Compounds having rip2 kinase inhibitory activity, pharmaceutical compositions containing the same, and uses thereof

Abstract

The present application belongs to the field of pharmaceutical chemistry, and particularly relates to a compound with RIP2 kinase inhibitory activity, a pharmaceutical composition containing the same, and application thereof. The present application provides a compound with the structure of formula (I), which is an effective inhibitor of RIP2 kinase and can be used for preventing and / or treating diseases and / or disorders responsive to RIP2 kinase.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to an aminoquinoline compound with NOD / RIP2 pathway inhibitory activity, compositions comprising the same, and the use of the aminoquinoline compound in medicine, particularly for the prevention and / or treatment of diseases (including various diseases such as tumors, autoimmune diseases, neurodegenerative diseases, metabolic diseases, and genetic diseases) that respond to RIP2 receptor inhibition. Background Technology

[0002] NOD-like receptors NOD1 and NOD2 (i.e., proteins 1 and 2 containing nucleotide-binding oligomerization domains) are pattern recognition receptors (PRRs). Activation of either NOD1 or NOD2 will trigger a cellular cascade reaction (Cell. Signal. 18(2006) 2223-2229). For example, under normal conditions, the leucine-rich domain (LPR) in NOD2 folds into the intermediate domain and is in a state of self-inhibition. However, when the LPR domain is recognized and bound by a muramyl dipeptide (MDP), the conformation of NOD2 will change, thereby activating itself (Science 300(2003) 1584-1587; J. Biol. Chem. 278(2003) 5509-5512), which in turn recruits and activates the downstream RIP2 through CARD-CRAD interactions. RIP2 undergoes autophosphorylation and ubiquitination by a series of E3 ubiquitin ligases, such as X chromosome-associated apoptosis inhibitor protein (XIAP) and cIAP1. Polyubiquitinated RIP2 oligomerizes, promoting polyubiquitination of NEMO / IKKγ and activation of transcription growth factor β-activated kinase 1 (TAK1). Activation of TAK1 further regulates the recruitment of TAB1 and TAB2 / 3, leading to activation of the MAPK kinase signaling pathway (ERK, p38, JNK) and NF-κB. For example, the TAB protein in the TAK1 / TAB2 / TAB3 complex binds to the k63 ubiquitin chain, allowing TAK1 to phosphorylate and activate the IKK complex. The activation of IKK promotes the phosphorylation of IκBα, causing the dissociation of IκB and the activation of the NF-κB pathway. Subsequently, the active heterodimer p65 / p50 translocates to the cell nucleus, activating the transcription of genes involved in immune responses, cell death pathways, and growth control (Clinical Immunology (2021), 223, 108648).

[0003] Receptor-interacting protein kinase 2 (RIP2, also known as RICK, CARDIAK, or CARD3) is a member of the receptor-interacting serine / threonine protein kinase family, encoded by the RIP2 gene located on human chromosome 8. This 61 kDa protein encodes a C-terminal casepase recruitment domain (CARD), an N-terminal kinase domain, and an intermediate domain (Curr. Biol. 8 (1998) 885-888). RIP2 plays a crucial role in the immune system, being regulated by the intracellular peptidoglycan sensors NOD1 and NOD2 (J. Immunol. 178 (2007) 2380-2386), triggering an innate immune response to defend against bacteria and infections. Initial studies suggested that RIP2 kinase activity was not essential for NF-κB pathway activation and cytokine production. However, transgenic mice lacking RIP2 protein did not respond to either NOD1 or NOD2 activating ligands, highlighting the important role of RIP2 kinase in NOD1 and NOD2 (J. Immunol. 2007, 178(4), 2380-2386). Once RIP2 is activated, serine 176 and tyrosine 474 undergo autophosphorylation. Phosphorylation of serine 176 is essential for RIP2 activity, while phosphorylation of tyrosine 474 can increase RIP2 activity but is not essential for signal transduction (Genes Dev. 24(2010), 2666-2677).

[0004] NOD / RIP2-dependent signaling pathway dysregulation is associated with numerous human diseases, including asthma, early-onset inflammatory bowel disease, sarcoidosis, Crohn's disease, multiple sclerosis, and Blau syndrome (a highly autoinflammatory disease). RIP2 is upregulated under pathological conditions such as sepsis and Alzheimer's disease. Furthermore, RIP2 plays a role in different cancer entities and serves as a prognostic marker, for example, in patients with inflammatory breast cancer (a rare and highly aggressive disease with a high mortality rate), where RIP2 is highly expressed (EMBO Mol. Med. 5 (2013) 1278-1295; Arthritis Rheum. 43 (2013) 125-130; Pediatr. Rheumatol. Online J. 12 (2014) 33; Scientific World Journal. 2016 (2016) 2597 376; J. Leukoc. Biol. 94 (2013) 927-932; Biochem. Biophys. Res. Commun. 281 (2001) 84-93; Cancers (Basel) 10 (2018)).

[0005] Since inhibiting RIP2 kinase activity can eliminate NOD1 / 2 downstream signaling, developing small molecule inhibitors that inhibit RIP2 kinase activity is expected to alleviate disease or pathological states caused by NF-κB or MAPK pathway activation, thereby producing preventive and / or therapeutic effects, and has broad clinical application prospects. Summary of the Invention

[0006] The problem the invention aims to solve

[0007] The purpose of this invention is to provide a compound having RIP2 inhibitory activity, pharmaceutical compositions comprising the compound, and applications thereof.

[0008] Solution for solving the problem

[0009] The objective of this invention is achieved through the following technical solution:

[0010] <First Aspect>

[0011] This invention provides a compound having RIP2 kinase inhibitory activity, or a pharmaceutically acceptable salt, ester, solvate, prodrug, isotope label, or isomer thereof, wherein the compound has the structure of formula (I):

[0012]

[0013] in,

[0014] n is selected from 0, 1, 2, and 3;

[0015] L is selected from single bond, -O-, -N(R) 6 )-and

[0016] Ring A is selected from 6- to 10-membered aryl and 5- to 10-membered heteroaryl;

[0017] R 1 Each is independently selected from hydrogen atom, deuterium atom, halogen, hydroxyl, amino, cyano, C 1-6 Alkyl, C 3-6 cycloalkyl, O(C) 1-6 Alkyl), NH(C) 1-6 Alkyl), N(C) 1-6 Alkyl)2, C 2-6 alkenyl and C 2-6 alkynyl group, the C 1-6 Alkyl, C 3-6 cycloalkyl, C 2-6 alkenyl and C 2-6 Each alkynyl group is independently and optionally surrounded by 1, 2, or 3 R groups. a replace;

[0018] R 2Selected from hydrogen atom, deuterium atom, C 1-3 Alkyl and C 3-6 cycloalkyl, the C 1-3 alkyl groups and the C 3-6 Each cycloalkyl group is independently and optionally surrounded by 1, 2, or 3 R groups. b replace;

[0019] R 3 Selected from hydrogen atom, halogen, hydroxyl, amino, C 1-6 Alkyl, O(C) 1-6 Alkyl), C 3-6 Cycloalkyl, 3- to 6-membered heterocycloalkyl, 5- to 10-membered aryl and 5- to 10-membered heteroaryl, wherein C 1-6 Alkyl, O(C) 1-6 Alkyl), C 3-6 Cycloalkyl, 3- to 6-membered heterocycloalkyl, 5- to 10-membered aryl, and 5- to 10-membered heteroaryl are each independently optionally bound by 1, 2, or 3 Rs. c replace;

[0020] R 4 Selected from C 1-3 Alkyl, the C 1-3 Alkyl groups may be optionally surrounded by 1, 2, or 3 R groups. d replace;

[0021] R 5 Selected from C 1-3 Alkyl, the C 1-3 Alkyl groups may be optionally surrounded by 1, 2, or 3 R's. e replace;

[0022] Or R 4 and R 5 Together with the phosphorus atoms attached to them, they form 5- to 6-membered heterocyclic alkyl groups or 5- to 8-membered heterocyclic alkenyl groups, each of which is independently and optionally surrounded by 1, 2, or 3 R atoms. d replace;

[0023] R 6 Selected from hydrogen atoms, C 1-3 Alkyl and C 3-6 cycloalkyl, wherein the C 1-3 alkyl groups and the C 3-6 Each cycloalkyl group is independently and optionally surrounded by 1, 2, or 3 R groups. f replace;

[0024] R 7 Selected from hydrogen atom, deuterium atom, fluorine atom, chlorine atom, bromine atom, hydroxyl group, amino group, cyano group, C 1-6 Alkyl, C 3-6 cycloalkyl, O(C) 1-6 Alkyl), NH(C)1-6 Alkyl), N(C) 1-6 Alkyl)2, C 2-6 alkenyl and C 2-6 alkynyl group, the C 1-6 Alkyl, C 3-6 cycloalkyl, C 2-6 alkenyl and C 2-6 Each alkynyl group is independently and optionally surrounded by 1, 2, or 3 R groups. a replace;

[0025] R a R b R d R e and R f Each is independently selected from fluorine, chlorine, bromine, iodine, hydroxyl, amino, CH3, and

[0026] R c Each is independently selected from deuterium, fluorine, chlorine, bromine, iodine, hydroxyl, amino, CH3, and Preferred atoms include fluorine, chlorine, bromine, iodine, hydroxyl, amino, and CH3. and

[0027] The 3- to 6-membered heterocyclic alkyl group, the 5- to 6-membered heterocyclic alkyl group, the 5- to 8-membered heterocyclic alkenyl group, and the 5- to 10-membered heteroaryl group each independently comprise 1, 2, or 3 cyclic heteroatoms or heteroatom groups, each of which is independently selected from O, S, NH, N, and P (=O).

[0028] Preferably, the compound, R 2 Selected from hydrogen atoms.

[0029] Preferably, in the compound, L is selected from single bonds, -O-, and...

[0030] Among them, R 6 The definition is as defined above.

[0031] Preferably, the compound, Selected from the following excerpt:

[0032] The following segments are preferred:

[0033]

[0034] More preferably, the compound, Selected from the following excerpt:

[0035] Preferred

[0036] Preferably, the compound, R 3 Selected from hydrogen atom, halogen, hydroxyl, amino, C 1-6 Alkyl, O(C) 1-6 Alkyl), C 3-6 Cycloalkyl, 3- to 6-membered heterocycloalkyl and 5- to 10-membered heteroaryl, wherein C 1-6 Alkyl, O(C) 1-6 Alkyl), C 3-6 Cycloalkyl, 3- to 6-membered heterocycloalkyl and 5- to 10-membered heteroaryl groups are each independently and optionally surrounded by 1, 2 or 3 R groups. c Replacement, preferred R 3 Selected from hydrogen atom, halogen, hydroxyl, amino, C 1-6 Alkyl, O(C) 1-6 alkyl), 3- to 6-membered heterocyclic alkyl and 5- to 10-membered heteroaryl, wherein C 1-6 Alkyl, O(C) 1-6 Alkyl groups, 3- to 6-membered heterocyclic alkyl groups, and 5- to 10-membered heteroaryl groups are each independently and optionally surrounded by 1, 2, or 3 R groups. c replace;

[0037] Among them, R c The definition is as defined above.

[0038] More preferably, the compound, R 3 Selected from hydrogen atom, fluorine atom, CH3, -OCD3, Preferred hydrogen atoms, CH3, Alternatively, hydrogen atoms, fluorine atoms, or -OCD3 may be preferred. Further optimization of fluorine atoms, -OCD3, Further optimization - OCD3,

[0039] Preferably, the compound, R 4 Selected from CH3, R 5 Selected from CH3.

[0040] Preferably, the compound, R 6 Selected from hydrogen atoms and C 1-3 Alkyl group, preferably with hydrogen atoms.

[0041] Preferably, the compound, R 7It is selected from hydrogen atoms, deuterium atoms, fluorine atoms, chlorine atoms and bromine atoms, with hydrogen atoms and fluorine atoms being preferred.

[0042] <Second aspect>

[0043] This invention provides the following compounds having RIP2 kinase inhibitory activity, or pharmaceutically acceptable salts, esters, solvates, prodrugs, isotope labels, or isomers thereof:

[0044]

[0045]

[0046] <Third aspect>

[0047] The present invention provides a pharmaceutical composition comprising a compound having RIP2 kinase inhibitory activity as described in the first aspect and the second aspect, or a pharmaceutically acceptable salt, ester, solvate, prodrug, isotope label, or isomer thereof.

[0048] Furthermore, the above-mentioned pharmaceutical composition also contains at least one pharmaceutically acceptable carrier.

[0049] <Fourth Aspect>

[0050] The present invention provides a pharmaceutical formulation comprising a compound having RIP2 kinase inhibitory activity according to the first aspect and the second aspect, or a pharmaceutically acceptable salt, ester, solvate, prodrug, isotope label, or isomer thereof, or a pharmaceutical composition according to the third aspect, wherein the pharmaceutical formulation is any one of tablets, capsules, injections, granules, powders, suppositories, pills, gels, powders, oral solutions, inhalers, suspensions, or dry suspensions.

[0051] <Fifth Aspect>

[0052] This invention provides a pharmaceutical combination comprising a compound having RIP2 kinase inhibitory activity as described in the first and second aspects, or a pharmaceutically acceptable salt, ester, solvate, prodrug, isotope label, or isomer thereof, or a pharmaceutical composition as described in the third aspect, or a pharmaceutical formulation as described in the fourth aspect, and one or more of antitumor drugs, anti-autoimmune disease drugs, anti-neurodegenerative disease drugs, anti-metabolic disease drugs, and hereditary disease drugs.

[0053] <Sixth Aspect>

[0054] The present invention provides the use of compounds having RIP2 kinase inhibitory activity according to the first aspect and the second aspect, or pharmaceutically acceptable salts, esters, solvates, prodrugs, isotope labels or isomers thereof, or pharmaceutical compositions according to the third aspect, or pharmaceutical formulations according to the fourth aspect, or pharmaceutical combinations according to the fifth aspect, in the preparation of medicaments for the prevention and / or treatment of diseases and / or conditions that are at least partially responsive to the RIP2 receptor.

[0055] The diseases and / or conditions mentioned include: uveitis, dermatitis, acute lung injury, type 2 diabetes mellitus, arthritis, ulcerative colitis, segmental ileitis, early-onset inflammatory bowel disease, extraintestinal inflammatory bowel disease, prevention of ischemia-reperfusion injury in solid organ transplantation, non-alcoholic fatty liver disease, autoimmune hepatitis, asthma, systemic lupus erythematosus, sarcoidosis, Wegener's granulomatosis and interstitial lung disease, pulmonary fibrosis, renal fibrosis, liver fibrosis, myocardial infarction, asthma, allergic pneumonia, interstitial lung disease, ankylosing spondylitis, multiple sclerosis, systemic sclerosis, polymyositis, rheumatoid arthritis, myasthenia gravis, juvenile diabetes mellitus, glomerulonephritis, autoimmune thyroiditis, graft rejection, Crohn's disease, Blau syndrome, scleroderma, psoriasis, stomatitis, retinitis pigmentosa, proliferative vitreoretinopathy, Best's vitrectomyelitis, eczema, urticaria. Vasculitis, eosinophilic fasciitis, wet and dry age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, diabetic macular erythema, uveitis, retinal vein occlusion, cystoid macular edema, glaucoma, Parkinson's disease, Alzheimer's disease, Huntington's disease, breast cancer, lung cancer, bladder cancer, pancreatic cancer, liver cancer, squamous cell carcinoma of the head and neck, thyroid cancer, sarcoma, osteosarcoma, desmoidoma, melanoma, prostate cancer, colorectal cancer. One or more of the following: ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, myeloma, lymphoma, mantle cell lymphoma, cutaneous T-cell lymphoma, chronic and non-progressive anemia, spontaneous or primary thrombocytosis, leukemia, acute leukemia, chronic leukemia, lymphocytic leukemia, myeloid leukemia, myelodysplastic syndrome, myeloproliferative disorders, brain tumor, astrocytoma, medulloblastoma, Schwann cell tumor, primary neuroectodermal tumor, or pituitary tumor.

[0056] <Seventh Aspect>

[0057] The present invention provides compounds having RIP2 kinase inhibitory activity according to the first aspect and the second aspect, or pharmaceutically acceptable salts, esters, solvates, prodrugs, isotope labels or isomers thereof, or pharmaceutical compositions according to the third aspect, or pharmaceutical formulations according to the fourth aspect, or pharmaceutical combinations according to the fifth aspect, for the prevention and / or treatment of diseases and / or conditions that are at least partially responsive to the RIP2 receptor.

[0058] <Eighth Aspect>

[0059] This invention provides a method for preventing and / or treating diseases and / or conditions that at least partially respond to the RIP2 receptor, comprising the steps of: administering a therapeutically effective amount of a compound having RIP2 kinase inhibitory activity according to the first aspect, the second aspect, or a pharmaceutically acceptable salt, ester, solvate, prodrug, isotope label, or isomer thereof, or a pharmaceutical composition according to the third aspect, or a pharmaceutical formulation according to the fourth aspect, or a combination of drugs according to the fifth aspect, to a patient in need.

[0060] Effects of the present invention

[0061] The compounds of the present invention having RIP2 kinase inhibitory activity can be used as effective inhibitors of RIP2 and can be used to prevent and / or treat diseases and / or conditions that respond to the RIP2 receptor. Attached Figure Description

[0062] Figure 1 This is a graph showing the test results of compound A6 in Example 19.

[0063] Figure 2 This describes the protective effect of compound A6 in Example 27 against MDP-induced peritonitis in mice.

[0064] Figure 3 This describes the protective effect of compound A24 in Example 27 against MDP-induced peritonitis in mice. Detailed Implementation

[0065] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0066] To more clearly describe the content of this invention, the terms involved are defined as follows:

[0067] The term "halogen" refers to fluorine, chlorine, bromine, or iodine, either alone or in combination, especially fluorine, chlorine, or bromine.

[0068] Term "C" 1-6 "Alkyl" refers to, alone or in combination, a saturated straight-chain or branched alkyl group comprising 1 to 6 (particularly 1 to 3) carbon atoms, including (but not limited to) methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, n-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, and 3,3'-dimethyl-2-butyl, etc. Preferably, "C 1-6 "alkyl" is any one of methyl, ethyl, n-propyl, isopropyl, and tert-butyl. Similarly, the term "C" 1-3 "Alkyl" alone or in combination refers to a saturated straight-chain or branched alkyl group containing 1 to 3 carbon atoms, including methyl, ethyl, propyl, isopropyl, etc.

[0069] Term "C" 2-6 "Alkenyl" refers to a straight-chain or branched hydrocarbon group, either alone or in combination, containing 2 to 6 carbon atoms (especially 2 to 4 carbon atoms) and having at least one unsaturated site (>C=C<), including (but not limited to) vinyl and allyl groups.

[0070] Term "C" 2-6 "Alynyl" refers to a straight-chain or branched monovalent hydrocarbon group that contains 2 to 6 carbon atoms (especially 2 to 3 carbon atoms) alone or in combination and has at least one alkynyl (-C≡C-) unsaturated site, including (but not limited to) ethynyl (-C≡CH) and propynyl (-CH2C≡CH).

[0071] The term "OC" 1-6 "alkyl" alone or in combination represents the group "C". 1-6 alkyl-O-", where "C" 1-6 "alkyl" indicates as defined above. "O(C)" 1-6 Alkyl groups include (but are not limited to) methoxy (-OCH3), ethoxy (-OCH2CH3), n-propoxy (-OCH2CH2CH3), isopropoxy (-OCH(CH3)2), n-butoxy (-OCH2CH2CH2CH3), sec-butoxy (-OCH(CH3)CH2CH3), isobutoxy (-OCH2CH(CH3)2), tert-butoxy (-OC(CH3)3), n-pentoxy (-OCH2CH2CH2CH2CH3), and neopentoxy (-OCH2C(CH3)3), etc.

[0072] The term "3- to 8-membered cycloalkyl" refers, alone or in combination, to a saturated or partially unsaturated monocyclic or polycyclic cycloalkyl group having 3 to 8 (particularly 3 to 6) carbon atoms, including (but not limited to) cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc. Similarly, the term "C 3-6 "Cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cycloalkyl group having 3 to 6 carbon atoms, either alone or in combination.

[0073] The term "3- to 8-membered heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic heterocyclic group comprising 3 to 8 (especially 3 to 6, more particularly 5 to 6) carbon atoms and heteroatoms or heteroatom groups for cyclization, wherein the heteroatoms or heteroatom groups are selected from N, NH, O, C(O), and S(O). m (Where m is 0, 1, or 2). "3- to 8-membered heterocyclic groups" include (but are not limited to) aziridinyl, azirrobutyl, oxobutyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, morpholinyl, piperazine, thiomorpholinyl, tetrahydropyranyl, 1,1-dioxothiomorpholinyl, butyrolactam, valproic acid, caprolactam, butyrolactone, valproic acid, or caprolactone, etc. Similarly, the term "3- to 6-membered heterocyclic alkyl" refers to a saturated monocyclic or polycyclic heterocyclic group containing 3 to 6 carbon atoms and heteroatoms or heteroatom groups selected from N, NH, O, C(O), and S(O). m (where m is 0, 1, or 2); the term "5- to 6-membered heterocyclic alkyl" refers to a saturated monocyclic or polycyclic heterocyclic group containing 5 to 6 carbon atoms for cyclization and heteroatoms or heteroatom groups selected from N, NH, O, C(O), and S(O). m (Where m is 0, 1, or 2). Similarly, the term "3- to 6-membered heterocyclic alkenyl" refers to a monocyclic or polycyclic heterocyclic group comprising 3 to 6 carbon atoms and heteroatoms or heteroatom groups for cyclization, and at least one carbon-carbon double bond, wherein the heteroatoms or heteroatom groups are selected from N, NH, O, C(O), and S(O). m (where m is 0, 1, or 2); the term "5- to 8-membered heterocyclic alkenyl" refers to a monocyclic or polycyclic heterocyclic group comprising 5 to 8 carbon atoms and heteroatoms or heteroatom groups for cyclization and at least one carbon-carbon double bond, wherein the heteroatoms or heteroatom groups are selected from N, NH, O, C(O), and S(O). m (where m is 0, 1 or 2).

[0074] The term "aryl" refers to any stable 6- to 10-membered monocyclic or bicyclic aromatic group, including (but not limited to) phenyl, naphthyl, tetrahydronaphthyl, 2,3-dihydroindenyl, or biphenyl. The hydrogen atoms on the "aryl" group are each optionally and independently replaced by one or more substituents described in this invention.

[0075] The term "heteroaryl" refers to an aromatic ring group formed by replacing a carbon atom in a ring with at least one heteroatom selected from sulfur, oxygen, or nitrogen. This aromatic ring group can be a 5- to 7-membered monocyclic or a 7- to 12-membered bicyclic group. In this invention, the number of heteroatoms in the heteroaryl group is preferably 1, 2, 3, or 4, such as thiophene, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyridine-2(1H)-keto, pyridin-4(1H)-keto, pyrroleyl, pyrazolyl, thiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, imidazolyl, tetrazolyl, isothiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, naphthyl, benzothiophene, indolyl, benzimidazolyl, benzothiazolyl, benzofuranyl, quinolinyl, isoquinolinyl, and quinazolinyl, etc. The hydrogen atoms on the “heteroaryl” group are each optionally and independently replaced by one or more substituents described in this invention. The term “5- to 10-membered heteroaryl” refers to a heteroaryl ring having 5 to 10 carbon atoms and heteroatoms, wherein the heteroaryl ring is as defined above.

[0076] The term "amino" alone or in combination represents primary amino (-NH2), secondary amino (-NH-) or tertiary amino.

[0077] The term "NH(C)" 1-6 "alkyl)" and the term "N(C)" 1-6 "alkyl)2" represents an amino group as defined above, either alone or in combination, wherein the hydrogen atom of the amino group is separated by one and two carbon atoms, respectively. 1-6 Alkyl groups are substituted, where "C" is a alkyl group. 1-6 "alkyl" means as defined above.

[0078] The term "pharmaceutically acceptable salt" indicates that the compounds of the present invention exist in the form of their pharmaceutical salts, including acid addition salts and base addition salts. Pharmaceutically acceptable salts are described in SMBerge's description of pharmaceutically acceptable salts in J. Pharmaceutical Sciences (Vol. 66: 1-19, 1977). In the present invention, a pharmaceutically acceptable non-toxic acid addition salt refers to a salt formed by the compounds of the present invention with an organic or inorganic acid, including (but not limited to) hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, and malic acid, etc. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed by the compounds of this invention with organic or inorganic bases, including (but not limited to) alkali metal salts, such as lithium, sodium, or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and organic base salts, such as ammonium salts or N-containing organic bases formed with N-groups.+ (C 1-6 Alkyl)4 salts, preferably lithium hydroxide, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, calcium carbonate, ammonia, triethylamine, or tetrabutylammonium hydroxide, etc. "Pharmaceutically acceptable salts" can be synthesized by conventional chemical methods.

[0079] The term "ester" is used to refer to organic esters, including monoesters, diesters, triesters, and more commonly polyesters.

[0080] The term "solvent" refers to an association formed by one or more solvent molecules with the compounds of this invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, etc.

[0081] The term "prodrug" refers to a chemical derivative of the compound of the present invention, which is converted into the compound represented by general formula I in vivo through a chemical reaction.

[0082] The term "isotope label" indicates that isotopes include (but are not limited to) [aspects of isotopes]. 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 17 O, 18 O, 18 F, 32 P, 35 S and 36 Cl, etc.

[0083] The term "isomer" encompasses all isomeric forms, including enantiomers, diastereomers, tautomers, and geometric isomers (including cis-trans isomers). Therefore, any single stereochemical isomer of the compound designed in this invention, or a mixture of its enantiomers, diastereomers, tautomers, or geometric isomers (or cis-trans isomers), is within the scope of this invention.

[0084] The term "independently" means that at least two groups (or ring systems) in a structure with the same or similar value ranges can have the same or different meanings under specific circumstances. For example, if substituent X and substituent Y are independently hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl, then when substituent X is hydrogen, substituent Y can be hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl; similarly, when substituent Y is hydrogen, substituent X can be hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl.

[0085] The term “optional” or “optionally” means that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0086] The term “prevention” refers to the complete or near-complete prevention of the occurrence of a disease or condition (e.g., infection, ischemia, or reperfusion injury) when a patient or subject is susceptible to or at risk of the disease or condition; prevention may also include suppression, i.e., preventing the development of the condition.

[0087] The term “treatment” means: 1) suppressing the disease; for example, suppressing the disease, symptoms or symptom of an individual experiencing or exhibiting the pathology or symptom of the disease (i.e., preventing further development of the pathology and / or symptom); or 2) improving the disease; for example, improving the disease, symptoms or symptom of an individual experiencing or exhibiting the pathology or symptom (i.e., reversing the pathology and / or symptom).

[0088] The term "therapeutic effective amount" refers to the amount of an active compound or agent that researchers, veterinarians, physicians, or other clinicians seek to elicit a biological or medical response in an tissue, system, animal, individual, or human.

[0089] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0090] In the following examples, all solvents and reagents used were of analytical or chemical purity; all solvents were redistilled before use; and all anhydrous solvents were processed according to standard or literature methods.

[0091] Column chromatography silica gel (100-200 mesh) and thin-layer chromatography silica gel (GF254) are products of Qingdao Marine Chemical Plant and Yantai Chemical Plant; unless otherwise specified, petroleum ether (60-90℃) / ethyl acetate (v / v) are used as eluents.

[0092] The colorimetric reagent is an ethanol solution of iodine or phosphomolybdic acid.

[0093] Unless otherwise specified, all extraction solvents were dried over anhydrous Na2SO4.

[0094] 1 HNMR was recorded using a Varian-400 nuclear magnetic resonance spectrometer, with TMS as an internal standard.

[0095] LC-MS was recorded using an Agilent Technologies 1100 high-performance liquid chromatography-ion trap mass spectrometry (LC-MSDTrap) system with a diode array detector (DAD) at detection wavelengths of 214 nm and 254 nm, and ion trap mass spectrometry (ESI source).

[0096] The HPLC column was an Agela Durashell C18 (4.6 × 50 mm, 3.5 μm); the mobile phase was 0.1% NH4HCO3 aqueous solution: acetonitrile (from 5:95 to 95:5 within 5 minutes); the flow rate was 1.8 mL / min.

[0097] The following abbreviations may be used in this invention: MeOH (methanol); Ph2O (diphenyl ether); dioxane (1,4-dioxane); Xantphos (4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene); Pd2(dba)3 (tris(dibenzylacetone)dipalladium); Et3N (triethylamine); con.HCl / conc.HCl (concentrated hydrochloric acid); sol.HCl (dilute hydrochloric acid); E tOH (ethanol); DCE (1,2-dichloroethane); DMF (N,N-dimethylformamide); i-PrOH (isopropanol); AcOH (acetic acid); (AcO)2O (acetic anhydride); DEAD (diethyl azodicarbonate); NBS (N-bromosuccinimide); DCM (dichloromethane); CDI (N,N-carbazide diimidazole); THF (tetrahydrofuran); PMBNH2 (p-methoxybenzylamine).

[0098] Example 1

[0099] Compound A1 is synthesized by the following method:

[0100]

[0101] 1. Synthesis of intermediate A1-1

[0102] p-Iodoaniline (1 g, 4.57 mmol) was dissolved in methanol (15 mL), and then 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (1.3 g, 6.85 mmol) was slowly added. The mixture was stirred at room temperature for 30 minutes, and a large amount of solid precipitated out. The mixture was filtered, and the filter cake was washed with ethanol (3 mL) and dried to obtain a pale white-yellow solid intermediate A1-1 (1.2 g, 70%). 1 H NMR (400MHz, DMSO-d6) δ 11.21 (d, J = 14.4Hz, 1H), 8.54 (d, J = 14.4Hz, 1H), 7.76 (d, J = 8.0Hz, 2H), 7.40 (d, J = 8.0Hz, 2H), 1.67 (s, 6H).

[0103] 2. Synthesis of intermediate A1-2

[0104] Diphenyl ether (140 mL) was added to a round-bottom flask, heated to 240 °C and stirred for 20 minutes to remove solvent and water. Then, A1-1 (1.2 g, 3.2 mmol) was added to the reaction mixture, and the mixture was stirred at 240 °C for 3 minutes. After cooling to room temperature, petroleum ether (80 mL) was added, and a solid precipitated. The solid was filtered, and the filter cake was slurried with ethyl acetate (20 mL), filtered, and dried to obtain a white solid intermediate A1-2 (850 mg, 98%). 1 H NMR (400MHz, DMSO-d6) δ11.87(s,1H),8.36(s,1H),7.97-7.87(m,2H),7.37(d,J=8.4Hz,1H),6.07(d,J=7.2Hz,1H).LC-MS(m / z):271.7[M+H] + .

[0105] 3. Synthesis of intermediate A1-3

[0106] A1-2 (200 mg, 0.74 mmol) was dissolved in phosphorus oxychloride (5 mL) and stirred at 110 °C for 15 minutes. The phosphorus oxychloride was removed by concentration, and the solid residue was dissolved in ethyl acetate (10 mL). The suspension was added dropwise to ice water (100 mL), and the pH was adjusted to 7 with saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate (60 mL * 2), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (petroleum ether / ethyl acetate (v / v) = 5 / 1) to give intermediate A1-3 (200 mg, 94%). 1 H NMR (400MHz, DMSO-d6) δ 8.87 (d, J = 4.0 Hz, 1H), 8.54 (s, 1H), 8.15 (d, J = 8.8 Hz, 1H), 7.88 (d, J = 8.8 Hz, 1H), 7.81 (d, J = 4.0 Hz, 1H).

[0107] 4. Synthesis of intermediate A1-4

[0108] Dissolve A1-3 (200 mg, 0.69 mmol) in 1,4-dioxane (5 mL), then add dimethylphosphine oxide (81 mg, 1.04 mmol), triethylamine (118 mg, 1.17 mmol), tris(dibenzylacetone)dipalladium (32 mg, 0.03 mmol), and 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (40 mg, 0.07 mmol) sequentially. Stir overnight at room temperature under nitrogen protection. Filter, concentrate, and purify by silica gel column chromatography (dichloromethane / methanol (v / v) = 50 / 1) to give intermediate A-4 (100 mg, 60%). 1H NMR (400MHz, DMSO-d6) δ8.96 (d, J = 4.4 Hz, 1H), 8.64 (d, J = 12.4 Hz, 1H), 8.27-8.13 (m, 2H), 7.89 (d, J = 4.4 Hz, 1H), 1.79 (s, 3H), 1.76 (s, 3H).

[0109] 5. Synthesis of final product A1

[0110] A1-4 (100 mg, 0.42 mmol) and 5-aminobenzothiazole (69 mg, 0.46 mmol) were dissolved in ethanol (3 mL), and one drop of concentrated hydrochloric acid was added. The mixture was stirred at 85 °C for 30 minutes, and a yellow solid precipitated out. After cooling to room temperature, the mixture was filtered and dried to obtain the final yellow solid product A1 (50 mg, yield 34%), which is the hydrochloride salt.

[0111] Compounds A2-A5 were synthesized according to the method of Example 1, except that the 5-aminobenzothiazole in step 5 was replaced with 3-amino-4-methylphenol, 5-fluoro-1H-indazole-3-amine, 4,5-dimethyl-1H-pyrazole-3-amine and 3-amino-4-fluorophenol, respectively.

[0112] Example 2

[0113] Compound A6 is synthesized by the following method:

[0114]

[0115] 1. Synthesis of intermediate A6-1

[0116] 4-Bromo-3-methoxyaniline (10 g, 49.49 mmol) was dissolved in ethanol (30 mL), and then 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (13.8 g, 72.24 mmol) was slowly added. The mixture was stirred at room temperature for 30 minutes, and a large amount of solid precipitated out. The mixture was filtered, and the filter cake was washed with ethanol (20 mL) and dried to obtain a pale yellow solid intermediate A6-1 (17.3 g, 98%). 1 H NMR (400MHz, DMSO-d6) δ11.23(d,J=14.4Hz,1H),8.64(d,J=14.8Hz,1H),7.59(d,J=8.8Hz,1H),7.43 (d, J=2.4Hz, 1H), 7.10 (dd, J=2.8Hz, J=8.8Hz, 1H), 3.90 (s, 3H), 1.67 (s, 6H). LC-MS (m / z): 353.9[MH] - .

[0117] 2. Synthesis of intermediate A6-2

[0118] Diphenyl ether (500 mL) was added to a round-bottom flask, heated to 240 °C and stirred for 20 minutes to remove solvent and water. Then, A6-1 (14.8 g, 41.55 mmol) was added to the reaction solution, and the mixture was stirred at 240 °C for 5 minutes. After cooling to room temperature, petroleum ether (200 mL) was added, and a solid precipitated. The solid was filtered and dried to obtain a white solid intermediate A6-2 (9.7 g, 92%). 1 H NMR (400MHz, DMSO-d6) δ11.69(s,1H),8.17(s,1H),7.86(d,J=7.6Hz,1H),7.05(s,1H),5.99(d,J=7.2Hz,1H),3.93(s,3H).LC-MS(m / z):254.0[M+H] + .

[0119] 3. Synthesis of intermediate A6-3

[0120] A6-2 (9.7 g, 38.18 mmol) was dissolved in phosphorus oxychloride (40 mL) and stirred at 110 °C for 30 minutes. The phosphorus oxychloride was removed by concentration, and the solid residue was dissolved in ethyl acetate (20 mL). The suspension was added dropwise to ice water (800 mL), and the pH was adjusted to 7 with saturated sodium bicarbonate solution. Extraction was performed with ethyl acetate (800 mL), and the organic phase was dried over anhydrous sodium sulfate and concentrated. The mixture was then slurried with ethyl acetate (50 mL), filtered, and dried to obtain a white solid intermediate, A6-3 (6.8 g, 65%). 1 H NMR (400MHz, DMSO-d6) δ8.81(d,J=4.8Hz,1H),8.37(s,1H),7.65(d,J=4.8Hz,1H),7.60(s,1H),4.04(s,3H).LC-MS(m / z):271.9[M+H] + .

[0121] 4. Synthesis of intermediate A6-4

[0122] A6-3 (6.8 g, 24.95 mmol) and 5-aminobenzothiazole (7.5 g, 49.90 mmol) were dissolved in isopropanol (200 mL), and concentrated hydrochloric acid (1 mL) was added. The mixture was stirred at 95 °C for 3 hours, and a solid precipitated out. After cooling to room temperature, the mixture was filtered. The filter cake was slurried with ethanol (50 mL), filtered, and dried to obtain a pale blue solid intermediate, A6-4 hydrochloride (7.0 g, 73%). 1H NMR (400MHz, DMSO-d6) δ14.25(s,1H),11.02(s,1H),9.53(s,1H),9.16(s,1H),8.46(d,J=6.8Hz,1H),8.37(d,J=8.4H z,1H),8.20(s,1H),7.59(d,J=8.4Hz,1H),7.53(s,1H),6.82(d,J=6.8Hz,1H),4.07(s,3H).LC-MS(m / z):386.0[M+H] + .

[0123] 5. Synthesis of final product A6

[0124] A6-4 (5.6 g, 14.50 mmol) was dissolved in 1,4-dioxane (25 mL), followed by the sequential addition of dimethylphosphine oxide (1.7 g, 21.75 mmol), triethylamine (2.49 g, 24.65 mmol), tris(dibenzylacetone)dipalladium (1.3 g, 1.45 mmol), and 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (0.8 g, 1.45 mmol). The mixture was microwaved at 125 °C for 4 hours under nitrogen protection. After filtration, the filtrate was adjusted to pH 7 with saturated sodium bicarbonate solution, extracted with a mixture of dichloromethane (300 mL) and methanol (50 mL), dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 15 / 1) to give the final product A6 (2.4 g, yield 43%).

[0125] Example 3

[0126] Compound A7 is synthesized by the following method:

[0127]

[0128] 1. Synthesis of intermediate A7-1

[0129] A6-3 (2.8 g, 10.27 mmol) was dissolved in 1,2-dichloroethane (10 mL), and boron tribromide (10.3 mL, 31 mmol) was added dropwise at room temperature. The mixture was then microwaved at 110 °C for 1 hour. After the reaction system cooled to room temperature, a saturated anhydrous sodium sulfite solution (20 mL) was added to quench the reaction, followed by the addition of water (50 mL). The mixture was extracted with dichloromethane (50 mL x 2), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 50 / 1) to give intermediate A7-1 (1.7 g, 64%). 1H NMR (400MHz, DMSO-d6) δ11.86(s,1H),8.87(d,J=5.2Hz,1H),8.42(s,1H),7.75(d,J=5.2Hz,1H),7.55(s,1H).LC-MS(m / z):257.7[M+H] + .

[0130] 2. Synthesis of intermediate A7-2

[0131] A7-1 (200 mg, 0.77 mmol) and potassium carbonate (215 mg, 1.55 mmol) were dissolved in N,N-dimethylformamide (3 mL), stirred at 80 °C for 30 minutes, and then 2-bromoethanol (193 mg, 1.55 mmol) was added, and stirring continued overnight. After the reaction was complete, the mixture was directly concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 50 / 1) to give intermediate A7-2 (150 mg, 64%). LC-MS (m / z): 301.7 [M+H] + .

[0132] 3. Synthesis of intermediate A7-3

[0133] A7-2 (150 mg, 0.50 mmol) and 5-aminobenzothiazole (93 mg, 0.62 mmol) were dissolved in isopropanol (3 mL), and one drop of concentrated hydrochloric acid was added. The mixture was stirred at 85 °C for 30 minutes, and a yellow solid precipitated out. After cooling to room temperature, the mixture was filtered and dried to obtain a yellow solid intermediate, A7-3 hydrochloride (120 mg, 58%). 1 H NMR (400MHz, DMSO-d6) δ14.27(s,1H),11.03(s,1H),9.53(s,1H),9.17(s,1H),8.45(d,J=6.4Hz,1H),8.37(d,J=8.4Hz,1H) ,8.20(s,1H),7.60(d,J=8.4Hz,1H),7.56(s,1H),6.81(d,J=6.8Hz,1H),4.28(s,2H),3.88(s,2H).LC-MS(m / z):415.6[M+H] + .

[0134] 4. Synthesis of final product A7

[0135] A7-3 hydrochloride (120 mg, 0.29 mmol) was dissolved in 1,4-dioxane (3 mL), followed by the sequential addition of dimethylphosphine oxide (34 mg, 0.43 mmol), triethylamine (50 mg, 0.49 mmol), tris(dibenzylacetone)dipalladium (26 mg, 0.03 mmol), and 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (18 mg, 0.03 mmol). The reaction mixture was microwaved at 125 °C for 1 hour under nitrogen protection. Saturated sodium bicarbonate aqueous solution was added to the reaction mixture, and the mixture was stirred at room temperature for 30 minutes. The solution was then concentrated and purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 20 / 1) to obtain the final product A7 (30 mg, yield 25%).

[0136] Example 4

[0137] Compound A8 is synthesized by the following method:

[0138]

[0139] 1. Synthesis of intermediate A8-1

[0140] A7-1 (200 mg, 0.78 mmol) and potassium carbonate (218 mg, 1.56 mmol) were dissolved in N,N-dimethylformamide (3 mL), and stirred at 80 °C for 30 minutes. Then, 1-bromo-2-methoxyethane (216 mg, 1.56 mmol) was added, and stirring continued overnight. After the reaction was complete, the mixture was directly concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 50 / 1) to give intermediate A8-1 (160 mg, 65%). LC-MS (m / z): 315.7 [M+H] + .

[0141] 2. Synthesis of intermediate A8-2

[0142] A8-1 (140 mg, 0.44 mmol) and 5-aminobenzothiazole (80 mg, 0.53 mmol) were dissolved in isopropanol (2 mL), and one drop of concentrated hydrochloric acid was added. The mixture was stirred at 95 °C for 30 minutes, and a yellow solid precipitated out. After cooling to room temperature, the mixture was filtered and dried to obtain a yellow solid intermediate, A8-2 hydrochloride (150 mg, 79%). 1H NMR (400MHz, DMSO-d6) δ14.30(s,1H),11.04(s,1H),9.53(s,1H),9.17(s,1H),8.45(d,J=6.0Hz,1H),8.37(d,J=8.4Hz,1H),8.20(s,1H ),7.60(d,J=8.4Hz,1H),7.55(s,1H),6.81(d,J=6.4Hz,1H),4.45-4.34(m,2H),3.89-3.75(m,2H),3.39(s,3H).LC-MS(m / z):427.6[MH] - .

[0143] 3. Synthesis of the final product A8

[0144] A8-2 (160 mg, 0.37 mmol) was dissolved in 1,4-dioxane (2 mL), followed by the addition of dimethylphosphine oxide (44 mg, 0.56 mmol), triethylamine (64 mg, 0.63 mmol), tris(dibenzylacetone)dipalladium (34 mg, 0.04 mmol), and 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (22 mg, 0.04 mmol). The mixture was microwaved at 125 °C for 1 hour under nitrogen protection. A saturated sodium bicarbonate aqueous solution was added to the reaction mixture, and the mixture was stirred at room temperature for 30 minutes. The solution was concentrated and purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 15 / 1) to obtain the final product A8 (16 mg, 10% yield).

[0145] Example 5

[0146] Compound A9 is synthesized by the following method:

[0147]

[0148] 1. Synthesis of intermediate A9-1

[0149] Dissolve A7-1 (200 mg, 0.77 mmol) and potassium carbonate (215 mg, 1.55 mmol) in N,N-dimethylformamide (3 mL), stir at 80 °C for 30 minutes, then add 3-bromomethyl-3-methyl-1-oxetane (257 mg, 1.55 mmol) and continue stirring overnight. After the reaction is complete, concentrate directly. Purify the residue by silica gel column chromatography (dichloromethane / methanol (v / v) = 50 / 1) to give intermediate A9-1 (180 mg, 68%).

[0150] 2. Synthesis of intermediate A9-2

[0151] A9-1 (180 mg, 0.52 mmol) and 5-aminobenzothiazole (94 mg, 0.63 mmol) were dissolved in isopropanol (3 mL), and one drop of concentrated hydrochloric acid was added. The mixture was stirred at 95 °C for 30 minutes, and a yellow solid precipitated out. After cooling to room temperature, the mixture was filtered and dried to obtain a yellow solid intermediate, A9-2 hydrochloride (150 mg, 63%). 1 H NMR (400MHz, DMSO-d6) δ14.18(s,1H),11.00(s,1H),9.53(s,1H),9.15(s,1H),8.46(d,J=6.8Hz,1H),8.38(d,J=8.4Hz,1H),8.20(s,1H) ,7.60(d,J=8.4Hz,1H),7.55(s,1H),6.83(d,J=6.4Hz,1H),4.65-4.55(m,2H),4.42-4.32(m,4H),1.48(s,3H).LC-MS(m / z):455.6[M+H] + .

[0152] 3. Synthesis of final product A9

[0153] A9-2 (150 mg, 0.33 mmol) was dissolved in 1,4-dioxane (3 mL), followed by the addition of dimethylphosphine oxide (38 mg, 0.49 mmol), triethylamine (56 mg, 0.56 mmol), tris(dibenzylacetone)dipalladium (30 mg, 0.03 mmol), and 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (19 mg, 0.03 mmol). The mixture was microwaved at 125 °C for 1 hour under nitrogen protection. A saturated sodium bicarbonate aqueous solution was added to the reaction mixture, and the mixture was stirred at room temperature for 30 minutes. The solution was concentrated and purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to obtain the final product A9 (26 mg, yield 17%).

[0154] Example 6

[0155] Compound A10 is synthesized by the following method:

[0156]

[0157] 1. Synthesis of intermediate A10-1

[0158] 4-Bromo-3-methoxyaniline (1 g, 4.95 mmol) was dissolved in 1,2-dichloroethane (7 mL), and boron tribromide (4.95 mL, 14.85 mmol) was added dropwise at room temperature. The mixture was then microwaved at 100 °C for 1 hour. After the reaction system cooled to room temperature, a saturated sodium sulfite solution (10 mL) was added to quench the reaction, followed by the addition of water (30 mL). The mixture was extracted with dichloromethane (30 mL x 2), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate (v / v) = 5 / 1) to give intermediate A10-1 (660 mg, 71%). LC-MS (m / z): 187.8 [M+H] + .

[0159] 2. Synthesis of intermediate A10-2

[0160] Dissolve A10-1 (660 mg, 3.5 mmol) in acetic acid (5 mL). Add acetic anhydride (396 mg, 3.9 mmol) dropwise to the reaction system at room temperature and continue stirring for 10 minutes. A white solid precipitates out. Add water (200 mL), filter, and obtain a white solid intermediate A10-2 (600 mg, 74%). 1 H NMR (400MHz, DMSO-d6) δ10.21(s,1H),9.93(s,1H),7.47(s,1H),7.33(d,J=8.8Hz,1H),6.86(d,J=8.4Hz,1H),2.01(s,3H).LC-MS(m / z):229.8[M+H] + .

[0161] 3. Synthesis of intermediate A10-3

[0162] A10-2 (600 mg, 2.61 mmol), triphenylphosphine (1.37 g, 5.22 mmol), and diethyl azodicarbonate (909 mg, 5.22 mmol) were dissolved in dry tetrahydrofuran (5 mL). Under nitrogen protection, the mixture was stirred at room temperature for 30 minutes. Then, 3-hydroxytetrahydrofuran (275 mg, 3.13 mmol) was added, and stirring continued for 2 hours. After the reaction was complete, the solution was concentrated and purified by silica gel column chromatography (petroleum ether / ethyl acetate (v / v) = 5 / 1) to give a white solid intermediate, crude A10-3 (960 mg). LC-MS (m / z): 299.7 [M+H] + .

[0163] 4. Synthesis of intermediate A10-4

[0164] 960 mg of crude intermediate A10-3 was dissolved in 15 mL of ethanol. Concentrated hydrochloric acid (4 mL) was added dropwise to the reaction mixture, and the mixture was stirred at 85 °C for 3 hours. After the reaction was complete, the solution was concentrated to obtain 620 mg of crude, colorless oily intermediate A10-4. LC-MS (m / z): 257.8 [M+H] + .

[0165] 5. Synthesis of intermediate A10-5

[0166] Dissolve A10-4 (620 mg, 2.4 mmol) in ethanol (15 mL), then slowly add 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (490 mg, 2.6 mmol), stir at room temperature for 10 minutes, and a solid precipitates out. Filter, wash the filter cake with ethanol (3 mL), and dry to obtain a pale white-yellow solid intermediate A10-5 (430 mg, 43%). 1 H NMR (400MHz, DMSO-d6) δ11.22(d,J=14.4Hz,1H),8.61(d,J=14.4Hz,1H),7.60(d,J=8.4Hz,1H),7.44(s,1H),7.12(d,J= 8.4Hz,1H),5.20(s,1H),3.98-3.74(m,4H),2.32-2.20(m,1H),2.04-1.95(m,1H),1.68(s,6H).LC-MS(m / z):409.6[MH] - .

[0167] 6. Synthesis of intermediate A10-6

[0168] Diphenyl ether (60 mL) was added to a round-bottom flask, heated to 240 °C and stirred for 20 minutes to remove solvent and water. Then, A10-5 (430 mg, 1.04 mmol) was added to the reaction solution, and the mixture was stirred at 240 °C for 5 minutes. After cooling to room temperature, petroleum ether (30 mL) was added, and a solid precipitated. The solid was filtered and dried to obtain a white solid intermediate, A10-6 (250 mg, 77%). 1 H NMR (400MHz, DMSO-d6) δ11.65(s,1H),8.17(s,1H),7.87(d,J=7.6Hz,1H),7.04(s,1H),5.99(d,J=7.6H z,1H),5.14(s,1H),3.99-3.77(m,4H),2.35-2.26(m,1H),2.11-2.02(m,1H).LC-MS(m / z):310.0[M+H] + .

[0169] 7. Synthesis of intermediate A10-7

[0170] A10-6 (250 mg, 0.81 mmol) was dissolved in phosphorus oxychloride (5 mL) and stirred at 110 °C for 30 minutes. After the reaction was complete, the solution was concentrated to obtain crude intermediate A10-7 (280 mg).

[0171] 8. Synthesis of intermediate A10-8

[0172] Crude A10-7 (220 mg, 0.67 mmol) and 5-aminobenzothiazole (111 mg, 0.74 mmol) were dissolved in isopropanol (5 mL), and one drop of concentrated hydrochloric acid was added. The mixture was stirred at 95 °C for 30 minutes, and a yellow solid precipitated out. The mixture was cooled to room temperature, filtered, and dried to obtain intermediate A10-8 hydrochloride (190 mg, 64%). 1 H NMR (400MHz, DMSO-d6) δ14.48(s,1H),11.12(s,1H),9.53(s,1H),9.22(s,1H),8.46(d,J=7.2Hz,1H),8.37(d,J=8.8Hz,1H),8.20(s,1H),7.65(s, 1H),7.59(d,J=8.4Hz,1H),6.81(d,J=7.2Hz,1H),5.27(s,1H),4.03-3.9 3(m,4H),2.45-2.37(m,1H),2.16-2.07(m,1H).LC-MS(m / z):442.0[M+H] + .

[0173] 9. Synthesis of the final product A10

[0174] A10-8 (190 mg, 0.43 mmol) was dissolved in 1,4-dioxane (3 mL), followed by the addition of dimethylphosphine oxide (40 mg, 0.52 mmol), triethylamine (65 mg, 0.65 mmol), tris(dibenzylacetone)dipalladium (37 mg, 0.04 mmol), and 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (23 mg, 0.04 mmol). The mixture was then microwaved at 125 °C for 1 hour under nitrogen protection. After filtration, the pH was adjusted to 7 with saturated sodium bicarbonate solution, and the solution was concentrated and purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 50 / 1) to obtain the final product A10 (45 mg, yield 24%).

[0175] Compounds A14-A15 were synthesized according to the method of Example 6, except that the 3-hydroxytetrahydrofuran in step 3 was replaced with (R)-3-hydroxytetrahydrofuran and (S)-3-hydroxytetrahydrofuran, respectively.

[0176] Example 7

[0177] Compound A11 is synthesized by the following method:

[0178]

[0179] 1. Synthesis of intermediate A11-1

[0180] 4-Nitroaniline (10 g, 72.5 mmol) was dissolved in dichloromethane (60 mL), and NBS (13 g, 72.5 mmol) was added. The mixture was stirred overnight at room temperature. A saturated sodium bicarbonate aqueous solution (50 mL) was added to the reaction solution and stirred for 5 minutes. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness to give a yellow solid product A11-1 (15 g, 96%). LC-MS (m / z): 216.7 [M+H] + .

[0181] 2. Synthesis of intermediate A11-2

[0182] Dissolve A11-1 (7.0 g, 32.3 mmol) in acetic acid (80 mL) and add it dropwise to a concentrated sulfuric acid solution (16 mL) containing sodium nitrite (2.4 g, 34.8 mmol) under ice bath conditions. After stirring for 4 hours, add an aqueous solution (60 mL) containing potassium iodide (16 g, 96.9 mmol) and iodine (3.5 g, 32.3 mmol) and stir overnight at room temperature. Add 200 mL of 15% NaOH aqueous solution to the reaction mixture. Extract the aqueous phase with ethyl acetate (300 mL), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain crude product A11-2 (10.0 g, 95%).

[0183] 3. Synthesis of intermediate A11-3

[0184] Crude A11-2 (10.0 g, 30.7 mmol) was dissolved in ethanol (60 mL), and ammonium chloride (8.2 g, 153 mmol) and iron powder (8.5 g, 153 mmol) were added. The mixture was stirred at 85 °C for 2 h. The mixture was filtered, the filtrate was concentrated, and the residue was subjected to silica gel column chromatography to obtain a yellow solid A11-3 (6.0 g, 65%). 1 H NMR (400MHz, CDCl3) δ8.45 (s, 1H), 8.08 (d, J = 8.8Hz, 1H), 7.85 (d, J = 8.4Hz, 1H), LC-MS (m / z): 297.6 [M+H] + .

[0185] 3. Synthesis of intermediate A11-4

[0186] Add A11-3 (6 g, 20 mmol) to ethanol (30 mL), then add 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (7.4 g mg, 40 mmol), and stir at room temperature for 2 h. Filter, and add the filter cake to diphenyl ether (30 mL), and stir at 220 °C for 3 min. Cool to room temperature, filter, and obtain filter cake A11-4 (4 g). LC-MS (m / z): 349.5 [M+H] + .

[0187] 4. Synthesis of intermediate A11-5

[0188] A11-4 (2 g, 5.75 mmol) was added to phosphorus oxychloride (20 mL), and the reaction was carried out at 110 °C for 2 hours. The reaction solution was concentrated to dryness under reduced pressure, and ethyl acetate (20 mL * 2) was added and evaporated to dryness. 5-Aminobenzothiazole (950 mg, 6.33 mmol) and isopropanol (15 mL) were added to the residue, and the mixture was heated to 95 °C and stirred overnight. The reaction solution was concentrated, and the pH was adjusted to 7-8 by adding saturated sodium bicarbonate aqueous solution (50 mL). The aqueous phase was extracted with ethyl acetate (100 mL), and the organic phase was concentrated. The residue was purified by silica gel column chromatography (dichloromethane:methanol (v / v) = 50:1) to give a yellow solid product A11-5 (950 mg, 35%). 1 H NMR(400MHz,DMSO-d6)δ9.44(s,1H),9.09(s,1H),8.48(d,J=5.6Hz,1H),8.24-8.21(m,2H),8.0 4(d,J=2.0Hz,1H),7.53(dd,J=4.2,2.0Hz,1H),7.01(d,J=5.6Hz,1H).LC-MS(m / z):481.4[M+H] + .

[0189] 5. Synthesis of intermediate A11-6

[0190] A11-5 (800 mg, 1.60 mmol), Pd2(dba)3 (73 mg, 0.08 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (93 mg, 0.16 mmol), dimethylphosphine oxide (187 mg, 2.40 mmol), and triethylamine (323 mg, 3.20 mmol) were added to 1,4-dioxane (10 mL), and the mixture was stirred overnight at 70 °C under nitrogen protection. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane:methanol (v / v) = 50:1) to give a yellow solid product A11-6 (240 mg, 33%). 1H NMR (400MHz, DMSO-d6) δ11.52(s,1H),9.53(s,1H),9.18(d,J=12.8Hz,1H),8.55(d,J=6.8Hz,1H),8.42-8.31(m,2 H),8.19(s,1H),7.59(d,J=8.4Hz,1H),6.92(d,J=6.4Hz,1H),2.00(s,3H),1.96(s,3H).LC-MS(m / z):432.0[M+H] + .

[0191] 6. Synthesis of the final product A11

[0192] A11-6 (60 mg, 0.14 mmol), Pd(dppf)Cl2 (10 mg, 0.014 mmol), 1-methyl-1H-pyrazole-4-ylboronic acid (35 mg, 0.28 mmol), and potassium carbonate (40 mg, 0.28 mmol) were suspended in 1,4-dioxane / water (10 mL / 0.5 mL) and stirred overnight at 100 °C under nitrogen protection. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 10 / 1) to give a blue solid product A11 (10 mg, yield 17%).

[0193] Compounds A12 and A27 were synthesized according to the method of Example 7, except that 1-methyl-1H-pyrazole-4-ylboronic acid in Example 7 was replaced with 1H-pyrazole-3-ylboronic acid and (3,5-dimethylisoxazole-4-yl)boronic acid, respectively.

[0194] Example 8

[0195] Compound A13 is synthesized by the following method:

[0196]

[0197] 1. Synthesis of the final product A13

[0198] A11-6 (50 mg, 0.12 mmol), Pd(dppf)Cl2 (10 mg, 0.014 mmol), tert-butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (90 mg, 0.24 mmol), and potassium carbonate (40 mg, 0.28 mmol) were suspended in 1,4-dioxane / water (10 mL / 0.5 mL) and stirred overnight at 100 °C under nitrogen protection. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane:methanol (v / v) = 10:1) to obtain a yellow solid crude product (50 mg). EtOH (2 mL) was added, followed by dropwise addition of HCl / EA (1.5 M, 2 mL). The mixture was stirred at room temperature for 2 h, and a solid precipitated. After the filter cake was dissolved in MeOH (3 mL), anhydrous diethyl ether (15 mL) was added, and a solid precipitated out. After filtration, a yellow solid product A13 (20 mg, 17%) was obtained, which was a hydrochloride.

[0199] Example 9

[0200] Compound A16 is synthesized by the following method:

[0201]

[0202] 1. Synthesis of intermediate A16-1

[0203] A7-1 (200 mg, 0.77 mmol) and potassium carbonate (211 mg, 1.54 mmol) were dissolved in N,N-dimethylformamide (3 mL), and stirred at 80 °C for 30 minutes. Then, 3-bromooxetine (213 mg, 1.54 mmol) was added, and stirring was continued overnight. After the reaction was complete, the mixture was directly concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 60 / 1) to give intermediate A16-1 (150 mg, 62%). 1 H NMR (400MHz, DMSO-d6) δ8.81(d,J=4.0Hz,1H),8.43(s,1H),7.68(d,J=4.4Hz,1H),7.25( s,1H),5.66-5.56(m,1H),5.11-5.03(m,2H),4.68-4.61(m,2H).LC-MS(m / z):314.0[M+H] + .

[0204] 2. Synthesis of intermediate A16-2

[0205] A16-1 (150 mg, 0.48 mmol) and 5-aminobenzothiazole (78 mg, 0.52 mmol) were dissolved in ethanol (2 mL), and one drop of concentrated hydrochloric acid was added. The mixture was stirred at 80 °C for 5 hours, and a yellow solid precipitated out. After cooling to room temperature, the mixture was filtered and dried to obtain a yellow solid intermediate, A16-2 hydrochloride (170 mg, 83%). 1 H NMR (400MHz, DMSO-d6) δ14.34(s,1H),11.08(s,1H),9.53(s,1H),9.22(s,1H),8.47(d,J=6.4Hz,1H),8.37(d,J=8.4Hz,1H),8.19(s,1H),7 .59(d,J=8.4Hz,1H),7.27(s,1H),6.82(d,J=6.4Hz,1H),5.62-5.52(m,1H),5.10-5.01(m,2H),4.76-4.65(m,2H).LC-MS(m / z):428.0[M+H] + .

[0206] 3. Synthesis of the final product A16

[0207] A16-2 (170 mg, 0.40 mmol) was dissolved in 1,4-dioxane (2 mL), followed by the addition of dimethylphosphine oxide (46 mg, 0.60 mmol), triethylamine (60 mg, 0.60 mmol), tris(dibenzylacetone)dipalladium (36 mg, 0.04 mmol), and 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (23 mg, 0.04 mmol). The mixture was reacted in a microwave oven at 125 °C for 2 hours under nitrogen protection. After filtration, the pH was adjusted to 7 with saturated sodium bicarbonate solution, and the solution was concentrated and purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 15 / 1) to give the final product A16 (30 mg, yield 18%).

[0208] Compound A21 was synthesized according to the method of Example 9, except that 3-bromooxyhexacyclobutane in step 1 of Example 9 was replaced with bromocyclobutane.

[0209] Example 10

[0210] Compound A18 is synthesized by the following method:

[0211]

[0212] 1. Synthesis of intermediate A18-1

[0213] (R)-(-)-glycerol acetal (1.0 g, 7.6 mmol) and carbon tetrabromide (3.2 g, 10.0 mmol) were dissolved sequentially in dichloromethane (50 mL). A dichloromethane solution (30 mL) of triphenylphosphine (2.0 g, 7.6 mmol) was added dropwise using a constant-pressure dropping funnel at room temperature, and the mixture was stirred overnight at room temperature. The solution was concentrated, and petroleum ether (50 mL) and 5% sodium bicarbonate aqueous solution (50 mL) were added. Stirring resulted in the precipitation of a white solid. The solution was filtered, and the filtrate was extracted with ethyl acetate (50 mL). The organic phase was washed with saturated sodium chloride aqueous solution (50 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate (v / v) = 10 / 1) to give a colorless oily intermediate A18-1 (340 mg, 23%).

[0214] 2. Synthesis of intermediate A18-2

[0215] Dissolve A7-1 (150 mg, 0.58 mmol) and potassium carbonate (160 mg, 1.16 mmol) in N,N-dimethylformamide (3 mL), stir at 80 °C for 30 minutes, then add A18-1 (340 mg, 1.75 mmol) and continue stirring overnight. Concentrate directly, and purify the residue by silica gel column chromatography (dichloromethane / methanol (v / v) = 50 / 1) to obtain a yellow solid intermediate A18-2 (130 mg, 20%). 1 H NMR(400MHz, DMSO-d6)δ8.81(d,J=4.8Hz,1H),8.39(s,1H),7.67–7.64(m,2H),4.54–4.48(m,1H),4.39–4.34(m, 1H),4.32–4.27(m,1H),4.17–4.13(m,1H),3.93–3.88(m,1H),1.40(s,3H),1.34(s,3H).LC-MS(m / z):371.9[M+H] + .

[0216] 3. Synthesis of intermediate A18-3

[0217] A18-2 (130 mg, 0.35 mmol) and 5-aminobenzothiazole (58 mg, 0.38 mmol) were dissolved in ethanol (3 mL), and one drop of 0.5 N hydrochloric acid was added. The mixture was stirred at 85 °C for 1.5 hours and concentrated. The residue was dissolved and clarified in a mixed solvent of dichloromethane (20 mL) and methanol (10 mL), and saturated sodium bicarbonate aqueous solution (1 mL) was added. After stirring, the mixture was directly concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 50 / 1) to give a yellow solid intermediate A18-3 (130 mg, 77%). 1H NMR (400MHz, DMSO-d6) δ9.42 (s, 1H), 9.24 (s, 1H), 8.75 (s, 1H), 8.43 (d, J = 5.2Hz, 1H), 8.19(d,J=8.8Hz,1H),8.01(d,J=1.6Hz,1H),7.52(dd,J=8.8Hz,J=2.0Hz,1H),7.43(s, 1H),6.92(d,J=5.6Hz,1H),4.54–4.48(m,1H),4.33–4.28(m,1H),4.27–4.22(m,1H),4. 18–4.13(m,1H),3.94–3.89(m,1H),1.41(s,3H),1.35(s,3H).LC-MS(m / z):486.0[M+H] + .

[0218] 4. Synthesis of intermediate A18-4

[0219] Intermediate A18-3 (130 mg, 0.27 mmol) was dissolved in 1,4-dioxane (2 mL), followed by the sequential addition of dimethylphosphine oxide (31 mg, 0.40 mmol), triethylamine (46 mg, 0.46 mmol), tris(dibenzylacetone)palladium (25 mg, 0.03 mmol), and 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (15 mg, 0.03 mmol). The mixture was reacted in a microwave oven at 125 °C for 2 hours under nitrogen protection. The residue was concentrated and purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 10 / 1) to give intermediate A18-4 (120 mg, 93%) as a yellow solid. 1 H NMR (300MHz, DMSO-d6) δ10.23(s,1H),9.45(s,1H),8.92(d,J=13.8Hz,1H),8.46(d,J=5.7Hz,1H) ,8.23(d,J=5.7Hz,1H),8.07(s,1H),7.54(dd,J=8.1Hz,J=1.2Hz,1H),7.41(d,J=3.9Hz,1H),6.90 (d,J=5.7Hz,1H),4.62–4.55(m,1H),4.38–4.36(m,1H),4.26–4.21(m,1H),4.18–4.12(m,1H),3. 88–3.82(m,1H),1.79(dd,J=13.8,4.2Hz,6H),1.40(s,3H),1.34(s,3H).LC-MS(m / z):484.1[M+H] + .

[0220] 5. Synthesis of the final product A18

[0221] Intermediate A18-4 (120 mg, 0.25 mmol) was dissolved in ethanol (5 mL), and ethyl acetate (3N) was added. The mixture was stirred at room temperature for 2 hours. A solid precipitated out. The solution was concentrated, and the residue was dissolved and clarified in a mixed solvent of dichloromethane (20 mL) and methanol (10 mL). The pH was adjusted to 7 with a saturated sodium bicarbonate aqueous solution, and the solution was directly concentrated. The residue was then... 18 C18 column purification (water / methanol (v / v) = 60 / 40) yielded a yellow solid final product A18 (90 mg, yield 82%).

[0222] Compound A19 was synthesized according to the method of Example 10, except that (R)-2,2-dimethyl-1,3-dioxacyclopentan-4-ylmethanol in step 1 of Example 10 was replaced with (S)-2,2-dimethyl-1,3-dioxacyclopentan-4-ylmethanol.

[0223] Example 11

[0224] Compound A20 is synthesized by the following method:

[0225]

[0226] 1. Synthesis of intermediate A20-1

[0227] 4-Bromo-3-fluoroaniline (500 mg, 2.63 mmol) was dissolved in ethanol (10 mL), and then 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (587 mg, 3.16 mmol) was slowly added. The mixture was stirred at room temperature for 2 hours, and a large amount of solid precipitated out. The mixture was filtered, and the filter cake was washed with methanol (10 mL) and dried to obtain a white solid intermediate A20-1 (820 mg, 91%). 1 H NMR(300MHz,DMSO-d6)δ11.24(d,J=14.1Hz,1H),8.58(d,J=14.4Hz,1H),7.84– 7.68(m,2H),7.42(dd,J=8.7,1.5Hz,1H),1.67(s,6H).LC-MS(m / z):342.0[MH] - .

[0228] 2. Synthesis of intermediate A20-2

[0229] Diphenyl ether (50 mL) was added to a round-bottom flask, heated to 240 °C and stirred for 30 minutes to remove solvent and water. Then, A20-1 (800 mg, 2.32 mmol) was added to the reaction solution, and the mixture was stirred at 240 °C for 3 minutes. After cooling to room temperature, petroleum ether (50 mL) was added, and a white solid precipitated. The precipitate was filtered, and the filter cake was dried to obtain crude white solid intermediate A20-2 (450 mg, 80%). 1 H NMR (300MHz, DMSO-d6) δ11.92(s,1H),8.28(d,J=7.8Hz,1H),7.96(d,J=7.2Hz,1H),7.47(d,J=9.6Hz,1H),6.08(d,J=7.8Hz,1H).LC-MS(m / z):242.0[M+H] + .

[0230] 3. Synthesis of intermediate A20-3

[0231] A20-2 (450 mg, 1.86 mmol) was dissolved in phosphorus oxychloride (3 mL) and stirred at 110 °C for 2 hours. The phosphorus oxychloride was removed by concentration, and the solid residue was dissolved in ethyl acetate (20 mL). The suspension was added dropwise to ice water (60 mL), and the pH was adjusted to 7 with saturated sodium bicarbonate solution. Extraction was performed with ethyl acetate (20 mL), and the organic phase was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate (v / v) = 50 / 1) to give a white solid intermediate A20-3 (274 mg, 57%). 1 H NMR (300MHz, DMSO-d6) δ8.90(d,J=4.8Hz,1H),8.52(d,J=7.2Hz,1H),7.08(d,J=9.6Hz,1H),7.82(d,J=4.8Hz,1H).LC-MS(m / z):260.0[M+H] + .

[0232] 4. Synthesis of intermediate A20-4

[0233] A20-3 (250 mg, 0.96 mmol) and 5-aminobenzothiazole (157 mg, 1.06 mmol) were dissolved in isopropanol (2 mL), and one drop of concentrated hydrochloric acid was added. The mixture was stirred at 95 °C for 1 hour, and a yellow solid precipitated out. The solid was then concentrated. The residue was dissolved in a mixed solvent of methanol (15 mL) and dichloromethane (30 mL), and the pH was adjusted to 7 with saturated sodium bicarbonate aqueous solution. The solution was then directly concentrated to give a white solid intermediate, A20-4 (320 mg, 89%). 1H NMR (300MHz, DMSO-d6) δ9.47–9.39(m,2H),8.92(d,J=7.8Hz,1H),8.49(d,J=5.1Hz,1H),8.21(d,J=8.7Hz,1H ),8.04(s,1H),7.78(d,J=10.2Hz,1H),7.54(d,J=7.8Hz,1H),6.99(d,J=4.8Hz,1H).LC-MS(m / z):373.9[M+H] + .

[0234] 5. Synthesis of final product A20

[0235] A20-4 (320 mg, 0.86 mmol) was dissolved in 1,4-dioxane (2 mL), followed by the sequential addition of dimethylphosphine oxide (100 mg, 1.28 mmol), triethylamine (130 mg, 1.28 mmol), tris(dibenzylacetone)palladium (78 mg, 0.09 mmol), and 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (49 mg, 0.09 mmol). The mixture was reacted in a microwave oven at 125 °C for 2 hours under nitrogen protection. The solution was then directly concentrated and purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 15 / 1) to give a yellow solid final product, A20 (117 mg, yield 37%).

[0236] Example 12

[0237] Compound A23 is synthesized by the following method:

[0238]

[0239] 1. Synthesis of intermediate A23-1

[0240] 2-Amino-4-methoxybenzoic acid (15.0 g, 89.7 mmol) was dissolved in N,N-dimethylformamide (50 mL). N-bromosuccinimide (16.0 g, 89.7 mmol) was added in portions at 0 °C, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete as detected by TLC, the reaction solution was concentrated, and ethyl acetate (200 mL) and petroleum ether (200 mL) were added to dissolve and slurry the mixture. The solid was filtered to obtain a gray solid intermediate A23-1 (18.0 g, 82%). 1 H NMR(400MHz, DMSO-d6)δ7.76(s,1H),6.41(s,1H),3.79(s,3H).LC-MS(m / z):246.0[M+H] + .

[0241] 2. Synthesis of intermediate A23-2

[0242] Nitromethane (21.9 g, 359.2 mmol) was added dropwise to an aqueous solution of sodium hydroxide (53.9 g, 1347.5 mmol) (50 mL) at room temperature. The mixture was then heated at 45 °C for 5 minutes with stirring, and the reaction solution was cooled to room temperature before adding nitromethane (21.9 g, 359.2 mmol) again. The reaction mixture was then stirred for 10 minutes, heated to 50 °C for another 5 minutes, cooled to room temperature, and poured into ice (500 g). The aqueous solution was acidified with concentrated hydrochloric acid to pH < 2 and immediately added to an aqueous suspension of A23-1 (22.0 g, 89.8 mmol) (300 mL), followed by concentrated hydrochloric acid (161.5 mL). The resulting suspension was stirred overnight and filtered to obtain a yellow solid intermediate A23-2 (24.0 g, 85%). 1 H NMR(300MHz,DMSO-d6)δ13.07(d,J=13.5Hz,1H),8.19(dd,J=13.2,6.3Hz,1H),8.0 9(s,1H),7.35(s,1H),6.82(d,J=6.3Hz,1H),3.99(s,3H).LC-MS(m / z):314.9[MH] - .

[0243] 3. Synthesis of intermediate A23-3

[0244] A23-2 (2.0 g, 6.3 mmol) was dissolved in N,N-dimethylformamide (300 mL), and N,N-carbonyldiimidazole (1.5 g, 9.45 mmol) was added. The mixture was heated at 60 °C overnight. After the reaction was completed by TLC monitoring, the reaction solution was concentrated, and acetonitrile (300 mL) was added. After the solid precipitated, it was filtered to obtain a brown solid intermediate A23-3 (1.4 g, 74.2%). 1 H NMR(300MHz,DMSO-d6)δ8.58(s,1H),7.76(s,1H),6.67(s,1H),6.64(s,1H),3.42(s,3H).LC-MS(m / z):299.0[M+H] + .

[0245] 4. Synthesis of intermediate A23-4

[0246] A23-3 (14.0 g, 46.9 mmol) was dissolved in phosphorus oxychloride (100 mL), and N,N-dimethylformamide (2 mL) was added. The mixture was heated at 110 °C overnight. After the reaction was complete, phosphorus oxychloride was removed by rotary evaporation, and ethyl acetate (20 mL × 2) was added and removed again by rotary evaporation. Dichloromethane (100 mL) and water (100 mL) were added to the residue, and the mixture was stirred for half an hour. The mixture was then separated. The aqueous phase was extracted with dichloromethane (200 mL × 2), and the organic phase was collected, dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography (dichloromethane) to give a white solid intermediate A23-4 (3.36 g, 23%). 1 H NMR (300MHz, CDCl3) δ9.25(s,1H),8.64(s,1H),7.51(s,1H),4.11(s,3H).

[0247] 5. Synthesis of intermediate A23-5

[0248] A23-4 (3.4 g, 10.6 mmol) was dissolved in a mixed solvent of isopropanol (100 mL) and water (25 mL), and iron powder (3.0 g, 53.2 mmol) and ammonium chloride (2.8 g, 53.2 mmol) were added. The mixture was reacted at 75 °C for 2 hours. After the reaction was complete, the mixture was filtered through diatomaceous earth, extracted with dichloromethane (100 mL × 3), and water (200 mL) was added. The organic phase was collected, dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography (petroleum ether / ethyl acetate (v / v) = 2 / 1) to give a brown solid intermediate A23-5 (2.5 g, 86%). 1 H NMR(300MHz,DMSO-d6)δ8.54(s,1H),8.04(s,1H),7.41(s,1H),5.96(s,2H),3.94(s,3H).LC-MS(m / z):287.0[M+H] + .

[0249] 6. Synthesis of intermediate A23-6

[0250] A23-5 (2.5 g, 8.74 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL), cooled to -10 °C, and nitroso tetrafluoroborate (1.1 g, 9.42 mmol) was added. The mixture was stirred at 0 °C for 50 minutes and then filtered to give a yellow solid. The solid was dried under infrared light and then added to decahydronaphthalene at 170 °C. After reacting for 5 minutes, heating was stopped. The decahydronaphthalene was removed by rotary evaporation, dissolved in dichloromethane, and purified by silica gel column chromatography (petroleum ether / ethyl acetate (v / v) = 4 / 1) to give a white solid intermediate A23-6 (435 mg, 17%). 1HNMR(300MHz, CDCl3)δ8.76(s,1H),8.39(s,1H),7.46(s,1H),4.05(s,3H).LC-MS(m / z):289.9[M+H] + .

[0251] 7. Synthesis of intermediate A23-7

[0252] Dissolve A23-6 (435 mg, 1.5 mmol) in ethanol (30 mL), add 1,3-benzothiazolamide (225 mg, 1.5 mmol), add 1 drop of concentrated hydrochloric acid, and react at 85 °C for 2 hours. After the reaction is complete, silica gel column chromatography (petroleum ether / ethyl acetate (v / v) = 2 / 1) gives white solid intermediate A23-7 (85 mg, 14%). 1 H NMR (300MHz, DMSO-d6) δ9.35(s,1H),9.24(s,1H),8.77(d,J=3.3Hz,1H),8.63(s,1H),8.04(d,J=8 .7Hz,1H),7.59(s,1H),7.50(s,1H),7.23(d,J=7.5Hz,1H),4.01(s,3H).LC-MS(m / z):403.9[M+H] + .

[0253] 8. Synthesis of final product A23

[0254] A23-7 (90 mg, 0.22 mmol) was dissolved in 1,4-dioxane (3 mL), and dimethylphosphine oxide (19 mg, 0.24 mmol), Pd2(dba)3 (20 mg, 0.02 mmol), Xantphos (13 mg, 0.02 mmol), and triethylamine (45 mg, 0.12 mmol) were added. The mixture was reacted under nitrogen protection in a microwave oven at 125 °C for 2 hours. After the reaction was complete, the mixture was purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 20 / 1). The crude product was then mixed with a mixture of ethyl acetate / diethyl ether (1 mL / 4 mL) and stirred until a yellow solid final product, A23 (14 mg, yield 16%), was obtained by filtration.

[0255] Example 13

[0256] Compound A24 is synthesized by the following method:

[0257]

[0258] 1. Synthesis of intermediate A24-1

[0259] 3-Trifluoromethoxyaniline (1.0 g, 5.6 mmol) was dissolved in N,N-dimethylformamide (3 mL), followed by N-bromosuccinimide (1.0 g, 5.6 mmol) dissolved in N,N-dimethylformamide (2 mL). The solutions were added dropwise to the reaction mixture using a constant pressure dropping funnel. The mixture was reacted at room temperature for 1 hour. Water (10 mL) was then added to the mixture, and the mixture was extracted with ethyl acetate (15 mL × 3). The organic phase was concentrated and purified by silica gel column chromatography (petroleum ether / ethyl acetate (v / v) = 5 / 1) to give a black oily intermediate A24-1 (1.3 g, 91%). 1 H NMR(300MHz, DMSO-d6)δ7.31(d,J=8.4Hz,1H),6.66(s,1H),6.51(d,J=6.9Hz,1H).LC-MS(m / z):255.9[M+H] + .

[0260] 2. Synthesis of intermediate A24-2

[0261] Intermediate A24-1 (0.99 g, 3.9 mmol) was dissolved in ethanol (6 mL), and 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (1.08 g, 5.8 mmol) was added. The mixture was stirred at room temperature for 10 minutes. A solid precipitated out, and the solid was filtered off. The filter cake was dried to obtain a white solid intermediate A24-2 (1.5 g, 94%). 1 H NMR (300MHz, CDCl3) δ11.26(d,J=13.8Hz,1H),8.57(d,J=13.8Hz,1H),7.71(d,J=8 .4Hz,1H),7.19(s,1H),7.10(d,J=8.1Hz,1H),1.77(s,6H).LC-MS(m / z):407.9[MH] - .

[0262] 3. Synthesis of intermediate A24-3

[0263] Diphenyl ether (20 mL) was heated at 240 °C for about 10 minutes, then intermediate A24-2 (1.5 g, 3.7 mol) was added and reacted for 1 minute. No bubbles were generated in the reaction system. The mixture was cooled to room temperature, and petroleum ether (20 mL) was added and stirred. After 10 minutes, the mixture was filtered, and the filter cake was washed with petroleum ether (10 mL) to obtain a pale yellow solid intermediate A24-3 (0.98 g, 86%). 1H NMR (300MHz, DMSO-d6) δ11.97(s,1H),8.35(s,1H),8.02(d,J=7.5Hz,1H),7.70(s,1H),6.13(d,J=7.5Hz,1H).LC-MS(m / z):307.9[M+H] + .

[0264] 4. Synthesis of intermediate A24-4

[0265] Intermediate A24-3 (0.98 g, 3.2 mmol) was dissolved in phosphorus oxychloride (10 mL), and the mixture was stirred at 110 °C for 3 hours. The reaction system was then concentrated, dissolved in ethyl acetate (10 mL), and then added dropwise to ice water (20 mL). The pH was adjusted to 7 with saturated sodium bicarbonate, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were combined, concentrated, and purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 50 / 1) to give intermediate A24-4 ​​(1.01 g, 96%) as a yellow solid. 1 H NMR(300MHz, DMSO-d6)δ8.96(d,J=4.8Hz,1H),8.60(s,1H),8.19(s,1H),7.90(d,J=4.8Hz,1H).LC-MS(m / z):325.9[M+H] + .

[0266] 5. Synthesis of intermediate A24-5

[0267] Intermediate A24-4 ​​(1.01 g, 3.09 mmol) was dissolved in ethanol (10 mL), and 5-aminobenzothiazole (0.47 g, 3.09 mmol) was added. One drop of concentrated hydrochloric acid was added to the reaction system to catalyze the reaction, and the mixture was reacted at 85 °C for 3 hours. The mixture was then cooled to room temperature, and a solid precipitated. The solid was filtered and washed with ethanol. The filter cake was dried to obtain a gray solid intermediate A24-5 (0.96 g, 70%), which was a hydrochloride salt. 1 H NMR (300MHz, DMSO-d6) δ14.95(s,1H),11.49(s,1H),9.54(s,1H),9.50(s,1H),8.59(d,J=6.9Hz,1H),8.38(d, J=8.4Hz,1H),8.29(s,1H),8.21(s,1H),7.61(d,J=8.4Hz,1H),6.94(d,J=6.9Hz,1H).LC-MS(m / z):439.9[M+H] + .

[0268] 6. Synthesis of final product A24

[0269] Intermediate A24-5 (956 mg, 2.00 mmol) was dissolved in 1,4-dioxane (10 mL), followed by the addition of dimethylphosphine oxide (254 mg, 3.26 mmol), triethylamine (373 mg, 3.69 mmol), tris(dibenzylacetone)palladium (199 mg, 0.2 mmol), and 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (126 mg, 0.2 mmol). The mixture was then microwaved at 125 °C for 1 hour under nitrogen protection. The solid precipitated, and the solid was filtered. The filter cake was dissolved in methanol (20 mL), and water (20 mL) was added. The pH was adjusted to 7 with saturated sodium bicarbonate. The mixture was extracted with dichloromethane (20 mL × 2), and the organic phase was concentrated. The mixture was purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 40 / 1) to obtain a pale blue crude product. The crude product was then stirred with ethanol (10 mL) overnight and filtered to obtain a white solid final product A24 (71 mg, yield 8%).

[0270] Compound A25 was synthesized according to the method of Example 13, except that 3-(trifluoromethoxy)aniline in step 1 of Example 13 was replaced with 3-(difluoromethoxy)aniline.

[0271] Example 14

[0272] Compound A26 is synthesized by the following method:

[0273]

[0274] 1. Synthesis of intermediate A26-1

[0275] 3-Pinnacol boronic acid aniline (4.0 g, 18.3 mmol), nickel diiodide (562 mg, 1.8 mmol), and trans-2-aminocyclohexanol acetate (274 mg, 1.8 mmol) were added to a microwave tube, followed by sodium bis(trimethylsilyl)amino (9.15 mL, 18.3 mmol) at 2 mmol / mL and dry isopropanol (20 mL). Under nitrogen protection, the mixture was stirred at room temperature for 10 minutes. 3-Iodoxetane (3.4 g, 18.3 mmol) was added, and the mixture was stirred at 120 °C for 2 hours under microwave irradiation. The reaction was quenched with water, and the organic phase was extracted with dichloromethane (50 mL * 2). The organic phases were combined, dried, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate (v / v) = 4 / 1) to give a yellow oily intermediate A26-1 (1.01 g, 37%). 1H NMR (300MHz, CDCl3) δ7.21–7.09(m,1H),6.81–6.71(m,2H),6.60(d,J=8.7Hz,1H),5.04(dd,J=8 .4,6.0Hz,2H),4.76(t,J=6.3Hz,2H),4.21–4.06(m,1H),3.70(s,2H).LC-MS(m / z):150.1[M+H] + .

[0276] 2. Synthesis of intermediate A26-2

[0277] Intermediate A26-1 (1.01 g, 6.7 mmol) was dissolved in acetonitrile (15 mL), and N-bromosuccinimide (961 mg, 5.4 mmol) in acetonitrile solution (5 mL) was added dropwise under ice bath conditions. The mixture was stirred at this temperature for 30 minutes. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate (v / v) = 4 / 1) to give intermediate A26-2 (913 mg, 60%) as a white solid. 1 H NMR (300MHz, CDCl3) δ7.31–7.24(m,1H),6.78(d,J=2.4Hz,1H),6.47(dd,J=8.7,2.7Hz,1H),5.05(dd ,J=7.8,6.0Hz,2H),4.77(t,J=6.6Hz,2H),4.59–4.45(m,1H),3.77(s,2H).LC-MS(m / z):228.0[M+H] + .

[0278] 3. Synthesis of intermediate A26-3

[0279] Intermediate A26-2 (913 mg, 4.0 mmol) was dissolved in ethanol (8 mL), and 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (893 mg, 4.8 mmol) was added. The mixture was stirred at room temperature for 30 minutes. A solid precipitated out, and the solid was filtered and washed with ethanol. The filter cake was dried to give a pale yellow solid intermediate A26-3 (1.18 g, 77%). 1 H NMR (300MHz, CDCl3) δ11.28(d,J=14.4Hz,1H),8.63(d,J=14.0Hz,1H),7.61(d,J=8.3Hz,1H),7.31–7 .27(m,1H),7.12–7.02(m,1H),5.17–5.07(m,2H),4.84–4.74(m,2H),4.67–4.52(m,1H),1.60(s,6H).

[0280] 4. Synthesis of intermediate A26-4

[0281] Diphenyl ether (30 mL) was added to a round-bottom flask, heated to 240 °C, and stirred for 20 minutes to remove solvent and water. A26-3 (1.18 g, 3.1 mmol) was slowly added, and the mixture was stirred at this temperature for 2 minutes. After cooling to room temperature, a solid precipitated out. The solid was filtered and washed with diethyl ether. The filter cake was dried to obtain a yellow solid intermediate, A26-4 (490 mg, 56%). 1 H NMR (300MHz, DMSO-d6) δ11.80(s,1H),8.19(s,1H),7.96(d,J=6.9Hz,1H),7.64(s,1H),6.07(d,J= 7.5Hz,1H),5.11–4.93(m,2H),4.68(t,J=6.3Hz,2H),4.63–4.47(m,1H).LC-MS(m / z):280.0[M+H] + .

[0282] 5. Synthesis of intermediate A26-5

[0283] Intermediate A26-4 (250 mg, 0.89 mmol) was dissolved in dichloromethane (4 mL), and pyridine (703 mg, 8.9 mmol) was added. Trifluoromethanesulfonic anhydride (1.25 g, 4.45 mmol) was added dropwise at 0 °C, and the mixture was stirred at this temperature for 30 minutes. The reaction solution was concentrated, and the residue was dissolved in 4 mL of dried 1,4-dioxane sieve and transferred to a two-necked flask. 5-Aminobenzothiazole (161 mg, 1.07 mmol), Pd2dba3 (82 mg, 0.09 mmol), Xantphos (52 mg, 0.09 mmol), and cesium carbonate (870 mg, 2.67 mmol) were added. Under nitrogen protection, the mixture was stirred at 100 °C for 10 minutes. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 20 / 1) to give gray solid intermediate A26-5 (130 mg, 35%). 1 H NMR (300MHz, DMSO-d6) δ9.43(s,1H),9.30(s,1H),8.77(s,1H),8.51(s,1H),8.20(d,J=8.1Hz,1H),8.02(s,1H),7.94(s,1 H),7.53(d,J=7.5Hz,1H),7.02(s,1H),5.11–4.96(m,2H),4.90–4.73(m,2H),4.71–4.53(m,1H).LC-MS(m / z):412.0[M+H] +

[0284] 6. Synthesis of the final product A26

[0285] Intermediate A26-5 (120 mg, 0.29 mmol) was dissolved in 1,4-dioxane (4 mL), and dimethylphosphine oxide (45 mg, 0.58 mmol), Pd2dba3 (27 mg, 0.03 mmol), Xantphos (17 mg, 0.03 mmol), and cesium carbonate (284 mg, 0.87 mmol) were added. The mixture was stirred at 130 °C for 2.5 hours under microwave irradiation. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 100 / 9) to obtain a crude product, which was then slurried in a mixed solvent of ethanol / diethyl ether (0.5 mL / 5 mL) to obtain a pale yellow solid final product A26 (20 mg, yield 17%).

[0286] Example 15

[0287] Compound A28 is synthesized by the following method:

[0288]

[0289] 1. Synthesis of intermediate A28-1

[0290] 5-Aminobenzothiazole (3.0 g, 20 mmol) was dissolved in acetonitrile (80 mL). A solution (20 mL) of 1-chloromethyl-4-fluoro-1,4-diazobicyclo2,2,2-octanebis(tetrafluoroborate) salt (7.0 g, 20 mmol) in acetonitrile was slowly added at 0 °C, and the mixture was stirred for 1 hour at this temperature. Water (200 mL) was added to the reaction solution, and the organic phase was extracted with dichloromethane (100 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 4 / 1) to give a yellow solid intermediate A28-1 (310 mg, 9.2%). 1 H NMR (300MHz, CDCl3) δ8.92 (s, 1H), 7.48 (d, J = 8.4Hz, 1H), 6.97 (t, J = 8.1Hz, 1H), 3.87 (s, 2H). LC-MS (m / z): 169.0 [M+H] + .

[0291] 2. Synthesis of intermediate A28-2

[0292] Intermediate A28-1 (37 mg, 0.22 mmol) was dissolved in ethanol (2 mL), and A6-3 (60 mg, 0.22 mmol) was added, followed by one drop of concentrated hydrochloric acid. The mixture was stirred at 85 °C for 4 hours. After cooling to room temperature, a solid precipitated out. The solid was filtered and washed with ethanol. The dried filter cake yielded a white solid intermediate A28-2 (68 mg, 73%). 1 H NMR(300MHz,DMSO-d6)δ14.01(s,1H),10.77(s,1H),9.55(s,1H),9.11(s,1H),8.55–8.43(m,1H),8.25 –8.14(m,1H),7.72–7.57(m,1H),7.48(s,1H),6.69–6.47(m,1H),4.08(s,3H).LC-MS(m / z):404.0[M+H] + .

[0293] 3. Synthesis of the final product A28

[0294] Intermediate A28-2 (68 mg, 0.18 mmol) was dissolved in 1,4-dioxane (2 mL), and dimethylphosphine oxide (21 mg, 0.27 mmol), Pd2dba3 (18 mg, 0.02 mmol), Xantphos (12 mg, 0.02 mmol), and triethylamine (36 mg, 0.36 mmol) were added. Under nitrogen protection, the mixture was stirred at 130 °C for 2 hours under microwave irradiation. Hydrochloric acid was neutralized with saturated sodium bicarbonate aqueous solution, and the organic phase was extracted with dichloromethane (10 mL * 3). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 20 / 1) to give a white solid final product (9 mg, yield 13%).

[0295] Example 16

[0296] Compound A29 is synthesized by the following method:

[0297]

[0298] 1. Synthesis of intermediate A29-1

[0299] Dissolve A16-1 (160 mg, 0.51 mmol) and A28-1 (86 mg, 0.51 mmol) in ethanol (4 mL). Add 1 drop of concentrated hydrochloric acid to ethanol (1 mL), and then add 1 drop of the solution to the reaction mixture. Stir the mixture overnight at 85 °C. Adjust the pH to 7 with saturated sodium bicarbonate solution. Extract the organic phase with dichloromethane (20 mL * 2). Combine the organic phases, dry with anhydrous sodium sulfate, concentrate the reaction solution, and purify the residue by silica gel column chromatography (dichloromethane / methanol (v / v) = 20 / 1) to obtain a pale yellow solid intermediate A29-1 (220 mg, 97%). 1 H NMR(300MHz,DMSO-d6)δ9.48(s,1H),9.15(s,1H),8.83(s,1H),8.48–8.28(m,1H),8.16–7.98(m,1H),7.68–7.44(m ,1H),7.01(s,1H),6.35(s,1H),5.69–5.45(m,1H),5.19–4.96(m,2H),4.77–4.53(m,2H).LC-MS(m / z):445.9[M+H] + .

[0300] 2. Synthesis of final product A29

[0301] Intermediate A29-1 (220 mg, 0.49 mmol) was dissolved in 1,4-dioxane (4 mL), and dimethylphosphorus oxide (76 mg, 0.98 mmol), Pd2dba3 (46 mg, 0.05 mmol), Xantphos (29 mg, 0.05 mmol), and cesium carbonate (319 mg, 0.98 mmol) were added. The mixture was stirred overnight at 110 °C. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 15 / 1) to give a crude product, which was then slurried with diethyl ether (10 mL) to give a white solid final product A29 (109 mg, 50% yield).

[0302] Table 1. Analytical structures and spectroscopic data of compounds A1-A29

[0303]

[0304]

[0305]

[0306]

[0307] Example 17

[0308] Compound B1 is synthesized by the following method:

[0309]

[0310] 1. Synthesis of intermediate B1-1

[0311] 2-Chloro-5-bromonicotinic acid (3 g, 12.7 mmol) and CDI (3.1 g, 19.1 mmol) were dissolved in N,N-dimethylformamide (30 mL). After stirring at room temperature for 1 hour, N,O-dimethylhydroxylamine hydrochloride (1.5 g, 15.3 mmol) and triethylamine (1.9 g, 19.1 mmol) were added, and stirring was continued at room temperature for 5 hours. After the reaction was complete, ethyl acetate (100 mL) was added, and the mixture was washed with water using saturated sodium bicarbonate solution (400 mL x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated. Purification by silica gel column chromatography (petroleum ether / ethyl acetate (v / v) = 2 / 1) yielded a yellow solid intermediate B1-1 (3.0 g, 85%). 1 H NMR(400MHz, CDCl3): δ8.50(s,1H),7.80(s,1H),3.52(s,3H),3.39(s,3H).LC-MS(m / z):279.0[M+H] + .

[0312] 2. Synthesis of intermediate B1-2

[0313] Intermediate B1-1 (2.6 g, 9.3 mmol) was dissolved in tetrahydrofuran (30 mL), and 3M methylmagnesium bromide tetrahydrofuran solution (3.4 mL, 10.2 mmol) was added dropwise at 0 °C, and the reaction was allowed to proceed at room temperature for 2 hours. The reaction was quenched by adding saturated ammonium chloride solution (10 mL), followed by the addition of water (200 mL) and extraction with ethyl acetate (200 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. The solution was purified by silica gel column chromatography (petroleum ether / ethyl acetate (v / v) = 15 / 1) to give colorless oily intermediate B1-2 (2.0 g, 92%). 1 H NMR(400MHz, CDCl3)δ8.55(s,1H),8.02(s,1H),2.70(s,3H).LC-MS(m / z):234.0[M+H] + .

[0314] 3. Synthesis of intermediate B1-3

[0315] Intermediate B1-2 (2.00 g, 8.55 mmol) was dissolved in N,N-dimethylformamide-dimethyl acetal (10 mL) and stirred at 110 °C for 3 hours. The reaction mixture was evaporated to dryness and then dissolved in N,N-dimethylacetamide (10 mL). p-Methoxybenzylamine (1.76 g, 12.82 mmol) and cesium carbonate (5.57 g, 17.09 mmol) were added, and the mixture was reacted overnight at 100 °C. Ethyl acetate (100 mL) was added, and the mixture was washed with water using saturated sodium chloride solution (400 mL * 3). The organic phase was dried over anhydrous sodium sulfate and concentrated. The solution was purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 120 / 1) to give intermediate B1-3 (700 mg, 24%) as a yellow solid. 1 H NMR (400MHz, CDCl3) δ8.81(d,J=2.4Hz,1H),8.75(d,J=2.4Hz,1H),7.72(d,J=8.0Hz,1H),7.23-7.20 (m,2H),6.87-6.85(m,2H),6.33(d,J=7.6Hz,1H),5.48(s,2H),3.78(s,3H).LC-MS(m / z):345.1[M+H] + .

[0316] 4. Synthesis of intermediate B1-4

[0317] Intermediate B1-3 (700 mg, 2.03 mmol) was dissolved in trifluoroacetic acid (2 mL) and reacted in a microwave oven at 110 °C for 2 hours. The reaction solution was evaporated to dryness, and the pH was adjusted to 7 with saturated sodium bicarbonate solution. Water (100 mL) was added, and the mixture was extracted with dichloromethane (50 mL) and methanol (10 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. Column chromatography (dichloromethane / methanol (v / v) = 50 / 1) yielded a yellow solid intermediate B1-4 (320 mg, 70%). 1 H NMR (400MHz, DMSO-d6) δ12.37(s,1H),8.85(s,1H),8.53(s,1H),8.02-7.96(m,1H),6.15(d,J=6.8Hz,1H).LC-MS(m / z):224.9[M+H] + .

[0318] 5. Synthesis of intermediate B1-5

[0319] B1-4 (300 mg, 1.33 mmol) was dissolved in phosphorus oxychloride (4 mL) and stirred at 110 °C for 1 hour. The solution was concentrated to obtain crude B1-5 (350 mg), a brown solid intermediate.

[0320] 6. Synthesis of intermediate B1-6

[0321] B1-5 (350 mg, 1.44 mmol) and 5-aminobenzothiazole (237 mg, 1.58 mmol) were dissolved in ethanol (3 mL), and one drop of concentrated hydrochloric acid was added. The mixture was stirred at 85 °C for 3 hours, resulting in the precipitation of a yellow solid. Water (30 mL) was added, and the pH was adjusted to 7 with a saturated sodium bicarbonate solution. The mixture was extracted with dichloromethane and methanol (30 mL / 10 mL * 2). The organic phase was dried over anhydrous sodium sulfate and concentrated. Column chromatography yielded a yellow solid intermediate B1-6 (105 mg, yield 20%) (dichloromethane / methanol (v / v) = 30 / 1). 1 HNMR(400MHz,DMSO-d6)δ11.78(s,1H),9.81(s,1H),9.55(s,1H),9.28(s,1H),8.55(d,J=5.6Hz,1H),8. 40(d,J=8.0Hz,1H),8.22(s,1H),7.62(d,J=8.4Hz,1H),6.96(d,J=6.4Hz,1H).LC-MS(m / z):357.0[M+H] + .

[0322] 7. Synthesis of final product B1

[0323] B1-6 (105 mg, 0.29 mmol) was dissolved in 1,4-dioxane (2 mL), followed by the sequential addition of dimethylphosphine oxide (34 mg, 0.44 mmol), triethylamine (45 mg, 0.44 mmol), tris(dibenzylacetone)dipalladium (27 mg, 0.03 mmol), and 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (17 mg, 0.03 mmol). The reaction mixture was microwaved at 125 °C for 2 hours under nitrogen protection. A saturated sodium bicarbonate aqueous solution was added to the reaction mixture, and the mixture was stirred at room temperature for 30 minutes. The solution was concentrated and purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 20 / 1) to obtain the final product B1 (10 mg, 10% yield).

[0324] Example 18

[0325] Compound B2 is synthesized by the following method:

[0326]

[0327] 1. Synthesis of intermediate B2-1

[0328] 2-Amino-4-methoxybenzonitrile (2.0 g, 13.5 mmol) was dissolved in acetonitrile (30 mL), and N-bromosuccinimide (2.7 g, 14.9 mmol) was dissolved in acetonitrile (10 mL) to form a suspension. The suspension was added dropwise to the reaction solution at 0 °C, and the mixture was allowed to return to room temperature. The mixture was stirred for 30 minutes, concentrated, and purified by silica gel column chromatography (petroleum ether / ethyl acetate (v / v) = 10 / 1) to give a pale yellow intermediate B2-1 (2.5 g, 82%). 1 H NMR(400MHz, CDCl3)δ7.52(s,1H),6.23(s,1H),4.48(s,2H),3.89(s,3H).LC-MS(m / z):228.0[M+H] + .

[0329] 2. Synthesis of intermediate B2-2

[0330] In a nitrogen-protected two-necked flask, 3M methylmagnesium bromide (20.5 mL, 61.5 mmol) and B2-1 (2.0 g, 8.8 mmol) dissolved in tetrahydrofuran (100 mL) were added dropwise to the reaction mixture under ice bath conditions. The mixture was brought to room temperature and stirred for 5 minutes, followed by stirring at 55 °C for 16 hours. After cooling to room temperature, 6N hydrochloric acid aqueous solution (20 mL) was slowly added to the reaction mixture, and the mixture was stirred for another 30 minutes. The hydrochloric acid was neutralized by adding saturated sodium bicarbonate aqueous solution, and the organic phase was extracted with dichloromethane (50 mL * 3). The extracts were combined and dried, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate (v / v) = 5 / 1) to obtain a pale gray intermediate B2-2 (700 mg, 33%). 1 H NMR(400MHz, CDCl3)δ7.83(s,1H),6.45(s,2H),6.08(s,1H),3.87(s,3H),2.51(s,3H).LC-MS(m / z):244.0[M+H] + .

[0331] 3. Synthesis of intermediate B2-3

[0332] Under ice bath conditions, B2-2 (700 mg) was dissolved in concentrated hydrochloric acid (10 mL), and the mixture was stirred at this temperature for 15 minutes to form a suspension. Sodium nitrite (221 mg, 3.2 mmol) was dissolved in water (1 mL) and added dropwise to the reaction solution. The mixture was stirred under ice bath conditions for 1.5 hours, then stirred overnight at room temperature, followed by reflux and stirring for 6 hours. After cooling to room temperature, the hydrochloric acid was neutralized with saturated sodium bicarbonate solution, and the organic phase was extracted with dichloromethane / methanol (5 / 1, 40 mL * 5). The extracts were combined and dried, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1) to give a white solid intermediate B2-3 (350 mg, 69%). 1H NMR(400MHz,DMSO-d6)δ13.43(s,1H),8.15(s,1H),7.73(s,1H),7.01(s,1H),3.98(s,3H).LC-MS(m / z):255.0[M+H] + .

[0333] 3. Synthesis of intermediate B2-4

[0334] Intermediate B2-3 (350 mg, 1.4 mmol) was dissolved in phosphorus oxychloride (4 mL). The mixture was stirred at 110 °C for 2 hours, concentrated, and ethyl acetate was added to form a suspension. The mixture was further concentrated, and the residue was dissolved in ethanol (5 mL). 5-Aminobenzothiazole (225 mg, 1.5 mmol) was added, and the mixture was stirred at 80 °C for 2 hours. After cooling to room temperature, a solid precipitated out. The solid was filtered and washed with ethanol, and dried to obtain a yellow solid intermediate B2-4 (290 mg, 54%), which was a hydrochloride salt. 1 H NMR (400MHz, DMSO-d6) δ15.79(s,1H),12.18(s,1H),9.54(s,1H),9.37(d,J=10.4Hz,1H),8.54(s,1H),8.39(d, J=8.0Hz,1H),8.30(s,1H),7.68(d,J=8.4Hz,1H),7.54(d,J=6.4Hz,1H),4.11(s,3H).LC-MS(m / z):386.9[M+H] + .

[0335] 4. Synthesis of final product B2

[0336] Intermediate B2-4 (130 mg, 0.34 mmol), dimethylphosphine oxide (52 mg, 0.68 mmol), Pd2(dba)3 (31 mg, 0.03 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (17 mg), and triethylamine (103 mg, 1.02 mmol) were dissolved in 1,4-dioxane (4 mL). Under nitrogen protection, the mixture was stirred at 120 °C for 4 hours under microwave irradiation. After cooling to room temperature, saturated sodium bicarbonate aqueous solution (20 mL) was added to the reaction solution, and the mixture was stirred at room temperature for 30 minutes. The organic phase was extracted with dichloromethane (10 mL * 3), the samples were combined, dried, and concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol (v / v) = 20 / 1) to obtain a pale yellow solid. The solid was slurried with ethanol / ether (1 / 5, 5 mL), dissolved in water (4 mL), and freeze-dried to obtain the yellow solid final product B2 (25 mg, yield 19%).

[0337] Table 2. Analytical structures and spectral data of compounds B1 and B2

[0338]

[0339] Example 19: Competitive Binding Experiment

[0340] In this embodiment, the binding affinity of compounds A1-A16, A20-A21, A24-A26 and B1 prepared in Examples 1-18 to RIP2 kinase was tested.

[0341] In the experiments, kinase-labeled T7 phage strains were prepared from E. coli hosts derived from strain BL21. E. coli were cultured to the logarithmic phase, infected with T7 phage, and cultured at 32°C with shaking until lysis. The lysate was centrifuged and filtered to remove cell debris; the remaining kinases were produced in HEK-293 cells and subsequently detected by qPCR using DNA labeling. Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands at room temperature for 30 minutes to generate affinity resins for kinase analysis. The ligand beads were blocked with excess biotin, and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, and 1 mM DTT) to remove unbound ligands and reduce nonspecific binding. The binding reaction was assembled by binding the kinase, ligand affinity beads, and testing the compound in 1x binding buffer (20% SeaBlock, 0.17x PBS, 0.05% Tween 20, 6 mM DTT). The test compounds were prepared as stock solutions in 100% DMSO at 111× concentration points. Kds were determined using a 3-fold compound dilution series at 11 concentration points and 3 DMSO control points. All compounds used for Kd measurements were distributed in 100% DMSO via acoustic transfer (non-contact distribution). The compounds were then directly diluted to the analytes to a final concentration of 0.9%. All reactions were performed on 384-well polypropylene plates in 0.02 mL volumes. The plates were incubated with shaking at room temperature for 1 hour, and the affinity beads were washed with wash buffer (1x PBS, 0.05% Tween 20). The affinity beads were then resuspended in elution buffer (1x PBS, 0.05% Tween 20, 0.5 μM non-biotinylated affinity ligand) and incubated with shaking at room temperature for 30 minutes. The kinase concentration in the elution buffer was determined by qPCR.

[0342] The standard dose-response curves were calculated using the Hill equation based on the binding constant (Kds). The binding affinity tests of compounds A1-A16, A20-A21, A24-A26, and B1 for RIP2 are shown in Table 3. The test curve for compound A6 is shown in the figure below. Figure 1 As shown.

[0343]

[0344] Table 3. Results of the test on the binding ability of the compounds to RIP2

[0345] A1 28 A2 89 A3 430 A4 89 A5 120 A6 3.1 A7 5.8 A8 2.0 A9 2.1 A10 1.8 A11 3 A12 2.3 A13 1.4 A14 3.0 A15 3.0 A16 3.8 A20 8.7 A21 1.5 A24 5.2 A25 6.4 A26 12 B1 3200

[0346] Table 3 shows that compounds A1-A16, A20-A21, and A24-A26 have a strong affinity for RIP2 kinase, while compound B1 has a weak affinity for RIP2 kinase.

[0347] Example 20: THP-1 cell experiment

[0348] Experimental steps:

[0349] The cells used were the acute monocytic leukemia cell line THP-1 (ATCC, catalog number TIB-202). TM The sterile incubator temperature is 37–38℃, the osmotic pressure is 260–320 mmol / L, the pH range is 7.2–7.4, and the carbon dioxide gas content is 5%.

[0350] THP-1 cells (1x10) 5 First, the cells were seeded in 96-well plates. One hour later, THP-1 cells were pre-incubated with the compound for 2 hours (the initial compound concentration was 20 mM, serially diluted with DMSO to concentrations of 0, 1, 3, and 10 μM; 1.1 μL of the compound was added to 100 μL of PBS, vortexed, and then added to each well at a concentration of 10 μL). Two hours later, 10 μL of MDP (final concentration 3 μg / μL) was added to each well. Six hours later, the cells were centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected. The IL-8 content in the supernatant was detected using an IL-8 ELISA kit (Lianke Biotechnology, catalog number EK108-96).

[0351] Table 4 shows the inhibitory effects of the compounds on interleukin-8.

[0352] A6 50 A7 25 A8 51 A9 55 A10 51 A11 37 A17 11 A18 12 A19 13 A22 60 A27 37 A28 63 A29 85 B2 0

[0353] Table 4 shows that compounds A6-A11, A17-A19, A22, and A27-29 can inhibit the release of interleukin-8 induced by MDP in THP-1 cells, while B2 has no significant inhibitory effect.

[0354] Example 21: Thermodynamic Solubility Test

[0355] Experimental steps:

[0356] 1. Add the compound to FaSSIF (pH 6.5) buffer to form a 4 mg / mL solution.

[0357] 2. Shake the sample tube for 1 hour, then allow it to equilibrate overnight at room temperature.

[0358] 3. Centrifuge the sample (10 min / 12000 rpm) to remove any undissolved precipitate.

[0359] 4. Transfer the supernatant to a new test tube.

[0360] 5. The peak area of ​​the supernatant after centrifugation was determined by LC / MS / MS.

[0361] 6. Measure the peak area of ​​the compound at concentrations of 20 nM, 200 nM, 800 nM, 4000 nM, 10000 nM, and 20000 nM, plot a standard curve, and obtain the solubility by linear regression based on the peak area obtained above.

[0362] Table 5. Results of thermodynamic solubility tests of compounds

[0363] A6 6115μM A7 5310μM A8 1038μM A9 283μM A13 4325μM

[0364] Table 5 shows that compounds A6-A9 and A13 have good thermodynamic solubility, with solubility exceeding 200 μM.

[0365] Example 22: Compound CYP Enzyme Inhibition Test

[0366] Experimental steps:

[0367] 1. Preheat 0.1M potassium phosphate buffer (K ​​buffer) to pH 7.4.

[0368] 2. Prepare the test compound and reference inhibitor (400×) in a 96-well plate:

[0369] 2.1. Transfer 8 μL of the 10 mM analyte to 12 μL of acetonitrile;

[0370] 2.2. Preparation of CYP1A2, CYP2C9, CYP2D6 cocktail inhibitor plus standard sample solution: 12μL 1mM α-naphthylflavonoid + 10μL 40mM sulfadiazine + 10μL 10mM quinidine + 8μL DMSO.

[0371] 2.3. Preparation of single inhibitor and standard sample solutions for CYP3A4 and CYP2C19: 8 μL of DMSO sample stock solution was added to 12 μL of acetonitrile; 4×NADPH cofactor solution was prepared (66.7 mg NADPH was added to 0.1 M potassium ion buffer, pH 7.4).

[0372] 4. Prepare 4× substrate (2 mL for each isomer).

[0373] 5. Prepare a 0.2 mg / mL human liver microsomal solution on ice (add 10 μL of liver microsomal solution (20 mg / mL) to 990 μL of K-buffer (0.1 M)).

[0374] 6. Add 400 μL of 0.2 mg / kg human microsomal solution to the detection well, and then add 2 μL of 400× analyte compound to the corresponding well.

[0375] 7. Add 200 μL of 0.2 mg / kg human microsome solution to a 96-well plate and then add 1 μL of reference inhibitor solution.

[0376] 8. Add the following solutions (in duplicate) to a 96-well plate.

[0377] 8.1. Add 30 μL of the 2× test compound and the reference compound to a 0.2 mg / mL human liver microsome solution;

[0378] 8.2. Add 15 μL of base solution.

[0379] 9. Incubate the 96-well plate with NADPH solution at 37°C for 5 minutes.

[0380] 10. Add 15 μL of preheated 8 mM NADPH solution to start the reaction.

[0381] 11.37℃ incubation test plates: 3A4 5 minutes; 1A2, 2B6, 2C8, 2C9, 2D6 10 minutes; 2C19 45 minutes.

[0382] 12. The reaction was terminated by adding 120 μL of acetonitrile solution containing IS.

[0383] 13. After cooling, vibrate on a shaker (IKA, MTS 2 / 4) for 10 minutes (600 rpm / min), then centrifuge at 5594 g for 15 minutes (reconstituted solution × 3R).

[0384] 14. Transfer 50 μL of supernatant from each well to a 96-well sample plate containing 50 μL of ultrapure water (Millipore, ZMQS50F01) for LC / MS / MS analysis.

[0385] Table 6. CYP450 Inhibition Experiment

[0386]

[0387] Table 7. CYP test results of compounds

[0388]

[0389] As shown in Table 7, compounds A6, A14-A16 of this invention do not have significant CYP inhibitory activity. Their inhibition rates against the tested CYP isoenzymes 1A2, 2C9, 2C19, 2D6, and CYP3A4 are all below 20%, indicating weak drug-drug interactions.

[0390] Example 23: Test of compound Caco-2

[0391] Experimental steps

[0392] Preheat HBSS buffer at 1.37℃.

[0393] 2. Take the compound from -20℃ and sonicate it for a few minutes (no less than 1 minute).

[0394] 3. Solvent preparation

[0395] 3.1. Donor buffer solution:

[0396] AB direction:

[0397] To prepare HBSS buffer with 0.3% DMSO and 5 μM LY (Lucifer Yellow): Add 150 μL DMSO and 50 μL LY (5 mM) to 50 mL HBSS buffer (pH 7.4).

[0398] HBSS buffer with 0.1% DMSO and 5 μM LY: Add 50 μL DMSO and 50 μL LY (5 mM) to 50 mL HBSS buffer (pH 7.4).

[0399] BA direction:

[0400] HBSS buffer with 0.3% DMSO: Add 150 μL of DMSO to 50 mL of HBSS buffer (pH 7.4).

[0401] HBSS buffer with 0.1% DMSO: Add 50 μL of DMSO to 50 mL of HBSS buffer (pH 7.4).

[0402] 3.2. Receptor buffer solution:

[0403] AB direction: Prepare HBSS buffer by adding 200 μL of 0.4% DMSO solution to 50 mL of HBS buffer (pH 7.4).

[0404] BA direction: Prepare HBSS buffer with 0.4% DMSO and 5uM LY: Add 200uL DMSO and 50uL LY (5mM) to 50mL HBSS buffer.

[0405] Table 8. Preparation of donor solution

[0406]

[0407] 4. Calculate TEER: Remove the cell culture plate from the incubator, wash the cell monolayer with HBSS buffer, and then measure the TEER value at Rm (room temperature).

[0408] 5. Centrifugation: Centrifuge (4000 rpm) for 5 min before adding the compound solution to the donor chamber.

[0409] 6. Administration: Add the solution according to the volumes listed in the table below (ensure that an additional 100 μL of donor sample can be prepared as T0).

[0410] Table 9. Volume of solution added

[0411]

[0412] 7. Top LY T0 Sample: Determine the LY concentration in the top chamber, and take 100 μL of sample from the top chamber onto an opaque plate as the LY concentration. T0 .

[0413] 8. Preheating: Preheat the top plate and base side plate at 37°C for about 5 minutes, then place the top plate on the base side plate to begin transport.

[0414] 9. Incubation: Keep the plate incubated at 37°C for 90 minutes.

[0415] 10. Prepare the standard curve: Prepare a 300 μM (20×) compound solution: Add 6 μL of the original sample solution to 194 μL of methanol / water (1:1) solution.

[0416] Table 10. Preparation of experimental solutions using methanol:water (1:1)

[0417] 300 100 400 → 60 60 100 200 → 20 20 100 400 → 4 4 100 400 → 0.8 0.8 100 300 → 0.2 0.2 100 100 → 0.1

[0418] Prepare 1× solution: 3 μL (20×) + 57 μL of 0.4% DMSO HBSS + 60 μL of acetonitrile and IS (Osamamethoxam or Imipramine) --- 120 μL (1×).

[0419] 11. Termination of transfer: After incubation for 90 minutes, separate the top plate from the base plate.

[0420] 12. Measure LY: Take 100 μL of sample from the base plate into the opaque plate.

[0421] 13. Measure LY using a fluorometer T0 -LY T90 The concentration.

[0422] 14. Dilute the donor or recipient sample with 0.4% DMSO HBSS and then mix with acetonitrile (Osamamethoxam or Imipramine) containing IS.

[0423] Table 11. Test results for compound Caco-2

[0424]

[0425] a: The test concentration was 2μM

[0426] Table 11 shows that compound A24 has good permeability and no obvious efflux. Compounds A6, A8, A9, A14, and A15 also have good permeability, but some efflux. Compounds A7, A13, and A16 have weak permeability. It can be seen that even a small change in the substituents of a compound can significantly alter its permeability and may even cause efflux.

[0427] Example 24: Test of plasma protein binding rate of compounds

[0428] Experimental steps:

[0429] Plasma protein binding rates in rats, dogs, and humans were assessed using a balanced dialysis method via Chemparter. In the experiment, the analyte was added to plasma to a concentration of 1 μM. The spiked plasma was placed on the donor side, and blank PBS buffer was placed on the receiver side. The dialysis block was capped and incubated on a shaker (60 rpm) at 37°C for 5 hours. Donors and recipients were collected after incubation. The plasma protein binding rate was calculated using the following formula:

[0430]

[0431] Table 12. Results of plasma protein binding rate test for compounds

[0432]

[0433] Table 12 shows that compounds A6, A14-A16, and A25 have moderate plasma protein binding rates in humans and dogs, while A24 has a high plasma protein binding rate in humans, rats, and dogs, and A25 has a high plasma protein binding rate in rats.

[0434] Example 25: Compound metabolic stability test

[0435] Experimental steps:

[0436] 1. Buffer preparation

[0437] Buffer A: 1.0L of 0.1M monobasic potassium phosphate buffer containing 1.0mM EDTA.

[0438] Buffer B: 1.0L of 0.1M dipotassium hydrogen phosphate buffer containing 1.0mM EDTA.

[0439] Buffer C: A 0.1 M potassium phosphate buffer, 1.0 mM EDTA, pH 7.4 was obtained by titrating 700 mL of buffer B with buffer A while monitoring with a pH meter.

[0440] 2. Prepare spiking solutions for the reference compound (Ketanserin) and the test compound.

[0441] 500 μM spiked solution: Add 10 μL of 10 mM DMSO stock solution to 190 μL of acetonitrile.

[0442] 1.5 μM spiked solution (0.75 mg / mL) in microsomes: Add 1.5 μL of 500 μM spiked solution and 18.75 μL of 20 mg / mL liver microsomes to 479.75 μL of buffer C.

[0443] 3. Prepare an NADPH stock solution (6 mM) by dissolving NADPH in buffer C.

[0444] 4. Dispense 30 μL of 1.5 μM spiked solution containing 0.75 mg / mL microsome solution onto the assay plates designated for different time points (0, 5, 15, 30, 45 minutes).

[0445] 5. For 0 minutes, add 135 μL of IS-containing ACN to the wells of the 0-minute plate, followed by 15 μL of NADPH stock solution (6 mM).

[0446] 6. Pre-incubate all other plates at 37°C for 5 minutes.

[0447] 7. Add 15 μL of NADPH stock solution (6 mM) to the plate to start the reaction and start timing.

[0448] 8. At 5 minutes, 15 minutes, 30 minutes and 45 minutes, add 135 μL of IS-containing ACN to the wells of the corresponding plates to terminate the reaction.

[0449] 9. After quenching, shake the plate on a vibrator (IKA, MTS 2 / 4) for 10 minutes (600 rpm / min), and then centrifuge at 5594 g for 15 minutes (Thermo Multifuge×3R).

[0450] 10. Transfer 50 μL of supernatant from each well to a 96-well sample plate containing 50 μL of ultrapure water (Millipore, ZMQS50F01) for LC / MS / MS analysis.

[0451] Table 13. Results of Compound Metabolic Stability Tests

[0452]

[0453] As shown in Table 13, compounds A6-A9 and A13-A16 of the present invention exhibit good metabolic stability in human, rat, and canine liver microsomes, with half-lives greater than 38 minutes in all three species.

[0454] Example 26: Pharmacokinetic Evaluation of Compounds

[0455] Experimental Objective 1: To test the pharmacokinetics of the compound in ICR mice.

[0456] Experimental steps:

[0457] The pharmacokinetic characteristics of the compounds after intravenous and oral administration were tested in rodents using a standard protocol. In the experiment, candidate compounds were prepared into clear solutions and administered to mice via single intravenous injection, while suspensions were used for oral administration. The intravenous solvent was 5% DMSO + 95% saline, and the oral solvent was 0.5% CMCNa. This project used 48 male ICR mice. 24 mice underwent intravenous administration at a dose of 2 mg / kg, and plasma samples were collected at 0 h (before administration) and at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h post-administration. The other 24 mice underwent oral gavage administration at a dose of 10 mg / kg, and plasma samples were collected at 0 h (before administration) and at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h post-administration. The collected samples were then analyzed by LC / MS / MS, and data were acquired. AUC represents the area under the plasma concentration-time curve from time zero to 24 hours. Cmax represents peak concentration; Tmax represents time to peak; T 1 / 2 The values ​​represent half-life; Cl represents clearance rate; Vd represents apparent volume of distribution; F represents bioavailability; po represents oral administration; iv represents intravenous administration.

[0458] Table 14. Results of mouse pharmacokinetic assays for the compounds

[0459]

[0460] Table 14 shows that compounds A6, A24, and A25 have excellent plasma exposure and bioavailability in mice.

[0461] Experimental Objective 2: To test the pharmacokinetics of the compound in SD rats.

[0462] Experimental steps:

[0463] The pharmacokinetic characteristics of the compounds after intravenous and oral administration were tested in rodents using a standard protocol. In the experiment, candidate compounds were prepared into clear solutions and administered to rats via single intravenous injection, while suspensions were used for oral administration. The intravenous solvent was 5% DMSO + 95% saline, and the oral solvent was 0.5% CMCNa. This project used nine male SD rats. Three rats received intravenous administration at a dose of 2 mg / kg, and plasma samples were collected at 0 h (before administration) and at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h post-administration. Three rats received oral gavage at a dose of 10 mg / kg, and three rats received oral gavage at a dose of 100 mg / kg. Plasma samples were collected at 0 h (before administration) and at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h post-administration. The collected samples were then analyzed by LC / MS / MS, and data were collected. AUC represents the area under the plasma concentration-time curve from time zero to 24 hours. Cmax represents the peak concentration; Tmax represents the time to peak concentration; T 1 / 2 The values ​​represent half-life; Cl represents clearance rate; Vd represents apparent volume of distribution; F represents bioavailability; po represents oral administration; iv represents intravenous administration.

[0464] Table 15. Results of rat pharmacokinetic assays of the compounds

[0465]

[0466] Table 15 shows that compounds A6 and A24 have excellent plasma exposure and bioavailability in rats.

[0467] Experimental Objective 3: To test the pharmacokinetics of the compound in a beagle dog.

[0468] Experimental steps:

[0469] The pharmacokinetic characteristics of the compound after intravenous and oral administration were tested in mammals using a standard protocol. In the experiment, the candidate compound was prepared as a clear solution and administered to beagle dogs via single intravenous injection, while a suspension was administered orally. The intravenous solvent was 5% DMSO + 95% saline, and the oral solvent was 0.5% CMCNa. Nine male beagle dogs were used in this project. Three beagle dogs received intravenous administration at a dose of 1 mg / kg, and plasma samples were collected at 0 h (before administration) and at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h post-administration. Three beagle dogs received oral gavage at a dose of 5 mg / kg, and three beagle dogs received oral gavage at a dose of 30 mg / kg. Plasma samples were collected at 0 h (before administration) and at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h post-administration. The collected samples were then analyzed by LC / MS / MS, and the data were collected. AUC represents the area under the plasma concentration-time curve from time zero to 24 hours. Cmax represents the peak concentration; Tmax represents the time to peak concentration; T 1 / 2 The values ​​represent half-life; Cl represents clearance rate; Vd represents apparent volume of distribution; F represents bioavailability; po represents oral administration; iv represents intravenous administration.

[0470] Table 16. Pharmacokinetic Results of Compound A6 in Beagle Dogs

[0471]

[0472] Table 17. Pharmacokinetic Results of Compound A24 in Beagle Dogs

[0473]

[0474] Tables 16 and 17 show that compounds A6 and A24 have excellent plasma exposure and bioavailability in dogs.

[0475] Example 27: In vivo efficacy study of an MDP (muramicyl dipeptide)-induced mouse peritonitis model

[0476] Experimental Objective: To investigate the activity of RIPK2 kinase inhibitors in an MDP-induced peritonitis model, and to clarify the blocking effect of the compounds on the NOD-RIPK2 signaling pathway by detecting the expression level of the downstream inflammatory cytokine IL-6. This aims to provide pharmacodynamic evidence for the treatment of RIPK2-related diseases.

[0477] Experimental Procedure: Mice were divided into four groups: a normal control group, a DMSO group, a positive control group (GSK2983559), and a compound group. The normal control group received no treatment. GSK2983559 was administered via gavage at a dose of 10 mg / kg, while the compound group received either 3 mg / kg or 10 mg / kg. Thirty minutes after administration, the animals were intraperitoneally injected with MDP (100 μg / mouse). Three hours later, blood was collected from the ocular vein after anesthesia. Serum IL-6 levels were detected by ELISA.

[0478] Experimental results: such as Figure 2 , Figure 3 As shown.

[0479] Experimental conclusions: Compared with the normal group, IL-6 levels were significantly increased after MDP modeling, indicating that downstream inflammatory pathways were activated and the model was successfully induced. IL-6 levels in all drug-treated groups decreased compared with the model group. Among them, A6 and A24 showed more significant decreases than the positive control GSK2983559 at the same drug dose, suggesting that these two compounds more effectively inhibited the activity of RIP2 kinase, blocked the activation of downstream inflammatory signals, and thus affected the synthesis and release of the cytokine IL-6.

Claims

1. The following compounds or their pharmaceutically acceptable salts that have RIP2 kinase inhibitory activity: , , , and .

2. A pharmaceutical composition comprising the compound having RIP2 kinase inhibitory activity as claimed in claim 1, or a pharmaceutically acceptable salt thereof.

3. A drug combination composition comprising the compound having RIP2 kinase inhibitory activity according to claim 1 or a pharmaceutically acceptable salt thereof, or the drug composition according to claim 2, and one or more of the following: antitumor drugs, anti-autoimmune disease drugs, anti-neurodegenerative disease drugs, anti-metabolic disease drugs, and hereditary disease drugs.

4. The use of the compound having RIP2 kinase inhibitory activity according to claim 1, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 2, or the pharmaceutical combination composition according to claim 3, in the preparation of a medicament for the prevention and / or treatment of diseases and / or conditions that are at least partially responsive to the RIP2 receptor.

5. The application according to claim 4, characterized in that: The diseases and / or conditions described are selected from dermatitis, acute lung injury, type II diabetes, arthritis, ulcerative colitis, segmental ileitis, early-onset inflammatory bowel disease, extraintestinal inflammatory bowel disease, prevention of ischemia-reperfusion injury in solid organ transplantation, non-alcoholic fatty liver disease, autoimmune hepatitis, asthma, systemic lupus erythematosus, sarcoidosis, Wegener's granulomatosis and interstitial lung disease, pulmonary fibrosis, renal fibrosis, liver fibrosis, myocardial infarction, asthma, allergic pneumonia, interstitial lung disease, ankylosing spondylitis, multiple sclerosis, systemic sclerosis, polymyositis, rheumatoid arthritis, myasthenia gravis, juvenile diabetes, glomerulonephritis, autoimmune thyroiditis, graft rejection, Crohn's disease, Blau syndrome, scleroderma, psoriasis, stomatitis, retinitis pigmentosa, proliferative vitreoretinopathy, and more. The following are considered as a possible diagnosis: Michelia nipponica macular degeneration, eczema, urticaria, vasculitis, eosinophilic fasciitis, wet and dry age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, diabetic macular erythema, uveitis, retinal vein occlusion, cystoid macular edema, glaucoma, Parkinson's disease, Alzheimer's disease, Huntington's disease, breast cancer, lung cancer, bladder cancer, pancreatic cancer, liver cancer, head and neck squamous cell carcinoma, thyroid cancer, sarcoma, osteosarcoma, desmoidoma, melanoma, prostate cancer, colorectal cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, myeloma, lymphoma, chronic and non-progressive anemia, spontaneous or primary thrombocytosis, leukemia, myelodysplastic syndrome, myeloproliferative disorders, brain tumor, astrocytoma, medulloblastoma, Schwann cell tumor, primary neuroectodermal tumor, or pituitary tumor, one or more of these.

6. The application according to claim 5, characterized in that: The lymphoma is selected from mantle cell lymphoma and cutaneous T-cell lymphoma.

7. The application according to claim 5, characterized in that: The leukemia is selected from acute leukemia, chronic leukemia, lymphocytic leukemia, and myeloid leukemia.

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