WO2023083283A1

WO2023083283A1 - Drug combination for treating tumor, and application thereof

Info

Publication number: WO2023083283A1
Application number: PCT/CN2022/131280
Authority: WO
Inventors: 薛黎婷; 古鹏; 陈平; 杨桂梅; 杨文清; 周峰; 唐任宏; 任晋生
Original assignee: 先声药业有限公司; 江苏先声药业有限公司
Priority date: 2021-11-12
Filing date: 2022-11-11
Publication date: 2023-05-19
Also published as: TW202333704A; CN118119620A

Abstract

A drug combination comprising a compound represented by formula (K) as a selective estrogen receptor downregulator (SERD) or a pharmaceutically acceptable salt thereof and a CDK4/6 inhibitor, a combined product or pharmaceutical composition containing the drug combination, and an application thereof.

Description

Combinations of drugs for treating tumors and their applications

Cross References to Related Applications

This application claims priority and rights to Chinese Patent Application No. 202111338407.1 filed with the State Intellectual Property Office of China on November 12, 2021, the disclosure of which is incorporated herein by reference in its entirety.

technical field

The disclosure belongs to the field of medicine, and relates to a drug combination of a compound represented by formula (K) as a selective estrogen receptor down-regulator (SERD) and a CDK4/6 inhibitor, a combination product or a drug combination comprising the drug combination substances, and their use in the treatment of tumors.

Background technique

Estrogen (E2) and estrogen alpha receptor (ERα) are important drivers of breast cancer development. More than 2/3 of breast cancer patients express ER transcription factors, and in most ER-positive patients, ER is still a key driver even in tumors that progress after early endocrine therapy, so ER is A major target for breast cancer therapy (Pharmacology & Therapeutics 186 (2018) 1–24). The purpose of endocrine therapy is to reduce the activity of ER. There are three main types, including selective estrogen receptor modulators (SERMs), such as tamoxifen (tamoxifen), which is an allosteric regulator of ER and inhibits its transcriptional activity after binding to ER. ; Aromatase inhibitors (aromatase inhibitors, AIs), by inhibiting the conversion of androgen into estrogen, reduce the level of estrogen in the body; and selective estrogen receptor down-regulators, such as fulvestrant (fulvestrant), not only as ER Antagonists inhibit its activity and also induce ER protein degradation. Although endocrine therapy is the first choice for patients with estrogen receptor-positive breast cancer, about 30% of patients will relapse after treatment, and almost all patients with metastatic breast cancer will develop drug resistance and progress.

Clinically, about 70-80% of breast cancers are positive for estrogen receptor (ER), the proliferation of such breast cancer cells is heavily dependent on ER, and 50% of breast cancer deaths are of this type. Early ER-positive breast cancer has a better prognosis, with a 5-year survival rate of over 90%. About 30% of patients who received postoperative endocrine therapy (TAM or AI drugs) relapsed within 10 years, but they could still receive standard endocrine therapy.

Fulvestrant is the first and only SERD drug clinically approved for the treatment of postmenopausal patients with ER-positive, metastatic breast cancer after progression on tamoxifen or an aromatase inhibitor. A number of research data show that patients treated with fulvestrant have not completely degraded ER in vivo. In addition, intramuscular injection caused obvious reactions such as pain, swelling, and redness at the injection site, and the absorption was slow and the exposure in vivo was limited. And other characteristics limit its clinical application, so patients with ER-positive breast cancer urgently need new treatment options.

Cyclin-dependent kinases 4 and 6 (CDK4/6) mediate the cell cycle transition from G0/G1 to S phase and promote cell proliferation. Palbociclib (Palbociclib, chemical name is 6-acetyl-8-cyclopentyl-5-methyl-2-[[5-(1-piperazinyl)-2-pyridyl]amino]pyrido[2 ,3-d]pyrimidin-7(8H)-one) as a CDK4/6 selective inhibitor, can be used for the treatment of cancer patients.

Contents of the invention

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

in,

R ¹ , R ² , R ³ , R ⁴ are independently selected from H, F, Cl, Br, I, CN, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy or C ₃ -C ₆ cycloalkane base;

X ₁ , X ₂ , X ₃ , X ₄ are independently selected from CR ⁶ or N;

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

R ⁵ is independently selected from C ₁ -C ₆ alkyl, the C ₁ -C ₆ alkyl is optionally substituted by 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 product, the combination product comprises the first pharmaceutical composition and the second pharmaceutical composition, and the first pharmaceutical composition comprises at least one compound represented by formula (K) or Its pharmaceutically acceptable salt and pharmaceutically acceptable excipients, the pharmaceutical composition II includes at least one CDK4/6 inhibitor and pharmaceutically acceptable excipients.

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

A first container comprising the first pharmaceutical composition as described above; and,

A second container comprising the pharmaceutical composition II as described above.

On the other hand, the present disclosure also provides a pharmaceutical composition, which comprises 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 of the above-mentioned pharmaceutical combinations, combination products or pharmaceutical compositions in the preparation of antitumor drugs.

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

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

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

On the other hand, the present disclosure also relates to the use of a compound represented by formula (K) or a pharmaceutically acceptable salt thereof in combination with a CDK4/6 inhibitor in the preparation of an antitumor drug.

Description of drawings

Figure 1 is the NOESY spectrum of the compound of formula (I).

Fig. 2 is the survival curve of the human ER-positive breast cancer MCF-7 brain orthotopic model mouse of the compound of formula (I).

Fig. 3 is a diagram of the body weight change of the human ER-positive breast cancer MCF-7 brain orthotopic model mouse with the compound of formula (I).

Fig. 4 is a graph showing the synergistic anti-proliferation effect of the compound of formula (I) combined with palbociclib on human breast cancer MCF-7 cells in vitro.

Fig. 5 is a diagram showing the anti-tumor growth effect of the compound of formula (I) and palbociclib in combination on human ER-positive breast cancer MCF-7 xenograft xenograft mouse model.

Fig. 6 is a diagram of body weight change of mice with subcutaneous xenograft tumor of human ER-positive breast cancer MCF-7 treated with compound of formula (I) and palbociclib.

Detailed ways

In order to facilitate the understanding of the disclosure herein, a more comprehensive description will be given below. However, these embodiments may be embodied in many different forms and are not limited to the specific examples described herein. Rather, these embodiments are provided in order to enable a thorough understanding of the disclosure herein.

Definitions and Explanations of Terms

Unless otherwise stated, the definitions of groups and terms recorded in the specification and claims, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in the examples, etc., Any combination and combination with each other is possible. Such combinations and combined group definitions and compound structures 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 or pharmaceutically acceptable salts thereof in the pharmaceutical combination can be administered simultaneously, and in some embodiments, the active ingredients or pharmaceutically acceptable salts thereof in the pharmaceutical combination can also be administered administered separately or sequentially.

The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable non-toxic acid or base salt, 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 and 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.

In this article

Indicates the junction site.

Graphical representations of racemic or enantiomerically pure compounds herein are from Maehr, J. Chem. Ed. 1985, 62:114-120. Unless otherwise specified, use wedge keys and dotted wedge keys (

and

) represents the absolute configuration of a stereocenter, with black real bonds and virtual bonds (

and

) represents the relative configuration of a stereocenter (such as the cis-trans configuration of an alicyclic compound).

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

The term "stereoisomer" refers to isomers resulting from differences in the arrangement of atoms in a molecule in space, including 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 specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E and Z geometric isomers, (-)- and (+)-enantiomers, (R)- and (S )-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic or other mixtures thereof, such as enantiomers or diastereomers Enriched mixtures, all of the above isomers and mixtures thereof are within the definition of compounds of the present disclosure. There may be additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms or asymmetric phosphorus atoms in substituents such as alkyl groups, and these isomers and their mixtures involved in all substituents are also included in Compounds of the disclosure are within the definition. The asymmetric atom-containing compounds of the present disclosure can be isolated in optically pure form or in racemic form, the optically active form can be resolved from a racemic mixture, or by using a chiral starting material or a chiral reagent synthesis.

The term "substituted" means that any one or more hydrogen atoms on the specified atom are replaced by substituents, as long as the valence of the specified atom is normal and the compound after substitution is stable.

The term "optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that description includes that said event or circumstance occurs and that it does not. For example, ethyl is "optionally" substituted with _halogen , meaning that the ethyl group can be unsubstituted ( _CH2CH3 ), monosubstituted ( _CH2CH2F _, _CH2CH2Cl _, etc.), polysubstituted ( _CHFCH2F , _CH2CHF2 , _CHFCH2Cl , _CH2CHCl2 _, _etc. ₎ or fully substituted ( _CF2CF3 , _CF2CCl3 , _CCl2CCl3 _, etc.) _. It will be appreciated by those skilled in the art that for any group containing one or more substituents, no sterically impossible and/or synthetically impossible substitution or substitution pattern is introduced.

When any variable (eg R ^a , R ^b ) occurs more than once in the composition or structure of a compound, its definition is independent at each occurrence. For example, if a group is substituted by 2 R ^b , each R ^b has independent options.

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

The term "alkyl" refers to a hydrocarbon group having the general formula C _n H _2n+1 , and the alkyl group may be straight or branched. The term "C ₁ -C ₁₀ alkyl" is understood to mean a straight-chain or branched saturated hydrocarbon group 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-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-di Methylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, etc.; the term "C ₁ -C ₆ alkyl" can be understood as 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, etc. The term "C ₁ -C ₃ alkyl" is understood to mean a linear or branched saturated alkyl group having 1 to 3 carbon atoms. The "C ₁ -C ₁₀ alkyl" may include "C ₁ -C ₆ alkyl" or "C ₁ -C ₃ alkyl", and the "C ₁ -C ₆ alkyl" may further include " C ₁ -C ₃ alkyl".

The term "alkoxy" refers to the group produced by the loss of the hydrogen atom on the hydroxyl group of straight-chain or branched alcohols, which can be understood as "alkyloxy" or "alkyl-O-". The term "C ₁ -C ₁₀ alkoxy" can be understood as "C ₁ -C ₁₀ alkyloxy" or "C ₁ -C ₁₀ alkyl-O-"; the term "C ₁ -C ₆ alkoxy" It can be understood as "C ₁ -C ₆ alkyloxy" or "C ₁ -C ₆ alkyl-O-". The "C ₁ -C ₁₀ alkoxy" may include "C ₁ -C ₆ alkoxy" and "C ₁ -C ₃ alkoxy" and other ranges, and the "C ₁ -C ₆ alkoxy""C ₁ -C ₃ alkoxy" may be further included.

The term "cycloalkyl" refers to a fully saturated carbocyclic ring in the form of a monocyclic ring, a fused ring, a bridged ring, or a spiro ring. 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 said cycloalkyl 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] decanyl, etc. The term "C ₃ -C ₁₀ cycloalkyl" may include "C ₃ -C ₆ cycloalkyl", and the term "C ₃ -C ₆ cycloalkyl" can be understood as representing a saturated monocyclic or bicyclic hydrocarbon ring, which has 3-6 carbon atoms, specific examples include but not limited to cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl and the like.

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

The term "heterocyclyl" refers to a fully saturated or partially saturated (not aromatic heteroaromatic as a whole) monocyclic, fused, spiro or bridged ring group containing 1-5 ring atoms Heteroatoms or heteroatom groups (ie, atomic groups containing heteroatoms), the "heteroatoms or heteroatom groups" include but are not limited to nitrogen atoms (N), oxygen atoms (O), sulfur atoms (S), phosphorus atoms (P) , boron atom (B), -S(=O) ₂ -, -S(=O)-, -P(=O) ₂ -, -P(=O)-, -NH-, -S(=O )(=NH)-, -C(=O)NH- or -NHC(=O)NH-, etc. The term "3-10 membered heterocyclic group" refers to a heterocyclic group with 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms, and its ring atoms contain 1-5 ring atoms independently selected from the above The heteroatom or heteroatom group. In particular, the heterocyclic group may include but not limited to: specific examples of 4-membered heterocyclic groups include but not limited to azetidinyl or oxetanyl; specific examples of 5-membered heterocyclic groups include but not limited to Not limited to tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydrooxazolyl or 2,5-dihydro-1H-pyrrole Specific examples of 6-membered heterocyclic groups include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, tetrahydro Pyridyl or 4H-[1,3,4]thiadiazinyl; specific examples of 7-membered heterocyclyl include, but are not limited to, diazepanyl. The heterocyclic group can also be a bicyclic group, wherein, specific examples of the 5,5-membered bicyclic group include, but are not limited to, hexahydrocyclopenta[c]pyrrol-2(1H)-yl; 5,6-membered bicyclic group Specific examples 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 heterocyclyl groups in this disclosure partially contain a benzene ring or a heteroaryl ring, the heterocyclyl groups as a whole are nonaromatic.

The term "3-10 membered heterocyclyloxy" means "3-10 membered heterocyclyl-O-".

The term "treating" means administering a compound or formulation described herein to prevent, improve 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, especially 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. arresting its development;

(iii) amelioration of a disease or disease state, even if the disease or disease state regresses.

The term "therapeutically effective amount" means (i) treating a particular disease, condition or disorder, (ii) alleviating, ameliorating or eliminating one or more symptoms of a particular disease, condition or disorder, or (iii) delaying The amount of a compound of the disclosure for the onset of one or more symptoms of a particular disease, condition or disorder. The amount of a compound of the disclosure that constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one skilled in the art. Based on its own knowledge and this 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 skilled 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 specific embodiments, the SERD and CDK4/6 inhibitors are administered orally.

In 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 can be administered simultaneously, separately or sequentially.

The term "pharmaceutically acceptable excipients" refers to those excipients that have no obvious stimulating effect on the organism and will not impair the biological activity and performance of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like.

The words "comprise", "comprise" or "comprise" and their English variants such as comprises or comprising should be understood in an open, non-exclusive sense, ie "including but not limited to".

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

Certain isotopically labeled compounds of the present application (eg, those labeled ^with3H ^and14C ) are useful in compound and/or substrate tissue distribution assays. Tritiated ( ^ie3H ) and carbon-14 ( ^ie14C ) isotopes are especially preferred for their ease of preparation and detectability. Positron-emitting isotopes, such as ¹⁵ O, ¹³ N, ¹¹ C, and ¹⁸ F, can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the present application can generally be prepared by following procedures similar to those disclosed in the Schemes and/or Examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

Furthermore, substitution with heavier isotopes such as deuterium (i.e. ² H) may confer certain therapeutic advantages resulting from greater metabolic stability (e.g. increased in vivo half-life or reduced dosage requirements), and thus in some cases The following may be preferred, where deuterium substitution may be partial or complete, partial deuterium substitution meaning at least one hydrogen is replaced by at least one deuterium.

The pharmaceutical composition of the present application can be prepared by combining the compound of the present application with suitable pharmaceutically acceptable auxiliary materials, for example, it can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders , granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols, etc.

Typical routes of administering a compound of the present application or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, Intramuscular, subcutaneous, intravenous administration.

The pharmaceutical composition of the present application can be produced by methods well known in the art, such as conventional mixing methods, dissolving methods, granulating methods, dragee-making methods, pulverizing methods, emulsifying methods, freeze-drying methods and the like.

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

Solid oral compositions can be prepared by conventional methods of mixing, filling or tabletting. It can be obtained, for example, by mixing the active compound with solid excipients, optionally milling the resulting mixture, adding other suitable excipients if desired, and processing the mixture into granules to obtain tablets Or the core of the sugar coating. Suitable auxiliary materials include but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, etc.

The pharmaceutical composition may also be adapted for parenteral administration as a suitable unit dosage form of sterile solutions, suspensions or lyophilized products.

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

in,

X ₁ , X ₂ , X ₃ , X ₄ are independently selected from CR ⁶ or N;

In some embodiments, R ¹ , R ² , R ³ , and R ⁴ in the compound represented by formula (K) are independently selected from H, F, Cl, Br, I, CN, or C ₁ -C ₆ alkyl.

In some embodiments, R ¹ , R ² , R ³ , and R ⁴ in the compound represented by formula (K) are independently selected from H, F, or methyl.

In some embodiments, R ¹ and R ² in the compound represented by formula (K) are independently selected from H, F or methyl.

In some embodiments, R ³ and R ⁴ in the compound represented by formula (K) are independently selected from H or methyl.

In some embodiments, R ³ and R ⁴ in the compound represented by formula (K) are independently selected from H.

In some embodiments, the structural unit in the compound shown in formula (K)

selected from

In some embodiments, the structural unit in the compound shown in formula (K)

selected from

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

In some embodiments, the compound represented by the formula (K) or a pharmaceutically acceptable salt thereof is selected from the compound represented by the formula (K-1) or a pharmaceutically acceptable salt thereof:

Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , X ₁ , X ₂ , X ₃ , and X ₄ are as defined above.

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

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

In some embodiments, the CDK4/6 inhibitor is selected from the group consisting of abemaciclib, ribociclib, palbociclib, 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 above compounds.

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

Further, the present disclosure provides a pharmaceutical combination, which comprises the compound represented by formula (I) or a pharmaceutically acceptable salt thereof and palbociclib or a pharmaceutically acceptable salt thereof.

The present disclosure provides a combination product, the combination product comprises the first pharmaceutical composition and the second pharmaceutical composition, the first pharmaceutical composition comprises at least one compound represented by formula (K) or its pharmaceutically acceptable salt and pharmaceutically acceptable excipients, the pharmaceutical composition II comprises at least one CDK4/6 inhibitor and pharmaceutically acceptable excipients.

In one embodiment of the present disclosure, the compound represented by formula (K) or a pharmaceutically acceptable salt thereof in the first pharmaceutical composition is selected from the compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

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

Further, the present disclosure provides a combination product, the combination product comprises the first pharmaceutical composition and the second pharmaceutical composition, the first pharmaceutical composition comprises the compound represented by formula (I) or its pharmaceutically acceptable salt and pharmaceutically acceptable excipients, the pharmaceutical composition II comprises palbociclib or a pharmaceutically acceptable salt thereof and pharmaceutically acceptable excipients.

The present disclosure also provides a kit comprising:

The present disclosure also provides a pharmaceutical composition, which comprises any of the above-mentioned pharmaceutical combinations and at least one pharmaceutically acceptable auxiliary material.

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

The compound represented by the formula (I) or a pharmaceutically acceptable salt thereof;

Palbociclib or a pharmaceutically acceptable salt thereof;

And, pharmaceutically acceptable excipients.

The present disclosure relates to the use of any of the above-mentioned pharmaceutical combinations, combination products or pharmaceutical compositions in the preparation of antitumor drugs.

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

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

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

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

Further, the present disclosure relates to the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof in combination with a CDK4/6 inhibitor in the preparation of an antitumor drug; in one embodiment of the present disclosure, the CDK4/6 The inhibitor is palbociclib 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 metastatic 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 represented by formula (I) or a pharmaceutically acceptable salt thereof is administered at a daily dose of about 1 mg to 1000 mg (calculated as free base).

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

The SERD compound herein has good antitumor activity in vivo and in vitro and druggability. In vivo experiments found that the SERD compound herein can significantly inhibit the tumor growth of ER-positive breast cancer mouse model, and significantly improve the survival period of ER-positive breast cancer brain orthotopic model mice. In addition, the SERD compound herein has one or more of the following technical benefits: high bioavailability, strong ER degradation ability, oral administration, and high ability to pass through the blood-brain barrier. Therefore, the SERD compounds herein have the potential to effectively treat ER-positive breast cancer (especially ER-positive breast cancer with brain metastases).

Compared with the single administration of any drug in the combination, the combination of the SERD compound herein and CDK4/6 inhibitors (such as palbociclib) produces better efficacy in reducing the growth of tumors or even eliminating tumors, showing excellent antitumor synergistic effect.

The chemical reactions of the embodiments of the present disclosure are carried out in suitable solvents suitable for the chemical changes of the present disclosure and the reagents and materials required therefor. In order to obtain the compounds of the present disclosure, it is sometimes necessary for those skilled in the art to modify or select synthetic steps or reaction schemes on the basis of existing embodiments.

The compounds of the present disclosure can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed herein, the embodiments formed by combining them with other chemical synthesis methods, and the methods described by those skilled in the art. Known equivalents, preferred embodiments include, but are not limited to, the examples of this disclosure.

Example

The present disclosure will be described in detail through examples below, but it does not imply any adverse limitation on the present disclosure. This article has described the content of this article in detail, and its specific embodiments are also disclosed. For those skilled in the art, various changes and modifications can be made to the specific embodiments without departing from the spirit and scope of the present disclosure. Improvements will be apparent. All reagents used herein are commercially available and used without further purification.

Unless otherwise specified, the ratios indicated for mixed solvents are volume mixing ratios.

Unless otherwise stated, % means weight percent wt%.

compound by hand or

The software is named, and the commercially available compounds adopt the supplier catalog name.

Compound structures were determined by nuclear magnetic resonance (NMR) and/or mass spectroscopy (MS). The unit of NMR shift is 10 ^-6 (ppm). The solvents determined by NMR are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is tetramethylsilane (TMS).

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

resolve resolution:

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

Dissolve tert-butylpyrrolidin-3-ylcarbamate (1.00g, 5.37mmol) in tetrahydrofuran (10mL), add sodium hydroxide solution (5mol·L ^-1 , 2.15mL) and 1-iodo-3 - Fluoropropane (1.06 g, 5.64 mmol). The reaction solution was stirred and reacted at 25°C for 16 hours. After the reaction of the raw materials was detected by TLC, the reaction solution was diluted with ethyl acetate, washed with 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 the organic phases were combined and dried over sodium sulfate. The organic phase was concentrated to dryness under reduced pressure, and then purified by column chromatography (silica, dichloromethane/methanol=100/ 1) The product tert-butyl(1-(3-fluoropropyl)pyrrolidin-3-yl)carbamate (0.81 g) was obtained.

¹ H 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

Dissolve tert-butyl(1-(3-fluoropropyl)pyrrolidin-3-yl)carbamate (0.81 g, 3.12 mmol) in 1,4-dioxane (9 mL), then add hydrochloric acid -1,4-dioxane solution (4moL·L ^-1 , 9mL), the reaction solution was a yellow transparent solution. The reaction solution was stirred at 25°C for 3 hours. After the reaction of the raw materials was detected by TLC, the reaction solution was concentrated to dryness under reduced pressure to obtain compound 1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride (0.71 g).

¹ H 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-(2,2,2-trifluoroethyl)propane-2-amine

Dissolve (2R)-1-(1H-indol-3-yl)propane-2-amine (600 mg, 3.44 mmol) and N,N-diisopropylethylamine (445.05 mg, 3.44 mmol) in 1, To 4-dioxane (10mL), add trifluoroethyl trifluoromethanesulfonate (1.20g, 5.17mmol) dissolved in 1,4-dioxane (5mL) at 25°C, and the reaction solution The reaction was stirred at 75°C 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 obtain the product (R)-1-(1H-indol-3-yl)-N -(2,2,2-Trifluoroethyl)propan-2-amine (0.69 g).

MS m/z(ESI):257.2[M+H] ⁺

¹ H NMR (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: (1S,3R)-1-(5-Bromopyridin-2-yl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9- Synthesis of Tetrahydro-1H-pyrido[3,4-b]indole

Dissolve (R)-1-(1H-indol-3-yl)-N-(2,2,2-trifluoroethyl)propan-2-amine (120.00 mg, 468.26 μmol) in toluene (2 mL) 5-bromo-pyridine-2-carbaldehyde (87.10mg, 468.26μmol) and acetic acid (562.40mg, 9.37mmol) were added, and the reaction solution was a yellow transparent solution

Stir at 90°C for 10 hours. After the completion of the reaction detected by LCMS, the reaction solution was cooled to room temperature, concentrated to dryness under reduced pressure, and purified by thin layer chromatography (silica, petroleum ether/ethyl acetate=4/1) to obtain (1S,3R)-1- (5-Bromopyridin-2-yl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4 -b] Indole (110.00 mg).

MS m/z(ESI):424.1,426.1[M+H] ⁺

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

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

MS m/z(ESI):490.2[M+H] ⁺

¹ H NMR (400MHz, METHANOL-d ₄ ) δ 7.88 (d, J = 2.76Hz, 1H), 7.46 (d, J = 7.78Hz, 1H), 7.25 (dd, J = 7.91, 3.89Hz, 2H) ,7.09-6.96(m,3H),4.97(s,1H),4.60(t,J=5.65Hz,1H),4.48(t,J=5.65Hz,1H),4.15(br s,1H),3.52 -3.36(m,3H),3.25-3.14(m,1H),3.09-2.88(m,6H),2.68(s,1H),2.51-2.39(m,1H),2.11-1.85(m,3H) ,1.20(d,J=6.53Hz,3H).

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

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

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

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

MS m/z(ESI):490.1[M+H] ⁺

¹ H 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,3R)-3-methyl-2-(2,2,2-trifluoroethyl )-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-amine (compound of formula (I)):

MS m/z(ESI):489.25,490.1[M+H] ⁺

¹ H 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).

Embodiment 3-2: the synthetic method 2 of formula (I) compound

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

Dissolve (S)-tert-butylpyrrolidin-3-ylaminomethyl ester (500.00mg, 2.68mmol) in tetrahydrofuran (10mL), add sodium hydroxide solution (5mol·L ^-1 , 1.07mL) and 1- Iodo-3-fluoropropane (529.88 mg, 2.82 mmol). The reaction solution was stirred and reacted at 25°C for 16 hours. After the reaction of the raw materials was detected by TLC, the reaction solution was diluted with ethyl acetate (50 mL), washed with 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 the organic phases were combined and dried over sodium sulfate. The organic phase was concentrated to dryness under reduced pressure, and then purified by column chromatography (silica, dichloromethane/methanol=100/ 1) The product (S)-tert-butyl(1-(3-fluoropropyl)pyrrolidin-3-yl)aminomethyl ester (480.00 mg) was obtained.

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

¹ H NMR (400MHz,DMSO-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: N-((S)-1-(3-fluoropropyl)pyrrolidin-3-yl)-6-(((1S,3R)-3-methyl-2-(2,2,2 Synthesis of -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-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro -1H-pyrido[3,4-b]indole (140.00 mg, 263.99 μmol) was dissolved in tetrahydrofuran (3 mL) and (S)-1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride was added salt (86.77 mg, 316.79 μmol), sodium tert-butoxide (152.22 mg, 1.58 mmol) and (2-dicyclohexylphosphine)-3,6-dimethoxy-2,4,6-triiso Propyl-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II) (23.93mg, 26.40μmol), the reaction solution was at 80°C under nitrogen atmosphere Stir for 4 hours. After the LCMS detection reaction was completed, the reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure, and purified by preparative liquid chromatography (Phenomenex Gemini C18 column, 3um silica, 30mm diameter, 75mm length); (using water (containing 0.225% formic acid) and acetonitrile (decreasingly polar mixtures as eluents) were purified to give the compound N-((S)-1-(3-fluoropropyl)pyrrolidin-3-yl)-6-(((1S ,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-1 -yl)pyridin-3-amine (37.79 mg).

MS m/z(ESI):365.1[M+H] ⁺ ;

¹ H 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 NMR) of the compound of formula (I):

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

Embodiment 3-3: the preparation of formula (I) compound succinate

Put 5.0 g of free compound of formula (I) and 100 ml of isopropyl ether into a 250 ml single-necked bottle, and stir at room temperature until the solid is completely dissolved. Add 1.21 g of succinic acid, stir the reaction solution at room temperature overnight, filter it with suction, and dry it in vacuum at room temperature for 3 hours to obtain 5.4 g of solid.

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

resolve resolution:

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

Dissolve (S)-tert-butylpyrrolidin-3-ylcarbamate (500.00mg, 2.68mmol) in tetrahydrofuran (10mL), add sodium hydroxide solution (5mol·L ^-1 , 1.07mL) and 1 - Iodo-3-fluoropropane (529.88 mg, 2.82 mmol). The reaction solution was stirred and reacted at 25°C for 16 hours. After the reaction of the raw materials was detected by TLC, the reaction solution was diluted with ethyl acetate (50 mL), washed with 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 the organic phases were combined and dried over sodium sulfate. The organic phase was concentrated to dryness under reduced pressure, and then purified by column chromatography (silica, dichloromethane/methanol=100/ 1) The product (S)-tert-butyl(1-(3-fluoropropyl)pyrrolidin-3-yl)carbamate (480.00 mg) was obtained.

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

Dissolve (S)-tert-butyl(1-(3-fluoropropyl)pyrrolidin-3-yl)carbamate (480.00 mg, 1.93 mmol) in 1,4-dioxane (3 mL) , and then hydrochloric acid-1,4-dioxane solution (4moL·L ^-1 , 4.94mL) was added, and the reaction solution was a yellow transparent solution. The reaction solution was stirred at 25°C for 3 hours. After the reaction of the raw materials was detected by TLC, the reaction solution was concentrated under reduced pressure to obtain compound (S)-1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride (450.00 mg).

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

(R)-1-(1H-indol-3-yl)-N-(2,2,2-trifluoroethyl)propane-2-amine (890mg, 3.47mmol) and 4-bromo-2, 6-Difluorobenzaldehyde (844.27mg, 3.82mmol) was dissolved in toluene (10mL) and acetic acid (2mL), and the reaction solution was stirred at 90°C 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 obtain the product (1S,3R)-1-(4-bromo-2,6-di Fluorophenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole ( 850mg).

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

(1S,3R)-1-(4-bromo-2,6-difluorophenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4, 9-tetrahydro-1H-pyridyl[3,4-b]indole (80.00 mg, 174.20 μmol) was dissolved in tetrahydrofuran (2 mL), and (S)-1-(3-fluoropropyl)pyrrolidine was added- 3-Amine hydrochloride (38.20mg, 209.04μmol) and sodium tert-butoxide (100.45mg, 1.05mmol) were stirred evenly, and tris(dibenzylideneacetone)dipalladium (31.90mg, 34.84 μmol) and (±)-2,2-bis(diphenylphosphino)-1,1'-binaphthyl (54.23 mg, 87.10 μmol). The reaction solution was stirred and reacted at 80° C. for 4 hours. After the LCMS detection of raw material reaction is complete, after the reaction solution is filtered, the filter cake is rinsed with tetrahydrofuran, the filtrate is concentrated to dryness under reduced pressure, and purified by preparative liquid chromatography (Phenomenex Gemini C18 column, 7 μ m silica, 50 mm diameter, 250 mm length, using Water (containing 0.225% formic acid) and a mixture of decreasing polarity of acetonitrile as eluent) to obtain compound (S)-N-(3,5-difluoro-4-((1S,3R)-3-methyl -2-(2,2,2-Trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-1 -(3-fluoropropyl)pyrrolidin-3-amine (4.69 mg).

MS m/z(ESI):525.2[M+H] ⁺

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

resolve resolution:

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

MS m/z(ESI):265.0[M+H] ⁺

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

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

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

MS m/z(ESI):165.2[M+H] ⁺

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

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

MS m/z(ESI):543.1[M+H] ⁺

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

resolve resolution:

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

Dissolve tert-butyl N-[trans-4-methylpyrrolidin-3-yl]carbamate (80 mg, 399.45 μmol) and potassium carbonate (110.41 mg, 798.89 μmol) in acetonitrile (8 mL), then 1-Fluoro-3-iodo-propane (90.11 mg, 479.34 μmol) was added, and the reaction solution was heated to 50° C. and stirred for 16 h. After the reaction was monitored by LCMS, the reaction solution was filtered, and the filtrate was concentrated to dryness and purified by column chromatography (silica, ethyl acetate/methanol=5/1) to obtain the compound tert-butyl N-[trans-1-(3- Fluoropropyl)-4-methyl-pyrrolidin-3-yl]carbamate (85mg).

MS m/z(ESI):261.1[M+H] ⁺

¹ H 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

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

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

Trans-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-amine hydrochloride (30 mg, 128.67 μmol), (1S,3R)-1-(4-bromo-2, 6-difluoro-phenyl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b ] indole (76.82 mg, 167.27 μmol) and cesium carbonate (167.69 mg, 514.68 μmol) were dissolved in dioxane (8 mL), then methanesulfonic acid (2-di-tert-butylphosphine-3,6-di Methoxy-2',4',6'-triisopropyl-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II)( tBuBrettphos-Pd-G3, 5.50 mg, 6.43 μmol). The reaction solution was replaced with nitrogen three times, then heated to 120° C. and stirred overnight. After the reaction was completed by LCMS monitoring, methanol (15 mL) was added to the reaction solution, filtered, and the filtrate was concentrated and purified by column chromatography (silica, petroleum ether/ethyl acetate=2/1) and preparative liquid chromatography (Phenomenex Synergi C18 Column: 4 μm silica, 30 mm diameter, 150 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile as eluent) to give compound trans-N-[3,5-difluoro-4 -[(1S,3R)-3-Methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b] Indol-1-yl]phenyl]-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-amine (1.35 mg).

MS m/z(ESI):539.3[M+H] ⁺

¹ H 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).

Tests for Biological Activity and Related Properties

Test Example 1: Detection of SERD compounds on the degradation of estrogen receptors in MCF7 cells

1. Purpose of the experiment

The purpose of this experiment is to determine the degradative activity of the SERD compound in this paper on the endogenously expressed estrogen receptor in MCF7 cells, and evaluate the activity of the compound according to the DC ₅₀ and the maximum degradation efficiency.

2. Experimental method

MCF7 cells (ATCC, HTB-22) were cultured with DMEM (Gibco, 11995-065) complete medium containing 10% fetal bovine serum. On the first day of the experiment, MCF7 cells were seeded in a 384-well plate at a density of 3000 cells/well using complete medium, and cultured in a 5% CO ₂ cell incubator at 37°C. The compound to be tested was dissolved in DMSO with a storage concentration of 10 mM, diluted with Echo 550 (Labcyte Inc.) and added to the cell culture plate. The initial concentration of each compound was 100 nM, 3-fold serial dilution, 9 concentration points, and the settings contained A blank control of 0.5% DMSO was used, and a double-well control was set at each concentration point. Incubate in a 37°C, 5% CO ₂ cell incubator for 24 hours. Add paraformaldehyde to the cell culture medium in each cell culture well, and the final concentration of paraformaldehyde is about 3.7% to fix the cells. After acting for 30 minutes, discard the supernatant and add 50 μL PBS to each well to wash once; add PBS (containing 0.5% v/v Tween-20) to treat cells for 30 minutes, wash once with PBS; add blocking solution (PBS containing 5% BSA, 0.05% Tween-20) and incubate at room temperature for 1 hour; remove blocking solution and add primary antibody mixture (anti-ER Monoclonal antibody, Estrogen Receptorα (D8H8) Rabbit mAb, GST, #8644S, 1:1000 dilution; anti-GAPDH monoclonal antibody, GAPDH (D4C6R) Mouse mAb, GST, #97166S, 1:2000 dilution) incubated at room temperature for 3 hours; Wash 3 times with PBST (PBS containing 0.05% Tween-20); add detection secondary antibody (800CW-goat anti-rabbit IgG, LI-COR, P/N:926-32211, 1:1000 dilution; 680RD-goat anti-mouse IgG , LI-COR, #925-68070, 1:1000 dilution), room temperature, incubate in the dark for 45 minutes; wash 3 times in PBST, use Odyssey CLx to read the fluorescence signal of each well, calculate Chanel 800(ER)/Chanel 680(GAPDH ) value. The wells treated with 0.1 μM fulvestrant were used as the 100% degradation control, and the degradation rate at each concentration point was calculated. XlLfit was used to analyze and process the data, and the degradation activity DC ₅₀ and the maximum degradation rate Imax of each compound were calculated. See Table 1 for data analysis.

Table 1 SERD compounds degrade activity of estrogen receptor in MCF7 cells

化合物编号Compound number	ER level DC ₅₀(nM) ER level DC ₅₀ (nM)	最大降解率maximum degradation rate

化合物1Compound 1	0.410.41	106％106%

化合物2Compound 2	8.58.5	92％92%
式(I)化合物Compound of formula (I)	0.150.15	104％104%

化合物5Compound 5	0.580.58	80％80%
化合物6Compound 6	0.500.50	90％90%

Test Example 2: Detection of the inhibitory effect of SERD compounds on the proliferation of MCF7 cells

1. Purpose of the experiment

The purpose of this experiment is to determine the inhibitory effect of the SERD compounds herein on the proliferation of MCF7 cells in vitro, and to evaluate the activity of the compounds according to the IC ₅₀ and the maximum inhibitory efficiency.

2. Experimental method

MCF7 cells (ATCC, HTB-22) were cultured with DMEM (Gibco, 11995-065) complete medium containing 10% fetal bovine serum. On the first day of the experiment, MCF7 cells were seeded in a 384-well plate at a density of 500 cells/well using complete medium, and cultured overnight at 37°C in a 5% CO ₂ cell incubator. The next day, add the compound to be tested for drug treatment, and use Echo550 (Labcyte Inc.) to dilute the compound solution with a storage concentration of 10 mM and transfer it to each cell culture well. The initial concentration of each compound in the cell is 100 nM , 3-fold serial dilution, 9 concentration points, a blank control containing 0.3% DMSO was set, and double-well controls were set at each concentration point. Culture in a 37°C, 5% CO ₂ cell incubator for 7 days, and take out the cell culture plate on the 8th day. join in

Luminescent Cell Viability Assay (Promega, G7573), after standing at room temperature for 10 minutes, read the luminescence signal value using a multi-labeled microplate reader EnVision (PerkinElmer), and use XLfit to calculate the inhibitory activity IC ₅₀ of each compound according to the concentration and luminescence signal value of the compound . Data analysis see Table 2

Table 2 Inhibitory activity of SERD compounds on MCF7 cell proliferation

化合物编号Compound number	MCF7细胞增殖抑制IC ₅₀(nM) MCF7 cell proliferation inhibition IC ₅₀ (nM)
化合物1 Compound 1	0.580.58
化合物2 Compound 2	1515
式(I)化合物Compound of formula (I)	0.270.27
化合物4Compound 4	0.230.23
化合物5 Compound 5	2.572.57
化合物6Compound 6	2.392.39

Test Example 3: Inhibition of SERD Compounds on CYP2C9 and CYP2D6 Enzyme Activities

The inhibition of the SERD compounds herein on CYP2C9 and CYP2D6 enzyme activities was determined by the following test method.

1. Test materials and instruments

1. Human liver microsomes (Corning 452117)

2. NADPH (Solarbio 705Y021)

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

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

5.AB Sciex Triple Quad 5500 liquid mass spectrometer

2. Test steps

1. Preparation of 100mM phosphate buffer solution (PBS): Weigh 7.098g Na ₂ HPO ₄ , add 500mL pure water and ultrasonically dissolve it as solution A. Weigh 3.400g of KH ₂ PO ₄ , add 250mL of pure water to ultrasonically dissolve, and make solution B. Place A solution on a stirrer and slowly add B solution until the pH value reaches 7.4 to prepare 100mM PBS buffer.

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

3. Take 2 μl of 20 mg/ml liver microsome solution, 1 μl of substrate working solution, 1 μl of compound working solution and 176 μl of PBS buffer, mix well, and pre-incubate in a 37°C water bath for 15 minutes. To the positive control group, 1 μl of diclofenac, dextromethorphan or midazolam working solution was added instead of compound working solution. At the same time, 10 mM NADPH solution was pre-incubated in a 37° C. water bath for 15 minutes. After 15 minutes, add 20 μl NADPH to each well to start the reaction and incubate at 37°C for 5 minutes (CYP2C9) or 20 minutes (CYP2D6). All incubation samples were set as double samples. After incubating for the corresponding time, 400ul ice methanol containing internal standard was added to all samples to terminate the reaction. Vortex to mix well, and centrifuge at 3220g, 4°C for 40 minutes. After centrifugation, transfer 100 μL of the supernatant to the sample plate, add 100 μL of ultrapure water and mix well for LC-MS/MS analysis.

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

Drug-drug interaction (DDI) refers to the physical or chemical changes produced by two or more drugs, and the changes in drug efficacy caused by these changes. Understanding drug interactions can provide patients with better pharmaceutical services, promote rational drug use, and maximize the avoidance of adverse reactions. Drug interactions are mainly metabolic interactions, which are mainly related to CYP450 enzymes involved in drug metabolism. The experimental results in Table 3 show that the SERD compounds herein have weak inhibitory ability to CYP450, which indicates that the SERD compounds herein have a low potential risk of DDI.

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

化合物编号Compound number	CYP2C9(μM)CYP2C9(μM)	CYP2D6(μM)CYP2D6(μM)
式(I)化合物Compound of formula (I)	>30>30	>30>30
化合物4Compound 4	2020	2727

Test Example 4: Determination of Plasma Protein Binding Rate of SERD Compounds

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

The protein binding rate of the SERD compound in the plasma of five species (human, monkey, dog, rat and mouse) was determined by the following test method.

1. Test materials and instruments

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

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

3. Positive control compound warfarin;

4. ABI QTrap 5500 liquid mass spectrometer.

2. Test steps

1. Concentration is the preparation of the buffer solution of 100mM sodium phosphate and 150mM NaCl: prepare the alkaline solution that concentration is 14.2g/L Na _HPO ₄ and 8.77g/L NaCl with ultrapure water, prepare concentration with ultrapure water An acidic solution of 12.0g/L NaH ₂ PO ₄ and 8.77g/L NaCl. Titrate the alkaline solution with an acidic solution to pH 7.4 to prepare a buffer solution with a concentration of 100mM sodium phosphate and 150mM NaCl.

2. Preparation of the dialysis membrane: Soak the dialysis membrane in ultrapure water for 60 minutes to separate the membrane into two pieces, then soak it in 20% ethanol for 20 minutes, and finally soak it in the buffer used for dialysis for 20 minutes.

3. Preparation of plasma: Thaw the frozen plasma quickly at room temperature, then centrifuge the plasma at 4°C and 3,220g for 10 minutes to remove clots, and collect the supernatant into a new centrifuge tube. The pH of the plasma was measured and recorded, using plasma with a pH of 7-8.

4. Preparation of compound-containing plasma samples: Dilute 10 mM stock solution of SERD compound or positive control compound with DMSO to obtain 200 μM working solution. Add 3 μl of 200 μM compound working solution to 597 μl of human, monkey, dog, rat or mouse plasma to obtain a plasma sample with a final concentration of 1 μM.

5. Equilibrium dialysis step: assemble the dialysis device according to the operating instructions. 120 μL of plasma samples containing 1 μM compound were added to one side of the dialysis membrane, and an equal volume of dialysate (phosphate buffered saline) was added to the other side. The experiment has two samples. Seal the dialysis plate, put it into the incubation device, and incubate at 37° C., 5% CO ₂ and about 100 rpm for 6 hours. After incubation, remove the sealant and pipette 50 μl from the buffer and plasma side of each well into a different well of a new plate. Add 50 μl of blank plasma to the phosphate buffer sample, add an equal volume of blank phosphate buffer to the plasma sample, and then add 300 μl of acetonitrile containing internal standard to precipitate protein. Vortex for 5 minutes and centrifuge at 3,220 g for 30 minutes at 4°C. Take 100 μl of supernatant to the injection plate, add 100 μL of ultrapure water and mix well for LC-MS/MS analysis.

The peak areas of the compounds on the buffer side and the plasma side were determined. The formula for calculating the plasma protein binding rate of a compound is as follows:

Free rate% = (the ratio of the peak area of the compound to the peak area of the internal standard _{on the buffer side} /the ratio of the peak area of the compound to the peak area of the internal standard _{on the plasma side} ) × 100

Binding rate%=100-free rate%

All data were calculated by Microsoft Excel software. Calculated plasma protein binding values of SERD compounds.

Table 4 The protein binding rate values of SERD compounds in human, dog, rat and mouse plasma

Test Example 5: Apparent Solubility of SERD Compounds in Phosphate Buffer at pH 7.4

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

1. Materials and Reagents

The compounds to be tested were prepared according to the methods described. The control drug progesterone was purchased from Sigma. Phosphate buffer with a pH value of 7.4 was prepared by our laboratory. Acetonitrile and methanol were purchased from Fisher. Other reagents were purchased from the market.

1.5ml flat-bottomed glass vial (BioTech Solutions); PTFE/silicone stopper (BioTech Solutions); PTFE-coated stir bar; MultiScreenHTS HV (0.45μm) 96well plate filter plate (Millipore, MSHVN4510or MSHVN4550) ; Eppendorf Thermomixer Comfort; Vacuum Manifold ORVMN96 (Orochem).

2. Experimental steps

1) Preparation of stock solution

10mM stock solutions of the test substance and the control drug progesterone were prepared in DMSO.

Apparent Solubility Determination Procedure

Take 30 μL of 10mM stock solution of the analyte and add it to the corresponding position of the corresponding 96-well plate in the specified order. Add 970 μL of pH 7.4 phosphate buffer to the corresponding vial of the sample plate. The experiments were performed in double parallel. Add a stir bar to each vial and cap with a Teflon/silicone stopper. Then put the sample tray into the Eppendorf Thermomixer Comfort, and vibrate for 2 hours at 1100 rpm at 25°C. After 2 hours, remove the stopper, remove the stir bar with a large magnet, and transfer the sample from the sample plate to the filter plate. Use a vacuum pump to generate negative pressure and filter the sample. 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 may be possible to dilute the sample diluent with a certain percentage of 50% ACN(IS).H ₂ O to obtain better peak shape. The dilution factor may be adjusted due to the solubility of the analyte or the strength of its liquid-mass response signal.

3) Sample analysis steps

Place the sampling plate into the sampling tray of the autosampler and evaluate the sample by LC/MS.

3. Experimental steps

All calculations were performed by Microsoft Excel. The analysis and quantification of the sample filtrate is accomplished by using LC/MS to identify and quantify the peaks of standard products of known concentration. Calculated apparent solubility values of SERD compounds in phosphate buffer at pH 7.4.

Table 5 Apparent Solubility Values of SERD Compounds in Phosphate Buffered Saline at pH 7.4

化合物编号Compound number	pH＝7.4表观溶解度(μM)pH=7.4 apparent solubility (μM)
式(I)化合物Compound of formula (I)	9292
化合物4Compound 4	<0.3<0.3

Test Example 6: Whether SERD compounds have potential inhibitory effect on voltage-gated potassium ion channel hERG

The hERG potassium channel is critical for the heart's normal electrical activity. Cardiac arrhythmias can be induced by blockade of hERG channels with various drugs. This side effect is a common cause of drug failure in preclinical safety trials, so minimization of hERG channel-blocking activity may be a desirable property of a drug candidate.

1. Materials and Reagents

1. Experimental Materials and Instruments

2. Cell Lines and Culture

HEK293 cell line stably expressing hERG ion channel (product number: K1236) was purchased from Invitrogen. The cell line was cultured in 85% DMEM, 10% dialyzed fetal bovine serum, 0.1mM non-essential amino acid solution, 100U/mL penicillin-streptomycin solution, 25mM HEPES, 5μg/mL blasticidin and 400μg/mL genetic in the culture medium of mycomycin. When the cell density increases to 40%-80% of the bottom area of the culture dish, trypsin is used for digestion and passage, and the passage is carried out three times a week. Before the experiment, the cells were cultured in a 6 cm dish at a density of 5×10 ⁵ , induced by adding 1 μg/mL doxycycline for 48 hours, and then the cells were digested and seeded on glass slides for subsequent manual patch clamp experiments.

3. Configuration of test compounds

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

2) The stock solution was diluted with DMSO at a ratio of 1:3 to other three intermediate concentration solutions, the concentrations were 3.33mM, 1.11mM and 0.37mM, respectively.

3) Before the start of the experiment, dilute the stock solution of the compound to be tested and the intermediate solution 1000 times with extracellular fluid to obtain a series of working solutions with concentrations of 10 μM, 3.33 μM, 1.11 μM and 0.37 μM, and dilute the 10 mM stock solution with extracellular fluid at the same time 333.33 times to get a 30 μM working solution. The content of DMSO in the working solution is 0.1-0.3% (volume ratio).

4) After the preparation of the working solution is completed, observe with the naked eye whether there is precipitation or turbidity in the working solution. If there is, it may be due to the poor solubility of the compound in the physiological solution, and it can be further ultrasonicated in a water bath for 30 minutes to improve the clarity of the solution.

5) Determine the potential inhibitory effect of the test substance on the hERG channel at 5 concentrations of 30 μM, 10 μM, 3.33 μM, 1.11 μM and 0.37 μM, fit the dose-effect curve and calculate the IC ₅₀ .

2. Experimental method

1. Place the small slide containing HEK293 cells in the culture dish in the perfusion tank of the micromanipulator.

2. Place the appropriate cell in the center of the field of view under an Olympus IX51, IX71 or IX73 inverted microscope, use a ×10 objective lens to find the tip of the glass electrode, and place it in the center of the field of view. Then use the micromanipulator to move the electrode down while adjusting the coarse focus helix so that the electrode slowly approaches the cell.

3. When it is close to the cell, switch to the ×40 objective lens for observation, and fine-tune the gear through the micromanipulator to make the electrode gradually approach the surface of the cell.

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

5. Compensate the instantaneous capacitive current Cfast in the voltage clamp mode. Then repeated brief negative pressure was applied to permeate the membrane, and finally the whole-cell recording mode was formed.

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

7. After the cell is stable, change the clamping voltage to -90mV, set the sampling frequency to 20kHz, and filter frequency to 10kHz. The detection condition of the leakage current is that the clamping voltage is changed to -80mV, and the duration is 500ms.

8. The hERG current test method is as follows: apply a depolarization command voltage for 4.8 seconds to depolarize the membrane potential from -80mV to +30mV, and then apply a repolarization voltage for 5.2 seconds to reduce the membrane potential to -50mV to remove channel loss. live, so that the hERG tail current can be observed. The peak value of the tail current is the magnitude of the hERG current.

9. The hERG current used to detect the test compound was continuously recorded for 120 seconds before administration to evaluate the stability of the hERG current produced by the test cells. Only stable cells within the acceptance range of the evaluation criteria can enter the subsequent compound detection.

10. Determination of the inhibitory effect of the test compound on the hERG current: firstly, the hERG current measured in the extracellular fluid containing 0.1% DMSO was used as the detection baseline. After the hERG current remained stable for at least 5 minutes, the solutions containing the compound to be tested were perfused sequentially around the cells from low to high concentrations. Wait about 5 minutes after each perfusion to allow the compound to fully act on the cells and simultaneously record the hERG current. After the recorded current tends to be stable, record the last 5 hERG current values, and take the average value as the final current value at a specific concentration. After the compound was tested, 150 nM Dofetilide was added to the same cell to completely inhibit the current, which served as a positive control for the cell. At the same time, the positive compound Duofilide was detected synchronously with the same patch clamp system before and after the end of the test compound experiment to ensure the reliability and sensitivity of the entire detection system.

3. Data analysis

The data is output by PatchMaster software and analyzed according to the following steps:

After perfusing the blank solvent or compound gradient solution, obtain 5 continuous current values stably, calculate the average value, and use them as "tail current size _blank " and "tail current size _compound "respectively;

The percent current inhibition is calculated by the following formula.

The dose-response curve was fitted by Graphpad Prism 8.0 software and the _IC50 value was calculated.

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

Table 6 Inhibitory activity of SERD compounds voltage-gated potassium ion channel hERG

化合物编号Compound number	hERG IC ₅₀(μM) hERG _IC50 (μM)
式(I)化合物Compound of formula (I)	1010
化合物4Compound 4	3.73.7

Test Example 7: Mouse Pharmacokinetic Evaluation of SERD Compounds

Experimental Materials

CD-1 mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. DMSO (dimethyl sulfoxide), HP-β-CD (hydroxypropyl-β-cyclodextrin), tetraethylene glycol (Tetraethylene Glycol), Captisol (SBE-β-CD, sulfobutyl-β -cyclodextrin) was purchased from Sigma. Acetonitrile was purchased from Merck (USA).

experimental method

Six female CD-1 mice (20-30g, 4-6 weeks) were randomly divided into 2 groups with 3 mice in each group. The 1st group tail vein injection administration test compound, dosage is 1mg/kg, vehicle is the aqueous solution (95%, v/v) of DMSO (5%, v/v) and 10% HP-β-CD, the 2nd group is oral The test compound was administered at a dose of 10 mg/kg in a vehicle of tetraethylene glycol (40%, v/v) and 7.5% aqueous solution of sulfobutyl-β-cyclodextrin (60%, v/v). Animals were fed and watered normally before the experiment. Venous blood was collected from mice in each group before administration and at 0.083 (intravenous injection group only), 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after administration. The collected whole blood samples were placed in K ₂ EDTA anticoagulant tubes, centrifuged for 5 minutes (12,000 rpm, 4° C.) and plasma was collected for testing.

Take 10 μL of mouse plasma sample, add 150 μL of acetonitrile solvent (including internal standard compound) to precipitate protein, vortex for 5 min, centrifuge (14,000 rpm) for 5 min, and dilute the supernatant with water containing 0.1% (v/v) FA for 2 times, quantitative detection was performed on LC-MS/MS system (AB Sciex Triple Quad 6500+). When measuring the sample concentration, obtain the CD-1 mouse plasma standard curve and quality control samples at the same time. For 10× diluted samples, take 2 μL sample and add 18 μL blank plasma, vortex for 0.5 min, then add 300 μL acetonitrile solvent (including internal standard compound) to precipitate protein, and the rest of the processing steps are the same as for undiluted samples.

The results of the PK test are shown below, the SERD compound exhibited good PK properties and oral bioavailability in mice.

Table 7 PK of SERD compounds in mice

Test Example 8: SERD Compounds Permeability of Blood Brain Barrier (BBB) in Rats

Drugs can pass through the blood-brain barrier of animals and have sufficient exposure in the brain is the key to the effectiveness of drugs on brain metastases. Therefore, by measuring drug concentrations in plasma and brain tissue after administration to animals, the effect of drugs in the brain Distribution, and then judge whether the drug can inhibit tumor growth in the brain orthotopic model.

Experimental Materials

SD female rats were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. MC (methylcellulose) was purchased from Sigma; acetonitrile was purchased from Merck (USA). PBS (phosphate buffered saline) was purchased from Sangon Biotech.

experimental method

Six female SD rats (200-300g, 6-8 weeks) were randomly divided into 2 groups with 3 rats in each group. The SERD compounds herein were administered separately in a vehicle of 0.5% methylcellulose aqueous solution. Animals were fed water normally before the experiment, fasted overnight, and resumed feeding four hours after administration. Plasma and brain tissue were collected from rats in each group 2 hours after administration. The collected whole blood samples were placed in K ₂ EDTA anticoagulant tubes, centrifuged for 5 minutes (12,000 rpm, 4°C) to collect plasma for testing; tissues were collected and blotted dry with filter paper, and samples were stored in a -80°C refrigerator for testing.

Take 10 μL of rat plasma sample, add 150 μL of acetonitrile solvent (including internal standard compound) to precipitate protein, vortex for 5 min, centrifuge (14,000 rpm) for 5 min, and dilute the supernatant with water containing 0.1% (v/v) FA for 2 times, quantitative detection was performed on LC-MS/MS system (AB Sciex Triple Quad 6500+). For 10× diluted samples, take 2 μL sample and add 18 μL blank plasma, vortex for 0.5 min, then add 300 μL acetonitrile solvent (including internal standard compound) to precipitate protein, and the rest of the processing steps are the same as for undiluted samples. SD rat plasma standard curve and plasma quality control samples were obtained at the same time when the plasma sample concentration was determined.

Rat brain tissue samples were first homogenized with 4 times the mass volume of PBS homogenate. Take 20 μL of brain tissue homogenate sample, add 20 μL of blank mouse plasma to dilute and mix, then add 600 μL of acetonitrile solvent (including internal standard compound) to precipitate protein, vortex for 5 min, centrifuge (14,000 rpm) for 5 min, supernatant with Diluted 2 times with 0.1% (v/v) FA in water, and carried out quantitative detection on LC-MS/MS system (AB Sciex Triple Quad 6500+). Compared with the prior art or other molecules disclosed herein, the compound of formula (I) of the present disclosure exhibits excellent blood-brain barrier penetration ability and high drug exposure in rat brain tissue.

The BBB test results are as follows:

Table 8 SERD compound rat blood-brain barrier penetration test

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

17β-estradiol tablets: Innovative Research of America, Cat No.: SE-121, 60-day release, 0.72mg/pellet

EMEM medium: ATCC, Cat No.: 30-2003

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

Pen Strep (Pen Strep): Gibco, Cat No.: 15240-122

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

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

D-PBS (phosphate buffered saline without calcium and magnesium ions): Hyclone, Cat.No.: SH30256.01

Matrigel: Corning, Cat. No.: 356237

experimental method

Animal information: NPG mice, female, 6-7 weeks old, weighing about 19-28 grams, were purchased from Beijing Weitongda Biotechnology Co., Ltd., and the mice were raised in an SPF-grade environment, and each cage was sent separately All animals had free access to a standard certified commercial laboratory diet and water ad libitum.

Cell culture: In vitro culture of human breast cancer MCF-7 cell line, the culture conditions are 10% fetal bovine serum, 1% Pen Strep, 10 μg/ml recombinant human insulin in EMEM (cell culture medium), 37°C, 5% CO ₂ incubator. Routine digestion with 0.25% trypsin-EDTA digestion solution once a week for passage. When the cell saturation is 80%-90% and the number reaches the requirement, collect the cells and count them.

Cell inoculation: Subcutaneously inoculate 0.1ml/(containing 1×10 ⁷ ) MCF-7 cell suspension (D-PBS: Matrigel, volume ratio 1:1) on the right back of each mouse, and inoculate In the first four days, 17β-estradiol tablets were subcutaneously inoculated. On the 24th day after cell inoculation, the patients were randomly divided into groups according to the tumor volume, and the grouping day was Day 0.

Administration: The dosage of the compound of formula (I) is 1, 3, or 10 mg/kg, administered orally (PO), and administered once a day (QD) for 3 weeks. There were 8 mice in the vehicle group and 6 mice in the administration group.

Tumor measurements and experimental indicators:

Tumor diameters were measured twice a week with vernier calipers. The formula for calculating the tumor volume is: V=0.5a×b ² , where a and b represent the long diameter and short diameter of the tumor, respectively. Mouse body weights were measured twice a week.

The antitumor efficacy of compounds was evaluated by tumor growth inhibition rate TGI (%). TGI (%)=[(1-(average tumor volume at the end of administration of a certain treatment group-average tumor volume at the beginning of administration of this treatment group)/(average tumor volume at the end of treatment of the solvent control group-at the beginning of treatment of the solvent control group Average tumor volume)]×100%.

Experimental results:

See Table 9. In the mouse subcutaneous transplanted tumor MCF-7 model, the compound of formula (I) has a significant inhibitory effect on tumor growth (P<0.01) at 1 mg/kg, 3 mg/kg, or 10 mg/kg orally administered once a day (P<0.01), and has It has a good dose-response relationship, and has the effect of shrinking tumors at the doses of 3mg/kg and 10mg/kg. Oral administration of the compound of formula (I) once a day at 10 mg/kg has a significant inhibitory effect on tumor growth (P<0.01), and has the effect of shrinking tumors. The compound of formula (I) did not significantly affect the body weight of the mice at the doses tried.

Table 9 MCF-7 subcutaneous tumor model tumor volume

Test Example 10: Inhibitory experiment of the compound of formula (I) on the growth of mouse MCF-7 brain orthotopic tumor model

Experimental reagents/instruments:

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

EMEM medium: ATCC, Cat No.: 30-2003

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

Pen Strep (Pen Strep): Gibco, Cat No.: 15240-122

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

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

Brain Stereotaxic Instrument: Reward, Cat No.: Standard/Digital Display/Single Arm/Mouse/68055

Micro injection pump: KDS, Cat No.: Legato130

Miniature Handheld Skull Drill: Reward, Cat No.:78001

experimental method:

Animal information: NPG mice, female, 6-8 weeks old, weighing about 17-29 grams, were purchased from Beijing Weitongda Biotechnology Co., Ltd., and the mice were raised in an SPF-grade environment, and each cage was sent separately All animals had free access to a standard certified commercial laboratory diet and water ad libitum.

Cell culture: In vitro culture of human breast cancer MCF-7 cell line, the culture conditions are 10% fetal bovine serum, 1% Pen Strep, 10 μg/ml recombinant human insulin in EMEM (cell culture medium), 37°C, 5% CO ₂ incubator. Routine digestion with 0.25% trypsin-EDTA digestion solution was performed twice a week for subculture. When the cell saturation is 80%-90% and the number reaches the requirement, collect the cells and count them.

Cell inoculation: 15μl/(containing 2×10 ⁶ ) MCF-7 cell suspension was inoculated into the mouse skull using a brain localizer, a micro-injection pump and a miniature hand-held cranial drill, and 17β was subcutaneously inoculated three days before cell inoculation - Estradiol tablets. On the 8th day after cell inoculation, the mice were randomly divided into groups according to their body weight, and the grouping day was Day 0.

Administration: Fulvestrant (Fulvestrant, AstraZeneca) dosage is 250mg/kg, subcutaneous injection (SC), administration (QW) once a week, the dosage of formula (I) compound is 30mg/kg , administered orally (PO), administered once a day (QD). There were 11 mice in the vehicle group and 8 mice in the administration group. All groups continued administration until the mice died, were euthanized due to poor condition or the experiment ended.

Experimental observation and end:

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

On Day 48, the experiment was over and all mice were euthanized.

Experimental results:

See Figure 2 and Figure 3. In the orthotopic MCF-7 model of the mouse brain, the body weight of the mice in the Fulvestrant group (250mg/kg administered subcutaneously once a week) continued to decrease, and the survival status of the mice was not significantly different from that of the solvent control group (median survival period, vehicle 29 days in the control group and 29.5 days in the Fulvestrant group). The mice in the compound group of formula (I) (30mg/kg administered orally once a day) had a stable body weight and no abnormal state. Until the end of the experiment, the mice in the compound group of formula (I) did not die. Compared with the solvent control or Fulvestrant, the formula (I) The compound has a significant inhibitory effect on MCF-7 brain orthotopic tumor model mice, and the survival period of the mice is significantly prolonged (P<0.01).

Test Example 11: The effect of the compound of formula (I) combined with palbociclib on the cell cycle of human breast cancer MCF-7

Cell information: human breast cancer MCF-7 cells were purchased from ATCC, and the culture conditions were DMEM (purchased from Gibco) + 10% FBS (purchased from Gibco) + 0.01 mg/ml human insulin (purchased from Yisheng) + 1% non-essential Amino acids (purchased from Gibco). Palbociclib was purchased from MCE.

Experimental method: when the fusion rate of the cultured cells reaches over 70%, digest the cells, adjust the cell density to 1.5E5/0.5mL/well, plant in a 24-well cell culture plate (Greiner), and culture overnight. Add 0.4mL medium to each well, add 50μL 6.4, 1.6 and 0.4μM palbociclib respectively, the final concentration is 320, 80, 20nM, add 50μL 0.2% DMSO medium to single drug well and cell control well. 50 μL of 200 nM compound of formula (I) was added to the combined well with a final concentration of 10 nM. Add 50 μL of 0.1% DMSO medium to the single drug well and the cell control well. After compound treatment for 40 hours, the cells were collected, fixed with 70% ethanol (Shanghai Test) at -20°C, and treated with 30 μg/mL propidium iodide (Invitrogen), 50 μg/mL ribonuclease A (Sigma) and 0.2 % Triton X-100 (Sigma) was stained at 37°C for 30 minutes in the dark. Use a flow cytometer (BD) to detect red fluorescence at an excitation wavelength of 488 nm, and simultaneously detect light scattering. Cell cycle analysis was performed using Flowjo.

Experimental results: The compound of formula (I) can cause cell cycle arrest and arrest cells in G1 phase. After combined with palbociclib, it can significantly increase the proportion of G1 phase arrest and reduce the proportion of S phase cells. The results are shown in Table 10.

Table 10 Formula (I) compound combined with palbociclib on MCF-7 cell cycle arrest

Test Example 12: Inhibitory effect of the compound of formula (I) combined with palbociclib on the proliferation of human breast cancer MCF-7 cells

Cell and compound information: human breast cancer MCF-7 was purchased from ATCC, and the culture conditions were DMEM (Gibco) + 10% FBS (Gibco) + 0.01 mg/ml human insulin (Yisheng) + 1% non-essential amino acids (Gibco). Palbociclib was purchased from MCE.

Experimental method: Human breast cancer MCF-7 was cultured. When the cell fusion rate reached over 70%, the cells were digested, and the density was adjusted to 500/20 μL/well, and the species was cultured overnight in a 384-well cell culture plate (Corning). Use Echo650 (Beckman) to transfer 120nL of 2-fold serial dilution of the compound of formula (I) (in the form of succinate) to the cell plate, and transfer 120nL of 2-fold serial dilution of palbociclib to the cell plate at the same time, add 40 μL of medium, After 7 days of compound treatment, CellTiter-Glo (Promega) was added to detect cell viability, cell wells were used as 100% viability control, medium wells were used as 0% viability control, combined analysis was carried out using Combinefit (a value greater than 10 indicated that a better combination was obtained) with synergistic effects).

Results: The compound of formula (I) combined with palbociclib has a synergistic effect on the proliferation inhibition of human breast cancer MCF-7 cells, and the results are shown in FIG. 4 .

Test Example 13: Combination pharmacodynamic study of human breast cancer MCF-7 xenograft subcutaneous xenograft mouse model

Experimental Materials:

Human breast cancer MCF-7 cells: ECACC, 86012803

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

EMEM medium: ATCC, Cat No.: 30-2003

Fetal bovine serum: ExCell; Cat No.: FND500

Double antibody: Gibco, Cat No.: 15240-062

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

DPBS: Corning, Cat. No.: 21-031-CVR

Matrigel: Corning, Cat.No.: 354234

Palbociclib: Simcere Pharmaceutical Co., Ltd. (generic), 125mg/capsule

experimental method:

Animal information: Balb/c nude mice, female, 6-8 weeks old, weighing about 18-22 grams, were purchased from Shanghai Lingchang Biotechnology Co., Ltd., and the mice were raised in an SPF-grade environment. Each cage Separate exhaust ventilation was provided and all animals had free access to a standard certified commercial laboratory diet and water ad libitum.

Cell culture: human breast cancer MCF-7 cell line was cultured in vitro, and the culture conditions were 10% fetal bovine serum and 1% double antibody in EMEM (cell culture medium), 37° C., 5% CO ₂ incubator. Routine digestion with 0.25% trypsin-EDTA digestion solution was performed twice a week for subculture. When the cell saturation is 80%-90% and the number reaches the requirement, collect the cells and count them.

Cell inoculation: Inoculate 0.2ml/(containing 1× ¹⁰⁷ ) MCF-7 cell suspension (DPBS: Matrigel, volume ratio 1:1) subcutaneously on the right back of each mouse, and inoculate 17β-estradiol tablets were subcutaneously inoculated two days before. On the 6th day after cell inoculation, when the average volume of the tumor reached 159.01 mm ³ , the administration began in groups, and the administration was randomly divided into groups according to the tumor volume. The day of grouping was Day 0.

Administration: The dosage of the compound of formula (I) (in the form of succinate, the dose is calculated as free base) is 1 mg/kg, administered orally (PO), once a day (QD)×25 times. The dosage of palbociclib is 40 mg/kg, administered orally (PO), administered once a day (QW) x 25 times. 8 mice per group.

Tumor measurements and experimental indicators:

Experimental results:

See Table 11, Figures 5 and 6. The vehicle group has 1 mouse body weight to drop more than 10%, palbociclib (40mg/kg) group has 4 mice body weight to drop more than 10%, palbociclib (40mg/kg) and formula (I) compound (1mg/kg) kg) combination group, there was one mouse whose body weight decreased by more than 10%. It can be seen that in this model, 40mg/kg of palbociclib has a certain effect on animal body weight. There was no morbidity or mortality in this model.

The test results showed that on the 24th day after the start of administration (Day 24), palbociclib administered orally once a day to tumor-bearing mice at 40 mg/kg showed a certain effect of inhibiting tumor growth (P<0.0001); 1mg/kg of the compound of formula (I) in tumor-bearing mice showed a significant effect of inhibiting tumor growth (P<0.0001); Compared with the vehicle control group and the corresponding single drug group, it showed a significantly enhanced tumor inhibitory effect.

Table 11 Tumor volume of MCF-7 subcutaneous tumor model

Unless expressly excluded or otherwise limited, every document cited herein, including any cross-referenced patent or patent application, or any patent application or patent from which this application claims priority, is hereby incorporated by reference in its entirety and into this article. Furthermore, to the extent that any meaning or definition of a term herein 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 and at least one CDK4/6 inhibitor, the selective estrogen receptor down-regulator being selected from compounds represented by formula (K) or a pharmaceutically acceptable salt thereof:

in,

R 1 , R 2 , R 3 , R 4 are independently selected from H, F, Cl, Br, I, CN, C 1 -C 6 alkyl, C 1 -C 6 alkoxy or C 3 -C 6 cycloalkane base;

X 1 , X 2 , X 3 , X 4 are independently selected from CR 6 or N;

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

R 5 is independently selected from C 1 -C 6 alkyl, and the C 1 -C 6 alkyl is optionally substituted by 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 represented by the formula (K) or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:
The pharmaceutical combination according to claim 1 or 2, wherein the CDK4/6 inhibitor is selected from the group consisting of abeciclib, ribociclib, palbociclib, 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 their pharmaceutically acceptable salts.
The pharmaceutical combination according to any one of claims 1 to 3, wherein the compound represented by formula (K) or a pharmaceutically acceptable salt thereof is selected from a compound of formula (I) or a pharmaceutically acceptable salt thereof :
The pharmaceutical combination according to any one of claims 1 to 4, wherein the CDK4/6 inhibitor is selected from palbociclib or a pharmaceutically acceptable salt thereof.
A combination product, the combination product comprising the first pharmaceutical composition and the second pharmaceutical composition, the first pharmaceutical composition comprising the compound shown in formula (K) as described in any one of claims 1 to 5 Or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable auxiliary material, the second pharmaceutical composition comprises at least one CDK4/6 inhibitor and a pharmaceutically acceptable auxiliary material.
The combination product according to claim 6, wherein the compound represented by formula (K) or a pharmaceutically acceptable salt thereof in the first pharmaceutical composition is selected from a compound represented by formula (I) or a pharmaceutically acceptable salt thereof acceptable salt.
The combination product according to claim 6 or 7, wherein the CDK4/6 inhibitor in the second pharmaceutical composition is selected from the group consisting of abeciclib, ribociclib, palbociclib, 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 their pharmaceutically acceptable salts.
A pharmaceutical composition, comprising the pharmaceutical combination according to any one of claims 1 to 5, and pharmaceutically acceptable auxiliary materials.
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 anti-tumor use.
The use according to claim 10, wherein the tumor is breast cancer.
The use according to claim 10, wherein the tumor is ER positive breast cancer.
The use according to claim 10, wherein the tumor is ER-positive brain metastatic breast cancer.
The use according to claim 10, wherein the tumor is ER-positive, HER-2-negative locally advanced or metastatic breast cancer.