Tetrahydrocycloheptaindazole compound salt form, crystal

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 is December 28, 2021.

[0002] [Technical field] The present invention relates to salt forms and crystals of tetrahydrocycloheptaindazole compounds and methods for their preparation, in particular to the use of the 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 has become the second most common cancer in the world and the most prevalent 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 developed into the most important biomarker for breast cancer. Using estrogen receptor expression as a discriminant indicator, breast cancer can be classified into estrogen receptor-positive breast cancer and estrogen receptor-negative breast cancer, among which estrogen receptor-positive breast cancer accounts 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 because of its minimal adverse effects and high efficacy. The first-line endocrine therapy is mainly aromatase inhibitors (AIs). Although the aromatase inhibitor letrozole has shown good efficacy in treating estrogen receptor-positive breast cancer, the problem of resistance of estrogen receptor-positive breast cancer to aromatase inhibitors has become increasingly significant with the use of the two drugs. Numerous studies have shown that estrogen receptors mutate in response to aromatase inhibitors, mainly the Y537X mutation, which allows the mutated estrogen receptor to maintain an operative conformation even in the absence of estrogen and continue to function as a receptor that promotes the proliferation of breast cancer cells. Fulvestrant is the only selective estrogen receptor down-modulator available on the market and has shown good efficacy in treating breast cancer resistant to hormone therapy. However, fulvestrant has many problems in the treatment of AI-resistant ER-mutated breast cancer. First, due to its poor pharmacokinetic properties, fulvestrant has almost zero oral bioavailability, and at the same time, fulvestrant has high blood clearance. For the above two reasons, the drug can only be administered by intramuscular injection. However, due to its strong lipophilic structure, fulvestrant has serious problems in tissue distribution even when administered intramuscularly, and its clinical symptoms are that only about 50% of breast cancer patients treated with fulvestrant showed clinical response. In addition, due to the poor pharmacokinetic properties, the currently approved dose of fulvestrant does not achieve complete degradation of ER, especially mutant ER, at tissue concentrations, and this treatment scheme is not optimal for AI-resistant ER-mutated breast cancer. Therefore, the development of drugs for ER-mutated breast cancer with better pharmacokinetic properties 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, has shown superior progression-free survival rates compared to standard treatments in phase III clinical trials, especially in patients with ESR1 mutations, and an NDA application is currently being submitted to the FDA. WO2018 / 077630A1 reports Camizestrant (AZD9833), a new generation non-covalent estrogen receptor degrader for the treatment of ER-positive breast cancer currently being developed by AstraZeneca, and phase III clinical trials of the molecule for the treatment of ER-positive HER2-negative breast cancer are ongoing. WO2019 / 245974A1 and WO2020 / 049150A1 report Giredestrant (GDC-9545) and Amcenestrant (SAR439859), new generation non-covalent estrogen receptor degraders currently being developed by Genetech and Sanofi, respectively, where Giredestrant is in Phase III clinical trials and has shown no advantage over existing therapies in its single-agent Phase II clinical trials, and Amcenestrant has also shown no advantage in Phase II clinical trials and is currently 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 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°.

[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 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: 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 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: 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 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: 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 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: 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 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: 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 an X-ray powder 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. 06±0.20°, 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 crystal of the compound represented by formula (III) is characterized in that its 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.

[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 A-type crystals of the compound represented by formula (III) are 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.

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

[0026] The present invention further provides a B-type crystal of the compound represented by formula (II), characterized in that its 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 crystal of the compound represented by formula (II) is characterized in that its 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°, 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 B-type 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 in that the powder X-ray diffraction spectrum is 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 curve is 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 in that the differential scanning calorimetry curve has an endothermic peak at 215.60±3°C.

[0038] In some embodiments of the present invention, the B-type crystals of the compound represented by formula (II) are characterized in that their differential scanning calorimetry curve is 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°, 19.201°, 20.201°, 21.201°, 22.201°, 23.201°, 24.201°, 25.201°, 26.201°, 27.201°, 28.201°, 29.201°, 30.201°, 31.201°, 32.201°, 33.201°, 34.201°, 35.201°, 36.201°, 37.201°, 38.201°, 39.201°, 40.201°, 41.201°, 42.201°, 43.201°, 44.201°, 45.201°, 46.201°, 47.201°, 48.201°, 49.201°, 50.201°, 51.201°, 52.201°, 53.201°, 54.201°, 55.201°, 56.201°, 57.201°, 58.201°, 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 C-type 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 in that its powder X-ray diffraction spectrum is 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 in that their differential scanning calorimetry curve is as shown in FIG.

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

[0051] The present invention further provides use of the above compound or 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 show 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 percentage of weight loss, Residue represents the residue, Residue percent represents the remaining rate, Integral represents the total amount of heat generated (endothermic), Normalized represents the standard amount of heat generated (endothermic), 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 the supplier's catalogue.

[0057] The 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] 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: 0.60mm Detector slit: 10.50mm Anti-scatter slit: 7.10mm Scanning range: 4~40deg Step angle: 0.02 deg Step width: 0.12 seconds Sample pan rotation speed: 15 rpm Differential Scanning Calorimeter (DSC) Method of the Present Invention Instrument model: TA Q2000 Differential Scanning Calorimeter Measurement method: The sample (~1 mg) is placed in a DSC aluminum crucible and the measurement is performed. The sample is heated from 30°C (room temperature) to 300°C (or 350°C) at a heating rate of 10°C / min under the conditions of 50 mL / min N2.

[0060] The Thermogravimetric Analysis (TGA) Method of the Present Invention Instrument model: TA Q5000IR Thermogravimetric Analyzer Measurement method: The measurement is performed by placing a sample (2-5 mg) in a TGA platinum crucible and heating the sample from room temperature to 350°C at a heating rate of 10°C / min under the 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 represented by 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 slowly evaporated at room temperature. The crystals were obtained the next day, and the crystal structure is shown in FIG. 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 C-type 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] FIG. 10 is 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 model-bearing mouse after administration of a test compound.

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

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

[0075] [Figure 13] The percentage (%) of body weight change in a human breast cancer MCF7 cell subcutaneous xenograft tumor-bearing mouse during the administration of type C crystals of the compound represented by formula (I).

[0076] FIG. 14 shows the percentage (%) of body weight change in mice bearing human breast cancer MCF7 cell subcutaneous xenograft tumor models during the administration of type C crystals of the compound represented by formula (I) compared with fulvestrant and palbociclib.

[0077] FIG. 15 shows the percentage (%) of body weight change in mice bearing 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 is a tumor growth curve of the B-type crystal of the compound of formula (II) in a subcutaneous xenograft tumor model mouse bearing MCF7 cells having an ESR1 D538G mutation.

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

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

[0081] FIG. 19 shows the percentage (%) of body weight change in mice bearing subcutaneous xenograft tumor models of MCF7 cells harboring ESR1 D538G mutation during 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 by examples, but does not mean to restrict the present invention. The compounds of the present invention can be prepared by various synthesis methods familiar to those skilled in the art, including the specific embodiments listed below, embodiments combined with other chemical synthesis methods, and equivalent alternative methods familiar to those skilled in the art, and preferred embodiments include, but are not limited to, the examples of the present invention. It is clear to those skilled in the art that various changes and modifications can be made in the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

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

[0084] [ka]

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

[0086] Step B: p-Toluenesulfonic acid monohydrate (166.26g, 874.08mmol) was added to a stirred mixture of 1-15 (100g, 291.36mmol) and tetrahydrofuran (700ml) at 25℃, and the mixture was stirred at 35℃ for 12 hours. The reaction solution was concentrated under reduced pressure until no liquid evaporated. The concentrated crude product was dissolved in acetonitrile (300ml) at 25℃, and then methoxycyclopentyl ether (1500ml) was added and stirred at 25℃ for 12 hours. The resulting suspension was filtered under reduced pressure using a Buchner funnel. The cake was washed with methoxycyclopentyl ether (50ml x 2), collected, and dried in vacuum to obtain compound 1-16.

[0087] Step C: Anhydrous potassium phosphate (112.00 g, 527.65 mmol) and 1-17 (32.64 g, 173.62 mmol) were added to a stirred suspension of 1-16 (100 g, 170.21 mmol) in acetonitrile (1 L) at 25° C., and the mixture was stirred at 40° C. for 12 hours under nitrogen gas protection. The reaction solution was concentrated under reduced pressure until the liquid stopped evaporating, and 2-methyltetrahydrofuran (500 ml x 3) and saturated aqueous sodium carbonate solution (400 ml) were added to the residue. Saturated saline (300 ml x 2) was added to the separated organic phase, and the separated organic phase was dried over anhydrous sodium sulfate, filtered, and 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 the protection of nitrogen gas, add n-butyllithium n-hexane solution (2.5mol / L, 28.21ml3) slowly dropwise to N,N-diisopropylamine (7.73g, 76.41mmol) in tetrahydrofuran (100ml), stir the reaction solution at 0°C for 0.5 hours, then cool to -70°C, add 1-1 (10g, 58.78mmol) in tetrahydrofuran (100ml) slowly dropwise, after the addition is completed, stir the reaction solution at -70°C for 0.5 hours, then add 1-2 (8.53g, 70.53mmol) in toluene (10ml) slowly dropwise to the reaction solution, after the addition is completed, stir the reaction solution at -70°C for 3 hours to react. The reaction solution was added with saturated aqueous ammonium chloride solution (200ml), extracted with ethyl acetate (100ml x 2), the organic phases were combined, washed with saturated aqueous ammonium chloride solution (200ml x 2) and saturated saline solution (200ml x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The product was purified by silica gel column chromatography (eluent: petroleum ether) to obtain 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: At 20°C under nitrogen gas protection, 9-borabicyclo[3.3.1]nonane (0.5mol / L, 32.45ml) was added to a solution of 1-3 (3.1g, 14.75mmol) in tetrahydrofuran (18ml), and the reaction solution was stirred at 60°C for 3 hours and then cooled to room temperature. Water (10ml), 1-4 (3.53g, 11.80mmol), tetrakis(triphenylphosphine)palladium(0) (1.7g, 1.47mmol) and potassium phosphate (4.7g, 22.12mmol) were added to the reaction solution in sequence. The mixture was stirred at 70°C for 16 hours under nitrogen gas protection. The resulting reaction solution was diluted with ethyl acetate (100ml), washed with saturated saline (200ml x 2), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a 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: Sodium hydroxide (2.15g, 53.83mmol) was added to a mixed solution of 1-5 (6.7g, 13.46mmol) in methanol (75ml) and water (25ml), and the mixed solution was stirred at 25°C for 16 hours. The resulting reaction solution was concentrated under reduced pressure to remove the solvent methanol, and the residue was diluted with water (150ml), then extracted with dichloromethane (100ml x 2), and the aqueous phase was adjusted to pH = 2 with hydrochloric acid (2mol / L), extracted with ethyl acetate (80ml x 4), and the combined organic phase was washed with saturated saline (200ml x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 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 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 solution (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 a crude product. Compound 1-8 was obtained by purification using silica gel column chromatography (eluent: petroleum ether / ethyl acetate=40 / 1-20 / 1).

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

[0099] Step G: At 25°C, potassium carbonate (7.78g, 56.27mmol), triphenylphosphine (1.48g, 5.63mol) and dichlorobis(triphenylphosphine)palladium(0) (1.84g, 2.63mmol) were added to a solution of 1-9 (25g, 37.51mmol) and bis(pinacolato)diboron (14.29g, 56.27mmol) in 1,4-dioxane (200ml). The mixture was stirred at 90°C for 12 hours under the protection of nitrogen gas. The resulting reaction solution was filtered through diatomaceous earth, diluted with water (40ml) and extracted with ethyl acetate (50ml x 3). The combined organic phase was washed with saturated brine (40ml x 1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give 30.43g 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: At 25°C, potassium hydroxide (1.95g, 34.71mmol) and tetrakis(triphenylphosphine)palladium(0) (364.67mg, 315.58μmol) were added to a mixture of 1-11 (1.91g, 6.31mmol) and 1-10 (3.12g, 6.31mmol) in tetrahydrofuran (30ml) and water (6ml). The mixture was stirred at 60°C for 12 hours under the protection of nitrogen gas. The resulting reaction solution was diluted with water (20ml) and extracted with dichloromethane (20ml x 3). The combined organic phase was washed with saturated saline (20ml x 1), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain the crude product. Compound 1-12 was obtained by purification by reverse phase silica gel column chromatography (gradient elution with 0.1% aqueous ammonia / acetonitrile). LCMS (ESI) m / z: 591.2 [M + H] +.

[0101] Step I: At 20°C, trifluoroacetic acid (60ml) was added to a solution of 1-12 (18g, 7.87mmol) in acetonitrile (180ml). The mixture was stirred at 80°C for 16 hours. The resulting reaction solution was concentrated under reduced pressure until the liquid stopped evaporating, and the pH was adjusted to 8-10 by adding aqueous sodium carbonate solution, and extracted with dichloromethane. The combined organic phase was washed with saturated saline, dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure to obtain a crude product. The compound represented by formula (I) was obtained by purifying the product by silica gel column chromatography (eluent: dichloromethane / methanol = 10 / 1). 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 represented by formula (I) was taken and 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, 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 represented by 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 represented by formula (I) was weighed and added to a 50 ml one-neck flask, and acetonitrile (9 ml) was added to dissolve it. After adding a magnet, the solution was placed on a magnetic heating stirrer (90°C) and stirred, and a solution of methanesulfonic acid (155 μl) in acetonitrile (1 ml) was added at 90°C and stirred. The mixture was slowly cooled to 20°C and stirred for 12 hours, and the resulting suspension was filtered and the cake was dried to obtain B-type crystals of the compound represented by formula (II).

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

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

[0106] 350 g of the B-type crystals of the compound represented by formula (II) obtained by the above-mentioned production method were weighed and added to a 5L one-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 mixed solution was slowly cooled to 25-30°C over 4-6 hours while stirring, and stirred at the same temperature for 12 hours to obtain a milky suspension. After filtering the reaction solution, a pale yellow cake was obtained, and the cake was thoroughly stirred with isopropyl acetate, washed twice (500 ml x 2), and the cake obtained by thermostatic suction filtration 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 filtered. Acetone (3.9 L) was further added to the obtained 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 represented by 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 C-type crystals of the compound represented by 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 Testing (1) ERα binding assay: evaluation of the activity of the compound represented by formula (I) in a radioligand binding assay 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 Testing 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 the IC using nonlinear, least squares regression methods. 50 The inhibition constants (K i ) is the IC 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 substitution does 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 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 media: DMEM (phenol red-free) containing 10% carbon-adsorbed serum, i.e., 450 mL of DMEM, 50 mL of carbon-adsorbed serum in 500 mL of cell culture medium. 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) and then mixed together. The reagents were 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, centrifuged at 1000 rpm / min for 5 min, the supernatant was removed, resuspended in pre-warmed experimental medium, counted, and the cell suspension was diluted with 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 in each well and cultured overnight at 37°C in a 5% CO2 incubator.

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

[0120] 3) After incubation was completed, 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 was completed, the luminescence signal was detected using EnVision.

[0121] Data Analysis: The data was 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 and IC of compound 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] Test conclusion: The compound of formula (I) has a significant antagonistic effect on the transcription function of HEK293 / GAL4 / ERα.

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

[0126] [Table 8]

[0127] Cell information and culture conditions:

[0128] [Table 9]

[0129] Preparation of test reagents: 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: A primary antibody (Estrogen Receptor alpha Monoclonal Antibody (SP1)) stock solution was diluted 1000-fold with a blocking buffer containing 0.100% Tween-20 to obtain a 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 uniformly.

[0133] Experimental steps: 1) Aspirate the complete medium from the culture flask, add an appropriate amount of trypsin to rinse, and then aspirate to remove residual serum.

[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 schematic of sample addition in 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 incubated in a 37°C, 5% CO2 incubator 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% TritonX-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 removed by tapping; in this manner, the plate was washed 5 times.

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

[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 the 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 mixture 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 an Odyssey Far Infrared Imaging System instrument.

[0156] Data analysis High control group mean: calculate the mean of the high control group; Low control group mean: calculate the mean 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 high control group = (standard deviation of high control group ÷ mean of high control group) × 100 Coefficient of variation (CV) of low control group = (standard deviation of low control group ÷ mean of low control group) × 100 Experimental window = high control group mean / low control group mean Z score = 1 - [3 x (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, CAMA-1 cells is evaluated, and the expression level of ERα protein is detected by ICW experiment after 4 hours of drug treatment.The results are shown below.

[0160] [Table 10]

[0161] Test 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 inhibition experiment Test materials:

[0163] [Table 11]

[0164] Cell information and culture conditions:

[0165] [Table 12]

[0166] Starvation medium: 1. CAMA-1: EMEM (without phenol red) 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 (without phenol red) with 10% carbon-adsorbed serum, i.e., 450 mL RPMI-1640, 50 mL carbon-adsorbed serum in 500 mL cell culture medium.

[0167] Experimental design The inhibitory effect of the compound represented by formula (I) on the proliferation of MCF-7, CAMA-1 and T-47D cells was analyzed. MCF-7 was tested with 10 concentration gradients set up with 3 replicates, and the compound concentration range was 100nM-0.0003815nM; CAMA-1 was tested with 9 concentration gradients set up with 3 replicates, and the compound concentration range was 100nM-0.001526nM; T-47D was tested with 9 concentration gradients set up with 3 replicates, and the compound concentration range was 2μM-0.00512nM, and two independent experiments were performed for each.

[0168] Experimental steps: MCF-7 cells: MCF-7 cells that reached 80% cell confluence were digested with trypsin, centrifuged, resuspended and counted. Then, cell suspensions were prepared using the corresponding culture medium, and 45 μL of 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 diluted 4x serially downwards to 10 concentrations using DMSO solution with a starting concentration of 20 μM. After culturing the cells overnight, the diluted compound was further diluted with the corresponding cell culture medium, and the diluted mixed solution was transferred to the corresponding cell plate, and the final concentration of the compound was diluted 4x downwards to 10 concentrations with a starting concentration of 100 nM, and the positive control well was 2 μM fulvestrant, mixed uniformly, centrifuged, and cultured at 37 ° C in a cell incubator containing 5% CO2 for the corresponding days in the above table.

[0169] T-47D cells: T-47D cells were starved for 3 days with 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, 40 μL / well of cell suspension was added to each well, and cultured at 37°C in a cell incubator with 5% CO2. The test compound formula (I) was diluted 5x downwards to 9 concentrations using DMSO solution with a starting concentration of 400 μM. After culturing the cells overnight, the diluted compounds were further diluted with the corresponding cell medium, and the diluted mixed solutions were transferred to the corresponding cell plates, respectively, with a final concentration of the compounds being 2 μM starting concentration and 5x downwards to 9 concentrations, with 2 μM fulvestrant for the positive control well, and 1 nM estradiol was added to the entire plate, mixed evenly, centrifuged, and cultured at 37°C in a cell incubator with 5% CO2 for the corresponding days in the above table.

[0170] CAMA-1 cells: CAMA-1 cells were starved for 3 days with 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 / well of cell suspension was added to each well and cultured at 37°C in a cell incubator with 5% CO2. The test compound of formula (I) was diluted 4× downwards to 9 concentrations with a starting concentration of 20 μM using DMSO solution. After culturing the cells overnight, the diluted compound was further diluted with the corresponding cell medium, and the diluted mixed solution was transferred to the corresponding cell plate, and the final concentration of the compound was diluted 4× downwards to 9 concentrations with a starting concentration of 100 nM, with 2 μM fulvestrant in the positive control well and 1 nM estradiol added to the entire plate, mixed evenly, centrifuged, and cultured at 37°C in a cell incubator with 5% CO2 for the corresponding days in the above table.

[0171] After the compounds were incubated for the above times, the cell culture plate was taken out, CTG reagent was added, mixed evenly, centrifuged, and incubated at room temperature for 10 minutes. The data was read on an Envision multi-label analyzer.

[0172] Data Analysis: The data was 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 obtain the IC 50 The IC values ​​were calculated. 50 Calculation formula: log(inhibitor) vs. response - Variable slope.

[0173] Test Results: This experiment analyzed the inhibitory effect of the test compound represented by 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) HCC1428 and MDA-MB-134VI cell proliferation inhibition experiment 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 a 10 concentration gradient.

[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% confluency in a T75 culture flask, trypsin was used to digest and collect the cells, and the cell passages for the first and second independent experiments were 8 and 6 generations, respectively.

[0182] 3) The cell densities of the first and second independent experiments, determined by centrifugation at 1000 rpm, resuspension in complete medium, counting, and measurement, were 3.60 × 10 per ml, respectively. 6 1.93 x 10 per cell and 1 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 8,000 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, and the final concentration of DMSO 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, equilibrate them to room temperature, and centrifuge them at 1000 rpm for 5 minutes. Discard 100 μL of cell culture supernatant from each well and add 100 μL of CellTiter Glo. Incubate in the dark at room temperature for 30 minutes.

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

[0188] Following the steps above, two independent replicate experiments were performed.

[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% confluency in a T75 culture flask, trypsin was used to digest and collect the cells, and 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, determined by centrifugation at 1000 rpm, resuspension in complete medium, counting, and measurement, were 1.30 × 10 per ml, respectively. 6 9.50 x 10 per cell and 1 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 concentration of DMSO 0.1%. DMSO wells were used as high control and medium only wells were used as low control.

[0194] 6) The cells were cultured in a 37℃, 5% CO2 incubator for 7 days.

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

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

[0197] Following the steps above, two independent replicate experiments were performed.

[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] The IC of the compound was calculated using the nonlinear fitting equation below. 50 (half inhibitory concentration) was obtained: Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -X)*HillSlope)) X: log 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 standard error and AVE is the mean.

[0201] Experimental Results This experiment analyzed the inhibitory effect of the compound represented by 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] Test conclusion: Compounds of formula (I) exhibit significant in vitro antiproliferative activity against MDA-MB-134VI and HCC1428 cells.

[0204] (6) Growth inhibition experiments 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 Culturing Tumor cells were cultured in an incubator at 37°C with 5% CO2. 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 solutions and compound treatment of cells 1) Preparation of 10X compound working solution: 199μL of cell culture medium was added to a V-bottom 96-well plate, and 1μL of compound was pipetted from a 2000X compound storage plate and added to the cell culture medium in the 96-well plate. 1μL of DMSO was added to the solvent control and blank control. Compound or DMSO was added and mixed uniformly with a pipette.

[0214] 2) Dosing: 15 μL of 10X compound working solution was taken and added to cell culture plates 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) Gently vortex 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 (equal to half the volume of cell culture medium in each well) of CellTiter-Glo working solution was added to 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 compounds were calculated in Excel, and then the inhibition curves were fitted using GraphPad Prism (6.02.328) software log(inhibitor) vs. response--Variable slope to obtain the related parameters.

[0227] Relative IC 50 is the concentration required to reduce the concentration to the half-way 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 50 is as shown in the table below.

[0229] [Table 18]

[0230] Test 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 generated by the probe substrate were detected by LC-MS / MS and IC 50 The values ​​were calculated. The solvent control sample was added with the same amount of solvent instead of the test sample, and the enzyme activity of the solvent control sample was set as 100%. The detailed information such as the concentration of human liver microsomes used in the experiment, the probe substrate concentration corresponding to each CYP, the incubation time, the positive control, and the metabolites are 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, with each test substance concentration in triplicate and each positive control inhibitor concentration in duplicate. The incubation reaction system included a mixed working solution of the corresponding isoenzyme probe substrate (at the designated concentration) and microsomes (0.100 mg protein / mL) and a working solution of the test substance or positive control inhibitor (at a series of final concentrations). All samples were preheated at 37°C for 10 min, and then the reaction was started by adding the cofactors. For the CYP3A metabolic reaction using midazolam as the probe substrate, the reaction time was 3 min, and all other reactions were 10 min.

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

[0237] Sample processing: After the incubation reaction system of each isozyme reached the corresponding reaction time, 200 μL of the stop solution containing the corresponding internal standard was added to the reaction plate to stop the reaction. After shaking thoroughly, it was placed in a centrifuge and centrifuged at 3220 × g for 20 minutes, and then the supernatant was taken out and diluted by adding the appropriate diluent at a predetermined ratio. The reaction plate was placed on a shaker and shaken to mix uniformly. Then, the metabolites of the probe substrate were analyzed using LC-MS / MS, and if the samples could not be analyzed immediately, the samples 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, and the value multiplied by 100% was taken as the percentage of the remaining activity of each isoenzyme. With the percentage of remaining activity as the ordinate and the inhibitor concentration as the abscissa, nonlinear regression analysis was performed using 3 or 4 parameters with SigmaPlot (V.14) software to obtain the IC of the test product and each positive inhibitor. 50 The IC values ​​were calculated by fitting with SigmaPlot software. 50 When the IC exceeds the maximum administered concentration (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 for CYP3A (using midazolam as a substrate). 50 The IC value was 8.38 μM, which is the IC for CYP3A (using testosterone as a substrate). 50 The value was 10.9 μM. The compounds of the present invention have weak inhibition against CYP enzymes, indicating a low risk of drug interactions.

[0243] (2) Pharmacokinetic study in mice Experimental Objective: Female Balb / c mice were used as test animals, and the plasma drug concentrations at different time points were measured after intravenous and intragastric administration of the test compound to the mice using LC / MS / MS method. The pharmacokinetic behavior of the C-type crystals of compound of formula (I) in the mouse body 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 out and made into a 1 mg / mL solution, stirred and sonicated until clear, and 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 the intravenous administration of the test compound to the 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 a commercially available anticoagulant tube with EDTA-K2 added beforehand. After the intragastric administration of the test compound, 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 a commercially available anticoagulant tube with EDTA-K2 added beforehand. Then, the plasma was removed after centrifugation (3,200 g, 4°C, 10 min), and the plasma was transferred to a pre-cooled centrifuge tube, flash frozen on dry ice, and stored in a -60°C or lower ultra-low temperature refrigerator until LC-MS / MS analysis was performed. The animals were fed 4 hours after administration. The content of the test compound in the plasma of the mice after intravenous and intragastric administration was measured using the 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 high tissue distribution, high oral absorption and availability, and show good pharmacokinetic properties in vivo.

[0251] (3) Pharmacokinetic study in rats Experimental Objective: Female Sprague-Dawley rats were used as test animals, and the test compounds were administered intravenously and intragastrically to the rats, and the plasma drug concentrations at different time points were measured using LC / MS / MS. The pharmacokinetic behavior of the B-type crystals of compound of formula (II) in the 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 out 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 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 into commercially available anticoagulant tubes containing EDTA-K2. After administration of the test compound to the intragastric administration group, 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 hours, respectively, and placed in a commercially available anticoagulant tube with EDTA-K2 added in advance. Then, the plasma was removed after centrifugation (3,200 g, 4 °C, 10 min), and the plasma was transferred to a pre-cooled centrifuge tube, flash frozen on dry ice, and stored in a -60 °C or lower ultra-low temperature refrigerator until LC-MS / MS analysis was performed. The animals were fed 4 hours after administration. The content of the test compound in the plasma of rats after intravenous and intragastric administration was measured using the 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 thus exhibit favorable pharmacokinetic properties in vivo.

[0259] (4) Pharmacokinetic study in dogs Experimental Objective: Female and male beagles were used as test animals, and the plasma drug concentrations at different time points were measured after intravenous and intragastric administration of the test compound to the beagles using LC / MS / MS. The pharmacokinetic behavior of the B-type crystals of compound of formula (II) in the 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 and stirred until clear; the solvent was 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 administration of the test compound to the intravenous administration group of beagles, 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 a commercially available anticoagulant tube with EDTA-K2 added in advance. After administration of the test compound to the intragastric administration group, 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 a commercially available anticoagulant tube with EDTA-K2 added in advance. Then, after centrifugation (3,200 g, 2-8 °C, 10 min), the plasma was removed, transferred to a pre-cooled centrifuge tube, flash frozen on dry ice, and stored in a -60 °C or lower ultra-low temperature refrigerator until LC-MS / MS analysis was performed. The animals were fed 4 hours after administration. An LC / MS / MS method was used to measure the content of the test compound in the plasma of beagles 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 beagle body, have a large tissue distribution, high oral absorption and availability, and show 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 mouse model with subcutaneous xenografted breast cancer MCF-7 cells Objective of the experiment: In this experiment, the in vivo efficacy of the C-type crystals of the compound represented by formula (I) was evaluated using a nude mouse model of human breast cancer MCF-7 cell subcutaneous xenograft tumor.

[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 monolayer 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 5% CO2 incubator at 37°C. Cells were routinely digested with trypsin-EDTA (Gibco) twice a week and subcultured. When the cell saturation was 80%-90% and the cell number reached the required number, the 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) on the left back, and 3 days later, 0.2 mL (1 × 10 7MCF-7 cells (containing Matrigel; Corning, volume ratio 1:1) were inoculated subcutaneously into the right dorsal region of each mouse, resulting in a mean tumor volume of 188 mm 3 When this was reached, the groups were divided and administered the drugs.

[0273] [Table 25]

[0274] Pharmaceutical manufacturing: An appropriate amount of compound of formula (I) sample was weighed out, and prepared into 0.1, 0.3, 1 and 3mg / mL solutions, respectively, vortexed for a period of time, and sonicated to form a clear solution, the solvent was 10% HP-β-CD. An appropriate amount of Palbociclib was weighed out, and prepared into 2.5mg / mL solutions, the pH was adjusted to 4.0, and vortexed for a period of time, and sonicated to form a clear solution, the solvent was 50mM sodium lactate buffer. Fulvestrant was used as a commercially available injection drug, Faslodex.

[0275] Tumor Measurements and Laboratory Endpoints: Tumor diameters were measured twice a week with a 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 proliferation rate T / C (%). Relative tumor proliferation rate T / C (%) = T RTV / C RTV ×100%(T RTV : Mean RTV of treatment group; C RTV RTV was calculated according to the tumor measurement results, and the formula was RTV = V t / V0, where V0 is the tumor volume measured at the time of 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)) x 100%.

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

[0279] After the experiment, the tumor weight was measured and T / C weight Calculate the percentage and T weight and C weight represent the tumor weights of the treatment and vehicle control groups, 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), they were tested with the Games-Howell method. p<0.05 was considered to indicate a significant difference.

[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, and the solvent control group was used as a 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 mean 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, the TGI was 85.3%, 100.9%, 104.6%, and 106.5%, respectively, the P values ​​were 0.003, 0.001, 0.001, and 0.001, respectively, compared with the control group, and the objective response rate (ORR) was 25% at the doses of 10 mg / kg and 30 mg / kg, so that the C-type crystals of the compound represented by formula (I) had significant tumor inhibitory effects at all of the above doses, and showed a predetermined dose-effect relationship with increasing dose.

[0283] [Table 26]

[0284] On day 21, the mean tumor volume in the fulvestrant (5 mg / mouse) and palbociclib (25 mg / kg) groups was 593 mm 3 and 781 mm 3 The T / C values ​​were 46.9% and 64.0%, respectively, the TGI was 60.9% and 42.8%, respectively, and the P values ​​compared with the control group were 0.019 and 0.219, respectively, so that the administration of fulvestrant alone had an antitumor effect, but the administration of palbociclib alone did not show an antitumor effect.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, and its disease control rate (DCR) reached 100%.

[0285] Palbociclib alone had no 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 ​​were 0.001, 0.002, and 0.012 compared with the control group, palbociclib, and the C-type crystals of the compound represented by formula (I) (3 mg / kg) group, 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 the C-type crystals of the compound represented by formula (I), which was a synergistic additive effect.

[0286] Test conclusion: Compared with the blank control group, the C-type crystals of the compound represented by formula (I) had significant tumor inhibitory effects at four doses of 1 mg / kg, 3 mg / kg, 10 mg / kg, or 30 mg / kg, with an effective dose of 1 mg / kg (TGI of 85.3%). When the C-type crystals of the compound represented by formula (I) (3 mg / kg) were used in combination with palbociclib, they showed a stronger antitumor effect than when administered alone. Tumor-bearing mice showed good tolerance to the C-type crystals of the compound represented by formula (I). The compounds of the present invention showed excellent tumor inhibitory effects in mouse pharmacodynamic models and had good clinical therapeutic potential.

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

[0288] Test materials:

[0289] [Table 27]

[0290] Experimental methods and steps: Cell culture preparation: Human breast cancer MCF7 cells carrying the ESR1 D538G mutation were routinely cultured in monolayer in vitro in EMEM medium supplemented with 10% fetal bovine serum, 1% Antibiotic-Antimycotic, 2 mM L-Glutamine, and 1% NEAA in a 5% CO2 incubator at 37°C. The cells were subcultured twice a week after routine digestion using trypsin-EDTA. When the cell saturation was 80%-90% and the cell number reached the required level, 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 6 MCF7 ESR1 D538G cells (PBS + Matrigel, volume ratio 1:1) were inoculated subcutaneously, 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: An appropriate amount of the B-type crystal sample of the compound of formula (II) was weighed out and made into a 0.5, 1.5 and 5.0 mg / mL solution, respectively, and vortexed and sonicated for a period to form a clear solution, the solvent being 10% HP-β-CD. An appropriate amount of palbociclib was weighed out and made into a 5 mg / mL solution, the pH was adjusted to 4.0, vortexed and sonicated for a period to form a clear solution, the solvent being 50 mM sodium lactate buffer.

[0295] Tumor Measurements and Laboratory Endpoints: Tumor diameters were measured twice a week with a 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 the compound is evaluated by TGI (%) or relative tumor proliferation rate T / C (%). Relative tumor proliferation rate T / C (%) = T RTV / C RTV ×100%(T RTV : Mean RTV of treatment group; C RTV RTV was calculated according to the tumor measurement results, and the formula was RTV = V t / V0, where V0 is the tumor volume measured at the time of 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, the tumor weight was measured and T / C weight Calculate the percentage and T weight and C weight represent the tumor weights of the treatment and vehicle control groups, respectively.

[0299] Statistical analysis was performed using GraphPad Prism software based on end-of-study RTV data. Comparisons between multiple groups were analyzed using one-way ANOVA, with p<0.05 considered to be significantly different and p>0.05 considered to be not significantly different.

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

[0301] [Table 29]

[0302] Thirty-one days 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) had 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 was 24.15%, 18.93% and 6.37%, respectively, and the TGI was 98.86%, 104.52% and 120.51%, respectively, and all p values ​​were <0.0001 compared with the blank control group. The B-type crystals of the compound represented by formula (II) had significant antitumor effects at the above doses, and the objective response rate showed good dose-dependence. The mean tumor volume of the group administered the B-type crystals of the compound represented by formula (II) in combination with palbociclib (50 mg / kg) was 31 mm 3The T / C was 4.67%, the TGI was 122.83%, the blank control group p value was <0.0001, and there was statistical significance compared with the group administered with the B-type crystal of the compound represented by formula (II) or palbociclib alone (p value <0.0001, synergy coefficient was 2.21), and 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 the B-type crystals of the compound represented by formula (II) have significant antitumor effects at doses of 5 mg / kg, 15 mg / kg and 50 mg / kg in a BALB / c nude mouse model of human breast cancer MCF7 cell subcutaneous xenograft tumors carrying the ESR1 D538G mutation. The B-type crystals of the compound represented by formula (II) have a synergistic antitumor effect when used in combination with palbociclib (50 mg / kg) at a dose of 5 mg / kg, which is equivalent to the antitumor effect of the B-type crystals of the compound represented by formula (II) at 50 mg / kg.

[0304] Test conclusion: The B-type crystals of the compound represented by formula (II) had an antitumor effect on the growth of subcutaneously transplanted tumors in MCF7 mice with ESR1 D538G mutation, and showed a certain dose-dependency, and when combined with palbociclib, showed a superior antitumor effect to that of single administration. During the experiment, the mice in each test group showed good drug tolerance. [Brief description of the drawings]

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

Claims

1. A compound represented by formula (III): [Formula 1]

2. A type crystal of the compound 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°. [Case 2]

3. The A-type crystal of the compound represented by formula (III) according to claim 2, 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°.

4. The A-type crystal of the compound represented by formula (III) according to claim 2, 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°.

5. The A-type crystal of the compound represented by formula (III) according to claim 4, 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°.

6. The A-type crystal of the compound represented by formula (III) according to claim 4, 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°.

7. The A-type crystal of the compound represented by formula (III) according to claim 4, 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°.

8. 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. The A-type crystal of the compound represented by formula (III) according to any one of claims 2 to 7, characterized in that it has diffraction peaks at 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°.

9. 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°, 33.198°. A type A crystal of the compound represented by formula (III) according to any one of claims 2 to 8.

10. A type A crystal of the compound represented by formula (III) according to any one of claims 2 to 8, characterized in that the powder X-ray diffraction spectrum is essentially as shown in Figure 1.

11. The thermogravimetric analysis curve is characterized by a weight loss of 0.198% at 150.000±3°C and a weight loss of 10.880% at 240.000±3°C. A type A crystal of the compound represented by formula (III) according to any one of claims 2 to 9.

12. 12. The A-type crystal of the compound represented by formula (III) according to claim 11, characterized in that the thermogravimetric analysis curve spectrum is as shown in FIG.

13. The A-type crystal of the compound represented by formula (III) according to any one of claims 2 to 9, 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.

14. 14. The A-type crystal of the compound represented by formula (III) according to claim 13, characterized in that the differential scanning calorimetric curve is as shown in FIG.

15. A B-type crystal 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°. [C3]

16. The B-type crystal of the compound represented by formula (II) according to claim 15, 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°.

17. The B-type crystal of the compound represented by formula (II) according to claim 16, 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°.

18. 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°, 18. The compound of formula (II) according to claim 17, characterized in that it has diffraction peaks at 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°.

19. A B-type crystal of the compound represented by formula (II) according to any one of claims 15 to 18, characterized in that the powder X-ray diffraction spectrum is essentially as shown in Figure 4.

20. The B-type crystal of the compound represented by formula (II) according to any one of claims 18 to 19, characterized in that the thermogravimetric analysis curve spectrum shows a weight loss of 0.085% at 200.000±3°C.

21. The B-type crystal of the compound represented by formula (II) according to claim 20, characterized in that the thermogravimetric analysis curve spectrum is as shown in FIG.

22. 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°. [C4]

23. 23. The C-type crystal of the compound represented by formula (I) according to claim 22, 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°.

24. The C-type crystal of the compound represented by formula (I) according to claim 23, 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°.

25. 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°, 22.062°, 23.062°, 24.062°, 25.062°, 26.062°, 27.062°, 28.062°, 29.062°, 30.062°, 31.062°, 32.062°, 33.062°, 34.062°, 35.062°, 36.062°, 37.062°, 38.062°, 39.062°, 40.062°, 41.062°, 42.062°, 43.062°, 44.062°, 45.062°, 46.062°, 47.062°, 48.062°, 49.062°, 50.062°, 51.062°, 52.062°, 53.062°, 54.062°, 55.062°, 56.062°, 57.062°, 58.062°, 59.062°, 60.062 25. The C-type crystal of the compound represented by formula (I) according to claim 24, 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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