Method for preparing heterocyclic compound

Abstract

Provided herein is a novel method of preparing heterocyclic compounds of formula (I) for activating T cells, NK cells and / or exhibiting anti-tumor activity. # imgabs0 #

Description

Technical Field

[0001] Provided herein is a novel method for preparing heterocyclic compounds of formula (I) for activating T cells, NK cells and / or exhibiting anti-tumor activity. Background Art

[0002] Casitas B lineage lymphoma proto-oncogene b (Cbl-b) is a member of the Cbl family ubiquitin ligase. Cbl family proteins have similar structural features, namely a highly conserved tyrosine kinase binding (TKB) domain that recognizes substrates and a C3HC4 RING (new gene of great interest) finger domain responsible for E3 ubiquitin ligase activity (Tsygankov, A., Cbl proteins. 2008, New York: Nova Science Publishers, vi, 210p). The first member of the Cbl family in mammalian cells, c-Cbl (Cbl or RNF55), was identified as a proto-oncoprotein in 1989 (Langdon, WY et al., Proc Natl Acad Sci USA, 1989. 86 (4): p. 1168-72), and Cbl-b (RNF56) and Cbl-c (Cbl-3 or RNF57) were subsequently cloned and characterized as having high sequence homology with c-Cbl at the N-terminus (Keane, MM et al., Oncogene, 1995. 10 (12): p. 2367-77; Kim, M. et al., Gene, 1999. 239 (1): p. 145-54). In the Cbl family, both c-Cbl and Cbl-b are well studied and are ubiquitously expressed in mammalian tissues. On the other hand, Cbl-c is less well known, has weaker E3 activity, and shows restricted expression in epithelial cells (Swaminathan, G. and AY Tsygankov, Journal of Cellular Physiology, 2006. 209(1): p. 21-43).

[0003] Cbl family members show similar autoinhibition and phosphorylation-induced activation mechanisms. Structural and biochemical studies reveal that inactive c-Cbl and Cbl-b adopt an autoinhibitory conformation in which the RING domain is positioned with low binding affinity to the E2 ubiquitin conjugating enzyme (UbcH5B). Tyrosine phosphorylation (Y371 of c-Cbl and Y363 of Cbl-b) induces large conformational changes to remove the masking of the TKB domain by the RING domain, thereby forming a surface to enhance the binding affinity of c-Cbl and Cbl-b to E2 (Kobashigawa, Y. et al., Proc Natl Acad Sci USA, 2011. 108(51): p. 20579-84; Dou, H. et al., Nat Struct Mol Biol, 2012. 19(2): p. 184-92). After activation, the structural rearrangement of c-Cbl and Cbl-b positions E2 toward the substrate and promotes the transfer of poly- or monoubiquitin to lysine residues. Ubiquitinated membrane proteins can further bind to the ESCRT complex, be internalized into endosomes, and attenuate receptor-mediated cell signaling (Ma, K. et al., 2013. pp. 219-244).

[0004] Among the three Cbl family members, Cbl-b has been well studied and has become an important intracellular checkpoint regulator in anti-tumor immunity. Cbl-b knockout mice show strong rejection of EL4, EG7, TC-1, B16F10 syngeneic transplanted tumors and spontaneous tumors induced by ATM knockout or UVB (Loeser, S. et al., J Exp Med, 2007. 204 (4): 879-91; Chiang, JY et al., J Clin Invest, 2007. 117 (4): 1029-36; Paolino, M. et al., Nature, 2014. 507 (7493): 508-12). It was found that CD8+ T cells and NK cells play a key role in eliminating tumors in Cbl-b knockout mice, as depletion of either cell population abolished Cbl-b knockout-induced tumor rejection (Loeser, S. et al., J Exp Med, 2007. 204(4): pp. 879-91; Paolino, M. et al., Nature, 2014. 507(7493): pp. 508-12). Cbl-b deficiency reduces the threshold of T cell activation and modulates the co-stimulatory pathway to enhance TCR signaling (Fang, D. and YC Liu, Nat Immunol, 2001. 2 (9): 870-5; Bachmaier, K. et al., Nature, 2000. 403 (6766): 211-6; Braun, M. et al., 2020. 53 (4): 805-823e15; Zhang, J. et al., 2002. 169 (5): 2236-40). Cbl-b depletion or E3 inactivation mutations have been shown to enhance NK cell function by blocking ligand-induced TAM receptor internalization (Paolino, M. et al., Nature, 2014. 507 (7493): 508-12). Bone marrow-derived dendritic cells (BMDCs) and macrophages without Cbl-b expression in mice also showed stronger activation after stimulation, which may also contribute to the tumor regression induced by Cbl-b deficiency (Abe, T. et al., Diabetes, 2013. 62(6): p. 1957-69; Wallner, S. et al., PLoS One, 2013. 8(6): p. e65178). In general, targeting Cbl-b is a potential new immunotherapy for cancer.

[0005] Although some literature has shown that knockout of both c-Cbl and Cbl-b can induce stronger T cell activation compared with single knockout of c-Cbl or Cbl-b, c-Cbl may not be the optimal immuno-oncology target because it functions as a tumor suppressor gene by downregulating activated receptor tyrosine kinases (RTKs). Clinically, heterozygous germline mutations (LOF) in CBL are associated with Noonan syndrome, RASopathy, and increase the risk of acute myeloid leukemia, myeloproliferative neoplasms, and juvenile myelomonocytic leukemia (Grand, FH et al., Blood, 2009. 113(24): 6182-92; Becker, H. et al., Blood, 2014. 123(12): 1883-6; Loh, ML et al., Blood, 2009. 114(9): 1859-63; Martinelli, S. et al., Am J Hum Genet, 2010. 87(2): 250-7). Compared with Cbl-b knockout, c-Cbl knockout altered the tumor microenvironment and promoted tumor growth in B16F10 and MC38 syngeneic models (Meyer, RD, D. Husain and N. Rahimi, Oncogene, 2011. 30 (19): pp. 2198-206; Lyle, C. et al., SciRep, 2019. 9 (1): p. 20257). Knockout of both c-Cbl and Cbl-b resulted in embryonic lethality in mice. Double knockout of hematopoietic c-Cbl and Cbl-b induced strong autoimmune diseases and early-onset lethal myeloproliferative diseases (Naramura, M. et al., Nat Immunol, 2002. 3 (12): p. 1192-9; Naramura, M. et al., Proc Natl Acad Sci USA, 2010. 107 (37): p. 16274-9). Therefore, selective inhibition of Cbl-b over c-Cbl should be achieved to avoid potential tumor-promoting effects and safety risks.

[0006] Although existing anti-CTLA-4 and anti-PD-1 therapies have shown clear clinical benefits in subsets of patients with various tumor types, there is still an unmet medical need for the development of novel immunotherapies to achieve robust and durable clinical anti-tumor efficacy. Preclinical data strongly suggest that there is great potential for the development of selective Cbl-b-targeted therapies to enhance anti-tumor immunity.

[0007] The present application provides novel heterocyclic compounds for inhibiting Cbl-b expression.

[0008] Citation or identification of any reference in this section shall not be construed as an admission that such reference is prior art to the present application. Summary of the invention

[0009] The present invention provides a method for preparing a compound of formula (I),

[0010]

[0011] or a pharmaceutically acceptable salt, tautomer, stereoisomer, isotopomer or enantiomer thereof,

[0012] Wherein, the method comprises:

[0013] a) amidating the compound of formula (II) with an amino reagent to obtain a compound of formula (IIa),

[0014]

[0015] b) reacting a compound of formula (IIa) with a compound of formula (III) via the halogen moiety of the compound of formula (III) to obtain a compound of formula (I);

[0016]

[0017] Where R 1 and R 2 are each independently hydrogen, substituted or unsubstituted C 1-8 Alkyl, or R 1 and R 2 With R 1 and R 2 The atoms to which they are attached together form a substituted or unsubstituted non-aromatic heterocyclic group;

[0018] R 3 It is hydrogen, cyanomethyl or methyl.

[0019] R 4 and R 5 are each independently hydrogen, substituted or unsubstituted C 1-8 Alkyl; and

[0020] X is a halogen moiety selected from F, Cl, Br or I.

[0021] In one embodiment, the amino reagent in step (a) is NH3 / MeOH.

[0022] In one embodiment, step (a) is carried out at a temperature of about 60°C to about 80°C; preferably 70°C.

[0023] In one embodiment, the reaction time of step (a) is 10-20 hours; preferably 15 hours.

[0024] In one embodiment, step (b) is carried out in a solvent, and the solvent is dioxane.

[0025] In one embodiment, a palladium catalyst and a base are provided in step (b), the palladium catalyst optionally having a phosphine ligand.

[0026] In one embodiment, the palladium catalyst is tBuXPhos Pd G3 and the phosphine ligand is tBuXPhos.

[0027] In one embodiment, the base in step (b) is tBuONa.

[0028] In one embodiment, step (b) is carried out at a temperature of about 90°C to about 110°C; preferably 100°C.

[0029] In one embodiment, the reaction time of step (b) is 10-14 hours; preferably 12 hours.

[0030] In one embodiment, R 1 and R 2 With R 1 and R 2 The atoms connected together form a substituted or unsubstituted non-aromatic heterocyclic group containing 1 nitrogen atom; preferably 3-methylpiperidin-1-yl.

[0031] In one embodiment, R 3 It is cyanomethyl or methyl.

[0032] In one embodiment, R 4 and R 5 Each is independently hydrogen, methyl, ethyl or methoxy.

[0033] In one embodiment, the compound of formula (I) is selected from

[0034]

[0035]

[0046] A more complete understanding of the embodiments of the present invention may be obtained by referring to the detailed description and examples, which are intended to illustrate non-limiting embodiments. DETAILED DESCRIPTION

[0036] definition

[0037] As used herein, the term "Cbl-b" refers to a Cbl-b protein. The term also includes naturally occurring variants of Cbl-b, including splice variants or allelic variants. The term also includes non-naturally occurring variants of Cbl-b, such as recombinant Cbl-b proteins or truncated variants thereof, which generally retain the binding ability (e.g., the ability to bind to an E2 enzyme) of naturally occurring Cbl-b or naturally occurring variants of Cbl-b.

[0038] As used herein and in the specification and claims that follow, the indefinite articles "a" and "an" and the definite article "the" include plural as well as singular referents, unless the context clearly dictates otherwise.

[0039] As used herein and unless otherwise indicated, the terms "about" and "approximately" when used in conjunction with the dosage, amount or weight percentage of the ingredients of the composition or dosage form refer to the dosage, amount or weight percentage recognized by those of ordinary skill in the art to provide a pharmacological effect equivalent to that obtained from the specified dosage, amount or weight percentage. In certain embodiments, when used in this context, the terms "about" and "approximately" encompass dosages, amounts or weight percentages within 30%, within 20%, within 15%, within 10% or within 5% of the specified dosage, amount or weight percentage.

[0040] As used herein and unless otherwise indicated, the terms "about" and "approximately", when used in conjunction with a value or range of values ​​provided to characterize a particular solid form (e.g., a particular temperature or temperature range, such as describing melting, dehydration, desolvation, or glass transition temperature; a change in mass, such as a change in mass as a function of temperature or humidity; solvent or water content, for example, expressed as mass or percentage; or peak position, such as in analysis by, for example, IR or Raman spectroscopy or XRPD), indicate that the value or range of values ​​may vary to an extent that is reasonable to one of ordinary skill in the art while still describing the solid form. Techniques used to characterize crystalline forms and amorphous solids include, but are not limited to, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), single crystal X-ray diffraction, vibrational spectroscopy (e.g., infrared (IR) and Raman spectroscopy), solid-state and solution nuclear magnetic resonance (NMR) spectroscopy, optical microscopy, hot stage optical microscopy, scanning electron microscopy (SEM), electron crystallography and quantitative analysis, particle size analysis (PSA), surface area analysis, solubility studies, and dissolution studies. In certain embodiments, the terms "about" and "approximately" when used in this context indicate that a value or range of values ​​may vary within 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1%, 0.5%, or 0.25% of the recited value or range of values. For example, in some embodiments, the value of an XRPD peak position may vary by as much as ±0.2° 2Θ (or ±0.2 degrees 2Θ) while still describing a specific XRPD peak.

[0041] "Alkyl" is a saturated, partially saturated or unsaturated straight or branched acyclic hydrocarbon having 1 to 10 carbon atoms, typically 1 to 8 carbons, or in some embodiments 1 to 6, 1 to 4, or 2 to 6 carbon atoms. Representative alkyl groups include -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; while saturated branched alkyl groups include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, -neopentyl, tert-pentyl, -2-methylpentyl, -3-methylpentyl, -4-methylpentyl, -2,3-dimethylbutyl, and the like. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, allyl, -CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, -C≡CH, -C≡C(CH3), -C≡C(CH2CH3), -CH2C≡CH, -CH2C≡C(CH3), and -CH2C≡C(CH7CH3), etc. The alkyl group may be substituted or unsubstituted. When alkyl groups described herein are referred to as "substituted," they may be substituted with any one or more substituents, such as those found in the exemplary compounds and embodiments disclosed herein; as well as halogen (chloro, iodo, bromo, or fluoro); alkyl; hydroxy; alkoxy; alkoxyalkyl; amino; alkylamino; carboxyl; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; acylamino; phosphonate; phosphine; thiocarbonyl; sulfonyl; sulfone; sulfonamide; ketone; aldehyde; ester; urea; carbamate; oxime; hydroxyamino; alkoxyamine; aralkyloxyamine; N-oxide; hydrazine; hydrazide; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; B(OH)2; or O(alkyl)aminocarbonyl.

[0042] "Cycloalkyl" is a saturated, partially saturated or unsaturated cyclic alkyl group having 3 to 10 carbon atoms, having a single ring or multiple condensed or bridged rings that may be optionally substituted with 1 to 3 alkyl groups. In some embodiments, the cycloalkyl group has 3 to 8 ring members, while in other embodiments, the number of ring carbon atoms is in the range of 3 to 5, 3 to 6, or 3 to 7. Cycloalkyl groups containing more than one ring may be fused, spirocyclic or bridged, or a combination thereof. Such cycloalkyl groups include, for example, monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, etc.; or polycyclic or bridged ring structures such as 1-bicyclo[1.1.1]pentyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, adamantyl, etc. Examples of unsaturated cycloalkyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, hexadienyl, etc. The cycloalkyl group may be substituted or unsubstituted. Such substituted cycloalkyl groups include, for example, cyclohexanol, etc.

[0043] "Aryl" is an aromatic carbocyclic group of 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple fused rings (e.g., naphthyl or anthracenyl). In some embodiments, the aryl group contains 6-14 carbons, and in other embodiments, the ring portion of the aryl group contains 6 to 12 or even 6 to 10 carbon atoms. Specific aryl groups include phenyl, biphenyl, naphthyl, etc. The aryl group may be substituted or unsubstituted. The phrase "aryl" also includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, etc.).

[0044] "Heterocyclyl" is an aromatic (also referred to as heteroaryl) or non-aromatic cycloalkyl group in which one to four ring carbon atoms are independently replaced by heteroatoms from the group consisting of O, S and N. In some embodiments, the heterocyclyl group includes 3 to 10 ring members, while other such groups have 3 to 5, 3 to 6 or 3 to 8 ring members. The heterocyclyl group may also be bonded to other groups at any ring atom (i.e., at any carbon atom or heteroatom of the heterocycle). The heterocyclyl group may be substituted or unsubstituted. The heterocyclyl group may include multiple condensed rings, including but not limited to bicyclic, tricyclic and tetracyclic rings and bridged or spirocyclic systems. The heterocyclyl group includes unsaturated, partially saturated and saturated ring systems, such as imidazolyl, imidazolinyl and imidazolidinyl (e.g., imidazolidin-4-one or imidazolidin-2,4-dionyl). The phrase heterocyclyl includes fused ring species, including those containing fused aromatic and non-aromatic groups, such as 1-aminotetralin and 2-aminotetralin, benzotriazolyl (e.g., 1H-benzo[d][1,2,3]triazolyl), benzimidazolyl (e.g., 1H-benzo[d]imidazolyl), 2,3-dihydrobenzo[1,4]dioxinyl and benzo[1,3]dioxolyl. The phrase also includes bridged polycyclic ring systems containing heteroatoms, such as, but not limited to, quinuclidine.Representative examples of heterocyclyl include, but are not limited to, aziridinyl, azetidinyl, azepanyl, oxetanyl, pyrrolidinyl, imidazolidinyl (e.g., imidazolidin-4-one or imidazolidin-2,4-dione), pyrazolidinyl, thiazolidinyl, tetrahydrophenylthio, tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, benzisoxazolyl (e.g., benzo[d]isoxazolyl), thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidinyl, piperazinyl (e.g., piperazin-2-one), morpholinyl, thiomorpholinyl, tetrahydropyranyl (e.g., tetrahydro-2H-pyranyl), tetrahydrothiopyranyl, oxathianyl, dioxyl, dithianyl, pyranyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, dihydropyridyl, dihydrodithiinyl, dihydrodisulfinyl, 1,4-dioxaspiro[4.5]decyl, homopiperazinyl, quinuclidinyl, indolyl (e.g., indolyl-2-one or isoindolyl-1-one), dihydroindolyl, isoindolyl, isoindolyl, azaindolyl (pyrrolopyridinyl or 1H-pyrrolo[2,3-b]pyridinyl), indazolyl, indolizinyl, benzotriazolyl (e.g., 1H-benzotriazolyl), 1H-benzo[d]imidazolyl, 1H-benzo[d]imidazolyl or 1H-benzo[d]imidazol-2(3H)-onyl, benzofuranyl, benzothiophenyl, benzothiazolyl, benzoxadiazolyl, benzoxazinyl, benzothiinyl, benzooxathiinyl, benzothiazinyl, benzoxazolyl (i.e., benzo[d]oxazolyl), benzothiazolyl, benzothiadiazole, benzo[1,3]dioxolyl, pyrazolopyridyl (e.g., 1H-pyrazolo[3,4-b]pyridyl, 1H-pyrazolo[4,3-b]pyridyl), imidazopyridyl (e.g., azabenzimidazolyl or 1H-imidazole Representative non-aromatic heterocyclic groups include, for example, 3,4-dihydroisoquinolin-1(2H)-one, quinolizinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, pteridinyl, thiazinyl, dihydrobenzothiazinyl, dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxinyl, tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, tetrahydropyrrolopyridinyl, tetrahydropyrazolopyridinyl, tetrahydroimidazopyridinyl, tetrahydrotriazolopyridinyl, tetrahydropyrimidin-2(1H)-one, and tetrahydroquinolinyl. Representative non-aromatic heterocyclic groups do not include fused ring species containing fused aromatic groups.Examples of non-aromatic heterocyclic groups include aziridinyl, azetidinyl, azepanyl, pyrrolidinyl, imidazolidinyl (e.g., imidazolidin-4-one or imidazolidin-2,4-dione), pyrazolidinyl, thiazolidinyl, tetrahydrothienyl, tetrahydrofuranyl, piperidinyl, piperazinyl (e.g., piperazin-2-one), morpholinyl, thiomorpholinyl, tetrahydropyranyl (e.g., tetrahydro-2H-pyranyl), tetrahydrothiopyranyl, oxathianyl, dithianyl, 1,4-dioxaspiro[4.5]decyl, homopiperazinyl, quinuclidinyl, or tetrahydropyrimidin-2(1H)-one. Representative substituted heterocyclic groups may be monosubstituted or substituted more than once, such as, but not limited to, pyridinyl or morpholinyl, 2-substituted, 3-substituted, 4-substituted, 5-substituted, or 6-substituted, or disubstituted with various substituents, such as those listed below.

[0045] "Heteroaryl" is an aryl ring system having one to four heteroatoms as ring atoms in a heteroaromatic ring system, wherein the remaining atoms are carbon atoms. In some embodiments, the heteroaryl group contains 3 to 6 ring atoms, and in other embodiments, the ring portion of the group contains 6 to 9 or even 6 to 10 atoms. Suitable heteroatoms include oxygen, sulfur and nitrogen. In certain embodiments, the heteroaryl ring system is monocyclic or bicyclic. Non-limiting examples include, but are not limited to, groups such as pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, benzisoxazolyl (e.g., benzo[d]isoxazolyl), thiazolyl, pyrrolyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, benzothienyl, furanyl, benzofuranyl, indolyl (e.g., indol-2-one or isoindolin-1-one), azaindolyl (pyrrolopyridinyl or 1H-pyrrolo[2,3-b]pyridinyl), indazolyl, benzimidazolyl (e.g., 1H-benzo[d]imidazolyl) ), imidazopyridyl (e.g., azabenzimidazolyl or 1H-imidazo[4,5-b]pyridyl), pyrazolopyridyl, triazolopyridyl, benzotriazolyl (e.g., 1H-benzo[d][1,2,3]triazolyl), benzoxazolyl (e.g., benzo[d]oxazolyl), benzothiazolyl, benzothiadiazolyl, isoxazolopyridyl, thionaphthyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolyl, isoquinolyl (e.g., 3,4-dihydroisoquinolin-1(2H)-one), tetrahydroquinolyl, quinoxalinyl, and quinazolinyl.

[0046] As used herein, "spirocycle" refers to two or more rings in which adjacent rings are connected by a single atom. The individual rings within a spirocycle may be the same or different. The individual rings in a spirocycle may be substituted or unsubstituted and may have different substituents than the other individual rings within a set of spirocycles.

[0047] "Cycloalkylalkyl" is a radical of the formula: -alkyl-cycloalkyl, wherein alkyl and cycloalkyl are as defined above. Substituted cycloalkylalkyl may be substituted at the alkyl, cycloalkyl, or both the alkyl and cycloalkyl portions of the radical. Representative cycloalkylalkyls include, but are not limited to, methylcyclopropyl, methylcyclobutyl, methylcyclopentyl, methylcyclohexyl, ethylcyclopropyl, ethylcyclobutyl, ethylcyclopentyl, ethylcyclohexyl, propylcyclopentyl, propylcyclohexyl, and the like.

[0048] An "aralkyl" group is a group of the formula: -alkyl-aryl, wherein the alkyl and aryl groups are as defined above. Substituted aralkyl groups may be substituted at the alkyl, aryl, or both the alkyl and aryl portions of the group. Representative aralkyl groups include, but are not limited to, benzyl and phenethyl groups and fused (cycloalkylaryl) alkyl groups, such as 4-ethyl-indanyl.

[0049] A "heterocyclylalkyl" group is a group of the formula: -alkyl-heterocyclyl, wherein the alkyl and heterocyclyl groups are defined above. Substituted heterocyclylalkyl groups may be substituted at the alkyl, heterocyclyl, or both alkyl and heterocyclyl portions of the group. Representative heterocyclylalkyl groups include, but are not limited to, 4-ethyl-morpholinyl, 4-propylmorpholinyl, furan-2-ylmethyl, furan-3-ylmethyl, pyridin-3-ylmethyl, tetrahydrofuran-2-ylethyl, and indol-2-ylpropyl.

[0050] "Halogen" is fluorine, chlorine, bromine or iodine.

[0051] A "hydroxyalkyl" group is an alkyl group as described above substituted with one or more hydroxy groups.

[0052] "Alkoxy" or "alkoxyl" is -O-(alkyl) where alkyl is defined above.

[0053] "Alkoxyalkyl" is -(alkyl)-O-(alkyl) where alkyl is as defined above.

[0054] An "amino" group is a group of the formula: -NH2.

[0055] An "alkylamino" group is a group of the formula: -NH-alkyl or -N(alkyl)2 wherein each alkyl group is independently as defined above.

[0056] A "carboxy" group is a group of the formula: -C(O)OH.

[0057] An "aminocarbonyl" group is a group of the formula: -C(O)N(R # )2、-C(O)NH(R # ) or -C(O)NH2, where each R #and R is independently substituted or unsubstituted alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl or heterocyclic as defined herein.

[0058] An "acylamino" group is a group of the formula: -NHC(O)(R # ) or -N(alkyl)C(O)(R # ), wherein each alkyl group and R # Independently as defined above.

[0059] A "sulfonylamino" group is a group of the formula: -NHSO2(R # ) or -N(alkyl)SO2(R # ), wherein each alkyl group and R # As defined above.

[0060] A "urea" group is a group of the formula: -N(alkyl)C(O)N(R # )2, -N(alkyl)C(O)NH(R # )、-N(alkyl)C(O)NH2、-NHC(O)N(R # )2、-NHC(O)NH(R # ) or -NH(CO)NHR # , where each alkyl group and R # Independently as defined above.

[0061] When groups described herein (other than alkyl) are referred to as "substituted", they may be substituted with any suitable substituent or substituents. Illustrative examples of substituents are those found in the exemplary compounds and embodiments disclosed herein, as well as halogen (chlorine, iodine, bromine or fluorine); alkyl; hydroxyl; alkoxy; alkoxyalkyl; amine; alkylamino; carboxyl; nitro; cyano; thiol; thioether; imine; imide; amidine; guanidine; enamine; aminocarbonyl; acylamino; phosphonate; phosphine; thiocarbonyl; sulfonyl; sulfone; sulfonamide; ketone; aldehyde; ester; urea; carbamate; oxime; hydroxyamino; alkoxyamine; aralkyloxyamine; N-oxide; hydrazine; hydrazide; hydrazone; azide; isocyanate; isothiocyanate; cyanate; thiocyanate; oxo (═O); B(OH)2, O(alkyl)aminocarbonyl; cyclo Alkyl, which may be monocyclic or fused or non-fused polycyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl), or heterocyclic, which may be monocyclic or fused or non-fused polycyclic (e.g., pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl or thiazinyl); monocyclic or fused or non-fused polycyclic aryl or heteroaryl (e.g., phenyl, naphthyl, pyrrolyl, indolyl, furanyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl, pyridyl, quinolyl, isoquinolyl, acridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, benzimidazolyl, benzothienyl or benzofuranyl); aryloxy; aralkyloxy; heterocyclyloxy; and heterocyclylalkoxy.

[0062] As used herein, the term "pharmaceutically acceptable salt(s)" refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic acids and bases and organic acids and bases. Suitable pharmaceutically acceptable base addition salts of compounds of formula (I) include, but are not limited to, those base addition salts well known in the art, see, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing, Easton PA (1990) or Remington: The Science and Practice of Pharmacy, 19th edition, Mack Publishing, Easton PA (1995).

[0063] Unless otherwise indicated, the term "stereoisomer" or "stereoisomerically pure" as used herein means that one stereoisomer of a compound is substantially free of other stereoisomers of the compound. For example, a stereoisomerically pure compound with one chiral center will be substantially free of the opposite enantiomer of the compound. A stereoisomerically pure compound with two chiral centers is substantially free of other diastereomers of the compound. A typical stereoisomerically pure compound contains greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of the compound, greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of other stereoisomers of the compound. Compounds can have chiral centers and can appear as racemates, individual enantiomers or diastereomers and mixtures thereof. All such isomeric forms are included in the embodiments disclosed herein, including mixtures thereof.

[0064] Embodiments disclosed herein encompass the use of stereoisomerically pure forms of such compounds, and the use of mixtures of these forms. For example, mixtures of enantiomers of a particular compound containing equal or unequal amounts can be used for methods and compositions disclosed herein. These isomers can be asymmetrically synthesized or split using standard techniques such as chiral columns or chiral splitting agents. See, for example, Jacques, J. et al., Enantiomers, Racemates and Resolutions (Wiley-Interscience, New York, 1981); Wilen, SH et al., Tetrahedron 33: 2725 (1977); Eliel, EL, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, SH, Tables of Resolving Agents and Optical Resolutions, p. 268 (ELEiel, Univ. of Notre Dame Press, Notre Dame, IN, 1972).

[0065] It should also be noted that the compound may include E and Z isomers, or mixtures thereof, as well as cis and trans isomers, or mixtures thereof. In certain embodiments, the compound is separated into E or Z isomers. In other embodiments, the compound is a mixture of E and Z isomers.

[0066] "Tautomers" refers to isomeric forms of a compound that are in equilibrium with each other. The concentrations of the isomeric forms will depend on the environment in which the compound is located, and may vary depending on, for example, whether the compound is a solid or in an organic or aqueous solution. For example, in aqueous solution, pyrazole may exhibit the following isomeric forms, which are referred to as tautomers of each other:

[0067]

[0068] It will be readily appreciated by those skilled in the art that various functional groups and other structures may exhibit tautomerism, and all tautomers of the compounds of formula (I) are within the scope of the present invention.

[0069] It should also be noted that compounds may contain unnatural proportions of atomic isotopes at one or more atoms. For example, a compound may contain an isotope such as tritium ( 3 H), iodine-125 ( 125 I), sulfur-35( 35 S) or carbon-14 ( 14 C), or can be radiolabeled with a radioactive isotope such as deuterium ( 2 H), carbon-13 ( 13 C) or nitrogen-15( 15 N) isotopically enriched. As used herein, an "isotopologue" is an isotopically enriched compound. The term "isotopically enriched" refers to an atom having an isotopic composition other than the natural isotopic composition of the atom. "Isotopically enriched" may also refer to an isotopic composition of at least one atom contained in a compound that is different from the natural isotopic composition of the atom. The term "isotopic composition" refers to the amount of each isotope present in a given atom. Radiolabeled and isotopically enriched compounds can be used as therapeutic agents (e.g., cancer and inflammatory therapeutic agents), research reagents (e.g., combined with assay reagents) and diagnostic agents (e.g., in vivo imaging agents). All isotopic changes of compounds as described herein (whether or not radioactive) are intended to be covered within the scope of the embodiments provided herein. In some embodiments, an isotopologue of a compound is provided, for example, an isotopologue is a deuterium, carbon-13 or nitrogen-15 enriched compound.

[0070] As used herein, "treating" means alleviating a disorder, disease or condition, or one or more symptoms associated with a disorder, disease or condition, or slowing or stopping the further progression or worsening of those symptoms, or alleviating or eradicating the cause of the disorder, disease or condition itself. In some embodiments, "treating" means all or partial relief of a disorder, disease or condition, or slowing or stopping the further development or worsening of those symptoms. In another embodiment, "treating" means all or partial relief of a disorder, disease or condition, or symptoms associated with a disorder, wherein the disorder is treatable or preventable by inhibiting Cbl-b.

[0071] As used herein, "prevention" means a method of delaying and / or hindering the onset, recurrence, or spread of all or part of a disorder, disease, or condition; a method of preventing a subject from developing a disorder, disease, or condition; or a method of reducing the risk of a subject developing a disorder, disease, or condition. In one embodiment, the disorder is a disorder that is treatable or preventable by inhibiting Cbl-b.

[0072] The term "effective amount" with respect to a compound means an amount capable of treating or preventing a condition, disease or disorder disclosed herein, or a symptom thereof.

[0073] The term "subject" includes animals, including but not limited to animals such as cows, monkeys, horses, sheep, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits or guinea pigs, in one embodiment mammals, in another embodiment humans.

[0074] Example

[0075] The following examples are intended to be exemplary only and should not be considered to be limited in any way. Unless otherwise stated, the experimental methods in the embodiments described below are conventional methods. Unless otherwise stated, reagents and materials are all commercially available. All solvents and chemicals used are of analytical grade or chemical purity. Solvents are all redistilled before use. Anhydrous solvents are all prepared according to standard methods or reference methods. Silica gel (100-200 mesh) for column chromatography and silica gel (GF254) for thin layer chromatography (TLC) can be purchased from Tsingdao Haiyang Chemical Co., Ltd. or Yantai Chemical Co., Ltd. in China; unless otherwise stated, all are eluted with petroleum ether (60°C-90°C) / ethyl acetate (v / v), and developed with ethanol solutions of iodine or molybdophosphoric acid. Unless otherwise stated, all extraction solvents are dried over anhydrous Na2SO4. 1H NMR spectra were recorded on a Bruck-400, Varian 400MR NMR spectrometer with TMS (tetramethylsilane) as internal standard. Coupling constants are given in Hertz. Peaks are reported as singlet (s), doublet (d), triplet (t), quartet (q), quintet (p), sextet (h), septet (hept), multiplet (m) or combinations thereof; br stands for broad. LC / MS data were recorded using an Agilent 1100, 1200 high performance liquid chromatograph-ion trap mass spectrometer (LC-MSD trap) equipped with a diode array detector (DAD) detecting at 214 nm and 254 nm and an ion trap (ESI source). All compound names, except reagents, are given by 18.0. or Chemaxon-Marvin JS generated. All mobile phase ratios for preparative HPLC are expressed as B% (organic phase) and the corresponding aqueous phase is A% = 100% - B%.

[0076] In the following examples, the following abbreviations are used.

[0077]

[0078]

[0079]

[0080]

[0081] Compound synthesis

[0082] Example 1: (S)-6-((3-methylpiperidin-1-yl)methyl)imidazo[1,2-a]pyridine-8-carboxamide

[0083]

[0084] Step 1: 6-vinylimidazolo[1,2-a]pyridine-8-carboxylic acid methyl ester

[0085]

[0086] To a solution of methyl 6-bromoimidazo[1,2-a]pyridine-8-carboxylate (10 g, 39.21 mmol) in dioxane (90 mL) / H2O (30 mL) was added potassium vinyl(trifluoro)borate (13.1 g, 98.01 mmol), K2CO3 (16.3 g, 117.6 mmol) and Pd(dppf)Cl2 (2.87 g, 3.92 mmol) at 15°C. The mixture was stirred at 100°C for 16 hours. The reactants were filtered and concentrated. The crude product was purified by column chromatography (SiO2, petroleum ether / EtOAc=5:1 to 0:1) to give the product (6.3 g, 79% yield). MS: M / e 203 (M+H) + ; 1 H NMR (400MHz, CDCl3-d) δ = 8.24 (s, 1H), 8.21 (d, J = 1.2Hz, 1H), 7.76 (s, 1H), 7.64 (s, 1H), 6.6 7(dd,J=12.0,16.0Hz,1H), 5.85(d,J=16.0Hz,1H), 5.42(d,J=12.0Hz,1H), 4.09(s,3H)ppm.

[0087] Step 2: Methyl 6-formylimidazo[1,2-a]pyridine-8-carboxylate

[0088]

[0089] To a solution of 6-vinyl imidazo[1,2-a]pyridine-8-carboxylic acid methyl ester (6.3 g, 31.1 mmol) in dioxane (125 mL) / H2O (25 mL) was added K2OsO4 2H2O (1.15 g, 3.1 mmol), NaIO4 (26.6 g, 124.6 mmol) in batches. The mixture was stirred at 25 ° C for 16 hours. The reactant was poured into H2O (100 mL) and extracted with EA (25 mL*3). The combined organic phases were concentrated to give a crude product. The crude product was purified by column chromatography (SiO2, petroleum ether / EtOAc=5:1 to 0:1) to give the product (4 g, 62% yield). MS: M / e 205 (M+H) + ; 1H NMR (400MHz, DMSO-d6) δ = 9.99 (s, 1H), 9.54 (d, J = 1.2Hz, 1H), 8.26 (d, J = 1.2Hz, 1H), 8.14 (d, J = 1.2Hz, 1H), 7.80 (d, J = 1.2Hz, 1H), 3.92 (s, 3H) ppm.

[0090] Step 3: (S)-6-((3-methylpiperidin-1-yl)methyl)imidazo[1,2-a]pyridine-8-carboxylic acid methyl ester

[0091]

[0092] To a solution of methyl 6-formyl imidazo[1,2-a]pyridine-8-carboxylate (1 g, 4.9 mmol), (S)-3-methylpiperidine hydrochloride (1.3 g, 9.8 mmol), Et3N (495 mg, 4.9 mmol) in DCM (10 mL) was added NaBH(OAc)3 (2.1 g, 9.8 mmol) in one portion. The mixture was stirred at 25 °C for 16 hours. The reactants were concentrated. The crude product was purified by column chromatography (SiO2, petroleum ether / EtOAc=1:1 to 0:1) to give the product (700 mg, 49% yield). MS: M / e 288 (M+H) + ; 1HNMR (400MHz, DMSO-d6) δ = 8.70 (s, 1H), 8.03 (s, 1H), 7.82 (s, 1H), 7.63 (s, 1H), 3.90 (s, 3H) 3. 51-3.43(m,2H),1.96-1.87(m,1H),1.96-1.86(m,1H),1.71-1.40(m,6H),0.92-0.78(m,4H)ppm.

[0093] Step 4: (S)-6-((3-methylpiperidin-1-yl)methyl)imidazo[1,2-a]pyridine-8-carboxamide

[0094]

[0095] A solution of (S)-ethyl 6-((3-methylpiperidin-1-yl)methyl)imidazo[1,2-a]pyridine-8-carboxylate (1.3 g, 4.3 mmol) in NH3 / MeOH (15 mL. 3M) was heated to 70°C in a sealed tube. The mixture was stirred at 70°C for 15 hours. The mixture was concentrated to give the crude product (650 mg, crude), which was used in the next step without purification. MS: M / e273 (M+H) + .

[0096] Example 2: (S)-N-(3-(1-(ethyl(methyl)carbamoyl)-3-methylcyclobutyl)phenyl)-6-((3-methylpiperidin-1-yl)methyl)imidazo[1,2-a]pyridine-8-carboxamide

[0097]

[0098] Step 1: 1-(3-Bromophenyl)-N-ethyl-N,3-dimethylcyclobutane-1-carboxamide

[0099]

[0100] DIEA (192.09 mg, 1.49 mmol) and HATU (339.07 mg, 891.74 umol) were added to a solution of 1-(3-bromophenyl)-3-methylcyclobutane-1-carboxylic acid (200 mg, 743.11 umol) in DMF (5 mL). The mixture was stirred at room temperature for 5 minutes, and then N-methylethylamine hydrochloride (106.53 mg, 1.11 mmol) was added. The mixture was stirred at room temperature for 20 hours. The mixture was poured into water (5 mL) and extracted with EA (5 mL * 2). The combined organic phase was washed with brine (30 mL), dried over Na2SO4, filtered and concentrated to give a residue, which was purified by preparative-TLC (PE: EA = 1: 1) to give the product (160 mg, 69% yield). MS (ESI) m / e [M + 1] 310.

[0101] Step 2: (S)-N-(3-(1-(ethyl(methyl)carbamoyl)-3-methylcyclobutyl)phenyl)-6-((3-methyl) 1-(2-piperidin-1-yl)methyl)imidazo[1,2-a]pyridine-8-carboxamide

[0102]

[0103] To a solution of 1-(3-bromophenyl)-N-ethyl-N,3-dimethylcyclobutane-1-carboxamide (50 mg, 161.17 umol) in dioxane (1 mL) was added (S)-6-((3-methylpiperidin-1-yl)methyl)imidazo[1,2-a]pyridine-8-carboxamide (43.89 mg, 161.17 umol), tBuONa (30.98 mg, 322.34 umol), tBuXPhos (6.84 mg, 16.12 umol) and tBuXPhos Pd G3 (12.80 mg, 16.12 umol). The mixture was stirred at 100° C. for 12 hours. The mixture was poured into water and extracted with EtOAc (1 mL). The combined organic phases were filtered and concentrated to give a residue, which was purified by preparative-HPLC (water (NH4HCO3)-ACN, Waters Xbridge BEH C18 100*30mm*10um, B%=55%-85%, 8 minutes) to give the product (7.11 mg). MS (ESI) m / e [M+1] 503; 1H NMR (400MHz, DMSO-d6) δ = 12.42 (s, 1H), 8.73 (s, 1H), 8.14 (s, 1H), 8.11 (s, 1H), 7.83-7.73 (m, 2H), 7. 72-7.66(m,1H),7.42(t,J=8.0Hz,1H),7.20(d,J=8.0Hz,1H),3.53(d,J=4.0Hz,2H),3.28-3.24(m,1 H),2.93(q,J=8.0Hz,1H),2.83-2.72(m,3H),2.30-2.20(m,2H),1.93(t,J=12.0Hz,2H),1.71-1.41( m,7H),1.23(s,1H),1.09-1.02(m,3H),1.02-0.95(m,2H),0.93-0.75(m,5H),0.64(t,J=8.0Hz,1H).

[0104] Example 3: (S)-N-(3-(3-methyl-1-(methylcarbamoyl)cyclobutyl)phenyl)-6-((3-methylpiperidin-1-yl)methyl)imidazo[1,2-a]pyridine-8-carboxamide

[0105]

[0106] Step 1: 1-(3-Bromophenyl)-N,3-dimethylcyclobutane-1-carboxamide

[0107]

[0108] DIEA (192.09 mg, 1.49 mmol) and HATU (339.07 mg, 891.74 umol) were added to a solution of 1-(3-bromophenyl)-3-methylcyclobutane-1-carboxylic acid (200 mg, 743.11 umol) in DMF (5 mL). The mixture was stirred at room temperature for 5 minutes, and then methylamine hydrochloride (75.26 mg, 1.11 mmol) was added. The mixture was stirred at room temperature for 20 hours. The mixture was poured into water (5 mL) and extracted with EA (5 mL * 2). The combined organic phases were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated to give a residue, which was purified by preparative-TLC (PE: EA = 1: 1) to give the product (150 mg, 71% yield). MS (ESI) m / e [M + 1] 284; 1H NMR (400 MHz, chloroform-d) δ=7.56 (s, 1H), 7.44-7.32 (m, 2H), 7.26-7.22 (m, 1H), 5.03 (s, 1H), 2.71 (d, J=4.0 Hz, 3H), 2.59-2.51 (m, 2H), 2.50-2.41 (m, 2H), 2.30–2.20 (m, 1H), 1.11 (d, J=8.0 Hz, 3H)

[0109] Step 2: (S)-N-(3-(3-methyl-1-(methylcarbamoyl)cyclobutyl)phenyl)-6-((3-methylpiperidinyl- 1-yl)methyl)imidazo[1,2-a]pyridine-8-carboxamide

[0110]

[0111] To a solution of 1-(3-bromophenyl)-N,3-dimethylcyclobutane-1-carboxamide (50 mg, 177.19 umol) in dioxane (1 mL) was added (S)-6-((3-methylpiperidin-1-yl)methyl)imidazo[1,2-a]pyridine-8-carboxamide (48.26 mg, 177.19 umol), tBuONa (34.06 mg, 354.38 umol), tBuXPhos (7.52 mg, 17.72 umol) and tBuXPhos Pd G3 (14.08 mg, 17.72 umol). The mixture was stirred at 100 ° C for 12 hours. The mixture was poured into water and extracted with EtOAc (1 mL). The combined organic phases were filtered and concentrated to give a residue, which was purified by preparative-HPLC (water (NH4HCO3)-ACN, Waters Xbridge BEH C18 100*30mm*10um, B%=45%-75%, 8 minutes) to give the product (13.06 mg). MS (ESI) m / e [M+1] 474. 1H NMR (400MHz, DMSO-d6) δ = 12.42 (s, 1H), 8.74 (s, 1H), 8.15 (s, 1H), 8.12 (s, 1H), 7.81 (d, J = 4.0Hz, 1H), 7. 76(d,J=8.0Hz,1H),7.69(s,1H),7.48(q,J=4.0Hz,1H),7.38(t,J=8.0Hz,1H),7.21(d,J=8.0Hz,1H),3. 57-3.49(m,2H),2.84-2.71(m,2H),2.62-2.58(m,1H),2.30-2.21(m,3H),2.17-2.08(m,1H),1.98-1.87 (m,2H),1.71-1.41(m,6H),1.27-1.13(m,1H),1.04(d,J=4.0Hz,3H),0.88(s,1H),0.82(d,J=8.0Hz,3H).

[0112] Example 4: (S)-N-(3-(1-(methoxy(methyl)carbamoyl)-3-methylcyclobutyl)phenyl)-6-((3-methylpiperidin-1-yl)methyl)imidazo[1,2-a]pyridine-8-carboxamide

[0113]

[0114] Step 1: 1-(3-Bromophenyl)-N-methoxy-N,3-dimethylcyclobutane-1-carboxamide

[0115]

[0116] The title compound was prepared under appropriate conditions according to a procedure similar to Example 2. MS (ESI) m / e [M+1] 312.

[0117] Step 2: (S)-N-(3-(1-(methoxy(methyl)carbamoyl)-3-methylcyclobutyl)phenyl)-6-((3- methylpiperidin-1-yl)methyl)imidazo[1,2-a]pyridine-8-carboxamide

[0118]

[0119] To a solution of 1-(3-bromophenyl)-N-methoxy-N,3-dimethylcyclobutane-1-carboxamide (50 mg, 160.15 umol) in dioxane (1 mL) was added (S)-6-((3-methylpiperidin-1-yl)methyl)imidazo[1,2-a]pyridine-8-carboxamide (43.62 mg, 160.15 umol), tBuONa (30.78 mg, 320.30 umol), tBuXPhos (6.80 mg, 16.02 umol) and tBuXPhos Pd G3 (12.72 mg, 16.02 umol). The mixture was stirred at 100 ° C for 12 hours. The mixture was poured into water and extracted with EtOAc (1 mL). The combined organic phases were filtered and concentrated to give a residue, which was purified by preparative-HPLC (water (NH4HCO3)-ACN, Phenomenex C18 75*30mm*3um, B%=40%-70%, 8 minutes) to give the product (4.33 mg). MS (ESI) m / e [M+1] 505; 1 H NMR (400MHz, DMSO-d6) δ = 12.44 (s, 1H), 8.73 (s, 1H), 8.15 (d, J = 4.0Hz, 1H), 8.11 (d, J = 1.2Hz, 1H) ,7.81(d,J=1.2Hz,1H),7.77(s,1H),7.67(d,J=8.0Hz,1H),7.42(t,J=8.0Hz,1H),7.18(d,J=8.0 Hz,1H),3.57-3.49(m,2H),3.17(s,3H),3.00(s,3H),2.83-2.73(m,2H),2.31-2.22(m,4H),1.97 -1.88(m,1H),1.72-1.44(m,6H),1.04(d,J=4.0Hz,3H),0.92-0.84(m,1H),0.82(d,J=8.0Hz,3H).

[0120] Example 5: (S)-N-(3-(3-(cyanomethyl)-1-(dimethylcarbamoyl)cyclobutyl)phenyl)-6-((3-methylpiperidin-1-yl)methyl)imidazo[1,2-a]pyridine-8-carboxamide

[0121]

[0122] Step 1: 1-(3-Bromophenyl)-3,3-dimethoxycyclobutane-1-carbonitrile

[0123]

[0124] 2-(3-bromophenyl)acetonitrile (2.07 g, 25.38 mmol) was added to a solution of NaH (4.08 g, 102.02 mmol, 60%) in DMF (200 mL) at 0 ° C. The mixture was stirred at 0 ° C for 5 minutes. Then 1,3-dibromo-2,2-dimethoxypropane (13.36 g, 51.01 mmol) was added at 0 ° C. The mixture was stirred at 60 ° C for 18 hours. Then NH4C was quenched and extracted with EtOAc (400 ml). The combined organic layer was washed with brine (200 mL) and dried over Na2SO4. After filtering and concentration, the resulting residue was purified by column chromatography (SiO2, PE: EA = 100 / 1, 4 / 1) to give the product (7 g, 46.34% yield).

[0125] Step 2: 1-(3-Bromophenyl)-3,3-dimethoxycyclobutane-1-carboxylic acid

[0126]

[0127] To a solution of 1-(3-bromophenyl)-3,3-dimethoxycyclobutane-1-carbonitrile (7g, 23.64mmol) in EtOH (70mL) was added NaOH (94.5ml, 10%). The mixture was stirred at 90°C for 12 hours. The ph was adjusted to about 5 by gradually adding HCl (3M). It was then extracted with EtOAc (400ml). The combined organic layers were washed with brine (200mL) and dried over Na2SO4. After filtering and concentrating, the residue was ground to give the product (5g, 67% yield). MS (ESI) m / e[M+1]285.

[0128] Step 3: 1-(3-Bromophenyl)-3,3-dimethoxy-N,N-dimethylcyclobutane-1-carboxamide

[0129]

[0130] To a solution of 1-(3-bromophenyl)-3,3-dimethoxycyclobutane-1-carboxylic acid (2g, 6.35mmol) in DMF (20mL) was added DIEA (2.07g, 25.38mmol), HATU (4.81g, 12.69mmol) and dimethylamine hydrochloride (2.46g, 19.04mmol). The mixture was stirred at room temperature for 18 hours. It was then quenched with H2O and extracted with EtOAc (20mL, 10mL). The combined organic layers were washed with brine (20mL) and dried over Na2SO4. After filtering and concentrating, the resulting residue was purified by column chromatography (SiO2, PE:EA=100 / 1, 1 / 1) to give the product (1.5g, 69% yield). MS (ESI) m / e[M+1]312. 1H NMR (400 MHz, chloroform-d) δ = 7.43 (t, J = 1.6 Hz, 1H), 7.29 (dd, J = 1.6, 7.7 Hz, 1H), 7.21-7.17 (m, 1H), 7.15-7.09 (m, 1H), 3.11 (d, J = 4.0 Hz, 6H), 2.97 (d, J = 16 Hz, 2H), 2.89 (s, 3H), 2.56-2.48 (m, 5H)

[0131] Step 4: 1-(3-Bromophenyl)-N,N-dimethyl-3-oxocyclobutane-1-carboxamide

[0132]

[0133] To a solution of 1-(3-bromophenyl)-3,3-dimethoxy-N,N-dimethylcyclobutane-1-carboxamide (1 g, 2.92 mmol) in acetone (10 mL) was added HCl (10 ml, 6 M). The mixture was stirred for 16 hours. The mixture was filtered to give the product (670 mg, 77% yield). MS (ESI) m / e [M+1] 298. 1 H NMR (400 MHz, CHLOROFORM-d) δ = 7.41-7.34 (m, 2H), 7.22-7.15 (m, 2H), 3.96-3.86 (m, 2H), 3.37-3.25 (m, 2H), 2.97 (s, 3H), 2.61 (s, 3H).

[0134] Step 5: 1-(3-bromophenyl)-3-(cyanomethylene)-N,N-dimethylcyclobutane-1-carboxamide

[0135]

[0136] To a solution of 1-(3-bromophenyl)-N,N-dimethyl-3-oxocyclobutane-1-formamide (670mg, 2.26mmol) in THF (3mL), diethyl (cyanomethyl)phosphonate (440mg, 2.49mmol) and t-BuOK (279mg, 2.49mmol) were added. The mixture was stirred at room temperature for 18 hours. It was then quenched with H2O and extracted with EtOAc (50mL). The combined organic layer was washed with brine (30mL) and dried over Na2SO4, filtered and concentrated to give a residue. The resulting residue was purified by column chromatography (SiO2, PE:EA=100 / 1, 1 / 1) to give the product (560mg, 69% yield). MS (ESI) m / e[M+1]321. 1H NMR (400 MHz, chloroform-d) δ=7.47-7.42 (m, 2H), 7.27-7.18 (m, 2H), 5.35-5.24 (m, 1H), 3.88 (m, 1H), 3.72-3.63 (m, 1H), 3.49-3.40 (m, 1H), 3.10-3.02 (m, 1H), 3.04-2.98 (m, 3H), 2.70-2.55 (m, 3H)

[0137] Step 6: 1-(3-bromophenyl)-3-(cyanomethyl)-N,N-dimethylcyclobutane-1-carboxamide

[0138]

[0139] To a solution of 1-(3-bromophenyl)-3-(cyanomethylene)-N,N-dimethylcyclobutane-1-carboxamide (560mg, 1.89mmol) in EtOH (1ml) and i-PrOH (3ml). Then NaBH4 (214.60mg, 5.67mmol) was added at 0°C. The mixture was stirred overnight at 60°C. Then quenched with NH4Cl and extracted with EtOAc (10mL). The combined organic layers were washed with brine (10mL) and dried over Na2SO4. After filtering and concentrating, the resulting residue was purified by column chromatography (SiO2, PE:EA=100 / 1, 1 / 3) to give the product (400mg, 71% yield). MS (ESI) m / e[M+1]323.

[0140] Step 7: (S)-N-(3-(3-(cyanomethyl)-1-(dimethylcarbamoyl)cyclobutyl)phenyl)-6-((3-methylcarbamoyl)cyclobutyl 1-(2-piperidin-1-yl)methyl)imidazo[1,2-a]pyridine-8-carboxamide

[0141]

[0142] To a solution of 1-(3-bromophenyl)-3-(cyanomethyl)-N,N-dimethylcyclobutane-1-carboxamide (50 mg, 155.66 umol) in DMF (1 mL) was added (S)-6-((3-methylpiperidin-1-yl)methyl)imidazo[1,2-a]pyridine-8-carboxamide (42.39 mg, 155.66 umol), tBuXPhos Pd-G3 (12.33 mg, 15.57 umol), tBuXPhos (6.61 mg, 15.57 umol) and NaOtBu (44.88 mg, 466.97 umol). The mixture was stirred at 100 ° C for 12 hours. The mixture was poured into water (1 mL) and extracted with DCM (2 mL). The combined organic phases were filtered and concentrated to give a residue, which was purified by preparative-HPLC (NH4HCO3)-CAN, Waters Xbridge BEH C18 100*30mm*10um, B%=45%-65%; 8 minutes) to give the product (8.52 mg). MS (ESI) m / e[M+1]513. 1 H NMR (400MHz, DMSO-d6)δ=12.42(s,1H),8.73(s,1H),8.15(s,1H),8.11(s,1H),7.85-7.78(m ,1H),7.73-7.60(m,2H),7.48-7.39(m,1H),7.28-7.23(m,0.3H),7.12(m,0.7H),3.55-3.50( m,2H),2.94-2.84(m,4H),2.83-2.72(m,4H),2.66(m,2H),2.59(m,1H),2.26-2.21(m,1H),2. 00-1.87(m,1H),1.70-1.58(m,4H),1.51-1.40(m,2H),1.31-1.19(m,1H),0.95-0.73(m,5H).

[0143] Determination

[0144] Biochemical assays

[0145] Cbl-b or Cbl biochemical assay protocol

[0146] The compounds disclosed herein are measured by stabilizing the inactive conformation of Cbl-b / Cbl and preventing the interaction of Cbl-b / Cbl with UbcH5B-Ub (E2 enzyme labeled with ubiquitin labeled with Bodipy-fluorescein) in a homogeneous time-resolved fluorescence-based assay. An assay performed in the absence of a Cbl-b / Cbl inhibitor, wherein phosphorylation of Cbl-b / Cbl by Src kinase allows UbcH5B-Ub to bind to Cbl-b / Cbl and allows Bodipy-fluorescein-labeled ubiquitin to approach streptavidin-terbium bound to N-terminally biotinylated Avi-labeled Cbl-b / Cbl. The resulting complex generates a FRET signal, indicating that Cbl-b / Cbl is active or not inhibited. An assay in the presence of a Cbl-b / Cbl inhibitor, wherein Cbl-b / Cbl is stabilized in an inactive conformation and cannot bind to UbcH5B-Ub. The lack of FRET signal indicates that Cbl-b / Cbl is inhibited by the Cbl-b / Cbl inhibitor.

[0147] Biochemical assay of co-expressed Cbl-b and BirA

[0148] In a homogeneous time-resolved fluorescence-based assay, the compounds disclosed herein were tested for stabilization of the inactive conformation of Cbl-b (aa36-427, internal) and for preventing the interaction of Cbl-b with UbcH5B-Ub. 0.6 Nm of recombinant co-expressed Cbl-b and BirA were pre-incubated with serial dilutions of the compounds (maximum concentration of 100 Mm, 3-fold serial dilutions, 16 points) in an assay buffer containing 50 mM HEPES (Ph 7.0), 100 mM NaCl, 0.01% BSA, 1 mM DTT, 0.01% TritonX-100 for 1.5 hours at room temperature. Then 12Nm internal UbcH5B-Ub conjugate protein, 120nM SRC (aa251-536, internal), 0.5mM ATP, 5mM MgCl2 and streptavidin-Tb cryptate (Cat. No. 610SATLB, Cisbio Bioassays) were added to the plate and further incubated for 2 hours at room temperature. HTRF signals (ex337 nm, em520 nm / 620 nm) were read on a BMG HERAstar FS instrument. The percentage inhibition of the interaction of Cbl-b with UbcH5B-Ub in the presence of increasing concentrations of compound was calculated based on the ratio of fluorescence at 520nm to fluorescence at 620nm. The IC of each compound was 1.54kJ / mL. 50 It was obtained by fitting the data to a four-parameter logistic equation using Dotmatics.

[0149] Biochemical assay of biotinylated Cbl-b

[0150] In a homogeneous time-resolved fluorescence-based assay, the compounds disclosed herein were tested for stabilization of the inactive conformation of Cbl-b (aa36-427, internal) and for preventing the interaction of Cbl-b with UbcH5B-Ub. 5.0 Nm recombinant biotinylated Cbl-b was pre-incubated with serial dilutions of the compounds (maximum concentration of 100 Mm, 3-fold serial dilutions, 16 points) in an assay buffer containing 50 mM HEPES (Ph 7.0), 100 mM NaCl, 0.01% BSA, 1 mM DTT, 0.01% TritonX-100 for 1.5 hours at room temperature. Then 25mM internal UbcH5B-Ub conjugate protein, 60nM SRC (aa251-536, internal), 0.5mM ATP, 5mM MgCl2 and streptavidin-Tb cryptate (Cat. No. 610SATLB, Cisbio Bioassays) were added to the plate and further incubated for 2 hours at room temperature. HTRF signals (ex 337nm, em 520nm / 620nm) were read on a BMG HERAstar FS instrument. The percentage inhibition of the interaction of Cbl-b with UbcH5B-Ub in the presence of increasing concentrations of compounds was calculated based on the ratio of the fluorescence at 520nm to the fluorescence at 620nm. The IC of each compound was 1.54kJ / mL. 50 It was obtained by fitting the data to a four-parameter logistic equation using Dotmatics.

[0151] Biochemical assay of co-expressed Cbl and BirA

[0152] In a homogeneous time-resolved fluorescence-based assay, the compounds disclosed herein were tested for stabilization of the inactive conformation of Cbl (aa 49-434, internal) and for preventing the interaction of Cbl-b with UbcH5B-Ub. 0.6 Nm of recombinant co-expressed Cbl-b and BirA were pre-incubated with serial dilutions of the compounds (maximum concentration of 100 Mm, 3-fold serial dilutions, 16 points) in an assay buffer containing 50 mM HEPES (Ph 7.0), 100 mM NaCl, 0.01% BSA, 1 mM DTT, 0.01% TritonX-100 for 1.5 hours at room temperature. Then 8 nM internal UbcH5B-Ub conjugate protein, 120 nM SRC (aa251-536, internal), 0.5 mM ATP, 5 mM MgCl2 and streptavidin-Tb cryptate (Cat. No. 610SATLB, Cisbio Bioassays) were added to the plate and further incubated for 2 hours at room temperature. HTRF signals (ex 337 nm, em 520 nm / 620 nm) were read on a BMG HERAstar FS instrument. The percentage inhibition of the interaction of Cbl with UbcH5B-Ub in the presence of increasing concentrations of compound was calculated based on the ratio of fluorescence at 520 nm to fluorescence at 620 nm. The IC of each compound was 1.5 ng / mL. 50 It was obtained by fitting the data to a four-parameter logistic equation using Dotmatics.

[0153] Biotinylated Cbl biochemical assay

[0154] In a homogeneous time-resolved fluorescence-based assay, the compounds disclosed herein were tested for stabilization of the inactive conformation of Cbl (aa49-434, internal) and for preventing the interaction of Cbl-b with UbcH5B-Ub. 5.0 nM recombinant biotinylated Cbl was pre-incubated with serial dilutions of the compounds (maximum concentration of 100 mM, 3-fold serial dilutions, 16 points) in an assay buffer containing 50 mM HEPES (Ph 7.0), 100 mM NaCl, 0.01% BSA, 1 mM DTT, 0.01% Triton X-100 for 1.5 hours at room temperature. Then 10 nM internal UbcH5B-Ub conjugate protein, 60 nM SRC (aa251-536, internal), 0.5 Mm ATP, 5 Mm MgCl2 and streptavidin-Tb cryptate (Cat. No. 610SATLB, Cisbio Bioassays) were added to the plate and further incubated for 2 hours at room temperature. HTRF signals (ex 337 nm, em 520 nm / 620 nm) were read on a BMG HERAstar FS instrument. The percentage inhibition of the interaction of Cbl with UbcH5B-Ub in the presence of increasing concentrations of compound was calculated based on the ratio of fluorescence at 520 nm to fluorescence at 620 nm. The IC of each compound was 1.5 ng / mL. 50 It was obtained by fitting the data to a four-parameter logistic equation using Dotmatics.

[0155] SPR assay

[0156] CBLB SPR

[0157] Surface plasmon resonance analysis was performed at room temperature using a BIAcore 8K system with a SA chip (Cytiva). For all measurements, HBS-EP buffer (Cytiva) consisting of 10mM HEPES, 150mM NaCl, 3mM EDTA and 0.05% (v / v) surfactant P20 was used as the running buffer. The CBLB protein (co-expressed with BirA biotin ligase) was fixed to the chip at a concentration of 500Nm. Then, gradient concentrations of compounds (0, 0.39, 0.78, 1.6, 3.1, 6.3, 13, 25, 50, 100 and 200nM) were flowed over the chip surface. After each cycle, the sensor surface was regenerated with 300mM NaCl. All binding kinetics were analyzed using the 1:1 kinetic mode using the BIA evaluation software.

[0158] CBL SPR

[0159] Surface plasmon resonance analysis was performed at room temperature using a BIAcore 8K system with a SA chip (Cytiva). For all measurements, HBS-EP buffer (Cytiva) consisting of 10mM HEPES, 150mM NaCl, 3mM EDTA and 0.05% (v / v) surfactant P20 was used as the running buffer. The CBL protein (co-expressed with BirA biotin ligase) was immobilized on the chip at a concentration of 500Nm. Then, gradient concentrations of compounds (0, 0.39, 0.78, 1.6, 3.1, 6.3, 13, 25, 50, 100 and 200nM) were flowed over the chip surface. After each cycle, the sensor surface was regenerated with 300mM NaCl. All binding kinetics were analyzed using the 1:1 kinetic mode using the BIA evaluation software.

[0160] The selected compounds showed good binding affinity in SPR assay.

[0161] Crystallization method

[0162] CBLB was incubated with the selected compounds for 1 h at 20 °C and the mixtures were subjected to initial crystal screening experiments by sitting-drop vapor diffusion at 293 K. Crystals were obtained under conditions consisting of 4% tacsimate Ph6.0, 12% PEG3350 or 0.2 M L-proline, 0.1 M hepes Ph7.2 / Ph7.5, 11% PEG3350.

[0163] Based on the crystal structure, ring A of the selected compounds of formula (I) forms a cation-π interaction with the guanidine side chain of Cbl-b Arg141. The significant binding energy of this interaction [Kumar K, Woo SM, Siu T, Cortopassi WA, Duarte F, Paton RS. Cation-π interactions in protein-ligand binding: theory and data-mining reveal different roles for lysine and arginine. Chem Sci. 2018 Jan 31;9(10):2655-2665] contributes to the high activity of our compound series.

[0164] Cell assay

[0165] Cellular autoubiquitination assay

[0166] HEK293 cells (ATCC) stably overexpressing Cbl-b or c-Cbl were maintained in DMEM medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 100 units / mL penicillin, and 0.1 mg / mL streptomycin (Thermo Fisher Scientific) in a humidified 37°C environment with 5% CO2.

[0167] Cell autoubiquitination was measured using MSD electrochemiluminescent immunoassay. Streptavidin-coated 384-well MSD plates (Meso Scale Discovery) were coated overnight with 2 μg / mL biotinylated anti-FLAG BioM2 mouse antibodies (Sigma) at 2°C to 8°C. On the second day, the plates were washed three times with 1×TBST and blocked with 5% BSA (Sigma) TBST. After 2 hours of compound treatment, cells were stimulated with 3Mm PV for 10 minutes. Cell lysates were then added to 25°C MSD plates, shaken at 450rpm for 1 hour, and then overnight at 2°C to 8°C to allow binding. The lysate was then discarded, and the plates were washed three times with 1×TBST, and incubated at 25°C with 1 μg / mL SULFO-TAG-labeled anti-ubiquitinated protein antibodies (Meso Scale Discovery) at 450rpm for 2.5 hours. The plate was washed again three times with 1× TBST and then MSDGOLD read buffer A (Meso Scale Discovery) was added. Signals were recorded at On a SECTOR S600 reader (Meso Scale Discovery).

[0168] Selected compounds showed good potency in autoubiquitination assays.

[0169] IL-2 production assay in Cbl-b or c-Cbl knockout Jurkat cells

[0170] Jurkat cells (ATCC) were infected with lentivirus expressing spCas9 and sgRNA targeting human Cbl-b or c-Cbl. Cell clones with stable knockout of Cbl-b or c-Cbl were established and maintained in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 100 units / mL penicillin and 0.1 mg / mL streptomycin (Thermo Fisher Scientific) at 37°C in a humidified environment with 5% CO2. The knockout efficiency of Cbl-b or c-Cbl in single cell clones was determined using genome sequencing and immunoblotting.

[0171] Cbl-b or c-Cbl knockout Jurkat cells were seeded into 96-well plates and then treated with compounds. After 2 hours of compound treatment, the cell suspension in each well was transferred to a high binding 96-well plate coated with anti-CD3 (OKT3, Thermo Fisher Scientific) for 24 hours at 37°C. The culture supernatant was collected and then measured for IL-2 concentration by a TR-FRET-based method as described in the manufacturer's manual (Cisbio). FRET signals were measured using a PHERAstar FSX reader (BMG Labtech).

[0172] Selected compounds showed good potency in the IL-2 production assay.

[0173] Human umbilical cord blood CD34 + Proliferation assay

[0174] Hematopoietic toxicity is one of the major limitations of full-dose use of anti-tumor agents, and it is difficult to produce drugs with strong anti-tumor effects and less hematopoietic toxicity (Kurtin S. Myeloid toxicity of cancer treatment. JAdv Pract Oncol. 2012; 3(4): 209-24.). Faced with this challenge, in vitro hematopoiesis models are increasingly used in hematopathology studies and preclinical safety studies of candidate drugs. Both colony-forming cell assays and hematopoietic stem cell proliferation assays successfully predicted the maximum tolerated dose of compounds (Pessina A, Albella B, Bayo M, Bueren J, Brantom P, Casati S, et al. Application of the CFU-GM assay to predict acute drug-induced neutropenia: an international blind trial to validate a prediction model for the maximum tolerated dose (MTD) of myelosuppressive xenobiotics. Toxicol Sci. 2003; 75 (2): 355-67., Clarke E, Pereira C, Chaney R, Woodside S, Eaves AC, Damen J. Toxicity testing using hematopoietic stem cell assays. Regen Med. 2007; 2 (6): 947-56). Moreover, we found that some c-Cbl or Cbl-b inhibitors abnormally increased hematopoietic stem cell proliferation. Therefore, a human umbilical cord blood CD34+ cell proliferation assay was established to assess the potential risk of compounds for hematopoietic toxicity.

[0175] Human umbilical cord blood CD34 + Cells (AllCells) were expanded for 7-12 days in IMDM medium (ATCC) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 100 units / mL penicillin, 0.1 mg / mL streptomycin (Thermo Fisher Scientific), 100 ng / mL recombinant human SCF (Peprotech), 50 ng / mL recombinant human TPO (Peprotech), 20 ng / mL recombinant human IL-3 (Peprotech), and 100 ng / mL recombinant human Flt-3L (Peprotech). Cells were grown in a 5%CO2 The cells were cultured in a humidified 37°C environment.

[0176] The CellTiter-Glo Luminescent Cell Viability Assay (Promega) was used to measure the activity of compounds in CD34 + Growth accelerating activity in cells. 5,000 cells were seeded in IMDM medium in each well of a 96-well plate, supplemented with 10% fetal bovine serum, 100 units / mL penicillin, 0.1 mg / mL streptomycin, and 10 ng / mL recombinant human SCF. After 7 days of exposure to the compounds disclosed herein, CellTiter-Glo reagent was added to each well. The mixture was mixed on an orbital shaker for 2 minutes to allow cell lysis, then incubated at room temperature for 10 minutes to stabilize the luminescent signal, which was measured using a PHERAstar FSX reader (BMG Labtech). The selected compounds showed low or moderate efficacy in a human cord blood CD34+ proliferation assay

[0177] In vitro plasma stability

[0178] Material

[0179]

[0180]

[0181] Process

[0182] The frozen plasma was thawed at 4°C and then centrifuged for 10 min.

[0183] One or more test compounds and positive controls were dissolved to 100× final concentration (0.1 mM) with DMSO, 2 μL of 0.1 mM control and test compound were added to 198 μL plasma, the samples were thoroughly mixed by brief vortexing, and then incubated in a 37°C water bath and gently shaken. The experiment will be performed in duplicate. The final organic solvent concentration in the assay should always be ≤1%. Aliquots of 20 Ul samples were taken from the plasma at 0 hours, 0.5 hours, 2 hours, and 4 hours to 200 Ul cold ACN (containing IS). The mixture was vortexed for 1 minute and centrifuged at 4000 rpm for 10 minutes. An aliquot of 30 μL supernatant was diluted with 120 μL ACN / H2O and then injected for LC-MS / MS analysis.

[0184] Data analysis

[0185] All calculations were performed using Microsoft Excel. Peak areas were determined from extracted ion chromatograms. Residual % was calculated using peak areas.

[0186] The selected compounds were stable in rat plasma.

[0187] A number of references have been cited, the disclosures of which are incorporated herein by reference in their entireties.

Claims

1. A method for preparing a compound of formula (I), or a pharmaceutically acceptable salt, tautomer, stereoisomer, isotopomer or enantiomer thereof, in, The method comprises: c) amidating the compound of formula (II) with an amino reagent to obtain a compound of formula (IIa), d) reacting a compound of formula (IIa) with a compound of formula (III) via the halogen moiety of the compound of formula (III) to obtain a compound of formula (I); Where R 1 and R 2 are each independently hydrogen, substituted or unsubstituted C 1-8 Alkyl, or R 1 and R 2 With R 1 and R 2 The atoms to which they are attached together form a substituted or unsubstituted non-aromatic heterocyclic group; R 3 It is hydrogen, cyanomethyl or methyl. R 4 and R 5 are each independently hydrogen, substituted or unsubstituted C 1-8 Alkyl; and X is a halogen moiety selected from F, Cl, Br or I.

2. The method of claim 1, wherein the amino reagent in step (a) is NH3 / MeOH.

3. The method of any one of claims 1-2, wherein step (a) is carried out at a temperature of about 60°C to about 80°C; preferably 70°C.

4. The method according to any one of claims 1 to 3, wherein the reaction time of step (a) is 10 to 20 hours; preferably 15 hours.

5. The method according to any one of claims 1 to 4, wherein step (b) is carried out in a solvent, and the solvent is dioxane.

6. The process according to any one of claims 1 to 5, wherein a palladium catalyst and a base are provided in step (b), the palladium catalyst optionally having a phosphine ligand.

7. The method of claim 6, wherein the palladium catalyst is tBuXPhos Pd G3 and the phosphine ligand is tBuXPhos.

8. The method of claim 6, wherein the base in step (b) is tBuONa.

9. The method of any one of claims 1 to 8, wherein step (b) is carried out at a temperature of about 90°C to about 110°C; preferably 100°C.

10. The method according to any one of claims 1 to 9, wherein the reaction time of step (b) is 10 to 14 hours; preferably 12 hours.

11. The method of any one of claims 1 to 10, wherein R1 and R 2 With R 1 and R 2 The atoms connected together form a substituted or unsubstituted non-aromatic heterocyclic group containing 1 nitrogen atom; preferably 3-methylpiperidin-1-yl.

12. The method of any one of claims 1 to 11, wherein R 3 It is cyanomethyl or methyl.

13. The method of any one of claims 1 to 12, wherein R 4 and R 5 Each is independently hydrogen, methyl, ethyl or methoxy.

14. The method of any one of claims 1 to 13, wherein the compound of formula (I) is selected from

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