TW202300145A - Pharmaceutical combination, kit containing same, and use thereof - Google Patents

Abstract

Disclosed are a pharmaceutical combination, a kit containing same, and the use thereof in the preparation of a drug for treating breast cancer. The pharmaceutical combination comprises a first active ingredient and a second active ingredient, wherein the first active ingredient is any one of or more of a compound having a structure represented by structural formula I, a stereoisomer thereof, a tautomer thereof, a polymorph thereof, a solvate thereof and a pharmaceutically acceptable salt thereof; and the second active ingredient is an estrogen receptor antagonist or an aromatase inhibitor. The above-mentioned first active ingredient and second active ingredient are co-administered in combination, the achieved co-treatment activity has a prominent improvement effect with respect to the separate administration of each active ingredient, and the synergistic effect is achieved.

Description

Pharmaceutical combination, kit comprising same and use thereof

The present invention relates to the field of breast cancer drugs, in particular, to a drug combination, a kit containing it and uses thereof.

Breast cancer has become the most threatening disease to women's health. Currently, there are at least 156 breast cancer drugs under research and on the market, 68% of which are targeted therapy drugs. A large number of studies have found that tumors are associated with cell cycle abnormalities. A large number of mutations in mitotic signaling proteins and defects in anti-mitotic signaling proteins in tumor cells lead to proliferation disorders; at the same time, most tumors have genomic instability (GIN) and chromosome instability (CIN). ), these three basic cell cycle defects are directly or indirectly caused by the loss of control of CDKs. Cyclin Dependent Kinase (CDK) inhibitors are increasingly becoming popular targets.

The function of CDKs is to phosphorylate and thereby activate or deactivate certain proteins. The catalytic step mediated by CDKs involves the transfer of phosphate from ATP to the substrate of the macromolecular enzyme. Several groups of compounds have been found (see eg Fischer, P.M. Curr. Opin. Drug Discovery Dev. 2001, 4, 623-634) to have antiproliferative properties due to CDK-specific ATP antagonism.

Currently, breast cancer drugs on the market include Palbociclib (PD-0332991), Ribociclib (LEE011) and Abemaciclib (LY2835219). However, the single drugs that have been marketed are still difficult to meet the clinical needs. In view of this, the present invention provides a combination of a CDK inhibitor and other pharmaceutical preparations, and it is expected that an ideal synergistic effect can be achieved through the combined use of the drug.

The main purpose of the present invention is to provide a drug combination, a kit containing it and its use in the preparation of drugs for treating breast cancer, so as to improve the effect of single drug use.

結構式I 其中，環A為芳基或雜芳基；Z選自CH ₂、NH、O或S；R ₁分別獨立地選自氫、鹵素、氰基、硝基、羥基、氨基、C _1-8烷基、C _1-8烷氧基、C _3-8環烷基、芳基、雜芳基、雜環基、雜環基-(CH ₂) _m-、芳基-C _1-6烷基-、雜芳基-C _1-6烷基-、NR ₁₂R ₁₃、NR ₁₂-C _1-6亞烷基-NR ₁₂R ₁₃、或雜環基-C(O)-，其中 C _1-8烷基、C _1-8烷氧基、C _3-8環烷基、芳基、雜芳基、雜環基、雜環基-(CH ₂) _m-、芳基-C _1-6烷基、雜芳基-C _1-6烷基或雜環基-C(O)-是各自未取代或者被至少一個選自鹵素、C _1-8烷基、C _3-8環烷基、雜環基、NR ₁₂R ₁₃、(CH ₂) _t-OH的取代基取代；R ₂和R ₃各自獨立地選自氫、羥基、氰基、硝基、氨基、鹵素、C _1-8烷基、C _1-8烷氧基、C _3-8環烷基、芳基、雜芳基或雜環基；其中 C _1-8烷基、C _1-8烷氧基、C _3-8環烷基、芳基、雜芳基或雜環基是各自未取代或者被至少一個選自鹵素、羥基、C _1-8烷基、C _3-8環烷基或雜環基的取代基取代；R ₁₂和R ₁₃各自獨立地選自氫、C _1-8烷基、芳基、雜芳基、雜環基或C _3-8環烷基；其中C _1-8烷基、芳基、雜芳基、雜環基或C _3-8環烷基是各自未取代或者被至少一個選自鹵素、羥基、C _1-8烷基、C _3-8環烷基或雜環基的取代基取代；m為 0、1、2、3 或 4；n為 0、1、2、3 或 4；t為 0、1、2、3 或 4，芳基為6到10員的單環或雙環的芳香環基團；雜芳基為5員或6員單環芳香族環系統，其由碳原子和1-4個選自N、O或S的雜原子組成；雜環基為由碳原子和1-3個選自N、O或S的雜原子組成的3-8員穩定飽和單環系統；第二活性成分，第二活性成分為雌激素受體拮抗劑或芳香化酶抑制劑。 In order to achieve the above object, according to one aspect of the present invention, a pharmaceutical combination is provided, comprising: a first active ingredient, the first active ingredient is a compound having the structure shown in structural formula I, its stereoisomer, its tautomer Any one or more of its body, its polymorph, its solvate and its pharmaceutically acceptable salt;

Structural formula I wherein, ring A is aryl or heteroaryl; Z is selected from CH ₂ , NH, O or S; R ₁ is independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, amino, C _{1 -8} alkyl, C _1-8 alkoxy, C _3-8 cycloalkyl, aryl, heteroaryl, heterocyclyl, heterocyclyl-(CH ₂ ) _m -, aryl-C _1-6 Alkyl-, heteroaryl-C _1-6 alkyl-, NR ₁₂ R ₁₃ , NR ₁₂ -C _1-6 alkylene-NR ₁₂ R ₁₃ , or heterocyclyl-C(O)-, where C _1-8 alkyl, C _1-8 alkoxy, C _3-8 cycloalkyl, aryl, heteroaryl, heterocyclyl, heterocyclyl-(CH ₂ ) _m -, aryl-C _{1- 6} Alkyl, heteroaryl-C _1-6 alkyl or heterocyclyl-C (O)- are each unsubstituted or replaced by at least one selected from halogen, C _1-8 alkyl, C _3-8 cycloalkyl , heterocyclyl, NR ₁₂ R ₁₃ , (CH ₂ ) _t -OH substituent substitution; R ₂ and R ₃ are each independently selected from hydrogen, hydroxyl, cyano, nitro, amino, halogen, C _1-8 Alkyl, C _1-8 alkoxy, C _3-8 cycloalkyl, aryl, heteroaryl or heterocyclic; where C _1-8 alkyl, C _1-8 alkoxy, C _3-8 Cycloalkyl, aryl, heteroaryl or heterocyclyl are each unsubstituted or substituted by at least one substituent selected from halogen, hydroxy, C _1-8 alkyl, C _3-8 cycloalkyl or heterocyclyl ; R ₁₂ and R ₁₃ are each independently selected from hydrogen, C _1-8 alkyl, aryl, heteroaryl, heterocyclyl or C _3-8 cycloalkyl; wherein C _1-8 alkyl, aryl, Heteroaryl, heterocyclyl or C _3-8 cycloalkyl are each unsubstituted or replaced by at least one substituent selected from halogen, hydroxyl, C _1-8 alkyl, C _3-8 cycloalkyl or heterocyclyl Substitution; m is 0, 1, 2, 3 or 4; n is 0, 1, 2, 3 or 4; t is 0, 1, 2, 3 or 4, aryl is a monocyclic or bicyclic ring with 6 to 10 members Aromatic ring group; heteroaryl is a 5-membered or 6-membered monocyclic aromatic ring system, which is composed of carbon atoms and 1-4 heteroatoms selected from N, O or S; heterocyclic group is composed of carbon atoms and a 3-8-membered stable saturated monocyclic system composed of 1-3 heteroatoms selected from N, O or S; the second active ingredient, the second active ingredient is an estrogen receptor antagonist or an aromatase inhibitor.

結構式I-A

結構式I-B                                 結構式I-4

結構式I-C                           結構式I-6

結構式I-D。 Further, the first active ingredient is a compound having any structure shown by structural formula IA to ID or structural formula I-4, structural formula I-6, its stereoisomer, its tautomer, its polymorphic form Any one or more of compounds, solvates and pharmaceutically acceptable salts thereof,

Structural formula IA

Structural Formula IB Structural Formula I-4

Structural Formula IC Structural Formula I-6

Structural ID.

結構式I-1                                 結構式I-2

結構式I-3

結構式I-5

結構式I-7。 Further, the first active ingredient is a compound having any one of structural formulas I-1 to I-3, structural formula I-5, and structural formula I-7, its stereoisomers, its tautomers, Any one or more of its polymorphs, its solvates and pharmaceutically acceptable salts thereof,

Structural formula I-1 Structural formula I-2

Structural formula I-3

Structural formula I-5

Structural formula I-7.

結構式I-1。 Further, the above-mentioned first active ingredient is any one or more of the compounds shown in the structural formula I-1 and pharmaceutically acceptable salts thereof,

Structural formula I-1.

Further, the above-mentioned pharmaceutically acceptable salts are the tartrate compound of the compound and the mesylate compound of the compound, preferably the first active ingredient is the compound or the tartrate compound of the compound.

Further, the above-mentioned tartrate compound has crystal form A, and the X-ray powder diffraction spectrum of crystal form A has characteristic peaks with diffraction angles 2θ of 4.4±0.2°, 23.6±0.2° and 26.9±0.2°, and the preferred crystal The X-ray powder diffraction pattern of type A has the characteristic peaks of 4.4±0.2°, 8.7±0.2°, 10.8±0.2°, 18.4±0.2°, 23.6±0.2° and 26.9±0.2° at diffraction angle 2θ, further The X-ray powder diffraction pattern of the preferred crystal form A has diffraction angles 2θ of 4.4±0.2°, 8.7±0.2°, 10.8±0.2°, 15.9±0.2°, 18.4±0.2°, 23.6±0.2° and 26.9° Characteristic peaks of ±0.2°.

Further, the above-mentioned estrogen receptor antagonist is selected from any one of fulvestrant and tamoxifen, and the aromatase inhibitor is selected from any one of letrozole and anastrozole.

Further, the above-mentioned first active ingredient and second active ingredient are administered simultaneously, separately or sequentially.

Further, the first active ingredient is a compound represented by structural formula I-1 or a pharmaceutically acceptable salt thereof; preferably, the daily dose of the compound represented by structural formula I-1 or a pharmaceutically acceptable salt thereof The dosage (based on the compound represented by structural formula I-1) ranges from 50 to 500 mg; more preferably, the daily dosage of the compound represented by structural formula I-1 or a pharmaceutically acceptable salt thereof (based on structural formula I The compound shown in -1) ranges from 200 to 500 mg; further preferably, the daily dosage of the compound shown in structural formula I-1 or a pharmaceutically acceptable salt thereof (based on the compound shown in structural formula I-1 Compound) range is 300~400mg.

Further, the second active ingredient is fulvestrant; preferably, the dosage of fulvestrant is 1-2000 mg each time, and the administration frequency is 1-2 times a day; more preferably, fulvestrant The dosage is 100-800 mg each time, and the administration frequency is 1-2 times a day; further preferably, the dosage of fulvestrant is 200-600 mg each time, and the administration frequency is 1-2 times a day.

Further, the second active ingredient is letrozole; preferably, the dose of letrozole is 1-200 mg each time, and the administration frequency is 1-2 times a day; more preferably, each dose of letrozole The dosage is 1-20 mg, and the dosing frequency is 1-2 times a day; further preferably, each dosage of letrozole is 1-5 mg, and the dosing frequency is 1-2 times a day.

Further, the second active ingredient is anastrozole; preferably, the dosage of anastrozole is 0.1-50 mg each time, and the administration frequency is 1-2 times a day; more preferably, each dosage of anastrozole The dosage is 1-25 mg, and the administration frequency is 1-2 times a day; further preferably, the dosage of anastrozole is 1-10 mg each time, and the administration frequency is 1-2 times a day.

Further, the above drug combination is used in the preparation of drugs for the treatment of breast cancer, preferably breast cancer is estrogen receptor positive (ER+) breast cancer, or/and human epidermal growth factor receptor 2 negative (HER2-) breast cancer Breast cancer may be locally advanced or metastatic.

According to another aspect of the present invention, there is provided a kit, which includes any one of the above-mentioned pharmaceutical combinations packaged in a container device, the first active ingredient and the second active ingredient in the pharmaceutical combination are administered simultaneously, separately or Apply sequentially.

According to another aspect of the present invention, there is provided a use of any one of the above drug combinations in the preparation of a drug for treating breast cancer.

Further, the aforementioned breast cancer is estrogen receptor positive (ER+) breast cancer, or/and human epidermal growth factor receptor 2 negative (HER2-) breast cancer, or locally advanced or metastatic breast cancer.

Applying the technical scheme of the present invention, the combination of the above-mentioned first active ingredient and the second active ingredient used in the present invention has an outstanding improvement in the joint therapeutic activity compared to the separate administration of each active ingredient. Therefore, the second achieved a synergistic effect.

It should be noted that, in the case of no conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the drawings and examples.

Unless otherwise stated, the basic terms used in the present invention are defined by the following meanings:

Unless otherwise stated, the terms "comprising" and "including" are used in this disclosure in an open-ended and non-limiting sense.

Unless otherwise stated in the present invention or clearly contradicted by the context, the terms "a" and "the" and similar recitations in describing the present invention (especially in the context of claims) should be understood to cover both singular and plural forms .

When the plural form of compounds, salts, etc. is used, this is considered to mean a single compound, salt, etc. at the same time.

In the term "pharmaceutical combination", the first active ingredient and the second active ingredient may be administered independently in separate formulations to be synergistically combined or by using different fixed combinations containing different amounts of the combination partners (i.e. simultaneously or at different time points) ) are combined for synergistic effect. For ease of administration, it may be presented in the form of a "kit" or "kit of parts" or a "combination preparation", in which case parts of the kit may, for example, be administered simultaneously or sequentially staggered in the case of a kit (ie administration of any part of the kit at different time points at the same or different time intervals). Combinations administered as combination preparations, the ratio of the total amounts of the first active ingredient and the second active ingredient may be varied, for example, to suit the needs of a subgroup of patients in need of treatment or the needs of an individual patient, in particular, for example, age or weight. sexual needs.

The term "pharmaceutical composition" is defined herein as a mixture or solution containing at least one therapeutic agent administered to an individual (eg, a mammal or a human) for the prevention or treatment of a particular disease or condition affecting the mammal.

The term "pharmaceutically acceptable" is defined in the present invention as those that are suitable within the scope of sound medical judgment for contacting tissues of an individual (such as a mammal or a human) without undue toxicity, irritation, allergic reaction and other complication problems and A compound, material, composition and/or dosage form with a reasonable benefit/risk ratio commensurate with it.

The term "co-administration" or "administration in combination" as used in the present invention is defined to encompass the administration of selected therapeutic agents to a single patient and is meant to include treatment regimens in which the agents are not necessarily administered by the same route or at the same time.

The term "treatment" as used in the present invention includes treatment that relieves, alleviates or reduces at least one symptom in a subject or affects the delay of disease progression. For example, treatment can be the elimination of one or several symptoms of a disorder or the complete eradication of a disorder (eg, cancer). Within the meaning of the present invention, the term "treatment" also means arresting, delaying the onset (ie the time before clinical manifestations of a disease occurs) and/or reducing the risk of disease development or progression. The term "protect" herein means preventing, delaying or treating (or all, as appropriate) the development or persistence or exacerbation of a disease in an individual.

The term "co-therapeutic activity" or "co-therapeutic effect" means that the therapeutic agents can act individually (in a chronologically staggered fashion, especially in a specific sequential fashion) and in accordance with the preferences of the warm-blooded animal (particularly human) being treated but still The administration is time-spaced to produce a (preferably synergistic) interaction (co-therapeutic effect). Whether this is the case can be determined by following blood levels showing that both compounds are present in the blood of the person to be treated at least during certain time intervals.

The term "pharmaceutically effective amount" or "clinically effective amount" or "therapeutically effective amount" of a combination of therapeutic agents refers to treatment with the combination sufficient to provide an observable improvement in the signs and symptoms observed at a clinical baseline of the disorder. amount.

Unless otherwise stated, "pharmaceutically acceptable salts" in the present invention include salts of acidic and basic groups that may exist in the compounds of the present invention. The compounds of the present invention are basic APIs that can form a wide variety of different salts with various inorganic and organic acids. Acids that can be used to prepare the pharmaceutically acceptable acid addition salts of the basic compounds of the present invention form non-toxic acid addition salts (i.e., salts containing pharmaceutically acceptable anions, such as acetate, benzoic acid Salt, bromide, chloride, citrate, fumarate, hydrobromide, hydrochloride, iodate, lactate, maleate, mandelate, nitrate, oxalate, water sylate, succinate and tartrate). The "pharmaceutically acceptable salts" of the present invention can also locate amorphous or crystalline materials in which the free base API and acid are ionized, or where the two components, the free base API and the acid, utilize mutual intermolecular interactions, Such as hydrogen bonding to produce a homogeneous crystalline material, or co-crystals formed by co-precipitation of free bases and acid groups. It should be understood that the salts of the present invention may also be mixtures of partially ionized and partially eutectic materials.

As analyzed in the prior art of the present invention, the present invention expects to achieve ideal synergistic effect when CDK inhibitors are used in combination with other pharmaceutical preparations. Based on this, the present invention provides a drug combination, a kit containing it and Its use in the preparation of medicines for treating breast cancer.

It should be noted that although CDK4/6 inhibitors include many structural types of compounds, the inventors of the present case found that not all CDK4/6 inhibitors can antagonize the above-mentioned second active ingredient - estrogen receptor Drugs or aromatase inhibitors produce synergistic effects, and even a certain degree of antagonism. In particular, the inventors found that the CDK4/6 inhibitors, which have considerable inhibitory activity against breast cancer when administered alone, often have very different effects when used in combination with the above-mentioned second active ingredient.

結構式I 其中，環A為芳基或雜芳基；Z選自CH ₂、NH、O或S；R ₁分別獨立地選自氫、鹵素、氰基、硝基、羥基、氨基、C _1-8烷基、C _1-8烷氧基、C _3-8環烷基、芳基、雜芳基、雜環基、雜環基-(CH ₂) _m-、芳基-C _1-6烷基-、雜芳基-C _1-6烷基-、NR ₁₂R ₁₃、NR ₁₂-C _1-6亞烷基-NR ₁₂R ₁₃、或雜環基-C(O)-，其中 C _1-8烷基、C _1-8烷氧基、C _3-8環烷基、芳基、雜芳基、雜環基、雜環基-(CH ₂) _m-、芳基-C _1-6烷基、雜芳基-C _1-6烷基或雜環基-C(O)-是各自未取代或者被至少一個選自鹵素、C _1-8烷基、C _3-8環烷基、雜環基、NR ₁₂R ₁₃、(CH ₂) _t-OH的取代基取代；R ₂和R ₃各自獨立地選自氫、羥基、氰基、硝基、氨基、鹵素、C _1-8烷基、C _1-8烷氧基、C _3-8環烷基、芳基、雜芳基或雜環基；其中 C _1-8烷基、C _1-8烷氧基、C _3-8環烷基、芳基、雜芳基或雜環基是各自未取代或者被至少一個選自鹵素、羥基、C _1-8烷基、C _3-8環烷基或雜環基的取代基取代；R ₁₂和R ₁₃各自獨立地選自氫、C _1-8烷基、芳基、雜芳基、雜環基或C _3-8環烷基；其中C _1-8烷基、芳基、雜芳基、雜環基或C _3-8環烷基是各自未取代或者被至少一個選自鹵素、羥基、C _1-8烷基、C _3-8環烷基或雜環基的取代基取代；m為 0、1、2、3 或 4；n為 0、1、2、3 或 4；t為 0、1、2、3 或 4，芳基為6到10員的單環或雙環的芳香環基團；雜芳基為5員或6員單環芳香族環系統，其由碳原子和1-4個選自N、O或S的雜原子組成；雜環基為由碳原子和1-3個選自N、O或S的雜原子組成的3-8員穩定飽和單環系統；第二活性成分為雌激素受體拮抗劑或芳香化酶抑制劑。 Based on the above background, the present invention provides a pharmaceutical combination, which comprises a first active ingredient and a second active ingredient, the first active ingredient is a compound having the structure shown in structural formula I, its stereoisomer, its mutual Any one or more of variants, polymorphs, solvates and pharmaceutically acceptable salts thereof;

Structural formula I wherein, ring A is aryl or heteroaryl; Z is selected from CH ₂ , NH, O or S; R ₁ is independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, amino, C _{1 -8} alkyl, C _1-8 alkoxy, C _3-8 cycloalkyl, aryl, heteroaryl, heterocyclyl, heterocyclyl-(CH ₂ ) _m -, aryl-C _1-6 Alkyl-, heteroaryl-C _1-6 alkyl-, NR ₁₂ R ₁₃ , NR ₁₂ -C _1-6 alkylene-NR ₁₂ R ₁₃ , or heterocyclyl-C(O)-, where C _1-8 alkyl, C _1-8 alkoxy, C _3-8 cycloalkyl, aryl, heteroaryl, heterocyclyl, heterocyclyl-(CH ₂ ) _m -, aryl-C _{1- 6} Alkyl, heteroaryl-C _1-6 alkyl or heterocyclyl-C (O)- are each unsubstituted or replaced by at least one selected from halogen, C _1-8 alkyl, C _3-8 cycloalkyl , heterocyclyl, NR ₁₂ R ₁₃ , (CH ₂ ) _t -OH substituent substitution; R ₂ and R ₃ are each independently selected from hydrogen, hydroxyl, cyano, nitro, amino, halogen, C _1-8 Alkyl, C _1-8 alkoxy, C _3-8 cycloalkyl, aryl, heteroaryl or heterocyclic; where C _1-8 alkyl, C _1-8 alkoxy, C _3-8 Cycloalkyl, aryl, heteroaryl or heterocyclyl are each unsubstituted or substituted by at least one substituent selected from halogen, hydroxy, C _1-8 alkyl, C _3-8 cycloalkyl or heterocyclyl ; R ₁₂ and R ₁₃ are each independently selected from hydrogen, C _1-8 alkyl, aryl, heteroaryl, heterocyclyl or C _3-8 cycloalkyl; wherein C _1-8 alkyl, aryl, Heteroaryl, heterocyclyl or C _3-8 cycloalkyl are each unsubstituted or replaced by at least one substituent selected from halogen, hydroxyl, C _1-8 alkyl, C _3-8 cycloalkyl or heterocyclyl Substitution; m is 0, 1, 2, 3 or 4; n is 0, 1, 2, 3 or 4; t is 0, 1, 2, 3 or 4, aryl is a monocyclic or bicyclic ring with 6 to 10 members Aromatic ring group; heteroaryl is a 5-membered or 6-membered monocyclic aromatic ring system, which is composed of carbon atoms and 1-4 heteroatoms selected from N, O or S; heterocyclic group is composed of carbon atoms and a 3-8-membered stable saturated monocyclic system composed of 1-3 heteroatoms selected from N, O or S; the second active ingredient is an estrogen receptor antagonist or an aromatase inhibitor.

The first active ingredient of the present invention is a CDK4/6 inhibitor, and the combination of the above-mentioned first active ingredient and the second active ingredient is co-administered, and the achieved co-therapeutic activity has a prominent improvement effect compared to the individual administration of each active ingredient , therefore, it shows that the two have achieved a synergistic effect. And the test process shows that the combination of the first active ingredient and the second active ingredient of the present invention has better drug sensitivity to breast cancer cells, and can play a better inhibitory effect in low-dose administration, which is also beneficial to avoid Side effects when large doses of the drug are administered.

結構式I-A

結構式I-B                                 結構式I-4

結構式I-C                           結構式I-6

結構式I-D。 The above-mentioned first active ingredient of the present invention has some differences in its synergistic effect when the second active ingredient is jointly administered. In some embodiments, the first active ingredient has structural formulas IA to ID or structural formula I-4, structural Any one or more of the compound of the structure shown in any one of formula I-6, its stereoisomer, its tautomer, its polymorph, its solvate and its pharmaceutically acceptable salt, in order to expect Further improve the synergistic effect.

Structural formula IA

Structural Formula IB Structural Formula I-4

Structural Formula IC Structural Formula I-6

Structural ID.

結構式I-1                                 結構式I-2

結構式I-3

結構式I-5

結構式I-7。 More preferably, the first active ingredient is a compound having any one of structural formulas I-1 to I-3, structural formula I-5, and structural formula I-7, its stereoisomers, and its tautomers , any one or more of its polymorphs, its solvates and pharmaceutically acceptable salts thereof:

Structural formula I-1 Structural formula I-2

Structural formula I-3

Structural formula I-5

Structural formula I-7.

The above-mentioned pharmaceutical combination of the present invention, especially when the above-mentioned pharmaceutical composition is used in the preparation of the medicine for treating breast cancer, the test shows that its synergistic effect is more prominent, such as breast cancer is estrogen receptor positive (ER+) breast cancer , or human epidermal growth factor receptor 2 negative (HER2-) breast cancer or locally advanced or metastatic breast cancer.

結構式I-1。 Especially when the first active ingredient is any one or more of the compounds represented by structural formula I-1 and pharmaceutically acceptable salts thereof, the comprehensive effect is more stable.

Structural formula I-1.

In some embodiments of the present invention, the above-mentioned pharmaceutically acceptable salt is preferably the tartrate compound of the compound and the mesylate compound of the compound, and the first active ingredient is preferably the above-mentioned compound or the tartrate compound of the above-mentioned compound.

In some embodiments of the present invention, the above-mentioned pharmaceutically acceptable salt is preferably the tartrate compound of the compound and the mesylate compound of the compound, and the first active ingredient is preferably the above-mentioned compound or the tartrate compound of the above-mentioned compound.

，結構式II。 The structural formula of the tartrate compound of a kind of above-mentioned compound is:

, structural formula II.

，結構式III。 The structural formula of a mesylate compound of above-mentioned compound A is:

, structural formula III.

The first active ingredients used in the present invention are all existing compounds in the prior art, for example, reference can be made to PCT patent application documents with publication numbers WO2018/113771A1 and WO2019242719A1, and the more specific preparation methods will not be repeated here.

Further, in order to improve the stability of the joint administration of the first active ingredient and the second active ingredient, preferably the above-mentioned tartrate compound has crystal form A, and the X-ray powder diffraction pattern of crystal form A has a diffraction angle 2θ of 4.4 Characteristic peaks of ±0.2°, 23.6±0.2° and 26.9±0.2°, the X-ray powder diffraction spectrum of the preferred crystal form A has diffraction angles 2θ of 4.4±0.2°, 8.7±0.2°, 10.8±0.2° , 18.4±0.2°, 23.6±0.2° and 26.9±0.2° characteristic peaks, and the X-ray powder diffraction spectrum of the better crystal form A has diffraction angles 2θ of 4.4±0.2°, 8.7±0.2°, 10.8 Characteristic peaks at ±0.2°, 15.9±0.2°, 18.4±0.2°, 23.6±0.2° and 26.9±0.2°. The tartrate compound with the above crystal form A has better solubility and more prominent stability, so its medicinal effect is more prominent.

In some embodiments of the present invention, the aforementioned estrogen receptor antagonist is selected from any one of fulvestrant and tamoxifen. The aromatase inhibitor is selected from any one of letrozole and anastrozole. The above-mentioned drugs are the active ingredients of the current first-line or second-line clinical commonly used drugs, so their safety is higher.

Although the optional ingredients of the above-mentioned second active ingredients are not as active ingredients in the existing commonly used drugs, when they are administered, the way of administration and the amount of administration are all different, in order to better realize the combination of each second active ingredient with the first For the purpose of synergistic effect of active ingredients, a corresponding range of clinical application can be given according to the individual needs of patients.

During clinical administration, patients can choose the simultaneous administration, separate administration or sequential administration of the first active ingredient and the second active ingredient according to the specific form of the drug combination. The specific dosage relationship of the above two types of active ingredients can be adjusted. Of course, in order to better exert synergistic effects, the preferred first active ingredient is a compound represented by structural formula I-1 or a pharmaceutically acceptable salt thereof; Preferably, the daily dosage of the compound represented by structural formula I-1 or a pharmaceutically acceptable salt thereof (based on the compound represented by structural formula I-1) ranges from 50 to 500 mg, more preferably from 200 to 500 mg , further preferably 300~400mg. In some embodiments, the second active ingredient is fulvestrant; preferably, the dosage of fulvestrant is 1-2000 mg each time, and the administration frequency is 1-2 times a day; more preferably, fluorine The dosage of fulvestrant is 100-800 mg each time, and the administration frequency is 1-2 times a day; further preferably, the dosage of fulvestrant is 200-600 mg each time, and the administration frequency is 1-2 times a day. 2 times. Alternatively, the second active ingredient is letrozole; preferably, the dosage of letrozole is 1-200 mg each time, and the dosing frequency is 1-2 times a day; more preferably, the dosage of letrozole The dose is 1-20 mg, and the dosing frequency is 1-2 times a day; further preferably, the dosage of letrozole is 1-5 mg each time, and the dosing frequency is 1-2 times a day. Alternatively, the second active ingredient is anastrozole; preferably, the dosage of anastrozole is 0.1-50 mg each time, and the dosage frequency is 1-2 times a day; more preferably, the dosage of anastrozole The dose is 1-25 mg, and the dosing frequency is 1-2 times a day; further preferably, the dosage of anastrozole is 1-10 mg each time, and the dosing frequency is 1-2 times a day.

Wherein, the above-mentioned pharmaceutical combination of the present invention can be administered after forming corresponding preparations with current active ingredients and pharmaceutically acceptable carriers. The aforementioned estrogen receptor antagonists, such as fulvestrant, are administered in the form of injections. Unless otherwise stated, they are prepared in a manner known per se, for example by various conventional mixing, comminution, direct compression, granulation, dragee coating, dissolution, lyophilization processes, melt granulation or processing techniques well known to those skilled in the art . It should be noted that the unit content of the combination partner contained in the individual dose of each dosage form does not necessarily constitute an effective amount by itself, because the required effective amount can be achieved by administering multiple dosage units.

The above-mentioned "pharmaceutically acceptable carrier" refers to a conventional pharmaceutical carrier suitable for desired pharmaceutical preparations, for example: diluents and excipients such as water, various organic solvents, etc.; fillers such as starch, sucrose, etc.; Binders such as cellulose derivatives, alginate, gelatin and polyvinylpyrrolidone (PVP); humectants such as glycerin; disintegrants such as agar, calcium carbonate and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; Surfactants such as cetyl alcohol; absorbent vehicles such as kaolin and bentonite; lubricants such as talc, calcium stearate, magnesium stearate and polyethylene glycols. In addition, other pharmaceutically acceptable auxiliary materials such as dispersants, stabilizers, thickeners, complexing agents, buffers, penetration enhancers, polymers, fragrances, sweeteners and dyes can be added therein. Excipients suitable for the desired dosage form and desired mode of administration are preferably used.

In another typical embodiment of the present invention, a use of any one of the above drug combinations in the preparation of a drug for treating breast cancer is provided.

The joint co-administration of the above-mentioned first active ingredient and the second active ingredient of the present invention has an outstanding improvement effect on the achieved co-therapeutic activity compared with the individual administration of each active ingredient, therefore, it shows that the two have achieved a synergistic effect .

In some embodiments, the aforementioned breast cancer is estrogen receptor positive (ER+) breast cancer, or human epidermal growth factor receptor 2 negative (HER2-) breast cancer, or locally advanced or metastatic breast cancer.

In another typical embodiment of the present invention, a kit is provided, the kit includes any one of the above pharmaceutical combinations packaged in a container device, the first active ingredient and the second active ingredient in the pharmaceutical combination are administered simultaneously , separately or sequentially. The above-mentioned kit is a convenient way to administer the pharmaceutical combination of the present invention, but it does not mean that the pharmaceutical combination of the present invention can only exist in the form of a kit.

The first active ingredient and the second active ingredient can be administered independently or by using different fixed combinations (ie simultaneously or at different points in time) containing different amounts of the combination partners. The parts of the kit can then, for example, be administered simultaneously or sequentially staggered (ie, any part of the kit is administered at different time points at the same or different time intervals). When administered as a combined preparation in the form of a kit, the ratio of the total amounts of the first active ingredient and the second active ingredient may be varied, for example to suit the needs of a subgroup of patients in need of treatment or the needs of an individual patient, such as in particular age or Weight specific needs.

In yet another typical embodiment of the present invention, a method for treating breast cancer with drug combination is provided. During treatment, the corresponding clinically administered dosage ranges of the first active ingredient and the second active ingredient can be administered according to the individual needs of the patient.

The beneficial effects of the present invention will be further described below in conjunction with examples and comparative examples.

（結構式II），晶型為晶型A，譜圖見圖1。第一活性成分參考WO2019242719A1的方法製備而成，氟維司群為市售。 [Example 1] Inhibitory effect of the first active ingredient in combination with fulvestrant on tumor cell proliferation The chemical formula of the first active ingredient is

(Structural formula II), the crystal form is crystal form A, and the spectrum is shown in Figure 1. The first active ingredient is prepared by referring to the method of WO2019242719A1, and fulvestrant is commercially available.

Methods: In this experiment, the DELFIA® Cell proliferation kit method of PerkinElmer was used to detect whether the combination of the first active ingredient and fulvestrant (Fulvestrant) has a synergistic effect on the inhibitory activity of T-47D cell proliferation to support clinical research. Combined drug strategy. This method utilizes the addition of BrdU (5-bromo-2'-deoxyuridine) into the cells as a DNA analog into the cell proliferation process, and then detects the amount of BrdU incorporated into the DNA through an immune response (anti-BrdU antibody) to respond to the cells level of proliferation. The culture conditions of the cells in this experiment simulated the estrogen stimulation under the pathological conditions of breast cancer. At the same time, the hormone factors that may exist in the serum were removed by activated carbon treatment to ensure the controllability of the system. Therefore, the combination of the first active ingredient and fulvestrant was selected under the condition of adding activated carbon-treated serum and β-estradiol to the medium to maximize the effect of the combination.

The specific experimental steps are: T-47D cells were cultured in phenol red-free DMEM medium, plus 10 μg/mL Human Insulin, 10% FBS and 1% double antibody. Cultured at 37°C, 5% CO ₂ . Routinely culture until the cell saturation is 80%~90%. When the number reaches the requirement, collect the cells. Cells were resuspended in medium treated with activated charcoal serum plus β-estradiol, counted, and prepared into a suitable density of cell suspension. Add the cell suspension to a 96-well plate, 100 µL per well, 3000 cells/well. The cells were cultured overnight in an incubator. The first active ingredient and fulvestrant were diluted with DMSO and diluted in culture medium for use. 24 hours after cell plating, add 90 µL of medium to each well, and then add 10 µL of the prepared compounds to the wells. The final concentration of the test compound is: test first active ingredient concentration: 60 nM. Test compound fulvestrant concentration: 150 nM. Combined drug concentration: 150 nM fulvestrant + 60 nM first active ingredient. Place the cell culture plate in the incubator for 96 hours. Dilute BrdU Labeling Reagent 10 times with culture medium, then add 2 μL to each well. Place the cell culture plate in the incubator overnight. Gently suck off the medium, add 100 μL of fixative to each well, and incubate at room temperature for 30 minutes. Discard the fixative, add 100 μL of 0.5 μg/mL Anti-BrdU-Eu antibody to each well, and incubate at room temperature for 60 minutes. Discard the antibody solution and wash 4 times with washing solution. Add 200 μL DELFIA Inducer to each well and incubate at room temperature for 30 minutes. Use Envision to read the fluorescent signal value.

Data analysis: The effect of compound drug combination was evaluated by the coefficient of drug in interaction (CDI). CDI is calculated according to the formula: CDI =AB/A×B, where AB is the ratio of cell well readings between the two-drug combination group and the DMSO control group, and A or B is the ratio of cell well readings between the drug alone group and the control group. If CDI<1, it proves that the nature of the two drugs is synergistic, and when CDI<0.7, the synergistic effect of the two drugs is very significant; if CDI=1, the nature of the two drugs is additive; if CDI>1, the nature of the two drugs is antagonistic.

Results: The experimental results of the combination of the first active ingredient and fulvestrant on the proliferation of T-47D cells showed that the CDI of the combination of 60 nM the first active ingredient and 150 nM fulvestrant was 0.59, according to the combined drug index Judgment criteria, indicating that the synergistic effect of the two drugs is very significant. The results are shown in Table 1 and Figure 2.

Conclusion: On T-47D cells, under estrogen-stimulated conditions simulating the pathological conditions of breast cancer patients, the combination index CDI of 60 nM the first active ingredient combined with 150 nM fulvestrant was 0.59, indicating that the two drugs synergistically The effect is very significant.

[Table 1] group Fluorescent reading %Ctrl CDI DMSO 20160±705.9 100.0±3.5 / first active ingredient 10783±425.6 53.5±2.1 / Fulvestrant 11053±248.0 54.8±1.2 / The first active ingredient + fulvestrant 3466±130.1 17.2±0.6 0.59 Note: Readings and %Ctrl are mean ± SEM characterization.

[Example 2]

In vivo pharmacodynamic study of tartrate salt of the first active ingredient with the aforementioned structural formula (II) or combined use of fulvestrant on human breast cancer MCF-7 cell subcutaneous xenograft tumor BALB/c nude mouse model

Methods: Balb/c nude mice were subcutaneously inoculated with 17β-estradiol tablets (0.18 mg, sustained release for 90 days) 3 days before cell inoculation. MCF-7 cells were inoculated subcutaneously on the back to establish the MCF-7 xenograft tumor animal model. The experiment was divided into solvent blank control group, first active ingredient 25 mg/kg group, first active ingredient 50 mg/kg group, fulvestrant 200 mg/kg group, first active ingredient 25 mg/kg+fulvestrant 200 mg/kg group, and the first active ingredient 50 mg/kg+fulvestrant 200 mg/kg group. 8 experimental animals in each group. The first active ingredient to be tested was intragastrically administered from the day of grouping, once a day, for a total of 29 days (QD × 29 days). Fulvestrant was administered subcutaneously from the day of grouping, once a week, and administered 5 times in total (QW x 5 times). The safety evaluation was carried out according to the animal body weight change and death, and the efficacy evaluation was carried out according to the relative tumor inhibition rate (TGI%).

The formula for calculating tumor volume is: V = 0.5a × b ² , where a and b represent the long and short diameters of the tumor, respectively. The antitumor efficacy of compounds was evaluated by TGI (%) or relative tumor proliferation rate T/C (%). TGI (%) reflects tumor growth inhibition rate. Calculation of TGI(%): TGI(%)=[(1-(Average tumor volume at the end of administration of a certain treatment group-Average tumor volume at the beginning of administration of this treatment group))/(Average tumor volume at the end of treatment of the solvent control group Volume - average tumor volume at the beginning of treatment in the solvent control group)] × 100%.

Relative tumor proliferation rate ΔT/ΔC(%): The calculation formula is as follows: ΔT/ΔC(%) = (Ti-T0)/(Vi-V0) x 100. Among them, V0 is the average tumor volume measured in the solvent control group during administration in groups (that is, D0), and Vi is the average tumor volume in the solvent control group at a certain measurement; Tumor volume, Ti is the average tumor volume of the administration group at a certain measurement.

[result]

1. Efficacy evaluation of compounds on MCF-7 cell xenograft tumor model:

On the 28th day after the mice were administered, the first active ingredient (25 mg/kg) group did not show a significant anti-tumor effect compared with the average tumor volume of the solvent control group, the p value was 0.1706, and the relative tumor proliferation rate ΔT/ΔC (%) was 62.02%, and the tumor growth inhibition rate TGI (%) was 37.98%. Compared with the average tumor volume of the solvent control group, the first active ingredient (50 mg/kg) group showed a significant anti-tumor effect, with a p value of 0.0020; the relative tumor proliferation rate ΔT/ΔC (%) was 28.97%; tumor growth inhibition The rate TGI (%) is 71.03%. Fulvestrant (200 mg/kg) group also showed a significant anti-tumor effect compared with the average tumor volume of the solvent control group, with a p value of 0.0001; the relative tumor proliferation rate ΔT/ΔC (%) was 11.13%; tumor growth inhibition The rate TGI (%) is 88.87%. The first active ingredient (25 mg/kg) + fulvestrant (200 mg/kg) group and the first active ingredient (50 mg/kg) + fulvestrant (200 mg/kg) group compared with the solvent control group The average tumor volume also showed significant anti-tumor effects, the p values were < 0.0001 and < 0.0001 respectively; the relative tumor proliferation rates △T/△C (%) were -12.05% and -12.80% respectively; the tumor growth inhibition rates TGI (%) are 112.05% and 112.80% respectively. The antitumor efficacy evaluation is shown in Table 2; the tumor growth curve is shown in Figure 3.

[Table 2] Evaluation of antitumor efficacy of the first active ingredient or combined use of fulvestrant on the MCF-7 xenograft tumor model (calculated based on the tumor volume on day 28 after administration) group solvent control first active ingredient first active ingredient Fulvestrant The first active ingredient + Fulvestrant The first active ingredient + Fulvestrant Dose (mg/kg) -- 25 mg/kg, QD 50 mg/kg, QD 200 mg/kg, QW 25 mg/kg, QD+ 200 mg/kg, QW 50 mg/kg, QD+ 200 mg/kg, QW Tumor volume (mm ³ ) ^a 0 days 153 ± 11 153 ± 11 153 ± 10 153 ± 10 153 ± 9 153 ± 8 3 days 231 ± 23 177 ± 13 183 ± 9 186 ± 19 157 ± 13 151 ± 13 7 days 300 ± 36 198 ± 14 184 ± 14 223 ± 43 158 ± 13 141 ± 16 10 days 348 ± 43 213 ± 14 195 ± 14 245 ± 50 151 ± 20 132 ± 17 14 days 430 ± 62 254 ± 20 193 ± 16 253 ± 58 133 ± 18 108 ± 17 17 days 496 ± 72 307 ± 23 205 ± 21 239 ± 61 102 ± 14 92 ± 15 21 days 585 ± 94 355 ± 30 230 ± 27 243 ± 77 98 ± 16 81 ± 14 24 days 667±127 416 ± 50 258 ± 40 223 ± 80 96 ± 18 75 ± 16 28 days 764 ± 158 532 ± 57 330 ± 60 221 ± 78 80 ± 18 75 ± 19 Relative tumor volume proliferation rate △T/△C (%) ^b - 62.02 28.97 11.13 -12.05 -12.80 Tumor growth inhibition rate TGI (%) ^b 37.98 71.03 88.87 112.05 112.80 p -value ^c - 0.1706 0.0020 0.0001 <0.0001 <0.0001 Note: a. Mean ± SEM, n=8. b. Tumor growth inhibition was calculated from ΔT/ΔC and TGI (TGI (%)=[1-(T ₂₈ -T ₀ )/(V ₂₈ -V ₀ )]×100). c. The p value is the comparative analysis of the tumor volume of the treatment group and the vehicle control group.

2. Safety evaluation of compounds on MCF-7 cell xenograft tumor model

In this model, as shown in Figure 4, all animals had no significant weight loss during the dosing period.

[in conclusion]

From the perspective of drug safety, tumor-bearing mice showed good tolerance to the first active ingredient at two doses of 25 mg/kg and 50 mg/kg or in combination with Fulvestrant (200 mg/kg) .

In terms of tumor volume and tumor weight, when the first active ingredient was used in combination with fulvestrant (200 mg/kg) at two doses of 25 mg/kg and 50 mg/kg, it also showed significant anti-tumor effects.

In terms of drug sensitivity, it can be seen from the data in the above table that the p- values of the first active ingredient at both doses of 25 mg/kg and 50 mg/kg can reach <0.0001, which is far lower than other CDK4/6 in the prior art Inhibitor administration p -value. This is enough to show that the combined use of the first active ingredient and the second active ingredient provided by the present invention has more significant drug sensitivity to the proliferation of breast cancer cells, and plays a better role in promoting the reduction of drug doses and side effects.

[Example 3 to Example 7, Comparative Example 1]

In Examples 3 to 8, different first active ingredients were combined with fulvestrant according to the dosage in the table to inhibit the proliferation of T47D tumor cells. The specific experimental process is as follows:

In this experiment, Abcam’s Brdu ELISA kit method was used to detect whether the combination of each first active ingredient and Fulvestrant (Fulvestrant) has a synergistic effect on the inhibitory activity of T47D cell proliferation to support the clinical combination strategy. This method utilizes the addition of BrdU (5-bromo-2'-deoxyuridine) into the cells as a DNA analog into the cell proliferation process, and then detects the amount of BrdU incorporated into the DNA through an immune response (anti-BrdU antibody) to respond to the cells level of proliferation. The culture conditions of the cells in this experiment simulated the estrogen stimulation under the pathological conditions of postmenopausal breast cancer. At the same time, activated carbon was used to remove the hormone factors that may exist in the serum to ensure the controllability of the system. Therefore, the combination of the first active ingredient and fulvestrant was selected under the condition of adding activated carbon-treated serum plus estradiol to the medium to maximize the combined drug effect.

The specific experimental steps are: T47D cells were cultured in phenol red-free RPMI-1640 medium, plus 10 μg/mL Human Insulin, 10% FBS and 1% double antibody. Cultured at 37°C, 5% CO ₂ . Routinely culture until the cell saturation is 80%-90%, and collect the cells when the number reaches the requirement. The cells were resuspended in the culture medium of activated charcoal-treated serum plus estradiol, counted, and prepared into a suitable density of cell suspension. The cell suspension was added to a 96-well plate and incubated overnight in a cell incubator. The first active ingredient and fulvestrant were diluted with DMSO and diluted in culture medium for use. 24 hours after the cells were plated, the prepared compounds were added to the wells. Place the cell culture plate in the incubator for 96 hours. 20 hours before the end of the experiment, 10 ul of 1X Brdu was added to each well, and 10 ul of phenol red-free 1640 medium containing 10% activated carbon adsorbed fetal bovine serum was added to the Background wells. Continue to cultivate. Gently aspirate the medium, add fixative to each well, and incubate at room temperature for 30 minutes. Discard the fixative, add 100 ul anti-BrdU monoclonal Detector Antibody to each well, and incubate at room temperature for 60 minutes. Discard the antibody solution and wash 4 times with washing solution. Add 100 ul 1X Peroxidase Goat Anti-Mouse IgG Conjugate to each well and incubate at room temperature for 30 minutes. Add 100 ul TMB to each well, incubate at room temperature for 30 minutes and read OD450.

Effect evaluation method: The effect of the combination of compounds was evaluated by Bliss independence model. Synergy scores were calculated using the Bliss independent model and the Loewe additive model. Above 5 indicates synergy, below -5 indicates antagonism.

The results are shown in Table 3.

。 [table 3] group first active ingredient concentration (first) second active ingredient Concentration (Second) Bliss_Score Example 3 Compound shown in structural formula I-1 180(nM) Fulvestrant 150.000(nM) 15.684297 Example 4 Compound shown in structural formula I-5 180(nM) Fulvestrant 150.000(nM) 10.203114 Example 6 Compound shown in structural formula I-3 180(nM) Fulvestrant 150.000(nM) 14.052328 Example 7 Compound shown in structural formula I-7 180(nM) Fulvestrant 150.000(nM) 6.2664433 Example 8 Compound shown in structural formula I-8 180(nM) Fulvestrant 150.000(nM) -6.242784 Note: structural formula I-8 is a compound

.

[Examples 9 to 11]

In Examples 9 to 11, different first active ingredients were combined with letrozole according to the dosage in the table to inhibit the proliferation of MCF-7 tumor cells. The specific experimental process is as follows.

In this experiment, Abcam’s Brdu ELISA kit method was used to detect whether the combination of different first active ingredients and letrozole (Letrozole) has a synergistic effect on the inhibitory activity of MCF-7 cell proliferation to support the clinical combination strategy. This method utilizes the addition of BrdU (5-bromo-2'-deoxyuridine) into the cells as a DNA analog into the cell proliferation process, and then detects the amount of BrdU incorporated into the DNA through an immune response (anti-BrdU antibody) to respond to the cells level of proliferation. The culture conditions of the cells in this experiment simulated the estrogen stimulation under the pathological conditions of postmenopausal breast cancer. At the same time, activated carbon was used to remove the hormone factors that may exist in the serum to ensure the controllability of the system. Therefore, the combination of different first active ingredients and letrozole was selected under the condition of adding activated carbon-treated serum plus androstenedione to the medium to maximize the combined drug effect.

The specific experimental steps are: MCF-7 cells were cultured in phenol red-free RPMI-1640 medium, plus 10 μg/mL Human Insulin, 10% FBS and 1% double antibody. Cultured at 37°C, 5% CO ₂ . Routinely culture until the cell saturation is 80%-90%, and collect the cells when the number reaches the requirement. Cells were resuspended in medium treated with activated charcoal and serum plus androstenedione, counted, and prepared into a suitable density of cell suspension. The cell suspension was added to a 96-well plate and incubated overnight in a cell incubator. The first active ingredient and letrozole were diluted with DMSO and diluted in culture medium for use. 24 hours after the cells were plated, the prepared compounds were added to the wells. Place the cell culture plate in the incubator for 96 hours. 20 hours before the end of the experiment, 10 ul of 1X Brdu was added to each well, and 10 ul of phenol red-free 1640 medium containing 10% activated carbon adsorbed fetal bovine serum was added to the Background wells. Continue to cultivate, gently suck off the medium, add fixative to each well, and incubate at room temperature for 30 minutes. Discard the fixative, add 100 ul anti-BrdU monoclonal Detector Antibody to each well, and incubate at room temperature for 60 minutes. Discard the antibody solution and wash 4 times with washing solution. Add 100 ul 1X Peroxidase Goat Anti-Mouse IgG Conjugate to each well and incubate at room temperature for 30 minutes. Add 100 ul TMB to each well, incubate at room temperature for 30 minutes and read OD450.

Effect evaluation method: The effect of the combination of compounds was evaluated by Bliss independence model. Synergy scores were calculated using the Bliss independent model and the Loewe additive model. Above 5 indicates synergy, below -5 indicates antagonism.

The results are shown in Table 4.

[Table 4] group first active ingredient concentration (first) second active ingredient Concentration (Second) Bliss_Score Example 9 Compound shown in structural formula I-1 20(nM) Letrozole 50.000(nM) 14.05 Example 10 Compound shown in structural formula I-1 180(nM) Letrozole 50.000(nM) 15.01 Example 11 Compound shown in structural formula I-5 180(nM) Letrozole 10.000(nM) 6.05

[Example 12]

A clinical trial of the tartrate salt of the first active ingredient (the compound represented by the aforementioned structural formula II) or the combination with fulvestrant.

[Inclusion criteria]

1. (Dose escalation stage) Medication alone: patients with advanced breast cancer diagnosed by histology or cytology, who cannot benefit from the existing standard treatment plan, and are not suitable for curative surgical resection or radiation therapy.

2. (Expansion of enrollment stage) Combined drug use: the following conditions must be met at the same time

Histology or cytology confirmed by the research center as HR-positive, HER-2-negative (if there is a needle biopsy for metastatic lesions, the results of metastatic lesions shall prevail), locally advanced or recurrent/metastatic female breast cancer, and it is not suitable for Surgical resection or radiation therapy for curative purposes.

Postmenopausal female patients in natural state; or premenopausal female patients who have previously undergone bilateral oophorectomy or medical castration to achieve postmenopausal state.

3. (Expansion of enrollment stage) Combined medication: ① For patients with recurrent and metastatic disease, no more than 1 line of chemotherapy is allowed. ②The following standards must be met at the same time: For patients who have received first-line endocrine therapy in the recurrent/metastasis stage, the progression-free time of continuous first-line endocrine therapy must be ≥ 6 months, and the disease progression has been confirmed by imaging; For patients who have received adjuvant endocrine therapy, it is necessary to meet the requirements of imaging-confirmed disease recurrence from the time of receiving adjuvant endocrine therapy (continuous medication for at least 2 years) to 12 months or less after the completion of adjuvant endocrine therapy.

Measurable lesions defined by RECIST V.1.1, tumor lesions that have previously received radiotherapy or other local treatments, are only considered as measurable lesions if there is a clear record of disease progression at the treatment site after completion of treatment.

[Dosing regimen]

(Dose escalation stage) Medication alone: 6 doses are designed in this stage, which are drug A (tartrate capsules of the compound represented by structural formula I-1) 50 mg/d, 100 mg/d, 200 mg/d, 300 mg /d, 400 mg/d and 500 mg/d. For oral administration, drug A capsules are administered once, followed by continuous administration once a day after elution for 7 days.

(Expansion of enrollment phase) Combination medication: drug A (tartrate capsules of the compound represented by structural formula I-1) 300 mg/d combined with fulvestrant 500 mg/28 d or drug A 400 mg/d combined with fulvestrant Group 500 mg/28d. The capsule of the first active ingredient is administered orally, once a day, and taken continuously. Fulvestrant 500 mg: once in the first cycle D1 and D15, once in the second cycle and every subsequent cycle D1, continuous slow intramuscular injection (1-2 min/5 mL) into the buttocks, one injection in each buttock. Every 28 days is a cycle.

[in conclusion]

As of February 25, 2022, of the 13 efficacy evaluable subjects in the 300 mg/d, 400 mg/d, and 500 mg/d dose groups administered alone (dose escalation phase), 1 subject exhibited a partial response (PR), the objective response rate (ORR) was 7.7%. (Expansion of enrollment stage) Among the 30 evaluable subjects in the combined drug group (the first active ingredient capsule 300 mg/d and 400 mg/d combined with fulvestrant), 17 subjects showed partial remission (PR ), the objective response rate (ORR) was 56.7%; among the 21 curative effect-evaluable subjects of drug A capsule 400 mg/d combined with fulvestrant, 14 subjects showed partial response (PR), the objective response rate (ORR) up to 66.7%.

The curative effect of drug A combined with fulvestrant (≥300 mg/d) was better than single drug A; at the same time, the historical data of fulvestrant in the treatment of advanced breast cancer that had relapsed or progressed after previous endocrine therapy (ORR: 9-21.3 %), the combined drug also showed superior curative effect.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

none

The description drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the schematic embodiments and descriptions of the present invention are used to explain the present invention, and do not constitute improper limitations to the present invention. In the schema: Fig. 1 shows the XRD spectrogram of the first active ingredient according to embodiment 1 of the present invention; Figure 2 shows the synergistic effect of the combination of the first active ingredient and fulvestrant according to Example 1 of the present invention on the inhibition of T-47D cell proliferation; Figure 3 shows the tumor growth curve of the MCF-7 cell xenograft tumor Balb/c nude mice in Example 2 after the first active ingredient or in combination with fulvestrant (Fulvestrant), and the data points represent the average value in the group Volume, error bars represent standard error (SEM); and Figure 4 shows the body weight changes of the Balb/c nude mice with MCF-7 cell xenograft tumors in Example 2 after the first active ingredient or in combination with fulvestrant (Fulvestrant), and the data points represent the average body weight in the group , error bars represent standard error (SEM).

Claims (16)

A pharmaceutical combination, comprising: a first active ingredient, the first active ingredient is a compound having a structure represented by structural formula I, its stereoisomer, its tautomer, its polymorph, its solvate and Any one or more of its pharmaceutically acceptable salts;

Structural formula I wherein, Ring A is aryl or heteroaryl; Z is selected from CH ₂ , NH, O or S; R ₁ is independently selected from hydrogen, halogen, cyano, nitro, hydroxyl, amino, C _{1 -8} alkyl, C _1-8 alkoxy, C _3-8 cycloalkyl, aryl, heteroaryl, heterocyclyl, heterocyclyl-(CH ₂ ) _m -, aryl-C _1-6 Alkyl-, heteroaryl-C _1-6 alkyl-, NR ₁₂ R ₁₃ , NR ₁₂ -C _1-6 alkylene-NR ₁₂ R ₁₃ , or heterocyclyl-C(O)-, where C _1-8 alkyl, C _1-8 alkoxy, C _3-8 cycloalkyl, aryl, heteroaryl, heterocyclyl, heterocyclyl-(CH ₂ ) _m -, aryl-C _{1- 6} Alkyl, heteroaryl-C _1-6 alkyl or heterocyclyl-C (O)- are each unsubstituted or replaced by at least one selected from halogen, C _1-8 alkyl, C _3-8 cycloalkyl , heterocyclyl, NR ₁₂ R ₁₃ , (CH ₂ ) _t -OH substituent substitution; R ₂ and R ₃ are each independently selected from hydrogen, hydroxyl, cyano, nitro, amino, halogen, C _1-8 Alkyl, C _1-8 alkoxy, C _3-8 cycloalkyl, aryl, heteroaryl or heterocyclic; where C _1-8 alkyl, C _1-8 alkoxy, C _3-8 Cycloalkyl, aryl, heteroaryl or heterocyclyl are each unsubstituted or substituted by at least one substituent selected from halogen, hydroxy, C _1-8 alkyl, C _3-8 cycloalkyl or heterocyclyl ; R ₁₂ and R ₁₃ are each independently selected from hydrogen, C _1-8 alkyl, aryl, heteroaryl, heterocyclyl or C _3-8 cycloalkyl; wherein C _1-8 alkyl, aryl, Heteroaryl, heterocyclyl or C _3-8 cycloalkyl are each unsubstituted or replaced by at least one substituent selected from halogen, hydroxyl, C _1-8 alkyl, C _3-8 cycloalkyl or heterocyclyl Substitution; m is 0, 1, 2, 3 or 4; n is 0, 1, 2, 3 or 4; t is 0, 1, 2, 3 or 4, and the aryl group is a monocyclic ring with 6 to 10 members or a bicyclic aromatic ring group; the heteroaryl is a 5-membered or 6-membered monocyclic aromatic ring system, which is composed of carbon atoms and 1-4 heteroatoms selected from N, O or S; the heteroaryl The ring group is a 3-8 membered stable saturated monocyclic ring system composed of carbon atoms and 1-3 heteroatoms selected from N, O or S; the second active ingredient is an estrogen receptor antagonist agents or aromatase inhibitors. The pharmaceutical combination as claimed in claim 1, wherein the first active ingredient is a compound having any one of structural formulas IA to ID or structural formula I-4, structural formula I-6, and its stereoisomers , any one or more of its tautomers, its polymorphs, its solvates and pharmaceutically acceptable salts thereof,

Structural formula IA

Structural Formula IB Structural Formula I-4

Structural Formula IC Structural Formula I-6

Structural ID. The pharmaceutical combination according to claim 1, wherein the first active ingredient is a compound having any one of structural formulas I-1 to I-3, structural formula I-5, and structural formula I-7, its Any one or more of stereoisomers, tautomers, polymorphs, solvates and pharmaceutically acceptable salts thereof,

Structural formula I-1 Structural formula I-2

Structural formula I-3

Structural formula I-5

Structural formula I-7. The pharmaceutical combination as claimed in claim 1, wherein the first active ingredient is any one or more of the compounds shown in structural formula I-1 and pharmaceutically acceptable salts thereof,

Structural formula I-1. The pharmaceutical combination as described in any one of claims 1 to 4, wherein the pharmaceutically acceptable salt is a tartrate compound of the compound and a mesylate compound of the compound, preferably the first An active ingredient is said compound or a tartrate salt compound of said compound. The drug combination as claimed in claim 5, wherein the tartrate compound has crystal form A, and the X-ray powder diffraction pattern of the crystal form A has a diffraction angle 2θ of 4.4±0.2°, 23.6±0.2° and a characteristic peak of 26.9±0.2°, preferably the X-ray powder diffraction pattern of the crystal form A has a diffraction angle 2θ of 4.4±0.2°, 8.7±0.2°, 10.8±0.2°, 18.4±0.2°, The characteristic peaks of 23.6±0.2° and 26.9±0.2°, further preferably, the X-ray powder diffraction pattern of the crystal form A has a diffraction angle 2θ of 4.4±0.2°, 8.7±0.2°, 10.8±0.2°, Characteristic peaks at 15.9±0.2°, 18.4±0.2°, 23.6±0.2° and 26.9±0.2°. The drug combination according to any one of claims 1 to 6, wherein the estrogen receptor antagonist is selected from any one of fulvestrant and tamoxifen, and the aromatase inhibitor is selected from Any one of letrozole and anastrozole. The pharmaceutical combination according to any one of claims 1 to 7, wherein the first active ingredient and the second active ingredient are administered simultaneously, separately or sequentially. The pharmaceutical combination according to claim 8, wherein the first active ingredient is a compound represented by structural formula I-1 or a pharmaceutically acceptable salt thereof; preferably, the compound represented by structural formula I-1 The daily dosage range of the compound or its pharmaceutically acceptable salt is 50~500 mg; more preferably, the daily dosage range of the compound represented by the structural formula I-1 or its pharmaceutically acceptable salt is 200 mg ~500mg; further preferably, the daily dosage range of the compound represented by structural formula I-1 or a pharmaceutically acceptable salt thereof is 300~400mg. The drug combination according to claim 8, wherein the second active ingredient is fulvestrant; preferably, the dosage of fulvestrant is 1-2000 mg each time, and the administration frequency is one day 1 to 2 times; more preferably, each dose of fulvestrant is 100 to 800 mg, and the administration frequency is 1 to 2 times a day; further preferably, each dose of fulvestrant The dosage is 200~600mg, and the administration frequency is 1~2 times a day. The drug combination as described in claim 8, wherein the second active ingredient is letrozole; preferably, the dosage of letrozole is 1-200 mg each time, and the administration frequency is 1-200 mg a day. 2 times; more preferably, each dose of letrozole is 1-20 mg, and the administration frequency is 1-2 times a day; further preferably, each dose of letrozole is 1-5 mg , The administration frequency is 1-2 times a day. The drug combination according to claim 8, wherein the second active ingredient is anastrozole; preferably, the dosage of anastrozole is 0.1-50 mg each time, and the administration frequency is 1-50 mg a day. 2 times; more preferably, the dosage of the anastrozole is 1-25 mg each time, and the dosage frequency is 1-2 times a day; further preferably, the dosage of the anastrozole is 1-10 mg each time , The administration frequency is 1-2 times a day. The drug combination as claimed in claim 1, wherein the drug combination is used in the preparation of drugs for the treatment of breast cancer, preferably the breast cancer is estrogen receptor positive (ER+) breast cancer, or/and human Epidermal growth factor receptor 2 negative (HER2-) breast cancer may be locally advanced or metastatic breast cancer. A kit, the kit comprising the pharmaceutical combination as described in any one of claims 1 to 13 packaged in a container device, the first active ingredient and the second active ingredient in the pharmaceutical combination are administered simultaneously, administered separately or sequentially. A use of the drug combination as described in any one of claims 1 to 13 in the preparation of drugs for treating breast cancer. The use as described in claim 15, wherein the breast cancer is estrogen receptor positive (ER+) breast cancer, or/and human epidermal growth factor receptor 2 negative (HER2-) breast cancer or localized Advanced or metastatic breast cancer.

Images