4: 3-(methyl-d3)-6-nitro-N-(4-(trifluoromethoxy)phenyl)-N-((2-( trimethylsilyl)ethoxy) methyl)- 3H-imidazo[4,5-b]pyridin-2-amine

To a solution of 3-(methyl-d3)-6-nitro-N-(4-(trifluoromethoxy) phenyl)-3H-imidazo[4,5-b] pyridin- 2-amine (2.8 g, 7.9 mmol) in dry THF (20 mL) was added NaH (943 mg, 23.6 mmol) at 0°C for 30 min, and then SEMCI (2 g, 11 .9 mmol) was added. The reaction mixture was stirred at rt for 3h. The reaction mixture was concentrated under reduced pressure. The residue was diluted with ethyl acetate (25 mL), and washed with H 2 0 (25 mL). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAc = 20/1 ) to give the desired product (3 g, 78%) as a yellow oil. LCMS (acidic 5 min): 3.86min [MH] + =487.2. Step 5: 3-(methyl-d3)-N2-(4-(trifluoromethoxy)phenyl)-N2-((2-(trimet hylsilyl) ethoxy) methyl)-3H- imidazo[4,5-b]pyridine-2, 6-diamine

To a solution of 3-(methyl-d3)-6-nitro-N-(4-(trifluoromethoxy)phenyl)-N-((2- (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridin-2-am ine (3 g, 6 mmol) in MeOH (30 ml.) was added zinc (2.3 g, 36 mmol) and NH CI (aq, 30 ml_). The reaction mixture was stirred at 60°C under nitrogen overnight. The reaction mixture was concentrated under reduced pressure. The residue was diluted with ethyl acetate (50 ml_), and washed with H 2 0 (30 ml_). The organic layer was separated, dried over sodium sulfate and concentrated. The residue was purified by column chromatography on silica gel (PE/EtOAc =20/1) to give the desired product (2.2 g, 81%) as a yellow oil. LCMS (acidic 5 min): 3.28 min [MH] + =457.2.

Step 6: N6-(2-chloropyrimidin-4-yl)-3-(methyl-d3)-N2-(4-(trifluorome thoxy) phenyl)-N2-((2- (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2,6 - diamine

A solution of 3-(methyl-d3)-N2-(4-(trifluoromethoxy)phenyl)-N2-((2-(trimet hylsilyl) ethoxy)methyl)- 3H-imidazo[4,5-b]pyridine-2, 6-diamine (2.1 g, 4.6 mmol) in 2-propanol (15 ml.) were added 2,4- dichloropyrimidine (1 .3 g, 9.2 mmol) and DIEA(1 .2 g, 9.2 mmol). The resulting mixture was heated to 100°C under nitrogen overnight. The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAC=10/1 ) to give the desired product (150 mg, 42%) as a yellow solid. LCMS (acidic 5 min): 3.70 min [MH] + =569.2. Step 7: N6-(2-chloropyrimidin-4-yl)-N6-methyl-3-(methyl-d3)-N2-(4-(t rifluoromethoxy) phenyl)- N2-((2-(trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyrid ine-2, 6-diamine

A solution of N6-(2-chloropyrimidin-4-yl)-3-(methyl-d3)-N2-(4-(trifluorome thoxy) phenyl)-N2-((2- (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2,6 - diamine (1.2 g, 2.1 mmol) in DMSO (10 ml.) was added CH 3 I (450 mg, 3.2 mmol) and Cs2CC>3 (2 g, 6.3 mmol). The mixture was stirred at 30 °C for 3h and partitioned between water and EtOAc. The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/EtOAC=20/1) to give the desired product (1.2 g, 86%) as a yellow oil. LCMS (acidic 5 min): 3.74 min [MH]+=584.1 .

Step 8: 2-(2-((4-((4-(methyl(3-(methyl-d3)-2-((4-(trifluoromethoxy)p henyl)amino)-3H-imidazo[4,5- b]pyridin-6-yl)amino)pyrimidin-2-yl)amino)-1 H-pyrazol-1-yl)methyl)-1 ,3-dioxan-5-yl)isoindoline- 1 ,3-dione

To a solution of N6-(2-chloropyrimidin-4-yl)-N6-methyl-3-(methyl-d3)-N2-(4- (trifluoromethoxy)phenyl)-N2-((2-(trimethylsilyl)ethoxy)meth yl)-3H-imidazo[4,5-b]pyridine-2,6- diamine (500 mg, 0.87 mmol) in 2-propanol (15 ml.) were added 2-(2-((4-amino-1 H-pyrazol-1- yl)methyl)-1 ,3-dioxan-5-yl)isoindoline-1 ,3-dione (300 mg, 0.95 mmol) and concentrated HCI (2 drops). The mixture was stirred at 80 °C under nitrogen overnight. The solid was collected by filtration, washed with diethyl ether and dried under reduced pressure to give the desired product (500 mg, 77%) as a yellow solid. LCMS (acidic 5 min): 2.72 min [MH] + =745.3.

Step 9: N6-(2-((1-((5-amino-1 ,3-dioxan-2-yl)methyl)-1 H-pyrazol-4-yl)amino) pyrimidin-4-yl)-N6- methyl-3-(methyl-d3)-N2-(4-(trifluoromethoxy)phenyl)-3H-imid azo[4,5-b]pyridine-2, 6-diamine A mixture of 2-(2-((4-((4-(methyl(3-(methyl-d3)-2-((4-(trifluoromethoxy)p henyl) amino)-3H- imidazo[4,5-b]pyridin-6-yl)amino)pyrimidin-2-yl)amino)-1 H-pyrazol-1 -yl)methyl)-1 ,3-dioxan-5- yl)isoindoline-1 ,3-dione (450 mg, 0.6 mmol) in MeNhb (2M in MeOH, 10 ml.) was stirred at RT overnight. The mixture was partitioned between EtOAc and water. The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by prep HPLC to give the desired product as TFA salt (1 .95 g, 57%) as a yellow solid. LCMS (acidic 5 min): 2.17 min [MH] + =615.3.

1 H NMR (400 MHz, DMSO-d 6 ) d 10.6 (s, 1 H), 9.62 (s, 1H), 8.50-8.06 (m, 4H), 8.05 (d, J = 8.8 Hz, 2H), 7.92-7.76 (m, 2H), 7.53 (br s, 1 H), 7.38 (d, J = 8.8 Hz, 2H), 5.90 (br s, 1 H), 4.80 (br s, 1 H), 4.30-4.00 (m, 4H), 3.70-3.56 (m, 4H), 3.38-3.26 (m, 2H).

Example 26 Step 1 : 3-methyl-6-nitro-N-(4-(trifluoromethyl)phenyl)-3H-imidazo[4, 5-b]pyridine-2-amine

To a solution of intermediate B (300 mg, 1.41 mmol), 4-(trifluoromethyl)benzenamine (275 mg, 1 .70 mmol) in isopropanol (20 ml.) was added toluene-4-sulfonic acid (25 mg, 0.145 mmol). The mixture was stirred at 80°C for 6 hours, and then concentrated under reduced pressure. The residue was partitioned between H 2 0 (20 ml.) and ethyl acetate (50 ml_). The organic layer was separated, dried over sodium sulfate and concentrated to give the desired product (400 mg, 86%) as a yellow solid. LCMS (acidic 5 min): 3.02 min [MH] + =338.1 . Step 2: 3-methyl-6-nitro-N-(4-(trifluoromethyl)phenyl)-N-((2-(trimet hylsilyl) ethoxy) methyl)-3H- imidazo[4,5-b]pyridin-2-amine

To a solution of N-(4-(trifluoromethyl)phenyl)-3-methyl-6-nitro-3H-imidazo [4,5-b]pyridin-2-amine (400 mg, 1.19 mmol) and SEMCI (396.8 mg, 2.38 mmol) in THF (20 mL) was added NaH (114.2 mg, 4.76 mmol). The mixture was stirred for 8 hours at room temperature, and then diluted with ethyl acetate (100 mL) and saturated NaHCC>3 solution (50 mL). The organic layer was separated, dried over sodium sulfate and concentrated to give the desired product (480 mg, 85%) as yellow oil. LCMS (acidic 5 min): 3.74 and 3.90 min [MH] + =468.1.

Step 3: 3-methyl-N2-(4-(trifluoromethyl)phenyl)-N2-((2-(trimethylsil yl) ethoxy)methyl)-3H- imidazo[4,5-b]pyridine-2, 6-diamine

To a solution of N-((2-(trimethylsilyl)ethoxy)methyl)-N-(4-(trifluoromethyl) phenyl)-3-methyl-6- nitro-3H-imidazo[4,5-b]pyridin-2-amine (480 mg, 1.03 mmol) in MeOH (30 ml.) and aqueous NH4CI (20 ml.) was added Zn (350 mg, 5.38 mmol) at 0 °C. The mixture was stirred at 60°C for 3 hours. The mixture was filtered and the filtering cake was washed with methanol. The combined filtrate was evaporated and the residue was diluted with ethyl acetate (50 ml.) and washed with H2O (25 ml_). The organic layer was separated, dried over sodium sulfate and concentrated to give the desired product (350 mg, 78%) as grey oil, which was used in the next step without further purification. LCMS (acidic 5 min): 3.35 and 3.50 min [MH] + =438.2. Step 4: N6,3-dimethyl-N2-(4-(trifluoromethyl)phenyl)-N2-((2-(trimeth ylsilyl) ethoxy) methyl)-3H- imidazo[4,5-b]pyridine-2, 6-diamine

To a solution of 3-methyl-N2-(4-(trifluoromethyl)phenyl)-N2-((2-(trimethylsil yl) ethoxy)methyl)-3H- imidazo[4,5-b]pyridine-2, 6-diamine (250 mg, 0.57 mmol), (CH 2 O) n (180 mg, 5.7 mmol) in MeOH

(25 ml.) was added NaOCH 3 (130 mg, 2.28 mmol). The mixture was stirred at 80°C for 4 hours. NaBhU (220 mg, 5.7 mmol) was added portionwise. The mixture was stirred for 2h at 70°C, then concentrated under reduced pressure. The reaction mixture was filtered and the filtering cake was washed with MeOH. The combined filtrates were evaporated and the residue was diluted with ethyl acetate (50 ml.) and washed with H 2 0 (25 ml_). The organic layer was separated, dried over sodium sulfate and concentrated to give the desired product (130 mg, 50%) as a yellow oil, which was used in the next step without further purification. LCMS (acidic 5 min): 3.46 and 3.00 min [MH] + =452.2.

Step 5: N-methyl-2-(4-((4-(methyl(3-methyl-2-((4-(trifluoromethyl)ph enyl)((2-(trimethyl silyl)ethoxy)methyl)amino)-3H-imidazo[4,5-b]pyridin-6-yl)ami no)pyrimidin-2-yl)amino)-1 H- pyrazol-1 -yl)acetamide

N6,3-dimethyl-N2-(4-(trifluoromethyl)phenyl)-N2-((2-(trim ethylsilyl)ethoxy)methyl)-3H- imidazo[4,5-b]pyridine-2, 6-diamine (130 mg, 0.31 mmol), K 2 CO 3 (86 mg, 0.062 mmol) and 2-(4- (4-chloropyrimidin-2-ylamino)-1 H- pyrazol-1-yl)-N-methylacetamide (165 mg, 0.62 mmol) in DMF (20 ml.) were stirred for 8 hours at 80°C. The mixture was diluted with ethyl acetate (100 ml.) and H 2 O (25 ml_). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by prep TLC (DCM:MeOH=10:1 ) to give the desired product (25 mg, 12%) as a brown solid . LCMS (acidic 5 min): 3.03 min [MH] + =682.3. Step 6: N-methyl-2-(4-((4-(methyl(3-methyl-2-((4-(trifluoromethyl)ph enyl)amino)-3H-imidazo[4,5- b]pyridin-6-yl)amino)pyrimidin-2-yl)amino)-1 H-pyrazol-1-yl)acetamide

To a solution of N-methyl-2-(4-(4-(methyl(3-methyl-2-((4- (trifluoromethyl)phenyl)((2- (trimethylsilyl)ethoxy)methyl)amino)-3H-imidazo[4,5-b]pyridi n-6-yl)amino)pyrimidin-2-ylamino)- 1 H-pyrazol-1-yl)acetamide (25 mg, 0.037 mmol) in MeOH (3 ml.) was added HCI (1 M, 1 ml), and then the mixture was stirred for 2 hours at 40°C. The mixture was diluted with ethyl acetate (10 ml.) and H 2 0 (2 ml_). The organic layer was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by prep TLC (DCM: MeOH=10:1 ) to give the desired product (5 mg, 25%) as a grey solid.

LCMS (acidic 5 min): 2.48 min [MH] + =552.2. 1 H NMR (400 MHz, DMSO-d 6 ) d 9.78 (s, 1 H), 9.06 (s, 1 H), 8.18 (d, J = 8.4 Hz, 2H), 8.07 (d, J = 2.0 Hz, 1 H), 7.84-7.70 (m,6H), 7.45 (s, 1 H), 5.7 (br s, 1 H), 4.56 (s, 2H), 3.82 (s,3H), 3.46 (s,3H), 2.60 (d, J= 4.4 Hz, 3H).

The following compounds were prepared according to the scheme above using the corresponding anilines:

Example 32 Step 1 : N6-(2-chloro-5-methoxypyrimidin-4-yl)-3-methyl-N2-(4-(triflu oromethoxy) phenyl)-N2-((2- (trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine-2, 6-diamine

A mixture of 3-methyl-N 2 -(4-(trifluoromethoxy)phenyl)-N 2 -((2-(trimethylsilyl) ethoxy)methyl)-3H- imidazo[4,5-b]pyridine-2, 6-diamine (550 mg, 1.2 mmol), 2,4-dichloro-5-methoxy pyrimidine (330 mg, 1 .8 mmol), DIEA (465 mg, 3.6 mmol) and IPA (20 ml.) was stirred at 70°C under ISh overnight. The mixture was concentrated under reduced pressure and extracted with DCM (50 ml_). The organic layer was washed with water (100 ml_), dried with Na 2 SC> 4 , concentrated and purified by column chromatography on silica gel (DCM/MeOH=100/1 -30/1 , V/V) to give the desired product (300 mg, 40 %) as a brown solid. LCMS (acidic 5 min): 2.81 min, 3.70 min; [MH] + =596.2.

Step 2: N 6 -(2-chloro-5-methoxypyrimidin-4-yl)-N 6 ,3-dimethyl-N 2 -(4-(trifluoromethoxy) phenyl)-N 2 - ((2-(trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine -2, 6-diamine

A mixture of N6-(2-chloro-5-methoxypyrimidin-4-yl)-3-methyl-N 2 -(4-(trifluoromethoxy) phenyl)-N 2 - ((2-(trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]pyridine -2, 6-diamine (100 mg, 0.168 mol), CH3I (71 mg, 0.5 mmol) and CS2CO3 (164 mg, 0.5 mmol) in DMF (3 ml.) was stirred at 0 °C. After 0.5 h, the mixture was allowed to warm to RT overnight. The mixture was extracted with DCM (50 ml_). The organic layer was washed with water (100 ml_), dried with Na 2 SC> 4 and concentrated under reduced pressure to give the desired product (100 mg, 98 %) as a brown solid. LCMS (acidic 5 min): 3.77 min; [MH] + =610.2.

Step 3: 2-(4-((5-methoxy-4-(methyl(3-methyl-2-((4-(trifluoromethoxy) phenyl) amino)-3H- imidazo[4,5-b]pyridin-6-yl)amino)pyrimidin-2-yl)amino)-1 H-pyrazol-1 -yl)-N-methyl acetamide

A mixture of N 6 -(2-chloro-5-methoxypyrimidin-4-yl)-N 6 ,3-dimethyl-N 2 -(4-

(trifluoromethoxy)phenyl)-N 2 -((2-(trimethylsilyl)ethoxy)methyl)-3H-imidazo[4,5-b]p yridine-2,6- diamine (100 mg, 0.164 mmol) and 2-(4-amino-1 H- pyrazol-1 -yl)-N-methylacetamide (51 mg, 0.328 mmol) in IPA (5 ml.) was added concentrated HCI (2 drops) and the mixture was stirred at 80°C under N 2 overnight. The mixture was concentrated under reduced pressure and partitioned between water and DCM (50 ml_). The organics were separated, washed with water (50 ml_), dried over Na 2 SC> 4 , concentrated under reduced pressure and purified by prep TLC (DCM/MeOH=10/1 ) to give the desired product (15 mg, 15 %) as a brown solid. LCMS (acidic 5 min): 2.88 min; [MH] + =597.9.

1 H NMR (400 MHz, MeOH-d 4 ) d 7.96 (s, 1H), 7.78 (d, J = 7.6 Hz, 2H), 7.64 (s, 2H), 7.57 (s, 1 H), 7.52 (s, 1 H), 7.29 (d, J= 7.2 Hz, 2H), 4.46 (s, 2H), 3.79 (s, 3H), 3.58 (s, 3H), 3.39 (s, 3H), 2.

Assay: Screening compounds for inhibition of TSQ induced necroptosis, 96 well plate format.

Cell Line ID: U937 human histiocytic leukemia cell line.

Cell Concentration (cells/well): Final cell density is 5000 cells per well.

Cell growth medium: HT -RPMI + 7.4% FBS. Cells are cultured in Corning 150cm 2 tissue culture flasks with vented caps at 37°C/5% CO2.

Incubation time (hours): 48 hours following addition of compounds and death stimuli

Compound concentration: 100nM DMSO final concentration (% v/v): 0.3%.

Compounds that are in the death stimulation cocktail and their final concentrations: hTNF-Fc (1 OOng/ml) - produced by standard procedures as shown in Bossen et al., J Biol Chem, 2006, 281 (20), 13964-13971.

Compound A (500nM) - Smac mimetic, Tetralogic and SYNthesis med chem. The Smac mimetic, Compound A, has been described previously in Vince JE, et al. (2007), Cell 131 (4):682-693.

Q-VD-Oph (10mM) - MP Biomedicals and R&D systems.

Assay experimental outline

The cellular assay was carried out according to the following steps:

1 . Each well was prepared by sequential addition of: a. DMSO (control; columns 1 -2 and 23-24) or compound in DMSO - addition was performed using acoustic transfer of nl volumes of stock compound to give final test concentrations of 36,12, 4, 1.3, 0.44, 0.148, 0.049, 0.016, 0.005 and 0.002 mM. All wells were backfilled with DMSO to a final total volume in the well of 10Onl. b. Following compound/DMSO addition in step (a), 40 mI_ of cell suspension (5x10 5 cells/mL) was added to provide a final cell concentration of 20,000 cells per well, and c. Following cell addition in step (b), 10mI_ of 5x TSQ cocktail (except to positive controls; columns 1 and 23) was added to each well.

2. After 48 hours, plate was removed from the 37°C incubator and equilibrated to room temperature for 45 minutes.

3. 15mI_ of room temperature CellTitre-Glo2™ (Promega™) was added to each well.

4. Shook plates for 2 minutes (~600rpm) and incubated at room temperature for 15 minutes to allow signal to stabilise.

5. Read luminescence readout on a plate reader.

Analysis

Percent viability was calculated for each compound according to equation (1 ):

% viability = 100 x ((RawData - NSA)/(TA-NSA) (1 ) wherein

RawData is the readout of any cell containing a compound of the invention

TA is the total activity provided by the luminescence readout from DMSO only wells (columns 2 and 24) = 100% viability

NSA is the non-specific activity provided by DMSO+TSQ wells (columns 1 and 23) = 0% viability

Curve fitting: 10-point titration curves are fitted with the 4-parameter logistic nonlinear regression model and the IC 5 o reported is the inflection point of the curve

Analysis: Data is loaded into Abase and normalised. 10 points titration curve are fitted with the 4 parameter logistic nonlinear regression model and the IC 5 o reported reflects the inflection point of the curve for curve fitting.

Interpretation of results: Assay involving the TSQ cocktail (T: TNF; S: Smac mimetic; Q: Q-VD-OPh): TSQ treatment ensures that cells specifically undergo necroptotic cell death. TNF activates the TNF receptor, Smac mimetic direct the signal away from proinflammatory signaling and toward the RIP1/RIP3- mediated cell death pathways, and Q-VD-OPh ensures that the apoptotic response is blocked leaving only the programmed necrosis response. The compounds’ activity (solution in DMSO) tested in this TSQ-induced assay is evaluated by determining the number of viable cells in culture by measuring the amount of ATP present as measured by CelltiterGlo.

Counter screen: In parallel, all compounds are tested for their ability to affect cell viability. The same U937 cells are treated with compound in DMSO without the TSQ cocktail. This counter screen enables evaluation of off-target effects. In this case, cell viability is measured by CelltiterGlo

The results of the screening of the compounds described above are shown below in Table 1 .

Binding affinity for MLKL and RIP1 was determined using the KINOMEscan™ technology developed by DiscoverX (USA; http://www.discoverx.com). The assay was conducted according to manufacturer instructions.

Protocol Description

Kinase assays. For most assays, kinase-tagged T7 phage strains were grown in parallel in 24- well blocks in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage from a frozen stock (multiplicity of infection = 0.4) and incubated with shaking at 32°C until lysis (90-150 minutes). The lysates were centrifuged (6,000 x g) and filtered (0.2 pm) to remove cell debris. The remaining kinases were produced in HEK-293 cells and subsequently tagged with DNA for qPCR detection. Streptavid in -coated magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1 % BSA, 0.05 % Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific phage binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test compounds in 1x binding buffer (20 % SeaBlock, 0.17x PBS, 0.05 % Tween 20, 6 mM DTT). Test compounds were prepared as 40x stocks in 100% DMSO and directly diluted into the assay. All reactions were performed in polypropylene 384-well plates in a final volume of 0.02 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (1x PBS, 0.05 % Tween 20). The beads were then re-suspended in elution buffer (1x PBS, 0.05 % Tween 20, 0.5 pM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR.

Compound Handling

An 11 -point 3-fold serial dilution of each test compound was prepared in 100% DMSO at 100x final test concentration and subsequently diluted to 1 x in the assay (final DMSO concentration = 1%). Most K D s were determined using a compound top concentration = 30,000 nM. If the initial K D determined was < 0.5 nM (the lowest concentration tested in the initial serial dilution), the measurement was repeated with a further 11 point 3-fold serial dilution starting at 3,000 nM.

Binding Constants (K D s)

K D for each test compound was calculated with a standard dose-response curve using the Hill equation (equation

Response = Backg

The Hill Slope was set to -1 .

Curves were fitted using a non-linear least square fit with the Levenberg-Marquardt algorithm.

Ref:

• Fabian, M.A. et al. A small molecule-kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol. 23, 329-336 (2005).

• Karaman, M.W. et al. A quantitative analysis of kinase inhibitor selectivity. Nat. Biotechnol. 26, 127-132 (2008).

• Hill, A.V. The possible effects of the aggregation of the molecules of hemoglobin on its dissociation curves. J. Physiol. (Lond.). 40, iv-vii (1910).

• Levenberg, K. A method for the solution of certain non-linear problems in least squares. Q. Appl. Math. 2, 164-168 (1944).

Table 1 : Table showing the results of cell based assays performed under assay 1 and binding data for compounds described above. A: IC 5 o < 100nM +++

100nM < IC50 < 500nM ++

IC50 > 500nM +

B: IC50 < 1 mM +++

1 mM < IO50 < 5mM ++

IC50 > 5mM +

C: K D < 250hM +++

250hM < K D < 1mM ++

KD > 1 mM +

D: K D < 10OhM +++

100hM < K D < 500hM++

500 h M < KD +

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.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.