Phosphorus-containing or sulfur-containing macrocyclic pyrazolopyrimidine compound and use thereof

Abstract

The present application relates to the field of biomedicine, and particularly relates to a small-molecule phosphorus-containing or sulfur-containing macrocyclic pyrazolopyrimidine compound, which has better immunomodulatory activity. Also provided in the present invention is the use of the small-molecule phosphorus-containing or sulfur-containing macrocyclic pyrazolopyrimidine compound in the prevention or treatment of TLR7-related diseases, and the use thereof as a vaccine adjuvant, a photodynamic therapeutic agent and a conjugated drug.

Description

Phosphorus- or sulfur-containing macrocyclic pyrazolopyrimidine compounds and uses thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the application with CN application number 202310768165.2 and application date June 27, 2023, and claims its priority. The content of the CN application is hereby introduced as a whole into this application. Technical Field

[0003] The present application relates to the field of biomedicine, and in particular to phosphorus- or sulfur-containing macrocyclic pyrazolopyrimidine compounds and uses thereof. Background Art

[0004] Pattern recognition receptors (PRRs) play a crucial role in maintaining the normal function of the innate immune system. Based on the homology of their protein domains, PRRs are divided into five families: Toll-like receptors (TLRs), C-type lectin receptors (CLRs), AIM2-like receptors (ALRs), RIG-I-like receptors (RLRs), and Nod-like receptors (NLRs). The first two families are transmembrane proteins, while the latter three families are cytoplasmic proteins.

[0005] TLRs are type I membrane proteins, primarily located on the cell membranes or endosomes of various immune cells. They recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), activating downstream signaling molecules in the immune system and initiating host defense responses. They can promote innate immune responses to eliminate pathogens and trigger adaptive immunity. As functional biomolecules, TLRs safeguard the immune systems of diverse hosts, from insects to humans. Ten TLR subtypes (TLRs1-10) have been identified in mammals. Based on cellular localization and ligand type, TLRs can be divided into two categories: the first category includes TLRs 1 / 2 / 4 / 5 / 6 / 10, and the second category includes TLRs 3 / 7 / 8 / 9. Class I TLRs are primarily located on the cell membrane, while class II TLRs are located in intracellular membrane structures such as endosomes, lysosomes, and the endoplasmic reticulum. Due to their crucial physiological functions in the immune system, TLRs are important drug targets, with significant application value in the treatment of infectious diseases, autoimmune diseases, cancer, and inflammatory disorders.

[0006] TLR7 is a member of the TLR family. Its activation initiates a myeloid differentiation factor 88 (MyD88)-dependent pathway, in which TLR7 collaborates with protein kinases, transcription factors, and adaptor proteins. Following ligand binding, TLR7 interacts with MyD88, which then recruits members of the interleukin-1 receptor-associated kinase (IRAK) family to form a large intracellular oligomeric signaling complex (the Myddosome). During the formation of this oligomeric signaling complex, IRAK4 activates the autophosphorylation of IRAK1. The released IRAK1 then interacts with tumor necrosis factor receptor-associated factor 6 (TRAF6). This cascade of signaling activates the NF-κB and mitogen-activated protein kinase (MAPK) pathways, leading to the production and release of proinflammatory cytokines and chemokines.

[0007] TLR7 plays an important role in inflammatory diseases, viral infections, cancer, vaccines, and autoimmune diseases. TLR7 agonists can be used to treat skin diseases and respiratory diseases, and can also be used as tumor immunomodulators, antiviral drugs, and vaccine adjuvants. Several TLR7 agonists are in clinical research, such as Resiquimod, which is in Phase II clinical trials for the treatment of bladder cancer, and Vesatolimod, which is in Phase II clinical trials for the treatment of HIV infection. Some small molecule TLR7 agonists that have entered the clinical stage (such as Guretolimod, PF-4878691, and GSK-2245035) have been forced to terminate clinical trials due to poor efficacy or adverse reactions. Currently, the only marketed small molecule compound in the field of TLR7 agonists is Imiquimod, which was approved in the United States in 1997 for the topical treatment of genital and perianal warts, and subsequently added indications such as basal cell carcinoma and actinic keratosis.

[0008] Summary of the Invention

[0009] The present invention aims to provide a novel, phosphorus- or sulfur-containing, macrocyclic pyrazolopyrimidine-based TLR7 small-molecule agonist with enhanced immunomodulatory activity. The present invention also provides the use of the TLR7 small-molecule agonist for preventing or treating TLR7-related diseases, as well as for use as a vaccine adjuvant and photodynamic therapy agent. The following inventions are provided:

[0010] Compound

[0011] In one aspect, the present application provides a compound having a structure as shown in formula (I), a pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope label or metabolite thereof,

[0012] in,

[0013] R 1 Selected from: hydrogen, halogen, C1-C6 alkyl;

[0014] Ring A is selected from a benzene ring, a 5-6 membered heteroaryl, a naphthalene ring and a bicyclic heteroaryl; optionally, Ring A is substituted by one or more substituents selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, halogen, -CH2R a 、-OR b 、-C(=O)R c , nitro, nitroso, cyano, hydroxyl, -SH, C2-C6 alkenyl, C2-C6 alkynyl, =O, -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)(C1-C6 alkyl), -NHC(=O)(C1-C3 alkyl), C3-C6 cycloalkyl, 3-6 membered heterocyclyl, phenyl, 5-6 membered heteroaryl, -(C1-C6 alkyl)OH, -(C1-C6 alkyl)NH2, -S(C1-C3 alkyl), -S(=O)(C1-C3 alkyl), -S(=O)(=O)(C1-C3 alkyl), -S(=O)(=O)NH(C1-C3 alkyl), -NR d S(=O)(=O)(C1-C6 alkyl), -P(=O)R e R f , sulfonic acid, -N=NPh, -Si(C1-C3 alkyl)3; R a 、R b 、R c 、R d 、R e 、R f Each independently selected from: hydrogen, -OH, -COOH, -C(=O)H, phenyl, C1-C6 alkyl, 3-6 membered heterocyclic group, -SiR x R y R z 、-NR x R y , -C(=O)(C1-C6 alkyl), halogen, C1-C6 alkoxy, -S(=O)(=O)(C1-C3 alkyl); R x 、R y 、R z Each is independently selected from hydrogen, C1-C6 alkyl, benzene ring; optionally, the 3-6 membered heterocyclic group is substituted by one or more C1-C6 alkyl groups;

[0015] R 5 、R 6 Each is independently hydrogen, C1-C6 alkyl, C1-C6 alkoxy, halogen, or halogenated C1-C6 alkyl;

[0016] Q is selected from -O-, -S-, and -NH-;

[0017] L 1 , L 2 Each independently selected from -(CH2) n -、-(CH2) n N(R 7 )-、-N(R 7 )(CH2) n -、-(CH2) n O-, -O(CH2) n -; n is selected from 0, 1, 2;

[0018] R 7 Selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, phenyl, phenoxy, substituted phenyl and substituted phenoxy, wherein the substituted phenyl is phenyl substituted by one or more groups selected from the following: halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, hydroxyl; in the substituted phenoxy, the phenyl ring is substituted by one or more groups selected from the following: halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, hydroxyl;

[0019] M is selected from the following groups:

[0020] Among them, R 8 Selected from hydrogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, phenyl, phenoxy, substituted phenyl and substituted phenoxy, wherein the substituted phenyl is phenyl substituted by one or more groups selected from the following: halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, hydroxyl; in the substituted phenoxy, the phenyl ring is substituted by one or more groups selected from the following: halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, hydroxyl.

[0021] In certain embodiments, R 1 Selected from: hydrogen, halogen.

[0022] In certain embodiments, R 1 For hydrogen.

[0023] In certain embodiments, Ring A is selected from a bicyclic heteroaryl substituted with one or more substituents, such as benzopyrrole, benzofuran, benzothiophene, benzoxazole, benzisoxazole, benzimidazole, benzotriazole, indazole, purine, indole, quinoline, pteridine, isoquinoline, quinazoline, quinoxaline, 1,8-naphthyridine, 1,5-naphthyridine, 9H-purine, 7H-purine, indane, indoline, 2,3-dihydrobenzofuran, 1,2,3,4-tetrahydronaphthalene, 1,2,3,4-tetrahydroquinoline, 3,4-dihydro-1H-chromene, 1,4-benzodioxane, benzomorpholine, 1,2,3,4-tetrahydroquinoxaline.

[0024] In certain embodiments, Ring A is selected from a benzene ring, a 5-6 membered heteroaryl ring, and optionally, Ring A is substituted with one or more substituents.

[0025] In certain embodiments, the substituents of Ring A are selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, halogen, -CH2R a 、-OR b 、-C(=O)R c , nitro, nitroso, cyano, hydroxy, -SH, =O, -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)(C1-C6 alkyl), -NHC(=O)(C1-C3 alkyl), -(C1-C6 alkyl)OH, -(C1-C6 alkyl)NH2, -S(C1-C3 alkyl), -S(=O)(C1-C3 alkyl), -S(=O)(=O)(C1-C3 alkyl), -S(=O)(=O)NH(C1-C3 alkyl), -NR d S(=O)(=O)(C1-C6 alkyl), -P(=O)R e R f , sulfonic acid group, -N=NPh, -Si(C1-C3 alkyl)3.

[0026] In certain embodiments, the substituents of Ring A are selected from C3-C6 cycloalkyl, 3-6 membered heterocyclyl, phenyl, 5-6 membered heteroaryl.

[0027] In certain embodiments, the substituents of Ring A are selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, halogen, -CH2R a 、-OR b 、-C(=O)R c .

[0028] In certain embodiments, R a 、R b 、R c 、R d 、R e 、R fEach independently selected from: hydrogen, -OH, -COOH, -C(=O)H, phenyl, C1-C6 alkyl, 3-6 membered heterocyclic group, -SiR x R y R z 、-NR x R y , R x 、R y 、R z Each is independently selected from hydrogen, C1-C6 alkyl, and benzene ring; optionally, the 3-6 membered heterocyclic group is substituted by one or more C1-C6 alkyl groups.

[0029] In certain embodiments, Ring A is a benzene ring optionally substituted with one or more substituents.

[0030] In certain embodiments, Ring A is a 5-6 membered heteroaryl group, wherein 1, 2 or 3 ring atoms of the 5-6 membered heteroaryl group are heteroatoms selected from nitrogen, oxygen and / or sulfur, wherein the nitrogen and / or sulfur are optionally substituted with oxo.

[0031] In certain embodiments, the substituents on Ring A are selected from hydrogen, halogen, -C(O)H, -CH2-OH, -CH2-(3-6 membered nitrogen-containing heterocyclyl), -CH2-NR x R y , -O-(3-6 membered nitrogen-containing heterocyclic group), C1-C6 alkoxy, wherein R x 、R y Each is independently selected from hydrogen, C1-C6 alkyl; optionally, the 3-6 membered nitrogen-containing heterocyclic group is substituted by one or more C1-C6 alkyl groups.

[0032] In certain embodiments, R 5 、R 6 Each is independently hydrogen or C1-C6 alkyl.

[0033] In certain embodiments, Q is -O-.

[0034] In certain embodiments, L 1 , L 2 Each independently selected from -(CH2) n -、-(CH2) n N(R 7 )-、-N(R 7 )(CH2) n -、-(CH2) n O-, -O(CH2) n -; n is selected from 0, 1. In certain embodiments, R 7 Selected from hydrogen, C1-C6 alkyl.

[0035] In certain embodiments, L1 , L 2 Each independently selected from -NH-, -NCH3-, -O-, -(CH2) n -, -CH2-NH-, -CH2-N(CH3)-, -N(CH3)-CH2-, -NH-CH2-, -CH2O-, -OCH2-; n is selected from 0, 1.

[0036] In certain embodiments, R 8 Selected from hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, substituted phenoxy, wherein the phenyl ring of the phenoxy is optionally substituted by halogen.

[0037] In certain embodiments, R 8 The present invention is selected from hydroxy, methyl, ethyl, methoxy, ethoxy, and substituted phenoxy groups, wherein the phenyl ring of the phenoxy group is optionally substituted by halogen.

[0038] In certain embodiments, the compounds of the present invention have a structure as shown in Formula (II):

[0039] in,

[0040] R 1 Selected from: hydrogen, halogen, C1-C6 alkyl;

[0041] R 2 、R 3 、R 4 Each is independently selected from: hydrogen, halogen, -C(O)H, -CH2-OH, -CH2-(3-6 membered nitrogen-containing heterocyclic group), -CH2-NR x R y , -O-(3-6 membered nitrogen-containing heterocyclic group), C1-C6 alkoxy, wherein R x 、R y Each is independently selected from hydrogen, C1-C6 alkyl; optionally, the 3-6 membered nitrogen-containing heterocyclic group is substituted by one or more C1-C6 alkyl groups;

[0042] R 5 、R 6 Each is independently hydrogen or C1-C6 alkyl;

[0043] L 1 , L 2 Each independently selected from -(CH2) n -、-(CH2) n N(R 7 )-、-N(R 7 )(CH2) n -、-(CH2) nO-, -O(CH2) n -; n is selected from 0, 1; R 7 Selected from hydrogen, C1-C6 alkyl;

[0044] M is selected from the following groups:

[0045] R 8 Selected from hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, substituted phenoxy, wherein the phenyl ring of the phenoxy is optionally substituted by halogen.

[0046] In certain embodiments, R 1 Selected from hydrogen and halogen.

[0047] In certain embodiments, R 2 For hydrogen.

[0048] In certain embodiments, R 2 、R 3 、R 4 are hydrogen respectively.

[0049] In certain embodiments, R 3 Selected from hydrogen, -CH2-OH, -CH2-(3-6 membered nitrogen-containing heterocyclic group), -CH2-NR x R y , C1-C6 alkoxy, -C(O)H, wherein R x 、R y Each is independently selected from hydrogen, C1-C6 alkyl; optionally, the 3-6 membered nitrogen-containing heterocyclic group is substituted by one or more C1-C6 alkyl groups.

[0050] In certain embodiments, R 3 Selected from hydrogen, -CH2-OH, -CH2-(5-6 membered nitrogen-containing heterocyclic group), -CH2-NR x R y , methoxy, -C(O)H, where R x 、R y Each is independently selected from hydrogen and methyl; optionally, the 5-6 membered nitrogen-containing heterocyclic group is substituted by one or more C1-C6 alkyl groups.

[0051] In certain embodiments, R 3 Selected from hydrogen, halogen, C1-C6 alkoxy.

[0052] In certain embodiments, R 3 For hydrogen.

[0053] In certain embodiments, R 4Selected from: hydrogen, halogen, -O-(3-6 membered nitrogen-containing heterocyclic group), hydroxyl, -C(O)H, -CH2-(3-6 membered nitrogen-containing heterocyclic group), C1-C6 alkoxy; optionally, the 3-6 membered nitrogen-containing heterocyclic group is substituted by one or more C1-C6 alkyl groups.

[0054] In certain embodiments, R 4 Selected from: hydrogen, halogen, -O-(5-6 membered nitrogen-containing heterocyclic group), hydroxyl, -C(O)H, -CH2-(5-6 membered nitrogen-containing heterocyclic group), methoxy; optionally, the 5-6 membered nitrogen-containing heterocyclic group is substituted by one or more C1-C6 alkyl groups.

[0055] In certain embodiments, R 4 Selected from: hydrogen, halogen, C1-C6 alkoxy, -CH2-(5-6 membered nitrogen-containing heterocyclic group), -CH2-NR x R y , where R x 、R y Each is independently selected from hydrogen, C1-C6 alkyl; optionally, the 3-6 membered nitrogen-containing heterocyclic group is substituted by one or more C1-C6 alkyl groups.

[0056] In certain embodiments, R 5 For hydrogen.

[0057] In certain embodiments, R 5 It is a methyl group.

[0058] In certain embodiments, R 6 For hydrogen.

[0059] In certain embodiments, R 6 It is a methyl group.

[0060] In certain embodiments, M is selected from the group consisting of:

[0061] R 9 is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, hydroxy. 9 It is a halogen.

[0062] In certain embodiments, the compounds of the present invention have a structure as shown in Formula (III), (IV), (V), (VI), (VII), or (VIII):

[0063] Among them, R 1 、R 2 、R 3 、R 4 、R 9As defined in any one of the above items respectively.

[0064] The hydroxyl groups and -NH- and -NH2 in the compounds of the present invention may optionally be protected by substituents, which may be hydroxyl or amino protecting groups commonly used in the art, such as Boc (tert-butyloxycarbonyl), TBDPS (tert-butyldiphenyl), THP (2-tetrahydropyranyl), etc. The compounds thus formed are also within the scope of protection of the present invention.

[0065] The compounds of the present invention may have a structure selected from the group consisting of:

[0066] Pharmaceutical compositions, uses, and disease prevention / treatment methods

[0067] In one aspect, the present application provides a pharmaceutical composition comprising a compound of the present invention, a pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or a metabolite thereof; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0068] The pharmaceutical compositions of the present invention can be formulated into any pharmaceutically acceptable dosage form and administered to patients in need of such treatment by oral, parenteral, rectal, pulmonary, topical, or transdermal routes. For oral administration, conventional solid preparations such as tablets, capsules, pills, and granules can be prepared; oral liquid preparations such as oral solutions, oral suspensions, and syrups can also be prepared. Suitable fillers, binders, disintegrants, lubricants, and the like can be added to oral preparations. For parenteral administration, injections can be prepared, including injection solutions, sterile powders for injection, and concentrated solutions for injection. Injections can be produced using conventional methods in the pharmaceutical field. Additives may be omitted or added depending on the properties of the drug. For rectal administration, suppositories can be prepared. For pulmonary administration, inhalants or sprays can be prepared. For topical administration, specific dosage forms and devices can be used, or creams, gels, and the like can be prepared. For transdermal administration, patches can be prepared.

[0069] In one aspect, the present application provides the use of a compound of the present invention, a pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or metabolite thereof in the preparation of a medicament, wherein the medicament is used as a TLR7 agonist for preventing or treating a disease associated with TLR7.

[0070] In one aspect, the present application provides a method for preventing or treating a TLR7-related disease in a subject, comprising administering to a subject in need thereof an effective amount of a compound of the present invention, a pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystalline form, isotopically labeled form thereof, or a metabolite thereof.

[0071] In some embodiments, the disease associated with TLR7 is selected from:

[0072] (1) tumors, including but not limited to: breast cancer, melanoma, meningioma, soft tissue sarcoma, salivary gland tumor, intraspinal tumor, mediastinal tumor, brain cancer, bone cancer, penile cancer, osteosarcoma, intracranial tumor, tongue cancer, maxillary sinus cancer, thyroid cancer, malignant lymphoma, multiple myeloma, pituitary adenoma, testicular tumor, non-Hodgkin's lymphoma, bladder cancer, leukemia, gastric cancer, nasopharyngeal cancer, laryngeal cancer, oral cancer, esophageal cancer, lung cancer, kidney cancer, cervical cancer, choriocarcinoma, vulvar cancer, skin cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, rectal cancer, colorectal cancer, Kaposi's sarcoma, non-melanoma skin cancer (including squamous cell carcinoma and basal cell carcinoma), hemangioma, glioma, non-Hodgkin's lymphoma (diffuse large B-cell lymphoma, B-cell non-Hodgkin's lymphoma), colon cancer, liver cancer, neuroblastoma, glioblastoma;

[0073] (2) diseases associated with viral infection, including but not limited to dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, tick-borne encephalitis virus, Kunjin virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus, Zika virus, HIV, HBV, HCV, HPV, RSV, SARS or influenza virus;

[0074] (3) Inflammatory diseases, respiratory diseases, skin diseases, gastrointestinal diseases, hepatobiliary diseases, nervous system diseases, musculoskeletal and connective tissue diseases, or autoimmune diseases, such as asthma, chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome (ARDS), ulcerative colitis, Crohn's disease, bronchitis, dermatitis, actinic keratosis, allergic rhinitis, psoriasis, scleroderma, urticaria, rheumatoid arthritis, multiple sclerosis, lupus, drug-induced allergic reactions, and rheumatism.

[0075] In the method, the compound of the present invention acts as a TLR7 agonist, and further, can act as an immunomodulator.

[0076] The compounds of the present invention have an immunoenhancing effect. Therefore, in one aspect, the present application provides the use of the compounds of the present invention, their pharmaceutically acceptable salts, prodrugs, stereoisomers, tautomers, hydrates, solvates, crystal forms, isotope labels, or metabolites thereof as vaccine adjuvants. In certain embodiments, the vaccine is used to prevent or treat diseases associated with any of the viral infections described above.

[0077] In one aspect, the present application provides use of a compound of the present invention, a pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance, or a metabolite thereof as a photodynamic therapy agent.

[0078] In one aspect, the present application provides the use of a compound of the present invention, a pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystalline form, isotope-labeled substance, or a metabolite thereof as a conjugate drug (e.g., an antibody immunostimulatory drug conjugate, ISAC). An antibody immunostimulatory drug conjugate (ISAC) is a monoclonal antibody that combines an antibody with an immunostimulatory agent to enhance the immune response by triggering the production of proinflammatory cytokines and chemokines. For example, BDC-1001 is an ISAC that contains an antibody targeting HER2 and a TLR7 / 8 agonist and is currently in clinical phase I / II for the treatment of solid tumors.

[0079] In the present application, the subject is preferably a mammal, such as bovine, equine, porcine, canine, feline, rodent, primate, such as human.

[0080] Preparation method

[0081] Route 1:

[0082] Intermediate 1-1 undergoes cyclization in the presence of a condensation reagent to give 1-2.

[0083] Route 2:

[0084] Intermediate 2-1 undergoes cyclization in the presence of a condensation reagent to give 2-2, which then undergoes a series of reactions to give 2-3.

[0085] Route 3:

[0086] Intermediate 3-1 undergoes cyclization under condensation reagent conditions to give 3-2.

[0087] Route 4:

[0088] Intermediate 4-1 undergoes cyclization in the presence of a condensing agent to give 4-2, which then undergoes a series of reactions to give 4-3, wherein X is selected from H, TBDPS, and TBS, and the remaining substituents are as described above.

[0089] Definition of terms

[0090] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures described herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0091] As used herein, the term "C1-C6 alkyl" refers to a group obtained by removing a hydrogen atom from a straight-chain or branched alkane containing 1 to 6 carbon atoms, specific examples of which include but are not limited to: methyl, ethyl, propyl, n-butyl, isobutyl, isopropyl, tert-butyl, n-pentyl, and n-hexyl. In the present invention, the preferred C1-C6 alkyl group is a C1-C4 alkyl group.

[0092] As used herein, the term "C2-C6 alkenyl" refers to a straight-chain or branched hydrocarbon group containing at least one carbon-carbon double bond and having 2-6 carbon atoms. Specific examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, and 1,4-hexadienyl. In the present invention, the preferred C2-C6 alkenyl group is C2-C4 alkenyl.

[0093] As used herein, the term "C2-C6 alkynyl" refers to a straight-chain or branched hydrocarbon group containing at least one triple bond and having 2-6 carbon atoms. Specific examples include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1,3-butadiynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, and 1,4-hexadiynyl. In the present invention, the preferred C2-C6 alkynyl group is a C2-C4 alkynyl group.

[0094] As used herein, the term "halogen" includes fluorine, chlorine, bromine and iodine. In the present invention, preferred halogens are fluorine, chlorine and bromine, and more preferred is bromine.

[0095] As used herein, the term "halogenated" refers to a group or compound in which hydrogen is replaced by one or more halogen atoms, including full halogenation and partial halogenation.

[0096] As used herein, the term "alkoxy" refers to a group formed in the form of alkyl-O-.

[0097] As used herein, the term "5-6 membered heteroaryl" refers to a monocyclic aromatic group containing 5-6 ring atoms and at least one ring atom being a heteroatom, wherein the heteroatom is a nitrogen atom, an oxygen atom and / or a sulfur atom. Specific examples include, but are not limited to, furyl, pyrrolyl, thienyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridyl, pyrimidinyl, pyridazinyl, and pyrazinyl.

[0098] As used herein, the term "bicyclic heteroaryl" refers to a bicyclic group in which two rings (at least one of which has aromatic character) are fused together and at least one ring atom is a heteroatom, which is a nitrogen atom, an oxygen atom and / or a sulfur atom, which nitrogen and / or sulfur are optionally substituted with oxo. Specific examples include, but are not limited to, benzopyrrole, benzofuran, benzothiophene, benzoxazole, benzisoxazole, benzimidazole, benzotriazole, indazole, purine, indole, quinoline, pteridine, isoquinoline, quinazoline, quinoxaline, 1,8-naphthyridine, 1,5-naphthyridine, 9H-purine, 7H-purine, indane, indoline, 2,3-dihydrobenzofuran, 1,2,3,4-tetrahydronaphthalene, 1,2,3,4-tetrahydroquinoline, 3,4-dihydro-1H-benzopyran, 1,4-benzodioxane, benzomorpholine, and 1,2,3,4-tetrahydroquinoxaline.

[0099] As used herein, the term "cycloalkyl" refers to a saturated or partially saturated monocyclic alkyl group that is not aromatic, preferably having 3 to 6 ring atoms, ie, a 3-6 membered saturated cycloalkyl group and a 3-6 membered partially saturated cycloalkyl group. Specific examples of 3-6 membered saturated cycloalkyl groups include, but are not limited to, cyclopropanyl (cyclopropyl), cyclobutanyl (cyclobutyl), cyclopentanyl (cyclopentyl), and cyclohexanyl (cyclohexyl); specific examples of 3-6 membered partially saturated cycloalkyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexa-1,3-dienyl, cyclohex-1,4-dienyl, cycloheptenyl, cyclohept-1,3-dienyl, cyclohept-1,4-dienyl, cyclohept-1,3,5-trienyl, cyclooctenyl, cyclooct-1,3-dienyl, cyclooct-1,4-dienyl, cyclooct-1,5-dienyl, cyclooct-1,3,5-trienyl, and cyclooctatetraenyl.

[0100] As used herein, the term "heterocyclyl" refers to a radical derived from a saturated or partially saturated monocyclic or condensed ring compound containing 3 to 14 ring atoms and at least one heteroatom (e.g., 1, 2, 3, 4, or 5 heteroatoms) by removing one hydrogen atom. The term "3-6 membered nitrogen-containing heterocyclyl" refers to a heterocyclyl containing 3, 4, 5, or 6 ring atoms, 1, 2, or 3 of which are nitrogen atoms, and optionally further containing 1 or 2 oxygen atoms or sulfur atoms.

[0101] As used herein, the term "pharmaceutically acceptable salt" refers to (i) a salt formed by an acidic functional group (e.g., -COOH) present in the compounds provided herein with a suitable inorganic or organic cation (base), and includes, but is not limited to, alkali metal salts, such as sodium salts, potassium salts, lithium salts, etc.; alkaline earth metal salts, such as calcium salts, magnesium salts, etc.; other metal salts, such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts, etc.; inorganic base salts, such as ammonium salts; organic base salts, such as tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazine salts, tetramethylamine salts, tris(hydroxymethyl)aminomethane salts. and (ii) salts formed by basic functional groups (e.g., -NH2) present in the compounds provided by the present invention and appropriate inorganic or organic anions (acids), including but not limited to hydrohalides, such as hydrofluorides, hydrochlorides, hydrobromides, hydroiodides, etc.; inorganic acid salts, such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkanesulfonates, such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, etc.; arylsulfonates, such as benzenesulfonates, p-toluenesulfonates, etc.; organic acid salts, such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, maleates, etc.; amino acid salts, such as glycine, trimethylglycine, arginine, ornithine, glutamate, aspartate, etc.

[0102] As used herein, the term "pharmaceutically acceptable ester" refers to an ester formed between the -COOH group present in the compounds provided herein and a suitable alcohol, or an ester formed between the -OH group present in the compounds provided herein and a suitable acid (e.g., a carboxylic acid or an oxygen-containing inorganic acid). Suitable ester groups include, but are not limited to, formates, acetates, propionates, butyrates, acrylates, ethylsuccinates, stearic acid esters, or palmitates. Esters can undergo hydrolysis in the presence of an acid or base to produce the corresponding acid or alcohol.

[0103] As used herein, the term "pharmaceutically acceptable amide" refers to an amide formed between -COOH present in the compounds provided herein and a suitable amine, or an amide formed between -NH- or -NH2 present in the compounds provided herein and a suitable acid (e.g., carboxylic acid, such as acetic acid).

[0104] As used herein, the term "solvate" refers to a substance formed by the association of a compound of the present invention with a solvent molecule. The solvent may be an organic solvent (e.g., methanol, ethanol, propanol, acetonitrile, etc.). For example, the compound of the present invention may form an ethanolate with ethanol. The compound of the present invention may also form a hydrate with water.

[0105] As used herein, the term "crystalline form" refers to the crystal structure of a substance. During crystallization, various factors may alter the intramolecular or intermolecular bonding patterns, resulting in different arrangements of molecules or atoms within the crystal lattice space, forming different crystal structures. The compounds of the present invention may exist in a single crystal structure or in multiple crystal structures, i.e., they may exhibit "polymorphism." The compounds of the present invention may exist in different crystalline forms.

[0106] As used herein, the term "stereoisomer" includes conformational isomers and configurational isomers, wherein the configurational isomers primarily include cis-trans isomers and optical isomers. The compounds of the present invention may exist in the form of stereoisomers, and therefore encompass all possible stereoisomeric forms, and any combination or any mixture thereof. For example, a single enantiomer, a single diastereomer, or a mixture thereof. When a compound of the present invention contains an olefin double bond, unless otherwise specified, it includes cis-isomers and trans-isomers, and any combination thereof.

[0107] As used herein, the term "tautomer" refers to a functional group isomer resulting from the rapid movement of an atom in a molecule between two positions, such as tautomers of the enol form and the keto form. If tautomers exist for the compounds of the present invention, they may exist in the form of a single tautomer or a mixture thereof, preferably in the form of a more stable tautomer as the main form.

[0108] As used herein, the term "prodrug" refers to a compound that can be converted into the compound of the present invention by reactions such as oxidation, reduction, hydrolysis, etc. in the subject's body. The prodrug itself may or may not have the biological activity of the compound of formula (I) (e.g., regulating glycolipid metabolism activity, anti-inflammatory activity, antioxidant activity). For example, a compound of formula (I) comprising a hydroxyl or carboxyl group can be administered in the form of an ester, which is hydrolyzed in the body to form a hydroxyl compound or a carboxyl compound. Similarly, a compound of formula (I) comprising an amino group can be acylated, alkylated, or phosphorylated to form, for example, an eicosanoylamino group, an alanylamino group, or a pivaloyloxymethylamino group for administration. Further information on the use of prodrugs can be found in Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (T Higuchi and W Stella) and Bioreversible Carriers in Drug Design, Pergamon Press, 1987 (ed. EB Roche, American Pharmaceutical Association). Some examples of prodrugs according to the present invention include: (i) if the compound of formula (I) contains a carboxylic acid functional group (-COOH), then its esters include, for example, replacing hydrogen with a (C1-C8)alkyl group; (ii) if the compound of formula (I) contains an alcohol functional group (-OH), then its ethers include, for example, replacing hydrogen with a (C1-C6)alkanoyloxymethyl group; and (iii) if the compound of formula (I) contains a primary or secondary amino functional group (-NH2 or -NHR, wherein R is not H), then its amides include, for example, replacing hydrogen with a (C1-C6)alkanoyloxymethyl group. 10 )alkanoyl replaces one or two hydrogens. In addition, certain compounds of formula (I) may themselves act as prodrugs of other compounds of formula (I).

[0109] As used herein, the term "pharmaceutically acceptable carrier or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, disintegrants, binders, surfactants, glidants, lubricants, pH adjusters, ionic strength enhancers, agents that maintain osmotic pressure, agents that delay absorption, diluents, antioxidants, colorants, flavorings, preservatives, taste-masking agents, etc. For example, non-limiting examples of disintegrants include sodium starch glycolate, sodium carboxymethylcellulose, calcium carboxymethylcellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methylcellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinized starch, and sodium alginate. Non-limiting examples of binders include microcrystalline cellulose, gelatin, sugar, polyethylene glycol, natural and synthetic gums, polyvinyl pyrrolidone, pregelatinized starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Non-limiting examples of diluents include lactose (monohydrate, spray-dried monohydrate, anhydrous, etc.), mannitol, xylitol, glucose, sucrose, sorbitol, microcrystalline cellulose, starch and calcium hydrogen phosphate dihydrate. Non-limiting examples of surfactants include sodium lauryl sulfate and polysorbate 80. Non-limiting examples of glidants include silicon dioxide and talc. Non-limiting examples of lubricants include magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate and a mixture of magnesium stearate and sodium lauryl sulfate. Non-limiting examples of pH regulators include, but are not limited to, phosphate buffers. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Preservatives include, but are not limited to, various antibacterial and antifungal agents such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like.

[0110] As used herein, the term "prevention" refers to the method implemented in order to prevent or delay the occurrence of disease or illness or symptom in a subject. As used herein, the term "treatment" refers to the method implemented in order to obtain beneficial or required clinical results. For the purposes of the present invention, beneficial or required clinical results include but are not limited to, alleviating symptoms, narrowing the scope of the disease, stabilizing (that is, no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease and alleviating symptoms (no matter in part or in whole), no matter it is detectable or undetectable. In addition, "treatment" can also refer to, compared with the expected survival (if not receiving treatment), extending the survival.

[0111] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, a disease prevention (e.g., metabolic disease or the symptom associated with a metabolic disease) effective amount refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., metabolic disease or the symptom associated with a metabolic disease); a disease treatment effective amount refers to an amount sufficient to cure or at least partially stop the disease and its complications of a patient already suffering from the disease. It is within the capabilities of those skilled in the art to determine such an effective amount. For example, the effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc. DETAILED DESCRIPTION

[0112] Nuclear Magnetic Resonance: In the following examples, the structures of the compounds were determined by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LCMS). -6 The units are given in ppm. NMR measurements were performed on a Bruker AVANCE III 500 MHz NMR instrument using deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD), etc. as the solvents, and tetramethylsilane (TMS) as the internal standard.

[0113] Analytical liquid chromatography: Analytical liquid chromatography was used to monitor the reaction system and analyze the purity of key intermediates and target products. Analytical liquid chromatography system: Agilent 1260 Infinity II (equipped with 1260 Binary Pump, Vial Sampler, MCT column oven, DAD WR detector); Column: Waters XBridge BEH C18 3.5 μm, 4.6 x 150 mm.

[0114] Preparative liquid chromatography: Small amounts of important intermediates and target products were isolated and purified using preparative liquid chromatography. The preparative liquid chromatography system used was an Agilent 1290 Infinity II (equipped with a 1290 Prep Bin Pump, a Prep ALS / FC injector / collector, and a 1260 VWD detector); a C18 reversed-phase silica column.

[0115] Liquid Chromatography-Mass Spectrometry: LC-MS was used for routine reaction system analysis and molecular weight determination of the target product. LC-MS system: Waters UPLC H-Class Ultra High Performance Liquid Chromatography-QDa Spectrometer; Column: Waters XBridge BEH C18 1.7 μm, 2.1 x 75 mm.

[0116] Liquid phase mobile phase ratios: Acidic conditions: A: 0.05% formic acid in water, B: acetonitrile; Alkaline conditions: A: 0.05% ammonia in water, B: acetonitrile; Neutral conditions: A: 5 mM NH₄HCO₃ in water, B: acetonitrile. The above mobile phase ratios are the default and can be adjusted based on the specific sample properties during analysis.

[0117] Rapid purification system: A rapid purification system was used for crude preparation and purification of compounds in conventional reaction systems. The instrument model was Biotage Isolera One, and the normal phase and reverse phase columns used were purchased from Santai Tech. The common specifications of normal phase silica gel columns are 12g, 25g, 40g, 80g, 120g, and 330g. The specific parameters are as follows: 40-63μm, The commonly used specifications of reversed-phase C18 columns are 40g, 80g, 120g and 330g, and the specific parameters are as follows: 20-45μm,

[0118] Thin layer chromatography: Use thin layer chromatography (TLC) silica gel plates to perform qualitative analysis of the reaction system or to separate and purify a small amount of sample. The product used for qualitative analysis by thin layer chromatography is Merck thin layer silica gel plates, with the following specifications: GF 254 , 20*20cm, glass plate, 0.25mm coating; the product used for separation and purification by thin layer chromatography is Leyan prepared silica gel plate, the specifications are as follows: GF 254 , 20*20cm, glass plate, 1mm coating.

[0119] Chemical reagents: The known starting materials of the present invention can be synthesized by using or according to the literature, patent methods, etc. known in the art, or can be purchased from chemical reagent companies such as Alfa Aesar, Acros, Sigma-Aldrich, TCI, Aikon, Anaiji, Aladdin, J&K, Bid Pharmaceuticals, Leyan, McLean, Shaoyuan (Accela), Titan (Adamas), Xiens, and Inokane.

[0120] Abbreviations

[0121] Protecting Group Abbreviations:

[0122] Bn benzyl

[0123] Boc tert-butyloxycarbonyl

[0124] Cbz benzyloxycarbonyl

[0125] MOM methoxymethyl

[0126] PMB p-methoxybenzyl

[0127] TBDPS tert-Butyldiphenylsilyl

[0128] TBS tert-Butyldimethylsilyl

[0129] THP 2-Tetrahydropyranyl

[0130] Tos p-Toluenesulfonyl

[0131] Reagent Abbreviations:

[0132] ACN acetonitrile

[0133] AIBN Azobisisobutyronitrile

[0134] Boc2O Di-tert-butyl dicarbonate

[0135] BPO Benzoyl Peroxide

[0136] tBuONa sodium tert-butoxide

[0137] CAM Ammonium Molybdate Cerium Sulfate Color Developer

[0138] CDI N,N'-Carbonyldiimidazole

[0139] m-CPBA m-chloroperbenzoic acid

[0140] DABCO triethylenediamine

[0141] DCE 1,2-dichloroethane

[0142] DCM dichloromethane

[0143] DEAD Diethyl azodicarboxylate

[0144] DIAD Diisopropyl azodicarboxylate

[0145] DIBAL-H Diisobutylaluminum hydride

[0146] DBU 1,8-diazobisspiro[5.4.0]undec-7-ene

[0147] DHP 3,4-dihydro-2H-pyran

[0148] DIEA N,N-Diisopropylethylamine

[0149] DMAP 4-dimethylaminopyridine

[0150] DMF N,N-dimethylformamide

[0151] DMSO dimethyl sulfoxide

[0152] EA Ethyl acetate

[0153] HMPA Hexamethylphosphoramide

[0154] HATU N,N,N′,N′-Tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphoric acid

[0155] urea

[0156] LAH lithium aluminum hydride

[0157] NBS N-bromosuccinimide

[0158] PE petroleum ether

[0159] PMA phosphomolybdic acid

[0160] PyBOP 1H-Benzotriazol-1-yloxytripyrrolidino hexafluorophosphate

[0161] TBAF Tetrabutylammonium fluoride

[0162] TBDPSCl tert-Butyldiphenylsilyl chloride

[0163] TBSCl tert-Butyldimethylsilyl chloride

[0164] TEA triethylamine

[0165] TFA trifluoroacetic acid

[0166] THF Tetrahydrofuran

[0167] TPSCl 2,4,6-Triisopropylbenzenesulfonyl chloride

[0168] TsCl p-Toluenesulfonyl chloride

[0169] TsOH p-Toluenesulfonic acid

[0170] Example

[0171] Example 1: Synthesis of Compound B-1

[0172] Step A: At room temperature, trifluoroacetic acid (1.5 mL) was added to a solution of 2-((4-amino-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethane-1-ol (1.5 g, 5.37 mmol) in methanol (15 mL). After the addition, the mixture was stirred at room temperature for 72 hours. After the reaction was completed, the mixture was set aside.

[0173] Step B: One-fifth of the reaction mixture from the previous step was concentrated to dryness and dissolved in N,N-dimethylformamide (10 mL). Potassium carbonate (746 mg, 5.40 mmol) was added and stirred for 30 minutes. Then, dimethyl (3-(bromomethyl)benzyl)phosphonate (633 mg, 2.16 mmol) was added. The mixture was heated to 60°C and stirred for 3 hours. The reaction mixture was filtered and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (SiO2, PE:EA = 89:11) to yield dimethyl (3-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)benzyl)phosphonate (110 mg, 0.27 mmol, 25%) as a white solid. ESI-MS m / z = 408.0 [M+H] + ;Calcu.=407.1.

[0174] Step C: To dimethyl (3-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)benzyl)phosphonate (110 mg, 0.27 mmol) dissolved in acetonitrile (2 mL) at room temperature was added lithium bromide (117 mg, 1.35 mmol). After complete addition, the mixture was heated to reflux and stirred for 5 hours. The mixture was concentrated under reduced pressure, and the crude product was purified by preparative HPLC. Preparation conditions: Waters XBridge C18 (19 mm × 250 mm, 5 μm); A: 0.05% formic acid in water, B: MeCN; 18 mL / min; 0-3 min, 5% B; 3-16.5 min, 15%-50% B; detector, UV 254 nm. The reaction afforded methyl hydrogen (3-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)benzyl)phosphonate (10 mg, 0.025 mmol, 9%) as a white solid product. 1 H NMR(500MHz,DMSO-d6)δppm 7.97(s,1H),7.12-7.23(m,4H),6.98-7.03(m,2H),5.32(s,2H),4.28(t,J=5 .0Hz, 2H), 3.68 (t, J = 5.0Hz, 2H), 3.44 (d, J = 10.0Hz, 3H), 2.35-2.37 (m, 2H). ESI-MS m / z=394.0[M+H] + ;Calcu.=393.1.

[0175] Step D: To N,N-dimethylformamide (2.5 mL) and 1,2-dichloroethane (13 mL) dissolved with methyl hydrogen (3-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)benzyl)phosphonate (8 mg, 0.02 mmol) at room temperature were added 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate (63.5 mg, 0.122 mmol) and N,N-diisopropylethylamine (11.0 mg, 0.085 mmol). After the addition was complete, the mixture was stirred at room temperature overnight and concentrated under reduced pressure. The crude product was subjected to preparative separation and purification by HPLC. Preparation conditions: Waters XBridge C18 (10 mm × 150 mm, 5 μm); A: 0.1% formic acid in water, B: MeCN; 18 mL / min; 0-3 min, 10% B; 3-20 min, 10%-30% B; detector, UV 254 nm. The reaction yielded compound B-1, a white solid product 1. 4 -amino-5-methoxy-1 1 H-6,9-dioxa-5-phospha-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzocyclononane-5-oxide (1 mg, 0.0026 mmol, 13%). 1 H NMR(500MHz,DMSO-d6)δppm7.90(s,1H),7.80(br s,1H),7.78-7.75(m,1H),7.49-7.50(m,1H),7.19-7.22(m,1H),7.14(br s,2H),5.38(d,J=15.0Hz,1H),5.30(d,J=15.0Hz,1H),4.85-4.89(m,1H),4.60 -4.65(m,1H),3.95-4.01(m,2H),3.55(d,J=10.0Hz,3H),3.21-3.29ppm(m,2H). ESI-MS m / z=376.0[M+H] + ;Calcu.=375.1.

[0176] Example 2: Synthesis of Compound B-2

[0177] Step A: 3,4-dihydro-2H-pyran (5.44 mL, 63.49 mmol) and p-toluenesulfonic acid (72.9 mg, 0.423 mmol) were added sequentially to a solution of 4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (8 g, 42.33 mmol) in ethyl acetate (300 mL) at room temperature. The reaction mixture was heated to 50°C and stirred for 3 hours, cooled to room temperature, and concentrated to give a crude product. The crude product was dissolved in ammonia solution (7.0 M methanol solution, 36 mL), sealed and heated to 50°C for 3 hours, cooled to room temperature, at which time the product precipitated. The product was diluted with water, filtered, and the solid was dried to give 6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (8.6 g, 33.9 mmol, 80%) as a white solid. ESI-MS m / z=254.0[M+H] + ;Calcu.=253.1.

[0178] Step B: To a solution of 6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (4.5 g, 17.74 mmol) in 1,4-dioxane (200 mL) were added 2-tert-butyldimethylsilyloxyethanol (31.3 g, 177.4 mmol) and sodium tert-butoxide (17.05 g, 177.4 mmol) at room temperature. The reaction mixture was heated to 110°C and reacted. The reaction mixture was allowed to stand for 6 hours, cooled to room temperature, filtered to remove insoluble matter, and the filtrate concentrated to give a yellow oily crude product. The crude product was separated on a silica gel column (SiO2, DCM:MeOH = 95:5) to give 2-((4-amino-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethan-1-ol (4.16 g, 14.89 mmol, 84%) as a pale yellow solid. ESI-MS m / z = 280.0 [M+H] + ;Calcu.=279.1.

[0179] Step C: To a solution of (3-aminophenyl)methanol (15 g, 121.8 mmol) in acetonitrile (200 mL) at room temperature were added N-acetylimidazole (13.75 g, 124.8 mmol) and 1,8-diazobisspiro[5.4.0]undec-7-ene (927 mg, 6.09 mmol) in sequence. The reaction mixture was reacted at room temperature for 18 hours and concentrated to give a crude product. The crude product was separated on a silica gel column (SiO2, DCM:MeOH = 96:4) to give (3-aminophenyl)acetic acid methyl ester (16.6 g, 100.5 mmol, 83%) as a white solid. ESI-MS m / z = 166.1 [M+H] +;Calcu.=165.1.

[0180] Step D: To a solution of 2-chlorophenyldichlorophosphine (12.3 g, 50.12 mmol) in 1,4-dioxane (50 mL) was slowly added dropwise (3-aminophenyl)acetic acid methyl ester (16.56 g, 100.2 mmol) at 0°C. After the addition was complete, the mixture was warmed to room temperature and the reaction was continued for 4 hours. The mixture was then cooled to 0°C again, and triethylamine (17.4 mL, 125.3 mmol) and 2-((4-amino-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethan-1-ol (3.5 g, 12.53 mmol) were added in sequence. After the addition was complete, the mixture was warmed to room temperature and the reaction was continued for 18 hours. The insoluble matter was removed by filtration, and the filtrate was concentrated to give a crude product. The crude product was separated on a silica gel column (SiO2, DCM:MeOH=95:5) to give benzyl 3-(((2-((4-amino-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethoxy)(2-chlorophenoxy)phosphoryl)amino)acetate (1.65 g, 2.67 mmol, 21%) as a colorless oil. ESI-MS m / z=617.1[M+H] + ;Calcu.=616.2。

[0181] Step E: To a solution of benzyl 3-(((2-((4-amino-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethoxy)(2-chlorophenoxy)phosphoryl)amino)acetate (1 g, 1.62 mmol) in methanol (30 mL) was added potassium carbonate (448.0 mg, 3.24 mmol) at 0°C. The reaction mixture was reacted at 0°C for 2 hours. Hydrochloric acid was added dropwise to the reaction system to adjust the pH to neutral. The crude product was concentrated to give a crude product, which was separated on a silica gel column (SiO2, DCM:MeOH = 97:3) to give 2-((4-amino-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethyl(2-chlorophenyl)(3-(hydroxymethyl)phenyl)phosphamide (870 mg, 1.513 mmol, 93%) as a colorless oil. ESI-MS m / z = 575.0 [M+H] + ;Calcu.=574.1.

[0182] Step F: To a solution of 2-((4-amino-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethyl(2-chlorophenyl)(3-(hydroxymethyl)phenyl)phosphamide (850 mg, 1.478 mmol) in methanol (10 mL) was added trifluoroacetic acid (2 mL, 26.12 mmol) at room temperature. The reaction mixture was reacted at room temperature for 24 hours and concentrated to give a crude product. The crude product was separated on a silica gel column (SiO2, DCM:MeOH=97:3) to give 2-((4-amino-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethyl(2-chlorophenyl)(3-(hydroxymethyl)phenyl)phosphamide (630 mg, 1.284 mmol, 87%) as a colorless oil. ESI-MS m / z=491.0 [M+H] + ;Calcu.=490.1。

[0183] Step G: To a solution of 2-((4-amino-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethyl(2-chlorophenyl)(3-(hydroxymethyl)phenyl)phosphamide (480 mg, 0.978 mmol) in tetrahydrofuran (450 mL) was added triphenylphosphine (2.56 g, 9.78 mmol) at room temperature. The reaction mixture was cooled to 0°C, and then a solution of diethyl azodicarboxylate (1.74 g, 9.98 mmol) in tetrahydrofuran (50 mL) was slowly added dropwise to the reaction system. After the addition was complete, the reaction mixture was warmed to room temperature and the reaction was continued for 18 hours. Water (5 mL) was added to quench the reaction and concentrated to obtain a crude product, which was then subjected to HPLC preparative separation under the following conditions: Waters XBridge C18 (19 mm × 250 mm, 5 μm); A: 0.5% formic acid in water, B: MeCN; 18 mL / min; 0-3 min, 20% B; 4-20 min, 35%-60% B; detector, UV 254 nm. The preparative fractions were lyophilized to obtain compound B-2, a white solid product 1. 4 -amino-5-(2-chlorophenoxy)-1 1 H-6,9-dioxa-4-aza-5-phospha-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzazepanonane-5-oxide (0.6 mg, 0.001 mmol, 0.1%). 1H NMR (500MHz, CD3OD) δppm 8.28-8.43(m,1H),7.83-7.90(m,1H),7.34-7.39(m,1H),7.24-7.27(m,1H),7.17-7.23(m,1H), 7.07-7.16(m,2H),7.02(d,J=7.8Hz,1H),6.74-6.77(m,1H),5.42-5.47(m,1H),5.31-5.36(m,4H),4.46-4.51(m,,2H). 31 P NMR (202MHz, CD3OD) δppm-1.47 (br s, 1P). ESI-MS m / z=473.2[M+H] + ;Calcu.=472.1。

[0184] Example 3: Synthesis of Compound B-3

[0185] Step A: To a solution of lithium aluminum hydride (2.54 g, 66.9 mmol) in tetrahydrofuran (50 mL) at 0°C, slowly add dimethyl 5-methoxyisophthalate (5.00 g, 22.3 mmol) in tetrahydrofuran (50 mL) dropwise. After complete addition, heat the mixture to 65°C and stir for 3 hours. After the reaction, cool to 0°C, then slowly add saturated ammonium chloride solution (50 mL) and water (50 mL). Extract with ethyl acetate three times (200 mL x 3). Combine the organic phases, wash once with saturated brine (200 mL), dry over anhydrous sodium sulfate, filter, and concentrate to dryness under reduced pressure. This reaction yields the product (5-methoxy-1,3-phenylene) dimethanol (3.52 g, 20.93 mmol, 94%) as a white solid.

[0186] Step B: Phosphorus tribromide (5.9 mL, 62.43 mmol) was added to (5-methoxy-1,3-phenylene)dimethanol (3.50 g, 20.81 mmol) dissolved in dichloromethane (50 mL) at 0°C. After complete addition, the mixture was warmed to room temperature and stirred for 3 hours. Methanol (5 mL) was slowly added to quench the reaction. Water (50 mL) was added and the mixture was extracted twice with dichloromethane (50 mL x 2). The combined organic phases were washed once with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a solid residue. The residue was separated on a silica gel column (SiO2, PE:EA = 90:10) to afford 1,3-bis(bromomethyl)-5-methoxybenzene (5.30 g, 18.03 mmol, 87%) as a white solid.

[0187] Step C: At room temperature, in a 100 mL sealed tube, 1,3-bis(bromomethyl)-5-methoxybenzene (5.00 g, 17.0 mmol) dissolved in toluene (15 mL) was added with methyldiethoxyphosphine (1.3 mL, 8.5 mmol). After complete addition, the mixture was heated to 110°C and stirred for 6 hours. The reaction solution was concentrated under reduced pressure to obtain the crude product. Separation on a silica gel column (SiO2, PE:EA = 90:10 and DCM:MeOH = 90:10) afforded ethyl 3-(bromomethyl)-5-methoxybenzyl)(methyl)phosphinate (1.98 g, 6.16 mmol, 72%) as a pale yellow solid. ESI-MS m / z = 320.8, 322.8 [M+H] + ;Calcu.=320.0,322.0.

[0188] Step D: To a solution of 2-((4-amino-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethan-1-ol (156 mg, 0.8 mmol) and ethyl (3-(bromomethyl)-5-methoxybenzyl)(methyl)phosphinate (513 mg, 1.6 mmol) in N,N-dimethylformamide (5 mL) at room temperature was added potassium carbonate (276 mg, 2.0 mmol). After the addition was complete, the mixture was heated to 60°C and stirred for 3 hours. The reaction mixture was filtered and concentrated to dryness under reduced pressure. The crude product was subjected to silica gel column chromatography (SiO2, PE:MeOH = 88:12) to afford the product (ethyl 3-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methoxybenzyl)(methyl)phosphinate (50 mg, 0.115 mmol, 14%) as a white solid. ESI-MS m / z = 436.0 [M+H] + ;Calcu.=435.2.

[0189] Step E: To ethyl (3-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methoxybenzyl)(methyl)phosphinate (50 mg, 0.115 mmol) dissolved in dichloromethane (4 mL) at 0°C was added trimethylsilyl bromide (0.3 mL, 2.3 mmol). After complete addition, the mixture was stirred at room temperature overnight. The reaction mixture was quenched by adding 1 mL of methanol, adjusted to neutral pH with triethylamine, and concentrated to dryness under reduced pressure. The crude product was separated by preparative HPLC using the following conditions: Waters XBridge C18 (19 mm × 250 mm, 5 μm); A: 0.1% formic acid in water, B: MeCN; 18 mL / min; 0-3.5 min, 5% B; 3.5-15 min, 5%-50% B; UV detector, 254 nm. The reaction yielded the product (3-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methoxybenzyl)(methyl)phosphinic acid (4.0 mg, 0.01 mmol, 9%) as a white solid. ESI-MS m / z = 407.9 [M+H] + ;Calcu.=407.1.

[0190] Step F: To 1,2-dichloroethane (8.7 mL) and N,N-dimethylformamide (1.3 mL) dissolved with (3-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methoxybenzyl)(methyl)phosphinic acid (4.0 mg, 0.01 mmol) at room temperature were added N,N-diisopropylethylamine (5.4 mg, 0.042 mmol) and 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate (31 mg, 0.06 mmol). After the addition was complete, the whole mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure to about 1 mL at room temperature, and the residue was subjected to HPLC preparative separation (preparative conditions: Waters XBridge C18 (19 mm × 250 mm, 5 μm); A: 0.1% formic acid in water, B: MeCN; 18 mL / min; 0-3 min, 5% B; 3-3.5 min, 5%-10% B; 3.5-15 min, 10%-70% B; detector, UV 254 nm). The reaction yielded compound B-3, a white solid product 1. 4 -Amino-3 5 -methoxy-5-methyl-1 1H-6,9-dioxa-5-phospha-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzocyclononane-5-oxide (0.49 mg, 0.0013 mmol, 13%). 1 H NMR(500MHz,DMSO-d6)δppm 7.90(s,1H),7.78(s,1H),7.53(s,1H),7.34(s,1H),7.22(s,2H),5.34-5.37(m,1H),5.25-5.28(m,1H),4.76-4.81(m, 1H), 4.58-4.62 (m, 1H), 3.90-3.93 (m, 1H), 3.81-3.83 (m, 1H), 3.67 (s, 3H), 3.14-3.17 (m, 2H), 1.29 (d, J = 14.0Hz, 3H). ESI-MS m / z=389.9[M+H] + ;Calcu.=389.1.

[0191] Example 4: Synthesis of Compound B-4

[0192] Step A: In a 35 mL pressure tube at room temperature, diethyl methylphosphite (540 mg, 3.97 mmol) was added to 1,3-bis(bromomethyl)-5-fluorobenzene (1.64 mg, 5.81 mmol) dissolved in toluene (10 mL). The mixture was stirred and allowed to react at 110°C for 3 hours. After the reaction was complete, the mixture was cooled to room temperature and, without further treatment, dried by spin drying. The sample was then purified by column chromatography (DCM:MeOH = 97:3). The solvent was then concentrated under reduced pressure to yield 800 mg of the product (ethyl 3-(bromomethyl)-5-fluorobenzyl)(methyl)phosphite (800 mg, 2.33 mmol, 40%) as a pale yellow oil. ESI-MS m / z = 308.8 [M+H] + ;Calcu.=308.0.

[0193] Step B: At room temperature, take a 100 mL round-bottom flask and add (3-(bromomethyl)-5-fluorobenzyl)(methyl)ethyl phosphite (600 mg, 1.94 mmol) to N,N-dimethylformamide (5 mL) dissolved with 2-((4-amino-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethane-1-ol (300 mg, 1.54 mmol) and potassium carbonate (457 mg, 3.31 mmol). After stirring evenly, the reaction was carried out at 40°C overnight. After the reaction was complete, the system was cooled to room temperature and, without further treatment, the sample was directly spin-dried and purified by column chromatography (DCM:MeOH = 92:8). The solvent was removed by concentration under reduced pressure to obtain the product (ethyl 3-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-fluorobenzyl)(methyl)phosphite (180 mg, 0.43 mmol, 28%) as a pale yellow oil. ESI-MS m / z = 424.1 [M+H] + ;Calcu.=423.1.

[0194] Step C: At room temperature, trimethylsilyl bromide (805 mg, 5.26 mmol) was added to a solution of ethyl (3-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-fluorobenzyl)(methyl)phosphite (180 mg, 0.43 mmol) in dichloromethane (10 mL). A drying tube was inserted and stirring was continued at room temperature for 72 hours. During this period, the reaction was monitored multiple times by LCMS. The reaction was stopped when the reaction product no longer continued to grow. After the reaction was completed, methanol (5 mL) was added to quench the reaction, and the sample was then spin-dried and directly sent for preparative liquid separation and purification. Preparation conditions: Waters XBridge Prep Shield RP18 OBD (19 mm × 250 mm, 5 μm); A: 0.1% formic acid in water; B: MeCN; 18 mL / min; 0-3 min, 5% B; 3-3.5 min, 5%-15% B; 3.5-15 min, 15%-70% B; 15-18 min, 70%-100% B; detector: UV 254 nm. Fractions were recovered and lyophilized to give the product (3-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-fluorobenzyl)(methyl)phosphonic acid (17 mg, 0.043 mmol, 10%) as a white solid. ESI-MS m / z = 395.9 [M+H] + ;Calcu.=395.1.

[0195] Step D: To a solution of (3-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-fluorobenzyl)(methyl)phosphonic acid (17 mg, 0.043 mmol) in 1,2-dichloroethane (10 mL) and N,N-dimethylformamide (1.5 mL) at room temperature was added 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (112 mg, 0.22 mmol) and N,N-diisopropylethylamine (28 mg, 0.22 mmol). After the addition was complete, the mixture was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to obtain a colorless oily crude product. The residue was subjected to HPLC preparative separation under the following conditions: Waters XBridge RP18 (19 mm × 250 mm, 5 μm); A: 0.1% formic acid in water, B: MeCN; 18 mL / min; 0-3 min, 5% B; 3-4 min, 5%-20% B; 4-15 min, 20%-60% B; 15-16 min, 60%-100% B; detector, UV 254 nm. The reaction yielded compound B-4 as a white solid. 4 -Amino-3 5 -Fluoro-5-methyl-1 1 H-6,9-dioxo-5-phospho-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzocyclononane-5-oxide (7.37 mg, 0.019 mmol, 44%). 1 H NMR(500MHz,DMSO-d6)δppm 7.93(s,1H),7.76-7.89(m,1H),7.51-7.60(m,2H),6.93-7.01(m,2H),5.40-5.45(m,1H),5.31-5.37(m,1H),4. 78-4.86(m,1H),4.61(m,1H),3.91-3.98(m,1H),3.82-3.90(m,1H),3.21-3.28(m,2H),1.31(d,J=14.0Hz,3H). ESI-MS m / z=377.9[M+H] + ;Calcu.=377.1.

[0196] Example 5: Synthesis of Compound B-5 / M-1

[0197] Step A: To a solution of sodium hydride (0.22 g, 8.99 mmol) in tetrahydrofuran (30 mL) was slowly added dropwise dimethyl phosphite (1.48 g, 13.49 mmol) in tetrahydrofuran (30 mL) at 0°C. After the addition, the mixture was stirred at room temperature for 15 minutes. Then, methyl 4-bromo-3-(bromomethyl)benzoate (2.77 g, 8.99 mmol) in tetrahydrofuran (60 mL) was added. The mixture was heated to reflux and stirred for 2 hours. Saturated ammonium chloride solution (30 mL) was slowly added to the reaction mixture, and the mixture was concentrated under reduced pressure at 45°C to remove tetrahydrofuran. The mixture was extracted three times with ethyl acetate (100 mL x 3). The organic phases were combined, washed twice with saturated brine (100 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting crude product was separated on a silica gel column (DCM:MeOH = 95:5). The reaction yielded a white product, methyl 4-bromo-3-((dimethoxyphosphoryl)methyl)benzoate (2.2 g, 6.53 mmol, 73%). ESI-MS m / z = 336.7, 338.7 [M+H] + ;Calcu.=336.0,338.0.

[0198] Step B: At room temperature, sodium borohydride (1.01 g, 26.7 mmol) was added portionwise to a solution of methyl 4-bromo-3-((dimethoxyphosphoryl)methyl)benzoate (1.80 g, 5.34 mmol) in tetrahydrofuran (25 mL). After complete addition, the mixture was heated to 65°C and stirred at this temperature for 0.5 hour. Methanol (25 mL) was then slowly added dropwise. After complete addition, the mixture was reacted at 65°C for 2 hours. The reaction mixture was cooled in an ice bath, and saturated ammonium chloride solution (50 mL) was slowly added. The mixture was concentrated under reduced pressure to remove tetrahydrofuran and methanol. Water (50 mL) was then added and the mixture was extracted three times with dichloromethane (100 mL x 3). The organic phases were combined, washed once with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. This reaction yielded dimethyl (2-bromo-5-(hydroxymethyl)benzyl)phosphonate (1.51 g, 4.88 mmol, 91%) as a pale yellow oily liquid, which was carried on to the next step without purification. ESI-MS m / z = 308.8, 310.8 [M+H] + ;Calcu.=308.0,310.0.

[0199] Step C: Phosphorus tribromide (0.5 mL, 5.33 mmol) was slowly added to dimethyl (2-bromo-5-(hydroxymethyl)benzyl)phosphonate (1.5 g, 4.85 mmol) dissolved in dichloromethane (25 mL) at 0°C. After complete addition, the mixture was stirred at this temperature for 10 minutes. Water (25 mL) was then slowly added to quench the reaction. The mixture was extracted three times with dichloromethane (25 mL x 3). The combined organic phases were washed once with saturated brine (25 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated on a silica gel column (DCM:MeOH = 95:5) to yield dimethyl (2-bromo-5-(bromomethyl)benzyl)phosphonate (840 mg, 2.26 mmol, 47%) as a white product. ESI-MS m / z = 370.7, 372.7, 374.7 [M+H] + ;Calcu.=369.9,371.9,373.9..

[0200] Step D: To a solution of 2-((4-amino-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethan-1-ol trifluoroacetate (340 mg, 1.1 mmol) in N,N-dimethylformamide (3 mL) at room temperature was added potassium carbonate (759 mg, 5.5 mmol). The mixture was stirred at room temperature for 30 minutes after the addition was complete. Then, a solution of dimethyl (2-bromo-5-(bromomethyl)benzyl)phosphonate (736 mg, 1.98 mmol) in N,N-dimethylformamide (2 mL) was added. The mixture was heated to 60°C and stirred for 3 hours. The reaction mixture was filtered and concentrated to dryness under reduced pressure to obtain the crude product, which was then separated on a silica gel column (SiO2, EA:MeOH = 88:12). The reaction yielded dimethyl (5-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromobenzyl)phosphonate (200 mg, 0.41 mmol, 37%) as a white solid product. ESI-MS m / z = 485.9, 487.9 [M+H] + ;Calcu.=485.0,487.0.。

[0201] Step E: To dimethyl (5-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromobenzyl)phosphonate (195 mg, 0.4 mmol) dissolved in acetonitrile (20 mL) at room temperature, lithium bromide (209 mg, 2.4 mmol) was added. After complete addition, the mixture was heated to reflux and stirred for 6 hours. The mixture was concentrated to dryness under reduced pressure at 40°C. The residue was dissolved in methanol (5 mL) and separated by reverse phase chromatography using an X-select C18 column (19 mm x 150 mm); mobile phase: water (containing 0.1% NH₄HCO₃) and acetonitrile; flow rate: 40 mL / min; gradient: acetonitrile from 0% to 100% over 20 minutes; detection wavelength: 254 nm. The mixture was lyophilized under reduced pressure. The reaction yielded methyl hydrogen (5-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromobenzyl)phosphonate (180 mg, 0.38 mmol, 95%) as a white solid. ESI-MS m / z = 471.8, 473.8 [M+H] + ;Calcu.=471.0,473.0.

[0202] Step F: At room temperature, N,N-diisopropylethylamine (0.26 mL, 1.6 mmol) and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (1.19 g, 2.28 mmol) were added to a solution of methyl hydrogen (5-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromobenzyl)phosphonate (180 mg, 0.38 mmol), N,N-dimethylformamide (50 mL) and 1,2-dichloroethane (330 mL). After the addition was complete, the mixture was stirred at room temperature overnight. The reaction solution was concentrated to dryness under reduced pressure to obtain a crude product, which was separated on a silica gel column (SiO2, EA:MeOH=88:12). The reaction gave a white product 1. 4 -Amino-3 4 -Bromo-5-methoxy-1 1 H-6,9-dioxa-5-phospha-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzocyclononane-5-oxide (105 mg, 0.23 mmol, 60%). ESI-MS m / z = 453.8, 455.8 [M+H] +; Calcu. = 453.0, 455.0.

[0203] Step G: At room temperature and under N2 protection, dissolve 1 4 -Amino-3 4 -Bromo-5-methoxy-1 1H-6,9-dioxa-5-phospha-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzocyclononane-5-oxide (18.2 mg, 0.04 mmol), potassium trifluoro(pyrrolidin-1-ylmethyl)borate (19.1 mg, 0.1 mmol), sodium carbonate (12.7 mg, 0.12 mmol), and 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (7.6 mg, 0.016 mmol) in 1,4-dioxane (1.6 mL) and water (0.4 mL) were added, followed by trisdibenzylideneacetone dipalladium (7.3 mg, 0.008 mmol). After the addition was complete, the mixture was heated to 90°C and stirred for 3 hours under protective atmosphere. The mixture was filtered and washed with a small amount of methanol. The filtrate was concentrated to about 1 mL, and the residue was subjected to HPLC preparative separation. Preparation conditions: YMC-Actus Triart C18 (19 mm × 250 mm, 5 μm); A: 0.1% formic acid in water, B: MeCN; 18 mL / min; 0-3 min, 10% B; 3-3.5 min, 10%-20% B; 3.5-17 min, 20%-65% B; detector, UV 254 nm. After purification, compound B-5 was obtained as a white solid product 1. 4 -amino-5-methoxy-3 4 -(pyrrolidin-1-ylmethyl)-1 1 H-6,9-dioxy-5-phospha-1-(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzazepinenonane-5-oxide, formate (2 mg, 0.004 mmol, 10%). 1 H NMR (500 MHz, DMSO-d6) δ ppm 8.23(s,1H),7.90(s,1H),7.70(s,1H),7.48(s,1H),7.16-7.17(m,1H),7. 08-7.10(m,1H),6.64(s,1H),5.32-5.37(m,1H),5.26-5.29(m,1H),4.73-4 .75(m,2H),3.96-3.97(m,2H),3.82(d,J=10.0Hz,2H),3.62-3.66(m,2H), 3.57(d,J=10.0Hz,3H),2.39-2.40(m,2H),2.34-2.36(m,2H),1.66(m,4H). ESI-MS m / z=459.0[M+H] + ;Calcu.=458.2.

[0204] Example 6: Synthesis of Compound B-6

[0205] Step A: To a thick-walled pressure tube, add methyl 3-(bromomethyl)-4-fluorobenzoate (3.8 g, 15.38 mmol), toluene (20 mL), and diethyl methylphosphite (3.5 mL, 23.07 mmol) in sequence. After complete addition, the reaction mixture was heated and stirred at 110°C for 3 hours. The solvent was concentrated under reduced pressure, and the resulting crude product was separated on a silica gel column (DCM:MeOH = 90:10). The reaction yielded the product, methyl 3-((ethoxy(methyl)phosphoryl)methyl)-4-fluorobenzoate (3.45 g, 12.58 mmol, 82%), as a yellow oil.

[0206] Step B: To methyl 3-((ethoxy(methyl)phosphoryl)methyl)-4-fluorobenzoate (3.45 g, 12.58 mmol) dissolved in tetrahydrofuran (25 mL) at 65°C, sodium borohydride (2.38 g, 62.90 mmol) was added, followed by the slow dropwise addition of methanol (25 mL). After stirring, the reaction mixture was stirred at 65°C for 2 hours. The reaction was quenched by the addition of saturated ammonium chloride, and the mixture was extracted with water and dichloromethane. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to afford the product, ethyl (2-fluoro-5-(hydroxymethyl)benzyl)(methyl)phosphinate (2.34 g, 9.50 mmol, 76%), as a yellow oil. ESI-MS m / z = 246.9 [M+H] + ;Calcu.=246.1.

[0207] Step C: Phosphorus tribromide (1 mL, 10.64 mmol) was added to ethyl (2-fluoro-5-(hydroxymethyl)benzyl)(methyl)phosphinate (2.34 g, 9.50 mmol) dissolved in dichloromethane (30 mL) at 0°C. After stirring, the reaction mixture was stirred at 0°C for 10 minutes. Water was added to the reaction system to quench the reaction, and the mixture was extracted with dichloromethane. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was then separated on a silica gel column (DCM:MeOH = 90:10) to yield the product, ethyl (5-(bromomethyl)-2-fluorobenzyl)(methyl)phosphinate (1.7 g, 5.50 mmol, 58%). ESI-MS m / z = 308.8, 310.8 [M+H] + ;Calcu.=308.0,310.0.

[0208] Step D: To a solution of 2-((4-amino-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethane-1-ol (66 mg, 0.34 mmol) and ethyl (5-(bromomethyl)-2-fluorobenzyl)(methyl)phosphinate (209 mg, 0.68 mmol) in N,N-dimethylformamide (5 mL) at room temperature was added potassium carbonate (93 mg, 0.68 mmol). After the addition was complete, the mixture was heated to 60°C and stirred for 3 hours. The reaction mixture was filtered and concentrated to dryness under reduced pressure. The crude product was subjected to silica gel column chromatography (SiO2, EA:MeOH = 88:12) to afford ethyl 5-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-fluorobenzyl)(methyl)phosphinate (27 mg, 0.064 mmol, 19%) as a white solid. ESI-MS m / z = 424.0 [M+H] + ;Calcu.=423.2.

[0209] Step E: To ethyl 5-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-fluorobenzyl)(methyl)phosphinate (25 mg, 0.059 mmol) dissolved in dichloromethane (2 mL) at 0°C was added trimethylsilyl bromide (180 mg, 1.18 mmol). After complete addition, the mixture was stirred at room temperature overnight. The reaction mixture was quenched by adding 0.6 mL of MeOH:H2O (83:17) and concentrated to dryness under reduced pressure. The crude product was separated by HPLC. Preparation conditions: Waters XBridge C18 (19 mm × 250 mm, 5 μm); A: 0.1% formic acid in water, B: MeCN; 18 mL / min; 0-3 min, 5% B; 3-3.5 min, 5%-10% B; 3.5-15 min, 10%-60% B; detector, UV 254 nm. The reaction yielded the product (5-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-fluorobenzyl)(methyl)phosphinic acid (5 mg, 0.013 mmol, 21%)) as a white solid. ESI-MS m / z = 395.9 [M+H] + ;Calcu.=395.1.

[0210] Step F: To a solution of (5-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-fluorobenzyl)(methyl)phosphinic acid (4 mg, 0.01 mmol) in 1,2-dichloroethane (8.7 mL) and N,N-dimethylformamide (1.3 mL) at room temperature was added N,N-diisopropylethylamine (5.5 mg, 0.042 mmol) and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (32 mg, 0.061 mmol). The mixture was stirred at room temperature overnight. The solution was concentrated under reduced pressure to approximately 1 mL at room temperature, and the residue was subjected to preparative HPLC separation. Preparation conditions: Waters XBridge C18 (19 mm × 250 mm, 5 μm); A: 0.1% formic acid in water, B: MeCN; 18 mL / min; 0-3 min, 5% B; 3-3.5 min, 5%-15% B; 3.5-15 min, 10%-55% B; detector, UV 254 nm). The reaction yielded compound B-6, a white solid product 1. 4 -Amino-3 4 -fluoro-5-methyl-11H-6,9-dioxa-5-phospha-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzocyclononane-5-oxide (1.5 mg, 0.004 mmol, 40%). 1 H NMR(500MHz,DMSO-d6)δppm 7.90(s,1H),7.70(s,1H),7.53(s,1H),7.20-7.23(m,1H),7.08-7.11(m,1H),6.60(s,1H),5.37-5.40(m,1H),5.29-5.32(m, 1H), 4.88-4.91 (m, 1H), 4.61-4.64 (m, 1H), 4.03-4.05 (m, 1H), 3.90-3.93 (m, 1H), 3.11-3.16 (m, 2H), 1.34 (d, J = 14.0Hz, 3H). ESI-MS m / z=377.9[M+H] + ;Calcu.=377.1.

[0211] Example 7: Synthesis of Compound B-7 / M-2

[0212] Step A: To a solution of 2-((4-amino-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethan-1-ol (4.00 g, 20.5 mmol) in N,N-dimethylformamide (10 mL) was added tert-butyldimethylsilyl chloride (4.63 g, 30.7 mmol) and imidazole (3.49 g, 51.2 mmol) at room temperature. The reaction mixture was reacted at room temperature for 2 hours. After completion of the reaction, the mixture was concentrated to obtain a crude product, which was then separated on a silica gel column (SiO2, DCM:MeOH = 95:5) to afford 6-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (5.60 g, 18.1 mmol, 88%) as a white solid. ESI-MS m / z = 310.0 [M+H] + ;Calcu.=309.2.

[0213] Step B: At room temperature, N-bromosuccinimide (17.48 g, 98.2 mmol) and azobisisobutyronitrile (1.08 g, 6.57 mmol) were added to a solution of methyl 4-bromo-3-methylbenzoate (15.0 g, 65.5 mmol) in carbon tetrachloride (300 mL). The reaction system was replaced with N2 protection. The reaction mixture was heated to 90°C and reacted for 8 hours. After the reaction was completed, the mixture was filtered and the insoluble matter was washed with ethyl acetate. The filtrate was dried to obtain a crude product, which was separated on a silica gel column (SiO2, PE:EA = 90:10) to obtain methyl 4-bromo-3-(bromomethyl)benzoate (12.5 g, 40.6 mmol, 62%). 1 H NMR (500MHz, DMSO-d6) δppm 8.13 (d, J = 2.1 Hz, 1H), 7.82 (dd, J = 8.3, 2.1 Hz, 1H), 7.67 (d, J = 8.3 Hz, 1H), 4.63 (s, 2H), 3.94 (s, 3H).

[0214] Step C: At room temperature, diethyl methylphosphite (5.30 g, 38.97 mmol) was added to a sealed tube containing a solution of methyl 4-bromo-3-(bromomethyl)benzoate (10.0 g, 32.47 mmol) in toluene. The reaction mixture was heated to 110°C and reacted for 3 hours. After completion of the reaction, the reaction mixture was cooled to room temperature and concentrated to obtain a crude product. The crude product was separated on a silica gel column (SiO2, DCM:MeOH = 95:5) to obtain methyl 4-bromo-3-((ethoxy(methyl)phosphoryl)methyl)benzoate (9.10 g, 27.15 mmol, 84%) as a colorless oil. ESI-MS m / z = 334.8 [M+H] + ;Calcu.=334.0.

[0215] Step D: Sodium borohydride (9.93 g, 262.6 mmol) was added to a solution of methyl 4-bromo-3-((ethoxy(methyl)phosphoryl)methyl)benzoate (8.80 g, 26.26 mmol) in tetrahydrofuran (100 mL) at room temperature. The reaction mixture was allowed to react at room temperature for 20 minutes. Methanol (100 mL) was then slowly added dropwise to the reaction system. After the addition, the reaction mixture was heated to 70°C and the reaction was continued for 6 hours. After the reaction, the reaction mixture was cooled to room temperature and concentrated to obtain a crude product. The crude product was separated on a silica gel column (SiO2, DCM:MeOH = 90:10) to obtain ethyl (2-bromo-5-(hydroxymethyl)benzyl)(methyl)phosphinate (6.60 g, 21.49 mmol, 82%) as a colorless oil. ESI-MS m / z = 306.8 [M+H] + ;Calcu.=306.0.

[0216] Step E: Tosyl chloride (14.90 g, 78.14 mmol) and 4-dimethylaminopyridine (239 mg, 1.954 mmol) were added sequentially to a solution of ethyl (2-bromo-5-(hydroxymethyl)benzyl)(methyl)phosphinate (6.00 g, 19.54 mmol) in 1,4-dioxane (200 mL) at room temperature. The reaction mixture was cooled to 0°C and the reaction system was replaced with N2 protection. Triethylamine (27 mL, 195.4 mmol) was slowly added dropwise to the reaction system. After the addition was complete, the reaction mixture was allowed to slowly warm to room temperature and the reaction was continued for 18 hours. After completion of the reaction, the reaction mixture was filtered and the filtrate was concentrated to obtain a crude product. The crude product was separated on a silica gel column (SiO2, DCM:MeOH=95:5) to obtain 4-bromo-3-((ethoxy(methyl)phosphoryl)methyl)benzyl-4-methylbenzenesulfonate (3.80 g, 8.24 mmol, 42%) as a colorless oil. ESI-MS m / z=324.7[M-135] + ;Calcu.=460.0.

[0217] Step F: 6-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (2.82 g, 9.10 mmol) and potassium carbonate (3.15 g, 22.75 mmol) were added sequentially to a solution of 4-bromo-3-((ethoxy(methyl)phosphoryl)methyl)benzyl-4-methylbenzenesulfonate (3.50 g, 7.59 mmol) in N,N-dimethylformamide (30 mL) at room temperature. The reaction mixture was heated to 60° C. and reacted for 3 hours. After the reaction, the reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was separated on a silica gel column (SiO2, DCM:MeOH=96:4) to obtain a colorless oily substance 5-((4-amino-6-(2-((tert-butyldimethylsilyl)oxy)ethoxy) -1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromobenzyl)(methyl)ethyl phosphinate (2.70 g, 4.51 mmol, 59%). ESI-MS m / z = 598.0 [M+H] + ;Calcu.=597.1。

[0218] Step G: At 0°C, trimethylsilyl bromide (7.67 g, 50.12 mmol) was slowly added dropwise to a solution of ethyl 5-((4-amino-6-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromobenzyl)(methyl)phosphinate (2.00 g, 3.34 mmol) in dichloromethane (100 mL). After the addition was complete, the reaction mixture was slowly warmed to room temperature and the reaction was continued for 18 hours. After the reaction was complete, methanol (50 mL) was added to the reaction system, and then 3 M sodium hydroxide solution was slowly added dropwise to adjust the pH of the reaction solution to neutral. The reaction solution was concentrated to obtain a crude product, which was dissolved in 20 mL of a mixed solvent (methanol:acetone = 1:1). The insoluble matter was removed by filtration, and the filtrate was concentrated to obtain a pale yellow crude product (5-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromobenzyl)(methyl)phosphinic acid (4.50 g, crude). The crude product was used directly in the next reaction without purification. ESI-MS m / z = 455.8 [M+H] + ;Calcu.=455.0.

[0219] Step H: 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (13.87 g, 26.65 mmol) and N,N-diisopropylethylamine (8.61 g, 66.63 mmol) were added sequentially to a mixed solvent of (5-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromobenzyl)(methyl)phosphinic acid (4.50 g, crude product) in dichloromethane (600 mL) and N,N-dimethylformamide (200 mL) at room temperature. The reaction mixture was reacted at room temperature for 48 hours. After the reaction was completed, water (20 mL) was added to the reaction system to quench the reaction. The reaction solution was concentrated to obtain a crude product, which was separated on a silica gel column (SiO2, DCM:MeOH=90:10) to obtain 1 4 -Amino-3 4 -Bromo-5-methyl-1 1 H-6,9-dioxa-5-phospha-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzocyclononane-5-oxide (380 mg, 0.867 mmol, 26% yield over two steps). 1 H NMR(500MHz,DMSO-d6)δppm 7.93(s,1H),7.90(s,1H),7.74-7.83(m,1H),7.54-7.55(d,J=5.0Hz,2H ),7.15-7.16(d,J=5.0Hz,1H),5.37-5.40(d,J=15.0Hz,1H),5.29-5.32 (d,J=15.0Hz,1H),4.99-5.04(m,1H),4.59-4.63(m,1H),4.10-4.14(m, 1H), 3.95-4.01 (m, 1H), 3.00-3.03 (m, 2H), 1.27-1.30 (d, J = 15.0Hz, 3H). ESI-MS m / z=437.8[M+H] + ;Calcu.=437.0.

[0220] Step I: Potassium trifluoro((pyrrolidin-1-yl)methyl)borate (87.2 mg, 0.456 mmol), sodium carbonate (72.6 mg, 0.685 mmol), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (43.5 mg, 0.091 mmol) and trisdibenzylideneacetone dipalladium (41.8 mg, 0.046 mmol) were added to 1 4 -Amino-3 4 -Bromo-5-methyl-1 1H-6,9-dioxa-5-phospha-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzocyclononane-5-oxide (100 mg, 0.228 mmol) was dissolved in 1,4-dioxane (4 mL) and water (1 mL). The reaction system was replaced with N2 protection. The reaction mixture was heated to 90°C for 3 hours. After the reaction, the reaction solution was cooled to room temperature and concentrated to obtain a crude product. The crude product was subjected to preparative separation under the following preparation conditions: Waters XBridge C18 (30 mm×250 mm, 5 μm); A: 0.1% formalin in water, B: MeCN; 18 mL / min; 0-3.5 min, 5% B; 3.5-15 min, 5%-50% B; 15-17 min, 50%-70% B; detector, UV 254 nm). The prepared fraction was lyophilized to obtain compound B-7, a white solid product 1 4 -amino-5-methyl-3 4 -(pyrrolidin-1-ylmethyl)-1 1 H-6,9-dioxo-5-phospha-1-(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzazepinenonane-5-oxide, formate salt (8 mg, 0.016 mmol, 7%). 1 H NMR(500MHz,DMSO-d6)δppm 8.24(s,1H),7.90(s,1H)7.77-7.79(m,1H),7.70(s,1H),7.51-7.53(m,1H),7.17(d,J=10.0Hz 1H),7.08(d,J=5.0Hz 1H),5.37(d,J=15.0Hz,1H),5.26-5.29(d,J=15.0Hz,1H),4.66-4.74(m,2H),3.82-3.95(m, 4H), 3.49-3.56 (m, 2H), 2.30-2.38 (m, 4H), 1.61-1.69 (m, 4H), 1.32-1.35 (d, J = 15.0Hz, 3H). ESI-MS m / z=443.0[M+H] + ;Calcu.=442.2.

[0221] Example 8: Synthesis of Compound B-8

[0222] Step A: To methyl 3-(bromomethyl)-4-methoxybenzoate (5 g, 19.3 mmol) dissolved in toluene (50 mL) was added diethyl methylphosphite (3.94 g, 28.95 mmol) at room temperature. After the addition, the mixture was heated to 110°C and stirred for 4 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was then separated on a silica gel column (SiO2, DCM:MeOH = 95:5). The reaction afforded methyl 3-((ethoxy(methyl)phosphoryl)methyl)-4-methoxybenzoate (5.02 g, 17.54 mmol, 91%) as a colorless oil. ESI-MS m / z = 286.9 [M+H] + ;Calcu.=286.1.

[0223] Step B: Diisobutylaluminum hydride (52.6 mL, 52.62 mmol) was added to methyl 3-((ethoxy(methyl)phosphoryl)methyl)-4-methoxybenzoate (5.02 g, 17.54 mmol) in dichloromethane (50 mL) at -30°C. After the addition, the mixture was stirred at -30°C for 3 hours. The reaction mixture was quenched with methanol and concentrated under reduced pressure to obtain the crude product. The crude product was separated on a silica gel column (SiO2, DCM:MeOH = 95:5). The reaction afforded the product (ethyl 5-(hydroxymethyl)-2-methoxybenzyl)(methyl)phosphinate (2.3 g, 8.91 mmol, 51%) as a colorless oil. ESI-MS m / z = 258.9 [M+H] + ;Calcu.=258.1.

[0224] Step C: To ethyl (5-(hydroxymethyl)-2-methoxybenzyl)(methyl)phosphinate (1.50 g, 5.81 mmol) dissolved in dichloromethane (15 mL) at 0°C, add thionyl chloride (1.24 g, 10.46 mmol). After the addition, the mixture was stirred at 0°C for 2 hours. Methanol was added to the reaction solution, and the mixture was concentrated under reduced pressure to obtain a crude product. The crude product was separated on a silica gel column (SiO2, DCM:MeOH = 95:5). The reaction yielded ethyl (5-(chloromethyl)-2-methoxybenzyl)(methyl)phosphinate (800 mg, 2.60 mmol, 45%) as a colorless oil. 1 H NMR(500MHz,DMSO-d6)δppm 7.28-7.32(m,2H),6.99(d,J=8.1Hz,1H),4.71(s,2H),3.94(q,J=7.2Hz,2H), 3.80 (s, 3H), 3.06-3.21 (m, 2H), 1.29 (d, J = 14.0Hz, 3H), 1.18 (t, J = 7.0Hz, 3H).

[0225] Step D: At room temperature, potassium carbonate (637 mg, 4.61 mmol) was added to N,N-dimethylformamide (10 mL) in which 2-((4-amino-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethane-1-ol (450 mg, 2.306 mmol) was dissolved. After the addition, the mixture was stirred at room temperature for 0.5 hours. Ethyl (5-(chloromethyl)-2-methoxybenzyl)(methyl)phosphinate (829 mg, 2.997 mmol) was then added and the temperature was raised to 60°C and the reaction was allowed to proceed overnight. The reaction solution was filtered through celite and then concentrated under reduced pressure to obtain a crude product. The crude product was dissolved in methanol (4 mL) and subjected to reverse phase separation. The chromatographic column was Santai SepaFlash C18 (Spherical C18, 20-45 μm, 80g); mobile phase: water (containing 0.05% TFA) and acetonitrile; flow rate: 40 ml / min; gradient: acetonitrile from 0% to 100% over 30 minutes; detection wavelength: 254 nm. Freeze-dried under reduced pressure to obtain the product (ethyl 5-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-methoxybenzyl)(methyl)phosphinate (90 mg, 0.186 mmol, 8%) as a colorless oil. ESI-MS m / z = 436.0 [M+H] + ;Calcu.=435.2.

[0226] Step E: At room temperature, sodium hydroxide (18.4 mg, 0.46 mmol) in water (1 mL) was added to a solution of ethyl (5-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-methoxybenzyl)(methyl)phosphinate (40 mg, 0.092 mmol) in methanol (1 mL). After the addition, the mixture was slowly warmed to 80°C and stirred for 8 hours. The reaction solution was directly subjected to reverse phase separation using a Santai SepaFlash C18 (Spherical C18, 20-45 μm, 40 g); mobile phase: water (containing 0.05% TFA) and acetonitrile; flow rate: 40 ml / min; gradient: acetonitrile from 0% to 100% over 30 minutes; detection wavelength: 254 nm. Freeze-dried under reduced pressure to obtain (5-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-methoxybenzyl)(methyl)phosphinic acid (24 mg, 0.059 mmol, 64%) as a white solid. ESI-MS m / z = 408.0 [M+H] + ;Calcu.=407.1.

[0227] Step F: To a solution of (5-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-methoxybenzyl)(methyl)phosphinic acid (24 mg, 0.059 mmol) and N,N-diisopropylethylamine (32 mg, 0.248 mmol) in N,N-dimethylformamide (8 mL) and 1,2-dichloroethane (53 mL) at room temperature was added 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (184 mg, 0.354 mmol). After the addition was complete, the mixture was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was then preparatively isolated under the following conditions: Waters XSelect C18 (30 mm × 250 mm, 5 μm); A: 0.1% formic acid in water, B: MeCN; 50 mL / min; 0-3 min, 5% B; 3-3.5 min, 5%-9% B; 3.5-15 min, 9%-32% B; detector, UV 254 nm). The product was lyophilized under reduced pressure to obtain compound B-8, 1 4 -Amino-3 4 -methoxy-5-methyl-1 1 H-6,9-dioxo-5-phospha-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzocyclononane-5-oxide (1.16 mg, 0.003 mmol, 5%). 1 H NMR(500MHz,DMSO-d6)δppm 8.15-8.19(m,1H),7.45-7.49(m,1H),7.26-7.29(m,1H),7.17-7.20(m,1H),7.10-7.14(m,1H),6.96-7.00(m,1H),5.31-5.34(m ,2H),4.29-4.40(m,1H),4.17-4.25(m,1H),4.04-4.13(m,1H),3.90-4.02(m,1H),3.75-3.81(m,3H),2.62-2.66(m,2H),1.30(br d,J=14.0Hz,3H). ESI-MS m / z=390.0[M+H] + ;Calcu.=389.1.

[0228] Example 9: Synthesis of Compound B-9

[0229] Step A: Potassium (dimethylamino)methyltrifluoroborate (37.6 mg, 0.228 mmol), sodium carbonate (29 mg, 0.274 mmol), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (17.4 mg, 0.036 mmol) and trisdibenzylideneacetone dipalladium (16.7 mg, 0.018 mmol) were added to 1 4 -Amino-3 4 -Bromo-5-methyl-1 1 H-6,9-dioxa-5-phospha-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzocyclononane-5-oxide (40 mg, 0.091 mmol) was dissolved in 1,4-dioxane (4 mL) and water (1 mL). The reaction system was replaced with N2 protection. The reaction mixture was heated to 90°C for 3 hours. After the reaction, the reaction solution was cooled to room temperature and concentrated to obtain a crude product. The crude product was subjected to preparative separation under the following conditions: Waters XBridge C18 (19 mm×250 mm, 5 μm); A: 0.1% formic acid in water, B: CH3CN; 18 mL / min; 0-3 min, 5% B; 3-15 min, 5%-50% B; 15-20 min, 50%-100% B; detector, UV 254 nm). The preparative fraction was lyophilized to obtain compound B-9, a white solid product 1. 4 -Amino-3 4 -((dimethylamino)methyl)-5-methyl-1 1 H-6,9-dioxy-5-phospha-1-(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzazepanonane-5-oxide, formate (5 mg, 0.011 mmol, 12%). 1 H NMR (500 MHz, DMSO-d6) δ ppm 8.17(s,1H),7.90(s,1H),7.78(brs,1H),7.71(s,1H),7.54(brs,1H),7.12-7. 14(m,1H),7.08-7.10(m,1H),5.38(d,J=15.0Hz,1H),5.28(d,J=15.0Hz,1H),4. 66-4.74(m,2H),3.83-3.94(m,2H),3.75(d,J=15.0Hz,1H),3.55-3.61(m,1H), 3.18-3.21(m,1H),3.10(d,J=15.0Hz,1H),2.12(s,6H),1.35(d,J=15.0Hz,3H). ESI-MS m / z=417.0[M+H]+ ;Calcu.=416.2.

[0230] Example 10: Synthesis of Compound B-10

[0231] Step A: To a solution of 6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine (230 mg, 1.36 mmol) and potassium carbonate (562 mg, 4.07 mmol) dissolved in N,N-dimethylformamide (3 mL) at 25°C was slowly added a solution of 1-(3-(bromomethyl)phenyl)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-N-methylmethanesulfonamide (592 mg, 1.36 mmol) in N,N-dimethylformamide (3 mL). After the addition was complete, the mixture was stirred at 25°C for 18 hours. The reaction mixture was concentrated under reduced pressure to obtain a solid residue, which was then separated on a silica gel column (SiO2, DCM:MeOH = 90:10). The reaction yielded a pale yellow solid product, 1-(3-((4-amino-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenyl)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-N-methylmethanesulfonamide (285 mg, 0.543 mmol, 40%). ESI-MS m / z = 525.0 [M+H] + ;Calcu.=524.2.

[0232] Step B: Acetic acid (46 μL, 0.8 mmol) was slowly added to a tetrabutylammonium fluoride solution (0.8 mL, 0.8 mmol) at 0-5°C until the pH of the solution reached 7.0. The above-prepared mixed solution was then slowly added dropwise to a solution of 1-(3-((4-amino-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenyl)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-N-methylmethanesulfonamide (280 mg, 0.533 mmol) dissolved in tetrahydrofuran (5 mL) at 0-5°C. After the addition, the mixture was stirred at 25°C for 3 hours. The reaction solution was directly concentrated under reduced pressure to obtain a solid residue, which was then separated on a silica gel column (SiO2, DCM:MeOH = 90:10). The reaction yielded a pale yellow solid product, 1-(3-((4-amino-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenyl)-N-(2-hydroxyethyl)-N-methylmethanesulfonamide (155 mg, 0.377 mmol, 71%). ESI-MS m / z = 410.9 [M+H] + ;Calcu.=410.1.

[0233] Step C: To 1-(3-((4-amino-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenyl)-N-(2-hydroxyethyl)-N-methylmethanesulfonamide (170 mg, 0.431 mmol) dissolved in acetonitrile (80 mL) was added cesium carbonate (674 mg, 2.069 mmol) and lithium iodide (277 mg, 2.069 mmol) at 25°C, and the whole mixture was heated to 80°C and stirred at 80°C for 6 hours. The solid residue was subjected to preparative HPLC separation under the following conditions: YMC-Actus Triart C18, 20*250; A: 5 mM ammonia in water; B: MeCN; 18 mL / min; 0-3 min, 5% B; 3-3.5 min, 5%-20% B; 3.5-15 min, 20%-60%; 15-15.5 min, 60%-100% B; detector, UV 254 nm. This reaction yielded B-10, a white solid product 1. 4 -amino-6-methyl-1 1 H-9-Oxa-5-thia-6-aza-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzazepanonane 5,5-dioxide (8.26 mg, 0.022 mmol, 5%). 1 H NMR(500MHz,DMSO-d6)δppm 7.94(s,1H),7.91(s,1H),7.58(m,2H),7.34(m,1H),7.27(m,1H),7.13(d,J =7.6Hz,1H),5.38(s,2H),4.58(m,4H),3.13(t,J=6.3Hz,2H),2.44(s,3H). ESI-MS m / z=375.0[M+H] + ;Calcu.=374.1.

[0234] Example 11: Synthesis of Compound B-11 / B-12

[0235] Step A: At 20 ° C, 4-bromo-3-(bromomethyl)benzoic acid methyl ester (4.0 g, 12.99 mmol) was dissolved in ethanol (40 mL). Thiourea (1.09 g, 14.29 mmol) was added, and the whole mixture was heated to 96 ° C and stirred at 96 ° C for 2 hours. It was then concentrated under reduced pressure to remove ethanol to obtain a white solid. Acetonitrile (18 mL) was then added to the white solid, and the whole mixture was cooled to 0-5 ° C. and 2N hydrochloric acid (18.2 mL, 36.4 mmol) was slowly added dropwise at 0-5 ° C. After the addition, the whole mixture was stirred at 0-5 ° C for 20 minutes. Then, N-chlorosuccinimide (6.94 g, 51.95 mmol) was added to the mixture in portions at 0-5 ° C until the solution was clear. After the addition, the whole mixture was stirred at 20 ° C for 20 minutes. The reaction mixture was concentrated under reduced pressure to remove most of the acetonitrile, then extracted twice with ethyl acetate (50 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a solid residue. The solid residue was separated on a silica gel column (SiO2, PE:EA = 75:25) to obtain methyl 4-bromo-3-((chlorosulfonyl)methyl)benzoate (3.15 g, crude) as a white solid.

[0236] Step B: To a solution of 2-(tert-butyldimethylsilyl)oxy)-N-methylethane-1-amine (2.18 g, 11.54 mmol), N,N-diisopropylethylamine (3.73 g, 28.85 mmol), and 4-dimethylaminopyridine (587 mg, 4.81 mmol) dissolved in dichloromethane (10 mL) was slowly added dropwise under N₂ at 25°C. The mixture was stirred at 25°C under N₂ for 18 hours. The reaction mixture was added with water (50 mL) and extracted twice with dichloromethane (20 mL x 2). The organic phases were combined, washed once with saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a solid residue. The solid residue was separated on a silica gel column (SiO2, PE:EA = 83:17). The reaction afforded methyl 4-bromo-3-((N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-N-methylaminosulfonyl)methyl)benzoate (1.52 g, 3.16 mmol, 24% yield over two steps) as a yellow oil. 1H NMR(500MHz,DMSO-d6)δppm 8.13(d,J=1.8Hz,1H),7.84(m,2H),4.67(m,2H),3.88(s,3H),3.66(t,J=5. 8Hz,2H),3.34(s,2H)3.17(t,J=5.8Hz,2H),2.87(s,3H),0.85(s,9H),0.04 (s,6H).

[0237] Step C: To a solution of methyl 4-bromo-3-((N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-N-methylaminosulfonyl)methyl)benzoate (1.52 g, 3.16 mmol) in tetrahydrofuran (10 mL) at 0-5°C, lithium aluminum hydride (96.04 mg, 2.53 mmol) was slowly added. The mixture was then stirred at 25°C for 0.5 hours. The reaction mixture was cooled to 0-5°C, and sodium sulfate nonahydrate was slowly added until no bubbles formed. The mixture was then stirred at room temperature for 30 minutes, filtered through celite, and the filtrate was collected and concentrated under reduced pressure to obtain a solid residue. The solid residue was separated on a silica gel column (SiO2, PE:EA = 80:20). The reaction afforded a yellow oily product, 1-(2-bromo-5-(hydroxymethyl)phenyl)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-N-methylmethanesulfonamide (1.90 g, crude product). ESI-MS m / z = 451.9 [M+H] + ;Calcu.=451.1.

[0238] Step D: To 1-(2-bromo-5-(hydroxymethyl)phenyl)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-N-methylmethanesulfonamide (1.90 g, crude) and triphenylphosphine (1.43 mg, 5.46 mmol) dissolved in acetonitrile (20 mL) at 0-5°C, slowly add N-bromosuccinimide (972 mg, 5.46 mmol), and continue stirring at 25°C for 2.5 hours. The reaction mixture is washed once with saturated aqueous sodium bicarbonate (50 mL) and once with saturated aqueous sodium chloride (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a solid residue. The solid residue is separated on a silica gel column (SiO2, PE:EA = 85:15). The reaction afforded a colorless, transparent oily product, 1-(2-bromo-5-(bromomethyl)phenyl)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-N-methylmethanesulfonamide (523 mg, 1.02 mmol, 32% yield over two steps). 1H NMR(500MHz,DMSO-d6)δppm 7.68(d,J=8.2Hz,1H),7.59(d,J=2.1Hz,1H),7.38(dd,J=8.2,2.1Hz,1H),4.72(s,2H),4.56 (s, 2H), 3.66 (t, J = 5.8Hz, 2H), 3.16 (t, J = 5.8Hz, 2H), 2.86 (s, 3H), 0.85 (s, 9H), 0.04 (m, 6H).

[0239] Step E: To a solution of 6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine (246 mg, 1.02 mmol) and potassium carbonate (421 mg, 3.04 mmol) in N,N-dimethylformamide (3 mL) was slowly added dropwise at 25°C. After completion of the addition, the mixture was stirred at 25°C for 15 hours. The reaction mixture was concentrated under reduced pressure to obtain a solid residue. The solid residue was separated on a silica gel column (SiO2, DCM:MeOH = 90:10). The reaction afforded the product 1-(5-((4-amino-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromophenyl)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-N-methylmethanesulfonamide (251 mg, 0.416 mmol, 41%) as a yellow oil. 1 H NMR(500MHz,DMSO-d6)δppm 8.28(m,2H),8.13(s,1H),7.64(d,J=8.3Hz,1H),7.31(d,J=1.6Hz,1H),7.15(dd,J=8.3,1.8Hz,1H),5.44 (s,2H),4.52(s,2H),3.59(t,J=5.8Hz,2H),3.04(t,J=5.7Hz,2H),2.77(s,3H),0.84(s,9H),0.02(s,6H). ESI-MS m / z=603.0[M+H] + ;Calcu.=602.1.

[0240] Step F: Tetrabutylammonium fluoride (0.54 mL, 0.54 mmol) was slowly added dropwise to a solution of 1-(5-((4-amino-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromophenyl)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-N-methylmethanesulfonamide (250 mg, 0.414 mmol) in tetrahydrofuran (5 mL) at 0-5°C. After completion of the addition, the mixture was stirred at 20°C for 1 hour. The reaction mixture was added with water (10 mL) and extracted twice with ethyl acetate (40 mL x 2). The organic phases were combined, washed once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a solid residue. The solid residue was separated on a silica gel column (SiO2, DCM:MeOH = 90:10). The reaction yielded an off-white solid product, 1-(5-((4-amino-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromophenyl)-N-(2-hydroxyethyl)-N-methylmethanesulfonamide (134 mg, 0.274 mmol, 66%). ESI-MS m / z = 488.8 [M+H] + ;Calcu.=488.0.

[0241] Step G: At 20°C, cesium carbonate (1.46g, 4.49mmol) and lithium iodide (301mg, 2.25mmol) were added to acetonitrile (30mL) in which 1-(5-((4-amino-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromophenyl)-N-(2-hydroxyethyl)-N-methylmethanesulfonamide (110mg, 0.225mmol) was dissolved. The whole mixture was then heated to 110°C and stirred at 110°C for 7 hours. The whole mixture was cooled to 20°C and stirred at 20°C for 14 hours. The reaction solution was filtered through celite, the filter cake was washed three times with acetonitrile (20*3), the filtrate was collected, and a solid residue was obtained by concentration under reduced pressure. The solid residue was separated by silica gel column (SiO2, DCM:MeOH=90:10). The reaction gave compound B-12, a white solid product 1 4 -Amino-3 4 -Bromo-6-methyl-1 1 H-9-oxa-5-thio-6-aza-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzazepane 5,5-dioxide (31 mg, 0.068 mmol, 30%). 1H NMR(500MHz,DMSO-d6)δppm 8.05(d,J=2.1Hz,1H),7.94(s,1H),7.61(m,3H),7.09(dd,J=8.2,2.1Hz,1H),5.38(s,2H),4.70(s,2H),4.46(br t,J=7.0Hz,2H), 3.04(br t,J=7.0Hz,2H), 2.72(s,3H). ESI-MS m / z=452.8[M+H] + ;Calcu.=452.0.

[0242] Step H: At 20°C, dissolve 1 4 -Amino-3 4 -Bromo-6-methyl-1 1 H-9-oxa-5-thio-6-aza-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzazepine 5,5-dioxide (10 mg, 0.022 mmol), potassium trifluoro(pyrrolidin-1-ylmethyl)borate (10.5 mg, 0.055 mmol) in 1,4-dioxane (4 mL) and water (1 mL) were added, and 2-dicyclohexylphosphine-2',4',6' -Triisopropylbiphenyl (3.2 mg, 0.007 mmol) and sodium carbonate (7.0 mg, 0.066 mmol), then the entire mixture was replaced with nitrogen three times, and then tris-dibenzylideneacetone palladium (3.0 mg, 0.003 mmol) was added to the mixture under nitrogen protection at 20°C. The entire mixture was replaced with nitrogen twice, heated to 90°C, and stirred at 90°C under nitrogen protection for 3 hours. The reaction solution was cooled to room temperature, filtered through celite, and concentrated under reduced pressure to obtain a light yellow product. The light yellow oil was subjected to HPLC preparative separation under the following preparation conditions: Waters XBridge Prep Shield RP18 5um OBD, 19*250; A: 5mM ammonia in water; B: MeCN; 16 mL / min; 0-3 min, 5% B; 3-15 min, 5%-95% B; detector, UV 254 nm. The reaction obtained compound B-12, a white solid product 1 4 -amino-6-methyl-3 4 -(pyrrolidin-1-ylmethyl)-1 1 H-9-Oxa-5-thia-6-aza-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzazepane 5,5-dioxide (0.50 mg, 0.001 mmol, 5%). 1H NMR(500MHz,DMSO-d6)δppm 7.98(m,1H),7.90(m,1H),7.66(m,2H),7.22(m,1H),7.11(m,1H),5.34(br s,2H),4.77(br s,2H),4.59(br d,J=5.6Hz,2H),3.63(m,2H),3.18(br d,J=6.4Hz,2H),2.55(s,3H),2.36(br s,4H),1.66(br s,4H). ESI-MS m / z=458.0[M+H] + ;Calcu.=457.2.

[0243] Example 12: Synthesis of Compound B-13 / M-3

[0244] Step A: To a solution of methyl 4-bromo-3-((chlorosulfonyl)methyl)benzoate (2.0 g, 3.05 mmol) and 2-((tert-butyldiphenylsilyl)oxy)ethane-1-amine (1.0 g, 3.339 mmol) in dichloromethane (100 mL) was added N,N-diisopropylethylamine (592 mg, 4.58 mmol) and DMAP (187 mg, 1.53 mmol) at 25°C. The mixture was stirred at room temperature for 3 hours. After completion of the reaction, the mixture was concentrated to obtain a solid residue. The residue was separated on a silica gel column (SiO2, PE:EA = 50:50) to obtain methyl 4-bromo-3-((N-(2-((tert-butyldiphenylsilyl)oxy)ethyl)sulfamoyl)methyl)benzoate (1.1 g, 61%) as a colorless oil. 1 H NMR(400MHz,DMSO-d6)δ8.10(d,1H),7.82(s,1H),7.61-7.65(m,4H),7.54(t,J=6.0Hz,1H),7.41- 7.48(m,6H),4.61(s,2H),3.86(s,3H),3.64(t,J=6.0Hz,2H),3.15(q,J=6.0Hz,2H),0.99(s,9H).

[0245] Step B: To a solution of methyl 4-bromo-3-((N-(2-(tert-butyldiphenylsilyl)oxy)ethyl)sulfamoyl)methyl)benzoate (1.1 g, 1.88 mmol) and di-tert-butyl dicarbonate (450 mg, 2.06 mmol) in tetrahydrofuran (40 mL) at 25°C, triethylamine (0.8 mL, 5.62 mmol) and 4-dimethylaminopyridine (45.8 mg, 0.375 mmol) were added. The mixture was stirred at room temperature for 3 hours. Upon completion of the reaction, the reaction mixture was concentrated to obtain a solid residue. The residue was separated on a silica gel column (SiO2, PE:EA=66:34) to give methyl 4-bromo-3-((N-(tert-butoxycarbonyl)-N-(2-((tert-butyldiphenylsilyl)oxy)ethyl)sulfamoyl)methyl)benzoate (1.0 g, 77%) as a colorless oily product. 1 H NMR (400MHz, DMSO-d6) δ7.56-7.58(m,5H),7.46-7.48(m,1H),7.45(t,J=2.0Hz,1H),7.39-7.44(m,5H),7.2 2(m,1H),5.08(s,2H),3.85(s,3H),3.57(t,J=5.0Hz,2H),3.46(t,J=5.0Hz,2H),1.48(s,9H),0.94(s,9H).

[0246] Step C: At 0°C, lithium aluminum hydride (55 mg, 1.45 mmol) was added portionwise to a solution of methyl 4-bromo-3-((N-(tert-butyloxycarbonyl)-N-(2-((tert-butyldiphenylsilyl)oxy)ethyl)sulfamoyl)methyl)benzoate (1.0 g, 1.448 mmol) in tetrahydrofuran (30 mL). After complete addition, the mixture was stirred at 0°C for 2 hours. After the reaction was complete, the reaction solution was kept at 0°C and then methanol (20 mL) was slowly added until no obvious bubbles appeared. The temperature was kept below 20°C during quenching. The mixture was then filtered through celite, and the filtrate was added with silica gel. The mixture was concentrated under reduced pressure to obtain a solid residue. The residue was separated on a silica gel column (SiO2, PE:EA = 50:50). The reaction afforded tert-butyl((2-bromo-5-(hydroxymethyl)benzyl)sulfonyl)(2-((tert-butyldiphenylsilyl)oxy)ethyl)carbamate (400 mg, 42%) as a white solid product. 1H NMR (400MHz, DMSO-d6) δ7.56-7.58(m,5H),7.46-7.48(m,1H),7.45(t,J=2.0Hz,1H),7.39-7.44(m,5H),7.22-7.24(m,1H),5.3 6(t,J=6.0Hz,1H),4.97(s,2H),4.44(d,J=5.0Hz,2H),3.57(t,J=5.0Hz,2H),3.54(t,J=5.0Hz,2H),1.47(s,9H),0.96(s,9H).

[0247] Step D: At 0°C, N-bromosuccinimide (161 mg, 0.905 mmol) was added portionwise to a solution of tert-butyl ((2-bromo-5-(hydroxymethyl)benzyl)sulfonyl)(2-((tert-butyldiphenylsilyl)oxy)ethyl)carbamate (400 mg, 0.604 mmol) and triphenylphosphine (237 mg, 0.905 mmol) in tetrahydrofuran (20 mL). After the addition was complete, the mixture was stirred at 0°C for 3 hours. After the reaction was complete, the residue was separated on a silica gel column (SiO2, PE:EA=84:16). The reaction afforded tert-butyl ((2-bromo-5-(bromomethyl)benzyl)sulfonyl)(2-((tert-butyldiphenylsilyl)oxy)ethyl)carbamate (400 mg, 91%) as an oil. ESI-MS m / z=723.9 [M+H] + ;Calcu.=723.1.

[0248] Step E: To a solution of tert-butyl ((2-bromo-5-(bromomethyl)benzyl)sulfonyl)(2-((tert-butyldiphenylsilyl)oxy)ethyl)carbamate (400 mg, 0.551 mmol) and 6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine (140 mg, 0.827 mmol) in N,N-dimethylformamide (5 mL) was added potassium carbonate (190 mg, 1.378 mmol) at room temperature. The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was cooled to room temperature or filtered through celite, and then water (50 mL) was added. The mixture was extracted three times with ethyl acetate (20 mL x 3). The organic phases were combined, washed once with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a solid residue. The residue was separated by TLC (SiO2, PE:EA = 80:20). The reaction afforded tert-butyl (5-((4-amino-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromobenzyl)sulfonyl)(2-(((tert-butyldiphenylsilyl)oxy)ethyl)carbamate (200 mg, 45%) as a white solid product. ESI-MS m / z = 813.1 [M+H] + ;Calcu.=812.2.

[0249] Step F: At 25°C, cesium carbonate (800 mg, 2.46 mmol) and lithium iodide (329 mg, 2.46 mmol) were added to tert-butyl (5-((4-amino-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromobenzyl)sulfonyl)(2-(((tert-butyldiphenylsilyl)oxy)ethyl)carbamate) dissolved in acetonitrile (10 mL). After the addition of lithium iodide (329 mg, 2.46 mmol), the mixture was stirred at 110°C for 20 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to obtain a solid residue, which was then subjected to HPLC preparation. Separation and preparation conditions: Waters XBridge C 18 (19 mm × 250 mm, 5 μm); A: 0.1% formic acid in water; B: MeCN; 18 mL / min; 0-3 min, 5% B; 3.5-15 min, 10%-100% B; detector, UV 254 nm, freeze-dried. A white solid product 1 was obtained. 4 -Amino-3 4 -Br-1 1 H-9-oxa-5-thia-6-aza-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzazepane 5,5-dioxide (15 mg, 14%). ESI-MS m / z = 438.9 [M+H]+ ;Calcu.=438.0.

[0250] Step G: At 25°C and under nitrogen protection, dissolve 1 4 -Amino-3 4 -Br-1 1 H-9-oxa-5-thia-6-aza-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzazepine 5,5-dioxide (15 mg, 0.034 mmol), potassium trifluoro(pyrrolidin-1-ylmethyl)borate (15.6 mg, 0.102 mmol), sodium carbonate (10.9 mg, 0.102 mmol), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (3.3 mg, 0.007 mmol) in 1,4-dioxane (4 mL), water (1 mL) was added with tris(dibenzylideneacetone)dipalladium (3.1 mg, 0.003 mmol). After the addition was complete, the mixture was stirred at 90°C overnight. After the reaction was complete, the reaction solution was directly concentrated and prepared. Preparation conditions: Waters XBridge C 18 (19 mm × 250 mm, 5 μm); A: 0.1% formic acid in water; B: MeCN; 18 mL / min; 0-3 min, 5% B; 3.5-15 min, 10%-100% B; detector, UV 254 nm, lyophilized. Compound B-13 was obtained as a white solid product, 14-amino-34-(pyrrolidin-1-ylmethyl)-11H-9-oxa-5-thia-6-aza-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzazepanonane 5,5-dioxide (0.8 mg, 5%). ESI-MS m / z = 443.9 [M+H] + ;Calcu.=443.2.

[0251] Example 13: Synthesis of Compound B-14

[0252] Step A: To a solid 4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (6.67 g, 35.29 mmol) was added aqueous ammonia (25%) (100 mL, 713.3 mmol) at room temperature. After the addition was complete, the mixture was stirred at room temperature overnight. The reaction mixture was then diluted with ethyl acetate (60 mL) and filtered through a Buchner funnel to afford a yellow solid. The resulting solid was then washed with petroleum ether:ethyl acetate (2:1) (300 mL) and dried to afford 6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine (5.62 g, 94%) as a yellow solid. ESI-MS m / z = 170.0 [M+H]+ ;Calcu.=169.0.

[0253] Step B: To a mixture of 6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine (1.905 g, 11.23 mmol) and 1-bromo-4-(bromomethyl)-2-nitrobenzene (4.25 g, 14.41 mmol) in tetrahydrofuran (25 mL) and N,N-dimethylformamide (40 mL) was added potassium carbonate (2.05 g, 11.23 mmol) at room temperature. The mixture was stirred at room temperature overnight. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (50 mL), filtered through a fritted funnel, and the solid residue was washed with ethyl acetate (50 mL). The filtrate was concentrated under reduced pressure. Finally, the crude product was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 30:70) to obtain 1-(4-bromo-3-nitrobenzyl)-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine (4.29 g, 54%) as a yellow solid. ESI-MS m / z = 382.8 [M+H] + ;Calcu.=382.0.

[0254] Step C: To a solution of 1-(4-bromo-3-nitrobenzyl)-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine (2.35 mg, 6.13 mmol) in tetrahydrofuran (240 mL) was added triethylamine (2.53 mL, 18.20 mmol) at room temperature. After the addition, the mixture was stirred at 0°C for 15 minutes. Then, di-tert-butyl dicarbonate (6.54 g, 29.98 mmol) and N,N-dimethyl-4-pyridinamine (52 mg, 0.43 mmol) were added to the reaction mixture. The mixture was allowed to warm to room temperature and stirred for 2 hours. Ethyl acetate (300 mL) was then added to dilute the reaction mixture. The organic phase was then washed with saturated ammonium chloride solution (60 mL), saturated sodium bicarbonate solution (60 mL), and saturated brine (60 mL). Then, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a dark brown liquid crude product of tert-butyl (1-(4-bromo-3-nitrobenzyl)-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)(tert-butoxycarbonyl)carbamate (3.58 g, crude product), which was used directly in the subsequent reaction.

[0255] Step D: To a mixed solvent of tetrahydrofuran (60 mL) and water (6 mL) in which tert-butyl (1-(4-bromo-3-nitrobenzyl)-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)(tert-butoxycarbonyl)carbamate (2.39 g, 4.09 mmol) was dissolved, acetic acid (4 mL) and iron powder (0.55 g, 0.198 mmol) were added at room temperature. After stirring, the reaction mixture was heated and stirred at 60°C for 5 hours. After the reaction solution was cooled, ethyl acetate (150 mL) was added to dilute the reaction solution, and the organic phase was washed with saturated ammonium chloride solution (35 mL), saturated sodium bicarbonate solution (35 mL), and saturated brine solution (35 mL). The organic phase was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a brown solid crude product. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 80:20) to give a white solid product, tert-butyl (1-(3-amino-4-bromobenzyl)-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)(tert-butoxycarbonyl)carbamate (1.08 g, two-step yield 32%). 1 H NMR (500MHz, DMSO-d6) δppm 8.29 (s, 1H), 7.28 (d, J = 8.0 Hz, 1H), 6.58 (s, 1H), 6.35 (br d, J = 8.0 Hz, 1H), 5.45 (s, 2H), 5.36 (s, 2H), 1.52 (s, 18H).

[0256] Step E: To a solution of tert-butyl (1-(3-amino-4-bromobenzyl)-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)(tert-butoxycarbonyl)carbamate (970 mg, 1.751 mmol) and triethylamine (738 μL, 5.309 mmol) in dichloromethane (40 mL) was added a solution of methyl 3-(chlorosulfonyl)propanoate (810 mg, 4.341 mmol) in dichloromethane (40 mL) at 0°C. After the addition was complete, the mixture was allowed to cool to room temperature and stirred for 1 hour. Ethyl acetate (150 mL) was added to dilute the mixture at 0°C, and saturated ammonium chloride (30 mL) was added to separate the layers. The resulting organic phase was then washed sequentially with saturated ammonium chloride (30 mL), saturated sodium bicarbonate (30 mL), and saturated brine (30 mL). Finally, the mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The reaction afforded methyl 3-(N-(5-((4-(bis(tert-butoxycarbonyl)amino)-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromophenyl)aminosulfonyl)propanoate (1203 mg, crude) as a brown solid product.

[0257] Step F: At 0°C, LiAlH4 (224 mg, 5.895 mmol) was slowly added portionwise to a solution of methyl 3-(N-(5-((4-(bis(tert-butoxycarbonyl)amino)-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromophenyl)aminosulfonyl)propanoate (1203 mg, 1.709 mmol) in tetrahydrofuran (50 mL). After the addition was complete, the mixture was warmed to room temperature and stirred for 1 hour. The reaction was quenched by the slow dropwise addition of methanol (20 mL) at 0°C. The mixture was then filtered through a celite-filled frit, and the reaction flask and celite were washed with ethyl acetate (100 mL) and methanol (15 mL). The resulting solution was concentrated under reduced pressure and purified by column chromatography on silica gel (SiO2, petroleum ether:ethyl acetate = 50:50). The reaction afforded a white solid product, tert-butyl (1-(4-bromo-3-((3-hydroxypropyl)sulfonamido)benzyl)-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)carbamate (820 mg, 81% yield over two steps). 1 H NMR(500MHz,DMSO-d6)δppm 11.27(s,1H),9.43(s,1H),8.52(s,1H),7.62(d,J=8.0Hz,1H),7.28-7.32(m,1H),6.95(d,J=8.0Hz,1H), 5.56(s,2H),4.65(t,J=5.0Hz,1H),3.43-3.49(m,2H),3.08-3.16(m,2H),1.80-1.88(m,2H),1.52(s,9H).

[0258] Step G: At 0°C, concentrated hydrochloric acid (36-38% wt) (3 mL) was added to a solution of tert-butyl (1-(4-bromo-3-((3-hydroxypropyl)sulfonamido)benzyl)-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)carbamate (401 mg, 0.70 mmol) in dioxane (10 mL). After the addition was complete, the mixture was allowed to cool to room temperature and stirred overnight. Ethyl acetate (100 mL) was added to dilute the reaction solution. Saturated sodium bicarbonate solution was slowly added with stirring at 0°C. The reaction solution was exothermic and bubbling profusely. Sodium bicarbonate solution was added until the bubbling subsided to neutralize the hydrochloric acid. Ethyl acetate (100 mL) was then added to dilute the solution. The organic phase was separated and washed with saturated sodium bicarbonate solution (20 mL) and then saturated brine (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give N-(5-((4-amino-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromophenyl)-3-hydroxypropane-1-sulfonamide (229 mg, 69%) as a white solid. ESI-MS m / z = 474.8 [M+H] + ;Calcu.=474.0.

[0259] Step H: Using a 120 mL pressure tube as the reaction vessel, cesium carbonate (619 mg, 1.90 mmol) was added to a solution of N-(5-((4-amino-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromophenyl)-3-hydroxypropane-1-sulfonamide (61 mg, 0.13 mmol) in acetonitrile (19 mL) at room temperature. After the addition was complete, the mixture was heated to 110°C and stirred for 18 hours. The mixture was filtered through a fritted funnel, and the solid was washed with ethyl acetate (80 mL) and methanol (15 mL). The filtrate was concentrated under reduced pressure and separated by preparative HPLC. Chromatographic conditions: Waters XBridge Prep Shield RP18 (19 mm × 250 mm, 5 μm); A: 5 mM NH4HCO3, B: ACN; 18 mL / min; 0-3 min, 10% B; 3-3.5 min, 10%-20% B; 3.5-15 min, 20%-90% B; 15-15.5 min, 90%-100% B; detector, UV 254 nm. The resulting fractions were lyophilized to yield compound B-14, a white solid product. 4 -Amino-3 4 -Br-1 1 H-9-oxa-5-thia-4-aza-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzocyclononane 5,5-dioxide (0.45 mg, 0.65%). 1H NMR(500MHz,DMSO-d6)δppm 12.07(s,1H),9.42(s,1H),7.80(br s,2H),7.63(d,J=8.0Hz,1H),7.38(d,J=2.0Hz,1H),6.99-7.05(m,1H),5.37(s,2H),4.49-4.54(m,2H),3.09-3.14(m,2H),1.81-1.87(m,2H). ESI-MS m / z=438.8[M+H] + ;Calcu.=438.0.

[0260] Example 14: Synthesis of Compound B-15

[0261] Step A: Using a 120 mL pressure tube as a reaction vessel, cesium carbonate (988 mg, 3.03 mmol) was added to a solution of N-(5-((4-amino-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromophenyl)-3-hydroxypropane-1-sulfonamide (153 mg, 0.32 mmol) in acetonitrile (30 mL) at room temperature. After the addition was complete, the mixture was temperature-controlled to 110°C and stirred for 48 hours. Afterwards, the mixture was filtered through a sand core funnel with silica gel, and the cesium carbonate solid was washed with ethyl acetate (100 mL), dichloromethane (100 mL) and methanol (15 mL). The filtrate was concentrated under reduced pressure to obtain a yellow solid. The obtained solid was dissolved with ethyl acetate (120 mL) and methanol (15 mL), and then washed with saturated ammonium chloride solution (15 mL*2) and saturated brine (15 mL). The filtrate was concentrated under reduced pressure to obtain a pale yellow solid crude product 1. 4 -Amino-3 4 -Br-1 1 H-9-oxa-5-thia-4-aza-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzocyclononane 5,5-dioxide (90 mg, crude). ESI-MS m / z = 438.8 [M+H] + ;Calcu.=438.0.

[0262] Step B: Under nitrogen protection, add 1 4 -Amino-3 4 -Br-1 1H-9-oxa-5-thia-4-aza-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzocyclononane 5,5-dioxide (43 mg, 0.098 mmol), potassium trifluoro(pyrrolidin-1-ylmethyl)borate (89 mg, 0.466 mmol), tris(dibenzylideneacetone)dipalladium (4.6 mg, 0.005 mmol), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (9.5 mg, 0.020 mmol), and sodium carbonate (32 mg, 0.302 mmol) were added. 1,4-dioxane (3.2 mL) and water (0.8 mL) were then added. After stirring, the mixture was deoxygenated three times to remove oxygen from the solvent. The mixture was then heated and stirred at 90°C for 4 hours under nitrogen. Afterwards, the mixture was filtered through a sand core funnel with a small amount of silica gel, and the solid was washed with ethyl acetate (60 mL) and methanol (20 mL). The resulting liquid was concentrated under reduced pressure to obtain an orange crude product, which was then separated and purified by preparative chromatography. The chromatographic conditions were as follows: Waters XBridge Prep Shield RP18 (19 mm × 250 mm, 5 μm); A: 5 mM NH4HCO3, B: ACN; 18 mL / min; 0-3 min, 10% B; 3-3.5 min, 10%-20% B; 3.5-18 min, 20%-80% B; 18-18.5 min, 80%-100% B; detector, UV 254 nm. After freeze-drying, compound B-15 was obtained, a white solid product 1 4 -Amino-3 4 -(pyrrolidin-1-ylmethyl)-1 1 H-9-oxa-5-thia-4-aza-1(1,6)-pyrazolo[3,4-d]pyrimidine-3(1,3)-benzocyclononane 5,5-dioxide (2.33 mg, 5% yield over two steps). 1 H NMR (500MHz, DMSO-d6) δppm8.13(s,1H),7.88(s,1H),7.70(br s,1H),7.48(br s,1H),7.13(d,J=8.0Hz,1H),6.92(br d,J=8.0Hz,1H),5.29(s,2H),4.65(t,J=6.0Hz,2H),3.73(s,2H),3.08-3.17(m,2H),2.44-2.46(m,4H),1.94-2.00(m,2H),1.70-1.74(m,4H). ESI-MS m / z=444.0[M+H] + ;Calcu.=443.2.

[0263] Test Example 1: Cell-level drug efficacy evaluation

[0264] Objective: To evaluate the TLR7 agonist activity of compounds using the HEK-BlueTM hTLR7 in vitro cell model

[0265] Experimental Materials:

[0266] 1. Cell line: HEK-BlueTM hTLR7 (InvivoGen)

[0267] 2 Reagents: QUANTI-BlueTMSolution (InvivoGen), DMEM (Invitrogen), FBS (HyClone), L-glutamine (GIBCO), TrypLE Express Enzyme (GIBCO), DPBS (GIBCO), NormocinTM (InvivoGen); Penicillin-Streptomycin (GIBCO)

[0268] 3. Instruments: Microplate reader (PerkinElmer, Multimode plate reader 2014), CO2 incubator (THERMO, STERI-CYCLEI160), cell counter (BECKMAN COULTER, vi-cell XR)

[0269] Experimental methods:

[0270] Step 1: Cell plating

[0271] a) Discard the culture medium in the T150 flask and add 10 mL of pre-warmed DPBS. Gently shake the flask to rinse the cells once. After discarding the DPBS, add 8.0 mL of TrypLE™ Express enzyme to the T150 flask and gently shake the flask to allow it to permeate all cells.

[0272] b) Discard the TrypLE™ Express enzyme in the culture flask and place it in an incubator for 1 to 2 minutes.

[0273] c) Add serum-containing medium to terminate digestion, collect the cell suspension, centrifuge at 300 rpm for 5 minutes at room temperature, and discard the supernatant.

[0274] d) Add fresh culture medium, mix the cells by pipetting, and then count the cells using a Vi-Cell XR cell counter.

[0275] e) Dilute the cells to a density of 6.25*10 5 cells / mL, 80 μL of diluted cell suspension was inoculated into a 96-well plate, i.e. 5*10 4cells / well.

[0276] f) Place the 96-well plate in an incubator and incubate overnight (5% CO 2 , 37° C.).

[0277] Step 2: Drug treatment

[0278] a) The test compound was prepared into a 10 mM stock solution in DMSO;

[0279] b) The test compound was diluted from 10 mM to 0.1 mM in culture medium, and then diluted 3-fold in culture medium containing 1% DMSO, for a total of 8 concentration gradients. DMSO was used as a negative control, and R848 and GS9620 were used as positive controls.

[0280] c) Add 20 μL of the diluted compound to a 96-well plate seeded with 80 μL of cells, diluting the drug 5-fold to a final DMSO concentration of 0.2% in each well. The final concentrations of the test compound and control compound were 20,000, 6,667, 2,222, 741, 247, 82, 27, and 9 nM.

[0281] d) Place the 96-well plate in a 5% CO2, 37°C incubator and incubate for 24 hours.

[0282] Step 3: Activity Detection

[0283] a) Transfer 10 μL of the cell supernatant after compound incubation to a 96-well transparent assay plate, then add 90 μL of QUANTI-Blue™ Solution to each well of the assay plate and incubate in a 5% CO2, 37°C incubator for 1 hour.

[0284] b) The absorbance of secreted alkaline phosphatase (SEAP) at 650 nm was detected using a microplate reader.

[0285] Data Analysis:

[0286] The activation fold was calculated using the following formula: activation fold = OD 650 Treatment group / OD 650 GraphPad Prism 7 software was used to fit the curves according to the concentrations of the compounds and the corresponding activation folds, and the EC 50 value.

[0287] Reference substances: R848 (InvivoGen) and GS-9620 (CAYMAN), the structures of which are shown in the figure below:

[0288] The experimental results are shown in the following table:

[0289] Test Example 2: Test of the agonist activity of the compounds of the present invention on human peripheral blood mononuclear cells

[0290] Human peripheral blood mononuclear cells (hPBMCs) represent the primary human immune cell population, including lymphocytes, monocytes, and dendritic cells. These cells express TLR7, TLR8, or TLR9 and respond to stimulation with their corresponding ligands. In this study, hPBMCs were stimulated with potential TLR7 agonists, and the levels of IFN-α, TNF-α, and IL-12p40 were measured to reflect the compounds' activation of the TLR7 receptor.

[0291] Experimental Materials:

[0292] 1. Cell line: hPBMCs (Sailybio). Culture conditions: RPMI 1640 medium + 10% (v / v) FBS + 1% Penicillin-Streptomycin, 5% CO2, 37°C.

[0293] 2. Reagents: RPMI 1640 medium (GIBCO), FBS (GIBCO), Penicillin-Streptomycin (GIBCO), Human IL-12 / IL-23p40 DuoSet ELISA (R&D), Human IFN-alpha 2 / IFNA2 DuoSet ELISA (R&D), Human TNF-alpha ELISA (R&D).

[0294] 3. Instruments: microplate reader (perkinElmer, Multimode plate reader 2014), CO2 incubator (THERMO, STERI-CYCLEI160), cell counter (BECKMAN COULTER, vi-cell XR).

[0295] Experimental methods:

[0296] g) Thaw frozen hPBMCs in a 37°C water bath and transfer to 1640 complete medium. Centrifuge at 500 rpm for 10 minutes at room temperature, discard the supernatant, resuspend the cells in 1640 complete medium, transfer to a culture flask, and culture overnight at 37°C, 5% CO2.

[0297] h) Collect hPBMCs after stable culture, count and dilute the cells to 1.11*10 7 cells / mL, 180 μL of diluted cell suspension was inoculated into a 96-well plate, i.e. 2*10 6cells / well, and the 96-well plate was placed in a constant temperature incubator and incubated for 24 h (5% CO2, 37°C).

[0298] i) The test compound was prepared into a 10 mM stock solution in DMSO and diluted to 0.03 mM (IFN-α assay) and 0.1 mM (TNF-α, IL-12p40 assay), respectively. The compound was then diluted into eight concentration gradients in culture medium containing 2% DMSO, with the final maximum compound concentration being 30 uM (IFN-α assay) and 100 uM (TNF-α, IL-12p40 assay).

[0299] j) 20 μL of the diluted compound was added to a 96-well cell culture plate, with the final DMSO concentration in each well being 0.2%.

[0300] k) After incubation at 37°C for 24 hours, the cell culture supernatant was collected by centrifugation, and the levels of IFN-α, TNF-α, and IL-12p40 in the supernatant were determined using ELISA kits.

[0301] l) Measure the light absorption at 450 nm with a microplate reader, and quantitatively determine the level of corresponding cytokine secretion based on the corresponding standard curve, and calculate the EC by fitting the curve. 50 The experimental results are shown in the following table:

Claims

1. A compound having a structure as shown in formula (I), a pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or a metabolite thereof, in, R 1 Selected from: hydrogen, halogen, C1-C6 alkyl; Ring A is selected from a benzene ring, a 5-6 membered heteroaryl, a naphthalene ring and a bicyclic heteroaryl; optionally, Ring A is substituted by one or more substituents selected from: hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, halogen, -CH2R a 、-OR b 、-C(=O)R c , nitro, nitroso, cyano, hydroxyl, -SH, C2-C6 alkenyl, C2-C6 alkynyl, =O, -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)(C1-C6 alkyl), -NHC(=O)(C1-C3 alkyl), C3-C6 cycloalkyl, 3-6 membered heterocyclyl, phenyl, 5-6 membered heteroaryl, -(C1-C6 alkyl)OH, -(C1-C6 alkyl)NH2, -S(C1-C3 alkyl), -S(=O)(C1-C3 alkyl), -S(=O)(=O)(C1-C3 alkyl), -S(=O)(=O)NH(C1-C3 alkyl), -NR d S(=O)(=O)(C1-C6 alkyl), -P(=O)R e R f , sulfonic acid, -N=NPh, -Si(C1-C3 alkyl)3; R a , R b , R c , R d , R e , R f Each is independently selected from: hydrogen, -OH, -COOH, -C(=O)H, phenyl, C1-C6 alkyl, 3-6 membered heterocyclic group, -SiR x R y R z 、-NR x R y , -C(=O)(C1-C6 alkyl), halogen, C1-C6 alkoxy, -S(=O)(=O)(C1-C3 alkyl); R x , R y , R z Each is independently selected from hydrogen, C1-C6 alkyl, benzene ring; Optionally, the 3-6 membered heterocyclic group is substituted by one or more C1-C6 alkyl groups; R 5 , R 6 Each is independently hydrogen, C1-C6 alkyl, C1-C6 alkoxy, halogen, halogenated C1-C6 alkyl; Q is selected from -O-, -S-, -NH-; L 1 , L 2 Each independently selected from -(CH2) n -、-(CH2) n N(R 7 )-、-N(R 7 )(CH2) n -、-(CH2) n O-, -O(CH2) n -; n is selected from 0, 1, 2; R 7 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, phenyl, phenoxy, substituted phenyl and substituted phenoxy, wherein the substituted phenyl is phenyl substituted by one or more groups selected from the following: halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, hydroxyl; in the substituted phenoxy, the benzene ring is substituted by one or more groups selected from the following: halogen halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, hydroxyl; M is selected from the following groups: Among them, R 8 Selected from hydrogen, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, phenyl, phenoxy, substituted phenyl and substituted phenoxy, wherein the substituted phenyl is phenyl substituted by one or more groups selected from the following: halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, hydroxyl; in the substituted phenoxy, the benzene ring is substituted by one or more groups selected from the following: halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, hydroxyl.

2. The compound of claim 1, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or metabolite form thereof, wherein: R 1 Selected from: hydrogen, halogen, C1-C6 alkyl; Preferably, R 1 is selected from hydrogen and halogen; Preferably, R 1 For hydrogen.

3. The compound of claim 1 or 2, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or metabolite form thereof, wherein: Ring A is selected from a benzene ring, a 5-6 membered heteroaryl group, and optionally, Ring A is substituted by one or more substituents; Preferably, the substituent is selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, halogen, -CH2R a 、-OR b 、-C(=O)R c ; Preferably, R a , R b , R c Each is independently selected from: hydrogen, -OH, C1-C6 alkyl, 3-6 membered heterocyclic group, -SiR x R y R z 、-NR x R y , R x , R y , R z Each is independently selected from hydrogen, C1-C6 alkyl, benzene ring; optionally, the 3-6 membered heterocyclic group is substituted by one or more C1-C6 alkyl groups.

4. The compound of any one of claims 1 to 3, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or metabolite form thereof, wherein: Ring A is a benzene ring optionally substituted by one or more substituents.

5. The compound of any one of claims 1 to 3, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or metabolite form thereof, wherein: Ring A is a 5-6 membered heteroaryl group, 1, 2 or 3 ring atoms of the 5-6 membered heteroaryl group are heteroatoms selected from nitrogen, oxygen and / or sulfur, and the nitrogen and / or sulfur are optionally substituted with oxo.

6. The compound of any one of claims 1 to 5, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or metabolite form thereof, wherein: The substituents on ring A are selected from hydrogen, halogen, -C(O)H, -CH2-OH, -CH2-(3-6 membered nitrogen-containing heterocyclic group), -CH2-NR x R y , -O-(3-6 membered nitrogen-containing heterocyclic group), C1-C6 alkoxy, wherein R x , R y Each is independently selected from hydrogen and C1-C6 alkyl; optionally, the 3-6 membered nitrogen-containing heterocyclic group is substituted by one or more C1-C6 alkyl groups.

7. The compound of any one of claims 1 to 6, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or metabolite form thereof, wherein: R 5 , R 6 Each is independently hydrogen or C1-C6 alkyl.

8. The compound of any one of claims 1 to 7, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or metabolite form thereof, wherein: Q is -O-.

9. The compound of any one of claims 1 to 8, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or metabolite form thereof, wherein: L 1 , L 2 Each independently selected from -(CH2) n -、-(CH2) n N(R 7 )-、-N(R 7 )(CH2) n -、-(CH2) n O-, -O(CH2) n -; n is selected from 0, 1; preferably, R 7 Selected from hydrogen, C1-C6 alkyl; Preferably, L 1 , L 2 Each independently selected from -NH-, -NCH3-, -O-, -(CH2) n -, -CH2-NH-, -CH2-N(CH3)-, -N(CH3)-CH2-, -NH-CH2-, -CH2O-, -OCH2-; n is selected from 0, 1.

10. The compound of any one of claims 1 to 9, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or metabolite form thereof, wherein: R 8 Selected from hydroxyl, C1-C6 alkyl, C1-C6 alkoxy (e.g. methoxy, ethoxy), substituted phenoxy, wherein the benzene ring on the phenoxy is optionally substituted by halogen; Preferably, R 8 The invention is selected from hydroxy, methyl, ethyl, methoxy, ethoxy and substituted phenoxy, wherein the benzene ring of the phenoxy is optionally substituted by halogen.

11. The compound according to any one of claims 1 to 10, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or metabolite form thereof, wherein the compound has a structure as shown in formula (II), in, R 1 Selected from: hydrogen, halogen, C1-C6 alkyl; R 2 , R 3 , R 4 Each is independently selected from: hydrogen, halogen, -C(=O)H, -CH2-OH, -CH2-(3-6 membered nitrogen-containing heterocyclic group), -CH2-NR x R y , -O-(3-6 membered nitrogen-containing heterocyclic group), C1-C6 alkoxy, wherein R x , R y Each is independently selected from hydrogen, C1-C6 alkyl; optionally, the 3-6 membered nitrogen-containing heterocyclic group is substituted by one or more C1-C6 alkyl groups; R 5 , R 6 Each is independently hydrogen or C1-C6 alkyl; L 1 , L 2 Each independently selected from -(CH2) n -、-(CH2) n N(R 7 )-、-N(R 7 )(CH2) n -、-(CH2) n O-, -O(CH2) n -; n is selected from 0, 1; R 7 Selected from hydrogen, C1-C6 alkyl; M is selected from the following groups: R 8 The invention is selected from hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, and substituted phenoxy, wherein the benzene ring of the phenoxy is optionally substituted by halogen.

12. The compound of claim 11, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or metabolite form thereof, wherein: R 2 is hydrogen; Preferably, R 2 , R 3 , R 4 are hydrogen respectively.

13. The compound of claim 11 or 12, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or metabolite form thereof, wherein: R 3 Selected from hydrogen, halogen, C1-C6 alkoxy.

14. The compound of any one of claims 11 to 13, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or metabolite form thereof, wherein: R 4 Selected from: hydrogen, halogen, C1-C6 alkoxy, -CH2-(5-6 membered nitrogen-containing heterocyclic group), -CH2-NR x R y , R x , R y Each is independently selected from hydrogen and C1-C6 alkyl; optionally, the 3-6 membered nitrogen-containing heterocyclic group is substituted by one or more C1-C6 alkyl groups.

15. The compound of any one of claims 1 to 14, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystalline form, isotope-labeled substance or metabolite thereof, having a structure as shown in formula (III), (IV), (V), (VI), (VII) or (VIII): in, R 1 , R 2 , R 3 , R 4 As defined in any one of claims 1 to 14, R 9 Selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, hydroxyl.

16. A compound according to any one of claims 1 to 15, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, a tautomer, a hydrate, a solvate, a crystal form, an isotope-labeled substance or a metabolite thereof, wherein the hydroxyl group, -NH- or -NH2 in the compound is protected by a substituent (e.g., Boc, TBDPS).

17. A compound according to any one of claims 1 to 16, a pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystalline form, isotope-labeled substance or a metabolite thereof, wherein the compound has a structure selected from the following:

18. A pharmaceutical composition comprising a compound according to any one of claims 1 to 17, a pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or a metabolite thereof; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

19. Use of the compound according to any one of claims 1 to 17, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or metabolite thereof in the preparation of a medicament, wherein the medicament is used as a TLR7 agonist for preventing or treating a disease associated with TLR7; Preferably, the disease associated with TLR7 is selected from: (1) Tumors, including but not limited to: breast cancer, melanoma, meningioma, soft tissue sarcoma, salivary gland tumors, intraspinal tumors, mediastinal tumors, brain cancer, bone cancer, penile cancer, osteosarcoma, intracranial tumors, tongue cancer, maxillary sinus cancer, thyroid cancer, malignant lymphoma, multiple myeloma, pituitary adenoma, testicular tumors, non-Hodgkin's lymphoma, bladder cancer, leukemia, gastric cancer, nasopharyngeal cancer, laryngeal cancer, oral cancer, esophageal cancer, lung cancer, kidney cancer, cervical cancer, choriocarcinoma, vulvar cancer, skin cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, rectal cancer, colorectal cancer, Kaposi's sarcoma, non-melanoma skin cancer (including squamous cell carcinoma and basal cell carcinoma), hemangioma, glioma, non-Hodgkin's lymphoma (diffuse large B-cell lymphoma, B-cell non-Hodgkin's lymphoma), colon cancer, liver cancer, neuroblastoma, glioblastoma; (2) diseases associated with viral infection, including but not limited to dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, tick-borne encephalitis virus, Kunjin virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Omsk hemorrhagic fever virus, bovine viral diarrhea virus, Zika virus, HIV, HBV, HCV, HPV, RSV, SARS or influenza virus; (3) Inflammatory diseases, respiratory diseases, skin diseases, gastrointestinal diseases, hepatobiliary diseases, nervous system diseases, musculoskeletal and connective tissue diseases, or autoimmune diseases, such as asthma, chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome (ARDS), ulcerative colitis, Crohn's disease, bronchitis, dermatitis, actinic keratosis, Allergic rhinitis, psoriasis, scleroderma, urticaria, rheumatoid arthritis, multiple sclerosis, lupus, drug-induced allergic reactions, rheumatism.

20. Use of the compound according to any one of claims 1 to 17, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or metabolite form thereof as a vaccine adjuvant.

21. Use of the compound according to any one of claims 1 to 17, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or metabolite form thereof as a photodynamic therapy agent.

22. Use of the compound of any one of claims 1 to 17, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope-labeled substance or metabolite form thereof as a conjugate drug (e.g., antibody immunostimulatory drug conjugate ISAC).

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