WO2024078488A1 - 二氢苯并噻喃类衍生物及其制备方法和用途 - Google Patents

二氢苯并噻喃类衍生物及其制备方法和用途 Download PDF

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WO2024078488A1
WO2024078488A1 PCT/CN2023/123758 CN2023123758W WO2024078488A1 WO 2024078488 A1 WO2024078488 A1 WO 2024078488A1 CN 2023123758 W CN2023123758 W CN 2023123758W WO 2024078488 A1 WO2024078488 A1 WO 2024078488A1
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acid
alkyl
mmol
palladium
mixture
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杨玉社
张丹
陆征宇
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中国科学院上海药物研究所
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    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • A61K31/397—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having four-membered rings, e.g. azetidine
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    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4155—1,2-Diazoles non condensed and containing further heterocyclic rings
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    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4433—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with oxygen as a ring hetero atom
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    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/453—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with oxygen as a ring hetero atom
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    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
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    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
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    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C07F5/025—Boronic and borinic acid compounds

Definitions

  • the present invention belongs to the field of medicine, and relates to a class of dihydrobenzothiopyran compounds, and their preparation methods and medical applications.
  • the present invention relates to dihydrobenzothiopyran derivatives represented by general formula (I), their racemates, enantiomers, diastereomers and mixtures thereof, their pharmaceutically acceptable salts and pharmaceutical compositions containing the compounds, and the use of the compounds for treating, preventing or diagnosing estrogen receptor-sensitive, estrogen receptor-mediated or dependent diseases or conditions, and the diseases are particularly preferably estrogen receptor-positive breast cancer.
  • Breast cancer is the world's leading cancer, with 2.26 million new cases worldwide in 2020. Although the five-year survival rate of early breast cancer exceeds 90%, it still causes nearly 700,000 deaths each year due to tumor metastasis and spread. More than 70% of breast cancer cases highly express estrogen receptors (ER). ER binds to estrogen, activates signaling pathways, and thus promotes the occurrence and development of breast cancer. It is the main driving factor for the occurrence and development of breast cancer.
  • ER estrogen receptors
  • Endocrine therapy is the first choice for the treatment of ER-positive breast cancer.
  • Standard treatment drugs include: (1) aromatase/estrogen synthase inhibitors, whose main mechanism is to inhibit the biosynthesis of endogenous estrogen.
  • Representative drugs include letrozole and anastrozole.
  • the main side effects of this type of drug are accelerated bone density loss and widespread acquired drug resistance; (2) estrogen receptor modulators (SERMs) that antagonize ER activity, namely tamoxifen.
  • SERMs estrogen receptor modulators
  • the disadvantage is that long-term use can lead to hormone-independent drug resistance, and the drug has a partial agonist effect, which increases the risk of endometrial hyperplasia, polyps and endometrial cancer.
  • SESDs Selective Estrogen Receptor Degraders
  • Intramuscular injection (a single intramuscular injection can only reach a maximum of 500 mg) is difficult to achieve the in vivo drug concentration required for optimal efficacy. It takes 3-6 months after administration to achieve a stable plasma concentration, and severe pain, swelling, redness and other reactions at the injection site also make the patient's compliance very poor, greatly limiting its clinical application.
  • Fulvestrant cannot pass through the blood-brain barrier and cannot be used to treat brain metastases, which is extremely difficult in the treatment of breast cancer. Therefore, the development of oral small molecule SERDs with good oral absorption, oral effectiveness, more ideal pharmacokinetic properties, and the ability to pass through the blood-brain barrier is a huge unmet clinical need.
  • SERDs/antagonists with public structures include LSZ-102 (US9322746), ZB716 (WO2016004166), RAD1901 (WO2016176666), SAR439859 (WO2018091153), AZD9833 (WO2019002442), and GDC-9545 (WO2016097072). So far, no orally effective small molecule SERDs have been approved for marketing.
  • the object of the present invention is to provide a SERD with novel structure, excellent ER antagonism and degradation activity, oral efficacy and good brain distribution.
  • R 1 is selected from OH, COOH, B(OH) 2 , halogen, C 1 -C 6 alkyl, halogenated C 1 -C 6 alkyl or C 1 -C 6 alkoxy
  • R 2 is selected from H, OH, COOH, halogen, cyano, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, halogenated C 1 -C 6 alkyl or hydroxy-substituted C 1 -C 6 alkyl; or R 1 , R 2 and the connected benzene ring form a benzo 5-6 membered heteroaryl group;
  • X is selected from S, S(O) 2 or O;
  • Ring A is selected from: C 3 -C 6 cycloalkyl, 5-8 membered heterocyclyl, C 6 -C 10 aryl or 5-8 membered heteroaryl;
  • each R 3 is independently selected from hydrogen, halogen, cyano, C 1 -C 6 alkylthio, C 1 -C 6 alkylCO-, C 1 -C 6 alkylSO 2 -, amino, -NH(C 1 -C 6 alkyl), -N(C 1 -C 6 alkyl)(C 1 -C 6 alkyl), -SO 2 NH 2 , -C(O)NH 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, halo-substituted C 1 -C 6 alkyl, halo-substituted C 1 -C 6 alkoxy or halo-substituted C 1 -C 6 alkylthio; o is 0 , 1, 2, 3 or 4;
  • Y 1 and Y 2 are independently selected from CR 4 or N;
  • each R 4 is independently selected from hydrogen, halogen, cyano, C 1 -C 6 alkylthio, C 1 -C 6 alkylCO-, C 1 -C 6 alkylSO 2 -, amino, -NH(C 1 -C 6 alkyl), -N(C 1 -C 6 alkyl)(C 1 -C 6 alkyl), -SO 2 NH 2 , -C(O)NH 2 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, halo-substituted C 1 -C 6 alkyl, halo-substituted C 1 -C 6 alkoxy or halo-substituted C 1 -C 6 alkylthio;
  • n 0, 1, 2, 3 or 4;
  • Z 1 -Z 2 is selected from OZ 2 , NH-Z 2 , SZ 2 , S(O)-Z 2 , S(O) 2 -Z 2 , O-(C 1 -C 6 alkylene)-Z 2 , O-(halogenated C 1 -C 6 alkylene)-Z 2 , NH-(C 1 -C 6 alkylene)-Z 2 or NH-(halogenated C 1 -C 6 alkylene)-Z 2 ;
  • Z 2 and Z 3 are independently selected from CH or N; n is 1, 2 or 3;
  • R 5 is C 1 -C 6 alkyl, optionally substituted by one or more substituents selected from the group consisting of halogen, cyano, hydroxy, carboxyl, amino, methoxy or -SO 2 CH 3 ;
  • ring A is selected from: C 3 -C 6 cycloalkyl, 5-8 membered heterocyclyl, C 6 -C 10 aryl or 5-8 membered heteroaryl; when X is O, ring A is selected from: C 3 -C 6 cycloalkyl, 5-8 membered heterocyclyl or 5-8 membered heteroaryl.
  • each R 3 is independently selected from hydrogen, halogen, cyano, C 1 -C 4 alkylthio, C 1 -C 4 alkylCO-, C 1 -C 4 alkylSO 2 -, amino, -NH(C 1 -C 4 alkyl), -N(C 1 -C 4 alkyl)(C 1 -C 4 alkyl), -SO 2 NH 2 , -C(O)NH 2 , C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halo-substituted C 1 -C 4 alkyl, halo-substituted C 1 -C 4 alkoxy or halo-substituted C 1 -C 4 alkylthio;
  • o 0, 1, 2, 3, or 4;
  • ring A is selected from: C 3 -C 6 cycloalkyl, 5-7 membered heterocyclyl, C 6 -C 10 aryl or 5-7 membered heteroaryl; when X is O, ring A is selected from: C 3 -C 6 cycloalkyl, 5-7 membered heterocyclyl or 5-7 membered heteroaryl.
  • Z 1 -Z 2 is selected from OZ 2 , NH-Z 2 , SZ 2 , S(O)-Z 2 , S(O) 2 -Z 2 , O-(C 1 -C 4 alkylene)-Z 2 , O-(halogenated C 1 -C 4 alkylene)-Z 2 , NH-(C 1 -C 4 alkylene)-Z 2 or NH-(halogenated C 1 -C 4 alkylene)-Z 2 ;
  • Z 2 and Z 3 are independently selected from CH or N;
  • n 1, 2 or 3;
  • R 5 is C 1 -C 4 alkyl, optionally substituted by one or more substituents selected from the group consisting of fluorine, chlorine, bromine, cyano, hydroxyl or carboxyl.
  • R 1 is selected from OH, COOH, B(OH) 2
  • R 2 is selected from H; or R 1 , R 2 and the connected benzene ring form
  • X is selected from S or S(O) 2 ;
  • Ring A is selected from: C 3 -C 6 cycloalkyl, 5-6 membered heterocyclyl, phenyl or 5-6 membered heteroaryl;
  • each R 3 is independently selected from hydrogen, fluorine, chlorine, bromine, cyano, C 1 -C 4 alkylthio, C 1 -C 2 alkylCO-, C 1 -C 2 alkylSO 2 -, amino, -NH(C 1 -C 2 alkyl), -N(C 1 -C 2 alkyl)(C 1 -C 2 alkyl), -SO 2 NH 2 , -C(O)NH 2 , C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halo-substituted C 1 -C 4 alkyl, halo-substituted C 1 -C 4 alkoxy or halo-substituted C 1 -C 4 alkylthio;
  • o 0, 1, 2, 3, or 4;
  • Y 1 and Y 2 are independently selected from CR 4 or N;
  • each R 4 is independently selected from hydrogen, fluorine, chlorine, bromine, cyano, C 1 -C 2 alkylthio, C 1 -C 2 alkylCO-, C 1 -C 2 alkylSO 2 -, amino, -NH(C 1 -C 2 alkyl), -N(C 1 -C 2 alkyl)(C 1 -C 2 alkyl), -SO 2 NH 2 , -C(O)NH 2 , C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halo-substituted C 1 -C 4 alkyl, halo-substituted C 1 -C 4 alkoxy or halo-substituted C 1 -C 4 alkylthio;
  • n 0, 1, 2 or 3;
  • Z 1 -Z 2 is selected from OZ 2 , NH-Z 2 , O-(C 1 -C 4 alkylene)-Z 2 , O-(halogenated C 1 -C 4 alkylene)-Z 2 , NH-(C 1 -C 4 alkylene)-Z 2 or NH-(halogenated C 1 -C 4 alkylene)-Z 2 ;
  • Z 2 and Z 3 are independently selected from CH or N;
  • n 1 or 2;
  • R 5 is C 1 -C 4 alkyl, optionally substituted by one or more substituents selected from the group consisting of fluorine, chlorine, bromine, cyano, hydroxyl or carboxyl.
  • R 1 is selected from OH, COOH, B(OH) 2
  • R 2 is selected from H; or R 1 , R 2 and the connected benzene ring form
  • ring A when X is S or S(O) 2 , ring A is selected from: C 3 -C 6 cycloalkyl, 5-7 membered heterocyclyl, phenyl or 5-7 membered heteroaryl; when X is O, ring A is selected from: C 3 -C 6 cycloalkyl, 5-7 membered heterocyclyl or 5-7 membered heteroaryl.
  • ring A when X is S or S(O) 2 , ring A is selected from: C 3 -C 6 cycloalkyl, 5-7 membered heterocyclyl, C 6 -C 10 aryl or 5-7 membered heteroaryl; in another preferred embodiment, ring A is selected from: phenyl, 5-6 membered heterocyclyl (having 1 or 2 heteroatoms selected from the following group: N, O) or 5-6 membered heteroaryl (having 1 or 2 N); in another preferred embodiment, ring A is selected from: phenyl, piperidinyl, pyridinyl, pyranyl, tetrahydropyranyl, pyrazolyl, pyrrolyl, imidazolyl, pyridazinyl, pyrimidinyl, pyrazinyl.
  • X is S or S(O) 2 ; and ring A is phenyl.
  • X is O;
  • Ring A is a 5-6 membered heterocyclic group (having 1 or 2 heteroatoms selected from the group consisting of N, O) or a 5-6 membered heteroaryl group (having 1 or 2 N).
  • X is O; and ring A is piperidinyl, pyridinyl, pyrazolyl, tetrahydropyranyl, pyranyl, pyrrolyl, imidazolyl, pyridazinyl, or pyrimidinyl.
  • each R 3 is independently selected from hydrogen, halogen, cyano, C 1 -C 4 alkylthio, C 1 -C 4 alkylCO-, C 1 -C 4 alkylSO 2 -, amino, -NH(C 1 -C 4 alkyl), -N(C 1 -C 4 alkyl)(C 1 -C 4 alkyl), -SO 2 NH 2 , -C(O)NH 2 , C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halo-substituted C 1 -C 4 alkyl, halo-substituted C 1 -C 4 alkoxy or halo-substituted C 1 -C 4 alkylthio; o is 0, 1, 2, 3 or 4.
  • R 3 is selected from hydrogen, halogen, cyano, thiomethyl, acetyl, mesyl, -NMe 2 , -SO 2 NH 2 , -C(O)NH 2 , C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halogenated C 1 -C 4 alkyl, halogenated C 1 -C 4 alkoxy or halogenated C 1 -C 4 alkylthio.
  • each R 3 is independently selected from hydrogen, fluorine, chlorine, bromine, CH 3 CO-, CH 3 SO 2 -, CH 3 CH 2 CO-, CH 3 CH 2 SO 2 -, amino, cyano, -NHCH 3 , -N(CH 3 ) 2 , -SO 2 NH 2 , -C(O)NH 2 , methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, methoxy, ethoxy, propoxy, butoxy, halogenated C 1 -C 2 alkyl, halogenated C 1 -C 4 alkoxy or halogenated C 1 -C 4 alkylthio; o is 0, 1, 2, 3 or 4.
  • Y 1 is CH, CF or N.
  • Y2 is CH or N.
  • the halogenated C 1 -C 2 alkyl group is selected from -CH 2 F, -CHF 2 , -CF 3 , -CH 2 CF 3 , and -CH 2 CH 2 F.
  • R 4 is selected from hydrogen, halogen, cyano, thiomethyl, acetyl, methanesulfonyl, -NMe 2 , -SO 2 NH 2 , -C(O)NH 2 , C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halogenated C 1 -C 4 alkyl, halogenated C 1 -C 4 alkoxy or halogenated C 1 -C 4 alkylthio; m is 0, 1, 2 or 3.
  • each R 4 is independently selected from hydrogen, fluorine, chlorine, and bromine; and m is 0, 1, 2 or 3.
  • Z 1 -Z 2 is selected from OZ 2 , NH-Z 2 , SZ 2 , S(O)-Z 2 , S(O) 2 -Z 2 , O-(C 1 -C 4 alkylene)-Z 2 , O-(halogenated C 1 -C 4 alkylene)-Z 2 , NH-(C 1 -C 4 alkylene)-Z 2 or NH-(halogenated C 1 -C 4 alkylene)-Z 2 .
  • the above-mentioned halogenated group refers to fluorinated, chlorinated or brominated group.
  • Z 1 -Z 2 is selected from OZ 2 , NH-Z 2 , -OCH 2 -Z 2 , -OCH 2 CH 2 -Z 2 , -OCH 2 CH 2 CH 2 -Z 2 , O-(fluoro-C 1 -C 2 alkylene)-Z 2 , NH-(C 1 -C 2 alkylene)-Z 2 , or NH-(fluoro-C 1 -C 2 alkylene)-Z 2 , O-(chloro-C 1 -C 2 alkylene)-Z 2 , NH-(C 1 -C 2 alkylene)-Z 2 , or NH-(chloro-C 1 -C 2 alkylene)-Z 2 .
  • Z 2 is CH, Z 3 is N; Z 2 is N, Z 3 is CH.
  • n 1, 2 or 3.
  • R 5 is -CH 2 F, -CHF 2 , -CF 3 , -CH 2 CN or -CH 2 CH 2 CH 2 F. In another preferred embodiment, R 5 is -CH 2 F.
  • ring A is selected from 5-6 membered cycloalkyl, 5-6 membered heterocycloalkyl or 5-6 membered heterocyclic aryl; preferably
  • the compound represented by the general formula (I) is a compound represented by the general formula (Ia):
  • R 1 , R 2 , R 3 , o, R 4 , m, Z 1 , Z 2 , n, Z 3 and R 5 are as defined above.
  • the compound represented by the general formula (I) has a compound represented by the general formula (Ib):
  • R 1 , R 2 , R 3 , o, R 4 , m, Z 1 , Z 2 , n, Z 3 and R 5 are as defined above.
  • the compound is the compound prepared in Example.
  • the pharmaceutically acceptable salt is a salt formed by a compound and an organic acid or an inorganic acid, wherein the organic acid is selected from L-tartaric acid, fumaric acid, L-malic acid, D-malic acid, citric acid, L-pyroglutamic acid, acetic acid, p-toluenesulfonate, benzenesulfonic acid, methanesulfonic acid, benzoic acid, lactic acid, mandelic acid, maleic acid, oxalic acid, and succinic acid; and the inorganic acid is selected from hydrochloric acid, phosphoric acid, sulfuric acid, and hydrobromic acid.
  • the organic acid is selected from L-tartaric acid, fumaric acid, L-malic acid, D-malic acid, citric acid, L-pyroglutamic acid, acetic acid, p-toluenesulfonate, benzenesulfonic acid, methanesulfonic acid, benzo
  • the second aspect of the present invention provides a method for preparing the compound represented by the general formula (I) described in the first aspect.
  • the preparation method comprises the following steps:
  • A3 undergoes a Suziki coupling reaction with an organic boron reagent to obtain A4, wherein the organic boron reagent is selected from: boric acid Pinacol borate
  • A6 is subjected to hydrogenation and hydrogenolysis reaction under the action of a palladium catalyst and hydrogen to obtain A7, wherein the palladium catalyst is selected from Pd/C and Pd(OH) 2 /C;
  • A8 undergoes nucleophilic substitution reaction and hydrolysis under alkaline conditions to obtain a compound of formula (I), wherein the base is selected from triethylamine, N,N-diisopropylethylamine, pyridine, carbonate, NaH, sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium methoxide, and sodium ethoxide;
  • X, R 4 , Y 2 , Y 1 , Ring A, R 3 , o, m, Z 3 , and R 5 are as defined above;
  • preparation method comprises the following steps:
  • LG is a leaving group selected from Br, Cl, OTf, OTs or OMs;
  • X, R 4 , Y 2 , Y 1 , Ring A, R 3 , o, m, Z 3 , and R 5 are as defined above;
  • R1 is -COOH
  • R2 is hydrogen
  • the preparation method comprises the following steps:
  • (ii1) B4 undergoes bromination reaction with a bromination reagent to obtain an alkenyl bromide B5, wherein the bromination reagent is selected from: pyridinium tribromide, N-bromosuccinimide;
  • (ii2) B5 undergoes a Suziki coupling reaction with an organic boron reagent to obtain B6, wherein the organic boron reagent is selected from: boric acid Boric acid pinacol ester
  • B6 is subjected to hydrogenation and hydrogenolysis reaction under the action of a palladium catalyst and hydrogen to obtain B7, wherein the palladium catalyst is selected from Pd/C and Pd(OH) 2 /C;
  • B8 undergoes nucleophilic substitution reaction and hydrolysis under alkaline conditions to obtain a compound of formula (I), wherein the base is selected from triethylamine, N,N-diisopropylethylamine, pyridine, carbonate, NaH, sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium methoxide, and sodium ethoxide;
  • the base is selected from triethylamine, N,N-diisopropylethylamine, pyridine, carbonate, NaH, sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium methoxide, and sodium ethoxide;
  • R 4 , Y 2 , Y 1 , Ring A, R 3 , o, m, Z 3 , and R 5 are as defined above, and R 6 is a C 1 -C 6 alkyl group;
  • the preparation method comprises the following steps:
  • the palladium catalyst is selected from [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride, tetrakis(triphenylphosphine)palladium, tris(dibenzylideneacetone)dipalladium, bis(dibenzylideneacetone)palladium, bistriphenylphosphinepalladium dichloride, di(tri-tert-butylphosphine)palladium, bis(tricyclohexylphosphine)palladium, and palladium acetate;
  • C2 is hydrolyzed under acidic conditions to obtain C3, wherein the acid is selected from sulfuric acid, hydrochloric acid, phosphoric acid, methanesulfonic acid, trifluoroacetic acid, acetic acid, and trifluoromethanesulfonic acid;
  • C3 undergoes Friedel-Crafts reaction under the action of an acid to obtain C4, wherein the acid is selected from the group consisting of trifluoromethanesulfonic acid, trifluoroacetic acid, Eaton's reagent, polyphosphoric acid, sulfuric acid, and hydrochloric acid;
  • C4 reacts with dihydropyran in the presence of an acid to obtain C5, wherein the acid includes p-toluenesulfonic acid, methanesulfonic acid, and p-toluenesulfonic acid pyridinium salt;
  • C6 reacts with an aromatic bromide in the presence of a palladium catalyst to obtain C7, wherein the palladium catalyst is selected from [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride, tetrakis(triphenylphosphine)palladium, tris(dibenzylideneacetone)dipalladium, bis(dibenzylideneacetone)palladium, bistriphenylphosphinepalladium dichloride, di(tri-tert-butylphosphine)palladium, bis(tricyclohexylphosphine)palladium, and palladium acetate;
  • the palladium catalyst is selected from [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride, tetrakis(triphenylphosphine)palladium, tris(dibenzylideneacetone)dipalladium,
  • bromination reaction with a bromination reagent to obtain an alkenyl bromide C8, wherein the bromination reagent is selected from: pyridinium tribromide, N-bromosuccinimide;
  • C9 undergoes hydrogenation and hydrogenolysis reaction under the action of a palladium catalyst and hydrogen to obtain C10, wherein the palladium catalyst is selected from Pd/C and Pd(OH) 2 /C;
  • (iii11) C11 is subjected to nucleophilic substitution reaction and hydrolyzed under acidic conditions to obtain a compound of formula (I), wherein the acid is selected from: trifluoromethanesulfonic acid, trifluoroacetic acid, sulfuric acid, hydrochloric acid, p-toluenesulfonic acid, and a solution of hydrogen chloride in an organic solvent (methanol, ethanol, isopropanol, ethyl acetate, diethyl ether or 1,4-dioxane);
  • an organic solvent methanol, ethanol, isopropanol, ethyl acetate, diethyl ether or 1,4-dioxane
  • X, R 4 , Y 2 , Y 1 , Ring A, R 3 , o, m, Z 3 and R 5 are as defined above.
  • the third aspect of the present invention provides a pharmaceutical composition comprising:
  • the pharmaceutical composition further comprises a glidant or a diluent.
  • the fourth aspect of the present invention provides the use of the compound represented by the general formula (I) described in the first aspect, or its racemate, enantiomer, diastereoisomer, or mixture thereof, or pharmaceutically acceptable salt, or the pharmaceutical composition described in the third aspect, for preparing a drug for treating, preventing or diagnosing estrogen receptor-related diseases, preferably, for preparing a drug for treating, preventing or diagnosing breast cancer, endometrial cancer, cervical cancer, skin cancer, prostate cancer, ovarian cancer, fallopian tube tumors, lung cancer, leukemia, osteoporosis, neurodegenerative diseases, cardiovascular diseases, lupus erythematosus, endometriosis and obesity.
  • the disease associated with estrogen receptor is an estrogen receptor-sensitive, estrogen receptor-mediated or dependent disease or condition.
  • the disease associated with estrogen receptor is selected from cancer, osteoporosis, neurodegenerative disease, cardiovascular disease, lupus erythematosus, endometriosis and obesity.
  • the disease associated with estrogen receptor is selected from breast cancer, endometrial cancer, cervical cancer, skin cancer, prostate cancer, ovarian cancer, fallopian tube tumors, lung cancer and leukemia.
  • the disease associated with estrogen receptor is selected from ER-positive breast cancer.
  • the disease associated with estrogen receptor is selected from ER-positive breast cancer brain metastasis.
  • the present invention provides a novel structure, excellent ER antagonism and degradation activity, and an orally effective SERD, which can treat, prevent or diagnose estrogen receptor-sensitive, estrogen receptor-mediated or dependent diseases or conditions, especially estrogen receptor-positive breast cancer.
  • the compound of the present invention has a good brain exposure and can be used for the treatment of patients with brain metastatic breast cancer, meeting this huge unmet clinical need.
  • Figure 1 is a graph showing the results of the uterine wet weight experiment, wherein A shows the average ratio of uterine weight to body weight in different experimental groups, *p ⁇ 0.05, **p ⁇ 0.01; B is a histological image of the cross-section of the uterus of each experimental group, and the height of the endometrial surface epithelium is marked with a black line.
  • FIG. 2 is a graph showing the results of the compound's growth inhibition experiment on the MCF-7 mouse subcutaneous tumor model, **p ⁇ 0.01.
  • the inventors of the present application have developed a dihydrobenzothiopyran derivative through extensive and in-depth research, having a structure shown in the general formula (I), as a selective estrogen receptor degrader and antagonist, for the treatment of estrogen receptor positive diseases, particularly estrogen receptor positive breast cancer.
  • the compound of the present invention has a good brain exposure and can be used for the treatment of patients with brain metastatic breast cancer. On this basis, the present invention was completed.
  • C 1 -C 6 refers to having 1, 2, 3, 4, 5 or 6 carbon atoms
  • C 1 -C 4 refers to having 1, 2, 3 or 4 carbon atoms
  • 3-6 membered refers to having 3, 4, 5 or 6 ring atoms
  • 5-8 membered refers to having 5, 6, 7 or 8 ring atoms, and so on.
  • Hydrotopes include all isotopes thereof. Isotopes should be understood to include atoms with the same atomic number but different mass numbers. For example, hydrogen isotopes include tritium and deuterium, carbon isotopes include 13 C and 14 C, oxygen isotopes include 16 O and 18 O, etc.
  • Halogen refers to fluorine, chlorine, bromine or iodine; "halo” refers to fluoro, chloro, bromo or iodo.
  • Cyano refers to -CN.
  • Alkyl refers to a group formed when a saturated hydrocarbon composed of only two elements, C and H, loses a hydrogen atom from any carbon atom, including straight-chain and branched aliphatic hydrocarbons. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and sec-butyl.
  • Alkylene refers to a straight or branched saturated aliphatic group, i.e., a divalent hydrocarbon group, having a specified number of carbon atoms and connecting at least two other groups.
  • the two groups connected to the alkylene group can be connected to the same or different atoms on the alkylene group.
  • a straight chain alkylene group can be a divalent group of -(CH2)n-, where n is 1, 2, 3, 4, 5 or 6.
  • Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene and hexylene.
  • the alkylene group can be substituted or unsubstituted.
  • Alkenyl refers to an alkyl group as defined above consisting of at least two carbon atoms and at least one carbon-carbon double bond, for example, ethenyl, 1-propenyl, 2-propenyl, and the like.
  • Cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent.
  • Polycyclic cycloalkanes include spirocyclic, fused-ring and bridged-ring cycloalkyls.
  • Alkoxy refers to -O-(alkyl) and -O-(cycloalkyl) wherein alkyl and cycloalkyl are as described above.
  • Alkylthio refers to -S-(alkyl) and -S-(cycloalkyl) wherein alkyl and cycloalkyl are as described above.
  • aryl means a hydrocarbon moiety comprising one or more aromatic rings.
  • C6-C12 aryl refers to an aromatic ring radical having 6 to 12 carbon atoms without heteroatoms on the ring, such as phenyl, naphthyl, etc.
  • C6-C10 aryl has a similar meaning. Examples of aryl include, but are not limited to, phenyl (Ph), naphthyl, pyrenyl, anthracenyl, and phenanthrenyl.
  • heterocyclyl refers to a saturated or unsaturated, non-aromatic cyclic group containing at least one (e.g., 1, 2, 3 or 4) ring heteroatom (e.g., N, O or S), such as piperidinyl, tetrahydropyranyl, tetrahydropyridinyl, Pyrrolinyl, dihydropyridinyl, dihydrofuranyl, dihydrothiophenyl, morpholinyl.
  • ring heteroatom e.g., N, O or S
  • heteroaryl refers to an aromatic cyclic group containing at least one (such as 1, 2, 3 or 4) ring heteroatoms (such as N, O or S), for example furanyl, pyrrolyl, thienyl, oxazolyl, imidazolyl, thiazolyl, pyridyl, quinolyl, isoquinolyl, indolyl, pyrimidinyl, pyranyl.
  • substitution means that one or more hydrogen atoms in a group are independently replaced by a corresponding number of substituents; the substitution is mono-substitution or poly-substitution, preferably, the poly-substitution is di-substitution, tri-substitution, tetra-substitution, or penta-substitution.
  • the di-substitution means having two substituents, and so on.
  • “Isomers” of the present invention refer to compounds having the same molecular formula but differing in nature or in the bond sequence of their atoms or in the spatial arrangement of their atoms.
  • Stereoisomers are isomers whose atoms differ in spatial arrangement.
  • Stereoisomers that are not mirror images of each other are diastereomers and stereoisomers that are non-superimposable mirror images of each other are enantiomers.
  • Chiral compounds can exist as a single enantiomer or as a mixture thereof. A mixture containing equal proportions of enantiomers is called a "racemic mixture".
  • pharmaceutically acceptable salt and “pharmaceutically acceptable salt” of the present invention refer to the salts of the compounds of the present invention, which are safe and effective when used in mammals and have the desired biological activity.
  • pharmaceutically acceptable salts and “pharmaceutically acceptable salts” refer to salts formed by the compound of formula I with an acid selected from the group consisting of hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, nitric acid, methanesulfonic acid, aminosulfonic acid, salicylic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, citric acid, acetic acid, lactic acid, tartaric acid, succinic acid, oxalic acid, pyruvic acid, malic acid, glutamic acid, p-toluenesulfonic acid, naphthalenes
  • the present invention is completed by conducting extensive compound design, synthesis, in vitro and in vivo biological activity testing, metabolism and other drugability studies.
  • the following examples are only specific descriptions of the present invention and do not constitute any limitation to the present invention. Those skilled in the art use the techniques, methods, or permutations and combinations of related methods known in the art, and minor changes made to the present invention are all within the scope of protection of the present invention.
  • the structure of the compound of the present invention is determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS).
  • the mass spectrum is measured by a Finnigan LTQ linear ion trap mass spectrometer.
  • the silica gel used for column chromatography separation is 200-300 mesh, and the ratio of the eluent is volume ratio.
  • Commercial raw materials and reagents used in the synthesis such as acids, bases, metal catalysts, bromination reagents, acylating reagents, arylboronic acids
  • Products purchased from reagent companies such as petroleum ether, ethyl acetate, methanol, dichloromethane, 1,4-dioxane, acetonitrile, etc. were directly used in the reaction without additional purification.
  • X is S
  • R 1 is OH
  • R 2 is hydrogen
  • Z 1 is -OCH 2 CH 2 -
  • Z 2 is N
  • the compound represented by the general formula (I), its racemate, enantiomer, diastereomer and mixture thereof, and its pharmaceutically acceptable salt can be prepared by the following route 1 :
  • R 3 , R 4 , R5 , Z 3 , m, n, and o are as described above;
  • Step 1 A1 reacts with PivCl in the presence of a base to obtain A2, wherein the base includes triethylamine, N,N-diisopropylethylamine, pyridine, or carbonate, and the solvent includes chlorinated hydrocarbons, ethers, esters, or acetonitrile, and the temperature is 0-50°C;
  • Step 2 A2 is reacted with trifluoromethanesulfonic anhydride under the action of pyridine to obtain trifluoromethanesulfonate A3, the solvent includes chlorinated hydrocarbons and ethers, and the temperature is 0°C to room temperature;
  • Step 3 A3 undergoes Suzuki coupling with an aryl boron reagent in the presence of a palladium catalyst and a base to obtain A4, wherein the palladium catalyst includes but is not limited to [1,1′-bis(diphenylphosphino)ferrocene] dichloropalladium, tetrakis(triphenylphosphine)palladium, tri(dibenzylideneacetone)dipalladium, bis(dibenzylideneacetone)palladium, bistriphenylphosphine palladium dichloride, di(tri-tert-butylphosphine)palladium, bis(tricyclohexylphosphine)palladium, palladium acetate, the aryl boron reagent includes but is not limited to boric acid, boric acid pinacol ester, boric acid neopentyl glycol ester, the base includes but is not limited to potassium acetate, sodium carbonate,
  • Step 4 A4 reacts with a bromination reagent to obtain an alkenyl bromide A5, wherein the bromination reagent includes pyridinium tribromide, N-bromosuccinimide, and bromine, and the solvent includes a halogenated hydrocarbon, acetonitrile, acetic acid, and methanol;
  • Step 5 A5 undergoes Suzuki coupling with an organic boron reagent in the presence of a palladium catalyst and a base to obtain A6, wherein the palladium catalyst includes but is not limited to [1,1′-bis(diphenylphosphino)ferrocene] dichloropalladium, tetrakis(triphenylphosphine)palladium, tri(dibenzylideneacetone)dipalladium, bis(dibenzylideneacetone)palladium, bistriphenylphosphinepalladium dichloride, di(tri-tert-butylphosphine)palladium, bis(tricyclohexylphosphine)palladium, palladium acetate, and the aryl boron reagent includes but is not limited to boric acid, boric acid pinacol ester, neopentyldiboron Alcohol esters, the base includes but is not limited to potassium acetate, sodium carbonate, potassium
  • Step 6 A6 is hydrogenated and hydrogenolyzed under the action of Pd/C or Pd(OH) 2 /C and hydrogen to obtain A7, wherein the solvent includes dioxane, tetrahydrofuran, toluene, N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, dichloroethane, ethanol, methanol, 1-50 atmospheres, and the temperature is 0-50°C;
  • Step 7 A7 is reacted with dibromoethane in the presence of a base to obtain A8, wherein the base includes triethylamine, N,N-diisopropylethylamine, pyridine, carbonate, NaH, sodium hydroxide, potassium hydroxide, lithium hydroxide, and the solvent includes dioxane, tetrahydrofuran, toluene, acetonitrile, acetone, dichloromethane, dichloroethane, ethanol, methanol, and the temperature is 20-80°C;
  • Step 8 A8 undergoes an amination reaction with the corresponding amine in the presence of a base and removes the protecting group to obtain a compound of formula (I), wherein the base includes triethylamine, N,N-diisopropylethylamine, pyridine, carbonate, NaH, sodium hydroxide, potassium hydroxide, lithium hydroxide, and the solvent includes 1,4-dioxane, tetrahydrofuran, toluene, acetonitrile, acetone, dichloromethane, dichloroethane, ethanol, methanol, and the temperature is 20-80°C;
  • X is S
  • R 1 is COOH
  • R 2 is hydrogen
  • Z 1 is -OCH 2 CH 2 -
  • Z 2 is N
  • the compound represented by the general formula (I), its racemate, enantiomer, diastereomer and mixture thereof, and its pharmaceutically acceptable salt can be prepared by the following route 2 :
  • R 3 , R 4 , R 5 , Z 3 , m, n, and o are as described above;
  • Step 1 A1 is deprotected under the action of a base to obtain B2, wherein the base includes sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium methoxide, sodium ethoxide, and the solvent includes water, 1,4-dioxane, tetrahydrofuran, ethanol, methanol, and the temperature is 20-80°C;
  • the base includes sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium methoxide, sodium ethoxide
  • the solvent includes water, 1,4-dioxane, tetrahydrofuran, ethanol, methanol, and the temperature is 20-80°C;
  • Step 2 B1 is reacted with trifluoromethanesulfonic anhydride in the presence of a base to obtain trifluoromethanesulfonate B2, wherein the base comprises trifluoromethanesulfonic anhydride.
  • the solvent includes 1,4-dioxane, tetrahydrofuran, dichloromethane, dichloroethane, ethanol, methanol, the temperature is 0-50°C;
  • Step 3 B2 undergoes carboxylation reaction with a carboxylating agent under the action of a palladium catalyst to obtain B3, wherein the palladium catalyst includes but is not limited to [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride, tetrakis(triphenylphosphine)palladium, tris(dibenzylideneacetone)dipalladium, bis(dibenzylideneacetone)palladium, bistriphenylphosphinepalladium dichloride, di(tri-tert-butylphosphine)palladium, bis(tricyclohexylphosphine)palladium, palladium acetate, the carboxylating agent includes formic acid, sodium formate, potassium formate, lithium formate, the solvent includes 1,4-dioxane, tetrahydrofuran, toluene, N,N-dimethylformamide, dimethyl sul
  • Step 4 B3 is subjected to esterification reaction to obtain B4, wherein the conditions include methanol/concentrated sulfuric acid, iodomethane/base, TMSCHN 2 /methanol, wherein the base includes NaH, carbonate, sodium hydroxide, potassium hydroxide;
  • Step 5-8 Prepare the compound of formula (I) from B4 by referring to the method for preparing the compound of formula (I) from A4 in route 1.
  • R 3 , R 4 , R 5 , Z 3 , m, n, and o are as described above;
  • Step 1 C1 reacts with methyl 3-mercaptopropionate under the action of a palladium catalyst and a base to obtain C2, wherein the palladium catalyst
  • the oxidizing agent includes but is not limited to [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium, tetrakis(triphenylphosphine)palladium, tri(dibenzylideneacetone)dipalladium, bis(dibenzylideneacetone)palladium, bistriphenylphosphinepalladium dichloropalladium, di(tri-tert-butylphosphine)palladium, bis(tricyclohexylphosphine)palladium, palladium acetate
  • the arylborone reagent includes but is not limited to boric acid, boric acid pinacol ester, boric acid neopentyl glycol ester
  • the base includes but is not limited to potassium
  • Step 2 C2 is hydrolyzed under acidic conditions to obtain C3, wherein the acid includes sulfuric acid, hydrochloric acid, phosphoric acid, methanesulfonic acid, trifluoroacetic acid, acetic acid, trifluoromethanesulfonic acid, and the solvent includes water, 1,4-dioxane, tetrahydrofuran, toluene, acetonitrile, acetone, and the temperature is room temperature-100°C;
  • Step 3 C3 undergoes Friedel-Crafts reaction under the action of a protonic acid to obtain C4, wherein the protonic acid includes but is not limited to polyphosphoric acid, Eaton's reagent, and trifluoromethanesulfonic acid;
  • Step 4 C4 reacts with 2,3-dihydropyran in the presence of p-toluenesulfonic acid or p-toluenesulfonic acid-pyridinium salt to obtain C5, wherein the solvent comprises halogenated hydrocarbon, toluene or acetonitrile;
  • Step 5 C5 reacts with p-toluenesulfonyl hydrazide to obtain hydrazone C6, wherein the solvent includes methanol, ethanol, toluene, or acetonitrile;
  • Step 6 C6 and an aromatic bromide undergo a carbene migration insertion reaction under the action of a palladium catalyst to obtain C7
  • the palladium catalyst includes but is not limited to [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride, tetrakis(triphenylphosphine)palladium, tri(dibenzylideneacetone)dipalladium, bis(dibenzylideneacetone)palladium, bistriphenylphosphinepalladium dichloride, di(tri-tert-butylphosphine)palladium, bis(tricyclohexylphosphine)palladium, palladium acetate
  • the base includes but is not limited to potassium acetate, sodium carbonate, potassium carbonate, cesium carbonate, potassium tert-butoxide, sodium tert-butoxide, lithium tert-butoxide, NaH
  • the solvent includes 1,4-dio
  • Steps 7-11 The subsequent route is similar to route 1.
  • the compound of general formula (I) is dissolved in an appropriate solvent, and 1 equivalent of acid is added, or 1 equivalent of acid is dissolved in the same solvent and then the acid solution is added dropwise to the solution of the compound of general formula (I), and stirred for 1 hour.
  • the powder is obtained after concentration (or freeze-dried), and the obtained powder is suspended in methyl tert-butyl ether, slurried for 30 minutes, filtered, and dried to obtain the corresponding salt.
  • the acid used is an organic acid or an inorganic acid, wherein the inorganic acid includes but is not limited to hydrochloric acid, phosphoric acid, sulfuric acid, hydrobromic acid, and nitric acid; the organic acid includes but is not limited to L-tartaric acid, fumaric acid, L-malic acid, D-malic acid, citric acid, L-pyroglutamic acid, acetic acid, p-toluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, benzoic acid, lactic acid, mandelic acid, maleic acid, oxalic acid, and succinic acid.
  • the inorganic acid includes but is not limited to hydrochloric acid, phosphoric acid, sulfuric acid, hydrobromic acid, and nitric acid
  • the organic acid includes but is not limited to L-tartaric acid, fumaric acid, L-malic acid, D-malic acid, citric acid, L-pyroglutamic
  • the solvent includes, but is not limited to, acetonitrile, acetone, ethyl acetate, dichloromethane, tetrahydrofuran, methanol, ethanol, isopropanol, 1,4-dioxane, chloroform, and ether.
  • A3 (6.2 g, 15.7 mmol) (reference patent: WO2018091153), 4-benzyloxyphenylboronic acid (4.3 g, 18.8 mmol), Pd(dppf)Cl 2 (0.65 g, 0.8 mmol) and cesium carbonate (10.1 g, 31.4 mmol) were weighed into a reaction bottle, 1,4-dioxane-water mixture (4:1 v/v, 100 mL) was added, and the mixture was reacted in an oil bath at 50°C under nitrogen protection for 1 hour. Water (50 mL) was added for dilution, and ethyl acetate (50 mL ⁇ 3) was used for extraction.
  • A4-a (5.9 g, 13.7 mmol) was dissolved in dichloromethane (200 mL), cooled in an ice-water bath, and tribromopyridinium salt (5.4 g, 13.7 mmol, 85% content) was added.
  • A5-a (0.8 g, 1.57 mmol), 2,4-difluorophenylboronic acid (0.3 g, 1.88 mmol), Pd(dppf)Cl 2 (128 mg, 0.16 mmol) and cesium carbonate (1.0 g, 3.14 mmol) were added to the reaction flask, and a mixture of 1,4-dioxane-water (4:1 v/v, 20 mL) was added, and the mixture was reacted in an oil bath at 80°C for 4 hours under nitrogen protection.
  • reaction solution was cooled, it was diluted with water (50 mL), extracted with ethyl acetate (50 mL ⁇ 2), and the organic phases were combined, washed with saturated sodium chloride solution (40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent.
  • A6-a (0.75 g, 88%) was obtained as a white solid by column chromatography separation (petroleum ether/ethyl acetate/dichloromethane 15:1).
  • A6-a (0.65 g) was dissolved in ethanol (15 mL), 20% Pd(OH) 2 /C (0.5 g) was added, and the mixture was reacted at 60°C in a hydrogen atmosphere (1 atm) for 8 h.
  • the crude product was filtered through celite and concentrated under reduced pressure to remove the solvent, and the crude product was separated by column chromatography (petroleum ether/ethyl acetate 3:1) to obtain a white solid A7-a (0.44 g, 81%).
  • A7-a (0.25 g, 0.55 mmol) was dissolved in acetonitrile (15 mL), 1,2-dibromoethane (2 mL) and potassium carbonate (380 mg, 2.75 mmol) were added, and the mixture was refluxed in an oil bath for 12 h. Insoluble matter was removed by filtration, and the solvent was removed by concentration under reduced pressure. The crude product was used directly in the next step without purification.
  • the crude product obtained in the previous step was dissolved in acetonitrile (15 mL), 3-fluoromethyl-azetidine hydrochloride (103 mg, 0.83 mmol) and potassium carbonate (380 mg, 2.75 mmol) were added, and after reflux in an oil bath for 4 h, the insoluble matter was filtered off, and the solvent was removed by concentration under reduced pressure.
  • the crude product was dissolved in methanol (5 mL), sodium methoxide (0.5 mL) was added, and the mixture was stirred at room temperature for 10 min, and then concentrated under reduced pressure to remove the solvent. The mixture was neutralized with 1N HCl to pH 7-8, and extracted with dichloromethane (15 mL ⁇ 3).
  • Example 1 (0.11 g, total yield of three steps was 42%).
  • A5-a (0.9 g, 1.77 mmol), 4-fluoro-2-methylphenylboronic acid (0.33 g, 2.12 mmol), Pd(PPh 3 ) 4 (204 mg, 0.17 mmol) and cesium carbonate (1.2 g, 3.54 mmol) were added to a reaction flask, and a mixture of 1,4-dioxane-water (4:1 v/v, 20 mL) was added, and the mixture was reacted in an oil bath at 80°C for 4 hours under nitrogen protection.
  • reaction solution was cooled, it was diluted with water (50 mL), extracted with ethyl acetate (50 mL ⁇ 2), the organic phases were combined, washed with saturated sodium chloride solution (40 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to remove the solvent, and separated by column chromatography (petroleum ether/ethyl acetate/dichloromethane 15:1) to obtain a white solid A6-b (0.70 g, 74%).
  • A6-b (0.65 g) was dissolved in ethanol (15 mL), 20% Pd(OH) 2 /C (0.5 g) was added, and the mixture was reacted under a hydrogen atmosphere (1 atm) at 60°C for 48 h.
  • the crude product was filtered through celite and concentrated under reduced pressure to remove the solvent, and the crude product was separated by column chromatography (petroleum ether/ethyl acetate 3:1) to obtain a white solid A7-b (0.23 g, 42%).
  • A7-b (0.22 g, 0.49 mmol) was dissolved in acetonitrile (15 mL), and 1,2-dibromoethane (2 mL) and potassium carbonate (340 mg, 2.4 mmol) were added. After the reactants were refluxed in an oil bath for 12 h, the insoluble matter was removed by filtration, and the solvent was removed by concentration under reduced pressure. The crude product was used directly in the next step without purification.
  • the crude product obtained in the previous step was dissolved in acetonitrile (15 mL), 3-fluoromethyl-azetidine hydrochloride (100 mg, 0.78 mmol) and potassium carbonate (340 mg, 2.4 mmol) were added, and after reflux in an oil bath for 4 h, the insoluble matter was filtered off, and the solvent was removed by concentration under reduced pressure.
  • the crude product was dissolved in methanol (5 mL), sodium methoxide (0.5 mL) was added, and the mixture was stirred at room temperature for 10 min, and then the solvent was removed by concentration under reduced pressure. The mixture was neutralized with 1N HCl to pH 7-8, and extracted with dichloromethane (15 mL ⁇ 3).
  • A5-a (0.9 g, 1.77 mmol), 2-fluoro-4-methoxyphenylboronic acid (0.36 g, 2.12 mmol), Pd(PPh 3 ) 4 (204 mg, 0.17 mmol) and cesium carbonate (1.2 g, 3.54 mmol) were added to a reaction flask, and a mixture of 1,4-dioxane-water (4:1 v/v, 20 mL) was added, and the mixture was reacted in an oil bath at 80°C for 4 hours under nitrogen protection.
  • reaction solution was cooled, it was diluted with water (50 mL), extracted with ethyl acetate (50 mL ⁇ 2), the organic phases were combined, washed with saturated sodium chloride solution (40 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to remove the solvent, and separated by column chromatography (petroleum ether/ethyl acetate/dichloromethane 15:1) to obtain a white solid A6-c (0.57 g, 58%).
  • A6-c (0.55 g) was dissolved in ethanol (15 mL), 20% Pd(OH) 2 /C (0.5 g) was added, and the mixture was reacted under a hydrogen atmosphere (1 atm) at 60°C for 24 h.
  • the crude product was filtered through celite and concentrated under reduced pressure to remove the solvent, and the crude product was separated by column chromatography (petroleum ether/ethyl acetate 3:1) to obtain a white solid A7-c (0.31 g, 67%).
  • A7-c (0.30 g, 0.49 mmol) was dissolved in acetonitrile (15 mL), and 1,2-dibromoethane (2 mL) and potassium carbonate (340 mg, 2.4 mmol) were added. After the reactants were refluxed in an oil bath for 12 h, the insoluble matter was removed by filtration, and the solvent was removed by concentration under reduced pressure. The crude product was used directly in the next step without purification.
  • the crude product obtained in the previous step was dissolved in acetonitrile (15 mL), and 3-fluoromethyl-azetidine hydrochloride (100 mg, 0.78 mmol) and potassium carbonate (340 mg, 2.4 mmol) were added. After refluxing in an oil bath for 4 h, the insoluble matter was filtered out and the solvent was removed by concentration under reduced pressure.
  • the crude product was dissolved in methanol (5 mL), sodium methoxide (0.5 mL) was added, and the mixture was stirred at room temperature for 10 min, then concentrated under reduced pressure to remove the solvent, neutralized with 1N HCl to pH 7-8, and extracted with dichloromethane (15 mL ⁇ 3).
  • A5-a (0.62 g, 1.2 mmol), 4-fluoro-2-methylphenylboronic acid (0.31 g, 1.8 mmol), Pd(PPh 3 ) 4 (138 mg, 0.12 mmol) and cesium carbonate (0.8 g, 2.4 mmol) were added to a reaction flask, and a mixture of 1,4-dioxane-water (4:1 v/v, 15 mL) was added, and the mixture was reacted in an oil bath at 80°C for 4 hours under nitrogen protection.
  • reaction solution was cooled, it was diluted with water (50 mL), extracted with ethyl acetate (50 mL ⁇ 2), the organic phases were combined, washed with saturated sodium chloride solution (40 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to remove the solvent, and separated by column chromatography (petroleum ether/ethyl acetate/dichloromethane 10:1) to obtain a white solid A6-d (0.54 g, 80%).
  • A6-d (0.52 g) was dissolved in a mixed solvent of ethanol/ethyl acetate (1:1 v/v, 15 mL), and 20% Pd(OH) 2 /C (0.4 g) was added, and the mixture was reacted at 60°C in a hydrogen atmosphere (1 atm) for 8 h.
  • the crude product was filtered through diatomaceous earth and concentrated under reduced pressure to remove the solvent, and the crude product was separated by column chromatography (petroleum ether/ethyl acetate 3:1) to obtain a white solid A7-d (0.37 g, 86%).
  • A7-d (0.18 g, 0.39 mmol) was dissolved in acetonitrile (10 mL), and 1,2-dibromoethane (2 mL) and potassium carbonate (340 mg, 2.4 mmol) were added.
  • the reactants were refluxed in an oil bath for 12 h, insoluble matter was removed by filtration, and the solvent was removed by concentration under reduced pressure. The crude product was used in the next step without purification.
  • the crude product obtained in the previous step was dissolved in acetonitrile (15 mL), 3-fluoromethyl-azetidine hydrochloride (65 mg, 0.52 mmol) and potassium carbonate (269 mg, 1.95 mmol) were added, and after reflux in an oil bath for 4 h, the insoluble matter was filtered off, and the solvent was removed by concentration under reduced pressure.
  • the crude product was dissolved in methanol (5 mL), sodium methoxide (0.5 mL) was added, and the mixture was stirred at room temperature for 10 min, and then concentrated under reduced pressure to remove the solvent. The mixture was neutralized with 1N HCl to pH 7-8, and extracted with dichloromethane (15 mL ⁇ 3).
  • A5-a (0.9 g, 1.77 mmol), 2,4-5-trifluorophenylboronic acid (0.37 g, 2.12 mmol), Pd(PPh 3 ) 4 (204 mg, 0.17 mmol) and cesium carbonate (1.2 g, 3.54 mmol) were added to a reaction flask, and a mixture of 1,4-dioxane-water (4:1 v/v, 20 mL) was added, and the mixture was reacted in an oil bath at 80°C for 4 hours under nitrogen protection.
  • reaction solution was cooled, it was diluted with water (50 mL), extracted with ethyl acetate (50 mL ⁇ 2), the organic phases were combined, washed with saturated sodium chloride solution (40 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to remove the solvent, and separated by column chromatography (petroleum ether/ethyl acetate/dichloromethane 15:1) to obtain a white solid A6-e (0.83 g, 84%).
  • A6-e (0.8 g) was dissolved in ethanol (15 mL), 20% Pd(OH) 2 /C (0.8 g) was added, and the mixture was reacted under a hydrogen atmosphere (1 atm) at 60°C for 24 h.
  • the crude product was filtered through celite and concentrated under reduced pressure to remove the solvent, and the crude product was separated by column chromatography (petroleum ether/ethyl acetate 3:1) to obtain a white solid A7-e (0.45 g, 67%).
  • A7-e (0.40 g, 0.85 mmol) was dissolved in acetonitrile (15 mL), and 1,2-dibromoethane (2 mL) and potassium carbonate (280 mg, 4.2 mmol) were added. After the reactants were refluxed in an oil bath for 12 h, the insoluble matter was removed by filtration, and the solvent was removed by concentration under reduced pressure. After separation by column chromatography (petroleum ether/ethyl acetate 10:1), 270 mg of a colorless oil was obtained.
  • the crude product obtained in the previous step was dissolved in acetonitrile (15 mL), and 3-fluoromethyl-azetidine hydrochloride (180 mg, 1.44 mmol) and potassium carbonate (550 mg, 4.0 mmol) were added. After refluxing in an oil bath for 4 h, the insoluble matter was filtered out and the solvent was removed. The crude product was dissolved in methanol (5 mL), sodium methoxide (0.5 mL) was added, and the mixture was stirred at room temperature for 10 min, then concentrated under reduced pressure to remove the solvent, neutralized with 1N HCl to pH 7-8, and extracted with dichloromethane (15 mL ⁇ 3).
  • A3 (1.0 g, 2.53 mmol), 3-fluoro-4-benzyloxyphenylboronic acid (0.6 g, 2.42 mmol), Pd(dppf)Cl 2 (0.25 g, 0.31 mmol) and cesium carbonate (1.8 g, 5.5 mmol) were weighed into a reaction bottle, 1,4-dioxane-water mixture (4:1 v/v, 100 mL) was added, and the mixture was reacted in an oil bath at 50°C under nitrogen protection for 1 hour.
  • A4-b (0.95 g, 2.12 mmol) was dissolved in dichloromethane (100 mL), cooled in an ice-water bath, and tribromopyridinium salt (0.83 g, 2.12 mmol, 85% content) was added.
  • A5-b (0.85 g, 1.62 mmol), 4-fluoro-2-methoxyphenylboronic acid (0.33 g, 1.94 mmol), Pd(PPh 3 ) 4 (128 mg, 0.16 mmol) and cesium carbonate (1.0 g, 3.14 mmol) were added to a reaction flask, and a mixture of 1,4-dioxane-water (4:1 v/v, 20 mL) was added, and the mixture was reacted in an oil bath at 80°C for 4 hours under nitrogen protection.
  • reaction solution was cooled, it was diluted with water (50 mL), extracted with ethyl acetate (50 mL ⁇ 2), the organic phases were combined, washed with saturated sodium chloride solution (40 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to remove the solvent, and separated by column chromatography (petroleum ether/ethyl acetate/dichloromethane 15:1) to obtain a white solid A6-f (0.71 g, 79%).
  • A6-a (0.32 g) was dissolved in ethanol (15 mL), 20% Pd(OH) 2 /C (0.35 g) was added, and the mixture was reacted at 60°C in a hydrogen atmosphere (1 atm) for 8 h.
  • the crude product was filtered through celite and concentrated under reduced pressure to remove the solvent, and the crude product was separated by column chromatography (petroleum ether/ethyl acetate 3:1) to obtain a white solid A7-f (0.17 g, 63%).
  • A7-a (165 mg, 0.55 mmol) was dissolved in acetonitrile (15 mL), 1,2-dibromoethane (2 mL) and potassium carbonate (380 mg, 2.75 mmol) were added, and after reflux in an oil bath for 12 h, the insoluble matter was filtered out and the solvent was removed by concentration under reduced pressure. The crude product was used directly in the next step without purification.
  • the crude product obtained in the previous step was dissolved in acetonitrile (15 mL), and 3-fluoromethyl-azetidine hydrochloride (103 mg, 0.83 mmol) and potassium carbonate (380 mg, 2.75 mmol) were added. After refluxing in an oil bath for 4 h, the insoluble matter was removed by filtration, and the solvent was removed by concentration under reduced pressure.
  • the crude product was dissolved in methanol (5 mL), and sodium methoxide (0.5 mL) was added. The mixture was stirred at room temperature for 10 min, and then concentrated under reduced pressure to remove the solvent. The mixture was neutralized with 1N HCl to pH 7-8, and extracted with dichloromethane (15 mL ⁇ 3).
  • A3 (3.0 g, 7.6 mmol), 4-(tert-butoxycarbonylamino)-phenylboronic acid (1.94 g, 9.1 mmol), Pd(dppf)Cl 2 (0.62 g, 0.76 mmol) and cesium carbonate (4.9 g, 14.2 mmol) were weighed into a reaction bottle, 1,4-dioxane-water mixture (4:1 v/v, 30 mL) was added, and the mixture was reacted in an oil bath at 50°C for 1 hour under nitrogen protection. Water (50 mL) was added for dilution, and the mixture was extracted with ethyl acetate (50 mL ⁇ 3).
  • A4-b (2.8 g, 6.4 mmol) was dissolved in dichloromethane (100 mL), pyridine (1.0 g, 12.8 mmol) was added, and the mixture was cooled in an ice-water bath, and pyridinium tribromide (2.5 g, 6.4 mmol, 85% content) was added. After stirring in an ice-water bath for 1 hour, the mixture was quenched with a saturated sodium bicarbonate solution (100 mL), and the liquids were separated.
  • aqueous phase was extracted with dichloromethane (50 mL), and the organic phases were combined, washed with a saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a crude product, which was separated by column chromatography (petroleum ether/ethyl acetate 10:1) to obtain a light yellow foam solid A5-c (2.0 g, 60%).
  • A5-c (1.0 g, 1.93 mmol), 4-fluoro-2-methoxyphenylboronic acid (0.39 g, 2.31 mmol), Pd(PPh 3 ) 4 (210 mg, 0.19 mmol) and cesium carbonate (1.3 g, 3.8 mmol) were added to a reaction flask, and a mixture of 1,4-dioxane and water (4:1 v/v, 20 mL) was added, and the mixture was reacted in an oil bath at 80°C for 4 hours under nitrogen protection.
  • reaction solution was cooled, it was diluted with water (50 mL), extracted with ethyl acetate (50 mL ⁇ 2), the organic phases were combined, washed with saturated sodium chloride solution (40 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to remove the solvent, and separated by column chromatography (petroleum ether/ethyl acetate 10:1) to obtain a white solid A6-g (0.91 g, 84%).
  • A6-g (0.9 g) was dissolved in ethanol (15 mL), 20% Pd(OH) 2 /C (0.85 g) was added, and the mixture was reacted at 60°C in a hydrogen atmosphere (1 atm) for 8 h.
  • the crude product was filtered through celite and concentrated under reduced pressure to remove the solvent, and the crude product was separated by column chromatography (petroleum ether/ethyl acetate 5:1) to obtain a white solid A7-g (0.8 g, 89%).
  • A7-g (0.76 g) was dissolved in 1,4-dioxane (10 mL), and hydrogen chloride (4M 1,4-dioxane solution, 4 mL) was added, and the mixture was reacted at room temperature for 2 h.
  • the solvent was removed by concentration under reduced pressure to obtain a crude product, which was then dissolved in dichloromethane (20 mL), then washed with a saturated sodium bicarbonate solution, separated, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was separated by column chromatography (petroleum ether/ethyl acetate 5:1) to obtain a white solid A8-g (0.58 g, 93%).
  • A8-g (0.56 g, 1.2 mmol) and N-tert-butyloxycarbonyl-3-azetidinone (0.25 g, 1.44 mmol) were dissolved in 1,2-dichloroethane (10 mL), 2 drops of acetic acid were added, and the mixture was stirred at room temperature for 10 minutes.
  • Sodium triacetoxyborohydride (0.76 g, 3.6 mmol) was added, and the mixture was reacted at room temperature for 12 h.
  • Saturated sodium bicarbonate solution (10 mL) was added, and the mixture was stirred for 10 minutes.
  • A9-g (340 mg, 0.54 mmol) was dissolved in 1,4-dioxane (5 mL), and hydrogen chloride (4M 1,4-dioxane solution, 2 mL) was added, and the reaction was carried out at room temperature for 2 h. The solvent was removed by concentration under reduced pressure to obtain a crude product, which was then dissolved in dichloromethane (20 mL), and then washed with saturated sodium bicarbonate solution, separated, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product.
  • the crude product was dissolved in DMF (5 mL), and DIPEA (140 mg, 1.09 mmol) and 3-fluoro-1-iodopropane (98 mg, 0.49 mmol) were added, and stirred at room temperature for 24 h. Water (30 mL) was added, and the mixture was extracted with ethyl acetate (20 mL ⁇ 2). The organic phase was washed with a saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The obtained crude product was dissolved in methanol (5 mL), and sodium methoxide (0.5 mL) was added.
  • A7-d (0.18 g, 0.39 mmol) was dissolved in acetonitrile (10 mL), and 1,2-dibromoethane (2 mL) and potassium carbonate (340 mg, 2.4 mmol) were added. After the reactants were refluxed in an oil bath for 12 h, the insoluble matter was removed by filtration, and the solvent was removed by concentration under reduced pressure to obtain a crude product A8-h (200 mg) which was directly used in the next step.
  • A9-h (139 mg, 0.23 mmol) was dissolved in acetonitrile (15 mL), and 3-fluoromethyl-azetidine hydrochloride (96 mg, 0.77 mmol) and potassium carbonate (165 mg, 1.2 mmol) were added. After refluxing in an oil bath for 4 h, the insoluble matter was removed by filtration, and the solvent was removed by concentration under reduced pressure. The crude product was dissolved in methanol (5 mL), and sodium methoxide (0.5 mL) was added. The mixture was stirred at room temperature for 10 min, and then concentrated under reduced pressure to remove the solvent. The mixture was neutralized with 1N HCl to pH 7-8, and extracted with dichloromethane (15 mL ⁇ 3).
  • Dissolve B1 (1.7 g, 4.9 mmol) in dichloromethane (50 mL), add triethylamine (1.0 g, 7.4 mmol), cool in an ice-water bath, and add tribromopyridinium salt (0.95 mL, 5.4 mmol).
  • reaction solution was cooled, it was diluted with water (50 mL), extracted with ethyl acetate (50 mL ⁇ 2), the organic phases were combined, washed with saturated sodium chloride solution (40 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to remove the solvent, and separated by column chromatography (petroleum ether/ethyl acetate 10:1) to obtain B6 (0.45 g, 82%).
  • the crude product obtained in the previous step was dissolved in acetonitrile (15 mL), 3-fluoromethyl-azetidine hydrochloride (140 mg, 1.1 mmol) and potassium carbonate (380 mg, 2.75 mmol) were added, and after reflux in an oil bath for 4 h, the insoluble matter was filtered off, and the solvent was removed by concentration under reduced pressure.
  • the crude product was dissolved in methanol (5 mL), 2M NaOH (1 mL) was added, and the reaction was carried out at 60°C for 10 min, and then the solvent was removed by concentration under reduced pressure. The mixture was neutralized with 1N HCl to pH 7-8, and extracted with dichloromethane (15 mL ⁇ 3).
  • B11 (0.70 g, 1.1 mmol), bipyralidin (0.42 g, 1.7 mmol), potassium acetate (0.33 g, 3.3 mmol), Pd(dppf)Cl 2 (90 mg, 0.11 mmol) and 1,4-dioxane (30 mL) were added to the reaction flask.
  • the reaction was placed in an oil bath at 100°C for 16 hours. After the reaction solution was cooled, it was concentrated under reduced pressure to remove the solvent to obtain a crude product, which was separated by column chromatography (petroleum ether/ethyl acetate 5:1) to obtain B12 (0.58 g, 86%).
  • B12 (0.50 g, 0.82 mmol) was dissolved in 1,4-dioxane (10 mL), and HCl (4M 1,4-dioxane solution, 4 mL) was added, and the mixture was reacted at room temperature for 30 min. The solvent was removed by concentration under reduced pressure to obtain a crude product, which was separated by column chromatography (petroleum ether/ethyl acetate 3:1) to obtain B13 (0.41 g, two-step yield 42%).
  • Eaton's reagent (CAS#: 39394-84-8; 40 mL) was added to the crude product C3 obtained in the previous step, and the mixture was reacted in an oil bath at 80°C for 0.5 hours.
  • the reaction solution was cooled to room temperature and poured into ice water, extracted with ethyl acetate (100 mL ⁇ 2), and the organic phase was washed with saturated sodium bicarbonate solution (100 mL) and saturated sodium chloride solution (100 mL) in sequence, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a crude product, and petroleum ether/ethyl acetate (2:1 v/v, 20 mL) was added and stirred, and filtered to obtain C4 (3.0 g, three-step yield 60%) as a yellow solid.
  • the crude product obtained in the previous step was dissolved in acetonitrile (15 mL), and 3-fluoromethyl-azetidine hydrochloride (100 mg, 0.8 mmol) and potassium carbonate (380 mg, 2.75 mmol) were added. After refluxing in an oil bath for 4 h, the insoluble matter was filtered out and the solvent was removed by concentration under reduced pressure.
  • the crude product was dissolved in isopropanol (6 mL), and HCl (4 M isopropanol solution, 2 mL) was added. The reaction was carried out at 60 ° C for 30 min, and then the solvent was removed by concentration under reduced pressure.
  • reaction solution was cooled, it was diluted with water (20 mL), extracted with ethyl acetate (50 mL ⁇ 2), the organic phases were combined, washed with saturated sodium chloride solution (40 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to remove the solvent, and separated by column chromatography (petroleum ether/ethyl acetate 5:1) to obtain C9-b (0.35 g, 80%).
  • the crude product was dissolved in isopropanol (6 mL), HCl (4M isopropanol solution, 2 mL) was added, and the reaction was carried out at 60°C for 1 h, and then the solvent was removed by concentration under reduced pressure, and the product was dissolved in dichloromethane (50 mL), neutralized with saturated sodium bicarbonate solution (20 mL), and separated.
  • Example 12 was obtained as a white solid (65 mg, the total yield of three steps was 44%).
  • Step 1 Weigh D1 (3.8 g, 10 mmol) (reference patent: WO2018091153), 4-benzyloxyphenylboronic acid (2.7 g, 11.8 mmol), Pd(dppf)Cl 2 (0.81 g, 1.0 mmol) and cesium carbonate (6.5 g, 20 mmol) in a reaction bottle, add 1,4-dioxane-water mixture (4:1 v/v, 50 mL), and react in a 50°C oil bath under nitrogen protection for 1 hour.
  • Step 2 Dissolve D2 (5.0 g, 12.0 mmol) in dichloromethane (200 mL), cool in an ice-water bath, add pyridinium tribromide (4.5 g, 12.0 mmol, 85% content).
  • Step 3 D3 (1.0 g, 2.03 mmol), N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester (0.75 g, 2.44 mmol), Pd(PPh 3 ) 4 (230 mg, 0.2 mmol) and cesium carbonate (1.6 g, 4.0 mmol) were added to a reaction flask, and a mixture of 1,4-dioxane-water (4:1 v/v, 20 mL) was added, and the mixture was reacted in an oil bath at 100°C for 4 hours under nitrogen protection.
  • reaction solution was cooled, it was diluted with water (50 mL), extracted with ethyl acetate (50 mL ⁇ 2), the organic phases were combined, washed with saturated sodium chloride solution (40 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to remove the solvent, and separated by column chromatography (petroleum ether/ethyl acetate 5:1) to obtain a white solid D4-a (1.1 g, 91%).
  • Step 4 D4-a (0.50 g, 0.84 mmol) was dissolved in 1,4-dioxane (5 mL), HCl (4M 1,4-dioxane solution, 2 mL) was added, and the mixture was stirred at room temperature for 30 min. The solvent was removed by concentration, and the crude product was dissolved in dichloromethane (100 mL), washed with saturated sodium bicarbonate solution, separated, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain the intermediate.
  • Step 5 D5-a (0.38 g) was dissolved in ethanol (15 mL), 20% Pd(OH) 2 /C (0.5 g) was added, and the mixture was reacted at 60°C in a hydrogen atmosphere (1 atm) for 8 h. The crude product was filtered through diatomaceous earth and concentrated under reduced pressure to remove the solvent, and then separated by column chromatography (petroleum ether/ethyl acetate 1:2) to obtain D6-a (0.21 g, 66%).
  • Step 6 D6-a (170 mg, 0.38 mmol) was dissolved in acetonitrile (15 mL), 1,2-dibromoethane (2 mL) and potassium carbonate (260 mg, 1.9 mmol) were added, and the insoluble matter was filtered out after refluxing in an oil bath for 12 h, and the solvent was removed by concentrating under reduced pressure to obtain a crude product.
  • Step 1 Dissolve D4-a (0.60 g, 1.01 mmol) in 1,4-dioxane (5 mL), add HCl (4M 1,4-dioxane solution, 2 mL), and stir at room temperature for 30 min. Concentrate to remove the solvent, dissolve the crude product in dichloromethane (100 mL), wash with saturated sodium bicarbonate solution, separate the liquids, dry the organic phase with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to remove the solvent to obtain the intermediate.
  • Step 2 Dissolve D5-b (0.54 g) in ethanol (15 mL), add 20% Pd(OH) 2 /C (0.5 g), and react for 8 h at 60°C in a hydrogen atmosphere (1 atm). Filter with diatomaceous earth and concentrate under reduced pressure to remove the solvent to obtain a crude product, which is separated by column chromatography (petroleum ether/ethyl acetate 1:1) to obtain D6-b (0.37 g, 80%).
  • Step 3 Dissolve D6-b (250 mg, 0.51 mmol) in acetonitrile (15 mL), add 1,2-dibromoethane (2 mL) and potassium carbonate (350 mg, 2.6 mmol), reflux in an oil bath for 12 h, filter out insoluble matter, and concentrate under reduced pressure to remove the solvent to obtain Crude product.
  • the obtained crude product was then dissolved in acetonitrile (15 mL), 3-fluoromethyl-azetidine hydrochloride (140 mg, 1.1 mmol) and potassium carbonate (350 mg, 2.6 mmol) were added, and after refluxing in an oil bath for 1 h, the insoluble matter was filtered off, and the solvent was removed by concentration under reduced pressure.
  • Step 1 D3 (1.0 g, 2.03 mmol), 3,6-dihydro-2H-pyran-4-boronic acid pinacol ester (0.52 g, 2.44 mmol), Pd(PPh 3 ) 4 (230 mg, 0.2 mmol) and cesium carbonate (1.6 g, 4.0 mmol) were added to a reaction flask, and a mixture of 1,4-dioxane-water (4:1 v/v, 20 mL) was added, and the mixture was reacted in an oil bath at 100° C. for 4 hours under nitrogen protection.
  • reaction solution was cooled, it was diluted with water (50 mL), extracted with ethyl acetate (50 mL ⁇ 2), the organic phases were combined, washed with saturated sodium chloride solution (40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent, and separated by column chromatography (petroleum ether/ethyl acetate 5:1) to obtain a white solid D5-c (0.86 g, 85%).
  • Step 2 D5-c (0.51 g) was dissolved in ethanol (15 mL), 20% Pd(OH) 2 /C (0.6 g) was added, and the mixture was reacted at 60°C in a hydrogen atmosphere (1 atm) for 8 h.
  • the crude product was filtered through diatomaceous earth and concentrated under reduced pressure to remove the solvent, and then separated by column chromatography (petroleum ether/ethyl acetate 3:1) to obtain D6-c (0.20 g, 47%).
  • Step 3 Dissolve D6-c (110 mg, 0.27 mmol) in acetonitrile (15 mL), add 1,2-dibromoethane (2 mL) and potassium carbonate (190 mg, 1.4 mmol), reflux in an oil bath for 12 h, filter out insoluble matter, and concentrate under reduced pressure to remove the solvent to obtain a crude product.
  • the crude product was then dissolved in acetonitrile (15 mL), 3-fluoromethyl-azetidine hydrochloride (80 mg, 0.64 mmol) and potassium carbonate (190 mg, 1.4 mmol) were added, and after refluxing in an oil bath for 2 h, the insoluble matter was filtered off, and the solvent was removed by concentration under reduced pressure.
  • the crude product was dissolved in methanol (5 mL), sodium methoxide (0.5 mL) was added, and the mixture was stirred at room temperature for 10 min, and then concentrated under reduced pressure to remove the solvent.
  • the mixture was neutralized with 1N HCl to pH 7-8, and extracted with dichloromethane (15 mL ⁇ 3). The organic phases were combined, and then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a crude product.
  • the crude product was separated by column chromatography (dichloromethane/methanol 20:1-10:1) to obtain Example 15 (110 mg, total yield of three steps was 93%).
  • Step 1 D3 (0.6 g, 1.2 mmol), 2-methoxy-5-pyridineboronic acid (0.25 g, 1.6 mmol), Pd(PPh 3 ) 4 (154 mg, 0.13 mmol) and cesium carbonate (0.9 g, 2.7 mmol) were added to a reaction flask, and a mixture of 1,4-dioxane-water (4:1 v/v, 20 mL) was added, and the mixture was reacted in an oil bath at 100°C for 4 hours under nitrogen protection.
  • reaction solution was cooled, it was diluted with water (50 mL), extracted with ethyl acetate (50 mL ⁇ 2), the organic phases were combined, washed with saturated sodium chloride solution (40 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to remove the solvent, and separated by column chromatography (petroleum ether/ethyl acetate 5:1) to obtain D5-d (0.61 g, 97%).
  • Step 2 Dissolve D5-c (0.4 g) in ethanol (15 mL), add 20% Pd(OH) 2 /C (0.4 g), and react under a hydrogen atmosphere (1 atm) at 60°C for 8 h. Filter with diatomaceous earth, concentrate under reduced pressure to remove the solvent to obtain a crude product, and separate by column chromatography (petroleum ether/ethyl acetate 2:1) to obtain D6-d (0.14 g, 42%).
  • Step 3 Dissolve D6-d (130 mg, 0.30 mmol) in acetonitrile (10 mL), add 1,2-dibromoethane (1 mL) and potassium carbonate (210 mg, 1.5 mmol), reflux in an oil bath for 12 h, filter out insoluble matter, and concentrate under reduced pressure to remove the solvent to obtain a crude product.
  • the crude product was then dissolved in acetonitrile (15 mL), 3-fluoromethyl-azetidine hydrochloride (90 mg, 0.72 mmol) and potassium carbonate (210 mg, 1.5 mmol) were added, and after refluxing in an oil bath for 2 h, the insoluble matter was removed by filtration, and the solvent was removed by concentration under reduced pressure.
  • the crude product was dissolved in methanol (5 mL), sodium methoxide (0.5 mL) was added, and the mixture was stirred at room temperature for 10 min, and then the solvent was removed by concentration under reduced pressure.
  • the mixture was neutralized to pH 7-8 with 1N HCl, and extracted with dichloromethane (15 mL ⁇ 3).
  • the organic phases were combined, and then dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a crude product.
  • the crude product was separated by column chromatography (dichloromethane/methanol 20:1-10:1) to obtain Example 16 (42 mg, total yield of three steps was 30%).
  • Step 1 D3 (1.0 g, 2.0 mmol), 1-methyl-1H-pyrazole-4-boronic acid (0.44 g, 0.35 mmol), Pd(PPh 3 ) 4 (260 mg, 0.22 mmol) and cesium carbonate (1.4 g, 4.3 mmol) were added to a reaction flask, and a mixture of 1,4-dioxane-water (4:1 v/v, 20 mL) was added, and the mixture was reacted in an oil bath at 80°C for 4 hours under nitrogen protection.
  • reaction solution was cooled, it was diluted with water (50 mL), extracted with ethyl acetate (50 mL ⁇ 2), the organic phases were combined, washed with saturated sodium chloride solution (40 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to remove the solvent, and separated by column chromatography (petroleum ether/ethyl acetate 2:1) to obtain D5-e (0.82 g, 81%).
  • Step 2 Dissolve D5-c (0.8 g) in ethanol (15 mL), add 20% Pd(OH) 2 /C (0.8 g), and react for 8 h at 60°C in a hydrogen atmosphere (1 atm). Filter with diatomaceous earth and concentrate under reduced pressure to remove the solvent to obtain a crude product, which is separated by column chromatography (petroleum ether/ethyl acetate 2:1) to obtain D6-e (0.57 g, 86%).
  • Step 3 Dissolve D6-e (220 mg, 0.54 mmol) in acetonitrile (10 mL), add 1,2-dibromoethane (2 mL) and potassium carbonate (373 mg, 2.7 mmol), reflux in an oil bath for 12 h, filter out insoluble matter, and concentrate under reduced pressure to remove the solvent to obtain a crude product.
  • the crude product was then dissolved in acetonitrile (15 mL), and 3-fluoromethyl-azetidine hydrochloride (140 mg, 1.12 mmol) and potassium carbonate (373 mg, 2.7 mmol) were added. After refluxing in an oil bath for 4 h, the insoluble matter was filtered out and the solvent was removed.
  • Example 18 The compound shown in Example 18 can be obtained by separating Example 4 by chiral preparative HPLC.
  • the NMR data of Example 18 is consistent with that of Example 4, ee>99%.
  • Example 19 The compound shown in Example 19 can be obtained by separating Example 5 by chiral preparative HPLC.
  • the NMR data of Example 19 are consistent with those of Example 5, ee>99%.
  • Example 20 The compound shown in Example 20 can be obtained by separating Example 4 by chiral preparative HPLC.
  • the NMR data of Example 20 is consistent with that of Example 4, ee>99%.
  • Example 21 A8-e was separated by chiral preparative HPLC to obtain the compound shown in Example 21.
  • the NMR data of Example 21 were consistent with those of Example 5, ee>99%.
  • Cis-3-(4-fluoro-2-methoxyphenyl)-4-(4-(2-(3-(fluoromethyl)azetidin-1-yl)ethoxy)phenyl)-7-hydroxythiochroman 50 mg, 0.1 mmol was dissolved in 1,4-dioxane (5 mL), followed by addition of a 1,4-dioxane solution of hydrogen chloride (4 M, 40 ⁇ M), and stirring at room temperature for 1 hour. The reaction solution was concentrated to remove the solvent, and then methyl tert-butyl ether (2 mL) was added and stirred at room temperature for 30 minutes, filtered, and the filter cake was dried to obtain a white powder (45 mg, 84%).
  • Cis-3-(4-fluoro-2-methoxyphenyl)-4-(4-(2-(3-(fluoromethyl)azetidin-1-yl)ethoxy)phenyl)-7-hydroxythiochroman 50 mg, 0.1 mmol
  • acetonitrile 5 mL
  • methanesulfonic acid 9.7 mg, 0.1 mmol
  • stirring room temperature for 1 hour.
  • water 10 mL
  • the mixture was lyophilized to obtain a white foamy solid (55 mg, 94%).
  • Test Example 1 Evaluation of the inhibitory activity of the compounds of the present invention on the proliferation of breast cancer MCF-7 cells
  • the purpose of this experiment is to determine the inhibitory effect of the compounds of the present invention on the in vitro proliferation of MCF-7 cells, a commonly used breast cancer estrogen receptor-positive cell, and to evaluate the in vitro activity of the compounds based on the IC 50 value.
  • CTG CellTiter-Glo
  • ATP is a key indicator of the metabolism of living cells.
  • the homogenous detection makes the cell lysis and the generated luminescent signal proportional to the amount of ATP present, and the amount of ATP is directly proportional to the number of cells in the culture.
  • the specific experimental method is as follows:
  • MCF-7 cells were cultured with DMEM + 10% FBS. The cells were cultured in a 37°C incubator with 5% CO 2. Cell passaging, recovery and cryopreservation were performed according to conventional methods. MCF-7 cells grew to a polymerization degree of about 80%, and the cells were trypsinized and inoculated into 96-well plates at 4 ⁇ 10 3 cells per well, with an inoculation volume of 90 ⁇ L per well, and cultured in a 37°C incubator overnight. The next day, a concentration gradient of drugs was added, 10 ⁇ L per well. The highest detection concentration of the compound was 10 ⁇ M, 4-fold dilution, and 10 concentrations.
  • the 96-well plate was placed in a 5% CO 2 , 37°C incubator and cultured for 6 days. 50 ⁇ L of CTG reagent was added to each well of the 96-well culture plate, shaken on a shaker for 5 minutes, placed at room temperature in the dark for 10 minutes, and 60 ⁇ L was transferred to a 384-well Opti-plate. The luminescence signal was read using a multifunctional microplate reader, and the signal intensity was used to characterize the number of living cells. The data of each drug-added group were normalized with the mean of the control group as 100%, and the data were processed and analyzed using GraphPad Prism software. The results are shown in Table 1.
  • the compounds of the present invention have a strong inhibitory effect on the proliferation of MCF-7 breast cancer cells, especially the compounds of Examples 18 and 19, whose anti-tumor activity is 7.6 times higher than that of the positive control fulvestrant and 15.6 times higher than that of SAR439859, reaching the sub-nanomolar level.
  • Test Example 2 Evaluation of the inhibitory activity of the compounds of the present invention on tamoxifen-resistant MCF-7 Tam1 cells
  • MCF-7 Tam1 cells are established by long-term exposure of MCF-7 cells to 4-hydroxytamoxifen, an active metabolite of tamoxifen, accompanied by long-term estrogen deprivation culture conditions, and have resistance to tamoxifen and aromatase inhibitors. This experiment will detect the inhibition of proliferation of the compounds of the present invention on tamoxifen-resistant cells MCF-7 Tam1, and evaluate the in vitro anti-tamoxifen-resistant breast cancer activity of the compounds based on the IC 50 value.
  • MCF-7 Tam1 cells were subcultured in DMEM medium containing penicillin (final concentration of 100U/mL), streptomycin (final concentration of 100 ⁇ g/mL), 10 ⁇ g/mL human insulin, 1 ⁇ M 4-hydroxytamoxifen and 10% FBS. When the cells were 90% confluent, the old medium was discarded and the cells were washed twice with 2ml PBS. After discarding PBS, 2mL 0.25% trypsin-0.02% EDTA mixed digestion solution was added and observed under a microscope for about 30s. When the cells became round, 2ml complete medium was quickly added to terminate the digestion. The cells were gently blown and collected.
  • inhibition rate (OD value of the control group - OD value of the drug group) / OD value of the control group.
  • Table 2 shows that the compounds of the present invention have a strong inhibitory effect on the proliferation of tamoxifen-resistant breast cancer cells MCF-7 Tam1, and their anti-tumor activity is similar to that of Fulvestrant and significantly better than that of SAR439859.
  • Test Example 3 Evaluation of the degradation activity of the compounds of the present invention on wild-type ER ⁇ in MCF-7 cells
  • the extent of ER degradation induced by the compounds described in the present invention was analyzed by a cell-based high-content imaging method.
  • MCF-7 cell culture medium 88% RPMI 1640, 10% FBS, 1% P/S and 1% GlutaMax
  • MCF-7 seeding medium 88% RPMI 1640, 10% FBS, 1% P/S and 1% GlutaMax.
  • Day 1 Dilute the cell suspension to 8.75 ⁇ 10 4 cells/mL with cell seeding medium, then drop 40 ⁇ L of cell suspension into each well of the assay plate and place in a 37°C CO 2 incubator.
  • Day 3 Dilute the compound 4-fold serially, transfer 500 ⁇ L, 10 concentration points. Dilute the compound with 20 ⁇ L of medium, transfer 10 ⁇ L to the plate with Bravo, and incubate in a 37°C incubator for 24 hours.
  • Day 4 Add 50 ⁇ L of 8% paraformaldehyde to the assay plate and place at room temperature for 40 minutes. Wash the assay plate twice with 100 ⁇ L PBS per well, then add 50 ⁇ L of 0.1% TritonX-100 in PBS to the assay plate and place at room temperature for 15 minutes.
  • the compounds of the present invention have a strong degradation effect on ER ⁇ , especially the compounds of Examples 1, 3, 18 and 19, whose ER degradation activity is about 2 times higher than that of the positive control fulvestrant and about 3 times higher than that of SAR439859, reaching the subnanomolar level.
  • Test Example 4 MCF-7 cell fluorescence reporter gene assay to detect the antagonistic activity of compounds against wild-type ER ⁇
  • ER ⁇ reporter gene construction HEK293/GAL4/ER ⁇
  • the cell line uses molecular cloning methods to express fusion proteins of the estrogen receptor LBD region (compound binding region) and the GAL4 DBD region (DNA binding region).
  • the ligand activates ER ⁇
  • the ER ⁇ -GAL4 fusion protein activates the expression of the downstream luciferase gene
  • the plate reader detects the chemiluminescent signal.
  • the ER ⁇ ligand stimulation concentration and the chemiluminescent signal are dose-dependent.
  • the HEK293/GAL4/ER ⁇ cell suspension was collected and centrifuged at 1000 rpm for 5 minutes. The supernatant was removed and resuspended with preheated culture medium (DMEM (without phenol red), containing 10% carbon-adsorbed serum, i.e., 500 mL of cell culture medium contains 450 mL of DMEM and 50 mL of carbon-adsorbed serum). After counting, the cell suspension was diluted with culture medium and inoculated into a 96-well cell culture plate at 40,000 cells/well. 80 ⁇ L of cell suspension was inoculated into each well and incubated overnight at 37°C in a 5% CO2 incubator.
  • DMEM preheated culture medium
  • 500 mL of cell culture medium contains 450 mL of DMEM and 50 mL of carbon-adsorbed serum.
  • the cell suspension was diluted with culture medium and inoculated into a 96-well cell culture plate at 40,000 cells/well
  • % inhibition rate 100-(RFU compound-RFU blank control)/(RFU negative control-RFU blank control) ⁇ 100%.
  • Negative control cells treated with agonists; blank control: cells not treated with agonists, and then Prism was used to plot and calculate the IC 50 value of the compound.
  • the compounds of the present invention have a strong antagonistic effect on wild-type ER ⁇ , which is superior to fulvestrant and clinical phase III drug SAR439859.
  • the antagonistic activity of the compounds of Examples 3, 4, 5, 18, and 19 on wild-type ER ⁇ is about 3 times higher than that of SAR439859, and more than 2 times higher than that of fulvestrant.
  • Test Example 5 Antagonistic effect of the compounds of the present invention on mutant ER ⁇ containing Y537S or D538G mutations
  • the mutant ER ⁇ has an increased affinity for estradiol and a reduced affinity for antagonists.
  • This experiment evaluates the in vitro antitumor activity of the compounds of the present invention against endocrine therapy-resistant breast cancer by detecting the antagonistic activity of the compounds against mutant ER ⁇ containing the main mutation hotspots Y537S and D538G.
  • SK-BR-3 Cell Culture Medium 89% 1640 without phenol red, 10% charcoal-stripped FBS and 1% GlutaMax
  • Day 1 1. Inoculate 80 ⁇ L of cell suspension and 30,000 cells into each well of the assay plate and incubate at 37°C 5% CO 2 for 24 hours.
  • Day 2 Prepare the transfection reagent and leave at room temperature for 15 minutes. Add 10 ⁇ L of transfection reagent to each well of the assay plate and incubate at 37°C 5% CO 2 for 24 hours.
  • Day 3 Add 10 ⁇ L of culture medium (100 nM ⁇ -estradiol, 10 ⁇ L of culture medium) to the assay plate and incubate at 37°C 5% CO 2 for 24 hours.
  • Day 4 1.
  • Example 4 has comparable antagonistic activity to Fulvestrant and SAR439859
  • Example 18 has 3.3-fold and 3.7-fold improvement over Fulvestrant and SAR439859, respectively
  • Example 19 has 4-fold and 4.5-fold improvement over Fulvestrant and SAR439859, respectively
  • Examples 18 and 19 have similar antagonistic activity to Fulvestrant, but have 5.7-fold and 5.2-fold improvement over SAR439859, respectively.
  • test examples 1 to 5 show that the compounds of the present invention have significant dual functions of antagonism/degradation of ER ⁇ , including significant antagonism of mutant ER ⁇ containing common mutation points, and significant antiproliferative activity against the proliferation of estrogen-dependent MCF-7 cells and tamoxifen-resistant MCF-7 Tam1 cells. Combining the results of these tests, it can be seen that the compounds of the present invention exhibit excellent in vitro antitumor efficacy, and are superior to the marketed drug Fulvestrant in multiple tests, and significantly superior to the control compound SAR439859.
  • Test Example 6 Evaluation of the pharmacokinetic properties of the compounds of the present invention in rats
  • the purpose of this experiment is to test the pharmacokinetic properties of the compounds of the present invention in rats.
  • the solvent is: 5% DMSO + 5% Solutol + 90% (0.5% MC), which is a colorless and clear dosing solution.
  • the dosing method and dosage are: oral, 10 mg/kg. SD rats were randomly divided into groups according to body weight, fasted but not watered for 12-14 hours one day before dosing, and fed 4 hours after dosing. 0.1 mL of blood was collected from each animal through the eye socket each time, anticoagulated with EDTAK2, and the collection time points were: 0, 5, 15, 30 minutes, 1, 2, 4, 6, 8, 24 hours after administration of the test substance. After blood sample collection, it was placed on ice and centrifuged within 30 minutes to separate plasma (centrifugation conditions: 5000 rpm, 10 minutes, 4°C).
  • the concentration of compounds in rat plasma was determined by LC-MS/MS.
  • the data acquisition and control system software was Analyst1.5.1 software (Applied Biosystem).
  • the peak integration method of the chromatogram sample was automatic integration; the ratio of the sample peak area to the internal standard peak area was used as an indicator and regressed with the concentration of the sample. Regression method: linear regression, weight coefficient is 1/X 2 .
  • Pharmacokinetic parameters were analyzed and processed by WinNonlin Professional v6.3 (Pharsight, USA) using non-compartmental model.
  • C max is the maximum measured blood drug concentration
  • AUC (0 ⁇ t) is calculated by the trapezoidal method
  • T max is the time when the blood drug concentration reaches the peak after administration.
  • Table 6 shows that the compounds of the present invention have good oral absorption and good drug exposure, which indicates that the drug efficacy in animals and clinical It can be administered orally.
  • Test Example 7 Evaluation of the effect of the compound on rat uterine weight gain and tissue distribution
  • Estrogen receptor modulators such as tamoxifen in endocrine therapy for ER-positive breast cancer increase the risk of endometrial hyperplasia, polyps, and endometrial cancer due to their partial agonist effects. Therefore, when developing selective estrogen receptor degraders (SERDs), this effect needs to be eliminated to make them complete ER antagonists.
  • SERMs selective estrogen receptor degraders
  • the ability of drugs to penetrate the blood-brain barrier of animals and have sufficient exposure in brain tissue is the key to their effectiveness against brain metastases. Therefore, by analyzing the distribution of drugs in animal plasma and brain after administration, it can be determined whether the drug has the potential to have an anti-tumor effect in the brain in situ tumor model.
  • the uterine wet weight experiment and the tissue distribution experiment of the compound involved in this test were carried out in the same batch of rats.
  • mice SPF female immature SD rats, 21 days old, provided by Changzhou Cavens Laboratory Animal Co., Ltd., raised in an SPF-grade breeding environment, with the indoor temperature controlled at 23 ⁇ 2°C, free access to food and water. A total of 30 animals. Adaptive breeding for 3 days before the experiment.
  • the rats were killed by carbon dioxide method, the uterus was dissected, irrelevant tissues were carefully removed, washed 2-3 times with D-Hanks solution to remove blood, drained and stored, and weighed.
  • HE staining was used to detect the thickness of the endometrium. Plasma and brain tissue were sampled and the concentrations of Example 18 and Example 19 in the plasma and brain tissue were analyzed.
  • HE experiment 1) Cut the fixed uterine tissue into 4 ⁇ m thick slices and place the slices in an oven for 1 hour; 2) Dewax the dried paraffin slices with conventional xylene, hydrate with gradient ethanol, and wash with distilled water; 3) Add hematoxylin to stain for 10-30 minutes, then wash away the hematoxylin stain with running water; 4) Fading with 1% hydrochloric acid ethanol until the slices turn red and lighter in color, and then put them in running water to restore them to blue. 5) Stain with eosin for 1 minute and rinse with running water; 6) Dehydrate and dry the slices with gradient alcohol, make them transparent with xylene, and seal them with neutral gum. 7) Randomly select a field of view and take pictures with a microscope (200 ⁇ ). The statistical differences between the data groups were analyzed using one-way ANOVA and Tukey’s test, and P values less than 0.05 were considered significant.
  • LC-MS/MS detection of drug concentration (1) Chromatographic conditions, chromatographic column: Waters BEH C18 column (50 mm ⁇ 2.1 mm, id 1.7 ⁇ m), column temperature 40°C; mobile phase A is ultrapure water (containing 0.1% formic acid), mobile phase B is methanol. Elution gradient: 0-1 min, AB (70:30), 1-5 min, AB (10:90), 5-6 min, AB (10:90), 6-6.1 min, AB (70:30), 6.1-9 min, AB (70:30). The elution time is 9 min, and the flow rate is 0.3 mL/min.
  • Standard curve The standard curves were respectively Accurately weigh the standard samples of Example 18 and Example 19, prepare a standard stock solution with a concentration of 1 mg/mL, dilute it to a standard working solution with a concentration of 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10000, 20000, 50000 ng/mL, add 5 ⁇ L of each concentration of standard working solution to 45 ⁇ L of blank matrix, and prepare a final concentration of 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450 , 50, 100, 200, 500, 1000, 2000, 5000 ng/mL standard samples, vortex mixed for 3 min, add 150 ⁇ L of
  • 17 ⁇ -ethinyl estradiol and 4-hydroxytamoxifen increased uterine wet weight, and epithelial cells showed a high columnar phenotype, while the epithelial cells of the blank control and Example 18 and Example 19 groups showed a low cubic phenotype, confirming that the example compound of the present invention is a complete antagonist and does not have the risk of endometrial cancer with estrogen receptor modulators such as tamoxifen.
  • the compounds of the present invention exhibit excellent blood-brain barrier penetration ability and high drug exposure in the brain tissue of rats, among which Example 18 has very good brain tissue exposure, with a B/P value of 14.1, which is much higher than other estrogen degraders disclosed so far, and the B/P value of Example 19 is as high as 29. These results indicate that the compounds of the present invention can be used to treat brain metastatic breast cancer.
  • Test Example 8 Growth inhibition experiment of the compound of the present invention on MCF-7 mouse subcutaneous tumor model
  • Test reagents fetal bovine serum (SH30070.03) (FBS, Hyclone, Logan, UT, USA); penicillin (I9532) (Sigma, St. Louis, MO, USA); streptomycin (85886) (Sigma, St. Louis, MO, USA); recombinant human insulin (91077C) (Sigma, St.
  • EMEM medium (30-2003) (ATCC, Rockville, MD, USA); trypsin (15090046) (Gibco, Grand Island, NY, USA); HBSS (H6648), DMSO (D8418), PEG400 (8074851000), PEG300 (8074841000), PBS (806552), Solutol HS-15 (42966) (Sigma, St.
  • mice Female athymic nude mice, provided by Changzhou Cavens Laboratory Animal Co., Ltd., Animal Certificate No.: SCXK(Su)2016-0010, were kept in an environment of 22 ⁇ 2°C and had free access to food and water.
  • MCF-7 cells were subcultured in EMEM medium containing penicillin (final concentration of 100U/mL), streptomycin (final concentration of 100 ⁇ g/mL), human recombinant insulin (final concentration of 0.01mg/mL) and 10% FBS.
  • penicillin final concentration of 100U/mL
  • streptomycin final concentration of 100 ⁇ g/mL
  • human recombinant insulin final concentration of 0.01mg/mL
  • FBS FBS
  • 2mL 0.25% trypsin-0.02% EDTA mixed digestion solution was added and observed under a microscope for about 30s.
  • 2ml complete medium was quickly added to terminate digestion. The cells were gently blown and collected. Centrifuge at 800rpm, 4°C for 5min, discard the supernatant, resuspend the cells with complete medium, culture in bottles, and change the medium every other day.
  • mice transplant tumor model 3 days before tumor implantation, estrogen pellets (0.36 mg estradiol, 60-day release) were implanted subcutaneously between the shoulder blades of mice using a sterile trocar. MCF-7 cells in the logarithmic growth phase were obtained, digested with trypsin, and resuspended into a 10 7 cells/mL cell suspension using a mixture of 50% HBSS and 50% Matrigel.
  • mice were subcutaneously injected with 200 ⁇ L of MCF-7 cell suspension in the right axillary mammary fat pad area, and the tumor volume (width 2 ⁇ length ⁇ ⁇ /6) and body weight were measured every 3 days; when the average tumor volume reached about 200 mm 3 , the mice were randomly divided into groups and gavage was administered.
  • Blank group 10% PEG300 + 25% of 20% Solutol + 65% PBS.
  • TGI (%) [(1-(the drug administration result of a certain treatment group (mean tumor volume at the end of dosing in the treatment group - mean tumor volume at the start of dosing in the treatment group)/(mean tumor volume at the end of dosing in the solvent control group - mean tumor volume at the start of dosing in the solvent control group)] ⁇ 100%.
  • the compounds of the present invention can treat or prevent various diseases related to estrogen by antagonizing/degrading estrogen receptors, such as cancer (breast cancer, ovarian cancer, colon cancer, prostate cancer, endometrial cancer), osteoporosis, neurodegenerative diseases, cardiovascular diseases, lupus erythematosus, endometriosis and obesity.
  • cancer breast cancer, ovarian cancer, colon cancer, prostate cancer, endometrial cancer
  • osteoporosis osteoporosis
  • cardiovascular diseases lupus erythematosus
  • endometriosis obesity.

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Abstract

本发明公开了一种二氢苯并噻喃类衍生物及其制备方法和用途。该二氢苯并噻喃类衍生物结构如通式(I)所示，各取代基的定义如说明书和权利要求书所述。本发明的二氢苯并噻喃类衍生物，作为选择性雌激素受体降解剂和拮抗剂，用于治疗雌激素受体阳性疾病。

Description

二氢苯并噻喃类衍生物及其制备方法和用途 技术领域
本发明属于医药领域，涉及一类二氢苯并噻喃类化合物，其制备方法和医药上的应用。特别地，本发明涉及通式(I)所示的二氢苯并噻喃类衍生物、其外消旋体、对映异构体、非对映异构体及其混合物、其可药用盐及含有所述化合物的药物组合物，以及使用所述化合物治疗、预防或诊断雌激素受体敏感的、雌激素受体介导的或依赖性的疾病或病症的用途，所述的疾病特别优选雌激素受体阳性乳腺癌。
背景技术
乳腺癌是全球第一大癌症，2020年全球新发病例高达226万例。尽管早期乳腺癌的5年生存期超过90％，但是由于肿瘤的转移和扩散等原因，每年仍然导致近70万人死亡。乳腺癌中超过70％的病例高表达雌激素受体(ER)。ER与雌激素结合，激活信号通路从而促进乳腺癌的发生、发展，是乳腺癌发生、发展最主要的驱动因素。
ER阳性乳腺癌的治疗首选疗法是内分泌疗法，标准治疗药物有：(1)芳香化酶/雌激素合成酶抑制剂，主要机制是抑制内源性雌激素的生物合成，代表性药物有来曲唑和阿那曲唑。这类药物的主要副作用是加速骨密度丧失和普遍存在获得性耐药；(2)拮抗ER活性的雌激素受体调节剂(Selective estrogen receptor modulators，SERMs)，即他莫昔芬，缺点是长期使用会导致不依赖激素的耐药，并且该药物由于呈现部分激动效应，会增加子宫内膜增生、息肉和子宫内膜癌的风险。尽管内分泌疗法取得了巨大的进步，但是30-50％的患者在内分泌治疗5年内会出现耐药进而发生疾病进展、转移，5年生存率仅20％左右，中位总生存时间也只有2～3年。以ESR1-LBD为主导的获得性耐药和以骨、脑、肝、肺和淋巴结转移为代表的远端转移是导致治疗抵抗和耐药病人的群体数量不断增加的重要原因，特别是脑转移(脑转移患者占10-15％)，目前还没有理想的治疗药物和治疗方法，患者预后很差，经临床药物治疗后的中位生存期仅为2-9个月。这些耐药的乳腺癌中，ER信号仍是一个关键驱动因子。因此，使用新方法靶向ER，阻断其信号通路仍然是开发治疗乳腺癌新型药物的关键。
雌激素受体降解剂(Selective Estrogen Receptor Degraders，SERDs)是一种全新的治疗方案。SERD通过与ER结合后，导致ER无法激活靶向基因的转录，并促使ER泛素化降 解，从而彻底阻断ER信号传导，抑制肿瘤细胞增殖，而且这一效果不受常见突变ESR1突变状态影响，因此能克服内分泌疗法所产生的获得性耐药及副作用，有望成为ER阳性乳腺癌的支柱疗法。氟维司群是目前唯一上市的SERD类药物，用于内分泌治疗后复发或疾病进展的ER+局部晚期或转移性绝经后乳腺癌患者，或既往未接受过内分泌治疗的绝经后HR+(孕激素受体阳性)/HER2-(人表皮生长因子受体-2阴性)局部进展期/转移性乳腺癌患者的治疗。氟维司群(优于雌激素受体调节剂)优异的抗肿瘤活性已被临床广泛认可，但由于其甾体类结构的特点，有溶解性差、吸收缓慢、体内暴露量低、生物利用度极低、代谢不稳定等问题，导致不能口服给药只能肌肉注射，而肌肉注射(单次肌注最高只能达到500mg)很难达到疗效最佳所需的体内药物浓度，给药后需要3-6个月时间来实现平稳的血浆浓度，且注射部位剧烈疼痛、肿胀、发红等反应也使病人顺应性很差，极大限制了其临床应用。此外，氟维司群不能透过血脑屏障，无法用于乳腺癌治疗中极为棘手的脑转移乳腺癌的治疗。因此，研发口服吸收良好、口服有效、药代动力学性质更理想、能透过血脑屏障的口服小分子SERD是巨大的临床未满足需求。
目前有多个候选SERDs药物分子处于临床研究不同阶段，公开结构的SERDs/拮抗剂包括LSZ-102(US9322746)、ZB716(WO2016004166)、RAD1901(WO2016176666)、SAR439859(WO2018091153)、AZD9833(WO2019002442)、GDC-9545(WO2016097072)。目前为止还没有口服有效的小分子SERDs获批上市。
目前对于内分泌疗法耐药的雌激素阳性乳腺癌患者的治疗选择还十分有限，仅有的氟维司群还存在很多问题，特别是对于脑转移乳腺癌患者几乎束手无策。
发明内容
本发明的目的在于提供一种结构新颖、具有优异的ER拮抗和降解活性、口服有效且有良好脑分布的SERD。
本发明的第一方面，提供一种通式(I)所示的化合物，或其外消旋体、对映异构体、非对映异构体、或其混合物形式、或可药用盐：
其中：R1选自OH、COOH、B(OH)2、卤素、C1-C6烷基、卤代C1-C6烷基或C1-C6烷氧基；R2选自H、OH、COOH、卤素、氰基、C1-C6烷基、C1-C6烷氧基、卤代C1-C6烷基或羟基取代的C1-C6烷基；或者R1、R2与相连的苯环形成苯并5-6元杂芳基；
X选自S、S(O)2或O；
环A选自：C3-C6环烷基、5-8元杂环基、C6-C10芳基或5-8元杂芳基；
各R3独立地选自氢、卤素、氰基、C1-C6烷硫基、C1-C6烷基CO-、C1-C6烷基SO2-、氨基、-NH(C1-C6烷基)、-N(C1-C6烷基)(C1-C6烷基)、-SO2NH2、-C(O)NH2、C1-C6烷基、C1-C6烷氧基、卤代C1-C6烷基、卤代C1-C6烷氧基或卤代C1-C6烷硫基；o为0、1、2、3或4；
Y1、Y2独立地选自CR4或N；
各R4独立地选自氢、卤素、氰基、C1-C6烷硫基、C1-C6烷基CO-、C1-C6烷基SO2-、氨基、-NH(C1-C6烷基)、-N(C1-C6烷基)(C1-C6烷基)、-SO2NH2、-C(O)NH2、C1-C6烷基、C1-C6烷氧基、卤代C1-C6烷基、卤代C1-C6烷氧基或卤代C1-C6烷硫基；
m为0、1、2、3或4；
Z1-Z2选自O-Z2、NH-Z2、S-Z2、S(O)-Z2、S(O)2-Z2、O-(C1-C6亚烷基)-Z2、O-(卤代C1-C6亚烷基)-Z2、NH-(C1-C6亚烷基)-Z2或NH-(卤代C1-C6亚烷基)-Z2；
Z2、Z3独立地选自CH或N；n为1、2或3；
R5为C1-C6烷基，任选被选自下组的一个或多个取代基取代：卤素、氰基、羟基、羧基、氨基、甲氧基或-SO2CH3；
前提是：X为S或S(O)2时，环A选自：C3-C6环烷基、5-8元杂环基、C6-C10芳基或5-8元杂芳基；X为O时，环A选自：C3-C6环烷基、5-8元杂环基或5-8元杂芳基。
在另一优选例中，各R3独立地选自氢、卤素、氰基、C1-C4烷硫基、C1-C4烷基CO-、C1-C4烷基SO2-、氨基、-NH(C1-C4烷基)、-N(C1-C4烷基)(C1-C4烷基)、-SO2NH2、-C(O)NH2、C1-C4烷基、C1-C4烷氧基、卤代C1-C4烷基、卤代C1-C4烷氧基或卤代C1-C4烷硫基；
o为0、1、2、3或4；
X为S或S(O)2时，环A选自：C3-C6环烷基、5-7元杂环基、C6-C10芳基或5-7元杂芳基；X为O时，环A选自：C3-C6环烷基、5-7元杂环基或5-7元杂芳基。
在另一优选例中，Z1-Z2选自O-Z2、NH-Z2、S-Z2、S(O)-Z2、S(O)2-Z2、O-(C1-C4亚烷基)-Z2、O-(卤代C1-C4亚烷基)-Z2、NH-(C1-C4亚烷基)-Z2或NH-(卤代C1-C4亚烷基)-Z2；
Z2、Z3独立地选自CH或N；
n为1、2或3；
R5为C1-C4烷基，任选被选自下组的一个或多个取代基取代：氟、氯、溴、氰基、羟基或羧基。
在另一优选例中，R1选自OH、COOH、B(OH)2，R2选自H；或者R1、R2与相连的苯环形成
X选自S或S(O)2；
环A选自：C3-C6环烷基、5-6元杂环基、苯基或5-6元杂芳基；
各R3独立地选自氢、氟、氯、溴、氰基、C1-C4烷硫基、C1-C2烷基CO-、C1-C2烷基SO2-、氨基、-NH(C1-C2烷基)、-N(C1-C2烷基)(C1-C2烷基)、-SO2NH2、-C(O)NH2、C1-C4烷基、C1-C4烷氧基、卤代C1-C4烷基、卤代C1-C4烷氧基或卤代C1-C4烷硫基；
o为0、1、2、3或4；
Y1、Y2独立地选自CR4或N；
各R4独立地选自氢、氟、氯、溴、氰基、C1-C2烷硫基、C1-C2烷基CO-、C1-C2烷基SO2-、氨基、-NH(C1-C2烷基)、-N(C1-C2烷基)(C1-C2烷基)、-SO2NH2、-C(O)NH2、C1-C4烷基、C1-C4烷氧基、卤代C1-C4烷基、卤代C1-C4烷氧基或卤代C1-C4烷硫基；
m为0、1、2或3；
Z1-Z2选自O-Z2、NH-Z2、O-(C1-C4亚烷基)-Z2、O-(卤代C1-C4亚烷基)-Z2、NH-(C1-C4亚烷基)-Z2或NH-(卤代C1-C4亚烷基)-Z2；
Z2、Z3独立地选自CH或N；
n为1或2；
R5为C1-C4烷基，任选被选自下组的一个或多个取代基取代：氟、氯、溴、氰基、羟基或羧基。
在另一优选例中，R1选自OH、COOH、B(OH)2，R2选自H；或者R1、R2与相连的苯环形成
在另一优选例中，X为S或S(O)2时，环A选自：C3-C6环烷基、5-7元杂环基、苯基或5-7元杂芳基；X为O时，环A选自：C3-C6环烷基、5-7元杂环基或5-7元杂芳基。
在另一优选例中，X为S或S(O)2时，环A选自：C3-C6环烷基、5-7元杂环基、C6-C10芳基或5-7元杂芳基；在另一优选例中，环A选自：苯基、5-6元杂环基(具有1或2个选自下组的杂原子：N、O)或5-6元杂芳基(具有1或2个N)；在另一优选例中，环A选自：苯基、哌啶基、吡啶基、吡喃基、四氢吡喃基、吡唑基、吡咯基、咪唑基、哒嗪基、嘧啶基、吡嗪基。
在另一优选例中，X为S或S(O)2；环A为苯基。
在另一优选例中，X为O；环A为5-6元杂环基(具有1或2个选自下组的杂原子：N、O)或5-6元杂芳基(具有1或2个N)。
在另一优选例中，X为O；环A为哌啶基、吡啶基、吡唑基、四氢吡喃基、吡喃基、吡咯基、咪唑基、哒嗪基、嘧啶基。
在另一优选例中，各R3独立地选自氢、卤素、氰基、C1-C4烷硫基、C1-C4烷基CO-、C1-C4烷基SO2-、氨基、-NH(C1-C4烷基)、-N(C1-C4烷基)(C1-C4烷基)、-SO2NH2、-C(O)NH2、C1-C4烷基、C1-C4烷氧基、卤代C1-C4烷基、卤代C1-C4烷氧基或卤代C1-C4烷硫基；o为0、1、2、3或4。
在另一优选例中，R3选自氢、卤素、氰基、硫甲基、乙酰基、甲磺酰基、-NMe2、-SO2NH2、-C(O)NH2、C1-C4烷基、C1-C4烷氧基、卤代C1-C4烷基、卤代C1-C4烷氧基或卤代C1-C4烷硫基。
在另一优选例中，各R3独立地选自氢、氟、氯、溴、CH3CO-、CH3SO2-、CH3CH2CO-、CH3CH2SO2-、氨基、氰基、-NHCH3、-N(CH3)2、-SO2NH2、-C(O)NH2、甲基、乙基、正丙基、异丙基、正丁基、异丁基、甲氧基、乙氧基、丙氧基、丁氧基、卤代C1-C2烷基、卤代C1-C4烷氧基或卤代C1-C4烷硫基；o为0、1、2、3或4。
在另一优选例中，Y1为CH、CF或N。
在另一优选例中，Y2为CH或N。
在另一优选例中，结构单元选自
在另一优选例中，卤代C1-C2烷基选自-CH2F、-CHF2、-CF3、-CH2CF3、-CH2CH2F。
在另一优选例中，R4选自氢、卤素、氰基、硫甲基、乙酰基、甲磺酰基、-NMe2、-SO2NH2、-C(O)NH2、C1-C4烷基、C1-C4烷氧基、卤代C1-C4烷基、卤代C1-C4烷氧基或卤代C1-C4烷硫基；m为0、1、2或3。
在另一优选例中，各R4独立地选自氢、氟、氯、溴；m为0、1、2或3。
在另一优选例中，Z1-Z2选自O-Z2、NH-Z2、S-Z2、S(O)-Z2、S(O)2-Z2、O-(C1-C4亚烷基)-Z2、O-(卤代C1-C4亚烷基)-Z2、NH-(C1-C4亚烷基)-Z2或NH-(卤代C1-C4亚烷基)-Z2。在另一优选例中，上述卤代是指氟代、氯代或溴代。
在另一优选例中，Z1-Z2选自O-Z2、NH-Z2、-OCH2-Z2、-OCH2CH2-Z2、-OCH2CH2CH2-Z2、O-(氟代C1-C2亚烷基)-Z2、NH-(C1-C2亚烷基)-Z2或NH-(氟代C1-C2亚烷基)-Z2、O-(氯代C1-C2亚烷基)-Z2、NH-(C1-C2亚烷基)-Z2或NH-(氯代C1-C2亚烷基)-Z2。
在另一优选例中，Z2为CH，Z3为N；Z2为N，Z3为CH。
在另一优选例中，n为1、2或3。
在另一优选例中，R5为-CH2F、-CHF2、-CF3、-CH2CN或-CH2CH2CH2F。在另一优选例中，R5为-CH2F。
在另一优选例中，结构单元选自
在另一优选例中，结构单元选自
在另一优选例中，X为O时，环A选自5～6元环烷基、5～6元杂环烷基或5～6元杂环芳基；优选
在另一优选例中，所述通式(I)所示的化合物为通式(Ia)所示的化合物：
R1、R2、R3、o、R4、m、Z1、Z2、n、Z3、R5的定义如前所述。
在另一优选例中，所述通式(I)所示的化合物具有通式(Ib)所示的化合物：
其中，R1、R2、R3、o、R4、m、Z1、Z2、n、Z3、R5的定义如前所述。
在另一优选例中，所述化合物为实施例制备的化合物。
在另一优选例中，所述可药用盐是化合物与有机酸或无机酸形成的盐，所述有机酸选自L-酒石酸、富马酸、L-苹果酸、D-苹果酸、柠檬酸、L-焦谷氨酸、醋酸、对甲苯磺酸盐、苯磺酸、甲磺酸、苯甲酸、乳酸、扁桃酸、马来酸、草酸、丁二酸；所述的无机酸选自盐酸、磷酸、硫酸、氢溴酸。
本发明的第二方面，提供第一方面所述的通式(I)所示的化合物的制备方法。
当R1为OH，R2为氢，Z1为OCH2CH2，Z2为N，所述制备方法包括以下步骤：

(i1)A3与有机硼试剂发生Suziki偶联反应得到A4，其中，所述的有机硼试剂选自：硼酸硼酸频那醇酯
(i2)A4与溴代试剂发生溴代反应得到烯基溴代物A5，其中，所述的溴代试剂选自：三溴化吡啶鎓、N-溴代丁二酰亚胺；
(i3)A5与有机硼试剂发生Suziki偶联反应得到A6，其中，所述的有机硼试剂选自：硼酸硼酸频那醇酯
(i4)A6在钯催化剂和氢气的作用下发生氢化和氢解反应得到A7，其中，所述的钯催化剂选自Pd/C、Pd(OH)2/C；
(i5)A7经亲核取代反应得到A8；
(i6)A8在碱性条件下发生亲核取代反应及水解得到式(I)化合物，所述的碱选自三乙胺、N，N-二异丙基乙胺、吡啶、碳酸盐、NaH、氢氧化钠、氢氧化钾、氢氧化锂、甲醇钠、乙醇钠；
X、R4、Y2、Y1、环A、R3、o、m、Z3、R5的定义同前所述；
或者所述制备方法包括以下步骤：
其中，A7与反应得到式(I)化合物，
LG为离去基团，选自Br、Cl、OTf、OTs或OMs；
X、R4、Y2、Y1、环A、R3、o、m、Z3、R5的定义同前所述；
或者R1为-COOH，R2为氢，所述制备方法包括以下步骤：
(ii1)B4与溴代试剂发生溴代反应得到烯基溴代物B5，其中，所述的溴代试剂选自：三溴化吡啶鎓、N-溴代丁二酰亚胺；
(ii2)B5与有机硼试剂发生Suziki偶联反应得到B6，其中，所述的有机硼试剂选自：硼酸硼酸频那醇酯
(ii3)B6在钯催化剂和氢气的作用下发生氢化和氢解反应得到B7，其中，所述的钯催化剂选自Pd/C、Pd(OH)2/C；
(ii4)B7经亲核取代反应得到B8；
(ii5)B8在碱性条件下发生亲核取代反应及水解得到式(I)化合物，所述的碱选自三乙胺、N，N-二异丙基乙胺、吡啶、碳酸盐、NaH、氢氧化钠、氢氧化钾、氢氧化锂、甲醇钠、乙醇钠；
X、R4、Y2、Y1、环A、R3、o、m、Z3、R5的定义同前所述，R6为C1-C6烷基；
或者R1、R2与相连的苯环形成时，所述制备方法包括以下步骤：

(iii1)C1与3-巯基丙酸甲酯在钯催化剂的作用下发生C-S偶联得到C2，其中，所述的钯催化剂选自[1，1′-双(二苯基膦)二茂铁]二氯化钯、四(三苯基膦)钯、三(二亚苄基丙酮)二钯、双(二亚苄基丙酮)钯、双三苯基磷二氯化钯、二(三叔丁基膦)钯、双(三环己基膦)钯、醋酸钯；
(iii2)C2在酸性条件下水解得到C3，所述的酸选自硫酸、盐酸、磷酸、甲磺酸、三氟乙酸、乙酸、三氟甲磺酸；
(iii3)C3在酸作用下发生傅克反应得到C4，所述酸选自：三氟甲磺酸、三氟乙酸、Eaton试剂、多聚磷酸、硫酸、盐酸；
(iii4)C4与二氢吡喃在酸作用件下反应得到C5，所述酸包括：对甲苯磺酸、甲磺酸、对甲苯磺酸吡啶盐；
(iii5)C5与对甲苯磺酰肼反应得到腙C6；
(iii6)C6与芳基溴代物在钯催化剂的作用下反应得到C7，其中，所述的钯催化剂选自[1，1′-双(二苯基膦)二茂铁]二氯化钯、四(三苯基膦)钯、三(二亚苄基丙酮)二钯、双(二亚苄基丙酮)钯、双三苯基磷二氯化钯、二(三叔丁基膦)钯、双(三环己基膦)钯、醋酸钯；
(iii7)C7与溴代试剂发生溴代反应得到烯基溴代物C8，其中，所述的溴代试剂选自：三溴化吡啶鎓、N-溴代丁二酰亚胺；
(iii8)C8与有机硼试剂发生Suziki偶联反应得到C9，其中，所述的有机硼试剂选自：硼酸硼酸频那醇酯
(iii9)C9在钯催化剂和氢气的作用下发生氢化和氢解反应得到C10，其中，所述的钯催化剂选自Pd/C、Pd(OH)2/C；
(iii10)C10经亲核取代反应得到C11；
(iii11)C11经亲核取代反应及在酸性条件下水解得到式(I)化合物，所述酸选自：三氟甲磺酸、三氟乙酸、硫酸、盐酸、对甲苯磺酸、氯化氢的有机溶剂(甲醇、乙醇、异丙醇、乙酸乙酯、乙醚或1，4-二氧六环)溶液；
X、R4、Y2、Y1、环A、R3、o、m、Z3、R5的定义同前所述。
本发明的第三方面，提供一种药物组合物，包含：
第一方面所述的通式(I)所示的化合物，或其外消旋体、对映异构体、非对映异构体、或其混合物形式、或可药用盐；和药学上可接受的载体。
在另一优选例中，所述药物组合物还包含助流剂或稀释剂。
本发明的第四方面，提供第一方面所述的通式(I)所示的化合物，或其外消旋体、对映异构体、非对映异构体、或其混合物形式、或可药用盐或者第三方面所述的药物组合物的用途，用于制备治疗、预防或诊断雌激素受体相关的疾病的药物，较佳地，用于制备治疗、预防或诊断乳腺癌、子宫内膜癌、宫颈癌、皮肤癌、前列腺癌、卵巢癌、输卵管肿瘤、肺癌、白血病、骨质疏松症、神经退行性疾病、心血管疾病、红斑狼疮、子宫内膜异位症及肥胖症的药物。
在另一优选例中，所述与雌激素受体相关的疾病为雌激素受体敏感的、雌激素受体介导的或依赖性的疾病或病症。
在另一优选例中，所述与雌激素受体相关的疾病选自癌症、骨质疏松症、神经退行性疾病、心血管疾病、红斑狼疮、子宫内膜异位症及肥胖症。
在另一优选例中，所述与雌激素受体相关的疾病选自乳腺癌、子宫内膜癌、宫颈癌、皮肤癌、前列腺癌、卵巢癌、输卵管肿瘤、肺癌和白血病。
在另一优选例中，所述与雌激素受体相关的疾病选自ER阳性乳腺癌。
在另一优选例中，所述与雌激素受体相关的疾病选自ER阳性乳腺癌脑转移。
本发明提供了一种结构新颖、具有优异的ER拮抗和降解活性、且口服有效的SERD，能够治疗、预防或诊断雌激素受体敏感的、雌激素受体介导的或依赖性的疾病或病症，特别雌激素受体阳性乳腺癌。此外，本发明化合物具有良好的脑暴露量，可用于脑转移性乳腺癌患者的治疗，满足此巨大的未满足的临床需求。
应理解，在本发明范围内，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。说明书中所揭示的各个特征，可以被任何提供相同、均等或相似目的的替代性特征取代。限于篇幅，在此不再一一赘述。
附图说明
图1为子宫湿重重量实验结果图，其中A示出不同实验组子宫重量与体重的平均比值，*p＜0.05，**p＜0.01；B为各实验组的子宫横切面组织学图像，子宫内膜表面上皮高度用黑线标示。
图2为化合物对MCF-7小鼠皮下肿瘤模型的生长抑制实验结果图，**p＜0.01。
具体实施方式
本申请的发明人经过广泛而深入地研究，研发出一种二氢苯并噻喃类衍生物，具有通式(I)所示的结构，作为选择性雌激素受体降解剂和拮抗剂，用于治疗雌激素受体阳性疾病，特别是雌激素受体阳性乳腺癌。此外，本发明化合物具有良好的脑暴露量，可用于脑转移性乳腺癌患者的治疗。在此基础上，完成了本发明。
术语
除非有相反陈述，否则下列用在说明书和权利要求书中的属于具有下述含义。
在本发明中，术语“C1-C6”是指具有1、2、3、4、5或6个碳原子，“C1-C4”是指具有1、2、3或4个碳原子，依此类推。“3-6元”是指具有3、4、5或6个环原子，“5-8元”是指具有5、6、7或8个环原子，依此类推。
本发明化合物中的“氢”、“碳”、“氧”包括其所有同位素。同位素应理解为包括具有相同原子数但具有不同质量数的哪些原子。举例来说，氢的同位素包括氚和氘，碳的同位素包括13C和14C，氧的同位素包括16O和18O等。
“卤素”指氟、氯、溴或碘；“卤代”指氟代、氯代、溴代或碘代。
“氰基”指-CN。
“羧基”指-C(＝O)OH。
“烷基”指仅由C和H两种元素组成的饱和碳氢化合物在任何碳原子失去一个氢原子后形成的基团，包括直链和支链的脂肪烃，非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基。
“亚烷基”是指具有指定的碳原子数并连接至少两个其他基团的直链或支链饱和脂肪族基团，即二价烃基团。连接到亚烷基的两个基团可以连接到亚烷基上相同的或不同原子。例如，直链亚烷基可以是-(CH2)n-的二价基团，其中n是1、2、3、4、5或6。代表性的亚烷基包括但不限于亚甲基、亚乙基、亚丙基、亚异丙基、亚丁基、亚异丁基、亚仲丁基、亚戊基和亚己基。亚烷基可以是取代或未取代的。
“烯基”指由至少两个碳原子和至少一个碳-碳双键组成的如上定义的烷基，例如乙烯基、1-丙烯基、2-丙烯基等。
“环烷基”指饱和或部分不饱和单环或多环环状烃取代基。多环环烷烃包括螺环、稠环和桥环的环烷基。
“烷氧基”指-O-(烷基)和-O-(环烷基)，其中烷基和环烷基的定义如上所述。
“烷硫基”指-S-(烷基)和-S-(环烷基)，其中烷基和环烷基的定义如上所述。
在本发明中，术语“芳基”表示包含一个或多个芳环的烃基部分。例如术语“C6-C12芳基”是指在环上不含杂原子的具有6至12个碳原子的芳香族环基，如苯基、萘基等。术语“C6-C10芳基”具有类似的含义。芳基的例子包括但不限于苯基(Ph)、萘基、芘基、蒽基和菲基。
在本发明中，术语“杂环基”表示包含至少一个(如1、2、3或4个)环杂原子(例如N，O或S)的饱和或不饱和的、非芳香性的环状基团，例如哌啶基、四氢吡喃基、四氢吡啶基、 吡咯啉基、二氢吡啶基、二氢呋喃基、二氢噻吩基、吗啉基。
在本发明中，术语“杂芳基”表示包含至少一个(如1、2、3或4个)环杂原子(例如N，O或S)的芳香性的环状基团，例如呋喃基、吡咯基、噻吩基、噁唑基、咪唑基、噻唑基、吡啶基、喹啉基、异喹啉基、吲哚基、嘧啶基、吡喃基。
“取代”指基团中的一个或多个氢原子独立地被相应数目的取代基所代替；所述取代为单取代或多取代，较佳地，所述多取代为二取代、三取代、四取代、或五取代。所述二取代就是指具有两个取代基，依此类推。
“独立地”指当取代基的个数超过一个时，这些取代基可以相同也可以不同；
本发明的“异构体”指具有相同分子式的但在性质上或在其原子的键序列上或在其原子的空间排列上不同的化合物。立体异构体是其原子在空间排列上不同的异构体。彼此不成镜像的立体异构体是非对映体并且互相是非重叠的镜像的立体异构体是对映体。手性化合物可以作为单一的对映体或其混合物存在。包含对映体的相等比例的混合物称作“外消旋混合物”。
本发明的“可药用盐”、“药学上可接受的盐”指本发明化合物的盐，这类盐用于哺乳动物体内时具有安全性和有效性，且具有应有的生物活性。在另一优选例中，“可药用盐”、““药学上可接受的盐”是指式I化合物同选自下组的酸形成的盐类：氢氟酸、盐酸、氢溴酸、磷酸、乙酸、草酸、硫酸、硝酸、甲磺酸、胺基磺酸、水杨酸、三氟甲磺酸、萘磺酸、马来酸、柠檬酸、醋酸、乳酸、酒石酸、琥珀酸、酢浆草酸、丙酮酸、苹果酸、谷氨酸、对甲苯磺酸、萘磺酸、乙磺酸、萘二磺酸、丙二酸、富马酸、丙酸、草酸、三氟乙酸、硬酯酸、扑酸、羟基马来酸、苯乙酸、苯甲酸、谷氨酸、抗坏血酸、对胺基苯磺酸、2-乙酰氧基苯甲酸和羟乙磺酸等；或者式I化合物与无机碱形成的钠盐、钾盐、钙盐、铝盐或铵盐；或者通式I化合物与有机碱形成的甲胺盐、乙胺盐或乙醇胺盐。
具体实施方式
本发明是进行了广泛的化合物设计、合成、体内外生物活性测试，代谢等成药性研究而完成。下面实施例仅仅是对本发明的具体的说明，并不对本发明构成任何限制，本领域技术人员利用本领域熟知的技术、方法，或相关方法的排列组合，对本发明所做的微小改变，均在本发明保护范围之内。
本发明化合物的合成方法
本发明化合物的结构通过核磁共振(NMR)或者质谱(MS)来确定。NMR数据在BRUKER AVANCE III 400或BRUKER AVANCE III 500或BRUKER AVANCE III 600核磁共振波谱仪上采集，测定溶剂为氘代二甲基亚砜(DMSO-d6)、氘代氯仿(CDCl3)或氘代甲醇(CD3OD)，化学位移以δ(ppm)表示，用于描述峰值信号的缩写如下：br＝宽信号，s＝单峰，d＝双重峰，dd＝双二重峰峰，t＝三重峰，q＝四重峰，m＝多重峰。质谱是用Finnigan LTQ线性离子阱质谱仪测定。柱层析分离所用硅胶为200-300目，洗脱剂的配比均为体积比。合成所用的商品化原料、试剂(如酸、碱、金属催化剂、溴代试剂、酰基化试剂、芳基硼酸 等)、溶剂(石油醚、乙酸乙酯、甲醇、二氯甲烷、1，4-二氧六环、乙腈等)等从试剂公司购买的产品直接用于反应，无需额外纯化。
在本发明化合物通式(I)的一优选例中，X为S，R1为OH，R2为氢，Z1为-OCH2CH2-，Z2为N；通式(I)所示的化合物其外消旋体、对映异构体、非对映异构体及其混合物、其药学上可接受的盐可通过以下路线1制备：
其中R3、R4、R5、Z3、m、n、o如前所述；
步骤1 A1在碱的作用下与PivCl反应得到A2，所述的碱包括三乙胺、N，N-二异丙基乙胺、吡啶、碳酸盐，溶剂包括氯代烃、醚类、酯类或乙腈，温度为0-50℃；
步骤2 A2在吡啶的作用下与三氟甲磺酸酐反应得到三氟甲磺酸酯A3，溶剂包括氯代烃、醚类，温度为0℃到室温；
步骤3 A3与芳基硼试剂在钯催化剂、碱存在条件下发生Suzuki偶联得到A4，其中所述的钯催化剂包括但不限于[1，1′-双(二苯基膦)二茂铁]二氯化钯、四(三苯基膦)钯、三(二亚苄基丙酮)二钯、双(二亚苄基丙酮)钯、双三苯基磷二氯化钯、二(三叔丁基膦)钯、双(三环己基膦)钯、醋酸钯，所述的芳基硼试剂包括但不限于硼酸、硼酸频那醇酯、硼酸新戊二醇酯，所述的碱包括但不限于乙酸钾、碳酸钠、碳酸钾、碳酸铯、1，8-二氮杂二环[5.4.0]十一碳-7-烯、三乙胺、N，N-二异丙基乙胺，所述的溶剂包括水、1，4-二氧六环、甲苯、N，N-二甲基甲酰胺、乙腈、乙醇、甲醇；
步骤4 A4与溴代试剂发生反应得到烯基溴代物A5，所述溴代试剂包括三溴吡啶鎓、N-溴代丁二酰亚胺、溴素，所述的溶剂包括卤代烃、乙腈、乙酸、甲醇；
步骤5 A5与有机硼试剂在钯催化剂、碱存在条件下发生Suzuki偶联得到A6，其中所述的钯催化剂包括但不限于[1，1′-双(二苯基膦)二茂铁]二氯化钯、四(三苯基膦)钯、三(二亚苄基丙酮)二钯、双(二亚苄基丙酮)钯、双三苯基磷二氯化钯、二(三叔丁基膦)钯、双(三环己基膦)钯、醋酸钯，所述的芳基硼试剂包括但不限于硼酸、硼酸频那醇酯、硼酸新戊二 醇酯，所述的碱包括但不限于乙酸钾、碳酸钠、碳酸钾、碳酸铯、1，8-二氮杂二环[5.4.0]十一碳-7-烯、三乙胺、N，N-二异丙基乙胺，所述的溶剂包括水、1，4-二氧六环、甲苯、N，N-二甲基甲酰胺、乙腈、乙醇、甲醇；
步骤6 A6在Pd/C或Pd(OH)2/C和氢气的作用下发生氢化和氢解反应得到A7，所述的溶剂包括二氧六环、四氢呋喃、甲苯、N，N-二甲基甲酰胺、二甲基亚砜、二氯甲烷、二氯乙烷、乙醇、甲醇，1-50个大气压，温度为0-50℃；
步骤7 A7与二溴乙烷在碱的作用下反应得到A8，所述的碱包括三乙胺、N，N-二异丙基乙胺、吡啶、碳酸盐、NaH、氢氧化钠、氢氧化钾、氢氧化锂，所述的溶剂包括二氧六环、四氢呋喃、甲苯、乙腈、丙酮、二氯甲烷、二氯乙烷、乙醇、甲醇，温度为20-80℃；
步骤8 A8与相应的胺在碱作用下发生胺化反应并脱除保护基得到式(I)化合物，所述的碱包括三乙胺、N，N-二异丙基乙胺、吡啶、碳酸盐、NaH、氢氧化钠、氢氧化钾、氢氧化锂，所述的溶剂包括1，4-二氧六环、四氢呋喃、甲苯、乙腈、丙酮、二氯甲烷、二氯乙烷、乙醇、甲醇，温度为20-80℃；
在本发明化合物通式(I)的一优选例中，X为S，R1为COOH，R2为氢，Z1为-OCH2CH2-，Z2为N；通式(I)所示的化合物其外消旋体、对映异构体、非对映异构体及其混合物、其药学上可接受的盐可通过以下路线2制备：
其中R3、R4、R5、Z3、m、n、o如前所述；
步骤1 A1在碱的作用下脱除保护基得到B2，所述的碱包括氢氧化钠、氢氧化钾、氢氧化锂、甲醇钠、乙醇钠，所述的溶剂包括水、1，4-二氧六环、四氢呋喃、乙醇、甲醇，温度为20-80℃；
步骤2 B1在碱的作用下与三氟甲磺酸酐反应得到三氟甲磺酸酯B2，所述的碱包括三 乙胺、N，N-二异丙基乙胺、吡啶、碳酸盐、NaH，所述的溶剂包括1，4-二氧六环、四氢呋喃、二氯甲烷、二氯乙烷、乙醇、甲醇，温度为0-50℃；
步骤3 B2与羧化试剂在钯催化剂作用下发生羧化反应得到B3，其中所述的钯催化剂包括但不限于[1，1′-双(二苯基膦)二茂铁]二氯化钯、四(三苯基膦)钯、三(二亚苄基丙酮)二钯、双(二亚苄基丙酮)钯、双三苯基磷二氯化钯、二(三叔丁基膦)钯、双(三环己基膦)钯、醋酸钯，所述的羧化试剂包括甲酸、甲酸钠、甲酸钾、甲酸锂，所述的溶剂包括1，4-二氧六环、四氢呋喃、甲苯、N，N-二甲基甲酰胺、二甲基亚砜、乙腈，温度为50-100℃；
步骤4 B3进行酯化反应得到B4，所述条件包括甲醇/浓硫酸、碘甲烷/碱、TMSCHN2/甲醇，其中所述的碱包括NaH、碳酸盐、氢氧化钠、氢氧化钾；
步骤5-8 从B4制备式(I)化合物参考路线一中A4制备式(I)化合物的方法。
在本发明化合物通式(I)的另一优选例中，当通式(I)所述化合物中R1，R2关环形成吡唑时，通式(I)所示的化合物其外消旋体、对映异构体、非对映异构体及其混合物、其药学上可接受的盐可通过路线3制备：
其中R3、R4、R5、Z3、m、n、o如前所述；
步骤1 C1在钯催化剂和碱的作用下与3-巯基丙酸甲酯反应得到C2，其中所述的钯催 化剂包括但不限于[1，1′-双(二苯基膦)二茂铁]二氯化钯、四(三苯基膦)钯、三(二亚苄基丙酮)二钯、双(二亚苄基丙酮)钯、双三苯基磷二氯化钯、二(三叔丁基膦)钯、双(三环己基膦)钯、醋酸钯，所述的芳基硼试剂包括但不限于硼酸、硼酸频那醇酯、硼酸新戊二醇酯，所述的碱包括但不限于乙酸钾、碳酸钠、碳酸钾、碳酸铯、1，8-二氮杂二环[5.4.0]十一碳-7-烯、三乙胺、N，N-二异丙基乙胺；
步骤2 C2在酸性条件下水解得到C3，所述的酸包括硫酸、盐酸、磷酸、甲磺酸、三氟乙酸、乙酸、三氟甲磺酸，所述的溶剂包括水、1，4-二氧六环、四氢呋喃、甲苯、乙腈、丙酮，温度为室温-100℃；
步骤3 C3在质子酸的作用下发生傅克反应得到C4，其中所述的质子酸的包括多但不限于聚磷酸、Eaton试剂、三氟甲磺酸；
步骤4 C4与2，3-二氢吡喃在对甲苯磺酸或对甲苯磺酸-吡啶盐的作用下发生反应得到C5，所述的溶剂包括卤代烃、甲苯、乙腈；
步骤5 C5与对甲苯磺酰肼反应得到腙C6，所述的溶剂包括甲醇、乙醇、甲苯、乙腈；
步骤6 C6和芳基溴代物在钯催化剂作用下发生卡宾迁移插入反应得到C7，所述的钯催化剂包括但不限于[1，1′-双(二苯基膦)二茂铁]二氯化钯、四(三苯基膦)钯、三(二亚苄基丙酮)二钯、双(二亚苄基丙酮)钯、双三苯基磷二氯化钯、二(三叔丁基膦)钯、双(三环己基膦)钯、醋酸钯，所述的碱包括但不限于乙酸钾、碳酸钠、碳酸钾、碳酸铯、叔丁醇钾、叔丁醇钠、叔丁醇锂、NaH，所述的溶剂包括1，4-二氧六环、甲苯、N，N-二甲基甲酰胺、乙腈；
步骤7-11后续路线与路线一类似。
本发明化合物的可药用盐的制备
将通式(I)化合物溶于适当溶剂中，加入1当量酸，或将1当量酸溶于相同溶剂中后再将酸溶液滴加至通式(I)化合物的溶液中，搅拌1小时。浓缩后得到粉末(或冻干后得到粉末)，将所得粉末悬浮于甲基叔丁基醚中，打浆30分钟，过滤，干燥得相应的盐。
其中所用酸为有机酸或无机酸，所述的无机酸包括但不限于盐酸、磷酸、硫酸、氢溴酸、硝酸；有机酸包括但不限于L-酒石酸、富马酸、L-苹果酸、D-苹果酸、柠檬酸、L-焦谷氨酸、醋酸、对甲苯磺酸、苯磺酸、甲磺酸、苯甲酸、乳酸、扁桃酸、马来酸、草酸、丁二酸。
其中所述的溶剂包括但不限于乙腈、丙酮、乙酸乙酯、二氯甲烷、四氢呋喃、甲醇、乙醇、异丙醇、1，4-二氧六环、氯仿、乙醚。
下面通过具体的制备实施例和测试例进一步说明本发明。
实施例1
Cis-3-(2，4-二氟苯基)-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)-7-羟基硫色满
合成路线：
第一步
称取A3(6.2g，15.7mmol)(参考专利：WO2018091153)、4-苄氧基苯硼酸(4.3g，18.8mmol)、Pd(dppf)Cl2(0.65g，0.8mmol)和碳酸铯(10.1g，31.4mmol)于反应瓶中，加入1，4-二氧六环-水混合物(4∶1v/v，100mL)，在氮气保护下在50℃油浴中反应1小时。加水(50mL)稀释，用乙酸乙酯(50mL×3)萃取，合并有机相，用饱和氯化钠溶液(100mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂，经柱层析分离(石油醚/乙酸乙酯/二氯甲烷10∶1∶1)得白色固体A4-a(5.9g，88％)。1H NMR(400MHz，CDCl3)δ7.44(ddd，J＝24.9，17.9，7.0Hz，5H)，7.24(d，J＝8.7Hz，2H)，7.15(d，J＝2.5Hz，1H)，7.09(d，J＝8.5Hz，1H)，7.01(d，J＝8.4Hz，2H)，6.84-6.74(m，1H)，6.02(t，J＝5.7Hz，1H)，5.13(s，2H)，3.46(d，J＝5.7Hz，2H)，1.40(s，9H).
第四步
将A4-a(5.9g，13.7mmol)用二氯甲烷溶解(200mL)，冷却在冰水浴中，加入三溴吡啶鎓盐(5.4g，13.7mmol，85％含量)。在冰水浴中搅拌1小时后，用饱和碳酸氢钠溶液(120mL)淬灭，分液，水相用二氯甲烷(100mL)萃取，合并有机相，并饱和氯化钠溶液(100mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗品，经柱层析分离(石油醚/乙酸乙酯/二氯甲烷10∶1∶1)得淡黄色固体A5-a(5.4g，77％)。1H NMR(400MHz，CDCl3)δ7.53-7.47(m，2H)，7.47-7.41(m，2H)，7.41-7.35(m，1H)，7.19-7.13(m，2H)，7.06(dt，J＝6.7，2.2Hz，3H)，6.74(d，J＝8.6Hz，1H)，6.69(dd，J＝8.6，2.3Hz，1H)，5.13(s，2H)，3.97(s，2H)，1.36(s，9H).
第五步
将A5-a(0.8g，1.57mmol)、2.4-二氟苯硼酸(0.3g，1.88mmol)、Pd(dppf)Cl2(128mg，0.16 mmol)和碳酸铯(1.0g，3.14mmol)加入反应瓶中，加入1，4-二氧六环-水混合物(4∶1v/v，20mL)，在氮气保护下在80℃油浴中反应4小时。反应液冷却后加水(50mL)稀释，用乙酸乙酯(50mL×2)萃取，合并有机相，用饱和氯化钠溶液(40mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂，经柱层析分离(石油醚/乙酸乙酯/二氯甲烷15∶1)得白色固体A6-a(0.75g，88％)。1H NMR(400MHz，CDCl3)δ7.44-7.30(m，5H)，7.12(d，J＝2.5Hz，1H)，6.95-6.82(m，4H)，6.82-6.77(m，2H)，6.77-6.69(m，2H)，6.57(td，J＝8.5，2.5Hz，1H)，5.01(s，2H)，3.65(s，2H)，1.35(s，9H).
第六步
将A6-a(0.65g)溶于乙醇中(15mL)，加入20％Pd(OH)2/C(0.5g)，在氢气氛围(1atm)、60℃下反应8h。用硅藻土过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(石油醚/乙酸乙酯3∶1)得白色固体A7-a(0.44g，81％)。1H NMR(400MHz，CDCl3)δ6.99-6.92(m，2H)，6.89-6.80(m，1H)，6.70-6.60(m，2H)，6.55(d，J＝8.5Hz，2H)，6.45(d，J＝8.5Hz，2H)，6.31-6.23(m，1H)，5.01(s，1H)，4.23(d，J＝3.4Hz，1H)，3.88(ddd，J＝12.8，12.8，2.8Hz，1H)，3.41(dd，J＝12.4，12.4Hz，1H)，2.73(d，J＝11.6Hz，1H)，1.35(s，9H).
第七步
将A7-a(0.25g，0.55mmol)溶于乙腈(15mL)中，加入1，2-二溴乙烷(2mL)和碳酸钾(380mg，2.75mmol)，在油浴中回流12h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物无需纯化直接用于下一步。
将上步所得粗产物溶于乙腈(15mL)中，加入3-氟甲基-氮杂环丁烷盐酸盐(103mg，0.83mmol)和碳酸钾(380mg，2.75mmol)，在油浴中回流4h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物用甲醇(5mL)溶解，加入甲醇钠(0.5mL)，室温下搅拌10min，随后减压浓缩除去溶剂，用1N HCl中和至pH 7-8，并用二氯甲烷(15mL×3)萃取，合并有机相，随后用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(二氯甲烷/甲醇30∶1～10∶1)得实施例1(0.11g，三步总收率为42％)。1H NMR(500MHz，CDCl3)δ6.89-6.82(m，1H)，6.76(d，J＝8.4Hz，1H)，6.69(d，J＝2.1Hz，1H)，6.65(t，J＝7.5Hz，1H)，6.54-6.40(m，5H)，6.30-6.24(m，1H)，4.52(dd，J＝47.4，5.1Hz，2H)，4.19(d，J＝3.0Hz，1H)，3.97-3.84(m，3H)，3.62(t，J＝7.7Hz，2H)，3.39(dd，J＝12.4，12.4Hz，1H)，3.28(q，J＝7.2Hz，2H)，3.03-2.87(m，3H)，2.67(d，J＝11.8Hz，1H).
实施例2
Cis-3-(4-氟-2-甲基苯基)-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)-7-羟基硫色满
合成路线：
第一步
将A5-a(0.9g，1.77mmol)、4-氟-2-甲基苯硼酸(0.33g，2.12mmol)、Pd(PPh3)4(204mg，0.17mmol)和碳酸铯(1.2g，3.54mmol)加入反应瓶中，加入1，4-二氧六环-水混合物(4∶1v/v，20mL)，在氮气保护下在80℃油浴中反应4小时。反应液冷却后加水(50mL)稀释，用乙酸乙酯(50mL×2)萃取，合并有机相，用饱和氯化钠溶液(40mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂，经柱层析分离(石油醚/乙酸乙酯/二氯甲烷15∶1)得白色固体A6-b(0.70g，74％)。1H NMR(400MHz，CDCl3)δ7.46-7.34(m，5H)，7.18(d，J＝2.4Hz，1H)，7.01-6.88(m，4H)，6.84-6.70(m，5H)，5.01(s，2H)，3.63(d，J＝14.3Hz，1H)，3.57(d，J＝14.3Hz，1H)，2.26(s，3H)，1.41(s，9H).
第二步
将A6-b(0.65g)溶于乙醇中(15mL)，加入20％Pd(OH)2/C(0.5g)，在氢气氛围(1atm)、60℃下反应48h。用硅藻土过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(石油醚/乙酸乙酯3∶1)得白色固体A7-b(0.23g，42％)。1H NMR(400MHz，CDCl3)δ7.28(s，1H)，7.04-6.91(m，3H)，6.70(dd，J＝8.4，2.4Hz，1H)，6.66-6.51(m，3H)，6.41(d，J＝8.3Hz，2H)，6.09(dd，J＝8.6，5.9Hz，1H)，4.75(s，1H)，4.14(d，J＝2.8Hz，2H)，3.78-3.69(m，1H)，3.45(dd，J＝12.3，12.3Hz，1H)，2.73(d，J＝11.7Hz，1H)，2.44(s，3H)，1.37(s，9H).
第三步
将A7-b(0.22g，0.49mmol)溶于乙腈(15mL)中，加入1，2-二溴乙烷(2mL)和碳酸钾(340mg，2.4mmol)。反应物在油浴中回流12h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物无需纯化直接用于下一步。
将上步所得粗产物溶于乙腈(15mL)中，加入3-氟甲基-氮杂环丁烷盐酸盐(100mg，0.78mmol)和碳酸钾(340mg，2.4mmol)，在油浴中回流4h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物用甲醇(5mL)溶解，加入甲醇钠(0.5mL)，室温下搅拌10min，随后减压浓缩除去溶剂，用1N HCl中和至pH 7-8，并用二氯甲烷(15mL×3)萃取，合并有机相，随后用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(二氯甲烷/甲醇30∶1～10∶1)得实施例2(137mg，三步总收率为59％)。1H NMR(500MHz，CDCl3)δ6.94(dd，J＝9.7，2.4Hz，1H)，6.77(d，J＝8.4Hz，1H)，6.70(d，J＝2.3Hz，1H)，6.62(td，J＝8.4，2.4Hz，1H)，6.51(d，J＝8.6Hz，2H)，6.45(dd，J＝8.3，2.4Hz，1H)，6.40(d，J＝8.4Hz，2H)，6.08(dd，J＝8.4，6.0Hz，1H)，4.52(dd，J＝47.3，5.3Hz，2H)，4.08(d，J＝3.6Hz，1H)，3.96-3.85(m，2H)，3.77-3.69(m，1H)，3.66-3.57(m，2H)，3.42(dd，J＝12.3，12.3Hz，1H)，3.32-3.25(m，2H)，3.02-2.86(m，3H)，2.65(d，J＝11.7Hz，1H)，2.44(s，3H).
实施例3
Cis-3-(2-氟-4-甲氧基苯基)-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)-7-羟基硫色满
合成路线：
第一步
将A5-a(0.9g，1.77mmol)、2-氟-4-甲氧基苯硼酸(0.36g，2.12mmol)、Pd(PPh3)4(204mg，0.17mmol)和碳酸铯(1.2g，3.54mmol)加入反应瓶中，加入1，4-二氧六环-水混合物(4∶1v/v，20mL)，在氮气保护下在80℃油浴中反应4小时。反应液冷却后加水(50mL)稀释，用乙酸乙酯(50mL×2)萃取，合并有机相，用饱和氯化钠溶液(40mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂，经柱层析分离(石油醚/乙酸乙酯/二氯甲烷15∶1)得白色固体A6-c(0.57g，58％)。1H NMR(400MHz，CDCl3)δ7.47-7.33(m，5H)，7.16(d，J＝2.4Hz，1H)，6.98(d，J＝8.6Hz，2H)，6.92(d，J＝8.6Hz，1H)，6.86-6.79(m，3H)，6.74(dd，J＝8.6，2.4Hz，1H)，6.57(dd，J＝12.0，2.4Hz，1H)，6.42(dd，J＝8.6，2.4Hz，1H)，5.03(s，2H)，3.76(s，3H)，3.68(s，2H)，1.39(s，9H).
第二步
将A6-c(0.55g)溶于乙醇中(15mL)，加入20％Pd(OH)2/C(0.5g)，在氢气氛围(1atm)、60℃下反应24h。用硅藻土过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(石油醚/乙酸乙酯3∶1)得白色固体A7-c(0.31g，67％)。1H NMR(400MHz，CDCl3)δ7.01-6.95(m，2H)，6.72-6.64(m，2H)，6.60-6.55(m，2H)，6.52-6.44(m，3H)，6.24(t，J＝8.6Hz，1H)，5.01(s，1H)，4.25(d，J＝3.3Hz，1H)，3.86(ddd，J＝12.8，2.9，2.9Hz，1H)，3.81(s，3H)，3.41(t，J＝12.4Hz，1H)，2.79-2.71(m，1H)，1.37(s，9H).
第三步
将A7-c(0.30g，0.49mmol)溶于乙腈(15mL)中，加入1，2-二溴乙烷(2mL)和碳酸钾(340mg，2.4mmol)。反应物在油浴中回流12h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物无需纯化直接用于下一步。
将上步所得粗产物溶于乙腈(15mL)中，加入3-氟甲基-氮杂环丁烷盐酸盐(100mg，0.78mmol)和碳酸钾(340mg，2.4mmol)，在油浴中回流4h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物用甲醇(5mL)溶解，加入甲醇钠(0.5mL)，室温下搅拌10min，随后减压浓缩除去溶剂，用1N HCl中和至pH 7-8，并用二氯甲烷(15mL×3)萃取，合并有机相，随后用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(二氯甲烷/甲醇 30∶1～10∶1)得实施例3(71mg，三步总收率为22％)。1H NMR(500MHz，CDCl3)δ6.74(d，J＝8.3Hz，1H)，6.67(d，J＝2.4Hz，1H)，6.64(dd，J＝12.0，2.5Hz，1H)，6.51(d，J＝8.4Hz，2H)，6.48-6.38(m，4H)，6.19(t，J＝8.6Hz，1H)，4.49(dd，J＝47.4，5.3Hz，2H)，4.17(d，J＝3.4Hz，1H)，3.95-3.80(m，3H)，3.78(s，3H)，3.58(t，J＝8.0，8.0Hz，1H)，3.35(t，J＝12.4Hz，1H)，3.25(q，J＝6.8Hz，2H)，2.97-2.82(m，3H)，2.66(d，J＝11.6Hz，1H).
实施例4
Cis-3-(4-氟-2-甲氧基苯基)-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)-7-羟基硫色满
合成路线：
第一步
将A5-a(0.62g，1.2mmol)、4-氟-2-甲基苯硼酸(0.31g，1.8mmol)、Pd(PPh3)4(138mg，0.12mmol)和碳酸铯(0.8g，2.4mmol)加入反应瓶中，加入1，4-二氧六环-水混合物(4∶1v/v，15mL)，在氮气保护下在80℃油浴中反应4小时。反应液冷却后加水(50mL)稀释，用乙酸乙酯(50mL×2)萃取，合并有机相，用饱和氯化钠溶液(40mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂，经柱层析分离(石油醚/乙酸乙酯/二氯甲烷10∶1)得白色固体A6-d(0.54g，80％)。1H NMR(400MHz，CDCl3)δ7.46-7.33(m，5H)，7.14(d，J＝2.5Hz，1H)，6.95-6.88(m，3H)，6.83-6.76(m，3H)，6.76-6.70(m，1H)，6.57(dd，J＝10.8，2.5Hz，1H)，6.37(td，J＝8.4，2.4Hz，1H)，5.02(s，2H)，4.00(br，1H)，3.82(s，3H)，3.25(br，1H)，1.38(s，9H).
第二步
将A6-d(0.52g)溶于乙醇/乙酸乙酯混合溶剂中(1∶1v/v，15mL)，加入20％Pd(OH)2/C(0.4g)，在氢气氛围(1atm)、60℃下反应8h。用硅藻土过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(石油醚/乙酸乙酯3∶1)得白色固体A7-d(0.37g，86％)。1H NMR(400MHz，CDCl3)δ7.00-6.95(m，2H)，6.71-6.64(m，2H)，6.55(d，J＝8.5Hz，2H)，6.41(d，J＝8.5Hz，3H)，6.16(dd，J＝8.5，6.6Hz，1H)，5.54(s，1H)，4.28(d，J＝3.3Hz，1H)，3.93(ddd，J＝12.9，2.8，2.8Hz，1H)，3.87(s，3H)，3.38(dd，J＝12.5，12.5Hz，1H)，2.69(d，J＝11.1Hz，1H)，1.38(s，9H).
第三步
将A7-d(0.18g，0.39mmol)溶于乙腈(10mL)中，加入1，2-二溴乙烷(2mL)和碳酸钾(340mg，2.4mmol)。反应物在油浴中回流12h后，过滤除去不溶物，减压浓缩除去溶剂，所得 粗产物无需纯化直接用于下一步。
将上步所得粗产物溶于乙腈(15mL)中，加入3-氟甲基-氮杂环丁烷盐酸盐(65mg，0.52mmol)和碳酸钾(269mg，1.95mmol)，在油浴中回流4h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物用甲醇(5mL)溶解，加入甲醇钠(0.5mL)，室温下搅拌10min，随后减压浓缩除去溶剂，用1NHCl中和至pH 7-8，并用二氯甲烷(15mL×3)萃取，合并有机相，随后用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(二氯甲烷/甲醇30∶1～10∶1)得实施例4(85mg，三步总收率为45％)。1H NMR(600MHz，CDCl3)δ6.75(d，J＝8.4Hz，1H)，6.66(d，J＝2.4Hz，1H)，6.63(dd，J＝10.9，2.4Hz，1H)，6.47(d，J＝8.7Hz，2H)，6.42(dd，J＝8.3，2.5Hz，1H)，6.41-6.34(m，3H)，6.13-6.07(m，1H)，4.49(dd，J＝47.4，5.3Hz，2H)，4.19(d，J＝3.7Hz，1H)，3.95-3.80(m，6H)，3.60-3.53(m，2H)，3.31(dd，J＝12.4，12.4Hz，1H)，3.26-3.20(m，2H)，2.96-2.82(m，3H)，2.58(d，J＝11.2Hz，1H).
实施例5
Cis-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)-3-(2，4，5-三氟苯基)-7-羟基硫色满
合成路线：
第一步
将A5-a(0.9g，1.77mmol)、2，4-5-三氟苯硼酸(0.37g，2.12mmol)、Pd(PPh3)4(204mg，0.17mmol)和碳酸铯(1.2g，3.54mmol)加入反应瓶中，加入1，4-二氧六环-水混合物(4∶1v/v，20mL)，在氮气保护下在80℃油浴中反应4小时。反应液冷却后加水(50mL)稀释，用乙酸乙酯(50mL×2)萃取，合并有机相，用饱和氯化钠溶液(40mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂，经柱层析分离(石油醚/乙酸乙酯/二氯甲烷15∶1)得白色固体A6-e(0.83g，84％)。1H NMR(400MHz，CDCl3)δ7.48-7.34(m，5H)，7.17(d，J＝2.4Hz，1H)，6.98-6.84(m，6H)，6.80-6.71(m，2H)，5.06(s，2H)，3.66(s，2H)，1.39(s，9H).
第二步
将A6-e(0.8g)溶于乙醇中(15mL)，加入20％Pd(OH)2/C(0.8g)，在氢气氛围(1atm)、60℃下反应24h。用硅藻土过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(石油醚/乙酸乙酯3∶1)得白色固体A7-e(0.45g，67％)。1H NMR(400MHz，CDCl3)δ7.04-6.92(m，3H)，6.71(dd，J＝8.4，2.4Hz，1H)，6.62(d，J＝8.5Hz，2H)，6.49(d，J＝8.5Hz，2H)，6.20-6.10(m，1H)，5.11(s，1H)，4.25(d，J＝3.4Hz，1H)，3.93-3.84(m，1H)，3.37(dd，J＝12.4，12.4Hz，1H)， 2.77-2.68(m，1H)，1.37(s，9H).
第三步
将A7-e(0.40g，0.85mmol)溶于乙腈(15mL)中，加入1，2-二溴乙烷(2mL)和碳酸钾(280mg，4.2mmol)。反应物在油浴中回流12h后，过滤除去不溶物，减压浓缩除去溶剂，经柱层析分离(石油醚/乙酸乙酯10∶1)得无色油状物270mg。
将上步所得粗产物溶于乙腈(15mL)中，加入3-氟甲基-氮杂环丁烷盐酸盐(180mg，1.44mmol)和碳酸钾(550mg，4.0mmol)，在油浴中回流4h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物用甲醇(5mL)溶解，加入甲醇钠(0.5mL)，室温下搅拌10min，随后减压浓缩除去溶剂，用1N HCl中和至pH 7-8，并用二氯甲烷(15mL×3)萃取，合并有机相，随后用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(二氯甲烷/甲醇30∶1～10∶1)得实施例5(158mg，2步总收率为68％)。1H NMR(500MHz，CDCl3)δ6.99-6.92(m，1H)，6.74(d，J＝8.4Hz，1H)，6.67(d，J＝2.4Hz，1H)，6.52(d，J＝8.7Hz，2H)，6.48-6.41(m，3H)，6.15-6.05(m，1H)，4.50(dd，J＝47.4，5.2Hz，2H)，4.17(d，J＝3.5Hz，1H)，3.95-3.83(m，3H)，3.58(t，J＝7.8Hz，2H)，3.35-3.22(m，3H)，2.99-2.83(m，3H)，2.63(d，J＝11.1Hz，1H).
实施例6
Cis-3-(4-氟-2-甲氧基苯基)-4-(3-氟-4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)-7-羟基硫 色满
合成路线：
第一步
称取A3(1.0g，2.53mmol)、3-氟-4-苄氧基苯硼酸(0.6g，2.42mmol)、Pd(dppf)Cl2(0.25g，0.31mmol)和碳酸铯(1.8g，5.5mmol)于反应瓶中，加入1，4-二氧六环-水混合物(4∶1v/v，100mL)，在氮气保护下在50℃油浴中反应1小时。加水(50mL)稀释，用乙酸乙酯(50mL× 3)萃取，合并有机相，用饱和氯化钠溶液(100mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂，经柱层析分离(石油醚/乙酸乙酯10∶1)得白色固体A4-b(0.97g，89％)。1H NMR(400MHz，CDCl3)δ7.52-7.33(m，5H)，7.16-6.95(m，5H)，6.79(dd，J＝8.5，2.5Hz，1H)，6.02(t，J＝5.7Hz，1H)，5.19(s，2H)，3.44(d，J＝5.8Hz，2H)，1.38(s，9H).
第二步
将A4-b(0.95g，2.12mmol)用二氯甲烷溶解(100mL)，冷却在冰水浴中，加入三溴吡啶鎓盐(0.83g，2.12mmol，85％含量)。在冰水浴中搅拌1小时后，用饱和碳酸氢钠溶液(50mL)淬灭，分液，水相用二氯甲烷(50mL)萃取，合并有机相，并饱和氯化钠溶液(50mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗品，经柱层析分离(石油醚/乙酸乙酯10∶1)得淡黄色固体A5-b(0.85g，76％)。1H NMR(400MHz，CDCl3)δ7.53-7.48(m，2H)，7.47-7.41(m，2H)，7.41-7.35(m，1H)，7.11-7.05(m，2H)，7.00(dd，J＝11.7，2.1Hz，1H)，6.93-6.88(m，1H)，6.72(s，2H)，5.21(s，2H)，3.95(s，2H)，1.36(s，9H).
第三步
将A5-b(0.85g，1.62mmol)、4-氟-2-甲氧基苯硼酸(0.33g，1.94mmol)、Pd(PPh3)4(128mg，0.16mmol)和碳酸铯(1.0g，3.14mmol)加入反应瓶中，加入1，4-二氧六环-水混合物(4∶1v/v，20mL)，在氮气保护下在80℃油浴中反应4小时。反应液冷却后加水(50mL)稀释，用乙酸乙酯(50mL×2)萃取，合并有机相，用饱和氯化钠溶液(40mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂，经柱层析分离(石油醚/乙酸乙酯/二氯甲烷15∶1)得白色固体A6-f(0.71g，79％)。1H NMR(400MHz，CDCl3)δ7.45-7.35(m，5H)，7.14(d，J＝2.4Hz，1H)，6.87(d，J＝8.6Hz，1H)，6.83-6.72(m，4H)，6.69-6.64(m，1H)，6.56(dd，J＝10.8，2.4Hz，1H)，6.38(td，J＝8.3，2.4Hz，1H)，5.10(s，2H)，3.81(s，3H)，1.38(s，9H).
第四步
将A6-a(0.32g)溶于乙醇中(15mL)，加入20％Pd(OH)2/C(0.35g)，在氢气氛围(1atm)、60℃下反应8h。用硅藻土过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(石油醚/乙酸乙酯3∶1)得白色固体A7-f(0.17g，63％)。1H NMR(400MHz，CDCl3)δ7.02-6.94(m，2H)，6.76-6.65(m，3H)，6.45(td，J＝8.3，2.5Hz，1H)，6.31(dd，J＝11.7，1.8Hz，1H)，6.25-6.17(m，2H)，5.22(s，1H)，4.28(d，J＝3.7Hz，1H)，3.99-3.91(m，1H)，3.88(s，3H)，3.37(dd，J＝12.5，12.5Hz，1H)，2.72(d，J＝12.0Hz，1H)，1.38(s，9H).
第五步
将A7-a(165mg，0.55mmol)溶于乙腈(15mL)中，加入1，2-二溴乙烷(2mL)和碳酸钾(380mg，2.75mmol)，在油浴中回流12h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物无需纯化直接用于下一步。
将上步所得粗产物溶于乙腈(15mL)中，加入3-氟甲基-氮杂环丁烷盐酸盐(103mg，0.83mmol)和碳酸钾(380mg，2.75mmol)，在油浴中回流4h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物用甲醇(5mL)溶解，加入甲醇钠(0.5mL)，室温下搅拌10min，随后减压浓缩除去溶剂，用1N HCl中和至pH 7-8，并用二氯甲烷(15mL×3)萃取，合并有机相，随后用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(二氯甲烷/甲醇30∶1～10∶1)得实施例6(98mg，三步总收率为56％)。1H NMR(500MHz，CDCl3)δ6.74(d，J＝ 8.3Hz，1H)，6.70-6.58(m，3H)，6.49-6.39(m，2H)，6.29-6.14(m，3H)，4.52(dd，J＝47.4，5.1Hz，2H)，4.20(s，1H)，4.06-3.81(m，6H)，3.68-3.55(m，2H)，3.36-3.27(m，3H)，3.01-2.86(m，3H)，2.64(d，J＝11.6Hz，1H).
实施例7
Cis-3-(4-氟-2-甲氧基苯基)-4-(4-(1-(3-氟丙基)氮杂环丁烷-3-基)氨基)苯基)-7-羟基硫色满
合成路线：
第一步
称取A3(3.0g，7.6mmol)、4-(叔丁氧基羰基氨基)-苯硼酸(1.94g，9.1mmol)、Pd(dppf)Cl2(0.62g，0.76mmol)和碳酸铯(4.9g，14.2mmol)于反应瓶中，加入1，4-二氧六环-水混合物(4∶1v/v，30mL)，在氮气保护下在50℃油浴中反应1小时。加水(50mL)稀释，用乙酸乙酯(50mL×3)萃取，合并有机相，用饱和氯化钠溶液(100mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂，经柱层析分离(石油醚/乙酸乙酯10∶1)得白色固体A4-c(3.1g，93％)。1H NMR(400MHz，CDCl3)δ7.36(d，J＝8.5Hz，2H)，7.21(d，J＝8.5Hz，2H)，7.11(d，J＝2.4Hz，1H)，7.04(d，J＝8.5Hz，1H)，6.75(dd，J＝8.5，2.4Hz，1H)，6.60(d，J＝6.1Hz，1H)，6.01(t，J＝5.7Hz，1H)，3.44(d，J＝5.7Hz，2H)，1.55(s，9H)，1.37(s，9H).
第二步
将A4-b(2.8g，6.4mmol)用二氯甲烷溶解(100mL)，加入吡啶(1.0g，12.8mmol)，冷却在冰水浴中，加入三溴吡啶鎓盐(2.5g，6.4mmol，85％含量)。在冰水浴中搅拌1小时后，用饱和碳酸氢钠溶液(100mL)淬灭，分液，水相用二氯甲烷(50mL)萃取，合并有机相，并饱和氯化钠溶液(50mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗品，经柱层析分离(石油醚/乙酸乙酯10∶1)得淡黄色泡沫状固体A5-c(2.0g，60％)。1H NMR(400MHz，CDCl3)δ7.43(d，J＝8.5Hz，2H)，7.13(d，J＝8.6Hz，2H)，7.04(d，J＝2.3Hz，1H)，6.74-6.63(m，3H)，3.95(s，2H)，1.55(s，9H)，1.35(s，9H).
第三步
将A5-c(1.0g，1.93mmol)、4-氟-2-甲氧基苯硼酸(0.39g，2.31mmol)、Pd(PPh3)4(210mg，0.19mmol)和碳酸铯(1.3g，3.8mmol)加入反应瓶中，加入1，4-二氧六环-水混合物(4∶1v/v，20mL)，在氮气保护下在80℃油浴中反应4小时。反应液冷却后加水(50mL)稀释，用乙酸乙酯(50mL×2)萃取，合并有机相，用饱和氯化钠溶液(40mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂，经柱层析分离(石油醚/乙酸乙酯10∶1)得白色固体A6-g(0.91g，84％)。1H NMR(400MHz，CDCl3)δ7.18(d，J＝7.9Hz，2H)，7.13(d，J＝2.4Hz，1H)，6.92(d，J＝8.0Hz，2H)，6.88(d，J＝8.6Hz，1H)，6.79(dd，J＝8.4，6.9Hz，1H)，6.70(dd，J＝8.6，2.4Hz，1H)，6.55(dd，J＝10.8，2.4Hz，1H)，6.51(s，1H)，6.35(td，J＝8.4，2.4Hz，1H)，3.99(br，1H)，3.81(s，3H)，3.25(br，1H)，1.52(s，9H)，1.37(s，9H).
第四步
将A6-g(0.9g)溶于乙醇中(15mL)，加入20％Pd(OH)2/C(0.85g)，在氢气氛围(1atm)、60℃下反应8h。用硅藻土过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(石油醚/乙酸乙酯5∶1)得白色固体A7-g(0.8g，89％)。1H NMR(400MHz，CDCl3)δ7.09(d，J＝8.2Hz，2H)，7.00-6.93(m，2H)，6.71-6.64(m，2H)，6.51-6.46(m，3H)，6.42(td，J＝8.3，2.4Hz，1H)，6.17(dd，J＝8.4，6.8Hz，1H)，4.30(d，J＝3.3Hz，1H)，3.95(dt，J＝12.9，2.7Hz，1H)，3.87(s，2H)，3.38(t，J＝12.5Hz，1H)，2.70(d，J＝11.1Hz，3H)，1.51(s，9H)，1.38(s，9H).
第五步
将A7-g(0.76g)溶于1，4-二氧六环中(10mL)，加入氯化氢(4M 1，4-二氧六环溶液，4mL)，室温下反应2h。减压浓缩除去溶剂得粗产品并用二氯甲烷(20mL)溶解，随后用饱和碳酸氢钠溶液洗，分液，有机相用无水硫酸钠干燥、过滤、浓缩得粗产物，经柱层析分离(石油醚/乙酸乙酯5∶1)得白色固体A8-g(0.58g，93％)。1H NMR(400MHz，CDCl3)δ7.02-6.93(m，2H)，6.71-6.63(m，2H)，6.46-6.40(m，3H)，6.34(d，J＝8.3Hz，2H)，6.20(dd，J＝8.5，6.7Hz，1H)，4.23(d，J＝3.2Hz，1H)，3.95-3.90(m，1H)，3.87(s，3H)，3.56(br，2H)，3.40(dd，J＝12.4，12.4Hz，1H)，2.68(d，J＝11.8Hz，1H)，1.37(s，9H).
第六步
将A8-g(0.56g，1.2mmol)和溶于N-叔丁氧羰基-3-氮杂环丁酮(0.25g，1.44mmol)溶解在1，2-二氯乙烷(10mL)中，加入2滴醋酸，室温下搅拌10分钟。加入三乙酰氧基硼氢化钠(0.76g，3.6mmol)，室温下反应12h。加入饱和碳酸氢钠溶液(10mL)，搅拌10分钟，用二氯甲烷萃取(20mL×2)，有机相用无水硫酸钠干燥、过滤、浓缩得粗产物，经柱层析分离(石油醚/乙酸乙酯5∶1)得白色固体A9-g(0.56g，76％)。1H NMR(400MHz，CDCl3)δ6.99-6.91(m，2H)，6.69-6.61(m，2H)，6.43-6.32(m，3H)，6.23(d，J＝8.2Hz，2H)，6.15(dd，J＝8.4，6.5Hz，1H)，4.26-4.18(m，3H)，4.10(d，J＝6.4Hz，1H)，3.85(s，4H)，3.72-3.64(m，2H)，3.36(dd，J＝12.4，12.4Hz，1H)，2.66(d，J＝11.0Hz，1H)，1.43(s，9H)，1.35(s，9H).
第七步
将A9-g(340mg，0.54mmol)溶于1，4-二氧六环中(5mL)，加入氯化氢(4M 1，4-二氧六环溶液，2mL)，室温下反应2h。减压浓缩除去溶剂得粗产品并用二氯甲烷(20mL)溶解，随后用饱和碳酸氢钠溶液洗，分液，有机相用无水硫酸钠干燥、过滤、浓缩得粗产物。将得 粗产物溶于DMF中(5mL)中，加入DIPEA(140mg，1.09mmol)和3-氟-1-碘代丙烷(98mg，0.49mmol)，在室温下搅拌24h。加入水(30mL)，用乙酸乙酯萃取(20mL×2)，有机相用饱和氯化钠溶液洗涤(20mL)、无水硫酸钠干燥、过滤、浓缩得粗产物。所得粗产物用甲醇(5mL)溶解，加入甲醇钠(0.5mL)，室温下搅拌10min，随后减压浓缩除去溶剂，用1N HCl中和至pH 7-8，并用二氯甲烷(15mL×3)萃取，合并有机相，随后用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(二氯甲烷/甲醇30∶1～10∶1)得无色油状物，冻干后得实施例7(98mg，三步总收率为56％)。1H NMR(500MHz，CDCl3)δ6.84(d，J＝8.4Hz，1H)，6.71-6.63(m，2H)，6.48-6.39(m，2H)，6.36(d，J＝8.2Hz，2H)，6.26(d，J＝8.2Hz，2H)，6.18(dd，J＝8.6，6.7Hz，1H)，4.50(dt，J＝47.1，5.8Hz，2H)，4.17(d，J＝3.2Hz，1H)，4.08(p，J＝6.1Hz，1H)，3.96-3.89(m，1H)，3.86(s，3H)，3.81-3.70(m，3H)，3.40(dd，J＝12.4，12.4Hz，1H)，3.04-2.96(m，2H)，2.74-2.61(m，3H)，1.88-1.75(m，2H).
实施例8
Cis-3-(4-氟-2-甲氧基苯基)-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)-7-羟基硫色满 -1，1-二氧化物
合成路线：
第一步
将A7-d(0.18g，0.39mmol)溶于乙腈(10mL)中，加入1，2-二溴乙烷(2mL)和碳酸钾(340mg，2.4mmol)。反应物在油浴中回流12h后，过滤除去不溶物，减压浓缩除去溶剂得粗产物A8-h(200mg)直接用于下一步。
第二步
将上述所得粗产物溶于乙醚中(8mL)中，将反应瓶置于冰水浴中，随后加入间氯过氧苯甲酸(141mg，0.78mmol)，搅拌6小时。用饱和亚硫酸钠溶液(5mL)淬灭反应，并用二氯甲烷萃取、无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(石油醚/乙酸乙酯5∶1)得A9-h(142mg，2步总收率为61％)。1H NMR(400MHz，CDCl3)δ7.76(d，J＝2.3Hz，1H)，7.23(dd，J＝8.5，2.4Hz，1H)，7.16(d，J＝8.6Hz，1H)，6.69(dd，J＝10.7，2.4Hz，1H)，6.64(d，J＝8.7Hz，2H)，6.44(td，J＝8.3，2.5Hz，1H)，6.36(d，J＝8.6Hz，2H)，6.08(dd，J＝8.3，6.6Hz，1H)，4.69-4.59(m，1H)，4.56(d，J＝4.1Hz，1H)，4.21(t，J＝6.2Hz，2H)，3.88(s，3H)，3.60(t，J＝6.2Hz，2H)，3.27(d，J＝13.5Hz，1H)，1.39(s，9H).
第二步
将A9-h(139mg，0.23mmol)溶于乙腈(15mL)中，加入3-氟甲基-氮杂环丁烷盐酸盐(96mg，0.77mmol)和碳酸钾(165mg，1.2mmol)，mmol)。在油浴中回流4h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物用甲醇(5mL)溶解，加入甲醇钠(0.5mL)，室温下搅拌10min，随后减压浓缩除去溶剂，用1N HCl中和至pH 7-8，并用二氯甲烷(15mL×3)萃取，合并有机相，随后用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(二氯甲烷/甲醇30∶1～10∶1)得实施例8(25mg，2步总收率为21％)。1H NMR(500MHz，CDCl3)δ7.34(d，J＝2.2Hz，1H)，7.28-7.25(m，1H)，6.99-6.89(m，2H)，6.65(dd，J＝10.8，2.4Hz，1H)，6.45-6.38(m，2H)，6.23(d，J＝8.6Hz，2H)，6.12-6.06(m，1H)，4.60(dd，J＝13.0，3.6Hz，1H)，4.54(d，J＝5.1Hz，1H)，4.49-4.40(m，2H)，3.94-3.71(m，4H)，3.64-3.54(m，2H)，3.30-3.22(m，2H)，3.16(d，J＝12.9Hz，1H)，3.01-2.85(m，3H)，2.25-2.19(m，1H)..
实施例9
Cis-3-(4-氟-2-甲氧基苯基)-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)硫色满-7-羧酸
合成路线：
第一步
A4-a(2.1g，4.9mmol)用甲醇(40mL)溶解，加入2M NaOH水溶液(5mL)，室温搅拌1h。调pH为7，用乙酸乙酯萃取(50mL×2)、无水硫酸钠干燥、过滤、减压浓缩除去溶剂，得白色固体B1(1.7g，100％)。LCMS：347.3[M+H].
第二步
将B1(1.7g，4.9mmol)用二氯甲烷(50mL)溶解，加入三乙胺(1.0g，7.4mmol)，冷却在冰水浴中，加入三溴吡啶鎓盐(0.95mL，5.4mmol)。在冰水浴中搅拌10min后，用水(100mL)淬灭，分液，水相用二氯甲烷(50mL)萃取，合并有机相，并饱和氯化钠溶液(50mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗品，经柱层析分离(石油醚/乙酸乙酯10∶1)得白色固体B2(2.3g，100％)。LCMS：479.1[M+H].
第三步
氮气氛围下，向反应瓶中加入甲酸钠(0.75g，14.4mmol)、乙酸酐(0.99g，9.6mmol)、DIPEA(1.25g，9.6mmol)和无水DMF(6mL)，室温下搅拌一小时后加入B2(2.3g，4.8mmol)、Pd(dppf)Cl2(0.4g，0.49mmol)、LiCl(0.62g，14.4mmol)和无水DMF(20mL)。将反应置于80℃油浴中反应24小时。反应液冷却后加乙酸乙酯(80mL)稀释，用1N HCl(100mL)洗涤三次，合并有机相，用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗产物。向所得粗产物中加入二氯甲烷(10mL)，搅拌5分钟，过滤取滤饼即得淡黄色固体B3(0.72g)，直接用于下一步。
第四步
将上述所得B3(0.72g)溶于甲醇中(20mL)，加入2浓硫酸(0.5mL)，在65℃下反应8h。减压浓缩除去溶剂得粗产品，随后用二氯甲烷(100mL)溶解，并依次用水(50mL)、饱和碳酸氢钠溶液(20mL)和饱和氯化钠(50mL)，有机相用无水硫酸钠干燥、过滤、浓缩得粗产物，经柱层析分离(石油醚/乙酸乙酯8∶1)得B4(0.78g，两步收率为42％)。1H NMR(400MHz，CDCl3)δ8.07(d，J＝1.8Hz，1H)，7.70(dd，J＝8.1，1.8Hz，1H)，7.51-7.34(m，5H)，7.23-7.18(m，2H)，7.13(d，J＝8.1Hz，1H)，7.03-6.98(m，2H)，6.15(t，J＝5.8Hz，1H)，5.12(s，2H)，3.93(s，3H)，3.47(d，J＝5.7Hz，2H).
第五步
将B4(0.75g，1.93mmol)用二氯甲烷(50mL)溶解，冷却在冰水浴中，加入三溴吡啶鎓盐(0.77g，2.03mmol)。在冰水浴中搅拌1小时后，用饱和碳酸氢钠溶液(100mL)淬灭，分液，水相用二氯甲烷(50mL)萃取，合并有机相，并饱和氯化钠溶液(50mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗品，经柱层析分离(石油醚/乙酸乙酯10∶1)得B5(0.50g，56％)。1H NMR(400MHz，CDCl3)δ8.00(d，J＝1.7Hz，1H)，7.63(dd，J＝8.3，1.7Hz，1H)，7.53-7.35(m，5H)，7.15(d，J＝8.7Hz，2H)，7.07(d，J＝8.7Hz，2H)，6.81(d，J＝8.3Hz，1H)，5.13(s，2H)，3.99(s，2H)，3.92(s，3H).
第六步
将B5(0.50g，1.07mmol)、4-氟-2-甲氧基苯硼酸(0.30g，1.6mmol)、Pd(PPh3)4(120mg，0.1mmol)和碳酸铯(0.7g，2.1mmol)加入反应瓶中，加入1，4-二氧六环-水混合物(4∶1v/v，15mL)，在氮气保护下在80℃油浴中反应4小时。反应液冷却后加水(50mL)稀释，用乙酸乙酯(50mL×2)萃取，合并有机相，用饱和氯化钠溶液(40mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂，经柱层析分离(石油醚/乙酸乙酯10∶1)得B6(0.45g，82％)。1H NMR(400MHz，CDCl3)δ8.11(s，1H)，7.67(dd，J＝8.2，1.3Hz，1H)，7.49-7.33(m，5H)，6.99(d，J＝ 8.2Hz，1H)，6.91(d，J＝8.1Hz，2H)，6.85-6.78(m，3H)，6.58(dd，J＝10.8，2.3Hz，1H)，6.39(td，J＝8.4，2.4Hz，1H)，5.03(s，2H)，4.08-3.87(m，4H)，3.82(s，3H)，3.31(br，1H).
第七步
将B6(0.44g，0.86mmol)溶于乙醇中(15mL)，加入20％Pd(OH)2/C(0.5g)，在氢气氛围(1atm)、50℃下反应8h。用硅藻土过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(石油醚/乙酸乙酯5∶1)得白色固体B7(0.26g，72％)。1H NMR(400MHz，CDCl3)δ7.99-7.92(m，1H)，7.61(dd，J＝8.0，1.7Hz，1H)，7.06(d，J＝8.1Hz，1H)，6.68(dd，J＝10.9，2.4Hz，1H)，6.58(d，J＝8.5Hz，2H)，6.48-6.36(m，3H)，6.18(dd，J＝8.4，6.8Hz，1H)，5.24(s，1H)，4.33(d，J＝3.2Hz，1H)，3.99-3.90(m，4H)，3.88(s，3H)，3.42(dd，J＝12.5，12.5Hz，1H)，2.74(d，J＝11.7Hz，1H).
第八步
将B7(230mg，0.54mmol)溶于乙腈(15mL)中，加入1，2-二溴乙烷(2mL)和碳酸钾(380mg，2.75mmol)，在油浴中回流12h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物无需纯化直接用于下一步。
将上步所得粗产物溶于乙腈(15mL)中，加入3-氟甲基-氮杂环丁烷盐酸盐(140mg，1.1mmol)和碳酸钾(380mg，2.75mmol)，在油浴中回流4h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物用甲醇(5mL)溶解，加入2M NaOH(1mL)，在60℃下反应10min，随后减压浓缩除去溶剂，用1N HCl中和至pH 7-8，并用二氯甲烷(15mL×3)萃取，合并有机相，随后用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(二氯甲烷/甲醇10∶1～8∶1)得实施例9(122mg，三步总收率为43％)。1H NMR(500MHz，CDCl3)δ7.94(s，1H)，7.59(d，J＝7.8Hz，1H)，6.97(d，J＝8.0Hz，1H)，6.66(dd，J＝10.8，2.0Hz，1H)，6.58(d，J＝8.5Hz，2H)，6.42(dd，J＝14.2，5.3Hz，3H)，6.20-6.11(m，1H)，5.03(s，1H)，4.52(dd，J＝47.3，3.9Hz，2H)，4.32(d，J＝2.9Hz，1H)，4.12-3.94(m，5H)，3.88(s，3H)，3.62(dt，J＝40.7，8.2Hz，2H)，3.43(dd，J＝12.5，12.5Hz，1H)，3.29-3.12(m，3H)，2.70(d，J＝11.5Hz，1H).
实施例10
Cis-(3-(4-氟-2-甲氧基苯基)-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)硫色满-7-硼酸
合成路线：

第一步
A7-d(1.6g，3.4mmol)和PMBCl(0.64g，4.1mmol)用丙酮(40mL)溶解，加入碳酸钾(0.95g，6.8mmol)、KI(57mg，0.34mmol)和四丁基溴化铵(0.1g)，在60℃下反应12h。过滤除去不溶物，经柱层析分离(石油醚/乙酸乙酯5∶1)得B10(1.7g，85％)。1H NMR(400MHz，CDCl3)δ7.33(d，J＝8.4Hz，2H)，6.98(d，J＝8.7Hz，2H)，6.91(d，J＝8.4Hz，2H)，6.73-6.62(m，4H)，6.50-6.35(m，3H)，6.18-6.10(m，1H)，4.90(s，2H)，4.28(d，J＝2.7Hz，1H)，3.98-3.85(m，4H)，3.82(s，3H)，3.38(dd，J＝12.5，12.5Hz，1H)，2.69(d，J＝11.6Hz，1H).
第二步
B10(1.6g，2.7mmol)溶于THF/MeOH(1∶2v/v，30mL)，加入NaOH水溶液(5M，4mL)，室温下搅拌30min。减压浓缩除去溶剂，用2N HCl调为中性，然后用乙酸乙酯(50mL×3)萃取、无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗品。将所得中间体溶于二氯甲烷(100mL)，加入三乙胺(0.54mL，4.1mmol)。冷却在冰水浴中，滴加三氟甲磺酸酐(0.6mL，3.2mmol)。搅拌10分钟后，加水淬灭，分液，有机相用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗产物，经柱层析分离(石油醚/乙酸乙酯5∶1)得B11(1.7g，98％)。1H NMR(400MHz，CDCl3)δ7.34(d，J＝8.4Hz，2H)，7.17(d，J＝2.5Hz，1H)，7.05(d，J＝8.6Hz，1H)，6.97-6.84(m，3H)，6.76-6.65(m，3H)，6.52-6.39(m，3H)，6.20-6.11(m，1H)，4.91(s，2H)，4.37-4.30(m，1H)，3.97-3.78(m，7H)，3.41(dd，J＝12.5，12.5Hz，1H)，2.74(d，J＝11.7Hz，1H).
第三步
氮气氛围下，向反应瓶中加入B11(0.70g，1.1mmol)、联硼酸频那醇酯(0.42g，1.7mmol)、醋酸钾(0.33g，3.3mmol)、Pd(dppf)Cl2(90mg，0.11mmol)和1，4-二氧六环(30mL)。将反应置于100℃油浴中反应16小时。反应液冷却后减压浓缩除去溶剂得粗产物，经柱层析分离(石油醚/乙酸乙酯5∶1)得B12(0.58g，86％)。1H NMR(400MHz，CDCl3)δ7.74(s，1H)，7.41(d，J＝7.5Hz，1H)，7.33(d，J＝8.5Hz，2H)，7.01(d，J＝7.6Hz，1H)，6.91(d，J＝8.5Hz，2H)，6.73-6.63(m，3H)，6.51-6.37(m，3H)，6.16(t，J＝7.5Hz，1H)，4.90(s，2H)，4.34-4.27(m，1H)，3.97(d，J＝12.9Hz，1H)，3.85(s，3H)，3.82(s，3H)，3.41(dd，J＝12.5，12.5Hz，1H)，2.70(d，J＝11.7Hz，1H)，1.37(s，12H).
第四步
将上述所得B12(0.50g，0.82mmol)溶于1，4-二氧六环(10mL)，加入HCl(4M 1，4-二氧六环溶液，4mL)，在室温下反应30min。减压浓缩除去溶剂得粗产品，经柱层析分离(石油醚/乙酸乙酯3∶1)得B13(0.41g，两步收率为42％)。1H NMR(400MHz，CDCl3)δ7.71(s，1H)，7.38(d，J＝7.6Hz，1H)，6.98(d，J＝7.6Hz，1H)，6.64(dd，J＝10.9，2.5Hz，1H)，6.54(d，J＝8.3Hz，2H)，6.44-6.37(m，3H)，6.15(dd，J＝8.5，6.7Hz，1H)，4.82(s，1H)，4.28(d，J＝3.3Hz，1H)，3.99-3.89(m，1H)，3.84(s，3H)，3.38(dd，J＝12.5，12.5Hz，1H)，2.68(d，J＝11.9Hz，1H)， 1.35(s，12H).
第五步
将B13(300mg，0.61mmol)溶于乙腈(15mL)中，加入1，2-二溴乙烷(2mL)和碳酸钾(420mg，3.0mmol)，在油浴中回流12h后，过滤除去不溶物，减压浓缩除去溶剂得粗产物。将所得粗产物溶于乙腈(15mL)中，加入3-氟甲基-氮杂环丁烷盐酸盐(140mg，1.1mmol)和碳酸钾(380mg，2.75mmol)，在油浴中回流4h后，过滤除去不溶物，减压浓缩除去溶剂得粗产物。将所得粗产物溶于乙腈(9mL)和水(9mL)中，浓盐酸(0.5mL)，室温下搅拌12h。反应液用二氯甲烷萃取、无水硫酸钠干燥、过滤后浓缩，经柱层析分离(二氯甲烷/甲醇30∶1～10∶1)得实施例10(51mg，三步总收率为16％)。1H NMR(500MHz，CDCl3)δ7.83(s，1H)，7.52(s，1H)，7.02-6.87(m，1H)，6.69-6.38(m，6H)，6.16(t，J＝7.9Hz，1H)，4.58-4.38(m，2H)，4.29(s，1H)，4.16-3.92(m，3H)，3.91-3.64(m，5H)，3.54-3.29(m，3H)，3.11-2.87(m，3H)，2.71(d，J＝10.8Hz，1H).
实施例11
Cis-3-(4-氟-2-甲氧基苯基)-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)-2，3，4，7-四氢硫 代吡喃[2，3-e]吲唑
合成路线：
第一步
4-溴吲唑(5.0g，25.4mmol)和2，3-二氢吡喃(6.9mL，71mmol)用二氯甲烷(100mL)溶解，加入对甲苯磺酸一水合物(0.48g，2.5mmol)，室温搅拌1h。反应液用饱和碳酸氢钠溶液(100mL)洗，分液，水相用二氯甲烷(100mL)萃取，合并有机相并无水硫酸钠干燥、过滤、减压浓缩除去溶剂，得白色固体为C1a和C1b混合物(7.1g，100％)。LCMS：281.2[M+H].
第二步
氮气氛围下，C1a和C1b混合物(7.0g，25mmol)、3-巯基丙酸甲酯(3.6g，30mmol)、Pd2(dba)3(2.3g，2.5mmol)、Xanphos(2.9g，5.0mmol)和DIPEA(8.0g，62.5mmol)称于反应瓶中，加入1，4-二氧六环(100mL)。在回流条件下反应72小时后，用硅藻土过滤、减压浓缩除去溶剂得粗品，用硅胶过滤(洗脱剂：石油醚/乙酸乙酯10∶1～3∶1)收集含C2a和C2b部分浓缩的粗产品，直接用于下一步。LCMS：321.3[M+H].
第三步
将上一步所得C2a和C2b混合物溶于1，4-二氧六环(20mL)，加入水(9mL)和浓盐酸(5mL)，在80℃油浴中反应2小时。减压浓缩除去溶剂得粗产物，加入甲苯(30mL)后浓缩，重复2次，得油状物C3，直接用于下一步。
第四步
向上一步所得C3粗产物中加入Eaton试剂(CAS#：39394-84-8；40mL)，在80℃油浴中反应0.5小时。将反应液冷却至室温后倒入冰水中，用乙酸乙酯(100mL×2)萃取，有机相依次用饱和碳酸氢钠溶液(100mL)和饱和氯化钠溶液(100mL)洗涤，用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗产物，加入石油醚/乙酸乙酯(2∶1v/v，20mL)搅拌，过滤得C4(3.0g，三步收率为60％)为黄色固体。1H NMR(400MHz，CDCl3)δ8.20(s，1H)，8.14(d，J＝8.9Hz，1H)，7.24(d，J＝8.9Hz，1H)，3.43-3.35(m，2H)，3.08-3.00(m，2H).
第五步
将C4(3.0g，14.7mmol)和2，3-二氢吡喃(2.8mL，29mmol)用四氢呋喃(50mL)溶解，加入对甲苯磺酸一水合物(0.31g，1.5mmol)，在油浴中回流12h。减压浓缩除去溶剂后用柱层析分离(石油醚/乙酸乙酯5∶1～3∶1)得C5(3.4g，81％)。1H NMR(400MHz，CDCl3)δ8.19-8.08(m，2H)，7.35(d，J＝9.0Hz，1H)，5.70(dd，J＝9.3，2.6Hz，1H)，4.08-3.97(m，1H)，3.81-3.68(m，1H)，3.43-3.32(m，2H)，3.09-2.96(m，2H)，2.59-2.44(m，1H)，2.22-2.01(m，2H)，1.88-1.62(m，4H).
第六步
将C5(1.8g，6.7mmol)和2，3-二氢吡喃(1.7g，9.1mmol)用乙醇(20mL)溶解，在油浴中回流6h。冷却到室温后，浓缩除去溶解。所得粗产物用1，4-二氧六环(100mL)溶解，加入4-苄氧基溴苯(2.2g，7.9mmol)、Pd2(dba)3(313mg，0.33mmol)、Xanphos(330mg，0.66mmol)和叔丁醇锂(1.2g，14.5mmol。在回流条件下反应2小时后，冷却至室温，用乙酸乙酯(100mL)稀释，随后用硅藻土过滤、减压浓缩除去溶剂得粗品，经柱层析分离(石油醚/乙酸乙酯4∶1)得C7(1.6g，55％)。1H NMR(400MHz，CDCl3)δ8.14(s，1H)，7.53-7.35(m，5H)，7.33-7.20(m，3H)，7.16(d，J＝8.7Hz，1H)，7.00(d，J＝8.5Hz，2H)，5.94(t，J＝5.8Hz，1H)，5.76-5.66 (m，1H)，5.12(s，2H)，4.12-4.00(m，1H)，3.81-3.70(m，1H)，3.51(d，J＝5.8Hz，2H)，2.67-2.51(m，1H)，2.24-2.05(m，2H)，1.85-1.64(m，3H).
第七步
将B4(500mg，1.1mmol)用二氯甲烷(50mL)溶解，冷却在冰水浴中，加入三溴吡啶鎓盐(450mg，1.2mmol)。在冰水浴中搅拌1小时后，用饱和碳酸氢钠溶液(100mL)淬灭，分液，水相用二氯甲烷(50mL)萃取，合并有机相，并饱和氯化钠溶液(50mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗品，经柱层析分离(石油醚/乙酸乙酯5∶1)得C8(460mg，78％)。1H NMR(400MHz，CDCl3)δ8.09(s，1H)，7.55-7.36(m，5H)，7.21-7.13(m，3H)，7.06(d，J＝8.7Hz，2H)，6.81(d，J＝8.9Hz，1H)，5.66(dd，J＝9.3，2.7Hz，1H)，5.13(s，2H)，4.07-3.97(m，3H)，3.77-3.68(m，1H)，2.59-2.46(m，1H)，2.19-2.03(m，2H)，1.82-1.66(m，3H).
第八步
将B5(0.90g，1.87mmol)、4-氟-2-甲氧基苯硼酸(0.30g，1.6mmol)、Pd(PPh3)4(202mg，0.17mmol)和碳酸铯(1.2g，3.7mmol)加入反应瓶中，加入1，4-二氧六环-水混合物(4∶1v/v，30mL)，在氮气保护下在80℃油浴中反应4小时。反应液冷却后加水(50mL)稀释，用乙酸乙酯(50mL×2)萃取，合并有机相，用饱和氯化钠溶液(40mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂，经柱层析分离(石油醚/乙酸乙酯5∶1)得C9-a(0.89g，92％)。1H NMR(500MHz，CDCl3)δ8.10(d，J＝0.9Hz，1H)，7.44-7.31(m，5H)，7.18(dd，J＝8.8，1.0Hz，1H)，6.96(d，J＝8.8Hz，1H)，6.88(d，J＝8.2Hz，2H)，6.82-6.73(m，3H)，6.55(dd，J＝10.9，2.4Hz，1H)，6.35(td，J＝8.4，2.4Hz，1H)，5.66(dd，J＝9.3，2.7Hz，1H)，5.00(s，2H)，4.09-3.89(m，2H)，3.80(s，3H)，3.76-3.68(m，1H)，3.37(s，1H)，2.62-2.44(m，1H)，2.22-2.02(m，2H)，1.82-1.62(m，3H).
第九步
将C9(0.8g，1.4mmol)溶于乙醇中(20mL)，加入20％Pd(OH)2/C(0.8g)，在氢气氛围(1atm)、50℃下反应24h。用硅藻土过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(石油醚/乙酸乙酯1∶1)得白色固体C10-a(0.32g，48％)。1H NMR(500MHz，DMSO-d6)δ9.14(s，1H)，8.02(s，1H)，7.29(dd，J＝8.6，2.3Hz，1H)，6.97-6.90(m，2H)，6.51(td，J＝8.5，2.5Hz，1H)，6.47-6.43(m，2H)，6.29-6.18(m，3H)，5.77(dd，J＝9.7，2.6Hz，1H)，4.32-4.28(m，1H)，3.89-3.82(m，4H)，3.81-3.76(m，1H)，3.73-3.65(m，1H)，3.43-3.34(m，1H)，2.90(d，J＝12.0Hz，1H)，2.43-2.30(m，1H)，2.05-1.98(m，1H)，1.96-1.90(m，1H)，1.78-1.66(m，1H)，1.60-1.50(m，J＝3.8，3.2Hz，2H).
第十步
将C10(200mg，0.408mmol)溶于乙腈(15mL)中，加入1，2-二溴乙烷(2mL)和碳酸钾(280mg，2.1mmol)，在油浴中回流12h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物直接用于下一步。
将上步所得粗产物溶于乙腈(15mL)中，加入3-氟甲基-氮杂环丁烷盐酸盐(100mg，0.8mmol)和碳酸钾(380mg，2.75mmol)，在油浴中回流4h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物用异丙醇(6mL)溶解，加入HCl(4M异丙醇溶液，2mL)，在60℃下反应30min，随后减压浓缩除去溶剂，用二氯甲烷(50mL)溶解、饱和碳酸氢钠溶液(20mL) 中和，分液，水相用二氯甲烷(20mL×2)萃取，合并有机相，随后用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(二氯甲烷/甲醇30∶1～10∶1)得白色固体实施例11(120mg，三步总收率为57％)。1H NMR(500MHz，CDCl3)δ10.80(s，1H)，8.12(s，1H)，7.04(d，J＝9.2Hz，1H)，6.91(d，J＝8.6Hz，1H)，6.65(dd，J＝10.9，2.5Hz，1H)，6.56(d，J＝8.8Hz，2H)，6.47-6.36(m，3H)，6.12(dd，J＝8.4，6.7Hz，1H)，4.48(dd，J＝47.4，5.6Hz，2H)，4.36(d，J＝3.6Hz，1H)，4.04(dt，J＝12.9，2.8Hz，1H)，3.90-3.81(m，5H)，3.52-3.45(m，3H)，3.14(t，J＝7.0Hz，2H)，2.91-2.76(m，4H).
实施例12
Cis-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)-3-(2，4，5-三氟苯基)-2，3，4，7-四氢硫代 吡喃[2，3-e]吲唑
合成路线：
第一步
将C8(0.40g，0.75mmol)、2，4，5-三氟苯硼酸(0.16g，0.90mmol)、Pd(PPh3)4(90mg，0.08mmol)和碳酸铯(0.50g，1.56mmol)加入反应瓶中，加入1，4-二氧六环-水混合物(4∶1v/v，15mL)，在氮气保护下在80℃油浴中反应4小时。反应液冷却后加水(20mL)稀释，用乙酸乙酯(50mL×2)萃取，合并有机相，用饱和氯化钠溶液(40mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂，经柱层析分离(石油醚/乙酸乙酯5∶1)得C9-b(0.35g，80％)。
第二步
将C9(0.35g，0.30mmol)溶于乙醇中(15mL)，加入20％Pd(OH)2/C(0.4g)，在氢气氛围(1atm)、50℃下反应8h。用硅藻土过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(石油醚/乙酸乙酯2∶1)得白色固体C10-b(145mg，49％)。1H NMR(400MHz，CDCl3)δ8.08(s，1H)，7.23(t，J＝8.3Hz，1H)，7.06-6.95(m，2H)，6.69-6.46(m，4H)，6.23-6.11(m，1H)，5.70(s，1H)，4.36(d，J＝3.8Hz，1H)，4.02(dd，J＝26.5，12.4Hz，1H)，3.82-3.71(m，2H)，3.47(t，J＝12.4Hz，1H)，2.85(d，J＝12.0Hz，1H)，2.66-2.52(m，1H)，2.24-2.02(m，2H)，1.72(d，J＝40.4Hz，3H).
第三步
将C10(140mg，0.282mmol)溶于乙腈(15mL)中，加入1，2-二溴乙烷(2mL)和碳酸钾(195 mg，1.41mmol)，在油浴中回流12h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物直接用于下一步。
将上步所得粗产物溶于乙腈(15mL)中，加入3-氟甲基-氮杂环丁烷盐酸盐(71mg，0.56mmol)和碳酸钾(195mg，1.41mmol)，在油浴中回流4h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物用异丙醇(6mL)溶解，加入HCl(4M异丙醇溶液，2mL)，在60℃下反应1h，随后减压浓缩除去溶剂，用二氯甲烷(50mL)溶解、饱和碳酸氢钠溶液(20mL)中和，分液，水相用二氯甲烷(20mL×2)萃取，合并有机相，随后用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(二氯甲烷/甲醇30∶1～10∶1)得白色固体实施例12(65mg，三步总收率为44％)。1H NMR(500MHz，CDCl3)δ11.14(s，1H)，8.14(s，1H)，7.07(d，J＝8.5Hz，1H)，6.99(q，J＝9.5Hz，1H)，6.91(d，J＝8.5Hz，1H)，6.64(d，J＝8.3Hz，2H)，6.53(d，J＝8.3Hz，2H)，6.14(q，J＝8.4Hz，1H)，4.51(dd，J＝47.4，5.5Hz，2H)，4.36(d，J＝3.1Hz，1H)，4.04-3.87(m，3H)，3.58-3.42(m，3H)，3.19(t，J＝7.0Hz，2H)，2.88(dt，J＝22.1，6.3Hz，4H).
实施例13
Cis-1-(4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)-7-羟基色满-3-基)哌啶-1-基乙酮
合成路线：：
第一步：称取D1(3.8g，10mmol)(参考专利：WO2018091153)、4-苄氧基苯硼酸(2.7g，11.8mmol)、Pd(dppf)Cl2(0.81g，1.0mmol)和碳酸铯(6.5g，20mmol)于反应瓶中，加入1，4-二氧六环-水混合物(4∶1v/v，50mL)，在氮气保护下在50℃油浴中反应1小时。加水(50mL)稀释，用乙酸乙酯(50mL×2)萃取，合并有机相，用饱和氯化钠溶液(100mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂，经柱层析分离(石油醚/乙酸乙酯20∶1)得白色固体D2(3.7g，90％)。1H NMR(400MHz，CDCl3)δ7.51-7.35(m，5H)，7.31-7.27(m，2H)，7.06-7.00(m，3H)，6.65(d，J＝2.3Hz，1H)，6.58(dd，J＝8.4，2.3Hz，1H)，5.74(t，J＝4.0Hz，1H)，5.13(s，2H)，4.86(d，J＝4.0Hz，2H)，1.37(s，9H).
第二步：将D2(5.0g，12.0mmol)用二氯甲烷溶解(200mL)，冷却在冰水浴中，加入三溴吡啶鎓盐(4.5g，12.0mmol，85％含量)。在冰水浴中搅拌1小时后，用饱和碳酸氢钠溶液(100mL)淬灭，分液，水相用二氯甲烷(100mL)萃取，合并有机相，并饱和氯化钠溶液(100mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗品，经柱层析分离(石油醚/乙酸乙酯10∶1)得白色固体D3(5.4g，85％)。1H NMR(400MHz，CDCl3)δ7.53-7.36(m，5H)，7.26-7.20(m，2H)，7.12-7.06(m，2H)，6.73(d，J＝8.4Hz，1H)，6.64(d，J＝2.3Hz，1H)，6.54(dd，J＝8.4，2.3Hz，1H)，5.14(s，2H)，5.05(s，2H)，1.37(s，9H).
第三步：将D3(1.0g，2.03mmol)、N-Boc-1，2，5，6-四氢吡啶-4-硼酸频哪醇酯(0.75g，2.44mmol)、Pd(PPh3)4(230mg，0.2mmol)和碳酸铯(1.6g，4.0mmol)加入反应瓶中，加入1，4-二氧六环-水混合物(4∶1 v/v，20mL)，在氮气保护下在100℃油浴中反应4小时。反应液冷却后加水(50mL)稀释，用乙酸乙酯(50mL×2)萃取，合并有机相，用饱和氯化钠溶液(40mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂，经柱层析分离(石油醚/乙酸乙酯5∶1)得白色固体D4-a(1.1g，91％)。1H NMR(400MHz，CDCl3)δ7.44(ddd，J＝25.8，19.8，7.2Hz，5H)，7.13(d，J＝8.5Hz，2H)，7.00(d，J＝8.6Hz，2H)，6.77(d，J＝8.4Hz，1H)，6.63(d，J＝2.3Hz，1H)，6.51(dd，J＝8.4，2.3Hz，1H)，5.50(s，1H)，5.12(s，2H)，4.90(s，2H)，3.89(s，2H)，3.27(s，2H)，1.80(s，2H)，1.47(s，9H)，1.36(s，9H).
第四步：将D4-a(0.50g，0.84mmol)溶于1，4-二氧六环中(5mL)，加入HCl(4M 1，4-二氧六环溶液，2mL)，室温下搅拌30min。浓缩除去溶剂，所得粗产物用二氯甲烷(100mL)溶解，用饱和碳酸氢钠溶液洗，分液，有机相用用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得中间体。所得中间体用二氯甲烷(10mL)溶解，加入三乙胺(170mg，1.7mmol)和乙酰氯(98mg，1.26mmol)，室温下搅拌30min。浓缩后的粗产物经柱层析分离(石油醚/乙酸乙酯1∶1)得D5-a(0.38g，两步收率为84％)。1H NMR(400MHz，CDCl3)δ7.52-7.34(m，5H)，7.15-7.08(m，2H)，7.04-6.97(m，2H)，6.76(d，J＝8.4Hz，1H)，6.63(d，J＝2.2Hz，1H)，6.52(dd，J＝8.4，2.3Hz，1H)，5.63-5.57(m，0.6H)&5.49-5.44(m，0.4H)(＝CH-)，5.12(s，1.2H)&5.12(s，0.8H)(-CH 2-O)，4.90(s，1.2H)&4.89(s，0.8H)(-CH2-O)(-CH 2-O)，4.07(q，J＝2.7Hz，1.2H)&3.93(q，J＝2.7Hz，0.8H)(-CH 2-N)，3.47(t，J＝5.6Hz，0.8H)&3.27(t，J＝5.5Hz，1.2H)(-CH 2-N)，2.06(s，1.2H)&2.04(s，1.8H)(CH 3CO-)，1.85-1.78(m，2H)，1.35(s，9H).
第五步：将D5-a(0.38g)溶于乙醇中(15mL)，加入20％Pd(OH)2/C(0.5g)，在氢气氛围(1atm)、60℃下反应8h。用硅藻土过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(石油醚/乙酸乙酯1∶2)得D6-a(0.21g，66％)。1H NMR(400MHz，CDCl3)δ6.97-6.88(m，3H)，6.81-6.75(m，2H)，6.61(d，J＝2.3Hz，1H)，6.52(dd，J＝8.3，1.8Hz，1H)，4.69-4.46(m，1H)，4.37-3.99(m，4H)，3.89-3.64(m，1H)，3.03-2.78(m，1H)，2.55-2.28(m，1H)，2.17-2.01(m，6H)，1.78-1.42(m，1H)，1.35(s，9H)，1.17-1.03(m，1H).
第六步：将D6-a(170mg，0.38mmol)溶于乙腈(15mL)中，加入1，2-二溴乙烷(2mL)和碳酸钾(260mg，1.9mmol)，在油浴中回流12h后，过滤除去不溶物，减压浓缩除去溶剂得粗产物。随后将所得粗产物溶于乙腈(15mL)中，加入3-氟甲基-氮杂环丁烷盐酸盐(95mg，0.76mmol)和碳酸钾(260mg，1.9mmol)，在油浴中回流1h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物用甲醇(5mL)溶解，加入甲醇钠(0.5mL)，室温下搅拌10min，随后 减压浓缩除去溶剂，用1NHCl中和至pH 7-8，并用二氯甲烷(15mL×3)萃取，合并有机相，随后用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(二氯甲烷/甲醇20∶1～10∶1)得实施例13(108mg，三步总收率为60％)。1H NMR(500MHz，CDCl3)δ6.95(dd，J＝8.7，3.2Hz，2H)，6.72-6.65(m，3H)，6.33(d，J＝2.5Hz，1H)，6.29(dd，J＝8.2，2.5Hz，1H)，4.68-4.41(m，3H)，4.21-3.90(m，6H)，3.86-3.58(m，4H)，3.36-3.25(m，2H)，3.01-2.76(m，4H)，2.50-2.26(m，1H)，2.16-1.91(m，6H)，1.13-1.00(m，1H).
实施例14
Cis-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)-3-(1-(甲基磺酰基)哌啶-4-基)-7-羟基 色满
合成路线：：
第一步：将D4-a(0.60g，1.01mmol)溶于1，4-二氧六环中(5mL)，加入HCl(4M 1，4-二氧六环溶液，2mL)，室温下搅拌30min。浓缩除去溶剂，所得粗产物用二氯甲烷(100mL)溶解，用饱和碳酸氢钠溶液洗，分液，有机相用用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得中间体。所得中间体用二氯甲烷(10mL)溶解，加入三乙胺(202mg，2.0mmol)和甲磺酸酐(186mg，1.5mmol)，室温下搅拌10min。浓缩后的粗产物经柱层析分离(石油醚/乙酸乙酯1∶1)得D5-b(0.57g，两步收率为99％)。1H NMR(400MHz，CDCl3)δ7.52-7.36(m，5H)，7.11(d，J＝8.7Hz，2H)，7.00(d，J＝8.7Hz，2H)，6.74(d，J＝8.4Hz，1H)，6.63(d，J＝2.3Hz，1H)，6.52(dd，J＝8.4，2.3Hz，1H)，5.58-5.52(m，1H)，5.12(s，2H)，4.88(s，2H)，3.79(q，J＝2.8Hz，2H)，3.18(t，J＝5.6Hz，2H)，2.68(s，3H)，1.97-1.90(m，2H)，1.36(s，9H).
第二步：将D5-b(0.54g)溶于乙醇中(15mL)，加入20％Pd(OH)2/C(0.5g)，在氢气氛围(1atm)、60℃下反应8h。用硅藻土过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(石油醚/乙酸乙酯1∶1)得D6-b(0.37g，80％)。1H NMR(400MHz，CDCl3)δ6.95(d，J＝8.5Hz，2H)，6.89(d，J＝8.4Hz，1H)，6.74(d，J＝8.5Hz，2H)，6.61(d，J＝2.3Hz，1H)，6.52(dd，J＝8.3，2.3Hz，1H)，4.25(dd，J＝11.0，2.9Hz，1H)，4.13(d，J＝4.7Hz，1H)，4.05(dd，J＝11.4Hz，1H)，3.80(d，J＝11.6，11.6Hz，1H)，3.67(d，J＝11.5Hz，1H)，2.74(s，3H)，2.62-2.51(m，1H)，2.47-2.37(m，1H)，2.19-2.04(m，2H)，1.52-1.44(m，1H)，1.38-1.26(m，11H)，1.23-1.12(m，1H).
第三步：将D6-b(250mg，0.51mmol)溶于乙腈(15mL)中，加入1，2-二溴乙烷(2mL)和碳酸钾(350mg，2.6mmol)，在油浴中回流12h后，过滤除去不溶物，减压浓缩除去溶剂得 粗产物。随后将所得粗产物溶于乙腈(15mL)中，加入3-氟甲基-氮杂环丁烷盐酸盐(140mg，1.1mmol)和碳酸钾(350mg，2.6mmol)，在油浴中回流1h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物用甲醇(5mL)溶解，加入甲醇钠(0.5mL)，室温下搅拌10min，随后减压浓缩除去溶剂，用1N HCl中和至pH 7-8，并用二氯甲烷(15mL×3)萃取，合并有机相，随后用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(二氯甲烷/甲醇20∶1～10∶1)得实施例14(140mg，三步总收率为54％)。1H NMR(600MHz，CDCl3)δ6.95(d，J＝8.7Hz，2H)，6.70-6.64(m，3H)，6.35(d，J＝2.4Hz，1H)，6.28(dd，J＝8.3，2.5Hz，1H)，4.51(dd，J＝47.4，5.2Hz，2H)，4.22-4.16(m，1H)，4.07(d，J＝4.8Hz，1H)，4.01-3.90(m，3H)，3.81(d，J＝12.5Hz，1H)，3.68(d，J＝11.6Hz，1H)，3.59(t，J＝7.7Hz，2H)，3.29-3.22(m，2H)，3.01-2.87(m，3H)，2.74(s，3H)，2.55(td，J＝12.0，2.6Hz，1H)，2.42(td，J＝11.5，3.4Hz，1H)，2.17(d，J＝13.9Hz，1H)，2.07(ddt，J＝12.4，8.8，4.5Hz，1H)，1.47(qd，J＝12.2，4.2Hz，1H)，1.37-1.27(m，2H)，1.15-1.08(m，1H).
实施例15
Cis-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)-3-(四氢-2H-吡喃-4-基)-7-羟基色满
合成路线：
第一步：将D3(1.0g，2.03mmol)、3，6-二氢-2H-吡喃-4-硼酸频哪醇酯(0.52g，2.44mmol)、Pd(PPh3)4(230mg，0.2mmol)和碳酸铯(1.6g，4.0mmol)加入反应瓶中，加入1，4-二氧六环-水混合物(4∶1 v/v，20mL)，在氮气保护下在100℃油浴中反应4小时。反应液冷却后加水(50mL)稀释，用乙酸乙酯(50mL×2)萃取，合并有机相，用饱和氯化钠溶液(40mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂，经柱层析分离(石油醚/乙酸乙酯5∶1)得白色固体D5-c(0.86g，85％)。1H NMR(400MHz，CDCl3)δ7.52-7.34(m，5H)，7.15(d，J＝8.7Hz，2H)，7.01(d，J＝8.7Hz，2H)，6.77(d，J＝8.4Hz，1H)，6.63(d，J＝2.1Hz，1H)，6.52(dd，J＝8.4，2.2Hz，1H)，5.55(s，1H)，5.12(s，2H)，4.91(s，2H)，4.15(d，J＝2.6Hz，2H)，3.59(t，J＝5.3Hz，2H)，1.84-1.76(m，2H)，1.36(s，9H).
第二步：将D5-c(0.51g)溶于乙醇中(15mL)，加入20％Pd(OH)2/C(0.6g)，在氢气氛围(1atm)、60℃下反应8h。用硅藻土过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(石油醚/乙酸乙酯3∶1)得D6-c(0.20g，47％)。1H NMR(400MHz，DMSO-d6)δ9.31(s，1H)，6.90(d，J＝8.3Hz，3H)，6.68(d，J＝8.5Hz，2H)，6.57(d，J＝2.3Hz，1H)，6.50(dd，J＝8.3，2.3Hz，1H)，4.28(dd，J＝10.9，3.1Hz，1H)，4.15(d，J＝4.3Hz，1H)，3.97(t，J＝11.7Hz，1H)，3.88-3.81(m， 1H)，3.72(d，J＝10.8Hz，1H)，3.21-3.11(m，1H)，3.10-3.01(m，1H)，2.00-1.92(m，2H)，1.38-1.22(m，12H)，1.19-1.16(m，1H).
第三步：将D6-c(110mg，0.27mmol)溶于乙腈(15mL)中，加入1，2-二溴乙烷(2mL)和碳酸钾(190mg，1.4mmol)，在油浴中回流12h后，过滤除去不溶物，减压浓缩除去溶剂得粗产物。随后将所得粗产物溶于乙腈(15mL)中，加入3-氟甲基-氮杂环丁烷盐酸盐(80mg，0.64mmol)和碳酸钾(190mg，1.4mmol)，在油浴中回流2h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物用甲醇(5mL)溶解，加入甲醇钠(0.5mL)，室温下搅拌10min，随后减压浓缩除去溶剂，用1N HCl中和至pH 7-8，并用二氯甲烷(15mL×3)萃取，合并有机相，随后用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(二氯甲烷/甲醇20∶1～10∶1)得实施例15(110mg，三步总收率为93％)。1H NMR(600MHz，CDCl3)δ6.94(d，J＝8.6Hz，2H)，6.70-6.64(m，3H)，6.35(d，J＝2.4Hz，1H)，6.27(dd，J＝8.3，2.4Hz，1H)，4.51(dd，J＝47.4，5.2Hz，2H)，4.20(dd，J＝10.9，2.7Hz，1H)，4.07(d，J＝4.4Hz，1H)，4.03-3.91(m，4H)，3.87(d，J＝11.1Hz，1H)，3.60(t，J＝7.7Hz，2H)，3.37-3.30(m，1H)，3.27(q，J＝8.1Hz，2H)，3.23-3.16(m，1H)，3.01-2.86(m，3H)，2.07-1.98(m，2H)，1.46(qd，J＝12.8，4.4Hz，lH)，1.32-1.24(m，2H)，1.14(d，J＝11.9Hz，1H).
实施例16
Cis-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)-3-(6-甲氧基吡啶-3-基)-7-羟基色满
合成路线：
第一步：将D3(0.6g，1.2mmol)、2-甲氧基-5-吡啶硼酸(0.25g，1.6mmol)、Pd(PPh3)4(154mg，0.13mmol)和碳酸铯(0.9g，2.7mmol)加入反应瓶中，加入1，4-二氧六环-水混合物(4∶1 v/v，20mL)，在氮气保护下在100℃油浴中反应4小时。反应液冷却后加水(50mL)稀释，用乙酸乙酯(50mL×2)萃取，合并有机相，用饱和氯化钠溶液(40mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂，经柱层析分离(石油醚/乙酸乙酯5∶1)得D5-d(0.61g，97％)。1H NMR(400MHz，CDCl3)δ7.91(d，J＝2.3Hz，1H)，7.51-7.35(m，5H)，7.16(dd，J＝8.6，2.5Hz，1H)，7.08(d，J＝8.7Hz，2H)，6.95(d，J＝8.7Hz，2H)，6.87(d，J＝8.5Hz，1H)，6.71(d，J＝2.1Hz，1H)，6.61-6.51(m，2H)，5.09(s，2H)，5.07(s，2H)，3.90(s，3H)，1.39(s，9H).
第二步：将D5-c(0.4g)溶于乙醇中(15mL)，加入20％Pd(OH)2/C(0.4g)，在氢气氛围(1atm)、60℃下反应8h。用硅藻土过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(石油醚/乙酸乙酯2∶1)得D6-d(0.14g，42％)。1H NMR(400MHz，CDCl3)δ7.56(d，J＝2.2Hz，1H)， 6.95(d，J＝8.4Hz，1H)，6.85(dd，J＝8.6，2.4Hz，1H)，6.70(d，J＝2.3Hz，1H)，6.60-6.47(m，6H)，4.38(dd，J＝10.9，10.9Hz，1H)，4.25-4.17(m，2H)，3.90(s，3H)，3.52(ddd，J＝11.2，5.3，3.6Hz，1H)，1.37(s，9H).
第三步：将D6-d(130mg，0.30mmol)溶于乙腈(10mL)中，加入1，2-二溴乙烷(1mL)和碳酸钾(210mg，1.5mmol)，在油浴中回流12h后，过滤除去不溶物，减压浓缩除去溶剂得粗产物。随后将所得粗产物溶于乙腈(15mL)中，加入3-氟甲基-氮杂环丁烷盐酸盐(90mg，0.72mmol)和碳酸钾(210mg，1.5mmol)，在油浴中回流2h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物用甲醇(5mL)溶解，加入甲醇钠(0.5mL)，室温下搅拌10min，随后减压浓缩除去溶剂，用1N HCl中和至pH 7-8，并用二氯甲烷(15mL×3)萃取，合并有机相，随后用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(二氯甲烷/甲醇20∶1～10∶1)得实施例16(42mg，三步总收率为30％)。1H NMR(400MHz，CDCl3)δ7.67(d，J＝2.4Hz，1H)，6.67(d，J＝8.4Hz，1H)，6.60(dd，J＝8.6，2.4Hz，1H)，6.56-6.48(m，5H)，6.43(d，J＝2.4Hz，1H)，6.33(dd，J＝8.3，2.5Hz，1H)，4.52(dd，J＝47.3，5.2Hz，2H)，4.29(dd，J＝11.1Hz，1H)，4.17-4.06(m，2H)，3.97-3.86(m，5H)，3.63(t，J＝8.3Hz，2H)，3.52(ddd，J＝11.5，5.4，3.5Hz，1H)，3.29(td，J＝7.5，4.9Hz，2H)，3.03-2.87(m，3H).
实施例17
Cis-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)-3-(1-甲基-1H-吡唑-4-基)-7-羟基色满
合成路线：
第一步：将D3(1.0g，2.0mmol)、1-甲基-1H-吡唑-4-硼酸(0.44g，0.35mmol)、Pd(PPh3)4(260mg，0.22mmol)和碳酸铯(1.4g，4.3mmol)加入反应瓶中，加入1，4-二氧六环-水混合物(4∶1 v/v，20mL)，在氮气保护下在80℃油浴中反应4小时。反应液冷却后加水(50mL)稀释，用乙酸乙酯(50mL×2)萃取，合并有机相，用饱和氯化钠溶液(40mL)洗涤、无水硫酸钠干燥、过滤、减压浓缩除去溶剂，经柱层析分离(石油醚/乙酸乙酯2∶1)得D5-e(0.82g，81％)。1H NMR(400MHz，CDCl3)δ7.53-7.35(m，5H)，7.18-7.09(m，4H)，6.97(s，1H)，6.71(d，J＝8.4Hz，1H)，6.67(s，1H)，6.64(d，J＝2.3Hz，1H)，6.51(dd，J＝8.4，2.3Hz，1H)，5.15(s，2H)，5.10(s，2H)，3.75(s，2H)，1.36(s，9H).
第二步：将D5-c(0.8g)溶于乙醇中(15mL)，加入20％Pd(OH)2/C(0.8g)，在氢气氛围(1atm)、60℃下反应8h。用硅藻土过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(石油醚/乙酸乙酯2∶1)得D6-e(0.57g，86％)。1H NMR(400MHz，CDCl3)δ6.99(s，1H)，6.93(d，J＝ 8.4Hz，1H)，6.70(s，1H)，6.68(d，J＝2.3Hz，1H)，6.64-6.54(m，5H)，4.35-4.22(m，3H)，3.80(s，3H)，3.49-3.40(m，1H)，1.37(s，9H).
第三步：将D6-e(220mg，0.54mmol)溶于乙腈(10mL)中，加入1，2-二溴乙烷(2mL)和碳酸钾(373mg，2.7mmol)，在油浴中回流12h后，过滤除去不溶物，减压浓缩除去溶剂得粗产物。随后将所得粗产物溶于乙腈(15mL)中，加入3-氟甲基-氮杂环丁烷盐酸盐(140mg，1.12mmol)和碳酸钾(373mg，2.7mmol)，在油浴中回流4h后，过滤除去不溶物，减压浓缩除去溶剂，所得粗产物用甲醇(5mL)溶解，加入甲醇钠(0.5mL)，室温下搅拌10min，随后减压浓缩除去溶剂，用1N HCl中和至pH 7-8，并用二氯甲烷(15mL×3)萃取，合并有机相，随后用无水硫酸钠干燥、过滤、减压浓缩除去溶剂得粗产品，经柱层析分离(二氯甲烷/甲醇25∶1～10∶1)得实施例17(119mg，三步总收率为50％)。1H NMR(600MHz，CDCl3)δ6.92(s，1H)，6.68-6.62(m，3H)，6.60-6.54(m，3H)，6.40(d，J＝2.4Hz，1H)，6.30(dd，J＝8.3，2.4Hz，1H)，4.51(dd，J＝47.4，5.3Hz，2H)，4.19-4.08(m，3H)，3.97-3.88(m，2H)，3.75(s，3H)，3.60(t，J＝7.5Hz，2H)，3.46(ddd，J＝9.6，5.4，3.8Hz，1H)，3.29-3.24(m，2H)，2.97-2.86(m，3H).
实施例18
(3S，4R)-3-(4-氟-2-甲氧基苯基)-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基-)7-羟基硫 色满
将实施例4用手性制备HPLC分离可得实施例18所示化合物，分离条件为：半制备型CHIRALCEL OD-H手性色谱柱(长度为250mm，内径为20mm，填料粒径为5μm)，流动相为异丙醇∶正己烷＝4∶6。实施例18的核磁数据与实施例4一致，ee＞99％。
实施例19
(3S，4R)-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)-3-(2，4，5-三氟苯基)-7-羟基硫色满
将实施例5用手性制备HPLC分离可得实施例19所示化合物，分离条件为：半制备型CHIRALCEL OD-H手性色谱柱(长度为250mm，内径为20mm，填料粒径为5μm)，流动相为异丙醇∶正己烷＝4∶6。实施例19的核磁数据与实施例5一致，ee＞99％。
实施例20
(3R，4S)-3-(4-氟-2-甲氧基苯基)-4-(4-(2-(3-(氟甲基)氮杂环丁烷-l-基)乙氧基)苯基-)7-羟基硫 色满
将实施例4用手性制备HPLC分离可得实施例20所示化合物，分离条件为：半制备型CHIRALCEL OD-H手性色谱柱(长度为250mm，内径为20mm，填料粒径为5μm)，流动相为异丙醇∶正己烷＝4∶6。实施例20的核磁数据与实施例4一致，ee＞99％。
实施例21
(3R，4S)-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)-3-(2，4，5-三氟苯基)-7-羟基硫色满
将A8-e用手性制备HPLC分离可得实施例21所示化合物，分离条件为：半制备型CHIRALCEL OD-H手性色谱柱(长度为250mm，内径为20mm，填料粒径为5μm)，流动相为异丙醇∶正己烷＝4∶6。实施例21的核磁数据与实施例5一致，ee＞99％。
实施例22
Cis-3-(4-氟-2-甲氧基苯基)-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)-7-羟基硫色满 盐酸盐
Cis-3-(4-氟-2-甲氧基苯基)-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)-7-羟基硫色满(50mg，0.1mmol)溶于1，4-二氧六环(5mL)，随后加入氯化氢的1，4-二氧六环溶液(4M，40μM)，室温搅拌1小时。将反应液浓缩除去溶剂后，加入甲基叔丁基醚(2mL)室温搅拌30分钟，过滤，滤饼抽干得白色粉末(45mg，84％)。1H NMR(400MHz，DMSO-d6)δ11.16(s，1H)，9.46(s，1H)，6.96(d，J＝10.7Hz，1H)，6.72(t，J＝8.1Hz，3H)，6.62(s，1H)，6.57-6.48(m，1H)，6.47-6.29(m，3H)，6.20(t，J＝7.4Hz，1H)，4.83-4.42(m，2H)，4.25-4.08(m，4H)，4.03-3.81(m，5H)，3.72(d，J＝12.9Hz，1H)，3.54-3.44(m，1H)，3.41-3.19(m，3H)，3.16-3.03(m，1H)，2.76(d，J＝12.0Hz，1H).
实施例23
Cis-3-(4-氟-2-甲氧基苯基)-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)-7-羟基硫色满 甲磺酸盐
Cis-3-(4-氟-2-甲氧基苯基)-4-(4-(2-(3-(氟甲基)氮杂环丁烷-1-基)乙氧基)苯基)-7-羟基硫色满(50mg，0.1mmol)溶于乙腈(5mL)，随后加入甲磺酸(9.7mg，0.1mmol)，室温搅拌1小时。加入水(10mL)后冻干得白色泡沫状固体(55mg，94％)。1H NMR(400MHz，CD3OD)δ6.87-6.81(m，1H)，6.76-6.67(m，3H)，6.63(d，J＝2.5Hz，1H)，6.49(d，J＝8.2Hz，2H)，6.46-6.34(m，2H)，6.18(t，J＝7.6Hz，1H)，4.74-4.56(m，2H)，4.51-4.24(m，5H)，4.20-4.10(m，3H)，3.95-3.81(m，4H)，3.70-3.64(m，1H)，3.61-3.55(m，1H)，3.43-3.35(m，1H)，2.72(s，3H)，2.70-2.61(m，1H).
生物学评价
以下结合测试例进一步说明本发明，但这些实施例并非意味着限制本发明的范围。
测试例1：本发明化合物对乳腺癌MCF-7细胞增殖的抑制活性评价
本实验的目的是测定本发明化合物对常用乳腺癌雌激素受体阳性细胞——MCF-7细胞的体外增殖抑制影响，根据IC50大小评价化合物的体外活性。
采用ATP法测试本发明化合物对MCF-7细胞增殖的抑制作用，具体采用CTG法。CellTiter-Glo简称CTG，是通过对ATP进行定量来测定培养物中活细胞数目的一种快速均质检测方法。ATP是活细胞新陈代谢的关键指标，均质的检测使得细胞裂解和产生的发光信号与存在的ATP量成正比，而ATP量直接与培养物中的细胞数量成正比。具体实验方法如下：
MCF-7细胞用DMEM+10％FBS培养。细胞置于37℃孵箱中5％CO2条件下培养。细胞传代、复苏及冻存均按常规方法进行。MCF-7细胞生长至80％左右聚合度，胰蛋白酶消化细胞，以每孔4×103个细胞接种到96孔板中，接种体积为每孔90μL，37℃孵箱中培养过夜。第二天，加入浓度梯度的药物，每孔10μL。化合物最高检测浓度为10μM，4倍稀释，10个浓度。将96孔板置于5％CO2，37℃孵箱中培养6天。96孔培养板中每孔加入50μL的CTG试剂，在振荡器上震荡5min，室温避光放置10min，取60μL转移至384孔Opti-plate。使用多功能酶标仪读取发光(luminescence)信号，信号强度用于表征活细胞数量。各加药组数据以对照组均值为100％进行标化，用GraphPad Prism软件对数据进行处理分析。结果参见表1。
表1本发明化合物体对MCF-7细胞增殖的抑制活性

从表1可知，本发明化合物对MCF-7乳腺癌细胞增殖有很强的抑制作用，特别是实施例18、19化合物，其抗肿活性比阳性对照氟维司群提高了7.6倍，比SAR439859提高了15.6倍，达到亚纳摩尔及水平。
测试例2：本发明化合物对他莫昔芬耐药细胞MCF-7 Tam1增殖抑制活性评价
MCF-7 Tam1细胞是通过MCF-7细胞长期暴露于他莫昔芬的活性代谢产物4-羟基他莫昔芬，同时伴有长期雌激素剥夺的培养条件而建立的，具有对他莫昔芬和芳香化酶抑制剂的抗性。本实验将检测本发明化合物对他莫昔芬耐药细胞MCF-7 Tam1增殖抑制，根据IC50值评价化合物的体外抗他莫昔芬耐药乳腺癌活性。
MCF-7 Tam1细胞传代培养，培养条件为含有青霉素(终浓度为100U/mL)、链霉素(终浓度为100μg/mL)、10μg/mL人胰岛素、1μM 4-羟基他莫昔芬以及10％FBS的DMEM培养基，当细胞融合至90％时，弃去旧培养基，用2ml PBS洗涤细胞2次，弃去PBS后加入2mL 0.25％胰蛋白酶-0.02％EDTA混合消化液，置显微镜下观察，约30s，当细胞变圆后迅速加入2ml完全培养基终止消化，轻轻吹打，收集细胞。800rpm，4℃，离心5min，弃去上清，用完全培养基重悬细胞，分瓶培养，隔天换液。化合物最高检测浓度为10μM，10倍稀释，8个浓度。使用适合进行化学发光检测的96孔板，每孔接种100μl细胞(1×104cells/ml)。按试验分组将各组细胞使用含对应浓度化合物的培养基孵育5天。取出细胞培养板，室温平衡10min。96孔板每孔加入100μl CTG检测试剂，室温孵育10min。酶标仪检测各组RFU值。抑制率计算方式为：抑制率＝(对照组OD值-给药组OD值)/对照组OD值。使用GraphPad计算各化合物对MCF-7Tam1细胞的IC50。
表2本发明化合物对他莫昔芬耐药细胞MCF-7 Tam1的抑制活性
表2表明，本发明化合物对对他莫昔芬耐药乳腺癌细胞MCF-7 Tam1增殖有很强的抑制作用，其抗肿瘤活性与氟维司群相似，明显优于SAR439859。
测试例3：本发明化合物体对MCF-7细胞内野生型ERα降解活性评价
通过基于细胞的高内涵成像方法分析本发明中所述的化合物引起的ER降解的程度。
MCF-7细胞培养基：88％RPMI 1640，10％FBS，1％P/S和1％GlutaMax；MCF-7播种培养基：88％RPMI 1640，10％FBS，1％P/S和1％GlutaMax.
第一天：用细胞播种培养基稀释细胞悬浮液至8.75×104cells/mL，随后将40μL细胞悬液滴入每孔检测板，置于37℃ CO2培养箱中。第三天：化合物稀释4倍连续稀释，转移500μL，10个浓度点。用20μL培养基稀释化合物，用Bravo转10μL至板上，在37℃培养箱中培养24小时。第四天：在测定板中加入50μL 8％多聚甲醛，室温下放置40分钟。每孔用100μL PBS洗涤检测板两次，然后在检测板中加入50μL 0.1％TritonX-100的PBS，室温下放置15分钟。每孔用100μL PBS洗涤检测板5次，然后加入含0.1％Tween 20的封闭缓冲液50μL，室温下放置1小时。弃掉检测板中的溶液，用阻断缓冲液按1∶1000的比例稀释一抗，加入25μL检测板，4℃孵育过夜。第五天：每孔用100μL PBS洗涤检测板5次，然后用阻断缓冲液稀释二抗(1∶1000)和DRAQ5(1∶2000)，加入25μL至检测板，室温孵育1小时。每孔用100μL PBS冲洗检测板5次，在Odyssey红外成像系统上读取检测板。然后将数据标准化，将800通道(ER)的综合强度除以700通道(DNA)的综合强度。背景减法是将所有归一化实验值减去归一化阴性对照井(无一抗体)的平均值。然后通过将每个实验值除以DMSO控制值的平均值(％响应＝(实验值/DMSO控制值)×100)计算归一化/背景减除数据的百分比响应。使用GraphPad Prism v6.02软件(GraphPad，San Diego，CA)生成剂量响应曲线、EC50和百分比ERα剩余值。
表3 本发明化合物对MCF-7细胞内野生型ERα降解活性
从表3可知，本发明化合物对ERα有很强的降解作用，特别是实施例1、3、18、19化合物，其ER降解活性比阳性对照氟维司群提高了大约2倍，比SAR439859提高了大约3倍，达到亚纳摩尔及水平。
测试例4：MCF-7细胞荧光报告基因实验检测化合物对野生型ERα拮抗活性
ERα报告基因构建原理：HEK293/GAL4/ERα，细胞株利用分子克隆方法将雌激素受体LBD区域(化合物结合区域)和GAL4 DBD区域(DNA结合区域)进行融合蛋白表达。当配体激活ERα时，ERα-GAL4融合蛋白启动下游luciferase基因表达，读板机检测到化学发光信号。ERα配体刺激浓度和化学发光信号呈剂量依赖关系。
将HEK293/GAL4/ERα细胞悬液收集起来，1000转/分钟离心5分钟，去掉上清，用预热的培养基(DMEM(无酚红)，含碳吸附血清10％，即500mL细胞培养基中包含450mLDMEM，50mL碳吸附血清)重悬，计数后用培养基稀释细胞悬液，按照40000细胞/孔接种到96孔细胞培养板中，每孔接种80μL细胞悬液，37℃，5％CO2培养箱，孵育过夜。 实验当天，每孔加入10μL化合物工作液到细胞板中，37℃，5％CO2培养箱，继续孵育1小时，再每孔加入10μL含有激动剂的培养基(10nM Estradiol)，Estradiol终浓度为1nM，DMSO终浓度为0.5％。细胞板在37℃，5％CO2培养箱中继续孵育24小时。结束孵育后，去掉细胞上清，细胞板每孔加入50μL Bright Glo检测试剂，25℃，孵育2分钟。结束孵育后，使用EnVision进行发光信号检测。
数据根据如下公式计算化合物处理后的抑制率：％抑制率＝100-(RFU化合物-RFU空白对照)/(RFU阴性对照-RFU空白对照)×100％。阴性对照：激动剂处理的细胞；空白对照：无激动剂处理的细胞，然后利用Prism作图计算化合物的IC50值。
表4 本发明代表性化合物对野生型ERα的体外拮抗活性
由表4的实验结果可知，本发明化合物对野生型ERα有很强的拮抗作用，优于氟维司群和临床III期药物SAR439859。其中实施例3、4、5、18、19化合物对野生型ERα的拮抗活性相比SAR439859提高约3倍，比氟维司群提高2倍以上。
测试例5：本发明化合物对含Y537S或D538G突变的突变型ERα的拮抗作用
15％-30％的乳腺癌患者在接受内分泌治疗后会出现ESR1配体结合区域的突变，以D538G及Y537S位点突变最为常见，特别是在雌激素受体阳性的转移性乳腺癌患者身上非常普遍，并且与较差的治疗效果有关。与野生型ERα不同，ERα配体结合域中的Y537S和D538G突变导致在没有配体的情况下自发募集共活化因子，例如过氧化物酶体增殖物激活受体-γ共激活因子和类固醇受体共激活因子，导致ERα的组成型激活，促进AF-2激动剂样构象的形成。在这种激活的构象中，突变的ERα对雌二醇的亲和力增加，而对拮抗剂的亲和力降低。本实验通过检测本发明化合物对含主要突变热点Y537S和D538G的突变型ERα的拮抗活性，评价化合物对内分泌疗法耐药乳腺癌的体外抗肿瘤活性。
SK-BR-3细胞培养基：89％1640不含酚红，10％活性炭处理FBS和1％GlutaMax
第一天：1.将80μL细胞悬液和30000个细胞接种到检测板的每孔中，37℃5％CO2孵育24小时。第二天：制备转染试剂，室温放置15分钟。测定板每孔加入10μL转染试剂，37℃5％CO2孵育24h。第三天：将10μL培养基(100nMβ-雌二醇，10μL培养基)加入检测板，37℃5％CO2孵育24h。第四天：1.从每孔中取出50μL培养基，将50μL荧光素酶检测试剂加入检测板中，室温振荡20分钟，然后在Envision上读数。2.将50μL Stop&Glo试剂加入检测板，室温振荡20分钟，然后在Envision上读数。使用XL-fit软件(供应商：ID Business Solutions Ltd.，软件版本：XL fit 5.0)分析数据，％Effect＝(样品值-空白对照)/(阳性对照-空白对照)*100。
表5 本发明化合物对突变型ERα的体外拮抗活性
表5数据表明，本发明的化合物对突变型ERα(Y537S或D538G)有很强的拮抗作用。其中，针对含Y537S突变的ERα突变体，实施例4与氟维司群和SAR439859拮抗活性相当，实施例18相对氟维司群和SAR439859分别有3.3倍和3.7倍的提高，而施例19相对氟维司群和SAR439859则分别有4倍和4.5倍的提高；对于含D538G突变的ERα突变体，实施例18和19与氟维司群的拮抗活性类似，但较SAR439859分别有5.7倍和5.2倍的提高。
测试例1至5结果显示，本发明化合物对ERα具有显著的拮抗/降解双重功能，包括对含常见突变点的突变型ERα表现出明显的拮抗作用，以及对雌激素依赖的MCF-7细胞和他莫昔芬耐药的MCF-7 Tam1细胞增殖均具有显著的抗增殖活性。综合这几项测试结果可知，本发明化合物表现出了优异的体外抗肿瘤药效，在多项测试中优于上市药物氟维司群，明显优于对照化合物SAR439859。
测试例6：本发明化合物的大鼠药代动力学性质评价
本实验目的为测试本发明化合物的大鼠药代动力学性质。
溶媒为：5％DMSO+5％Solutol+90％(0.5％MC)，呈无色澄清给药溶液。给药方式及剂量为：口服，10mg/kg。SD大鼠按体重随机分组，给药前1天禁食不禁水12～14h，给药后4h给食。每只动物每次通过眼眶取0.1mL血液，EDTAK2抗凝，采集时间点为：给予受试物后0，5，15，30min，1，2，4，6，8，24h。血液样本采集后置于冰上，并于30分钟之内离心分离血浆(离心条件：5000转/分钟，10分钟，4℃)。分析前存放于-80℃。利用LC-MS/MS测定大鼠血浆中化合物的浓度。数据采集及控制系统软件为Analyst1.5.1软件(Applied Biosystem)。图谱样品峰积分方式为自动积分；采用样品峰面积和内标峰面积的比值作为指标，和样品的浓度进行回归。回归方式：线性回归，权重系数为1/X2。药代动力学参数用WinNonlin Professional v6.3(Pharsight，USA)用非房室模型分析处理。Cmax为实测的最大血药浓度，血药浓度-时间曲线下面积AUC(0→t)由梯形法计算得到，Tmax为给药后血药浓度达峰时间。实验数据用“均数±标准差”(Mean±SD，n≥3)或“均数”(Mean，n＝2)表示。
表6本发明实施例实施例18和19化合物的药代动力学参数
表6表明，本发明化合物，口服吸收良好，药物暴露量好，预示动物体内药效和临床 可以口服给药。
测试例7：化合物对大鼠子宫增重的影响评价和组织分布情况
ER阳性乳腺癌内分泌疗法中的雌激素受体调节剂(SERM)如他莫昔芬，由于呈现部分激动效应，会增加子宫内膜增生、息肉和子宫内膜癌的风险。因此，开发选择性雌激素受体降解剂(SERD)的时候需消除这一效应，使之为ER完全拮抗剂。通过考察化合物对大鼠子宫重量(以子宫湿重/大鼠体重的形式表示)和子宫内膜的影响可以判断化合物是否为完全拮抗剂，这对安全性评价具有重要意义。另一方面，药物能穿透动物的血脑屏障、在脑组织中有足够的暴露量，是其对脑转移病灶有效的关键，因此通过分析给药后动物血浆和脑部的药物分布情况，可以判断药物是否具有在脑原位肿瘤模型中起到抗肿瘤效果的潜力。
本测试所涉及的子宫湿重实验及化合物的组织分布实验在同一批大鼠中进行。
试验动物：SPF级雌性不成熟SD大鼠，21天龄，由常州卡文斯实验动物有限公司提供，饲养于SPF级饲养环境中，室内温度控制在23±2℃，自由饮食和摄水。动物总数30只。实验前适应性饲养3天。
实验分组：空白组：每天口服2％吐温-80/0.5％羟甲基纤维素(10mg/kg)，连续3天。空白+17α-乙炔雌二醇组：每天口服17α-乙炔雌二醇(0.1mg/kg)，连续3天。4-羟基他莫昔芬组：每天口服4-羟基他莫昔芬组(60mg/kg)，连续3天。实施例18组：每天口服实施例18(10mg/kg)，连续3天。实施例19组：每天口服实施例16(10mg/kg)，连续3天。
最后一次给药4h后，二氧化碳法处死大鼠，解剖取子宫，仔细剔除无关组织，用D-Hanks液洗涤2～3次，洗去血液，沥干水分保存，称取重量。HE染色检测子宫内膜厚度。对血浆和脑组织进行取样，分析血浆和脑组织中实施例18、实施例19的浓度。
HE实验：1)将固定好的子宫组织切成4μm厚的切片，将切片放在烘箱中1h；2)将干燥的石蜡切片进行常规二甲苯脱蜡，下行梯度乙醇水化，蒸馏水洗；3)加入苏木精染色10min～30min，然后用流水洗去苏木精染液；4)1％盐酸乙醇褪色，见切片变红，颜色较浅即可，在放入自来水流水中使其恢复蓝色。5)伊红染0色1min，流水冲洗；6)切片经梯度酒精脱水干燥，二甲苯透明，中性树胶封片。7)随机选择一个视野使用显微镜(200×)拍照。数据组间统计学差异采用one-way ANOVA和Tukey’s检验，P值小于0.05认为有显著性差异。
LC-MS/MS检测药物浓度：(1)色谱条件，色谱柱：Waters BEH C18 column(50mm×2.1mm，id 1.7μm)液相色谱柱，柱温40℃；流动相A为超纯水(含0.1％甲酸)，流动相B为甲醇。洗脱梯度为：0～1min，A-B(70∶30)，1-5min，A-B(10∶90)，5～6min，A-B(10∶90)，6-6.1min，A-B(70∶30)，6.1-9min，A-B(70∶30)。时间为9min，流速为0.3mL/min。(2)质谱条件：采用LC-MS/MS进行测定，离子源为ESI源，正离子模式检测；加热毛细管温度为450℃；CAD为4；气帘气为11；GS1(N2)为40；GS2(N2)为40；扫描方式为多级反应监测(MRM)用于定量分析的离子反应分别为m/z 498.80→139.30(实施例18)，m/z 504.70→116.20(实施例19)。(3)标准曲线：分别准 确称取实施例18、实施例19标准品，配制成浓度为1mg/mL的标准储备液，将其稀释为浓度为10、20、50、100、200、500、1000、2000、5000、10000、20000、50000ng/mL的标准品工作液，在45μL空白基质中分别加入5μL的各浓度标准品工作液，配制成终浓度分别为1、2、5、10、20、50、100、200、500、1000、2000、5000ng/mL的标准样品，涡旋混合3min，加入150μL含内标(4-氯苯丙氨酸)的甲醇，涡旋振荡3min，18000rpm离心5min，取上清150μL转移至新的Eppendorf管中，18000rpm离心5min，取上清100μL转移至进样瓶中，5μL进样。(4)样品中实施例18、19浓度测定取50μL待测血浆样本至Eppendorf管中，涡旋混合3min，加入150μL含内标(4-氯苯丙氨酸)的甲醇，涡旋振荡3min，18000rpm离心5min，取上清150μL转移至新的Eppendorf管中，18000rpm离心5min，取上清100μL转移至进样瓶中，5μL进样分析。
子宫湿重实验结果如下：
表7本发明代表性化合物对未成熟大鼠子宫重量的影响
实验结果如表7和图1所示，17α-乙炔雌二醇组和4-羟基他莫昔芬(他莫昔芬体内的活性代谢形式)组的子宫重量与体重的比值是空白组的3倍和2倍，表明对子宫组织的ERα有激动作用。但是本发明实施例18、实施例19均展现出了有效的减小子宫重量的作用，使子宫湿重与体重的比值降低到空白组的60％左右，表明对ERα有拮抗/反向激动作用。此外，用苏木精和伊红对子宫内膜进行染色(图1，B)，以进行组织学评估。17α-乙炔雌二醇和4-羟基他莫昔芬增加了子宫湿重，上皮细胞呈高柱状表型，而空白对照和实施例18、实施例19组的上皮细胞呈现低立方表型，证实了本发明的实施例化合物是一个完全的拮抗剂，没有他莫昔芬等雌激素受体调节剂发生宫内膜癌的风险。
表8本发明代表性化合物的大鼠组织分布情况(PO-10mg/kg，QD)
由表8可知，本发明化合物表现出优秀的血脑屏障穿透能力，在大鼠的脑组织中药物暴露量高，其中实施例18具有很好的脑组织暴露量，B/P值达到14.1，远高于目前为止公开的其他雌激素降解剂，而实施例19的B/P值更是达到29。这些结果表明本发明化合物可用于脑转移乳腺癌的治疗。
测试例8：本发明化合物对MCF-7小鼠皮下肿瘤模型的生长抑制实验
试验试剂：胎牛血清(SH30070.03)(FBS，Hyclone，Logan，UT，USA)；青霉素(I9532)(Sigma，St.Louis，MO，USA)；链霉素(85886)(Sigma，St.Louis，MO，USA)；重组人胰岛素(91077C)(Sigma，St.Louis，MO，USA)；EMEM培养基(30-2003)(ATCC，Rockville，MD， USA)；胰蛋白酶(15090046)(Gibco，Grand Island，NY，USA)；HBSS(H6648)、DMSO(D8418)、PEG400(8074851000)、PEG300(8074841000)、PBS(806552)、Solutol HS-15(42966)(Sigma，St.Louis，MO，USA)；Matrigel Matrix(BD Bioscience，USA)；雌激素微丸(0.36mg雌二醇，60天释放)(SE-121)(Innovative Research of America，Florida，USA)；PVDF膜(0.45μm)(Millipore，Schwalbach，德国)；StarSignal Western Protein Marker(10-200kDa)(M227-01)(GenStar，北京，中国)；丽春红、吐温20、丙烯酰胺、十二烷基硫酸钠、PMSF(Solon，OH，USA)；蛋白印迹膜再生液(ZN1923，Biolab，北京，中国)；蛋白裂解液(RIPA)、1.5 mol/L Tris HCl(pH6.8)、1.5mol/L Tris HCl(pH8.8)(Beyotime，上海，中国)；ECL发光液(Thermo Fisher Scientific，Pittsburgh，PA，USA)；Anti-ERα抗体(21244-1-AP)购自Proteintech公司(Proteintech，湖北，中国)；Goat Anti-Rabbit IgG H&L(HRP)(ab6721)购自Abcam公司(Abcam，Cambridge，UK)。
试验动物：雌性无胸腺裸鼠，由常州卡文斯实验动物有限公司提供，动物合格证号：SCXK(苏)2016-0010，饲养于22±2℃的环境中，自由摄食和饮水。
细胞的培养条件：MCF-7细胞传代培养，培养条件为含有青霉素(终浓度为100U/mL)、链霉素(终浓度为100μg/mL)、human recombinant insulin(终浓度为0.01mg/mL)以及10％FBS的EMEM培养基，当细胞融合至90％时，弃去旧培养基，用2ml PBS洗涤细胞2次，弃去PBS后加入2mL 0.25％胰蛋白酶-0.02％EDTA混合消化液，置显微镜下观察，约30s，当细胞变圆后迅速加入2ml完全培养基终止消化，轻轻吹打，收集细胞。800rpm，4℃，离心5min，弃去上清，用完全培养基重悬细胞，分瓶培养，隔天换液。
小鼠移植瘤模型构建：在肿瘤植入前3天，用无菌的套管针将雌激素微丸(0.36mg雌二醇，60天释放)植入小鼠肩胛骨之间的皮下。取对数生长期的MCF-7细胞，使用胰蛋白酶将MCF-7细胞消化，使用50％HBSS和50％Matrigel混合液将MCF-7细胞重悬成107cells/mL细胞悬液。每只小鼠于右侧腋窝乳腺脂肪垫区域皮下注射200μL MCF-7细胞悬液，每3天测量肿瘤体积(宽2×长×π/6)和体重；肿瘤平均体积达到约200mm3时，将小鼠随机分组并灌胃给药。
空白组：10％PEG300+25％of20％Solutol+65％PBS，小鼠MCF-7移植瘤模型建立且肿瘤平均体积达到约200mm3后，每日灌胃给药，灌胃体积为0.1ml/10g，连续给药21天(N＝8)；
LSZ102(15mg/kg)组：小鼠MCF-7移植瘤模型建立且肿瘤平均体积达到约200mm3后，每日灌胃给药LSZ102，给药剂量为15mg/kg，灌胃体积为0.1mL/10g，连续给药21天(N＝8)；LSZ-102是诺华公司开发的处于临床研究阶段的选择性雌激素受体降解剂，是最早进入临床研究的口服SERD之一；
实施例4(5mg/kg)组：小鼠MCF-7移植瘤模型建立且肿瘤平均体积达到约200mm3后，每日灌胃给药实施例4，给药剂量为5mg/kg，灌胃体积为0.1mL/10g，连续给药21天(N＝8)；
造模开始每3天早上固定时间称量各组小鼠体重并测量小鼠肿瘤体积大小(宽2×长×π/6)；化合物的抑瘤疗效用肿瘤生长抑制率TGI(％)来评价。TGI(％)＝[(1-(某处理组给药结 束时平均瘤体积-该处理组开始给药时平均瘤体积)/(溶剂对照组给药结束时平均瘤体积-溶剂对照组开始给药时平均瘤体积)]×100％。
表9 本发明代表性化合物皮下移植瘤模型肿瘤体积(p.o.，QD)
实验结果(表9和图2)显示，在小鼠皮下移植瘤MCF-7模型中，与空白组相比，本发明化合物实施例4在5mg/kg一天一次口服给药时对肿瘤生长具有显著抑制作用，TGI为78％，与临床I期药物LSZ102三倍剂量(15mg/kg)下体内抗肿瘤效果相当。由此可见，本发明化合物表现出很强的体内抗肿瘤活性，其抗肿瘤活性大约是对照药LSZ102的3倍。
以上结果显示本发明的化合物可以通过拮抗/降解雌激素受体来治疗或预防与雌激素有关的多种疾病，例如癌症(乳腺癌、卵巢癌、结肠癌、前列腺癌、子宫内膜癌)，骨质疏松症，神经退行性疾病，心血管疾病，红斑狼疮，子宫内膜异位症及肥胖症等。
尽管以上已经对本发明作了详细描述，但是本领域技术人员理解，在不偏离本发明的精神和范围内的前提下可以对本发明进行各种修改和改变。本发明的权利范围并不限于上文所作的详细描述，而是如在所附权利要求中阐述的那样。

Claims (10)

  1. 一种通式(I)所示的化合物，或其外消旋体、对映异构体、非对映异构体、或其混合物形式、或可药用盐：
    式中：
    R1选自OH、COOH、B(OH)2、卤素、C1-C6烷基、卤代C1-C6烷基或C1-C6烷氧基；
    R2选自H、OH、COOH、卤素、氰基、C1-C6烷基、C1-C6烷氧基、卤代C1-C6烷基或羟基取代的C1-C6烷基；或者R1、R2与相连的苯环形成苯并5-6元杂芳基；
    X选自S、S(O)2或O；
    环A选自：C3-C6环烷基、5-8元杂环基、C6-C10芳基或5-8元杂芳基；
    各R3独立地选自氢、卤素、氰基、C1-C6烷硫基、C1-C6烷基CO-、C1-C6烷基SO2-、氨基、-NH(C1-C6烷基)、-N(C1-C6烷基)(C1-C6烷基)、-SO2NH2、-C(O)NH2、C1-C6烷基、C1-C6烷氧基、卤代C1-C6烷基、卤代C1-C6烷氧基或卤代C1-C6烷硫基；
    o为0、1、2、3或4；
    Y1、Y2独立地选自CR4或N；
    各R4独立地选自氢、卤素、氰基、C1-C6烷硫基、C1-C6烷基CO-、C1-C6烷基SO2-、氨基、-NH(C1-C6烷基)、-N(C1-C6烷基)(C1-C6烷基)、-SO2NH2、-C(O)NH2、C1-C6烷基、C1-C6烷氧基、卤代C1-C6烷基、卤代C1-C6烷氧基或卤代C1-C6烷硫基；
    m为0、1、2、3或4；
    Z1-Z2选自O-Z2、NH-Z2、S-Z2、S(O)-Z2、S(O)2-Z2、O-(C1-C6亚烷基)-Z2、O-(卤代C1-C6亚烷基)-Z2、NH-(C1-C6亚烷基)-Z2或NH-(卤代C1-C6亚烷基)-Z2；
    Z2、Z3独立地选自CH或N；
    n为1、2或3；
    R5为C1-C6烷基，任选被选自下组的一个或多个取代基取代：卤素、氰基、羟基、羧基、氨基、甲氧基或-SO2CH3；
    前提是，X为S或S(O)2时，环A选自：C3-C6环烷基、5-8元杂环基、C6-C10芳基或5-8元杂芳基；X为O时，环A选自：C3-C6环烷基、5-8元杂环基或5-8元杂芳基。
  2. 如权利要求1所述的通式(I)所示的化合物，或其外消旋体、对映异构体、非对映异构体、或其混合物形式、或可药用盐，其特征在于，所述通式(I)所示的化合物为通式(Ia)所示的化合物：
    R1、R2、R3、o、R4、m、Z1、Z2、n、Z3、R5的定义如权利要求1所述。
  3. 如权利要求1所述的通式(I)所示的化合物或可药用盐，其特征在于，所述通式(I)所示的化合物为通式(Ib)所示的化合物：
    其中，R1、R2、R3、o、R4、m、Z1、Z2、n、Z3、R5的定义如权利要求1所述。
  4. 如权利要求1所述的通式(I)所示的化合物，或其外消旋体、对映异构体、非对映异构体、或其混合物形式、或可药用盐，其特征在于，
    各R3独立地选自氢、卤素、氰基、C1-C4烷硫基、C1-C4烷基CO-、C1-C4烷基SO2-、氨基、-NH(C1-C4烷基)、-N(C1-C4烷基)(C1-C4烷基)、-SO2NH2、-C(O)NH2、C1-C4烷基、C1-C4烷氧基、卤代C1-C4烷基、卤代C1-C4烷氧基或卤代C1-C4烷硫基；
    o为0、1、2、3或4；
    X为S或S(O)2时，环A选自：C3-C6环烷基、5-7元杂环基、C6-C10芳基或5-7元杂芳基；X为O时，环A选自：C3-C6环烷基、5-7元杂环基或5-7元杂芳基。
  5. 如权利要求1所述的通式(I)所示的化合物，或其外消旋体、对映异构体、非对映异构体、或其混合物形式、或可药用盐，其特征在于，
    Z1-Z2选自O-Z2、NH-Z2、S-Z2、S(O)-Z2、S(O)2-Z2、O-(C1-C4亚烷基)-Z2、O-(卤代C1-C4亚烷基)-Z2、NH-(C1-C4亚烷基)-Z2或NH-(卤代C1-C4亚烷基)-Z2；
    Z2、Z3独立地选自CH或N；
    n为1、2或3；
    R5为C1-C4烷基，任选被选自下组的一个或多个取代基取代：氟、氯、溴、氰基、羟基、羧基、氨基、甲氧基或-SO2CH3。
  6. 如权利要求1所述的通式(I)所示的化合物，或其外消旋体、对映异构体、非对映异构体、或其混合物形式、或可药用盐，其特征在于，R1选自OH、COOH、B(OH)2，R2选自H；或者R1、R2与相连的苯环形成
    X选自S或S(O)2；环A选自：C3-C6环烷基、5-6元杂环基、苯基或5-6元杂芳基；
    各R3独立地选自氢、氟、氯、溴、氰基、C1-C4烷硫基、C1-C2烷基CO-、C1-C2烷基SO2-、氨基、-NH(C1-C2烷基)、-N(C1-C2烷基)(C1-C2烷基)、-SO2NH2、-C(O)NH2、C1-C4烷基、C1-C4烷氧基、卤代C1-C4烷基、卤代C1-C4烷氧基或卤代C1-C4烷硫基；
    o为0、1、2、3或4；
    Y1、Y2独立地选自CR4或N；
    各R4独立地选自氢、氟、氯、溴、氰基、C1-C2烷硫基、C1-C2烷基CO-、C1-C2烷基SO2-、氨基、-NH(C1-C2烷基)、-N(C1-C2烷基)(C1-C2烷基)、-SO2NH2、-C(O)NH2、C1-C4烷基、C1-C4烷氧基、卤代C1-C4烷基、卤代C1-C4烷氧基或卤代C1-C4烷硫基；
    m为0、1、2或3；
    Z1-Z2选自O-Z2、NH-Z2、O-(C1-C4亚烷基)-Z2、O-(卤代C1-C4亚烷基)-Z2、NH-(C1-C4亚烷基)-Z2或NH-(卤代C1-C4亚烷基)-Z2；
    Z2、Z3独立地选自CH或N；
    n为1或2；
    R5为C1-C4烷基，任选被选自下组的一个或多个取代基取代：氟、氯、溴、氰基、羟基或羧基。
  7. 如权利要求1所述的通式(I)所示的化合物，或其外消旋体、对映异构体、非对映异构体、或其混合物形式、或可药用盐，其特征在于，选自下组：

  8. 如权利要求1所述的通式(I)所示的化合物的制备方法，其特征在于，R1为OH，R2为氢，Z1为OCH2CH2，Z2为N，所述制备方法包括以下步骤：
    (i1)A3与有机硼试剂发生Suziki偶联反应得到A4，其中，所述的有机硼试剂选自：硼酸硼酸频那醇酯
    (i2)A4与溴代试剂发生溴代反应得到烯基溴代物A5，其中，所述的溴代试剂选自：三溴化吡啶鎓、N-溴代丁二酰亚胺；
    (i3)A5与有机硼试剂发生Suziki偶联反应得到A6，其中，所述的有机硼试剂选自：硼酸硼酸频那醇酯
    (i4)A6在钯催化剂和氢气的作用下发生氢化和氢解反应得到A7，其中，所述的钯催化剂选自Pd/C、Pd(OH)2/C；
    (i5)A7经亲核取代反应得到A8；
    (i6)A8在碱性条件下发生亲核取代反应及水解得到式(I)化合物，所述的碱选自三乙胺、N，N-二异丙基乙胺、吡啶、碳酸盐、NaH、氢氧化钠、氢氧化钾、氢氧化锂、甲醇钠、乙醇钠；
    X、R4、Y2、Y1、环A、R3、o、m、Z3、R5的定义同前所述；
    或者所述制备方法包括以下步骤：
    其中，A7与反应得到式(I)化合物，
    LG为离去基团，选自Br、Cl、OTf、OTs或OMs；
    X、R4、Y2、Y1、环A、R3、o、m、Z3、R5的定义同前所述；
    或者R1为-COOH，R2为氢，所述制备方法包括以下步骤：
    (ii1)B4与溴代试剂发生溴代反应得到烯基溴代物B5，其中，所述的溴代试剂选自：三溴化吡啶鎓、N-溴代丁二酰亚胺；
    (ii2)B5与有机硼试剂发生Suziki偶联反应得到B6，其中，所述的有机硼试剂选自：硼酸硼酸频那醇酯
    (ii3)B6在钯催化剂和氢气的作用下发生氢化和氢解反应得到B7，其中，所述的钯催化剂选自Pd/C、Pd(OH)2/C；
    (ii4)B7经亲核取代反应得到B8；
    (ii5)B8在碱性条件下发生亲核取代反应及水解得到式(I)化合物，所述的碱选自三乙胺、N，N-二异丙基乙胺、吡啶、碳酸盐、NaH、氢氧化钠、氢氧化钾、氢氧化锂、甲醇钠、乙醇钠；
    X、R4、Y2、Y1、环A、R3、o、m、Z3、R5的定义同前所述，R6为C1-C6烷基；
    或者R1、R2与相连的苯环形成时，所述制备方法包括以下步骤：
    (iii1)C1与3-巯基丙酸甲酯在钯催化剂的作用下发生C-S偶联得到C2，其中，所述的钯催化剂选自[1，1′-双(二苯基膦)二茂铁]二氯化钯、四(三苯基膦)钯、三(二亚苄基丙酮)二钯、双(二亚苄基丙酮)钯、双三苯基磷二氯化钯、二(三叔丁基膦)钯、双(三环己基膦)钯、醋酸钯；
    (iii2)C2在酸性条件下水解得到C3，所述的酸选自硫酸、盐酸、磷酸、甲磺酸、三氟乙酸、乙酸、三氟甲磺酸；
    (iii3)C3在酸作用下发生傅克反应得到C4，所述酸选自：三氟甲磺酸、三氟乙酸、Eaton试剂、多聚磷酸、硫酸、盐酸；
    (iii4)C4与二氢吡喃在酸作用件下反应得到C5，所述酸包括：对甲苯磺酸、甲磺酸、对甲苯磺酸吡啶盐；
    (iii5)C5与对甲苯磺酰肼反应得到腙C6；
    (iii6)C6与芳基溴代物在钯催化剂的作用下反应得到C7，其中，所述的钯催化剂选自[1，1′-双(二苯基膦)二茂铁]二氯化钯、四(三苯基膦)钯、三(二亚苄基丙酮)二钯、双(二亚苄基丙酮)钯、双三苯基磷二氯化钯、二(三叔丁基膦)钯、双(三环己基膦)钯、醋酸钯；
    (iii7)C7与溴代试剂发生溴代反应得到烯基溴代物C8，其中，所述的溴代试剂选自：三溴化吡啶鎓、N-溴代丁二酰亚胺；
    (iii8)C8与有机硼试剂发生Suziki偶联反应得到C9，其中，所述的有机硼试剂选自： 硼酸硼酸频那醇酯
    (iii9)C9在钯催化剂和氢气的作用下发生氢化和氢解反应得到C10，其中，所述的钯催化剂选自Pd/C、Pd(OH)2/C；
    (iii10)C10经亲核取代反应得到C11；
    (iii11)C11经亲核取代反应及在酸作用下水解得到式(I)化合物，所述酸选自：三氟甲磺酸、三氟乙酸、硫酸、盐酸、对甲苯磺酸、氯化氢的有机溶剂(甲醇、乙醇、异丙醇、乙酸乙酯、乙醚或1，4-二氧六环)溶液；
    X、R4、Y2、Y1、环A、R3、o、m、Z3、R5的定义同前所述。
  9. 一种药物组合物，其特征在于，包含：
    如权利要求1所述的通式(I)所示的化合物，或其外消旋体、对映异构体、非对映异构体、或其混合物形式、或可药用盐；和药学上可接受的载体。
  10. 如权利要求1所述的通式(I)所示的化合物，或其外消旋体、对映异构体、非对映异构体、或其混合物形式、或可药用盐或者权利要求9所述的药物组合物的用途，其特征在于，用于制备治疗、预防或诊断雌激素受体相关的疾病的药物，较佳地，用于制备治疗、预防或诊断乳腺癌、子宫内膜癌、宫颈癌、皮肤癌、前列腺癌、卵巢癌、输卵管肿瘤、肺癌、白血病、骨质疏松症、神经退行性疾病、心血管疾病、红斑狼疮、子宫内膜异位症及肥胖症的药物。
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