Salts, crystal forms and preparation methods of pyrrolidine compounds

Abstract

This disclosure provides a pharmaceutically acceptable salt of a compound of formula (I) with excellent physicochemical and / or pharmaceutical properties, its crystallization, its preparation method, and its use, wherein the pharmaceutically acceptable salt is selected from sulfates, phosphates, benzoates, succinates, adipates, fumarates, L-malates, and citrates, preferably benzoates, succinates, and adipates.

Description

Technical Field

[0001] This disclosure pertains to the pharmaceutical field and relates to pharmaceutically acceptable salts of pyrrolidine compounds, crystallization of said pharmaceutically acceptable salts, and corresponding preparation methods and uses. Prior Technology

[0002] Estrogen (E2) and estrogen α receptor (ERα) are important drivers of breast cancer development and progression. More than two-thirds of breast cancer patients express ER transcription factors, and ER remains a key driver in most ER-positive patients, even in tumors that have progressed after early endocrine therapy. Therefore, ER is a major target for breast cancer treatment (Pharmacology & Therapeutics 186 (2018) 1–24). Endocrine therapy aims to reduce ER activity, primarily through three classes: selective estrogen receptor modulators (SERMs), such as tamoxifen, which is an allosteric regulator of ER and inhibits its transcriptional activity upon binding; aromatase inhibitors (AIs), which reduce estrogen levels in the body by inhibiting the conversion of androgens to estrogens; and selective estrogen receptor downregulators, such as fulvestrant, which not only acts as an ER antagonist but also induces ER protein degradation. Although endocrine therapy is the first-line treatment for estrogen receptor-positive breast cancer patients, about 30% of patients will relapse after treatment, and almost all patients with metastatic breast cancer will develop drug resistance and progress.

[0003] Clinically, approximately 70-80% of breast cancers test positive for estrogen receptor (ER). The proliferation of these breast cancer cells is heavily dependent on ER, and this subtype accounts for 50% of breast cancer deaths. Early-stage ER-positive breast cancer has a relatively good prognosis, with a 5-year survival rate exceeding 90%. About 30% of patients who undergo postoperative endocrine therapy (TAM or AI drugs) experience recurrence within 10 years, but can still receive standard endocrine therapy.

[0004] Fulvestrant is the first and only clinically approved SERD (Sequencing Agent of Disease) drug for the treatment of postmenopausal patients with ER-positive metastatic breast cancer who have progressed after treatment with tamoxifen or aromatase inhibitors. Multiple studies have shown that ER degradation is not fully achieved in patients treated with fullvestrant. Furthermore, the intramuscular injection method causes significant reactions such as pain, swelling, and redness at the injection site, and its slow absorption and limited exposure restrict its clinical application. Therefore, new treatment options are urgently needed for patients with ER-positive breast cancer.

[0005] PCT / CN2021 / 093736 (application date May 14, 2021) describes a compound N-((S)-1-(3-fluoropropyl)pyrrolidine-3-yl)-6-((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridine-3-amine (compound of formula (I)), which is a selective estrogen receptor downregulator. In vitro and in vivo models show that compound (I) has a broad inhibitory effect on ER-positive cells. Summary of the Invention

[0006] This disclosure provides pharmaceutically acceptable salts of compounds of formula (I), The pharmaceutically acceptable salt is selected from sulfates, phosphates, benzoates, succinates, adipates, fumarates, L-malates, and citrates.

[0007] This disclosure also provides the adipate C crystal form of the compound of formula (I) above, wherein the X-ray powder diffraction pattern of the C crystal form, expressed as a diffraction angle 2θ, has diffraction peaks at 4.46±0.2º, 6.30±0.2º, 8.90±0.2º, and 19.10±0.2º.

[0008] This disclosure also provides the benzoate B crystal form of the compound of formula (I) above, wherein the X-ray powder diffraction pattern of the B crystal form, expressed in terms of diffraction angle 2θ, has diffraction peaks at 7.19±0.2º, 10.01±0.2º, 11.39±0.2º, and 18.78±0.2º.

[0009] This disclosure also provides the succinate A crystal form of the compound of formula (I) above, wherein the X-ray powder diffraction pattern of the A crystal form, expressed as a diffraction angle 2θ, has diffraction peaks at 7.13±0.2º, 9.30±0.2º, 11.29±0.2º, 15.51±0.2º, and 19.89±0.2º.

[0010] This disclosure also provides a method for preparing a pharmaceutically acceptable salt of the compound of formula (I) above, the method comprising the step of reacting the compound of formula (I) with the corresponding acid to form a salt.

[0011] This disclosure also provides a method for preparing the above-mentioned C crystal form, comprising the following steps: (1-a) Mix adipic acid and solvent; (2-a) Mix the compound of formula (I) with a solvent; (3-a) Mix the mixture from step (1-a) with the mixture from step (2-a); (4-a) The mixture of the adipic acid and the compound of formula (I) is stirred to allow crystallization; (5-a) Filter out the solid, collect the filter cake, and dry it.

[0012] This disclosure also provides a method for preparing the above-mentioned B crystal form, comprising the following steps: (1-b) Mix the compound of formula (I) with a solvent; (2-b) Add benzoic acid to the mixture from step (1-b); (3-b) The mixture of the compound of formula (I) and the benzoic acid is stirred to allow crystallization; (4-b) Filter out and collect the solids.

[0013] This disclosure also provides a method for preparing the above-mentioned crystal form A, comprising the following steps: (1-c) Mix the compound of formula (I) with a solvent; (2-c) Add succinic acid to the mixture from step (1-c); (3-c) The mixture of the compound of formula (I) and the succinic acid is stirred to precipitate a solid; (4-c) Filter out and collect the solids.

[0014] This disclosure also provides a pharmaceutical composition comprising a pharmaceutically acceptable salt or crystal form of a compound of formula (I) above, and pharmaceutically acceptable excipients.

[0015] This disclosure also provides the use of pharmaceutically acceptable salts of compounds of formula (I), the crystal forms described above, or the pharmaceutical compositions described above in the preparation of medicaments for the prevention or treatment of estrogen receptor-related diseases. Simple Explanation of the Diagram

[0016] Details of one or more embodiments of the subject matter described herein are set forth in the following drawings and description. Other features, aspects, and advantages of the subject matter of this specification will become apparent from the description, drawings, and claims, wherein: Figure 1 shows the NOESY spectrum of compound (I). Figure 2 shows the XRPD pattern of the adipate C crystal form of compound (I). Figure 3 shows the DSC spectrum of the adipate C crystal form of compound (I). Figure 4 shows the TGA spectrum of the adipate C crystal form of compound (I). Figure 5 shows the XRPD pattern of the benzoate B crystal form of compound (I). Figure 6 shows the DSC spectrum of the benzoate B crystal form of compound (I). Figure 7 shows the TGA spectrum of the benzoate B crystal form of compound (I). Figure 8 shows the XRPD pattern of succinate A, a crystal form of compound (I). Figure 9 shows the DSC spectrum of succinate A, a crystal form of compound (I). Figure 10 shows the TGA spectrum of succinate A, a crystal form of compound (I). Figure 11 is a stereoscopic ellipsoidal diagram of the crystal form of succinate A of compound (I). Figure 12 is a stacked diagram of the unit cell structure of succinate A, a compound of formula (I). Figure 13. Anti-tumor growth effect of compound (I) succinate in MCF-7 xenograft mouse model. Figure 14. Effect of compound (I) succinate on body weight in mice with MCF-7 xenograft subcutaneous tumors. Figure 15 shows the anti-tumor growth effect of compound (I) succinate in a T47D xenograft mouse model. Figure 16. Changes in body weight in mice with T47D xenografts caused by the effect of succinate (Formula I). Figure 17. Survival curve of MCF-7 xenogeneic brain tumor-bearing mice with succinate of formula (I). Figure 18 shows the survival curve of MCF-7 xenogeneic brain tumor-bearing mice with compound (I). Figure 19 shows the effect of compound (I) on the body weight of MCF-7 xenografted brain tumor-bearing mice. Implementation

[0017] This disclosure provides a pharmaceutically acceptable salt of a compound of formula (I) with excellent physicochemical and / or pharmaceutical properties, and its crystallization.

[0018] This disclosure provides pharmaceutically acceptable salts of the compounds represented by formula (I), wherein the pharmaceutically acceptable salts are selected from sulfates, phosphates, benzoates, succinates, adipates, fumarates, L-malates, and citrates, preferably benzoates, succinates, adipates, fumarates, L-malates, and citrates, and more preferably benzoates, succinates, and adipates.

[0019] In some embodiments, in the pharmaceutically acceptable salt of the compound of formula (I), the molar ratio of the compound of formula (I) to the acid molecule is about 0.5 to 2.

[0020] In some embodiments, the molar ratio of the compound of formula (I) to the acid molecule in the pharmaceutically acceptable salt of the compound of formula (I) is about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2.0.

[0021] In some embodiments, the molar ratio of the compound of formula (I) to the acid molecule in the pharmaceutically acceptable salt of the compound of formula (I) is about 0.5, 1.0, 1.5 and 2.0.

[0022] In some embodiments, the succinate of the compound of formula (I) is shown as that of formula (II), wherein x is selected from 0.5 to 2.

[0023] In some embodiments, the benzoate of the compound of formula (I) is shown as that of formula (III), wherein y is selected from 0.5 to 2.

[0024] In some embodiments, the adipate salt of the compound of formula (I) is shown as that of formula (IV), wherein z is selected from 0.5 to 2.

[0025] In some implementations, x, y, and z are independently selected from 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0.

[0026] In some implementations, x, y, and z are independently selected from 0.8, 0.9, 1.0, 1.1, and 1.2.

[0027] In some implementations, x, y, and z are independently selected from 1.0.

[0028] This disclosure also provides a method for preparing a pharmaceutically acceptable salt of the compound of formula (I) above, comprising: a step of forming a salt of the compound of formula (I) with an acid, wherein the acid is selected from sulfuric acid, phosphoric acid, benzoic acid, succinic acid, adipic acid, fumaric acid, L-malic acid and citric acid, preferably benzoic acid, succinic acid, adipic acid, fumaric acid, L-malic acid and citric acid, and more preferably benzoic acid, succinic acid and adipic acid.

[0029] The solvent used in the salt formation reaction described in this disclosure is selected from at least one of cyclohexane, n-heptane, toluene, chloroform, diethyl ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, acetone, methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, methanol, ethanol, isopropanol, n-butanol, and acetonitrile, preferably at least one of isopropyl ether, methyl tert-butyl ether, methanol, isopropanol, and tetrahydrofuran.

[0030] In an optional embodiment, the method for preparing the pharmaceutically acceptable salt of the compound of formula (I) above further includes one or a combination of steps such as evaporating a solvent or stirring to crystallize, filtering, and drying.

[0031] This disclosure also provides the crystal form of the adipate of the compound of formula (I).

[0032] In some embodiments, the adipate salt of compound (I) is in anhydrous form.

[0033] This disclosure provides the C crystal form of the compound adipate of formula (I) above, wherein the C crystal form has diffraction peaks at 4.46±0.2º, 6.30±0.2º, 8.90±0.2º and 19.10±0.2º in the X-ray powder diffraction pattern expressed as diffraction angle 2θ.

[0034] In some embodiments, the C-type crystal has diffraction peaks at 4.46±0.2º, 6.30±0.2º, 7.03±0.2º, 8.90±0.2º, 16.45±0.2º, 17.78±0.2º, 19.10±0.2º, and 21.03±0.2º in its X-ray powder diffraction pattern expressed as diffraction angle 2θ.

[0035] In some embodiments, the C-type crystal, in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ, is located at 4.46±0.2º, 6.30±0.2º, 7.03±0.2º, 8.90±0.2º, 10.65±0.2º, 11.95±0.2º, 12.55±0.2º, 13.34±0.2º, 14.20±0.2º, 16.02±0.2º, and 16.45±0.2º. .2º, 17.78±0.2º, 18.33±0.2º, 19.10±0.2º, 19.77±0.2º, 20.12±0.2º, 21.03± There are diffraction peaks at 0.2º, 21.62±0.2º, 24.30±0.2º, 25.97±0.2º, 27.39±0.2º and 27.98±0.2º.

[0036] In some embodiments, the C-type crystal, in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ, is located at 4.46±0.2º, 6.30±0.2º, 7.03±0.2º, 8.90±0.2º, 10.65±0.2º, 11.95±0.2º, 12.55±0.2º, 12.96±0.2º, 13.34±0.2º, 14.20±0.2º, 15.20±0.2º, 16.02±0.2º, 16.45±0.2º, 17.78±0.2º, 18.33±0.2º, and 19.10±0. .2º, 19.77±0.2º, 20.12±0.2º, 21.03±0.2º, 21.62±0.2º, 22.44±0.2º, 22.52±0.2º, 22.54±0.2º, 23.35±0.2º, There are diffraction peaks at 23.98±0.2º, 24.30±0.2º, 24.86±0.2º, 25.37±0.2º, 25.97±0.2º, 27.39±0.2º, 27.98±0.2º and 28.81±0.2º.

[0037] In some embodiments, the C-type crystal has an X-ray powder diffraction pattern that is substantially consistent with that in Figure 2.

[0038] In some embodiments, the DSC spectrum of the C crystal form has an endothermic peak at 149.89±5℃.

[0039] In some implementations, the DSC spectrum of the C crystal form is substantially consistent with that in Figure 3.

[0040] This disclosure also provides a method for preparing the adipate C crystal form of the compound of formula (I), including a salt formation reaction step of the compound of formula (I) with adipic acid.

[0041] In some embodiments of this disclosure, the preparation method of the above-mentioned C-type crystal includes the following steps: (1-a) Mix adipic acid and solvent; (2-a) Mix the compound of formula (I) with a solvent; (3-a) Mix the mixture from step (1-a) with the mixture from step (2-a); (4-a) The mixture of the adipic acid and the compound of formula (I) is stirred to allow crystallization; (5-a) Filter out the solid, collect the filter cake (i.e. the solid), and dry it.

[0042] In some embodiments, the solvents in steps (1-a) and (2-a) are selected from at least one of cyclohexane, n-heptane, toluene, chloroform, diethyl ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, acetone, methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, methanol, ethanol, isopropanol, n-butanol, and acetonitrile, preferably at least one of isopropyl ether, methyl tert-butyl ether, methanol, isopropanol, and tetrahydrofuran, more preferably isopropanol.

[0043] In some embodiments, the molar ratio of the compound of formula (I) to adipic acid is 1:2 to 2:1, preferably 1:1.

[0044] In some implementations, step (1-a) is performed under heating conditions.

[0045] In some embodiments, step (1-a) is performed at a heating temperature of about 40-80°C, preferably 60°C.

[0046] In some embodiments, step (4-a) is carried out at 15-30°C, preferably at 20-25°C.

[0047] This disclosure also provides the crystal form of the benzoate of the compound of formula (I).

[0048] In some embodiments, the benzoate of compound (I) is in anhydrous form.

[0049] This disclosure provides the benzoate B crystal form of the compound of formula (I), wherein the X-ray powder diffraction pattern of the B crystal form, expressed as diffraction angle 2θ, has diffraction peaks at 7.19±0.2º, 10.01±0.2º, 11.39±0.2º, and 18.78±0.2º.

[0050] In some embodiments, the B crystal form has diffraction peaks at 7.19±0.2º, 10.01±0.2º, 11.39±0.2º, 18.78±0.2º and 20.09±0.2º in its X-ray powder diffraction pattern expressed as diffraction angle 2θ.

[0051] In some embodiments, the X-ray powder diffraction pattern of the B crystal form, expressed as a diffraction angle 2θ, shows diffraction peaks at 7.19±0.2º, 10.01±0.2º, 11.39±0.2º, 18.78±0.2º, 19.01±0.2º, 19.66±0.2º, 19.91±0.2º, and 20.09±0.2º.

[0052] In some embodiments, the B crystal form, in its X-ray powder diffraction pattern expressed as diffraction angle 2θ, exhibits diffraction peaks at 4.69±0.2º, 6.34±0.2º, 7.19±0.2º, 10.01±0.2º, 11.39±0.2º, 18.78±0.2º, 19.01±0.2º, 19.66±0.2º, 19.91±0.2º, and 20.09±0.2º.

[0053] In some embodiments, the B crystal form, in its X-ray powder diffraction pattern expressed as diffraction angle 2θ, exhibits diffraction peaks at 4.69±0.2º, 6.34±0.2º, 7.19±0.2º, 10.01±0.2º, 11.11±0.2º, 11.39±0.2º, 14.78±0.2º, 18.78±0.2º, 19.01±0.2º, 19.25±0.2º, 19.66±0.2º, 19.91±0.2º, 20.09±0.2º, 20.80±0.2º, and 21.66±0.2º.

[0054] In some embodiments, the X-ray powder diffraction pattern of the B crystal form, expressed as a diffraction angle 2θ, shows diffraction peaks at 4.69±0.2º, 6.34±0.2º, 7.19±0.2º, 9.83±0.2º, 10.01±0.2º, 11.11±0.2º, 11.39±0.2º, 14.78±0.2º, 18.78±0.2º, 19.01±0.2º, 19.25±0.2º, 19.66±0.2º, 19.91±0.2º, 20.09±0.2º, 20.80±0.2º, and 21.66±0.2º.

[0055] In some embodiments, the B crystal form, in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ, is located at angles of 4.69±0.2º, 4.91±0.2º, 6.34±0.2º, 7.19±0.2º, 9.37±0.2º, 9.83±0.2º, 10.01±0.2º, 10.32±0.2º, 11.11±0.2º, 11.39±0.2º, 12.06±0.2º, 12.69±0.2º, 13.52±0.2º, 14.07±0.2º, 14.36±0.2º, and 14.78±0.2º. 2º, 17.30±0.2º, 18.78±0.2º, 19.01±0.2º, 19.25±0.2º, 19.66±0.2º, 19.91±0.2º, 20.09±0.2º, 20.80±0.2º, 2 There are diffraction peaks at 1.15±0.2º, 21.66±0.2º, 22.35±0.2º, 23.57±0.2º, 23.70±0.2º, 24.63±0.2º, 25.68±0.2º, and 27.05±0.2º.

[0056] In some embodiments, the X-ray powder diffraction pattern of the B crystal form is substantially consistent with that in Figure 5.

[0057] In some embodiments, the DSC spectrum of the B crystal form has an endothermic peak at 150.08±5℃.

[0058] In some implementations, the DSC spectrum of the B crystal form is substantially consistent with that in Figure 6.

[0059] This disclosure also provides a method for preparing the benzoate B crystal form of the compound of formula (I), including a salt formation reaction step of the compound of formula (I) with benzoic acid.

[0060] In some embodiments of this disclosure, the preparation method of the above-mentioned B crystal form includes the following steps: (1-b) Mix the compound of formula (I) with a solvent; (2-b) Add benzoic acid to the mixture from step (1-b); (3-b) The mixture of the compound of formula (I) and the benzoic acid is stirred to allow crystallization; (4-b) Filter out and collect the solids.

[0061] In some embodiments, the solvent in step (1-b) is selected from at least one of cyclohexane, n-heptane, toluene, chloroform, diethyl ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, acetone, methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, methanol, ethanol, isopropanol, n-butanol, and acetonitrile, preferably at least one of isopropyl ether, methyl tert-butyl ether, methanol, isopropanol, and tetrahydrofuran, more preferably isopropyl ether.

[0062] In some embodiments, the molar ratio of the compound of formula (I) to benzoic acid is 1:2 to 2:1, preferably 1:1.

[0063] In some embodiments, step (1-b) is performed at 15-30°C, preferably at 20-25°C.

[0064] In some embodiments, the stirring in step (3-b) is carried out under heating conditions, preferably at a heating temperature of about 40-68°C, more preferably at 60-68°C.

[0065] Furthermore, the method for preparing the benzoate B crystal form of compound (I) further includes the following steps: (5-b) Mix the solid collected in step (4-b) with the solvent; (6-b) Stir the resulting mixture; (7-b) Filter out and collect the solids, and dry them.

[0066] In some embodiments, the solvent in step (5-b) is selected from at least one of isopropyl ether, methyl tert-butyl ether, methanol, isopropanol and tetrahydrofuran, preferably isopropyl ether.

[0067] In some embodiments, step (6-b) is performed at 15-30°C, preferably at 20-25°C.

[0068] This disclosure also provides the crystal form of the succinate of compound (I).

[0069] In some implementations, the succinate of formula (I) is in anhydrous form.

[0070] This disclosure provides a succinate A crystal form of compound (I), wherein the A crystal form has diffraction peaks at 7.13±0.2º, 9.30±0.2º, 11.29±0.2º, 15.51±0.2º, and 19.89±0.2º in its X-ray powder diffraction pattern expressed as diffraction angle 2θ.

[0071] In some embodiments, the A crystal form, in its X-ray powder diffraction pattern expressed as diffraction angle 2θ, exhibits diffraction peaks at 7.13±0.2º, 9.30±0.2º, 11.29±0.2º, 15.51±0.2º, 17.10±0.2º, 19.89±0.2º, 20.26±0.2º, 20.91±0.2º, 21.51±0.2º, 22.69±0.2º, 23.58±0.2º, and 25.42±0.2º.

[0072] In some embodiments, the A crystal form, in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ, is located at 5.01±0.2º, 7.13±0.2º, 9.30±0.2º, 10.09±0.2º, 11.29±0.2º, 11.73±0.2º, 13.75±0.2º, 14.30±0.2º, 15.18±0.2º, 15.51±0.2º, 16.00±0.2º, 17.10±0.2º, 18.25±0.2º, 1 8.69±0.2º, 19.89±0.2º, 20.26±0.2º, 20.91±0.2º, 21.12±0.2º, 21.51±0.2º, 22.69±0.2º, 23. There are diffraction peaks at 05±0.2º, 23.58±0.2º, 24.58±0.2º, 25.42±0.2º, 26.66±0.2º, 27.24±0.2º and 29.47±0.2º.

[0073] In some embodiments, the A crystal form, in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ, exhibits the following diffraction angles: 5.01±0.2º, 7.13±0.2º, 9.30±0.2º, 10.09±0.2º, 11.29±0.2º, 11.73±0.2º, 13.75±0.2º, 14.30±0.2º, 15.18±0.2º, 15.51±0.2º, 16.00±0.2º, 17.10±0.2º, 18.25±0.2º, 18.69±0.2º, 19.89±0.2º, 2 0.26±0.2º, 20.91±0.2º, 21.12±0.2º, 21.51±0.2º, 22.69±0.2º, 23.05±0.2º, 23.58±0.2º, 24.58±0.2º, 25. There are diffraction peaks at 42±0.2º, 26.66±0.2º, 27.24±0.2º, 28.65±0.2º, 29.47±0.2º, 30.90±0.2º, 32.31±0.2º, and 34.45±0.2º.

[0074] In some embodiments, the X-ray powder diffraction pattern of the A-type crystal is substantially consistent with that in Figure 8.

[0075] In some embodiments, the DSC spectrum of the A crystal form has an endothermic peak at 186.82±5℃.

[0076] In some implementations, the DSC spectrum of the A crystal form is substantially consistent with that in Figure 9.

[0077] In some embodiments, the cell parameters of the A crystal form are a = 24.6731(4) Å, b = 24.6731(4) Å, c = 10.2575(3) Å, α = 90°, β = 90°, γ = 90°.

[0078] This disclosure also provides a method for preparing succinate A crystal form of compound (I), comprising the following steps: (1-c) Mix the compound of formula (I) with a solvent; (2-c) Add succinic acid to the mixture from step (1-c); (3-c) The mixture of the compound of formula (I) and the succinic acid is stirred to precipitate a solid; (4-c) Filter out and collect the solids.

[0079] In some embodiments, the solvent in step (1-c) is selected from at least one of cyclohexane, n-heptane, toluene, chloroform, diethyl ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, acetone, methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, methanol, ethanol, isopropanol, n-butanol, and acetonitrile, preferably at least one of isopropyl ether, methyl tert-butyl ether, methanol, isopropanol, and tetrahydrofuran, more preferably isopropyl ether.

[0080] In some embodiments, the molar ratio of the compound of formula (I) to succinic acid is 1:2 to 2:1, preferably 1:1.

[0081] In some embodiments, step (3-c) is carried out at 15-30°C, preferably at 20-25°C.

[0082] In some implementations, the reaction time for step (3-c) is 1-48 hours, preferably 3-24 hours, and more preferably 8-16 hours.

[0083] Furthermore, the method for preparing the succinate A crystal form of compound (I) further includes the following steps: (5-c) Mix the solid collected in step (4-c) with the solvent; (6-c) Stir the resulting mixture to allow crystallization; (7-c) Filter out and collect the solids, and dry them.

[0084] In some embodiments, the solvent in step (5-c) is selected from at least one of methanol, ethanol, isopropanol, tetrahydrofuran, and water, preferably a mixture of ethanol and water.

[0085] In some embodiments, the volume ratio of ethanol to water is approximately 1:1 to 50:1, preferably 2:1 to 20:1; in some specific embodiments of this disclosure, the volume ratio of ethanol to water is approximately 10:1, 9:1, 8:1, 7:1, 6:1, 5:1 or 4:1.

[0086] In some embodiments, step (6-c) is carried out at 15-30°C, preferably at 20-25°C.

[0087] In some embodiments, the stirring and crystallization time in step (6-c) is 0.5-48 hours, preferably 1-12 hours, and more preferably 2-4 hours.

[0088] This disclosure also provides pharmaceutical compositions comprising a pharmaceutically acceptable salt or crystal form of a compound of formula (I) above and pharmaceutically acceptable excipients.

[0089] This disclosure also relates to the use of pharmaceutically acceptable salts or crystal forms of compounds of formula (I) above, or the pharmaceutical compositions thereof, in the preparation of medicaments for the prevention or treatment of estrogen receptor-related diseases.

[0090] Furthermore, this disclosure relates to the use of pharmaceutically acceptable salts or crystal forms of compounds of formula (I) above, or pharmaceutical compositions thereof, in the prevention or treatment of estrogen receptor-related diseases.

[0091] Furthermore, this disclosure relates to pharmaceutically acceptable salts or crystal forms of the compounds of formula (I) above, or pharmaceutical compositions thereof, for the prevention or treatment of estrogen receptor-related diseases.

[0092] This disclosure also relates to a method for treating estrogen receptor-related diseases, the method comprising administering a therapeutically effective dose of a pharmaceutically acceptable salt or crystal form of the compound of formula (I) above, the pharmaceutical composition above, or a pharmaceutical preparation comprising a pharmaceutically acceptable salt or crystal form of the compound of formula (I) above of this disclosure.

[0093] This disclosure also relates to the use of pharmaceutically acceptable salts or crystal forms of compounds of formula (I) above, or the pharmaceutical compositions thereof, in the preparation of medicaments for the prevention or treatment of tumors.

[0094] Furthermore, this disclosure relates to the use of pharmaceutically acceptable salts or crystal forms of compounds of formula (I) above, or pharmaceutical compositions thereof, in the prevention or treatment of tumors.

[0095] Furthermore, this disclosure relates to pharmaceutically acceptable salts or crystal forms of the compounds of formula (I) above, or pharmaceutical compositions thereof, for the prevention or treatment of tumors.

[0096] This disclosure also relates to a method of treating tumors, the method comprising administering a therapeutically effective dose of a pharmaceutically acceptable salt or crystal form of the compound of formula (I) above, the pharmaceutical composition above, or a pharmaceutical preparation comprising a pharmaceutically acceptable salt or crystal form of the compound of formula (I) above of this disclosure.

[0097] In some implementations, the estrogen receptor-related diseases include, but are not limited to, tumors.

[0098] In some implementations, the estrogen receptor-related disease or tumor is breast cancer.

[0099] In some implementations, the estrogen receptor-related disease or tumor is ER-positive breast cancer.

[0100] In some implementations, the estrogen receptor-related disease or tumor is ER-positive breast cancer with brain metastases.

[0101] In some implementations, the individual is a cancer patient.

[0102] In some implementations, the individual is a breast cancer patient.

[0103] In some implementations, the individual is an ER-positive breast cancer patient.

[0104] In some implementations, the individual is an ER-positive breast cancer patient with brain metastases.

[0105] Technical effect

[0106] The pharmaceutically acceptable salts of the compounds of formula (I) disclosed herein and their crystallization have at least one of the following advantages:

[0107] The pharmaceutically acceptable salts of the compounds of formula (I) disclosed herein and their crystals have at least one of the following advantages: good water solubility, high purity, good stability, and low hygroscopicity;

[0108] Compared to free bases or other salt forms, the benzoate, succinate, or adipate salts of compounds of formula (I) and their crystal forms can effectively suppress the formation of isomers and / or have better thermal stability;

[0109] The pharmaceutically acceptable salts or crystal forms of the compounds of formula (I) provided in this disclosure also have at least one of the advantages of simple preparation method, mild crystallization conditions, high crystallinity, good solubility, good stability and non-hygroscopicity, making them suitable for preparation into desired pharmaceutical compositions. []

[0110] Definitions and Explanations

[0111] The pharmaceutically acceptable salt of the compound of formula (I) disclosed herein is administered daily at a dose of 0.01 mg / kg to 200 mg / kg body weight, preferably 0.05 mg / kg to 50 mg / kg body weight, more preferably 0.1 mg / kg to 30 mg / kg body weight, in the form of single or separate doses.

[0112] Typical routes of administration for the pharmaceutically acceptable salts or their crystalline forms or polymorphs, or combinations thereof, or pharmaceutical compositions thereof, disclosed herein include, but are not limited to, oral, rectal, local, inhalation, parenteral, sublingual, vaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0113] In an optional embodiment, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These excipients enable the compounds of this application to be formulated into tablets, pills, lozenges, sugar-coated tablets, capsules, liquids, gels, pastes, suspensions, etc., for oral administration to patients.

[0114] Solid oral compositions can be prepared using conventional mixing, filling, or tableting methods. For example, they can be obtained by mixing the active compound with solid excipients, optionally milling the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into granules to obtain the core of a tablet or sugar-coated formulation. Suitable excipients include, but are not limited to, binders, diluents, disintegrants, lubricants, glidants, sweeteners, or flavoring agents.

[0115] The term “about” is used in this disclosure to mean approximately, around. When the term “about” is used in conjunction with a digit range, the range is modified by expanding the upper and lower limits of the stated digit range. Unless otherwise stated, the term “about” is used herein to modify the upper and lower limits of the stated value by a digit value with a 10% deviation.

[0116] Unless otherwise stated, the terms “comprise,” “comprise,” or “comprise,” and their English variations such as “comprises” or “comprising,” shall be understood in an open, non-exclusive sense, meaning “including but not limited to.”

[0117] Throughout this specification, the terms "some embodiments," "optional embodiments," or "implementation" refer to specific reference elements, structures, or features that are relevant to the description in at least one embodiment. Therefore, the phrases "some embodiments," "optional embodiments," or "implementation" appearing in different places throughout the specification do not necessarily all refer to the same embodiment. Furthermore, specific elements, structures, or features may be combined in one or more embodiments in any suitable manner.

[0118] The room temperature mentioned in this disclosure refers to 20 ± 5 °C.

[0119] The range "m~n" as described in this disclosure is a shortened representation of any combination of real numbers between m and n, where m and n are real numbers. The numerical range "0.5~2" includes, but is not limited to, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0, which have been listed herein.

[0120] The "X-ray powder diffraction pattern" described in this disclosure was obtained using Cu-Kα radiation measurements.

[0121] The “X-ray powder diffraction pattern or XRPD pattern” mentioned in this disclosure refers to the X-ray powder diffraction pattern obtained when X-rays are incident on an atomic surface of a crystal or part of a crystal sample with a lattice spacing of d at a grazing angle θ (the complementary angle of the incident angle, also known as the Bragg angle) with a grazing angle θ.

[0122] The crystal form disclosed in this disclosure is a substantially pure crystal. The term "substantially pure" as used in this application means that the purity of the crystal form is at least 85 wt%, preferably at least 90 wt%, and more preferably at least 95 wt%.

[0123] For the same compound and the same crystal form, the peak positions of their XRPD spectra are generally similar, but the relative intensity error may be large. It should also be noted that in the identification of mixtures, factors such as decreased content may cause the absence of some diffraction lines. In this case, it is not necessary to rely on all diffraction peaks observed in the high-purity sample; even a single diffraction peak may be characteristic for a given crystal.

[0124] The “2θ or 2θ angle” mentioned in this disclosure refers to the diffraction angle, where θ is the Bragg angle, and the unit is º or degrees; the error range of 2θ for each diffraction peak is ±0.20º.

[0125] 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.

[0126] The drying temperature described in this disclosure is generally 20 °C to 100 °C, preferably 25 °C to 70 °C, more preferably 40 °C to 60 °C, and can be dried under normal pressure or reduced pressure. Preferably, drying is carried out under reduced pressure.

[0127] For those skilled in the art, the determination of the molar ratio of the disclosed compounds to acid molecules is subject to a certain degree of error; generally, ±10% is within a reasonable error range. The error may vary to some extent depending on the context in which it is used, but this variation shall not exceed ±10%, preferably ±5%.

[0128] The term "treatment" means administering the compound or preparation described in this application to improve or eliminate a disease or one or more symptoms related to said disease, and includes: (i) Suppress the disease or disease state, that is, curb its development; (ii) Relieve the disease or disease state, even if the disease or disease state subsides.

[0129] The term "prevention" means administering the compound or preparation described in this application to prevent a disease or one or more symptoms associated with said disease, and includes:

[0130] To prevent the occurrence of disease or disease state in objects (e.g., mammals), especially when such objects are susceptible to the disease state but have not yet been diagnosed with it.

[0131] The term "therapeuticly effective dose" refers to the amount of the disclosed compound used to treat a particular disease, condition, or disorder; (ii) to reduce, improve, or eliminate one or more symptoms of a particular disease, condition, or disorder; or (iii) to delay the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of the disclosed compound constituting a "therapeuticly effective dose" varies depending on the compound, the disease state and its severity, the route of administration, and the age of the mammal to be treated, but may routinely be determined by a person skilled in the art based on their own knowledge and the content of this disclosure.

[0132] The term "pharmaceuticalally acceptable excipient" refers to excipients that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0133] In this application, the terms "individual" or "patient" include both mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates (e.g., chimpanzees and other apes and monkeys); livestock, such as cattle, horses, sheep, goats, and pigs; domesticated animals, such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs. Examples of non-human mammals include, but are not limited to, birds and fish.

[0134] The testing conditions of the instruments used in this experiment are as follows: 1. Differential Scanning Calorimeter (DSC) Instrument Model: TA DSC2500 Purge gas: Nitrogen Sample tray: Aluminum tray, non-sealed cap Method: Linear heating Heating rate: 10 °C / min Temperature range: 30 °C ~ Set endpoint temperature 2. X-ray Powder Diffraction (XRPD) Instrument Model: Bruker D8 Focus Ray: Cu Kα,Kα1(Å):1.54060; Kα2(Å):1.54439 Kα1 / Kα2:2 Slit (°): 2.5 Scanning mode: θ / 2θ, scanning range: 3-40° Dwell time (seconds): 0.12 Scan step size (°2θ): 0.01 Voltage: 40 kV, Current: 40 mA 3. Thermogravimetric Analysis (TGA) Instrument Model: TA TGA550 Purge gas: Nitrogen Sample tray: Platinum, open Method: Linear heating Heating rate: 10 °C / min Temperature range: 30 °C ~ Set endpoint temperature 4. High-performance liquid chromatography (HPLC) for saturation solubility test Chromatographic column: Water XBridge C18 (4.6*100 mm, 3.5 μm) Mobile phase A: 0.01 mol / L ammonium chloride solution (pH 10.0) Mobile phase B: Acetonitrile Isocratic conditions: mobile phase A:B = 50:50, isocratic for 15 min Flow rate: 1.5 mL / min Wavelength: 260 nm Column temperature: 35℃ Injection volume: 10 µl Diluent: Acetonitrile 5. High-performance liquid chromatography (HPLC) for the detection of isomer impurities Chromatographic column: CHIRALPAK AD-H (4.6*250mm, 5μm) Mobile phase A: n-hexane Mobile phase B: 0.1% diethylamine-ethanol Isocratic conditions: Mobile phase A:Mobile phase B = 70:30, isocratic operation for 15 minutes. Flow rate: 1.0 mL / min Wavelength: 260 nm Column temperature: 35℃ Injection volume: 10 µl Diluent: 0.1% diethylamine-ethanol High-performance liquid chromatography (HPLC) for stability testing of pharmaceutically acceptable salts of compound (I) Chromatographic column: Water XBridge C18 (4.6*150mm, 3.5μm) Mobile phase A: 0.01 mol / L ammonium chloride solution (adjusted to pH 10.0 with ammonia water) Mobile phase B: Acetonitrile Flow rate: 1.0 mL / min Wavelength: 285 nm Column temperature: 35℃ Injection volume: 20 µl

[0135] Those skilled in the art will recognize that measurements of XRPD peak positions and / or intensities for a given crystalline form of the same compound will vary within a range of error. The 2θ value allows for an appropriate range of error. Typically, the range of error is expressed in "±". For example, 5.92 ± 0.2º 2θ represents a range of approximately 6.12 to 5.72. Depending on sample preparation techniques, calibration techniques applied to the instrument, human operational bias, etc., those skilled in the art will recognize that an appropriate range of error for XRPD diffraction angles (2θ) can be approximately ±0.20º, ±0.15º, ±0.10º, ±0.05º, or less. When used to describe XRPD plots, the terms "substantially consistent" or "substantially as shown" refer to a plot that includes at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 90%, or at least 99% of diffraction angles within a standard deviation of ±0.2º 2θ.

[0136] Because those skilled in the art recognize that measurements from DSC thermograms of a given crystalline form of the same compound will vary within error tolerances, the single-peak value (expressed in degrees Celsius) allows for an appropriate range of error. Typically, this range is expressed in "±". For the same crystalline form of the same compound, in successive analyses, errors in thermal transition temperature and melting point are typically within approximately ±5.0 °C. For example, a peak value of "186.82 ± 5 °C" indicates a range from 181.82 to 191.82. Depending on sample preparation techniques, calibration techniques applied to the instrument, human operational bias, etc., those skilled in the art recognize that an appropriate range of error for a single-peak value can be ±5.0, ±4.0, ±3.0, ±2.0, or less.

[0137] The lux (legal symbol lx) is the unit of luminance. An object uniformly illuminated has an luminous flux of 1 lumen over an area of ​​1 square meter, and its luminance is 1 lux.

[0138] Example []

[0139] The following embodiments detail the technical solutions of the invention, but the scope of protection of this disclosure includes, but is not limited to, these embodiments.

[0140] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The unit of NMR shift is 10⁻⁶ (ppm). The solvents used for NMR determination are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is tetramethylsilane (TMS); "IC 50" refers to the half-maximum inhibition concentration, which is the concentration at which half of the maximum inhibition effect is achieved.

[0141] Example 1: Synthesis of N-((S)(1-(3-fluoropropyl)pyrrolidine-3-yl)-6-((1S, 3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-amine (compound of formula (I))

[0142] Synthesis method:

[0143] Step 1: Synthesis of (S)-tert-butyl(1-(3-fluoropropyl)pyrrolidine-3-yl)aminomethyl ester

[0144] (S)-tert-butylpyrrolidone-3-ylaminomethyl ester (500.00 mg, 2.68 mmol) was dissolved in tetrahydrofuran (10 mL), and sodium hydroxide solution (5 mol·L⁻¹, 1.07 mL) and 1-iodo-3-fluoropropane (529.88 mg, 2.82 mmol) were added. The reaction mixture was stirred at 25 °C for 16 hours. After the reaction was complete as determined by TLC, the reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated ammonium chloride solution (10 mL). The aqueous and organic phases were collected separately. The aqueous phase was extracted three times with ethyl acetate (20 mL). All organic phases were combined, dried over sodium sulfate, concentrated under reduced pressure, and then purified by column chromatography (silicon dioxide, dichloromethane / methanol). [=]100 / 1) yielded product (S)-tert-butyl(1-(3-fluoropropyl)pyrrolidine-3-yl)aminomethyl ester (480.00 mg).

[0145] 1H NMR (400 MHz, METHANOL- d 4) δ 4.58-4.53 (m, 1H), 4.46-4.40 (m, 1H), 4.14-4.04 (m, 1H), 2.93-2.85 (m, 1H), 2.77-2.67 (m, 1H), 2.61 (dd, J =7.78, 5.52 Hz, 3H), 2.47-2.40 (m, 1H), 2.29-2.17 (m, 1H), 1.99-1.82 (m, 2H), 1.71-1.61 (m, 1H), 1.45 (s, 9H).

[0146] Step 2: Synthesis of (S)-1-(3-fluoropropyl)pyrrolidine-3-amine hydrochloride

[0147] (S)-tert-butyl(1-(3-fluoropropyl)pyrrolidine-3-yl)aminomethyl ester (480.00 mg, 1.93 mmol) was dissolved in 1,4-dioxane (3 mL), and then 1,4-dioxane hydrochloride solution (4 mol·L⁻¹, 4.94 mL) was added. The reaction solution was a yellow, transparent solution. The reaction solution was stirred at 25 °C for 3 hours. After the reaction was confirmed to be complete by TLC, the reaction solution was concentrated under reduced pressure to obtain compound (S)-1-(3-fluoropropyl)pyrrolidine-3-amine hydrochloride (450.00 mg).

[0148] 1H NMR (400 MHz, DMSO- d 6) δ 8.80-8.42 (m, 3H), 4.62 (s, 1H), 4.51 (s, 1H), 4.12-3.45 (m, 3H), 3.17 (br s, 3H), 2.35-1.99 (m, 4H).

[0149] Step 3: Synthesis of (R)-1-(1H-indol-3-yl)-N-(2,2,2-trifluoroethyl)propane-2-amine

[0150] (2R)-1-(1H-indol-3-yl)propane-2-amine (600 mg, 3.44 mmol) and N,N-diisopropylethylamine (445.05 mg, 3.44 mmol) were dissolved in 1,4-dioxane (10 mL). Trifluoroethyl trifluoromethanesulfonate (1.20 g, 5.17 mmol) dissolved in 1,4-dioxane (5 mL) was added at 25 °C. The reaction mixture was stirred at 75 °C for 16 hours. The reaction mixture was concentrated to dryness under reduced pressure. Then, it was purified by column chromatography (silicon dioxide, petroleum ether / ethyl acetate = 3 / 1) to give the product (R)-1-(1H-indol-3-yl)-N-(2,2,2-trifluoroethyl)propane-2-amine (0.69 g).

[0151] MS m / z (ESI): 257.2 [M+H] +;

[0152] 1H NMR (400MHz, METHANOL-d 4) δ 7.56-7.54 (d, J= 8.0 Hz, 1H), 7.36-7.34 (d, J= 8.0 Hz, 1H), 7.12-7.08 (m, 2H), 7.03-7.00 (m, 1H), 3.26-3.23 (m, 2H), 3.12-3.10 (m, 1H), 2.93-2.88 (m, 1H), 2.80-2.78 (m, 1H), 1.11 (d, J= 6.0 Hz, 3H)

[0153] Step 4: Synthesis of (1S,3R)-1-(5-bromopyridin-2-yl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole

[0154] (R)-1-(1H-indol-3-yl)-N-(2,2,2-trifluoroethyl)propane-2-amine (120.00 mg, 468.26 μmol) was dissolved in toluene (2 mL), followed by the addition of 5-bromo-pyridine-2-carboxaldehyde (87.10 mg, 468.26 μmol) and acetic acid (562.40 mg, 9.37 mmol). The reaction solution was a yellow, transparent solution. The reaction solution was stirred at 90 °C for 10 hours. After the reaction was detected by LCMS, the reaction solution was cooled to room temperature, concentrated to dryness under reduced pressure, and purified by thin-layer chromatography (silicon dioxide, petroleum ether / ethyl acetate = 4 / 1) to obtain (1S,3R)-1-(5-bromopyridin-2-yl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (110.00 mg).

[0155] MS m / z (ESI): 424.1, 426.1 [M+H] +.

[0156] 1H NMR (400 MHz, METHANOL- d 4) δ 8.59 (s, 1H), 8.01-7.94 (m, 1H), 7.54 (d, J =8.53 Hz, 1H), 7.46 (d, J =7.78 Hz, 1H), 7.30 (d, J =8.03 Hz, 1H), 7.08 (d, J =7.59 Hz, 1H), 7.03-6.96 (m, 1H), 5.08 (s, 1H), 3.58-3.46 (m, 1H), 3.38-3.34 (m, 1H), 3.13-2.99- (m, 1H), 2.80 (d, J =4.52 Hz, 1H), 2.70-2.61 (m, 1H), 1.26 (d, J =6.78 Hz, 3H).

[0157] Step 5: Synthesis of N-((S)-1-(3-fluoropropyl)pyrrolidine-3-yl)-6-(((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-amine (compound of formula (I))

[0158] (1S,3R)-1-(5-bromopyridin-2-yl)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole (140.00 mg, 263.99 μmol) was dissolved in tetrahydrofuran (3 mL), and (S)-1-(3-fluoropropyl)pyrrolidine-3-amine hydrochloride (86.77 mg, 316.79 μmol), sodium tert-butoxide (152.22 mg, 1.58 mmol), and methanesulfonic acid (2-dicyclohexylphosphine)-3,6-dimethoxy-2,4,6-triisopropyl-1,1-biphenyl)(2-amino-1,1-biphenyl-2-yl)palladium(II) (23.93 mg, 26.40 μmol) were added. The reaction mixture was stirred at 80 °C for 4 hours under nitrogen atmosphere. After the reaction was detected by LCMS, the solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. Then, it was purified by preparative liquid chromatography (Phenomenex Gemini C18 column, 3 μm silicon dioxide, 30 mm diameter, 75 mm length). The compound N-((S)-1-(3-fluoropropyl)pyrrolidine-3-yl)-6-(((1S,3R)-3-methyl-2-(2,2,2-trifluoroethyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)pyridin-3-amine (37.79 mg) was obtained.

[0159] MS m / z (ESI): 365.1 [M+H] +.

[0160] 1H NMR (400 MHz, METHANOL- d 4) δ 7.89 (d, J =2.76 Hz, 1H), 7.46 (d, J =7.78 Hz, 1H), 7.25 (d, J =8.53 Hz, 2H), 7.09-7.03 (m, 2H), 7.02-6.97 (m, 1H), 4.98 (s, 1H), 4.60 (t, J =5.65 Hz, 1H), 4.49 (t, J =5.65 Hz, 1H), 4.18 (br s, 1H), 3.51-3.35 (m, 4H), 3.14-2.99 (m, 5H), 2.92 (dd, J =15.18, 4.89 Hz, 1H), 2.65 (dd, J =16.06, 6.78 Hz, 1H), 2.53-2.42 (m, 1H), 2.12-1.92 (m, 3H), 1.20 (d, J =6.78 Hz, 3H).

[0161] Identification of the absolute configuration of compounds of formula (I)

[0162] The NOESY spectrum (Figure 1) shows that the methyl hydrogen at the 3-position and the hydrogen at the 1-position of compound (I) have a significant NOE effect, proving that they are on the same side. The relative configuration of the pyridyl group at the 1-position and the methyl group at the 3-position on the 6-membered guanidine ring is trans. Since the absolute configuration of the carbon atom at the 3-position is R, the absolute configuration of the carbon atom at the 1-position is S.

[0163] Example 2: Preparation of sulfate of compound (I)

[0164] 500 mg of compound (I) and 10 ml of isopropyl ether were added to a 50 ml single-necked flask and stirred at room temperature until the solid was completely dissolved. 100 mg of concentrated sulfuric acid was added, and the mixture was heated to reflux and kept at this temperature for 1 h. The mixture was then cooled to room temperature and stirred overnight. The mixture was filtered and dried under vacuum at 40 °C for 3 h to obtain 0.58 g of solid.

[0165] Example 3: Preparation of phosphate of compound (I)

[0166] 0.5 g of compound (I) and 10 ml of isopropyl ether were added to a 50 ml single-necked flask and stirred at room temperature until the solid was completely dissolved. 118 mg of phosphoric acid was added, and the mixture was heated to reflux and kept at this temperature for 1 h. The mixture was then cooled to room temperature and stirred overnight. The mixture was filtered and dried under vacuum at 40 °C for 3 h to obtain 0.53 g of solid.

[0167] Example 4: Preparation of fumarate of compound (I)

[0168] 0.5 g of compound (I) and 10 ml of isopropanol were added to a 50 ml single-necked flask and stirred at room temperature until the solid was completely dissolved. 118.5 mg of fumaric acid was added, and the reaction solution was heated to reflux and kept at this temperature for 1 h. The mixture was then cooled to room temperature and stirred overnight. The mixture was filtered and dried under vacuum at 40 °C for 3 h to obtain 0.3 g of solid.

[0169] Example 5: Preparation of L-malate of compound (I)

[0170] Add 50 mg of compound (I) and 1 ml of isopropyl ether to a 5 ml centrifuge tube, stir at room temperature until the solid is completely dissolved, add 13.7 mg of L-malic acid, stir at room temperature overnight, filter, and dry the solid under vacuum at room temperature for 3 h to obtain 30 mg of solid.

[0171] Example 6: Preparation of citrate of compound (I)

[0172] Add 50 mg of compound (I) and 1 ml of methyl tert-butyl ether to a 5 ml centrifuge tube, stir at room temperature until the solid is completely dissolved, add 21.5 mg of citric acid, stir at room temperature for 3 h, filter, and dry the solid under vacuum at room temperature for 3 h to obtain 26 mg of solid.

[0173] Example 7: Preparation of the C crystal form of compound (I) adipate

[0174] 0.7 g of adipic acid and 19 ml of isopropanol were added to a 100 ml single-necked flask and heated to 60 °C until the solid dissolved completely. 2.36 g of compound (I) and 19 ml of isopropanol were added to the same flask and stirred at room temperature until dissolved. Then, the adipic acid-isopropanol solution was added dropwise, and the mixture was stirred overnight at room temperature. Crystallization occurred, and the mixture was filtered and dried under vacuum at room temperature for 3 hours to obtain 1.66 g of solid. The obtained solid was confirmed to be in crystalline form (C-type) by X-ray powder diffraction (XRPD). The XRPD pattern of the C-type is shown in Figure 2, its DSC pattern in Figure 3, and its TGA pattern in Figure 4. The peak value of its DSC endothermic peak is around 149.89 °C. The positions of its XRPD diffraction peaks are shown in Table 1 below. Table 1 Peak number 2. θ value [o or degree] d[Å] Relative Intensity (%) 1 4.458 19.80532 62.02% 2 6.300 14.01734 100.00% 3 7.025 12.57291 11.55% 4 8.899 9.92934 21.43% 5 10.647 8.30269 6.37% 6 11.947 7.40198 4.27% 7 12.551 7.04674 3.82% 8 12.961 6.82520 3.07% 9 13.342 6.63099 4.26% 10 14.200 6.23215 3.74% 11 15.204 5.82268 1.97% 12 16.023 5.52687 9.86% 13 16.451 5.38418 12.52% 14 17.777 4.98527 12.54% 15 18.325 4.83751 3.54% 16 19.098 4.64343 21.85% 17 19.766 4.48806 8.68% 18 20.123 4.40924 7.01% 19 21.030 4.22097 11.66% 20 21.620 4.10711 4.33% 21 22.443 3.95833 7.66% 22 22.524 3.94430 7.75% 23 22.542 3.94124 7.45% 24 23.352 3.80631 2.95% 25 23.977 3.70841 1.84% 26 24.300 3.65994 5.85% 27 24.860 3.57867 5.44% 28 25.374 3.50730 4.11% 29 25.973 3.42779 5.47% 30 27.393 3.25328 6.39% 31 27.976 3.18672 3.71% 32 28.807 3.09673 1.87%

[0175] 1H NMR (400 MHz, DMSO) δ 10.60 (s, 1H), 7.85 (d, J = 2.7 Hz, 1H), 7.39 (t, J = 9.5 Hz, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.11 – 6.86 (m, 4H), 5.99 (d, J = 6.8 Hz, 1H), 4.91 (s, 1H), 4.53 (t, J = 6.0 Hz, 1H), 4.42 (t, J = 6.0 Hz, 1H), 3.85 (d, J = 3.2 Hz, 1H), 3.55 (dq, J = 20.2, 10.0 Hz, 1H), 3.35 – 3.25 (m, 1H), 3.01 (dq, J = 19.3, 9.6 Hz, 1H), 2.81 (dd, J = 9.2, 7.0 Hz, 1H), 2.71 – 2.52 (m, 3H), 2.49 – 2.41 (m, 3H), 2.34 (dd, J = 9.3, 4.5 Hz, 1H), 2.25 – 2.13 (m, 5H), 1.87 – 1.72 (m, 2H), 1.61 – 1.52 (m, 1H), 1.52 – 1.41 (m, 4H), 1.13 (d, J = 6.7 Hz, 3H).

[0176] Example 8: Preparation of the B crystal form of compound (I)

[0177] 3.0 g of compound (I) and 100 ml of isopropyl ether were added to a 250 ml single-necked flask and stirred at room temperature until the solid was completely dissolved. 0.75 g of benzoic acid was added and stirred at room temperature overnight. The mixture was then filtered. The filter cake and 30 ml of isopropyl ether were added to a 100 ml single-necked flask and stirred at room temperature for 1 h to induce crystallization. The crystals were then filtered and dried under vacuum at room temperature for 3 h to obtain 3.11 g of solid. The obtained solid was confirmed to be crystalline (B-type) by X-ray powder diffraction. The XRPD pattern of the crystalline solid is shown in Figure 5, its DSC pattern is shown in Figure 6, and its TGA pattern is shown in Figure 7. The peak value of the DSC endothermic peak is around 150.08 °C. The positions of the XRPD diffraction peaks are shown in Table 2 below. Table 2 Peak sequence number 2. θ value [o or degree] d[Å] Relative strength (%) 1 4.692 18.81760 38.49% 2 4.905 18.00028 15.27% 3 6.343 13.92354 32.41% 4 7.193 12.27979 84.17% 5 9.370 9.43108 9.39% 6 9.827 8.99308 20.60% 7 10.007 8.83218 53.01% 8 10.319 8.56606 9.43% 9 11.111 7.95703 21.44% 10 11.388 7.76420 51.80% 11 12.064 7.33060 6.73% 12 12.688 6.97112 12.47% 13 13.516 6.54578 3.97% 14 14.067 6.29085 16.11% 15 14.359 6.16354 15.27% 16 14.781 5.98852 27.88% 17 17.303 5.12098 9.90% 18 18.780 4.72137 100.00% 19 19.007 4.66536 61.65% 20 19.249 4.60735 46.38% 21 19.659 4.51223 62.54% 22 19.912 4.45531 59.93% 23 20.091 4.41613 85.94% 24 20.796 4.26793 31.48% 25 21.146 4.19817 18.68% 26 21.657 4.10014 31.29% 27 22.350 3.97456 9.67% 28 23.573 3.77102 16.86% 29 23.702 3.75082 19.76% 30 24.634 3.61104 9.39% 31 25.681 3.46609 6.73% 32 27.048 3.29390 7.99%

[0178] 1H NMR (400 MHz, DMSO) δ 10.60 (s, 1H), 7.98 – 7.91 (m, 2H), 7.85 (d, J = 2.7 Hz, 1H), 7.62 (t, J = 7.4 Hz, 1H), 7.50 (t, J = 7.6 Hz, 2H), 7.40 (d, J = 7.6 Hz, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.09 – 6.99 (m, 2H), 6.97 – 6.89 (m, 2H), 6.00 (d, J = 6.8 Hz, 1H), 4.90 (s, 1H), 4.54 (t, J = 6.0 Hz, 1H), 4.42 (t, J = 6.0 Hz, 1H), 3.85 (s, 2H), 3.01 (dt, J = 25.3, 9.6 Hz, 2H), 2.82 (dd, J = 9.2, 6.9 Hz, 1H), 2.71 – 2.54 (m, 3H), 2.45 (dd, J = 11.9, 7.3 Hz, 3H), 2.35 (dd, J = 9.4, 4.5 Hz, 1H), 2.19 (dt, J = 13.4, 7.8 Hz, 1H), 1.91 – 1.73 (m, 2H), 1.55 (dt, J = 12.7, 6.3 Hz, 1H), 1.13 (d, J = 6.7 Hz, 3H).

[0179] Example 9: Preparation of succinate, a compound of formula (I)

[0180] 5.0 g of compound (I) and 100 ml of isopropyl ether were placed in a 250 ml single-necked flask and stirred at room temperature until the solid was completely dissolved. 1.21 g of succinic acid was added, and the mixture was stirred at room temperature overnight. The mixture was then filtered and dried under vacuum at room temperature for 3 h to obtain 5.4 g of succinate of compound (I).

[0181] Example 10: Preparation of the crystal form of compound (I) succinate A

[0182] Example 10-1: The succinate obtained in Example 9 was dissolved in methanol and concentrated under reduced pressure to obtain a foamy solid. 500 mg of the foamy solid was weighed and added to a 10 ml single-necked flask with 4.5 ml of ethanol and 0.5 ml of water. The mixture was stirred at room temperature until the solid dissolved. After stirring for 2 hours, a solid precipitated. The solid was filtered and dried under vacuum at room temperature for 3 hours to obtain 0.22 g of succinate A crystal form of compound (I). The obtained solid was confirmed by X-ray powder diffraction. The XRPD pattern of the crystalline solid is shown in Figure 8, its DSC pattern in Figure 9, and its TGA pattern in Figure 10. The peak value of the DSC endothermic peak is around 186.82 °C. The positions of the XRPD diffraction peaks are shown in Table 3 below. Table 3 Peak number 2. θ value [o or degree] d[Å] Relative Intensity (%) 1 5.009 17.62809 2.60% 2 7.126 12.39530 28.15% 3 9.303 9.49916 18.36% 4 10.094 8.75593 6.21% 5 11.288 7.83226 46.04% 6 11.734 7.53556 4.28% 7 13.752 6.43413 5.95% 8 14.295 6.19071 6.23% 9 15.181 5.83144 5.59% 10 15.508 5.70918 16.05% 11 15.995 5.53652 8.84% 12 17.100 5.18115 12.94% 13 18.253 4.85637 4.70% 14 18.689 4.74414 4.38% 15 19.894 4.45944 100.00% 16 20.258 4.37996 13.01% 17 20.908 4.24534 14.71% 18 21.123 4.20252 7.07% 19 21.514 4.12706 10.75% 20 22.688 3.91620 11.49% 21 23.050 3.85536 5.98% 22 23.579 3.77018 10.22% 23 24.581 3.61866 3.74% 24 25.417 3.50146 14.28% 25 26.662 3.34073 5.88% 26 27.235 3.27173 6.09% 27 28.650 3.11327 1.93% 28 29.472 3.02833 4.89% 29 30.895 2.89201 3.40% 30 32.307 2.76879 3.35% 31 34.449 2.60131 1.59%

[0183] 1H NMR (400 MHz, DMSO) δ 10.59 (s, 1H), 7.86 (d, J = 2.6 Hz, 1H), 7.40 (d, J = 7.7 Hz, 1H), 7.26 (d, J = 7.9 Hz, 1H), 7.08 (d, J = 8.5 Hz, 1H), 7.02 (t, J = 7.5 Hz, 1H), 6.98 – 6.91 (m, 2H), 6.01 (d, J = 6.4 Hz, 1H), 4.91 (s, 1H), 4.54 (t, J = 6.0 Hz, 1H), 4.42 (t, J = 6.0 Hz, 1H), 3.89 (s, 1H), 3.54 (dq, J = 20.1, 10.0 Hz, 1H), 3.31 (dq, J = 13.3, 6.5 Hz, 1H), 3.02 (dq, J = 19.3, 9.6 Hz, 1H), 2.89 (dd, J = 9.4, 6.9 Hz, 1H), 2.67 (dt, J = 8.8, 6.4 Hz, 2H), 2.59 – 2.52 (m, 4H), 2.43 (dd, J = 9.2, 4.1 Hz, 1H), 2.40 (s, 4H), 2.21 (td, J = 13.7, 7.8 Hz, 1H), 1.90 – 1.75 (m, 2H), 1.59 (td, J = 12.6, 7.0 Hz, 1H), 1.14 (d, J = 6.7 Hz, 3H).

[0184] The succinate A crystal form of compound (I) can also be prepared by the following embodiment: 100 mg of the above-mentioned foamy solid and 500 μL of solvent (see Table 4) are added to a 5 ml centrifuge tube, stirred at room temperature for 1-24 hours, the solid is filtered out, and then dried under vacuum at room temperature. See Table 4 for details. Table 4 Implementation Examples solvent Crystal form 10-2 Isopropyl ether Crystal type A 10-3 Methyl tert-butyl ether Crystal type A

[0185] [Example]

[11] [:Mode] [(I)] [Study on Single Crystals of Succinate Compounds] []

[0186] Single crystals of succinate of formula (I) can be prepared by the following method: about 10 mg of the above-mentioned succinate of formula (I) (prepared by Example 9) is placed in a 3 mL sample bottle, 1500 µL of isopropyl ether and 900 µL of tetrahydrofuran are added, and after ultrasonic dissolution, the bottle mouth is sealed with a sealing film and a small hole is punched in the sealing film with a needle. After standing at room temperature for 3 days, crystals are precipitated. The obtained single crystal sample is identified as crystal type A by X-ray powder diffraction.

[0187] Single-crystal X-ray diffraction data were collected and the single-crystal structure was analyzed. The single-crystal structure data are shown in Table 5.

[0188] Based on the single-crystal diffraction results, the single crystal belongs to the tetragonal crystal system, space group I 4. Cell parameters: a = 24.6731(4) Å, b = 24.6731(4) Å, c = 10.2575(3) Å, α = 90°, β = 90°, γ = 90°, cell volume V = 6244.4(3) Å 3. Reliability factors R1 = 0.0891, wR2 = 0.2852, S = 0.992. The stoichiometric formula is C 30H 37F 4N 5O 4, the calculated molecular weight of the asymmetric unit is 607.64, a single unit cell contains 8 asymmetric units, each asymmetric unit contains 1 free base molecule and 1 succinic acid molecule, as shown in Figures 11 and 12.

[0189] The absolute configuration of succinate A, the crystal form of compound (I), is confirmed as follows: Table 5: Single-crystal X-ray diffraction data Chemical formula C 30H 37F 4N 5O 4 Chemical formula weight 607.64 Crystal system Quartet Space Group I 4 a,Å a = 24.6731(4) b,Å b = 24.6731(4) c,Å c = 10.2575(3) α,deg 90° β,deg 90° γ,deg 90° Volume, Å 3 6244.4(3) Z 8 D calc, g cm⁻³ 1.293 Temperature, K 190.0 Radiation (wavelength) Ga Kα(1.34139) instrument BRUKER D8 VENTURE range of h,k,l -32<=h<=29, -31<=k<=32, -11<=l<=13 θ range 2.203-60.581 program SHELXL-2014 Data collected 8778 Unique data 7042 R(F o) 0.0891 RW(F o 2) 0.2852 Goodness of fit 0.992 Absolute configuration determination Flack parameter -0.09(10)

[0190] Testing of biological activity and related properties

[0191] Test Example 1: Saturated solubility test of compound of formula (I) and its pharmaceutically acceptable salt

[0192] Experimental instruments: rotary water bath constant temperature shaker (Taicang Wenerhuijin, model DSHZ-300A); high performance liquid chromatograph (Agilent 1260).

[0193] Experimental method: Accurately weigh about 10 mg of the test sample and place it in a 10 ml centrifuge tube. Add 1 ml of water and shake in a water bath constant temperature shaker (37℃, 150 rpm). If the sample is completely dissolved, add another 10 mg of sample and shake for about 24 hours. Take the supernatant, dilute it appropriately with acetonitrile, and perform HPLC detection. Determine its concentration according to the external standard method.

[0194] Experimental results: The saturated solubility of the test compounds is shown in Table 6. It can be seen that the 24-hour saturated solubility of compounds (I) succinate, benzoate and adipate is significantly better than that of free base. Table 6: Results of Saturated Solubility Test compound samples solvent Solubility at 24 hours (mg / ml) Formula (I) compound succinate (Example 10-1) water 3.27 Compound (I) benzoate (Example 8) 0.70 Compound (I) adipate (Example 7) 2.48 Compound of formula (I) (Example 1) 0.05

[0195] Test Example 2: Impurity Investigation Test of Compound (I) and its Pharmaceutical Salt Isomers

[0196] Experimental Methods: Compound (I) succinate (Example 10-1, crystal form A), compound (I) adipic acid salt (Example 7, crystal form C), and compound (I) (Example 1) were all packaged using two layers of polyethylene and one layer of pharmaceutical composite film. The changes in the content of isomer impurities of compound (I) under conditions of high temperature, light, high humidity, and accelerated reaction were investigated. The results are shown in Table 7. The isomer impurities are isomer 1 and isomer 2, with structures shown below. ND indicates that the impurity was not detected.

[0197] Experimental Results: Under high temperature, light, accelerated, and high humidity conditions, the isomer impurities of compounds (I) succinate and (I) adipate did not show significant growth. However, under 5 days of high temperature conditions, the content of isomer 1 impurity in the free state of compound (I) showed a significant increase. Furthermore, a new isomer 2 impurity was detected in the free state of compound (I) under high temperature, light, accelerated, and high humidity conditions. This experiment demonstrates that the above-mentioned salt forms of the compounds exhibit superior thermal stability and can more effectively suppress the formation of isomer impurities compared to the free base. Table 7: Results of the isomer impurity investigation test Test sample Layout conditions Isomer 1 content % Isomer 2 content % Formula (I) compound succinate 0 o'clock 0.20 ND Compound (I) adipate 0.17 ND Compound of formula (I) 0.03 ND Formula (I) compound succinate 60℃, 8 days 0.23 ND 40℃ / 75%RH, 8 days 0.26 ND 92.5%RH, 8 days 0.25 ND 4500 lux, 8 days 0.23 ND Compound (I) adipate 60℃, 8 days 0.16 ND 40℃ / 75%RH, 8 days 0.23 ND 92.5%RH, 8 days 0.25 ND 4500 lux, 8 days 0.21 ND Compound of formula (I) 60℃, 5 days 0.12 0.48 40℃ / 75%RH, 5 days 0.04 0.10 92.5%RH, 5 days 0.04 0.05 4500 lux, 5 days 0.04 0.34

[0198] Test Example 3: Stability Study of Pharmaceutical Salts of Compound (I)

[0199] Experimental method: Succinate (Example 10-1, crystal form A), benzoate (Example 8, crystal form B), and adipate (Example 7, crystal form C) of formula (I) were packaged with two layers of polyethylene and one layer of pharmaceutical composite film. The changes in the number and total amount of impurities under conditions such as high temperature, light, acceleration and high humidity were investigated. The results are shown in Table 8.

[0200] Experimental results: Succinate, benzoate and adipate of formula (I) can all control the total impurity content well. In particular, succinate and adipate of formula (I) show excellent performance in terms of both total impurity content and impurity growth rate under conditions such as high temperature, light, acceleration and high humidity. Table 8: Results of Stability Tests Test sample Layout conditions Number of impurities and total impurity content Formula (I) compound succinate 0 o'clock 3 / 1.1% 60℃, 5 days 6 / 1.5% 40℃ / 75%RH, 5 days 3 / 1.2% 92.5%RH, 5 days 3 / 1.3% 4500 lux, 5 days 8 / 1.6% Compound (I) benzoate 0 o'clock 6 / 2.6% 60℃, 4 days 7 / 3.6% 40℃ / 75%RH, 4 days 8 / 2.9% 92.5%RH, 4 days 7 / 2.8% 4500 lux, 4 days 7 / 2.9% Compound (I) adipate 0 o'clock 1 / 0.69% 60℃, 5 days 4 / 1.2% 40℃ / 75%RH, 5 days 3 / 0.87% 92.5%RH, 5 days 2 / 0.82% 4500 lux, 5 days 3 / 1.0% []

[0201] [Test Example] [4] [:Mode] [(I)] [Pharmacokinetic evaluation of compounds and their pharmaceutically acceptable salts in rats]

[0202] Experimental materials: Female SD rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. MC (methylcellulose) was purchased from Sigma. Acetonitrile was purchased from Merck (USA).

[0203] Experimental methods:

[0204] Twelve female SD rats (200-300g, 6-8 weeks old) were randomly divided into four groups of three. Each group was orally administered compound (I), compound (I) benzoate, compound (I) succinate, and compound (I) adipic acid salt, respectively, at a dose of 24 mg / kg, in a 0.5% methylcellulose aqueous solution. Before the experiment, the rats were given normal water and fasted overnight. Food was resumed 4 hours after drug administration. Blood samples were collected from the jugular vein of each group before drug administration and at 0.25, 0.5, 1, 2, 4, 6, 8, 24, and 48 hours after drug administration. The collected whole blood samples were placed in K2EDTA anticoagulant tubes, centrifuged for 5 min (12000 rpm, 4ºC), and plasma was collected for analysis.

[0205] Take 10 μL of rat plasma sample [,] Add 150 μL of acetonitrile solvent (containing internal standard compound) to precipitate the protein, vortex for 5 min, centrifuge (14000 rpm) for 5 min, and dilute the supernatant 2-fold with water containing 0.1% (v / v) FA. Quantitative detection was performed using an LC-MS / MS system (AB Sciex Triple Quad 6500+). SD rat plasma standard curves and quality control samples were used concurrently with the sample concentration determination. For 10x diluted samples, 2 μL of sample was added to 18 μL of blank plasma, vortexed for 0.5 min, and then 300 μL of acetonitrile solvent (containing internal standard compound) was added to precipitate the protein. The remaining processing steps were the same as for the undiluted sample.

[0206] Experimental results:

[0207] Compared to the free base, compounds (I) benzoate, (I) succinate and (I) adipate all exhibit superior PK characteristics and good oral bioavailability. Table 9. Rat PK test results of compounds of formula (I) and their pharmaceutically acceptable salts. PK parameters Compound of formula (I) Free base Benzoate Succinate adipic acid salt T max(h) 5.50 5.00 5.50 5.50 C max(ng / mL) 692 825 961 961 AUC last(h*ng / mL) 19188 23834 25893 22760 F% - 124 135 119

[0208] Test Example 5: In vitro inhibition of breast cancer cell proliferation by compound (I) succinate A crystal form.

[0209] Experimental materials: MCF-7, CAMA-1, HCC1500, T47D, and SK-BR-3 cells were purchased from ATCC. EFM-19 and the MCF-7 ESR1 Y537S mutant strain were purchased from KORBIA Biotechnology. Cell culture conditions followed the ATCC or KORBIA product instructions. Culture media were purchased from Invitrogen, and serum was purchased from Gibico. MCF-7 culture medium consisted of DMEM + 10% FBS + 0.01 mg / ml human insulin (Yisheng) + 1% non-essential amino acids. CAMA-1 culture medium consisted of EMEM + 10% FBS. HCC1500 and EFM-19 culture medium consisted of RPMI-1640 + 10% FBS. T47D culture medium consisted of RPMI-1640 + 10% HI-FBS + 2 units / mL bovine insulin (Solarbio). MCF-7 ESR1 Y537S medium consisted of MEM + 10% FBS + 1 mM sodium pyruvate + 1% non-essential amino acids. SK-BR-3 medium consisted of McCoy's 5A + 10% FBS. Fulvestrant was purchased from MCE. The succinate A crystal form of compound (I) was prepared according to the method in Example 10-1.

[0210] Experimental methods: When the cell confluence rate reached 70% or higher, the cells were digested, and the cell densities of MCF-7, T47D, MCF ESR1 Y537S were adjusted to 500 / 20 μL / well, HCC1500 cells to 2000 / 20 μL / well, and CAMA-1, EFM-19, and SK-BR-3 cells to 1000 / 20 μL / well. The cells were then seeded in 384-well cell culture plates (Corning) and cultured overnight. 120 nL of the 3-fold diluted compound was transferred to cell culture plates using an Echo 650 (Beckman) apparatus, followed by 40 μL of the appropriate culture medium. After 7 days of compound treatment, CellTiter-Glo (Promega) was added to detect cell viability. Cell wells served as 0% inhibition controls, and wells treated with 0.1 μM fulvestrant served as 100% inhibition controls (for MCF-7 ESR1 Y537S, wells treated with 2 μM fulvestrant served as 100% inhibition controls; for SK-BR-3, wells treated with culture medium served as 100% inhibition controls). The IC50 and maximum inhibition rate were calculated using IDBS XLfit with 4-parameter fitting.

[0211] Experimental results: The succinate A crystal form of compound (I) showed strong inhibitory effects on the proliferation of different ER-positive breast cancer cell lines, with an IC50 of <1 nM. The IC50 for the proliferation inhibition of the ESR1 Y537S mutant was 27.6 nM, while it had no inhibitory effect on the proliferation of ER-negative cells SK-BR-3 at the highest concentration of 2 μM. Table 10 Inhibitory effect of compound (I) succinate A crystal form on breast cancer cell proliferation cell lines IC 50_nM Maximum inhibition rate % MCF7 0.78 99.59 MCF-7 ESR1 Y537S 27.60 100.96 HCC1500 0.94 90.32 CAMA-1 0.38 97.66 EFM-19 0.40 110.99 T47D 0.55 94.29 SK-BR-3 (ER -) >2000 -2.5

[0212] Test Example 6: Effect of compound (I) succinate A crystal form on the degradation of ER protein in MCF-7 and MCF-7 ESR1 Y537S mutant strains.

[0213] Experimental materials: MCF-7 was purchased from ATCC, and the MCF-7 ESR1 Y537S mutant strain was purchased from Kebai Biotechnology. Cell culture conditions were in accordance with the ATCC or Kebai product instructions. Culture medium was purchased from Invitrogen, and serum was purchased from Gibico. MCF-7 culture medium consisted of DMEM + 10% FBS + 0.01 mg / ml human insulin (Yisheng) + 1% non-essential amino acids. CAMA-1 culture medium consisted of EMEM + 10% FBS. MCF-7 ESR1 Y537S culture medium consisted of MEM + 10% FBS + 1 mM sodium pyruvate + 1% non-essential amino acids. Fulvestrant was purchased from MCE. The succinate A crystal form of compound (I) was prepared according to the method in Example 10-1.

[0214] Experimental Methods: When the cell confluence reached 70% or higher, the cells were digested, and the cell density was adjusted to 5000 / 20 μL / well. The cells were then seeded in 384-well cell culture plates (Greiner) and cultured overnight. 100 nL of a 3-fold diluted compound was transferred to the cell culture plates using an Echo 650 (Beckman). After 24 hours, 20 μL of 8% formaldehyde (Shanghai Test) was added for fixation at room temperature for 20 minutes. Then, 50 μL of ice-cold methanol (Millipore) was added for permeabilization for 10 minutes. Finally, 50 μL of Licor blocking buffer was added for blocking at room temperature for 1 hour. Finally, 20 μL of ER antibody (CST) and GAPDH antibody (Abcam) were added, and the cells were incubated overnight at 4°C. After washing away the primary antibody, add IRDye 800 goat anti-rabbit IgG antibody and IRDye 680 goat anti-mouse IgG antibody (LI-COR) and incubate in the dark for 45 minutes. Wash away the secondary antibody, centrifuge at 1200 rpm for 1 minute at a time to remove residual liquid, and detect the 800 nM and 680 nM signals using a Sapphire RGBNIR (Azure). Perform quantitative fluorescence analysis using AzureSpot to calculate the Chanel 800 (ER) / Chanel 680 (GAPDH) ratio. Cell wells were used as 0% inhibition controls, wells treated with 0.1 μM fulvestrant for MCF-7 cells were used as 100% inhibition controls, and wells treated with 2 μM fulvestrant for MCF-7 ESR1 Y537S cells were used as 100% degradation controls. 4-parameter fitting was performed using IDBS XLfit to calculate DC 50 and maximum degradation rate data.

[0215] Experimental results: The succinate A crystal form of compound (I) showed high ER degradation activity against both MCF-7 and ESR1 Y537S mutant strains. Table 11 shows the degradation effects of succinate A crystal form of compound (I) on MCF-7 and ESR1 Y537S mutant ER. cell lines DC 50_nM Maximum degradation rate % MCF7 0.73 105.09 MCF-7 ESR Y537S 47.32 99.57

[0216] Test Example 7: Pharmacodynamic Study of Compound (I) Succinate in a Mouse Model of Human ER-Positive Breast Cancer MCF-7 Xenograft Subcutaneous Tumor

[0217] Experimental materials: Human breast cancer MCF-7 cells: ECACC, 86012803 17β-Estradiol Tablets: Innovative Research of America, Cat No.: SE-121, 60-day release, 0.18 mg / pellet EMEM culture medium: ATCC, Cat No.: 30-2003 Fetal bovine serum: ExCell; Cat No.: FND500 Bismuth subsalicylate: Gibco, Cat No.: 15240-062 0.25% Trypsin-EDTA: Gibco, Cat No.: 25200-072 DPBS:Corning, Cat. No.: 21-031-CVR Substrate adhesive: Corning, Cat. No.: 354234 Fulvestrant injection: AstraZeneca, 250 mg / 5 mL / vial

[0218] Experimental methods:

[0219] Animal Information: Balb / c nude mice, female, 6-8 weeks old, weighing approximately 18-22 grams. The animals were purchased from Shanghai Lingchang Biotechnology Co., Ltd. The mice were housed in an SPF-grade environment with individual ventilation in each cage. All animals had free access to standard certified commercial laboratory food and water.

[0220] Cell culture: Human breast cancer MCF-7 cell line was cultured in vitro under the following conditions: EMEM (cell culture medium) with 10% fetal bovine serum and 1% penicillin-drug antibiotics, at 37°C in a 5% CO2 incubator. Cells were passaged twice a week using 0.25% trypsin-EDTA digestion solution. When cell confluence reached 80%-90% and the required number was achieved, cells were harvested and counted.

[0221] Cell inoculation: 0.2 ml of MCF-7 cell suspension (containing 1 × 10⁷ cells) (DPBS:Matrix gel, volume ratio 1:1) was subcutaneously inoculated into the right posterior back of each mouse. Two days prior to cell inoculation, 17β-estradiol tablets were subcutaneously administered. On day 6 post-inoculation, when the average tumor volume reached 173.65 mm³, drug administration was initiated based on tumor volume, with grouping determined by randomization. Day 0 was the day of group administration.

[0222] Administration: The succinate of formula (I) (Example 10-1, crystal form A) was administered at a dose of 0.3, 1, 3, or 10 mg / kg (based on free base) orally (PO) once daily (QD) x 21 times. Fulvestrant was administered at a dose of 250 mg / kg subcutaneously (SC) once weekly (QW) x 4 times. Eight mice were used in each group.

[0223] Tumor measurements and experimental indicators:

[0224] The tumor diameter was measured twice weekly using calipers. The tumor volume was calculated using the formula: V = 0.5axb², where a and b represent the major and minor diameters of the tumor, respectively. The mouse's body weight was measured twice weekly.

[0225] The antitumor efficacy of the compound was evaluated using the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume at the end of treatment - mean tumor volume at the start of treatment) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the start of treatment in the solvent control group)] x 100%.

[0226] [Experimental Results:] []

[0227] See Table 12, Figures 13 and 14. Although a few mice experienced a weight loss exceeding 10%, this was unrelated to the dose of the test compound (I) succinate, and the weight of the mice in the test drug group was not significantly lower than that in the solvent control group. Considering that the estrogen patch in this model may affect the mouse's condition and weight, these individual weight losses were not related to the test compound (I) succinate. No mice died in any of the experimental groups.

[0228] The results showed that, on Day 20 after the start of administration, subcutaneous injection of 250 mg / kg of Fulvestrant (a control compound) into human breast cancer MCF-7 tumor-bearing mice once a week had a weak inhibitory effect on tumor growth in the MCF-7 xenograft tumor model (P<0.05). Oral administration of 0.3 mg / kg of the test compound (I) succinate to tumor-bearing mice once a day showed a certain inhibitory effect on tumor growth compared to the solvent control group (P<0.0001). Oral administration of 1 mg / kg, 3 mg / kg, or 10 mg / kg of the test compound (I) succinate to tumor-bearing mice once a day showed a significant inhibitory effect on tumor growth compared to the solvent control group (P<0.0001), exhibiting a good dose-dependent effect. At doses of 1 mg / kg and above, tumor regression began. In the human breast cancer MCF-7 subcutaneous tumor model, the compound (I) succinate at doses of 1 mg / kg and above (PO, QD) showed significantly superior antitumor activity compared to the control Fulvestrant (250 mg / kg, SC, QW) (P<0.0001). [surface] [12 MCF-7] [Tumor volume in the subcutaneous tumor model] [] [Test compound] [dose] [mg / kg] [Route of administration] [Dosage frequency] [Mean tumor volume ()] [mm, 3 , ] [)] [TGI] [(] [%] [)] [0] [sky] [2] [sky] [6] [sky] [9] [sky]

[13] [sky]

[16] [sky]

[20] [sky] Solvent control / PO QD 174 222 290 368 507 679 780 / Fulvestrant 250 SC QW 174 213 267 305 367 483 605 28.84 Formula (I) compound succinate 0.3 PO QD 174 199 236 278 340 396 506 45.22 Formula (I) compound succinate 1 PO QD 174 192 129 112 92 83 75 116.33 Formula (I) compound succinate 3 PO QD 174 180 96 76 55 38 33 123.26 Formula (I) compound succinate 10 PO QD 174 188 72 57 56 37 29 123.80

[0229] [Test Example] [8] [Mode] [(I)] [People who consume the compound succinate] [ER] [Positive breast cancer] [T47D] [Pharmacodynamic Study of a Mouse Model of Xenograft Subcutaneous Tumor] []

[0230] [Experimental Materials:] [] Human breast cancer xxT47D cells, which are T47D (ATCC, HTB-133) cells that have undergone two in vivo passages. 17β-Estradiol Tablets: Innovative Research of America, Cat No.: SE-121, 60-day release, 0.18 mg / pellet RPMI-1640 culture medium: Gibco, Cat No.: C22400500BT Fetal bovine serum: HyClone; Cat No.: SV30087.03 Bovine Insulin: Source Leaf, Cat. No.: 11070-73-8 Bismuth subsalicylate: Gibco, Cat No.: 15240-062 0.25% Trypsin-EDTA: Gibco, Cat No.: 25200072 DPBS:Corning, Cat. No.: 21-031-CVR Substrate adhesive: Corning, Cat. No.: 354234 Fulvestrant injection: AstraZeneca, 250 mg / 5 mL / vial

[0231] [Experimental Method:] []

[0232] Animal Information: Balb / c nude mice, female, 6-8 weeks old, weighing approximately 18-22 grams. The animals were purchased from Shanghai Lingchang Biotechnology Co., Ltd. The mice were housed in an SPF-grade environment with individual ventilation in each cage. All animals had free access to standard certified commercial laboratory food and water.

[0233] Cell culture: Human breast cancer xxT47D cells were cultured in vitro under the following conditions: RPMI-1640 medium supplemented with 10% fetal bovine serum, 0.2 Units / mL bovine insulin, and 1% penicillin-dextrose antibody, incubated at 37ºC in a 5% CO2 incubator. Cells were passaged twice a week using trypsin-EDTA digestion. When cell confluence reached 80%–90% and the desired number was achieved, cells were harvested and counted.

[0234] Cell inoculation: 0.2 mL (containing 1 × 10⁷ cells) of xxT47D cell suspension (DPBS:Matrix gel, volume ratio 1:1) was subcutaneously inoculated into the right posterior back of each mouse. Two days prior to cell inoculation, 17β-estradiol tablets were subcutaneously administered. On day 6 post-inoculation, when the average tumor volume reached 137.09 mm³, grouping and drug administration began, with mice randomly assigned to groups based on tumor volume. Day 0 was the day of grouping and drug administration.

[0235] Administration: The succinate of formula (I) (Example 10-1, crystal form A) was administered orally at a dose of 0.3, 1, or 3 mg / kg, once daily (QD) x 28 times. Fulvestrant was administered subcutaneously at a dose of 250 mg / kg, once weekly (QW) x 4 times. Eight mice were used in each group.

[0236] Tumor measurements and experimental indicators:

[0237] The tumor diameter was measured twice weekly using calipers. The tumor volume was calculated using the formula: V = 0.5axb², where a and b represent the major and minor diameters of the tumor, respectively. The mouse's body weight was measured twice weekly.

[0238] The antitumor efficacy of the compound was evaluated using the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume at the end of treatment - mean tumor volume at the start of treatment) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the start of treatment in the solvent control group)] x 100%.

[0239] [Experimental Results:]

[0240] See Table 13, Figure 15 and Figure 16.

[0241] The results showed that, on day 28 after the start of administration, subcutaneous injection of 250 mg / kg of the control compound Fulvestrant into human breast cancer T47D tumor-bearing mice once a week showed a certain anti-tumor growth effect compared with the solvent control group (P<0.0001). Oral administration of 0.3 mg / kg, 1 mg / kg, or 3 mg / kg of the test compound (I) succinate into tumor-bearing mice once a day showed a certain or significant inhibitory effect on tumor growth compared with the solvent control group (P<0.0001), exhibiting a good dose-dependent effect. In the human breast cancer T47D subcutaneous tumor model, compound (I) succinate at doses of 1 mg / kg and above (PO, QD) showed significantly superior anti-tumor activity compared with the control compound Fulvestrant (250 mg / kg, SC, QW) (P<0.0001). [surface] [13 T47D] [Tumor volume in the subcutaneous tumor model] [] Test compound dose mg / kg Administration method Dosage frequency Mean tumor volume (mm³) TGI (%) 0 days 4 days 7 days 11 days 14 days 18 days 21 days 25 days 28 days Solvent control / PO QD 137 245 355 473 580 700 796 931 1078 / Fulvestrant 250 SC QW 137 220 317 372 424 474 466 557 627 48.35 Formula (I) compound succinate 0.3 PO QD 137 217 318 412 505 544 501 596 703 39.87 Formula (I) compound succinate 1 PO QD 137 228 261 271 245 271 220 289 349 77.47 Formula (I) compound succinate 3 PO QD 137 207 208 196 185 197 176 225 277 85.04 []

[0242] [Test Example] [9] [Mode] [(I)] [People who consume the compound succinate] [ER] [Positive breast cancer] [MCF-7] [Pharmacodynamic Study of a Mouse Model of Xenograft Brain Tumor] []

[0243] [Experimental Materials] [] Human breast cancer MCF-7 cells: ATCC, HTB-22 17β-estradiol tablets: Innovative Research of America, SE-121, 60-day release, 0.72 mg / pellet EMEM culture medium: ATCC, Cat No.: 30-2003 Fetal bovine serum: Gibco, Cat No.: 10099-141C Recombinant human insulin: Shanghai Yisheng Biotechnology Co., Ltd., Cat No.: 40112ES60 Penicillin / Streptomycin (P / S): Gibco, Cat No.: 15140-122 0.25% Trypsin-EDTA: Gibco, Cat No.: 25200-072 DPBS:Hyclone, Cat. No.: SH30256.01 Fulvestrant injection: AstraZeneca, 250 mg / 5 mL / vial

[0244] [Experimental Methods]

[0245] Animal Information: NPG mice, female, 6-7 weeks old, weighing approximately 17-23 grams. The animals were purchased from Beijing Vitonda Biotechnology Co., Ltd. The mice were housed in an SPF-grade environment with individual ventilation in each cage. All animals had free access to standard certified commercial laboratory food and water.

[0246] Cell culture: Human breast cancer MCF-7 cell line was cultured in vitro under the following conditions: EMEM (cell culture medium) with 10% fetal bovine serum, 10 μg / ml recombinant human insulin, and 1% P / S, at 37°C in a 5% CO2 incubator. Cells were passaged once or twice a week using 0.25% trypsin-EDTA digestion. Cells were harvested and counted when the desired cell count was reached and the cells were in the logarithmic growth phase.

[0247] Cell inoculation: 0.015 ml / (containing 2 × 10⁶ cells) of MCF-7 cell suspension was orally inoculated into the brain of each mouse (1 mm anterior to the anterior fontanelle, 2 mm to the right of the midline, and 3 mm below the level of the dura mater of the skull). Three days prior to cell inoculation, 17β-estradiol tablets were subcutaneously administered. On day 8 after cell inoculation, grouping and administration began, with animals randomly assigned to groups based on body weight. Day 0 was the day of grouping.

[0248] Administration: The succinate of formula (I) (Example 10-1, crystal form A) was administered orally (PO) once daily (QD) at doses of 3, 10, or 30 mg / kg, continuing until Day 60. Fulvestrant was administered subcutaneously (SC) once weekly (QW) at doses of 250 mg / kg, continuing until Day 60. Eight mice were used in each group.

[0249] Weight measurement and experimental indicators: The mice were weighed twice a week. The antitumor efficacy of the compound was evaluated using median survival.

[0250] [Experimental Results:] []

[0251] See Table 14 and Figure 17. Compared with the solvent control group, once-weekly subcutaneous injection of 250 mg / kg Fulvestrant in MCF-7 brain orthotopic tumor model animals did not significantly improve the survival rate of the animals. However, once-daily oral administration of 10 or 30 mg / kg succinate of formula (I) in MCF-7 brain orthotopic tumor model animals significantly improved the survival rate of mice (P<0.001), improved the condition of the animals, and the efficacy was significantly stronger than that of Fulvestrant (P<0.001). [surface] [14 MCF-7] [Effects of different treatment groups on mouse survival rate in orthotopic brain model] [] [Test compound] [dose] [mg / kg] [Route of administration] [Dosage frequency] [Median survival (days)] [No.]

[60] [sky] [(Day 60)] [Percentage of surviving mice] Solvent control / PO QD 26.5 1 / 8 Fulvestrant 250 SC QW 27 1 / 8 Formula (I) compound succinate 3 PO QD 31 3 / 8 Formula (I) compound succinate 10 PO QD - 8 / 8 Formula (I) compound succinate 30 PO QD - 8 / 8 [, , ]

[0252] [Test Example]

[10] [:Mode] [(I)] [Compound Pairs] [MCF7] [Study on the Degradation Effect of Intracellular Estrogen Receptors] [] 1. Experimental Objective The purpose of this experiment is to determine the degradation activity of compound (I) on endogenously expressed estrogen receptors in MCF7 cells, and to evaluate the activity of the compound based on DC 50 and maximum degradation efficiency. 2. Experimental Methods MCF7 cells (ATCC, HTB-22) were cultured in DMEM (Gibco, 11995-065) complete medium containing 10% fetal bovine serum. On day 1, MCF7 cells were seeded at a density of 3000 cells / well in 384-well plates using complete medium and cultured at 37ºC in a 5% CO2 cell incubator. The test compound was dissolved in DMSO at a storage concentration of 10 mM, diluted with Echo 550 (Labcyte Inc.), and added to the cell culture plates. The initial concentration of the test compound was 100 nM, with 3-fold serial dilutions, resulting in 9 concentration points. A blank control containing 0.5% DMSO was included, and duplicate wells were set up for each concentration point. The cells were cultured at 37ºC in a 5% CO2 cell incubator for 24 hours. Add paraformaldehyde to each cell culture well to the cell culture medium, with a final paraformaldehyde concentration of approximately 3.7% to fix the cells. After 30 minutes of treatment, discard the supernatant and wash each well once with 50 μL of PBS. Add PBS containing 0.5% v / v Tween-20 and treat the cells for 30 minutes, followed by washing with PBS once. Add blocking buffer (PBS containing 5% BSA and 0.05% Tween-20) and incubate at room temperature for 1 hour. Remove the blocking buffer and add a primary antibody mixture (anti-ER monoclonal antibody, Estrogen Receptor α (D8H8) Rabbit mAb, GST, #8644S, 1:1000 dilution; anti-GAPDH monoclonal antibody, GAPDH (D4C6R) Mouse mAb, GST, #97166S, 1:2000 dilution) and incubate at room temperature for 3 hours. Finally, incubate with PBST (PBS containing 0.05% BSA and 0.05% Tween-20). Wash three times with Tween-20; add detection secondary antibodies (800CW-goat anti-rabbit IgG, LI-COR, P / N: 926-32211, 1:1000 dilution; 680RD-goat anti-mouse IgG, LI-COR, #925-68070, 1:1000 dilution), incubate at room temperature in the dark for 45 minutes; wash three times with PBST, read the fluorescence signal of each well using an Odyssey CLx, and calculate the Chanel 800(ER) / Chanel 680 (GAPDH) values. Wells treated with 0.1 μM fulvestrant were used as a 100% degradation control, and the degradation rate at each concentration point was calculated. Data were analyzed using XLfit to calculate the degradation activity DC50 and maximum degradation rate Imax of the compounds. Data analysis is shown in Table 15. [surface]

[15] [Mode] [(I)] [Compound Pairs] [MCF7] [Intracellular estrogen receptor degradation activity] [] Compound numbering ER level DC 50 (nM) Maximum degradation rate Compound of formula (I) 0.15 104% []

[0253] [Test Example]

[11] [:Mode] [(I)] [Compound Pairs] [MCF7] [Study on the inhibitory effect on cell proliferation] 1. Experimental Objective The purpose of this experiment was to determine the inhibitory effect of compound (I) on the in vitro proliferation of MCF7 cells and to evaluate the activity of the compound based on IC50. 2. Experimental Methods MCF7 cells (ATCC, HTB-22) were cultured in DMEM (Gibco, 11995-065) complete medium containing 10% fetal bovine serum. On day 1, MCF7 cells were seeded at a density of 500 cells / well in 384-well plates using complete medium and cultured overnight at 37ºC in a 5% CO2 cell incubator. On day 2, the test compound was added for treatment. A 10 mM solution of the compound was diluted and transferred to each well using an Echo550 (Labcyte Inc.). The initial concentration of the test compound in the cells was 100 nM, serially diluted 3-fold, resulting in 9 concentration points. A blank control containing 0.3% DMSO was included, and duplicate wells were set at each concentration point. Cells were cultured at 37ºC in a 5% CO2 cell incubator for 7 days. On day 8, the cell culture plates were removed. Add CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7573), incubate at room temperature for 10 minutes, and then read the luminescence signal value using an EnVision (PerkinElmer) multilabel microplate reader. Calculate the IC50 of the inhibitory activity of each compound using XLfit based on the compound concentration and luminescence signal value. 3. Data analysis is shown in Table 16. Table 16 Inhibitory activity of compound (I) on MCF7 cell proliferation Compound numbering MCF-7 cell proliferation inhibition IC50 (nM) Compound of formula (I) 0.27

[0254] [Test Example]

[12] [:Mode] [(I)] [Compound in] [pH] [Value is] [7.4] Apparent solubility of [in phosphate buffer] []

[0255] For an orally administered compound to reach its site of action and for effective absorption in the intestines, the compound needs to be in solution. Therefore, compounds with high inherent solubility may be more suitable for pharmaceutical use.

[0256] I. Materials and Reagents

[0257] The test compound was prepared according to the described method. Progesterone, the control drug, was purchased from Sigma. Phosphate buffer (pH 7.4) was prepared in our laboratory. Acetonitrile and methanol were purchased from Fisher. Other reagents were purchased commercially.

[0258] 1.5 ml flat-bottomed glass vial (BioTech Solutions); PTFE / silicone bottle stopper (BioTech Solutions); PTFE-coated stir bar; MultiScreenHTS HV (0.45 µm) 96 well plate filter (Millipore, MSHVN4510 or MSHVN4550); Eppendorf Thermomixer Comfort; Vacuum Manifold ORVMN96 (Orochem).

[0259] II. Experimental Procedure

[0260] Preparation of stock solution

[0261] Prepare 10 mM stock solutions of the analyte and the control drug progesterone using DMSO.

[0262] Apparent solubility determination steps

[0263] Add 30 µL of 10 mM analyte stock solution to the corresponding wells of the 96-well plate in the specified order. Add 970 µL of pH 7.4 phosphate buffer to the corresponding vial of the sample plate. The experiment is double-replicated. Add a stir bar to each vial and cap it with a PTFE / silicone stopper. Then place the sample tray in an Eppendorf Thermomixer Comfort and shake at 1100 rpm at 25°C for 2 hours. After 2 hours, remove the stopper, remove the stir bar with a large magnet, and then transfer the sample from the sample plate to the filter plate. Filter the sample using a vacuum pump to create negative pressure. Transfer 5 µL of the filtrate to a new sample plate, and then add 5 µL of DMSO and 490 µL of 50% ACN(IS).H₂O (acetonitrile:water = 1:1 internal standard). Depending on the peak shape, the sample diluent may be diluted with a certain proportion of 50% ACN(IS).H₂O to obtain a better peak shape. The dilution factor may be adjusted depending on the solubility of the analyte or the strength of its liquid chromatography response signal.

[0264] Sample analysis steps

[0265] The sample plate is placed into the sample tray of the autosampler, and the sample is evaluated by liquid chromatography-mass spectrometry.

[0266] III. Experimental Procedure

[0267] All calculations were performed using Microsoft Excel. Analysis and quantification of the sample filtrate were accomplished through qualitative and quantitative analysis of standard peaks of known concentrations using LC-MS. The apparent solubility of compound (I) in phosphate buffer at pH 7.4 was calculated. Table 17 Apparent solubility values ​​of compound (I) in phosphate buffer at pH 7.4 Compound numbering pH = 7.4 Apparent solubility (µM) Compound of formula (I) 92

[0268] [Test Example]

[13] [:Mode] [(I)] [The compound's effect on the blood-brain barrier in rats ()] [BBB] [Through ability] []

[0269] The ability of a drug to cross the blood-brain barrier and achieve sufficient exposure in the brain is crucial for its effectiveness against brain metastases. Therefore, by measuring the drug concentration in plasma and brain tissue after administration to animals, we can assess the distribution of the drug in the brain and determine whether the drug can inhibit tumor growth in an in situ brain model.

[0270] Experimental materials

[0271] Female SD rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. MC (methylcellulose) was purchased from Sigma; acetonitrile was purchased from Merck (USA). PBS (phosphate buffered saline) was purchased from Sangon Biotech.

[0272] Experimental methods

[0273] Six female SD rats (200-300g, 6-8 weeks old) were randomly divided into two groups of three each. Each group was administered compound (I) and 0.5% methylcellulose aqueous solution as solvent. Before the experiment, the rats were given normal water and fasted overnight. Feeding resumed four hours after drug administration. Plasma and brain tissue were collected from each group two hours after drug administration. Whole blood samples were placed in K2EDTA anticoagulant tubes, centrifuged for 5 min (12,000 rpm, 4ºC), and plasma was collected for analysis. Tissue samples were blotted dry with filter paper and stored at -80°C for analysis.

[0274] Take 10 μL of rat plasma sample, add 150 μL of acetonitrile solvent (containing internal standard compound) to precipitate protein, vortex for 5 min, centrifuge (14,000 rpm) for 5 min, and dilute the supernatant 2-fold with water containing 0.1% (v / v) FA. Quantitatively detect the protein using an LC-MS / MS system (AB Sciex Triple Quad 6500+). For 10x diluted samples, take 2 μL of sample and add 18 μL of blank plasma, vortex for 0.5 min, add 300 μL of acetonitrile solvent (containing internal standard compound) to precipitate protein, and follow the same steps as the undiluted sample. A standard curve of SD rat plasma and plasma quality control samples were used concurrently with the determination of plasma sample concentrations.

[0275] Rat brain tissue samples were first homogenized with 4 times their volume of PBS homogenate. 20 μL of the homogenate was diluted with 20 μL of blank mouse plasma, and then 600 μL of acetonitrile (containing an internal standard compound) was added to precipitate the protein. After vortexing for 5 min, the sample was centrifuged (14,000 rpm) for 5 min. The supernatant was diluted 2-fold with water containing 0.1% (v / v) FA and quantitatively analyzed using an LC-MS / MS system (AB Sciex Triple Quad 6500+). The compound of formula (I) exhibited excellent blood-brain barrier permeability, resulting in high drug exposure in rat brain tissue.

[0276] The BBB test results are shown below: Table 18. Blood-brain barrier permeation experiment of compound (I) in rats. Compound numbering Dosage mg / kg plasma drug concentration ng / ml Brain drug concentration ng / g B / P ratio Compound of formula (I) 100 QD 2617 10560 4.2

[0277] [Test Example]

[14] [:Mode] [(I)] [Compound Pairs] [MCF-7] [Growth inhibition experiment in a mouse subcutaneous tumor model] []

[0278] Experimental reagents Human breast cancer MCF-7 cells: ATCC, HTB-22 17β-Estradiol tablets: Innovative Research of America, Cat No.: SE-121, 60-day release, 0.72 mg / pellet EMEM culture medium: ATCC, Cat No.: 30-2003 Fetal bovine serum: Gbico; Cat No.: 1099-141C Penicillin (Pen Strep): Gibco, Cat No.: 15240-122 Recombinant human insulin: Shanghai Yisheng, Cat. No.: 40112ES60 0.25% Trypsin-EDTA: Gibco, Cat No.: 25200-072 D-PBS (Calcium and Magnesium-Free Phosphate Buffer): Hyclone, Cat. No.: SH30256.01 Matrigel: Corning, Cat. No.: 356237

[0279] Experimental methods

[0280] Animal Information: NPG mice, female, 6-7 weeks old, weighing approximately 19-28 grams. The animals were purchased from Beijing Vitonda Biotechnology Co., Ltd. The mice were housed in an SPF-grade environment with individual ventilation in each cage. All animals had free access to standard certified commercial laboratory food and water.

[0281] Cell culture: Human breast cancer MCF-7 cell line was cultured in vitro under the following conditions: EMEM (cell culture medium) with 10% fetal bovine serum, 1% PenStrip, and 10 μg / ml recombinant human insulin, incubated at 37°C in a 5% CO2 incubator. Cells were passaged once a week using a routine digestion process with 0.25% trypsin-EDTA digestion solution. When cell saturation reached 80%-90% and the required number was achieved, cells were harvested and counted.

[0282] Cell inoculation: 0.1 ml of MCF-7 cell suspension (D-PBS: Matrigel, volume ratio 1:1) was subcutaneously injected into the right posterior back of each mouse, and 17β-estradiol tablets were subcutaneously injected four days prior to cell inoculation. On day 24 after cell inoculation, mice were randomly assigned to groups based on tumor volume, with the grouping day designated as Day 0.

[0283] Administration: The compound of formula (I) was administered orally (PO) once daily (QD) for 3 weeks at a dose of 1, 3, or 10 mg / kg. Eight mice were in the solvent group and six mice were in the administration group.

[0284] Tumor measurements and experimental indicators:

[0285] The tumor diameter was measured twice weekly using calipers. The tumor volume was calculated using the formula: V = 0.5axb², where a and b represent the major and minor diameters of the tumor, respectively. The mouse's body weight was measured twice weekly.

[0286] The antitumor efficacy of the compound was evaluated using the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume at the end of treatment - mean tumor volume at the start of treatment) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the start of treatment in the solvent control group)] x 100%.

[0287] Experimental results:

[0288] See Table 19. In the mouse subcutaneous xenograft MCF-7 model, compound (I) showed significant inhibitory effects on tumor growth (P<0.01) at once-daily oral administration of 1 mg / kg, 3 mg / kg, or 10 mg / kg, with a good dose-response relationship. Compound (I) also showed tumor shrinkage at doses of 3 mg / kg and 10 mg / kg. Compound (I) at once-daily oral administration of 10 mg / kg showed significant inhibitory effects on tumor growth (P<0.01) and also showed tumor shrinkage. Compound (I) did not significantly affect mouse body weight at the tested doses. Table 19 Tumor Volume in the MCF-7 Subcutaneous Tumor Model Test compound dose mg / kg Administration method Dosage frequency Mean tumor volume (mm³) TGI (%) 0 days 21 days Solvent control / PO QD 179 403 / Compound of formula (I) 1 PO QD 183 207 89.08 Compound of formula (I) 3 PO QD 169 136 114.71 Compound of formula (I) 10 PO QD 177 91 138.17

[0289] [Test Example]

[15] [:Mode] [(I)] [Compound's effect on mice] [MCF-7] [Experiment on the inhibition of brain tumor growth in an orthotopic model] []

[0290] Experimental reagents / instruments: Human breast cancer MCF-7 cells: ATCC, HTB-22 17β-Estradiol tablets: Innovative Research of America, Cat No.: SE-121, 60-day release, 0.72 mg / pellet EMEM culture medium: ATCC, Cat No.: 30-2003 Fetal bovine serum: Gibco, Cat. No.: 1099-141C Penicillin (Pen Strep): Gibco, Cat No.: 15240-122 Recombinant human insulin: Shanghai Yisheng, Cat. No.: 40112ES60 0.25% Trypsin-EDTA: Gibco, Cat No.: 25200-072 Stereotype Analyzer: Reward, Cat No.: Standard / Digital Display / Single Arm / Mouse / 68055 Microinjection pump: KDS, Cat No.: Legato130 Miniature Handheld Skull Drill: Rayward, Cat No.: 78001 []

[0291] Experimental methods:

[0292] Animal Information: NPG mice, female, 6-8 weeks old, weighing approximately 17-29 grams. The animals were purchased from Beijing Vitonda Biotechnology Co., Ltd. The mice were housed in an SPF-grade environment with individual ventilation in each cage. All animals had free access to standard certified commercial laboratory food and water.

[0293] Cell culture: Human breast cancer MCF-7 cell line was cultured in vitro under the following conditions: EMEM (cell culture medium) with 10% fetal bovine serum, 1% PenStrip, and 10 μg / ml recombinant human insulin, incubated at 37°C in a 5% CO2 incubator. Cells were passaged twice a week using 0.25% trypsin-EDTA digestion solution. When cell saturation reached 80%-90% and the required number was achieved, cells were harvested and counted.

[0294] Cell inoculation: 15 μl / (containing 2×10⁶) MCF-7 cell suspension was inoculated intracranially into mice using a brain localization device, a microinfusion pump, and a mini handheld skull drill. Three days prior to cell inoculation, mice were subcutaneously injected with 17β-estradiol tablets. On day 8 after cell inoculation, mice were randomly assigned to groups based on their body weight, with the grouping day designated as Day 0.

[0295] Administration: Fulvestrant (AstraZeneca) was administered at a dose of 250 mg / kg, subcutaneously (SC), once weekly (QW); compound (I) was administered orally at a dose of 30 mg / kg, once daily (QD). Eleven mice were in the solvent group and eight mice were in the drug group. All mice were continuously administered the drug until death, euthanized due to poor condition, or the experiment was terminated.

[0296] Experimental observations and conclusion:

[0297] Mice were weighed twice a week, and their survival status was observed.

[0298] The experiment ended on day 48, and all mice were euthanized.

[0299] Experimental results:

[0300] See Figures 18 and 19. In the mouse orthotopic brain tumor (MCF-7) model, mice in the Fulvestrant group (administered subcutaneously once a week) experienced a continuous decrease in body weight, and their survival status was not significantly different from that of the solvent control group (median survival: 29 days in the solvent control group, 29.5 days in the Fulvestrant group). Mice in the compound (I) group (administered orally once a day) maintained stable body weight and showed no abnormalities. No mice in the compound (I) group died until the experimental endpoint. Compared with the solvent control or Fulvestrant, the compound had a significant inhibitory effect on the MCF-7 orthotopic brain tumor model mice, and the survival time of the mice was significantly prolonged (P<0.01).

[0301] [Cross-references to related applications] This application claims priority and benefit to Chinese Patent Application No. 202111339152.0, filed on November 12, 2021 with the China Intellectual Property Office, the disclosure of which is incorporated herein by reference in its entirety.

[0302] The foregoing outlines components of several embodiments to enable those skilled in the art to better understand the concepts of the embodiments of the present invention. Those skilled in the art should understand that the embodiments of the present invention can be used as a basis to design or modify other processes and structures to achieve the same purpose and / or benefits as the embodiments described herein. Those skilled in the art should also understand that these equivalent structures do not depart from the spirit and scope of the present invention, and various changes, substitutions, and other options can be made therein without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

[0303]

Claims

1. A pharmaceutically acceptable salt of a compound of formula (I), wherein, The pharmaceutically acceptable salts are selected from sulfates, phosphates, benzoates, succinates, adipates, fumarates, L-malates, and citrates.

2. A pharmaceutically acceptable salt of the compound of formula (I) according to claim 1, wherein the pharmaceutically acceptable salt of the compound of formula (I) is selected from its benzoate, succinate and adipate.

3. A pharmaceutically acceptable salt of the compound of formula (I) according to claim 1, wherein the succinate of the compound of formula (I) is shown in formula (II), wherein, x is selected from 0.5 to 2.

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

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

6. A pharmaceutically acceptable salt of the compound of formula (I) according to claim 3, wherein x is selected from 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2.

0.

7. A pharmaceutically acceptable salt of the compound of formula (I) according to claim 4, wherein y is selected from 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2.

0.

8. A pharmaceutically acceptable salt of the compound of formula (I) according to claim 5, wherein z is selected from 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2.

0.

9. An adipate C crystal form of a compound of formula (I), wherein the C crystal form has diffraction peaks at 4.46±0.2º, 6.30±0.2º, 8.90±0.2º, and 19.10±0.2º in an X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

10. The C-type according to claim 9, wherein: The X-ray powder diffraction pattern of the C crystal form, expressed as a diffraction angle 2θ, shows diffraction peaks at 4.46±0.2º, 6.30±0.2º, 7.03±0.2º, 8.90±0.2º, 16.45±0.2º, 17.78±0.2º, 19.10±0.2º, and 21.03±0.2º.

11. The C-form according to claim 9 or 10, wherein the DSC spectrum of the C-form has an endothermic peak at 149.89±5℃.

12. A benzoate B crystal form of a compound of formula (I), wherein the X-ray powder diffraction pattern of the B crystal form, expressed as a diffraction angle 2θ, has diffraction peaks at 7.19±0.2º, 10.01±0.2º, 11.39±0.2º, and 18.78±0.2º.

13. The B crystal form according to claim 12, wherein: The X-ray powder diffraction pattern of the B crystal form, expressed as a diffraction angle 2θ, shows diffraction peaks at 7.19±0.2º, 10.01±0.2º, 11.39±0.2º, 18.78±0.2º, 19.01±0.2º, 19.66±0.2º, 19.91±0.2º, and 20.09±0.2º.

14. The B crystal form according to claim 12 or 13, wherein the DSC spectrum of the B crystal form has an endothermic peak at 150.08±5℃.

15. A succinate crystal form A of a compound of formula (I), wherein the X-ray powder diffraction pattern of the crystal form A, expressed as a diffraction angle 2θ, has diffraction peaks at 7.13±0.2º, 9.30±0.2º, 11.29±0.2º, 15.51±0.2º, and 19.89±0.2º.

16. Crystal form A according to claim 15, wherein: The X-ray powder diffraction pattern of the A crystal form, expressed as a diffraction angle 2θ, shows diffraction peaks at 7.13±0.2º, 9.30±0.2º, 11.29±0.2º, 15.51±0.2º, 17.10±0.2º, 19.89±0.2º, 20.26±0.2º, 20.91±0.2º, 21.51±0.2º, 22.69±0.2º, 23.58±0.2º, and 25.42±0.2º.

17. Crystal form A according to claim 15, wherein: In the X-ray powder diffraction pattern of the A crystal form, expressed as a diffraction angle 2θ, the diffraction angles are 5.01±0.2º, 7.13±0.2º, 9.30±0.2º, 10.09±0.2º, 11.29±0.2º, 11.73±0.2º, 13.75±0.2º, 14.30±0.2º, 15.18±0.2º, 15.51±0.2º, 16.00±0.2º, 17.10±0.2º, and 18.

2. 5±0.2º, 18.69±0.2º, 19.89±0.2º, 20.26±0.2º, 20.91±0.2º, 21.12±0.2º, 21.51±0.2º, 22.69±0 .2º, 23.05±0.2º, 23.58±0.2º, 24.58±0.2º, 25.42±0.2º, 26.66±0.2º, 27.24±0.2º and 29.47±0.2º There are diffraction peaks.

18. Crystal form A according to claim 15, wherein: The X-ray powder diffraction pattern of the A-type crystal is basically consistent with that in Figure 8.

19. According to any one of claims 15 to 18, the A crystal form has an endothermic peak at 186.82 ± 5 °C in its DSC spectrum.

20. The A crystal form according to any one of claims 15 to 18, wherein the cell parameters of the A crystal form are a = 24.6731(4) Å, b = 24.6731(4) Å, c = 10.2575(3) Å, α = 90°, β = 90°, γ = 90°.

21. A method for preparing a pharmaceutically acceptable salt of any one of the compounds of formula (I) in claims 1 to 8, said preparation method comprising the step of reacting the compound of formula (I) with a corresponding acid to form a salt.

22. The preparation method according to claim 21, wherein the solvent for the salt-forming reaction is selected from at least one of cyclohexane, n-heptane, toluene, chloroform, diethyl ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, acetone, methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, methanol, ethanol, isopropanol, n-butanol, and acetonitrile.

23. The preparation method according to claim 21 or 22, wherein the preparation method further comprises one or a combination of stirring crystallization, filtration and drying.

24. A method for preparing the C-type crystal form according to any one of claims 9 to 11, comprising the following steps: (1-a) mixing adipic acid and a solvent; (2-a) mixing a compound of formula (I) and a solvent; (3-a) mixing the mixture of step (1-a) with the mixture of step (2-a); (4-a) stirring the resulting mixture of the adipic acid and the compound of formula (I) to induce crystallization; (5-a) filtering out the crystals, collecting the filter cake, and drying; wherein, The solvents in steps (1-a) and (2-a) are selected from at least one of cyclohexane, n-heptane, toluene, chloroform, diethyl ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, acetone, methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, methanol, ethanol, isopropanol, n-butanol, and acetonitrile.

25. The preparation method according to claim 24, wherein, The solvents in steps (1-a) and (2-a) are selected from at least one of isopropyl ether, methyl tert-butyl ether, methanol, isopropanol and tetrahydrofuran; the molar ratio of the compound of formula (I) to adipic acid is 1:2 to 2:1; step (1-a) is carried out under heating conditions at a temperature of about 40-80°C; step (4-a) is carried out at 15-30°C.

26. A method for preparing crystal form B according to any one of claims 12-14, comprising the following steps: (1-b) mixing a compound of formula (I) and a solvent; (2-b) adding benzoic acid to the mixture of step (1-b); (3-b) stirring the resulting mixture of the compound of formula (I) and the benzoic acid to induce crystallization; (4-b) filtering and collecting the solid; wherein, The solvent in step (1-b) is selected from at least one of cyclohexane, n-heptane, toluene, chloroform, diethyl ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, acetone, methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, methanol, ethanol, isopropanol, n-butanol, and acetonitrile.

27. The preparation method according to claim 26, wherein, The solvent in step (1-b) is selected from at least one of isopropyl ether, methyl tert-butyl ether, methanol, isopropanol, and tetrahydrofuran; the molar ratio of the compound of formula (I) to benzoic acid is 1:2 to 2:1; step (1-b) is carried out at 15-30°C; the stirring in step (3-b) is carried out under heating conditions at a temperature of approximately 40-68°C; optionally, the method for preparing the B crystal form further includes the following steps: (5-b) mixing the solid collected in step (4-b) with the solvent; (6-b) stirring the resulting mixture; (7-b) filtering out and collecting the solid, and drying it; wherein the solvent in step (5-b) is selected from at least one of isopropyl ether, methyl tert-butyl ether, methanol, isopropanol, and tetrahydrofuran; step (6-b) is carried out at 15-30°C.

28. A method for preparing crystal form A according to any one of claims 15-20, comprising the following steps: (1-c) mixing a compound of formula (I) and a solvent; (2-c) adding succinic acid to the mixture of step (1-c); (3-c) stirring the resulting mixture of the compound of formula (I) and the succinic acid to precipitate a solid; (4-c) filtering and collecting the solid; wherein, The solvent in step (1-c) is selected from at least one of cyclohexane, n-heptane, toluene, chloroform, diethyl ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, acetone, methyl acetate, ethyl acetate, isopropyl acetate, butyl acetate, methanol, ethanol, isopropanol, n-butanol, and acetonitrile.

29. The preparation method according to claim 28, wherein, The solvent in step (1-c) is selected from at least one of isopropyl ether, methyl tert-butyl ether, methanol, isopropanol, and tetrahydrofuran; the molar ratio of the compound of formula (I) to succinic acid is 1:2 to 2:1; step (3-c) is carried out at 15-30°C; the reaction time of step (3-c) is 1-48 hours; optionally, the method for preparing the A crystal form further includes the following steps: (5-c) mixing the solid collected in step (4-c) with the solvent; (6-c) stirring the resulting mixture to crystallize; (7-c) filtering out and collecting the solid, and drying it; the solvent in step (5-c) is selected from at least one of methanol, ethanol, isopropanol, tetrahydrofuran, and water; step (6-c) is carried out at 15-30°C; the reaction time of step (6-c) is 0.5-48 hours.

30. The preparation method according to claim 29, wherein, The solvent in step (1-c) is isopropyl ether; the molar ratio of the compound of formula (I) to succinic acid is 1:1; the reaction time in step (3-c) is 3-24 hours; the solvent in step (5-c) is selected from a mixed solvent of ethanol / water, wherein the volume ratio of ethanol / water is approximately 1:1 to 50:

1.

31. A pharmaceutical composition comprising a pharmaceutically acceptable salt of a compound of formula (I) as claimed in any one of claims 1 to 8 or a crystal form as claimed in any one of claims 9 to 20, and a pharmaceutically acceptable excipient.

32. Use of a pharmaceutically acceptable salt of the compound of formula (I) according to any one of claims 1 to 8, the crystal form according to any one of claims 9 to 20, or the pharmaceutical composition according to claim 31 in the preparation of a medicament for the prevention or treatment of estrogen receptor-related diseases.

33. The use as described in claim 32, wherein, The estrogen receptor-related disease mentioned is a tumor.

34. According to the use described in claim 33, wherein, The tumor is breast cancer.

35. The use as described in claim 33, wherein, The tumor is an ER-positive breast cancer.

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