Pyrrolidine compounds and uses thereof

Abstract

Disclosed are a pyrrolidine compound represented by formula (I) or a pharmaceutically acceptable salt thereof, a pharmaceutical composition comprising the same, and the use of the pharmaceutical composition as a selective estrogen receptor degrader (SERD) in the prevention or treatment of estrogen receptor-related diseases. [Formula 1] JPEG2023526568000088.jpg8392

Description

[Technical Field]

[0001] This application claims priority to a prior application entitled "Pyrrolidine Compounds and Uses Thereof" and bearing patent application number CN202010414013.9, filed with the State Intellectual Property Administration on May 15, 2020. The above-mentioned prior application is incorporated herein by reference in its entirety.

[0002] The present invention relates to novel pyrrolidine compounds or pharmaceutically acceptable salts thereof, pharmaceutical compositions containing same, and their use as selective estrogen receptor degraders (SELFs) in the prevention or treatment of associated diseases. [Background technology]

[0003] Estrogen (E2) and the estrogen alpha receptor (ERα) are key drivers of breast cancer development and progression. More than two-thirds of patients with breast cancer express the ER transcription factor, and in the majority of ER-positive patients, the ER remains a major driver of tumor progression even after early endocrine therapy. Therefore, ER is an important target for breast cancer therapy (Pharmacology & Therapeutics 186(2018)1-24). The goal of endocrine therapy is to reduce ER activity. There are three main types of therapy: selective estrogen receptor modulators (SERMs) such as tamoxifen, which are allosteric modulators of ER and inhibit its transcriptional activity after binding to the ER; aromatase inhibitors (AIs), which reduce estrogen levels in the body by inhibiting the conversion of androgens to estrogen; and selective estrogen receptor downregulators such as fulvestrant, which act as ER antagonists, not only inhibiting ER activity but also inducing ER proteolysis. Endocrine therapy is the first-line treatment for patients with estrogen receptor-positive breast cancer, but approximately 30% of patients experience recurrence after treatment, and almost all patients with metastatic breast cancer develop drug resistance, resulting in disease progression. There are two main mechanisms of resistance to endocrine therapy: one is centered on the estrogen receptor signaling pathway itself, including activating mutations, amplification, and fusion with other genes of the estrogen receptor-encoding gene ESR1, and dysregulation of estrogen receptor coregulators and downstream cell cycle regulators; the other involves 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] Clinical findings indicate that approximately 70-80% of breast cancer patients are estrogen receptor (ER) positive. The proliferation of such breast cancer cells is highly dependent on ER, and this subtype accounts for 50% of breast cancer deaths. Early-stage ER-positive breast cancer has a better prognosis, with a 5-year survival rate exceeding 90%. Approximately 30% of patients who receive adjuvant endocrine therapy (TAM or AI drugs) experience recurrence within 10 years, but they are still able to undergo standard endocrine therapy. Nevertheless, acquired drug resistance, primarily caused by ESR1-LBD mutations, and distant metastases (including bone, brain, liver, lung, and lymph node metastases, with approximately 10%-15% of patients having brain metastases) can emerge, leading to increased resistance to treatment. In patients with advanced metastatic breast cancer, brain metastases develop slower than lung, liver, and bone metastases, and their prognosis is poorer, with a median survival time of only 2-9 months after clinical drug treatment.

[0005] Fulvestrant is the first and only SERD drug clinically approved for the treatment of postmenopausal patients with ER-positive metastatic breast cancer whose disease progresses after treatment with tamoxifen or an aromatase inhibitor. Currently, AstraZeneca (see Patent Application WO 2018077630A1) and Genentech (see Patent Application WO 2019245974A1) have also disclosed a series of novel SERD compounds and their corresponding medical uses. Data from multiple studies have shown that ER degradation was not completely achieved in patients treated with fulvestrant. Furthermore, its clinical applications are significantly limited by its characteristics, such as obvious reactions at the injection site caused by intramuscular injection, such as pain, swelling, and redness, slow absorption, and limited exposure in the body (fulvestrant cannot penetrate the blood-brain barrier, and the maximum dose of a single intramuscular injection is only 500 mg; its pharmacodynamic properties and intramuscular administration route limit the maximum dose that can be administered to patients). Therefore, new treatment options are urgently needed for patients with ER-positive breast cancer. Summary of the Invention [Means for solving the problem]

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

[0007] [ka]

[0008] During the ceremony, R 1 , R 2 , R 3 , and R 4 are 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 CR 6 or N, R 6 are H, F, Cl, Br, I, OH, CN, C1-C 10 Alkyl, C3-C 10 Cycloalkyl, 3-10 membered heterocyclyl, C1-C 10 Alkoxy, C3-C 10 cycloalkyloxy, or 3- to 10-membered heterocyclyloxy; Y is selected from O or NH; R 5 are independently selected from C1-C6 alkyl, and C1-C6 alkyl is R a and optionally replaced by R a is selected from F, Cl, Br, I, OH, CN, C1-C6 alkyl, C1-C6 alkoxy, or C3-C6 cycloalkyl; However, the compound represented by formula (I) is

[0009] [ka]

[0010] provided that it does not contain

[0011] In some embodiments, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the compounds represented by the following formula (II) or a pharmaceutically acceptable salt thereof:

[0012] [ka]

[0013] 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.

[0014] [ka]

[0015] In some embodiments, R 1 , R 2 , R 3 , and R 4 are independently selected from H, F, Cl, Br, I, CN, or C1-C6 alkyl.

[0016] In some embodiments, R 1 , R 2 , R 3 , and R 4 are independently selected from H, F, Cl, Br, I, CN, or C1-C3 alkyl.

[0017] In some embodiments, R 1 , R 2 , R 3 , and R 4 are independently selected from H, F, Cl, Br, I, CN, or methyl.

[0018] In some embodiments, R 1 , R 2 , R 3 , and R 4are independently selected from H, F, Cl, Br, I, or C1-C3 alkyl.

[0019] In some embodiments, R 1 , R 2 , R 3 , and R 4 are independently selected from H, F, Cl, Br, I, or methyl.

[0020] In some embodiments, R 1 , R 2 , R 3 , and R 4 are independently selected from H, F, or methyl.

[0021] In some embodiments, R 1 and R 2 are independently selected from H, F, or methyl.

[0022] In some embodiments, R 3 and R 4 is independently selected from H or methyl.

[0023] In some embodiments, R 3 and R 4 is independently selected from H.

[0024] In some embodiments, the structural unit

[0025] [ka]

[0026] teeth,

[0027] [ka]

[0028] is selected from.

[0029] In some embodiments, the structural unit

[0030] [ka]

[0031] teeth,

[0032] [ka]

[0033] is selected from.

[0034] In some embodiments, the structural unit

[0035] [ka]

[0036] teeth,

[0037] [ka]

[0038] is selected from.

[0039] In some embodiments, the structural unit

[0040] [ka]

[0041] teeth,

[0042] [ka]

[0043] is selected from.

[0044] In some embodiments, the structural unit

[0045] [ka]

[0046] teeth,

[0047] [ka]

[0048] is selected from.

[0049] In some embodiments, the structural unit

[0050] [ka]

[0051] teeth,

[0052] [ka]

[0053] is selected from.

[0054] In some embodiments, the structural unit

[0055] [ka]

[0056] teeth,

[0057] [ka]

[0058] is selected from.

[0059] In some embodiments, X1, X2, X3, and X4 are CR 6 or N, and at least two of the X1, X2, X3, and X4 groups are CR 6 is selected from.

[0060] In some embodiments, X1, X2, X3, and X4 are CR 6 or N, and at least three of the X1, X2, X3, and X4 groups are CR 6 is selected from.

[0061] In some embodiments, R 6 is selected from H, F, Cl, Br, I, CN, C1-C3 alkyl, or C1-C3 alkoxy.

[0062] In some embodiments, R 6 is selected from H, F, Cl, Br, I, CN, or C1-C3 alkoxy.

[0063] In some embodiments, R 6 is selected from H, F, Cl, Br, I, CN, or methoxyl.

[0064] In some embodiments, R 6 is selected from H, F, Cl, Br, I, or CN.

[0065] In some embodiments, R 6 is selected from H, F, Cl, Br, or I.

[0066] In some embodiments, R 6 is selected from H or F.

[0067] In some embodiments, the structural unit

[0068] [ka]

[0069] teeth,

[0070] [ka]

[0071] is selected from.

[0072] In some embodiments, the structural unit

[0073] [ka]

[0074] teeth, [ka]

[0075] is selected from.

[0076] In some embodiments, R 5 is selected from C1-C3 alkyl, and C1-C3 alkyl is R a is optionally replaced by

[0077] In some embodiments, R a is selected from F, Cl, Br, I, OH, or CN.

[0078] In some embodiments, R a is selected from F, OH, or CN.

[0079] In some embodiments, R a is selected from F or OH.

[0080] In some embodiments, R 5 is selected from CH2CF3, CH2CHF2, CH2CF2CH2OH, or CH2CF2CH2CN.

[0081] In some embodiments, R 5 is selected from CH2CF3 or CH2CF2CH2OH.

[0082] In some embodiments, Y is selected from NH.

[0083] In some embodiments, the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:

[0084] [ka]

[0085] In some embodiments, the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:

[0086] [ka]

[0087] In some embodiments, the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:

[0088] [ka]

[0089] In some embodiments, the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:

[0090] [ka]

[0091] In some embodiments, the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:

[0092] [ka]

[0093] 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 adjuvant.

[0094] Furthermore, the present invention relates to the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in the preparation of a medicament for preventing or treating an estrogen receptor-associated disease.

[0095] 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 the prevention or treatment of an estrogen receptor-associated disease.

[0096] Furthermore, the present invention relates to the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in the prevention or treatment of an estrogen receptor-associated disease.

[0097] Furthermore, the present invention relates to a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for the prevention or treatment of an estrogen receptor-associated disease.

[0098] The present invention also relates to a method for treating an estrogen receptor-associated disease, comprising administering to a patient a therapeutically effective amount of a pharmaceutical preparation comprising a compound represented by formula (I) of the present invention or a pharmaceutically acceptable salt thereof.

[0099] In a preferred embodiment of the present invention, the estrogen receptor-associated disease includes, but is not limited to, tumors.

[0100] In a preferred embodiment of the present invention, the estrogen receptor-associated disease is breast cancer.

[0101] In a preferred embodiment of the present invention, the estrogen receptor-associated disease is ER-positive breast cancer.

[0102] In a preferred embodiment of the present invention, the estrogen receptor-associated disease is ER-positive breast cancer brain metastasis.

[0103] In a preferred embodiment of the invention, the patient is a patient with breast cancer.

[0104] In a preferred embodiment of the invention, the patient is one with ER-positive breast cancer.

[0105] In a preferred embodiment of the invention, the patient is a patient with ER-positive breast cancer brain metastasis.

[0106] The compounds of the present invention have good antitumor activity in vitro and in vivo and drug discovery potential. In vivo experiments have found that the compounds of the present invention can significantly inhibit tumor growth in a mouse model of ER-positive breast cancer and significantly improve survival in an intracranial mouse model of ER-positive breast cancer. Furthermore, the compounds of the present invention have high bioavailability and a strong ability to degrade ER, can be administered orally, have good blood-brain barrier permeability, and have the potential to effectively treat ER-positive breast cancer (especially ER-positive breast cancer brain metastasis).

[0107] Definitions and Explanations of Terms Unless otherwise specified, the definitions of groups and terms described in the present specification and claims, including their definitions as examples, exemplary definitions, preferred definitions, definitions set forth in tables, and definitions of specific compounds in the examples, can be combined and incorporated with each other. The group definitions and compound structures obtained after such combinations and incorporations are intended to be within the scope described in the present specification.

[0108] As used herein,

[0109] [ka]

[0110] indicates the linkage site.

[0111] The term "pharmaceutically acceptable salts" refers to pharmaceutically acceptable salts of non-toxic acids or bases, including salts of inorganic acids and bases and salts of organic acids and bases.

[0112] The term "stereoisomer" refers to isomers created as a result of different spatial arrangements of atoms in molecules, including cis and trans isomers, enantiomers and diastereomers.

[0113] The compounds of the present invention may contain asymmetric atoms (optical centers) such as carbon, sulfur, nitrogen, and phosphorus atoms, or asymmetric double bonds. Racemates, enantiomers, diastereomers, and geometric isomers are encompassed within the scope of the present invention.

[0114] Graphical representations of racemic or enantiomerically pure compounds herein are derived from Maehr, J. Chem. Ed. 1985, 62:114-120. Unless otherwise indicated, solid wedge bonds and dashed wedge bonds

[0115] [ka]

[0116] is used to denote the absolute configuration of a stereocenter, and a straight solid bond and a straight dashed bond

[0117] [ka]

[0118] is used to represent the cis or trans configuration of an alicyclic compound. Where compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless otherwise specified, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are encompassed within the scope of the invention.

[0119] The compounds of the present invention may exist in particular 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, as well as other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, and all of these are within the scope of the present invention. Additional asymmetric carbon atoms, sulfur atoms, nitrogen atoms, or phosphorus atoms may be present in substituents such as alkyl groups. All of these isomers and mixtures thereof are encompassed within the scope of the present invention. Compounds of the present application containing asymmetric atoms can be isolated into optically active-pure or racemic forms. Optically active-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, the following:

[0120] [ka]

[0121] The term "tautomer" refers to a functional isomer resulting from the rapid movement of atoms at two positions within a molecule. The compounds of the present invention may exhibit tautomerism. Tautomeric compounds may exist in two or more interconverting forms. Because tautomers generally exist in equilibrium, attempts to separate a single tautomer usually result in the formation of a mixture whose chemical and physical properties are consistent with the mixture of compounds. The position of the equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones, such as acetaldehyde, the ketone form predominates, and in phenols, the enol form predominates. The present invention encompasses all tautomeric forms of the compounds.

[0122] The term "pharmaceutical composition" refers to a mixture of one or more compounds described herein or physiologically / pharmaceutically acceptable salts or prodrugs thereof with other chemical components, such as physiologically / pharmaceutically acceptable adjuvants. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0123] The term "substituted" means that any one or more hydrogen atoms on the specified atom are replaced by a substituent, provided that the valence state of the specified atom is normal and the substituted compound is stable. If the substituent is oxo (i.e., =0), it means that two hydrogen atoms are replaced, although this would not occur on an aromatic group.

[0124] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes the occurrence and non-occurrence of the event or circumstance. For example, the phrase "ethyl is 'optionally' substituted with halogen" means that ethyl can be unsubstituted (CH2CH3), monosubstituted (such as CH2CH2F), polysubstituted (such as CHFCH2F, CH2CHF2), or fully substituted (CF2CF3). It will be understood by those skilled in the art that with respect to groups containing one or more substituents, any substitution or substitution pattern that is sterically impractical and / or synthetically infeasible is not intended to be introduced into such groups.

[0125] Any variable (R a , R b When any group (such as ) occurs more than one time in any constituent or structure of a compound, its definition on each occurrence is independent. For example, when a group is made up of two R b When substituted by b has independent options.

[0126] When the number of linking groups is 0, such as -(CH2)0-, it means that the linking group is a bond.

[0127] When one of the variables is selected from a chemical bond or absence, it means that the two groups to which it is attached are directly linked, for example, if L represents a bond in ALZ, it means that the structure is actually AZ.

[0128] When the linking direction of the linking group mentioned in the present specification is not specified, the linking direction is arbitrary. For example,

[0129] [ka]

[0130] L in 1 When is selected from "C1-C3 alkylene-O", L 1 is the ring Q and R 1 and "Ring Q-C1-C3 alkylene-OR" 1 " can be formed, and the rings Q and R 1 The ring QO-C1-C3 alkylene-R 1 " can also be formed.

[0131] The term "halo" or "halogen" refers to fluoro, chloro, bromo, and iodo.

[0132] C in this specification m ~C nmeans that it has an integer number of carbon atoms in the range of m to n. For example, "C1 to C 10 " means that the group can have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms.

[0133] The term "alkyl" refers to a group of the general formula C, which may be straight or branched. n H 2n+1 The term "C1-C" refers to a hydrocarbon group. 10 "Alkyl" shall be understood to denote a linear or branched saturated monovalent hydrocarbon radical having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. For example, said alkyl may be 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, and preferably "C1-C 10 "Alkyl" may include "C1-C6 alkyl" or "C1-C3 alkyl", where "C1-C6 alkyl" shall be understood to denote a linear or branched saturated monovalent hydrocarbon radical having 1, 2, 3, 4, 5, or 6 carbon atoms, and "C1-C3 alkyl" shall be understood to denote a linear or branched saturated monovalent hydrocarbon radical having 1, 2, or 3 carbon atoms.

[0134] Term “C1~C 10 Alkoxy is C1-C 10 Alkyloxy" or "C1-C 10 It is understood that the alkyl group is "C1 to C alkyl-O-" and preferably "C1 to C 10 "Alkoxy" may include "C1-C6 alkoxy" or "C1-C3 alkoxy".

[0135] The term “C3~C10 The term "cycloalkyl" shall be understood to denote a saturated monovalent mono- 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 group, such as a decalin ring. The term "C3-C6 cycloalkyl" shall be understood to denote a saturated monovalent mono- or bicyclic hydrocarbon ring having 3 to 6 carbon atoms.

[0136] The term “C3~C 10 Cycloalkyloxy is C3-C 10 It can be understood that the term "cycloalkyl-O-" is used, and preferably "C3 to C 10 "Cycloalkyloxy" may include "C3-C6 cycloalkyloxy".

[0137] The term "heterocyclyl" refers to a fully saturated or partially saturated (as opposed to heteroaromatic groups which are aromatic as a whole) monovalent, monocyclic, fused-ring, spirocyclic, or bridged-ring radical in which ring atoms contain 1 to 5 heteroatoms or heteroatomic groups (i.e., atomic groups containing heteroatoms), where "heteroatoms or heteroatomic groups" include, but are not limited to, nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), boron (B), =0, =S, -ON=, -C(=O)O-, -C(=O)-, -C(=S)-, -S(=O)-, -S(=O)-, and optionally substituted -NH-, -S(=O)(=NH)-, -C(=O)NH-, -C(=NH)-, -S(=O)NH-, S(=O)NH-, -NHC(=O)NH-, and the like. The term "3- to 10-membered heterocyclyl" refers to a heterocyclyl group containing 3, 4, 5, 6, 7, 8, 9, or 10 ring atoms, wherein the ring atoms contain 1 to 5 heteroatoms or heteroatom groups independently selected from the heteroatoms or heteroatom groups described above. Specific examples of 3-membered heterocyclyls include, but are not limited to, epoxypropyl or azacyclopropyl. Examples of 4-membered heterocyclyls include, but are not limited to, azetidinyl and oxetanyl. Examples of 5-membered heterocyclyls 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 heterocyclyls include, but are not limited to, tetrahydropyranyl, piperidyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, tetrahydropyridyl, or 4H-[1,3,4]thiadiazinyl. Examples of 7-membered heterocyclyls include, but are not limited to, diazepanyl.The heterocyclyl may be a bicyclic group, specifically, an example of a 5,5-membered bicyclic group includes, but is not limited to, hexahydrocyclopenta[c]pyrrol-2(1H)-yl, and an example of a 5,6-membered bicyclic group includes, but is not limited to, hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl, 5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazinyl, or 5,6,7,8-tetrahydroimidazo[1,5-a]pyrazinyl. Optionally, the heterocyclyl may be a benzo-fused ring group of the above-mentioned 4- to 7-membered heterocyclyl, such as, but not limited to, dihydroisoquinolinyl. Optionally, a 3- to 10-membered heterocyclyl can be a "3- to 10-membered heterocycloalkyl," and "3- to 10-membered heterocycloalkyl" can further encompass ranges such as "3- to 7-membered heterocycloalkyl" or "5- to 6-membered heterocycloalkyl." Some of the bicyclic heterocyclyl groups herein partially contain one benzene ring or one heteroaromatic ring, but the heterocyclyl group as a whole is still non-aromatic.

[0138] The term "3 to 10-membered heterocyclyloxy" refers to "3 to 10-membered heterocyclyl-O-".

[0139] The term "treatment" refers to the administration of a compound or preparation of the present application to prevent, ameliorate, or eliminate a disease or one or more symptoms associated with a disease; (i) preventing the onset of a disease or condition in a mammal, particularly where the mammal is susceptible to the condition but has not been diagnosed with the condition; (ii) inhibiting a disease or condition, i.e., suppressing its occurrence; (iii) alleviation of the disease or condition, i.e., resolution of the disease or condition; Includes:

[0140] The term "therapeutically effective amount" refers to an amount of a compound of the present invention that (i) treats or prevents a particular disease, condition, or disorder, (ii) attenuates, 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 described herein. The amount of a compound of the present invention that constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, the method of administration, and the age of the mammal being treated, but can be routinely determined by one of ordinary skill in the art according to their own knowledge and this disclosure.

[0141] The term "adjuvant" refers to a pharmaceutically acceptable inactive ingredient. Examples of types of the term "excipient" include, but are not limited to, binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, etc. Excipients can improve the handling properties of pharmaceutical preparations, i.e., by increasing flowability and / or cohesion, making the preparation more suitable for direct compression. Examples of typical "pharmaceutically acceptable carriers" suitable for use in the above-mentioned preparations include carbohydrates, starches, celluloses and their derivatives, which are adjuvants commonly used in pharmaceutical preparations.

[0142] The term "pharmaceutically acceptable adjuvant" refers to an adjuvant that does not have a significant irritating effect on organisms and does not impair the biological activity and properties of the active compound. Suitable adjuvants are well known to those skilled in the art and include carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0143] The word "comprise" and its variations such as "comprises" or "comprising" are to be understood in an open and non-exclusive sense, i.e., "including but not limited to."

[0144] The compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed in combination with other chemical synthetic methods, and equivalent alternative embodiments known to those skilled in the art, and preferred embodiments include, but are not limited to, the examples of the present invention.

[0145] The present application also includes isotopically labeled compounds of the present application that are identical to those enumerated herein, except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes that can be incorporated into the compounds of the present application include, respectively: 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 There are isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as Cl.

[0146] Certain isotopically labeled compounds of the present application (e.g., 3 H and 14 C) are useful in compound and / or substrate tissue distribution assays. 3 H) and carbon-14 (i.e. 14 C) Isotopes are particularly preferred for their ease of preparation and detectability. 15 O. 13 N, 11 C, and 18Positron-emitting isotopes such as F are useful in positron emission tomography (PET) studies to investigate substrate occupancy. Isotopically labeled compounds of the present application can generally be prepared following procedures similar to those disclosed in the following schemes and / or examples by substituting isotopically labeled reagents for non-isotopically labeled reagents.

[0147] Furthermore, heavier isotopes (deuterium, i.e. 2 H) may confer certain therapeutic benefits resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosing requirements, and therefore may be preferred in some circumstances, although deuterium substitution may be partial or complete, with partial deuterium substitution meaning that at least one hydrogen is replaced by at least one deuterium.

[0148] The pharmaceutical compositions of the present application can be prepared by combining the compounds of the present application with suitable pharmaceutically acceptable auxiliaries. For example, the pharmaceutical compositions of the present application 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.

[0149] Typical routes of administration of the compounds of the present application, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, include, but are not limited to, oral, rectal, topical, by inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0150] The pharmaceutical compositions of the present application can be manufactured by conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, and lyophilizing methods which are well known in the art.

[0151] In some embodiments, the pharmaceutical composition is in oral form.For oral administration, the pharmaceutical composition can be prepared by mixing the active compound with pharmaceutically acceptable excipients known in the art.Such excipients allow the compound of the present application to be formulated into tablets, pills, lozenges, sugar-coated tablets, capsules, liquids, gels, syrups, suspensions, etc. for oral administration to patients.

[0152] Solid oral compositions can be prepared by conventional blending, filling, or tablet-forming methods.For example, it can be obtained by mixing the active compound with a solid excipient, optionally pulverizing the resulting mixture, adding other suitable excipients as needed, and processing the mixture into granules to obtain tablets or sugar-coated tablet cores.Suitable excipients include, but are not limited to, binders, diluents, disintegrants, lubricants, glidants, sweeteners, or flavoring agents.

[0153] The pharmaceutical compositions may also be suitable for parenteral administration, such as sterile solutions, suspensions, or lyophilized products in appropriate unit dosage forms.

[0154] The daily administration dose of the compound of general formula I for all administration methods described herein is 0.01 mg / kg to 100 mg / kg body weight, preferably 0.05 mg / kg to 50 mg / kg body weight, more preferably 0.1 mg / kg to 30 mg / kg body weight, in single or divided dose form.

[0155] The chemical reactions described in specific embodiments of the present invention are completed in suitable solvents, which must be suitable for the chemical transformations of the present invention and the reagents and materials required thereby. In order to obtain the compounds of the present invention, those skilled in the art may need to modify or select synthetic steps or reaction schemes based on existing embodiments. [Brief explanation of the drawings]

[0156] [Figure 1] 1 is a NOESY spectrum of compound 3. [Figure 2] 1 is a schematic diagram of the tumor growth curve of the MCF-7 subcutaneous tumor model in Test Example 9. [Figure 3] 1 is a diagram showing changes in body weight in the MCF-7 subcutaneous tumor model in animals in Test Example 9. [Figure 4] 10 is a diagram showing the survival curve of the MCF-7 intracranial model in mice in Test Example 10. [Figure 5] 1 is a diagram showing changes in body weight in the MCF-7 intracranial model in mice in Test Example 10. DETAILED DESCRIPTION OF THE INVENTION

[0157] The technical solutions of the present invention are illustrated in more detail in the following examples, and the scope of protection of the present invention includes but is not limited to these.

[0158] The structure of the compound is determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The NMR shifts are -6 The NMR analysis solvents are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is tetramethylsilane (TMS). 50 " refers to the half-maximal inhibitory concentration, the concentration at which half of the maximum inhibitory effect is achieved. [Example]

[0159] 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-pyridino[3,4-b]indol-1-yl)pyridin-3-amine (Compound 1)

[0160] [ka]

[0161] Synthesis method:

[0162] [ka]

[0163] Step 1: Synthesis of tert-butyl (1-(3-fluoropropyl)pyrrolidin-3-yl)carbamate

[0164] [ka]

[0165] Dissolve tert-butyl pyrrolidin-3-ylcarbamate (1.00 g, 5.37 mmol) in tetrahydrofuran (10 mL) and add sodium hydroxide solution (5 mol L -1 , 2.15 mL) and 1-iodo-3-fluoropropane (1.06 g, 5.64 mmol) were added. The reaction solution was stirred at 25 °C for 16 hours. After the reaction of the raw material was completed as detected by TLC, the reaction solution was diluted with ethyl acetate and then washed with saturated ammonium chloride solution, and the aqueous and organic phases were collected separately. The aqueous phase was extracted three times with ethyl acetate (50 mL), and then all the organic phases were combined and dried over sodium sulfate. The organic phase was concentrated to dryness under reduced pressure and then purified by column chromatography (silica, dichloromethane / methanol = 100 / 1) to give the product tert-butyl (1-(3-fluoropropyl)pyrrolidin-3-yl)carbamate (0.81 g). 1 H NMR(400MHz, methanol-d4)δ 4.57(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).

[0166] Step 2: Synthesis of 1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride

[0167] [ka]

[0168] Dissolve tert-butyl (1-(3-fluoropropyl)pyrrolidin-3-yl)carbamate (0.81 g, 3.12 mmol) in 1,4-dioxane (9 mL), and then add hydrochloric acid-1,4-dioxane solution (4 mol L -1 9 mL) was added, and the mixture was reacted to obtain a yellow transparent solution. The reaction solution was stirred at 25° C. for 3 hours. After the reaction of the raw materials was completed as detected by TLC, the reaction solution was concentrated to dryness under reduced pressure to obtain the compound 1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride (0.71 g). 1 H NMR (400 MHz, methanol-d₄) δ 4.68 (t, J = 5.52 Hz, 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).

[0169] Step 3: Synthesis of (R)-1-(1H-indol-3-yl)-N-(2,2,2-trifluoroethyl)propan-2-amine

[0170] [ka]

[0171] (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), and trifluoroethyl trifluoromethanesulfonate (1.20 g, 5.17 mmol) dissolved in 1,4-dioxane (5 mL) was added at 25 °C. The reaction solution was stirred at 75 °C for 16 hours. The reaction solution was concentrated to dryness under reduced pressure. The crude product was then purified by column chromatography (silica, petroleum ether / ethyl acetate = 3 / 1) to give the product (R)-1-(1H-indol-3-yl)-N-(2,2,2-trifluoroethyl)propan-2-amine (0.69 g). MS m / z(ESI):257.2[M+H] + ; 1 H NMR (400 MHz, methanol-d₄) δ 7.56-7.54 (d, J = 8.0 Hz, 1H), 7.36-7.34 (d, J = 8.0 Hz, 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 = 6.0 Hz, 3H).

[0172] Step 4: Synthesis of (1S,3R)-1-(5-bromopyridin-2-yl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyridino[3,4-b]indole

[0173] [ka]

[0174] (R)-1-(1H-indol-3-yl)-N-(2,2,2-trifluoroethyl)propan-2-amine (120.00 mg, 468.26 μmol) was dissolved in toluene (2 mL), and 5-bromopyridine-2-formaldehyde (87.10 mg, 468.26 μmol) and acetic acid (562.40 mg, 9.37 mmol) were added to the solution, which was a yellow, transparent solution. The reaction solution was stirred at 90° C. for 10 hours. After completion of the reaction as detected by LCMS, the reaction solution was cooled to room temperature, concentrated to dryness 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-pyridino[3,4-b]indole (110.00 mg). MS m / z(ESI):424.1, 426.1[M+H] + . 1 H NMR(400MHz, methanol-d4)δ 8.59(s,1H),8.01-7.94(m,1H),7.54(d,J=8.53Hz,1H),7.46(d,J=7.78Hz,1H),7.30(d,J=8.03Hz,1H),7.08(d,J=7.59Hz,1H),7.03-6.96(m, 1H),5.08(s,1H),3.58-3.46(m,1H),3.38-3.34(m,1H),3.13-2.99-(m, 1H),2.80(d,J=4.52Hz,1H),2.70-2.61(m,1H),1.26(d,J=6.78Hz,3H).

[0175] 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-pyridino[3,4-b]indol-1-yl)pyridin-3-amine

[0176] [ka]

[0177] (1S,3R)-1-(5-bromopyridin-2-yl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyridino[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 hydroxide, and HCl were added. Thorium tert-butoxide (149.50 mg, 1.56 mmol) and (2-dicyclohexylphosphino)-3,6-dimethoxyl-2,4,6-triisopropyl-1,1-biphenyl)(2-amino-1,1-biphenyl-2-yl)palladium(II) methanesulfonate (23.50 mg, 25.93 μmol) were added to the reaction mixture, which became a brown, cloudy solution under a nitrogen atmosphere. The reaction mixture was stirred at 80° C. for 4 hours. After completion of the reaction as detected by LCMS, the reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure and purified by preparative liquid chromatography (Phenomenex Gemini C18 column, 3 μm silica, diameter 30 mm, length 75 mm) (using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile as eluents) to give the compound N-(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyridino[3,4-b]indol-1-yl)pyridin-3-amine (22.23 mg). MS m / z(ESI):490.2[M+H] + . 1H NMR(400MHz, methanol-d4)δ 7.88(d,J=2.76Hz,1H),7.46(d,J=7.78Hz,1H),7.25(dd,J=7.91,3.89Hz,2H),7.09 -6.96(m,3H),4.97(s,1H),4.60(t,J=5.65Hz,1H),4.48(t,J=5.65Hz,1H),4.15(br s,1H),3.52-3.36(m,3H),3.25-3.14(m,1H),3.09-2.88(m,6H),2.68(s,1H),2.51-2.39(m,1H),2.11-1.85(m,3H),1.20(d,J=6.53Hz,3H).

[0178] 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-pyridino[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-pyridino[3,4-b]indol-1-yl)pyridin-3-amine (Compound 3)

[0179] [ka]

[0180] Racemic N-(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyridino[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% aqueous ammonia) and water as eluents) and preparative liquid chromatography (Phenomenex Gemini Purification on a C18 column, 3 μm silica, 30 mm diameter, 75 mm length, using decreasingly polar mixtures of water (containing 0.05% aqueous ammonia) and acetonitrile as eluents, gave N-((R)(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyridino[3,4-b]i)-2H-pyridino[3,4-b]i. N-((S)(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyridino[3,4-b]indol-1-yl)pyridin-3-amine (8.22 mg, retention time of 2.627 min) 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-pyridino[3,4-b]indol-1-yl)pyridin-3-amine (10.85 mg, retention time of 2.817 min).

[0181] N—((R)(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyridino[3,4-b]indol-1-yl)pyridin-3-amine (compound 2): MS m / z(ESI):490.1[M+H] + 1H NMR (400 MHz, methanol-d4) δ 7.89 (d, J = 2.76 Hz, 1H), 7.48-7.44 (m, 1H), 7.29-7.24 (m, 2H), 7.09-7.04 (m, 2H), 7.00 (s, 1H), 4.98 (s, 1H), 4.64-4.60 (m, 1H), 4.52-4.48 (m, 1H), 4.25-4.16 (m, 1H),3.54-3.36(m,4H),3.25-3.09(m,4H),3.05(s,1H),2.89(d,J=4.52Hz,1H), 2.70-2.60(m,1H),2.55-2.42(m,1H),2.16-1.94(m,3H),1.20(d,J=6.78Hz,3H).

[0182] N-((S)(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyridino[3,4-b]indol-1-yl)pyridin-3-amine (compound 3): MS m / z(ESI):489.25,490.1[M+H] + 1 H NMR(400MHz, methanol-d4)δ 7.90-7.88(m,1H),7.48-7.43(m,1H),7.29-7.23(m,2H),7.09-7.03(m,2H),7.03-6.97(m,1H),4.99 -4.97(m,1H),4.63-4.59(m,1H),4.52-4.47-(m,1H),4.24-4.16(m,1H),3.52-3.36(m,4H),3.15(br s,4H),3.05-2.99(m,1H),2.96-2.88(m,1H),2.68(s,1H),2.57-2.43(m,1H),2.12-1.94(m,3H),1.20(d,J=6.53Hz,3H).

[0183] N-((S)-1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyridino[3,4-b]indol-1-yl)pyridin-3-amine (Compound 3) can also be obtained by the following synthetic method.

[0184] [ka]

[0185] Step 1: Synthesis of (S)-tert-butyl (1-(3-fluoropropyl)pyrrolidin-3-yl)carbamate

[0186] [ka]

[0187] (S)-tert-butyl pyrrolidin-3-ylcarbamate (500.00 mg, 2.68 mmol) was dissolved in tetrahydrofuran (10 mL) and diluted with sodium hydroxide solution (5 mol L -1 , 1.07 mL) and 1-iodo-3-fluoropropane (529.88 mg, 2.82 mmol) were added. The reaction solution was stirred at 25 °C for 16 hours. After the reaction of the raw material was completed as detected by TLC, the reaction solution was diluted with ethyl acetate (50 mL) and then washed with saturated ammonium chloride solution (10 mL), and the aqueous and organic phases were collected separately. The aqueous phase was extracted three times with ethyl acetate (20 mL), and then all the organic phases were combined and dried over sodium sulfate. The organic phase was concentrated to dryness under reduced pressure and then purified by column chromatography (silica, dichloromethane / methanol = 100 / 1) to give the product (S)-tert-butyl (1-(3-fluoropropyl)pyrrolidin-3-yl)carbamate (480.00 mg). 1H NMR(400MHz, methanol-d4)δ 4.58-4.53(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).

[0188] Step 2: Synthesis of (S)-1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride

[0189] [ka]

[0190] (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 -1 When 4.94 mL of hexane was added, the reaction solution became a yellow, transparent solution. The reaction solution was stirred at 25° C. for 3 hours. After the reaction of the raw materials was completed as detected by TLC, the reaction solution was concentrated under reduced pressure to obtain compound (S)-1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride (450.00 mg). 1 H NMR(400MHz,DMSO-d6)δ 8.80-8.42(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).

[0191] 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-pyridino[3,4-b]indol-1-yl)pyridin-3-amine

[0192] [ka]

[0193] (1S,3R)-1-(5-bromopyridin-2-yl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyridino[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 (2-dicyclohexylphosphino)-3,6-dimethoxyl-2,4,6-triisopropyl-1,1-biphenyl)(2-amino-1,1-biphenyl-2-yl)palladium(II) methanesulfonate (23.93 mg, 26.40 μmol) were added, and the reaction solution was stirred at 80° C. under a nitrogen atmosphere for 4 hours. After completion of the reaction as detected by LCMS, the reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure and purified by preparative liquid chromatography (Phenomenex Gemini C18 column, 3 μm silica, diameter 30 mm, length 75 mm) (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-pyridino[3,4-b]indol-1-yl)pyridin-3-amine (37.79 mg). MS m / z(ESI):365.1[M+H] + 1H NMR(400MHz, methanol-d4)δ 7.89(d,J=2.76Hz,1H),7.46(d,J=7.78Hz,1H),7.25(d,J=8.53Hz,2H),7.09-7.03(m,2H) ,7.02-6.97(m,1H),4.98(s,1H),4.60(t,J=5.65Hz,1H),4.49(t,J=5.65Hz,1H),4.18(br s,1H),3.51-3.35(m,4H),3.14-2.99(m,5H),2.92(dd,J=15.18,4.89Hz,1H),2.65(dd ,J=16.06,6.78Hz,1H),2.53-2.42(m,1H),2.12-1.92(m,3H),1.20(d,J=6.78Hz,3H).

[0194] Identification of the absolute configuration of compound 3 Identification by 2D NMR:

[0195] [ka]

[0196] The NOESY spectrum (Figure 1) showed that the methyl hydrogen at position 3 of compound 3 had a clear NOE effect with the hydrogen at position 1, proving that the two were on the same side. Since the pyridyl at position 1 and the methyl at position 3 on the six-membered piperidine ring are trans, and the absolute configuration of the carbon atom at position 3 is known to be R, the absolute configuration of the carbon atom at position 1 is S.

[0197] 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-pyridino[3,4-b]indol-1-yl)phenyl)-1-(3-fluoropropyl)pyrrolidin-3-amine (Compound 4)

[0198] [ka]

[0199] Synthesis method:

[0200] [ka]

[0201] Step 1: Synthesis of (S)-tert-butyl (1-(3-fluoropropyl)pyrrolidin-3-yl)carbamate

[0202] [ka]

[0203] (S)-tert-butyl pyrrolidin-3-ylcarbamate (500.00 mg, 2.68 mmol) was dissolved in tetrahydrofuran (10 mL) and diluted with sodium hydroxide solution (5 mol L -1 , 1.07 mL) and 1-iodo-3-fluoropropane (529.88 mg, 2.82 mmol) were added. The reaction solution was stirred at 25 °C for 16 hours. After the reaction of the raw material was completed as detected by TLC, the reaction solution was diluted with ethyl acetate (50 mL) and then washed with saturated ammonium chloride solution (10 mL), and the aqueous and organic phases were collected separately. The aqueous phase was extracted three times with ethyl acetate (20 mL), and then all the organic phases were combined and dried over sodium sulfate. The organic phase was concentrated to dryness under reduced pressure and then purified by column chromatography (silica, dichloromethane / methanol = 100 / 1) to give the product (S)-tert-butyl (1-(3-fluoropropyl)pyrrolidin-3-yl)carbamate (480.00 mg). 1 H NMR(400MHz, methanol-d4)δ 4.58-4.53(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).

[0204] Step 2: Synthesis of (S)-1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride

[0205] [ka]

[0206] (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 -1 When 4.94 mL of hexane was added, the reaction solution became a yellow, transparent solution. The reaction solution was stirred at 25° C. for 3 hours. After the reaction of the raw materials was completed as detected by TLC, the reaction solution was concentrated under reduced pressure to obtain compound (S)-1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride (450.00 mg). 1 H NMR(400MHz,DMSO-d6)δ 8.80-8.42(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).

[0207] 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-pyridino[3,4-b]indole

[0208] [ka]

[0209] (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), and the reaction solution was stirred at 90 °C for 6 hours. The reaction solution was concentrated to dryness under reduced pressure. The reaction solution was then purified by column chromatography (silica, petroleum ether / ethyl acetate = 20 / 1) to give the product (1S,3R)-1-(4-bromo-2,6-difluorophenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyridino[3,4-b]indole (850 mg). 1 H NMR(400MHz, methanol-d4)δ 7.45-7.43(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).

[0210] 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-pyridino[3,4-b]indol-1-yl)phenyl)-1-(3-fluoropropyl)pyrrolidin-3-amine

[0211] [ka]

[0212] (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, the mixture was stirred homogeneously, and then tris(dibenzalacetone)dipalladium (31.90 mg, 34.84 μmol) and (±)-2,2-bis(diphenylphosphino)-11-binaphthyl (54.23 mg, 87.10 μmol) were added under a nitrogen atmosphere. The reaction solution was stirred at 80 ° C. for 4 hours. After the reaction of the raw materials was completed as detected by LCMS, the reaction solution was filtered, and the filter cake was then rinsed with tetrahydrofuran. The filtrate was concentrated to dryness under reduced pressure and purified by preparative liquid chromatography (Phenomenex Gemini C18 column, 7 μm silica, diameter 50 mm, length 250 mm, using a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity) to obtain the compound (S)—N-(3,5-difluoro-4-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyridino[3,4-b]indol-1-yl)phenyl)-1-(3-fluoropropyl)pyrrolidin-3-amine (4.69 mg). MS m / z(ESI):525.2[M+H] + 1H NMR(400MHz, methanol-d4)δ 8.49(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).

[0213] 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-pyridino[3,4-b]indol-1-yl)phenyl)-4-fluoro-1-(3-fluoropropyl)pyrrolidin-3-amine (Compound 5)

[0214] [ka]

[0215] Synthesis method:

[0216] [ka]

[0217] Step 1: Synthesis of tert-butyl (trans-4-fluoro-1-(3-fluoropropyl)pyrrolidin-3-yl)carbamate

[0218] [ka]

[0219] 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., and 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. The crude product was then purified by column chromatography (silica, ethyl acetate / methanol=10 / 1) to give the product tert-butyl (trans-4-fluoro-1-(3-fluoropropyl)pyrrolidin-3-yl)carbamate (0.15 g). MS m / z (ESI): 265.0 [M + H] + ; 1 H NMR (400 MHz, chloroform-d) δ 4.87 (br s, 1H), 4.59 (t, J = 5.9 Hz, 1H), 4.48 (t, J = 5.9 Hz, 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).

[0220] Step 2: Synthesis of trans-4-fluoro-1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride

[0221] [ka]

[0222] 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), and 4 M dioxane hydrochloric acid (2.13 mL) was added. 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). MS m / z(ESI):165.2[M+H] + .

[0223] 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-pyridino[3,4-b]indol-1-yl)phenyl)-4-fluoro-1-(3-fluoropropyl)pyrrolidin-3-amine

[0224] [ka]

[0225] (1S,3R)-1-(4-bromo-2,6-difluorophenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyridino[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 tertiary pentanol (5 mL), and methanesulfonic acid (2-dicyclohexylphosphino-3,6-dimethoxyl-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) were added. After being replaced with nitrogen three times, the reaction solution was stirred for 13 hours at 120° C. The reaction solution was cooled to room temperature and poured into water (10 mL), the solution was stirred for 10 minutes, extracted twice with ethyl acetate (20 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The crude 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% aqueous 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-pyridino[3,4-b]indol-1-yl)phenyl)-4-fluoro-1-(3-fluoropropyl)pyrrolidin-3-amine (27.5 mg). MS m / z(ESI):543.1[M+H] + ; 1H NMR (400 MHz, methanol-d4) δ 7.42(d,J=7.5Hz,1H),7.22(d,J=7.5Hz,1H),7.09-6.93(m,2H),6.30-6.16( m,2H),5.25(s,1H),4.58(t,J=5.8Hz,1H),4.46(t,J=5.8Hz,1H),4.11-3.87( m,1H),3.67-3.53(m,1H),3.43-3.34(m,3H),3.19-2.92(m,3H),2.81-2.55( m,4H),2.29(dd,J=6.9,9.7Hz,1H),2.02-1.83(m,2H),1.19(d,J=6.4Hz,3H).

[0226] Example 6: Synthesis of trans-N-[3,5-difluoro-4-[(1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-1,3,4,9-tetrahydropyridino[3,4-b]indol-1-yl]phenyl]-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-amine (Compound 6)

[0227] [ka]

[0228] Synthesis method:

[0229] [ka]

[0230] Step 1: Synthesis of tert-butyl N-[trans-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-yl]carbamate

[0231] [ka]

[0232] 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), and then 1-fluoro-3-iodo-propane (90.11 mg, 479.34 μmol) was added. The reaction solution was heated to 50 ° C. and stirred for 16 hours. Completion of the reaction was monitored by LCMS. The reaction solution was filtered, and the filtrate was concentrated to dryness and purified by column chromatography (silica, ethyl acetate / methanol = 5 / 1) to obtain compound tert-butyl N-[trans-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-yl]carbamate (85 mg). MS m / z(ESI):261.1[M+H] + 1 H NMR (400MHz, methanol-d4)δ 4.66(br d,J=2.4Hz,1H),4.61-4.42(m,2H),4.05-3.93(m,1H),3.71-3.56(m,2H),3.17(br d,J=9.4Hz,1H),2.90(br s,2H),2.54(br s,1H),2.19(br d,J=5.2Hz,2H),2.07-1.89(m,1H),1.47(s,9H),1.24-1.11(m,3H).

[0233] Step 2: Synthesis of trans-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-amine hydrochloride

[0234] [ka]

[0235] Tert-butyl N-[trans-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-yl]carbamate (80 mg, 307.28 μmol) was dissolved in dioxane (2 mL), and then 4 M dioxane hydrochloric acid (1.54 mL) was added, and the reaction solution was stirred at room temperature overnight. The reaction solution was concentrated to dryness to obtain compound trans-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-amine hydrochloride (70 mg). 1 H NMR (400 MHz, methanol-d₄) δ 4.73–4.64 (m, 1H), 4.61–4.50 (m, 1H), 4.23–4.06 (m, 1H), 4.02–3.63 (m, 5H), 3.54–3.42 (m, 1H), 3.01–2.58 (m, 1H), 2.32–2.12 (m, 2H), 1.37–1.27 (m, 3H).

[0236] Step 3: Synthesis of trans-N-[3,5-difluoro-4-[(1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-1,3,4,9-tetrahydropyridino[3,4-b]indol-1-yl]phenyl]-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-amine

[0237] [ka]

[0238] 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-tetrahydropyridino[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), and then tBuBrettphos-Pd-G3 (5.50 mg, 6.43 μmol) was added. The reaction solution was purged with nitrogen three times, then heated to 120 °C, and stirred overnight. Completion of the reaction was monitored by LCMS. Methanol (15 mL) was added to the reaction solution, the mixture was filtered, and 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, diameter 30 mm, length 150 mm, 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-tetrahydropyridino[3,4-b]indol-1-yl]phenyl]-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-amine (1.35 mg). MS m / z(ESI):539.3[M+H] + ; 1H NMR(400MHz, methanol-d4)δ 7.42(d,J=7.6Hz,1H),7.21(d,J=8.8Hz,1H),7.08-6.86(m,2H),6.21(d,J=11.6Hz,2H),5.24(s,1H),4.62-4.58(m,1H),4.52-4.45(m ,1H),3.73-3.65(m,1H),3.62-3.47(m,3H),3.23-3.15(m,2H),3.08-2.95(m,2H),2.86-2.54(m,2H),2.41-2.00(m,3H),1.42-1.25(m 2H),1.23(dd,J=3.2,Hz,6.8Hz,3H),1.19(d,J=6.4Hz,3H).

[0239] 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-pyridino[3,4-b]indol-2(9H)-yl)-2,2-difluoropropan-1-ol

[0240] [ka]

[0241] Synthesis method

[0242] [ka]

[0243] Step 1: Synthesis of 3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl trifluoromethanesulfonate

[0244] [ka]

[0245] 3-((tert-Butyldiphenylsilyl)oxy)-2,2-difluoropropan-1-ol (1 g, 2.85 mmol) and 2,6-dimethylpyridine (366.88 mg, 3.42 mmol) were dissolved in anhydrous dichloromethane (15 mL), and trifluoromethanesulfonic anhydride (885.52 mg, 3.14 mmol) was added dropwise in an ice-water bath. The reaction solution was stirred at 25 °C for 16 hours. After completion of the reaction as monitored by TLC (silica, petroleum ether:ethyl acetate = 10:1), the reaction solution was washed successively with water (10 mL), hydrochloric acid (1 mol / L, 10 mL), and saturated sodium carbonate solution (10 mL). After the collected organic phase was dried over sodium sulfate, the organic phase was 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). 1 H NMR (400MHz, methanol-d4) δ 7.66(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).

[0246] Step 2: Synthesis of (R)—N-(1-(1H-indol-3-yl)prop-2-yl)-3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropan-1-amine

[0247] [ka]

[0248] 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), and then N-ethyl-N-isopropylpropan-2-amine (600.81 mg, 4.65 mmol) was added. The reaction solution was stirred at 80° C. for 16 hours. The completion of the reaction was monitored by LCMS, and the reaction solution was concentrated and purified by column chromatography (silica, petroleum ether / ethyl acetate=100 / 17) to give the product (R)-N-(1-(1H-indol-3-yl)prop-2-yl)-3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropan-1-amine (662.00 mg). MS m / z(ESI):507.2[M+H] + ; 1 H NMR(400MHz,chloroform-d)δ 7.72-7.63(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),7.0 1(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).

[0249] 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-pyridino[3,4-b]indole

[0250] [ka]

[0251] (R)—N-(1-(1H-indol-3-yl)prop-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), and then acetic acid (355.55 mg, 5.92 mmol, 338.62 μL) was added. The reaction solution was stirred at 90° C. for 16 hours. Completion of the reaction was monitored by LCMS and TLC (petroleum ether:ethyl acetate=10:1), and the reaction solution was concentrated to dryness under reduced pressure. The reaction concentrate was diluted with ethyl acetate (5 mL) and 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 to dryness under reduced pressure, 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-pyridino[3,4-b]indole (350.00 mg). MS m / z(ESI):708.9[M+H] + ; 1 H NMR (400 MHz, chloroform-d) δ 7.65-7.59 (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).

[0252] 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-pyridino[3,4-b]indol-1-yl)-3,5-difluorophenyl)-1-(3-fluoropropyl)pyrrolidin-3-amine

[0253] [ka]

[0254] (1S,3R)-1-(4-bromo-2,6-difluorophenyl)-2-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyridino[3,4-b]indole (300.00 mg, 422.72 μmol) and (S)-1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride (129 mg, 50 The reaction mixture (7.27 μmol) was dissolved in tetrahydrofuran (10 mL), and then sodium tert-butoxide (243.75 mg, 2.54 mol) and 2,2-bis(diphenylphosphino)-1,1-binaphthyl (131.61 mg, 211.36 μmol) were added. Tris(dibenzylideneacetone)dipalladium (77.42 mg, 84.54 μmol) was added to the reaction mixture under a nitrogen atmosphere. The reaction mixture was stirred at 80° C. for 16 hours. After completion of the reaction as monitored by LCMS, the reaction mixture was concentrated to dryness under reduced pressure. The reaction concentrate was diluted with ethyl acetate (10 mL) and then washed three times with water (10 mL). The collected aqueous phase was extracted three times with ethyl acetate (20 mL). The organic phase was dried over sodium sulfate, concentrated to dryness under reduced pressure, and then purified by thin layer chromatography (silica, petroleum ether / tetrahydrofuran=1 / 1, 1% aqueous ammonia) to give (S)—N-(4-((1S,3R)-2-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyridino[3,4-b]indol-1-yl)-3,5-difluorophenyl)-1-(3-fluoropropyl)pyrrolidin-3-amine (54.00 mg). MS m / z (ESI): 400.5 [M + H] +

[0255] 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-pyridino[3,4-b]indol-2(9H)-yl)-2,2-difluoropropan-1-ol

[0256] [ka]

[0257] (S)—N-(4-((1S,3R)-2-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyridino[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 tetrahydrofuran solution (1 mol / L, 129.04 μL) was added. The reaction solution was stirred at 25° C. for 5 hours. Completion of the reaction was monitored by TLC (silica, dichloromethane:methanol=10:1). Then, 5 mL of water was added to the reaction solution, and the solution 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. Then, the organic phase was purified by thin layer chromatography (silica, dichloromethane:methanol=10:1) to obtain the compound 3-((1S,3R)-1-(2,6-difluoro-4-(((S)-1-(3-fluoropropyl)pyrrolidin-3-yl)amino)phenyl)-3-methyl-3,4-dihydro-1H-pyridino[3,4-b]indol-2(9H)-yl)-2,2-difluoropropan-1-ol (32.00 mg). MS m / z (ESI): 559.1 [M + Na] + 1H NMR(400MHz, methanol-d4)δ 7.38(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),2.7 0-2.51(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).

[0258] Testing of biological activity and related properties Test Example 1: Detection of the degrading effect of the compound of the present invention on estrogen receptors in MCF7 cells 1. Purpose of the experiment The purpose of the experiment was to measure the degradation activity of the compounds of the present invention against endogenously expressed estrogen receptors in MCF7 cells and to compare the activity of the compounds with DCs. 50 and to evaluate according to maximum degradation efficiency.

[0259] 2. Experimental Method MCF7 cells (ATCC, HTB-22) were cultured in complete DMEM (Gibco, 11995-065) medium containing 10% fetal bovine serum. On the first day of the experiment, MCF7 cells were seeded into 384-well plates at a density of 3,000 cells / well using complete medium and cultured in a 5% CO2 cell incubator at 37°C. The compounds to be tested were dissolved in DMSO at a stock concentration of 10 mM, and the solution was diluted using an Echo 550 (Labcyte Inc.) and added to the cell culture plate. Each compound, with a starting concentration of 100 nM for treatment, was subjected to a 3-fold gradient dilution with nine concentration points. A blank control containing 0.5% DMSO was set up, and duplicate wells were set up at each concentration point as controls. The cultures were cultured in a 5% CO2 cell incubator at 37°C for 24 hours. Paraformaldehyde was added to the cell culture medium in each cell culture well to a final concentration of approximately 3.7% to fix the cells. After incubating the mixture for 30 minutes, the supernatant was discarded, and 50 μL of PBS was added to each well for one wash. The cells were treated with PBS containing 0.5% v / v Tween-20 for 30 minutes and then washed once with PBS. Blocking solution (PBS containing 5% BSA and 0.05% Tween-20) was added, and the mixture was incubated at room temperature for 1 hour. The blocking solution was removed, and a mixture of primary antibodies (anti-ER mAb, estrogen receptor α (D8H8) rabbit mAb, GST, 8644S, diluted 1:1000, and anti-GAPDH mAb, GAPDH (D4C6R) mouse mAb, GST, 97166S, diluted 1:2000) was added, and the mixture was incubated at room temperature for 3 hours. The cultures were washed three times with PBST (PBS containing 0.05% Tween-20). Secondary detection antibodies (800CW-goat anti-rabbit IgG, LI-COR, P / N: 926-32211, 1:1000 dilution; 680RD-goat anti-mouse IgG, LI-COR, P / N: 925-68070, 1:1000 dilution) were added, and the mixture was incubated at room temperature in the dark for 45 minutes. After washing three times with PBST, the fluorescence signal of each well was read using an Odyssey CLx, and the values of Channel 800 (ER) / Channel 680 (GAPDH) were calculated.Wells treated with 0.1 μM fulvestrant were used as a 100% degradation control to calculate the degradation rate at each concentration point. Data were analyzed and processed by XlLfit to calculate the degradation activity DC of each compound. 50 and the maximum degradation rate Imax was calculated. See Table 1 for data analysis.

[0260] [Table 1]

[0261] Test Example 2: Detection of the inhibitory effect of the compound of the present invention on the proliferation of MCF7 cells 1. Purpose of the experiment The purpose of the experiment was to measure the inhibitory effect of the compounds of the present invention on the proliferation of MCF7 cells in vitro and to evaluate the IC 50 and to evaluate the activity of the compounds according to their maximum inhibitory efficiency.

[0262] 2. Experimental Method MCF7 cells (ATCC, HTB-22) were cultured in complete DMEM (Gibco, 11995-065) medium containing 10% fetal bovine serum. On the first day of the experiment, MCF7 cells were seeded into a 384-well plate at a density of 500 cells / well using complete medium and cultured overnight in a 37°C, 5% CO2 cell incubator. The next day, the compounds to be tested were added for drug treatment. Compound solutions with a stock concentration of 10 mM were diluted using an Echo550 (Labcyte Inc.) and transferred to each cell culture well. Each compound, with a starting concentration of 100 nM for treatment in cells, was subjected to a 3-fold gradient dilution to create nine concentration points. A blank control containing 0.3% DMSO was set up, and duplicate wells were set up as controls at each concentration point. The cultures were cultured in a 37°C, 5% CO2 cell incubator for 7 days, and the cell culture plates were removed on day 8. CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7573) was added, and the plate was left to stand at room temperature for 10 minutes. The luminescence signal value was then read using a multilevel microplate reader, EnVision (PerkinElmer), and the inhibitory activity IC of each compound was calculated. 50 was calculated according to the compound concentration and luminescence signal value using XLfit.

[0263] 3. See Table 2 for data analysis.

[0264] [Table 2]

[0265] Test Example 3: Inhibitory effect of the compounds of the present invention on CYP2C9 and CYP2D6 enzyme activity The inhibition of CYP2C9 and CYP2D6 enzyme activity by the compounds of the present invention was measured by the following test method.

[0266] I. Test Materials and Equipment 1. Human liver microsomes (Corning 452117) 2. NADPH (Solarbio 705Y021) 3. Positive substrates diclofenac (Sigma SLBV3438), dextromethorphan (TRC 3-EDO-175-1), and midazolam (Cerilliant FE01161704) 4. Positive inhibitors: sulfaphenazole (D. Ehrenstorfer GmbH 109012), quinidine (TCI WEODL-RE), and ketoconazole (Sigma 100M1091V) 5.AB Sciex Triple Quad 5500 Liquid Chromatography-Mass Spectrometry

[0267] II. Testing process 1. Preparation of 100 mM phosphate buffered saline (PBS): 7.098 g of NaHPO was weighed, 500 mL of pure water was added, and the mixture was subjected to ultrasonic dissolution to obtain solution A. 3.400 g of KHPO was weighed, 250 mL of pure water was added, and the mixture was subjected to ultrasonic dissolution to obtain solution B. Solution A was placed on a stirrer, and solution B was slowly added until the pH reached 7.4 to prepare 100 mM PBS buffer solution.

[0268] 2. Preparation of 10 mM NADPH solution in 100 mM PBS buffer. 10 mM stock solutions of compounds of the present invention were diluted with DMSO to obtain 200x concentrations of compound working solutions (6000, 2000, 600, 200, 60, 20, 0 μM). 200x concentrations of positive inhibitor working solutions (sulfaphenazole, 1000, 300, 100, 30, 10, 3, 0 μM, quinidine / ketoconazole, 100, 30, 10, 3, 1, 0.3, 0 μM) were obtained by diluting the positive inhibitor stock solutions with DMSO. 200x concentrations of substrate working solutions (120 μM diclofenac, 400 μM dextromethorphan, and 200 μM midazolam) were prepared in water, acetonitrile, or acetonitrile / methanol.

[0269] 3.2 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 were mixed uniformly and pre-incubated in a 37°C water bath for 15 minutes. To the positive control group, 1 μl of diclofenac, dextromethorphan, or midazolam working solution was added instead of the compound working solution. Simultaneously, 10 mM NADPH solution was pre-incubated in a 37°C water bath for 15 minutes. After 15 minutes, 20 μl of NADPH was added to each well to initiate the reaction, and the reaction mixture was incubated at 37°C for 5 minutes (CYP2C9) or 20 minutes (CYP2D6). Duplicate samples were set up for all incubation samples. After the corresponding incubation time, the reaction was stopped by adding 400 μl of iced methanol containing the internal standard to all samples. The mixture was mixed uniformly by vortexing and centrifuged at 3220 g for 40 minutes at 4° C. After centrifugation, 100 μL of the supernatant was transferred to a loading plate, and 100 μL of ultrapure water was added and mixed uniformly for LC-MS / MS analysis.

[0270] IC of the compounds of the present invention against CYP2C9 and CYP2D6 50 Values were calculated with Excel XLfit 5.3.1.3.

[0271] [Table 3]

[0272] Test Example 4: Measurement of plasma protein binding rate of the compound of the present invention Human plasma protein binding is a major factor controlling the amount of free (unbound) drug available for target binding and plays an important role in a drug's observed in vivo efficacy. Thus, among compounds with similar potency and exposure levels, compounds with a high free fraction (low levels of plasma protein binding) may exhibit increased efficacy.

[0273] The protein binding rates of the compounds of the present invention in the plasma of five species (human, monkey, dog, rat, and mouse) were measured by the following test method.

[0274] I. Test Materials and Equipment 1. Human plasma (BioIVT), Beagle dog plasma (BioIVT), SD rat plasma (BioIVT), CD-1 mouse plasma (BioIVT), 2. 96-well equilibrium dialysis plate (HTDialysis LLC, Gales Ferry, CT, HTD96B), equilibrium dialysis membrane (MWCO 12-14K, No. 1101), 3. Positive control compound warfarin, 4. ABI QTrap 5500 liquid chromatography-mass spectrometry.

[0275] II. Testing process 1. Preparation of a buffer solution containing 100 mM sodium phosphate and 150 mM NaCl: An alkaline solution containing 14.2 g / L NaHPO and 8.77 g / L NaCl was prepared using ultrapure water, and an acidic solution containing 12.0 g / L NaHPO and 8.77 g / L NaCl was prepared using ultrapure water. The alkaline solution was titrated to pH 7.4 with the acidic solution to prepare a buffer solution containing 100 mM sodium phosphate and 150 mM NaCl. 2. Preparation of dialysis membrane: The dialysis membrane was soaked in ultrapure water for 60 minutes to separate the membrane into two pieces, which were then soaked in 20% ethanol for 20 minutes, and finally soaked in the buffer solution used for dialysis for 20 minutes. 3. Preparation of plasma: Frozen plasma was rapidly thawed at room temperature, then centrifuged at 3,220 g for 10 minutes at 4°C to remove clots, and the supernatant was collected in a new centrifuge tube. The pH of the plasma was measured and recorded, and plasma with a pH of 7 to 8 was used. 3. Preparation of compound-containing plasma samples: A 10 mM stock solution of a compound of the invention or a positive control compound was diluted with DMSO to obtain a 200 μM working solution. 3 μl of the 200 μM compound working solution was added to 597 μl of human, monkey, dog, rat, or mouse plasma to obtain plasma samples with a final concentration of 1 μM. 4. Equilibrium dialysis step: The dialysis apparatus was assembled according to the operating instructions. 120 μL of plasma sample containing 1 μM compound was added to one side of the dialysis membrane, and an equal volume of dialysate (phosphate buffer) was added to the other side. Duplicate samples were set up for the experiment. The dialysis plate was sealed, placed in an incubator, and incubated at 37°C, 5% CO2, and approximately 100 rpm for 6 hours. After incubation was completed, the sealing film was removed, and 50 μL of each well was pipetted from the buffer and plasma sides, respectively, into another well of a new plate. 50 μL of blank plasma was added to the phosphate buffer sample, and an equal volume of blank phosphate buffer was added to the plasma sample. Then, 300 μL of acetonitrile containing the internal standard was added to precipitate the proteins. The mixture was vortexed for 5 minutes and centrifuged at 3,220 g for 30 minutes at 4°C. 100 μL of the supernatant was placed in a loading plate, and 100 μL of ultrapure water was added and mixed uniformly for LC-MS / MS analysis.

[0276] The peak areas of the compound in the buffer solution and plasma were measured. The formula for calculating the plasma protein binding rate of the compound is as follows: Free rate % = (internal standard peak area 緩衝液側 Ratio of compound peak area to internal standard peak area 血漿側 (ratio of compound peak area to Binding rate %=100-Free rate %

[0277] All data were calculated using Microsoft Excel software. The plasma protein binding rate values of the compounds of the present invention were calculated.

[0278] [Table 4]

[0279] Test Example 5: Apparent solubility of the compound of the present invention in phosphate buffer solution at pH 7.4 In order for an orally administered compound to reach the site of action and be effectively absorbed by the intestine, the compound is expected to be in a dissolved form; therefore, compounds with high intrinsic solubility may be more suitable for pharmaceutical use.

[0280] I. Materials and Reagents The compounds to be tested were prepared according to the described method. The control drug progesterone was purchased from Sigma. Phosphate buffer solution of pH 7.4 was prepared in our laboratory. Acetonitrile and methanol were purchased from Fisher. Other reagents were purchased commercially.

[0281] 1.5 ml flat-bottom glass vials (BioTech Solutions), polytetrafluoroethylene / silicone stoppers (BioTech Solutions), polytetrafluoroethylene-coated stir bars, MultiScreenHTS HV (0.45 μm) 96-well plate filter plate (Millipore, MSHVN4510 or MSHVN4550), Eppendorf Thermomixer Comfort, vacuum manifold ORVMN96 (Orochem).

[0282] II. Experimental Procedure 1) Preparation of stock solutions 10 mM stock solutions of the substances to be tested and the control drug progesterone were prepared in DMSO.

[0283] 2) Apparent solubility measurement process Thirty microliters of a 10 mM stock solution of the substance to be tested was added to the corresponding location in the corresponding 96-well plate in the order indicated. 970 μL of phosphate buffer (pH 7.4) was added to the corresponding vial in the sample plate. Experiments were performed in parallel in duplicate. A stir bar was added to each vial, and a polytetrafluoroethylene / silicone stopper was placed over it. The sample tray was then placed in an Eppendorf Thermomixer Comfort and shaken at 1100 rpm for 2 hours at 25°C. After 2 hours, the stopper was removed, the stir bar was sucked out with a large magnet, and the sample was then transferred from the sample plate to a filter plate. A vacuum pump was used to create negative pressure, and the sample was filtered. Five microliters of the filtrate was transferred to a new sample plate, followed by the addition of 5 μL of DMSO and 490 μL of 50% ACN(IS).H2O (internal standard acetonitrile:water = 1:1). Depending on the peak shape, it may be possible to dilute the sample diluent with a certain ratio of 50% ACN(IS).H2O for better peak shape. The dilution fold can be adjusted depending on the solubility of the substance to be tested or the signal strength of its response to liquid chromatography-mass spectrometry.

[0284] 3) Sample analysis process The loading plate was placed in the loading tray of the autosampler and the samples were evaluated by liquid chromatography-mass spectrometry analysis.

[0285] III. Experimental Procedure All calculations were performed using Microsoft Excel. Analysis and quantification of sample filtrates were achieved by characterizing and quantifying peaks of known concentrations of standards using liquid chromatography-mass spectrometry. The apparent solubility of the compounds of the present invention in phosphate buffer (pH 7.4) was calculated.

[0286] [Table 5]

[0287] Test Example 6: Whether the compounds of the present invention have a potential inhibitory effect on the voltage-gated potassium ion channel hERG The hERG potassium channel is crucial for normal electrical activity in the heart. Arrhythmias can be induced by blockade of the hERG channel by various drugs. Such side effects are a common cause of drug failure in preclinical safety testing; therefore, minimization of hERG channel blocking activity may be a desirable property for drug candidates.

[0288] I. Materials and Reagents

[0289] [Table 6-1]

[0290] 2. Cell Lines and Culture The HEK293 cell line (catalog number K1236) stably expressing the hERG ion channel was purchased from Invitrogen. The cell line was cultured in a medium containing 85% DMEM, 10% dialyzed fetal bovine serum, 0.1 mM non-essential amino acid solution, 100 U / mL penicillin-streptomycin solution, 25 mM HEPES, 5 μg / mL blasticidin, and 400 μg / mL geneticin. When the cell density increased to 40%–80% of the bottom area of the culture dish, trypsin was used for digestion and passage, and the cells were passaged three times a week. Before the experiment, cells were cultured at 5 × 10 in a 6 cm culture dish. 5 The cells were cultured at a density of 1000 kJ / mL and induced by adding 1 μg / mL doxycycline for 48 h, and then the cells were digested and plated onto glass slides for subsequent manual patch clamp experiments.

[0291] 3. Preparation of Compounds to be Tested 1) According to the SOP-ADMET-MAN-007 standard operating procedure, the compound to be tested was dissolved in DMSO to prepare a stock solution with a final concentration of 10 mM. 2) The stock solution was subjected to gradient dilution with DMSO at a ratio of 1:3 to give solutions with three other intermediate concentrations of 3.33 mM, 1.11 mM, and 0.37 mM, respectively. 3) Before the start of the experiment, the stock and intermediate solutions of the compounds to be tested were diluted 1000-fold with extracellular solution to obtain working solutions with concentrations of 10 μM, 3.33 μM, 1.11 μM, and 0.37 μM. Furthermore, the 10 mM stock solution was diluted 333.33-fold with extracellular solution to obtain a working solution with a concentration of 30 μM. The DMSO content in the working solutions was 0.1-0.3% (volume ratio). (Note: The content of DMSO in the working solution must be controlled within 1% (volume ratio) to avoid cytotoxicity.) 4) After the preparation of the working solution was completed, visual observation was carried out to check whether there was any precipitate or cloudy material in the working solution. If there was any precipitate or cloudy material, which was probably due to the low solubility of the compound in physiological solution, it could then be further subjected to ultrasonic treatment in a water bath for 30 minutes to improve the clarity of the solution. 5) The potential inhibitory effect of the test substance on the hERG channel was measured at five concentrations: 30 μM, 10 μM, 3.33 μM, 1.11 μM, and 0.37 μM, and the dose-effect curve was fitted to determine the IC 50 was calculated.

[0292] II. Experimental Methods 1. A small glass slide containing HEK293 cells in a culture dish was placed in the perfusion tank of a micromanipulator. 2. Prepare an appropriate amount of cells and place them in the center of the field of view under an Olympus IX51, IX71, or IX73 inverted microscope. Use a 10x objective to find the tip of the glass electrode and place it in the center of the field of view. Then, use a micromanipulator to move the electrode downward while adjusting the coarse focus knob so that the electrode can slowly approach the cells. 3. When approaching the cell, a 40x objective lens is used for observation, and a micromanipulator is used for precise adjustment so that the electrode can gradually approach the cell surface. 4. Negative pressure was applied to form a gigaseal between the electrode tip and the cell membrane, with a resistance greater than 1 GΩ. 5. The instantaneous capacitive current Cfast was compensated in voltage-clamp mode, and then short negative pressures were repeatedly applied to rupture the membrane, thereby finally forming the whole-cell recording mode. 6. Under a fixed membrane potential of -60 mV, the slow capacitive current Cslow, cell membrane capacitance (Cm), and input membrane resistance (Ra) were compensated for, respectively. After the cells were stabilized, the clamp voltage was changed to -90 mV, the sampling frequency was set to 20 kHz, and the filtering frequency was set to 10 kHz. The leak current detection conditions were: clamp voltage was changed to -80 mV, and the time lapse was 500 ms. 8. The hERG current was measured as follows: a depolarizing voltage was applied for 4.8 seconds to depolarize the membrane potential from -80 mV to +30 mV, followed by a repolarizing voltage for 5.2 seconds to reduce the membrane potential to -50 mV and remove channel inactivation, resulting in the observation of the hERG tail current. The peak value of the tail current represents the magnitude of the hERG current. 9. The hERG current used to detect the compound to be tested was continuously recorded for 120 seconds before administration to evaluate the stability of the hERG current produced by the test cells. Only stable cells within the acceptable range of the evaluation criteria could be used for subsequent compound testing. 10. Measurement of the inhibitory effect of the test compound on hERG current: First, hERG current measured in an extracellular solution containing 0.1% DMSO was used as the detection baseline. After the hERG current remained stable for at least 5 minutes, solutions containing the test compound were perfused around the cells in increasing concentrations. Approximately 5 minutes were allowed after each perfusion to allow the compound to fully act on the cells, and the hERG current was simultaneously recorded. After the recorded current stabilized, the last five hERG current values were recorded, and the average value was used as the final current value at a particular concentration. After the compound test was completed, 150 nM dofetilide was added to the same cells to completely inhibit the current, which served as a positive control for the cells. Furthermore, the positive compound dofetilide was detected using the same patch clamp system both before and after the test compound experiment to ensure the reliability and sensitivity of the entire detection system.

[0293] III. Data Analysis Data was exported by PatchMaster software and analyzed according to the following steps: 1) After perfusion of blank solvent or compound gradient solution, the average of five consecutive current values obtained in a stable state was defined as the "tail current value." ブランク " and "Tail current value 化合物 " is calculated as follows. 2) The percent current inhibition was calculated by the following formula:

number

[0294] The inhibition of hERG by compounds of the present invention is shown in Table 6 below.

[0295] [Table 6-2]

[0296] Test Example 7: Pharmacokinetic evaluation of the compounds of the present invention in mice Experimental materials CD-1 mice were purchased from Beijing Vital River 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).

[0297] Experimental Method Six female CD-1 mice (20-30 g, 4-6 weeks) were randomized into two groups, with three mice per group. 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), 7.5% Captisol (w / v) in water. Animals were provided with regular food and water before the experiment. Blood was sampled from the vein of each group of mice at pre-dose and 0.083 (intravenous injection group only), 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after dosing. Collected whole blood samples were placed in K2EDTA anticoagulant tubes and centrifuged (12,000 rpm, 4°C) for 5 minutes. Plasma was then collected for subsequent detection.

[0298] A 10 μL sample of mouse plasma was collected and 150 μL of acetonitrile solvent (containing the internal standard compound) was added to precipitate the protein. The mixture was vortexed for 5 minutes and then centrifuged (14,000 rpm) for 5 minutes. The supernatant was diluted 2-fold with water containing 0.1% (v / v) FA and injected into an LC-MS / MS system (AB Sciex Triple Quad 6500+) for quantitative detection. A calibration standard curve was prepared in CD-1 mouse plasma along with quality control samples, and plasma concentrations were measured. For the 10-fold diluted sample, 2 μL of sample was collected and 18 μL of blank plasma was added. The mixture was vortexed for 0.5 minutes and 300 μL of acetonitrile solvent (containing the internal standard compound) was added to precipitate the protein. Other processing steps were identical to those for the undiluted sample.

[0299] The PK test results are shown below, and the compounds of the present invention show good PK properties and oral bioavailability in mice.

[0300] [Table 7]

[0301] Test Example 8: Blood-brain barrier (BBB) permeability of the compound of the present invention in rats Sufficient exposure of a drug to the brain through penetration through the blood-brain barrier of an animal is essential for achieving drug efficacy against brain metastatic lesions. Therefore, drug distribution in the brain can be assessed by measuring drug concentrations in the plasma and brain tissue of animals after administration to determine whether a drug can inhibit tumor growth in an intracranial model.

[0302] Experimental materials Female SD rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Methylcellulose (MC) was purchased from Sigma, and acetonitrile was purchased from Merck (USA). Phosphate-buffered saline (PBS) was purchased from Sangon Biotech Co., Ltd.

[0303] Experimental Method Six female SD rats (200-300 g, 6-8 weeks) were randomized into two groups, with three rats per group. Each rat received a compound of the present invention, and the vehicle was a 0.5% aqueous methylcellulose solution. The animals were given water as usual, fasted overnight before the experiment, and resumed feeding 4 hours after administration. Plasma and brain tissue were collected from rats in each group 2 hours after administration. The collected whole blood samples were placed in K2EDTA anticoagulant tubes and centrifuged (12,000 rpm, 4°C) for 5 minutes, and then the plasma was collected for subsequent detection. After tissue collection, the tissues were dried using filter paper, and the samples were stored in a -80°C freezer for subsequent detection.

[0304] A 10 μL sample of rat plasma was collected and 150 μL of acetonitrile solvent (containing the internal standard compound) was added to precipitate the protein. The mixture was vortexed for 5 minutes and then centrifuged (14,000 rpm) for 5 minutes. The supernatant was diluted 2-fold with water containing 0.1% (v / v) FA and injected into an LC-MS / MS system (AB Sciex Triple Quad 6500+) for quantitative detection. For the 10-fold diluted sample, 2 μL of the sample was collected and 18 μL of blank plasma was added. The mixture was vortexed for 0.5 minutes and 300 μL of acetonitrile solvent (containing the internal standard compound) was added to precipitate the protein. Other processing steps were identical to those for the undiluted sample. A calibration standard curve was prepared in SD rat plasma along with plasma quality control samples to measure plasma concentrations.

[0305] Brain tissue samples from rats were first homogenized with four times the mass volume of PBS homogenate. 20 μL of the brain tissue homogenate sample was collected and 20 μL of blank mouse plasma was added. The mixture was diluted and mixed uniformly, and 600 μL of acetonitrile solvent (containing the internal standard compound) was added to precipitate the protein. The mixture was vortexed for 5 minutes and then centrifuged (14,000 rpm) for 5 minutes. The supernatant was diluted two-fold with water containing 0.1% (v / v) FA and injected into an LC-MS / MS system (AB Sciex Triple Quad 6500+) for quantitative detection. Compound No. Example 3 of the present invention exhibited excellent blood-brain barrier permeability and high drug exposure in rat brain tissue.

[0306] The results of the BBB test are shown below.

[0307] [Table 8]

[0308] Test Example 9: Growth inhibition test of the compound of the present invention in a mouse MCF-7 subcutaneous tumor model Experimental Reagents Human breast cancer MCF-7 cells: ATCC, HTB-22 17β-estradiol tablets: Innovative Research of America, catalog number: SE-121, 60-day release, 0.72 mg / pellet EMEM medium: ATCC, Catalog number: 30-2003 Fetal Bovine Serum: Gbico, Catalog Number: 1099-141C Penicillin (Pen Strep): Gibco, Catalog Number: 15240-122 Recombinant human insulin: Shanghai Yeasen Biotechnology Co., Ltd. Catalog number: 40112ES60 0.25% Trypsin-EDTA: Gibco, Catalog Number: 25200-072 D-PBS (phosphate buffered saline without Ca and Mg ions): Hyclone, Catalog Number: SH30256.01 Matrigel: Corning, Catalog Number: 356237

[0309] Experimental Method Animal information: NPG mice (female, 6-7 weeks, weight: approximately 19-28 g) were purchased from Beijing Vitalstar Biotechnology Co., Ltd. Mice were fed in an SPF environment, and each cage position was individually ventilated. All animals had free access to standard certified commercial laboratory chow and water.

[0310] Cell culture: Human breast cancer MCF-7 cell line was 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 a 5% CO2 incubator at 37°C. Conventional digestion with 0.25% trypsin-EDTA digestion solution was performed once a week for passage. When cell saturation reached 80%-90% and the required number was reached, cells were harvested and counted.

[0311] Cell inoculation: 0.1ml / (1×10 7 MCF-7 cell suspension (containing 17β-estradiol) in a 1:1 volume ratio of D-PBS to Matrigel was subcutaneously inoculated into the right back of each mouse. Four days before cell inoculation, the mice were subcutaneously inoculated with 17β-estradiol tablets. 24 days after cell inoculation, the mice were randomized into treatment groups according to tumor volume. The day of group division was day 0.

[0312] Administration: Compound No. Example 1 was administered orally (PO) at a dose of 1, 3, or 10 mg / kg once daily (QD) for 3 weeks. Compound No. Example 2 was administered orally (PO) at a dose of 10 mg / kg once daily (QD) for 3 weeks. Eight mice were in the vehicle group and six mice were in the treatment group.

[0313] Tumor Measurements and Laboratory Parameters: Tumor diameters were measured twice a week using a vernier caliper. Tumor volume was calculated using the formula V = 0.5a × b 2 where a and b represent the long and short diameters of the tumor, respectively. The mice were weighed twice a week.

[0314] The tumor-inhibiting efficacy of the compounds was evaluated using tumor growth inhibition (TGI) (%): TGI (%) = [(1 - (mean tumor volume at the end of treatment in a particular treatment group - mean tumor volume at the start of treatment in a treatment group) / (mean tumor volume at the end of treatment in the vehicle control group - mean tumor volume at the start of treatment in the vehicle control group)] x 100%.

[0315] Test Results: See Table 9, Figures 2 and 3. In the MCF-7 model of subcutaneously transplanted tumors in mice, Compound No. Example 3 of the present invention had a significant inhibitory effect on tumor growth (P<0.01) when orally administered once daily at 1 mg / kg, 3 mg / kg, or 10 mg / kg, demonstrating a good dose-response relationship. When administered at doses of 3 mg / kg and 10 mg / kg, the compound exhibited a tumor-shrinking effect. Compound No. Example 3 of the present invention had a significant inhibitory effect on tumor growth (P<0.01) when orally administered once daily at 10 mg / kg, demonstrating a tumor-shrinking effect. Compounds No. Example 3 and Example 7 did not significantly affect mouse body weight at the tested doses.

[0316] [Table 9]

[0317] Test Example 10: Growth inhibition test of the compound of the present invention in a mouse MCF-7 intracranial tumor model Experimental Reagents / Equipment: Human breast cancer MCF-7 cells: ATCC, HTB-22 17β-estradiol tablets: Innovative Research of America, catalog number: SE-121, 60-day release, 0.72 mg / pellet EMEM medium: ATCC, Catalog number: 30-2003 Fetal bovine serum: Gibco, Catalog number: 1099-141C Penicillin (Pen Strep): Gibco, Catalog Number: 15240-122 Recombinant human insulin: Shanghai Yeasen Biotechnology Co., Ltd. Catalog number: 40112ES60 0.25% Trypsin-EDTA: Gibco, Catalog Number: 25200-072 Stereotaxic instrument: RWD Life Technology Co., Ltd. Catalog number: Standard / Digital / Single Arm / Mouse / 68055 Microinjection pump: KDS, Catalog number: Legato130 Small Handheld Cranial Drill: RWD Life Technology Co., Ltd. Catalog Number: 78001

[0318] Experimental Method: Animal information: NPG mice (female, 6-8 weeks, weight: approximately 17-29 g) were purchased from Beijing Vitalstar Biotechnology Co., Ltd. Mice were fed in an SPF environment, and each cage position was individually ventilated. All animals had free access to standard certified commercial laboratory chow and water.

[0319] Cell culture: Human breast cancer MCF-7 cell line was 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 a 5% CO2 incubator at 37°C. Conventional digestion with 0.25% trypsin-EDTA digestion solution was performed twice a week for passage. When cell saturation reached 80%-90% and the required number was reached, cells were harvested and counted.

[0320] Cell inoculation: 15μl / (2×10 6MCF-7 cell suspensions containing 17β-estradiol were inoculated intracerebrally into mice using a stereotaxic apparatus, a microinjection pump, and a small handheld skull drill. Three days before cell inoculation, mice were subcutaneously inoculated with 17β-estradiol tablets. Eight days after cell inoculation, mice were randomized into groups for treatment according to their body weight. The day of group division was designated day 0.

[0321] Administration: Fulvestrant (AstraZeneca) was administered subcutaneously (SC) at a dose of 250 mg / kg once a week (QW), and Compound No. Example 3 was administered orally (PO) at a dose of 30 mg / kg once a day (QD). Eleven mice were in the vehicle group, and eight mice were in the treatment group. Mice in all groups were continuously administered until death, when mice were euthanized due to poor condition or termination of the experiment.

[0322] Experimental Observations and Evaluation Items: The mice were weighed twice a week and their survival status was monitored.

[0323] At the end of the experiment on day 48, all mice were euthanized.

[0324] Test Results: See Figures 4 and 5. In the MCF-7 intracranial tumor model in mice, mice in the fulvestrant group (250 mg / kg subcutaneously administered once a week) continued to lose weight, and their survival status was not significantly different from that of the vehicle control group (median survival time: 29 days for the vehicle control group, 29.5 days for the fulvestrant group). Mice in the group administered with Compound No. Example 3 of the present invention (30 mg / kg orally administered once a day) had stable weight and showed no abnormalities until the end of the experiment. No deaths occurred in mice in the group administered with the compound of Example 3 of the present invention. Compared with the vehicle control or fulvestrant, the compound of Example 3 had a significant inhibitory effect in the MCF-7 intracranial tumor model in mice, and the survival time of the mice was significantly longer (P<0.01).

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, 【Chemical 1】 During the ceremony, R １ , R ２ , R ３ , and R ４ are H, F, Cl, Br, I, CN, C １ ~C ６ Alkyl, C １ ~C ６ Alkoxy, or C ３ ~C ６ cycloalkyl; Structural Unit 【Chemistry 2】 Y is selected from O or NH; R ５ is C １ ~C ６ alkyl, and １ ~C ６ Alkyl is R ａ and optionally replaced by R ａ are F, Cl, Br, I, OH, CN, C １ ~C ６ Alkyl, C １ ~C ６ Alkoxy, or C ３ ~C ６ A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein: R is 1 or 2; R is 2 or 3; R is 3 or 4; R is 4 or 5; R is 5 or 6; R is 6 or 7; R is 7 or 8; R is 8 or 9; R is 9 or 10; R is 10 or 11;

2. R １ , R ２ , R ３ , and R ４ is H, F, Cl, Br, I, CN, or C １ ~C ３ 2. The compound of formula (I) of claim 1, or a pharmaceutically acceptable salt thereof, wherein: R is independently selected from alkyl;

3. R ５ is C １ ~C ３ alkyl, wherein C １ ~C ３ Alkyl is R ａ 2. The compound of formula (I) of claim 1, or a pharmaceutically acceptable salt thereof, optionally substituted by:

4. The structural unit 【Chemistry 3】 teeth, 【Chemistry 4】 2. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, selected from:

5. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is the following compound or a pharmaceutically acceptable salt thereof: 【Chemistry 5】

6. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is the following compound or a pharmaceutically acceptable salt thereof: 【Chemistry 6】

7. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable adjuvant.

8. Use of the compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 7, in the preparation of a medicament for preventing or treating an estrogen receptor-related disease.

9. A pharmaceutical composition for preventing or treating an estrogen receptor-related disease, comprising the compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable adjuvant.

Images