NZ739511B2 - Pyrazole pyrimidine derivative and uses thereof - Google Patents

Abstract

The present invention provides pyrazole pyrimidine derivatives of formula (I) which inhibit Casein kinase I (CKI) and/or lnterleukin-1 receptor-associated kinase 1 (IRAKI) and methods of their manufacture, compositions comprising them and uses thereof in methods of treating malignant disease and disorders and methods for treating inflammatory diseases and disorders.

Description

PYRAZOLE PYRIMIDINE DERIVATIVE AND USES THEREOF TECHNOLOGICAL FIELD The present invention provides pyrazole pyrimidine derivatives and uses thereof in methods of treating malignant disease and disorders and methods for treating in?ammatory diseases and disorders.

BACKGROUND The casein kinase 1 family (CKl, or CKI) are serine/threonine kinases with six s (isoforms) in humans: (1, yl, y2, y3, 5 and a. They differ in length and sequence of the N-terminal (9— 76 amino acids) and especially the C-terminal (24— 200 amino acids) non-catalytic domain (Schittek and Sinnberg, Molecular Cancer 2014, ).

CK15 and CKla are 98% identical in their kinase domain and 53% identical in their C-terminal regulatory domain (Fish KJ et al. J Biol Chem 1995, 875— . Whereas, there is some redundancy with respect to CKl substrate phosphorylation, most CKl isoforms have distinct biological roles. The wide range of CKl substrates shows that the CKl family members are involved in multiple cellular processes, from regulation of membrane trafficking, cytokinesis, vesicular ort, ribosome biogenesis, DNA repair, signal transduction pathways, apoptosis and in the circadian rhythm schild U et al. Cell Signal 2005, 17:675—689; Cheong JK and Virshup DM. Int J Biochem Cell Biol 2011, 43:465—469; Zemp I, et al. J Cell Sci 2014, 127:1242—1253).

CKla plays a role in the mitotic e ion during cell division and in DNA repair mechanisms and participates in RNA metabolism (Knippschild U et al. Cell Signal 2005, 17:675—689). It butes to the activation of mTOR via sustained degradation of the endogenous mTOR inhibitor DEPTOR (Duan S et al. Mol Cell 2011, 44:317—324).

CKla has a major role in regulation of the Wnt/B-catenin signaling pathway. The inventors of this application have shown that CKla is a key component of the B-catenin destruction complex. When the Wnt receptors are not engaged, CKla phosphorylates B- catenin at serine residue S45, which is necessary for the g phosphorylation of another kinase, GSK3 (Amit et al. Genes Dev. 2002 16: 1066- 1076).

B-catenin phosphorylation by GSK3 at residues T41, S37 and S33, generates a ubiquitination degron, ting the E3 SCF-B-TrCP, leading to the ubiquitination and degradation of B-catenin rs H and Nusse R Cell 2012, 149: 1192-1205). The inventors have further shown that inducible ablation of CKla in the mouse gut epithelium triggers a massive epithelial Wnt response, which surprisingly did not alter intestinal homeostasis, with only little enhanced proliferation and no tumorigenesis (Elyada et al.

Nature 2011, 470: 409-413). This is dissimilar to the consequences of acute ablation of other ents of the B-catenin destruction complex, such as APC, which results in loss of homeostasis and tumorigenesis (O.J. Sansom, O.J. et al. Genes Dev. 2004, 18:1385—1390).

The inventors of the present application have found that the reason for homeostasis maintenance following CKla ablation is that parallel to Wnt activation, CKla ablation induces several tumor-suppressor ys, among which are DNA damage response (DDR), cellular ence and p53 pathway activation (Elyada E et al. Nature 2011, 470: 409-413, Pribluda A et al. Cancer Cell 2013, 24: 1-5). s the molecular mechanisms underlying the tion of these anti- stic pathways are still elusive, the inventors have found that that CKla ablation induces disproportionally minor DNA damage, with no signs of ATM activation, indicating that CKlot-induced DDR and p53 activation likely entail uncommon lar mechanisms (Burstain I et al, unpublished). In addition, the inventors have found that CKla on s in the induction of a new type of an in?ammatory se, denoted parain?ammation, which is confined to the epithelium, with no common signs of in?ammatory response (in?ammatory cell infiltration, calor, rubor, tumor and dolor) (Pribluda A et al. Cancer Cell 2013, 24: 1-5, Lasry A and Ben-Neriah Y 2015, Trends in Immunology, V01. 36: 217-228). Parain?ammation cooperates with WT p53 02429524\45-Ol tion in suppressing tumorigenesis, yet switches to a tumor promoting mechanism in the absence of functional p53 Pribluda A et al. Cancer Cell 2013, 24: 1-5, Aran et al., Genome Biol. 2016 Jul 8;17(1):145).

Whereas it is already established that CKla is a major regulator of p53, the inventors have also found that the combined ablation of CKIES and CKla in the gut epithelium also s in p53 activation, which may synergize with CKla-induced p53 activation.

IRAKl was identified as a therapeutic target for MDS, and certain subsets of AML and triple negative breast cancer (Garrett W. Rhyasen et al, 2013, Cancer Cell 24, 90—104, Rhyasen GW, Bolanos L, Starczynowski DT, 2013, Exp Hematol. 41:1005-7, Zhen Ning Wee et al, 2015, NATURE ICATIONS, 6:8746).

IRAKl mRNA is over-expressed in ~20-30% of MDS patients and the IRAKl protein is dramatically over-expressed and is hyperactivated in a majority of MDS marrow sample examined. IRAKl is a serine/threonine kinase that mediates signals elicited from Toll- like receptor (TLR) and Interleukin-l Receptor (ILlR). Following receptor tion, IRAKl becomes phosphorylated which then leads to recruitment of TRAF6, resulting in TRAF6 activation of NF-KB and JNK pathways. The molecular source of IRAKl overexpression and/ or hyperactivation in MDS (or AML) is not conclusive. It is thought that over-expression of TLR or necessary cofactors in MDS clones may result in c IRAKl activation even in the absence of infection. Small molecule inhibitors targeting IRAKl /4 Inhibitor, Amgen Inc.) have been originally developed for autoimmune and in?ammatory diseases. Given that IRAKl is ctivated (i.e., phosphorylated) in MDS but not normal marrow cells, Starczynowski and colleagues showed that IRAK- Inhibitor treatment (IRAKl/4, Amgen) and the knockdown of IRAKl resulted in dramatic impairment of MDS cell proliferation, progenitor function, and viability in vitro and in vivo. Yu and colleagues showed that IRAKl overexpression confers triple negative breast cancer cells (TNBC) growth advantage through NF-KB-related cytokine ion and metastatic TNBC cells exhibit gain of IRAKl dependency, resulting in high susceptibility to genetic and pharmacologic tion of IRAKl. Paclitaxel ent of TNBC cells s strong IRAKl phosphorylation, an increase in in?ammatory cytokine expression, enrichment of cancer stem cells and acquired resistance to axel 02429524\45-0 l treatment. Pharmacologic inhibition of IRAKl was able to reverse paclitaxel resistance by triggering massive apoptosis. IRAKl was also found to be a DEK riptional target and is essential for head and neck cancer cell survival (Adams AK et al.

Oncotarget. 2015, 22; 6(41): 43395 - 43407) and also as potential target in the treatment of in?ammatory- and immune-related ers (Bahia MS et al. Cell Signal. 2015 Jun;27(6):1039-55).

The inventors have thus found that compounds of the invention are able to inhibit IRAKl, an important upstream regulator of the NF-kB pathway which plays an important role in hematological malignancies.

GENERAL PTION The present invention provides a compound having the general formula (1), ing any stereoisomer or salt thereof: R3 R4 NH <\ / R5 R7 / wherein R1 and R2 are each independently selected from H, straight or branched C1 — Cg alkyl, ht or branched C1 — C5 alkoxy, straight or branched C1 — C5 acyl, C5 — C15 aryl, C3 — C7 heteroaryl each optionally substituted by at least one of halide, hydroxyl, ester, ether, C5 — C15 aryl, C3 — C7 heteroaryl, and amide; or R1 and R2 er with the nitrogen atom they are connected to form a 4 — 7 membered saturated, unsaturated or aromatic ring that may optionally e at least one of N, O, NH, C=N, C=O or $02 and can optionally be substituted with at least one of 02429524\45-Ol ht or branched C1 — C5 alkyl, C5 — C15 aryl, C3 — C7 heteroaryl, hydroxyl, halide and cyano; R3 and R4 are each independently selected from H, straight or branched C1 — Cg alkyl optionally substituted by at least one of halide, hydroxyl, alkoxy, C5 — C15 aryl, C3 — C7 aryl, ester and amide; or R1 or R2 together with R3 and the carbon and nitrogen atom they are each connected to form a 4 — 7 membered saturated unsaturated or aromatic ring that may ally include at least one of N, NH, O, C=N, C=O, $02, and can ally be substituted with at least one of straight or branched C1 — C5 alkyl, C5 — C15 aryl, C3 — C7 aryl, hydroxyl, carbonyl, and halide; R5 and Rg are each independently selected from H, halide, straight or branched C1 — Cg alkyl, straight or branched C2 — Cg alkenyl, straight or ed C2 — Cg all R6 is selected from straight or branched C1 — Cg alkyl, straight or branched C2 — Cg alkenyl, straight or branched C2 — Cg alkynyl, C5 — C10 cycloalkyl, saturated or unsaturated 4 — 6 membered heterocyclyle; optionally tuted by at least one of straight or branched C1 — Cg alkyl, C3 — C7 cycloalkyl, 4 — 6 membered heterocyclyle, C5 — C15 aryl, C3 — C7 aryl, halide, hydroxyl, C1 — C5 alkyl ; R7 is selected from straight or branched C1 — C8 alkyl, ht or branched C2 — Cg alkenyl, straight or branched C2 — Cg alkynyl; substituted by at least one C3 — C7 cycloalkyl, 4 — 6 membered heterocyclyle, C5 — C15 aryl, C3 — C7 heteroaryl, halide, hydroxyl, C1 — C5 alkyl .

The present invention provides a compound haVing the general formula (1), including any isomer or salt thereof wherein: R1 and R2 are each independently selected from H, straight or branched C1 — Cg alkyl, straight or branched C2 — C8 alkenyl, straight or branched C2 — C8 alkynyl, straight or branched C1 — C5 alkoxy, ht or branched C1 — C5 acyl, C5 — C15 aryl, C3 — C7 heteroaryl each optionally substituted by at least one of halide, hydroxyl, ester, ether, C5 — C15 aryl, C3 — C7 heteroaryl and amide; or R1 and R2 together with the nitrogen atom they are connected to form a 4 — 7 membered saturated unsaturated or aromatic ring that may optionally include at least one of N, O, NH, C=N, C=O or $02 and can optionally be substituted with at least one of 02429524\45-Ol straight or branched C1 — C5 alkyl, straight or branched C2 — C5 alkenyl, straight or branched C2 — C5 alkynyl, C5 — C15 aryl, C3 — C7 heteroaryl, hydroxyl, halide and cyano; R3 and R4 are each independently selected from H, straight or branched C1 — Cg alkyl, straight or branched C2 — Cg alkenyl, straight or branched C2 — Cg alkynyl, optionally tuted by at least one of halide, hydroxyl, alkoxy, ester, C5 — C15 aryl, C3 — C7 heteroaryl and amide; or R1 or R2 together with R3 and the carbon and nitrogen atom they are connected to form a 4 — 7 membered saturated unsaturated or ic ring that may optionally include at least one of N, NH, O, C=N, C=O, $02, and can optionally be substituted with at least one of straight or branched C1 — C5 alkyl, straight or branched C2 — C5 alkenyl, straight or branched C2 — C5 alkynyl, C5 — C15 aryl, C3 — C7 heteroaryl, hydroxyl, yl, and halide; R5 and R8 are each independently selected from H, halide, straight or branched C1 — Cg alkyl, straight or branched C2 — Cg alkenyl, straight or branched C2 — Cg alkynyl, optionally substituted by at least one halide (in some embodiments CF3); R6 is selected from straight or branched C1 — C8 alkyl, straight or branched C2 — Cg alkenyl, straight or branched C2 — Cg alkynyl, C5 — C10 cycloalkyl, saturated or unsaturated 4 — 6 membered heterocyclyle; ally substituted by at least one of straight or branched C1 — C8 alkyl, C3 — C7 cycloalkyl, 4 — 6 membered heterocyclyle, C5 — C15 aryl, C3 — C7 heteroaryl, halide, hydroxyl, C1 — C5 alkyl halid; R7 is selected from straight or branched C1 — Cg alkyl, straight or ed C2 — Cg l, straight or branched C2 — C8 alkynyl; substituted by at least one C3 — C7 cycloalkyl, 4 — 6 ed heterocyclyle, C5 — C15 aryl, C3 — C7 heteroaryl, halide, hydroxyl, C1 — C5 alkyl halide.

In some embodiments, R1 and R2 are each independently selected from H, straight or branched C1 — Cg alkyl optionally tuted by at least one of halide, yl, ester and amide.

In some embodiments, R1 and R2 are each independently selected from H, straight or branched C1 — C5 alkoxy optionally tuted by at least one of halide, hydroxyl, ester and amide. 02429524\45-Ol In some embodiments, R1 and R2 are each independently selected from H, C1 — C5 acyl, optionally substituted by at least one of halide, hydroxyl, ester,ether and amide.

In other ments, R1 and R2 are each independently selected from H, C5 — C15 aryl, optionally substituted by at least one of halide, hydroxyl, ester,ether and amide.

In some embodiments, at least one of R1 and R2 is H.

In some embodiments, R4 is H. In some embodiments, R3 and R4 are H.

In some embodiments, R5 is selected from H, Cl and straight or branched C1 — C4 alkyl. In some embodiments, R5 is H. In some embodiments, R8 is ed from H, Cl and straight or branched C1 — C4 alkyl. In some embodiments, R8 is H. In some further embodiment one of R5 or R8 is H (i.e. only one of R5 or R8 is H, in other words one of R5 or R8 is different than H).

In some embodiments, R6 is selected from straight or branched C1 — Cg alkyl, C5 — C10 cycloalkyl, saturated or unsaturated 4 — 6 membered heterocyclyle; and R7 is ed from straight or branched C1 — C8 alkyl, substituted by at least one C3 — C7 cycloalkyl, 4 — 6 membered heterocyclyle, C5 — C15 aryl, C3 — C7 heteroaryl, halide, hydroxyl, C1 — C5 alkyl halide.

In some embodiments, R6 is selected from a straight or branched C1 — C8 alkyl, C5 — C10 cycloalkyl, 4 — 6 membered saturated heterocyclyle.

In some embodiments, R7 is a ht or ed C1 — C8 alkyl substituted by at least one of C3 — C7 cycloalkyl and hydroxyl.

In some embodiments, R6 is selected from straight or branched C1 — C8 alkyl, saturated or unsaturated 4 — 6 ed heterocyclyle, each optionally substituted by at least one of ht or branched C1 — Cg alkyl, C3 — C7 cycloalkyl, halide, hydroxyl, CF3.

In some embodiments, R7 is a ht or branched C1 — C8 alkyl substituted by at least one C3 — C7 cycloalkyl. 02429524\45-Ol In some embodiments, R1 and R2 together with the nitrogen atom they are connected to form a 4 — 7 membered saturated ring optionally including at least one of N or O, NH, C=N, C=O or $02 (i.e. in addition to the N atom R1 and R2 are connected to) and can optionally be substituted with at least one of straight or branched C1 — C5 alkyl, hydroxyl, halide and cyano.

In some embodiments, R1 and R2 together with the nitrogen atom they are connected to form a 4 — 7 membered saturated ring.

In some ments, R1 and R2 er with the nitrogen atom they are connected to form a 4 — 7 membered saturated ring including at least one of N or O (in addition to the N atom R1 and R2 are connected to).

In further embodiments, R1 and R2 together with the nitrogen atom they are connected to form a 4 — 7 ed aromatic ring optionally including at least one of N or O (in addition to the N atom R1 and R2 are ted to).

In some ments, R1 or R2 together with R3 and the carbon and nitrogen atom they are connected to form a 4 — 7 membered saturated ring that optionally includes at least one of N, NH, O, C=O, $02, and can ally be substituted with at least one of straight or branched C1 — C5 alkyl, hydroxyl, carbonyl, and halide.

In some ments, R1 or R2 together with R3 and the carbon and nitrogen atom they are connected to form a 4 — 7 membered saturated ring that includes at least one of NH, O, or C=O.

In some embodiments, the compound of the invention is selected from the following: 02429524\45-Ol A29-1 A29 0\ A19-4 0 02429524\45-0 1 A19 OH 02429524\45-01 A30-1 A30-2 02429524\45-01 _ 12 _ "H N‘ ,0 W \N \ (\N / A65 \é, A68 i H, /" O "FM / A71 cuN H N :N‘ "‘0’ If; Cl A74 ©/\ "'Hx‘ "N H \ \hlr/N\ A75 \ CI H N :N‘ N \ NO, W/ CI A76 / HYK 1)" . .J / H H" A80 g H N W A81 N’ CE "0’ 1/_N /NN‘ \ H H N\ \ A82 H 02429524\45-01 In some ments, the compound of the invention is: 02429524\45-01 The term "straight or branched C1 — C8 alkyl" should be understood to encompass a hydrocarbon saturated chain, which can be ht or branched, comprising 1, 2, 3, 4, , 6, 7, or 8 carbon atoms.

The term "straight or branched C2 — C8 alkenyl" or "straight or branched C2 — C5 alkenyl" should be understood to encompass a hydrocarbon chain having at least one double bond between any two carbon atoms in the chain, which can be straight or branched, comprising 2, 3, 4, 5, 6, 7, or 8 carbon atoms or 2, 3, 4, 5 carbon atoms, tively.

The term ght or branched C2 — C8 alkynyl" should be understood to encompass a hydrocarbon chain haVing at least one triple bond between any two carbon atoms in the chain, which can be straight or branched, comprising 2, 3, 4, 5, 6, 7, or 8 carbon atoms.

The term "straight or branched C1 — C5 alkoxy" should be tood to encompass an —OR9 moiety wherein R9 is a straight or branched C1 — C5 alkyl. 02429524\45-0 l The term "halide" should be understood to encompass any halogen radical selected from —F, -Br, -Cl, -1.

The term "C1 — C5 alkyl halide " should be understood to ass any ht or branched alkyl chain having between 1 to 5 carbon atoms being substituted by at least one halogen radical selected from —F, -Br, -Cl, -1, at any point one the straight or branched chain. In some embodiments alkyl halide includes one halogen; in other embodiments alkyl halide includes two halogen atoms (the same or different); in other embodimebts, alkyl halide includes three halogen atoms (the same or different) and so on.

The term "hydroxyl" should be understood to encompass —OH.

The term "ester" should be understood to encompass any of OR10 or -OC(=O)R10 wherein R10 is a straight or branched C1 — Cg alkyl.

The term "amide" should be understood to encompass any of — C(=O)NR11R12', (=O)R12' wherein R11 and R12' are each independantly H or a straight or branched C1 — Cg alkyl.

The term "ether" should be understood to encompass any of —R130R14' or —OR15' wherein R13 is selected from a straight or branched C1 — Cg alkylene and R14' and R15' are each indpependantly selected from a straight or ed C1 — C8 alkyl.

The term ght or branched C1 — C5 acyl" should be understood to encompass any —C(=O)R16 wherein R16 is C1 — C5 stright or branched alkyl.

The term "C5 — C15 aryl" should be understood to ass any single or fused aromatic ring system comprising 5 to 7 carbon atoms. Examples include, but are not limited to phenyl, pentalenyl, naphatalenyl, anthracenyl, and any combinations thereof.

The term "C3 — C7 heteroaryl" should be understood to encompass any single or fused aromatic ring system comprising 5 to 7 carbon atoms and at least one heteroatom selected from N, O and S. Examples include, but are not limited to furanyl, benzofuranyl, 02429524\45-Ol isobenzofuranyl, pyrrolynyl, indolynyl, isoindolinyl, thiophenyl, hiophenyl, banzo[c]thiophenyl, imidazolyl, benzimidazolyl, purinyl, pyrazolyl, indazolyl, oxazolyl, benzoxazolyl, isoxazolyl, benzisoxazolyl, thiasolyl, benzothiazolyl, pyridinyl, auinolinyl, isoquinolinyl, pyromodinyl, quinzolinyl, pyridazinyl, cinnolinyl and any combinations When referring to the embodiment wherein R1 and R2 together with the nitrogen atom they are connected to form a 4 — 7 membered ted unsaturated or aromatic ring should be understood to relate to any ring that may be formed haVing 4, 5, 6, or 7 members including said nitrogen atom. Said ring can be saturated, i.e. haVing all sigma bonds, unsaturated, i.e. haVing at least one double or at least one triple bond or any ations thereof or aromatic, i.e. a ring system that possess aromatic character, cyclically conjugated molecular ring system with a stability (due to delocalization ) icantly greater than that of a hypothetical localized structure (e. g. Kekule structure).

For example, said ring can be selected from piperidinyl, pyrrolidinyl, azetidinyl and so forth.

In some embodiments said ring may optionally include (within the ring members) at least one of N, O, NH, C=N, C=O or $02. In some further embodiments, said ring may be optionally substituted (on the ring system by substitution of an -H atom on said ring) with at least one of straight or branched C1 — C5 alkyl, yl, halide and cyano (-CN).

When referring to the embodiments wherein R1 or R2 er with R3 and the carbon and nitrogen atom they are ted to form a 4 — 7 membered saturated unsaturated or aromatic ring should be understood to relate to any ring that may be formed haVing 4, 5, 6, or 7 s including said nitrogen atom. This ring forms a spiro bi-ring system with the cyclohexyl ring in the backbone of nd of formula I. Said ring can be saturated, i.e. haVing all sigma bonds, or unsaturated, i.e. haVing at least one double or at least one triple bond or any ations thereof. In some embodiments, the ring is an aromatic ring In some embodiments, said ring optionally includes at least one of N, NH, O, C=N, C=O, 802 within the ring formation. In some further embodiments, said ring is 02429524\45-Ol optionally substituted (on the ring system by substitution of an -H atom on said ring) with at least one of straight or ed C1 — C5 alkyl, hydroxyl, carbonyl (-C(=O)R n R is H or C1 — C5 straight or branched alkyl), and halide.

The term "C5 — C10 cycloalkyl" or the term "C3 — C7 cycloalkyl" should be understood to encompass a saturated (i.e. the ring containing only sigma bonds between its members) hydrocarbon ring that comprises 5, 6, 7, 8, 9, or 10 carbon atoms or 3, 4, 5, 6, or 7 carbon atoms respectively.

The term "saturated 0r aromatic 4 — 6 membered heterocyclyle" , unsaturated should be understood to encompass a saturated (i.e. the ring containing only sigma bonds between its members) unsaturated or aromatic (i.e. the ring containing at least one double bond or at least one triple bond or any ations thereof) ring containing 4, 5, or 6 members at least one of which is a atom selected from N, O, S, P.

The term "optionally substituted" as used herein means that the groups in question are either unsubstituted or tuted with one or more of the substituents specified.

When the groups in question are substituted with more than one substituent the substituents may be the same or different.

Certain of the compounds described herein may contain one or more chiral center, or may otherwise be capable of existing as two enantiomers or several diastereomers.

Accordingly, the compounds of this invention include also es of enantiomers as well as purified enantiomers or enantiomerically enriched mixtures. The compounds of this invention include also mixtures of diastereomers, as well as purified diastereomers or diastereomerically ed mixtures.

The invention also includes any salt of a compound of formula (1), including any pharmaceutically acceptable salt, wherein a compound of the invention has a net charge (either positive or negative) and at least one counter ion (having a counter negative or positive charge) is added o to form said salt. The phrase aceutically acceptable salt(s)", as used herein, means those salts of nds of the invention that are safe and effective for pharmaceutical use in mammals and that possess the desired 02429524\45-Ol ical activity. Pharmaceutically acceptable salts e salts of acidic or basic groups present in compounds of the invention. Pharmaceutically acceptable acid addition salts include, but are not d to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid ate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, ate, onate, saccharate, formate, te, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e., l,l'-methylene-bis-(2-hydroxynaphthoate)) salts. Certain compounds of the invention can form pharmaceutically acceptable salts with various amino acids. Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts. For a review on pharmaceutically acceptable salts see BERGE ET AL., 66 J. PHARM. SCI. 1-19 (1977), incorporated herein by reference.

The invention further provides a ition comprising at least one compound as defined herein above and below (according to l formula I).

The present invention also relates to pharmaceutical compositions comprising a compound of the subject invention in admixture with pharmaceutically acceptable auxiliaries, and optionally other therapeutic agents. The auxiliaries must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipients thereof. ceutical compositions include those suitable for oral, rectal, nasal, topical ding transdermal, buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and ermal) administration or administration via an implant. The compositions may be prepared by any method well known in the art of pharmacy.

Such methods include the step of bringing in association compounds of the invention or combinations f with any auxiliary agent. The auxiliary agent(s), also named accessory ingredient(s), include those conventional in the art, such as carriers, 02429524\45-Ol fillers, binders, diluents, disintegrants, lubricants, colorants, ?avouring agents, anti- oxidants, and wetting agents.

Pharmaceutical compositions suitable for oral administration may be presented as discrete dosage units such as pills, tablets, dragees or capsules, or as a powder or es, or as a solution or suspension. The active ingredient may also be presented as a bolus or paste. The compositions can further be processed into a suppository or enema for rectal administration.

The invention further includes a pharmaceutical composition, as hereinbefore described, in combination with packaging material, including instructions for the use of the ition for a use as hereinbefore described.

For parenteral administration, suitable itions include aqueous and non- s sterile injection. The compositions may be presented in ose or multi-dose containers, for example sealed vials and ampoules, and may be stored in a freeze-dried (lyophilised) condition requiring only the on of sterile liquid carrier, for example water, prior to use. For transdermal administration, e.g. gels, patches or sprays can be contemplated. itions or formulations le for pulmonary administration e. g. by nasal inhalation include fine dusts or mists which may be generated by means of metered dose rized aerosols, nebulisers or ators.

The exact dose and regimen of administration of the composition will necessarily be ent upon the therapeutic or nutritional effect to be achieved and may vary with the particular formula, the route of administration, and the age and condition of the individual subject to whom the composition is to be administered.

The invention further provides a compound as defined herein above and below ding to general formula I), for use in therapy.

The term "treatment" or "therapy" as used herein means the management and care of a patient for the purpose of combating a disease, er or condition. The term is intended to include the delaying of the progression of the disease, disorder or condition, 02429524\45-Ol the alleviation or relief of symptoms and complications, and/or the cure or elimination of the disease, disorder or condition. The patient to be d is preferably a mammal, in particular a human being.

It should be understood that the dosage ranges set forth above are exemplary only and are not intended to limit the scope of this invention. The therapeutically ive amount for each active compound can vary with factors including but not limited to the ty of the compound used, stability of the active compound in the t's body, the severity of the conditions to be alleviated, the total weight of the patient treated, the route of administration, the ease of tion, distribution, and excretion of the active compound by the body, the age and sensitivity of the patient to be treated, and the like, as will be apparent to a skilled artisan. The amount of stration can be adjusted as the various factors change over time.

For oral delivery, the active compounds can be incorporated into a formulation that includes pharmaceutically acceptable carriers such as binders (e. g., gelatin, cellulose, gum tragacanth), ents (e.g., starch, e), lubricants (e.g., magnesium stearate, silicon dioxide), disintegrating agents (e.g., alginate, Primogel, and corn starch), and sweetening or ?avoring agents (e.g., glucose, sucrose, saccharin, methyl salicylate, and peppermint). The formulation can be orally delivered in the form of enclosed gelatin capsules or compressed s. Capsules and tablets can be prepared in any conventional techniques. The capsules and tablets can also be coated with various gs known in the art to modify the ?avors, tastes, colors, and shapes of the capsules and tablets. In addition, liquid carriers such as fatty oil can also be included in capsules. le oral formulations can also be in the form of suspension, syrup, chewing gum, wafer, elixir, and the like. If d, conventional agents for modifying ?avors, tastes, colors, and shapes of the special forms can also be included. In addition, for convenient administration by enteral feeding tube in patients unable to swallow, the active compounds can be dissolved in an acceptable lipophilic vegetable oil vehicle such as olive oil, corn oil and saf?ower oil. 02429524\45-Ol The active compounds can also be administered parenterally in the form of solution or suspension, or in lyophilized form capable of conversion into a solution or sion form before use. In such formulations, diluents or pharmaceutically acceptable carriers such as sterile water and physiological saline buffer can be used. Other conventional solvents, pH buffers, stabilizers, anti-bacteria agents, surfactants, and antioxidants can all be included. For example, useful components include sodium chloride, acetates, citrates or phosphates buffers, glycerin, dextrose, fixed oils, methyl parabens, polyethylene glycol, propylene glycol, sodium bisulfate, benzyl alcohol, ascorbic acid, and the like. The parenteral formulations can be stored in any conventional containers such as vials and ampoules.

Routes of topical administration include nasal, bucal, mucosal, rectal, or vaginal applications. For topical administration, the active compounds can be formulated into lotions, creams, ointments, gels, powders, pastes, sprays, sions, drops and aerosols.

Thus, one or more thickening agents, humectants, and stabilizing agents can be included in the formulations. es of such agents include, but are not limited to, polyethylene glycol, ol, xanthan gum, petrolatum, beeswax, or l oil, lanolin, squalene, and the like. A special form of topical stration is delivery by a transdermal patch.

Methods for preparing transdermal s are sed, e.g., in Brown, et al. (1988) Ann. Rev. Med. 392221-229 which is incorporated herein by nce.

Subcutaneous implantation for sustained e of the active compounds may also be a suitable route of administration. This entails al procedures for ting an active compound in any suitable formulation into a subcutaneous space, 6. g., beneath the anterior abdominal wall. See, e.g., Wilson et al. (1984) J. Clin. Psych. -247.

Hydrogels can be used as a carrier for the sustained release of the active compounds.

Hydrogels are generally known in the art. They are typically made by crosslinking high molecular weight biocompatible polymers into a network, which swells in water to form a gel like material. In some instances, hydrogels are biodegradable or biosorbable. For purposes of this invention, els made of polyethylene glycols, collagen, or poly(glycolic-co-L-lactic acid) may be useful. See, e.g., Phillips et al. (1984) J.

Pharmaceut. Sci, 73: 720. 02429524\45-Ol The invention further provides a compound as d herein above and below (according to general formula I), for use in the inhibition of and least one of Casein kinase I (CKI) and Interleukin-l receptor-associated kinase 1 (IRAKl). In some embodiments, a compound as defined herein above and below ding to general formula I), is used in the inhibition of CKI and IRAKl. Under the above embodiments, the use of a compound of the invention as defined herein above and below (according to formula 1) enables the treatment of diseases, disorders, symptoms and conditions associated with at least one of CKI and IRAKl (or in some embodiments, both CKI and IRAKl). Under such aspects the invention provides treatment of es, disorders, symptoms and conditions associated with the inhibition of at least one of CKI and IRAKl (or in some embodiments, both CKI and .

In another one of its aspects the invention provides a compound as defined herein above and below (according to general a I), for use in the inhibition of Interleukin- 1 receptor-associated kinase 1 (IRAKl).

The invention further provides a nd as defined herein above and below (according to general formula I), for use in the inhibition of Casein kinase I (CKI).

The term "Casein kinase I" should be understood to encompass a protein kinases family that are serine/threonine-selective enzymes that on as regulators of signal transduction ys in most eukaryotic cell types. CKl isoforms are involved in Wnt signaling, circadian rhythms, nucleo-cytoplasmic shuttling of transcription factors, DNA repair, p53 activation and DNA transcription.

The term "Interleukin-1 receptor-associated kinase 1" should be understood to encompass an enzyme encoded by the IRAK] gene which was found to be an important upstream regulator of the NF-kB pathway involved in disease pathways of logical malignancies, such as multiple myeloma, MDS, leukemia and ma, breast cancer, head and neck , in?ammatory and immune related disorders and others. 02429524\45-Ol When referring to the "inhibition" of said enzyme, it should be understood to encompass any qualitative or quantitative decrease in the activity of said enzyme due to direct or indirect binding of at least one compound of the invention to said enzyme.

The ion r provides a compound as defined herein above and below (according to general formula I), for use in the treatment of a condition, symptom or disease associated with a malignant condition.

In some embodiments, said malignant condition is cancer. In other embodiments, said cancer has either WT, or mutant p53 (mutations that deactivate p53 typical of cancer conditions). In further embodiments, said cancer is ed from leukemia, malignant melanoma, breast cancer, prostate cancer and colorectal cancer. In some embodiments, said cancer has WT p53.

The invention further provides a compound as defined herein above and below, for use in the ent of cancer having WT p53, wherein said WT p53 is a biomarker for the said compound efficacy. Thus, under this aspect WT p53 serves as a measurable indicator to the efficacy of cancer treatment of the compound or composition comprising a compound of the invention. The invnetion further provides a method of treating cancer having WT p53 in a subject in need thereof, wherein said WT p53 is a biomarker for the said compound efficacy In some embodiments, said use further comprises an induction of cancer immunotherapy response. Thus, in some embodiments of the invention a compound or a composition sing a compound of the ion provides both treatment of cancer (anti-tumor, anti-malignant activity) and cancer immunotherapy response.

In some embodiments, said malignant condition is selected from hematological malignancies (Multiple Myeloma, Myelodysplastic me (MDS), Acute Myeloid Leukemia (AML), Melanoma and ER-negative breast cancer, Diffuse Large B cell lymphoma (DLBCL), Chronic enous ia (CML), Chronic lymphocytic leukemia (CLL), head and neck , and any combinations thereof. 02429524\45-Ol In r one of its aspects the invnetion provides a compound as d herein above and below for use in inducing anti-tumor response. In some embodiments, said anti-tumor response ses cancer immunotherapy response.

The term "induced anti-tumor response" should be understood to encompass any qualitative or quantitative chemotherapy of cancerous tumors.

The term "cancer immunotherapy response" should be understood to ass any qualitative or tative cancer immunotherapy induction of the subject's own immune system to fight the cancerous cells. Typically, immunotherapies can be categorized as active, passive or hybrid (active and e), and are designed to exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system of a subject, known as tumour-associated antigens ; they are often proteins or other macromolecules (e. g. carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapies enhance existing anti-tumor responses.

In some embodiments, said cancer immunotherapy reasponse relates to the change in the expression of an immune checkpoint molecules on tumor cell, on an n presenting cell, on a T cell or on a Natural Killer (NK) cell.

In some embodiments, said cancer immunotherapy reasponse s to the reduction in the expression of an immune checkpoint molecule on tumor cell that s suppression of the anti-tumor activity of a T cell.

In some embodiments, said cancer immunotherapy reasponse relates to the reduction in the expression of the checkpoint protein PD-Ll. In some other embodiments, said immunotherapy response relates to the inhibition of PD-Ll. In some further aspcts, a compound of the invention is used in a method of inhibiting PD-Ll.

The invention further provides a compound as defined herein above and below (according to general formula I), for use in the ent of an in?ammatory and immune related disorder including a condition, symptom or disease associated therewith. 02429524\45-Ol When referring to "inflammatory and immune related disorders" it should be understood to relate to any type of disorder (including conditions, symptoms and diseases associated therewith) that are ble with eukin-l receptor associated kinase inhibitors. It has been shown for example that IRAKl is an indispensable element of IL- Rs and TLR pathways that can regulate the al levels of cytokines, and therefore can be employed to manage immune- and in?ammation-related disorders such as for example rheumatoid arthritis, atory bowel disease, psoriasis, gout, asthma and cancer (Bahia MS et al. Cell . 2015 Jun;27(6):lO39-55).

The invention further provides a method of treating a condition, symptom or disease associated with a malignant condition in a subject in need f, said method comprising the step of administering to said subject at least one compound as defined herein above and below (according to general formula I).

The invention further provides a method of inhibiting at least one of Casein kinase I (CKI) and Interleukin-l receptor-associated kinase 1 (IRAKl) in a subject in need thereof comprising the step of administrating to said subject at least one compound as d herein above and below (according to general formula I).

In another one of its aspects the invention provides a method of inhibiting Interleukin-l receptor-associated kinase 1 ) in a subject in need thereof comprising the step of administrating to said subject at least one compound as defined herein above and below (according to l formula I).

In some embodiments a method of the invnetion further comprising inducing cancer immunotherapy response in said subject.

In another one of its aspects the ion provides a method for inducing an cancer immunotherapy response in a subject in need thereof, said method comprising the step of stering to said subject at least one compound as disclosed herein above and below. 02429524\45-Ol The invention further es a method of treating an inflammatory and immune related disorder including a condition, symptom or disease associated therewith; said method ses administering to a subject in need thereof a compound as defined herein above and below (according to general a I).

The invention also provides a method of inhibiting Casein kinase I in a subject in need thereof comprising the step of administrating to said subject at least one compound as defined herein above and below (according to general formula I).

The invention also provides for the use of a nd as defined herein above and below (according to general a I), or a stereoisomer or salt thereof, in the manufacture of a medicament for treating a condition, symptom or disease associated with a ant condition. In certain embodiments the malignant condition is a cancer, e.g. a cancer which has WT p53. In certain embodiments WT p53 is a biomarker for the efficacy of said compound.

In certain embodiments the cancer is leukemia, multiple myeloma, malignant melanoma, breast cancer, prostate cancer, or colorectal cancer. In certain embodiments the malignant condition is multiple a, ysplastic syndrome (MDS), acute myeloid leukemia (AML), melanoma, ER-negative breast cancer, diffuse large B cell lymphoma (DLBCL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), or head and neck cancer.

The invention also provides for the use of a compound as defined herein above and below (according to general formula I), or a stereoisomer or salt thereof, in the manufacture of a medicament for inducing an immunotherapy response.

The ion also provides for the use of a compound as defined herein above and below (according to general formula I), or a isomer or salt thereof, in the manufacture of a ment for treating an inflammatory and immune related disorder.

BRIEF DESCRIPTION OF THE DRAWINGS In order to better understand the subject matter that is sed herein and to exemplify how it may be d out in practice, embodiments will now be described, by way of nonlimiting example only, with reference to the accompanying drawings, in which: (followed by 26A) s 1A and 1B show a dose response of the ted compounds of the invention (Fig. 1A compounds A36, A39, A6, A14, A35, A51, A29, A19-4, A41, A28, A42, A43, A46; Fig. 1B compounds A30-1, A30-2, A51, A60, A64, A65) - screen in RKO colorectal cell line.

RKO cells were incubated for 16 hours at 370C with indicated concentrations of the compounds and analyzed by Western Blot. Shown are ß-catenin and p53 stabilization and phosphorylation of H2AX (?H2AX), a marker of DNA damage, indicative of CKIa kinase tion. Note that whereas ß-catenin and p53 stabilization, and H2AX phosphorylation is a common effect of most compounds, some compounds do not stabilize ß-catenin, s close analogues (e.g., A19- 4), only stabilize ß-catenin. CKIa n levels serves as a loading control.

Figures 2A-2D show that the CKIa inhibitor A14 induces apoptosis of bone marrow cells isolated from CML blast crisis mice in a dose dependent manner (ex vivo). Fig. 2A is a schematic representation of the experimental procedure; Incubation with A14 or DMSO for 8 hours. Fig. 2B shows a dramatic reduction of the leukemic cell number following A14 treatment – a dose response curve. Fig. 2C shows the increased percentage of apoptotic cells (Annexin5+/7AAD-) in a dose dependent manner. Fig. 2D shows the increased tage of total death (7AAD+) cells in a dose dependent manner. (followed by 27) Figures 3A-3C demonstrate that A14 significantly reduces the leukemia cell burden in the peripheral blood and bone marrow in vivo in CML blast crisis mice. Fig. 3A is a schematic representation of the experimental procedure, inoculating BM cells from a CML blast crisis mouse (GFP+ cells) to normal C57Bl/6 mice and daily treatment (oral administration) with A14 or vehicle alone. Fig. 3B shows the percentage of GFP+ leukemia cells in peripheral blood at day 9 following treatment; A14 (N=6) ed to the vehicle treated mice (N=6). Fig. 3C shows the percentage of GFP+ leukemia cells in the bone marrow at day 9 following treatment: A14 (N=6) compared to the vehicle treated mice (N=6).

Figures 4A-4F show the te blood count at day 9 following A14 treatment of CML blast crisis mice: Fig. 4A shows the absolute number of White blood cells (WBC) in peripheral blood (109/L) (N=5). Fig. 4B shows the absolute number of Lymphocytes ) in peripheral blood (109/L) (N=5). Fig. 4C shows the absolute number of Monocytes (Mono) in peripheral blood (109/L) (N=5). Fig. 4D shows the absolute number of Granulocytes (Gran) in peripheral blood (109/L). Fig. 4E shows the red blood counts (RBC, 1012/L). Fig. 4F shows the hemoglobin level (HGB, g/L).

Figure 5 shows representative photographs of blood smears from A14 treated compared to vehicle-treated CML blast crisis mouse at day 9 following BM transplantation.

Figure 6 shows a schematic entation of preparation of the AML mouse model.

Figures 7A-7D show the inhibitory effect of A14 on the progression of AML. Fig. 7A is a schematic entation of the mental procedures. Fig. 7B demonstrates the percentage of GFP+ leukocytes in peripheral blood (PB) of A14 d compared to vehicle treated mice. Figs. 7C and 7D shows representative Flow Cytometry analysis plots of the GFP+ leukocytes in PB one day before (Fig. 7C) and three days (Fig. 7D) following A14 treatment. 02429524\45-Ol Figures 8A-8G show the complete blood count at day 9 after the treatment of AML mice with A14 of the invention. Fig. 8A shows the absolute number of white blood cells (WBC) in peripheral blood (109/L). Fig. 8B shows the absolute number of lymphocytes (Lymph) in peripheral blood ). Fig. 8C shows the absolute number of monocytes (Mono) in peripheral blood (109/L). Fig. 8D shows the absolute number of granulocytes (Gran) in peripheral blood (109/L). Fig. 8E shows the red blood counts (RBC, 1012/L). Fig. 8F shows the hemoglobin (g/L). Fig. 8G shows platelets (PLT) in eral blood (109/L).

Figure 9 shows representative photographs of blood smears from A14 compared to vehicle-treated AML mice at day 9 following the first A14 treatment.

Figure 10 shows IRAKl inhibition by the inhibitor compounds of the invention, A51 and A14. Cells were harvested and analyzed by Western blot. Blots were incubated with the following dies: Phospho-IRAKl (Thr209), (A1074, AssayBiothechnology; 1/ 1,000), Phospho-IKKa/B (Ser176/180) (16A6, Cell Signaling; 1/ 1,000), IKKa (2682 , cell signaling; 00), IKK B (2370 cell signaling; 1/1,1000), o-c-Jun (Ser 63) (9261, cell signaling; 1/1,1000),p53 (DO-1&1801 hybridoma mix; dilution of 1:20 of supernatants from each), CKIa (C-19; 1/1,000; Santa Cruz Biotechnology) and phospho- histoneH2AX (S139; 1/1,000; ore). Secondary dies were HRP-linked goat anti-mouse, goat anti-rabbit and rabbit anti-goat antibodies (all 1/10,000; Jackson).

Inhibition of Phospho-IRAKl, Phospho-IKKa/B and Phospho-c-Jun indicate IRAKl inhibition. p53 stabilization and phosphorylation of H2AX (yH2AX), a marker of DNA damage, indicative of CKIa kinase inhibition. CKIa n levels serves as a loading control.

Figures E relate to the experimental results achieved from a single-dose treatment of CKI inhibitor A51 in AML mice. Fig. 11A schematically shows the preparation of AML mice and their treatment with A51 (20mg/Kg) at day 30 from induction of the e (Leukemia inoculation). Figs 11B — 11E show the effect of A51 after 16hrs from treatment (in comparison with ent with vehicle alone) in reduction of the total leukemia cells in the blood: Fig. 11B shows the reduction in WBC in the peripheral blood (PB), Fig. 11C shows the shrinking of the leukemic spleen and Figs. 11D 02429524\45-0 l and 11E show the reduction of proportion of leukemic blasts (GFP+ cells) in the peripheral blood (PB) and bone marrow (BM), tively.

Figures 12A and 12B show es of spleen and bone from treated AML mice.

Actual reduction in spleen size (splenomegaly) after treatment with A51, as disclosed above (Fig. 12A) and opaque bones turned to normal color ing a single dose treatment (Fig. 12B).

Figures 13A-13E show the s of in vitro treatment effects of CKI inhibitors on AML cells isolated from the bone marrow of leukemic mice. Shown are the percentage of dead cells (7AAD+) and the effects of the inhibitors on the expression of the major immune checkpoint protein PD-Ll in the leukemic cells; tor treatment was in 10 or 100nM for several time points (5 hours, — Fig. 13A, 6 hours,— Figs. 13B and 13C, and 9 hours Figs. 13D and 13E).

DETAILED DESCRIPTION OF EMBODIMENTS Pre aration 0 E c clo r0 [ dimeth lamina 2- meth lthio rimidin lbut—3-en0ne CoreA and 4- 5- c clo r0 lmeth l- 1H- ra 0l l meth lthio rimidine CoreB S N I / \lr \ WHHCI /N M —O> kELNIO\ N / 3A "YN DMFDMA I /s N o HATUTEADCM LDA,THF,—78"C 80 "oz h /\N|r / CoreA —>/S\NfN;NH2NH2H2090 °c /Core B Step 1: N, O-Dimethylhydroxylamine hydrochloride (25.14 g, 257.69 mmol, 1.72 eq), HATU (56.97 g, 149.82 mmol, 1.00 eq) and TEA (45.48 g, 449.46 mmol, 3.00 eq) were added to a solution of 2-cyclopropylacetic acid (15.00 g, 149.82 mmol, 1.00 eq) in DCM (500 mL) at 0 CC, and then the e was stirred at 30 CC for 3 h. The resulting mixture was poured into water (500 mL). The aqueous washing phase was extracted with DCM (3*250 mL). The combined organic layers were dried over anhydrous NazSO4, filtered and concentrated. The residue was purified by column 02429524\45-0 l chromatography (SiOz, petroleum PE): ethyl acetate (EA) = 50:1 to 10: 1) to give the desired compound 2 (13.20 g, 82.97 mmol, 55.4% yield) as colorless liquid. 1H C13, 400 MHz) 5 3.65 (s, 3H), 3.17 (s, 3H), 2.33 (d, J: 6.8 Hz, 2H), 1.09-1.06 (m, 1H), 0.54—0.50 (m, 2H), 0.16-0.14 (m, 2H).

Step 2: To a on of 4-methylmethylsulfanyl-pyrimidine (9.00 g, 64.19 mmol, 1.00 eq) in THF (500 mL) was added LDA (2 M, 48.46 mL, 1.51 eq) at -78°C.

After stirring for 1 h, a solution of compound 2 (13.79 g, 96.29 mmol, 1.50 eq) in THF (500 mL) was added drop wise at -78 CC and then the reaction mixture was stirred at -78 CC for 4 h. Quenched with saturated aq. NH4Cl (100 mL), the aqueous phase was extracted with ethyl acetate (3 X 50 mL). The combined organic layers were washed with brine, dried over anhydrous NazSO4, filtered and concentrated. The residue was crystallized from petroleum ether/ethyl acetate to afford the desired compound 4 (13.60 g, 55.06 mmol, 85.8% yield) as a yellow solid.

LCMS: RT = 0.629 min, m/z 223.0 [M+H]+.

Step 3: A solution of compound 4 (13.60 g, 61.18 mmol, 1.00 eq) in DMF-DMA (51.42 g, 2.45 mol, 40 eq) was d at 90 CC for 2 h. The solvent was removed in vaccum. The residue was purified by column chromatography on silica gel to give Core A (10.60 g, 36.30 mmol, 59.3% yield) as a yellow solid.

LCMS: RT = 0.634 min, m/z 278.2 [M+H]+ 1H NMR(CDC13, 400 MHz) 5 8.38 (d, J = 6.8 Hz, 1H), 7.62 (s, 1H), 6.96 (s, 1H), .87 (m, 6H), 2.56 (s, 3H), 2.38 (d, J = 8.8 Hz, 2H), 1.04—1.02 (m, 1H), 0.52-0.46 (m, 2H), 0.09—0.04 (m, 2H).

Step 4: A solution of Core A (10.60 g, 38.21 mmol, 1.00 eq) and hydrazine hydrate (6.75 g, 114.63 mmol, 3.00 eq) in ethanol (200 mL) was d at 90 CC for 3 h.

The solvent was removed in vacuum. The residue was purified by column chromatography on silca gel to afford Core B (9.00 g, 35.81 mmol, 93.7% yield) as a light yellow solid.

LCMS: RT = 2.018 min, m/z 247.1 [M+H]+ 02429524\45-0 l 1H NMR(CDC13, 400 MHz) 5 8.43 (d, J = 5.2 Hz, 1H), 8.04 (s, 1H), 7.10 (d, J = .2 Hz, 1H), 3.12 (d, J = 7.2 Hz, 2H), 2.60 (s, 3H), 1.19-1.14(m, 1H), 0.66 (q, J = 5.2 Hz, 2H), 0.32 (q, J = 5.2 Hz, 2H).

Pre aration 0 4- 5- c clo r0 lmeth l-I-meth l-IH- razol l lsultonyllgyrimid ine (Core C 2 /S\fN\ MeNHNH N 2 /3\f \ N / 90°C N CoreA 5 + / 1 N s N / / Y \ Step 1: A solution of Core A (6.20 g, 22.35 mmol, 1.00 eq) and methylhydrazine (8.00 g, 69.46 mmol, 3.11 eq) in ethanol (100 mL) was stirred at 90 CC for 16 hrs. The solvent was removed in . The residue was purified by pre-HPLC (basic condition) to afford compound 5 (1.80 g, 6.84 mmol, 30.6% yield) as a yellow solid and the isomer 5A (2.00 g, 7.30 mmol, 32.6% yield) as a yellow oil.

Compound 5: LCMS: RT = 2.551 min, m/z 261.1 [M+H]+ 1H NMR(CDC13, 400 MHz) 5 8.38 (d, J = 5.2 Hz, 1H), 7.90 (s, 1H), 7.11 (d, J = .2 Hz, 1H), 3.93 (s, 3H), 3.24 (d, J: 6.4 Hz, 2H), 2.62 (s, 3H), 1.12 — 1.09 (m, 1H), 0.54 - 0.49 (m, 2H), 0.32-0.28 (m, 2H).

Regioisomer 5A: LCMS: RT = 2.486 min, m/z 261.1 [M+H]+ 1H NMR(CDC13, 400 MHz) 5 8.41 (d, J = 5.2 Hz, 1H), 7.90 (s, 1H), 7.04 (d, J = .2 Hz, 1H), 3.92 (s, 3H), 2.96 (d, J: 6.4 Hz, 2H), 2.61 (s, 3H), 1.20 — 1.17 (m, 1H), 0.50 - 0.45 (m, 2H), 0.26 — 0.22 (m, 2H).

Step 2: To a solution of compound 1 (1.50 g, 5.76 mmol, 1.00 eq) in DCM (20 mL) was added m-CPBA (2.98 g, 17.28 mmol, 3.00 eq) at 0 CC and stirred at 30 0C for 02429524\45-0 l 2 hrs. The resulting mixture was washed with NaHS03 (2 x 100 mL), saturate aq.

NaHCO3 (100 mL) and brine, dried over anhydrous NazSO4, filtered and concentrated.

The residue was purified by column chromatography on silica gel (petroleum ether: ethyl acetate = 10:1 to 1:1) to give the nd Core C (1.50 g, 5.08 mmol, 88.2% yield) as a yellow solid.

LCMS: RT = 1.891 min, m/z 293.0 [M+H]+ 1H C13, 400 MHz) 5 8.74 (d, J: 5.6 Hz, 1H), 7.97 (s, 1H), 7.58 (d, J: .6 Hz, 1H), 3.94 (s, 3H), 3.37 (s, 3H), 3.25 (d, J: 6.8 Hz, 2H), 1.14 — 1.11 (m, 1H), 0.52 - 0.49 (m, 2H), 0.37 — 0.35 (m, 2H). l res or the re aration o A-n: MethodA: preparation of compound n_6 A solution of Core B (25 mmol, 1.00 eq) in DMF (50 mL) was cooled to 0 CC, and NaH (1.50 eq) was added. After stirred at 0 CC for 1 h, RBr (1.60 eq) was added and the reaction mixture was stirred at 20 CC for 15 hrs. Water (10.00 mL) was added and extracted with ethyl acetate (3*10 mL). The combined organic layers were washed with water (10 mL) and brine (10 mL), dried over NazSO4, filtered and concentrated to give the crude product, which was purified to give compound n_6.

Method B: preparation of compound n_7 To a solution of compound n_6 (10 mmol, 1.00 eq) in dichloromethane (30 mL) was added m-CPBA (3.00 eq) at 0 CC and the reaction mixture was stirred at 20-30 0C for 5 h. The resulting mixture was washed with NaHSO3 (2 x 50.00 mL), saturate aq. 02429524\45-0 l NaHC03 (50 mL) and brine (10 mL), dried over anhydrous NazSO4, filtered and concentrated to give compound n_7.

Method C: preparation of compound A-n A mixture of compound n_7 (10 mmol, 1.0 eq), DIEA (10.00 eq) and trans amino- cyclohexanol (1.0~3.0 eq) in DMSO (80 mL) was heated at 160 CC for 3 hrs. The reaction mixture was cooled to ambient temperature and poured into water (100 mL) and then extracted with dichloromethane (3X50 ml). The combined layers were washed with brine, dried over anhydrous NazSO4, filtered and concentrated. The residue was purified by pre-HPLC (basic ion) to afford nd A-n.

Method D: preparation of compound A-n The on of compound n_7 (10 mmol, 1.00 eq) and trans-cyclohexane-1,4- diamine (2.0~4.0 eq) in dioxane (40 mL) was stirred at 130°C for 2h with microwave.

The mixture was filtered and the filtrate was purified by pre-HPLC (basic condition) to give A-n.

Pre aration 0 1r 4r -N1- 4- 5- c clo ro lmeth l-I-iso ro l-IH- razol- 4- l n-Z- lc clohexane-I 4-diamine A-35 N‘ J\ N\ NH2 H N mCPBA N Br S N , _> :NN\< H2N\0 —> / Y \ O, \f \ NaH’DMF N e MW Step 1: Compound 35_6 was synthesized according to the procedure described in method A and it was ed as a yellow solid after HPLC (TFA condition) purification.

Yield: 17.1%; LCMS: RT = 1.959 min, m/z 289.1 [M+H]+ 02429524\45-01 1H NMR(CDC13, 400 MHz) 5 8.49-8.47 (d, J = 2.8 Hz, 1H), 8.06 (s, 1H), 7.21— 7.20 (d, J = 5.6 Hz, 1H), .60 (t, J: 6.6 Hz, 1H), 3.26-3.24 (d, J: 6.4 Hz, 2H), 2.65 (s, 3H), 1.56 (s, 3H), 1.55 (s, 3H), 1.07—1.04 (m, 1H), 0.54—0.51 (m, 2H), 0.30-0.28 (m, Step 2: Compound 35_7 was synthesized according to the procedure described in method B and it was obtained as a yellow solid after purification h TLC. Yield: 57.8%; LCMS: RT = 0.711 min, m/z 321.1 [M+H]+ Step 3: Compound A-35 was synthesized according to the procedure described in method D with trans-cyclohexane-l, ine (4.0 eq) and it was obtained as a white solid. Yield: 19.7% LCMS: RT = 1.223 min, m/z 355.3 [M+H]+ 1H NMR(CDC13, 400 MHz) 5 8.17-8.16 (d, J = 5.2 Hz, 1H), 7.89 (s, 1H), 6.72— 6.71 (d, J = 5.2 Hz, 1H), 4.85-4.83 (d, J = 8.8 Hz, 1H), 4.60-4.53 (m, 1H), 3.23—3.22 (d, J = 6.4 Hz, 2H), 2.20—2.18 (d, J = 10 Hz, 2H), 2.02—1.99 (m, 2 H), 1.54 (s, 3H),1.52 (s, 3H), 1.40—1.27 (m, 4H), 1.08-1.06 (m, 1H), 0.52—0.48 (m, 2H), 0.28—0.26 (m, 2H).

Pre aration 0 1R 4R -N1- 4- 1-c clo ent [ c clo r0 lmeth l-IH- ra 0l l rimidin-Z- l c clohexane-I 4-diamine A-36 H2NVO’NHZ dioxane, MW HZN‘"0’ Step 1: Compound 36_6 was synthesized according to the procedure described in method A and it was ed as a yellow oil after purification by prep_HPLC (TFA ion). Yield: 14.7% 02429524\45-0 l _ 35 _ LCMS: RT = 2.228 min, m/z 315.2 [M+H]+ 1H C13, 400 MHz) 5 8.31-8.30 (d, J = 5.6 Hz, 1H), 7.84 (s, 1H), 7.03— 7.01 (d, J = 5.2 Hz, 1H), 4.67—4.63 (m, 1H), 3.20-3.18 (d, J = 6.4 Hz, 2H), 2.52 (s, 3H), 2.05—2.02 (m, 7H), 1.65—1.62 (m, 2H), 0.98 (m, 1H), 0.43—0.39 (m, 2H), 0.23—0.20 (m, Step 2: Compound 36_7 was made according to the procedure described in method B and it was obtained as a yellow solid after purification through column chromatography. Yield: 66.6% LCMS: RT = 0.707 min, m/z 347.2 [M+H]+ Step 3: Compound A-36 was made according to the ure described in method D with trans-cyclohexane-l, 4-diamine (2.0 eq) and it was obtained as a whit solid after purification by PLC (TFA condition). Yield: 41.35% LCMS: RT = 0.591 min, m/z 381.4 [M+H]+ 1H NMR (CD30D, 400 MHz) 5 8.26 (s, 1H), 8.08-8.06 (d, J = 6.4 Hz, 1H), 7.27— 7.25 (d, J = 6.8 Hz, 1H), 4.15 (s, 1H), 3.33 (s, 3H), 3.20 (s, 1H), 2.25—1.99 (m, 11H), 1.75 (m, 2H), 1.64-1.58 (m, 4H), 1.16 (m, 1H), .55 (m, 2H), 0.36—0.35 (m, 1H).

Pre aration 0 1R 4R -N1- 4- 5- c clo r0 lmeth l tetrah dro-ZH- l c clohexane-I 4-diamine A-39 HZN"0"" dloxane,' MW v HZN‘ O; Step 1: To a solution of Core B (700.00 mg, 2.84 mmol, 1.00 eq) in MeCN (14 mL) was added CSzCO3 (1.85 g, 5.68 mmol, 2.00 eq) at 0°C. After 30 min, 4- 02429524\45-0 l bromotetrahydropyran (703.03 mg, 4.26 mmol, 1.50 eq) was added. The mixture was stirred at 100°C for 16 h in a sealed tube. The reaction e was filtered and the filtrate was concentrated. The crude t was purified by prep-HPLC (TFA) to give compound 39_6 (45.0 mg, 136.18 umol, 4.8% yield) as a yellow solid.

LCMS: RT = 0.702 min, m/z 331.1 [M+H]+ 1H NMR(CDC13, 400 MHz) 5 8.42-8.40 (d, J = 5.6 Hz, 1H), 7.94 (s, 1H), 7.11— 7.10 (d, J = 5.2 Hz, 1H), 4.37—4.33 (m, 1H), 4.19—4.15 (m, 1H), .54 (m, 2H), 3.28- 3.26 (d, J: 6.4 Hz, 2H), 2.6 (s, 3H), 2.44—2.40 (m, 2H), 1.87-1.84 (m, 2H), 1.05—1.02611, 1H), 0.55-0.50 (m, 2H), 0.33—0.30 (m, 2H).

Step 2: Compound 39_7 was made according to the procedure described in method B and it was obtained as a yellow solid after prep TLC purification. Yield: 72.6% LCMS: RT = 0.607 min, m/z 363.1 [M+H]+ Step 3: Compound A-39 was made according to the procedure described in method C with trans-cyclohexane-l,4-diamine (3.0 eq) without DIEA and it was obtained as a yellow solid after purification through prep-HPLC (basic condition). Yield: 40.2% LCMS: RT = 1.104 min, m/z 397.4 [M+H]+ 1H NMR(CDC13, 400 MHz) 5 8.17-8.16 (d, J = 4.4 Hz, 1H), 7.89 (s, 1H), 6.72- 6.70 (d, J = 5.2 Hz, 1H), 5.12—5.07 (m, 1H), 4.37—4.34 (m, 1H), 4.18-4.15 (m, 2H), 3.84 (s, 1H), 3.59—3.53 (m, 2H), 3.26-3.25 (d, J: 6.4 Hz, 2H), 2.77 (s, 1H), .40 (m, 2H), 2.16 (s, 2H), 1.93 (s, 2H), 1.87-1.83 (m, 2H), 1.32—1.27 (m, 4H), 1.05 (s, 1H), .49 (m, 2H), 0.29—0.27 (m, 2H). 02429524\45-0 l Pre aration 0 1R 4R -N1- 4- 5- c clo r0 lmeth l 0xetan l -1H- razol l rimidin-Z- lc clohexane-I 4-diamine A-43 HZNVO’NHZ (1'onane, MW \-0’ Step 1: nd 43_6 was synthesized according to the procedure bed in method A and it was ed as a yellow solid after purification by prep-HPLC (TFA condition). Yield: 17.9% LCMS: RT = 0.664 min, m/z 303.1 [M+H]+ 1H NMR(CDC13, 400 MHz) 5 8.44-8.42 (d, J = 5.2 Hz, 1H), 8.04 (s, 1H), 7.13— 7.12 (d, J = 5.6 Hz, 1H), 5.89-5.34 (m, 1H), 5.28-5.25 (t, J: 6.6 Hz, 2H), 5.03-5.00 (t, J = 7.0 Hz, 2H), 3.21—3.20 (d, J = 6.4 Hz, 2H), 2.6 (s, 3H), 0.97—0.94 (m, 1H), 0.51-0.46 (m, 2H), 0.25—0.22 (m, 2H).

Step 2: Compound 43_7 was synthesized according to the procedure described in method B and it was obtained as a yellow solid after prep TLC purification. Yield: 79.6% LCMS: RT = 0.566 min, m/z 335.1 [M+H]+ Step 3: Compound A-43 was sized according to the procedure described in method D with trans-cyclohexane-l, 4-diamine (3.0 eq) and it was obtained as a white gum after purification by prep-HPLC (TFA condition). Yield: 24.7%; LCMS: RT = 0.987 min, m/z 369.3 [M+H]+ 1H NMR (CD30D, 400 MHz) 5 8.40 (s, 1H), 8.13-8.11 (d, J: 6.8 Hz, 1H), 7.31— 7.29 (d, J: 7.2 Hz, 1H), 5.83-5.80 (t, J: 7.0 Hz, 1H), 5.15—5.12 (t, J: 6.2 Hz, 2H), 5.06- .03 (t, J = 7.0 Hz, 2H), 4.12 (s, 1H), 3.28-3.26 (d, J = 6.4 Hz, 2H), 3.19 (s, 1H), 2.23— 2.17 (m, 4H), 1.62—1.60 (m, 4H), 1.06 (s, 1H), 0.53—0.52 (m, 2H), 0.28-0.27 (m, 2H). 02429524\45-01 Pre aration 0 R -tetrah dro uran l methanesul onate 41 4 HOD—’MsCIDCM Et3N O To a solution of trahydrofuranol (500.00 mg, 5.68 mmol, 1.00 eq) in DCM (5 mL) were added TEA (1.15 g, 11.36 mmol, 2.00 eq) and esulfonyl chloride (650.11 mg, 5.68 mmol, 1.00 eq) at 0°C. The mixture was stirred at 20°C for 1 hr. The mixture was diluted with water (20 mL) and extracted with DCM (10 mL*2). The combined organic layers were concentrated to give 41_4 (900.00 mg, 5.42 mmol, 95.3% yield) as a colorless liquid. 1H C13, 400 MHz) 5 5.33—5.30 (m, 1H), 4.06-3.90 (m, 4H), 3.05 (s, 3H), 2.28-2.23 (m, 2H).

Pre aration 0 IR 45 -N1- 4- 5- c clo r0 lmeth l S -tetrah dro uran- 3- l-IH- ra 0l l rimidin-Z- lc clohexane-I 4-diamine A-41 H2N"OM —> | dioxane, MW Hng‘OH\rN Step 1: Compound 41_6 was synthesized according to the procedure described in method A and it was ed as a yellow solid after purification by prep-HPLC (TFA condition). Yield: 13.1% 1H NMR (CDC13, 400 MHz) 5 8.42-8.40 (d, J = 4.4 Hz, 1H), 7.94 (s, 1H), 7.11— 7.10 (d, J = 7.2 Hz, 1H), 5.04—5.00 (m, 1H), 4.27—4.03 (m, 4H), .27 (d, J = 8.4 Hz, 2H), 2.6 (s, 3H), 2.48-2.42 (m, 2H), 1.07—1.02 (m, 1H), 0.53—0.49 (m, 1H), 0.30-0.28 (m, 02429524\45-01 Step 2: Compound 41_7 was synthesized according to the procedure described in method B and it was obtained as a yellow solid after prep TLC purification. Yield: 57.5% LCMS: RT = 0.585 min, m/z 349.2 [M+H]+ Step 3: Compound A-41 was sized according to the procedure bed in method D with trans-cyclohexane-l, 4-diamine (4.0 eq) and it was obtained as a white gummy solid after purification by prep HPLC (TFA condition). Yield: 17.2% LCMS: RT = 0.969 min, m/z 383.3 [M+H]+ 1H NMR (CD3OD, 400 MHZ) 5 8.26 (s, 1H), 8.10-8.09 (d, J: 6.8 Hz, 1H), 7.25- 7.23 (d, J: 6.8 Hz, 1 H), 5.28-5.23 (m, 1H), 4.22-4.12 (m, 3H), 3.99-3.95 (m, 2H), 3.35- 3.33 (d, J = 6.8 Hz, 2H), 3.19 (s, 1H), 2.47-2.44 (m, 2H), 2.35-2.17 (m, 4H), .57 (m, 4H), 1.14 (s, 1H), 0.58-0.56 (m, 2H), 0.36 (m, 2H).

Pre aration 0 S -tetrah dro uran l methanesul onate 42 4 Ho" DCM, Et3N M50" Compound 42_4 was made in the same way as 41_4 from (S)-tetrahydrofuran 01 and it was a ess liquid. Yield: 90.0% 1H NMR(CDC13, 400 MHz) 5 5.33-5.30 (m, 1H), 4.04-3.86 (m, 4H), 3.05 (s, 3H), 2.27—2.22 (m, 2H). 02429524\45-0 l Pre aration 0 1R 4R -N1- 4- 5- c clo r0 lmeth [ R -tetrah dro uran- 3- l-IH- razol l rimidin-Z- lc ane-I 4-diamine A-42 H2N"O’N" dioxane, MW H2N‘"0’ Step 1: Compound 42_6 was synthesized according to the procedure described in method A and it was obtained as a yellow solid after purification by prep HPLC (TFA condition). Yield: 21.4% LCMS: RT = 0.692 min, m/z 317.1 [M+H]+ 1H NMR (CDClg, 400 MHz) 5 8.42-8.40 (d, J = 4.4 Hz, 1H), 7.95 (s, 1H), 7.12— 7.10 (d, J = 7.2 Hz, 1H), 5.04—5.00 (m, 1H), 4.27—4.03 (m, 4H), 3.29—3.27 (d, J = 8.4 Hz, 2H), 2.6 (s, 3H), .42 (m, 2H), 1.06-1.03 (m, 1H), 0.55—0.50 (m, 1H), 0.31—0.29 (m, Step 2: Compound 42_7 was synthesized according to the procedure bed in method B and it was obtained as a yellow solid after prep TLC purification. Yield: 83.2% LCMS: RT = 0.587 min, m/z 349.1 [M+H]+ Step 3: Compound A-42 was synthesized according to the procedure described in method D and it was obtained as a white gummy solid after purification by prep HPLC (TFA condition). Yield: 51.5% LCMS: RT = 1.078 min, m/z 383.3 [M+H]+ 1H NMR , 400 MHz) 5 8.26 (s, 1H), 8.10-8.08 (d, J: 6.8 Hz, 1H), 7.28- 7.26 (d, J: 6.8 Hz, 1H), 5.27—5.24 (m, 1H), 4.22—4.13 (m, 3H), 3.99-3.96 (m, 2H), 3.35- 3.31 (t, J: 6.8 Hz, 2H), 3.19 (s, 1H), .44 (m, 2H), 2.36-2.17 (m, 4H), 1.63-1.58 (m, 4H), 1.15 (s, 1H), 0.58-0.56 (m, 2H), 0.36 (m, 2H). 02429524\45-0 l Pre aration 0 1R 4R -N1- 4- 5- c clo r0 lmeth l tetrah dro-ZH- l-IH- ra 0l l rimidin-Z- lc clohexane-I 4-diamine A-38 O NH2NHCbz Pd(OH)2 H2 0 CoreA o —, ":12 2'?" —> NaBHgCN MeOH MeOH EtOH 38_1 38_2 38_3 0‘ o /N\ o m—CPBA / N .4sz0’ —> | —> DCM N dioxaneMW HZN‘" Step 1: The mixture of 38_1 (1.20 g, 11.99 mmol, 1.40 eq) and benzyl N- aminocarbamate (1.42 g, 8.56 mmol, 1.00 eq) in MeOH (15 mL) was stirred at 30°C for 2 h. NaBH3CN (2.69 g, 42.82 mmol, 5.00 eq) was added. The resulting mixture was d at 30 CC for 16 h. The mixture was concentrated and diluted with water (50 mL) and EA (50 mL). The organic layer was concentrated and ed by column chromatography (PE:EA=10:1~2:1) to give 38_2 (1.50 g, 5.99 mmol, 70% yield) as a colorless oil.

LCMS: RT = 0.568 min, m/z 273.2 [M+H]+ 1H NMR(CDC13, 400 MHZ) 5 7.40-7.35 (m, 5H), 6.25 (s, 1H), 5.26-5.14 (m, 2H), 3.86-3.73 (m, 3H), 3.48-3.30 (m, 2H), 1.91-1.85 (m, 2H), 1.59-1.46(m, 2H).

Step 2: To a solution of 38_2 (1.60 g, 6.39 mmol, 1.00 eq) in MeOH (15 mL) was added Pd(OH)2 (179.54 mg, 1.28 mmol, 0.20 eq). The mixture was stirred at 20°C under H2 (l5psi) for 16 h. The mixture was filtered and the filtrate was trated to give 38_3 (450 mg, crude) as a colorless oil, which was used in the next step directly without purification.

Step 3: Compound 38_6 was made from Core A and compound 38_4 according to the same procedure as described for the sis of Core B and it was obtained as a yellow oil after purification by prep-HPLC (TFA condition). Yield: 40.3% LCMS: RT = 0.851 min, m/z 331.1 [M+H]+ 02429524\45-0 l 1H NMR (CDC13, 400 MHz) 5 8.41-8.40 (d, J = 5.2 Hz, 1H), 7.92 (s, 1H), 7.10— 7.09 (d, J: 5.2 Hz, 1H), 4.34—4.33 (m, 1H), .00 (m, 2H), 3.87-3.81 (t, J = 10.8 Hz, 1H), 3.51—3.50 (m, 1H), 3.29—3.19 (m, 2H), 2.60 (s, 3H), 2.35 (m, 1H), 2.13 (m, 1H), 1.89-1.85 (m, 2H), 1.06-1.03 (m, 1H), 0.53—0.51 (m, 2H), 0.31 (m, 2H).

Step 4: Compound 38_7 was sized according to the procedure described in method B and it was obtained as a yellow solid after prep TLC cation. Yield: 56.1% LCMS: RT = 0.739 min, m/z 363.1 [M+H]+ Step 5: Compound A-38 was synthesized according to the procedure described in method D with trans-cyclohexane-l, 4-diamine (4.0 eq) and it was obtained as a yellow solid after cation by prep HPLC (basic condition). Yield: 40% LCMS: RT = 1.154 min, m/z 397.3 [M+H]+ 1H NMR (CDC13, 400 MHz) 5 .17 (d, J = 5.2 Hz, 1H), 7.86 (s, 1H), 6.70- 6.69 (d, J = 5.2 Hz, 1H), 4.88-4.86 (m, 1H), 4.32—4.29 (m, 1H), 4.02—4.00 (t, J = 5.6 Hz, 2H), 3.86-3.81 (m, 2H), 3.51—3.49 (t, J = 3.00 Hz, 1H), 3.27—3.19 (m, 2H), 2.81 (s, 1H), 2.34—2.31 (m, 1H), 2.19-2.16 (m, 3H), 2.00—1.97 (m, 4H),1.38—1.27 (m, 4H), 1.06 (s, 1H), 0.52-0.50 (m, 2H), 0.28 (m, 2H) General procedure [or the synthesis of A-n Method E The reaction mixture of Core C (1.00 eq) and amine n (4.00 eq) in DMSO (8 mL) was stirred at 160 CC for 3 h. The reaction mixture was cooled to rt and poured onto ice-H20 (20 mL). The aqueous layer was extracted with EA (50 mL*3). The combined organic layers were washed with brine (50 mL*3), dried over NazSO4, filtered and concentrated. The residue was purified by column chromatography to give A-n. 02429524\45-01 _ 43 _ Method F The reaction mixture of Core C (1.00 eq), TBAF (2.00 eq), K2CO3 (4.00 eq) and amine n (4.00 eq) in DMSO (10 mL) was stirred at 160 CC for 3 h. The reaction mixture was cooled to 15 CC and poured into H20 (20 mL). The s layer was ted EA (20 mL*3). The combined organic layers were washed with brine (20 mL*3), dried over NazSO4, filtered and concentrated. The residue was purified by Pre- HPLC (HCl condition) to give A-n.

Method G The reaction mixture of amine n (1.20 eq) and Core C (1.00 eq) in dioxane (3 mL) was d at 130°C under microwave for 1.5 h. The reaction mixture was poured into H20 (20 mL). The aqueous layer was extracted EA (20 mL*3). The combined organic layers were washed with brine (20 mL*3), dried over NazSO4, filtered and concentrated. The residue was purified by Pre-HPLC (basic condition) to give A-n.

Pre aration 0 1R 4R -N1- 4- 5- c clo r0 lmeth l-I-meth l-IH- ra 0l dimeth lc clohexane-I 4-diamine A-28 O // \ HQNVUNHZ DMSO, 160 00,3 h HzN‘ Step 1: Compound A-14 was prepared according to the procedure described in method G and it was obtained as a yellow solid. Yield: 38.4% LCMS: RT = 2.043 min, m/z 327.2 [M+H]+. 1H NMR(CDC13, 400 MHz) 5 8.17 (d, J: 5.3 Hz, 1H), 7.83 (s, 1H), 6.70 (d, J: .3 Hz, 1H), 4.91 (s, 1H), 3.93 — 3.85 (m, 4H), 3.21 (d, J: 6.3 Hz, 2H), 2.74 (s, 1H), 2.16 (d, J : 4.0 Hz, 2H), 1.94 (d, J : 6.7 Hz, 2H), 1.30 — 1.25 (m, 4H), 1.11 — 1.10 (m, 1H), 0.50 - 0.46 (m, 1H), 0.27 — 0.24 (m, 1H) 02429524\45-0 l Step 2: To a mixture ofA-14 (18.52 mg, 122.53 umol, 1.00 eq) in Et0H (500 ML) was added 2,3-dihydrobenzotriazolylmethanol (18.52 mg, 122.53 umol, 1.00 eq) in one portion at 0°C. The mixture was stirred at 15 CC for 1 hr and NaBH4 (9.27 mg, 245.06 umol, 2.00 eq) was added. The resulting mixture was stirred at 15 CC for 1 h, and poured into H20 (10 mL). The aqueous layer was extracted DCM (20 mL*3). The combined organic layer was washed with brine, dried over NaSO4 and concentrated. The residue was purified by prep HPLC (basic condition) to give A-28 (5.00 mg, 14.10 umol, 11.5% yield, 100% purity) as a white solid.

LCMS: RT = 2.535 min, m/z 355.2 [M+H]+ 1H NMR(CDC13, 400 MHz) 5 8.10 (d, J = 5.2 Hz, 1H), 7.76 (s, 1H), 6.63 (d, J = .2 Hz, 1H), 4.78 (d, J: 3.6 Hz, 1H), 3.81 (s, 3H), 3.77 — 3.71 (m, 1H), 3.13 (d, J: 6.4 Hz, 2H), 2.27 (s, 6H), 2.17 (d, J: 12.4 Hz, 2H), 1.93 (d, J: 11.6 Hz, 2H), 1.36-1.31 (m, 2H), 1.23 — 1.17 (m, 2H), 1.04—1.02 (m, 1H), 0.44 — 0.39 (m, 2H), 0.20-0.18 (m, 2H).

Pre aration 0 8-amin0-1 3-diazas iro 4.5 -Z 4-di0ne h drochloride {amine-192 O 0 0 >\"NH . y H H HCI/dloxane H —> —> NHBoc o CNHBOC M80" 0 NH2HC| 19 2 amine-19 Step 1: A solution of NaCN (2.75 g, 56.02 mmol, 2.39 eq) in H20 (10 mL) was added to the mixture of compound 19_1 (5.00 g, 23.44 mmol, 1.00 eq) and (NH4)2C03 (4.96 g, 51.57 mmol, 2.20 eq) in Et0H (25 mL) and H20 (25 mL). The reaction mixture was d at 10 CC for 16 hours and then at 70 CC for another 24 hours. TLC (PE:EA=3:1) showed that reactant was consumed (Rf=0.55) and a major spot fromed (Rf=0.25). The reaction mixture was allowed to cool down and filtered. The filter cake was washed with H20 (100 mL) and dried. Compound 19_2 (4.00 g, 14.12 mmol, 60.2% yield) was obtained as a white solid. 1H NMR d6, 400 MHz) 8 10.51 (s, 1H), 8.48 (s, 1H), 6.70-6.72 (d, J = 8.0 Hz, 1H), 3.16 (s, 1H), .76 (m, 2H), 1.65-1.64 (d, J: 4.0 Hz, 2H), 1.62-1.52 (m, 2H), 1.60-1.59 (m, 1H), 1.37 (s, 11H). 02429524\45-Ol Step 2: To the mixture of compound 19_2 (1.00 g, 3.53 mmol, 1.00 eq) in MeOH (10 mL) was added HCl/dioxane (4 M, 10 mL, 11.33 eq) at 0 CC and the reaction e was stirred at 10 CC for 16 hour. LCMS showed the consumption of reactant. The reaction mixture was trated to give Amine-19 0 mg, 3.19 mmol, 90.3% yield, HCl) as a white solid. 1H NMR (DMSO-d6, 400 MHz) 8 8.52 (s, 1H), 8.21 (s, 3H),3.06 (s, 1H), 1.96- 1.89 (m, 1H), 1.76-1.59 (m, 6H).

Pre aration 0 8- 4- 5- c clo r0 lmeth lmeth l-IH- razol l rimidin-Z- l amino -1 3-dia as iro 4.5 decane-Z 4-di0ne A19 4 and I-amino- yl]amin0 chclohexyllmethanol (A492 HNy‘NH mNHQHCI H amine-19 T O N method E A19_4 Step 1: Compound A19_4 was prepared according to the ure described in method E with amine-19 (4 eq) and DIEA (4 eq) and it was as a white solid after purification by prep HPLC (basic condition). Yield: 72.2% LCMS: RT =0.573 min, m/z 397.2 [M+H]+ 1H NMR (CD30D, 400 MHz) 5 8.22 (s, 1H), 8.14-8.09 (m, 1H), 7.24 (d, J = 5.2 Hz, 1H), 5.19 (d, J = 6.8 Hz, 1H), 4.21 (s, 1H), 3.94 (s, 3H), 3.30 (d, J = 6.8 Hz, 2H), 2.20 (d, J = 6.0 Hz, 2H), 2.09—2.05 (t, 2H), 1.86 (d, J = 14.8 Hz, 2H),1.69-1.65 (m, 2H), 1.25—1.21 (m, 1H), 0.60 (d, J = 7.6 Hz, 2H), 0.39—0.35 (m, 2H), 02429524\45-0 l Step 2: Compound A19_4 (95.00 mg, 240.23 umol, 1.00 eq) was added to NaOH/Hzo (3 M, 640.60 uL, 8.00 eq) and the reaction mixture was stirred at 120 CC for 16 h. The reaction mixture was cooled to 15 CC and poured into H20 (20 mL). The aqueous layer was adjusted to pH=7 with 2N HCl solution. The mixture was concentrated. The crude t was ated with MeOH (50 mL*3). The filtrate was concentrated to give 19_5 (300.00 mg, crude) as a white solid.

LCMS: RT =1.148 min, m/z 371.2 [M+H]+ 1H NMR (CD30D, 400 MHz) 5 8.15 (d, J: 5.2 Hz, 1H), 7.95—7.93 (m, 1H), 6.89- 6.85 (m, 1H), 4.03 (s, 1H), 3.91 (s, 3H), 3.28 (s, 2H), 2.32—2.25 (m, 2H), 2.16—2.13 (d, J = 9.6 Hz, 2H), 1.99—1.95 (d, J = 9.6 Hz, 2H) ,1.57—1.54 (d, J = 14Hz, 2H), 1.37 (s, 1H), 0.51-0.48 (m, 2H), 0.31—0.27 (m, 2H), Step 3: To the e of 19_5 (330.00 mg, 890.81 umol, 1.00 eq) in THF (500 ML) was added BH3 (1M, 7.13 mL, 8.00 eq) and the on mixture was stirred at 100°C for 16 h. The reaction mixture was cooled to 15 CC and poured into MeOH (20 mL). The mixture was concentrated. The crude product was purified by Pre-HPLC (basic condition) to give A-19 (23.00 mg, 64.52 umol, 7.2% yield, 100% purity) as a white solid.

LCMS: RT =1.060 min, m/z 357.2 [M+H]+ 1H NMR (CD30D, 400 MHz) 5 8.12 (d, J = 5.6 Hz, 1H), 7.93 (s, 1H), 6.85-6.82 (m, 1H), 3.88-3.80 (m, 4H), 3.28 (s, 2H), 3.39 (s, 2H), 3.27 (d, J: 6.4 Hz, 2H), 2.03—1.94 (m, 3H) ,1.68-1.58 (m, 6H), 1.13 (d, J = 5.2 Hz, 1H), 0.50—0.47 (m, 2H), 0.30—0.28 (m, Pre aration 0 8- 4- 5- c clo r0 lmeth th l-IH- ra ol l rimidin-Z- l amino 0xaa as iro 4.5 decan-Z-one A-26 To the mixture of A-19 (20.00 mg, 56.11 umol, 1.00 eq) in THF (500 ML) was added CDI (9.1 mg, 56.11 umol, 1.00 eq) and the reaction mixture was stirred at 25 CC 02429524\45-0 l for 16 h. The reaction mixture was concentrated. The crude product was purified by prep HPLC (basic condition) to give A-26 (4.00 mg, 10.46 umol, 18.6% yield, 100% purity) as a white solid.

LCMS: RT =2.210 min, m/z 383.2 [M+H]+ 1H NMR , 400 MHz) 5 8.19 (s, 1H), 7.84 (s, 1H), 6.74 (s, 1H), 5.97 (s, 1H), 5.02 (s, 1H), 4.15 (s, 1H), 3.89 (s, 4H), 3.17 (s, 2H), 2.16-2.13 (d, J: 12.4 Hz, 2H), 1.98-1.95 (d, J: 13.2 Hz, 2H), 1.72-1.68 (d, J: 12.8 Hz, 2H) ,1.48-1.45 (d, J: 10.08Hz, 2H), 1.10 (s, 1H), 0.49 (s, 2H), 0.24 (s, 2H).

Pre aration 0 1R 4R -N1- 4- 5- c clo r0 lmeth l-I-meth l-IH- ra 0l l rimidin-Z- l-N4-meth lc clohexane-I 4-diamine A-27 1. LAH, THF 2. 80020, THF 27_Boc A-27 Step 1: nd 27_2 was made ing to the procedure descibed in method E with amine-27 (1.0 eq) and DIEA (10 eq) at 160°C for 6 h and it was ed as a yellow solid after purification by prep HPLC (TFA). Yield: 41.1% 1H NMR(CDC13, 400 MHz) 5 8.18 (d, J: 5.2 Hz, 1H), 7.83 (s, 1H), 6.71 (d, J: .6 Hz, 1H), 4.87 (s, 1H), 4.41 (s, 1H), 3.89 — 3.82 (m, 4H), 3.49 (s, 1H), 3.19 (d, J: 6.3 Hz, 2H), 2.19 — 2.05 (m, 4H), 1.46 (s, 9H), 1.35 — 1.29 (m, 7H), 1.26 (m, 2H), 1.23 (m, 2H), 0.50 — 0.46 (m, 2H), 0.26 — 0.24 (m, 2H) Step 2: To a solution of 27_2 (100.00 mg, 234.44 umol, 1.00 eq) in THF (4.00 mL) was added LiAlH4 (26.69 mg, 703.32 umol, 3.00 eq) at 0°C. The mixture was stirred at 70°C for 3 h. The mixture was cooled to 0°C and then quenched with aq.NH4Cl (3 drops). The resulting solution was dried over NazSO4 and then concentrated. The residue 02429524\45-0 l was failed to be purified by prep-HPLC. So A-27 (70 mg, crude) will be purified after protected with Boc group. To the solution of A-27 (crude) in THF (1 mL) was added (BocZ)2 (200 mg, 2 eq). After stirring for 1 hour at 20 0C, the reaction mixture was purified by LC (EA) to get 27_B0c (30 mg, 29.13% yield total), which was used for next step directly.

Step 3: To a solution of 27_B0c (30.00 mg, 68.09 umol, 1.00 eq) in DCM (100 ML) was added oxane (4 M, 85 uL, 5.00 eq) in one portion at 0°C and the mixture was stirred at 15 CC for 5 h. The reaction mixture was concentrated to give A-27 (12.00 mg, 30.88 umol, 45.35% yield, 97% purity, HCl) as a light yellow solid. Yield: 45.4% LCMS: RT =2.558 min, m/z 341.2 [M+H]+ 1H NMR , 400MHz) 5 8.26 (s, 1H), 8.14 (d, J = 6.4 Hz, 1H), 7.31 (d, J: 6.4Hz, 1H), 4.19 — 4.12 (m, 1H), 3.95 (s, 3H), 3.71 (s, 2H), 3.17 (s, 1H),2.76 (s, 3H), 2.31— 2.20 (m, 4H), 1.65 (m, 4H), 1.21 (s, 1H), 0.51 (s, 2H), 0.37 (d, J = 3.2 Hz, 2H).

Pre aration 0 N- 1R 4R amin0c clohex l meth0x acetamide h drochloride amine-29 "\NHZHCI | "‘N\B NH2 MeO\/i 00 O HCI/dioxane HN Boc\ ’ \ #0 #0 29 1 29 2 amine-29 Step 1: To a mixture of 29_1 (658.25 mg, 3.07 mmol, 1.0 eq) and 2- methoxyacetyl chloride (500 mg, 4.61 mmol, 1.5 eq) in DCM (5 mL) was added DIEA (310.82 mg, 3.07 mmol, 1.00 eq) in at 0°C. Then the reaction mixture was stirred at 15 CC for 2 hours. The reaction mixture was concentrated under vacuum. The residue was purified by column chromatography to give 29_2 (490.00 mg, 1.71 mmol, 55.7% yield) as a white solid. 1H NMR(CDC13, 400 MHZ) 5 3.80 (s, 1H), 3.74-3.67 (m, 1H), 3.40 (s, 1H), 3.33 (s, 3H), .87 (m, 4H), 1.37 (s, 9H), 1.22-1.12 (m, 3H) Step 2: Compound 29-2 was deprotected in a similar manner as shown in step 3 of the synthesis of A27 and Amine-29 was obtained as a white solid. Yield: 91.9% 02429524\45-0 l 1H NMR (CD30D, 400MHz) 4.05 (s, 1H), 374 (s, 2H), 3.70 - 3.69 (In, 1H), 3.36 (s, 3H), 3.28-3.27 (In, 1H), 3.09-3.07 (In, 1H), 2.07 - 2.04 (In, 2H), 1.94 - 1.92 (In, 2H), 1.56 - 1.40 (In, 4H) Pre n 0 N- 1R 4R 4- 5- c clo r0 lmeth l-I-meth l-IH- razol- 4- l rimidin-Z- lamina c clohex th0x acetamide A29 1 N .n 2 o\ "O \S \ method E T \ HN N —> 0' + (g HN OMe *0 Core C amine-29 A29_1 Compound A29_1 was made according to the procedure ed in method E with amine-29 (1.0 eq) and DIEA (4 eq) and it was obtained as a light yellow solid after purification by prep HPLC (basic condition). Yield: 27.4% LCMS: RT = 2.535 min, m/z 355.2 [M+H]+ 1H NMR(CDC13, 400 MHz) 5 8.20 (d, J: 5.2 Hz, 1H), 7.85 (s, 1H), 6.73 (d, J: .2 Hz, 1H), 6.40 (d, J: 8.0 Hz, 1H), 4.92 (s, 1H), 3.91 (m, 7H), 3.45 (m, 3H), 3.27— 3.20 (m, 2H), 2.21 (s, 2H), 2.10 (s, 2H), 1.45 — 1.36 (m, 4H), 1.13—1.12 (m, 1H), 0.53 — 0.48 (m, 2H), 0.28-0.26 (q, J : 4.9 Hz, 2H) Pre aration 0 1R 4R -N1- 4- 5- c clo r0 lmeth l-I-meth l-IH- razol l rimidin-Z- l -N4- 2-meth0x eth lc clohexane-I 4-diamine A-29 A29_1 A-29 To a mixture of LAH (40.00 mg, 1.05 mmol, 10.50 eq) in THF (4 InL) was added a solution of A29_1 (40.00 mg, 100.38 umol, 1.00 eq) in THF (1 mL) at 15°C. The mixture was stirred at 70 CC for 36 hr. The reaction mixture was cooled down and poured 02429524\45-0 l into H20 (0.1 mL). 1N NaOH (0.1 mL) was added and filtered. The filtrate was concentrated. The crude t was purified by Pre-HPLC (basic condition) to give A- 29 (4.00 mg, 9.88 umol, 9.8% yield, 95.0 % purity) as a yellow solid.

LCMS: RT = 2.535 min, m/z 355.2 [M+H]+ 1H NMR (CDC13, 300 MHz) 5 8.17 (d, J: 6.8 Hz, 1 H), 7.82 (s, 1 H), 6.76 — 6.69 (m, 1 H), 4.88 (d, J = 10.4 Hz, 1 H), 3.89 (s, 4 H), .50 (m, 2 H), 3.38 (s, 3 H), 3.22 (d, J = 8.40 Hz, 2 H) 2.84-2.81 (m, 2H), 2.49 (s, 1H) 2.19 (s, 2H), 2.01 (s, 1H) 1.38 (s, 4H),1.07(s, 1H) 0.49—0.43 (m, 2H) ,0.25 — 0.20 (m, 2 H) Pre aration 0 1R 4R i eridin-I- lc clohexanamine h drochloride {amine-472 NHBoc NHZ .—’O’NHBoc '\/\/\/ w—m N‘"0’HCI H2N" K2003 MeCN reflux HC' dioxane 47_1 47_2 amine-47_ Step 1: A mixture of 47_1 (200.00 mg, 933.27 umol, 1.00 eq), 1, 5-diiodopentane (302.32 mg, 933.27 umol, 1.00 eq) and K2C03 (515.95 mg, 3.73 mmol, 4.00 eq) in MeCN (10 mL) was stirred at 90 CC for 16 hours. The mixture was filtered and the filtrate was trated to give 47_2 (300 mg, crude) as a white solid and it was used in next step directly.

Step 2: Crude 47_2 was deprotected in a similar manner as shown in step 3 of the synthesis of A27 and 47 was ed as a white solid. 1H NMR (D20, 400 MHz) 5 3.55-3.10 (m, 6H), 2.16-2.08 (m, 4H), 1.59—1.41 (m, 10H).

Pre aration 0 4- 5- c clo r0 lmeth l-I-meth l-IH- razol l-N- 1R 4R i eridin-I- lc clohex l rimidin-Z-amine A-47 Core C amine—47 A-47 02429524\45-01 Compound A-47 was prepared according to the procedure described in method F with amine-47 and it was obtained as a white solid after cation by prep HPLC (HCl condition). Yield: 49.3% LCMS: RT = 1.530 min, m/z 395.2 [M+H]+ 1H NMR (CD30D, 400 MHz) 5 8.21 (s, 1H), 8.06 (d, J: 6.8 Hz, 1H), .21 (m, 1H), 4.10 (s, 1H), 3.88 (s, 3H), 3.49 (d, J: 11.2 Hz, 2H), 3.23 (s, 2H), 3.06-3.00 (m, 4H), 2.26 (d, J = 10 Hz, 4H), 1.96-1.57 (m, 10H), 1.15 (s, 1H), 0.50 (s, 2H), 0.31—0.30 (m, 2H) Pre aration 0 1R 4R m0r c clohexanamine h drochloride {amine-48 2 NHBoc NH 0NHB0C Br\/\o’\/Br HCI/dioxane \' —>0’ "O’HCf H2N‘ K2C03, MeCN, reflux FN‘ FN‘ 48 1 (K) 48 2 Ox) amine-48 Crude amine-48 was synthesized in a similar manner as described for the synthesis of amine -47 and it was obtained as a yellow solid. 1H NMR (D20, 400 MHz) 5 3. 87 (s, 4H), 324—3. 11 (m, 6H), 220—2. 12 (m, 4H), 1.59—1.39 (m, 5H).

Pre aration 0 4- 5- c clo r0 lmeth l-I-meth l-IH- ra 0l l-N- 1r 4r m0r c clohex l rimidin-Z-amine A-48 N H2 N\ @CI method F + QN\" —> Core C amine-48 A'48 Compound A-48 was prepared according to the procedure described in method F with amine-48 with TBAF (1.0 eq) and it was obtained as a white solid after purification by prep HPLC (HCl condition). Yield: 51.2% LCMS: RT = 2.095 min, m/z 397.3 [M+H]+ 02429524\45-0 l 1H NMR (CDgOD, 400 MHz) 5 8.16 (s, 1H), 8.01 (s, 1H), 7.19 (s, 1H), 4.06-3.97 (m, 3H), 3.83-3.78 (m, 5H), 3.51—3.42 (m, 2H), 3.24 (s, 1H), 3.14-3.12(m, 1H), 2.29— 2.19 (m, 4H), 1.69-1.63 (m, 2H), 1.54-1.48 (m, 2H), , 1H),0.45 (s, 2H), 0.25 (s, Pre aration 0 1R 4R rrolidin-I- l c clohexanamine h drochloride 1amine-492 NHB00 NH 0’NHBOC BFW\Br HCI2 HCI/dioxane v. . 1. N N" HZN‘ K2C03, MeCN, reflux C} C, 49_1 49 2 amine-49 Crude amine-49 was synthesized in a similar manner as described for the synthesis of amine -47 and it was obtained as a white solid. 1H NMR (D20, 400 MHZ) 5 3.55—3.53 (m, 3H), .99 (m, 4H), 2.21—2.09 (m, 2H), 2.06—1.99 (m, 7H), 1.49—1.40 (m, 4H).

Pre aration 0 4- 5- c clo r0 lmeth l-I-meth l-IH- razol l-N- 1R 4R rrolidin-I- lc clohex l rimidin-Z-amine A-49 Core C amine-49 A-49 Compound A-49 was prepared according to the procedure bed in method F with amine-49 and it was obtained as a white solid after purification by prep HPLC (HCl condition).Yield: 89.3% LCMS: RT = 2.607 min, m/z 381.3 [M+H]+ 1H NMR(CDC13, 400 MHz) 5 8.16 (s, 1H), 8.01 (s, 1H), 7.19 (s, 1H), 4.06 (s, 1H), 3.83 (s, 3H), 3.08 (s, 2H), .48 (m, 13H), 1.09 (s, 1H), 0.45 (s, 2H), 0.25 (s, 02429524\45-Ol Pre aration 0 1R 4R azetidin-I— l c clohexanamine h drochloride (amine-502 NHBoc (0]:NHBoc B Br HCI/dioxane CV10; HZN" K2003 MeCN reflux C/w 50 2 amine-50 Crude amine-50 was synthesized in a r manner as described for the synthesis of amine -47 and it was ed as a white solid 1H NMR (CDClg, 400 MHz) 5 4.15-4.08 (m, 2H), 3.65-3.12 (m, 1H), 2.28-2.03 (m, 6H), 1.50—1.42 (m, 4H), 1.26—1.20 (m, 1H).

Pre aration 0 N- 1R 4R a etidin-I- l c clohex l 5- c clo r0 lmeth l-I-meth l-IH- razol l rimidin-Z-amine A-50 0°"? C amine-50 A-50 Compound A-50 was prepared according to the procedure described in method F with 50 and it was obtained as a white solid after purification by prep HPLC (Basic condition). Yield: 16% LCMS: RT = 2.494 min, m/z 367.3 [M+H]+ 1H NMR (CDC13, 400 MHz) 5 8.17 (d, J: 6.8 Hz, 1H), 7.88 (s, 1H), 6.70 (d, J: 6.8 Hz, 1H), 4.86 (d, J = 11.6 Hz, 1H), 3.91 (m, 4H), 3.25—3.20 (m, 5H), 2.31—2.07 (m, 6H), 2.05—2.02 (m, 2H), 1.24—1.10 (m, 5H), 0.47—0.43 (m, 2H), 0.25—0.22 (s, 2H).

Pregaration of (IR, 4R 2-N115-(cyclobutylmethyl 2methyl-1H-erazol ngerimidin-Z-yl2cyclohexanediamine (A-45 2 02429524\45-Ol /s N HHCI o ‘u’j /s N 0 /N CN COZH / ‘o/ 3A \f \ DMFDMA _> —> 0 N’ \—> N / HC|(6M) —>/§\rN/\o CDI/DCM l LDA/THF 80 c 2h 45 1 45 2 45 4 45_5 MeNHNH2 /SYN\ mCPBA ElOH method D YN/\ Step 1: A solution of obutylacetonitrile (1.00 g, 10.51 mmol, 1.00 eq) in HCl (6 M, 10.00 mL, 5.71 eq) was stirred at 120°C for 16 h. The mixture was diluted with water (50 mL) and extracted with EA (20 mL*2). The combined organic layer was washed with water (40 mL), dried and concentrated to give 45_1 (750 mg, 6.57 mmol, 62.5% yield) as a colorless liquid 1H NMR (CDCl3, 400 MHz) 5 .68 (m, 1H), 2.47—2.45 (d, J = 8.0 Hz, 2H), .15 (m, 2H), 1.90-1.88 (m, 2H), 1.75—1.70 (m, 2H).

Step 2-4: Compound 45_5 was synthesized in a similar manner as decribed for the synthesis of Core A in scheme 1.1.

Compound 45_2 was obtained as a colorless liquid. 1H NMR(CDC13, 400 MHz) 5 3.68 (s, 3H), 3.16 (s, 3H), 3.75—3.71 (m, 1H), 2.55— 2.53 (m, 2H), 2.16-2.13 (m, 3H), 1.89-1.87 (m, 2H), 1.73-1.68 (m, 2H).

Compound 45_4 was obtained as a yellow oil LCMS: RT = 0.795 min, m/z 237.1 [M+H]+ Compound 45_5 was obtained as a dark brown solid: 1H NMR (CDClg, 400 MHz) 5 8.39-8.38 (d, J: 4.8 Hz, 1H), 8.02 (s, 1H), 7.59 (s, 1H), 6.96-6.92(m, 1H), 2.96 (s, 3H), 2.89 (m, 5H), 2.72-2.68 (m, 1H), 2.57 (s, 3H), 2.10— 2.05 (m, 2H), 1.87-1.82(m, 4H).

Step 5-6: nd 45_7 was synthesized in a similar manner as decribed for the synthesis of Core C in scheme 1.2.

Compound 45_6 was obtained as a yellow oil after purification by prep HPLC (TFA condition). Yield: 21.2%; 02429524\45-0 l 1H NMR(CDC13, 400 MHz) 5 841—839 (d, J = 7.2 Hz, 1H), 7.88 (s, 1H), 7.09— 7.07 (d, J = 6.8 Hz, 1H), 3.89 (s, 3H), .33 (d, J = 9.6 Hz, 2H), 2.72—2.67 (m, 1H), 2.6 (s, 3H), 2.02—1.98 (m, 2H), 1.84-1.76 (m, 4H).

Compound 45_7 was obtained as a yellow oil after cation by pre-TLC.

Yield: 86.6% ; LCMS: RT = 0.706 min, m/z 307.2 [M+H]+ Step 7: Compound A-45 was made according to the procedure described in method D with trans-cyclohexane-l, 4-diamine (4.0 eq) and it was obtained as a yellow solid after purification by prep HPLC (basic condition). Yield: 21.9%; LCMS: RT = 0.539 min, m/z 341.3 [M+H]+; 1H NMR(CDC13, 400 MHz) 5 8.18-8.17 (d, J = 5.2 Hz, 1H), 7.82 (s, 1H), 6.69- 6.68 (d, J = 5.2 Hz, 1H), 4.85—4.83 (d, J = 8.0 Hz, 1H), 3.91—3.84 (m, 4H), 3.33—3.31 (d, J = 7.6 Hz, 2H), 2.75—2.68 (m, 2H), 2.18—2.17 (m, 2H), 2.00—1.93 (m, 4H), .77 (m, 4H), 1.31—1.26 (m, 4H).

Pre aration 0 1R 4R -N1- 4- 5- c clo en lmeth l-I-meth l-IH- razol l rimidin-Z- l c clohexane-I 4-diamine A-46 HHCI o /N o "\fjj/ /s N\ COzH / ‘0/ N / 3A T DMFDMA I —> N’O\ N / O—> /S\r \ CDI/DCM 80 l LDA/THF C,2h NI / 461 462 464 46_5 N N ’\ ,0 7‘ 0’ 7‘ N‘ N‘ N‘ MeNHNH2 /s N\ \ mchA \ H211" \ —> I —>\oleN\O I —> HYN\ EtOH DCM method D I N / N / N / HN‘"2 466 467 A-46 Compound A-46 was synthesized in a manner similar as described for the sis of compound A-45.

Compound 46_2 was obtained as a colorless oil. 1H NMR(CDC13, 400 MHz) 5 3.64 (s, 3H), 3.14 (s, 3H), 2.41—2.40 (t, J = 7.2 Hz, 2H), .25 (m, 1H), 1.82—1.80 (m, 2H), 1.59—1.51 (m, 4H), 1.15 — 1.13 (m, 2H). 02429524\45-01 _ 56 _ Compound 46_5 was obtained as a yellow oil.

LCMS: RT = 1.294 min, m/z 306.2 [M+H]+ Compound 46_6 was obtained as a yellow oil after cation by prep TLC.

Yield: 26.5% LCMS: RT = 0.866 min, m/z 289.1 [M+H]+ 1H NMR (CDClg, 400 MHz) 5 8.42-8.41 (d, J = 5.2 Hz, 1H), 7.90 (s, 1H), 7.10— 7.09 (d, J = 5.2 Hz, 1H), 3.90 (s, 3H), 3.26-3.24 (d, J = 7.6 Hz, 2H), 2.61 — 2.60 (d, J = 4.8 Hz, 3H), 2.21 (m, 1H), 1.72 — 1.67 (m, 4H), 1.27 — 1.26 (m, 2H), 1.25 (m, 3H).

Compound 46_7 was obtained as a white solid after purification by prep TLC.

Yield: 44.0% LCMS: RT = 1.219 min, m/z 321.2 [M+H]+ Compound A-46 was obtained as a yellow solid after purification by prep HPLC (basic condition). Yield: 52.7% LCMS: RT = 2.381 min, m/z 355.2 [M+H]+ 1H NMR (CD30D, 400 MHz) 5 8.16 (s, 1H), 8.07-8.03 (t, J = 8.0 Hz, 1H), 7.20— 7.18 (d, J = 6.8 Hz, 1H), 4.06-4.02 (m, 1H), 3.88 (s, 3H), 3.31—3.30 (m, 2H), 3.16 (s, 1H), 2.17—2.15 (m, 5H), 1.67 (m, 4H), 1.56 (m, 6H), 1.30—1.25 (m, 2H) Pre aration 0 1R 4R -N1- 5-chl0r0 5- c clo r0 lmeth l-I-meth l-IH- razol l rimidin-Z- lc clohexane-I 4-diamine A-51 n- F z -78°C 6' 5E N\ "UN: CI51_3 ClYN\ \ I YN Amphos)2 dioxaneMW a.q Na2C03(2M),DME O’HNI 130°C 1 5 h HZN‘ 02429524\45-0 l Step 1: To a solution of nd N-methylpyrazole (51_1, 8.00 g, 97.44 mmol, 1.00 eq) in THF (160 mL) was added drop-wise n-BuLi (2.5 M, 46.77 mL, 1.20 eq) at - 78°C. After the resulting mixture was stirred for 1 h at that temperature, the solution of ropanecarbaldehyde (8.20 g, 116.93 mmol, 1.20 eq) in THF (80 mL) was added drop-wise. And then the reaction mixture was stirred at 20°C for 16 h. TLC (PE:EA = 2:1) showed reactant 1 (Rf = 0.3) was consumed and product (Rf = 0.05) was .

The mixture was poured into aqueous NH4Cl (300 mL) and stirred for 10 min. The aqueous phase was ted with ethyl acetate (100 mL*2). The combined organic phase was washed with brine (100 mL), dried over anhydrous NazSO4, filtered and concentrated in vacuum. The residue was purified by column chromatography (SiOz, PE:EA = 8:1~0:1) to afford compound 51_3 (12.00 g, 78.85 mmol, 80.9% yield, 100% purity) as a colorless oil.

LCMS: RT = 0.118 min, m/z 153.1 [M+H]+ Step 2: The reaction mixture of compound 51_3 (9.00 g, 59.14 mmol, 1.00 eq), TFA (40.46 g, 354.84 mmol, 26.27 mL, 6.00 eq) and Et3SiH (41.26 g, 354.84 mmol, 56.52 mL, 6.00 eq) in DCM (900 mL) was stirred at 40°C for 36h. The mixture was adjusted to pH = 8 with s NaHC03 and separated. The organic layer was concentrated and purified by prep HPLC (basic ion) to give compound 51_5 (2.10 g, 15.42 mmol, 26.1% yield) as a dark brown oil.

LCMS: RT = 0.565 min, m/z 137.1 [M+H]+ Step 3: To a solution of compound 51_5 (2.10 g, 15.42 mmol, 1.00 eq) in DCM (21 mL) was added NBS (3.02 g, 16.96 mmol, 1.10 eq) at 0°C. The mixture was stirred at 20°C for 2h. The e was concentrated and purified by column chromatography (SiOz, PE:EA = 20:1) to give compound 51_6 (3.00 g, 13.95 mmol, 90.5% yield) as a yellow oil.

LCMS: RT = 0.784 min, m/z 217.1 [M+H]+ 1H NMR (CDCl3, 400 MHz) 5 7.39 (s, 1 H), 3.87 (s, 3 H), 2.65-2.63 (d, J = 8.8 Hz, 2 H), 0.98-0.94 (m, 1H), 0.55—0.51 (m, 2H), 0.29—0.25 (m, 2H).

Step 4: To a solution of compound 51_6 (3.00 g, 13.95 mmol, 1.00 eq) in THF (60 mL) was added n-BuLi (2 M, 10.46 mL, 1.50 eq) drop-wise at -78°C. After sitrring for 0.5h at that temperature, a solution of 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2- 02429524\45-0 l dioxaborolane (5.19 g, 27.90 mmol, 2.00 eq) in THF (6 mL) was added. The resulting mixture was warmed to 20°C and stirred for 0.5h. TLC (PE:EA = 5:1) showed nt (Rf = 0.6) was consumed and product (Rf = 0.5) was formed. The mixture was quenched with saturated NH4Cl (50 mL) and extracted with EA (100 mL). The orgainic layer was concentrated and ed by column chromatography (SiOz, PE:EA = 20: 1~10: 1) to give compound 51_ 7 (3.30 g, 11.40 mmol, 81.7% yield, 90.5% purity) as a colorless oil.

LCMS: RT = 0.801 min, m/z 263.2 [M+H]+ 1H NMR (CDCl3, 400 MHz) 5 7.67 (s, 1 H), 3.85 (s, 3 H), 2.82-2.81 (d, J = 6.8 Hz, 2 H),1.30 (s, 12H), .90 (m, 1H), 0.45—0.42 (m, 2H), 0.29—0.27 (m, 2H).

Step 5: To a solution of compound 51_ 7 (500.00 mg, 1.91 mmol, 1.00 eq) in DME (10 mL) were added 2, 4, 5-trichloropyrimidine (51_8, 420.40 mg, 2.29 mmol, 1.20 eq), Na2C03 (2 M, 2.10 mL, 2.20 eq) and catalyst PdClz(Amphos)2 (67.62 mg, 95.50 umol, 0.05 eq) under nitrogen. The resulting e was stirred at 85 CC for 2hr under nitrogen. TLC (PE:EA = 5: 1) showed reactant (Rf = 0.4) was consumed and product (Rf = 0.5) was formed. The mixture was diluted with water (50 mL) and extracted with EA (50ml). The organic layer was concentrated and purified by silica gel column (PE:EA = : 1) to afford compound 51_9 (350.0 mg, 1.05 mmol, 55% yield, 85% purity) as a yellow LCMS: RT = 0.819 min, m/z 283.1 [M+H]+ Step 6: The reaction mixture of compound 51_9 (300.00 mg, 1.06 mmol, 1.00 eq) and trans-cyclohexane-l, 4-diamine (484.17 mg, 4.24 mmol, 4.00 eq) in dioxane (4.5 mL) was stirred at 130°C for 2h under ave. The e was filtered and concentrated. The crude was purified by prep-HPLC (HCl condition) to give A-51 (80.00 mg, 200.04 umol, 18.9% yield, 99.3% , HCl) as a yellow solid.

LCMS: RT = 2.817 min, m/z 361.1 [M+H]+ 1H NMR (DMSO-de, 400 MHz) 5 8.37 (s, 1 H), 8.29 (s, 3 H), 8.08 (s, 1 H), 3.86 (s, 3 H), 3.70-3.68 (m, 2 H), 3.06-3.05 (d, J = 6.4 Hz, 2H), 2.96 (s, 1H), 2.03—1.95 (m, 4H), 1.49—1.32 (m, 4H), 0.98 (s, 1H), 0.42—0.40 (m, 2H), 0.16 (m, 2H). 02429524\45-0 l Pre aration 0 1R 4R -N1- 4- 5- c clo r0 lmeth l-I-meth l-IH- razol lmeth l rimidin-Z- lc clohexane-I 4-diamine A-52 CI N Cl on NH2 N / N, N\ H2N"0 N\ 52_8 CI | \rN\ PdC|2(Amphos)2 N dioxgneMW H NO’HN a.q Na2C03(2M),DME 130 c 1.5 h 2 52 9 A52 Step 1: To a solution of compound 51_7 (500.00 mg, 1.91 mmol, 1.00 eq) in DME (10 mL) were added compound 52_8 (373.60 mg, 2.29 mmol, 1.20 eq), Na2C03 (a.q) (2 M, 2.1 mL, 2.20 eq) and catalyst PdClz(Amphos)2 (67.62 mg, 95.50 umol, 0.05 eq) under nitrogen. The resulting e was stirred at 85°C for 2 h under en. TLC (PE:EA = 5: 1) showed reactant (Rf = 0.4) was ed and product (Rf = 0.25) was . The mixture was diluted with water (50 mL) and extracted with EA (50 mL). The organic layer was concentrated and purified by column chromatography (SiOz, PE:EA = :1~5:1) to give compound 52_9 (350.00 mg, 1.15 mmol, 60.3% yield, 86.5% purity) as a yellow oil.

LCMS: RT = 0.761 min, m/z 263.2 [M+H]+ Step 2: The reaction mixture of compound 52_9 (350.00 mg, 1.33 mmol, 1.00 eq) and trans-cyclohexane-l, 4-diamine (607.49 mg, 5.32 mmol, 4.00 eq) in dioxane (5 mL) was stirred at 130°C for 2 h under microwave. TLC (PE:EA = 1:1) showed reactant (Rf = 0.6) was consumed and product (Rf = 0.05) was formed. The mixture was filtered and concentrated. The crude was purified by prep-HPLC (HCl condition) and then prep- HPLC (basic condition) to give A-52 (30.0 mg, 88.00 umol, 6.6% yield, 99.8% purity) as a yellow solid.

LCMS: RT = 2.429 min, m/z 341.2 [M+H]+ 1H NMR (CDC13, 400 MHz) 8.09 (s, 1H), 7.67 (s, 1H), 4.72—4.70 (d, J = 8.4Hz, 1H), 3.9l(s, 3H), 3.82-3.80 (m, 1H), .00 (d, J = 6.4Hz, 2H), 2.79-2.76 (m, 1H), 2.22 (s, 3H), 2.14—1.93 (m, 4H), 1.32—1.23 (m, 4H), 0.97 (m, 1H),0.45-0.42 (m, 2H), 0.12— 0.10 (m, 2H). ation of (1R34RZ-NI-(S-chl0r0g5-(cycl02rogylmethyl2methyl-1H- rd 0l l rimidin-Z- lmeth lc clohexane-I ine A-30 1 and IS 45 - 02429524\45-0 1 N1- 5-chl0r0 5- c clo r0 lmeth l-I-meth l-IH- 4- l rimidin-Z- l meth lc clohexane-I 4-diamine A-30 2 O NHBn Pd(OH)2 H BnNH2 CoreD N N N \ NaBH4 MeOH T \ DMSO DIEA N / 300"" C' HBoc HBoc HBoc 0°C 30_1 so_2 Amine-30 30_3 UT' N A-30_2 Step 1: To a e of compound 30_1 (900.00 mg, 3.96 mmol, 1.00 eq) and BnNHz (424.26 mg, 3.96 mmol, 432.92 uL, 1.00 eq) in DCM (18.00 mL) were added AcOH (237.77 mg, 3.96 mmol, 226.45 uL, 1.00 eq) and NaBH(OAc)3 (1.68 g, 7.92 mmol, 2.00 eq). The resulting mixture was stirred at 20°C for 2 hours. The mixture was quenched with H20 (50 mL) and separated. The organic layer was concentrated. The residue was purified by column chromatography on A1203 (DCM:MeOH=20:l) to give compound 30_2 (650.00 mg, 2.03 mmol, 51.3% yield, 99.6% purity) as a red oil.

LCMS: RT = 1.520 min, m/z 319.2 [M+H]+ 1H NMR (CDCl3, 400 MHz) 5 .31 (m, 5 H), 4.43—4.35 (m, 1 H), 3.81 (d, 1:10.23 Hz, 2 H), 2.62-2.50 (m, 1H), 2.12—2.09 (m, 1H), 1.82-1.79 (m, 4H), 1.43(s, 9H), 1.38-1.27 (m, 7H).

Step 2: To a solution of compound 30_2 (350.00 mg, 1.10 mmol, 1.00 eq) in MeOH (3.5 mL) was added Pd(OH)2 (35.00 mg). The mixture was stirred at 20°C for 16 hours under H2 (16 psi). TLC (DCM:MeOH=1021) showed reactant (Rf=0.6) consumed and product (Rf=0.3) formed. The mixture was filtered and the mother liqud was concentrated to give amine-30 (220.00 mg, 963.5 umol, 87.6% yield) as a red oil.

MS: m/z 229.2 [M+H]+ 1H NMR (CDCl3, 400 MHz) 5 4.43—4.33 (m, 1 H), 3.82-2.66 (m, 1 H), 2.08 (s, 1H), .66 (m, 4H), 1.43 (s, 9H), .27 (m, 7H). 02429524\45-0 l Step 3: To a solution of Amine-30 (300.00 mg, 917.99 umol, 1.00 eq) and Core D (230.57 mg, 1.01 mmol, 1.10 eq) in DMSO (4 mL) were added DIEA (480.97 uL, 2.75 mmol, 3.00 eq) and TBAF (48.00 mg, 2.75 mmol, 0.20 eq). The e was stirred at 160°C for 3 hours. The mixture was separated between EA (50 mL) and H20 (50 mL).

The c layer was concentrated to give 30_3 (400.00 mg, crude) as a red oil.

LCMS: RT = 1.081 min, m/z 475.2 [M+H]+ Step 4: To a solution of 30_3 (400.00 mg, 842.05 umol, 1.00 eq) in DCM (4 mL) was added TFA (800 ML) at 0°C. The ing mixture was stirred at 20°C for 1 hour.

TLC (PE:EA=2:1) showed nt (Rf=0.6) consumed and product (Rf=0.05) formed.

The mixture was concentrated. The residue was purified by prep-HPLC (HCl condition) to give A-30_1 (55.00 mg, 127.60 umol, 15.1% yield, 95.4% purity, HCl) and A-30_2 (40.00 mg, 95.31 umol, 11.3% yield, 98.0% purity, HCl) as yellow solid.

LCMS(A-30_1): RT = 2.495 min, m/z 375.2 [M+H]+ 1H ak-1 (MeOD, 400 MHz) 5 8.49 (s, 1 H), 8.45 (s, 1 H), 4.10 (s, 1 H), 3.96 (s, 3H), 3.23 (d, J=6.4 Hz, 2H), 2.11-2.10(m, 2H), 2.07-1.94(m, 2H), 1.84-1.74 (m, 4H), 1.47 (s, 3H), 1.09-1.08(m, 1H),0.55-0.51 (m, 2H), 0.28-0.27 (m, 2H).

LCMS(A-30_2): RT = 2.527 min, m/z 375.2 [M+H]+ 1H NMR_Peak-2 (MeOD, 400 MHz) 5 8.47 (s, 2 H), 4.19 (s, 1 H), 3.96 (s, 3H), 3.19 (d, J=6.4 Hz, 2H), 2.05—2.199 (m, 2H), 1.87-1.78 (m, 2H), 1.42 (s, H), 1.08-1.07 (m, 1H), 0.54—0.52 (m, 2H), 0.30-0.28 (m, 2H). 02429524\45-0 l Pre n 0 1R 4R -N1- 4- 5- c clobut lmeth l is0 r0 l-IH- razol l rimidin-Z- l c clohexane-I 4-diamine A53 /s N\ /O\H/ \1 \lei/ / / DMSO DCMCDI LDA/THF s N o / \r \ H2NNYHCI 'J / DMFDM /S /"I\ N~< EtOHTEA 80°C so 00, 2h / 53 5 53 7 dioxane 130 °C, 1.5 h Step 1: To a mixture of NaCN (13.81 g, 281.82 mmol, 1.40 eq) in DMSO (240 mL) was added 53_1 (30.00 g, 201.30 mmol, 22.56 mL, 1.00 eq) dropwise at 60°C. The mixture was ketp at 75°C for 16hr. The mixture was cool down and diluted with water (500 mL). The solution was ted with EtOAc (200 mL*3). The combined organic layers were washed with brine (100 mL*3) and dried. The organic layer was concentrated to give 53_2 (15.00 g, 157.66 mmol, 78.3% yield) as a light yellow liquid. 1H NMR (CDCl3, 400 MHz) 5 2.64-2.63 (m, 1 H), 2.42—2.41 (d, J=6.4 Hz, 2 H), 2.20—2.17 (m, 2 H), 1.90-1.83 (m, 4H).

Step 2: A solution of 53_2 (15.00 g, 157.66 mmol, 1.00 eq) in HCl (6 M, 150 mL, .71 eq) was stirred at 120°C for 16 hours. The mixture was diluted with water (500 mL) and extracted with EtOAc (200mL*2). The combined organic layers were washed with water (400 mL). The organic layer was dried and trated to give 53_3 (16.00 g, 140.18 mmol, 88.9% yield) as a colorless liquid. 1H NMR (CDCl3, 400 MHz) 5 2.69-2.68 (m, 1 H), 2.47—2.45 (m, 2 H), 2.172 15 (m, 2H), 1.89-1.87 (m, 2H), 1.75—1.70 (m, 2H).

Step 3: To a solution of 53_3 (16.00 g, 140.18 mmol, 1.00 eq) and N- methoxymethanamine (20.51 g, 210.27 mmol, 1.50 eq, HCl) in DCM (160 mL) was added CDI (45.46 g, 280.36 mmol, 2.00 eq) at 0°C in portions. The mixture was stirred 02429524\45-0 l at 20°C for 16 hours. The mixture was diluted with water (50 mL) and extracted with DCM(30 mL*3). The combined organic layer was dried and concentrated to give crude product. The crude product was purified by silica gel column (PE:EA=50: 1~10: 1) to give 53_4 (12.00 g, 76.33 mmol, 54.4% yield) as a colorless liquid. 1H NMR , 400 MHz) 5 3.68-3.67 (d, J=2.4 Hz, 3 H), 3.16 (s, 3 H), 2.77— 2.73 (m, 1H), 2.54—2.53 (m, 2H), 2.16-2.13 (m, 2H), 1.88-1.86 (m, 2H), 1.73—1.72 (m, 2H), 1.70-1.68 (m, 2H).

Step 4: To a solution of 53_4 (4.50 g, 32.09 mmol, 1.00 eq) in THF (225 mL) was added LDA (2 M, 24.07 mL, 1.50 eq) at -78°C. After stirred for 1 hr, a solution of 2- cyclobutyl-N-methoxy-N-methyl-acetamide (6.05 g, 38.51 mmol, 1.20 eq) in THF (120 mL) was added into it at -78°C. The resulting mixture was d at -78°C for 4 hr, quenched with saturated NH4Cl (200 mL), and the aqueous phase was extracted with ethyl acetate (200 mL*3). The combined layers were washed with brine (200 mL), dried over anhydrous , filtered and trated to give 53_5 (10.00 g, crude) as a yellow LCMS: RT = 0.788 min, m/z 237.1 [M+H]+ Step 5: The solution of 53_5 (10.00 g, 42.31 mmol, 1.00 eq) in DMF- DMA(201.68 g, 1.69 mol, 224.09 mL, 40.00 eq) was stirred at 90°C for 2 hr. The mixture was concentrated to give crude product. The crude product was purified by silica gel column(DCM:MeOH=1:0~10:1) to give 53_6 (8.80 g, crude) as a black brown solid.

Step 6: To a solution of 53_6 (800.00 mg, 2.75 mmol, 1.00 eq) in EtOH (12 mL) was added isopropylhydrazine (364.95 mg, 3.30 mmol, 1.20 eq, HCl) and TEA (333.93 mg, 3.30 mmol, 457.43 uL, 1.20 eq). The e was stirred at 90°C for 1 hr.

The mixture was concentrated. The crude was purified by pre-HPLC(TFA) to give 53_7 (600.00 mg, 1.88 mmol, 68.5% yield, 95% purity) as a yellow oil.

LCMS: RT = 0.901 min, m/z 303.2 [M+H]+ Step 7: To a on of 53_7 (600.00 mg, 1.98 mmol, 1.00 eq) in DCM (9 mL) was added m-CPBA (1.01 g, 4.96 mmol, 85% purity, 2.50 eq) at 0°C. The mixture was stirred at 20°C for 3 hr. The reaction was quenched with saturated a.q NaSzO3 (50 mL) and extracted with DCM (20 mL*2). The combined organic layers were washed with a.q NaHCO3 (40 mL). The organic layer was dried and concentrated. The crude was 02429524\45-0 l purified by prep-TLC (DCM: 0:1)(Rf=0.6) to give 53_8 (500.00 mg, 1.50 mmol, 75.5% yield) as a yellow oil.

LCMS: RT = 0.803 min, m/z 335.1 [M+H]+ Step 8: To a solution of 53_8 (500.00 mg, 1.50 mmol, 1.00 eq) in dioxane (7.5 mL) was added trans-cyclohexane-1,4-diamine (685.14 mg, 6.00 mmol, 4.00 eq). The e was stirred at 130°C for 2 hr in the microwave. The mixture was ed and concentrated. The crude was purified by prep HPLC(base) and prep HPLC(HCl) to give A-53 (250.00 mg, 615.9 umol, 41% yield, 99.8% purity, HCl) as a yellow solid.

LCMS: RT = 2.57 min, m/z 369.2 [M+H]+ 1H NMR (DMSO, 400 MHz) 5 8.88-8.87 (m, 1 H), 8.38-8.25 (m, 5 H), 7.30-7.28 (m, 1H), 4.78-4.76 (m, 1H), 3.36-3.34 (m, 2H), 3.05 (s, 1H), 2.61 (s, 1H), 2.05(m, 4H), 1.92—1.91 (m, 2H),1.78 (m, 4H), 1.47—1.39 (m, 10H).

Pre aration 0 1R 4R -N1- 4- 1-meth l 1-meth lc clo r0 lmeth l-IH- ra 0l l rimidin-Z- l c clohexane-I 4-diamine A56 N N ’ ’ /H HCI o 1;"\ ng/—> R N‘ \ NH2NH2H20 N/O\ —> N—BS> Br | BuLi KOH 120°C 56 1 56_2 2 ’NN~ /Yj' ’"N- O\B \ n-BuLi,THF,-78°C (5 Pd(amphos) CI2 / DCM a. q Na2003(2M) DME 56 5 56 6 Sygher/ l‘" 0’" N H2N N dioxane, 130 0C 0‘0 \lJr/ 56_7 A-56 Step 1: To a solution of 1-methylcyclopropanecarboxylic acid (4.00 g, 39.95 mmol, 1.00 eq) and CD1 (7.13 g, 43.95 mmol, 1.10 eq) in DCM (40 mL) was added N- methoxymethanamine (4.68 g, 47.94 mmol, 1.20 eq, HCl) at 0°C in portions. The mixture was stirred at 20°C for 16 hr. The mixture was diluted with water (50 mL) and extracted with DCM (20 mL*3). The ed organic layers were dried and concentrated to give 02429524\45-0 l crude product. The crude product was purified by silica gel column (PE:EA=30:1~10:1) to give 56_1 (3.30 g, 23.05 mmol, 57.7% yield) as a colorless liquid. 1H NMR (CDCl3, 400 MHz) 5 3.73 (s, 3 H), 3.23 (s, 3 H), 1.37 (s, 3H), 1.05—1.03 (m, 2H), 0.58-0.55 (m, 2H).

Step 2: To a solution of 1-methylpyrazole (1.70 g, 20.71 mmol, 1.72 mL, 1.00 eq) in THF (35.00 mL) was added n-BuLi (2.5 M, 9.94 mL, 1.20 eq) at -78°C. Stirred for 1hr. Compound 56_1 (3.26 g, 22.78 mmol, 1.10 eq) in THF (35 mL) was added to it at - 78°C. The resulting mixture was stirred at 20°C for 1hr. The mixture was quenched with saturated NH4Cl (20 mL) and extracted with EtOAc (20 mL*2). The organic layer was concentrated and ed by silica gel column (PE:EA=1:0~20:1) to give 56_2 (2.50 g, 13.41 mmol, 64.8% yield, 88.1% purity) as a yellow oil.

LCMS: RT = 0.609 min, m/z 165.1 [M+H]+ Step 3: The reaction mixture of 56_2 (2.00 g, 12.18 mmol, 1.00 eq) and KOH (2.73 g, 48.72 mmol, 4.00 eq) in NHzNH2.H2O (2.57 g, 48.72 mmol, 2.49 mL, 95% purity, 4.00 eq) and diglycol (40 mL) was heated to 110 °C for 1.5 hr, then at 200°C for r 1 hour with Dean-Stark. The mixture was diluted with water (50 mL) and extracted with MTBE (50 mL*2). The combined organic layers were concentrated to give 56_3 (1.10 g, crude) as a colorless oil.

LCMS: RT = 0.639 min, m/z 151.1 [M+H]+ Step 4: To a solution of 56_3 (1.10 g, 7.32 mmol, 1.00 eq) in DCM (11 mL) was added NBS (1.30 g, 7.32 mmol, 1.00 eq) at 0°C. The e was stirred at 20°C for 1 hr. The mixture was trated. The crude was purified by silica gel column(PE:EA=1:0~50:1) to give 56_4 (1.45 g, 5.65 mmol, 77.1% yield, 89.2% purity) as a colorless oil.

LCMS: RT = 0.835 min, m/z 229.0 [M+H]+ 1H NMR (CDCl3, 400 MHz) 5 7.39 (s, 1 H), 3.85 (s, 3 H), 2.73 (s, 2H), 1.05 (s, 3H), .31 (m, 4H).

Step 5: To a solution of 56_4 (1.45 g, 6.33 mmol, 1.00 eq) in THF (29 mL) was added n-BuLi (2 M, 4.75 mL, 1.50 eq) at -78°C. After 30 min, 2-isopropoxy-4,4,5,5- tetramethyl-1,3,2-dioxaborolane (2.36 g, 12.66 mmol, 2.59 mL, 2.00 eq) in THF (2.5 mL) 02429524\45-0 l was added to it. The resulting mixture was warmed to 20°C and stirred for 0.5 hr. The mixture was quenched with saturated NH4Cl (50 mL) and extracted with EtOAc (100 mL). The orgainic layer was trated and purified by silica gel column(PE:EA=20:1~10:1) to give 56_5 (1.25 g, 4.11 mmol, 64.9% yield, 90.7% purity) as a colorless oil.

LCMS: RT = 0.928 min, m/z 277.1 [M+H]+ Step 6: To a solution of 56_5 (500.00 mg, 1.81 mmol, 1.00 eq) in DME (10 mL) was added 4-chloromethylsulfanyl-pyrimidine (290.79 mg, 1.81 mmol, 210.72 uL, 1.00 eq), NA2CO3 (2 M, 1.99 mL, 2.20 eq) and 4-ditert-butylphosphanyl- N,N- dimethyl-aniline;dichloropalladium (64.09 mg, 90.50 umol, 64.09 uL, 0.05 eq) under nitrogen. The resulting mixture was stirred at 85°C for 2 hr under nitrogen. The e was diluted with water (50 mL) and extracted with EtOAc (50 mL). The organic layer was concentrated and purified by silica gel column (PE:EA=5:1) to give 56_6 (350.00 mg, 1.14 mmol, 63% yield, 89% purity) as a yellow oil.

LCMS: RT = 0.829 min, m/z 275.1 [M+H]+ Step 7: To a solution of 56_6 (350.00 mg, 1.28 mmol, 1.00 eq) in DCM (5.5 mL) was added MCPBA (690.28 mg, 3.20 mmol, 80% purity, 2.50 eq) at 0°C. The e was stirred at 20°C for 2 hr. The mixture was quenched with aqueous NazSzO3 (100 mL) and extracted with DCM (50 mL*2). The combined organic layers were concentrated and purified by silica gel column(PE:EA=1:1) to give 56_7 (200.00 mg, 617.72 umol, 48.6% yield, 94.6% purity) as a yellow solid.

LCMS: RT = 0.653 min, m/z 307.1 [M+H]+ Step 8: The mixture of 56_7 (200.00 mg, 652.78 umol, 1.00 eq) and trans- exane-1,4-diamine (298.17 mg, 2.61 mmol, 4.00 eq) in e (3 mL) was stirred at 130°C for 2 hr in the microwave. The mixture was filtered and trated. The crude was purified by prep-HPLC(base) to give A-56 (50.00 mg, 144.85 umol, 22.2% yield, 98.6 % purity) as a yellow solid.

LCMS: RT = 1.999 min, m/z 341.2 [M+H]+ 1H NMR (CDCl3, 400 MHz) 5 8.18-8.17 (d, J=5.2 Hz, 1 H), 7.82 (s, 1 H), 6.69- 6.67 (d, J=5.2 Hz, 1H), .85 (d, J=5.6 Hz, 1H), 3.88 (s, 3H), 3.84 (s, 1H), 3.38 (s, 02429524\45-0 l 2H), 2.75—2.73 (m, 1H), 2.17-2.16 (m, 2H), 1.94—1.92 (m, 2H), .27 (m, 4H), 1.08 (s, 3 H), 0.31—0.25 (m, 4H).

Pre aration 0 1R 4R -N1- 4- 1-meth lne0 ent l-IH- ra 01 1 rimidin-Z- l c ane -1 4-diamine A57 / H/ O COZH 0/ N‘ \ NH2NH2 H20 7< —> b_—> —> DCM,CD| BUU KOH,200°C 571 572 O& _N /s N\ CI /Lo’é\0 0% \ N\ NJ m—CPBA n-BuLi,THF,-78°C Pd(amphos)C|2 DCM aq Na2003, DME H2NOONHZ dioxane 130°C H2N‘‘0’:TN\ Compound A-57 was synthesized in a manner similar as described for the sis of compound A-56.

LCMS: RT = 1.558 min, m/z 343.2 [M+H]+ 1H NMR (MeOD, 400 MHz) 5 8.20-8.13 (m, 2 H), 7.29—7.27 (d, J=5.2 Hz, 1H), 4.12—4.09 (m, 1H), 3.92 (s, 3H), 3.34 (s, 1H), 3.21 (s, 1H), 2.20 (m, 4H), 1.63 (m, 4H), 0.98 (s, 9 H). 02429524\45-01 Pre aration 0 4- 2- 1R 4R amin0c clohex lamina rimidin l meth l- 1H- razol-S- l c clo r0 [methanol A58 n—BuLi,THF,- /Sj'\|/\N/j/CI /S Pd(amphos)C|2 a.q Na2C03(2M),DME H N"ONHZ H dioxane, N‘"U W/ 130 °C,MW H2 Step 1 to Step 6: Intermediate 58_6 was synthesized from compound 51_3 in a manner similar as described for the synthesis of A-56.

LCMS: RT = 0.676 min, m/z 457.4 [M+H]+ Step 7: To a solution of 58_6 (300.00 mg, 656.89 umol, 1.00 eq) in THF (300 ML) was added TBAF.3H20 (414.51 mg, 1.31 mmol, 2.00 eq) at 20°C. The mixture was stirred for 30 min. The mixture was filtered and rtrated. The crude product was purified by prep-HPLC(base) to give A-58 (27.00 mg, 76.89 umol, 11.7% yield, 97.5% ) as a yellow solid.

LCMS: RT = 2.29 min, m/z 343.2 [M+H]+ 1H NMR (CDC13, 400 MHz) 5 8.23-8.22 (d, J=5.2 Hz, 1 H), 7.86 (s, 1 H), 6.81- 6.80 (d, J=5.2 Hz, 1H), 5.09 (s, 1H), 4.32—4.30 (m, 1H), 3.89 (s, 3H), .79 (m, 1H), 2.93-2.88 (m, 1H), 2.20-2.06 (m, 4H), 1.52—1.31 (m, 5H), .28 (m, 4H). 02429524\45-0 l Pre aration 0 1R 4R -N1- r0 5- c clo r0 lmeth 0 r0 [- IH- razol- 4- l rimidin-Z- lc clohexane-I 4-diamine A59 E/N\ —>Br ?—>\<*0a}? 10vol TFA DCM n- BuLi,THF, DCM -78°C :NN‘< YJ' 0"" N CI (;|\r (ERIN—MH2N‘ k] N N\< dioxaneMW N"U T \ Pd(amphos)CI2 "I a- q Na200pg(2M) DME 130°C 15 h H2N‘ 59_5 A-59 Compound A-59 was synthesized in a manner similar as described for the synthesis of compound A-51.

LCMS: RT = 2.478 min, m/z 389.2 [M+H]+ 1H NMR (MeOD, 400 MHZ) 5 8.42-8.36 (m, 2 H), 4.82-4.75 (m, 1H), 4.00 (s, 1H), 3.21-3.20 (s, 3H), 2.20-2.15 (m, 4H), 1.60-1.56 (m, 4H), 1.51 (s, 6H), 1.05 (s, 1H), 0.55-0.53 (m, 2H), 0.28 (m, 2H).

Pre aration 0 1R 4R -N1- 4- 5- c clo r0 lmeth l-I-meth l-IH- 4- l tri uorometh l rimidin-Z- l c clohexane-I ine A60 CI N CI Til MeSNa /813:0 CF3 E120.ZnCI2 CF3 DioxaneMW160°C Step 1: To a solution of 60_1 (2.00 g, 9.22 mmol, 1.00 eq) in THF (40 mL) was added ZnClz-EtzO (1 M, 11.06 mL, 1.20 eq) at 0°C under nitrogen protection. The mixture was stirred for 2 hours at 0°C. Methylsulfanylsodium (646.23 mg, 9.22 mmol, 02429524\45-0 1 587.48 uL, 1.00 eq) was added. The resulting e was stirred at 20°C for 16 hours.

TLC (pure PE) showed reactant 1 (Rf=0.5) consumed and product (Rf=0.3) formed. The mixture was quenched with 1M HCl (20 mL) and concentrated. The aqueous layer was extracted with DCM (20 mL*3). The combined organic layer was concentrated and purified by silica gel column (PE: EA=1 :0~50: 1) to give 60_2 (1.00 g, 1.97 mmol, 21.4% yield, 45.1% purity) as colorless oil.

LCMS: RT = 0.794 min, m/z 228.9 [M+H]+ 1H NMR , 400 MHz) 5 8.67 (s, 1H), 2.62 (s, 3H).

Step 2: To a solution of 51_7 (574.11 mg, 2.19 mmol, 1.00 eq) in THF (10 mL) were added 60_2 (500.00 mg, 2.19 mmol, 1.00 eq), K3PO4 (1 M, 4.38 mL, 2.00 eq) and AdanuPBiphenyl (50.00 mg) under nitrogen. The resulting mixture was d at 85°C for 2 hours under nitrogen. TLC (PE:EA=1:1) showed reactant (Rf=0.6) consumed and product (R=0.5) . The mixture was concentrated and diluted with H20 (50 mL).

The s layer was extracted with EA (20 mL*2). The organic layer was concentrated.

The residue was purified by silica gel column (PE:EA=20:1~5:1) to give 60_3 (300.00 mg, 611.96 umol, 27.9% yield, 66.9% ) as a yellow solid.

LCMS: RT = 0.927 min, m/z 329.0 [M+H]+ 1H NMR (CDCl3, 400 MHz) 5 8.75 (s, 1H), 7.75 (s, 1H), 3.94 (s, 3H), 2.95 (d, J=6.8Hz, 2H), 2.57 (s, 3H), .94 (m, 1H), 0.48-0.44 (m, 2H), 0.17—0.13 (m, 2H).

Step 3: To a solution of 60_3 (400.00 mg, 1.22 mmol, 1.00 eq) in DCM (7 mL) was added m-CPBA (657.92 mg, 3.05 mmol, 80% purity, 2.50 eq) at 0°C. The mixture was stirred at 20°C for 1 hour. TLC eOH=20:1) showed reactant (Rf=0.6) consumed and product (Rf=0.5) formed. The mixture was quenched with a.q NazS2O3 (100 mL) and extracted with DCM (50 mL*2). The organic layer was concentrated and purified by silica gel column (PE:EA=1 : 1) to give 60_4 (300.00 mg, 738.23 umol, 60.5% yield, 88.7% purity) as a yellow solid.

LCMS: RT = 0.72 min, m/z 361.0 [M+H]+ Step 4: The mixture of 60_4 0 mg, 555.02 umol, 1.00 eq) and trans- cyclohexane-1, 4-diamine (253.51 mg, 2.22 mmol, 4.00 eq) in dioxane (3 mL) was stirred at 130°C for 2 hours under microwave. The mixture was filtered and the filtrate was 02429524\45-0 l concentrated. The residue was purified by prep-HPLC (HCl condition) to give A-60 (80.00 mg, 185.66 umol, 33.5% yield, 100% purity, HCl) as a yellow solid.

LCMS: RT = 2.417 min, m/z 395.2 [M+H]+ 1H NMR (MeOH, 400 MHz) 5 8.66 (s, 1H), 7.90 (s, 1H), 4.15 (s, 1H), 3.97 (s, 3H), 3.20 (s, 1H), 3.10 (s, 2H), 2.18 (s, 4H), 1.62 (s, 4H), 1.05 (s, 1H), 0.53 (s, 2H), 0.25 (s, 2H).

Pre aration 0 N- 1R 4R 1H- ra 0l l c clohex l chl0r0 5- c clo r0 lmeth l-I-meth l-IH- razol l rimidin-Z-amine A64 NHBoc NHBoc NHBoc ODCMMsCI , DMSODIEA / OH (ESL)032003 DMFH/NE%> $51} HQH’JIELVY 3 h 160 °C CN 64_1 64_2 64_3 amine-64 A-64 Step 1: To a solution of 64_1 (2.00 g, 9.29 mmol, 1.00 eq) in DCM (20 mL) were added TEA (1.88 g, 18.58 mmol, 2.58 mL, 2.00 eq) and methanesulfonyl chloride (1.06 g, 9.29 mmol, 719.03 uL, 1.00 eq) at 0°C. The e was stirred at 20°C for 2 hours. The mixture was diluted with H20 (30 mL) and extracted with DCM (20 mL*2). The combined organic layers were dried and concentrated to give 64_2 (2.50 g, 8.52 mmol, 91.7% yield) as a white solid. 1H NMR(CDC13, 400 MHz) 5 4.88 (s, 1 H), 4.48 (s, 1 H), 3.52 (s, 1 H), 3.01 (s, 3H), 2.07—2.03 (m, 2H), 1.83—1.82 (m, 2H), 1.73-1.70(m, 2H), 1.61-1.55 (m, 7H), 1.448 (s, 9H).

Step 2: To a on of 1H-pyrazole (195.25 mg, 2.87 mmol, 1.20 eq) in MeCN (7.00 mL) were added CS2CO3 (1.56 g, 4.78 mmol, 2.00 eq) and 64_2 (700.00 mg, 2.39 mmol, 1.00 eq). The mixture was stirred at 100 CC for 2 hours. The mixture was diluted with H20 (20 mL) and extracted with EtOAc (10 mL*3). The combined organic layers were washed with brine (20 mL) and trated. The residue was purified by pre- HPLC (basic ion) to give 64_ 3(200.00 mg, 732.25 umol, 30.6% yield, 97.1% purity) as a yellow solid. 02429524\45-0 l _ 72 _ MS: m/z RT = 0.888 min, 266.0 [M+H]+ Step 3: To a solution of 64_3 0 mg, 678.35 umol, 1.00 eq) in DCM (2 mL) was added TFA (400.00 uL) at 0°C. The mixture was d at 20°C for 1 hour. The mixture was diluted with H20 (30 mL) and extracted with DCM (10 mL*2). The aqueous layer was lyophilizd to give 64 0 mg, 633.91 umol, 93. 5% yield, 98.3% purity, TFA) as colorless oil.

LCMS: RT = 0.272 min, m/z 166.2 [M+H]+ Step 4: To a solution of 51_9 (100.00 mg, 353.16 umol, 1.00 eq) in DMSO (1.5 mL) were added amine-64 (95.48 mg, 529.74 umol, 1.50 eq) and DIEA (182.57 mg, 1.41 mmol, 246.71 uL, 4.00 eq). The resulting e was stirred at 160°C for 3 hours, filtered and the mother liquid was concentrated. The residue was purified by pre-HPLC (basic condition) and then prep-HPLC (HCl condition) to give A-64 (10.00 mg, 23.42 umol, 6.6% yield, 100% purity) as a yellow solid.

LCMS: RT = 1.981 m/Z 412.2[M+H]+ 1H NMR (MeOD, 400 MHz) 5 8.41-8.37 (m, 2 H), 7.83 (s, 1 H), 7.67 (s, 1 H),6.39 (s, 1H), 4.36 (s, 1H), 4.12-4.08 (m, 1H), 3.95 (s, 3H), 3.25—3.23 (d, J=6.4Hz, 2H), 2.26- 2.23 (m, 4H), 2.05-2.02(m, 2H), 1.99-1.96 (m, 2H), 1.73-1.67(m, 2H), 1.09 (s, 1H), 0.55— 0.50 (m, 2H), 0.27-0.26 (m, 2H).

H NHBoc NHz NHBoc l/N : : [\lJ TFA 51- _. _.

CsZCOBDMF DCM 0"TN DMSODIEA OMs gj g} 3 h 160 00 QN 64_2 65_1 amine-65 "5 Compound A-65 was synthesized in a manner similar as described for the synthesis of compound A-64 as a yellow solid.

LCMS: RT = 1.461 min, m/z 412.2 [M+H]+ 02429524\45-0 l 1H NMR (MeOD, 400 MHz) 5 9.10 (s, 1 H), 8.42 (s, 1 H), 7.81 (s, 1 H), 7.62 (s, 1 H), 4.55—4.49 (m, 1H), 4.13 (s, 1H), 3.96 (s, 3H), 3.25—3.24 (d, J=6.4Hz, 2H), 2.36-2.28 (m, 4H), 2.08-1.98 (m, 2H), 1.80-1.74 (m, 2H), 1.09 (s, 1H), 0.53—0.51 (m, 2H), 0.27— 0.26 (m, 2H).

Pre aration 0 1R 4R -N1- 5-chl0r0 5- c clo r0 lmeth th l-IH- ra 0l l ri midin-Z- l-N4- hen lc clohexane-I 4-diamine A-68 NHBoc QIHBOC NHz H TFA 51 9 —> *— 0 PMWe HN DMSO DIEA Xantphos 16 h 1 3h,160 cD toluene ,NH NH Ph Ph/ 68 1 68 A-68 Step 1: To a solution of tert-butyl ((1R,4R)aminocyclohexyl)carbamate (500.00 mg, 2.33 mmol, 1.00 eq) and iodobenzene (713.01 mg, 3.49 mmol, 389.63 uL, 1.50 eq) in THF (15 mL) were added t—BuOK (784.35 mg, 6.99 mmol, 3.00 eq) and RuPhos Indoline (100.00 mg). The resulting mixture was stirred at 80°C for 12 hours under nitrogen. The mixture was filtered and the mother liquid was trated. The residue was purified by silica gel column (PE:EA=10:1~4:1) to give 68_1 (300.00 mg, 869.39 umol, 37.3% yield, 84.1% purity) as a white solid.

LCMS: RT = 1.024 min, m/z 291.0 [M+H]+ Step 2 and step 3: Compound A-68 was synthesized in a manner similar as described for the synthesis of nd A-64 as a yellow solid.

LCMS: RT = 1.194 min, m/z 437.2 [M+H]+ 1H NMR (MeOD, 400 MHZ) 5 8.29 (s, 1 H), 8.03 (s, 1 H), 7.75-7.34 (m, 5 H), 3.86 (s, 3H), 3.05 (d, J=6.4Hz, 2H), 2.00-1.97 (m, 4H), 1.55 (s, 2H), 1.37-1.29 (m, 2H), 0.97 (s, 1H), 0.39 (s, 2H), 0.14 (s, 2H). 02429524\45-0 l Pre aration 0 5R 8R 5-chl0r0 5- c clo r0 lmeth lmeth l-1H- razol l rimidin-Z- l amino azas iro 4.5 decan-Z-one A-71 CIYN DMSODIEA N TBAF 160°C 0W3]Yr: amine1 To a on of amine-71 (180.00 mg, 879.34 umol, 1.00 eq, HCl) and 51_9 (248.99 mg, 879.34 umol, 1.00 eq) in DMSO (2.7 mL) were added DIEA (614.30 uL, 3.52 mmol, 4.00 eq) and TBAF (1 M, 175.87 uL, 0.20 eq). The mixture was stirred at 160°C for 3 hours. The mixture was cooled down and filtered. The solid were washed with MeOH (10 mL) at room temperature and then EtOAc (3 mL) at 50°C to give A-71 (40.00 mg, 90.39 umol, 11.4% yield, 93.7% purity) as a white solid.

LCMS: RT = 2.836 min, m/z 415.2 [M+H]+ 1H NMR (DMSO, 400 MHz) 5 8.26 (s, 1 H), 8.00 (s, 1 H), 7.72 (s, 1 H), 7.20 (s, 1 H), 3.85 (s, 3H), 3.70-3.68 (m, 1H), 3.06 (d, z, 2H), 2.19—2.15 (m, 2H), 1.86- 1.80 (m, 5H), 1.64-1.60 (m, 3H), 1.49—1.41 (m, 5H), 0.97 (s, 1H), 0.40 (s, 2H), 0.13 (s, To a solution of A-51 (200.00 mg, 554.20 umol, 1.00 eq) and benzaldehyde (58.81 mg, 554.20 umol, 56.01 uL, 1.00 eq) were added ACOH (33.28 mg, 554.20 umol, 31.70 uL, 1.00 eq) and NaBH3CN (69.65 mg, 1.11 mmol, 2.00 eq). The e was sitrred at °C for 16 hours. The mixture was quenched with aqueous NaHC03 (1 mL) and 02429524\45-0 l concentrated. The residue was purified by PLC (column: Phenomenex Gemini mm*10um; mobile phase: [water (0.05% ammonia hydroxide V/V)-ACN];B%: %,12min) to give A-74 (100.00 mg, 217.80 umol, 39.3% yield, 98.2% purity) as a pink solid.

LCMS: RT = 2.74 min, m/z 451.2 [M+H]+ 1H NMR (CDCl3, 400 MHz) 5 8.20-8.13 (m, 2 H), 7.34—7.33 (m, 5 H), 4.87 (d, J=8.4Hz, 1H), 3.91 (s, 3H), 3.84 (s, 3H), 3.07-3.06 (m, 2H), 2.58-2.52 (m, 1H), 2.16-2.02 (m, 4H), 1.32—1.22 (m, 4H), 1.01—0.99 (m, 1H), 0.49—0.45 (m, 2H), 0.18-0.16 (m, 2H). diamineA-75 HN—N /N N‘ O WU/ \\ H N\ \N‘ NaBH3CN HZN" vU W/ A-51 A-75 To a solution of A-51 (200.00 mg, 554.20 umol, 1.00 eq) and 1H-pyrazole carbaldehyde (53.25 mg, 554.20 umol, 1.00 eq) were added AcOH (34.86 uL, 609.62 umol, 1.10 eq) and NaBH3CN (69.65 mg, 1.11 mmol, 2.00 eq). The mixture was sitrred at 15°C for 16 hours. The mixture was quenched with aqueous NaHCO3 (1 mL) and concentrated. The residue was purified by prep-HPLC (column: enex Gemini 150*25mm*10um; mobile phase: [water (0.05% ammonia ide V/V)-ACN]; B%: 39%-39%,12min) to give A-75 (4.00 mg, 9.04 umol, 1.6% yield, 99.7% purity) as a yellow solid.

LCMS: RT = 2.95 min, m/z 441.2 [M+H]+ 1H NMR (CDCl3, 400 MHz) 5 8.21-8.12 (m, 2 H), 7.61 (s, 2 H), 4.90 (d, J=8.4Hz, 1H), 3.91 (s, 3H), 3.85-3.80 (m, 3H), 3.06-3.05 (m, 2H), 2.70 (m, 1H), 2.19—2.07 (m, 4H), 1.42—1.23 (m, 4H), 1.09 (m, 1H), 0.50—0.45 (m, 2H), 0.18-0.14 (m, 2H). 02429524\45-0 l Pre aration 0 1R 4R -N1- 5-chl0r0 5- c clo r0 lmeth l-I-meth l-IH- 4- l rimidin-Z- l-N4- ridin lmeth lc clohexane-I 4-diamine A-76 N N "Na Cl 0%" \‘ a 3 a"\‘\ | / H2N" CI A-51 UT To a solution of A-51 (200.00 mg, 554.20 umol, 1.00 eq) and pyridinecarbalde hyde (59.36 mg, 554.20 umol, 52.07 uL, 1.00 eq) were added AcOH (33.28 mg, 554.20 umol, 31.70 uL, 1.00 eq) and NaBH3CN (69.65 mg, 1.11 mmol, 2.00 eq). The mixture was sitrred at 15°C for 16 hours. The mixture was quenched with aqueous NaHC03 (1 mL) and concentrated. The residue was purified by prep-HPLC (column: Phenomenex Gemini 150*25mm*10um;mobile phase: [water (0.05% ammonia hydroxide V/V)- ACN];B%: 40%-58%,12min) to give A-76(100.00 mg, 219.09 umol, 39.5% yield, 98.8% purity) as a yellow solid.

LCMS: RT = 2.381 min, m/z 452.2 [M+H]+ 1H NMR (CDC13, 400 MHz) 51 (m, 2 H), 7.69 (d, J=8.0Hz, 1H), 7.27 (s, 1 H), 4.87 (d, J=8.0Hz, 1H), 3.91 (s, 3H), 3.85-3.81 (m, 3H), 3.07-3.06 (m, 2H), 2.56- 2.51 (m, 1H), 2.16-2.01 (m, 4H), 1.30—1.22 (m, 4H), 1.09 (m, 1H), 0.50-0.46 (m, 2H), .16 (m, 2H).

Pre aration 0 1R 4R -N1- 1H- razol lmeth l-N4- 4- 5- c clo r0 lmeth l-I-meth l-IH- ra 0l l rimidin-Z- lc clohexane-I 4- diamine (A802 H l N m, "TM / (:r N :Jr \ ACOH,MeOH / "2"". / \ \ ilk/fly A-14 O N’ A-80 To a solution of A-14 (200.00 mg, 612.67 umol, 1.00 eq) and 1H-pyrazole carbaldehyde (58.87 mg, 612.67 umol, 1.00 eq) were added AcOH (36.79 mg, 612.67 02429524\45-0 l umol, 35.04 uL, 1.00 eq) and NaBH3CN (77.00 mg, 1.23 mmol, 2.00 eq). The mixture was sitrred at 15 CC for 16 hours. The mixture was quenched with s NaHCO3 (1 mL) and trated. The residue was purified by PLC (basic condition) to give A-80 (20.00 mg, 48.90 umol, 8% yield, 99.4% purity) as a yellow solid.

LCMS: RT = 2.418 min, m/z 407.2 [M+H]+ 1H NMR(CDC13, 400 MHz) 5 8.17 (d, J=5.2Hz, 1H), 7.83 (s, 1H), 7.58 (s, 1H), 6.71 (d, J=6.2Hz, 1H), 4.88 (d, J=7.6Hz, 1H), 3.89 (s, 4H), 3.81 (s, 2H), 3.21 (d, J=6.0Hz, 2H), 2.61 (s, 1H), 2.21—2.03 (m, 4H), 1.36-1.25 (m, 4H), 1.09 (s, 1H), 0.48-0.46 (m, 2H), 0.25—0.24 (m, 2H).

Pre aration 0 1R 4R -N1- 4- 5- c clo r0 lmeth l meth l-IH- ra 0l l rimidin-Z- l-N4- 1-meth l-IH- ra 0l lmeth lc clohexane-I 4-diamine H I N \ \o NaBH30N H «U N\ / O\"er\ / _‘ AcOH,MeOH \ler/ H2N‘" / ‘MjAH A-14 N" A-81 To a solution of A-14 (200.00 mg, 612.67 umol, 1.00 eq) and compound 81_1 (67.46 mg, 612.67 umol, 1.00 eq) were added AcOH (36.79 mg, 612.67 umol, 35.04 uL, 1.00 eq) and NaBHgCN (77.00 mg, 1.23 mmol, 2.00 eq). The mixture was sitrred at 15 CC for 16hours. The e was quenched with a.q NaHC03 (1 mL) and concentrated. The residue was purified by prep-HPLC (basic condition) to give A-81 (40.00 mg, 87.81 umol, 14.3% yield, 92% purity) as a yellow solid.

LCMS: RT = 2.469 min, m/z 421.2 [M+H]+ 1H NMR(CDC13, 400 MHz) 5 8.17 (d, J=5.2Hz, 1H), 7.82 (s, 1 H), 7.57 (s, 1 H), 7.51 (s, 1 H), 6.7 (d, J=6.2Hz, 1H), 4.84 (d, J=7.6Hz, 1H), 3.89-3.85 (m, 8H), 3.19 (d, J=6.0Hz, 2H), 2.73 (s, 1H), 2.25-2.12(m, 4H), 1.54 (m, 1 H), 1.29—1.23 (m, 2H), 1.07 (s, 1H), 0.50-0.46 (m, 2H), 0.26-0.24 (m, 2H). 02429524\45-0 l diamine (A-822 y M H I H N /}\l —>NaBH3CN U 1 \ \ \\‘ / \ Cl H2N CI A-51 AO HMJN A-82 To a solution of A-51 (200.00 mg, 554.20 umol, 1.00 eq) and 1H-pyrazole carbaldehyde (53.25 mg, 554.20 umol, 1.00 eq) were added AcOH (31.7 uL, 554.20 umol, 1.00 eq) and N (69.65 mg, 1.11 mmol, 2.00 eq). The mixture was sitrred at 15 CC for 16 hours. The mixture was quenched with a.q NaHCO3 (1 mL) and concentrated. The residue was purified by prep-HPLC (basic condition) to give A-82 (35.00 mg, 79.28 umol, 14.3% yield, 99.9% purity) as a yellow solid.

LCMS: RT = 2.867 min, m/z 441.2 [M+H]+ 1H NMR (CDC13, 400 MHz) 5 8.20 (s, 1H), 8.13 (s, 1H), 7.53 (s, 1H), 6.22 (s, 1H), 4.91 (d, J=7.6Hz, 1H), 3.93 (s, 2H), 3.91 (s, 3H), 3.81 (s, 1H), 3.49 (s, 1H), 3.07 (d, J=5.6Hz, 2H), 2.56-2.53 (m, 1H), 2.16-2.02 (m, 4H), .32 (m, 4H), 1.05 (s, 1H), 0.48-0.45 (m, 2H), 0.18-0.16 (m, 2H). diamine (A83 2 I g" Ti(i—PrO)4,NaBH3CN k] N :{N N N \ \ O I; O —> Hy/j/K N/ DCE,TEA 0’ \hlr/ \ H‘" Cl H2N‘ H A-51 83_1 N/ A-83 To a solution of A-51(100.00 mg, 251.67 umol, 1.00 eq, HCl), compound 83_1 (27.71 mg, 251.67 umol, 1.00 eq) were added TEA (503.34 umol, 69.77 uL, 2.00 eq) and 02429524\45-0 l Ti(i-PrO)4 (149 uL, 503.34 umol, 2.00 eq). The mixture was stirred at 80 0C for 12 hours.

Then NaBHgCN (39.54 mg, 629.18 umol, 2.50 eq) was added. The resulting mixture was stirred at 15 0C for 4 hours. The mixture was quenched with a.q NaHCO3 (50 mL) and extracted with DCM (50 mL*2). The organic layer was concentrated and purified by prep- HPLC (basic condition) to give A-83 (20.00 mg, 43.96 umol, 17.5% yield) as a yellow solid.

LCMS: RT = 1.897 m/Z 455.2[M+H]+ 1H NMR (CDClg, 400 MHz) 5 8.19 (s, 1H), 8.12 (s, 1H), 7.52 (s, 2H), 4.90 (d, J=7.6Hz, 1H), 4.05—4.00 (m, 1H), 3.91 (s, 3H), 3.78-3.76 (m, 1H), 3.06 (d, J=5.6Hz, 2H), .46 (m, 1H), 2.11-2.08 (m, 4H), 1.40-1.38 (m, 3H), 1.25-1.19(m, 4H), 1.05 (s, 1H), 0.47—0.44 (m, 2H), 0.16-0.15 (m, 2H).

Pre n 0 N- 1R 4R 5-chl0r0 5- c clo r0 lmeth l-I-meth [- 1H- razol l rimidin-Z- lamina c clohex l-1H- razolecarb0xamide A87 ( 0 1 Mn N EDCI,HOBt / N I): \ OH I \ H HTN\ + N’ DCM,D|EA hi / HZN‘" CI HMNVUo N/ A-87 To a solution of compound 87_1 (46.59 mg, 415.65 umol, 1.00 eq) in DCM (4 mL) were added DIEA (181.5 uL, 1.04 mmol, 2.50 eq), EDCI (95.62 mg, 498.78 umol, 1.20 eq) and HOBt (11.23 mg, 83.13 umol, 0.20 eq) at 0 CC. The reaction mixture was stirred at 0 CC for 0.5 hour. The solution of A-51 (150.00 mg, 415.65 umol, 1.00 eq) in DCM (4.5 mL) was added. The resulting e was stirred at 20 CC for 16 hours. The mixture was diluted with H20 (50 mL) and ted with DCM (40 mL*3). The organic layer was concentrated and ed by prep-HPLC (HCl condition) to give A-87 (80.00 mg, 160.61 umol, 38.6% yield, 98.6% purity, HCl) as a yellow solid.

LCMS: RT = 2.391 m/Z 455.2[M+H]+ 02429524\45-0 l 1H NMR (MeOD, 400 MHZ) 5 8.48 (s, 1H), 8.39 (s, 1H), 8.16 (s, 2H), 4.04 (s, 1H), 3.96 (s, 3H), 3.93-3.90 (In, 1H), 3.24 (d, J=6.4Hz, 2H), 2.18-2.04 (m, 4H), 1.67- , 4H), 1.18 (s, 1H), 0.57-0.55 (In, 2H), 0.30-0.29 (In, 2H).

Pre aration 0 1R 4R -N1- 5-chl0r0 5- c clo r0 lmeth l-I-meth l-IH- ra 0l l rimidin-Z- l-N4- 5-meth l-IH- ra 0l lmeth lc clohexane-I 4- diamine (A-912 I (Y0 4 kl I N N I)" U I" / "(II"; H2N\" / Cl ACOH MeOH H‘ A-51 A-91 To a on of A-51 (150.00 mg, 415.65 umol, 1.00 eq) and 5-methyl-1H- pyrazolecarbaldehyde (54.92 mg, 498.78 umol, 1.20 eq) in MeOH (1.5 mL) were added AcOH (23.77 uL, 415.65 umol, 1.00 eq) and NaBH3CN (78.36 mg, 1.25 mmol, 3.00 eq). The mixture was stirred at 15 CC for 16 hours. The mixture was quenched with a.q NaHCO3 (10 mL) and extracted with EA (20 mL*2). The combined organic layers were concentrated and purified by prep-HPLC (HCl condition) to give A-91 (20.00 mg, 36.22 umol, 8.7% yield, 89% purity, HCl) as a white solid.

LCMS: RT = 2.425 m/z 455.2[M+H]+ 1HNMR (MeOD, 400 MHz) 5 8.44-8.41 (m, 2H), 8.23 (s, 1H), 4.26 (s, 2H), 4.13— 4.06 (m, 1H), 3.95 (s, 3H), 3.24 (d, J=6.4Hz, 2H), 2.51 (s, 3H), 2.40—2.24 (m, 4H), 1.76- 1.61 (m, 4H), 1.09 (s, 1H), .53 (m, 2H), 0.28-0.27 (m, 2H). 02429524\45-0 l Pre aration 0 1R 4R -N1- 5-chl0r0 5- c clo r0 lmeth l-I-meth l-IH- 2 2 2-tri h l c clohexane-I 4-diamine A94 M F30/\o/ \CF3 I "1‘ H I )9 HTN\ / N , \"lr/\ TEA "I / H ng CI THF, 80°C Fell/\M‘" CI A-51 A-94 To a on of A-51 (150.00 mg, 415.65 umol, 1.00 eq) in THF (4.5 mL) were added TEA (144.04 uL, 1.04 mmol, 2.50 eq) and 2,2,2-tri?uoroethyl tri?uoromethanesulfonate (115.77 mg, 498.78 umol, 1.20 eq). The mixture was d at 50 CC for 16 hours. The mixture was concentrated and purified by prep-HPLC (HCl condition) to give A-94 (80.00 mg, 163.38 umol, 39.3% yield, 97.9% purity, HCl) as a yellow solid.

LCMS: RT = 2.137 min, m/z 443.2 [M+H]+ 1H NMR (MeOD, 400 MHz) 5 8.45 (m, 2H), 4.15—4.09 (m, 2H), 4.09 (s, 1H), 3.95 (s, 3H), 3.39-3.36 (m, 1H), 3.22—3.21 (m, 2H), 2.33—2.25 (m, 4H), 1.71-1.56 (m, 4H), 1.09 (s, 1H), 0.54—0.52 (m, 2H), 0.28-0.27 (m, 2H).

Pre aration 0 Pre aration 0 1R 4R -N1- 1H- razol lmeth l -N4- 5- chl0r0 5- c clo r0 lmeth l-I-meth l-IH- ra 0l l rimidin-Z- l-NI- 2 22- HWHNO N \r \[\i / Y \ | [\i / CI MeZSNZESSDC HW'WU CI A-75 A-95 To a on of A-75 (150.00 mg, 340.16 umol, 1.00 eq) in MeCN (4.5 mL) were added CszCO3 (221.66 mg, 680.32 umol, 2.00 eq) and 2,2,2-tri?uoroethyl tri?uoromethanesulfonate (118.43 mg, 510.24 umol, 1.50 eq). The e was stirred at 70 CC for 16 hours. The mixture was filtered; the filtrate was concentrated and purified 02429524\45-0 l by prep-HPLC (HCl condition) to give A-95 (20.00 mg, 35.7 umol, 10.5% yield, 100% purity, HCl) as a white solid.

LCMS: RT = 2.285 m/Z 523.2[M+H]+ 1H NMR (MeOD, 400 MHz) 5 8.41-8.39 (m, 2H), 8.02 (s, 1H), 7.76 (s, 1H), 5.03— 4.97 (m, 2H), 4.23 (s, 2H), 4.12—4.04 (m, 1H), 3.95 (s, 3H), 3.21 (d, J=6.4Hz, 3H), 2.34— 2.23 (m, 4H), 1.65-1.53 (m, 4H), 1.09 (s, 1H), 0.54—0.52 (m, 2H), 0.27-0.26 (m, 2H). diamine (A-962 HQNHNTi(i-PrO)4 NaBHaCN HYN mi); DCE TEA \\ K(\tH\hf A-96 To a solution of A-75 (50.00 mg, 113.4 umol, 1.00 eq) and compound 1H- pyrazolecarbaldehyde (32.69 mg, 340.17 umol, 3.00 eq) were added TEA (31.44 uL, 226.78 umol, 2.00 eq) and Ti(Oi-Pr)4 (67.1 uL, 226.78 umol, 2.00 eq). The mixture was stirred at 80 CC for 12 hours. NaBHgCN (21.38 mg, 340.17 umol, 3.00 eq) was added and the resulting e was stirred at 15 CC for 4 hours. The reaction was ed with a.q NaHC03 (1 mL) and ed. The filtrate was purified by prep-HPLC (TFA and then basic condition) to give A-96 (7.00 mg, 13.10 umol, 11.6% yield, 97.5% purity) as a white solid.

LCMS: RT = 2.382 m/z 521.3[M+H]+ 1H NMR (MeOD, 400 MHz) 5 8.23 (m, 2H), 8.03 (s, 1H), 7.78 (s, 4H), 4.47—4.29 (m, 4H), 3.91-3.87 (m, 4H), 3.12 (d, J=6.4Hz, 2H), 2.27—2.22 (m, 4H), 1.94-1.85 (m, 2H), 1.46-1.36 (m, 2H), 1.01—1.00 (m, 1H), 0.45—0.44 (m, 2H), 0.14—0.13 (m, 2H). 02429524\45-0 l Pre aration 0 1R 4R -N1- 4- 5- c clo r0 lmeth l-I-meth l-IH- razol l uoro rimidin-Z- lc clohexane-I 4-diamine A-86 2 Y/F Pd((amphos)C|2 CIYN dioxane 160°CMW TN a. q Na2C03(2M) DME Step 1: To a on of compound 51_7 (800 mg, 3.05 mmol, 1.00 eq) in DME (16 mL) were added compound 2 (509.54 mg, 3.05 mmol, 1.00 eq), Na2C03 (2 M, 4.6 mL, 3.00 eq) and 4—ditert-butylphosphanyl-N,N-dimethyl-aniline;dichloropalladium (108.04 mg, 152.50 umol, 108 uL, 0.05 eq) under nitrogen. The resulting mixture was stirred at 85 CC for 2 hours under nitrogen. The mixture was diluted with H20 (50 mL) and extracted with EA (50 mL). The organic layer was concentrated to give compound 86_1 (1.20 g, crude) as a yellow oil.

LCMS: RT = 1.819 min, m/z 267.1 [M+H]+ Step 2: The mixture of compound 86_1 (1.10 g, 4.12 mmol, 1.00 eq) and compound 4 (1.88 g, 16.48 mmol, 4.00 eq) in dioxane (16 mL) was stirred at 130 CC for 8 hours under microwave. The mixture was filtered and concentrated. The residue was ed by pre-HPLC (HCl condition) to give A-86 (1.30 g, crude) as a yellow solid. A- 86 (300 mg) was purified again by prep-HPLC (HCl ion) to give A-86 (20.00 mg, 52.51 umol, 12.1% yield, 100% purity, HCl) as a yellow solid.

LCMS: RT = 2.321 min, m/z 345.2 [M+H]+ 1H NMR (MeOD, 400 MHz) 5 8.37-8.36 (m, 1 H), 8.23-8.22 (m, 1 H), 4.07—4.03 (m, 1H), 3.96 (s, 3H), 3.21 (d, z, 2H), 2.24-2.18 (m, 4H), 1.68-1.57 (m, 2H), 1.19 (s, 1H), 0.57—0.55 (m, 2H), 0.38-0.34 (m, 2H). 02429524\45-0 l Pre aration 0 1R 4R -N1- 1H- razol l meth l -N4- 4- 5- c clo r0 lmeth l-I-meth l-IH- razol l uoro rimidin-Z- lc clohexane- 1,4-diamine (A-85 2 r H I /N H I N N’ H N NaBH3CN,HOAc U\Alr/ HZNN0’ WM/ F MEANN A-86 A-85 To a solution of A-86 (300.00 mg, 783.90 umol, 1.00 eq) and 1H-pyrazole carbaldehyde (82.86 mg, 862.29 umol, 1.10 eq) in MeOH (3 mL) were added AcOH (49.31 uL, 862.29 umol, 1.10 eq) and NaBH3CN (98.52 mg, 1.57 mmol, 2.00 eq). The mixture was sitrred at 15 CC for 16 hours. The mixture was quenched with a.q NaHC03 (1 mL) and concentrated. The e product was purified by prep-HPLC (basic ion) to give A-85 (50.00 mg, 111.53 umol, 14.2% yield, 94.7% purity) as a yellow solid.

LCMS: RT = 2.472 min, m/z 425.3 [M+H]+ 1H NMR (CDClg, 400 MHz) 5 .09 (m, 1 H), 8.02-8.01 (m, 1 H), 7.60 (s, 1H), 4.80 (d, J=8.4Hz, 1H), 3.91 (s, 3H), 3.84 (m, 3H), 3.22—3.21 (m, 2H), 2.68 (s, 1H), 2.21-2.06 (m, 4H), 1.41—1.35 (m, 2H), 1.30—1.24 (m, 3H), 1.10—1.09 (m, 1H), 0.49—0.45 (m, 2H), 0.24—0.22 (m, 2H).

Biological experimental grocedures Primary Screen in RKO cells for compounds having typical CKIu inhibitory activity (B-catenin and p53 ization and histone H2AX phosphorylation; see Elyada et al, Nature 2011 Feb 17; 470(7334):409-13; Pribluda et al, Cancer Cell 2013 Aug 12; 24(2):242-56). RKO colorectal cells were incubated with 10 uM of each of the nds for 16 hours at 37°C. Cells were washed with PBS and cell pellets were incubated with ice cold protein lysis buffer containing protease inhibitor cocktail (1/200; chem) and phosphatase inhibitors (20mM p-nitrophenyl phosphate (PNPP), 20mM B-glycerophosphate and 300nM okadaic acid). Western blot analysis was med by 02429524\45-0 l means of standard techniques. Blots were incubated with antibodies detecting B-catenin (1/2,500; BD Transduction), p53 (DO-1&1801 hybridoma mix; dilution of 1:20 of supernatants from each), CKIa (C-19; 1/ 1,000; Santa Cruz Biotechnology) and phospho- histoneH2AX ($139; 1/ 1,000; Millipore). Secondary antibodies were HRP-linked goat anti-mouse, goat anti-rabbit and rabbit anti-goat antibodies (all 1/10,000; n). Blots were developed using ECL (GE Healthcare).

Dose Response assay of the most active nds. Active compounds were further analyzed in a dose-response experiment (Figures 1A and 1B). Similar to the y screen, RKO cells were incubated with descending concentrations of each of the active compounds for 16 hours at 370C. Cell t isolation and Western Blot analysis were similar to the primary screen. tion of a mouse model of CML blast crisis and inhibitor s in this model. To generate the BCR-ABL—inducible chronic myeloid leukemia (CML) model, bone marrow (BM) cells from 10 weeks wild type mouse were extracted and enriched for cKit expressing cells ep #18757) and incubated overnight at 370C in RPMI growth medium supplemented with 15% FCS, L-Glutamine, Pen/Strep (Biological Industries, Israel) and stem cell factor (SCF), IL-3, IL-6 and TPO tech). The culture was then infected with R-ABL-IRES-GFP retrovirus construct containing supernatant medium for 4 hours, then growth medium was added and infected cells were incubated at 370C for additional 24h. The culture was then injected I.V. into sublethally irradiated (500 rad) mice. Upon observing a fast steady increase of GFP-expressing cells in the peripheral blood of inoculated mice (by FACS) and rising numbers of leukocyte and immature cells (detected by Wright-Giemsa stained blood films), mice were sacrificed and their BM cells were transferred to new sublethally irradiated WT hosts; each such transfer was termed disease generation. By the fourth transfer, the hosts were no longer sublethally irradiated prior to disease transfer. Blast crisis development was readily detectable by a highly abnormal number of blast cells, more than 30% of white blood cells (WBC) in the eral blood (PB), and short time intervals between ers. CKI inhibitor studies were performed on late generation diseases, in which PB blasts were easily detectable, with no host irradiation and a short tion time (up to 12 days). Mice were monitored daily for cachexia, weight loss, lethargy, and ruff coats, and moribund mice were sacrificed upon moribund. 02429524\45-0 l For evaluating CKIa inhibition effect on CML, a selective CKIa inhibitor (A14) was administered by oral gavage once a day at a dose of 10 mg/kg, starting from 24h after BM transplantation (BMT) (Figure 3A). The inhibitor was dissolved in 1% methyl cellulose with 0.1% Tween 80 and 0.2% Poly-ethylene glycol (Vehicle). Control mice were treated with the vehicle only.

Ex vivo inhibitor s (Figures 2A and 13A-E). Freshly isolated BM from AML (13A-E) or CML blast crisis (2A) carrying mice were grown in RPMI supplemented with 15% FCS, L-Glutamine, Pen/Strep, Hepes, Sodium Pyruvate and non- essential amino acids (Biological Industries, Israel). CKI tors (A14, A51, A75 or A86) were dissolved in DMSO and added to the tissue culture medium at the indicated concentrations; control cultures were treated with DMSO only. Several hours following treatment (as indicated in each experiment), cells were harvested and counted manually using a camera and rd inverted light microscope. Dead cells were excluded using Trypan Blue (Sigma). AnnexinV-PE (MBL), 7AAD ) and PD-Ll (BioLegend) staining was evaluated by FACS according to manufacturer's recommendation. tion of a mouse model of acute myeloid leukemia (AML) (Figure 6) and treatment with the CKIu inhibitor A14 (Figures 7, 8 and 9) or A51 (Figures 11 and 12). To generate the MLL-AF9 Acute myeloid leukemia (AML) model, bone marrow (BM) cells from 10 week old wild type mouse were extracted and enriched for cKit sing cells (EasySep ) and incubated overnight in RPMI supplemented with % FCS L-Glutamine, Pen/Strep (Biological Industries, Israel) and stem cell factor (SCF), IL-3, IL-6 and TPO (Peprotech). The culture was infected with MSCV-MLL-AF9- IRES-GFP retrovirus uct containing supernatant medium for 4 hours, then growth medium was added and ed cells were incubated at 370C for additional 24h. . The culture was then injected I.V. into sub lethally irradiated (500 rad) mice. Upon detectable steady increase of GFP expressing cells in the mice peripheral blood ed by FACS analysis) and rise in leukocyte numbers and re cells (detected by Wright-Giemsa d blood films), mice were sacrificed and their BM was transferred (1St BMT) to sublethally irradiated WT hosts. Upon emergence of AML disease mice were sacrificed and 50,000 BM cells were transplanted (2nd BMT) into WT host mice. GFP sing cells were monitored in the peripheral blood and upon detecting >10% GFP+ in PB (day 11 after BMT) mice were treated with A14 inhibitor (Figure 7A). A14 was administered 02429524\45-01 by oral gavage once a day at a dose of 20mg/kg for 3 days followed by g/day for 6 more days. The inhibitor was dissolved in 1% methyl cellulose with 0.1% Tween 80 and 0.2% Poly-ethylene glycol (Vehicle). Control mice were d with the vehicle only. Mice were monitored daily for cachexia, lethargy, and ruff coats, and moribund mice were sacrificed. For a single dose experiment (Figure 11A) A51 was administered by oral gavage at a single dose of 20mg/kg and mice were sacrificed 16 hours following treatment.

FACS analysis. All assays were performed on BD's equipment: FACS caliber, FACS ARIA sorter or LSR II machines. For immunostaining, cells were suspended in a 1% BSA/PBS buffer with 5uM EDTA. Cells were then analyzed by using Annexin V PE Apootosis Detection Kit (eBioscience), 7-AAD (TONBO biosciences) and PE anti- mouse CD274 (B7-H1, PD-L1) antibody (clone 2, BioLegend); Assays were performed according to the manufacturer’s instruction. Monoclonal antibodies specific for CD16 and CD32 (Miltenyi ) were used for blockade of Fc receptors before staining.

Complete blood counts. eral venous blood was obtained from the mouse facial vein using standard techniques and analyzed using the auto hematology analyzer BC-2800 (Mindray) per manufacturer’s instructions.

Table 1 provides quantified information for compounds of the ion in activation of p53 and DNA damage response (DDR) and nin stabilization as an indicator of Wnt pathway activation. p53 activation was determined according to the degree of protein ization in several Western blot assays (Western blot examples are shown in Figs 1A and 1B). For example, A43 stabilized p53 significantly above the non- treatment control at 6 uM with no activity at 2 uM (Fig 1A, lower right panel) and thus received an average value of + for p53 activation. In contrast, A35 started stabilizing p53 at 0.2 uM, with maximal stabilization at 1 uM (Fig 1A, upper right panel) and thus received an average value of +++ for p53 activation. A19-4, neither stabilized p53, nor induced yH2AX (a DNA damage response [DDR] tor), but stabilized B-catenin at 2uM, similarly to the best B-catenin stabilizing compounds (Fig 1A, lower left panel) and thus ed a value of +++ for nin/Wnt tion. 02429524\45-0 l Table 1: p53, DNA damage response and Wnt/B-catenin activation of compounds of the invention Compound Structure Name Mass DDR [3— (M+H+) & p53 Catenin activity activit A14 (1R,4R)-N1-(4- Calc’d for (5 ' C18H27N5: (cyclopropylmet 327 .2; hy1)methy1- Found: 1H-pyrazol 327 .2 y1)pyrimidin lohexane- 1 ,4—diamine A29-1 N-(( 1R,4R) Calc’d for ((4'(5' C21H31N6 (cyclopropylmet 02: 399.2: hy1)methy1- Found: 39 1H-pyrazol 9.2 imidin y1)amino)cycloh methoxyacetami A27 (1R,4R)-N1-(4- Calc’d for (5 ' C19H29N62 (cyclopropylmet 341 .2.; hy1)methy1- Found: 34 1H-pyrazol 1.2 y1)pyrimidin yl) -N4- methylcyclohex ane- 1,4—diamine A28 (1R,4R)-N1-(4- Calc’d for (5 ' C20H31N62 (cyclopropylmet 355 . 3; hy1)methy1- Found: 35 azol 5 .2 y1)pyrimidin yl) -N4,N4- dimethylcyclohe xane- 1 ,4- diamine A36 (1R,4R)-N1-(4- Calc’d for ( 1 -cyclopenty1- C22H33N6: - 38 1 .3; (cyclopropylmet Found: 38 hy1)- 1H- 1 .4 pyrazol y1)pyrimidin yl)cyclohexane- 1 ,4—diamine A39 (1R,4R)-N1-(4- Calc’d for (5 - C22H33N6 propylmet O: 397. 3; hy1) Found:39 (tetrahydro-2H- 7 .4 02429524\45-0 1 _ 89 _ pyrany1)-1H- pyrazol y1)pyrimidin yl)cyclohexane- amine A29 (1R,4R)-N1-(4- Calc’d for (5' C21H33N6 (cyclopropylmet 0: 385.3; hy1)methy1- Found: 38 1H-pyrazol 5.2 y1)pyrimidin y1)-N4-(2- methoxyethyl)c xane-1,4- diamine A19-4 8-((4-(5- Calc’d for (cyclopropylmet C20H25N7 hy1)methy1- 02: 396.2; 1H-pyrazol Foundz39 y1)pyrimidin 6.2 y1)amino)-1,3- diazaspiro[4.5]d ecane-2,4-dione A35 (1R,4R)-N1-(4- Calc’d for (5- C20H31N5: (cyclopropylmet 355.3; - Foundz35 isopropyl-IH- 5.3 pyrazol y1)pyrimidin yl)cyclohexane- 1,4-diamine A41 (1r,4S)-N1-(4- Calc’d for (5- C21H31N6 propylmet 0: 383.3; hy1)((S)- Foundz38 tetrahydrofuran- 3.3 3-y1)-1H- pyrazol y1)pyrimidin yl)cyclohexane- 1,4-diamine A42 (1R,4R)-N1-(4- Calc’d for (5' C21H31N6 (cyclopropylmet 0: 383.3; hy1)((R)- Foundz38 tetrahydrofuran- 3.2 3-y1)-1H- pyrazol y1)pyrimidin yl)cyclohexane- 1,4-diamine A43 (1R,4R)-N1-(4- Calc’d for (5' C20H29N6 propylmet 0: 369.2; hy1)(oxetan- Found: 36 3-y1)-1H- 02429524\45-0 1 pyrazol y1)pyrimidin yl)cyclohexane- 1,4—diamine A46 (1R,4R)-N1-(4— (5 - C20H31N5: (cyclopentylmet 355.3; hy1)methy1- Found: 35 1H-pyrazol y1)pyrimidin yl)cyclohexane- 1,4—diamine A38 (1R,4R)-N1-(4— Calc’d for (5 ' C22H33N6 (cyclopropylmet 0: 397.3; hy1) Foundz39 (tetrahydro-2H- 7.3 pyrany1)-1H- pyrazol y1)pyrimidin yl)cyclohexane- 1,4—diamine A45 (1R,4R)-N1-(4— Calc’d for (5 ' N62 (cyclobutylmeth 341.2; y1)methy1- Foundz34 1H-pyrazol 1.3 y1)pyrimidin yl)cyclohexane- 1,4—diamine A19 (1-amino((4— Calc’d for (5' C19H29N6 (cyclopropylmet O: hy1)methyl- 357.2; 1H-pyrazol Foundz35 y1)pyrimidin 7.2 y1)amino)cycloh ethanol A26 8-((4—(5- Calc’d for + + (cyclopropylmet C20H27N5 hyl)methy1— Oz: 1H-pyrazol 383.2; y1)pyrimidin Foundz38 no) 3.2 oxa-l- azaspiro[4.5]dec anone A47 4-(5- Calc’d for ++ + (cyclopropylmet C23H35N62 hy1)methyl- 395.3; 1H-pyrazol Found: 39 y1)-N-((1R,4R)- 5.2 eridin y1)cyclohexyl)p yrimidin amine 02429524\45-0 1 A48 4-(5- Calc’d for (cyclopropylmet C22H33N6 hyl)methy1— O: 1H-pyrazol 397.3; yl)-N-((1R,4R)- 39 4- 7.3 morpholinocycl )pyrimidi namine A49 4-(5- Calc’d for (cyclopropylmet C22H33N62 hy1)methyl- 381.3; azol Found: 38 yl)-N-((1R,4R)- 1.3 4-(pyrrolidin— 1- yl)cyclohexy1)p yrimidin amine A50 N-(( 1R,4R) Calc’d for (azetidin-l- C21H31N61 yl)cyclohexy1)- 367.3; 4-(5- Found:36 (cyclopropylmet 7.3 hy1)methyl- 1H-pyrazol y1)pyrimidin amine A51 (1R,4R)-N1-(5- Chloro(5- (cyclopropylmet hy1)methyl- 1H-pyrazol y1)pyrimidin y1)cyclohexane- 1,4—diamine A52 )-N1-(4— Calc’d for (5 - C19H29N5: (cyclopropylmet 341.2; hy1)methy1- Found: 34 1H-pyrazol 1.2 yl) -5 - methylpyrimidi y1)cyclohexane- amine A53 (1R,4R)-N1-(4— Calc’d for (5 ' C21 H33N62 (cyclobutylmeth 369. 3; y1)isopropy1— Found: 36 1H-pyrazol 9.2 y1)pyrimidin y1)cyclohexane- 1,4—diamine A58 (4-(2—(((1R,4R)- Calc’d for 4- C18H27N6 aminocyclohexy O: 1)amino)pyrimid 343 .2; iny1) Found: 34 methyl- 1H- 3.2 02429524\45-0 1 pyrazol y1)(cyclopr0py1) methanol A59 (1R,4R)-N-1(5- Calc’d for ++ Chloro(5- (cyclopropylmet hy1)- 1- isopropyl- 1H- pyrazol y1)pyrimidin y1)cyclohexane- 1,4—diamine A56 (1R,4R)-N1-(4- Calc’d for NA (1-methy1((1- C19H29N6' methylcyclopro 341.2; pyl)methy1)- 1H- Found: 34 pyrazol- 1 .2 4-y1)pyrimidin- y1)cyclohexane- 1,4—diamine A57 (1R,4R)-N1-(4- Calc’d for (l-methyl-S- C19H31N5. \ neopentyl- 1H- 343.3; pyrazol Found: 34 imidin yl)cyclohexane - 1,4—diamine INA A30-1 (1R,4R)-N1-(5- Calc’d for ++++ ++ Chloro(5- (cyclopropylmet : hy1)methyl- ' 1H-pyrazol : yl) pyrimidin . yl) methylcyclohex ane- 1,4—diamine A30-2 (1s,4s)-N1-(5- ++ H N‘ N \ Chloro(5- T \[J / (cyclopropylmet hy1)methy1- H2N Cl 1H-pyrazol y1)pyrimidin yl) methylcyclohex ane- 1,4—diamine A60 /N (1R,4R)-N1-(4- Calc’d for ++ +/- H N‘ N \ (5' C19H26F3 \r \ (cyclopropylmet N5: \. [J / hy1)methyl- 395.2; H2N\ CFs 1H-pyrazol 39 yl)-S- 5.2 oromethyl idin y1)cyclohexane- 1,4—diamine 02429524\45-0 1 A64 N-((1R,4R) Calc’d for (lH-pyrazol-lyl )cyclohexyl)- -Chloro(5- propylmet hy1)- 1-methy1— 1H-pyrazol y1)pyrimidin amine A65 N-((1R,4R) ( lH-imidazol- 1- yl)cyclohexyl)- -Chloro(5- (cyclopropylmet hy1)- y1— 1H-pyrazol y1)pyrimidin amine A68 )-N1-(5- Chloro(5- (cyclopropylmet hy1)- 1-methy1— 1H-pyrazol y1)pyri Z- y1)-N4- phenylcyclohex ane- 1,4-diamine A71 (5r,8r)((5- Calc’d for Chloro(5- C21H28C1 (cyclopropylmet N50: hy1)- 1-methy1— 415 .2; 1H-pyrazol Found: y1)pyrimidin 415.2 y1)amino) azaspiro[4.5]dec an-Z-one A74 (1R,4R)-N1- Calc’d for benzyl-N4-(5- Chloro(5- (cyclopropylmet hy1)- 1-methy1— 1H-pyrazol y1)pyrimidin yl)cyclohexane- 1,4-diamine A75 (1R,4R)-N1- ((1H-pyrazol hyl)-N4- (5-chloro(5- (cyclopropylmet hy1)- 1-methy1— 1H-pyrazol y1)pyrimidin yl)cyclohexane- 1,4-diamine 02429524\45-01 A76 (1R,4R)-N1-(5- Calc’d for Chloro(5- (cyclopropylmet hy1)- 1-methy1- azol yl) pyrimidin yl) yridin- ylmethyl)cycloh exane- 1,4- diamine A80 ( 1r, 4r)-N1- Calc’d for yrazol N82 yl)methy1)-N4- 407.3; (4-(5- Found: (cyclopropylmet 407.2 hy1)- 1-methy1- 1H-pyrazol y1)pyrimidin y1)cyclohexane- 1,4—diamine A81 ( 1R,4R)-N1- Calc’d for ((1H-pyrazol C23H33N82 yl)methy1)-N4- 421.3; (5-chloro(5- Found: (cyclopropylmet 421.2 hy1)- 1-methy1- 1H-pyrazol y1)pyrimidin y1)cyclohexane- 1,4—diamine A82 ( 1R,4R)-N1- ((1H-pyrazol yl)methy1)-N4- (5-chloro(5- (cyclopropylmet hy1)- 1-methy1- 1H-pyrazol y1)pyrimidin y1)cyclohexane- 1,4—diamine A83 (1R,4R)-N1-(1- (1H-pyrazol y1)ethy1)-N4-(5- Chloro(5- (cyclopropylmet hy1)- 1-methy1- 1H-pyrazol y1)pyrimidin y1)cyclohexane- 1,4—diamine A87 ,4R) Calc’d for ((5-chloro(5 - C22H28C1 propylmet N802 hy1)- 1-methy1- 455.2; 1H-pyrazol Found: y1)pyrimidin y1)amino)cycloh exyl) - 1H- 02429524\45-0 1 pyrazole amide A91 (1R,4R)-N1-(5- Calc’d for Chloro(5- (cyclopropylmet hy1)methy1- azol y1)pyrimidin yl)-N4-((5- methyl- 1H- pyrazol y1)methyl)cyclo hexane-1,4- diamine A94 (1R,4R)-N1-(5- Calc’d for Chloro(5- C20H27C1F (cyclopropylmet hy1)methy1- 1H-pyrazol y1)pyrimidin yl)-N4-(2,2,2- tri?uoroethyl)cy clohexane-1,4- diamine A95 ( 1R,4R)-N1- Calc’d for ((1H-pyrazol C1F yl)methy1)-N4- (5-chloro(5- (cyclopropylmet hy1)- 1-methy1— 3.2 1H-pyrazol y1)pyrimidin yl)-N1 -(2,2,2- tri?uoroethyl)cy clohexane-1,4- A96 (1R,4R)-N1,N1- Calc’d for bis(( 1H- pyrazol yl)methy1)-N4- (5-chloro(5- (cyclopropylmet 1.3 hy1)- 1-methy1— 1H-pyrazol y1)pyrimidin lohexane- 1,4-diamine A86 (1R,4R)-N1-(4- Calc’d for (5 - C18H25FN (cyclopropylmet 6. hy1)- 1-methy1— 345 .2; 1H-pyrazol Found: yl) -5 - 345 .2 ?uoropyrimidin yl)cyclohexane- 1,4-diamine 02429524\45-01 A85 (1R,4R)-N1- Calc’d for (( lH-pyrazol C22H30FN yl)methyl)-N4- 5: (4-(5- 425.3; (cyclopropylmet Found: hyl)methyl- 425.3 lH-pyrazol yl) fluoropyrimidin lohexane- l,4-diamine + indicates low, yet significant activation at a compound concentration of 6 MM; +++ indicates l B-catenin or p53 stabilization at >2 uM; ++++ indicates l activation of p53 and DDR at >0.5uM, +++++ indicates maximal activation of p53 and DDR at >0.1uM.

IRAKl tion with compounds of the invention. RKO cells were incubated for 16 hours at 370C with the ted concentrations of compounds of the invention A51 (1 uM) and A14 (2uM) (Figure 10). At the indicated time points, RKO were treated with TNFa (100 units/ml). Cells were harvested and analyzed by Western blot. Blots were incubated with the following antibodies: Phospho-IRAKl (Thr209), (A1074, AssayBiothechnology; 1/ 1,000), Phospho-IKKa/B (Ser176/180) (16A6, Cell Signaling; 1/ , IKKa (2682 cell signaling; , 1/ 1,1000), IKKB (2370 cell signaling; 1/1,1000), Phospho-c-Jun (Ser 63) (9261, cell signaling; 00), p53 (DO-1&1801 hybridoma mix; on of 1:20 of supernatants from each), CKIa (C-19; 1/1,000; Santa Cruz Biotechnology) and phospho-histoneH2AX ($139; 1/ 1,000; Millipore). Secondary antibodies were HRP-linked goat anti-mouse, goat anti-rabbit and rabbit anti-goat antibodies (all 1/10,000; Jackson). Blots were developed using ECL (GE Healthcare).

Figure 10, shows the inhibition of phosphorylation of IRAKl as well as Phospho-IKKa/B and Phospho-c-Jun, indicative of IRAKl kinase Inhibition. Also shown is p53 stabilization and phosphorylation of H2AX (yH2AX), a marker of DNA damage, indicative of CKIa kinase inhibition. CKIa protein levels serves as a loading control.

Kinome affinity scan for A51 (WXL5846, see Table 2 , shows that the key targets of A51 includes the entire CKI family members and IRAKl, with a few control kinases. KINOMEscanTM is based on a competition binding assay that quantitatively measures the ability of a compound to compete with an immobilized, active-site directed 02429524\45-0 l ligand. The assay is performed by combining three components: DNA-tagged kinase; immobilized ligand; and a test compound. The ability of the test compound to compete with the immobilized ligand is measured Via quantitative PCR of the DNA tag; % Ctrl=0 (zero) indicates a complete inhibition of the kinase tested by a concentration of luM inhibitor (Fabian, MA. et al. A small molecule-kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol. 23, 6 (2005) and Karaman, M.W. et al. A quantitative analysis of kinase inhibitor selectiVity. Nat. Biotechnol. 26, 127-132 (2008)).

Table 2: Kinome af?nity scan for A51 (WXL5846) ERKl 94 """"""" ERK271 """""""""""""""""""""""$1"""""""""""" iiiiiiiiiiiiiiiiiiiiiii FGFR294 iiiiiiiiiiiE GFR3100 iiiiiiiiiiiiiii "REES;"""""""""""""""""39"""""""""""" iiiiiiiiiiiiiiiiiiiiiiiiiiii53iiiiiiiiiiiiiiiiiiiiiii GSK3A iiiiiiiiiiiE iiiiiiiiiiiii iiiiiiii KKbeta77 iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii§3iiiiiiiiiiiiiiiiiiiiiiii iiiiiiiiiiiii RAKlO """""""""""""""""""""69"""""""""""" IRAK4 02429524\45-0 l _ 98 _ """"E SNK1A119 """""""""""""""6‘3"""""""""""" CSNKlAlL """""""""""" CSNK1D28 """""E SNKlEOl """""E SK1G207 """"""""""""""""""""iE"""""""""""" CSNK2A1 Table 3 shows the Kd measurements for the interaction of A14 WVXL-4085) and A51 (WXL-5846) with IRAKl Table 3: Kd of A14 (WXL-4085) and A51 (WXL-5846) with IRAKl Target 85-02N WXL-5846 A14 A51 Gene Symbol Kd (nM) Kd (nM) IRAKI 11 2 9 Conclusion. IRAKl as a superior target in a kinome scan shows zero binding of IRAKl to its target in the presence of the inhibitors. Pyrazole pyrimidine compounds of the invention A51 and A14 showed excellent binding Kd to IRAKl. The compounds also showed inhibition of IRAKl activation and inhibition of the activation of the IRAKl target IKK a B kinase) in RKO cells Western blot analysis). It is to be noted that compound A51 showed complete (100%) inhibition of o- (active) IRAKl at a concentration of 1 uM in the RKO cell line. As a ison, Garrett W. Rhyasen et al. showed that Amgen's IRAK1-4 inhibitor used in the treatment of MDS and breast cancer ted only 70% 0f IRAK1/4 in cell lines at 10 uM (Garrett W. Rhyasen et al, 2013, Cancer Cell 24, , see especially Figure 2 therein). Thus, compounds of the invention, such as A51, are found to be excellent tors of IRAKl, an important upstream regulator of the NF-kB pathway which plays an important role in hematological 02429524\45-Ol malignancies (including among others multiple myeloma, MDS, leukemia and lymphoma, head and neck cancer and breast cancer). -dose treatment effect of a CKI inhibitor in AML mice and PD-Ll expression. AML mice were prepared by inoculating 9 oncogene-transduced bone marrow cells to C57/BL6 mice. MLL-AF9 fusion represents one of a poor prognosis human AML induced by chromosomal translocation. 30 days after leukemia inoculation, the recipient mice have high white blood cells (WBC) counts (x10 higher than a normal mouse), and harbor >95% leukemia blasts in the bone marrow and 50% of the peripheral WBC in these mice are AML blasts. These mice have splenomegaly and their bones are pale and fragile due to the acute leukemia (Fig. 12). Oral treatment with A51 (20mg/Kg) for 16hrs results in e ion of the total leukemia cells in the blood (Fig. 11B), shrinking of the leukemic spleen (Fig. 11C and Fig. 12A), 50% and >90% reduction of proportion of leukemic blasts (GFP+ cells) in the bone marrow and blood, respectively (Fig. 11D and Fig. 11E). Opaque bones turned to normal color following a single dose treatment (Fig. 12B).

In Vitro treatment effects of CKI inhibitors on AML cells isolated from the bone marrow of leukemic mice. Shown are the percentage of dead cells (7AAD+) following 10 or lOOnM tor-treatment, at 6 and 9 hours after treatment (Fig. 13B and 13D). DMSO treatment resulted in the inhibitors on the ia expression of the major immune checkpoint n PD- Ll by ?ow cytometry analysis: reduction of the mean ?uorescence intensity (MFI) at 5hrs, and a decrease in the fraction of PD-Ll-positive leukemia cells after inhibitor ent in comparison to DMSO-treated cells at 6 and 9 hours (decrease expressed by % of DMSO control) (Fig. 13A, 13C and 13E). 02429524\45-0 l

Claims (52)

What we claim is:

1. A compound having formula (I), or a stereoisomer or salt thereof: R3 R4 R2 N NH R5 R1 N wherein: R1 and R2 are each independently H; or straight or branched C1 – C8 alkyl, straight or branched C1 – C5 alkoxy, straight or branched C1 – C5 acyl, or C3 – C7 heteroaryl, each optionally tuted by at least one of halo, hydroxyl, C5 – C15 aryl, and C3 – C7 heteroaryl; R1 and R2 together with the nitrogen atom they are connected to form a 4 – 7 membered saturated, unsaturated, or ic ring that optionally includes at least one of N, O, NH, C=N, C=O, and SO2; and is optionally substituted with at least one of straight or branched C1 – C5 alkyl, C5 – C15 aryl, C3 – C7 heteroaryl, yl, halo, and cyano; R3 and R4 are each independently H; or straight or branched C1 – C8 alkyl, optionally substituted by at least one of halo, hydroxyl, C1 – C5 alkoxy, C5 – C15 aryl, and C3 – C7 aryl; or R1 or R2 together with R3 and the carbon and nitrogen atoms they are each connected to form a 4 – 7 membered saturated, rated, or aromatic ring that optionally includes at least one of N, NH, O, C=N, C=O, and SO2; and is optionally substituted with at least one of straight or branched C1 – C5 alkyl, C5 – C15 aryl, C3 – C7 heteroaryl, hydroxyl, and halo; R5 and R8 are each independently H or halo; or straight or branched C1 – C8 alkyl, straight or branched C2 – C8 alkenyl, or straight or branched C2 – C8 alkynyl; each optionally substituted by at least one halo; R6 is straight or branched C1 – C8 alkyl, straight or branched C2 – C8 alkenyl, straight or ed C2 – C8 l, C5 – C10 cycloalkyl, or saturated or unsaturated 4 – 6 membered heterocyclyl; each optionally substituted by at least one of straight or ed C1 – C8 alkyl, C3 – C7 cycloalkyl, 4 – 6 membered heterocyclyl, C5 – C15 aryl, C3 – C7 heteroaryl, halo, hydroxyl, and C1 – C5 haloalkyl; and R7 is straight or branched C1 – C8 alkyl, straight or ed C2 – C8 alkenyl, or straight or branched C2 – C8 alkynyl; each substituted by at least one C3 – C7 cycloalkyl, 4 – 6 membered heterocyclyl, C5 – C15 aryl, C3 – C7 heteroaryl, halo, hydroxyl, or C1 – C5 haloalkyl.

2. The nd, or the stereoisomer or salt thereof according to claim 1, wherein R1 and R2 are each independently H; or ht or branched C1 – C8 alkyl, optionally tuted by at least one of halo, C5 – C15 aryl, C3 – C7 heteroaryl, and hydroxyl.

3. The compound, or the stereoisomer or salt thereof according to claim 1, n R1 and R2 are each independently H; or straight or branched C1 – C5 alkoxy, optionally substituted by at least one of halo and hydroxyl.

4. The compound, or the stereoisomer or salt thereof according to claim 1, wherein R1 and R2 are each independently H; or C1 – C5 acyl, ally substituted by at least one of halo and hydroxyl.

5. The compound, or the stereoisomer or salt thereof according to any one of the ing claims, wherein R4 is H.

6. The compound, or the stereoisomer or salt thereof according to any one of the preceding claims, wherein R3 and R4 are each H.

7. The compound, or the stereoisomer or salt thereof according to any one of the preceding claims, wherein R5 is H, Cl, or straight or branched C1 – C4 alkyl.

8. The compound, or the stereoisomer or salt thereof according to any one of the preceding claims, wherein R5 is H.

9. The compound, or the stereoisomer or salt thereof according to any one of the preceding claims, wherein R8 is H, Cl, or ht or branched C1 – C4 alkyl.

10. The compound, or the stereoisomer or salt thereof according to any one of the preceding , wherein R8 is H.

11. The compound, or the stereoisomer or salt thereof ing to any one of the preceding claims, wherein one of R5 or R8 is H.

12. The compound, or the stereoisomer or salt thereof according to any one of the preceding , wherein at least one of R1 and R2 is H.

13. The compound, or the stereoisomer or salt thereof according to any one of the preceding claims, wherein R6 is straight or ed C1 – C8 alkyl, C5 – C10 cycloalkyl, or saturated or unsaturated 4 – 6 membered heterocyclyl; and R7 is straight or ed C1 – C8 alkyl, substituted by at least one C3 – C7 cycloalkyl, 4 – 6 membered heterocyclyl, C5 – C15 aryl, C3 – C7 heteroaryl, halo, hydroxyl, or C1 – C5 haloalkyl.

14. The compound, or the stereoisomer or salt thereof ing to any one of the preceding claims, wherein R6 is straight or branched C1 – C8 alkyl, C5 – C10 cycloalkyl, or 4 – 6 membered saturated heterocyclyl.

15. The compound, or the stereoisomer or salt thereof according to any one of the preceding claims, wherein R7 is straight or branched C1 – C8 alkyl substituted by at least one of C3 – C7 cycloalkyl and hydroxyl.

16. The compound, or the stereoisomer or salt thereof according to any one of the ing claims, wherein R6 is straight or branched C1 – C8 alkyl, or saturated, unsaturated or aromatic 4 – 6 membered heterocyclyl; each ally substituted by at least one of ht or branched C1 – C8 alkyl, C3 – C7 cycloalkyl, halo, hydroxyl, and CF3.

17. The compound, or the stereoisomer or salt thereof according to any one of the preceding claims, n R7 is a straight or branched C1 – C8 alkyl substituted by at least one C3 – C7 cycloalkyl.

18. The compound, or the stereoisomer or salt thereof according to any one of the preceding claims, wherein R1 and R2 together with the nitrogen atom they are connected to form a 4 – 7 membered saturated ring that optionally includes at least one of N, O, NH, C=N, C=O, and SO2; and is optionally substituted with at least one of straight or branched C1 – C5 alkyl, hydroxyl, halo, and cyano.

19. The compound, or the stereoisomer or salt thereof according to any one of the preceding claims, wherein R1 and R2 together with the nitrogen atom they are connected to form a 4 – 7 membered saturated ring.

20. The compound, or the stereoisomer or salt thereof according to any one of the preceding claims, wherein R1 and R2 together with the nitrogen atom they are connected to form a 4 – 7 membered saturated ring that includes at least one of N and O.

21. The compound, or the isomer or salt thereof according to any one of the preceding claims, wherein R1 and R2 together with the nitrogen atom they are connected to form a 4 – 7 membered ic ring that optionally includes at least one of N and O.

22. The compound, or the isomer or salt thereof ing to any one of the preceding claims, n R1 or R2 together with R3 and the carbon and nitrogen atom they are connected to form a 4 – 7 membered saturated ring that optionally includes at least one of N, NH, O, C=O, and SO2; and is optionally substituted with at least one of straight or branched C1 – C5 alkyl, yl, and halo.

23. The compound, or the stereoisomer or salt thereof ing to any one of the preceding claims, wherein R1 or R2 together with R3 and the carbon and nitrogen atom they are connected to form a 4 – 7 membered saturated ring that es at least one of NH, O, and C=O.

24. A compound, or the stereoisomer or salt thereof selected from: H N N N A29-1 O A19-4 H N N N A30-1 H2N A30-2 H N N N F3C N Cl A94 H A86 and A85 .

25. A pharmaceutical composition comprising a compound, or the stereoisomer or salt thereof according to any one of claims 1 to 24.

26. A compound, or the stereoisomer or salt thereof according to any one of claims 1 to 24, for use in therapy.

27. A compound, or the stereoisomer or salt f according to any one of claims 1 to 24, for use in the inhibition of least one of Casein kinase I (CKI) and interleukin-1 receptor-associated kinase 1 (IRAK1).

28. A compound, or the isomer or salt thereof according to any one of claims 1 to 24, for use in the inhibition of Casein kinase I (CKI).

29. A compound, or the stereoisomer or salt thereof according to any one of claims 1 to 24, for use in the inhibition of interleukin-1 receptor-associated kinase 1 (IRAK1).

30. A compound, or the stereoisomer or salt thereof ing to any one of claims 1 to 24, for use in inducing anti-tumor response.

31. A compound, or the stereoisomer or salt thereof for use according to claim 30, wherein said anti-tumor response comprises cancer therapy response.

32. A compound, or the stereoisomer or salt thereof according to any one of claims 1 to 24, for use in the treatment of a ion, symptom or disease associated with a malignant condition.

33. A compound, or the stereoisomer or salt thereof for use according to claim 32, wherein said malignant condition is cancer.

34. A compound, or the isomer or salt thereof for use according to claim 32, wherein said malignant condition is multiple myeloma, myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), ma, ative breast , e large B cell lymphoma (DLBCL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), or head and neck cancer.

35. A compound, or the stereoisomer or salt thereof for use according to claim 33, wherein said cancer has WT p53.

36. A compound, or the stereoisomer or salt thereof according to any one of claims 1 to 24, for use in the treatment of cancer having WT p53, wherein said WT p53 is a biomarker for said compound efficacy.

37. A nd, or the stereoisomer or salt thereof for use according to claim 33, wherein said cancer is multiple myeloma, leukemia, malignant ma, breast cancer, prostate cancer, or colorectal cancer.

38. A compound, or the isomer or salt thereof according to any one of claims 1 to 24, for use in the treatment of an inflammatory and immune d disorder.

39. Use of a compound, or the stereoisomer or salt thereof ing to any one of claims 1 to 24 in the manufacture of a medicament for treating a condition, symptom or disease ated with a malignant condition.

40. The use according to claim 39, wherein said malignant condition is cancer.

41. The use according to claim 40, n said cancer has WT p53.

42. The use according to claim 41, wherein said WT p53 is a biomarker for said compound efficacy.

43. The use ing to claim 40, wherein said cancer is leukemia, multiple myeloma, malignant melanoma, breast , prostate cancer, or colorectal cancer.

44. The use according to claim 39, wherein said malignant condition is multiple myeloma, myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), melanoma, ER-negative breast cancer, diffuse large B cell lymphoma (DLBCL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), or head and neck cancer.

45. Use of a nd, or the stereoisomer or salt thereof according to any one of claims 1 to 24 in the manufacture of a medicament for inducing an immunotherapy response.

46. Use of a compound, or the stereoisomer or salt thereof according to any one of claims 1 to 24 in the manufacture of a medicament treating an inflammatory and immune related disorder.

47. A compound, or the stereoisomer or salt thereof according to claim 1 substantially as herein described or exemplified.

48. A ceutical composition according to claim 25 substantially as herein described or exemplified.

49. The use according to claim 39 substantially as herein described or exemplified.

50. The use according to claim 42 substantially as herein described or exemplified.

51. The use according to claim 45 substantially as herein described or exemplified.

52. The use according to claim 46 substantially as herein described or exemplified. ¢H< N.onN.onN.on I S ¢H< 2%: mm< o< Hmmammammamm mm< ¢m< Ta? om< a? NoNoNoNoNo Lntnmxgw 33%me mwgw+au..m mg umvmm KWNE» $53K“ g? ?g?gzg mmm wwmm X?wx» Nuom< m H h@# IOM< m h@# 2%: mo< m oo< hm; L??g? £53?an mmm gym gnu X?wI» 3< 5me mN DN o 95mm o; 3 ULSMW— Wm ww M <~ AI UN 95m: ULSMW— Hug—OD m=8 omm _ :25 5w on< 3< \\\\\\\\v\\\\\\m§w?d<§ 59:5. £3 o Sargon £wa5. «2 >m_o\wuco $3.8 3.4. 3 cosmb??Eua. +++++++++ LLLLlr—LLIE onwhmmVMNH be; o_o_:o> on O O N ‘— 33m: 90L X Sllaa INS IBIOJ. m_m_._u 8d u! +d:l9 % _U mm 95m: 52m 95m: 3 3 23m: 225% :90 mass: .3:_|_ a ”E