WO2025241971A1 - 作为cd73抑制剂衍生物及其应用 - Google Patents

作为cd73抑制剂衍生物及其应用

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Publication number
WO2025241971A1
WO2025241971A1 PCT/CN2025/095011 CN2025095011W WO2025241971A1 WO 2025241971 A1 WO2025241971 A1 WO 2025241971A1 CN 2025095011 W CN2025095011 W CN 2025095011W WO 2025241971 A1 WO2025241971 A1 WO 2025241971A1
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Prior art keywords
optionally substituted
compound
cancer
mmol
indazol
Prior art date
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PCT/CN2025/095011
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English (en)
French (fr)
Inventor
李洪林
钱旭红
徐玉芳
赵振江
朱丽丽
张国真
石存剑
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
East China University of Science and Technology
East China Normal University
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East China University of Science and Technology
East China Normal University
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Application filed by East China University of Science and Technology, East China Normal University filed Critical East China University of Science and Technology
Publication of WO2025241971A1 publication Critical patent/WO2025241971A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65583—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system each of the hetero rings containing nitrogen as ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4192—1,2,3-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
    • C07F9/6571—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms
    • C07F9/657163—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms the ring phosphorus atom being bound to at least one carbon atom
    • C07F9/657172—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms the ring phosphorus atom being bound to at least one carbon atom the ring phosphorus atom and one oxygen atom being part of a (thio)phosphinic acid ester: (X = O, S)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
    • C07F9/6571—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms
    • C07F9/6574—Esters of oxyacids of phosphorus

Definitions

  • This invention relates to the field of medicinal chemistry; specifically, it relates to novel compounds, their synthesis methods, and their application as CD73 inhibitors in the preparation of drugs for tumor-related diseases.
  • Adenosine interferes with cell proliferation, apoptosis, and angiogenesis. It fosters immune tolerance in the tumor microenvironment and promotes tumor development.
  • CD73 is a membrane-associated enzyme fixed to the cell surface via C-terminal glycosylated phosphatidylinositol (GPI). It is ubiquitous in lymphocytes, endothelial cells, and epithelial cells, and is highly expressed in the colon, kidney, and brain. In normal tissues, acute elevations of CD73-dependent adenosine primarily serve to protect ischemic and inflamed tissues, playing physiological roles in ion transport, barrier function maintenance, endothelial cell homeostasis, and cardioprotection.
  • GPI C-terminal glycosylated phosphatidylinositol
  • CD73 can be detached from the membrane by phosphatidylinositol-specific phospholipases or proteolytic enzymes, producing soluble CD73 that retains its catalytic activity.
  • This protein consists of two identical polypeptide chains linked by a hinge to form a non-covalent homodimer, which exists in both open and closed conformations, and its conformation is altered by domain rotation.
  • extracellular adenosine triphosphate (ATP) is hydrolyzed by CD39 into adenosine monophosphate (AMP).
  • AMP adenosine monophosphate
  • CD73 catalyzes the synthesis of adenosine and phosphate.
  • ATP is a pro-inflammatory mediator.
  • ADA Adenosine deaminase
  • adenosine limits the pro-apoptotic effects of extracellular ATP and affects the immune activity of immune cells in the body. This contributes to tumor growth, metastasis, and survival, and induces immune escape from tumor cells. Therefore, CD39 and CD73 have become popular targets for cancer treatment. Research on their inhibitors is increasing.
  • CD39 inhibitors are less extensive than that on CD73 inhibitors as targets for tumor suppressor drugs. This may be because the expression characteristics of CD39 in non-tumor cells limit the effective anti-tumor targeting of CD39 inhibitors. Studies have also shown that CD39 activity is reversible in the presence of nucleoside diphosphate kinase (NDPK) and adenosine monophosphate kinase. In contrast, CD73 activity is almost irreversible. Therefore, CD73 has become a key target leading to immunosuppression, and its inhibition is considered a potential better treatment for cancer.
  • NDPK nucleoside diphosphate kinase
  • adenosine monophosphate kinase adenosine monophosphate kinase
  • This invention provides a compound of general formula (I), its tautomers, or pharmaceutical salts:
  • Another object of the present invention is to provide a pharmaceutical composition comprising the above-described compounds.
  • Another object of the present invention is to provide the use of the above-mentioned compounds in the preparation of medicaments for treating CD73-related diseases or inhibiting CD73.
  • the present invention provides a compound of general formula I or a pharmaceutically acceptable salt thereof:
  • A is an optionally substituted benzene ring, or a 5- or 6-membered heterocycle containing nitrogen, oxygen, and sulfur;
  • M is an optional substituted C8-C12 aryl, heteroaryl, or heterocyclic group
  • Z1 is selected from the group consisting of: O, S, CR1 , R2 , or NR3 , wherein R1 , R2 , and R3 are independently selected from: hydrogen, halogen, optionally substituted C1-C10 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C5-C6 aryl, optionally substituted C5-C6 heterocyclic group containing one or two heteroatoms independently selected from N, O, or S;
  • Z 2 is selected from the group consisting of: CR 4 or N, wherein R 4 is hydrogen, halogen, optionally substituted C1-C10 alkyl, optionally C3-C8 cycloalkyl, optionally substituted C5-C6 aryl, or optionally substituted C5-C6 heterocyclic group containing one or two heteroatoms independently selected from N, O or S.
  • R 4 is hydrogen, halogen, optionally substituted C1-C10 alkyl, optionally C3-C8 cycloalkyl, optionally substituted C5-C6 aryl, or optionally substituted C5-C6 heterocyclic group containing one or two heteroatoms independently selected from N, O or S.
  • X is selected from CR5 R6 , NR7 , O, S, carbonyl, sulfone or sulfoxide, wherein R5 , R6 and R7 are independently selected from: H, halogen, optionally substituted C1-C10 alkyl, optionally substituted C3-C8 cycloalkyl;
  • Y is selected from CR8, R9 , NR10 , O, S, carbonyl, sulfone, or sulfoxide, wherein R8 , R9 , and R10 are independently selected from: H, halogen, optionally substituted C1-C10 alkyl, optionally substituted C3-C8 cycloalkyl; CH2 , NH, O, S, carbonyl, sulfone, or sulfoxide;
  • Q1 and Q2 are each independently selected from the following group: hydrogen, optionally substituted C1-C10 alkyl (including but not limited to hydroxymethyl, hydroxyethyl, optionally substituted (e.g., deuterated) C1-C3 alkoxymethyl, optionally substituted C1-C3 alkoxyethyl), optionally substituted C3-C8 cycloalkyl, optionally substituted C5-C6 aryl, optionally substituted C5-C6 heterocyclic, optionally substituted phenyl C1-C5 alkyl, optionally substituted C5-C6 heterocyclic C1-C5 alkyl containing 1, 2, 3 or 4 heteroatoms independently selected from N, O or S, hydroxyl, optionally substituted C1-C3 formyl (including but not limited to hydroxyformyl, C1-C3 alkoxyformyl);
  • W1 and W2 are independently selected from: NH, O, or S;
  • P1 is H, optionally substituted C8-C12 aryl and optionally substituted C8-C12 aromatic heterocyclic groups, optionally substituted C1-C10 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C1-C10 alkylformyloxymethyl; or, W1P1 as a whole constitutes an amino acid linked by a phosphoramide bond, an esterified amino acid linked by a phosphoramide bond, or a phosphate prodrug group.
  • P2 is H, optionally substituted C8-C12 aryl and optionally substituted C8-C12 aromatic heterocyclic groups, optionally substituted C1-C10 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C1-C10 alkylformyloxymethyl;
  • W2P2 as a whole consists of amino acids linked by phosphoramide bonds, esterified amino acids linked by phosphoramide bonds, and phosphate prodrug groups.
  • P1 and P2 can form optionally substituted 5-8 membered cyclic ethers, lactones, or lactams.
  • M, Z1, Z2, X, Y, Q1, Q2, W1, W2, P1, and P2 are as described above.
  • M, Q1, Q2, W1, W2, P1, and P2 are as described above.
  • M is as follows:
  • B is an optionally substituted C5-C6 aromatic ring or an optionally substituted C5-C6 heterocycle containing N, O or S;
  • A1, A2, A3, A4, and A5 are each independently selected from CH or N;
  • R3 is selected from: H, halogen (F, Cl or Br), nitro, cyano, optionally substituted C1-C10 alkyl (e.g., trifluoromethyl, hydroxymethyl), optionally substituted C3-C8 cycloalkyl, optionally substituted C5-C6 heterocyclic C1-C5 alkyl (e.g., benzyl), optionally substituted C1-C5 alkoxy, optionally substituted C1-C5 alkoxyformyl, optionally substituted amino, optionally substituted C1-C5 alkylformylamino;
  • n is selected from 1 to 5; preferably an integer from 1 to 3.
  • B is an optional substituted C5-C6 aromatic ring or an optional substituted C5-C6 heterocycle containing N, O or S;
  • A1, A2, A3, A4, and A5 are each independently selected from CH or N;
  • R3 is selected from: H, halogen (F, Cl or Br), nitro, cyano, optionally substituted C1-C10 alkyl (e.g., trifluoromethyl, hydroxymethyl), optionally substituted C3-C8 cycloalkyl, optionally substituted C5-C6 heterocyclic C1-C5 alkyl (e.g., benzyl), optionally substituted C1-C5 alkoxy, optionally substituted C1-C5 alkoxyformyl, optionally substituted amino, optionally substituted C1-C5 alkylformylamino;
  • n is selected from 1 to 5, preferably an integer from 1 to 3;
  • Q1, Q2, W1, W2, P1, and P2 are as described above.
  • A1, R3, Q1, Q2, W1, W2, P1, P2 and n are as described above.
  • the present invention provides compounds selected from the group consisting of:
  • the compound is selected from the following compounds:
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the compound described in the first aspect or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition is a dosage form suitable for oral administration, including but not limited to tablets, solutions, suspensions, capsules, granules, and powders.
  • the present invention provides the use of the compounds described in the first aspect in the preparation of CD73 inhibitors.
  • the CD73 inhibitor is a drug for treating or preventing CD73-mediated diseases, or for inhibiting CD73.
  • the CD73-mediated disease is cancer.
  • the cancer is selected from the group consisting of: the cancer including but not limited to breast cancer, multiple myeloma, and bladder cancer as preferred, and the cancer selected from breast cancer, multiple myeloma, bladder cancer, endometrial cancer, gastric cancer, cervical cancer, rhabdomyosarcoma, non-small cell lung cancer, small cell lung cancer, pleomorphic lung cancer, ovarian cancer, esophageal cancer, melanoma, colorectal cancer, hepatocellular carcinoma, head and neck tumors, cholangiocarcinoma, myelodysplastic syndrome, malignant glioma, prostate cancer, thyroid cancer, Schwann cell carcinoma, squamous cell carcinoma of the lung, lichenoid keratosis, synovial sarcoma, skin cancer, adenocarcinoma, testicular cancer, or liposarcoma.
  • the cancer including but not limited to breast cancer, multiple myeloma, and bladder cancer as preferred
  • the present invention provides a method for treating or preventing CD73-mediated diseases, the method comprising administering a therapeutically or preventively effective amount of the compound of the first aspect or the pharmaceutical composition of the second aspect to a subject requiring treatment or prevention of a CD73-mediated disease.
  • the CD73-mediated disease is cancer.
  • the cancer includes, but is not limited to, breast cancer, multiple myeloma, and bladder cancer.
  • the cancer is selected from breast cancer, multiple myeloma, bladder cancer, endometrial cancer, gastric cancer, cervical cancer, rhabdomyosarcoma, non-small cell lung cancer, small cell lung cancer, pleomorphic lung cancer, ovarian cancer, esophageal cancer, melanoma, colorectal cancer, hepatocellular carcinoma, head and neck tumors, cholangiocarcinoma, myelodysplastic syndrome, malignant glioma, prostate cancer, thyroid cancer, Schwann cell carcinoma, squamous cell carcinoma of the lung, lichenoid keratosis, synovial sarcoma, skin cancer, adenocarcinoma, testicular cancer, or liposarcoma.
  • Figure 1 shows the workflow for in vivo pharmacodynamic evaluation
  • Figure 2 shows the cytotoxicity evaluation results of compounds 16, 31, and 33 on HEK293 and L02.
  • alkyl refers to a saturated branched, straight, or cycloalkyl group with a carbon chain length of 1-10 carbon atoms.
  • Preferred alkyl groups include those with lengths of 1-5, 1-2, 1-6, 1-4, or 3-8 carbon atoms.
  • Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and heptyl.
  • An alkyl group may be substituted with one or more substituents, such as halogens or haloalkyl groups.
  • an alkyl group may be substituted with 1-4 fluorine atoms, or it may be substituted with a fluoroalkyl group.
  • alkenyl generally refers to a monovalent hydrocarbon group having at least one double bond, typically containing 2-8 carbon atoms, preferably 2-6 carbon atoms, and can be straight-chain or branched.
  • alkenyl groups include, but are not limited to, vinyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, etc.
  • ester group generally refers to a carboxylic acid derivative having at least one ester group, typically containing 3-8 carbon atoms, preferably 3-6 carbon atoms, and can be straight-chain or branched.
  • ester groups include, but are not limited to, methyl formate, ethyl formate, methyl acetate, ethyl acetate, propyl acetate, etc.
  • hydroxyl group refers to a branched or straight-chain alcohol with a carbon chain length of 1-10 carbon atoms, typically containing 1-10 carbon atoms, preferably 1-6 carbon atoms, and can be straight-chain or branched.
  • ester hydroxyl groups include, but are not limited to, 1-hydroxy-n-butyl, 1-hydroxy-isobutyl, etc.
  • amide refers to a group with the structural formula "-R'-NH-C(O)-R", where R' can be selected from hydrogen or alkyl, and R can be selected from alkyl, alkenyl, alkynyl , alkyl substituted with NRcRd , alkenyl substituted with NRcRd , alkynyl substituted with NRcRd , alkyl substituted with halogen, and alkenyl substituted with cyano, where Rc and Rd can be selected from alkyl and alkenyl.
  • aryl refers to a monocyclic, bicyclic, or tricyclic aromatic group containing 6 to 14 carbon atoms, including phenyl, naphthyl, phenanthryl, anthracene, indyl, genyl, tetrahydronaphthyl, dihydroindyl, etc.
  • the aryl group may optionally be substituted by 1 to 5 (e.g., 1, 2, 3, 4, or 5) substituents selected from the following: halogen, C1-4 aldehyde, C1-6 alkyl, cyano, nitro, amino, amide, hydroxy, hydroxymethyl, halogen-substituted alkyl (e.g., trifluoromethyl), halogen-substituted alkoxy (e.g., trifluoromethoxy), carboxyl, C1-4 alkoxy, ethoxyformyl, N( CH3 ) and C1-4 acyl, heterocyclic, or heteroaryl, etc.
  • substituents selected from the following: halogen, C1-4 aldehyde, C1-6 alkyl, cyano, nitro, amino, amide, hydroxy, hydroxymethyl, halogen-substituted alkyl (e.g., trifluoromethyl), halogen-substituted alkoxy (e.g., tri
  • heterocyclic group includes, but is not limited to, 5- or 6-membered heterocyclic groups containing 1-3 heteroatoms selected from O, S or N, including but not limited to furanyl, thiophene, pyrrole, pyrrolidinyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, pyranyl, pyridinyl, pyrimidinyl, pyrazinyl, piperidinyl, morpholinyl, etc.
  • aromatic heterocyclic group refers to a group containing 5-14 ring atoms, with 6, 10, or 14 electrons shared in the ring system.
  • the ring atoms are carbon atoms and 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur.
  • Useful aromatic heterocyclic groups include piperazinyl, morpholinyl, piperidinyl, pyrrolyl, thiopheneyl, furanyl, pyranyl, pyrroleyl, imidazolyl, pyrazolyl, pyridinyl, including but not limited to pyrimidinyl.
  • the aromatic heterocyclic group may optionally be substituted by 1 to 5 (e.g., 1, 2, 3, 4 or 5) substituents selected from the following: halogen, C1-4 aldehyde, C1-6 straight or branched alkyl, cyano, nitro, amino, hydroxy, hydroxymethyl, halogen-substituted alkyl (e.g., trifluoromethyl), halogen-substituted alkoxy (e.g., trifluoromethoxy), carboxyl, C1-4 alkoxy, ethoxyformyl, N( CH3 ) and C1-4 acyl.
  • substituents selected from the following: halogen, C1-4 aldehyde, C1-6 straight or branched alkyl, cyano, nitro, amino, hydroxy, hydroxymethyl, halogen-substituted alkyl (e.g., trifluoromethyl), halogen-substituted alkoxy (e.g., trifluoromethoxy),
  • alkoxy group refers to an alkyl-substituted alkoxy group.
  • Preferred alkoxy groups are those with 1-6 carbon atoms, more preferably alkoxy groups with 1-3 carbon atoms. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, etc.
  • An alkoxy group may be substituted with one or more substituents, such as halogens or haloalkyl groups.
  • an alkoxy group may be an alkyl group substituted with 1-4 fluorine atoms, or the alkyl group may be an alkyl group substituted with a fluorinated alkyl group.
  • halogen refers to fluorine, chlorine, bromine, or iodine.
  • “optionally substituted” means that the modified substituent may be optionally substituted by 1 to 5 (e.g., 1, 2, 3, 4 or 5) substituents selected from the following: halogen, C1-4 aldehyde, C1-6 straight or branched alkyl, cyano, nitro, amino, hydroxy, hydroxymethyl, halogen-substituted alkyl (e.g., trifluoromethyl), halogen-substituted alkoxy (e.g., trifluoromethoxy), carboxyl, C1-4 alkoxy, ethoxyformyl, N( CH3 ) and C1-4 acyl.
  • 1 to 5 e.g., 1, 2, 3, 4 or 5
  • substituents selected from the following: halogen, C1-4 aldehyde, C1-6 straight or branched alkyl, cyano, nitro, amino, hydroxy, hydroxymethyl, halogen-substituted alkyl (e.g., trifluoromethyl), hal
  • amino acids linked by amide bonds refers to amino acids whose amino groups are linked to phosphate groups to form phosphoramide linkages, thereby attaching the amino acid to the main structure of the compound.
  • Esterified amino acids linked by amide bonds refers to esterified amino acids whose amino groups are linked to phosphoric acid to form phosphoramide bonds, thereby attaching the esterified amino acid to the main structure of the compound.
  • Phosphoprodrug group refers to a prodrug group introduced into a drug containing a phosphate group to improve polarity.
  • the present invention further provides compounds represented by general formulas II-V or pharmaceutically acceptable salts thereof:
  • the present invention provides compounds selected from the group consisting of:
  • the compound is selected from the following compounds:
  • the present invention provides a pharmaceutical composition containing a therapeutically effective amount of the compounds of the present invention or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier or excipient.
  • Examples of pharmaceutically acceptable salts of the compounds of the present invention include, but are not limited to, inorganic and organic acid salts, such as hydrochloride, hydrobromide, sulfate, citrate, lactate, tartrate, maleate, fumarate, mandelate, and oxalate; and inorganic and organic base salts formed with bases such as sodium hydroxyl, tris(hydroxymethyl)aminomethane (TRIS, aminobutanetriol), and N-methylglucosamine.
  • inorganic and organic acid salts such as hydrochloride, hydrobromide, sulfate, citrate, lactate, tartrate, maleate, fumarate, mandelate, and oxalate
  • inorganic and organic base salts formed with bases such as sodium hydroxyl, tris(hydroxymethyl)aminomethane (TRIS, aminobutanetriol), and N-methylglucosamine.
  • compositions of the present invention can be formulated into dosage forms suitable for various routes of administration, including but not limited to those formulated for parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, intrathecal, intracranial, nasal, or topical administration for the treatment of tumors and other diseases.
  • a dosage is an amount of medicine that effectively improves or eliminates one or more symptoms.
  • an effective amount is an amount sufficient to improve or, in some way, alleviate the symptoms associated with the disease.
  • Such a dosage may be administered as a single dose or may be administered according to an effective treatment regimen.
  • a dosage may cure the disease, but administration is usually intended to improve the symptoms of the disease. Repeated administration is generally required to achieve the desired symptom improvement.
  • the dosage of the medicine will be determined based on the patient's age, health and weight, the type of concurrent treatment, the frequency of treatment, and the desired therapeutic benefit.
  • the pharmaceutical formulations of this invention can be administered to any mammal, provided they can obtain the therapeutic effects of the compounds of this invention. Humans are the most important of these mammals.
  • the compounds or pharmaceutical compositions thereof of the present invention can be used to treat a variety of diseases mediated by CD73.
  • the cancers include, but are not limited to, breast cancer, multiple myeloma, and bladder cancer, preferably selected from breast cancer, multiple myeloma, bladder cancer, endometrial cancer, gastric cancer, cervical cancer, rhabdomyosarcoma, non-small cell lung cancer, small cell lung cancer, pleomorphic lung cancer, ovarian cancer, esophageal cancer, melanoma, colorectal cancer, hepatocellular carcinoma, head and neck tumors, cholangiocarcinoma, myelodysplastic syndrome, malignant glioma, prostate cancer, thyroid cancer, Schwann cell carcinoma, squamous cell carcinoma of the lung, lichen keratosis, synovial sarcoma, skin cancer, adenocarcinoma, testicular cancer, or liposarcoma.
  • the pharmaceutical formulations of the present invention can be manufactured using known methods. For example, they can be manufactured by conventional mixing, granulation, tableting, dissolving, or freeze-drying processes.
  • solid excipients and active compounds can be combined, and the mixture can be selectively ground. If desired or necessary, appropriate excipients can be added, and the granular mixture can be processed to obtain tablets or tablet cores.
  • Suitable excipients include sugars such as lactose or sucrose, mannitol or sorbitol; cellulose preparations or calcium phosphates, such as tricalcium phosphate or dicalcium phosphate; and binders, such as starch pastes, including corn starch, wheat starch, rice starch, potato starch, gelatin, astragalus gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone. If necessary, disintegrants, such as the starches mentioned above, as well as carboxymethyl starch, croscarmellose, agar, or alginate or its salts, such as sodium alginate, can be added.
  • sugars such as lactose or sucrose, mannitol or sorbitol
  • cellulose preparations or calcium phosphates such as tricalcium phosphate or dicalcium phosphate
  • binders such as starch pastes, including corn starch, wheat starch,
  • Adjuvants especially flow conditioners and lubricants, include silica, talc, stearates such as calcium magnesium stearate, stearic acid, or polyethylene glycol.
  • a suitable coating that resists gastric juices can be provided to the tablet core.
  • a concentrated sugar solution can be applied. This solution may contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, varnish solutions, and suitable organic solvents or solvent mixtures.
  • a suitable cellulose solution such as cellulose acetate phthalate or hydroxypropyl methylcellulose phthalate, can be used. Dyes or pigments can be added to the coating of the tablet or lozenge core, for example, to identify or characterize combinations of active ingredient dosages.
  • This invention also includes the use of the compounds of this invention in the preparation of medicaments for the prevention or treatment of CD73-mediated diseases or for inhibiting CD73 activity.
  • the compound provided by this invention is a compound with a completely new structure
  • the compound provided by this invention exhibits excellent inhibitory activity against CD73
  • the compounds provided by this invention lay the foundation for the development of drugs that can inhibit CD73, and have great prospects for industrialization and commercialization, as well as market value and significant economic benefits.
  • the reagents and raw materials used in this invention are all commercially available.
  • the synthesis method is referenced in RSC Adv 2018, 8(24), 13121-13128. Specifically: Take a 1L reaction flask, add 600mL of deionized water, add NaNO2 (21.83g, 316.4mmol), and sonicate to aid dissolution. After complete dissolution, add small amounts of 6-nitroindole (5.13g, 3.16mmol) in portions to suspend the product. Stir for 5min, then slowly add 6N hydrochloric acid (46.5mL, 284.7mmol) dropwise using a constant pressure dropping funnel for 30-40min. After the addition is complete, react at room temperature for 2h until the reaction is complete. Filter the reaction solution, wash the filter cake twice with 100mL of water, once with 50mL of EA, and once with 100mL of PE. Dry to obtain 1-1 4.8g. Yellow solid; no further purification is required. Product yield: 79.4%.
  • the reaction solution was evaporated to dryness to obtain a brownish-yellow oily substance. Upon addition of 60 mL of acetonitrile, a solid precipitated immediately. The product 1-2 (3.54 g) was obtained by filtration. The product, a brownish-yellow solid, required no further purification and the yield was 57.2%.
  • Step 4 ((6-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)methyl)phosphonate diethyl ester (1-4)
  • Step 5 ((6-amino-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)methyl)phosphonate diethyl ester (1-5)
  • Step 6 ((6-((5-chloro-3-fluoro-2-nitrophenyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)methyl)phosphonate diethyl ester (1-6)
  • Step 7 ((6-((5-chloro-3-fluoro-2-aminophenyl)amino)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)methyl)phosphonate diethyl ester (1-7)
  • Step 8 ((6-(6-chloro-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)methyl)phosphonate diethyl ester (1-8)
  • Step 9 ((6-(6-chloro-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1H-indazol-3-yl)methoxy)methyl)phosphonate diethyl ester (1-9)
  • Step 10 (((6-(6-chloro-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1H-indazol-3-yl)methoxy)methyl)phosphonic acid (1)
  • Step 3 ((6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)methyl)phosphonate diethyl ester (2-3)
  • Step 4 ((1-(tetrahydro-2H-pyran-2-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)-1H-indazol-3-yl)methoxy)methyl)phosphonate diethyl ester (2-4)
  • Step 5 ((6-(5-chloropyrazolo[1,5-a]pyrimidin-3-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)methyl)phosphonate diethyl ester (2-5)
  • the organic phase was separated, washed twice with water, once with saturated brine, and dried over anhydrous sodium sulfate.
  • the crude product was obtained by rotary evaporation, and the residue was loaded onto a column to give 80 mg of a yellow solid, with a yield of 19%.
  • Step 6 ((6-(5-chloropyrazolo[1,5-a]pyrimidin-3-yl)-1H-indazole-3-yl)methoxy)methyl)phosphonate diethyl ester (2-6)
  • Step 7 (((6-(5-chloropyrazolo[1,5-a]pyrimidin-3-yl)-1H-indazole-3-yl)methoxy)methyl)phosphonic acid (2)
  • Step 1 3-(((tert-butyldimethylsiloxy)methyl)-6-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (27-1)
  • Step 2 3-(((tert-butyldimethylsiloxy)methyl)-6-amino-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (27-2)
  • Step 3 N-(5-bromo-3-fluoro-2-nitrophenyl)-3-(((tert-butyldimethylsiloxy)methyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole-6-amine (27-3)
  • Step 4 5-Bromo-N- 1- (3-(((tert-butyldimethylsilyl)oxy)methyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)-3-fluorobenzene-1,2-diamine (27-4)
  • Step 5 6-Bromo-1-(3-(((tert-butyldimethylsilyl)oxy)methyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)-4-fluoro-1H-benzo[d][1,2,3]triazole (27-5)
  • Step 6 (6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methanol(27-6)
  • Step 7 2-((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)-2-(diethoxyphosphoryl)ethyl acetate (27-7)
  • Step 8 (1-((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)-2-hydroxyethyl)phosphonate diethyl ester (27-8)
  • Step 9 (1-((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1H-indazol-3-yl)methoxy)-2-hydroxyethyl)phosphonate diethyl ester (27-9)
  • the reaction was quenched with saturated sodium bicarbonate solution, extracted with EA, and the organic phase was collected. The mixture was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain the crude product.
  • the crude product was purified by silica gel column chromatography to obtain diethyl phosphonate (1-((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1H-indazol-3-yl)methoxy)-2-hydroxyethyl)phosphonate, a yellow oil (50 mg, 92.20 ⁇ mol), yield 79.12%).
  • Step 10 (1-((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1H-indazol-3-yl)methoxy)-2-hydroxyethyl)phosphonic acid (27)
  • Step 1 (1-((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)-2-methoxyethyl)phosphonate diethyl ester (28-1)
  • reaction was monitored by TLC until it was complete.
  • the reaction was quenched with sodium bicarbonate, extracted with ethyl acetate, and the organic phase was collected.
  • the organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation.
  • Step 1 Ethyl 2-((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)-2-(diethoxyphosphoryl)-3-methoxypropionate (29-1)
  • the solution was stirred at this temperature for 2 hours, and the reaction was monitored by TLC until complete.
  • the reaction was quenched by adding saturated ammonium chloride aqueous solution, extracted with ethyl acetate, and the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation.
  • Step 2 (2-((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)-1-ethoxy-3-methoxypropane-2-yl)phosphonate diethyl ester (29-2)
  • reaction was monitored by TLC to be complete.
  • the reaction was quenched with saturated ammonium chloride solution, extracted with ethyl acetate and water, and the organic phase was collected. The organic phase was dried over anhydrous sodium sulfate, concentrated, and mixed.
  • the crude product was purified by silica gel column chromatography to obtain a yellow oily substance (95 mg, 138.00 ⁇ mol) of diethyl phosphonate (2-((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)-1-ethoxy-3-methoxypropane-2-yl)phosphonate, with a yield of 75.12%).
  • Step 1 2-((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)-2-(diethoxyphosphoryl)propionate (30-1)
  • Step 1 Ethylhydro(((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1H-indazol-3-yl)methoxy)methyl)phosphonate (33-1)
  • Step 2 Ethyl phenyl(((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1H-indazol-3-yl)methoxy)methyl)phosphonate (33-2)
  • Step 3 ((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1H-indazol-3-yl)methoxy)methyl)phosphonic acid phenylhydrogen ester (33-3)
  • Step 4 ((((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1H-indazol-3-yl)methoxy)methyl)(phenoxy)phosphoryl)-D-alanine isopropyl ester (33)
  • Step 4 Ethyl 2-((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)-2-(diethoxyphosphoryl)-3-(2-((2-(trimethylsilyl)ethoxy)methyl)-2H-tetrazol-5-yl)propionate (34-5)
  • Step 5 (2-((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)-1-ethoxy-1-oxo-3-(2-((2-(trimethylsilyl)ethoxy)methyl)-2H-tetraazol-5-yl)prop-2-yl)phosphonate triethylammonium salt (34-6)
  • Step 6 (2-((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1H-indazol-3-yl)methoxy)-1-ethoxy-1-oxo-3-(2H-tetrazol-5-yl)prop-2-yl)phosphonate triethylammonium salt (compound 34)
  • the crude compound 34-6 obtained in the previous step was dissolved in methanol (1 mL), and 2 mL of hydrochloric acid methanol solution was added. The reaction was monitored by TLC until complete. The reaction solution was evaporated to dryness and purified by preparative liquid chromatography to obtain a pale yellow solid compound 34 (40 mg, 0.07 mmol), with a yield of 50%.
  • Step 1 Diethyl(2-((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)-1-hydroxy-3-(2-((2-(trimethylsilyl)ethoxy)methyl)-2H-tetrazol-5-yl)propyl-2-yl)phosphonate (35-1)
  • Step 1 Diethyl(2-((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)-1-(2-((2-(trimethylsilyl)ethoxy)methyl)-2H-tetrazol-5-yl)-3-((2-(2-(trimethylsilyl)ethoxy)methyl)-2H-tetrazol-5-yl)methoxy)propyl-2-yl)phosphonate (36-1)
  • Steps 2 and 3 are the same as in Synthesis Example 34.
  • Step 1 Ethyl 2-((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)-2-(diethoxyphosphoryl)-3-(2-(trimethylsilyl)ethoxy)propionate (37-1)
  • Steps 2, 3, 4, and 5 are the same as in Synthesis Example 36.
  • the synthesis method is the same as in Example 37.
  • Step 1 (6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methanethiol (42-1)
  • Step 2 (((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methyl)thio)methyl)phosphonate diethyl ester (42-2)
  • Step 3 (((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methyl)sulfonyl)methyl)phosphonate diethyl ester (42-3)
  • Step 4 (((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1H-indazol-3-yl)methyl)sulfonyl)methyl)phosphonic acid (compound 42)
  • Step 1 Ethyl 2-((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)-2-(diethoxyphosphoryl)-3-(4-fluorophenyl)propionate (44-1)
  • Step 2 (2-((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)-1-ethoxy-3-(4-fluorophenyl)-1-oxopropane-2-yl)phosphonic acid (44-2)
  • Step 3 (2-((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1H-indazol-3-yl)methoxy)-1-ethoxy-3-(4-fluorophenyl)-1-oxopropane-2-yl)phosphonate triethylammonium salt (compound 44)
  • Step 1 Diethyl(2-((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)-1-(methoxy-d3)-3-(2-((2-(trimethylsilyl)ethoxy)methyl)-2H-tetraazol-5-yl)prop-2-yl)phosphonate (46-1)
  • Step 1 ((((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2-methylpropionate) (48-2)
  • Step 2 ((((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1H-indazol-3-yl)methoxy)methyl)phosphoryl)bis(oxy))bis(methylene)bis(2-methylpropionate) (Compound 48)
  • Step 1 ((((6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)methoxy)methyl)(hydroxy)phosphoryl)oxy)methyl isobutyrate (49-1)
  • Step 2 is the same as step 2 in synthesis example 48.
  • Step 1 (6-(6-bromo-4-fluoro-1H-benzo[d][1,2,3]triazol-1-yl)-1H-indazol-3-yl)methanol (compound 50)
  • Test Example 1 The enzyme activity assay scheme for CD73 small molecule compounds at the molecular and cellular levels disclosed in this patent.
  • AMP is dephosphated under the catalysis of CD73 to produce adenosine.
  • the phosphate and molybdate ions then form a phosphomolybdic acid complex under acidic conditions, which in turn forms a green complex with malachite green.
  • Higher enzyme activity results in higher inorganic phosphate content and stronger absorbed signal intensity.
  • Adding an inhibitor weakens the enzyme activity, thus indirectly reflecting the inhibitory activity of the inhibitor.
  • the ratio of Regent A to Regent B is 100:1. After preparation, it is stable for more than 12 hours in the dark.
  • the components are as follows: 1mM CaCl2, 1mM MgCl2, 200mM NaCl, 10mM KCl, 100mM Tris-HCl, pH 8.0.
  • Plate preparation Add 70 ⁇ l of protein to each sample well and positive well, and 70 ⁇ l of buffer to each blank well.
  • Cell Plating When the cell density of MDA-MB-231 reaches 80%-90% and cell viability reaches 95% or higher, compound screening can be performed. Remove the cell culture dish from the CO2 cell culture incubator, discard the culture medium, add 2 mL of D-hank’s solution to wash away residual culture medium and dead cells, discard the D-hank’s solution, add 500 ⁇ L of 0.25% trypsin to the cell surface, and incubate at 37°C for one minute to digest the cells. After digestion, add 2 mL of fresh 1640 complete culture medium, and use a pipette to completely remove the cells from the cell culture dish. Transfer the cell suspension to a 15 mL sterile centrifuge tube and centrifuge at 1000 rpm for three minutes.
  • CD73 molecular and cellular enzyme activity assays show that the activity levels are represented by A, B, C, and D, where A: IC50 ⁇ 0.5 nM, B: 0.5 nM ⁇ IC50 ⁇ 1 nM, C: 1 nM ⁇ IC50 ⁇ 100 nM, and D: IC50 > 100 nM.
  • Test Example 2 In vivo pharmacodynamic evaluation
  • This invention evaluates the in vivo pharmacodynamic properties of compounds with CD73 enzyme activity of B or higher at the molecular or cellular level, and finds that these compounds all possess excellent in vivo pharmacodynamic properties.
  • Preparation of tumor cell suspension Thaw the purchased matrix gel at 4°C and pre-cool it with a sterilized 1mL pipette tip box. Collect the cells after centrifugation, discard the supernatant, and resuspend them in serum-free RPIM1640 medium. Centrifuge again and repeat this washing process three times to remove FBS from the complete medium. After discarding the supernatant in the last wash, resuspend the cells in an appropriate volume of serum-free RPIM1640 medium and mix with a pipette. Transfer 10 ⁇ L to a 1.5mL centrifuge tube, add 10 ⁇ L of trypan blue and mix.
  • Inject 10 ⁇ L into a cell counting chamber insert the counting chamber into the cell counter, and start counting after focusing. Adjust the cell density to twice the target density with medium according to the count density, then add an equal volume of matrix gel and mix by pipetting. Place the prepared cell suspension on ice for tumor inoculation.
  • Tumor inoculation A 1 mL sterile syringe was used for tumor inoculation. First, the cell suspension was transferred to the syringe, air bubbles were removed, and then 100 ⁇ L of cell suspension was subcutaneously injected into the right nape of the neck of C57BL/6 mice. Based on the previous experience of the research group in constructing this model, 5 ⁇ 104 E.G7-OVA mouse lymphoma cells were inoculated into each mouse. After tumor inoculation, the condition of the mice was observed daily. When the tumor reached about 100 mm3 , the drug was administered. Throughout the study, body weight and tumor volume were measured every other day.
  • TGI tumor growth inhibition rate
  • This invention evaluated the cytotoxicity of compounds with CD73 enzyme activity results of B or above at the molecular or cellular level, and found that these compounds all have good safety and drug-like properties.
  • the compounds of the present invention are found to be effective inhibitors of CD73, laying the foundation for the treatment of CD73-mediated cancers.

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Abstract

本发明涉及作为CD73抑制剂衍生物及其应用。具体而言，本发明涉及式I所示化合物、含有式I化合物的药物组合物及所述化合物在制备治疗CD73相关疾病或抑制CD73的药物中的用途。

Description

作为CD73抑制剂衍生物及其应用 技术领域
本发明涉及药物化学领域；具体地说，本发明新型化合物，其合成方法及其作为CD73抑制剂在制备肿瘤相关疾病的药物中的应用。
背景技术
腺苷通过激活肿瘤细胞、内皮细胞或免疫细胞表达的腺苷受体，以旁分泌和自分泌的方式调节免疫和炎症细胞的功能。如巨噬细胞、树突状细胞、髓系抑制细胞、T细胞和自然杀伤细胞(NK细胞)。ADO干扰细胞增殖、凋亡和血管生成。它使肿瘤微环境具有免疫耐受性，并对肿瘤的发展起到促进作用。
研究表明，体内抑制CD73可以显著抑制细胞外腺苷水平。胞外腺苷的产生主要是由于CD39和CD73的协同作用。CD73是一种膜相关酶，通过C末端的糖基化磷脂酰肌醇(GPI)固定在细胞表面。它普遍存在于淋巴细胞、内皮细胞和上皮细胞中，在结肠、肾脏和脑中高度表达。在正常组织中，依赖CD73表达的腺苷急性升高主要用于保护缺血和炎症组织，在离子转运、屏障功能维持、内皮细胞动态平衡和心肌保护中发挥生理作用。在不同的组织和细胞类型中，CD73可以通过磷脂酰肌醇特异性磷脂酶或蛋白水解酶从膜上脱落，产生保持催化活性的可溶性CD73。该蛋白质由两条相同的多肽链组成，通过铰链连接形成一个非共价的同源二聚体，该二聚体同时存在于开放和封闭的构象中，通过结构域的旋转来改变构象。首先，胞外的三磷酸腺苷(ATP)被CD39水解为一磷酸腺苷(AMP)。CD73催化合成腺苷和磷酸。三磷酸腺苷是一种促炎介质。细胞外ATP和腺苷的动态平衡是免疫动态平衡的重要组成部分。受损和垂死的细胞释放三磷酸腺苷，通过与P2X和P2Y嘌呤受体结合激活免疫细胞，产生促炎效应。腺苷脱氨酶(ADA)是一种负性调节因子，可导致腺苷的快速失活。因此，细胞外CD39-CD73-ADA系统维持的腺苷平衡是维持免疫平衡的关键。然而，当体内存在恶性肿瘤或细胞时，细胞外的ATP会被CD39和CD73大量水解成腺苷，超出ADA的水解范围，这导致腺苷水平持续增加。反过来，腺苷限制了细胞外ATP的促凋亡作用，并影响体内免疫细胞的免疫活性。这有助于肿瘤的生长、转移和存活，并诱导肿瘤细胞的免疫逃逸。因此，CD39和CD73成为肿瘤治疗的热门靶点。对它们的抑制剂的研究正在增加。
然而，对CD39抑制剂的研究比对CD73抑制剂作为肿瘤抑制药物靶点的研究要少。这可能是由于CD39在非肿瘤细胞中的表达特性限制了CD39抑制剂的有效抗肿瘤靶向性。也有研究表明，CD39的活性在核苷二磷酸激酶(NDPK)和腺苷酸激酶的存在下是可逆的。相比之下，CD73的活性几乎是不可逆转的。因此，CD73已成为导致免疫抑制的关键靶点，其抑制也被认为是一种更好的肿瘤治疗方法。
发明内容
本发明提供一种通式(Ⅰ)所示的化合物、其互变异构体或药用盐：
本发明的另一目的是提供包含上述化合物的药物组合物。
本发明还有一目的是提供上述化合物在制备治疗CD73相关疾病或抑制CD73的药物中的用途。
在第一方面，本发明提供通式I所示的化合物或其药学上可接受的盐：
其中，
A为任选取代的苯环、含氮、氧和硫的5或6元杂环；
M为任选取代的C8-C12芳基或杂芳基或杂环基；
Z1选自下组：O、S、CR1R2或NR3，其中R1、R2和R3独立选自：氢、卤素、任选取代的C1-C10烷基、任选取代的C3-C8环烷基、任选取代的C5-C6芳基、任选取代的含有1或2个独立选自N、O或S的杂原子的C5-C6杂环基；
Z2选自下组：CR4或N，其中R4为氢、卤素、任选取代的C1-C10烷基、任选C3-C8环烷基、任选取代的C5-C6芳基、任选取代的含有1或2个独立选自N、O或S的杂原子的C5-C6杂环基；
X选自CR5R6、NR7、O、S、羰基、砜基或亚砜基，其中R5、R6和R7独立选自：H、卤素、任选取代的C1-C10烷基、任选取代的C3-C8环烷基；
Y选自CR8R9、NR10、O、S、羰基、砜基或亚砜基，其中R8、R9和R10独立选自：H、卤素、任选取代的C1-C10烷基、任选取代的C3-C8环烷基；CH2、NH、O、S、羰基、砜基或亚砜基；
Q1和Q2各自独立选自下组：氢、任选取代的C1-C10烷基(包括但不限于羟甲基、羟乙基、任选取代的(例如氘代的)C1-C3烷氧基甲基、任选取代的C1-C3烷氧基乙基)、任选取代的C3-C8环烷基、任选取代的C5-C6芳基、任选取代的C5-C6杂环基、任选取代的苯基C1-C5烷基、任选取代的含有1、2、3或4个独立选自N、O或S的杂原子的C5-C6杂环基C1-C5烷基、羟基、任选取代的C1-C3甲酰基(包括但不限于羟基甲酰基、C1-C3烷氧基甲酰基)；
W1和W2独立选自：NH、O或S；
P1为H、任选取代的C8-C12芳基及任选取代的C8-C12芳杂环基、任选取代的C1-C10烷基、任选取代的C3-C8环烷基、任选取代的C1-C10烷基甲酰氧基甲基；或者，W1P1整体构成以磷酰胺键相连的氨基酸、以磷酰胺键相连的酯化氨基酸、磷酸前药基团；
P2为H、任选取代的C8-C12芳基及任选取代的C8-C12芳杂环基、任选取代的C1-C10烷基、任选取代的C3-C8环烷基、任选取代的C1-C10烷基甲酰氧基甲基；W2P2整体构成以磷酰胺键相连的氨基酸、以磷酰胺键相连的酯化氨基酸、磷酸前药基团；
或者P1、P2可以形成任选取代的5-8元环醚、内酯、内酰胺。
在具体的实施方式中，所述化合物如通式II所示：
其中，M、Z1、Z2、X、Y、Q1、Q2、W1、W2、P1、P2如上所述。
在具体的实施方式中，所述化合物如通式Ⅲ所示：
其中，M、Q1、Q2、W1、W2、P1、P2如上所述。
在优选的实施方式中，M如下所示：
其中，B为任选取代的C5-C6芳环、任选取代的含N、O或S的C5-C6杂环；
A1、A2、A3、A4、A5各自独立选自CH或N；
R3选自：H、卤素(F、Cl或Br)、硝基、氰基、任选取代的C1-C10烷基(例如三氟甲基、羟甲基)、任选取代的C3-C8环烷基、任选取代的C5-C6杂环基C1-C5烷基(例如苄基)、任选取代的C1-C5烷氧基、任选取代的C1-C5烷氧基甲酰基、任选取代的氨基、任选取代的C1-C5烷基甲酰氨基；
n选自1-5；优选1-3的整数。
在具体的实施方式中，所述化合物如通式Ⅳ所示：
其中，B为任选取代的C5-C6芳环、任选取代的含N、O或S的C5-C6杂环；
A1、A2、A3、A4、A5各自独立选自CH或N；
R3选自：H、卤素(F、Cl或Br)、硝基、氰基、任选取代的C1-C10烷基(例如三氟甲基、羟甲基)、任选取代的C3-C8环烷基、任选取代的C5-C6杂环基C1-C5烷基(例如苄基)、任选取代的C1-C5烷氧基、任选取代的C1-C5烷氧基甲酰基、任选取代的氨基、任选取代的C1-C5烷基甲酰氨基；
n选自1-5，优选1-3的整数；
Q1、Q2、W1、W2、P1、P2如上所述。
在具体的实施方式中，所述化合物如通式Ⅴ所示：
其中，A1、R3、Q1、Q2、W1、W2、P1、P2和n如上所述。
在具体的实施方式中，本发明提供选自下组的化合物或其药学上可接受的盐：


优选地，所述化合物选自以下化合物：
在第二方面，本发明提供一种药物组合物，所述药物组合物含有第一方面所述的化合物或其药学上可接受的盐，以及药学上可接受的载体或赋形剂。
在优选的实施方式中，所述药物组合物是适于口服的剂型，包括但不限于片剂、溶液剂、混悬液、胶囊剂、颗粒剂、粉剂。
在第三方面，本发明提供第一方面所述的化合物在制备CD73抑制剂中的用途。
在具体的实施方式中，所述CD73抑制剂是治疗或预防CD73介导的疾病，或抑制CD73的药物。
在具体的实施方式中，所述CD73介导的疾病为癌症。
在具体的实施方式中，所述癌症选自下组：所述癌症包括但不限于乳腺癌、多发性骨髓瘤、膀胱癌作为优选，所述癌症选自乳腺癌、多发性骨髓瘤、膀胱癌、子宫内膜癌、胃癌、宫颈癌、横纹肌肉瘤、非小细胞肺癌、小细胞肺癌、多形性肺癌、卵巢癌、食管癌、黑色素瘤、结肠直肠癌、肝细胞瘤、头颈部肿瘤、肝胆管细胞癌、骨髓增生异常综合征、恶性胶质瘤、前列腺癌、甲状腺癌、徐旺氏细胞瘤、肺鳞状细胞癌、苔薛样角化病、滑膜肉瘤、皮肤癌、腺癌、睾丸癌或脂肪肉瘤。
在第四方面，本发明提供治疗或预防CD73介导的疾病的方法，所述方法包括将治疗或预防有效量的第一方面所述的化合物或第二方面所述的药物组合物给予需要治疗或预防CD73介导的疾病的受试者。
在优选的实施方式中，所述CD73介导的疾病为癌症。
在优选的实施方式中，所述癌症包括但不限于：乳腺癌、多发性骨髓瘤、膀胱癌作为优选，所述癌症选自乳腺癌、多发性骨髓瘤、膀胱癌、子宫内膜癌、胃癌、宫颈癌、横纹肌肉瘤、非小细胞肺癌、小细胞肺癌、多形性肺癌、卵巢癌、食管癌、黑色素瘤、结肠直肠癌、肝细胞瘤、头颈部肿瘤、肝胆管细胞癌、骨髓增生异常综合征、恶性胶质瘤、前列腺癌、甲状腺癌、徐旺氏细胞瘤、肺鳞状细胞癌、苔薛样角化病、滑膜肉瘤、皮肤癌、腺癌、睾丸癌或脂肪肉瘤。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了体内药效学评价的流程；
图2显示了化合物16、31、33对HEK293与L02的细胞毒性评价结果。
具体实施方式
经过广泛而深入的研究，本发明人首次意外地发现一种结构全新的CD73抑制活性的化合物；所述化合物对CD73具有优异的抑制活性；从而为开发能抑制靶向CD73的药物奠定了新的物质基础，具备极大的产业化和商品化前景以及市场价值，经济效益显著。在此基础上完成了本发明。
术语定义
本文中涉及到的一些基团定义如下：
本文中，“烷基”指碳链长度为1-10个碳原子的饱和的支链或直链或环烷基，优选的烷基包括长1-5个、1-2个、1-6个、1-4个、3-8个碳原子不等的烷基。烷基的例子包括但不限于：甲基、乙基、正丙基、异丙基、正丁基、异丁基、庚基等。烷基可以被1个或多个取代基取代，例如被卤素或卤代烷基取代。例如，烷基可以是被1-4个氟原子取代的烷基，或者烷基可以是被氟代烷基取代的烷基。
本文中，“链烯基”通常表示具有至少一个双键的单价烃基，通常含有2-8个碳原子，优选含有2-6个碳原子，可以是直链或支链。链烯基的例子包括但不限于乙烯基、丙烯基、异丙烯基、丁烯基、异丁烯基、己烯基等等。
本文中，“酯基”通常表示具有至少一个酯基的羧酸衍生物，通常含有3-8个碳原子，优选含有3-6个碳原子，可以是直链或支链。酯基的例子包括但不限于甲酸甲酯、甲酸乙酯、乙酸甲酯、乙酸乙酯、乙酸丙酯等等。
本文中，“羟基”指碳链长度为1-10个碳原子的支链或直链醇，通常含有1-10个碳原子，优选含有1-6个碳原子，可以是直链或支链。酯羟基的例子包括但不限于1-羟基正丁基、1-羟基异丁基等等。
本文中，“酰氨基”指结构式为“-R’-NH-C(O)-R”的基团，其中，R’可选自氢或烷基，R可选自烷基、链烯基、炔基、被NRcRd取代的烷基、被NRcRd取代的链烯基和NRcRd取代的炔基、被卤素取代的烷基、被氰基取代的链烯基，其中，Rc和Rd可选自烷基和链烯基。
本文中，“芳基”指含有6到14个碳原子的单环、双环或三环芳族基团，包括苯基、萘基、菲基、蒽基、茚基、茀基、四氢化萘基、二氢化茚基等。芳基可任选地被1-5个(例如，1、2、3、4或5个)选自以下的取代基取代：卤素、C1-4醛基、C1-6烷基、氰基、硝基、氨基、酰胺基、羟基、羟甲基、卤素取代的烷基(例如三氟甲基)、卤素取代的烷氧基(例如三氟甲氧基)、羧基、C1-4烷氧基、乙氧甲酰基、N(CH3)和C1-4酰基等、杂环基或杂芳基等。
本文中，“杂环基”包括但不限于含有1-3个选自O、S或N的杂原子的5元或6元杂环基团，包括但不限于呋喃基、噻吩基、吡咯基、吡咯烷基、吡唑基、咪唑基、三唑基、噁唑基、吡喃基、吡啶基、嘧啶基、吡嗪基、哌啶基、吗啉基等。
本文中，“芳杂环基”是指含有5-14个环原子，并且有6个、10个或14个电子在环体系上共用。而且所含环原子是碳原子和从氧、氮、硫中任选的1-3个杂原子。有用的芳杂环基包括哌嗪基、吗啉基、哌啶基、吡咯烷基、噻吩基、呋喃基、吡喃基、吡咯基、咪唑基、吡唑基、吡啶基、包括但不限制于嘧啶基等。芳杂环基可任选地被1-5个(例如，1、2、3、4或5个)选自以下的取代基取代：卤素、C1-4醛基、C1-6直链或支链烷基、氰基、硝基、氨基、羟基、羟甲基、卤素取代的烷基(例如三氟甲基)、卤素取代的烷氧基(例如三氟甲氧基)、羧基、C1-4烷氧基、乙氧甲酰基、N(CH3)和C1-4酰基。
本文中，“烷氧基”指被烷基取代的氧基。优选的烷氧基是长1-6个碳原子的烷氧基，更优选为长1-3个碳原子的烷氧基。烷氧基的例子包括但不限于：甲氧基、乙氧基、丙氧基等。烷氧基可以被1个或多个取代基取代，例如被卤素或卤代烷基取代。例如，烷氧基可以是被1-4个氟原子取代的烷基，或者烷基可以是被氟代烷基取代的烷基。
本文中，“卤素”指氟、氯、溴或碘。
本文中，“任选取代的”指其所修饰的取代基可任选地被1-5个(例如，1、2、3、4或5个)选自以下的取代基取代：卤素、C1-4醛基、C1-6直链或支链烷基、氰基、硝基、氨基、羟基、羟甲基、卤素取代的烷基(例如三氟甲基)、卤素取代的烷氧基(例如三氟甲氧基)、羧基、C1-4烷氧基、乙氧甲酰基、N(CH3)和C1-4酰基。
在本文中，“以酰胺键相连的氨基酸”是指氨基酸的氨基与磷酸形成磷酰胺连接，从而将氨基酸连接在化合物的主体结构上。
“以酰胺键相连的酯化氨基酸”是指酯化的氨基酸的氨基与磷酸形成磷酰胺连接，从而将酯化氨基酸连接在化合物的主体结构上。
“磷酸前药基团”是指在含磷酸基团的药物中为改善极性而引入的前药基团。
本发明的化合物
在本文中，本发明提供通式I所示的化合物或其药学上可接受的盐：
其中的取代基的定义如上所述。
在优选的实施方式中，本发明进一步提供通式II-V所示的化合物或其药学上可接受的盐：
其中的取代基的定义如上所述。
在具体的实施方式中，本发明提供选自下组的化合物或其药学上可接受的盐：


优选地，所述化合物选自以下化合物：

在本发明的化合物的基础上，本发明提供一种药物组合物，该组合物含有治疗有效量的本发明的化合物或其药学上可接受的盐，以及药学上可接受的载体或赋形剂。
本发明化合物的药学上可接受的盐的例子包括但不限于无机和有机酸盐，例如盐酸盐、氢溴酸盐、硫酸盐、柠檬酸盐、乳酸盐、酒石酸盐、马来酸盐、富马酸盐、扁桃酸盐和草酸盐；以及与碱例如钠羟基、三(羟基甲基)胺基甲烷(TRIS，胺丁三醇)和N-甲基葡糖胺形成的无机和有机碱盐。
本发明的药物组合物可被配制成适合各种给药途径的制剂形式，包括但不限于被配制成用于肠外，皮下，静脉，肌肉，腹腔内，透皮，口腔，鞘内，颅内，鼻腔或外用途径给药的形式，用于治疗肿瘤和其他疾病。给药量是有效地改善或消除一个或多个病症的药量。对于特定疾病的治疗，有效量是足以改善或以某些方式减轻与疾病有关的症状的药量。这样的药量可作为单一剂量施用，或者可依据有效的治疗方案给药。给药量也许可治愈疾病，但是给药通常是为了改善疾病的症状。一般需要反复给药来实现所需的症状改善。药的剂量将根据病人的年龄，健康与体重，并行治疗的种类，治疗的频率，以及所需治疗效益来决定。
本发明的药物制剂可以给予任何哺乳动物，只要他们能获得本发明化合物的治疗效果。在这些哺乳动物中最为重要的是人类。
本发明的化合物或其药物组合物可用于治疗各种由CD73介导参与的疾病。所述癌症包括但不限于：乳腺癌、多发性骨髓瘤、膀胱癌作为优选，所述癌症选自乳腺癌、多发性骨髓瘤、膀胱癌、子宫内膜癌、胃癌、宫颈癌、横纹肌肉瘤、非小细胞肺癌、小细胞肺癌、多形性肺癌、卵巢癌、食管癌、黑色素瘤、结肠直肠癌、肝细胞瘤、头颈部肿瘤、肝胆管细胞癌、骨髓增生异常综合征、恶性胶质瘤、前列腺癌、甲状腺癌、徐旺氏细胞瘤、肺鳞状细胞癌、苔薛样角化病、滑膜肉瘤、皮肤癌、腺癌、睾丸癌或脂肪肉瘤。
本发明的药物制剂可用已知的方式制造。例如，由传统的混合，制粒，制锭，溶解，或冷冻干燥过程制造。制造口服制剂时，可结合固体辅料和活性化合物，选择性研磨混合物。如果需要或必要时加入适量助剂后，加工颗粒混合物，获得片剂或锭剂芯。
合适的辅料特别是填料，例如糖类如乳糖或蔗糖，甘露醇或山梨醇；纤维素制剂或钙磷酸盐，例如磷酸三钙或磷酸氢钙；以及粘结剂，例如淀粉糊，包括玉米淀粉，小麦淀粉，大米淀粉，马铃薯淀粉，明胶，黄芪胶，甲基纤维素，羟丙基甲基纤维素，羧甲基纤维素钠，或聚乙烯吡咯烷酮。如果需要，可增加崩解剂，比如上面提到的淀粉，以及羧甲基淀粉，交联聚乙烯吡咯烷酮，琼脂，或褐藻酸或其盐，如海藻酸钠。辅助剂特别是流动调节剂和润滑剂，例如，硅石，滑石，硬脂酸盐类，如镁硬脂酸钙，硬脂酸或聚乙二醇。如果需要，可以給锭剂核芯提供可以抵抗胃液的合适包衣。为此，可以应用浓缩糖类溶液。这个溶液可以含有阿拉伯树胶，滑石，聚乙烯吡咯烷酮，聚乙二醇和/或二氧化钛，漆溶液和合适的有机溶剂或溶剂混合物。为了制备耐胃液的包衣，可使用适当的纤维素溶液，例如醋酸纤维素邻苯二甲酸或羟丙基甲基纤维素邻苯二甲酸。可向药片或锭剂核芯的包衣加入染料或色素。例如，用于识别或为了表征活性成分剂量的组合。
本发明也包括本发明化合物在制备预防或治疗CD73介导的疾病或抑制CD73活性的药物中的用途。
本发明的优点：
1.本发明提供的化合物是一种结构全新的化合物；
2.本发明提供的化合物对CD73具有优异的抑制活性；
3.本发明提供的化合物为开发能抑制靶向CD73的药物奠定了基础，具备极大的产业化和商品化前景以及市场价值，经济效益显著。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
本发明所用试剂和原料均市售可得。
实施例1：CD73抑制剂合成
(((6-(6-溴-4-氯-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物1)
步骤1：6-硝基-1H-吲唑-3-甲醛(1-1)
合成方法参考文献RSC Adv 2018,8(24),13121-13128.具体如下：取1L的反应瓶，加入600mL去离子水，加入NaNO2(21.83g，316.4mmol)，超声下助溶。完全溶解后，分批少量加入6-硝基吲哚(5.13g，3.16mmol)使其混悬，搅拌5min后，使用恒压滴液漏斗缓慢滴加6N盐酸(46.5mL,284.7mmol)持续30-40min。滴加完毕后室温反应2h至反应完全。将反应液抽滤，滤饼用100mL水洗两次，50mL EA淋洗一次，100mL PE洗一次。干燥得1-1 4.8g。黄色固体，产物无需进一步纯化。产物收率79.4％。
1H NMR(400MHz,DMSO-d6)δ14.78(s,1H),10.25(s,1H),8.59(d,J＝1.9Hz,1H),8.32(dd,J＝8.9,0.7Hz,1H),8.16(dd,J＝8.9,2.0Hz,1H).LC-MS/ESI[M-H]-190.10.
步骤2：6-硝基-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-甲醛(1-2)
将化合物1-1(4.3g,22.5mmol)置于250mL反应瓶中，加入50mL二氯甲烷溶解。于0℃冰浴下滴加甲磺酸(0.146mL，2.25mmol)。再将3,4-2H-吡喃(5.14mL，56.2mmol)用20mL二氯甲烷稀释并滴入上述反应液中。撤去冰浴，室温反应8h后未反应完全，后补加3,4-2H-吡喃1.5mL，1.5h后反应完全。将反应液旋干得棕褐色油状物，加入60mL乙腈立即有固体析出。抽滤得产物1-2 3.54g。产物无需进一步纯化，褐黄色固体，产物收率57.2％。
1H NMR(400MHz,DMSO-d6)δ10.24(s,1H),8.93(d,J＝1.9Hz,1H),8.61–7.91(m,2H),6.34(dd,J＝9.3,2.5Hz,1H),3.90(dd,J＝6.7,4.2Hz,2H),2.47–2.33(m,1H),2.09(ddt,J＝13.6,8.4,3.9Hz,2H),1.87–1.72(m,1H),1.64(p,J＝4.4Hz,2H).
步骤3：(6-硝基-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲醇(1-3)
将化合物1-2(16.5g,59.9mmol)用200mL THF溶解，于0℃下分批加入NaBH4(3.4g89.9mmol).撤去冰浴，室温反应30min，反应完全后用200mL饱和氯化铵溶液猝灭反应，在无气泡生成后用100mL EA萃取3次，合并有机相。用水洗涤一次，饱和NaCl洗一次。使用无水Na2SO4干燥有机层，过滤并浓缩得1-3 14g，类白色固体。产物无需进一步纯化。产物收率84.2％。
1H NMR(400MHz,DMSO-d6)δ8.71(d,J＝2.0Hz,1H),8.10(d,J＝8.8Hz,1H),8.01(dd,J＝9.0,2.0Hz,1H),6.08(dd,J＝9.7,2.4Hz,1H),5.51(t,J＝5.8Hz,1H),4.84(d,J＝5.8Hz,2H),3.92–3.78(m,2H),2.39(tdd,J＝13.4,9.4,3.4Hz,1H),2.09–1.94(m,2H),1.77(tdd,J＝16.3,9.4,4.8Hz,1H),1.59(ddt,J＝9.2,6.8,3.9Hz,2H).LC-MS/ESI[M+H]+278.10.
步骤4：((6-硝基-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸二乙酯(1-4)
取NaH(3.98g,99.54mmol)于烘干的双口瓶中，加入无水DMF 20mL并降温至0℃，在N2氛围下注入(6-硝基-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲醇(23.00g，82.95mmol)的DMF溶液100mL，反应1h后，再注入对甲苯磺酰氧甲基膦酸二乙酯(26.74g，82.95mmol)的DMF溶液50mL。10min后撤去冰浴并室温反应过夜。用500mL EA稀释反应液，加入水500mL，分离有机相。有机相用水洗三次，饱和NaCl洗一次。使用无水Na2SO4干燥有机层。浓缩后残渣用柱色谱纯化得1-4 14.5g。产物为无色油状物，收率41％。
1H NMR(400MHz,DMSO-d6)δ8.76(d,J＝1.8Hz,1H),8.21–7.90(m,2H),6.12(dd,J＝9.6,2.4Hz,1H),4.98(s,2H),4.04(p,J＝7.3Hz,4H),3.94–3.73(m,4H),2.44–2.33(m,1H),2.07–1.98(m,2H),1.82–1.71(m,1H),1.61–1.57(m,2H),1.21(t,J＝7.0Hz,6H).
步骤5：((6-氨基-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸二乙酯(1-5)
取化合物1-4(12.00g，28.08mmol)、还原铁粉(7.84g，140.39mmol)和氯化铵(6.01g，112.31mmol)于反应瓶中，加入150mL乙醇和150mL去离子水，N2氛围下85℃反应4h。反应结束后，加入500mL EA稀释反应液，过硅藻土。有机相用水洗涤一次，饱和NaCl洗一次。使用无水Na2SO4干燥有机层。浓缩后残渣用柱色谱纯化得1-5 8.8g。褐色油，收率79％。
1H NMR(400MHz,DMSO-d6)δ7.43(d,J＝8.5Hz,1H),6.58(s,1H),6.54(dd,J＝8.6,1.5Hz,1H),5.52(dd,J＝9.7,2.5Hz,1H),5.40(s,2H),4.74(s,2H),4.04(p,J＝7.3Hz,4H),3.89–3.80(m,3H),3.68–3.61(m,1H),2.39–2.30(m,1H),2.10–1.83(m,2H),1.76–1.68(m,1H),1.58–1.53(m,2H),1.21(t,J＝7.0Hz,6H).
步骤6：((6-((5-氯-3-氟-2-硝基苯基)氨基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸二乙酯(1-6)
取化合物1-5(0.80g，2.01mmol)和5-氯-1，3-二氟-2-硝基苯(0.39g，2.01mmol)于反应瓶中，用10mL DMSO溶解，再加入10mL三乙胺，100℃反应24h。用100mL EA稀释反应液，加入水50mL，分离有机相。有机相用水洗三次，饱和NaCl洗一次。使用无水Na2SO4干燥有机层。浓缩后残渣用柱色谱纯化得1-6 0.5g。产物为红褐色固体，收率43％。
1H NMR(400MHz,DMSO-d6)δ9.14(s,1H),7.83(d,J＝8.6Hz,1H),7.56(d,J＝1.7Hz,1H),7.14(dd,J＝10.7,2.1Hz,1H),7.11(dd,J＝8.7,1.8Hz,1H),6.95(t,J＝1.9Hz,1H),5.77(dd,J＝9.7,2.4Hz,1H),4.88(s,2H),4.05(p,J＝7.1Hz,4H),3.90–3.85(m,3H),3.76–3.67(m,1H),2.40–2.31(m,1H),2.12–1.90(m,2H),1.77–1.66(m,1H),1.62–1.50(m,2H),1.21(t,J＝7.1Hz,6H).
步骤7：((6-((5-氯-3-氟-2-氨基苯基)氨基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸二乙酯(1-7)
取化合物1-6(0.45g，0.79mmol)、还原铁粉(0.22g，3.94mmol)和氯化铵(0.17g，3.15mmol)于反应瓶中，加入10mL乙醇和10mL去离子水，N2氛围下85℃反应4h。反应结束后，加入50mL EA稀释反应液，过硅藻土。有机相用水洗涤一次，饱和NaCl洗一次。使用无水Na2SO4干燥有机层。浓缩后产物无需进一步纯化直接进行下一步反应。
步骤8：((6-(6-氯-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸二乙酯(1-8)
取化合物1-7(280mg，0.52mmol)于反应瓶中，加入5mL乙腈溶解。于常温下滴加亚硝酸异戊酯(104μL，0.77mmol)。将反应瓶移置65℃油浴反应30min，冷却室温，浓缩后残渣用柱色谱纯化得1-8 220mg，白色固体，收率77％。
1H NMR(400MHz,DMSO-d6)δ8.29(dd,J＝1.8,0.7Hz,1H),8.15(dd,J＝8.6,0.7Hz,1H),7.93(d,J＝1.5Hz,1H),7.70(dd,J＝8.6,1.7Hz,1H),7.64(dd,J＝10.1,1.5Hz,1H),6.04(dd,J＝9.7,2.4Hz,1H),5.01(s,2H),4.06(dq,J＝8.2,7.1Hz,4H),3.95(d,J＝8.4Hz,2H),3.91–3.84(m,1H),3.80–3.74(m,1H),2.48–2.38(m,1H),2.20–1.95(m,2H),1.81–1.71(m,1H),1.60–1.55(m,2H),1.24(t,J＝7.1Hz,6H).
步骤9：((6-(6-氯-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸二乙酯(1-9)
取化合物1-8(200mg，0.36mmol)于反应瓶中，加入3mL二氯甲烷溶解。室温搅拌中滴加三氟乙酸1.5mL，继续搅拌2h。反应结束后用饱和碳酸钠溶液调至PH＝10，用50mL EA萃取，分离有机相。有机相用水洗两次，饱和食盐水洗一次，无水硫酸钠干燥。旋干，残渣上柱。得黄色固体1-9 105mg。收率62％。
1H NMR(400MHz,DMSO-d6)δ13.45(s,1H),8.13(dd,J＝8.6,0.7Hz,1H),8.05–8.01(m,1H),7.95(d,J＝1.5Hz,1H),7.64(d,J＝1.7Hz,1H),7.62(t,J＝1.6Hz,1H),5.01(s,2H),4.05(dq,J＝8.2,7.0Hz,4H),3.93(d,J＝8.4Hz,2H),1.23(t,J＝7.0Hz,6H).
步骤10：(((6-(6-氯-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(1)
取化合物1-9(75mg，0.16mmol)于反应瓶中，加入2mL 1,2-二氯乙烷溶解，N2保护下注入三甲基溴硅烷(317μL，2.40mmol)后，将反应升温至40℃反应过夜。反应结束后加入2mL甲醇，旋干反应液，残渣用甲醇重结晶得白色固体粉末35mg，收率53％。
1H NMR(400MHz,DMSO-d6)δ8.16(d,J＝8.6Hz,1H),8.01(d,J＝1.7Hz,1H),7.95(s,1H),7.78–7.55(m,2H),4.99(s,2H),3.67(d,J＝8.8Hz,2H).LC-MS/ESI[M-H]-410.00.
(((6-(5-氯吡唑并[1,5-a]嘧啶-3-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物2)
步骤1：6-溴-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-甲醛(2-1)
将化合物6-溴-1H-吲唑-3-甲醛(10.00g,44.44mmol)置于250mL反应瓶中，加入100mL二氯甲烷溶解，将3,4-2H-吡喃(10.14mL，111.09mmol)用20mL二氯甲烷稀释并滴入上述反应液中。再滴加甲磺酸(0.29mL，4.44mml)，反应迅速由浑浊转为黑色，10min后反应完全。旋干反应液，加入30mL EA，搅拌5min后抽滤，滤饼用20mL PE洗两次，干燥后得2-1 9g白色固体，收率65％。
1H NMR(400MHz,DMSO-d6)δ10.17(s,1H),8.26(d,J＝1.6Hz,1H),8.08(d,J＝8.6Hz,1H),7.57(dd,J＝8.6,1.6Hz,1H),6.09(dd,J＝9.5,2.2Hz,1H),4.03–3.72(m,2H),2.43–2.33(m,1H),2.16–1.99(m,2H),1.81–1.70(m,1H),1.64–1.58(m,2H).
步骤2：(6-溴-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲醇(2-2)
将化合物2-1(6.20g，20.05mmol)用100mL THF溶解，于0℃下分批加入NaBH4(1.14g，30.08mmol).撤去冰浴，室温反应30min，反应完全后用200mL饱和氯化铵溶液猝灭反应，在无气泡生成后用100mL EA萃取3次，合并有机相。用水洗涤一次，饱和NaCl洗一次。使用无水Na2SO4干燥有机层，过滤并浓缩得SCJ-17003 5.7g，白色固体。产物无需进一步纯化。产物收率91％。
1H NMR(400MHz,DMSO-d6)δ8.00(d,J＝1.4Hz,1H),7.81(dd,J＝8.6,0.6Hz,1H),7.31(dd,J＝8.5,1.6Hz,1H),5.82(dd,J＝9.9,2.5Hz,1H),5.37(t,J＝5.9Hz,1H),4.76(d,J＝5.9Hz,2H),3.88–3.84(m,1H),3.78–3.72(m,1H),2.40–2.30(m,1H),2.09–1.87(m,2H),1.77–1.66(m,1H),1.59–1.53(m,2H).
步骤3：((6-溴-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸二乙酯(2-3)
取NaH(0.80g,20.15mmol)于烘干的双口瓶中，加入无水DMF 10mL并降温至0℃，在N2氛围下注入2-2(5.70g，18.32mmol)的DMF溶液20mL，反应1h后，再注入对甲苯磺酰氧甲基膦酸二乙酯(6.49g，20.15mmol)的DMF溶液10mL。10min后撤去冰浴并室温反应过夜。用200mL EA稀释反应液，加入水200mL，分离有机相。有机相用水洗三次，饱和NaCl洗一次。使用无水Na2SO4干燥有机层。浓缩后残渣用柱色谱纯化得2-3 2.0g。产物为无色油状物，收率23.7％。
NMR(400MHz,DMSO-d6)δ8.05(d,J＝1.3Hz,1H),7.80(d,J＝8.4Hz,1H),7.36(dd,J＝8.5,1.6Hz,1H),5.87(dd,J＝9.8,2.5Hz,1H),4.90(s,2H),4.02(dq,J＝8.2,7.1Hz,4H),3.91–3.83(m,3H),3.79–3.72(m,1H),2.43–2.25(m,1H),2.11–1.85(m,2H),1.81–1.48(m,3H),1.21(t,J＝7.1Hz,6H).
步骤4：((1-(四氢-2H-吡喃-2-基)-6-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸二乙酯(2-4)
取化合物2-3(2.00g，4.34mmol)于反应瓶中，加联硼酸频哪醇酯(1.65g，6.50mmol)，Pd(dppf)Cl2(0.32g，0.43mmol)和醋酸钾(0.85g，8.67mmol)，Ar换气三次后加入1,4-二氧六环后回流反应2h，TLC监测反应完全。将反应冷却至室温后加入100mL水，用150mL EA萃取，分离有机相，有机相用水洗两次，饱和食盐水洗一次，无水硫酸钠干燥。旋干得粗品，残渣上柱，得黄色固体1.0g，收率45％。
1H NMR(400MHz,DMSO-d6)δ8.01(s,1H),7.85(dd,J＝8.1,0.9Hz,1H),7.48(d,J＝8.1Hz,1H),5.98(dd,J＝9.6,2.5Hz,1H),4.91(s,2H),4.02(dq,J＝8.2,7.0Hz,5H),3.88(d,J＝8.4Hz,2H),3.86–3.75(m,2H),2.46–2.32(m,1H),2.09–1.90(m,2H),1.83–1.73(m,1H),1.63–1.47(m,2H),1.33(s,12H),1.25–1.17(m,6H).
步骤5：((6-(5-氯吡唑并[1,5-a]嘧啶-3-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸二乙酯(2-5)
取化合物2-4(400mg，0.79mmol)于反应瓶中，加5-氯-3-碘吡唑并[1,5-a]嘧啶(242mg，0.87mmol)、Pd(dppf)Cl2(57mg，0.079mmol)和磷酸钾(334mg，1.57mmol)，氮气换气三次后加入1,4-二氧六环10ml及水2mL后回流反应过夜，TLC监测反应完全。将反应冷却至室温后加入80mL水，用50mL EA萃取，分离有机相，有机相用水洗两次，饱和食盐水洗一次，无水硫酸钠干燥。旋干得粗品，残渣上柱得黄色固体80mg，收率19％。
1H NMR(400MHz,DMSO-d6)δ9.24(d,J＝7.3Hz,1H),8.96(s,1H),8.44(t,J＝1.1Hz,1H),7.98(dd,J＝8.4,1.3Hz,1H),7.90(dd,J＝8.5,0.8Hz,1H),7.23(d,J＝7.3Hz,1H),5.86(dd,J＝9.9,2.4Hz,1H),4.91(s,2H),4.04(dq,J＝8.3,7.1Hz,4H),3.97(d,J＝11.6Hz,1H),3.91(d,J＝8.4Hz,2H),3.86–3.72(m,1H),2.48–2.35(m,1H),2.11–1.98(m,2H),1.85–1.72(m,1H),1.68–1.54(m,2H),1.22(t,J＝7.0Hz,6H).
步骤6：((6-(5-氯吡唑并[1,5-a]嘧啶-3-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸二乙酯(2-6)
取化合物2-5(75mg，0.14mmol)于反应瓶中，加入4mL二氯甲烷溶解。室温搅拌中滴加三氟乙酸2mL，继续搅拌3h。反应结束后用饱和碳酸钠溶液调至PH＝10，用50mL EA萃取，分离有机相。有机相用水洗两次，饱和食盐水洗一次，无水硫酸钠干燥。旋干，残渣上柱。得黄色固体55mg。收率87％。
1H NMR(400MHz,DMSO-d6)δ13.12(s,1H),9.25(d,J＝7.2Hz,1H),8.94(s,1H),8.30(t,J＝1.1Hz,1H),7.95–7.65(m,2H),7.23(d,J＝7.2Hz,1H),4.92(s,2H),4.03(dq,J＝8.1,7.1Hz,4H),3.89(d,J＝8.4Hz,2H),1.22(t,J＝7.0Hz,6H).
步骤7：(((6-(5-氯吡唑并[1,5-a]嘧啶-3-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(2)
取化合物2-6(50mg，0.11mmol)于反应瓶中，加入2mL 1,2-二氯乙烷溶解，N2保护下注入三甲基溴硅烷(0.22mL，1.67mmol)后，将反应升温至40℃反应过夜。反应结束后加入2mL甲醇，旋干反应液，残渣用甲醇重结晶得淡黄色固体粉末21mg，收率48％。
1H NMR(400MHz,DMSO-d6)δ13.05(s,1H),9.24(d,J＝7.2Hz,1H),8.93(s,1H),8.28(s,1H),7.92(d,J＝8.4Hz,1H),7.80(dd,J＝8.5,1.4Hz,1H),7.23(d,J＝7.3Hz,1H),4.91(s,2H),3.63(d,J＝8.8Hz,2H).LC-MS/ESI[M-H]-392.05.
(((6-(6-氯咪唑并[1,2-b]哒嗪-3-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物3)
合成方法同实例2。淡黄色固体粉末44mg，收率81％。1H NMR(600MHz,DMSO-d6)δ13.25(s,1H),8.50(s,1H),8.46(d,J＝1.4Hz,1H),8.37(d,J＝9.5Hz,1H),8.00(d,J＝8.5Hz,1H),7.77(dd,J＝8.5,1.5Hz,1H),7.52(dd,J＝9.4,1.5Hz,1H),4.94(s,2H),3.65(d,J＝8.8Hz,2H).LC-MS/ESI[M-H]-392.05.
(((6-(6-氯咪唑并[1,2-a]嘧啶-3-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物4)
合成方法同实例2。淡黄色固体粉末10mg，收率38％。1H NMR(600MHz,DMSO-d6)δ13.20(s,1H),9.25(d,J＝2.5Hz,1H),8.63(d,J＝2.5Hz,1H),8.10(s,1H),8.07–8.00(m,1H),7.85(t,J＝1.1Hz,1H),7.45(dd,J＝8.3,1.4Hz,1H),4.95(s,2H),3.63(d,J＝8.9Hz,2H).LC-MS/ESI[M-H]-392.05.
(((6-(5-氯噻吩并[3,2-b]吡啶-3-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物5)
合成方法同实例2。淡黄色固体粉末36mg，收率82％。1H NMR(400MHz,DMSO-d6)δ13.17(s,1H),8.67(d,J＝8.6Hz,1H),8.60(s,1H),8.36(t,J＝1.1Hz,1H),7.96(d,J＝8.4Hz,1H),7.73–7.65(m,1H),7.58(d,J＝8.5Hz,1H),4.94(s,2H),3.64(d,J＝8.9Hz,2H).LC-MS/ESI[M-H]-408.00.
(((6-(5-氯呋喃并[3,2-b]吡啶-3-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物6)
合成方法同实例2。淡黄色固体粉末36mg，收率36％。1H NMR(400MHz,DMSO-d6)δ13.19(s,1H),9.07(s,1H),8.55(s,1H),8.25(d,J＝8.7Hz,1H),7.96(d,J＝8.4Hz,1H),7.76(dd,J＝8.5,1.3Hz,1H),7.56(d,J＝8.6Hz,1H),4.93(s,2H),3.64(d,J＝8.9Hz,2H).LC-MS/ESI[M-H]-392.00.
(((6-溴-1H,1'H-[3,6'-联吲唑]-3'-基)甲氧基)甲基)膦酸(化合物7)
合成方法同实例2。产物为白色固体。1H NMR(400MHz,DMSO-d6)δ13.41(s,1H),13.07(s,1H),8.06(d,J＝8.7Hz,1H),8.03(d,J＝1.3Hz,1H),7.98(d,J＝8.4Hz,1H),7.85(d,J＝1.6Hz,1H),7.80–7.74(m,1H),7.38(dd,J＝8.6,1.7Hz,1H),4.94(s,2H),3.64(d,J＝8.9Hz,2H).LC-MS/ESI[M-H]-435.00.
(((6-(5-氨基-1H-吡唑并[4,3-b]吡啶-3-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物8)
合成方法同实例2。产物为淡褐色固体。1H NMR(600MHz,Methanol-d4)δ8.24(dd,J＝9.4,2.4Hz,1H),8.06(dd,J＝8.5,3.0Hz,1H),7.94(s,1H),7.58(dd,J＝8.3,1.4Hz,1H),7.03(d,J＝9.5Hz,1H),5.06(s,2H),3.85(d,J＝9.3Hz,2H).LC-MS/ESI[M-H]-373.00.
(((6-(5-乙酰基-4,5,6,7-四氢噻吩并[3,2-c]吡啶-2-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物9)
合成方法同实例2。产物为淡褐色固体。1H NMR(400MHz,DMSO-d6)δ13.01(s,1H),7.86(dd,J＝8.5,2.8Hz,1H),7.63(s,1H),7.38(dd,J＝9.9,6.5Hz,2H),4.88(s,2H),4.56(d,J＝13.8Hz,2H),3.79(t,J＝5.7Hz,1H),3.75(t,J＝5.7Hz,1H),3.61(d,J＝8.8Hz,2H),2.90(t,J＝5.7Hz,1H),2.77(t,J＝5.7Hz,1H),2.12(s,3H).LC-MS/ESI[M-H]-420.10.
(((6-(2-氧代-1,2-二氢吡啶-3-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物10)
合成方法同实例2。产物为白色固体。1H NMR(400MHz,DMSO-d6)δ13.02(s,1H),8.07(s,1H),7.85(d,J＝8.5Hz,1H),7.79(dd,J＝7.0,2.0Hz,1H),7.54–7.31(m,2H),6.35(t,J＝6.7Hz,1H),4.92(s,2H),3.63(d,J＝8.9Hz,2H).LC-MS/ESI[M-H]-334.00.
(((6-(1-甲基-6-氧代-1,6-二氢哒嗪-3-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物11)
合成方法同实例2。产物为白色固体。1H NMR(400MHz,DMSO-d6)δ13.23(s,1H),8.18(d,J＝9.7Hz,1H),8.04–7.90(m,2H),7.71(dd,J＝8.6,1.5Hz,1H),7.09(d,J＝9.7Hz,1H),4.94(s,2H),3.79(s,3H),3.65(d,J＝8.9Hz,2H).LC-MS/ESI[M-H]-349.05.
(((6-(6-甲基哒嗪-3-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物12)
合成方法同实例2。产物为白色固体。1H NMR(400MHz,DMSO-d6)δ13.31(s,1H),8.50(d,J＝8.8Hz,1H),8.30(s,1H),8.05(d,J＝8.5Hz,1H),7.97–7.88(m,2H),4.97(s,2H),3.66(d,J＝8.9Hz,2H),2.75(s,3H).LC-MS/ESI[M-H]-333.05.
(((6-(6-溴哒嗪-3-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物13)
合成方法同实例2。产物为白色固体。1H NMR(400MHz,DMSO-d6)δ13.32(s,1H),8.34(d,J＝9.0Hz,1H),8.29(s,1H),8.13(d,J＝9.0Hz,1H),8.03(d,J＝8.5Hz,1H),7.96–7.87(m,1H),4.95(s,2H),3.64(d,J＝8.8Hz,2H).LC-MS/ESI[M-H]-396.95.
(((6-([1,2,4]三唑并[4,3-a]吡啶-5-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物14)
合成方法同实例2。甲醇重结晶，产物为白色固体。1H NMR(400MHz,DMSO-d6)δ13.26(s,1H),9.35(s,1H),9.07(s,1H),8.02(d,J＝8.1Hz,1H),7.96(s,2H),7.85(s,1H),7.51(d,J＝8.1Hz,1H),4.95(s,2H),3.66(s,2H).LC-MS/ESI[M-H]-358.05.
(((6-(3-(三氟甲基)-[1,2,4]三唑并[4,3-a]吡啶-5-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物15)
合成方法同实例2。产物为白色固体。1H NMR(400MHz,DMSO-d6)δ13.22(s,1H),8.76(s,1H),8.17(d,J＝9.6Hz,1H),8.08(dd,J＝9.5,1.6Hz,1H),8.02(d,J＝8.4Hz,1H),7.90(d,J＝1.5Hz,1H),7.54(dd,J＝8.4,1.5Hz,1H),4.95(s,2H),3.64(d,J＝8.9Hz,2H).LC-MS/ESI[M-H]-426.05.
(((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物16)
合成方法同实例1。白色固体(90mg,197.3μmol)，产率89.44％。1H NMR(600MHz,DMSO-d6)δ13.38(s,1H),8.16(d,J＝8.6Hz,1H),8.07(d,J＝1.4Hz,1H),8.01(d,J＝1.7Hz,1H),7.71(dd,J＝9.8,1.4Hz,1H),7.60(dd,J＝8.6,1.8Hz,1H),6.37(s,2H),4.99(s,2H),3.67(d,J＝8.8Hz,2H).LC-MS/ESI[M-H]-454.00.
(((6-(6-溴-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物17)
合成方法同实例1。白色固体。LC-MS/ESI[M-H]-436.00.
(((6-(6-氰基-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物18)
合成方法同实例1。白色固体。1H NMR(400MHz,DMSO-d6)δ8.58(d,J＝1.1Hz,1H),8.18(d,J＝8.4Hz,1H),8.06(dd,J＝3.9,1.7Hz,1H),7.97(dd,J＝10.3,1.1Hz,1H),7.64(dd,J＝8.6,1.8Hz,1H),5.00(s,2H),3.68(d,J＝8.9Hz,2H).LC-MS/ESI[M-H]-401.05.
(((6-(6-(三氟甲基)-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物19)
合成方法同实例1。白色固体。1H NMR(400MHz,DMSO-d6)δ13.41(s,1H),8.48(d,J＝8.7Hz,1H),8.38–8.29(m,1H),8.18(d,J＝8.6Hz,1H),8.06(d,J＝1.7Hz,1H),7.86(dd,J＝8.8,1.6Hz,1H),7.66(dd,J＝8.6,1.8Hz,1H),5.00(s,2H),3.68(d,J＝8.8Hz,2H).LC-MS/ESI[M-H]-426.05.
(((6-(6-溴-5-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物20)
合成方法同实例1。白色固体。1H NMR(400MHz,DMSO-d6)δ8.42(d,J＝5.8Hz,1H),8.32(d,J＝8.5Hz,1H),8.16(d,J＝8.6Hz,1H),8.02(d,J＝1.8Hz,1H),7.62(dd,J＝8.6,1.8Hz,1H),4.99(s,2H),3.68(s,2H).LC-MS/ESI[M-H]-454.00.
(((6-(6-溴-7-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物21)
合成方法同实例1。白色固体。1H NMR(400MHz,DMSO-d6)δ8.11(d,J＝8.6Hz,1H),8.07–7.97(m,2H),7.77(dd,J＝8.9,5.9Hz,1H),7.56(dt,J＝8.6,2.1Hz,1H),4.99(s,2H),3.68(d,J＝8.8Hz,2H).LC-MS/ESI[M-H]-454.00.
(((6-(6-溴-5-氯-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物22)
合成方法同实例1。白色固体。1H NMR(400MHz,DMSO-d6)δ13.40(s,1H),8.63(s,1H),8.46(s,1H),8.16(d,J＝8.6Hz,1H),8.02(d,J＝1.8Hz,1H),7.62(dd,J＝8.6,1.8Hz,1H),4.99(s,2H),3.67(d,J＝8.8Hz,2H).LC-MS/ESI[M-H]-470.00.
(((6-(5,6-二氯-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物23)
合成方法同实例1。白色固体。1H NMR(600MHz,DMSO-d6)δ13.38(s,1H),8.65(s,1H),8.35(s,1H),8.16(d,J＝8.5Hz,1H),8.01(d,J＝1.8Hz,1H),7.62(dd,J＝8.5,1.8Hz,1H),4.99(s,2H),3.67(d,J＝8.8Hz,2H).13C NMR(151MHz,DMSO-d6)δ145.14,142.66,141.03,134.14,132.37,132.03,128.17,122.75,122.29,121.34,116.61,113.43,105.79,67.07(d,JC-O-C-P＝12.5Hz),66.09(d,JC-P＝161.2Hz).LC-MS/ESI[M-H]-426.00.
(((6-(5-溴-3H-[1,2,3]三唑并[4,5-b]吡啶-3-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物24)
合成方法同实例1。白色固体。1H NMR(600MHz,DMSO-d6)δ13.42(s,1H),8.70(d,J＝8.6Hz,1H),8.35(d,J＝1.8Hz,1H),8.16(d,J＝8.6Hz,1H),7.89(dd,J＝8.7,1.8Hz,1H),7.84(d,J＝8.6Hz,1H),4.99(s,2H),3.67(d,J＝8.8Hz,2H).LC-MS/ESI[M-H]-437.00.
(((6-(6-溴-1H-[1,2,3]三唑并[4,5-b]吡啶-1-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物25)
合成方法同实例1。白色固体。1H NMR(600MHz,DMSO-d6)δ12.88(s,1H),8.24(s,1H),7.84–7.79(m,2H),7.59(d,J＝2.0Hz,1H),7.17(d,J＝1.8Hz,1H),6.98(dd,J＝8.6,1.9Hz,1H),4.86(s,2H),3.62(d,J＝8.8Hz,2H).13C NMR(151MHz,DMSO-d6)δ147.96,142.22,139.67,131.24,126.99,125.64,122.05,118.63,115.34,105.26,99.32,67.21(d,JC-O-C-P＝12.6Hz),66.10(d,JC-P＝161.5Hz).LC-MS/ESI[M-H]-437.00.
(((6-(6-氯-1H-[1,2,3]三唑并[4,5-c]吡啶-1-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(化合物26)
合成方法同实例1。白色固体。1H NMR(400MHz,DMSO-d6)δ13.40(s,1H),9.49(d,J＝1.1Hz,1H),8.19(d,J＝1.1Hz,1H),8.17(d,J＝8.5Hz,1H),8.04(d,J＝1.8Hz,1H),7.64(dd,J＝8.6,1.9Hz,1H),4.99(s,2H),3.67(d,J＝8.8Hz,2H).LC-MS/ESI[M-H]-393.00.
(1-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-2-羟乙基)膦酸(化合物27)
步骤1：3-(((叔丁基二甲基硅氧基)甲基)-6-硝基-1-(四氢-2H-吡喃-2-基)-1H-吲唑(27-1)
取6-硝基-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-甲醇25g，90.16mmol于双口瓶中，加入无水DMF溶解，加入(30.69g，450.81mmol)咪唑，N2保护，置换气多次，置于冰浴中，待温度降至0℃后加入(33.97g，225.40mmol)叔丁基二甲基氯硅烷，转移至室温反应，约半小时后TLC监测反应完全。旋转蒸发除去DMF，乙酸乙酯和水萃取，浓缩有机相，加入少量乙酸乙酯和二氯甲烷打浆，析出3-(((叔丁基二甲基硅烷基)氧基)甲基)-6-硝基-1-(四氢-2H-吡喃-2-基)-1H-吲唑白色块状固体(29.14g，79.84mmol),产率88.55％。
1H NMR(400MHz,DMSO-d6)δ8.74–8.70(m,1H),8.05–7.98(m,2H),6.09(dd,J＝9.5,2.4Hz,1H),5.05(s,2H),3.84(tdd,J＝15.6,11.5,4.7Hz,2H),2.43–2.29(m,1H),2.01(tq,J＝16.5,3.8Hz,2H),1.82–1.70(m,1H),1.58(ddt,J＝12.2,8.7,3.8Hz,2H),0.86(s,9H),0.09(s,6H).
步骤2：3-(((叔丁基二甲基硅氧基)甲基)-6-氨基-1-(四氢-2H-吡喃-2-基)-1H-吲唑(27-2)
取3-(((叔丁基二甲基硅烷基)氧基)甲基)-6-硝基-1-(四氢-2H-吡喃-2-基)-1H-吲唑(29g,74.42mmol)于反应瓶中，加入3g 10％Pd/C，用甲醇及二氯甲烷溶解，H2置换多次，室温反应。约30分钟后，TLC监测反应完全。硅藻土抽滤后，收集滤液，旋转蒸发除去溶剂，得到3-(((叔丁基二甲基硅烷基)氧基)甲基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-6-胺红褐色油状液体(24.85g，68.73mmol)，无需纯化直接投下一步，产率92.35％。
1H NMR(400MHz,DMSO-d6)δ7.40(d,J＝8.6Hz,1H),6.59(d,J＝1.8Hz,1H),6.54(dd,J＝8.6,1.8Hz,1H),5.51(dd,J＝9.7,2.5Hz,1H),5.37(s,2H),4.85(s,2H),3.93–3.83(m,1H),3.71–3.59(m,1H),2.35(dq,J＝12.7,5.5,4.2Hz,1H),2.03(d,J＝15.6Hz,1H),1.89(d,J＝13.1Hz,1H),1.72(d,J＝10.1Hz,1H),1.56(s,2H),0.88(s,9H),0.08(d,J＝2.1Hz,6H).
步骤3：N-(5-溴-3-氟-2-硝基苯基)-3-(((叔丁基二甲基硅氧基)甲基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-6-胺(27-3)
取3-(((叔丁基二甲基硅烷基)氧基)甲基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-6-胺(15.5g，42.87mmol)于反应瓶中，加入2，5-二氟-4溴硝基苯(11.22g，47.16mmol)，三乙胺75ml，DMSO 75ml溶解，于100℃下加热回流。TLC监测反应无剩余后，乙酸乙酯和水进行萃取，收集有机相，无水硫酸钠干燥，浓缩得粗产物，经硅胶柱层析(PE/EA＝10/1)分离，得到N-(5-溴-3-氟-2-硝基苯基)-3-(((叔丁基二甲基硅烷基)氧基)甲基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-6-胺黄褐色油状液体(4.665g，258.37,8.08mmol)，产率18.78％。
1H NMR(400MHz,DMSO-d6)δ9.23(s,1H),7.91(d,J＝8.6Hz,1H),7.67(s,1H),7.39(dd,J＝10.3,2.1Hz,1H),7.27–7.16(m,2H),5.91–5.83(m,1H),5.10(d,J＝3.1Hz,2H),4.01(d,J＝11.5Hz,1H),3.83(d,J＝12.3Hz,1H),2.50(d,J＝14.6Hz,1H),2.13(s,1H),2.08(d,J＝13.3Hz,1H),1.86(s,1H),1.70(s,2H),1.02(d,J＝3.2Hz,9H),0.24(d,J＝3.4Hz,6H).
步骤4：5-溴-N1-(3-(((叔丁基二甲基硅烷基)氧基)甲基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-6-基)-3-氟苯-1,2-二胺(27-4)
取N-(5-溴-3-氟-2-硝基苯基)-3-(((叔丁基二甲基硅烷基)氧基)甲基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-6-胺(4.6g,7.94mmol),氯化铵(4.25g，79.37mmol)于反应瓶中，加入10ml水和40ml乙醇使其溶解混悬，于搅拌中缓缓加入铁粉(2.22g，39.69mmol)，于85℃下加热回流，约一小时后反应完全。硅藻土抽滤除去铁粉，收集滤液，EA和水进行萃取，旋转蒸发除去溶剂，浓缩得到粗产品，经硅胶柱层析分离得到5-溴-N1-(3-(((叔丁基二甲基硅烷基)氧基)甲基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-6-基)-3-氟苯-1,2-二胺黄色油状液体(4.247g，7.73mmol)，产率97.36％。
1H NMR(400MHz,DMSO-d6)δ7.70(s,1H),7.60(d,J＝8.7Hz,1H),7.03(dd,J＝9.0,1.9Hz,2H),6.97(d,J＝1.8Hz,1H),6.81(dd,J＝8.7,1.8Hz,1H),5.60(dd,J＝9.5,2.6Hz,1H),4.98(s,2H),4.89(s,2H),3.85(d,J＝11.3Hz,1H),3.64(ddd,J＝11.3,9.4,3.9Hz,1H),2.39–2.25(m,1H),2.05–1.85(m,2H),1.71(d,J＝11.6Hz,1H),1.54(t,J＝4.6Hz,2H),0.87(s,9H),0.08(d,J＝1.5Hz,6H).
步骤5：6-溴-1-(3-(((叔丁基二甲基硅烷基)氧基)甲基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-6-基)-4-氟-1H-苯并[d][1,2,3]三唑(27-5)
取5-溴-N1-(3-(((叔丁基二甲基硅烷基)氧基)甲基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-6-基)-3-氟苯-1,2-二胺(4.2g,7.64mmol)于反应瓶中，加入50ml乙腈溶解，滴加亚硝酸异戊酯(1.34g，11.46mmol)，于65℃下加热搅拌。约1小时后TLC监测反应完全，旋转蒸发除去溶剂，粗产物经硅胶柱层析(PE/EA＝12/1)纯化得6-溴-1-(3-(((叔丁基二甲基硅烷基)氧基)甲基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-6-基)-4-氟-1H-苯并[d][1,2,3]三唑，黑色油状液体(2.186g，3.90mmol),产率51.03％。
1H NMR(400MHz,DMSO-d6)δ8.27(d,J＝1.8Hz,1H),8.12–8.05(m,2H),7.75(dd,J＝9.9,1.4Hz,1H),7.69(dd,J＝8.5,1.8Hz,1H),6.03(dd,J＝9.6,2.3Hz,1H),5.15–5.05(m,2H),3.89(d,J＝11.6Hz,1H),3.78(dt,J＝12.0,6.8Hz,1H),2.44(d,J＝11.6Hz,1H),2.03(d,J＝15.7Hz,2H),1.77(s,1H),1.66–1.56(m,2H),0.92(s,9H),0.15(d,J＝1.5Hz,6H).
步骤6：(6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲醇(27-6)
取6-溴-1-(3-(((叔丁基二甲基硅烷基)氧基)甲基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-6-基)-4-氟-1H-苯并[d][1,2,3]三唑(3.63g,6.48mmol)，四丁基氟化铵(1M)(19.43ml,19.43mmol)于反应瓶中，加入50ml四氢呋喃溶解，于室温下搅拌反应，约30分钟后TLC监测反应完全，EA和水萃取，收集有机相，无水硫酸钠干燥，浓缩得(6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲醇，黑色油状物(1.748g,3.92mmol)，产率60.48％，无需纯化直接投下一步。
步骤7：2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-(二乙氧基磷酰基)乙酸乙酯(27-7)
取(((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸(1g,2.24mmol)于反应瓶中，加入10mL甲苯溶解混悬，滴加2-重氮-2-(二乙氧基磷酰基)乙酸乙酯(673mg,2.69mmol)，加入10mg二聚醋酸铑，在100℃下加热回流过夜，TLC监测反应完全后，旋转蒸发除去甲苯，粗产物经硅胶柱层析分离纯化得2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-(二乙氧基磷酰基)乙酸乙酯，褐色油状物(965mg,1.44mmol),产率64.43％。
1H NMR(400MHz,DMSO-d6)δ8.34–8.24(m,2H),8.07(d,J＝1.5Hz,1H),7.75(ddd,J＝8.3,6.9,1.6Hz,2H),6.06(dt,J＝9.6,2.1Hz,1H),5.13–4.97(m,2H),4.89(dd,J＝19.2,1.6Hz,1H),4.13–4.06(m,6H),3.91–3.75(m,2H),2.41(d,J＝20.0Hz,1H),2.08–1.99(m,2H),1.77(d,J＝12.6Hz,1H),1.61(q,J＝9.0,6.5Hz,2H),1.25–1.22(m,9H).
步骤8：(1-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-羟乙基)膦酸二乙酯(27-8)
取2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-(二乙氧基磷酰基)乙酸乙酯(207mg,309.67μmol)于反应瓶中，加入2mL THF溶解，置于冰浴中搅拌，待温度降至0℃后滴加LiBH4的四氢呋喃溶液(2M，620μL,1.24mmol)，转移至室温反应。约2小时后，TLC监测反应完全，饱和氯化铵溶液淬灭，乙酸乙酯和水萃取，收集有机相，无水硫酸钠干燥，旋转蒸发除去溶剂，得到(1-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-羟乙基)膦酸二乙酯，黄色油状液体(152mg,242.65μmol)产率78.36％。
1H NMR(400MHz,DMSO-d6)δ8.27(d,J＝8.4Hz,2H),8.05(d,J＝1.4Hz,1H),7.75(dd,J＝9.9,1.4Hz,1H),7.69(dd,J＝8.5,1.7Hz,1H),6.04(d,J＝8.7Hz,1H),5.13(t,J＝8.8Hz,3H),4.12–4.04(m,5H),3.88(s,1H),3.81(d,J＝8.5Hz,2H),3.66(dd,J＝12.8,6.7Hz,1H),2.44(s,1H),2.06(s,2H),1.77(s,1H),1.60(s,2H),1.24(ddd,J＝7.1,2.9,1.3Hz,6H).
步骤9：(1-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-2-羟乙基)膦酸二乙酯(27-9)
取(1-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-羟乙基)膦酸二乙酯(73mg,116.54μmol)于反应瓶中，加入2mL二氯甲烷溶解，置于冰浴中搅拌，待温度降至0℃后滴加1mL三氟乙酸，约30分钟TLC监测反应完全。饱和碳酸氢钠溶液淬灭，EA萃取，收集有机相，无水硫酸钠干燥，旋转蒸发除去溶剂得到粗产物。粗产物经硅胶柱层析分离纯化，得到(1-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-2-羟乙基)膦酸二乙酯，黄色油状物(50mg,92.20μmol)，产率79.12％。
步骤10：(1-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-2-羟乙基)膦酸(27)
取(1-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-羟乙基)膦酸二乙酯(50mg,92.20μmol)于反应瓶中，加入2mL无水1,2-二氯乙烷溶解，氮气保护，反复置换气多次。置于冰浴中，待温度降至0℃后，缓缓滴加三甲基溴硅烷(424mg,2.77mmol)，移至室温反应，TLC监测反应完全后，旋转蒸发除去溶剂，粗产物经二氯甲烷和乙酸乙酯打浆纯化，得到(1-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-2-羟乙基)膦酸，黄色固体(16mg,32.91μmol)，产率35.69％。
1H NMR(600MHz,DMSO-d6)δ13.28(s,1H),8.30(d,J＝8.6Hz,1H),8.05(d,J＝1.4Hz,1H),7.98(d,J＝1.8Hz,1H),7.72(dd,J＝9.9,1.4Hz,1H),7.57(dd,J＝8.5,1.8Hz,1H),5.21(d,J＝11.2Hz,1H),5.06(d,J＝11.2Hz,1H),3.82–3.79(m,2H),3.75–3.72(m,2H),3.60(ddd,J＝11.9,8.9,4.4Hz,2H).
(1-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-2-甲氧基乙基)膦酸(化合物28)
步骤1：(1-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-甲氧基乙基)膦酸二乙酯(28-1)
取(1-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-羟乙基)膦酸二乙酯(136mg,265μmol)于反应瓶中，加入1,8-双二甲氨基萘(228mg,1.06mmol)，三甲基氧鎓四氟硼酸(118mg,795μmol)以及分子筛，用2.5mL无水二氯甲烷溶解，N2保护，室温反应，2小时后TLC监测反应完全，碳酸氢钠淬灭，乙酸乙酯萃取，收集有机相，无水硫酸钠干燥，旋转蒸发除去溶剂，粗产物经硅胶柱层析提纯，得到(1-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-甲氧基乙基)膦酸二乙酯，白色固体产物(68mg,106μmol)，产率40.07％。
1H NMR(400MHz,DMSO-d6)δ8.29(d,J＝1.8Hz,1H),8.21(dd,J＝8.6,2.6Hz,1H),8.06(d,J＝1.4Hz,1H),7.73(ddd,J＝18.8,9.2,1.5Hz,2H),6.07–6.00(m,1H),5.24–4.99(m,2H),4.24(ddt,J＝10.4,8.2,2.4Hz,1H),4.10(dq,J＝9.4,7.2Hz,4H),3.89(dd,J＝11.1,4.0Hz,1H),3.79(dt,J＝11.8,6.6Hz,1H),3.73–3.58(m,2H),3.33(s,3H),2.48–2.33(m,1H),2.03(dt,J＝16.3,5.6Hz,2H),1.61(d,J＝8.0Hz,2H),1.25(tt,J＝3.1,1.5Hz,6H).
步骤2与3的合成同实例27的步骤9和10。产率40.44％。1H NMR(600MHz,DMSO-d6)δ13.30(s,1H),8.22(d,J＝8.4Hz,1H),8.05(s,1H),7.99(d,J＝1.7Hz,1H),7.72(d,J＝9.8Hz,1H),7.58(d,J＝8.5Hz,1H),5.20(d,J＝11.3Hz,1H),5.02(d,J＝11.2Hz,1H),3.93–3.86(m,2H),3.68(d,J＝11.5Hz,2H),3.56(m,J＝11.7,8.9,3.3Hz,2H),3.27(s,3H).
(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-1-羟基-3-甲氧基丙-2-基)膦酸(化合物29)
步骤1：2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-(二乙氧基磷酰基)-3-甲氧基丙酸乙酯(29-1)
取2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-(二乙氧基磷酰基)乙酸乙酯(130mg,194.48μmol)于反应瓶中，加入2mL无水THF溶解，N2保护，将体系置于-15℃中，缓缓滴加双(三甲基硅基)氨基钠(253μL,253μmol),搅拌30分钟后,加入四丁基碘化铵(36mg,97.24μmol),随后立即逐滴加入氯甲基甲基醚(56μL,680.67μmol)。将溶液在该温度下搅拌2小时，TLC监测反应完全，加入饱和氯化铵水溶液猝灭，乙酸乙酯萃取，收集有机相，无水硫酸钠干燥，旋转蒸发除去溶剂，粗产物通过硅胶柱层析分离纯化，得到2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-(二乙氧基磷酰基)-3-甲氧基丙酸乙酯，黄色油状产物(137mg,131.93μmol)，产率67.84％。
1H NMR(400MHz,DMSO-d6)δ8.36(d,J＝8.6Hz,1H),8.26(s,1H),8.05(s,1H),7.71(dd,J＝15.7,9.2Hz,2H),6.03(dd,J＝9.6,2.3Hz,1H),5.17(qd,J＝10.8,3.1Hz,2H),4.28(q,J＝7.1,6.6Hz,2H),4.15–4.05(m,6H),4.02(t,J＝5.4Hz,2H),3.91–3.73(m,2H),3.36(s,3H),2.43(d,J＝11.8Hz,1H),1.99(s,2H),1.76(s,1H),1.58(s,2H),1.28–1.17(m,13H).
步骤2：(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-1-乙氧基-3-甲氧基丙烷-2-基)膦酸二乙酯(29-2)
取2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-(二乙氧基磷酰基)-3-甲氧基丙酸乙酯(129mg,181.05μmol)于反应瓶中，加入二氯化钙(61mg,543.15μmol)，乙醇溶解，置于冰浴中，待温度降至0℃后向溶液中缓缓加入硼氢化钠(21mg,543.15μmol)，转移至室温反应，约半小时后，TLC监测反应完全，饱和氯化铵溶液淬灭，乙酸乙酯和水萃取，收集有机相，无水硫酸钠干燥，浓缩拌样，粗产物经硅胶柱层析分离纯化，得到(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-1-乙氧基-3-甲氧基丙烷-2-基)膦酸二乙酯黄色油状物(95mg,138.00μmol),产率75.12％。
1H NMR(400MHz,DMSO-d6)δ8.29–8.22(m,2H),8.03(d,J＝1.4Hz,1H),7.74–7.63(m,2H),6.00(d,J＝9.2Hz,1H),5.21–5.08(m,3H),4.15–3.97(m,6H),3.93–3.71(m,7H),2.47–2.33(m,1H),2.01(d,J＝18.1Hz,3H),1.75(s,1H),1.58(s,2H),1.31–1.14(m,9H).
步骤3与4的合成同实例27的步骤9和10。产率54.45％。1H NMR(600MHz,DMSO-d6)δ13.23(s,1H),8.29(d,J＝8.6Hz,1H),8.05(d,J＝1.4Hz,1H),7.96(s,1H),7.71(d,J＝9.9,1.4Hz,1H),7.55(d,J＝8.5,1.8Hz,1H),5.21–5.13(m,2H),4.03(q,J＝7.1Hz,1H),3.87–3.81(m,4H),3.77(d,J＝10.5,8.6Hz,2H),3.34(s,3H).
(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-1-羟基丙-2-基)膦酸(化合物30)
步骤1：2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-(二乙氧基磷酰基)丙酸乙酯(30-1)
取2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-(二乙氧基磷酰基)乙酸乙酯(350mg,523.59μmol)与反应瓶中，加入碳酸铯(683mg,2.09mmol)，DMF溶解，于搅拌中加入碘甲烷(372mg,2.62mmol)，40℃下反应，TLC监测反应完全后，乙酸乙酯和水萃取，收集有机相，无水硫酸钠干燥，浓缩，粗产物经硅胶柱层析分离纯化，得到2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-(二乙氧基磷酰基)丙酸乙酯黄色液体产物(266mg,389.75μmol)，产率74.44％。
1H NMR(400MHz,DMSO-d6)δ8.35(dd,J＝8.5,3.2Hz,1H),8.29(s,1H),8.06(t,J＝1.8Hz,1H),7.74(m,J＝14.2,9.3,1.6Hz,2H),6.04(d,J＝9.3Hz,1H),5.07–4.93(m,2H),4.29(p,J＝6.9Hz,3H),3.91–3.75(m,2H),3.66(d,J＝11.1Hz,1H),2.44(d,J＝14.9Hz,1H),1.77(d,J＝15.9Hz,5H),1.60(s,3H),1.39–1.31(m,9H).
步骤2、3与4的合成同实例29.产率70.84％.1H NMR(600MHz,DMSO-d6)δ13.22(s,1H),8.31(d,J＝8.5Hz,1H),8.04(d,J＝1.4Hz,1H),7.96(d,J＝1.8Hz,1H),7.71(dd,J＝9.9,1.4Hz,1H),7.54(dd,J＝8.5,1.8Hz,1H),5.10(s,2H),3.94–3.90(m,1H),3.69–3.61(m,2H),3.45–3.41(m,1H),1.43(d,J＝14.6Hz,3H).
(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-1-羟基-3-苯基丙-2-基)膦酸(化合物31)
取2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-(二乙氧基磷酰基)乙酸乙酯(27-7,150mg,224.40μmol)于反应瓶中，加入碳酸铯(146mg,448.79μmol)，DMF溶解，搅拌中加入溴化苄(115mg,673.19μmol)室温反应，TLC监测反应完全后，乙酸乙酯和水萃取，收集有机相，无水硫酸钠干燥，浓缩，粗产物经硅胶柱层析分离纯化得产物2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-(二乙氧基磷酰基)-3-苯基丙酸乙酯黄色油状液体(31-1 90mg,118.64μmol)，产率53％。
1H NMR(400MHz,DMSO-d6)δ8.26(s,1H),8.05(d,J＝4.7Hz,1H),7.83–7.73(m,2H),7.51(d,J＝8.7Hz,1H),7.25–7.13(m,5H),6.03(d,J＝9.7Hz,1H),5.33–5.18(m,2H),4.29–4.20(m,7H),3.84(d,J＝33.5Hz,2H),3.46(d,J＝13.4Hz,1H),2.43(s,1H),2.05(s,2H),1.81(d,J＝31.1Hz,1H),1.60(s,2H),1.34(m,J＝7.2,3.5Hz,9H).
步骤2、3与4的合成同实例29.产率80％.1H NMR(600MHz,DMSO-d6)δ8.06–8.01(m,2H),7.96(d,J＝1.8Hz,1H),7.71(dd,J＝9.8,1.4Hz,1H),7.48(dd,J＝8.5,1.8Hz,1H),7.39–7.33(m,2H),7.26–7.13(m,3H),5.31(d,J＝10.9Hz,1H),5.18(d,J＝10.9Hz,1H),3.72(t,J＝11.1Hz,1H),3.64–3.58(m,1H),3.27(dd,J＝14.0,10.9Hz,1H),3.18(dd,J＝14.0,8.0Hz,1H).13C NMR(151MHz,DMSO-d6)δ153.19,151.46,143.83,141.07,136.95,136.89,136.41,136.36,135.56,135.44,133.89,131.49,131.42,128.03,127.87,126.48,123.62,122.47,122.17,122.12,116.33,113.96,113.82,111.23,111.19,106.01,80.22,79.22,61.66,61.62,60.83,36.35,36.31.LC-MS/ESI[M-H]-630.1
(1-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-2-乙氧基-2-氧乙基)膦酸(化合物32)
产率96.29％。1H NMR(400MHz,DMSO-d6)δ13.39(s,1H),8.34(d,J＝8.6Hz,1H),8.06(s,1H),7.98(s,1H),7.71(d,J＝10.0Hz,1H),7.58(d,J＝8.7Hz,1H),5.19(d,J＝11.4Hz,1H),4.94(d,J＝11.3Hz,1H),4.28(d,J＝19.2Hz,1H),3.79(d,J＝9.5Hz,4H),1.04(t,J＝7.0Hz,3H).
((((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)(苯氧基)磷酰)-D-丙氨酸异丙酯(化合物33)
步骤1：乙基氢(((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸酯(33-1)
取化合物1-9(220mg,0.43mmol)，一水合氢氧化锂(180mg,4.20mmol)于反应瓶中，加入5mL THF和1.2mL水，室温反应2d后，加入20mL水稀释反应液，用1N盐酸调pH＝5作用，50mL EA萃取。收集有机相，无水硫酸钠干燥，旋转蒸发除去溶剂，得33-1白色固体150mg，产物无需进一步纯化。产物收率72.1％.
1H NMR(400MHz,DMSO-d6)δ13.50(s,1H),8.21(d,J＝8.5Hz,1H),8.13(d,J＝1.4Hz,1H),8.08(d,J＝1.8Hz,1H),7.78(dd,J＝10.0,1.4Hz,1H),7.67(dd,J＝8.6,1.8Hz,1H),5.05(s,2H),4.02(p,J＝7.2Hz,2H),3.81(d,J＝8.6Hz,2H),1.25(t,J＝7.1Hz,3H).
步骤2：苯基(((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸乙酯(33-2)
取化合物33-1(100mg,0.21mmol)于反应瓶中，加入无水DCM 1mL，注入氯化亚砜(45μL,0.62mmol)后，再滴入一滴DMF催化。1h后旋干反应液，加入苯酚(39mg,0.41mmol)，用1mL无水DCM重新溶解反应物，滴入三乙胺(200μL,1.45mmol),室温反应2h后，旋转蒸发除去溶剂，残渣上柱得33-2白色固体55mg。产物收率56.2％.
1H NMR(400MHz,DMSO-d6)δ13.52(s,1H),8.17(d,J＝8.6Hz,1H),8.10(dd,J＝10.6,1.6Hz,2H),7.78(dd,J＝9.9,1.4Hz,1H),7.67(dd,J＝8.6,1.8Hz,1H),7.47–7.36(m,2H),7.30–7.15(m,3H),5.11(d,J＝1.7Hz,2H),4.25(tt,J＝8.3,6.5Hz,2H),4.17(dd,J＝8.1,2.8Hz,2H),1.31(t,J＝7.1Hz,3H).
步骤3：((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)膦酸苯氢酯(33-3)
取化合物33-2(60mg,0.11mmol)于反应瓶中，加入DCM 2mL，注入TMS-Br(71μL,0.55mmol)后室温反应过夜，加入2mL甲醇,旋转蒸发除去溶剂，加入EA打浆，抽滤得33-3白色固体50mg。无需进一步纯化，产物收率87.7％.
步骤4：((((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)(苯氧基)磷酰)-D-丙氨酸异丙酯(33)
取化合物33-3(100mg,0.19mmol)于反应瓶中，加入无水DCM 1mL，注入草酰氯(48μL,0.56mmol)后，再滴入一滴DMF催化，反应迅速放泡。1h后旋干反应液，加入L-丙氨酸异丙酯盐酸盐(63mg,0.38mmol)，用1mL无水DCM重新溶解反应物，滴入无水三乙胺(104μL,0.75mmol),室温反应过夜后，旋转蒸发除去溶剂，残渣上柱得33白色固体32mg。
产物收率26.4％.1H NMR(400MHz,DMSO-d6)δ13.50(s,1H),8.16(d,J＝7.1Hz,1H),8.05(d,J＝8.9Hz,2H),7.75(d,J＝9.8Hz,1H),7.69–7.56(m,1H),7.35(t,J＝8.0Hz,2H),7.18(q,J＝7.7,6.9Hz,3H),5.77(q,J＝11.9Hz,1H),5.06(d,J＝11.2Hz,2H),4.84(dt,J＝11.9,5.9Hz,1H),4.02–3.79(m,3H),1.29–1.09(m,9H).LC-MS/ESI[M-H]-643.05
2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-1-羟基-3-(2H-四唑-5-基)丙-2-基)膦酸(34)
步骤1：2-((2-(三甲基硅基)乙氧基)甲基)-2H-四唑-5-羧酸乙酯(34-2)
将化合物34-1(10.00g,70.36mmol)溶于THF，冰浴条件下加入NaH(2.03g,84.43mmol)，随后加入SEM-Cl(14.08g,84.43mmol)。室温反应，TLC监测反应完全，加水淬灭反液，乙酸乙酯萃取，得到中间体34-2(20.00g,73.43mmol)，产率98％。
1H NMR(400MHz,DMSO-d6)δ6.14(s,2H),4.49(q,J＝7.0Hz,2H),3.73(t,J＝7.5Hz,2H),1.40(t,J＝5.8Hz,3H),0.92(t,J＝9.4Hz,2H),-0.00(s,9H).
步骤2：(2-((2-(三甲基硅基)乙氧基)甲基)-2H-四唑-5-基)甲醇(34-3)
将化合物34-2(1.00g,4.30mmol)溶于MeOH，冰浴条件下加入NaBH4(0.33g,8.60mmol)。室温反应，TLC监测反应完全，加水淬灭反液，乙酸乙酯萃取，得到中间体34-3(0.80g,3.47mmol)，产率80％。
1H NMR(400MHz,DMSO-d6)δ5.99(s,2H),4.74(d,J＝6.0Hz,2H),3.76–3.67(m,2H),0.90(d,J＝8.1Hz,2H),-0.00(s,9H).
步骤3：5-(溴甲基)-2-((2-(三甲基硅基)乙氧基)甲基)-2H-四唑(34-4)
在-40℃条件下，将化合物34-3(0.20g,0.87mmol),PPh3(0.46g,1.74mmol)溶于DCM，后加入NBS(0.31g,1.74mmol)。TLC监测反应完全，加水淬灭反液，乙酸乙酯萃取，柱层析纯化，得到中间体34-4(0.40g,0.17mmol)，产率40％。
1H NMR(400MHz,DMSO-d6)δ5.99(s,2H),4.92(s,2H),3.67(t,J＝8.1Hz,2H),0.86(t,J＝8.1Hz,2H),-0.05(s,9H).
步骤4：乙基2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-(二乙氧基磷酰基)-3-(2-((2-(三甲基硅基)乙氧基)甲基)-2H-四唑-5-基)丙酸酯(34-5)
将化合物27-7(0.27g,0.40mmol)溶于甲苯(5mL)，冰浴条件下加入TBAI(75mg,0.20mmol)，随后加入中间体34-4(0.23g,0.80g)，50％ KOH溶液(1mL),室温反应，TLC监测反应完全，加饱和氯化铵溶液淬灭反应液，乙酸乙酯萃取，得到黄色油状中间体34-5(0.15g,0.17mmol)，产率42％。
1H NMR(400MHz,DMSO-d6)δ8.36(d,J＝1.9Hz,1H),8.17–8.02(m,2H),7.86(dd,J＝9.9,1.4Hz,1H),7.69(dt,J＝8.6,1.6Hz,1H),6.15–5.98(m,3H),5.49(dd,J＝10.8,4.6Hz,1H),5.30(d,J＝11.1Hz,1H),4.47–4.29(m,5H),4.15(d,J＝7.1Hz,1H),4.02–3.85(m,3H),3.85–3.76(m,1H),3.70(ddd,J＝9.2,7.5,2.0Hz,2H),2.58–2.47(m,1H),2.14(s,2H),1.86(d,J＝9.2Hz,1H),1.70(s,2H),1.29-1.40(m,9H),0.96–0.81(m,2H),0.00(s,9H).
步骤5：(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-1-乙氧基-1-氧代-3-(2-((2-(三甲基硅基)乙氧基)甲基)-2H-四唑-5-基)丙-2-基)膦酸三乙基铵盐(34-6)
将化合物34-5(117mg,0.13mmol)溶于CH3CN(5mL)，后加入TEA(0.27g,2.65mmol)，TMSBr(0.30g,1.95mmol)，室温反应，TLC监测反应结束，旋干反应液得到中间体34-6，无需纯化。
步骤6：(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-1-乙氧基-1-氧代-3-(2H-四唑-5-基)丙-2-基)膦酸三乙基铵盐(化合物34)
将上一步得到的粗品化合物34-6，溶于甲醇(1mL)，加入2mL盐酸甲醇溶液，TLC监测反应完全，旋干反应液，制备液相纯化得到淡黄色固体化合物34(40mg,0.07mmol),收率50％。
m/z(ESI,-ve ion)＝608.0,[M-H]-,1H NMR(400MHz,DMSO)δ13.25(s,1H),10.32(s,1H),8.01(d,J＝1.3Hz,1H),7.90(d,J＝9.5Hz,2H),7.76–7.67(m,1H),7.41(d,J＝8.7Hz,1H),5.21(d,J＝10.5Hz,1H),5.06(d,J＝10.6Hz,1H),4.26(t,J＝9.0Hz,1H),4.16(d,J＝10.3Hz,1H),3.87(d,J＝16.3Hz,1H),3.72(d,J＝7.9Hz,1H),3.22(s,1H),3.17(s,1H),3.10–3.02(m,4H),1.24(t,J＝7.1Hz,3H),1.20(t,J＝7.3Hz,6H)..13C NMR(151MHz,DMSO)δ171.52,152.26(d,JCF＝261.2Hz),143.64,140.96,136.30,135.42(d,JCF＝18.1Hz),133.70,123.69,122.35,122.08(d,JCF＝7.1Hz),116.02,113.83(d,JCF＝18.3Hz),111.07,105.76,82.33,62.59,61.05,45.69,29.48,14.47,8.80.
(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-1-羟基-3-(2H-四唑-5-基)丙-2-基)膦酸三乙基铵盐(化合物35)
步骤1:二乙基(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-1-羟基-3-(2-((2-(三甲基硅基)乙氧基)甲基)-2H-四唑-5-基)丙-2-基)膦酸酯(35-1)
将中间体27-2(0.34g,0.40mmol)溶于乙醇(5mL)，冰浴条件下加入CaCl2(75mg,0.20mmol)，NaBH4(67mg,1.79mmol)。室温反应，TLC监测反应完全，加饱和氯化铵溶液淬灭反应液，乙酸乙酯萃取，得到中间体35-1(0.53g,0.63mmol)，产率86％。
1H NMR(400MHz,DMSO)δ8.33(d,J＝1.9Hz,1H),8.29–8.23(m,1H),8.11(d,J＝1.4Hz,1H),7.83(dd,J＝9.9,1.4Hz,1H),7.70(dd,J＝8.6,1.8Hz,1H),6.10(dd,J＝9.7,2.3Hz,1H),6.03(s,2H),5.40(dd,J＝5.1,3.6Hz,2H),4.21–4.06(m,6H),3.96(d,J＝11.5Hz,1H),3.90–3.83(m,1H),3.72(t,J＝8.0Hz,2H),3.63(dt,J＝12.3,3.2Hz,2H),2.50(m,1H),2.14(m,2H),1.84(m,1H),1.68(m,2H),1.28(m,6H),0.94–0.88(m,2H),0.00(s,9H).
步骤2、3同合成实例34
产率36％，淡黄色固体。
m/z(ESI,-ve ion)＝566.0[M-H]-。1H NMR(400MHz,DMSO-d6)δ13.25(s,1H),10.32(s,1H),8.01(d,J＝1.3Hz,1H),7.90(d,J＝9.5Hz,2H),7.70(dd,J＝9.9,1.3Hz,1H),7.41(d,J＝8.7Hz,1H),5.21(d,J＝10.5Hz,1H),5.06(d,J＝10.6Hz,1H),4.27(d,J＝10.7Hz,1H),4.20–4.13(m,1H),3.87(d,J＝16.8Hz,1H),3.72(d,J＝7.9Hz,1H),3.17(s,1H),3.06(s,1H),1.24(t,J＝7.1Hz,3H).13C NMR(151MHz,DMSO)δ152.31(d,JCF＝259.7Hz),143.99,140.97,136.35,135.48(d,JCF＝18.1Hz),133.83,123.48,122.41,122.11(d,JCF＝9.1Hz),116.19,113.93,113.80(d,JCF＝259.7Hz),111.19,105.87,78.83,77.86(d,JPC＝146.5Hz),62.31,60.20,46.10,9.05.
(2-((6-(6-溴-4-氟-(1-((2H-四唑-5-基)甲氧基)-2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-3-(2H-四唑-5-基)丙-2-基)膦酸三乙基铵盐(化合物36)
步骤1:二乙基(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-1-(2-((2-(三甲基硅基)乙氧基)甲基)-2H-四唑-5-基)-3-((2-((2-(三甲基硅基)乙氧基)甲基)-2H-四唑-5-基)甲氧基)丙-2-基)膦酸酯(36-1)
冰浴条件下，将化合物35-1(0.10g,0.12mmol)，34-4(0.17g,0.60mmol)溶于DMF(4mL)，随后加入NaH(60％,12mg,0.30mmol)，冰浴条件下反应30min，反应完全后用饱和氯化铵溶液淬灭反应液，乙酸乙酯萃取，有机层用饱和氯化钠溶液洗涤，无水硫酸镁干燥，减压干燥，柱层析纯化(PE:EA＝20:80)，得淡黄色油状物36-1(0.25g,0.24mmol)，产率：64％。
1H NMR(400MHz,DMSO-d6)δ8.39–8.33(m,1H),8.24(dd,J＝8.6,4.1Hz,1H),8.13(d,J＝1.4Hz,1H),7.85(dd,J＝9.9,1.3Hz,1H),7.69(dd,J＝8.5,1.8Hz,1H),6.12(dd,J＝9.8,2.4Hz,1H),6.09(s,2H),6.06(s,2H),5.43(dd,J＝10.3,5.4Hz,1H),5.32(dd,J＝10.8Hz,1H),5.06(t,J＝2.6Hz,2H),4.33–4.25(m,1H),4.21–4.11(m,6H),3.98(d,J＝10.6Hz,1H),3.93–3.84(m,1H),3.77(d,J＝7.6Hz,2H),3.73(d,J＝7.5Hz,2H),3.66–3.55(m,1H),2.58–2.47(m,1H),2.22–2.12(m,2H),1.95–1.82(m,1H),1.70(s,2H),1.31–1.29(m,3H),1.28–1.27(m,3H),0.98–0.88(m,4H),0.03(s,9H),-0.00(s,9H).
步骤2、3同合成实例34。
产率：64％，淡黄色固体。m/z(ESI,-ve ion)＝608.0,1H NMR(400MHz,DMSO-d6)δ13.21(s,1H),9.10(s,0.2H),8.02(d,J＝1.3Hz,1H),7.98–7.86(m,2H),7.70(dd,J＝9.9,1.4Hz,1H),7.38(dd,J＝8.7,1.7Hz,1H),5.32(d,J＝10.9Hz,1H),5.08(d,J＝11.0Hz,1H),4.99(dd,2H),4.12(t,1H),4.0(t,1H),3.56(s,1H),3.39(s,1H),3.09(d,J＝7.1Hz,1H),1.17(t,J＝7.3Hz,2H).13C NMR(151MHz,DMSO)δ155.37,152.32(d,JCF＝261.2Hz),143.70,140.96,136.35(d,JCF＝3.0Hz),135.49(d,JCF＝18.1Hz),133.82,123.32,122.33,122.14(d,JCF＝7.6Hz),116.19,113.88(d,JCF＝21.1Hz),111.19(d,JCF＝6.0Hz),105.88,78.83(d,JPC＝3.0Hz),72.87,63.37,60.91,46.23,9.06
(1-((2H-四唑-5-基)甲氧基)-2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-3-羟基丙-2-基)膦酸三乙基铵盐(化合物37)
步骤1:乙基2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-(二乙氧基磷酰基)-3-(2-(三甲基硅基)乙氧基)丙酸酯(37-1)
于-15℃条件下，将化合物27-2(1.50mmol,1.00g)溶于THF(25mL)，缓慢滴加NaHMDS(1N,2mL)。25min后，加入TBAI(四丁基碘化铵)(0.75mmol,0.28g)，随后立刻向反应体系中加入SEM-Cl(2-(三甲基硅基)乙氧基甲基氯)(4.4mmol,0.75g)，反应液于-15℃继续反应2.5h。反应完全后，用饱和氯化铵淬灭，乙酸乙酯萃取，柱层析纯化(PE:EA＝120:80)得到黄色油状物37-1(0.60g，0.76mmol)，产率：51％。
1H NMR(400MHz,DMSO-d6)δ8.41(td,J＝8.5,1.0Hz,1H),8.29(d,J＝1.8Hz,1H),8.04(t,J＝1.7Hz,1H),7.76(dd,J＝9.9,1.4Hz,1H),7.70(dd,J＝8.6,1.8Hz,1H),6.05(dd,J＝9.7,2.4Hz,1H),5.29–5.17(m,2H),4.30(qd,J＝6.9,1.6Hz,2H),4.20–4.05(m,6H),3.90(d,J＝11.3Hz,1H),3.80(td,J＝11.1,6.9Hz,1H),3.66(m,1H),3.54(td,J＝9.3,7.1Hz,1H),2.48–2.37(m,1H),2.07(s,2H),1.77(d,J＝6.9Hz,1H),1.61(s,2H),1.32–1.23(m,9H),0.90(m,2H),-0.00(s,9H).
步骤2、3、4、5同合成实例36。
(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-1,3-二羟基丙-2-基)膦酸三乙基铵盐(化合物38)
步骤1、2参考合成实例37。
产率50％，淡黄色固体。m/z(ESI,-ve ion)＝596.0[M-H]-。1H NMR(600MHz,DMSO)δ13.24(s,1H),9.26(s,1H),8.34(d,J＝8.5Hz,1H),8.04(d,J＝1.4Hz,1H),7.96(d,J＝1.8Hz,1H),7.71(dd,J＝9.8,1.4Hz,1H),7.54(dd,J＝8.5,1.8Hz,1H),5.21(s,2H),3.96–3.88(m,4H),3.12–3.07(m,8H),1.19(t,J＝7.3Hz,12H).13C NMR(151MHz,DMSO)δ152.32(d,JCF＝243.9Hz),144.15,141.08,136.40(d,JCF＝7.1Hz),135.49(d,JCF＝16.9Hz),133.92,123.85,122.69,122.15(d,JCF＝8.46Hz),116.40,113.90(d,JCF＝18.3Hz),111.22(d,JCF＝4.2Hz),106.01,79.67(d,JPC＝139.6Hz),60.95(d,JPC＝9.1Hz),60.38,46.07,9.02.
(1-((2H-四唑-5-基)甲氧基)-2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-3-(2-甲氧基乙氧基)丙-2-基)膦酸三乙基铵盐(化合物39)
合成方法同实例37。
产率50％，淡黄色固体。m/z(ESI,-ve ion)＝654.1[M-H]-。1H NMR(600MHz,DMSO)δ13.21(s,1H),9.67(s,1H),8.26(d,J＝8.5Hz,1H),8.02(s,1H),7.92(s,1H),7.69(d,J＝10.0Hz,1H),7.43(d,J＝8.5Hz,1H),5.27(d,J＝10.7Hz,1H),5.12(d,J＝10.8Hz,1H),4.96(q,J＝12.0Hz,2H),4.08(dd,J＝11.3,4.4Hz,1H),4.05–4.01(m,1H),4.00–3.97(m,1H),3.83(dd,J＝11.4,3.4Hz,1H),3.63–3.58(m,2H),3.47(t,J＝4.8Hz,2H),3.08(q,J＝7.3Hz,6H),1.18(t,J＝7.3Hz,11H).13C NMR(151MHz,DMSO)δ155.70,152.31(d,JCF＝261.2Hz),143.99,141.02,136.34(d,JCF＝7.6Hz),135.48(d,JCF＝18.1Hz),133.84,123.62(d,JCF＝15.1Hz),122.55,122.14(d,JCF＝7.6Hz),116.20,113.87(d,JCF＝15.1Hz),111.16(d,JCF＝4.2Hz),105.89,81.57(d,JPC＝151.0Hz),72.65,71.75(d,JPC＝10.6Hz),70.72(d,JPC＝6.0Hz),69.86(d,JPC＝10.6Hz),63.86,60.62,58.62,46.01,8.98.
(1-(苄氧基)-2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-3-(2-甲氧基乙氧基)丙-2-基)膦酸三乙基铵盐(化合物40)
合成方法参考实例39。
产率45％，淡黄色固体。m/z(ESI,-ve ion)＝662.0[M-H]-。1H NMR(400MHz,DMSO)δ13.25(s,1H),8.39(s,1H),7.93(d,J＝15.3Hz,2H),7.77–7.15(m,7H),5.25(s,2H),4.56(s,2H),4.02–3.84(m,4H),3.59(s,4H),3.16–2.76(m,23H),1.47–0.84(m,33H).13CNMR(151MHz,DMSO)δ152.33(d,JCF＝261.3Hz),144.70,141.07,139.28,135.55,133.81,129.08,128.57,127.89,127.68,122.08,115.90,113.94,111.08,105.82,73.12,71.79(d,JPC＝25.7Hz),70.68,60.58,58.60,51.89,45.95,8.87.
(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-1-羟基-3-(2-甲氧基乙氧基)丙-2-基)膦酸三乙基铵盐(化合物41)
合成方法参考实例39。
产率：40％，淡黄色固体。m/z(ESI,-ve ion)＝572.0[M-H]-。1H NMR(400MHz,DMSO)δ13.23(s,1H),8.34(d,J＝8.6Hz,1H),8.03(d,J＝1.5Hz,1H),7.95(d,J＝2.0Hz,1H),7.70(d,J＝10.0Hz,1H),7.52(dd,J＝8.6,1.8Hz,1H),5.23(d,J＝10.8Hz,1H),5.16(d,J＝10.1Hz,1H),3.98(dd,J＝10.7,6.3Hz,1H),3.92–3.81(m,3H),3.69–3.58(m,2H),3.53–3.44(m,2H),3.26(s,3H),3.09(q,J＝7.2Hz,1H),1.18(t,J＝7.3Hz,2H).13C NMR(101MHz,DMSO)δ152.32(d,JCF＝261.6Hz),144.12,141.09,136.38(d,JCF＝8.1Hz),135.49(d,JCF＝19.2Hz),133.91,123.90,122.70,122.11(d,JCF＝8.1Hz),116.33,113.86(d,JCF＝20.2Hz),111.17,105.94,79.59(d,JPC＝151.5Hz),71.73,70.81,61.30,60.53,58.63,46.09,9.04.
(((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲基)磺酰基)甲基)膦酸(化合物42)
步骤1:(6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲硫醇(42-1)
氮气氛围下，将偶氮二甲酸二异丙酯(DIAD,0.66g,3.24mmol)，三苯基磷(PPh3,0.85g,3.24mmol)溶于THF(10mL),5分钟后，加入化合物27-4(0.50g,1.08mmol)和硫代乙酸(AcSH,0.25g 3.24mmol),回流反应过夜，反应结束三乙胺(TEA,100μL)淬灭反应液，旋干混合液，后加水复溶，乙酸乙酯萃取，有机相用饱和氯化钠洗涤，无水硫酸镁干燥，有机相旋干后溶解于甲醇(6mL)，后加入三乙胺(1mL)与二硫代苏糖醇(0.17g,1.08mmol),搅拌3小时后，柱层析纯化，得到淡黄色油状化合物42-1(0.12g,0.27mmol)，产率:25％。
m/z(ESI,-ve ion)＝460.0[M-H]-,m/z(ESI,+ve ion)＝378.0[M+H]+。1H NMR(400MHz,DMSO)δ8.30–8.16(m,2H),8.05(d,J＝1.3Hz,1H),7.74(dd,J＝9.9,1.5Hz,1H),7.66(dd,J＝8.6,1.8Hz,1H),5.98(dd,J＝9.8,2.3Hz,1H),4.18(d,J＝7.8Hz,2H),3.88(d,J＝12.1Hz,1H),3.82–3.70(m,1H),3.16(t,J＝7.8Hz,1H),2.46–2.38(m,1H),2.12–2.02(m,2H),1.74(t,J＝6.2Hz,1H),1.64–1.51(m,2H).
步骤2:(((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲基)硫代)甲基)膦酸二乙酯(42-2)
将中间体42-1(0.34g,0.74mmol,1.0eq)溶于DMF(5mL)，并于-10℃条件下搅拌30min,随后加入碘甲基磷酸二乙酯(0.31g,1.11mmol,1.5eq)，加毕，反应15min后室温反应1h，反应完毕用饱和氯化铵淬灭，乙酸乙酯萃取，有机层饱和氯化钠洗涤，无水硫酸镁干燥，浓缩，柱层析(梯度洗脱PE:EA＝50:50–100％ EA)，得到淡黄色油状物42-2(0.10g,0.16mmol),产率：22％。
m/z(ESI,+ve ion)＝612.2[M+H]+。1H NMR(400MHz,DMSO)δ8.26(s,1H),8.19(d,J＝8.5Hz,1H),8.05(d,J＝1.5Hz,1H),7.74(d,J＝9.8Hz,1H),7.68(dd,J＝8.6,1.8Hz,1H),6.01(d,J＝7.4Hz,1H),4.36(s,2H),4.10–4.02(m,6H),2.92(d,J＝12.8Hz,2H),2.51–2.53(m,1H),2.08–2.04(m,2H),1.77–1.74(m,1H),1.60–1.58(m,2H),1.25–1.24(m,6H).
步骤3:(((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲基)磺酰基)甲基)膦酸二乙酯(42-3)
将中间体42-2(0.10g,0.16mmol,1.0eq)溶于MeOH:H2O:THF(1:1:2,1mL,1mL,2mL)混合溶剂，随后加入oxone(0.56g,1.63mmol,10.0eq)，过夜反应，反应液NaHCO3淬灭，乙酸乙酯萃取，有机层饱和氯化钠洗涤，无水硫酸镁干燥，浓缩，制备TLC板纯化,得到淡黄色固体42-3(60mg,0.10mmol)，产率：58％。
m/z(ESI,-ve ion)＝642.0[M-H]-。1H NMR(400MHz,DMSO)δ8.32(d,J＝2.3Hz,1H),8.18(d,J＝8.6Hz,1H),8.07(d,J＝1.5Hz,1H),7.74(dd,J＝8.5,1.7Hz,2H),6.08(dd,J＝9.5,2.3Hz,1H),5.24(s,2H),4.34(dd,J＝16.7,2.1Hz,2H),4.22–4.12(m,4H),4.08–3.99(m,1H),3.95–3.87(m,1H),2.45–2.42(m,1H),2.10–2.04(m,2H),1.80–1.75(m,1H),1.63–1.56(m,2H),1.30(t,J＝7.0Hz,6H).
步骤4:(((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲基)磺酰基)甲基)膦酸(化合物42)
将化合物42-3(0.06g,0.09mmol,1eq)溶于DCE(5mL)，随后加入TMSI(0.28g,1.40mmol)，室温反应24h，旋干反应液，甲醇打浆，得到白色固体42(20mg,0.16mmol),m/z(ESI,-ve ion)＝501.9[M-H]-。
1H NMR(600MHz,DMSO)δ13.64(s,1H),8.20(d,J＝8.6Hz,1H),8.13–8.01(m,2H),7.70(d,J＝10.3Hz,1H),7.66(d,J＝8.6Hz,1H),5.22(s,2H),3.87(d,JPC＝16.1Hz,2H).13CNMR(151MHz,DMSO)δ152.30(d,JCF＝243.9Hz),140.98,136.31(d,JCF＝5.6Hz),135.51(d,JCF＝16.9Hz),135.00,134.17,123.47,123.17,122.23(d,JCF＝7.1Hz),117.23,113.94(d,JCF＝18.3Hz),111.23(d,JCF＝4.2Hz),106.30,52.49,51.33(d,JPC＝121.3Hz).
(((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲基)硫代)甲基)膦酸(化合物43)
合成方法参考实例42。收率：55％，白色固体。m/z(ESI,-ve ion)＝470.0[M-H]-。1HNMR(600MHz,DMSO)δ13.32(s,1H),8.17(d,J＝8.5Hz,1H),8.08(s,1H),7.98(d,J＝1.8Hz,1H),7.71(d,J＝9.8Hz,1H),7.59(dd,J＝8.5,1.8Hz,1H),4.33(s,2H),2.64(d,JPC＝13.3Hz,2H).13C NMR(151MHz,DMSO)δ152.31(d,JFC＝242.5Hz),143.21,141.10,136.33,135.51(d,JFC＝16.9Hz),134.05,122.71,122.21,121.89,116.51,113.92(d,JFC＝18.3Hz),111.27,106.21,28.34,27.58(d,JPC＝132.5Hz).
(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-1-乙氧基-3-(4-氟苯基)-1-氧代丙烷-2-基)膦酸(化合物44)
步骤1:乙基2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-2-(二乙氧基磷酰基)-3-(4-氟苯基)丙酸酯(44-1)
将化合物27-2(0.20g,0.29mmol,1.0eq)，碳酸铯(0.28g,0.87mmol,3.0eq)溶于DMF，后加入4-F-苄溴(0.12g,0.60mmol,2.0eq)，室温过夜，反应液饱和氯化铵溶液淬灭，乙酸乙酯萃取。柱层析纯化，得到黄色油状物44-1(0.18g,0.23mmol),产率80％，m/z(ESI,+ve ion)＝776.2[M+H]+。
1H NMR(400MHz,DMSO)δ8.25(d,J＝1.8Hz,1H),8.04(dd,J＝4.2,1.4Hz,1H),7.82(dd,J＝8.6,2.3Hz,1H),7.74(dd,J＝9.9,1.4Hz,1H),7.53(ddd,J＝8.6,4.8,1.7Hz,1H),7.21–7.11(m,2H),7.03(td,J＝8.9,2.9Hz,2H),6.02(dd,J＝9.9,2.7Hz,1H),5.22(dd,J＝12.4,5.6Hz,2H),4.29–4.18(m,6H),3.92–3.74(m,2H),3.29–3.20(m,2H),2.46–2.37(m,1H),2.11–2.00(m,2H),1.83–1.71(m,1H),1.64–1.52(m,2H),1.36–1.28(m,6H),1.24(t,J＝7.0Hz,3H).
步骤2:(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-1-乙氧基-3-(4-氟苯基)-1-氧代丙烷-2-基)膦酸(44-2)
将中间体44-1(50mg,0.06mmol,1.0eq)溶于DCE(2mL)，随后加入三乙胺(0.12g,1.20mmol,20.0eq)和TMSI(2.96g,19.35mmol,15eq)，室温反应24h，旋干反应液，无需进一步纯化，直接投下一步，m/z(ESI,-ve ion)＝868.2[M-H]-。
步骤3：(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-1-乙氧基-3-(4-氟苯基)-1-氧代丙烷-2-基)膦酸三乙基铵盐(化合物44)
将上一步得到的粗品中间体44-2溶于甲醇(2mL)，后加入con.HCl(0.25mL)，室温反应2d，旋干反应液，水洗固体，得到淡黄色固体44(25mg,0.04mmol)，产率：66％，m/z(ESI,-ve ion)＝634.0[M-H]-。
1H NMR(600MHz,DMSO)δ13.21(s,1H),9.78(s,1H),8.07–7.99(m,1H),7.93(dd,J＝5.2,3.2Hz,2H),7.69(dd,J＝9.8,1.3Hz,1H),7.39–7.31(m,1H),7.12(dd,J＝8.4,5.6Hz,2H),6.95(t,J＝8.6Hz,2H),5.44(d,J＝11.0Hz,1H),5.37(d,J＝11.1Hz,1H),4.07-4.13(m,1H),4.16-4.20(m,1H),3.54(t,J＝10.1Hz,1H),3.26(dd,J＝14.1,7.3Hz,1H),3.08(q,J＝7.3Hz,2H),1.19(t,J＝7.3Hz,7H).13C NMR(151MHz,DMSO)δ171.06,161.55(d,JCF＝227.0Hz),152.33(d,JCF＝243.9Hz),144.33,141.19,136.24,135.52(d,JCF＝16.9Hz),133.82,132.39(d,JCF＝7.1Hz),124.31,122.45,122.13(d,JCF＝7.1Hz),115.64,114.80(d,JCF＝18.3Hz),113.88(d,JCF＝19.6Hz),111.19,105.70,84.37,83.43,63.58,60.92(d,JPC＝375.1Hz),45.99,29.48,14.56,9.00.
(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-1-(4-氟苯基)-3-羟基丙-2-基)膦酸(化合物45)
步骤1参考实例44步骤1，步骤2参考实例35步骤1，步骤3参考实例42步骤4。
(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-1-(甲氧基-d3)-3-(2H-四唑-5-基)丙-2-基)膦酸三乙基铵盐(化合物46)
步骤1：二乙基(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)-1-(甲氧基-d3)-3-(2-((2-(三甲基硅基)乙氧基)甲基)-2H-四唑-5-基)丙-2-基)膦酸酯(46-1)
冰浴条件下，将化合物35-1(0.22g,0.26mmol,1eq)，CD3I(0.38g,2.62mmol,10eq)溶于DMF(5mL)，随后加入NaH(60％,32mg,0.79mmol,3.0eq)，冰浴条件下反应3h，反应完全后用饱和氯化铵溶液淬灭反应液，乙酸乙酯萃取，有机层用饱和氯化钠溶液洗涤，无水硫酸镁干燥，减压干燥，柱层析纯化(PE:EA＝30:70)，得淡黄色油状物46-1(0.18g,1.29mmol)，产率：95％。
1H NMR(400MHz,DMSO)δ8.35(d,J＝1.8Hz,1H),8.24–8.19(m,1H),8.12(d,J＝1.4Hz,1H),7.84(dd,J＝9.9,1.5Hz,1H),7.72(dd,J＝8.6,1.8Hz,1H),6.10(d,J＝9.0Hz,1H),6.05(s,2H),5.46–5.37(m,1H),5.28(d,J＝10.8Hz,1H),4.27–4.10(m,4H),4.01–3.81(m,4H),3.73(t,J＝8.2Hz,3H),3.63–3.56(m,1H),2.58–2.40(m,1H),2.15–2.07(m,2H),1.94–1.75(m,-1H),1.74–1.63(m,2H),1.31–1.27(m,6H),0.92(t,J＝8.9Hz,2H),0.00(s,9H).
步骤2、3参考合成实例34。
产率50％，白色固体。m/z(ESI,-ve ion)＝583.0[M-H]-。1H NMR(400MHz,DMSO)δ13.25(s,1H),8.11–8.00(m,2H),7.94(d,J＝1.8Hz,1H),7.70(dd,J＝9.9,1.4Hz,1H),7.48(dd,J＝8.6,1.8Hz,1H),5.35(d,J＝11.0Hz,1H),5.09(d,J＝10.8Hz,1H),3.94(dd,J＝10.8,7.7Hz,1H),3.77(dd,J＝10.8,5.7Hz,1H),3.55(dd,J＝15.3,12.1Hz,1H),3.44(dd,J＝15.2,11.2Hz,1H),3.10(q,J＝7.2Hz,1H),1.18(t,J＝7.9Hz,2H).13C NMR(101MHz,DMSO)δ152.33(d,JCF＝261.6Hz),143.67,141.06,136.41,135.59,133.93,123.45,122.40,122.09,116.35,113.88(d,JCF＝20.2Hz),111.17,105.94,78.80,77.27(d,JPC＝77.5Hz),71.97,61.01,46.18,27.17,9.08.
(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-1-甲氧基-3-(2H-四唑-5-基)丙-2-基)膦酸三乙基铵盐(化合物47)
操作同实例46。
产率73％，淡黄色固体。ESI,-ve ion)＝583.0[M-H]-。H NMR(400MHz,DMSO)δ8.10–7.97(m,2H),7.92(d,J＝1.7Hz,1H),7.70(dd,J＝9.9,1.4Hz,1H),7.46(dd,J＝8.6,1.8Hz,1H),5.35(d,J＝10.9Hz,1H),5.03(d,J＝10.9Hz,1H),4.00(dd,J＝11.0,6.4Hz,1H),3.75(dd,J＝11.0,4.4Hz,1H),3.60(t,J＝15.1Hz,1H),3.39(dd,J＝14.9,11.3Hz,1H),3.33(s,3H),3.10(q,J＝7.2Hz,1H),1.18(t,J＝7.2Hz,3H).13C NMR(101MHz,DMSO)δ159.41,152.50(d,JCF＝299.0Hz),143.77,141.04,136.40,135.50(d,JCF＝19.2Hz),133.90,123.48,122.42,122.09,116.30,113.87(d,JCF＝20.2Hz),111.18,105.89,78.14(d,JPC＝152,5Hz),72.09,60.94,59.15,46.12,27.34,9.05.
((((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)磷酰基)双(氧基))双(亚甲基)双(2-甲基丙酸酯)(化合物48)
步骤1：((((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)甲基)磷酰基)双(氧基))双(亚甲基)双(2-甲基丙酸酯)(48-2)
将化合物48-1(0.20g,0.37mmol)、CsCO3(0.18g,11mmol)溶于NMP(10mL)，80℃反应3min，后加入氯甲基异丁酸酯(3mL,11mmol)，10min后，饱和氯化铵淬灭反应液，乙酸乙酯萃取反应液，柱层析纯化(PE:EA＝50:50)得到淡黄色油状物48-2(0.33g,0.44mmol)，产率：82％。
m/z(ESI,+ve ion)＝740.1[M+H]+。1H NMR(400MHz,DMSO)δ8.28(s,1H),8.13(d,J＝8.4Hz,1H),8.02(s,1H),7.73(d,J＝9.9Hz,1H),7.67(d,J＝8.6Hz,1H),6.03(d,J＝8.3Hz,1H),5.66(s,2H),5.62(s,2H),5.01(s,2H),4.05(d,J＝8.4Hz,2H),3.94–3.86(m,1H),3.81–3.75(m,1H),2.60–2.54(m,2H),2.46–2.37(m,1H),2.08–2.01(m,2H),1.82–1.73(m,1H),1.62–1.55(m,2H),1.08(s,6H),1.07(s,6H).
步骤2：((((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)磷酰基)双(氧基))双(亚甲基)双(2-甲基丙酸酯)(化合物48)
室温条件下，将化合物48-2(0.33g,0.44mmol)溶于DCM(10mL)，后加入TFA(1.50g,13.16mmol)，反应结束旋干反应液，柱层析纯化(PE:EA＝50:50)得到淡黄色油状物48(0.16g,0.24mmol)，产率：55％。
m/z(ESI,+ve ion)＝656.2[M+H]+。1H NMR(400MHz,DMSO)δ13.47(s,1H),8.12(d,J＝8.6Hz,1H),8.03(d,J＝7.3Hz,2H),7.71(d,J＝11.4Hz,1H),7.61(dd,J＝8.6,1.8Hz,1H),5.65(s,2H),5.62(s,2H),5.01(s,2H),4.02(d,J＝8.2Hz,2H),2.59–2.54(m,2H),1.08(d,J＝1.5Hz,6H),1.06(d,J＝1.5Hz,6H).13C NMR(101MHz,DMSO)δ175.23,152.35(d,JCF＝261.6Hz),142.10,141.08,136.35(d,JCF＝8.1Hz),135.53(d,JCF＝19.2Hz),134.09,122.51,122.36,122.23,116.99,113.92(d,JCF＝20.2Hz),111.18,106.30,81.86,67.43,64.10(d,JPC＝163.6Hz),33.45,18.75.
((((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)(羟基)磷酰基)氧基)甲基异丁酸酯三乙基铵盐(化合物49)
步骤1：((((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1-(四氢-2H-吡喃-2-基)-1H-吲唑-3-基)甲氧基)甲基)(羟基)磷酰基)氧基)甲基异丁酸酯(49-1)
将化合物48-1(0.20g,0.37mmol)、TEA(0.97g,9.62mmol)溶于NMP(10mL)，80℃反应3min，后加入氯甲基异丁酸酯(3mL,11mmol)，10min后，饱和氯化铵淬灭反应液，乙酸乙酯萃取反应液，无需纯化，直接投下一步。
m/z(ESI,-ve ion)＝746.1[M-H]-，m/z(ESI,+ve ion)＝748.2[M+H]+。
步骤2同合成实例48步骤2。
产率：40％，淡黄色油状物。m/z(ESI,-ve ion)＝554.0[M-H]-。1H NMR(400MHz,DMSO)δ13.41(s,1H),8.15(d,J＝8.5Hz,1H),8.06(d,J＝1.5Hz,1H),8.01(d,J＝2.1Hz,1H),7.71(dd,J＝9.9,1.4Hz,1H),7.60(dd,J＝8.5,1.9Hz,1H),5.54(d,J＝12.6Hz,2H),4.99(s,2H),3.78(d,J＝8.4Hz,2H),3.19–3.14(m,6H),2.55–2.52(m,1H),1.05(d,J＝7.0Hz,6H),0.94(t,J＝7.3Hz,11H).13C NMR(151MHz,DMSO)δ175.84,152.29(d,JCF＝261.2Hz),143.38,141.14,136.23,135.49(d,JCF＝18.1Hz),133.74,123.16,122.48,122.13(d,JCF＝7.6Hz),116.21,113.86(d,JCF＝21.1Hz),111.25(d,JCF＝6.0Hz),106.05,83.57(d,JPC＝4.5Hz),68.53(d,JPC＝154.0Hz),66.85,66.78(d,JPC＝4.5Hz),57.96,33.68,18.97,13.95.
(6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲醇(化合物50)
步骤1：(6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲醇(化合物50)
将化合物8d-1(0.40g,0.90mmol)溶于甲醇(2mL)，后加入浓盐酸(2mL)，室温反应12h，析出固体，抽滤，滤饼水洗，得到红色固体化合物50(0.16g,0.24mmol)，产率：93％。m/z(ESI,-ve ion)＝360.0[M-H]-。1H NMR(400MHz,DMSO)δ8.16(d,J＝8.6Hz,1H),8.05(d,J＝1.4Hz,1H),7.98(d,J＝1.8Hz,1H),7.70(dd,J＝9.9,1.6Hz,1H),7.57(dd,J＝8.7,1.8Hz,1H),4.88(s,2H).13C NMR(101MHz,DMSO)δ152.37(d,JCF＝261.6Hz),146.64,141.22,136.35(d,JCF＝7.1Hz),135.56(d,JCF＝18.2Hz),133.91,123.12,122.25,122.15(d,JCF＝4.04Hz),116.31,113.94(d,JCF＝20.2Hz),111.23(d,JCF＝5.1Hz),106.04,57.20.
(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-1-羟基-3-(4-(三氟甲基)苯基)丙-2-基)膦酸(化合物51)
合成方法参考实例45。产率：78％，淡黄色固体。m/z(ESI,-ve ion)＝642.0[M-H]-。1H NMR(600MHz,DMSO)δ13.26(s,1H),8.10–8.00(m,2H),7.97(s,1H),7.71(d,J＝11.3Hz,1H),7.59(d,J＝8.4Hz,2H),7.56(d,J＝8.5Hz,2H),7.46(dd,J＝8.5,1.8Hz,1H),5.31(d,J＝10.9Hz,1H),5.19(d,J＝10.9Hz,1H),3.81–3.69(m,2H),3.30(d,J＝11.1Hz,2H).13CNMR(151MHz,DMSO)δ152.34(d,JCF＝261.2Hz),143.82,142.33,141.09,136.36(d,JCF＝7.6Hz),135.51(d,JCF＝18.1Hz),133.93,132.21,127.71,127.35(q,JCF＝9.1Hz),124.83(q,JCF＝329.9Hz),124.64(q,JCF＝96.6Hz),122.45,122.15(d,JCF＝9.1Hz),116.23,113.91(d,JCF＝19.6Hz),111.17(d,JCF＝4.5Hz),106.01,79.91(d,JPC＝152.5Hz),61.16(d,JPC＝7.6Hz),60.79,36.81.
(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-1-(3-氟苯基)-3-羟基丙-2-基)膦酸(化合物52)
合成方法参考实例45。产率：78％，淡黄色固体。m/z(ESI,-ve ion)＝592.0[M-H]-。1H NMR(600MHz,DMSO)δ8.09–8.01(m,2H),7.97(d,J＝2.5Hz,1H),7.70(d,J＝9.9Hz,1H),7.49(dd,J＝8.5,1.8Hz,1H),7.29–7.23(m,1H),7.22–7.15(m,2H),7.03–6.98(m,1H),5.31(d,J＝10.9Hz,1H),5.19(d,J＝10.9Hz,1H),3.76(dd,J＝11.7,9.1Hz,1H),3.69(dd,J＝14.3,11.7Hz,1H),3.30–3.19(m,2H).13C NMR(151MHz,DMSO)δ162.14(d,JCF＝241.6Hz),152.18(d,JCF＝261.2Hz),143.80,141.09,139.96(d,JCF＝9.1Hz),136.38(d,JCF＝7.1Hz),135.50(d,JCF＝16.9Hz),133.91,129.59(d,JCF＝9.1Hz),127.59,123.53,122.46,122.15(d,JCF＝9.1Hz),118.04(d,JCF＝21.1Hz),116.32,113.88(d,JCF＝19.6Hz),113.23(d,JCF＝19.6Hz),111.17(d,JCF＝4.5Hz),106.03,79.82(d,JPC＝149.5Hz),61.37,60.75,36.40.
(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-1-羟基-3-(4-(三氟甲氧基)苯基)丙-2-基)膦酸(化合物53)
合成方法参考实例45。产率：56％，淡黄色固体。m/z(ESI,-ve ion)＝658.0[M-H]-。1H NMR(600MHz,DMSO)δ13.25(s,1H),8.15–7.90(m,3H),7.70(d,J＝9.9Hz,1H),7.49(d,J＝8.6Hz,3H),7.20(d,J＝8.4Hz,2H),5.32(d,J＝10.9Hz,1H),5.20(d,J＝10.9Hz,1H),3.80–3.74(m,1H),3.72–3.66(m,1H),3.26(dd,J＝10.7,4.9Hz,2H).13C NMR(151MHz,DMSO)δ152.33(d,JCF＝261.2Hz),147.27,143.81,141.09,136.69(d,JCF＝9.1Hz),136.34(d,JCF＝7.6Hz),135.50(d,JCF＝18.1Hz),133.92,133.15,123.56,122.30(d,JCF＝42.3Hz),121.42,120.44,119.73,116.23,113.89(d,JCF＝19.6Hz),111.15(d,JCF＝4.5Hz),105.99,79.79(d,JPC＝151.0Hz),61.26(d,JPC＝7.6Hz),60.79,36.04.
(1-([1,1'-联苯]-4-基)-2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-3-羟基丙-2-基)膦酸(化合物54)
合成方法参考实例45。产率：48％，淡黄色固体。m/z(ESI,-ve ion)＝650.1[M-H]-。1H NMR(600MHz,DMSO)δ8.08–8.00(m,2H),7.96(d,J＝1.9Hz,1H),7.70(d,J＝11.3Hz,1H),7.62(d,J＝7.9Hz,2H),7.51(d,J＝8.4Hz,2H),7.49–7.43(m,3H),7.40(t,J＝7.7Hz,2H),7.30(t,J＝7.4Hz,1H),5.34(d,J＝10.8Hz,1H),5.22(d,J＝10.9Hz,1H),3.78(dd,J＝12.0,6.0Hz,1H),3.69(dd,J＝15.6,11.6Hz,1H),3.31(dd,J＝14.1,11.1Hz,1H),3.24(dd,J＝14.0,8.6Hz,1H).13C NMR(151MHz,DMSO)δ152,.32(d,JCF＝261.2Hz),143.89,141.09,140.55,138.30,136.35(d,JCF＝6.0Hz),135.49(d,JCF＝18.1Hz),133.89,132.02,129.29,127.56,126.90,126.28,123.65,122.49,122.15(d,JCF＝9.1Hz),116.33,113.89(d,JCF＝19.6Hz),111.17,105.97,80.35,79.35,61.22(d,JPC＝107.2Hz),36.09.
(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-1-(4-氯苯基)-3-羟基丙-2-基)膦酸(化合物55)
合成方法参考实例45。产率：54％，淡黄色固体。m/z(ESI,-ve ion)＝608.1[M-H]-。1H NMR(600MHz,DMSO)δ8.07–8.01(m,2H),8.00–7.95(m,1H),7.70(dd,J＝9.8,1.4Hz,1H),7.49(dd,J＝8.5,1.8Hz,1H),7.41–7.35(m,2H),7.30–7.24(m,2H),5.29(dd,J＝10.1,0.5Hz,1H),5.17(dd,J＝11.0,1.2Hz,1H),3.75(dd,J＝11.7,8.8Hz,1H),3.69(dd,J＝14.1,11.7Hz,1H),3.26–3.21(m,1H),3.21–3.18(m,1H).13C NMR(151MHz,DMSO)δ152.33(d,JCF＝261.2Hz),143.84,141.09,136.35(d,JCF＝7.6Hz),136.16(d,JCF＝9.1Hz),135.52(d,JCF＝18.3Hz),133.93,133.26,131.22,127.86,123.61,122.44,122.17(d,JCF＝7.1Hz),116.21,113.90(d,JCF＝12.1Hz),111.18(d,JCF＝4.5Hz),105.99,79.80(d,JPC＝152.5Hz),61.20(d,JPC＝7.6Hz),60.79,36.10.
(2-((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)-1-(4-氰基苯基)-3-羟基丙-2-基)膦酸(化合物56)
合成方法参考实例45。
产率：56％，淡黄色固体。m/z(ESI,-ve ion)＝599.0[M-H]-。1H NMR(600MHz,DMSO)δ13.25(s,1H),8.15–7.90(m,3H),7.70(d,J＝9.9Hz,1H),7.49(d,J＝8.6Hz,3H),7.20(d,J＝8.4Hz,2H),5.32(d,J＝10.9Hz,1H),5.20(d,J＝10.9Hz,1H),3.80–3.74(m,1H),3.72–3.66(m,1H),3.26(dd,J＝10.7,4.9Hz,2H).13C NMR(151MHz,DMSO)δ152.33(d,JCF＝261.2Hz),147.27,143.81,141.09,136.69(d,JCF＝9.1Hz),136.34(d,JCF＝7.6Hz),135.50(d,JCF＝18.1Hz),133.92,133.15,123.56,122.30(d,JCF＝42.3Hz),121.42,120.44,119.73,116.23,113.89(d,JCF＝19.6Hz),111.15(d,JCF＝4.5Hz),105.99,79.79(d,JPC＝151.0Hz),61.26(d,JPC＝7.6Hz),60.79,36.04.
(4S)-2-(((6-(6-溴-4-氟-1H-苯并[d][1,2,3]三唑-1-基)-1H-吲唑-3-基)甲氧基)甲基)-4-(2-氯苯基)-1,3,2-二氧杂膦-2-氧化物(化合物57)
称取化合物x-1(120mg，0.26mmol)、(S)-1-(3-氯苯基)丙烷-1,3-二醇(147mg，0.79mmol)和二环己基甲烷二胺(163mg，0.79mmol)于25mL反应瓶中，加入5mL N,N-二甲基甲酰胺和1mL吡啶溶解后，于70℃反应4h，待反应完全后，旋干溶剂，加乙酸乙酯后有白色固体析出，抽滤后往滤液中滴加1M HCl溶液,用乙酸乙酯和饱和食盐水萃取3次，有机相用无水硫酸钠干燥，经PLC制备板分离得到白色固体粉末42mg，产率为26％。
1H NMR(600MHz,DMSO-d6)δ13.44(s,1H),8.11(d,J＝8.5Hz,1H),8.02(dd,J＝9.4,1.6Hz,2H),7.71(dd,J＝9.8,1.4Hz,1H),7.56(dd,J＝8.5,1.8Hz,1H),7.47(d,J＝2.0Hz,1H),7.40–7.32(m,3H),5.69(dt,J＝10.8,2.4Hz,1H),5.13–4.93(m,2H),4.54(tt,J＝11.2,3.7Hz,1H),4.38(dddd,J＝15.3,10.9,4.6,2.3Hz,1H),4.05(qd,J＝14.3,8.5Hz,2H),2.23–2.06(m,2H).LC-MS/ESI[M+H]+:606.00.
测试例：
生物活性测试
测试例1：本专利公开的CD73小分子化合物分子水平及细胞水平酶活测试方案
实验原理：AMP在CD73的催化下脱掉磷酸生成腺苷，磷酸根和钼酸根在酸性条件下生成磷钼杂多酸缔合物，然后又与孔雀绿形成的绿色复合物。酶的活力越高，产生的无机磷酸含量越高，波吸收的信号强度越强，加入抑制剂后，酶活力会有所减弱，由此可以间接反应抑制剂的抑制活性。
分子水平测活方案：
测活体系：
1.配孔雀绿显色剂。
Regent A与Regent B的比例为100:1，配制好以后避光稳定12小时以上。
2.配底物(AMP，250μM，约15×终浓度)
称量18.26mg AMP(M＝365.2)于烧杯中，用超纯水定容至200ml，搅拌使其完全溶解，然后每管1.5ml分装，保存于-20℃冰箱中。
3.配制分子水平测活缓冲液(0.22μm滤膜过滤)：
一般一次配制200ml。各成分如下：1mM CaCl2，1mM MgCl2，200mM NaCl，10mM KCl，100mM Tris-HCl，pH 8.0。
4.配制空白对照(0.5％DMSO buffer)
5.配药，待测化合物3倍梯度稀释，得到11个浓度。
5.稀释蛋白(15ng/ml)
6.铺板：样品孔和阳性孔每孔加入70μl蛋白，空白孔加70μl buffer。
7.样品孔加药，每孔加入5μl，室温孵育30min，阳性和空白孔加0.5％DMSO buffer。
8.加底物(AMP)在烘箱中孵育30min
9.加孔雀绿试剂室温显色30min
10.酶标仪620nm处读数
11.计算酶促反应初速度V0，抑制剂的活性测试则在上述反应中加入不同浓度的抑制剂，计算酶促反应初速度V1，化合物的抑制率则由公式(1-V1/V0)×100％计算。每次实验做三组平行对照。
细胞水平：
测活体系：
1.配孔雀绿显色剂。同分子水平。
2.配底物(AMP，250μM，约10×终浓度)。同分子水平。
3.配制细胞水平测活缓冲液(0.22μm滤膜过滤)：
一般一次配制200ml。各成分如下：20mM HEPES，pH7.4，137mM NaCl2，5.4mM KCl，1.3mM CaCl，4.2mM NaHCO3，0.1％glucose。
4.配空白对照(0.5％DMSO buffer)。
5.配药，待测化合物：3倍梯度稀释，得到11个浓度。
6.取细胞铺板：当MDA-MB-231的细胞密度达80％-90％、细胞活力达到95％以上时，即可进行化合物筛选。将细胞培养皿从二氧化碳细胞培养箱取出，弃去培养基，加入2mL D-hank’s清洗残留的培养基以及死细胞，弃去D-hank’s后，向细胞表面滴加500μL 0.25％胰酶，放入37℃细胞培养箱消化细胞一分钟，细胞消化下来以后加入2mL新鲜的1640完全培养基，用移液枪将细胞完全从细胞培养皿上吹打下来，将细胞悬液转移至15mL无菌离心管中，1000rpm离心三分钟。弃去培养基，加入4mL不含磷酸根的活性测试buffer重悬细胞，然后1000rpm离心三分钟，重复上述操作三次。最后一次洗涤完以后，加入1mL活性测试buffer重悬细胞，吹打均匀后取10μL细胞悬液到EP管中，加入10μL 0.4％台盼蓝染液吹打均匀，取10μL上述混合溶液到细胞计数板中，将细胞计数板插入细胞计数仪进行计数并记下细胞浓度。将确定了细胞密度的细胞悬液用活性测试buffer进行稀释，在96孔板中样品以及阳性对照孔每孔加入80μl，3000个细胞。空白孔加相应量的buffer。
7.样品孔每孔加药10μl，烘箱孵育30min，
8.每孔加底物(AMP)10μl，烘箱孵育30min
9.孵育结束后，用空白离心机230×g，离心5min，然后取上清转移到平底96孔板中，之后每孔加入20μl孔雀绿显色剂，室温显色30min。
10.酶标仪620nm处读数。
11.计算酶促反应初速度V0，抑制剂的活性测试则在上述反应中加入不同浓度的抑制剂，计算酶促反应初速度V1，化合物的抑制率则由公式(1-V1/V0)×100％计算。每次实验做三组平行对照。
CD73分子水平及细胞水平酶活测试结果(化合物34–41、44、46–47、49以三乙基铵盐进行了试验)活性水平以A、B、C、D表示，A:IC50≤0.5nM，B:0.5nM<IC50≤1nM，C:1nM<IC50≤100nM，D:IC50>100nM。








测试例2:体内药效学评价
本发明对CD73在分子水平或细胞水平酶活测试结果为B或B以上的化合物进行体内药效学性质评价，发现这些化合物均具备优异的体内药效学性质。
1扩增E.G7-OVA小鼠T淋巴瘤细胞
将本课题组的细胞库中冻存的小鼠T淋巴瘤细胞(E.G7-OVA)进行复苏。首先打开水浴锅，等温度升至37℃，立即将细胞放入，水浴约5分钟。水浴的同时，打开生物安全柜，准备好复苏要用的RPIM1640完全培养基，并转移4mL培养基到15mL尖底离心管中。待细胞液融化后，喷洒酒精后放入生物安全柜，用移液枪将细胞液转移到加有培养基的15ml离心管中，用1mL移液枪缓慢吹打混匀，然后1000rpm离心3分钟。弃去上清，向细胞沉淀中加入1mL完全培养基重悬。根据冻存细胞多少，转移到合适大小的培养皿，补加合适体积的培养基。然后将培养皿呈十字型晃动使细胞在培养皿中均匀分布。随后，将培养皿置于细胞培养箱中进行培养。2天后，观察细胞状态，对细胞进行换液或传代。待细胞长势较好时，按照2天一传、1:3的比例进行传代。
2造模(E.G7-OVA皮下瘤模型)
(1)制备肿瘤细胞悬液：事先将购买的基质胶放在4℃冰箱化冻并同时放一盒已灭菌的1mL枪头盒以预冷，将扩增了足够数量的细胞通过离心弃上清，收集起来，用无血清RPIM1640培养基重悬，再离心，如此重复洗涤3遍以去掉完全培养基中的FBS。最后一遍弃掉上清后，用适当体积的无血清RPIM1640培养基重悬，用移液枪混匀。取10μL转移到1.5mL离心管中，然后向离心管中加入10μL台盼蓝混匀，取10μL注入到细胞计数板中，随后将计数板插入细胞计数仪中，等聚焦后，开始计数。根据计数密度，再用培养基调整细胞密度到目标密度的2倍，然后加入等体积的基质胶，吹打混匀。将制备好的细胞悬液置于冰上，以备接瘤；
(2)接瘤：接瘤时采用1mL无菌注射器，首先将细胞悬液转移到注射器中，赶走气泡，然后向C57BL/6小鼠的右侧颈背部皮下注射100μL细胞悬液。通过课题组前期该模型构造经验，每只小数接种5×104个E.G7-OVA小鼠淋巴瘤细胞。接瘤后，每天观察小鼠状态，当肿瘤达到约100mm3时，进行给药。在整个研究过程中每隔一天测量一次体重和肿瘤体积。肿瘤生长抑制率(TGI)按公式计算：TGI(％)＝[1-(Ti-T0)/(Ci-C0)]×100，其中Ti为某天给药组化合物治疗后的平均肿瘤体积，T0为给药组初始平均肿瘤体积，Ci为某天对照组溶剂治疗后的平均肿瘤体积，C0为对照组初始平均肿瘤体积。
3药物处理方案
各个化合物的给药方案及剂量如下表所示
各给药组体内抗肿瘤效果汇总如下表所示

4.体内药效结论：专利所述化合物31、35与PD-1抗体RMP1-14联用可显著抑制T淋巴瘤异植小鼠肿瘤模型的体内增殖，其它高活性化合物也表现出良好的体内药效学活性。
测试例3
本发明对CD73在分子水平或细胞水平酶活测试结果为B或B以上的化合物进行了细胞毒性评价，发现这些化合物均具备较好的安全性和成药性。
化合物16、31、33在100nM的水平对人胚胎肾细胞293(HEK293)、正常人肝细胞(L-02)未表现出显著的细胞毒性，表明该系列化合物具有较好的安全性和成药性。
因此，经分子水平活性测试后，本发明的化合物是能够良好抑制CD73的化合物，为由CD73介导的癌症的治疗奠定了基础。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (11)

  1. 通式I所示的化合物或其药学上可接受的盐：
    其中，
    A为任选取代的苯环、含氮、氧和硫的5或6元杂环；
    M为任选取代的C8-C12芳基或杂芳基或杂环基；
    Z1选自下组：O、S、CR1R2或NR3，其中R1、R2和R3独立选自：氢、卤素、任选取代的C1-C10烷基、任选取代的C3-C8环烷基、任选取代的C5-C6芳基、任选取代的含有1或2个独立选自N、O或S的杂原子的C5-C6杂环基；
    Z2选自下组：CR4或N，其中R4为氢、卤素、任选取代的C1-C10烷基、任选C3-C8环烷基、任选取代的C5-C6芳基、任选取代的含有1或2个独立选自N、O或S的杂原子的C5-C6杂环基；
    X选自CR5R6、NR7、O、S、羰基、砜基或亚砜基，其中R5、R6和R7独立选自：H、卤素、任选取代的C1-C10烷基、任选取代的C3-C8环烷基；
    Y选自CR8R9、NR10、O、S、羰基、砜基或亚砜基，其中R8、R9和R10独立选自：H、卤素、任选取代的C1-C10烷基、任选取代的C3-C8环烷基；CH2、NH、O、S、羰基、砜基或亚砜基；
    Q1和Q2各自独立选自下组：氢、任选取代的C1-C10烷基(包括但不限于羟甲基、羟乙基、任选取代的(例如氘代的)C1-C3烷氧基甲基、任选取代的C1-C3烷氧基乙基)、任选取代的C3-C8环烷基、任选取代的C5-C6芳基、任选取代的C5-C6杂环基、任选取代的苯基C1-C5烷基、任选取代的含有1、2、3或4个独立选自N、O或S的杂原子的C5-C6杂环基C1-C5烷基、羟基、任选取代的C1-C3甲酰基(包括但不限于羟基甲酰基、C1-C3烷氧基甲酰基)；
    W1和W2独立选自：NH、O或S；
    P1为H、任选取代的C8-C12芳基及任选取代的C8-C12芳杂环基、任选取代的C1-C10烷基、任选取代的C3-C8环烷基、任选取代的C1-C10烷基甲酰氧基甲基；或者，W1P1整体构成以磷酰胺键相连的氨基酸、以磷酰胺键相连的酯化氨基酸、磷酸前药基团；
    P2为H、任选取代的C8-C12芳基及任选取代的C8-C12芳杂环基、任选取代的C1-C10烷基、任选取代的C3-C8环烷基、任选取代的C1-C10烷基甲酰氧基甲基；W2P2整体构成以磷酰胺键相连的氨基酸、以磷酰胺键相连的酯化氨基酸、磷酸前药基团；
    或者P1、P2可以形成任选取代的5-8元环醚、内酯、内酰胺。
  2. 如权利要求1所述的化合物或其药学上可接受的盐，其特征在于，所述化合物如通式II所示：
    其中，M、Z1、Z2、X、Y、Q1、Q2、W1、W2、P1、P2如权利要求1所述。
  3. 如权利要求2所述的化合物或其药学上可接受的盐，其特征在于，所述化合物如通式Ⅲ所示：
    其中，M、Q1、Q2、W1、W2、P1、P2如权利要求2所述。
  4. 如权利要求3所述的化合物或其药学上可接受的盐，其特征在于，所述化合物如通式Ⅳ所示：
    其中，B为任选取代的C5-C6芳环、任选取代的含N、O或S的C5-C6杂环；
    A1、A2、A3、A4、A5各自独立选自CH或N；
    R3选自：H、卤素(F、Cl或Br)、硝基、氰基、任选取代的C1-C10烷基(例如三氟甲基、羟甲基)、任选取代的C3-C8环烷基、任选取代的C5-C6杂环基C1-C5烷基(例如苄基)、任选取代的C1-C5烷氧基、任选取代的C1-C5烷氧基甲酰基、任选取代的氨基、任选取代的C1-C5烷基甲酰氨基；
    n选自1-5，优选1-3的整数；
    Q1、Q2、W1、W2、P1、P2如权利要求3所述。
  5. 如权利要求4所述的化合物或其药学上可接受的盐，其特征在于，所述化合物如通式Ⅴ所示：
    其中，A1、R3、Q1、Q2、W1、W2、P1、P2和n如权利要求4所述。
  6. 选自下组的化合物或其药学上可接受的盐：



    优选地，所述化合物选自以下化合物：

  7. 一种药物组合物，所述药物组合物含有权利要求1-6中任一项所述的化合物或其药学上可接受的盐，以及药学上可接受的载体或赋形剂。
  8. 权利要求1-6中任一项所述的化合物在制备CD73抑制剂中的用途。
  9. 如权利要求8所述的用途，其特征在于，所述CD73抑制剂是治疗或预防CD73介导的疾病，或抑制CD73的药物。
  10. 如权利要求9所述的用途，其特征在于，所述CD73介导的疾病为癌症。
  11. 如权利要求10所述的用途，其特征在于，所述癌症选自下组：所述癌症包括但不限于乳腺癌、多发性骨髓瘤、膀胱癌作为优选，所述癌症选自乳腺癌、多发性骨髓瘤、膀胱癌、子宫内膜癌、胃癌、宫颈癌、横纹肌肉瘤、非小细胞肺癌、小细胞肺癌、多形性肺癌、卵巢癌、食管癌、黑色素瘤、结肠直肠癌、肝细胞瘤、头颈部肿瘤、肝胆管细胞癌、骨髓增生异常综合征、恶性胶质瘤、前列腺癌、甲状腺癌、徐旺氏细胞瘤、肺鳞状细胞癌、苔薛样角化病、滑膜肉瘤、皮肤癌、腺癌、睾丸癌或脂肪肉瘤。
PCT/CN2025/095011 2024-05-23 2025-05-15 作为cd73抑制剂衍生物及其应用 Pending WO2025241971A1 (zh)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6017903A (en) * 1996-09-27 2000-01-25 Guilford Pharmaceuticals Inc. Pharmaceutical compositions and methods of treating a glutamate abnormality and effecting a neuronal activity in an animal using NAALADase inhibitors
WO2001002411A1 (en) * 1999-07-06 2001-01-11 Methylgene Inc. SULFONAMIDOMETHYL PHOSPHONATE INHIBITORS OF β-LACTAMASE
CN117777107A (zh) * 2023-12-26 2024-03-29 华东理工大学 Cd73抑制性化合物及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6017903A (en) * 1996-09-27 2000-01-25 Guilford Pharmaceuticals Inc. Pharmaceutical compositions and methods of treating a glutamate abnormality and effecting a neuronal activity in an animal using NAALADase inhibitors
WO2001002411A1 (en) * 1999-07-06 2001-01-11 Methylgene Inc. SULFONAMIDOMETHYL PHOSPHONATE INHIBITORS OF β-LACTAMASE
CN117777107A (zh) * 2023-12-26 2024-03-29 华东理工大学 Cd73抑制性化合物及其应用

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