WO2022127904A1

WO2022127904A1 - Pharmaceutically acceptable salt of indazole derivative, and crystalline form and preparation method therefor

Info

Publication number: WO2022127904A1
Application number: PCT/CN2021/139125
Authority: WO
Inventors: 王林; 邵启云; 冯君; 贺峰; 赵苗苗; 杜振兴
Original assignee: 江苏恒瑞医药股份有限公司; 上海恒瑞医药有限公司
Priority date: 2020-12-18
Filing date: 2021-12-17
Publication date: 2022-06-23
Also published as: TW202241879A; CN116615418A

Abstract

Provided are a pharmaceutically acceptable salt of an indazole derivative, and a crystalline form and a preparation method therefor. Particularly, provided are a crystal form of a pharmaceutically acceptable salt of a compound represented by formula (I), and a preparation method therefor. The provided crystal form of the pharmaceutically acceptable salt of the compound represented by formula (I) has good stability and can be better used for clinical treatment.

Description

A kind of pharmaceutically acceptable salt of indazole derivatives, crystalline form and preparation method thereof

This application claims the priority of Chinese patent application 202011510169.3 with an application date of 2020/12/18. This application cites the full text of the above Chinese patent application.

technical field

The present disclosure relates to a pharmaceutically acceptable salt, a crystalline form and a preparation method of an indazole derivative, belonging to the field of medicine.

Background technique

Breast cancer is one of the most common malignant tumors in women. According to the 2012 GLOBALCAN statistics (CA CANCER J CLIN 2015; 65:87–108), there are about 1.7 million new cancer cases and 520,000 deaths worldwide every year. Both morbidity and mortality rank first among female malignant tumors. According to the 2017 China Cancer Registry Annual Report released by the National Cancer Center, breast cancer ranks first in the incidence of female malignant tumors, with about 279,000 new cases each year, and an annual increase of about 2%.

About 70% of breast cancer patients are estrogen receptor (ER) positive breast cancer. Endocrine therapy plays an important role in the treatment of this part of breast cancer patients. Endocrine therapy is mainly divided into three categories, namely aromatase inhibitor (AI), which can inhibit the conversion of androgens into estrogen, reduce the level of estrogen in the body, and selective estrogen receptor modulator (selective estrogen receptor modulator). , SERM), antagonizes estrogen receptor activity, and selective estrogen receptor degrader (SERD), which can not only antagonize estrogen receptor activity, but also promote receptor degradation (Pharmacol Ther .2017 Dec 28). Although endocrine therapy is the treatment of choice for estrogen receptor-positive breast cancer, about 30% of patients receiving adjuvant therapy will relapse, and almost all patients with metastatic breast cancer will develop drug resistance and progress. The mechanisms of resistance to endocrine therapy are mainly divided into two categories. One is focused on the estrogen receptor signaling pathway itself, including the activating mutation, amplification, and fusion with other genes of the gene ESR1 encoding the estrogen receptor. Other mechanisms include the activation of signaling pathways that cross-react with the estrogen receptor signaling pathway, such as the growth factor receptor pathway, etc. (Nat Rev Clin Oncol. 2015 Oct. 2015). ; 12(10):573-83).

Two studies in 2013 detected ESR1 gene mutations in 11 to 55% of estrogen receptor-positive metastatic breast cancer patients who had received aromatase inhibitor therapy. Further research found that the mutant receptor can occur independently of estrogen. Phosphorylation and transcriptional effects enable estrogen-dependent MCF7-inoculated tumors to grow independent of estrogen in vivo, and mutant receptors render SERM tamoxifen and SERD fulvestrant activity decreased. Therefore, ESR1 gene mutation may be one of the mechanisms of drug resistance in estrogen-positive breast cancer (Nat Rev Clin Oncol. 2015 Oct;12(10):573-83 and Nat Genet 2013;45:1439-45). In subsequent studies, a certain proportion of ESR1 gene mutations were found in patients with estrogen receptor-positive metastatic breast cancer, and the mutation rate was about 30%. In the BOLERO-2 clinical trial, ER Y537S and ER D538G mutations were found in 29% of the ctDNA of patients with estrogen receptor-positive metastatic breast cancer that progressed after AIs therapy. In the exemestane alone group, both progression free survival (PFS) and overall survival (OS) were shorter in patients with mutations than in patients without mutations [Nat Genet 2013; 45:1446-51].

In summary, ESR1 gene mutations mostly occur in metastatic estrogen receptor-positive breast cancer patients who have progressed after AIs therapy. These patients are no longer sensitive to AIs therapy. Therefore, it is necessary to develop estrogen receptors targeting ESR1 gene mutations. antagonist.

The first-in-class estrogen receptor covalent binding antagonist H3B-6545 developed by Eisai Company has strong inhibitory activity on wild-type and mutant estrogen receptors, and can bind to the receptor through covalent binding. To exert longer-term efficacy, clinical phase I and II trials are currently underway. Currently, there are WO2016196346 and WO2016196342 patents for estrogen receptor antagonists with mutations in the ESR1 gene.

PCT/CN2020/096744 provides an indazole derivative whose chemical name is (E)-1-morpholinyl-4-((1-(((5-((Z)-4,4,4 -Trifluoro-1-(3-fluoro-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)methyl)cyclopropyl ) amino) but-2-en-1-one (Formula I), providing patients with a new treatment option.

SUMMARY OF THE INVENTION

The present disclosure provides a pharmaceutically acceptable salt of a compound represented by formula (I), the pharmaceutically acceptable salt is selected from maleate or hydrochloride,

In an optional embodiment, the present disclosure provides a maleic acid salt of a compound shown in formula (I), wherein the mol ratio of compound shown in formula (I) to maleic acid is 1:2-2:1.

In an optional embodiment, the present disclosure provides a maleate salt of a compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to maleic acid is 1:1.

The present disclosure also provides a method for preparing the pharmaceutically acceptable salt of the aforementioned formula (I), comprising: the step of forming a salt between the compound of formula (I) and an acid selected from maleic acid, hydrochloric acid or their solution, the solvent used in the salt-forming reaction is selected from water, methanol, n-propanol, isopropanol, ethanol, isopropyl ether, tetrahydrofuran, isopropyl acetate, acetone, butanone, methyl tert-butyl ether, acetonitrile, One or more of 1,4-dioxane, ethyl acetate and n-hexane.

The present disclosure further provides Form I of the maleate salt of the compound represented by formula (I), whose X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 16.448, 16.956, 19.332, 20.135, 21.645, 22.257 and 22.696 .

Further, the I crystal form of the maleate salt of the compound shown in the formula (I), wherein the mol ratio of the compound shown in the formula (I) and maleic acid is 1:2-2:1, preferably 1: 1.

The present disclosure further provides Form I of the maleate salt of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 7.853, 16.448, 16.956, 19.332, 20.135, 20.835, 21.645, 22.257, 22.696 and a characteristic peak at 25.879.

The present disclosure further provides Form I of the maleate salt of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 6.643, 7.853, 16.448, 16.956, 18.671, 19.332, 20.135, 20.835, 21.645 , 22.257, 22.696, 25.879 and 29.015 have characteristic peaks.

The present disclosure further provides Form II of the maleate salt of the compound represented by formula (I), whose X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 7.440, 15.005, 15.503, 17.599, 18.763, 20.471 and 26.259 .

Further, the II crystal form of the maleate of the compound shown in the formula (I), wherein the mol ratio of the compound shown in the formula (I) and maleic acid is 1:2-2:1, preferably 1: 1.

The present disclosure further provides Form II of the maleate salt of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 7.440, 8.724, 15.005, 15.503, 17.599, 18.136, 18.763, 20.471, 26.259 and a characteristic peak at 28.925.

The present disclosure further provides Form II of the maleate salt of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 7.440, 8.724, 15.005, 15.503, 17.599, 18.136, 18.763, 20.471, 22.600 , 23.556, 24.643, 26.259 and 28.925 have characteristic peaks.

The present disclosure further provides Form III of the maleate salt of the compound represented by formula (I), whose X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 6.830, 16.440, 17.358, 19.295, 19.919, 20.946 and 26.340 .

Further, the III crystal form of the maleate of the compound shown in the formula (I), wherein the mol ratio of the compound shown in the formula (I) and maleic acid is 1:2-2:1, preferably 1: 1.

The present disclosure further provides Form III of the maleate salt of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 6.830, 7.916, 16.440, 17.358, 19.295, 19.919, 20.946, 23.702, 25.820 and characteristic peaks at 26.340.

The present disclosure further provides Form III of the maleate salt of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 6.830, 7.916, 14.150, 16.440, 17.358, 19.295, 19.919, 20.946, 22.943 , 23.702, 25.820, 26.340 and 29.177 have characteristic peaks.

Another aspect of the present disclosure provides a method for preparing the I crystal form of the maleate salt of the compound represented by formula (I), the method comprising the following steps:

1) compound shown in formula (I), maleic acid are dissolved in at least one solvent selected from ethanol, acetone, methanol or water,

2) Crystallization.

Further, the crystalline form I of the maleate of the compound shown in the formula (I) prepared above, wherein the mol ratio of the compound shown in the formula (I) to the maleic acid is 1:2-2:1, preferably 1 :1.

Another aspect of the present disclosure provides a method for preparing the II crystal form of the maleate salt of the compound represented by the formula (I), the method comprising the following steps:

1) compound shown in formula (I), maleic acid are dissolved in at least one solvent selected from ethanol, isopropyl ether, n-hexane and butanone,

2) Crystallization.

Further, the crystalline form II of the maleate of the compound represented by the formula (I) prepared above, wherein the mol ratio of the compound represented by the formula (I) to the maleic acid is 1:2-2:1, preferably 1 :1.

Another aspect of the present disclosure provides a method for preparing the maleate salt form III of the compound represented by formula (I), the method comprising raising the temperature of the maleate salt form I of the compound represented by formula (I) to 110°C.

Further, the III crystal form of the maleate of the compound shown in the formula (I) prepared above, wherein the mol ratio of the compound shown in the formula (I) to the maleic acid is 1:2-2:1, preferably 1 :1.

The present disclosure provides a hydrochloride salt of a compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to hydrochloric acid is 1:2-2:1.

In an optional embodiment, the present disclosure provides a hydrochloride salt of the compound represented by formula (I), wherein the molar ratio of the compound represented by formula (I) to hydrochloric acid is 1:1.

The present disclosure further provides a crystal form of the hydrochloride salt of the compound represented by formula (I), whose X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 6.253, 12.578, 13.583, 18.151, 19.174, 20.027 and 26.978.

Further, in the crystal form a of the hydrochloride salt of the compound represented by formula (I), the molar ratio of the compound represented by formula (I) to hydrochloric acid is 1:2-2:1, preferably 1:1.

The present disclosure further provides a crystal form of the hydrochloride salt of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 6.253, 8.647, 11.210, 12.578, 13.583, 18.151, 19.174, 20.027, 26.676 and There is a characteristic peak at 26.978.

The present disclosure further provides a crystal form of the hydrochloride salt of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 6.253, 8.647, 11.210, 12.578, 13.583, 18.151, 19.174, 20.027, 24.105, There are characteristic peaks at 25.004, 25.375, 26.676 and 26.978.

The present disclosure further provides the b crystal form of the hydrochloride salt of the compound represented by formula (I), whose X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 12.613, 15.871, 16.013, 17.839, 18.144, 19.186 and 20.074.

Further, in the crystal form b of the hydrochloride salt of the compound represented by formula (I), the molar ratio of the compound represented by formula (I) to hydrochloric acid is 1:2-2:1, preferably 1:1.

The present disclosure further provides the b crystal form of the hydrochloride salt of the compound represented by formula (I), the X-ray powder diffraction pattern of which is 12.613, 15.871, 16.013, 17.839, 18.144, 19.186, 20.074, 20.773, 21.186 and There is a characteristic peak at 26.977.

Further, in the b crystal form of the hydrochloride of the compound shown in the formula (I), the mol ratio of the compound shown in the formula (I) and hydrochloric acid is 1:2-2:1, preferably 1:1.

The present disclosure further provides the b crystal form of the hydrochloride salt of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 12.613, 15.871, 16.013, 17.839, 18.144, 19.186, 20.074, 20.773, 21.186, There are characteristic peaks at 22.512, 24.181, 26.599 and 26.977.

The present disclosure further provides the crystal form c of the hydrochloride salt of the compound represented by formula (I), whose X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 15.515, 17.137, 19.743, 20.471, 21.525, 23.442 and 25.987.

The present disclosure further provides the crystal form c of the hydrochloride salt of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 7.353, 13.066, 14.042, 15.515, 17.137, 19.743, 20.471, 21.525, 23.442 and There is a characteristic peak at 25.987.

The present disclosure further provides the crystal form c of the hydrochloride salt of the compound represented by formula (I), whose X-ray powder diffraction pattern at 2θ angles is 6.970, 7.353, 9.527, 13.066, 14.042, 15.515, 17.137, 19.743, 20.471, There are characteristic peaks at 21.525, 23.442, 25.987 and 29.252.

Another aspect of the present disclosure provides a method for preparing the a-crystal form of the hydrochloride salt of the compound represented by formula (I), the method comprising the following steps:

1) the compound shown in formula (I) is dissolved in at least one solvent selected from ethanol, ether, butanone, n-hexane or isopropanol,

2) Add hydrochloric acid and crystallize out.

Further, in the crystal form a of the hydrochloride salt of the compound represented by formula (I) prepared above, the molar ratio of the compound represented by formula (I) to hydrochloric acid is 1:2-2:1, preferably 1:1.

Another aspect of the present disclosure provides a method for preparing the b crystal form of the hydrochloride salt of the compound represented by formula (I), the method comprising the following steps:

1) compound shown in formula (I) is dissolved in at least one solvent selected from isopropanol, isopropyl ether, n-hexane, ethyl acetate and acetonitrile,

2) Add hydrochloric acid and crystallize out.

Further, in the crystal form b of the hydrochloride salt of the compound represented by formula (I) prepared above, the molar ratio of the compound represented by formula (I) to hydrochloric acid is 1:2-2:1, preferably 1:1.

Another aspect of the present disclosure provides a method for preparing the crystal form c of the hydrochloride salt of the compound represented by formula (I), the method comprising the following steps:

1) the compound shown in formula (I) is dissolved in at least one solvent selected from acetone, methanol and water,

2) Add aqueous hydrochloric acid solution to crystallize out.

In an optional embodiment, the aforementioned crystalline form of the pharmaceutically acceptable salt of the compound represented by formula (I), wherein the error range of the 2θ angle is ±0.2.

In certain embodiments, the preparation method of the crystal form described in the present disclosure further comprises the steps of filtration, washing or drying.

The present disclosure also provides a pharmaceutical composition prepared from the pharmaceutically acceptable salt or the crystal form of the pharmaceutically acceptable salt of the compound represented by the aforementioned formula (I).

The present disclosure also provides a pharmaceutical composition, comprising the following components: i) a pharmaceutically acceptable salt of the compound represented by the aforementioned formula (I) or a crystalline form of a pharmaceutically acceptable salt, and ii) optionally selected from a pharmaceutically acceptable salt acceptable carrier, diluent or excipient.

The present disclosure also provides a method for preparing a pharmaceutical composition, comprising the steps of mixing the aforementioned component i) and component ii).

The present disclosure also provides a pharmaceutically acceptable salt of the compound represented by the aforementioned formula (I) or a crystal form of a pharmaceutically acceptable salt of the compound represented by the aforementioned formula (I), or the aforementioned composition, or prepared by the aforementioned method Use of the obtained composition in the preparation of estrogen receptor modulators.

The present disclosure also provides the pharmaceutically acceptable salt of the compound represented by the aforementioned formula (I) or the crystal form of the pharmaceutically acceptable salt of the aforementioned compound represented by the formula (I) or the aforementioned composition or prepared by the aforementioned method. Use of the composition in the preparation of a medicament for the prevention and/or treatment of an estrogen receptor-mediated or dependent disease or condition, preferably the estrogen receptor-mediated or dependent disease or condition is cancer, more Breast cancer, ovarian cancer, endometrial cancer, prostate cancer or uterine cancer are preferred, and breast cancer is most preferred.

The "2θ or 2θ angle" mentioned in this disclosure refers to the diffraction angle, and θ is the Bragg angle, in degrees or degrees; the error range of each characteristic peak 2θ is ±0.20, which can be -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, -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.

"Crystallization" in the present disclosure includes, but is not limited to, stirring crystallization, beating crystallization and volatile crystallization.

The preparation method of the crystal form described in the present disclosure also includes steps such as filtration and drying.

The drying temperature mentioned in the present disclosure is generally 25°C-100°C, preferably 40°C-70°C, and drying under normal pressure or under reduced pressure is possible.

Description of drawings

Fig. 1. Form I XRPD spectrum of the maleate salt of the compound represented by formula (I);

Figure 2. Form II XRPD spectrum of the maleate salt of the compound represented by formula (I);

Figure 3. Form III XRPD spectrum of the maleate salt of the compound represented by formula (I);

Figure 4. Form a XRPD spectrum of the hydrochloride of the compound represented by formula (I);

Figure 5. B crystal form XRPD spectrum of the hydrochloride salt of the compound represented by formula (I);

Figure 6. Form c XRPD spectrum of the hydrochloride salt of the compound represented by formula (I).

Detailed ways

The present invention will be explained in more detail below with reference to the embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and do not limit the essence and scope of the present invention.

The test conditions of the instruments used in the experiment:

The structures of the compounds were determined by nuclear magnetic resonance (NMR) or/and mass spectrometry (MS). NMR shifts ([delta]) are given in units of 10<" ⁶ > (ppm). NMR was measured by Bruker AVANCE-400 nuclear magnetic instrument or Bruker AVANCE NEO 500M, and the solvent was deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated chloroform (CDCl ₃ ), deuterated methanol (CD ₃ OD) , the internal standard is tetramethylsilane (TMS).

The MS was measured with an Agilent 1200/1290 DAD-6110/6120 Quadrupole MS LC/MS instrument (manufacturer: Agilent, MS model: 6110/6120 Quadrupole MS).

waters ACQuity UPLC-QD/SQD (manufacturer: waters, MS model: waters ACQuity Qda Detector/waters SQ Detector)

THERMO Ultimate 3000-Q Exactive (Manufacturer: THERMO, MS Model: THERMO Q Exactive)

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

Chiral HPLC analysis was determined using an Agilent 1260 DAD high performance liquid chromatograph.

HPLC preparations used Waters 2545-2767, Waters 2767-SQ Detector2, Shimadzu LC-20AP and Gilson GX-281 preparative chromatographs.

Chiral preparations were performed using a Shimadzu LC-20AP preparative chromatograph.

Ion chromatography used Thermo Scientific Dionex Intergrion, column type: Dionex IonPacTM AS11-HC (4 μm, 4×250 cm).

XRPD is X-ray powder diffraction detection: the measurement is carried out with a BRUKER D8Discover X-ray diffractometer, specific information collected: Cu anode (40kV, 40mA), Cu-Kα rays

Scanning mode: θ/2θ, scanning range (2θ range): 3～50°.

DSC is differential scanning calorimetry: METTLER TOLEDO DSC 3+ differential scanning calorimeter is used for measurement, the heating rate is 10°C/min, and the specific temperature range refers to the corresponding spectrum (mostly 25-300 or 25-350°C), nitrogen purge Speed 50mL/min.

TGA is thermogravimetric analysis: METTLER TOLEDO TGA 2 type thermogravimetric analyzer was used for detection, the heating rate was 10°C/min, the specific temperature range was referred to the corresponding spectrum (mostly 25-300°C), and the nitrogen purge rate was 50mL/min.

DVS is dynamic moisture adsorption: SMS DVS Advantage is used for detection. At 25°C, the humidity changes from 50%-95%-0%-95%-50% in steps of 10% (the last step is 5%) (the specific humidity range is The corresponding spectrum shall prevail, and most of the methods are listed here), and the judgment standard is that dm/dt is not more than 0.002%.

There is no special description in the examples, and the solution refers to an aqueous solution.

There is no special description in the examples, and the reaction temperature is room temperature, which is 20°C to 30°C.

The monitoring of the reaction progress in the embodiment adopts thin layer chromatography (TLC), the developing solvent used in the reaction, the eluent system of the column chromatography used for purifying the compound and the developing solvent system of the thin layer chromatography method include: A: Dichloromethane/methanol system, B: n-hexane/ethyl acetate system, the volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of basic or acidic reagents such as triethylamine and acetic acid can also be added for adjustment.

Example 1. Compound of formula I (E)-1-morpholinyl-4-((1-(((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H -Indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)methyl)cyclopropyl)amino)but-2-en-1-one preparation

The first step: tert-butyl (1-(((5-iodopyridin-2-yl)oxy)methyl)cyclopropyl)carbamate 1c

Sodium hydride (0.4 g, 10.7 mmol) was dissolved in N,N-dimethylformamide (20 mL), and tert-butyl 1-(hydroxymethyl)cyclopropylcarbamate 1b (1.0 g, 5.3 mmol) was added at room temperature , prepared by the well-known method "Journal of Organic Chemistry, 2002, 67(11), 3965-3968"), after the addition, 2-fluoro-5-iodopyridine 1a (1.8g, 8.0mmol) was slowly added. The reaction was stopped after stirring at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by thin layer chromatography with developing solvent system B to obtain the title product 1c (2.4 g), yield: 86%.

MS m/z(ESI): 391.0[M+1]

The second step: (Z)-(1-(((5-(4,4,4-trifluoro-1-(3-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H -Indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)methyl)cyclopropyl)carbamate tert-butyl ester 1f

3-Fluoro-1-(tetrahydro-2H-pyran-2-yl)-5-(4,4,4-trifluorobut-1-yn-1-yl)-1H-indazole 1d(1.8 g, 5.5 mmol, prepared by the method disclosed in Example 3 on page 84 of the specification in patent application WO2018098305), dissolved in methyltetrahydrofuran (40 mL), added with double pinacol borate (1.7 g, 6.6 mmol), Platinum tetrakistriphenylphosphine (137 mg, 0.1 mmol) was purged with argon three times, and the temperature was raised to 85° C. and stirred for 3 hours. Cool to room temperature, add compound 1c (2.0 g, 5.2 mmol), bistriphenylphosphonium palladium dichloride (741 mg, 1.1 mmol), cesium carbonate (3.6 g, 11.0 mmol) and water (1 mL), and stir at room temperature overnight. Iodobenzene 1e (1.2 g, 6.1 mmol), potassium hydroxide (1.5 g, 27.6 mmol) were added, the argon was pumped for 3 times, the temperature was raised to 85° C., stirred for 2 hours, and then cooled to room temperature to stop the reaction. The reaction solution was concentrated under reduced pressure, and the residue was purified by thin layer chromatography with developing solvent system B to obtain the title product 1f (3.0 g), yield: 88%.

MS m/z(ESI): 667.2[M+1]

The third step: (Z)-(1-(((5-(4,4,4-trifluoro-1-(3-fluoro-1-(tetrahydro-2H-pyran-2-yl)-1H -Indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)methyl)cyclopropyl)-1-amine 1g

Compound 1f (1.8 g, 2.7 mmol) was dissolved in dichloromethane (15 mL), trifluoroacetic acid (3 mL) was added, and the reaction was stirred at room temperature for 5 hours to stop the reaction. The reaction solution was concentrated under reduced pressure, and the reaction solution was adjusted to about pH 8 with saturated sodium bicarbonate solution (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the title product 1g crude product (1.4g), yield: 89%, the product was directly carried to the next step without purification.

The fourth step: (E)-1-morpholinyl-4-((1-(((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1-(tetrafluoro) Hydro-2H-pyran-2-yl)-1H-indazol-5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)methyl)cyclopropane base)amino)but-2-en-1-one 1i

Compound 1 g (1.7 g, 2.8 mmol) was dissolved in N,N-dimethylformamide (20 mL), diisopropylethylamine (1.1 g, 8.5 mmol) was added at room temperature, and (E)-4- Bromo-1-morpholinobut-2-en-1-one for 1 h (0.7 g, 2.8 mmol, prepared by the method disclosed in Example 15 on page 65 of the specification in patent application US2016347717), and the reaction was stirred for 2 hours. Stop and cool the reaction, add saturated sodium bicarbonate solution (15 mL), extract with ethyl acetate (50 mL×2), combine the organic phases, wash with saturated sodium chloride solution (50 mL×4), dry over anhydrous sodium sulfate, filter , the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with developing solvent system A to give the title product 1i (1.3 g), yield: 65%.

MS m/z(ESI): 720.2[M+1]

The fifth step: (E)-1-morpholinyl-4-((1-(((5-((Z)-4,4,4-trifluoro-1-(3-fluoro-1H-indazole) -5-yl)-2-phenylbut-1-en-1-yl)pyridin-2-yl)oxy)methyl)cyclopropyl)amino)but-2-en-1-one I

Compound 1i (2.0 g, 2.8 mmol) was dissolved in methanol (5 mL), hydrochloric acid (12 N, 10 mL) was added and the reaction was stirred for 3 hours. The reaction was stopped and cooled, the reaction solution was concentrated, saturated sodium bicarbonate solution (15 mL) was added, extracted with dichloromethane (50 mL×4), the organic phases were combined, washed with water (30 mL×3) and saturated sodium chloride solution ( 50 mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with developing solvent system A to obtain the title product 1 (1.3 g), yield: 73%.

MS m/z(ESI): 636.2[M+1];

¹ H NMR (400MHz, CD ₃ OD) 7.65(d,2H), 7.49(d,1H), 7.30-7.22(m,7H), 6.82-6.76(m,1H), 6.60-6.52(m,2H) , 4.15(s, 2H), 3.62-3.39(m, 12H), 0.76-0.64(m, 4H). The crystal form of the product is amorphous as determined by X-ray powder diffraction.

Test Example 1: Determination of Inhibitory Effect of Compound of Formula I on Estrogen Receptor Reporter Gene Activity

1. The purpose of the experiment

The purpose of this experiment is to test the inhibitory effect of the disclosed compounds on the activity of the estrogen receptor reporter gene, and to evaluate the in vitro activity of the compounds according to the IC ₅₀ size.

2. Experimental method

MCF7 cells (ATCC, HTB-22) expressing the estrogen receptor response element-controlled luciferase reporter gene ERE-luc (synthesized by Jinweizhi Biotechnology Co., Ltd.) MCF7/ERE-luc containing 10% fetal bovine serum and 500 μg /ml G418 in MEM (GE Healthcare, SH30024.01) medium for culture. On the first day of the experiment, MCF7/ERE-luc cells were seeded in a 96-well plate at a density of 30,000 cells/well using incomplete MEM medium containing 10% activated carbon-treated fetal bovine serum (BioSun, BS-0004-500). 100 μl of cell suspension per well was placed in a cell culture incubator at 37 °C, 5% CO ₂ overnight. The next day, 10 μl of β-estradiol prepared in incomplete medium and different concentrations of the compounds to be tested were added to each well. The final concentration of β-estradiol was 0.1 nM, and the final concentration of the compounds was 10 times starting from 10 μM. For the 9 concentration points of the gradient dilution, set a blank control containing 0.5% DMSO, and place it in a cell incubator at 37 °C and 5% CO ₂ for 20 hours. On the third day, the 96-well plate was taken out, and 100 μl of ONE-Glo ^™ Luciferase Assay system (Promega, E6110) was added to each well to detect the activity of luciferase. After 3 minutes at room temperature until the cells were fully lysed, the multi-labeled microplate was used for enzyme labeling. The luminescence signal value was read by an instrument (PerkinElmer, VICTOR 3), and the _IC50 value of the inhibitory activity of the compound was calculated according to the concentration of the compound and the luminescence signal value with Graphpad Prism software.

3. Test results

The inhibitory effect of the compounds in the present disclosure on the activity of the estrogen receptor reporter gene was determined by the above experiments, and the chemiluminescence signal value was plotted with the logarithmic concentration of the compound using Graghpad Prism, and the _IC50 value of the compound of formula I was determined to be 1 nM .

Therefore, the disclosed compounds have a significant inhibitory effect on the estrogen receptor reporter gene.

Test Example 2: Inhibitory effect of the compounds of the present disclosure on the proliferation of MCF7 cells

1. The purpose of the experiment

The purpose of this experiment is to determine the inhibitory activity of the disclosed compounds on the proliferation of MCF7 cells, and to evaluate the in vitro activity of the compounds according to the IC ₅₀ size.

2. Experimental method

MCF7 cells (ATCC, HTB-22) were cultured in MEM (GE Healthcare, SH30024.01) complete medium containing 10% fetal bovine serum. On the first day of the experiment, MCF7 cells were seeded in a 96-well plate at a density of 3,000 cells/well in complete medium, 100 μl of cell suspension per well, and placed in a cell incubator at 37°C and 5% CO ₂ overnight. The next day, the medium was aspirated, and each well was replaced with 135 μl of MEM incomplete medium containing 2% fetal bovine serum. At the same time, 15 μl of different concentrations of the compounds to be tested prepared in incomplete medium were added to each well. The final concentration of the compounds was 9 concentration points of 4-fold serial dilution starting from 100 nM, set up blank control containing 0.5% DMSO, and placed in a cell incubator at 37 °C, 5% CO ₂ for 144 hours. On the eighth day, remove the 96-well cell culture plate and add 150 μl to each well

Luminescent Cell Viability Assay (Promega, G7573), after 10 minutes at room temperature, use a multi-label microplate microplate reader (PerkinElmer, VICTOR 3) to read the luminescence signal value, and use Graphpad Prism software to calculate according to the compound concentration and luminescence signal value _IC50 values for compound inhibitory activity.

3. Data analysis

Using Graghpad Prism to plot the chemiluminescence signal value and the logarithmic concentration of the compound, the IC ₅₀ value of the compound was obtained as 0.5 nM, and the result showed that the compound of the present disclosure has a significant inhibitory effect on the proliferation of MCF7 cells.

Test Example 3: Experimental Biological Evaluation of Expression of ERα Mutant MCF7 Cell Proliferation Inhibition

1. The purpose of the experiment

The purpose of this experiment was to determine the inhibitory activity of the disclosed compounds on the proliferation of MCF7 cells expressing ERα mutants.

2. Experimental method

Site-directed mutagenesis and cell line construction

The mutants ERαY537S and ERαD538G of human estrogen receptorα (ERα) protein were obtained by double-primer PCR using the cDNA (Accession No. NM000125) of the wild-type ESR1 gene as a template for site-directed mutagenesis. The primer sequences used for mutation are as follows (the underlined nucleotides are the sites of mutation): Y537S: F-AAG AAC GTG GTG CCC CTC T C T GAC CTG CTG CTG GAG ATG; R-CAT CTC CAG CAG CAG GTC A G A GAG GGG CAC CAC GTT CTT; D538G: F-AAC GTG GTG CCC CTC TAT G G C CTG CTG CTG GAG ATG CTG; R-CAG CAT CTC CAG CAG CAG G C C ATA GAG GGG CAC CAC GTT. The cDNA of mutant ESR1 was cloned into the target lentiviral vector pCDH-CMV-MCS-EF1-Puro. The lentiviral plasmids carrying the mutant ESR1 gene sequences and lentiviral packaging plasmids were then transfected into HEK-293T cells (ATCC, CRL-3216) by Lipofectamine 3000 Transfection Reagent (ThermoFisher Scientific, Cat# L3000075). 48 hours after transfection, the virus-containing medium supernatant was filtered and ultracentrifuged to obtain the virus pellet, resuspended and dissolved with an appropriate amount of medium, added to MCF7 cells (ATCC, HTB-22), and added to the final concentration Incubate overnight with 8 μg/ml polybrene. Two days after transfection, 1 μg/ml puromycin was added to the cell culture medium for resistance screening, and about two weeks later, the MCF7 cell line capable of stably expressing ERαY537S and ERαD538G mutants was obtained.

Cell proliferation inhibition assay

MCF7 cells expressing ERα mutants were cultured in MEM (GE Healthcare, SH30024.01) complete medium containing 10% fetal bovine serum. On the first day of the experiment, cells were seeded in a 96-well plate at a density of 3,000 cells/well in complete medium, 100 μl of cell suspension per well, and cultured overnight in a cell incubator at 37°C, 5% CO ₂ . The next day, the medium was aspirated, and each well was replaced with 135 μl of MEM incomplete medium containing 2% fetal bovine serum. At the same time, 15 μl of different concentrations of the compounds to be tested prepared in incomplete medium were added to each well. The final concentration of the compounds was 9 concentration points of 4-fold serial dilution starting from 100 nM, set up blank control containing 0.5% DMSO, and placed in a cell incubator at 37 °C, 5% CO ₂ for 144 hours. On the eighth day, remove the 96-well cell culture plate and add 150 μl to each well

Luminescent Cell Viability Assay (Promega, G7573), after 10 minutes at room temperature, use a multi-label microplate microplate reader (PerkinElmer, VICTOR 3) to read the luminescence signal value, and use Graphpad Prism software to calculate according to the compound concentration and luminescence signal value The IC ₅₀ value of the inhibitory activity of the compound, the IC ₅₀ of the inhibitory effect of the compound of the present disclosure on the proliferation of MCF7 D538G cells expressing ERα mutant is 2nM, and the IC ₅₀ of the inhibitory effect on the proliferation of MCF7ERα Y537S cells expressing ERα mutant is 3nM, the results show that the present disclosure The compound has obvious inhibitory effect on the proliferation of MCF7 cells expressing ERα mutant.

Test Example 4. BALB/C nude mice pharmacokinetic test of the disclosed compounds

1. Abstract

Taking BALB/C nude mice as test animals, the drug concentration in the plasma of BALB/C nude mice at different times after intragastric administration of the compound of formula I was determined by LC/MS/MS method. The pharmacokinetic behavior of the compound of formula I of the present disclosure in BALB/C nude mice was studied, and its pharmacokinetic characteristics were evaluated.

2. Test plan

2.1 Test drug

Compounds of formula I.

2.2 Experimental animals

36 BALB/C nude mice, female, were divided into 4 groups on average, and 9 mice were divided into 1 group. They were purchased from Jisijie Laboratory Animal Co., Ltd., and the animal production license number was SCXK (Shanghai) 2013-0006.

2.3 Drug preparation

Weigh an appropriate amount of sample, add 5% volume of DMSO, 5% volume of Tween 80 and 90% volume of normal saline to prepare 0.1 mg/mL colorless clear and transparent liquid.

2.4 Administration

After a night of fasting, the mice were administered by intragastric administration respectively, and the administration volume was 0.2 ml/10 g, and the administration dose of the compound of formula I was 30 mg/kg.

3. Operation

36 Balb/C nude mice, female; fasted overnight and administered by gavage. At 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 11.0, 24.0 h after administration, 0.1 ml of blood was collected (3 animals at each time point), placed in a heparinized test tube, centrifuged at 3500 rpm for 10 min to separate plasma, and stored at -20°C save. Determination of the content of the test compound in the plasma of nude mice after intragastric administration of different concentrations of drugs: take 25 μL of nude mouse plasma at each time after administration, add 40 μL of internal standard solution camptothecin (100 ng/mL), 200 μL of acetonitrile, and vortex. Spin and mix for 5 minutes, centrifuge for 10 minutes (4000 rpm), and take 0.5 μL of the supernatant from the plasma sample for LC/MS/MS analysis.

4. Pharmacokinetic parameter results of BALB/C nude mice

The pharmacokinetic parameters of the compounds of formula I of the present disclosure are as follows:

Conclusion: The disclosed compounds have good pharmacokinetic absorption and have obvious pharmacokinetic absorption effect.

Test Example 5. Biological evaluation of estrogen receptor ERα wild type and ERα Y537S mutant covalent modification

1. The purpose of the experiment

The purpose of this experiment was to determine the covalent modification of the estrogen receptor ERα wild type and ERα Y537S mutant by the compounds of the present disclosure.

2. Experimental method

The ligand binding domain (LBD, ligand binding domain, aa296-554) of estrogen receptor ERα wild type and ERα Y537S mutant was expressed and purified from E. coli. Add 2 μM ERα wild-type or ERα Y537S mutant protein and 10 μM compound to a buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM TCEP, 5% glycerol, mix well, and incubate at 4°C for 24 hours. High-resolution mass spectrometry detection. Or add 1μM ERα wild-type or ERα Y537S mutant protein and 3μM compound to a buffer containing 50mM Tris-HCl, pH7.5, 150mM NaCl, 1mM TCEP, 5% glycerol, mix well, and incubate at 37°C for 15 minutes. for high-resolution mass spectrometry. In the spectrum of mass spectrometry detection results, the peak whose molecular weight is the sum of the protein and the compound is the covalently modified product, and the percentage of covalent modification is calculated by calculating the ratio of the unbound compound protein to the total protein.

Covalent modification ratio after 24 hours of covalent modification:

Conclusion: The test compounds have good covalent modification effect on ERα wild type or ERα Y537S mutant protein.

Embodiment 2, the preparation of maleate salt I crystal form

Weigh 10 mg of the compound of formula (I) and 3.0 mg of maleic acid ligand, add 500 ul of ethanol solvent and stir to dissolve, stir for 2 days, slowly volatilize, gradually precipitate a solid, filter, and vacuum dry the solid, the obtained product ¹ H-NMR shows the compound The molar ratio to maleic acid is 1:1.

Through X-ray powder diffraction detection, the product is defined as crystal form I, the XRPD spectrum is shown in Figure 1, and the peak positions are shown in Table 1.

Table 1. Maleate Form I Peak Positions

DSC spectrum shows endothermic peaks at 61.47°C and 115.48°C; TGA spectrum shows 2.81% weight loss at 25°C-140°C.

Embodiment 3, the preparation of maleate salt I crystal form

Weigh 10mg of the compound of formula (I) and 3.0mg of maleic acid ligand, add 500ul of 10% water/acetone solvent and stir to dissolve, stir for 2 days, slowly volatilize, gradually separate out a solid, filter, and dry the solid in vacuo. NMR data show that the salt The molar ratio of the compound represented by Chinese formula (I) to maleic acid is 1:1. It was detected as Form I by X-ray powder diffraction.

Embodiment 4, the preparation of maleate salt I crystal form

Weigh 10mg of the compound of formula (I) and 3.0mgq maleic acid ligand, add 500ul 10% water/methanol solvent and stir to dissolve, stir for 2 days, slowly volatilize, and gradually precipitate a solid, filter, and vacuum dry the solid, and the nuclear magnetic data show that the salt The molar ratio of the compound represented by Chinese formula (I) to maleic acid is 1:1. It was detected as Form I by X-ray powder diffraction.

Example 5. Preparation of maleate II crystal form

The compound represented by formula I (25 mg, 39.33 umol) was added to 1.5 mL of a mixed solvent of ethanol and n-hexane (V/V=1:1), stirred, and 0.5 mL of maleic acid (4.57 mg, 39.33 umol) butanone was added dropwise The solution was stirred to dissolve, and 0.5 mL of n-hexane was added and stirred. After 0.5 hours, a white turbid liquid gradually formed, stirred at room temperature for 16 hours, filtered, and the filter cake was collected and dried in vacuo to obtain the title product (25 mg, yield: 100%) .

The obtained product was characterized by ¹ H-NMR, and the nuclear magnetic data showed that the molar ratio of the main component and maleic acid in the salt was 1:1.

¹ H NMR (400MHz, CD ₃ OD) δ 7.73-7.64 (m, 2H), 7.52 (dd, 1H), 7.32 (dd, 2H), 7.26 (d, 4H), 7.22 (dt, 1H), 6.82 (d,1H),6.75-6.63(m,2H),6.27(s,2H),4.41(s,2H),4.01(d,2H),3.69-3.61(m,8H),3.50-3.35(m ,2H),1.19-1.17(m,2H),1.16-1.05(m,2H).

Through X-ray powder diffraction detection, the product is defined as crystal form II, as shown in Figure 2, and the peak positions are shown in Table 2.

The DSC spectrum shows an endothermic peak at 136.02°C; the TGA spectrum shows a weight loss of 0.58% at 25°C-85°C, 3.73% at 85°C-150°C, and 4.39% at 150°C-210°C.

Table 2. Peak positions of maleate II crystal form

Example 6. Preparation of maleate III crystal form

The crystal form of compound I represented by formula I was heated to 110° C. by DSC, and detected by X-ray powder diffraction. The crystal form of the product was transformed and defined as crystal form III, as shown in FIG. The obtained product was characterized by ¹ H-NMR, and the nuclear magnetic data showed that the molar ratio of the main component and maleic acid in the salt was 1:1.

Table 3. Peak positions of maleate III crystal form

Embodiment 7, the preparation of hydrochloride a crystal form

The compound shown in formula I (25 mg, 39.33 μmol) was added to a mixed solvent of 2 mL of ethanol and ether (V/V=1:1), stirred, and dissolved, and 3.28 μL of concentrated hydrochloric acid (12 M, 39.33 mmol, 3.28 μL was slowly added dropwise, 35%), a white turbid liquid appeared, which gradually turned into a viscous state, continued stirring, and gradually formed a white turbid liquid after 0.5 hours, stirred at room temperature for 16 hours, filtered, collected the filter cake, and vacuum-dried to obtain the title product (23 mg, collected rate: 80%). Through X-ray powder diffraction detection, the product is defined as crystal form a, as shown in Figure 4, and the peak positions are shown in Table 4.

Table 4. Peak positions of hydrochloride a crystal form

Embodiment 8, the preparation of hydrochloride a crystal form

The compound represented by formula I (1.5 g, 2.36 mmol) was added to 10 mL of butanone, stirred, heated to 60 ° C, dissolved, and then added 0.5 mL of n-hexane clear liquid, cooled to 40 ° C, and slowly added dropwise 229.36 μL concentrated Hydrochloric acid (270.41 mg, 2.60 mmol, 35%) was dissolved, then cooled to room temperature and stirred at room temperature for 24 hours. No solid was precipitated. The compound hydrochloride salt of formula I was added (Example 11), and gradually A white solid was precipitated, stirred for 16 hours, and gradually formed a white cloudy liquid, which was filtered, and the filter cake was collected and dried in vacuo to obtain the title product (1 g, yield: 63%).

The chloride ion content was 4.99% as detected by ion chromatography, indicating that the molar ratio of the main component and hydrochloric acid in the salt was 1:1.

The product was found to be crystal form a by X-ray powder diffraction.

The DSC spectrum shows an endothermic peak at 173.48°C; the TGA spectrum shows a weight loss of 2.81% at 25°C-170°C and a weight loss of 2.07% at 170°C-200°C. DVS test shows that under normal storage conditions (ie, 25°C, 60%RH), the sample has a hygroscopic weight gain of about 0.89%; under accelerated experimental conditions (ie, 70%RH), the hygroscopic weight gain of the sample is about 1.02%; under extreme conditions At low temperature (90% RH), the hygroscopic weight gain was about 1.49%. After the DVS test, the crystal form was re-measured, and the crystal form did not change.

Embodiment 9, the preparation of hydrochloride a crystal form

The compound shown in formula I (25 mg, 39.33 μmol) was added to 2 mL of isopropanol, stirred to dissolve, and 3.28 μL of concentrated hydrochloric acid (12 M, 39.33 mmol, 35%) was added dropwise, and a white turbid liquid appeared, which gradually became viscous. , continued stirring, and gradually formed a white cloudy liquid after 0.5 hours, stirred at room temperature for 16 hours, filtered, collected the filter cake, and dried in vacuo to obtain the title product (23 mg, yield: 80%). According to X-ray powder diffraction, the product is crystal form a.

Example 10. Preparation of hydrochloride a crystal form

The compound shown in formula I (25 mg, 39.33 μmol) was added to 2 mL of butanone, stirred to dissolve, and 3.28 μL of concentrated hydrochloric acid (12 M, 39.33 mmol, 35%) was added dropwise, and a white turbid liquid appeared, which gradually became viscous, Continue stirring, after 0.5 hours, a white cloudy liquid gradually formed, stirred at room temperature for 16 hours, filtered, collected the filter cake, and dried in vacuo to obtain the title product (23 mg, yield: 80%). According to X-ray powder diffraction, the product is crystal form a.

Example 11. Preparation of hydrochloride salt b crystal form

The compound represented by formula I (1.2 g, 1.89 mmol) was added to 30 mL of a mixed solvent of isopropanol, isopropyl ether and n-hexane (V/V/V=1:1:1), stirred, dissolved, and added dropwise. 173.05 μL of concentrated hydrochloric acid (12M, 2.07 mmol, 35%) was dissolved, stirred at room temperature for 24 hours, a white cloudy liquid was gradually formed, filtered, the filter cake was collected, and dried in vacuo to obtain the title product (1.1 g, yield: 86.69%) ).

The chloride ion content was 5.04% as detected by ion chromatography, indicating that the molar ratio of the main component and hydrochloric acid in the salt was 1:1.

Through X-ray powder diffraction detection, the product is defined as crystal form b, the XRPD spectrum is shown in Figure 5, and the peak positions are shown in Table 5.

The DSC spectrum shows that the endothermic peak is not obvious, and the exothermic peak peak is 183.54°C;

DVS test shows that under normal storage conditions (ie, 25°C, 60% RH), the sample has a hygroscopic weight gain of about 0.84%; under accelerated experimental conditions (ie, 70% RH), the hygroscopic weight gain of the sample is about 0.99%; under extreme conditions At low temperature (90% RH), the hygroscopic weight gain was about 1.8%. The desorption process and the adsorption process of this sample were basically coincident during the humidity change from 0% to 95% RH. After the DVS test, the crystal form was re-measured, and the crystal form did not change.

Table 5. Peak positions of hydrochloride salt form b

Example 12. Preparation of hydrochloride salt b crystal form

The compound represented by formula I (50 mg, 78.66 umol) was added to a mixed solvent of 2 mL of n-hexane and ethyl acetate (V/V=1:1), stirred to dissolve, and 6.55 μL of concentrated hydrochloric acid (12M, 78.66 μL) was slowly added dropwise. mmol, 35%), a white turbid liquid appeared, which gradually became viscous, continued stirring, and gradually formed a white turbid liquid after 0.5 hours, stirred at room temperature for 16 hours, filtered, collected the filter cake, and vacuum-dried to obtain the title product (45mg , yield: 85.12%). The product was found to be crystal form b by X-ray powder diffraction.

Example 13. Preparation of hydrochloride salt b crystal form

The compound represented by formula I (50 mg, 78.66 μmol) was added to 2 mL of acetonitrile, stirred, and dissolved, and 7.21 μL of concentrated hydrochloric acid (12M, 86.5 mmol, 35%) was slowly added dropwise, still dissolved, and stirred at room temperature for 24 hours. After that, a white cloudy liquid gradually formed, filtered, and the filter cake was collected and dried in vacuo to obtain the title product (40 mg, yield: 75.66%). The product was found to be crystal form b by X-ray powder diffraction.

Example 14. Preparation of hydrochloride salt c crystal form

Weigh 15 mg of the compound represented by formula I, add 500 μl of solvent, stir to clarify, add 26 μL of 1M hydrochloric acid aqueous solution without precipitation, and slowly volatilize to obtain the product, which is detected by X-ray powder diffraction, and the product is defined as crystal form c. The XRPD spectrum is shown in Figure 6, and the peak positions are shown in Table 6.

The DSC spectrum shows that the endothermic peaks are 94.07°C and 147.06°C; the TGA spectrum shows that the weight loss is 5.30% at 25°C-120°C.

DVS test shows that under normal storage conditions (ie, 25°C, 60% RH), the sample has a hygroscopic weight gain of about 8.64%; under accelerated experimental conditions (ie, 70% RH), the hygroscopic weight gain of the sample is about 9.32%; under extreme conditions (90% RH), the hygroscopic weight gain was about 12.32%. The desorption process and the adsorption process of this sample were basically coincident during the humidity change from 0% to 95% RH. After the DVS test, the crystal form was re-measured, and the crystal form did not change.

Table 6. Peak positions of hydrochloride salt form c

Embodiment 15. Experiment on influencing factors of hydrochloride crystal form a

Take the compound of formula 1 hydrochloride crystal form a in an open clean weighing bottle, and investigate the conditions under high temperature (40°C, 60°C), light (4500lx ± 500lx), high humidity (90% ± 5% RH, 75% ± The stability of the sample under the condition of 5% RH), the sampling period is 30 days, and the results are shown in Table 7 below.

Table 7. 30-day test results of the influence factors of the compound of formula I hydrochloride crystal form a

The experimental results show that the hydrochloride crystal form a of the compound of formula I has good stability in physical and chemical properties under the conditions of illumination, 40° C., 60° C., 75% RH and 90% RH.

Embodiment 16. Long-term accelerated stability experiment of hydrochloride crystal form a

The long-term (25°C, 60%RH) and accelerated (40°C, 75%RH) stability of the compound hydrochloride crystal form a represented by formula I was investigated for 6 months, and the results are shown in Table 8 below.

Table 8

The results showed that the hydrochloride crystal form a sample was placed under long-term (25°C, 60%RH) and accelerated (40°C, 75%RH) conditions for 6 months, and the physicochemical stability was good.

Claims

A pharmaceutically acceptable salt of a compound shown in formula (I), the pharmaceutically acceptable salt is selected from maleate and hydrochloride,
A maleate of compound shown in formula (I) according to claim 1, the maleate of compound shown in described formula (I), wherein compound shown in formula (I) and maleic acid The molar ratio is 1:2-2:1, preferably 1:1.
The maleate of the compound represented by the formula (I) according to claim 2, the I crystal form of the maleate of the compound represented by the formula (I), wherein the compound represented by the formula (I) and the maleate The molar ratio of lylic acid is 1:1, and its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 16.448, 16.956, 19.332, 20.135, 21.645, 22.257 and 22.696, and the X-ray powder diffraction pattern is preferably at 2θ. The angles are 7.853, 16.448, 16.956, 19.332, 20.135, 20.835, 21.645, 22.257, 22.696 and 25.879, there are characteristic peaks, more preferably the X-ray powder diffraction pattern is at 2θ angles of 6.643, 7.853, 16.448, 16.952, 18.671, 19.3 , 20.135, 20.835, 21.645, 22.257, 22.696, 25.879 and 29.015 have characteristic peaks.
The maleate salt of the compound represented by the formula (I) according to claim 2, the II crystal form of the maleate salt of the compound represented by the formula (I), wherein the compound represented by the formula (I) and the maleate The molar ratio of lylic acid is 1:1, and its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 7.440, 15.005, 15.503, 17.599, 18.763, 20.471 and 26.259, and the X-ray powder diffraction pattern is preferably at 2θ. Characteristic peaks at angles of 7.440, 8.724, 15.005, 15.503, 17.599, 18.136, 18.763, 20.471, 26.259 and 28.925, more preferably X-ray powder diffraction pattern at 2θ angles of 7.440, 8.724, 15.005, 15.503, 17.5969, 18.133 , 18.763, 20.471, 22.600, 23.556, 24.643, 26.259 and 28.925 have characteristic peaks.
The maleate salt of the compound represented by the formula (I) according to claim 2, the III crystal form of the maleate salt of the compound represented by the formula (I), wherein the compound represented by the formula (I) and the maleate The molar ratio of lylic acid is 1:1, and its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 6.830, 16.440, 17.358, 19.295, 19.919, 20.946 and 26.340, preferably the X-ray powder diffraction pattern is at 2θ. The angles are 6.830, 7.916, 16.440, 17.358, 19.295, 19.919, 20.946, 23.702, 25.820 and 26.340, there are characteristic peaks, more preferably the X-ray powder diffraction pattern is at 2θ angles of 6.830, 7.916, 14.150, 16.2940, 17.358, 19.2958, 19.340 , 19.919, 20.946, 22.943, 23.702, 25.820, 26.340 and 29.177 have characteristic peaks.
A method for preparing the I crystal form of the maleate of the compound shown in the formula (I) according to claim 3, the method comprises the following steps:

1) compound shown in formula (I), maleic acid are dissolved in at least one solvent selected from ethanol, acetone, methanol or water,

2) Crystallization.
A method for preparing the II crystal formation of the maleate of the compound shown in the formula (I) according to claim 4, the method comprises the following steps:

1) compound shown in formula (I), maleic acid are dissolved in at least one solvent selected from ethanol, isopropyl ether, n-hexane and butanone,

2) Crystallization.
A method for preparing the III crystal formation of the maleate of the compound shown in the formula (I) according to claim 5, the method comprises heating the maleate I crystal formation of the compound shown in the formula (I) to 110°C step.
A kind of hydrochloride of compound shown in formula (I) according to claim 1, the hydrochloride of compound shown in described formula (I), wherein the mol ratio of compound shown in formula (I) and hydrochloric acid is 1:2-2:1, preferably 1:1.
The hydrochloride of the compound represented by the formula (I) according to claim 9, the a crystal form of the hydrochloride of the compound represented by the formula (I), wherein the mole of the compound represented by the formula (I) and hydrochloric acid The ratio is 1:1, and its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 6.253, 12.578, 13.583, 18.151, 19.174, 20.027 and 26.978. Characteristic peaks at 8.647, 11.210, 12.578, 13.583, 18.151, 19.174, 20.027, 26.676 and 26.978, more preferably X-ray powder diffraction pattern at 2θ angles of 6.253, 8.647, 11.210, 12.578, 13.583, 18.151, 19.174 , 24.105, 25.004, 25.375, 26.676 and 26.978 have characteristic peaks.
The hydrochloride of the compound represented by the formula (I) according to claim 9, the b crystal form of the hydrochloride of the compound represented by the formula (I), wherein the mole of the compound represented by the formula (I) and hydrochloric acid The ratio is 1:1, and its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 12.613, 15.871, 16.013, 17.839, 18.144, 19.186 and 20.074. Characteristic peaks at 15.871, 16.013, 17.839, 18.144, 19.186, 20.074, 20.773, 21.186 and 26.977, more preferably X-ray powder diffraction pattern at 2θ angles of 12.613, 15.871, 16.013, 17.839, 18.074, 20.7736, 20 , 21.186, 22.512, 24.181, 26.599 and 26.977 have characteristic peaks.
The hydrochloride of the compound represented by the formula (I) according to claim 9, the crystal form c of the hydrochloride of the compound represented by the formula (I), its X-ray powder diffraction pattern at the 2θ angle is: There are characteristic peaks at 15.515, 17.137, 19.743, 20.471, 21.525, 23.442 and 25.987, and the preferred X-ray powder diffraction pattern is at 2θ angles of 7.353, 13.066, 14.042, 15.515, 17.137, 19.743, 20.471, 21.525, 23.47871, 21.525, 23.4 There are characteristic peaks, more preferably the X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 6.970, 7.353, 9.527, 13.066, 14.042, 15.515, 17.137, 19.743, 20.471, 21.525, 23.442, 25.987 and 29.252.
A method for preparing a crystal form of the hydrochloride of the compound shown in the formula (I) according to claim 10, the method comprises the following steps:

1) the compound shown in formula (I) is dissolved in at least one solvent selected from ethanol, ether, butanone, n-hexane or isopropanol,

2) Add hydrochloric acid and crystallize out.
A method for preparing the b crystal form of the hydrochloride of the compound shown in the formula (I) according to claim 11, the method comprises the following steps:

1) compound shown in formula (I) is dissolved in at least one solvent selected from isopropanol, isopropyl ether, n-hexane, ethyl acetate and acetonitrile,

2) Add hydrochloric acid and crystallize out.
A method for preparing the c crystal form of the hydrochloride of the compound shown in the formula (I) according to claim 12, the method comprises the following steps:

1) the compound shown in formula (I) is dissolved in at least one solvent selected from acetone, methanol and water,

2) Add aqueous hydrochloric acid solution to crystallize out.
The crystalline form of the maleate salt of the compound represented by the formula (I) according to claims 3-5, and the crystalline form of the hydrochloride salt of the compound represented by the formula (I) according to claims 10-12, wherein, The error range of the 2θ angle is ±0.2.
The pharmaceutically acceptable salt of the compound represented by formula (I) according to claim 1, 2, 9,

Or according to the crystal form of the pharmaceutically acceptable salt of the compound represented by the formula (I) according to claims 3-5, 10-12, 16, or prepared according to the method described in claims 6-8, 13-15 The composition prepared by any one of the crystal forms of the pharmaceutically acceptable salt of the compound represented by the formula (I) or a mixture thereof.
A pharmaceutical composition comprising the following components:

i) the pharmaceutically acceptable salt of the compound represented by formula (I) according to claim 1, 2, 9,

Or according to the crystal form of the pharmaceutically acceptable salt of the compound represented by the formula (I) according to claims 3-5, 10-12, 16, or prepared according to the method described in claims 6-8, 13-15 Any one of the crystalline forms of the pharmaceutically acceptable salts of the compound of formula (I) or a mixture thereof; and

ii) one or more pharmaceutically acceptable carriers, diluents or excipients.
A method for preparing a pharmaceutical composition, comprising the pharmaceutically acceptable salt of the compound shown in the formula (I) according to claims 1, 2, 9, or according to claims 3-5, 10-12, 16 The crystal form of the pharmaceutically acceptable salt of the compound represented by the formula (I), or the pharmaceutically acceptable salt of the compound represented by the formula (I) prepared according to the methods described in claims 6-8 and 13-15 The step of admixing any one of the crystalline forms of the salt or a mixture thereof with a pharmaceutically acceptable carrier, diluent or excipient.
The pharmaceutically acceptable salt of the compound represented by formula (I) according to claim 1, 2, 9,

Or according to the crystal form of the pharmaceutically acceptable salt of the compound represented by the formula (I) described in claims 3-5, 10-12, 16, or prepared according to the method described in claims 6-8, 13-15 Any one of the crystalline forms of the pharmaceutically acceptable salt of the compound represented by the formula (I) or a mixture thereof, or the composition according to claim 17, 18 in the preparation of prevention and/or treatment estrogen receptor-mediated Use in the medicament of a disease or disorder of or dependence.
The pharmaceutically acceptable salt of the compound represented by the formula (I) according to claims 1, 2 and 9, or the compound represented by the formula (I) according to claims 3-5, 10-12 and 16. The crystal form of the pharmaceutically acceptable salt, or any one of the crystal forms of the pharmaceutically acceptable salt of the compound represented by the formula (I) prepared according to the method of claims 6-8, 13-15, or a mixture thereof , or use of the composition according to claim 17, 18 in the preparation of a medicament for preventing and/or treating an estrogen receptor-mediated or dependent disease or condition, preferably the estrogen receptor-mediated The disease or condition of or dependence is cancer, more preferably breast cancer, ovarian cancer, endometrial cancer, prostate cancer or uterine cancer, most preferably breast cancer.