Pyrrolidine compound and application thereof

Abstract

Disclosed are a pyrrolidine compound represented by formula (I) or a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the pyrrolidine compound or the pharmaceutically acceptable salt thereof, and a use of the pyrrolidine compound or the pharmaceutically acceptable salt thereof as a selective estrogen receptor degradation agent (SERD) in prevention or treatment of estrogen receptor related diseases.

Description

[0001] This application claims priority to a prior application filed with the State Intellectual Property Office of China on May 15, 2020, with patent application number CN202010414013.9, entitled “Pyrrolidine compounds and their uses.” The entire text of the prior application is incorporated herein by reference. Technical Field

[0002] The present invention relates to a novel pyrrolidine compound or a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the same, and use of the pyrrolidine compound as a selective estrogen receptor degrader or downregulator (SERD) in preventing or treating related diseases. Background Art

[0003] Estrogen (E2) and estrogen receptor α (ERα) are important drivers of breast cancer development and progression. ER transcription factors are expressed in more than two-thirds of breast cancer patients, and in most ER-positive patients, ER remains a key driver, even in tumors that progress after early endocrine therapy. 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] Clinically, approximately 70-80% of breast cancers test positive for estrogen receptors (ERs). This type of breast cancer cell proliferation is heavily dependent on ERs, and 50% of breast cancer deaths occur in this subtype. Early-stage ER-positive breast cancer has a good prognosis, with a 5-year survival rate exceeding 90%. Approximately 30% of patients who receive postoperative endocrine therapy (TAM or AI drugs) experience recurrence within 10 years, but can still receive standard endocrine therapy. Despite this, acquired drug resistance, primarily caused by ESR1-LBD mutations, and the emergence of distant metastases (bone, brain, liver, lung, lymph nodes, etc.) (approximately 10-15% of patients with brain metastases) have led to patients becoming resistant to treatment, resulting in a growing number of drug-resistant patients. Among patients with advanced metastatic breast cancer, brain metastases develop later than lung, liver, and bone metastases, and have a poorer prognosis. The median survival after clinical drug treatment is only 2 to 9 months.

[0005] Fulvestrant is the first and only clinically approved SERD drug for the treatment of postmenopausal patients with ER-positive, metastatic breast cancer after progression on tamoxifen or aromatase inhibitors. Currently, AstraZeneca (see patent application WO2018077630A1) and Genentech (see patent application WO2019245974A1) have also disclosed a series of novel SERD compounds and corresponding medical uses. Data from multiple studies have shown that ER degradation is not completely achieved in patients treated with fulvestrant. In addition, the intramuscular injection method causes obvious reactions such as pain, swelling, and redness at the injection site, and its slow absorption and limited in vivo exposure (it cannot pass through the blood-brain barrier, and a single intramuscular injection can only reach a maximum of 500 mg. Its pharmacodynamic characteristics and intramuscular administration route limit the maximum dose that can be given to patients). These characteristics greatly limit its clinical application. Therefore, patients with ER-positive breast cancer are in urgent need of new treatment options. Summary of the Invention

[0006] The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof:

[0007]

[0008] in,

[0009] R 1 、R 2 、R 3 、R 4 Independently selected from H, F, Cl, Br, I, CN, C1-C6 alkyl, C1-C6 alkoxy or C3-C6 cycloalkyl;

[0010] X1, X2, X3, X4 are independently selected from CR 6 or N;

[0011] R 6Selected from H, F, Cl, Br, I, OH, CN, C1-C 10 Alkyl, C3-C 10 Cycloalkyl, 3-10 membered heterocyclic group, C1-C 10 Alkoxy, C3-C 10 Cycloalkyloxy or 3-10 membered heterocyclyloxy;

[0012] Y is selected from O or NH;

[0013] R 5 independently selected from C1-C6 alkyl, said C1-C6 alkyl being optionally replaced by R a replace;

[0014] R a is selected from F, Cl, Br, I, OH, CN, C1-C6 alkyl, C1-C6 alkoxy or C3-C6 cycloalkyl;

[0015] Provided that the compound represented by formula (I) does not contain

[0016] In some embodiments, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the compound represented by formula (II) or a pharmaceutically acceptable salt thereof:

[0017]

[0018] In some embodiments, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the compound represented by formula (III) or a pharmaceutically acceptable salt thereof:

[0019]

[0020] In some embodiments, R 1 、R 2 、R 3 、R 4 Independently selected from H, F, Cl, Br, I, CN or C1-C6 alkyl.

[0021] In some embodiments, R 1 、R 2 、R 3 、R 4 Independently selected from H, F, Cl, Br, I, CN or C1-C3 alkyl.

[0022] In some embodiments, R 1 、R 2 、R 3 、R 4 are independently selected from H, F, Cl, Br, I, CN or methyl.

[0023] In some embodiments, R 1 、R 2 、R 3 、R 4 Independently selected from H, F, Cl, Br, I or C1-C3 alkyl.

[0024] In some embodiments, R 1 、R 2 、R 3 、R 4 are independently selected from H, F, Cl, Br, I or methyl.

[0025] In some embodiments, R 1 、R 2 、R 3 、R 4 are independently selected from H, F or methyl.

[0026] In some embodiments, R 1 、R 2 are independently selected from H, F or methyl.

[0027] In some embodiments, R 3 、R 4 are independently selected from H or methyl.

[0028] In some embodiments, R 3 、R 4 Independently selected from H.

[0029] In some embodiments, the structural unit Selected from

[0030] In some embodiments, the structural unit Selected from

[0031] In some embodiments, the structural unit Selected from

[0032] In some embodiments, the structural unit Selected from

[0033] In some embodiments, the structural unit Selected from

[0034] In some embodiments, the structural unit Selected from

[0035] In some embodiments, the structural unit Selected from

[0036] In some embodiments, X1, X2, X3, and X4 are independently selected from CR 6 or N, and at least two of the X1, X2, X3, and X4 groups are selected from CR 6 .

[0037] In some embodiments, X1, X2, X3, and X4 are independently selected from CR 6 or N, and at least three of the X1, X2, X3, and X4 groups are selected from CR 6 .

[0038] In some embodiments, the R 6 is selected from H, F, Cl, Br, I, CN, C1-C3 alkyl or C1-C3 alkoxy.

[0039] In some embodiments, the R 6 is selected from H, F, Cl, Br, I, CN or C1-C3 alkoxy.

[0040] In some embodiments, the R 6 is selected from H, F, Cl, Br, I, CN or methoxy.

[0041] In some embodiments, the R 6 is selected from H, F, Cl, Br, I or CN.

[0042] In some embodiments, the R 6 is selected from H, F, Cl, Br or I.

[0043] In some embodiments, the R 6 Selected from H or F.

[0044] In some embodiments, the structural unit Selected from

[0045] In some embodiments, the structural unit Selected from

[0046] In some embodiments, R 5 is selected from C1-C3 alkyl, wherein the C1-C3 alkyl is optionally replaced by R a replace.

[0047] In some embodiments, the R ais selected from F, Cl, Br, I, OH or CN.

[0048] In some embodiments, the R a is selected from F, OH or CN.

[0049] In some embodiments, the R a Selected from F or OH.

[0050] In some embodiments, R 5 Selected from CH2CF3, CH2CHF2, CH2CF2CH2OH or CH2CF2CH2CN.

[0051] In some embodiments, R 5 Selected from CH2CF3 or CH2CF2CH2OH.

[0052] In some embodiments, Y is selected from NH.

[0053] In some embodiments, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or pharmaceutically acceptable salts thereof:

[0054]

[0055] In some embodiments, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or pharmaceutically acceptable salts thereof:

[0056]

[0057] In some embodiments, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or pharmaceutically acceptable salts thereof:

[0058]

[0059]

[0060] In some embodiments, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or pharmaceutically acceptable salts thereof:

[0061]

[0062]

[0063] In some embodiments, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or pharmaceutically acceptable salts thereof:

[0064]

[0065]

[0066] 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 excipient.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] Furthermore, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for preventing or treating estrogen receptor-related diseases.

[0071] The present invention also relates to a method for treating estrogen receptor-related diseases, which comprises administering to a patient a therapeutically effective dose of a pharmaceutical preparation comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof according to the present invention.

[0072] In a preferred embodiment of the present invention, the estrogen receptor-related diseases include but are not limited to tumors.

[0073] In a preferred embodiment of the present invention, the estrogen receptor-related disease is breast cancer.

[0074] In a preferred embodiment of the present invention, the estrogen receptor-related disease is ER-positive breast cancer.

[0075] In a preferred embodiment of the present invention, the estrogen receptor-related disease is ER-positive breast cancer brain metastasis.

[0076] In a preferred embodiment of the present invention, the patient is a breast cancer patient.

[0077] In a preferred embodiment of the present invention, the patient is an ER-positive breast cancer patient.

[0078] In a preferred embodiment of the present invention, the patient is a patient with ER-positive breast cancer brain metastasis.

[0079] The compounds of the present invention exhibit excellent in vitro and in vivo anti-tumor activity and drugability. In vivo studies have shown that the compounds of the present invention can significantly inhibit tumor growth in a mouse model of ER-positive breast cancer and significantly improve the survival of mice with an orthotopic brain model of ER-positive breast cancer. Furthermore, the compounds of the present invention have high bioavailability, strong ER degradation, oral administration, and high blood-brain barrier crossability, demonstrating their potential as effective treatments for ER-positive breast cancer, particularly brain metastases.

[0080] Definitions and Explanations of Terms

[0081] Unless otherwise indicated, the definitions of groups and terms in the present specification and claims, including definitions provided as examples, exemplary definitions, preferred definitions, definitions in tables, and definitions of specific compounds in the Examples, may be arbitrarily combined and coupled with one another. The resulting group definitions and compound structures shall fall within the scope of the present specification.

[0082] In this article Indicates the attachment site.

[0083] The term "pharmaceutically acceptable salts" refers to salts of pharmaceutically acceptable non-toxic acids or bases, including salts of inorganic acids and bases, and organic acids and bases.

[0084] The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.

[0085] The compounds of the present invention may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms (optical centers) or asymmetric double bonds. Racemates, enantiomers, diastereomers, and geometric isomers are all within the scope of the present invention.

[0086] 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. represents the cis-trans configuration of alicyclic compounds. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless specified otherwise, they are intended to include both E and Z geometric isomers. Likewise, all tautomeric forms are encompassed within the scope of the present invention.

[0087] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms, or asymmetric phosphorus atoms may be present in substituents such as alkyl groups. All of these isomers and their mixtures are included within the scope of the present invention. The compounds of the present application containing asymmetric atoms can be isolated in optically pure form or in racemic form. Optically pure forms can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents. Non-limiting examples of stereoisomers include, but are not limited to:

[0088]

[0089] 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.

[0090] The term "pharmaceutical composition" refers to a mixture of one or more compounds described herein or their physiologically / pharmaceutically acceptable salts or prodrugs with other chemical components, such as physiologically / pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate administration of the compound to an organism.

[0091] 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.

[0092] 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.

[0093] When any variable (such as R a 、R b ) appears more than once in the composition or structure of a compound, its definition is independent in each case. For example, if a group is represented by two R b is replaced, then each R b There are independent options.

[0094] When the number of a linking group is 0, such as -(CH2)0-, it means that the linking group is a bond.

[0095] When one of the variables is selected from a chemical bond or does not exist, it means that the two groups it connects are directly connected. For example, when L in ALZ represents a bond, it means that the structure is actually AZ.

[0096] When the linking group mentioned in this article does not specify its connection direction, its connection direction is arbitrary. L in 1 When selected from "C1-C3 alkylene-O", L 1 You can connect the rings Q and R in the same direction as reading from left to right. 1 Constitute "ring Q-C1-C3 alkylene-OR 1 ", you can also connect rings Q and R in the opposite direction of reading order from left to right 1 Constitute the "ring QO-C1-C3 alkylene-R 1 ”.

[0097] The term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

[0098] In this article, C m -C n , means an integer number of carbon atoms in the range mn. For example, "C1-C 10 ” means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, or 10 carbon atoms.

[0099] The term "alkyl" refers to a group of the formula C n H 2n+1 The term "C1-C 10 “Alkyl” is understood to mean a linear or branched, saturated, monovalent hydrocarbon radical having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. The alkyl radical 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, etc.; preferably, “C1-C 10 "Alkyl" may include "C1-C6 alkyl" or "C1-C3 alkyl", "C1-C6 alkyl" should be understood to mean a straight-chain or branched saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5, or 6 carbon atoms, and "C1-C3 alkyl" should be understood to mean a straight-chain or branched saturated monovalent hydrocarbon group having 1, 2, or 3 carbon atoms.

[0100] The term "C1-C 10 "Alkoxy" can be understood as "C1-C 10 Alkyloxy" or "C1-C 10 Alkyl-O-", preferably, "C1-C 10 The "alkoxy" may include a "C1-C6 alkoxy" or a "C1-C3 alkoxy".

[0101] The term "C3-C 10 The term "cycloalkyl" is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 10 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl, or a bicyclic hydrocarbon ring such as decalin. The term "C3-C6 cycloalkyl" is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 6 carbon atoms.

[0102] The term "C3-C 10 "Cycloalkyloxy" can be understood as "C3-C 10 Cycloalkyl-O-", preferably, "C3-C 10 The “cycloalkyloxy” may include a “C3-C6 cycloalkyloxy”.

[0103] The term "heterocyclyl" refers to a fully saturated or partially saturated (heteroaromatic group that is not aromatic as a whole) monovalent monocyclic, fused, spiro or bridged ring group containing 1 to 5 heteroatoms or heteroatomic groups (i.e., heteroatom-containing atomic groups) 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. The term "3-10 membered heterocyclyl" refers to a heterocyclyl group having 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms, and containing 1 to 5 heteroatoms or heteroatom groups independently selected from the above-mentioned heteroatoms. In particular, examples of 3-membered heterocyclyl groups include, but are not limited to, glycidyl or aziridine; examples of 4-membered heterocyclyl groups include, but are not limited to, azetidinyl and oxetanyl; examples of 5-membered heterocyclyl groups include, but are not limited to, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydrooxazole or 2,5-dihydro-1H-pyrrolyl; examples of 6-membered heterocyclyl groups include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, tetrahydropyridinyl or 4H-[1,3,4]thiadiazinyl; Examples of 7-membered heterocyclic groups include, but are not limited to, diazepanyl; The heterocyclic group may also be a bicyclic group, wherein examples of 5,5-membered bicyclic groups include, but are not limited to, a hexahydrocyclopenta[c]pyrrol-2(1H)-yl ring, and examples of 5,6-membered bicyclic groups include, but are not limited to, a hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl ring, a 5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazinyl ring, or a 5,6,7,8-tetrahydroimidazo[1,5-a]pyrazine. Optionally, the heterocyclic group may be a benzo-fused ring group of the above 4-7-membered heterocyclic groups, examples of which include, but are not limited to, dihydroisoquinolinyl. Optionally, the 3-10 membered heterocyclic group may be a "3-10 membered heterocycloalkyl"; "3-10 membered heterocycloalkyl" may further include "3-7 membered heterocycloalkyl" or "5-6 membered heterocycloalkyl" and the like; although some bicyclic heterocyclic groups in the present application partially contain a benzene ring or a heteroaromatic ring, the heterocyclic group as a whole is still non-aromatic.

[0104] The term "3-10 membered heterocyclyloxy" refers to "3-10 membered heterocyclyl-O-".

[0105] The term "treatment" means administering the compound or formulation described herein to prevent, improve or eliminate a disease or one or more symptoms associated with the disease, and includes:

[0106] (i) preventing a disease or disease state from occurring in a mammal, particularly where such mammal is susceptible to the disease state but has not yet been diagnosed as having the disease state;

[0107] (ii) inhibiting the disease or disease state, i.e., curbing its development;

[0108] (iii) ameliorating the disease or condition, i.e., causing regression of the disease or condition.

[0109] 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.

[0110] 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.

[0111] The term "pharmaceutically acceptable excipient" refers to an excipient that is non-irritating to organisms and does not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art and include, for example, carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.

[0112] The words “comprise,” “comprise,” or “comprises,” and variations thereof such as comprises or comprising, should be construed in an open, non-exclusive sense, ie, “including but not limited to.”

[0113] 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.

[0114] 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.

[0115] 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.

[0116] In addition, the use of heavier isotopes such as deuterium (i.e. 2 H)) substitution may offer certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and may therefore be preferred in certain circumstances, wherein deuterium substitution may be partial or complete, partial deuterium substitution meaning that at least one hydrogen is replaced by at least one deuterium.

[0117] 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.

[0118] 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.

[0119] 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, sugar-coated pill making methods, grinding methods, emulsification methods, freeze-drying methods, etc.

[0120] 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.

[0121] 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.

[0122] The pharmaceutical composition may also be suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in appropriate unit dosage forms.

[0123] In all administration methods described herein, the compound of formula I is administered at a daily dosage of 0.01 to 100 mg / kg body weight, preferably 0.05 to 50 mg / kg body weight, more preferably 0.1 to 30 mg / kg body weight, in the form of single or divided doses.

[0124] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1 is the NOESY spectrum of compound 3;

[0126] Figure 2 This is a tumor growth curve of the MCF-7 subcutaneous tumor model in Test Example 9;

[0127] Figure 3 This is a graph showing weight changes in animals in the MCF-7 subcutaneous tumor model in Test Example 9;

[0128] Figure 4 This is a survival curve of mice in the MCF-7 brain orthotopic model in Test Example 10;

[0129] Figure 5 This is a graph showing weight changes in MCF-7 brain in situ model mice in Test Example 10. DETAILED DESCRIPTION

[0130] The following examples illustrate the technical solutions of the invention in detail, but the protection scope of the present invention includes but is not limited to them.

[0131] 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 determination are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is tetramethylsilane (TMS); "IC 50 ” refers to the half-maximal inhibitory concentration, which is the concentration at which half of the maximum inhibitory effect is achieved.

[0132] Example 1: Synthesis of N-(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-amine (Compound 1)

[0133]

[0134] Synthesis method:

[0135]

[0136] Step 1: Synthesis of tert-butyl (1-(3-fluoropropyl)pyrrolidin-3-yl)carbamate

[0137]

[0138] Tert-butylpyrrolidin-3-ylcarbamate (1.00 g, 5.37 mmol) was dissolved in tetrahydrofuran (10 mL), and sodium hydroxide solution (5 mol·L -1, 2.15mL) and 1-iodo-3-fluoropropane (1.06g, 5.64mmol). The reaction solution was stirred at 25°C for 16 hours. After TLC detection of the complete reaction of the raw materials, the reaction solution was diluted with ethyl acetate and washed with saturated ammonium chloride solution, and the aqueous phase and organic phase were collected separately. After the aqueous phase was extracted three times with ethyl acetate (50mL), all organic phases were combined, dried over sodium sulfate, and the organic phase was concentrated under reduced pressure and then purified by column chromatography (silica, dichloromethane / methanol=100 / 1) to obtain the product tert-butyl (1-(3-fluoropropyl) pyrrolidin-3-yl) carbamate (0.81g).

[0139] 1 H NMR(400MHz,METHANOL-d4)δ4.57(t,J=5.77Hz,1H),4.45(t,J=5.77Hz,1H),4.11(br d,J=7.78Hz,1H),3.05-2.97(m,1H),2.93-2.81(m,1H),2.80-2.69(m,3H),2.66-2.5 6(m,1H),2.30-2.20(m,1H),2.04-1.87(m,2H),1.78-1.68(m,1H),1.51-1.38(m,9H).

[0140] Step 2: Synthesis of 1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride

[0141]

[0142] Tert-butyl (1-(3-fluoropropyl) pyrrolidin-3-yl) carbamate (0.81 g, 3.12 mmol) was dissolved in 1,4-dioxane (9 mL), and then a hydrochloric acid-1,4-dioxane solution (4 mol L) was added. -1 , 9 mL) to form a yellow transparent solution. The reaction solution was stirred at 25°C for 3 hours. After TLC analysis of the reaction, the reaction solution was concentrated under reduced pressure to dryness to obtain the compound 1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride (0.71 g).

[0143] 1 H NMR (400MHz, METHANOL-d4) δ4.68(t,J=5.52Hz,1H),4.56(s,1H),4.30-3.79(m,3H),3.68(s,1H),3.48(br s,2H),3.31-3.21(m,1H),2.82-2.46(m,1H),2.32-2.14(m,3H).

[0144] Step 3: Synthesis of (R)-1-(1H-indol-3-yl)-N-(2,2,2-trifluoroethyl)propan-2-amine

[0145]

[0146] (2R)-1-(1H-indol-3-yl)propan-2-amine (600 mg, 3.44 mmol) and N,N-diisopropylethylamine (445.05 mg, 3.44 mmol) were dissolved in 1,4-dioxane (10 mL). Trifluoroethyl trifluoromethanesulfonate (1.20 g, 5.17 mmol) dissolved in 1,4-dioxane (5 mL) was added at 25°C. The reaction mixture was stirred at 75°C for 16 hours. The reaction mixture was concentrated to dryness under reduced pressure and then purified by column chromatography (silica, petroleum ether / ethyl acetate = 3 / 1) to obtain (R)-1-(1H-indol-3-yl)-N-(2,2,2-trifluoroethyl)propan-2-amine (0.69 g).

[0147] MSm / z(ESI):257.2[M+H] + ;

[0148] 1 H NMR(400MHz, METHANOL-d4)δ7.56-7.54(d,J=8.0Hz,1H),7.36-7.34(d,J=8.0Hz,1H),7.12-7.08(m,2H),7.03- 7.00(m,1H),3.26-3.23(m,2H),3.12-3.10(m,1H),2.93-2.88(m,1H),2.80-2.78(m,1H),1.11(d,J=6.0Hz,3H)

[0149] Step 4: Synthesis of (1S,3R)-1-(5-bromopyridin-2-yl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole

[0150]

[0151] (R)-1-(1H-indol-3-yl)-N-(2,2,2-trifluoroethyl)propane-2-amine (120.00 mg, 468.26 μmol) was dissolved in toluene (2 mL), and 5-bromopyridine-2-carboxaldehyde (87.10 mg, 468.26 μmol) and acetic acid (562.40 mg, 9.37 mmol) were added. The reaction mixture became a yellow transparent solution. The reaction mixture was stirred at 90°C for 10 hours. After the reaction was completed, the reaction solution was cooled to room temperature, concentrated under reduced pressure, and purified by thin layer chromatography (silica, petroleum ether / ethyl acetate = 4 / 1) to give (1S,3R)-1-(5-bromopyridin-2-yl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (110.00 mg).

[0152] MSm / z(ESI):424.1,426.1[M+H] + .

[0153] 1 H NMR(400MHz,METHANOL-d4)δ8.59(s,1H),8.01-7.94(m,1H),7.54(d,J=8.53H z,1H),7.46(d,J=7.78Hz,1H),7.30(d,J=8.03Hz,1H),7.08(d,J=7.59Hz,1H), 7.03-6.96(m,1H),5.08(s,1H),3.58-3.46(m,1H),3.38-3.34(m,1H),3.13-2. 99-(m,1H),2.80(d,J=4.52Hz,1H),2.70-2.61(m,1H),1.26(d,J=6.78Hz,3H).

[0154] Step 5: Synthesis of N-(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-amine

[0155]

[0156] (1S,3R)-1-(5-bromopyridin-2-yl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (110.00 mg, 259.28 μmol) was dissolved in tetrahydrofuran (2 mL), and 1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride (68.18 mg, 311.13 μmol), sodium tert-butoxide (149.50 mg, 1.56 mmol) and methanesulfonic acid (2-dicyclohexylphosphine)-3,6-dimethoxy-2,4,6-triisopropyl-1,1-biphenyl)(2-amino-1,1-biphenyl-2-yl)palladium(II) (23.50 mg, 25.93 μmol) were added. The reaction solution was a brown turbid solution under nitrogen atmosphere. The reaction mixture was stirred at 80°C for 4 hours. After completion of the reaction as determined by LCMS, the mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The mixture was then purified by preparative liquid chromatography (Phenomenex Gemini C18 column, 3 μm silica, 30 mm diameter, 75 mm length) using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile as eluents to afford N-(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-amine (22.23 mg).

[0157] MSm / z(ESI):490.2[M+H] + .

[0158] 1 H NMR (400MHz, METHANOL-d4) δ7.88(d,J=2.76Hz,1H),7.46(d,J=7.78Hz,1H),7.25(dd,J=7.91,3.8 9Hz,2H),7.09-6.96(m,3H),4.97(s,1H),4.60(t,J=5.65Hz,1H),4.48(t,J=5.65Hz,1H),4.15(br s,1H),3.52-3.36(m,3H),3.25-3.14(m,1H),3.09-2.88(m,6H),2.68(s,1H),2.51-2.39(m,1H),2.11-1.85(m,3H),1.20(d,J=6.53Hz,3H).

[0159] Example 2: Synthesis of N-((R)(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-amine (Compound 2) and Example 3: Synthesis of N-((S)(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-amine (Compound 3)

[0160]

[0161] The racemate N-(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-amine (80.00 mg, 153.61 μmol) was subjected to chiral separation (DAICEL CHIRALPAK AY-H column, 5 μm silica, 30 mm diameter, 250 mm length, using decreasingly polar mixtures of isopropanol (containing 0.1% ammonia) and water as eluents) and purification by preparative liquid chromatography (Phenomenex Gemini The product was purified on a C18 column, 3 μm silica, 30 mm diameter, 75 mm length, using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile as eluent to afford N-((R)(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-1-yl)-4-nitropropane-2-ylpyrido[3,4-b]indole-1 ... -yl)pyridin-3-amine (8.22 mg, retention time 2.627 minutes) and N-((S)(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-amine (10.85 mg, retention time 2.817 minutes).

[0162] N-((R)(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-amine (Compound 2):

[0163] MSm / z(ESI):490.1[M+H] +

[0164] 1 H NMR(400MHz,METHANOL-d4)δ7.89(d,J=2.76Hz,1H),7.48-7.44(m,1H),7.29-7.24(m,2 H),7.09-7.04(m,2H),7.00(s,1H),4.98(s,1H),4.64-4.60(m,1H),4.52-4.48(m,1H), 4.25-4.16(m,1H),3.54-3.36(m,4H),3.25-3.09(m,4H),3.05(s,1H),2.89(d,J=4.52H z,1H),2.70-2.60(m,1H),2.55-2.42(m,1H),2.16-1.94(m,3H),1.20(d,J=6.78Hz,3H).

[0165] N-((S)(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-amine (Compound 3):

[0166] MSm / z(ESI):489.25,490.1[M+H] +

[0167] 1 H NMR(400MHz,METHANOL-d4)δ7.90-7.88(m,1H),7.48-7.43(m,1H),7.29-7.23(m,2H),7.09-7.03(m,2H),7.03-6.9 7(m,1H),4.99-4.97(m,1H),4.63-4.59(m,1H),4.52-4.47-(m,1H),4.24-4.16(m,1H),3.52-3.36(m,4H),3.15(br s,4H),3.05-2.99(m,1H),2.96-2.88(m,1H),2.68(s,1H),2.57-2.43(m,1H),2.12-1.94(m,3H),1.20(d,J=6.53Hz,3H).

[0168] N-((S)-1-(3-fluoropropyl)pyrrolidin-3-yl)-6-(((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-amine (Compound 3) can also be obtained by the following synthetic method:

[0169]

[0170] Step 1: Synthesis of (S)-tert-butyl (1-(3-fluoropropyl)pyrrolidin-3-yl)aminomethyl ester

[0171]

[0172] (S)-tert-Butylpyrrolidin-3-ylaminomethyl ester (500.00 mg, 2.68 mmol) was dissolved in tetrahydrofuran (10 mL), and sodium hydroxide solution (5 mol·L -1 , 1.07mL) and 1-iodo-3-fluoropropane (529.88mg, 2.82mmol). The reaction solution was stirred at 25°C for 16 hours. After TLC detection of the complete reaction of the raw materials, the reaction solution was diluted with ethyl acetate (50mL), washed with saturated ammonium chloride solution (10mL), and the aqueous phase and organic phase were collected separately. After the aqueous phase was extracted three times with ethyl acetate (20mL), all organic phases were combined, dried over sodium sulfate, and the organic phase was concentrated under reduced pressure and then purified by column chromatography (silica, dichloromethane / methanol=100 / 1) to obtain the product (S)-tert-butyl (1-(3-fluoropropyl) pyrrolidin-3-yl) aminomethyl ester (480.00mg).

[0173] 1 H NMR(400MHz,METHANOL-d4)δ4.58-4.53(m,1H),4.46-4.40(m,1H),4.14-4.04(m,1H),2.93-2.85(m,1H),2.77-2.67(m,1H ),2.61(dd,J=7.78,5.52Hz,3H),2.47-2.40(m,1H),2.29-2.17(m,1H),1.99-1.82(m,2H),1.71-1.61(m,1H),1.45(s,9H).

[0174] Step 2: Synthesis of (S)-1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride

[0175]

[0176] (S)-tert-Butyl (1-(3-fluoropropyl) pyrrolidin-3-yl) aminomethyl ester (480.00 mg, 1.93 mmol) was dissolved in 1,4-dioxane (3 mL), and then a hydrochloric acid-1,4-dioxane solution (4 mol L) was added. -1, 4.94 mL) to give a yellow, transparent solution. The reaction solution was stirred at 25°C for 3 hours. After TLC analysis of the complete reaction, the reaction solution was concentrated under reduced pressure to yield compound (S)-1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride (450.00 mg).

[0177] 1 H NMR (400MHz, DMSO-d6) δ8.80-8.42(m,3H),4.62(s,1H),4.51(s,1H),4.12-3.45(m,3H),3.17(br s,3H),2.35-1.99(m,4H).

[0178] Step 3: Synthesis of N-((S)-1-(3-fluoropropyl)pyrrolidin-3-yl)-6-(((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-amine

[0179]

[0180] (1S,3R)-1-(5-bromopyridin-2-yl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (140.00 mg, 263.99 μmol) was dissolved in tetrahydrofuran (3 mL), and (S)-1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride (86.77 mg, 316.79 μmol), sodium tert-butoxide (152.22 mg, 1.58 mmol) and methanesulfonic acid (2-dicyclohexylphosphine)-3,6-dimethoxy-2,4,6-triisopropyl-1,1-biphenyl)(2-amino-1,1-biphenyl-2-yl)palladium(II) (23.93 mg, 26.40 μmol) were added. The reaction mixture was stirred at 80°C for 4 hours under nitrogen atmosphere. After the reaction was completed, the product was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and then purified by preparative liquid chromatography (Phenomenex Gemini C18 column, 3 μm silica, 30 mm diameter, 75 mm length); (using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile as eluents) to give the compound N-((S)-1-(3-fluoropropyl)pyrrolidin-3-yl)-6-(((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-amine (37.79 mg).

[0181] MSm / z(ESI):365.1[M+H] +

[0182] 1 H NMR (400MHz, METHANOL-d4) δ7.89(d,J=2.76Hz,1H),7.46(d,J=7.78Hz,1H),7.25(d,J=8.53Hz,2H),7.0 9-7.03(m,2H),7.02-6.97(m,1H),4.98(s,1H),4.60(t,J=5.65Hz,1H),4.49(t,J=5.65Hz,1H),4.18(br s,1H),3.51-3.35(m,4H),3.14-2.99(m,5H),2.92(dd,J=15.18,4.89Hz,1H),2.65(dd ,J=16.06,6.78Hz,1H),2.53-2.42(m,1H),2.12-1.92(m,3H),1.20(d,J=6.78Hz,3H).

[0183] Absolute configuration identification of compound 3

[0184] 2D NMR identification:

[0185]

[0186] NOESY spectrum( Figure 1 ) showed that the methyl hydrogen at position 3 of compound 3 and the hydrogen at position 1 had an obvious NOE effect, proving that the two were on the same side, and the relative configuration of the pyridyl at position 1 and the methyl at position 3 on the 6-membered piperidine ring was trans. It is known that the absolute configuration of the carbon atom at position 3 is R, so the absolute configuration of the carbon atom at position 1 is S.

[0187] Example 4: Synthesis of (S)-N-(3,5-difluoro-4-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-1-(3-fluoropropyl)pyrrolidin-3-amine (Compound 4)

[0188]

[0189] Synthesis method:

[0190]

[0191] Step 1: Synthesis of (S)-tert-butyl (1-(3-fluoropropyl)pyrrolidin-3-yl)carbamate

[0192] (S)-tert-Butylpyrrolidin-3-ylcarbamate (500.00 mg, 2.68 mmol) was dissolved in tetrahydrofuran (10 mL), and sodium hydroxide solution (5 mol·L -1 , 1.07mL) and 1-iodo-3-fluoropropane (529.88mg, 2.82mmol). The reaction solution was stirred at 25°C for 16 hours. After TLC detection showed that the reaction of the raw materials was complete, the reaction solution was diluted with ethyl acetate (50mL) and washed with saturated ammonium chloride solution (10mL), and the aqueous phase and organic phase were collected separately. After the aqueous phase was extracted three times with ethyl acetate (20mL), all organic phases were combined, dried over sodium sulfate, and the organic phase was concentrated under reduced pressure and then purified by column chromatography (silica, dichloromethane / methanol=100 / 1) to obtain the product (S)-tert-butyl (1-(3-fluoropropyl) pyrrolidin-3-yl) carbamate (480.00mg).

[0193] 1 H NMR(400MHz,METHANOL-d4)δ4.58-4.53(m,1H),4.46-4.40(m,1H),4.14-4.04(m,1H),2.93-2.85(m,1H),2.77-2.67(m,1H ),2.61(dd,J=7.78,5.52Hz,3H),2.47-2.40(m,1H),2.29-2.17(m,1H),1.99-1.82(m,2H),1.71-1.61(m,1H),1.45(s,9H).

[0194] Step 2: Synthesis of (S)-1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride

[0195]

[0196] (S)-tert-Butyl (1-(3-fluoropropyl) pyrrolidin-3-yl) carbamate (480.00 mg, 1.93 mmol) was dissolved in 1,4-dioxane (3 mL), and then a hydrochloric acid-1,4-dioxane solution (4 mol L) was added. -1 , 4.94 mL) to give a yellow, transparent solution. The reaction solution was stirred at 25°C for 3 hours. After TLC analysis of the complete reaction, the reaction solution was concentrated under reduced pressure to yield compound (S)-1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride (450.00 mg).

[0197] 1H NMR (400MHz, DMSO-d6) δ8.80-8.42(m,3H),4.62(s,1H),4.51(s,1H),4.12-3.45(m,3H),3.17(br s,3H),2.35-1.99(m,4H).

[0198] Step 3: Synthesis of (1S,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

[0199]

[0200] (R)-1-(1H-indol-3-yl)-N-(2,2,2-trifluoroethyl)propan-2-amine (890 mg, 3.47 mmol) and 4-bromo-2,6-difluorobenzaldehyde (844.27 mg, 3.82 mmol) were dissolved in toluene (10 mL) and acetic acid (2 mL). The reaction mixture was stirred at 90°C for 6 hours. The reaction mixture was concentrated to dryness under reduced pressure and then purified by column chromatography (silica, petroleum ether / ethyl acetate = 20 / 1) to obtain the product (1S,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 (850 mg).

[0201] 1 H NMR(400MHz,METHANOL-d4)δ7.45-7.43(d,J=7.60Hz,1H),7.24-7.20(m,3H),7.05-6.99(m,2H),5.36(s ,1H),3.57-3.54(m,1H),3.46-3.40(m,1H),3.01-2.95(m,2H),2.70-2.65(m,1H),1.20(d,J=6.4Hz,3H).

[0202] Step 4: Synthesis of (S)-N-(3,5-difluoro-4-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-1-(3-fluoropropyl)pyrrolidin-3-amine

[0203]

[0204] (1S,3R)-1-(4-bromo-2,6-difluorophenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyridyl[3,4-b]indole (80.00 mg, 174.20 μmol) was dissolved in tetrahydrofuran (2 mL). (S)-1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride (38.20 mg, 209.04 μmol) and sodium tert-butoxide (100.45 mg, 1.05 mmol) were added and stirred until uniform. Tris(dibenzylideneacetone)dipalladium (31.90 mg, 34.84 μmol) and (±)-2,2-bis(diphenylphosphino)-11-binaphthyl (54.23 mg, 87.10 μmol) were then added under nitrogen. The reaction mixture was stirred at 80°C for 4 hours. After LCMS detection, the reaction mixture was filtered, the filter cake was rinsed with tetrahydrofuran, and the filtrate was concentrated to dryness under reduced pressure. The mixture was purified by preparative liquid chromatography (Phenomenex Gemini C18 column, 7 μm silica, 50 mm diameter, 250 mm length, using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile as eluents) to obtain compound (S)-N-(3,5-difluoro-4-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-1-(3-fluoropropyl)pyrrolidin-3-amine (4.69 mg).

[0205] MSm / z(ESI):525.2[M+H] +

[0206] 1 H NMR(400MHz,METHANOL-d4)δ8.49(s,1H),7.42(d,J=7.3Hz,1H),7.21(d,J=7.8Hz,1H),7.08-6.94 (m,2H),6.19(d,J=11.5Hz,2H),5.24(s,1H),4.60(t,J=5.6Hz,1H),4.48(t,J=5.6Hz,1H),4.11(br s,1H),3.62-3.53(m,1H),3.21(br s,1H),3.09-2.94(m,6H),2.63(dd,J=15.3,4.3Hz,1H),2.51-2.38(m,1H),2 .12-1.99(m,2H),1.96-1.85(m,1H),1.39-1.29(m,2H),1.19(d,J=6.5Hz,3H)

[0207] Example 5: Synthesis of trans-N-(3,5-difluoro-4-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-4-fluoro-1-(3-fluoropropyl)pyrrolidin-3-amine (Compound 5)

[0208]

[0209] Synthesis method:

[0210]

[0211] Step 1: Synthesis of tert-butyl (trans-4-fluoro-1-(3-fluoropropyl)pyrrolidin-3-yl)carbamate

[0212]

[0213] 1-Fluoro-3-iodopropane (151.86 mg, 807.87 μmol) and tert-butyl (trans-4-fluoropyrrolidin-3-yl) carbamate (150 mg, 734.43 μmol) were dissolved in acetonitrile (4 mL). Potassium carbonate (203.00 mg, 1.47 mmol) was added at 25°C. The reaction solution was stirred at 60°C for 13 hours. The reaction solution was cooled to 25°C, filtered, and concentrated to dryness under reduced pressure. Purification by column chromatography (silica, ethyl acetate / methanol = 10 / 1) afforded the product tert-butyl (trans-4-fluoro-1-(3-fluoropropyl)pyrrolidin-3-yl) carbamate (0.15 g).

[0214] MSm / z(ESI):265.0[M+H] + ;

[0215] 1 H NMR(400MHz,CHLOROFORM-d)δ4.87(br s,1H),4.59(t,J=5.9Hz,1H),4.48(t,J=5.9Hz,1H),4.16(br s,1H),3.29-3.05(m,1H),3.01-2.87(m,1H),2.78-2.41(m,4H),2.02-1.81(m,2H),1.48(s,9H).

[0216] Step 2: Synthesis of trans-4-fluoro-1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride

[0217]

[0218] Tert-butyl ((trans-4-fluoro-1-(3-fluoropropyl)pyrrolidin-3-yl)carbamate (150 mg, 567.51 μmol) was dissolved in 1,4-dioxane (2 mL), 4 M dioxane hydrochloride (2.13 mL) was added, and the reaction solution was stirred at 25°C for 13 hours. The reaction solution was concentrated to obtain the product, trans-4-fluoro-1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride (0.12 g).

[0219] MSm / z(ESI):165.2[M+H] + .

[0220] Step 3: Synthesis of trans-N-(3,5-difluoro-4-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-4-fluoro-1-(3-fluoropropyl)pyrrolidin-3-amine

[0221]

[0222] (1S,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 (140 mg, 304.84 μmol) and trans-4-fluoro-1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride (86.74 mg, 365.81 μmol) were dissolved in To tert-amyl alcohol (5 mL) was added methanesulfonic acid (2-dicyclohexylphosphino-3,6-dimethoxy-2,4,6-triisopropyl-1,1-biphenyl)(2-amino-1,1-biphenyl-2-yl) palladium(II) (25.50 mg, 30.48 μmol) and cesium carbonate (595.95 mg, 1.83 mmol). After nitrogen substitution three times, the reaction solution was stirred at 120°C for 13 hours. The reaction solution was cooled to room temperature and poured into water (10 mL) and stirred for 10 minutes. The solution was extracted twice with ethyl acetate (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The product was then purified by column chromatography (silica, petroleum ether / ethyl acetate = 2 / 1) and preparative liquid chromatography (Phenomenex Gemini-NX column: 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile as eluents) to give the product trans-N-(3,5-difluoro-4-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-4-fluoro-1-(3-fluoropropyl)pyrrolidin-3-amine (27.5 mg).

[0223] MSm / z(ESI):543.1[M+H] + ;

[0224] 1 H NMR(400MHz,METHANOL-d4)δ7.42(d,J=7.5Hz,1H),7.22(d,J=7.5Hz,1H),7.09-6.9 3(m,2H),6.30-6.16(m,2H),5.25(s,1H),4.58(t,J=5.8Hz,1H),4.46(t,J=5.8Hz,1H ),4.11-3.87(m,1H),3.67-3.53(m,1H),3.43-3.34(m,3H),3.19-2.92(m,3H),2.81 -2.55(m,4H),2.29(dd,J=6.9,9.7Hz,1H),2.02-1.83(m,2H),1.19(d,J=6.4Hz,3H).

[0225] Example 6: Synthesis of trans-N-[3,5-difluoro-4-[(1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-1,3,4,9-tetrahydropyrido[3,4-b]indol-1-yl]phenyl]-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-amine (Compound 6)

[0226]

[0227] Synthesis method:

[0228]

[0229] Step 1: Synthesis of tert-butyl N-[trans-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-yl]carbamate

[0230]

[0231] Tert-butyl N-[trans-4-methylpyrrolidin-3-yl]carbamate (80 mg, 399.45 μmol) and potassium carbonate (110.41 mg, 798.89 μmol) were dissolved in acetonitrile (8 mL), followed by the addition of 1-fluoro-3-iodopropane (90.11 mg, 479.34 μmol). The reaction mixture was heated to 50°C and stirred for 16 h. LCMS monitored the reaction completion. The reaction mixture was filtered, the filtrate concentrated to dryness, and purified by column chromatography (silica, ethyl acetate / methanol = 5 / 1) to yield tert-butyl N-[trans-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-yl]carbamate (85 mg).

[0232] MSm / z(ESI):261.1[M+H] +

[0233] 1 H NMR (400MHz, METHANOL-d4) δ4.66(br d,J=2.4Hz,1H),4.61-4.42(m,2H),4.05-3.93(m,1H),3.71-3.56(m,2H),3.17(br d,J=9.4Hz,1H),2.90(br s,2H),2.54(brs,1H),2.19(br d,J=5.2Hz,2H),2.07-1.89(m,1H),1.47(s,9H),1.24-1.11(m,3H).

[0234] Step 2: Synthesis of trans-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-amine hydrochloride

[0235]

[0236] Tert-butyl N-[trans-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-yl]carbamate (80 mg, 307.28 μmol) was dissolved in dioxane (2 mL), followed by the addition of 4 M hydrochloric acid in dioxane (1.54 mL). The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated to dryness to yield trans-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-amine hydrochloride (70 mg).

[0237] 1 H NMR(400MHz,METHANOL-d4)δ4.73-4.64(m,1H),4.61-4.50(m,1H),4.23-4.06(m,1H),4.0 2-3.63(m,5H),3.54-3.42(m,1H),3.01-2.58(m,1H),2.32-2.12(m,2H),1.37-1.27(m,3H)

[0238] Step 3: Synthesis of trans-N-[3,5-difluoro-4-[(1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-1,3,4,9-tetrahydropyrido[3,4-b]indol-1-yl]phenyl]-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-amine

[0239]

[0240] Trans-1-(3-Fluoropropyl)-4-methyl-pyrrolidin-3-amine hydrochloride (30 mg, 128.67 μmol), (1S,3R)-1-(4-bromo-2,6-difluoro-phenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-1,3,4,9-tetrahydropyrido[3,4-b]indole (76.82 mg, 167.27 μmol), and cesium carbonate (167.69 mg, 514.68 μmol) were dissolved in dioxane (8 mL), followed by the addition of tBuBrettphos-Pd-G3 (5.50 mg, 6.43 μmol). The reaction mixture was purged with nitrogen three times, heated to 120°C, and stirred overnight. LCMS monitored the reaction for completion. Methanol (15 mL) was added to the reaction solution, and the mixture was filtered. The filtrate was concentrated and purified by column chromatography (silica, petroleum ether / ethyl acetate = 2 / 1) and preparative liquid chromatography (Phenomenex Synergi C18 column: 4 μm silica, 30 mm diameter, 150 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile as eluents) to give the compound trans-N-[3,5-difluoro-4-[(1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-1,3,4,9-tetrahydropyrido[3,4-b]indol-1-yl]phenyl]-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-amine (1.35 mg).

[0241] MSm / z(ESI):539.3[M+H] + ;

[0242] 1 H NMR (400MHz, METHANOL-d4) δ7.42(d,J=7.6Hz,1H),7.21(d,J=8.8Hz,1H),7.08-6.86(m,2H),6.21(d,J=11.6Hz,2H),5.24(s,1H),4.62-4.58(m,1H) ,4.52-4.45(m,1H),3.73-3.65(m,1H),3.62-3.47(m,3H),3.23-3.15(m,2 H),3.08-2.95(m,2H),2.86-2.54(m,2H),2.41-2.00(m,3H),1.42-1.25(m 2H), 1.23 (dd, J=3.2, Hz, 6.8Hz, 3H), 1.19 (d, J=6.4Hz, 3H).

[0243] Example 7: Synthesis of 3-((1S,3R)-1-(2,6-difluoro-4-(((S)-1-(3-fluoropropyl)pyrrolidin-3-yl)amino)phenyl)-3-methyl-3,4-dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)-2,2-difluoropropan-1-ol

[0244]

[0245] Synthesis method

[0246]

[0247] Step 1: Synthesis of 3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl trifluoromethanesulfonate

[0248]

[0249] 3-((tert-Butyldiphenylsilyl)oxy)-2,2-difluoropropan-1-ol (1 g, 2.85 mmol) and 2,6-lutidine (366.88 mg, 3.42 mmol) were dissolved in anhydrous dichloromethane (15 mL). Trifluoromethanesulfonic anhydride (885.52 mg, 3.14 mmol) was added dropwise in an ice-water bath. The reaction mixture was stirred at 25°C for 16 hours. After the reaction was completed as monitored by TLC (silica, petroleum ether: ethyl acetate = 10:1), the reaction solution was washed sequentially with water (10 mL), hydrochloric acid (1 mol / L, 10 mL) and saturated sodium carbonate solution (10 mL). The collected organic phase was dried over sodium sulfate, concentrated to dryness under reduced pressure, and then purified by column chromatography (silica, petroleum ether / ethyl acetate = 100 / 15) to give the product 3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl trifluoromethanesulfonate (880.00 mg).

[0250] 1 H NMR (400MHz, METHANOL-d4) δ7.66 (d, J = 7.0Hz, 4H), 7.50-7.36 (m, 6H), 4.96 (t, J = 12.2Hz, 2H), 3.90 (t, J = 12.0Hz, 2H), 1.06 (s, 9H).

[0251] Step 2: Synthesis of (R)-N-(1-(1H-indol-3-yl)propan-2-yl)-3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropan-1-amine

[0252]

[0253] 3-((tert-Butyldiphenylsilyl)oxy)-2,2-difluoropropyl trifluoromethanesulfonate (822.49 mg, 1.70 mmol) and (R)-1-(1H-indol-3-yl)propan-2-amine (270 mg, 1.55 mmol) were dissolved in 1,4-dioxane (10 mL), followed by the addition of N-ethyl-N-isopropylpropan-2-amine (600.81 mg, 4.65 mmol). The reaction mixture was stirred at 80°C for 16 hours. LCMS monitored the reaction completion, and the reaction solution was concentrated and purified by column chromatography (silica, petroleum ether / ethyl acetate = 100 / 17) to obtain the product (R)-N-(1-(1H-indol-3-yl)propan-2-yl)-3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropan-1-amine (662.00 mg).

[0254] MSm / z(ESI):507.2[M+H] + ;

[0255] 1 H NMR(400MHz,CHLOROFORM-d)δ7.72-7.63(m,4H),7.61(d,J=7.9Hz,1H),7.49-7.33(m,7H),7.20(dt,J=1.1,7.6Hz,1H),7.14-7 .09(m,1H),7.01(d,J=2.3Hz,1H),3.89-3.76(m,2H),3.27-3.05(m,3H),2.94-2.77(m,2H),1.13(d,J=6.3Hz,3H),1.06(s,9H)

[0256] Step 3: Synthesis of (1S,3R)-1-(4-bromo-2,6-difluorophenyl)-2-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole

[0257]

[0258] (R)-N-(1-(1H-indol-3-yl)propan-2-yl)-3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropan-1-amine (300 mg, 592.07 μmol) and 4-bromo-2,6-difluorobenzaldehyde (143.93 mg, 651.27 μmol) were dissolved in toluene (5 mL), followed by the addition of acetic acid (355.55 mg, 5.92 mmol, 338.62 uL). The reaction mixture was stirred at 90°C for 16 hours. The reaction was monitored for completion by LCMS and TLC (petroleum ether:ethyl acetate = 10:1), and the reaction mixture was concentrated to dryness under reduced pressure. The reaction concentrate was diluted with ethyl acetate (5 mL), then washed three times with brine (5 mL). The collected aqueous phase was extracted three times with ethyl acetate (10 mL). The organic phase was dried over sodium sulfate and concentrated under reduced pressure. It was then purified by thin layer chromatography (silica, petroleum ether / ethyl acetate = 10 / 1) to give (1S,3R)-1-(4-bromo-2,6-difluorophenyl)-2-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (350.00 mg).

[0259] MSm / z(ESI):708.9[M+H] + ;

[0260] 1 H NMR(400MHz,CHLOROFORM-d)δ7.65-7.59(m,4H),7.52(br d,J=6.5Hz,1H),7.44-7.37(m,6H),7.25-7.20(m,1H),7.12(m,J=5.6,7 .2Hz,2H),6.95(d,J=8.3Hz,2H),5.28(s,1H),4.01-3.88(m,1H),3.69-3 .54(m,2H),3.35-3.20(m,1H),2.98(dd,J=4.6,14.7Hz,1H),2.83-2.69( m,1H),2.60(dd,J=3.8,15.6Hz,1H),1.15(d,J=6.5Hz,3H),1.04(s,9H).

[0261] Step 4: Synthesis of (S)-N-(4-((1S,3R)-2-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3,5-difluorophenyl)-1-(3-fluoropropyl)pyrrolidin-3-amine

[0262]

[0263] (1S,3R)-1-(4-bromo-2,6-difluorophenyl)-2-(3-(tert-butyldiphenylsilyl)oxy)

[0264] (S)-1-(3-Fluoropropyl)pyrrolidin-3-amine hydrochloride (129 mg, 507.27 μmol) and (S)-1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride (100 mg, 422.72 μmol) were dissolved in tetrahydrofuran (10 mL). Sodium tert-butoxide (243.75 mg, 2.54 mol) and 2,2-bis(diphenylphosphino)-1,1-binaphthyl (131.61 mg, 211.36 μmol) were then added to the reaction system under a nitrogen atmosphere. The reaction mixture was stirred at 80°C for 16 hours. After completion of the reaction, the reaction mixture was concentrated to dryness under reduced pressure, as monitored by LCMS. The reaction concentrate was diluted with ethyl acetate (10 mL), then washed three times with water (10 mL). The collected aqueous phase was extracted three times with ethyl acetate (20 mL). The organic phase was dried over sodium sulfate and concentrated to dryness under reduced pressure. The product was then purified by thin layer chromatography (silica, petroleum ether / tetrahydrofuran = 1 / 1, 1% ammonia water) to give (S)-N-(4-((1S,3R)-2-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3,5-difluorophenyl)-1-(3-fluoropropyl)pyrrolidin-3-amine (54.00 mg).

[0265] MSm / z(ESI):400.5[M+H] +

[0266] Step 5: Synthesis of 3-((1S,3R)-1-(2,6-difluoro-4-(((S)-1-(3-fluoropropyl)pyrrolidin-3-yl)amino)phenyl)-3-methyl-3,4-dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)-2,2-difluoropropan-1-ol

[0267]

[0268] (S)-N-(4-((1S,3R)-2-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3,5-difluorophenyl)-1-(3-fluoropropyl)pyrrolidin-3-amine (50.00 mg, 64.52 μmol) was dissolved in tetrahydrofuran (2 mL), and tetrabutylammonium fluoride solution in tetrahydrofuran (1 mol / L, 129.04 μL) was added. The reaction mixture was stirred at 25°C for 5 hours. The reaction was monitored for completion by TLC (silica, dichloromethane:methanol = 10:1). Afterwards, 5 mL of water was added to the reaction mixture, and the mixture was stirred at room temperature for 10 minutes. The organic phase was washed three times with brine (5 mL), and the collected aqueous phase was extracted three times with ethyl acetate (10 mL). The organic phase was dried over sodium sulfate and concentrated to dryness under reduced pressure. It was then purified by thin layer chromatography (silica, dichloromethane:methanol=10:1) to give compound 3-((1S,3R)-1-(2,6-difluoro-4-(((S)-1-(3-fluoropropyl)pyrrolidin-3-yl)amino)phenyl)-3-methyl-3,4-dihydro-1H-pyrido[3,4-b]indol-2(9H)-yl)-2,2-difluoropropan-1-ol (32.00 mg).

[0269] MS m / z (ESI): 559.1 [M+Na] +

[0270] 1 H NMR(400MHz, METHANOL-d4)δ7.38(d,J=7.3Hz,1H),7.18(d,J=7.5Hz,1H),7.02-6.91(m,2H),6.14(d, J=11.8Hz,2H),5.15(s,1H),4.53(t,J=5.8Hz,1H),4.42(t,J=5.6Hz,1H),3.99-3.91(m,1H),3.86-3. 72(m,1H),3.66-3.57(m,1H),3.52-3.39(m,1H),3.20-3.08(m,1H),3.00-2.91(m,2H),2.84-2.73(m, 2H),2.70-2.51(m,5H),2.37-2.26(m,1H),1.98-1.83(m,2H),1.76-1.65(m,1H),1.14(d,J=6.5Hz,3H)

[0271] Testing of biological activity and related properties

[0272] Test Example 1: Detection of the effect of the compounds of the present invention on the degradation of estrogen receptors in MCF7 cells

[0273] 1. Purpose of the experiment

[0274] The purpose of this experiment is to determine the degradation activity of the compounds of the present invention on the endogenously expressed estrogen receptor in MCF7 cells. 50 and maximum degradation efficiency to evaluate the activity of the compounds.

[0275] 2. Experimental Methods

[0276] MCF7 cells (ATCC, HTB-22) were cultured in complete DMEM (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 incubator. Test compounds were dissolved in DMSO to a stock concentration of 10 mM, diluted using an Echo 550 (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 continued at 37°C in a 5% CO2 incubator for 24 hours. Paraformaldehyde was added to the cell culture medium in each well to a final concentration of approximately 3.7% to fix the cells. After 30 minutes, the supernatant was discarded and each well was washed once with 50 μL of PBS. The cells were treated with PBS containing 0.5% v / v Tween-20 for 30 minutes and washed once with PBS. Blocking solution (5% BSA, 0.05% Tween-20 in PBS) 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, GST, #8644S, 1:1000 dilution; anti-GAPDH monoclonal antibody, GAPDH (D4C6R) Mouse mAb, GST, #97166S, 1:2000 dilution) was added and incubated at room temperature for 3 hours. The cells were washed with PBST (PBS containing 0.05% The cells were washed three times with 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. The cells were washed three times with PBST and the fluorescence signal of each well was read using an Odyssey CLx. The Chanel 800 (ER) / Chanel 680 (GAPDH) values ​​were calculated. The degradation rate at each concentration point was calculated, using the 0.1 μM fulvestrant-treated well as the 100% degradation control. The data were analyzed using XlLfit to calculate the degradation activity DC of each compound. 50 And the maximum degradation rate Imax. Data analysis is shown in Table 1

[0277] Table 1 Degradation activity of the compounds of the present invention on estrogen receptor in MCF7 cells

[0278] Compound number <![CDATA[ER level DC 50 (nM)]]> Maximum degradation rate Example 1 0.41 106％ Example 2 8.5 92％ Example 3 0.15 104％ Example 4 0.04 99％ Example 5 0.58 80％ Example 6 0.50 90％ Example 7 0.06 99％

[0279] Test Example 2: Detection of the inhibitory effect of the compound of the present invention on MCF7 cell proliferation

[0280] 1. Purpose of the experiment

[0281] The purpose of this experiment is to determine the inhibitory effect of the compounds of the present invention on the proliferation of MCF7 cells in vitro. 50 The activity of the compound was evaluated by the maximum inhibitory efficiency.

[0282] 2. Experimental Methods

[0283] 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. Culture was carried out at 37°C in a 5% CO2 cell culture incubator for 7 days. On the eighth day, the cell culture plate was removed. Add 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 .

[0284] 3. Data analysis see Table 2

[0285] Table 2 Inhibitory activity of the compounds of the present invention on MCF7 cell proliferation

[0286]

[0287]

[0288] Test Example 3: Inhibitory effect of the compounds of the present invention on CYP2C9 and CYP2D6 enzyme activities

[0289] The inhibition of the compounds of the present invention on the activities of CYP2C9 and CYP2D6 enzymes was determined using the following test method.

[0290] 1. Test materials and instruments

[0291] 1. Human liver microsomes (Corning 452117)

[0292] 2. NADPH (Solarbio 705Y021)

[0293] 3. Positive substrates: diclofenac (Sigma SLBV3438), dextromethorphan (TRC 3-EDO-175-1), and midazolam (Cerilliant FE01161704)

[0294] 4. Positive inhibitors sulfaphenazole (D. Ehrenstorfer GmbH 109012), quinidine (TCI WEODL-RE) and ketoconazole (Sigma 100M1091V)

[0295] 5. AB Sciex Triple Quad 5500 LC / MS

[0296] 2. Test steps

[0297] 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.

[0298] 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.

[0299] 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 sample with 10 mM NADPH solution in a 37°C water bath for 15 minutes. After 15 minutes, add 20 μl of NADPH to each well to initiate the reaction, and incubate at 37°C for 5 minutes (CYP2C9) or 20 minutes (CYP2D6). 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.

[0300] The IC values ​​of the compounds of the present invention for CYP2C9 and CYP2D6 were calculated by Excel XLfit 5.3.1.3. 50 value.

[0301] Table 3 IC values ​​of the compounds of the present invention against CYP2C9 and CYP2D6 50 value

[0302] Example No. CYP2C9 (μM) CYP2D6 (μM) Example 3 >30 >30 Example 4 20 27 Example 7 8 23

[0303] Test Example 4: Determination of Plasma Protein Binding Rate of the Compounds of the Invention

[0304] Human plasma protein binding is a key factor controlling the amount of free (unbound) drug available for binding to the target and plays an important role in the observed in vivo efficacy of drugs. Therefore, for compounds with similar potency and exposure levels, compounds with a high free fraction (low levels of plasma protein binding) can exhibit enhanced efficacy.

[0305] The protein binding rate of the compounds of the present invention in the plasma of five species (human, monkey, dog, rat and mouse) was determined using the following test method.

[0306] 1. Test materials and instruments

[0307] 1. Human plasma (BioIVT), beagle dog plasma (BioIVT), SD rat plasma (BioIVT), CD-1 mouse plasma (BioIVT);

[0308] 2. 96-well equilibrium dialysis plate (HTDialysis LLC, Gales Ferry, CT, HTD96B), equilibrium dialysis membrane (MWCO 12-14K, #1101);

[0309] 3. Positive control compound warfarin;

[0310] 4.ABIQTrap 5500 liquid spectrometer.

[0311] 2. Test steps

[0312] 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.

[0313] 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.

[0314] 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.

[0315] 3. Preparation of compound-containing plasma samples: Dilute a 10 mM stock solution of the compound of the 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 human, monkey, dog, rat, or mouse plasma to obtain a final concentration of 1 μM in the plasma sample.

[0316] 4. 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 buffered saline) 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 buffered saline sample, and add an equal volume of blank phosphate buffered saline to the plasma sample. 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 sample injection plate and add 100 μL of ultrapure water to mix thoroughly. Prepare for LC-MS / MS analysis.

[0317] 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:

[0318] Free rate % = (ratio of compound peak area to internal standard peak area 缓冲液侧 / Ratio of compound peak area to internal standard peak area 血浆侧 )×100

[0319] Binding rate % = 100 - free rate %

[0320] All data were calculated using Microsoft Excel software. The calculated plasma protein binding values ​​of the compounds of the present invention were obtained.

[0321] Table 4 Protein binding rate of the compounds of the present invention in human, dog, rat and mouse plasma

[0322]

[0323]

[0324] Test Example 5: Apparent solubility of the compound of the present invention in phosphate buffer at pH 7.4

[0325] In order for an orally administered compound to reach the site of action and for efficient absorption from the intestine, the compound needs to be in solution, and therefore compounds with high intrinsic solubility may be more suitable for pharmaceutical use.

[0326] 1. Materials and Reagents

[0327] The test compounds were prepared according to the described methods. The control drug, progesterone, was purchased from Sigma. The pH 7.4 phosphate buffer was prepared in our laboratory. Acetonitrile and methanol were purchased from Fisher. Other reagents were purchased commercially.

[0328] 1.5 mL flat-bottom glass vials (BioTech Solutions); Teflon / silicone stoppers (BioTech Solutions); Teflon-coated stirring bars; MultiScreen HTS HV (0.45 μm) 96-well plate filter plates (Millipore, MSHVN4510 or MSHVN4550); Eppendorf Thermomixer Comfort; Vacuum Manifold ORVMN96 (Orochem).

[0329] 2. Experimental steps

[0330] 1) Preparation of stock solution

[0331] Prepare 10 mM stock solutions of the test substance and control drug progesterone in DMSO.

[0332] 2) Apparent solubility determination steps

[0333] Dispense 30 μL of 10 mM analyte stock solution into the corresponding positions of a 96-well plate in the designated order. Add 970 μL of pH 7.4 phosphate buffer to the corresponding vials in the sample plate. Perform the experiment in duplicate. Add a stir bar to each vial and cap with a Teflon / silicone stopper. The sample plate is then placed in an Eppendorf Thermomixer Comfort and shaken at 1100 rpm at 25°C for 2 hours. After 2 hours, remove the stoppers, remove the stir bars with a large magnet, and transfer the samples from the sample plate to a filter plate. Filter the samples using a vacuum pump to generate negative pressure. Transfer 5 μL of the filtrate to a new sample plate, followed by the addition of 5 μL of DMSO and 490 μL of 50% ACN(IS).H2O (1:1 internal standard acetonitrile:water). Depending on peak shape, the sample diluent may be diluted with 50% ACN(IS).H2O to improve peak shape. The dilution factor may be adjusted depending on the solubility of the analyte or the strength of its liquid mass response signal.

[0334] 3) Sample analysis steps

[0335] The sample plate was placed into the sample tray of the autosampler and the samples were evaluated by liquid chromatography-mass spectrometry (LC-MS).

[0336] 3. Experimental steps

[0337] All calculations were performed using Microsoft Excel. Analysis and quantification of the sample filtrates were performed by qualitative and quantitative analysis of standard peaks of known concentrations using a liquid chromatography mass spectrometer. The apparent solubility values ​​of the compounds of the invention in phosphate buffer at pH 7.4 were calculated.

[0338] Table 5 Apparent solubility values ​​of the compounds of the present invention in phosphate buffer at pH 7.4

[0339] Example No. pH=7.4 Apparent solubility (μM) Example 3 92 Example 4 <0.3 Example 7 4

[0340] Test Example 6: Whether the compound of the present invention has a potential inhibitory effect on the voltage-gated potassium ion channel hERG

[0341] hERG potassium channels are essential for normal electrical activity in the heart. Cardiac arrhythmias can be induced by a variety of drugs that block hERG channels. This side effect is a common cause of drug failure in preclinical safety trials, so minimizing hERG channel blocking activity may be a desirable characteristic of a drug candidate.

[0342] 1. Materials and Reagents

[0343] 1. Experimental Materials and Instruments

[0344]

[0345]

[0346] 2. Cell Lines and Culture

[0347] HEK293 cell line stably expressing hERG ion channel (Cat. No. K1236) was purchased from Invitrogen. The cell line was cultured in a medium containing 85% DMEM, 10% dialyzed fetal bovine serum, 0.1mM non-essential amino acid solution, 100U / mL penicillin-streptomycin solution, 25mM HEPES, 5μg / mL blasticidin, and 400μg / mL geneticin. When the cell density grew to 40% to 80% of the bottom area of ​​the culture dish, trypsin was used for digestion and passage, and passage was performed three times a week. Before the experiment, cells were plated at 5×10 5 The cells were cultured at a density of 100 μg / mL in 6 cm culture dishes and induced with 1 μg / mL doxycycline for 48 h. The cells were then digested and seeded on glass slides for subsequent manual patch clamp experiments.

[0348] 3. Preparation of test compounds

[0349] 1) According to the standard operating procedure SOP-ADMET-MAN-007, the test compound was dissolved in DMSO and prepared into a stock solution with a final concentration of 10 mM.

[0350] 2) The stock solution was diluted with DMSO in a 1:3 gradient to form three other intermediate concentration solutions, with concentrations of 3.33 mM, 1.11 mM, and 0.37 mM, respectively.

[0351] 3) Prior to the experiment, the test compound stock solution and intermediate solution were diluted 1000-fold with extracellular fluid to produce a series of working solutions with concentrations of 10 μM, 3.33 μM, 1.11 μM, and 0.37 μM. Simultaneously, the 10 mM stock solution was diluted 333.33-fold with extracellular fluid to produce a 30 μM working solution. The DMSO content in the working solutions was 0.1-0.3% (v / v).

[0352] (Note: The DMSO content in the working solution should be controlled within 1% (volume ratio) to avoid cytotoxicity.)

[0353] 4) After preparing the working solution, visually inspect it for precipitation or turbidity. If so, this may be due to poor solubility of the compound in physiological solutions. Further sonication in a water bath for 30 minutes may be recommended to improve solution clarity.

[0354] 5) Determine the potential inhibitory effect of the test substance on hERG channels at five concentrations of 30 μM, 10 μM, 3.33 μM, 1.11 μM and 0.37 μM, fit the dose-effect curve and calculate the IC50.

[0355] 2. Experimental Methods

[0356] 1. Place the small glass slide containing HEK293 cells in the culture dish into the perfusion tank on the microscope stage.

[0357] 2. Using an Olympus IX51, IX71, or IX73 inverted microscope, center the field of view on an appropriate cell. Using a ×10 objective lens, locate the tip of the glass electrode and position it in the center of the field of view. Then, use the micromanipulator to lower the electrode while adjusting the coarse focus knob to gradually bring the electrode closer to the cell.

[0358] 3. When approaching the cell, switch to a ×40 objective lens for observation and use the micromanipulator to fine-tune the gear to gradually bring the electrode closer to the cell surface.

[0359] 4. Apply negative pressure to form a seal with a resistance greater than 1 GΩ between the electrode tip and the cell membrane.

[0360] 5. Compensate for the transient capacitive current Cfast in voltage clamp mode. Repeatedly apply short negative pressures to rupture the membrane, ultimately establishing whole-cell recording mode.

[0361] 6. Under the condition that the membrane potential is clamped at -60 mV, the slow capacitive current Cslow, cell membrane capacitance (Cm) and input membrane resistance (Ra) are compensated separately.

[0362] 7. After the cells stabilize, change the clamp voltage to -90 mV, set the sampling frequency to 20 kHz, and the filter frequency to 10 kHz. Leakage current detection conditions are: clamp voltage to -80 mV, time duration 500 ms.

[0363] 8. The hERG current measurement method is as follows: a 4.8-second depolarization command voltage is applied to depolarize the membrane potential from -80 mV to +30 mV. Then, a 5.2-second repolarization voltage is applied to reduce the membrane potential to -50 mV to eliminate channel inactivation, allowing the hERG tail current to be observed. The peak value of the tail current is the magnitude of the hERG current.

[0364] 9. hERG currents used to detect test compounds were recorded continuously for 120 seconds before administration to assess the stability of hERG currents generated by the test cells. Only cells that were stable within the acceptable range of evaluation criteria were allowed to proceed to subsequent compound testing.

[0365] 10. Determine the inhibitory effect of the test compound on the hERG current: First, the hERG current measured in the extracellular solution containing 0.1% DMSO is used as the detection baseline. After the hERG current remains stable for at least 5 minutes, the solution containing the test compound is perfused around the cells in sequence from low concentration to high concentration. After each perfusion, wait for about 5 minutes to allow the compound to fully act on the cells and record the hERG current simultaneously. After the recorded current tends to stabilize, record the last 5 hERG current values, and take the average value as the final current value at a specific concentration. After the compound is tested, add 150nM dofetilide to the same cell to completely inhibit its current as a positive control for the cell. At the same time, the positive compound dofetilide is synchronously detected using the same patch clamp system before and after the end of the test compound experiment to ensure the reliability and sensitivity of the entire detection system.

[0366] 3. Data Analysis

[0367] The data were exported by PatchMaster software and analyzed according to the following steps:

[0368] 1) After perfusing the blank solvent or compound gradient solution, the average value of the 5 consecutive current values ​​obtained was calculated and used as the "tail current magnitude" 空白 ” and “tail current size 化合物 ”

[0369] 2) The current suppression percentage is calculated using the following formula.

[0370]

[0371] 3) The dose-effect curve was fitted using Graphpad Prism 8.0 software and the IC was calculated. 50 The inhibition of hERG by the compounds of the present invention is shown in Table 6 below.

[0372] Table 6 Inhibitory activity of the compounds of the present invention on voltage-gated potassium channel hERG

[0373] Example No. <![CDATA[hERG IC 50 (μM)]]> Example 3 10 Example 4 3.7 Example 7 8.4

[0374] Test Example 7: Pharmacokinetic Evaluation of the Compounds of the Invention in Mice

[0375] Experimental Materials

[0376] CD-1 mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. DMSO (dimethyl sulfoxide), HP-β-CD (hydroxypropyl-β-cyclodextrin), tetraethylene glycol, and Captisol (SBE-β-CD, sulfobutyl-β-cyclodextrin) were purchased from Sigma. Acetonitrile was purchased from Merck (USA).

[0377] Experimental methods

[0378] Six female CD-1 mice (20-30 g, 4-6 weeks) were randomly divided into two groups of three. Group 1 received the test compound via tail vein injection at a dose of 1 mg / kg in 5% DMSO in 10% HP-β-CD in water. Group 2 received the test compound orally at a dose of 10 mg / kg in 40% Tetraethylene Glycol (v / v) and 7.5% Captisol (w / v) in water. The animals were fed and watered normally before the experiment. Venous blood samples were collected from each group of mice before dosing and at 0.083 (iv group only), 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after dosing. Whole blood samples were placed in K2EDTA anticoagulant tubes and centrifuged for 5 minutes (12,000 rpm, 4°C) to obtain plasma for analysis.

[0379] Take 10 μL of mouse plasma sample and add 150 μL of acetonitrile (containing an internal standard compound) to precipitate proteins. After vortexing for 5 minutes, centrifuge at 14,000 rpm for 5 minutes. The supernatant is diluted 2-fold with water containing 0.1% (v / v) FA and quantified on an LC-MS / MS system (AB Sciex Triple Quad 6500+). A CD-1 mouse plasma standard curve and quality control samples are also included when determining sample concentrations. For the 10x diluted sample, take 2 μL and add 18 μL of blank plasma. After vortexing for 0.5 minutes, add 300 μL of acetonitrile (containing an internal standard compound) to precipitate proteins. All other processing steps are the same as for the undiluted sample.

[0380] The PK test results are shown below. The compound of the present invention exhibits good PK properties and oral bioavailability in mice:

[0381] Table 7 PK of the compounds of the present invention in mice

[0382]

[0383] Test Example 8: Blood-brain barrier (BBB) ​​permeability of the compounds of the present invention in rats

[0384] The key to the drug's effectiveness against brain metastases is that the drug can pass through the animal's blood-brain barrier and be exposed to a sufficient amount in the brain. Therefore, by measuring the drug concentration in the animal's plasma and brain tissue after administration, the distribution of the drug in the brain can be evaluated, and then it can be determined whether the drug can inhibit tumor growth in the in situ brain model.

[0385] Experimental Materials

[0386] Female SD rats were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. MC (methylcellulose) was purchased from Sigma, acetonitrile was purchased from Merck (USA), and PBS (phosphate buffered saline) was purchased from Sangon Biotechnology.

[0387] Experimental methods

[0388] Six female SD rats (200-300 g, 6-8 weeks) were randomly divided into two groups, with three rats in each group. The compound of the present invention and the solvent 0.5% methylcellulose aqueous solution were administered respectively. The animals were fed with water normally before the experiment and fasted overnight. Food was resumed four hours after administration. Plasma and brain tissue were collected from each group of rats 2 hours after administration. The collected whole blood samples were placed in K2EDTA anticoagulant tubes and centrifuged for 5 minutes (12,000 rpm, 4°C) to obtain plasma for testing; after tissue collection, they were dried with filter paper and stored in a -80 degree refrigerator for testing.

[0389] 10 μL of rat plasma sample was added to 150 μL of acetonitrile (containing an internal standard compound) to precipitate proteins. After vortexing for 5 minutes, the sample was centrifuged (14,000 rpm) for 5 minutes. The supernatant was diluted 2-fold with water containing 0.1% (v / v) FA and quantified on an LC-MS / MS system (AB Sciex Triple Quad 6500+). For the 10x diluted sample, 2 μL of the sample was added to 18 μL of blank plasma. After vortexing for 0.5 minutes, 300 μL of acetonitrile (containing an internal standard compound) was added to precipitate proteins. All other processing steps were the same as for the undiluted sample. A standard curve using SD rat plasma and a plasma quality control sample were also run during plasma sample concentration determination.

[0390] Rat brain tissue samples were first homogenized with 4 times the mass volume of PBS. A 20 μL sample of brain tissue homogenate was diluted and mixed with 20 μL of blank mouse plasma. Protein was then precipitated with 600 μL of acetonitrile (containing an internal standard compound). After vortexing for 5 minutes, the mixture was centrifuged (14,000 rpm) for 5 minutes. The supernatant was diluted 2-fold with water containing 0.1% (v / v) FA and quantitatively analyzed using an LC-MS / MS system (AB Sciex Triple Quad 6500+). Example 3, compound of the present invention, demonstrated excellent blood-brain barrier permeability, with high drug exposure in rat brain tissue.

[0391] The BBB test results are as follows:

[0392] Table 8 Rat blood-brain barrier penetration test of the compounds of the present invention

[0393]

[0394] Test Example 9: Growth inhibition experiment of the compound of the present invention on MCF-7 mouse subcutaneous tumor model

[0395] Experimental reagents

[0396] Human breast cancer MCF-7 cells: ATCC, HTB-22

[0397] 17β-estradiol tablets: Innovative Research of America, Cat No.: SE-121, 60-day release, 0.72 mg / pellet EMEM culture medium: ATCC, Cat No.: 30-2003

[0398] Fetal bovine serum: Gbico; Cat No.: 1099-141C

[0399] Penicillin (Pen Strep): Gibco, Cat No.: 15240-122

[0400] Recombinant human insulin: Shanghai Yisheng, Cat.No.: 40112ES60

[0401] 0.25% Trypsin-EDTA: Gibco, Cat No.: 25200-072

[0402] D-PBS (Calcium- and magnesium-free phosphate buffered saline): Hyclone, Cat. No.: SH30256.01

[0403] Matrigel:Corning,Cat.No.:356237

[0404] Experimental methods

[0405] Animal information: NPG mice, female, 6-7 weeks old, weighing approximately 19-28 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.

[0406] Cell Culture: Human breast cancer MCF-7 cells were cultured in vitro in EMEM (cell culture medium) supplemented with 10% fetal bovine serum, 1% Pen-Strep, and 10 μg / ml recombinant human insulin in an incubator at 37°C and 5% CO2. Cells were routinely digested and passaged weekly using 0.25% trypsin-EDTA. Cells were harvested and counted when cell saturation reached 80%-90% and the desired number of cells was reached.

[0407] Cell inoculation: 0.1 ml / (containing 1×10 7MCF-7 cell suspension (D-PBS:Matrigel, 1:1 volume ratio) was subcutaneously inoculated on the right back of each mouse. Four days prior to cell inoculation, 17β-estradiol tablets were administered subcutaneously. On day 24 after cell inoculation, mice were randomly assigned to groups based on tumor volume for dosing, with group assignment designated as Day 0.

[0408] Administration: Compound Example 1 was administered at a dose of 1, 3, or 10 mg / kg, orally (PO), once daily (QD) for 3 weeks. Compound Example 2 was administered at a dose of 10 mg / kg, orally (PO), once daily (QD) for 3 weeks. The vehicle group consisted of 8 mice and the drug group consisted of 6 mice. Tumor measurements and experimental indicators:

[0409] Tumor diameter was measured twice a week using a vernier caliper. Tumor volume was calculated using the formula: V = 0.5a x b 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.

[0410] The anti-tumor efficacy of the compound was evaluated using the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume of a treatment group at the end of dosing - mean tumor volume of the treatment group at the start of dosing) / (mean tumor volume of the solvent control group at the end of treatment - mean tumor volume of the solvent control group at the start of treatment)] x 100%.

[0411] Experimental results:

[0412] See Table 9, Figure 2 and Figure 3 In the mouse subcutaneous transplant tumor MCF-7 model, the compound Example 3 of the present invention has a significant inhibitory effect on tumor growth (P<0.01) when administered orally once a day at 1 mg / kg, 3 mg / kg, or 10 mg / kg, and has a good dose-response relationship, with the effect of shrinking tumors at doses of 3 mg / kg and 10 mg / kg. The compound Example 3 of the present invention has a significant inhibitory effect on tumor growth (P<0.01) when administered orally once a day at 10 mg / kg, and has the effect of shrinking tumors. Compound Example 3 and Example 7 did not significantly affect the weight of mice at the doses tried.

[0413] Table 9 Tumor volume of MCF-7 subcutaneous tumor model

[0414]

[0415] Test Example 10: Inhibitory effect of the compound of the present invention on the growth of MCF-7 brain orthotopic tumor model in mice Experimental reagents / instruments:

[0416] Human breast cancer MCF-7 cells: ATCC, HTB-22

[0417] 17β-estradiol tablets: Innovative Research of America, Cat No.: SE-121, 60-dayrelease, 0.72mg / pellet

[0418] EMEM culture medium: ATCC, Cat No.: 30-2003

[0419] Fetal bovine serum: Gibco, Cat. No.: 1099-141C

[0420] Penicillin (Pen Strep): Gibco, Cat No.: 15240-122

[0421] Recombinant human insulin: Shanghai Yisheng, Cat.No.: 40112ES60

[0422] 0.25% Trypsin-EDTA: Gibco, Cat No.: 25200-072

[0423] Stereotaxic instrument: Rayward, Cat No.: Standard / Digital Display / Single Arm / Mouse / 68055

[0424] Microsyringe pump: KDS, Cat No.: Legato130

[0425] Micro handheld cranial drill: Rayward, Cat No.: 78001

[0426] Experimental methods:

[0427] Animal information: NPG mice, female, 6-8 weeks old, weighing approximately 17-29 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.

[0428] Cell Culture: Human breast cancer MCF-7 cells were cultured in vitro in EMEM (cell culture medium) supplemented with 10% fetal bovine serum, 1% Pen-Strep, and 10 μg / ml recombinant human insulin in an incubator at 37°C and 5% CO2. Cells were routinely digested and passaged twice weekly using 0.25% trypsin-EDTA. Cells were harvested and counted when cell saturation reached 80%-90% and the required number of cells was reached.

[0429] Cell inoculation: 15 μl / (containing 2×10 6MCF-7 cell suspensions were inoculated intracranially into mice using a brain positioning device, a microinjection pump, and a micro-handheld cranial drill. Three days prior to cell inoculation, 17β-estradiol tablets were administered subcutaneously. Eight days after cell inoculation, mice were randomly divided into groups based on body weight and administered the drug. Grouping was designated Day 0.

[0430] Dosing: Fulvestrant (AstraZeneca) was administered at a dose of 250 mg / kg subcutaneously (SC) once weekly (QW), and Example 3 was administered at a dose of 30 mg / kg orally (PO) once daily (QD). The vehicle group consisted of 11 mice, and the drug group consisted of 8 mice. All groups continued to receive drug until death, euthanasia due to poor condition, or termination of the experiment.

[0431] Experimental observation and conclusion:

[0432] The body weight of mice was measured twice a week, and the survival status of mice was observed.

[0433] The experiment ended on Day 48, and all mice were euthanized.

[0434] Experimental results:

[0435] See Figure 4 and Figure 5 In the mouse brain orthotopic MCF-7 model, the weight of mice in the Fulvestrant group (administered 250 mg / kg subcutaneously once a week) continued to decrease, and the survival status of the mice was not significantly different from that of the solvent control group (median survival time, 29 days in the vehicle control group, 29.5 days in the Fulvestrant group). The weight of mice in the Example 3 compound group of the present invention (administered orally at 30 mg / kg once a day) was stable and there were no abnormalities. Until the end of the experiment, no mice in the Example 3 compound group of the present invention died. Compared with the solvent control or Fulvestrant, the Example 3 compound had a significant inhibitory effect on the MCF-7 brain orthotopic tumor model mice, and the survival time of the mice was significantly prolonged (P<0.01).

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof: in, R 1 、R 2 、R 3 、R 4 Independently selected from H, F, Cl, Br, I, CN, C1-C6 alkyl, C1-C6 alkoxy or C3-C6 cycloalkyl; X1, X2, X3, and X4 are independently selected from CR 6 or N; R 6 Selected from H, F, Cl, Br, I, OH, CN, C1-C 10 Alkyl, C3-C 10 Cycloalkyl, 3-10 membered heterocyclic group, C1-C 10 Alkoxy, C3-C 10 Cycloalkyloxy or 3-10 membered heterocyclyloxy; Y is selected from O or NH; R 5 independently selected from C1-C6 alkyl, said C1-C6 alkyl being optionally replaced by R a replace; R a is selected from F, Cl, Br, I, OH, CN, C1-C6 alkyl, C1-C6 alkoxy or C3-C6 cycloalkyl; Provided that the compound represented by formula (I) does not contain 2. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: R 1 、R 2 、R 3 、R 4 Independently selected from H, F, Cl, Br, I, CN or C1-C3 alkyl.

3. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X1, X2, X3, and X4 are independently selected from CR 6 or N, and at least two of the X1, X2, X3, and X4 groups are selected from CR 6 .

4. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: R 6 is selected from H, F, Cl, Br, I, CN, C1-C3 alkyl or C1-C3 alkoxy.

5. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: R 5 is selected from C1-C3 alkyl, wherein the C1-C3 alkyl is optionally replaced by R a replace.

6. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Structural unit Selected from 7. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the compound represented by formula (II) or a pharmaceutically acceptable salt thereof:

8. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or pharmaceutically acceptable salts thereof:

9. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or pharmaceutically acceptable salts thereof:

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 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 estrogen receptor-related diseases; Preferably, the estrogen receptor-related disease is a tumor. More preferably, the tumor is breast cancer, More preferably, the tumor is ER-positive breast cancer, More preferably, the tumor is ER-positive breast cancer brain metastasis.

12. A method for preventing or treating an estrogen receptor-related disease, comprising administering to a subject in need thereof 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; Preferably, the estrogen receptor-related disease is a tumor. More preferably, the tumor is breast cancer, More preferably, the tumor is ER-positive breast cancer, More preferably, the tumor is ER-positive breast cancer brain metastasis.

13. A pharmaceutical composition for preventing or treating estrogen receptor-related diseases, comprising the compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient; Preferably, the estrogen receptor-related disease is a tumor. More preferably, the tumor is breast cancer, More preferably, the tumor is ER-positive breast cancer, More preferably, the tumor is ER-positive breast cancer brain metastasis.

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