CN118119620A

CN118119620A - Pharmaceutical composition for treating tumors and application thereof

Info

Publication number: CN118119620A
Application number: CN202280069694.7A
Authority: CN
Inventors: 薛黎婷; 古鹏; 陈平; 杨桂梅; 杨文清; 周峰; 唐任宏; 任晋生
Original assignee: Nanjing Zaiming Pharmaceutical Co ltd
Current assignee: Nanjing Zaiming Pharmaceutical Co ltd
Priority date: 2021-11-12
Filing date: 2022-11-11
Publication date: 2024-05-31
Also published as: TW202333704A; WO2023083283A1

Abstract

A pharmaceutical combination comprising a compound of formula (K) or a pharmaceutically acceptable salt thereof as a selective estrogen receptor down-regulator (SERD) and a CDK4/6 inhibitor, a combination product or pharmaceutical composition comprising said pharmaceutical combination, and uses thereof.

Description

Pharmaceutical composition for treating tumors and application thereof

Cross Reference to Related Applications

The present application claims priority and equity to chinese patent application number 202111338407.1 filed on day 11 and 12 of 2021 to the chinese national intellectual property agency, the disclosure of which is incorporated herein by reference in its entirety.

Technical Field

The present disclosure is in the field of medicine, and relates to a pharmaceutical combination of a compound of formula (K) and a CDK4/6 inhibitor as a selective estrogen receptor down-regulator (SERD), a combination product or pharmaceutical composition comprising said pharmaceutical combination, and their use for treating tumors.

Background

Estrogen (E2) and estrogen alpha receptor (erα) are important drivers of the development and progression of breast cancer. In breast cancer patients, more than 2/3 of the patients express ER transcription factors, and in most ER-positive patients, ER is a key driver even in tumors that progress after early endocrine treatment, and therefore ER is a major target for breast cancer treatment (Pharmacology & Therapeutics 186 (2018) 1-24). Endocrine therapy aims at reducing ER activity, and there are three main classes, including Selective Estrogen Receptor Modulators (SERMs), such as tamoxifen (tamoxifen), which are allosteric modulators of ER and inhibit their transcriptional activity upon binding to ER; aromatase inhibitors (aromatase inhibitors, AIs) reduce in vivo estrogen levels by inhibiting the conversion of androgens to estrogens; and selective estrogen receptor down-regulators such as fulvestrant (fulvestrant), which not only inhibit the activity of ER antagonists, but also have the effect of inducing ER protein degradation. Although endocrine therapy is the first choice for estrogen receptor positive breast cancer patients, about 30% of patients relapse after treatment and almost all metastatic breast cancer patients develop drug resistance and progress.

Clinically, about 70-80% of breast cancers are positive for Estrogen Receptor (ER), proliferation of such breast cancer cells is severely dependent on ER, and 50% of breast cancer death cases are of this type. Early ER-positive breast cancer has better prognosis, and the survival rate of 5 years exceeds 90 percent. Recurrence occurs in about 30% of patients with post-operative endocrine therapy (TAM or AI medications) within 10 years, but still can receive standard endocrine therapy.

Fulvestrant is the first and only SERD-type drug clinically approved for use in post-menopausal patients with ER-positive, metastatic breast cancer following the progression of tamoxifen or aromatase inhibitors. The study data show that ER degradation cannot be completely realized in patients treated by fulvestrant, in addition, obvious reactions such as pain, swelling, redness and the like at injection positions caused by intramuscular injection are slow in absorption, limited in-vivo exposure and the like, so that clinical application of the patients is limited, and new treatment options are needed for ER-positive breast cancer patients.

Cyclin dependent kinases 4 and 6 (CDKs 4/6) mediate the transition of the cell cycle from G0/G1 to S phase, promoting cell proliferation. Piperacillin Bai Xili (Palbociclib, chemical name 6-acetyl-8-cyclopentyl-5-methyl-2- [ [5- (1-piperazinyl) -2-pyridinyl ] amino ] pyrido [2,3-d ] pyrimidin-7 (8H) -one) as a CDK4/6 selective inhibitor, is useful in the treatment of cancer patients.

Disclosure of Invention

In one aspect, the present disclosure provides a pharmaceutical combination comprising at least one selective estrogen receptor down-regulator (SERD) selected from a compound of formula (K) or a pharmaceutically acceptable salt thereof, and at least one CDK4/6 inhibitor:

wherein,

R ¹、R ²、R ³、R ⁴ is independently selected from H, F, cl, br, I, CN, C ₁-C ₆ alkyl, C ₁-C ₆ alkoxy, or C ₃-C ₆ cycloalkyl;

X ₁、X ₂、X ₃、X ₄ is independently selected from CR ⁶ or N;

R ⁶ is selected from H, F, cl, br, I, OH, CN, C ₁-C ₁₀ alkyl, C ₃-C ₁₀ cycloalkyl, 3-10 membered heterocyclyl, C ₁-C ₁₀ alkoxy, C ₃-C ₁₀ cycloalkyloxy, or 3-10 membered heterocyclyloxy;

R ⁵ is independently selected from C ₁-C ₆ alkyl, said C ₁-C ₆ alkyl optionally substituted with R ^a;

r ^a is selected from F, cl, br, I, OH, CN, C ₁-C ₆ alkyl, C ₁-C ₆ alkoxy or C ₃-C ₆ cycloalkyl.

In another aspect, the present disclosure provides a combination comprising a pharmaceutical composition I comprising at least one compound of formula (K), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant, and a pharmaceutical composition II comprising at least one CDK4/6 inhibitor and a pharmaceutically acceptable adjuvant.

In another aspect, the present disclosure also provides a kit comprising:

A first container comprising a first pharmaceutical composition as described above; and, a step of, in the first embodiment,

A second container comprising a second pharmaceutical composition as described above.

In another aspect, the present disclosure also provides a pharmaceutical composition comprising any of the above pharmaceutical combinations, and at least one pharmaceutically acceptable excipient.

In another aspect, the present disclosure relates to the use of any one of the above pharmaceutical combinations, combinations or pharmaceutical compositions for the preparation of an antitumor drug.

In another aspect, the present disclosure relates to the use of any one of the above pharmaceutical combinations, combinations or pharmaceutical compositions for anti-tumor.

In another aspect, the present disclosure relates to any one of the above pharmaceutical combinations, combinations or pharmaceutical compositions for use in anti-tumor.

In another aspect, the present disclosure relates to an anti-tumor method comprising administering to a patient in need thereof a therapeutically effective amount of any one of the above pharmaceutical combinations, or pharmaceutical compositions.

In another aspect, the present disclosure also relates to the use of a compound of formula (K) or a pharmaceutically acceptable salt thereof in combination with a CDK4/6 inhibitor in the manufacture of an antitumor medicament.

Drawings

FIG. 1 is a NOESY pattern of a compound of formula (I).

FIG. 2 is a graph of survival of a model mouse in situ of human ER-positive breast cancer MCF-7 brain of a compound of formula (I).

FIG. 3 is a graph showing the weight change of mice in an in situ model of human ER-positive breast cancer MCF-7 brain of the compound of formula (I).

FIG. 4 is a graph showing the synergistic in vitro antiproliferative effect of a combination of a compound of formula (I) and piperaquine Bai Xili on human breast cancer MCF-7 cells.

FIG. 5 is a graph showing the effect of a combination of a compound of formula (I) and piperaquine Bai Xili on anti-tumor growth in a mouse model of human ER-positive breast cancer MCF-7 xenograft.

FIG. 6 is a graph showing the weight change of mice with a subcutaneous engrafted tumor of human ER-positive breast cancer MCF-7 xenograft with a combination of a compound of formula (I) and piperaquine Bai Xili.

Detailed Description

To facilitate an understanding of the disclosure herein, a more complete description is provided below. These embodiments may, however, be embodied in many different forms and are not limited to the specific examples described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Definition and description of terms

Unless otherwise indicated, the radical and term definitions recited in the specification and claims, including as examples, exemplary definitions, preferred definitions, definitions recited in tables, definitions of specific compounds in the examples, and the like, may be arbitrarily combined and coupled with each other. Such combinations and combined group definitions and structures of compounds should fall within the scope of the description.

The term "pharmaceutical combination" refers to a combination of two or more active ingredients or pharmaceutically acceptable salts thereof. In some embodiments, the active ingredients in the pharmaceutical combination or pharmaceutically acceptable salts thereof may be administered simultaneously, and in some embodiments, the active ingredients in the pharmaceutical combination or pharmaceutically acceptable salts thereof may also be administered separately or sequentially.

The term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of non-toxic acids or bases, including salts of inorganic acids and bases, organic acids and bases, such as succinate.

The term "pharmaceutical composition" refers to a mixture of one or more active ingredients of the present disclosure with pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to facilitate administration of a compound of the present disclosure, or a pharmaceutical combination thereof, to a subject.

Herein, a method of manufacturing a semiconductor deviceRepresenting the ligation site.

The graphic representation of racemates or enantiomerically pure compounds herein is from Maehr, J.chem. Ed.1985, 62:114-120. Unless otherwise indicated, wedge-shaped keys and virtual wedge-shaped keys are usedAnd) Representing the absolute configuration of a three-dimensional center by using black real keys and virtual keysAnd) Representing the relative configuration of a stereocenter (e.g., the cis-trans configuration of a alicyclic compound).

The term "tautomer" refers to a functional group isomer that results from the rapid movement of an atom in a molecule at two positions. Compounds of the present disclosure may exhibit tautomerism. Tautomeric compounds may exist in two or more interconvertible species. Tautomers generally exist in equilibrium and attempts to isolate individual tautomers often result in a mixture whose physicochemical properties are consistent with the mixture of compounds. The location of the equilibrium depends on the chemical nature of the molecule. For example, among many aliphatic aldehydes and ketones such as acetaldehyde, the ketone type predominates; whereas, among phenols, the enol form is dominant. The present disclosure encompasses all tautomeric forms of the compounds.

The term "stereoisomers" refers to isomers arising from the spatial arrangement of atoms in a molecule, and includes cis-trans isomers, enantiomers and diastereomers.

The compounds of the present disclosure may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms, or asymmetric double bonds, and thus the compounds of the present disclosure may exist in particular geometric or stereoisomeric forms. Particular geometric or stereoisomeric forms may be cis and trans isomers, E and Z geometric isomers, (-) -and (+) -enantiomers, (R) -and (S) -enantiomers, diastereomers, (D) -isomers, (L) -isomers, and racemic or other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which fall within the definition of compounds of the disclosure. Additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms, or asymmetric phosphorus atoms may be present in the substituents such as alkyl groups, and all such isomers and mixtures thereof referred to in the substituents are included within the definition of compounds of the present disclosure. The asymmetric atom containing compounds of the present disclosure may be isolated in optically active pure form or in racemic form, which may be resolved from racemic mixtures, or synthesized by using chiral starting materials or chiral reagents.

The term "substituted" means that any one or more hydrogen atoms on a particular atom is substituted with a substituent, provided that the valence of the particular atom is normal and the substituted compound is stable.

The term "optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, ethyl "optionally" substituted with halogen means that ethyl can be unsubstituted (CH ₂CH ₃), monosubstituted (CH ₂CH ₂F、CH ₂CH ₂ Cl, etc.), polysubstituted (CHFCH ₂F、CH ₂CHF ₂、CHFCH ₂Cl、CH ₂CHCl ₂, etc.), or fully substituted (CF ₂CF ₃、CF ₂CCl ₃、CCl ₂CCl ₃, etc.). It will be appreciated by those skilled in the art that for any group comprising one or more substituents, no substitution or pattern of substitution is introduced that is sterically impossible and/or synthetic.

When any variable (e.g., R ^a、R ^b) occurs more than once in the composition or structure of a compound, its definition in each case is independent. For example, if one group is substituted with 2R ^b, then each R ^b has an independent option.

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

The term "alkyl" refers to a hydrocarbon group of the formula C _nH _2n+1, which may be straight or branched. The term "C ₁-C ₁₀ alkyl" is understood to mean a straight-chain or branched saturated hydrocarbon radical having 1,2,3,4, 5,6,7,8, 9 or 10 carbon atoms. Specific examples of the alkyl group include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-ethylpropyl, 1, 2-dimethylpropyl, neopentyl, 1-dimethylpropyl, and 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3-dimethylbutyl, 2-dimethylbutyl, 1-dimethylbutyl, 2, 3-dimethylbutyl, 1, 3-dimethylbutyl, or 1, 2-dimethylbutyl, and the like; the term "C ₁-C ₆ alkyl" is understood to mean an alkyl group having 1 to 6 carbon atoms, specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, and the like. The term "C ₁-C ₃ alkyl" is understood to mean a straight-chain or branched saturated alkyl group having 1 to 3 carbon atoms. The "C ₁-C ₁₀ alkyl" may include the ranges of "C ₁-C ₆ alkyl" or "C ₁-C ₃ alkyl", etc., and the "C ₁-C ₆ alkyl" may further include "C ₁-C ₃ alkyl".

The term "alkoxy" refers to a group generated by the loss of a hydrogen atom on a hydroxyl group of a straight or branched chain alcohol, and is understood to be "alkyloxy" or "alkyl-O-". The term "C ₁-C ₁₀ alkoxy" may be understood as "C ₁-C ₁₀ alkyloxy" or "C ₁-C ₁₀ alkyl-O-"; the term "C ₁-C ₆ alkoxy" is understood to mean "C ₁-C ₆ alkyloxy" or "C ₁-C ₆ alkyl-O-". The "C ₁-C ₁₀ alkoxy" may include the ranges of "C ₁-C ₆ alkoxy" and "C ₁-C ₃ alkoxy", etc., and the "C ₁-C ₆ alkoxy" may further include "C ₁-C ₃ alkoxy".

The term "cycloalkyl" refers to a carbocycle that is fully saturated and exists as a single ring, fused ring, bridged ring, or spiro ring, etc. Unless otherwise indicated, the carbocycle is typically a 3 to 10 membered ring. The term "C ₃-C ₁₀ cycloalkyl" is understood to mean a saturated monocyclic, fused, spiro or bridged ring having 3 to 10 carbon atoms. Specific examples of the cycloalkyl group include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl (bicyclo [2.2.1] heptyl), bicyclo [2.2.2] octyl, adamantyl, spiro [4.5] decyl, and the like. The term "C ₃-C ₁₀ cycloalkyl" may include "C ₃-C ₆ cycloalkyl" and the term "C ₃-C ₆ cycloalkyl" is understood to mean a saturated mono-or bicyclic hydrocarbon ring having 3 to 6 carbon atoms, specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

The term "C ₃-C ₁₀ cycloalkyloxy" may be understood as "C ₃-C ₁₀ cycloalkyl-O-", preferably "C ₃-C ₁₀ cycloalkyloxy" may comprise "C ₃-C ₆ cycloalkyloxy".

The term "heterocyclyl" refers to a fully saturated or partially saturated (not aromatic in nature as a whole) monocyclic, fused, spiro, or bridged ring group containing 1 to 5 heteroatoms or groups of heteroatoms (i.e., groups of heteroatoms) in the ring atoms, including but not limited to nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), boron (B), -S (=o) ₂-、-S(＝O)-、-P(＝O) ₂ -, -P (=o) -, -NH-, -S (=o) (=nh) -, -C (=o) NH-, or-NHC (=o) NH-, or the like. The term "3-10 membered heterocyclic group" means a heterocyclic group having 3,4,5, 6,7,8, 9 or 10 ring atoms and containing 1 to 5 heteroatoms or groups of heteroatoms independently selected from the group consisting of the above. In particular, the heterocyclic groups may include, but are not limited to: specific examples of 4-membered heterocyclyl groups include, but are not limited to, azetidinyl or oxetanyl; specific examples of 5-membered heterocyclyl groups include, but are not limited to, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4, 5-dihydro-oxazolyl, or 2, 5-dihydro-1H-pyrrolyl; specific examples of 6 membered heterocyclyl groups include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, tetrahydropyridinyl, or 4H- [1,3,4] thiadiazinyl; specific examples of 7-membered heterocyclyl groups include, but are not limited to, diazepinyl. The heterocyclic group may also be a bicyclic group, wherein specific examples of 5,5 membered bicyclic groups include, but are not limited to, hexahydrocyclopenta [ c ] pyrrol-2 (1H) -yl; specific examples of 5,6 membered bicyclo groups include, but are 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. Although some bicyclic heterocycles in this disclosure contain one benzene ring or one heteroaryl ring in part, the heterocyclic group as a whole is not aromatic.

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

The term "treating" means administering a compound or formulation of the application to prevent, ameliorate or eliminate a disease or one or more symptoms associated with the disease, and includes:

(i) Preventing the occurrence of a disease or disease state in a mammal, particularly when such mammal is susceptible to the disease state, but has not been diagnosed as having the disease state;

(ii) Inhibiting a disease or disease state, i.e., inhibiting its progression;

(iii) The disease or condition is alleviated, even if the disease or condition subsides.

The term "therapeutically effective amount" means an amount of a compound of the present disclosure that (i) treats a particular disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein. The amount of a compound of the present disclosure that constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and severity thereof, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one of ordinary skill in the art based on his own knowledge and the present disclosure.

The terms "subject" or "patient" are used interchangeably herein. In some embodiments, the term "subject" or "patient" is a mammal. In some embodiments, the subject or patient is a mouse. In some embodiments, the subject or patient is a human.

The term "administering" means physically introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Routes of administration of SERD and CDK4/6 inhibitors include, but are not limited to, oral, parenteral, intravenous, transdermal, sublingual, intramuscular, and subcutaneous administration. In some particular embodiments, the SERD and CDK4/6 inhibitors are administered orally.

In the pharmaceutical combinations or combination products of the present disclosure, the SERD and CDK4/6 inhibitors may be in separate or single formulations. When the SERD and CDK4/6 inhibitors are in separate formulations, the SERD and CDK4/6 inhibitors may be administered simultaneously, separately or sequentially.

The term "pharmaceutically acceptable excipients" refers to those excipients which do not significantly stimulate the organism and which do not impair the biological activity and properties of the active compound. Suitable excipients are well known to the person skilled in the art, such as carbohydrates, waxes, water soluble and/or water swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like.

The words "comprise", "comprising" or "includes" and variations thereof such as include or comprise are to be interpreted in an open, non-exclusive sense, i.e. "including but not limited to".

The application also includes isotopically-labeled compounds of the application which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic weight or mass number different from the atomic weight or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as ²H、 ³H、 ¹¹C、 ¹³C、 ¹⁴C、 ¹³N、 ¹⁵N、 ¹⁵O、 ¹⁷O、 ¹⁸O、 ³¹P、 ³²P、 ³⁵S、 ¹⁸F、 ¹²³I、 ¹²⁵I and ³⁶ Cl, respectively, and the like.

Certain isotopically-labeled compounds of the present application (e.g., those labeled with ³ H and ¹⁴ C) are useful in compound and/or substrate tissue distribution assays. Tritiated (i.e., ³ H) and carbon-14 (i.e., ¹⁴ C) isotopes are particularly preferred for their ease of preparation and detectability. Positron emitting isotopes such as ¹⁵O、 ¹³N、 ¹¹ C and ¹⁸ F are useful in Positron Emission Tomography (PET) studies to determine substrate occupancy. Isotopically-labeled compounds of the present application can generally be prepared by following procedures analogous to those disclosed in the schemes and/or examples below by substituting an isotopically-labeled reagent for an non-isotopically-labeled reagent.

Furthermore, substitution with heavier isotopes such as deuterium (i.e., ² H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements), and hence may be preferred in certain circumstances, wherein deuterium substitution may be partial or complete, partial deuterium substitution being that at least one hydrogen is substituted by at least one deuterium.

The pharmaceutical compositions of the present application may be prepared by combining the compounds of the present application with suitable pharmaceutically acceptable excipients, for example, in solid, semi-solid, liquid or gaseous formulations such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres, aerosols and the like.

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

The pharmaceutical compositions of the present application may be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, freeze-drying, and the like.

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

The solid oral compositions may be prepared by conventional mixing, filling or tabletting methods. For example, it can be obtained by the following method: the active compound is mixed with solid auxiliary materials, the resulting mixture is optionally milled, if desired with other suitable auxiliary materials, and the mixture is then processed to granules, giving a tablet or dragee core. Suitable excipients include, but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, and the like.

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

The present disclosure provides a pharmaceutical combination comprising at least one selective estrogen receptor down-regulator (SERD) selected from a compound of formula (K) or a pharmaceutically acceptable salt thereof, and at least one CDK4/6 inhibitor:

wherein,

X ₁、X ₂、X ₃、X ₄ is independently selected from CR ⁶ or N;

In some embodiments, R ¹、R ²、R ³、R ⁴ in the compound of formula (K) is independently selected from H, F, cl, br, I, CN or C ₁-C ₆ alkyl.

In some embodiments, R ¹、R ²、R ³、R ⁴ in the compound of formula (K) is independently selected from H, F or methyl.

In some embodiments, R ¹、R ² in the compound of formula (K) is independently selected from H, F or methyl.

In some embodiments, R ³、R ⁴ in the compound of formula (K) is independently selected from H or methyl.

In some embodiments, R ³、R ⁴ in the compound of formula (K) is independently selected from H.

In some embodiments, the structural unit in the compound of formula (K)Selected from the group consisting of

In some embodiments, R ⁵ in the compound of formula (K) is selected from CH ₂CF ₃.

In some embodiments, the compound of formula (K) or a pharmaceutically acceptable salt thereof is selected from the group consisting of compounds of formula (K-1):

Wherein ,R ¹、R ²、R ³、R ⁴、R ⁵、X ₁、X ₂、X ₃、X ₄ is as defined above.

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

In some embodiments, the compound of formula (K) or a pharmaceutically acceptable salt thereof is selected from the group consisting of compounds of formula (I):

In some embodiments, the CDK4/6 inhibitor is selected from the group consisting of abbe-cili (abemaciclib), rebaudinib (ribociclib), piperaquine Bai Xili 、alvociclib、lerociclib、trilaciclib、voruciclib、AT-7519、FLX-925、INOC-005、BPI-1178、PD-0183812、NSC-625987、CGP-82996、PD-171851、SHR-6390、BPI-16350,, and pharmaceutically acceptable salts of the foregoing.

In some embodiments, the CDK4/6 inhibitor is selected from piperaquine Bai Xi or a pharmaceutically acceptable salt thereof.

Further, the present disclosure provides a pharmaceutical combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and piperaquine Bai Xi or a pharmaceutically acceptable salt thereof.

The present disclosure provides a combination comprising a I-th pharmaceutical composition comprising at least one compound of formula (K) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable adjuvant, and a II-th pharmaceutical composition comprising at least one CDK4/6 inhibitor and a pharmaceutically acceptable adjuvant.

In one embodiment of the present disclosure, the compound of formula (K) or a pharmaceutically acceptable salt thereof in the I-th pharmaceutical composition is selected from the group consisting of compounds of formula (I) or a pharmaceutically acceptable salt thereof.

In one embodiment of the present disclosure, the CDK4/6 inhibitor in the II pharmaceutical composition is selected from piperylene Bai Xi or a pharmaceutically acceptable salt thereof.

Further, the present disclosure provides a combination comprising a first pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable adjuvant, and a second pharmaceutical composition comprising piper Bai Xi or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable adjuvant.

The present disclosure also provides a kit comprising:

The present disclosure also provides a pharmaceutical composition comprising any of the above pharmaceutical combinations, and at least one pharmaceutically acceptable excipient.

In a preferred embodiment of the present disclosure, the pharmaceutical composition comprises:

the compound shown in the formula (I) or pharmaceutically acceptable salt thereof;

Bai Xi li or a pharmaceutically acceptable salt thereof;

And pharmaceutically acceptable excipients.

The present disclosure relates to the use of any one of the above pharmaceutical combinations, combination products or pharmaceutical compositions for the preparation of an antitumor drug.

The present disclosure relates to the use of any one of the above pharmaceutical combinations, combination products or pharmaceutical compositions for anti-tumor.

The present disclosure relates to any one of the above pharmaceutical combinations, combinations or pharmaceutical compositions for use in anti-tumor.

The present disclosure relates to an anti-tumor method comprising administering to a patient in need thereof a therapeutically effective amount of any one of the above pharmaceutical combinations, combinations or pharmaceutical compositions.

The disclosure also relates to the use of a compound of formula (K) or a pharmaceutically acceptable salt thereof in combination with a CDK4/6 inhibitor in the manufacture of an antitumor medicament.

Further, the present disclosure relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with a CDK4/6 inhibitor for the preparation of an antitumor drug; in one embodiment of the present disclosure, the CDK4/6 inhibitor is piperaquine Bai Xi or a pharmaceutically acceptable salt thereof.

In one embodiment of the present disclosure, the tumor is breast cancer.

In one embodiment of the present disclosure, the tumor is ER positive breast cancer.

In one embodiment of the present disclosure, the tumor is ER positive brain metastasis breast cancer.

In one embodiment of the present disclosure, the tumor is ER-positive, HER-2 negative locally advanced or metastatic breast cancer.

In some embodiments of the present disclosure, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered at a daily dose of about 1mg to 1000mg (as the free base).

In some embodiments of the present disclosure, the piperaquine Bai Xi or pharmaceutically acceptable salt thereof is administered in a daily dose of about 50mg to 500mg (as free base).

The SERD compounds disclosed herein have good in vivo and in vitro antitumor activity and drug resistance. In vivo experiments show that the SERD compound can obviously inhibit the tumor growth of an ER-positive breast cancer mouse model and obviously improve the survival time of the ER-positive breast cancer brain in-situ model mouse. In addition, the SERD compounds herein have one or more of the following technical benefits: has high bioavailability, strong ER degradation capability, can be orally taken, and has high blood brain barrier passing capability. The SERD compounds herein thus have the potential to be effective in the treatment of ER-positive breast cancers (particularly ER-positive breast cancer brain metastases).

The combination of a SERD compound herein with a CDK4/6 inhibitor (e.g., piper Bai Xili) results in better efficacy in reducing tumor growth or even eliminating tumors, exhibiting superior anti-tumor synergy relative to either drug administered alone in this combination.

The chemical reactions of the embodiments of the present disclosure are accomplished in a suitable solvent that is compatible with the chemical changes of the present disclosure and the reagents and materials needed. Modifications or choices of synthesis steps or reaction schemes based on the existing embodiments are sometimes required by those skilled in the art in order to obtain the compounds of the present disclosure.

The compounds of the present disclosure may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth herein, embodiments formed by combining them with other chemical synthetic methods, and equivalent alternatives well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present disclosure.

Examples

The present disclosure is described in detail below by way of examples, but is not meant to be limiting in any way. Having described the disclosure in detail, specific embodiments thereof are also disclosed, it will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments without departing from the spirit and scope of the disclosure. All reagents used herein are commercially available and can be used without further purification.

Unless otherwise indicated, the ratio of the mixed solvent is a volume mixing ratio.

Unless otherwise indicated,% refers to weight percent wt%.

The compounds being obtained by hand or by handSoftware naming, commercial compounds are referred to by vendor catalog names.

The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) and/or Mass Spectrometry (MS). The unit of NMR shift was 10 ^-6 (ppm). The solvent for NMR measurement is deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is Tetramethylsilane (TMS).

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

The synthesis method comprises the following steps:

step 1: synthesis of tert-butyl (1- (3-fluoropropyl) pyrrolidin-3-yl) carbamate

Tert-butylpyrrolidin-3-ylcarbamate (1.00 g,5.37 mmol) was dissolved in tetrahydrofuran (10 mL) and sodium hydroxide solution (5 mol. L ^-1, 2.15 mL) and 1-iodo-3-fluoropropane (1.06 g,5.64 mmol) were added. The reaction mixture was stirred at 25℃for 16 hours. After the TLC detection of the completion of the reaction of the starting materials, the reaction solution was diluted with ethyl acetate, washed with a saturated ammonium chloride solution, and the aqueous phase and the organic phase were collected, respectively. After the aqueous phase was extracted three times with ethyl acetate (50 mL), all organic phases were combined, dried over sodium sulfate, and 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).

¹H NMR(400MHz,METHANOL-d ₄)δ4.57(t,J＝5.77Hz,1H),4.45(t,J＝5.77Hz,1H),4.11(br d,J＝7.78Hz,1H),3.05-2.97(m,1H),2.93-2.81(m,1H),2.80-2.69(m,3H),2.66-2.56(m,1H),2.30-2.20(m,1H),2.04-1.87(m,2H),1.78-1.68(m,1H),1.51-1.38(m,9H).

Step 2: synthesis of 1- (3-fluoropropyl) pyrrolidin-3-amine hydrochloride

Tert-butyl (1- (3-fluoropropyl) pyrrolidin-3-yl) carbamate (0.81 g,3.12 mmol) was dissolved in 1, 4-dioxane (9 mL), then 1, 4-dioxane hydrochloride solution (4 moL L ^-1, 9 mL) was added and the reaction solution was a yellow transparent solution. The reaction solution was stirred at 25℃for 3 hours. After the completion of the reaction of the starting materials by TLC, the reaction solution was concentrated under reduced pressure to dryness to give the compound 1- (3-fluoropropyl) pyrrolidin-3-amine hydrochloride (0.71 g).

¹H NMR(400MHz,METHANOL-d ₄)δ4.68(t,J＝5.52Hz,1H),4.56(s,1H),4.30-3.79(m,3H),3.68(s,1H),3.48(br s,2H),3.31-3.21(m,1H),2.82-2.46(m,1H),2.32-2.14(m,3H).

Step 3: synthesis of (R) -1- (1H-indol-3-yl) -N- (2, 2-trifluoroethyl) propan-2-amine

(2R) -1- (1H-indol-3-yl) propan-2-amine (600 mg,3.44 mmol) and N, N-diisopropylethylamine (445.05 mg,3.44 mmol) were dissolved in 1, 4-dioxane (10 mL), trifluoroethyl triflate (1.20 g,5.17 mmol) dissolved in 1, 4-dioxane (5 mL) was added at 25℃and the reaction was stirred at 75℃for 16 hours. The reaction solution was concentrated to dryness under reduced pressure, and 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-trifluoroethyl) propan-2-amine (0.69 g).

MS m/z(ESI):257.2[M+H] ⁺

¹H NMR(400MHz,METHANOL-d ₄)δ7.56-7.54(d,J＝8.0Hz,1H),7.36-7.34(d,J＝8.0Hz,1H),7.12-7.08(m,2H),7.03-7.00(m,1H),3.26-3.23(m,2H),3.12-3.10(m,1H),2.93-2.88(m,1H),2.80-2.78(m,1H),1.11(d,J＝6.0Hz,3H).

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

(R) -1- (1H-indol-3-yl) -N- (2, 2-trifluoroethyl) propan-2-amine (120.00 mg, 468.26. Mu. Mol) was dissolved in toluene (2 mL), and 5-bromo-pyridine-2-carbaldehyde (87.10 mg, 468.26. Mu. Mol) and acetic acid (562.40 mg,9.37 mmol) were added thereto, and the reaction solution was a yellow transparent solutionStirring is carried out at 90℃for 10 hours. After completion of LCMS detection reaction, 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 (1 s,3 r) -1- (5-bromopyridin-2-yl) -3-methyl-2- (2, 2-trifluoroethyl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole (110.00 mg).

MS m/z(ESI):424.1,426.1[M+H] ⁺

¹H NMR(400MHz,METHANOL-d ₄)δ8.59(s,1H),8.01-7.94(m,1H),7.54(d,J＝8.53Hz,1H),7.46(d,J＝7.78Hz,1H),7.30(d,J＝8.03Hz,1H),7.08(d,J＝7.59Hz,1H),7.03-6.96(m,1H),5.08(s,1H),3.58-3.46(m,1H),3.38-3.34(m,1H),3.13-2.99-(m,1H),2.80(d,J＝4.52Hz,1H),2.70-2.61(m,1H),1.26(d,J＝6.78Hz,3H).

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

(1S, 3R) -1- (5-bromopyridin-2-yl) -3-methyl-2- (2, 2-trifluoroethyl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole (110.00 mg, 259.28. Mu. Mol) was dissolved in tetrahydrofuran (2 mL), 1- (3-fluoropropyl) pyrrolidin-3-amine hydrochloride (68.18 mg, 311.13. Mu. Mol), sodium t-butoxide (149.50 mg,1.56 mmol) and methanesulfonic acid (2-dicyclohexylphosphine) -3, 6-dimethoxy-2, 4, 6-triisopropyl-1, 1 '-biphenyl) (2-amino-1, 1' -biphenyl-2-yl) palladium (II) (23.50 mg, 25.93. Mu. Mol) were added, and the reaction solution was a brown turbid solution under nitrogen atmosphere. The reaction solution was stirred at 80℃for 4 hours. After completion of LCMS detection reaction, 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 C column, 3 μm silica, 30mm diameter, 75mm length); purification (using a decreasing polarity mixture of water (containing 0.225% formic acid) and acetonitrile as eluent) afforded the compound N- (1- (3-fluoropropyl) pyrrolidin-3-yl) -6- ((1 s,3 r) -3-methyl-2- (2, 2-trifluoroethyl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indol-1-yl) pyridin-3-amine (22.23 mg).

MS m/z(ESI):490.2[M+H] ⁺

¹H NMR(400MHz,METHANOL-d ₄)δ7.88(d,J＝2.76Hz,1H),7.46(d,J＝7.78Hz,1H),7.25(dd,J＝7.91,3.89Hz,2H),7.09-6.96(m,3H),4.97(s,1H),4.60(t,J＝5.65Hz,1H),4.48(t,J＝5.65Hz,1H),4.15(br s,1H),3.52-3.36(m,3H),3.25-3.14(m, 1H),3.09-2.88(m,6H),2.68(s,1H),2.51-2.39(m,1H),2.11-1.85(m,3H),1.20(d,J＝6.53Hz,3H).

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

Example 3-1: synthesis method 1 of N- (S) (1- (3-fluoropropyl) pyrrolidin-3-yl) -6- ((1S, 3R) -3-methyl-2- (2, 2-trifluoroethyl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indol-1-yl) pyridin-3-amine (compound of formula (I))

Racemate N- (1- (3-fluoropropyl) pyrrolidin-3-yl) -6- ((1S, 3R) -3-methyl-2- (2, 2-trifluoroethyl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indol-1-yl) pyridin-3-amine (80.00 mg, 153.61. Mu. Mol) was purified by chiral separation (DAICEL CHIRALPAK AY-H column, 5 μm silica, 30mm diameter, 250mm length, using isopropanol (0.1% aqueous ammonia) and a mixture of decreasing polarity of water as eluent) and preparative liquid chromatography purification (Phenomenex Gemini C column, 3 μm silica, 30mm diameter, 75mm length, using a mixture of decreasing polarity of water (0.05% aqueous ammonia) and acetonitrile as eluent) to give N- (R) (1- (3-fluoropropyl) pyrrolidin-3-yl) -6- ((1S, 3R) -3-methyl-2- (2, 2-trifluoroethyl) -2,3, 4-tetrahydro-1-pyrido [ 3-H-1-yl ] pyridin-3-yl) 1- (2, 3-trifluoro-3, 4-1-2, 4-pyrido-3-H-yl) amine, the retention time was 2.627 minutes) and N- (S) (1- (3-fluoropropyl) pyrrolidin-3-yl) -6- ((1S, 3 r) -3-methyl-2- (2, 2-trifluoroethyl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indol-1-yl) pyridin-3-amine (10.85 mg, retention time 2.817 minutes).

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

MS m/z(ESI):490.1[M+H] ⁺

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

n- (S) (1- (3-fluoropropyl) pyrrolidin-3-yl) -6- ((1S, 3 r) -3-methyl-2- (2, 2-trifluoroethyl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indol-1-yl) pyridin-3-amine (compound of formula (I):

MS m/z(ESI):489.25,490.1[M+H] ⁺

¹H NMR(400MHz,METHANOL-d ₄)δ7.90-7.88(m,1H),7.48-7.43(m,1H),7.29-7.23(m,2H),7.09-7.03(m,2H),7.03-6.97(m,1H),4.99-4.97(m,1H),4.63-4.59(m,1H),4.52-4.47-(m,1H),4.24-4.16(m,1H),3.52-3.36(m,4H),3.15(br s,4H),3.05-2.99(m,1H),2.96-2.88(m,1H),2.68(s,1H),2.57-2.43(m,1H),2.12-1.94(m,3H),1.20(d,J＝6.53Hz,3H).

Example 3-2: synthesis method 2 of compound of formula (I)

Step 1: synthesis of (S) -tert-butyl (1- (3-fluoropropyl) pyrrolidin-3-yl) aminomethyl ester

(S) -tert-butylpyrrolidin-3-ylaminomethyl ester (500.00 mg,2.68 mmol) is dissolved in tetrahydrofuran (10 mL) and sodium hydroxide solution (5 mol. Times.L ^-1, 1.07 mL) and 1-iodo-3-fluoropropane (529.88 mg,2.82 mmol) are added. The reaction mixture was stirred at 25℃for 16 hours. After the completion of the reaction of the starting materials by TLC, the reaction solution was diluted with ethyl acetate (50 mL), and then washed with a saturated ammonium chloride solution (10 mL), and the aqueous phase and the organic phase were collected, respectively. After the aqueous phase was extracted three times with ethyl acetate (20 mL), all organic phases were combined, dried over sodium sulfate, and 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-d ₄)δ4.58-4.53(m,1H),4.46-4.40(m,1H),4.14-4.04(m,1H),2.93-2.85(m,1H),2.77-2.67(m,1H),2.61(dd,J＝7.78,5.52Hz,3H),2.47-2.40(m,1H),2.29-2.17(m,1H),1.99-1.82(m,2H),1.71-1.61(m,1H),1.45(s,9H).

Step 2: synthesis of (S) -1- (3-fluoropropyl) pyrrolidin-3-amine hydrochloride

(S) -tert-butyl (1- (3-fluoropropyl) pyrrolidin-3-yl) aminomethyl ester (480.00 mg,1.93 mmol) was dissolved in 1, 4-dioxane (3 mL) and then 1, 4-dioxane hydrochloride solution (4 moL L ^-1, 4.94 mL) was added and the reaction solution was a yellow transparent solution. The reaction solution was stirred at 25℃for 3 hours. After the completion of the reaction of the starting materials by TLC, the reaction solution was concentrated under reduced pressure to give the compound (S) -1- (3-fluoropropyl) pyrrolidin-3-amine hydrochloride (450.00 mg).

¹H NMR(400MHz,DMSO-d ₆)δ8.80-8.42(m,3H),4.62(s,1H),4.51(s,1H),4.12-3.45(m,3H),3.17(br s,3H),2.35-1.99(m,4H).

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

(1S, 3R) -1- (5-bromopyridin-2-yl) -3-methyl-2- (2, 2-trifluoroethyl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole (140.00 mg, 263.99. Mu. Mol) was dissolved in tetrahydrofuran (3 mL), and (S) -1- (3-fluoropropyl) pyrrolidin-3-amine hydrochloride (86.77 mg, 316.79. Mu. Mol), sodium t-butoxide (152.22 mg,1.58 mmol) and methanesulfonic acid (2-dicyclohexylphosphine) -3, 6-dimethoxy-2, 4, 6-triisopropyl-1, 1 '-biphenyl) (2-amino-1, 1' -biphenyl-2-yl) palladium (II) (23.93 mg, 26.40. Mu. Mol) were added to stir the reaction mixture under nitrogen atmosphere at 80℃for 4 hours. After completion of LCMS detection reaction, 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 C column, 3um silica, 30mm diameter, 75mm length); purification (using a decreasing polarity mixture of water (containing 0.225% formic acid) and acetonitrile as eluent) afforded the compound N- ((S) -1- (3-fluoropropyl) pyrrolidin-3-yl) -6- (((1S, 3 r) -3-methyl-2- (2, 2-trifluoroethyl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indol-1-yl) pyridin-3-amine (37.79 mg).

MS m/z(ESI):365.1[M+H] ⁺；

¹H NMR(400MHz,METHANOL-d ₄)δ7.89(d,J＝2.76Hz,1H),7.46(d,J＝7.78Hz,1H),7.25(d,J＝8.53Hz,2H),7.09-7.03(m,2H),7.02-6.97(m,1H),4.98(s,1H),4.60(t,J＝5.65Hz,1H),4.49(t,J＝5.65Hz,1H),4.18(br s,1H),3.51-3.35(m,4H), 3.14-2.99(m,5H),2.92(dd,J＝15.18,4.89Hz,1H),2.65(dd,J＝16.06,6.78Hz,1H),2.53-2.42(m,1H),2.12-1.92(m,3H),1.20(d,J＝6.78Hz,3H).

Absolute configuration identification (two-dimensional nuclear magnetism) of compounds of formula (I):

The NOESY pattern (FIG. 1) shows that the methyl hydrogen at the 3-position and the hydrogen at the 1-position of the compound of the formula (I) have obvious NOE effect, which proves that the two are on the same side, the relative configuration of the pyridyl group at the 1-position and the methyl group at the 3-position on the 6-membered piperidine ring is trans, the absolute configuration of the carbon atom at the 3-position is known as R, and therefore, the absolute configuration of the carbon atom at the 1-position is S.

Examples 3-3: preparation of succinate salt of Compound of formula (I)

5.0G of the compound of formula (I) in free form, 100ml of isopropyl ether are placed in a 250ml single-necked flask and stirred at room temperature until the solid is completely dissolved. 1.21g of succinic acid was added, and the reaction solution was stirred overnight at room temperature, filtered off with suction, and dried under vacuum at room temperature for 3 hours to give 5.4g of solid.

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

The synthesis method comprises the following steps:

Step 1: synthesis of (S) -tert-butyl (1- (3-fluoropropyl) pyrrolidin-3-yl) carbamate

(S) -tert-butylpyrrolidin-3-ylcarbamate (500.00 mg,2.68 mmol) was dissolved in tetrahydrofuran (10 mL), and sodium hydroxide solution (5 mol. L ^-1, 1.07 mL) and 1-iodo-3-fluoropropane (529.88 mg,2.82 mmol) were added. The reaction mixture was stirred at 25℃for 16 hours. After the completion of the reaction of the starting materials by TLC, the reaction solution was diluted with ethyl acetate (50 mL), and then washed with a saturated ammonium chloride solution (10 mL), and the aqueous phase and the organic phase were collected, respectively. After the aqueous phase was extracted three times with ethyl acetate (20 mL), all organic phases were combined, dried over sodium sulfate, and 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).

Step 2: synthesis of (S) -1- (3-fluoropropyl) pyrrolidin-3-amine hydrochloride

(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 hydrochloric acid-1, 4-dioxane solution (4 moL L ^-1, 4.94 mL) was added and the reaction solution was a yellow transparent solution. The reaction solution was stirred at 25℃for 3 hours. After the completion of the reaction of the starting materials by TLC, the reaction solution was concentrated under reduced pressure to give the compound (S) -1- (3-fluoropropyl) pyrrolidin-3-amine hydrochloride (450.00 mg).

Step 3: synthesis of (1S, 3R) -1- (4-bromo-2, 6-difluorophenyl) -3-methyl-2- (2, 2-trifluoroethyl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole

(R) -1- (1H-indol-3-yl) -N- (2, 2-trifluoroethyl) propan-2-amine (890mg, 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℃for 6 hours. The reaction solution was concentrated to dryness under reduced pressure, and then purified by column chromatography (silica, petroleum ether/ethyl acetate=20/1) to give the product (1 s,3 r) -1- (4-bromo-2, 6-difluorophenyl) -3-methyl-2- (2, 2-trifluoroethyl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole (850 mg).

¹H NMR(400MHz,METHANOL-d ₄)δ7.45-7.43(d,J＝7.60Hz,1H),7.24-7.20(m,3H),7.05-6.99(m,2H),5.36(s,1H),3.57-3.54(m,1H),3.46-3.40(m,1H),3.01-2.95(m,2H),2.70-2.65(m,1H),1.20(d,J＝6.4Hz,3H).

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

(1S, 3R) -1- (4-bromo-2, 6-difluorophenyl) -3-methyl-2- (2, 2-trifluoroethyl) -2,3,4, 9-tetrahydro-1H-pyridinyl [3,4-b ] indole (80.00 mg, 174.20. Mu. Mol) was dissolved in tetrahydrofuran (2 mL), and (S) -1- (3-fluoropropyl) pyrrolidin-3-amine hydrochloride (38.20 mg, 209.04. Mu. Mol) and sodium t-butoxide (100.45 mg,1.05 mmol) were added and after stirring uniformly, tris (dibenzylideneacetone) dipalladium (31.90 mg, 34.84. Mu. Mol) and (. + -.) -2, 2-bis (diphenylphosphino) -1,1' -binaphthyl (54.23 mg, 87.10. Mu. Mol) were added under nitrogen. The reaction mixture was stirred at 80℃for 4 hours. After LCMS detection of complete reaction of the starting materials, the reaction solution was filtered, the filter cake was rinsed with tetrahydrofuran, the filtrate was concentrated to dryness under reduced pressure, and purified by preparative liquid chromatography (Phenomenex Gemini C column, 7 μm silica, 50mm diameter, 250mm length, using a decreasing polarity mixture of water (containing 0.225% formic acid) and acetonitrile as eluent) to give the compound (S) -N- (3, 5-difluoro-4- ((1S, 3 r) -3-methyl-2- (2, 2-trifluoroethyl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indol-1-yl) phenyl) -1- (3-fluoropropyl) pyrrolidin-3-amine (4.69 mg).

MS m/z(ESI):525.2[M+H] ⁺

¹H NMR(400MHz,METHANOL-d ₄)δ8.49(s,1H),7.42(d,J＝7.3Hz,1H),7.21(d,J＝7.8Hz,1H),7.08-6.94(m,2H),6.19(d,J＝11.5Hz,2H),5.24(s,1H),4.60(t,J＝5.6Hz,1H),4.48(t,J＝5.6Hz,1H),4.11(br s,1H),3.62-3.53(m,1H),3.21(br s,1H),3.09-2.94(m,6H),2.63(dd,J＝15.3,4.3Hz,1H),2.51-2.38(m,1H),2.12-1.99(m,2H),1.96-1.85(m,1H),1.39-1.29(m,2H),1.19(d,J＝6.5Hz,3H)

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

The synthesis method comprises the following steps:

Step 1: synthesis of tert-butyl (trans-4-fluoro-1- (3-fluoropropyl) pyrrolidin-3-yl) carbamate

1-Fluoro-3-iodopropane (151.86 mg, 807.87. Mu. Mol) and tert-butyl (trans-4-fluoropyrrolidin-3-yl) carbamate (150 mg, 734.43. Mu. Mol) were dissolved in acetonitrile (4 mL), potassium carbonate (203.00 mg,1.47 mmol) was added at 25℃and the reaction solution was stirred at 60℃for 13 hours. The reaction solution was cooled to 25 ℃, filtered, and concentrated to dryness under reduced pressure. 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] ⁺

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

Step 2: synthesis of trans-4-fluoro-1- (3-fluoropropyl) pyrrolidin-3-amine hydrochloride

Tert-butyl (trans-4-fluoro-1- (3-fluoropropyl) pyrrolidin-3-yl) carbamate (150 mg, 567.51. Mu. Mol) was dissolved in 1, 4-dioxane (2 mL), 4M hydrochloric acid-1, 4-dioxane (2.13 mL) was added, and the reaction mixture was stirred at 25℃for 13 hours. The reaction solution was concentrated to give the product trans-4-fluoro-1- (3-fluoropropyl) pyrrolidin-3-amine hydrochloride (0.12 g).

MS m/z(ESI):165.2[M+H] ⁺

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

(1S, 3R) -1- (4-bromo-2, 6-difluorophenyl) -3-methyl-2- (2, 2-trifluoroethyl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole (140 mg, 304.84. Mu. Mol) and trans-4-fluoro-1- (3-fluoropropyl) pyrrolidin-3-amine hydrochloride (86.74 mg, 365.81. Mu. Mol) were dissolved in t-amyl alcohol (5 mL), methanesulfonic acid (2-dicyclohexylphosphino-3, 6-dimethoxy-2, 4, 6-triisopropyl-1, 1 '-biphenyl) (2-amino-1, 1' -biphenyl-2-yl) palladium (II) (25.50 mg, 30.48. Mu. Mol) and cesium carbonate (595.95 mg,1.83 mmol) were added thereto, and after three times of nitrogen substitution, the reaction solution was stirred at 120℃for 13 hours. The reaction solution was cooled to room temperature and poured into water (10 mL) and stirred for 10 minutes, ethyl acetate (20 mL) was extracted 2 times, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated to dryness under reduced pressure, and then purified by column chromatography (silica, petroleum ether/ethyl acetate=2/1) and preparative liquid chromatography (PhenomenexGemini-NX column: 3 μm silica, 30mm diameter, 75mm length; using a mixture of water (containing 0.05% aqueous ammonia) and acetonitrile with decreasing polarity as eluent) to give the product trans-N- (3, 5-difluoro-4- ((1 s,3 r) -3-methyl-2- (2, 2-trifluoroethyl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indol-1-yl) phenyl) -4-fluoro-1- (3-fluoropropyl) pyrrolidin-3-amine (27.5 mg).

MS m/z(ESI):543.1[M+H] ⁺

¹H NMR(400MHz,METHANOL-d ₄)δ7.42(d,J＝7.5Hz,1H),7.22(d,J＝7.5Hz,1H),7.09-6.93(m,2H),6.30-6.16(m,2H),5.25(s,1H),4.58(t,J＝5.8Hz,1H),4.46(t,J＝5.8Hz,1H),4.11-3.87(m,1H),3.67-3.53(m,1H),3.43-3.34(m,3H),3.19-2.92(m,3H),2.81-2.55(m,4H),2.29(dd,J＝6.9,9.7Hz,1H),2.02-1.83(m,2H),1.19(d,J＝6.4Hz,3H).

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

The synthesis method comprises the following steps:

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

Tert-butyl N- [ trans-4-methylpyrrolidin-3-yl ] carbamate (80 mg, 399.45. Mu. Mol) and potassium carbonate (110.41 mg, 798.89. Mu. Mol) were dissolved in acetonitrile (8 mL), and then 1-fluoro-3-iodo-propane (90.11 mg, 479.34. Mu. Mol) was added thereto, and the reaction mixture was heated to 50℃and stirred for 16 hours. After completion of LCMS monitoring, the reaction was filtered and the filtrate was concentrated to dryness and purified by column chromatography (silica, ethyl acetate/methanol=5/1) to give the compound tert-butyl N- [ trans-1- (3-fluoropropyl) -4-methyl-pyrrolidin-3-yl ] carbamate (85 mg).

MS m/z(ESI):261.1[M+H] ⁺

¹H NMR(400MHz,METHANOL-d ₄)δ4.66(br d,J＝2.4Hz,1H),4.61-4.42(m,2H),4.05-3.93(m,1H),3.71-3.56(m,2H),3.17(br d,J＝9.4Hz,1H),2.90(br s,2H),2.54(br s,1H),2.19(br d,J＝5.2Hz,2H),2.07-1.89(m,1H),1.47(s,9H),1.24-1.11(m,3H).

Step 2: synthesis of trans-1- (3-fluoropropyl) -4-methyl-pyrrolidin-3-amine hydrochloride

Tert-butyl N- [ trans-1- (3-fluoropropyl) -4-methyl-pyrrolidin-3-yl ] carbamate (80 mg, 307.28. Mu. Mol) was dissolved in dioxane (2 mL), then 4M hydrochloric acid-1, 4-dioxane (1.54 mL) was added, and the reaction was stirred at room temperature overnight. The reaction solution was concentrated to dryness to give the compound trans-1- (3-fluoropropyl) -4-methyl-pyrrolidin-3-amine hydrochloride (70 mg).

¹H NMR(400MHz,METHANOL-d ₄)δ4.73-4.64(m,1H),4.61-4.50(m,1H),4.23-4.06(m,1H),4.02-3.63(m,5H),3.54-3.42(m,1H),3.01-2.58(m,1H),2.32-2.12(m,2H),1.37-1.27(m,3H).

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

Trans-1- (3-fluoropropyl) -4-methyl-pyrrolidin-3-amine hydrochloride (30 mg, 128.67. Mu. Mol), (1S, 3R) -1- (4-bromo-2, 6-difluoro-phenyl) -3-methyl-2- (2, 2-trifluoroethyl) -2,3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole (76.82 mg, 167.27. Mu. Mol) and cesium carbonate (167.69 mg, 514.68. Mu. Mol) were dissolved in dioxane (8 mL), and methanesulfonic acid (2-di-tert-butylphosphine-3, 6-dimethoxy-2 ',4',6 '-triisopropyl-1, 1' -biphenyl) (2 '-amino-1, 1' -biphenyl-2-yl) palladium (II) (tBuBrettphos-Pd-G3, 5.50mg, 6.43. Mu. Mol) was then added. The reaction solution was replaced with nitrogen three times, and then heated to 120 ℃ and stirred overnight. After completion of LCMS monitoring the reaction, methanol (15 mL) was added to the reaction solution, filtered, and the filtrate concentrated and purified by column chromatography (silica, petroleum ether/ethyl acetate=2/1) and preparative liquid chromatography (Phenomenex Synergi C column: 4 μm silica, 30mm diameter, 150mm length; using a decreasing polarity mixture of water (containing 0.225% formic acid) and acetonitrile as eluent) to give the compound trans-N- [3, 5-difluoro-4- [ (1 s,3 r) -3-methyl-2- (2, 2-trifluoroethyl) -2,3,4, 9-tetrahydro-1H-pyrido [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-d ₄)δ7.42(d,J＝7.6Hz,1H),7.21(d,J＝8.8Hz,1H),7.08-6.86(m,2H),6.21(d,J＝11.6Hz,2H),5.24(s,1H),4.62-4.58(m,1H),4.52-4.45(m,1H),3.73-3.65(m,1H),3.62-3.47(m,3H),3.23-3.15(m,2H),3.08-2.95(m,2H),2.86-2.54(m,2H),2.41-2.00(m,3H),1.42-1.25(m 2H),1.23(dd,J＝3.2,Hz,6.8Hz,3H),1.19(d,J＝6.4Hz,3H).

Testing of biological Activity and related Properties

Test example 1: detection of degradation effect of SERD (surface enhanced Raman Scattering) compound on estrogen receptor in MCF7 cells

1. Purpose of experiment

The purpose of this experiment was to determine the degradation activity of the SERD compounds herein on endogenously expressed estrogen receptors within MCF7 cells and to evaluate the activity of the compounds based on DC ₅₀ and maximum degradation efficiency.

2. Experimental method

MCF7 cells (ATCC, HTB-22) were cultured in complete medium of DMEM (Gibco, 11995-065) containing 10% fetal bovine serum. On the first day of the experiment, MCF7 cells were seeded at a density of 3000 cells/well in 384 well plates, 37 ℃,5% co ₂ cell incubator. Test compounds were dissolved in DMSO, stored at 10mM, diluted with Echo 550 (labyte inc.) and added to cell culture plates, each compound treated at an initial concentration of 100nm, 3-fold gradient dilution, 9 concentration points, a blank control containing 0.5% DMSO was set, and a double-well control was set at each concentration point. The cells were incubated at 37℃in a 5% CO ₂ cell incubator for 24 hours. Adding paraformaldehyde into the cell culture solution to obtain final concentration of paraformaldehyde of about 3.7% for fixing cells, allowing the cells to act for 30 min, discarding supernatant, and adding 50 μl of PBS for washing once per well; cells were treated with PBS (0.5% v/v Tween-20) for 30 min and washed once with PBS; blocking solution (5% BSA in PBS, 0.05% Tween-20) was added and incubated for 1 hour at room temperature; adding primary antibody mixture (anti-ER monoclonal antibody, estrogen Receptor α (D8H 8) Rabbit mAb, GST, # 864S, 1:1000 dilution; anti-GAPDH monoclonal antibody, GAPDH (D4C 6R) Mouse mAb, GST, #97166S,1:2000 dilution) into the deblocking solution, and incubating for 3 hours at room temperature; wash 3 times with PBST (0.05% Tween-20 in PBS); adding detection secondary antibody (800 CW-goat anti-rabbit IgG, LI-COR, P/N:926-32211,1:1000 dilution; 680 RD-goat anti-mouse IgG, LI-COR, #925-68070,1:1000 dilution), and incubating at room temperature in the absence of light for 45 min; PBST was washed 3 times and each well fluorescent signal was read using Odyssey CLx, and Chanel (ER)/Chanel 680 (GAPDH) values were calculated. Degradation rates at each concentration point were calculated using 0.1 μm fulvestrant treated wells as 100% degradation control, and degradation activity DC ₅₀ and maximum degradation rate Imax for each compound were calculated using XlLfit analysis of the treatment data. The data analysis is shown in Table 1.

Degradation Activity of Compounds of Table 1 SERD on the intracellular estrogen receptor of MCF7

Numbering of compounds	ER level DC ₅₀(nM)	Maximum degradation rate
Compound 1	0.41	106％
Compound 2	8.5	92％
A compound of formula (I)	0.15	104％
Compound 5	0.58	80％
Compound 6	0.50	90％

Test example 2: detection of inhibition effect of SERD (surface enhanced Raman Scattering) compound on proliferation of MCF7 cells

1. Purpose of experiment

The purpose of this experiment was to determine the inhibitory effect of the SERD compounds herein on MCF7 cell proliferation in vitro, and to evaluate the activity of the compounds based on IC ₅₀ and the maximum inhibition efficiency.

2. Experimental method

MCF7 cells (ATCC, HTB-22) were cultured in complete medium of DMEM (Gibco, 11995-065) containing 10% fetal bovine serum. On the first day of the experiment, MCF7 cells were seeded at 500/well in 384-well plates using complete medium, 37 ℃,5% co ₂ cell incubator overnight. The following day, test compounds were added for drug treatment, 10mM stock concentration of compound solution was diluted with Echo550 (Labcyte Inc.) and transferred into each cell culture well, each compound was diluted at 100nM in the initial concentration of treatment in the cell, 3-fold gradient, 9 concentration points, a blank control containing 0.3% DMSO was set, and a double well control was set at each concentration point. Cell culture plates were removed on day 8 after 7 days in a 5% CO ₂ cell incubator at 37 ℃. Adding inLuminescent Cell Viability Assay (Promega, G7573), after 10 minutes of standing at room temperature, the luminescence signal value was read using a multi-label microplate reader EnVision (PerkinElmer), and the inhibitory activity IC ₅₀ of each compound was calculated from the concentration of the compound and the luminescence signal value using XLfit. The data analysis is shown in Table 2

Inhibitory Activity of Compounds of Table 2 SERD on MCF7 cell proliferation

Numbering of compounds	MCF7 Inhibition of cell proliferation IC ₅₀(nM)
Compound 1	0.58
Compound 2	15
A compound of formula (I)	0.27
Compound 4	0.23
Compound 5	2.57
Compound 6	2.39

Test example 3: inhibition of CYP2C9, CYP2D6 enzymatic Activity by SERD Compounds

The inhibition of CYP2C9, CYP2D6 enzymatic activity by the SERD compounds herein was determined using the following assay.

1. Test material and instrument

1. Human liver microsome (Corning 452117)

2.NADPH(Solarbio 705Y021)

3. The positive substrates diclofenac (Sigma SLBV 3438), dextromethorphan (TRC 3-EDO-175-1) and midazolam (CERILLIANT FE 01161704)

4. Sulfobenzopyrazoles (D. Ehrensterfer GmbH 109012), quinidine (TCI WEODL-RE) and ketoconazole (Sigma 100M 1091V), positive inhibitors

5.AB Sciex Triple Quad 5500 liquid chromatography mass spectrometer

2. Test procedure

Preparation of 1.100 mM Phosphate Buffer (PBS): 7.098g of Na ₂HPO ₄ was weighed, and 500mL of pure water was added for ultrasonic dissolution to obtain solution A. 3.400g KH ₂PO ₄ was weighed out and added with 250mL pure water for ultrasonic dissolution as solution B. Solution A was placed on a stirrer and solution B was slowly added until the pH reached 7.4 to prepare 100mM PBS buffer.

2. A10 mM NADPH solution was prepared with 100mM PBS buffer. The 10mM SERD compound stock solution was diluted with DMSO to give 200 Xconcentration of compound working solution (6000, 2000, 600, 200, 60, 20, 0. Mu.M). The positive inhibitor stock solution was diluted with DMSO to give 200 x concentration of positive inhibitor working solution (sulfabenzene, 1000, 300, 100, 30, 10, 3, 0 μm; quinidine/ketoconazole, 100, 30, 10, 3, 1, 0.3, 0 μm). Substrate working solutions (120. Mu.M diclofenac, 400. Mu.M dextromethorphan, and 200. Mu.M midazolam) were prepared at 200 Xconcentration with water, acetonitrile or acetonitrile/methanol.

3. Mu.l of a 20mg/ml liver microsome solution, 1. Mu.l of a substrate working solution, 1. Mu.l of a compound working solution and 176. Mu.l of PBS buffer were taken, mixed well and placed in a 37℃water bath for pre-incubation for 15 minutes. The positive control group was added with 1 μl of diclofenac, dextromethorphan, or midazolam working solution instead of the compound working solution. While 10mM NADPH solution was pre-incubated together in a 37℃water bath for 15 minutes. After 15 minutes, 20. Mu.l of NADPH was added to each well, the reaction was started and incubated at 37℃for 5 minutes (CYP 2C 9) or 20 minutes (CYP 2D 6). All incubation samples were double-sampled. After incubation for the corresponding time, the reaction was terminated by adding 400ul of ice methanol containing internal standard to all samples. Vortex mixing, 3220g, 4 ℃ centrifugation 40 minutes. After centrifugation, 100. Mu.L of the supernatant was transferred to a sample plate, and 100. Mu.L of ultrapure water was added and mixed for LC-MS/MS analysis.

IC ₅₀ values of SERD compounds for CYP2C9 and CYP2D6 were calculated by Excel XLfit 5.3.1.3.

Drug-drug interactions (DDI) refers to physical or chemical changes of 2 or more drugs and changes in drug effects due to these changes. Knowing the drug interactions can provide better pharmaceutical services for patients and promote reasonable medication, and maximally avoid adverse reactions. Drug interactions are dominated by metabolic interactions, which are mainly related to CYP450 enzymes involved in drug metabolism. The experimental results of table 3 demonstrate that the SERD compounds herein have poor inhibition of CYP450, suggesting that the SERD compounds herein have less potential for DDI.

IC ₅₀ values of Table 3 SERD compounds for CYP2C9 and CYP2D6

Numbering of compounds	CYP2C9(μM)	CYP2D6(μM)
A compound of formula (I)	>30	>30
Compound 4	20	27

Test example 4: plasma protein binding rate assay for SERD compounds

Human plasma protein binding is a key factor in controlling the amount of free (unbound) drug available to bind to a target, playing an important role in the observed in vivo efficacy of the drug. Thus, compounds with high free fraction (low levels of plasma protein binding) may exhibit enhanced efficacy for compounds with similar potency and exposure levels.

The protein binding rate of SERD compounds in plasma of 5 species (human, monkey, canine, rat and mouse) was determined using the following assay.

1. Test material and instrument

1. Human plasma (BioIVT), beagle plasma (BioIVT), SD rat plasma (BioIVT), CD-1 mouse plasma (BioIVT);

2.96 well equilibrium dialysis plate (HTDIALYSIS LLC, GALES FERRY, CT, HTD B), equilibrium dialysis membrane (MWCO 12-14K, # 1101);

3. The positive control compound warfarin;

ABI QTrap 5500 liquid Mass Spectrometry.

2. Test procedure

1. Preparation of buffer with concentration of 100mM sodium phosphate and 150mM NaCl: an alkaline solution having a concentration of 14.2g/L Na ₂HPO ₄ and 8.77g/L NaCl was prepared with ultrapure water, and an acidic solution having a concentration of 12.0g/L NaH ₂PO ₄ and 8.77g/L NaCl was prepared with ultrapure water. The alkaline solution was titrated with an acidic solution to a pH of 7.4 to prepare a buffer with a concentration of 100mM sodium phosphate and 150mM NaCl.

2. Preparation of a dialysis membrane: the dialysis membrane was immersed in ultrapure water for 60 minutes to separate the membrane into two pieces, then immersed in 20% ethanol for 20 minutes, and finally immersed in a buffer solution for dialysis for 20 minutes.

3. Preparation of plasma: frozen plasma was thawed quickly at room temperature, then the plasma was centrifuged at 3,220g for 10 minutes at 4 ℃ to remove the clot, and the supernatant was collected into a new centrifuge tube. The pH of the plasma was measured and recorded, and plasma with pH 7-8 was used.

4. Preparation of compound-containing plasma samples: 200. Mu.M working solution was obtained by diluting a stock of 10mM SERD compound or positive control compound with DMSO. 597. Mu.l of human, monkey, dog, rat or mouse plasma was added with 3. Mu.l of 200. Mu.M working solution of the compound to give a plasma sample having a final concentration of 1. Mu.M.

5. An equilibrium dialysis step: the dialysis device is assembled according to the instructions. On one side of the dialysis membrane 120. Mu.L of a plasma sample containing 1. Mu.M compound was added and on the other side an equal volume of dialysis fluid (phosphate buffer) was added. The test was performed with double samples. The plate was sealed and placed in an incubator, and incubated at 37℃for 6 hours with 5% CO ₂ and at about 100 rpm. After the incubation, the sealing film was removed and 50 μl was pipetted from the buffer and plasma side of each well into a different well of a new plate. To the phosphate buffer sample, 50. Mu.l of blank plasma was added, to the plasma sample, an equal volume of blank phosphate buffer was added, and then 300. Mu.l of acetonitrile containing internal standard precipitated protein was added. Vortex for 5min and centrifuge at 3,220g for 30 min at 4 ℃. 100. Mu.l of the supernatant was taken out to a sample plate, and 100. Mu.l of ultrapure water was added and mixed well for LC-MS/MS analysis.

The peak areas of the compounds on the buffer side and the plasma side were determined. The plasma protein binding rate of the compound was calculated as follows:

Percent freeness = (ratio of compound peak area to internal standard peak area _{Buffer side}/ratio of compound peak area to internal standard peak area _{Plasma side}) ×100

Binding%100-free%

All data were calculated by Microsoft Excel software. Plasma protein binding values for the obtained SERD compounds were calculated.

Protein binding values of compounds of Table 4 SERD in human, canine, rat and mouse plasma

Test example 5: apparent solubility of SERD Compounds in phosphate buffer at pH 7.4

In order for an oral compound to reach the site of action and for efficient absorption in the intestinal tract, the compound needs to be in solution, and thus a compound having high intrinsic solubility may be more suitable for pharmaceutical use.

1. Materials and reagents

The test compounds were prepared according to the methods described. Control progesterone was purchased from Sigma. phosphate buffer at pH 7.4 was prepared by the present laboratory. Acetonitrile and methanol were purchased from Fisher. Other reagents are commercially available.

1.5Ml flat bottom glass vial (BioTech Solutions); a polytetrafluoroethylene/silicone bottle stopper (BioTech Solutions); coating stirring rods with polytetrafluoroethylene; multiScreenHTS HV (0.45 μm) 96well plate filter plates (Millipore, MSHVN4510or MSHVN 4550); eppendorf Thermomixer Comfort; vacuum Manifold ORVMN96 (Orochem).

2. Experimental procedure

1) Preparation of stock solution

10MM stock solutions of the test and control progesterone were prepared with DMSO.

Apparent solubility determination step

30. Mu.L of 10mM stock solution of the test substance was added to the corresponding positions of the corresponding 96-well plates in the order given. 970. Mu.L of phosphate buffer at pH 7.4 was added to the corresponding vial of the sample plate. The experiment was double parallel. A stirring rod was added to each vial and covered with a teflon/silicone stopper. The sample pan was then placed into Eppendorf Thermomixer Comfort and oscillated at 25 degrees for 2 hours at a speed of 1100 revolutions. After 2 hours, the stopper was removed, the stirring rod was pulled away with a large magnet, and then the sample was transferred from the sample plate to the filter plate. Negative pressure was generated by a vacuum pump and the sample was filtered. Transfer 5 μl of filtrate to a new sample plate, then add 5 μl DMSO and 490 μl 50% acn (IS). H ₂ O (internal standard acetonitrile: water=1:1). Depending on the peak shape, it IS possible to dilute the sample dilution with a proportion of 50% acn (IS) H ₂ O to obtain a better peak shape. The dilution ratio may be adjusted according to the solubility of the analyte or the strength of the liquid response signal.

3) Sample analysis step

The sample plate was placed in the sample pan of the autosampler and the samples were evaluated by liquid analysis.

3. Experimental procedure

All calculations were performed by Microsoft Excel. Analysis and quantification of the sample filtrate is accomplished by qualitative and quantitative determination of standard peaks of known concentration using a liquid. The apparent solubility value of the SERD compound in phosphate buffer at pH 7.4 was calculated.

Apparent solubility values of the compounds of Table 5 SERD in phosphate buffer at pH 7.4

Numbering of compounds	Ph=7.4 apparent solubility (μm)
A compound of formula (I)	92
Compound 4	<0.3

Test example 6: whether SERD compounds have potential inhibitory effects on voltage-gated potassium channel hERG

The hERG potassium channel is critical for the normal electrical activity of the heart. Arrhythmia can be induced by blocking hERG channels with multiple drugs. This side effect is a common cause of drug failure in preclinical safety tests, and therefore minimization of hERG channel blocking activity may be an ideal property for drug candidates.

1. Materials and reagents

1. Experimental materials and instruments

2. Cell lines and cultures

HEK293 cell line (cat# K1236) stably expressing the hERG ion channel was purchased from Invitrogen corporation. The cell line was cultured in a medium containing 85% DMEM, 10% dialyzed fetal calf serum, 0.1mM non-essential amino acid solution, 100U/mL penicillin-streptomycin solution, 25mM HEPES, 5. Mu.g/mL blasticidin and 400. Mu.g/mL geneticin. When the cell density is increased to 40% -80% of the bottom area of the culture dish, digestion and passage are carried out by trypsin, and the passage is carried out three times per week. Prior to the experiment, cells were cultured in 6cm dishes at a density of 5X 10 ⁵, induced for 48 hours with the addition of 1. Mu.g/mL doxycycline, and then the cells were digested and inoculated onto slides for subsequent manual patch clamp experiments.

3. Test compound configuration

1) Test compounds were dissolved in DMSO and formulated as stock solutions at a final concentration of 10mM according to SOP-ADMET-MAN-007 standard protocol.

2) Stock solutions were diluted in DMSO at 1:3 ratio to three other intermediate concentration solutions at 3.33mM, 1.11mM and 0.37mM, respectively.

3) Before the start of the experiment, stock solutions of the test compounds and the intermediate solutions were diluted 1000-fold with extracellular fluid to give working solutions of serial concentrations of 10. Mu.M, 3.33. Mu.M, 1.11. Mu.M and 0.37. Mu.M, while 10mM stock solution was diluted 333.33-fold with extracellular fluid to give working solutions of 30. Mu.M. The DMSO content of the working solution is 0.1-0.3% (volume ratio).

4) After the working solution is prepared, whether sediment or turbidity exists in the working solution is observed by naked eyes. If present, the compound may be further sonicated in a water bath for 30 minutes to improve the clarity of the solution, possibly due to poor solubility of the compound in the physiological solution.

5) The potential inhibition of hERG channel by the test substance at 5 concentrations of 30. Mu.M, 10. Mu.M, 3.33. Mu.M, 1.11. Mu.M and 0.37. Mu.M was determined and a dose response curve was fitted and IC ₅₀ was calculated.

2. Experimental method

1. The slide with HEK293 cells in the petri dish was placed in a perfusion channel of a micromanipulation station.

2. The appropriate cells were centered in the field of view under an Olympus IX51, IX71 or IX73 inverted microscope, and the tip of the glass electrode was found using a x 10 objective lens and centered in the field of view. The electrode is then moved down using the micromanipulator while the coarse focusing helix is adjusted so that the electrode approaches the cell slowly.

3. When approaching the cells quickly, the objective lens is changed into a multiplied by 40 to observe, and the micromanipulator is used for fine tuning, so that the electrodes gradually approach the surfaces of the cells.

4. Negative pressure is given to form a seal with a resistance higher than 1G omega between the electrode tip and the cell membrane.

5. The instantaneous capacitance current Cfast is compensated in the voltage clamp mode. And then repeatedly giving short negative pressure to rupture membranes to finally form a whole-cell recording mode.

6. The slow capacitance current Cslow, cell membrane capacitance (Cm) and input membrane resistance (Ra) were compensated for under membrane potential clamping at-60 mV.

7. After the cells were stabilized, the clamping voltage was changed to-90 mV, the sampling frequency was set to 20kHz, and the filtration frequency was 10kHz. The detection condition of the leakage current is that the clamp voltage is changed to-80 mV, and the time period is 500ms.

The herg current test method is as follows: the application of a 4.8 second depolarization command voltage depolarizes the membrane potential from-80 mV to +30mV, followed by a momentary application of a 5.2 second repolarization voltage to drop the membrane potential to-50 mV to remove channel deactivation, resulting in the observation of hERG tail current. The peak of the tail current is the magnitude of hERG current.

9. The hERG currents used to detect test compounds were recorded for 120 seconds prior to dosing to assess the stability of the test cells to produce hERG currents. Only stable cells within the acceptance range of the evaluation criteria can enter subsequent compound detection.

10. Determining the inhibition of hERG current by the test compound: first, hERG current measured in extracellular fluid containing 0.1% dmso was used as a test baseline. Solutions containing the test compound were perfused around the cells sequentially from low to high concentration after hERG current remained stable for at least 5 minutes. Wait about 5 minutes after each perfusion end to allow the compound to act adequately on the cells and record hERG current simultaneously. The last 5 hERG current values were recorded after the current to be recorded tended to stabilize and their average value was taken as the current value at the specific concentration at which it was finally obtained. After the compounds were tested, 150nM of Duofilede (Dofetilide) was added to the same cells and their currents were completely inhibited as positive controls for the cells. Meanwhile, the positive compound, i.e. the multi-Feilide, is synchronously detected by the same patch clamp system before and after the test of the test compound is finished, so that the reliability and the sensitivity of the whole detection system are ensured.

3. Data analysis

The data is output by PATCHMASTER software and analyzed as follows:

After filling a blank solvent or a compound gradient solution, 5 continuous current values are obtained stably, and an average value is obtained and is respectively used as a tail current size _{Blank space} and a tail current size _{Compounds of formula (I)};

the percent current suppression is calculated by the following formula.

The dose response curves were fitted by GRAPHPAD PRISM 8.0.0 software and IC ₅₀ values calculated.

The inhibition of hERG by SERD compounds is shown in table 6 below.

Table 6 SERD inhibition of voltage-gated potassium channel hERG by compounds

Numbering of compounds	hERG IC ₅₀(μM)
A compound of formula (I)	10
Compound 4	3.7

Test example 7: mouse pharmacokinetic evaluation of SERD compounds

Experimental materials

CD-1 mice were purchased from Peking Virtualia laboratory animal technologies Co. DMSO (dimethyl sulfoxide), HP-beta-CD (hydroxypropyl-beta-cyclodextrin), tetraethylene glycol (TETRAETHYLENE GLYCOL), captisol (SBE-beta-CD, sulfobutyl-beta-cyclodextrin) were purchased from Sigma. Acetonitrile was purchased from Merck (USA).

Experimental method

Female CD-1 mice were divided into 6 groups (20-30 g,4-6 weeks) at random, 3 per group. Group 1 tail intravenous administration of test compound at a dose of 1mg/kg, vehicle of DMSO (5%, v/v) and 10% aqueous hp- β -CD (95%, v/v), group 2 oral administration of test compound at a dose of 10mg/kg, vehicle of tetraethylene glycol (40%, v/v) and 7.5% aqueous sulfobutyl- β -cyclodextrin (60%, v/v). The animals were fed water normally prior to the experiment. Each group of mice was subjected to intravenous blood sampling at 0.083 (intravenous only) 0.25, 0.5, 1,2, 4, 6, 8 and 24h before and after dosing. The collected whole blood sample was placed in K ₂ EDTA anticoagulation tube, and after centrifugation for 5min (12,000 rpm,4 ℃) plasma was taken for testing.

A10. Mu.L sample of mouse plasma was taken, 150. Mu.L of acetonitrile solvent (containing an internal standard compound) was added to precipitate the protein, and after vortexing for 5min, the solution was centrifuged (14,000 rpm) for 5min, and the supernatant was diluted 2-fold with water containing 0.1% (v/v) FA and quantitatively detected in an LC-MS/MS system (AB Sciex Triple Quad 6500+). CD-1 mouse plasma standard curve and quality control samples were obtained simultaneously when determining the sample concentration. For 10 Xdiluted samples, 2. Mu.L of the sample was added to 18. Mu.L of blank plasma, and after vortexing for 0.5min, 300. Mu.L of acetonitrile solvent (containing the internal standard compound) was added to precipitate the protein, and the rest of the treatment steps were the same as without diluting the sample.

PK test results as shown below, SERD compounds showed good PK properties and oral bioavailability in mice.

PK of Table 7 SERD Compounds in mice

Test example 8: SERD Compounds Blood Brain Barrier (BBB) penetration ability in rats

The fact that the drug can penetrate through the blood brain barrier of animals and has enough exposure in the brain is the key that the drug is effective for brain metastasis focus, so that the distribution situation of the drug in the brain can be estimated by measuring the concentration of the drug in blood plasma and brain tissues after animal administration, and then whether the drug can play a role in inhibiting tumor growth in a brain in-situ model is judged.

Experimental materials

SD female rats were purchased from beijing velariwa laboratory animal technologies limited. MC (methylcellulose) was purchased from Sigma; acetonitrile was purchased from Merck (USA). PBS (phosphate buffered saline) was purchased from an organism.

Experimental method

Female SD rats were divided into 2 groups at random (200-300 g,6-8 weeks), 3 per group. SERD compounds herein were administered separately, with vehicle 0.5% aqueous methylcellulose. Animals were fed normal water before the experiment, fasted overnight, and fed back four hours after administration. Plasma and brain tissue were collected 2h after dosing for each group of rats. Placing the collected whole blood sample in a K ₂ EDTA anticoagulation tube, centrifuging for 5min (12,000 rpm,4 ℃) and taking plasma to be measured; after tissue collection, the tissue is sucked by filter paper, and the sample is stored in a refrigerator at-80 ℃ to be tested.

10. Mu.L of rat plasma sample was taken, 150. Mu.L of acetonitrile solvent (containing an internal standard compound) was added to precipitate the protein, and after vortexing for 5min, the solution was centrifuged (14,000 rpm) for 5min, and the supernatant was diluted 2-fold with water containing 0.1% (v/v) FA and quantitatively detected in an LC-MS/MS system (AB Sciex Triple Quad 6500+). For 10 Xdiluted samples, 2. Mu.L of the sample was added to 18. Mu.L of blank plasma, and after vortexing for 0.5min, 300. Mu.L of acetonitrile solvent (containing the internal standard compound) was added to precipitate the protein, and the rest of the treatment steps were the same as without diluting the sample. SD rat plasma standard curve and plasma quality control sample were obtained simultaneously when determining plasma sample concentrations.

Rat brain tissue samples were first homogenized with 4 volumes of PBS homogenate. Taking 20 mu L of brain tissue homogenate sample, adding 20 mu L of blank mouse plasma, diluting and uniformly mixing, adding 600 mu L of acetonitrile solvent (containing an internal standard compound) to precipitate protein, carrying out centrifugation (14,000 rpm) for 5min after vortex for 5min, diluting supernatant with water containing 0.1% (v/v) FA for 2 times, and carrying out quantitative detection on the supernatant in an LC-MS/MS system (AB Sciex Triple Quad 6500+). The compounds of formula (I) of the present disclosure exhibit excellent blood brain barrier penetration ability with respect to the prior art or other molecules disclosed herein, and have high drug exposure in brain tissue of rats.

The BBB test results are shown below:

Table 8 SERD Compound rat blood brain Barrier permeation experiments

Test example 9: growth inhibition experiment of SERD compound on MCF-7 mouse subcutaneous tumor model

Experimental reagent

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

17 Beta-estradiol tablet: innovative Research of America Cat No. SE-121,60-DAY RELEASE,0.72mg/pellet

EMEM broth: ATCC, cat No. 30-2003

Fetal bovine serum: gbico; cat No. 1099-141C

Green streptomycin (Pen Strep): gibco, cat No. 15240-122

Recombinant human insulin: assist in the sea, cat.no.:40112ES60

0.25% Pancreatin-EDTA: gibco, cat No.:25200-072

D-PBS (calcium-free magnesium phosphate buffer): hyclone, cat.No.: SH30256.01

Matrigel:Corning,Cat.No.:356237

Experimental method

Animal information: NPG mice, females, 6-7 weeks, weighing approximately 19-28 grams, were purchased from Beijing Vietnam Biotechnology Inc., and were kept in SPF-grade environment with each cage individually ventilated, and all animals were free to obtain standard certified commercial laboratory diet and free drinking water.

Cell culture: the human breast cancer MCF-7 cell strain is cultured in vitro under the conditions that 10% fetal bovine serum, 1% pen strep,10 mug/ml recombinant human insulin and 5% CO ₂ incubator at 37 ℃ are added into EMEM (cell culture solution). Passaging was performed by conventional digestion with 0.25% pancreatin-EDTA digest once a week. When the saturation of the cells is 80% -90% and the number reaches the requirement, the cells are collected and counted.

Cell inoculation: 0.1 ml/(1X 10 ⁷) MCF-7 cell suspension (D-PBS: matrigel, volume ratio 1:1) was inoculated subcutaneously on the right back of each mouse and 17 beta-estradiol pieces were inoculated subcutaneously four days before cell inoculation. On Day 24 after cell inoculation, the doses were randomly grouped according to tumor volume, day 0.

Administration: the compound of formula (I) is administered at a dose of 1,3, or 10mg/kg, orally (PO), once daily (QD). Times.3 weeks. Vehicle group 8 mice and dosing group 6 mice.

Tumor measurement and experimental index:

Tumor diameters were measured twice weekly with vernier calipers. The calculation formula of the tumor volume is: v=0.5a×b ², a and b represent the long and short diameters of the tumor, respectively. Mice body weight was measured twice weekly.

The tumor-inhibiting effect of the compound was evaluated by tumor growth inhibition ratio TGI (%). TGI (%) = [ (1- (mean tumor volume at the end of dosing of a treatment group-mean tumor volume at the beginning of dosing of a treatment group)/(mean tumor volume at the end of treatment of a solvent control group-mean tumor volume at the beginning of treatment of a solvent control group) ]x100%.

Experimental results:

See table 9. In the mouse subcutaneous transplantation tumor MCF-7 model, the compound of the formula (I) has a remarkable inhibition effect (P < 0.01) on tumor growth by oral administration of 1mg/kg,3mg/kg or 10mg/kg once a day, has a better dose response relationship, and has the effect of shrinking tumors at doses of 3mg/kg and 10 mg/kg. The compound of formula (I) has a significant inhibitory effect on tumor growth (P < 0.01) and has tumor-reducing effect by oral administration once a day of 10 mg/kg. The compound of formula (I) did not significantly affect mouse body weight at the dose tried.

Table 9 MCF-7 tumor volumes of subcutaneous tumor model

Test example 10: inhibition experiment of Compound of formula (I) on growth of mouse MCF-7 brain in situ tumor model

Experimental reagents/instruments:

human breast cancer MCF-7 cells: ATCC, HTB-22

EMEM broth: ATCC, cat No. 30-2003

Fetal bovine serum: gibco, cat.No.:1099-141C

Green streptomycin (Pen Strep): gibco, cat No. 15240-122

Recombinant human insulin: assist in the sea, cat.no.:40112ES60

0.25% Pancreatin-EDTA: gibco, cat No.:25200-072

Brain stereotactic instrument: ruiword, cat No.: standard/digital display/Single arm/mouse/68055

Microinjection pump: KDS, cat No.: legato130

Miniature hand-held cranial drill: ruiword, cat No.:78001

The experimental method comprises the following steps:

Animal information: NPG mice, females, 6-8 weeks, weighing approximately 17-29 grams, were purchased from Beijing Vietnam Biotechnology Inc., and were kept in SPF-grade environment with each cage individually ventilated, and all animals were free to obtain standard certified commercial laboratory diet and free drinking water.

Cell culture: the human breast cancer MCF-7 cell strain is cultured in vitro under the conditions that 10% fetal bovine serum, 1% pen strep,10 mug/ml recombinant human insulin and 5% CO ₂ incubator at 37 ℃ are added into EMEM (cell culture solution). Passaging was performed twice a week with a conventional digestion treatment with 0.25% pancreatin-EDTA digest. When the saturation of the cells is 80% -90% and the number reaches the requirement, the cells are collected and counted.

Cell inoculation: mu.l/(containing 2X 10 ⁶) MCF-7 cell suspension was inoculated into the cranium of mice using a brain locator, a microinjection pump and a micro hand-held cranial drill, and 17 beta-estradiol pieces were inoculated subcutaneously three days before cell inoculation. On Day 8 after cell inoculation, the mice were dosed randomly on Day 0 according to body weight of the mice.

Administration: fulvestat (Fulvestrant, african) was administered at a dose of 250mg/kg, subcutaneously (SC), once a week (QW), and the compound of formula (I) was administered at a dose of 30mg/kg, orally (PO), once a day (QD). Vehicle group 11 mice and dosing group 8 mice. All groups continued to be dosed until mice died, euthanized due to poor status or the end of the experiment.

Experimental observation and end:

the mice were measured twice weekly for body weight and observed for survival.

Day 48 ended the experiment and all mice were euthanized.

Experimental results:

See fig. 2 and 3. In the mice brain in situ MCF-7 model, the weight of Fulvestrent mice (250 mg/kg administered subcutaneously once a week) continued to decrease, and the survival status of the mice did not significantly differ from the solvent control group (median survival, 29 days in the solvent control group, 29.5 days in the Fulvestrent group). Mice in the compound group of the formula (I) (30 mg/kg once a day oral administration) have stable body weight, no abnormality in state, no death of the mice in the compound group of the formula (I) occurs until the experimental end point, and the compound of the formula (I) has obvious inhibition effect on MCF-7 brain in-situ tumor model mice relative to solvent control or Fulvestat, and the survival period of the mice is obviously prolonged (P < 0.01).

Test example 11: effect of the Compound of formula (I) in combination with piperaquine Bai Xili on the MCF-7 cell cycle of human breast cancer

Cell information: human breast cancer MCF-7 cells were purchased from ATCC under the conditions of DMEM (purchased from Gibco) +10% FBS (purchased from Gibco) +0.01mg/ml human insulin (purchased from Santa next) +1% non-essential amino acids (purchased from Gibco). Pepper Bai Xili was purchased from MCE.

The experimental method comprises the following steps: when the cell fusion rate reaches over 70%, cells are digested, the cell density is adjusted to 1.5E5/0.5 mL/well, and the cells are cultured in a 24-well cell culture plate (Greiner) overnight. 0.4mL of medium was added to each well, 50. Mu.L 6.4,1.6 and 0.4. Mu.M of piperaquine Bai Xili were added, respectively, at a final concentration of 320, 80, 20nM, and 50. Mu.L of 0.2% DMSO medium was added to the single well and the cell control well. 50. Mu.L of 200nM compound of formula (I) was added to the conjugate well at a final concentration of 10nM. Single drug wells and cell control wells were added with 50. Mu.L of 0.1% DMSO medium. After 40 hours of compound treatment, cells were collected, fixed with 70% ethanol (Shanghai) at-20℃and stained with 30. Mu.g/mL propidium iodide (Invitrogen), 50. Mu.g/mL ribonuclease A (Sigma) and 0.2% Triton X-100 (Sigma) at 37℃for 30 minutes in the absence of light. Red fluorescence was detected at excitation wavelength 488nm using a flow cytometer (BD) while light scattering was detected. Cell cycle was analyzed using Flowjo.

Experimental results: the compound of formula (I) can cause cell cycle arrest, arrest cells in G1 phase, and can obviously improve G1 phase arrest proportion and reduce S phase cell proportion after being combined with the piperaquine Bai Xili, and the result is shown in Table 10.

TABLE 10 effects of Compounds of formula (I) on MCF-7 cell cycle arrest in combination with piperaquine Bai Xili

Test example 12: proliferation inhibition of human breast cancer MCF-7 cells by the combination of a compound of formula (I) and piperaquine Bai Xili

Cell and compound information: human breast cancer MCF-7 was purchased from ATCC under the conditions of DMEM (Gibco) +10% FBS (Gibco) +0.01mg/ml human insulin (next holy) +1% nonessential amino acids (Gibco). Pepper Bai Xili was purchased from MCE.

The experimental method comprises the following steps: when the cell fusion rate of the cultured human breast cancer MCF-7 reaches more than 70%, cells are digested, and the density is adjusted to 500/20 mu L/well seed is cultured in 384-well cell culture plates (Corning) overnight. The compound of formula (I) (in the form of succinate) was transferred to the cell plate using an Echo650 (Beckman) 2-fold gradient of 120nL, while the compound was transferred to the cell plate 120nL 2-fold gradient of piperaquine Bai Xi, and 40 μl of medium was added, and after 7 days of compound treatment, cellTiter-Glo (Promega) was added to examine the cell viability, the cell wells served as 100% viability control, the medium wells served as 0% viability control, and the combination analysis was performed using Combenefit (a number greater than 10 indicated that a better combination synergy was achieved).

Results: the synergistic effect of the compound of formula (I) in combination with piperaquine Bai Xili on the proliferation inhibition of human breast cancer MCF-7 cells is shown in figure 4.

Test example 13: combined pharmaceutical study of human breast cancer MCF-7 xenograft subcutaneous tumor mouse model

Experimental materials:

human breast cancer MCF-7 cells: ECACC,86012803

17 Beta-estradiol tablet: innovative Research of America Cat No. SE-121,60-DAY RELEASE,0.18mg/pellet

EMEM broth: ATCC, cat No. 30-2003

Fetal bovine serum: an ExCell; cat No. FND500

Double antibody: gibco, cat No. 15240-062

0.25% Pancreatin-EDTA: gibco, cat No.:25200-072

DPBS：Corning,Cat.No.:21-031-CVR

Matrigel Corning, cat.No.:354234

Piperacillin Bai Xili (Palbociclib): miao medicine Co., ltd (imitation), 125 mg/capsule

The experimental method comprises the following steps:

Animal information: balb/c nude mice, females, 6-8 weeks, weighing about 18-22 grams, animals were purchased from Shanghai Ling Biotechnology Inc., and were kept in SPF-grade environment with each cage individually ventilated and all animals were free to obtain standard certified commercial laboratory diet and free drinking water.

Cell culture: the human breast cancer MCF-7 cell strain is cultured in vitro under the condition that 10% fetal bovine serum, 1% double antibody and 5% CO ₂ incubator are added into EMEM (cell culture solution). Passaging was performed twice a week with a conventional digestion treatment with 0.25% pancreatin-EDTA digest. When the saturation of the cells is 80% -90% and the number reaches the requirement, the cells are collected and counted.

Cell inoculation: 0.2 ml/(1X 10 ⁷) of MCF-7 cell suspension (DPBS: matrigel, volume ratio 1:1) was inoculated subcutaneously on the right back of each mouse and 17 beta-estradiol pieces were inoculated subcutaneously two days before cell inoculation. On Day 6 after cell inoculation, the drug administration was started when the average tumor volume reached 159.01mm ³, and was randomly grouped according to tumor volume, day 0.

Administration: the compound of formula (I) (succinate form, dose in free base) was administered at a dose of 1mg/kg, orally (PO), once daily (QD). Times.25. The dose of the drug administered to the piperazine Bai Xili was 40mg/kg, and the drug was orally administered (PO) once daily (QW). Times.25 times. 8 mice per group.

Tumor measurement and experimental index:

Experimental results:

See table 11, fig. 5 and fig. 6. The solvent group had more than 10% weight loss in 1 mouse, the piperaquine Bai Xili (40 mg/kg) group had more than 10% weight loss in 4 mice, and the combination of piperaquine Bai Xili (40 mg/kg) and the compound of formula (I) (1 mg/kg) had more than 10% weight loss in 1 mouse. It can be seen that in this model, 40mg/kg of piperaquine Bai Xili has a certain effect on animal weight. There were no morbidity or mortality in this model.

The test result shows that the drug administration of the piperaquine Bai Xi with the concentration of 40mg/kg orally to the tumor-bearing mice once a Day shows a certain effect of inhibiting tumor growth (P < 0.0001) on the 24 th Day (Day 24) after the beginning of the drug administration; oral administration of 1mg/kg of the compound of formula (I) to tumor-bearing mice once a day showed a remarkable tumor growth inhibition effect (P < 0.0001); the combination of 40mg/kg of piperaquine Bai Xili and 1mg/kg of the compound of formula (I) showed a significantly enhanced anti-tumor effect relative to both the vehicle control group and the corresponding single drug group.

Table 11 MCF-7 tumor volumes in subcutaneous tumor model

Each document cited herein, including any cross-referenced patent or patent application, any patent application or patent for which the application claims priority, is hereby incorporated by reference in its entirety unless expressly excluded or otherwise limited. Furthermore, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term herein shall govern.

While particular embodiments of the present disclosure have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the disclosure.

Claims

A pharmaceutical combination comprising at least one selective estrogen receptor down-regulator selected from the group consisting of compounds of formula (K) or pharmaceutically acceptable salts thereof, and at least one CDK4/6 inhibitor:

wherein,

R ¹、R ²、R ³、R ⁴ is independently selected from H, F, cl, br, I, CN, C ₁-C ₆ alkyl, C ₁-C ₆ alkoxy, or C ₃-C ₆ cycloalkyl;

X ₁、X ₂、X ₃、X ₄ is independently selected from CR ⁶ or N;

R ⁶ is selected from H, F, cl, br, I, OH, CN, C ₁-C ₁₀ alkyl, C ₃-C ₁₀ cycloalkyl, 3-10 membered heterocyclyl, C ₁-C ₁₀ alkoxy, C ₃-C ₁₀ cycloalkyloxy, or 3-10 membered heterocyclyloxy;

R ⁵ is independently selected from C ₁-C ₆ alkyl, said C ₁-C ₆ alkyl optionally substituted with R ^a;

r ^a is selected from F, cl, br, I, OH, CN, C ₁-C ₆ alkyl, C ₁-C ₆ alkoxy or C ₃-C ₆ cycloalkyl.
The pharmaceutical combination according to claim 1, wherein the compound of formula (K) or a pharmaceutically acceptable salt thereof is selected from the following compounds or pharmaceutically acceptable salts thereof:
a pharmaceutical combination according to claim 1 or 2 wherein the CDK4/6 inhibitor is selected from the group consisting of arbeli, rebaudinib, pip Bai Xili 、alvociclib、lerociclib、trilaciclib、voruciclib、AT-7519、FLX-925、INOC-005、BPI-1178、PD-0183812、NSC-625987、CGP-82996、PD-171851、SHR-6390、BPI-16350, and pharmaceutically acceptable salts thereof.
A pharmaceutical combination according to any one of claims 1 to 3, wherein the compound of formula (K) or a pharmaceutically acceptable salt thereof is selected from compounds of formula (I):
the pharmaceutical combination according to any one of claims 1 to 4, wherein the CDK4/6 inhibitor is selected from piper Bai Xi or a pharmaceutically acceptable salt thereof.
A combination comprising a I-th pharmaceutical composition comprising a compound of formula (K) or a pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 5 and a pharmaceutically acceptable adjuvant, and a II-th pharmaceutical composition comprising at least one CDK4/6 inhibitor and a pharmaceutically acceptable adjuvant.
The combination according to claim 6, wherein the compound of formula (K) or a pharmaceutically acceptable salt thereof in the I-th pharmaceutical composition is selected from the group consisting of compounds of formula (I) or a pharmaceutically acceptable salt thereof.
The combination product according to claim 6 or 7, wherein the CDK4/6 inhibitor in the II pharmaceutical composition is selected from the group consisting of arbeli, rebaudinib, pip Bai Xili 、alvociclib、lerociclib、trilaciclib、voruciclib、AT-7519、FLX-925、INOC-005、BPI-1178、PD-0183812、NSC-625987、CGP-82996、PD-171851、SHR-6390、BPI-16350, and pharmaceutically acceptable salts thereof.
A pharmaceutical composition comprising the pharmaceutical combination of any one of claims 1 to 5, and a pharmaceutically acceptable adjuvant.
The pharmaceutical combination according to any one of claims 1 to 5, the combination product according to any one of claims 6 to 8 and the pharmaceutical composition according to claim 9 for antitumor use.
The use of claim 10, wherein the tumor is breast cancer.
The use of claim 10, wherein the tumor is ER positive breast cancer.
The use of claim 10, wherein the tumor is ER-positive brain-metastatic breast cancer.
The use of claim 10, wherein the tumor is ER positive, HER-2 negative locally advanced or metastatic breast cancer.