WO2020114477A1 - 作为免疫调节剂的化合物及其制备方法和应用 - Google Patents

作为免疫调节剂的化合物及其制备方法和应用 Download PDF

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WO2020114477A1
WO2020114477A1 PCT/CN2019/123514 CN2019123514W WO2020114477A1 WO 2020114477 A1 WO2020114477 A1 WO 2020114477A1 CN 2019123514 W CN2019123514 W CN 2019123514W WO 2020114477 A1 WO2020114477 A1 WO 2020114477A1
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陆洪福
邢唯强
彭建彪
郭海兵
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上海济煜医药科技有限公司
江西济民可信集团有限公司
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Priority to CN201980009309.8A priority Critical patent/CN111630029B/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D205/00—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom
    • C07D205/02—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings
    • C07D205/04—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • 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
    • A61K31/397—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having four-membered rings, e.g. azetidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/10—Anti-acne agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to compounds represented by formula (II) and their pharmaceutically effective salts, and the use of the compounds as S1P1 agonists.
  • Sphingosine-1-phosphate is an amphoteric biological signaling molecule belonging to lysophospholipid (LP). S1P can activate complex downstream signals by acting on five G protein-coupled receptor subtypes, sphingosine-1-phosphate receptors (S1PR 1-5 ), thereby regulating important physiological and biochemical functions. S1P binds to different S1P receptors to regulate different physiological functions, and plays an important role in maintaining the health of the body and the occurrence of diseases.
  • LP lysophospholipid
  • S1P1 receptor agonists interfere with lymphocyte traffic king, sequestering them in lymph nodes and other secondary lymphoid tissues. This leads to a decrease in peripheral circulating lymphocytes, and the clinical value of lymphocyte isolation is to exclude them from the sight of inflammation and/or autoimmune reactions in surrounding tissues.
  • This isolation of lymphocytes (for example, in lymph nodes) is believed to be the result of the simultaneous action of: agonist-driven functional antagonism of S1P1 receptors on T cells (thus reducing the outflow of S1P mobilized T cells from lymph nodes Ability) and sustained agonism of the S1P1 receptor on the lymph node endothelium (thereby improving the barrier function against lymphocyte migration). Therefore, S1P 1 receptor agonists reduce the body's autoimmunity by preventing the transport of lymphocytes, and thus can be used as an immunosuppressant to treat various body immune diseases.
  • S1P1 agonist Fingolimod Fingolimod, FTY720
  • Fingolimod FTY720
  • MS Multiple Scleorosis
  • FTY720 is a non-selective S1P receptor agonist.
  • the combination of FTY720 and S1P3 in the body often leads to a series of important side effects, such as bradycardia, which greatly limits its therapeutic immunity
  • the scope of application in the field of disease Therefore, the discovery of the second generation of highly selective S1P1 agonists, making it a therapeutic drug for immune diseases with better efficacy, fewer side effects, and wider application has become one of the hot spots in drug research.
  • shortening the half-life of S1P1 receptor agonists in the body is also the goal of discovering second-generation S1P agonists.
  • a longer half-life will result in the continued suppression of lymphocyte trafficking and a decrease in the number of peripheral blood lymphocytes, thereby making the drug user's immune function low and increasing the risk of viral infection.
  • the half-life of S1P1 receptor agonists such as FTY720 in the human body is as long as 6 to 9 days, so even if the drug is stopped, it will take a long time for the number of lymphocytes to return to normal.
  • the present invention provides a compound represented by formula (II) or a pharmaceutically acceptable salt thereof,
  • n is selected from 1, 2, and 3;
  • n is selected from 1 and 2;
  • y is selected from 1 and 2;
  • R 1 is selected from H, halogen, OH, NH 2 , CN, C 1-6 alkyl, C 3-6 cycloalkyl, 3-7 membered heterocycloalkyl, and C 1-6 heteroalkyl, the C 1-6 alkyl, C 3-6 cycloalkyl, 3-7 membered heterocycloalkyl or C 1-6 heteroalkyl are optionally substituted with 1, 2 or 3 R;
  • R 2 is selected from H, halogen, OH, NH 2 , CN and C 1-6 alkyl, the C 1-6 alkyl is optionally substituted by 1, 2 or 3 R;
  • Ring A is selected from C 3-7 cycloalkyl, 3-7 membered heterocycloalkyl and C 3-7 cycloalkenyl, said C 3-7 cycloalkyl, 3-7 membered heterocycloalkyl or C 3 -7 cycloalkenyl is optionally substituted with 1, 2 or 3 R;
  • L 1 is selected from
  • L 2 is selected from single bonds, O and S;
  • T 1 is selected from N and CH;
  • T 2 is selected from N and CH;
  • T 3 is selected from N and CH;
  • R is independently selected from H, F, Cl, Br, I, OH, NH 2 , C 1-3 alkyl, and CF 3 ;
  • the C 1-6 heteroalkyl group and the 3-7 membered heterocycloalkyl group contain 1, 2, or 3 heteroatoms or heteroatom groups independently selected from NH, O, and S.
  • the aforementioned R is independently selected from H, F, Cl, Br, I, OH, NH 2 , CH 3 , CH 2 CH 3 and CF 3 , and other variables are as defined in the present invention.
  • the above R 1 is selected from H, F, Cl, Br, OH, NH 2 , CN, C 1-3 alkyl, C 3-6 cycloalkyl, and 3-7 membered heterocycloalkyl And C 1-3 alkoxy, the C 1-3 alkyl, C 3-6 cycloalkyl, 3-7 membered heterocycloalkyl or C 1-3 alkoxy is optionally substituted by 1, 2 or 3 R is substituted, other variables are as defined in the present invention.
  • R 1 is selected from H, F, Cl, Br, OH, NH 2 , CN, CH 3 , CH 2 CH 3 , CF 3 , Other variables are as defined in the present invention.
  • R 2 is selected from H, F, Cl, Br, OH, NH 2 , CN, CH 3 and CH 2 CH 3 , the CH 3 or CH 2 CH 3 is optionally Or 3 R substitutions, other variables are as defined in the present invention.
  • R 2 is selected from H, F, Cl, Br, OH, NH 2 , CN, CH 3 , CH 2 CH 3 and CF 3 , and other variables are as defined in the present invention.
  • the above L 1 is selected from Other variables are as defined in the present invention.
  • the aforementioned ring A is selected from Said It can be optionally substituted with 1, 2 or 3 R, and other variables are as defined in the present invention.
  • the aforementioned ring A is selected from Other variables are as defined in the present invention.
  • the above compound or a pharmaceutically acceptable salt thereof is selected from
  • n T 1 , T 2 , T 3 , R, L 2 , R 1 , R 2 and ring A are as defined above.
  • the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
  • n is selected from 1, 2, and 3;
  • n is selected from 1 and 2;
  • R 1 is selected from H, halogen, OH, NH 2 , CN, C 1-6 alkyl, C 3-6 cycloalkyl, 3-7 membered heterocycloalkyl, and C 1-6 heteroalkyl, the C 1-6 alkyl, C 3-6 cycloalkyl, 3-7 membered heterocycloalkyl or C 1-6 heteroalkyl are optionally substituted with 1, 2 or 3 R;
  • R 2 is selected from H, halogen, OH, NH 2 , CN and C 1-6 alkyl, the C 1-6 alkyl is optionally substituted by 1, 2 or 3 R;
  • Ring A is selected from C 3-7 cycloalkyl, 3-7 membered heterocycloalkyl and C 3-7 cycloalkenyl, said C 3-7 cycloalkyl, 3-7 membered heterocycloalkyl or C 3 -7 cycloalkenyl is optionally substituted with 1, 2 or 3 R;
  • L 1 is selected from
  • L 2 is selected from single bonds, O and S;
  • T 1 is selected from N and CH;
  • T 2 is selected from N and CH;
  • T 3 is selected from N and CH;
  • R is independently selected from H, F, Cl, Br, I, OH, NH 2 , C 1-3 alkyl, and CF 3 ;
  • the C 1-6 heteroalkyl group and the 3-7 membered heterocycloalkyl group contain 1, 2, or 3 heteroatoms or heteroatom groups independently selected from NH, O, and S.
  • the aforementioned R is independently selected from H, F, Cl, Br, I, OH, NH 2 , CH 3 , CH 2 CH 3 and CF 3 , and other variables are as defined in the present invention.
  • the above R 1 is selected from H, F, Cl, Br, OH, NH 2 , CN, C 1-3 alkyl, C 3-6 cycloalkyl, and 3-7 membered heterocycloalkyl And C 1-3 alkoxy, the C 1-3 alkyl, C 3-6 cycloalkyl, 3-7 membered heterocycloalkyl or C 1-3 alkoxy is optionally substituted by 1, 2 or 3 R is substituted, other variables are as defined in the present invention.
  • R 1 is selected from H, F, Cl, Br, OH, NH 2 , CN, CH 3 , CH 2 CH 3 , CF 3 , Other variables are as defined in the present invention.
  • R 2 is selected from H, F, Cl, Br, OH, NH 2 , CN, CH 3 and CH 2 CH 3 , the CH 3 or CH 2 CH 3 is optionally Or 3 R substitutions, other variables are as defined in the present invention.
  • R 2 is selected from H, F, Cl, Br, OH, NH 2 , CN, CH 3 , CH 2 CH 3 and CF 3 , and other variables are as defined in the present invention.
  • the above L 1 is selected from Other variables are as defined in the present invention.
  • the aforementioned ring A is selected from Said It can be optionally substituted with 1, 2 or 3 R, and other variables are as defined in the present invention.
  • the aforementioned ring A is selected from Other variables are as defined in the present invention.
  • the present invention also provides a compound of the formula or a pharmaceutically acceptable salt thereof,
  • the present invention also provides a pharmaceutical composition containing the above compound or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the present invention also provides the use of the above compound or a pharmaceutically acceptable salt thereof or the above pharmaceutical composition in the preparation of a medicament for preventing and/or treating S1P1 receptor-related diseases.
  • the above uses, wherein the S1P1 receptor-related diseases are selected from Ulcerative Colitis, Crohn's Disease, Multiple Sclerosis, Systemic Lupus erythematosus (Systemic lupus erythematosus), lupus nephritis (Lupus nephritis), rheumatoid arthritis (Rheumatoid arthritis), primary biliary cholangitis (Primary) Biliary Cholangitis, allergic dermatitis (Atopic Dermatitis), cerebral hemorrhage ( Intracerebral, Hemorrhage, Graft Versus Host Disease, Psoriasis, Type I Diabetes, Acne, Microbial Infection or Microbial Disease and Viral Infection or Viral Disease.
  • S1P1 receptor-related diseases are selected from Ulcerative Colitis, Crohn's Disease, Multiple Sclerosis, Systemic Lupus erythematosus (Systemic lupus erythematosus),
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms, which are within the scope of reliable medical judgment and are suitable for use in contact with human and animal tissues Without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salt refers to a salt of a compound of the present invention, prepared from a compound having a specific substituent and a relatively non-toxic acid or base found in the present invention.
  • base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts.
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in solution or a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Bisulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, trifluoroacetic acid, maleic acid, malonic acid, benzoic acid, succinic acid, Suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; also includes amino acids (such as arginine Etc.), and salts of organic acids such as glucuronic acid. Certain compounds of the present invention contain basic and acidic functional groups and can be converted to any
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing acid radicals or bases by conventional chemical methods. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.
  • the compounds of the present invention may exist in specific geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers Isomers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, all of which belong to this Within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in the substituents such as alkyl. All these isomers and mixtures thereof are included in the scope of the present invention.
  • tautomer or “tautomeric form” means that at room temperature, isomers of different functional groups are in dynamic equilibrium and can quickly convert to each other. If tautomers are possible (as in solution), the chemical equilibrium of tautomers can be achieved.
  • proton tautomers also known as prototropic tautomers
  • proton tautomers include interconversion through proton migration, such as keto-enol isomerization and imine-ene Amine isomerization.
  • Valence tautomer (valence tautomer) includes some recombination of bond-forming electrons for mutual conversion.
  • keto-enol tautomerization is the interconversion between two tautomers of pentane-2,4-dione and 4-hydroxypent-3-en-2-one.
  • the compound of the present invention may contain unnatural proportions of atomic isotopes in one or more atoms constituting the compound.
  • compounds can be labeled with radioisotopes, such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C).
  • the hydrogen can be replaced by heavy hydrogen to form a deuterated drug.
  • the bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have reduced toxic and side effects and increased drug stability. , Strengthen efficacy, prolong the biological half-life of drugs and other advantages.
  • substituted means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include heavy hydrogen and hydrogen variants, as long as the valence state of the particular atom is normal and the compound after substitution is stable of.
  • substituent which may include heavy hydrogen and hydrogen variants, as long as the valence state of the particular atom is normal and the compound after substitution is stable of.
  • optionally substituted means that it may or may not be substituted. Unless otherwise specified, the type and number of substituents may be arbitrary on the basis that they are chemically achievable.
  • any variable (such as R) appears more than once in the composition or structure of a compound, its definition in each case is independent.
  • R when any variable (such as R) appears more than once in the composition or structure of a compound, its definition in each case is independent.
  • the group can be optionally substituted with at most two Rs, and R in each case has independent options.
  • combinations of substituents and/or variants thereof are only allowed if such combinations will produce stable compounds.
  • substituents listed do not indicate which atom they are connected to the substituted group, such substituents can be bonded through any of their atoms, for example, pyridyl as a substituent can be through any one of the pyridine rings The carbon atom is attached to the substituted group.
  • connection direction is arbitrary, for example,
  • the linking group L in the middle is -CH 2 O-, then -CH 2 O- can be formed by connecting phenyl and cyclopentyl in the same direction as the reading order from left to right It can also be formed by connecting phenyl and cyclopentyl in the opposite direction to the reading order from left to right
  • Combinations of the linking groups, substituents, and/or variants thereof are only allowed if such combinations will produce stable compounds.
  • the number of atoms on a ring is usually defined as the number of members of the ring.
  • “3-6 membered ring” refers to a “ring” in which 3-6 atoms are arranged around.
  • C 1-6 alkyl is used to indicate a linear or branched saturated hydrocarbon group composed of 1 to 6 carbon atoms.
  • the C 1-6 alkyl group includes C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-4 , C 6 and C 5 alkyl groups; etc.; Is monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine).
  • C 1-6 alkyl examples include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl , S-butyl and t-butyl), pentyl (including n-pentyl, isopentyl and neopentyl), hexyl and so on.
  • C 1-3 alkyl is used to indicate a linear or branched saturated hydrocarbon group composed of 1 to 3 carbon atoms.
  • the C 1-3 alkyl group includes C 1-2 and C 2-3 alkyl groups, etc.; it may be monovalent (such as methyl), divalent (such as methylene), or polyvalent (such as methine) .
  • Examples of C 1-3 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.
  • heteroalkyl by itself or in combination with another term means a stable linear or branched alkyl radical consisting of a certain number of carbon atoms and at least one heteroatom or heteroatom group or a combination thereof.
  • the heteroatom is selected from B, O, N, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized.
  • the heteroalkyl is a C 1-6 heteroalkyl; In other embodiments, the heteroalkyl is a heteroalkyl C 1- 3.
  • heteroatom or heteroatom group can be located at any internal position of the heteroalkyl group, including the attachment position of the alkyl group to the rest of the molecule, but the term "alkoxy" is a conventional expression and refers to the connection to the rest of the molecule through an oxygen atom Those alkyl groups.
  • C 1-3 alkoxy refers to those alkyl groups containing 1 to 3 carbon atoms connected to the rest of the molecule through one oxygen atom.
  • the C 1-3 alkoxy group includes C 1-2 , C 2-3 , C 3 and C 2 alkoxy groups and the like.
  • Examples of C 1-3 alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.
  • C 3-7 cycloalkyl means a saturated cyclic hydrocarbon group consisting of 3 to 7 carbon atoms, which is a monocyclic and bicyclic ring system, and the C 3-7 cycloalkyl includes C 4-7 , C 5-7 , C 3-5 , C 4-5 and C 5-6 cycloalkyl, etc.; it may be monovalent, divalent or multivalent.
  • Examples of C 3-6 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
  • the term "3-7 membered heterocycloalkyl" by itself or in combination with other terms means a saturated cyclic group consisting of 3 to 7 ring atoms with 1, 2, 3 or 4 ring atoms Are heteroatoms independently selected from O, S, and N, and the rest are carbon atoms, wherein nitrogen atoms are optionally quaternized, and nitrogen and sulfur heteroatoms may be optionally oxidized (ie, NO and S(O) p , p Is 1 or 2). It includes single-ring and double-ring systems, wherein the double-ring system includes spiro ring, parallel ring and bridge ring.
  • the hetero atom may occupy the connection position of the heterocyclic alkyl group to the rest of the molecule.
  • the 3-7 membered heterocycloalkyl group includes 3-6 membered, 4-7 membered, 4 membered, 5 membered, 6 membered, and 7 membered heterocycloalkyl groups.
  • 3-7 membered heterocycloalkyl examples include, but are not limited to, azetidinyl, oxetanyl, thiatanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl ( (Including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl, etc.), tetrahydrofuranyl (including tetrahydrofuran-2-yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2- Piperidinyl and 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), Dioxanyl, dithianyl, isoxazolidinyl, isothiazolidinyl,
  • C 3-7 cycloalkenyl means a partially unsaturated cyclic hydrocarbon group consisting of 3 to 7 carbon atoms containing at least one carbon-carbon double bond, which includes monocyclic and bicyclic System, in which the bicyclic system includes spiro ring, bicyclic ring and bridge ring, any ring in this system is non-aromatic.
  • the C 3-7 cycloalkenyl group includes C 3-6 , C 3-5 , C 4-7 , C 4-8 , C 4-6 , C 4-5 , C 5-7 or C 5-6 rings Alkenyl and the like; it can be monovalent, divalent or multivalent.
  • C 3-8 cycloalkenyl examples include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like.
  • C n-n+m or C n -C n+m includes any specific case of n to n+m carbons, for example, C 1-12 includes C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , and C 12 , and also includes any range from n to n+m, for example, C 1- 12 includes C 1-3 , C 1-6 , C 1-9 , C 3-6 , C 3-9 , C 3-12 , C 6-9 , C 6-12 , and C 9-12, etc.; similarly, n yuan to n +m member means that the number of atoms in the ring is n to n+m, for example, 3-12 member ring includes 3 member ring, 4 member ring, 5 member ring, 6 member ring, 7 member ring, 8 member ring, 9 member ring , 10-membered ring, 11-membered
  • leaving group refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (eg, an affinity substitution reaction).
  • substituent groups include triflate; chlorine, bromine, and iodine; sulfonate groups such as mesylate, tosylate, p-bromobenzenesulfonate, and p-toluenesulfonate Ester, etc.; acyloxy, such as acetoxy, trifluoroacetoxy, etc.
  • protecting group includes but is not limited to "amino protecting group", “hydroxy protecting group” or “mercapto protecting group”.
  • amino protecting group refers to a protecting group suitable for preventing side reactions at the amino nitrogen position.
  • Representative amino protecting groups include, but are not limited to: formyl; acyl, such as alkanoyl (such as acetyl, trichloroacetyl, or trifluoroacetyl); alkoxycarbonyl, such as tert-butoxycarbonyl (Boc) ; Arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-di -(4'-methoxyphenyl) methyl; silyl, such as trimethylsilyl (TMS) and tert-butyld
  • hydroxyl protecting group refers to a protecting group suitable for preventing side reactions of hydroxyl groups.
  • Representative hydroxy protecting groups include, but are not limited to: alkyl groups, such as methyl, ethyl, and tert-butyl; acyl groups, such as alkanoyl groups (such as acetyl); arylmethyl groups, such as benzyl (Bn), p-methyl Oxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl, such as trimethylsilyl (TMS) and tert-butyl Dimethylsilyl (TBS) and so on.
  • alkyl groups such as methyl, ethyl, and tert-butyl
  • acyl groups such as alkanoyl groups (such as acetyl)
  • arylmethyl groups such as benzyl (Bn), p-methyl Oxybenzyl (
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by the combination with other chemical synthesis methods and well known to those skilled in the art Equivalently, preferred embodiments include but are not limited to the embodiments of the present invention.
  • the solvent used in the present invention is commercially available.
  • DIAD diisopropyl azodicarboxylate
  • Xphos stands for 2-dicyclohexylphosphonium-2,4,6-triisopropylbiphenyl
  • LiHMDS stands for hexamethyldisilazyl Lithium amide
  • NIS stands for N-iodosuccinimide
  • Pd 2 (dba) 3 stands for tris(dibenzylideneacetone) dipalladium
  • THF stands for tetrahydrofuran
  • DIAD stands for diisopropyl azodicarboxylate
  • PPh 3 represents triphenylphosphine
  • AntPhos represents 4-(9-anthracenyl)-3-(tert-butyl)-2,3-dihydrobenzo[d][1,3]oxygen, phosphinopentyl conjugate
  • DIEA represents N,N-diisopropylethylamine
  • HOA represents N,N
  • Figure 1 shows the effect of compound 2 in the rat peripheral blood lymphocyte (PBL) reduction test.
  • reaction solution was quenched with 200 ml of water, 150 mL of ethyl acetate was added, the organic layer was separated by extraction, and the aqueous layer was further extracted with ethyl acetate (150 mL ⁇ 2). The combined organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a residue. The residue was purified by silica gel chromatography to obtain intermediate I-6.
  • the residue was diluted with ethyl acetate (30 mL), washed with water (15 mL) and saturated brine (30 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product.
  • the crude product was purified by silica gel chromatography to obtain the target compound I-22.
  • triphenylphosphine (1.13g, 4.31mmol) was dissolved in tetrahydrofuran (6mL), followed by diisopropyl azodicarboxylate (578mg, 2.86mmol), intermediate 1-27 (421mg, 1.74 mmol) and 2-fluoro-4-hydroxybenzaldehyde (200 mg, 1.43 mmol).
  • the reaction mixture was stirred at room temperature overnight under the protection of argon.
  • the reaction mixture was added with water (20 mL) and extracted with ethyl acetate (60 mL), the aqueous phase was extracted with ethyl acetate (60 mL ⁇ 2), and the organic phases were combined.
  • intermediate I-29 (900 mg, 3.17 mmol) and Pd/C (10% w/w, 90 mg) were added to a 100 mL stainless steel reactor, followed by methanol (10.0 mL). Replace the gas in the cylinder with hydrogen and pressurize the hydrogen to 40 atm. The reaction mixture was heated to 50°C under this pressure, and the reaction was stirred at this temperature for 10 hours. After cooling the reaction solution to room temperature, it was filtered through celite, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain the target compound I-30, which was directly used in the next reaction without purification.
  • intermediate I-30 700 mg, 2.45 mmol was dissolved in anhydrous tetrahydrofuran (10.0 mL). After the reaction solution was lowered to 0°C under the protection of nitrogen, lithium tetrahydroaluminum (1M THF solution) (6.13mL, 6.13mmol) was slowly added dropwise. After the dropwise addition, the reaction mixture was raised to room temperature, and the reaction was stirred at room temperature for 10 hours. Diethyl ether (10.0 mL) was added to the reaction mixture and cooled to 0° C., water (4.00 mL) was added, and then aqueous sodium hydroxide solution (6.00 mL, 10% w/w, aq.) was added.
  • methyl 4-bromo-3-(trifluoromethyl)benzoate 100 mg, 0.35 mmol
  • cyclobutylboronic acid 70 mg, 0.70 mmol
  • Palladium acetate 4.5 mg, 0.02 mmol
  • AntPhos 11.1 mg, 0.03 mmol
  • potassium phosphate 297 mg, 1.40 mmol
  • lithium tetrahydroaluminum 291.4 mg, 7.68 mmol was added in batches to a solution of intermediate 1-33 (660 mgl) in tetrahydrofuran (15 mL).
  • the reaction mixture was reacted at room temperature for 16 hours.
  • the reaction solution was cooled to 0°C, and water (2 mL) was added to quench the reaction.
  • the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product.
  • the crude product was isolated and purified by reverse phase preparation to obtain intermediate 1-34.
  • triphenylphosphine 115 mg, 0.44 mmol was added to a solution (5 mL) of intermediate 1-34 (50 mg, 0.22 mmol) and 2-fluoro-4-hydroxybenzaldehyde (46 mg, 0.33 mmol) in tetrahydrofuran. in.
  • the reaction system was replaced with argon three times.
  • Diisopropyl azodicarboxylate 90 mg, 0.44 mmol was slowly added dropwise to the above reaction solution at 0°C under the protection of argon, and reacted at this temperature for 30 minutes. Then the reaction system was warmed to room temperature and reacted for 5 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude product.
  • the crude product was purified by silica gel chromatography to obtain intermediate I-35.
  • reaction solution was diluted with water (10 mL) and extracted with ethyl acetate (10 mL ⁇ 3), and the organic phases were combined.
  • the organic phase was washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain intermediate I-36, and the product was directly used in the next reaction without purification.
  • the reaction solution was cooled to room temperature, water (50 mL) and ethyl acetate (50 mL) were added in sequence, the layers were separated, the organic phase was separated, the aqueous phase was extracted with ethyl acetate (50 mL ⁇ 2), the organic phases were combined, and saturated common salt was used It was washed with water (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography to obtain intermediate I-37.
  • the intermediate I-48 (380mg, 1.39mmol) was dissolved in tetrahydrofuran (5mL), the reaction system was cooled to -40 °C, under the protection of argon, diisobutyl aluminum hydride in toluene solution (1.5M, 3.71mL, 5.56mmol). After the addition was completed, the reaction solution was stirred at -40°C for half an hour, and then stirred at room temperature overnight. The reaction solution was cooled to 0°C, slowly quenched with water (1 mL), the mixture was poured into water (50 mL), and extracted with ethyl acetate (20 mL ⁇ 3).
  • intermediate I-55 (3.5 g, 12.23 mmol) was dissolved in ethanol (80 mL), and palladium on carbon (100 mg, 10% w/w) was slowly added.
  • the reaction mixture was stirred at 60°C and 10 atmospheres for 72 hours. After cooling the reaction solution to room temperature, the palladium carbon in the mixture was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a crude product.
  • the crude product was separated and purified by silica gel chromatography to obtain the target intermediate 1-56.
  • intermediate I-57 200 mg, 0.768 mmol
  • 2-fluoro-4-hydroxybenzaldehyde 161 mg, 1.15 mmol
  • triphenylphosphine 404 mg, 1.54 was added sequentially mmol
  • diisopropyl azodicarboxylate 311 mg, 1.54 mmol
  • the reaction mixture was stirred at 30°C for 5 hours.
  • the reaction mixture was poured into water (20 mL), and extracted with ethyl acetate (5 mL ⁇ 4).
  • the organic phases were combined, washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, and filtered.
  • the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product.
  • the crude product was separated and purified by silica gel chromatography to obtain intermediate I-58.
  • the crude product was dissolved in methanol (60 mL), and concentrated sulfuric acid (5.00 mL) was added dropwise. After the dropwise addition, the reaction mixture was stirred and reacted at 70°C for 16 hours. After cooling the reaction liquid to room temperature, the organic solvent was concentrated under reduced pressure to obtain a concentrate. Water (100 mL) was added to the concentrate, and extracted with ethyl acetate (80 mL ⁇ 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-60.
  • intermediate I-60 500 mg, 2.92 mmol was dissolved in acetonitrile (20 mL).
  • Intermediate I-13 (1.08 g, 3.52 mmol) and cesium carbonate (2.85 g, 8.75 mmol) were added sequentially.
  • the reaction mixture was stirred at room temperature for 16 hours.
  • the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product.
  • the crude product was separated and purified by silica gel chromatography to obtain intermediate 1-61.
  • intermediate I-62 (200 mg, 0.541 mmol) was dissolved in dichloromethane (10 mL), and Dess-Martin oxidant (413 mg, 0.974 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-63.
  • the intermediate 1-68 (594 mg, 1.42 mmol) was dissolved in methanol (10 mL), and platinum dioxide (60.0 mg) was added. The reaction mixture was replaced with hydrogen three times, and the reaction was stirred at room temperature under a hydrogen atmosphere for 3 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography to obtain intermediate 1-69.
  • reaction solution was cooled to room temperature, diluted with water (20 mL), and extracted with ethyl acetate (15 mL ⁇ 3).
  • the organic phases were combined, and the organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered.
  • the filtrate was concentrated under reduced pressure to obtain a crude product, and the crude product was purified by silica gel chromatography to obtain intermediate I-72.
  • reaction solution was cooled to room temperature, diluted with water (20 mL), and extracted with ethyl acetate (15 mL ⁇ 3).
  • the organic phases were combined, and the organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered.
  • the filtrate was concentrated under reduced pressure to obtain a crude product, and the crude product was purified by silica gel chromatography to obtain intermediate I-76.
  • the intermediate 1-66 (700 mg, 1.77 mmol) was dissolved in acetonitrile (10 mL), and 2-iodopropane (3.0 g, 17.6 mmol) and cesium carbonate (1.73 g, 5.31 mmol) were added sequentially.
  • the reaction mixture was stirred at 45°C overnight.
  • the organic solvent was removed by concentration under reduced pressure to obtain a crude product.
  • the crude product was separated and purified by silica gel chromatography to obtain intermediate I-77.
  • the intermediate 1-77 (770 mg, 1.76 mmol) was dissolved in tetrahydrofuran (5 mL), and diisobutylaluminum hydride (7 mL, 1 M in hexane, 7.00 mmol) was added.
  • the reaction mixture was reacted at -45°C with stirring for 1.5 hours.
  • the reaction was quenched by the addition of saturated aqueous ammonium chloride solution (20 mL). Ethyl acetate (60 mL) was added for liquid separation and extraction, and the aqueous phase was extracted with ethyl acetate (60 mL ⁇ 2) to obtain an organic phase.
  • the intermediate 1-78 was dissolved in 1,2-dichloroethane (6 mL), and manganese dioxide (426 mg, 4.9 mmol) was added. The reaction mixture was stirred at 70°C overnight under the protection of argon, cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to obtain crude product I-79. The crude product was used in the next reaction without further purification.
  • Diisopropylamine (612mg, 6.05mmol) was dissolved in anhydrous tetrahydrofuran (20.0mL), under the protection of nitrogen, it was cooled to -78 °C, and then slowly added n-BuLi (3.61mL, 1.6M in hexane , 5.78 mmol). At this temperature, the reaction was stirred for 0.75 hour. Then, at -78°C, intermediate I-81 (2.00 g, 5.50 mmol) was slowly dropped into the system, and the reaction was stirred at this temperature for 0.5 hour. Finally, methyl iodide (937 mg, 6.60 mmol) was slowly added to the system at -78°C.
  • reaction solution was slowly warmed to room temperature and stirred overnight.
  • the reaction mixture was quenched with saturated ammonium chloride solution (15.0 mL). After adding saturated brine (150 mL), it was extracted with ethyl acetate (100 mL ⁇ 2).
  • the organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product.
  • the crude product was separated and purified by silica gel chromatography to obtain intermediate I-82.
  • Intermediate 1-83 (650 mg, 1.99 mmol) and TBAF (1.04 g, 3.98 mmol) were dissolved in anhydrous tetrahydrofuran (15.0 mL), and reacted at 25°C for 10 hours. The reaction solution was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate 1-84.
  • reaction solution was heated to 100°C and stirred for 8 hours, then cooled to room temperature, water (50 mL) was added, extracted with ethyl acetate (50 mL*3), the organic phases were combined, washed with saturated brine (50 mL), dried over It was dried over sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was separated and purified by silica gel chromatography to obtain intermediate 1-89.
  • reaction solution was heated to 40°C and stirred for 1 hour, then water (50 mL) was added at 40°C, extracted with ethyl acetate (50 mL*3), the organic phases were combined, washed with saturated brine (30 mL), dried over Sodium sulfate was dried, filtered, the filtrate was concentrated to dryness under reduced pressure, and the residue was separated and purified by silica gel chromatography to obtain intermediate I-90.
  • reaction solution was cooled to room temperature, water (5 mL) was added to the mixture, extracted with ethyl acetate (20 mL x 3), the organic phases were combined, concentrated under reduced pressure and separated and purified by silica gel chromatography to obtain intermediate I-94.
  • intermediate I-96 (900 mg, 3.47 mmol) and intermediate I-45 (879 mg, 3.82 mmol) were dissolved in N,N-dimethylformamide (15.0 mL), and then cesium carbonate (3.39 g, 10.4 mmol). The reaction mixture was stirred at 50°C for 3 hours. The reaction system was cooled to room temperature, poured into water (50 mL), and extracted with ethyl acetate (20 mL ⁇ 4). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-97.
  • the intermediate 1-97 (700 mg, 1.71 mmol) was dissolved in tetrahydrofuran (15.0 mL), and then lithium tetrahydroaluminum (71.3 mg, 1.88 mmol) was added at 0°C.
  • the reaction mixture was warmed to 10°C and the reaction was stirred for 2 hours.
  • Water/tetrahydrofuran (71 mg/20 mL) was added to the reaction system, the system was stirred at room temperature for 10 minutes, sodium hydroxide solution (71 mg, 10% w/w) was added, and the mixture was stirred at room temperature for 30 minutes and filtered.
  • the filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product.
  • the crude product was separated and purified by silica gel chromatography to obtain intermediate I-98.
  • the intermediate 1-98 (520 mg, 1.37 mmol) was dissolved in dichloroethane (15.0 mL), and then manganese dioxide (956 mg, 11.0 mmol) was added. The reaction mixture was stirred at 80°C for 3 hours. The reaction system was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated and purified by silica gel chromatography to obtain intermediate I-99.
  • the intermediate 1-59 (60.0 mg, 0.125 mmol) was dissolved in ethanol/water (1 mL/1 mL), and lithium hydroxide monohydrate (15.7 mg, 0.375 mmol) was added. The reaction mixture was stirred and reacted at 10°C for 2 hours. The reaction solution was directly separated and purified by preparative HPLC to obtain compound 17.
  • the cell line used for the test is CHO-K1 EDG1 ⁇ -Arrestin Cell Line, supplier: DiscoverX, article number: 93-0207C2.
  • the test evaluates the inhibitory effect of the compound on S1P1-mediated forskolin-induced cAMP activity.
  • the cell line used for the test is CHO-K1 EDG1 ⁇ -Arrestin Cell Line, supplier: DiscoverX, article number: 93-0207C2. According to the supplier's instructions, the cells were added to the test plate at 25 ⁇ L per cell suspension containing 5000 cells and incubated at 37°C for 20 hours. Ten concentrations of 4-fold diluted compounds were added to the cell culture medium and incubated at 37°C for 90 minutes. Prepare the test solution, 12 ⁇ L per well, incubate at room temperature for 60 minutes, and read the plate with Envision. Data by nonlinear regression analysis to determine EC ⁇ -arrestin activity 50. The experimental results are shown in Table 2.
  • CHO-K1 DEG1 cells ( CHO-K1 EDG1 ⁇ -Arrestin Cell Line, supplier: DiscoverX, article number: 93-0207C2), remove the medium (F12 medium 1000mL, 10% FBS, 800 ⁇ g/mL G418, 300 ⁇ g/mL Hygromycin, 1% GlutaMax and 1 %Pen/Strep), rinse the cells with 2ml DPBS, add 5mL cell dispersion solution (Invitrogen-13151014) to disperse the cells, incubate for 1 to 2 minutes in a 37°C incubator, tap the flask to make the cells fall off, and add 5mL growth medium Pipette gently to fully suspend cells. Vi-Cell was used for cell counting.
  • the cells, culture conditions and cell collection conditions used in this experiment are the same as in Experimental Example 3.
  • mice Male Sprague-Dawley rats weighing 200-220g. Feeding environment: temperature 23 ⁇ 2°C, relative humidity 40-70%, lighting time is 7 o'clock in the morning, and 7 o'clock in the evening; animals are free to feed common feed and sterilized drinking water. All animal experiments have been approved by the animal ethics committee; all animal experiment operations comply with the relevant SOP requirements of the animal house. The animals were kept adaptively for one week before the experiment.
  • Animals are administered orally with a volume of 10 mL/kg.
  • the vehicle administered was 0.5% DMSO + 0.5% MC.
  • Animals were anesthetized with isoflurane 5 hours after drug administration, and 100-150 ⁇ l of peripheral blood was collected through the orbits in EP tubes, placed on ice, and analyzed for lymphocyte count detection by XT-2000i automatic blood analyzer within 30 minutes. ; Another 20 ⁇ l whole blood, 40 ⁇ l DDW dilution, liquid nitrogen quick-freeze, blood compound concentration test.
  • test compound 2 reduced the peripheral blood lymphocyte (PBL) count in rats 5 hours after administration, as shown in FIG. 1.
  • IC50 is 94.6 nM (Compound 2).

Abstract

本发明公开了式(Ⅱ)所示化合物及其药效上可接受的盐，以及该化合物作为S1P1激动剂的应用。

Description

作为免疫调节剂的化合物及其制备方法和应用
本申请主张如下优先权：
CN201811488085.7，申请日2018年12月6日；
CN201910414361.3，申请日2019年5月17日；
CN201911161765.2，申请日2019年11月22日。
技术领域
本发明涉及式(Ⅱ)所示化合物及其药效上可接受的盐，以及该化合物作为S1P1激动剂的应用。
背景技术
鞘氨醇-1-磷酸(Sphingosine-1-phosphate，S1P)是属于溶血磷脂(lysophospholipid，LP)的一种两性生物信号分子。S1P可通过作用于5种G蛋白偶联受体亚型——鞘氨醇-1-磷酸受体(S1PR 1-5)激活复杂的下游信号，从而调节重要的生理生化功能。S1P与不同的S1P受体结合可调节不同的生理功能，在维持机体健康以及疾病发生过程中起着重要的作用。
S1P1受体激动剂干扰淋巴细胞归巢(lymphocyte traffic king)，将它们隔离(sequestering)在淋巴结和其它二级淋巴组织中。这导致外周循环淋巴细胞减少，淋巴细胞隔离在临床上的价值是将它们从周围组织中的炎症和/或自身免疫反应视域中排除。这种对淋巴细胞的隔离(例如在淋巴结中)被认为是以下同时作用的结果：由激动剂驱动的对T细胞上S1P1受体的功能性拮抗作用(因此降低S1P动员T细胞从淋巴结中流出的能力)和对淋巴结内皮上S1P1受体的持续激动作用(从而提高对抗淋巴细胞迁移的屏障功能)。因此，S1P 1受体激动剂通过阻止淋巴细胞的运输来降低人体自身免疫能力，因而可以作为免疫抑制剂用于治疗各种身免疫性疾病。
其中S1P1激动剂芬戈莫德(Fingolimod，FTY720)被FDA批准用于复发性多发性硬化症(Multiple Scleorosis，MS)的治疗，为免疫性疾病的治疗开辟了新的治疗领域。尽管FTY720具有临床功效，但它是一个非选择性的S1P受体激动剂，FTY720在体内与S1P3的结合往往会导致一系列重要的副作用，如心动过缓等，从而大大限制了其治疗免疫性疾病领域的应用范围。因此，发现第二代高选择性S1P1激动剂，使之成为疗效更好、副作用更小以及应用范围更广的免疫性疾病治疗药物成为了药物研究的热点之一。
除了提高靶点选择性外，缩短药物即S1P1受体激动剂在体内的半衰期也是发现第二代S1P激动剂的目标。作为免疫抑制剂药物，较长半衰期会导致淋巴细胞的运输被持续抑制，外周血淋巴细胞数减少，从而使得用药者免疫功能低下，增加病毒性感染的风险。在发生感染的情况下，往往需要停止药物使得外周血淋巴细胞数尽快恢复到正常水平，以便能够快速恢复人体免疫功能。其中S1P1受体激动剂如FTY720在人体内的半衰期长达6～9天，因此即便停止服用该药物，需要在很长时间内淋巴细胞数才能恢复正常。
因此，目前本领域仍然需要开发新型的具有S1P1受体选择性、半衰期较短的S1P1受体激动剂，以克服现有疗法的缺陷。
发明内容
本发明提供了式(Ⅱ)所示化合物或其药学上可接受的盐，
Figure PCTCN2019123514-appb-000001
其中，
m选自1、2和3；
n选自1和2；
y选自1和2；
R 1选自H、卤素、OH、NH 2、CN、C 1-6烷基、C 3-6环烷基、3～7元杂环烷基和C 1-6杂烷基，所述C 1-6烷基、C 3-6环烷基、3～7元杂环烷基或C 1-6杂烷基任选被1、2或3个R取代；
R 2选自H、卤素、OH、NH 2、CN和C 1-6烷基，所述C 1-6烷基任选被1、2或3个R取代；
环A选自C 3-7环烷基、3～7元杂环烷基和C 3-7环烯基，所述C 3-7环烷基、3～7元杂环烷基或C 3-7环烯基任选被1、2或3个R取代；
L 1选自
Figure PCTCN2019123514-appb-000002
L 2选自单键、O和S；
T 1选自N和CH；
T 2选自N和CH；
T 3选自N和CH；
R分别独立地选自H、F、Cl、Br、I、OH、NH 2、C 1-3烷基和CF 3；
所述C 1-6杂烷基和3～7元杂环烷基包含1、2或3个独立选自NH、O和S的杂原子或杂原子团。
本发明的一些方案中，上述R分别独立地选自H、F、Cl、Br、I、OH、NH 2、CH 3、CH 2CH 3和CF 3，其它变量如本发明所定义。
本发明的一些方案中，上述R 1选自H、F、Cl、Br、OH、NH 2、CN、C 1-3烷基、C 3-6环烷基、3～7元杂环烷基和C 1-3烷氧基，所述C 1-3烷基、C 3-6环烷基、3～7元杂环烷基或C 1-3烷氧基任选被1、2或3个R取代，其它变量如本发明所定义。
本发明的一些方案中，上述R 1选自H、F、Cl、Br、OH、NH 2、CN、CH 3、CH 2CH 3、CF 3、
Figure PCTCN2019123514-appb-000003
Figure PCTCN2019123514-appb-000004
其它变量如本发明所定义。
本发明的一些方案中，上述R 2选自H、F、Cl、Br、OH、NH 2、CN、CH 3和CH 2CH 3，所述CH 3或CH 2CH 3任选被1、2或3个R取代，其它变量如本发明所定义。
本发明的一些方案中，上述R 2选自H、F、Cl、Br、OH、NH 2、CN、CH 3、CH 2CH 3和CF 3，其它变量如本发明所定义。
本发明的一些方案中，上述L 1选自
Figure PCTCN2019123514-appb-000005
其它变量如本发明所定义。
本发明的一些方案中，上述环A选自
Figure PCTCN2019123514-appb-000006
所述
Figure PCTCN2019123514-appb-000007
任选被1、2或3个R取代，其它变量如本发明所定义。
本发明的一些方案中，上述环A选自
Figure PCTCN2019123514-appb-000008
其它变量如本发明所定义。
本发明的一些方案中，上述结构单元
Figure PCTCN2019123514-appb-000009
选自
Figure PCTCN2019123514-appb-000010
Figure PCTCN2019123514-appb-000011
其它变量如本发明所定义。
本发明的一些方案中，上述结构单元
Figure PCTCN2019123514-appb-000012
选自
Figure PCTCN2019123514-appb-000013
其它变量如本发明所定义。
本发明的一些方案中，上述化合物或其药学上可接受的盐，其选自
Figure PCTCN2019123514-appb-000014
其中，
m、n、T 1、T 2、T 3、R、L 2、R 1、R 2、环A如上述所定义。
本发明提供了式(Ⅰ)所示化合物或其药学上可接受的盐，
Figure PCTCN2019123514-appb-000015
其中，
m选自1、2和3；
n选自1和2；
R 1选自H、卤素、OH、NH 2、CN、C 1-6烷基、C 3-6环烷基、3～7元杂环烷基和C 1-6杂烷基，所述C 1-6烷基、C 3-6环烷基、3～7元杂环烷基或C 1-6杂烷基任选被1、2或3个R取代；
R 2选自H、卤素、OH、NH 2、CN和C 1-6烷基，所述C 1-6烷基任选被1、2或3个R取代；
环A选自C 3-7环烷基、3～7元杂环烷基和C 3-7环烯基，所述C 3-7环烷基、3～7元杂环烷基或C 3-7环烯基 任选被1、2或3个R取代；
L 1选自
Figure PCTCN2019123514-appb-000016
L 2选自单键、O和S；
T 1选自N和CH；
T 2选自N和CH；
T 3选自N和CH；
R分别独立地选自H、F、Cl、Br、I、OH、NH 2、C 1-3烷基和CF 3；
所述C 1-6杂烷基和3～7元杂环烷基包含1、2或3个独立选自NH、O和S的杂原子或杂原子团。
本发明的一些方案中，上述R分别独立地选自H、F、Cl、Br、I、OH、NH 2、CH 3、CH 2CH 3和CF 3，其它变量如本发明所定义。
本发明的一些方案中，上述R 1选自H、F、Cl、Br、OH、NH 2、CN、C 1-3烷基、C 3-6环烷基、3～7元杂环烷基和C 1-3烷氧基，所述C 1-3烷基、C 3-6环烷基、3～7元杂环烷基或C 1-3烷氧基任选被1、2或3个R取代，其它变量如本发明所定义。
本发明的一些方案中，上述R 1选自H、F、Cl、Br、OH、NH 2、CN、CH 3、CH 2CH 3、CF 3、
Figure PCTCN2019123514-appb-000017
Figure PCTCN2019123514-appb-000018
其它变量如本发明所定义。
本发明的一些方案中，上述R 2选自H、F、Cl、Br、OH、NH 2、CN、CH 3和CH 2CH 3，所述CH 3或CH 2CH 3任选被1、2或3个R取代，其它变量如本发明所定义。
本发明的一些方案中，上述R 2选自H、F、Cl、Br、OH、NH 2、CN、CH 3、CH 2CH 3和CF 3，其它变量如本发明所定义。
本发明的一些方案中，上述L 1选自
Figure PCTCN2019123514-appb-000019
其它变量如本发明所定义。
本发明的一些方案中，上述环A选自
Figure PCTCN2019123514-appb-000020
所述
Figure PCTCN2019123514-appb-000021
任选被1、2或3个R取代，其它变量如本发明所定义。
本发明的一些方案中，上述环A选自
Figure PCTCN2019123514-appb-000022
Figure PCTCN2019123514-appb-000023
其它变量如本发明所定义。
本发明的一些方案中，上述结构单元
Figure PCTCN2019123514-appb-000024
选自
Figure PCTCN2019123514-appb-000025
Figure PCTCN2019123514-appb-000026
其它变量如本发明所定义。
本发明的一些方案中，上述结构单元
Figure PCTCN2019123514-appb-000027
选自
Figure PCTCN2019123514-appb-000028
其它变量如本发明所定义。
本发明还提供了下式化合物或其药学上可接受的盐，
Figure PCTCN2019123514-appb-000029
Figure PCTCN2019123514-appb-000030
本发明还提供了一种药物组合物，所述的药物组合物含有上述化合物或其药学上可药用盐，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
本发明还提供了上述化合物或其可药用盐或上述药物组合物在制备预防和/或治疗用作S1P1受体相关疾病的药物中的用途。
本发明的一些方案中，上述用途，其中所述的S1P1受体相关疾病选自溃疡性结肠炎(Ulcerative colitis)、克罗恩病(Crohn’s disease)、多发性硬化症(Multiple sclerosis)、系统性红斑狼疮(Systemic lupus erythematosus)、狼疮性肾炎(Lupus nephritis)、类风湿性关节炎(Rheumatoid arthritis)、原发性胆汁胆管炎(Primary Biliary Cholangitis)、过敏性皮肤炎(Atopic Dermatitis)、脑出血(Intracerebral hemorrhage)、移植物抗宿主病(Graft versus Host Disease)、牛皮癣(Psoriasis)、I型糖尿病(Type I diabetes)、痤疮(Acne)、微生物感染或微生物疾病及病毒感染或病毒疾病。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物的中性形式接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机胺或镁盐或类似的盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在溶液或合适的惰性溶剂中用足够量的酸与这类化合物的中性形式接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸，碳酸氢根，磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如乙酸、丙酸、异丁酸、三氟乙酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐。本发明的某些特定的化 合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。
本发明的化合物可以存在特定的。除非另有说明，术语“互变异构体”或“互变异构体形式”是指在室温下，不同官能团异构体处于动态平衡，并能很快的相互转化。若互变异构体是可能的(如在溶液中)，则可以达到互变异构体的化学平衡。例如，质子互变异构体(proton tautomer)(也称质子转移互变异构体(prototropic tautomer))包括通过质子迁移来进行的互相转化，如酮-烯醇异构化和亚胺-烯胺异构化。价键异构体(valence tautomer)包括一些成键电子的重组来进行的相互转化。其中酮-烯醇互变异构化的具体实例是戊烷-2,4-二酮与4-羟基戊-3-烯-2-酮两个互变异构体之间的互变。
本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚( 3H)，碘-125( 125I)或C-14( 14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。“任选”或“任选地”指的是随后描述的事件或状况可能但不是必需出现的，并且该描述包括其中所述事件或状况发生的情况以及所述事件或状况不发生的情况。
术语“被取代的”是指特定原子上的任意一个或多个氢原子被取代基取代，可以包括重氢和氢的变体，只要特定原子的价态是正常的并且取代后的化合物是稳定的。术语“任选被取代的”是指可以被取代，也可以不被取代，除非另有规定，取代基的种类和数目在化学上可以实现的基础上可以是任意的。
当任何变量(例如R)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。因此，例如，如果一个基团被0-2个R所取代，则所述基团可以任选地至多被两个R所取代，并且每种情况下的R都有独立的选项。此外，取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
当其中一个变量选自单键时，表示其连接的两个基团直接相连，比如
Figure PCTCN2019123514-appb-000031
中L 2代表单键时表示该结构实际上是
Figure PCTCN2019123514-appb-000032
当所列举的取代基中没有指明其通过哪一个原子连接到被取代的基团上时，这种取代基可以通过其任何原子相键合，例如，吡啶基作为取代基可以通过吡啶环上任意一个碳原子连接到被取代的基团 上。
当所列举的连接基团没有指明其连接方向，其连接方向是任意的，例如，
Figure PCTCN2019123514-appb-000033
中连接基团L为-CH 2O-，此时-CH 2O-既可以按与从左往右的读取顺序相同的方向连接苯基和环戊基构成
Figure PCTCN2019123514-appb-000034
也可以按照与从左往右的读取顺序相反的方向连接苯基和环戊基构成
Figure PCTCN2019123514-appb-000035
所述连接基团、取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
除非另有规定，环上原子的数目通常被定义为环的元数，例如，“3-6元环”是指环绕排列3-6个原子的“环”。
除非另有规定，术语“C 1-6烷基”用于表示直链或支链的由1至6个碳原子组成的饱和碳氢基团。所述C 1-6烷基包括C 1-5、C 1-4、C 1-3、C 1-2、C 2-6、C 2-4、C 6和C 5烷基等；其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C 1-6烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异丙基)、丁基(包括n-丁基，异丁基，s-丁基和t-丁基)、戊基(包括n-戊基，异戊基和新戊基)、己基等。
除非另有规定，术语“C 1-3烷基”用于表示直链或支链的由1至3个碳原子组成的饱和碳氢基团。所述C 1-3烷基包括C 1-2和C 2-3烷基等；其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C 1-3烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异丙基)等。
术语“杂烷基”本身或者与另一术语联合，表示由一定数目碳原子和至少一个杂原子或杂原子团组成的，稳定的直链或支链的烷基原子团或其组合物。在一些实施方案中，杂原子选自B、O、N和S，其中氮和硫原子任选地被氧化，氮杂原子任选地被季铵化。在另一些实施方案中，杂原子团选自-C(＝O)O-、-C(＝O)-、-C(＝S)-、-S(＝O)、-S(＝O) 2-、-C(＝O)N(H)-、-N(H)-、-C(＝NH)-、-S(＝O) 2N(H)-和-S(＝O)N(H)-。在一些实施方案中，所述杂烷基为C 1-6杂烷基；在另一些实施方案中，所述杂烷基为C 1- 3杂烷基。杂原子或杂原子团可以位于杂烷基的任何内部位置，包括该烷基与分子其余部分的连接位置，但术语“烷氧基”属于惯用表达，是指通过一个氧原子连接到分子的其余部分的那些烷基基团。杂烷基的实例包括但不限于-OCH 3、-OCH 2CH 3、-OCH 2CH 2CH 3、-OCH 2(CH 3) 2、-CH 2-CH 2-O-CH 3、-NHCH 3、-N(CH 3) 2、-NHCH 2CH 3、-N(CH 3)(CH 2CH 3)、-CH 2-CH 2-NH-CH 3、-CH 2-CH 2-N(CH 3)-CH 3、-SCH 3、-SCH 2CH 3、-SCH 2CH 2CH 3、-SCH 2(CH 3) 2、-CH 2-S-CH 2-CH 3、-CH 2-CH 2、-S(＝O)-CH 3、-CH 2-CH 2-S(＝O) 2-CH 3、和至多两个杂原子可以是连续的，例如-CH 2-NH-OCH 3。
除非另有规定，术语“C 1-3烷氧基”表示通过一个氧原子连接到分子的其余部分的那些包含1至3个碳原子的烷基基团。所述C 1-3烷氧基包括C 1-2、C 2-3、C 3和C 2烷氧基等。C 1-3烷氧基的实例包括但不限于甲氧基、乙氧基、丙氧基(包括正丙氧基和异丙氧基)等。
除非另有规定，“C 3-7环烷基”表示由3至7个碳原子组成的饱和环状碳氢基团，其为单环和双环体系，所述C 3-7环烷基包括C 4-7、C 5-7、C 3-5、C 4-5和C 5-6环烷基等；其可以是一价、二价或者多价。C 3-6环烷基的实例包括，但不限于，环丙基、环丁基、环戊基、环己基等。
除非另有规定，术语“3-7元杂环烷基”本身或者与其他术语联合分别表示由3至7个环原子组成的饱和环状基团，其1、2、3或4个环原子为独立选自O、S和N的杂原子，其余为碳原子，其中氮原 子任选地被季铵化，氮和硫杂原子可任选被氧化(即NO和S(O) p，p是1或2)。其包括单环和双环体系，其中双环体系包括螺环、并环和桥环。此外，就该“3-6元杂环烷基”而言，杂原子可以占据杂环烷基与分子其余部分的连接位置。所述3-7元杂环烷基包括3-6元、4-7元、4元、5元、6元和7元杂环烷基等。3-7元杂环烷基的实例包括但不限于氮杂环丁基、氧杂环丁基、硫杂环丁基、吡咯烷基、吡唑烷基、咪唑烷基、四氢噻吩基(包括四氢噻吩-2-基和四氢噻吩-3-基等)、四氢呋喃基(包括四氢呋喃-2-基等)、四氢吡喃基、哌啶基(包括1-哌啶基、2-哌啶基和3-哌啶基等)、哌嗪基(包括1-哌嗪基和2-哌嗪基等)、吗啉基(包括3-吗啉基和4-吗啉基等)、二噁烷基、二噻烷基、异噁唑烷基、异噻唑烷基、1,2-噁嗪基、1,2-噻嗪基、六氢哒嗪基、高哌嗪基或高哌啶基等。
除非另有规定，“C 3-7环烯基”表示包含至少一个碳-碳双键的由3至7个碳原子组成的部分不饱和的环状碳氢基团，其包括单环和双环体系，其中双环体系包括螺环、并环和桥环，此体系的任意环都是非芳香性的。所述C 3-7环烯基包括C 3-6、C 3-5、C 4-7、C 4-8、C 4-6、C 4-5、C 5-7或C 5-6环烯基等；其可以是一价、二价或者多价。C 3-8环烯基的实例包括但不限于，环丙烯基、环丁烯基、环戊烯基、环戊二烯基、环己烯基、环己二烯基等。
除非另有规定，C n-n+m或C n-C n+m包括n至n+m个碳的任何一种具体情况，例如C 1-12包括C 1、C 2、C 3、C 4、C 5、C 6、C 7、C 8、C 9、C 10、C 11、和C 12，也包括n至n+m中的任何一个范围，例如C 1- 12包括C 1-3、C 1-6、C 1-9、C 3-6、C 3-9、C 3-12、C 6-9、C 6-12、和C 9-12等；同理，n元至n+m元表示环上原子数为n至n+m个，例如3-12元环包括3元环、4元环、5元环、6元环、7元环、8元环、9元环、10元环、11元环、和12元环，也包括n至n+m中的任何一个范围，例如3-12元环包括3-6元环、3-9元环、5-6元环、5-7元环、6-7元环、6-8元环、和6-10元环等。
术语“离去基团”是指可以被另一种官能团或原子通过取代反应(例如亲和取代反应)所取代的官能团或原子。例如，代表性的离去基团包括三氟甲磺酸酯；氯、溴、碘；磺酸酯基，如甲磺酸酯、甲苯磺酸酯、对溴苯磺酸酯、对甲苯磺酸酯等；酰氧基，如乙酰氧基、三氟乙酰氧基等等。
术语“保护基”包括但不限于“氨基保护基”、“羟基保护基”或“巯基保护基”。术语“氨基保护基”是指适合用于阻止氨基氮位上副反应的保护基团。代表性的氨基保护基包括但不限于：甲酰基；酰基，例如链烷酰基(如乙酰基、三氯乙酰基或三氟乙酰基)；烷氧基羰基，如叔丁氧基羰基(Boc)；芳基甲氧羰基，如苄氧羰基(Cbz)和9-芴甲氧羰基(Fmoc)；芳基甲基，如苄基(Bn)、三苯甲基(Tr)、1,1-二-(4'-甲氧基苯基)甲基；甲硅烷基，如三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)等等。术语“羟基保护基”是指适合用于阻止羟基副反应的保护基。代表性羟基保护基包括但不限于：烷基，如甲基、乙基和叔丁基；酰基，例如链烷酰基(如乙酰基)；芳基甲基，如苄基(Bn)，对甲氧基苄基(PMB)、9-芴基甲基(Fm)和二苯基甲基(二苯甲基，DPM)；甲硅烷基，如三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)等等。
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
本发明所使用的溶剂可经市售获得。
本发明采用下述缩略词：DIAD代表偶氮二甲酸二异丙酯；Xphos代表2-二环己基磷-2,4,6-三异丙基联苯；LiHMDS代表六甲基二硅基胺基锂；NIS代表N-碘代丁二酰亚胺；Pd 2(dba) 3代表三(二亚苄基 丙酮)二钯；THF代表四氢呋喃；DIAD代表偶氮二甲酸二异丙酯；PPh 3代表三苯基膦；AntPhos代表4-(9-蒽基)-3-(叔丁基)-2,3-二氢苯并[d][1,3]氧,膦戊轭；DIEA代表N,N-二异丙基乙胺；HOAc代表乙酸；DMF代表N,N-二甲基甲酰胺；DIBAL-H代表二异丁基氢化铝；TBAF代表四丁基氟化铵。
化合物依据本领域常规命名原则或者使用
Figure PCTCN2019123514-appb-000036
软件命名，市售化合物采用供应商目录名称。
附图说明
图1为化合物2在大鼠外周血淋巴细胞(PBL)降低测试中的作用。
具体实施方式
下面通过实施例对本申请进行详细描述，但并不意味着存在对本申请而言任何不利的限制。本文已经详细地描述了本申请，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本申请精神和范围的情况下针对本申请具体实施方式进行各种变化和改进将是显而易见的。
中间体的制备
参考例1：中间体I-1的制备
Figure PCTCN2019123514-appb-000037
将4-溴-3-三氟甲基苯甲酸甲酯(6.32g,22.32mmol)溶解于二氧六环/水(4:1,30mL)中，加入环戊基-1-烯-1-硼酸(3.0g,26.79mmol)，四三苯基膦钯(1.3g,1.12mmol)和碳酸钾(9.2g,67mmol)。将反应液于110℃，氩气保护下搅拌15小时。待冷却至室温后，将溶剂浓缩蒸干得残留物，残留物经硅胶色谱法纯化后得中间体I-1。
1H NMR(400MHz,CDCl 3)δ8.32(d,J＝1.5Hz,1H),8.11(dd,J＝8.0,1.5Hz,1H),7.37(d,J＝8.0Hz,1H),5.83–5.77(m,1H),3.95(s,3H),2.74–2.63(m,2H),2.58–2.51(m,2H),2.08–1.99(m,2H).
参考例2：中间体I-2的制备
Figure PCTCN2019123514-appb-000038
将中间体I-1(1.0g,3.7mmol)溶解于20mL甲醇中，加入钯碳(50mg,w/w＝10％)。将反应混合物于氢气氛围下，室温搅拌24小时。反应液过滤，滤液浓缩得中间体I-2粗产物。该粗产物未经纯化直接用于下一步反应。
1H NMR(400MHz,CDCl 3)δ8.27(d,J＝1.3Hz,1H),8.13(dd,J＝8.3,1.3Hz,1H),7.54(d,J＝8.3Hz,1H),3.93(s,3H),3.47–3.36(m,1H),2.16–2.07(m,2H),1.93–1.82(m,2H),1.82–1.70(m,2H),1.69–1.55(m,2H).
参考例3：中间体I-3的制备
Figure PCTCN2019123514-appb-000039
将中间体I-2(950mg，3.5mmol)溶解于15mL四氢呋喃中，于-20℃氩气保护下，逐滴加入四氢锂铝的四氢呋喃溶液(1.5M,7mL,10.5mmol)。滴加完成后，将反应液于-20℃搅拌1小时。将反应液温度升至0℃后，加入饱和氯化铵水溶液(4mL)和乙酸乙酯(5mL)。将反应液过滤，滤液浓缩得中间体I-3。该粗产物未经纯化，直接用于下一步反应。
参考例4：中间体I-4的制备
Figure PCTCN2019123514-appb-000040
将中间体I-3(300mg,1.2mmol)和2-氟-4-羟基苯甲醛(201mg,1.44mmol)溶解于四氢呋喃(55mL)中，冰水浴冷却下，加入三苯基膦(644mg,2.4mmol)和DIAD(484mg,2.4mmol)，并于室温搅拌过夜。向反应液中加入水(20mL)和乙酸乙酯(60mL)后分离有机相，水层用乙酸乙酯萃取(60mL×2)。将合并的有机层经饱和食盐水洗涤，无水硫酸钠干燥后过滤浓缩，得残留物。残留物经硅胶色谱法纯化后得中间体I-4。
1H NMR(400MHz,CDCl 3)δ10.22(s,1H),7.84(t,J＝8.4Hz,1H),7.64(s,1H),7.57–7.47(m,2H),6.90–6.81(m,1H),6.72(dd,J＝12.2,2.1Hz,1H),5.11(s,2H),3.44–3.33(m,1H),2.17–2.05(m,2H),1.92–1.80(m,2H),1.79–1.69(m,2H),1.65–1.58(m,2H).
参考例5：中间体I-5的制备
Figure PCTCN2019123514-appb-000041
将2,4-二羟基苯甲酸甲酯(5.0g,29.8mmol)溶解于丙酮中(100mL)，加入碳酸钾(8.2g,59.5mmol)，将反应液于室温下搅拌2小时后，加入苄溴(5.6g,32.7mmol)，并将反应液于60℃继续搅拌16小时。待冷却至室温后，将反应液过滤，滤液浓缩得残留物，残留物经硅胶色谱法纯化后得中间体I-5。
LC-MS(ESI)[M+H] +259.1.
1H NMR(400MHz,DMSO-d 6)δ10.78(s,1H),7.76–7.67(m,1H),7.47–7.28(m,5H),6.66–6.55(m,2H),5.17(s,2H),3.86(s,3H).
参考例6：中间体I-6的制备
Figure PCTCN2019123514-appb-000042
将中间体I-5(6.28g,24.3mmol)溶解于四氢呋喃(100mL)中，于-78℃下逐滴加入LiHMDS(31.6mL,31.6mmol，1M的四氢呋喃溶液)。滴加完成后，将反应温度缓慢升至-40℃，搅拌2小时后，加入N,N-双三氟甲磺酰基苯胺(9.54g,26.7mmol)。将反应温度升至室温，并搅拌16小时。反应液用200毫升水淬灭,加入150mL乙酸乙酯，萃取分出有机层，水层继续用乙酸乙酯(150mL×2)萃取。合并的有机层经饱和食盐水洗涤，无水硫酸钠干燥后过滤浓缩，得残留物。残留物经硅胶色谱法纯化后得中 间体I-6。
参考例7：中间体I-7的制备
Figure PCTCN2019123514-appb-000043
向中间体I-6(7.5g,19.2mmol)的四氢呋喃溶液中(100mL)，加入乙烯基氟硼酸钾(2.83g,21.2mmol)，磷酸钾(12.2g,57.7mmol)，醋酸钯(86.3mg,0.39mmol)和Xphos(275mg,0.58mmol)，并将反应液在氩气保护下，于75℃搅拌4小时。待冷却至室温后，将反应液用200毫升水稀释，并用乙酸乙酯萃取(100mL×3)。合并的有机层经饱和食盐水洗涤，无水硫酸钠干燥后过滤浓缩，得残留物。残留物经硅胶色谱法纯化后得中间体I-7。
LC-MS(ESI)[M+H] +269.1.
参考例8：中间体I-8的制备
Figure PCTCN2019123514-appb-000044
将中间体I-7(500mg,1.87mmol)溶解于甲醇(20mL)中，加入钯碳(100mg,10％w/w)，并将反应液于氢气氛围下室温搅拌16小时。将反应液过滤，滤液浓缩得残留物，残留物经硅胶色谱法纯化后得中间体I-8。
LC-MS(ESI)[M+H] +181.1.
参考例9：中间体I-9的制备
Figure PCTCN2019123514-appb-000045
将中间体I-3(2.5g,10.25mmol)溶解于10mL二氯亚砜中，并于50℃下加热2小时。待冷却至室温后，将反应液浓缩得残留物。将残留物溶解于乙酸乙酯(10mL)中，依次用饱和碳酸氢钠水溶液(10mL)和食盐水(10mL)洗涤。经无水硫酸钠干燥后，过滤浓缩得中间体I-9。
1H NMR(400MHz,CDCl 3)δ7.53(s,1H),7.49–7.36(m,2H),4.50(s,2H),3.35–3.23(m,1H),2.07–1.95(m,2H),1.84–1.72(m,2H),1.71–1.59(m,2H),1.57–1.44(m,2H).
参考例10：中间体I-10的制备
Figure PCTCN2019123514-appb-000046
将中间体I-8(310mg,1.72mmol)溶解于N,N-二甲基甲酰胺(20mL)中，加入中间体I-9(589.1mg,2.24mmol)和碳酸铯(1.12g,3.44mmol)。将混合液于室温下搅拌16小时。反应液用50毫升水淬灭，并用乙酸乙酯(30mL×3)萃取。合并的有机层经饱和食盐水洗涤，无水硫酸钠干燥后过滤浓缩， 得残留物。残留物经硅胶色谱法纯化后得中间体I-10。
参考例11：中间体I-11的制备
Figure PCTCN2019123514-appb-000047
将中间体I-10(670mg,1.65mmol)溶解于四氢呋喃(20mL)中，于-40℃下滴加二异丁基铝氢(1.5M甲苯溶液，3.3ml,4.95mmol)。滴加完毕后，将反应液于室温下搅拌16小时。反应液用50毫升水淬灭，并用乙酸乙酯(30mL×3)萃取。合并的有机层经饱和食盐水洗涤，无水硫酸钠干燥后过滤浓缩，得残留物。残留物经硅胶色谱法纯化后得中间体I-11。
LC-MS(ESI)[M-17] +361.1.
参考例12：中间体I-12的制备
Figure PCTCN2019123514-appb-000048
将中间体I-11(400mg,1.06mmol)溶解于1,2-二氯乙烷(20mL)中，加入二氧化锰(921.6mg,10.6mmol)，并将反应液于70℃搅拌16小时。待冷却至室温后，将反应液过滤，滤液蒸干得残留物。残留物经硅胶色谱法纯化后得中间体I-12。
LC-MS(ESI)[M+H] +377.1.
参考例13：中间体I-13的制备
Figure PCTCN2019123514-appb-000049
将中间体I-3(5.0g,20.47mmol)溶解于10mL 40％HBr/H 2O溶液中，并于100℃下加热3小时。待冷却至室温后，将反应液浓缩得残留物。向残留物中加入100mL水，乙酸乙酯(50mL×3)萃取。合并的有机层经食盐水(50mL)洗涤，无水硫酸钠干燥后，过滤浓缩得残留物。残留物经硅胶色谱法纯化后得中间体I-13。
1H NMR(400MHz,MeOH-d 4)δ7.65(d,J＝1.7Hz,1H),7.60(dd,J＝8.2,1.6Hz,1H),7.53(d,J＝8.2Hz,1H),4.58(s,2H),3.41–3.32(m,1H),2.10–2.01(m,2H),1.94–1.83(m,2H),1.78–1.67(m,2H),1.67–1.57(m,2H).
参考例14：中间体I-14的制备
Figure PCTCN2019123514-appb-000050
将4-羟基苯甲醛(122mg,1mmol)溶解于乙腈(3mL)中，加入中间体I-13(306mg,1mmol)和碳酸铯(650mg,2mmol)。混合液于室温下搅拌3小时后，将反应液过滤。滤液浓缩得残留物。残留 物经硅胶色谱法纯化后得中间体I-14。
参考例15：中间体I-15的制备
Figure PCTCN2019123514-appb-000051
将2-氯-4-羟基苯甲醛(200mg,1.28mmol)和中间体I-13(473mg,1.54mmol)溶解于乙腈(4mL)中，加入碳酸铯(1.25g,3.84mmol)，并将反应液于室温搅拌过夜。向反应液中加入水(10mL)和乙酸乙酯(30mL)并分离有机相，水相用乙酸乙酯萃取(30mL×2)。合并的有机层经食盐水(50mL)洗涤，无水硫酸钠干燥后，过滤浓缩得残留物。残留物经硅胶色谱法纯化后得中间体I-15。
1H NMR(400MHz,CDCl 3)δ10.34(s,1H),7.90(d,J＝8.7Hz,1H),7.65(s,1H),7.58–7.48(m,2H),7.02(d,J＝2.4Hz,1H),6.98–6.93(m,1H),5.11(s,2H),3.44–3.33(m,1H),2.15–2.06(m,2H),1.92–1.80(m,2H),1.79–1.69(m,2H),1.66–1.57(m,2H).
参考例16：中间体I-16的制备
Figure PCTCN2019123514-appb-000052
将中间体I-13(200mg,0.65mmol)和4-羟基-2-甲基苯甲醛(80.2mg,0.59mmol)溶解于乙腈(10mL)中，加入碳酸铯(423.8mg,1.30mmol)，并将反应液于室温搅拌2小时。将反应液过滤，滤液浓缩得残留物。残留物经硅胶色谱法纯化后得中间体I-16。
LC-MS(ESI)[M-H] -361.2.
参考例17：中间体I-17的制备
Figure PCTCN2019123514-appb-000053
将中间体I-13(460.0mg,1.50mmol)和4-羟基-2-溴苯甲醛(201.0mg,1.00mmol)溶解于乙腈(10mL)中，加入碳酸钾(414.1mg,3.00mmol)，并将反应液于50℃搅拌16小时。将反应液冷却至室温后，向反应液中加入水(10mL)，并用乙酸乙酯萃取(15mL×3)。合并的有机层经食盐水洗涤，无水硫酸钠干燥后，过滤浓缩得残留物。残留物经硅胶色谱法纯化后得中间体I-17。
LC-MS(ESI)[M-H] -425.1
参考例18：中间体I-18的制备
Figure PCTCN2019123514-appb-000054
向中间体I-17(120.0mg,0.28mmol)的N,N-二甲基甲酰胺(10mL)溶液中，加入氰化锌(65.3mg,0.56mmol)，四三苯基膦钯(50mg,0.04mmol)，并将反应液于氩气保护下，140℃搅拌3小时。待 冷却至室温后，将反应液用25mL水稀释，并用乙酸乙酯萃取(20mL×2)。合并的有机层经食盐水洗涤，无水硫酸钠干燥后，过滤浓缩得残留物。残留物经硅胶色谱法纯化后得中间体I-18。
LC-MS(ESI)[M-H] –372.2.
1H NMR(400MHz,CDCl 3)δ10.23(s,1H),8.01(d,J＝8.7Hz,1H),7.65(s,1H),7.57–7.49(m,2H),7.34(d,J＝2.5Hz,1H),7.30(dd,J＝8.7,2.5Hz,1H),5.18(s,2H),3.45–3.31(m,1H),2.16–2.07(m,2H),1.93–1.82(m,2H),1.80–1.69(m,2H),1.65–1.58(m,2H).
参考例19：中间体I-19的制备
Figure PCTCN2019123514-appb-000055
将2-羟基-3-三氟甲基吡啶(5.00g，30.66mmol)和NIS(6.90g,30.66mmol)溶于N,N-二甲基甲酰胺和乙腈的混合溶剂(60.0mL，1:1)中。将反应混合物升温至80℃，并在该温度下搅拌反应3小时。将反应混合物冷却至25℃，加入碳酸氢钠溶液(35.0mL，1M)并搅拌五分钟，用二氯甲烷(500mLx2)萃取。合并有机相，减压浓缩除去有机溶剂得到粗产品。粗产品加水(25.0mL)搅拌后过滤，滤饼真空干燥得到中间体I-19粗产物，产品未经进一步纯化直接用于下一步反应。
参考例20：中间体I-20的制备
Figure PCTCN2019123514-appb-000056
将中间体I-19(6.3g，21.80mmol)溶于三氯氧磷(15.0mL)中。将反应混合物升温至100℃，并在该温度下搅拌反应10小时。将反应液冷却至室温后缓慢加入冰水(200mL)中，用碳酸钠调节溶液的pH至中性，再用乙酸乙酯(400mL×2)萃取。合并有机相，用无水硫酸钠干燥，过滤，滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶柱(PE:EA＝100:1to 10:1)分离纯化得到中间体I-20。
1H NMR(400MHz,CDCl 3)δ8.78(s,1H),8.28(s,1H).
参考例21：中间体I-21的制备
Figure PCTCN2019123514-appb-000057
将中间体I-20(2.10g,6.83mmol)，乙酸钠(1.12g,13.66mmol)，醋酸钯(76.78mg，0.342mmol)和1,1′-双(二苯基膦)二茂铁(114mg，0.205mmol)溶于无水甲醇(25.0mL)中。将反应混合物升温至80℃并在该温度下于CO氛围下反应10小时。将反应液冷却到25℃后通过硅藻土过滤，滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法纯化得到中间体I-21。
LC-MS(ESI)[M+H] +240.1,[M+H+41] +281.1.
1H NMR(400MHz,CD 3OD)δ9.13(d,J＝1.8Hz,1H),8.64(d,J＝1.8Hz,1H),3.99(s,3H).
参考例22：中间体I-22的制备
Figure PCTCN2019123514-appb-000058
将中间体I-21(500mg,2.09mmol)，环戊烯-1-硼酸(281mg,2.51mmol),三环己基膦(58.6mg,0.209mmol),碳酸铯(2.04g,6.27mmol)和Pd 2(dba) 3溶于二氧六环/已腈/水(12.00mL,2.5:2.5:1)的混合溶液中，在氮气保护下将反应液升至100℃并在该温度下反应8小时。将反应液冷却到室温后用硅藻土过滤，滤液减压浓缩除去有机溶剂。残余物用乙酸乙酯(30mL)稀释，经水(15mL)和饱和食盐水(30mL)洗涤，无水硫酸钠干燥后过滤。滤液减压浓缩除去有机溶剂得粗产品。粗产品用硅胶色谱法纯化得到目标化合物I-22。
LC-MS(ESI)[M+H+41] +299.2
参考例23：中间体I-23的制备
Figure PCTCN2019123514-appb-000059
将中间体I-22(300mg,1.17mmol)和PtO 2(53.1mg)溶于乙酸乙酯(10.0mL)中。反应混合物在25℃和氢气氛围下反应10小时。反应液用硅藻土过滤，滤液减压浓缩除去有机溶剂得到中间体I-23。
LC-MS(ESI)[M+H+41] +301.2.
参考例24：中间体I-24的制备
Figure PCTCN2019123514-appb-000060
将中间体I-23(270mg,1.04mmol)溶于四氢呋喃(4.00mL)中。混合物冷却到0℃后在氮气保护缓慢滴加BH 3(3.12mL,1M in THF，3.12mmol)。然后升至25℃继续反应2小时。将反应液再次冷却到0℃后用甲醇(4.00mL)淬灭。把反应液减压浓缩除去有机溶剂，残余物用乙酸乙酯(30.0mL)稀释后用水(15.0mL)洗涤。有机相用无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到中间体I-24的粗产物，该粗产品未经纯化直接用于下一步反应。
LC-MS(ESI)[M+H] +246.1
参考例25：中间体I-25的制备
Figure PCTCN2019123514-appb-000061
将中间体I-24(130mg,粗产物),PPh 3(170mg,0.649mmol)和2-氟-4-羟基苯甲醛(53.5mg,0.382mmol)溶于四氢呋喃(3.00mL)中，然后冷却到0℃后在氮气保护下缓慢滴加DIAD(116mg,0.573mmol)。反应混合物升温至25℃继续反应10小时。反应液加入水(10.0mL)并用乙酸乙酯(15.0mL×2)萃取。合并有机相，用无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品用硅胶色谱法分离纯化得到中间体I-25。
LC-MS(ESI)[M+H+41] +409.2.
参考例26：中间体I-26的制备
Figure PCTCN2019123514-appb-000062
将中间体I-25(25.0mg,68.06μmol)和3-氮杂环丁烷甲酸甲酯盐酸盐(10.32mg,68.06μmol)溶于甲醇(1.00mL)中，然后加入DIEA(8.80mg,68.06μmol)和乙酸(8.17mg,136.12μmol)。反应混合物在25℃氮气保护下反应3小时。然后加入NaBH 3CN(4.28mg，68.06μmol)，反应混合物继续在25℃搅拌10小时。将反应液加入水(10.0mL)，用乙酸乙酯(15.0mL)萃取。有机相减压浓缩除去有机溶剂得中间体I-26粗产物，该粗产品未经纯化直接用于下一步反应。
LC-MS(ESI)[M+H] +467.2
参考例27：中间体I-27的制备
Figure PCTCN2019123514-appb-000063
在-45℃将中间体I-1(1.0g，3.70mmol)溶于四氢呋喃(10mL)中，加入二异丁基氢化铝的甲苯溶液(9.9mL,1.5M,14.85mmol)。将反应混合物在-45℃搅拌下反应2小时。在0℃下，加入饱和氯化铵水溶液来淬灭反应(20mL)，过滤。滤液加入乙酸乙酯(60mL)分液萃取，水相用乙酸乙酯(60mL×2)提取得到有机相。合并有机相，用饱和食盐水洗(50mL)，无水硫酸钠干燥，过滤。滤液减压浓缩得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-27。
1H NMR(400MHz,CDCl 3)δ7.65(s,1H),7.46(d,J＝7.8Hz,1H),7.28(d,J＝7.8Hz,1H),5.72(br.s,1H),4.73(s,2H),2.69–2.59(m,2H),2.55–2.46(m,2H),2.09–1.97(m,2H).
参考例28：中间体I-28的制备
Figure PCTCN2019123514-appb-000064
在0℃中，将三苯基磷(1.13g,4.31mmol)溶于四氢呋喃(6mL)中，依次加入偶氮二甲酸二异丙酯(578mg，2.86mmol)，中间体I-27(421mg，1.74mmol)和2-氟-4-羟基苯甲醛(200mg，1.43mmol)。反应混合物在氩气保护下室温搅拌过夜。反应混合物加入水(20mL)并用乙酸乙酯(60mL)分液萃取，水相用乙酸乙酯(60mL×2)萃取，合并有机相。有机相用饱和食盐水洗(50mL)，无水硫酸钠干燥，过滤。滤液减压浓缩除得到粗产品。粗产品经硅胶色谱法纯化得到中间体I-28。
1H NMR(400MHz,CDCl 3)δ10.22(s,1H),7.85(t,J＝8.8Hz,1H),7.70(s,1H),7.54–7.49(m,1H),7.33(d,J＝8.8Hz,1H),6.86(dd,J＝8.8,2.2Hz,1H),6.73(dd,J＝12.2,2.3Hz,1H),5.79–5.71(br.s,1H),5.14(s,2H),2.72–2.61(m,2H),2.56–2.47(m,2H),2.10–1.98(m,2H).
参考例29：中间体I-29的制备
Figure PCTCN2019123514-appb-000065
在室温下，将甲基-4-溴-3-(三氟甲基)苯甲酸甲酯(2g，7.07mmol)，环己烯-1-基硼酸(1.07g，8.48mmol)和碳酸钾(2.93g,21.2mmol)溶于二氧六环(20.0mL)和水(4.00mL)中，然后加入Pd(PPh 3) 4(409mg,0.354mmol)。在氮气保护下，将反应混合物升温至110℃，并在该温度下搅拌反应5小时。将反应液冷却至室温，加入水(200mL)，用乙酸乙酯(300mL×2)萃取。合并有机相，用饱和食盐水(200mL)洗，无水硫酸钠干燥，过滤，滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法纯化后得到中间体I-29。
1H NMR(400MHz,CD 3OD)δ8.25(d,J＝1.5Hz,1H),8.15(dd,J＝8.0,1.4Hz,1H),7.39(d,J＝8.0Hz,1H),5.60(s,1H),3.94(s,3H),2.28–2.14(m,4H),1.82–1.67(m,4H).
参考例30：中间体I-30的制备
Figure PCTCN2019123514-appb-000066
在室温下，将中间体I-29(900mg，3.17mmol)和Pd/C(10％w/w,90mg)加入到一个100mL的不锈钢反应釜中，然后加入甲醇(10.0mL)。将钢瓶中的气体用氢气置换，并把氢气加压到40atm。在该压力下将反应混合物升温至50℃，并在该温度下搅拌反应10小时。将反应液冷却至室温后，用硅藻土过滤，滤液减压浓缩除去有机溶剂得到目标化合物I-30，该产品未经纯化直接用于下一步反应。
1H NMR(400MHz,CD 3OD)δ8.22(d,J＝1.4Hz,1H),8.16(dd,J＝8.3,1.5Hz,1H),7.67(d,J＝8.2Hz,1H),3.93(s,3H),2.97(dd,J＝11.6,10.8Hz,1H),1.84(ddd,J＝23.6,11.9,3.3Hz,5H),1.61–1.36(m,5H).
参考例31：中间体I-31的制备
Figure PCTCN2019123514-appb-000067
在室温下，将中间体I-30(700mg,2.45mmol)溶于无水四氢呋喃(10.0mL)中。在氮气保护下将反应液降至0℃后，缓慢滴加四氢铝锂(1M THF溶液)(6.13mL，6.13mmol)。滴加完毕后，反应混合升至室温，并在室温下搅拌反应10小时。向反应混合物中加入乙醚(10.0mL)并冷却到0℃，加入水(4.00mL),然后加入氢氧化钠水溶液(6.00mL，10％w/w，aq.)。再加入水(18.0mL)并搅拌10分钟。加入无水硫酸钠到该体系并继续搅拌10分钟。然后过滤，减压浓缩除去有机溶剂得到粗产品。经硅胶色谱法分离纯化得到中间体I-31。
1H NMR(400MHz,CD 3OD)δ7.61(s,1H),7.55–7.47(m,2H),4.60(d,J＝6.1Hz,2H),2.97–2.85(m,1H),1.90–1.72(m,5H),1.58–1.33(m,5H).
参考例32：中间体I-32的制备
Figure PCTCN2019123514-appb-000068
在室温下，将中间体I-31(400mg,1.55mmol)，2-氟-4-羟基苯甲醛(217mg,1.55mmol)和PPh 3(1.22g,4.65mmol)溶于无水四氢呋喃(10.0mL)中，在氮气保护下将反应液降至0℃后。缓慢滴加DIAD(471mg,2.33mmol)，滴加完毕后，将反应液升到室温后，然后在室温下反应10小时。向反应混合物中加入水(100mL)，用乙酸乙酯(150mL×2)萃取。合并有机相，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品硅胶色谱法分离纯化得到中间体I-32。
1H NMR(400MHz,CD 3OD)δ10.12(s,1H),7.88–7.76(m,1H),7.73–7.62(m,2H),7.61–7.54(m,1H),7.00–6.88(m,2H),5.19(d,J＝8.4Hz,2H),3.02–2.86(m,1H),1.92–1.72(m,5H),1.59–1.33(m,5H).
参考例33：中间体I-33的制备
Figure PCTCN2019123514-appb-000069
在室温下，将4-溴-3-(三氟甲基)苯甲酸甲酯(100mg，0.35mmol)和环丁基硼酸(70mg,0.70mmol)溶于二甲苯中(4mL)中，然后将醋酸钯(4.5mg,0.02mmol)，AntPhos(11.1mg,0.03mmol)和磷酸钾(297mg,1.40mmol)加入上述体系。反应体系用氩气置换三次，并在氩气保护下于140℃下反应16小时。将反应液冷却至室温，加入水(15mL)。用乙酸乙酯萃取(15mL×2)，合并有机相，有机相用饱和食盐水(20mL)洗，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法纯化得到中间体I-33。
1H NMR(400MHz,CDCl 3)δ8.26(s,1H),8.18(d,J＝8.1Hz,1H),7.66(d,J＝8.2Hz,1H),4.11–3.81(m,4H),2.46–2.32(m,2H),2.28–2.15(m,2H),2.12–1.98(m,1H),1.93–1.84(m,1H).
参考例34：中间体I-34的制备
Figure PCTCN2019123514-appb-000070
在0℃下，将四氢铝锂(291.4mg，7.68mmol)分批次加入到中间体I-33(660mgl)的四氢呋喃(15mL)溶液中。反应混合物室温反应16小时。将反应液冷却至0℃，加入水(2mL)淬灭反应。反应液过滤，滤液减压浓缩除去有机溶剂得到粗产品。粗产品经反相制备分离纯化得到中间体I-34。
1H NMR(400MHz,CDCl 3)δ7.64–7.49(m,3H),4.72(s,2H),3.94–3.81(m,1H),2.41–2.29(m,2H),2.25–2.12(m,2H),2.09–1.96(m,1H),1.92–1.80(m,1H).
参考例35：中间体I-35的制备
Figure PCTCN2019123514-appb-000071
在室温下，将三苯基磷(115mg，0.44mmol)加入到中间体I-34(50mg,0.22mmol)和2-氟-4-羟基苯甲醛(46mg,0.33mmol)的四氢呋喃溶液(5mL)中。反应体系用氩气置换三次。在氩气保护下偶氮二甲酸二异丙酯(90mg，0.44mmol)于0℃下缓慢滴加至上述反应溶液中，并于该温度下反应30分钟。然后将反应体系升温至室温反应5小时。将反应混合物减压浓缩得到粗产品。粗产品经硅胶色谱法纯化得到中间体I-35。
LC-MS(ESI)[M-H] -351.1.
参考例36：中间体I-36的制备
Figure PCTCN2019123514-appb-000072
在室温下，将中间体I-35(55.0mg，0.156mmol)和氮杂环丁烷-3-甲酸甲酯盐酸盐(23.6mg，0.156mmol)溶于甲醇(3mL)中，将N，N-二异丙基乙胺(20.2mg,0.156mmol)和醋酸(18.7mg，0.311mmol)依次加入至上述体系中。反应混合物室温反应3小时，然后将氰基硼氢化钠(14.7mg，0.234mmol)加入反应混合物中，并于室温下继续反应16小时。反应液用水(10mL)稀释，并用乙酸乙酯萃取(10mL×3)，合并有机相。有机相用饱和食盐水(15mL)洗，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到中间体I-36，产品未经纯化直接用于下一步反应。
LC-MS(ESI)[M+H] +452.3.
参考例37：中间体I-37制备
Figure PCTCN2019123514-appb-000073
将4-羟基-3-(三氟甲基)苯甲酸甲酯(950mg，4.32mmol)溶于N,N-二甲基甲酰胺(15mL)中，向反应体系中依次加入碘代环戊烷(1270mg，6.48mmol)，碳酸铯(4223mg，12.96mmol)。将反应混合物升温至45℃，并在该温度下搅拌反应16小时。将反应液冷却至室温，依次加入水(50mL)和乙酸乙酯(50mL)，萃取分层，分出有机相，水相用乙酸乙酯(50mL×2)萃取，合并有机相，用饱和食盐水(20mL)洗，无水硫酸钠干燥，过滤。滤液减压浓缩得到粗产品。粗产品经硅胶色谱法纯化得到中间体I-37。
LC-MS(ESI)[M+H] +289.1。
1H NMR(400MHz,CDCl 3)δ8.24(d,J＝2.0Hz,1H),8.15(dd,J＝8.7,2.1Hz,1H),7.01(d,J＝8.9Hz,1H),4.96–4.92(m,1H),3.91(s,3H),1.95–1.92(m,2H),1.92–1.88(m,2H),1.87–1.79(m,2H),1.70–1.63(m,2H).
参考例38：中间体I-38制备
Figure PCTCN2019123514-appb-000074
将中间体I-37(1.19g，4.13mmol)溶于无水四氢呋喃(50mL)中，在-40℃下向反应混合物中滴加DIBAL(5.51mL,8.26mmol，1.5M甲苯溶液)。滴加完毕，将反应混合物升温至室温，并在该温度下搅拌反应16小时。依次加入水(100mL)和乙酸乙酯(50mL)，萃取分层，分出有机相，水相用乙酸乙酯(50mL×2)萃取产品，合并有机相，用饱和食盐水(20mL)洗，无水硫酸钠干燥，过滤。滤液减压浓缩得到粗产品。粗产品经硅胶色谱法纯化得到中间体I-38。
1H NMR(400MHz,CDCl 3)δ7.55(d,J＝1.8Hz,1H),7.45(dd,J＝8.5,1.8Hz,1H),6.97(d,J＝8.5Hz,1H),4.89–4.85(m,1H),4.64(s,2H),1.92–1.89(m,2H),1.88–1.84(m,2H),1.84–1.76(m,2H),1.65–1.61(m,2H).
参考例39：中间体I-39制备
Figure PCTCN2019123514-appb-000075
将三苯基膦(606mg，2.31mmol)溶于无水四氢呋喃(15mL)中，加入DIAD(467mg，2.31mmol)。混合物降至0℃，并在该温度下搅拌5分钟，然后依次加入中间体I-38(400mg，1.54mmol)和2-氟-4-羟基苯甲醛(216mg，1.54mmol)。反应混合物缓慢升至室温并在该温度下搅拌反应16小时。反应混合物减压浓缩除去有机溶剂，残余物经柱层析(硅胶，乙酸乙酯/石油醚＝0-30％)分离纯化得到化合物I-39。
LC-MS(ESI)[M-H] -381.0.
1H NMR(400MHz,CDCl 3)δ10.21(s,1H),7.84(t,J＝8.4Hz,1H),7.61(d,J＝1.9Hz,1H),7.50(dd,J＝8.6,2.0Hz,1H),7.02(d,J＝8.6Hz,1H),6.85(dd,J＝8.8,2.2Hz,1H),6.71(dd,J＝12.3,2.3Hz,1H),5.05(s,2H),4.92–4.86(m,1H),1.94–1.90(m,2H),1.90–1.86(m,2H),1.86–1.80(m,2H),1.68–1.61(m,2H).
参考例40：中间体I-40制备
Figure PCTCN2019123514-appb-000076
将中间体I-39(155mg，0.405mmol)溶于甲醇(10.0mL)中，在搅拌下依次加入3-甲酸甲酯氮杂环丁烷盐酸盐(61.4mg，0.405mmol)，DIEA(52.3mg，0.405mmol)，HOAc(48.6mg，0.810mmol)。反应混合物在室温下搅拌反应4小时后，加入NaBH 3CN(38.2mg，0.608mmol)，反应混合物在室温下搅拌反应16小时。依次加入水(50mL)和乙酸乙酯(20mL)，萃取分层，分出有机相，水相用乙酸乙 酯(20mL×2)萃取产品，合并有机相，用饱和食盐水(10mL)洗，无水硫酸钠干燥，过滤。滤液减压浓缩得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-40。
LC-MS(ESI)[M+H] +482.2.
1H NMR(400MHz,CDCl 3)δ7.59(d,J＝1.9Hz,1H),7.50(dd,J＝8.6,1.9Hz,1H),7.24(d,J＝8.6Hz,1H),7.02–6.98(m,1H),6.75(dd,J＝8.5,2.4Hz,1H),6.68(dd,J＝11.6,2.4Hz,1H),4.96(s,2H),4.90–4.86(m,1H),3.77–3.73(m,2H),3.73(s,3H),3.72–3.68(m,2H),3.52(t,J＝7.9Hz,2H),3.42(dd,J＝15.4,7.8Hz,1H),1.94–1.91(m,2H),1.90–1.88(m,2H),1.84–1.81(m,2H),1.66–1.61(m,2H).
参考例41：中间体I-41制备
Figure PCTCN2019123514-appb-000077
将2-氟-4-氰基苯甲醛(2.00g，13.41mmol)和氮杂环丁烷-3-甲酸甲酯盐酸盐(4.07g,26.82mmol)溶于甲醇(30mL)中。降温至10℃，然后依次加入DIPEA(3.47g,26.82mmol)和乙酸(2.42g，40.23mmol)，反应液在10℃搅拌反应2小时，加入氰基硼氢化钠(1.69g，26.82mmol)并继续搅拌1h，将反应液倒入饱和碳酸氢钠(100mL)中，用二氯甲烷(50mLx3)萃取，有机相合并，用饱和食盐水(50mL)洗涤，经无水硫酸钠干燥，过滤。滤液减压浓缩，残余物经硅胶色谱法分离纯化得到中间体I-41。
LC-MS(ESI)[M+H] +249.2
参考例42：中间体I-42制备
Figure PCTCN2019123514-appb-000078
将中间体I-41(1.00g，4.03mmol)和Ni(100mg)加入到乙酸/水(3:2，10mL)中，在氩气保护下，反应混合物在50℃下搅拌反应72小时。将反应液冷却至室温后，抽滤，滤液用乙酸乙酯(30mLx3)萃取，合并有机相，用饱和食盐水(30mL)洗涤，无水硫酸钠干燥，过滤。滤液减压浓缩得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-42。
LC-MS(ESI)[M+H] +254.2.
参考例43：中间体I-43制备
Figure PCTCN2019123514-appb-000079
将3-三氟甲基-4-溴苯酚(1.0g,4.1mmol)溶解于乙腈(20mL)中，依次加入苄溴(1.05g,6.15mmol)，碳酸钾(0.8g,5.79mmol)和碘化钾(66mg,0.4mmol)。将反应液于90℃下搅拌反应10小时。将反应液冷却至室温，倒入100mL水中，并用石油醚萃取(30mL×3)。合并有机相，用饱和食盐水洗涤，无水硫酸钠干燥，过滤。滤液减压浓缩得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-43。
1H NMR(400MHz,DMSO-d 6)δ7.78(d,J＝8.8Hz,1H),7.52–7.31(m,6H),7.26(dd,J＝8.8,3.0Hz,1H),5.21(s,2H).
参考例44：中间体I-44制备
Figure PCTCN2019123514-appb-000080
将中间体I-43(500mg,1.5mmol)溶于二氧六环/水(4:1,10mL)的混合液中，加入环戊烯硼酸(203mg,1.8mmol)，四三苯基膦钯(87mg,0.075mmol)和碳酸钾(621mg,4.5mmol)。将反应液于氩气氛围下，80℃加热搅拌10小时。将反应液冷却至室温，加入100mL水中，并用乙酸乙酯萃取(20mL×3)。合并有机相，用饱和食盐水洗涤，无水硫酸钠干燥，过滤。滤液减压浓缩得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-44。
1HNMR(400MHz,DMSO-d 6)7.64–7.13(m,8H),5.75–5.57(br.s,1H),5.20(s,2H),2.63–2.54(m,2H),2.49–2.42(m,2H),2.00–1.90(m,2H).
参考例45：中间体I-45制备
Figure PCTCN2019123514-appb-000081
向中间体I-44(450mg,1.4mmol)的乙醇溶液(10mL)中加入钯碳(20mg)，并于氢气氛围下，在80℃加热搅拌10小时。冷却至室温后，将反应液过滤，并在滤液中加入二氧化铂(20mg)。将反应液于氢气氛围下，在80℃加热搅拌10小时。将反应液过滤，滤液经硅胶色谱法分离纯化得到中间体I-45。
1HNMR(400MHz,DMSO-d 6)δ9.86(s,1H),7.39(d,J＝8.4Hz,1H),7.05–6.94(m,2H),3.19–3.06(m,1H),2.01–1.73(m,4H),1.69–1.44(m,4H).
参考例46：中间体I-46制备
Figure PCTCN2019123514-appb-000082
将中间体I-42(150mg,0.592mmol)、中间体I-45(136mg，0.592mmol)和三苯基磷(186mg，0.710mmol)溶于无水四氢呋喃(3mL)中，将反应液降至0℃后，氩气保护下搅拌20分钟，然后缓慢滴加DIAD(144mg，0.710mmol)。滴加完毕后，反应混合物升温至室温搅拌反应2小时。将混合物浓缩干，残余物经硅胶色谱法分离纯化得到中间体I-46。
LC-MS(ESI)[M+H] +466.1.
参考例47：中间体I-47制备
Figure PCTCN2019123514-appb-000083
2-呋喃硼酸(1.48g，13.25mmol)，3-三氟甲基-4-溴苯甲酸甲酯(2.50g，8.83mmol)，[1,1'-双(二苯基膦基)二茂铁]二氯化钯(644mg，0.88mmol)和三乙胺(1.79g，17.66mmol)混合于N,N-二甲基甲酰胺(50mL)中，反应液在氮气保护下85℃下搅拌过夜。将反应液降至室温后倒入水(500mL)中，用乙酸乙酯萃取(50mL×2)。合并有机相，用饱和食盐水(150mL)洗，无水硫酸钠干燥，过滤。滤液减压浓缩得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-47。
1H NMR(400MHz,Chloroform-d)δ8.42(d,J＝1.7Hz,1H),8.21(dd,J＝8.3,1.7Hz,1H),7.89(d,J＝8.2Hz,1H),7.59(d,J＝1.8Hz,1H),6.88(d,J＝3.4Hz,1H),6.55(dd,J＝3.5,1.8Hz,1H),3.96(s,3H).
参考例48：中间体I-48制备
Figure PCTCN2019123514-appb-000084
中间体I-47(2.00g，7.40mmol)和二氧化铂(84mg，0.37mmol)混合于乙酸乙酯(20mL)中，在一个大气压的氢气氛围下，反应液在35℃下搅拌20小时。将反应液过滤，滤液减压浓缩得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-48。
1H NMR(400MHz,Chloroform-d)δ8.28(s,1H),8.18(dd,J＝8.2,1.8Hz,1H),7.80(d,J＝8.2Hz,1H),5.25(t,J＝7.4Hz,1H),4.22–4.16(m,1H),4.00–3.94(m,1H),3.93(s,3H),2.43(dq,J＝13.3,6.8Hz,1H),2.07–1.99(m,2H),1.71–1.60(m,1H).
参考例49：中间体I-49制备
Figure PCTCN2019123514-appb-000085
将中间体I-48(380mg，1.39mmol)溶于四氢呋喃(5mL)中，将反应体系降温至-40℃，在氩气保护下，滴加二异丁基氢化铝的甲苯溶液(1.5M,3.71mL,5.56mmol)。加料完毕后，反应液在-40℃下搅拌反应半小时，然后室温搅拌过夜。将反应液降温至0℃，用水(1mL)缓慢地淬灭，混合物倒入水(50mL)中，用乙酸乙酯(20mL×3)萃取。合并有机相，并用饱和食盐水(30mL)洗，无水硫酸钠干燥，过滤。滤液减压浓缩得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-49。
1H NMR(400MHz,Chloroform-d)δ7.69(d,J＝8.1Hz,1H),7.62(s,1H),7.54(d,J＝8.1Hz,1H),5.26–5.18(m,1H),4.73(s,2H),4.22–4.15(m,1H),4.00–3.93(m,1H),2.46–2.35(m,1H),2.08–1.99(m,2H),1.78(s,1H),1.70–1.61(m,1H).
参考例50：中间体I-50制备
Figure PCTCN2019123514-appb-000086
中间体I-49(230mg，0.93mmol)，2-氟-4-羟基苯甲醛(157mg，1.12mmol)和三苯基膦(341mg，1.30mmol)混合于四氢呋喃(5mL)中，将反应体系降至0℃后，加入偶氮二甲酸二异丙酯(263mg，1.30mmol)，反应液在0℃下搅拌反应半小时，然后升至室温，在室温下搅拌过夜。减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-50。
LC-MS(ESI)[M+H+41] +410.2.
参考例51：中间体I-51制备
Figure PCTCN2019123514-appb-000087
将化合物3-呋喃硼酸(1.97g，17.61mmol)溶于DMF(30.0mL)中，依次加入3-三氟甲基-4-溴苯甲酸甲酯(3.84g，13.57mmol)、碳酸铯(8.83g，27.10mmol)和[1,1'-双(二苯基膦基)二茂铁]二氯化钯二氯甲烷络合物(335mg，0.410mmol)。反应液在氩气保护下升温至90℃搅拌反应16小时，将水(60mL)和乙酸乙酯(60mL)加入到反应液中，分液，水相用乙酸乙酯(60mL x 2)萃取，合并有机相，并用饱和食盐水(10mL)洗涤，无水硫酸钠干燥，过滤。滤液减压浓缩至干，粗品经硅胶色谱法分离纯化得到中间体I-51。
1H NMR(400MHz,Chloroform-d)δ8.42(d,J＝1.7Hz,1H),8.19(dd,J＝8.0,1.7Hz,1H),7.60(d,J＝1.5Hz,1H),7.54–7.48(m,2H),6.62–6.56(m,1H),3.97(s,3H).
参考例52：中间体I-52制备
Figure PCTCN2019123514-appb-000088
将中间体I-51(3.11g，11.51mmol)溶于甲醇(100mL)中，加入Pd/C(500mg，10％w/w)，反应混合物在氢气氛围下室温搅拌16小时。反应液过滤，滤液浓缩至干。粗品经硅胶色谱法分离纯化得中间体I-52。
1H NMR(400MHz,Chloroform-d)δ8.30(d,J＝1.8Hz,1H),8.20–8.15(m,1H),7.62(d,J＝8.3Hz,1H),4.18–4.04(m,2H),3.94(s,3H),3.92–3.82(m,3H),2.53–2.40(m,1H),2.02–1.91(m,1H).
参考例53：中间体I-53制备
Figure PCTCN2019123514-appb-000089
将中间体I-52(1.00g，3.65mmol)溶于无水四氢呋喃(25mL)中，降温至-40℃，氩气保护下缓慢滴加二异丁基氢化铝的甲苯溶液(1.5M，9.73mL，14.60mmol)，滴加完毕升温至室温搅拌2小时，反应完毕后将混合物降温至0℃，加入水(100mL)淬灭反应，加入乙酸乙酯(50mL)，分液，水相用乙酸乙酯(50mL x 2)萃取，有机相合并，经食盐水(20mL x 2)洗涤，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂，残余物经硅胶色谱法分离纯化得中间体I-53。
LC-MS(ESI)[M-H] -245.0.
参考例54：中间体I-54制备
Figure PCTCN2019123514-appb-000090
将中间体I-53(123mg，0.500mmol)溶于干燥四氢呋喃(2.00mL)中，氩气保护下加入2-氟-4-羟基苯甲醛(70.1mg，0.500mmol)和三苯基磷(157mg，0.600mmol)，室温搅拌20分钟后，降温至0℃，缓慢滴加偶氮二甲酸二异丙酯(121mg，0.600mmol)，滴加完毕反应液升至室温搅拌3小时，反应液减压浓缩至干。浓缩物中加入乙酸乙酯(10mL)，分别用水(10mLx3)洗，饱和食盐水(10mL)洗涤，无水硫酸钠干燥，过滤。滤液减压浓缩干后经硅胶色谱法分离纯化得中间体I-54。
LC-MS(ESI)[M-H] -：367.0
参考例55：中间体I-55制备
Figure PCTCN2019123514-appb-000091
在10℃下，将4-溴-3-(三氟甲基)苯甲酸甲酯(3.5g，12.37mmol)和3,6-二氢-2H-吡喃-4-硼酸频哪醇酯(3.11g，14.8mmol)溶于二氧六环/水(50mL,4/1)中,然后加入四三苯基磷钯(710mg,0.614mmol)和碳酸钾(5.1g,36.9mmol)。反应混合物在氩气保护下110℃搅拌反应3小时。向反应液中加水(200mL)，并用石油醚(50mL×4)萃取。合并有机相，并用饱和食盐水(50mL)洗，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得中间体I-55粗产物。该粗产品未经纯化直接用于下一步反应。
1H NMR(400MHz,CDCl 3)δ8.34(d,J＝1.4Hz,1H),8.16(dd,J＝8.0,1.4Hz,1H),7.35(t,J＝6.6Hz,1H),5.69(s,1H),4.28(q,J＝2.7Hz,2H),3.95(s,3H),3.92(t,J＝5.4Hz,2H),2.36(dt,J＝7.2,2.5Hz,2H).
参考例56：中间体I-56制备
Figure PCTCN2019123514-appb-000092
在18℃下，将中间体I-55(3.5g，12.23mmol)溶于乙醇(80mL)中，缓慢加入钯碳(100mg，10％w/w)。反应混合物在60℃和10个大气压下搅拌反应72小时。将反应液冷却至室温后，过滤除去混合物中的钯碳，滤液减压浓缩得到粗产品。粗产品经硅胶色谱法分离纯化得到目中间体I-56。
1H NMR(400MHz,CDCl 3)δ8.31(s,1H),8.19(d,J＝8.2Hz,1H),7.57(d,J＝8.2Hz,1H),4.10(dd,J＝11.5,4.1Hz,2H),3.94(s,3H),3.56(td,J＝11.8,1.7Hz,2H),3.25(t,J＝11.8Hz,1H),1.88(dd,J＝12.6,4.2Hz,2H),1.75–1.67(m,2H).
参考例57：中间体I-57制备
Figure PCTCN2019123514-appb-000093
将中间体I-56(2.0g,6.94mmol)溶于四氢呋喃中，在-60℃下将二异丁基氢化铝溶液(13.9mL,1.5M甲苯溶液)缓慢滴加到上述混合物中。反应混合物在-30℃下搅拌反应3小时后，将反应混合物倒入稀盐酸(100mL，2N)中，并用乙酸乙酯(50mL×3)萃取。合并有机相，用饱和碳酸氢钠(30mL)洗涤后，再用饱和食盐水(50mL)洗，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-57。
1H NMR(400MHz,CDCl 3)δ7.64(s,1H),7.53(d,J＝8.2Hz,1H),7.46(d,J＝8.1Hz,1H),4.71(s,2H),4.07(dd,J＝11.5,4.2Hz,2H),3.54(td,J＝11.9,1.6Hz,2H),3.18(t,J＝11.8Hz,1H),2.03(d,J＝4.8Hz,1H),1.85(qd,J＝12.4,4.3Hz,2H),1.69(d,J＝10.6Hz,2H).
参考例58：中间体I-58制备
Figure PCTCN2019123514-appb-000094
在10℃下，将中间体I-57(200mg,0.768mmol)和2-氟-4-羟基苯甲醛(161mg,1.15mmol)溶于四氢呋喃(3mL)，依次加入三苯基磷(404mg，1.54mmol)和偶氮二甲酸二异丙酯(311mg,1.54mmol)。反应混合物在30℃下搅拌反应5小时。将反应混合物倒入水(20mL)中，并用乙酸乙酯(5mL×4)萃取。合并有机相，并用饱和食盐水(5mL)洗，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-58。
1H NMR(400MHz,CDCl 3)δ10.22(s,1H),7.85(t,J＝8.4Hz,1H),7.69(s,1H),7.59(d,J＝8.1Hz,1H),7.53(d,J＝7.9Hz,1H),6.86(dd,J＝8.8,2.3Hz,1H),6.72(dd,J＝12.2,2.3Hz,1H),5.13(s,2H),4.09(dd,J＝11.6,4.3Hz,2H),3.56(td,J＝11.9,1.9Hz,2H),3.21(t,J＝11.9Hz,1H),1.87(dd,J＝12.6,4.1Hz,2H),1.72(dd,J＝12.9,1.8Hz,2H).
参考例59：中间体I-59制备
Figure PCTCN2019123514-appb-000095
在10℃下，将中间体I-58(90.0mg,0.235mmol)溶于甲醇(3mL)，依次加入3-甲酸甲酯氮杂环丁烷盐酸盐(71.2mg,0.470mmol)，二异丙基乙胺(60.7mg,0.470mmol)和冰乙酸(42.3mg,0.705mmol)。反应混合物在10℃下搅拌反应2小时。向反应体系中加入氰基硼氢化钠(29.5mg,0.470mmol)。反应混合物在10℃下继续搅拌反应1小时。将反应混合物倒入饱和碳酸氢钠溶液(20mL)，并用乙酸乙酯(8mL×3)萃取。合并有机相，用饱和食盐水(5mL)洗，无水硫酸钠干燥，过滤。滤液减压浓 缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-59。
LC-MS(ESI)[M+H] +482.2.
参考例60：中间体I-60制备
Figure PCTCN2019123514-appb-000096
在室温下，将3,5-二氟吡啶甲酸(3.10g，19.5mmol)溶于氢氧化锂水溶液(2N，97.5mL，195mmol)中。反应混合物在100℃下搅拌反应16小时。将反应液冷却至室温后，加入浓盐酸(12M)将反应混合物pH值调至4。反应混合物减压浓缩除去有机溶剂得到粗产品。
在室温下，将粗产品溶于甲醇(60mL)，滴加浓硫酸(5.00mL)。滴加完毕，反应混合物在70℃下搅拌反应16小时。将反应液冷却至室温后，减压浓缩除去有机溶剂得到浓缩物。往浓缩物中加水(100mL)，并用乙酸乙酯(80mL×3)萃取。合并有机相，用饱和食盐水(50mL)洗涤，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-60。
LC-MS(ESI)[M+H] +171.9.
参考例61：中间体I-61制备
Figure PCTCN2019123514-appb-000097
在室温下，将中间体I-60(500mg,2.92mmol)溶于乙腈(20mL)中。依次加入中间体I-13(1.08g，3.52mmol)，碳酸铯(2.85g,8.75mmol)。加料完毕后，反应混合物在室温下搅拌反应16小时。将反应混合物过滤，滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-61。
LC-MS(ESI)[M+H] +398.1.
参考例62：中间体I-62制备
Figure PCTCN2019123514-appb-000098
在0℃下，将中间体I-61(998mg，2.51mmol)的无水四氢呋喃(10mL)溶液滴加到氢化铝锂的四氢呋喃悬浊液(5.00mL，5.00mmol，1M)中。滴加完毕后，反应混合物在0℃下搅拌反应1小时。反应液在0℃下加饱和氯化铵淬灭(40mL)，并用乙酸乙酯(50mL×3)萃取。合并有机相，用饱和食盐水(40mL)洗涤，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-62。
LC-MS(ESI)[M+H] +370.0.
参考例63：中间体I-63制备
Figure PCTCN2019123514-appb-000099
在室温下，将中间体I-62(200mg，0.541mmol)溶于二氯甲烷(10mL)中，加入Dess-Martin氧化剂(413mg，0.974mmol)。反应混合物在室温下搅拌反应1小时。将反应混合物过滤，滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-63。
LC-MS(ESI)[M+H] +368.0.
参考例64：中间体I-64制备
Figure PCTCN2019123514-appb-000100
在室温下，将中间体I-63(158mg，0.430mmol)溶于甲醇(3mL)和二氯甲烷(3mL)中，依次加入氮杂环丁烷-3-甲酸甲酯盐酸盐(65.2mg，0.430mmol)、三乙胺(43.5mg，0.430mmol)和醋酸(51.6mg，0.859mmol)。反应混合物在40℃下搅拌反应5小时后，加入氰基硼氢化钠(54.0mg，0.859mmol)，并在40℃下继续搅拌反应2小时。将反应混合物冷却至室温后，减压浓缩除去有机溶剂得到浓缩物。往浓缩物中加水(20mL)，二氯甲烷(10mL×3)萃取。合并有机相，用饱和食盐水(20mL)洗涤，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-64。
LC-MS(ESI)[M+H] +467.1。
参考例65：中间体I-65制备
Figure PCTCN2019123514-appb-000101
中间体I-13(200mg，0.65mmol),4-羟基-2-甲氧基苯甲醛(99mg，0.65mmol)和碳酸钾(180mg，1.30mmol)混合于乙腈(5mL)中，反应液在50℃下搅拌过夜。将反应液降至室温后过滤，滤液减压浓缩得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-65。
LC-MS(ESI)[M+H] +379.2。
参考例66：中间体I-66制备
Figure PCTCN2019123514-appb-000102
在室温下，将2，4-二羟基苯甲酸甲酯(4.50g，26.8mmol)和中间体I-13(8.23g,26.8mmol)溶于丙酮中(50mL)中，然后将碳酸钾(7.41g,53.6mmol)加入上述体系。反应体系50℃下搅拌反 应4小时。将反应液冷却至室温，过滤。滤液加入水(60mL)稀释，并用乙酸乙酯萃取(50mL×3)。合并有机相，有机相用饱和食盐水(50mL)洗涤，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法纯化得到中间体I-66。
LC-MS(ESI)[M-H] -393.0。
参考例67：中间体I-67制备
Figure PCTCN2019123514-appb-000103
在0℃下，将三乙胺(1.02g，10.1mmol)加入中间体I-66(2.00g,5.07mmol)的二氯甲烷(30mL)溶液中，然后将三氟甲磺酸酐(2.15g,7.62mmol)缓慢加入到上述反应体系中。反应混合物升至室温，并在室温下搅拌反应3小时。反应液用二氯甲烷(30mL)稀释，并依次用水(30mL)和饱和食盐水(30mL)洗涤，有机相用无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法纯化得到中间体I-67。
LC-MS(ESI)[M-H] -525.2。
参考例68：中间体I-68制备
Figure PCTCN2019123514-appb-000104
在室温下，将四(三苯基膦)钯(164mg，0.142mmol)和碳酸钠(453mg,4.27mmol)加入到中间体I-67(750mg,1.42mmol)及异丙烯基硼酸频哪醇酯(958mg,5.70mmol)的二氧六环和水的混合溶液中(24mL,5:1)中。反应体系用氩气置换三次。在氩气保护下于90℃反应3小时。将反应液冷却至室温，加入水(20mL)稀释，并用乙酸乙酯萃取(20mL×3)。合并有机相，有机相用饱和食盐水(20mL)洗涤，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法纯化得到中间体I-68。
LC-MS(ESI)[M+H] +419.2。
参考例69：中间体I-69制备
Figure PCTCN2019123514-appb-000105
在室温下，将中间体I-68(594mg,1.42mmol)溶于甲醇(10mL)中，加入二氧化铂(60.0mg)。反应混合物用氢气置换三次，并于氢气氛围下室温搅拌反应3小时。将反应混合物过滤，滤液减压浓缩得到粗产品。粗产品经硅胶色谱法纯化得到中间体I-69。
LC-MS(ESI)[M+H] +421.2。
参考例70：中间体I-70制备
Figure PCTCN2019123514-appb-000106
在-30℃下，将DIBAL-H(3.78mL,1M in hexane，3.78mmol)加入到中间体I-69(530mg,1.26mmol)的四氢呋喃(15mL)溶液中。反应液于该温度下继续反应2小时。反应完毕后，于0℃下用水(10mL)和氢氧化钠水溶液(10mL，1M)淬灭。过滤，滤液用乙酸乙酯萃取(10mL×3)。合并有机相，有机相用饱和食盐水(20mL)洗涤，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到中间体I-70。
LC-MS(ESI)[M-H] -391.3。
参考例71：中间体I-71制备
Figure PCTCN2019123514-appb-000107
在氩气保护下，中间体I-70(494mg,1.26mmol)及二氧化锰(548mg,6.30mmol)的1,2-二氯乙烷(20mL)的混合溶液于70℃下反应5小时。冷却至室温，过滤。滤液减压浓缩得到粗产品。粗产品经硅胶色谱法纯化得到中间体I-71。
LC-MS(ESI)[M+H] +391.2。
参考例72：中间体I-72制备
Figure PCTCN2019123514-appb-000108
在室温下，将中间体I-71(120mg，0.307mmol)和氮杂环丁烷-3-甲酸甲酯盐酸盐(93.1mg,0.614mmol)溶于甲醇(10mL)中，将N,N-二异丙基乙胺(79.4mg,0.614mmol)和醋酸(55.3mg，0.921mmol)依次加入至上述体系中。反应混合物于室温反应8小时。然后将氰基硼氢化钠(38.6mg，0.614mmol)加入反应混合物中，并于50℃下继续反应16小时。反应液冷却至室温，用水(20mL)稀释，并用乙酸乙酯萃取(15mL×3)。合并有机相，有机相用饱和食盐水(20mL)洗，无水硫酸钠干燥，过滤。滤液减压浓缩得到粗产品，粗产品经硅胶色谱法纯化得到中间体I-72。
LC-MS(ESI)[M+H] +490.4。
参考例73：中间体I-73制备
Figure PCTCN2019123514-appb-000109
在室温下，将四(三苯基膦)钯(49.2mg，0.0426mmol)和碳酸钠(452mg,4.26mmol)加入到中间体I-67(750mg,1.42mmol)及环丙基硼酸(366mg,4.26mmol)的二氧六环和水的混合溶液中(36mL,5:1)中。反应体系用氩气置换三次。在氩气保护下于100℃反应5小时。将反应液冷却至室温。加入水(20mL)稀释，并用乙酸乙酯萃取(20mL×3)。合并有机相，有机相用饱和食盐水(20mL)洗涤，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法纯化得到中间体I-73。
LC-MS(ESI)[M-H] -417.2。
参考例74：中间体I-74制备
Figure PCTCN2019123514-appb-000110
在-30℃下，将DIBAL-H(3.72mL,1M in hexane，3.72mmol)加入到中间体I-73(520mg,1.24mmol)的四氢呋喃(10mL)溶液中。反应液于该温度下继续反应1小时。反应完毕后，于0℃下用水(5mL)和氢氧化钠水溶液(5mL，1M)淬灭。过滤，滤液用乙酸乙酯萃取(10mL×3)。合并有机相，有机相用饱和食盐水(20mL)洗涤，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到中间体I-74，产品未经进一步纯化直接用于下一步反应。
LC-MS(ESI)[M+H-18] +373.2。
参考例75：中间体I-75制备
Figure PCTCN2019123514-appb-000111
在氩气保护下，中间体I-74(484mg,1.24mmol)及二氧化锰(539mg,6.20mmol)的1,2-二氯乙烷(15mL)的混合溶液于70℃下反应3小时。冷却至室温，过滤。滤液减压浓缩得到粗产品。粗产品经硅胶色谱法纯化得到中间体I-75。
LC-MS(ESI)[M+H] +389.2。
参考例76：中间体I-76制备
Figure PCTCN2019123514-appb-000112
在室温下，将中间体I-75(200mg，0.515mmol)和氮杂环丁烷-3-甲酸甲酯盐酸盐(156mg，1.03 mmol)溶于甲醇(10mL)中，将N,N-二异丙基乙胺(133mg,1.03mmol)和醋酸(93.1mg，1.55mmol)依次加入至上述体系中。反应混合物于室温反应5小时。然后将氰基硼氢化钠(64.7mg，1.03mmol)加入反应混合物中，并于50℃下继续反应16小时。反应液冷却至室温，用水(20mL)稀释，并用乙酸乙酯萃取(15mL×3)。合并有机相，有机相用饱和食盐水(20mL)洗，无水硫酸钠干燥，过滤。滤液减压浓缩得到粗产品，粗产品经硅胶色谱法纯化得到中间体I-76。
LC-MS(ESI)[M+H] +488.3。
参考例77：中间体I-77制备
Figure PCTCN2019123514-appb-000113
在室温下，将中间体I-66(700mg，1.77mmol)溶于乙腈(10mL)中,依次加入2-碘丙烷(3.0g，17.6mmol)和碳酸铯(1.73g，5.31mmol)。将反应混合物在45℃搅拌下过夜。减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-77。
1H NMR(400MHz,CDCl 3)δ7.82(d,J＝9.1Hz,1H),7.65(s,1H),7.55(d,J＝8.2Hz,1H),7.49(d,J＝8.2Hz,1H),6.58–6.53(m,2H),5.07(s,2H),4.56–4.47(m,1H),3.84(s,3H),3.42–3.32(m,1H),2.14–2.05(m,2H),1.89–1.58(m,6H),1.36(d,J＝6.1Hz,6H).
参考例78：中间体I-78制备
Figure PCTCN2019123514-appb-000114
在-45℃，将中间体I-77(770mg，1.76mmol)溶于四氢呋喃(5mL)中，加入二异丁基氢化铝(7mL，1M in hexane,7.00mmol)。将反应混合物在-45℃搅拌下反应1.5小时。在0℃下，通过加入饱和氯化铵水溶液来淬灭反应(20mL)。加入乙酸乙酯(60mL)分液萃取，水相用乙酸乙酯(60mL×2)提取得到有机相。合并有机相，用饱和食盐水(50mL)洗，无水硫酸钠干燥，过滤。滤液减压浓缩得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-78。
1H NMR(400MHz,CDCl 3)δ7.66(s,1H),7.55(d,J＝8.6Hz,1H),7.48(d,J＝8.1Hz,1H),7.16(d,J＝8.2Hz,1H),6.55–6.52(m,1H),6.49(dd,J＝8.2,2.3Hz,1H),5.03(s,2H),4.60(s,2H),4.58–4.50(m,1H),3.42–3.32(m,1H),2.14–2.06(m,2H),1.90–1.59(m,6H),1.35(d,J＝6.1Hz,6H).
参考例79：中间体I-79制备
Figure PCTCN2019123514-appb-000115
在室温下，将中间体I-78溶于1,2二氯乙烷(6mL)中，加入二氧化锰(426mg，4.9mmol)。反应混合物在氩气保护下70℃搅拌过夜，降至室温，过滤。滤液减压浓缩得到粗产品I-79。粗产品没有进一步纯化直接用于下一步反应。
1H NMR(400MHz,CDCl 3)δ10.32(s,1H),7.82(d,J＝8.7Hz,1H),7.66(s,1H),7.55(d,J＝8.4Hz,1H),7.50(d,J＝8.2Hz,1H),6.61–6.57(m,1H),6.51(s,1H),5.10(s,2H),4.64–4.57(m,1H),3.42–3.34(m,1H),2.15–2.07(m,2H),1.92–1.61(m,6H),1.38(d,J＝6.0Hz,6H).
参考例80：中间体I-80制备
Figure PCTCN2019123514-appb-000116
在室温下，将中间体I-79和氮杂环丁烷-3-羧酸甲酯盐酸盐(59mg，0.39mmol)溶于甲醇(3mL)中，依次加入N,N-二异丙基乙胺(50mg，0.39mmol)和冰乙酸(47mg，0.78mmol)。反应混合物在室温搅拌下反应6小时后，将氰基硼氢化钠(49mg，0.78mmol)加入到反应体系中继续在室温搅拌下过夜。反应体系中加入水(10mL)和乙酸乙酯(30mL)分液萃取，水相用乙酸乙酯(30mL×2)提取得到有机相。合并有机相，用饱和食盐水(20mL)洗，无水硫酸钠干燥，过滤。滤液减压浓缩得到粗产品I-80。粗产品没有进一步纯化直接用于下一步反应。
LC-MS(ESI)[M+H] +506.6
参考例81：中间体I-81制备
Figure PCTCN2019123514-appb-000117
将4-溴-2-氯苯酚(5.00g，24.1mmol),咪唑(4.11g,60.3mmol)和三异丙氯硅烷(5.57g,28.9mmol)溶于DCM(40.0mL)中。反应混合物在25℃下搅拌反应10小时。反应液用二氯甲烷(200mL)稀释后用饱和食盐水(300mL)洗涤。有机相减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-81。
1H NMR(400MHz,CD 3OD)δ7.51(d,J＝2.4Hz,1H),7.30(dd,J＝8.8,2.4Hz,1H),6.88(d,J＝8.7Hz,1H),1.33(m,3H),1.13(d,J＝7.4Hz,18H).
参考例82：中间体I-82制备
Figure PCTCN2019123514-appb-000118
将二异丙胺(612mg,6.05mmol)溶到无水四氢呋喃(20.0mL)中，在氮气的保护下，把其冷却至-78℃，然后缓慢滴加n-BuLi(3.61mL,1.6M in hexane,5.78mmol)。并在该温度下搅拌反应0.75小时。然后继续在-78℃下，将中间体I-81(2.00g，5.50mmol)缓慢滴到该体系，在该温度下搅拌反应0.5小时。最后将碘甲烷(937mg,6.60mmol)在-78℃下缓慢加入该体系。反应液缓慢升至室温并搅拌过夜。反应混合物用饱和氯化铵溶液(15.0mL)淬灭该反应后加入饱和食盐水(150mL),并用乙酸乙酯(100 mL×2)萃取。合并有机相，并用无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-82。
1H NMR(400MHz,CDCl 3)δ7.28(d,J＝8.8Hz,1H),6.65(d,J＝8.8Hz,1H),2.50(s,3H),1.30(m,3H),1.12(d,J＝7.4Hz,18H).
参考例83：中间体I-83制备
Figure PCTCN2019123514-appb-000119
将中间体I-82(1.00g,2.65mmol)溶于无水四氢呋喃(15.0mL)中。在氮气的保护下，把其冷却至-78℃，然后缓慢滴加正丁基锂(1.99mL,1.6M正己烷溶液，3.18mmol)。并在该温度下搅拌反应1小时。并在-78℃下再将无水N，N-二甲基甲酰胺(290.6mg,3.975mmol)缓慢滴到该溶液，并在该温度下搅拌反应2小时后用饱和氯化铵溶液(10.0mL)淬灭该反应。加入水(40mL),并用乙酸乙酯(60mL×3)萃取。合并有机相，并用无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-83。
1H NMR(400MHz,CDCl 3)δ10.14(s,1H),7.62(d,J＝8.5Hz,1H),6.88(d,J＝8.5Hz,1H),2.74(s,3H),1.35(m,3H),1.13(d,J＝7.4Hz,18H).
参考例84：中间体I-84制备
Figure PCTCN2019123514-appb-000120
将中间体I-83(650mg,1.99mmol)和TBAF(1.04g,3.98mmol)溶于无水四氢呋喃(15.0mL)中，并在25℃下反应10小时。将反应液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-84。
1H NMR(400MHz,CDCl 3)δ10.12(s,1H),7.70(d,J＝8.5Hz,1H),7.03(d,J＝8.5Hz,1H),2.74(s,3H).
参考例85：中间体I-85制备
Figure PCTCN2019123514-appb-000121
将中间体I-84(80.0mg,0.469mmol),中间体I-13(173mg,0.563mmol)和碳酸钾(195mg,1.41mmol)溶于乙腈(5.00mL)中，把温度升至80℃，并在该温度下反应10小时。反应液冷却至室温，加入水(20.0mL)并用乙酸乙酯(15mL×2)萃取。合并有机相，用饱和食盐水(10.0mL)洗，有机相用无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品用硅胶色谱法分离纯化得到中间体I-85。
1H NMR(400MHz,CDCl 3)δ10.15(s,1H),7.73(d,J＝8.6Hz,1H),7.69(s,1H),7.62(d,J＝8.2Hz,1H),7.51(d,J＝8.2Hz,1H),6.96(d,J＝8.6Hz,1H),5.22(s,2H),3.38(m,1H),2.76(s,3H),2.10(m,2H),1.86(m,2H),1.74(m,2H),1.62(m,2H).
参考例86：中间体I-86制备
Figure PCTCN2019123514-appb-000122
将中间体I-85(80.0mg,0.202mmol),3-氮杂环丁烷甲酸甲酯盐酸盐(30.6mg,0.202mmol)和DIEA(26.1mg,0.202mmol)溶于甲醇(3.00mL)中，然后加入乙酸(24.26mg,0.404mmol)。将体系内的气体用氮气置换，在25℃下反应3小时。然后加入氰基硼氢化钠(12.7mg,0.202mmol),在25℃下继续反应10小时。将反应液加入水(10.0mL),并用乙酸乙酯(15.0mL×2)萃取。合并有机相并用无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂的中间体I-86.该粗产品未经纯化直接用于下一步反应。LC-MS(ESI)[M+H] +496.2
参考例87：中间体I-87制备
Figure PCTCN2019123514-appb-000123
将邻甲基对溴苯甲酸(5.00g，23.3mmol)溶于N,N-二甲基甲酰胺(50.0mL)中，依次加入氯代丁二酰亚胺(3.11g，23.3mmol)和醋酸钯(523mg，2.33mmol)。反应液升温至100℃搅拌反应24小时。将反应液冷却至室温后，加入水(200mL)，混合液用乙酸乙酯(40mL*3)萃取。合并有机相，并用饱和食盐水(40mL)洗涤，经无水硫酸钠干燥，过滤。滤液减压浓缩至干，残余物经硅胶色谱法分离纯化得中间体I-87。
1H NMR(400MHz,Methanol-d 4)δ7.49(s,1H),7.43–7.41(s,1H),2.34(s,3H).
参考例88：中间体I-88制备
Figure PCTCN2019123514-appb-000124
将中间体I-87(4.50g)溶于N,N-二甲基甲酰胺(40.0mL)中，依次加入碘甲烷(5.11g，36.0mmol)和碳酸钾(7.46g，54.0mmol)。反应液升温至40℃搅拌反应2小时。在40℃加入水(200mL)，混合液用乙酸乙酯(40mL*3)萃取。合并有机相，并用饱和食盐水(40mL)洗涤，经无水硫酸钠干燥，过滤。滤液减压浓缩至干，残余物经硅胶色谱法分离纯化得中间体I-88。
1H NMR(400MHz,Chloroform-d)δ7.41(s,1H),7.29–7.27(s,1H),3.94(s,3H),2.30(s,3H).
参考例89：中间体I-89制备
Figure PCTCN2019123514-appb-000125
将中间体I-88(3.30g，12.5mmol)溶于二甲亚砜(40.0mL)中，依次加入联硼酸频那醇酯(4.14 g，16.3mmol)、[1,1'-双(二苯基膦基)二茂铁]二氯化钯(0.915g，1.25mmol)和醋酸钾(3.68g，37.5mmol)。将反应液升温至100℃搅拌反应8小时后，冷却至室温，加入水(50mL)，用乙酸乙酯(50mL*3)萃取，有机相合并，用饱和食盐水(50mL)洗涤，经无水硫酸钠干燥，过滤，滤液减压浓缩至干，残余物经硅胶色谱法分离纯化得中间体I-89。
1H NMR(400MHz,Chloroform-d)δ7.64(s,1H),7.52(s,1H),3.95–3.93(s,3H),2.31(s,3H),1.26(s,12H).
参考例90：中间体I-90的制备
Figure PCTCN2019123514-appb-000126
将中间体I-89(3.31g，10.7mmol)溶于四氢呋喃(30mL)中，加入氢氧化钠溶液(1M，10.7mL，10.7mmol)，搅拌下缓慢加入双氧水(30％w/w，2.41g，21.2mmol)。将反应液升温至40℃搅拌反应1小时，然后在40℃下加入水(50mL)，用乙酸乙酯(50mL*3)萃取，有机相合并，用饱和食盐水(30mL)洗涤，经无水硫酸钠干燥，过滤，滤液减压浓缩至干，残余物经硅胶色谱法分离纯化得中间体I-90。
LC-MS(ESI)[M-H] -199.0.
参考例91：中间体I-91的制备
Figure PCTCN2019123514-appb-000127
将中间体I-90(602mg，3.00mmol)溶于乙腈(10.0mL)中，依次加入中间体I-13(1.38g，4.50mmol)和碳酸铯(1.95g，6.00mmol)。反应液升温至40℃搅拌反应4小时，将反应液将至室温，过滤，滤液浓缩干，残余物经硅胶色谱法分离纯化得中间体I-91。
LC-MS(ESI)[M-H] -425.1.
参考例92：中间体I-92的制备
Figure PCTCN2019123514-appb-000128
将中间体I-91(996mg，2.33mmol)溶于四氢呋喃(10.0mL)中，降温至-40℃在氩气保护下缓慢滴加DIBAL-H的正己烷溶液(1M，6.99mL)，滴加完毕后升温至室温继续搅拌4小时。将反应液倒入1N冰盐酸溶液中(50mL)，用乙酸乙酯(40mL*3)萃取，有机相合并，用饱和食盐水(40mL)洗涤，经无水硫酸钠干燥，过滤，滤液减压浓缩至干得中间体I-92直接用于下一步反应。
LC-MS(ESI)[M-H] -397.1.
参考例93：中间体I-93的制备
Figure PCTCN2019123514-appb-000129
将中间体I-92(720mg，1.81mmol)溶于二氯乙烷(5.00mL)中，加入二氧化锰(785mg，9.03mmol)。将反应液升温至60℃搅拌反应5小时，热过滤，滤液减压浓缩至干，残余物经硅胶色谱法分离纯化得中间体I-93。
LC-MS(ESI)[M-H] -395.1.
参考例94：中间体I-94的制备
Figure PCTCN2019123514-appb-000130
将中间体I-93(140mg，0.353mmol)和吖叮酸甲酯盐酸盐(161mg，1.06mmol)溶于甲醇(3.00mL)中，加入冰乙酸(0.50mL)，反应液升温至50℃，氩气保护下搅拌反应20小时，加入氰基硼氢化钠(88.7mg，1.41mmol)后，继续在50℃搅拌2小时。将反应液冷却至室温，混合物中加入水(5mL)，经乙酸乙酯(20mL x 3)萃取，有机相合并，真空减压浓缩并硅胶色谱法分离纯化得到中间体I-94。
LC-MS(ESI)[M+H] +496.2。
参考例95：中间体I-95的制备
Figure PCTCN2019123514-appb-000131
在0℃下，向4-甲基水杨酸甲酯(6.00g，36.1mmol)的四氢呋喃(100mL)溶液中，加入60％氢化钠(2.17g,54.2mmol)。反应混合物在10℃下搅拌反应30分钟。在10℃下，向反应混合物中加入碘甲烷(7.69g,54.2mmol)，加料完毕，反应混合物升温至20℃并搅拌反应8小时。反应混合物用饱和氯化铵(400mL)淬灭后，用乙酸乙酯(100mL×4)萃取。合并有机相，用饱和食盐水(200mL)洗，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-95。
1H NMR(400MHz,CDCl 3)δ7.72(d,J＝8.4Hz,1H),6.79(d,J＝6.8Hz,2H),3.90(s,3H),3.87(s,3H),2.38(s,3H).
参考例96：中间体I-96的制备
Figure PCTCN2019123514-appb-000132
在室温下，向中间体I-95(3.00g，16.6mmol)的四氯化碳(30.0mL)溶液中，加入N-溴代丁二酰亚胺(2.95g，16.6mmol)和过氧化苯甲酰(40.2mg，1.66mmol)。反应混合物在80℃下搅拌反应8小时。将反应体系冷却至室温，倒入水(100mL)中，并用乙酸乙酯(50mL×4)萃取。合并有机相，用饱和食盐水(50mL)洗，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-96。
1H NMR(400MHz,CDCl 3)δ7.77(d,J＝8.3Hz,1H),7.00(d,J＝1.6Hz,2H),4.46(s,2H),3.93(s,3H),3.89(s,3H).
参考例97：中间体I-97的制备
Figure PCTCN2019123514-appb-000133
在室温下，将中间体I-96(900mg,3.47mmol)和中间体I-45(879mg,3.82mmol)溶于N,N-二甲基甲酰胺(15.0mL)，然后加入碳酸铯(3.39g,10.4mmol)。反应混合物在50℃下搅拌反应3小时。将反应体系冷却至室温，倒入水(50mL)中，并用乙酸乙酯(20mL×4)萃取。合并有机相，用饱和食盐水(20mL)洗，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-97。
1H NMR(400MHz,CDCl 3)δ7.82(d,J＝7.8Hz,1H),7.37(d,J＝8.7Hz,1H),7.20(d,J＝2.8Hz,1H),7.13–6.98(m,3H),5.09(s,2H),3.92(s,3H),3.89(s,3H),3.38–3.24(m,1H),2.13–1.98(m,2H),1.89–1.64(m,4H),1.53(m,2H).
参考例98：中间体I-98的制备
Figure PCTCN2019123514-appb-000134
在0℃下，将中间体I-97(700mg,1.71mmol)溶于四氢呋喃(15.0mL)，然后在0℃下加入四氢铝锂(71.3mg，1.88mmol)。反应混合物升温至10℃并搅拌反应2小时。向反应体系加入水/四氢呋喃(71mg/20mL)，体系在室温下搅拌10分钟，加入氢氧化钠溶液(71mg，10％w/w)，混合物在室温下搅拌30分钟，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-98。
1H NMR(400MHz,CDCl 3)δ7.36(d,J＝8.8Hz,1H),7.29(d,J＝7.6Hz,1H),7.21(d,J＝2.8Hz,1H),7.08(dd,J＝8.7,2.7Hz,1H),7.02–6.94(m,2H),5.05(s,2H),4.69(d,J＝6.4Hz,2H),3.89(s,3H),3.37–3.23(m,1H),2.11–2.01(m,2H),1.83(m,2H),1.71(m,2H),1.51(m,2H).
参考例99：中间体I-99的制备
Figure PCTCN2019123514-appb-000135
在室温下，将中间体I-98(520mg,1.37mmol)溶于二氯乙烷(15.0mL)，然后加入二氧化锰(956mg,11.0mmol)。反应混合物在80℃下搅拌反应3小时。将反应体系冷却至室温，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-99。
1H NMR(400MHz,CDCl 3)δ10.45(s,1H),7.85(d,J＝8.1Hz,1H),7.38(d,J＝8.7Hz,1H),7.20(d,J＝2.6Hz,1H),7.08(m,3H),5.11(s,2H),3.95(s,3H),3.38–3.23(m,1H),2.05(s,2H),1.83(s,2H),1.76–1.66(m,2H),1.55–1.47(m,2H).
参考例100：中间体I-100的制备
Figure PCTCN2019123514-appb-000136
在中间体I-99(150mg,0.396mmol)的甲醇溶液中(2.0mL)分别加入3-吖啶酸甲酸甲酯盐酸盐(120mg,0.792mmol)，二异丙基乙胺(102mg,0.792mmol)和冰乙酸(71.5mg,1.19mmol)。在室温下搅拌1小时后，向上述反应体系中加入三乙酰氧基硼氢化钠(168mg,0.792mmol)，然后继续搅拌8小时。向上述反应体系中倒入水(10mL)，用乙酸乙酯(5mL×4)萃取。合并有机相，用饱和食盐水(10mL)洗，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-100。
LC-MS(ESI)[M+H] +478.2.
参考例101：中间体I-101的制备
Figure PCTCN2019123514-appb-000137
将中间体I-38(200mg，0.768mmol)溶于四氢呋喃(10.0mL)中。在0℃下向反应体系中依次加入4-羟基-2-甲氧基-苯甲醛(140mg，0.922mmol)，三苯基膦(302mg，1.15mmol)和DIAD(233mg，1.15mmol)。反应混合物在室温下搅拌反应3小时。反应液减压浓缩得到粗产品，粗产品经硅胶色谱法分离纯化得到中间体I-101。
LC-MS(ESI)[M+H] +395.2。
参考例102：中间体I-102的制备
Figure PCTCN2019123514-appb-000138
将中间体I-31(1.00g，3.87mmol)混合于40％氢溴酸水溶液(10mL)中，反应混合物在100℃下搅拌反应2小时。将反应混合物冷却至室温，二氯甲烷萃取(10mL×2)。合并有机相，用饱和碳酸氢钠水溶液(20mL)洗，饱和食盐水(20mL)洗，无水硫酸钠干燥，过滤，滤液浓缩除去有机溶剂得 到粗产品。粗产品经硅胶色谱法分离纯化得到中间体I-102。
1H NMR(400MHz,Chloroform-d)δ7.61(d,J＝1.9Hz,1H),7.51(dd,J＝8.1,2.0Hz,1H),7.43(d,J＝8.1Hz,1H),4.48(s,2H),2.98–2.86(m,1H),1.90–1.73(m,5H),1.49–1.25(m,5H)。
参考例103：中间体I-103的制备
Figure PCTCN2019123514-appb-000139
将4-羟基-2-甲氧基-苯甲醛(109mg，0.716mmol)溶于乙腈(10mL)中。向反应体系中依次加入中间体I-102(230mg，0.716mmol)和碳酸钾(198mg，1.43mmol)。反应混合物在45℃下搅拌反应3小时。反应液减压浓缩得到粗产品，粗产品经硅胶色谱法分离纯化得到中间体I-103。
LC-MS(ESI)[M+H] +393.6。
1H NMR(400MHz,DMSO-d 6)δ10.18(s,1H),7.77(s,1H),7.73(d,J＝8.3Hz,1H),7.68(d,J＝5.8Hz,1H),7.66(d,J＝5.2Hz,1H),6.81(d,J＝2.2Hz,1H),6.75(dd,J＝8.6,1.8Hz,1H),5.28(s,2H),3.90(s,3H),2.82(t,J＝11.4Hz,1H),1.85–1.78(m,2H),1.74–1.66(m,3H),1.59–1.48(m,2H),1.41–1.28(m,3H).
实施例的制备：
实施例1：化合物1的制备
Figure PCTCN2019123514-appb-000140
将中间体I-4(93mg,0.25mmol)和3-羧酸环丁胺(51mg,0.5mmol)混悬于四氢呋喃和甲醇的混合液(5mL，1:1)中，加入氰基硼氢化钠(47mg,0.75mmol)和醋酸(0.2mL)后，将反应液于室温搅拌过夜。蒸去大部分反应液，向残留物中加入水(10mL)和乙酸乙酯(30mL)后分离有机相，水层用乙酸乙酯萃取(30mL×2)。将合并的有机层经饱和食盐水洗涤，无水硫酸钠干燥后过滤浓缩，得残留物。残留物经制备HPLC纯化后得化合物1。
LC-MS(ESI)[M+H] +452.1.
1H NMR(400MHz,MeOH-d 4)δ7.69–7.57(m,3H),7.46–7.41(m,1H),6.98–6.90(m,2H),5.17(s,2H),4.42(s,2H),4.36–4.23(m,4H),3.66–3.59(m,1H),3.39–3.34(m,1H),2.10–2.02(m,2H),1.95–1.85(m,2H),1.79–1.70(m,2H),1.68–1.59(m,2H).
实施例2：化合物2的制备
Figure PCTCN2019123514-appb-000141
将中间体I-12(110mg,0.293mmol)和3-羧酸环丁胺(44.3mg,0.439mmol)混悬于四氢呋喃(18mL)和甲醇(18mL)的混合液中，加入醋酸(0.01mL)并将反应液于室温下搅拌16小时。加入氰基 硼氢化钠(55.4mg,0.882mmol)，并继续室温搅拌3小时。向反应液中加入50mL水，用乙酸乙酯萃取(30mL×3)。将合并的有机层经饱和食盐水洗涤，无水硫酸钠干燥后过滤浓缩，得残留物。残留物经制备HPLC纯化后得化合物2。
LC-MS(ESI)[M+H] +462.2.
1H NMR(400MHz,DMSO-d 6)δ7.75–7.67(m,2H),7.67–7.61(m,1H),7.14(d,J＝8.4Hz,1H),6.85–6.80(m,1H),6.79–6.73(m,1H),5.11(s,2H),3.46(s,2H),3.28–3.22(m,3H),3.18–3.11(m,3H),2.61(q,J＝7.2Hz,2H),2.04–1.95(m,2H),1.88–1.79(m,2H),1.71–1.56(m,4H),1.13(t,J＝7.5Hz,3H).
实施例3：化合物3的制备
Figure PCTCN2019123514-appb-000142
将中间体I-14(210mg,0.60mmol)和3-羧酸环丁胺(60mg,0.60mmol)混悬于于四氢呋喃和甲醇的混合液(1:4,20mL)中，加入醋酸(1mL)并将反应液于室温下搅拌2小时。加入氰基硼氢化钠(226mg,3.6mmol)，并继续搅拌16小时。将反应液蒸干，向残留物中加入乙酸乙酯(10mL)和水(10mL)，将有机层分离，浓缩得残留物。残留物经制备HPLC纯化后得化合物3。
LC-MS(ESI)[M+H] +434.3.
1H NMR(400MHz,CDCl 3)δ7.62(s,1H),7.55–7.44(m,2H),7.36(d,J＝8.3Hz,2H),6.95(d,J＝8.4Hz,2H),5.00(s,2H),4.30–3.90(m,6H),3.48–3.30(m,2H),2.15–1.99(m,2H),1.92–1.77(m,2H),1.78–1.65(m,2H),1.64–1.52(m,2H).
实施例4：化合物4的制备
Figure PCTCN2019123514-appb-000143
将中间体I-15(100mg,0.26mmol)和3-羧酸环丁胺(53mg,0.52mmol)混悬于四氢呋喃和甲醇的混合液(9mL,1:3)中。加入氰基硼氢化钠(193mg,3.12mmol)和醋酸(0.5mL)，并将反应液于室温搅拌两天。向反应液中加入0.5mL盐酸(2N)，并经制备HPLC纯化后得化合物4。
LC-MS(ESI)[M+H] +468.1.
1H NMR(400MHz,MeOH-d 4)δ7.68(s,1H),7.66–7.62(m,1H),7.59(d,J＝8.2Hz,1H),7.49(d,J＝8.6Hz,1H),7.21(d,J＝2.5Hz,1H),7.06(dd,J＝8.6,2.6Hz,1H),5.16(s,2H),4.41(s,2H),4.24–4.12(m,4H),3.44–3.35(m,2H),2.10–2.02(m,2H),1.94–1.86(m,2H),1.78–1.70(m,2H),1.68–1.59(m,2H).
实施例5：化合物5的制备
Figure PCTCN2019123514-appb-000144
将中间体I-16(180mg,0.50mmol)和3-羧酸环丁胺(50.5mg,0.50mmol)混悬于四氢呋喃和甲醇的混合液(1:1,10mL)中，加入醋酸(0.5mL)。室温下搅拌2小时后，加入氰基硼氢化钠(94.5mg,1.50mmol)，并将反应液于室温搅拌16小时。加入20mL水和20mL乙酸乙酯，萃取分出有机层，水层继续用乙酸乙酯萃取(20mL×2)。将合并的有机层经饱和食盐水洗涤，浓缩，得残留物。残留物经制备HPLC纯化后得化合物5。
LC-MS(ESI)[M+H] +448.3.
H NMR(400MHz,MeOH-d 4)δ7.66(s,1H),7.65-7.55(m,2H),7.32(d,J＝8.5Hz,1H),6.96(d,J＝2.5Hz,1H),6.91(dd,J＝8.4,2.6Hz,1H),5.13(s,2H),4.35(s,2H),4.25–4.13(m,4H),3.45–3.33(m,2H),2.40(s,3H),2.11–2.02(m,2H),1.95–1.85(m,2H),1.79–1.59(m,4H).
实施例6：化合物6的制备
Figure PCTCN2019123514-appb-000145
将中间体I-18((55.0mg,0.15mmol)和3-羧酸环丁胺(22.2mg,0.22mmol)混悬于四氢呋喃和甲醇的混合液(5mL/5mL)中。加入氰基硼氢化钠(27.7mg,0.44mmol)和醋酸(3滴)，并将反应液于室温搅拌16小时。用盐酸(1N)将反应液pH调至3后，将反应液蒸干，残留物经制备HPLC纯化后得化合物6。
LC-MS(ESI)[M+H] +459.2.
1H NMR(400MHz,MeOH-d 4)δ7.69(s,1H),7.67–7.63(m,1H),7.62–7.55(m,2H),7.49(d,J＝2.7Hz,1H),7.38(dd,J＝8.7,2.7Hz,1H),5.20(s,2H),4.31(s,2H),4.14–4.00(m,4H),3.45–3.35(m,2H),2.12–2.03(m,2H),1.95–1.86(m,2H),1.79–1.70(m,2H),1.69–1.59(m,2H).
实施例7：化合物7的制备
Figure PCTCN2019123514-appb-000146
将中间体I-26(30mg,粗产物)和氢氧化锂一水合物(5.34mg,127.35μmol)溶于四氢呋喃(0.500mL)，甲醇(0.500mL)和水(0.200mL)中。反应混合物在25℃下搅拌反应10小时后，直接把反应液减压浓缩除去有机溶剂和水得到粗产品。粗产品用制备HPLC纯化后得化合物7。
LC-MS(ESI)[M+H] +453.2.
1H NMR(400MHz,CD 3OD)δ8.82(s,1H),8.10(s,1H),7.46(m,1H),7.09–6.88(m,2H),5.23(s,2H),4.34(s,2H),4.16(m,4H),3.57-3.46(m,1H),3.44-3.37(m,1H),2.11-1.96(m,2H),1.96-1.82(m,4H),1.81-1.65(m,2H).
实施例8：化合物8的制备
Figure PCTCN2019123514-appb-000147
在室温下，将中间体I-28(140mg,0.38mmol)和氮杂环丁烷-3-羧酸甲酯盐酸盐(58mg，0.38mmol)溶于甲醇(3mL)中，依次加入N,N-二异丙基乙胺(49mg，0.38mmol)和冰乙酸(46mg，0.766mmol)。反应混合物在室温搅拌下反应6小时后，将氰基硼氢化钠(48mg，0.76mmol)加入到反应体系中，并继续在室温搅拌下过夜。向反应液中加入四氢呋喃/水(2mL，4：1)，加入氢氧化锂单水合物(32.0mg，0.76mmol)。反应混合物在室温搅拌下反应2小时后。减压浓缩除去有机溶剂得到粗产品。经制备HPLC纯化得到化合物8。
LC-MS(ESI)[M+H] +450.2
1H NMR(400MHz,CDCl 3)δ7.66(s,1H),7.59–7.39(m,2H),7.32–7.27(m,1H),6.80(d,J＝8.0Hz,1H),6.72(d,J＝11.0Hz,1H),5.71(s,1H),5.01(s,2H),4.51–3.94(m,6H),3.62–3.49(m,1H),2.68–2.57(m,2H),2.55–2.44(m,2H),2.08–1.93(m,2H).
实施例9：化合物9的制备
Figure PCTCN2019123514-appb-000148
在室温下，将中间体I-32(90.0mg,0.24mmol)，3-吖丁啶羧酸(121mg,1.20mmol)和冰乙酸(18.7mg,0.312mmol)溶于四氢呋喃(2.00mL)和甲醇(2.00mL)中。在氮气保护下，反应混合物在50℃下搅拌反应10小时后。加入NaBH 3CN(60mg,0.96mmol)，然后在25℃下继续搅拌反应5小时。直接把反应液用1N盐酸溶液调节pH到5-6，然后减压浓缩除去有机溶剂和水得到粗产品。粗产品经制备HPLC分离纯化得到化合物9。
LC-MS(ESI)[M+H] +466.2.
1H NMR(400MHz,CD 3OD)δ7.69(s,1H),7.64(d,J＝8.2Hz,1H),7.58(d,J＝8.1Hz,1H),7.46(t,J＝8.5Hz,1H),6.95(d,J＝8.8Hz,2H),5.17(s,2H),4.53–4.22(m,6H),3.74–3.63(m,1H),2.93(m,1H),1.92–1.82(m,2H),1.81–1.74(m,2H),1.60–1.49(m,2H),1.46–1.30(m,4H).
实施例10：化合物10的制备
Figure PCTCN2019123514-appb-000149
在室温下，将氢氧化锂一水合物(16.7mg，0.399mmol)的水(1mL)溶液加入到中间体I-36(60.0mg,0.133mmol)的四氢呋喃溶液(3mL)中。反应混合物在室温下搅拌反应3小时后，用稀盐酸(1M)调节pH至4～6。反应混合物滤膜过滤后，用制备HPLC纯化得到化合物10。
LC-MS(ESI)[M+H] +438.2.
1H NMR(400MHz,CD 3OD)δ7.75–7.66(m,3H),7.44(t,J＝8.4Hz,1H),6.97–6.93(m,2H),5.18(s,2H),4.34(s,2H),4.17(d,J＝8.4Hz,4H),3.95–3.86(m,1H),3.44–3.35(m,1H),2.40–2.29(m,2H),2.29–2.19(m,2H),2.12–2.00(m,1H),1.94–1.87(m,1H).
实施例11：化合物11的制备
Figure PCTCN2019123514-appb-000150
将中间体I-40(137mg，0.285mmol)溶于四氢呋喃(1.00mL)和甲醇(1.00mL)中，加入氢氧化锂一水合物(23.9mg，0.570mmol)的水(1.00mL)溶液。反应混合物在室温下搅拌反应1小时后，用稀盐酸(1N)将反应混合物pH值调至6.0。加入乙酸乙酯(10.0mL)萃取分液，分出有机相，水相以乙酸乙酯(10.0mL×2)萃取，合并有机相，用饱和食盐水(10mL)洗，无水硫酸钠干燥，过滤。滤液减压浓缩除去溶剂得到粗产品。粗产品用制备HPLC分离纯化得到化合物11。
LC-MS(ESI)[M+H] +468.2.
1H NMR(400MHz,CD 3OD)δ7.66–7.57(m,2H),7.42(t,J＝8.4Hz,1H),7.16(d,J＝8.4Hz,1H),6.98–6.88(m,2H),5.09(s,2H),4.99–4.95(m,1H),4.36(s,2H),4.19(d,J＝8.4Hz,4H),3.46–3.38(m,1H),1.99–1.74(m,6H),1.74–1.60(m,2H).
实施例12：化合物12的制备
Figure PCTCN2019123514-appb-000151
将中间体I-46(150mg,0.322mmol)溶于四氢呋喃(3.00mL)中，加入氢氧化锂一水合物(40.5mg，0.966mmol)的水(0.5mL)溶液。反应混合物在室温下搅拌反应2小时后，加入稀盐酸(1M)将反应混合物pH值调至5。反应混合物用乙酸乙酯(5mL×2)萃取，合并有机相，用饱和食盐水(5mL)洗，无水硫酸钠干燥，过滤。减压浓缩除去有机溶剂得到粗产品。粗产品用制备HPLC分离纯化得到化合物12。
LC-MS(ESI)[M+H] +452.2.
1H NMR(400MHz,Methanol-d 4)δ7.57–7.44(m,2H),7.40–7.29(m,2H),7.24–7.16(m,2H),5.18(s,2H),4.40(s,2H),4.17(d,J＝8.3Hz,4H),3.41(m,1H),3.28(m,1H),2.09–1.95(m,2H),1.94–1.79(m,2H),1.77–1.64(m,2H),1.66–1.49(m,2H).
实施例13：化合物13的制备
Figure PCTCN2019123514-appb-000152
在室温下，将中间体I-4(100mg,0.27mmol)和哌啶-4-羧酸(70mg，0.54mmol)加入到1,2二氯乙烷/四氢呋喃(2.5mL，4：1)的混合溶剂中，加入冰乙酸(0.5mL)。反应混合物在50℃下搅拌反应2小时后，降至室温。在0℃下，加入醋酸硼氢化钠(170mg，0.81mmol)。反应混合物在室温下继续搅拌过夜。减压浓缩除去有机溶剂得到粗产品，加入稀盐酸(2N，1mL)。用制备HPLC分离纯化得到化合物13。
LC-MS(ESI)[M+H] +480.6.
1H NMR(400MHz,CDCl 3)δ7.63(s,1H),7.60–7.44(m,3H),6.80(dd,J＝8.6,2.3Hz,1H),6.70(dd,J＝11.5,2.4Hz,1H),5.03(s,2H),4.00(s,2H),3.42–3.33(m,1H),3.33–3.19(m,2H),2.70–2.46(m,2H),2.40–2.29(m,1H),2.19–2.04(m,4H),2.04-1.91(m,2H),1.91–1.81(m,2H),1.80–1.68(m,2H),1.66–1.54(m,2H).
实施例14：化合物14的制备
Figure PCTCN2019123514-appb-000153
将中间体I-4(183mg，0.50mmol)溶于1,2-二氯乙烷中(2.50mL)中，依次加入3-甲酸哌啶(65.0mg，0.50mmol)和乙酸(0.100mL)，反应液升温至50℃搅拌16h，加入氰基硼氢化钠(158mg，2.50mmol)并搅拌4h，将反应液减压浓缩至干，加入甲醇(5mL)溶解后经制备HPLC纯化得到化合物14。
LC-MS(ESI)[M+H] +480.3。
1H NMR(400MHz,CD 3OD)δ7.68(s,1H),7.65(d,J＝8.3Hz,1H),7.59(d,J＝8.2Hz,1H),7.45(t,J＝8.7Hz,1H),6.97–6.89(m,2H),5.16(s,2H),4.18(s,2H),3.41–3.34(m,1H),3.26–2.94(br.s,4H),2.67–2.55(m,1H),2.11–2.01(m,2H),1.98–1.84(m,4H),1.84–1.68(m,4H),1.68–1.59(m,2H).
实施例15：化合物15的制备
Figure PCTCN2019123514-appb-000154
中间体I-50(100mg,0.271mmol)，3-吖啶羧酸(82.5mg,0.816mmol)和醋酸(0.5mL)混合于四氢呋喃(15mL)和甲醇(15mL)的混合溶剂中，反应液在40℃下搅拌反应3小时。然后加入氰基硼氢化钠(51.3mg,0.816mmol)，反应液在40℃下搅拌过夜。将反应混合物过滤，滤液减压浓缩得到粗品。粗品用制备HPLC分离纯化得到化合物15。
LC-MS(ESI)[M+H] +454.1。
1H NMR(400MHz,MeOH-d 4)δ7.81–7.68(m,3H),7.45(t,J＝8.4Hz,1H),7.01–6.92(m,2H),5.26–5.15(m,3H),4.36(s,2H),4.27–4.11(m,5H),3.99–3.93(m,1H),3.45–3.38(m,1H),2.46–2.37(m,1H),2.15 –2.01(m,2H),1.74–1.65(m,1H)。
实施例16：化合物16的制备
Figure PCTCN2019123514-appb-000155
将中间体I-54(100mg，0.271mmol)溶于甲醇/四氢呋喃(1:1，10mL)中，加入3-吖丁啶羧酸(82.2mg，0.813mmol)和冰乙酸(0.500mL)，反应液升温至40℃搅拌过夜，然后加入氰基硼氢化钠(51.1mg，0.813mmol)并在40℃搅拌2小时，加入冰乙酸(1mL)淬灭过量硼氢化钠，反应液过滤后经制备HPLC分离纯化得到化合物16。
LC-MS(ESI)[M+H] +454.2.
1H NMR(400MHz,CD 3OD)δ7.73(s,1H),7.70(d,J＝8.3Hz,1H),7.63(d,J＝8.2Hz,1H),7.43(t,J＝8.4Hz,1H),6.98–6.91(m,2H),5.18(s,2H),4.35(s,2H),4.18(d,J＝8.4Hz,4H),4.14–4.09(m,1H),4.08–4.02(m,1H),3.93–3.87(m,1H),3.83–3.73(m,2H),3.43–3.36(m,1H),2.48–2.39(m,1H),2.05–1.96(m,1H).
实施例17：化合物17的制备
Figure PCTCN2019123514-appb-000156
在10℃下，将中间体I-59(60.0mg,0.125mmol)溶于乙醇/水(1mL/1mL)中，加入一水合氢氧化锂(15.7mg,0.375mmol)。反应混合物10℃下搅拌反应2小时后。反应液直接用制备HPLC分离纯化得到化合物17。
LC-MS(ESI)[M+H] +468.2.
1H NMR(400MHz,DMSO-d 6)δ7.80–7.66(m,3H),7.25(t,J＝8.6Hz,1H),6.88(dd,J＝12.0,2.3Hz,1H),6.82(dd,J＝8.4,2.3Hz,1H),5.16(s,2H),3.97(dd,J＝11.2,3.7Hz,2H),3.48–3.40(m,4H),3.33(t,J＝6.7Hz,2H),3.17–3.04(m,4H),1.88–1.76(m,2H),1.62–1.54(m,2H).
实施例18：化合物18的制备
Figure PCTCN2019123514-appb-000157
在室温下，将中间体I-64(70.0mg,0.150mmol)溶于四氢呋喃(2mL)和甲醇(0.5mL)中，加入氢氧化锂一水合物(12.6mg，0.300mmol)的水(0.5mL)溶液。反应混合物在室温下搅拌反应1小时后，加入稀盐酸(1M)将反应混合物pH值调至5。反应混合物减压浓缩除去有机溶剂得到粗产品。 粗产品用制备HPLC分离纯化得到化合物18。
LC-MS(ESI)[M+H] +453.2.
1H NMR(400MHz,DMSO-d 6)δ8.19(d,J＝1.8Hz,1H),7.76–7.69(m,2H),7.66(d,J＝8.1Hz,1H),7.51(dd,J＝11.6,2.3Hz,1H),5.24(s,2H),3.60(d,J＝2.1Hz,2H),3.39(t,J＝7.5Hz,2H),3.26(m,3H),3.19–3.09(m,1H),2.06–1.94(m,2H),1.90–1.78(m,2H),1.73–1.53(m,4H).
实施例19：化合物19的制备
Figure PCTCN2019123514-appb-000158
中间体I-65(250mg,0.66mmol)，氮杂环丁烷-3-羧酸(200mg,1.98mmol)和醋酸(1mL)混合于甲醇和四氢呋喃的混合溶剂中(60mL，1:1)。反应混合物在40℃下搅拌反应3小时。然后向反应体系中加入氰基硼氢化钠(124mg,1.98mmol)的甲醇(2mL)溶液，继续在40℃下搅拌过夜。减压浓缩除去有机溶剂得到粗品。粗品用制备HPLC分离纯化得到化合物19。
LC-MS(ESI)[M+H] +464.2。
1H NMR(400MHz,CD 3OD)δ7.70(s,1H),7.66(d,J＝8.1Hz,1H),7.60(d,J＝8.2Hz,1H),7.32(d,J＝8.4Hz,1H),6.74(d,J＝2.2Hz,1H),6.67(dd,J＝8.4,2.2Hz,1H),5.17(s,2H),4.38-4.19(m,6H),3.91(s,3H),3.44-3.33(m,2H),2.13-2.03(m,2H),1.97-1.86(m,2H),1.81-1.56(m,4H).
实施例20：化合物20的制备
Figure PCTCN2019123514-appb-000159
在室温下，将中间体I-4(100mg,0.27mmol)和吡咯烷-3-羧酸(62mg，0.54mmol)加入到1,2二氯乙烷/四氢呋喃(2mL，4:1)的混合溶剂中，加入冰乙酸(0.5mL)。反应混合物在50℃下搅拌反应7小时后，降至室温。在0℃下加入醋酸硼氢化钠(170mg，0.81mmol)。反应混合物在室温下继续搅拌反应过夜，减压浓缩除去有机溶剂得到粗产品。向粗产品中加入2N稀盐酸(1mL)，用制备HPLC分离纯化得到化合物20。
LC-MS(ESI)[M+H] +466.3.
1H NMR(400MHz,DMSO-d 6)δ7.73–7.67(m,2H),7.65(d,J＝8.1Hz,1H),7.29(t,J＝8.6Hz,1H),6.88(dd,J＝11.9,2.4Hz,1H),6.83(dd,J＝8.4,2.4Hz,1H),5.15(s,2H),3.53(s,2H),3.29–3.21(m,1H),2.90–2.84(m,1H),2.64–2.72(m,1H),2.60–2.54(m,1H),2.49–2.39(m,2H),2.04–1.96(m,2H),1.95–1.88(m,2H),1.88–1.78(m,2H),1.72–1.56(m,4H).
实施例21：化合物21的制备
Figure PCTCN2019123514-appb-000160
在冰浴下，将氢氧化锂一水合物(12.8mg，0.305mmol)的水(1mL)溶液加入到中间体I-72(50.0mg,0.102mmol)的四氢呋喃溶液(3mL)中。反应混合物在室温下搅拌反应3小时后，用稀盐酸(1M)调节pH至5～6。反应混合物滤膜过滤后，用制备HPLC纯化得到化合物21。
LC-MS(ESI)[M+H] +476.3.
1H NMR(400MHz,CD 3OD)δ7.70(s,1H),7.66(d,J＝8.3Hz,1H),7.59(d,J＝8.2Hz,1H),7.31(d,J＝8.5Hz,1H),7.03(d,J＝2.6Hz,1H),6.92(dd,J＝8.5,2.7Hz,1H),5.17(s,2H),4.38(s,2H),4.16(d,J＝8.4Hz,4H),3.43–3.35(m,2H),3.23–3.16(m,1H),2.12–2.04(m,2H),1.96–1.86(m,2H),1.80–1.69(m,2H),1.69-1.56(m,2H),1.26(d,J＝6.8Hz,6H).
实施例22：化合物22的制备
Figure PCTCN2019123514-appb-000161
在冰水浴下，将氢氧化锂一水合物(11.6mg，0.276mmol)的水(1mL)溶液加入到中间体I-76(45.0mg,0.0923mmol)的四氢呋喃溶液(3mL)中。反应混合物在室温下搅拌反应3小时后，用稀盐酸(1M)调节pH至5～6。反应混合物滤膜过滤后，用制备HPLC纯化得到化合物22。
LC-MS(ESI)[M+H] +474.3.
1H NMR(400MHz,CD 3OD)δ7.67(s,1H),7.64(d,J＝8.3Hz,1H),7.58(d,J＝8.2Hz,1H),7.31(d,J＝8.5Hz,1H),6.90(dd,J＝8.5,2.6Hz,1H),6.69(d,J＝2.6Hz,1H),5.13(s,2H),4.52(s,2H),4.21(d,J＝8.3Hz,4H),3.46–3.35(m,2H),2.16–2.00(m,3H),1.98–1.86(m,2H),1.81–1.58(m,4H),1.14–1.02(m,2H),0.77–0.65(m,2H).
实施例23：化合物23的制备
Figure PCTCN2019123514-appb-000162
在室温下，将中间体I-80(190mg,0.38mmol)粗产品溶于四氢呋喃/水(2mL，4:1)中，加入氢氧化锂单水合物(32.0mg，0.76mmol)。反应混合物在室温搅拌下反应2小时后。减压浓缩除去有机溶剂得到粗产品。用制备HPLC分离纯化得到化合物23。
LC-MS(ESI)[M+H] +492.3.
1H NMR(400MHz,CD 3OD)δ7.69(s,1H),7.65(d,J＝8.3Hz,1H),7.59(d,J＝8.2Hz,1H),7.29(d,J＝8.4Hz,1H),6.70(d,J＝2.2Hz,1H),6.65(dd,J＝8.4,2.3Hz,1H),5.17(s,2H),4.75–4.66(m,1H),4.27(s,2H), 4.25–4.15(m,4H),3.43–3.34(m,2H),2.13–2.03(m,2H),1.97–1.85(m,2H),1.80–1.69(m,2H),1.69-1.57(m,2H),1.37(d,J＝6.0Hz,6H).
实施例24：化合物24的制备
Figure PCTCN2019123514-appb-000163
将中间体I-86(90.0mg,181.46μmol)和氢氧化锂一水合物(22.84mg,554.38μmol)溶于四氢呋喃(3.00mL)和水(0.500mL)中。反应混合物在25℃下搅拌反应10小时后。直接把反应液减压浓缩除去有机溶剂和水得到粗产品。粗产品用制备HPLC分离纯化得到化合物24。
LC-MS(ESI)[M+H] +482.2.
1H NMR(400MHz,CD 3OD)δ7.74(s,1H),7.67(d,J＝8.1Hz,1H),7.59(d,J＝8.1Hz,1H),7.33(d,J＝8.5Hz,1H),7.08(d,J＝8.5Hz,1H),5.24(s,2H),4.40(s,2H),4.16(d,J＝8.3Hz,4H),3.46–3.34(m,2H),2.47(s,3H),2.15–2.00(m,2H),1.97–1.83(m,2H),1.81–1.55(m,4H).
实施例25：化合物25的制备
Figure PCTCN2019123514-appb-000164
将中间体I-94(55.7mg,0.112mmol)溶于甲醇(2.00mL)中，加入氢氧化锂一水合物(14.1mg，0.336mmol)和水(0.500mL)。反应混合物在30℃下搅拌反应2小时。反应液经制备HPLC分离纯化得到化合物25。
LC-MS(ESI)[M+H] +482.2.
1H NMR(400MHz,Methanol-d 4)δ7.67(s,1H),7.64(d,J＝8.3Hz,1H),7.59(d,J＝8.3Hz,1H),7.09(d,J＝2.7Hz,1H),6.99(d,J＝2.5Hz,1H),5.15(s,2H),4.63(s,2H),4.42(d,J＝8.6Hz,4H),3.76–3.65(m,1H),3.41–3.33(m,1H),2.48(s,3H),2.14–2.00(m,2H),1.97–1.83(m,2H),1.82–1.67(m,2H),1.70–1.56(m,2H)
实施例26：化合物26的制备
Figure PCTCN2019123514-appb-000165
在室温下，将中间体I-100(100mg,0.209mmol)溶于甲醇/水(1mL/1mL)中，加入一水合氢氧化锂(17.5mg,0.418mmol)。反应混合物室温下搅拌反应2小时后。用制备HPLC分离纯化得到实施例26。
LC-MS(ESI)[M+H] +464.2.
1H NMR(400MHz,DMSO-d 6)δ7.52(d,J＝8.7Hz,1H),7.27(dd,J＝8.7,2.4Hz,1H),7.24–7.17(m,2H),7.05(s,1H),6.98(d,J＝7.6Hz,1H),5.11(s,2H),3.78(s,3H),3.50(s,2H),3.42–3.37(m,2H),3.22–3.12(m,4H),2.00–1.90(m,2H),1.87–1.76(m,2H),1.70–1.49(m,4H).
实施例27：化合物27的制备
Figure PCTCN2019123514-appb-000166
将中间体I-101(150mg，0.380mmol)溶于四氢呋喃/无水甲醇(1:1，40.0mL)中，依次加入3-吖丁啶羧酸(154mg，1.52mmol)和冰乙酸(0.500mL)。反应混合物在40℃下搅拌反应16小时。加入氰基硼氢化钠(71.6mg，1.14mmol)，反应混合物在40℃下搅拌反应16小时。加入水(80mL)淬灭反应后用乙酸乙酯(30.0mL×3)萃取。合并有机相，用饱和食盐水洗，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品用制备HPLC分离纯化得到化合物27.
LC-MS(ESI)[M+H] +480.3。
1H NMR(400MHz,DMSO-d 6)δ7.70–7.65(m,2H),7.27(d,J＝9.2Hz,1H),7.10(d,J＝8.3Hz,1H),6.60(d,J＝2.2Hz,1H),6.55(dd,J＝8.3,2.2Hz,1H),5.05(s,2H),5.04–5.00(m,1H),3.74(s,3H),3.43(s,2H),3.38–3.33(m,2H),3.20–3.10(m,3H),1.96–1.85(m,2H),1.80–1.60(m,6H).
实施例28：化合物28的制备
Figure PCTCN2019123514-appb-000167
将中间体I-103(120mg，0.306mmol)溶于四氢呋喃/无水甲醇(1:1，40.0mL)中，依次加入3-吖丁啶羧酸(92.8mg，0.918mmol)和冰乙酸(0.500mL)。反应混合物在40℃下搅拌反应16小时。加入氰基硼氢化钠(57.7mg，0.918mmol)，反应混合物在40℃下搅拌反应16小时。加入水(80mL)淬灭反应后用乙酸乙酯(30mL×3)萃取。合并有机相，用饱和食盐水洗，无水硫酸钠干燥，过滤。滤液减压浓缩除去有机溶剂得到粗产品。粗产品用制备HPLC分离纯化得到化合物28。
LC-MS(ESI)[M+H] +478.3。
1H NMR(400MHz,DMSO-d 6)δ7.73(s,1H),7.70(d,J＝8.3Hz,1H),7.64(d,J＝8.0Hz,1H),7.10(d,J＝8.3Hz,1H),6.62(d,J＝2.1Hz,1H),6.55(dd,J＝8.3,2.1Hz,1H),5.12(s,2H),3.75(s,3H),3.44(s,2H),3.39–3.33(m,2H),3.21–3.11(m,3H),2.82(m,1H),1.86–1.78(m,2H),1.75–1.66(m,3H),1.59–1.48(m,2H),1.40–1.27(m,3H).
实验例1：S1P1介导的cAMP抑制作用
测试所用细胞株为
Figure PCTCN2019123514-appb-000168
CHO-K1 EDG1 β-Arrestin Cell Line，供货商：DiscoverX，货号：93-0207C2。测试通过化合物对S1P1介导的毛喉萜(Forskolin)诱导cAMP活性的抑制作用进行评价。
每次测试将不同浓度的化合物和终浓度0.6μM的毛喉萜加入测试板孔，1000rpm离心10秒。取一支冻存细胞，用HBSS缓冲液洗两次，重悬。按每孔5000细胞加入测试板，震荡20秒，1000rpm离心 10秒，室温孵育60分钟。加入抗cAMP-Eu2+-Cryptate和cAMP-d2，震荡20秒，1000rpm离心10秒，室温孵育60分钟，用Envision读板。通过非线性回归分析数据以确定化合物对毛喉萜诱导的cAMP的抑制的EC 50。实验结果如表1所示。
表1化合物对S1P1受体介导的cAMP抑制作用的激活
化合物编号 S1P1 cAMP EC 50(nM) 化合物编号 S1P1 cAMP EC 50(nM)
化合物1 <0.05 化合物10 0.29
化合物2 <0.05 化合物11 0.098
化合物3 0.16 化合物12 0.075
化合物4 0.12 化合物19 0.19
化合物5 0.09 化合物20 0.064
化合物6 0.30 化合物22 0.15
化合物7 0.36 化合物25 0.065
化合物8 0.033 化合物26 0.17
化合物9 0.048 化合物28 0.11
实验数据表明，本发明化合物对S1P1介导的cAMP抑制作用显示出很好的激活特性。
实验例2：S1P1介导的β-Arrestin报告基因激活作用
测试所用细胞株为
Figure PCTCN2019123514-appb-000169
CHO-K1 EDG1 β-Arrestin Cell Line，供货商：DiscoverX，货号：93-0207C2。实验操作按照供应商说明，按每孔25μL细胞悬液含5000细胞将细胞加到测试板上，37℃培养20小时。4倍稀释的10个浓度化合物加入细胞培液中，37℃培养90分钟。配制检测液，每孔12μL，室温孵育60分钟，Envision读板。通过非线性回归分析数据以测定β-arrestin活性的EC 50。实验结果如表2所示。
表2化合物对S1P1受体介导的β-arrestin活性的激活作用
化合物编号 S1P1 β-arrestin EC 50(nM) 化合物编号 S1P1 β-arrestin EC 50(nM)
化合物1 1.7 化合物9 1.93
化合物2 2.9 化合物10 1.57
化合物3 1.28 化合物12 0.43
化合物4 1.22 化合物19 0.92
化合物5 0.98 化合物20 1.55
化合物6 1.95 化合物26 1.45
化合物8 1.96 化合物28 1.62
实验数据表明，本发明化合物对S1P1介导的β-arrestin表现出良好的激活作用。
实验例3：本发明化合物的S1P1受体的内化效应实验
1.CHO-K1 DEG1细胞(
Figure PCTCN2019123514-appb-000170
CHO-K1 EDG1 β-Arrestin Cell Line，供货商：DiscoverX，货号：93-0207C2)，除去培养基(F12 medium 1000mL,10％FBS,800μg/mL G418,300μg/mL Hygromycin,1％GlutaMax and 1％Pen/Strep)，用2ml DPBS冲洗细胞，加入5mL细胞分散液(Invitrogen-13151014)分散细胞，37℃培养箱孵育1～2分钟，轻拍培养瓶使细胞脱落，并加入5mL生长培养基，移液管轻 轻吹打使细胞充分悬浮。使用Vi-Cell进行细胞计数。室温下以1000rpm离心5分钟，轻轻倒出上清液，并将细胞重新悬浮在FACS缓冲液中，使其浓度为每毫升1.5e6个细胞。2.在384孔板中用DMSO稀释S1P和化合物，并将500nL体积转移到96V孔板(Cat#Axygen-WIPP02280)中。3.将50μL细胞加入96孔板；4.将96孔板在37℃5％CO2培养箱中孵育2h。5.在室温下以1500rpm离心细胞5分钟，除去上清液。6.向重悬细胞中加入100μl FACS缓冲液，以1500rpm离心5min，并除去上清液。7.用FACS缓冲液200倍稀释抗人S1P1/EDG-1-Alex647(R&D-FAB1864R)和抗IgG2B-Alex647(R&D-IC0041R)的抗体。8.在96孔中加入50μL抗体，并将板转移至4℃30分钟。9.将细胞在4℃以1500rpm离心5分钟，除去上清液。10.将100μL FACS缓冲液添加到重悬的细胞中。11.将细胞在4℃下以1500rpm离心5分钟，除去上清液。12.洗涤后，将细胞重悬于每孔50μL FACS缓冲液中。13.使用iQue Screener PLUS-VBR读取细胞样品。实验结果如表3所示。
表3化合物对S1P1受体的内化效应实验
化合物编号 S1P1内化EC 50(nM) 化合物编号 S1P1内化EC 50(nM)
化合物1 0.037 化合物3 <0.01
化合物2 0.18 CBP-307 0.48
实验数据表明，本发明化合物对S1P1受体表现出很好的内化激活作用。
实验例4：针对S1P3受体激动剂活性的测定
本实验所用细胞，培养条件及细胞收集条件同实验例3。
1)在测定板的每个孔中加入25μL(5000个细胞)的细胞悬液，37℃下孵育20小时。(2)4倍系列稀释的化合物以获得10个剂量，37℃孵育90分钟。(3)向检测板的每个孔中添加12μL检测试剂，在23℃下孵育60分钟。(4)Envision读数。实验结果如表4所示。
表4针对S1P3受体激动剂活性的测定
Figure PCTCN2019123514-appb-000171
实验数据表明，本发明化合物对S1P3受体表现出很好的选择性。
实验例5：本发明化合物的体内药代动力学实验
本实验例对大鼠通过静脉注射和口服给药进行了体内药代动力学评价。
实验方法和条件：雄性Sprague Dawley大鼠，分别静脉注射单次给予待测化合物1mg/Kg(溶剂5％DMSO/15％Solutol/80％Saline)和口服灌胃给药1mg/Kg(溶剂0.5％MC)，给药后5min,15min,30min,1hr,2hr,4hr,6hr,8hr,24hr经颌下静脉采血，每个样品采集约0.20mL，肝素钠抗凝，采集后放置冰上，并于1小时之内离心分离血浆待测。血浆中血药浓度的检测采用液相串联质谱法(LC/MS/MS)，测得浓度运用Phoenix WinNonlin软件计算药代动力学参数。实验结果如表5和表6所示。
表5：口服给药(1mg/kg)的药代动力学
化合物 T 1/2(hr) C max(ng/mL) AUC inf(ng*hr/mL) F(％)
化合物2 10.2 100 2034 45.3
表6：静脉注射给药(1mg/kg)的药代动力学
化合物 T 1/2(hr) AUC inf(ng*hr/mL) Cl(L/hr/kg)
化合物2 8.1 4486 0.23
实验数据表明，本发明化合物在大鼠体内表现出较好的代谢稳定性。
实验例6：本发明化合物在大鼠外周淋巴细胞降低(Peripheral Lymphocyte Lowering，PLL)测定中的作用。
雄性Sprague-Dawley大鼠，体重200-220g。饲养环境：温度23±2℃，相对湿度40-70％，照明时间早上7点开灯，晚上7点关灯；动物自由饲喂普通饲料和灭菌饮用水。所有动物实验都获得了动物伦理委员会的批准；所有动物实验操作都遵守动物房相关SOP要求。动物在试验前适应性饲养一周。
动物口服给药，给药体积10mL/kg。给药溶媒为0.5％DMSO+0.5％MC。在给药后5小时动物用异氟烷麻醉，且通过眼眶来收取100～150μl外周血于EP管，冰块上放置，30min内用XT-2000i全自动血液分析仪进行血液分析淋巴细胞计数检测；另外20μl全血，40μl DDW稀释，液氮速冻，进行血液化合物浓度检测。
结果显示给药后5小时测试化合物2使大鼠外周血淋巴细胞(PBL)计数降低，如图1所示。IC50为94.6nM(化合物2)。

Claims (15)

  1. 式(Ⅱ)所示化合物或其药学上可接受的盐，
    Figure PCTCN2019123514-appb-100001
    其中，
    m选自1、2和3；
    n选自1和2；
    y选自1和2；
    R 1选自H、卤素、OH、NH 2、CN、C 1-6烷基、C 3-6环烷基、3～7元杂环烷基和C 1-6杂烷基，所述C 1-6烷基、C 3-6环烷基、3～7元杂环烷基或C 1-6杂烷基任选被1、2或3个R取代；
    R 2选自H、卤素、OH、NH 2、CN和C 1-6烷基，所述C 1-6烷基任选被1、2或3个R取代；
    环A选自C 3-7环烷基、3～7元杂环烷基和C 3-7环烯基，所述C 3-7环烷基、3～7元杂环烷基或C 3-7环烯基任选被1、2或3个R取代；
    L 1选自
    Figure PCTCN2019123514-appb-100002
    L 2选自单键、O和S；
    T 1选自N和CH；
    T 2选自N和CH；
    T 3选自N和CH；
    R分别独立地选自H、F、Cl、Br、I、OH、NH 2、C 1-3烷基和CF 3；
    所述C 1-6杂烷基和3～7元杂环烷基包含1、2或3个独立选自NH、O和S的杂原子或杂原子团。
  2. 根据权利要求1所述化合物或其药学上可接受的盐，其中，R 1选自H、F、Cl、Br、OH、NH 2、CN、C 1-3烷基、C 3-6环烷基、3～7元杂环烷基和C 1-3烷氧基，所述C 1-3烷基、C 3-6环烷基、3～7元杂环烷基或C 1-3烷氧基任选被1、2或3个R取代。
  3. 根据权利要求2所述化合物或其药学上可接受的盐，其中，R 1选自H、F、Cl、Br、OH、NH 2、CN、CH 3、CH 2CH 3、CF 3、
    Figure PCTCN2019123514-appb-100003
  4. 根据权利要求1～3任意一项所述化合物或其药学上可接受的盐，其中，R 2选自H、F、Cl、Br、OH、NH 2、CN、CH 3和CH 2CH 3，所述CH 3或CH 2CH 3任选被1、2或3个R取代。
  5. 根据权利要求4所述化合物或其药学上可接受的盐，其中，R 2选自H、F、Cl、Br、OH、NH 2、CN、CH 3、CH 2CH 3和CF 3。
  6. 根据权利要求1～3任意一项所述化合物或其药学上可接受的盐，其中，L 1选自
    Figure PCTCN2019123514-appb-100004
    Figure PCTCN2019123514-appb-100005
  7. 根据权利要求1～3任意一项所述化合物或其药学上可接受的盐，其中，环A选自
    Figure PCTCN2019123514-appb-100006
    Figure PCTCN2019123514-appb-100007
    所述
    Figure PCTCN2019123514-appb-100008
    任选被1、2或3个R取代。
  8. 根据权利要求7任意一项所述化合物或其药学上可接受的盐，其中，环A选自
    Figure PCTCN2019123514-appb-100009
    Figure PCTCN2019123514-appb-100010
  9. 根据权利要求1或8所述化合物或其药学上可接受的盐，其中，结构单元
    Figure PCTCN2019123514-appb-100011
    选自
    Figure PCTCN2019123514-appb-100012
    Figure PCTCN2019123514-appb-100013
  10. 根据权利要求1～3任意一项所述化合物或其药学上可接受的盐，其中，结构单元
    Figure PCTCN2019123514-appb-100014
    选自
    Figure PCTCN2019123514-appb-100015
  11. 根据权利要求1任意一项所述化合物或其药学上可接受的盐，其选自
    Figure PCTCN2019123514-appb-100016
    其中，
    m、n、T 1、T 2、T 3、R、L 2如权利要求1所定义；
    R 1如权利要求1～3所定义；
    R 2如权利要求1、4、5所定义；
    环A如权利要求1、7、8所定义。
  12. 下式化合物或其药学上可接受的盐，
    Figure PCTCN2019123514-appb-100017
    Figure PCTCN2019123514-appb-100018
  13. 一种药物组合物，所述的药物组合物含有如权利要求1～12任意一项所述化合物或其药学上可药用盐，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
  14. 根据权利要求1～12任意一项所述化合物或其可药用盐或根据权利要求13所述的药物组合物在制备 预防和/或治疗用作S1P1受体相关疾病的药物中的用途。
  15. 根据权利要求14所述的用途，其中所述的S1P1受体相关疾病选自溃疡性结肠炎、克罗恩病、多发性硬化症、系统性红斑狼疮、狼疮性肾炎、类风湿性关节炎、原发性胆汁胆管炎、过敏性皮肤炎、脑出血、移植物抗宿主病、牛皮癣、I型糖尿病、痤疮、微生物感染或微生物疾病及病毒感染或病毒疾病。
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WO2004103306A2 (en) * 2003-05-19 2004-12-02 Irm Llc Immunosuppressant compounds and compositions
CN1787817A (zh) * 2003-05-19 2006-06-14 Irm责任有限公司 免疫抑制剂化合物及组合物
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WO2004103306A2 (en) * 2003-05-19 2004-12-02 Irm Llc Immunosuppressant compounds and compositions
CN1787817A (zh) * 2003-05-19 2006-06-14 Irm责任有限公司 免疫抑制剂化合物及组合物
CN1921863A (zh) * 2004-02-24 2007-02-28 Irm责任有限公司 免疫抑制剂化合物和组合物
CN102387704A (zh) * 2009-02-10 2012-03-21 雅培制药有限公司 S1p5受体的激动剂和拮抗剂，和其用法

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