A salt form of a tricyclic tetrahydroisoquinoline derivative

Abstract

The present disclosure relates to a salt form of a tricyclic tetrahydroisoquinoline derivative. Specifically, the present disclosure relates to different salt forms of a compound represented by formula (I) and a preparation method thereof. The salt form of the compound represented by formula (I) provided by the present disclosure has good stability and can be better used for clinical treatment.

Description

[0001] This application claims priority to Chinese patent application 2021107799053, filed on July 9, 2021. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This disclosure relates to a salt form of a tricyclic tetrahydroisoquinoline derivative, its preparation method, and its pharmaceutical uses, belonging to the pharmaceutical field. Background Technology

[0003] In the CSCO Breast Cancer Diagnosis and Treatment Guidelines (2020 Edition), fulvestrant was revised to a Level I recommendation for hormone receptor-positive advanced breast cancer patients who have not undergone endocrine therapy, and fulvestrant combined with a CDK4 / 6 inhibitor was newly added as a Level II recommendation. Furthermore, fulvestrant combined with a CDK4 / 6 inhibitor has become a Level I recommendation for patients who have failed non-steroidal AI (NSAI) and steroidal AI (SAI) therapy. As the only currently approved SERD, fulvestrant is approved in injectable form, which presents inconvenience for patients. Therefore, finding new and more effective SERDs not only has significant market value but also provides breast cancer patients with greater choice and convenience in medication. Currently, Sanofi's SAI439859, as a new generation of orally administered SERD inhibitor, is in Phase III clinical trials. Additionally, Roche's GDC-9545, AstraZeneca's AZD-9833, and Radius' RAD1901 have all entered Phase III trials. Published patent applications for selective estrogen receptor-mediated modulators include WO2014165723, WO2014151899, WO2014141292, WO2014191726, WO2015092634, WO2014135834, WO2014106848 and EP1113007.

[0004] PCT application WO2021139756A discloses a tricyclic tetrahydroisoquinoline derivative of formula (I) for use as an estrogen receptor downregulator (SERD). To meet the drug requirements, it is necessary to study its salts and crystal forms.

[0005] Summary of the Invention

[0006] This disclosure provides a pharmaceutically acceptable salt of the compound represented by formula (I), said pharmaceutically acceptable salt being selected from phosphates, sulfates, tartrates, citrates, hydrochlorides, hydrobroms, methanesulfonates, formates, acetates, succinates, maleates, malates, or p-toluenesulfonates.

[0007]

[0008] In some embodiments, a pharmaceutically acceptable salt of the compound represented by formula (I) provided in this disclosure is used, wherein the molar ratio of the compound represented by formula (I) to the acid molecule is selected from 1:5 to 5:1.

[0009] In some embodiments, a pharmaceutically acceptable salt of the compound represented by formula (I) provided in this disclosure is used, wherein the molar ratio of the compound represented by formula (I) to the acid molecule is 1:1.

[0010] In some embodiments, the compound represented by formula (I) provided in this disclosure is a pharmaceutically acceptable salt, wherein the molar ratio of the compound represented by formula (I) to the acid molecule is 1:3.

[0011] In some embodiments, this disclosure provides a phosphate of the compound shown in formula (I), wherein the molar ratio of the compound shown in formula (I) to the phosphate molecule is 1:3.

[0012] This disclosure provides a crystal form I of the phosphate of the compound shown in formula (I), wherein the molar ratio of the compound shown in formula (I) to the phosphate molecule is 1:3, and the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ has characteristic peaks at 6.1, 13.5, 19.2, and 22.3.

[0013] In some embodiments, the I crystal form of the phosphate of the compound shown in formula (I), wherein the molar ratio of the compound shown in formula (I) to the phosphate molecule is 1:3, has characteristic peaks at 6.1, 13.5, 13.9, 19.2, 21.8, 22.3, and 23.4 in the X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0014] In some embodiments, the I crystal form of the phosphate of the compound shown in formula (I), wherein the molar ratio of the compound shown in formula (I) to the phosphate molecule is 1:3, has characteristic peaks at 6.1, 12.4, 13.5, 13.9, 15.0, 16.3, 16.9, 19.2, 20.8, 21.8, 22.3, 23.4, 24.1, 24.6, 26.2, 27.5, and 30.0 in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0015] In some embodiments, the phosphate of the compound shown in formula (I) is in crystal form I, wherein the molar ratio of the compound shown in formula (I) to the phosphate molecule is 1:3, and the X-ray powder diffraction pattern expressed in terms of diffraction angle 2θ is shown in the attached figure. Figure 2 As shown.

[0016] In some embodiments, the phosphate of the compound shown in formula (I) is in crystal form I, wherein the molar ratio of the compound shown in formula (I) to the phosphate molecule is 1:3, the crystal system is triclinic, and the axial length is... Axial angle α = 89.575(2)°; β = 89.859(2)°; γ = 60.745(2)°; cell volume

[0017] This disclosure provides a method for preparing a phosphate of the compound shown in formula (I) or a crystal form I of the phosphate of the compound shown in formula (I), comprising the step of reacting the compound shown in formula (I) with phosphoric acid.

[0018] This disclosure provides a method for preparing the I crystal form of the phosphate of the compound shown in formula (I), comprising the following steps:

[0019] 1) A solution of the compound shown in formula (I) in solvent A, wherein solvent A is selected from ketone solvents, ester solvents or ether solvents;

[0020] 2) Prepare a solution of phosphoric acid in solvent B, wherein solvent B is selected from water or alcohol solvents;

[0021] 3) The compound shown in formula (I) crystallizes out after being mixed with solvent A solution of phosphoric acid in solvent B solution.

[0022] In some embodiments, a solution of solvent A of the compound of formula (I) is prepared, wherein solvent A is selected from acetone, ethyl acetate or methyl tert-butyl ether.

[0023] In some embodiments, a solution of phosphoric acid in solvent B is prepared, wherein solvent B is selected from methanol, ethanol, or water.

[0024] This disclosure provides a sulfate of the compound shown in formula (I), wherein the molar ratio of the compound shown in formula (I) to sulfuric acid molecules is 1:1.

[0025] This disclosure provides a crystal form A of the sulfate of the compound shown in formula (I), wherein the molar ratio of the compound shown in formula (I) to sulfuric acid molecules is 1:1, and the X-ray powder diffraction pattern expressed as a diffraction angle 2θ has characteristic peaks at 6.6, 7.9, 15.1, 20.7, and 22.1.

[0026] In some embodiments, the sulfate of the compound shown in formula (I) has crystal form A, and the molar ratio of the compound shown in formula (I) to sulfuric acid molecules is 1:1. The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, has characteristic peaks at 6.6, 7.9, 13.4, 14.5, 15.1, 19.3, 19.8, 20.7, 22.1, 24.8, 25.4, 25.7, and 27.6.

[0027] In some embodiments, the sulfate of the compound shown in formula (I) has crystal form A, and the molar ratio of the compound shown in formula (I) to sulfuric acid molecules is 1:1. The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, has characteristic peaks at 6.6, 7.9, 11.6, 13.4, 14.5, 15.1, 15.9, 18.1, 19.3, 19.8, 20.7, 22.1, 24.0, 24.8, 25.4, 25.7, 27.2, 27.6, 28.3, and 29.2.

[0028] In some embodiments, the sulfate of the compound shown in formula (I) is in crystal form A, and the molar ratio of the compound shown in formula (I) to sulfuric acid molecules is 1:1. The X-ray powder diffraction pattern expressed as a diffraction angle 2θ is shown in the attached figure. Figure 8 As shown.

[0029] In some embodiments, the sulfate of the compound shown in formula (I) is in crystal form A, the molar ratio of the compound shown in formula (I) to sulfuric acid molecules is 1:1, it is monoclinic, and its axial length is... Axial angle β = 90°; Crystal volume

[0030] This disclosure provides a method for preparing the sulfate of the compound shown in formula (I) or the A crystal form of the sulfate of the compound shown in formula (I), comprising the step of reacting the compound shown in formula (I) with sulfuric acid.

[0031] This disclosure provides a method for obtaining the A crystal form of the sulfate of the compound shown in formula (I), comprising the following steps:

[0032] 1) A solution of the compound shown in formula (I) in solvent A, wherein solvent A is selected from ketone solvents, ester solvents, nitrile solvents or alcohol solvents;

[0033] 2) Prepare a sulfuric acid solution in solvent B, wherein solvent B is selected from water, nitrile solvents or alcohol solvents;

[0034] 3) The compound shown in mixed formula (I) crystallizes out after being mixed with solvent A solution of sulfuric acid in solvent B solution.

[0035] In some embodiments, a solution of solvent A of the compound shown in formula (I) is prepared, wherein solvent A is selected from acetone, ethyl acetate, acetonitrile, methanol or ethanol.

[0036] In some embodiments, a solution of sulfuric acid in solvent B is prepared, wherein solvent B is selected from methanol, acetonitrile, ethanol or water.

[0037] This disclosure provides an amorphous form of the citrate salt of the compound shown in formula (I).

[0038] This disclosure provides a method for preparing a citrate salt of the compound shown in formula (I), comprising the step of reacting the compound shown in formula (I) with citric acid to form a salt.

[0039] This disclosure provides an amorphous L-tartrate salt of the compound shown in formula (I).

[0040] This disclosure provides a method for preparing an L-tartrate salt of the compound shown in formula (I), comprising the step of reacting the compound shown in formula (I) with L-tartrate to form a salt.

[0041] This disclosure provides a method for preparing the hydrochloride salt of the compound shown in formula (I), comprising the step of reacting the compound shown in formula (I) with hydrochloric acid to form a salt.

[0042] This disclosure provides a method for preparing the hydrobromide salt of the compound shown in formula (I), comprising the step of reacting the compound shown in formula (I) with hydrobromic acid to form a salt.

[0043] This disclosure provides a method for preparing a methanesulfonate of the compound shown in formula (I), comprising the step of reacting the compound shown in formula (I) with methanesulfonic acid to form a salt.

[0044] This disclosure provides a method for preparing a formate salt of the compound shown in formula (I), comprising the step of reacting the compound shown in formula (I) with formic acid to form a salt.

[0045] This disclosure provides a method for preparing an acetate of the compound shown in formula (I), comprising the step of reacting the compound shown in formula (I) with acetic acid to form a salt.

[0046] This disclosure provides a method for preparing a succinate of the compound shown in formula (I), comprising the step of reacting the compound shown in formula (I) with succinic acid.

[0047] This disclosure provides a method for preparing a maleate salt of the compound shown in formula (I), comprising the step of reacting the compound shown in formula (I) with maleic acid to form a salt.

[0048] This disclosure provides a method for preparing the malate of the compound shown in formula (I), comprising the step of reacting the compound shown in formula (I) with malic acid to form a salt.

[0049] This disclosure provides a method for preparing p-toluenesulfonate of the compound shown in formula (I), comprising the step of reacting the compound shown in formula (I) with p-toluenesulfonic acid to form a salt.

[0050] In some embodiments, the I crystal form of the phosphate of the compound of formula (I) provided in this disclosure or the A crystal form of the sulfate of the compound of formula (I) has an error range of ±0.2 for the characteristic peak 2θ angle in the X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0051] On the other hand, this disclosure provides a pharmaceutical composition comprising the following ingredients:

[0052] 1) Phosphates, sulfates, tartrates, citrates, hydrochlorides, hydrobroms, methanesulfonates, formates, acetates, succinates, maleates, malates, or p-toluenesulfonates of the compound shown in formula (I), crystal form I of the phosphate of the compound shown in formula (I) or crystal form A of the sulfate of the compound shown in formula (I), amorphous form of the citrate of the compound shown in formula (I), amorphous form of the L-tartrate of the compound shown in formula (I), or mixtures thereof; and

[0053] 2) Choose any pharmaceutically acceptable carrier, diluent or excipient.

[0054] On the other hand, this disclosure provides a method for preparing a pharmaceutical composition, comprising:

[0055] 1) Phosphates, sulfates, tartrates, citrates, hydrochlorides, hydrobroms, methanesulfonates, formates, acetates, succinates, maleates, malates, or p-toluenesulfonates of the compound shown in formula (I), crystal form I of the phosphate of the compound shown in formula (I) or crystal form A of the sulfate of the compound shown in formula (I), amorphous form of the citrate of the compound shown in formula (I), amorphous form of the L-tartrate of the compound shown in formula (I), or mixtures thereof; and

[0056] 2) Any pharmaceutically acceptable method of mixing carriers, diluents, or excipients.

[0057] In another aspect, this disclosure provides the use of the phosphate, sulfate, tartrate, citrate, hydrochloride, hydrobromide, methanesulfonate, formate, acetate, succinate, maleate, malate, or p-toluenesulfonate of the compound shown in formula (I), the I crystal form of the phosphate of the compound shown in formula (I), or the A crystal form of the sulfate of the compound shown in formula (I), the amorphous form of the citrate of the compound shown in formula (I), the amorphous form of the L-tartrate of the compound shown in formula (I), or mixtures or compositions thereof, in the preparation of estrogen receptor downregulators (SERDs).

[0058] This disclosure also provides the use of phosphates, sulfates, tartrates, citrates, hydrochlorides, hydrobroms, methanesulfonates, formates, acetates, succinates, maleates, malates, or p-toluenesulfonates of the compounds shown in formula (I), crystal form I of the phosphates of the compounds shown in formula (I) or crystal form A of the sulfates of the compounds shown in formula (I), amorphous forms of citrates of the compounds shown in formula (I), amorphous forms of L-tartrates of the compounds shown in formula (I), or mixtures or compositions thereof, in the preparation of medicaments for the prevention and / or treatment of cancer, wherein the cancers are preferably selected from breast cancer, endometrial cancer, uterine cancer, cervical cancer, skin cancer, prostate cancer, ovarian cancer, fallopian tube tumors, hemophilia, and leukemia.

[0059] This disclosure, in another aspect, provides phosphates, sulfates, tartrates, citrates, hydrochlorides, hydrobroms, methanesulfonates, formates, acetates, succinates, maleates, malates, or p-toluenesulfonates of the compounds shown in formula (I), crystal form I of the phosphate of the compound shown in formula (I) or crystal form A of the sulfate of the compound shown in formula (I), amorphous form of the citrate of the compound shown in formula (I), amorphous form of the L-tartrate of the compound shown in formula (I), or mixtures thereof, or compositions thereof, for the preparation of preventive and / or therapeutic agents for estrogen receptor-mediated or dependent diseases or Use in a medicament for a condition; preferably, the estrogen receptor-mediated or dependent disease or condition is selected from cancer, central nervous system defects, cardiovascular system defects, hematological system defects, immune and inflammatory diseases, susceptibility to infection, metabolic defects, neurological defects, mental defects, and reproductive defects; preferably, the cancer is selected from breast cancer, endometrial cancer, uterine cancer, cervical cancer, skin cancer, prostate cancer, ovarian cancer, fallopian tube tumors, hemophilia, and leukemia; more preferably, the cancer is selected from breast cancer, ovarian cancer, endometrial cancer, prostate cancer, and uterine cancer.

[0060] The "2θ or 2θ angle" mentioned in this disclosure refers to the diffraction angle, where θ is the Bragg angle, and the unit is ° or degree; the error range of 2θ for each characteristic peak is ±0.20, specifically -0.20, -0.19, -0.18, -0.17, -0.16, -0.15, -0.14, -0.13, -0.12, -0.11, -0.10, -0.09, -0.08, and -0.07. -0.06, -0.05, -0.04, -0.03, -0.02, -0.01, 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20.

[0061] The “crystallization” described in this disclosure includes, but is not limited to, crystallization by stirring, crystallization by cooling, crystallization by dissolution, and crystallization by volatilization.

[0062] The “differential scanning calorimetry or DSC” described in this disclosure refers to measuring the temperature difference and heat flow difference between the sample and the reference material during the sample heating or isothermal process, in order to characterize all physical and chemical changes related to thermal effects and obtain phase transition information of the sample.

[0063] The drying temperature described in this disclosure is generally 25℃-100℃, preferably 40℃-70℃, and can be dried under normal pressure or reduced pressure.

[0064] The values ​​in this disclosure are instrument measurements or calculated values ​​after instrument measurement, and are subject to a certain degree of error. Generally speaking, ±10% is within the reasonable error range. Of course, the context in which the value is used needs to be considered. For example, for the content of total impurities, the value is defined as having an error variation of no more than ±10% after measurement, and can be ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%, preferably ±5%.

[0065] The starting material used in the crystal form preparation method disclosed herein can be any form of compound shown in Formula I, including but not limited to: amorphous, arbitrary crystal form, hydrate, solvate, etc.

[0066] The “crystallization” described in this disclosure includes, but is not limited to, crystallization by stirring, crystallization by cooling, crystallization by pulping, and crystallization by volatilization. Attached Figure Description

[0067] Figure 1 XRPD spectra of the amorphous compound shown in formula (I);

[0068] Figure 2 XRPD spectrum of phosphate I crystal form of the compound shown in formula (I);

[0069] Figure 3 TGA spectrum of phosphate I crystal form of the compound shown in formula (I);

[0070] Figure 4 DSC spectrum of phosphate I crystal form of the compound shown in formula (I);

[0071] Figure 5 XRPD spectra of phosphate I crystal form of the compound shown in formula (I) after DVS;

[0072] Figure 6 DVS spectrum of phosphate I crystal form of the compound shown in formula (I);

[0073] Figure 7Single crystal spectrum of phosphate I, the compound shown in formula (I);

[0074] Figure 8 XRPD spectrum of the sulfate of the compound shown in formula (I) in crystal form A;

[0075] Figure 9 TGA spectrum of the sulfate of the compound shown in formula (I) in crystal form A;

[0076] Figure 10 DSC spectrum of the sulfate of the compound shown in formula (I) in crystal form A;

[0077] Figure 11 DVS spectrum of the sulfate of the compound shown in formula (I) in crystal form A;

[0078] Figure 12 Single crystal spectra of the sulfate of the compound shown in formula (I) in crystal form A;

[0079] Figure 13 XRPD spectra of the amorphous tartrate salt of the compound shown in formula (I);

[0080] Figure 14 XRPD spectrum of the amorphous citrate of the compound shown in formula (I). Detailed Implementation

[0081] The present invention will be further described in detail through the following embodiments and experimental examples. These embodiments and experimental examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0082] Test conditions of the instruments used in the experiment:

[0083] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) were expressed in 10⁻¹⁰. -6 The unit (ppm) is given. NMR determination was performed using a Bruker AVANCE-400 NMR spectrometer or a Bruker AVANCE NEO 500M. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS).

[0084] MS measurements were performed using an Agilent 1200 / 1290 DAD-6110 / 6120 Quadrupole MS LC-MS system (manufacturer: Agilent, MS model: 6110 / 6120 Quadrupole MS), a Waters ACQuity UPLC-QD / SQD system (manufacturer: Waters, MS model: Waters ACQuity Qda Detector / Waters SQ Detector), and a THERMO Ultimate 3000-Q Exactive system (manufacturer: THERMO, MS model: THERMO Q Exactive).

[0085] High-performance liquid chromatography (HPLC) analysis was performed using an Agilent HPLC 1200DAD, an Agilent HPLC 1200VWD, and a Waters HPLC e2695-2489 high-performance liquid chromatograph.

[0086] Chiral HPLC analysis was performed using an Agilent 1260 DAD high-performance liquid chromatograph.

[0087] High performance liquid chromatography (HPLC) was performed using Waters 2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP, and Gilson-281 preparative chromatographs.

[0088] Chiral preparation was performed using a Shimadzu LC-20AP preparative chromatograph.

[0089] The CombiFlash rapid preparation system uses a CombiFlash Rf200 (TELEDYNE ISCO).

[0090] XRPD (X-ray Powder Diffraction) was used for analysis: measurements were performed using a BRUKER D8 DISCOVER X-ray diffractometer. Specific data collected included: Cu anode (40 kV, 40 mA), Cu-Kα rays. Scanning mode: θ / 2θ, scanning range (2θ range): 3~48°.

[0091] DSC is differential scanning calorimetry: measurements were performed using a METTLER TOLEDO DSC 3+ differential scanning calorimeter, with a heating rate of 10℃ / min and the specific temperature range referred to the corresponding spectrum (25-240℃). Nitrogen purging rate was 50mL / min.

[0092] TGA was a thermogravimetric analysis: the analysis was performed using a METTLER TOLEDO TGA 2 thermogravimetric analyzer, with a heating rate of 10℃ / min and the specific temperature range referred to the corresponding spectrum (30-350℃). The nitrogen purging rate was 20mL / min.

[0093] DVS stands for Dynamic Moisture Adsorption: The detection method is SMS DVS Advantage, with humidity changes of 50%-95%-0%-95%-50% at 25℃, in 10% increments (the final step is 5%) (the specific humidity range is subject to the corresponding spectrum; the methods listed here are the most commonly used methods). The judgment criterion is dm / dt ≤ 0.002%.

[0094] Thin-layer chromatography silica gel plates are Yantai Huanghai HSGF254 or Qingdao GF254. The silica gel plates used in thin-layer chromatography (TLC) have a diameter of 0.15 mm to 0.2 mm, and the diameter of the silica gel plates used for thin-layer chromatography separation and purification products is 0.4 mm to 0.5 mm.

[0095] Silica gel column chromatography generally uses Yantai Huanghai silica gel with a mesh size of 200-300 as the carrier.

[0096] The average inhibition rate and IC50 value of the kinase were determined using a NovoStar microplate reader (BMG GmbH, Germany).

[0097] The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, AccelaChemBio Inc, and Darui Chemicals.

[0098] Unless otherwise specified in the examples, all reactions can be carried out under an argon or nitrogen atmosphere.

[0099] Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

[0100] A hydrogen atmosphere refers to a reaction flask connected to a hydrogen balloon with a volume of approximately 1L.

[0101] The pressurized hydrogenation reaction was performed using a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

[0102] The hydrogenation reaction is usually carried out under vacuum, filled with hydrogen gas, and repeated 3 times.

[0103] The microwave reaction was performed using a CEM Discover-S 908860 microwave reactor.

[0104] Unless otherwise specified in the examples, "solution" refers to an aqueous solution.

[0105] Unless otherwise specified in the examples, the reaction temperature was room temperature, and the reaction time was 20 to 30 samples.

[0106] The reaction process in the examples was monitored using thin-layer chromatography (TLC). The developing solvent used in the reaction, the eluent system used for column chromatography to purify the compounds, and the developing solvent system for TLC included: A: dichloromethane / methanol system, B: n-hexane / ethyl acetate system, and C: petroleum ether / ethyl acetate system. The volume ratio of the solvent was adjusted according to the polarity of the compounds, and small amounts of basic or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0107] Example 1: Preparation of the compound shown in Formula 1 (Preparation method of Example 13 in application with priority: 202010680491.4)

[0108] 2,2-Difluoro-3-((5S,7R)-5-(5-((1-(3-fluoropropyl)azacyclobut-3-yl)amino)pyridin-2-yl)-7-methyl-7,8-dihydro-[1,3]dioxapentanol[4,5-g]isoquinoline-6(5H)-yl-2,2-d2)prop-1-ol

[0109]

[0110] first step

[0111] (R)-N-(1-(3,4-di(benzyloxy)phenyl)prop-2-yl)-3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoroprop-1-amine 1b

[0112] Compound 1a (1.0 g, 3.0 mmol) was dissolved in dioxane (20 mL), and diisopropylethylamine (1.2 g, 9.0 mmol) and 3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyltrifluoromethanesulfonate (1.0 g, 2.0 mmol, prepared by a known method, “Bioorganic & Medicinal Chemistry Letters, 2018, 28(14), 2528-2532”) were added. The reaction was carried out under argon protection and stirred in an oil bath at 80 °C for 20 hours. The reaction mixture was cooled, concentrated under reduced pressure, and extracted with ethyl acetate (100 mL × 2) by adding saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography using solvent system B to give title compound 1b (1.7 g), yield: 84%. MS m / z (ESI): 680.2 [M+1].

[0113] Step 2

[0114] (R)-4-(2-((3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)amino)propyl)benzene-1,2-diphenol 1c

[0115] Compound 1b (1.4 g, 2.0 mmol) was dissolved in methanol (10 mL) under argon protection. Palladium hydroxide on carbon (0.2 g) was added, and hydrogen was added by a hydrogen balloon and stirred for 3 hours. After stirring was stopped, the mixture was filtered, and the filtrate was concentrated under reduced pressure to give the title compound 1c (0.9 g), yield: 94%.

[0116] Step 3

[0117] (1S,3R)-1-(5-bromopyridin-2-yl)-2-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-3-methyl-1,2,3,4-tetrahydroisoquinoline-6,7-diol1d

[0118] Compound 1c (0.8 g, 1.6 mmol) was dissolved in toluene (10 mL), and acetic acid (0.2 g, 3.2 mmol) and 5-bromopyridaldehyde (0.6 g, 3.2 mmol) were added. The reaction mixture was stirred in an oil bath at 80 °C for 16 hours, and then the reaction was stopped. The reaction mixture was cooled, concentrated under reduced pressure, and water (10 mL) was added. A saturated sodium bicarbonate solution (20 mL) was slowly added to adjust the pH of the reaction mixture to approximately 8. The mixture was extracted with ethyl acetate (10 mL × 2), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography using solvent system B to give the title compound 1d (256 mg), in 75% yield.

[0119] MS m / z(ESI): 667.1 [M+1].

[0120] Step 4

[0121] (5S,7R)-5-(5-bromopyridin-2-yl)-6-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-7-methyl-5,6,7,8-tetrahydro-[1,3]dioxapentano[4,5-g]isoquinoline-2,2-d21e

[0122] Compound 1d (350 mg, 0.5 mmol) was dissolved in N,N-dimethylformamide (10 mL), and dibromodideuterium methane (277 mg, 1.6 mmol) and cesium carbonate (512 mg, 1.6 mmol) were added. The reaction was stirred in an oil bath at 70 °C for 16 hours, and then the reaction was stopped. The reaction was cooled, concentrated under reduced pressure, and water (10 mL) was added. The mixture was extracted with ethyl acetate (10 mL × 2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography using solvent system B to give the title compound 1e (170 mg), yield: 48%.

[0123] MS m / z(ESI): 681.1 [M+1].

[0124] Step 5

[0125] 6-((5S,7R)-6-(3-((tert-butyldiphenylsilyl)oxy)-2,2-difluoropropyl)-7-methyl-5,6,7,8-tetrahydro-[1,3]dioxapentanecyclo[4,5-g]isoquinoline-5-yl-2,2-d2)-N-(1-(3-fluoropropyl)azacyclobut-3-yl)pyridine-3-amine 1f

[0126] Compound 1e (170 mg, 0.25 mmol) was dissolved in dioxane (10 mL), and compound 1-(3-fluoropropyl)azacyclobut-3-amine (prepared by the method disclosed in Example 1 on page 50 of patent application "WO2019228443") (44 mg, 0.3 mmol), 2,2'-bis-(diphenylphosphino)-1,1'-binaphthyl (12 mg, 0.02 mmol), tris(dibenzylideneacetone)dipalladium (20 mg, 0.02 mmol), and sodium tert-butoxide (29 mg, 0.3 mmol) were added under argon protection. The reaction was carried out in an oil bath at 80 °C with stirring for 16 hours, and then the reaction was stopped. The reaction mixture was cooled, concentrated, and saturated sodium bicarbonate solution (10 mL) was added. The mixture was extracted with ethyl acetate (10 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography with solvent system B to give the title compound 1f (47 mg) in 51% yield.

[0127] Step 6

[0128] 2,2-Difluoro-3-((5S,7R)-5-(5-((1-(3-fluoropropyl)azacyclobut-3-yl)amino)pyridin-2-yl)-7-methyl-7,8-dihydro-[1,3]dioxapentanol[4,5-g]isoquinoline-6(5H)-yl-2,2-d2)prop-1-ol

[0129] Compound 1f (103 mg, 0.14 mmol) was dissolved in dichloromethane (5 mL), and a 1 M tetrabutylammonium fluoride tetrahydrofuran solution (1 mL) was added dropwise under ice bath. After the addition was complete, the mixture was stirred at room temperature for 1.5 hours, concentrated under reduced pressure, and extracted with ethyl acetate (5 mL × 3) by adding saturated sodium bicarbonate solution. The organic phases were combined, washed with saturated sodium chloride solution (5 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography using solvent system B to give title compound 1 (11 mg), yield 42%.

[0130] MS m / z(ESI):495.2[M+1].

[0131] 1 H NMR(400MHz,CD3OD)7.80(d,1H),7.04(d,1H),6.94(dd,1H),6.61(s,1H),6. 19(s,1H),4.64(br,2H),4.54-4.52(m,1H),4.45-4.42(m,1H),4.15-4.10(m ,1H),3.85-3.83(m,2H),3.76-3.68(m,1H),3.64-3.56(m,1H),3.14-2.97(m ,4H),2.78-2.68(m,3H),2.59-2.52(m,1H),1.84-1.74(m,2H),1.05(d,3H).

[0132] X-ray powder diffraction analysis showed that the product was amorphous, and the XRPD spectrum is shown below. Figure 1 .

[0133] Biological evaluation

[0134] The present disclosure is further described and explained below with reference to test examples, but these embodiments are not intended to limit the scope of the present disclosure.

[0135] Test Example 1: Inhibition of the binding of the disclosed compound to E and ER.

[0136] The disclosed compound inhibits the binding of estrogen (E) to estrogen receptor (ER), thereby blocking the binding of the E-ER complex to the estrogen response element (ERE), and subsequently blocking the expression of downstream luciferase protein. The inhibitory effect of the disclosed compound on the binding of E to ER was tested by the following method.

[0137] 1. Experimental Objective

[0138] The purpose of this experiment was to test the inhibitory effect of the compound on the binding of E to ER, based on IC50. 50 Size is used to evaluate the in vitro activity of compounds.

[0139] 2. Experimental Methods

[0140] ERE was cloned upstream of the luciferase gene, and MCF-7 / ERE-luciferase monoclonal cells were selected by transfection with MCF-7 cells (TCHu74, Chinese Academy of Sciences Type Culture Collection). MCF-7 / ERE-luciferase cells were seeded in 96-well plates with MEM (hyclone, SH30024.01B) medium containing 10% charcoal-stripped FBS (Moregate, FBSF), 1% sodium pyruvate (Sigma, S8636), 1% non-essential amino acids (Sigma, M7145), and 500 μg / ml G418 at a seeding density of 30,000 cells / well. Cells were cultured at 37°C under 5% CO2. The drug was prepared as a 20 mM stock solution, serially diluted 10-fold with 100% DMSO, and then diluted 20-fold with culture medium. After culturing cells for 24 hours, remove the culture medium and add 0.1 nM estradiol (Sigma, E2758) and 10 μL of the drug diluted in the culture medium to each well. For the control group, add DMSO. Gently shake to mix. Incubate at 37°C in a 5% CO2 incubator. After 24 hours, discard the cell culture medium and add 50 μL / well of the prepared luciferase substrate (Promega, E6110). Incubate at room temperature in the dark for 10-15 minutes and measure the chemiluminescence signal value.

[0141] 3. Test Results

[0142] The inhibitory effect of the compound in this disclosure on the binding of E and ER was determined through the above experiments. The chemiluminescence signal value was analyzed against the logarithmic concentration of the compound using a Graphpad Prism analyzer to obtain the compound's IC50 value. 50 The values ​​are shown in Table 1.

[0143] Table 1. Inhibitory effect of the compounds in this disclosure on the binding of E to ER

[0144] Compound numbering <![CDATA[IC 50 (nM)]]> 1 0.69

[0145] Conclusion: The compound to be protected in this disclosure has a significant inhibitory effect on the binding of E and ER.

[0146] Test Example 2: Inhibitory effect of the disclosed compound on the proliferation of MCF-7 cells

[0147] 1. Experimental Objective

[0148] The purpose of this experiment was to test the inhibitory effect of the disclosed compound on the proliferation activity of MCF-7 cells using the ATP method, based on the IC50 assay. 50 Size is used to evaluate the in vitro activity of compounds.

[0149] 2. Experimental Methods

[0150] MCF-7 cells (TCHu74, Chinese Academy of Sciences Type Culture Collection) were seeded in 96-well plates in MEM (hyclone, SH30024.01B) medium containing 10% FBS (Gibco, 10099-141), 1% sodium pyruvate (Sigma, S8636), and 1% non-essential amino acids (Sigma, M7145). The seeding density was 4,000 cells / well, and the cells were cultured at 37°C and 5% CO2. The compound was prepared as a 20 mM stock solution, serially diluted to a final concentration of 1000X with 100% DMSO, and then diluted 20-fold with medium containing 2% FBS. After 24 hours of culture, the culture medium was removed, and 90 μL of culture medium containing 2% FBS and 10 μL of drug were added to each well. The control group was added with 10 μL of DMSO. The mixture was gently shaken and mixed, while the blank group contained only 100 μL of 2% FBS culture medium. The mixture was placed in an incubator at 37°C and 5% CO2. After 72 hours, 50 μL of the mixed Cell Titer-Glo (Promega, G7571) was added to each well, shaken and mixed, and incubated at room temperature for 10 minutes. The chemiluminescence signal value was then measured.

[0151] 3. Data Analysis

[0152] The chemiluminescence signal value was plotted against the logarithmic concentration of the compound using Graphpad Prism to obtain the IC50 of the compound. 50 The values ​​are shown in Table 2.

[0153] Table 2. Inhibitory effect of the disclosed compounds on MCF-7 cell proliferation

[0154] Compound numbering <![CDATA[IC 50 (nM)]]> 1 0.29

[0155] Test Example 3: Experimental biological evaluation of the inhibitory effect of the disclosed compound on the proliferation of ERα mutant MCF7 cells.

[0156] 1. Experimental Objective

[0157] The purpose of this experiment is to determine the inhibitory activity of the disclosed compound on the proliferation of ERα mutant MCF7 cells.

[0158] 2. Experimental Methods

[0159] Site-directed mutagenesis and cell line construction

[0160] The mutant ERα (ERα) protein, Y537S, was obtained by site-directed mutagenesis using two-primer PCR with wild-type ESR1 gene cDNA (Accession No. NM000125) as a template. The primer sequences used for mutation are as follows (underlined nucleotides indicate the mutation sites): Y537S: F-AAG AAC GTGGTG CCC CTC TCT GAC CTG CTG CTG GAG ATG (SEQ ID NO:1); R-CAT CTC CAG CAG CAGGTC AGA GAG GGG CAC CAC GTT CTT (SEQ ID NO:2). The mutant ESR1 cDNA was cloned into the target lentiviral vector pCDH-CMV-MCS-EF1-Puro. The lentiviral plasmid carrying the mutant ESR1 gene sequence and the lentiviral packaging plasmid were then transfected into HEK-293T cells (ATCC, CRL-3216) using Lipofectamine 3000 Transfection Reagent (ThermoFisherScientific, Cat#L3000075). Forty-eight hours post-transfection, the virus-containing culture supernatant was filtered, ultracentrifuged to obtain the viral pellet, resuspended in an appropriate amount of culture medium, and added to MCF7 cells (ATCC, HTB-22). Polybrene was added to a final concentration of 8 μg / ml and incubated overnight. Two days after transfection, 1 μg / ml puromycin was added to the cell culture medium for resistance selection. Approximately two weeks later, an MCF7 cell line stably expressing the ERαY537S mutant was obtained.

[0161] Cell proliferation inhibition experiment

[0162] MCF7 cells expressing the ERα mutant were cultured in MEM (GE Healthcare, SH30024.01) complete medium containing 10% fetal bovine serum. On day 1, cells were seeded at a density of 3,000 cells / well in 96-well plates using complete medium, with 100 μL of cell suspension per well, and incubated overnight at 37°C with 5% CO2. On day 2, the medium was aspirated, and each well was replaced with 135 μL of MEM incomplete medium containing 2% fetal bovine serum. Simultaneously, 15 μL of different concentrations of the test compound prepared in incomplete medium was added to each well. The final concentrations of the compound were determined by a 4-fold serial dilution starting from 100 nM, with a blank control containing 0.5% DMSO. The plates were incubated at 37°C with 5% CO2 for 144 hours. On day 8, the 96-well cell culture plates were removed, and 150 μL of CellTiter- ... The Luminescent CellViability Assay (Promega, G7573) was incubated at room temperature for 10 minutes. The luminescence signal values ​​were then read using a multi-label microplate reader (PerkinElmer, VICTOR 3). The IC50 of the compound's inhibitory activity was calculated using Graphpad Prism software based on the compound's concentration and luminescence signal value. 50 The values ​​are shown in Table 3.

[0163] 3. Test Results

[0164] Table 3. IC50 values ​​of the inhibitory effect of the disclosed compounds on the proliferation of ERα mutant MCF7 cells. 50 value

[0165] Compound numbering <![CDATA[IC 50 (nM)]]> 1 1.36

[0166] Conclusion: The compound to be protected in this disclosure has a significant inhibitory effect on the proliferation of MCF7 cells expressing the ERα mutant.

[0167] Test Example 4: Degradation effect of the disclosed compound on ERα

[0168] 1. Experimental Objective

[0169] The method for testing the degradation effect of the disclosed compounds on ERα is used to determine the degradation effect of the disclosed compounds on ERα.

[0170] 2. Experimental Methods

[0171] ERα-positive breast cancer cell line MCF-7 was cultured in DMEM / F12 medium (HyClone, SH30023.01) containing 10% fetal bovine serum (Corning, 35-010-CV). On the first day of the experiment, after cell digestion, the cells were washed once with phenol red-free DMEM / F12 medium (ThermoFisher, 11039-021) containing 5% activated charcoal-treated fetal bovine serum (BIOSUN, BS-0004-500), resuspended, and counted. The cell density was adjusted to 1.79 × 10⁶ cells / year. 5Cells / mL. 280 μL of cell suspension was added to each well of a 48-well plate (Corning, 3548), and the cells were incubated overnight at 37°C in a 5% CO2 incubator. On the second day, the compound was serially diluted with DMSO and further diluted with phenol red-free DMEM / F12 medium containing 5% activated charcoal-treated fetal bovine serum. 20 μL of the diluted compound was added to each well of the 48-well plate, resulting in final concentrations of 3000, 300, 30, 3, 0.3, 0.03, and 0.003 nM. The 48-well plates were incubated for 16 to 18 hours. 96-well plates were coated with the capture antibody from the Human ERα / NR3A1 Total Protein Assay Kit (R&D, DYC5715-5). The capture antibody was prepared at 1 μg / mL with PBS, and 100 μL was added to each well of a 96-well plate (Corning, 3590), which was then incubated overnight at 26°C. On the third day of the experiment, the antibody-coated 96-well plates were washed once with PBS, and 200 μL of PBS containing 1% BSA was added to each well. The plates were then incubated at 37°C for 1.5 hours for blocking. The cell culture supernatant was discarded, and the cells were washed once with PBS. 60 μL of cell lysis buffer was added to each well. The cell lysis buffer consisted of PBS containing 6M urea, 1mM EDTA, 0.5% Triton X-100, 1mM PMSF, and a protease inhibitor (Roche, 04693159001). The cells were lysed on ice for 15 minutes, and then 300 μL of PBS containing 1mM EDTA and 0.5% Triton X-100 was added to each well to dilute the urea to 1M. The blocking buffer in the 96-well plates was discarded, and 100 μL of the diluted cell lysis buffer was added to each well. The plates were incubated at 37°C for 2 hours, and then washed five times with PBS. Dilute the biotinylated detection antibody to 0.4 μg / mL with PBS containing 1% BSA, then add 100 μL of the antibody to each well and incubate at 37°C for 1 hour. Wash the plate five times, then add 100 μL of avidin-HRP diluted 200-fold with PBS containing 1% BSA to each well and incubate at 37°C for 30 minutes. Wash the plate five times again, then add 100 μL of TMB substrate to each well and incubate at room temperature until a blue color appears. Add 100 μL of stop solution to each well. Read the OD450 signal using a Pherastar multi-plate reader. Calculate the IC50 of the inhibitory activity of the compound using Graphpad Prism software. 50 Value. The maximum degradation rate of the compound is the ratio of the residual ERα level in cells after treatment with the 3000 nM compound to the residual ERα level in cells after treatment with 3000 nM fulvestrant.

[0172] 3. Test Results

[0173] EC measured by the degradation of ERα by the disclosed compound 50 The values ​​are shown in Table 4.

[0174] Table 4. Degradation effect of the compounds disclosed herein on ERα

[0175] Example number <![CDATA[EC 50 (nM)]]> Emax degradation (%) Fulvestrant 0.06 100 1 0.38 107

[0176] Conclusion: The compound to be protected in this disclosure has a significant degradation effect on ERα.

[0177] Test Example 5: Inhibitory effect of the disclosed compound on the enzyme activity of the midazolam metabolite site in human liver microsomes.

[0178] The enzyme activity of the disclosed compound at the midazolam metabolite site of human liver microsomes was determined using the following experimental method:

[0179] I. Experimental Materials and Instruments

[0180] 1. Phosphate buffer (20×PBS, purchased from Sangon Biotech).

[0181] 2. NADPH (ACROS, A2646-71-1),

[0182] 3. Human liver microsomes (Corning Gentest, Cat No. 452161, Lot No. 9050002, Donor, 36)

[0183] 4. ABI QTrap 4000 LC-MS / MS (AB Sciex)

[0184] 5. ZORBAX Extend-C18, 3×50mm, 3.5μm (Agilent Technologies, USA)

[0185] 6. CYP probe substrate (midazolam, TRC, M343000 / 3μM) and positive control inhibitor (ketoconazole, SIGMA, Cat No. K1003-100MG).

[0186] II. Experimental Procedure

[0187] Prepare 100 mM PBS buffer. Use this buffer to prepare 0.25 mg / mL microsomal solutions, 7.5 mM MgCl2, and 5 mM NADPH solutions. Dilute the 30 mM stock solution with DMSO to obtain a series of solutions I with concentrations of 30 mM, 10 mM, 3 mM, 1 mM, 0.3 mM, 0.03 mM, 0.003 mM, and 0 mM. Then, dilute the above series of solutions I 200 times with phosphate-buffered saline (PBS) to obtain a series of test solutions II (150, 50, 15, 5, 1.5, 0.15, 0.015, and 0 μM). Dilute with PBS to a concentration of 15 μM for midazolam working solution.

[0188] Take 40 μL of 0.25 mg / mL microparticle solution prepared in 7.5 mM MgCl2, and 20 μL each of 15 μM midazolam working solution and compound working solutions (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM), and mix thoroughly. The positive control group is replaced with ketoconazole at the same concentration. Simultaneously, pre-incubate with 5 mM NADPH solution at 37 °C for 5 minutes. After 5 minutes, add 20 μL of NADPH to each well, start the reaction, and incubate for 30 minutes. All incubated samples are duplicated. After 30 minutes, add 250 μL of acetonitrile containing the internal standard to all samples, mix well, shake at 800 rpm for 10 minutes, and then centrifuge at 3700 rpm for 10 minutes. Take 100 μL of the supernatant, mix with 80 μL of ultrapure water, and transfer to LC-MS / MS for analysis.

[0189] The IC50 value for the drug at the CYP3A4 midazolam metabolite site was calculated using Graphpad Prism. 50 The values ​​are shown in Table 5.

[0190] Table 5. IC50 values ​​of the compounds disclosed herein at the midazolam metabolite site in human liver microsomes CYP3A4. 50 value

[0191] Compound numbering <![CDATA[IC 50 (μM)]]> 1 15.38

[0192] Conclusion: The compound to be protected in this disclosure has a weak inhibitory effect on the midazolam metabolic site of human liver microsomes CYP3A4, exhibiting better safety, suggesting that no metabolic drug interaction based on the midazolam metabolic site of CYP3A4 will occur.

[0193] Test Example 6: Inhibitory effect of the disclosed compound on the enzyme activity of the CYP3A4 testosterone metabolic site in human liver microsomes.

[0194] The enzyme activity of the disclosed compound at the human liver microsomal CYP3A4 testosterone metabolism site was determined using the following experimental method:

[0195] I. Experimental Materials and Instruments

[0196] 1. Phosphate buffer (20×PBS, purchased from Sangon Biotech).

[0197] 2. NADPH (ACROS, A2646-71-1),

[0198] 3. Human liver microsomes (Corning Gentest, Cat No. 452161, Lot No. 905002, Donor 36)

[0199] 4. ABI QTrap 4000 LC-MS / MS (AB Sciex)

[0200] 5. ZORBAX Extend-C18, 3×50mm, 3.5μm (Agilent Technologies, USA)

[0201] 6. CYP probe substrate (testosterone, Wokai, CAS No. [58-22-0] / 75μM), and positive control inhibitor (ketoconazole, SIGMA, Cat No. K1003-100MG).

[0202] II. Experimental Procedure

[0203] Prepare 100 mM PBS buffer. Using this buffer, prepare 0.25 mg / ml microsomal solutions, 7.5 mM MgCl2, and 5 mM NADPH solutions. Dilute the 30 mM stock solution with DMSO to obtain a series of solutions I with concentrations of 30 mM, 10 mM, 3 mM, 1 mM, 0.3 mM, 0.03 mM, 0.003 mM, and 0 mM. Then, dilute these solutions I 200-fold with phosphate-buffered saline (PBS) to obtain a series of test solutions II (150, 50, 15, 5, 1.5, 0.15, 0.015, and 0 μM). Dilute with PBS to a testosterone working solution concentration of 375 μM.

[0204] Take 40 μL of a 0.25 mg / mL microsome solution prepared in 7.5 mM MgCl2, and then take 20 μL each of 375 μM testosterone working solution and compound working solutions (150, 50, 15, 5, 1.5, 0.15, 0.015, 0 μM), and mix thoroughly. The positive control group is replaced with ketoconazole at the same concentration. Simultaneously, pre-incubate the mixture with 5 mM NADPH solution at 37 °C for 5 minutes. After 5 minutes, add 20 μL of NADPH to each well, start the reaction, and incubate for 30 minutes. After 30 minutes, add 250 μL of acetonitrile containing the internal standard to all samples, mix well, shake at 800 rpm for 10 minutes, and then centrifuge at 3700 rpm for 10 minutes. Take 100 μL of the supernatant, mix with 80 μL of ultrapure water, and transfer to LC-MS / MS for analysis.

[0205] The IC50 value for the drug at the CYP3A4 testosterone metabolite site was calculated using Graphpad Prism. 50 The values ​​are shown in Table 6.

[0206] Table 6. IC50 of the compounds disclosed herein on the testosterone metabolism site of human liver microsomal CYP3A4 50 value

[0207] Compound numbering <![CDATA[IC 50 (μM)]]> 1 >30

[0208] Conclusion: The compound to be protected in this disclosure exhibits weaker inhibition of the testosterone metabolism site of human liver microsome CYP3A4, demonstrating better safety.

[0209] Test Example 7: Time-dependent inhibition of the enzyme activity of the disclosed compound on the midazolam metabolite site of human liver microsomes.

[0210] The time-dependent inhibitory effect of this compound on the midazolam metabolite site in human liver microsomes was determined using the following experimental method:

[0211] I. Experimental Materials and Instruments

[0212] 1. Phosphate buffer (20×PBS, purchased from Sangon Biotech).

[0213] 2. NADPH (ACROS, A2646-71-1),

[0214] 3. Human liver microsomes (Corning Gentest, Cat No. 452161, Lot No. 9050002, Donor, 36)

[0215] 4. ABI QTrap 4000 LC-MS / MS (AB Sciex)

[0216] 5. ZORBAX Extend-C18, 3×50mm, 3.5μm (Agilent Technologies, USA)

[0217] 6. CYP probe substrate (midazolam, TRC, M343000 / 3μM) and positive control inhibitor (verapamil, Adamas Reagent Co., Ltd., Cat No. 25904A).

[0218] II. Experimental Procedure

[0219] Prepare 100 mM PBS buffer. Use this buffer to prepare 0.25 mg / ml microsomal solutions, 7.5 mM MgCl2, and 5 mM NADPH solutions. Dilute the 30 mM stock solution with DMSO to obtain a series of solutions I with concentrations of 30 mM, 10 mM, 3 mM, 1 mM, 0.3 mM, 0.1 mM, 0.03 mM, and 0 mM. Then, dilute the above series of solutions I 200 times with phosphate-buffered saline (PBS) to obtain a series of test solutions II (150, 50, 15, 5, 1.5, 0.5, 0.15, and 0 μM). Dilute with PBS to a concentration of 15 μM for midazolam working solution.

[0220] Shake the prepared test series solutions well and dispense 20 μL into the corresponding reaction plates (+NADPH, T0, and -NADPH groups), with three replicates. Add 40 μL of liver microsome working solution to each 96-well plate, 20 μL of the corresponding substrate solution to the T0 plate, and 20 μL of NADPH to both the T0 and +NADPH groups. Start the incubation period and place the plates in a water bath at 37°C for 30 min. After incubation, remove the T0 plate and terminate the reaction with 250 μL of a solution containing the internal standard ACN. Add 20 μL of the corresponding substrate solution to the +NADPH group and 20 μL of the corresponding substrate solution and 20 μL of NADPH to the -NADPH group. Start the incubation period and place the plates in a water bath at 37°C for 30 min. After incubation, remove the plates and terminate the reaction with 250 μL of a solution containing the internal standard ACN. Then shake at 800 rpm for 10 min and centrifuge at 4000 rpm for 15 min. Take 100 μL of the supernatant, mix it with 80 μL of ultrapure water, and then transfer it to LC-MS / MS for analysis.

[0221] The IC50 value for the drug at the CYP3A4 midazolam metabolite site was calculated using Graphpad Prism. 50 The values ​​are shown in Table 7.

[0222] Table 7. IC50 values ​​of the compounds disclosed herein at the midazolam metabolite site in human liver microsomes CYP3A4.50 Value and IC 50 Shift multiple

[0223]

[0224] Conclusion: The compound to be protected in this disclosure has a weak inhibitory effect on the midazolam metabolic site of human liver microsomes CYP3A4, exhibiting better safety, suggesting that no metabolic drug interaction based on the midazolam metabolic site of CYP3A4 will occur.

[0225] Test Example 8: Time-dependent inhibition of the enzyme activity of the disclosed compound on the human liver microsomal CYP3A4 testosterone metabolic site.

[0226] The time-dependent inhibitory effect of this compound on the enzyme activity of the human liver microsomal CYP3A4 testosterone metabolism site was determined using the following experimental method:

[0227] I. Experimental Materials and Instruments

[0228] 1. Phosphate buffer (20×PBS, purchased from Sangon Biotech).

[0229] 2. NADPH (ACROS, A2646-71-1),

[0230] 3. Human liver microsomes (Corning Gentest, Cat No. 452161, Lot No. 905002, Donor 36)

[0231] 4. ABI QTrap 4000 LC-MS / MS (AB Sciex)

[0232] 5. ZORBAX Extend-C18, 3×50mm, 3.5μm (Agilent Technologies, USA)

[0233] 6. CYP probe substrate (testosterone, Wokai, CAS No. [58-22-0] / 75μM), and positive control inhibitor (verapamil, Adamas Reagent Co Ltd, Cat No. 25904A).

[0234] II. Experimental Procedure

[0235] Prepare 100 mM PBS buffer. Use this buffer to prepare 0.25 mg / mL microsomal solutions, 7.5 mM MgCl2, and 5 mM NADPH solutions. Dilute the 30 mM stock solution with DMSO to obtain a series of solutions I with concentrations of 30 mM, 10 mM, 3 mM, 1 mM, 0.3 mM, 0.1 mM, 0.03 mM, and 0 mM. Then, dilute the above series of solutions I 200 times with phosphate-buffered saline (PBS) to obtain a series of test solutions II (150, 50, 15, 5, 1.5, 0.5, 0.15, and 0 μM). Dilute with PBS to a concentration of 15 μM for midazolam working solution.

[0236] Shake the prepared test series solutions well and dispense 20 μL into the corresponding reaction plates (+NADPH, T0, and -NADPH groups), with three replicates. Add 40 μL of liver microsome working solution to each 96-well plate, 20 μL of the corresponding substrate solution to the T0 plate, and 20 μL of NADPH to both the T0 and +NADPH groups. Start the incubation period and place the plates in a water bath at 37°C for 30 min. After incubation, remove the T0 plate and terminate the reaction with 250 μL of a solution containing the internal standard ACN. Add 20 μL of the corresponding substrate solution to the +NADPH group, and add 20 μL of the corresponding substrate solution and 20 μL of NADPH to the -NADPH group. Start the incubation period and place the plates in a water bath at 37°C for 30 min. After incubation, remove the plates and terminate the reaction with 250 μL of a solution containing the internal standard ACN. Then shake at 800 rpm for 10 min and centrifuge at 4000 rpm for 15 min. Take 100 μL of the supernatant, mix it with 80 μL of ultrapure water, and then transfer it to LC-MS / MS for analysis.

[0237] The IC50 value for the drug at the CYP3A4 testosterone metabolite site was calculated using Graphpad Prism. 50 The values ​​are shown in Table 8.

[0238] Table 8. IC50 of the compounds disclosed herein on the testosterone metabolism site of human liver microsomal CYP3A4 50 Value and IC 50 shift multiplier

[0239]

[0240] Conclusion: The compound to be protected in this disclosure exhibits weaker inhibition of the testosterone metabolism site of human liver microsomes CYP3A4, demonstrating better safety and suggesting that no CYP3A4-based metabolic drug interactions will occur.

[0241] Test Example 9

[0242] 1. Experimental Objective

[0243] The blocking effect of the disclosed compound on hERG potassium current was tested using a fully automated patch clamp on stable cell lines transfected with hERG potassium channels.

[0244] 2. Experimental Methods

[0245] 2.1 Experimental Materials and Instruments

[0246] 2.1.1 Experimental Materials:

[0247]

[0248] 2.1.2 Experimental Apparatus:

[0249]

[0250]

[0251] 2.2 Fully Automated Patch Clamp Experiment Procedure

[0252] HEK293-hERG stable cell lines were passaged at a density of 1:4 in MEM / EBSS medium (10% FBS, 400 μg / ml G418, 1% MEM non-essential amino acid solution (100×), 1% sodium pyruvate solution). Automated patch-clamp experiments were performed within 48-72 hours of culture. On the day of the experiment, cells were digested with 0.25% trypsin, centrifuged to collect cells, and resuspended in extracellular fluid (140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM D glucose monohydrate, 10 mM Hepes, pH 7.4, 298 mOsm) to prepare a cell suspension. The cell suspension was placed on the cell bank of the Patchliner instrument. The Patchliner instrument used a negative pressure controller to add cells onto the chip (NPC-16), and the negative pressure attracted individual cells to the wells of the chip. Once the whole-cell mode is established, the instrument will generate the hERG current according to the set hERG current-voltage program, and then automatically perfuse the compound from low to high concentration. Data collection and analysis are performed using HEAK Patchmaster, HEAK EPC10 patch-clamp amplifier (Nanion), Pathliner software, and Pathcontrol HT software to analyze the current at each compound concentration and the blank control current.

[0253] 2.3 Test Results

[0254] The blocking effect of this disclosed compound on the hERG potassium current was determined through the above experiments, and the measured IC 50 The values ​​are shown in Table 9.

[0255] Table 9. IC50 of the blocking effect of the compounds of this disclosure on hERG potassium current 50

[0256] Compound numbering <![CDATA[IC 50 (μM)]]> 1 >30

[0257] Note: IC 50 ≥30μM indicates no inhibitory activity; 30>IC 50 ≥10μM indicates weak inhibitory activity; 10>IC 50 ≥1 μM indicates moderate inhibitory activity; IC50 50 <1μM indicates strong inhibitory activity.

[0258] Conclusion: The compound to be protected in this disclosure has no inhibitory activity against hERG and exhibits better safety.

[0259] Example 2: Preparation of phosphate I crystal form of the compound shown in formula (I)

[0260] Approximately 450 mg of the compound shown in formula (I) was dissolved in 10 mL of acetone, and 1.25 mL of 1.46 M phosphate ethanol solution was added. After heating and cooling for 24 h, the sample was centrifuged and dried under vacuum at 40 °C to obtain a solid. X-ray powder diffraction analysis determined the solid to be crystal form I. The XRPD spectrum is shown below. Figure 2 As shown, the positions of its characteristic peaks are shown in Table 10, and the TGA spectrum (attached) Figure 3 The results show that crystal form I loses 0.38% of its weight between 30-115℃; DSC spectrum (attached) Figure 4 The XRPD spectrum after DVS shows that crystal form I has an endothermic peak at 188.98℃; (see attached image). Figure 5 The crystal form remained unchanged; the DVS spectrum is attached. Figure 6 The single crystal spectrum is attached. Figure 7 The molar ratio of free base to phosphate is 1:3; axial length Axial angle α = 89.575(2)°; β = 89.859(2)°; γ = 60.745(2)°; crystal volume

[0261] Table 10. Characteristic peaks of crystal form I

[0262]

[0263] Example 3: Preparation of phosphate I crystal form of the compound shown in formula (I)

[0264] About 15 mg of the compound shown in formula (I) was dissolved in 0.2 mL of ethyl acetate, and 41 μL of 1.46 M ethanolic phosphoric acid solution was added. After heating and cooling for 24 h, the mixture was centrifuged and dried to obtain a solid, which was identified as crystal form I by X-ray powder diffraction.

[0265] Example 4: Preparation of the A crystal form of the sulfate of the compound shown in formula (I)

[0266] Approximately 15 mg of the compound shown in formula (I) was dissolved in 0.2 mL of ethanol, and 16.3 μL of 1.84 M sulfuric acid ethanol solution was added. After heating and cooling for 24 h, the mixture was centrifuged and dried to obtain a solid. X-ray powder diffraction analysis identified the product as crystal form A. The XRPD spectrum is shown below. Figure 8 The positions of its characteristic peaks are shown in Table 11, and the TGA spectrum is attached. Figure 9 The results show that crystal form A loses 6.84% of its weight between 30-90℃; DSC spectrum (attached) Figure 10 The DVS spectrum shows that crystal form A has two endothermic peaks, with peak values ​​of 68.13℃ and 146.66℃; the sample deliquesces after DVS; the DVS spectrum is shown below. Figure 11 The single crystal spectrum is attached. Figure 12 The molar ratio of free base to sulfate is 1:1; axial length Axial angle β = 90°; Crystal volume

[0267] Table 11. Characteristic peaks of crystal form A

[0268]

[0269]

[0270] Example 5: Preparation of the A crystal form of the sulfate of the compound shown in formula (I)

[0271] Approximately 15 mg of the compound shown in formula (I) was dissolved in 0.2 mL of ethyl acetate, and 16.3 μL of 1.84 M sulfuric acid ethanol solution was added. After heating and cooling for 24 h, the solid was centrifuged and dried. X-ray powder diffraction analysis showed that it was crystal form A.

[0272] Example 6: Preparation of the A crystal form of the sulfate of the compound shown in formula (I)

[0273] Approximately 148 mg of the compound shown in formula (I) was dissolved in 1 mL of acetone, and 0.163 mL of 1.84 M sulfuric acid ethanol solution was added. After heating and cooling for 40 h, the solid was centrifuged and dried. X-ray powder diffraction analysis showed that it was crystal form A.

[0274] Example 7: Preparation of the A crystal form of the sulfate of the compound shown in formula (I)

[0275] Approximately 296 mg of the compound shown in formula (I) was dissolved in 1 mL of methanol, and 0.33 mL of 1.84 M sulfuric acid methanol solution was added. After heating and cooling for 24 h, the solid was centrifuged and dried. X-ray powder diffraction analysis showed that it was crystal form A.

[0276] Example 8: Preparation of the amorphous form of L-tartrate of the compound shown in formula (I)

[0277] 25 mg of the compound shown in formula (I) was added to 0.5 mL of isopropanol and stirred until dissolved. 8 mg of L-tartaric acid was diluted with 0.5 mL of isopropanol and added dropwise to the above reaction solution. The mixture was stirred under nitrogen protection at room temperature to form a white turbid liquid. After stirring for 16 hours, the mixture was filtered, the filter cake was collected, and vacuum dried to obtain a white solid. X-ray powder diffraction analysis showed that the product was amorphous. The XRPD spectrum is shown below. Figure 13 .

[0278] Example 9: Preparation of amorphous citrate

[0279] 25 mg of the compound shown in formula (I) was added to 0.5 mL of isopropanol and stirred until dissolved. 10 mg of citric acid was diluted with 0.5 mL of isopropanol and added dropwise to the above reaction solution. The mixture was stirred under nitrogen protection at room temperature to form a white turbid liquid. After stirring for 16 hours, the mixture was filtered, the filter cake was collected, and vacuum dried to obtain a white solid. X-ray powder diffraction analysis showed that the product was amorphous. The XRPD spectrum is shown below. Figure 14 .

[0280] Example 10: Preparation of hydrochloride

[0281] Weigh about 15 mg of the compound shown in formula (I) and dissolve it in 0.2 mL of ethanol. Add 50 μL of 1.2 M hydrochloric acid ethanol solution, heat and cool for 24 h while stirring, and evaporate at room temperature to obtain an oily gel-like solid.

[0282] Example 11: Preparation of hydrobromide

[0283] Weigh about 15 mg of the compound shown in formula (I) and dissolve it in 0.2 mL of ethyl acetate. Add 80 μL of 0.75 M hydrobromic acid ethanol solution, heat and stir for 24 h, and evaporate at room temperature to obtain an oily gel-like solid.

[0284] Example 12: Preparation of methanesulfonate

[0285] Weigh about 15 mg of the compound shown in formula (I) and dissolve it in 0.2 mL of ethyl acetate. Add 39 μL of 1.53 M aqueous methanesulfonic acid solution, heat and stir for 24 h, and evaporate at room temperature to obtain an oily gel-like solid.

[0286] Example 13: Preparation of Formate

[0287] Weigh about 15 mg of the compound shown in formula (I) and dissolve it in 0.2 mL of acetone. Add 22.6 μL of 2.65 M formic acid aqueous solution, heat and cool for 24 h while stirring, and evaporate at room temperature to obtain an oily gel-like solid.

[0288] Example 14: Preparation of acetate

[0289] Weigh about 15 mg of the compound shown in formula (I) and dissolve it in 0.2 mL of ethanol. Add 34.2 μL of 1.75 M aqueous acetic acid solution, heat and cool for 24 h while stirring, and allow it to evaporate at room temperature to obtain an oily gel-like solid.

[0290] Example 15: Preparation of Succinate

[0291] Weigh about 15 mg of the compound shown in formula (I) and dissolve it in 0.2 mL of acetone. Add 36 μL of 0.83 M succinic acid ethanol solution, heat and cool for 24 h while stirring, and evaporate at room temperature to obtain an oily gel-like solid.

[0292] Example 16: Preparation of maleate

[0293] Weigh about 15 mg of the compound shown in formula (I) and dissolve it in 0.2 mL of ethyl acetate. Add 30 μL of 1 M maleic acid aqueous solution, heat and cool for 24 h while stirring, and evaporate at room temperature to obtain an oily gel-like solid.

[0294] Example 17: Preparation of malate

[0295] Weigh about 15 mg of the compound shown in formula (I) and dissolve it in 0.2 mL of ethyl acetate. Add 30 μL of 1 M malic acid aqueous solution, heat and cool for 24 h while stirring, and evaporate at room temperature to obtain an oily gel-like solid.

[0296] Example 18: Preparation of p-Toluenesulfonate

[0297] Weigh approximately 15 mg of the compound shown in formula (I) and dissolve it in 0.2 mL of ethanol. Add 10.28 mg of p-toluenesulfonic acid.

[0298] Add 60 μL of purified water, heat and cool for 24 h while stirring, and allow to evaporate at room temperature to obtain an oily gel-like solid.

[0299] Example 19, Hygroscopicity Study

[0300] Using Surface Measurement Systems intrinsic, at 25°C and humidity starting from 50%, the humidity range was investigated from 0% to 95%, with a step size of 10%. The judgment criterion was that the mass change dM / dT for each gradient was ≤0.002%, TMAX 360 min, and two cycles were performed.

[0301] Table 12. Hygroscopicity Study Data

[0302]

[0303] Example 20: Stability Study of Influencing Factors

[0304] The free amorphous, phosphate I crystal form, and sulfate A crystal form samples were laid out in open positions to investigate their stability under light (4500 Lux), high temperature (40℃, 60℃), and high humidity (RH 75%, RH 92.5%) conditions. The sampling period was 30 days.

[0305] Table 13. Influencing Factors and Results of the 30-Day and 1-Month Trials

[0306]

[0307]

[0308] Conclusion: The influencing factor experiment showed that phosphate crystal form I exhibited good physical and chemical stability after 30 days under the influencing factor conditions. Sulfate crystal form A showed good physical and chemical stability under conditions other than light exposure, but deliquesced under high humidity. Amorphous form deliquesced under high humidity, but was physically stable under other conditions; its chemical stability was slightly worse under high temperature and light exposure.

[0309] Experimental Example 21: Long-term / Accelerated Stability

[0310] The stability of the free amorphous state, phosphate I crystal form, and sulfate A crystal form was investigated under conditions of 25℃ / 60%RH and 40℃ / 75%RH, respectively.

[0311] Table 14. Long-term / Accelerated Stability of Free Base Amorphous Form

[0312]

[0313]

[0314] Table 15. Long-term / accelerated stability of phosphate I crystal form

[0315]

[0316] Table 16. Long-term / accelerated stability of sulfate A crystal form

[0317]

[0318] Conclusion: The stability results after 3 months of long-term accelerated testing showed that the phosphate I crystal form and the sulfate A crystal form had good physicochemical stability, which was slightly better than that of the free amorphous state.

Claims

1. A pharmaceutically acceptable salt of a compound of formula (I), said pharmaceutically acceptable salt being selected from phosphates, sulfates, tartrates, citrates, hydrochlorides, hydrobroms, methanesulfonates, formates, acetates, succinates, maleates, malates, and p-toluenesulfonates. 。 2. A pharmaceutically acceptable salt of the compound of formula (I) according to claim 1, wherein the molar ratio of the compound of formula (I) to the acid molecule is selected from 5:1 to 1:

5.

3. A pharmaceutically acceptable salt of the compound of formula (I) according to claim 2, wherein the molar ratio of the compound of formula (I) to the acid molecule is selected from 1:1 or 1:

3.

4. A pharmaceutically acceptable salt of the compound of formula (I) according to claim 3, wherein the molar ratio of the compound of formula (I) to phosphate molecules is 1:

3.

5. A phosphate of formula (I) according to claim 1, wherein the molar ratio of the compound of formula (I) to phosphate molecules is 1:3, and the diffraction angle is 2... θ The X-ray powder diffraction pattern, expressed as an angle, shows characteristic peaks at 6.1, 13.5, 19.2, and 22.

3. 。 6. The I crystal form of the phosphate of the compound of formula (I) according to claim 5, at a diffraction angle of 2... θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 6.1, 13.5, 13.9, 19.2, 21.8, 22.3, and 23.

4.

7. The I crystal form of the phosphate of the compound of formula (I) according to claim 5, at a diffraction angle of 2... θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 6.1, 12.4, 13.5, 13.9, 15.0, 16.3, 16.9, 19.2, 20.8, 21.8, 22.3, 23.4, 24.1, 24.6, 26.2, 27.5, and 30.

0.

8. A pharmaceutically acceptable salt of the compound of formula (I) according to claim 2, wherein the molar ratio of the compound of formula (I) to sulfuric acid molecules is 1:

1.

9. A crystal form A of the sulfate of the compound of formula (I) according to claim 1, wherein the molar ratio of the compound of formula (I) to sulfuric acid molecules is 1:1, and the diffraction angle is 2... θ The X-ray powder diffraction pattern, expressed as an angle, shows characteristic peaks at 6.6, 7.9, 15.1, 20.7, and 22.1 Å. 。 10. The sulfate form of the compound of formula (I) according to claim 9, at a diffraction angle of 2... θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 6.6, 7.9, 13.4, 14.5, 15.1, 19.3, 19.8, 20.7, 22.1, 24.8, 25.4, 25.7, and 27.

6.

11. The sulfate form of the compound of formula (I) according to claim 9, at a diffraction angle of 2... θ The X-ray powder diffraction pattern, expressed in terms of angle, shows characteristic peaks at 6.6, 7.9, 11.6, 13.4, 14.5, 15.1, 15.9, 18.1, 19.3, 19.8, 20.7, 22.1, 24.0, 24.8, 25.4, 25.7, 27.2, 27.6, 28.3, and 29.

2.

12. The crystal form of a pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 5-7 or 9-11, wherein the error range of the 2θ angle is ±0.

2.

13. A method for preparing a phosphate of the compound of formula (I) according to any one of claims 1-4, or a crystal form I of the phosphate of the compound of formula (I) according to any one of claims 5-7, comprising the step of reacting the compound of formula (I) with phosphoric acid.

14. A method for preparing the I crystal form of a phosphate of the compound of formula (I) according to any one of claims 5-7, comprising the following steps: 1) A solution of the compound shown in formula (I) in solvent A, wherein solvent A is selected from ketone solvents, ester solvents or ether solvents; 2) Prepare a solution of phosphoric acid in solvent B, wherein solvent B is selected from water or alcohol solvents; 3) The compound shown in formula (I) crystallizes out after being mixed with solvent A solution of phosphoric acid in solvent B solution.

15. The method for preparing the I crystal form of the phosphate of the compound of formula (I) according to claim 14, wherein solvent A is selected from acetone, ethyl acetate or methyl tert-butyl ether; and solvent B is selected from methanol, ethanol or water.

16. A method for preparing the sulfate of the compound of formula (I) according to any one of claims 1-3 or 8, or the A crystal form of the sulfate of the compound of formula (I) according to any one of claims 9-11, comprising the step of reacting the compound of formula (I) with sulfuric acid.

17. A method for preparing the A crystal form of the sulfate of the compound of formula (I) according to any one of claims 9-11, comprising the following steps: 1) A solution of the compound shown in formula (I) in solvent A, wherein solvent A is selected from ketone solvents, ester solvents or alcohol solvents; 2) Prepare a solution of sulfuric acid in solvent B, wherein solvent B is selected from water or alcohol solvents; 3) The compound shown in the mixed formula (I) crystallizes out after being mixed with solvent A solution and sulfuric acid solvent B solution.

18. The method for preparing the A crystal form of the sulfate of the compound shown in formula (I) according to claim 17, wherein solvent A is selected from acetone, ethyl acetate, methanol or ethanol; and solvent B is selected from methanol, ethanol or water.

19. A method for preparing a pharmaceutically acceptable salt of the compound of formula (I) according to claim 1, comprising the step of reacting the compound of formula (I) with tartaric acid, citric acid, hydrochloric acid, hydrobromic acid, methanesulfonic acid, formic acid, acetic acid, succinic acid, maleic acid, malic acid or p-toluenesulfonic acid to form a salt.

20. A pharmaceutical composition comprising the following ingredients: 1) A pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 1-4, 8, or a crystal form of a pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 5-7, 9-12, or a mixture thereof; and 2) Any pharmaceutically acceptable carrier or excipient may be selected.

21. A method for preparing a pharmaceutical composition, comprising: 1) A pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 1-4, 8, or a crystal form of a pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 5-7, 9-12, or a mixture thereof; and 2) A step of mixing any pharmaceutically acceptable carrier or excipient.

22. The use of a pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 1-4, 8, or a crystal form of a pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 5-7, 9-12, or a mixture thereof, or the use of the pharmaceutical composition of claim 20 in the preparation of an estrogen receptor modulator.

23. The use according to claim 22, wherein it is used in the preparation of an estrogen receptor downregulator.

24. The use of a pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 1-4, 8, or a crystal form of a pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 5-7, 9-12, or a mixture thereof, or the use of the pharmaceutical composition of claim 20 in the preparation of a medicament for the prevention and / or treatment of cancer.

25. The use according to claim 24, wherein the cancer is selected from breast cancer, endometrial cancer, uterine cancer, cervical cancer, skin cancer, prostate cancer, ovarian cancer, fallopian tube tumors, hemophilia, and leukemia.

26. The use of a pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 1-4, 8, or a crystal form of a pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 5-7, 9-12, or a mixture thereof, or the use of the pharmaceutical composition of claim 20 in the preparation of a medicament for the prevention and / or treatment of estrogen receptor-mediated or dependent diseases or conditions.

27. The use according to claim 26, wherein the estrogen receptor-mediated or dependent disease or condition is selected from cancer, central nervous system defects, cardiovascular system defects, blood system defects, immune and inflammatory diseases, susceptibility to infection, metabolic defects, neurological defects, mental defects, and reproductive defects.

28. The use according to claim 27, wherein the cancer is selected from breast cancer, endometrial cancer, uterine cancer, cervical cancer, skin cancer, prostate cancer, ovarian cancer, fallopian tube tumors, hemophilia, and leukemia.

29. The use according to claim 28, wherein the cancer is selected from breast cancer, ovarian cancer, endometrial cancer, prostate cancer, and uterine cancer.

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