WO2021228210A1 - 吡咯烷类化合物及其应用 - Google Patents

吡咯烷类化合物及其应用 Download PDF

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WO2021228210A1
WO2021228210A1 PCT/CN2021/093736 CN2021093736W WO2021228210A1 WO 2021228210 A1 WO2021228210 A1 WO 2021228210A1 CN 2021093736 W CN2021093736 W CN 2021093736W WO 2021228210 A1 WO2021228210 A1 WO 2021228210A1
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compound
pharmaceutically acceptable
acceptable salt
formula
tumor
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PCT/CN2021/093736
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English (en)
French (fr)
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古鹏
刘磊
张国宝
周峰
唐任宏
任晋生
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江苏先声药业有限公司
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Priority to JP2023513900A priority Critical patent/JP2023526568A/ja
Priority to CA3178707A priority patent/CA3178707A1/en
Priority to AU2021269612A priority patent/AU2021269612A1/en
Priority to US17/925,102 priority patent/US20230348459A1/en
Priority to CN202180035358.6A priority patent/CN115836068A/zh
Priority to EP21804022.8A priority patent/EP4151636A4/en
Publication of WO2021228210A1 publication Critical patent/WO2021228210A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Definitions

  • the present invention relates to a novel pyrrolidine compound or a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing them, and a selective estrogen receptor degrading agent (Selective Estrogen Receptor Degrader or Downregulator, SERD) related to prevention or treatment Use in disease.
  • a selective estrogen receptor degrading agent Selective Estrogen Receptor Degrader or Downregulator, SERD
  • Estrogen (E2) and estrogen alpha receptor (ER ⁇ ) are important driving factors for the development of breast cancer. More than two-thirds of breast cancer patients express ER transcription factors, and in most ER-positive patients, ER is still a key driving factor even in tumors that progress after early endocrine therapy. Therefore, ER is A major target for breast cancer treatment (Pharmacology & Therapeutics 186 (2016) 1-24).
  • the purpose of endocrine therapy is to reduce ER activity.
  • SERMs selective estrogen receptor modulators
  • tamoxifen which is an allosteric modulator of ER, which inhibits its transcriptional activity after binding to ER.
  • AIs Aromatase inhibitors
  • fulvestrant selective estrogen receptor down-regulators
  • the antagonist inhibits its activity and also has the effect of inducing ER protein degradation.
  • endocrine therapy is the first choice for patients with estrogen receptor-positive breast cancer, about 30% of patients will relapse after treatment, and almost all patients with metastatic breast cancer will develop resistance and progress.
  • One is focused on the estrogen receptor signaling pathway itself, including the activation mutation, amplification, and fusion with other genes of the gene encoding the estrogen receptor, estrogen receptor.
  • Co-regulators and downstream control of cell cycle factors, etc.; another type of mechanism includes the activation of signal pathways that cross-react with the estrogen receptor signaling pathway, such as the growth factor receptor pathway (Oncol Ther, 2017, 5:17) –29).
  • Fulvestrant is the first and only SERD drug clinically approved for the treatment of postmenopausal patients with ER-positive, metastatic breast cancer after the progression of tamoxifen or aromatase inhibitors.
  • AstraZeneca see patent application WO2018077630A1
  • Genentech see patent application WO2019245974A1
  • Data from a number of studies have shown that patients treated with Fulvestrant have not fully achieved ER degradation.
  • intramuscular injection has caused obvious reactions such as pain, swelling, and redness at the injection site, as well as slow absorption and limited exposure in the body.
  • the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof:
  • R 1 , R 2 , R 3 , R 4 are independently selected from H, F, Cl, Br, I, CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy or C 3 -C 6 cycloalkane base;
  • X 1 , X 2 , X 3 , and X 4 are independently selected from CR 6 or N;
  • R 6 is selected from H, F, Cl, Br, I, OH, CN, C 1 -C 10 alkyl, C 3 -C 10 cycloalkyl, 3-10 membered heterocyclic group, C 1 -C 10 alkoxy Group, C 3 -C 10 cycloalkyloxy or 3-10 membered heterocyclyloxy;
  • Y is selected from O or NH
  • R 5 is independently selected from C 1 -C 6 alkyl, said C 1 -C 6 alkyl optionally substituted with R a;
  • R a is selected from F, Cl, Br, I, OH, CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy or C 3 -C 6 cycloalkyl;
  • the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the compound represented by formula (II) or a pharmaceutically acceptable salt thereof:
  • the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the compound represented by formula (III) or a pharmaceutically acceptable salt thereof:
  • R 1 , R 2 , R 3 , R 4 are independently selected from H, F, Cl, Br, I, CN, or C 1 -C 6 alkyl.
  • R 1 , R 2 , R 3 , R 4 are independently selected from H, F, Cl, Br, I, CN, or C 1 -C 3 alkyl.
  • R 1 , R 2 , R 3 , R 4 are independently selected from H, F, Cl, Br, I, CN, or methyl.
  • R 1 , R 2 , R 3 , R 4 are independently selected from H, F, Cl, Br, I, or C 1 -C 3 alkyl.
  • R 1 , R 2 , R 3 , R 4 are independently selected from H, F, Cl, Br, I, or methyl.
  • R 1 , R 2 , R 3 , and R 4 are independently selected from H, F, or methyl.
  • R 1 and R 2 are independently selected from H, F, or methyl.
  • R 3 and R 4 are independently selected from H or methyl.
  • R 3 and R 4 are independently selected from H.
  • X 1 , X 2 , X 3 , X 4 are independently selected from CR 6 or N, and at least 2 of X 1 , X 2 , X 3 , and X 4 groups are selected from CR 6 .
  • X 1 , X 2 , X 3 , X 4 are independently selected from CR 6 or N, and at least 3 of X 1 , X 2 , X 3 , and X 4 groups are selected from CR 6 .
  • the R 6 is selected from H, F, Cl, Br, I, CN, C 1 -C 3 alkyl or C 1 -C 3 alkoxy.
  • the R 6 is selected from H, F, Cl, Br, I, CN, or C 1 -C 3 alkoxy.
  • the R 6 is selected from H, F, Cl, Br, I, CN, or methoxy.
  • the R 6 is selected from H, F, Cl, Br, I, or CN.
  • the R 6 is selected from H, F, Cl, Br, or I.
  • the R 6 is selected from H or F.
  • R 5 is selected from C 1 -C 3 alkyl, said C 1 -C 3 alkyl optionally substituted with R a.
  • the R a is selected from F, Cl, Br, I, OH or CN.
  • the R a is selected from F, OH or CN.
  • the R a is selected from F or OH.
  • R 5 is selected from CH 2 CF 3 , CH 2 CHF 2 , CH 2 CF 2 CH 2 OH, or CH 2 CF 2 CH 2 CN.
  • R 5 is selected from CH 2 CF 3 or CH 2 CF 2 CH 2 OH.
  • Y is selected from NH.
  • the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:
  • the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:
  • the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:
  • the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:
  • the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:
  • the present invention also provides a pharmaceutical composition, which comprises a compound represented by formula (I) or a pharmaceutically acceptable salt thereof and pharmaceutically acceptable auxiliary materials.
  • the present invention relates to the use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in the preparation of a medicine for preventing or treating estrogen receptor-related diseases.
  • the present invention relates to a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for use in the prevention or treatment of estrogen receptor related diseases.
  • the present invention relates to the use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in the prevention or treatment of estrogen receptor related diseases.
  • the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for preventing or treating estrogen receptor-related diseases.
  • the present invention also relates to a method for treating estrogen receptor-related diseases, which comprises administering to a patient a therapeutically effective dose of a pharmaceutical preparation containing the compound of formula (I) of the present invention or a pharmaceutically acceptable salt thereof.
  • the estrogen receptor related diseases include but are not limited to tumors.
  • the estrogen receptor related disease is breast cancer.
  • the estrogen receptor related disease is ER-positive breast cancer.
  • the estrogen receptor related disease is brain metastasis of ER-positive breast cancer.
  • the patient is a breast cancer patient.
  • the patient is an ER-positive breast cancer patient.
  • the patient is an ER-positive breast cancer brain metastasis patient.
  • the compound of the present invention has good anti-tumor activity in vivo and in vitro and drug-making properties. In vivo experiments have found that the compound of the present invention can significantly inhibit tumor growth in ER-positive breast cancer mouse model mice, and significantly improve the survival time of ER-positive breast cancer brain orthotopic model mice. In addition, the compound of the present invention has high bioavailability, strong ER degradation ability, can be taken orally, and has high ability to pass through the blood-brain barrier, and has the potential to effectively treat ER-positive breast cancer (especially brain metastasis of ER-positive breast cancer).
  • pharmaceutically acceptable salts refers to pharmaceutically acceptable salts of non-toxic acids or bases, including salts of inorganic acids and bases, and organic acids and bases.
  • stereoisomers refers to isomers produced by the different arrangements of atoms in the molecule in space, including cis-trans isomers, enantiomers and diastereomers.
  • the compound of the present invention may have an asymmetric atom such as a carbon atom, a sulfur atom, a nitrogen atom, a phosphorus atom (optical center), or an asymmetric double bond. Racemates, enantiomers, diastereomers, and geometric isomers are all included in the scope of the present invention.
  • the compounds of the present invention may exist in specific geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers Conformers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as enantiomers or diastereomer-enriched mixtures, all of these mixtures belong to Within the scope of the present invention.
  • Additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms, or asymmetric phosphorus atoms may be present in substituents such as alkyl groups.
  • the asymmetric atom-containing compound of the present application can be isolated in an optically pure form or a racemic form.
  • the optically active pure form can be resolved from the racemic mixture or synthesized by using chiral raw materials or chiral reagents.
  • stereoisomers include, but are not limited to:
  • tautomer refers to an isomer of a functional group resulting from the rapid movement of an atom in a molecule at two positions.
  • the compounds of the present invention may exhibit tautomerism.
  • Tautomeric compounds can exist in two or more mutually convertible species.
  • Tautomers generally exist in an equilibrium form, and an attempt to separate a single tautomer usually produces a mixture whose physical and chemical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties of the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the ketone type is dominant; in phenol, the enol type is dominant.
  • the present invention encompasses all tautomeric forms of the compound.
  • pharmaceutical composition means a mixture of one or more of the compounds described in the text or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically/pharmaceutically acceptable excipients .
  • the purpose of the pharmaceutical composition is to facilitate the administration of the compound to the organism.
  • substituted means that any one or more hydrogen atoms on a specific atom are replaced by a substituent, as long as the valence of the specific atom is normal and the substituted compound is stable.
  • it means that two hydrogen atoms are replaced, and the oxo will not occur on the aromatic group.
  • the term “optional” or “optionally” means that the event or situation described later can occur or not occur, and the description includes occurrence of said event or situation and non-occurrence of said event or situation.
  • the ethyl group is "optionally" substituted by halogen, meaning that the ethyl group can be unsubstituted (CH 2 CH 3 ), monosubstituted (such as CH 2 CH 2 F), or polysubstituted (such as CHFCH 2 F, CH 2 CHF 2 etc.) or completely substituted (CF 2 CF 3 ).
  • CH 2 CH 3 unsubstituted
  • monosubstituted such as CH 2 CH 2 F
  • polysubstituted such as CHFCH 2 F, CH 2 CHF 2 etc.
  • CF 2 CF 3 completely substituted
  • any variable e.g., R a, R b
  • its definition on each occurrence is independent. For example, if a group is replaced by 2 R b , then each R b has independent options.
  • linking group When the number of a linking group is 0, such as -(CH 2 ) 0 -, it means that the linking group is a bond.
  • linking group involved in this article does not indicate its linking direction
  • its linking direction is arbitrary.
  • L 1 in “C 1 -C 3 alkylene-O" is selected from “C 1 -C 3 alkylene-O”
  • L 1 can connect ring Q and R 1 in the same direction as the reading order from left to right to form “ring QC 1 -C 3 alkylene-OR 1 "
  • rings Q and R 1 in the opposite direction of the reading order from left to right to form “ring QOC 1 -C 3 alkylene-R 1 ".
  • halo or halogen refers to fluorine, chlorine, bromine and iodine.
  • C m -C n in this context means having an integer number of carbon atoms in the range of mn.
  • C 1 -C 10 means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, or 10 carbon atoms.
  • alkyl refers to a hydrocarbon group of the general formula C n H 2n+1 , and the alkyl group may be linear or branched.
  • C 1 -C 10 alkyl should be understood to mean a linear or branched saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.
  • the alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl Group, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, etc.; preferably, "C 1 -C 10 "Alkyl" may include "C 1 -C 6 alkyl
  • C 1 -C 10 alkoxy can be understood as “C 1 -C 10 alkyloxy” or “C 1 -C 10 alkyl-O-", preferably, "C 1 -C 10 alkoxy”
  • the radical may include "C 1 -C 6 alkoxy” or "C 1 -C 3 alkoxy”.
  • C 3 -C 10 cycloalkyl should be understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring, which has 3 to 10 carbon atoms. Such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl, or a bicyclic hydrocarbon group such as decalin ring.
  • C 3 -C 6 cycloalkyl should be understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring, which has 3 to 6 carbon atoms.
  • C 3 -C 10 cycloalkyloxy can be understood as “C 3 -C 10 cycloalkyl-O-", preferably, "C 3 -C 10 cycloalkyloxy” may include "C 3 -C 6 cycloalkyloxy”.
  • 3--10 membered heterocyclic group means a heterocyclic group having 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms, and its ring atoms contain 1-5 independently selected from the above The heteroatom or heteroatom group.
  • 3-membered heterocyclic groups include but are not limited to glycidyl or azetidinyl
  • 4-membered heterocyclic groups include, but are not limited to, azetidinyl and oxetanyl
  • Examples of 5-membered heterocyclic groups include, but are not limited to, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydrooxazole or 2, 5-Dihydro-1H-pyrrolyl
  • 6-membered heterocyclic groups include, but are not limited to, tetrahydropyranyl, piperidinyl,
  • the heterocyclic group may be a benzo-fused ring group of the aforementioned 4-7 membered heterocyclic group, and examples include, but are not limited to, dihydroisoquinolinyl and the like.
  • the 3-10 membered heterocyclic group may be a "3-10 membered heterocycloalkyl"; the “3-10 membered heterocycloalkyl” may further include a "3-7 membered heterocycloalkyl” or In the scope of "5-6 membered heterocycloalkyl", although some bicyclic heterocyclic groups in this application partially contain a benzene ring or a heteroaromatic ring, the heterocyclic group is still non-aromatic as a whole.
  • treatment means administering the compound or formulation described in this application to prevent, ameliorate or eliminate a disease or one or more symptoms associated with the disease, and includes:
  • terapéuticaally effective amount means (i) treatment or prevention of a particular disease, condition or disorder, (ii) reduction, amelioration or elimination of one or more symptoms of a particular disease, condition or disorder, or (iii) prevention or delay
  • the amount of the compound of the present invention that constitutes a “therapeutically effective amount” varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but it can be routinely determined by those skilled in the art. Determined by its own knowledge and this disclosure.
  • excipients refers to pharmaceutically acceptable inert ingredients.
  • examples of types of the term “excipient” include, without limitation, binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, and the like. Excipients can enhance the handling characteristics of the pharmaceutical preparation, that is, make the preparation more suitable for direct compression by increasing fluidity and/or adhesion.
  • examples of typical "pharmaceutically acceptable carriers” suitable for the above formulations are: sugars, starches, cellulose and its derivatives and other auxiliary materials commonly used in pharmaceutical formulations.
  • pharmaceutically acceptable excipients refers to those excipients that have no obvious stimulating effect on the organism and will not damage the biological activity and performance of the active compound.
  • Suitable auxiliary materials are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like.
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and those well known to those skilled in the art Equivalent alternatives, preferred implementations include but are not limited to the embodiments of the present invention.
  • the present application also includes compounds of the present application that are the same as those described herein, but have one or more atoms replaced by an isotope-labeled atom having an atomic weight or mass number different from the atomic weight or mass number commonly found in nature.
  • isotopes that can be incorporated into the compounds of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.
  • isotope-labeled compounds of the application can be used in compound and/or substrate tissue distribution analysis. Tritiated (ie 3 H) and carbon-14 (ie 14 C) isotopes are especially preferred due to their ease of preparation and detectability. Positron emission isotopes such as 15 O, 13 N, 11 C, and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy.
  • PET positron emission tomography
  • the isotopically-labeled compounds of the present application can be prepared by the following procedures similar to those disclosed in the schemes and/or examples below, by replacing non-isotopically-labeled reagents with isotope-labeled reagents.
  • substitution with heavier isotopes can provide certain therapeutic advantages resulting from higher metabolic stability (for example, increased in vivo half-life or reduced dosage requirements), and therefore in certain situations
  • deuterium substitution can be partial or complete, and partial deuterium substitution refers to the substitution of at least one hydrogen by at least one deuterium.
  • the pharmaceutical composition of the present application can be prepared by combining the compound of the present application with suitable pharmaceutically acceptable excipients, for example, can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, and powders. , Granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.
  • Typical routes for administering the compound of the present application or a pharmaceutically acceptable salt or pharmaceutical composition thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, Intramuscular, subcutaneous, and intravenous administration.
  • the pharmaceutical composition of the present application can be manufactured by methods well known in the art, such as conventional mixing method, dissolution method, granulation method, sugar-coated pill method, grinding method, emulsification method, freeze-drying method, etc.
  • the pharmaceutical composition is in an oral form.
  • the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These auxiliary materials enable the compound of the present application to be formulated into tablets, pills, lozenges, sugar-coated agents, capsules, liquids, gels, slurries, suspensions, etc., for oral administration to patients.
  • the solid oral composition can be prepared by conventional mixing, filling or tableting methods. For example, it can be obtained by the following method: mixing the active compound with solid excipients, optionally grinding the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into granules to obtain tablets Or the core of the dragee.
  • suitable excipients include but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, and the like.
  • the pharmaceutical composition may also be suitable for parenteral administration, such as a sterile solution, suspension or lyophilized product in a suitable unit dosage form.
  • the daily dose is 0.01 to 100 mg/kg body weight, preferably 0.05 to 50 mg/kg body weight, more preferably 0.1 to 30 mg/kg body weight, in single or divided doses form.
  • Figure 1 is the NOESY spectrum of compound 3
  • Figure 2 is a graph showing the tumor growth curve of the MCF-7 subcutaneous tumor model in Test Example 9;
  • Figure 3 is a graph showing changes in the body weight of the MCF-7 subcutaneous tumor model animal in Test Example 9;
  • Figure 4 is the survival curve of the MCF-7 brain orthotopic model mouse in Test Example 10;
  • Fig. 5 is a graph showing changes in body weight of MCF-7 brain orthotopic model mice in Test Example 10.
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS).
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • the unit of NMR shift is 10 -6 (ppm).
  • the solvents measured by NMR are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is tetramethylsilane (TMS);
  • TMS tetramethylsilane
  • IC 50 refers to the half inhibitory concentration, which refers to the half of the maximum inhibitory effect concentration.
  • Step 1 Synthesis of tert-butyl (1-(3-fluoropropyl)pyrrolidin-3-yl) carbamate
  • Step 3 Synthesis of (R)-1-(1H-indol-3-yl)-N-(2,2,2-trifluoroethyl)propane-2-amine
  • Step 5 N-(1-(3-Fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl) ) Synthesis of -2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-amine
  • reaction solution was stirred at 80°C for 4 hours. After the reaction is detected by LCMS, it is cooled to room temperature and filtered. After the filtrate is concentrated under reduced pressure, it is purified by preparative liquid chromatography (Phenomenex Gemini C18 column, 3um silica, 30mm diameter, 75mm length); (using water (containing 0.225% formic acid) ) And acetonitrile as the eluent) was purified to obtain the compound N-(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl -2-(2,2,2-Trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-amine (22.23mg).
  • Example 2 N-((R)(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2 -Trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-amine (Compound 2) Synthesis and Examples 3: N-((S)(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-tri Synthesis of fluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-amine (compound 3)
  • Step 1 Synthesis of (S)-tert-butyl(1-(3-fluoropropyl)pyrrolidin-3-yl)carbamate
  • Step 3 N-((S)-1-(3-fluoropropyl)pyrrolidin-3-yl)-6-(((1S,3R)-3-methyl-2-(2,2,2 -Trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridine-3-amine
  • Step 1 Synthesis of (S)-tert-butyl(1-(3-fluoropropyl)pyrrolidin-3-yl)carbamate
  • Step 3 (1S,3R)-1-(4-bromo-2,6-difluorophenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3, Synthesis of 4,9-Tetrahydro-1H-pyrido[3,4-b]indole
  • Step 4 (S)-N-(3,5-Difluoro-4-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3, Synthesis of 4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-1-(3-fluoropropyl)pyrrolidin-3-amine
  • Step 1 Synthesis of tert-butyl (trans-4-fluoro-1-(3-fluoropropyl)pyrrolidin-3-yl) carbamate
  • Step 3 Trans-N-(3,5-Difluoro-4-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4 Synthesis of ,9-Tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-4-fluoro-1-(3-fluoropropyl)pyrrolidin-3-amine
  • reaction solution was cooled to room temperature, poured into water (10 mL) and stirred for 10 minutes, extracted with ethyl acetate (20 mL) twice, the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure.
  • Step 1 Synthesis of tert-butyl N-[trans-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-yl]carbamate
  • Step 3 Trans-N-[3,5-Difluoro-4-[(1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-1,3,4 Synthesis of ,9-Tetrahydropyrido[3,4-b]indol-1-yl]phenyl]-1-(3-fluoropropyl)-4-methyl-pyrrolidine-3-amine
  • trans-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-amine hydrochloride (30mg, 128.67 ⁇ mol), (1S,3R)-1-(4-bromo-2, 6-Difluoro-phenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-1,3,4,9-tetrahydropyrido[3,4-b]indole (76.82mg, 167.27 ⁇ mol) and cesium carbonate (167.69mg, 514.68 ⁇ mol) were dissolved in dioxane (8mL), and then tBuBrettphos-Pd-G3 (5.50mg, 6.43 ⁇ mol) was added.
  • Example 7 3-((1S,3R)-1-(2,6-difluoro-4-(((S)-1-(3-fluoropropyl)pyrrolidin-3-yl)amino)benzene Yl)-3-methyl-3,4-dihydro-1H-pyrido[3,4-b]indole-2(9H)-yl)-2,2-difluoropropane-1-ol
  • Step 1 Synthesis of 3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl triflate
  • Step 2 (R)-N-(1-(1H-indol-3-yl)prop-2-yl)-3-((tert-butyldiphenylsilyl)oxy)-2,2 -Synthesis of difluoropropan-1-amine
  • Step 3 (1S,3R)-1-(4-bromo-2,6-difluorophenyl)-2-(3-((tert-butyldiphenylsilyl)oxy)-2, Synthesis of 2-difluoropropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole
  • Step 4 (S)-N-(4-((1S,3R)-2-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl) -3-Methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3,5-difluorophenyl)-1-(3 -Fluoropropyl) pyrrolidin-3-amine synthesis
  • reaction solution was stirred and reacted at 80°C for 16 hours. After the completion of the reaction monitored by LCMS, the reaction solution was concentrated to dryness under reduced pressure.
  • the reaction concentrate was diluted with ethyl acetate (10 mL), and then washed three times with water (10 mL). The collected aqueous phase was extracted three times with ethyl acetate (20 mL). The organic phase was dried over sodium sulfate and concentrated under reduced pressure.
  • Step 5 3-((1S,3R)-1-(2,6-difluoro-4-(((S)-1-(3-fluoropropyl)pyrrolidin-3-yl)amino)phenyl )-3-Methyl-3,4-dihydro-1H-pyrido[3,4-b]indole-2(9H)-yl)-2,2-difluoropropane-1-ol
  • reaction solution was stirred and reacted at 25°C for 5 hours.
  • Test Example 1 Detection of the degradation effect of the compound of the present invention on the estrogen receptor in MCF7 cells
  • the purpose of this experiment is to determine the degradation activity of the compound of the present invention on the endogenously expressed estrogen receptor in MCF7 cells, and to evaluate the activity of the compound based on the DC 50 and the maximum degradation efficiency.
  • MCF7 cells (ATCC, HTB-22) were cultured in DMEM (Gibco, 11995-065) complete medium containing 10% fetal bovine serum. On the first day of the experiment, MCF7 cells were seeded in a 384-well plate at a density of 3000 cells/well using a complete medium, and cultured in a cell incubator at 37°C and 5% CO 2. The compounds to be tested were dissolved in DMSO at a storage concentration of 10mM, diluted with Echo 550 (Labcyte Inc.) and added to the cell culture plate.
  • the initial concentration of each compound treatment was 100nM, with a 3-fold dilution, 9 concentration points, and setting containing A blank control of 0.5% DMSO, and a double-well control for each concentration point. Incubate for 24 hours in a 37°C, 5% CO 2 cell incubator. Add paraformaldehyde to each cell culture well. The final concentration of paraformaldehyde is about 3.7% to fix the cells.
  • Test Example 2 Detection of the inhibitory effect of the compound of the present invention on the proliferation of MCF7 cells
  • MCF7 cells (ATCC, HTB-22) were cultured in DMEM (Gibco, 11995-065) complete medium containing 10% fetal bovine serum. On the first day of the experiment, MCF7 cells were seeded in a 384-well plate at a density of 500 cells/well using complete medium, and cultured overnight in a 37°C, 5% CO 2 cell incubator. The next day, add the test compound for drug treatment, use Echo550 (Labcyte Inc.) to dilute the compound solution with a storage concentration of 10mM and transfer it to each cell culture well.
  • Echo550 (Labcyte Inc.)
  • the initial concentration of each compound in the cell is 100nM , 3-fold gradient dilution, 9 concentration points, set a blank control containing 0.3% DMSO, and set a double-well control for each concentration point.
  • 37°C, 5% CO 2 cell incubator was cultured for 7 days, and on the eighth day, the cell culture plate was taken out.
  • join in Luminescent Cell Viability Assay Promega, G7573
  • use XLfit to calculate the inhibitory activity IC 50 of each compound based on the compound concentration and luminescence signal value .
  • Test Example 3 The inhibitory effect of the compound of the present invention on the enzyme activity of CYP2C9 and CYP2D6
  • the inhibition of CYP2C9 and CYP2D6 enzyme activity by the compounds of the present invention was determined by the following test method.
  • IC 50 values of the compounds of the present invention for CYP2C9 and CYP2D6 are calculated by Excel XLfit 5.3.1.3.
  • Test Example 4 Determination of the plasma protein binding rate of the compound of the present invention
  • Human plasma protein binding is a key factor that controls the amount of free (unbound) drug that can be used to bind to the target, and plays an important role in the observed in vivo efficacy of the drug. Therefore, for compounds with similar potency and exposure levels, compounds with high free fractions (low levels of plasma protein binding) can exhibit enhanced potency.
  • the protein binding rate of the compound of the present invention in the plasma of 5 species was determined by the following test method.
  • 96-well balanced dialysis plate (HTDialysis LLC, Gales Ferry, CT, HTD96B), balanced dialysis membrane (MWCO 12-14K, #1101);
  • Preparation of buffer solution with a concentration of 100 mM sodium phosphate and 150 mM NaCl prepare an alkaline solution with a concentration of 14.2 g/L Na 2 HPO 4 and 8.77 g/L NaCl with ultra-pure water, and use ultra-pure water to prepare a concentration of An acidic solution of 12.0g/L NaH 2 PO 4 and 8.77g/L NaCl. Titrate the alkaline solution with an acidic solution to a pH of 7.4 to prepare a buffer solution with a concentration of 100 mM sodium phosphate and 150 mM NaCl.
  • Preparation of the dialysis membrane soak the dialysis membrane in ultrapure water for 60 minutes to separate the membrane into two pieces, then soak it in 20% ethanol for 20 minutes, and finally soak it in the buffer for dialysis for 20 minutes.
  • Preparation of plasma Thaw the frozen plasma quickly at room temperature, then centrifuge the plasma at 4°C and 3,220g for 10 minutes to remove clots, and collect the supernatant in a new centrifuge tube. Measure and record the pH of plasma, using plasma with a pH of 7-8.
  • the peak areas of the compound on the buffer side and the plasma side were measured.
  • the formula for calculating the plasma protein binding rate of the compound is as follows:
  • Free rate% (the ratio of compound peak area to internal standard peak area buffer side /compound peak area to internal standard peak area ratio plasma side ) ⁇ 100
  • Table 4 The protein binding rate value of the compound of the present invention in human, dog, rat and mouse plasma
  • Test Example 5 Apparent solubility of the compound of the present invention in a phosphate buffer with a pH of 7.4
  • test compound was prepared according to the recorded method.
  • the reference drug progesterone was purchased from Sigma.
  • Phosphate buffer with a pH of 7.4 is prepared by our laboratory.
  • Acetonitrile and methanol were purchased from Fisher.
  • Other reagents are purchased from the market.
  • DMSO DMSO was used to prepare 10 mM stock solutions of the test substance and the reference drug progesterone.
  • Example number pH 7.4 apparent solubility ( ⁇ M) Example 3 92 Example 4 ⁇ 0.3 Example 7 4
  • Test Example 6 Whether the compound of the present invention has a potential inhibitory effect on the voltage-gated potassium ion channel hERG
  • hERG potassium channels are essential for the normal electrical activity of the heart. Arrhythmia can be induced by blocking hERG channels with a variety of drugs. This side effect is a common cause of drug failure in preclinical safety trials, so the minimization of hERG channel blocking activity may be an ideal feature of candidate drugs.
  • the HEK293 cell line (Cat. No.: K1236) stably expressing the hERG ion channel was purchased from Invitrogen.
  • the cell line was cultured with 85% DMEM, 10% dialysis fetal bovine serum, 0.1mM non-essential amino acid solution, 100U/mL penicillin-streptomycin solution, 25mM HEPES, 5 ⁇ g/mL blasticidin and 400 ⁇ g/mL genetic In the medium of mycin.
  • trypsin is used for digestion and passage, and passage is performed three times a week.
  • the cells were cultured in a 6cm petri dish at a density of 5 ⁇ 10 5 , and 1 ⁇ g/mL doxycycline was added to induce 48 hours, and then the cells were digested and seeded on glass slides for subsequent manual patch clamp experiments.
  • test compound is dissolved in DMSO and prepared into a stock solution with a final concentration of 10 mM.
  • the detection condition of the leakage current is that the clamping voltage is converted to -80mV, and the time course is 500ms.
  • the hERG current test method is as follows: apply a depolarization command voltage for 4.8 seconds to depolarize the membrane potential from -80mV to +30mV, and then apply a repolarization voltage for 5.2 seconds to reduce the membrane potential to -50mV to remove channel loss. So that the hERG tail current can be observed.
  • the peak value of the tail current is the magnitude of the hERG current.
  • the hERG current used to detect the test compound was continuously recorded for 120 seconds before administration to evaluate the stability of the hERG current produced by the test cell. Only stable cells within the acceptance range of the evaluation criteria can enter the subsequent compound testing.
  • the hERG current measured in the extracellular fluid containing 0.1% DMSO is used as the detection baseline. After the hERG current remains stable for at least 5 minutes, the solution containing the test compound is sequentially perfused around the cells from a low concentration to a high concentration. After each perfusion, wait for about 5 minutes to allow the compound to fully act on the cells and simultaneously record the hERG current. After the recording current stabilizes, record the last 5 hERG current values, and take the average value as the final current value at a specific concentration. After testing the compound, add 150nM Dofilide to the same cell to completely inhibit its current, as a positive control for the cell. At the same time, the positive compound Dofilide uses the same patch clamp system for simultaneous detection before and after the end of the test compound experiment to ensure the reliability and sensitivity of the entire detection system.
  • the data is output by PatchMaster software and analyzed according to the following steps:.
  • Example 3 10 Example 4 3.7
  • Test Example 7 Evaluation of the pharmacokinetics of the compound of the present invention in mice
  • CD-1 mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • DMSO dimethyl sulfoxide
  • HP- ⁇ -CD hydroxypropyl- ⁇ -cyclodextrin
  • Tetraethylene Glycol tetraethylene glycol
  • Captisol SBE- ⁇ -CD, sulfobutyl- ⁇ -Cyclodextrin
  • Acetonitrile was purchased from Merck (USA).
  • mice 6 female CD-1 mice (20-30g, 4-6 weeks) were randomly divided into 2 groups, 3 mice in each group.
  • the first group was given the test compound by tail vein injection at a dose of 1 mg/kg with a vehicle of 5% DMSO in 10% HP- ⁇ -CD in water, and the second group was given orally with the test compound at a dose of 10 mg/kg with a vehicle of 40% Tetraethylene Glycol ( v/v), 7.5% Captisol(w/v) in water. Feed and water normally before the animal experiment.
  • Mice in each group were subjected to intravenous blood sampling at 0.083 (intravenous injection group only), 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after administration. The collected whole blood samples were placed in a K 2 EDTA anticoagulation tube, and after centrifugation for 5 min (12,000 rpm, 4° C.), plasma was taken for testing.
  • the PK test results are as follows.
  • the compound of the present invention shows good PK properties and oral bioavailability in mice:
  • Test Example 8 The blood-brain barrier (BBB) penetration ability of the compound of the present invention in rats
  • Drugs can penetrate the animal’s blood-brain barrier and have sufficient exposure in the brain is the key to the drug’s effectiveness on brain metastases. Therefore, by measuring the drug concentration in the plasma and brain tissue of the animal after administration, the drug in the brain can be evaluated. Distribution, and then determine whether the drug can inhibit tumor growth in the brain orthotopic model.
  • mice Six female SD rats (200-300g, 6-8 weeks) were randomly divided into 2 groups with 3 rats in each group.
  • the compounds of the present invention were administered separately, and the solvent was 0.5% methylcellulose aqueous solution.
  • the animals were fed with water normally, fasted overnight, and resumed feeding four hours after the administration.
  • Each group of rats collected plasma and brain tissue 2h after administration.
  • the collected whole blood samples were placed in a K 2 EDTA anticoagulation tube, and centrifuged for 5 min (12,000 rpm, 4° C.) to take plasma for testing; the tissues were collected and blotted dry with filter paper, and the samples were stored in a -80 degree refrigerator for testing.
  • Rat brain tissue samples were first homogenized with 4 times the mass volume of PBS homogenate. Take 20 ⁇ L of brain tissue homogenate sample, add 20 ⁇ L of blank mouse plasma to dilute and mix, then add 600 ⁇ L of acetonitrile solvent (including internal standard compound) to precipitate the protein, vortex for 5min, centrifuge (14,000rpm) for 5min, supernatant containing 0.1%(v/v) FA was diluted 2 times with water, and quantitatively detected in the LC-MS/MS system (AB Sciex Triple Quad 6500+).
  • Example 3 of the compound of the present invention exhibits excellent blood-brain barrier permeability, and drug exposure is relatively high in the brain tissue of rats.
  • the BBB test results are as follows:
  • Test Example 9 Growth inhibition experiment of the compound of the present invention on MCF-7 mouse subcutaneous tumor model
  • Human breast cancer MCF-7 cells ATCC, HTB-22
  • EMEM culture medium ATCC, Cat No.: 30-2003
  • Pancreatin-EDTA Gibco, Cat No.: 25200-072
  • D-PBS calcium-magnesium ion-free phosphate buffer
  • mice Female, 6-7 weeks old, weighing about 19-28 grams, the animals were purchased from Beijing Weitongda Biotechnology Co., Ltd. The mice were kept in an SPF environment, and each cage was sent out separately Wind, all animals have free access to standard certified commercial laboratory food and free drinking water.
  • Cell culture Human breast cancer MCF-7 cell line is cultured in vitro, and the culture conditions are EMEM (cell culture medium) with 10% fetal bovine serum, 1% Pen Strep, 10 ⁇ g/ml recombinant human insulin, 37°C, 5% CO 2 Incubator. Use 0.25% pancreatin-EDTA digestion solution for routine digestion and passage once a week. When the cell saturation is 80%-90% and the number reaches the requirement, the cells are collected and counted.
  • Cell inoculation 0.1ml/(containing 1 ⁇ 10 7 ) MCF-7 cell suspension (D-PBS: Matrigel, volume ratio 1:1) was subcutaneously inoculated on the right back of each mouse, and the cells were inoculated 17 ⁇ -estradiol tablets were inoculated subcutaneously in the first four days. On the 24th day after cell inoculation, drugs were randomly divided into groups according to the tumor volume, and the grouping day was Day 0.
  • the administration dose of Compound Example 1 is 1, 3, or 10 mg/kg, orally administered (PO), once a day (QD) x 3 weeks.
  • the dosage of Compound Example 2 is 10 mg/kg, oral administration (PO), once a day (QD) x 3 weeks.
  • the tumor diameter was measured with vernier calipers twice a week.
  • the body weight of the mice was measured twice a week.
  • TGI tumor growth inhibition rate
  • the compound Example 3 of the present invention was orally administered at 1 mg/kg, 3 mg/kg, or 10 mg/kg once a day, which had a significant inhibitory effect on tumor growth (P ⁇ 0.01), and It has a good dose-response relationship and has the effect of shrinking tumors at the doses of 3mg/kg and 10mg/kg.
  • the compound Example 3 of the present invention was orally administered at 10 mg/kg once a day, which has a significant inhibitory effect on tumor growth (P ⁇ 0.01), and has the effect of shrinking tumors.
  • Compound Example 3 and Example 7 did not significantly affect the body weight of mice at the doses tried.
  • Test Example 10 Inhibition experiment of the compound of the present invention on the growth of mouse MCF-7 brain orthotopic tumor model
  • Human breast cancer MCF-7 cells ATCC, HTB-22
  • EMEM culture medium ATCC, Cat No.: 30-2003
  • Fetal Bovine Serum Gibco, Cat. No.: 1099-141C
  • Pancreatin-EDTA Gibco, Cat No.: 25200-072
  • Brain stereotaxic device Reward, Cat No.: standard type/digital display/single arm/mouse/68055
  • Micro syringe pump KDS, Cat No.: Legato130
  • Mini handheld skull drill Reward, Cat No.: 78001
  • mice Female, 6-8 weeks old, weighing about 17-29 grams, the animals were purchased from Beijing Weitongda Biotechnology Co., Ltd. The mice were kept in an SPF environment, and each cage was sent out separately Wind, all animals have free access to standard certified commercial laboratory food and free drinking water.
  • Cell culture Human breast cancer MCF-7 cell line is cultured in vitro, and the culture conditions are EMEM (cell culture medium) with 10% fetal bovine serum, 1% Pen Strep, 10 ⁇ g/ml recombinant human insulin, 37°C, 5% CO 2 Incubator. Use 0.25% pancreatin-EDTA digestion solution twice a week for routine digestion and passage. When the cell saturation is 80%-90% and the number reaches the requirement, the cells are collected and counted.
  • Fulvestrant Fulvestrant, AstraZeneca
  • SC subcutaneous injection
  • QW subcutaneous injection
  • PO Oral administration
  • QD once a day
  • mice The body weight of the mice was measured twice a week, and the survival status of the mice was observed.
  • mice in the Fulvestrant group 250 mg/kg administered subcutaneously once a week continued to lose weight, and there was no significant difference between the survival status of the mice and the solvent control group (median survival time, vehicle The control group was 29 days, and the Fulvestrant group was 29.5 days).
  • the mice in the compound group of Example 3 of the present invention (30 mg/kg orally administered once a day) had stable body weight and no abnormal state. Until the end of the experiment, none of the mice in the compound group of Example 3 of the present invention died.
  • the compound of Example 3 has a significant inhibitory effect on MCF-7 brain orthotopic tumor model mice, and the survival period of the mice is significantly prolonged (P ⁇ 0.01).

Abstract

公开了一种如式（I）所示吡咯烷类化合物或其药学可接受的盐，含有它们的药物组合物及其作为选择性雌激素受体降解剂（SERD）在预防或治疗雌激素受体相关疾病中的用途。

Description

吡咯烷类化合物及其应用
本申请要求2020年05月15日向中国国家知识产权局提交的，专利申请号为CN202010414013.9，发明名称为“吡咯烷类化合物及其应用”的在先申请的优先权。上述在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明涉及一种新型的吡咯烷类化合物或其药学可接受的盐，含有它们的药物组合物以及作为选择性雌激素受体降解剂(Selective Estrogen Receptor Degrader or Downregulator,SERD)在预防或治疗相关疾病中的用途。
背景技术
雌激素(E2)及雌激素α受体(ERα)是乳腺癌发生发展的重要驱动因子。在乳腺癌患者中有超过2/3的患者表达ER转录因子，并且在大多数ER阳性患者中，即使经过早期的内分泌治疗后进展的肿瘤中，ER仍是一个关键的驱动因子，因此ER是乳腺癌治疗的一个主要靶点(Pharmacology & Therapeutics 186(2018)1–24)。内分泌治疗目的是降低ER活性，主要有三类，包括选择性雌激素受体调节剂(SERMs)，比如他莫昔芬(tamoxifen)，是ER的别构调节剂，同ER结合后抑制其转录活性；芳香化酶抑制剂(aromatase inhibitors,AIs),通过抑制雄激素转化为雌激素，减低体内雌激素水平；以及选择性雌激素受体下调剂，比如氟维司群(fulvestrant)，不仅作为ER的拮抗剂抑制其活性，还具有诱导ER蛋白降解的作用。虽然内分泌治疗是雌激素受体阳性乳腺癌患者的首选，但是约有30％的治疗后病人会发生复发，并且几乎所有的转移性乳腺癌患者都会产生耐药而发生进展。内分泌治疗产生耐药的机制主要有两类，一是集中在雌激素受体信号通路本身，包括编码雌激素受体的基因ESR1的激活突变，扩增，与其他基因的融合，雌激素受体共调节因子和下游控制细胞周期因子的失调等；另一类机制包括与雌激素受体信号通路有交叉反应的信号通路的激活，比如生长因子受体通路等(Oncol Ther，2017,5:17–29)。
临床上，约70-80％的乳腺癌检测雌激素受体(ER)呈阳性，这类乳腺癌细胞的增殖严重依赖ER，且50％的乳腺癌死亡病例均为该类分型。早期ER阳性乳腺癌预后较好，5年生存率超过90％。术后内分泌治疗(TAM或AI药物)的病人10年内约30％出现复发，但仍然可以接受标准的内分泌治疗。尽管如此，以ESR1-LBD突变为主导致的获得性耐药以及远端转移(骨脑肝肺淋巴结等)的出现(其中脑转移患者约10-15％)，患者开始对治疗抵抗，导致耐药病人的群体数量不断增加。在晚期转移性乳腺癌患者中，脑转移较肺、肝、骨转移晚，预后较差，经临床药物治疗后的中位生存期仅为2～9个月。
氟维司群是首个也是唯一经临床批准用于他莫昔芬或芳香化酶抑制剂进展后治疗ER阳性、转移性乳腺癌的绝经后患者的SERD类药物。目前，阿斯利康(参见专利申请WO2018077630A1)及基因泰克公司(参见专利申请WO2019245974A1)也公开了一系列结构新颖的SERD类化合物及相应的医药用途。多项研究数据显示经氟维司群治疗的患者体内并未能完全实现ER的降解，此外肌肉注射方式造成的注射部位疼痛、肿胀、发红等明显反应，且吸收缓慢、体内暴露量受限(不能透过血脑屏障，单次肌注最高只能达到500mg，其药效动力学特征和肌内给药途径限制了其可给予患者的最高剂量)等特点极大限制了其临床应用，因此ER阳性乳腺癌患者亟需新的治疗选择。
发明内容
本发明提供一种式(I)所示化合物或其药学上可接受的盐：
Figure PCTCN2021093736-appb-000001
其中，
R 1、R 2、R 3、R 4独立选自H、F、Cl、Br、I、CN、C 1-C 6烷基、C 1-C 6烷氧基或C 3-C 6环烷基；
X 1、X 2、X 3、X 4独立地选自CR 6或N；
R 6选自H、F、Cl、Br、I、OH、CN、C 1-C 10烷基、C 3-C 10环烷基、3-10元杂环基、C 1-C 10烷氧基、C 3-C 10环烷基氧基或3-10元杂环基氧基；
Y选自O或NH；
R 5独立选自C 1-C 6烷基，所述C 1-C 6烷基任选被R a取代；
R a选自F、Cl、Br、I、OH、CN、C 1-C 6烷基、C 1-C 6烷氧基或C 3-C 6环烷基；
条件是，式(I)所示化合物不包含
Figure PCTCN2021093736-appb-000002
在一些实施方案中，所述式(I)所示的化合物或其药学可接受的盐，选自式(II)所示化合物或其药学可接受的盐：
Figure PCTCN2021093736-appb-000003
在一些实施方案中，所述式(I)所示的化合物或其药学可接受的盐，选自式(III)所示化合物或其药学可接受的盐：
Figure PCTCN2021093736-appb-000004
在一些实施方案中，R 1、R 2、R 3、R 4独立选自H、F、Cl、Br、I、CN或C 1-C 6烷基。
在一些实施方案中，R 1、R 2、R 3、R 4独立选自H、F、Cl、Br、I、CN或C 1-C 3烷基。
在一些实施方案中，R 1、R 2、R 3、R 4独立选自H、F、Cl、Br、I、CN或甲基。
在一些实施方案中，R 1、R 2、R 3、R 4独立选自H、F、Cl、Br、I或C 1-C 3烷基。
在一些实施方案中，R 1、R 2、R 3、R 4独立选自H、F、Cl、Br、I或甲基。
在一些实施方案中，R 1、R 2、R 3、R 4独立选自H、F或甲基。
在一些实施方案中，R 1、R 2独立选自H、F或甲基。
在一些实施方案中，R 3、R 4独立选自H或甲基。
在一些实施方案中，R 3、R 4独立选自H。
在一些实施方案中，结构单元
Figure PCTCN2021093736-appb-000005
选自
Figure PCTCN2021093736-appb-000006
Figure PCTCN2021093736-appb-000007
在一些实施方案中，结构单元
Figure PCTCN2021093736-appb-000008
选自
Figure PCTCN2021093736-appb-000009
Figure PCTCN2021093736-appb-000010
在一些实施方案中，结构单元
Figure PCTCN2021093736-appb-000011
选自
Figure PCTCN2021093736-appb-000012
Figure PCTCN2021093736-appb-000013
在一些实施方案中，结构单元
Figure PCTCN2021093736-appb-000014
选自
Figure PCTCN2021093736-appb-000015
Figure PCTCN2021093736-appb-000016
在一些实施方案中，结构单元
Figure PCTCN2021093736-appb-000017
选自
Figure PCTCN2021093736-appb-000018
Figure PCTCN2021093736-appb-000019
在一些实施方案中，结构单元
Figure PCTCN2021093736-appb-000020
选自
Figure PCTCN2021093736-appb-000021
Figure PCTCN2021093736-appb-000022
在一些实施方案中，结构单元
Figure PCTCN2021093736-appb-000023
选自
Figure PCTCN2021093736-appb-000024
Figure PCTCN2021093736-appb-000025
在一些实施方案中，X 1、X 2、X 3、X 4独立地选自CR 6或N，且X 1、X 2、X 3、X 4基团中的至少2个选自CR 6。
在一些实施方案中，X 1、X 2、X 3、X 4独立地选自CR 6或N，且X 1、X 2、X 3、X 4基团中的至少3个选自CR 6。
在一些实施方案中，所述R 6选自H、F、Cl、Br、I、CN、C 1-C 3烷基或C 1-C 3烷氧基。
在一些实施方案中，所述R 6选自H、F、Cl、Br、I、CN或C 1-C 3烷氧基。
在一些实施方案中，所述R 6选自H、F、Cl、Br、I、CN或甲氧基。
在一些实施方案中，所述R 6选自H、F、Cl、Br、I或CN。
在一些实施方案中，所述R 6选自H、F、Cl、Br或I。
在一些实施方案中，所述R 6选自H或F。
在一些实施方案中，结构单元
Figure PCTCN2021093736-appb-000026
选自
Figure PCTCN2021093736-appb-000027
在一些实施方案中，结构单元
Figure PCTCN2021093736-appb-000028
选自
Figure PCTCN2021093736-appb-000029
在一些实施方案中，R 5选自C 1-C 3烷基，所述C 1-C 3烷基任选被R a取代。
在一些实施方案中，所述R a选自F、Cl、Br、I、OH或CN。
在一些实施方案中，所述R a选自F、OH或CN。
在一些实施方案中，所述R a选自F或OH。
在一些实施方案中，R 5选自CH 2CF 3、CH 2CHF 2、CH 2CF 2CH 2OH或CH 2CF 2CH 2CN。
在一些实施方案中，R 5选自CH 2CF 3或CH 2CF 2CH 2OH。
在一些实施方案中，Y选自NH。
在一些实施方案中，所述式(I)所示的化合物或其药学可接受的盐，选自以下化合物或其药学可接受的盐：
Figure PCTCN2021093736-appb-000030
Figure PCTCN2021093736-appb-000031
在一些实施方案中，所述式(I)所示的化合物或其药学可接受的盐，选自以下化合物或其药学可接受的盐：
Figure PCTCN2021093736-appb-000032
在一些实施方案中，所述式(I)所示的化合物或其药学可接受的盐，选自以下化合物或其药学可接受的盐：
Figure PCTCN2021093736-appb-000033
Figure PCTCN2021093736-appb-000034
在一些实施方案中，所述式(I)所示的化合物或其药学可接受的盐，选自以下化合物或其药学可接受的盐：
Figure PCTCN2021093736-appb-000035
Figure PCTCN2021093736-appb-000036
在一些实施方案中，所述式(I)所示的化合物或其药学可接受的盐，选自以下化合物或其药学可接受的盐：
Figure PCTCN2021093736-appb-000037
本发明还提供药物组合物，其包含式(I)所示化合物或其药学可接受的盐和药学上可接受的辅料。
进一步，本发明涉及式(I)所示的化合物或其药学上可接受的盐，或其药物组合物在制备预防或者治疗雌激素受体相关疾病的药物中的用途。
进一步，本发明涉及式(I)所示的化合物或其药学上可接受的盐，或其 药物组合物，用于预防或者治疗雌激素受体相关疾病。
进一步，本发明涉及式(I)所示的化合物或其药学上可接受的盐，或其药物组合物在预防或者治疗雌激素受体相关疾病中的用途。
进一步，本发明涉及预防或者治疗雌激素受体相关疾病的式(I)化合物或其药学上可接受的盐，或其药物组合物。
本发明还涉及治疗雌激素受体相关疾病的方法，该方法包括给以患者治疗上有效剂量的包含本发明所述的式(I)化合物或其药学上可接受的盐的药物制剂。
本发明的一个优选实施方案中，所述的雌激素受体相关疾病包括但不限于肿瘤。
本发明的一个优选实施方案中，所述的雌激素受体相关疾病为乳腺癌。
本发明的一个优选实施方案中，所述的雌激素受体相关疾病为ER阳性乳腺癌。
本发明的一个优选实施方案中，所述的雌激素受体相关疾病为ER阳性乳腺癌脑转移。
本发明的一个优选实施方案中，所述患者为乳腺癌患者。
本发明的一个优选实施方案中，所述患者为ER阳性乳腺癌患者。
本发明的一个优选实施方案中，所述患者为ER阳性乳腺癌脑转移患者。
本发明化合物具有良好的体内外抗肿瘤活性以及成药性。体内试验发现本发明化合物能够显著抑制ER阳性乳腺癌小鼠模型肿瘤生长，以及显著提高ER阳性乳腺癌脑原位模型小鼠的生存期。另外，本发明化合物生物利用度高、ER降解能力强、可口服、通过血脑屏障能力高，具有有效治疗ER阳性乳腺癌(特别是ER阳性乳腺癌脑转移)的潜力。
术语定义和说明
除非另有说明，本发明说明书和权利要求书中记载的基团和术语定义，包括其作为实例的定义、示例性的定义、优选的定义、表格中记载的定义、实施例中具体化合物的定义等，可以彼此之间任意组合和结合。这样的组合和结合后的基团定义及化合物结构，应当属于本发明说明书记载的范围内。
本文中
Figure PCTCN2021093736-appb-000038
表示连接位点。
术语“药学上可接受的盐”是指药学上可接受的无毒酸或碱的盐，包括无机酸和碱、有机酸和碱的盐。
术语“立体异构体”是指由分子中原子在空间上排列方式不同所产生的异构体，包括顺反异构体、对映异构体和非对映异构体。
本发明的化合物可以具有不对称原子如碳原子、硫原子、氮原子、磷原子(光学中心)或不对称双键。外消旋体、对映异构体、非对映异构体、几何异构体都包括在本发明的范围之内。
本文中消旋体或者对映体纯的化合物的图示法来自Maehr， J.Chem.Ed.1985，62:114-120。除非另有说明，用楔形键和虚楔键
Figure PCTCN2021093736-appb-000039
表示一个立体中心的绝对构型，用黑实键和虚键
Figure PCTCN2021093736-appb-000040
表示脂环化合物的顺反构型。当本文所述化合物含有烯属双键或其它几何不对称中心，除非另有规定，它们包括E、Z几何异构体。同样地，所有的互变异构形式均包括在本发明的范围之内。
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子、不对称硫原子、不对称氮原子或不对称磷原子。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。本申请的含有不对称原子的化合物可以以光学活性纯的形式或外消旋形式被分离出来。光学活性纯的形式可以从外消旋混合物拆分，或通过使用手性原料或手性试剂合成。立体异构体的非限制性实例包括但不限于：
Figure PCTCN2021093736-appb-000041
Figure PCTCN2021093736-appb-000042
术语“互变异构体”是指因分子中某一原子在两个位置迅速移动而产生的官能团异构体。本发明化合物可表现出互变异构现象。互变异构的化合物可以存在两种或多种可相互转化的种类。互变异构体一般以平衡形式存在，尝试分离单一互变异构体时通常产生一种混合物，其理化性质与化合物的混合物是一致的。平衡的位置取决于分子内的化学特性。例如，在很多脂族醛和酮如乙醛中，酮型占优势；而在酚中，烯醇型占优势。本发明包含化合物的所有互变异构形式。
术语“药物组合物”表示一种或多种文本所述化合物或其生理学/药学上可接受的盐或前体药物与其它化学组分的混合物，其它组分例如生理学/药学上可接受的辅料。药物组合物的目的是促进化合物对生物体的给药。
术语“被取代”是指特定原子上的任意一个或多个氢原子被取代基取代，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧代(即＝O)时，意味着两个氢原子被取代，氧代不会发生在芳香基上。
术语“任选”或“任选地”是指随后描述的事件或情况可以发生或不发生，该描述包括发生所述事件或情况和不发生所述事件或情况。例如，乙基“任选”被卤素取代，指乙基可以是未被取代的(CH 2CH 3)、单取代的(如CH 2CH 2F)、多取代的(如CHFCH 2F、CH 2CHF 2等)或完全被取代的(CF 2CF 3)。本领域技术人员可理解，对于包含一个或多个取代基的任何基团，不会引入任何在空间上不可能存在和/或不能合成的取代或取代模式。
当任何变量(例如R a、R b)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。例如，如果一个基团被2个R b所取代，则每个R b都有独立的选项。
当一个连接基团的数量为0时，比如-(CH 2) 0-，表示该连接基团为键。
当其中一个变量选自化学键或不存在时，表示其连接的两个基团直接相连，比如A-L-Z中L代表键时表示该结构实际上是A-Z。
当本文中涉及到的连接基团若没有指明其连接方向，则其连接方向是任意的。例如当结构单元
Figure PCTCN2021093736-appb-000043
中的L 1选自“C 1-C 3亚烷基-O”时，此时L 1既可以按照与从左到右的读取顺序相同的方向连接环Q和R 1构成“环Q-C 1-C 3亚烷基-O-R 1”，也可以按照从左到右的读取顺序相反的方向连接环Q和R 1构成“环Q-O-C 1-C 3亚烷基-R 1”。
术语“卤”或“卤素”是指氟、氯、溴和碘。
本文中的C m-C n，是指具有m-n范围中的整数个碳原子。例如“C 1-C 10”是指该基团可具有1个碳原子、2个碳原子、3个碳原子、4个碳原子、5个碳原子、6个碳原子、7个碳原子、8个碳原子、9个碳原子或10个碳原子。
术语“烷基”是指通式为C nH 2n+1的烃基，该烷基可以是直链或支链的。术语“C 1-C 10烷基”应理解为表示具有1、2、3、4、5、6、7、8、9或10个碳原子的直链或支链饱和一价烃基。所述烷基是例如甲基、乙基、丙基、丁基、戊基、己基、异丙基、异丁基、仲丁基、叔丁基、异戊基、2-甲基丁基、1-甲基丁基、1-乙基丙基、1，2-二甲基丙基、新戊基、1，1-二甲基丙基、4-甲基戊基、3-甲基戊基、2-甲基戊基、1-甲基戊基、2-乙基丁基、1-乙基丁基、3，3-二甲基丁基、2，2-二甲基丁基、1，1-二甲基丁基、2，3-二甲基丁基、1，3-二甲基丁基或1，2-二甲基丁基等；优选地，“C 1-C 10烷基”可以包含“C 1-C 6烷基”或“C 1-C 3烷基”，“C 1-C 6烷基”应理解为表示具有1、2、3、4、5、6个碳原子的直链或支链饱和一价烃基，“C 1-C 3烷基”应理解为表示具有1、2、3个碳原子的直链或支链饱和一价烃基。
术语“C 1-C 10烷氧基”可理解为“C 1-C 10烷基氧基”或“C 1-C 10烷基-O-”，优选地，“C 1-C 10烷氧基”可以包含“C 1-C 6烷氧基”或“C 1-C 3烷氧基”。
术语“C 3-C 10环烷基”应理解为表示饱和的一价单环或双环烃环，其具有3～10个碳原子。如环丙基、环丁基、环戊基、环己基、环庚基、环辛基、环壬基或环癸基，或者是双环烃基如十氢化萘环。术语“C 3-C 6环烷基”应理解为表示饱和的一价单环或双环烃环，其具有3～6个碳原子。
术语“C 3-C 10环烷基氧基”可理解为“C 3-C 10环烷基-O-”，优选地，“C 3-C 10环烷基氧基”可以包含“C 3-C 6环烷基氧基”。
术语“杂环基”是指完全饱和的或部分饱和的(整体上不是具有芳香性的杂芳族)一价单环、并环、螺环或桥环基团，其环原子中含有1-5个杂原子或杂原子团(即含有杂原子的原子团)，所述“杂原子或杂原子团”包括但不限于氮原子(N)、氧原子(O)、硫原子(S)、磷原子(P)、硼原子(B)，＝O,＝S,-O-N＝,-C(＝O)O-,-C(＝O)-,-C(＝S)-,-S(＝O) 2-,-S(＝O)-,以及任选被取代的-NH-,-S(＝O)(＝NH)-,-C(＝O)NH-,-C(＝NH)-,-S(＝O) 2NH-,S(＝O)NH-,-NHC(＝O)NH-等。术语“3-10元杂环基”意指环原子数目为3、4、5、6、7、8、9或10的杂环基，且其环原子中含有1-5个独立选自上文所述的杂原子或杂原子团。特别地，3元杂环基的实例包括但不限于环氧丙基或氮杂环丙基；4元杂环基的实例包括但不限于氮杂环丁烷基、氧杂环丁烷基；5元杂环基的实例包括但不限于四氢呋喃基、二氧杂环戊烯基、吡咯烷基、咪唑烷基、吡唑烷基、吡咯啉基、4,5-二氢噁唑或2,5-二氢-1H-吡咯基；6元杂环基的实例包括但不限于四氢吡喃基、哌啶基、吗啉基、二噻烷基、硫代吗啉基、哌嗪基、三噻烷基、四氢吡啶基或4H-[1,3,4]噻二嗪基；7元杂环基的实例包括但不限于二氮杂环庚烷基；所述杂环基还可以是双环基，其中，5,5元双环基实例包括但不限于六氢环戊并[c]吡咯-2(1H)-基环，5,6元双环基实例包括但不限于六氢吡咯 并[1,2-a]吡嗪-2(1H)-基环、5,6,7,8-四氢-[1,2,4]三唑并[4,3-a]吡嗪基环或5,6,7,8-四氢咪唑并[1,5-a]吡嗪。任选地，所述杂环基可以是上述4-7元杂环基的苯并稠合环基，实例包括但不限于二氢异喹啉基等。任选地，所述3-10元杂环基可以是“3-10元杂环烷基”；“3-10元杂环烷基”进一步可以包含“3-7元杂环烷基”或“5-6元杂环烷基”等范围；本申请中尽管有些双环类杂环基部分地含有一个苯环或一个杂芳环，但所述杂环基整体上仍是无芳香性的。
术语“3-10元杂环基氧基”是指“3-10元杂环基-O-”。
术语“治疗”意为将本申请所述化合物或制剂进行给药以预防、改善或消除疾病或与所述疾病相关的一个或多个症状，且包括：
(i)预防疾病或疾病状态在哺乳动物中出现，特别是当这类哺乳动物易患有该疾病状态，但尚未被诊断为已患有该疾病状态时；
(ii)抑制疾病或疾病状态，即遏制其发展；
(iii)缓解疾病或疾病状态，即使该疾病或疾病状态消退。
术语“治疗有效量”意指(i)治疗或预防特定疾病、病况或障碍，(ii)减轻、改善或消除特定疾病、病况或障碍的一种或多种症状，或(iii)预防或延迟本文中所述的特定疾病、病况或障碍的一种或多种症状发作的本发明化合物的用量。构成“治疗有效量”的本发明化合物的量取决于该化合物、疾病状态及其严重性、给药方式以及待被治疗的哺乳动物的年龄而改变，但可例行性地由本领域技术人员根据其自身的知识及本公开内容而确定。
术语“辅料”是指可药用惰性成分。术语“赋形剂”的种类实例非限制性地包括粘合剂、崩解剂、润滑剂、助流剂、稳定剂、填充剂和稀释剂等。赋形剂能增强药物制剂的操作特性，即通过增加流动性和/或粘着性使制剂更适于直接压缩。适用于上述制剂的典型的“药学上可接受的载体”的实例为：糖类，淀粉类，纤维素及其衍生物等在药物制剂中常用到的辅料。
术语“药学上可接受的辅料”是指对有机体无明显刺激作用，而且不会损害该活性化合物的生物活性及性能的那些辅料。合适的辅料是本领域技术人员熟知的，例如碳水化合物、蜡、水溶性和/或水可膨胀的聚合物、亲水性或疏水性材料、明胶、油、溶剂、水等。
词语“包括(comprise)”、“含有(comprise)”或“包含(comprise)”及其英文变体例如comprises或comprising应理解为开放的、非排他性的意义，即“包括但不限于”。
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
本申请还包括与本文中记载的那些相同的，但一个或多个原子被原子量或质量数不同于自然中通常发现的原子量或质量数的原子置换的同位素标记的本申请化合物。可结合到本申请化合物的同位素的实例包括氢、碳、氮、 氧、磷、硫、氟、碘和氯的同位素，诸如分别为 2H、 3H、 11C、 13C、 14C、 13N、 15N、 15O、 17O、 18O、 31P、 32P、 35S、 18F、 123I、 125I和 36Cl等。
某些同位素标记的本申请化合物(例如用 3H及 14C标记的那些)可用于化合物和/或底物组织分布分析中。氚化(即 3H)和碳-14(即 14C)同位素对于由于它们易于制备和可检测性是尤其优选的。正电子发射同位素，诸如 15O、 13N、 11C和 18F可用于正电子发射断层扫描(PET)研究以测定底物占有率。通常可以通过与公开于下文的方案和/或实施例中的那些类似的下列程序，通过同位素标记试剂取代未经同位素标记的试剂来制备同位素标记的本申请化合物。
此外，用较重同位素(诸如氘(即 2H))取代可以提供某些由更高的代谢稳定性产生的治疗优点(例如增加的体内半衰期或降低的剂量需求)，并且因此在某些情形下可能是优选的，其中氘取代可以是部分或完全的，部分氘取代是指至少一个氢被至少一个氘取代。
本申请的药物组合物可通过将本申请的化合物与适宜的药学上可接受的辅料组合而制备，例如可配制成固态、半固态、液态或气态制剂，如片剂、丸剂、胶囊剂、粉剂、颗粒剂、膏剂、乳剂、悬浮剂、栓剂、注射剂、吸入剂、凝胶剂、微球及气溶胶等。
给予本申请化合物或其药学上可接受的盐或其药物组合物的典型途径包括但不限于口服、直肠、局部、吸入、肠胃外、舌下、阴道内、鼻内、眼内、腹膜内、肌内、皮下、静脉内给药。
本申请的药物组合物可以采用本领域众所周知的方法制造，如常规的混合法、溶解法、制粒法、制糖衣药丸法、磨细法、乳化法、冷冻干燥法等。
在一些实施方案中，药物组合物是口服形式。对于口服给药，可以通过将活性化合物与本领域熟知的药学上可接受的辅料混合，来配制该药物组合物。这些辅料能使本申请的化合物被配制成片剂、丸剂、锭剂、糖衣剂、胶囊剂、液体、凝胶剂、浆剂、悬浮剂等，用于对患者的口服给药。
可以通过常规的混合、填充或压片方法来制备固体口服组合物。例如，可通过下述方法获得：将所述的活性化合物与固体辅料混合，任选地碾磨所得的混合物，如果需要则加入其它合适的辅料，然后将该混合物加工成颗粒，得到了片剂或糖衣剂的核心。适合的辅料包括但不限于：粘合剂、稀释剂、崩解剂、润滑剂、助流剂、甜味剂或矫味剂等。
药物组合物还可适用于肠胃外给药，如合适的单位剂型的无菌溶液剂、混悬剂或冻干产品。
本文所述的通式Ⅰ化合物的所有施用方法中，每天给药的剂量为0.01到100mg/kg体重，优选为0.05到50mg/kg体重，更优选0.1到30mg/kg体重，以单独或分开剂量的形式。
本发明具体实施方式的化学反应是在合适的溶剂中完成的，所述的溶剂须适合于本发明的化学变化及其所需的试剂和物料。为了获得本发明的化合物， 有时需要本领域技术人员在已有实施方式的基础上对合成步骤或者反应流程进行修改或选择。
附图说明
图1为化合物3的NOESY图谱；
图2为测试例9中的MCF-7皮下瘤模型肿瘤生长曲线图；
图3为测试例9中的MCF-7皮下瘤模型动物体重变化图；
图4为测试例10中的MCF-7脑原位模型小鼠生存曲线图；
图5为测试例10中的MCF-7脑原位模型小鼠体重变化图。
具体实施方式
以下实施例详细说明发明的技术方案，但本发明的保护范围包括但不限于此。
化合物的结构是通过核磁共振(NMR)和/或质谱(MS)来确定的。NMR位移的单位为10 -6(ppm)。NMR测定的溶剂为氘代二甲基亚砜、氘代氯仿、氘代甲醇等，内标为四甲基硅烷(TMS)；“IC 50”指半数抑制浓度，指达到最大抑制效果一半时的浓度。
实施例1：N-(1-(3-氟丙基)吡咯烷-3-基)-6-((1S，3R)-3-甲基-2-(2，2，2-三氟乙基)-2，3，4，9-四氢-1H-吡啶并[3，4-b]吲哚-1-基)吡啶-3-胺(化合物1)的合成
Figure PCTCN2021093736-appb-000044
合成方法：
Figure PCTCN2021093736-appb-000045
步骤1：叔丁基(1-(3-氟丙基)吡咯烷-3-基)氨基甲酸酯的合成
Figure PCTCN2021093736-appb-000046
将叔丁基吡咯烷-3-基氨基甲酸酯(1.00g，5.37mmol)溶于四氢呋喃(10mL)中，加入氢氧化钠溶液(5mol﹒L -1，2.15mL)和1-碘-3-氟丙烷(1.06g，5.64mmol)。反应液于25℃搅拌反应16小时。TLC检测原料反应完全后，将反应液用乙酸乙酯稀释后，用饱和氯化铵溶液洗涤，分别收集水相和有机相。水相用乙酸乙酯(50mL)萃取三次后，将所有有机相合并，用硫酸钠干燥后，有机相减压浓缩干，然后柱层析纯化(二氧化硅，二氯甲烷/甲醇＝100/1)得到产物叔丁基(1-(3-氟丙基)吡咯烷-3-基)氨基甲酸酯(0.81g)。
1H NMR(400MHz,METHANOL-d 4)δ4.57(t,J＝5.77Hz,1H),4.45(t,J＝5.77Hz,1H),4.11(br d,J＝7.78Hz,1H),3.05-2.97(m,1H),2.93-2.81(m,1H),2.80-2.69(m,3H),2.66-2.56(m,1H),2.30-2.20(m,1H),2.04-1.87(m,2H),1.78-1.68(m,1H),1.51-1.38(m,9H).
步骤2：1-(3-氟丙基)吡咯烷-3-胺盐酸盐的合成
Figure PCTCN2021093736-appb-000047
将叔丁基(1-(3-氟丙基)吡咯烷-3-基)氨基甲酸酯(0.81g，3.12mmol)溶于1，4-二氧六环(9mL)中，然后加入盐酸-1，4-二氧六环溶液(4moL﹒L -1，9mL)，反应为黄色透明溶液。反应液在25℃下搅拌3小时。TLC检测 原料反应完全后，将反应溶液减压浓缩干得化合物1-(3-氟丙基)吡咯烷-3-胺盐酸盐(0.71g)。
1H NMR(400MHz,METHANOL-d 4)δ4.68(t,J＝5.52Hz,1H),4.56(s,1H),4.30-3.79(m,3H),3.68(s,1H),3.48(br s,2H),3.31-3.21(m,1H),2.82-2.46(m,1H),2.32-2.14(m,3H).
步骤3：(R)-1-(1H-吲哚-3-基)-N-(2,2,2-三氟乙基)丙烷-2-胺的合成
Figure PCTCN2021093736-appb-000048
将(2R)-1-(1H-吲哚-3-基)丙烷-2-胺(600mg，3.44mmol)和N,N-二异丙基乙胺(445.05mg，3.44mmol)溶于1,4-二氧六环(10mL)中，在25℃下加入溶在1,4-二氧六环(5mL)中的三氟甲磺酸三氟乙酯(1.20g，5.17mmol)，反应液于75℃搅拌反应16小时。将反应液减压浓缩至干。然后柱层析纯化(二氧化硅，石油醚/乙酸乙酯＝3/1)得到产物(R)-1-(1H-吲哚-3-基)-N-(2,2,2-三氟乙基)丙烷-2-胺(0.69g)。
MS m/z(ESI):257.2[M+H] +；
1H NMR(400MHz,METHANOL-d 4)δ7.56-7.54(d,J＝8.0Hz,1H),7.36-7.34(d,J＝8.0Hz,1H),7.12-7.08(m,2H),7.03-7.00(m,1H),3.26-3.23(m,2H),3.12-3.10(m,1H),2.93-2.88(m,1H),2.80-2.78(m,1H),1.11(d,J＝6.0Hz,3H)
步骤4：(1S，3R)-1-(5-溴吡啶-2-基)-3-甲基-2-(2，2，2-三氟乙基)-2，3，4，9-四氢-1H-吡啶并[3，4-b]吲哚的合成
Figure PCTCN2021093736-appb-000049
将(R)-1-(1H-吲哚-3-基)-N-(2，2，2-三氟乙基)丙烷-2-胺(120.00mg，468.26μmol)溶于甲苯(2mL)中，加入5-溴-吡啶-2-甲醛(87.10mg，468.26μmol)和乙酸(562.40mg，9.37mmol)，反应液为黄色透明溶液。反应液在90℃下搅拌10小时。LCMS检测反应完成后，将反应溶液冷却至室温后，减压浓缩干，经薄层层析纯化(二氧化硅，石油醚/乙酸乙酯＝4/1)得(1S，3R)-1-(5-溴吡啶-2-基)-3-甲基-2-(2，2，2-三氟乙基)-2，3，4，9-四氢-1H-吡啶并[3，4-b]吲哚(110.00mg)。
MS m/z(ESI):424.1,426.1[M+H] +。
1H NMR(400MHz,METHANOL-d 4)δ8.59(s,1H),8.01-7.94(m,1H),7.54 (d,J＝8.53Hz,1H),7.46(d,J＝7.78Hz,1H),7.30(d,J＝8.03Hz,1H),7.08(d,J＝7.59Hz,1H),7.03-6.96(m,1H),5.08(s,1H),3.58-3.46(m,1H),3.38-3.34(m,1H),3.13-2.99-(m,1H),2.80(d,J＝4.52Hz,1H),2.70-2.61(m,1H),1.26(d,J＝6.78Hz,3H).
步骤5：N-(1-(3-氟丙基)吡咯烷-3-基)-6-((1S，3R)-3-甲基-2-(2，2，2-三氟乙基)-2，3，4，9-四氢-1H-吡啶并[3，4-b]吲哚-1-基)吡啶-3-胺的合成
Figure PCTCN2021093736-appb-000050
将(1S，3R)-1-(5-溴吡啶-2-基)-3-甲基-2-(2，2，2-三氟乙基)-2，3，4，9-四氢-1H-吡啶并[3，4-b]吲哚(110.00mg，259.28μmol)溶于四氢呋喃(2mL)中，加入1-(3-氟丙基)吡咯烷-3-胺盐酸盐(68.18mg，311.13μmol)，叔丁醇钠(149.50mg，1.56mmol)和甲烷磺酸(2-二环己基膦)-3，6-二甲氧基-2，4，6-三异丙基-1，1-联苯)(2-氨基-1，1-联苯-2-基)钯(II)(23.50mg，25.93μmol)，在氮气环境下反应液为棕色混浊溶液。反应液在80℃下搅拌4小时。LCMS检测反应完成后，冷却至室温，过滤，滤液减压浓缩后，经过制备液相色谱纯化(Phenomenex Gemini C18柱，3um二氧化硅，30mm直径，75mm长度)；(使用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液)纯化得到化合物N-(1-(3-氟丙基)吡咯烷-3-基)-6-((1S，3R)-3-甲基-2-(2，2，2-三氟乙基)-2，3，4，9-四氢-1H-吡啶并[3，4-b]吲哚-1-基)吡啶-3-胺(22.23mg)。
MS m/z(ESI):490.2[M+H] +。
1H NMR(400MHz,METHANOL-d 4)δ7.88(d,J＝2.76Hz,1H),7.46(d,J＝7.78Hz,1H),7.25(dd,J＝7.91,3.89Hz,2H),7.09-6.96(m,3H),4.97(s,1H),4.60(t,J＝5.65Hz,1H),4.48(t,J＝5.65Hz,1H),4.15(br s,1H),3.52-3.36(m,3H),3.25-3.14(m,1H),3.09-2.88(m,6H),2.68(s,1H),2.51-2.39(m,1H),2.11-1.85(m,3H),1.20(d,J＝6.53Hz,3H).
实施例2：N-((R)(1-(3-氟丙基)吡咯烷-3-基)-6-((1S，3R)-3-甲基-2-(2，2，2-三氟乙基)-2，3，4，9-四氢-1H-吡啶并[3，4-b]吲哚-1-基)吡啶-3-胺(化合物2)的合成和实施例3：N-((S)(1-(3-氟丙基)吡咯烷-3-基)-6-((1S，3R)-3-甲基-2-(2，2，2-三氟乙基)-2，3，4，9-四氢-1H-吡啶并[3，4-b]吲哚-1-基)吡啶-3-胺(化合物3)的合成
Figure PCTCN2021093736-appb-000051
将消旋体N-(1-(3-氟丙基)吡咯烷-3-基)-6-((1S，3R)-3-甲基-2-(2，2，2-三氟乙基)-2，3，4，9-四氢-1H-吡啶并[3，4-b]吲哚-1-基)吡啶-3-胺(80.00mg，153.61μmol)经过手性分离(DAICEL CHIRALPAK AY-H柱，5μm二氧化硅，30mm直径，250mm长度，使用异丙醇(含有0.1％氨水)和水的极性递减的混合物作为洗脱液)和制备液相色谱纯化(Phenomenex Gemini C18柱，3μm二氧化硅，30mm直径，75mm长度，使用水(含有0.05％氨水)和乙腈的极性递减的混合物作为洗脱液)纯化得到N-((R)(1-(3-氟丙基)吡咯烷-3-基)-6-((1S，3R)-3-甲基-2-(2，2，2-三氟乙基)-2，3，4，9-四氢-1H-吡啶并[3，4-b]吲哚-1-基)吡啶-3-胺(8.22mg，保留时间为2.627分钟)和N-((S)(1-(3-氟丙基)吡咯烷-3-基)-6-((1S，3R)-3-甲基-2-(2，2，2-三氟乙基)-2，3，4，9-四氢-1H-吡啶并[3，4-b]吲哚-1-基)吡啶-3-胺(10.85mg，保留时间为2.817分钟)。
N-((R)(1-(3-氟丙基)吡咯烷-3-基)-6-((1S，3R)-3-甲基-2-(2，2，2-三氟乙基)-2，3，4，9-四氢-1H-吡啶并[3，4-b]吲哚-1-基)吡啶-3-胺(化合物2):
MS m/z(ESI):490.1[M+H] +
1H NMR(400MHz,METHANOL-d 4)δ7.89(d,J＝2.76Hz,1H),7.48-7.44(m,1H),7.29-7.24(m,2H),7.09-7.04(m,2H),7.00(s,1H),4.98(s,1H),4.64-4.60(m,1H),4.52-4.48(m,1H),4.25-4.16(m,1H),3.54-3.36(m,4H),3.25-3.09(m,4H),3.05(s,1H),2.89(d,J＝4.52Hz,1H),2.70-2.60(m,1H),2.55-2.42(m,1H),2.16-1.94(m,3H),1.20(d,J＝6.78Hz,3H).
N-((S)(1-(3-氟丙基)吡咯烷-3-基)-6-((1S，3R)-3-甲基-2-(2，2，2-三氟乙基)-2，3，4，9-四氢-1H-吡啶并[3，4-b]吲哚-1-基)吡啶-3-胺(化合物3)：
MS m/z(ESI):489.25,490.1[M+H] +
1H NMR(400MHz,METHANOL-d 4)δ7.90-7.88(m,1H),7.48-7.43(m,1H),7.29-7.23(m,2H),7.09-7.03(m,2H),7.03-6.97(m,1H),4.99-4.97(m,1H),4.63-4.59(m,1H),4.52-4.47-(m,1H),4.24-4.16(m,1H),3.52-3.36(m,4H),3.15(br s,4H),3.05-2.99(m,1H),2.96-2.88(m,1H),2.68(s,1H),2.57-2.43(m,1H),2.12-1.94(m,3H),1.20(d,J＝6.53Hz,3H).
N-((S)-1-(3-氟丙基)吡咯烷-3-基)-6-(((1S,3R)-3-甲基-2-(2,2,2-三氟乙基)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚-1-基)吡啶-3-胺(化合物3)还可通过以下合成方法获得：
Figure PCTCN2021093736-appb-000052
步骤1：(S)-叔丁基(1-(3-氟丙基)吡咯烷-3-基)氨基甲酯的合成
Figure PCTCN2021093736-appb-000053
将(S)-叔丁基吡咯烷-3-基氨基甲酯(500.00mg，2.68mmol)溶于四氢呋喃(10mL)中，加入氢氧化钠溶液(5mol﹒L -1，1.07mL)和1-碘-3-氟丙烷(529.88mg，2.82mmol)。反应液于25℃搅拌反应16小时。TLC检测原料反应完全后，将反应液用乙酸乙酯(50mL)稀释后，用饱和氯化铵溶液(10mL)洗涤，分别收集水相和有机相。水相用乙酸乙酯(20mL)萃取三次后，将所有有机相合并，用硫酸钠干燥后，有机相减压浓缩干，然后柱层析纯化(二氧化硅，二氯甲烷/甲醇＝100/1)得到产物(S)-叔丁基(1-(3-氟丙基)吡咯烷-3-基)氨基甲酯(480.00mg)。
1H NMR(400MHz,METHANOL-d 4)δ4.58-4.53(m,1H),4.46-4.40(m,1H),4.14-4.04(m,1H),2.93-2.85(m,1H),2.77-2.67(m,1H),2.61(dd,J＝7.78,5.52Hz,3H),2.47-2.40(m,1H),2.29-2.17(m,1H),1.99-1.82(m,2H),1.71-1.61(m,1H),1.45(s,9H).
步骤2：(S)-1-(3-氟丙基)吡咯烷-3-胺盐酸盐的合成
Figure PCTCN2021093736-appb-000054
将(S)-叔丁基(1-(3-氟丙基)吡咯烷-3-基)氨基甲酯(480.00mg，1.93mmol) 溶于1，4-二氧六环(3mL)中，然后加入盐酸-1，4-二氧六环溶液(4moL﹒L -1，4.94mL)，反应液为黄色透明溶液。反应液在25℃下搅拌3小时。TLC检测原料反应完全后，将反应溶液减压浓缩得化合物(S)-1-(3-氟丙基)吡咯烷-3-胺盐酸盐(450.00mg)。
1H NMR(400MHz,DMSO-d 6)δ8.80-8.42(m,3H),4.62(s,1H),4.51(s,1H),4.12-3.45(m,3H),3.17(br s,3H),2.35-1.99(m,4H).
步骤3：N-((S)-1-(3-氟丙基)吡咯烷-3-基)-6-(((1S,3R)-3-甲基-2-(2,2,2-三氟乙基)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚-1-基)吡啶-3-胺的合成
Figure PCTCN2021093736-appb-000055
将(1S,3R)-1-(5-溴吡啶-2-基)-3-甲基-2-(2,2,2-三氟乙基)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚(140.00mg，263.99μmol)溶于四氢呋喃(3mL)中加入(S)-1-(3-氟丙基)吡咯烷-3-胺盐酸盐(86.77mg，316.79μmol)，叔丁醇钠(152.22mg，1.58mmol)和甲烷磺酸(2-二环己基膦)-3，6-二甲氧基-2，4，6-三异丙基-1，1-联苯)(2-氨基-1，1-联苯-2-基)钯(II)(23.93mg，26.40μmol)在氮气环境下反应液在80℃下搅拌4小时。LCMS检测反应完成后，冷却至室温，过滤，滤液减压浓缩后，经过制备液相色谱纯化(Phenomenex Gemini C18柱，3um二氧化硅，30mm直径，75mm长度)；(使用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液)纯化得到化合物N-((S)-1-(3-氟丙基)吡咯烷-3-基)-6-(((1S,3R)-3-甲基-2-(2,2,2-三氟乙基)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚-1-基)吡啶-3-胺(37.79mg)。
MS m/z(ESI):365.1[M+H] +
1H NMR(400MHz,METHANOL-d 4)δ7.89(d,J＝2.76Hz,1H),7.46(d,J＝7.78Hz,1H),7.25(d,J＝8.53Hz,2H),7.09-7.03(m,2H),7.02-6.97(m,1H),4.98(s,1H),4.60(t,J＝5.65Hz,1H),4.49(t,J＝5.65Hz,1H),4.18(br s,1H),3.51-3.35(m,4H),3.14-2.99(m,5H),2.92(dd,J＝15.18,4.89Hz,1H),2.65(dd,J＝16.06,6.78Hz,1H),2.53-2.42(m,1H),2.12-1.92(m,3H),1.20(d,J＝6.78Hz,3H).
化合物3的绝对构型鉴定
二维核磁鉴定：
Figure PCTCN2021093736-appb-000056
NOESY图谱(图1)显示，化合物3的3位上的甲基氢与1位上的氢有明显的NOE效应，证明两者在同侧，1位上的吡啶基与3位上的甲基在6元哌啶环上的相对构型为反式，已知3位碳原子的绝对构型为R，因此1位碳原子的绝对构型为S。
实施例4：(S)-N-(3,5-二氟-4-((1S，3R)-3-甲基-2-(2,2,2-三氟乙基)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚-1-基)苯基)-1-(3-氟丙基)吡咯烷-3-胺(化合物4)的合成
Figure PCTCN2021093736-appb-000057
合成方法：
Figure PCTCN2021093736-appb-000058
步骤1：(S)-叔丁基(1-(3-氟丙基)吡咯烷-3-基)氨基甲酸酯的合成
Figure PCTCN2021093736-appb-000059
将(S)-叔丁基吡咯烷-3-基氨基甲酸酯(500.00mg，2.68mmol)溶于四 氢呋喃(10mL)中，加入氢氧化钠溶液(5mol﹒L -1，1.07mL)和1-碘-3-氟丙烷(529.88mg，2.82mmol)。反应液于25℃搅拌反应16小时。TLC检测原料反应完全后，将反应液用乙酸乙酯(50mL)稀释后，用饱和氯化铵溶液(10mL)洗涤，分别收集水相和有机相。水相用乙酸乙酯(20mL)萃取三次后，将所有有机相合并，用硫酸钠干燥后，有机相减压浓缩干，然后柱层析纯化(二氧化硅，二氯甲烷/甲醇＝100/1)得到产物(S)-叔丁基(1-(3-氟丙基)吡咯烷-3-基)氨基甲酸酯(480.00mg)。
1H NMR(400MHz,METHANOL-d 4)δ4.58-4.53(m,1H),4.46-4.40(m,1H),4.14-4.04(m,1H),2.93-2.85(m,1H),2.77-2.67(m,1H),2.61(dd,J＝7.78,5.52Hz,3H),2.47-2.40(m,1H),2.29-2.17(m,1H),1.99-1.82(m,2H),1.71-1.61(m,1H),1.45(s,9H).
步骤2：(S)-1-(3-氟丙基)吡咯烷-3-胺盐酸盐的合成
Figure PCTCN2021093736-appb-000060
将(S)-叔丁基(1-(3-氟丙基)吡咯烷-3-基)氨基甲酸酯(480.00mg，1.93mmol)溶于1，4-二氧六环(3mL)中，然后加入盐酸-1，4-二氧六环溶液(4moL﹒L -1，4.94mL)，反应液为黄色透明溶液。反应液在25℃下搅拌3小时。TLC检测原料反应完全后，将反应溶液减压浓缩得化合物(S)-1-(3-氟丙基)吡咯烷-3-胺盐酸盐(450.00mg)。
1H NMR(400MHz,DMSO-d 6)δ8.80-8.42(m,3H),4.62(s,1H),4.51(s,1H),4.12-3.45(m,3H),3.17(br s,3H),2.35-1.99(m,4H).
步骤3：(1S,3R)-1-(4-溴-2,6-二氟苯基)-3-甲基-2-(2,2,2-三氟乙基)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚的合成
Figure PCTCN2021093736-appb-000061
将(R)-1-(1H-吲哚-3-基)-N-(2,2,2-三氟乙基)丙烷-2-胺(890mg，3.47mmol)和4-溴-2,6-二氟苯甲醛(844.27mg，3.82mmol)溶于甲苯(10mL)和乙酸(2mL)中，反应液于90℃搅拌反应6小时。将反应液减压浓缩至干。然后柱层析纯化(二氧化硅，石油醚/乙酸乙酯＝20/1)得到产物(1S,3R)-1-(4-溴-2,6-二氟苯基)-3-甲基-2-(2,2,2-三氟乙基)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚(850mg)。
1H NMR(400MHz,METHANOL-d 4)δ7.45-7.43(d,J＝7.60Hz,1H), 7.24-7.20(m,3H),7.05-6.99(m,2H),5.36(s,1H),3.57-3.54(m,1H),3.46-3.40(m,1H),3.01-2.95(m,2H),2.70-2.65(m,1H),1.20(d,J＝6.4Hz,3H).
步骤4：(S)-N-(3,5-二氟-4-((1S，3R)-3-甲基-2-(2,2,2-三氟乙基)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚-1-基)苯基)-1-(3-氟丙基)吡咯烷-3-胺的合成
Figure PCTCN2021093736-appb-000062
将(1S，3R)-1-(4-溴-2,6-二氟苯基)-3-甲基-2-(2,2,2-三氟乙基)-2,3,4,9-四氢-1H-吡啶基[3,4-b]吲哚(80.00mg，174.20μmol)溶于四氢呋喃(2mL)中，加入(S)-1-(3-氟丙基)吡咯烷-3-胺盐酸盐(38.20mg，209.04μmol)和叔丁醇钠(100.45mg，1.05mmol)搅拌均匀后，在氮气范围下加入三(二苯亚甲基丙酮)二钯(31.90mg，34.84μmol)和(±)-2，2-双(二苯膦基)-11-联萘(54.23mg，87.10μmol)。反应液于80℃搅拌反应4小时。LCMS检测原料反应完全后，将反应液过滤后，用四氢呋喃淋洗滤饼，滤液减压浓缩干，经过制备液相色谱纯化(Phenomenex Gemini C18柱，7μm二氧化硅，50mm直径，250mm长度，使用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液)纯化得到化合物(S)-N-(3,5-二氟-4-((1S，3R)-3-甲基-2-(2,2,2-三氟乙基)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚-1-基)苯基)-1-(3-氟丙基)吡咯烷-3-胺(4.69mg)。
MS m/z(ESI):525.2[M+H] +
1H NMR(400MHz,METHANOL-d 4)δ8.49(s,1H),7.42(d,J＝7.3Hz,1H),7.21(d,J＝7.8Hz,1H),7.08-6.94(m,2H),6.19(d,J＝11.5Hz,2H),5.24(s,1H),4.60(t,J＝5.6Hz,1H),4.48(t,J＝5.6Hz,1H),4.11(br s,1H),3.62-3.53(m,1H),3.21(br s,1H),3.09-2.94(m,6H),2.63(dd,J＝15.3,4.3Hz,1H),2.51-2.38(m,1H),2.12-1.99(m,2H),1.96-1.85(m,1H),1.39-1.29(m,2H),1.19(d,J＝6.5Hz,3H)
实施例5：反式-N-(3,5-二氟-4-((1S,3R)-3-甲基-2-(2,2,2-三氟乙基)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚-1-基)苯基)-4-氟-1-(3-氟丙基)吡咯烷-3-胺(化合物5)的合成
Figure PCTCN2021093736-appb-000063
合成方法：
Figure PCTCN2021093736-appb-000064
步骤1：叔丁基(反式-4-氟-1-(3-氟丙基)吡咯烷-3-基)氨基甲酸酯的合成
Figure PCTCN2021093736-appb-000065
将1-氟-3-碘丙烷(151.86mg，807.87μmol)和叔丁基(反式-4-氟吡咯烷-3-基)氨基甲酸酯(150mg，734.43μmol)溶于乙腈(4mL)中，在25℃下加入碳酸钾(203.00mg，1.47mmol)，反应液在60℃下搅拌反应13小时。将反应液冷却至25℃，过滤，减压浓缩至干。然后柱层析纯化(二氧化硅，乙酸乙酯/甲醇＝10/1)得到产物叔丁基(反式-4-氟-1-(3-氟丙基)吡咯烷-3-基)氨基甲酸酯(0.15g)。
MS m/z(ESI):265.0[M+H] +；
1H NMR(400MHz,CHLOROFORM-d)δ4.87(br s,1H),4.59(t,J＝5.9Hz,1H),4.48(t,J＝5.9Hz,1H),4.16(br s,1H),3.29-3.05(m,1H),3.01-2.87(m,1H),2.78-2.41(m,4H),2.02-1.81(m,2H),1.48(s,9H).
步骤2：反式-4-氟-1-(3-氟丙基)吡咯烷-3-胺盐酸盐的合成
Figure PCTCN2021093736-appb-000066
将叔丁基((反式--4-氟-1-(3-氟丙基)吡咯烷-3-基)氨基甲酸酯(150mg，567.51μmol)溶于1,4-二氧六环(2mL)中，加入4M盐酸二氧六环(2.13mL)，反应液于25℃搅拌反应13小时。将反应液浓缩得到产物反式-4-氟-1-(3-氟丙基)吡咯烷-3-胺盐酸盐(0.12g)。
MS m/z(ESI):165.2[M+H] +。
步骤3：反式-N-(3,5-二氟-4-((1S,3R)-3-甲基-2-(2,2,2-三氟乙基)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚-1-基)苯基)-4-氟-1-(3-氟丙基)吡咯烷-3-胺的合成
Figure PCTCN2021093736-appb-000067
将(1S,3R)-1-(4-溴-2,6-二氟苯基)-3-甲基-2-(2,2,2-三氟乙基)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚(140mg，304.84μmol)和反式-4-氟-1-(3-氟丙基)吡咯烷-3-胺盐酸盐(86.74mg，365.81μmol)溶于叔戊醇(5mL)中，加入甲磺酸(2-二环己基膦基-3,6-二甲氧基-2,4,6-三异丙基-1,1-联苯)(2-氨基-1,1-联苯-2-基)钯(II)(25.50mg，30.48μmol)和碳酸铯(595.95mg，1.83mmol)，氮气置换三次后反应液120℃搅拌反应13小时。将反应液冷却至室温并倒入水(10mL)中并搅拌10分钟，乙酸乙酯(20mL)萃取2次，有机相用无水硫酸钠干燥，过滤，减压浓缩至干。然后柱层析纯化(二氧化硅，石油醚/乙酸乙酯＝2/1)和制备液相色谱纯化(PhenomenexGemini-NX柱：3μm二氧化硅，30mm直径，75mm长度；使用水(含有0.05％氨水)和乙腈的极性递减的混合物作为洗脱液)纯化得到产物反式-N-(3,5-二氟-4-((1S,3R)-3-甲基-2-(2,2,2-三氟乙基)-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚-1-基)苯基)-4-氟-1-(3-氟丙基)吡咯烷-3-胺(27.5mg)。
MS m/z(ESI):543.1[M+H] +；
1H NMR(400MHz,METHANOL-d 4)δ7.42(d,J＝7.5Hz,1H),7.22(d,J＝7.5Hz,1H),7.09-6.93(m,2H),6.30-6.16(m,2H),5.25(s,1H),4.58(t,J＝5.8Hz,1H),4.46(t,J＝5.8Hz,1H),4.11-3.87(m,1H),3.67-3.53(m,1H),3.43-3.34(m,3H),3.19-2.92(m,3H),2.81-2.55(m,4H),2.29(dd,J＝6.9,9.7Hz,1H),2.02-1.83(m,2H),1.19(d,J＝6.4Hz,3H).
实施例6：反式-N-[3,5-二氟-4-[(1S,3R)-3-甲基-2-(2,2,2-三氟乙基)-1,3,4,9-四氢吡啶并[3,4-b]吲哚-1-基]苯基]-1-(3-氟丙基)-4-甲基-吡咯烷-3-胺(化合物6)的合成
Figure PCTCN2021093736-appb-000068
合成方法：
Figure PCTCN2021093736-appb-000069
步骤1：叔丁基N-[反式-1-(3-氟丙基)-4-甲基-吡咯烷-3-基]氨基甲酸酯的合成
Figure PCTCN2021093736-appb-000070
将叔丁基N-[反式-4-甲基吡咯烷-3-基]氨基甲酸酯(80mg，399.45μmol)和碳酸钾(110.41mg，798.89μmol)溶解在乙腈(8mL)中，然后加入1-氟-3-碘-丙烷(90.11mg，479.34μmol)，反应液升温到50℃搅拌反应16h。LCMS监测反应完毕。反应液过滤，滤液浓缩到干经柱层析纯化(二氧化硅，乙酸乙酯/甲醇＝5/1)得化合物叔丁基N-[反式-1-(3-氟丙基)-4-甲基-吡咯烷-3-基]氨基甲酸酯(85mg)。
MS m/z(ESI):261.1[M+H] +
1H NMR(400MHz,METHANOL-d 4)δ4.66(br d,J＝2.4Hz,1H),4.61-4.42(m,2H),4.05-3.93(m,1H),3.71-3.56(m,2H),3.17(br d,J＝9.4Hz,1H),2.90(br s,2H),2.54(br s,1H),2.19(br d,J＝5.2Hz,2H),2.07-1.89(m,1H),1.47(s,9H),1.24-1.11(m,3H).
步骤2：反式-1-(3-氟丙基)-4-甲基-吡咯烷-3-胺盐酸盐的合成
Figure PCTCN2021093736-appb-000071
将叔丁基N-[反式-1-(3-氟丙基)-4-甲基-吡咯烷-3-基]氨基甲酸酯(80mg，307.28μmol)溶解在二氧六环(2mL)中，然后加入4M盐酸二氧六环(1.54mL)，反应液室温搅拌过夜。反应液浓缩到干得化合物反式-1-(3-氟丙基)-4-甲基-吡咯烷-3-胺盐酸盐(70mg)。
1H NMR(400MHz,METHANOL-d 4)δ4.73-4.64(m,1H),4.61-4.50(m,1H),4.23-4.06(m,1H),4.02-3.63(m,5H),3.54-3.42(m,1H),3.01-2.58(m,1H),2.32-2.12(m,2H),1.37-1.27(m,3H)
步骤3：反式-N-[3,5-二氟-4-[(1S,3R)-3-甲基-2-(2,2,2-三氟乙基)-1,3,4,9-四氢吡啶并[3,4-b]吲哚-1-基]苯基]-1-(3-氟丙基)-4-甲基-吡咯烷-3-胺的合成
Figure PCTCN2021093736-appb-000072
将反式-1-(3-氟丙基)-4-甲基-吡咯烷-3-胺盐酸盐(30mg，128.67μmol)，(1S,3R)-1-(4-溴-2,6-二氟-苯基)-3-甲基-2-(2,2,2-三氟乙基)-1,3,4,9-四氢吡啶并[3,4-b]吲哚(76.82mg，167.27μmol)和碳酸铯(167.69mg，514.68μmol)溶解在二氧六环(8mL)中，然后加入tBuBrettphos-Pd-G3(5.50mg，6.43μmol)。反应液用氮气置换三次，然后加热到120℃后搅拌过夜。LCMS监测反应完毕。向反应液中加入甲醇(15mL)，过滤，滤液浓缩经柱层析(二氧化硅，石油醚/乙酸乙酯＝2/1)和制备液相色谱纯化(Phenomenex Synergi C18柱：4um二氧化硅，30mm直径，150mm长度；使用水(含有0.225％甲酸)和乙腈的极性递减的混合物作为洗脱液)得到化合物反式-N-[3,5-二氟-4-[(1S,3R)-3-甲基-2-(2,2,2-三氟乙基)-1,3,4,9-四氢吡啶并[3,4-b]吲哚-1-基]苯基]-1-(3-氟丙基)-4-甲基-吡咯烷-3-胺(1.35mg)。
MS m/z(ESI):539.3[M+H] +；
1H NMR(400MHz,METHANOL-d 4)δ7.42(d,J＝7.6Hz,1H),7.21(d,J＝8.8Hz,1H),7.08-6.86(m,2H),6.21(d,J＝11.6Hz,2H),5.24(s,1H),4.62-4.58(m,1H),4.52-4.45(m,1H),3.73-3.65(m,1H),3.62-3.47(m,3H),3.23-3.15(m,2H),3.08-2.95(m,2H),2.86-2.54(m,2H),2.41-2.00(m,3H),1.42-1.25(m 2H),1.23(dd,J＝3.2,Hz,6.8Hz,3H),1.19(d,J＝6.4Hz,3H).
实施例7：3-((1S，3R)-1-(2,6-二氟-4-(((S)-1-(3-氟丙基)吡咯烷-3-基)氨基)苯基)-3-甲基-3，4-二氢-1H-吡啶并[3,4-b]吲哚-2(9H)-基)-2,2-二氟丙烷-1-醇的合成
Figure PCTCN2021093736-appb-000073
合成方法
Figure PCTCN2021093736-appb-000074
步骤1：3-((叔-丁基二苯基甲硅烷基)氧基)-2,2-二氟丙基三氟甲磺酸酯的合成
Figure PCTCN2021093736-appb-000075
将3-((叔-丁基二苯基甲硅烷基)氧基)-2,2-二氟丙烷-1-醇(1g，2.85mmol)和2,6-二甲基吡啶(366.88mg，3.42mmol)溶于无水二氯甲烷(15mL)中，在冰水浴的环境下滴加三氟甲磺酸酐(885.52mg，3.14mmol)。反应液在25℃ 下搅拌反应16小时。TLC(二氧化硅，石油醚：乙酸乙酯＝10:1)监测反应完成后，将反应液用依次用水(10mL)、盐酸(1mol/L，10mL)和饱和碳酸钠溶液(10mL)洗涤，收集的有机相用硫酸钠干燥后，有机相减压浓缩干，然后柱层析纯化(二氧化硅，石油醚/乙酸乙酯＝100/15)得到产物3-((叔-丁基二苯基甲硅烷基)氧基)-2,2-二氟丙基三氟甲磺酸酯(880.00mg)。
1H NMR(400MHz,METHANOL-d 4)δ7.66(d,J＝7.0Hz,4H),7.50-7.36(m,6H),4.96(t,J＝12.2Hz,2H),3.90(t,J＝12.0Hz,2H),1.06(s,9H).
步骤2：(R)-N-(1-(1H-吲哚-3-基)丙-2-基)-3-((叔丁基二苯基甲硅烷基)氧基)-2,2-二氟丙-1-胺的合成
Figure PCTCN2021093736-appb-000076
将3-((叔-丁基二苯基甲硅烷基)氧基)-2,2-二氟丙基三氟甲磺酸酯(822.49mg，1.70mmol)和(R)-1-(1H-吲哚-3-基)丙烷-2-胺(270mg,1.55mmol)溶于1,4-二氧六环(10mL)中，然后加入N-乙基-N-异丙基丙烷-2-胺(600.81mg，4.65mmol)。反应液在80℃下搅拌反应16小时。LCMS监测反应完成，反应液浓缩后经柱层析纯化(二氧化硅，石油醚/乙酸乙酯＝100/17)得到产物(R)-N-(1-(1H-吲哚-3-基)丙-2-基)-3-((叔丁基二苯基甲硅烷基)氧基)-2,2-二氟丙-1-胺(662.00mg)。
MS m/z(ESI):507.2[M+H] +；
1H NMR(400MHz,CHLOROFORM-d)δ7.72-7.63(m,4H),7.61(d,J＝7.9Hz,1H),7.49-7.33(m,7H),7.20(dt,J＝1.1,7.6Hz,1H),7.14-7.09(m,1H),7.01(d,J＝2.3Hz,1H),3.89-3.76(m,2H),3.27-3.05(m,3H),2.94-2.77(m,2H),1.13(d,J＝6.3Hz,3H),1.06(s,9H)
步骤3：(1S,3R)-1-(4-溴-2,6-二氟苯基)-2-(3-((叔-丁基二苯基甲硅烷基)氧基)-2,2-二氟丙基)-3-甲基-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚的合成
Figure PCTCN2021093736-appb-000077
将(R)-N-(1-(1H-吲哚-3-基)丙-2-基)-3-((叔丁基二苯基甲硅烷基)氧基)-2,2-二氟丙-1-胺(300mg，592.07μmol)和4-溴-2,6-二氟苯甲醛(143.93mg，651.27μmol)溶于甲苯(5mL)，然后加入乙酸(355.55mg，5.92mmol，338.62uL)。反应液在90℃下搅拌反应16小时。LCMS和TLC (石油醚：乙酸乙酯＝10：1)监测反应完成，反应液减压浓缩干。反应浓缩液用乙酸乙酯(5mL)稀释,然后用食盐水(5mL)洗涤三次，收集到的水相用乙酸乙酯(10mL)萃取三次，有机相用硫酸钠干燥后减压浓缩干，然后经薄层层析纯化(二氧化硅，石油醚/乙酸乙酯＝10/1)得(1S,3R)-1-(4-溴-2,6-二氟苯基)-2-(3-((叔-丁基二苯基甲硅烷基)氧基)-2,2-二氟丙基)-3-甲基-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚(350.00mg)。
MS m/z(ESI):708.9[M+H] +；
1H NMR(400MHz,CHLOROFORM-d)δ7.65-7.59(m,4H),7.52(br d,J＝6.5Hz,1H),7.44-7.37(m,6H),7.25-7.20(m,1H),7.12(m,J＝5.6,7.2Hz,2H),6.95(d,J＝8.3Hz,2H),5.28(s,1H),4.01-3.88(m,1H),3.69-3.54(m,2H),3.35-3.20(m,1H),2.98(dd,J＝4.6,14.7Hz,1H),2.83-2.69(m,1H),2.60(dd,J＝3.8,15.6Hz,1H),1.15(d,J＝6.5Hz,3H),1.04(s,9H).
步骤4：(S)-N-(4-((1S,3R)-2-(3-((叔-丁基二苯基甲硅烷基)氧基)-2,2-二氟丙基)-3-甲基-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚-1-基)-3,5-二氟苯基)-1-(3-氟丙基)吡咯烷-3-胺的合成
Figure PCTCN2021093736-appb-000078
将(1S,3R)-1-(4-溴-2,6-二氟苯基)-2-(3-((叔-丁基二苯基甲硅烷基)氧基)-2,2-二氟丙基)-3-甲基-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚(300.00mg，422.72μmol)和(S)-1-(3-氟丙基)吡咯烷-3-胺盐酸盐(129mg，507.27μmol)溶于四氢呋喃(10mL)，然后加入叔丁醇钠(243.75mg，2.54mol)和2,2-双(二苯膦基)-1,1-联萘(131.61mg，211.36μmol),三(二亚苄基丙酮)二钯(77.42mg，84.54μmol)在氮气环境下加入该反应体系。反应液在80℃下搅拌反应16小时。LCMS监测反应完成后，反应液减压浓缩干。反应浓缩液用乙酸乙酯稀释(10mL),然后用水(10mL)洗涤三次，收集到的水相用乙酸乙酯(20mL)萃取三次，有机相用硫酸钠干燥后减压浓缩干，然后经薄层层析纯化(二氧化硅，石油醚/四氢呋喃＝1/1，1％氨水)得(S)-N-(4-((1S,3R)-2-(3-((叔-丁基二苯基甲硅烷基)氧基)-2,2-二氟丙基)-3-甲基-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚-1-基)-3,5-二氟苯基)-1-(3-氟丙基)吡咯烷-3-胺(54.00mg)。
MS m/z(ESI):400.5[M+H] +
步骤5：3-((1S,3R)-1-(2,6-二氟-4-(((S)-1-(3-氟丙基)吡咯烷-3-基)氨基)苯基)-3-甲基-3,4-二氢-1H-吡啶并[3,4-b]吲哚-2(9H)-基)-2,2-二氟丙烷-1-醇的合成
Figure PCTCN2021093736-appb-000079
将(S)-N-(4-((1S,3R)-2-(3-((叔-丁基二苯基甲硅烷基)氧基)-2,2-二氟丙基)-3-甲基-2,3,4,9-四氢-1H-吡啶并[3,4-b]吲哚-1-基)-3,5-二氟苯基)-1-(3-氟丙基)吡咯烷-3-胺(50.00mg，64.52μmol)溶于四氢呋喃(2mL),加入四丁基氟化铵四氢呋喃溶液(1mol/L，129.04uL)。反应液在25℃下搅拌反应5个小时。TLC(二氧化硅，二氯甲烷：甲醇＝10：1)监测反应完成，然后向反应液中加入5mL水，在室温下搅拌10分钟。有机相用食盐水(5mL)洗涤三次，收集到的水相用乙酸乙酯(10mL)萃取三次，有机相用硫酸钠干燥后减压浓缩干，然后经薄层层析纯化(二氧化硅，二氯甲烷：甲醇＝10：1)得化合物3-((1S,3R)-1-(2,6-二氟-4-(((S)-1-(3-氟丙基)吡咯烷-3-基)氨基)苯基)-3-甲基-3,4-二氢-1H-吡啶并[3,4-b]吲哚-2(9H)-基)-2,2-二氟丙烷-1-醇(32.00mg)。
MS m/z(ESI):559.1[M+Na] +
1H NMR(400MHz,METHANOL-d 4)δ7.38(d,J＝7.3Hz,1H),7.18(d,J＝7.5Hz,1H),7.02-6.91(m,2H),6.14(d,J＝11.8Hz,2H),5.15(s,1H),4.53(t,J＝5.8Hz,1H),4.42(t,J＝5.6Hz,1H),3.99-3.91(m,1H),3.86-3.72(m,1H),3.66-3.57(m,1H),3.52-3.39(m,1H),3.20-3.08(m,1H),3.00-2.91(m,2H),2.84-2.73(m,2H),2.70-2.51(m,5H),2.37-2.26(m,1H),1.98-1.83(m,2H),1.76-1.65(m,1H),1.14(d,J＝6.5Hz,3H)
生物学活性及相关性质的测试
测试例1：本发明化合物对MCF7细胞内雌激素受体降解效果检测
1.实验目的
本实验的目的是测定本发明化合物对MCF7细胞内内源表达的雌激素受体的降解活性，根据DC 50及最大降解效率评价化合物的活性。
2.实验方法
MCF7细胞(ATCC,HTB-22)用含10％胎牛血清的DMEM(Gibco，11995-065)完全培养基进行培养。实验第一天，使用完全培养基将MCF7细胞以3000个/孔的密度种于384孔板，37℃，5％CO 2细胞培养箱培养。待测化合物溶解于DMSO,储存浓度为10mM,用Echo 550(Labcyte Inc.)稀释并加入细胞培养板内，各化合物处理的起始浓度为100nM，3倍梯度稀释，9个浓度点，设置含0.5％DMSO的空白对照，各浓度点设双复孔对照。37℃，5％CO 2细胞培养箱培养24小时。各细胞培养孔内加入多聚甲醛至细胞培养液 内，多聚甲醛终浓度约3.7％以固定细胞，作用30分钟后，弃上清，加入50μL PBS每孔洗涤一次；加入PBS含0.5％v/v Tween-20处理细胞30分钟，PBS洗涤一次；加入封闭液(PBS内含5％BSA，0.05％Tween-20)室温孵育1小时；去封闭液加入一抗混合液(抗-ER单抗，Estrogen Receptorα(D8H8)Rabbit mAb,GST,#8644S,1：1000稀释；抗-GAPDH单抗，GAPDH(D4C6R)Mouse mAb,GST,#97166S，1：2000稀释)室温孵育3小时；用PBST(PBS内含0.05％Tween-20)洗涤3次；加入检测二抗(800CW-羊抗兔IgG，LI-COR，P/N:926-32211,1:1000稀释；680RD-羊抗鼠IgG，LI-COR，#925-68070，1：1000稀释)，室温，避光孵育45分钟；PBST洗涤3次，使用Odyssey CLx读取各孔荧光信号，计算Chanel 800(ER)/Chanel 680(GAPDH)数值。以0.1μM fulvestrant处理孔作为100％降解对照，计算各浓度点的降解率，使用XlLfit分析处理数据，计算各化合物的降解活性DC 50及最大降解率Imax。数据分析见表1
表1 本发明化合物对MCF7细胞内雌激素受体降解活性
化合物编号 ER level DC 50(nM) 最大降解率
实施例1 0.41 106％
实施例2 8.5 92％
实施例3 0.15 104％
实施例4 0.04 99％
实施例5 0.58 80％
实施例6 0.50 90％
实施例7 0.06 99％
测试例2：本发明化合物对MCF7细胞增殖的抑制效果检测
1.实验目的
本实验的目的是测定本发明化合物对MCF7细胞体外增殖的抑制影响，根据IC 50及最大抑制效率评价化合物的活性。
2.实验方法
MCF7细胞(ATCC,HTB-22)用含10％胎牛血清的DMEM(Gibco，11995-065)完全培养基进行培养。实验第一天，使用完全培养基将MCF7细胞以500个/孔的密度种于384孔板，37℃，5％CO 2细胞培养箱过夜培养。第二天，加入待测化合物进行药物处理，采用Echo550(Labcyte Inc.)将储存浓度为10mM的化合物溶液进行稀释及转移至各细胞培养孔内，各化合物在细胞内的处理起始浓度为100nM，3倍梯度稀释，9个浓度点，设置含0.3％DMSO 的空白对照，各浓度点设双复孔对照。37℃，5％CO 2细胞培养箱培养7天，第八天，取出细胞培养板。加入
Figure PCTCN2021093736-appb-000080
Luminescent Cell Viability Assay(Promega,G7573),室温放置10分钟后，使用多标记酶标仪EnVision(PerkinElmer)读取发光信号值，用XLfit根据化合物的浓度和发光信号值计算各化合物的抑制活性IC 50。
3.数据分析见表2
表2 本发明化合物对MCF7细胞增殖的抑制活性
Figure PCTCN2021093736-appb-000081
测试例3：本发明化合物对CYP2C9、CYP2D6酶活性的抑制作用
本发明化合物对CYP2C9、CYP2D6酶活性的抑制采用如下试验方法测定。
一、试验材料及仪器
1.人肝微粒体(Corning 452117)
2.NADPH(Solarbio 705Y021)
3.阳性底物双氯芬酸(Sigma SLBV3438)、右美沙芬(TRC 3-EDO-175-1)和咪达唑仑(Cerilliant FE01161704)
4.阳性抑制剂磺胺苯吡唑(D.Ehrenstorfer GmbH 109012)、奎尼丁(TCI WEODL-RE)和酮康唑(Sigma 100M1091V)
5.AB Sciex Triple Quad 5500液质联用仪
二、试验步骤
1.100mM磷酸缓冲液(PBS)的配制：称取7.098g Na 2HPO 4，加入500mL纯水超声溶解，作为溶液A。称取3.400g KH 2PO 4，加入250mL纯水超声溶解，作为溶液B。将A溶液放置在搅拌器上缓慢加入B溶液直到pH值达到7.4配制成100mM的PBS缓冲液。
2.用100mM的PBS缓冲液配制10mM的NADPH溶液。用DMSO稀释10mM的本发明化合物储备液得到200×浓度的化合物工作液(6000、2000、600、200、60、20、0μM)。用DMSO稀释阳性抑制剂储备液得到200×浓度的阳性抑制剂工作液(磺胺苯吡唑，1000、300、100、30、10、3、0μM；奎尼丁/酮康唑，100、30、10、3、1、0.3、0μM)。用水、乙腈或乙腈/甲醇配制200×浓度的底物工作液(120μM双氯芬酸、400μM右美沙芬和200μM咪达唑仑)。
3.取2μl 20mg/ml的肝微粒体溶液、1μl底物工作液、1μl化合物工作液和176μl PBS缓冲液，混合均匀，置于37℃水浴中预孵育15分钟。阳性对照组加入1μl双氯芬酸、右美沙芬或咪达唑仑工作液代替化合物工作液。同时将10mM的NADPH溶液一起在37℃水浴中预孵育15分钟。15分钟后，取20μl NADPH加入到各个孔中，启动反应，37℃下孵育5分钟(CYP2C9)或20分钟(CYP2D6)。所有孵育样品设双样本。孵育相应时间后向所有样本中加入400ul含内标的冰甲醇终止反应。涡旋混匀，3220g、4℃离心40分钟。离心结束后转移100μL上清液到进样板，加入100μL超纯水混匀，用于LC-MS/MS分析。
经Excel XLfit 5.3.1.3计算得到本发明化合物对CYP2C9和CYP2D6的IC 50值。
表3 本发明化合物对CYP2C9和CYP2D6的IC 50值
实施例编号 CYP2C9(μM) CYP2D6(μM)
实施例3 >30 >30
实施例4 20 27
实施例7 8 23
测试例4：本发明化合物的血浆蛋白结合率测定
人血浆蛋白结合是控制可用于结合至靶标的游离(未结合)药物量的关键因素，在观察到的药物体内功效中起重要作用。因此，对于具有相似效力和暴露水平的化合物，具有高游离分数(低水平的血浆蛋白结合)的化合物可以展现出增强的功效。
本发明化合物在5个种属(人、猴、犬、大鼠和小鼠)血浆中的蛋白结合率采用如下试验方法测定。
一、试验材料及仪器
1.人血浆(BioIVT)、比格犬血浆(BioIVT)、SD大鼠血浆(BioIVT)、CD-1小鼠血浆(BioIVT)；
2.96孔平衡透析板(HTDialysis LLC,Gales Ferry,CT，HTD96B)，平衡 透析膜(MWCO 12-14K,#1101)；
3.阳性对照化合物华法林；
4.ABI QTrap 5500液质联用仪。
二、试验步骤
1.浓度为100mM磷酸钠盐和150mM NaCl的缓冲液的配制：用超纯水配制浓度为14.2g/L Na 2HPO 4和8.77g/L NaCl的碱性溶液，用超纯水配制浓度为12.0g/L NaH 2PO 4和8.77g/L NaCl的酸性溶液。用酸性溶液滴定碱性溶液至pH值为7.4配制成浓度为100mM磷酸钠盐和150mM NaCl的缓冲液。
2.透析膜的准备：将透析膜浸泡在超纯水中60分钟以便将膜分离成两片，然后用20％乙醇浸泡20分钟，最后用透析所用缓冲液浸泡20分钟。
3.血浆的准备：将冷冻的血浆迅速在室温下解冻，然后将血浆在4℃、3,220g下离心10分钟去除凝块，并将上清收集到新的离心管中。测定和记录血浆的pH值，使用pH值为7-8的血浆。
3.含化合物的血浆样品的配制：用DMSO稀释10mM的本发明化合物或阳性对照化合物的储备液得到200μM的工作液。597μl人、猴、犬、大鼠或小鼠血浆中加入3μl 200μM的化合物工作液得到终浓度为1μM的血浆样品。
4.平衡透析步骤：按照操作说明将透析装置组装起来。在透析膜的一侧加入120μL含1μM化合物的血浆样品，另一侧加入等体积的透析液(磷酸盐缓冲液)。试验设双样本。封上透析板，放入孵育装置，在37℃、5％CO 2及约100rpm转速下孵育6小时。孵育结束后，去除封膜，从每个孔的缓冲液和血浆侧吸取50μl到新板的不同孔中。在磷酸盐缓冲液样品中加入50μl空白血浆，在血浆样品中加入等体积的空白磷酸盐缓冲液，然后加入300μl含内标的乙腈沉淀蛋白。涡旋5分钟，在4℃、3,220g下离心30分钟。取100μl上清液至进样板，加入100μL超纯水混匀，用于LC-MS/MS分析。
测定化合物在缓冲液侧和血浆侧的峰面积。计算化合物的血浆蛋白结合率公式如下：
游离率％＝(化合物峰面积与内标峰面积比值 缓冲液侧/化合物峰面积与内标峰面积比值 血浆侧)×100
结合率％＝100-游离率％
所有的数据均通过Microsoft Excel软件进行计算。计算得到的本发明化合物的血浆蛋白结合率值。
表4 本发明化合物在人、犬、大鼠和小鼠血浆中的蛋白结合率值
Figure PCTCN2021093736-appb-000082
Figure PCTCN2021093736-appb-000083
测试例5：本发明化合物在pH值为7.4的磷酸盐缓冲液中的表观溶解度
为了使口服化合物到达作用部位，并且为了肠道的有效吸收，该化合物需处于溶液状态，因此具有高固有溶解度的化合物可能更适合于药物用途。
一、材料和试剂
待测化合物按记载方法制备。对照药孕酮从Sigma购买。pH值为7.4的磷酸盐缓冲液由本实验室配制。乙腈和甲醇从Fisher购买。其他试剂从市场购买。
1.5毫升平底玻璃小瓶(BioTech Solutions)；聚四氟乙烯/硅有机树脂瓶塞(BioTech Solutions)；聚四氟乙烯包被搅拌棒；MultiScreenHTS HV(0.45μm)96 well plate过滤板(Millipore,MSHVN4510 or MSHVN4550)；Eppendorf Thermomixer Comfort；Vacuum Manifold ORVMN96(Orochem)。
二、实验步骤
1)储备液的配制
用DMSO配制待测物和对照药孕酮的10mM储备液。
2)表观溶解度测定步骤
取30μL 10mM待测物储备液，以指定顺序加到对应96孔板的对应位置。在样品板的对应小瓶加入970μL的pH值7.4的磷酸盐缓冲液。实验为双平行。在每个小瓶中加一根搅拌棒，并盖上聚四氟乙烯/硅有机树脂瓶塞。随后将样品盘放进Eppendorf Thermomixer Comfort，以1100转的转速在25度条件下震荡2个小时。2小时后，去除瓶塞，用一块大磁铁吸走搅拌棒，然后从样品板转移样品至过滤板。用真空泵产生负压，过滤样品。转移5μL滤液到新的样品板，然后加入5μL DMSO和490μL 50％ACN(IS).H 2O(内标乙腈： 水＝1:1)。根据峰形情况，可能用一定比例的50％ACN(IS).H 2O来稀释样品稀释液以获得更好的峰形。稀释倍数可能因待测物溶解性的大小或其液质响应信号强弱而调整。
3)样品分析步骤
将进样板放进自动进样器的进样盘中，通过液质分析评估样品。
三、实验步骤
通过Microsoft Excel进行所有的计算。样品滤液的分析和定量，是通过使用液质对已知浓度的标准品峰的定性和定量完成的。计算得到的本发明化合物在PH值为7.4的磷酸盐缓冲液中的表观溶解度值。
表5 本发明化合物在pH值为7.4的磷酸盐缓冲液中的表观溶解度值
实施例编号 pH＝7.4表观溶解度(μM)
实施例3 92
实施例4 <0.3
实施例7 4
测试例6：本发明化合物对电压门控钾离子通道hERG是否有潜在抑制作用
hERG钾通道对心脏正常的电活动至关重要。心律失常可以通过多种药物阻断hERG通道来诱导。这种副作用是临床前安全性试验中药物失效的常见原因，因此hERG通道阻断活性的最小化可能是候选药物的理想特性。
一、材料和试剂
1.实验材料和仪器
Figure PCTCN2021093736-appb-000084
Figure PCTCN2021093736-appb-000085
2.细胞系和培养
稳定表达hERG离子通道的HEK293细胞株(货号：K1236)购自Invitrogen公司。该细胞株培养于含85％DMEM、10％透析胎牛血清、0.1mM非必需氨基酸溶液、100U/mL青霉素-链霉素溶液、25mM HEPES、5μg/mL杀稻瘟菌素和400μg/mL遗传霉素的培养基中。待细胞密度增长至培养皿底面积的40％～80％时，采用胰蛋白酶进行消化传代，每周传代三次。在实验前，细胞按照5×10 5的密度培养在6cm培养皿中，加入1μg/mL强力霉素诱导48小时，然后将细胞消化并接种在玻片上以备后续的手动膜片钳的实验。
3.待测化合物配置
1)按照SOP-ADMET-MAN-007标准操作规程，待测化合物用DMSO溶解并配制成终浓度为10mM的储备液。
2)用DMSO将储备液以1:3比例梯度稀释成其他三个中间浓度溶液，浓度分别为3.33mM、1.11mM和0.37mM。
3)实验开始前，用细胞外液将待测化合物储备液及中间溶液稀释1000倍得到系列浓度为10μM、3.33μM、1.11μM和0.37μM的工作溶液，同时用细胞外液将10mM储备液稀释333.33倍得到30μM的工作溶液。工作溶液中DMSO的含量为0.1-0.3％(体积比)。
(注：工作溶液中DMSO的含量应控制在1％(体积比)以内，避免细胞毒性。)
4)工作液配制完成后，肉眼观察工作液中是否有沉淀或者浑浊。如有，可能是由于化合物在生理溶液中溶解性不佳所致，可将其进一步水浴超声30分钟，以改善溶液的澄清度。
5)测定测试物在30μM、10μM、3.33μM、1.11μM和0.37μM这5个浓度下对hERG通道的潜在抑制作用并拟合量效曲线以及计算IC50。
二、实验方法
1.将培养皿中载有HEK293细胞的小玻片放置于显微操作台的灌流槽中。
2.在Olympus IX51，IX71或IX73倒置显微镜下将合适的细胞调置于视野中央，使用×10倍物镜找到玻璃电极的尖端，并置于视野的中央。然后使用微操纵器下移电极，同时调整粗准焦螺旋，使电极慢慢接近细胞。
3.当快接近细胞时，转换为×40倍物镜进行观察，通过微操纵器微调档，使电极逐渐接近细胞的表面。
4.给予负压，使电极尖与细胞膜之间形成电阻高于1GΩ的封接。
5.在电压钳模式下对瞬时电容电流Cfast进行补偿。然后重复给予短促的负压进行破膜，最终形成全细胞记录模式。
6.在膜电位钳制于-60mV的条件下，对缓慢电容电流Cslow、细胞膜电容(Cm)和输入膜电阻(Ra)分别进行补偿。
7.细胞稳定后，将钳制电压改为-90mV，采样频率设置为20kHz，过滤频率为10kHz。漏电流的检测条件为钳制电压转为-80mV，时程500ms。
8.hERG电流测试方法如下：施加4.8秒去极化命令电压将膜电位从-80mV去极化至+30mV，然后瞬间施加5.2秒的复极化电压使膜电位降至-50mV以去除通道失活，从而得以观察到hERG尾电流。尾电流的峰值为hERG电流的大小。
9.用于检测待测化合物的hERG电流在给药前均被持续记录120秒以评估受试细胞产生hERG电流的稳定性。只有在评价标准接受范围以内的稳定细胞才能进入后续化合物检测。
10.测定待测化合物对hERG电流的抑制作用：首先将在含0.1％DMSO的细胞外液中测定得到的hERG电流作为检测基线。在hERG电流保持稳定至少5分钟后将含有待测化合物的溶液从低浓度到高浓度依次灌注于细胞周围。每次灌流结束后等待约5分钟以使化合物充分作用于细胞并同步记录hERG电流。待记录电流趋于稳定后记录最后5个hERG电流值，并取其平均值作为其最终在特定浓度下的电流值。在测试完化合物后，加入150nM多菲莱德至同一个细胞上，将其电流完全抑制，作为该细胞的阳性对照。同时，阳性化合物多菲莱德在测试化合物实验结束前后用同一膜片钳系统进行同步检测，以确保整个检测系统的可靠性和灵敏性。
三、数据分析
数据由PatchMaster软件输出，按以下步骤进行分析：。
1)灌注空白溶剂或化合物梯度溶液后，稳定得到的5个连续。电流值，求取平均值，分别作为“尾电流大小 空白”和“尾电流大小 化合物”
2)电流抑制百分率通过以下公式进行计算。
Figure PCTCN2021093736-appb-000086
3)量效曲线通过Graphpad Prism 8.0软件进行拟合并计算IC 50值。
本发明化合物对hERG的抑制情况见下表6
表6 本发明化合物电压门控钾离子通道hERG的抑制活性
实施例编号 hERG IC 50(μM)
实施例3 10
实施例4 3.7
实施例7 8.4
测试例7：本发明化合物的小鼠药代动力学评价
实验材料
CD-1小鼠购自北京维通利华实验动物技术有限公司。DMSO(二甲基亚砜)、HP-β-CD(羟丙基-β-环糊精)、Tetraethylene Glycol(三缩四乙二醇),Captisol(SBE-β-CD,磺丁基-β-环糊精)购自Sigma。乙腈购自Merck(USA)。实验方法
雌性CD-1小鼠6只(20-30g，4-6周)，随机分成2组，每组3只。第1组尾静脉注射给予测试化合物，剂量为1mg/kg，溶媒5％DMSO in 10％HP-β-CD in water，第2组口服给予测试化合物，剂量10mg/kg，溶媒40％Tetraethylene Glycol(v/v),7.5％Captisol(w/v)in water。动物实验前正常喂食喂水。每组小鼠于给药前及给药后0.083(仅静脉注射组)、0.25、0.5、1、2、4、6、8和24h进行静脉采血。收集的全血样品置于K 2EDTA抗凝管中，离心5min后(12,000rpm，4℃)取血浆待测。
取小鼠血浆样品10μL，加入150μL乙腈溶剂(其中含内标化合物)沉淀蛋白，涡旋5min后，离心(14,000rpm)5min，上清液用含0.1％(v/v)FA的水稀释2倍，于LC-MS/MS系统(AB Sciex Triple Quad 6500+)进行定量检测。在测定样品浓度时随行CD-1小鼠血浆标准曲线和质控样品。对10x稀释样品，取2μL样品加入18μL的空白血浆，涡旋0.5min后，加入300μL乙腈溶剂(其中含内标化合物)沉淀蛋白，其余处理步骤同不稀释样品。
PK测试结果如下所示，本发明化合物在小鼠中表现出良好的PK性质和口服生物利用度：
表7 本发明化合物在小鼠中的PK
Figure PCTCN2021093736-appb-000087
Figure PCTCN2021093736-appb-000088
测试例8：本发明化合物在大鼠中血脑屏障(BBB)透过能力
药物能透过动物的血脑屏障在脑中有足够的暴露量是药物对脑转移病灶有效的关键，因此通过测量动物给药后血浆和脑组织中的药物浓度，可以评估药物在脑中的分布情况，进而判断药物能否在脑原位模型中起到抑制肿瘤生长的效果。
实验材料
SD雌性大鼠购自北京维通利华实验动物技术有限公司。MC(甲基纤维素)购自Sigma；乙腈购自Merck(USA)。PBS(磷酸盐缓冲盐)购自生工生物。实验方法
雌性SD大鼠6只(200-300g，6-8周)，随机分成2组，每组3只。分别给予本发明化合物，溶媒0.5％甲基纤维素水溶液。动物实验前正常喂水，禁食过夜，给药后四小时恢复给食。每组大鼠于给药后2h收集血浆和脑组织。收集的全血样品置于K 2EDTA抗凝管中，离心5min后(12,000rpm，4℃)取血浆待测；组织采集后用滤纸吸干，样品保存在-80度冰箱待测。
取大鼠血浆样品10μL，加入150μL乙腈溶剂(其中含内标化合物)沉淀蛋白，涡旋5min后，离心(14,000rpm)5min，上清液用含0.1％(v/v)FA的水稀释2倍，于LC-MS/MS系统(AB Sciex Triple Quad 6500+)进行定量检测。对10x稀释样品，取2μL样品加入18μL的空白血浆，涡旋0.5min后，加入300μL乙腈溶剂(其中含内标化合物)沉淀蛋白，其余处理步骤同不稀释样品。在测定血浆样品浓度时随行SD大鼠血浆标准曲线和血浆质控样品。
大鼠脑组织样品先用4倍质量体积的PBS匀浆液进行匀浆。取脑组织匀浆液样品20μL，加入20μL空白小鼠血浆进行稀释混匀再加入600μL乙腈溶剂(其中含内标化合物)沉淀蛋白，涡旋5min后，离心(14,000rpm)5min，上清液用含0.1％(v/v)FA的水稀释2倍，于LC-MS/MS系统(AB Sciex Triple Quad 6500+)进行定量检测。本发明化合物实施例3表现出优秀的血脑屏障透过能力，在大鼠的脑组织中药物暴露量较高。
BBB测试结果如下所示：
表8 本发明化合物大鼠血脑屏障透过实验
Figure PCTCN2021093736-appb-000089
Figure PCTCN2021093736-appb-000090
测试例9：本发明化合物对MCF-7小鼠皮下肿瘤模型的生长抑制实验
实验试剂
人乳腺癌MCF-7细胞：ATCC,HTB-22
17β-雌二醇片：Innovative Research of America,Cat No.:SE-121,60-day release,0.72mg/pellet
EMEM培养液：ATCC,Cat No.:30-2003
胎牛血清：Gbico；Cat No.:1099-141C
青链霉素(Pen Strep)：Gibco,Cat No.:15240-122
重组人胰岛素：上海翊圣，Cat.No.：40112ES60
0.25％胰酶-EDTA：Gibco,Cat No.:25200-072
D-PBS(无钙镁离子磷酸盐缓冲液)：Hyclone,Cat.No.:SH30256.01
Matrigel:Corning,Cat.No.:356237
实验方法
动物信息：NPG小鼠，雌性，6-7周，体重约19-28克，动物购自北京维通达生物技术有限公司，将小鼠饲养在SPF级的环境中，每个笼位单独送排风，所有动物都可以自由获取标准认证的商业实验室饮食和自由饮水。
细胞培养：人乳腺癌MCF-7细胞株体外培养，培养条件为EMEM(细胞培养液)中加入10％胎牛血清，1％Pen Strep，10μg/ml重组人胰岛素，37℃、5％CO 2孵箱。一周一次用0.25％胰酶-EDTA消化液进行常规消化处理传代。当细胞饱和度为80％-90％，数量达到要求时，收取细胞，计数。
细胞接种：将0.1ml/(含1×10 7)MCF-7细胞悬液(D-PBS：Matrigel，体积比为1:1)皮下接种于每只小鼠的右后背，并于细胞接种前四天皮下接种17β-雌二醇片。在接种细胞后第24天，依据肿瘤体积随机分组给药，分组当天为Day 0。
给药：化合物实施例1的给药剂量为1，3，或10mg/kg，口服给药(PO)，每天一次给药(QD)x3周。化合物实施例2的给药剂量为10mg/kg，口服给药(PO)，每天一次给药(QD)x3周。溶媒组8只小鼠，给药组6只小鼠。肿瘤测量和实验指标：
每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a x b 2，a和b分别表示肿瘤的长径和短径。每周两次测量小鼠体重。
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)/(溶剂对照组治疗结束时平均瘤体积-溶剂对照组开始治疗时平均瘤体积)]x100％。
实验结果：
见表9，图2和图3。在小鼠皮下移植瘤MCF-7模型中，本发明化合物实施例3在1mg/kg,3mg/kg,或10mg/kg一天一次口服给药对肿瘤生长具有显著抑制作用(P<0.01)，且具有较好的剂量反应关系，在3mg/kg和10mg/kg剂量下具有缩小肿瘤的效果。本发明化合物实施例3在10mg/kg一天一次口服给药对肿瘤生长具有显著抑制作用(P<0.01)，且具有缩小肿瘤的效果。化合物实施例3和实施例7在所尝试剂量下未显著影响小鼠体重。
表9 MCF-7皮下瘤模型肿瘤体积
Figure PCTCN2021093736-appb-000091
测试例10：本发明化合物对小鼠MCF-7脑原位肿瘤模型生长的抑制实验
实验试剂/仪器：
人乳腺癌MCF-7细胞：ATCC,HTB-22
17β-雌二醇片：Innovative Research of America,Cat No.:SE-121,60-day release,0.72mg/pellet
EMEM培养液：ATCC,Cat No.:30-2003
胎牛血清：Gibco，Cat.No.:1099-141C
青链霉素(Pen Strep)：Gibco,Cat No.:15240-122
重组人胰岛素：上海翊圣，Cat.No.：40112ES60
0.25％胰酶-EDTA：Gibco,Cat No.:25200-072
脑立体定位仪：瑞沃德，Cat No.:标准型/数显/单臂/小鼠/68055
微量注射泵：KDS，Cat No.:Legato130
微型手持颅钻：瑞沃德，Cat No.:78001
实验方法：
动物信息：NPG小鼠，雌性，6-8周，体重约17-29克，动物购自北京维通达生物技术有限公司，将小鼠饲养在SPF级的环境中，每个笼位单独送排风，所有动物都可以自由获取标准认证的商业实验室饮食和自由饮水。
细胞培养：人乳腺癌MCF-7细胞株体外培养，培养条件为EMEM(细胞培养液)中加入10％胎牛血清，1％Pen Strep，10μg/ml重组人胰岛素，37℃、 5％CO 2孵箱。一周两次用0.25％胰酶-EDTA消化液进行常规消化处理传代。当细胞饱和度为80％-90％，数量达到要求时，收取细胞，计数。
细胞接种：将15μl/(含2×10 6)MCF-7细胞悬液利用脑定位仪，微量注射泵和微型手持颅钻，接种于小鼠颅内，并于细胞接种前三天皮下接种17β-雌二醇片。在接种细胞后第8天，依据小鼠体重随机分组给药，分组当天为Day 0。
给药：Fulvestrant(氟维司群，阿斯利康)给药剂量为250mg/kg，皮下注射(SC)，每周一次给药(QW)，化合物实施例3的给药剂量为30mg/kg，口服给药(PO)，每天一次给药(QD)。溶媒组11只小鼠，给药组8只小鼠。所有组持续给药直到小鼠死亡，因状态差安乐死或者实验结束。
实验观察和结束：
每周两次测量小鼠体重，并观察小鼠生存状态。
Day 48结束实验，安乐死所有小鼠。
实验结果：
见图4和图5。在小鼠脑原位MCF-7模型中，Fulvestrant组小鼠(250mg/kg一周一次皮下注射给药)体重持续下降，小鼠的生存状况和溶剂对照组无明显差别(中位生存期，溶媒对照组29天，Fulvestrant组29.5天)。本发明实施例3化合物组小鼠(30mg/kg一天一次口服给药)体重平稳，状态无异常，直到实验终点，本发明实施例3化合物组小鼠均未出现死亡，相对于溶剂对照或者Fulvestrant，实施例3化合物对MCF-7脑原位肿瘤模型小鼠具有显著的抑制作用，小鼠的生存期显著的延长(P<0.01)。

Claims (13)

  1. 式(I)所示化合物或其药学上可接受的盐：
    Figure PCTCN2021093736-appb-100001
    其中，
    R 1、R 2、R 3、R 4独立选自H、F、Cl、Br、I、CN、C 1-C 6烷基、C 1-C 6烷氧基或C 3-C 6环烷基；
    X 1、X 2、X 3、X 4独立地选自CR 6或N；
    R 6选自H、F、Cl、Br、I、OH、CN、C 1-C 10烷基、C 3-C 10环烷基、3-10元杂环基、C 1-C 10烷氧基、C 3-C 10环烷基氧基或3-10元杂环基氧基；
    Y选自O或NH；
    R 5独立选自C 1-C 6烷基，所述C 1-C 6烷基任选被R a取代；
    R a选自F、Cl、Br、I、OH、CN、C 1-C 6烷基、C 1-C 6烷氧基或C 3-C 6环烷基；
    条件是，式(I)所示化合物不包含
    Figure PCTCN2021093736-appb-100002
  2. 根据权利要求1所述的式(I)所示化合物或其药学上可接受的盐，其特征在于，R 1、R 2、R 3、R 4独立选自H、F、Cl、Br、I、CN或C 1-C 3烷基。
  3. 根据权利要求1所述的式(I)所示化合物或其药学上可接受的盐，其特征在于，X 1、X 2、X 3、X 4独立地选自CR 6或N，且X 1、X 2、X 3、X 4基团中的至少2个选自CR 6。
  4. 根据权利要求1所述的式(I)所示化合物或其药学上可接受的盐，其特征在于，R 6选自H、F、Cl、Br、I、CN、C 1-C 3烷基或C 1-C 3烷氧基。
  5. 根据权利要求1所述的式(I)所示化合物或其药学上可接受的盐，其特征在于，R 5选自C 1-C 3烷基，所述C 1-C 3烷基任选被R a取代。
  6. 根据权利要求1所述的式(I)所示化合物或其药学上可接受的盐，其 特征在于，结构单元
    Figure PCTCN2021093736-appb-100003
    选自
    Figure PCTCN2021093736-appb-100004
    Figure PCTCN2021093736-appb-100005
  7. 根据权利要求1所述的式(I)所示化合物或其药学上可接受的盐，其特征在于，所述式(I)所示化合物或其药学上可接受的盐选自式(II)所示化合物或其药学可接受的盐：
    Figure PCTCN2021093736-appb-100006
  8. 根据权利要求1所述的式(I)所示化合物或其药学上可接受的盐，其特征在于，所述式(I)所示化合物或其药学上可接受的盐选自以下化合物或其药学可接受的盐：
    Figure PCTCN2021093736-appb-100007
    Figure PCTCN2021093736-appb-100008
  9. 根据权利要求1所述的式(I)所示化合物或其药学上可接受的盐，其特征在于，所述式(I)所示化合物或其药学上可接受的盐选自以下化合物或其药学可接受的盐：
    Figure PCTCN2021093736-appb-100009
  10. 一种药物组合物，所述组合物包含权利要求1至9任一项的化合物或其药学上可接受的盐，以及药学上可接受的辅料。
  11. 权利要求1至9任一项的化合物或其药学上可接受的盐、或权利要求10所述的药物组合物在制备预防或者治疗雌激素受体相关疾病的药物中的用途；
    优选地，所述雌激素受体相关疾病为肿瘤，
    更优选地，所述肿瘤为乳腺癌，
    更优选地，所述肿瘤为ER阳性乳腺癌，
    更优选地，所述肿瘤为ER阳性乳腺癌脑转移。
  12. 一种预防或治疗雌激素受体相关疾病的方法，所述方法包括向有此 需要的对象施用权利要求1至9任一项所述的化合物或其药学上可接受的盐、或权利要求10所述的药物组合物；
    优选地，所述雌激素受体相关疾病为肿瘤，
    更优选地，所述肿瘤为乳腺癌，
    更优选地，所述肿瘤为ER阳性乳腺癌，
    更优选地，所述肿瘤为ER阳性乳腺癌脑转移。
  13. 一种用于预防或治疗雌激素受体相关疾病的药物组合物，所述药物组合物包含权利要求1至9任一项所述的化合物或其药学上可接受的盐，以及药学上可接受的辅料；
    优选地，所述雌激素受体相关疾病为肿瘤，
    更优选地，所述肿瘤为乳腺癌，
    更优选地，所述肿瘤为ER阳性乳腺癌，
    更优选地，所述肿瘤为ER阳性乳腺癌脑转移。
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