WO2023083292A1 - Salt of pyrrolidine compound, crystal form thereof, and preparation method therefor - Google Patents

Abstract

A pharmaceutically acceptable salt of the compound of formula (I), a crystal thereof, a preparation method therefor, and a use thereof. The pharmaceutically acceptable salt is selected from sulfate, phosphate, benzoate, succinate, adipate, fumarate, L-malate, and citrate, preferably benzoate, succinate, and adipate.

Description

Salt, crystal form and preparation method of pyrrolidine compound

Cross References to Related Applications

This application claims the priority and rights of Chinese Patent Application No. 202111339152.0 filed with the State Intellectual Property Office of China on November 12, 2021, the disclosure of which is incorporated herein by reference in its entirety.

technical field

The disclosure belongs to the field of medicine, and relates to pharmaceutically acceptable salts of pyrrolidine compounds, crystals of the pharmaceutically acceptable salts, and corresponding preparation methods and applications.

Background technique

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

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

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

PCT/CN2021/093736 (application date May 14, 2021) describes a compound N-((S)-1-(3-fluoropropyl)pyrrolidin-3-yl)-6-((1S, 3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-1- Base) pyridin-3-amine (compound of formula (I)), the compound of formula (I) is a selective estrogen receptor down-regulator, and the in vivo and in vitro models show that the compound of (I) has a wide range of inhibitory effects on ER-positive cells.

Contents of the invention

The present disclosure provides pharmaceutically acceptable salts of compounds of formula (I),

Wherein, the pharmaceutically acceptable salt is selected from sulfate, phosphate, benzoate, succinate, adipate, fumarate, L-malate and citrate.

The present disclosure also provides the crystal form C of the adipate salt of the compound of the above formula (I). In the X-ray powder diffraction pattern represented by the diffraction angle 2θ of the crystal form C, there are 4.46±0.2°, 6.30±0.2°, 6.30±0.2°, There are diffraction peaks at 8.90±0.2° and 19.10±0.2°.

The present disclosure also provides the crystal form B of the benzoate salt of the compound of the above formula (I). In the X-ray powder diffraction pattern represented by the diffraction angle 2θ of the crystal form B, there are 7.19±0.2°, 10.01±0.2°, There are diffraction peaks at 11.39±0.2° and 18.78±0.2°.

The present disclosure also provides the crystal form A of the succinate salt of the compound of the above formula (I). In the X-ray powder diffraction pattern represented by the diffraction angle 2θ of the crystal form A, at 7.13±0.2°, 9.30±0.2°, 11.29 There are diffraction peaks at ±0.2°, 15.51±0.2°, and 19.89±0.2°.

The present disclosure also provides a method for preparing a pharmaceutically acceptable salt of the compound of formula (I), the preparation method comprising the step of reacting the compound of formula (I) with a corresponding acid to form a salt.

The present disclosure also provides a method for preparing the above crystal form C, comprising the following steps:

(1-a) mixing adipic acid and a solvent;

(2-a) mixing the compound of formula (I) and a solvent;

(3-a) mixing the mixture of step (1-a) and the mixture of step (2-a);

(4-a) Stir the resulting mixture mixed with the adipic acid and the compound of formula (I), and crystallize;

(5-a) The solid was filtered out, and the filter cake was collected and dried.

The present disclosure also provides a method for preparing the above crystal form B, comprising the following steps:

(1-b) mixing the compound of formula (I) and a solvent;

(2-b) adding benzoic acid to the mixture of step (1-b);

(3-b) Stir the resulting mixture mixed with the compound of formula (I) and the benzoic acid, and crystallize;

(4-b) The solid was filtered off and collected.

The present disclosure also provides a method for preparing the above crystal form A, comprising the following steps:

(1-c) mixing the compound of formula (I) and a solvent;

(2-c) adding succinic acid to the mixture of step (1-c);

(3-c) stirring the mixture obtained by mixing the compound of formula (I) and the succinic acid to precipitate a solid;

(4-c) The solid was filtered off and collected.

The present disclosure also provides a pharmaceutical composition, which comprises a pharmaceutically acceptable salt of the compound of formula (I) or the above crystal form, and pharmaceutically acceptable auxiliary materials.

The present disclosure also provides the use of the pharmaceutically acceptable salt of the compound of the above formula (I), the above crystal form or the above pharmaceutical composition in the preparation of a drug for preventing or treating estrogen receptor-related diseases.

Description of drawings

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

Fig. 2 is the XRPD spectrum of the crystal form C of adipate salt of the compound of formula (I).

Fig. 3 is the DSC spectrum of the crystal form C of adipate salt of the compound of formula (I).

Fig. 4 is the TGA spectrum of the crystal form C of adipate salt of the compound of formula (I).

Fig. 5 is the XRPD spectrum of the benzoic acid salt B crystal form of the compound of formula (I).

Fig. 6 is the DSC spectrum of the crystal form B of the benzoate salt of the compound of formula (I).

Fig. 7 is the TGA spectrum of the benzoic acid salt B crystal form of the compound of formula (I).

Fig. 8 is the XRPD spectrum of the crystal form A of succinate salt of the compound of formula (I).

Fig. 9 is the DSC spectrum of the crystal form A of the compound succinate of formula (I).

Fig. 10 is the TGA spectrum of the crystal form A of succinate salt of the compound of formula (I).

Fig. 11 is a three-dimensional structural ellipsoid diagram of crystal form A of succinate salt of the compound of formula (I).

Fig. 12 is a packing diagram of the unit cell structure of the crystal form A of the compound succinate of formula (I).

Fig. 13 is a picture of anti-tumor growth effect of compound succinate of formula (I) on MCF-7 xenograft subcutaneous xenograft tumor mouse model.

Fig. 14 is a graph showing the change in body weight of mice with MCF-7 xenogeneic xenograft tumors transplanted with succinate of the compound of formula (I).

Fig. 15 is the effect diagram of anti-tumor growth of compound succinate of formula (I) on T47D xenograft subcutaneous xenograft tumor mouse model.

Fig. 16 is a graph showing the change in body weight of T47D xenograft subcutaneously transplanted tumor mice with succinate of the compound of formula (I).

Fig. 17 The survival curve of the compound succinate of formula (I) on MCF-7 heterogeneous brain orthotopic tumor-bearing mice.

Fig. 18 is a graph showing the survival curve of the compound of formula (I) on MCF-7 xenogeneic brain orthotopic tumor-bearing mice.

Fig. 19 is a graph showing the change in body weight of MCF-7 heterogeneous brain orthotopic tumor-bearing mice by the compound of formula (I).

Detailed ways

The present disclosure provides a pharmaceutically acceptable salt of a compound of formula (I) with excellent physical and chemical properties and/or pharmaceutical properties and crystals thereof,

The present disclosure provides a pharmaceutically acceptable salt of the compound represented by formula (I), wherein the pharmaceutically acceptable salt is selected from sulfate, phosphate, benzoate, succinate, adipate, fumaric acid salt, L-malate and citrate, preferably benzoate, succinate, adipate, fumarate, L-malate and citrate, more preferably benzoate, succinate salts and adipates.

In some embodiments, in the pharmaceutically acceptable salt of the compound of formula (I), the molar ratio of the compound of formula (I) to the acid molecule is about 0.5-2.

In some embodiments, in the pharmaceutically acceptable salt of the compound of formula (I), the molar ratio of the compound of formula (I) to the acid molecule is about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3 , 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0.

In some embodiments, in the pharmaceutically acceptable salt of the compound of formula (I), the molar ratio of the compound of formula (I) to the acid molecule is about 0.5, 1.0, 1.5 and 2.0.

In some embodiments, the succinate salt of the compound of formula (I) above is represented by formula (II), wherein, x is selected from 0.5-2,

In some embodiments, the benzoate salt of the compound of formula (I) above is represented by formula (III), wherein, y is selected from 0.5 to 2,

In some embodiments, the adipate salt of the compound of formula (I) above is represented by formula (IV), wherein, z is selected from 0.5-2,

In some embodiments, x, y, z are independently selected from 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0.

In some embodiments, x, y, z are independently selected from 0.8, 0.9, 1.0, 1.1 and 1.2.

In some embodiments, x, y, z are independently selected from 1.0.

The present disclosure also provides a method for preparing a pharmaceutically acceptable salt of the compound of formula (I), comprising: the step of forming a salt of the compound shown in formula (I) with an acid, wherein the acid is selected from sulfuric acid, phosphoric acid, benzoic acid, succinic acid, Adipic acid, fumaric acid, L-malic acid and citric acid, preferably benzoic acid, succinic acid, adipic acid, fumaric acid, L-malic acid and citric acid, more preferably benzoic acid, succinic acid and adipic acid.

The solvent used in the salt-forming reaction described in the present disclosure is selected from cyclohexane, n-heptane, toluene, chloroform, ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, acetone, methyl acetate , ethyl acetate, isopropyl acetate, butyl acetate, methanol, ethanol, isopropanol, n-butanol and acetonitrile, preferably isopropyl ether, methyl tert-butyl ether, methanol, isopropanol and tetrahydrofuran at least one of the

In an optional embodiment, the method for preparing a pharmaceutically acceptable salt of the compound of formula (I) further includes one or a combination of solvent volatilization, stirring crystallization, filtration, drying, etc.

The present disclosure also provides a crystalline form of the adipate salt of the compound of formula (I).

In some embodiments, the crystalline form of the adipate salt of the compound of formula (I) is an anhydrate.

The present disclosure provides the C crystal form of the adipate salt of the compound of formula (I), the C crystal form, in the X-ray powder diffraction pattern represented by the diffraction angle 2θ, at 4.46±0.2°, 6.30±0.2°, There are diffraction peaks at 8.90±0.2° and 19.10±0.2°.

In some embodiments, in the X-ray powder diffraction pattern represented by the diffraction angle 2θ, the crystal form C is at 4.46±0.2°, 6.30±0.2°, 7.03±0.2°, 8.90±0.2°, 16.45±0.2 °, 17.78±0.2°, 19.10±0.2° and 21.03±0.2° have diffraction peaks.

In some embodiments, in the X-ray powder diffraction pattern represented by the diffraction angle 2θ, the crystal form C is at 4.46±0.2°, 6.30±0.2°, 7.03±0.2°, 8.90±0.2°, 10.65±0.2 °, 11.95±0.2°, 12.55±0.2°, 13.34±0.2°, 14.20±0.2°, 16.02±0.2°, 16.45±0.2°, 17.78±0.2°, 18.33±0.2°, 19.10±0.2°, 19.77±0.2 °, 20.12±0.2°, 21.03±0.2°, 21.62±0.2°, 24.30±0.2°, 25.97±0.2°, 27.39±0.2° and 27.98±0.2° have diffraction peaks.

In some embodiments, in the X-ray powder diffraction pattern represented by the diffraction angle 2θ, the crystal form C is at 4.46±0.2°, 6.30±0.2°, 7.03±0.2°, 8.90±0.2°, 10.65±0.2 °, 11.95±0.2°, 12.55±0.2°, 12.96±0.2°, 13.34±0.2°, 14.20±0.2°, 15.20±0.2°, 16.02±0.2°, 16.45±0.2°, 17.78±0.2°, 18.33±0.2 °, 19.10±0.2°, 19.77±0.2°, 20.12±0.2°, 21.03±0.2°, 21.62±0.2°, 22.44±0.2°, 22.52±0.2°, 22.54±0.2°, 23.35±0.2°, 23.98±0.2 There are diffraction peaks at 24.30±0.2°, 24.86±0.2°, 25.37±0.2°, 25.97±0.2°, 27.39±0.2°, 27.98±0.2° and 28.81±0.2°.

In some embodiments, the X-ray powder diffraction pattern of the crystal form C is substantially consistent with that in FIG. 2 .

In some embodiments, the DSC spectrum of the crystal form C has an endothermic peak at 149.89±5°C.

In some embodiments, the DSC spectrum of Form C is substantially the same as that of FIG. 3 .

The present disclosure also provides a method for preparing the crystal form C of adipate salt of the compound of formula (I), comprising the step of reacting the compound of formula (I) with adipic acid to form a salt.

In some schemes of the present disclosure, the preparation method of the above crystal form C includes the following steps:

(1-a) mixing adipic acid and a solvent;

(2-a) mixing the compound of formula (I) and a solvent;

(3-a) mixing the mixture of step (1-a) and the mixture of step (2-a);

(4-a) Stir the resulting mixture mixed with the adipic acid and the compound of formula (I), and crystallize;

(5-a) Filter out the solid, collect the filter cake (ie the solid), and dry.

In some embodiments, the solvent in step (1-a) and step (2-a) is selected from cyclohexane, n-heptane, toluene, chloroform, diethyl ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran , at least one of ethylene glycol dimethyl ether, acetone, methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, methanol, ethanol, isopropanol, n-butanol and acetonitrile, preferably isopropyl ether, At least one of methyl tert-butyl ether, methanol, isopropanol and tetrahydrofuran, more preferably isopropanol.

In some embodiments, the molar ratio of the compound of formula (I) to adipic acid is 1:2-2:1, preferably 1:1.

In some embodiments, the step (1-a) is performed under heating.

In some embodiments, the step (1-a) is carried out at a heating temperature of about 40-80°C, preferably 60°C.

In some embodiments, the step (4-a) is carried out at 15-30°C, preferably at 20-25°C.

The present disclosure also provides a crystalline form of the benzoate salt of the compound of formula (I).

In some embodiments, the crystalline form of the benzoate salt of the compound of formula (I) is an anhydrate.

The present disclosure provides the crystal form B of the benzoate salt of the compound of formula (I). In the X-ray powder diffraction pattern represented by the diffraction angle 2θ, the crystal form B is at 7.19±0.2°, 10.01±0.2°, 11.39± There are diffraction peaks at 0.2° and 18.78±0.2°.

In some embodiments, in the X-ray powder diffraction pattern represented by the diffraction angle 2θ of the B crystal form, there are ° has a diffraction peak.

In some embodiments, in the X-ray powder diffraction pattern represented by the diffraction angle 2θ of the B crystal form, at 7.19±0.2°, 10.01±0.2°, 11.39±0.2°, 18.78±0.2°, 19.01±0.2° , 19.66±0.2°, 19.91±0.2°, 20.09±0.2° have diffraction peaks.

In some embodiments, in the X-ray powder diffraction pattern represented by the diffraction angle 2θ of the B crystal form, at 4.69±0.2°, 6.34±0.2°, 7.19±0.2°, 10.01±0.2°, 11.39±0.2 °, 18.78±0.2°, 19.01±0.2°, 19.66±0.2°, 19.91±0.2° and 20.09±0.2° have diffraction peaks.

In some embodiments, in the X-ray powder diffraction pattern represented by the diffraction angle 2θ of the B crystal form, at 4.69±0.2°, 6.34±0.2°, 7.19±0.2°, 10.01±0.2°, 11.11±0.2 °, 11.39±0.2°, 14.78±0.2°, 18.78±0.2°, 19.01±0.2°, 19.25±0.2°, 19.66±0.2°, 19.91±0.2°, 20.09±0.2°, 20.80±0.2°, 21.66±0.2 ° has a diffraction peak.

In some embodiments, in the X-ray powder diffraction pattern represented by the diffraction angle 2θ of the B crystal form, at 4.69±0.2°, 6.34±0.2°, 7.19±0.2°, 9.83±0.2°, 10.01±0.2° , 11.11±0.2°, 11.39±0.2°, 14.78±0.2°, 18.78±0.2°, 19.01±0.2°, 19.25±0.2°, 19.66±0.2°, 19.91±0.2°, 20.09±0.2°, 20.80±0.2° , There is a diffraction peak at 21.66±0.2°.

In some embodiments, in the X-ray powder diffraction pattern represented by the diffraction angle 2θ of the B crystal form, at 4.69±0.2°, 4.91±0.2°, 6.34±0.2°, 7.19±0.2°, 9.37±0.2 °, 9.83±0.2°, 10.01±0.2°, 10.32±0.2°, 11.11±0.2°, 11.39±0.2°, 12.06±0.2°, 12.69±0.2°, 13.52±0.2°, 14.07±0.2°, 14.36±0.2 °, 14.78±0.2°, 17.30±0.2°, 18.78±0.2°, 19.01±0.2°, 19.25±0.2°, 19.66±0.2°, 19.91±0.2°, 20.09±0.2°, 20.80±0.2°, 21.15±0.2 °, 21.66±0.2°, 22.35±0.2°, 23.57±0.2°, 23.70±0.2°, 24.63±0.2°, 25.68±0.2°, 27.05±0.2° have diffraction peaks.

In some embodiments, the X-ray powder diffraction pattern of the crystal form B is substantially consistent with FIG. 5 .

In some embodiments, the DSC spectrum of the crystal form B has an endothermic peak at 150.08±5°C.

In some embodiments, the DSC spectrum of Form B is substantially consistent with FIG. 6 .

The present disclosure also provides a preparation method of the crystal form B of the benzoate salt of the compound of formula (I), comprising the step of reacting the compound of formula (I) with benzoic acid to form a salt.

In some schemes of the present disclosure, the preparation method of the above-mentioned B crystal form includes the following steps:

(1-b) mixing the compound of formula (I) and a solvent;

(2-b) adding benzoic acid to the mixture of step (1-b);

(3-b) Stir the resulting mixture mixed with the compound of formula (I) and the benzoic acid, and crystallize;

(4-b) The solid was filtered off and collected.

In some embodiments, the solvent in step (1-b) is selected from cyclohexane, n-heptane, toluene, chloroform, diethyl ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, ethylene glycol dimethyl ether , acetone, methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, methanol, ethanol, isopropanol, n-butanol and acetonitrile, preferably isopropyl ether, methyl tert-butyl ether, At least one of methanol, isopropanol and tetrahydrofuran, more preferably isopropyl ether.

In some embodiments, the molar ratio of the compound of formula (I) to benzoic acid is 1:2-2:1, preferably 1:1.

In some embodiments, the step (1-b) is carried out at 15-30°C, preferably at 20-25°C.

In some embodiments, the stirring in step (3-b) is carried out under heating conditions, preferably the heating temperature is about 40-68°C, more preferably 60-68°C.

Further, the preparation method of the benzoate B crystal form of the compound of formula (I) also includes the following steps:

(5-b) mixing the solid collected in step (4-b) with a solvent;

(6-b) stirring the resulting mixture;

(7-b) was filtered off and the solid was collected and dried.

In some embodiments, the solvent in step (5-b) is selected from at least one of isopropyl ether, methyl tert-butyl ether, methanol, isopropanol and tetrahydrofuran, preferably isopropyl ether.

In some embodiments, the step (6-b) is carried out at 15-30°C, preferably at 20-25°C.

The present disclosure also provides a crystalline form of the succinate salt of the compound of formula (I).

In some embodiments, the crystalline form of the succinate salt of the compound of formula (I) is an anhydrate.

The present disclosure provides the crystal form A of succinate salt of the compound of formula (I). In the X-ray powder diffraction pattern represented by the diffraction angle 2θ, the crystal form A is at 7.13±0.2°, 9.30±0.2°, 11.29±0.2 °, 15.51±0.2°, 19.89±0.2° have diffraction peaks.

In some embodiments, in the X-ray powder diffraction pattern represented by the diffraction angle 2θ, the crystal form A is at 7.13±0.2°, 9.30±0.2°, 11.29±0.2°, 15.51±0.2°, 17.10±0.2 °, 19.89±0.2°, 20.26±0.2°, 20.91±0.2°, 21.51±0.2°, 22.69±0.2°, 23.58±0.2°, 25.42±0.2° have diffraction peaks.

In some embodiments, in the X-ray powder diffraction pattern represented by the diffraction angle 2θ, the crystal form A is at 5.01±0.2°, 7.13±0.2°, 9.30±0.2°, 10.09±0.2°, 11.29±0.2 °, 11.73±0.2°, 13.75±0.2°, 14.30±0.2°, 15.18±0.2°, 15.51±0.2°, 16.00±0.2°, 17.10±0.2°, 18.25±0.2°, 18.69±0.2°, 19.89±0.2 °, 20.26±0.2°, 20.91±0.2°, 21.12±0.2°, 21.51±0.2°, 22.69±0.2°, 23.05±0.2°, 23.58±0.2°, 24.58±0.2°, 25.42±0.2°, 26.66±0.2 °, 27.24±0.2° and 29.47±0.2° have diffraction peaks.

In some embodiments, in the X-ray powder diffraction pattern represented by the diffraction angle 2θ, the crystal form A is at 5.01±0.2°, 7.13±0.2°, 9.30±0.2°, 10.09±0.2°, 11.29±0.2 °, 11.73±0.2°, 13.75±0.2°, 14.30±0.2°, 15.18±0.2°, 15.51±0.2°, 16.00±0.2°, 17.10±0.2°, 18.25±0.2°, 18.69±0.2°, 19.89±0.2 °, 20.26±0.2°, 20.91±0.2°, 21.12±0.2°, 21.51±0.2°, 22.69±0.2°, 23.05±0.2°, 23.58±0.2°, 24.58±0.2°, 25.42±0.2°, 26.66±0.2 °, 27.24±0.2°, 28.65±0.2°, 29.47±0.2°, 30.90±0.2°, 32.31±0.2°, 34.45±0.2° have diffraction peaks.

In some embodiments, the X-ray powder diffraction pattern of Form A is substantially consistent with FIG. 8 .

In some embodiments, the DSC spectrum of the crystal form A has an endothermic peak at 186.82±5°C.

In some embodiments, the DSC spectrum of Form A is substantially consistent with FIG. 9 .

In some embodiments, the unit cell parameters of the A crystal form are

α=90°, β=90°, γ=90°.

The present disclosure also provides a method for preparing the succinate A crystal form of the compound of formula (I), comprising the following steps:

(1-c) mixing the compound of formula (I) and a solvent;

(2-c) adding succinic acid to the mixture of step (1-c);

(3-c) stirring the mixture obtained by mixing the compound of formula (I) and the succinic acid to precipitate a solid;

(4-c) The solid was filtered off and collected.

In some embodiments, the solvent in step (1-c) is selected from cyclohexane, n-heptane, toluene, chloroform, diethyl ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, ethylene glycol dimethyl ether , acetone, methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, methanol, ethanol, isopropanol, n-butanol and acetonitrile, preferably isopropyl ether, methyl tert-butyl ether, At least one of methanol, isopropanol and tetrahydrofuran, more preferably isopropyl ether.

In some embodiments, the molar ratio of the compound of formula (I) to succinic acid is 1:2-2:1, preferably 1:1.

In some embodiments, the step (3-c) is carried out at 15-30°C, preferably at 20-25°C.

In some embodiments, the reaction time of the step (3-c) is 1-48 hours, preferably 3-24 hours, more preferably 8-16 hours.

Further, the preparation method of the succinate A crystal form of the compound of formula (I) also includes the following steps:

(5-c) mixing the solid collected in step (4-c) with a solvent;

(6-c) stirring and crystallizing the resulting mixture;

(7-c) was filtered off and the solid was collected and dried.

In some embodiments, the solvent in step (5-c) is at least one selected from methanol, ethanol, isopropanol, tetrahydrofuran and water, preferably a mixed solvent of ethanol/water.

In some embodiments, the volume ratio of ethanol/water is about 1:1～50:1, preferably 2:1～20:1; in some specific embodiments of the present disclosure, the volume ratio of ethanol/water is about 10 :1, 9:1, 8:1, 7:1, 6:1, 5:1 or 4:1.

In some embodiments, the step (6-c) is carried out at 15-30°C, preferably at 20-25°C.

In some embodiments, the stirring crystallization time of the step (6-c) is 0.5-48 hours, preferably 1-12 hours, more preferably 2-4 hours.

The present disclosure also provides a pharmaceutical composition, which comprises a pharmaceutically acceptable salt of the compound of formula (I) above or the crystal form above and pharmaceutically acceptable excipients.

The present disclosure also relates to the use of the pharmaceutically acceptable salt of the compound of formula (I) above or the above crystal form, or the above pharmaceutical composition in the preparation of drugs for preventing or treating estrogen receptor-related diseases.

Further, the present disclosure relates to the use of a pharmaceutically acceptable salt of the compound of formula (I) above or the above crystal form, or the above pharmaceutical composition in the prevention or treatment of estrogen receptor-related diseases.

Further, the present disclosure relates to a pharmaceutically acceptable salt of the compound of the above formula (I) or the above crystal form, or the above pharmaceutical composition for preventing or treating estrogen receptor-related diseases.

The present disclosure also relates to a method for treating estrogen receptor-related diseases, the method comprising administering to an individual a therapeutically effective dose of the pharmaceutically acceptable salt of the compound of formula (I) or the above-mentioned crystal form, the above-mentioned pharmaceutical composition, or the above-mentioned Pharmaceutically acceptable salts of the compound of formula (I) or pharmaceutical preparations of the above crystal forms.

The present disclosure also relates to the use of the pharmaceutically acceptable salt of the compound of the above formula (I) or the above crystal form, or the above pharmaceutical composition in the preparation of drugs for preventing or treating tumors.

Further, the present disclosure relates to the use of a pharmaceutically acceptable salt of the compound of formula (I) or the above crystal form, or the above pharmaceutical composition in the prevention or treatment of tumors.

Further, the present disclosure relates to a pharmaceutically acceptable salt of the compound of formula (I) or the above crystal form, or the above pharmaceutical composition for preventing or treating tumors.

The present disclosure also relates to a method for treating tumors, the method comprising administering to an individual a therapeutically effective dose of a pharmaceutically acceptable salt of the above-mentioned compound of formula (I) or the above-mentioned crystal form, the above-mentioned pharmaceutical composition or a compound containing the above-mentioned formula (I) of the present disclosure Pharmaceutically acceptable salts or pharmaceutical preparations of the above crystal forms.

In some embodiments, the estrogen receptor-related diseases include but are not limited to tumors.

In some embodiments, the estrogen receptor-related disease or tumor is breast cancer.

In some embodiments, the estrogen receptor-related disease or tumor is ER-positive breast cancer.

In some embodiments, the estrogen receptor-related disease or tumor is brain metastases from ER-positive breast cancer.

In some embodiments, the individual is a cancer patient.

In some embodiments, the individual is a breast cancer patient.

In some embodiments, the individual is a patient with ER-positive breast cancer.

In some embodiments, the individual is a patient with brain metastases from ER-positive breast cancer.

technical effect

The pharmaceutically acceptable salts of the compound of formula (I) and crystals thereof provided by the present disclosure have at least one of the following advantages:

The pharmaceutically acceptable salts of the compounds of formula (I) and their crystals provided by the present disclosure have at least one of the advantages of good water solubility, high purity, good stability, and low hygroscopicity;

Compared with free base or other salt forms, benzoate, succinate or adipate and crystal forms thereof of the compound of formula (I) can also effectively inhibit the generation of isomers, and/or have better thermal stability;

The pharmaceutically acceptable salt of the compound of formula (I) or its crystal form provided by the present disclosure also has at least one of the advantages of simple preparation method, mild crystallization conditions, high crystallinity, good solubility, good stability, and difficult to absorb moisture, etc., It is suitable for preparing the desired pharmaceutical composition.

Definition and Description

The daily dosage of the pharmaceutically acceptable salt of the compound of formula (I) of the present disclosure is 0.01 mg/kg to 200 mg/kg body weight, preferably 0.05 mg/kg to 50 mg/kg body weight, more preferably 0.1 mg/kg to 30 mg/kg body weight. kg body weight, in single or divided doses.

Typical routes of administration of the pharmaceutically acceptable salts of the present disclosure or their crystalline forms or their polymorphs, or their polymorphic compositions, or their pharmaceutical compositions include, but are not limited to, oral, rectal, topical , inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration.

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

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

The term "about" is used in this disclosure to mean approximately, around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the limits of the stated numerical range. Unless otherwise indicated, the term "about" is used herein to modify the upper and lower limits of the stated value by a numerical value of 10% deviation.

Unless otherwise stated, the terms "comprise", "comprise" or "comprise" and their English variants such as comprises or comprising are to be understood in an open, non-exclusive sense, ie "including but not limited to".

References throughout this specification to "some embodiments", "alternative embodiments" or "embodiments" means that at least one embodiment includes specific reference elements, structures or features described in relation to that embodiment . Thus, appearances of the phrases "some embodiments," "alternative embodiments," or "an embodiment" in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, particular elements, structures or characteristics may be combined in any suitable manner in one or more embodiments.

The room temperature mentioned in the present disclosure refers to 20±5°C.

The range "m∼n" described in the present disclosure represents an abbreviated representation of any combination of real numbers between m and n, where both m and n are real numbers. The numerical range "0.5~2" includes but is not limited to 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 .

The "X-ray powder diffraction pattern" described in this disclosure is measured using Cu-Kα radiation.

"X-ray powder diffraction pattern or XRPD pattern" described in the present disclosure refers to according to Bragg's formula 2d Sinθ=nλ (wherein, λ is the wavelength of X-ray,

The order n of diffraction is any positive integer, generally take the first-order diffraction peak, n=1), when the X-ray is incident on the crystal or part of the crystal sample at the grazing angle θ (the complementary angle of the incident angle, also known as the Bragg angle) On an atomic plane with a d-lattice plane spacing, the Bragg equation can be satisfied, and thus this group of X-ray powder diffraction patterns has been measured.

The crystalline forms disclosed in the present disclosure are substantially pure crystals. The term "substantially pure" as used herein means that the crystalline form has a purity of at least 85 wt%, preferably at least 90 wt%, more preferably at least 95 wt%.

For the same crystal form of the same compound, the peak positions of their XRPD patterns are generally similar, and the relative intensity error may be large. It should also be pointed out that in the identification of mixtures, due to the decrease of content and other factors, some diffraction lines will be missing. At this time, there is no need to rely on all the diffraction peaks observed in high-purity samples, and even one diffraction peak may also affect the given is characteristic for a given crystal.

The "2θ or 2θ angle" mentioned in the present disclosure refers to the diffraction angle, θ is the Bragg angle, and the unit is ° or degree; the error range of each diffraction peak 2θ is ±0.20°.

"Differential scanning calorimetry or DSC" in this disclosure refers to the measurement of the temperature difference and heat flow difference between the sample and the reference object during the heating or constant temperature of the sample to characterize all the physical changes related to thermal effects and Chemical changes, to obtain the phase transition information of the sample.

The drying temperature described in the present disclosure is generally 20°C-100°C, preferably 25°C-70°C, more preferably 40°C-60°C, and can be dried under normal pressure or reduced pressure. Preferably, drying is performed under reduced pressure.

For those skilled in the art, there is a certain degree of error in the determination of the molar ratio of the disclosed compound to the acid molecule, and generally speaking, ±10% is within a reasonable error range. There is a degree of error that will vary with the context in which it is used, but the error will vary by no more than ±10%, preferably ±5%.

The term "treating" means administering a compound or formulation described herein to improve or eliminate a disease or one or more symptoms associated with the disease, and includes:

(i) inhibiting a disease or disease state, i.e. arresting its development;

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

The term "prevention" means administering a compound or formulation described herein to prevent a disease or one or more symptoms associated with the disease, and includes:

Preventing a disease or disease state from occurring in a subject (eg, a mammal), particularly when such a subject is susceptible to the disease state but has not yet been diagnosed as having the disease state.

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

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

As used herein, the term "individual" or "patient" includes mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates (such as chimpanzees and other apes and monkeys); livestock such as cattle, horses, sheep, goats, pigs; domesticated animals , such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs. Examples of non-human mammals include, but are not limited to, birds, fish, and the like.

The test condition of the instrument used in this disclosure experiment:

1. Differential Scanning Calorimeter (DSC)

Instrument model: TA DSC2500

Purge gas: Nitrogen

Sample pan: aluminum pan, non-sealed gland

Method: linear heating

Heating rate: 10°C/min

Temperature range: 30 ℃ ~ set the end temperature

2. X-ray Powder Diffraction (XRPD)

Instrument model: Bruker D8 Focus

Rays: Cu Kα, Kα1

: 1.54060; Kα2

: 1.54439

Kα1/Kα2:2

Slit (°): 2.5

Scanning mode: θ/2θ, scanning range: 3-40°

Dwell time (seconds): 0.12

Scan step size (°2θ): 0.01

Voltage: 40kV, Current: 40mA

3. Thermogravimetric Analysis (TGA)

Instrument model: TA TGA550

Purge gas: Nitrogen

Sample tray: Platinum, open

Method: linear heating

Heating rate: 10°C/min

Temperature range: 30 ℃ ~ set the end temperature

4. High performance liquid chromatography (HPLC) for saturation solubility test

Chromatographic column: Water XBridge C18 (4.6*100mm, 3.5μm)

Mobile phase A: 0.01mol/L ammonium chloride solution (pH10.0)

Mobile Phase B: Acetonitrile

Isocratic conditions: mobile phase A:B=50:50, isocratic for 15min

Flow rate: 1.5mL/min

Wavelength: 260nm

Column temperature: 35°C

Injection volume: 10μl

Diluent: Acetonitrile

5. High performance liquid chromatography (HPLC) for detection of isomer impurities

Chromatographic column: CHIRALPAK AD-H (4.6*250mm, 5μm)

Mobile Phase A: n-Hexane

Mobile phase B: 0.1% diethylamine-ethanol

Isocratic conditions: mobile phase A: mobile phase B = 70:30, run isocratically for 15 minutes

Flow rate: 1.0mL/min

Wavelength: 260nm

Column temperature: 35°C

Injection volume: 10μl

Diluent: 0.1% diethylamine-ethanol

The high performance liquid chromatography (HPLC) of formula (I) compound pharmaceutically acceptable salt stability investigation test

Chromatographic column: Water XBridge C18 (4.6*150mm, 3.5μm)

Mobile phase A: 0.01mol/L ammonium chloride solution (adjust the pH to 10.0 with ammonia water)

Mobile Phase B: Acetonitrile

Flow rate: 1.0mL/min

Wavelength: 285nm

Column temperature: 35°C

Injection volume: 20μl

Those skilled in the art recognize that measurements of XRPD peak positions and/or intensities for a given crystalline form of the same compound will vary within error. The 2Θ values allow for an appropriate margin of error. Usually, the margin of error is represented by "±". For example, 5.92 ± 0.2° 2Θ represents a range of about 6.12 to 5.72. Depending on the sample preparation technique, the calibration technique applied to the instrument, human operator bias, etc., one skilled in the art recognizes that suitable error ranges for XRPD diffraction angles (2Θ) may be about ±0.20°, ±0.15°, ±0.10° , ±0.05° or less. When used to describe an XRPD pattern, the term "substantially identical" or "substantially as shown" means comprising at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 90% Or a pattern of diffraction peaks with at least 99% of the diffraction angles within a standard deviation of ±0.2° 2Θ.

As one skilled in the art recognizes, measurements of a DSC thermogram for a given crystalline form of the same compound will vary within a margin of error. Single-peak peak values (expressed in degrees Celsius) allow for a modest margin of error. Usually, the margin of error is represented by "±". For the same crystalline form of the same compound, thermal transition temperatures and melting points are typically within about ±5.0°C in consecutive analyses. For example, the peak value of "186.82±5°C" indicates that it is in the range of 181.82 to 191.82. Depending on the sample preparation technique, the calibration technique applied to the instrument, human operator bias, etc., one skilled in the art will recognize that suitable error ranges for unimodal peaks may be ±5.0, ±4.0, ±3.0, ±2.0 or less.

Lux (lux, legal symbol lx) is the unit of luminance. When the luminous flux obtained by an object uniformly illuminated by light is 1 lumen on an area of 1 square meter, its illuminance is 1 lux.

Example

The following examples describe the technical solutions of the invention in detail, but the protection scope of the present disclosure includes but is not limited thereto.

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

Example 1: 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 (formula (I) compound)

resolve resolution:

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

Dissolve (S)-tert-butylpyrrolidin-3-ylaminomethyl ester (500.00mg, 2.68mmol) in tetrahydrofuran (10mL), add sodium hydroxide solution (5mol·L ^-1 , 1.07mL) and 1- Iodo-3-fluoropropane (529.88 mg, 2.82 mmol). The reaction solution was stirred and reacted at 25°C for 16 hours. After the reaction of the raw materials was detected by TLC, the reaction solution was diluted with ethyl acetate (50 mL), washed with saturated ammonium chloride solution (10 mL), and the aqueous phase and the organic phase were collected respectively. After the aqueous phase was extracted three times with ethyl acetate (20 mL), all the organic phases were combined and dried over sodium sulfate. The organic phase was concentrated to dryness under reduced pressure, and then purified by column chromatography (silica, dichloromethane/methanol=100/ 1) The product (S)-tert-butyl(1-(3-fluoropropyl)pyrrolidin-3-yl)aminomethyl ester (480.00 mg) was obtained.

¹ H NMR (400MHz, METHANOL-d ₄ )δ4.58-4.53(m,1H),4.46-4.40(m,1H),4.14-4.04(m,1H),2.93-2.85(m,1H),2.77 -2.67(m,1H),2.61(dd,J＝7.78,5.52Hz,3H),2.47-2.40(m,1H),2.29-2.17(m,1H),1.99-1.82(m,2H),1.71 -1.61(m,1H),1.45(s,9H).

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

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

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

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

Dissolve (2R)-1-(1H-indol-3-yl)propane-2-amine (600 mg, 3.44 mmol) and N,N-diisopropylethylamine (445.05 mg, 3.44 mmol) in 1, To 4-dioxane (10mL), add trifluoroethyl trifluoromethanesulfonate (1.20g, 5.17mmol) dissolved in 1,4-dioxane (5mL) at 25°C, and the reaction solution The reaction was stirred at 75°C for 16 hours. The reaction solution was concentrated to dryness under reduced pressure. Then column chromatography purification (silica, petroleum ether/ethyl acetate = 3/1) to obtain the product (R)-1-(1H-indol-3-yl)-N-(2,2,2-tri Fluoroethyl)propan-2-amine (0.69g).

MS m/z(ESI):257.2[M+H] ⁺ ;

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

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

Dissolve (R)-1-(1H-indol-3-yl)-N-(2,2,2-trifluoroethyl)propan-2-amine (120.00 mg, 468.26 μmol) in toluene (2 mL) 5-bromo-pyridine-2-carbaldehyde (87.10mg, 468.26μmol) and acetic acid (562.40mg, 9.37mmol) were added, and the reaction solution was a yellow transparent solution. The reaction solution was stirred at 90° C. for 10 hours. After the completion of the reaction detected by LCMS, the reaction solution was cooled to room temperature, concentrated to dryness under reduced pressure, and purified by thin layer chromatography (silica, petroleum ether/ethyl acetate=4/1) to obtain (1S,3R)-1- (5-bromopyridin-2-yl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4 -b] Indole (110.00 mg).

MS m/z (ESI): 424.1, 426.1 [M+H] ⁺ .

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

Step 5: N-((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 (compound of formula (I))

(1S,3R)-1-(5-bromopyridin-2-yl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro -1H-pyrido[3,4-b]indole (140.00 mg, 263.99 μmol) was dissolved in tetrahydrofuran (3 mL), and (S)-1-(3-fluoropropyl)pyrrolidin-3-amine salt was added salt (86.77mg, 316.79μmol), sodium tert-butoxide (152.22mg, 1.58mmol) and methanesulfonic acid (2-dicyclohexylphosphine)-3,6-dimethoxy-2,4,6-tri Isopropyl-1,1-biphenyl)(2-amino-1,1-biphenyl-2-yl)palladium(II) (23.93 mg, 26.40 μmol), the reaction solution was stirred at 80°C for 4 hours under nitrogen atmosphere . After the LCMS detection reaction is completed, cool to room temperature, filter, and the filtrate is concentrated under reduced pressure, and purified by preparative liquid chromatography (Phenomenex Gemini C18 column, 3um silica, 30mm diameter, 75mm length); (using water (containing 0.225% formic acid ) and a mixture of decreasing polarity of acetonitrile as eluent) to obtain compound N-((S)-1-(3-fluoropropyl)pyrrolidin-3-yl)-6-(((1S,3R) -3-Methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl) Pyridin-3-amine (37.79 mg).

MS m/z (ESI): 365.1 [M+H] ⁺ .

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

Absolute Configuration Identification of Compounds of Formula (I)

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

Embodiment 2: the preparation of formula (I) compound sulfate

Add 500mg of the compound of formula (I) and 10ml of isopropyl ether into a 50ml single-necked bottle, stir at room temperature until the solid is completely dissolved, add 100mg of concentrated sulfuric acid, heat up to the reflux of the reaction solution, keep the reaction for 1h, cool down to room temperature and stir overnight, and filter with suction. The solid was dried under vacuum at 40°C for 3 hours to obtain 0.58 g of solid.

Embodiment 3: the preparation of formula (I) compound phosphate

Add 0.5g of the compound of formula (I) and 10ml of isopropyl ether into a 50ml single-necked bottle, stir at room temperature until the solid is completely dissolved, add 118mg of phosphoric acid, heat up to the reflux of the reaction solution, keep the reaction for 1h, cool down to room temperature and stir overnight, and filter with suction. The solid was dried under vacuum at 40°C for 3 hours to obtain 0.53 g of solid.

Embodiment 4: the preparation of formula (I) compound fumarate

Add 0.5g of the compound of formula (I) and 10ml of isopropanol into a 50ml single-necked bottle, stir at room temperature until the solid is completely dissolved, add 118.5mg of fumaric acid, heat up to the reflux of the reaction solution, keep the reaction for 1h, lower it to room temperature and stir overnight, After suction filtration, the solid was dried under vacuum at 40°C for 3 hours to obtain 0.3 g of solid.

Embodiment 5: the preparation of formula (I) compound L-malate

Add 50 mg of the compound of formula (I) and 1 ml of isopropyl ether into a 5 ml centrifuge tube, stir at room temperature until the solid is completely dissolved, add 13.7 mg of L-malic acid, stir at room temperature overnight, filter with suction, and dry the solid in vacuum at room temperature for 3 hours to obtain 30 mg of solid.

Embodiment 6: the preparation of formula (I) compound citrate

Add 50 mg of compound of formula (I) and 1 ml of methyl tert-butyl ether into a 5 ml centrifuge tube, stir at room temperature until the solid is completely dissolved, add 21.5 mg of citric acid, stir at room temperature for 3 h, filter with suction, and dry the solid in vacuum at room temperature for 3 h to obtain 26 mg of solid .

Embodiment 7: the preparation of formula (I) compound adipate C crystal form

Add 0.7g of adipic acid and 19ml of isopropanol into a 100ml single-necked bottle, raise the temperature to 60°C, and dissolve the solid. Add 2.36g of the compound of formula (I) and 19ml of isopropanol into a 100ml single-necked bottle, stir at room temperature to dissolve, then drop into adipate isopropanol solution, stir at room temperature overnight, crystallize, filter with suction, and dry in vacuum at room temperature for 3h to obtain 1.66 g solid. The obtained solid was confirmed to be a crystal form (C crystal form) by X-ray Powder Diffraction (XRPD). , its DSC endothermic peak is around 149.89°C, and its XRPD diffraction peak position is shown in Table 1 below.

Table 1

¹ H NMR (400MHz, DMSO) δ10.60(s, 1H), 7.85(d, J=2.7Hz, 1H), 7.39(t, J=9.5Hz, 1H), 7.26(d, J=8.0Hz, 1H), 7.11–6.86(m, 4H), 5.99(d, J=6.8Hz, 1H), 4.91(s, 1H), 4.53(t, J=6.0Hz, 1H), 4.42(t, J=6.0 Hz,1H),3.85(d,J=3.2Hz,1H),3.55(dq,J=20.2,10.0Hz,1H),3.35–3.25(m,1H),3.01(dq,J=19.3,9.6Hz ,1H),2.81(dd,J＝9.2,7.0Hz,1H),2.71–2.52(m,3H),2.49–2.41(m,3H),2.34(dd,J＝9.3,4.5Hz,1H), 2.25–2.13(m,5H),1.87–1.72(m,2H),1.61–1.52(m,1H),1.52–1.41(m,4H),1.13(d,J＝6.7Hz,3H).

Embodiment 8: the preparation of formula (I) compound benzoate B crystal form

Add 3.0g of compound of formula (I) and 100ml of isopropyl ether into a 250ml single-necked bottle, stir at room temperature until the solid is completely dissolved, add 0.75g of benzoic acid, stir at room temperature overnight, filter with suction, add 100ml of filter cake and 30ml of isopropyl ether Stir in a one-necked flask at room temperature for 1 h, crystallize, filter with suction, and vacuum-dry the obtained filter cake at room temperature for 3 h to obtain 3.11 g of solid. The obtained solid is confirmed to be a crystal form (B crystal form) by X-ray powder diffraction spectrum. The XRPD spectrum of the crystalline solid is shown in Figure 5, its DSC spectrum is shown in Figure 6, and the TGA spectrum is shown in Figure 7. Its DSC endothermic peak peak is at 150.08 Around ℃, the XRPD diffraction peak positions are shown in Table 2 below.

Table 2

¹ H NMR (400MHz,DMSO)δ10.60(s,1H),7.98–7.91(m,2H),7.85(d,J=2.7Hz,1H),7.62(t,J=7.4Hz,1H), 7.50(t, J=7.6Hz, 2H), 7.40(d, J=7.6Hz, 1H), 7.26(d, J=8.0Hz, 1H), 7.09–6.99(m, 2H), 6.97–6.89(m ,2H),6.00(d,J=6.8Hz,1H),4.90(s,1H),4.54(t,J=6.0Hz,1H),4.42(t,J=6.0Hz,1H),3.85(s ,2H),3.01(dt,J=25.3,9.6Hz,2H),2.82(dd,J=9.2,6.9Hz,1H),2.71–2.54(m,3H),2.45(dd,J=11.9,7.3 Hz, 3H), 2.35(dd, J=9.4, 4.5Hz, 1H), 2.19(dt, J=13.4, 7.8Hz, 1H), 1.91–1.73(m, 2H), 1.55(dt, J=12.7, 6.3Hz, 1H), 1.13(d, J=6.7Hz, 3H).

Embodiment 9: the preparation of formula (I) compound succinate

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

Embodiment 10: Preparation of crystal form A of compound succinate of formula (I)

Example 10-1: The succinate obtained in Example 9 was dissolved in methanol, and concentrated under reduced pressure to obtain a foamy solid. Weigh 500 mg of foamy solid, add ethanol (4.5 ml) and water (0.5 ml) into a 10 ml single-necked bottle, stir at room temperature, the solid dissolves, keep stirring for 2 hours to precipitate a solid, filter it with suction, and dry the obtained solid in vacuum at room temperature for 3 hours to obtain 0.22 g Crystal form A of the succinate salt of the compound of formula (I). The obtained solid was confirmed by X-ray powder diffraction spectrum. The XRPD spectrum of the crystalline solid is shown in Figure 8, its DSC spectrum is shown in Figure 9, and its TGA spectrum is shown in Figure 10. As shown in Table 3 below.

table 3

¹ H NMR (400MHz, DMSO) δ10.59(s, 1H), 7.86(d, J=2.6Hz, 1H), 7.40(d, J=7.7Hz, 1H), 7.26(d, J=7.9Hz, 1H), 7.08(d, J=8.5Hz, 1H), 7.02(t, J=7.5Hz, 1H), 6.98–6.91(m, 2H), 6.01(d, J=6.4Hz, 1H), 4.91( s,1H),4.54(t,J=6.0Hz,1H),4.42(t,J=6.0Hz,1H),3.89(s,1H),3.54(dq,J=20.1,10.0Hz,1H), 3.31(dq, J=13.3,6.5Hz,1H),3.02(dq,J=19.3,9.6Hz,1H),2.89(dd,J=9.4,6.9Hz,1H),2.67(dt,J=8.8, 6.4Hz, 2H), 2.59–2.52(m, 4H), 2.43(dd, J＝9.2, 4.1Hz, 1H), 2.40(s, 4H), 2.21(td, J＝13.7, 7.8Hz, 1H), 1.90–1.75(m,2H),1.59(td,J=12.6,7.0Hz,1H),1.14(d,J=6.7Hz,3H).

The crystal form A of compound succinate of formula (I) can also be prepared by the following examples: add 100 mg of the above-mentioned foamy solid and 500 μL of solvent (see Table 4) to a 5 ml centrifuge tube, stir at room temperature for 1-24 hours, and filter out solid and dried under vacuum at room temperature. See Table 4 for details.

Table 4

Example	solvent	crystal form
10-2	isopropyl ether	Form A
10-3	methyl tert-butyl ether	Form A

Embodiment 11: Research on single crystal of compound succinate of formula (I)

A single crystal of the succinate of the compound of formula (I) can be prepared by the following method: take about 10 mg of the succinate of the compound of the formula (I) (prepared in Example 9) in a 3 mL sample bottle, add 1500 μL of isopropyl ether and 900 μL tetrahydrofuran, after ultrasonic dissolution, seal the bottle mouth with a parafilm and pierce a small hole in the parafilm with a needle, leave it at room temperature for 3 days, and precipitate crystals, and the obtained single crystal sample is detected by X-powder diffraction as A crystal form.

Single crystal X-ray diffraction data collection was performed on the obtained single crystal and its single crystal structure was analyzed. Its single crystal structure data are shown in Table 5.

According to the analysis of the single crystal diffraction results, the single crystal belongs to the tetragonal crystal system and the space group I 4. Cell parameters:

α=90°, β=90°, γ=90°, unit cell volume

Reliability factor R1=0.0891, wR2=0.2852, S=0.992. The stoichiometric formula is C ₃₀ H ₃₇ F ₄ N ₅ O ₄ , and the calculated molecular weight of the asymmetric unit is 607.64. There are 8 asymmetric units in a single unit cell, and each asymmetric unit contains 1 free base molecule and 1 succinic acid molecules, as shown in Figure 11 and Figure 12.

Confirm that the absolute configuration of formula (I) compound succinate A crystal form is:

Table 5: Single crystal X-ray diffraction data

Tests for Biological Activity and Related Properties

Test example 1, formula (I) compound and its pharmaceutically acceptable salt saturation solubility test

Experimental equipment: rotary water bath constant temperature oscillator (Taicang Wener Huijin, model DSHZ-300A); high performance liquid chromatography (Agilent 1260).

Experimental method: Accurately weigh about 10 mg of the sample to be tested, put it in a 10 ml centrifuge tube, add 1 ml of water, and shake in a water bath constant temperature shaker (37 ° C, 150 rpm). If the sample is completely dissolved, continue to add 10 mg of the sample and shake for about After 24 hours, take the supernatant, perform HPLC detection after appropriate dilution with acetonitrile, and determine its concentration by the external standard method.

Experimental results: the saturated solubility of the compounds to be tested is shown in Table 6. It can be seen that the 24h saturated solubility of the compound of formula (I) succinate, benzoate and adipate is significantly better than that of the free base.

Table 6: Saturated Solubility Test Results

Test example 2, investigation test of isomer impurity of formula (I) compound and pharmaceutically acceptable salt thereof

Test method: formula (I) compound succinate (embodiment 10-1, A crystal form), formula (I) compound adipate (embodiment 7, C crystal form), formula (I) compound (implementation Example 1) are packaged with two layers of polyethylene and one layer of pharmaceutical composite film, focusing on the changes in the content of isomer impurities of the compound of formula (I) under conditions such as high temperature, light, high humidity, and acceleration. The results are shown in Table 7 shown. The isomer impurities are isomer 1 and isomer 2, respectively, and the structures are shown below. N.D means the impurity was not detected.

Test result: formula (I) compound succinate and formula (I) compound adipate are under high temperature, light, acceleration and high humidity conditions, and its isomer impurity all does not increase obviously, the free state formula (I) The content of the isomer 1 impurity of the compound increased significantly under the high temperature condition for 5 days. In addition, the compound of formula (I) in the free state is detected as a new impurity of isomer 2 under high temperature, light, acceleration and high humidity conditions. This test proves that the above-mentioned salt form of the compound has better thermal stability, and can more effectively suppress the generation of isomer impurities compared with the free base.

Table 7: Test results of isomer impurity investigation

Test example 3, formula (I) compound pharmaceutically acceptable salt stability investigation test

Test method: the formula (I) compound succinate (embodiment 10-1, A crystal form), the formula (I) compound benzoate (embodiment 8, B crystal form), the formula (I) compound hexadiene Salt (embodiment 7, C crystal form) all adopts two layers of polyethylene, one layer of pharmaceutical composite film packing, investigates the change of impurity amount and impurity total amount under conditions such as high temperature, illumination, acceleration and high humidity, the result As shown in Table 8.

Test result: formula (I) compound succinate, formula (I) compound benzoate and formula (I) compound adipate all can control total impurity content preferably, especially formula (I) compound succinic acid The salt and the adipate salt of the compound of formula (I) perform well in both aspects of total impurity content and impurity growth rate under conditions such as high temperature, light, acceleration and high humidity.

Table 8: Stability investigation test results

Test Example 4: Rat Pharmacokinetic Evaluation of Compounds of Formula (I) and Pharmaceutically Acceptable Salts thereof

Experimental Materials:

SD female rats were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

MC (methylcellulose) was purchased from Sigma.

Acetonitrile was purchased from Merck (USA).

experimental method:

Twelve female SD rats (200-300g, 6-8 weeks) were randomly divided into 4 groups, with 3 rats in each group. Oral administration of formula (I) compound, formula (I) compound benzoate, formula (I) compound succinate and formula (I) compound adipate, dose 24mg/kg, vehicle 0.5% methylcellulose aqueous solution. Animals were fed water normally before the experiment, fasted overnight, and food supply was resumed 4 hours after administration. Rats in each group were collected from the jugular vein before administration and at 0.25, 0.5, 1, 2, 4, 6, 8, 24h and 48h after administration. The collected whole blood samples were placed in K ₂ EDTA anticoagulant tubes, centrifuged for 5 minutes (12000 rpm, 4° C.) and plasma was collected for testing.

Take 10 μL of rat plasma sample, add 150 μL of acetonitrile solvent (including internal standard compound) to precipitate protein, vortex for 5 minutes, centrifuge (14000rpm) for 5 minutes, and dilute the supernatant 2 times with water containing 0.1% (v/v) FA , quantitatively detected on LC-MS/MS system (AB Sciex Triple Quad 6500+). SD rat plasma standard curve and quality control samples were accompanied by the determination of sample concentration. For 10x diluted samples, take 2 μL sample and add 18 μL blank plasma, vortex for 0.5 min, add 300 μL acetonitrile solvent (including internal standard compound) to precipitate protein, and the rest of the processing steps are the same as for undiluted samples.

Experimental results:

Compared with free base, formula (I) compound benzoate, formula (I) compound succinate and formula (I) compound adipate have achieved more excellent PK characteristics, and have good oral bioavailability .

The rat PK test result of table 9 formula (I) compound and pharmaceutically acceptable salt thereof

Test Example 5 Study on In vitro Proliferation Inhibition of Formula (I) Compound Succinate A Crystal Form on Breast Cancer Cells

Experimental materials: MCF-7, CAMA-1, HCC1500, T47D, and SK-BR-3 cells were purchased from ATCC, and EFM-19 and MCF-7 ESR1 Y537S mutant strains were purchased from Kebai Biotech. The cell culture conditions refer to ATCC or Kebai product instructions, the medium is purchased from Invitrogen, and the serum is purchased from Gibico. MCF-7 medium is DMEM+10%FBS+0.01mg/ml human insulin (Yisheng)+1% non-essential amino acid. CAMA-1 medium is EMEM+10% FBS. HCC1500 and EFM-19 medium are RPMI-1640+10% FBS. T47D medium is RPMI-1640+10% HI-FBS+2 unit/mL bovine insulin (Solarbio). MCF-7 ESR1 Y537S medium is MEM + 10% FBS + 1mM sodium pyruvate + 1% non-essential amino acids. SK-BR-3 medium is McCoy's 5A+10% FBS. Fulvestrant was purchased from MCE. The crystal form A of succinate salt of the compound of formula (I) was prepared according to the method in Example 10-1.

Experimental method: when the fusion rate of the cultured cells reaches more than 70%, digest the cells, adjust the density of MCF-7, T47D, MCF ESR1 Y537S cells to 500/20 μL/well, adjust the density of HCC1500 cells to 2000/20 μL/well, CAMA-1, EFM-19 and SK-BR-3 cells were adjusted to a density of 1000/20 μL/well and cultured overnight in a 384-well cell culture plate (Corning). Use Echo650 (Beckman) to transfer 120nL of 3-fold diluted compound to the cell plate, add 40 μL of corresponding medium, after 7 days of compound treatment, add CellTiter-Glo (Promega) to detect cell viability, the cell well is used as 0% inhibition control, 0.1 μM fluorine Wells treated with fulvestrant were used as 100% inhibition control (wells treated with 2 μM fulvestrant for MCF-7 ESR1 Y537S were used as 100% inhibition control, and medium wells were used as 100% inhibition control for SK-BR-3). Using IDBS XLfit to perform 4-parameter fitting to calculate IC ₅₀ and maximum inhibition rate data.

Experimental results: the succinate A crystal form of the compound of formula (I) showed strong proliferation inhibitory effects on different ER-positive breast cancer cell lines, with IC ₅₀ <1nM, and the IC ₅₀ for proliferation inhibition of ESR1 Y537S mutant strains was 27.6nM, SK-BR-3 has no inhibitory effect on the proliferation of ER-negative cells at the highest concentration of 2 μM.

Table 10 formula (I) compound succinate A crystal form inhibits breast cancer cell proliferation

cell line	IC50_nM	Maximum inhibition rate%
MCF7	0.78	99.59
MCF-7 ESR1 Y537S	27.60	100.96
HCC1500	0.94	90.32
CAMA-1	0.38	97.66
EFM-19	0.40	110.99
T47D	0.55	94.29
SK-BR-3(ER - )	>2000	-2.5

Test example 6 formula (I) compound succinate A crystal form to MCF-7 and MCF-7 ESR1 Y537S mutant strain ER protein

Degradation

Experimental materials: MCF-7 was purchased from ATCC, and the MCF-7 ESR1 Y537S mutant strain was purchased from Kebai Biotech. The cell culture conditions refer to ATCC or Kebai product instructions, the medium is purchased from Invitrogen, and the serum is purchased from Gibico. MCF-7 medium is DMEM+10%FBS+0.01mg/ml human insulin (Yisheng)+1% non-essential amino acid. CAMA-1 medium is EMEM+10% FBS. MCF-7 ESR1 Y537S medium is MEM + 10% FBS + 1mM sodium pyruvate + 1% non-essential amino acids. Fulvestrant was purchased from MCE. The crystal form A of succinate salt of the compound of formula (I) was prepared according to the method in Example 10-1.

Experimental method: when the fusion rate of the cultured cells reaches more than 70%, digest the cells, adjust the cell density to 5000/20 μL/well, seed in a 384-well cell culture plate (Greiner) and culture overnight. Use Echo650 (Beckman) to transfer 100nL 3-fold diluted compound to the cell plate, add 20μL 8% formaldehyde (Shanghai test) after 24 hours to fix at room temperature for 20 minutes, add 50μL ice methanol (Millipore) to permeate the membrane for 10 minutes, add 50μL Licor blocking The buffer was blocked at room temperature for 1 hour, and 20 μL of ER antibody (CST) and GAPDH antibody (Abcam) were added to incubate overnight at 4°C. Wash off the primary antibody, add IRDye 800 goat anti-rabbit IgG antibody and IRDye 680 goat anti-mouse IgG antibody (LI-COR) and incubate in the dark for 45 minutes, wash off the secondary antibody, invert 1200 rpm and centrifuge for 1 minute to remove residual liquid, use Sapphire RGBNIR (Azure) detects 800nM and 680nM signals. Use AzureSpot for fluorescence quantitative analysis, calculate Chanel 800(ER)/Chanel 680(GAPDH) value, cell wells as 0% inhibition control, MCF-7 use 0.1μM fulvestrant treatment wells as 100% inhibition control, MCF-7 ESR1 Y537S wells were treated with 2 μM fulvestrant as 100% degradation. IDBS XLfit was used to perform 4-parameter fitting to calculate DC ₅₀ and maximum degradation rate data.

Experimental results: The succinate A crystal form of the compound of formula (I) showed a high ER degradation effect on MCF-7 and ESR1 Y537S mutant strains.

Table 11 Formula (I) compound succinate A crystal form on MCF-7 and ESR1 Y537S mutant strain ER degradation

cell line	DC 50_nM	Maximum degradation rate%
MCF7	0.73	105.09
MCF-7 ESR Y537S	47.32	99.57

Pharmacodynamic study of human ER-positive breast cancer MCF-7 xenograft subcutaneous xenograft tumor mouse model of test example 7 formula (I) compound succinate

Experimental Materials:

Human breast cancer MCF-7 cells: ECACC, 86012803

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

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

Fetal bovine serum: ExCell; Cat No.: FND500

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

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

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

Matrigel: Corning, Cat.No.: 354234

Fulvestrant injection (Fulvestrant): AstraZeneca, 250mg/5mL/bottle

experimental method:

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

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

Cell inoculation: Inoculate 0.2ml/(containing 1× ¹⁰⁷ ) MCF-7 cell suspension (DPBS: Matrigel, volume ratio 1:1) subcutaneously on the right back of each mouse, and inoculate 17β-estradiol tablets were subcutaneously inoculated two days before. On the 6th day after cell inoculation, when the average tumor volume reached 173.65 mm ³ , group administration began, and random grouping was administered according to the tumor volume. The day of group administration was Day 0 (Day 0).

Administration: the administration dose of formula (I) compound succinate (Example 10-1, A crystal form) is 0.3, 1, 3 or 10 mg/kg (calculated as free base), orally administered (PO), Once-daily dosing (QD) x21 times. The dosage of fulvestrant (Fulvestrant) is 250 mg/kg, subcutaneous injection (SC), once a week (QW) x 4 times. 8 mice per group.

Tumor measurements and experimental indicators:

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

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

Experimental results:

See Table 12, Figure 13 and Figure 14. Although individual mice have more than 10% weight loss, it has nothing to do with the dose of the test product formula (I) compound succinate, and the body weight of the mice in the test drug group is not particularly significant compared with the vehicle control group. Considering that the model is affected by the estrogen patch and may affect the state and body weight of the mice, the weight loss of these individual mice has nothing to do with the test product formula (I) compound succinate. No mouse died in each group of the experiment.

The test results show that on the 20th day (Day 20) after the start of administration, subcutaneous injection of Fulvestrant 250 mg/kg into human breast cancer MCF-7 tumor-bearing mice once a week has a significant effect on the tumor growth of the MCF-7 xenograft tumor model. Weak inhibitory effect (P<0.05). Orally administered 0.3mg/kg of the test product formula (I) compound succinate to tumor-bearing mice once a day, compared with the vehicle control group, showed a certain effect of inhibiting tumor growth (P<0.0001), once a day orally Giving tumor-bearing mice 1mg/kg, 3mg/kg or 10mg/kg of the test product formula (I) compound succinate, compared with the vehicle control group, showed a significant effect of inhibiting tumor growth (P<0.0001) , and showed a good dose dependence, at the dose of 1mg/kg and above, the tumor began to regress. On human breast cancer MCF-7 subcutaneous tumor model, formula (I) compound succinate shows significantly better than reference substance Fulvestrant (250mg/kg, SC) at a dose of 1mg/kg and above (PO, QD). , QW) antitumor activity (P<0.0001).

Table 12 MCF-7 subcutaneous tumor model tumor volume

Test Example 8 Pharmacodynamic study of compound succinate of formula (I) on human ER-positive breast cancer T47D xenograft subcutaneous xenograft tumor mouse model

Experimental Materials:

Human breast cancer xxT47D cells are T47D (ATCC, HTB-133) cells that have been passaged twice in vivo

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

RPMI-1640 culture medium: Gibco, Cat No.: C22400500BT

Fetal bovine serum: HyClone; Cat No.:SV30087.03

Bovine insulin: Yuanye, Cat.No.:11070-73-8

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

0.25% Trypsin-EDTA: Gibco, Cat No.: 25200072

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

Matrigel: Corning, Cat.No.: 354234

Fulvestrant injection (Fulvestrant): AstraZeneca, 250mg/5mL/bottle

experimental method:

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

Cell culture: Human breast cancer xxT47D cells were cultured in vitro. The culture conditions were RPMI-1640 medium plus 10% fetal bovine serum, 0.2 Units/mL bovine insulin, 1% double antibody, and cultured in a 5% CO ₂ incubator at 37°C. Routine digestion with trypsin-EDTA was performed twice a week for passaging. When the confluence of the cells is 80%-90% and the number reaches the requirement, the cells are collected and counted.

Cell inoculation: Inoculate 0.2mL (containing 1× ¹⁰⁷ ) xxT47D cell suspension (DPBS: Matrigel, volume ratio 1:1) subcutaneously on the right back of each mouse, and two days before cell inoculation Subcutaneous inoculation of 17β-estradiol tablets. On the 6th day after cell inoculation, when the average volume of the tumor reached 137.09 mm ³ , group administration began, and the group was randomly assigned according to the tumor volume, and the day of group administration was Day 0 (Day 0).

Administration: The dosage of the compound succinate of formula (I) (Example 10-1, A crystal form) is 0.3, 1 or 3mg/kg, administered orally (PO), administered once a day (QD) x28 Second-rate. The dosage of fulvestrant (Fulvestrant) is 250 mg/kg, subcutaneous injection (SC), once a week (QW) x 4 times. 8 mice per group.

Tumor measurements and experimental indicators:

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

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

Experimental results:

See Table 13, Figure 15 and Figure 16.

The test results showed that on the 28th day (Day 28) after the start of administration, subcutaneous injection of 250mg/kg Fulvestrant to human breast cancer T47D tumor-bearing mice once a week showed a certain anti-tumor effect compared with the vehicle control group. The effect of growth (P<0.0001). Once a day orally administered to tumor-bearing mice 0.3mg/kg, 1mg/kg or 3mg/kg of the test product formula (I) compound succinate, compared with the vehicle control group, there is a certain or significant effect of inhibiting tumor growth ( P<0.0001), and showed a good dose dependence. On human breast cancer T47D subcutaneous tumor model, formula (I) compound succinate shows significantly better than reference substance Fulvestrant (250mg/kg, SC, QW) under the dose (PO, QD) of 1mg/kg and above. ) antitumor activity (P<0.0001).

Table 13 T47D subcutaneous tumor model tumor volume

Test Example 9 Pharmacodynamic study of compound succinate of formula (I) on human ER-positive breast cancer MCF-7 xenograft brain orthotopic transplantation mouse model

Experimental Materials

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

17β-estradiol tablets: Innovative Research of America, SE-121, 60-day release, 0.72mg/pellet

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

Fetal bovine serum: Gibco, Cat No.: 10099-141C

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

Penicillin (P/S): Gibco, Cat No.: 15140-122

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

DPBS: Hyclone, Cat. No.: SH30256.01

Fulvestrant injection (Fulvestrant): AstraZeneca, 250mg/5mL/bottle

experimental method

Animal information: NPG mice, female, 6-7 weeks old, weighing about 17-23 grams, were purchased from Beijing Weitongda Biotechnology Co., Ltd., and the mice were raised in an SPF-grade environment, and each cage was sent separately All animals had free access to a standard certified commercial laboratory diet and water ad libitum.

Cell culture: In vitro culture of human breast cancer MCF-7 cell line, the culture conditions are 10% fetal bovine serum, 10 μg/ml recombinant human insulin and 1% P/S in EMEM (cell culture medium), 37 ° C, 5% CO ₂ incubators. Passage with routine digestion with 0.25% trypsin-EDTA once or twice a week. When the number reaches the requirement and the cells are in the logarithmic growth phase, the cells are collected and counted.

Cell inoculation: 0.015ml/(containing 2×10 ⁶ ) MCF-7 cell suspension was inoculated in the brain of each mouse (1mm in front of bregma, 2mm on the right side of the raphe, 3mm below the dura mater plane of the skull) , and 17β-estradiol tablets were subcutaneously inoculated three days before cell inoculation. On the 8th day after cell inoculation, group administration began, and animals were randomly divided into groups according to body weight, and the day of grouping was Day 0.

Administration: the dosage of the compound succinate of formula (I) (Example 10-1, crystal form A) is 3, 10 or 30 mg/kg, administered orally (PO), administered once a day (QD), Dosing continued until Day 60. The dosage of fulvestrant (Fulvestrant) was 250mg/kg, subcutaneously injected (SC), once a week (QW), and continued until Day 60. 8 mice per group.

Body weight measurements and experimental indicators:

Mice were weighed twice a week.

The antitumor efficacy of compounds was evaluated by the median survival time.

Experimental results:

See Table 14 and Figure 17. Compared with the solvent control group, giving MCF-7 brain orthotopic tumor model animals 250 mg/kg Fulvestrant subcutaneously once a week did not significantly improve the survival rate of the animals, while oral administration of MCF-7 brain orthotopic tumor model animals once a day for 10 or 30mg/kg formula (I) compound succinate can significantly increase the survival rate of mice (P<0.001), improve the state of animals, and the drug effect is significantly stronger than that of Fulvestrant (P<0.001).

Table 14 Effect of MCF-7 brain orthotopic model of each treatment group on the survival rate of mice

Test Example 10: Research on the effect of compounds of formula (I) on the degradation of estrogen receptors in MCF7 cells

1. Purpose of the experiment

The purpose of this experiment is to measure the degradation activity of the compound of formula (I) on the endogenously expressed estrogen receptor in MCF7 cells, and evaluate the activity of the compound according to DC ₅₀ and maximum degradation efficiency.

2. Experimental method

MCF7 cells (ATCC, HTB-22) were cultured with DMEM (Gibco, 11995-065) complete medium containing 10% fetal bovine serum. On the first day of the experiment, MCF7 cells were seeded in a 384-well plate at a density of 3000 cells/well using complete medium, and cultured in a 5% CO ₂ cell incubator at 37°C. The compound to be tested was dissolved in DMSO with a storage concentration of 10mM, diluted with Echo 550 (Labcyte Inc.) and added to the cell culture plate, the initial concentration of the compound to be tested was 100nM, 3-fold serial dilution, 9 concentration points, set A blank control containing 0.5% DMSO was used, and a double-well control was set at each concentration point. Incubate in a 37°C, 5% CO ₂ cell incubator for 24 hours. Add paraformaldehyde to the cell culture medium in each cell culture well. The final concentration of paraformaldehyde is about 3.7% to fix the cells. After 30 minutes of action, discard the supernatant and add 50 μL PBS to each well to wash once; add PBS containing 0.5% v /v Tween-20 to treat cells for 30 minutes, wash once with PBS; add blocking solution (5% BSA, 0.05% Tween-20 in PBS) and incubate at room temperature for 1 hour; remove blocking solution and add primary antibody mixture (anti-ER monoclonal antibody , Estrogen Receptorα (D8H8) Rabbit mAb, GST, #8644S, 1:1000 dilution; anti-GAPDH monoclonal antibody, GAPDH (D4C6R) Mouse mAb, GST, #97166S, 1:2000 dilution) at room temperature for 3 hours; with PBST ( PBS containing 0.05% Tween-20) and washed 3 times; add detection secondary antibody (800CW-goat anti-rabbit IgG, LI-COR, P/N:926-32211, 1:1000 dilution; 680RD-goat anti-mouse IgG, LI -COR, #925-68070, 1:1000 dilution), room temperature, incubate in the dark for 45 minutes; wash 3 times with PBST, use Odyssey CLx to read the fluorescence signal of each well, and calculate the value of Chanel 800(ER)/Chanel 680(GAPDH) . The wells treated with 0.1 μM fulvestrant were used as the 100% degradation control, and the degradation rate at each concentration point was calculated. XlLfit was used to analyze the processing data, and the degradation activity _DC50 and the maximum degradation rate Imax of the compound were calculated. See Table 15 for data analysis.

Table 15 formula (I) compound is to MCF7 intracellular estrogen receptor degradation activity

Compound number	ER level DC 50 (nM)	maximum degradation rate
Compound of formula (I)	0.15	104%

Test Example 11: Study on the inhibitory effect of the compound of formula (I) on the proliferation of MCF7 cells

1. Purpose of the experiment

The purpose of this experiment is to determine the inhibitory effect of the compound of formula (I) on the proliferation of MCF7 cells in vitro, and to evaluate the activity of the compound according to IC ₅₀ .

2. Experimental method

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

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

3. Data analysis see Table 16

The inhibitory activity of table 16 formula (I) compound to MCF7 cell proliferation

Compound number	MCF-7 cell proliferation inhibition IC 50 (nM)
Compound of formula (I)	0.27

Test Example 12: Apparent Solubility of Compound of Formula (I) in Phosphate Buffered Saline at pH 7.4

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

1. Materials and Reagents

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

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

2. Experimental steps

Preparation of stock solution

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

Apparent Solubility Determination Procedure

Take 30 μL of 10mM stock solution of the analyte and add it to the corresponding position of the corresponding 96-well plate in the specified order. Add 970 μL of pH 7.4 phosphate buffer to the corresponding vial of the sample plate. The experiments were performed in double parallel. Add a stir bar to each vial and cap with a Teflon/silicone stopper. Then put the sample tray into the Eppendorf Thermomixer Comfort, and vibrate for 2 hours at 1100 rpm at 25°C. After 2 hours, remove the stopper, remove the stir bar with a large magnet, and transfer the sample from the sample plate to the filter plate. Use a vacuum pump to generate negative pressure and filter the sample. Transfer 5 μL of filtrate to a new sample plate, then add 5 μL DMSO and 490 μL 50% ACN(IS).H ₂ O (internal standard acetonitrile:water=1:1). Depending on the peak shape, it may be possible to dilute the sample diluent with a certain percentage of 50% ACN(IS).H ₂ O to obtain better peak shape. The dilution factor may be adjusted due to the solubility of the analyte or the strength of its liquid-mass response signal.

Sample Analysis Procedure

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

3. Experimental steps

All calculations were performed by Microsoft Excel. The analysis and quantification of the sample filtrate is accomplished by using LC/MS to identify and quantify the peaks of standard products of known concentration. The calculated apparent solubility value of the compound of formula (I) in phosphate buffer solution with a pH value of 7.4.

The apparent solubility value of the compound of table 17 formula (I) in the phosphate buffer saline buffered saline that pH value is 7.4

Compound number	pH=7.4 apparent solubility (μM)
Compound of formula (I)	92

Test Example 13: The blood-brain barrier (BBB) penetration ability of the compound of formula (I) in rats

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

Experimental Materials

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

experimental method

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

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

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

The BBB test results are as follows:

Table 18 formula (I) compound rat blood-brain barrier penetration test

Test Example 14: Growth inhibition experiment of the compound of formula (I) on MCF-7 mouse subcutaneous tumor model

experimental reagent

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

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

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

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

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

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

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

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

Matrigel: Corning, Cat. No.: 356237

experimental method

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

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

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

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

Tumor measurements and experimental indicators:

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

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

Experimental results:

See Table 19. In the subcutaneous transplanted tumor MCF-7 model in mice, the compound of formula (I) has a significant inhibitory effect on tumor growth (P<0.01) at 1 mg/kg, 3 mg/kg, or 10 mg/kg orally administered once a day, and has It has a good dose-response relationship, and it has the effect of shrinking tumors at the doses of 3mg/kg and 10mg/kg. Oral administration of the compound of formula (I) once a day at 10 mg/kg has a significant inhibitory effect on tumor growth (P<0.01), and has the effect of shrinking tumors. The compound of formula (I) did not significantly affect the body weight of mice at the doses tried.

Table 19 MCF-7 subcutaneous tumor model tumor volume

Test Example 15: Inhibitory experiment of the compound of formula (I) on the growth of mouse MCF-7 brain orthotopic tumor model

Experimental reagents/instruments:

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

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

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

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

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

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

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

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

Micro injection pump: KDS, Cat No.: Legato130

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

experimental method:

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

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

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

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

Experimental observation and end:

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

On the 48th day (Day 48), the experiment was terminated, and all mice were euthanized.

Experimental results:

See Figure 18 and Figure 19. In the orthotopic MCF-7 model of the mouse brain, the body weight of the mice in the Fulvestrant group (250mg/kg administered subcutaneously once a week) continued to decrease, and the survival status of the mice was not significantly different from that of the solvent control group (median survival period, vehicle 29 days in the control group and 29.5 days in the Fulvestrant group). The mice in the compound group of formula (I) (30 mg/kg administered orally once a day) had a stable body weight and no abnormal state. Until the end of the experiment, the mice in the compound group of formula (I) did not die. Compared with the solvent control or Fulvestrant, the compound It has a significant inhibitory effect on MCF-7 brain orthotopic tumor model mice, and the survival period of mice is significantly prolonged (P<0.01).

Claims (26)

1. A pharmaceutically acceptable salt of a compound of formula (I),

   

   Wherein, the pharmaceutically acceptable salt is selected from sulfate, phosphate, benzoate, succinate, adipate, fumarate, L-malate and citrate.
2. The pharmaceutically acceptable salt of the compound of formula (I) according to claim 1, which is selected from the group consisting of benzoate, succinate and adipate.
3. The pharmaceutically acceptable salt of the compound of formula (I) according to claim 1, the succinate of the compound of formula (I) is shown in formula (II), wherein, x is selected from 0.5～2,

   
4. According to the pharmaceutically acceptable salt of the compound of formula (I) according to claim 1, the benzoate salt of the compound of formula (I) is shown in formula (III), wherein, y is selected from 0.5～2,

   
5. According to the pharmaceutically acceptable salt of the compound of formula (I) according to claim 1, the adipate salt of the compound of formula (I) is shown in formula (IV), wherein, z is selected from 0.5～2,

   
6. The pharmaceutically acceptable salt of the compound of formula (I) according to claim 3, wherein x is selected from 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2.0; preferably, x is selected from 0.8, 0.9, 1.0, 1.1 and 1.2.
7. The pharmaceutically acceptable salt of the compound of formula (I) according to claim 4, wherein y is selected from 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2.0; preferably, y is selected from 0.8, 0.9, 1.0, 1.1 and 1.2.
8. The pharmaceutically acceptable salt of the compound of formula (I) according to claim 5, wherein z is selected from 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2.0; preferably z is selected from 0.8, 0.9, 1.0, 1.1 and 1.2.
9. The adipate salt C crystal form of the compound of formula (I),

   

   In the X-ray powder diffraction pattern represented by the diffraction angle 2θ of the crystal form C, there are diffraction peaks at 4.46±0.2°, 6.30±0.2°, 8.90±0.2°, and 19.10±0.2°.
10. The C crystal form according to claim 9, wherein:

    In the X-ray powder diffraction pattern represented by the diffraction angle 2θ of the crystal form C, at 4.46±0.2°, 6.30±0.2°, 7.03±0.2°, 8.90±0.2°, 16.45±0.2°, 17.78±0.2°, Diffraction peaks at 19.10±0.2° and 21.03±0.2°; or

    In the X-ray powder diffraction pattern of the crystal form C represented by diffraction angle 2θ, at 4.46±0.2°, 6.30±0.2°, 7.03±0.2°, 8.90±0.2°, 10.65±0.2°, 11.95±0.2°, 12.55±0.2°, 13.34±0.2°, 14.20±0.2°, 16.02±0.2°, 16.45±0.2°, 17.78±0.2°, 18.33±0.2°, 19.10±0.2°, 19.77±0.2°, 20.12±0.2°, Diffraction peaks at 21.03±0.2°, 21.62±0.2°, 24.30±0.2°, 25.97±0.2°, 27.39±0.2° and 27.98±0.2°; or

    The X-ray powder diffraction pattern of the C crystal form is basically consistent with that in FIG. 2 .
11. According to the crystal form C according to claim 9 or 10, the DSC spectrum of the crystal form C has an endothermic peak at 149.89±5°C.
12. The benzoate B crystal form of the compound of formula (I),

    

    In the X-ray powder diffraction pattern represented by the diffraction angle 2θ of the crystal form B, there are diffraction peaks at 7.19±0.2°, 10.01±0.2°, 11.39±0.2°, and 18.78±0.2°.
13. The B crystal form according to claim 12, wherein:

    In the X-ray powder diffraction pattern represented by the diffraction angle 2θ of the B crystal form, at 7.19±0.2°, 10.01±0.2°, 11.39±0.2°, 18.78±0.2°, 19.01±0.2°, 19.66±0.2°, Diffraction peaks at 19.91±0.2° and 20.09±0.2°; or

    In the X-ray powder diffraction pattern represented by the diffraction angle 2θ of the B crystal form, at 4.69±0.2°, 6.34±0.2°, 7.19±0.2°, 9.83±0.2°, 10.01±0.2°, 11.11±0.2°, 11.39±0.2°, 14.78±0.2°, 18.78±0.2°, 19.01±0.2°, 19.25±0.2°, 19.66±0.2°, 19.91±0.2°, 20.09±0.2°, 20.80±0.2°, 21.66±0.2° have diffraction peaks; or

    In the X-ray powder diffraction pattern represented by the diffraction angle 2θ of the B crystal form, at 4.69±0.2°, 4.91±0.2°, 6.34±0.2°, 7.19±0.2°, 9.37±0.2°, 9.83±0.2°, 10.01±0.2°, 10.32±0.2°, 11.11±0.2°, 11.39±0.2°, 12.06±0.2°, 12.69±0.2°, 13.52±0.2°, 14.07±0.2°, 14.36±0.2°, 14.78±0.2°, 17.30±0.2°, 18.78±0.2°, 19.01±0.2°, 19.25±0.2°, 19.66±0.2°, 19.91±0.2°, 20.09±0.2°, 20.80±0.2°, 21.15±0.2°, 21.66±0.2°, Diffraction peaks at 22.35±0.2°, 23.57±0.2°, 23.70±0.2°, 24.63±0.2°, 25.68±0.2°, 27.05±0.2°; or

    The X-ray powder diffraction pattern of the crystal form B is basically consistent with that in FIG. 5 .
14. According to the crystal form B according to claim 12 or 13, the DSC spectrum of the crystal form B has an endothermic peak at 150.08±5°C.
15. The crystal form A of the succinate salt of the compound of formula (I),

    

    In the X-ray powder diffraction pattern represented by the diffraction angle 2θ of the crystal form A, there are diffraction peaks at 7.13±0.2°, 9.30±0.2°, 11.29±0.2°, 15.51±0.2°, and 19.89±0.2°.
16. A crystal form according to claim 15, wherein:

    In the X-ray powder diffraction pattern of the crystal form A represented by diffraction angle 2θ, at 7.13±0.2°, 9.30±0.2°, 11.29±0.2°, 15.51±0.2°, 17.10±0.2°, 19.89±0.2°, Diffraction peaks at 20.26±0.2°, 20.91±0.2°, 21.51±0.2°, 22.69±0.2°, 23.58±0.2°, 25.42±0.2°; or

    In the X-ray powder diffraction pattern of the crystal form A represented by the diffraction angle 2θ, at 5.01±0.2°, 7.13±0.2°, 9.30±0.2°, 10.09±0.2°, 11.29±0.2°, 11.73±0.2°, 13.75±0.2°, 14.30±0.2°, 15.18±0.2°, 15.51±0.2°, 16.00±0.2°, 17.10±0.2°, 18.25±0.2°, 18.69±0.2°, 19.89±0.2°, 20.26±0.2°, 20.91±0.2°, 21.12±0.2°, 21.51±0.2°, 22.69±0.2°, 23.05±0.2°, 23.58±0.2°, 24.58±0.2°, 25.42±0.2°, 26.66±0.2°, and Diffraction peak at 29.47±0.2°; or

    The X-ray powder diffraction pattern of the crystal form A is basically consistent with that in FIG. 8 .
17. According to the crystal form A according to claim 15 or 16, the DSC spectrum of the crystal form A has an endothermic peak at 186.82±5°C.
18. According to the crystal form A described in any one of claims 15-17, the unit cell parameters of the crystal form A are:

    

    

    α=90°, β=90°, γ=90°.
19. A method for preparing a pharmaceutically acceptable salt of a compound of formula (I) according to any one of claims 1 to 8, said preparation method comprising the step of reacting the compound of formula (I) with a corresponding acid to form a salt.
20. According to the preparation method described in claim 19, the solvent of the salt-forming reaction is selected from cyclohexane, n-heptane, toluene, chloroform, ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, ethylene glycol dimethyl At least one of ether, acetone, methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, methanol, ethanol, isopropanol, n-butanol and acetonitrile.
21. According to the preparation method described in claim 19 or 20, the preparation method further comprises the steps of one or a combination of stirring and crystallization, filtering and drying.
22. The method for preparing the C crystal form described in any one of claims 9-11, comprising the following steps:

    (1-a) mixing adipic acid and a solvent;

    (2-a) mixing the compound of formula (I) and a solvent;

    (3-a) mixing the mixture of step (1-a) and the mixture of step (2-a);

    (4-a) Stir the resulting mixture mixed with the adipic acid and the compound of formula (I), and crystallize;

    (5-a) filtering out the crystals, collecting the filter cake, and drying;

    Preferably, the solvent in step (1-a) and step (2-a) is selected from cyclohexane, n-heptane, toluene, chloroform, diethyl ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, ethylene di At least one of alcohol dimethyl ether, acetone, methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, methanol, ethanol, isopropanol, n-butanol and acetonitrile, more preferably isopropyl ether, methyl At least one of tert-butyl ether, methanol, isopropanol and tetrahydrofuran, most preferably isopropanol;

    Preferably, the molar ratio of the compound of formula (I) to adipic acid is 1:2 to 2:1, more preferably the molar ratio is 1:1;

    Preferably, the step (1-a) is carried out under heating conditions, more preferably the heating temperature is about 40-80°C;

    Preferably, the step (4-a) is carried out at 15-30°C.
23. The method for preparing the B crystal form described in any one of claims 12-14, comprising the following steps:

    (1-b) mixing the compound of formula (I) and a solvent;

    (2-b) adding benzoic acid to the mixture of step (1-b);

    (3-b) Stir the resulting mixture mixed with the compound of formula (I) and the benzoic acid, and crystallize;

    (4-b) filtering out and collecting the solid;

    Preferably, the solvent in step (1-b) is selected from cyclohexane, n-heptane, toluene, chloroform, ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, acetone, At least one of methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, methanol, ethanol, isopropanol, n-butanol and acetonitrile, more preferably isopropyl ether, methyl tert-butyl ether, methanol, At least one of isopropanol and tetrahydrofuran, most preferably isopropyl ether;

    Preferably, the molar ratio of the compound of formula (I) to benzoic acid is 1:2 to 2:1, more preferably the molar ratio is 1:1;

    Preferably, the step (1-b) is carried out at 15-30°C;

    Preferably, the stirring in step (3-b) is carried out under heating conditions, more preferably the heating temperature is about 40-68°C, most preferably 60-68°C;

    Optionally, the preparation method of the B crystal form also includes the following steps:

    (5-b) mixing the solid collected in step (4-b) with a solvent;

    (6-b) stirring the resulting mixture;

    (7-b) filter out and collect solid, dry;

    Preferably, the solvent in step (5-b) is selected from at least one of isopropyl ether, methyl tert-butyl ether, methanol, isopropanol and tetrahydrofuran, preferably isopropyl ether;

    Preferably, step (6-b) is carried out at 15-30°C.
24. The method for preparing the crystal form A described in any one of claims 15-18, comprising the following steps:

    (1-c) mixing the compound of formula (I) and a solvent;

    (2-c) adding succinic acid to the mixture of step (1-c);

    (3-c) stirring the mixture obtained by mixing the compound of formula (I) and the succinic acid to precipitate a solid;

    (4-c) filtering out and collecting the solid;

    Preferably, the solvent in step (1-c) is selected from cyclohexane, n-heptane, toluene, chloroform, ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, acetone, At least one of methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, methanol, ethanol, isopropanol, n-butanol and acetonitrile, more preferably isopropyl ether, methyl tert-butyl ether, methanol, At least one of isopropanol and tetrahydrofuran, most preferably isopropyl ether;

    Preferably, the molar ratio of the compound of formula (I) to succinic acid is 1:2 to 2:1, more preferably the molar ratio is 1:1;

    Preferably, the step (3-c) is carried out at 15-30°C;

    Preferably, the reaction time of the step (3-c) is 1-48 hours, more preferably 3-24 hours;

    Optionally, the preparation method of the A crystal form also includes the following steps:

    (5-c) mixing the solid collected in step (4-c) with a solvent;

    (6-c) stirring and crystallizing the resulting mixture;

    (7-c) filter out and collect solid, dry;

    Preferably, the solvent in step (5-c) is selected from at least one of methanol, ethanol, isopropanol, tetrahydrofuran and water, more preferably a mixed solvent of ethanol/water;

    Preferably, the volume ratio of ethanol/water is about 1:1-50:1, more preferably 2:1-20:1, most preferably 10:1, 9:1, 8:1, 7:1, 6 :1, 5:1 or 4:1;

    Preferably, the step (6-c) is carried out at 15-30°C;

    Preferably, the reaction time of the step (6-c) is 0.5-48 hours.
25. A pharmaceutical composition, which comprises the pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 1-8 or the crystal form according to any one of claims 9-18, and pharmaceutically acceptable auxiliary materials.
26. The pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 1 to 8, the crystal form according to any one of claims 9 to 18, or the pharmaceutical composition according to claim 25 is used for preventing or treating Use in drugs for estrogen receptor-related diseases;

    Preferably, the estrogen receptor-related disease is a tumor;

    More preferably, the tumor is breast cancer;

    More preferably, the tumor is ER-positive breast cancer;

    More preferably, the tumor is brain metastases from ER-positive breast cancer.

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