Estrogen receptor modulator compounds and their uses

Abstract

This application provides a compound or a pharmaceutically acceptable salt thereof having estrogen receptor modulating activity or function, wherein the compound has the structure of Formula (I) and possesses the substituents and structural features described herein. This application also provides a pharmaceutical composition comprising the compound of Formula (I) or a pharmaceutically acceptable salt thereof, and the use of the compound of Formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating estrogen receptor-related diseases. #imgabs0#

Description

[0001] This application claims priority to three prior applications: patent application number 202010536528.6, filed with the State Intellectual Property Office on June 12, 2020, entitled “Estrogen Receptor Modulator Compounds and Uses Thereof”; patent application number 202010572513.5, filed with the State Intellectual Property Office on June 22, 2020, entitled “Estrogen Receptor Modulator Compounds and Uses Thereof”; and patent application number 202110573837.5, filed with the State Intellectual Property Office on May 25, 2021, entitled “Estrogen Receptor Modulator Compounds and Uses Thereof”. The full texts of the three applications are incorporated herein by reference. Technical Field

[0002] The present invention relates to an estrogen receptor modulator compound or a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the compound, and use of the compound in preventing or treating estrogen receptor-related diseases. Background Art

[0003] Estrogen (E2) and estrogen receptor α (ERα) are important drivers of breast cancer development. More than two-thirds of breast cancer patients express ER transcription factors, and in most ER-positive patients, even in tumors that progress after early endocrine therapy, ER remains a key driver. Therefore, ER is a major target for breast cancer treatment (Pharmacology & Therapeutics 186(2018)1-24). Endocrine therapy aims to reduce ER activity. There are three main types of endocrine therapy: selective estrogen receptor modulators (SERMs), such as tamoxifen, which are allosteric modulators of ER that inhibit its transcriptional activity upon binding to ER; aromatase inhibitors (AIs), which reduce estrogen levels in the body by inhibiting the conversion of androgens to estrogens; and selective estrogen receptor downregulators, such as fulvestrant, which not only act as ER antagonists to inhibit its activity but also induce ER protein degradation. Although endocrine therapy is the first choice for patients with estrogen receptor-positive breast cancer, approximately 30% of patients will relapse after treatment, and nearly all patients with metastatic breast cancer will develop resistance and progress. There are two main mechanisms of endocrine therapy resistance. One is centered on the estrogen receptor signaling pathway itself, including activating mutations, amplifications, fusions with other genes in the gene encoding the estrogen receptor, ESR1, and dysregulation of estrogen receptor coregulators and downstream cell cycle control factors. The other mechanism involves the activation of signaling pathways that cross-react with the estrogen receptor signaling pathway, such as growth factor receptor pathways (Oncol Ther, 2017, 5:17–29).

[0004] Fulvestrant is the first and only selective estrogen receptor downregulator (SERD) drug clinically approved for the treatment of postmenopausal patients with ER-positive, metastatic breast cancer after progression on tamoxifen or aromatase inhibitors. In addition, AstraZeneca (see patent application WO2018077630A1) and Genentech (see patent application WO2019245974A1) have also disclosed a series of structurally novel SERD compounds and corresponding medical uses. Multiple research data have shown that ER degradation is not completely achieved in patients treated with fulvestrant, but this may be due to the limited dose of fulvestrant (up to 500 mg, mainly due to its pharmacodynamic characteristics and intramuscular administration route, which limit the maximum dose that can be given to patients). Therefore, SERD drugs with higher bioavailability, greater ER antagonism, greater ER degradation, and convenient oral administration that can be used in premenopausal patients with high estrogen levels are urgently needed in clinical practice. Summary of the Invention

[0005] In order to solve at least one of the above technical problems, the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof:

[0006]

[0007] in,

[0008] X 1 、X 2 、X 3 、X 4 Independently selected from CR 7 or N;

[0009] R 7 is selected from H, F, Cl, Br, I, OH, CN, C1-C6 alkyl, C3-C6 cycloalkyl, 3-6 membered heterocyclyl, C1-C6 alkoxy, C3-C6 cycloalkyloxy or 3-6 membered heterocyclyloxy;

[0010] Het is selected from

[0011] R 1 、R 2 、R 9 independently selected from hydrogen, OH, F, Cl, Br, I, C1-C6 alkyl or C2-C8 alkenyl;

[0012] Or, R 1 、R 2The carbon atoms to which it is connected together form a C3-C6 cycloalkyl group or a 3-6 membered heterocycloalkyl group, wherein the C3-C6 cycloalkyl group or the 3-6 membered heterocycloalkyl group is optionally replaced by R a replace;

[0013] R 5 is selected from C1-C6 alkyl, C2-C8 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl or 3-6 membered heterocycloalkyl, wherein the C1-C6 alkyl, C2-C8 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl or 3-6 membered heterocycloalkyl is optionally substituted with one or more groups selected from the group consisting of deuterium, F, Cl, Br, I, CN, OH, OCH3 and SO2CH3;

[0014] R 6 is selected from H, C1-C6 alkyl, C2-C8 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl or 3-6 membered heterocycloalkyl, wherein the C1-C6 alkyl, C2-C8 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl or 3-6 membered heterocycloalkyl is optionally substituted with one or more groups selected from the group consisting of F, Cl, Br, I, CN, OH, OCH3 and SO2CH3;

[0015] Or, R 1 or R 2 One and R 6 and the C and N to which they are connected together form a 3-6 membered heterocyclic group;

[0016] R 3 Selected from H, CN, COOH, C(O)OR b 、OC(O)R b 、CONH2、C(O)NR b R c 、SO2R b 、SO2NR b R c , C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, C3-C6 heterocyclic, C6-C 10 Aryl or C5-C 10 Heteroaryl, the C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclic, C6-C 10 Aryl or C5-C 10 Heteroaryl is optionally substituted by one or more R d replace;

[0017] R 4is selected from H, F, Cl, Br, I, OH, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl or C3-C6 heterocyclyl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl or C3-C6 heterocyclyl is optionally substituted with one or more groups selected from the group consisting of F, Cl, Br, I, CN, OH, OCH3 and SO2CH3;

[0018] Ring Q is selected from C6-C 10 Aryl or 5-10 membered heteroaryl, the C6-C 10 Aryl or 5-10 membered heteroaryl is optionally substituted by one or more R 8 replace;

[0019] R 8 Selected from F, Cl, Br, I, OH, CN, COOH, C(O)OR b 、OC(O)R b 、CONH2、C(O)NR b R c NR c C(O)R b 、SO2R b 、SO2NR b , C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, 3-6 membered heterocyclic group, C6-C 10 Aryl or 5-10 membered heteroaryl, the C3-C6 cycloalkyl, 3-6 membered heterocyclic group, C6-C 10 The aryl or 5-10 membered heteroaryl group is optionally substituted by one or more groups selected from the group consisting of F, Cl, Br, I, OH, CN, C1-C6 alkyl, and C1-C6 alkoxy;

[0020] R a Selected from halogen, OH, CN, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl or 3-6 membered heterocyclic group, the C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 The cycloalkyl or 3-6 membered heterocyclic group is optionally substituted by halogen;

[0021] R b independently selected from C1-C6 alkyl, C2-C8 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl, 3-6 membered heterocyclyl, C6-C 10 Aryl or 5-10 membered heteroaryl;

[0022] R cindependently selected from H, C1-C6 alkyl, C2-C8 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl, 3-6 membered heterocyclyl, C6-C 10 Aryl or 5-10 membered heteroaryl;

[0023] R d independently selected from F, Cl, Br, I, OH, CN, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl or 3-6 membered heterocyclyl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl or 3-6 membered heterocyclyl is optionally substituted with one or more groups selected from F, Cl, Br, I or OH;

[0024] n is 0, 1, or 2;

[0025] m is 1, 2, 3 or 4;

[0026] p is 1 or 2;

[0027] The conditions are: (1) When n is 0 or 1, R 1 is not H, F or methyl; (2) when m is 1 or 2, R 9 Not for H.

[0028] In some embodiments, R 5 Selected from C1-C6 alkyl, C2-C8 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl or 3-6 membered heterocycloalkyl, wherein the C1-C6 alkyl, C2-C8 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl or 3-6 membered heterocycloalkyl is optionally substituted by one or more groups selected from the following: F, Cl, Br, I, CN, OH, OCH3 and SO2CH3.

[0029] In some embodiments, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound represented by formula (Ia) or a pharmaceutically acceptable salt thereof:

[0030]

[0031] In some embodiments, the Het is selected from

[0032] In some embodiments, the Het is selected from where R 1 、R 2 The carbon atoms to which it is connected together form a C3-C6 cycloalkyl group or a 3-6 membered heterocycloalkyl group, wherein the C3-C6 cycloalkyl group or the 3-6 membered heterocycloalkyl group is optionally replaced by R a replace.

[0033] In some embodiments, Het is selected from where R1 、R 2 The carbon atoms to which it is connected together form a C3-C6 cycloalkyl group or a 3-6 membered heterocycloalkyl group, wherein the C3-C6 cycloalkyl group or the 3-6 membered heterocycloalkyl group is optionally replaced by R a Substitution; said n is 1.

[0034] In some embodiments, the Het is selected from where R 1 、R 2 Together with the carbon atom to which it is attached, it forms cyclopropane.

[0035] In some embodiments, the Het is selected from where R 1 or R 2 One and R 6 and the C and N to which they are attached together form a 3-6 membered heterocyclic group.

[0036] In some embodiments, the Het is selected from where R 1 or R 2 One and R 6 and the C and N to which they are connected together form a 3-6 membered heterocyclic group; wherein n is 1.

[0037] In some embodiments, Het is selected from where R 1 or R 2 One and R 6 Together with the C and N to which they are connected, they form a piperidine ring.

[0038] In some embodiments, the Het is selected from

[0039] In some embodiments, the R 9 is selected from hydrogen, OH, F, Cl, Br, I or C1-C6 alkyl.

[0040] In some embodiments, the R 9 Selected from hydrogen or CH3.

[0041] In some embodiments, said m is selected from 1.

[0042] In some embodiments, m is selected from 3.

[0043] In some embodiments, m is selected from 4.

[0044] In some embodiments, the Het is selected from

[0045] In some embodiments, p is 1.

[0046] In some embodiments, the Het is selected from

[0047] In some embodiments, the Het is selected from

[0048] In some embodiments, the Het is selected from

[0049] In some embodiments, the R 3 Selected from C1-C6 alkyl, CN, COOH, C(O)OR b 、OC(O)R b 、CONH2、C(O)NR b R c or C6-C 10 Aryl; the C1-C6 alkyl or C6-C 10 The aryl group is optionally substituted with one or more R d Replacement, R d independently selected from F, Cl, Br, I, OH, CN or C1-C6 alkyl optionally substituted by one or more groups selected from F, Cl, Br, I or OH.

[0050] In some embodiments, the R 3 Selected from C1-C6 alkyl or C6-C 10 Aryl; the C1-C6 alkyl or C6-C 10 The aryl group is optionally substituted with one or more R d Replacement, R d independently selected from F, Cl, Br, OH or C1-C6 alkyl optionally substituted by one or more groups selected from F, Cl, Br or OH.

[0051] In some embodiments, the R 3 Selected from C1-C3 alkyl or phenyl; the C1-C3 alkyl or phenyl is optionally replaced by one or more R d Replacement, R d independently selected from F, OH or trifluoromethyl.

[0052] In some embodiments, the R 3 Selected from 4-trifluoromethylphenyl, -CH2CF3,

[0053] In some embodiments, the R 4 is selected from C1-C6 alkyl groups optionally substituted by F, Cl, Br or I.

[0054] In some embodiments, the R 4 It is a methyl group.

[0055] In some embodiments, the R 5 is a C1-C6 alkyl group optionally substituted by deuterium, F, Cl, Br or I.

[0056] In some embodiments, the R 5 is a C1-C6 alkyl group optionally substituted by F, Cl, Br or I.

[0057] In some embodiments, the R 5 It is 1,1-dideutero-3-fluoropropyl, 3-fluoropropyl or 2-fluoroethyl.

[0058] In some embodiments, the R 5 is 3-fluoropropyl or 2-fluoroethyl.

[0059] In some embodiments, the X1 is selected from CR 7 or N, X2, X3, X4 are all CR 7 .

[0060] In some embodiments, X1 is selected from CH or N, and X2, X3, and X4 are all CH.

[0061] In some embodiments, the R 7 is selected from H, F, Cl, Br or I.

[0062] In some embodiments, the R 6 Selected from H or C1-C6 alkyl.

[0063] In some embodiments, when Het is combined with NR 6 When the connected atom is a chiral carbon, the configuration is (S)-configuration.

[0064] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (II) or a pharmaceutically acceptable salt thereof:

[0065]

[0066] where X 1 、X 2 、X 3 、X 4 、Het、R 3 、R 4 、R 6 As defined in formula (I);

[0067] Y is selected from NR 10 , O or S;

[0068] R 10Selected from H, C1-C6 alkyl, C2-C8 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl or 3-6 membered heterocycloalkyl, wherein the C1-C6 alkyl, C2-C8 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl or 3-6 membered heterocycloalkyl is optionally substituted by one or more groups selected from the following: F, Cl, Br, I, CN, OH, OCH3 and SO2CH3.

[0069] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (IIa) or a pharmaceutically acceptable salt thereof:

[0070]

[0071] where X 1 、X 2 、X 3 、X 4 、Het、R 3 、R 4 、R 6 As defined in formula (I);

[0072] Y is selected from NR 10 , O or S;

[0073] R 10 Selected from H, C1-C6 alkyl, C2-C8 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl or 3-6 membered heterocycloalkyl, wherein the C1-C6 alkyl, C2-C8 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl or 3-6 membered heterocycloalkyl is optionally substituted by one or more groups selected from the following: F, Cl, Br, I, CN, OH, OCH3 and SO2CH3.

[0074] In some embodiments, Y is selected from NR 10 .

[0075] In some embodiments, R 10 Selected from H, C1-C6 alkyl, C3-C6 cycloalkyl or 3-6 membered heterocycloalkyl.

[0076] In some embodiments, R 10 For H.

[0077] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (III) or a pharmaceutically acceptable salt thereof:

[0078]

[0079] where X 1 、X 2 、X 3 、X4 、Het、R 3 、R 4 、R 6 As defined in formula (I).

[0080] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (IIIa) or a pharmaceutically acceptable salt thereof:

[0081]

[0082] where X 1 、X 2 、X 3 、X 4 、Het、R 3 、R 4 、R 6 As defined in formula (I).

[0083] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (IV) or a pharmaceutically acceptable salt thereof:

[0084]

[0085] where X 1 、X 2 、X 3 、X 4 、Het、R 3 、R 4 、R 6 、R 8 As defined in formula (I).

[0086] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (IVa) or a pharmaceutically acceptable salt thereof:

[0087]

[0088] where X 1 、X 2 、X 3 、X 4 、Het、R 3 、R 4 、R 6 、R 8 As defined in formula (I).

[0089] In some embodiments, the compound of formula (IV) wherein R 8 Selected from C6-C 10 Aryl or 5-10 membered heteroaryl, the C6-C 10The aryl or 5-10 membered heteroaryl is optionally substituted by one or more groups selected from the group consisting of F, Cl, Br, I, OH, CN, C1-C6 alkyl, and C1-C6 alkoxy.

[0090] In some embodiments, the compound of formula (IV) wherein R 8 is selected from phenyl or 5-6 membered heteroaryl, wherein the phenyl or 5-6 membered heteroaryl is optionally substituted by one or more groups selected from the following groups: F, Cl, Br, I, OH, C1-C3 alkyl, C1-C3 alkoxy.

[0091] In some embodiments, the compound of formula (IV) wherein R 8 is selected from pyrazole, wherein the pyrazole is optionally substituted with one or more methyl groups.

[0092] In some embodiments, the compound of formula (IV) wherein R 8 Selected from 1H-pyrazol-4-yl or 1-methyl-1H-pyrazol-4-yl.

[0093] In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or pharmaceutically acceptable salts thereof:

[0094]

[0095]

[0096]

[0097] In one aspect, the compounds described herein exist as racemic mixtures or in enantiomerically enriched or enantiomerically pure forms. In certain embodiments, the compounds described herein are prepared as their individual stereoisomers by reacting the racemic mixture of the compound with an optically active resolving agent to form a pair of diastereomeric compounds / salts, separating the diastereoisomers and recovering optically pure enantiomers. In some embodiments, the resolution of enantiomers is carried out using covalent diastereomeric derivatives of the compounds described herein. In another embodiment, diastereoisomers are separated by separation / resolution techniques based on solubility differences. In certain embodiments, the compounds described herein are prepared as their individual stereoisomers by enzymatic resolution. In some embodiments, the resolution of individual stereoisomers is carried out using lipase or esterase. In some embodiments, the resolution of individual stereoisomers is carried out by asymmetric deacylation catalyzed by lipase or esterase. In other embodiments, separation of stereoisomers is performed by chromatography, or by forming diastereomeric salts and separating by recrystallization or chromatography, or any combination thereof. In some embodiments, stereoisomers are obtained by stereoselective synthesis.

[0098] The present invention also provides a pharmaceutical composition comprising a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient.

[0099] In some embodiments, the pharmaceutical composition is formulated for intravenous injection, subcutaneous injection, oral administration, or topical administration.

[0100] In some embodiments, the pharmaceutical composition is a tablet, pill, capsule, liquid, suspension, gel, dispersion, solution, emulsion, ointment, or lotion.

[0101] Furthermore, 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 drug for preventing or treating estrogen receptor-related diseases.

[0102] Furthermore, 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 preventing or treating estrogen receptor-related diseases.

[0103] Furthermore, 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 preventing or treating estrogen receptor-related diseases.

[0104] Furthermore, the present invention relates to a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for preventing or treating estrogen receptor-related diseases.

[0105] The present invention also relates to a method for preventing or treating estrogen receptor-related diseases, which comprises administering to a subject in need thereof a preventive or therapeutically effective dose of a compound represented by formula (I) of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0106] In some embodiments of the present invention, the estrogen receptor-related diseases include but are not limited to cancer and autoimmune diseases.

[0107] In some embodiments of the present invention, the estrogen receptor-related disease is preferably a tumor.

[0108] Definitions and Explanations of Terms

[0109] Unless otherwise indicated, the terms used in this application have the following meanings. The definitions of groups and terms described in this application, including their definitions as examples, exemplary definitions, preferred definitions, definitions described in tables, and definitions of specific compounds in the examples, may be combined and coupled with each other in any manner. A specific term should not be considered as undefined or unclear unless specifically defined, but should be understood according to its common meaning in the art. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0110] In this article Indicates the junction site.

[0111] The diagrammatic representations of racemates or enantiomerically pure compounds herein are from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise indicated, wedge and dotted wedge keys are used. To indicate the absolute configuration of a stereocenter, use black real and imaginary bonds. Indicates the relative configuration of a stereocenter (such as the cis-trans configuration of an alicyclic compound).

[0112] The term "tautomer" refers to functional isomers resulting from the rapid shift of an atom between two positions in a molecule. The compounds of the present invention may exhibit tautomerism. Tautomeric compounds can exist as two or more interconvertible species. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer usually result in a mixture with physical and chemical properties consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones, such as acetaldehyde, the keto form predominates, while in phenols, the enol form predominates. The present invention encompasses all tautomeric forms of the compounds.

[0113] The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers, diastereomers and conformational isomers.

[0114] The compounds of the present invention may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms or asymmetric double bonds, and thus the compounds of the present invention may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E and Z geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures or other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the definition of the compounds of the present invention and mixtures thereof. Additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms or asymmetric phosphorus atoms may be present in substituents such as alkyl groups, and all of these isomers and mixtures thereof involved in the substituents are also within the definition of the compounds of the present invention. The compounds of the present invention containing an asymmetric atom can be isolated in optically pure or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.

[0115] The term "substituted" refers to the replacement of any one or more hydrogen atoms on a particular atom by a substituent, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is an oxo (i.e., =O), it means that two hydrogen atoms are replaced. Oxo does not occur on aromatic groups.

[0116] The term "optionally" or "optionally" means that the event or circumstance described subsequently may or may not occur, and the description includes both the occurrence of the event or circumstance and the non-occurrence of the event or circumstance. For example, an ethyl group is "optionally" substituted with a halogen, meaning that the ethyl group may be unsubstituted (CH2CH3), monosubstituted (such as CH2CH2F), polysubstituted (such as CHFCH2F, CH2CHF2, etc.), or fully substituted (CF2CF3). It will be understood by those skilled in the art that for any group containing one or more substituents, no substitution or substitution pattern that would be sterically impossible and / or incomposable to synthesize will be introduced.

[0117] In this article, C m-n , means that the moiety has an integer number of carbon atoms in a given range. For example, "C 1-6 ” means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.

[0118] When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. For example, if a group is substituted with two R's, each R has an independent option.

[0119] When the number of a linking group is 0, such as -(CH2)0-, it means that the linking group is a bond.

[0120] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0121] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of a non-toxic acid or base, including salts of inorganic acids and bases, and organic acids and bases. Examples of pharmaceutically acceptable salts include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, and salts with basic or acidic amino acids.

[0122] The term "solvate" refers to an association or complex of one or more solvent molecules with a compound of the present invention. Examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate (EtOAc), acetic acid (AcOH), and ethanolamine.

[0123] The term "pharmaceutical composition" refers to a mixture of one or more compounds described herein, or physiologically / pharmaceutically acceptable salts or prodrugs thereof, with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the compound to an organism. The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present application to an organism.

[0124] The term "substituted" refers to the replacement of any one or more hydrogen atoms on a particular atom by a substituent, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is an oxo (i.e., =O), it means that two hydrogen atoms are replaced. Oxo does not occur on aromatic groups.

[0125] The term "optionally" or "optionally" means that the event or circumstance described subsequently may or may not occur, and the description includes both the occurrence of the event or circumstance and the non-occurrence of the event or circumstance. For example, an ethyl group is "optionally" substituted with a halogen, meaning that the ethyl group may be unsubstituted (CH2CH3), monosubstituted (such as CH2CH2F), polysubstituted (such as CHFCH2F, CH2CHF2, etc.), or fully substituted (CF2CF3). It will be understood by those skilled in the art that for any group containing one or more substituents, no substitution or substitution pattern that would be sterically impossible and / or incomposable to synthesize will be introduced.

[0126] The term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

[0127] The term "hydroxy" refers to an -OH group.

[0128] The term "cyano" refers to a -CN group.

[0129] The term "mercapto" refers to a -SH group.

[0130] The term "amino" refers to a -NH2 group.

[0131] The term "nitro" refers to a -NO2 group.

[0132] The term "alkyl" refers to a group of the formula C n H 2n+1 The alkyl group can be straight or branched. For example, the term "C1-C6 alkyl" is understood to mean a straight or branched saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5 or 6 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, 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. Similarly, the alkyl portion of the alkoxy group (i.e., alkyl) has the same definition as above. This article describes "C 1-6 The alkyl group may contain a C 1-3 Alkyl" and other ranges.

[0133] The term "alkenyl" refers to a linear or branched unsaturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms and having at least one double bond. Non-limiting examples of alkenyl groups include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, 1,3-butadienyl, and the like. The term "C2-C8 alkenyl" is understood to mean a linear or branched monovalent hydrocarbon group containing one or more double bonds and having 2, 3, 4, 5, 6, 7, or 8 carbon atoms.

[0134] The term "alkynyl" refers to a linear or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, having at least one triple bond. Non-limiting examples of alkynyl groups include, but are not limited to, ethynyl (-C≡CH), 1-propynyl (-C≡C-CH3), 2-propynyl (-CH2-C≡CH), 1,3-butadiynyl (-C≡CC≡CH), and the like. The term "C2-C4 alkynyl" should be understood to mean a linear or branched monovalent hydrocarbon group containing one or more triple bonds and having 2, 3, or 4 carbon atoms.

[0135] The term "alkoxy" refers to an -O-alkyl group. According to the present invention, suitable alkoxy groups are C 1-6 Alkoxy, such as C 1-5 Alkoxy, C 1-4 Alkoxy, C 1-3 Alkoxy includes methoxy, ethoxy, propoxy, isopropoxy, isobutoxy, sec-butoxy, etc. For example, the term "C1-C6 alkoxy" can be understood as "C1-C6 alkyloxy" or "C1-C6 alkyl-O-", such as "C1-C6 alkoxy" can include "C1-C3 alkoxy".

[0136] The term "cycloalkyl" refers to a fully saturated carbocyclic ring that can exist as a monocyclic, bridged, or spirocyclic ring. Unless otherwise indicated, the carbocyclic ring is typically a 3- to 10-membered ring. Non-limiting examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, adamantyl, and the like. The term "C3-C6 cycloalkyl" should be understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 6 carbon atoms. Such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or cyclodecyl, or a bicyclic hydrocarbon ring such as a decalin ring.

[0137] The term "cycloalkyloxy" can be understood as "cycloalkyl-O-". For example, the term "C 3-6 "Cycloalkyloxy" can be understood as "C 3-6 Cycloalkyl-O-".

[0138] The term "heterocyclyl" refers to a fully saturated or partially unsaturated (heteroaromatic group that is not aromatic as a whole) monovalent monocyclic, fused, bridged or spirocyclic group containing 1 to 5 heteroatoms or heteroatomic groups (i.e., heteroatomic groups containing heteroatoms) in the ring atoms, wherein the "heteroatoms or heteroatomic groups" include but are not limited to nitrogen atom (N), oxygen atom (O), sulfur atom (S), phosphorus atom (P), boron atom (B), =O, =S, -ON=, -C(=O)O-, -C(=O)-, -C(=S)-, -S(=O)2-, -S(=O)-, and optionally substituted -NH-, -S(=O)(=NH)-, -C(=O)NH-, -C(=NH)-, -S(=O)2NH-, S(=O)NH-, -NHC(=O)NH-, etc. Unless otherwise indicated, the heterocycle is typically a 3- to 7-membered ring containing 1 to 3 heteroatoms (e.g., 1 or 2 heteroatoms) independently selected from sulfur, oxygen, and / or nitrogen. Non-limiting examples of heterocyclyl groups include, but are not limited to, oxiranyl, tetrahydrofuranyl, dihydrofuranyl, pyrrolidinyl, N-methylpyrrolidinyl, dihydropyrrolyl, piperidinyl, piperazinyl, pyrazolidinyl, 4H-pyranyl, morpholinyl, thiomorpholinyl, tetrahydrothiophenyl, and the like.

[0139] The term "3-6 membered heterocyclyl" or "3-6 membered heterocyclyloxy" refers to a saturated or partially saturated monovalent monocyclic or bicyclic hydrocarbon ring containing 1, 2 or 3 heteroatoms selected from N, O and S. In particular, the heterocyclic group may include, but is not limited to: a 3-membered ring, such as an oxirane ring; a 4-membered ring, such as an azetidinyl group, an oxetane group; a 5-membered ring, such as a tetrahydrofuranyl group, a dioxolyl group, a pyrrolidinyl group, an imidazolidinyl group, a pyrazolidinyl group, a pyrrolinyl group, a 4,5-dihydrooxazole group, or a 2,5-dihydro-1H-pyrrolyl group; or a 6-membered ring, such as a tetrahydropyranyl group, a piperidinyl group, a morpholinyl group, a dithianyl group, a thiomorpholinyl group, a piperazinyl group, or a trithianyl group; or a partially saturated 6-membered ring, such as a tetrahydropyridinyl group, a piperidinyl group, a morpholinyl group, a dithianyl group, a thiomorpholinyl group, a piperazinyl group, a trithianyl group, a tetrahydropyridinyl group, or a 4H-[1,3,4]thiadiazinyl group. According to the present invention, the heterocyclic group as a whole is non-aromatic. The “3- to 6-membered heterocyclyl” may include a “5- to 6-membered heterocyclyl”, and the “3- to 6-membered heterocyclyloxy” may include a “5- to 6-membered heterocyclyloxy”.

[0140] The term "heterocycloalkyl" refers to a monovalent cyclic group that is fully saturated and can exist as a monocyclic, fused, bridged or spirocyclic ring. Unless otherwise indicated, the heterocycle is typically a 3 to 7 membered ring containing 1, 2 or 3 heteroatoms (such as 1 or 2 heteroatoms) independently selected from sulfur, oxygen and / or nitrogen. Examples of 3-membered heterocycloalkyl groups include, but are not limited to, oxirane, thioethane, and nitroethane groups. Non-limiting examples of 4-membered heterocycloalkyl groups include, but are not limited to, azetidinyl, oxetanyl, and thietanyl groups. Examples of 5-membered heterocycloalkyl groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidinyl, imidazolidinyl, and tetrahydropyrazolyl groups. Examples of 6-membered heterocycloalkyl groups include, but are not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, piperazinyl, 1,4-thioxanyl, 1,4-dioxanyl, thiomorpholinyl, 1,3-dithianyl, and 1,4-dithianyl groups. Examples of 7-membered heterocycloalkyl groups include, but are not limited to, azepanyl, oxetanyl, and thiepanyl groups. Examples of the 5-6-membered heterocycloalkyl groups containing B formed herein include, but are not limited to

[0141] The "3-6 membered heterocycloalkyl" in the term "3-6 membered heterocycloalkyl" or "3-6 membered heterocycloalkyloxy" refers to a heterocycloalkyl group having 3, 4, 5 or 6 ring atoms. "3-6 membered heterocycloalkyl" may include "5-6 membered heterocycloalkyl", and "3-6 membered heterocycloalkyloxy" may include "5-6 membered heterocycloalkyloxy".

[0142] The term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated π electron system. For example, an aryl group may have 6-20 carbon atoms, 6-14 carbon atoms, or 6-12 carbon atoms. Non-limiting examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, and 1,2,3,4-tetrahydronaphthalene. The term "C6-C 10 "Aryl" is understood to mean a monovalent aromatic or partially aromatic monocyclic or bicyclic hydrocarbon ring having 6 to 10 carbon atoms. In particular, a ring having 6 carbon atoms ("C6 aryl"), such as phenyl; or a ring having 9 carbon atoms ("C9 aryl"), such as indanyl or indenyl, or a ring having 10 carbon atoms ("C 10 "aryl"), for example tetrahydronaphthyl, dihydronaphthyl or naphthyl.

[0143] The term "heteroaryl" refers to a monocyclic or fused polycyclic ring system containing at least one ring atom selected from N, O, and S, with the remaining ring atoms being C, and having at least one aromatic ring. Exemplary heteroaryl groups have a single 4- to 8-membered ring, particularly a single 5- to 8-membered ring, or multiple fused rings containing 6 to 14, particularly 6 to 10, ring atoms. Non-limiting examples of heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, quinolyl, isoquinolyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, and the like. The term "5- to 10-membered heteroaryl" is understood to include monovalent monocyclic or bicyclic aromatic ring systems having 5 to 10 ring atoms and containing 1 to 5 heteroatoms independently selected from N, O, and S. The term "5-10 membered heteroaryl" is to be understood as including monovalent monocyclic or bicyclic aromatic ring systems having 5, 6, 7, 8, 9 or 10 ring atoms, in particular 5 or 6 or 9 or 10 ring atoms, and which contain 1 to 5 or 1 to 3 heteroatoms independently selected from N, O and S and which, in each case, may additionally be benzo-fused. In particular, heteroaryl is selected from thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, thiadiazolyl and the like and benzo derivatives thereof, for example benzofuranyl, benzothienyl, benzothiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl , indolyl, isoindolyl, etc.; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, etc., and benzo derivatives thereof, such as quinolyl, quinazolinyl, isoquinolyl, etc.; or acinyl, indolizinyl, purinyl, etc. and benzo derivatives thereof; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, etc. The term "5-6 membered heteroaryl" refers to an aromatic ring system having 5 or 6 ring atoms, and containing 1-3 or 1-2 heteroatoms independently selected from N, O and S. Examples include, but are not limited to, thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl or triazinyl. "5-10 membered heteroaryl" may include "5-6 membered heteroaryl".

[0144] The term "therapeutically effective amount" means an amount of a compound of the present invention that (i) treats or prevents a particular disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder as described herein. The amount of a 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 can be routinely determined by one skilled in the art based on their knowledge and this disclosure.

[0145] The term "excipient" refers to a pharmaceutically acceptable inert ingredient. Examples of the term "excipient" include, but are not limited to, binders, disintegrants, lubricants, glidants, stabilizers, fillers, and diluents. Excipients enhance the handling characteristics of pharmaceutical formulations by increasing their fluidity and / or adhesion, making them more suitable for direct compression. Typical examples of "pharmaceutically acceptable carriers" suitable for the above-mentioned formulations include sugars, starches, cellulose, and their derivatives, which are commonly used excipients in pharmaceutical formulations.

[0146] The words “comprise,” “contain,” or “include” and their English variations such as comprises, contains, includes, comprising, containing, or including should be understood as having an open, non-exclusive meaning, i.e., “including but not limited to.”

[0147] The present application also includes isotopically labeled compounds of the present application that are identical to those described herein, but in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of 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 et al.

[0148] Certain isotope-labeled compounds of the present application (e.g.3 H and 14 C-labeled) can be used in compound and / or substrate tissue distribution assays. 3 H) and carbon-14 (i.e. 14 C) isotopes are particularly preferred due to their ease of preparation and detectability. Positron emitting 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. Isotopically labeled compounds of the present application can generally be prepared by following procedures analogous to those disclosed in the Schemes and / or Examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0149] The pharmaceutical compositions of the present application can be prepared by combining the compounds of the present application with suitable pharmaceutically acceptable excipients, and can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols, etc.

[0150] Typical routes of administration of the compounds of the present invention, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, vaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0151] The pharmaceutical composition of the present application can be manufactured by methods well known in the art, such as conventional mixing methods, dissolution methods, granulation methods, emulsification methods, freeze-drying methods, etc.

[0152] In some embodiments, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These excipients enable the compounds of the present application to be formulated into tablets, pills, lozenges, dragees, capsules, liquids, gels, slurries, suspensions, and the like for oral administration to a patient.

[0153] Solid oral compositions can be prepared by conventional mixing, filling, or tableting methods. For example, they can be prepared by mixing the active compound with a solid excipient, optionally grinding the resulting mixture, adding other suitable excipients as needed, and then granulating the mixture to obtain a tablet or dragee core. Suitable excipients include, but are not limited to, binders, diluents, disintegrants, lubricants, glidants, sweeteners, or flavoring agents.

[0154] The pharmaceutical composition may also be suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in appropriate unit dosage forms.

[0155] In all administration methods of the compounds of general formula (I) described herein, the daily dosage is 0.01 to 200 mg / kg body weight, preferably 0.05 to 50 mg / kg body weight, more preferably 0.1 to 30 mg / kg body weight, in the form of single or divided doses.

[0156] The term "IC 50 IC is the half-maximal inhibitory concentration, which represents the concentration of a specific compound required to achieve 50% inhibition of a biological process in vitro. 50 The value can be converted logarithmically to pIC 50 Value (-log IC 50 ), where higher values indicate exponentially greater potency.

[0157] 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, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0158] The chemical reactions of the present invention are carried out in suitable solvents that are compatible with the chemical transformations of the present invention and the reagents and materials required. To obtain the compounds of the present invention, it may sometimes be necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0159] For the sake of clarity, the present application is further illustrated with examples, but the examples do not limit the scope of the present application. All reagents used in the present application are commercially available and can be used without further purification. DETAILED DESCRIPTION

[0160] The following examples illustrate the technical solutions of the present invention in detail, but the scope of protection of the present invention includes but is not limited to these. The present invention has been described in detail herein, and specific embodiments thereof have been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention. All reagents used in this application are commercially available and can be used without further purification.

[0161] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The units of NMR shifts are 10 -6(ppm). The solvents for NMR measurement are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is tetramethylsilane (TMS). 50 ” refers to the half-maximal inhibitory concentration, which is the concentration at which half of the maximum inhibitory effect is achieved.

[0162] Example 1: 3-(((1R,3R)-1-(2,6-difluoro-4-(((S)-5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-yl)amino)phenyl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropan-1-ol

[0163]

[0164] Step 1: Synthesis of tert-butyl (S)-(5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-yl)carbamate

[0165]

[0166] Add tert-butyl (S)-(5-azaspiro[2.4]hept-7-yl)carbamate (2.12 g, 10.0 mmol) to 40 mL of acetonitrile (MeCN), followed by potassium carbonate (2.76 g, 20.0 mmol) and 3-fluoro-1-iodopropane (2.26 g, 12.0 mmol). Heat to 80°C and stir for 6 hours until the reaction is complete. Filter the reaction mixture, wash the filter cake with acetonitrile, and combine the filtrates for column chromatography to obtain the title compound.

[0167] Step 2: Synthesis of (S)-5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-amine

[0168]

[0169] tert-Butyl (S)-(5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-yl)carbamate (1.00 g, 3.68 mmol) was added to 10 mL of dichloromethane, followed by 5 mL of a 4 mol / L hydrogen chloride solution in dioxane. The reaction was stirred at room temperature for 2 hours, and the reaction was complete. The reaction solution was concentrated, and the pH was adjusted to 7-8 by adding saturated aqueous sodium carbonate. The solution was extracted with dichloromethane, and the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated to obtain the title compound.

[0170] Step 3: Synthesis of 3-(((1R,3R)-1-(2,6-difluoro-4-(((S)-5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-yl)amino)phenyl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropan-1-ol

[0171]

[0172] 3-((1R,3R)-1-(4-bromo-2,6-difluorophenyl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropan-1-ol was prepared according to Example 340 of WO2016097072A1. 3-((1R,3R)-1-(4-bromo-2,6-difluorophenyl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropan-1-ol (470 mg, 1.00 mmol), methanesulfonic acid (2-dicyclohexylphosphine)-3,6-dimethoxy-2,4,6-triisopropyl-1,1-biphenyl)(2-amino-1,1-biphenyl-2-yl)palladium(II) (Brettphos Pd G3) (90.0 mg, 0.10 mmol), sodium tert-butoxide (t-BuONa) (192 mg, 2.00 mmol), (S)-5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-amine (172 mg, 1.00 mmol) were added to 5 mL of toluene, and the mixture was heated to 80°C under argon and stirred for 4 hours until the reaction was complete. The reaction solution was added to 50 mL of water, and the mixture was extracted with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain the title compound.

[0173] 1H NMR(400MHz,DMSO-d6)δ10.61(s,1H),7.46-7.45(m,1H),7.28-7.26(m,1H),7.09-7.00(m,2H),6.23-6 .31(m,2H),5.36-5.33(m,1H),5.12(s,1H),4.65-4.62(m,1H),4.52-4.50(m,1H),3.80-3.69(m,2H),3. 53-3.48(m,2H),3.40-3.37(m,1H),3.22-3.11(m,3H),2.92-2.87(m,1H),2.72-2.63(m,3H),2.50-2.42 (m,3H),1.91-1.85(m,2H),1.16-1.14(m,3H),0.85-0.75(m,1H),0.72-0.68(m,2H),0.59-0.52(m,1H).

[0174] LC / MS (m / z, MH + ):563.3.

[0175] Example 2: (S)-N-(3,5-difluoro-4-((1R,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-amine

[0176]

[0177] (1R,3R)-1-(4-bromo-2,6-difluorophenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole was prepared according to Example 304 of WO2016097072A1. Then, referring to Step 3 of Example 1 above, except that 3-((1R,3R)-1-(4-bromo-2,6-difluorophenyl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropan-1-ol in Step 3 of Example 1 was replaced with (1R,3R)-1-(4-bromo-2,6-difluorophenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole, the title compound was prepared by the same method.

[0178] 1H NMR(400MHz,DMSO-d6)δ10.58(s,1H),7.39-7.38(m,1H),7.21-7.19(m,1H),7.02-6.92(m,3H),6.23-6 .20(m,2H),5.09(s,1H),4.56-4.53(m,1H),4.44-4.41(m,1H),3.75-3.70(m,1H),3.47-3.41(m,2H),3. 35-3.32(m,1H),3.05-3.01(m,1H),2.97-2.91(m,1H),2.84-2.78(m,1H),2.62-2.55(m,2H),2.48-2.38 (m,3H),1.85-1.74(m,2H),1.12-1.10(m,3H),0.74-0.70(m,1H),0.64-0.61(m,2H),0.48-0.43(m,1H).

[0179] LC / MS (m / z, MH + ):551.3.

[0180] Example 3: 2,2-difluoro-3-((1S,3R)-1-(5-(((S)-5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-yl)amino)pyridin-2-yl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)propan-1-ol

[0181]

[0182] (R)-3-(((1-(1H-indol-3-yl)propan-2-yl)amino)-2,2-difluoropropane-1-ol was prepared according to Example 340 of WO2016097072A1. (R)-3-(((1-(1H-indol-3-yl)propan-2-yl)amino)-2,2-difluoropropane-1-ol (2.68 g, 10.0 mmol) and 5-bromo-2-pyridinecarboxaldehyde (1.84 g, 10.0 mmol) were added to 20 mL of toluene (Tol), and 1 mL of acetic acid (AcOH) was added. The mixture was heated to 90°C and stirred for 12 hours until the reaction was complete. The reaction solution was concentrated and diluted with 100 mL of water. The pH was adjusted to 8-9 with saturated sodium bicarbonate solution, and the mixture was extracted with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography. Then, the above step 3 of Example 1 was referred to, except that the 3-((1R,3R)-1-(5-bromo-pyridin-2-yl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropane-1-ol in Example 1 was replaced by The title compound was prepared by the same method: replacing 3-((1R,3R)-1-(5-bromo-pyridin-2-yl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropan-1-ol with 5-bromo-pyridin-2-yl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropan-1-ol.

[0183] 1 H NMR(400MHz,DMSO-d6)δ10.60(s,1H),7.91-7.90(m,1H),7.40-7.35(m,1H),7.25-7.20(m,2H),7. 00-6.96(m,4H),5.10(s,1H),4.56-4.53(m,1H),4.44-4.41(m,1H),3.79-3.69(m,2H),3.53-3.45( m,2H),3.40-3.32(m,1H),3.22-3.17(m,3H),2.92-2.80(m,1H),2.70-2.63(m,3H),2.50-2.46(m,3 H),1.94-1.85(m,2H),1.16-1.10(m,3H),0.82-0.75(m,1H),0.75-0.68(m,2H),0.60-0.52(m,1H).

[0184] LC / MS (m / z, MH + ):528.3.

[0185] Example 4: (S)-5-(3-fluoropropyl)-N-(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-yl)-5-azaspiro[2.4]hept-7-amine

[0186]

[0187] The preparation method is similar to that of Example 3, except that the starting material (R)-3-(((1-(1H-indol-3-yl)propan-2-yl)amino)-2,2-difluoropropan-1-ol in Example 3 is replaced by (R)-1-(1H-indol-3-yl)-N-(2,2,2-trifluoroethyl)propan-2-amine, and the title compound is prepared in the same manner.

[0188] 1 H NMR (400MHz, DMSO-d6) δ10.58(s,1H),7.90-7.88(m,1H),7.42-7.40(m,1H),7.27-7.25(m,1H),7.05-6.96 (m,4H),4.89(s,1H),4.56-4.53(m,1H),4.44-4.41(m,1H),3.75-3.70(m,1H),3.47-3.41(m,1H),3.35 -3.32(m,1H),3.04-3.00(m,1H),2.96-2.89(m,1H),2.84-2.78(m,1H),2.62-2.55(m,2H),2.48-2.38( m,3H),1.85-1.74(m,2H),1.14-1.13(m,3H),0.75-0.73(m,1H),0.64-0.61(m,2H),0.48-0.43(m,1H).

[0189] 13 C NMR(400MHz,DMSO-d6)δ147.24,143.56,136.57,133.91,132.84,126.62,126 .51(q,J=278.2Hz),122.64,120.60,118.67,118.15,117.57,111.09,107.61, 82.29(d,J=161.4Hz),64.30,62.33,61.42,55.99,51.69(d,J=5.7Hz),48.66 ,48.24(q,J=29.9Hz),28.95(d,J=19.3Hz),26.16,25.32,17.64,13.28,7.94.

[0190] LC / MS (m / z, MH + ):516.3.

[0191] Example 5: 3-((6S,8R)-6-(2,6-difluoro-4-(((S)-5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-yl)amino)phenyl)-8-methyl-3,6,8,9-tetrahydro-7H-pyrazolo[4,3-f]isoquinolin-7-yl)-2,2-difluoropropan-1-ol

[0192]

[0193] Step 1: Synthesis of (7S)-N-(4-((6S,8R)-7-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl)-3,5-difluorophenyl)-5-(3-fluoropropyl)-5-azaspiro[2.4]heptylamine

[0194]

[0195] According to the preparation method of Example 5 of WO2018077630A1, (6S, 8R)-6-(4-bromo-2,6-difluorophenyl)-7-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline was prepared. Then, (6S, 8R)-6-(4-bromo-2,6-difluorophenyl)-7-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline (793 mg, 1.00 mmol) and Brettphos Pd were added. G3 (90.0 mg, 0.10 mmol), sodium tert-butoxide (192 mg, 2.00 mmol), and (S)-5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-amine (172 mg, 1.00 mmol) were added to 5 mL of toluene. The mixture was heated to 80°C under argon and stirred for 6 hours. The reaction was completed. The reaction solution was added to 50 mL of water and extracted with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain the title compound.

[0196] Step 2: Synthesis of 3-((6S,8R)-6-(2,6-difluoro-4-(((S)-5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-yl)amino)phenyl)-8-methyl-3,6,8,9-tetrahydro-7H-pyrazolo[4,3-f]isoquinolin-7-yl)-2,2-difluoropropan-1-ol

[0197]

[0198] (7S)-N-(4-((6S,8R)-7-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl)-3,5-difluorophenyl)-5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-amine (400 mg, 0.50 mmol) was added to 10 mL of methanol (MeOH), followed by 5 mL of a 4 mol / L hydrogen chloride solution in dioxane. The reaction was stirred at room temperature for 1 hour until completion. The reaction solution was concentrated, saturated aqueous sodium carbonate solution was added to adjust the pH to 7-8, and the mixture was extracted with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the title compound.

[0199] 1 H NMR(400MHz,DMSO-d6)δ12.97(s,1H),8.05(s,1H),7.22-7.20(m,1H),6.71-6.69(m,1H),6.20-6 .14(m,3H),5.28-5.25(m,1H),5.07(s,1H),4.56-4.53(m,1H),4.44-4.41(m,1H),3.72-3.64(m,2 H),3.54-3.50(m,1H),3.18-3.00(m,3H),2.90-2.85(m,1H),2.67-2.60(m,2H),2.48-2.37(m,6H ),1.85-1.74(m,2H),1.03-1.02(m,3H),0.75-0.70(m,1H),0.64-0.60(m,2H),0.47-0.42(m,1H).

[0200] LC / MS (m / z, MH + ):564.3.

[0201] Example 6: 2,2-difluoro-3-(((6S,8R)-6-(5-(((S)-5-(3-fluoropropyl)-5-azaspiro[2.4]heptyl-7-yl)amino]pyridin]-2-yl)-8-methyl-3,6,8,9-tetrahydro-7H-pyrazolo[4,3-f]isoquinolin-7-yl)propan-1-ol

[0202]

[0203]

[0204] The starting material (6S, 8R)-6-(5-bromopyridin-2-yl)-7-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline was prepared according to the preparation method of Example 16 of WO2018077630A1. Then refer to the preparation method of Example 5, except that the (6S,8R)-6-(4-bromo-2,6-difluorophenyl)-7-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline in Step 1 of Example 5 is replaced with (6S,8R)-6-(5-bromopyridin-2-yl)-7-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline, and the title compound is prepared in the same manner.

[0205] 1 H NMR(400MHz,DMSO-d6)δ13.06(s,1H),8.04(s,1H),7.84-7.83(m,1H),7.24-7.22(m,1H),6.89-6 .79(m,3H),5.70-5.68(m,1H),4.92(s,1H),4.55-4.52(m,1H),4.43-4.40(m,1H),3.72-3.64(m,2 H),3.54-3.50(m,1H),3.18-3.00(m,3H),2.90-2.85(m,1H),2.67-2.60(m,2H),2.48-2.37(m,6H ),1.85-1.74(m,2H),1.03-1.02(m,3H),0.75-0.70(m,1H),0.64-0.60(m,2H),0.47-0.42(m,1H).

[0206] LC / MS (m / z, MH + ):529.3.

[0207] Example 7: (S)-5-(3-fluoropropyl)-N-(6-((6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl)pyridin-3-yl)-5-azaspiro[2.4]hept-7-amine

[0208]

[0209] The starting material (6S, 8R)-6-(5-bromopyridin-2-yl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline was prepared according to the preparation method of Example 17 of WO2018077630A1. The preparation method of Example 5 was used as described above, except that the (6S,8R)-6-(4-bromo-2,6-difluorophenyl)-7-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline in Step 1 of Example 5 was replaced with (6S,8R)-6-(5-bromopyridin-2-yl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline. The title compound was prepared by the same method.

[0210] 1 H NMR(400MHz,DMSO-d6)δ12.99(s,1H),8.06(s,1H),7.83(s,1H),7.23-7.21(m,1H),6.98-6.88(m,2H),6.81-6 .79(m,1H),5.71-5.69(m,1H),4.92(s,1H),4.55-4.52(m,1H),4.43-4.40(m,1H),3.75-3.70(m,1H),3.52-3. 45(m,2H),3.09-3.05(m,2H),3.00-2.94(m,1H),2.87-2.81(m,1H),2.65-2.60(m,1H),2.48-2.45(m,3H),2.3 9-2.35(m,1H),1.85-1.72(m,2H),1.09-1.08(m,3H),0.77-0.72(m,1H),0.64-0.59(m,2H),0.45-0.40(m,1H).

[0211] LC / MS (m / z, MH + ):517.3.

[0212] Example 8: (S)-N-(3,5-difluoro-4-((6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl)phenyl)-5-(3-fluoropropyl)-5-azaspiro[2.4]heptan-7-amine

[0213]

[0214] According to the first and second steps of Example 27 of WO2019223715A1, (6S,8R)-6-(4-bromo-2,6-difluorophenyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline was prepared. Then, referring to the preparation method of Example 5, the title compound was obtained by replacing (6S,8R)-6-(4-bromo-2,6-difluorophenyl)-7-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline with (6S,8R)-6-(4-bromo-2,6-difluorophenyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline in Step 1 of Example 5.

[0215] 1 H NMR(400MHz,DMSO-d6)δ13.05(s,1H),8.11(s,1H),7.29-7.27(m,1H),6.80-6.78(m,1H),6.26-6 .21(m,3H),5.20(s,1H),4.62-4.59(m,1H),4.50-4.47(m,1H),3.76-3.73(m,1H),3.54-3.51(m,1 H),3.27-3.20(m,2H),3.08-3.06(m,1H),2.97-2.93(m,2H),2.68-2.66(m,1H),2.54-2.42(m,4H ),1.89-1.79(m,2H),1.14-1.13(m,3H),0.78-0.75(m,1H),0.70-0.66(m,2H),0.53-0.50(m,1H).

[0216] LC / MS (m / z, MH + ):552.2.

[0217] Example 9: (S)-N-(4-((6S,8R)-7-(2,2-difluoropropyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl)-3,5-difluorophenyl)-5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-amine

[0218]

[0219] Step 1: Synthesis of (R)-3-(2-(((2,2-difluoropropyl)amino)propyl)-2-methylaniline

[0220]

[0221] (R)-N-(1-(3-bromo-2-methylphenyl)propan-2-yl)-2,2-difluoropropan-1-amine (14.3 g), benzophenone imine (10.0 g), tris(dibenzylideneacetone)dipalladium (Pd2(dba)3) (0.42 g), 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (BINAP) (0.57 g), and sodium tert-butoxide (6.60 g) were added to 60 mL of toluene. The mixture was heated to 80°C under argon and stirred for 5 hours to complete the reaction. The reaction solution was spin-dried, and 100 mL of dichloromethane and dilute hydrochloric acid solution (2N, 100 mL) were added. After vigorous stirring for 2 hours, the reaction was completed. The layers were separated, the dichloromethane layer was discarded, and the aqueous layer was adjusted to pH 10-11 with 5N sodium hydroxide aqueous solution. The mixture was extracted with ethyl acetate, and the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated to obtain 12.0 g of a crude oil.

[0222] Step 2: Synthesis of (1S,3R)-1-(4-bromo-2,6-difluorophenyl)-2-(2,2-difluoropropyl)-3,5-dimethyl-1,2,3,4-tetrahydroisoquinolin-6-amine

[0223]

[0224] (R)-3-(2-(((2,2-difluoropropyl)amino)propyl)-2-methylaniline (12.0 g) and 4-bromo-2,6-difluorobenzaldehyde (13.0 g) were added to 50 mL of acetic acid, 5 mL of water was added, and the mixture was stirred at 60°C. The reaction was monitored by TLC. After completion of the reaction, the reaction solution was spin-dried, 100 ml of water was added, and a saturated aqueous sodium bicarbonate solution was added to adjust the pH to 8-9. The mixture was extracted with ethyl acetate, and the organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography with petroleum ether:ethyl acetate = 10:1 (V:V) to obtain 13.9 g of an oil.

[0225] Step 3: Synthesis of (6S,8R)-6-(4-bromo-2,6-difluorophenyl)-7-(2,2-difluoropropyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline

[0226]

[0227] (1S,3R)-1-(4-bromo-2,6-difluorophenyl)-2-(2,2-difluoropropyl)-3,5-dimethyl-1,2,3,4-tetrahydroisoquinolin-6-amine (3.56 g) was added to 40 mL of propionic acid, cooled to -20°C, and a 2N aqueous solution of sodium nitrite (560 mg) was added dropwise. After stirring for 1 hour, the reaction was completed. 50 mL of ethyl acetate was added to the reaction solution, and then a saturated sodium bicarbonate solution was slowly added dropwise to adjust the pH to 8-9. The reaction was extracted with ethyl acetate, and the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated to obtain 4.00 g of an oil.

[0228] Step 4: Synthesis of (6S,8R)-6-(4-bromo-2,6-difluorophenyl)-7-(2,2-difluoropropyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline

[0229]

[0230] (6S,8R)-6-(4-bromo-2,6-difluorophenyl)-7-(2,2-difluoropropyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline (4.00 g) was added to 100 mL of dichloromethane, 4-methylpyridinium benzenesulfonate (PPTS) (100 mg) was added, and 6 mL of 3,4-dihydro-2H-pyran (DHP) was added. The mixture was heated to 50°C and stirred for 10 hours until the reaction was complete. The reaction solution was concentrated and then purified by column chromatography with petroleum ether:ethyl acetate = 10:1 (V:V) to obtain 1.90 g of an oil.

[0231] Step 5: Synthesis of (S)-N-(4-((6S,8R)-7-(2,2-difluoropropyl)-8-methyl-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl)-3,5-difluorophenyl)-5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-amine

[0232]

[0233] The preparation method of Reference Example 5 was used, except that the (6S,8R)-6-(4-bromo-2,6-difluorophenyl)-7-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline in Step 1 of Example 5 was replaced with (6S,8R)-6-(4-bromo-2,6-difluorophenyl)-8-methyl-3-(tetrahydro-2H-pyran-2-yl)-7-(2,2-difluoropropyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinoline. The title compound was prepared in the same manner.

[0234] 1 H NMR(400MHz,DMSO-d6)δ12.97(s,1H),8.05(s,1H),7.23-7.21(m,1H),6.72-6.70(m,1H),6.22-6.19( m,3H),5.07(s,1H),4.57-4.52(m,1H),4.42-4.39(m,1H),3.75-3.70(m,1H),3.52-3.45(m,2H),3.09- 3.05(m,2H),3.00-2.94(m,1H),2.87-2.81(m,1H),2.65-2.60(m,1H),2.48-2.45(m,3H),2.39-2.35( m,1H),1.85-1.72(m,2H),1.46-1.36(m,3H),1.03-1.01(m,3H),0.70-0.58(m,3H),0.45-0.42(m,1H).

[0235] LC / MS (m / z, MH + ):548.3.

[0236] Example 10: (S)-N-(3,5-difluoro-4-((1S,3R)-3-methyl-6-(1H-pyrazol-4-yl)-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinolin-1-yl)phenyl)-5-(3-fluoropropyl)-5-azaspiro[2.4]heptane-7-amine

[0237]

[0238] Step 1: Synthesis of 5-bromo-2-(diethoxymethyl)-1,3-difluorobenzene

[0239]

[0240] 4-Bromo-2,6-difluorobenzaldehyde (1.69 g, 7.66 mmol) was added to 17 mL of ethanol, followed by triethoxymethane (1.13 g, 7.66 mmol) and 1 drop of concentrated sulfuric acid. The mixture was heated to 60°C and stirred for 2 hours until the reaction was complete. The reaction solution was dried to obtain the title compound.

[0241] Step 2: Synthesis of (S)-2,6-difluoro-4-((5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-yl)amino)benzaldehyde

[0242]

[0243] 5-Bromo-2-(diethoxymethyl)-1,3-difluorobenzene (2.26 g, 7.66 mmol), 5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-amine (1.1 g, 6.4 mmol), Brettphos Pd G3 (580.0 mg, 0.06 mmol), and sodium tert-butoxide (1.47 g, 15.31 mmol) were added to 30 mL of toluene. The mixture was heated to 80°C and stirred under argon for 15 hours until the reaction was complete. The reaction solution was added to 50 mL of water and extracted with ethyl acetate. The organic layers were combined and dried over anhydrous sodium sulfate. The organic layers were concentrated and added with 20 mL of tetrahydrofuran. 2 mol / L dilute hydrochloric acid solution was added and the reaction was stirred at room temperature for 2 hours until the reaction was complete. 50 mL of water was added and the mixture was extracted with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the title compound.

[0244] Step 3: Synthesis of (1S,3R)-1-(2,6-difluoro-4-((S)-(5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-yl)amino)phenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinolin-6-ol

[0245]

[0246] 2,6-Difluoro-4-((5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-yl)amino)benzaldehyde (0.95 g, 3.05 mmol) and (R)-3-(2-(((2,2,2-trifluoroethyl)amino)propyl)phenol (0.71 g, 3.05 mmol) were added to 20 mL of toluene / acetic acid = 10 / 1 system, heated to 80°C and stirred for 15 hours. After completion of the reaction, the reaction solution was concentrated and purified by column chromatography (petroleum ether / ethyl acetate gradient elution) to obtain the title compound.

[0247] Step 4: Synthesis of (1S,3R)-1-(2,6-difluoro-4-((S)-(5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-yl)amino)phenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinolin-6-yl trifluoromethanesulfonate

[0248]

[0249] (1S,3R)-1-(2,6-difluoro-4-((5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-yl)amino)phenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinolin-6-ol (1.10 g, 2.09 mmol), bis(trifluoromethanesulfonyl)aniline (PhNTf2) (1.49 g, 4.17 mmol), and triethylamine (TEA) (0.63 g, 6.26 mmol) were added to 10 mL of dichloromethane (DCM) and stirred at room temperature for 12 hours. The reaction was complete after 100 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain the title compound.

[0250] Step 5: Synthesis of (S)-N-(3,5-difluoro-4-((1S,3R)-3-methyl-6-(1H-pyrazol-4-yl)-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinolin-1-yl)phenyl)-5-(3-fluoropropyl)-5-azaspiro[2.4]heptan-7-amine

[0251]

[0252] (1S,3R)-1-(2,6-difluoro-4-((5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-yl)amino)phenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinolin-6-yltrifluoromethanesulfonic acid (200 mg, 0.30 mmol), 1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (Pd(dppf)Cl2) (22.0 mg, 0.03 mmol), cesium carbonate (198 mg) , 0.61mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate (89mg, 0.30mmol) were added to 5mL of 1,4-dioxane / water = 10 / 1 system, heated to 90°C under argon protection and stirred for 14 hours, after which the reaction was completed. The reaction solution was concentrated and 10mL of dichloromethane was added, followed by 2mL of trifluoroacetic acid. After stirring at room temperature for 2 hours, the reaction was completed. The reaction solution was concentrated and column chromatography was performed to obtain the title compound.

[0253] 1 H NMR(400MHz,DMSO-d6)δ8.20(s,1H),7.99(s,2H),7.33-7.26(m,2H),6.71-6.69(m,1H) ,6.22-6.16(m,3H),5.04(s,1H),4.56-4.53(m,1H),4.44-4.41(m,1H),3.73-3.68(m,2 H),3.18-3.02(m,3H),2.83-2.92(m,2H),2.68-2.56(m,2H),2.47-2.36(m,3H),1.84-1 .74(m,2H),1.03-1.02(m,3H),0.72-0.69(m,1H),0.64-0.60(m,2H),0.47-0.45(m,1H).

[0254] LC / MS (m / z, MH + ):578.2.

[0255] Example 11: 3-((1R,3R)-1-(2,6-difluoro-4-((4aR,7aR)-6-(3-fluoropropyl)octahydro-1H-pyrrolo[3,4-b]pyridin-1-yl)phenyl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropan-1-ol

[0256]

[0257] The preparation method is similar to that in step 3 of Example 1, except that the (S)-5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-amine in step 3 of Example 1 is replaced by (4aR, 7aR)-6-(3-fluoropropyl)octahydro-1H-pyrrolo[3,4-b]pyridine, and the title compound is prepared in the same manner.

[0258] 1H NMR(400MHz,DMSO-d6)δ10.56(s,1H),7.40-7.38(m,1H),7.20-7.18(m,1H),7.02-6.92(m,2H),6. 49-6.46(m,2H),5.30-5.26(m,1H),5.11(s,1H),4.53-4.50(m,1H),4.41-4.31(m,2H),3.74-3.62( m,1H),3.49-3.41(m,3H),3.18-3.05(m,1H),2.85-2.80(m,2H),2.77-2.60(m,4H),2.58-2.56(m,2 H),2.48-2.40(m,2H),2.24-2.20(m,1H),1.80-1.68(m,4H),1.55-1.49(m,2H),1.09-1.07(m,3H).

[0259] LC / MS (m / z, MH + ):577.3.

[0260] Example 12: (S)-5-(3-fluoropropyl)-N-(6-((1S,3R)-3-methyl-6-(1H-pyrazol-4-yl)-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydroisoquinolin-1-yl)pyridin-3-yl)-5-azaspiro[2.4]hept-7-amine

[0261]

[0262]

[0263] The preparation method is similar to that of Example 10, except that the starting material 5-bromo-2-(diethoxymethyl)-1,3-difluorobenzene in step 2 of Example 10 is replaced with 5-bromo-2-(diethoxymethyl)-pyridine, and steps 2 to 5 are reacted in the same manner to obtain the title compound.

[0264] 1H NMR(400MHz,DMSO-d6)δ8.73(s,1H),7.85(s,2H),7.67-7.59(m,2H),7.39-7.26(m ,2H),6.71-6.69(m,1H),6.22-6.16(m,2H),4.94(s,1H),4.55-4.41(m,2H),3.73- 3.68(m,2H),3.18-3.02(m,3H),2.83-2.92(m,2H),2.68-2.56(m,2H),2.47-2.36( m,3H),1.86-1.72(m,3H),1.05-0.96(m,1H),0.75-0.57(m,2H),0.38-0.49(m,1H).

[0265] LC / MS (m / z, MH + ):543.2.

[0266] Example 13: (S)-N-(3,5-difluoro-4-((1S,3R)-3-methyl-6-(1-methyl-1H-pyrazol-4-yl)-2-(4-(trifluoromethyl)phenyl)-1,2,3,4-tetrahydroisoquinolin-1-yl)phenyl)-5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-amine

[0267]

[0268] Step 1: Synthesis of (R)-N-(1-(3-(phenoxyphenyl)propan-2-yl)-4-(trifluoromethyl)aniline

[0269]

[0270] (R)-1-(3-phenoxyphenyl)propane-2-amine (2.27 g), p-bromobenzotrifluoride (2.25 g), tris(dibenzylideneacetone)dipalladium (900 mg), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (Xphos) (920 mg), and cesium carbonate (6.50 g) were added to 50 mL of 1,4-dioxane. The mixture was heated to 100°C under nitrogen and the reaction was complete within 6 hours. The reaction mixture was purified by column chromatography (petroleum ether / ethyl acetate gradient elution) to obtain the title compound.

[0271] Step 2: Synthesis of (R)-N-(1-(3-(phenoxyphenyl)propan-2-yl)-4-(trifluoromethyl)aniline

[0272]

[0273] (R)-N-(1-(3-(phenoxyphenyl)propan-2-yl)-4-(trifluoromethyl)aniline (1.00 g) was added to 20 mL of methanol, and palladium on carbon (100 mg) was added. After hydrogen was introduced, the mixture was stirred at room temperature. The reaction was completed after 4 hours, and the mixture was filtered with suction, and the filtrate was concentrated to obtain the title compound.

[0274] Step 3: Synthesis of (S)-N-(3,5-difluoro-4-((1S,3R)-3-methyl-6-(1-methyl-1H-pyrazol-4-yl)-2-(4-(trifluoromethyl)phenyl)-1,2,3,4-tetrahydroisoquinolin-1-yl)phenyl)-5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-amine

[0275]

[0276] The preparation method is similar to that of Example 10, except that (R)-3-(2-(((2,2,2-trifluoroethyl)amino)propyl)phenol in Step 3 of Example 10 is replaced with (R)-3-(2-(((4-(trifluoromethyl)phenyl)amino)propyl)phenol, and then Steps 2 to 5 are carried out in the same manner to obtain the title compound.

[0277] 1 H NMR(400MHz,DMSO-d6)δ8.10(s,1H),7.83(s,1H),7.48-7.36(m,4H),7.17-7.15(m,1H), 7.01-6.99(m,2H),6.53(s,1H),6.19-6.15(m,2H),5.92(s,1H),4.52-4.51(m,1H),4.40- 4.37(m,1H),3.85(s,1H),3.54-3.47(m,4H),2.79-2.50(m,3H),2.34-2.33(m,2H),1.91 -1.79(m,2H),0.92-0.91(m,2H),0.65-0.64(m,1H),0.590.57(m,2H),0.43-0.42(m,1H).

[0278] LC / MS (m / z, MH + ):654.3.

[0279] Example 14: 3-((1R,3R)-1-(2,6-difluoro-4-(((S)-1-(3-fluoropropyl)piperidin-3-yl)amino)phenyl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropan-1-ol

[0280]

[0281] The preparation method is similar to step 3 of Example 1, except that (S)-5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-amine in step 3 of Example 1 is replaced by (S)-1-(3-fluoropropyl)piperidin-3-amine to obtain the title compound.

[0282] 1 H NMR(400MHz,DMSO-d6)δ10.66(s,1H),7.43-7.37(m,2H),7.03-6.90(m,2H),6. 25-6.17(m,3H),5.79-5.75(m,1H),5.35(s,1H),4.55-4.52(m,1H),4.43-4.39( m,1H),3.60-3.57(m,2H),3.31-3.30(m,3H),2.98-2.78(m,4H),2.72-2.60(m,2 H),2.39-2.32(m,2H),2.05-1.95(m,1H),1.84-1.64(m,6H),0.89-0.77(m,3H).

[0283] LC / MS (m / z, MH + ):551.2

[0284] Example 15: 3-((1R,3R)-1-(2,6-difluoro-4-(((S)-1-(3-fluoropropyl)azepan-3-yl)amino)phenyl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropan-1-ol

[0285]

[0286] The preparation method is similar to that of Example 1, except that (S)-(5-azaspiro[2.4]heptane-7-yl)carbamic acid tert-butyl ester in step 1 of Example 1 is replaced by (S)-azepan-3-ylcarbamic acid tert-butyl ester, and steps 1 to 3 of Example 1 are reacted in the same manner to prepare the title compound.

[0287] 1H NMR(400MHz,DMSO-d6)δ10.53(s,1H),7.38-7.36(m,1H),7.20-7.18(m,1H),7. 00-6.91(m,2H),6.19-6.06(m,3H),5.27-5.24(m,1H),5.04(s,1H),4.55-4.53( m,1H),4.43-4.42(m,1H),3.44-3.38(m,3H),2.83-2.80(m,2H),2.72-2.50(m,6 H),1.99-1.96(m,2H),1.80-1.60(m,7H),1.55-1.50(m,3H),1.07-1.06(m,3H).

[0288] LC / MS (m / z, MH + ):565.2.

[0289] Example 16: N-(3,5-difluoro-4-((6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl)phenyl)-1-(3-fluoropropyl)-3-methylazetidin-3-amine

[0290]

[0291] Step 1: Synthesis of 1-(3-fluoropropyl)-3-methylazetidin-3-amine

[0292]

[0293] 1-(3-fluoropropyl)-3-amino-3-methylacridine was prepared by referring to the method of step 1 and step 2 of Example 1, except that the (S)-(5-azaspiro[2.4]hept-7-yl)carbamic acid tert-butyl ester in step 1 of Example 1 was replaced with 3-(Boc-amino)-3-methylazetidine.

[0294] Step 2: Synthesis of N-(3,5-difluoro-4-((6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl)phenyl)-1-(3-fluoropropyl)-3-methylazetidin-3-amine

[0295]

[0296] The title compound was prepared by referring to the preparation method of Example 8, except that the (S)-5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-amine in Example 8 was replaced by 1-(3-fluoropropyl)-3-amino-3-methylacridine. The last two steps of Example 8 were carried out in the same manner to obtain the title compound.

[0297] 1 H NMR(400MHz,DMSO-d6)δ13.00(s,1H),8.06(s,1H),7.24-7.22(m,1H),6.77-6.7 5(m,1H),6.63(s,1H),5.94-5.90(m,2H),5.17(s,1H),4.52-4.49(m,1H),4.40- 4.37(m,1H),3.51-3.44(m,2H),3.32-3.30(m,3H),3.19-3.14(m,1H),2.98-2.8 8(m,4H),2.48-2.46(m,1H),1.69-1.60(m,2H),1.46(s,3H),1.10-1.08(m,3H).

[0298] LC / MS (m / z, MH + ):526.2.

[0299] Example 17: 3-(((1R,3R)-1-(2,6-difluoro-4-((((1R,4R,5S)-2-(3-fluoropropyl)-2-azabicyclo[2.1.1]hexan-5-yl)amino)phenyl)-3-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)-2,2-difluoropropan-1-ol

[0300]

[0301] The preparation method is similar to that of Example 1, except that the (7S)-5-(benzyl)-5-azaspiro[2.4]hept-7-ylcarbamic acid tert-butyl ester in step 1 of Example 1 is replaced with ((1R, 4R, 5S)-2-azabicyclo[2.1.1]hexyl-5-yl)benzylcarbamate to prepare the intermediate ((1R, 4R, 5S)-2-(3-fluoropropyl)-2-azabicyclo[2.1.1]hexyl-5-yl)benzylcarbamate. Benzyl ((1R, 4R, 5S)-2-(3-fluoropropyl)-2-azabicyclo[2.1.1]hexyl-5-yl)carbamate (290 mg, 1.00 mmol) was added to 10 mL of methanol, and 10 mg of Pd / C was added. After hydrogen was introduced and stirred at room temperature for 5 hours, the reaction was completed. The reaction solution was filtered and concentrated to obtain the intermediate (1R, 4R, 5S)-2-(3-fluoropropyl)-2-azabicyclo[2.1.1]hexan-5-amine. The title compound was prepared by the same method as in Example 1, Step 3, except that (S)-5-(3-fluoropropyl)-5-azaspiro[2.4]hept-7-amine in Step 3 of Example 1 was replaced with (1R, 4R, 5S)-2-(3-fluoropropyl)-2-azabicyclo[2.1.1]hexan-5-amine.

[0302] 1 H NMR(400MHz,DMSO-d6)δ10.39(s,1H),7.37-7.32(m,2H),6.98-6.88(m,2H),6.35(s,1H),6.21-6 .18(m,1H),5.95(s,1H),5.67-5.65(m,1H),4.66-4.63(m,1H),4.54-4.51(m,1H),4.34-4.36(m,1 H),4.27-4.20(m,1H),3.41-3.40(m,1H),3.31-3.24(m,2H),2.93-2.91(m,1H),2.81-2.67(m,4H ),2.59-2.55(m,3H),2.19-2.16(m,1H),1.88-1.77(m,2H),1.29-1.20(m,2H),1.07-1.06(m,3H).

[0303] LC / MS (m / z, MH + ):549.2.

[0304] Example 18: (S)-5-(3-fluoropropyl-1,1-d2)-N-(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-yl)-5-azaspiro[2.4]hept-7-amine

[0305]

[0306] Step 1: Synthesis of tert-butyl (S)-(5-(3-fluoropropionyl)-5-azaspiro[2.4]hept-7-yl)carbamate

[0307]

[0308] Add tert-butyl (S)-(5-azaspiro[2.4]heptane-7-yl)carbamate (1.06 g) to 10 mL of N,N-dimethylformamide, and add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.60 g), 3-fluoropropionic acid (1.00 g), and N,N-diisopropylethylamine (2.50 g). After stirring at room temperature for 4 hours, the reaction is complete. The reaction solution is added to 100 mL of water and extracted with ethyl acetate. The organic layers are combined, concentrated, and purified by column chromatography (dichloromethane / methanol gradient elution) to obtain the title compound.

[0309] Step 2: Synthesis of (S)-1-(7-amino-5-azaspiro[2.4]heptyl-5-yl)-3-fluoropropan-1-one

[0310]

[0311] Add tert-butyl (S)-(5-(3-fluoropropionyl)-5-azaspiro[2.4]hept-7-yl)carbamate (1.00 g) to 10 mL of a 4 mol / L hydrogen chloride 1,4-dioxane solution. Stir at room temperature for 1 hour and the reaction is complete. Concentrate the reaction solution, add saturated sodium bicarbonate solution, and extract with ethyl acetate. Combine the organic layers, dry over anhydrous sodium sulfate, concentrate the organic layer, and directly use it in the next step.

[0312] Step 3: Synthesis of (S)-5-(3-fluoropropyl-1,1-d2)-5-azaspiro[2.4]hept-7-amine

[0313]

[0314] (S)-1-(7-amino-5-azaspiro[2.4]heptyl-5-yl)-3-fluoropropane-1-one (400 mg) was added to 20 mL of anhydrous tetrahydrofuran, and lithium aluminum hydride (AlD4Li) (100 mg) was added. The mixture was heated to 70°C under nitrogen protection. The reaction was completed after 4 hours. The reaction solution was concentrated and used directly in the next step.

[0315] Step 4: Synthesis of (S)-5-(3-fluoropropyl-1,1-d2)-N-(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-yl)-5-azaspiro[2.4]hept-7-amine

[0316]

[0317] (1S,3R)-1-(5-bromopyridin-2-yl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyridyl[3,4-b]indole (200 mg), methanesulfonic acid (2-dicyclohexylphosphine)-3,6-dimethoxy-2,4,6-triisopropyl-1,1-biphenyl)(2-amino-1,1-biphenyl-2-yl)palladium(II) (Brettphos Pd G3) (45.0 mg), sodium tert-butoxide (192 mg), and (S)-5-(3-fluoropropyl-1,1-d2)-5-azaspiro[2.4]hept-7-amine (100 mg) were added to 5 mL of toluene. The mixture was heated to 80° C. under argon protection and stirred for 4 hours. The reaction was completed. The reaction solution was purified by column chromatography (dichloromethane / methanol gradient elution) to obtain the title compound.

[0318] 1 H NMR(400MHz,DMSO-d6)δ10.58(s,1H),7.90-7.88(m,1H),7.42-7.40(m,1H),7.27-7.25(m,1H) ,7.05-6.96(m,4H),5.75(d,J=8.8Hz,1H),4.89(s,1H),4.56-4.53(m,1H),4.44-4.41(m,1H),3 .77-3.70(m,1H),3.59-3.41(m,1H),3.35-3.25(m,1H),3.04-2.89(m,2H),2.75-2.36(m,5H), 1.85-1.71(m,2H),1.14-1.12(m,3H),0.75-0.73(m,1H),0.64-0.61(m,2H),0.48-0.43(m,1H).

[0319] LC / MS (m / z, MH + ):518.3.

[0320] Biological activity and related properties test examples

[0321] Test Example 1: Detection of the effect of the compounds of the present invention on estrogen receptor degradation in MCF7 cells

[0322] Principle of the test: Determine the degradation activity of the compounds of the present invention on the estrogen receptor endogenously expressed in MCF7 cells. 50 and maximum degradation efficiency to evaluate the activity of the test compound.

[0323] Test method:

[0324] MCF7 cells (purchased from ATCC, HTB-22) were cultured in complete DMEM (purchased from Gibco, 11995-065) supplemented with 10% fetal bovine serum. On the first day of the experiment, MCF7 cells were seeded at a density of 3,000 cells / well in a 384-well plate using complete medium and incubated at 37°C in a 5% CO2 cell culture incubator. Test compounds were dissolved in DMSO to a stock concentration of 10 mM, diluted using Echo550 (purchased from Labcyte Inc.), and added to the cell culture plates. Each compound treatment started at 100 nM and was serially diluted three-fold across nine concentrations. A blank control containing 0.5% DMSO was included, and duplicate wells were used for each concentration. Incubation was performed at 37°C in a 5% CO2 cell culture incubator for 24 hours. Paraformaldehyde was added to the cell culture medium in each cell culture well to a final concentration of approximately 3.7% to fix the cells. After 30 minutes, the supernatant was discarded and 50 μL PBS was added to each well for washing. PBS containing 0.5% v / v Tween-20 was added to treat the cells for 30 minutes and washed once with PBS. Blocking solution (homemade, PBS containing 5% BSA, 0.05% Tween-20) was added and incubated at room temperature for 1 hour. The blocking solution was removed and a mixture of primary antibodies (anti-ER monoclonal antibody, Estrogen Receptor α (D8H8) Rabbit mAb, purchased from CST, #8644S, diluted 1:1000; anti-GAPDH monoclonal antibody, GAPDH (D4C6R) Mouse mAb) was added. The cells were incubated with mAb (CST, #97166S, 1:2000 dilution) at room temperature for 3 hours; washed three times with PBST (homemade, PBS containing 0.05% Tween-20); secondary detection antibodies (800CW - goat anti-rabbit IgG, LI-COR, P / N: 926-32211, 1:1000 dilution; 680RD - goat anti-mouse IgG, LI-COR, #925-68070, 1:1000 dilution) were added and incubated at room temperature in the dark for 45 minutes; washed three times with PBST, and the fluorescence signal of each well was read using the Odyssey CLx. Data were processed using XLfit, and the degradation activity DC of each compound was calculated based on the compound concentration and fluorescence signal value. 50 .

[0325] Test results:

[0326] Under the experimental conditions, the test compound has good degradation activity on ER at the cellular level. The test results of the test compound's corresponding ER activity are shown in Table 1.

[0327] Table 1 Results of in vitro ER activity test of test compounds based on cell level

[0328]

[0329]

[0330] Test Example 2: Detection of the inhibitory effect of the compound of the present invention on T47D cell proliferation

[0331] Principle of the test: Determine the inhibitory effect of the compound of the present invention on the proliferation of T47D cells in vitro. 50 The activity of the compound was evaluated by the maximum inhibitory efficiency.

[0332] Test method:

[0333] T47D (T-47D) cells (purchased from ATCC, HTB-133) were cultured with RPMI-1640 (purchased from Gibco, A10491-01) complete medium containing 10% fetal bovine serum. On the first day of the experiment, T47D cells were seeded in a 384-well plate at a density of 500 cells / well using complete medium and cultured overnight at 37°C in a 5% CO2 cell culture incubator. On the second day, the compound to be tested was added for drug treatment. The compound solution with a storage concentration of 10mM was diluted and transferred to each cell culture well using Echo550 (purchased from Labcyte Inc.). The starting concentration of each compound in the cell was 100nM, with 3-fold gradient dilution and 10 concentration points. A blank control containing 0.3% DMSO was set up, and a double-dip control was set up for each concentration point. Cultured at 37°C, 5% CO2 cell culture incubator for 7 days. On the eighth day, the cell culture plate was removed. Add CellTiter- Luminescent Cell Viability Assay (purchased from Promega, G7573) was placed at room temperature for 10 minutes, and then the luminescence signal value was read using a multi-label microplate reader EnVision (purchased from PerkinElmer). The inhibitory activity IC of each compound was calculated based on the concentration and luminescence signal value using XLfit. 50 .

[0334] Test results:

[0335] Under the experimental conditions, the test compound showed good inhibitory activity against T47D breast cancer cells. The results of the inhibitory activity of the test compound on T47D cell proliferation are shown in Table 2.

[0336] Table 2 Inhibitory activity of compounds on T47D cell proliferation

[0337] Test compound <![CDATA[T47D IC 50 (nM)]]> Example 1 0.28 Example 2 0.44 Example 4 0.27 Example 5 0.04 Example 6 0.42 Example 7 0.57 Example 8 0.31 Example 9 0.42 Example 10 0.42 Example 11 0.21 Example 14 0.44 Example 16 0.21 Example 17 0.30

[0338] Test Example 3: Detection of the inhibitory effect of the compound of the present invention on MCF7 cell proliferation

[0339] Experimental principle: Determine the inhibitory effect of the compound of the present invention on the proliferation of MCF7 cells in vitro. 50 The maximum inhibitory efficiency was used to evaluate the activity of the compound.

[0340] Test method:

[0341] MCF7 cells (ATCC, HTB-22) were cultured with 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 at 37°C in a 5% CO2 cell culture incubator. On the second day, the compound to be tested was added for drug treatment. The compound solution with a storage concentration of 10mM was diluted and transferred to each cell culture well using Echo550 (Labcyte Inc.). The starting concentration of each compound in the cell was 100nM, with 3-fold gradient dilution and 9 concentration points. A blank control containing 0.3% DMSO was set up, and a double-dip control was set up for each concentration point. Cultured at 37°C in a 5% CO2 cell culture incubator for 7 days. On the eighth day, the cell culture plate was removed. Add CellTiter- Luminescent Cell Viability Assay (Promega, G7573) was placed at room temperature for 10 minutes, and the luminescence signal value was read using a multi-label microplate reader EnVision (PerkinElmer). The inhibitory activity IC of each compound was calculated based on the compound concentration and luminescence signal value using XLfit. 50 and maximum inhibition rate I max , the results are shown in Table 3.

[0342] Table 3 Inhibitory activity of compounds on MCF7 cell proliferation

[0343] Compound number <![CDATA[IC50 for inhibiting MCF-7 cell proliferation 50 (nM)]]> Maximum inhibition rate (%) Example 4 0.07 105.79 Example 8 0.08 105.71

[0344] Test Example 4: Pharmacokinetic properties of the compounds of the present invention

[0345] Principle: Using mice as test animals, the LC / MS / MS method is used to determine the drug concentration in the plasma of mice at different time points after oral and intravenous administration of the compound of the present invention. This is to study the pharmacokinetic behavior of the compound of the present invention in mice and evaluate its pharmacokinetic characteristics.

[0346] Test method:

[0347] 4.1 Experimental Animals

[0348] Eighteen healthy adult female BALB / c mice were divided into six groups, with three mice in each group, and gavage was performed. The mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. with animal production license number: SCXK (Zhejiang) 2019-0001.

[0349] 4.2 Drug preparation

[0350] A certain amount of drug was weighed and dissolved in DMSO 5% + PG 20% + anhydrous ethanol 5% + solutol 10% + water 60% to prepare 10 mg / ml for oral administration.

[0351] 4.3 Administration

[0352] BALB / c mice were fasted overnight and then gavage-administered with the drug at a dose of 10 mg / kg and a dosing volume of 1 mL / kg.

[0353] 4.4 Operation

[0354] After oral administration, mice were given blood (40 μL) from the orbit at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 24 h after administration. 5 μL of EDTA-K2 was used for anticoagulation. The plasma was separated by centrifugation at 12,000 rpm and 4°C for 5 min and stored at -20°C.

[0355] To determine the concentration of the test compound in mouse plasma after oral administration of various drug concentrations: thaw samples at room temperature and vortex for 1 minute. Quantitatively transfer 15 μL to a 2 ml 96-well plate, add 150 μL of internal standard precipitant, and shake (1200 rpm for 3 minutes). Centrifuge (4000 rpm for 15 minutes), transfer 100 μL of the supernatant to a 1 ml 96-well plate, dry under nitrogen, add 100 μL of a reconstitution solution (1:9 acetonitrile / water), shake well (900 rpm for 3 minutes), and inject 20 μL for analysis. LC / MS / MS conditions: mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile, column: ACE C18 5 μm (3.0 mm x 50 mm), column temperature: 35°C, flow rate: 0.5 ml / min.

[0356] Test results:

[0357] Under the experimental conditions, the test compound showed good pharmacokinetic properties, and the results are shown in Table 4.

[0358] Table 4 Pharmacokinetic parameters of the compounds after single oral administration in mice

[0359]

[0360] Test Example 5: Brain-to-blood ratio test after oral administration of the compound of the present invention to mice

[0361] 1. Experimental purpose:

[0362] After oral administration of the compound of the present invention to mice, the drug entry into the brain and the brain-to-blood ratio were measured to evaluate the brain penetration of the compound.

[0363] 2. Experimental methods:

[0364] 2.1 Investigational Drugs

[0365] GDC-9545 (Compound 340 in Table 1 of CN107108611A, prepared according to the patented method), the compound of Example 4, and the compound of Example 8.

[0366] 2.2 Experimental animals

[0367] Eighteen healthy adult female BALB / c mice were divided into six groups with three mice in each group. The specific grouping is shown in the following table. The mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. with animal production license number: SCXK (Zhejiang) 2019-0001.

[0368]

[0369] 2.3 Drug preparation

[0370] A certain amount of the test compound was weighed and dissolved in 40% tetraethylene glycol (7.5% Captisol) + 60% water to prepare 1 mg / mL for oral administration.

[0371] 2.4 Administration

[0372] BALB / c mice were fasted overnight and then gavage-administered with the drug at a dose of 10 mg / kg and a volume of 10 mL / kg.

[0373] 2.5 Sample collection and testing

[0374] Mice were administered via gavage and euthanized 2 and 4 hours after administration. Brain tissue and whole blood were collected. Whole blood was anticoagulated with EDTA-K2 and centrifuged at 12,000 rpm at 4°C for 5 minutes to separate plasma, which was then stored at -20°C. Brain tissue samples were homogenized in PBS and precipitated with acetonitrile. The concentration of the test compound in brain tissue was determined by liquid chromatography-mass spectrometry. The linear range was 1 to 1,000 ng / mL.

[0375] 2.6 Experimental Results

[0376] The brain-to-blood ratio parameters of each test compound are shown in Table 5.

[0377] Table 5 Brain-to-blood ratio parameters of the test compounds

[0378]

[0379] From the above results, it can be seen that the compound of the present invention can penetrate the blood-brain barrier of mice and enter the brain tissue.

[0380] Test Example 6: Determination of Plasma Protein Binding Rate of the Compounds of the Invention

[0381] 1. Test materials and instruments

[0382] 1. CD-1 mouse plasma (BioIVT)

[0383] 2. 96-well equilibrium dialysis plate (HTDialysis LLC, Gales Ferry, CT, HTD96B), equilibrium dialysis membrane (MWCO 12-14K, #1101)

[0384] 3. Positive control compound warfarin

[0385] 4. ABI QTrap 5500 LC / MS

[0386] 2. Test steps

[0387] 1. Preparation of a 100mM sodium phosphate and 150mM NaCl buffer: Prepare an alkaline solution of 14.2g / L Na2HPO4 and 8.77g / L NaCl using ultrapure water. Prepare an acidic solution of 12.0g / L NaH2PO4 and 8.77g / L NaCl using ultrapure water. Titrate the alkaline solution with the acidic solution to a pH of 7.4 to prepare a 100mM sodium phosphate and 150mM NaCl buffer.

[0388] 2. Preparation of 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 dialysis buffer for 20 minutes.

[0389] 3. Plasma Preparation: Thaw frozen plasma quickly at room temperature. Centrifuge the plasma at 3,220 g for 10 minutes at 4°C to remove clots. Collect the supernatant into a fresh centrifuge tube. Measure and record the pH of the plasma; use plasma with a pH of 7-8.

[0390] 4. Preparation of plasma samples containing compounds: Dilute 10 mM stock solution of the compound of the present invention or positive control compound with DMSO to obtain a 200 μM working solution. Add 3 μl of the 200 μM compound working solution to 597 μl of mouse plasma to obtain a plasma sample with a final concentration of 1 μM.

[0391] 5. Equilibrium Dialysis Step: Assemble the dialysis apparatus according to the operating instructions. Add 120 μL of plasma sample containing 1 μM compound to one side of the dialysis membrane and an equal volume of dialysate (phosphate buffer) to the other side. Perform a two-sample test. Seal the dialysis plate and place it in an incubator. Incubate for 6 hours at 37°C, 5% CO2, and approximately 100 rpm. After the incubation period, remove the seal and pipette 50 μL from the buffer and plasma sides of each well into separate wells of a new plate. Add 50 μL of blank plasma to the phosphate buffer sample, add an equal volume of blank phosphate buffer to the plasma sample, and then add 300 μL of acetonitrile containing an internal standard to precipitate the protein. Vortex for 5 minutes and centrifuge at 3,220 g for 30 minutes at 4°C. Transfer 100 μL of the supernatant to the injection plate and add 100 μL of ultrapure water to mix thoroughly for LC-MS / MS analysis.

[0392] Measure the peak area of the compound on the buffer side and the plasma side. The formula for calculating the plasma protein binding rate of the compound is as follows:

[0393] % free rate = (ratio of compound peak area to internal standard peak area on the buffer side / ratio of compound peak area to internal standard peak area on the plasma side) * 100%

[0394] % bound = 1-% free

[0395] All data were calculated using Microsoft Excel. The calculated plasma protein binding values of the compounds of the present invention are shown in Table 6.

[0396] Table 6 Protein binding rate of the compounds of the present invention in CD-1 mouse plasma

[0397] Example No. % Binding Rate Example 4 98.77

[0398] Test Example 7: Determination of membrane permeability and transport properties of the compounds of the present invention

[0399] The membrane permeability and transport properties of the compounds of the present invention were determined using the following test methods.

[0400] 1. Test materials and instruments

[0401] 1. Caco-2 cells (ATCC)

[0402] 2. HEPES (Solarbio 804D049), Penicillin / Streptomycin (Solarbio 20200109) and Trypsin / EDTA (Solarbio), PBS (Solarbio 20200620)

[0403] 3. Fetal bovine serum (FBS) (Sigma WXBD0055V), fluorescein (Sigma MKCJ3738), NaHCO3 (SigmaSLBZ4647)

[0404] 4. Hank's balanced salt solution (Gibco 2085528) and non-essential amino acids (NEAA) (Gibco 2211548), Trypsin / EDTA (Gibco 2120732)

[0405] 5. High glucose DMEM (Corning 20319014)

[0406] 6.HTS Transwell-96Well Permeable(Corning,3391)

[0407] 7. Resistance tester (Millipore, ERS-2)

[0408] 8. Vision (Nexcelom Bioscience)

[0409] 9. Infinite 200PRO Microplate Reader (Tecan, Infinite M200PRO)

[0410] 10. Positive control compounds: Metoprolol (Sinopharm 100084-201403), Erythromycin (MCE84550), and Cimetidine (Sinopharm 100158-201406)

[0411] 11. ABI QTrap 5500 LC / MS

[0412] 2. Test steps

[0413] 1. Caco-2 cell culture

[0414] 1) Preparation of transport buffer (HBSS containing 25 mM HEPES, pH 7.4): Accurately weigh 5.958 g HEPES and 0.35 g NaHCO₃, dissolve in 900 mL of pure water, then add 100 mL of 10× HBSS, stir well, adjust the pH to 7.4, and filter.

[0415] 2) Preparation of Caco-2 cell culture medium: FBS, penicillin, streptomycin, kanamycin, and NEAA were added to high-glucose DMEM (containing L-glutamine) medium to prepare a cell culture medium containing 10% FBS, 0.1 mg / mL streptomycin, 100 units of penicillin, 0.6 μg / mL kanamycin, and 1×NEAA.

[0416] 3) Culture cells in a T-75 flask in a 37°C, 5% CO2 incubator. Discard the medium when cells reach 80-90% confluence. Rinse the cells with 5 mL of PBS, add 1.5 mL of Trypsin / EDTA, and incubate in a 37°C incubator for 5-10 minutes until the cells detach like a quicksand. Finally, neutralize the Trypsin / EDTA with FBS-containing medium.

[0417] 4) Centrifuge the cell suspension at 120 x g for 10 minutes and discard the supernatant.

[0418] 5) Add cell culture medium to resuspend the cells and adjust the density to 6.86×10 5 cells / mL of cell suspension.

[0419] 2. Caco-2 Cell Seeding

[0420] 1) Add 50 μL of culture medium to each well of the Transwell chamber, add 25 mL of culture medium to the lower layer, and place in a 37°C, 5% CO2 incubator to preheat for 1 hour.

[0421] 2) Add 50 μL of cell suspension to each well of the preheated Transwell chamber, and the final seeding density is 2.4×10 5 cells / cm 2 .

[0422] 3) Culture for 14-18 days, changing the medium every other day. Change the medium within 48 hours of initial plating. The medium must be changed the day before the experiment.

[0423] 3. Assessing Monolayer Cell Membrane Integrity

[0424] 1) After 14 days of culture, cells were confluent and differentiated and ready for transport experiments.

[0425] 2) Use a resistance meter to measure the resistance of the single-layer membrane and record the resistance of each hole.

[0426] 3) After the measurement is completed, re-incubate the Transwell culture plate

[0427] 4) Calculate the TEER value: TEER value = TEER (Ω) measured value × membrane area (cm 2 )

[0428] The resistance of the cell membrane is less than 230Ω·cm 2 , indicating that the cell monolayer membrane has poor density and cannot be used for the experiment.

[0429] 4. Transport Experiment

[0430] 1) Dilute a 10 mM stock solution of the compound of the present invention or the positive control compound with DMSO to obtain a 2 mM stock solution, and then dilute the 2 mM stock solution with transport buffer to obtain a 10 μM working solution of the compound of the present invention or the positive control compound.

[0431] 2) Remove the Caco-2 cell plate from the incubator, wash the Transwell plate twice with preheated transport buffer, and incubate it in a 37°C incubator for 30 minutes.

[0432] 3) To measure the rate of compound transport from the apical to the basolateral side (A→B), add 108 μL of compound working solution to the top of the Transwell chamber. Immediately, remove 8 μL of sample from the top and transfer it to 72 μL of transport buffer. Add 240 μL of stop solution containing an internal standard to terminate transport, which serves as the initial apical sample. Simultaneously, add 300 μL of transport buffer to the receiving end (basolateral side). This experiment is performed in duplicate.

[0433] 4) To measure the transport rate of the compound from the basolateral to the apical side (B→A), add 308 μL of compound working solution to the receiving end (basolateral side). Immediately, remove 8 μL of sample from the basolateral side and transfer it to 72 μL of transport buffer. Add 240 μL of stop solution containing an internal standard to terminate transport, which serves as the initial basolateral sample. Simultaneously, add 300 μL of transport buffer to the apical side of the Transwell chamber. This experiment is performed in duplicate.

[0434] 5) Place the cell culture plate in a 37°C CO2 incubator and incubate for 2 hours.

[0435] 6) After the transport experiment is complete, transfer 8 μL of sample from the dosing end (i.e., the top end in the A→B direction and the base end in the B→A direction) to 72 μL of transport buffer. Add 240 μL of stop solution containing an internal standard to terminate transport. Transfer 80 μL of sample from the receiving end (i.e., the base end in the A→B direction and the top end in the B→A direction) to 240 μL of stop solution containing an internal standard. Vortex at 1000 rpm for 10 minutes and centrifuge at 3,220 g for 30 minutes. Transfer 100 μL of the supernatant to a sample injection plate and add 100 μL of ultrapure water to mix thoroughly. Prepare for LC-MS / MS analysis.

[0436] 7) After the transport experiment, measure fluorescence. Prepare a 10 mM Lucifer Yellow stock solution with water and dilute to 100 μM with transport buffer. Add 100 μL of Lucifer Yellow solution to the top of the Transwell chamber and 300 μL of transport buffer to the basolateral end. Incubate in a 37°C CO2 incubator for 30 minutes. Transfer 80 μL of the solution from the basolateral end to a 96-well plate and measure cell fluorescence using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 530 nm (to assess membrane integrity).

[0437] The fluorescence value of Caco-2 cell monolayer was calculated using the following formula:

[0438] LY Leakage={I acceptor ×0.3 / (I acceptor ×0.3+I donor ×0.1)}×100%

[0439] I acceptor Refers to the fluorescence density on the receiving side (0.3 mL), I donor Refers to the fluorescence density of the drug-administered side (0.1 mL). LY > 1.0% indicates poor monolayer membrane tightness, and the corresponding results will be excluded from the evaluation.

[0440] The peak areas of the compound at the administration side and the receiving side were measured. The apparent permeability coefficient (P app , unit: cm / s) and efflux ratio (ER):

[0441] P app ={V A ×[drug] acceptor / (Area×incubation time×[drug] initial donor}

[0442] V A is the volume of the receiving end solution (A→B is 0.3 mL, B→A is 0.1 mL); Area is the Transwell-96 well plate membrane area (0.143 cm2 );incubation time is the incubation time (unit: s); [drug] acceptor is the drug concentration at the receiving side, [drug] initial donor is the initial drug concentration on the dosing side.

[0443]

[0444] P app(B-A) is the apparent permeability coefficient from the base to the top; P app(A-B) is the apparent permeability from the top to the base.

[0445] The calculated apparent permeability coefficients and efflux ratios of the compounds of the present invention are shown in Table 7.

[0446] Table 7 Apparent permeability coefficient and efflux ratio of the compounds of the present invention

[0447]

[0448] Test Example 8: Inhibitory Effects of the Compounds of the Present Invention on CYP2C9, CYP2D6, and CYP3A4 Enzyme Activities

[0449] The inhibition of the compounds of the present invention on the activities of CYP2C9, CYP2D6 and CYP3A4 enzymes was determined using the following test method.

[0450] 1. Test materials and instruments

[0451] 1. Human liver microsomes (Corning 452117)

[0452] 2.Na2HPO4 (Sigma SLBZ6180)

[0453] 3.KH2PO4 (Sigma SLBT6559)

[0454] 4. NADPH (Solarbio 705Y021)

[0455] 5. NADPH (Solarbio 705Y021)

[0456] 6. Positive substrates: diclofenac (Sigma SLBV3438), dextromethorphan (TRC 3-EDO-175-1), and midazolam (Cerilliant FE01161704)

[0457] 7. Positive inhibitors sulfaphenazole (D. Ehrenstorfer GmbH 109012), quinidine (TCI WEODL-RE) and ketoconazole (Sigma 100M1091V)

[0458] 8. AB Sciex Triple Quad 5500 LC / MS

[0459] 2. Test steps

[0460] Preparation of 100 mM phosphate buffer (PBS): Weigh 7.098 g of Na₂HPO₄ and dissolve it in 500 mL of pure water by sonication to prepare Solution A. Weigh 3.400 g of KH₂PO₄ and dissolve it in 250 mL of pure water by sonication to prepare Solution B. Place Solution A on a stirrer and slowly add Solution B until the pH reaches 7.4 to prepare 100 mM PBS buffer.

[0461] 2. Prepare a 10 mM NADPH solution in 100 mM PBS buffer. Dilute a 10 mM stock solution of the compound of the invention with DMSO to obtain 200× concentration working solutions of the compound (6000, 2000, 600, 200, 60, 20, 0 μM). Dilute a positive inhibitor stock solution with DMSO to obtain 200× concentration working solutions of the positive inhibitor (sulfaphenazole, 1000, 300, 100, 30, 10, 3, 0 μM; quinidine / ketoconazole, 100, 30, 10, 3, 1, 0.3, 0 μM). Prepare a 200× concentration working solution of the substrate (120 μM diclofenac, 400 μM dextromethorphan, and 200 μM midazolam) in water, acetonitrile, or acetonitrile / methanol.

[0462] 3. Mix 2 μl of 20 mg / ml liver microsome solution, 1 μl of substrate working solution, 1 μl of compound working solution, and 176 μl of PBS buffer, and preincubate in a 37°C water bath for 15 minutes. For the positive control, add 1 μl of diclofenac, dextromethorphan, or midazolam working solution instead of the compound working solution. Simultaneously, preincubate the samples in a 37°C water bath for 15 minutes with 10 mM NADPH solution. After 15 minutes, add 20 μl of NADPH to each well to initiate the reaction. Incubate at 37°C for 5 minutes (CYP2C9), 20 minutes (CYP2D6), or 5 minutes (CYP3A4). All incubations should be performed in duplicate. After the appropriate incubation time, terminate the reaction by adding 400 μl of ice-cold methanol containing the internal standard to all samples. Vortex to mix thoroughly and centrifuge at 3220 g at 4°C for 40 minutes. After centrifugation, 100 μL of supernatant was transferred to the sample injection plate, and 100 μL of ultrapure water was added and mixed for LC-MS / MS analysis.

[0463] The IC values of the compounds of the present invention against CYP2C9, CYP2D6 and CYP3A4 were calculated using Excel XLfit 5.3.1.3. 50 See Table 8 for values.

[0464] Table 8 IC values of the compounds of the present invention against CYP2C9, CYP2D6 and CYP3A4 50 value

[0465]

[0466] Test Example 9: Growth inhibition experiment of compound on MCF-7 xenograft tumor

[0467] Experimental reagents:

[0468] Human breast cancer MCF-7 cells: ECACC-86012803

[0469] 17β-estradiol tablets: Innovative Research of America, Cat No.: SE-121, 60-day release, 0.18 mg / pellet

[0470] EMEM culture medium: ATCC, Cat No.: 30-2003

[0471] Fetal bovine serum: Hyclone; Cat No.: SV30087.03

[0472] Antibiotic-Antimycotic: Gibco, Cat No.: 15240-062

[0473] 0.25% Trypsin-EDTA: Gibco, Cat No.: 25200-072

[0474] DPBS:Corning,Cat No.:21-031-CVR

[0475] Matrigel: Corning, Cat No.: 354234

[0476] Experimental methods:

[0477] Animal information: Balb / c nude mice, female, 6-8 weeks old, weighing approximately 18-22 g, were purchased from Shanghai Lingchang Biotechnology Co., Ltd. The mice were housed in an SPF-grade environment with separate ventilation in each cage. All animals had free access to standard certified commercial laboratory diet and free drinking water.

[0478] Cell Culture: Human breast cancer MCF-7 cells were cultured in vitro in EMEM (cell culture medium) supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic in a 37°C, 5% CO2 incubator. Twice weekly, cells were routinely digested and passaged using 0.25% trypsin-EDTA. When cell saturation reached 80%-90% and the required number of cells was reached, cells were harvested and counted.

[0479] Cell inoculation: 0.2 ml / (containing 1×10 7 MCF-7 cell suspension (DPBS plus Matrigel, 1:1 volume ratio) was subcutaneously inoculated on the right back of each mouse. Two days prior to cell inoculation, 17β-estradiol tablets were administered subcutaneously. Six days after cell inoculation, mice were randomly divided into groups based on tumor volume and administered the drug. Grouping was designated Day 0.

[0480] Administration: The compound was administered at a dose of 3 mg / kg or 10 mg / kg, orally (PO), once daily (QD) x 3 weeks, or PO, QD x 3 weeks, with 6 mice per group.

[0481] Tumor measurements and experimental parameters:

[0482] Tumor diameter was measured twice a week using a vernier caliper. Tumor volume was calculated using the formula: V = 0.5 axb 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The body weight of mice was measured twice a week.

[0483] The antitumor efficacy of the compound was evaluated by tumor growth inhibition rate (TGI).

[0484] TGI (%) = [(1-(average tumor volume of a treatment group at the end of drug administration-average tumor volume of the treatment group at the beginning of drug administration) / (average tumor volume of the solvent control group at the end of treatment-average tumor volume of the solvent control group at the beginning of treatment)] x 100%.

[0485] The experimental results are shown in Table 9.

[0486] Table 9 Tumor volume of MCF-7 subcutaneous tumor model

[0487]

[0488] Test Example 10: Growth inhibition experiment of compounds on T47D xenograft tumors

[0489] Experimental reagents:

[0490] Human breast cancer T47D cells: ATCC, HTB-133

[0491] 17β-estradiol tablets: Innovative Research of America, Cat No.: SE-121, 60-day release, 0.36 mg / pellet

[0492] RPMI1640 culture medium: Gibco, Cat No.: 22400-089

[0493] Fetal bovine serum: Gbico; Cat No.: 10099-141C

[0494] Penicillin (Pen Strep): Gibco, Cat No.: 15240-122

[0495] 0.25% Trypsin-EDTA: Gibco, Cat No.: 25200-072

[0496] D-PBS (Calcium- and magnesium-free phosphate buffered saline): Hyclone, Cat. No.: SH30256.01

[0497] Matrigel: Corning, Cat No.: 356237

[0498] Bovine insulin: Shanghai Yisheng, Cat No.: 40107ES60

[0499] Experimental methods:

[0500] Animal information: NPG mice, female, 6-8 weeks old, weighing approximately 18-22 g, were purchased from Beijing Weitongda Biotechnology Co., Ltd. The mice were housed in an SPF-grade environment with separate ventilation in each cage. All animals had free access to standard certified commercial laboratory diet and free drinking water.

[0501] Cell Culture: Human breast cancer T47D cells were cultured in RPMI 1640 (cell culture medium) supplemented with 10% fetal bovine serum, 8 μg / ml bovine insulin, and 1% pen-strep in a 37°C, 5% CO2 incubator. Cells were routinely digested and passaged weekly using 0.25% trypsin-EDTA. Cells were harvested and counted when confluence reached 80%-90% and the desired number of cells was reached.

[0502] Cell inoculation: 0.1 ml / (containing 1×10 7 Each mouse was subcutaneously inoculated with a suspension of T47D cells (DPBS plus Matrigel, 1:1 volume ratio) on the right flank of the back. Two days prior to cell inoculation, mice were subcutaneously administered a 17β-estradiol tablet. On day 29 after cell inoculation, mice were randomly assigned to groups based on tumor volume for dosing, with group assignment designated Day 0.

[0503] Administration: Positive drug Fulvestrant ( Fulvestrant injection (AstraZeneca) was administered at a dose of 250 mg / kg subcutaneously (SC) once weekly (QW) for 4 weeks, and the compound was administered at a dose of 3 mg / kg or 10 mg / kg orally (PO) once daily (QD) for 4 weeks. Six mice were included in each group.

[0504] Tumor measurements and experimental parameters:

[0505] Tumor diameter was measured twice a week using a vernier caliper. Tumor volume was calculated using the formula: V = 0.5 axb 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The body weight of mice was measured twice a week.

[0506] The antitumor efficacy of the compound was evaluated by tumor growth inhibition rate (TGI).

[0507] TGI (%) = [(1-(average tumor volume of a treatment group at the end of drug administration-average tumor volume of the treatment group at the beginning of drug administration) / (average tumor volume of the solvent control group at the end of treatment-average tumor volume of the solvent control group at the beginning of treatment)] x 100%.

[0508] The experimental results are shown in Table 10.

[0509] Table 10 Tumor volume of T-47D subcutaneous tumor model

[0510]

[0511] Test Example 11: Langendorff isolated heart perfusion experiment

[0512] Experimental reagents:

[0513] Glucose, General-reagent, catalog number: G2410A

[0514] EDTA, Sinopharm, catalog number: 10009617

[0515] Heparin sodium salt, Sinopharm, catalog number: 63007131

[0516] Sodium pentobarbital, Solarbio, catalog number: P8410

[0517] Test compounds: compound of Example 4, compound of Example 8, GDC-9545 (Compound 340 in Table 1 of CN107108611A, prepared according to the patented method), AZD-9833 (Compound 17 in Example 1 of CN109843888A, prepared according to the patented method).

[0518] Preparation of isolated heart:

[0519] Adult Hartley guinea pigs weighing 250-300 g were anesthetized with sodium pentobarbital (40 mg / kg, ip) intraperitoneally for 5-10 minutes and heparinized (500 U / kg, ip) before anesthesia. The thoracic cavity was rapidly opened, and the heart, containing a portion of the superior vena cava and a long aortic arch, was immediately isolated from the guinea pig's chest and immersed in pre-chilled benchtop solution (benefits in mM: 131 NaCl, 4.0 KCl, 1.8 CaCl₂, 1.2 MgSO₄, 11.1 glucose, 24.9 NaHCO₃, 1.2 KH₂PO₄ (pH 7.4)). The aorta of the isolated guinea pig heart was quickly inserted into the perfusion needle of a Langendorff perfusion apparatus and secured. Retrograde perfusion was then performed with benchtop solution saturated with 95% O₂ and 5% CO₂ (pH 7.35±0.05) at 36.5±1°C, maintaining a constant flow of 15–20 ml / min. Two recording electrodes were placed on either side of the ventricular endocardium to generate a bipolar transverse electrocardiogram.

[0520] Electrical signal recording:

[0521] ECG (bipolar ventricular electrocardiogram) was recorded using a pair of surface electrodes on the epicardium. One electrode was placed on the right ventricle near the atrioventricular annulus, and the other on the surface of the left ventricle. Their positions were adjusted to obtain a large, clear T wave. An indifferent electrode was placed at the aortic root. After stabilization for 30 minutes, the positions of the recording electrodes were not adjusted during the entire experiment.

[0522] All signals were amplified and digitized by an amplifier and recorded at a sampling rate of 1 kHz before being saved to a hard disk for offline analysis. All data were recorded in real time by a PowerLab isolated heart system.

[0523] Experimental plan:

[0524] After 30 minutes of stabilization (results after 30 minutes of stabilization serve as the solvent negative control), all hearts meeting the experimental conditions were perfused with compound-containing perfusate. The same experimental procedure was repeated three times, with each heart perfused with increasing concentrations of the following perfusate (1.1, 3.3, and 10 μM). Following perfusion, the heart was washed with normal benchtop buffer for 20 minutes. Data were collected at sinus rhythm for evaluation of the effects of the test compound.

[0525] Parameter analysis:

[0526] ECG results show the overall electrical activity of the heart. The following parameters are derived from analysis of the experimentally recorded ECG: RR interval, PR interval, QRS complex, QT interval, and QTc (Bazett QTc = QT / RR).

[0527] The experimental results are shown in Table 11.

[0528] Table 11 Heart rate change results of Langendorff test

[0529]

[0530] Note: * indicates significant difference.

[0531] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A compound represented by formula (II) or a pharmaceutically acceptable salt thereof: in: X 1 、X 2 、X 3 、X 4 Independently selected from CR 7 or N; R 7 is selected from H, F, Cl, Br or I; Het is selected from R 1 、R 2 Together with the carbon atom to which it is attached, it forms a cyclopropane; R 5 is a C1-C6 alkyl group optionally substituted by deuterium, F, Cl, Br or I; R 6 Selected from H or C1-C6 alkyl; Or, R 1 or R 2 One and R 6 and their respective connected C and N together form a piperidine ring, R 1 or R 2 The other one is H; R 3 is selected from C1-C6 alkyl, the C1-C6 alkyl is optionally substituted by one or more R d Replacement, R d C1-C6 alkyl independently selected from F, Cl, Br, OH or optionally substituted by one or more groups selected from F, Cl, Br or OH; R 4 is selected from C1-C6 alkyl optionally substituted by F, Cl, Br or I; n is 1; p is 1; Y is selected from NR 10 ; R 10 For H.

2. A compound represented by formula (III) or a pharmaceutically acceptable salt thereof: in: X 1 、X 2 、X 3 、X 4 Independently selected from CR 7 or N; R 7 is selected from H, F, Cl, Br or I; Het is selected from R 1 、R 2 Together with the carbon atom to which it is attached, it forms a cyclopropane; R 5 is a C1-C6 alkyl group optionally substituted by deuterium, F, Cl, Br or I; R 6 Selected from H or C1-C6 alkyl; Or, R 1 or R 2 One and R 6 and their respective connected C and N together form a piperidine ring, R 1 or R 2 The other one is H; R 3 is selected from C1-C6 alkyl, the C1-C6 alkyl is optionally substituted by one or more R d Replacement, R d C1-C6 alkyl independently selected from F, Cl, Br, OH or optionally substituted by one or more groups selected from F, Cl, Br or OH; R 4 is selected from C1-C6 alkyl optionally substituted by F, Cl, Br or I; n is 1; p is 1.

3. A compound represented by formula (IV) or a pharmaceutically acceptable salt thereof: in: X 1 、X 2 、X 3 、X 4 Independently selected from CR 7 or N; R 7 is selected from H, F, Cl, Br or I; Het is selected from R 1 、R 2 Together with the carbon atom to which it is attached, it forms a cyclopropane; R 5 is a C1-C6 alkyl group optionally substituted by deuterium, F, Cl, Br or I; R 6 Selected from H or C1-C6 alkyl; Or, R 1 or R 2 One and R 6 and their respective connected C and N together form a piperidine ring, R 1 or R 2 The other one is H; R 3 Selected from C1-C6 alkyl or C6-C 10 Aryl; the C1-C6 alkyl or C6-C 10 The aryl group is optionally substituted with one or more R d Replacement, R d C1-C6 alkyl independently selected from F, Cl, Br, OH or optionally substituted by one or more groups selected from F, Cl, Br or OH; R 4 is selected from C1-C6 alkyl optionally substituted by F, Cl, Br or I; n is 1; p is 1; R 8 is selected from phenyl or 5-6 membered heteroaryl, wherein the phenyl or 5-6 membered heteroaryl is optionally substituted by one or more groups selected from the following groups: F, Cl, Br, I, OH, C1-C3 alkyl, C1-C3 alkoxy.

4. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the Het is selected from where R 1 、R 2 The carbon atom to which it is connected forms a cyclopropane, n is 1, R 5 is a C1-C6 alkyl group optionally substituted by deuterium, F, Cl, Br or I.

5. The compound of formula (I) according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein the Het is selected from where R 1 or R 2 With R 6 Together with the C and N connected to each other, they form a piperidine ring, R 1 or R 2 The other one is H.

6. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the Het is selected from p is 1, R 5 is a C1-C6 alkyl group optionally substituted by deuterium, F, Cl, Br or I.

7. The compound of formula (I) according to claim 6 or a pharmaceutically acceptable salt thereof, wherein the Het is selected from R 5 is a C1-C6 alkyl group optionally substituted by deuterium, F, Cl, Br or I.

8. The compound of formula (I) according to claim 7 or a pharmaceutically acceptable salt thereof, wherein the Het is selected from R 5 is a C1-C6 alkyl group optionally substituted by deuterium, F, Cl, Br or I.

9. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from one of the following structures:

10. A pharmaceutical composition comprising the compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

11. Use of the compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 10, in the preparation of a medicament for preventing or treating an estrogen receptor-related disease, wherein the estrogen receptor-related disease is a tumor.

Images