WO2021000855A1

WO2021000855A1 - Malt1 inhibitors and uses thereof

Info

Publication number: WO2021000855A1
Application number: PCT/CN2020/099233
Authority: WO
Inventors: Wenge Zhong; Xiaotian Zhu; Song Feng; Xianqiang SUN; Min Yang
Original assignee: Qilu Regor Therapeutics Inc.
Priority date: 2019-07-01
Filing date: 2020-06-30
Publication date: 2021-01-07
Also published as: TW202115077A

Abstract

Provided herein are compounds represented by structural formula (I): or pharmaceutically acceptable salts thereof useful for treating an autoimmune disorder, an inflammatory disease, or a cancer.

Description

[Rectified under Rule 91, 23.07.2020] MALT1 INHIBITORS AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to International Patent Application No. PCT/CN2019/094154, filed on July 1, 2019. The entire contents of the aforementioned application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Paracaspases are members of the C14 family of cysteine proteases. They are proteases related to caspases present in animals and slime mold, in contrast to metacaspases, which are present in plants, fungi, and protists. Paracaspases are more similar to caspases than metacaspases are, indicating that the paracaspases diverged from caspases from a common metacaspase ancestor.

Paracaspase was first identified in a recurrent t (11; 18) (q21; q21) chromosomal translocation associated with a subset of MALT (Mucosa Associated Lymphoid Tissue) lymphoma. This chromosomal translocation is the most common mutation identified in MALT lymphoma, and the translocation leads to the formation of a fusion oncoprotein consisting of the N-terminus of the caspase inhibitor protein c-IAP2 (also known as Baculoviral IAP repeat-containing protein 3) and the C-terminus of a human paracaspase called MALT1.

MALT1 (Mucosa Associated Lymphoid Tissue lymphoma translocation protein 1) is the only paracaspase in human. It has both scaffold functions and protease functions, and it transduces signals from cell surface receptors in both immune cells (such as B and T lymphocytes, NK lymphocytes, as well as myeloid and mast cells) and non-immune cells. In immune cells, MALT1 functions downstream of immunereceptors (such as the B cell receptor (BCR) and the T cell receptor (TCR) ) with an ITAM (Immunoreceptor Tyrosine-based Activation Motif) sequence. In non-immune cells, MALT1 functions downstream of certain G Protein Coupled Receptors (GPCRs) and the EGFR (Her2/neu) . The scaffold function of MALT1 contributes to the assembly of other signaling proteins into signaling complexes called CBM (see below) , while its cysteine protease function is responsible for cleaving a number of target proteins, at least in some immune cells. The MALT1 proteolytic activity appears essential for T cell activation and also the development of B cell lymphomas.

For example, upon TCR /BCR activation, MALT1 becomes the active subunit of the so-called CBM complex consisting of multiple subunits of three proteins: CARD11 (CAspase Recruitment Domain family member 11, also known as CARMA1) , BCL10 (B-Cell CLL/Lymphoma 10) and MALT1. In general, however, MALT1 and its partner BCL10 bind to different members of CARD (caspase recruitment domain) -containing CARMA (CARD-containing Membrane Associated guanylate kinase) family of proteins, depending on the cell lineage. In particular, the CBM complex formed upon antigen receptor stimulation (via TCR or BCR pathway) in the lymphocytes involves CARMA1/CARD11, whereas CARD9 interacts with MALT1 downstream of Toll like or C-type lectin receptors. Meanwhile, the CBM complex of MALT1-BCL10-CARD10 links signaling via GPCR and NF-κB activation in non-immune cells (McAllister-Lucas et al., PNAS 104: 139-44, 2007) . On the other hand, CARD14 interacts with MALT1 and BCL10 in keratinocytes.

MALT1 has an N-terminal death domain (DD) with unknown function, followed by two immunoglobulin-like domains (Ig) required for BCL10 binding. The central caspase-like domain share low sequence homology with caspase, but adopts significantly similar folding structure as the caspases, with the active site residues being H415 and C464. The caspase-like domain is followed by a third Ig domain that contains K644, a monoubiquitination site that controls protease activity. MALT1 also contains two binding motifs for the ubiquitin ligase TRAF6 (tumor necrosis factor receptor-associated factor 6) . TRAF6 polyubiquitinates MALT1 on multiple C-terminal lysine residues, generating K63-linked ubiquitin chains that can in turn promote activation of the inhibitor of NF-κB kinase (IKK) complex through recruitment of the IKK-activating kinase TAK1 via the adaptor proteins TAB 2/3.

MALT1 is a key mediator of the classical NF-κB signaling pathway, and acts as a central protein involved in many diseases directly or indirectly involving the inflammatory transcription factor NF-kB. MALT1 affects NF-κB signaling through two distinct mechanisms. First, through its scaffolding function, MALT1 recruits NF-κB signaling proteins such as TRAF6, TAB-TAK1, or ΝΕΜΟ-ΙKKα/β. Second, through its Cys protease function, MALT1 cleaves and deactivates negative regulators of NF-κB signaling, such as RelB, A20, or CYLD. The ultimate endpoint of MALT1 activity is the nuclear translocation of the NF-κB transcription factor complex and activation of NF-κB signaling (Jaworski et al., Cell Mol Life Science 73: 459-473, 2016) , leading to the production of interleukin-2 (IL-2) , and in the activation and proliferation of T and B lymphocytes.

The c-IAP2-MALT1 fusion is a potent activator of the NF-κB pathway (Rosebeck et al., World J Biol Chem 7: 128-137, 2016) . It mimics ligand-bound TNF receptor, and promotes TRAF2-dependent ubiquitination of RIP 1, which acts as a scaffold for activating canonical NF-κB signaling. Furthermore, the cIAP2-MALT1 fusion has been shown to cleave and generate a stable, constitutively active fragment of NF-κB-inducing kinase (NIK) , thereby activating the non-canonical NF-κB pathway (Rosebeck et al., Science 331: 468-472, 2011) .

MALT1 functions as an intracellular signaling protein in innate (e.g., natural killer or NK cells, dendritic cells (DC) , and mast cells) as well as adaptive immune cells (e.g., T cells and B cells) . Certain publications appear to suggest the important role of MALT1 and its proteolytic function in signaling cascades triggered by innate cell receptors like Dectin receptors, and in signaling cascades triggered by G-protein coupled receptors (GPCR) in many cell types. Accordingly, MALT1 is of interest in the mechanism of autoimmune and inflammatory pathologies. Additionally, it was noted that constitutive (dysregulated) MALT1 activity is associated with MALT lymphoma and Activated B Cell-like Diffuse Large B Cell Lymphoma (ABC-DLBCL) .

Indeed, constitutive activation of NF-κB signaling is the hallmark of ABC-DLBCL. DLBCL (Diffuse large B-cell lymphoma) is a cancer of B cells and is the most common form of non-Hodgkin’s lymphoma (NHL) in adults, accounting for approximately 25%of lymphoma cases. DLBCL is an aggressive tumor which can arise in almost any part of the body. Typically, DLBCL arises from normal B cells, but it can also represent a malignant transformation of other types of lymphoma or leukemia with underlying immunodeficiency being a significant risk factor. There are two major biologically distinct molecular subtypes of DLBCL: germinal center B-cell (GCB) and activated B-cell (ABC) . ABC-DLBCL is derived from B cells that are in the process of differentiating from germinal center B cells to plasma cells. Approximately 40%of all DLBCL is ABC-DLBCL, the more aggressive form of DLBCL. Typically, patients diagnosed with the ABC subtype have poorer outcomes than GCB patients. NF-κB pathway activation in ABC-DLBCL is driven by mutations of signaling components, such as CD79A/B, CARD11, MYD88, or A20 (Staudt, Cold Spring Harb Perspect Biol 2010, 2; Lim et al., Immunol Rev 246: 359-378, 2012) . Despite advances in treatment, one third of DLBCL patients either do not respond or relapse within a short time.

MALT1 has also been reported to be involved in several other disease pathologies, e.g., different types of oncological disorders including lung adenocarcinoma (Jiang et al., Cancer Research 71: 2183-2192, 2011; Pan et al., Oncogene 1: 10, 2015) , breast cancer (Pan et al., Mol Cancer Res 14: 93-102, 2016) , mantle cell lymphoma (Penas et al., Blood 115: 2214-2219, 2010; Rahal et al., Nature Medicine 20: 87-95, 2014) , marginal zone lymphoma (Remstein et al., Am J Pathol 156: 1183-1188, 2000; Baens et al., Cancer Res 66: 5270-5277, 2006; Ganapathi et al., Oncotarget 1: 10, 2016; Bennett et al., Am J of Surgical Pathology 1: 7, 2016) , cutaneous T cell lymphomas such as Sezary syndrome (Qin et al., Blood 98: 2778-2783, 2001; Doebbeling et al., J of Exp and Clin Cancer Res 29: 1-5, 2010) , primary effusion lymphoma (Bonsignore et al., Leukemia 31: 614-624, 2017) , pancreatic cancer (WO2016/193339A1) , certain types of chronic lymphocytic leukemia with CARD11 mutation, and also certain subtypes of GCB-DLBCL type of lymphomas that involve MALT1.

In addition to lymphomas, MALT1 has been shown to play a critical role in innate and adaptive immunity (Jaworski et al., Cell Mol Life Sci. 2016) .

MALT1 scaffold and protease functions are essential for the development of peritoneal B1 B cells, marginal zone (MZ) B cells, and natural regulatory T cells (nTreg) . Polarization of naive CD4 ⁺ T cells into the Th17 subset of T helper cells is also heavily dependent on MALT1 protease function.

For example, MALT1 has been found to play a key role in mouse experimental allergic encephalomyelitis, a mouse model of multiple sclerosis (McGuire et al., J. Neuroinflammation 11: 124, 2014) . It has been shown that MALT1 protease inhibitor can attenuate disease onset and progression of mouse experimental allergic encephalomyelitis (McGuire et al., J. Neuroinflammation 11: 124, 2014) . Mice expressing catalytically inactive MALT1 mutant showed loss of marginal zone B cells and B1 B cells, as well as general immune deficiency characterized as decreased T and B cell activation and proliferation. However, those mice also developed spontaneous multi-organ autoimmune inflammation at the age of 9 to 10 weeks. It is still poorly understood why MALT1 protease dead knock-in mice show a break of tolerance while conventional MALT1 KO mice do not. One hypothesis suggests the unbalanced immune homeostasis in MALT1 protease dead knock-in mice may be caused by incomplete deficiency in T and B cell but severe deficiency of immunoregulatory cells (Jaworski et al., EMBO J. 2014; Gewies et al., Cell Reports 2014; Bornancin et al., J. Immunology 2015; Yu et al., PLOS One 2015) . Similarly, MALT1 deficiency in humans has been associated with combined immunodeficiency disorder (McKinnon et al., J. Allergy Clin. Immunol. 133: 1458-1462, 2014; Jabara et al., J. Allergy Clin. Immunol. 132: 151-158, 2013; Punwani et al., J. Clin. Immunol. 35: 135-146, 2015) . Given the difference between genetic mutation and pharmacological inhibition, a phenotype of MALT1 protease dead knock-in mice might not resemble that of patients treated with MALT1 protease inhibitors. A reduction of immunosuppressive T cells by MALT1 protease inhibition may be beneficial to cancer patients by potentially increasing antitumor immunity.

In any event, dysregulation of MALT1 activity plays a role in the development of diseases such as MALT1-dependent inflammatory and/or autoimmune diseases (e.g., rheumatoid arthritis (RA) , multiple sclerosis (MS) , psoriasis, systemic lupus, Sjogren’s syndrome, and Hashimoto’s thyroiditis) . Targeting an immunomodulatory protein can have direct and indirect benefits in a variety of inflammatory disorders of multiple organs, for example, in treating psoriasis (Lowes et al., Ann Review Immunology 32: 227-255, 2014; Afonina et al., EMBO Reports 1-14, 2016; Howes et al., Biochem J 1-23, 2016) , multiple sclerosis (Jabara et al., J Allergy Clin Immunology 132: 151-158, 2013; McGuire et al., J of Neuroinflammation 11: 1-12, 2014) , rheumatoid arthritis, Sjogren’s syndrome (Streubel et al., Clin Cancer Research 10: 476-480, 2004; Sagaert et al., Modern Pathology 19: 225-232, 2006) , ulcerative colitis (Liu et al., Oncotarget 1-14, 2016) , MALT lymphomas of different organs (Suzuki et al., Blood 94: 3270-3271, 1999; Akagi et al., Oncogene 18: 5785-5794, 1999) and different types of allergic disorders resulting from chronic inflammation.

Inhibitors of MALT1 activity have been identified as potential therapeutics. Rebaud et al. (Nat Immunol 9 (3) : 272-81, 2008) describes a warhead-equipped substrate analogue zVRPRfmk, while Lim et al. (J Med Chem 58 (21) : 8591-8502, 2015) describes the small molecule MALT1 inhibitor MI2. Nagel et al. (Cancer Cell 22 (6) : 825-837, 2012) describes another small molecule inhibitor mepazine. One characteristic of these MALT1 inhibitors is that the compounds are proposed for autoimmune or inflammatory pathways, or cancers dependent on dysregulated NF-κB pathway activity. Other inhibitors of MALT1 proteolytic activity have been described with activity in preclinical lymphoma models (Vincendeau et al., Int. J. Hematoi. Oncol. 2: 409, 2013) .

Similarly, Novartis (WO2015/181747) discloses a genus of small molecule MALT1 inhibitors, and assays those compounds in a MALT1 biochemical assay, and also an NF-κB reporter gene assay driven by ectopic expression of the cIAP2-MALT1 fusion protein typical of MALT-lymphomas, and an IL2 promoter-driven reporter gene assay. Also see WO2017/081641 (Novartis) directed to a subset of the same class of compounds.

Through the use of BTK inhibitors such as ibrutinib, clinical proof-of-concept studies have shown that inhibiting the NF-κB signaling can be efficacious in treating ABC-DLBCL. MALT1 functions downstream of BTK in the NF-κB signaling pathway. Thus, a MALT1 inhibitor can target ABC-DLBCL patients either not responding or have acquired resistance to BTK inhibitors such as ibrutinib, mainly in patients with CARD11 mutations.

Small molecule inhibitors of MALT1 protease have demonstrated efficacy in preclinical models of ABC-DLBCL (Fontan et al., Cancer Cell 22: 812-824, 2012; Nagel et al., Cancer Cell 22: 825-837, 2012; Fontan et al., Clin Cancer Res 19: 6662-68, 2013) . Further, covalent catalytic site and allosteric inhibitors of MALT1 protease function have been described, suggesting that inhibitors of this protease may be useful as pharmaceutical agents (Demeyer et al., Trends Mol Med 22: 135-150, 2016) .

Thus, there is a need for MALT1 inhibitory compounds for treating diseases or disorders involving MALT1 activation, such as cancers as well as immunological and inflammatory disorders that depend on MALT1-NF-kB activation.

SUMMARY OF THE INVENTION

Described herein are compounds of Formulae (I) , (II-A) , or (II-B) , and the compounds of the examples (collectively referred to herein as “the compounds of the invention” ) , that inhibit the activity of MALT1, and pharmaceutically acceptable salts thereof.

In one aspect, the invention provides a compound represented by structural formula (I) :

or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein

ring A is

Z is O, NR ₆, or S;

A ₁ and A ₂ are each independently CR ₁ or N;

each instance of R ₁ is hydrogen; halogen; -OH; CN; -COOC _1-6 alkyl; C _1-6 alkoxy optionally substituted by halogen; C _1-6 alkoxy carbonyl; phenyl; amino; N, N-di-C _1-6 alkyl amino; C _1-6 alkyl optionally substituted by halogen, phenyl, or a 5-6 membered heterocyclic ring having 1 to 3 heteroatoms selected from N and O which ring is optionally substituted by C _1-6 alkyl; Rh; ORh; a 5-6 membered heteroaryl ring having 1 to 3 heteroatoms selected from N and O said ring being optionally substituted by amino, C _1-6 alkyl optionally substituted by amino or hydroxy or by N-mono or N, N-di-C _1-6 alkylamino carbonyl; wherein

Rh is a 5-6 membered heterocyclyl ring having 1 to 4 heteroatoms selected from N, O and S, said ring being optionally substituted by C _1-6 alkyl, -OH, or oxo;

each instance of R ₂ is hydrogen; halogen; CN; -COOC _1-6 alkyl; C _1-6 alkoxy optionally substituted by halogen; C _1-6 alkoxy carbonyl; amino; N, N-di-C _1-6 alkyl amino; C _1-6 alkyl optionally substituted by halogen, -OH, phenyl, or a 5-6 membered heterocyclic ring having 1 to 2 heteroatoms selected from N and O which ring is optionally substituted by C _1-6 alkyl; Rh; ORh; a 5-6 membered heteroaryl ring having 1 to 3 heteroatoms selected from N and O said ring being optionally substituted by amino, C _1-6 alkyl optionally substituted by amino or hydroxy, or by N-mono or N, N-di-C _1-6 alkylamino carbonyl;

each instance of R ₃ is H; deuterium; halogen; CN; -OH; -COOH; -NR _aR _b; -SR _c; -SO ₂R _c; -SO ₂NR _c; -C (=O) NR _aR _b; C _1-6 alkoxy optionally substituted by halogen, -OH, C _1-6 alkyl, -NH ₂, -NHC (=O) C _1-6 alkyl, N-di-C _1-6 alkyl amino, or N-mono-C _1-6 alkyl amino; C _1-6 alkyl optionally substituted by halogen, C _2-6 alkenyl, -OH, -NH ₂, -NHC (=O) C _1-6 alkyl, N-di-C _1-6 alkyl amino, N-mono-C _1-6 alkyl amino, -O-Rg, Rg, phenyl, or C _1-6 alkoxy wherein said alkoxy optionally substituted by halogen, -OH, C _1-6 alkoxy, N, N-di-C _1-6 alkyl amino, Rg or phenyl; C _3-6 cycloalkyl optionally substituted by halogen, -OH, C _1-6 alkyl, N, N-di-C _1-6 alkyl amino or C _1-6alkoxy-C _1-6 alkyl; phenyl optionally substituted by halo or C _1-6 alkoxy; a 5-6 membered heteroaryl ring having 1 to 3 heteroatoms selected from N and O said ring being optionally substituted by C _1-6 alkyl which may be optionally substituted by amino or -OH; Rg; or N, N-di-C _1-6 alkyl amino carbonyl; wherein

Rg is a 3-6 membered heterocyclic ring having 1-3 heteroatoms selected from N and O said ring being optionally substituted by –OH, -NH ₂, C _1-6 alkyl, C _1-6 alkoxy-C _1-6 alkyl, or C _1-6 alkoxy-carbonyl;

R _a is independently H or C _1-6 alkyl optionally substituted by C _1-6 alkoxy, and R _b is independently H, C _1-6 alkyl, -COC _1-6 alkyl, -SO ₂C _1-6 alkyl, C _3-6 cycloalkyl or 3-6 membered heterocyclic ring having 1-3 heteroatoms selected from N and O said ring being optionally substituted by C _1-6 alkyl, C _1-6 alkoxy-C _1-6 alkyl, or C _1-6 alkoxy-carbonyl, or

R _a and R _b, together with the nitrogen atom attached to, form a 4-6 membered heterocyclic ring having 1-3 heteroatoms selected from N, O, and S said ring being optionally substituted by -OH, -NH ₂, N-di-C _1-6 alkyl amino, N-mono-C _1-6 alkyl amino, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 haloalkoxy, O-cyclopropyl, C _1-6 alkoxy-C _1-6 alkyl, or C _1-6 alkyl-carbonyl;

each instance of R _c is C _1-6 alkyl or C _3-6 cycloalkyl;

wherein the alkyl represented by R _a, R _b, or R _c or in the group represented by R _a, R _b, or R _c is optionally substituted by halogen, -OH, C _1-2 alkoxy, or C _3-4 cycloalkyl;

each instance of R ₄ is H, deuterium, halogen, CN, C _1-6 alkyl, or C _1-6 haloalkyl;

each instance of R ₄’ is H, deuterium, F, or Cl;

each instance of R ₅ is H, deuterium, C _1-6 alkyl, or C _1-6 haloalkyl; and

R ₆ is H; OH; C _1-6 alkyl optionally substituted by halogen, OH, or C _1-6 alkoxy; or C _3-6 cycloalkyl optionally substituted by halogen, OH, or C _1-6 alkoxy.

Provided herein are pharmaceutical compositions comprising an effective amount of the compounds of the invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

Also provided is a combination therapy comprising a therapeutically effective amount of the compounds of the invention, or a pharmaceutically acceptable salt thereof, and one or more therapeutically active co-agents.

The present invention further provides a method of inhibiting MALT1 activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compounds of the invention, or a pharmaceutically acceptable salt thereof.

In certain embodiments of the methods of the invention, the subject has a disease or condition, such as an autoimmune disorder, an inflammatory disease, or a cancer, and wherein the disease or condition is treated.

In certain embodiments of the methods of the invention, the disease or condition is rheumatoid arthritis (RA) , multiple sclerosis (MS) , systemic lupus erythematosus (SLE) , a vasculitic condition, an allergic disease, an airway disease (such as asthma and chronic obstructive pulmonary disease (COPD) ) , a condition caused by delayed or immediate type hypersensitivity, anaphylaxis, acute or chronic transplant rejection, a graft versus host disease, a cancer of hematopoietic origin or solid tumor, including chronic myelogenous leukemia (CML) , myeloid leukemia, non-Hodgkin lymphoma (NHL) , or a B cell lymphoma.

Certain embodiments disclose a compound of the present invention, or a pharmaceutically acceptable salt thereof, for use as a medicament, such as a medicament acting as a MALT1 inhibitor.

The present disclosure also provides a use of the compound of the invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same in any of the methods of the invention described above. In one embodiment, provided is the compound of the invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same for use in any of the method of the invention described herein. In another embodiment, provided is use of the compound of the invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same for the manufacture of a medicament for any of the method of the invention described.

BRIEF DESACRIPTION OF THE DRAWINGS

FIG. 1 shows dose-response curves for representative compounds of the invention in the GLOSENSOR ^TM assay described in Biological Example 1. Note that certain compounds are partial inhibitors, with maximum inhibition of MALT1 paracaspase activity at about 70-80%.

FIG. 2 shows dose-response curves for representative compounds of the invention in the IL-2 production assay as described in Biological Example 2. Note that certain compounds are partial inhibitors, with maximum inhibition of MALT1 paracaspase activity at about 50-80%.

DETAILED DESCRIPTION OF THE INVENTION

1. Overview

Described herein are compounds of Formulae (I) , (II-A) , or (II-B) , and the compounds of the examples (collectively referred to herein as “the compounds of the invention” ) , that inhibit the activity of MALT1, and pharmaceutically acceptable salts thereof. Also disclosed are method of using the same to inhibit MALT1 activity, in order to treat a disease or condition as described herein, including an autoimmune disorder, an inflammatory disease, or a cancer.

2. Definitions

As used herein, the term “a, ” “an, ” “the” and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.

The term “halo” as used herein means halogen and includes chloro, fluoro, bromo and iodo.

The term “alkyl” used alone or as part of a larger moiety, such as “alkoxy” or “haloalkyl” and the like, means saturated aliphatic straight-chain or branched monovalent hydrocarbon radical. Unless otherwise specified, an alkyl group typically has 1-4 or 1-6 carbon atoms, i.e. (C ₁-C ₄) alkyl or (C ₁-C ₆) alkyl. Here, a “ (C ₁-C ₄) alkyl” group means a radical having from 1 to 4 carbon atoms in a linear or branched arrangement. Examples include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, etc.

The term “C _1-6 alkylene” refers to divalent fully saturated branched or straight-chain monovalent hydrocarbon radical having 1 to 6 carbon atoms. Similarly, the terms “C _1-4 alkylene, ” “C _1-3 alkylene, ” and “C _1-2 alkylene” are to be construed accordingly. Representative examples of C _1-6 alkylene include, but are not limited to, methylene, ethylene, n-propylene, isopropylene, n-butylene, sec-butylene, iso-butylene, tert-butylene, n-pentylene, isopentylene, neopentylene, and n-hexylene.

The term “C ₁-C ₆ alkyl optionally substituted by hydroxyl” refers to C ₁-C ₆ alkyl as defined above which may be substituted by one or more hydroxy. Examples include, but are not limited to, hydroxymethyl, hydroxyethyl, 1, 2-dihydroxyethyl, 2, 3-dihyroxy-propyl, and the like.

As used herein, the term “di C _1-6 alkylamino” refers to a moiety of the formula -N (R _a) -R _a where each R _a is a C _1-6 alkyl, which may be the same or different, as defined above, in analogy thereto the term “mono C _1-6 alkylamino” which refers to a moiety of the formula -N (H) -R _a where R _a is a C _1-6 alkyl, as defined above.

The term “alkenyl” means branched or straight-chain monovalent hydrocarbon radical containing at least one double bond. Alkenyl may be mono or polyunsaturated, and may exist in the E or Z configuration. Unless otherwise specified, an alkenyl group typically has 2-6 carbon atoms, i.e. (C ₂-C ₆) alkenyl. For example, “ (C ₂-C ₆) alkenyl” means a radical having from 2-6 carbon atoms in a linear or branched arrangement.

The term “alkynyl” means branched or straight-chain monovalent hydrocarbon radical containing at least one triple bond. Unless otherwise specified, an alkynyl group typically has 2-6 carbon atoms, i.e., (C ₂-C ₆) alkynyl. For example, “ (C ₂-C ₆) alkynyl” means a radical having from 2-6 carbon atoms in a linear or branched arrangement.

The term “alkoxy” means an alkyl radical attached through an oxygen linking atom, represented by -O-alkyl. For example, “C ₁-C ₆ alkoxy” refers to -O-C ₁-C ₆ alkyl, wherein alkyl is defined herein above, and “ (C ₁-C ₄) alkoxy” includes methoxy, ethoxy, propoxy, and butoxy, etc. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, cyclopropyloxy-, cyclohexyloxy-and the like. Typically, alkoxy groups have about 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 1 to 2 carbon atoms.

The terms “haloalkyl” and “haloalkoxy” means alkyl or alkoxy, as the case may be, substituted with one or more halogen atoms. Examples of haloalkyl, include, but are not limited to, trifluoromethyl, trichloromethyl, pentafluoroethyl and the like.

Thus the term “C _1-6 alkyl optionally substituted by halogen” refers to C ₁-C ₆ alkyl as defined above which may be substituted by one or more halogens. Examples include, but are not limited to, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2, 2, 2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, 3-bromo-2-fluoropropyl, and 1-bromomethyl-2-bromoethyl.

The term “cycloalkyl” as employed herein includes saturated cyclic, bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 14 carbons containing the indicated number of rings and carbon atoms (for example a C ₃-C ₁₄ monocyclic, C ₄-C ₁₄ bicyclic, C ₅-C ₁₄ tricyclic, or C ₆-C ₁₄ polycyclic cycloalkyl) . In some embodiments “cycloalkyl” is a monocyclic cycloalkyl. Examples of monocyclic cycloalkyl groups include cyclopentyl (C ₅) , cyclohexyl (C ₆) , cyclopropyl (C ₃) cyclobutyl (C ₄) , cycloheptyl (C ₇) and cyclooctyl (C ₈) . In some embodiments “cycloalkyl” is a bicyclic cycloalkyl. Examples of bicyclic cycloalkyls include bicyclo [1.1.0] butane (C ₄) , bicyclo [1.1.1] pentane (C ₅) , spiro [2.2] pentane (C ₅) , bicyclo [2.1.0] pentane (C ₅) , bicyclo [2.1.1] hexane (C ₆) , bicyclo [3.3.3] undecane (C ₁₁) , decahydronaphthalene (C ₁₀) , bicyclo [4.3.2] undecane (C ₁₁) , spiro [5.5] undecane (C ₁₁) and bicyclo [4.3.3] dodecane (C ₁₂) . In some embodiments “cycloalkyl” is a tricyclic cycloalkyl. Examples of tricyclic cycloalkyls include adamantine (C ₁₂) . Unless otherwise described, a “cycloalkyl” has from three to six carbon atoms and is monocyclic.

The term “aryl group” used alone or as part of a larger moiety as in “aralkyl, ” “aralkoxy, ” or “aryloxyalkyl, ” means a carbocyclic aromatic ring. The term “aryl” may be used interchangeably with the terms “aryl ring, ” “carbocyclic aromatic ring, ” “aryl group, ” and “carbocyclic aromatic group. ” Typically, aryl is monocyclic, bicyclic or tricyclic aryl having 6-20 carbon atoms, typically 6-14 ring carbon atoms. Furthermore, the term “aryl” as used herein, refers to an aromatic substituent which can be a single aromatic ring, or multiple aromatic rings that are fused together. Examples includes phenyl, naphthyl, anthracenyl, 1, 2-dihydronaphthyl, 1, 2, 3, 4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl and the like.

A “substituted aryl group” is substituted at any one or more substitutable ring atom, which is a ring carbon atom bonded to a hydrogen. A substituted aryl is typically substituted by 1-5 (such as one, or two, or three) substituents independently selected from the group consisting of: hydroxyl, thiol, cyano, nitro, C ₁-C ₄ alkyl, C ₁-C ₄ alkenyl, C ₁-C ₄ alkynyl, C ₁-C ₄ alkoxy, C ₁-C ₄ thioalkyl, C ₁-C ₄ alkenyloxy, C ₁-C ₄ alkynyloxy, halogen, C ₁-C ₄ alkylcarbonyl, carboxy, C ₁-C ₄ alkoxycarbonyl, amino, C ₁-C ₄ alkylamino, di-C ₁-C ₄ alkylamino, C ₁-C ₄ alkylaminocarbonyl, di-C ₁-C ₄ alkylaminocarbonyl, C ₁-C ₄ alkylcarbonylamino, C ₁-C ₄ alkylcarbony, C ₁-C ₄ alkyl amino, sulfonyl, sulfamoyl, alkylsulfamoyl, and C ₁-C ₄ alkylaminosulfonyl, where each of the afore-mentioned hydrocarbon groups (e.g., alkyl, alkenyl, alkynyl, alkoxy residues) may be further substituted by one or more residues independently selected at each occurrence from halogen, hydroxyl or C ₁-C ₄ alkoxy groups.

The term “heterocyclyl group” or “heterocyclic group” means a monocyclic, non-aromatic (including partially saturated) ring with preferably 3 to 10-members containing preferably 1-4 ring heteroatoms, or a polycyclic ring with ring with preferably 7 to 20 members and from preferably 1 to 4 ring heteroatoms, wherein the polycyclic ring having one or more monocyclic non-aromatic heterocyclic ring fused with one or more aromatic or heteroaromatic ring. The heterocyclyl group typically has 3 to 7, 3 to 24, 4 to 16, 5 to 10, or 5 or 6 ring atoms; wherein optionally one to four, especially one or two ring atoms are a heteroatom (the remaining ring atoms therefore being carbon) . Each heteroatom is independently selected from nitrogen, quaternary nitrogen, oxidized nitrogen (e.g., NO) ; oxygen; and sulfur, including sulfoxide and sulfone. The heterocyclic group can be attached at a heteroatom or a carbon atom. Examples of heterocycles include tetrahydrofuran (THF) , dihydrofuran, 1, 4-dioxane, morpholine, 1, 4-dithiane, piperazine, piperidine, 1, 3-dioxolane, imidazoisdine, imidazoline, pyrroline, pyrrolidine, tetrahydropyran, dihydropyran, oxathiolane, dithiolane, 1, 3-dioxane, 1, 3-dithiane, oxathiane, thiomorpholine, and the like.

The heterocyclyl group can include fused or bridged rings as well as spirocyclic rings. In one embodiment, the heterocyclyl group is a bicyclic ring having a monocyclic non-aromatic heterocyclic ring fused with a phenyl group. Exemplary polycyclic heterocyclic group includes tetrahydroisoquinolinyl (such as 1, 2, 3, 4-tetrahydroisoquinolin-7-yl, 2-methyl-1, 2, 3, 4-tetrahydroisoquinolin-7-yl, 1, 2, 3, 4-tetrahydroisoquinolin-6-yl and 2-methyl-1, 2, 3, 4-tetrahydroisoquinolin-6-yl) , isoindolinyl (such as 2-ethylisoindolin-5-yl, 2-methylisoindolin-5-yl) , indolinyl, tetrahydrobenzo [f] oxazepinyl (e.g., 2, 3, 4, 5-tetrahydrobenzo [f] [1, 4] oxazepin-7-yl) .

The term “heterocycle, ” “heterocyclyl, ” or “heterocyclic” whether saturated or partially unsaturated, also refers to rings that are optionally substituted. A substituted heterocyclyl may be a heterocyclyl group independently substituted by 1-4, such as one, or two, or three, or four substituents.

In some embodiments, a heterocyclyl group is a 3-14 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ( “3-14 membered heterocyclyl” ) .

The term “heteroaryl, ” “heteroaromatic, ” “heteroaryl ring, ” “heteroaryl group, ” “heteroaromatic ring, ” and “heteroaromatic group, ” used alone or as part of a larger moiety as in “heteroaralkyl” or “heteroarylalkoxy, ” refers to aromatic ring groups having 5 to 14 ring atoms selected from carbon and at least one (typically 1 to 4, more typically 1 or 2) heteroatoms (e.g., oxygen, nitrogen or sulfur) . “Heteroaryl” includes monocyclic rings and polycyclic (e.g., bi-or thi-cyclic) rings in which a monocyclic heteroaromatic ring is fused to one or more other carbocyclic aromatic or heteroaromatic rings. As such, “5-14 membered heteroaryl” includes monocyclic, bicyclic or tricyclic ring systems.

Examples of monocyclic 5-6 membered heteroaryl groups include furanyl (e.g., 2-furanyl, 3-furanyl) , imidazolyl (e.g., N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl) , isoxazolyl (e.g., 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl) , oxadiazolyl (e.g., 2-oxadiazolyl, 5-oxadiazolyl) , oxazolyl (e.g., 2-oxazolyl, 4-oxazolyl, 5-oxazolyl) , pyrazolyl (e.g., 3-pyrazolyl, 4-pyrazolyl) , pyrrolyl (e.g., 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl) , pyridyl (e.g., 2-pyridyl, 3-pyridyl, 4-pyridyl) , pyrimidinyl (e.g., 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl) , pyridazinyl (e.g., 3-pyridazinyl) , thiazolyl (e.g., 2-thiazolyl, 4-thiazolyl, 5-thiazolyl) , triazolyl (e.g., 2-triazolyl, 5-triazolyl) , tetrazolyl (e.g., tetrazolyl) , thienyl (e.g., 2-thienyl, 3-thienyl) , pyrimidinyl, pyridinyl and pyridazinyl.

Typically, the heteroaryl is a 5-10 membered ring system (e.g., 5-6 membered monocycle or an 8-10 membered bicycle) or a 5-6 membered ring system. Typical heteroaryl groups include 2-or 3-thienyl, 2-or 3-furyl, 2-or 3-pyrrolyl, 2-, 4-, or 5-imidazolyl, 3-, 4-, or 5-pyrazolyl, 2-, 4-, or 5-thiazolyl, 3-, 4-, or 5-isothiazolyl, 2-, 4-, or 5-oxazolyl, 3-, 4-, or 5- isoxazolyl, 3-or 5-1, 2, 4-triazolyl, 4-or 5-1, 2, 3-triazolyl, tetrazolyl, 2-, 3-, or 4-pyridyl, 3-or 4-pyridazinyl, 3-, 4-, or 5-pyrazinyl, 2-pyrazinyl, and 2-, 4-, or 5-pyrimidinyl.

Examples of polycyclic aromatic heteroaryl groups include carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, isoquinolinyl, indolyl, isoindolyl, acridinyl, or benzisoxazolyl.

Thus the term “heteroaryl” also refers to a group in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Non-limiting examples include 1-, 2-, 3-, 5-, 6-, 7-, or 8-indolizinyl, 1-, 3-, 4-, 5-, 6-, or 7-isoindolyl, 2-, 3-, 4-, 5-, 6-, or 7-indolyl, 2-, 3-, 4-, 5-, 6-, or 7-indazolyl, 2-, 4-, 5-, 6-, 7-, or 8-purinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, or 9-quinolizinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinoliyl, 1-, 3-, 4-, 5-, 6-, 7-, or 8-isoquinoliyl, 1-, 4-, 5-, 6-, 7-, or 8-phthalazinyl, 2-, 3-, 4-, 5-, or 6-naphthyridinyl, 2-, 3-, 5-, 6-, 7-, or 8-quinazolinyl, 3-, 4-, 5-, 6-, 7-, or 8-cinnolinyl, 2-, 4-, 6-, or 7-pteridinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, or 8-4aH carbazolyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, or 8-carbzaolyl, 1-, 3-, 4-, 5-, 6-, 7-, 8-, or 9-carbolinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9-, or 10-phenanthridinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, or 9-acridinyl, 1-, 2-, 4-, 5-, 6-, 7-, 8-, or 9-perimidinyl, 2-, 3-, 4-, 5-, 6-, 8-, 9-, or 10-phenathrolinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, or 9-phenazinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9-, or 10-phenothiazinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9-, or 10-phenoxazinyl, 2-, 3-, 4-, 5-, 6-, or 1-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-benzisoqinolinyl, 2-, 3-, 4-, or thieno [2, 3-b] furanyl, 2-, 3-, 5-, 6-, 7-, 8-, 9-, 10-, or 11-7H-pyrazino [2, 3-c] carbazolyl, 2-, 3-, 5-, 6-, or 7-2H-furo [3, 2-b] -pyranyl, 2-, 3-, 4-, 5-, 7-, or 8-5H-pyrido [2, 3-d] -o-oxazinyl, 1-, 3-, or 5-1H-pyrazolo [4, 3-d] -oxazolyl, 2-, 4-, or 5-4H-imidazo [4, 5-d] -thiazolyl, 3-, 5-, or 8-pyrazino [2, 3-d] pyridazinyl, 2-, 3-, 5-, or 6-imidazo [2, 1-b] -thiazolyl, 1-, 3-, 6-, 7-, 8-, or 9-furo [3, 4-c] cinnolinyl, 1-, 2-, 3-, 4-, 5-, 6-, 8-, 9-, 10-, or 11-4H-pyrido [2, 3-c] carbazolyl, 2-, 3-, 8-, or 7-imidazo [1, 2-b] [1, 2, 4] triazinyi, 7-benzo [b] thienyl, 2-, 4-, 5-, 6-, or 7-benzoxazolyl, 2-, 4-, 5-, 6-, or 7-benzimidazolyl, 2-, 4-, 4-, 5-, 6-, or 7-benzothiazolyl, 1-, 2-, 4-, 5-, 6-, 7-, 8-, or 9-benzoxapinyl, 2-, 4-, 5-, 6-, 7-, or 8-benzoxazinyl, 1-, 2-, 3-, 5-, 6-, 7-, 8-, 9-, 10-, or 11-1H-pyrrolo [1, 2-b] [2] benzazapinyl.

Typical fused heteroary groups include, but are not limited to 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolinyl, 1-, 3-, 4-, 5-, 6-, 7-, or 8-isoquinolinyl, 2-, 3-, 4-, 5-, 6-, or 7-indolyl, 2-, 3-, 4-, 5-, 6-, or 7-benzo [b] thienyl, 2-, 4-, 5-, 6-, or 7-benzoxazolyl, 2-, 4-, 5-, 6-, or 7-benzimidazolyl, and 2-, 4-, 5-, 6-, or 7-benzothiazolyl.

As used herein, the term a pyridin or a pyridyl optionally substituted by hydroxy e.g. 2-pyridyl, 3-pyridyl, or 4-pyridyh refers to a respective hydroxy-pyridin or hydroxy-pyridyl and may include its tautomeric form such as a respective pyridone or pyridon-yi.

As used herein the term pyridin or pyridyl optionally substituted by oxo e.g. 2-pyridyl, 3-pyridyl, or 4-pyridyl, refers to a respective pyridone or pyridon-yl and may include its tautomeric form such as a respective hydroxy-pyridin or hydroxy-pyridyl, provided said tautomeric form may be obtainable. Pyridin or pyridyl optionally substituted by oxo may further refer to a respective pyridine-N-oxide or pyridyl-N-oxide.

A “substituted heteroaryl group” is substituted at any one or more substitutable ring atom, which is a ring carbon or ring nitrogen atom bonded to a hydrogen.

The term “bridged bicyclic group” refers to a ring system which includes two rings that share at least three adjacent ring atoms.

As used herein, many moieties (e.g., alkyl, alkylene, cycloalkyl, aryl, heteroaryl, or heterocyclyl) are referred to as being either “substituted” or “optionally substituted. ” When a moiety is modified by one of these terms, unless otherwise noted, it denotes that any portion of the moiety that is known to one skilled in the art as being available for substitution can be substituted, which includes one or more (e.g., one to three) substituents. Where if more than one substituent is present, then each substituent may be independently selected. Such means for substitution are well-known in the art and/or taught by the instant invention. The optional substituents can be any substituents that are suitable to attach to the moiety.

Where suitable substituents are not specifically enumerated, exemplary substituents include, but are not limited to: (C ₁-C ₅) alkyl, (C ₁-C ₅) hydroxyalkyl, (C ₁-C ₅) haloalkyl, (C ₁-C ₅) alkoxy, (C ₁-C ₅) haloalkoxy, halogen, hydroxyl, cyano, amino, -CN, -NO ₂, -OR ^c1, -NR ^a1R ^b1, -S (O) _iR ^a1, -NR ^a1S (O) _iR ^b1, -S (O) _iNR ^a1R ^b1, -C (=O) OR ^a1, -OC (=O) OR ^a1, -C (=S) OR ^a1, -O (C=S) R ^a1, -C (=O) NR ^a1R ^b1, -NR ^a1C (=O) R ^b1, -C (=S) NR ^a1R ^b1, -C (=O) R ^a1, -C (=S) R ^a1, NR ^a1C (=S) R ^b1, -O (C=O) NR ^a1R ^b1, -NR ^a1 (C=S) OR ^b1, -O (C=S) NR ^a1R ^b1, -NR ^a1 (C=O) NR ^a1R ^b1, -NR ^a1 (C=S) NR ^a1R ^b1, phenyl, or 5-6 membered heteroaryl. Each R ^a1 and each R ^b1 are independently selected from –H and (C ₁-C ₅) alkyl, optionally substituted with hydroxyl or (C ₁-C ₃) alkoxy; R ^c1 is –H, (C ₁-C ₅) haloalkyl or (C ₁-C ₅) alkyl, wherein the (C ₁-C ₅) alkyl is optionally substituted with hydroxyl or (C ₁-C ₃) alkoxy.

The compounds described herein may exist in various tautomeric forms. The term “tautomers” or “tautomeric” refers to two or more interconvertible compounds/substituents resulting from at least one formal migration of a hydrogen atom and at least one change in valency (e.g., a single bond to a double bond, a triple bond to a single bond, or vice versa) . Exemplary tautomerizations include keto-to-enol, amide-to-imide, lactam-to-lactim, enamine-to-imine, and enamine-to- (a different enamine) tautomerizations. The present teachings encompass compounds in the form of tautomers, which includes forms not depicted structurally. All such isomeric forms of such compounds are expressly included. If a tautomer of a compound is aromatic, this compound is aromatic.

It is to be understood that when a compound herein is represented by a structural formula or designated by a chemical name herein, all other tautomeric forms which may exist for the compound are encompassed by the structural formula.

The compounds of any one of the formulae described above may exhibit one or more kinds of isomerism (e.g. optical, geometric or tautomeric isomerism) . The compounds of any one of the formulae described above may also be isotopically labelled. Such variation is implicit to the compounds of any one of the formulae described above defined as they are by reference to their structural features and therefore within the scope of the present disclosure.

Compounds of any one of the formulae described above containing one or more asymmetric carbon atoms can exist as two or more stereoisomers. Where a compound of any one of the formulae described above contains an alkenyl or alkenylene group, geometric cis/trans (or Z/E) isomers are possible. Where structural isomers are interconvertible via a low energy barrier, tautomeric isomerism ( “tautomerism” ) can occur. This can take the form of proton tautomerism in compounds of any one of the formulae described above containing, for example, an imino, keto, or oxime group, or so-called valence tautomerism in compounds which contain an aromatic moiety. It follows that a single compound may exhibit more than one type of isomerism.

Compounds having one or more chiral centers can exist in various stereoisomeric forms. Stereoisomers are compounds that differ only in their spatial arrangement. Stereoisomers include all diastereomeric, enantiomeric, and epimeric forms as well as racemates and mixtures thereof. The term “geometric isomer” refers to compounds having at least one double bond, wherein the double bond (s) may exist in cis (also referred to as syn or entgegen (E) ) or trans (also referred to as anti or zusammen (Z) ) forms as well as mixtures thereof. When a disclosed compound is named or depicted by structure without indicating stereochemistry, it is understood that the name or the structure encompasses one or more of the possible stereoisomers, or geometric isomers, or a mixture of the encompassed stereoisomers or geometric isomers.

When a geometric isomer is depicted by name or structure, it is to be understood that the named or depicted isomer exists to a greater degree than another isomer, that is that the geometric isomeric purity of the named or depicted geometric isomer is greater than 50%, such as at least 60%, 70%, 80%, 90%, 99%, or 99.9%pure by weight. Geometric isomeric purity is determined by dividing the weight of the named or depicted geometric isomer in the mixture by the total weight of all of the geomeric isomers in the mixture.

Racemic mixture means 50%of one enantiomer and 50%of is corresponding enantiomer. When a compound with one chiral center is named or depicted without indicating the stereochemistry of the chiral center, it is understood that the name or structure encompasses both possible enantiomeric forms (e.g., both enantiomerically-pure, enantiomerically-enriched or racemic) of the compound. When a compound with two or more chiral centers is named or depicted without indicating the stereochemistry of the chiral centers, it is understood that the name or structure encompasses all possible diasteriomeric forms (e.g., diastereomerically pure, diastereomerically enriched and equimolar mixtures of one or more diastereomers (e.g., racemic mixtures) of the compound.

Enantiomeric and diastereomeric mixtures can be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Enantiomers and diastereomers also can be obtained from diastereomerically-or enantiomerically-pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

When a compound is designated by a name or structure that indicates a single enantiomer, unless indicated otherwise, the compound is at least 60%, 70%, 80%, 90%, 99%or 99.9%optically pure (also referred to as “enantiomerically pure” ) . Optical purity is the weight in the mixture of the named or depicted enantiomer divided by the total weight in the mixture of both enantiomers.

When the stereochemistry of a disclosed compound is named or depicted by structure, and the named or depicted structure encompasses more than one stereoisomer (e.g., as in a diastereomeric pair) , it is to be understood that one of the encompassed stereoisomers or any mixture of the encompassed stereoisomers is included. It is to be further understood that the stereoisomeric purity of the named or depicted stereoisomers at least 60%, 70%, 80%, 90%, 99%or 99.9%by weight. The stereoisomeric purity in this case is determined by dividing the total weight in the mixture of the stereoisomers encompassed by the name or structure by the total weight in the mixture of all of the stereoisomers.

The pharmaceutically acceptable salts of compounds of any one of the formulae described above may also contain a counterion which is optically active (e.g. d-lactate or l-lysine) or racemic (e.g. dl-tartrate or dl-arginine) .

Cis/trans isomers may be separated by conventional techniques well known to those skilled in the art, for example, chromatography and fractional crystallisation.

Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC) . Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound of any one of the formulae described above contains an acidic or basic moiety, a base or acid such as 1-phenylethylamine or tartaric acid. The resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted to the corresponding pure enantiomer (s) by means well known to a skilled person. Chiral compounds of any one of the formulae described above (and chiral precursors thereof) may be obtained in enantiomerically-enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50%by volume of isopropanol, typically from 2%to 20%, and from 0 to 5%by volume of an alkylamine, typically 0.1%diethylamine. Concentration of the eluate affords the enriched mixture. Chiral chromatography using sub-and supercritical fluids may be employed. Methods for chiral chromatography useful in some embodiments of the present disclosure are known in the art (see, for example, Smith, Roger M., Loughborough University, Loughborough, UK; Chromatographic Science Series (1998) , 75 (Supercritical Fluid Chromatography with Packed Columns) , pp. 223-249 and references cited therein) . Columns can be obtained from Chiral Technologies, Inc, West Chester, Pa., USA, a subsidiary of

Chemical Industries, Ltd., Tokyo, Japan.

It must be emphasized that the compounds of any one of the formulae described above have been drawn herein in a single tautomeric form, all possible tautomeric forms are included within the scope of the present disclosure.

The present disclosure also includes all pharmaceutically acceptable isotopically-labeled compounds of any one of the formulae described above wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number which predominates in nature.

Examples of isotopes suitable for inclusion in the compounds of the present disclosure include isotopes of hydrogen, such as ²H and ³H, carbon, such as ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³⁶Cl, fluorine, such as ¹⁸F, iodine, such as ¹²³I and ¹²⁵I, nitrogen, such as ¹³N and ¹⁵N, oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorus, such as ³²P, and sulfur, such as ³⁵S.

Certain isotopically-labelled compounds of any one of the formulae described above, for example those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e., ³H, and carbon-14, i.e., ¹⁴C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e., ²H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.

Isotopically-labeled compounds of any one of the formulae described above can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.

Pharmaceutically acceptable solvates in accordance with the present disclosure include those wherein the solvent of crystallization may be isotopically substituted, e.g., D ₂O, d ₆-acetone, d ₆-DMSO.

Any formula given herein is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds, such as 2H. Further, substitution with deuterium (i.e., 2H or D) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements or an improvement in therapeutic index, if is understood that deuterium in this context is regarded as a substituent of a compound of the formula (I) . The concentration of such a heavier isotope, specifically deuterium, may be defined by the isotopic enrichment factor. The term “isotopic enrichment factor” as used herein means the ratio between the isotopic abundance and the natural abundance of a specified isotope. If a substituent in a compound of this invention is denoted deuterium, such compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5%deuterium incorporation at each designated deuterium atom) , at least 4000 (60%deuterium incorporation) , at least 4500 (67.5%deuterium incorporation) , at least 5000 (75%deuterium incorporation) , at least 5500 (82.5%deuterium incorporation) , at least 6000 (90%deuterium incorporation) , at least 6333.3 (95%deuterium incorporation) , at least 6466.7 (97%deuterium incorporation) , at least 6600 (99%deuterium incorporation) , or at least 6633.3 (99.5%deuterium incorporation) .

The compounds of this invention can exist in free form for treatment, or where appropriate, as a pharmaceutically acceptable salt form.

As used herein, the terms “salt” or “salts” refers to an acid addition or base addition salt of a compound of the invention. “Salts” include in particular “pharmaceutically acceptable salts. ” The term “pharmaceutically acceptable salts” refers to salts that retain the biological effectiveness and properties of the compounds of this invention and, which typically are not biologically or otherwise undesirable, in many cases, the compounds of the present invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyi groups or groups similar thereto.

Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids, e.g., acetate, aspartate, benzoate, besylate, bromide/hydrobromide, bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate, chloride/hydrochloride, chlortheophylionate, citrate, ethandisulfonate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hydroiodide/iodide, isothionate, lactate, lactobionate, laurylsuifate, maiate, maleate, malonate, mandelate, mesylate, methylsulphate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphafe/dihydrogen phosphate, poiygaiacturonafe, propionate, stearate, succinate, subsalicylate, tartrate, tosyiate and trifiuoroacetate salts.

Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XI I of the periodic table, in certain embodiments, the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium and magnesium salts.

Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Certain organic amines include isopropylamine, benzathine, choiinate, diethanoiamine, diethyiamine, lysine, meglumine, piperazine and tromethamine.

The pharmaceutically acceptable salts of the present invention can be synthesized from a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate or the like) , or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Generally, use of non-aqueous media like ether, ethyl acetate, ethanol, isopropanoi, or acetonitrile is desirable, where practicable. Lists of additional suitable salts can be found, e.g., in “Remington’s Pharmaceutical Sciences, ” 20th ed., Mack Publishing Company, Easton, PA, (1985) ; and in “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002) .

The terms “composition” and “formulation” are used interchangeably.

A “subject” is a mammal, preferably a human, but can also be an animal in need of veterinary treatment, e.g., companion animals (e.g., dogs, cats, and the like) , farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like) .

As used herein, a subject is “in need of” a treatment if such subject would benefit biologically, medically or in quality of life from such treatment.

The term “administer, ” “administering, ” or “administration” refers to methods introducing a compound of the invention, or a composition thereof, in or on a subject. These methods include, but are not limited to, intraarticular (in the joints) , intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, subcutaneous, orally, topically, intrathecally, inhalationally, transdermally, rectally, and the like. Administration techniques that can be employed with the agents and methods described herein are found in e.g., Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington’s, Pharmaceutical Sciences (current edition) , Mack Publishing Co., Easton, Pennsylvania.

As used herein, the term “inhibit, ” “inhibition” or “inhibiting” refers to the reduction or suppression of a given condition, symptom, or disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

The terms “treatment, ” “treat, ” and “treating” refer to reversing, alleviating, or inhibiting the progress of a disease described herein. In some embodiments, treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed (i.e., therapeutic treatment) . In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (i.e., prophylactic treatment) (e.g., in light of a history of symptoms and/or in light of exposure to a pathogen) . Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.

The terms “condition, ” “disease, ” and “disorder” are used interchangeably.

Generally, an effective amount of a compound taught herein varies depending upon various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art. An effective amount of a compound of the present teachings may be readily determined by one of ordinary skill by routine methods known in the art.

The term “an effective amount” means an amount when administered to the subject which results in beneficial or desired results, including clinical results, e.g., inhibits, suppresses or reduces the symptoms of the condition being treated in the subject as compared to a control. For example, an effective amount can be given in unit dosage form (e.g., from 1 mg to about 50 g per day, e.g., from 1 mg to about 5 grams per day) .

The term “a therapeutically effective amount” of a compound of the present invention refers to an amount of the compound of the present invention that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc. In one non-limiting embodiment, the term “a therapeutically effective amount” refers to the amount of the compound of the present invention that, when administered to a subject, is effective to (1) at least partially alleviating, inhibiting, preventing and/or ameliorating a condition, or a disorder or a disease (i) mediated by MALT1, or (ii) associated with MALT1 activity, or (iii) characterized by activity (normal or abnormal) of MALT1; or (2) reducing or inhibiting the activity of MALT1; or (3) reducing or inhibiting the expression of MALT1; or (4) modifying the protein levels of MALT1. In another non-limiting embodiment, the term “a therapeutically effective amount” refers to the amount of the compound of the present invention that, when administered to a cell, or a tissue, or a non-cellular biological material, or a medium, is effective to at least partially reducing or inhibiting the activity of MALT1; or reducing or inhibiting the expression of MALT1 partially or completely.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and another examples, or exemplary language (e.g. “such as” ) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed.

The general chemical terms used in the formulae above have their usual meanings.

As used herein, “h” or “hr” refers to hour or hours, “min” refers to minutes or minutes, “MCL” refers to mantle cell lymphoma, “AML” refers to acute myeloid leukemia, “CML” refers to chronic myeloid leukemia, “Boc” refers to N-tert-butoxycarbonyl, “EA” refers to ethyl acetate, “DCM” refers to dichloromethane, “DMSO” refers to dimethylsulfoxide, “DMA” refers to dimethylacetamide, “THF” refers to tetrahydrofuran, “MtBE” refers to methyl tert-butyl ether, “TEA” refers to triethylamine, “FBS” refers to fetal bovine serum, “PBS” refers to phosphate buffered saline, “BSA” refers to bovine serum albumin, “RT” refers to room temperature, “mpk” means milligrams per kilogram, “po” refers to per os (oral) , “qd” means once daily dosing, “HPLC” means high pressure liquid chromatography, “q2d” means a single dose every 2 days, “q2dx10” means a single dose every 2 days times 10, “VSMC” refers to vascular smooth muscle cell and “XRD” refers to X-ray diffraction.

As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents) , isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington’s Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329) . Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

Furthermore, the compounds of the present invention, including their salts, can also be obtained in the form of their hydrates, or include other solvents used for their crystallization. The compounds of the present invention may inherently or by design form solvates with pharmaceutically acceptable solvents (including water) ; therefore, it is intended that the invention embrace both solvated and unsolvated forms. The term “solvate” refers to a molecular complex of a compound of the present invention (including pharmaceutically acceptable salts thereof) with one or more solvent molecules. Such solvent molecules are those commonly used in the pharmaceutical art, which are known to be innocuous to the recipient, e.g., water, ethanol, and the like. The term “hydrate” refers to the complex where the solvent molecule is water.

The compounds of the present invention, including salts, hydrates and solvates thereof, may inherently or by design form polymorphs. in another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition can be formulated for particular routes of administration such as oral administration, parenteral administration, and rectal administration, etc. In addition, the pharmaceutical compositions of the present invention can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or suppositories) , or in a liquid form (including without limitation solutions, suspensions or emulsions) . The pharmaceutical compositions can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc.

Typically, the pharmaceutical compositions are tablets or gelatin capsules comprising the active ingredient together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyieneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymefhylceiluiose and/or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners. Tablets may be either film coated or enteric coated according to methods known in the art.

Suitable compositions for oral administration include an effective amount of a compound of the invention in the form of tablets, lozenges, aqueous or oily suspensions, dispersibie powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use are prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets may contain the active ingredient in admixture with nontoxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients are, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets are uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. Formulations for oral use can be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.

Certain injectable compositions are aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. Said compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. Said compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1-75%, or contain about 1-50%, of the active ingredient. Suitable compositions for transdermal application include an effective amount of a compound of the invention with a suitable carrier. Carriers suitable for transdermal delivery include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. For example, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound of the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.

Suitable compositions for topical application, e.g., to the skin and eyes, include aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations, e.g., for delivery by aerosol or the like. Such topical delivery systems will in particular be appropriate for dermal application, e.g., for the treatment of skin cancer, e.g., for prophylactic use in sun creams, lotions, sprays and the like. They are thus particularly suited for use in topical, including cosmetic, formulations well-known in the art. Such may contain solubiiizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

As used herein a topical application may also pertain to an inhalation or to an intranasal application. They may be conveniently delivered in the form of a dry powder (either alone, as a mixture, for example a dry blend with lactose, or a mixed component particle, for example with phospholipids) from a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray, atomizer or nebuliser, with or without the use of a suitable propellant.

The present invention further provides anhydrous pharmaceutical compositions and dosage forms comprising the compounds of the present invention as active ingredients, since water may facilitate the degradation of certain compounds.

Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. An anhydrous pharmaceutical composition may be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions are packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials) , blister packs, and strip packs.

The invention further provides pharmaceutical compositions and dosage forms that comprise one or more agents that reduce the rate by which the compound of the present invention as an active ingredient will decompose. Such agents, which are referred to herein as “stabilizers, ” include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers, etc.

3. Compounds

Disclosed herein are embodiments of a compound having the general structure of Formula (I) .

In a first embodiment of the invention, provided is a compound represented by Formula (I) or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein

ring A is

Z is O, NR ₆, or S;

A ₁ and A ₂ are each independently CR ₁ or N;

each instance of R ₂ is hydrogen; halogen; CN; -COOC _1-6 alkyl; C _1-6 alkoxy optionally substituted by halogen; C _1-6 alkoxy carbonyl; amino; N, N-di-C _1-6 alkyl amino; C _1-6 alkyl optionally substituted by halogen, -OH, phenyl, or a 5-6 membered heterocyclic ring having 1 to 2 heteroatoms selected from N and O which ring is optionally substituted by C _1-6 alkyl; Rh;ORh; a 5-6 membered heteroaryl ring having 1 to 3 heteroatoms selected from N and O said ring being optionally substituted by amino, C _1-6 alkyl optionally substituted by amino or hydroxy, or by N-mono or N, N-di-C _1-6 alkylamino carbonyl;

Rg is a 3-6 membered heterocyclic ring having 1-3 heteroatoms selected from N and O said ring being optionally substituted by -OH, -NH ₂, C _1-6 alkyl, C _1-6 alkoxy-C _1-6 alkyl, or C _1-6 alkoxy-carbonyl;

each instance of R _c is C _1-6 alkyl or C _3-6 cycloalkyl;

each instance of R ₄’ is H, deuterium, F, or Cl;

each instance of R ₅ is H, deuterium, C _1-6 alkyl, or C _1-6 haloalkyl; and

In a second embodiment of the invention, provided is a compound of formula (I) or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein Z is O, and the remaining variables are as defined in the first embodiment.

In a third embodiment of the invention, provided is a compound of formula (I) , or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein ring A is

and the remaining variables are as defined in the first and/or second embodiment.

In a fourth embodiment of the invention, provided is a compound of formula (I) , or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein each instance of R ₃ is H; halogen; CN; -OH; -COOH; -NR _aR _b; -SR _c; -SO ₂R _c; -SO ₂NR _c; -C (=O) NR _aR _b; C _1-4 alkoxy optionally substituted by halogen, -OH, C _1-6 alkyl, -NH ₂, -NHC (=O) C _1-6 alkyl, N-di-C _1-6 alkyl amino, or N-mono-C _1-6 alkyl amino; C _1-4 alkyl optionally substituted by halogen, C _1-2 alkoxy, N-di-C _1-6 alkyl amino, or N-mono-C _1-6 alkyl amino; C _3-6 cycloalkyl; or Rg, and the remaining variables are as defined in the first, second and/or third embodiments.

In a fifth embodiment, the compound of formula (I) is represented by structural formula (II-A) or (II-B) :

or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein each instance of R ₄’ is H or F, and the remaining variables are as defined in the first, second, third and/or fourth embodiments.

In a sixth embodiment, the compound of formula (I) , (II-A) , or (II-B) , or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein

each instance of R ₃ is H; halogen; CN; -OH; -COOH; -NR _aR _b; -SR _c; -SO ₂R _c; -SO ₂NR _c; -C (=O) NR _aR _b; C _1-4 alkoxy optionally substituted by halogen, -OH, C _1-4 alkyl, -NH ₂, N-di-C _1-4 alkyl amino, or N-mono-C _1-4 alkyl amino; C _1-4 alkyl optionally substituted by halogen, C _1-2 alkoxy, N-di-C _1-4 alkyl amino, or N-mono-C _1-4 alkyl amino; C _3-6 cycloalkyl; morpholinyl; oxetanyl; or azetidinyl optionally substituted by methyl;

R _a is independently H or C _1-4 alkyl optionally substituted by C _1-4 alkoxy;

R _b is independently H, C _1-4 alkyl, -COC _1-4 alkyl, -SO ₂C _1-4 alkyl, C _3-6 cycloalkyl or 3-6 membered heterocyclic ring having 1-3 heteroatoms selected from N and O said ring being optionally substituted by C _1-4 alkyl, C _1-4 alkoxy-C _1-4 alkyl, or C _1-4 alkoxy-carbonyl, or

R _a and R _b, together with the nitrogen atom attached to, form a 4-6 membered heterocyclic ring having 1-3 heteroatoms selected from N, O, and S said ring being optionally substituted by -OH, C _1-4 alkyl, C _1-4 haloalkyl, C _1-4 alkoxy, C _1-4 haloalkoxy, or C _1-4 alkoxy-C _1-4 alkyl, and the remaining variables are as defined in the first, second, third, fourth and/or fifth embodiments.

In a seventh embodiment, the compound of formula (I) , (II-A) , or (II-B) , or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein each instance of R ₄ is H,halogen, C _1-2 alkyl optionally substituted with halogen, each instance of R ₄’ is H, and the remaining variables are as defined in the first, second, third, fourth, fifth and/or sixth embodiments.

In an eighth embodiment, the compound of formula (I) , (II-A) , or (II-B) , or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein R ₂ is H, Cl, CN, or C _1-6 alkyl optionally substituted with halo or -OH, and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, and/or seventh embodiments.

In a ninth embodiment, the compound of formula (I) , (II-A) , or (II-B) , or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein R ₁ is H; halogen; -OH; CN; C _1-6 alkoxy optionally substituted by halogen; C _1-6 alkoxy carbonyl; phenyl; N, N-di-C _1-6 alkyl amino; C _1-6 alkyl optionally substituted by halogen or phenyl; a 5-6 membered heteroaryl ring containing 1 to 3 N atoms said ring being optionally substituted by C _1-6 alkyl optionally substituted by amino or hydroxy or by mono-or di-N-C _1-6 alkylamino carbonyl; O-Rh; or Rh; wherein Rh is a 5-6 membered heterocyclyl containing 1 to 4 heteroatoms selected from N, O and S said ring being optionally substituted by C _1-6 alkyl, OH, or oxo, and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, and/or eighth embodiments.

In a tenth embodiment, the compound of formula (I) , (II-A) , or (II-B) , or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein R ₁ is H; halogen; C _1-4 alkyl optionally substituted by halogen; C _1-4 alkoxy; or 5 -6 membered heteroaryl containing 1 to 3 N atoms, and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, and/or ninth embodiments.

In an eleventh embodiment, the compound of formula (I) , (II-A) , or (II-B) , or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein each instance of R ₃ is bromo, methyl, ethyl, propyl, isopropyl, COOH, -CH (CH ₃) OCH ₃, -CH (CH ₃) OCH ₂CH ₃,

-CH (CH ₃) N (CH ₃) ₂, -CH ₂OCH ₃, cyclopropyl, morpholinyl, -S (CH ₃) , -N (CH ₃) ₂, -N (CH ₃) (CH ₂CH ₃) , -N (CH ₂CH ₃) ₂, -N (CH ₃) (isopropyl) , -N (CH ₃) (cyclopropyl) , -N (CH ₂CH ₃) (cyclopropyl) , -N (CH ₃) (SO ₂CH ₃) , -N (CH ₂CH ₂OCH ₃) (cyclopropyl) , or -N (CH ₃) (CH ₂CH ₂OCH ₃) , and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, and/or tenth embodiments.

In a twelfth embodiment, the compound of formula (I) , (II-A) , or (II-B) , or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein each instance of R ₃ is -CH (CH ₃) OCH ₃,

ethyl, or cyclopropyl, and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, and/or tenth embodiments.

In a thirteenth embodiment, the compound of formula (I) , (II-A) , or (II-B) , or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein R ₄ is H, F, Cl, Br, CH ₃, or CF ₃, and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, and/or twelfth embodiments.

In a fourteenth embodiment, the compound of formula (I) , (II-A) , or (II-B) , or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein R ₂ is Cl or CF ₃, and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and/or thirteenth embodiments.

In a fifteenth embodiment, the compound of formula (I) , (II-A) , or (II-B) , or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein R ₁ is H, 1, 2, 3, -triazole, -OCH ₃, or CF ₃, and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth and/or fourteenth embodiments.

In a sixteenth embodiment, the compound of formula (I) , (II-A) , or (II-B) , or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein R ₄ is H or Cl, and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, and/or fifteenth embodiments.

In a seventeenth embodiment, the compound of formula (I) , (II-A) , or (II-B) , or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein

is

and the remaining variables are as defined in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, and/or sixteenth embodiments.

In one embodiment, the compound or a pharmaceutically acceptable salt thereof is selected from the compounds of formula (I) , (II-A) , or (II-B) , or in the Examples, as described herein.

1. A compound represented by structural formula (I) :

or a pharmaceutically acceptable salt thereof, wherein

ring A is

Z is O, N, or S;

A ₁ and A ₂ are each independently CR ₁ or N;

each instance of R ₁ is halogen; CN; -COOC _1-6 alkyl; C _1-6 alkoxy optionally substituted by halogen; C _1-6 alkoxy carbonyl; amino; N, N-di-C _1-6 alkyl amino; C _1-6 alkyl optionally substituted by halogen, phenyl, or a 5-6 membered heterocyclic ring having 1 to 2 heteroatoms selected from N and O which ring is optionally substituted by C _1-6alkyl; Rh; ORh; a 5-6 membered heteroaryl ring having 1 to 3 heteroatoms selected from N and O said ring being optionally substituted by amino, C _1-6 alkyl optionally substituted by amino or hydroxy, or by N-mono or N, N-di-C _1-6 alkylamino carbonyl; wherein

Rh is a 5-6 membered heterocyclyl ring having 1 to 4 heteroatoms selected from N, O and S, said ring being optionally substituted by C _1-6 alkyl, -OH, or oxo,

each instance of R ₂ is halogen; CN; -COOC _1-6 alkyl; C _1-6 alkoxy optionally substituted by halogen; C _1-6 alkoxy carbonyl; amino; N, N-di-C _1-6 alkyl amino; C _1-6 alkyl optionally substituted by halogen, phenyl, or a 5-6 membered heterocyclic ring having 1 to 2 heteroatoms selected from N and O which ring is optionally substituted by C _1-6 alkyl; Rh; ORh; a 5-6 membered heteroaryl ring having 1 to 3 heteroatoms selected from N and O said ring being optionally substituted by amino, C _1-6 alkyl optionally substituted by amino or hydroxy, or by N-mono or N, N-di-C _1-6 alkylamino carbonyl;

each instance of R ₃ is H; deuterium; halo; -OH; NR _aR _b; -SR _c; -SO ₂R _c; -SO ₂NR _c; C _1-6 alkoxy optionally substituted by halogen, -OH, C _1-6 alkyl, -NH ₂, N-di-C _1-6 alkyl amino, or N-mono-C _1-6 alkyl amino; C _1-6 alkyl optionally substituted by halogen, C _2-6 alkenyl, -OH, -NH ₂, N-di-C _1-6 alkyl amino, N-mono-C _1-6 alkyl amino, O-Rg, Rg, phenyl, or by C _1-6 alkoxy wherein said alkoxy optionally substituted by halogen, -OH, C _1-6 alkoxy, N, N-di-C _1-6 alkyl amino, Rg or phenyl; C _3-6 cycloalkyl optionally substituted by halogen, -OH, C _1-6 alkyl, N, N-di-C _1-6 alkyl amino or C _1-6alkoxy-C _1-6 alkyl; phenyl optionally substituted by halo or C _1-6 alkoxy; a 5-6 membered heteroaryl ring having 1 to 3 heteroatoms selected from N and O said ring being optionally substituted by C _1-6 alkyl which may be optionally substituted by amino or -OH; Rg; or N, N-di-C _1-6 alkyl amino carbonyl; wherein

R _g is a 3-6 membered heterocyclic ring having 1-3 heteroatoms selected from N and O said ring being optionally substituted by –OH, -NH ₂, C _1-6 alkyl, C _1-6 alkoxy-C _1-6 alkyl, or C _1-6 alkoxy-carbonyl;

R _a is independently H or C _1-6 alkyl, and R _b is independently H, C _1-6 alkyl, -COC _1-6 alkyl, -SO ₂C _1-6 alkyl, C _3-6 cycloalkyl or 3-6 membered heterocyclic ring having 1-3 heteroatoms selected from N and O said ring being optionally substituted by C _1-6 alkyl, C _1-6 alkoxy-C _1-6 alkyl, or C _1-6 alkoxy-carbonyl, or

each instance of R _c is C _1-6 alkyl or C _3-6 cycloalkyl;

each instance of R ₄ is H, deuterium, halogen, CN, C _1-6 alkyl, or C _1-6 haloalkyl; and

each instance of R ₅ is H, deuterium, C _1-6 alkyl, or C _1-6 haloalkyl.

2. The compound of paragraph 1 or a pharmaceutically acceptable salt thereof, wherein each instance of R ₃ is H; halo; -OH; NR _aR _b; -SR _c; -SO ₂R _c; -SO ₂NR _c; C _1-4 alkoxy optionally substituted by halogen, -OH, C _1-6 alkyl, -NH ₂, N-di-C _1-6 alkyl amino, or N-mono-C _1-6 alkyl amino; C _1-4 alkyl optionally substituted by halo or C _1-2 alkoxy; C _3-6 cycloalkyl; or Rg.

3. The compound of

paragraph

1 or 2, or a pharmaceutically acceptable salt thereof, wherein Z is O.

4. The compound of any one of paragraphs 1-3, or a pharmaceutically acceptable salt thereof, wherein R ₃ is H; halo; -OH; NR _aR _b; -SR _c; -SO ₂R _c; -SO ₂NR _c; C _1-4 alkoxy optionally substituted by halogen, -OH, C _1-6 alkyl, -NH ₂, N-di-C _1-6 alkyl amino, or N-mono-C _1-6 alkyl amino; methyl, ethyl, isopropyl, C _1-4 haloalkyl; morpholinyl; oxetanyl; or azetidinyl optionally substituted by methyl.

5. The compound of any one of paragraphs 1-4, or a pharmaceutically acceptable salt thereof, wherein ring A is

6. The compound of any one of paragraphs 1-5, wherein the compound is represented by structural formula (II-A) or (II-B) :

or a pharmaceutically acceptable salt thereof.

7. The compound of any one of paragraphs 1-6, or a pharmaceutically acceptable salt thereof, wherein each instance of R ₄ is H, halogen, C _1-2 alkyl optionally substituted with halo.

8. The compound of any one of paragraphs 1-7, or a pharmaceutically acceptable salt thereof, wherein R ₂ is H, Cl, CN, or C _1-6 alkyl optionally substituted with halo or -OH.

9. The compound of any one of paragraphs 1-8, or a pharmaceutically acceptable salt thereof, wherein R ₁ is H; halogen; -OH; CN; C _1-6 alkoxy optionally substituted by halogen;

C _1-6 alkoxy carbonyl; phenyl; N, N-di-C _1-6 alkyl amino; C _1-6 alkyl optionally substituted by halogen or phenyl; a 5-6 membered heteroaryl ring containing 1 to 3 N atoms said ring being optionally substituted by C _1-6 alkyl optionally substituted by amino or hydroxy, or by mono-or di-N-C _1-6 alkylamino carbonyl; O-Rh; or Rh; wherein Rh is a 5-6 membered heterocyclyl containing 1 to 4 heteroatoms selected from N, O and S said ring being optionally substituted by C _1-6 alkyl, OH, or oxo.

10. The compound of any one of paragraphs 1-9, or a pharmaceutically acceptable salt thereof, wherein R ₁ is H; halogen; C _1-4 alkyl optionally substituted by halogen; or 5 -6 membered heteroaryl containing 1 to 3 N atoms.

11. The compound of any one of paragraphs 1-10, or a pharmaceutically acceptable salt thereof, wherein each instance of R ₃ is H, ethyl, isopropyl, cyclopropyl, morpholinyl, N (CH ₃) (cyclopropyl) or N (CH ₃) (CH ₂CH ₂OCH ₃) .

12. The compound of any one of paragraphs 1-10, or a pharmaceutically acceptable salt thereof, wherein each instance of R ₃ is H, F, or Cl, preferably H or F.

13. The compound of any one of paragraphs 1-12, or a pharmaceutically acceptable salt thereof, wherein R ₄ is H, F, Cl, Br, CH ₃, or CF ₃.

14. The compound of any one of paragraphs 1-13, or a pharmaceutically acceptable salt thereof, wherein R ₂ is Cl or CF ₃.

15. The compound of any one of paragraphs 1-14, or a pharmaceutically acceptable salt thereof, wherein R ₁ is H, 1, 2, 3, -triazole, or CF ₃.

16. The compound of any one of paragraphs 1-15, or a pharmaceutically acceptable salt thereof, wherein R ₄ is H or Cl.

17. The compound of any one of paragraphs 1-16, or a pharmaceutically acceptable salt thereof, wherein

is

In certain embodiments, the compound of the invention is a partial inhibitor of MALT1, in that a maximum of about 50-85%, or a maximum of about 60-80%, or a maximum of about 70-80%, or a maximum of about 75%of the activity of MALT1 is inhibited. MALT1 activity inhibition can be assessed using any of the art-recognized methods, such as those described in the Biological Examples, particularly Example 1 (Glosensor assay or variations thereof) and/or Example 2 (IL-2 production) . In certain embodiments, MALT1 activity inhibition is assessed using the Glosensor assay as described in Biological Example 1.

4. Treatable Diseases

In certain embodiments, the compounds of the invention can be used to treat diseases or indications in which the activity of a CBM complex (such as one comprising CARMA1/BCL10/MALT1, or CARD9/BCL10/MALT1) and/or MALT1 activity is upregulated or overly active (e.g., activating CARD11 mutation, overexpression of wild-type MALT1, gain-of-function activating mutation of MALT1, loss-of-function of MALT1 suppressor, etc. ) .

Thus in certain embodiments, the compounds of the invention can be used to treat diseases or indications in which the NF-κB signaling pathway is activated or dysregulated. While not wishing to be bound by any particular theory, it is believed that blockade or inhibition of MALT1 directly down-regulates the NF-κB pathway in such diseases or indications (e.g., cancers) , resulting in treatment.

In certain other embodiment, while still not wishing to be bound by any particular theory, it is further believed that the subject MALT1 inhibitors may act independent of the direct inhibition of dysregulated NF-κB pathway, such as those in certain tumor cells. Rather, inhibition of the paracaspase activity of MALT1 by the subject compounds affects a number of components of the immune system independent of or in addition to the NF-κB pathway, and broadly regulates specified T cell populations of a subject, such as the Th17 cells, or natural regulatory T cells (nTreg) or Tregs, either but not the other, or both. Thus certain compounds of the invention act as immunomodulatory agents to fine tune the activity of specific T cell populations, and indirectly expand the range of treatable cancers and other autoimmune /inflammatory diseases or indications by administration of a subject MALT1 inhibitor, irrespective of whether the cancers /autoimmune /inflammatory diseases or indications have dysregulated NF-κB pathway activity.

In certain embodiments, the compounds of the invention preferentially or selectively inhibit Th17 cells over Tregs (e.g., the ratio of IC ₅₀s for Treg over Th17 is over 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, or 100) . In certain embodiments, the compounds of the invention does not substantially inhibit Tregs.

Conditions and disorders characterized by disregulated NF-kB activation include autoimmune, immunological, and/or inflammatory disorders, allergic disorders, respiratory disorders, and oncological disorders.

In an aspect, the present invention provides a method of inhibiting MALT1 activity in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound of the present invention.

In another related aspect, the compounds of the present invention can be used in the manufacture of a medicament for inhibiting MALT1 activity.

According to any of the above related aspects of the present invention, the subject has a disease or condition, such as an autoimmune disorder, an inflammatory disease, or a cancer, and wherein the disease or condition is treated.

In certain embodiments, the disease or condition is rheumatoid arthritis (RA) , multiple sclerosis (MS) , systemic lupus erythematosus (SLE) , a vasculitic condition, an allergic disease, an airway disease (such as asthma and chronic obstructive pulmonary disease (COPD) ) , a condition caused by delayed or immediate type hypersensitivity, anaphylaxis, acute or chronic transplant rejection, a graft versus host disease, a cancer of hematopoietic origin or solid tumor, including chronic myelogenous leukemia (CML) , myeloid leukemia, non-Hodgkin lymphoma (NHL) , or a B cell lymphoma.

In one embodiment, the treatable disease or disorder is a cancer (oncological disorder) .

Oncological disorders treatable by the compounds of the invention may, inter alia, include cancers of hematopoietic origin or solid tumors, carcinoma, sarcoma, lymphoma, leukemia and germ ceil tumors, e.g. adenocarcinoma, bladder cancer, clear cell carcinoma, skin cancer, brain cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, bladder cancer, brain tumours, breast cancer, gastric cancer, germ cell tumours, glioblastoma, hepatic adenomas, Hodgkin's lymphoma, liver cancer, kidney cancer, lung cancer, ovarian cancer, dermal tumors, prostate cancer, renal cell carcinoma, stomach cancer, medullobiastoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, marginal zone lymphoma, cutaneous T-cell lymphoma, other B cell lymphomas, melanoma, mucosa-associated lymphoid tissue (MALT) lymphoma, chronic myelogenous leukemia, myeloid leukemia, multiple myeloma, plasma cell neoplasm, lentigo maligna melanomas, and acral lentiginous melanoma.

In certain embodiments, the cancer is leukemia or lymphoma.

In certain embodiments, the leukemia is chronic lymphocytic leukemia (CLL) , such as CLL with CARD11 mutation.

In certain embodiments, the lymphoma is Mucosa Associated Lymphoid Tissue lymphoma (MALT) .

In certain embodiments, the lymphoma is mantle cell lymphoma. In certain embodiments, the lymphoma is marginal zone lymphoma. In certain embodiments, the lymphoma is cutaneous T cell lymphomas such as Sezary syndrome. In certain embodiments, the lymphoma is primary effusion lymphoma.

In certain embodiments, the lymphoma is non-Hodgkin’s lymphoma (NHL) . In certain embodiments, the lymphoma is DLBCL (Diffuse large B-cell lymphoma) . In certain embodiments, the lymphoma is germinal center B-cell (GCB) DLBCL. In certain embodiments, the lymphoma is activated B-cell (ABC) DLBCL (such as those with activating mutations in CARD11) .

In certain embodiments, the cancer is lung adenocarcinoma.

In certain embodiments, the cancer is breast cancer.

In certain embodiments, the cancer is pancreatic cancer.

Autoimmune and/or inflammatory disorders treatable by the compounds of the invention may, inter alia, be selected from arthritis, ankylosing spondylitis (AS) , inflammatory bowel disease (IBD) , ulcerative colitis (UC) , gastritis, pancreatitis, Crohn's disease (CD) , celiac disease, multiple sclerosis (MS) , systemic lupus erythematosus (SLE) , rheumatic fever, gout, organ or transplant rejection, acute or chronic graft-versus-host disease, chronic allograft rejection, Behcet's disease, uveitis, psoriasis, dermatitis, atopic dermatitis, dermatomyositis, myasthena gravis, Grave's disease, Hashimoto thyroiditis, Sjogren's syndrome, and blistering disorders (e.g, pemphigus vulgaris) , antibody-mediated vasculitis syndromes, including ANCA-associated vasculitis, Hennoch-Schonlein Purpura, and immune-complex vasculitis (either primary or secondary to infection or cancers) .

In certain embodiments, the treatable disease or disorder is an autoimmune disease or disorder, or an inflammatory disease or disorder.

In certain embodiments, the disease is rheumatoid arthritis (RA) .

In certain embodiments, the disease is psoriasis.

In certain embodiments, the disease is ulcerative colitis (UC) .

In certain embodiments, the disease is multiple sclerosis (MS) .

In certain embodiments, the disease is allergic encephalomyelitis.

In certain embodiments, the disease is systemic lupus, Sjogren’s syndrome, or Hashimoto’s thyroiditis.

In certain embodiments, the disease or disorder is an allergic disorder resulting from chronic inflammation.

Allergic disorder treatable by the compounds of the invention may, inter alia, include: contact dermatitis, celiac disease, asthma, hypersensitivity to house dust mites, pollen and related allergens, Berylliosis (or chronic beryllium disease (CBD) ) .

Respiratory disorders treatable by the compounds of the invention may, inter alia, include: asthma, bronchitis, chronic obstructive pulmonary disease (COPD) , cystic fibrosis, pulmonary oedema, pulmonary embolism, pneumonia, puimonary sarcoidosis, silicosis, pulmonary fibrosis, respiratory failure, acute respiratory distress syndrome, primary puimonary hypertension and emphysema.

In other embodiments, the compounds of the invention may also be useful in the treatment of BENTA ( “B cell Expansion with NF-κB and T cell Anergy” ) disease, lupus nephritis, and polymyositis.

In certain embodiments, the autoimmune disease or disorder, or the inflammatory disease or disorder is treated by further administering an immunosuppressive agent, such as cyclosporine, rapamycin, methotrexate and the like.

5. Combination Therapy

The compounds of the invention may be used in combination therapy with one or more additional /secondary therapeutic agents suitable for treating a disease or indication treatable by the subject compounds.

Thus in certain embodiments, methods of the invention using compounds of the invention may additionally comprise administering to the subject in need thereof a further therapeutic agent. The further therapeutic agent may be: (i) an immunomodulatory agent which blocks or inhibits an immune system checkpoint, which checkpoint may or may not be a component of the NF-κB pathway; and/or (ii) an agent which directly stimulates an immune effector response, such as a cytokine, or a tumor specific adoptively transferred T cell population, or an antibody specific for a protein expressed by a tumor cell; and/or (iii) a composition comprising a tumor antigen or immunogenic fragment thereof; and/or (iv) a chemotherapeutic agent.

The compound of the invention may be administered either simultaneously with, before, or after the further therapeutic agent. The MALT1 inhibitor may be administered separately, by the same or different route of administration, or together in the same pharmaceutical composition as the further therapeutic agent.

As used herein, “co-administration” or “combined administration” or the like are meant to encompass administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.

The term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term “fixed combination” means that the active ingredients, e.g., a subject compound and a co-agent, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the active ingredients, e.g., a subject compound and a co-agent, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the compounds in the body of the patient.

In one embodiment, the invention provides a product comprising a compound of the invention, such as a subject compound or any subgroup thereof, and at least one other therapeutic agent as a combined preparation for simultaneous, separate or sequential use in therapy. Products provided as a combined preparation include a composition comprising the compound of the invention or any subgroup thereof and the other therapeutic agent (s) together in the same pharmaceutical composition, or the subject compound or any subgroup thereof and the other therapeutic agent (s) in separate form, e.g., in the form of a kit.

In one embodiment, the invention provides a pharmaceutical composition for use in therapy comprising a subject compound or any subgroup thereof and an additional immunomodulatory agent or a composition comprising a tumor antigen or immunogenic fragment thereof. Optionally, the pharmaceutical composition may comprise a pharmaceutically acceptable excipient.

In one embodiment, the invention provides a kit comprising two or more separate pharmaceutical compositions, at least one of which contains a subject compound, and another contains a second therapeutic agent discussed herein. In one embodiment, the kit comprises means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is a blister pack, as typically used for the packaging of tablets, capsules and the like. The kit of the invention may be used for administering different dosage forms, for example, oral and parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another. To assist compliance, the kit of the invention typically comprises directions for administration.

It will be appreciated that many of the further therapeutic agents used in the methods of the invention may be biologicals requiring intravenous, intraperitoneal or depot administration. In a further embodiment, the compound of the invention is orally administered and the further therapeutic agent is administered parenterally, for example intravenously, intraperitoneally or as a depot.

In certain embodiments, the second /additional therapeutic agent is an immune system checkpoint. Effector T cell activation is normally triggered by the TCR recognizing antigenic peptide presented by the MHC complex. The type and level of activation achieved is then determined by the balance between signals which stimulate and signals which inhibit the effector T cell response. “Immune system checkpoint” is used herein to refer to any molecular interaction which alters the balance in favor of inhibition of the effector T cell response. That is, a molecular interaction which, when it occurs, negatively regulates the activation of an effector T cell. Such an interaction might be direct, such as the interaction between a ligand and a cell surface receptor which transmits an inhibitory signal into an effector T cell. Or it might be indirect, such as the blocking or inhibition of an interaction between a ligand and a cell surface receptor which would otherwise transmit an activatory signal into the effector T cell, or an interaction which promotes the upregulation of an inhibitory molecule or cell, or the depletion by an enzyme of a metabolite required by the effector T cell, or any combination thereof.

Examples of immune system checkpoints include: a) The interaction between indoleamine 2, 3-dioxygenase (ID01) and its substrate; b) The interaction between PD1 and PD-L1 and/or PD1 and PD-L2; c) The interaction between CTLA-4 and CD86 and/or CTLA-4 and CD80; d) The interaction between B7-H3 and/or B7-H4 and their respective ligands; e) The interaction between HVEM and BTLA; f) The interaction between GAL9 and TIM3; g) The interaction between MHC class I or II and LAG 3; and h) The interaction between MHC class I or II and KIR; i) The interaction between OX40 (CD134) and OX40L (CD252) ; j) The interaction between CD40 and CD40L (CD154) ; k) The interaction between 4-1 BB (CD137) and ligands including 4-1 BBL; l) The interaction between GITR and ligands including GITRL.

A representative checkpoint for the purposes of the present invention is checkpoint (b) , namely the interaction between PD1 and either of its ligands PD-L1 and PD-L2. PD1 is expressed on effector T cells. Engagement with either ligand results in a signal which downregulates activation. The ligands are expressed by some tumors. PD-L1 in particular is expressed by many solid tumors, including melanoma. These tumors may therefore down regulate immune mediated anti-tumor effects through activation of the inhibitory PD1 receptors on T cells. By blocking the interaction between PD1 and one or both of its ligands, a checkpoint of the immune response may be removed, leading to augmented anti-tumor T cell responses. Therefore, PD1 and its ligands are examples of components of an immune system checkpoint which may be targeted in the method of the invention.

Another checkpoint for the purposes of the present invention is checkpoint (c) , namely the interaction between the T cell receptor CTLA-4 and its ligands, the B7 proteins (B7-1 and B7-2) . CTLA-4 is ordinarily upregulated on the T cell surface following initial activation, and ligand binding results in a signal which inhibits further/continued activation. CTLA-4 competes for binding to the B7 proteins with the receptor CD28, which is also expressed on the T cell surface but which upregulates activation. Thus, by blocking the CTLA-4 interaction with the B7 proteins, but not the CD28 interaction with the B7 proteins, one of the normal check points of the immune response may be removed, leading to augmented anti-tumor T cell responses. Therefore, CTLA-4 and its ligands are examples of components of an immune system checkpoint which may be targeted in the method of the invention.

An “immunomodulatory agent” as used herein includes any agent which, when administered to a subject, blocks or inhibits the action of an immune system checkpoint, resulting in the upregulation of an immune effector response in the subject, typically a T cell effector response, which may comprise an anti-tumor T cell effector response.

The immunomodulatory agent used in the method of the present invention may block or inhibit any of the immune system checkpoints described above. The agent may be an antibody or any other suitable agent which results in said blocking or inhibition. The agent may thus be referred to generally as an inhibitor of a said checkpoint.

An “antibody” as used herein includes whole antibodies and any antigen binding fragment (i.e., “antigen-binding portion” ) or single chains thereof. An antibody may be a polyclonal antibody or a monoclonal antibody, and may be produced by any suitable method. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include a Fab fragment, a F (ab’) ₂ fragment, a Fab’ fragment, a F _d fragment, a Fv fragment, a dAb fragment, and an isolated complementarity determining region (CDR) . Single chain antibodies such as scFv and heavy chain antibodies such as VHH and camel antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody.

In certain embodiments, antibodies which block or inhibit the CTLA-4 interaction with B7 proteins include ipilumumab, tremelimumab, or any of the antibodies disclosed in WO2014/207063. Other molecules include polypeptides, or soluble mutant CD86 polypeptides. In certain embodiments, the antibody is Ipilumumab.

In certain embodiments, antibodies which block or inhibit the PD1 interaction with PD-L1 include Nivolumab, Pembrolizumab, Lambrolizumab, Pidilzumab, BGB-A317 and AMP-224. In certain embodiments, the antibody is Nivolumab or pembrolizumab. Anti-PD-L1 antibodies include atezolizemab, avelumab or durvalumab, MEDI-4736 and MPDL3280A.

In certain embodiments, antibodies which block or inhibit the interaction between 4-1 BB and its ligand include utomilumab.

Other suitable inhibitors include small molecule inhibitors (SMI) , which are typically small organic molecules. In certain embodiments, inhibitors of ID01 include Epacadostat (INCB24360) , Indoximod, GDC-0919 (NLG919) and F001287. Other inhibitors of ID01 include 1-methyltryptophan (1-MT) .

As used herein, “an agent which directly stimulates an immune effector response” means any suitable agent, but typically refers to a cytokine or chemokine (or an agent which stimulates production of either) , a tumor specific adoptively transferred T cell population, or an antibody specific for a protein expressed by a tumor cell.

The cytokine may be an interferon selected from IFNα, ΙFΝβ, IFNγ and IFNA, or an interleukin, such as IL-2. The chemokine may be an inflammatory mediator, for example selected from CXCL9, 10, and 11, which attract T cells expressing CXCR3. The agent which stimulates production of a cytokine or chemokine may be an adjuvant suitable for administration to humans. One example is Bacille Calmette-Guerin (BCG) , which is typically administered intravesical (i.e. urethral catheter) for treatment of bladder cancer. A typical dosage regime of BCG for bladder cancer is once per week for six weeks, but given its long safety history it is also administered indefinitely as maintenance. BCG has been shown to stimulate immune responses to bladder cancer. BCG has also been used as an adjuvant in combination with compositions which comprise tumor antigens (i.e. with cancer vaccines) , particularly for colon cancer when it is administered typically intradermally. Such uses of BCG are also envisaged in the present invention. The tumor specific adoptively transferred T cell population directly increases the size of the tumor specific T cell population in an individual, and may be generated by any suitable means. However, typically the process involves isolating tumor specific T cells from a tumor sample taken from a patient, and selectively culturing those cells before returning the expanded population of tumor-specific T cells to the patient. Alternatively, a tumor specific T cell population may be produced by genetic engineering of the T cell receptor locus, followed by expansion of the altered cell.

Antibodies specific for proteins expressed by a tumor cell typically stimulate immune activity by binding to the tumor cell and promoting destruction of the cell via antibody-dependent cell-mediated cytotoxicity (ADCC) . Examples of antibodies of this type include anti-CD20 antibodies such as ofatumumab or rituximab, and anti-CD152 antibodies such as alemtuzumab.

Thus in certain exemplary embodiments, the compounds of the invention may be used in combination with a calcineurin inhibitor, e.g., cyclosporin A or FK 506; a mTOR inhibitor, e.g., rapamycin, 40-0- (2-hydroxyethyl) -rapamycin, biolimus-7 or biolimus-9; an ascomycin having immunosuppressive properties, e.g., ABT-281, ASM981; a corticosteroid; cyclophosphamide; azathioprene; methotrexate; leflunomide; mizoribine; mycophenolic acid or salt; mycophenolate mofetil; IL-1β inhibitor.

In another embodiment, compounds of the invention are combined with a co-agent which are PI3 Kinase inhibitors.

In another embodiment, compounds of the invention are combined with co-agent that influence BTK (Bruton's tyrosine kinase) .

For the treatment of oncological diseases, compounds of the invention may be used in combination with B-cell modulating agents, e.g., Rituximab, BTK or Syk inhibitors, inhibitors of PKC, PI3 kinases, PDK, PIM, JAK and mTOR and BH3 mimetics.

6. Compound Screening /Assay Methods

The compounds of the invention inhibit the paracaspase activity of MALT1, which enzymatic activity can be assayed directly using numerous biochemical assays, such as the GLOSENSOR ^TM assay described in Example 1. IC ₅₀ values of any compounds can be determined accordingly over a range of inhibitor concentrations.

In Example 1 (incorporated herein by its entirety, but only briefly described here to avoid redundancy) , MALT1 inhibitor activity can be assessed using a GLOSENSOR ^TM (Promega Corp., Madison, WI) split luciferase system. In this system, the firefly luciferase is genetically modified and split into two distinct domains, linked by a short MALT1 substrate peptide (such as a cleavage site sequence of a MALT1 substrate -RelB) , as described in Fontan et al. (J Clin Invest. 128 (10) : 4397-4412, 2018, incorporated herein by reference) . The luciferase is inactive in this configuration. Upon MALT1 activation, such as stimulating cells with a functional MALT1 using PMA/IO (ionomycin) , the MALT1 substrate peptide is cleaved, causing a conformation change that reestablishes a functional luciferase protein. When a suitable luciferase substrate, such as the Bright Glo substrate of Promega Corp., is provided, a luminescent signal can be detected. The presence of MALT1 inhibitor in this system reduced the luminescent signal, in a manner that is proportional to the concentration of the MALT1 inhibitor. Thus IC ₅₀ values of the inhibitor can be determined over a range of inhibitor concentrations. This assay can be done in any suitable cell line, such as the Raji lymphoma cells. The GLOSENSOR ^TM construct encoding the luciferase enzyme can be stably integrated into the genome of the cell line.

Example 2 (incorporated herein by its entirety, but only briefly described here to avoid redundancy) describes an alternative or additional assay to assess MALT1 protease inhibition by a subject compound. This assay is based on the ability of such inhibitors to antagonize NF-κB signaling through MALT1 activation that leads to IL-2 production (as a downstream event of MALT1 activation) . Briefly, a suitable cell line capable of producing IL-2 upon stimulation, such as the Jurkat cells (immortalized human T lymphocytes) , can be stimulated to produce IL-2 using the T Cell TRANSACT ^TM reagent (Miltenyi Biotec) , which has been developed as a ready-to-use reagent to activate and expand human T cells via CD3 and CD28 co-stimulation. IL-2 so produced can be collected from cell supernatant, and the amount of IL-2 can be determined using standard ELISA assay based on a standard curve generated using a series of known concentrations of IL-2. The presence of MALT1 inhibitor in this system reduced the amount of IL-2 produced, in a manner that is proportional to the concentration of the MALT1 inhibitor. Thus IC ₅₀ values of the inhibitor can be determined over a range of inhibitor concentrations.

One or both assays can be /were used to determine the IC ₅₀ values of the subject compounds.

Additional assays may also be used to assess the ability of any particular MALT1 inhibitor of the invention to inhibit the paracaspase activity of MALT1, or to screen for compounds possessing MALT1 inhibitory activity.

For example, in one assay, MALT1 proteolytic activity can be assayed using a fluorescent probe covalently attached to a short MALT1 substrate peptide. The probe is not fluorescent in this state. Upon cleavage of the substrate peptide by activated MALT1, the probe is released and becomes fluorescent, which can be measured using a fluorometer. The presence of the MALT1 inhibitor reduces such fluorescent signal over a range of inhibitor concentrations.

In particular, the C-domain of MALT1 (amino acids 329-824) can be used as a MALT1 surrogate for this inhibitor screening. The readout parameter can be the increase of fluorescence lifetime over time, proportional to enzyme activity. The assay employs a short peptide substrate for MALT1, labeled with the single fluorophore PT14 as a fluorescence lifetime probe sensitive to the cleavage state of the substrate (PT14: 6- (9-oxo-9H-acridin-10-yl) -hexanoate, AssayMetrics, UK) . The peptide substrate has the following sequence: Ac-Trp-Leu-Arg-Ser-Arg-Cys (PT14) -NH ₂ (Product number BS-91 17, Biosyntan, Germany) . Here, Ac stands for an acetyl group, Cys (PT14) is a cysteine residue with the fluorophore PT14 conjugated to the cysteine sulfhydryl group via a maieimide group; C-terminus of the peptide is amidated; the scissile bond is between Arg and the terminal Cys. The assay buffer consists of 200 mM Tris/HCI at pH 7.5, 0.8 M Na citrate, 100 μΜ EGTA, 100 μΜ DTT and 0.05 % (w/v) CHAPS. Kinetic characterization of the enzymatic reaction can be used to determine a Michaelis Constant (K _M) and a k _cat value. The assay can be run in 384-well plate format using black microtiter round well plates (Product number 95040020, Thermo Electron Oy, Finland) . Test compounds can be dissolved in 100% (v/v) DMSO or a mixture containing 90% (v/v) DMSO and 10% (v/v) water at a stock concentration of 100 mM. Serial dilutions of test compounds can be done using either 100% (v/v) DMSO or a mixture containing 90% (v/v) DMSO and 10% (v/v) water.

For measuring compound inhibition, 0.25 μL of test compound is mixed with 12.5 μL of enzyme in wells of the 384-well plates, and the mixture is incubated for 60 minutes at room temperature (22℃) . Then 12.5 μL of substrate is added, and the enzymatic reaction is allowed to proceed for 60 minutes at room temperature (22℃) . The total assay volume is thus 25.25 μL, and the final assay concentrations for enzyme and substrate are 2.5 nM and 1 μΜ, respectively. The increase in assay signal over time is expected to be linear for at least 60 minutes at the assay conditions, and directly proportional to the concentration of active enzyme up to at least 2.5 nM. The DMSO content is between 0.9 and 1 % (v/v) . The final assay concentrations of the test compounds ranged typically from 100 μΜ to 1 nM in a serial dilution series using a dilution factor of 3.16 (i.e., half-logarithmic dilution steps) . As controls, reactions are performed in multiple wells either by only adding DMSO instead of test compound, leading to an uninhibited enzymatic reaction (i.e., 0%inhibition) , or by adding assay buffer without enzyme mixed with DMSO, which is the equivalent of a fully inhibited reaction (i.e., 100%inhibition) . The fluorescence lifetimes are recorded using a microtiter plate reader such as the TECAN Ultra Evolution FLT instrument with fluorescence excitation at 405 nm and emission recording at 450 nm. The fluorescence lifetimes can be transformed to percentage inhibitions using the above mentioned controls as reference (for 0 and 100%inhibition) . The IC ₅₀ value is calculated from the plot of percentage inhibition versus inhibitor concentration using non-linear regression analysis software (Origin, OriginLab Corporation, USA) . The data are fitted using a 4 Parameter Logistic Model, characterized by the following equation:

y = A2 + (A1-A2) / (1+ (x /IC ₅₀) ^ p)

where y is the %-inhibition at the inhibitor concentration, A1 is the lowest inhibition value, and A2 is the maximum inhibition value. The exponent, p, is the Hill coefficient.

In another embodiment, MALT1 inhibition can also be assessed using a reporter gene (such as luciferase) under the control of a promoter responsive to NF-kB activation. For example, a construct encoding the cIAP2-MALT1 fusion (a constitutively active MALT1 protease) can be stably integrated into a host cell (such as HEK293) genome, and the activated NF-kB signaling can be read out using a luciferase under the control of a NF-kB response element in the promoter. The presence of MALT1 inhibitor will down-regulate MALT1-stimulated NF-kB signaling, as reflected by reduced luciferase reporter gene activity.

More specifically, this cIAP2-MALT1-driven NF-κB reporter gene assay (RGA) is conducted in a suitable cell such as HEK293. In this assay, the fusion protein cIAP2-MALT1 is driving constitutive NF-kB activation in the MALT-type of B cell lymphoma. To monitor the activity of MALT1 inhibitors on NF-kB signaling, a stably transfected HEK293 cell line is established, in which cell line the activated cIAP2-MALT1 fusion protein is constitutively expressed, and the firefly luciferase reporter gene is under the control of NF-kB response elements. In this system, inhibition of MALT1 protease activity reduces NF-kB signaling, which is reported as less luciferase activity. Inhibition of luciferase gene expression can be measured using a luciferase activity detection assay. Briefly, 1.8 × 10 ⁴ cells/90 μL/well are seeded in a sterile, white-walled, clear-bottom tissue-culture-treated 96-well mircoplates (Costar, Cat-No 3903) . After overnight incubation at 37℃, 10 μL of 10 × 3-fold serial compound dilutions prepared initially in DMSO, followed by a 1: 100 intermediated dilution in cell culture medium, is added to the cells using liquid handling robotics (Velocity Bravo 11, Agilent) . Unless otherwise mentioned, compound start concentration can be set at 10 μΜ, and the final vehicle concentration can be 0.1%DMSO in all wells. After 24-hour compound incubation, cell viability is assessed in a first step 3 hrs following addition of 10 μL 135 μgmL resazurin sodium salt (SIGMA Cat-Nr R7017) dissolved in phosphate-buffered saline. Following quantification of reduced resazurin at excitation/emission wavelengths of 540/590nm on a multipurpose microplate reader (e.g., Infinite M200Pro, TECAN) , cells are subjected to quantification of luciferase expression levels following incubation with 70 μL ONEGlow (Promega, Cat-No. E6120) homogenous assay buffer for 20 min at room temperature. Light emission is recorded on a multipurpose microplate reader (e.g., Infinite M200Pro, TECAN) in luminescence detection mode. Raw data are processed using an Excel analysis template. The effect of a particular test compound concentration on NF-kB activity is expressed as luciferase signal (Relative Light Units) normalized to cell viability by means of division with the reduced resazurin signal (Relative Fluorescence Units) . The value obtained for vehicle-treated cells is set as 100%. Absolute (50%reduction relative to vehicle control) and relative (inflection point) IC ₅₀ values (μΜ) are determined using 4-parametric curve-fitting (XLfit, V4.3.2) . In addition, %normalized NF-κB signal and %cell viability at the highest compound concentration are tested.

In another embodiment, MALT1 inhibition can also be assessed using a Human IL-2 promoter reporter gene assay (RGA) in Jurkat cells. Similar to the assay in Example 2, Jurkat cells can be stimulated by anti-CD28 mAb and PMA to produce IL-2. Unlike Example 2, the produced IL-2 is indirectly measured using a reporter gene (such as luciferase) under the control of an IL-2-responsive promoter. Thus the amount of IL-2 produced is proportional to the measured luciferase activity.

In this assay, a transfected Jurkat clone can be propagated in RPMI 1640 medium supplemented with 10 %heat inactivated fetal calf serum, 50 μΜ 2-mercaptoethanol and 1 mg/ml Geneticin. The cell concentration should not exceed 1 × 10 ⁶ /ml during culturing. The cells should not exceed passage 30. Prior to the assay the cells are washed and prepared to the concentration of 2 × 10 ⁶ cells/mL. Compound dilutions are made as 2×-concentrated solutions, then diluted 1/2 by addition to cells. About 250 μL of compound dilution and 250 μL of cells are mixed together in wells of a 96-deep well plate. Cells /compounds premix are incubated for 30 min. at 37℃ and 5%CO ₂ directly in the deep well plate. After pre-incubation of cells with compounds, cells are stimulated with anti-CD28 mAb at 3 g/mL +PMA at 1 μg/mL. Both co-stimulants are diluted in culture medium at a 10×-concentrated solution. 10 μL of co-stimulants are pipetted into the white 96-well plates and 100 μL of cell/compound mix is immediately added in duplicates. The cells are stimulated for 5.5 h at 37℃ and 5%CO ₂. After cell stimulation, 50 μL of BriteLifePlus reagent (Perkin Elmer) is added to each well and the bioluminescence is measured with a Wallac EnVision reader (Perkin Elmer) .

Any of the assays described above can be scaled up for large scale or high throughput screening. Using any of the assays described above, IC ₅₀ values of the subject compounds can be determined. In certain embodiments, the IC ₅₀ values of the subject compounds are determined using the methods described in Example 1 and/or Example 2.

7. Pharmaceutical Compositions

The invention provides pharmaceutical compositions which comprise any one of the compounds described herein, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the formulation and/or administration of an active agent to and/or absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the subject. Non-limiting examples of pharmaceutically acceptable carriers and excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer’s solution) , alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with or interfere with the activity of the compounds provided herein. One of ordinary skill in the art will recognize that other pharmaceutical carriers and excipients are suitable for use with disclosed compounds.

These compositions optionally further comprise one or more additional therapeutic agents. Alternatively, a compound of the invention may be administered to a patient in need thereof in combination with the administration of one or more other therapeutic regimens (e.g. Gleevec or other kinase inhibitors, interferon, bone marrow transplant, farnesyl transferase inhibitors, bisphosphonates, thalidomide, cancer vaccines, hormonal therapy, antibodies, radiation, etc) . For example, additional therapeutic agents for conjoint administration or inclusion in a pharmaceutical composition with a compound of this invention may be another one or more anticancer agents.

As described herein, the compositions of the present invention comprise a compound of the invention together with a pharmaceutically acceptable carrier, which, as used herein, includes any and all solvents, diluents, or other vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington’s Pharmaceutical Sciences, Fifteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1975) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the compounds of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component (s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

8. Formulations

This invention also encompasses a class of compositions comprising the active compounds of this invention in association with one or more pharmaceutically-acceptable carriers and/or diluents and/or adjuvants (collectively referred to herein as “carrier” materials) and, if desired, other active ingredients.

In certain embodiments, the invention provides a pharmaceutical formulation for treating cancer, in particular the cancers described herein, comprising a compound of the present invention or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier.

In certain embodiments, the invention provides a pharmaceutical formulation for treating a cancer selected from the group consisting of those described herein in treatable disease section, comprising a compound of the present invention or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier.

The active compounds of the present invention may be administered by any suitable route, preferably in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the treatment intended. The compounds and compositions of the present invention may, for example, be administered orally, mucosally, topically, rectally, pulmonarily such as by inhalation spray, or parentally including intravascularly, intravenously, intraperitoneally, subcutaneously, intramuscularly, intrasternally and infusion techniques, in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles.

The pharmaceutically active compounds of this invention can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, including humans and other mammals.

For oral administration, the pharmaceutical composition may be in the form of, for example, a tablet, capsule, suspension or liquid. The pharmaceutical composition is preferably made in the form of a dosage unit containing a particular amount of the active ingredient.

Examples of such dosage units are tablets or capsules. For example, a suitable daily dose for a human or other mammal may vary depending on the condition of the patient and other factors, but, once again, can be determined using routine methods.

The amount of compounds which are administered and the dosage regimen for treating a disease condition with the compounds and/or compositions of this invention depends on a variety of factors, including the age, weight, sex and medical condition of the subject, the type of disease, the severity of the disease, the route and frequency of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods. As mentioned previously, the daily dose can be given in one administration or may be divided between 2, 3, 4 or more administrations.

For therapeutic purposes, the active compounds of this invention are ordinarily combined with one or more adjuvants, excipients or carriers appropriate to the indicated route of administration. If administered per os, the compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted or encapsulated for convenient administration. Such capsules or tablets may contain a controlled-release formulation as may be provided in a dispersion of active compound in hydroxypropylmethyl cellulose.

In the case of skin conditions, it may be preferable to apply a topical preparation of compounds of this invention to the affected area two to four times a day. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable for administration to the eye, ear, or nose. For topical administration, the active ingredient may comprise from 0.001%to 10%w/w, e.g., from 1%to 2%by weight of the formulation, although it may comprise as much as 10%w/w, but preferably not more than 5%w/w, and more preferably from 0.1%to 1%of the formulation.

The compounds of this invention can also be administered by a transdermal device. Preferably transdermal administration will be accomplished using a patch either of the reservoir and porous membrane type or of a solid matrix variety. In either case, the active agent is delivered -continuously from the reservoir or microcapsules through a membrane into the active agent permeable adhesive, which is in contact with the skin or mucosa of the recipient. If the active agent is absorbed through the skin, a controlled and predetermined flow of the active agent is administered to the recipient. In the case of microcapsules, the encapsulating agent may also function as the membrane. The oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner.

While the phase may comprise merely an emulsifier, it may comprise a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier (s) with or without stabilizer (s) make-up the so-called emulsifying wax, and the wax together with the oil and fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations. Emulsifiers and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate, sodium lauryl sulfate, glyceryl distearate alone or with a wax, or other materials well known in the art.

The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is very low. Thus, the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono-or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters may be used. These may be used alone or in combination depending on the properties required.

Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.

Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredients are dissolved or suspended in suitable carrier, especially an aqueous solvent for the active ingredients.

The active ingredients are preferably present in such formulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10%and particularly about 1.5%w/w.

Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions may be prepared from sterile powders or granules using one or more of the carriers or diluents mentioned for use in the formulations for oral administration or by using other suitable dispersing or wetting agents and suspending agents. The compounds may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, tragacanth gum, and/or various buffers. Other adjuvants and modes of administration are well and widely known in the pharmaceutical art. The active ingredient may also be administered by injection as a composition with suitable carriers including saline, dextrose, or water, or with cyclodextrin (i.e. Captisol) , cosolvent solubilization (i.e. propylene glycol) or micellar solubilization (i.e. Tween 80) .

The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed, including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

For pulmonary administration, the pharmaceutical composition may be administered in the form of an aerosol or with an inhaler including dry powder aerosol.

Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable nonirritating excipient such as cocoa butter and polyethylene glycols that are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.

The pharmaceutical compositions may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc. Tablets and pills can additionally be prepared with enteric coatings. Such compositions may also comprise adjuvants, such as wetting, sweetening, flavoring, and perfuming agents. Pharmaceutical compositions of this invention comprise a compound of the formulas described herein or a pharmaceutically acceptable salt thereof; an additional agent selected from a kinase inhibitory agent (small molecule, polypeptide, antibody, etc. ) , an immunosuppressant, an anticancer agent, an anti-viral agent, antiinflammatory agent, antifungal agent, antibiotic, or an anti-vascular hyperproliferation compound; and any pharmaceutically acceptable carrier, adjuvant or vehicle.

Alternate compositions of this invention comprise a compound of the formulae described herein or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier, adjuvant or vehicle. Such compositions may optionally comprise one or more additional therapeutic agents, including, for example, kinase inhibitory agents (small molecule, polypeptide, antibody, etc. ) , immunosuppressants, anti-cancer agents, anti-viral agents, antiinflammatory agents, antifungal agents, antibiotics, or anti-vascular hyperproliferation compounds.

The term “pharmaceutically acceptable carrier or adjuvant” refers to a carrier or adjuvant that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound. Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, selfemulsifying drug delivery systems (SEDDS) such as d-atocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Cyclodextrins such as u-, P-, and y-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2 and 3-hydroxypropyl- cyclodextrins, or other solubilized derivatives may also be advantageously used to enhance delivery of compounds of the formulae described herein.

The pharmaceutical compositions may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions and/or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying and/or suspending agents.

If desired, certain sweetening, flavoring and/or coloring agents may be added. The pharmaceutical compositions may comprise formulations utilizing liposome or microencapsulation techniques, various examples of which are known in the art.

The pharmaceutical compositions may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents, examples of which are also well known in the art.

9. Treatment Kits

One aspect of the present invention relates to a kit for conveniently and effectively carrying out the methods or uses in accordance with the present invention. In general, the pharmaceutical pack or kit comprises one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Such kits are especially suited for the delivery of solid oral forms such as tablets or capsules. Such a kit preferably includes a number of unit dosages, and may also include a card having the dosages oriented in the order of their intended use. If desired, a memory aid can be provided, for example in the form of numbers, letters, or other markings or with a calendar insert, designating the days in the treatment schedule in which the dosages can be administered. Optionally associated with such container (s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

The following representative examples contain important additional information, exemplification and guidance which can be adapted to the practice of this invention in its various embodiments and the equivalents thereof. These examples are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit its scope. Indeed, various modifications of the invention, and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art upon review of this document, including the examples which follow and the references to the scientific and patent literature cited herein.

The contents of the cited references are incorporated herein by reference to help illustrate the state of the art.

In addition, for purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 ^th Ed., inside cover. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in “Organic Chemistry, ” Thomas Sorrell, University Science Books, Sausalito: 1999, and “Organic Chemistry, ” Morrison &Boyd (3d Ed) , the entire contents of both of which are incorporated herein by reference.

10. Synthesis Schemes

The compounds of Formula I can be prepared by one of ordinary skill in the art following art recognized techniques and procedures. More specifically, compounds of Formula I can be prepared as set forth in the schemes, methods, and examples set forth below. It will be recognized by one of skill in the art that the individual steps in the following schemes may be varied to provide the compounds of Formula I. The reagents and starting materials are readily available to one of ordinary skill in the art. All substituents, unless otherwise specified, are as previously defined.

EXAMPLES

The followings are the abbreviations used and meaning thereof in the specification:

EtOAc/EA: Ethyl acetate

DCM: Dichloromethane

ACN: Acetonitrile

THF: Tetrahydrofuran

DMSO: Dimethyl sulfoxide

MeOH: Methanol

EtOH: Ethanol

DMF: N, N-Dimethylformamide

DMA: N, N-Dimethylacetamide

DMF DMA: N, N-Dimethylformamide dimethylacetal

NCS: N-Chlorosuccinimide

NBS: N-Bromosuccinimide

NIS: N-Iodosuccinimide

Pd-C : Palladium on Carbon

LDA: Lithium diisopropylamide

TFA: Trifluoroacetic acid

PTSA: p-Toluene sulfonic acid

DIBAL-H: Diisobutylaluminum hydride

LAH : Lithium aluminum hydride

PE: Petroleum Ether

Py: Pyridine

DPPA : Diphenylphosphoryl azide

CDI : 1, 1-Carbonyl diimidazole

TEA: Triethylamine

DIPEA: N, N-Diisopropylethylamine

DMAP: 4- (Dimethylamino) pyridine

EDCI: N- (3-Dimethylaminopropyl) -N-ethyl carbodiimide hydrochloride

HOBT: 1-Hydroxybenzo triazole

TfOH: Trifluoromethane sulfonic acid

dppf: 1, 1 Ferrocenediyl-bis (diphenylphosphine)

DAST: (Diethylamino) sulfur trifluoride

Pd ₂ (dba) ₃: Tris (dibenzylideneacetone) dipalladium (0)

Boc: tert-Butoxycarbonyl

Ac: Acetyl

TMSI : Trimethyl silyl iodi de

TBAI: Tetrabutylammonium iodide

PPh ₃: Triphenyl phosphine

dba: Dibenzylideneacetone

BINAP: 2, 2'-Bis (diphenyl phosphino) -1, 1'-bi naphthyl

MsCl: Methane sulfonyl chloride

TsCl: Toluene sulfonyl chloride

DMAP: 4-Dimethyl aminopyridine

LiHMDS: Lithium bis (trimethyl silyl) amide

NaHMDS: Sodium bis (trimethyl silyl) amide

DMS: dimethyl sulfide

DAST: Diethylamino sulfur trifluoride

DME: dimethoxyethane

DCE: Dichloroethane

NMR: Nuclear Magnetic Resonance

LC-MS: Liquid Chromatography Mass Spectrometry

ESI-MS: Electro Spray Ionization Mass Spectrometry:

GCMS: Gas Chromatography Mass Spectrometry

TLC: Thin Layer Chromatography

TCR: T cell receptor

BCR: B cell receptor

CARD: Caspase activation and recruitment domain

mM: millimolar

μM: micromolar

mL: microliter

ng: nanogram

nM: nanomolar

nm: nanometer

IC ₅₀: Half maximal inhibitory concentration

OD: Optical density

A. Biological Examples

Example 1. Compound Screening on Raji/GLOSENSOR ^TM Stable Cell Line

This example provides an assay that enables high-throughput screening /assessment of MALT1 inhibitor activity in living cells. This assay can be used, for example, to identify compounds with potent in vivo activity against MALT1, to identify MALT1 inhibitors that are more potent than other MALT1 inhibitors, to identify MALT1 inhibitors that are partial (vs. full) inhibitors that can inhibit a maximum of about 70-80% (but not 100%) of MALT1 proteolytic activity, and to study structure-function relationship among structurally related MALT1 inhibitors, etc.

The basic principle of the assay described herein is based on Fontan et al. (J Clin Invest. 128 (10) : 4397-4412, 2018, incorporated herein by reference) , which describes a robust and sensitive method for the detection of intracellular MALT1 paracaspase activity, and the effect of any potential MALT1 inhibitors on MALT1 activity. The method is based on the GLOSENSOR ^TM (Promega Corp., Madison, WI) split luciferase system that utilizes a bioluminescent chimeric protein composed of a genetically modified form of firefly luciferase split into 2 distinct domains, by insertion of a cAMP-binding protein moiety. Binding of cAMP induces a conformational change that reestablishes a functional luciferase protein resulting in luminescence. Instead of inserting a cAMP-binding protein moiety, Fontan engineered a reporter so that this conformational change was caused by MALT1-induced proteolytic cleavage, and hence luciferase activity would be a surrogate of endogenous MALT1 protease activity. More specifically, Fontan used the cleavage site sequence of RelB -a natural MALT1 substrate -in their GLOSENSOR ^TM construct, such that the encoded luciferase would be specifically activated by MALT1. The GLOSENSOR ^TM construct was tested in the Raji lymphoma cells, which are easily transducible and suitable for such assays, since they activate MALT1 upon stimulation with PMA and ionomycin (IO) .

Thus in the assay described herein below, a required number of Raji cells (e.g., 5 ×10 ⁵ cell/mL) were grown in a T75 flask overnight in the absence of antibiotics, and the cells were allowed to reach 70-90%confluence the following day. Cells of the optimal density were harvested the following day, and were counted to verify. After washing the harvested cells in pre-warmed PBS (without Ca ²⁺ and Mg ²⁺) , the Raji cells were resuspended in the appropriate Resuspension Buffer (included with Neon ^TM Kits, Thermo fisher, #MPK1025) at a final density of about 1 × 10 ⁷ cells/mL.

An appropriate volume of GLOSENSOR ^TM plasmid (0.5 μg per well for Raji cells) was then added into the cell suspension. For the EGFP control group, 0.5 μg of the EGFP plasmid was added to the cell suspension separately.

Twenty-four different electroporation conditions were tested, by varying pulse voltage, pulse width, and pulse number, and Condition 16 in the table below (Pulse Voltage: 1400 V; Pulse width: 20 msec.; pulse number: 2) was chosen for electroporation. An equal amount of EGFP plasmids were also electroporated into Raji cells to evaluate transfection efficiency.

Two days after electroporation, Raji cells electroporated with the EGFP construct were harvested, and expression of EGFP was determined by FACS analysis. Electroporation efficiency was determined by gating FITC ⁺ ratio.

Also two days after electroporation, Raji cells electroporated with (or without) the GLOSENSOR ^TM construct were harvested and then seeded to 96-well plates at a density of about 10,000 cells per well. Cells were stimulated with PMA (200 ng/mL) /Ionomycin (1 μM) for 2 hours at 37℃ to activate MALT1 protease activity. BRIGHT GLO ^TM (Promega Corp., Madison, WI) substrate was then added to each well to detect luciferase signal. Raji cells successfully electroporated by the GLOSENSOR ^TM construct and expressed MALT1, upon PMA/IO stimulation of MALT1 activity, exhibited strong luciferase signal after MALT1 cleaved the GLOSENSOR ^TM fusion protein to reconstitute luciferase activity.

To establish Raji cells with the GLOSENSOR ^TM construct stably integrated into the genome, electroporated Raji cells were harvested 2 days post electroporation, and then re-plated in 24-well plates with G418 media to select for cells with stably integrated GLOSENSOR ^TM construct (which expresses neomycin resistant gene) . Cell culture medium was changed every three days by spinning cells down at 1,500 rpm for 5 min each time. After 13-14 days, any surviving cells (presumably with stably integrated GLOSENSOR ^TM construct) were pooled and frozen down before assessing luciferase activity using Bright-Glo substrate.

Single cell cloning of any Raji cells with stably integrated GLOSENSOR ^TM construct were obtained by first diluting the pool of stable Raji cells to a density of about 1 cell per 100 μL, before about 100 μL of cell suspension (containing on average 1 single cell) was pipetted into each 96-well plate wells. The cells were allowed to grow for 3-4 weeks to grow into single cell clones.

Each single cell clone can be tested for MALT1 activity after PMA/IO stimulation. Specifically, cells can be plated at a density of about 20,000 cells /well in 96-well plate wells, before stimulation by adding 200 ng/mL PMA plus 1 μM Ionomycin. As control, equal volume of media without PMA and IO was added to control wells. Further, unelectroporated Raji cells were used as vehicle control, which generated negative signal under both stimulation and un-stimulation conditions. Cell clones with assay window bigger than 7 were chosen as positive clones.

Such positive Raji stable cell clones were further tested by IC ₅₀ determination using a reference compound of the invention. Specifically, about 20,000 cells were plated into each well of a 96-well plate in 40 μL. Meanwhile, a reference compound (RGT005-001, which is disclosed as Example 10 in WO2015/181747) in 200 μM starting concentration were serially diluted 3-fold to 10-point doses before each dose went into contact with the plated cells. After 30 min incubation at 37℃, cells were contacted with PMA/IO (or equal volume of media control) to stimulate MALT1 activity for another 60 min, before Bright Glo luciferase substrate was added to measure liciferase activity. Inhibition of MALT1 activity in the presence of the reference compound can be determined as the inhibition rate.

Any other test compounds can be similarly serially diluted, such that Raji stable cell lines with integrated GLOSENSOR ^TM constructs can come into contact with different concentrations of potential MALT1 inhibitors to assess IC ₅₀ values for each test compound, preferably in high throughput format. MALT1 with high inhibitory activity can be identified using this assay.

Conditions and equipments for a representative assay experiment are listed below.

Materials and Media

1. Reagents &consumables

*RT = room temperature

2. Equipments

3. Media and solutions

Complete medium for Raji

Raji: RPM1640: 90%; FBS: 10%; Penicillin/Streptomycin: 1%

Complete medium for Raji/GLOSENSOR ^TM

Raji: RPM1640: 90%; FBS: 10%; Penicillin/Streptomycin: 1%; G418: 1,500 μg/mL

Freezing medium

FBS: 90%; DMSO: 10%

Procedures for Stable Cell Line Generation

1. Transfection /Electroporation

Day 1: Preparing Cells

The required number of cells (5 × 10 ⁵ cell/mL in T75 flask) were seeded so that the cell confluence reached 70–90%the following day. No antibiotics were included in the culture medium.

Day 2: Electroporation using Neon ^TM Transfection System (Thermo fisher#MPK5000)

a. Harvest cell suspension. Count cells by mixing 20 μL of cell suspension with 20 μL of trypan blue using Countstar machine. Centrifuge cells at 1,000 rpm for 5 min.

b. Remove the supernatant and resuspend the cell pellet in pre-warmed PBS (without Ca ²⁺ and Mg ²⁺) . Centrifuge cells at 1,000 rpm for 5 min.

c. Resuspend the cell pellet in the appropriate Resuspension Buffer (included with Neon ^TM Kits, ThermoFisher, #MPK1025) at a final density of 1 × 10 ⁷ cells/mL for Raji.

d. Add appropriate volume of GLOSENSOR ^TM plasmid into above cell suspension (0.5 μg per well for Raji cells) . For the EGFP group, add 0.5 μg of EGFP plasmid to the cell suspension sepeartely.

e. Before plasmid transfection, pipet culture medium without antibiotics to 24-well plate, 500 μL/well. Pre-warm the plate at 37℃ for a while.

f. Electroporate Raji with GLOSENSOR ^TM plasmid using Condition 16 (Pulse Voltage: 1400 V; Pulse width: 20 msec.; pulse number: 2) . Equal amount of EGFP was also electroporated into Raji cells to evaluate transfection efficiency. Note: A total of 24 transfection conditions (see below) were tested, and Condition 16 was chosen as the optimal condition) .

Sample	Pulse voltage	Pulse width	Pulse no.
1	0	0	0
2	1400	20	1
3	1500	20	1
4	1600	20	1
5	1700	20	1
6	1100	30	1
7	1200	30	1
8	1300	30	1
9	1400	30	1
10	1000	40	1
11	1100	40	1
12	1200	40	1
13	1100	20	2
14	1200	20	2
15	1300	20	2
16	1400	20	2
17	850	30	2
18	950	30	2
19	1050	30	2
20	1150	30	2
21	1300	10	3
22	1400	10	3
23	1500	10	3
24	1600	10	3

Note: pulse voltage in volts, and pulse width in msec.

Day 4: Evaluation of transfection /electrpoporation efficiency

Harvest cells in EGFP electroporated well, run FACS. Evaluating transfection efficiency by gating FITC ⁺ ratio.

Day 4: Evaluation of transient transfection signal

Harvest cells with or without GLOSENSOR ^TM transfection. Seed cells to 96-well plate, 10,000 cells per well. Cells were stimulated with PMA (200 ng/mL) /Ionomycin (1 μM) for 2 hours at 37 degree. Bright Glo was then added to detect the luciferase signal.

Day 4-Day 17: Antibiotic treatment

a. On day 4, transfer cells in 24-well plate to T25 flask. Add 1,500 μg/mL G418 in order to get stable clones of Raji/GLOSENSOR ^TM.

b. Cell culture medium was changed every three days by spinning down at 1,500 rpm for 5 min.

c. On Day 17, harvest the pool stable cells. Freeze them and evaluate the pool’s signal using Bright-Glo.

2. Preparation of single cell clone

a. Pooled stably transfected cells were diluted to 1 cell /100 μL.

b. Pipet 100 μL of cell suspension to 10 of 96-well plates.

c. Let cells grow for 3-4 weeks. Mark the clones that grew up from one single cell.

3. Primary identification of positive clones in Raji/GLOSENSOR ^TM using Bright-Glo

a. Observe cell status of each stable clone under microscope. Gave a rough evaluation of cell density for each one.

b. Plate cells to 96-well plate (Corning #3610) , 90 μL/well, about 2 × 10 ⁴ cells/well.

c. For stimulated groups, transfer 10 μL of 200 ng/mL PMA plus 1 μM Ionomycin to the 96-well plate.

For unstimulated groups, transfer 10 μL of medium to each well of the 96-well plate. Take unelectroporated Raji cells as vehicle control, which is supposed to generate negative signal under stimulation and un-stimulation.

d. Incubate the plate at 37℃, 5%CO ₂ incubator for 2 hrs.

e. Add 100 μL/well of Bright-Glo to the plate.

f. Agitate the plates on plate shaker (～300 rpm) for 5 min at room temperature.

g. Read luminescence signal in Envision plate reader (96-well USL) .

h. Process the data. Check the assay window of each clone between stimulated and un-stimulated group. Chose the clones with assay window bigger than 7 as the positive ones.

4. Validation of Positive Clones by IC ₅₀ Determination of the Reference Compound RGT005-001

a. Seed cells to 96-well plate (Corning #3610) , 40 μL/well, 20,000 cells/well.

b. Compound preparation:

i) Dilute RGT005-001 (stock 10 mM in DMSO) into 200 μM using DMSO (add 1 μL stock into 49 μL DMSO to obtain 200 μM) .

ii) 200 μM RGT005-001 was taken as the top dose. Then dilute it in 3-fold (mix 10 μL top dose solution with 20 μL DMSO) to 10-point doses.

iii) Dilute compound serially using pre-warmed medium (2 μL compound solution mix by 198 μL medium) to obtain 2× compound.

iv) Treat cells with 40 μL of compound solution in 96-well plate.

v) Shake it at 300 rpm for 2 min. Incubate assay plates at 37 degree, 5%CO ₂ for 30 min.

vi) For High/Low controls, add 40 μL medium solution (containing 1%DMSO) into plate.

c. 30 min later, transfer 20 μL of 5× PMA/Ionomycin to the 96-well plate.

d. For High control, add 20 μL PMA/Ionomycin into plate. For Low control, add 20 μL medium into plate.

e. Incubate the plate at 37℃, 5%CO ₂ incubator for 1 hr.

f. Add 100 μL/well of Bright-Glo to the assay plate.

g. Agitate the plates on plate shaker (～300 rpm) for 5 min at room temperature.

h. Read the luminescence signal at Envision plate reader (96-well USL) .

i. Process data to obtain inhibition rate by using the equation: (Reading of High control well-Reading of Compound treated well) *100%/Reading of High control well-Reading of Low control well) . Fit the inhibition rate against different concentrations of compound. Generate IC ₅₀ using non-linear analysis in GraphPad prism.

5. Mycoplasma detection

Mycoplasma detection was performed using MYCOALERT ^TM PLUS Mycoplasma Detection Kit (ordering information: Lonza, LT07-710) , and the assay strictly followed the manual supplied by the vendor.

Procedures for Compound Screening on Raji/GLOSENSOR ^TM Cell Line

Seeding cells on 96-well plate:

Seeding cells to 96-well plate (Corning #3610) , total 2 × 10 ⁴ cells/well (40 μL/well) .

Note: Cell number and cell viability are recorded every time.

Compound serial dilution

For reference compound RGT005-001:

a. Dilute RGT005-001 (stock 10 mM in DMSO) into 200 μM using DMSO (add 1 μL stock into 49 μL DMSO to obtain 200 μM) .

b. Then serially dilute it in 3-fold using DMSO (mix 10 μL top dose solution with 20 μL DMSO) to 10-point doses.

c. Mix compound solution (100×) with pre-warmed medium to obtain 2× compound solution (2 μL compound solution mixed by 198 μL medium) . Final DMSO concentration is 1% (2×) .

For test compounds:

a. Dilute test compounds (stock 10 mM in DMSO) into 2 mM using DMSO (add 3 μL stock into 12 μL DMSO to obtain 2 mM) .

b. Then 3-fold serially dilute 2 mM of test compounds in duplicate using DMSO (mix 5 μL top dose solution with 10 μL DMSO) to 10-point doses.

c. Mix compound solution (100×) using pre-warmed medium to obtain 2× compound solution (2 μL compound solution mix by 198 μL medium) . Final DMSO concentration is 1% (2×) .

Raji/GLOSENSOR ^TM cell pretreatment with compounds

a. Treat Raji/GLOSENSOR ^TM with different concentrations of compounds as the platemap shown below, 40 μL/well.

b. Shake the plate at 300 rpm for 2 min. Incubate assay plates at 37℃, 5%CO ₂ for 30 min.

c. For High/Low control, add 40 μL medium solution (containing 1%DMSO) into plate.

PMA/Ionomycin stimulation for 1hr

a. Prepare 1 μg/mL PMA plus 5 μM Ionomycin mixture using pre-warmed medium (8 μL PMA plus 8 μL Ionomycin in 7984 μL medium) .

b. After 30-min treatment of compounds, transfer 20 μL of PMA/Ionomycin mixture into the 96-well plate (expect positive control) .

c. For Low control, add 20 μL medium into wells.

d. Incubate the plate at 37℃, 5%CO ₂ incubator for 1 hr.

Luciferase signal detection and data analysis

a. Add 100 μL/well of Bright-Glo to the assay plate.

b. Agitate the plates on plate shaker (～300 rpm) for 5min at room temperature.

c. Read the luminescence signal in Envision plate reader (96-well USL) .

d. Process data to inhibition rate using the equation: (Reading of High control well-Reading of Compound treated well) × 100% (Reading of High control well-Reading of Low control well. Fit the inhibition rate against different concentrations of compound. Generate IC ₅₀ using non-linear analysis in GraphPad prism.

Glosensor dose-response plots for exemplary compounds of the invention are presented in FIG. 1.

Example 2. Compound Efficacy Study Regarding IL-2 Production in Jurkat Cells

This example demonstrates that the subject compounds affects IL-2 production in Jurkat cells. Meanwhile, the ability of a test compound to affect IL-2 production in the same system can be tested using ELISA.

Jurkat cells are chosen for this assay since they are immortalized human T lymphocytes capable of producing interleukin 2 (IL-2) . Jurkat cells can be stimulated to produce IL-2 using the T Cell TRANSACT ^TM reagent (Miltenyi Biotec) , which has been developed as a ready-to-use reagent to activate and expand human T cells via CD3 and CD28. Its polymeric nanomatrix structure consists of a colloidal polymeric nanomatrix conjugated to humanized CD3 and CD28 agonists, and ensures gentle and efficient activation of T cells while maintaining high viability.

Jurkat cells stimulated to produce IL-2 can be centrifuged to collect IL-2-containing supernatant, and the amount of IL-2 produced can be measured using standard ELISA assay using anti-IL-2 antibodies. The presence of MALT1 inhibitors reduced the amount of IL-2 production by Jurkat cells, and the IC ₅₀ value for each MALT1 inhibitor can be quantitatively measured using this assay.

An exemplary assay protocol is provided herein below.

Materials and Equipment:

1. Reagents:

2. Equipments &Supplies:

3. Procedures

Procedures for Jurkat cell thawing and seeding:

Cell Growth Medium: RPMI1640 (ATCC, Cat #30-2001) ; 10%FBS (Gibco, Cat #10091148) ; 1%PenStrep (Gibco, Cat #15140) ; and Beta-ME (Gibco, Cat#21985023) .

1. Sub-culturing

This cell line is normally split twice weekly at a 1: 3 ratio dilutions.

1) Cell cultures were maintained either by addition of fresh medium /replacement of medium, or by centrifugation followed by subsequent resuspension at 3-4 ×10 ⁵ cells/mL.

2) Cells were subcultured when cell concentration /density reached 1×10 ⁶ cells/mL. Cell concentration was controlled to not exceed 3 × 10 ⁶ cells/mL.

3) Medium was renewed once every 2 to 3 days.

2. Refresh the medium before the assay day

3. Seeding cells in 96-well plate (Day 1)

1) Cells were harvest by centrifugation and by discarding the cell growth medium. Cell number was counted, and the total number of cells needed for the assay was calculated.

2) Cell suspension was diluted with an appropriate volume of the Cell Culture Media.

3) Cell suspension was then dispensed into a sterile, disposable reservoir.

4) Then 1 × 10 ⁵ cells/well (50 μL/well) of the cell suspension were transferred into 96-well plate (cell plate: corning-3788) .

5) The plates were incubated in a humidified air, 5%CO ₂ incubator at 37℃ for 1 hour.

Procedures for compound and Transact preparation (Day 1) :

1. Compound and DMSO preparation and addition:

Compound serial dilution (source plate)

1) Compounds were solubilized in 100%DMSO to a concentration of 10 mM, and then diluted to 1 mM with DMSO: 5 μL of 10 mM compound + 45 μL DMSO =50 μL 1 mM compounds into Row A.

2) About 40 μL of DMSO was added into B1-H10 wells. Then pipettor was used for serial dilution of the compounds, by transferring 20 μL compounds into 40 μL DMSO (8 points, 3-fold, B2-H9) . By this means, a series of compound concentrations diluted in DMSO were obtained.

2× Compound dose preparation (inter plate: Corning-3599) :

1) 95 μL of RPMI 1640 medium (RPMI1640 + 10%FBS + 1%PS + 0.055 mM 2-ME) was added with Pipettor into 96-well inter plate 1, then 5 μL of compounds was transferred from the source plate into this Inter plate1 (A2-H9) and mixed;

2) 5 μL of DMSO from the source plate was transferred into this Inter plate1 (A1-H1, A10-H10) by Pipettor and mixed well.

3) 240 μL of RPMI 1640 medium (RPMI1640 + 10%FBS + 1%PS + 0.055mM 2-ME) was added with Pipettor into 96-well inter plate 2 (A1-H10) , then 10 μL of compounds or DMSO were transferred from inter plate1 into this Inter plate2 (A1-H10) by Pipettor and mixed well to obtain the final 2× compounds concentration.

4) 50 μL/well of working concentration of DMSO and compound solution were added to cell plate (Corning-3788) . The final conc of compound was 1 μM, and the final DMSO concentration in cell culture was 0.1%. The plate was then incubated at 37℃ in a 5%CO ₂ incubator for 0.5 hour.

2. TRANSACT ^TM reagent: stored at 4℃

1) The TRANSACT ^TM reagent working solutions were diluted freshly from stock solutions with RPMI 1640 medium (RPMI1640 + 10%FBS + 1%PS + 0.055 mM Beta-ME) each time before use. Samples were without dilution.

Stimuli	Stock	1	Working	Final Conc.	Dilution Fold	Vol. (μL)
Transact	100%	2%	1.00%	50	360
Medium					17,640
Total					18,000

2) Add 100 μL/well of 2× Transact to each well.

3) Incubate at 37℃ in a 5%CO ₂ incubator for 24 hours.

Procedures for supernatant collection

Samples collection (Day 2)

1) After incubation in a 37℃, 5%CO ₂ incubator for 24 hours, centrifuging the cell plates at 1,000 rpm for 10 min. Collect 100 μL/well supernatant, then perform IL-2 ELISA assay. The supernatant can be stored at -80℃ and IL-2 ELISA assay can be performed the following day. Supernatant were diluted 3-fold if necessary to ensure that the assay did not exceed the linear range of IL-2 standard curve.

Procedures for IL-2 ELISA (Day 2) :

Day 1 for coating plates:

1) Micro-wells were coated with 100 μL per well of Capture Antibody diluted in Coating Buffer. Lot-specific instruction/analysis certificate was used as a guide for recommended antibody coating dilution. Plates were sealed, and incubated overnight at 4℃.

Day 2 for IL-2 ELISA:

1) Wells were aspirated and washed 3 times with ≥ 300 μL/well of Wash Buffer. After last wash, the plate was inverted and blotted on absorbent paper to remove any residual buffer.

2) Plates were blocked with ≥ 200 μL/well Assay Diluent, and then incubated at RT for 1 hour.

3) Aspiration/washing were repeated as in step 2.

4) Standard and sample dilutions were prepared in Assay Diluent.

IL-2 standard stock preparation: 1 mL of deionized water was added to vial (235 ng/vial) to reach a stock conc. of 235 ng/mL. Standard stock was aliquoted at 50 μL/vial, and was frozen at -80℃.

The STD curve was diluted from the stock on the day of assay with 500 pg/ml top, 2-fold, 8 doses (including 0 pg/ml) . After dilution, 100 μL/well STD was transferred to ELISA plate (column 12) .

	Stock 1 (pg/ml)	Stock 2 (pg/ml)	final (pg/ml)	fold	Add
IL-2 (μL)	235,000	23,500 (10+90)	500	47	8
Diluent (μL)					368
total (μL)					376

5) 100 μL of each standard, sample, and control was Pipetted into appropriate wells. Plate was sealed and incubated for 2 hours at RT.

6) Aspiration /washing were repeated as in step 2, but with 5 total washes.

7) 100 μL of Working Detector (Detection Antibody + SAv-HRP reagent) was added to each well. Plate was sealed and incubated for 1 hour at RT.

8) Aspiration /washing were repeated as in step 2, but with 7 total washes.

NOTE: In this final wash step, wells were soaked in wash buffer for 30 seconds to 1 minute for each wash.

9) 100 μL of Substrate Solution was added to each well. The plate (without plate sealer) was incubated for 30 minutes at room temperature in the dark.

10) 50 μL of Stop Solution was added to each well.

11) Absorbance was read at 450 nm within 30 minutes of stopping reaction. If wavelength correction is available, absorbance at 570 nm was subtracted from absorbance at 450 nm.

Data Analysis:

The percent (%) inhibition at each compound concentration was calculated based on and relative to the signal in the HPE and ZPE wells contained within each assay plate. The HPE wells were used as 0%inhibition, and the ZPE wells that did not contain any compound but rather DMSO (final concentration = 0.1%) were used as 100%inhibition. The concentrations and %inhibition values for tested compounds were plotted, and the concentration of compound required for 50%inhibition (IC ₅₀) was determined with a Three-parameter logistic dose response equation. The endpoint value (IC ₅₀) for the reference peptide/compound was evaluated in each experiment as a quality control measure. If the endpoint value was within 3-fold of the expected value, the experiment was deemed acceptable.

The cellular data obtained from exemplary compounds are listed in the Table 1 below. The IC ₅₀ values are indicated as "++++" , for values less than or equal to 100 nM; "+++" , for values less than or equal to 500 nM; "++" , for values less than or equal to 1 μM; and "+" , for values greater than 1 μM, respectively.

Table 1

IL-2 Jurkat cell Transact dose-response plots for exemplary compounds of the invention are presented in FIG. 2.

Example 3. Inhibitory Activity Against Treg vs. Th17

CD4 ⁺ T cells play a critical role in regulating the immune system to combat foreign pathogens, including bacterial or viral infection as well as cancer. Proper function of the CD4 ⁺ T cell compartment relies on an adjustable equilibrium among various T cell subsets, among which Th17 cells and regulatory T cells (Tregs) play important roles in regulating autoimmunity and cancer. The delicate balance between Th17 and Treg cells is not only crucial for maintaining a healthy, functioning immune environment, but also has major therapeutic implications for disease treatment. In other words, purposefully offsetting the Th17/Treg balance could be effective in treating diseases associated with disruption of Th17/Treg balance and homeostasis.

Th17 cells are vital for host defense against pathogens, and have been implicated in causing autoimmune disorders and cancer, while Tregs are required for self-tolerance and defense against autoimmunity, and often correlate with cancer progression. This example demonstrates that the compounds of the invention have differential inhibitory effect on Th17 and Treg T cells, based on assays that measure inhibitory function of the subject compounds against Th17 or Treg function, including stimulated expansion of differentiated Th17 or Treg cells.

A. Th17 Assay –7-day culture for generating polarized human Th17-like cells

CD4 ⁺

T cells were differentiated to human Th17-like cells over a 7-day ex vivo culture, by resuspending the

T cells in media containing anti-CD28 co-stimulatory antibody (to provide the “second” activation signal) , a mixture of cytokines (such as IL-6 (essential for Th17 differentiation) , IL-1β (promotes Th17 differentiation) , TGF-β1 (essential for Th17 differentiation) , IL-23 (required to sustain the Th17 phenotype) ) , and the neutralizing antibodies anti-IL-4 (to inhibit

T cell polarization into Th2 cells) and anti-IFN-γ (to inhibit

T cell polarization into Th1 cells) , before plating the cells on tissue culture wells pre-coated with the anti-CD3 antibody (to provide the “first” activation signal) . The differentiated Th17-like cells were harvested, and then re-plated with anti-CD3 and anti-CD28 co-stimulation in cytokine mixture, to measure IL-17A production, in the presence and absence of decreasing concentrations of test compounds, in order to assess the abilities of the test compounds to inhibit IL-17A production and/or Th17 function.

The

(Lanthanide Chelate Excite) TR-FRET technology was used to assess IL-17A production using a standard curve. In brief, the

TR-FRET assay is a combination of time resolved fluorescence (TRF) with those of

Resonance Energy Transfer (FRET) for detection and quantitation in high throughput format. It uses a long-lifetime fluorescent lanthanide chelates (Ulight) that allows for a delay in measurement between excitation and emissions. The Ulight fluorophore is conjugated to a first anti-IL-17A antibody (i.e., the ULight labeled anti-hIL17A antibody) , for use with a

Europium W-1024-ITC chelate-labeled second anti-IL-17A antibody (i.e., the Eu-labeled anti-hIL17A antibody) . When both antibodies bind to IL-17A simultaneously, excitation of the Eu moiety leads to emission of a detectable light from the Ulight fluorophore through TRF-FRET. The amount of the IL-17A in the reaction was proportional to the detectable signal. A standard curve generated by using a series of known concentrations of IL-17A sample was used to measure IL-17A concentration in a test sample.

For FACS analysis, Th17-like cells were stimulated by PMA/ionomycin to promote cytokine production, and brefeldin-A was used to disrupt intracellular transportation to retain the synthesized cytokine intracellularly for FACS using fluorescently labeled anti-IL-17A Ab.

Reagents:

Equipment &Supplies:

Differentiation Assay Protocol

1. Dilute anti-CD3 antibody to 10 μg/mL with sterile PBS.

2. Coat 24-well Flat bottom tissue culture plates with 300 μL of diluted anti-CD3 antibody. Seal the plate and incubate it at 4℃ overnight for antibody crosslinking.

3. Wash the coated plate with 1,000 μL of sterile PBS twice on Day 2.

4. Resuspend 1 vial of

(CD4 ⁺, CD45RA ⁺) T cells (Allcells) in X-Vivo 15 medium containing anti-CD28 antibody (1 μg/mL) , IL-6 (10 ng/mL) , IL-1β (10 ng/mL) , TGF-β1 (5 ng/mL) , IL-23 (10 ng/mL) , and the neutralizing antibodies anti-IL-4 (10 μg/mL) and anti-IFN-γ (10 μg/mL) , at a density of 1.0 × 10 ⁵ cells/mL.

QC criteria for Donor selection:

PBMC QC: Fresh; CD3 ⁺, 40-60%; CD4 ⁺/CD8 ⁺, 1.5-2.

CD4 ⁺

T cell QC: Fresh;

Sorting method: double negative selection

Live cell: >95%

CD3 ⁺CD4 ⁺CD45RA ⁺: >95%

5. Add 1 mL of T cell suspension to 24-well plate pre-coated with anti-CD3 antibody. Culture the cells for 7 days in 37℃ and 5%CO ₂ incubator.

6. On day 7:

Harvest the cells to run IL-17A FACS assay, and seed cells in 96-well plate for compound test as following (TOTAL 150 μL assay system) :

1) Seed resuspended cells to 96-well plates pre-coated with anti-CD3 antibody (2×10 ⁴ cells /well, 100 μL /well, 2×10 ⁵ cells /mL) in X-vivo medium.

2) Incubate 1 hour in incubator.

3) Treat cells with different concentrations of serially diluted compounds (serial 3-fold dilutions from 10,000 nM to 4.57 nM) prepared in X-vivo medium for 72 hours (37℃, 5%CO ₂) , in the presence of all the antibodies and cytokines used in the differentiation protocol to maintain the Th17 phenotype (50 μL /well in 96-well plate wells) .

4) For High control wells, prepare 0.2%DMSO in X-vivo medium with the antibodies and cytokines (50 μL /well in 96-well plate) .

5) For Low control wells, prepare 0.2%DMSO in X-vivo medium without the antibodies and cytokines (50 μL /well in 96-well plate) .

6) After 72 hrs, collect 50 μL of supernatant from each well for HTRF assay, and test cells in FACS assay.

FACS Assay procedure

1) Prepare PMA (Final: 50 ng/mL) and ionomycin (Final: 1 μg/mL) and eBioscience ^TM Brefeldin A in X-vivo medium without the antibody and cytokines for all wells.

2) Add 50 μL/well PMA/ionomycin (P/I) and eBioscience ^TM Brefeldin A Solution (1,000×) to all wells. Re-stimulate the cells for 6 hours.

3) Harvest the cells for FACS analysis.

4) Wash the cells once with 1 mL PBS, Centrifuge at 300 g for 5 min.

5) Resuspend the cells in 1 mL PBS. Add 1 μL of the reconstituted fluorescent reactive dye (prepared before) -APC Cy7 to 1 mL cell suspension and mix well. Incubate at room temperature for 20 min, protect from light, Centrifuge at 300 g for 5 min.

6) Wash the cells once with 1 ml PBS, Centrifuge at 300 g for 5 min.

7) Resuspend the cells in 100 μL staining buffer. Stain the cells with CD4 ⁺-PE antibodies at RT for 30 minutes in the dark, Centrifuge at 300 g for 5 min.

8) Wash the cells twice in 500 μL staining buffer, Centrifuge at 300 g for 5 min.

9) Resuspend the cells in 100 μL fixation buffer at 4℃ for 20 minutes.

10) Wash the cells twice in 1*Perm/Wash buffer.

11) Prepare anti-IL-17A in 1*Perm/Wash buffer, add 100 μL anti-IL-17A in at 4℃ for 40 minutes in the dark.

12) Wash the cells twice with 1*Perm/Wash buffer and resuspend them in stain buffer before flow cytometric analysis.

13) Once the staining is completed, acquire the cells for data analysis.

14) Report percent of IL-17A positive cell.

IL-17A

Assay procedure

1. Prepare 1× Ultra HiBlock Buffer: add 400 μL 5× HiBlock Buffer to 1,600 μL H ₂O

2. prepare hIL-17A analyte standard dilutions: reconstitute with H ₂O to create a 10 μg/mL solution. Prepare standard 3-fold serial dilutions, with the highest concentration being 1.00E-06 g/mL of hIL-17A in 15 μL, and the lowest concentration being 3.00E-12 g/mL of hIL-17A in 15 μL (change tips between each standard dilution) .

3. Prepare 4× MIX Eu-labeled anti-hIL17A antibody (1.2 nM) + ULight labeled anti-hIL17A antibody (12 nM) : add 1.32 μL of 500 nM Eu-labeled anti-hIL-17A antibody and 13.2 μL of 500 nM Ulight labeled anti-hIL-17A antibody

4. In a white 384 plate:

A: add 15 μL of each analyte standard dilution or 15 μL of sample, centrifuge at 1,000 rpm for 1 min.

B: add 5 μL of a 4× MIX Eu-labeled anti-hIL-17A antibody (0.3 nM final) + Ulight labeled anti-hIL-17A antibody (3 nM final) , centrifuge at 1,000 rpm for 1 min.

5. Incubation 120 min at RT (room temperature)

6. Read using LANCE TRF Laser (in TR-FRET mode)

B. Treg Differentiation –6-day culture for generating polarized human Treg cells

CD4 ⁺

T cells were differentiated to human Treg cells over a 6-day ex vivo culture, using the CellXVivo Human Treg Cell Differentiation Kit (Cat. No. CDK006) from the R&D Systems, Inc. (Minneapolis, MN) according to the manufacture’s recommendation. The kit contains optimized proteins and reagents to drive efficient differentiation of

CD4 ⁺ T cells into FoxP3 ⁺CD25 ⁺ Treg cells. After 6 days of culturing, the differentiated Tregs were confirmed by FACS assay for CD4 ⁺CD25 ⁺ and FOXP3 ⁺ expression.

Specifically, for FACS analysis, Tregs were stained with fluorophore phycoerythrin (PE) -conjugated anti-CD4 antibody (CD4 ⁺-PE) and fluorophore allophycocyanin (APC) -conjugated anti-CD25 antibody (CD25 ⁺-APC) for surface CD4 and CD25 expression, before cells were fixed and permeated for detecting FOXP3 expression using anti-FOXP3-FITC antibody. The stained cells were subjected to flow cytometric analysis to determine the percentage of FOXP3 ⁺ positive cell.

Meanwhile, the differentiated Tregs were harvested, and then re-plated with anti-CD3 and anti-CD28 co-stimulation in cytokine mixture, to measure IL-10 production by Tregs, in the presence and absence of decreasing concentrations of test compounds, in order to assess the abilities of the test compounds to inhibit IL-10 production. IL-10 production was measured using the IL-10 ELISA Kit (Cat. No. 430604) from Biolegend (San Diego, CA) according to the manufacture’s protocol.

Materials and equipment:

Reagents:

Equipments &Supplies:

Differentiation assay procedure:

1. Reconstitute Human Treg Reagent 1 and Human Treg Reagent 2 each with 125 μL of Reconstitution Buffer 1, to generate 400× stocks.

2. Reconstitute Human Treg Reagent 4 with 125 μL of Reconstitution Buffer 2 to generate a 400× stock.

3. Add 125 μL each of

Human Treg Reagents

1, 2, and 4 (400× stocks) , and 50 μL of Human Treg Reagent 3 (1,000×) to 49.6 mL of X-VIVO 15 serum-free medium.

4. Coat 24-well Flat bottom tissue culture plate with 300 μL diluted anti-CD3 antibody. Seal the plate and incubate at 4℃ overnight for antibody crosslinking.

5. Wash the coated plate with 1,000 μL wash buffer twice on Day 2.

6. Resuspend 1 vial of

T cells (CD4 ⁺, CD45RA ⁺; Allcells) in X-Vivo 15 medium, with the Human Treg Reagents prepared in step 3, at a density of 1 × 10 ⁵ cells/mL.

QC criteria for Donor selection:

PBMC QC: Fresh; CD3 ⁺, 40-60%; CD4 ⁺/CD8 ⁺, 1.5-2.

CD4 ⁺

T cell QC: Fresh;

Sorting method: double negative selection

Live cell: >95%

CD3 ⁺CD4 ⁺CD45RA ⁺: >95%

7. Add 1 mL cells to 24-well plate pre-coated with anti-CD3 antibody.

8. Incubate the plate in 37℃ and 5%CO ₂ incubator for 6 days.

9. On day 6, harvest the cells to measure Foxp3 ⁺CD25 ⁺CD4 ⁺ T cells by FACS assay, and seed cells in 96-well plate for compound test (TOTAL 150 μL assay system) :

1) Resuspend and seed cells to 96-well plates pre-coated with anti-CD3 antibody (1×10 ⁴ cells/well, 100 μL/well, 1×10 ⁵ cells/mL) in X-vivo medium.

2) Incubate 1 hour in incubator (37℃, 5%CO ₂) .

3) Treat cells with different concentrations of serial dilution of compounds (serial 3-fold dilutions from 10,000 nM to 4.57 nM) prepared in X-vivo medium for 72 hours (37℃, 5%CO ₂) , in the presence of the Human Treg Reagents prepared in step 3 in the differentiation protocol (50 μL/well to 96-well plate) .

4) For High control wells, 0.2%DMSO is prepared in X-vivo medium, with the Human Treg Reagents prepared in step 3 in the differentiation protocol (50 μL/well to 96-well plate) .

5) For Low control wells, 0.2%DMSO is prepared in X-vivo medium, without the Human Treg Reagents prepared in step 3 in the differentiation protocol (50 μL/well to 96-well plate) .

6) After 72 hr, collect 120 μL supernatant to test IL-10 production by Treg cells using ELISA assay, and collect cells for FACS assay to confirm CD4 ⁺CD25 ⁺FoxP3 ⁺ expression on Tregs (High control and Low control only) and CTG assay (compound wells plus High/Low control) .

FACS assay procedure:

1. Wash the cells once with 1 mL PBS, Centrifuge at 300 g for 5 min.

2. Resuspend the cells in 1 mL PBS. Add 1 μL of the reconstituted fluorescent reactive dye (prepared before) -APC Cy7 to 1 mL cell suspension and mix well. Incubate at room temperature for 20 min, protect from light, Centrifuge at 300 g for 5 min.

3. Wash the cells once with 1 mL PBS, Centrifuge at 300 g for 5 min.

4. Resuspend the cells in 100 μL staining buffer. Stain the cells with fluorophore phycoerythrin (PE) -conjugated anti-CD4 antibody (CD4 ⁺-PE) and fluorophore allophycocyanin (APC) -conjugated anti-CD25 antibody (CD25 ⁺-APC) at RT for 30 minutes in the dark.

5. Wash the cells twice in 500 μL stain buffer, Centrifuge at 300 g for 5 min.

6. Resuspend the cells in 500 μL Fixation/Permeabilization buffer (4×, dilute with Fixation/Permeabilization dilute) at 4℃ for 30 minutes. Centrifuge at 300 g for 5 min at 4℃.

7. Add 500 μL 1×Permeabilization wash buffer (dilute by ddH ₂O from 10×) to resuspend. Centrifuge at 300 g for 5 min at 4℃.

8. Add 100 μL anti-FOXP3-FITC antibody premix (prepared in 1×Permeabilization wash buffer) to samples.

9. Incubate at 4℃ for 45 min in the dark.

10. Add 500 μl 1×Permeabilization wash buffer. Centrifuge, remove supernatant and vortex. Repeat once.

11. Resuspend them in stain buffer before flow cytometric analysis.

12. Once the staining is completed, acquire the cells, percent of FOXP3 ⁺ positive cell was reported.

CTG assay procedure:

1. Thaw one vial of aliquoted CTG reagent at RT and equilibrate it to room temperature prior to use.

2. Equilibrate the assay plate and at room temperature for approximately 20 minutes.

3. Harvest 120 μL supernatant to another plate to run IL-10 ELISA assay.

4. Add 30 μL/well of CTG reagent to cell assay plate.

5. Incubate for 15 min at RT on a plate shaker at 300 rpm, avoid light exposure.

6. Luminescence signal was read on plate reader Envision.

Data analysis

Inhibition rate of the compound is calculated according to the formula below:

%inhibition=100-100* (Luminescence value-low Luminescence value) / (High Luminescence value-Low Luminescence value) .

ELISA assay Procedure:

● Reagent Preparation:

1. Dilute 5× Coating Buffer A to 1× with deionized water. For one plate, dilute 2.4 mL 5×Coating Buffer A in 9.6 mL deionized water.

2. Dilute pre-titrated capture antibody 1: 200 in 1× Coating Buffer A. For one plate, dilute 60 μL capture antibody in 11.94 mL 1× Coating Buffer A.

3. Dilute 5× Assay Diluent A to 1× with PBS (pH 7.4) . For 50 mL, dilute 10 mL 5× Assay Diluent A in 40 mL PBS.

4. Lyophilized vials are under vacuum pressure. Reconstitute lyophilized standard with 0.2 mL of 1× Assay Diluent A. Allow the reconstituted standard to sit for 15 minutes at room temperature, then mix gently prior to making dilutions.

5. Prior to use, prepare 1,000 μL of the top standard at a concentration of 250 pg/mL from the stock solution in 1× Assay Diluent A (refer to Lot-Specific Certificate of Analysis/ELISA MAX ^TM Deluxe Set Protocol) .

Perform six two-fold serial dilutions of the 250 pg/mL top standard with 1× Assay Diluent A in separate tubes. After diluting, the human IL-10 standard concentrations are 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.3 pg/mL, 15.6 pg/mL, 7.8 pg/mL, and 3.9 pg/mL, respectively. 1× Assay Diluent A serves as the zero standard (0 pg/mL) .

6. Dilute the pre-titrated Biotinylated Detection Antibody 1: 200 in 1× Assay Diluent A. For one plate, dilute 60 μL Detection Antibody in 11.94 mL 1× Assay Diluent A.

7. Dilute Avidin-HRP 1: 1000 in 1× Assay Diluent A. For one plate, dilute 12 μL Avidin-HRP in 11.99 mL 1× Assay Diluent A.

8. TMB Substrate Solution is a mixture of equal volumes of Substrate Solution A and Substrate Solution B. Mix the two components immediately prior to use. For one plate, mix 5.5 mL Substrate Solution A with 5.5 mL of Substrate Solution B in a clean container (solution should be clear and colorless) .

9. Samples: For cell culture supernatant samples, the end user may need to determine the dilution factors in a preliminary experiment. Serum or plasma samples should be tested initially without any dilution. If dilution is required, samples should be diluted in 1×Assay Diluent A before adding to the wells.

● ELISA assay:

1. One day prior to running the ELISA, dilute Capture Antibody in 1× Coating Buffer A as described in Reagent Preparation. Add 100 μL of this Capture Antibody solution to all wells of a 96-well plate provided in this set. Seal plate and incubate overnight (16-18 hrs) between 2℃ and 8℃.

2. Bring all reagents to room temperature (RT) prior to use. It is strongly recommended that all standards and samples be run in duplicate or triplicate. A standard curve is required for each assay.

3. Wash plate 4 times with at least 300 μL Wash Buffer per well and blot residual buffer by firmly tapping plate upside down on absorbent paper. All subsequent washes should be performed similarly.

4. To block non-specific binding and reduce background, add 200 μL 1× Assay Diluent A per well.

5. Seal plate and incubate at RT for 1 hour with shaking on a plate shaker (e.g. 500 rpm with a 0.3 cm circular orbit) . All subsequent incubation with shaking should be performed similarly

6. While plate is being blocked, prepare the appropriate sample dilutions (if necessary) and standards.

7. Wash plate 4 times with Wash Buffer.

8. Add 100 μL/well of standards or samples to the appropriate wells. If dilution is required, samples should be diluted in 1× Assay Diluent A before adding to the wells.

9. Seal plate and incubate at RT for 2 hours with shaking.

10. Wash plate 4 times with Wash Buffer.

11. Add 100 μL of diluted Detection Antibody solution to each well, seal plate and incubate at RT for 1 hour with shaking.

12. Wash plate 4 times with Wash Buffer.

13. Add 100 μL of diluted Avidin-HRP solution to each well, seal plate and incubate at RT for 30 minutes with shaking.

14. Wash plate 5 times with Wash Buffer. For this final wash, soak wells in Wash Buffer for 30 seconds to 1 minute for each wash. This will help minimize background.

15. Add 100 μL of freshly mixed TMB Substrate Solution and incubate in the dark for 30 minutes. Positive wells should turn blue in color. It is not necessary to seal the plate during this step.

16. Stop reaction by adding 100 μL of Stop Solution to each well. Positive wells should turn from blue to yellow.

17. Read absorbance at 450 nm within 15 minutes. If the reader can read at 570 nm, the absorbance at 570 nm can be subtracted from the absorbance at 450 nm.

Using the assays above, the inhibitory effect on Treg cells by several representative compounds of the invention (and in several cases, a comparator compound) are summarized in the tables below. Since assays conducted on different batches may not necessarily be directly comparable to one another, only compounds assayed in the same batches were directly compared in the same tables. Note that the comparator compound is a partial inhibitor for Treg inhibition, in that the maximum achievable inhibition is about 55-65%at the highest concentration.

* Max. stands for maximum inhibition

** Max. Conc. is the highest tested compound concentration.

Likewise, the inhibitory effect on Th17 cells by several representative compounds of the invention (and in several cases, a comparator compound) are summarized in the tables below.

Compound ID	Th17 Assay IC ₅₀ (nM)
RGT005-001 (Ref)	30.9
Compound 64	224
Compound 65 or 66 (Peak 1)	36.3
Compound 66 or 65 (Peak 2)	311
Compound 79	148
Compound 3	174
Compound 16	116
Compound 2	64
Compound 8	1680
Compound 46	219
Compound 67	81.3
Compound 62 or 63 (Peak 1)	179
Compound 63 or 62 (Peak 2)	580
Compound 70	205

Example 4. Mouse Pharmacokinetic (PK) Data

Pharmacokinetic data for representative compounds of the invention and a comparator compound were obtained in male CD1 mice. Specifically, male CD1 mice in each group were fed with either a low dose of 5 mg/kg compound p.o., or a high dose of 30 mg/kg of the same compound p.o.. PK data including AUC _last (hr*ng/mL) and bioavailability (F%) were obtained for various compounds of the invention and the comparator. AUC fold increase was calculated based on the AUC of high dose vs. that of the low dose of the same compound.

* N.D. Not Determined

B. Synthetic Examples

Equipment Description

NMR spectra were measured with a Varian Mercury spectrometer operating at 400 MHz ( ¹H) , 376 MHz ( ¹⁹F) or 75 MHz ( ¹³C) . Solvents used for samples are specified in the experimental procedures for each compound. Chemical shifts are expressed in parts per million (ppm, δunits) . Coupling constants are in units of hertz (Hz) . Splitting patterns describe apparent multiplicities and are designated as s (singlet) , d (doublet) , t (triplet) , q (quartet) , quint (quintet) , m (multiplet) , br (broad) .

The following system was used for LCMS: Agilent 6120 (Binary Gradient Module pump) , XBridge analytical column C ₁₈, 5 μm, 4.6 × 50 mm, 25 ℃, 5 μL injection volume, 2 mL/min, with a gradient of acetonitrile in aqueous 0.1%Ammonium acetate according to the following timings:

Experimental procedures: All reactions were conducted under an atmosphere of dry nitrogen unless specified otherwise. TLC plates were visualised with u.v. light. Flash chromatography refers to column chromatography over silica gel (40-60 μm) using glass columns. Alternatively, automated chromatography was performed using Biotage SP1 or Biotage Isolera systems with u.v. detection at 220 or 254 nm and employing Biotage normal phase or reverse phase silica cartridges. Further details can be found under the relevant experimental procedure.

Scheme 1

Synthesis of the compounds of formula I

Compounds of the formula I can be prepared from imidazole intermediates as described in Scheme 1. Reacting an imidazole derivative 1 such as (aminooxy) diphenylphosphine oxide, in the presence of a base, such as sodium hydride in a solvent, such as DMF, affords the aminated imidazole derivative 2. Compound 2 can be N-protected via reaction with di-tert-butyldicarbonate to afford a compound 3. Compound 3 can be brominated or chlorinated with a brominating or chlorinating agent, such as bromine or N-bromosuccinimide, in a solvent, such as DMF, to afford a compound 4 as the major product. Compound 4 can react with ethyl acetate in the presence of a base, such as potassium tert-butoxide in a solvent such as THF, to afford a compound 5 . Reacting Compound 5 with l , l-dimethoxy-N, N-dimethylmethanamine (DMF-DMA) in a solvent such as DCM, at room temperature affords cyclized product 6 . Reaction of compound 6 with a chlorinating agent, such as phosphorus oxy chloride/bromide affords compound 7. Compound 7 can be reacted with boronic acids of the formula R ₁B (OH) ₂ or boronate esters under Suzuki coupling conditions, to afford compound 8. The compound 8 can be hydrolyzed with hydrolyzing agents, such as lithium hydroxide or sodium hydroxide, in a protonic solvent, such as methanol or ethanol to afford acid derivative 9. The imidazole pyridazine acid 9 undergoes Curtis rearrangement in the presence of diphenyl phosphoryl azide (DPPA) and treated with pyridine amine 10 to afford the compound of formula I.

Scheme 2

Synthesis of the compounds of formula II

The alkylation of the compounds 6 with alkyl halides such as methyl iodide, ethyl iodide, propyl bromide, by using a suitable base such as sodium hydride, lithium hexamethyldisilane, cesium carbonate, potassium carbonate in one or more solvents such as DMF, DMA, THF, toluene or mixture thereof to afford imidazolepyridazine ether analogs 11, which was treated with LiOH to afford the acid 12. The imidazole pyridazine acid 9 undergoes Curtis rearrangement in the presence of diphenyl phosphoryl azide (DPPA) and treated with pyridine amine 10 to afford the compounds of formula II.

Scheme 3

Synthesis of the compounds of formula III

The compounds of formula III can be prepared by following the methods described in scheme 3. The Buchwald-Hartwig amination of bromide 7 with primary or secondary amine R ₅R ₆NH in the presence of a suitable Pd catalyst such as Pd ₂dba ₃, Pd (dppf) Cl ₂, Pd (OAc) ₂, a suitable ligand such as BINAP, xantphos, triphenylphosphine and an appropriate base such as t-BuONa or t-BuOK, in solvents like tetrahydrofuran, dioxane, or toluene eventually affords the compounds of formula III.

Scheme 4

The synthesis of the compounds of formula IV

The compounds of formula IV can be prepared by following the methods depicted in scheme 4. The hydrogenation of compounds 7 under Pd/C and hydrogen gives compound 15, which undergoes hydrolysis with lithium hydroxide and followed by Curtis rearrangement, and quenched with amino pyridine 10 or derivatives eventually afford the compounds of formula IV.

Scheme 5

The synthesis of the compounds of formula V

The compounds of formula V can be prepared from the imidazole pyridazine ester 7 as depicted in scheme 5. Hydrolysis of the ester 7 under alkaline conditions (such as K ₂CO ₃, LiOH, NaOH etc. ) in solvents like THF, water, methanol or a mixture (s) yields corresponding carbocylic acid 17 , which undergoes Curtis rearrangement in the presence of DPPA and a tertiary amine base and trapping of the isocyanate derivative gives the desired product 10. The subsequent Stille coupling of bromide 18 with organotin compounds gives the corresponding alkynes compound 19, which was treated with aqueous HCl and affords the ketone 20; after reduced with Sodium borohydride to the corresponding alcohol 21, treated with thionyl chloride, then quenched with different alcohols, the ether 22 was obtained, which is subject to carbomate 23 and yields the urea compound V.

Intermediate 6c and 7c

Step 1

To a solution of ethyl 1H-imidazole-2-carboxylate (10 g, 71.36 mmol) in THF (200 mL) was added NaH (3.55 g, 92.76 mmol, 60%purity) at 0 ℃, the resulting solution was warmed up to room temperature and stirred for 2 hours, and then cooled to 0 ℃, the solution of O-diphenylphosphorylhydroxylamine (23.30 g, 99.90 mmol) in THF (200 mL) was added dropwise into the mixture. After 20 mins, the ice bath was removed, and the resulting mixture was stirred at room temperature for 12 hours. TLC showed a new spot was formed; the reaction was quenched with water until it became a clear solution, and concentrated to dryness in vacuo. The crude product was suspended in Ethyl acetate (700 mL) with stirring for 30 min and filtered. The filtrate was concentrated and purified by flash column chromatography (DCM: Methanol 30: 1) to afford ethyl 1-aminoimidazole-2-carboxylate (10.32 g, 66.51 mmol, 93.21%yield) as a colorless oil. LC-MS: m/z 156 [M+H] ⁺.

Step 2

To a stirred solution of ethyl 1-aminoimidazole-2-carboxylate (10 g, 64.45 mmol) in DMF (60 mL) was added DMAP (1.18 g, 9.67 mmol) and Di-tert-butyl dicarbonate (18.29 g, 83.79 mmol, 19.23 mL) . The reaction mixture was stirred at 85 ℃ for 4 h and upon completion of the reaction, the mixture was poured into water and extracted with EtOAc (2 × 600 mL) . The combined organic phase was washed with brine (100 mL) and dried over anhydrous Na ₂SO ₄, filtered and concentrated in vacuo. The crude was purified by flash column chromatography (Petroleum ether: Ethyl acetate = 5: 1) to afford the ethyl 1- (tert-butoxycarbonylamino) imidazole-2-carboxylate (13.2 g, 51.71 mmol, 80.23%yield) as a yellow solid. LC-MS: m/z 256 [M+H] ⁺.

Step 3

To a suspension of ethyl 1- (tert-butoxycarbonylamino) imidazole-2-carboxylate (3.5 g, 13.71 mmol) in DMF (15 mL) was added slowly 1-chloropyrrolidine-2, 5-dione (1.83 g, 13.71 mmol, 1.11 mL) in DMF (5 mL) . The reaction mixture was stirred at room temperature for 6h and upon completion of the reaction, the reaction was added a saturated sodium bicarbonate and extracted with EtOAc (2 ×150 mL) . The combined organic phase was washed with brine (100 mL) and dried over anhydrous Na ₂SO ₄, filtered and concentrated under vacuo. The crude was purified by flash column chromatography (Petroleum ether/ethyl acetate= 10: 1) to afford the ethyl 1- (tert-butoxycarbonylamino) -4-chloro-imidazole-2-carboxylate (1.9 g, 6.56 mmol, 47.83%yield) as a colorless oil. LC-MS: m/z 290 [M+H] ⁺.

Step 4

To a solution of ethyl 1- (tert-butoxycarbonylamino) -4-chloro-imidazole-2-carboxylate (3.7 g, 12.77 mmol) in THF (10 mL) was added dropwise potassium 2-methylpropan-2-olate (1 M, 38.31 mL) at 0 ℃ for 5 minutes, Dry ethyl acetate (2.81 g, 31.93 mmol, 3.12 mL) was added into the mixture and stirred at 0 ℃ for 15 minutes. The ice bath was removed and the resulting mixture was stirred at room temperature for 2 hours. TLC showed spot was formed, the reaction was cooled 0℃ and treated with 1.0 N HCl via dropwise addition until the suspension was completely dissolved, and then it was extracted with EtOAc (2 ×40 mL) . The combined organic phase was washed with brine (100 mL) and dried over anhydrous Na ₂SO ₄, filtered and concentrated to afford ethyl (Z) -3- [1- (tert-butoxycarbonylamino) -4-chloro-imidazol-2-yl] -3-hydroxy-prop-2-enoate (4.24 g, crude) as a yellow solid. LC-MS: m/z 332 [M+H] ⁺.

Step 5

To a solution of ethyl (Z) -3- [1- (tert-butoxycarbonylamino) -4-chloro-imidazol-2-yl] -3-hydroxy-prop-2-enoate (4.24 g, 12.78 mmol) in DCM (30 mL) was added 1, 1-dimethoxy-N, N-dimethyl-methanamine (12.18 g, 102.25 mmol, 13.69 mL) . The reaction mixture was stirred at room temperature for 3h and upon completion of the reaction, the mixture was concentrated to give crude, which was recrystallization with ethyl acetate to give ethyl 2-chloro-8-hydroxy-imidazo [1, 2-b] pyridazine-7-carboxylate (2.4 g, 9.93 mmol, 77.72%yield) as a white solid. LC-MS: m/z 242 [M+H] ⁺.

Step 6

To a stirred solution of ethyl 2-chloro-8-hydroxy-imidazo [1, 2-b] pyridazine-7-carboxylate (6 g, 24.83 mmol) in 1, 4-Dioxane (20 mL) at room temperature was added Phosphorus oxybromide (17.80 g, 62.08 mmol, 6.31 mL) , the reaction mixture was stirred at 110 ℃ for 2h and upon completion of the reaction, the reaction mixture was cooled to room temperature, poured into water and extracted with EtOAc (2 ×150 mL) . The combined organic phase was washed with brine (100 mL) and dried over anhydrous Na ₂SO ₄, filtered and concentrated in vacuo. The crude was purified by flash column chromatography (Petroleum ether/ethyl acetate = 7: 1) to afford ethyl 8-bromo-2-chloro-imidazo [1, 2-b] pyridazine-7-carboxylate (3.3 g, 10.84 mmol, 43.64%yield) as a yellow solid. LC-MS: m/z 304 [M+H] ⁺.

Example 1. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8-cyclopropylimidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a solution of ethyl 8-bromo-2-chloroimidazo [1, 2-b] pyridazine-7-carboxylate 7c (700.0 mg, 2.31 mmol) in toluene (42 mL) was added cyclopropylboronic acid (397.4 mg, 4.62 mmol) , tricyclohexylphosphine (646.8 mg, 2.31 mmol) and potassium phosphate (1.22 g, 5.775 mmol) . The suspension was degassed and exchanged with N ₂ two times. The palladium (II) acetate (129.4 mg, 0.5775 mmol) was added into the mixture. The reaction mixture was heated to 100 ℃ for 12 hours. The starting material was consumed and the desired mass was detected from LC-MS. The mixture was concentrated under reduced pressure and the result residue was purified with flash column chromatography (eluting with ethyl acetate in petroleum from 1%to 15%) to give the ethyl 2-chloro-8-cyclopropylimidazo [1, 2-b] pyridazine-7-carboxylate (330.0 mg, 50.3%yield) as yellow solid. LC-MS: m/z 266 [M+H] ⁺.

Step 2

To a solution of ethyl 2-chloro-8-cyclopropylimidazo [1, 2-b] pyridazine-7-carboxylate (352 mg, 1.328 mmol) in THF (8 mL) was added the solution of lithium hydroxide monohydrate (111.6 mg, 2.657 mmol) in water (2.5 mL) . The mixture was stirred at 30 ℃ for 4 hours. The starting material was consumed and the desired mass was detected from LC-MS. It was concentrated to remove the solvent; the residue was acidified with HCl (1 M) to pH = 3～4. The mixture was extracted with EtOAc and the organic phase was dried, concentrated to give 2-chloro-8-cyclopropylimidazo [1, 2-b] pyridazine-7-carboxylic acid (310 mg, 98.5%yield) as yellow solid. LC-MS: m/z 238 [M+H] ⁺.

Step 3

To a solution of 2-chloro-8-cyclopropylimidazo [1, 2-b] pyridazine-7-carboxylic acid (150 mg, 0.6329 mmol) in 1, 4-dioxane (10 mL) was added diphenylphosphonic azide (209 mg, 0.7595 mmol) and triethylamine (320.2 mg, 3.164 mmol) . The resulting mixture was stirred at room temperature for 30 minutes, then 5-chloro-6- (triazol-2-yl) pyridin-3-amine (185.1 mg, 0.9494 mmol) was added and the mixture was heated to 100℃ for 2 hours. The mixture solution was poured into water and extracted with ethyl acetate twice. The organic phase was dried over Na ₂SO ₄, concentrated and the result residue was purified by flash column chromatography (eluting with ethyl acetate in petroleum from 10%to 80%) to give the 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8-cyclopropylimidazo [1, 2-b] pyridazin-7-yl) urea (64 mg) as a white solid. LC-MS: m/z 430 [M+H] ⁺. ¹H NMR (DMSO-d6, 400 MHz) : δ 9.78 (s, 1 H) , 8.99 (s, 1 H) , 8.72 (s, 1 H) , 8.58 (d, J = 2.4 Hz, 1 H) , 8.48 (d, J = 2.4 Hz, 1 H) , 8.34 (s, 1 H) , 8.16 (s, 2 H) , 2.17-2.14 (m, 1 H) , 1.73-1.71 (m, 2 H) , 1.16-1.14 (m, 2 H) .

Example 2. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8-isopropylimidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a solution of ethyl 8-bromo-2-chloro-imidazo [1, 2-b] pyridazine-7-carboxylate (250 mg, 820.94 μmol) , 2-isopropenyl-4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolane 7c (179.3 mg, 1.07 mmol) in toluene (3 mL) and water (0.3 mL) were added potassium phosphate (522.8 mg, 2.46 mmol) and bis (triphenylphosphine) palladium (II) chloride (57.6 mg, 82.09 μmol) . The mixture was stirred at 100 ℃ for 24 h under N ₂. LC-MS show some starting material was left, and bis (triphenylphosphine) palladium (II) chloride (57.6 mg, 82.09 μmol) was added again. The mixture was stirred at 100 ℃ under N ₂ for another 24 h. After cooling down to room temperature, the reaction mixture was filtered and the filtrate was concentrated to give a residue, which was purified by flash column chromatography (eluting with PE/EA = 10/1) to afford ethyl 2-chloro-8-isopropenyl-imidazo [1, 2-b] pyridazine-7-carboxylate (57 mg, 26%yield) as a yellow solid and 100 mg of ethyl 8-bromo-2-chloro-imidazo [1, 2-b] pyridazine-7-carboxylate was recovered. LC-MS: m/z 266 [M+H] ⁺.

Step 2

To a solution of ethyl 2-chloro-8-isopropenyl-imidazo [1, 2-b] pyridazine-7-carboxylate (68 mg, 255.93 μmol) in methanol (5 mL) was added PtO ₂ (15 mg, 255.93 μmol) . The reaction mixture was stirred at room temperature overnight under H ₂, and then filtrated, concentrated under vacuo. The crude product was purified by flash column chromatography (eluting with PE/EA = 10/1) to afford ethyl 2-chloro-8-isopropyl-imidazo [1, 2-b] pyridazine-7-carboxylate (32 mg, 47%yield) as clean oil. LC-MS: m/z 268 [M+H] ⁺

Step 3

To a solution of ethyl 2-chloro-8-isopropyl-imidazo [1, 2-b] pyridazine-7-carboxylate (32 mg, 119.53 μmol) in THF (2 mL) was added lithium hydroxide monohydrate (20.1 mg, 478.13 μmol) in water (0.4 mL) . The mixture was stirred at room temperature for 5 h. Upon removal of solvent, the residue was acidified with HCl (2 M) to pH = 3～4. The mixture was extracted by EtOAc and the organic phase was dried and concentrated to give 2-chloro-8-isopropyl-imidazo [1, 2-b] pyridazine-7-carboxylic acid (28.6 mg, quant. ) as a white solid, which was used directly for next step. LC-MS: m/z 240 [M+H] ⁺.

Step 4

To a solution of 2-chloro-7-isopropyl-pyrazolo [1, 5-a] pyrimidine-6-carboxylic acid (28.6 mg, 119.34 μmol) in 1, 4-dioxane (6 mL) was added diphenylphosphonic azide (37.1 mg, 143.20 μmol) and triethylamine (60.4 mg, 596.68 μmol) . The resulting solution was stirred at room temperature for 30 minutes, then 5-chloro-6- (triazol-2-yl) pyridin-3-amine (28.0 mg, 143.20 μmol) was added and the mixture was stirred at 100 ℃ for 2 hour. Solvent was removed and the residue was purified by flash column chromatography (eluting with DCM: MeOH = 30 : 1) and prep-HPLC to give 1- (2-chloro-7-isopropyl-pyrazolo [1, 5-a] pyrimidin-6-yl) -3- [5-chloro-6- (triazol-2-yl) -3-pyridyl] urea (8 mg, 18.51 μmol, 15.51%yield) as a white solid. LC- MS: m/z 432.0 [M+H] ⁺. ¹H NMR (DMSO-d6, 400 MHz) : δ 9.72 (s, 1H) , 8.91 (s, 1H) , 8.71 (s, 1H) , 8.57 (d, J = 2.4 Hz, 1H) , 8.47 (d, J = 2.4 Hz, 1H) , 8.39 (s, 1H) , 8.16 (s, 2H) , 3.51 –3.44 (m, 1H) , 1.48 (d, J = 6.8 Hz, 6H) .

Example 3. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8-ethylimidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a solution of ethyl 8-bromo-2-chloro-imidazo [1, 2-b] pyridazine-7-carboxylate 7c (600 mg, 1.97 mmol) , 4, 4, 5, 5-tetramethyl-2-vinyl-1, 3, 2-dioxaborolane (910 mg, 5.91 mmol) and tricyclohexylphosphine (552 mg, 1.97 mmol) in 1, 4-dioxane (20 mL) were added potassium phosphate (1.25 g, 5.91 mmol) and palladium (II) acetate (110 mg, 492.57 μmol) . The suspension was stirred at 100 ℃ for 2 h under N ₂. After cooling down to room temperature, the reaction mixture was filtered and the filtrate was concentrated to give a residue, which was purified by flash column chromatography (eluting with PE/EA = 10/1) to afford ethyl 2-chloro-8-vinyl-imidazo [1, 2-b] pyridazine-7-carboxylate (230 mg, 46%yield) as a yellow solid. LC-MS: m/z 252 [M+H] ⁺.

Step 2

To a solution of ethyl 2-chloro-8-vinyl-imidazo [1, 2-b] pyridazine-7-carboxylate 8d (230 mg, 913.90 μmol) in methanol (5 mL) was added PtO ₂ (41.5 mg, 182.78 μmol) . The reaction mixture was stirred at room temperature for 3 h under H ₂, and then filtrated, concentrated under vacuo. The crude product was purified by flash column chromatography (eluting with PE/EA = 10/1) to afford ethyl 2-chloro-8-ethyl-imidazo [1, 2-b] pyridazine-7-carboxylate (170 mg, 73%yield) as clean oil. LC-MS: m/z 254 [M+H] ⁺

Step 3

To a solution of ethyl 2-chloro-8-ethyl-imidazo [1, 2-b] pyridazine-7-carboxylate 8e (170 mg, 670.12 μmol) in THF (2 mL) and ethanol (1 mL) was added lithium hydroxide monohydrate (112.5 mg, 2.68 mmol) in water (1 mL) . The mixture was stirred at room temperature for 4 h. Upon removal of solvent, the residue was acidified with HCl (2 M) to pH = 3～4. The mixture was extracted by EtOAc and the organic phase was dried and concentrated to give 2-chloro-8-ethyl-imidazo [1, 2-b] pyridazine-7-carboxylic acid (150 mg, 99%yield) as a white solid, which was used directly for next step. LC-MS: m/z 226 [M+H] ⁺.

Step 4

To a solution of 2-chloro-7-ethyl-pyrazolo [1, 5-a] pyrimidine-6-carboxylic acid 9c (100 mg, 443.20 μmol) in 1, 4-dioxane (6 mL) were added diphenylphosphonic azide (137.8 mg, 531.84 μmol) and triethylamine (224.2 mg, 2.22 mmol) . The resulting solution was stirred at room temperature for 30 minutes, then 5-chloro-6- (triazol-2-yl) pyridin-3-amine (130.0 mg, 664.80 μmol) was added and the mixture was stirred at 100 ℃ for 2 hour. Solvent was removed and the residue was purified by flash column chromatography (eluting with DCM: MeOH = 30 : 1) and prep-HPLC to give 1- (2-chloro-7-ethyl-pyrazolo [1, 5-a] pyrimidin-6-yl) -3- [5-chloro-6- (triazol-2-yl) -3-pyridyl] urea (68 mg, 37%yield) . LC-MS: m/z 418.0 [M+H] ⁺ ¹H NMR (DMSO-d6, 400 MHz) : δ 9.78 (brs, 1H) , 8.93 (brs, 1H) , 8.90 (s, 1H) , 8.58 (d, J =2.4 Hz, 1H) , 8.48 (d, J = 2.4 Hz, 1H) , 8.38 (s, 1H) , 8.16 (s, 2H) , 2.95 (q, J = 7.2 Hz, 2H) , 1.26 (t, J = 7.6 Hz, 3H) .

Example 4. Synthesis of 1- (8-bromo-2-chloroimidazo [1, 2-b] pyridazin-7-yl) -3- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) urea

Step 1

To a solution of ethyl 8-bromo-2-chloroimidazo [1, 2-b] pyridazine-7-carboxylate 9d (114 mg, 0.3762 mmol) in THF (4 mL) was added the solution of lithium hydroxide monohydrate (23.7 mg, 0.5644 mmol) in water (0.5 mL) . The mixture was stirred at 30 ℃ for 4 hours. The starting material was consumed and the desired mass was detected from LC-MS. The resulting mixture was concentrated to remove the solvents, the residue was acidified with HCl (1 M) to pH = 3～4. The mixture was extracted with EtOAc and the organic phase was dried, concentrated to give 8-bromo-2-chloroimidazo [1, 2-b] pyridazine-7-carboxylic acid (96 mg, 92.8%) as a yellow solid. LC-MS: m/z 276 [M+H] ⁺.

Step 2

To a solution of 8-bromo-2-chloroimidazo [1, 2-b] pyridazine-7-carboxylic acid (96 mg, 0.3491 mmol) in 1, 4-dioxane (6 mL) was added diphenylphosphonic azide (115.3 mg, 0.4189 mmol) and triethylamine (176.6 mg, 1.745 mmol) . The resulting mixture was stirred at room temperature for 30 minutes, then 5-chloro-6- (triazol-2-yl) pyridin-3-amine (102.1 mg, 0.5236 mmol) was added and the mixture was heated to 100 ℃ for 2 hours. The mixture solution was poured into water and extracted with ethyl acetate twice. The organic was dried over Na ₂SO ₄, concentrated and the result residue was purified by flash column chromatography (eluting with ethyl acetate in petroleum from 10%to 80%) to give the 1- (8-bromo-2-chloroimidazo [1, 2-b] pyridazin-7-yl) -3- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) urea (10.2 mg) as a white solid. LC-MS: m/z 468 [M+H] ⁺. ¹H NMR (DMSO-d6, 400 MHz) : δ 10.19 (s, 1 H) , 9.13 (s, 1 H) , 9.06 (s, 1 H) , 8.57 (d, J = 1.6 Hz, 1 H) , 8.54 (s, 1 H) , 8.49 (d, J =2.0 Hz, 1 H) , 8.17 (s, 2 H) .

Example 5. Synthesis of 1- (2-chloro-8-cyclopropylimidazo [1, 2-b] pyridazin-7-yl) -3- (2-(trifluoromethyl) pyridin-4-yl) urea

Step 1

To a solution of 2-chloro-8-cyclopropylimidazo [1, 2-b] pyridazine-7-carboxylic acid 9a (50 mg, 0.211 mmol) in 1, 4-dioxane (2 mL) was added diphenylphosphonic azide (69.7 mg, 0.2532 mmol) and triethylamine (106.8 mg, 1.055 mmol) . The resulting mixture was stirred at room temperature for 30 minutes, then 2- (trifluoromethyl) pyridin-4-amine 10b (51.3 mg, 0.317 mmol) was added and the mixture was heated to 100℃ for 2 hours. The mixture solution was poured into water and extracted with ethyl acetate twice. The organic was dried over Na ₂SO ₄, concentrated and the result residue was purified by flash column chromatography (eluting with ethyl acetate in petroleum from 10%to 80%) to give the 1- (2-chloro-8-cyclopropylimidazo [1, 2-b] pyridazin-7-yl) -3- (2- (trifluoromethyl) pyridin-4-yl) urea (5.2 mg) as a white solid. LC-MS: m/z 397 [M+H] ⁺. ¹H NMR (DMSO-d6, 400 MHz) : δ 9.89 (s, 1 H) , 8.95 (s, 1 H) , 8.68 (s, 1 H) , 8.55 (d, J = 5.6 Hz, 1 H) , 8.34 (s, 1 H) , 8.07 (d, J = 1.6 Hz, 1 H) , 7.63-7.62 (m, 1 H) , 2.16-2.12 (m, 1 H) , 1.71-1.69 (m, 2 H) , 1.14-1.12 (m, 2 H) .

Example 6. Synthesis of 1- (2-chloro-8-cyclopropylimidazo [1, 2-b] pyridazin-7-yl) -3- (6- (trifluoromethyl) pyridin-3-yl) urea

Step 1

To a solution of 2-chloro-8-cyclopropylimidazo [1, 2-b] pyridazine-7-carboxylic acid 9a (50 mg, 0.211 mmol) in 1, 4-dioxane (3 mL) was added diphenylphosphonic azide (69.7 mg, 0.2532 mmol) and triethylamine (106.8 mg, 1.055 mmol) . The resulting mixture was stirred at room temperature for 30 minutes, then 6- (trifluoromethyl) pyridin-3-amine 10c (51.3 mg, 0.3165 mmol) was added and the mixture was heated to 100℃ for 2 hours. The mixture solution was poured into water and extracted with ethyl acetate twice. The organic phase was dried over Na ₂SO ₄, concentrated and the resulting residue was purified by flash column chromatography (eluting with ethyl acetate in petroleum from 10%to 100%) to give the 1- (2-chloro-8-cyclopropylimidazo [1, 2-b] pyridazin-7-yl) -3- (6- (trifluoromethyl) pyridin-3-yl) urea (12.7 mg) as a white solid. LC-MS: m/z 397 [M+H] ⁺. ¹H NMR (DMSO-d6, 400 MHz) : δ 9.69 (s, 1 H) , 8.88 (s, 1 H) , 8.76 (d, J = 2.0 Hz, 1 H) , 8.72 (s, 1 H) , 8.33 (s, 1 H) , 8.26-8.23 (m, 1 H) , 7.84 (d, J = 8.8 Hz, 1 H) , 2.16-2.12 (m, 1 H) , 1.72-1.68 (m, 2 H) , 1.15-1.11 (m, 2 H) .

Example 7. Synthesis of 1- (2-bromo-8-cyclopropylimidazo [1, 2-b] pyridazin-7-yl) -3- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) urea

Step 1

To a suspension of ethyl 1- (tert-butoxycarbonylamino) imidazole-2-carboxylate 3 (3.57 g, 13.99 mmol) in DMF (30 mL) was added slowly 1-bromopyrrolidine-2, 5-dione (2.49 g, 13.99 mmol, 1.19 mL) in DMF (10 mL) . The reaction mixture was stirred at room temperature for 12 hours and upon completion of the reaction as judged by TLC, the reaction mixture was added a saturated sodium bicarbonate and extracted with EtOAc (2 ×150 mL) . The combined organic phase was washed with brine (100 mL) and dried over anhydrous Na ₂SO ₄, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography (Petroleum ether/ethyl acetate, 10: 1) to afford ethyl 4-bromo-1- (tert-butoxycarbonylamino) imidazole-2-carboxylate (2.5 g, 7.48 mmol, 53.49%yield) as a colorless oil. LC-MS: m/z 334.1 [M+H] ⁺.

Step 2

To a solution of ethyl 4-bromo-1- (tert-butoxycarbonylamino) imidazole-2-carboxylate 4d (2.5 g, 7.48 mmol) in THF (4 mL) was added dropwise potassium 2-methylpropan-2-olate (1 M, 22.44 mL) at 0 ℃ for 5 minutes, dry ethyl acetate (1.65 g, 18.70 mmol, 1.83 mL) was added into the mixture and stirring was continued at 0 ℃ for 15 minutes. The ice bath was removed and the resulting mixture was stirred at room temperature for 2 hours. TLC showed spot was formed, the reaction was cooled 0 ℃ and treated with 1.0 N HCl via dropwise addition until the suspension was completely dissolved, and extracted with EtOAc (2 ×40 mL) . The combined organic phase was washed with brine (100 mL) and dried over anhydrous Na ₂SO ₄, filtered and concentrated to afford ethyl (Z) -3- [4-bromo-1- (tert-butoxycarbonylamino) imidazol-2-yl] -3-hydroxy-prop-2-enoate (2.26 g, crude) as a yellow solid. LC-MS: m/z 376.1 [M+H] ⁺.

Step 3

To a solution of ethyl (Z) -3- [4-bromo-1- (tert-butoxycarbonylamino) imidazol-2-yl] -3-hydroxy-prop-2-enoate 5d (2.26 g, 6.01 mmol) in DCM (10 mL) was added 1, 1-dimethoxy-N, N-dimethyl-methanamine (2.86 g, 24.03 mmol, 3.22 mL) . The reaction mixture was stirred at room temperature for 3 h and upon completion of the reaction, the mixture was concentrated to give ethyl 2-bromo-8-hydroxy-imidazo [1, 2-b] pyridazine-7-carboxylate (1.72 g, crude) as a yellow solid. LC-MS: m/z 286.1 [M+H] ⁺.

Step 4

To a stirred solution of ethyl 2-bromo-8-hydroxy-imidazo [1, 2-b] pyridazine-7-carboxylate 6d (1.72 g, 6.01 mmol) in 1, 4-dioxane (20 mL) at room temperature was added phosphorus oxybromide (4.31 g, 15.03 mmol, 1.53 mL) . The reaction mixture was stirred at 110 ℃ for 3 h and upon completion of the reaction, the reaction mixture was cooled to room temperature, and then it was poured into water and extracted with EtOAc (2 ×50 mL) . The combined organic phase was washed with brine (50 mL) and dried over anhydrous Na ₂SO ₄, filtered and concentrated in vacuo to afford a crude, which was purified by flash column chromatography (Petroleum ether/ethyl acetate, 10: 1) to afford the ethyl 2, 8-dibromoimidazo [1, 2-b] pyridazine-7-carboxylate (1.22 g, 3.50 mmol, 58.15%yield) as a yellow solid. LC-MS: m/z 348.1 [M+H] ⁺.

Step 5

To a solution of ethyl 2, 8-dibromoimidazo [1, 2-b] pyridazine-7-carboxylate 7d (200 mg, 573.10 μmol) , cyclopropylboronic acid (147.7 mg, 1.72 mmol) in Toluene (5 mL) was added tricyclohexylphosphane (160.7 mg, 573.10 μmol) , tripotassium phosphate (304.1 mg, 1.43 mmol) and diacetoxypalladium (25.7 mg, 114.62 μmol) . The suspension was stirred at 100 ℃ for 4 hours. The mixture was filtered through a pad of Celite and the filtrate was diluted with water (5 mL) and extract with Ethyl acetate (10mL) . The organic phase was washed with brine, dried, and concentrated to obtain the residue, which was purified by silica-gel column chromatography eluting with Petroleum ether/Ethyl acetate (8/1) to give ethyl 2-bromo-8-cyclopropyl-imidazo [1, 2-b] pyridazine-7-carboxylate (118 mg, 380.47 μmol, 66.39%yield) as a white solid. LC-MS: m/z 310.1 [M+H] ⁺.

Step 6

To a solution of ethyl 2-bromo-8-cyclopropyl-imidazo [1, 2-b] pyridazine-7-carboxylate 8f (118 mg, 380.47 μmol) in Ethanol (4 mL) at 0 ℃ was added lithium hydroxide monohydrate (1 M, 1.90 mL) . The mixture was stirred at room temperature for 10 h. Upon removal of solvent, the residue was acidified with HCl (2 M) to pH = 3～4. The mixture was diluted with H ₂O (10 mL) and extracted with ethyl acetate (10 mL x 2) . The combined organic layers were washed brine (10 mL) , dried over anhydrous sodium sulfate, filtered and concentrated to give 2-bromo-8-cyclopropyl-imidazo [1, 2-b] pyridazine-7-carboxylic acid (107 mg, crude) as a white solid. LC-MS: m/z 287 [M+H] ⁺.

Step 7

To a solution of 2-bromo-8-cyclopropyl-imidazo [1, 2-b] pyridazine-7-carboxylic acid 9e (107 mg, 379.31 μmol) in 1, 4-dioxane (7 mL) was added triethylamine (191.9 mg, 1.90 mmol, 264.34 uL) and [azido (phenoxy) phosphoryl] oxybenzene (125.3 mg, 455.2 μmol, 98.63 uL) at room temperature. The mixture was stirred for 30min. Then 5-chloro-6- (triazol-2-yl) pyridin-3-amine 10a (96.4 mg, 493.10 μmol) was added and the reaction mixture was stirred at 100 ℃ for 2 h. The mixture was concentrated to obtain the residue, which was purified by prep-TLC (Dichloromethane : Methanol=20: 1) to give 1- (2-bromo-8-cyclopropyl-imidazo [1, 2-b] pyridazin-7-yl) -3- [5-chloro-6- (triazol-2-yl) -3-pyridyl] urea (37.1 mg, 78.15 μmol, 20.60%yield) as a yellow solid. LC-MS: (ESI) m/z 474 [M+H] ⁺. ¹H NMR (400 MHz, DMSO-d ₆) δ 9.86 (s, 1H) , 9.04 (s, 1H) , 8.71 (s, 1H) , 8.58 (s, 1H) , 8.48 (s, 1H) , 8.38 (s, 1H) , 8.16 (d, J = 2.0 Hz, 2H) , 2.21-2.14 (m, 1H) , 1.76-1.72 (m, 2H) , 1.17-1.14 (m, 2H) .

Example 8. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (8-cyclopropylimidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a solution of ethyl 1- ( (tert-butoxycarbonyl) amino) -1H-imidazole-2-carboxylate 3 (1.5 g, 5.882 mmol) in THF (4.5 mL) at 0 ℃, was added dropwise a cold (pre-cooled at 0 ℃) potassium tert-butoxide (1 M, 17.6 mL) at 0 ℃ for 5 minutes. Dry ethyl acetate (1.29 g, 14.7 mmol, 1.43 mL) which was pre-cooled at 0 ℃, was added dropwise. Stirring was continued at 0℃ for 15 minutes. The ice bath was removed, and the resulting mixture was stirred at room temperature for 2 hours. The starting material was consumed and the desired mass was detected from LC-MS. The reaction mixture was cooled 0℃ and acidified with 1.0 N HCl via dropwise addition until complete dissolution of the suspension. It was extracted with ethyl acetate (2 ×40 mL) . The combined organic phase was washed with brine (100 mL) and dried over anhydrous Na ₂SO ₄, filtered and concentrated. The result residue was purified by flash column chromatography (eluting with ethyl acetate in petroleum from 0%to 20%) to give the (Z) -ethyl 3- (1- ( (tert-butoxycarbonyl) amino) -1H-imidazol-2-yl) -3-hydroxyacrylate (380 mg, 21.8%yield) as faint yellow solid. LC-MS: m/z 298 [M+H] ⁺.

Step 2

To a solution of (Z) -ethyl 3- (1- ( (tert-butoxycarbonyl) amino) -1H-imidazol-2-yl) -3-hydroxyacrylate 5a (380 mg, 1.279 mmol) in DCM (10 mL) was added 1, 1-dimethoxy- N, N-dimethyl-methanamine (762 mg, 6.397 mmol) . The reaction mixture was stirred at rt for 13 hours. The starting material was consumed and the desired mass was detected from LC-MS. The mixture was concentrated and the residue was purified with flash chromatography (eluting with Methanol/Dichloromethane=10/1) to get the ethyl 8-hydroxyimidazo [1, 2-b] pyridazine-7-carboxylate (122 mg, 45.9%yield) as yellow oil-solid. LC-MS: m/z 208 [M+H] ⁺.

Step 3

To a solution of ethyl 8-hydroxyimidazo [1, 2-b] pyridazine-7-carboxylate 6a (122 mg, 0.5894 mmol) in 1, 4-Dioxane (8 mL) at rt was added Phosphorus oxybromide (506.9 mg, 1.768 mmol) . The reaction mixture was stirred at 110℃ for 2 hours . The starting material was consumed, and the desired mass was detected from LC-MS. The reaction mixture was cooled to rt and poured into water, extracted with ethyl acetate (2 ×15 mL) . The combined organic phase was washed with brine (10 mL) and dried over anhydrous Na ₂SO ₄, filtered and concentrated. The crude product was purified by flash column chromatography (eluting with ethyl acetate in petroleum from 1%to 25%) to afford the ethyl 8-bromoimidazo [1, 2-b] pyridazine-7-carboxylate (110 mg, 69.4%yield) as a white solid. LC-MS: m/z 270 [M+H] ⁺.

Step 4

To a solution of ethyl 8-bromoimidazo [1, 2-b] pyridazine-7-carboxylate 7a (110.0 mg, 0.4089 mmol) in toluene (5 mL) was added cyclopropylboronic acid (70.3 mg, 0.8178 mmol) , tricyclohexylphosphine (114.5 mg, 0.4089 mmol) and potassium phosphate (216.7 mg, 1.022 mmol) . The suspension was degassed and exchanged with N ₂ two times. The palladium (II) acetate (18.3 mg, 0.08178 mmol) was added into the mixture. The reaction mixture was heated to 100 ℃ for 12 hours. The starting material was consumed and the desired mass was detected from LC-MS. The mixture was concentrated under reduced pressure and the result residue was purified with flash column chromatography (eluting with ethyl acetate in petroleum from 1%to 15%) to give the ethyl 8-cyclopropylimidazo [1, 2-b] pyridazine-7-carboxylate (34.0 mg, 36%yield) as a yellow solid. LC-MS: m/z 232 [M+H] ⁺.

Step 5

To a solution of ethyl 8-cyclopropylimidazo [1, 2-b] pyridazine-7-carboxylate 8g (67 mg, 0.29 mmol) in THF (4 mL) was added the solution of lithium hydroxide monohydrate (30.4 mg, 0.7251 mmol) in water (0.7 mL) . The mixture was stirred at 30 ℃ for 4 hours. The starting material was consumed and the desired mass was detected from LC-MS. It was concentrated to remove the solvents, the residue was acidified with HCl (1 M) to pH = 3～4. The mixture was extracted with ethyl acetate and the organic phase was dried, concentrated to give 8-cyclopropylimidazo [1, 2-b] pyridazine-7-carboxylic acid (55 mg, 93.4%yield) as white solid. LC-MS: m/z 204 [M+H] ⁺.

Step 6

To a solution of 8-cyclopropylimidazo [1, 2-b] pyridazine-7-carboxylic acid 9f (55 mg, 0.2709 mmol) in 1, 4-dioxane (4 mL) was added diphenylphosphonic azide (89.5 mg, 0.3251 mmol) and triethylamine (137.1 mg, 1.354 mmol) . The resulting mixture was stirred at room temperature for 30 minutes, then 5-chloro-6- (triazol-2-yl) pyridin-3-amine 10a (79.2 mg, 0.4064 mmol) was added and the mixture was heated to 100℃ for 2 hours. The mixture solution was poured into water and extracted with ethyl acetate twice. The organic was dried over Na ₂SO ₄, concentrated and the result residue was purified by flash column chromatography (eluting with ethyl acetate in petroleum from 10%to 80%) to give the 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (8-cyclopropylimidazo [1, 2-b] pyridazin-7-yl) urea (9.1 mg) as a white solid. LC-MS: m/z 396 [M+H] ⁺. ¹H NMR (DMSO-d6, 400 MHz) : δ 9.73 (s, 1 H) , 8.93 (s, 1 H) , 8.58-8.57 (m, 2 H) , 8.47 (d, J = 2.4 Hz, 1 H) , 8.15 (s, 2 H) , 8.13 (d, J = 1.2 Hz, 1 H) , 7.61 (d, J = 1.2 Hz, 1 H) , 2.22-2.18 (m, 1 H) , 1.89-1.86 (m, 2 H) , 1.15-1.10 (m, 2 H) .

Example 9. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (8-propylimidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a solution of 2-chloro-8-cyclopropylimidazo [1, 2-b] pyridazine-7-carboxylic acid 9a (60 mg, 0.2532 mmol) in methanol (3 mL) was added Pd/C (30 mg) . The reaction mixture was stirred at room temperature for 3 hours under H ₂. The starting material was consumed and the desired mass was detected from LC-MS. It was filtered and the filtrated was concentrated under reduced pressure. The crude product was purified by flash column chromatography (eluting with ethyl acetate in petroleum from 0%to 80%) to give the 8-propylimidazo [1, 2-b] pyridazine-7-carboxylic acid (50 mg, 96.3%yield) as white solid. LC-MS: m/z 206 [M+H] ⁺.

Step 2

To a solution of 8-propylimidazo [1, 2-b] pyridazine-7-carboxylic acid 9g (50 mg, 0.2439 mmol) in 1, 4-dioxane (4 mL) was added diphenylphosphonic azide (87.2 mg, 0.3171 mmol) and triethylamine (123.4 mg, 1.219 mmol) . The resulting mixture was stirred at room temperature for 30 minutes, then 5-chloro-6- (triazol-2-yl) pyridin-3-amine 10a (71.3 mg, 0.366 mmol) was added and the mixture was heated to 100℃ for 2 hours. The mixture solution was poured into water and extracted with ethyl acetate twice. The organic was dried over Na ₂SO ₄, concentrated and the result residue was purified by flash column chromatography (eluting with ethyl acetate in petroleum from 10%to 80%) to give the 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (8-propylimidazo [1, 2-b] pyridazin-7-yl) urea (15.7 mg) as a white solid. LC-MS: m/z 398 [M+H] ⁺. ¹H NMR (DMSO-d6, 400 MHz) : δ 9.75 (s, 1 H) , 8.77 (s, 1 H) , 8.76 (s, 1 H) , 8.57 (d, J = 2.4 Hz, 1 H) , 8.48 (d, J = 2.4 Hz, 1 H) , 8.18 (d, J = 1.2 Hz, 1 H) , 8.15 (s, 2 H) , 7.67 (d, J = 1.6 Hz, 1 H) , 2.98-2.94 (m, 2 H) , 1.73-1.69 (m, 2 H) , 0.99-0.96 (m, 3 H) .

Example 10. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloroimidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a solution of ethyl 8-bromo-2-chloroimidazo [1, 2-b] pyridazine-7-carboxylate 7c (50 mg, 0.165 mmol) in ethanol (8 mL) was added Pd/C (5 mg) . The reaction mixture was stirred at room temperature for 3 hours under H ₂. The starting material was consumed and the desired mass was detected from LC-MS. It was filtered and the filtrated was concentrated under reduced pressure. The crude product was purified by flash column chromatography (eluting with ethyl acetate in petroleum from 0%to 30%) to give the ethyl 2-chloroimidazo [1, 2-b] pyridazine-7-carboxylate (25 mg, 67.4%yield) as white solid. LC-MS: m/z 226 [M+H] ⁺.

Step 2

To a solution of ethyl 2-chloroimidazo [1, 2-b] pyridazine-7-carboxylate 15c (42 mg, 0.1867 mmol) in THF (2 mL) was added the solution of lithium hydroxide monohydrate (15.7 mg, 0.373 mmol) in water (0.3 mL) . The mixture was stirred at 30 ℃ for 4 hours. The starting material was consumed and the desired mass was detected from LC-MS. The resulting mixture was concentrated to remove the solvents; the residue was acidified with HCl (1 M) to pH = 3～4. The mixture was extracted with ethyl acetate and the organic phase was dried, concentrated to give the 2-chloroimidazo [1, 2-b] pyridazine-7-carboxylic acid (35 mg, 95.1%) as yellow solid. LC-MS: m/z 198 [M+H] ⁺.

Step 3

To a solution of 2-chloroimidazo [1, 2-b] pyridazine-7-carboxylic acid 16c (35 mg, 0.1777 mmol) in 1, 4-dioxane (4 mL) was added diphenylphosphonic azide (58.7 mg, 0.213 mmol) and triethylamine (89.9 mg, 0.8885 mmol) . The resulting mixture was stirred at room temperature for 30 minutes, then 5-chloro-6- (triazol-2-yl) pyridin-3-amine 10a (52 mg, 0.267 mmol) was added and the mixture was heated to 100℃ for 2 hours. The mixture solution was poured into water and extracted with ethyl acetate twice. The organic was dried over Na ₂SO ₄, concentrated and the result residue was purified by flash column chromatography (eluting with ethyl acetate in petroleum from 10%to 100%) to give the 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloroimidazo [1, 2-b] pyridazin-7-yl) urea (10.2 mg) as a white solid. LC-MS: m/z 390 [M+H] ⁺. ¹H NMR (DMSO-d6, 400 MHz) : δ 9.87 (s, 1 H) , 9.75 (s, 1 H) , 8.63-8.61 (m, 2 H) , 8.46 (d, J = 2.4 Hz, 1 H) , 8.34 (s, 1 H) , 8.16-8.13 (m, 3 H) .

Example 11. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2, 8-dichloroimidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a solution of ethyl 2-chloro-8-hydroxyimidazo [1, 2-b] pyridazine-7-carboxylate 6c (200 mg, 0.8299 mmol) in THF (4 mL) was added the solution of lithium hydroxide monohydrate (69.7 mg, 1.66 mmol) in water (1.6 mL) . The mixture was stirred at 70 ℃ for 14 hours. The starting material was consumed and the desired mass was detected from LC-MS. It was concentrated to remove the solvent, the residue was acidified with HCl (1 M) to pH = 3～4. The mixture was extracted with ethyl acetate and the organic phase was dried, concentrated to give the 2-chloro-8-hydroxyimidazo [1, 2-b] pyridazine-7-carboxylic acid (160 mg, 90.5%) as white solid. LC-MS: m/z 214 [M+H] ⁺.

Step 2

The solution of 2-chloro-8-hydroxyimidazo [1, 2-b] pyridazine-7-carboxylic acid 9h (187.0 mg, 929.1 μmol) in POCl ₃ (6 mL) was heated at 120 ℃ for 12 hours. The starting material was consumed and the desired mass was detected from LC-MS. The reaction solution was concentrated and the residue was quenched with water (10 mL) and extracted with DCM (25 mL x 2) . The organic layer was washed with brine (20 mL) , dried over anhydrous Na ₂SO ₄ and concentrated under reduced pressure. The residue was purified by flash chromatography (eluting with methanol/dichloromethane=10/1) to get the crude product of 2, 8-dichloroimidazo [1, 2-b] pyridazine-7-carboxylic acid (150 mg, crude) as a white solid. LC-MS: m/z 232 [M+H] ⁺.

Step 3

To a solution of 2, 8-dichloroimidazo [1, 2-b] pyridazine-7-carboxylic acid 9i (50 mg, 0.2164 mmol) in 1, 4-dioxane (5 mL) was added diphenylphosphonic azide (71.5 mg, 0.2597 mmol) and triethylamine (109.5 mg, 1.082 mmol) . The resulting mixture was stirred at room temperature for 30 minutes, then 5-chloro-6- (triazol-2-yl) pyridin-3-amine 10a (63.3 mg, 0.3246 mmol) was added and the mixture was heated at 100℃ for 2 hours. The mixture solution was poured into water and extracted with ethyl acetate twice. The organic was dried over Na ₂SO ₄, concentrated and the result residue was purified by flash column chromatography (eluting with ethyl acetate in petroleum from 10%to 100%) to give the 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2, 8-dichloroimidazo [1, 2-b] pyridazin-7-yl) urea (3.5 mg) as a white solid. LC-MS: m/z 424 [M+H] ⁺. ¹H NMR (DMSO-d6, 400 MHz) : δ 10.11 (s, 1 H) , 9.25 (s, 2 H) , 8.57 (d, J = 2.4 Hz, 1 H) , 8.52 (s, 1 H) , 8.48 (d, J = 2.4 Hz, 1 H) , 8.16 (s, 2 H) .

Example 12. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8-methylimidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a solution of ethyl 8-bromo-2-chloro-imidazo [1, 2-b] pyridazine-7-carboxylate 7c (200 mg, 656.76 μmol) , methylboronic acid (147.4 mg, 2.46 mmol) and tricyclohexylphosphine (230.2 mg, 820.94 μmol) in 1, 4-dioxane (5 mL) were added potassium phosphate (522.8 mg, 2.46 mmol) and palladium (II) acetate (92.2 mg, 410.47 μmol) . The suspension was stirred at 100 ℃ for 5 h under N ₂. After cooling down to room temperature, the reaction mixture was filtered and the filtrate was concentrated to give a residue, which was purified by flash column chromatography (eluting with PE/EA = 10/1) to give ethyl 2-chloro-8-methyl-imidazo [1, 2-b] pyridazine-7-carboxylate (68 mg, 283.74 μmol, 43.20%yield) as a white solid. LC-MS: m/z 240 [M+H] ⁺.

Step 2

To a solution of ethyl 2-chloro-8-methyl-imidazo [1, 2-b] pyridazine-7-carboxylate 8h (85 mg, 354.67 μmol) in THF (3 mL) was added lithium hydroxide monohydrate (29.8 mg, 709.34 μmol) in water (0.7 mL) . The mixture was stirred at room temperature for 5 h. Upon removal of the solvents, the residue was acidified with HCl (2 M) to pH = 3～4. The mixture was extracted by EtOAc and the organic phase was dried and concentrated to give 2-chloro-8-methyl-imidazo [1, 2-b] pyridazine-7-carboxylic acid (74 mg, 99%yield) as a white solid. LC-MS: m/z 212 [M+H] ⁺.

Step 3

To a solution of 2-chloro-7-methyl-pyrazolo [1, 5-a] pyrimidine-6-carboxylic acid 9j (74 mg, 349.71 μmol) in 1, 4-dioxane (6 mL) were added diphenylphosphonic azide (108.8 mg, 419.65 μmol) and triethylamine (176.9 mg, 1.75 mmol) . The resulting solution was stirred at room temperature for 30 minutes, then 5-chloro-6- (triazol-2-yl) pyridin-3-amine 10a (102.6 mg, 524.56 μmol) was added and the mixture was stirred at 100 ℃ for 2 hours. Solvent was removed and the residue was purified by flash column chromatography (eluting with DCM: MeOH = 30 : 1) and prep-HPLC to give 1- (2-chloro-7-methyl-pyrazolo [1, 5-a] pyrimidin-6-yl) -3- [5-chloro-6- (triazol-2-yl) -3-pyridyl] urea (33 mg, 23%yield) . LC-MS: m/z 403.8. ¹H NMR (DMSO, 400 MHz) : δ 9.79-9.12 (m, 2H) , 8.89 (s, 1H) , 8.58 (d, J = 1.6 Hz, 1H) , 8.48 (d, J = 2.0 Hz, 1H) , 8.37 (s, 1H) , 8.16 (s, 2H) , 2.47 (s, 3H) .

Synthesis of compounds of formula II

Example 13. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8-methoxyimidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a stirred solution of ethyl 2-chloro-8-hydroxy-imidazo [1, 2-b] pyridazine-7-carboxylate 6c (100 mg, 0.41 mmol) and potassium carbonate (171 mg, 1.24 mmol) in DMF (5 mL) at room temperature was added iodomethane (176.2 mg, 1.24 mmol, 77.29 uL) . The reaction mixture was stirred at room temperature for 2 hours, and upon completion of the reaction, the mixture was concentrated under vacuo. The crude product was purified by flash column chromatography (SiO ₂, DCM/MeOH = 10: 1) to afford the ethyl 2-chloro-8-methoxy- imidazo [1, 2-b] pyridazine-7-carboxylate (90 mg, 352.03 μmol, 85%yield) as a yellow solid. LC-MS: m/z 256.1 [M+H] ⁺

Step 2

To a stirred solution of ethyl 2-chloro-8-methoxy-imidazo [1, 2-b] pyridazine-7-carboxylate 11a (90 mg, 352.03 μmol) in EtOH (2 mL) and H ₂O (2 mL) was added LiOH H ₂O (73.9 mg, 1.76 mmol) . The reaction mixture was stirred at room temperature for 3 hours and upon completion of the reaction, the mixture was diluted with EtOAc (5 mL) and adjust pH = 2 with 1N HCl. The layers were separated, and the aqueous layer was extracted with EtOAc (2 × 5 mL) . The combined organic phase was washed with brine (100 mL) and dried over anhydrous Na ₂SO ₄, filtered and concentrated in vacuo. The crude product 2-chloro-8-methoxy-imidazo [1, 2-b] pyridazine-7-carboxylic acid (73 mg, 320.73 μmol, 91%yield) was a yellow solid and used for the next step directly. LC-MS: m/z 226.0 [M-H] ⁺.

Step 3

To a stirred solution of 2-chloro-8-methoxy-imidazo [1, 2-b] pyridazine-7-carboxylic acid 12a (65 mg, 285.54 μmol) in 1, 4-dioxane (4 mL) solution was added diphenylphosphoryl azide (102.2 mg, 371.20 μmol, 80.44 uL) , Et ₃N (1.43 mmol) . The reaction mixture was stirred at room temperature for 30 min, then 5-chloro-6- (triazol-2-yl) pyridin-3-amine 10a (111.7 mg, 571.08 μmol) as added and the mixture was stirred for 2 h at 100 ℃, and upon completion of the reaction, the mixture was diluted with EtOAc (4 mL) , the organic phase was washed with brine (100 mL) and dried over anhydrous Na ₂SO ₄, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography (SiO ₂, DCM/MeOH = 20: 1) to afford the 1- (2-chloro-8-methoxy-imidazo [1, 2-b] pyridazin-7-yl) -3- [5-chloro-6- (triazol-2-yl) -3-pyridyl] urea (10.3 mg, 24.51 μmol, 8%yield) as a white solid. LC-MS: m/z 408.0 [M+H] ⁺. ¹H NMR (400 MHz, DMSO-d ₆) : 10.27 (s, 1H) , 9.15 (s, 1H) , 8.73 (s, 1H) , 8.49-8.46 (m, 3H) , 8.15 (s, 2H) , 4.25 (s, 3H) .

Example 14. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8-ethoxyimidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a stirred solution of ethyl 2-chloro-8-hydroxy-imidazo [1, 2-b] pyridazine-7-carboxylate 6c (200 mg, 827.71 μmol) in DMF (5 mL) at room temperature was added iodoethane (387.3 mg, 2.48 mmol, 199.63 uL) . The reaction mixture was stirred at 80 ℃ for 2 hours under microwave radiation , upon completion of the reaction, the mixture was concentrated under vacuo. The crude product was purified by flash column chromatography (SiO ₂, DCM/MeOH = 10: 1) to afford the ethyl 2-chloro-8-ethoxy-imidazo [1, 2-b] pyridazine-7-carboxylate (103 mg, 381.93 μmol, 46%yield) as yellow solid. LC-MS: m/z 270.1 [M+H] ⁺

Step 2

To a stirred solution of ethyl 2-chloro-8-ethoxy-imidazo [1, 2-b] pyridazine-7-carboxylate 11b (103 mg, 381.93 μmol) in EtOH (2 mL) and H ₂O (2 mL) was added LiOH H ₂O (73.9 mg, 1.76 mmol) . The reaction mixture was stirred at room temperature for 3 hours, and upon completion of the reaction, the mixture was diluted with EtOAc (5 mL) and adjust pH = 2 with 1N HCl. The layers were separated, and the aqueous layer was extracted with EtOAc (2 × 5 mL) . The combined organic phase was washed with brine (100 mL) and dried over anhydrous Na ₂SO ₄, filtered and concentrated in vacuo. The crude product 2-chloro-8-ethoxy-imidazo [1, 2-b] pyridazine-7-carboxylic acid (70 mg, 289.70 μmol, 76%yield) was a yellow solid and used for the next step directly. LC-MS: m/z 240.0 [M-H] ⁺

Step 3

To a stirred solution of 2-chloro-8-ethoxy-imidazo [1, 2-b] pyridazine-7-carboxylic acid 12b (70 mg, 289.70 μmol) in 1, 4-dioxane (4 mL) was added diphenylphosphoryl azide (159.5 mg, 579.40 μmol, 125.55 uL) , Et ₃N (146.3 mg, 1.45 mmol) . The reaction mixture was stirred at room temperature for 30 min, then 5-chloro-6- (triazol-2-yl) pyridin-3-amine 10a (113.3 mg, 579.40 μmol) was added and the mixture was stirred for 2 h at 100 ℃, and upon completion of the reaction, the mixture was diluted with EtOAc (4 mL) , the organic phase was washed with brine (100 mL) and dried over anhydrous Na ₂SO ₄, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography (SiO ₂, DCM/MeOH = 20: 1) to afford the 1- (2-chloro-8-ethoxy-imidazo [1, 2-b] pyridazin-7-yl) -3- [5-chloro-6- (triazol-2-yl) -3-pyridyl] urea (8.6 mg, 19.80 μmol, 6.84%yield) as a white solid. LC-MS: m/z 434.0 [M+H] ⁺. ¹H NMR (400 MHz, DMSO-d ₆) : 10.27-10.26 (m, 1H) , 9.14 (s, 1H) , 8.76 (s, 1H) , 8.50-8.47 (m, 3H) , 8.15 (s, 2H) , 4.79 (q, J = 6.8 Hz, 2H) , 1.40 (t, J = 6.8 Hz, 3H) .

Example 15. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8-isopropoxyimidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

o a stirred solution of ethyl 2-chloro-8-hydroxy-imidazo [1, 2-b] pyridazine-7-carboxylate 6c (350 mg, 1.45 mmol) and cesium carbonate (1.42 g, 4.35 mmol) in DMF (5 mL) at room temperature was added 2-bromopropane (1.78 g, 14.48 mmol, 248.30 uL) . The resulting mixture was stirred at 120℃ for 2 hours under microwave radiation, and upon completion of the reaction, the mixture was concentrated in vacuo. The crude product was purified by flash column chromatography (SiO ₂, DCM/MeOH = 10: 1) to afford the ethyl 2-chloro-8-isopropoxy-imidazo [1, 2-b] pyridazine-7-carboxylate (82 mg, 289.03 μmol, 20%yield) as a yellow oil. LC-MS: m/z 84.1 [M+H] ⁺.

Step 2

To a stirred solution of ethyl 2-chloro-8-isopropoxy-imidazo [1, 2-b] pyridazine-7-carboxylate 11c (82 mg, 289.03 μmol) in EtOH (2 mL) and H ₂O (2 mL) was added LiOH H ₂O (60.70 mg, 1.45 mmol) . The reaction mixture was stirred at room temperature for 3 hours, and upon completion of the reaction, the mixture was diluted with EtOAc (5 mL) and adjust pH = 2 with 1N HCl. The layers were separated, and the aqueous layer was extracted with EtOAc (2 × 5 mL) . The combined organic phase was washed with brine (100 mL) and dried over anhydrous Na ₂SO ₄, filtered and concentrated in vacuo. The crude product 2-chloro-8-isopropoxy-imidazo [1, 2-b] pyridazine-7-carboxylic acid (67 mg, 262.07 μmol, 91%yield) was a yellow solid and used for the next step directly. LC-MS: m/z 254.0 [M-H] ⁺.

Step 3

To a stirred solution of 2-chloro-8-isopropoxy-imidazo [1, 2-b] pyridazine-7-carboxylic acid 12c (21.2 mg, 82.77 μmol) in 1, 4-dioxane (4 mL) solution was added diphenylphosphoryl azide (45.6 mg, 165.54 μmol, 35.87 uL) , Et ₃N (41.80 mg, 413.85 μmol) . The reaction mixture was stirred at room temperature for 30 min, then 5-chloro-6- (triazol-2-yl) pyridin-3-amine 10a (32.4 mg, 165.54 μmol) was added and the mixture was stirred for 2 h at 100 ℃ and upon completion of the reaction, the mixture was diluted with EtOAc (4 mL) , the organic phase was washed with brine (100 mL) and dried over anhydrous Na ₂SO ₄, filtered and concentrated under vacuo. The crude product was purified by flash column chromatography (Silica gel, DCM/MeOH = 20: 1) to afford the 1- (2-chloro-8-isopropoxy-imidazo [1, 2-b] pyridazin-7-yl) -3- [5-chloro-6- (triazol-2-yl) -3-pyridyl] urea (11.3 mg, 25.21 μmol, 30%yield) as a white solid. LC-MS: m/z 448.0 [M+H] ⁺. ¹H NMR (400 MHz, DMSO-d ₆) : 10.29 (s, 1H) , 9.15 (s, 1H) , 8.78 (s, 1H) , 8.50-8.47 (m, 3H) , 8.15 (s, 2H) , 3.35-3.29 (m, 1H) 1.68 (d, J = 6.4 Hz, 2H) .

Synthesis of compounds of formula III

Example 16. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (cyclopropyl (methyl) amino) imidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a solution of ethyl 8-bromo-2-chloro-imidazo [1, 2-b] pyridazine-7-carboxylate 7c (200 mg, 656.76 μmol) in THF (4 mL) was added N-methylcyclopropanamine (141.3 mg, 1.31 mmol, 364.05 uL, HCl) . The suspension was stirred at room temperature for 3 hours. LCMS showed most of reactant was consumed. The solvent was removed to give ethyl 2-chloro-8- [cyclopropyl (methyl) amino] imidazo [1, 2-b] pyridazine-7-carboxylate (186 mg, 631.07 μmol, 96.09%yield) as a yellow solid. LC-MS: m/z 295.1 [M+H] ⁺.

Step 2

To a solution of ethyl 2-chloro-8- [cyclopropyl (methyl) amino] imidazo [1, 2-b] pyridazine-7-carboxylate 13a (186 mg, 631.07 μmol) in Ethanol (4 mL) at 0 ℃ was added lithium hydroxide monohydrate (1 M, 1.89 mL) . The mixture was stirred at room temperature for 10 h. Upon removal of solvent, the residue was acidified with HCl (2 M) to pH = 3～4. The suspension was filtered and the filter cake was dried in vacuum to give 2-chloro-8- [cyclopropyl (methyl) amino] imidazo [1, 2-b] pyridazine-7-carboxylic acid (168 mg, 629.96 μmol, 99.82%yield) as a white solid.

Step 3

To a solution of 2-chloro-8- [cyclopropyl (methyl) amino] imidazo [1, 2-b] pyridazine-7-carboxylic acid 14a (157 mg, 588.71 μmol) in 1, 4-Dioxane (7 mL) were added triethylamine (297.9 mg, 2.94 mmol, 410.27 uL) and [azido (phenoxy) phosphoryl] oxybenzene (194.42 mg, 706.46 μmol, 153.08 uL) at room temperature. The mixture was stirred for 30 mins. Then 5- chloro-6- (triazol-2-yl) pyridin-3-amine 10a (149.70 mg, 765.33 μmol) was added and the reaction mixture was stirred at 100 ℃ for 2 h. The mixture was concentrated to obtain the residue, which was purified by prep-TLC (Dichloromethane : Methanol=20: 1) to give 1- [2-chloro-8- [cyclopropyl (methyl) amino] imidazo [1, 2-b] pyridazin-7-yl] -3- [5-chloro-6- (triazol-2-yl) -3-pyridyl] urea (42.7 mg, 92.97 μmol, 15.79%yield) as a white solid. LC-MS: m/z 459 [M+H] ⁺. ¹H NMR (DMSO-d ₆, 400 MHz) δ 9.73 (s, 1H) , 8.55-8.47 (m, 4H) , 8.23 (d, J =6.4 Hz, 1H) , 8.15 (s, 2H) , 3.22-3.17 (m, 4H) , 0.67-0.51 (m, 4H) .

Example 17. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (dimethylamino) imidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a solution of ethyl 8-bromo-2-chloro-imidazo [1, 2-b] pyridazine-7-carboxylate 7c (300 mg, 985.13 μmol) in THF (10 mL) was added N-methylmethanamine (2 M, 2.46 mL) . The suspension was stirred at room temperature for 1h. TLC showed most of reactant was consumed and one main spot was present. The mixture was concentrated to obtain the residue, which was purified by silica-gel column chromatography eluting with Petroleum ether/Ethyl acetate (10/1) to give ethyl 2-chloro-8- (dimethylamino) imidazo [1, 2-b] pyridazine-7-carboxylate (250 mg, 930.41 μmol, 94.45%yield) as a yellow solid. LC-MS: m/z 269.1 [M+H] ⁺.

Step 2

To a solution of ethyl 2-chloro-8- (dimethylamino) imidazo [1, 2-b] pyridazine-7-carboxylate 13b (100 mg, 372.16 μmol) in ethanol/water (5/5 mL) at 0 ℃ was added lithium hydroxide monohydrate (1 M, 1.12 mL) . The mixture was stirred at room temperature for 10h.

Upon removal of solvent, the residue was acidified with HCl (2 M) to pH = 3～4. The suspension was filtered and the filter cake was dried in vacuum to give 2-chloro-8- (dimethylamino) imidazo [1, 2-b] pyridazine-7-carboxylic acid (89.6 mg, crude) as a yellow solid, which was used directly for next step.

Step 3

o a solution of 2-chloro-8- (dimethylamino) imidazo [1, 2-b] pyridazine-7-carboxylic acid 14b (82 mg, 340.75 μmol) in 1, 4-Dioxane (7 mL) was added triethylamine (172.4 mg, 1.70 mmol, 237.47 uL) and [azido (phenoxy) phosphoryl] oxybenzene (112.5 mg, 408.90 μmol, 88.61 uL) at room temperature. And then the mixture was stirred for 30min. Then 5-chloro-6- (triazol-2-yl) pyridin-3-amine (86.7 mg, 442.97 μmol) was added and the reaction mixture was stirred at 100 ℃ for 3 h. The mixture was concentrated to obtain the residue, which was purified by prep-HPLC to give 1- [2-chloro-8- (dimethylamino) imidazo [1, 2-b] pyridazin-7-yl] -3- [5-chloro-6- (triazol-2-yl) -3-pyridyl] urea (7.0 mg, 16.16 μmol, 4.74%yield) as a white solid. LC-MS: m/z 433 [M+H] ⁺. ¹H NMR (400 MHz, DMSO-d ₆) δ 9.75 (s, 1H) , 8.57 (d, J = 1.6 Hz, 2H) , 8.46 (d, J = 1.6 Hz, 1H) , 8.32 (s, 1H) , 8.23 (s, 1H) , 8.15 (s, 2H) , 3.24 (s, 6H) .

Example 18. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8-morpholinoimidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a mixture of ethyl 8-bromo-2-chloro-imidazo [1, 2-b] pyridazine-7-carboxylate 7c (100.0 mg, 328.4 μmol) in THF (3 mL) was added morpholine (57.2 mg, 656.8 μmol) at 25 ℃. The reaction mixture was stirred at 25 ℃ overnight. The solid was filtered off and filtrate was concentrated under vacuo. The residue was purified by column chromatography (eluting with PE/EA=10/1) to get ethyl 2-chloro-8-morpholino-imidazo [1, 2-b] pyridazine-7-carboxylate (82 mg, 80.36%yield) as a yellow solid. LC-MS: m/z 311 [M+H] ⁺.

Step 2

To a solution of ethyl 2-chloro-8-morpholino-imidazo [1, 2-b] pyridazine-7-carboxylate 13c (60 mg, 193.09 μmol) in THF (6 mL) and Ethanol (1 mL) was added lithium hydroxide monohydrate (81.0 mg, 1.93 mmol) in water (1 mL) . The mixture was stirred at 60 ℃ for 48 hours. Upon removal of the solvents, the residue was acidified with HCl (2 M) to pH = 3～4. The mixture was extracted by EtOAc and the organic phase was dried, concentrated to give 2-chloro-8-morpholino-imidazo [1, 2-b] pyridazine-7-carboxylic acid (54.6 mg, quant. ) as a white solid, which was used directly for next step. LC-MS: m/z 267 [M+H] ⁺.

Step 3

To a solution of 2-chloro-7-morpholino-pyrazolo [1, 5-a] pyrimidine-6-carboxylic acid 14c (54 mg, 191.03 μmol) in 1, 4-Dioxane (6 mL) was added diphenylphosphonic azide (59.4 mg, 229.23 μmol, 46.79 μL) and triethylamine (96.6 mg, 955.13 μmol, 133.13 μL) . The resulting suspension was stirred at room temperature for 30 minutes. Then 5-chloro-6- (triazol-2-yl) pyridin-3-amine (56.1 mg, 286.54 μmol) was added and the mixture was stirred at 100 ℃for 2 hours. Solvent was removed and the residue was purified by flash column chromatography (eluting with DCM: MeOH = 30 : 1) and prep-HPLC to give 1- (2-chloro-7-morpholino-pyrazolo [1, 5-a] pyrimidin-6-yl) -3- [5-chloro-6- (triazol-2-yl) -3-pyridyl] urea (18 mg, 19.83%yield) as a white solid. LC-MS: m/z 475.0 [M+H] ⁺. ¹H NMR (DMSO-d6, 400 MHz) : δ 9.89 (brs, 1H) , 8.58 (d, J = 2.4 Hz, 2H) , 8.50 (s, 1H) , 8.48 (d, J = 2.4 Hz, 1H) , 8.29 (s, 1H) , 8.15 (s, 2H) , 3.78 (t, J = 4.0 Hz, 4H) , 3.78 (t, J = 4.0 Hz, 4H) .

Example 19. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- ( (2-methoxyethyl) (methyl) amino) imidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a solution of ethyl 8-bromo-2-chloro-imidazo [1, 2-b] pyridazine-7-carboxylate 7c (90 mg, 0.3 mmol) in THF (1 mL) was added 2-methoxy-N-methyl-ethanamine (131.7 mg, 1.48 mmol) at room temperature. The resulting mixture was stirred at room temperature for 2 h. TLC showed that the reaction was completed. The reaction mixture was concentrated and purified by flash silica gel chromatography (0～10%EtOAc in PE) to give ethyl 2-chloro-8- [2-methoxyethyl (methyl) amino] imidazo [1, 2-b] pyridazine-7-carboxylate (90 mg, 97%yield) as a white solid. LC-MS: m/z 313.1 [M+H] ⁺.

Step 2

A mixture of ethyl 2-chloro-8- [2-methoxyethyl (methyl) amino] imidazo [1, 2-b] pyridazine-7-carboxylate 13d (100 mg, 319.7 μmol) and lithium hydroxide (153.2 mg, 6.4 mmol) in THF (3 mL) , Water (2 mL) and methanol (2 mL) was stirred at 50 ℃ for 12 h. LC-MS showed that the reaction was completed. The solvent was removed under reduced pressure and the residue was dissolved in water (10 mL) and extracted with EA (5 mLx2) . The aqueous was acidized with 1 M HCl to pH ～ 6, extracted with DCM (10 mLx5) and the combined organic layers were dried over anhydrous Na ₂SO _4, filtered and concentrated to give 2-chloro-8- [2-methoxyethyl (methyl) amino] imidazo [1, 2-b] pyridazine-7-carboxylic acid (70 mg, 77%yield) as yellow solid, which was used in next step directly. LC-MS: m/z 285.1 [M+H] ⁺.

Step 3

To a solution of 2-chloro-8- [2-methoxyethyl (methyl) amino] imidazo [1, 2-b] pyridazine-7-carboxylic acid 14d (70 mg, 245.9 μmol) in 1, 4-dioxane was added [azido (phenyl) phosphoryl] benzene (77.7 mg, 319.6 μmol) and triethylamine (124.4 mg, 1.23 mmol) at room temperature. The resulting mixture was stirred for 30 min. 5-Chloro-6- (triazol-2-yl) pyridin-3-amine 10a (72.1 mg, 368.8 μmol) was added and the mixture was stirred at 100 ℃ for 2 hours. LC-MS showed that the reaction was completed, diluted with Ethyl acetate (20 mL) , washed with brine (10mLx2) . The organic layer was concentrated and purified by Prep-TLC (eluting with DCM: MeOH = 20: 1) to give 1- [2-chloro-8- [2-methoxyethyl (methyl) amino] imidazo [1, 2-b] pyridazin-7-yl] -3- [5-chloro-6- (triazol-2-yl) -3-pyridyl] urea (14.8 mg, 13%yield) as a white solid. LC-MS: m/z 477.0 [M+H] ⁺. ¹H NMR (400 MHz, DMSO-d ₆) : δ 9.79 (s, 1 H) , 8.57 (d, J = 2.4 Hz, 1H) , 8.50 (s, 1 H) , 8.47 (d, J = 2.0 Hz, 1 H) , 8.42 (s, 1 H) , 8.25 (s, 1 H) , 8.15 (s, 2 H) , 3.90 (t, J = 5.2 Hz, 2 H) , 3.55 (t, J = 5.6 Hz, 2 H) , 3.19-3.18 (m, 6 H) .

Example 20. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (7-cyclopropyl-2-methyl-2H-pyrazolo [4, 3-b] pyridin-6-yl) urea

Step 1

To a solution of methyl 4-amino-1-methyl-1H-pyrazole-3-carboxylate (2.0 g, 12.9 mmol) in DMF (120 mL) at room temperature was added di-tert-butyl dicarbonate (3.38 g, 15.44 mmol) . The reaction mixture was heated to 60 ℃ for 5 hours. The starting material was consumed and the desired mass was detected from LC-MS. The reaction mixture was poured into water and extracted with ethyl acetate for two times. The combined organic phase was dried over Na ₂SO ₄, washed with brine, filtered and concentrated. The resulting residue was purified by silical-gel column chromatography eluting with ethyl acetate in petroleum ether from 0%to 50%to give the methyl 4- (tert-butoxycarbonylamino) -1-methyl-pyrazole-3-carboxylate (2.5 g, 75.98%yield) . LC-MS: m/z 256 [M+H] ⁺.

Step 2

To a solution of methyl 4- (tert-butoxycarbonylamino) -1-methyl-pyrazole-3-carboxylate (2.5 g, 9.79 mmol) in Tetrahydrofuran (24 mL) was added a solution of Lithium hydroxide (586.4 mg, 24.48 mmol) in water (5 mL) at room temperature. The reaction mixture was stirred at room temperature for 4 hours. The starting material was consumed completely and the desired mass was detected from LC-MS. It was concentrated and acidified with HCl (1 N) to pH = 3～4. Then the reaction mixture was filtered and the filtrate cake was washed with water. And then it was dried over vacuo to give 4- (tert-butoxycarbonylamino) -1-methyl-pyrazole-3-carboxylic acid (2.3 g, 97.35%yield) as a white solid. LC-MS: m/z 242 [M+H] ⁺.

Step 3

To a solution of 4- (tert-butoxycarbonylamino) -1-methyl-pyrazole-3-carboxylic acid (2.3 g, 9.53 mmol) in Acetonitrile (8 mL) and Tetrahydrofuran (8 mL) was added 1, 1‘-Carbonyldiimidazole (2.32 g, 14.30 mmol) . The resulting reaction mixture was stirred at room temperature for 2 hours and magnesium chloride (907.6 mg, 9.53 mmol) , Triethylamine (2.89 g, 28.60 mmol, 3.99 mL) were added. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was poured into water and extracted with ethyl acetate two times. The combined organic phase was dried over Na ₂SO ₄, washed with brine, filtered and concentrated. The resulting residue was purified by silical-gel column chromatography eluting with ethyl acetate in petroleum ether from 0%to 30%to give the ethyl 3- [4- (tert-butoxycarbonylamino) -1-methyl-pyrazol-3-yl] -3-oxo-propanoate (2.26 g, 76.14%yield) . LC-MS: m/z 312 [M+H] ⁺.

Step 4

To a solution of ethyl 3- [4- (tert-butoxycarbonylamino) -1-methyl-pyrazol-3-yl] -3-oxo-propanoate (1.0 g, 3.21 mmol) in Dichloromethane (15 mL) at room temperature was added N, N-Dimethylformamide dimethyl acetal (3.06 g, 25.70 mmol, 3.44 mL) by dropwise. The reaction mixture was stirred at room temperature overnight. The starting material was consumed and the desired mass was detected from LC-MS. The reaction mixture was concentrated and the residue was purified with silical-gel column chromatography eluting with methanol in dichloromethane from 0%to 10%to give the ethyl 7-hydroxy-2-methyl-pyrazolo [4, 3-b] pyridine-6-carboxylate (603 mg, 84.87%yield) . LC-MS: m/z 222 [M+H] ⁺.

Step 5

A solution of ethyl 7-hydroxy-2-methyl-pyrazolo [4, 3-b] pyridine-6-carboxylate (300 mg, 1.36 mmol) in Phosphorus oxychloride (20.79 g, 135.62 mmol) was heated to 80 ℃ for 3 hours. LC-MS showed the starting material was consumed and the desired mass was detected. The reaction mixture was concentrated and the residue was purified by silical-gel column chromatography eluting with ethyl acetate in petroleum ether from 0%to 50%to give the ethyl 7-chloro-2-methyl-pyrazolo [4, 3-b] pyridine-6-carboxylate (300 mg, 92.30%yield) . LC-MS: m/z 240 [M+H] ⁺.

Step 6

To a solution of ethyl 7-chloro-2-methyl-pyrazolo [4, 3-b] pyridine-6-carboxylate (300 mg, 1.25 mmol) in N, N-Dimethylformamide (12 mL) at room temperature was added cyclopropylboronic acid (1.08 g, 12.52 mmol) and Sodium carbonate (398.0 mg, 3.76 mmol) . The reaction mixture was degassed and exchanged with N ₂ for two times. Dichlorobis [di-tert-butyl (4-dimethylaminophenyl) phosphine] palladium (177.3 mg, 250.4 μmol) was added into the mixture and the resulting reaction mixture was heated to 100 ℃ overnight. The starting material was consumed and the desired mass was detected from LC-MS. The reaction mixture was poured into water and extracted with ethyl acetate twice. The combined organic phase was washed with brine, dried over Na ₂SO ₄, filtered and concentrated. The resulting residue was purified by silica-gel column chromatography eluting with ethyl acetate in petroleum ether from 0%to 20%to give the ethyl 7-cyclopropyl-2-methyl-pyrazolo [4, 3-b] pyridine-6-carboxylate (70 mg, 22.80%yield) . LC-MS: m/z 246 [M+H] ⁺.

Step 7

To a solution of ethyl 7-cyclopropyl-2-methyl-pyrazolo [4, 3-b] pyridine-6-carboxylate (85 mg, 346.6 μmol) in Tetrahydrofuran (6 mL) at 0 ℃ was added Lithium hydroxide (20.75 mg, 866.37 μmol) in water (1 mL) dropwise. The resulting reaction mixture was stirred at room temperature for 4 hours. The starting material was consumed and the desired mass was detected from LC-MS. The reaction solution was acidified with HCl (1 N) to pH = 3～4. And then it was extracted with ethyl acetate twice. The combined organic phase was washed with brine, dried over Na ₂SO ₄, filtered and concentrated to give the 7-cyclopropyl-2-methyl-pyrazolo [4, 3-b] pyridine-6-carboxylic acid (68 mg, 90.33%yield) as a yellow solid, which was used directly for next step without further purification. LC-MS: m/z 218 [M+H] ⁺.

Step 8

To a solution of 7-cyclopropyl-2-methyl-pyrazolo [4, 3-b] pyridine-6-carboxylic acid (68 mg, 0.3134 mmol) in 1, 4-dioxane (6 mL) was added diphenylphosphonic azide (103.5 mg, 0.376 mmol) and triethylamine (158.6 mg, 1.567 mmol) . The resulting mixture was stirred at room temperature for 30 minutes, then 5-chloro-6- (triazol-2-yl) pyridin-3-amine (91.6 mg, 0.47 mmol) was added and the mixture was heated to 100℃ for 2 hours. The reaction solution was poured into water and extracted with ethyl acetate twice. The organic was dried over Na ₂SO ₄, concentrated and the resulting residue was purified by flash column chromatography (eluting with ethyl acetate in petroleum from 10%to 80%) to give the 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (7-cyclopropyl-2-methyl-2H-pyrazolo [4, 3-b] pyridin-6-yl) urea (13.1 mg, 10.21%yield) as a white solid. LC-MS: m/z 410 [M+H] ⁺. ¹H NMR (DMSO-d ₆, 400 MHz) : δ 9.70 (s, 1 H) , 8.74 (s, 1 H) , 8.56 (d, J = 2.0 Hz, 1 H) , 8.51 (s, 1 H) , 8.47 (d, J = 2.0 Hz, 1 H) , 8.42 (s, 1 H) , 8.14 (s, 2 H) , 4.15 (s, 3 H) , 2.23-2.19 (m, 1 H) , 1.71-1.67 (m, 2 H) , 1.1-1.06 (m, 2 H) .

Example 21. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (methyl (oxetan-3-yl) amino) imidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a solution of ethyl 8-bromo-2-chloro-imidazo [1, 2-b] pyridazine-7-carboxylate 7c (150.0 mg, 492.6 μmol) in THF (3 mL) was added oxetan-3-amine (72.0 mg, 985.1 μmol) . The mixture was stirred at room temperature overnight. After the reaction was completed, the solid was filtered off and the filtrate was concentrated. The residue was purified by column chromatography (eluting with PE/EA=10/1) to give ethyl 2-chloro-8- (oxetan-3- ylamino) imidazo [1, 2-b] pyridazine-7-carboxylate (140.0 mg, 95.8%yield) as a white solid. LC-MS: m/z 297 [M+H] ⁺.

Step 2

To a solution of ethyl 2-chloro-8- (oxetan-3-ylamino) imidazo [1, 2-b] pyridazine-7-carboxylate 13e (140 mg, 471.84 μmol) in DMF (3 mL) was added NaH (90.4 mg, 2.4 mmol, 60%purity) at 0 ℃. The resulting solution was warmed up to room temperature and stirred for 0.5 hour. Then iodomethane (401.8 mg, 2.8 mmol) was added dropwise into the mixture. The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with water and extracted with ethyl acetate (70 mL) . The combined organic phase was washed with brine (100 mL) and dried over anhydrous Na ₂SO ₄, filtered and concentrated in vacuo. The residue was purified by flash column chromatography to afford ethyl 2-chloro-8- [methyl (oxetan-3-yl) amino] imidazo [1, 2-b] pyridazine-7-carboxylate (60.0 mg, 40.9%yield) as a colorless oil. LC-MS: m/z 311 [M+H] ⁺.

Step 3

To a solution of ethyl 2-chloro-8- [methyl (oxetan-3-yl) amino] imidazo [1, 2-b] pyridazine-7-carboxylate 13f (60.0 mg, 193.1 μmol) in THF (2 mL) and Ethanol (1 mL) was added Lithium hydroxide monohydrate (81.0 mg, 1.9 mmol) in water (1 mL) . The mixture was stirred at 50 ℃ for 48 hours. Upon removal of solvent, the residue was acidified with HCl (2 M) to pH = 3～4. The mixture was extracted by EtOAc and the combined organic phase was dried over anhydrous Na ₂SO ₄, filtered and concentrated in vacuo to give 2-chloro-8- [methyl (oxetan-3-yl) amino] imidazo [1, 2-b] pyridazine-7-carboxylic acid (54.5 mg, 99.8%yield) as a white solid, which was used directly for next step. LC-MS: m/z 283 [M+H] ⁺.

Step 4

To a solution of 2-chloro-7- [methyl (oxetan-3-yl) amino] pyrazolo [1, 5-a] pyrimidine-6-carboxylic acid 14e (55.0 mg, 194.6 μmol) in 1, 4-Dioxane (6 mL) was added diphenylphosphonic azide (60.5 mg, 233.5 μmol) and triethylamine (98.4 mg, 972.8 μmol) . The resulting solution was stirred at room temperature for 30 minute, then 5-chloro-6- (triazol-2-yl) pyridin-3-amine (57.1 mg, 291.9 μmol) was added and the mixture was stirred at 100 ℃ for 2 hours. Solvent was removed and the residue was purified by flash column chromatography (eluting with DCM: MeOH = 30 : 1) and prep-HPLC to give the title compound 1- [2-chloro-7- [methyl (oxetan-3-yl) amino] pyrazolo [1, 5-a] pyrimidin-6-yl] -3- [5-chloro-6- (triazol-2-yl) -3-pyridyl] urea (6 mg, 6.5%yield) . LC-MS: m/z 475.0 [M+H] ⁺. ¹H NMR (DMSO-d ₆, 400 MHz) : δ 8.92 (s, 1H) , 8.72-8.69 (m, 2H) , 8.58 (d, J = 2.0 Hz, 1H) , 8.49 (d, J = 2.4 Hz, 1H) , 8.29 (s, 1H) , 8.16 (s, 2H) , 5.08 (t, J = 6.4 Hz, 1H) , 4.73 (t, J = 6.4 Hz, 2H) , 4.63 (t, J = 6.4 Hz, 2H) , 3.08 (s, 3H) .

Example 22. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (3-chloro-8-cyclopropylimidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a suspension of ethyl 1- (tert-butoxycarbonylamino) imidazole-2-carboxylate 3 (1.75 g, 6.86 mmol) in DMF (10 mL) was added slowly 1-chloropyrrolidine-2, 5-dione (0.92 g, 6.86 mmol) in DMF (5 mL) . The reaction mixture was stirred at room temperature for 6 h and upon completion of the reaction, the reaction mixture was added a saturated sodium bicarbonate and extracted with EtOAc (2 ×100 mL) . The combined organic phase was washed with brine (50 mL) and dried over anhydrous Na ₂SO ₄, filtered and concentrated under vacuo. The crude was purified by flash column chromatography (Petroleum ether/ethyl acetate 10: 1) to afford the ethyl 1- ( (tert-butoxycarbonyl) amino) -5-chloro-1H-imidazole-2-carboxylate (1.0 g, 3.46 mmol, 50.4%yield) as a colorless oil. LC-MS: m/z 290 [M+H] ⁺.

Step 2

To a solution of ethyl 1- ( (tert-butoxycarbonyl) amino) -5-chloro-1H-imidazole-2-carboxylate 4 (1.0 g, 3.45 mmol) in THF (20 mL) was added dropwise potassium 2-methylpropan-2-olate (1 M, 10.35 mL) at 0 ℃ for 5 minute, dry ethyl acetate (760.27 g, 8.63 mmol, 0.84 mL) was added into the mixture and it was stirred at 0 ℃ for 15 minute. The ice bath was removed and the resulting mixture was stirred at room temperature for 2 hours. TLC showed spot was formed, the reaction was cooled 0 ℃ and treated with 1.0 N HCl via dropwise addition until the suspension was completely dissolved. The resulting solution was extracted with EtOAc (2 ×40 mL) . The combined organic phase was washed with brine (100 mL) and dried over anhydrous Na ₂SO ₄, filtered and concentrated to afford (Z) -ethyl 3- (1- ( (tert-butoxycarbonyl) amino) -5-chloro-1H-imidazol-2-yl) -3-hydroxyacrylate (720 mg, crude) as a yellow solid. LC-MS: m/z 332 [M+H] ⁺.

Step 3

To a solution of (Z) -ethyl 3- (1- ( (tert-butoxycarbonyl) amino) -5-chloro-1H-imidazol-2-yl) -3-hydroxyacrylate 5e (720 mg, 2.17 mmol) in DCM (20 mL) was added 1, 1-dimethoxy-N, N-dimethyl-methanamine (1.94 g, 16.28 mmol, 2.18 mL) . The reaction mixture was stirred at room temperature for 3 h and upon completion of the reaction, the mixture was concentrated to give crude, which was recrystallized in Ethyl acetate to afford ethyl 3-chloro-8-hydroxyimidazo [1, 2-b] pyridazine-7-carboxylate (380 mg, 1.57 mmol, 72.46%yield) as a white solid. LC-MS: m/z 242 [M+H] ⁺.

Step 4

To a stirred solution of ethyl 3-chloro-8-hydroxyimidazo [1, 2-b] pyridazine-7-carboxylate 6e (380 mg, 1.57 mmol) in 1, 4-Dioxane (6 mL) at room temperature was added Phosphorus oxybromide (1.35 g, 4.72 mmol, 0.48 mL) . The reaction mixture was stirred at 110 ℃ for 2 h. Upon completion of the reaction, the reaction mixture was cooled to room temperature, the reaction mixture was poured into water and extracted with EtOAc (2 ×10 mL) . The combined organic phase was washed with brine (10 mL) and dried over anhydrous Na ₂SO ₄, filtered and concentrated in vacuo. The crude was purified by flash column chromatography (Petroleum ether/ethyl acetate = 7: 1) to afford ethyl 8-bromo-3-chloroimidazo [1, 2-b] pyridazine-7-carboxylate (220 mg, 722.43 μmol, 45.94%yield) as a yellow solid. LC-MS: m/z 304 [M+H] ⁺.

Step 5

To a solution of ethyl 8-bromo-3-chloroimidazo [1, 2-b] pyridazine-7-carboxylate 7e (220 mg, 722.43 μmol) , cyclopropylboronic acid (62.1 mg, 722.43 mmol) in Toluene (4 mL) were added tricyclohexylphosphane (40.5 mg, 144.49 μmol) , tripotassium phosphate (383.4 mg, 1.81 mmol) and diacetoxypalladium (16.2 mg, 72.24 μmol) . The suspension was stirred at 100 ℃ for 4 h. TLC (Petroleum ether : Ethyl acetate = 5 : 1) showed most of starting material were consumed and one main spot was present. The reaction mixture was filtered through a pad of Celite. The filtrate was diluted with water (5 mL) and extract with Ethyl acetate (10 mL) . The organic phase was washed with brine, dried over Na ₂SO ₄, and concentrated to obtain the residue, which was purified by silica-gel column chromatography eluting with Petroleum ether/Ethyl acetate (8/1) to give ethyl 3-chloro-8-cyclopropyl-imidazo [1, 2-b] pyridazine-7-carboxylate (136 mg, 511.86 μmol, 70.85%yield) as a yellow solid. LC-MS: m/z 266 [M+H] ⁺.

Step 6

To a solution of ethyl 3-chloro-8-cyclopropyl-imidazo [1, 2-b] pyridazine-7-carboxylate 8i (136 mg, 511.86 μmol) in Ethanol/water (5/5 mL) at 0 ℃ was added lithium hydroxide monohydrate (1 M, 2.55 mL) . The resulting reaction mixture was stirred at room temperature for 10 h. Upon removal of solvent, the residue was acidified with HCl (2 M) to pH = 3～4. The suspension was filtered and the filter cake was dried in vacuo to give 3-chloro-8-cyclopropylimidazo [1, 2-b] pyridazine-7-carboxylic acid (110.0 mg, crude) as a yellow solid, which was used directly for next step.

Step 7

To a solution of 3-chloro-8-cyclopropyl-imidazo [1, 2-b] pyridazine-7-carboxylic acid 9k (110 mg, 462.88 μmol) in 1, 4-Dioxane (4 mL) was added triethylamine (233.8 mg, 2.31 mmol, 321.20 uL) and diphenylphosphonic azide (165.6 mg, 601.75 μmol, 130.40uL) at room temperature, the mixture was stirred for 30 min. Then 5-chloro-6- (triazol-2-yl) pyridin-3-amine (90.5 mg, 462.88 μmol) was added and the reaction mixture was stirred at 100 ℃ for 3 h. The mixture was concentrated to obtain the residue, which was purified by prep-HPLC to give 1- (3-chloro-8-cyclopropyl-imidazo [1, 2-b] pyridazin-7-yl) -3- [5-chloro-6- (triazol-2-yl) -3-pyridyl] urea (35.4 mg, 82.28 μmol, 17.78%yield) as a white solid. LC-MS: m/z 430 [M+H] ⁺. ¹H NMR (400 MHz, DMSO-d ₆) δ 9.75 (s, 1H) , 9.00 (s, 1H) , 8.78 (s, 1H) , 8.58 (d, J = 2.4Hz, 1H) , 8.48 (d, J = 2.4Hz, 1H) , 8.15 (s, 2H) , 7.75 (s, 1H) , 2.23-2.18 (m, 1H) , 1.85-1.81 (m, 2H) , 1.19-1.14 (m, 2H) .

Example 23. Synthesis of 1- (8- (azetidin-1-yl) -2-chloroimidazo [1, 2-b] pyridazin-7-yl) -3- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) urea

Step 1

To a solution of ethyl 8-bromo-2-chloroimidazo [1, 2-b] pyridazine-7-carboxylate 7c (100 mg, 0.33 mmol) in THF (5 mL) at 0 ℃ was added azetidine hydrochloride (92.6 mg, 0.99 mmol) and triethylamine (100.2 mg, 0.99 mmol) . The resulting reaction mixture was stirred at room temperature overnight. The starting material was consumed and the desired mass was detected from LC-MS. The reaction mixture was concentrated and the resulting residue was purified by flash column chromatography (eluting with ethyl acetate in petroleum from 0%to 30%) to give the ethyl 8- (azetidin-1-yl) -2-chloroimidazo [1, 2-b] pyridazine-7-carboxylate (91 mg, 98.5%yield) as a white solid. LC-MS: m/z 281 [M+H] ⁺.

Step 2

To a solution of ethyl 8- (azetidin-1-yl) -2-chloroimidazo [1, 2-b] pyridazine-7-carboxylate 13g (91 mg, 0.33 mmol) in THF (5 mL) and Ethanol (5 mL) was added the solution of lithium hydroxide monohydrate (136.5 mg, 3.25 mmol) in water (3.2 mL) . The reaction mixture was stirred at 70 ℃ for 4 hours. The starting material was consumed and the desired mass was detected from LC-MS. After removing solvent under vacuo, the residue was acidified with HCl (1 M) to pH = 3～4. And then it was extracted with ethyl acetate, the combined organic phase was dried over Na ₂SO ₄, filtered and concentrated to give 8- (azetidin-1-yl) -2-chloroimidazo [1, 2-b] pyridazine-7-carboxylic acid (81 mg, 98.9%yield) as white solid, which was used for next step directly without further purification. LC-MS: m/z 253 [M+H] ⁺.

Step 3

To a solution of 8- (azetidin-1-yl) -2-chloroimidazo [1, 2-b] pyridazine-7-carboxylic acid 14f (81 mg, 0.32 mmol) in 1, 4-dioxane (5 mL) was added diphenylphosphonic azide (115 mg, 0.42 mmol) and triethylamine (162.6 mg, 1.61 mmol) . The resulting mixture was stirred at room temperature for 30 minute, then 5-chloro-6- (triazol-2-yl) pyridin-3-amine (87.7 mg, 0.45 mmol) was added and the mixture was heated to 100 ℃ for 2 hours. The reaction mixture was poured into water and extracted with ethyl acetate twice. The combined organic phase was dried over Na ₂SO ₄, concentrated and the resulting residue was purified by flash column chromatography (eluting with ethyl acetate in petroleum from 10%to 80%) to give the 1- (8- (azetidin-1-yl) -2-chloroimidazo [1, 2-b] pyridazin-7-yl) -3- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) urea (15.8 mg) as a purple solid. LC-MS: m/z 445 [M+H] ⁺. ¹H NMR (DMSO-d ₆, 400 MHz) : δ 9.68 (s, 1 H) , 8.58 (s, 1 H) , 8.45 (d, J = 2.4 Hz, 1 H) , 8.19 (s, 1 H) , 8.15 (s, 1 H) , 8.13 (s, 2 H) , 7.91 (s, 1 H) , 4.62 (t, J = 7.6 Hz, 4 H) , 2.38-2.34 (m, 2 H) .

Example 24. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (pyrrolidin-1-yl) imidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a solution of ethyl 8-bromo-2-chloro-imidazo [1, 2-b] pyridazine-7-carboxylate 7c (100.0 mg, 328.4 μmol) in 1, 4-dioxane (6 mL) was added pyrrolidine (23.4 mg, 328.4 μmol) , cesium carbonate (321.2 mg, 985.2 μmol) , tris (dibenzylideneacetone) dipalladium (0) (30.1 mg, 32.84 μmol) and 4, 5-bis (diphenylphosphino) -9, 9-dimethylxanthene (38.0 mg, 65.68 μmol) . The reaction mixture was stirred at 100 ℃ for 2 hours under N ₂. The reaction mixture was cooled to room temperature, concentrated in vacuo to afford a residue, which was purified by flash column chromatography (eluting with PE/EA=10/1) to afford ethyl 2-chloro-8-pyrrolidin-1-yl-imidazo [1, 2-b] pyridazine-7-carboxylate (85 mg, 87.8%yield) as a yellow solid. LC-MS: m/z 295 [M+H] ⁺.

Step 2

To a solution of ethyl 2-chloro-8-pyrrolidin-1-yl-imidazo [1, 2-b] pyridazine-7-carboxylate 13h (85.0 mg, 288.4 μmol) in EtOH (4 mL) and H ₂O (4 mL) was added lithium hydroxide monohydrate (121.1 mg, 2.88 mmol) . The reaction mixture was stirred at 70 ℃ for 16 hours. The reaction mixture was diluted with EtOAc (5 mL) and adjusted pH = 6 with HCl (1N) . The aqueous was extracted with EtOAc (2 × 5 mL) and the combined organic phase was washed with brine (100 mL) , dried over anhydrous Na ₂SO ₄, filtered and concentrated in vacuo to give crude 2-chloro-8-pyrrolidin-1-yl-imidazo [1, 2-b] pyridazine-7-carboxylic acid (70 mg, 91.0%yield) as a yellow solid, which was used directly for next step without further purification.

Step 3

To a solution of 2-chloro-8-pyrrolidin-1-yl-imidazo [1, 2-b] pyridazine-7-carboxylic acid 14g (70 mg, 262.5 μmol) in 1, 4-dioxane (4 mL) was added diphenylphosphoryl azide (93.9 mg, 341.23 μmol) , triethylamine (132.6 mg, 1.3 mmol) . The reaction mixture was stirred at room temperature for 30 min. Then 5-chloro-6- (triazol-2-yl) pyridin-3-amine (66.8 mg, 341.2 μmol) was added and the mixture was stirred at 100 ℃ for 2 h. The reaction mixture was cooled to room temperature, solvent was removed and the residue was purified by flash column chromatography (eluting with DCM: MeOH = 20 : 1) to afford 1- (2-chloro-8-pyrrolidin-1-yl-imidazo [1, 2-b] pyridazin-7-yl) -3- [5-chloro-6- (triazol-2-yl) -3-pyridyl] urea (25 mg, 20.7%yield) as a white solid. LC-MS: m/z 459.0 [M+H] ⁺. ¹H NMR (DMSO-d ₆, 400 MHz) : δ 8.58 (s, 1H) , 8.45 (d, J = 2.4 Hz, 1H) , 8.17 (s, 1H) , 8.14 (s, 2H) , 7.94 (s, 1H) , 4.01 (t, J = 6.0 Hz, 4H) , 1.87 (t, J = 6.4 Hz, 4H) .

Example 25. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (ethyl (methyl) amino) imidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a solution of ethyl 8-bromo-2-chloroimidazo [1, 2-b] pyridazine-7-carboxylate 7c (100 mg, 0.33 mmol) in THF (4 mL) at 0 ℃ was added N-methylethanamine (68.2 mg, 1.16 mmol) . The reaction mixture was stirred at room temperature overnight. The starting material was consumed and the desired mass was detected from LC-MS. The reaction mixture was concentrated and the resulting residue was purified by flash column chromatography (eluting with ethyl acetate in petroleum from 0%to 30%) to give the ethyl 2-chloro-8- (ethyl (methyl) amino) imidazo [1, 2-b] pyridazine-7-carboxylate (90 mg, 96.7%yield) as a faint yellow solid. LC-MS: m/z 283 [M+H] ⁺.

Step 2

To a solution of ethyl 2-chloro-8- (ethyl (methyl) amino) imidazo [1, 2-b] pyridazine-7-carboxylate 13i (90 mg, 0.32 mmol) in THF (5 mL) and ethanol (5 mL) was added the solution of lithium hydroxide monohydrate (67 mg, 1.60 mmol) in water (1.6 mL) . The mixture was stirred at 70 ℃ for 4 hours. The starting material was consumed and the desired mass was detected from LC-MS. The solvent was removed under vacuo to afford a residue, which was acidified with HCl (1 N) to pH = 3～4. The resulting mixture was extracted with ethyl acetate and the combined organic phase was washed with brine, dried over Na ₂SO ₄, filtrated and concentrated to give 2-chloro-8- (ethyl (methyl) amino) imidazo [1, 2-b] pyridazine-7-carboxylic acid (81 mg, 99.9%yield) as a faint yellow solid, which was used directly for next step. LC-MS: m/z 255 [M+H] ⁺.

Step 3

To a solution of 2-chloro-8- (ethyl (methyl) amino) imidazo [1, 2-b] pyridazine-7-carboxylic acid 14h (81 mg, 0.3189 mmol) in 1, 4-dioxane (5 mL) was added diphenylphosphonic azide (114.1 mg, 0.4146 mmol) and triethylamine (161.3 mg, 1.594 mmol) . The resulting mixture was stirred at room temperature for 30 minute, then 5-chloro-6- (triazol-2-yl) pyridin-3-amine 10a (87 mg, 0.4465 mmol) was added and the mixture was heated at 100℃ for 2 hours. The mixture solution was poured into water and extracted with ethyl acetate twice. The combined organic phase was dried over Na ₂SO ₄, filtrated and concentrated, the residue was purified by flash column chromatography (eluting with ethyl acetate in petroleum from 10%to 80%) to give the 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (ethyl (methyl) amino) imidazo [1, 2-b] pyridazin-7-yl) urea (7.4 mg, 5.2%) as a white solid. LC-MS: m/z 447 [M+H] ⁺. ¹H NMR (DMSO-d ₆, 400 MHz) : δ 9.77 (s, 1 H) , 8.56 (d, J = 2.4 Hz, 2 H) , 8.46 (d, J = 2.4 Hz, 1 H) , 8.42 (s, 1 H) , 8.23 (s, 1 H) , 8.14 (s, 2 H) , 3.70 (q, J = 7.2, 14.4 Hz, 2 H) , 3.13 (s, 3 H) , 1.16 (t, J = 7.2 Hz, 3 H) .

Example 26. Synthesis of 1- (2-chloro-8- (cyclopropyl (methyl) amino) imidazo [1, 2-b] pyridazin-7-yl) -3- (2- (trifluoromethyl) pyridin-4-yl) urea

Step 1

To a solution of ethyl 8-bromo-2-chloro-imidazo [1, 2-b] pyridazine-7-carboxylate 7c (200 mg, 656.76 μmol) in THF (4 mL) was added N-methylcyclopropanamine (141.3 mg, 1.31 mmol, 364.0 uL, HCl) . The suspension was stirred at room temperature for 3 hours. LCMS showed most of starting material was consumed. The solvent was removed to give ethyl 2-chloro-8- [cyclopropyl (methyl) amino] imidazo [1, 2-b] pyridazine-7-carboxylate (186 mg, 631.07 μmol, 96.09%yield) as a yellow solid. LC-MS: m/z 295.1 [M+H] ⁺.

Step 2

To a solution of ethyl 2-chloro-8- [cyclopropyl (methyl) amino] imidazo [1, 2-b] pyridazine-7-carboxylate 13a (186 mg, 631.07 μmol) in Ethanol (4 mL) at 0 ℃ was added lithium hydroxide monohydrate (1 M, 1.89 mL) . The mixture was stirred at room temperature for 10 hours. Upon removal of the solvents, the residue was acidified with HCl (2 M) to pH =3～4. The suspension was filtered and the filter cake was dried in vacuo to give 2-chloro-8- [cyclopropyl (methyl) amino] imidazo [1, 2-b] pyridazine-7-carboxylic acid (168 mg, 629.96 μmol, 99.82%yield) as a white solid, which was used directly in next step.

Step 3

To a solution of 2-chloro-8- [cyclopropyl (methyl) amino] imidazo [1, 2-b] pyridazine-7-carboxylic acid (168 mg, 629.96 μmol) in 1, 4-Dioxane (7 mL) was added triethylamine (318.7 mg, 3.15 mmol, 439.02 uL) and diphenylphosphonic azide (208.0 mg, 755.95 μmol, 163.81 uL) at room temperature. The resulting reaction mixture was stirred at room temperature for 30 min. Then 2- (trifluoromethyl) pyridin-4-amine (132.76 mg, 818.95 μmol) was added and the reaction mixture was stirred at 100 ℃ for 2 h. The reaction mixture was concentrated to afford the residue, which was purified by prep-TLC (eluting with Dichloromethane: Methanol = 20: 1) to give 1- [2-chloro-8- [cyclopropyl (methyl) amino] imidazo [1, 2-b] pyridazin-7-yl] -3- [2- (trifluoromethyl) -4-pyridyl] urea (13.4 mg, 31.47 μmol, 5.00%yield) as a white solid. LC-MS: m/z 426 [M+H] ⁺. ¹H NMR (400 MHz, DMSO-d ₆) δ 9.83 (s, 1H) , 8.53 (d, J = 5.6 Hz, 1H) , 8.48 (s, 2H) , 8.24 (s, 1H) , 8.04 (d, J = 2.0 Hz, 1H) , 7.64-7.62 (m, 1H) , 3.21 (s, 3H) , 3.18-3.14 (m, 1H) , 0.65-0.62 (m, 2H) , 0.51-0.49 (m, 2H) .

Example 27. Synthesis of 1- (2-chloro-8- (isopropyl (methyl) amino) imidazo [1, 2-b] pyridazin-7-yl) -3- (2- (trifluoromethyl) pyridin-4-yl) urea

Step 1

To a solution of ethyl 8-bromo-2-chloro-imidazo [1, 2-b] pyridazine-7-carboxylate 7c (250.0 mg, 820.9 μmol) in THF (5 mL) was added N-methylpropan-2-amine (600.4 mg, 8.21 mmol) . The resulting reaction mixture was stirred at room temperature overnight. After the reaction was completed, the solid was filtered off and filtrate was concentrated under vacuo. The residue was purified by column chromatography (eluting with PE/EA=10/1) to get ethyl 2-chloro-8- (isopropyl (methyl) amino) imidazo [1, 2-b] pyridazine-7-carboxylate (82 mg, 80.36%yield) as a yellow solid. LC-MS: m/z 297.1 [M+H] ⁺.

Step 2

To a solution of ethyl 2-chloro-8- (isopropyl (methyl) amino) imidazo [1, 2-b] pyridazine-7-carboxylate 13j (210.0 mg, 707.7 μmol) in THF (4 mL) and H ₂O (4 mL) was added lithium hydroxide monohydrate (297 mg, 7.08 mmol) . The reaction mixture was stirred at 60 ℃ for 48 h. Upon removal of solvent, the residue was acidified with HCl (2 N) to pH = 3～4. The resulting mixture was extracted by EtOAc, the combined organic phase was dried over Na ₂SO ₄, filtrated and concentrated to give ethyl 2-chloro-8- (isopropyl (methyl) amino) imidazo [1, 2-b] pyridazine-7-carboxylate (171.0 mg, quant. ) as a white solid, which was used directly for next step. LC-MS: m/z 297.1 [M+H] ⁺.

Step 3

To a solution of 2-chloro-8- (isopropyl (methyl) amino) imidazo [1, 2-b] pyridazine-7-carboxylic acid 14i (171 mg, 636.4 μmol) in 1, 4-Dioxane (4 mL) was added diphenylphosphonic azide (227.7 mg, 229.23 μmol, 827.3 uL) and triethylamine (321.4 mg, 3.18 mmol) . The resulting suspension was stirred at room temperature for 30 mins. Then 2- (trifluoromethyl) pyridin-4-amine (103.2 mg, 636.40 μmol) was added and the resulting reaction mixture was stirred at 100 ℃ for 2 hours. Solvent was removed under vacuo to afford residue, which was purified by flash column chromatography (eluting with DCM: MeOH = 30 : 1) and prep-HPLC to give 1- (2-chloro-8- (isopropyl (methyl) amino) imidazo [1, 2-b] pyridazin-7-yl) -3- (2- (trifluoromethyl) pyridin-4-yl) urea (46.0 mg, 16.9%yield) as a white solid. LC-MS: m/z 428.0 [M+H] ⁺. ¹H NMR (400 MHz, DMSO-d ₆) : δ 9.88 (brs, 1H) , 8.53 (d, J = 5.6 Hz, 1H) , 8.42-8.40 (m, 2H) , 8.24-8.23 (m, 1H) , 8.06-8.05 (m, 1H) , 7.65-7.63 (m, 1H) , 4.52-4.47 (m, 1H) , 2.95 (s, 3H) , 1.22 (d, J = 6.4 Hz, 6H) .

Example 28. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (cyclopropyl (2-methoxyethyl) amino) imidazo [1, 2-b] pyridazin-7-yl) urea

Example 28 was prepared in analogy to the procedure described for the preparation of example 21 by using N- (2-methoxyethyl) cyclopropanamine as the starting material instead of oxetan-3-amine. LC-MS: m/z 503.0 [M+H] ⁺. ¹H NMR (400 MHz, DMSO-d ₆) : δ 10.06 (s, 1H) , 8.61 (s, 1H) , 8.56 (d, J = 2.4Hz, 1H) , 8.48 (d, J = 2.0 Hz, 1H) , 8.40 (s, 1H) , 8.25 (s, 1H) , 8.15 (s, 2H) , 3.90 (t, J = 5.6 Hz, 2H) , 3.52 (t, J = 5.6 Hz, 2H) , 3.33-3.31 (m, 1H) , 3.14 (s, 3H) , 0.68-0.66 (m, 2H) , 0.51-0.49 (m, 2H) .

Example 29. Synthesis of (S) -1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (3-methylmorpholino) imidazo [1, 2-b] pyridazin-7-yl) urea

Example 29 was prepared in analogy to the procedure described for the preparation of example 21 by using (3S) -3-methylmorpholine as the starting material instead of oxetan-3-amine. LC-MS: m/z 489.0 [M+H] ⁺ _; ¹H NMR (400 MHz, DMSO-d6) : δ 10.29 (brs, 1H) , 9.16 (s, 1H) , 8.73 (s, 1H) , 8.57 (d, J = 2.4 Hz, 1H) , 8.51 (d, J = 2.4 Hz, 1H) , 8.35 (s, 1H) , 8.16 (s, 2H) , 4.16-4.12 (m, 1H) , 3.90-3.79 (m, 3H) , 3.49-3.43 (m, 2H) , 3.09-3.06 (m, 1H) , 0.86-0.85 (m, 3H) .

Example 30. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- ( (2S, 6R) -2, 6-dimethylmorpholino) imidazo [1, 2-b] pyridazin-7-yl) urea

Example 30 was prepared in analogy to the procedure described for the preparation of example 21 by using (2S, 6R) -2, 6-dimethylmorpholine as the starting material instead of oxetan-3-amine. LCMS m/z 503 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.85 (s, 1H) , 8.58 (d, J = 2.4 Hz, 1H) , 8.52 (s, 1H) , 8.50 (s, 1H) , 8.47 (d, J = 2.4 Hz, 1H) , 8.27 (s, 1H) , 8.78 (s, 1H) , 8.15 (s, 2H) , 3.82-3.78 (m, 4H) , 2.99-2.93 (m, 2H) , 1.11 (d, J = 2.4 Hz, 6H) .

Example 31. Synthesis of (S) -1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (3- (methoxymethyl) morpholino) imidazo [1, 2-b] pyridazin-7-yl) urea

Example 31 was prepared in analogy to the procedure described for the preparation of example 21 by using (3S) -3- (methoxymethyl) morpholine as the starting material instead of oxetan-3-amine. LC-MS (ESI) m/z 518.9 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d ₆) δ 10.09 (s, 1H) , 8.78 (s, 1H) , 8.58-8.57 (m, 1H) , 8.50-8.49 (m, 2H) , 8.31 (s, 1H) , 8.15 (s, 1H) , 8.04 (s, 1H) , 4.39 (s, 1H) , 3.91-3.84 (m, 2H) , 3.79-3.75 (m, 1H) , 3.71-3.65 (m, 1H) , 3.50-3.39 (m, 4H) , 3.08 (s, 3H) .

Example 32. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (1, 1-dioxidothiomorpholino) imidazo [1, 2-b] pyridazin-7-yl) urea

Example 32 was prepared in analogy to the procedure described for the preparation of example 21 by using 1, 4-thiazinane 1, 1-dioxide as the starting material instead of oxetan-3-amine. LC-MS: m/z 523 [M+H] ⁺; ¹H NMR (400 MHz; DMSO-d6) : δ 10.02 (br, 1H) , 8.79 (s, 1H) , 8.79 (s, 1H) , 8.59 (d, J = 2.4 Hz, 1H) , 8.49 (d, J = 2.4 Hz, 1H) , 8.36 (s, 1H) , 8.16 (s, 2H) , 3.86 (d, J = 5.2 Hz, 4H) , 3.42 (s, 4H) .

Example 33. Synthesis of (S) -1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (3-methoxypyrrolidin-1-yl) imidazo [1, 2-b] pyridazin-7-yl) urea

Example 33 was prepared in analogy to the procedure described for the preparation of example 21 by using (3S) -3-methoxypyrrolidine as the starting material instead of oxetan-3-amine. LC-MS: m/z 489 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d6) : δ 9.79 (br, 1H) , 8.59 (s, 1H) , 8.45 (d, J = 2.0 Hz, 2H) , 8.19 (s, 1H) , 8.14 (s, 2H) , 7.95 (s, 1H) , 4.16-4.01 (m, 5) , 3.17 (s, 3H) , 2.06-2.05 (m, 1) , 1.94-1.89 (m, 1) .

Example 34. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (methylthio) imidazo [1, 2-b] pyridazin-7-yl) urea

Example 34 was prepared in analogy to the procedure described for the preparation of example 21 by using sodium methanethiolate as the starting material instead of oxetan-3-amine. LC-MS: m/z 436.0 [M+H] ⁺; ¹HNMR (400 MHz, DMSO-d6) : δ 10.28 (s, 1H) , 9.02 (s, 1H) , 8.98 (s, 1H) , 8.56 (d, J = 2.4 Hz, 1H) , 8.47 (d, J = 2.4 Hz, 1H) , 8.41 (s, 1H) , 8.16 (s, 2H) , 2.82 (s, 3H) .

Example 35. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (cyclopropyl (ethyl) amino) imidazo [1, 2-b] pyridazin-7-yl) urea

Example 35 was prepared in analogy to the procedure described for the preparation of example 21 by using N-ethylcyclopropanamine as the starting material instead of oxetan-3-amine. LC-MS: m/z 473 [M+H] ⁺; ¹H NMR (400 MHz; DMSO-d ₆) : δ 9.83 (s, 1H) , 8.67 (s, 1H) , 8.55 (d, J = 2.4 Hz, 1H) , 8.48 (d, J = 2.4 Hz, 2H) , 8.26 (s, 1H) , 8.15 (s, 2H) , 3.71 (q, J =7.2 Hz, 2H) , 3.26-3.22 (m, 1H) , 1.12 (t, J = 7.2 Hz, 3H) , 0.69-0.65 (m, 2H) , 0.51-0.48 (m, 2H) .

Example 36. Synthesis of 1- (2-chloro-8- (ethyl (2-methoxyethyl) amino) imidazo [1, 2-b] pyridazin-7-yl) -3- (2- (trifluoromethyl) pyridin-4-yl) urea

Example 36 was prepared in analogy to the procedure described for the preparation of example 21 by using N-ethyl-2-methoxy-ethanamine as the starting material instead of oxetan-3-amine. LC-MS: m/z 458.1 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d ₆) δ 10.13 (s, 1H) , 8.76 (s, 1H) , 8.55 (d, J = 4.8 Hz, 2H) , 8.27 (s, 1H) , 8.07 (d, J = 1.6 Hz, 1H) , 7.63-7.61 (m, 1H) , 3.67 (t, J = 5.6 Hz, 2H) , 3.55 (q, J = 7.2 Hz, 2H) , 3.41 (t, J = 5.6 Hz, 2H) , 3.12 (s, 3H) , 1.01 (t, J = 7.2 Hz, 3H) .

Example 37. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (ethyl (2-methoxyethyl) amino) imidazo [1, 2-b] pyridazin-7-yl) urea

Example 37 was prepared in analogy to the procedure described for the preparation of example 21 by using N-ethyl-2-methoxy-ethanamine as the starting material instead of oxetan-3-amine.

LC-MS: m/z 490.9 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d ₆) δ 10.13 (s, 1H) , 8.80 (s, 1H) , 8.63 (s, 1H) , 8.56 (d, J = 2.4 Hz, 1H) , 8.48 (d, J = 2.4 Hz, 1H) , 8.27 (s, 1H) , 8.15 (s, 2H) , 3.68 (t, J = 6.0 Hz, 2H) , 3.57 (q, J = 7.2 Hz, 2H) , 3.43 (t, J = 6.0 Hz, 2H) , 3.15 (s, 3H) , 1.03 (t, J = 7.2 Hz, 3H) .

Example 38. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (diethylamino) imidazo [1, 2-b] pyridazin-7-yl) urea

Example 38 was prepared in analogy to the procedure described for the preparation of example 21 by using N-ethylethanamine as the starting material instead of oxetan-3-amine. LC-MS: m/z 461.0 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d ₆) : δ 10.51 (brs, 1H) , 8.50 (s, 1H) , 8.78 (s, 1H) , 8.57 (d, J = 2.4 Hz, 1H) , 8.48 (d, J = 2.4 Hz, 1H) , 8.26 (s, 1H) , 8.15 (s, 2H) , 3.55 (q, J = 7.2 Hz, 4H) , 1.05 (t, J = 7.2 Hz, 6H) .

Example 39. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (8-morpholinoimidazo [1, 2-b] pyridazin-7-yl) urea

Example 39 was prepared in analogy to the procedure described for the preparation of example 21 by using morpholine as the starting material instead of oxetan-3-amine.

LC-MS: m/z 441.1; ¹H NMR (400 MHz, DMSO-d ₆) : δ 9.80 (brs, 1H) , 8.59 (d, J = 2.4 Hz, 1H) , 8.48 (d, J = 2.4 Hz, 2H) , 8.39 (s, 1H) , 8.15 (s, 2H) , 8.09 (d, J = 1.2 Hz, 1H) , 7.60 (d, J =1.2 Hz, 1H) , 3.78 (t, J = 4.8 Hz, 4H) , 3.69 (t, J = 4.8 Hz, 4H) .

Example 40. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (3-methoxyazetidin-1-yl) imidazo [1, 2-b] pyridazin-7-yl) urea

Example 40 was prepared in analogy to the procedure described for the preparation of example 21 by using 3-methoxyazetidine hydrochloride as the starting material instead of oxetan-3-amine. LC-MS: m/z 475.0 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d6) : δ 8.59 (s, 1H) , 8.45 (d, J = 2.4 Hz, 1H) , 8.19 (s, 1H) , 8.14 (s, 2H) , 7.96 (s, 1H) , 4.81-4.77 (m, 2H) , 4.41-4.39 (m, 2H) , 4.34-4.30 (m, 1H) , 3.25 (s, 3H) .

Example 41. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (3-methoxy-3-methylazetidin-1-yl) imidazo [1, 2-b] pyridazin-7-yl) urea

Example 41 was prepared in analogy to the procedure described for the preparation of example 21 by using 3-methoxy-3-methyl-azetidine hydrochloride as the starting material instead of oxetan-3-amine. LC-MS: m/z 489.0 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d6) : δ8.59 (s, 1H) , 8.45 (d, J = 2.0 Hz, 1H) , 8.19 (s, 1H) , 8.14 (s, 2H) , 7.96 (s, 1H) , 4.51 (d, J =10.0 Hz, 2H) , 4.42 (d, J = 9.6 Hz, 2H) , 3.21 (s, 3H) , 1.46 (s, 3H) .

Example 42. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (piperazin-1-yl) imidazo [1, 2-b] pyridazin-7-yl) urea

Example 42 was prepared in analogy to the procedure described for the preparation of example 21 by using tert-butyl piperazine-1-carboxylate as the starting material instead of oxetan-3-amine. LC-MS m/z 474 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d ₆) δ 10.13 (s, 1H) , 8.76 (s, 1H) , 8.59 (t, J = 2.0 Hz, 1H) , 8.47 (d, J = 2.0 Hz, 1H) , 8.43 (s, 1H) , 8.26 (s, 1H) , 8.14 (s, 2H) , 3.54 (s, 4H) , 2.91 (s, 4H) , 1.90 (d, J = 1.2Hz, 1H) .

Example 43. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (4-methylpiperazin-1-yl) imidazo [1, 2-b] pyridazin-7-yl) urea

Example 43 was prepared in analogy to the procedure described for the preparation of example 21 by using 1-methylpiperazine as the starting material instead of oxetan-3-amine. LCMS m/z 488 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d ₆) δ 10.14 (s, 1H) , 8.80 (s, 1H) , 8.59 (s, 1H) , 8.46 (s, 1H) , 8.38 (s, 1H) , 8.25 (s, 1H) , 8.14 (s, 2H) , 3.61 (s, 4H) , 2.22 (s, 4H) , 1.90 (s, 3H) .

Example 44. Synthesis of 1- (2-chloro-8- (dimethylamino) imidazo [1, 2-b] pyridazin-7-yl) -3- (5-methylpyridin-3-yl) urea

Example 44 was prepared in analogy to the procedure described for the preparation of example 21 by using N-methylmethanamine as the starting material instead of oxetan-3-amine. LC-MS (ESI+) [ (M+H) +] : 406.0; ¹H NMR (400 MHz, DMSO-d6) δ 10.58 (br s, 1H) , 8.80 (s, 1H) , 8.23 (s, 1H) , 8.20 (s, 1H) , 7.63 (s, 1H) , 3.23 (s, 6H) .

Example 45. Synthesis of 1- (2-chloro-6- (trifluoromethyl) pyridin-4-yl) -3- (2-chloro-8- (ethyl (2-methoxyethyl) amino) imidazo [1, 2-b] pyridazin-7-yl) urea

Example 45 was prepared in analogy to the procedure described for the preparation of example 21 by using N-ethyl-2-methoxy-ethanamine and 2-chloro-6- (trifluoromethyl) pyridin-4-amine--methane as the starting materials instead of oxetan-3-amine and 5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-amine. LC-MS: m/z 492.0 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d ₆) δ 10.26 (s, 1H) , 8.65 (d, J = 6.4 Hz, 2H) , 8.28 (s, 1H) , 7.94 (d, J = 1.4 Hz, 1H) , 7.84 (d, J = 1.4 Hz, 1H) , 3.71 (t, J = 5.6 Hz, 2H) , 3.58 (q, J =7.2 Hz, 2H) , 3.42 (t, J = 5.6 Hz, 2H) , 3.13 (s, 3H) , 1.02 (t, J = 7.2 Hz, 3H) .

Example 46. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (8-isopropylimidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a solution of ethyl 8-bromoimidazo [1, 2-b] pyridazine-7-carboxylate 7a (300 mg, 1.11 mmol) in toluene (4 mL) was added isopropenylboronic acid (95.4 mg, 1.11 mmol) , tricyclohexylphosphine (62.3 mg, 0.222 mmol) and Potassium phosphate (589.5 mg, 2.78 mmol) . The suspension was degassed and exchanged with N ₂ two times. The Palladium (II) acetate (24.9 mg, 0.111 mmol) was added into the mixture. The reaction mixture was stirred at 100 ℃ for 12 hours. The starting material was consumed and the desired mass was detected from LC-MS. The mixture was concentrated in vacuo and the resulting residue was purified with flash column chromatography (eluting with PE/EA = 10/1) to give the ethyl 8-isopropenylimidazo [1, 2-b] pyridazine-7-carboxylate (100.5 mg, 39.13%yield) as a yellow solid.

LC-MS: m/z 232.1 [M+H] ⁺;

Step 2

To a solution of ethyl 8-isopropenylimidazo [1, 2-b] pyridazine-7-carboxylate 8j (100 mg, 0.432 mmol) in methanol (5 mL) was added Platinum (IV) oxide (15 mg, 0.256 mmol) . The reaction mixture was stirred at room temperature overnight under hydrogen atmosphere. It was filtrated and concentrated under vacuum. The crude was purified by flash column chromatography (eluting with PE/EA = 10/1) to afford product ethyl 8-isopropylimidazo [1, 2-b] pyridazine-7-carboxylate (94 mg, 93.19 %yield) as a white solid.

LC-MS: m/z 234.1 [M+H] ⁺

Step 3

To a solution of ethyl 8-isopropylimidazo [1, 2-b] pyridazine-7-carboxylate 8k (92 mg, 0.394 mmol) in THF (2 mL) was added lithium hydroxide monohydrate (82.8 mg, 1.97 mmol) in water (0.4 mL) . The mixture was stirred at room temperature for 5 hours. Upon removal of solvent, the residue was acidified with HCl (2 M) to pH = 3～4. The mixture was extracted by EtOAc and the combined organic phase was dried and concentrated to give 8-isopropylimidazo [1, 2-b] pyridazine-7-carboxylic acid (80 mg, 98.84%yield) as a yellow solid .

LC-MS: m/z 206.1 [M+H] ⁺.

Step 4

To a solution of 8-isopropylimidazo [1, 2-b] pyridazine-7-carboxylic acid 9l (80 mg, 0.390 mmol) in 1, 4-dioxane (6 mL) was added diphenylphosphonic azide (128.74 mg, 0.468 mmol) and triethylamine (196.87 mg, 1.95 mmol) . The resulting solution was stirred at room temperature for 30 minutes, then 5-chloro-6- (triazol-2-yl) pyridin-3-amine (76.3 mg, 0.389 mmol) was added and the mixture was stirred at 100 ℃ for 2 hours. After removing solvent under vacuum, the residue was purified by flash column chromatography (eluting with DCM: MeOH = 30 : 1) to give 1- [5-chloro-6- (triazol-2-yl) -3-pyridyl] -3- (8-isopropylimidazo [1, 2-b] pyridazin-7-yl) urea (14.8 mg, 9.54%yield) as a white solid.

LC-MS: m/z 398.1 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d6) : δ 9.67 (s, 1H) , 8.81 (s, 1H) , 8.59-8.57 (m, 2 H) , 8.47-8.46 (m, 1 H) , 8.18 (d, J = 1.2 Hz, 1H) , 8.15 (s, 2 H) , 7.70 (d, J =1.2 Hz, 1H) , 3.52-3.47 (m, 1H) , 1.51 (d, J = 6.8 Hz, 6H) .

Example 47. Synthesis of 1- (8-isopropylimidazo [1, 2-b] pyridazin-7-yl) -3- (2- (trifluoromethyl) pyridin-4-yl) urea

Example 47 was prepared in analogy to the procedure described for the preparation of example 46 by using 2- (trifluoromethyl) pyridin-4-amine as the starting material instead of 5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-amine.

LC-MS: m/z 365.1 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d6) : δ 9.78 (s, 1H) , 8.77 (s, 1H) , 8.56-8.54 (m, 2 H) , 8.18 (d, J = 1.2 Hz, 1H) , 8.06-8.05 (m, 1 H) , 7.70 (d, J = 1.2 Hz, 1H) , 7.63-7.61 (m, 1 H) , 3.51-3.44 (m, 1H) , 1.50 (d, J = 7.2 Hz, 6H) .

Example 48. Synthesis of 1- (5-chloro-6-methoxypyridin-3-yl) -3- (8-isopropylimidazo [1, 2-b] pyridazin-7-yl) urea

Example 48 was prepared in analogy to the procedure described for the preparation of example 46 by using 5-chloro-6-methoxy-pyridin-3-amine as the starting material instead of 5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-amine.

LC-MS: m/z 361.1 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d6) : δ 9.14 (s, 1H) , 8.62-8.59 (m, 2H) , 8.15-8.13 (m, 3H) , 7.67-7.66 (m, 1H) , 3.91 (s, 3H) , 3.53-3.46 (m, 1H) , 1.50 (d, J = 7.2 Hz, 6H) .

Example 49. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (8-ethylimidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

o a solution of ethyl 2-chloro-8-ethyl-imidazo [1, 2-b] pyridazine-7-carboxylate 8e (150 mg, 0.591 mmol) in Methanol (5 mL) was added Pd/C (71.8 mg, 591.29 μmol) and the reaction mixture was degassed and purged with H ₂ for several times and stirred under H ₂ atmosphere at room temperature for 3 hours. The mixture was filtered and the filter cake was washed with ethyl acetate. The combined organic layers were concentrated to afford ethyl 8-ethylimidazo [1, 2-b] pyridazine-7-carboxylate (129 mg, crude) as a yellow oil which was used in the next step without further purification.

LC-MS: m/z 220.1 [M+H] ⁺.

Step 2

To a solution of ethyl 8-ethylimidazo [1, 2-b] pyridazine-7-carboxylate 8e2 (129 mg, 0.588 mmol) in THF (3 mL) at 0 ℃ was added lithium hydroxide (74.1 mg, 1.77 mmol) . The mixture was stirred at rt for 12 hours. Upon removal of the solvents, the residue was acidified with HCl (2 M) to pH = 3～4. The suspension was filtered and the filter cake was dried in vacuum to give 8-ethylimidazo [1, 2-b] pyridazine-7-carboxylic acid (112 mg, crude) as a yellow solid.

LC-MS: m/z 192.1 [M+H] ⁺.

Step 3

To a solution of 8-ethylimidazo [1, 2-b] pyridazine-7-carboxylic acid 9c2 (147 mg, 0.769 mmol) in 1, 4-dioxane (7 mL) was added N, N-diethylethanamine (389.0 mg, 3.84 mmol) and [azido (phenoxy) phosphoryl] oxybenzene (253.9 mg, 0.922 mmol) at room temperature. The mixture was stirred at room temperature for 30 mins. Then 5-chloro-6- (triazol-2-yl) pyridin-3-amine (195.5 mg, 0.999 mmol) was added and the reaction mixture was stirred at 100 ℃ for 2 hours. TLC (Dichloromethane : Methanol=20: 1, UV) showed the most starting material was consumed. The mixture was concentrated to obtain the residue, which was purified by Prep-TLC (Dichloromethane: Methanol=20: 1) to give 1- [5-chloro-6- (triazol-2-yl) -3-pyridyl] -3- (8-ethylimidazo [1, 2-b] pyridazin-7-yl) urea (45.4 mg, 15.38%yield) as a white solid.

LC-MS: m/z 384.0 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d ₆) δ 9.74 (s, 1H) , 8.81 (s, 1H) , 8.78 (s, 1H) , 8.57 (d, J = 2.4 Hz, 1H) , 8.48 (d, J = 2.4 Hz, 1H) , 8.19-8.16 (m, 3H) , 7.68 (d, J = 2.4 Hz, 1H) , 2.99 (q, J = 7.2 Hz, 2H) , 1.28 (t, J = 7.2 Hz, 1H) .

Example 50. 1- [5-chloro-6- (triazol-2-yl) -3-pyridyl] -3- (3-fluoro-8-isopropyl-imidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a mixture of ethyl 8-bromoimidazo [1, 2-b] pyridazine-7-carboxylate 7a (540 mg, 2.00 mmol) in mixed solvent of H ₂O (2.5 mL) and 1, 4-dioxane (10 mL) were added potassium phosphate (1.27 g, 6.00 mmol) , 2-isopropenyl-4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolane (403.2 mg, 2.40 mmol) and [1, 1'-bis (diphenylphosphino) ferrocene] dichloropalladium (II) (146.3 mg, 199.94 μmol) . The resulting mixture was degassed with N ₂, stirred under reflux for 8 hours, cooled to room temperature and filtered through silica gel. The solid cake was washed with ethyl acetate (20 mL x 3) , and the filtrate was concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (elution with EtOAc/petroleum ether, 0～50%, v/v) to afford ethyl 8-isopropenylimidazo [1, 2-b] pyridazine-7-carboxylate (250 mg, 54.07%yield) as a white solid. LCMS (ESI ⁺) [ (M+H) ⁺] : 231.8.

Step 2

A mixture of ethyl 8-isopropenylimidazo [1, 2-b] pyridazine-7-carboxylate 8l (250 mg, 1.08 mmol) and palladium on activated carbon (0.1 g, 1.08 mmol) in MeOH (5 mL) was stirred under H ₂ atmosphere for 2 hours at room temperature and filtered through a pad of Celite. The solid cake was washed with MeOH, and the filtrate was concentrated in vacuo to afford crude ethyl 8-isopropylimidazo [1, 2-b] pyridazine-7-carboxylate (180 mg, 71.38%yield) . LCMS (ESI ⁺) [ (M+H) ⁺] : 233.7.

Step 3

To a solution of ethyl 8-isopropylimidazo [1, 2-b] pyridazine-7-carboxylate 8m (90 mg, 385.83 μmol) in MeCN (5 mL) was added Selecfluor (205.02 mg, 578.74 μmol) in portions at 5 ℃～10 ℃. The mixture was stirred for 4 hours at this temperature, poured into H ₂O (20 mL) , and extracted with ethyl acetate (15 mL x 2) . The combined organic phase was washed with brine (20 mL) , dried over anhydrous Na ₂SO ₄, and filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel (elution with ethyl acetate in petroleum ether from 10%to 50%) to afford ethyl 3-fluoro-8-isopropyl-imidazo [1, 2-b] pyridazine-7-carboxylate (23 mg, 23.73%yield) as a white solid. LCMS (ESI ⁺) [ (M+H) ⁺] : 251.6.

Step 4

To a solution of ethyl 3-fluoro-8-isopropyl-imidazo [1, 2-b] pyridazine-7-carboxylate 8n (23 mg, 91.54 μmol) in THF (0.5 mL) was added a solution of lithium hydroxide monohydrate (11.52 mg, 274.62 μmol) in H ₂O (0.2 mL) . The mixture was stirred at room temperature overnight and concentrated in vacuo. The residue was acidified with HCl aqueous solution (2M) to pH 3～4. The resulting mixture was extracted with ethyl acetate (15 mL x 2) , and the combined organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated in vacuo to give crude 3-fluoro-8-isopropyl-imidazo [1, 2-b] pyridazine-7-carboxylic acid (18 mg, 88.10%yield) . LCMS (ESI ⁺) [ (M+H) ⁺] : 223.8.

Step 5

To a solution of 3-fluoro-8-isopropyl-imidazo [1, 2-b] pyridazine-7-carboxylic acid 9m (18 mg, 80.64 μmol) in 1, 4-dioxane (4 mL) were added diphenylphosphonic azide (31.4 mg, 120.97 μmol, 24.69 uL) and TEA (24.5 mg, 241.93 μmol, 33.72 uL) . The resulting mixture was stirred at room temperature for 30 minutes, followed by the addition of 5-chloro-6- (triazol-2-yl) pyridin-3-amine (15.8 mg, 80.64 μmol) . The mixture was stirred at 100 ℃ overnight, cooled to room temperature, poured into water (10 mL) and extracted with ethyl acetate (15 mL x 2) . The combined organic phase was dried over anhydrous Na ₂SO ₄ and filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column chromatography on silica gel (elution with ethyl acetate in petroleum ether from 10%to 80%) and prep-HPLC to give 1- [5-chloro-6- (triazol-2-yl) -3-pyridyl] -3- (3-fluoro-8-isopropyl- imidazo [1, 2-b] pyridazin-7-yl) urea (4.0 mg, 11.93%yield) as a white solid. LCMS (ESI ⁺) [ (M+H) ⁺] : 416. ¹H NMR (400 MHz, DMSO-d ₆) δ 8.97 (s, 1H) , 8.69 (s, 1H) , 8.59 (d, J = 2.4 Hz, 1H) , 8.47 (d, J = 2.4 Hz, 1H) , 8.16 (s, 2H) , 7.53 (d, J = 7.2 Hz, 1H) , 3.50 (dt, J = 13.6, 6.8 Hz, 1H) , 1.51 (d, J = 6.8 Hz, 6H) .

Example 51. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (trifluoromethyl) imidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a stirred solution of cuprous chloride (195.1 mg, 1.97 mmol) , potassium tert-butoxide 7c (220.7 mg, 1.97 mmol) was added 1, 10-phenanthroline (355.06 mg, 1.97 mmol) in DMF (3.0 mL) , degassed and refilled with N ₂ three times, the dark red mixture was stirred at room temperature for 30 mins, then trimethyl (trifluoromethyl) silane (280.6 mg, 1.97 mmol, 313.03 uL) was slowly added. The resulting mixture was further stirred at room temperature for 1h and ethyl 8-bromo-2-chloro-imidazo [1, 2-b] pyridazine-7-carboxylate 7c (300 mg, 985.13 μmol) was expeditiously added , degassed and refilled with N ₂ three times, heated at 50℃ for 18 hours and judged by the LCMS to be complete, then cooled to room temperature, diluted with MTBE and filtered through a pad of celite pad. The celite pad was washed with MTBE and the combined organic layer was washed with brine, and dried over Na ₂SO ₄ , After filtration and evaporation of the solvent, the crude mixture was purified by silica gel column chromatography using EA/PE=0: 100-1: 50 as eluent to give ethyl 2-chloro-8- (trifluoromethyl) imidazo [1, 2-b] pyridazine-7-carboxylate (80 mg, 272.45 μmol, 27.66%yield) , LC-MS: m/z 294 [M+H] ⁺

Step 2

To a stirred solution of ethyl 2-chloro-8- (trifluoromethyl) imidazo [1, 2-b] pyridazine-7-carboxylate 8o (80 mg, 272.45 μmol) in THF (2.0 mL) was added LiOH (0.82 mL, 1 M) , the resulting mixture was stirred at room temperature overnight and judged by LCMS to be complete. Then 3M hydrochloric acid was added to adjust pH= 2-3 and extracted with EA (20 mL) , the organic phases were washed with brine (5 mL) , dried over Na ₂SO ₄, filtered and concentrated in vacuo to yield crude 2-chloro-8- (trifluoromethyl) imidazo [1, 2-b] pyridazine-7-carboxylic acid (80 mg, 301.23 μmol, 110.56%yield) , the crude product was directly used in the next step without further purification. LC-MS: m/z 266 [M+H] ⁺

Step 3

To a stirred solution of 2-chloro-8- (trifluoromethyl) imidazo [1, 2-b] pyridazine-7-carboxylic acid 9n (80 mg, 301.23 μmol) in 1, 4-Dioxane (2.0 mL) , added TEA (91.4 mg, 903.69 μmol, 125.96 uL) and DPPA (124.4 mg, 451.85 μmol, 97.91 uL) . the reaction mixture was stirred at room temperature for 30 mins, then 5-chloro-6- (triazol-2-yl) pyridin-3-amine 10a (88.4 mg, 451.85 μmol) was added, the reaction mixture was stirred at 100℃ overnight, and judged by the LCMS to be complete, the mixture was cooled to room temperature and evaporation of the solvent, the crude mixture was purified by Prep-HPLC to give 1- [5-chloro-6- (triazol-2-yl) -3-pyridyl] -3- [2-chloro-8- (trifluoromethyl) imidazo [1, 2-b] pyridazin-7-yl] urea (20 mg, 43.65 μmol, 14.49%yield) , LC-MS: m/z 459 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H) , 9.26 (s, 1H) , 9.07 (s, 1H) , 8.61 (s, 1H) , 8.56 (t, J = 0.5 Hz, 1H) , 8.47 (t, J = 1.2 Hz, 1H) , 8.17 (s, 2H) .

Example 52. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (methoxymethyl) imidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a stirred solution of 2- (bromomethyl) -4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolane (300 mg, 1.36 mmol) in MeOH (6 mL) was added CH ₃ONa (73.4 mg, 1.36 mmol) . The reaction mixture was stirred at 25℃ for overnight, added ice water (1 mL) , and extracted with EtOAc (40 mL) , the organic phases were washed with brine (5mL) and dried over Na ₂SO ₄, filtered and concentrated in vacuo to give crude 2- (methoxymethyl) -4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolane (240 mg, 1.40 mmol, 102.73%yield) as a white solid.

Step 2

To a stirred solution of ethyl 8-bromo-2-chloro-imidazo [1, 2-b] pyridazine-7-carboxylate (339.88 mg, 1.12 mmol) in 1, 4-Dioxane (5.0 mL) , H ₂O (0.5 mL) was added 2- (methoxymethyl) -4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolane (240 mg, 1.40 mmol) , K ₃PO ₄ (888.42 mg, 4.19 mmol) , degassed and refilled with N ₂ three times, added Pd (dppf) Cl ₂ ^. DCM (113.93 mg, 139.51 μmol) and degassed and refilled with N ₂ three times, the mixture was heated at 90℃ for 4 hours and judged by the LCMS to be complete, then cooled, diluted with EA and washed with brine, and dried over Na ₂SO ₄. After filtration and evaporation of the solvent, the crude mixture was purified by silica gel column chromatography using EA/PE=0: 100-1: 10 as eluent to give ethyl 2-chloro-8- (methoxymethyl) imidazo [1, 2-b] pyridazine-7-carboxylate (80 mg, 296.64 μmol, 21.26%yield) as a white solid. LC-MS: m/z 270 [M+H] ⁺ .

Step 3

To a stirred solution of ethyl 2-chloro-8- (methoxymethyl) imidazo [1, 2-b] pyridazine-7-carboxylate 8p (80 mg, 296.64 μmol) in THF (4.0 mL) , was added LiOH (0.5 M, 1.2 ml) at 0 ℃ , stirred 2 h at 0 ℃ and judged by the LCMS to be complete, Then 3M hydrochloric acid was added to adjust pH= 2-3 and extracted with EA (20 mL) , the organic phases were washed with brine (5 mL) and dried over Na ₂SO ₄, filtered and concentrated in vacuo to give crude 2-chloro-8- (methoxymethyl) imidazo [1, 2-b] pyridazine-7-carboxylic acid (60 mg, 248.31 μmol, 83.71%yield) , LC-MS: m/z 242 [M+H] ⁺ , the crude product was directly used in the next step without further purification.

Step 4

To a stirred solution of 2-chloro-8- (methoxymethyl) imidazo [1, 2-b] pyridazine-7-carboxylic acid 9o (60 mg, 248.31 μmol) in 1, 4-Dioxane (4 mL) was added TEA (75.4 mg, 744.94 μmol, 103.83 uL) and DPPA (90.6 mg, 372.47 μmol) . the reaction mixture was stirred at room temperature for 2 h. Then 5-chloro-6- (triazol-2-yl) pyridin-3-amine (58.3 mg, 297.98 μmol) was added, and the reaction mixture was stirred 2 hours at 100℃ and judged by the LCMS to be complete. The mixture was cooled to room temperature and evaporation of the solvent, the crude mixture was purified by Prep-HPLC to give 1- [2-chloro-8- (methoxymethyl) imidazo [1, 2-b] pyridazin-7-yl] -3- [5-chloro-6- (triazol-2-yl) -3-pyridyl] urea (14.0 mg, 32.24 μmol) , LC-MS: m/z 435 [M+H] ⁺ ; ¹H NMR (400 MHz, DMSO-d6) δ 9.05 (s, 1H) , 8.49 (d, J = 2.3 Hz, 1H) , 8.42 (d, J = 2.3 Hz, 1H) , 8.34 (s, 1H) , 8.09 (s, 2H) , 4.81 (s, 2H) , 3.32 (s, 3H) .

Example 53. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (8- (methoxymethyl) imidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a solution of ethyl 8-isopropylimidazo [1, 2-b] pyridazine-7-carboxylate 7a (1.5 g, 5.55 mmol) in THF (20 mL) was added lithium hydroxide monohydrate (699.2 mg, 16.66 mmol) in water (10 mL) . The mixture was stirred at room temperature for 5 hours. Upon removal of solvent, the residue was acidified with HCl (2 M) to pH = 3～4. And the mixture was extracted by EtOAc and the combined organic phases were dried and concentrated to give 8-bromoimidazo [1, 2-b] pyridazine-7-carboxylic acid (1.25 g, 92.99%yield) as a yellow solid.

LC-MS: m/z 241.1 [M+H] ⁺.

Step 2

To a stirred solution of 8-bromoimidazo [1, 2-b] pyridazine-7-carboxylic acid 17a (1.25 g, 5.16 mmol) in Toluene (15 mL) and 2-methylpropan-2-ol (10 mL) was added triethylamine (2.61 g, 25.82 mmol) , Diphenylphosphoryl azide (1.71 g, 6.20 mmol, 1.34 mL) , tert-butoxycarbonyl anhydride (1.69 g, 7.75 mmol, 1.78 mL) . The reaction mixture was stirred at 100 ℃ for 4 hours. After removing solvent under vacuum, the residue was purified by flash column chromatography (eluting with hexanes/ethyl acetate = 1: 1) to give tert-butyl N- (8-bromoimidazo [1, 2-b] pyridazin-7-yl) carbamate (820 mg, 41.43%yield) as a white solid.

LC-MS: m/z 313.1 [M+H] ⁺.

Step 3

To a stirred solution of tert-butyl N- (8-bromoimidazo [1, 2-b] pyridazin-7-yl) carbamate 18a (800 mg, 2.55 mmol) in MeOH (10 mL) was added triethylamine (1.29 g, 12.77 mmol) , [1, 1'-Bis (diphenylphosphino) ferrocene] dichloropalladium (II) (186.9 mg, 0.255 mmol) . The resulting reaction mixture was stirred at 100℃ under CO (3 MPa) for 16 hours. The reaction mixture was cooled to room temperature. After removing solvent under vacuum, the residue was purified by flash column chromatography (eluting with hexanes/ethyl acetate =1: 1) to give methyl 7- (tert-butoxycarbonylamino) imidazo [1, 2-b] pyridazine-8-carboxylate (460 mg, 61.60%yield) as a yellow solid.

LC-MS: m/z 293.1 [M+H] ⁺.

Step 4

To a stirred solution of methyl 7- (tert-butoxycarbonylamino) imidazo [1, 2-b] pyridazine-8-carboxylate 19a (460 mg, 1.57 mmol) in MeOH (10 mL) was added sodium borohydride (297 mg, 7.87 mmol) in portions at 0℃, the resulting reaction mixture was stirred at 25℃ for 16 hours. The reaction was quenched by saturated NH ₄Cl and the mixture was extracted with EtOAc (10 mL) , the combined organic phase was washed with brine (5 mL) and dried over anhydrous Na ₂SO ₄, filtered and concentrated in vacuo. The crude was purified by flash column chromatography (DCM/MeOH=20: 1) to afford the tert-butyl N- [8- (hydroxymethyl) imidazo [1, 2-b] pyridazin-7-yl] carbamate (300 mg, 72.13%yield) as a yellow solid.

LC-MS: m/z 265.1 [M+H] ⁺.

Step 5

To a stirred solution of tert-butyl N- [8- (hydroxymethyl) imidazo [1, 2-b] pyridazin-7-yl] carb amate 19b (300 mg, 1.14 mmol) in MeOH (5.00 mL) was added thionyl chloride (1.35 g, 11.35 mmol) at 0 ℃, then the resulting reaction mixture was stirred at 60 ℃ for 16 hours. After removing solvent under vacuum, the residue was purified by flash column chromatography (eluting with DCM/MeOH=10: 1) to afford the methyl 7-aminoimidazo [1, 2-b] pyridazine-8-carboxylate (90 mg, 41.05%yield) as a yellow solid.

LC-MS: m/z 179.1 [M+H] ⁺.

Step 6

To a stirred solution of 5-chloro-6- (triazol-2-yl) pyridin-3-amine 10a (90 mg, 468.33 μmol) in Toluene (10 mL) was added triethylamine (153 mg, 1.52 mmol) , bis (trichloromethyl) carbonate (74.9 mg, 0.252 mmol) . The reaction mixture was stirred at 100 ℃ for 2 hours, then the solution of 8- (methoxymethyl) imidazo [1, 2-b] pyridazin-7-amine 19b (90 mg, 0.505 mmol) in DCM (2.00 mL) was added at 0 ℃, the reaction mixture was stirred at room temperature for 2 hours. After removing solvent under vacuum, the residue was purified by flash column chromatography (eluting with DCM: MeOH = 30: 1) to give 1- [5-chloro-6- (triazol-2-yl) -3-pyridyl] -3- [8- (methoxymethyl) imidazo [1, 2-b] pyridazin-7-yl] urea (17.8 mg, 8.82%yield) as a white solid.

LC-MS: m/z 400.1 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d6) : δ 10.30 (s, 1H) , 9.04 (s, 1H) , 8.77 (s, 1H) , 8.55 (d, J = 2.4 Hz, 1H) , 8.50 (d, J = 2.4 Hz, 1H) , 8.22 (d, J = 1.2 Hz, 1H) , 8.16 (s, 2H) , 7.69 (d, J = 1.2 Hz, 1H) , 4.94 (s, 2H) , 3.39 (s, 3H) .

Example 54. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (cyclopropylamino) imidazo [1, 2-b] pyridazin-7-yl) urea

Example 54 was prepared in analogy to the procedure described for the preparation of example 21 by using cyclopropanamine as the starting material instead of oxetan-3-amine.

LC-MS: m/z 445.0 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d ₆) δ 9.68 (s, 1H) , 8.58 (d, J = 2.4 Hz, 1H) , 8.44 (d, J = 2.4 Hz, 1H) , 8.36 (s, 1H) , 8.19 (s, 1H) , 8.14 (s, 2H) , 8.06 (s, 1H) , 7.18 (s, 1H) , 3.36-3.29 (m, 1H) , 0.76-0.69 (m, 4H) .

Example 55. Synthesis of N- (7- (3- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) ureido) imidazo [1, 2-b] pyridazin-8-yl) acetamide

Step 1

To a solution of ethyl 8-bromoimidazo [1, 2-b] pyridazine-7-carboxylate 7a (4 g, 14.81 mmol) in THF (50 mL) and water (30 mL) was added LiOH (1.24 g, 29.62 mmol) . The resulting mixture was stirred at room temperature for 10 mins. TLC showed that the reaction was complete. The mixture was acidified with 1 M HCl to pH = 6 and extracted with EtOAc. The combined organic layers were dried, filtered and concentrated to give 8-bromoimidazo [1, 2-b] pyridazine-7-carboxylic acid, (3.26 g, 13.47 mmol, 91%yield) as a yellow solid. LC-MS: m/z 241.9 [M+H] ⁺.

Step 2

A mixture of 8-bromoimidazo [1, 2-b] pyridazine-7-carboxylic acid 17a (3.26 g, 13.47 mmol) , [azido (phenyl) phosphoryl] benzene (3.93 g, 16.16 mmol) , N, N-diethylethanamine (6.81 g, 67.35 mmol, 9.39 mL) , Boc ₂O (14.70 g, 67.35 mmol) in toluene (25 mL) and 2-methylpropan-2-ol (25 mL) was degassed and refilled with N ₂ (three times) . After the mixture was heated at 100℃ for 12 hours, LCMS showed that the reaction was complete. The mixture was concentrated, and the residue was diluted with EA (100 mL) , washed with brine (40 mLx2) , and dried. The organic layer was concentrated and the crude product was purified by flash silica gel chromatography (Petroleum ether /ethyl acetate = 2/1) to give tert-butyl N- (8-bromoimidazo [1, 2-b] pyridazin-7-yl) carbamate (2.3 g, 55%yield) as a yellow solid. LC-MS: m/z 315.0 [M+H] ⁺.

Step 3

To a solution of tert-butyl N- (8-bromoimidazo [1, 2-b] pyridazin-7-yl) carbamate 18a (850 mg, 2.71 mmol) in N, N-dimethylformamide (15 mL) was added Cs ₂CO ₃ (1.77 g, 5.43 mmol) . The resulting mixture was stirred at 80 ℃ for 2 hours. TLC showed that the reaction was complete. The mixture was diluted with EA (50 mL) and filtered. The filtrate was washed with sat. NH ₄Cl (20 mL) , 1 M LiCl (20 mLx2) , and concentrated. The residue was purified by silica gel column chromatography (Petroleum ether /ethyl acetate = 3/1) to give tert-butyl N- (8-bromoimidazo [1, 2-b] pyridazin-7-yl) -N- [ (4-methoxyphenyl) methyl] carbamate (1.1 g, 94%yield) as a white solid. LC-MS: m/z 433.1 [M+H] ⁺.

Step 4

A mixture of tert-butyl N- (8-bromoimidazo [1, 2-b] pyridazin-7-yl) -N- [ (4-methoxyphenyl) methyl] carbamate 18b (1.1 g, 2.54 mmol) , acetamide (299 mg, 5.08 mmol) , Cs ₂CO ₃ (1.65 g, 5.08 mmol) , Pd ₂ (dba) ₃ (465 mg, 507.73 μmol) and Xantphos (587 mg, 1.02 mmol) in 1, 4-dioxane (20 mL) was degassed and backfilled with N ₂ (three times) . After the mixture was heated at 100 ℃ for 12 hours, LCMS showed that the reaction was complete. The precipitate was filtered off and the filtrate was concentrated. The residue was purified by Prep-TLC to give tert-butyl N- (8-acetamidoimidazo [1, 2-b] pyridazin-7-yl) -N- [ (4-methoxyphenyl) methyl] carbamate (600 mg, 57%yield) as a yellow solid. LC-MS: m/z 412.2 [M+H] ⁺.

Step 5

To a solution of tert-butyl N- (8-acetamidoimidazo [1, 2-b] pyridazin-7-yl) -N- [ (4-methoxyphenyl) methyl] carbamate 19c (600 mg, 1.46 mmol) in TFA (5 mL) was stirred at room temperature for 12 hours. The mixture was concentrated, and the residue was diluted with DCM (30 mL) , washed with sat. NaHCO ₃ (15 mLx2) , and then concentrated to give the crude product which was purified by silica gel chromatography (DCM/MeOH = 20/1) to give N- (7-aminoimidazo [1, 2-b] pyridazin-8-yl) acetamide (180 mg, 65%yield) as a yellow solid. LC-MS: m/z 192.1 [M+H] ⁺.

Step 6

A solution of N- (7-aminoimidazo [1, 2-b] pyridazin-8-yl) acetamide 20b (50 mg, 0.261 mmol) and phenyl N- [5-chloro-6- (triazol-2-yl) -3-pyridyl] carbamate (99 mg, 0.314 mmol) in DMF was stirred at 80 ℃ for 12 h. LCMS showed that the reaction was completed. The mixture was diluted with EtOAc (15 mL) , washed with 1 M LiCl and concentrated. The residue was purified by Prep-HPLC to give N- [7- [ [5-chloro-6- (triazol-2-yl) -3-pyridyl] carbamoylamino] imidazo [1, 2-b] pyridazin-8-yl] acetamide (23.8 mg, 22%yield) as a white solid. LC-MS: m/z 413.1 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d ₆) : δ 10.90 (s, 1H) , 10.41 (s, 1H) , 8.83 (s, 1H) , 8.54 (d, J = 2.4 Hz, 1H) , 8.45 (d, J = 2.4 Hz, 1H) , 8.26 (s, 1H) , 8.15 (s, 3H) , 7.70 (s, 1H) , 2.30 (s, 3H) .

Example 56. Synthesis of 2-chloro-7- (3- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) ureido) -N, N-dimethylimidazo [1, 2-b] pyridazine-8-carboxamide

Step 1

To a stirred solution of methyl 7- (tert-butoxycarbonylamino) -2-chloro-imidazo [1, 2-b] pyridazine-8-carboxylate 19d1 (200 mg, 612.12 μmol) in THF (4.0 mL) , was added LiOH (1.0 M, 1.8 mL) , stirred 2 h and judged by the LCMS to be complete, then 3M HCl was added to adjust pH= 2-3 and extracted with EA (40 mL) , the organic phases were washed with brine (5mL) and dried over Na ₂SO ₄, filtered and concentrated in vacuo to give 7- (tert-butoxycarbonylamino) -2-chloro-imidazo [1, 2-b] pyridazine-8-carboxylic acid (190 mg, 607.59 μmol, 99.26%yield) , LC-MS: m/z 313 [M+H] ⁺ , the crude product was directly used in the next step without further purification.

Step 2

To a stirred solution of 7- (tert-butoxycarbonylamino) -2-chloro-imidazo [1, 2-b] pyridazine-8-carboxylic acid 19d2 (190 mg, 607.59 μmol) in DMF (2.0 mL) was added dimethylamine hydrochloride (99.1 mg, 1.22 mmol) , HATU (462 mg, 1.22 mmol) , DIEA (471 mg, 3.65 mmol) , stirred 4.0 h and judged by the LCMS to be complete, added 4.0 mL water and extracted with EA (40 mL) , the organic phase were washed with brine (5mL) and dried over Na ₂SO ₄, filtered and concentrated in vacuo, the crude mixture was purified by silica gel column chromatography using EA/PE=0: 100-1: 10 as eluent to give tert-butyl N- [2-chloro-8- (dimethylcarbamoyl) imidazo [1, 2-b] pyridazin-7-yl] carbamate (150 mg, 441.47 μmol, 72.66%yield) , LC-MS: m/z 340 [M+H] ⁺.

Step 3

To a stirred solution of tert-butyl N- [2-chloro-8- (dimethylcarbamoyl) imidazo [1, 2-b] pyridazin-7-yl] carbamate 19d3 (150 mg, 441.47 μmol) was added HCl/EA (1.0 M, 5 mL) , the resulting mixture was stirred overnight and was adjusted pH =9-10 with Na ₂CO ₃ and extracted with EA (40 mL) , the organic phases were washed with brine (5mL) and dried over Na ₂SO ₄, filtered and concentrated in vacuo to give 7-amino-2-chloro-N, N-dimethyl-imidazo [1, 2-b] pyridazine-8-carboxamide (100 mg, 417.26 μmol, 94.52%yield) , LC-MS: m/z 240 [M+H] ⁺, the crude product was directly used in the next step without further purification.

Step 4

To a stirred solution of 7-amino-2-chloro-N, N-dimethyl-imidazo [1, 2-b] pyridazine-8-carboxamide 20c (50 mg, 208.63 μmol) in DMF (4.0 mL) was added CDI (120 mg, 834.51 μmol) and TEA (126.7 mg, 1.25 mmol, 174.47 uL) , the reaction mixture was heated at 80℃ for 4 hours and cooled to 40 ℃. Thereto 5-chloro-6- (triazol-2-yl) pyridin-3-amine (40.8 mg, 208.63 μmol) was added. The mixture was heated at 80 ℃ for overnight, cooled to room temperature and diluted with EA (40 mL) , the organic phases were washed with brine (5mL) and dried over Na ₂SO ₄, filtered and concentrated in vacuo, the crude mixture was purified by Prep-HPLC to give 2-chloro-7- [ [5-chloro-6- (triazol-2-yl) -3-pyridyl] carbamoylamino] -N, N-dimethyl-imidazo [1, 2-b] pyridazine-8-carboxamide (6.0 mg, 13.01 μmol, 6.23%yield) . LC-MS: m/z 462 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d6) δ 9.16 (s, 1H) , 8.55 (d, J = 2.3 Hz, 1H) , 8.47 (d, J = 2.3 Hz, 1H) , 8.44 (s, 1H) , 8.16 (s, 2H) , 3.12 (s, 3H) , 2.89 (s, 3H) .

Example 57. Synthesis of 2-chloro-7- (3- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) ureido) imidazo [1, 2-b] pyridazine-8-carboxylic acid

Step 1

To a microwave vial was added ethyl 8-bromo-2-chloro-imidazo [1, 2-b] pyridazine-7-carboxylate 7c (300 mg, 985.13 μmol) , diethyl oxalate (172.8mg, 1.18 mmol, 159.96 uL) , N, N-dimethylpyridin-4-amine (144.4 mg, 1.18 mmol) and EtOH (6.0 mL) , the reaction mixture was stirred under N ₂ for 10 mins, bis (triphenylphosphine) palladium (II) chloride (69.2 mg, 98.51 μmol) was added and heated at 120 ℃ for 0.5 hour under microwave irradiation. The mixture was allowed to cool to room temperature. After filtration and evaporation of the solvent, the crude mixture was purified by silica gel column chromatography using EA/PE=0: 100-100: 0 as eluent to give 2-chloro-7-ethoxycarbonyl-imidazo [1, 2-b] pyridazine-8-carboxylic acid (190 mg, 704.64 μmol, 71.53%yield) , LC-MS: m/z 270 [M+H] ⁺ .

Step 2

To a stirred solution of 2-chloro-7-ethoxycarbonyl-imidazo [1, 2-b] pyridazine-8-carboxylic acid 8q (100 mg, 370.86 μmol) in THF (2.0 mL) , was added LiOH (1.0 M, 1.0 ml) , the mixture was stirred for 5 hours and judged by the LCMS to be complete, then 3M hydrochloric acid was added to adjust pH= 2-3 and extracted with EA (40mL) , the organic phase was washed with brine (5 mL) , dried over Na ₂SO ₄, filtered and concentrated in vacuo to afford 2-chloroimidazo [1, 2-b] pyridazine-7, 8-dicarboxylic acid (85 mg, 351.84 μmol, 94.87%yield) , LC-MS: m/z 242 [M+H] ⁺, the crude product was directly used in the next step without further purification.

Step 3

To a stirred solution of 2-chloroimidazo [1, 2-b] pyridazine-7, 8-dicarboxylic acid 9q (85 mg, 351.84 μmol) in 1, 4-Dioxane (2.0 mL) , was added DPPA (128.3 mg, 527.76 μmol) and TEA (106.8 mg, 1.06 mmol, 147.12 uL) . the reaction mixture was stirred at room temperature for 2 hours. Then 5-chloro-6- (triazol-2-yl) pyridin-3-amine (82.6 mg, 422.21 μmol) was added and heated at 100 ℃ for 4 hours and judged by the LCMS to be complete, added 1.0 mL ice water and extracted with EA (40 mL) , the organic phase was washed with brine (5mL) and dried over Na ₂SO ₄, filtered and concentrated in vacuo , the crude mixture was purified by Prep-HPLC to give 2-chloro-7- [ [5-chloro-6- (triazol-2-yl) -3-pyridyl] carbamoylamino] imidazo [1, 2-b] pyridazine-8-carboxylic acid (9.0 mg, 20.73 μmol, 5.89%yield) as a white solid, LC-MS: m/z 435 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d6) δ9.11 (d, J = 8.6 Hz, 1H) , 8.47 (d, J = 2.3 Hz, 1H) , 8.42 (d, J = 2.3 Hz, 1H) , 8.31 (d, J = 10.2 Hz, 1H) , 8.09 (s, 2H) .

Example 58. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (imidazo [1, 2-b] pyridazin-7-yl) urea

Example 58 was prepared in analogy to the procedure described for the preparation of example 10 by using 8-bromoimidazo [1, 2-b] pyridazine-7-carboxylate as the starting material.

LC-MS: m/z 356.0; ¹H NMR (400 MHz, DMSO-d ₆) : δ 9.79 (s, 1H) , 8.59 (s, 1H) , 8.62-8.59 (m, 2H) , 8.47-8.46 (m, 1H) , 8.17-8.16 (m, 4H) , 7.66 (s, 1H) .

Example 59. Synthesis of 1- (8-bromo-2-chloroimidazo [1, 2-b] pyridazin-7-yl) -3- (5-chloro-6-methoxypyridin-3-yl) urea

Example 59 was prepared in analogy to the procedure described for the preparation of example 10 by using 8-bromoimidazo [1, 2-b] pyridazine-7-carboxylate as the starting material.

LC-MS: m/z 430.8 [M+H] ⁺.

Example 60. Synthesis of 1- (8-amino-2-chloroimidazo [1, 2-b] pyridazin-7-yl) -3- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) urea

Example 60 was prepared in analogy to the procedure described for the preparation of example 10 by using 8-bromoimidazo [1, 2-b] pyridazine-7-carboxylate as the starting material.

LC-MS: m/z 405 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d ₆) δ 10.20 (s, 1H) , 8.85 (s, 1H) , 8.60 (d, J = 2.2 Hz, 1H) , 8.45 (d, J = 2.2 Hz, 1H) , 8.19 (s, 1H) , 8.17 (s, 1H) , 8.14 (s, 2H) , 6.98 (s, 2H) .

Examples 61-63. Synthesis of 1- [5-chloro-6- (triazol-2-yl) -3-pyridyl] -3- [8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl] urea / (S) -1- [5-chloro-6- (triazol-2-yl) -3-pyridyl] -3- [8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl] urea / (R) -1- [5-chloro-6- (triazol-2-yl) -3-pyridyl] -3- [8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl] urea

Step 1

To a solution of tert-butyl N- (8-bromoimidazo [1, 2-b] pyridazin-7-yl) carbamate 18a (10.36 g, 33.08 mmol) in N, N-dimethylformamide (50 mL) was added tributyl (1-ethoxyvinyl) stannane (23.90 g, 66.17 mmol, 22.33 mL) at room temperature, followed by bis (triphenylphosphine) palladium (II) chloride (2.32 g, 3.31 mmol) under N ₂. The resulting reaction mixture was stirred at 100 ℃ for 3 hours. After the reaction was complete, the mixture was concentrated and the residue was purified by flash column chromatography (Petroleum ether : Ethyl acetate 3: 1) to give tert-butyl N- [8- (1-ethoxyvinyl) imidazo [1, 2-b] pyridazin-7-yl] carbamate (10.06 g, 99 %yield) as a yellow oil.

LC-MS: m/z 305 [M+H] ⁺.

Step 2

To a solution of tert-butyl N- [8- (1-ethoxyvinyl) imidazo [1, 2-b] pyridazin-7-yl] carbamate 19e (2 g, 6.57 mmol) in dioxane (20 mL) was added HCl/dioxane (20 mL) . The mixture was stirred at 80 ℃ for 2 hours, and then the solvent was removed in vacuo. Then HCl/water (20 mL) was added and the mixture was stirred at 80 ℃ for 2 hours. Then the mixture was basified by Na ₂CO ₃ (aq) to pH～8, and the solid was filtered, dried to give the compound 1- (7-aminoimidazo [1, 2-b] pyridazin-8-yl) ethanone (1.15 g, 99.33%yield) as a yellow solid.

LC-MS: m/z 177 [M+H] ⁺

Step 3

To a solution of 1- (7-aminoimidazo [1, 2-b] pyridazin-8-yl) ethanone 20d (4.8 g, 27.25 mmol) in methanol (60 mL) was added sodium borohydride (2.06 g, 54.49 mmol, 1.93 mL) at 0 ℃. The mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction mixture was diluted with water (50 mL) and extracted with EtOAc (3 × 250 mL) . The combined organic phase was dried over anhydrous Na ₂SO ₄, filtered and concentrated in vacuo. The crude was purified by flash chromatography (DCM/MeOH 15: 1) to afford 1- (7-aminoimidazo [1, 2-b] pyridazin-8-yl) ethanol (3.84 g, 79 %yield) as a gray solid.

LC-MS: m/z 179 [M+H] ⁺.

Step 4

To a solution of 1- (7-aminoimidazo [1, 2-b] pyridazin-8-yl) ethanol 21a (4 g, 22.45 mmol) in methanol (45.00 mL) was added thionyl chloride (2.4 mL) at room temperature. The mixture was stirred at 50 ℃ for overnight. After the reaction was complete, the reaction mixture was concentrated and purified by flash chromatography (DCM/MeOH 30: 1) to afford 8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-amine (3.6 g, 83%yield) as a yellow solid.

LC-MS: m/z 193 [M+H] ⁺.

Step 5

To a solution of 8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-amine 22a (10 g, 52.02 mmol) in THF (200 mL) was added phenyl N- [5-chloro-6- (triazol-2-yl) -3-pyridyl] carbamate (17.25 g, 54.63 mmol) at room temperature. The mixture was stirred at 80 ℃ for 3 hours and LCMS showed most of starting material was consumed. The solvents were removed and the crude was purified by flash chromatography (DCM/MeOH 30: 1) to give 1- [5-chloro-6- (triazol-2-yl) -3-pyridyl] -3- [8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl] urea (61) (15 g, 69.67%yield) as a white solid.

Compound (61) was chirally separated by Prep-Chiral-SFC with the following conditions (Daicel Chiralpak, IG 4.6mm*250mm*5um, 5 to 50%Hex in EtOH, 1.0 mL/min) . The fractions were evaporated to dryness to afford the desired compounds.

Peak 1: (S) -1- [5-chloro-6- (triazol-2-yl) -3-pyridyl] -3- [8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl] urea (62) or (R) -1- [5-chloro-6- (triazol-2-yl) -3-pyridyl] -3- [8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl] urea (63) .

Retention time: 6.146 mins; ee (%) : >99%,

(0.5, CH ₃OH) ;

Peak 2: (R) -1- [5-chloro-6- (triazol-2-yl) -3-pyridyl] -3- [8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl] urea (63) or (S) -1- [5-chloro-6- (triazol-2-yl) -3-pyridyl] -3- [8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl] urea (62) .

Retention time: 7.681 mins, ee: >99%;

(0.5, CH ₃OH) ;

LC-MS: m/z 414 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d6) : δ 10.60 (s, 1H) , 9.12 (s, 1H) , 8.81 (s, 1H) , 8.55 (d, J = 2.4 Hz, 1H) , 8.50 (d, J = 2.4 Hz, 1H) , 8.20 (d, J = 1.6 Hz, 1H) , 8.16 (s, 2H) , 7.67 (d, J = 1.2 Hz, 1H) , 5.33 (q, J = 6.8 Hz, 1H) , 3.34 (s, 3H) , 1.56 (d, J = 6.4 Hz, 3H) .

Examples 64-66: Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl) urea / (S) -1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl) urea / (R) -1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl) urea

Step 1:

To a solution of ethyl 8-bromo-2-chloroimidazo [1, 2-b] pyridazine-7-carboxylate 7c (31 g, 102.3 mmol) in THF (500 mL) was added the solution of lithium hydroxide monohydrate (8.6 g, 204.6 mmol) in water (200 mL) . The resulting mixture was stirred at room temperature for 10 minutes. The reaction mixture was quenched with HCl (1 N) to pH = 3～4, and it was extracted with EtOAc and the combined organic phase was dried, concentrated to give 8-bromo-2-chloroimidazo [1, 2-b] pyridazine-7-carboxylic acid (28 g, 99.6%) as a yellow solid.

LC-MS: m/z 276 [M+H] ⁺.

Step 2:

To a solution of 8-bromo-2-chloroimidazo [1, 2-b] pyridazine-7-carboxylic acid 17c (28 g, 101.8 mmol) in tert-Butanol (280 mL) and toluene (300 mL) was added diphenylphosphoryl azide (39.2 g, 142.5 mmol) , Triethylamine (51.5 g, 509 mmol) and Di-tert-butyl dicarbonate (66.58 g, 305.4 mmol) . The reaction mixture was stirred at 100 ℃ for 4 hours. LC-MS showed the starting material was consumed and the desired mass was detected. After concentrated and the resulting residue was purified by flash column chromatography (eluting with ethyl acetate in petroleum from 0%to 10%) to give the tert-butyl (8-bromo-2-chloroimidazo [1, 2-b] pyridazin-7-yl) carbamate (19.4 g, yield: 55.1%) as a yellow solid.

LC-MS: m/z 347 [M+H] ⁺.

Step 3:

To a solution of tert-butyl (8-bromo-2-chloroimidazo [1, 2-b] pyridazin-7-yl) carbamate 18c (7.75 g, 22.4 mmol) in N, N-Dimethylformamide (100 mL) was added tributyl (1-ethoxyvinyl) stannane (16.22 g, 44.8 mmol) at room temperature. The resulting mixture was degassed and exchanged with N ₂ twice and Bis (triphenylphosphine) palladium (II) chloride (3.14 g, 4.48 mmol) was added. The reaction mixture was stirred at 100 ℃ for 4 hours. LC-MS showed the starting material was consumed and the desired mass was detected. After concentrated and the resulting residue was purified by silica-gel column chromatography eluting with ethyl acetate in petroleum ether from 0%to 10%to give the tert-butyl (2-chloro-8- (1-ethoxyvinyl) imidazo [1, 2-b] pyridazin-7-yl) carbamate (5.9 g, yield: 77.9%) as a white solid.

LC-MS: m/z 339 [M+H] ⁺.

Step 4:

To a solution of tert-butyl (2-chloro-8- (1-ethoxyvinyl) imidazo [1, 2-b] pyridazin-7-yl) carbamate 19f (5.9 g, 17.46 mmol) in 1, 4-Dioxane (100 mL) was added HCl/1, 4-Dioxane (30 mL) . The reaction mixture was stirred at 80 ℃ for 2 hours. The starting material was consumed and the desired mass was detected from LC-MS. The reaction mixture was concentrated and the residue was basified with triethylamine to pH ～ 7-8. After concentrated and the residue was purified by silica-gel column chromatography eluting with ethyl acetate in petroleum ether from 0%to 30%to give the 1- (7-amino-2-chloroimidazo [1, 2-b] pyridazin-8-yl) ethanone (2.0 g, yield: 54.5%) as a yellow solid.

LC-MS: m/z 211 [M+H] ⁺.

Step 5:

To a solution of 1- (7-amino-2-chloroimidazo [1, 2-b] pyridazin-8-yl) ethanone 20e (2.0 g, 9.52 mmol) in methanol (50 mL) was added NaBH ₄ (1.08 g. 28.57 mmol) at 0 ℃. The resulting mixture was stirred at room temperature for 4 hours. After the reaction was complete, the mixture was concentrated and the residue was purified by flash chromatography eluting with Methanol/Dichloromethane=10/1 to afford 1- (7-amino-2-chloroimidazo [1, 2-b] pyridazin-8-yl) ethanol (1.94 g, yield: 96%) as a yellow solid.

LC-MS: m/z 213 [M+H] ⁺.

Step 6:

To a solution of 1- (7-amino-2-chloroimidazo [1, 2-b] pyridazin-8-yl) ethanol 21c (1.94 g, 9.151 mmol) in methanol (60 mL) was added SOCl ₂ (12 mL) at 0 ℃. The resulting mixture was stirred at 50 ℃ for overnight. The starting material was consumed and the desired mass was detected from LC-MS. The resulting mixture was concentrated and the residue was purified by silica-gel column chromatography eluting with ethyl acetate in petroleum ether from 0%to 30%to give the 2-chloro-8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-amine (1.7 g, yield: 82.1%) as a yellow solid.

LC-MS: m/z 227 [M+H] ⁺.

Step 7:

To a solution of 2-chloro-8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-amine 22c (300 mg, 1.327 mmol) in N, N-Dimethylformamide (14 mL) was added phenyl (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) carbamate 23a (752.6 mg, 2.389 mmol) . The resulting mixture was heated at 90 ℃ for 8 hours under microwave radiation. The starting material was consumed and the desired mass was detected from LC-MS. The mixture was concentrated and the residue was purified by flash chromatography (eluting with Methanol/Dichloromethane: 20/1) to get the 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl) urea (64) (401 mg, yield: 67.6%) as a white solid.

Compound (64) was chirally separated by Prep-Chiral-SFC with the following conditions (Daicel Chiralpak, IG 4.6mm*250mm*5um, 5 to 50%Hex in EtOH, 1.0 mL/min) . The fractions were evaporated to dryness to afford the desired compounds.

Peak 1: (S) -1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl) urea (65) or (R) -1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl) urea (66) .

Retention time: 10.280 mins, ee (%) : >99%.

Peak 2: (R) -1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl) urea (66) or (S) -1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl) urea (65) .

Retention time: 11.856 mins, ee (%) : >99%.

LC-MS: m/z 448 [M+H] ⁺. LC-MS: m/z 448 [M+H] ⁺; ¹H NMR (400 MHz; DMSO-d6) : δ 10.62 (s, 1 H) , 9.21 (s, 1 H) , 8.85 (s, 1 H) , 8.55 (d, J = 2.4 Hz, 1 H) , 8.49 (d, J = 2.0 Hz, 1 H) , 8.39 (s, 1 H) , 8.15 (s, 2 H) , 5.19-5.24 (m, 1 H) , 3.34 (s, 3 H) , 1.55 (d, J = 6.8 Hz, 3 H) .

Example 67. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (2-chloro-8- (1- (dimethylamino) ethyl) imidazo [1, 2-b] pyridazin-7-yl) urea

Step 1

To a solution of ethyl 8-acetyl-2-chloro-imidazo [1, 2-b] pyridazine-7-carboxylate 8r (50 mg, 0.187 mmol) in methanol (3 mL) was added N-methylmethanamine (25.3 mg, 0.56 mmol, ) and sodium cyanoborohydride (23.5 mg, 0.374 mmol) . The reaction mixture was stirred at room temperature for overnight. LC-MS showed the starting material was consumed and the desired mass was detected. The reaction mixture was concentrated and the result residue was purified by silica-gel column chromatography eluting with ethyl acetate in petroleum ether from 0%to 100%to give the 2-chloro-8- [1- (dimethylamino) ethyl] imidazo [1, 2-b] pyridazine-7-carboxylic acid (36 mg, 71.72%yield) .

LC-MS: m/z 269 [M+H] ⁺.

Step 2

To a solution of 2-chloro-8- [1- (dimethylamino) ethyl] imidazo [1, 2-b] pyridazine-7-carboxylic acid 9r (36 mg, 0.134 mmol) in 1, 4-dioxane (3 mL) was added Diphenylphosphoryl azide (44.3 mg, 0.161 mmol) and Triethylamine (67.8 mg, 0.67 mmol) . The reaction mixture was stirred at room temperature for 30 minutes and then 5-chloro-6- (triazol-2-yl) pyridin-3-amine (39.3 mg, 0.2 mmol) was added. The reaction mixture was heated to 100 ℃ for 2 hours. LC-MS showed the starting material was consumed and the desired mass was detected. It was concentrated and purified by silica-gel column chromatography eluting with ethyl acetate in petroleum ether from 0%to 100%to give the 1- [2-chloro-8- [1- (dimethylamino) ethyl] imidazo [1, 2-b] pyridazin-7-yl] -3- [5-chloro-6- (triazol-2-yl) -3-pyridyl] urea (5.7 mg, 9.22%yield) as a white solid

LC-MS: m/z 461 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d ₆) : δ 10.59 (s, 2H) , 9.24 (s, 1H) , 8.60 (d, J = 2.0 Hz, 1H) , 8.50 (d, J = 2.4 Hz, 1H) , 8.35 (s, 1H) , 8.15 (s, 2H) , 4.10-4.05 (m, 1H) , 2.30 (s, 6H) , 1.39 (d, J = 6.4 Hz, 3H) .

Example 68. Synthesis of N- [7- [ [5-chloro-6- (triazol-2-yl) -3-pyridyl] carbamoylamino] imidazo [1, 2-b] pyridazin-8-yl] acetamide

Example 68 was prepared in analogy to the procedure described for the preparation of Example 3 by using 2- (trifluoromethyl) pyridin-4-amine as the starting material.

LC-MS: m/z 385 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d6) : δ 9.99 (brs, 1H) , 8.95 (brs, 1H) , 8.86 (s, 1H) , 8.56 (d, J = 5.2 Hz, 1H) , 8.38 (s, 1H) , 8.07 (d, J = 1.6 Hz, 1H) , 7.62 (dd, J = 2.0, 5.6 Hz, 1H) , 2.93 (q, J = 7.6 Hz, 2H) , 1.24 (t, J = 7.6 Hz, 3H) .

Examples 69-71. Synthesis of 1- (5-chloro-6-methoxy-3-pyridyl) -3- [8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl] urea / (S) -1- (5-chloro-6-methoxy-3-pyridyl) -3- [8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl] urea / (R) -1- (5-chloro-6-methoxy-3-pyridyl) -3- [8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl] urea

Examples 69-71 were prepared in analogy to the procedure described for the preparation of Examples 61-63 by using 5-chloro-6-methoxy-pyridin-3-amine as one of starting materials.

The title compound 1- (5-chloro-6-methoxy-3-pyridyl) -3- [8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl] urea (69) was obtained (30 mg, 21.04%yield) as a white solid.

LC-MS: m/z 377 [M+H] ⁺; ¹H NMR (400 MHz; DMSO-d6) : δ 9.95 (s, 1H) , 9.12 (s, 1H) , 8.59 (s, 1H) , 8.17 (d, J = 1.6 Hz, 2H) , 8.13 (d, J = 2.4 Hz, 1H) , 7.63 (d, J = 0.8 Hz, 1H) , 5.33-5.30 (m, 1H) , 3.92 (s, 3H) , 3.31 (s, 3H) , 1.53 (d, J = 6.4 Hz, 3H) .

Compound (69) was chirally separated by Prep-Chiral-SFC with the following conditions (Column: Eclipse XDB-C18; column size: 4.6*150mm*5um, mobile phase: B (CAN) , A (0.1%TFA) ) . The fractions were evaporated to dryness to afford the desired compounds.

Peak 1: (S) -1- (5-chloro-6-methoxy-3-pyridyl) -3- [8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl] urea (70) or (R) -1- (5-chloro-6-methoxy-3-pyridyl) -3- [8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl] urea (71) .

Retention time: 9.290 mins, ee > 99%.

Peak 2: (R) -1- (5-chloro-6-methoxy-3-pyridyl) -3- [8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl] urea (71) or (S) -1- (5-chloro-6-methoxy-3-pyridyl) -3- [8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl] urea (70) .

Retention time: 9.305 mins, ee > 99%.

LC-MS: m/z 377 [M+H] ⁺; ¹H NMR (400 MHz; DMSO-d6) : δ 9.95 (s, 1H) , 9.11 (s, 1H) , 8.59 (s, 1H) , 8.16 (d, J = 1.6 Hz, 2H) , 8.13 (d, J = 2.4 Hz, 1H) , 7.63 (d, J = 0.8 Hz, 1H) , 5.33-5.30 (m, 1H) , 3.92 (s, 3H) , 3.31 (s, 3H) , 1.53 (d, J = 6.4 Hz, 3H) .

Example 72. Synthesis of 1- (8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl) -3- (2- (trifluoromethyl) pyridin-4-yl) urea

Example 72 was prepared in analogy to the procedure described for the preparation of Example 61 by using 6- (trifluoromethyl) pyridin-3-amine as the starting material.

LC-MS: m/z 381 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d6) : δ 10.63 (s, 1H) , 9.08 (s, 1H) , 8.77 (s, 1H) , 8.58 (d, J = 5.6 Hz, 1H) , 8.20 (d, J = 1.2 Hz, 1H) , 8.07 (d, J = 1.6 Hz, 1H) , 7.67 (d, J = 1.2 Hz, 1H) , 7.63 (dd, J = 2.0, 5.6Hz, 1H) , 5.31 (q, J = 6.8 Hz, 1H) , 3.32 (s, 3H) , 1.54 (d, J = 6.8 Hz, 3H) .

Example 73. Synthesis of 1- (2- (difluoromethyl) pyridin-4-yl) -3- (8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl) urea

Example 73 was prepared in analogy to the procedure described for the preparation of example 61 by using 6- (difluoromethyl) pyridin-3-amine as the starting material.

LC-MS: m/z 363 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d6) : δ 10.49 (s, 1H) , 9.08 (s, 1H) , 8.73 (s, 1H) , 8.50 (d, J = 6.0 Hz, 1H) , 8.20 (d, J = 1.2 Hz, 1H) , 7.89 (d, J = 1.6 Hz, 1H) , 7.66 (d, J = 1.2 Hz, 1H) , 7.54 (d, J = 5.2 Hz, 1H) , 5.31 (q, J = 6.8 Hz, 1H) , 3.32 (s, 3H) , 1.55 (d, J = 6.8 Hz, 3H) .

Examples 74-76. Synthesis of 1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (8- (1-ethoxyethyl) imidazo [1, 2-b] pyridazin-7-yl) urea/ (S) -1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (8- (1-ethoxyethyl) imidazo [1, 2-b] pyridazin-7-yl) urea / (R) -1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (8- (1-ethoxyethyl) imidazo [1, 2-b] pyridazin-7-yl) urea

Examples 74-76 were prepared in analogy to the procedure described for the preparation of Examples 61-63 by using different starting material.

LC-MS: m/z 428 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d6) : δ 10.58 (s, 1H) , 9.11 (s, 1H) , 8.76 (s, 1H) , 8.57 (d, J = 2.4 Hz, 1H) , 8.50 (d, J = 2.0 Hz, 1H) , 8.20 (d, J = 0.8 Hz, 1H) , 8.16 (s, 2H) , 7.66 (d, J = 1.2 Hz, 1H) , 5.44 (q, J = 6.4 Hz, 1H) , 3.53 (m, 2H) , 1.56 (d, J = 6.4 Hz, 3H) , 1.21 (q, J= 8.8 Hz, 3H) .

Compound (74) was chirally separated by Prep-Chiral-SFC with the following conditions (Column: Daicel Chiralpak, IG 4.6mm*250mm*5um, 5 to 50%Hex in EtOH, 1.0 mL/min) . The fractions were evaporated to dryness to afford the desired compounds.

Peak 1: (S) -1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (8- (1-ethoxyethyl) imidazo [1, 2-b] pyridazin-7-yl) urea (75) or (R) -1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (8- (1-ethoxyethyl) imidazo [1, 2-b] pyridazin-7-yl) urea (76) .

Retention time: 6.18 mins; ee > 99%.

Peak 2: (R) -1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (8- (1-ethoxyethyl) imidazo [1, 2-b] pyridazin-7-yl) urea (76) or (S) -1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (8- (1-ethoxyethyl) imidazo [1, 2-b] pyridazin-7-yl) urea (75) .

Retention time: 7.70 mins; ee > 99%.

Example 77. Synthesis of (S) -1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl) urea

A mixture of 1- [5-chloro-6- (triazol-2-yl) -3-pyridyl] -3- [8- [ (1S) -1-methoxyethyl] imidazo [1, 2-b] pyridazin-7-yl] urea (100 mg, 241.65 μmol, obtained from peak 1 of Examples 61-63) , silve r carbonate (2.0 mg, 12.08 μmol) , and selectfluor (34.2 mg, 96.66 μmol) in acetonitrile (10 m L) was stirred in a microwave tube. The reaction tube was sealed, heated at 85 ℃ under micr owave radiation, and stirred for 1 hour. The reaction mixture was filtered through a pad of Ce lite with CH ₂Cl ₂, and the combined organic layers were concentrated in vacuo. The resulting mixture was purified by silica gel chromatography (eluting with MeOH in CH ₂Cl ₂ from 0 to 2 0%) to afford crude and then pre-HPLC (Mobile Phase: ACN-H ₂O (0.05%HCO ₂H) ; Gradien t: 40) to give 1- [5-chloro-6- (triazol-2-yl) -3-pyridyl] -3- [3-fluoro-8- [ (1S) -1-methoxyethyl] imid azo [1, 2-b] pyridazin-7-yl] urea (12.5 mg, 28.95 μmol, 11.98%yield) . LC-MS: m/z 431.8 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d ₆) δ 10.64 (s, 1H) , 9.18 (s, 1H) , 8.84 (s, 1H) , 8.56 (d, J = 2.3 Hz, 1H) , 8.51 (d, J = 2.3 Hz, 1H) , 8.17 (s, 2H) , 7.50 (d, J = 7.1 Hz, 1H) , 5.28 (q, J = 6.7 Hz, 1H) , 3.33 (s, 3H) , 1.56 (d, J = 6.7 Hz, 3H) .

Example 78. Synthesis of (R) -1- (5-chloro-6- (2H-1, 2, 3-triazol-2-yl) pyridin-3-yl) -3- (3-fluoro-8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl) urea

Example 78 was prepared in analogy to the procedure described for the preparation of Example 77 by using 1- [5-chloro-6- (triazol-2-yl) -3-pyridyl] -3- [8- [ (1R) -1-methoxyethyl] imidazo [1, 2-b] pyridazin-7-yl] urea (obtained from peak 2 of Examples 61-63) as starting material.

LC-MS: m/z 431.8 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d ₆) δ 10.63 (s, 1H) , 9.19 (s, 1H) , 8. 82 (s, 1H) , 8.56 (d, J = 2.3 Hz, 1H) , 8.51 (d, J = 2.3 Hz, 1H) , 8.17 (s, 2H) , 7.52 (d, J = 7.0 Hz, 1H) , 5.28 (q, J = 6.7 Hz, 1H) , 3.34 (s, 3H) , 1.56 (d, J = 6.7 Hz, 3H) .

Example 79. Synthesis of 1- (2-chloro-8- (1-methoxyethyl) imidazo [1, 2-b] pyridazin-7-yl) -3- (2- (trifluoromethyl) pyridin-4-yl) urea

Example 78 was prepared in analogy to the procedure described for the preparation of Example 61 by using 2- (trifluoromethyl) pyridin-4-amine as starting material.

LC-MS: m/z 415 [M+H] ⁺; ¹H NMR (400 MHz; DMSO-d6) : δ 10.68 (s, 1H) , 9.17 (s, 1H) , 8.83 (s, 1H) , 8.58 (d, J = 5.6 Hz, 1H) , 8.40 (s, 1H) , 8.06 (s, 1H) , 7.63-7.61 (m, 1H) , 5.22-5.17 (m, 1H) , 3.32 (s, 3H) , 1.53 (d, J = 6.8 Hz, 3H) .

Example 80. Synthesis of 1- [2-chloro-8- [methyl (methylsulfonyl) amino] imidazo [1, 2-b] pyridazin-7-yl] -3- [5-chloro-6- (triazol-2-yl) -3-pyridyl] urea

Step 1

A mixture of ethyl 8-bromo-2-chloro-imidazo [1, 2-b] pyridazine-7-carboxylate 7c (300 mg, 985.13 μmol) , methanesulfonamide (140.6 mg, 1.48 mmol) and dicesium carbonate (641.9 mg, 1.97 mmol) in 1, 4-dioxane (10 mL) was degassed and refilled with N ₂ (three times) . The mixture was heated at 90 ℃ and stirred for 12 hours. TLC showed the reaction was complete. The mixture was diluted with EA (50 mL) , washed with sat. NH ₄Cl (15 mL x 2) and concentrated. The residue was purified by flash silica-gel chromatography (eluting with hexane /ethyl acetate = 1/1) to give ethyl 2-chloro-8- (methanesulfonamido) imidazo [1, 2-b] pyridazine-7-carboxylate (230 mg, yield: 73%) as white solid.

LC-MS: m/z 319.0 [M+H] ⁺.

Step 2

To a solution of ethyl 2-chloro-8- (methanesulfonamido) imidazo [1, 2-b] pyridazine-7-carboxylate 8s (120 mg, 0.376 mmol) in N, N-dimethylformamide (5 mL) was added potassium carbonate (104 mg, 0.753 mmol) and iodomethane (267 mg, 1.88 mmol) at room temperature. The resulting mixture was stirred at 80 ℃ for 2 hours. LC-MS showed the reaction was completed. The mixture was diluted with ethyl acetate (20 mL) , washed with 1 N LiCl and concentrated. The residue was purified by flash silica-gel column chromatography (eluting with hexanes /ethyl acetate = 2 /1) to give ethyl 2-chloro-8- [methyl (methylsulfonyl) amino] imidazo [1, 2-b] pyridazine-7-carboxylate (120 mg, yield: 96%) as a white solid.

LC-MS: m/z 333.0 [M+H] ⁺.

Step 3

To a solution of ethyl 2-chloro-8- [methyl (methylsulfonyl) amino] imidazo [1, 2-b] pyridazine-7-carboxylate 8t (120 mg, 0.36 mmol) in THF (3 mL) and Water (1 mL) was added lithium hydroxide (34.5 mg, 1.44 mmol) and the reaction was stirred at room temperature for 12 hours. TLC showed the reaction was completed. The solvent was removed under reduced pressure and the residue was dissolved in water (10 mL) . The solution was acidified with 1 N HCl to pH=6.0. The formed precipitate was filtered and dried to give 2-chloro-8- [methyl (methylsulfonyl) amino] imidazo [1, 2-b] pyridazine-7-carboxylic acid (85 mg, 77%yield) as a yellow solid which was used for the next step directly.

LC-MS: m/z 305.1 [M+H] ⁺.

Step 4

To a solution of 2-chloro-8- [methyl (methylsulfonyl) amino] imidazo [1, 2-b] pyridazine-7-carboxylic acid 9s (85 mg, 0.279 mmol) in 1, 4-dioxane (5 mL) was added [azido (phenoxy) phosphoryl] oxybenzene (99.8 mg, 0.363 mmol) and N, N-diethylethanamine (141.1 mg, 1.39 mmol) at room temperature. The resulting mixture was stirred at this temperature for 30 min. Then 5-chloro-6- (triazol-2-yl) pyridin-3-amine (81.9 mg, 0.418 mmol) was added and the mixture was stirred at 100 ℃ for 2 hours. LC-MS showed the reaction was complete and quenched with EA (20 mL) , washed with sat. NH ₄Cl (10 mL x 2) . The organic layer was concentrated and the residue was purified by Prep-TLC (DCM: MeOH=20: 1) to give crude product, which was purified by Prep-HPLC to give 1- [2-chloro-8- [methyl (methylsulfonyl) amino] imidazo [1, 2-b] pyridazin-7-yl] -3- [5-chloro-6- (triazol-2-yl) -3-pyridyl] urea (8.9 mg, 6%yield) as a white solid.

LC-MS: m/z 497.0 [M+H] ⁺; ¹H NMR (400 MHz, DMSO-d ₆) : δ 10.50 (s, 1H) , 9.43 (s, 1H) , 9.01 (s, 1H) , 8.55 (d, J = 2.4 Hz, 1H) , 8.50 (d, J = 2.0 Hz, 1H) , 8.48 (s, 1H) , 8.17 (s, 2H) , 3.40 (s, 3H) , 3.33 (s, 3H) .

Claims

A compound represented by structural formula (I) :

or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein

ring A is

Z is O, NR ⁶, or S;

A ₁ and A ₂ are each independently CR ₁ or N;

each instance of R ₁ is hydrogen; halogen; -OH; CN; -COOC _1-6 alkyl;C _1-6 alkoxy optionally substituted by halogen; C _1-6 alkoxy carbonyl; phenyl; amino;N, N-di-C _1-6 alkyl amino; C _1-6 alkyl optionally substituted by halogen, phenyl, or a 5-6 membered heterocyclic ring having 1 to 3 heteroatoms selected from N and O which ring is optionally substituted by C _1-6 alkyl; Rh; ORh; a 5-6 membered heteroaryl ring having 1 to 3 heteroatoms selected from N and O said ring being optionally substituted by amino, C _1-6 alkyl optionally substituted by amino or hydroxy or by N-mono or N, N-di-C _1-6 alkylamino carbonyl; wherein

Rh is a 5-6 membered heterocyclyl ring having 1 to 4 heteroatoms selected from N, O and S, said ring being optionally substituted by C _1-6 alkyl, -OH, or oxo,

each instance of R ₂ is hydrogen; halogen; CN; -COOC _1-6 alkyl; C _1-6 alkoxy optionally substituted by halogen; C _1-6 alkoxy carbonyl; amino; N, N-di-C _1-6 alkyl amino; C _1-6 alkyl optionally substituted by halogen, -OH, phenyl, or a 5-6 membered heterocyclic ring having 1 to 2 heteroatoms selected from N and O which ring is optionally substituted by C _1-6 alkyl; Rh; ORh; a 5-6 membered heteroaryl ring having 1 to 3 heteroatoms selected from N and O said ring being optionally substituted by amino, C _1-6 alkyl optionally substituted by amino or hydroxy, or by N-mono or N, N-di-C _1-6 alkylamino carbonyl;

each instance of R ₃ is H; deuterium; halogen; CN; -OH; -COOH; -NR _aR _b;-SR _c; -SO ₂R _c; -SO ₂NR _c; -C (=O) NR _aR _b; C _1-6 alkoxy optionally substituted by halogen, -OH, C _1-6 alkyl, -NH ₂, -NHC (=O) C _1-6 alkyl, N-di-C _1-6 alkyl amino, or N-mono-C _1-6 alkyl amino; C _1-6 alkyl optionally substituted by halogen, C _2-6 alkenyl, -OH, -NH ₂, -NHC (=O) C _1-6 alkyl, N-di-C _1-6 alkyl amino, N-mono-C _1-6 alkyl amino, -O-Rg, Rg, phenyl, or C _1-6 alkoxy wherein said alkoxy optionally substituted by halogen, -OH, C _1-6 alkoxy, N, N-di-C _1-6 alkyl amino, Rg or phenyl; C _3-6 cycloalkyl optionally substituted by halogen, -OH, C _1-6 alkyl, N, N-di-C _1-6 alkyl amino or C _1-6alkoxy-C _1-6 alkyl; phenyl optionally substituted by halo or C _1-6 alkoxy; a 5-6 membered heteroaryl ring having 1 to 3 heteroatoms selected from N and O said ring being optionally substituted by C _1-6 alkyl which may be optionally substituted by amino or -OH; Rg; or N, N-di-C _1-6 alkyl amino carbonyl; wherein

Rg is a 3-6 membered heterocyclic ring having 1-3 heteroatoms selected from N and O said ring being optionally substituted by –OH, -NH ₂, C _1-6 alkyl, C _1-6 alkoxy-C _1-6 alkyl, or C _1-6 alkoxy-carbonyl;

R _a is independently H or C _1-6 alkyl optionally substituted by C _1-6 alkoxy, and R _b is independently H, C _1-6 alkyl, -COC _1-6 alkyl, -SO ₂C _1-6 alkyl, C _3-6 cycloalkyl or 3-6 membered heterocyclic ring having 1-3 heteroatoms selected from N and O said ring being optionally substituted by C _1-6 alkyl, C _1-6 alkoxy-C _1-6 alkyl, or C _1-6 alkoxy-carbonyl, or

R _a and R _b, together with the nitrogen atom attached to, form a 4-6 membered heterocyclic ring having 1-3 heteroatoms selected from N, O, and S said ring being optionally substituted by -OH, -NH ₂, N-di-C _1-6 alkyl amino, N-mono-C _1-6 alkyl amino, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 haloalkoxy, O-cyclopropyl, C _1-6 alkoxy-C _1-6 alkyl, or C _1-6 alkyl-carbonyl;

each instance of R _c is C _1-6 alkyl or C _3-6 cycloalkyl;

wherein the alkyl represented by R _a, R _b, or R _c or in the group represented by R _a, R _b, or R _c is optionally substituted by halogen, -OH, C _1-2 alkoxy, or C _3-4 cycloalkyl;

each instance of R ₄ is H, deuterium, halogen, CN, C _1-6 alkyl, or C _1-6 haloalkyl;

each instance of R ₄’ is H, deuterium, F, or Cl;

each instance of R ₅ is H, deuterium, C _1-6 alkyl, or C _1-6 haloalkyl; and

R ₆ is H; OH; C _1-6 alkyl optionally substituted by halogen, OH, or C _1-6 alkoxy; or C _3-6 cycloalkyl optionally substituted by halogen, OH, or C _1-6 alkoxy.
The compound of claim 1, or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein Z is O.
The compound of claim 1 or 2, or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein ring A is
The compound of any one of claims 1-3, or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein each instance of R ₃ is H; halogen; CN; -OH; -COOH; -NR _aR _b; -SR _c; -SO ₂R _c; -SO ₂NR _c; -C (=O) NR _aR _b; C _1-4 alkoxy optionally substituted by halogen, -OH, C _1-6 alkyl, -NH ₂, -NHC (=O) C _1-6 alkyl, N-di-C _1-6 alkyl amino, or N-mono-C _1-6 alkyl amino; C _1-4 alkyl optionally substituted by halogen, C _1-2 alkoxy, N-di-C _1-6 alkyl amino, or N-mono-C _1-6 alkyl amino; C _3-6 cycloalkyl; or Rg.
The compound of any one of claims 1-4, wherein the compound is represented by structural formula (II-A) or (II-B) :

or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein each instance of R ₄’ is H or F.
The compound of any one of claims 1-5, or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein

each instance of R ₃ is H; halogen; CN; -OH; -COOH; -NR _aR _b; -SR _c; -SO ₂R _c;-SO ₂NR _c; -C (=O) NR _aR _b; C _1-4 alkoxy optionally substituted by halogen, -OH, C _1-4 alkyl, -NH ₂, N-di-C _1-4 alkyl amino, or N-mono-C _1-4 alkyl amino; C _1-4 alkyl optionally substituted by halogen, C _1-2 alkoxy, N-di-C _1-4 alkyl amino, or N-mono-C _1-4 alkyl amino; C _3-6 cycloalkyl; morpholinyl; oxetanyl; or azetidinyl optionally substituted by methyl;

R _a is independently H or C _1-4 alkyl optionally substituted by C _1-4 alkoxy;

R _b is independently H, C _1-4 alkyl, -COC _1-4 alkyl, -SO ₂C _1-4 alkyl, C _3-6 cycloalkyl or 3-6 membered heterocyclic ring having 1-3 heteroatoms selected from N and O said ring being optionally substituted by C _1-4 alkyl, C _1-4 alkoxy-C _1-4 alkyl, or C _1-4 alkoxy-carbonyl, or

R _a and R _b, together with the nitrogen atom attached to, form a 4-6 membered heterocyclic ring having 1-3 heteroatoms selected from N, O, and S said ring being optionally substituted by -OH, C _1-4 alkyl, C _1-4 haloalkyl, C _1-4 alkoxy, C _1-4 haloalkoxy, or C _1-4 alkoxy-C _1-4 alkyl.
The compound of any one of claims 1-6, or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein each instance of R ₄ is H, halogen, C _1-2 alkyl optionally substituted with halo, and each instance of R ₄’ is H.
The compound of any one of claims 1-7, or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein R ₂ is H, Cl, CN, or C _1-6 alkyl optionally substituted with halogen or -OH.
The compound of any one of claims 1-8, or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein each instance of R ₁ is H; halogen; -OH; CN; C _1-6 alkoxy optionally substituted by halogen; C _1-6 alkoxy carbonyl; phenyl; N, N-di-C _1-6 alkyl amino; C _1-6 alkyl optionally substituted by halogen or phenyl; a 5-6 membered heteroaryl ring containing 1 to 3 N atoms said ring being optionally substituted by C _1-6 alkyl optionally substituted by amino or hydroxy or by mono-or di-N-C _1-6 alkylamino carbonyl; ORh; or Rh; wherein Rh is a 5-6 membered heterocyclyl containing 1 to 4 heteroatoms selected from N, O and S said ring being optionally substituted by C _1-6 alkyl, -OH, or oxo.
The compound of any one of claims 1-9, or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein R ₁ is H; halogen; C _1-4 alkyl optionally substituted by halogen; C _1-4 alkoxy; or 5 -6 membered heteroaryl containing 1 to 3 N atoms.
The compound of any one of claims 1-10, or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein each instance of R ₃ is bromo, methyl, ethyl, propyl, isopropyl, COOH, -CH (CH ₃) OCH ₃, -CH (CH ₃) OCH ₂CH ₃,

-CH (CH ₃) N (CH ₃) ₂, -CH ₂OCH ₃, cyclopropyl, morpholinyl, -S (CH ₃) , -N (CH ₃) ₂, -N (CH ₃) (CH ₂CH ₃) , -N (CH ₂CH ₃) ₂, -N (CH ₃) (isopropyl) , -N (CH ₃) (cyclopropyl) , -N (CH ₂CH ₃) (cyclopropyl) , -N (CH ₂CH ₂OCH ₃) (cyclopropyl) , -N (CH ₃) (SO ₂CH ₃) , or -N (CH ₃) (CH ₂CH ₂OCH ₃) .
The compound of any one of claims 1-10, or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein each instance of R ₃ is -CH (CH ₃) OCH ₃,

ethyl, or cyclopropyl.
The compound of any one of claims 1-12, or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein R ₄ is H, F, Cl, Br, CH ₃, or CF ₃.
The compound of any one of claims 1-13, or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein R ₂ is Cl or CF ₃.
The compound of any one of claims 1-14, or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein R ₁ is H, 1, 2, 3, -triazole, -OCH ₃, or CF ₃.
The compound of any one of claims 1-15, or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein R ₄ is H or Cl.
The compound of any one of claims 1-16, or a pharmaceutically acceptable salt, or a stereoisomer thereof, wherein
is
A pharmaceutical composition comprising an effective amount of the compound of any one of claims 1-17 or a pharmaceutically acceptable salt, or a stereoisomer thereof, and a pharmaceutically acceptable carrier.
A combination comprising a therapeutically effective amount of a compound of any one of claims 1-17, or a pharmaceutically acceptable salt, or a stereoisomer thereof, and one or more therapeutically active co-agents.
A method of inhibiting MALT1 activity in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound of any one of claim 1-17, or a pharmaceutically acceptable salt, or a stereoisomer thereof; or the pharmaceutical composition of claim 18; or the combination of claim 19.
The method of claim 20, wherein the subject has a disease or condition, such as an autoimmune disorder, an inflammatory disease, or a cancer, and wherein the disease or condition is treated upon inhibition of MALT1 activity.
The method of claim 21, wherein the disease or condition is rheumatoid arthritis (RA) , multiple sclerosis (MS) , systemic lupus erythematosus (SLE) , a vasculitic condition, an allergic disease, an airway disease (such as asthma and chronic obstructive pulmonary disease (COPD) ) , a condition caused by delayed or immediate type hypersensitivity, anaphylaxis, acute or chronic transplant rejection, a graft versus host disease, a cancer of hematopoietic origin or solid tumor, including chronic myelogenous leukemia (CML) , myeloid leukemia, non-Hodgkin lymphoma (NHL) , or a B cell lymphoma.
The method of claim 21 or 22, wherein the disease or condition is treated by selectively or preferentially inhibiting Th17 compared to Treg.
A compound according to any one of claims 1-17, or a pharmaceutically acceptable salt, or a stereoisomer thereof, for use as a medicament, such as a medicament acting as a MALT1 inhibitor.