TW202333704A - Drug combination for treating tumor, and application thereof - Google Patents

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

Drug combinations for treating tumors and their applications

The present disclosure belongs to the field of medicine and relates to a pharmaceutical 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 pharmaceutical combination containing the pharmaceutical combination substances, and their use in the treatment of tumors.

Estrogen (E2) and estrogen alpha receptor (ERα) are important driving factors in the development of breast cancer. More than two-thirds of breast cancer patients express ER transcription factors, and in the majority of ER-positive patients, ER remains a key driver of tumors that progress even after early endocrine therapy, so ER is A major target for breast cancer treatment (Pharmacology & Therapeutics 186 (2018) 1–24). The purpose of endocrine therapy is to reduce ER activity. There are three main categories, including selective estrogen receptor modulators (SERMs), such as tamoxifen, which is an allosteric regulator of ER and inhibits its transcriptional activity after binding to ER. ; Aromatase inhibitors (AIs), which reduce estrogen levels in the body by inhibiting the conversion of androgens into estrogen; and selective estrogen receptor downregulators, such as fulvestrant, which not only act 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 estrogen receptor (ER) positive. The proliferation of this type of breast cancer cells depends heavily on ER, and 50% of breast cancer death cases are of this type. Early-stage ER-positive breast cancer has a better prognosis, with a 5-year survival rate of over 90%. About 30% of patients who receive postoperative endocrine therapy (TAM or AI drugs) relapse within 10 years, but they can 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. Multiple research data show that patients treated with fulvestrant fail to completely degrade ER. In addition, intramuscular injection causes obvious reactions such as pain, swelling, and redness at the injection site, slow absorption, and limited body exposure. Its characteristics limit its clinical application, so patients with ER-positive breast cancer are in urgent need of 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 (chemical name: 6-acetyl-8-cyclopentyl-5-methyl-2-[[5-(1-pyridinyl)-2-pyridyl]amino]pyrido[ 2,3-d]pyrimidine-7(8H)-one), as a CDK4/6 selective inhibitor, can be used for the treatment of cancer patients.

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 the group consisting of compounds represented by formula (K) or its pharmaceutically acceptable salt: (K) Wherein, R ¹ , R ² , R ³ and 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 cycloalkyl} ; X _{1 ,} X ₂ ^, X _{3 ,} Alkyl, C ₃ -C ₁₀ cycloalkyl, 3-10 membered heterocyclyl, C ₁ -C ₁₀ alkoxy, C ₃ -C ₁₀ cycloalkyloxy or 3-10 membered heterocyclyloxy; R ⁵ is independently selected from C _{1 -C 6} alkyl, which 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.

On the other hand, the present disclosure provides a combination product, the combination product includes a first pharmaceutical composition and a second pharmaceutical composition, and the first pharmaceutical composition includes at least one compound represented by formula (K) or its pharmaceutical composition. Acceptable salts and pharmaceutically acceptable auxiliary materials, the second pharmaceutical composition includes at least one CDK4/6 inhibitor and pharmaceutically acceptable auxiliary materials.

On the other hand, the present disclosure also provides a medicine kit containing: A first container containing the first pharmaceutical composition as described above; and, A second container containing the II pharmaceutical composition as described above. On the other hand, the present disclosure also provides a pharmaceutical composition, which includes any of the above pharmaceutical combinations and at least one pharmaceutically acceptable excipient.

On the other hand, the present disclosure relates to the use of any of the above pharmaceutical combinations, combination products or pharmaceutical compositions in the preparation of anti-tumor drugs.

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

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

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

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

In order to facilitate understanding of the disclosure herein, a more complete description is provided below. However, these embodiments may be implemented in many different forms and are not limited to the specific examples described herein. Rather, these embodiments are provided so that a thorough and complete understanding of the disclosure herein will be provided.

Definitions and explanations of terms

Unless otherwise stated, the definitions of groups and terms recorded in the description and claims, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in the examples, etc., can be Any combination and combination with each other. Such combinations and combined group definitions and compound structures should fall within the scope described in the specification.

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

The term "pharmaceutically acceptable salts" refers to pharmaceutically acceptable salts of nontoxic acids or bases, including salts of inorganic acids and bases, organic acids and bases, such as succinates.

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 pharmaceutical compositions is to facilitate administration to a subject of a compound of the present disclosure, or a pharmaceutical combination thereof.

In this article " ” indicates the connection site.

The schematic representation of racemic or enantiopure compounds herein is taken from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise stated, wedge and virtual wedge bonds ( and ) represents the absolute configuration of a stereocenter, using black real and imaginary bonds ( and ) represents the relative configuration of a stereocenter (such as the cis-trans configuration of an alicyclic compound).

The term "tautomer" refers to a functional group isomer 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 yield a mixture whose physical and chemical 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 is dominant; in phenols, the enol form is dominant. This disclosure includes all tautomeric forms of the compounds.

The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, 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 therefore 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. These isomers and their mixtures involved in all substituents are also included in Within the definition of the compounds of this disclosure. Compounds of the present disclosure containing asymmetric atoms can be isolated in optically active pure form or in a racemic form, the optically active pure form can be resolved from a racemic mixture, or by using chiral starting materials or chiral reagents synthesis.

The term "substituted" means that any one or more hydrogen atoms on a specific atom are replaced by a substituent, as long as the valence state of the specific atom is normal and the substituted compound is stable.

The term "optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes both the occurrence and absence of the stated event or circumstance. For example, the ethyl group is "optionally" substituted by halogen, which means that the ethyl group can be unsubstituted (CH ₂ CH ₃ ), monosubstituted (CH ₂ CH ₂ F, CH ₂ CH ₂ Cl, etc.), or polysubstituted. (CHFCH ₂ F, CH ₂ CHF ₂ , CHFCH ₂ Cl, CH ₂ CHCl _2, etc.) or completely substituted (CF ₂ CF ₃ , CF ₂ CCl ₃ , CCl ₂ CCl _3, etc.). It will be understood by those skilled in the art that any substitution or substitution pattern that is sterically impossible and/or cannot be synthesized will not be introduced for any group containing one or more substituents.

When any variable (e.g., R ^a , R ^b ) occurs more than once in the composition or structure of a compound, its definition in each instance is independent. For example, if a group is replaced by 2 R ^b , there are separate options for each R ^b .

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

The term "alkyl" refers to a hydrocarbon group of the general formula C _n H _2n+1 , which 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 radical having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. Specific examples of the alkyl group include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2- Methylbutyl, 1-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 to mean an alkyl group with 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 straight-chain 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 other ranges, and the "C ₁ -C ₆ alkyl" may further include " C ₁ -C ₃ alkyl”.

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

The term "cycloalkyl" refers to a fully saturated carbocyclic ring that exists in the form of a single ring, a fused ring, a bridged ring or a spiro ring. Unless otherwise indicated, the carbocyclic ring is generally 3 to 10 membered. The term "C ₃ -C ₁₀ cycloalkyl" is understood to mean a saturated monocyclic, paracyclic, spirocyclic or bridged ring having 3 to 10 carbon atoms. Specific examples of the cycloalkyl group include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl (bicyclo[2.2 .1] heptyl), bicyclo[2.2.2]octyl, adamantyl, spiro[4.5]decyl, etc. The term "C ₃ -C ₁₀ cycloalkyl" may include "C ₃ -C ₆ cycloalkyl", and the term "C ₃ -C ₆ cycloalkyl" may be understood to mean a saturated monocyclic or bicyclic hydrocarbon ring having 3 to 6 carbon atoms, specific examples include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, etc.

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

The term "heterocyclyl" refers to a fully saturated or partially saturated (not heteroaromatic as a whole) monocyclic, fused ring, spirocyclic or bridged ring group containing 1 to 5 ring atoms. Heteroatom or heteroatom group (that is, an atomic group containing heteroatoms), the "heteroatom or heteroatom group" includes but is not limited to nitrogen atom (N), oxygen atom (O), sulfur atom (S), phosphorus atom (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 heterocyclyl" refers to a heterocyclyl with a number of ring atoms of 3, 4, 5, 6, 7, 8, 9 or 10, and its ring atoms contain 1 to 5 independently selected from the above The heteroatom or heteroatom group. In particular, the heterocyclyl group may include, but is not limited to: specific examples of 4-membered heterocyclyl groups include, but are not limited to, azetidinyl or oxetanyl; specific examples of 5-membered heterocyclyl groups include but Not limited to tetrahydrofuryl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydroxazolyl or 2,5-dihydro-1H-pyrrole group; specific examples of 6-membered heterocyclic groups include, but are not limited to, tetrahydropyranyl, pyridinyl, morpholinyl, dithialkyl, thiomorpholinyl, pyrazinyl, tristhialkyl, tetrahydropyranyl Pyridyl or 4H-[1,3,4]thiadiazinyl; specific examples of 7-membered heterocyclyl include but are not limited to diazepanyl. The heterocyclic group may also be a bicyclic group, wherein specific examples of 5,5-membered bicyclic groups include but are not limited to hexahydrocyclopenta[c]pyrrole-2(1H)-yl; 5,6-membered bicyclic groups. Specific examples include, but are not limited to, hexahydropyrro[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 the present disclosure partially contain a benzene ring or a heteroaromatic ring, the heterocyclyl groups are still non-aromatic as a whole.

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, ameliorate, or eliminate a disease or one or more symptoms associated with the disease, and includes: (i) prevent the occurrence of a disease or disease state in mammals, particularly where such mammals are susceptible to, but have not yet been diagnosed with, that disease state; (ii) inhibit a disease or disease state, that is, arrest its progression; (iii) Alleviation of a disease or condition, i.e. resolution of the disease or condition.

The term "therapeutically effective amount" means (i) treating a specified disease, condition, or disorder, (ii) alleviating, ameliorating, or eliminating one or more symptoms of a specified disease, condition, or disorder, or (iii) delaying the onset of a specified disease, condition, or disorder described herein. An amount of a compound of the disclosure that is responsible for the onset of one or more symptoms of a disease, condition or disorder. The amount of a compound of the present disclosure that constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one skilled in the art. based on their 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 human.

The term "administering" means physically introducing a composition comprising a therapeutic agent to a subject using any of a variety of methods and delivery systems known to those skilled in the art. Routes of administration for 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 inhibitor are in separate formulations, the SERD and CDK4/6 inhibitor can be administered simultaneously, separately, or sequentially.

The term "pharmaceutically acceptable excipients" refers to those excipients that have no obvious irritating effect on the organism and do 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, etc.

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

The present application also includes compounds of the present application that are the same as those described herein, but are isotopically labeled in which one or more atoms are replaced by an atom having an atomic weight or mass number different from that typically 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 respectively 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 with ³ H and ¹⁴ C) can be used in compound and/or substrate tissue distribution analyses. Tritiated (i.e. ³ H) and carbon-14 (i.e. ¹⁴ C) isotopes are particularly preferred due to their ease of preparation and detectability. Positron-emitting isotopes such as ¹⁵ O, ¹³ N, ¹¹ C, and ¹⁸ F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the present application can generally be prepared by substituting an isotopically labeled reagent for a non-isotopically labeled reagent by the following formula similar to those disclosed in the Schemes and/or Examples below.

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

The pharmaceutical compositions of the present application can be prepared by combining the compounds of the present application with appropriate pharmaceutically acceptable excipients, for example, they 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 administration of the compounds of the present application or pharmaceutically acceptable salts thereof or pharmaceutical compositions thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, Intramuscular, subcutaneous, and intravenous administration.

The pharmaceutical composition of the present application can be manufactured by methods well known in the art, such as conventional mixing methods, dissolving methods, granulation methods, sugar-coated pill making methods, grinding methods, emulsification methods, freeze-drying methods, etc.

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

Solid oral compositions may be prepared by conventional mixing, filling or tableting methods. For example, it can be obtained by the following method: mixing the active compound with solid excipients, optionally grinding the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into granules to obtain tablets Or sugar-coated core. Suitable excipients include but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, etc.

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

The present disclosure provides a pharmaceutical combination, which comprises at least one selective estrogen receptor downregulator (SERD) and at least one CDK4/6 inhibitor. The SERD is selected from the group consisting of compounds represented by formula (K) or pharmaceuticals thereof. Acceptable salt: (K) Wherein, R ¹ , R ² , R ³ and 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 cycloalkyl} ; X _{1 ,} X ₂ ^, X _{3 ,} Alkyl, C ₃ -C ₁₀ cycloalkyl, 3-10 membered heterocyclyl, C ₁ -C ₁₀ alkoxy, C ₃ -C ₁₀ cycloalkyloxy or 3-10 membered heterocyclyloxy; R ⁵ is independently selected from C _{1 -C 6} alkyl, which 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 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 represented by formula (K) Selected from , , or .

In some embodiments, the structural unit in the compound represented by formula (K) Selected from or .

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

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

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

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

Furthermore, the present disclosure provides a pharmaceutical combination, which comprises a 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 includes a first pharmaceutical composition and a second pharmaceutical composition. The first pharmaceutical composition includes at least one compound represented by formula (K) or a pharmaceutically acceptable salt thereof. and pharmaceutically acceptable auxiliary materials. The second pharmaceutical composition includes at least one CDK4/6 inhibitor and pharmaceutically acceptable auxiliary materials.

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 group consisting of compounds represented by formula (I) or a pharmaceutically acceptable salt thereof.

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

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

The present disclosure also provides a pill kit containing: A first container containing the first pharmaceutical composition as described above; and, A second container containing the II pharmaceutical composition as described above.

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

In a preferred embodiment of the present disclosure, the pharmaceutical composition comprises: The compound 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 pharmaceutical combinations, combination products or pharmaceutical compositions in the preparation of anti-tumor drugs.

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

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

The present disclosure relates to anti-tumor methods, which methods include administering a therapeutically effective amount of any of the above pharmaceutical combinations, combination products or pharmaceutical compositions to a patient in need thereof.

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

Further, the present disclosure relates to the use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof in combination with a CDK4/6 inhibitor in the preparation of anti-tumor drugs; 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 (based on free base).

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

The SERD compounds herein have good in vitro and in vivo anti-tumor activity and druggability. In vivo tests have found that the SERD compounds in this article can significantly inhibit tumor growth in ER-positive breast cancer mouse models, and significantly improve the survival of ER-positive breast cancer brain orthotopic model mice. In addition, the SERD compounds herein have 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 brain metastases from ER-positive breast cancer).

The combination of a SERD compound herein and a CDK4/6 inhibitor (such as palbociclib) produces superior efficacy in reducing tumor growth or even eliminating tumors compared to either drug in the combination given alone. synergistic anti-tumor effect.

The chemical reactions of the specific embodiments of the present disclosure are completed in a suitable solvent, and the solvent must be suitable for the chemical changes of the present disclosure and the required reagents and materials. In order to obtain the compounds of the present disclosure, those skilled in the art sometimes need to modify or select synthetic steps or reaction procedures based on 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, embodiments formed by their combination with other chemical synthesis methods, and those well known to those skilled in the art. Equivalent alternatives and preferred implementations include but are not limited to the embodiments of the present disclosure.

Example

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

Unless otherwise stated, the ratios expressed for mixed solvents are volumetric mixing ratios.

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

Compounds were named manually or using ChemDraw® software, and commercially available compounds were named using supplier catalog names.

The structure of the compound is determined by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS). The unit of NMR shift is 10 ^-6 (ppm). The solvents used for NMR measurement are deuterated dimethyl styrene, 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-trifluoroethyl) 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-yl carbamate (1.00g, 5.37mmol) in tetrahydrofuran (10mL), add sodium hydroxide solution (5mol﹒L ^-1 , 2.15mL) and 1-iodo-3 -Fluoropropane (1.06g, 5.64mmol). The reaction solution was stirred at 25°C for 16 hours. After TLC detects that the raw material reaction is complete, the reaction solution is diluted with ethyl acetate, washed with saturated ammonium chloride solution, and the aqueous phase and organic phase are collected respectively. After the aqueous phase was extracted three times with ethyl acetate (50 mL), all organic phases were combined, dried over sodium sulfate, concentrated to dryness under reduced pressure, and then purified by column chromatography (silica, dichloromethane/methanol = 100/ 1) Obtain the product tert-butyl (1-(3-fluoropropyl)pyrrolidin-3-yl) carbamate (0.81g).

¹ H NMR (400 MHz, METHANOL- d ₄ ) δ 4.57 (t, J = 5.77 Hz, 1H), 4.45 (t, J = 5.77 Hz, 1H), 4.11 (br d, J = 7.78 Hz, 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 (0.81g, 3.12mmol) in 1,4-dioxane (9mL), then add hydrochloric acid -1,4-dioxane solution (4moL﹒L ^-1 , 9mL), the reaction solution is a yellow transparent solution. The reaction solution was stirred at 25°C for 3 hours. After TLC detects that the reaction of the raw materials is complete, the reaction solution is concentrated to dryness under reduced pressure to obtain compound 1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride (0.71g).

¹ H NMR (400 MHz, METHANOL- d ₄ ) δ 4.68 (t, J = 5.52 Hz, 1H), 4.56 (s, 1H), 4.30-3.79 (m, 3H), 3.68 (s, 1H), 3.48 ( br s, 2H), 3.31-3.21 (m, 1H), 2.82-2.46 (m, 1H), 2.32-2.14 (m, 3H).

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 (600mg, 3.44mmol) and N,N-diisopropylethylamine (445.05mg, 3.44mmol) in 1, To 4-dioxane (10 mL), add trifluoroethyl trifluoromethanesulfonate (1.20 g, 5.17 mmol) dissolved in 1,4-dioxane (5 mL) 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)propane-2-amine (0.69 g).

MS m/z (ESI): 257.2 [M+H] ⁺

¹ H NMR (400MHz, METHANOL-d ₄ ) δ 7.56-7.54 (d, J = 8.0 Hz, 1H), 7.36-7.34 (d, J = 8.0 Hz, 1H), 7.12-7.08 (m, 2H), 7.03 -7.00 (m, 1H), 3.26-3.23 (m, 2H), 3.12-3.10 (m, 1H), 2.93-2.88 (m, 1H), 2.80-2.78 (m, 1H), 1.11 (d, J = 6.0 Hz, 3H).

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)propane-2-amine (120.00 mg, 468.26 μmol) in toluene (2 mL) , add 5-bromo-pyridine-2-carboxaldehyde (87.10 mg, 468.26 μmol) and acetic acid (562.40 mg, 9.37 mmol), and the reaction solution becomes a yellow transparent solution. The reaction solution was stirred at 90°C for 10 hours. After the LCMS detection reaction is completed, the reaction solution is 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 (110.00mg).

MS m/z (ESI):424.1, 426.1 [M+H] ⁺

¹ H NMR (400 MHz, METHANOL- d ₄ ) δ 8.59 (s, 1H), 8.01-7.94 (m, 1H), 7.54 (d, J = 8.53 Hz, 1H), 7.46 (d, J = 7.78 Hz, 1H), 7.30 (d, J = 8.03 Hz, 1H), 7.08 (d, J = 7.59 Hz, 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 = 4.52 Hz, 1H), 2.70-2.61 (m, 1H), 1.26 (d, J = 6.78 Hz , 3H).

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

(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, 311.13 μmol), sodium tert-butoxide (149.50 mg, 1.56 mmol) and methanesulfonic acid (2-dicyclohexylphosphine)-3,6-dimethoxy-2,4,6-triisopropyl- 1,1'-biphenyl) (2-amino-1,1'-biphenyl-2-yl)palladium (II) (23.50 mg, 25.93 μmol), the reaction solution was a brown turbid solution under nitrogen atmosphere. The reaction solution was stirred at 80°C for 4 hours. After the LCMS detection reaction is completed, the reaction solution is cooled to room temperature, filtered, and the filtrate is concentrated under reduced pressure and purified by preparative liquid chromatography (Phenomenex Gemini C18 column, 3 μm silica, 30 mm diameter, 75 mm length); (use water ( Compound N-(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3 was purified using a mixture of decreasing polarity containing 0.225% formic acid) and acetonitrile as eluent). -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 (400 MHz, METHANOL- d ₄ ) δ 7.88 (d, J = 2.76 Hz, 1H), 7.46 (d, J = 7.78 Hz, 1H), 7.25 (dd, J = 7.91, 3.89 Hz, 2H) , 7.09-6.96 (m, 3H), 4.97 (s, 1H), 4.60 (t, J = 5.65 Hz, 1H), 4.48 (t, J = 5.65 Hz, 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 = 6.53 Hz, 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)) Synthesis method 1

The racemate N-(1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl) (80.00 mg, 153.61 μmol) was chiral separated (DAICEL CHIRALPAK AY-H column, 5 μm silica, 30 mm diameter, 250 mm length, using decreasingly polar mixtures of isopropyl alcohol (containing 0.1% ammonia) and water as eluent) and preparative liquid chromatography purification (Phenomenex Gemini C18 column , 3μm silica, 30mm diameter, 75mm length, purified using water (containing 0.05% ammonia) and a mixture of decreasing polarity of acetonitrile as eluent to obtain N-(R)(1-(3-fluoropropyl)pyrrole Alk-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyridine and [3,4-b]indol-1-yl)pyridin-3-amine (8.22 mg, retention time 2.627 min) and N-(S)(1-(3-fluoropropyl)pyrrolidine-3 -base)-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 (400 MHz, METHANOL- d ₄ ) δ 7.89 (d, J = 2.76 Hz, 1H), 7.48-7.44 (m, 1H), 7.29-7.24 (m, 2H), 7.09-7.04 (m, 2H ), 7.00 (s, 1H), 4.98 (s, 1H), 4.64-4.60 (m, 1H), 4.52-4.48 (m, 1H), 4.25-4.16 (m, 1H), 3.54-3.36 (m, 4H ), 3.25-3.09 (m, 4H), 3.05 (s, 1H), 2.89 (d, J = 4.52 Hz, 1H), 2.70-2.60 (m, 1H), 2.55-2.42 (m, 1H), 2.16- 1.94 (m, 3H), 1.20 (d, J = 6.78 Hz, 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 (400 MHz, METHANOL- d ₄ ) δ 7.90-7.88 (m, 1H), 7.48-7.43 (m, 1H), 7.29-7.23 (m, 2H), 7.09-7.03 (m, 2H), 7.03 -6.97 (m, 1H), 4.99-4.97 (m, 1H), 4.63-4.59 (m, 1H), 4.52-4.47- (m, 1H), 4.24-4.16 (m, 1H), 3.52-3.36 (m , 4H), 3.15 (br s, 4H), 3.05-2.99 (m, 1H), 2.96-2.88 (m, 1H), 2.68 (s, 1H), 2.57-2.43 (m, 1H), 2.12-1.94 ( m, 3H), 1.20 (d, J = 6.53 Hz, 3H).

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

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

Dissolve (S)-tert-butylpyrrolidin-3-ylcarbamomethyl 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 at 25°C for 16 hours. After the complete 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 organic phase were collected respectively. After the aqueous phase was extracted three times with ethyl acetate (20 mL), all the organic phases were combined, dried over sodium sulfate, concentrated to dryness under reduced pressure, and then purified by column chromatography (silica, dichloromethane/methanol = 100/ 1) Obtain product (S)-tert-butyl (1-(3-fluoropropyl)pyrrolidin-3-yl)carbamomethyl ester (480.00 mg).

¹ H NMR (400 MHz, METHANOL- d ₄ ) δ 4.58-4.53 (m, 1H), 4.46-4.40 (m, 1H), 4.14-4.04 (m, 1H), 2.93-2.85 (m, 1H), 2.77 -2.67 (m, 1H), 2.61 (dd, J = 7.78, 5.52 Hz, 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

Dissolve (S)-tert-butyl (1-(3-fluoropropyl)pyrrolidin-3-yl) aminomethyl ester (480.00 mg, 1.93 mmol) in 1,4-dioxane (3 mL), Then add hydrochloric acid-1,4-dioxane solution (4moL﹒L ^-1 , 4.94mL), and the reaction solution becomes a yellow transparent solution. The reaction solution was stirred at 25°C for 3 hours. After TLC detects that the reaction of the raw materials is complete, the reaction solution is concentrated under reduced pressure to obtain compound (S)-1-(3-fluoropropyl)pyrrolidin-3-amine hydrochloride (450.00 mg).

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.80-8.42 (m, 3H), 4.62 (s, 1H), 4.51 (s, 1H), 4.12-3.45 (m, 3H), 3.17 (br s, 3H), 2.35-1.99 (m, 4H).

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 (140.00mg, 263.99μmol) was dissolved in tetrahydrofuran (3mL) 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 methanesulfonic acid (2-dicyclohexylphosphine)-3,6-dimethoxy-2,4,6-triiso Propyl-1,1'-biphenyl) (2-amino-1,1'-biphenyl-2-yl)palladium(II) (23.93 mg, 26.40 μmol), the reaction solution was heated at 80°C under nitrogen atmosphere Stir for 4 hours. After the LCMS detection reaction is completed, the reaction solution is cooled to room temperature, filtered, and the filtrate is concentrated under reduced pressure and purified by preparative liquid chromatography (Phenomenex Gemini C18 column, 3um silica, 30mm diameter, 75mm length); (use water ( Compound N-((S)-1-(3-fluoropropyl)pyrrolidin-3-yl)-6-(((1S)) was purified using a mixture of decreasing polarity containing 0.225% formic acid) and acetonitrile as eluent. ,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 (400 MHz, METHANOL- d ₄ ) δ 7.89 (d, J = 2.76 Hz, 1H), 7.46 (d, J = 7.78 Hz, 1H), 7.25 (d, J = 8.53 Hz, 2H), 7.09 -7.03 (m, 2H), 7.02-6.97 (m, 1H), 4.98 (s, 1H), 4.60 (t, J = 5.65 Hz, 1H), 4.49 (t, J = 5.65 Hz, 1H), 4.18 ( br s, 1H), 3.51-3.35 (m, 4H), 3.14-2.99 (m, 5H), 2.92 (dd, J = 15.18, 4.89 Hz, 1H), 2.65 (dd, J = 16.06, 6.78 Hz, 1H ), 2.53-2.42 (m, 1H), 2.12-1.92 (m, 3H), 1.20 (d, J = 6.78 Hz, 3H).

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

The NOESY spectrum (Figure 1) shows that the methyl hydrogen at position 3 and the hydrogen at position 1 of the compound of formula (I) have obvious NOE effects, proving that they are on the same side, and the pyridyl group at position 1 and the hydrogen at position 3 The relative configuration of the methyl group on the 6-membered pyridine ring is trans. It is known that the absolute configuration of the carbon atom at position 3 is R, so the absolute configuration of the carbon atom at position 1 is S.

Example 3-3: Preparation of succinate of compound of formula (I)

Put 5.0g of the free compound of formula (I) and 100ml of isopropyl ether into a 250ml single-neck bottle, and stir at room temperature until the solid is completely dissolved. 1.21g of succinic acid was added, the reaction solution was stirred at room temperature overnight, filtered with suction, and dried under vacuum at room temperature for 3 hours to obtain 5.4g 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-yl carbamate (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 at 25°C for 16 hours. After the complete 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 organic phase were collected respectively. After the aqueous phase was extracted three times with ethyl acetate (20 mL), all the organic phases were combined, dried over sodium sulfate, concentrated to dryness under reduced pressure, and then purified by column chromatography (silica, dichloromethane/methanol = 100/ 1) Obtain the product (S)-tert-butyl (1-(3-fluoropropyl)pyrrolidin-3-yl) carbamate (480.00 mg).

¹ H NMR (400 MHz, METHANOL- d ₄ ) δ 4.58-4.53 (m, 1H), 4.46-4.40 (m, 1H), 4.14-4.04 (m, 1H), 2.93-2.85 (m, 1H), 2.77 -2.67 (m, 1H), 2.61 (dd, J = 7.78, 5.52 Hz, 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

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

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.80-8.42 (m, 3H), 4.62 (s, 1H), 4.51 (s, 1H), 4.12-3.45 (m, 3H), 3.17 (br s, 3H), 2.35-1.99 (m, 4H).

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

Combine (R)-1-(1H-indol-3-yl)-N-(2,2,2-trifluoroethyl)propane-2-amine (890 mg, 3.47 mmol) and 4-bromo-2, 6-Difluorobenzaldehyde (844.27 mg, 3.82 mmol) was dissolved in toluene (10 mL) and acetic acid (2 mL), and the reaction solution was stirred at 90°C for 6 hours. The reaction solution was concentrated to dryness under reduced pressure, 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 ) δ 7.45-7.43 (d, J = 7.60 Hz, 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 = 6.4 Hz, 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- After 3-amine hydrochloride (38.20mg, 209.04μmol) and sodium tert-butoxide (100.45mg, 1.05mmol) were stirred evenly, tris(diphenylmethylacetone)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 at 80°C for 4 hours. After LCMS detects that the raw material reaction is complete, the reaction solution is filtered, and 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 μm silica, 50 mm diameter, 250 mm length, use 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 ) δ 8.49 (s, 1H), 7.42 (d, J = 7.3 Hz, 1H), 7.21 (d, J = 7.8 Hz, 1H), 7.08-6.94 (m, 2H ), 6.19 (d, J = 11.5 Hz, 2H), 5.24 (s, 1H), 4.60 (t, J = 5.6 Hz, 1H), 4.48 (t, J = 5.6 Hz, 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, 4.3 Hz, 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 = 6.5 Hz, 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

resolve resolution:

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

Dissolve 1-fluoro-3-iodopropane (151.86 mg, 807.87 μmol) and tert-butyl (trans-4-fluoropyrrolidin-3-yl) carbamate (150 mg, 734.43 μmol) in acetonitrile (4 mL) , add potassium carbonate (203.00 mg, 1.47 mmol) at 25°C, and stir the reaction solution at 60°C for 13 hours. The reaction solution was cooled to 25°C, filtered, and concentrated to dryness under reduced pressure. Then column chromatography purified (silica, ethyl acetate/methanol = 10/1) to obtain the product tert-butyl (trans-4-fluoro-1-(3-fluoropropyl)pyrrolidin-3-yl) amino Formate (0.15g).

MS m/z (ESI): 265.0 [M+H] ⁺

¹ H NMR (400MHz, CHLOROFORM-d) δ 4.87 (br s, 1H), 4.59 (t, J = 5.9 Hz, 1H), 4.48 (t, J = 5.9 Hz, 1H), 4.16 (br s, 1H) , 3.29-3.05 (m, 1H), 3.01-2.87 (m, 1H), 2.78-2.41 (m, 4H), 2.02-1.81 (m, 2H), 1.48 (s, 9H).

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 ), add 4M hydrochloric acid-1,4-dioxane (2.13 mL), and stir the reaction solution 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 (0.12g).

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) (25.50 mg, 30.48 μmol) and cesium carbonate (595.95 mg, 1.83 mmol), replaced with nitrogen three times Afterwards, 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 (10 mL) and stirred for 10 minutes. Extracted twice with ethyl acetate (20 mL). The organic phase was dried with anhydrous sodium sulfate, filtered, concentrated to dryness under reduced pressure, and then column chromatographed. 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 (a mixture of decreasing polarity as eluent) was purified to obtain the product trans-N-(3,5-difluoro-4-((1S,3R)-3-methyl-2-(2,2,2-tri Fluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)-4-fluoro-1-(3-fluoropropyl )pyrrolidin-3-amine (27.5 mg).

MS m/z (ESI): 543.1 [M+H] ⁺

¹ H NMR (400MHz, METHANOL-d ₄ ) δ 7.42 (d, J = 7.5 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.09-6.93 (m, 2H), 6.30-6.16 (m , 2H), 5.25 (s, 1H), 4.58 (t, J = 5.8 Hz, 1H), 4.46 (t, J = 5.8 Hz, 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, 9.7 Hz, 1H), 2.02-1.83 (m, 2H), 1.19 (d, J = 6.4 Hz, 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 (Compound 6) Synthesis

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 (85 mg).

MS m/z (ESI): 261.1[M+H] ⁺

¹ H NMR (400MHz, METHANOL-d ₄ ) δ4.66 (br d, J = 2.4 Hz, 1H), 4.61-4.42 (m, 2H), 4.05-3.93 (m, 1H), 3.71-3.56 (m, 2H), 3.17 (br d, J = 9.4 Hz, 1H), 2.90 (br s, 2H), 2.54 (br s, 1H), 2.19 (br d, J = 5.2 Hz, 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 add 4M hydrochloric acid-1,4-dioxane (1.54mL), and stir the reaction solution at room temperature overnight. The reaction solution was concentrated to dryness to obtain the compound trans-1-(3-fluoropropyl)-4-methyl-pyrrolidin-3-amine hydrochloride (70 mg).

¹ H NMR (400MHz, METHANOL-d ₄ ) δ 4.73-4.64 (m, 1H), 4.61-4.50 (m, 1H), 4.23-4.06 (m, 1H), 4.02-3.63 (m, 5H), 3.54- 3.42 (m, 1H), 3.01-2.58 (m, 1H), 2.32-2.12 (m, 2H), 1.37-1.27 (m, 3H).

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), and 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 monitored by LCMS, 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 water (containing 0.225% formic acid) and acetonitrile with decreasing polarity mixtures as eluents) to obtain the 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]indola 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 ) δ 7.42 (d, J = 7.6 Hz, 1H), 7.21 (d, J = 8.8 Hz, 1H), 7.08-6.86 (m, 2H), 6.21 (d, J = 11.6 Hz, 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, 6.8 Hz, 3H), 1.19 (d, J = 6.4 Hz, 3H).

Testing of biological activity and related properties

Test Example 1 : Detection of the degradation effect of SERD compounds on estrogen receptors in MCF7 cells 1. Experimental purpose The purpose of this experiment is to determine the degradation activity of the SERD compounds on the endogenously expressed estrogen receptors in MCF7 cells. According to DC ₅₀ and Maximum degradation efficiency evaluates the activity of a compound. 2. Experimental methods MCF7 cells (ATCC, HTB-22) were cultured in DMEM (Gibco, 11995-065) complete medium containing 10% fetal calf 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 culture medium and cultured in a 37ºC, 5% CO ₂ cell incubator. The compound to be tested was dissolved in DMSO, the storage concentration was 10 mM, diluted with Echo 550 (Labcyte Inc.) and added to the cell culture plate. The starting concentration of each compound treatment was 100 nM, 3-fold gradient dilution, 9 concentration points, Set up a blank control containing 0.5% DMSO, and set up duplicate well controls at each concentration point. Incubate in a 37ºC, 5% _CO2 cell incubator for 24 hours. Add paraformaldehyde to the cell culture medium in each cell culture well. The final concentration of paraformaldehyde is about 3.7% to fix the cells. After 30 minutes, discard the supernatant and add 50 μL PBS to each well for washing once; add PBS (containing 0.5 % v/v Tween-20), 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 the blocking solution and add primary antibody mixture (anti- -ER monoclonal antibody, Estrogen Receptor α (D8H8) Rabbit mAb, GST, #8644S, 1:1000 dilution; anti-GAPDH monoclonal antibody, GAPDH(D4C6R) Mouse mAb, GST, #97166S, 1:2000 dilution) incubated at room temperature 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, diluted 1:1000), incubate at room temperature for 45 minutes in the dark; wash with PBST 3 times, use Odyssey CLx to read the fluorescence signal of each well, and calculate Chanel 800 (ER)/ Chanel 680 (GAPDH) value. Use the wells treated with 0.1 μM fulvestrant as a 100% degradation control to calculate the degradation rate at each concentration point. Use XlLfit to analyze the processing data and calculate the degradation activity DC ₅₀ and maximum degradation rate Imax of each compound. See Table 1 for data analysis. Table 1 Activity of SERD compounds on estrogen receptor degradation in MCF7 cells Compound number ER level DC ₅₀ (nM) maximum degradation rate Compound 1 0.41 106% Compound 2 8.5 92% Compounds of formula (I) 0.15 104% Compound 5 0.58 80% Compound 6 0.50 90%

Test Example 2 : Detection of the inhibitory effect of SERD compounds on MCF7 cell proliferation 1. Experimental purpose The purpose of this experiment is to determine the inhibitory effect of the SERD compounds in this article on MCF7 cell proliferation in vitro, and evaluate the activity of the compound based on IC ₅₀ and maximum inhibitory efficiency. 2. Experimental methods MCF7 cells (ATCC, HTB-22) were cultured in DMEM (Gibco, 11995-065) complete medium containing 10% fetal calf 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 culture medium and cultured overnight in a 37ºC, 5% CO ₂ cell culture incubator. The next day, the compound to be tested was added for drug treatment. The compound solution with a storage concentration of 10 mM was diluted and transferred to each cell culture well using Echo550 (Labcyte Inc.). The starting concentration of each compound in the cells was 100 nM, 3-fold gradient dilution, 9 concentration points, set a blank control containing 0.3% DMSO, and set a duplicate well control at each concentration point. Incubate in a 37ºC, 5% _CO2 cell culture incubator for 7 days, and remove the cell culture plate on the 8th day. Add CellTiter-Glo ^® Luminescent Cell Viability Assay (Promega, G7573), leave it at room temperature for 10 minutes, use a multi-label microplate reader EnVision (PerkinElmer) to read the luminescence signal value, and use XLfit to calculate each compound based on the concentration and luminescence signal value. Inhibitory activity _IC50 of the compound. Data analysis is shown in Table 2 Table 2 Inhibitory activity of SERD compounds on MCF7 cell proliferation Compound number MCF7 cell proliferation inhibition IC ₅₀ (nM) Compound 1 0.58 Compound 2 15 Compounds of formula (I) 0.27 Compound 4 0.23 Compound 5 2.57 Compound 6 2.39

test case 3 : SERD compound pair CYP2C9 , CYP2D6 Inhibition of enzyme activity

The inhibition of CYP2C9 and CYP2D6 enzyme activities by the SERD compounds in this article was measured using the following test methods.

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 sulfapyrazole (D. Ehrenstorfer GmbH 109012), quinidine (TCI WEODL-RE) and ketoconazole (Sigma 100M1091V)

5. AB Sciex Triple Quad 5500 LC/MS

2. Test steps

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

2. Prepare a 10 mM NADPH solution using 100 mM PBS buffer. Dilute the 10 mM SERD compound stock solution with DMSO to obtain 200x concentration compound working solutions (6000, 2000, 600, 200, 60, 20, 0 μM). Dilute the positive inhibitor stock solution with DMSO to obtain a 200x concentration positive inhibitor working solution (sulfapyrazole, 1000, 300, 100, 30, 10, 3, 0 μM; quinidine/ketoconazole, 100, 30, 10, 3, 1, 0.3, 0 μM). Prepare a 200x concentration substrate working solution (120 μM diclofenac, 400 μM dextromethorphan, and 200 μM midazolam) 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 evenly, and pre-incubate in a 37 °C water bath for 15 minutes. In the positive control group, 1 μl of diclofenac, dextromethorphan or midazolam working solution was added to replace the compound working solution. At the same time, pre-incubate 10 mM NADPH solution together 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, add 400 ul of ice-cold methanol containing internal standard to all samples to terminate the reaction. Vortex to mix, and centrifuge at 3220g, 4°C for 40 minutes. After centrifugation, transfer 100 μL of the supernatant to the injection plate, add 100 μL of ultrapure water and mix well for LC-MS/MS analysis.

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

Drug-drug interaction (DDI) refers to the physical or chemical changes produced by two or more drugs, as well as the changes in drug efficacy caused by these changes. Understanding drug interactions can provide patients with better pharmaceutical services and promote rational drug use, minimizing the occurrence 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 compound in this article has weak inhibitory ability on CYP450, indicating that the potential risk of DDI in the SERD compound in this article is small. Table 3 IC ₅₀ values of SERD compounds against CYP2C9 and CYP2D6 Compound number CYP2C9 (μM) CYP2D6 (μM) Compounds of formula (I) >30 >30 Compound 4 20 27

test case 4 : SERD Determination of plasma protein binding of compounds

Human plasma protein binding is a key factor in 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. Therefore, for compounds with similar potency and exposure levels, compounds with high free fractions (low levels of plasma protein binding) may exhibit enhanced efficacy.

The protein binding rate of SERD compounds in the plasma of 5 species (human, monkey, dog, rat and mouse) was determined using 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. Preparation of a buffer solution with a concentration of 100 mM sodium phosphate and 150 mM NaCl: Use ultrapure water to prepare an alkaline solution with a concentration of 14.2 g/L Na ₂ HPO ₄ and 8.77 g/L NaCl. An acidic solution with a concentration of 12.0 g/L NaH ₂ PO ₄ and 8.77 g/L NaCl. Titrate the alkaline solution with the acidic solution to a pH of 7.4 to prepare a buffer solution with a concentration of 100 mM sodium phosphate and 150 mM NaCl.

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

3. Plasma preparation: Thaw the frozen plasma quickly at room temperature, then centrifuge the plasma at 4°C and 3,220 g for 10 minutes to remove clots, and collect the supernatant into a new centrifuge tube. Determine and record the pH of plasma, use plasma with a pH of 7-8.

4. Preparation of compound-containing plasma samples: Dilute 10 mM SERD compound or positive control compound stock solution with DMSO to obtain a 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 steps: Assemble the dialysis device according to the operating instructions. Add 120 μL of plasma sample containing 1 μM compound to one side of the dialysis membrane, and add an equal volume of dialysate (phosphate buffer) to the other side. The test is set up with two samples. Seal the dialysis plate, place it in an incubator, and incubate for 6 hours at 37°C, 5% _CO2 , and approximately 100 rpm. After the incubation, remove the sealing film and pipet 50 μl from the buffer and plasma sides of each well into different wells of a new plate. Add 50 μl of blank plasma to the phosphate buffer sample, add an equal volume of blank phosphate buffer to the plasma sample, and then add 300 μl of acetonitrile containing the internal standard to precipitate the protein. Vortex for 5 minutes and centrifuge at 3,220 g for 30 minutes at 4°C. Take 100 μl of the supernatant to the injection plate, add 100 μL of ultrapure water, mix well, and use for LC-MS/MS analysis.

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

Free rate % = (ratio of compound peak area to internal standard peak area _{buffer side} /ratio of compound peak area to internal standard peak area _{plasma side} ) x100

Binding rate%=100-free rate%

All data are calculated using Microsoft Excel software. Calculated plasma protein binding values for SERD compounds. Table 4 Protein binding rate values of SERD compounds in human, dog, rat and mouse plasma Compound number species Protein binding rate (%) Compounds of formula (I) people 98.8 cynomolgus monkey 97.8 Beagles 98.7 SD rat 97.1 CD-1 mice 98.1 Compound 4 people 99.8 cynomolgus monkey 99.8 Beagles 99.8 SD rat 99.8 CD-1 mice 99.8

test case 5 : SERD compound in pH The value is 7.4 Apparent solubility in phosphate buffer

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 documented methods. 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.

1.5 ml flat-bottomed glass vial (BioTech Solutions); PTFE/silicone stopper (BioTech Solutions); PTFE-coated stir rod; MultiScreenHTS HV (0.45 µm) 96 well plate (Millipore, MSHVN4510 or MSHVN4550); Eppendorf Thermomixer Comfort; Vacuum Manifold ORVMN96 (Orochem).

2. Experimental steps 1) Preparation of stock solutions Use DMSO to prepare 10 mM stock solutions of the test substance and the control drug progesterone. 2) In the apparent solubility determination step, take 30 µL of 10 mM stock solution of the analyte and add it to the corresponding position of the 96-well plate in the specified order. Add 970 µL of pH 7.4 phosphate buffer to the corresponding vial on the sample plate. The experiment is double parallel. Add a stir rod to each vial and secure with a Teflon/silicone stopper. The sample tray was then placed in an Eppendorf Thermomixer Comfort and shaken at 1100 rpm at 25°C for 2 hours. After 2 hours, remove the cork, use a large magnet to remove the stir bar, 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 the 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, the sample diluent may be diluted with a certain proportion of 50% ACN(IS).H ₂ O to obtain better peak shape. The dilution factor may be adjusted based on the solubility of the analyte or the strength of its liquid response signal. 3) Sample analysis step: Place the injection plate into the injection tray of the automatic sampler, and evaluate the sample through liquid mass analysis.

3. Experimental steps

All calculations were performed via Microsoft Excel. The analysis and quantification of the sample filtrate is accomplished by using liquid quality to identify and quantify the standard peaks of known concentrations. Calculated apparent solubility values of SERD compounds in phosphate buffer at pH 7.4. Table 5 Apparent solubility values of SERD compounds in phosphate buffer at pH 7.4 Compound number pH = 7.4 Apparent solubility (µM) Compounds of formula (I) 92 Compound 4 <0.3

test case 6 : SERD Compounds for voltage-gated potassium channels AHr whether Potentially inhibitory

hERG potassium channels are critical for normal electrical activity of the heart. Arrhythmias can be induced by blocking hERG channels with a variety of 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 for drug candidates.

1. Materials and reagents 1. Experimental materials and instruments Experimental materials Supplier(item number) 6/3.5 cm cell culture dish Shanghai Huayasi Chuang Biotechnology Co., Ltd. (150288/153066) Dialyzed fetal bovine serum Shanghai Bohan Biotechnology Co., Ltd. (BS-0005-500) DMEM medium Thermo Fisher Scientific (China) Co., Ltd. (10569) HEPES Thermo Fisher Scientific (China) Co., Ltd. (15630080) trypsin Thermo Fisher Scientific (China) Co., Ltd. (2192509) Penicillin-streptomycin solution Thermo Fisher Scientific (China) Co., Ltd. (15140-122) MEM non-essential amino acid solution Thermo Fisher Scientific (China) Co., Ltd. (11140) Geneticin (G418) Thermo Fisher Scientific (China) Co., Ltd. (11811031) blasticidin Thermo Fisher Scientific (China) Co., Ltd. (R21001) polylysine Thermo Fisher Scientific (China) Co., Ltd. (P4832) Dophired Beijing Ypres Technology Development Co., Ltd. (D525700) doxycycline Sigma Aldrich (Shanghai) Trading Co., Ltd. (D9891) carbon dioxide incubator Thermo Fisher Scientific (China) Co., Ltd. (371) Drawing instrument American Sutter Company (P-97) micromanipulator American Siskiyou Company (MC1000e) micromanipulator American Sutter Company (ROE-200; MP285) amplifier German HEKA Company (EPC10) microscope Olympus China Co., Ltd. (IX51/71/73) perfusion system American ALA Company (VM8 gravity drug delivery system)

2. Cell lines and culture

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

3. Configuration of compounds to be tested

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

2) Use DMSO to dilute the stock solution in a 1:3 gradient into three other intermediate concentration solutions, with concentrations of 3.33 mM, 1.11 mM and 0.37 mM respectively.

3) Before starting the experiment, dilute the stock solution and intermediate solution of the compound to be tested 1000 times with extracellular solution to obtain a series of working solutions with concentrations of 10 μM, 3.33 μM, 1.11 μM and 0.37 μM. At the same time, use extracellular solution to dilute the 10 mM stock solution. The solution was diluted 333.33 times to obtain 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 fluid is completed, visually observe whether there is any precipitation or turbidity in the working fluid. If so, it may be due to poor solubility of the compound in the physiological solution. It can be further sonicated 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 hERG channels at five concentrations: 30 μM, 10 μM, 3.33 μM, 1.11 μM and 0.37 μM, fit the dose-effect curve and calculate IC ₅₀ .

2. Experimental methods 1. Place the small slide containing HEK293 cells in the culture dish into the perfusion tank of the microscope operating table. 2. Place the appropriate cells in the center of the field of view under an Olympus IX51, IX71 or IX73 inverted microscope, use a ×10x 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 downward, and at the same time adjust the coarse focus screw to make the electrode slowly approach the cells. 3. When approaching the cell, switch to the ×40 objective lens for observation, and use the micromanipulator to fine-tune the position so that the electrode gradually approaches 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 transient capacitance current Cfast in voltage clamp mode. Then, short periods of negative pressure are applied repeatedly to rupture the membrane, and finally a whole-cell recording mode is formed. 6. Under the condition that the membrane potential is clamped at -60 mV, the slow capacitive current Cslow, cell membrane capacitance (Cm) and input membrane resistance (Ra) are compensated respectively. 7. After the cells are stable, change the clamping voltage to -90 mV, set the sampling frequency to 20 kHz, and the filtering frequency to 10 kHz. The detection conditions for leakage current are that the clamping voltage changes to -80 mV and the time duration is 500 ms. 8. The hERG current test method is as follows: Apply a depolarization command voltage for 4.8 seconds to depolarize the membrane potential from -80 mV to +30 mV, and then instantaneously apply a repolarization voltage for 5.2 seconds to reduce the membrane potential to below -50 mV. Removal of channel inactivation allowed observation of hERG tail currents. 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 generated by the test cells. Only stable cells within the acceptance range of the evaluation criteria can enter subsequent compound testing. 10. Determine the inhibitory effect of the compound to be tested on hERG current: First, the hERG current measured in extracellular fluid containing 0.1% DMSO is used as the detection baseline. After the hERG current remains stable for at least 5 minutes, the solution containing the compound to be tested is perfused around the cells sequentially from low concentration to high concentration. Wait about 5 minutes after each perfusion to allow the compound to fully act on the cells and to record hERG current simultaneously. After the recorded current stabilizes, record the last 5 hERG current values, and take their average value as the final current value at a specific concentration. After testing the compound, 150 nM Dofetilide was added to the same cell to completely inhibit its current, which served as a positive control for the cell. At the same time, the positive compound Dophilide was detected simultaneously using the same patch clamp system before and after the end of the test compound experiment to ensure the reliability and sensitivity of the entire detection system.

3. Data analysis

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

After instilling the blank solvent or compound gradient solution, the five continuous current values obtained stably are averaged and used as the "tail current size _blank " and "tail current size _compound "respectively;

The current suppression percentage is calculated using the following formula.

The dose-response curve was fitted by Graphpad Prism 8.0 software and the IC ₅₀ 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 channel hERG Compound number hERG IC ₅₀ (μM) Compounds of formula (I) 10 Compound 4 3.7

test case 7 : SERD Mouse pharmacokinetic evaluation of compounds

Experimental materials

CD-1 mice were purchased from Beijing Vitong Lihua Experimental Animal Technology Co., Ltd. DMSO (dimethylsulfoxide), 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 methods

Six female CD-1 mice (20-30g, 4-6 weeks) were randomly divided into 2 groups, 3 mice in each group. Group 1 was administered the test compound via tail vein injection at a dose of 1 mg/kg, and the solvent was DMSO (5%, v/v) and 10% HP-β-CD aqueous solution (95%, v/v). Group 2 The test compound was administered orally at a dose of 10 mg/kg, and the solvent was tetraethylene glycol (40%, v/v) and 7.5% sulfobutyl-β-cyclodextrin aqueous solution (60%, v/v). Animals were fed and watered normally before experiments. 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, 4ºC) and the plasma was taken for testing.

Take 10 μL of mouse plasma sample, add 150 μL of acetonitrile solvent (containing internal standard compounds) to precipitate the protein, vortex for 5 minutes, centrifuge (14,000 rpm) for 5 minutes, and use the supernatant with 0.1% (v/v) FA. It was diluted 2 times with water and quantitatively detected on the 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, add 2 μL of sample to 18 μL of blank plasma. After vortexing for 0.5 min, add 300 μL of acetonitrile solvent (which contains internal standard compounds) to precipitate the protein. The remaining processing steps are the same as for the non-diluted sample.

The PK test results are shown below. The SERD compounds showed good PK properties and oral bioavailability in mice. Table 7 PK of SERD compounds in mice Compound number Medication/dose mg/kg Maximum drug concentration ng/ml Time to reach peak drug concentration h Drug half elimination time h Drug exposure ng*h/ml Oral bioavailability (%) Compounds of formula (I) IV-1 mpk 98 -- 13 414 68% PO-10 mpk 219 3 9 2804 Compound 4 IV-1 mpk 78 -- twenty one 639 60% PO-10 mpk 222 5 19 3853 PO-10 mpk 555 1 4 5447

test case 8 : SERD Compounds on the blood-brain barrier in rats ( BBB ) through ability

The key to the drug being effective against brain metastases is that the drug can pass through the blood-brain barrier of animals and have sufficient exposure in the brain. Therefore, by measuring the drug concentration in the plasma and brain tissue after administration to animals, the distribution of the drug in the brain can be evaluated. situation, and then determine whether the drug can inhibit tumor growth in the brain orthotopic model.

Experimental materials

SD female rats were purchased from Beijing Vitong 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 methods

Six female SD rats (200-300g, 6-8 weeks) were randomly divided into 2 groups, 3 rats in each group. The SERD compound of this article and the solvent 0.5% methylcellulose aqueous solution were administered respectively. The animals were fed with water normally before the experiment, fasted overnight, and resumed food 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, 4ºC), and the plasma was collected for testing. After tissue collection, blot dry with filter paper, and the samples were stored in a -80 ºC refrigerator for testing.

Take 10 μL of rat plasma sample, add 150 μL of acetonitrile solvent (containing internal standard compounds) to precipitate the protein, vortex for 5 minutes, centrifuge (14,000 rpm) for 5 minutes, and use the supernatant with 0.1% (v/v) FA. It was diluted 2 times with water and quantitatively detected on the LC-MS/MS system (AB Sciex Triple Quad 6500+). For 10× diluted samples, add 2 μL of sample to 18 μL of blank plasma. After vortexing for 0.5 min, add 300 μL of acetonitrile solvent (which contains internal standard compounds) to precipitate the protein. The remaining processing steps are the same as for the non-diluted sample. When measuring the concentration of plasma samples, obtain the SD rat plasma standard curve and plasma quality control samples at the same time.

Rat brain tissue samples were first homogenized with 4 times the quality volume of PBS homogenate. Take 20 μL of brain tissue homogenate sample, add 20 μL of blank mouse plasma, dilute and mix, then add 600 μL of acetonitrile solvent (which contains internal standard compounds) to precipitate the protein, vortex for 5 minutes, and centrifuge (14,000 rpm) for 5 minutes. The supernatant was diluted 2-fold with water containing 0.1% (v/v) FA and quantitatively detected on an LC-MS/MS system (AB Sciex Triple Quad 6500+). Relative to the prior art or other molecules disclosed herein, the compounds of formula (I) of the present disclosure exhibit excellent blood-brain barrier penetration and high drug exposure in the brain tissue of rats.

The BBB test results are as follows: Table 8 Rat blood-brain barrier penetration test of SERD compounds Compound number Dosage mg/kg Plasma drug concentration ng/ml Brain drug concentration ng/g B/P ratio Compounds of formula (I) 100 QD 2617 10560 4.2

test case 9 : SERD compound pair MCF-7 Growth inhibition experiment of mouse subcutaneous tumor model

Experimental reagents Human breast cancer MCF-7 cells: ATCC, HTB-22 17β-estradiol tablets: Innovative Research of America, Cat No.: SE-121, 60-day release, 0.72 mg/pellet EMEM culture medium: ATCC, Cat No.: 30-2003 Fetal bovine serum: Gbico; Cat No.: 1099-141C 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 (calcium and magnesium ion-free phosphate buffer saline): Hyclone, Cat. No.: SH30256.01 Matrigel: Corning, Cat. No.: 356237

Experimental methods

Animal information: NPG mice, female, 6-7 weeks old, weighing about 19-28 grams. The animals were purchased from Beijing Weitongda Biotechnology Co., Ltd. The mice were raised in an SPF-level 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: Human breast cancer MCF-7 cell line was cultured in vitro. The culture conditions were EMEM (cell culture medium) added with 10% fetal calf serum, 1% Pen Strep, 10 μg/ml recombinant human insulin, 37°C, 5% CO ₂ incubators. Perform routine digestion and passaging with 0.25% trypsin-EDTA digestion solution once a week. When the cell saturation is 80%-90% and the number reaches the requirement, collect the cells and count them.

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

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

Tumor measurements and experimental indicators:

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

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

Experimental results:

See Table 9. In the mouse subcutaneous xenograft tumor MCF-7 model, oral administration of the compound of formula (I) at 1 mg/kg, 3 mg/kg, or 10 mg/kg once a day had a significant inhibitory effect on tumor growth (P<0.01) , and has a good dose-response relationship, and has the effect of shrinking tumors at doses of 3 mg/kg and 10 mg/kg. Oral administration of the compound of formula (I) at 10 mg/kg once a day has a significant inhibitory effect on tumor growth (P<0.01) and has the effect of shrinking tumors. Compounds of formula (I) did not significantly affect mouse body weight at the doses attempted. Table 9 Tumor volume of MCF-7 subcutaneous tumor model test compound Dosemg/kg Dosing method Dosing frequency Average tumor volume (mm ³ ) TGI (%) 0 days 21 days Solvent control / PO QD 179 403 / Compounds of formula (I) 1 PO QD 183 207 89.08 Compounds of formula (I) 3 PO QD 169 136 114.71 Compounds of formula (I) 10 PO QD 177 91 138.17

test case 10 :Mode (I) compounds in mice MCF-7 Growth inhibition experiment of brain orthotopic tumor model

Experimental reagents/instruments: Human breast cancer MCF-7 cells: ATCC, HTB-22 17β-estradiol tablets: Innovative Research of America, Cat No.: SE-121, 60-day release, 0.72 mg/pellet EMEM culture medium: ATCC, Cat No.: 30-2003 Fetal bovine serum: Gibco, Cat. No.: 1099-141C 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 Microsyringe pump: KDS, Cat No.: Legato130 Miniature handheld cranial drill: Ryward, Cat No.: 78001

Experimental method:

Animal information: NPG mice, female, 6-8 weeks old, weighing about 17-29 grams. The animals were purchased from Beijing Weitongda Biotechnology Co., Ltd. The mice were raised in an SPF-level 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: Human breast cancer MCF-7 cell line was cultured in vitro. The culture conditions were EMEM (cell culture medium) added with 10% fetal calf serum, 1% Pen Strep, 10 μg/ml recombinant human insulin, 37°C, 5% CO ₂ incubators. Perform routine digestion and passaging with 0.25% trypsin-EDTA digestion solution twice a week. When the cell saturation is 80%-90% and the number reaches the requirement, collect the cells and count them.

Cell inoculation: Inoculate 15 μl/(containing 2×10 ⁶ ) MCF-7 cell suspension into the brain of mice using a brain positioning instrument, a microinjection pump and a micro hand-held cranial drill, and inoculate subcutaneously three days before cell inoculation. 17β-estradiol tablets. On the 8th day after the cells were inoculated, the mice were randomly divided into groups for administration according to their body weight, and the grouping day was Day 0.

Dosage: Fulvestrant (AstraZeneca) is administered at a dose of 250 mg/kg, subcutaneously (SC), once weekly (QW), and the compound of formula (I) is administered at a dose of 30 mg /kg, administered orally (PO), once daily (QD). There were 11 mice in the vehicle group and 8 mice in the drug administration group. Administration continued in all groups until the mice died, were euthanized due to poor condition, or the experiment ended.

Experimental observations and conclusion:

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

The experiment ended on Day 48 and all mice were euthanized.

Experimental results:

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

test case 11 :Mode (I) Compound combined with palbociclib on human breast cancer MCF-7 cell cycle effects

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

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

Experimental results: The compound of formula (I) can cause cell cycle arrest and arrest cells in the G1 phase. When combined with palbociclib, it can significantly increase the proportion of G1 phase arrest and reduce the proportion of cells in the S phase. The results are shown in Table 10. Table 10 The arresting effect of compound of formula (I) combined with palbociclib on MCF-7 cell cycle test compound Compound concentration (nM) G1 period% S period% G2 phase% cell control / 56.4 22.2 20.5 52.4 21.7 24.8 Compounds of formula (I) 10 66.6 19 13.9 Palbociclib 20 60.7 21.7 17.2 Palbociclib 80 67.9 17.7 14 Palbociclib 320 84.3 14.1 0 Compound of formula (I) + palbociclib 10+20 70.4 21.8 6.77 Compound of formula (I) + palbociclib 10+80 81.4 8.45 8.76 Compound of formula (I) + palbociclib 10+320 91.9 3.56 3.51

test case 12 :Mode (I) Compound combined with palbociclib on human breast cancer MCF-7 cell proliferation inhibition

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 more than 70%, the cells were digested, adjusted to a density of 500/20 μL/well, and seeded in a 384-well cell culture plate (Corning) for overnight culture. Use Echo650 (Beckman) to transfer 120 nL of 2-fold serial dilution of compound of formula (I) (succinate form) to the cell plate, and simultaneously transfer 120 nL of 2-fold serial dilution of palbociclib to the cell plate, and add 40 μL Culture medium, after compound treatment for 7 days, add CellTiter-Glo (Promega) to detect cell viability. The cell wells are used as 100% viability control, and the culture medium wells are used as 0% viability control. Combined analysis is performed using Combenefit (a value greater than 10 indicates better results). synergistic effect).

Results: The compound of formula (I) combined with palbociclib had a synergistic effect on inhibiting the proliferation of human breast cancer MCF-7 cells. The results are shown in Figure 4.

test case 13 : human breast cancer MCF-7 Study on the pharmacodynamics of combined use in xenograft subcutaneous tumor 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, 0.18 mg/pellet EMEM culture medium: ATCC, Cat No.: 30-2003 Fetal bovine serum: ExCell; Cat No.: FND500 Double resistance: 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), 125 mg/capsule

Experimental method:

Animal information: Balb/c nude mice, female, 6-8 weeks old, weighing about 18-22 grams, animals were purchased from Shanghai Lingchang Biotechnology Co., Ltd., and the mice were raised in an SPF-level environment, each cage It is individually ventilated and all animals have free access to a standard certified commercial laboratory diet and water ad libitum.

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

Cell inoculation: 0.2 ml/(containing 1×10 ⁷ cells) MCF-7 cell suspension (DPBS: Matrigel, volume ratio 1:1) was subcutaneously inoculated into the right back of each mouse, and before cell inoculation Inoculate 17β-estradiol tablets subcutaneously two days later. On the 6th day after the cells were inoculated, ^when the average tumor volume reached 159.01 mm, drug administration was started in groups. The drugs were randomly divided into groups according to the tumor volume. The grouping day was Day 0.

Administration: The dosage of the compound of formula (I) (succinate form, dosage is based on free base) is 1 mg/kg, administered orally (PO), once daily (QD) × 25 times. The dosage of palbociclib is 40 mg/kg, administered orally (PO), once daily (QW) × 25 times. 8 mice per group.

Tumor measurements and experimental indicators:

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

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

Experimental results:

See Table 11, Figure 5 and Figure 6. One mouse in the solvent group had a weight loss of more than 10%, and 4 mice in the palbociclib (40 mg/kg) group had a weight loss of more than 10%. Palbociclib (40 mg/kg) and the compound of formula (I) ( 1 mg/kg) combination group, one mouse lost more than 10% of its body weight. It can be seen that in this model, 40 mg/kg palbociclib has a certain impact 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), 40 mg/kg of palbociclib was orally administered to tumor-bearing mice once a day and showed a certain inhibitory effect on tumor growth (P<0.0001); Administration of 1 mg/kg of compound of formula (I) to tumor-bearing mice showed a significant inhibitory effect on tumor growth (P<0.0001); 40 mg/kg of palbociclib and 1 mg/kg of compound of formula (I) combined The treatment group showed significantly enhanced anti-tumor effect compared with the vehicle control group and the corresponding single drug group. Table 11 Tumor volume of MCF-7 subcutaneous tumor model test compound Dosemg/kg Dosing method Dosing frequency Average tumor volume (mm ³ ) TGI (%) 0 days 3 days 7 days 10 days 14 days 17 days 21 days 24 days Solvent control / PO QD 159 228 358 475 563 643 828 746 / Palbociclib 40 PO QD 159 220 223 265 288 322 384 314 73.62 Compounds of formula (I) 1 PO QD 159 212 203 203 180 196 208 170 98.18 Palbociclib + compound of formula (I) 40 +1 PO+PO QD+QD 159 197 150 123 98 78 67 38 120.71

[Cross-reference to related applications] This application claims the priority and rights of Chinese Patent Application No. 202111338407.1 submitted to the National Intellectual Property Administration of China on November 12, 2021. The disclosure content of the application is incorporated herein by reference in its entirety.

Unless expressly excluded or otherwise limited, every document cited herein, including any cross-referenced patent or patent application, or any patent application or patent to which this application claims priority, is hereby incorporated by reference in its entirety and Enter 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.

The components of several embodiments are summarized above so that those with ordinary skill in the technical field to which the present invention belongs can better understand the concepts of the embodiments of the present invention. It should be understood by those of ordinary skill in the technical field that the embodiments of the present invention can be used as a basis to design or modify other processes and structures to achieve the same purposes and/or achieve the same results as the embodiments introduced herein. benefits. Those with ordinary skill in the technical field to which the present invention belongs should also understand that these equivalent structures do not deviate from the spirit and scope of the present invention, and that various modifications can be made without departing from the spirit and scope of the present invention. Changes, Substitutions and Alternatives. Therefore, the protection scope of the present invention shall be determined by the appended patent application scope.

without

The details of one or more embodiments of the subject matter described in this specification are set forth in the accompanying drawings and the description below. Other features, aspects and advantages of the subject matter of this specification will become apparent from the description, drawings and claims, among which: Figure 1 is the NOESY spectrum of the compound of formula (I). Figure 2 is a survival curve of human ER-positive breast cancer MCF-7 brain orthotopic model mouse using the compound of formula (I). Figure 3 is a graph showing changes in body weight of mice using the MCF-7 brain orthotopic model of human ER-positive breast cancer with the compound of formula (I). Figure 4 is a diagram showing the in vitro anti-proliferation synergistic effect of the combination of the compound of formula (I) and palbociclib on human breast cancer MCF-7 cells. Figure 5 is a graph showing the anti-tumor growth effect of the compound of formula (I) combined with palbociclib on the human ER-positive breast cancer MCF-7 xenograft subcutaneous tumor mouse model. Figure 6 is a graph showing body weight changes in mice with human ER-positive breast cancer MCF-7 xenograft subcutaneous transplantation tumors that were combined with the compound of formula (I) and palbociclib.

Claims (14)

A pharmaceutical combination comprising at least one selective estrogen receptor down-regulating agent and at least one CDK4/6 inhibitor, the selective estrogen receptor down-regulating agent selected from the group consisting of compounds represented by formula (K) or pharmaceuticals thereof Acceptable salt: (K) Wherein, R ¹ , R ² , R ³ and 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 cycloalkyl} ; X _{1 ,} X ₂ ^, X _{3 ,} Alkyl, C ₃ -C ₁₀ cycloalkyl, 3-10 membered heterocyclyl, C ₁ -C ₁₀ alkoxy, C ₃ -C ₁₀ cycloalkyloxy or 3-10 membered heterocyclyloxy; R ⁵ is independently selected from C _{1 -C 6} alkyl, which 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 formula (K) or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof: or . The drug 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 the group consisting of compounds 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 includes a first pharmaceutical composition and a second pharmaceutical composition, the first pharmaceutical composition includes a compound represented by formula (K) as described in any one of claims 1 to 5, or Its pharmaceutically acceptable salt and pharmaceutically acceptable auxiliary materials, the second pharmaceutical composition includes at least one CDK4/6 inhibitor and pharmaceutically acceptable auxiliary materials. 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 the group consisting of compounds 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 II 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 described in any one of claims 1 to 5, and pharmaceutically acceptable excipients. A pharmaceutical combination described in any one of claims 1 to 5, a combination product described in any one of claims 6 to 8, and a pharmaceutical composition described in 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 metastasis breast cancer. The use according to claim 10, wherein the tumor is ER-positive, HER-2-negative locally advanced or metastatic breast cancer.