Crystals of tetrahydrocycloheptaindazole compounds

Abstract

Disclosed are salt forms and crystals of a series of tetrahydrocycloheptaindazole compounds and methods for their preparation, in particular the use of the salt forms and crystals of the compound of formula (I) in the manufacture of medicaments for treating related diseases. [Formula 1] JPEG2024546191000044.jpg68169

Description

Detailed Description of the Invention

[0001] This application claims priority to: CN202111631128.4, filing date: December 28, 2021.

[0002] [Technical Field] The present invention relates to salt forms and crystals of tetrahydrocycloheptaindazole compounds and methods for preparing the same, and in particular to the use of salt forms and crystals of the compound of formula (I) in the manufacture of medicaments for treating related diseases.

[0003] [Background technology] According to WHO statistics, breast cancer is the second most common cancer in the world and the most common cancer among women. After years of research, the role of the estrogen-estrogen receptor signaling pathway in the development of breast cancer has been identified, and estrogen receptor (ER) has become the most important biomarker for breast cancer. Using estrogen receptor expression as a discriminant, breast cancer can be classified into estrogen receptor-positive and estrogen receptor-negative breast cancer, with estrogen receptor-positive breast cancer accounting for more than 70% of all breast cancer patients.

[0004] Endocrine therapy (ET), which targets the estrogen-estrogen receptor signaling pathway in breast cancer cells, has become the first-line treatment for estrogen receptor-positive breast cancer due to its minimal adverse effects and high efficacy. First-line endocrine therapy is primarily an aromatase inhibitor (AI). The aromatase inhibitor letrozole has shown good efficacy in treating estrogen receptor-positive breast cancer; however, the use of these two drugs has led to an increasingly significant problem of resistance to aromatase inhibitors in estrogen receptor-positive breast cancer. Numerous studies have shown that estrogen receptors respond to aromatase inhibitors by mutating, primarily the Y537X mutation, which allows the mutated estrogen receptor to maintain an active conformation in the absence of estrogen and continue to function as a receptor that promotes breast cancer cell proliferation. Fulvestrant is the only selective estrogen receptor downmodulator available on the market and has shown good efficacy in treating breast cancer resistant to hormone therapy. However, fulvestrant faces many challenges in the treatment of AI-resistant, ER-mutated breast cancer. First, due to its poor pharmacokinetic properties, fulvestrant has nearly zero oral bioavailability and high blood clearance. For these two reasons, the drug can only be administered via intramuscular injection. However, due to its strong lipophilic structure, fulvestrant suffers from significant tissue distribution issues even when administered intramuscularly. Clinically, only approximately 50% of breast cancer patients treated with fulvestrant demonstrate clinical responses. Furthermore, due to its insufficient pharmacokinetic properties, the currently approved dose of fulvestrant does not achieve complete degradation of ER, especially mutant ER, at tissue concentrations, making this treatment scheme suboptimal for AI-resistant, ER-mutated breast cancer. Therefore, the development of a drug with better pharmacokinetic properties for ER-mutated breast cancer remains an unmet medical need.

[0005] [ka]

[0006] Elacestrant (RAD1901, Garner, F. et al., Anticancer Drugs, 2015, 26, 948-956), a selective estrogen receptor degrader developed by Radius and Menarini, demonstrated superior progression-free survival rates compared with standard treatments in Phase III clinical trials, particularly in patients with ESR1 mutations, and an NDA application has been submitted to the FDA. WO2018 / 077630A1 reports that AstraZeneca is currently developing camizestrant (AZD9833), a new generation noncovalent estrogen receptor degrader for the treatment of ER-positive breast cancer, and Phase III clinical trials are underway for the treatment of ER-positive, HER2-negative breast cancer. WO2019 / 245974A1 and WO2020 / 049150A1 report that Giredestrant (GDC-9545) and Amcenestrant (SAR439859), new generation non-covalent estrogen receptor degraders, are currently being developed by Genetech and Sanofi, respectively. Giredestrant is currently undergoing Phase III clinical trials, but its Phase II single-agent clinical trials have shown no advantages compared to existing treatments. Amcenestrant also showed no advantages in Phase II clinical trials, and the companies have announced that the studies will now be discontinued.

[0007] Summary of the Invention The present invention provides a compound represented by formula (III):

[0008] [ka]

[0009] The present invention further provides a type A crystal of the compound represented by formula (III), characterized in that its powder X-ray diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 20.92±0.20°, 22.08±0.20°, and 24.70±0.20°.

[0010] [ka]

[0011] In some embodiments of the present invention, the A-type crystal of the compound represented by formula (III) is characterized in that its powder X-ray diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 12.30±0.20°, 15.64±0.20°, 17.00±0.20°, 18.26±0.20°, 20.92±0.20°, 22.08±0.20°, 22.62±0.20°, and 24.70±0.20°.

[0012] In some embodiments of the present invention, the type A crystal of the compound represented by formula (III) is characterized in that its powder X-ray diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 5.18±0.20°, 5.92±0.20°, 8.12±0.20°, 12.30±0.20°, 15.64±0.20°, 17.00±0.20°, 18.26±0.20°, 20.92±0.20°, 22.08±0.20°, and 24.70±0.20°.

[0013] In some embodiments of the present invention, the type A crystal of the compound represented by formula (III) is characterized in that its powder X-ray diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 5.18±0.20°, 5.92±0.20°, 8.12±0.20°, 12.30±0.20°, 15.64±0.20°, 17.00±0.20°, 18.26±0.20°, 19.54±0.20°, 20.92±0.20°, 22.08±0.20°, 22.62±0.20°, and 24.70±0.20°.

[0014] In some embodiments of the present invention, the type A crystal of the compound represented by formula (III) is characterized in that its powder X-ray diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 5.18±0.20°, 5.92±0.20°, 8.12±0.20°, 12.30±0.20°, 15.64±0.20°, 17.00±0.20°, 18.26±0.20°, 19.54±0.20°, 20.92±0.20°, 22.08±0.20°, 24.70±0.20°, and 25.48±0.20°.

[0015] In some embodiments of the present invention, the type A crystal of the compound represented by formula (III) is characterized in that its powder X-ray diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 5.18±0.20°, 5.92±0.20°, 8.12±0.20°, 12.30±0.20°, 15.64±0.20°, 17.00±0.20°, 18.26±0.20°, 19.54±0.20°, 20.92±0.20°, 22.08±0.20°, 22.62±0.20°, 24.70±0.20°, and 25.48±0.20°.

[0016] In some embodiments of the present invention, the A-type crystal of the compound represented by formula (III) has a powder X-ray diffraction spectrum exhibiting the following 2θ angles: 5.18±0.20°, 5.92±0.20°, 8.12±0.20°, 12.30±0.20°, 15.64±0.20°, 16.42±0.20°, 17.00±0.20°, 17.66±0.20°, 18.26±0.20°, 19.54±0.20°, 20.00±0.20°, 21.00±0.20°, 22.00±0.20°, 23.00±0.20°, 24.00±0.20°, 25.00±0.20°, 26.00±0.20°, 27.00±0.20°, 28.00±0.20°, 29.00±0.20°, 30.00±0.20°, 31.00±0.20°, 32.00±0.20°, 33.00±0.20°, 34.00±0.20°, 35.00±0.20°, 36.00±0.20°, 37.00±0.20°, 38.00±0.20°, 39.00±0.20°, 40.00±0.20°, 41.00±0.20°, 42.00±0.20°, 43.00±0.20°, 44.00±0.20°, 45.00±0.20°, 46.00± It is characterized by having characteristic diffraction peaks at 0.06±0.20°, 20.92±0.20°, 22.08±0.20°, 22.62±0.20°, 23.32±0.20°, 23.86±0.20°, 24.70±0.20°, 25.48±0.20°, 26.68±0.20°, 28.50±0.20°, 29.54±0.20°, 31.58±0.20°, and 33.20±0.20°.

[0017] In some embodiments of the present invention, the type A crystal of the compound represented by formula (III) is characterized in that its powder X-ray diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 5.181°, 5.920°, 8.119°, 12.299°, 15.638°, 16.418°, 17.002°, 17.660°, 18.261°, 19.539°, 20.061°, 20.919°, 22.080°, 22.621°, 23.320°, 23.861°, 24.700°, 25.483°, 26.681°, 28.498°, 29.542°, 31.578°, and 33.198°.

[0018] In some embodiments of the present invention, the A-type crystal of the compound represented by formula (III) is characterized in that its powder X-ray diffraction spectrum is essentially as shown in FIG.

[0019] In some embodiments of the present invention, the XRPD spectral analysis data of the Form A crystal is as shown in Table 1.

[0020] [Table 1]

[0021] In some embodiments of the present invention, the A-type crystal of the compound represented by formula (III) has a powder X-ray diffraction spectrum at the following 2θ angles: and / or 5.18±0.20°, and / or 5.92±0.20°, and / or 8.12±0.20°, and / or 12.30±0.20°, and / or 15.64±0.20°, and / or 16.42±0.20°, and / or 17.00±0.20°, and / or 17.66±0.20°, and / or 18.26±0.20°, and / or 19.54±0.20°, and / or 20. and / or 20.92±0.20°, and / or 22.08±0.20°, and / or 22.62±0.20°, and / or 23.32±0.20°, and / or 23.86±0.20°, and / or 24.70±0.20°, and / or 25.48±0.20°, and / or 26.68±0.20°, and / or 28.50±0.20°, and / or 29.54±0.20°, and / or 31.58±0.20°, and / or 33.20±0.20°.

[0022] In some embodiments of the present invention, the A-type crystals of the compound represented by formula (III) are characterized in that their thermogravimetric analysis curves show a weight loss of 0.198% at 150.000±3°C and a weight loss of 10.880% at 240.000±3°C.

[0023] In some embodiments of the present invention, the A-type crystal of the compound represented by formula (III) is characterized in that its thermogravimetric analysis curve is as shown in FIG.

[0024] In some embodiments of the present invention, the Type A crystal of the compound represented by formula (III) is characterized in that its differential scanning calorimetry curve has endothermic peak onsets at 175.87±5°C, 214.68±5°C, and 292.11±5°C.

[0025] In some embodiments of the present invention, the type A crystal of the compound represented by formula (III) is characterized by its differential scanning calorimetry curve as shown in FIG.

[0026] The present invention further provides type B crystals of the compound represented by formula (II), characterized in that the powder X-ray diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 14.24±0.20°, 22.18±0.20°, and 23.78±0.20°.

[0027] [ka]

[0028] In some embodiments of the present invention, the B-type crystals of the compound represented by formula (II) are characterized in that their powder X-ray diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 6.50±0.20°, 14.24±0.20°, 15.80±0.20°, 18.18±0.20°, 22.18±0.20°, 23.78±0.20°, and 25.30±0.20°.

[0029] In some embodiments of the present invention, the B-type crystals of the compound represented by formula (II) are characterized in that their powder X-ray diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 6.50±0.20°, 7.86±0.20°, 14.24±0.20°, 15.80±0.20°, 16.92±0.20°, 18.18±0.20°, 19.66±0.20°, 20.76±0.20°, 22.18±0.20°, 23.78±0.20°, 25.30±0.20°, and 26.12±0.20°.

[0030] In some embodiments of the present invention, the B-type crystal of the compound represented by formula (II) has a powder X-ray diffraction spectrum exhibiting the following 2θ angles: 6.499°, 7.860°, 9.739°, 10.257°, ​​11.862°, 12.255°, 13.021°, 14.240°, 15.417°, 15.797°, 16.541°, 16.920°, 17.558°, 18.182°, 18.439°, 18.702°, 19.660°, 20.381°, 20.761°, 21.494°, and 21.647°. , 22.180°, 23.781°, 24.099°, 24.498°, 25.304°, 26.118°, 26.821°, 27.239°, 28.579°, 28.924°, 29.302°, 29.881°, 30.278°, 30.681°, 30.938°, 31.764°, 32.978°, 34.260°, 35.101°, 35.419°, 35.761°, 36.597°, 37.083°, 37.540°, and 38.423°.

[0031] In some embodiments of the present invention, the XRPD spectral analysis data of the B-type crystals is as shown in Table 2.

[0032] [Table 2]

[0033] In some embodiments of the present invention, the type B crystal of the compound represented by formula (II) has a powder X-ray diffraction spectrum at the following 2θ angles: and / or 6.50±0.20°, and / or 7.86±0.20°, and / or 9.74±0.20°, and / or 10.26±0.20°, and / or 11.86±0.20°, and / or 12.26±0.20°, and / or 13.02±0.20°, and / or 14.24±0.20°, and / or 15.42±0.20°, and / or 15.80±0.20°, and / or 16.54±0.20°, and / or 16.92±0.20°, and / or 17.56±0.20°, and / or 18.18±0.20°, and / or 18.44±0.20°, and / or 18.70±0.20°, and / or 19.66±0.20°, and / or 20.38±0.20°, and / or 20.76±0.20°, and / or 21.49±0.20°, and / or 21.65±0.20°, and / or 22.18±0.20°, and / or 23.78±0.20°, and / or 24.10±0.20°, and / or 24.50±0.20°, and / or 25.30±0.20°, and / or 26.12±0.20°, and / or 26.82±0.20°, and / or 27.24±0.20°, and / or 28.58±0.20°, and / or 28.92±0.20°, and / or 29.30±0.20°, and / or 29.88±0.20°, and / or 30.28±0.20°, and / or 30.68±0 0.20°, and / or 30.94±0.20°, and / or 31.76±0.20°, and / or 32.98±0.20°, and / or 34.26±0.20°, and / or 35.10±0.20°, and / or 35.42±0.20°, and / or 35.76±0.20°, and / or 36.60±0.20°, and / or 37.08±0.20°, and / or 37.54±0.20°, and / or 38.42±0.20°.

[0034] In some embodiments of the present invention, the B-type crystals of the compound represented by formula (II) are characterized by having a powder X-ray diffraction spectrum essentially as shown in FIG.

[0035] In some embodiments of the present invention, the B-type crystals of the compound represented by formula (II) are characterized in that the thermogravimetric analysis curve shows a weight loss of 0.085% at 200.000±3°C.

[0036] In some embodiments of the present invention, the B-type crystals of the compound represented by formula (II) are characterized in that their thermogravimetric analysis curves are as shown in FIG.

[0037] In some embodiments of the present invention, the B-type crystals of the compound represented by formula (II) are characterized by having an endothermic peak at 215.60±3°C in its differential scanning calorimetry curve.

[0038] In some embodiments of the present invention, the B-type crystals of the compound represented by formula (II) are characterized by a differential scanning calorimetry curve as shown in FIG.

[0039] The present invention further provides a C-type crystal of the compound represented by formula (I), characterized in that its powder X-ray diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 4.90±0.20°, 15.82±0.20°, and 22.26±0.20°.

[0040] [ka]

[0041] In some embodiments of the present invention, the C-type crystal of the compound represented by formula (I) is characterized in that its powder X-ray diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 4.90±0.20°, 9.82±0.20°, 15.82±0.20°, 17.48±0.20°, 18.64±0.20°, 22.26±0.20°, 23.74±0.20°, and 29.36±0.20°.

[0042] In some embodiments of the present invention, the C-type crystal of the compound represented by formula (I) is characterized in that its powder X-ray diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 30±0.20°, 4.90±0.20°, 9.82±0.20°, 11.06±0.20°, 14.20±0.20°, 15.82±0.20°, 17.48±0.20°, 18.64±0.20°, 22.26±0.20°, 23.74±0.20°, 24.46±0.20°, and 29.36±0.20°.

[0043] In some embodiments of the present invention, the C-type crystal of the compound represented by formula (I) has a powder X-ray diffraction spectrum exhibiting the following 2θ angles: 3.301°, 4.901°, 7.898°, 9.319°, 9.819°, 11.061°, 14.200°, 14.721°, 15.197°, 15.821°, 16.457°, 17.481°, 18.101°, 19.101°, 20.101°, 21.101°, 22.101°, 23.101°, 24.101°, 25.101°, 26.101°, 27.101°, 28.101°, 29.101°, 30.101°, 31.101°, 32.101°, 33.101°, 34.101°, 35.101°, 36.101°, 37.101°, 38.101°, 39.101°, 40.101°, 41.101°, 42.101°, 43.101°, 44.101°, 45.101°, 46.101°, 47.101°, 48.101°, 49.101°, 50.101°, 51.101°, 52.101°, 53.101°, 54.101°, 55.101°, 56.101°, 57.101°, 58.101°, It is characterized by having characteristic diffraction peaks at 8.642°, 19.762°, 20.961°, 21.382°, 22.259°, 23.740°, 24.461°, 25.761°, 26.295°, 26.943°, 27.518°, 29.358°, 30.075°, 31.317°, 31.916°, 34.598°, and 37.661°.

[0044] In some embodiments of the present invention, the XRPD spectral analysis data of the Form C crystal is as shown in Table 3.

[0045] [Table 3]

[0046] In some embodiments of the present invention, the C-type crystal of the compound represented by formula (I) has a powder X-ray diffraction spectrum at the following 2θ angles: and / or 3.30±0.20°, and / or 4.90±0.20°, and / or 7.90±0.20°, and / or 9.32±0.20°, and / or 9.82±0.20°, and / or 11.06±0.20°, and / or 14.20±0.20°, and / or 14.72±0.20°, and / or 15.20±0.20°, and / or 15.82±0.20°, and / or 16.46±0.20°, and / or 17.48±0.20°, and / or 18.10±0.20°, and / or 18.64±0.20°, and / or 19.76±0.20°, and / or 20.96±0.20°, and / or 21.38±0.20°, and / or 22.26±0.20°, and / or 23.74±0.20°, and / or 24.46±0.20°, and / or 25.76±0.20°, and / or 26.30±0.20°, and / or 26.94±0.20°, and / or 27.52±0.20°, and / or 29.36±0.20°, and / or 30.08±0.20°, and / or 31.32±0.20°, and / or 31.92±0.20°, and / or 34.60±0.20°, and / or 37.66±0.20°.

[0047] In some embodiments of the present invention, the C-type crystal of the compound represented by formula (I) is characterized by having a powder X-ray diffraction spectrum essentially as shown in FIG.

[0048] In some embodiments of the present invention, the C-type crystals of the compound represented by formula (I) are characterized in that their differential scanning calorimetry curve has an endothermic peak onset at 124.14±5°C, an exothermic peak onset at 199.34±5°C, and an exothermic peak onset at 278.70±5°C.

[0049] In some embodiments of the present invention, the C-type crystals of the compound represented by formula (I) are characterized by a differential scanning calorimetry curve as shown in FIG.

[0050] The present invention further provides a crystal of the compound represented by formula (II), characterized in that its crystal structure is as shown in FIG.

[0051] The present invention further provides use of the above compound and a crystal thereof in the manufacture of a medicament for treating an ESR1-positive tumor.

[0052] [Technical effect] The compound of the present invention has good PK properties and oral absorption rate, and its crystals are stable.

[0053] The compounds of the present invention exhibit excellent activity against wild-type and mutant ESR1-positive tumor models and show excellent efficacy against various types of ESR1-positive cells. + / HER2 - This could potentially provide a more effective treatment for patients with locally advanced or metastatic breast cancer.

[0054] [Definitions and Explanations] The present invention uses the following abbreviations: DMSO stands for dimethyl sulfoxide, TsOH stands for p-toluenesulfonic acid, PBS stands for 0.9% sodium chloride phosphate buffer, THP stands for tetrahydropyranyl, and Nf stands for perfluoro-1-n-butyl.

[0055] Weight loss represents the weight loss, Weight percent loss represents the weight loss percentage, Residue represents the residue, Residue percent represents the residual rate, Integral represents the total amount of heat (heat) released (endotherm), Normalized represents the standard amount of heat (heat) released (endotherm), Peak represents the peak value, Onset represents the initial melting temperature, Endset represents the final melting temperature, Left limit represents the temperature of the left boundary, and Right limit represents the temperature of the right boundary.

[0056] Compounds are named according to conventional naming principles in the art or using ChemDraw® software, commercially available compounds are named in supplier catalogs.

[0057] X-ray powder diffractometer (XRPD) method of the present invention Instrument model: Bruker D8 advance X-ray diffractometer Measurement method: Approximately 10 to 20 mg of sample is used for XRPD detection.

[0058] The detailed XRPD parameters are as follows: X-ray tube: Cu, kα, (λ=1.54056Å).

[0059] Tube voltage: 40 kV, tube current: 40 mA Divergence slit: 0.60 mm Detector slit: 10.50 mm Anti-scatter slit: 7.10 mm Scanning range: 4~40° Step angle: 0.02 deg Step width: 0.12 seconds Sample pan rotation speed: 15 rpm Differential Scanning Calorimetry (DSC) Method of the Present Invention Instrument model: TA Q2000 differential scanning calorimeter Measurement method: A sample (approximately 1 mg) is placed in a DSC aluminum crucible and heated from 30°C (room temperature) to 300°C (or 350°C) at a heating rate of 10°C / min under conditions of 50 mL / min N2.

[0060] The Thermogravimetric Analysis (TGA) Method of the Present Invention Instrument model: TA Q5000IR Thermogravimetric Analyzer Measurement method: A sample (2-5 mg) is placed in a TGA platinum crucible and heated from room temperature to 350°C at a heating rate of 10°C / min under conditions of 25 mL / min N2, or until the weight is reduced by 20%.

[0061] The single crystal diffraction method of the present invention Sample incubation: 2 mg of the compound of formula (II) was dissolved in 400 μL of methanol / ethyl acetate (1:1) at room temperature. The sample solution was placed in a 1 mL semi-sealed sample vial and allowed to slowly evaporate at room temperature. The crystals were obtained the next day, as shown in Figure 9.

[0062] Instrument model: Rigaku Oxford Diffraction XtaLAB Synergy-S four-circle diffractometer equipped with a HyPix-6000HE area detector.

[0063] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] XRPD pattern of type A crystal of the compound represented by formula (III).

[0064] [Figure 2] TGA pattern of type A crystals of the compound represented by formula (III).

[0065] [Figure 3] DSC pattern of type A crystals of the compound represented by formula (III).

[0066] [Figure 4] XRPD pattern of type B crystals of the compound represented by formula (II).

[0067] [Figure 5] TGA pattern of type B crystals of the compound represented by formula (II).

[0068] [Figure 6] DSC pattern of type B crystals of the compound represented by formula (II).

[0069] [Figure 7] XRPD pattern of type C crystals of the compound represented by formula (I).

[0070] [Figure 8] DSC pattern of type C crystals of the compound represented by formula (I).

[0071] [Figure 9] A schematic diagram of the single crystal structure of the compound represented by formula (II).

[0072] [Figure 10] Tumor growth curve of type C crystal of the compound of formula (I) in a human breast cancer MCF7 cell subcutaneous xenograft tumor-bearing mouse model after administration of the test compound.

[0073] FIG. 11 shows tumor growth curves of the compound of formula (I) type C crystals compared to fulvestrant and palbociclib in mice bearing a human breast cancer MCF7 cell subcutaneous xenograft tumor model after administration of the test compound.

[0074] FIG. 12 shows the tumor growth curve of the C-type crystals of the compound of Formula (I) in combination with palbociclib in a human breast cancer MCF7 cell subcutaneous xenograft tumor model-bearing mice after administration of the test compound.

[0075] [FIG. 13] Percentage change (%) in body weight of mice bearing a human breast cancer MCF7 cell subcutaneous xenograft tumor model during the administration of type C crystals of the compound represented by formula (I).

[0076] [Figure 14] Percentage change (%) in body weight of mice bearing a human breast cancer MCF7 cell subcutaneous xenograft tumor model during the administration of type C crystals of the compound of formula (I) compared with fulvestrant and palbociclib.

[0077] FIG. 15 shows the percentage change (%) in body weight of mice bearing a human breast cancer MCF7 cell subcutaneous xenograft tumor model during the administration of type C crystals of the compound of Formula (I) in combination with palbociclib.

[0078] FIG. 16 shows the tumor growth curve of the B-type crystal of the compound of formula (II) in a subcutaneous xenograft tumor model mouse bearing MCF7 cells harboring the ESR1 D538G mutation.

[0079] FIG. 17 shows the tumor growth curve of the B-type crystals of the compound of formula (II) in combination with palbociclib in mice bearing a subcutaneous xenograft tumor model of MCF7 cells harboring the ESR1 D538G mutation.

[0080] FIG. 18 shows the percentage (%) of weight change in mice bearing a subcutaneous xenograft tumor model of MCF7 cells harboring the ESR1 D538G mutation during the administration of type B crystals of the compound of formula (II).

[0081] FIG. 19 shows the percentage change (%) in body weight of mice bearing a subcutaneous xenograft tumor model of MCF7 cells harboring the ESR1 D538G mutation during the administration of type B crystals of the compound of Formula (II) in combination with palbociclib.

[0082] [Mode for Carrying Out the Invention] The present invention will be specifically described below using examples, but this does not mean to limit the present invention in any way. The compounds of the present invention can be prepared by various synthetic methods familiar to those skilled in the art, including the specific embodiments listed below, embodiments combined with other chemical synthetic methods, and equivalent alternative methods familiar to those skilled in the art. Preferred embodiments include, but are not limited to, the examples of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0083] Example 1: Preparation of compounds of formula (I) Preparation of intermediate compounds 1-11:

[0084] [ka]

[0085] Step A: To a stirred solution of 1-13 (100.0 g, 420.38 mmol) in N-methylpyrrolidone (1000 mL) at 25 °C, triethylamine (87.77 mL, 630.57 mmol) and 1-14 (78.30 g, 420.38 mmol) were added sequentially. The mixture was stirred at 130 °C (internal temperature) for 12 h under nitrogen gas protection. After cooling to 25 °C, the reaction solution was stirred, water (1000 mL) was added, and stirring was continued at 25 °C for 1 h. The resulting suspension was vacuum filtered through a Buchner funnel, and the cake was washed with water (200 mL × 2). The cake was collected and dried under vacuum to obtain compound 1-15.

[0086] Step B: To a stirred solution of 1-15 (100 g, 291.36 mmol) in tetrahydrofuran (700 mL) at 25 °C, p-toluenesulfonic acid monohydrate (166.26 g, 874.08 mmol) was added, and the mixture was stirred at 35 °C for 12 hours. The reaction solution was concentrated under reduced pressure until no more liquid evaporated. The concentrated crude product was dissolved in acetonitrile (300 mL) at 25 °C, followed by the addition of methoxycyclopentyl ether (1500 mL) and stirring at 25 °C for 12 hours. The resulting suspension was filtered under reduced pressure using a Buchner funnel. The cake was washed with methoxycyclopentyl ether (50 mL × 2), collected, and dried in vacuo to give compound 1-16.

[0087] Step C: To a stirred suspension of 1-16 (100 g, 170.21 mmol) in acetonitrile (1 L) at 25 °C, anhydrous potassium phosphate (112.00 g, 527.65 mmol) and 1-17 (32.64 g, 173.62 mmol) were added and the mixture was stirred at 40 °C for 12 h under nitrogen gas protection. The reaction solution was concentrated under reduced pressure until no more liquid was evaporated, and 2-methyltetrahydrofuran (500 ml × 3) and saturated aqueous sodium carbonate solution (400 ml) were added to the residue. Saturated brine (300 ml × 2) was added to the separated organic phase, which was then dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and dried to give compound 1-11. LCMS (ESI) m / z: 303 / 305 [M+H] + .

[0088] Preparation of Compounds of Formula (I):

[0089] [ka]

[0090] Step A: At 0°C under nitrogen gas protection, a solution of n-butyllithium in n-hexane (2.5 mol / L, 28.21 ml) was slowly added dropwise to a solution of N,N-diisopropylamine (7.73 g, 76.41 mmol) in tetrahydrofuran (100 ml). The reaction solution was stirred at 0°C for 0.5 hours, then cooled to -70°C. A solution of 1-1 (10 g, 58.78 mmol) in tetrahydrofuran (100 ml) was slowly added dropwise. After the addition was complete, the reaction solution was stirred at -70°C for 0.5 hours. Next, a solution of 1-2 (8.53 g, 70.53 mmol) in toluene (10 ml) was slowly added dropwise to the reaction solution. After the addition was complete, the reaction solution was stirred at -70°C for 3 hours. The reaction mixture was added with saturated aqueous ammonium chloride (200 ml) and extracted with ethyl acetate (100 ml × 2). The combined organic phases were washed with saturated aqueous ammonium chloride (200 ml × 2) and saturated brine (200 ml × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The product was purified by silica gel column chromatography (eluent: petroleum ether) to give compound 1-3.

[0091] Compound 1-3: 1 HNMR (400 MHz, CDCl3) δ = 5.82 - 5.59 (m, 1H), 5.15 (d, J = 1.0 Hz, 1H), 5.13 - 5.09 (m, 1H), 4.19 (q, J = 7.1 Hz, 2H), 2.82 - 2.74 (m, 1H), 2.69 - 2.53 (m, 1H), 2.50 - 2.40 (m, 1H), 2.39 - 2.30 (m, 1H), 2.30 - 2.17 (m, 1H), 1.32 - 1.27 (m, 3H).

[0092] Step B: Under nitrogen gas protection at 20 °C, 9-borabicyclo[3.3.1]nonane (0.5 mol / L, 32.45 ml) was added to a solution of 1-3 (3.1 g, 14.75 mmol) in tetrahydrofuran (18 ml). The reaction solution was stirred at 60 °C for 3 h and then cooled to room temperature. Water (10 ml), 1-4 (3.53 g, 11.80 mmol), tetrakis(triphenylphosphine)palladium(0) (1.7 g, 1.47 mmol), and potassium phosphate (4.7 g, 22.12 mmol) were added to the reaction solution, followed by stirring at 70 °C for 16 h under nitrogen gas protection. The resulting reaction solution was diluted with ethyl acetate (100 ml), washed with saturated brine (200 ml × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. Purification by silica gel column chromatography (eluent: petroleum ether / ethyl acetate=100 / 1 to 0 / 1) gave compound 1-5. LCMS (ESI) m / z: 431.1 [M + H] + .

[0093] Step C: To a mixture of 1-5 (6.7 g, 13.46 mmol) in methanol (75 ml) and water (25 ml) was added sodium hydroxide (2.15 g, 53.83 mmol), and the mixture was stirred at 25° C. for 16 hours. The resulting reaction mixture was concentrated under reduced pressure to remove the solvent methanol. The residue was diluted with water (150 ml) and extracted with dichloromethane (100 ml × 2). The aqueous phase was adjusted to pH 2 with hydrochloric acid (2 mol / L) and extracted with ethyl acetate (80 ml × 4). The combined organic phase was washed with saturated brine (200 ml × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 1-6.

[0094] Compound 1-6: 1HNMR (400 MHz, CDCl3) δ = 7.34 (d, J = 4.3 Hz, 2H), 6.99 - 6.90 (m, 1H), 5.59 (td, J = 2.4, 9.3 Hz, 1H), 4.04 (br d, J = 11.0 Hz, 1H), 3.73 (dt, J = 2.9, 10.9 Hz, 1H), 2.97 (br t, J = 7.1 Hz, 2H), 2.82 - 2.72 (m, 1H), 2.72 - 2.56 (m, 1H), 2.56 - 2.42 (m, 1H), 2.28 - 2.13 (m, 2H), 2.06 - 1.99 (m, 1H), 1.79 - 1.58 (m, 7H). LCMS (ESI) m / z: 403.1 [M + H] +.

[0095] Step D: 1-6 (2.7 g, 6.54 mmol) was dissolved in polyphosphoric acid (30 ml), and the mixture was stirred at 110 °C for 16 hours. The resulting reaction solution was diluted with water (300 ml) and extracted with ethyl acetate (100 ml × 3). The combined organic phase was washed with saturated aqueous sodium carbonate (200 ml × 2) and saturated brine (200 ml), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 1-7. LCMS (ESI) m / z: 301.1 [M + H] +

[0096] Step E: 1-7 (2.7 g, 8.99 mmol) was dissolved in dichloromethane (50 mL), and p-toluenesulfonic acid monohydrate (855.26 mg, 4.5 mmol) and 3,4-dihydro-2H-pyran (1.13 g, 13.49 mmol) were added. The mixture was stirred at 25 °C for 16 hours. The reaction solution was concentrated under reduced pressure to obtain the crude product. The product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 40 / 1 to 20 / 1) to obtain compound 1-8.

[0097] Compound 1-8: 1HNMR (400 MHz, CDCl3) δ = 7.81 (dd, J = 5.4, 8.9 Hz, 1H), 7.38 (td, J = 2.0, 8.8 Hz, 1H), 5.59 (td, J = 2.4, 9.2 Hz, 1H), 4.06 - 3.95 (m, 1H), 3.81 - 3.64 (m, 2H), 3.36 - 3.23 (m, 1H), 3.22 - 2.95 (m, 2H), 2.53 - 2.38 (m, 1H), 2.35 - 2.20 (m, 2H), 2.19 - 2.08 (m, 2H), 2.06 - 2.00 (m, 1H), 1.80 - 1.65 (m, 5H)). LCMS (ESI) m / z: 385.1 [M + H] +.

[0098] Step F: At −40°C under nitrogen gas protection, a solution of 1-8 (40 g, 104.07 mmol) in tetrahydrofuran (266 mL) was added dropwise to a solution of potassium bis(trimethylsilyl)amide in tetrahydrofuran (1 mol / L, 114.47 mL, 114.47 mmol). After the addition was complete, the mixture was stirred at −40°C for 1 hour. Perfluorobutanesulfonyl fluoride (20.11 mL, 114.47 mmol) was added dropwise to the reaction solution at −40°C, and the mixture was slowly warmed to 25°C and stirred for 12 hours. The resulting reaction solution was quenched by adding saturated aqueous ammonium chloride (1300 mL) at 25°C. The mixture was diluted with 500 mL of water and extracted with ethyl acetate (600 mL × 3). The combined organic phase was washed with saturated brine (200 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product (79.13 g). The crude product was stirred with ethanol (160 ml) at 25° C. for 12 hours, filtered and washed to give compound 1-9.

[0099] Step G: To a solution of 1-9 (25 g, 37.51 mmol) and bis(pinacolato)diboron (14.29 g, 56.27 mmol) in 1,4-dioxane (200 mL) at 25 °C, potassium carbonate (7.78 g, 56.27 mmol), triphenylphosphine (1.48 g, 5.63 mol), and dichlorobis(triphenylphosphine)palladium(0) (1.84 g, 2.63 mmol) were added. The mixture was stirred at 90 °C for 12 h under nitrogen gas protection. The resulting reaction solution was filtered through diatomaceous earth, diluted with water (40 mL), and extracted with ethyl acetate (50 mL × 3). The combined organic phase was washed with saturated brine (40 mL × 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 30.43 g of crude product. Acetonitrile (150 ml) and water (37.5 ml) were added, heated to 50° C. to dissolve, cooled to 25° C., and stirred for 12 hours. The resulting suspension was filtered under reduced pressure, and the cake was collected to give compound 1-10. LCMS (ESI) m / z: 495.2 [M+H] + .

[0100] Step H: To a solution of 1-11 (1.91 g, 6.31 mmol) and 1-10 (3.12 g, 6.31 mmol) in tetrahydrofuran (30 mL) and water (6 mL) was added potassium hydroxide (1.95 g, 34.71 mmol) and tetrakis(triphenylphosphine)palladium(0) (364.67 mg, 315.58 μmol) at 25 °C. The mixture was stirred at 60 °C for 12 h under nitrogen gas protection. The resulting reaction solution was diluted with water (20 mL) and extracted with dichloromethane (20 mL × 3). The combined organic phase was washed with saturated brine (20 mL × 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The product was purified by reverse-phase silica gel column chromatography (gradient elution with 0.1% aqueous ammonia / acetonitrile) to give compound 1-12. LCMS (ESI) m / z: 591.2 [M + H] +.

[0101] Step I: At 20°C, trifluoroacetic acid (60 ml) was added to a solution of 1-12 (18 g, 7.87 mmol) in acetonitrile (180 ml). The mixture was stirred at 80°C for 16 hours. The resulting reaction solution was concentrated under reduced pressure until no more liquid was evaporated. The pH was adjusted to 8-10 by adding aqueous sodium carbonate, and the mixture was extracted with dichloromethane. The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1) to obtain the compound represented by formula (I). LCMS (ESI) m / z: 507.3 [M+H] + .

[0102] Example 2: Preparation of A-type crystals of the compound represented by formula (III) 5 g of the compound of formula (I) was dissolved in 30 ml of acetonitrile. The mixture was heated to 90°C, fumaric acid (1.26 g) was added, and the mixture was stirred for 1 hour. The mixture was then slowly cooled to 20°C and stirred for 12 hours. The resulting suspension was filtered, and the cake was dried to obtain type A crystals of the compound of formula (III). 1 H NMR (400 MHz, DMSO-d6) δ = 12.60 (s, 1H), 8.01 (d, J = 1.2 Hz, 1H), 7.71 (d, J = 0.8 Hz, 1H), 7.61 (br d, J = 6.4 Hz, 1H), 7.28 (dd, J = 2.4, 8.8 Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H), 6.64 (s, 2H), 4.61 (t, J = 6.0 Hz, 1H), 4.50 (t, J = 6.0 Hz, 1H), 4.46 - 4.35 (m, 1H), 3.54 (q, J = 11.6 Hz, 2H), 3.13 - 2.94 (m, 4H), 2.85 - 2.70 (m, 4H), 2.45 - 2.24 (m, 3H), 2.10 (br t, J = 6.8 Hz, 2H), 2.03 - 1.87 (m, 2H), 1.86 - 1.74 (m, 1H).

[0103] Example 3: Preparation of B-type crystals of the compound represented by formula (II) 1 g of the compound of formula (I) was weighed and added to a 50 ml single-neck flask, and acetonitrile (9 ml) was added to dissolve it. After adding a magnet, the solution was placed on a magnetically heated stirrer (90 ° C) and stirred. At 90 ° C, a solution of methanesulfonic acid (155 μl) in acetonitrile (1 ml) was added and stirred. The mixture was slowly cooled to 20 ° C and stirred for 12 hours. The resulting suspension was filtered, and the cake was dried to obtain type B crystals of the compound of formula (II).

[0104] 1 g of the compound of formula (I) was weighed and added to a 50 ml single-neck flask, and ethyl acetate (10 ml) was added to dissolve it. After adding a magnet, the solution was placed on a magnetic stirrer (90 ° C) and stirred. At 90 ° C, a solution of methanesulfonic acid (155 μl) in ethyl acetate (1 ml) was added and stirred for 1 hour. The mixture was slowly cooled to 20 ° C and stirred for 12 hours. The resulting suspension was filtered, and the cake was dried to obtain type B crystals of the compound of formula (II).

[0105] 1 g of the compound of formula (I) was weighed and added to a 50 ml single-neck flask, and isopropyl acetate (9 ml) was added to dissolve it. After adding a magnet, the solution was placed on a magnetically heated stirrer (20 ° C) and stirred. At 20 ° C, a solution of methanesulfonic acid (155 μl) in isopropyl acetate (1 ml) was added and stirred. The mixture was slowly cooled to 4 ° C and stirred for 17 hours. The resulting suspension was filtered, and the cake was dried to obtain type B crystals of the compound of formula (II).

[0106] 350 g of the B-type crystals of the compound represented by formula (II) obtained by the above-mentioned preparation method were weighed and placed in a 5-L single-neck flask. Dimethyl sulfoxide (455 ml) was added and the mixture was heated to 60-70°C until completely dissolved. Isopropyl acetate (3 L) was added dropwise at 60-70°C over 1 hour and stirred at the same temperature for 1 hour. The mixture was slowly cooled to 25-30°C over 4-6 hours with stirring and then stirred at the same temperature for 12 hours to obtain a milky white suspension. The reaction solution was filtered to obtain a pale yellow cake. The cake was thoroughly stirred with isopropyl acetate, washed twice (500 ml x 2), and subjected to thermostatic suction filtration. The resulting cake was vacuum-dried to obtain a crude product. Acetone (4.6 L) was added, and the mixture was stirred at 55-60°C for 16 hours and then filtered. Acetone (3.9 L) was further added to the resulting cake, and the mixture was stirred at 55-60°C for 16 hours, filtered, and the cake was dried to obtain B-type crystals of the compound represented by formula (II). 1H NMR (400 MHz, DMSO-d6) δ = 12.55 (s, 1H), 9.84 - 9.66 (m, 1H), 8.03 (br d, J = 6.8 Hz, 1H), 7.72 (s, 1H), 7.70 - 7.53 (m, 1H), 7.24 (dd, J = 2.0, 8.7 Hz, 1H), 6.81 (d, J = 8.8 Hz, 1H), 4.60 (q, J = 5.6 Hz, 1H), 4.52 - 4.43 (m, 2H), 4.05 - 3.62 (m, 2H), 3.61 - 3.37 (m, 4H), 3.29 (br s, 2H), 3.23 - 2.99 (m, 1H), 2.96 (br t, J = 6.8 Hz, 2H), 2.40 - 2.31 (m, 5H), 2.14 - 1.94 (m, 6H).

[0107] Example 4: Preparation of C-type crystals of the compound represented by formula (I) 4 g of the compound of formula (I) was dissolved in 12 ml of isopropyl acetate, and then 30 ml of n-heptane was slowly added with stirring. After stirring at 25°C for 12 hours, the resulting suspension was filtered and the cake was dried to obtain type C crystals of the compound of formula (I). 1H NMR (400 MHz, CD3OD) δ = 7.92 (d, J = 1.3 Hz, 1H), 7.72 (d, J = 1.3 Hz, 1H), 7.18 (dd, J = 2.4, 8.8 Hz, 1H), 6.85 (d, J = 8.8 Hz, 1H), 4.55 (t, J = 5.9 Hz, 1H), 4.47 - 4.37 (m, 2H), 3.41 - 3.33 (m, 2H), 3.08 (br t, J = 7.0 Hz, 2H), 2.98 (dd, J = 7.1, 9.9 Hz, 1H), 2.79 (dt, J = 6.3, 8.6 Hz, 1H), 2.68 - 2.58 (m, 3H), 2.54 (dd, J = 4.9, 10.0 Hz, 1H), 2.46 - 2.30 (m, 3H), 2.19 - 2.11 (m, 2H), 2.00 - 1.84 (m, 2H), 1.82 - 1.71 (m, 1H). LCMS (ESI) m / z: 507.2 [M+H] + .

[0108] Experimental Example 1: Biological Test (1) ERα binding test: activity evaluation of the compound represented by formula (I) in a radioligand binding test Experimental material: estrogen ERα Source: Human recombinant insect Sf9 cells Ligand: 0.5nM 3 H]estradiol Solvent: 1.0% DMSO Nonspecific ligand: 1.0 μM diethylstilbestrol Incubation time / temperature: 2 hours @25℃ Specific binding: 85% Incubation buffer: 10 mM Tris-HCl, pH 7.4, 0.1% BSA, 10% glycerol, 1 mM DTT Quantitative methods: Radioligand binding Kd: 0.2nM Criteria for significance: ≥ 50% maximal activation or inhibition Bmax: 1400 pmol / mg protein Experimental Method: The methods used in this study were adapted from the scientific literature (Obourn, JD et al., Biochemistry, 1993, 32, 6229-6236).

[0109] Data Analysis: MathIQ TM (ID Business Solutions Ltd., UK) software was used to measure IC using nonlinear, least squares regression. 50 The inhibitory constant (K i ) is the IC of the test compound measured experimentally 50 , the radioligand concentration and ligand K used in the study D The historical values ​​of nH (experimentally determined at Eurofins Panlabs, Inc.) were calculated using the Cheng and Prusoff equation (Cheng, Y., Prusoff, WH, Biochem. Pharmacol. 22:3099-3108, 1973). As shown, the Hill coefficient (nH), which defines the slope of the competitive binding curve, was calculated using MathIQ. TM Hill coefficients were calculated using the IC. Significant deviations from a value of 1 may indicate that the bond substitutions in question do not conform to the laws of mass action at a single binding site. 50 , Ki and / or n H If the data does not include the standard error of the mean (SEM), the data is not sufficiently quantitative and should not be used to calculate the data (K i ,I C 50 , n H ) should be interpreted with caution.

[0110] [Table 4]

[0111] Conclusion: The compounds of the present invention have strong binding activity to ERα in vitro.

[0112] (2) HEK293 / GAL4 / ERα antagonism experiment Test materials:

[0113] [Table 5]

[0114] Cell information and culture conditions:

[0115] [Table 6]

[0116] Experimental medium: DMEM (phenol red-free) containing 10% carbon-adsorbed serum, i.e., 500 mL of cell culture medium contains 450 mL of DMEM and 50 mL of carbon-adsorbed serum. Bright Glo detection reagent: Bright-Glo TM The Luciferase Assay kit contains two reagents: Bright-Glo TM Buffer Bright-Glo TM Substrate Before use, the two reagents were equilibrated to room temperature (25°C), then mixed and used after they were completely dissolved. Unused Bright Glo was stored at -20°C for continued use.

[0117] Experimental Design: To analyze the antagonistic effect of the compound of formula (I) on ERα, triplicate wells were set up and the compound concentrations ranged from 200 nM to 0.00256 nM.

[0118] Experimental steps: 1) The HEK293 / GAL4 / ERα cell suspension was collected and centrifuged at 1000 rpm for 5 minutes. The supernatant was removed and resuspended in pre-warmed experimental medium. After counting, the cell suspension was diluted with the experimental medium. 40,000 cells were seeded per well in a 96-well cell culture plate, and 80 μL of the cell suspension was seeded into each well. The cells were then cultured overnight at 37°C in a 5% CO2 incubator.

[0119] 2) On the day of the experiment, 10 μL of compound working solution was added to each well of the cell plate according to the compound placement map, and the plate was incubated for 1 hour in an incubator at 37°C with 5% CO2. Then, 10 μL of assay medium containing 1 nM estradiol was added to each well. 10 μL of assay medium was added to the blank control well, for a final estradiol concentration of 1 nM. The plate was incubated for 24 hours in an incubator at 37°C with 5% CO2.

[0120] 3) After incubation, the cell supernatant was removed, and 50 μL of Bright Glo detection reagent was added to each well of the cell plate and incubated for 2 minutes at 25°C. After incubation, the luminescence signal was detected using EnVision.

[0121] Data Analysis: The data were used to calculate the ERa inhibition rate after compound treatment according to the following formula: % inhibition rate = 100 - (RFU 化合物 -RFU ブランク対照 ) / (RFU 陰性対照 -RFU ブランク対照 ) × 100%. Negative control: cells treated with 1 nM estradiol; blank control: cells not treated with estradiol. Plotted in Prism to determine IC of compounds. 50 values ​​were calculated.

[0122] Experimental results IC of antagonistic activity of the compound represented by formula (I) against HEK293 / GAL4 / ERα 50 is as shown in Table 7 below.

[0123] [Table 7]

[0124] Testing Conclusion: The compound of formula (I) has a significant antagonistic effect on the transcriptional function of HEK293 / GAL4 / ERα.

[0125] (3) ERα degradation experiments in MCF-7, T-47D, and CAMA-1 cells Test materials:

[0126] [Table 8]

[0127] Cell information and culture conditions:

[0128] [Table 9]

[0129] Test reagent production: Preparation of TritonX-100: TritonX-100 was diluted 100-fold with DPBS to obtain 1.00% TritonX-100.

[0130] Preparation of blocking buffer containing 0.100% Tween 20: Tween 20 was diluted 1000 times with blocking buffer to obtain 0.100% Tween 20 blocking buffer.

[0131] Preparation of primary antibody dilution: The primary antibody (Estrogen Receptor alpha Monoclonal Antibody (SP1)) stock solution was diluted 1000-fold with blocking buffer containing 0.100% Tween-20 to obtain the primary antibody dilution.

[0132] Preparation of secondary antibody and DRAQ5 mixed solution: Secondary antibody (LI-COR-926-32211, diluted 1:1000) and DRAQ5 (DR51000, diluted 1:2000) were diluted in blocking buffer containing 0.100% Tween-20 and mixed to homogenize.

[0133] Experimental steps: 1) Complete medium was aspirated from the culture flask, an appropriate amount of trypsin was added to rinse, and residual serum was then removed by aspiration.

[0134] 2) Add an appropriate amount of trypsin to the culture flask, rinse twice, then aspirate. Place the culture flask in an incubator for 2-3 minutes, remove it, and shake the culture flask to detach the cells.

[0135] 3) Cells were resuspended in fresh complete medium and counted in a cell counter.

[0136] 4) The cell suspension was arranged according to plating density.

[0137] 5) 30.0 μL of the cell suspension was added to each well of a 384-well plate.

[0138] 6) The experimental well plate was placed in a centrifuge and centrifuged at 1000 rpm for 1 minute.

[0139] 7) The experimental well plate was cultured overnight in a cell culture incubator.

[0140] 8) According to the sample addition schematic of the experimental well plate (see 3.5.1), 20.0 μL of diluted test sample was added to each well. The final concentration of DMSO in the reaction system was 1.00%.

[0141] 9) The experimental well plate was placed in a centrifuge and centrifuged at 1000 rpm for 1 minute.

[0142] 10) The well plate was cultured in an incubator at 37°C with 5% CO2 for 4 hours.

[0143] 11) The plate was taken out and equilibrated at room temperature for 10 minutes, after which the medium was removed from the well plate.

[0144] 12) 100 μL of 4.00% PFA was added to each well and fixed at room temperature for 40 minutes.

[0145] 13) The liquid in the plate was removed by tapping, and 100 μL of 1×PBS was added to each well using a multidrop, and the plate was washed twice.

[0146] 14) 50 μL of ice-cold PBS containing 0.1% Triton X-100 was added for permeabilization, and the cells were left at room temperature for 15 minutes.

[0147] 15) The liquid in the plate was removed by tapping, and 100 μL of PBS was added to each well of the plate using a multidrop, and then removed by tapping. In this way, the plate was washed five times.

[0148] 16) 50 μL of blocking buffer (LI-COR-927-40000) containing 0.1% Tween-20 was added to each well, and the wells were blocked at room temperature for 1 hour.

[0149] 17) The buffer was removed from the plate by tapping.

[0150] 18) 25 μL of diluted primary antibody solution was added to each well of a 384-well plate. The plate was incubated overnight at 4°C.

[0151] 19) The primary antibody on the plate was removed by tapping, and the plate was washed five times by adding PBS to the multidrop.

[0152] 20) 25 μL of a mixed solution of secondary antibody and DRAQ5 was added to each well and incubated at room temperature for 1 hour.

[0153] 21) PBS was added to the multidrop and the plate was washed five times.

[0154] 22) Wash the plate twice with ddH2O and tap dry.

[0155] 23) Plates were read using the Odyssey Far Infrared Imaging System instrument.

[0156] Data analysis High control group mean: calculate the mean value of the high control group; Low control group mean: calculate the mean value of the low control group; High control group standard deviation (SD): calculate the standard deviation of the high control group; Low control group standard deviation (SD): calculate the standard deviation of the low control group.

[0157] Coefficient of variation (CV) of the high control group = (standard deviation of the high control group ÷ mean value of the high control group) × 100 Coefficient of variation (CV) of the low control group = (standard deviation of the low control group ÷ mean value of the low control group) × 100 Experimental window = High control group mean / Low control group mean Z score = 1 - [3 × (high control group standard deviation + low control group standard deviation) ÷ (high control group mean - low control group mean)] Inhibition rate (%) = (1 - (signal value of experimental well - mean value of low control group) / (mean value of high control group - mean value of low control group)) x 100 I C 50 Values ​​were calculated using the data analysis software Graph Pad Prism 7.0.

[0158] Analytical quality control criteria: experimental window >2, Z-score >0.5, consistency of reference / positive compound data.

[0159] Test Results: In this experiment, the effect of the compound of formula (I) on the expression of ERα protein in MCF-7, T-47D and CAMA-1 cells was evaluated, and the expression level of ERα protein was detected by ICW experiment after 4 hours of drug treatment. The results are shown below.

[0160] [Table 10]

[0161] Testing Conclusion: The compound represented by formula (I) has a significant degrading effect on ERα in various breast cancer cells.

[0162] (4) MCF-7, T-47D and CAMA-1 cell proliferation inhibitory effect experiment Test materials:

[0163] [Table 11]

[0164] Cell information and culture conditions:

[0165] [Table 12]

[0166] Starvation medium: 1. CAMA-1: EMEM (phenol red-free) containing 10% carbon-adsorbed serum, i.e., 500 mL of cell culture medium contains 440 mL of EMEM, 50 mL of carbon-adsorbed serum, 5 mL of NEAA, and 5 mL of 100 mM sodium pyruvate; 2. T-47D: RPMI-1640 (phenol red-free) containing 10% carbon-adsorbed serum, i.e., 450 mL of RPMI-1640 and 50 mL of carbon-adsorbed serum in 500 mL of cell culture medium.

[0167] Experimental design The inhibitory effect of the compound of formula (I) on the proliferation of MCF-7, CAMA-1, and T-47D cells was analyzed. MCF-7 cells were tested at 10 concentration gradients with three replicates, with the compound concentration ranging from 100 nM to 0.0003815 nM. CAMA-1 cells were tested at 9 concentration gradients with three replicates, with the compound concentration ranging from 100 nM to 0.001526 nM. T-47D cells were tested at 9 concentration gradients with three replicates, with the compound concentration ranging from 2 μM to 0.00512 nM. Two independent experiments were performed for each.

[0168] Experimental steps: MCF-7 cells: MCF-7 cells reaching 80% cell confluence were digested with trypsin, centrifuged, resuspended, and counted. Cell suspensions were then prepared using the corresponding culture medium. 45 μL of the cell suspension was added to each well of a 384-well plate and cultured at 37°C in a cell incubator containing 5% CO2. The test compound of Formula (I) was serially diluted 4x downward in DMSO solution to 10 concentrations, starting at a concentration of 20 μM. After overnight cell culture, the diluted compound was further diluted with the corresponding cell culture medium. The diluted mixture was transferred to the corresponding cell plate, and the final compound concentration was 100 nM starting at a concentration of 100 nM, followed by 4x downward dilution. Positive control wells contained 2 μM fulvestrant. The cells were mixed uniformly, centrifuged, and cultured at 37°C in a cell incubator containing 5% CO2 for the number of days indicated in the table.

[0169] T-47D cells: T-47D cells were starved for 3 days in starvation medium, digested with trypsin, centrifuged, resuspended, and counted. After preparing cell suspensions using the corresponding medium, the cells were transferred to a 384-well plate, and 40 μL of cell suspension was added to each well. The cells were then cultured at 37°C in a cell incubator with 5% CO2. The test compound (I) was diluted 5x downward using DMSO solution to nine concentrations, starting at 400 μM. After overnight cell culture, the diluted compound was further diluted with the corresponding cell culture medium. The diluted mixture was transferred to the corresponding cell plate, and the final compound concentration was 5x downward to nine concentrations, starting at 2 μM. Positive control wells contained 2 μM fulvestrant, and the entire plate was supplemented with 1 nM estradiol. The mixture was then mixed evenly, centrifuged, and cultured at 37°C in a cell incubator with 5% CO2 for the number of days indicated in the table above.

[0170] CAMA-1 cells: CAMA-1 cells were starved for 3 days in starvation medium, digested with trypsin, centrifuged, resuspended, and counted. After preparing cell suspensions using the corresponding medium, the cells were transferred to a 96-well plate, and 80 μL of cell suspension was added to each well. The cells were then cultured at 37°C in a cell incubator containing 5% CO2. The test compound of Formula (I) was diluted 4x downward using DMSO solution to nine concentrations, starting at a starting concentration of 20 μM. After overnight cell culture, the diluted compound was further diluted with the corresponding cell culture medium. The diluted mixture was transferred to the corresponding cell plate, and the final compound concentration was 4x downward to nine concentrations, starting at a starting concentration of 100 nM. Positive control wells contained 2 μM fulvestrant, and the entire plate was supplemented with 1 nM estradiol. The mixture was then mixed evenly, centrifuged, and cultured at 37°C in a cell incubator containing 5% CO2 for the number of days indicated in the table.

[0171] After the compound incubation time, the cell culture plate was removed, CTG reagent was added, the mixture was mixed evenly, centrifuged, and incubated at room temperature for 10 minutes. The data were read using an Envision multilabel analyzer.

[0172] Data Analysis: The data were used to calculate cell viability after compound treatment according to the following formula: % inhibition = 100 - (RFU 化合物 -RFU 陽性対照 ) / (RFU 陰性対照 -RFU 陽性対照 ) × 100%. Positive control: cells treated with 2 μM fulvestrant; negative control: cells treated with 0.5% DMSO, then plotted in Prism5 to determine the IC of the compound. 50 The IC value was calculated. 50 Calculation formula: log(inhibitor) vs. response - Variable slope.

[0173] Test Results: This experiment analyzed the inhibitory effect of the test compound of formula (I) on the proliferation of MCF-7, CAMA-1 and T-47D cells, and two independent experimental evaluations were performed with three replicates.

[0174] IC of compounds for inhibition of cell proliferation 50 is as shown in the table below.

[0175] [Table 13]

[0176] Experimental Conclusion Compounds of formula (I) exhibit significant in vitro antiproliferative activity in MCF-7, CAMA-1 and T-47D cells.

[0177] (5) Cell proliferation inhibition experiments on HCC1428 and MDA-MB-134VI cells Test materials:

[0178] [Table 14]

[0179] Experimental Design: The starting detection concentration of the compound represented by formula (I) was 200 nM, and the compound was diluted 4-fold into 10 concentration gradients.

[0180] Experimental steps MDA-MB-134VI cells: 1) MDA-MB-134VI cells were cultured in DMEM complete growth medium containing 20% ​​FBS and 1% PenStrep.

[0181] 2) When the cells reached 80-90% confluence in a T75 culture flask, trypsin was used to digest and collect the cells. The cell passages for the primary and secondary independent experiments were 8 and 6 generations, respectively.

[0182] 3) The cell densities of the first and second independent experiments were determined by centrifuging the cells at 1000 rpm, resuspending them in complete medium, and counting them to be 3.60 × 10 per ml, respectively. 6 1.93 x 10 cells per ml 6 The cells were single cells, and the survival rates exceeded 95% in all cases.

[0183] 4) Cell suspensions were prepared in complete medium. In two experiments, the density of the cell suspensions was 4.00 × 10 per ml. 4 The cells were seeded at 8000 cells / 195 μL / well in a 96-well round-bottom plate and cultured overnight in an incubator at 37°C and 5% CO 2 .

[0184] 5) On day 2, 5 μL of diluted compound was added to the wells, and the final DMSO concentration was adjusted to 0.1%. DMSO wells were used as high controls, and medium-only wells were used as low controls.

[0185] 6) MDA-MB-134VI cells were cultured in an incubator at 37°C with 5% CO2 for 10 days.

[0186] 7) Remove the cells from the incubator, allow them to equilibrate to room temperature, and then centrifuge them at 1000 rpm for 5 minutes. Discard 100 μL of the cell culture supernatant from each well, and add 100 μL of CellTiter Glo. Incubate the cells in the dark at room temperature for 30 minutes.

[0187] 8) Luminescence values ​​were read using BMG.

[0188] Two independent replicate experiments were performed according to the steps above.

[0189] HCC1428 cells: 1) HCC1428 cells were cultured in RPMI1640 complete growth medium containing 10% FBS and 1% PenStrep.

[0190] 2) When the cells reached 80-90% confluence in a T75 culture flask, they were digested and collected using trypsin. The cell passages for the first and second independent experiments were 7 and 13 generations, respectively.

[0191] 3) The cell densities of the first and second independent experiments were determined by centrifuging the cells at 1000 rpm, resuspending them in complete medium, and counting them to be 1.30 × 10 per ml, respectively. 6 9.50 x 10 cells per ml 5 The cells were single cells, and the survival rates exceeded 95% in all cases.

[0192] 4) Cell suspensions were prepared in complete medium. In two experiments, the density of the cell suspensions was 1.25 × 10 per ml. 4 The cells were seeded at 2500 cells / 195 μL / well in a 96-well round-bottom plate and cultured overnight in an incubator at 37°C and 5% CO 2 .

[0193] 5) 5 μL of diluted compound was added to make the final DMSO concentration 0.1%. DMSO wells were used as high controls, and medium-only wells were used as low controls.

[0194] 6) The cells were cultured in an incubator at 37°C with 5% CO2 for 7 days.

[0195] 7) Remove the cells from the incubator, allow them to equilibrate to room temperature, and then centrifuge them at 1000 rpm for 5 minutes. Discard 100 μL of the cell culture supernatant from each well, and add 100 μL of CellTiter Glo. Incubate the cells in the dark at room temperature for 30 minutes.

[0196] 8) Luminescence values ​​were read using BMG.

[0197] Two independent replicate experiments were performed according to the steps above.

[0198] Data analysis The reading value of the negative control was set as an inhibition rate of 0%, and the reading value of the positive control was set as an inhibition rate of 100%, and the inhibition rate of each test solution was calculated.

[0199]

number

[0200] IC of the compound using the nonlinear fitting equation below 50 (half inhibitory concentration) was obtained: Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)*HillSlope)) X: logarithmic value of compound concentration Y: Compound inhibition rate (%inh) Z' factor calculation formula: Z'=1-3*(SD_H+SD_L) / (Ave_H-Ave_L) SD is the standard error and AVE is the mean.

[0201] Experimental results This experiment analyzed the inhibitory effect of the compound of formula (I) on the proliferation of MDA-MB-134VI and HCC1428 cells, and determined the IC of the compound that inhibited cell proliferation. 50 is as shown in the table below.

[0202] [Table 15]

[0203] Testing Conclusion: The compound of formula (I) exhibits significant in vitro antiproliferative activity against MDA-MB-134VI and HCC1428 cells.

[0204] (6) Growth inhibitory effect of MCF-7 mutant cell lines MCF-7 ESR1-Y537S and MCF-7 ESR1-D538G Test materials:

[0205] [Table 16]

[0206] Cell information and culture conditions:

[0207] [Table 17]

[0208] Experimental methods and steps: 1. Cell Culture Tumor cells were cultured in an incubator at 37°C with 5% CO. They were passaged periodically, and cells in the logarithmic growth phase were used for plating.

[0209] 2. Cell Plating 1) Cells were stained with trypan blue and viable cells were counted.

[0210] 2) The cells were washed once with PBS and then resuspended in phenol red-free RPMI1640 + 10% FBS medium, and the concentration was adjusted to an appropriate concentration.

[0211] 3) 135 μL of cell suspension was added to each well of the culture plate, and medium without cells was added to blank control wells.

[0212] 4) The culture plate was incubated overnight in an incubator at 37°C, 5% CO2, and 100% relative humidity.

[0213] 3. Preparation of 10X Compound Working Solution and Compound Treatment of Cells 1) Preparation of 10X compound working solution: 199 μL of cell culture medium was added to a V-bottom 96-well plate, and 1 μL of compound was pipetted from a 2000X compound stock plate and added to the cell culture medium in the 96-well plate. 1 μL of DMSO was added to the solvent control and blank control. Compound or DMSO was added and mixed evenly with a pipette.

[0214] 2) Administration: 15 μL of 10X compound working solution was taken and added to the cell culture plate as shown in Table 1. 15 μL of DMSO-cell medium mixed solution was added to the solvent control and blank control. The final concentration of DMSO was 0.05%.

[0215] 3) The 96-well cell plate was returned to the incubator for culture, and the cells were detected after 7 days of culture.

[0216] 4. CellTiter-Glo Luminescent Cell Viability Detection The following steps were performed according to the instructions of the Promega CellTiter-Glo Luminescent Cell Viability Detection Kit (Promega-G7573).

[0217] 1) CellTiter-Glo buffer was thawed and left at room temperature.

[0218] 2) The CellTiter-Glo substrate was left at room temperature.

[0219] 3) CellTiter-Glo buffer was added to the bottle of CellTiter-Glo substrate to dissolve the substrate, producing a CellTiter-Glo working solution.

[0220] 4) Vortex gently to completely dissolve the substrate.

[0221] 5) The cell culture plate was removed and allowed to equilibrate to room temperature for 30 minutes.

[0222] 6) 75 μL of CellTiter-Glo working solution was added to each well (equivalent to half the volume of cell culture medium in each well). The cell plate was wrapped in aluminum foil to protect it from light.

[0223] 7) The culture plate was shaken on an orbital shaker for 2 minutes to induce cell lysis.

[0224] 8) The culture plate was left at room temperature for 10 minutes to allow the luminescence signal to stabilize.

[0225] 9) Luminescent signals were detected using a 2104 EnVision plate reader.

[0226] Data Analysis: The inhibition rate of the test compound was calculated using the following formula (Inhibition rate, IR): IR (%) = (1-(RLU) 化合物 -RLU ブランク対照 ) / (RLU 溶媒対照 -RLU ブランク対照 )) × 100%. The inhibition rates of different concentrations of the compounds were calculated using Excel, and then the inhibition curves were fitted using GraphPad Prism (6.02.328) software, log (inhibitor) vs. response—variable slope, to obtain the relevant parameters.

[0227] Relative IC 50 is the concentration required to reduce the concentration to the halfway point between the upper and lower plateaus of the curve, and is the relative IC 90 is the concentration required to reduce the concentration to the 90% point between the upper and lower plateaus of the curve.

[0228] Test Results: This experiment analyzed the inhibitory effect of the test compound represented by formula (I) on the proliferation of two mutant MCF-7 cells, and determined the IC value of the compound for inhibiting cell proliferation. 50 is as shown in the table below.

[0229] [Table 18]

[0230] Testing Conclusion: The compound of formula (I) exhibits significant in vitro antiproliferative activity against ESR1-Y537S and ESR1-D538G mutant MCF-7 cells.

[0231] Experimental Example 2: Evaluation of DMPK properties (1) Studies on the inhibition of cytochrome P450 isozymes Test materials:

[0232] [Table 19]

[0233] Experimental methods and steps: The probe substrate, test substance, human liver microsomes, and cofactors were incubated together, and the concentrations of metabolites produced by the probe substrate were detected by LC-MS / MS and analyzed by IC 50 The values ​​were calculated. The solvent control sample was added with the same volume of solvent instead of the test sample, and the enzyme activity of the solvent control sample was set to 100%. The detailed information used in the experiment, such as the concentration of human liver microsomes, the probe substrate concentration corresponding to each CYP, the incubation time, the positive control, and the metabolites, is shown in the table below.

[0234] [Table 20]

[0235] Incubation of test substances and positive control inhibitors: All samples were incubated in a water bath at 37°C. Each concentration of the test substance was replicated in triplicate, and each concentration of the positive control inhibitor was replicated in duplicate. The incubation reaction system included a mixed working solution of the corresponding isozyme probe substrate (at the designated concentration) and microsomes (0.100 mg protein / mL), as well as a working solution of the test substance or positive control inhibitor (at a range of final concentrations). All samples were preheated at 37°C for 10 minutes, after which the cofactors were added to initiate the reaction. For the CYP3A metabolic reaction using midazolam as the probe substrate, the reaction time was 3 minutes; all other reactions were 10 minutes.

[0236] Vehicle control incubations: The solvent control data served as a reference value for calculating the activity of each isozyme in the incubation reaction system. The solvent controls were divided into two types: (1) a solvent control for the test substance, with three parallel incubation reactions containing a working mixture of the corresponding isozyme probe substrate (at the designated concentration) and microsomes (0.100 mg / mL) in DMSO:methanol (v:v, 1:9); and (2) a solvent control for the positive control inhibitor, with two parallel incubation reactions containing a working mixture of the corresponding isozyme probe substrate (at the designated concentration) and microsomes (0.100 mg protein / mL) in DMSO:methanol (v:v, 1:9). All samples were preheated at 37°C for 10 min, and then the reaction was initiated by adding cofactors. The reaction time for the CYP3A metabolic reaction using midazolam as the probe substrate was 3 min, while all other reactions were 10 min.

[0237] Sample processing: After the incubation reaction for each isozyme reached the corresponding reaction time, 200 μL of stop solution containing the corresponding internal standard was added to the reaction plate to terminate the reaction. After thorough shaking, the plate was placed in a centrifuge and centrifuged at 3220 × g for 20 minutes. The supernatant was then removed and diluted with the appropriate diluent at the specified ratio. The reaction plate was placed on a shaker and shaken to ensure uniform mixing. Metabolites of the probe substrate were then analyzed using LC-MS / MS. If samples could not be analyzed immediately, they were stored at 2–8°C until use.

[0238] After protein precipitation of the incubated samples, the metabolites of the probe substrate were quantitatively analyzed using LC-MS / MS.

[0239] Data Analysis: The activity of each CYP isoenzyme was reflected by the production rate of each probe substrate metabolite. The activity of each isoenzyme in the solvent control culture reaction system without the addition of the test substance or positive inhibitor was set as 100%, and the production rate of the probe substrate metabolite in the presence of different concentrations of the test substance or positive inhibitor was compared with the production rate of the solvent control metabolite. The value obtained by multiplying by 100% was used as the percentage of residual activity of each isoenzyme. Nonlinear regression analysis was performed using 3 or 4 parameters with SigmaPlot (V.14) software, with the percentage of residual activity as the vertical axis and the inhibitor concentration as the horizontal axis, to determine the IC40 of the test product and each positive inhibitor. 50 The IC values ​​were calculated by fitting using SigmaPlot software. 50 When the IC exceeds the maximum dose (100 μM) or when fitting reveals that the IC 50 If IC 50 Values ​​were labeled as ">100 μM."

[0240]

number

[0241] A four-parameter equation was used when the minimum enzyme activity was within ±10%, otherwise a three-parameter equation was used.

[0242] Test Results: IC of the compound of formula (I) for CYP1A2 50 The IC value was 97.2 μM, which is the IC value for CYP3A (using midazolam as a substrate). 50 The IC value was 8.38 μM, indicating that it inhibits CYP3A (using testosterone as a substrate). 50 The value was 10.9 μM. The compounds of the present invention have weak inhibition of CYP enzymes, indicating a low risk of drug interactions.

[0243] (2) Pharmacokinetics in mice Experimental Objective: Female Balb / c mice were used as test animals. After intravenous and intragastric administration of the test compound, plasma drug concentrations were measured at different time points using LC / MS / MS. The pharmacokinetic behavior of the C-type crystals of compound of formula (I) in mice was studied, and its pharmacokinetic properties were evaluated.

[0244] Experimental scheme: Test animals: Animals were purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0245] Pharmaceutical manufacturing: An appropriate amount of sample was weighed and made into a 1 mg / mL solution, stirred until clear, and sonicated; the solvent was 15% HP-β-CD.

[0246] Administration: After overnight fasting, the IV group was intravenously administered with a dose of 5 mg / kg, and the PO group was intragastrically administered with a dose of 10 mg / kg.

[0247] Experimental Procedure: After intravenous administration of the test compound to female Balb / c mice, 30 μL of blood was collected from the saphenous vein at 0.0833, 0.25, 0.5, 1, 2, 4, 8, and 24 hours and placed in commercially available anticoagulant tubes pre-loaded with EDTA-K2. After intragastric administration, 30 μL of blood was collected from the jugular vein at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours and placed in commercially available anticoagulant tubes pre-loaded with EDTA-K2. Plasma was then removed by centrifugation (3,200 g, 4°C, 10 minutes). The plasma was transferred to pre-cooled centrifuge tubes, flash-frozen on dry ice, and stored in a -60°C or colder ultra-low temperature refrigerator until LC-MS / MS analysis. Animals were fed 4 hours after administration. The content of the test compound in the plasma of mice after intravenous and intragastric administration was measured using an LC / MS / MS method. The linear range of the method was 2.00-2000 nM.

[0248] Test Results:

[0249] [Table 21]

[0250] The C-type crystals of the compound represented by formula (I) are metabolically stable in mice, have a large tissue distribution, and exhibit high oral absorption and availability, and have good pharmacokinetic properties in vivo.

[0251] (3) Pharmacokinetics in rats Experimental Objective: Female Sprague-Dawley rats were used as test animals. After intravenous and intragastric administration of the test compound, plasma drug concentrations were measured at different time points using LC / MS / MS. The pharmacokinetic behavior of the B-type crystals of compound of formula (II) in rats was studied, and its pharmacokinetic properties were evaluated.

[0252] Experimental scheme: Test animals: Animals were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0253] Pharmaceutical manufacturing: An appropriate amount of sample was weighed and made into a 0.2 mg / mL solution, stirred until clear, and the solvent was 20% SBE-β-CD.

[0254] Administration: After overnight fasting, the IV group was intravenously administered with a dose of 1 mg / kg, and the PO group was intragastrically administered with a dose of 2 mg / kg.

[0255] Experimental Procedure: After intravenous administration of the test compound to each of the female Sprague-Dawley rats, 200 μL of blood was collected from the jugular vein at 0.0830, 0.250, 0.500, 1.00, 2.00, 4.00, 6.00, 8.00, 24.0, 32.0, and 48.0 hours, and placed in a commercially available anticoagulant tube containing EDTA-K2. After intragastric administration of each test compound, 200 μL of blood was collected from the jugular vein at 0.250, 0.500, 1.00, 2.00, 4.00, 6.00, 8.00, 24.0, 32.0, and 48.0 h, respectively. The blood was placed in a commercially available anticoagulant tube containing pre-prepared EDTA-K2. Plasma was then extracted by centrifugation (3,200 g, 4°C, 10 min). The plasma was transferred to a pre-cooled centrifuge tube, flash-frozen on dry ice, and stored in a -60°C or colder ultra-low temperature refrigerator until LC-MS / MS analysis. Animals were fed 4 h after administration. The content of the test compound in rat plasma after intravenous and intragastric administration was measured using an LC / MS / MS method. The linear range of the method was 2.00–2000 nM.

[0256] Test Results:

[0257] [Table 22]

[0258] The B-type crystals of the compound represented by formula (II) are metabolically stable in rats, have a large tissue distribution, and exhibit high oral absorption and availability, and have good pharmacokinetic properties in vivo.

[0259] (4) Pharmacokinetics in dogs Experimental Objective: Female and male beagles were used as test animals. After intravenous and intragastric administration of the test compound, plasma drug concentrations were measured at different time points using LC / MS / MS. The pharmacokinetic behavior of the B-type crystals of compound of formula (II) in beagles was studied, and its pharmacokinetic properties were evaluated.

[0260] Experimental scheme: Test animals: Animals were purchased from Beijing Marshall Biotechnology Co., Ltd.

[0261] Pharmaceutical manufacturing: Appropriate amounts of sample were weighed and made into 0.2 and 0.3 mg / mL solutions, which were stirred until clear, in a solvent of 20% SBE-β-CD.

[0262] Administration: After overnight fasting, the IV group was intravenously administered with a dose of 0.3 mg / kg, and the PO group was intragastrically administered with a dose of 1 mg / kg.

[0263] Experimental Procedure: After intravenous administration of the test compound to beagle mice, 800 μL of blood was collected from a peripheral vein at 0.25, 0.5, 1, 2, 4, 6, 8, 24, 32, 48, 72, and 96 hours, and placed in commercially available anticoagulant tubes pre-loaded with EDTA-K2. After intragastric administration of the test compound, 800 μL of blood was collected from a peripheral vein at 0.25, 0.5, 1, 2, 4, 6, 8, 24, 32, 48, 72, and 96 hours, and placed in commercially available anticoagulant tubes pre-loaded with EDTA-K2. Plasma was then removed by centrifugation (3,200 g, 2-8°C, 10 minutes). The plasma was transferred to pre-cooled centrifuge tubes, flash-frozen on dry ice, and stored in a -60°C or colder ultra-low temperature refrigerator until LC-MS / MS analysis. Animals were fed 4 hours after administration. The LC / MS / MS method was used to measure the content of the test compound in beagle plasma after intravenous and intragastric administration. The linear range of the method was 2.00-2000 nM.

[0264] Test Results:

[0265] [Table 23]

[0266] The B-type crystals of the compound represented by formula (II) of the present invention are metabolically stable in the body of a beagle, have a large tissue distribution, high oral absorption and availability, and have good pharmacokinetic properties in vivo.

[0267] Experimental Example 3: Evaluation of drug efficacy in the body (1) In vivo drug efficacy study in BALB / c nude mice with subcutaneous xenografted breast cancer MCF-7 cells Experimental Objective: In this experiment, the in vivo efficacy of the C-type crystals of the compound of formula (I) was evaluated using a nude mouse model with subcutaneous xenograft tumors of human breast cancer MCF-7 cells.

[0268] Test materials:

[0269] [Table 24]

[0270] Experimental methods and steps: Cell culture preparation: Human breast cancer MCF-7 cells were routinely cultured in vitro in monolayers in EMEM (EBSS; ATCC) medium supplemented with 10% fetal bovine serum (Cellmax), 2 mM glutamine (Gibco), and 1% non-essential amino acids (NEAA; Gibco) in a 37°C, 5% CO2 incubator. Cells were subcultured twice weekly using trypsin-EDTA (Gibco) for routine digestion. When cell saturation reached 80%-90% and the required cell number was reached, cells were harvested, counted, and inoculated.

[0271] Experimental animals were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Zhejiang branch).

[0272] Tumor cell inoculation and grouping Cell inoculation: Each mouse was subcutaneously inoculated with an estrogen tablet (0.36 mg / tablet; IRA) in the left dorsal region. Three days later, 0.2 mL (1 × 10 7MCF-7 cells (containing Matrigel; CORNING, volume ratio 1:1) were subcutaneously inoculated into the right dorsal region of each mouse, resulting in a mean tumor volume of 188 mm 3 When the dose reached 100 mg / kg, the groups were divided and administered the drug.

[0273] [Table 25]

[0274] Pharmaceutical manufacturing: Appropriate amounts of compound of formula (I) samples were weighed and prepared into 0.1, 0.3, 1, and 3 mg / mL solutions, respectively. The solutions were vortexed briefly and sonicated to form clear solutions in 10% HP-β-CD. An appropriate amount of palbociclib was weighed and prepared into 2.5 mg / mL solutions, the pH adjusted to 4.0, vortexed briefly, and sonicated to form clear solutions in 50 mM sodium lactate buffer. Fulvestrant was used in the form of a commercially available injection, Faslodex.

[0275] Tumor Measurements and Laboratory Indicators: Tumor diameters were measured twice a week with a vernier caliper. Tumor volume was calculated using the formula: V = 0.5a × b 2 where a and b represent the long and short diameters of the tumor, respectively.

[0276] The antitumor effect of the compound was evaluated by TGI (%) or relative tumor growth rate T / C (%). Relative tumor growth rate T / C (%) = T RTV / C RTV ×100%(T RTV : Mean RTV of treatment group; C RTV (The mean RTV value of the control group.) The relative tumor volume (RTV) was calculated according to the tumor measurement results, using the formula RTV = V t / V0, where V0 is the tumor volume measured at the time of group dosing (i.e., D0), and V t is the tumor volume at a particular measurement, and T RTV and C RTV data were taken on the same day.

[0277] TGI (%) reflects the tumor growth inhibition rate: TGI (%) = [1 - (mean tumor volume at the end of treatment for a particular treatment group - mean tumor volume at the start of treatment for that treatment group) / (mean tumor volume at the end of treatment for the control group - mean tumor volume at the start of treatment for the control group)) × 100%.

[0278] The Q value of the combination index was calculated according to King's formula. The criteria were: a Q value between 0.85 and 1.15 indicated synergy (+), >1.15 to 2.0 indicated potentiation (++), <0.85 to 0.55 indicated antagonism (-), and <0.55 indicated significant antagonism (--); Q = Ea + b / (Ea + Eb - Ea × Eb), where Ea + b is the tumor inhibition rate in the combination group, and Ea and Eb are the tumor inhibition rates in the single-administration groups, respectively.

[0279] After the experiment, tumor weight was measured and T / C weight Calculate the percentage and T weight and C weight represents the tumor weight of the treatment group and the vehicle control group, respectively.

[0280] Statistical analysis was performed using SPSS software based on the RTV data at the end of the study. Comparisons between groups were analyzed using one-way ANOVA, and when variances were not homogeneous (significant differences in F values), the Games-Howell test was used. A p<0.05 was considered significant.

[0281] Test Results: In this experiment, the efficacy of the C-type crystals of compound of formula (I) was evaluated in a human breast cancer xenograft tumor model, with the solvent control group as the reference. The tumor volumes of each group at different time points and the corresponding statistical analysis results are shown in Table 14 and Figure 10, and the changes in mouse body weight during the administration period are shown in Figure 11.

[0282] On the 21st day after administration, the average tumor volume in the blank control group was 1225 mm 3The compound represented by formula (I) was administered at four doses of 1 mg / kg, 3 mg / kg, 10 mg / kg, and 30 mg / kg, and the average tumor volume was 340 mm 3 , 179mm 3 , 140mm 3 , and 121mm 3 The T / C values ​​were 27.2%, 14.8%, 11.3%, and 9.7%, respectively, and the TGI were 85.3%, 100.9%, 104.6%, and 106.5%, respectively. The P values ​​compared with the control group were 0.003, 0.001, 0.001, and 0.001, respectively. The objective response rate (ORR) was 25% at doses of 10 mg / kg and 30 mg / kg. Therefore, the C-type crystal of the compound represented by formula (I) had a significant tumor inhibitory effect at all of the above doses, and showed a predetermined dose-effect relationship as the dose increased.

[0283] [Table 26]

[0284] On day 21, the mean tumor volume in the fulvestrant (5 mg / mouse) and palbociclib (25 mg / kg) monotherapy groups was 593 mm 3 and 781 mm 3 The T / C values ​​were 46.9% and 64.0%, respectively, and the TGI values ​​were 60.9% and 42.8%, respectively. Compared with the control group, the P values ​​were 0.019 and 0.219, respectively. Therefore, administration of fulvestrant alone had an antitumor effect, but administration of palbociclib alone did not. Compared with the above two reference compounds, the C-type crystal of the compound represented by formula (I) showed a stronger antitumor effect at 3 mg / kg, with a disease control rate (DCR) of 100%.

[0285] Administration of palbociclib alone did not have an antitumor effect, but when the compound represented by formula (I) (3 mg / kg) was administered in combination with palbociclib, the mean tumor volume was 79 mm 3The T / C value was 6.1%, the TGI was 110.5%, and the P values ​​compared with the control group, palbociclib, and the C-type crystals of the compound represented by formula (I) (3 mg / kg) group were 0.001, 0.002, and 0.012, respectively. The objective response rate (ORR) reached 87.5%, and the Q value calculated by Jin's formula was 1.10(+). Therefore, the combined administration showed a stronger antitumor effect than the single administration of C-type crystals of the compound represented by formula (I), and exhibited a synergistic additive effect.

[0286] Testing Conclusion: Compared with the blank control group, the C-type crystals of the compound of formula (I) exhibited significant tumor-inhibiting effects at all four doses: 1 mg / kg, 3 mg / kg, 10 mg / kg, and 30 mg / kg, with an effective dose of 1 mg / kg (TGI 85.3%). When the C-type crystals of the compound of formula (I) (3 mg / kg) were used in combination with palbociclib, they exhibited a more potent antitumor effect than when administered alone. Tumor-bearing mice showed good tolerance to the C-type crystals of the compound of formula (I). The compounds of the present invention exhibited excellent tumor-inhibiting effects in a mouse pharmacodynamic model, demonstrating their potential for clinical treatment.

[0287] (2) In vivo drug efficacy study in a mouse model of subcutaneous xenograft tumors of human breast cancer MCF-7 cells harboring the ESR1 D538G mutation Experimental Objective: A subcutaneous xenograft tumor model was established in BALB / c nude mice using human breast cancer MCF7 cells harboring the ESR1 D538G mutation to evaluate the in vivo antitumor effect of the B-type crystals of compound (II).

[0288] Test materials:

[0289] [Table 27]

[0290] Experimental methods and steps: Cell culture preparation: Human breast cancer MCF7 cells harboring the ESR1 D538G mutation were cultured in vitro in monolayers in EMEM medium supplemented with 10% fetal bovine serum, 1% antibiotic-antimycotic, 2 mM L-glutamine, and 1% NEAA at 37°C in a 5% CO2 incubator. Cells were subcultured twice weekly after routine digestion using trypsin-EDTA. When cell saturation reached 80%-90% and the required cell number was reached, the cells were harvested, counted, and inoculated.

[0291] Experimental animals: Animals were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0292] Tumor cell inoculation and grouping Cell inoculation: Inject 0.2 mL (10 × 10) of PBS into the right dorsal region of each mouse. 6 MCF7 ESR1 D538G cells (PBS + Matrigel, volume ratio 1:1) were subcutaneously inoculated, and the average tumor volume was 147 mm 3 When the rats reached the age of 18, they were divided into groups and administered. The experimental grouping and administration method are shown in the table below. The first day of administration was designated as day 0.

[0293] [Table 28]

[0294] Pharmaceutical manufacturing: Appropriate amounts of the Type B crystal samples of the compound of Formula (II) were weighed and prepared into 0.5, 1.5, and 5.0 mg / mL solutions, respectively, which were vortexed briefly and sonicated to form clear solutions in 10% HP-β-CD.Appropriate amounts of palbociclib were weighed and prepared into 5 mg / mL solutions, the pH of which was adjusted to 4.0, vortexed briefly, and sonicated to form clear solutions in 50 mM sodium lactate buffer.

[0295] Tumor Measurements and Laboratory Indicators: Tumor diameters were measured twice a week with a vernier caliper. Tumor volume was calculated using the formula: V = 0.5a × b 2 where a and b represent the long and short diameters of the tumor, respectively.

[0296] The antitumor effect of a compound is evaluated by TGI (%) or relative tumor growth rate T / C (%). Relative tumor growth rate T / C (%) = T RTV / C RTV ×100%(T RTV : Mean RTV of treatment group; C RTV (The mean RTV value of the negative control group is used.) The relative tumor volume (RTV) was calculated based on the tumor measurement results using the formula RTV = V t / V0, where V0 is the tumor volume measured at the time of group dosing (i.e., D0), and V t is the tumor volume at a particular measurement, and T RTV and C RTV data were taken on the same day.

[0297] TGI (%) reflects the tumor growth inhibition rate: TGI (%) = [1 - (mean tumor volume at the end of treatment for a particular treatment group - mean tumor volume at the start of treatment for that treatment group) / (mean tumor volume at the end of treatment for the vehicle control group - mean tumor volume at the start of treatment for the vehicle control group)] x 100%.

[0298] After the experiment, tumor weight was measured and T / C weight Calculate the percentage and T weight and C weight represents the tumor weight of the treatment group and the vehicle control group, respectively.

[0299] Statistical analysis was performed using GraphPad Prism software based on the end-of-study RTV data. Comparisons between multiple groups were analyzed using one-way ANOVA, with p<0.05 considered significant and p>0.05 considered non-significant.

[0300] Test Results: In this experiment, the efficacy of the B-type crystals of compound of formula (II) was evaluated in a human breast cancer MCF7 cell xenograft tumor model carrying the ESR1 D538G mutation, with a blank control group as the reference. The tumor volumes and statistical analysis of each group at different time points are shown in Table 12 and Figure 29, and the changes in mouse body weight during the administration period are shown in Figure 13.

[0301] [Table 29]

[0302] On the 31st day after group allocation, the mean tumor volume in the blank control group was 654 mm 3 The mean tumor volume in the palbociclib (50 mg / kg) monotherapy group was 295 mm 3 The T / C value was 45.47%, the TGI was 70.91%, and the p-value was <0.0001 compared to the blank control group, confirming that administration of palbociclib alone had a significant antitumor effect. The B-type crystals of the compound represented by formula (II) showed an average tumor volume of 153 mm at doses of 5 mg / kg, 15 mg / kg, and 50 mg / kg, respectively. 3 , 124mm 3 and 43mm 3 The T / C values ​​were 24.15%, 18.93%, and 6.37%, respectively, and the TGI values ​​were 98.86%, 104.52%, and 120.51%, respectively, with p values ​​of <0.0001 compared to the blank control group. The B-type crystals of the compound represented by formula (II) had significant antitumor effects at all of the above doses, and the objective response rate showed good dose-dependence. The mean tumor volume in the group administered with the combined B-type crystals of the compound represented by formula (II) (5 mg / kg) and palbociclib (50 mg / kg) was 31 mm 3The T / C was 4.67% and the TGI was 122.83%, with a p value of <0.0001 for the blank control group, which was statistically significant compared to the group administered with either type B crystals of the compound of formula (II) or palbociclib alone (p value <0.0001, synergy coefficient 2.21). The objective response rate of the combined administration group reached 100%. Therefore, the combined administration had a more significant antitumor effect than the single administration.

[0303] The above results suggest that in a BALB / c nude mouse model with subcutaneous xenograft tumors of human breast cancer MCF7 cells harboring the ESR1 D538G mutation, the B-type crystals of the compound represented by formula (II) exhibited significant antitumor effects at doses of 5 mg / kg, 15 mg / kg, and 50 mg / kg. The B-type crystals of the compound represented by formula (II) exhibited a synergistic antitumor effect when used in combination with palbociclib (50 mg / kg) at a dose of 5 mg / kg, which was equivalent to the antitumor effect of the B-type crystals of the compound represented by formula (II) at 50 mg / kg.

[0304] Testing Conclusion: The B-type crystals of the compound of formula (II) showed antitumor activity against the growth of subcutaneously transplanted tumors in MCF7 mice harboring the ESR1 D538G mutation, demonstrating a dose-dependent effect. When combined with palbociclib, the B-type crystals demonstrated superior antitumor activity compared to monotherapy. During the experiment, mice in each test group showed good tolerance to the drug. [Brief explanation of the drawings]

[0305] [Figure 1] 1 is an XRPD pattern of type A crystal of the compound represented by formula (III). [Figure 2] 1 is a TGA pattern of type A crystals of the compound represented by formula (III). [Figure 3] 1 is a DSC pattern of type A crystals of the compound represented by formula (III). [Figure 4] 1 is an XRPD pattern of type B crystals of the compound represented by formula (II). [Figure 5] 1 is a TGA pattern of type B crystals of the compound represented by formula (II). [Figure 6] 1 is a DSC pattern of type B crystals of the compound represented by formula (II). [Figure 7] 1 is an XRPD pattern of type C crystals of the compound represented by formula (I). [Figure 8] 1 is a DSC pattern of type C crystals of the compound represented by formula (I). [Figure 9] FIG. 1 is a schematic diagram of the single crystal structure of the compound represented by formula (II). [Figure 10] 1 shows a tumor growth curve of the C-type crystal of the compound of formula (I) in a human breast cancer MCF7 cell subcutaneous xenograft tumor-bearing mouse model after administration of the test compound. [Figure 11] 1 shows tumor growth curves of crystalline form C of compound of formula (I) compared to fulvestrant and palbociclib in mice bearing a human breast cancer MCF7 cell subcutaneous xenograft tumor model after administration of the test compound. [Figure 12] 1 is a tumor growth curve of Form C crystals of compound of Formula (I) in combination with palbociclib in a human breast cancer MCF7 cell subcutaneous xenograft tumor model tumor-bearing mice after administration of the test compound. [Figure 13] 1 shows the percentage (%) of change in body weight of a tumor-bearing mouse model of human breast cancer MCF7 cells subcutaneous xenograft tumor during the administration of type C crystals of the compound represented by formula (I). [Figure 14] 1 shows the percentage change (%) in body weight of mice bearing a human breast cancer MCF7 cell subcutaneous xenograft tumor model during the administration of type C crystals of the compound of formula (I) compared with fulvestrant and palbociclib. [Figure 15] 1 shows the percentage change (%) in body weight of mice bearing a human breast cancer MCF7 cell subcutaneous xenograft tumor model during the administration of Type C crystals of the compound of Formula (I) in combination with palbociclib. [Figure 16] 1 shows a tumor growth curve of the B-type crystal of the compound of formula (II) in a subcutaneous xenograft tumor model of MCF7 cells bearing an ESR1 D538G mutation. [Figure 17]1 shows the tumor growth curve of the B-type crystals of the compound of formula (II) in combination with palbociclib in a mouse model of subcutaneous xenograft tumors of MCF7 cells harboring the ESR1 D538G mutation. [Figure 18] 1 shows the percentage (%) of weight change in mice bearing a subcutaneous xenograft tumor model of MCF7 cells harboring the ESR1 D538G mutation during the administration of type B crystals of the compound of formula (II). [Figure 19] 1 shows the percentage change in body weight (%) in mice bearing a subcutaneous xenograft tumor model of MCF7 cells harboring the ESR1 D538G mutation during the administration of type B crystals of the compound of Formula (II) in combination with palbociclib.

Claims

1. A type A crystal of the fumarate salt represented by formula (III), characterized in that the powder X-ray diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 20.92±0.20°, 22.08±0.20°, and 24.70±0.20°. 【Chemistry 1】

2. 2. The A-type crystal of the fumarate salt represented by formula (III) according to claim 1, characterized in that the powder X-ray diffraction spectrum has diffraction peaks at the following 2θ angles: 12.30±0.20°, 15.64±0.20°, 17.00±0.20°, 18.26±0.20°, 20.92±0.20°, 22.08±0.20°, 22.62±0.20°, and 24.70±0.20°.

3. 2. The type A crystal of the fumarate salt represented by formula (III) according to claim 1, characterized in that the powder X-ray diffraction spectrum has diffraction peaks at the following 2θ angles: 5.18±0.20°, 5.92±0.20°, 8.12±0.20°, 12.30±0.20°, 15.64±0.20°, 17.00±0.20°, 18.26±0.20°, 20.92±0.20°, 22.08±0.20°, and 24.70±0.20°.

4. 4. The type A crystal of the fumarate salt represented by formula (III) according to claim 3, characterized in that the powder X-ray diffraction spectrum has diffraction peaks at the following 2θ angles: 5.18±0.20°, 5.92±0.20°, 8.12±0.20°, 12.30±0.20°, 15.64±0.20°, 17.00±0.20°, 18.26±0.20°, 19.54±0.20°, 20.92±0.20°, 22.08±0.20°, 22.62±0.20°, and 24.70±0.20°.

5. 4. The type A crystal of fumarate salt represented by formula (III) according to claim 3, characterized in that the powder X-ray diffraction spectrum has diffraction peaks at the following 2θ angles: 5.18±0.20°, 5.92±0.20°, 8.12±0.20°, 12.30±0.20°, 15.64±0.20°, 17.00±0.20°, 18.26±0.20°, 19.54±0.20°, 20.92±0.20°, 22.08±0.20°, 24.70±0.20°, and 25.48±0.20°.

6. 4. The type A crystal of fumarate salt represented by formula (III) according to claim 3, characterized in that the powder X-ray diffraction spectrum has diffraction peaks at the following 2θ angles: 5.18±0.20°, 5.92±0.20°, 8.12±0.20°, 12.30±0.20°, 15.64±0.20°, 17.00±0.20°, 18.26±0.20°, 19.54±0.20°, 20.92±0.20°, 22.08±0.20°, 22.62±0.20°, 24.70±0.20°, and 25.48±0.20°.

7. Powder X-ray diffraction spectrum shows the following 2θ angles: 5.18±0.20°, 5.92±0.20°, 8.12±0.20°, 12.30±0.20°, 15.64±0.20°, 16.42±0.20°, 17.00±0.20°, 17.66±0.20°, 18.26±0.20°, 19.54±0.20°, 20.06±0.20°, 20.92±0.20°, 22.08±0.20°, 22.6 7. The type A crystal of fumarate represented by formula (III) according to any one of claims 1 to 6, characterized by having diffraction peaks at the following angles: 23.2±0.20°, 23.32±0.20°, 23.86±0.20°, 24.70±0.20°, 25.48±0.20°, 26.68±0.20°, 28.50±0.20°, 29.54±0.20°, 31.58±0.20°, and 33.20±0.20°.

8. 7. The type A crystal of the fumarate salt represented by formula (III) according to any one of claims 1 to 6, characterized in that the powder X-ray diffraction spectrum has diffraction peaks at the following 2θ angles: 5.181°, 5.920°, 8.119°, 12.299°, 15.638°, 16.418°, 17.002°, 17.660°, 18.261°, 19.539°, 20.061°, 20.919°, 22.080°, 22.621°, 23.320°, 23.861°, 24.700°, 25.483°, 26.681°, 28.498°, 29.542°, 31.578°, and 33.198°.

9. The A-type crystal of the fumarate salt represented by formula (III) according to any one of claims 1 to 6, characterized in that the thermogravimetric analysis curve shows a weight loss of 0.198% at 150.000±3°C and a weight loss of 10.880% at 240.000±3°C.

10. The A-type crystals of the fumarate salt represented by formula (III) according to any one of claims 1 to 6, characterized in that the differential scanning calorimetry curve has endothermic peak onsets at 175.87±5°C, 214.68±5°C, and 292.11±5°C.

11. A B-type crystal of the methanesulfonate salt represented by formula (II), characterized in that the powder X-ray diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 14.24±0.20°, 22.18±0.20°, and 23.78±0.20°. 【Chemistry 2】

12. 12. The B-type crystal of methanesulfonate salt represented by formula (II) according to claim 11, characterized in that the powder X-ray diffraction spectrum has diffraction peaks at the following 2θ angles: 6.50±0.20°, 14.24±0.20°, 15.80±0.20°, 18.18±0.20°, 22.18±0.20°, 23.78±0.20°, and 25.30±0.20°.

13. 13. The type B crystal of methanesulfonate salt represented by formula (II) according to claim 12, characterized in that the powder X-ray diffraction spectrum has diffraction peaks at the following 2θ angles: 6.50±0.20°, 7.86±0.20°, 14.24±0.20°, 15.80±0.20°, 16.92±0.20°, 18.18±0.20°, 19.66±0.20°, 20.76±0.20°, 22.18±0.20°, 23.78±0.20°, 25.30±0.20°, and 26.12±0.20°.

14. The powder X-ray diffraction spectrum shows the following 2θ angles: 6.499°, 7.860°, 9.739°, 10.257°, ​​11.862°, 12.255°, 13.021°, 14.240°, 15.417°, 15.797°, 16.541°, 16.920°, 17.558°, 18.182°, 18.439°, 18.702°, 19.660°, 20.381°, 20.761°, 21.494°, 21.647°, 22.180°, 23.781°, 24.099°, 24.498°, 25.

14. The B-type crystal of methanesulfonate salt represented by formula (II) according to claim 13, characterized by having diffraction peaks at 304°, 26.118°, 26.821°, 27.239°, 28.579°, 28.924°, 29.302°, 29.881°, 30.278°, 30.681°, 30.938°, 31.764°, 32.978°, 34.260°, 35.101°, 35.419°, 35.761°, 36.597°, 37.083°, 37.540°, and 38.423°.

15. The B-type crystals of methanesulfonate salt represented by formula (II) according to claim 14, characterized in that the thermogravimetric analysis curve spectrum shows a weight loss of 0.085% at 200.000±3°C.

16. A C-type crystal of the compound represented by formula (I), characterized in that the powder X-ray diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 4.90±0.20°, 15.82±0.20°, and 22.26±0.20°. 【Transformation 3】

17. 17. A C-type crystal of the compound of formula (I) according to claim 16, characterized in that the powder X-ray diffraction spectrum has diffraction peaks at the following 2θ angles: 4.90±0.20°, 9.82±0.20°, 15.82±0.20°, 17.48±0.20°, 18.64±0.20°, 22.26±0.20°, 23.74±0.20°, and 29.36±0.20°.

18. 18. A C-type crystal of the compound of formula (I) according to claim 17, characterized in that the powder X-ray diffraction spectrum has diffraction peaks at the following 2θ angles: 30±0.20°, 4.90±0.20°, 9.82±0.20°, 11.06±0.20°, 14.20±0.20°, 15.82±0.20°, 17.48±0.20°, 18.64±0.20°, 22.26±0.20°, 23.74±0.20°, 24.46±0.20°, and 29.36±0.20°.

19. The powder X-ray diffraction spectrum shows the following 2θ angles: 3.301°, 4.901°, 7.898°, 9.319°, 9.819°, 11.061°, 14.200°, 14.721°, 15.197°, 15.821°, 16.457°, 17.481°, 18.101°, 18.642°, 19.762°, 20.961°, 21.382°, 22.

19. A C-type crystal of the compound of formula (I) according to claim 18, characterized in that it has diffraction peaks at 2.259°, 23.740°, 24.461°, 25.761°, 26.295°, 26.943°, 27.518°, 29.358°, 30.075°, 31.317°, 31.916°, 34.598°, and 37.661°.

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