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

Abstract

The invention relates to the field of biological medicine, in particular to a micromolecular phosphorus-containing or sulfur-containing macrocyclic pyrazolopyrimidine compound which has better immunomodulatory activity, and further provides application of the micromolecular phosphorus-containing or sulfur-containing macrocyclic pyrazolopyrimidine compound in prevention or treatment of TLR7-related diseases and application of the micromolecular phosphorus-containing or sulfur-containing macrocyclic pyrazolopyrimidine compound in treatment of TLR7-related diseases. The invention also discloses application of the compound as a vaccine adjuvant, a photodynamic therapeutic agent and a coupling drug.

Description

Technical Field

[0001] 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

[0002] Pattern recognition receptors (PRRs) play a vital role in maintaining the normal function of the innate immune system. According to the homology of the protein domain, PRRs are divided into five families: Toll-like receptors (TLR), C-type lectin receptors (CLR), AIM2-like receptors (ALR), RIG-I-like receptors (RLR) and Nod-like receptors (NLR). The first two families are transmembrane proteins, while the last three families are cytoplasmic proteins.

[0003] TLRs are type I membrane proteins, mainly distributed on the cell membrane surface or inclusion body membrane of various immune cells. TLRs can recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), activate downstream signaling molecules of the immune system, and initiate host defense responses. They can promote innate immune responses to eliminate pathogens and trigger adaptive immunity. As functional biological molecules, TLRs guard the immune systems of different types of hosts, from insects to humans. Ten TLR subtypes (TLR1-10) have been identified in mammals. According to cell localization and ligand type, TLRs can be divided into two categories. The first category includes TLR1 / 2 / 4 / 5 / 6 / 10, and the second category includes TLR3 / 7 / 8 / 9. The first type of TLRs are mainly distributed on the cell membrane, and the second type of TLRs are distributed in the intracellular membrane structures, such as endosomes, lysosomes, endoplasmic reticulum, etc. Due to the important physiological functions of TLRs in the immune system, TLRs are important drug targets and have important application value in infectious diseases, autoimmune diseases, cancer, and inflammatory diseases.

[0004] TLR7 is a member of the TLR family. The activation of TLR7 initiates the myeloid differentiation factor 88 (MyD88)-dependent pathway, in which TLR7 works in concert with protein kinases, transcription factors, and adaptor proteins. After TLR7 binds to the ligand, it interacts with MyD88, which then recruits members of the interleukin 1 receptor-associated kinase (IRAK) family to form a large intracellular oligomeric signaling complex (Myddosome). During the formation of this oligomeric signaling complex, IRAK4 activates the autophosphorylation of IRAK1, and the released IRAK1 then interacts with tumor necrosis factor receptor-associated factor 6 (TRAF6). After the signal is transmitted step by step, it activates the NF-κB pathway and the mitogen-activated protein kinase (MAPK) pathway, leading to the production and release of proinflammatory cytokines and chemokines.

[0005] 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. Multiple TLR7 agonists are in clinical research, for example, Resiquimod is in the second phase of clinical trials for the treatment of bladder cancer, and Vesatolimod is in the second phase of clinical trials for the treatment of HIV infection. Some small molecule TLR7 agonists that have entered the clinical stage (such as Guretolimod, PF-4878691, GSK-2245035) were forced to terminate clinical trials due to poor efficacy or adverse reactions. At present, the only small molecule compound on the market 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. Summary of the invention

[0006] The present invention aims to provide a novel phosphorus- or sulfur-containing macrocyclic pyrazolopyrimidine TLR7 small molecule agonist with better immunomodulatory activity. The present invention also provides the use of the TLR7 small molecule agonist for preventing or treating diseases related to TLR7, as well as the use as a vaccine adjuvant and photodynamic therapy agent, thereby providing the following inventions:

[0007] Compound

[0008] 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 a metabolite thereof,

[0009]

[0010] in,

[0011] R 1 Selected from: hydrogen, halogen, C1-C6 alkyl;

[0012] 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;

[0013] R 5 , R 6 Each is independently hydrogen, C1-C6 alkyl, C1-C6 alkoxy, halogen, halogenated C1-C6 alkyl;

[0014] Q is selected from -O-, -S-, -NH-;

[0015] 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;

[0016] 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;

[0017] M is selected from the following groups:

[0018]

[0019] 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.

[0020] In certain embodiments, R 1 Selected from: hydrogen, halogen.

[0021] In certain embodiments, R 1 For hydrogen.

[0022] 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-benzopyran, 1,4-benzodioxane, benzomorpholine, 1,2,3,4-tetrahydroquinoxaline.

[0023] In certain embodiments, ring A is selected from a benzene ring, a 5-6 membered heteroaryl, and optionally, ring A is substituted with one or more substituents.

[0024] In certain embodiments, the substituents of Ring A are selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, halogen, -CH2R a 、-ORb 、-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, -N=NPh, -Si(C1-C3 alkyl)3.

[0025] In certain embodiments, the substituents of Ring A are selected from C3-C6 cycloalkyl, 3-6 membered heterocyclyl, phenyl, 5-6 membered heteroaryl.

[0026] 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 .

[0027] In certain embodiments, 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 , 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.

[0028] In certain embodiments, Ring A is a benzene ring optionally substituted with one or more substituents.

[0029] In certain embodiments, Ring A is a 5-6 membered heteroaryl, wherein 1, 2 or 3 ring atoms of the 5-6 membered heteroaryl are heteroatoms selected from nitrogen, oxygen and / or sulfur, and the nitrogen and / or sulfur are optionally substituted with oxo.

[0030] 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 and C1-C6 alkyl; optionally, the 3-6 membered nitrogen-containing heterocyclic group is substituted by one or more C1-C6 alkyl groups.

[0031] In certain embodiments, R 5 , R 6 Each is independently hydrogen or C1-C6 alkyl.

[0032] In certain embodiments, Q is -O-.

[0033] 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.

[0034] In certain embodiments, 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.

[0035] In certain embodiments, 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.

[0036] In certain embodiments, 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.

[0037] In certain embodiments, the compounds of the present invention have a structure as shown in Formula (II):

[0038]

[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) n O-, -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]

[0046] 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.

[0047] In certain embodiments, R 1 Selected from hydrogen and halogen.

[0048] In certain embodiments, R 2 For hydrogen.

[0049] In certain embodiments, R 2, R 3 , R 4 are hydrogen respectively.

[0050] 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 and C1-C6 alkyl; optionally, the 3-6 membered nitrogen-containing heterocyclic group is substituted by one or more C1-C6 alkyl groups.

[0051] In certain embodiments, R 3 Selected from hydrogen, -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.

[0052] In certain embodiments, R 3 Selected from hydrogen, halogen, C1-C6 alkoxy.

[0053] In certain embodiments, R 3 For hydrogen.

[0054] In certain embodiments, R 4 Selected 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.

[0055] 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.

[0056] 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 yEach 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.

[0057] In certain embodiments, R 5 For hydrogen.

[0058] In certain embodiments, R 5 It is methyl.

[0059] In certain embodiments, R 6 For hydrogen.

[0060] In certain embodiments, R 6 It is methyl.

[0061] In certain embodiments, M is selected from the following groups:

[0062]

[0063] R 9 is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, hydroxy. 9 It is a halogen.

[0064] In certain embodiments, the compounds of the invention have a structure as shown in Formula (III), (IV), (V), (VI), (VII) or (VIII):

[0065]

[0066] Among them, R 1 , R 2 , R 3 , R 4 , R 9 As defined in any of the above items respectively.

[0067] The hydroxyl groups and -NH- and -NH2 in the compounds of the present invention may be optionally 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 protection scope of the present invention.

[0068] The compounds of the present invention may have a structure selected from the following:

[0069]

[0070] Pharmaceutical composition, use, and disease prevention / treatment methods

[0071] 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.

[0072] The pharmaceutical composition of the present invention can be prepared into any pharmaceutically acceptable dosage form and applied to patients who need such treatment by oral, parenteral, rectal, pulmonary, topical or transdermal administration. When used for oral administration, it can be prepared into conventional solid preparations, such as tablets, capsules, pills, granules, etc.; it can also be prepared into oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc. When made into oral preparations, suitable fillers, binders, disintegrants, lubricants, etc. can be added. When used for parenteral administration, it can be prepared into injections, including injections, sterile powders for injection and concentrated solutions for injection. When made into injections, conventional methods in the existing pharmaceutical field can be used for production. When preparing injections, additives may not be added, or suitable additives may be added according to the properties of the drug. When used for rectal administration, it can be prepared into suppositories, etc. When used for pulmonary administration, it can be prepared into inhalants or sprays, etc. When used for topical administration, it can be used in specific dosage forms and devices or prepared into creams, gels, etc. When used for transdermal administration, it can be prepared into patches.

[0073] 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 their metabolite forms for preparing drugs, which are used as TLR7 agonists for preventing or treating diseases associated with TLR7.

[0074] In one aspect, the present application provides a method for preventing or treating a TLR7-related disease in a subject, the method 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, isotope-labeled substance or a metabolite thereof.

[0075] In some embodiments, the disease associated with TLR7 is selected from:

[0076] (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;

[0077] (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;

[0078] (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.

[0079] In the method, the compound of the present invention acts as a TLR7 agonist, and further, can act as an immunomodulator.

[0080] 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 their metabolites as vaccine adjuvants. In certain embodiments, the vaccine is used to prevent or treat diseases associated with any of the viral infections described above.

[0081] In one aspect, the present application provides use of the compound of the present invention, its pharmaceutically acceptable salt, prodrug, stereoisomer, tautomer, hydrate, solvate, crystal form, isotope label or metabolite form thereof as a photodynamic therapy agent.

[0082] In one aspect, the present application provides the use of a compound of the present invention, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, a tautomer, a hydrate, a solvate, a crystalline form, an isotope label or a metabolite thereof as a conjugated drug (e.g., an antibody immunostimulatory conjugate drug ISAC). An antibody immunostimulatory conjugate drug (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 includes an antibody targeting HER2 and a TLR7 / 8 agonist, and is currently in clinical phase I / II for the treatment of solid tumors.

[0083] In the present application, the subject is preferably a mammal, such as bovine, equine, porcine, canine, feline, rodent, primate, such as human.

[0084] Preparation method

[0085] Route 1:

[0086]

[0087] Intermediate 1-1 undergoes cyclization under condensation reagent conditions to give 1-2.

[0088] Route 2:

[0089]

[0090] Intermediate 2-1 undergoes cyclization under condensation reagent conditions to obtain 2-2, and 2-2 undergoes a series of reactions to obtain 2-3.

[0091] Route 3:

[0092]

[0093] Intermediate 3-1 undergoes cyclization under condensation reagent conditions to give 3-2.

[0094] Route 4:

[0095]

[0096] Intermediate 4-1 undergoes cyclization under condensation reagent conditions to obtain 4-2, and 4-2 undergoes a series of reactions to obtain 4-3, wherein X is selected from H, TBDPS, TBS, and the remaining substituents are as described above.

[0097] Definition of terms

[0098] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the laboratory operation steps involved in this article are all conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0099] As used herein, the term "C1-C6 alkyl" refers to a group obtained by removing a hydrogen atom from a straight 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, n-hexyl. In the present invention, the preferred C1-C6 alkyl is a C1-C4 alkyl.

[0100] As used herein, the term "C2-C6 alkenyl" refers to a straight 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 is C2-C4 alkenyl.

[0101] As used herein, the term "C2-C6 alkynyl" refers to a straight 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 is C2-C4 alkynyl.

[0102] 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.

[0103] As used herein, the term "halogenated" means that hydrogen on a group or compound is replaced by one or more halogen atoms, including full halogenation and partial halogenation.

[0104] As used herein, the term "alkoxy" refers to a group formed in the form of alkyl-O-.

[0105] As used herein, the term "5-6 membered heteroaryl" refers to a monocyclic group with aromaticity 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.

[0106] As used herein, the term "bicyclic heteroaryl" refers to a bicyclic group in which two rings (at least one of which has aromaticity) 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, and the 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.

[0107] As used herein, the term "cycloalkyl" refers to a saturated or partially saturated monocyclic cyclic 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.

[0108] As used herein, the term "heterocyclyl" refers to a group obtained by removing a hydrogen atom from a saturated or partially saturated monocyclic or condensed ring compound containing 3-14 ring atoms and at least one heteroatom (e.g., 1, 2, 3, 4 or 5 heteroatoms). 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, the heterocyclyl further contains 1 or 2 oxygen atoms or sulfur atoms.

[0109] 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 by the present invention and 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), and including but not limited to, hydrohalides, such as hydrofluorides, hydrochlorides, hydrobromides, hydroiodides, etc.; inorganic acid salts, such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkane sulfonates, such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, etc.; aryl sulfonates, 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.

[0110] As used herein, the term "pharmaceutically acceptable ester" refers to an ester formed by -COOH present in the compound provided by the present invention and a suitable alcohol, or an ester formed by -OH present in the compound provided by the present invention 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 reactions to generate corresponding acids or alcohols in the presence of acids or bases.

[0111] As used herein, the term "pharmaceutically acceptable amide" refers to an amide formed by -COOH present in the compounds provided herein and a suitable amine, or an amide formed by -NH- or -NH2 present in the compounds provided herein and a suitable acid (e.g., carboxylic acid, such as acetic acid).

[0112] As used herein, the term "solvate" refers to a substance formed by the association of the compounds of the present invention with solvent molecules. The solvent may be an organic solvent (e.g., methanol, ethanol, propanol, acetonitrile, etc.), for example, the compounds of the present invention may form ethanolates with ethanol. The compounds of the present invention may also form hydrates with water.

[0113] As used herein, the term "crystal form" refers to the crystal structure of a substance. When a substance crystallizes, various factors may affect the intramolecular or intermolecular bonding mode, causing molecules or atoms to be arranged differently in the lattice space, forming different crystal structures. The compounds of the present invention may exist in one crystal structure or in multiple crystal structures, i.e., have "polymorphism". The compounds of the present invention may exist in different crystal forms.

[0114] As used herein, the term "stereoisomer" includes conformational isomers and configurational isomers, wherein the configurational isomers mainly include cis-trans isomers and optical isomers. The compounds of the present invention can exist in the form of stereoisomers, and therefore encompass all possible stereoisomer forms, and any combination or any mixture thereof. For example, a single enantiomer, a single diastereomer or a mixture of the above. When the compounds of the present invention contain an olefin double bond, unless otherwise specified, it includes cis-isomers and trans-isomers, and any combination thereof.

[0115] 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 component.

[0116] As used herein, the term "prodrug" refers to a compound that can be converted into the present invention in the subject by reactions such as oxidation, reduction, hydrolysis, etc. The prodrug itself may or may not have the biological activity of the compound of formula (I) (e.g., regulating glucose and lipid metabolism activity, anti-inflammatory activity, antioxidant activity). For example, a compound of formula (I) including a hydroxyl group or a carboxyl group can be administered in the form of an ester, which is hydrolyzed and converted into a hydroxyl compound or a carboxyl compound in the body. Similarly, a compound of formula (I) including an amino group undergoes acylation, alkylation or phosphorylation to form, for example, eicosanoylamino, alanylamino, pivaloyloxymethylamino compounds 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).

[0117] 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 a subject and an 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, flavoring agents, preservatives, taste masking agents, etc. For example, non-limiting examples of disintegrants include sodium starch glycolate, sodium carboxymethylcellulose, calcium carboxymethylcellulose, cross-linked sodium carboxymethylcellulose, cross-linked 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. Reagents that maintain osmotic pressure include but are not limited to sugar, NaCl and the like. Reagents 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.

[0118] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical result. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., 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 (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to, compared with the expected survival period (if not receiving treatment), extending the survival period.

[0119] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain a 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, prevent, 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 prevent the disease and its complications of a patient who has a disease. It is entirely 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

[0120] 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). NMR chemical shifts (δ) are expressed in 10 -6 The units are given in ppm. NMR measurements were performed using a Bruker AVANCE III 500 MHz NMR instrument, with deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD) and the internal standard being tetramethylsilane (TMS).

[0121] Analytical liquid chromatography: Use analytical liquid chromatography to monitor the reaction system and analyze the purity of important 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); Chromatographic column: Waters XBridge BEH C18 3.5um, 4.6*150mm.

[0122] Preparative liquid chromatography: Use preparative liquid chromatography to separate and purify a small amount of important intermediates and target products. Preparative liquid chromatography system: Agilent 1290 Infinity II (equipped with 1290 Prep Bin Pump, Prep ALS / FC injector / collector, 1260 VWD detector); C18 reverse phase silica gel column.

[0123] Liquid chromatography-mass spectrometry: LCMS is used for routine reaction system detection and molecular weight signal determination of target products. LC-MS system: Waters UPLC H-Class ultra-high performance liquid chromatography-QDa coupling instrument; chromatographic column: Waters XBridge BEH C181.7um, 2.1*75mm.

[0124] Liquid phase mobile phase ratio: Acidic conditions: A: 0.05% formic acid aqueous solution, B: acetonitrile; Alkaline conditions: A: 0.05% ammonia aqueous solution, B: acetonitrile; Neutral conditions: A: 5mM NH4HCO3 aqueous solution, B: acetonitrile. The above data are the default mobile phase ratios, which can be appropriately adjusted according to the properties of the actual samples when performing analysis and testing.

[0125] Rapid purification system: A rapid purification system is used for rough preparation and purification of compounds in conventional reaction systems. The instrument model is Biotage Isolera One, and the normal phase column and reverse phase column used with it are 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 reverse phase C18 columns are 40g, 80g, 120g and 330g, and the specific parameters are as follows: 20-45μm,

[0126] Thin layer chromatography: Use thin layer chromatography (TLC) silica gel plates to conduct 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.

[0127] 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, Aicon, Anergy, Aladdin, J&K, Bid Pharmaceuticals, Leyan, McLean, Accela, Adamas, Xiens, and Inokane.

[0128] Abbreviations

[0129] Protecting Group Abbreviations:

[0130] Bn Benzyl

[0131] Boc tert-Butyloxycarbonyl

[0132] Cbz benzyloxycarbonyl

[0133] MOM Methoxymethyl

[0134] PMB p-Methoxybenzyl

[0135] TBDPS tert-Butyldiphenylsilyl

[0136] TBS tert-Butyldimethylsilyl

[0137] THP 2-Tetrahydropyranyl

[0138] Tos p-Toluenesulfonyl

[0139] Reagent Abbreviations:

[0140] ACN Acetonitrile

[0141] AIBN Azobisisobutyronitrile

[0142] Boc2O Di-tert-butyl dicarbonate

[0143] BPO Benzoyl Peroxide

[0144] tBuONa Sodium tert-butoxide

[0145] CAM Ammonium molybdate cerium sulfate color developer

[0146] CDI N,N'-Carbonyldiimidazole

[0147] m-CPBA m-Chloroperbenzoic acid

[0148] DABCO Triethylenediamine

[0149] DCE 1,2-Dichloroethane

[0150] DCM Dichloromethane

[0151] DEAD Diethyl azodicarboxylate

[0152] DIAD Diisopropyl azodicarboxylate

[0153] DIBAL-H Diisobutylaluminum hydride

[0154] DBU 1,8-diazobispiro[5.4.0]undec-7-ene

[0155] DHP 3,4-Dihydro-2H-pyran

[0156] DIEA N,N-Diisopropylethylamine

[0157] DMAP 4-dimethylaminopyridine

[0158] DMF N,N-Dimethylformamide

[0159] DMSO Dimethyl sulfoxide

[0160] EA Ethyl acetate

[0161] HMPA Hexamethylphosphoramide

[0162] HATU N,N,N′,N′-Tetramethyl-O-(7-azabenzotriazol-1-yl)urea hexafluorophosphate

[0163] LAH Lithium Aluminum Hydride

[0164] NBS N-Bromosuccinimide

[0165] PE Petroleum Ether

[0166] PMA Phosphomolybdic Acid

[0167] PyBOP 1H-Benzotriazol-1-yloxytripyrrolidino hexafluorophosphate

[0168] TBAF Tetrabutylammonium fluoride

[0169] TBDPSCl tert-Butyldiphenylsilyl chloride

[0170] TBSCl tert-Butyldimethylsilyl chloride

[0171] TEA Triethylamine

[0172] TFA Trifluoroacetic acid

[0173] THF Tetrahydrofuran

[0174] TPSCl 2,4,6-Triisopropylbenzenesulfonyl chloride

[0175] TsCl p-Toluenesulfonyl chloride

[0176] TsOH p-Toluenesulfonic acid

[0177] Example

[0178] Example 1: Synthesis of Compound B-1

[0179]

[0180] Step A: At room temperature, trifluoroacetic acid (1.5 mL) was added to methanol (15 mL) in which 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) was dissolved. After the addition, the whole mixture was stirred at room temperature for 72 hours and set aside after the reaction was completed.

[0181] Step B: 1 / 5 of the reaction solution in 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 after the addition was complete, and then (3-(bromomethyl)benzyl)phosphonic acid dimethyl ester (633 mg, 2.16 mmol) was added, and the whole mixture was heated to 60°C and stirred for 3 hours. The reaction solution was filtered, and concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column separation (SiO2, PE:EA=89:11). The reaction obtained a white solid (3-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)benzyl)phosphonic acid dimethyl ester (110 mg, 0.27 mmol, 25%). ESI-MS m / z=408.0[M+H] + ;Calcu.=407.1.

[0182] Step C: At room temperature, lithium bromide (117 mg, 1.35 mmol) was added to acetonitrile (2 mL) in which dimethyl (3-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)benzyl)phosphonate (110 mg, 0.27 mmol) was dissolved. After the addition was complete, the entire mixture was heated to reflux and stirred for 5 hours. The mixture was concentrated under reduced pressure, and the crude product was subjected to HPLC preparation, separation and purification. 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, UV254 nm. The reaction gave 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)δppm7.97(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.

[0183] Step D: At room temperature, 1H-benzotriazol-1-yloxytripyrrolidinoyl hexafluorophosphate (63.5 mg, 0.122 mmol) and N,N-diisopropylethylamine (11.0 mg, 0.085 mmol) were added to N,N-dimethylformamide (2.5 mL) and 1,2-dichloroethane (13 mL) in which methyl hydrogen (3-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)benzyl)phosphonate (8 mg, 0.02 mmol) was dissolved. After the addition was complete, the whole mixture was stirred at room temperature overnight, concentrated under reduced pressure, and the crude product was subjected to HPLC preparation, separation and purification. Preparation conditions: Waters XBridge C18 (10mm×150mm, 5μm); A: 0.1% formic acid in water, B: MeCN; 18mL / min; 0~3min, 10% B; 3~20min, 10%~30% B; detector, UV 254nm. 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)δppm 7.90(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.

[0184] Example 2: Synthesis of Compound B-2

[0185]

[0186] Step A: At room temperature, 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). The reaction mixture was heated to 50°C and stirred for 3 hours, cooled to room temperature, and concentrated to obtain a crude product. The crude product was dissolved in an 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, diluted with water, filtered, and the solid was dried to obtain a white solid product 6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (8.6 g, 33.9 mmol, 80%). ESI-MS m / z=254.0[M+H] + ;Calcu.=253.1.

[0187] 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) was 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 stirred for 6 hours, cooled to room temperature, filtered to remove insoluble matter, and concentrated to obtain a yellow oily crude product. The crude product was separated by silica gel column (SiO2, DCM: MeOH = 95: 5) to obtain a light yellow solid product 2-((4-amino-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethane-1-ol (4.16 g, 14.89 mmol, 84%). ESI-MS m / z = 280.0 [M+H] + ;Calcu.=279.1.

[0188] Step C: At room temperature, N-acetylimidazole (13.75 g, 124.8 mmol) and 1,8-diazobispiro[5.4.0]undec-7-ene (927 mg, 6.09 mmol) were added to a solution of (3-aminophenyl)methanol (15 g, 121.8 mmol) in acetonitrile (200 mL) in sequence. The reaction mixture was reacted at room temperature for 18 hours and concentrated to obtain a crude product. The crude product was separated on a silica gel column (SiO2, DCM:MeOH=96:4) to obtain a white solid product (3-aminophenyl)acetic acid methyl ester (16.6 g, 100.5 mmol, 83%). ESI-MS m / z=166.1[M+H] + ;Calcu.=165.1.

[0189] 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 heated to room temperature and the reaction was continued for 4 hours. Then the mixture was 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)ethane-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 obtain a crude product. The crude product was separated by silica gel column (SiO2, DCM:MeOH=95:5) to obtain colorless oily 3-(((2-((4-amino-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethoxy)(2-chlorophenoxy)phosphoryl)amino)acetic acid benzyl ester (1.65 g, 2.67 mmol, 21%). ESI-MS m / z=617.1[M+H] + ;Calcu.=616.2.

[0190] 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 allowed to react 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 and separated by silica gel column (SiO2, DCM: MeOH = 97: 3) to obtain a colorless oily product 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%). ESI-MS m / z = 575.0 [M+H] + ;Calcu.=574.1.

[0191] Step F: Trifluoroacetic acid (2 mL, 26.12 mmol) was added 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)phosphoramide (850 mg, 1.478 mmol) in methanol (10 mL) 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). The reaction gave a colorless oily product 2-((4-amino-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethyl(2-chlorophenyl)(3-(hydroxymethyl)phenyl)phosphoramide (630 mg, 1.284 mmol, 87%). ESI-MS m / z=491.0[M+H] + ;Calcu.=490.1.

[0192] 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 the crude product was concentrated to obtain a crude product, which was subjected to HPLC preparation and separation. Preparation 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 preparation fraction was 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%). 1HNMR(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.

[0193] Example 3: Synthesis of Compound B-3

[0194]

[0195] Step A: At 0°C, in tetrahydrofuran (50 mL) dissolved with lithium aluminum hydride (2.54 g, 66.9 mmol), tetrahydrofuran (50 mL) dissolved with 5-methoxyisophthalic acid dimethyl ester (5.00 g, 22.3 mmol) was slowly added dropwise. After the addition, the whole mixture was heated to 65°C and stirred for 3 hours. After the reaction was completed, it was cooled to 0°C, and then saturated ammonium chloride solution (50 mL) and water (50 mL) were slowly added. It was extracted with ethyl acetate three times (200 mL*3), the organic phases were combined, washed once with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The reaction obtained the product (5-methoxy-1,3-phenylene) dimethanol (3.52 g, 20.93 mmol, 94%) as a white solid.

[0196] Step B: At 0°C, phosphorus tribromide (5.9 mL, 62.43 mmol) was added to dichloromethane (50 mL) in which (5-methoxy-1,3-phenylene) dimethanol (3.50 g, 20.81 mmol) was dissolved. After the addition was complete, the entire mixture was heated to room temperature and stirred for 3 hours. The reaction solution was slowly added with methanol (5 mL) to quench the reaction, water (50 mL) was added, and the mixture was extracted twice with dichloromethane (50 mL*2). 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, which was separated by a silica gel column (SiO2, PE:EA=90:10). The reaction obtained a white solid product, 1,3-bis(bromomethyl)-5-methoxybenzene (5.30 g, 18.03 mmol, 87%).

[0197] Step C: At room temperature, in a 100 mL sealed tube, 1,3-bis(bromomethyl)-5-methoxybenzene (5.00 g, 17.0 mmol) was dissolved in toluene (15 mL), and methyl diethoxyphosphine (1.3 mL, 8.5 mmol) was added. After the addition, the whole mixture was heated to 110°C and stirred for 6 hours under protection. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was separated by a silica gel column (SiO2, PE:EA=90:10 and DCM:MeOH=90:10). The reaction obtained a light yellow solid product (3-(bromomethyl)-5-methoxybenzyl)(methyl) ethyl phosphinate (1.98 g, 6.16 mmol, 72%). ESI-MS m / z=320.8,322.8[M+H] + ;Calcu.=320.0,322.0.

[0198] Step D: At room temperature, potassium carbonate (276 mg, 2.0 mmol) was added to N,N-dimethylformamide (5 mL) in which 2-((4-amino-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethane-1-ol (156 mg, 0.8 mmol) and ethyl (3-(bromomethyl)-5-methoxybenzyl)(methyl)phosphinate (513 mg, 1.6 mmol) were dissolved. After the addition was complete, the mixture was heated to 60°C and stirred for 3 hours. The reaction solution was filtered, concentrated to dryness under reduced pressure, and the crude product was subjected to silica gel column chromatography (SiO2, PE:MeOH=88:12). The reaction gave the product (3-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methoxybenzyl)(methyl)ethyl phosphinate (50 mg, 0.115 mmol, 14%) as a white solid. ESI-MS m / z=436.0[M+H] + ;Calcu.=435.2.

[0199] Step E: At 0°C, trimethylsilyl bromide (0.3 mL, 2.3 mmol) was added to dichloromethane (4 mL) in which 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) was dissolved. After the addition, the whole mixture was stirred at room temperature overnight. 1 mL of methanol was added to the reaction solution to quench the reaction, and the pH was adjusted to neutral with triethylamine. The reaction solution was concentrated to dryness under reduced pressure, and 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; 0-3.5 min, 5% B; 3.5-15 min, 5%-50% B; detector, UV 254 nm). The reaction gave 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.

[0200] Step F: At room temperature, N,N-diisopropylethylamine (5.4 mg, 0.042 mmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphate (31 mg, 0.06 mmol) were added to 1,2-dichloroethane (8.7 mL) and N,N-dimethylformamide (1.3 mL) in which (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) was dissolved. After the addition was complete, the whole mixture was stirred at room temperature overnight. The mixture was concentrated to about 1 mL under reduced pressure at room temperature, and the residue was subjected to HPLC preparation 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%-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)δppm7.90(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.

[0201] Example 4: Synthesis of Compound B-4

[0202]

[0203] Step A: At room temperature, take a 35 mL pressure tube, add diethyl methylphosphite (540 mg, 3.97 mmol) to toluene (10 mL) in which 1,3-bis(bromomethyl)-5-fluorobenzene (1.64 mg, 5.81 mmol) is dissolved, and the system is stirred evenly and reacted at 110°C for 3 hours. After the reaction is completed, the system is cooled to room temperature, and without additional treatment, the sample is directly spin-dried and purified by column chromatography (DCM: MeOH = 97: 3), and the solvent is removed by concentration under reduced pressure to obtain 800 mg of a light yellow oily product (3-(bromomethyl)-5-fluorobenzyl)(methyl)ethyl phosphite (800 mg, 2.33 mmol, 40%). ESI-MS m / z = 308.8 [M+H] + ;Calcu.=308.0.

[0204] Step B: At room temperature, take a 100 mL round-bottom flask, 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), stir well, and react at 40 ° C overnight. After the reaction was completed, the system was cooled to room temperature, and without additional treatment, the sample was directly spin-dried and purified by column chromatography (DCM: MeOH = 92: 8), and the solvent was removed by concentration under reduced pressure to obtain a light yellow oily product (3-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-fluorobenzyl)(methyl)phosphite ethyl ester (180 mg, 0.43 mmol, 28%). ESI-MS m / z = 424.1 [M+H] + ;Calcu.=423.1.

[0205] Step C: At room temperature, trimethylsilyl bromide (805 mg, 5.26 mmol) was added to dichloromethane (10 mL) in which (3-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-fluorobenzyl)(methyl)phosphite ethyl ester (180 mg, 0.43 mmol) was dissolved, and a drying tube was inserted, followed by continued stirring at room temperature for 72 hours. During this period, the reaction was monitored several times by LCMS, and 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 then the sample was directly sent to preparative liquid phase separation and purification after spin drying. Preparation conditions: Waters XBridge Prep ShieldRP18 OBD (19mm×250mm, 5μm); A: 0.1% formic acid in water; B: MeCN; 18mL / min; 0~3min, 5% B; 3~3.5min, 5%~15% B; 3.5~15min, 15%~70% B; 15~18min, 70%~100% B; detector, UV 254nm. The fractions were recovered and freeze-dried to obtain a white solid product (3-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-fluorobenzyl)(methyl)phosphonic acid (17mg, 0.043mmol, 10%). ESI-MS m / z=395.9[M+H] + ;Calcu.=395.1.

[0206] Step D: 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (112 mg, 0.22 mmol) and N,N-diisopropylethylamine (28 mg, 0.22 mmol) were added to 1,2-dichloroethane (10 mL) and N,N-dimethylformamide (1.5 mL) in which (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) was dissolved at room temperature. After the addition was complete, the whole mixture was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to obtain a colorless oily crude product, and the residue was subjected to HPLC preparation and separation. Preparation conditions: WatersXBridge RP18 (19mm×250mm, 5μm); A: 0.1% formic acid in water, B: MeCN; 18mL / min; 0-3min, 5% B; 3-4min, 5%-20% B; 4-15min, 20%-60% B; 15-16min, 60%-100% B; detector, UV 254nm. The reaction obtained compound B-4, a white solid 1 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.

[0207] Example 5: Synthesis of Compound B-5 / M-1

[0208]

[0209] Step A: At 0°C, dimethyl phosphite (1.48 g, 13.49 mmol) dissolved in tetrahydrofuran (30 mL) was slowly added dropwise to sodium hydride (0.22 g, 8.99 mmol) dissolved in tetrahydrofuran (30 mL). 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) dissolved in tetrahydrofuran (60 mL) was added. The whole mixture was heated to reflux and stirred for 2 hours. Saturated ammonium chloride solution (30 mL) was slowly added to the reaction solution, and tetrahydrofuran was removed by concentrating under reduced pressure at 45°C. The mixture was extracted three times with ethyl acetate (100 mL*3), and the organic phases were combined and washed twice with saturated brine (100 mL*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained crude product was separated by silica gel column (DCM: MeOH = 95:5). The reaction gave 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.

[0210] Step B: At room temperature, sodium borohydride (1.01 g, 26.7 mmol) was added in batches to tetrahydrofuran (25 mL) in which methyl 4-bromo-3-((dimethoxyphosphoryl)methyl)benzoate (1.80 g, 5.34 mmol) was dissolved. After the addition was complete, the entire mixture was heated to 65°C and stirred at this temperature for 0.5 hours. Then methanol (25 mL) was slowly added dropwise and reacted at 65°C for 2 hours. The reaction solution was cooled in an ice bath, saturated ammonium chloride solution (50 mL) was slowly added, and tetrahydrofuran and methanol were removed by concentration under reduced pressure, and then water (50 mL) was added, and the mixture was extracted three times with dichloromethane (100 mL*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. The reaction obtained a light yellow oily liquid (2-bromo-5-(hydroxymethyl)benzyl)phosphonic acid dimethyl ester (1.51 g, 4.88 mmol, 91%), which was directly used for the next step without purification. ESI-MS m / z=308.8,310.8[M+H] + ;Calcu.=308.0,310.0.

[0211] Step C: At 0°C, slowly add phosphorus tribromide (0.5 mL, 5.33 mmol) to dichloromethane (25 mL) in which (1.5 g, 4.85 mmol) of (2-bromo-5-(hydroxymethyl)benzyl)phosphonic acid dimethyl ester is dissolved. After the addition, the whole mixture is stirred at temperature for 10 minutes, and then slowly add water (25 mL) to quench the reaction. The mixture is extracted with dichloromethane three times (25 mL*3). The organic phases are combined, washed once with saturated brine (25 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product is separated on a silica gel column (DCM:MeOH=95:5). The reaction yields a white product (840 mg, 2.26 mmol, 47%) of (2-bromo-5-(bromomethyl)benzyl)phosphonic acid dimethyl ester. ESI-MS m / z=370.7, 372.7, 374.7 [M+H] + ;Calcu.=369.9,371.9,373.9..

[0212] Step D: At room temperature, potassium carbonate (759 mg, 5.5 mmol) was added to N,N-dimethylformamide (3 mL) in which trifluoroacetate (340 mg, 1.1 mmol) of 2-((4-amino-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethane-1-ol was dissolved. After the addition, the mixture was stirred at room temperature for 30 minutes. 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 whole mixture was heated to 60°C and stirred for 3 hours. The reaction solution was filtered, concentrated to dryness under reduced pressure to obtain a crude product, which was separated by silica gel column (SiO2, EA:MeOH=88:12). The reaction gave a white solid product (5-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromobenzyl)phosphonic acid dimethyl ester (200 mg, 0.41 mmol, 37%). ESI-MS m / z=485.9, 487.9 [M+H] + ;Calcu.=485.0,487.0.

[0213] Step E: At room temperature, lithium bromide (209 mg, 2.4 mmol) was added to acetonitrile (20 mL) in which dimethyl (5-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromobenzyl)phosphonate (195 mg, 0.4 mmol) was dissolved. After the addition, the mixture was heated to reflux and stirred for 6 hours. The mixture was concentrated to dryness under reduced pressure at 40°C, and the residue was dissolved in methanol (5 mL) and subjected to reverse phase separation. The column was X select C18 (19 mm x 150 mm); the mobile phase was water (containing 0.1% NH4HCO3) and acetonitrile; the flow rate was 40 mL / min; the gradient was acetonitrile from 0% to 100% in 20 minutes; the detection wavelength was 254 nm. The mixture was freeze-dried 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.

[0214] Step F: At room temperature, N,N-diisopropylethylamine (0.26mL, 1.6mmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphate (1.19g, 2.28mmol) 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 (180mg, 0.38mmol), N,N-dimethylformamide (50mL) and 1,2-dichloroethane (330mL). After the addition, the whole 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 by 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.

[0215] Step G: At room temperature and under N2 protection, 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 (18.2mg, 0.04mmol), potassium trifluoro(pyrrolidin-1-ylmethyl)borate (19.1mg, 0.1mmol), sodium carbonate (12.7mg, 0.12mmol), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (7.6mg, 0.016mmol) in 1,4-dioxane (1.6mL) and water (0.4mL), tridibenzylideneacetone dipalladium (7.3mg, 0.008mmol) was added, and after the addition, the whole mixture was heated to 90℃ and stirred for 3 hours under protection. Filtered with suction, washed with a small amount of methanol, the filtrate was concentrated to about 1mL, and the residue was subjected to HPLC preparative separation. Preparation conditions: YMC-Actus Triart C18 (19mm×250mm, 5μm); A: 0.1% formic acid in water, B: MeCN; 18mL / min; 0~3min, 10% B; 3~3.5min, 10%~20% B; 3.5~17min, 20%~65% B; detector, UV 254nm. After purification, compound B-5 was obtained, 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(500MHz,DMSO-d6)δppm8.23(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.

[0216] Example 6: Synthesis of Compound B-6

[0217]

[0218] Step A: In a thick-walled pressure-resistant tube, methyl 3-(bromomethyl)-4-fluorobenzoate (3.8 g, 15.38 mmol), toluene (20 mL) and diethyl methylphosphite (3.5 mL, 23.07 mmol) were added in sequence. After the addition, the reaction mixture was heated and stirred at 110°C for 3 hours. The solvent was removed by concentration under reduced pressure, and the resulting crude product was separated by silica gel column (DCM: MeOH = 90: 10). The reaction obtained the product methyl 3-((ethoxy(methyl)phosphoryl)methyl)-4-fluorobenzoate (3.45 g, 12.58 mmol, 82%) as a yellow oil.

[0219] Step B: At 65°C, sodium borohydride (2.38 g, 62.90 mmol) was added to tetrahydrofuran (25 mL) in which methyl 3-((ethoxy(methyl)phosphoryl)methyl)-4-fluorobenzoate (3.45 g, 12.58 mmol) was dissolved, and then methanol (25 mL) was slowly added dropwise. After stirring evenly, the reaction mixture was stirred at 65°C for 2 hours. Saturated ammonium chloride was added to quench the reaction, and water and dichloromethane were added for extraction. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the product (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.

[0220] Step C: At 0°C, phosphorus tribromide (1 mL, 10.64 mmol) was added to dichloromethane (30 mL) in which (2-fluoro-5-(hydroxymethyl)benzyl)(methyl)phosphinate (2.34 g, 9.50 mmol) was dissolved. After stirring evenly, the reaction mixture was stirred at 0°C for 10 minutes. Water was added to the reaction system to quench the reaction, and the dichloromethane was extracted and the organic phases were combined. The mixture was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was separated on a silica gel column (DCM:MeOH=90:10). The reaction obtained the product (5-(bromomethyl)-2-fluorobenzyl)(methyl)phosphinate (1.7 g, 5.50 mmol, 58%) as a yellow solid. ESI-MS m / z=308.8,310.8[M+H] + ;Calcu.=308.0,310.0.

[0221] Step D: At room temperature, potassium carbonate (93 mg, 0.68 mmol) was added to N,N-dimethylformamide (5 mL) in which 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) were dissolved. After the addition, the whole mixture was heated to 60°C and stirred for 3 hours. The reaction solution was filtered, concentrated to dryness under reduced pressure, and the crude product was subjected to silica gel column chromatography (SiO2, EA:MeOH=88:12). The reaction gave a white solid product (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%). ESI-MS m / z=424.0[M+H] + ;Calcu.=423.2.

[0222] Step E: At 0°C, trimethylsilyl bromide (180 mg, 1.18 mmol) was added to dichloromethane (2 mL) in which 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) was dissolved. After the addition was complete, the entire mixture was stirred at room temperature overnight. 0.6 mL (MeOH:H2O=83:17) was added to the reaction solution to quench the reaction, and the reaction was concentrated to dryness under reduced pressure. The crude product was separated by HPLC. Preparation conditions: Waters XBridge C18 (19mm×250mm, 5μm); A: 0.1% formic acid in water, B: MeCN; 18mL / min; 0~3min, 5% B; 3~3.5min, 5%~10% B; 3.5~15min, 10%~60% B; detector, UV254nm. The reaction gave the product (5-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-fluorobenzyl)(methyl)phosphinic acid (5mg, 0.013mmol, 21%) as a white solid. ESI-MS m / z=395.9[M+H] + ;Calcu.=395.1.

[0223] Step F: At room temperature, N,N-diisopropylethylamine (5.5 mg, 0.042 mmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphate (32 mg, 0.061 mmol) were added to 1,2-dichloroethane (8.7 mL) and N,N-dimethylformamide (1.3 mL) in which (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) was dissolved. 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. Preparation conditions: Waters XBridge C18 (19mm×250mm, 5μm); A: 0.1% formic acid in water, B: MeCN; 18mL / min; 0~3min, 5% B; 3~3.5min, 5%~15% B; 3.5~15min, 10%~55% B; detector, UV 254nm). 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.

[0224] Example 7: Synthesis of Compound B-7 / M-2

[0225]

[0226] Step A: At room temperature, tert-butyldimethylsilyl chloride (4.63 g, 30.7 mmol) and imidazole (3.49 g, 51.2 mmol) were added to a solution of 2-((4-amino-1H-pyrazolo[3,4-d]pyrimidin-6-yl)oxy)ethane-1-ol (4.00 g, 20.5 mmol) in N,N-dimethylformamide (10 mL) in sequence. The reaction mixture was reacted at room temperature for 2 hours. After the reaction, it was concentrated to obtain a crude product. The crude product was separated by silica gel column (SiO2, DCM:MeOH=95:5) to obtain a white solid 6-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (5.60 g, 18.1 mmol, 88%). ESI-MS m / z=310.0[M+H] + ;Calcu.=309.2.

[0227] 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 the reaction was carried out for 8 hours. After the reaction was completed, the reaction was filtered, the insoluble matter was washed with ethyl acetate, and the filtrate was dried to obtain a crude product. The crude product 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).

[0228] Step C: At room temperature, diethyl methylphosphite (5.30 g, 38.97 mmol) was added to a sealed tube of a toluene solution of methyl 4-bromo-3-(bromomethyl)benzoate (10.0 g, 32.47 mmol), and the reaction mixture was heated to 110°C and reacted for 3 hours. After the reaction, the reaction mixture was cooled to room temperature, and the reaction solution was concentrated to obtain a crude product. The crude product was separated by a silica gel column (SiO2, DCM: MeOH = 95:5) to obtain a colorless oily substance 4-bromo-3-((ethoxy(methyl)phosphoryl)methyl)benzoate (9.10 g, 27.15 mmol, 84%). ESI-MS m / z = 334.8 [M+H] + ;Calcu.=334.0.

[0229] Step D: Sodium borohydride (9.93 g, 262.6 mmol) was added to a tetrahydrofuran (100 mL) solution of methyl 4-bromo-3-((ethoxy(methyl)phosphoryl)methyl)benzoate (8.80 g, 26.26 mmol) at room temperature. The reaction mixture was reacted at room temperature for 20 minutes, and then methanol (100 mL) was slowly added dropwise to the reaction system. After the addition, the reaction mixture was heated to 70°C and continued to react for 6 hours. After the reaction was completed, the reaction mixture was cooled to room temperature and concentrated to obtain a crude product. The crude product was separated by a silica gel column (SiO2, DCM:MeOH=90:10) to obtain colorless oily (2-bromo-5-(hydroxymethyl)benzyl)(methyl)ethyl phosphinate (6.60 g, 21.49 mmol, 82%). ESI-MS m / z=306.8[M+H] + ;Calcu.=306.0.

[0230] Step E: At room temperature, p-toluenesulfonyl 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). The reaction mixture was cooled to 0°C, the reaction system was replaced with N2 protection, and triethylamine (27 mL, 195.4 mmol) was slowly added dropwise to the reaction system. After the addition, the reaction mixture was slowly warmed to room temperature and the reaction was continued for 18 hours. After the reaction was completed, 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 a colorless oily substance 4-bromo-3-((ethoxy(methyl)phosphoryl)methyl)benzyl-4-methylbenzenesulfonate (3.80 g, 8.24 mmol, 42%). ESI-MS m / z=324.7[M-135] + ;Calcu.=460.0.

[0231] 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 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. Heat to 60°C and react for 3 hours. After the reaction, the reaction mixture is cooled to room temperature, filtered, and the filtrate is concentrated to obtain a crude product. The crude product is separated by 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)phosphinate (2.70 g, 4.51 mmol, 59%). ESI-MS m / z=598.0[M+H] + ;Calcu.=597.1.

[0232] 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 completed, methanol (50 mL) was added to the reaction system, and then 3M sodium hydroxide solution was slowly added dropwise to adjust the pH of the reaction solution to neutral. The crude product was concentrated to obtain a crude product. The crude product was dissolved in 20 mL of a mixed solvent (methanol:acetone = 1:1), and the insoluble matter was removed by filtration. The filtrate was concentrated to obtain a light 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 product). The crude product was directly used in the next step without purification. ESI-MS m / z = 455.8 [M+H] + ;Calcu.=455.0.

[0233] Step H: At room temperature, 1H-benzotriazol-1-yloxytripyrrolidino 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). 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. The crude product was separated by 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, two-step yield 26%). 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.

[0234] 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 tris(dibenzylideneacetone)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 a mixed solution of 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 and reacted 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. The preparation conditions were: Waters XBridge C18 (30 mm×250 mm, 5 μm); A: 0.1% formic acod 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 freeze-dried 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 (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=10.0Hz1H),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.

[0235] Example 8: Synthesis of Compound B-8

[0236]

[0237] Step A: At room temperature, diethyl methylphosphite (3.94 g, 28.95 mmol) was added to toluene (50 mL) in which methyl 3-(bromomethyl)-4-methoxybenzoate (5 g, 19.3 mmol) was dissolved. After the addition, the whole 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 separated by silica gel column (SiO2, DCM:MeOH=95:5). The reaction obtained a colorless oily product, methyl 3-((ethoxy(methyl)phosphoryl)methyl)-4-methoxybenzoate (5.02 g, 17.54 mmol, 91%). ESI-MS m / z=286.9[M+H] + ;Calcu.=286.1.

[0238] Step B: At -30°C, 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). After the addition, the entire mixture was stirred at -30°C for 3 hours. Methanol was added to the reaction solution to quench it, and it was concentrated under reduced pressure to obtain a crude product. The crude product was separated by silica gel column (SiO2, DCM:MeOH=95:5). The reaction gave a colorless oily product (ethyl 5-(hydroxymethyl)-2-methoxybenzyl)(methyl)phosphinate (2.3 g, 8.91 mmol, 51%). ESI-MS m / z=258.9[M+H] + ;Calcu.=258.1.

[0239] Step C: At 0°C, add thionyl chloride (1.24 g, 10.46 mmol) to dichloromethane (15 mL) in which (5-(hydroxymethyl)-2-methoxybenzyl)(methyl)phosphinate (1.50 g, 5.81 mmol) is dissolved. After the addition, the entire mixture is stirred at 0°C for 2 hours. Methanol is added to the reaction solution, and the mixture is concentrated under reduced pressure to obtain a crude product. The crude product is separated by a silica gel column (SiO2, DCM:MeOH=95:5). The reaction yields a colorless oily product (5-(chloromethyl)-2-methoxybenzyl)(methyl)phosphinate (800 mg, 2.60 mmol, 45%). 1 H NMR(500MHz,DMSO-d6)δppm7.28-7.32(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).

[0240] 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 whole mixture was stirred at room temperature for 0.5 hours. Then (5-(chloromethyl)-2-methoxybenzyl)(methyl) ethyl phosphinate (829 mg, 2.997 mmol) was added thereto, and the temperature was raised to 60°C and reacted overnight. The reaction solution was filtered through diatomaceous earth 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 increased from 0% to 100% in 30 minutes; detection wavelength: 254nm. Freeze-dried under reduced pressure to obtain a colorless oily product (5-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-methoxybenzyl)(methyl)phosphinate (90mg, 0.186mmol, 8%). ESI-MS m / z=436.0[M+H] + ;Calcu.=435.2.

[0241] Step E: At room temperature, sodium hydroxide (18.4 mg, 0.46 mmol) in water (1 mL) was added to methanol (1 mL) in which 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) was dissolved. After the addition, the whole mixture was slowly warmed to 80°C and stirred for 8 hours. The reaction solution was directly subjected to reverse phase separation, chromatographic column: Santai SepaFlash C18 (Spherical C18, 20-45 μm, 40g); mobile phase: water (containing 0.05% TFA) and acetonitrile; flow rate: 40 ml / min; gradient: acetonitrile increased from 0% to 100% in 30 minutes; detection wavelength: 254nm. Freeze-dried under reduced pressure to obtain a white solid (5-((4-amino-6-(2-hydroxyethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-methoxybenzyl)(methyl)phosphinic acid (24mg, 0.059mmol, 64%). ESI-MS m / z=408.0[M+H] + ;Calcu.=407.1.

[0242] Step F: At room temperature, 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (184 mg, 0.354 mmol) was added to N,N-dimethylformamide (8 mL) and 1,2-dichloroethane (53 mL) in which (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) were dissolved. After the addition was complete, the whole mixture was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was prepared and separated 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 freeze-dried 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)δppm8.15-8.19(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.

[0243] Example 9: Synthesis of Compound B-9

[0244]

[0245] Step A: Potassium (dimethylamino)methyl trifluoroborate (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 tris(dibenzylideneacetone)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 a mixed solution of 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 and reacted 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 (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, UV254 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)-benzazepinenonane-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.

[0246] Example 10: Synthesis of Compound B-10

[0247]

[0248] Step A: At 25°C, 1-(3-(bromomethyl)phenyl)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-N-methylmethanesulfonamide (592 mg, 1.36 mmol) dissolved in N,N-dimethylformamide (3 mL) was slowly added to 6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine (230 mg, 1.36 mmol) and potassium carbonate (562 mg, 4.07 mmol) at N,N-dimethylformamide (3 mL). After the addition was complete, the entire mixture was stirred at 25°C for 18 hours. The reaction solution was concentrated under reduced pressure to obtain a solid residue, which was separated by a silica gel column (SiO2, DCM:MeOH=90:10). The reaction gave 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.

[0249] Step B: At 0-5°C, acetic acid (46 μL, 0.8 mmol) was slowly added to a tetrabutylammonium fluoride solution (0.8 mL, 0.8 mmol) until the pH of the solution was 7.0, and then, at 0-5°C, 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) was dissolved in tetrahydrofuran (5 mL), and the above-prepared mixed solution was slowly added dropwise. After the addition, the whole mixture was stirred at 25°C for 3 hours. The reaction solution was directly concentrated under reduced pressure to obtain a solid residue, and the solid residue was separated by a silica gel column (SiO2, DCM:MeOH=90:10). The reaction gave 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.

[0250] Step C: Cesium carbonate (674 mg, 2.069 mmol) and lithium iodide (277 mg, 2.069 mmol) were added to acetonitrile (80 mL) in which 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) was dissolved 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, preparation conditions: YMC-Actus Triart C18, 20*250; A: 5mM ammonia in water; B: MeCN; 18mL / min; 0~3min, 5% B; 3~3.5min, 5%~20% B; 3.5~15min, 20%~60% 15~15.5min, 60%~100% B; detector, UV 254nm. The 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.

[0251] Example 11: Synthesis of Compound B-11 / B-12

[0252]

[0253] Step A: At 20°C, thiourea (1.09 g, 14.29 mmol) was added to ethanol (40 mL) in which methyl 4-bromo-3-(bromomethyl)benzoate (4.0 g, 12.99 mmol) was dissolved, and then the whole mixture was heated to 96°C and stirred at 96°C for 2 hours, and then concentrated under reduced pressure to remove ethanol to obtain a white solid, and then acetonitrile (18 mL) was added to the white solid, and then 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, and then N-chlorosuccinimide (6.94 g, 51.95 mmol) was added to the mixture in batches 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 solution was concentrated under reduced pressure to remove most of the acetonitrile, and then extracted twice with ethyl acetate (50 mL*2). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a solid residue. The solid residue was separated by a silica gel column (SiO2, PE:EA=75:25) to obtain a white solid product, 4-bromo-3-((chlorosulfonyl)methyl)benzoic acid methyl ester (3.15 g, crude product).

[0254] Step B: At 25°C and N2 protection, 2-((tert-butyldimethylsilyl)oxy)-N-methylethane-1-amine (2.18g, 11.54mmol), N,N-diisopropylethylamine (3.73g, 28.85mmol) and 4-dimethylaminopyridine (587mg, 4.81mmol) dissolved in dichloromethane (10mL) were slowly added dropwise. After the addition, the whole mixture was stirred at 25°C and N2 protection for 18 hours. Water (50mL) was added to the reaction solution, and it was extracted twice with dichloromethane (20mL*2). The organic phases were combined, washed once with saturated sodium chloride aqueous solution (50mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a solid residue. The solid residue was separated by silica gel column (SiO2, PE:EA=83:17). The reaction gave a yellow oily product, methyl 4-bromo-3-((N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-N-methylaminosulfonyl)methyl)benzoate (1.52 g, 3.16 mmol, two-step yield 24%). 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).

[0255] Step C: At 0-5°C, lithium aluminum hydride (96.04 mg, 2.53 mmol) was slowly added to tetrahydrofuran (10 mL) in which methyl 4-bromo-3-((N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-N-methylaminosulfonyl)methyl)benzoate (1.52 g, 3.16 mmol) was dissolved, and the whole mixture was stirred at 25°C for 0.5 hours. The reaction solution was cooled to 0-5°C, and then sodium sulfate nonahydrate was slowly added to the reaction solution until no bubbles were generated, and then the whole mixture was stirred at room temperature for 30 minutes, and then filtered through diatomaceous earth, and the filtrate was collected and concentrated under reduced pressure to obtain a solid residue. The solid residue was separated by silica gel column (SiO2, PE:EA=80:20). The reaction gave 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.

[0256] Step D: At 0-5°C, N-bromosuccinimide (972 mg, 5.46 mmol) was slowly added to acetonitrile (20 mL) in which 1-(2-bromo-5-(hydroxymethyl)phenyl)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-N-methylmethanesulfonamide (1.90 g, crude product) and triphenylphosphine (1.43 mg, 5.46 mmol) were dissolved, and then stirring was continued at 25°C for 2.5 hours. The reaction solution was washed once with a saturated aqueous sodium bicarbonate solution (50 mL), washed once with a saturated aqueous 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 by 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, two-step yield 32%). 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).

[0257] Step E: At 25°C, 1-(2-bromo-5-(bromomethyl)phenyl)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-N-methylmethanesulfonamide (523 mg, 1.02 mmol) was slowly added dropwise to N,N-dimethylformamide (3 mL) in which 6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine (246 mg, 1.02 mmol) and potassium carbonate (421 mg, 3.04 mmol) were dissolved. After the addition was complete, the mixture was stirred at 25°C for 15 hours. The reaction solution was directly concentrated under reduced pressure to obtain a solid residue. The solid residue was separated by silica gel column (SiO2, DCM:MeOH=90:10). The reaction afforded a yellow oily 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%). 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.

[0258] Step F: Tetrabutylammonium fluoride (0.54 mL, 0.54 mmol) was slowly added dropwise to tetrahydrofuran (5 mL) in which 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) was dissolved at 0-5°C. After the addition was complete, the whole mixture was stirred at 20°C for 1 hour. Water (10 mL) was added to the reaction solution, and the mixture was extracted twice with ethyl acetate (40 mL*2). The two organic phases were combined, washed once with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a solid residue. The solid residue was separated by a silica gel column (SiO2, DCM:MeOH=90:10). The reaction gave 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.

[0259] Step G: At 20°C, cesium carbonate (1.46 g, 4.49 mmol) and lithium iodide (301 mg, 2.25 mmol) were added to acetonitrile (30 mL) in which 1-(5-((4-amino-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromophenyl)-N-(2-hydroxyethyl)-N-methylmethanesulfonamide (110 mg, 0.225 mmol) was dissolved, and then the whole mixture was 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 diatomaceous earth, 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 obtained 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.

[0260] 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 with 2-dicyclohexylphosphine-2',4',6' -Triisopropylbiphenyl (3.2 mg, 0.007 mmol) and sodium carbonate (7.0 mg, 0.066 mmol), then the whole mixture was replaced with nitrogen three times, and then tridibenzylideneacetone dipalladium (3.0 mg, 0.003 mmol) was added to the mixture under nitrogen protection at 20 ° C, and then the whole mixture was replaced with nitrogen twice, heated to 90 ° C, and stirred for 3 hours at 90 ° C and nitrogen protection. The reaction solution was cooled to room temperature, then filtered through diatomaceous earth, and concentrated under reduced pressure after filtration to obtain a light yellow substance. The light yellow oil was subjected to HPLC preparation and separation, preparation conditions: Waters XBridge PrepShield RP18 5um OBD, 19*250; A: 5mM ammonia in water; B: MeCN; 16mL / min; 0~3min, 5% B; 3~15min, 5%~95% B; detector, UV 254nm. 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.

[0261] Example 12: Synthesis of Compound B-13 / M-3

[0262]

[0263] Step A: At 25°C, N,N-diisopropylethylamine (592 mg, 4.58 mmol) and DMAP (187 mg, 1.53 mmol) were added to dichloromethane (100 mL) in which 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) were dissolved. After the addition of DMAP (187 mg, 1.53 mmol), the whole mixture was stirred at room temperature for 3 hours. After the reaction was complete, it was directly concentrated to obtain a solid residue. The residue was separated by silica gel column (SiO2, PE:EA=50:50) to obtain a colorless oil product, methyl 4-bromo-3-((N-(2-((tert-butyldiphenylsilyl)oxy)ethyl)sulfamoyl)methyl)benzoate (1.1 g, 61%). 1 HNMR(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).

[0264] Step B: At 25°C, triethylamine (0.8mL, 5.62mmol) and 4-dimethylaminopyridine (45.8mg, 0.375mmol) were added to tetrahydrofuran (40mL) in which methyl 4-bromo-3-((N-(2-((tert-butyldiphenylsilyl)oxy)ethyl)sulfamoyl)methyl)benzoate (1.1g, 1.88mmol) and di-tert-butyl dicarbonate (450mg, 2.06mmol) were dissolved. The whole mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was directly concentrated to obtain a solid residue. The residue was separated by silica gel column (SiO2, PE:EA=66:34) and the reaction gave 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 HNMR (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(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).

[0265] Step C: At 0°C, lithium aluminum hydride (55 mg, 1.45 mmol) was added in batches to tetrahydrofuran (30 mL) in which methyl 4-bromo-3-((N-(tert-butyloxycarbonyl)-N-(2-((tert-butyldiphenylsilyl)oxy)ethyl)sulfamoyl)methyl)benzoate (1.0 g, 1.448 mmol) was dissolved. After the addition was complete, the entire mixture was stirred at 0°C for 2 hours. After the reaction was complete, the reaction solution was temperature controlled at 0°C, and then methanol (20 mL) was slowly added until no obvious bubbles appeared in the reaction solution. The temperature was controlled not to exceed 20°C during quenching, and then filtered through diatomaceous earth, silica gel was added to the filtrate, and the solid residue was concentrated under reduced pressure. The residue was separated by silica gel column (SiO2, PE:EA=50:50). The reaction gave 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).

[0266] Step D: At 0°C, N-bromosuccinimide (161 mg, 0.905 mmol) was added in batches to tetrahydrofuran (20 mL) in which 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) were dissolved. After the addition was complete, the entire mixture was stirred at 0°C for 3 hours. After the reaction was complete, the residue was separated by silica gel column (SiO2, PE:EA=84:16). The reaction gave 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.

[0267] Step E: At room temperature, potassium carbonate (190 mg, 1.378 mmol) was added to N,N-dimethylformamide (5 mL) in which 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) were dissolved. After the addition was complete, the whole mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction solution was controlled to room temperature or filtered through diatomaceous earth, and then water (50 mL) was added. The mixture was extracted three times with ethyl acetate (20 mL×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 gave a white solid product, 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%). ESI-MS m / z=813.1 [M+H] + ;Calcu.=812.2.

[0268] Step F: At 25°C, cesium carbonate (800 mg, 2.46 mmol) and lithium iodide (329 mg, 2.46 mmol) were added to acetonitrile (10 mL) in which 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, 0.246 mmol) was dissolved. After the addition of lithium iodide (329 mg, 2.46 mmol), the whole 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 prepared by HPLC. Separation and preparation conditions: Waters XBridge C 18 (19mm×250mm, 5μm); A: 0.1% formic acid in water; B: MeCN; 18mL / min; 0~3min, 5% B; 3.5~15min, 10%~100% B; detector, UV 254nm, freeze-dried. A white solid product 1 was obtained 4 -Amino-3 4 -Bromo-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, 14%). ESI-MS m / z=438.9[M+H]+ ;Calcu.=438.0.

[0269] Step G: At 25°C under nitrogen protection, dissolve 1 4 -Amino-3 4 -Bromo-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), tri(dibenzylideneacetone)dipalladium (3.1 mg, 0.003 mmol) was added, and the whole mixture was stirred at 90°C overnight. After the reaction was complete, the reaction solution was directly concentrated and prepared. Preparation conditions: Waters XBridgeC 18 (19mm×250mm, 5μm); A: 0.1% formic acid in water; B: MeCN; 18mL / min; 0~3min, 5% B; 3.5~15min, 10%~100% B; detector, UV 254nm, freeze-dried. Compound B-13, 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 (0.8mg, 5%) was obtained. ESI-MS m / z=443.9[M+H] + ;Calcu.=443.2.

[0270] Example 13: Synthesis of Compound B-14

[0271]

[0272] Step A: Add ammonia water (25%) (100 mL, 713.3 mmol) to the solid 4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (6.67 g, 35.29 mmol) at room temperature. After the addition, the whole mixture was stirred at room temperature overnight. After that, the reaction solution was diluted with ethyl acetate (60 mL) and filtered with a Buchner funnel to obtain a yellow solid. Then, the obtained solid was washed with petroleum ether:ethyl acetate = 2:1 (300 mL) and dried to obtain a yellow solid product 6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine (5.62 g, 94%). ESI-MS m / z = 170.0 [M+H] + ;Calcu.=169.0.

[0273] Step B: At room temperature, potassium carbonate (2.05 g, 11.23 mmol) was added to a mixed solvent of tetrahydrofuran (25 mL) and N,N-dimethylformamide (40 mL) in which 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) were dissolved. After the addition, the entire mixture was controlled at room temperature and stirred overnight. After the reaction was completed, ethyl acetate (50 mL) was added to the reaction solution for dilution, filtered through a sand core 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 a yellow solid product 1-(4-bromo-3-nitrobenzyl)-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine (4.29 g, 54%). ESI-MS m / z = 382.8 [M+H] + ;Calcu.=382.0.

[0274] Step C: At room temperature, triethylamine (2.53 mL, 18.20 mmol) was added to tetrahydrofuran (240 mL) in which 1-(4-bromo-3-nitrobenzyl)-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine (2.35 mg, 6.13 mmol) was dissolved. After the addition, the whole mixture was stirred at 0°C for 15 min. 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, and the temperature was naturally raised to room temperature and stirred for 2 hours. Then, ethyl acetate (300 mL) was added to dilute the reaction solution, and then the organic phase was washed with saturated ammonium chloride solution (60 mL), saturated sodium bicarbonate solution (60 mL), and saturated saline solution (60 mL). Then, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain 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 directly used in subsequent reactions.

[0275] Step D: At room temperature, acetic acid (4 mL) and iron powder (0.55 g, 0.198 mmol) were added 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. After stirring evenly, 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 then the organic phase was washed with saturated ammonium chloride solution (35 mL), saturated sodium bicarbonate solution (35 mL), and saturated saline solution (35 mL). Then, the organic phase was 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 HNMR (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).

[0276] Step E: At 0°C, a solution of methyl 3-(chlorosulfonyl)propanoate (810 mg, 4.341 mmol) in dichloromethane (40 mL) was added 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). After the addition, the whole mixture was cooled to room temperature and stirred for 1 hour. At 0°C, ethyl acetate (150 mL) was added to dilute, and saturated ammonium chloride solution (30 mL) was added to separate the liquids. The obtained organic phase was then washed with saturated ammonium chloride solution (30 mL), saturated sodium bicarbonate solution (30 mL), and saturated brine (30 mL), and finally dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The reaction afforded a brown solid product, 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 product).

[0277] Step F: At 0°C, LiAlH4 (224 mg, 5.895 mmol) was slowly added in batches to tetrahydrofuran (50 mL) in which 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) was dissolved. After the addition, the whole mixture was warmed to room temperature and stirred for 1 hour. At 0°C, methanol (20 mL) was slowly added dropwise to quench the reaction. Then, the mixture was filtered through a sand core funnel with diatomaceous earth, and the reaction flask and diatomaceous earth were washed with ethyl acetate (100 mL) and methanol (15 mL). The resulting liquid was concentrated under reduced pressure and purified by column chromatography with 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, two-step yield 81%). 1H 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).

[0278] Step G: At 0°C, concentrated hydrochloric acid (36-38% wt) (3 mL) was added to dioxane (10 mL) in which 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) was dissolved. After the addition, the whole mixture was controlled to room temperature and stirred overnight. Ethyl acetate (100 mL) was added to dilute the reaction solution, and saturated sodium bicarbonate solution was slowly added under stirring at 0°C. The reaction solution was exothermic and bubbled a lot; sodium bicarbonate solution was added until the solution stopped bubbling to neutralize the hydrochloric acid. After that, ethyl acetate (100 mL) was added to dilute the solution, and the organic phase was separated by separation, and the organic phase was washed with saturated sodium bicarbonate solution (20 mL) and saturated brine (20 mL) in turn. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a white solid product, N-(5-((4-amino-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-2-bromophenyl)-3-hydroxypropane-1-sulfonamide (229 mg, 69%). ESI-MS m / z=474.8[M+H] + ;Calcu.=474.0.

[0279] Step H: Using a 120 mL pressure tube as a reaction vessel, add cesium carbonate (619 mg, 1.90 mmol) to acetonitrile (19 mL) in which 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) is dissolved at room temperature. After the addition, the entire mixture is heated to 110°C and stirred for 18 hours. The mixture is filtered through a sand core funnel, the solid is washed with ethyl acetate (80 mL) and methanol (15 mL), the filtrate is concentrated under reduced pressure, and sent to preparative HPLC for separation. Chromatographic conditions: Waters XBridge Prep Shield RP18 (19mm×250mm, 5μm); A: 5mM NH4HCO3, B: ACN; 18mL / min; 0~3min, 10% B; 3~3.5min, 10%~20% B; 3.5~15min, 20%~90% B; 15~15.5min, 90%~100% B; detector, UV 254nm. The obtained fractions were lyophilized to obtain compound B-14, a white solid product 1 4 -Amino-3 4 -Bromo-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%). 1 H 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.

[0280] Example 14: Synthesis of Compound B-15

[0281]

[0282] Step A: Using a 120 mL pressure tube as a reaction container, add cesium carbonate (988 mg, 3.03 mmol) to an acetonitrile solution (30 mL) containing 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) at room temperature. After the addition is complete, the entire mixture is 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 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 -Bromo-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 product). ESI-MS m / z=438.8[M+H] + ;Calcu.=438.0.

[0283] Step B: Under nitrogen protection, add 1 4 -Amino-3 4 -Bromo-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-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (9.5 mg, 0.020 mmol), sodium carbonate (32 mg, 0.302 mmol). Then, 1,4-dioxane (3.2 mL) and water (0.8 mL) were added. After stirring evenly, deoxygenation was performed three times to remove oxygen from the solvent. Then, the mixture was heated and stirred at 90°C for 4 hours under nitrogen protection. Afterwards, filter through a sand core funnel with a small amount of silica gel, wash the solid with ethyl acetate (60 mL) and methanol (20 mL), concentrate the obtained liquid under reduced pressure to obtain an orange crude product, and separate and purify the obtained solid by preparative chromatography. The chromatographic conditions are as follows: Waters XBridge PrepShield 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 for two steps). 1 H NMR(500MHz,DMSO-d6)δppm 8.13(s,1H),7.88(s,1H),7.70(brs,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.

[0284] Test Example 1: Evaluation of drug efficacy at the cellular level

[0285] Objective: To evaluate the TLR7 agonist activity of compounds using the HEK-BlueTMhTLR7 in vitro cell model

[0286] Experimental Materials:

[0287] 1 Cell line: HEK-BlueTMhTLR7 (InvivoGen)

[0288] 2 Reagents: QUANTI-BlueTMSolution (InvivoGen), DMEM (Invitrogen), FBS (HyClone), L-glutamine (GIBCO), TrypLE Express Enzyme (GIBCO), DPBS (GIBCO), NormocinTM (InvivoGen); Penicillin-Streptomycin (GIBCO)

[0289] 3 Instruments: Microplate reader (PerkinElmer, Multimode plate reader 2014), CO2 incubator (THERMO, STERI-CYCLEI160), cell counter (BECKMAN COULTER, vi-cell XR)

[0290] Experimental methods:

[0291] Step 1: Cell plating

[0292] a) Discard the culture medium in the T150 flask, add 10 mL of pre-warmed DPBS, and gently shake the flask to rinse the cells once. After discarding the DPBS, add 8.0 mL of TrypLETM Express enzyme to the T150 flask and gently shake the flask to infiltrate all cells.

[0293] b) Discard the TrypLETM Express enzyme in the culture bottle and place it in the incubator for 1 to 2 minutes.

[0294] c) Add serum-containing culture medium to terminate digestion, collect the cell suspension, centrifuge at 300r at room temperature for 5 minutes, and discard the supernatant.

[0295] d) Add fresh culture medium, mix the cells by pipetting, and then count the cells using a vi-cell XR cell counter.

[0296] e) Dilute the cells to a density of 6.25*10 5 cells / mL, 80 μL of the diluted cell suspension was inoculated into a 96-well plate, i.e., 5*10 4cells / well.

[0297] f) Place the 96-well plate in an incubator and incubate overnight (5% CO 2 , 37° C.).

[0298] Step 2: Drug treatment

[0299] a) The test compound was prepared into a 10 mM stock solution in DMSO;

[0300] b) The compound to be tested was diluted from 10 mM to 0.1 mM with culture medium, and then the compound was diluted 3-fold with culture medium containing 1% DMSO, for a total of 8 concentration gradients, DMSO was used as negative control, and R848 and GS9620 were used as positive controls.

[0301] c) Take 20 μL of the diluted compound and add it to a 96-well plate inoculated with 80 μL of cells, dilute the drug 5 times, and the final concentration of DMSO in each well is 0.2%. The final concentrations of the test compound and the control compound are 20000, 6667, 2222, 741, 247, 82, 27, and 9 nM.

[0302] d) Place the 96-well plate in a 5% CO2, 37°C incubator and incubate for 24 hours.

[0303] Step 3: Activity Detection

[0304] a) Take 10 μL of the cell supernatant after compound incubation and transfer it to a 96-well transparent detection plate, then add 90 μL of QUANTI-BlueTM Solution to each well of the detection plate, and incubate in a 5% CO2, 37°C incubator for 1 hour.

[0305] b) The light absorption of secretory alkaline phosphatase (SEAP) at 650 nm was detected using an ELISA reader.

[0306] Data Analysis:

[0307] The activation factor was calculated using the following formula: activation factor = OD 650 Treatment group / OD 650 The GraphPadPrism 7 software was used to fit the curve according to the concentration of the compound and the corresponding activation factor, and the EC 50 value.

[0308] Reference substances: R848 (InvivoGen) and GS-9620 (CAYMAN), the structures are shown below:

[0309]

[0310] The experimental results are shown in the following table:

[0311]

[0312]

[0313] Test Example 2: Test of the agonistic activity of the compounds of the present invention on human peripheral blood mononuclear cells

[0314] Human peripheral blood mononuclear cells (hPBMCs) represent the main human immune cells in the blood, including lymphocytes, monocytes and dendritic cells. These cells express TLR7, TLR8 or TLR9 and respond to stimulation by the corresponding ligands. In this experiment, hPBMCs were stimulated with potential TLR7 agonists, and the levels of IFN-α, TNF-α and IL-12p40 were detected to reflect the activation of the compounds on the TLR7 receptor.

[0315] Experimental Materials:

[0316] 1. Cell line: hPBMCs (sailybio), culture conditions: RPMI 1640 medium + 10% (v / v) FBS + 1% double antibody (Penicillin-Streptomycin), 5% CO2, 37°C.

[0317] 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).

[0318] 3. Instruments: microplate reader (perkinElmer, Multimode plate reader 2014), CO2 incubator (THERMO, STERI-CYCLEI160), cell counter (BECKMAN COULTER, vi-cell XR).

[0319] Experimental methods:

[0320] g) The frozen hPBMCs were thawed in a 37°C water bath and transferred to 1640 complete medium.

[0321] After centrifugation at 500r for 10 minutes at room temperature, the supernatant was discarded, the cells were resuspended in 1640 complete medium and transferred to a culture flask, and cultured overnight at 37°C and 5% CO2.

[0322] h) Collect hPBMCs after stable culture, count and dilute the cells to 1.11*10 7 cells / mL, 180 μL of the diluted cell suspension was inoculated into a 96-well plate, i.e. 2*10 6 cells / well, and placed the 96-well plate in a constant temperature incubator for 24 h (5% CO2, 37°C).

[0323] i) The test compound was prepared into a 10 mM stock solution with DMSO and diluted to 0.03 mM (IFN-α detection) and 0.1 mM (TNF-α, IL-12p40 detection), respectively. Then, the compound was diluted into 8 concentration gradients with culture medium containing 2% DMSO. The final maximum concentration of the compound was 30 uM (IFN-α detection) and 100 uM (TNF-α, IL-12p40 detection), respectively.

[0324] j) 20 μL of the diluted compound was added to a 96-well cell culture plate, and the final concentration of DMSO in each well was 0.2%.

[0325] 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 an ELISA detection kit.

[0326] l) The light absorption at 450 nm was measured by an enzyme-labeled instrument, and the level of secretion of the corresponding cytokines was quantitatively determined according to the corresponding standard curve, and the EC was calculated by fitting the curve. 50 The experimental results are shown in the following table:

[0327]

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 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; 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, and 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).

Images