Crystal form of a polysubstituted benzene ring compound maleate salt, method for preparing the same, and use thereof

Abstract

A crystal form of a polysubstituted benzene ring compound maleate, a preparation method thereof and application thereof. The crystal form of the polysubstituted benzene ring compound salt as shown in formula (II) has good physical and chemical stability, high solubility, is not easy to absorb moisture, has good fluidity, more formulation process selection, is more suitable for industrialized production, and has important value for optimization and development of drugs.

Description

[0001] This application claims priority to Chinese patent application 2020110887642, filed on October 13, 2020. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to the crystal forms of maleate salts of polysubstituted benzene ring compounds, their preparation methods, and their applications. Background Technology

[0003] A multi-substituted benzene ring compound, the structure of which is shown in Formula I.

[0004]

[0005] This compound, disclosed in patent WO2020020374A1, is an EZH2 (Zeste homolog enhancer 2) inhibitor that can be used to prevent or treat EZH2-mediated diseases, including brain cancer, thyroid cancer, cardiac sarcoma, lung cancer, oral cancer, stomach cancer, and various other cancers.

[0006] The phenomenon that a substance can exist in two or more different crystal structures is called polymorphism. For pharmaceuticals, this polymorphism can affect drug absorption, thereby impacting bioavailability and resulting in varying clinical efficacy and toxic side effects. Therefore, developing advantageous crystal forms of polysubstituted benzene ring compound salts, as shown in Formula I, is of great significance.

[0007] In drug development, solubility and hygroscopicity are both factors that need to be considered. However, these two indicators are usually correlated. Active ingredients that are easily soluble in water may also absorb moisture, so substances with high solubility are generally more hygroscopic. Therefore, developing compounds with high solubility and low hygroscopicity is of great significance for drug development. Summary of the Invention

[0008] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies, such as the low solubility and hygroscopicity of polysubstituted benzene ring compounds (as shown in Formula I) in drug preparation. This invention provides a crystal form of a polysubstituted benzene ring compound salt (as shown in Formula II), its preparation method, and its applications. This crystal form exhibits good physicochemical stability, high solubility, low hygroscopicity, good flowability, more formulation process options, and is more suitable for industrial production, thus possessing significant value for drug optimization and development.

[0009] The present invention provides a crystal form A of a maleate salt of a polysubstituted benzene ring compound as shown in Formula II, and the X-ray powder diffraction pattern expressed in 2θ angle shows diffraction peaks at 7.39±0.2°, 8.78±0.2°, 13.99±0.2°, 15.21±0.2°, 15.73±0.2°, 17.75±0.2°, 18.51±0.2° and 21.06±0.2°.

[0010]

[0011] The crystal form A of the polysubstituted benzene ring compound maleate as shown in Formula II, whose X-ray powder diffraction pattern is expressed in 2θ angles, may also have diffraction peaks at one or more of the following 2θ angles: 8.51±0.2° and 16.06±0.2°.

[0012] The crystal form A of the polysubstituted benzene ring compound maleate as shown in Formula II, whose X-ray powder diffraction pattern is expressed in 2θ angles, may also have diffraction peaks at one or more of the following 2θ angles: 18.20±0.2° and 20.68±0.2°.

[0013] The polysubstituted benzene ring compound maleate crystal form A, as shown in Formula II, has an X-ray powder diffraction pattern expressed at 2θ angles, which also shows diffraction peaks at one or more of the following 2θ angles: 22.81±0.2° and 25.28±0.2°.

[0014] The crystal form A of the polysubstituted benzene ring compound maleate as shown in Formula II, its X-ray powder diffraction pattern expressed in 2θ angle, and its diffraction peaks and relative intensities are also shown in Table 1.

[0015] Table 1

[0016] Diffraction peak number 2θ angle (°) Relative strength (%) 1 7.39±0.2 42.64 2 8.51±0.2 12.59 3 8.78±0.2 37.91 4 11.79±0.2 4.22 5 12.73±0.2 3.84 6 13.99±0.2 56.77 7 15.21±0.2 100.00 8 15.73±0.2 88.27 9 16.06±0.2 17.68 10 17.75±0.2 67.49 11 17.91±0.2 32.21 12 18.20±0.2 25.80

[0017] 13 18.51±0.2 42.40 14 19.13±0.2 1.87 15 19.80±0.2 1.17 16 20.68±0.2 24.70 17 21.06±0.2 66.84 18 21.32±0.2 13.09 19 22.11±0.2 9.65 20 22.81±0.2 22.28 21 23.07±0.2 5.24 22 23.66±0.2 9.98 23 23.84±0.2 8.90 24 24.41±0.2 5.27 25 24.68±0.2 12.31 26 25.28±0.2 57.69 27 25.62±0.2 23.51 28 26.23±0.2 10.60 29 26.68±0.2 16.79 30 26.84±0.2 12.91 31 28.06±0.2 20.35 32 29.11±0.2 3.72 33 29.41±0.2 10.37 34 29.67±0.2 5.16 35 29.99±0.2 16.33 36 30.86±0.2 3.90 37 32.44±0.2 1.66 38 33.38±0.2 4.28 39 34.46±0.2 1.45 40 35.33±0.2 2.75 41 36.50±0.2 2.36 42 37.08±0.2 4.26 43 38.33±0.2 4.65

[0018] The X-ray powder diffraction pattern of the polysubstituted benzene ring compound maleate as shown in Formula II, represented by the 2θ angle, is also substantially as follows: Figure 1 As shown.

[0019] In this invention, the X-ray powder diffraction patterns are all obtained using the Kα spectral lines of a Cu target.

[0020] In this invention, the differential scanning calorimetry (DSC) of the maleate crystal form A of the polysubstituted benzene ring compound as shown in Formula II may have major endothermic peaks at 185.3℃±3℃ and 204.4℃±3℃.

[0021] In this invention, the DSC of crystal form A of the polysubstituted benzene ring compound maleate as shown in Formula II can also be substantially as follows: Figure 2 As shown.

[0022] In this invention, the thermogravimetric analysis (TGA) diagram of crystal form A of the polysubstituted benzene ring compound maleate as shown in Formula II can also be substantially as follows: Figure 3 As shown.

[0023] The present invention also provides a method for preparing crystal form A of the maleate of the polysubstituted benzene ring compound as shown in Formula II, wherein the method is method 1, method 2, method 3, method 4, method 5 or method 6:

[0024] Method 1: In solvent A, react the polysubstituted benzene ring compound as shown in Formula I with maleic acid;

[0025] Solvent A is selected from one or more of methyl isobutyl ketone, 2-methyltetrahydrofuran, and ethyl acetate;

[0026] Method 2: Form a suspension solution of the polysubstituted benzene ring compound maleate crystal form B as shown in Formula II in solvent B, and stir.

[0027] Solvent B is selected from one or more of alcohol solvents, ester solvents, ketone solvents, ether solvents, benzene solvents, and nitrile solvents;

[0028] Method 3: Prepare a saturated solution of maleate salt of polysubstituted benzene ring compound as shown in Formula II in a mixed solvent of dimethyl sulfoxide and ethyl acetate, and allow it to crystallize.

[0029] Method 4: Dissolve the crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II in solvent C, add solvent D, and crystallize.

[0030] The solvent C is selected from one or more of methanol, dimethyl sulfoxide, and chloroform. When the solvent C is methanol, the solvent D is ethyl acetate; when the solvent C is dimethyl sulfoxide, the solvent D is isopropyl acetate; when the solvent C is chloroform, the solvent D is one or more of ethyl acetate, acetonitrile, and butanone.

[0031] Method 5: Perform a gas-liquid permeation crystallization experiment on the crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II;

[0032] The positive solvent is methanol, and the antisolvent is selected from one or more of acetone, ethyl acetate, and tetrahydrofuran;

[0033] Method 6: Place the crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II in the environment of solvent E and perform a gas-solid permeation experiment.

[0034] The solvent E is one or more of methanol, acetonitrile, acetone, dimethyl sulfoxide, isopropanol, and dimethylformamide.

[0035] In methods 2-6 above, the X-ray powder diffraction pattern of the crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II, expressed at an angle of 2θ, is essentially as follows: Figure 4 As shown.

[0036] In Method 1, the reaction temperature can be conventional in the art, preferably room temperature.

[0037] In Method 1, the reaction time can be conventional in the art, preferably 2-4 days.

[0038] In Method 1, the molar ratio of the polysubstituted benzene ring compound as shown in Formula I to the maleic acid can be conventional in the art, preferably 1:(1-1.2), for example 1:1.07.

[0039] In Method 1, the volume-to-mass ratio of solvent A to the polysubstituted benzene ring compound as shown in Formula I can be conventional in the art, preferably 15-40 ml / g, for example 17.6 ml / g.

[0040] In method 2, the stirring can be conventional in the art, and is preferably magnetic stirring.

[0041] In method 2, the stirring temperature can be conventional in the art, preferably 0-50℃, for example, room temperature, 50℃, or temperature cycling at 50℃-5℃ and 0.1℃ / min. When the stirring temperature is 50℃, the stirring time is 5-8 days; when the stirring temperature is room temperature, the stirring time is 25-35 days; when the stirring temperature is temperature cycling, the temperature cycling conditions are 50℃-5℃, 0.1℃ / min, 2-4 cycles.

[0042] In method 2, the alcohol solvent can be conventional in the art, preferably ethanol and / or isopropanol.

[0043] In Method 2, the ester solvent can be conventional in the art, preferably ethyl acetate and / or isopropyl acetate.

[0044] In Method 2, the ketone solvent can be conventional in the art, preferably one or more of acetone, butanone, and methyl isobutyl ketone.

[0045] In Method 2, the ether solvent can be conventional in the art, preferably one or more of anisole, cyclopentyl methyl ether and methyl tert-butyl ether, more preferably anisole and / or cyclopentyl methyl ether.

[0046] In method 2, the benzene solvent can be conventional in the art, and toluene is preferred.

[0047] In method 2, the nitrile solvent can be conventional in the art, preferably acetonitrile.

[0048] In Method 2, the volume-to-mass ratio of solvent B to the crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II can be conventional in the art, preferably 20-50 ml / g, for example 25 ml / g.

[0049] In method 3, the volume ratio of dimethyl sulfoxide and ethyl acetate in the mixed solvent can be conventional in the art, preferably 1:(2-5), for example 1:3.

[0050] In Method 3, the volume-to-mass ratio of the mixed solvent to the crystal form B of the maleate of the polysubstituted benzene ring compound as shown in Formula II can be conventional in the art, preferably 20-50 ml / g, for example 25 ml / g.

[0051] In method 4, the solvent D can be added in a conventional manner in the art, preferably by dripping.

[0052] In method 4, the volume ratio of solvent D to solvent C can be conventional in the art, preferably (7-15):1, for example 9:1.

[0053] In method 5, the temperature of the gas-liquid permeation experiment can be conventional in the art, preferably room temperature.

[0054] In method 5, the volume ratio of the antisolvent to the normal solvent can be conventional in the art, preferably (7-15):1, for example 8:1.

[0055] In Method 6, the temperature of the gas-solid permeation experiment can be conventional in the art, preferably room temperature.

[0056] In Method 6, the duration of the gas-solid permeation experiment can be conventional in the art, preferably 20-50 days, for example, 30 days.

[0057] In method 6, the volume-to-mass ratio of solvent E to the crystal form B of the polysubstituted benzene ring compound maleate as shown in formula II can be conventional in the art, preferably 100-400 ml / g, for example 200 ml / g.

[0058] In methods 3-5, the solution preparation method can refer to conventional preparation methods in the art, such as the preparation method of saturated solution. Preferably, the saturated solution is filtered, preferably by membrane filtration.

[0059] In methods 3-5, the crystallization can be a conventional method for such operations in the art.

[0060] For example, in method 3, the crystallization is preferably carried out by slow cooling, which is more preferably from 50°C to 5°C, and the slow cooling rate is 0.1-0.5°C / min, more preferably 0.1°C / min. If the solution is still clear, it is transferred to room temperature for evaporation.

[0061] For example, in method 4, preferably, a solid precipitates after the addition of solvent D; if no solid precipitates, the temperature is lowered to 5°C; if no solid still precipitates, the temperature is then increased to room temperature for evaporation or to vacuum drying at 50°C.

[0062] For example, in method 5, the crystallization method is preferably room temperature crystallization or vacuum drying at 50°C.

[0063] Methods 1-6 may further include the following post-processing steps: separation and drying. The separation method may be conventional in the art, preferably centrifugation or filtration. The drying method may be conventional in the art, preferably vacuum drying.

[0064] Preferably, the crystal form B of the maleate of the polysubstituted benzene ring compound as shown in Formula II is prepared by the following method: dissolving the polysubstituted benzene ring compound as shown in Formula I with maleic acid in ethanol, cooling to crystallize, and adding methyl tert-butyl ether.

[0065] In the method for preparing crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II, the molar ratio of the polysubstituted benzene ring compound as shown in Formula I to the maleic acid can be conventional in the art, preferably 1:(0.95-0.98), for example 1:0.978.

[0066] In the method for preparing crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II, the mass ratio of ethanol to the polysubstituted benzene ring compound as shown in Formula I can be conventional in the art, preferably (7-9):1, for example 6.9:1.

[0067] In the method for preparing crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II, the cooling crystallization temperature can be conventional in the art, preferably 15-25°C, for example 20°C.

[0068] In the method for preparing crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II, the mass ratio of the methyl tert-butyl ether to the polysubstituted benzene ring compound as shown in Formula I can be conventional in the art, preferably (12-15):1, for example 13:1.

[0069] The method for preparing the polysubstituted benzene ring compound maleate crystal form B as shown in Formula II may further include the following post-processing steps: filtration and drying, wherein the filtration can be performed under conventional conditions and procedures in the art, and preferably, the filter cake obtained after filtration is further washed with methyl tert-butyl ether; the drying can be performed under conventional conditions and procedures in the art.

[0070] The present invention also provides a pharmaceutical composition comprising crystal form A of the maleate of the polysubstituted benzene ring compound as shown in Formula II, and a pharmaceutically acceptable carrier.

[0071] The polysubstituted benzene ring compound maleate crystal form A as shown in Formula II of the present invention, or the above-described pharmaceutical composition, can be used to treat and / or prevent EZH2-mediated diseases. Preferably, the EZH2-mediated diseases include: cancer, pulmonary hypertension, myelofibrosis, human immunodeficiency virus (HIV) disease, graft-versus-host disease (GVHD), Weaver syndrome, psoriasis, and liver fibrosis. More preferably, the EZH2-mediated disease is cancer.

[0072] Preferably, the cancer includes metastatic or malignant tumors.

[0073] Preferably, the cancers include brain cancer, thyroid cancer, cardiac sarcoma, lung cancer, oral cancer, stomach cancer, liver cancer, kidney cancer, pancreatic cancer, esophageal cancer, nasopharyngeal cancer, laryngeal cancer, colorectal cancer, breast cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, melanoma, glioblastoma, lymphoma, leukemia, adrenocortical neuroblastoma, skin cancer, astrocytoma, etc.

[0074] In this invention, the crystal form A of the maleate of the polysubstituted benzene ring compound as shown in Formula II can also be used in combination with one or more other active ingredients; when used in combination, the active ingredients can be separate compositions for simultaneous or separate administration via the same or different routes of administration during treatment, or they can be administered together in the same pharmaceutical composition.

[0075] In this invention, there are no special restrictions on the method of administration of the pharmaceutical composition. Various dosage forms can be selected for administration according to the patient's age, gender, other conditions and symptoms. For example, tablets, pills, solutions, suspensions, emulsions, granules or capsules can be administered orally. Injections can be administered alone or mixed with injection delivery solutions (such as glucose solutions and amino acid solutions) for intravenous injection. Suppositories are administered rectally.

[0076] In some instances, the crystal form A of the maleate of the polysubstituted benzene ring compound as shown in Formula II does not undergo transformation when formulated with one or more pharmaceutically acceptable carriers and / or excipients and / or diluents.

[0077] In this invention, room temperature refers to 10-35℃.

[0078] In this invention, the water activity a of the solvent used is... w <0.2.

[0079] In this invention, "prevention" refers to "protection". "Prevention" means a reduction in the risk of acquiring or developing a disease or disorder (i.e., resulting in at least one clinical symptom of the disease not developing in a subject who may have been exposed to a disease-causing agent or a pre-existing predisposition to the disease).

[0080] In this invention, "treatment" means improving a disease or disorder (i.e., stopping the disease or reducing its manifestations, the degree or severity of its clinical symptoms); or improving at least one physical parameter that may not be perceived by the subject; or slowing the progression of the disease.

[0081] The crystal forms of this invention can be identified using one or more solid-state analysis methods, such as X-ray powder diffraction, single-crystal X-ray diffraction, infrared absorption spectroscopy, differential scanning calorimetry, and thermogravimetric analysis. Those skilled in the art know that the peak intensity and / or peak characteristics of X-ray powder diffraction may vary depending on experimental conditions. Furthermore, due to differences in instrument precision, the measured 2θ values ​​may have an error of approximately ±0.2°. The relative intensity of the peaks depends more on certain properties of the sample being measured, such as crystal size and purity, than on the peak position; therefore, the measured peak intensity may deviate by approximately ±20%. Despite experimental errors, instrumental errors, and orientation bias, those skilled in the art can still obtain sufficient information for identifying various crystal forms from the X-ray powder diffraction data provided in this patent. In infrared spectroscopy, differences in instrument performance, the degree of grinding during sample preparation, and the degree of water absorption can all affect the shape of the spectrum and the position of the absorption peaks to some extent. In DSC measurements, the initial temperature, maximum temperature, and heat of fusion of the endothermic peak obtained by actual measurement have a certain degree of variability, depending on the heating rate, crystal shape and purity, and other measurement parameters.

[0082] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0083] The reagents and raw materials used in this invention are all commercially available.

[0084] The positive and progressive effects of this invention are as follows: the method for preparing crystal form A of the polysubstituted benzene ring compound maleate as shown in Formula II provided by this invention is simple and has good physicochemical stability (no crystal form transformation or decrease in chemical purity occurred after being left open at 60°C for one day and at 25°C / 60%RH and 40°C / 75%RH for one week), good solubility (sourness reaches 6 mg / ml in water and SGF, and 4 mg / ml in FaSSIF and FeSSIF), and is not easily hygroscopic (moisture adsorption is 0.4% at 25°C / 80%RH, with almost no hygroscopicity), and good flowability (when D90 is controlled at 200 μm, the angle of repose of a single API is about 40°). As a raw material, it is more conducive to the development of formulation processes and has important value. Attached Figure Description

[0085] Figure 1 The image shows the X-ray powder diffraction pattern of crystal form A of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0086] Figure 2 Differential scanning calorimetry (DSC) of crystal form A of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0087] Figure 3 Thermogravimetric analysis (TGA) diagram of crystal form A of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0088] Figure 4 The image shows the X-ray powder diffraction pattern of crystal form B of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0089] Figure 5 The image shows the X-ray powder diffraction pattern of free crystal form A of the polysubstituted benzene ring compound as shown in Formula I.

[0090] Figure 6 The image shows the X-ray powder diffraction pattern of the free crystal form B of the polysubstituted benzene ring compound as shown in Formula I.

[0091] Figure 7 The image shows the X-ray powder diffraction pattern of crystal form C of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0092] Figure 8 The image shows the X-ray powder diffraction pattern of crystal form D of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0093] Figure 9 The image shows the X-ray powder diffraction pattern of crystal form E of the polysubstituted benzene ring compound maleate as shown in Formula II.

[0094] Figure 10The image shows the X-ray powder diffraction pattern of crystal form A of the polysubstituted benzene ring compound succinate as shown in Formula III.

[0095] Figure 11 The image shows the X-ray powder diffraction pattern of crystal form A of the polysubstituted benzene ring compound hydrobromide as shown in Figure IV.

[0096] Figure 12 The image shows the X-ray powder diffraction pattern of the free crystal form D of the polysubstituted benzene ring compound represented by Formula I.

[0097] Figure 13 The image shows the X-ray powder diffraction pattern of the free crystal form C of the polysubstituted benzene ring compound represented by Formula I.

[0098] Figure 14 The image shows the X-ray powder diffraction pattern of the free crystal form E of the polysubstituted benzene ring compound represented by Formula I.

[0099] Figure 15 The image shows the X-ray powder diffraction pattern of the free crystal form F of the polysubstituted benzene ring compound represented by Formula I.

[0100] Figure 16 The image shows the X-ray powder diffraction pattern of the free crystal form G of the polysubstituted benzene ring compound represented by Formula I.

[0101] Figure 17 The image shows the X-ray powder diffraction pattern of the free crystal form H of the polysubstituted benzene ring compound represented by Formula I.

[0102] Figure 18 The image shows the X-ray powder diffraction pattern of free crystal form I of the polysubstituted benzene ring compound represented by formula I. Detailed Implementation

[0103] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0104] The instruments used in the following embodiments are shown in Table 2:

[0105] Table 2

[0106]

[0107] Powder X-ray diffraction analysis (XRPD):

[0108] XRPD images were acquired using an Empyrean and PANalytical X'Pert3 powder diffractometer, and the scanning parameters are shown in Table 3.

[0109] Table 3

[0110]

[0111]

[0112] Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC):

[0113] TGA and DSC plots were acquired using a Discovery 5500 thermogravimetric analyzer and a Discovery 2500 differential scanning calorimeter, respectively. The parameters are shown in Table 4.

[0114] Table 4

[0115] parameter TGA DSC method linear heating linear heating Sample tray Aluminum tray, open Aluminum disc, pressure cap Temperature range Ambient temperature -350℃ 25℃-300℃ Scan rate (°C / min) 10 50 Protective gas Nitrogen Nitrogen

[0116] Dynamic moisture adsorption (DVS)

[0117] Dynamic moisture adsorption (DVS) curves were acquired using the DVSIntrinsic data acquisition system of SMS (Surface Measurement Systems). Relative humidity at 25°C was corrected for the deliquescence points of LiCl, Mg(NO3)2, and KCl. DVS test parameters are listed in Table 5.

[0118] Table 5

[0119] parameter Setting value temperature 25℃ Sample volume 10-20mg Protective gas and flow rate <![CDATA[N2,200mL / min]]> dm / dt 0.002% / min Minimum dm / dt equilibrium time 10min Maximum balancing time 180min RH range 0%RH-95%RH-0%RH RH gradient 10％

[0120] Hydrogen NMR (Liquid NMR) 1 H Solution NMR)

[0121] The proton NMR spectra were acquired on a Bruker 400M NMR spectrometer, using DMSO-d6 as the solvent.

[0122] Ultra-high performance liquid chromatography (UPLC)

[0123] The purity and solubility of the samples in the experiment were tested by Waters ultra-high performance liquid chromatography, under the conditions shown in Table 6:

[0124] Table 6

[0125]

[0126] Example 1: Preparation of crystal form A of the polysubstituted benzene ring compound as shown in Formula I (refer to WO2020 / 020374A1)

[0127] At room temperature, DIPEA (1.9 g, 15.1 mmol, 3 eq) was added to 10 mL of a DMF solution containing 3-aminomethyl-4-methyl-6-methylpyridin-2(1H)-one hydrochloride (1 g, 5.3 mmol, 1.05 eq), EDC·HCl (1.2 g, 6.6 mmol, 1.25 eq), HOBt·H2O (0.97 g, 6.6 mmol, 1.25 eq), and 3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methyl-5-(trans-3-(piperidin-1-yl)cyclobutoxy)benzoic acid (2.1 g, 5.0 mmol, 1 eq). After the reaction was complete, 80 mL of water was added, the mixture was filtered, and the filter cake was dried to obtain 1.7 g of the product. N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl-3-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-2-methyl-5-(trans-3-(piperidin-1-yl)cyclobutoxy)benzamide. m / z(ES+), [M+H] + =551.40; HPLC tR=6.901min. 1 H NMR (400MHz, DMSO-d6): δ11.45(s,1H),8.02(t,J=4.8Hz,1H),6.59(d,J=1.9Hz,1H ),6.38(d,J=2.0Hz,1H),5.85(s,1H),4.70-4.63(m,1H),4.25(d,J=4.0Hz,2H),3. 83(d,J=12Hz,2H),3.24(d,J=8Hz,2H),3.00-2.85(m,3H),2.32-2.23(m,10H),2.1 0(d,J=8Hz,8H),1.6-1.59(m,2H),1.50(m,6H),1.38(s,2H),0.78(t,J=8.0Hz,1H).

[0128] The sample obtained by X-ray powder diffraction pattern determination is the free crystal form A of a polysubstituted benzene ring compound as shown in Formula I. The X-ray powder diffraction pattern is as follows. Figure 5 As shown.

[0129] Example 2: Preparation of crystal form A of maleate of polysubstituted benzene ring compound as shown in Formula II

[0130] Weigh 397.4 mg of the polysubstituted benzene ring compound A as shown in Formula I obtained in Example 1 into a 20-mL glass vial; add 87.5 mg of maleic acid; add 7.0 mL of ethyl acetate to form a suspension, and stir magnetically at room temperature for about 3 days; filter to separate the obtained solid, and vacuum dry overnight at room temperature to obtain 443.8 mg of solid sample.

[0131] The sample obtained by X-ray powder diffraction analysis was crystal form A of maleate, a polysubstituted benzene ring compound as shown in Formula II. Its X-ray powder diffraction pattern, expressed at 2θ angle, and the diffraction peaks, relative intensities, interplanar spacing, peak heights, and left half-width (FWHM Left) are also shown in Table 7. The X-ray powder diffraction pattern is as follows: Figure 1 As shown.

[0132] Table 7

[0133] Diffraction peak number 2θ angle (°) Relative strength (%) d(A) Peak height (cts) Left half peak width [°2θ] 1 7.39 42.64 11.96 874.60 0.1023 2 8.51 12.59 10.39 258.19 0.1279 3 8.78 37.91 10.08 777.54 0.0768 4 11.79 4.22 7.51 86.66 0.0768 5 12.73 3.84 6.95 78.75 0.0768 6 13.99 56.77 6.33 1164.38 0.1279 7 15.21 100.00 5.83 2051.22 0.1023 8 15.73 88.27 5.63 1810.70 0.1023 9 16.06 17.68 5.52 362.71 0.0768 10 17.75 67.49 5.00 1384.39 0.1023 11 17.91 32.21 4.95 660.71 0.0512 12 18.20 25.80 4.88 529.20 0.1023 13 18.51 42.40 4.79 869.71 0.1535 14 19.13 1.87 4.64 38.29 0.2047 15 19.80 1.17 4.48 24.10 0.1535 16 20.68 24.70 4.29 506.60 0.1279 17 21.06 66.84 4.22 1371.13 0.1023 18 21.32 13.09 4.17 268.57 0.1023 19 22.11 9.65 4.02 197.99 0.1535 20 22.81 22.28 3.90 456.96 0.1023 21 23.07 5.24 3.86 107.47 0.1535 22 23.66 9.98 3.76 204.75 0.0768 23 23.84 8.90 3.73 182.56 0.1023 24 24.41 5.27 3.65 108.03 0.0768 25 24.68 12.31 3.61 252.44 0.1535 26 25.28 57.69 3.52 1183.41 0.1279 27 25.62 23.51 3.48 482.25 0.1279 28 26.23 10.60 3.40 217.40 0.1023

[0134] 29 26.68 16.79 3.34 344.30 0.1023 30 26.84 12.91 3.32 264.74 0.1023 31 28.06 20.35 3.18 417.33 0.0768 32 29.11 3.72 3.07 76.27 0.1023 33 29.41 10.37 3.04 212.76 0.1279 34 29.67 5.16 3.01 105.94 0.1023 35 29.99 16.33 2.98 334.96 0.0768 36 30.86 3.90 2.90 79.96 0.1279 37 32.44 1.66 2.76 34.05 0.6140 38 33.38 4.28 2.68 87.81 0.0768 39 34.46 1.45 2.60 29.72 0.1535 40 35.33 2.75 2.54 56.49 0.1535 41 36.50 2.36 2.46 48.41 0.1535 42 37.08 4.26 2.42 87.29 0.0768 43 38.33 4.65 2.35 95.32 0.1023

[0135] Figure 2 In the DSC spectrum, endothermic signals were observed at 185.3 °C and 204.4 °C in crystal form A of the polysubstituted benzene ring compound maleate as shown in Formula II.

[0136] Figure 3 In the TGA spectrum, the crystal form A of the polysubstituted benzene ring compound maleate, as shown in Formula II, loses 1.58% weight at 160 °C.

[0137] Crystal form A of maleate, a polysubstituted benzene ring compound as shown in Formula II. 1 The H NMR data are as follows: 1 H NMR (400MHz, DMSO-d6): δ11.45(s,1H),9.22(brs,1H),8.03(t,J=4.8Hz,1H),6.62(d,J=2.4Hz,1H),6. 42(d,J=2.4Hz,1H),6.02(s,2H),5.86(s,1H),4.80(t,J=6.4Hz,1H),4.25(d,J=4.8Hz,2H),3.83(d,J= 11.2Hz,3H),3.32–3.20(m,4H),2.99(dd,J=6.8,14.0Hz,2H),2.97–2.92(m,1H),2.70–2.67(m,4H),2. 40–2.38(m,2H),2.20(s,3H),2.11(s,3H),2.10(s,1H),1.81–1.46(m,10H),0.79(t,J=6.8Hz,3H)ppm.

[0138] The results showed that in the crystal form A of the maleate of the polysubstituted benzene ring compound as shown in Formula II, the molar ratio of the polysubstituted benzene ring compound as shown in Formula I to maleic acid was 1:1, the molar ratio of the free state of the polysubstituted benzene ring compound as shown in Formula I to ethyl acetate was 1:0.08, and the residual mass fraction of ethyl acetate was about 1.0%.

[0139] Example 3: Preparation of crystal form A of maleate of polysubstituted benzene ring compound as shown in Formula II

[0140] Compared to Example 2, the only difference is the type of solvent; in Example 2, ethyl acetate was replaced with methyl isobutyl ketone and 2-methyltetrahydrofuran, respectively.

[0141] The X-ray powder diffraction patterns of the samples obtained by this method were compared with those of the samples in Example 2, and it was determined that they were all crystal form A of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0142] Example 4: Preparation of crystal form B of maleate of polysubstituted benzene ring compound as shown in Formula II

[0143] 19.89 g of crystal form A of the polysubstituted benzene ring compound shown in Formula I was added to a reaction vessel, along with 137.4 g of anhydrous ethanol. The mixture was stirred and heated to 75°C, maintaining the internal temperature at 75°C. 4.0 g of maleic acid was added to the reaction vessel through a feeding funnel, and the mixture was stirred until dissolved. The internal temperature of the reaction vessel was lowered to 20°C, and the mixture was stirred until a large amount of solid precipitated. The internal temperature was maintained at 20°C, and 257.8 g of methyl tert-butyl ether was added. After the addition was complete, the mixture was stirred for 0.5–3 hours. The solution was placed in a filter press. After filtering the mother liquor, the filter cake was washed with 60.1 g of methyl tert-butyl ether, filtered dry, and a solid wet product was obtained. After drying, 21.16 g of solid sample was obtained.

[0144] The sample, as determined by X-ray powder diffraction, is crystal form B of maleate, a polysubstituted benzene ring compound as shown in Formula II. Its X-ray powder diffraction pattern, expressed at 2θ angle, is shown below. Figure 4 As shown.

[0145] Example 5: Preparation of crystal form A of maleate of polysubstituted benzene ring compound as shown in Formula II

[0146] Weigh approximately 20 mg of maleate crystal form B, the polysubstituted benzene ring compound shown in Formula II obtained in Example 4, into an HPLC vial. Add 0.5 mL of isopropanol. Place the resulting suspension at 50 °C and magnetically stir (-1000 rpm). After 6 days, centrifuge to obtain the sample.

[0147] The X-ray powder diffraction pattern of the sample obtained by this method was compared with the pattern of the sample in Example 2, and it was determined to be crystal form A of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0148] Example 6: Preparation of crystal form A of maleate of polysubstituted benzene ring compound as shown in Formula II

[0149] Compared to Example 5, the only difference is the type of solvent; the isopropanol in Example 5 is replaced with isopropanol / water (a) w=0.2), butanone, isopropyl acetate, anisole, acetonitrile, dichloromethane / cyclopentyl methyl ether (v:v=1:4), toluene, methyl isobutyl ketone, tetrahydrofuran, 1,4-dioxane, acetone, ethanol, methyl isobutyl ketone / isopropyl acetate (v:v=1:1), isopropanol / methyl tert-butyl ether (v:v=1:1) or acetonitrile / toluene (v:v=1:1).

[0150] The X-ray powder diffraction patterns of the samples obtained by this method were compared with those of the samples in Example 2, and it was determined that they were all crystal form A of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0151] Example 7: Preparation of crystal form A of maleate of polysubstituted benzene ring compound as shown in Formula II

[0152] Compared with Example 5, the only difference is the temperature. The 50℃ in Example 5 is replaced with a temperature cycle, that is, 50℃-5℃, 0.1℃ / min, 3 cycles.

[0153] The X-ray powder diffraction patterns of the samples obtained by this method were compared with those of the samples in Example 2, and it was determined that they were all crystal form A of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0154] Example 8: Preparation of crystal form A of maleate of polysubstituted benzene ring compound as shown in Formula II

[0155] Compared with Example 5, the difference lies in the temperature and reaction time. The 50°C in Example 5 is replaced with room temperature, and the reaction time of 6 days is replaced with 30 days.

[0156] The X-ray powder diffraction patterns of the samples obtained by this method were compared with those of the samples in Example 2, and it was determined that they were all crystal form A of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0157] Example 9: Preparation of crystal form A of maleate of polysubstituted benzene ring compound as shown in Formula II

[0158] Weigh 20 mg of maleate (crystal form B), the polysubstituted benzene ring compound of formula II obtained in Example 4, into a 3-mL vial. Add 0.5 mL of a mixed solution of dimethyl sulfoxide and ethyl acetate (v:v = 1:3). Stir at 50°C for approximately 2 hours, then filter (using a 0.45 μm PTFE membrane). Collect the filtrate and place it in a biochemical incubator. Cool the filtrate from 50°C to 5°C at a rate of 0.1°C / min. Transfer the sample to room temperature to allow it to evaporate. Filter the precipitated solid to obtain the final sample.

[0159] The X-ray powder diffraction pattern of the sample obtained by this method was compared with the pattern of the sample in Example 2, and it was determined to be crystal form A of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0160] Example 10: Preparation of crystal form A of maleate of polysubstituted benzene ring compound as shown in Formula II

[0161] Weigh out the crystal form B of the maleate salt of the polysubstituted benzene ring compound as shown in Formula II obtained in Example 4, and prepare nearly saturated stock solutions using the positive solvents in Table 8. Add 1 mL of the stock solution to a 20-mL vial, and add the corresponding antisolvents in Table 8 while stirring magnetically. Add dropwise while stirring until a solid precipitates. In Table 8, "*" indicates that no solid was obtained after adding approximately 9 mL of antisolvent; the solid was then obtained at 5°C. The specific positive and antisolvents are shown in Table 8.

[0162] Table 8

[0163]

[0164] The X-ray powder diffraction patterns of the samples obtained by this method were compared with those of the samples in Example 2, and it was determined that they were all crystal form A of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0165] Example 11: Preparation of crystal form A of maleate of polysubstituted benzene ring compound as shown in Formula II

[0166] Weigh out crystal form B of the maleate salt of the polysubstituted benzene ring compound shown in Formula II obtained in Example 4, and prepare a nearly saturated stock solution with methanol as the normal solvent. Add 0.5 mL of the stock solution to a 3-mL vial. Take another 20-mL vial and add about 4 mL of the antisolvent acetone to it. Place the 3-mL vial open over the 20-mL vial, then seal the 20-mL vial and let it stand at room temperature. When solid precipitation is observed, collect the solid and perform XRPD testing.

[0167] The X-ray powder diffraction pattern of the sample obtained by this method was compared with the pattern of the sample in Example 2, and it was determined to be crystal form A of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0168] Example 12: Preparation of crystal form A of maleate of polysubstituted benzene ring compound as shown in Formula II

[0169] Compared with Example 11, the only difference is the type of antisolvent. In Example 11, the antisolvent acetone is replaced with ethyl acetate or tetrahydrofuran.

[0170] The X-ray powder diffraction pattern of the sample obtained by this method was compared with the pattern of the sample in Example 2, and it was determined to be crystal form A of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0171] Comparative Example 1: Compared with Example 5, the only difference is the type of solvent. The isopropanol in Example 5 was replaced with the solvents shown in Table 9. "*" indicates that the mixture was stirred, clarified, and then evaporated at room temperature to obtain a solid.

[0172] Table 9

[0173] serial number Solvent (v:v) result 1 <![CDATA[IPA / H2O(a w =0.4)]]> <![CDATA[Polymorph C of maleate * > 2 <![CDATA[IPA / H2O(a w =0.6)]]> <![CDATA[Polymorph C of maleate * > 3 <![CDATA[IPA / H2O(a w =0.8)]]> <![CDATA[Polymorph C of maleate * > 4 <![CDATA[H2O]]> Maleate crystal form C

[0174] X-ray powder diffraction (XRD) analysis revealed that samples 1-4 all exhibited crystal form C of maleate, a polysubstituted benzene ring compound as shown in Formula II. The XRD patterns are shown below. Figure 7 As shown:

[0175] Comparative Example 2: Compared with Example 7, the only difference is the type of solvent, which is replaced with water.

[0176] The X-ray powder diffraction patterns of the samples obtained by this method were compared with those of the samples in Comparative Example 1, and it was determined that they were all crystal form C of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0177] Comparative Example 3: Compared with Example 8, the only difference is the type of solvent. The solvent in Example 8 was replaced with the solvents shown in the table below, where "*" indicates that a solid was obtained by evaporation at room temperature. The results are shown in Table 10:

[0178] Table 10

[0179] serial number Solvent (v:v) result 1 <![CDATA[Acetone / H2O(a w =0.4)]]> Maleate crystal form C 2 <![CDATA[Acetone / H2O(a w =0.6)]]> Maleate crystal form C 3 <![CDATA[Acetone / H2O(a w =0.8)]]> <![CDATA[Polymorph C of maleate * > 4 <![CDATA[H2O]]> Maleate crystal form C 5 <![CDATA[CHCl3 / n-Heptane(1:4)]]> Maleate crystal form B

[0180] X-ray powder diffraction (XPD) analysis revealed that sample number 5 was crystal form B of maleate, a polysubstituted benzene ring compound as shown in Formula II. XPD patterns of samples numbered 1-4 were compared with those of the sample in Comparative Example 1, confirming that they were all crystal form C of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0181] Comparative Example 4: Compared with Example 9, the only difference is the type of solvent. The solvent in Example 9, a mixture of dimethyl sulfoxide and ethyl acetate (v:v = 1:3), was replaced with the solvents shown in Table 11. "*" indicates that after cooling to 5°C, the solid precipitated directly. The results are shown in Table 11.

[0182] Table 11

[0183] serial number Solvent (v:v) result 1 <![CDATA[MeOH / H2O]]> <![CDATA[Polymorph C of maleate * > 2 EtOH / ACN Maleate crystal form C 3 NMP / THF (1:3) Maleate crystal form C 4 <![CDATA[CHCl3 / Acetone(1:2)]]> <![CDATA[Polymorph C of maleate * >

[0184] The X-ray powder diffraction patterns of samples No. 1-4 were compared with those of the sample in Comparative Example 1, and it was determined that they were all crystal form C of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0185] Comparative Example 5: Compared with Example 10, the only difference is the type of solvent. The positive solvent and the anti-solvent in Example 10 are replaced with the solvents shown in Table 12, where ** indicates that the solid is obtained by clarification at 5°C and evaporation at room temperature. # The solution was clarified at 5℃. Due to the high boiling point of the solvent, it was dried under vacuum at 50℃ to obtain a solid. The specific results are shown in Table 12.

[0186] Table 12

[0187]

[0188]

[0189] The X-ray powder diffraction patterns of samples No. 1-11 were compared with those of sample No. 1 in Comparative Example 1 and were determined to be crystal form C of maleate of polysubstituted benzene ring compound as shown in Formula II; the X-ray powder diffraction pattern of sample No. 12 was compared with that of sample No. 5 in Comparative Example 3 and was determined to be crystal form B of maleate of polysubstituted benzene ring compound as shown in Formula II.

[0190] Comparative Example 6: Compared with Example 11, the only difference is the type of solvent. The positive and negative solvents in Example 11 are replaced with the solvents shown in Table 13, where... * Note: Due to the high boiling point of the solvent, the solid was obtained by vacuum drying at 50℃. The results are shown in Table 13.

[0191] Table 13

[0192]

[0193] The X-ray powder diffraction patterns of samples No. 1-6 were compared with those of the sample in Comparative Example 1, and it was determined that they were all crystal form C of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0194] Comparative Example 7: The crystal form B of the maleate of the polysubstituted benzene ring compound as shown in Formula II obtained in Example 4 was weighed into a 3-mL vial. Approximately 4 mL of the solvent shown in the table below was added to a 20-mL vial. The 3-mL vial was placed open inside the 20-mL vial, and then the 20-mL vial was sealed. After being left at room temperature for 30 days, XRPD testing was performed, and the results are shown in Table 14.

[0195] Table 14

[0196] serial number solvent result 1 <![CDATA[H2O]]> Maleate crystal form C 2 DCM Maleate crystal form D

[0197] 3 EtOH Maleate crystal form B 4 THF Maleate crystal form B 5 IPAc Maleate crystal form B 6 EtOAc Maleate crystal form B

[0198] The X-ray powder diffraction pattern of sample No. 1 was compared with that of sample No. 1 in Comparative Example 1, and both were determined to be crystal form C of maleate, a polysubstituted benzene ring compound as shown in Formula II. X-ray powder diffraction analysis of sample No. 2 determined it to be crystal form D of maleate, a polysubstituted benzene ring compound as shown in Formula II. The X-ray powder diffraction pattern is shown below. Figure 8 As shown; the X-ray powder diffraction patterns of samples No. 3-6 were compared with those of sample No. 5 in Comparative Example 3, and it was determined that they were all crystal form B of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0199] Comparative Example 8: Approximately 20 mg of maleate (crystal form B) of the polysubstituted benzene ring compound (as shown in Formula II) obtained in Example 4 was weighed into a 3-mL vial. Approximately 4 mL of saturated salt solution (40-90% RH) was added to a 20-mL vial. The 3-mL vial was placed open inside the 20-mL vial, and then the 20-mL vial was sealed. After being left at room temperature for 30 days, XRPD testing was performed. The results are shown in Table 15.

[0200] Table 15

[0201] serial number solvent result 1 <![CDATA[K2CO3 saturated (-40% RH)]]> Maleate crystal form B 2 <![CDATA[Mg(NO3)2 saturated (-50% RH)]]> Maleate crystal form B 3 NaCl saturated solution (-70% RH) Maleate crystal form C 4 <![CDATA[KNO3 saturated solution (-90% RH)]]> Maleate crystal form C

[0202] The X-ray powder diffraction patterns of samples No. 1-2 were compared with those of sample No. 5 in Comparative Example 3, and it was determined that they were all crystal form B of maleate, a polysubstituted benzene ring compound as shown in Formula II; the X-ray powder diffraction patterns of samples No. 3-4 were compared with those of sample No. 1 in Comparative Example 1, and it was determined that they were all crystal form C of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0203] Comparative Example 9: 20 mg of maleate crystal form B of the polysubstituted benzene ring compound (as shown in Formula II) obtained in Example 3 was weighed into a 3-mL glass vial. 1.0-2.0 mL of the solvent listed in the table below was added to dissolve the solution. After filtration (using a PTFE membrane with a pore size of 0.45 μm), the filtrate was collected. The vial containing the clear solution was sealed with a sealing film, and several small holes were punched in the sealing film. The solution was then allowed to evaporate slowly at room temperature. When solid precipitated, the solid was collected and subjected to XRPD testing. The results are shown in Table 16.

[0204] Table 16

[0205] serial number Solvent (v:v) result 1 <![CDATA[CHCl3 / THF(2:1)]]> Maleate crystal form B 2 MeOH Maleate crystal form C 3 MeOH / EtOAc(2:1) Maleate crystal form C 4 <![CDATA[EtOH / H2O(4:1)]]> Maleate crystal form C 5 MeOH / ACN(2:1) Maleate crystal form C 6 DCM / Acetone (2:1) Maleate crystal form D 7 <![CDATA[CHCl3]]> Maleate crystal form D

[0206] 8 DCM Maleate crystal form D

[0207] The X-ray powder diffraction patterns of sample No. 1 were compared with those of sample No. 5 in Comparative Example 3, and were confirmed to be crystal form B of maleate, a polysubstituted benzene ring compound as shown in Formula II. The X-ray powder diffraction patterns of samples No. 2-5 were compared with those of sample No. 1 in Comparative Example 7, and were confirmed to be crystal form C of maleate, a polysubstituted benzene ring compound as shown in Formula II. The X-ray powder diffraction patterns of samples No. 6-8 were compared with those of sample No. 2 in Comparative Example 7, and were confirmed to be crystal form D of maleate, a polysubstituted benzene ring compound as shown in Formula II.

[0208] Comparative Example 10: The polysubstituted benzene ring compound maleate of Formula II obtained in Example 2, in crystal form A, was heated to 187°C under N2 protection. The sample obtained by X-ray powder diffraction was crystal form E of the polysubstituted benzene ring compound maleate of Formula II. The X-ray powder diffraction pattern is shown below. Figure 9 As shown.

[0209] Comparative Example 11: Preparation of Crystal Form A of Succinate of Polysubstituted Benzene Ring Compound as shown in Formula III

[0210]

[0211] Weigh 395.8 mg of crystal form A of the polysubstituted benzene ring compound (as shown in Formula I) obtained in Example 1 into a 20-mL glass vial; add 88.0 mg of succinic acid; add 7.0-mL of ethyl acetate to form a suspension, and stir magnetically at room temperature for about 3 days; filter to separate the obtained solid, and dry under vacuum at room temperature overnight to obtain 418.9 mg of solid. X-ray powder diffraction analysis determined that the sample was crystal form A of the polysubstituted benzene ring compound succinate (as shown in Formula III). The X-ray powder diffraction pattern is as follows. Figure 10 As shown, 1 The H NMR data are as follows: 1H NMR (400MHz, DMSO-d6): δ11.51(s,1H),8.10(t,J=5.2Hz,1H),6.65(d,J=2.4Hz,1H),6.44(d,J=2 .4Hz,1H),5.89(s,1H),4.89–4.84(m,1H),4.27(d,J=5.2Hz,2H),3.85(d,J=10.4Hz,2H),3.33(t, J=10.8Hz,2H),3.01(dd,J=6.8,13.6Hz,2H),2.93–2.89(m,1H),2.85–2.76(m,1H),2.42(s,4H)2 .42–2.35(m,4H),2.22(s,3H),2.13(d,J=4.0,6H),1.63–1.46(m,10H),0.81(t,J=7.2Hz,3H)ppm.

[0212] Comparative Example 12: Preparation of Crystal Form A of Hydrobromide of Polysubstituted Benzene Ring Compound as shown in Formula IV

[0213]

[0214] Weigh 400.7 mg of the polysubstituted benzene ring compound (crystal form A) obtained in Example 1 (as shown in Formula I) into a 20 mL glass vial, and add 3.5 mL of ethyl acetate and mix. Weigh 156.7 mg of hydrobromic acid and add 3.5 mL of ethyl acetate and mix. Mix the two ethyl acetate portions to form a suspension, and stir magnetically at room temperature for about 3 days. Centrifuge to separate the obtained solid, and vacuum dry overnight at room temperature to obtain 318.6 mg of solid. X-ray powder diffraction pattern analysis shows that the sample is the polysubstituted benzene ring compound (crystal form A) of hydrobromide (as shown in Formula IV). The X-ray powder diffraction pattern is as follows. Figure 11 As shown, 1 The H NMR data are as follows: 1H NMR (400MHz, DMSO-d6): δ11.51(s,1H),8.10(t,J=5.2Hz,1H),6.65(d,J=2.4Hz,1H),6.44(d,J =2.4Hz,1H),5.89(s,1H),4.89–4.84(m,1H),4.27(d,J=5.2Hz,2H),3.85(d,J=10.4Hz,2H),3. 33(t,J=10.8Hz,2H),3.01(dd,J=6.8,13.6Hz,2H),2.93–2.89(m,1H),2.85–2.76(m,4H),2.42 –2.35(m,2H),2.22(s,3H),2.13(d,J=4.0,6H),1.87–1.46(m,10H),0.81(t,J=7.2Hz,3H)ppm.

[0215] Comparative Example 13: Preparation of the free crystal form D of the polysubstituted benzene ring compound as shown in Formula I

[0216] 400.7 mg of crystal form A of the polysubstituted benzene ring compound as shown in Formula I obtained in Example 1 was weighed and stirred in 7 mL of isopropyl acetate at room temperature for 5 days. The resulting solid was separated by filtration and dried under vacuum at room temperature overnight to obtain 330.0 mg of solid. X-ray powder diffraction analysis determined that the sample was in the free crystal form D as shown in Formula I. The X-ray powder diffraction pattern is shown below. Figure 12 As shown, 1 The H NMR data are as follows: 1 H NMR (400MHz, DMSO-d6): δ11.48(s,1H),8.05(t,J=5.2Hz,1H),6.59(d,J=2.4Hz,1H),6.38(d,J= 2.4Hz,1H),5.68(s,1H),4.68–4.68(m,1H),4.24(d,J=5.2Hz,2H),3.82(d,J=10.4Hz,2H),3.23 (t,J=10.8Hz,2H),2.98(dd,J=6.8,13.6Hz,2H),2.93–2.92(m,1H),2.91–2.90(m,1H),2.29–2. 26(m,8H),2.19(s,3H),2.11(s,3H),2.10(s,3H),1.61–1.39(m,10H),0.79(t,J=7.2Hz,3H)ppm.

[0217] Comparative Example 14: Screening of free polymorphs of polysubstituted benzene ring compounds as shown in Formula I

[0218] Compared with Comparative Example 12, the only difference is the solvent and temperature. Isopropyl acetate and room temperature in the Comparative Example were replaced with the solvent and temperature in Table 17, respectively. The results are shown in Table 17:

[0219] Table 17

[0220]

[0221]

[0222] The samples obtained by X-ray powder diffraction pattern determination are the free crystal forms A, B, C, E, F, G, and I of the polysubstituted benzene ring compound as shown in Formula I. The X-ray powder diffraction patterns of the obtained samples are as follows: Figure 5-6 , Figure 13-16 and Figure 18 As shown.

[0223] Comparative Example 15: Screening of free polymorphs of polysubstituted benzene ring compounds as shown in Formula I

[0224] Compared to Comparative Example 13, No. 5, the only difference is that the free crystal form G of the polysubstituted benzene ring compound as shown in Formula I obtained in Example 13, No. 5, is heated to 160°C to obtain the free crystal form H of the polysubstituted benzene ring compound as shown in Formula I. The X-ray powder diffraction pattern of the obtained sample is shown below. Figure 17 As shown.

[0225] Example 1: Polymorph screening of maleate salts of multi-substituted benzene ring compounds as shown in Formula II

[0226] As described in Examples 5-12 and Comparative Examples 1-10, a total of 5 crystal forms were found during the screening and crystal form identification process, which were named crystal forms A, B, C, D and E of maleate, respectively. Among them, A, B and E are amorphous forms, and C and D are hydrate crystal forms. Among them, crystal form E of maleate was only obtained at high temperature and transformed back into crystal form A of maleate when cooled to room temperature.

[0227] To further explore the interconversion relationships between different crystal forms and screen out more stable crystal forms, suspension competition experiments were conducted on the amorphous forms A and B of maleate, and suspension competition experiments with different water activities were conducted on the amorphous form A of the dominant maleate and the hydrate crystal forms C and D of maleate.

[0228] Competition experiment of maleate crystal form A and B suspensions: Four 5-ml vials were taken, and 2ml each of IPA and IPAc were added to each. Excess maleate crystal form B was added, and the solutions were stirred at room temperature / 50℃ for 24 hours, then filtered to obtain saturated solutions of maleate IPA and IPAc. Approximately 10mg each of maleate crystal form A and B were added to each saturated solution, and the solutions were suspended and stirred at room temperature / 50℃ for 3 days before XRPD testing. The results are shown in Table 18.

[0229] Table 18

[0230]

[0231] The results showed that all systems yielded maleate crystal form A, which was more stable than crystal form B at room temperature / 50°C.

[0232] Suspension competition experiment of maleate amorphous form A with maleate hydrate crystal forms C and D: Take 6 5-ml vials and add 2ml of different water activities (a) to each vial. w To obtain saturated solutions, an excess of maleate crystal form B was added to an ethanol solution containing (0 / 0.2 / 0.4 / 0.6 / 0.8 / 1) and stirred at room temperature for 24 hours. The solution was then filtered. Approximately 10 mg of maleate crystal forms A / C / D was added to each saturated solution, and the solution was suspended and stirred at room temperature for 2 days before XRPD testing. Alternatively, two 5-ml vials were prepared, each containing 2 ml of water, followed by an excess of maleate crystal form B. The vials were then stirred at 50℃ / 80℃ for 20 hours / 2 hours, respectively, and filtered to obtain saturated solutions. Approximately 10 mg of maleate crystal forms A / C / D was added to each saturated solution, and the solution was suspended and stirred at 50℃ / 80℃ for 1 day / 3.5 hours before XRPD testing. The results are shown in Table 19.

[0233] Table 19

[0234]

[0235] The results showed that maleate crystal form A was obtained when the water activity was less than or equal to 0.2, and maleate crystal form C was obtained when the water activity was greater than or equal to 0.4 and when it was in water at 50℃ / 80℃. Both maleate crystal form C and maleate crystal form D were converted to crystal form A when suspended and stirred in an ethanol solution with a water activity of 0.2 or anhydrous ethanol at room temperature.

[0236] Based on the above examples and suspension competition experiments, it was found that among the five crystal forms of maleate, crystal form E is obtained only at high temperatures and reverts to crystal form A upon cooling to room temperature. Crystal forms A and B have similar solubilities, but crystal form A has better solid-state properties, being a granular solid (while crystal form B is a powdery solid). Furthermore, crystal form A is more stable than crystal form B, and crystal form B will revert to crystal form A under various solvents and conditions. Crystal forms C and D readily revert to crystal form A, and crystal form C, being a hydrate, reverts to crystal form A after drying at 170°C. Therefore, among the five crystal forms of maleate, crystal form A is the most stable.

[0237] Example 2: Physicochemical Stability

[0238] Appropriate amounts of maleate salt (crystal form A) of the polysubstituted benzene ring compound shown in Formula II were weighed and left exposed at 60°C for one day, and at 25°C / 60%RH and 40°C / 75%RH for one week. The solid samples after different conditions were tested for purity by HPLC to assess chemical stability, and for crystal form by XRPD to assess physical stability. The evaluation results are summarized in Table 20.

[0239] Table 20

[0240]

[0241] The results showed that under the three test conditions, the polysubstituted benzene ring compound maleate, as shown in Formula II, did not undergo crystal transformation or decrease in chemical purity, indicating that it has good physical and chemical stability.

[0242] Example 3: Hygroscopicity

[0243] Through salt form screening experiments, multiple salt forms of the polysubstituted benzene ring compound shown in Formula I were obtained. Considering crystallinity, TGA weight loss, DSC thermal signal, and salt molar ratio, crystal form A of maleate (as shown in Formula II), succinate (as shown in Formula III), and hydrobromide (as shown in Formula IV) were selected. The following effect experiments were then conducted:

[0244] Approximately 10 mg each of the following polysubstituted benzene ring compounds were taken: maleate (as shown in Formula II), succinate (as shown in Formula III), hydrobromide (as shown in Formula IV), and free form D of the polysubstituted benzene ring compound (as shown in Formula I). ​​The samples were dried at 25°C and 0% RH. After the rate of mass change over time was less than 0.002%, the hygroscopic characteristics of the samples were tested when the humidity changed from 0% RH to 95% RH, and the dehumidification characteristics were tested when the humidity changed from 0% RH to 95% RH and back to 0% RH. The humidity change step was 10% RH. The balance was considered to be in equilibrium when the mass change rate dm / dt was less than 0.002%. The equilibrium standard during the testing process was a mass change rate less than 0.002% / minute within 10 minutes. The maximum equilibrium time was 3 hours. The isothermal adsorption / desorption water characteristics under these test conditions were determined, and the samples after DVS testing were subjected to XRPD detection. The results are shown in Table 21.

[0245] Table 21

[0246]

[0247] The results showed that among the four crystal forms, maleate crystal form A had the lowest hygroscopicity, with a water adsorption of 0.4% at 25℃ / 80%RH, indicating almost no hygroscopicity, and the crystal form did not change after DVS testing.

[0248] Example 4: Dynamic Solubility

[0249] The dynamic solubility of four samples—maleate (as shown in Formula II), succinate (as shown in Formula III), hydrobromide (as shown in Formula IV), and free crystalline form D of a polysubstituted benzene ring compound (as shown in Formula I)—was tested in three biological solvents (SGF, FaSSIF, and FeSSIF) and water at 37°C. In the experiment, 45 mg of the solid was mixed with 4.5 mL of solvent in 5 mL centrifuge tubes, which were then sealed and fixed on a rotating disk at 40 rpm. The rotating disk was placed in a 37°C incubator. Samples were taken at four time points: 1, 2, 4, and 24 hours after equilibration. At each sampling point, the concentration and pH of the filtrate were measured, and the crystalline form of the solid was determined.

[0250] The results showed that in water, succinate crystal form A was highly soluble (>8.44 mg / mL), maleate crystal form A and hydrobromide crystal form A had similar solubilities (6.99 mg / mL and 4.26 mg / mL, respectively), while the free crystal form D had low solubility (0.016 mg / mL).

[0251] In SGF, all three salt forms are soluble (>7.83 mg / mL), while the free crystal form D has relatively low solubility (6.98 mg / mL).

[0252] In FaSSIF, the solubility of succinate crystal form A and maleate crystal form A is relatively high (5.39 mg / mL and 4.15 mg / mL), while the solubility of hydrobromide crystal form A and free crystal form D is relatively low (1.39 mg / mL and 0.42 mg / mL).

[0253] In FeSSIF, succinate crystal form A, maleate crystal form A and free crystal form D are highly soluble (>7.35 mg / mL), while hydrobromide crystal form A has relatively low solubility (5.33 mg / mL).

[0254] In summary, the results show that, in terms of solubility in the four media, maleate crystal form A and succinate crystal form A have relatively better solubility. Both achieve a solubility of 6 mg / ml in water and SGF, meeting the dissolution sink conditions for formulations up to 600 mg, and exhibiting complete in vitro release. In FaSSIF and FeSSIF, the solubility reaches 4 mg / ml. However, considering the overall hygroscopicity, succinate crystal form A has higher hygroscopicity, which is unfavorable for subsequent formulation processes. Maleate crystal form A, on the other hand, has lower hygroscopicity. When the D90 is controlled at 200 μm, the API exhibits excellent flowability, with a single API angle of repose around 40°. Dry-mix direct compression processes can be considered for both forms, making it more suitable as an active pharmaceutical ingredient for formulation development.

Claims

1. A crystal form A of a maleate salt of a polysubstituted benzene ring compound as shown in Formula II, characterized in that, The X-ray powder diffraction pattern, expressed in terms of 2θ angle, shows diffraction peaks at 7.39±0.2°, 8.78±0.2°, 13.99±0.2°, 15.21±0.2°, 15.73±0.2°, 17.75±0.2°, 18.51±0.2°, and 21.06±0.2°. 。 2. The crystal form A of the maleate salt of the polysubstituted benzene ring compound as shown in Formula II as described in claim 1, characterized in that, The X-ray powder diffraction pattern, expressed in terms of 2θ angle, also shows diffraction peaks at one or more of the following 2θ angles: 8.51±0.2° and 16.06±0.2°; And / or, the differential scanning calorimetry (DSC) plot of maleate of the polysubstituted benzene ring compound as shown in Formula II shows major endothermic peaks at 185.3℃±3℃ and 204.4℃±3℃. And / or, the thermogravimetric analysis diagram of crystal form A of the polysubstituted benzene ring compound maleate as shown in Formula II is basically as shown in Figure 3.

3. The crystal form A of the maleate salt of the polysubstituted benzene ring compound as shown in Formula II as described in claim 1, characterized in that, The X-ray powder diffraction pattern, expressed in terms of 2θ angle, also shows diffraction peaks at one or more of the following 2θ angles: 18.20±0.2° and 20.68±0.2°; And / or, the DSC of crystal form A of the polysubstituted benzene ring compound maleate as shown in Formula II is essentially as shown in Figure 2.

4. The crystal form A of the maleate salt of the polysubstituted benzene ring compound as shown in Formula II as described in claim 1, characterized in that, The X-ray powder diffraction pattern, expressed in terms of 2θ angle, also shows diffraction peaks at one or more of the following 2θ angles: 22.81±0.2° and 25.28±0.2°.

5. The crystal form A of the maleate salt of the polysubstituted benzene ring compound as shown in Formula II according to any one of claims 1-4, characterized in that, The X-ray powder diffraction pattern expressed in terms of 2θ angle is basically as shown in Figure 1.

6. The method for preparing crystal form A of the maleate salt of the polysubstituted benzene ring compound as shown in Formula II according to any one of claims 1-5, characterized in that, The preparation method is one of the following methods: method 1, method 2, method 3, method 4, method 5, or method 6: Method 1: In solvent A, react the polysubstituted benzene ring compound as shown in Formula I with maleic acid; Solvent A is selected from one of methyl isobutyl ketone, 2-methyltetrahydrofuran, and ethyl acetate; Method 2: Form a suspension solution of the polysubstituted benzene ring compound maleate crystal form B as shown in Formula II in solvent B, and stir. Solvent B is selected from isopropanol, isopropanol / water, butanone, isopropyl acetate, anisole, acetonitrile, dichloromethane / cyclopentyl methyl ether, toluene, methyl isobutyl ketone, tetrahydrofuran, 1,4-dioxane, acetone, ethanol, methyl isobutyl ketone / isopropyl acetate, isopropanol / methyl tert-butyl ether, or acetonitrile / toluene; wherein, in the isopropanol / water, a w It is 0.2; Method 3: Prepare a saturated solution of maleate salt of polysubstituted benzene ring compound as shown in Formula II in a mixed solvent of dimethyl sulfoxide and ethyl acetate, and allow it to crystallize. Method 4: Dissolve the crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II in solvent C, add solvent D, and crystallize. The solvent C is selected from one or more of methanol, dimethyl sulfoxide, and chloroform. When the solvent C is methanol, the solvent D is ethyl acetate; when the solvent C is dimethyl sulfoxide, the solvent D is isopropyl acetate; when the solvent C is chloroform, the solvent D is one or more of ethyl acetate, acetonitrile, and butanone. Method 5: Perform a gas-liquid permeation crystallization experiment on the crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II; The positive solvent is methanol, and the antisolvent is selected from acetone, ethyl acetate, and tetrahydrofuran. Method 6: Place the crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II in the environment of solvent E and perform a gas-solid permeation experiment. The solvent E is one of methanol, acetonitrile, acetone, dimethyl sulfoxide, isopropanol, and dimethylformamide; In methods 2-6, the X-ray powder diffraction pattern of the crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II, expressed at an angle of 2θ, is essentially as shown in Figure 4.

7. The method for preparing crystal form A of the polysubstituted benzene ring compound maleate as shown in Formula II as described in claim 6, characterized in that, In Method 1, the reaction temperature is room temperature; And / or, in method 1, the reaction time is 2-4 days; And / or, in method 1, the molar ratio of the polysubstituted benzene ring compound as shown in formula I to the maleic acid is 1:(1-1.2). And / or, in method 1, the volume-to-mass ratio of solvent A to the polysubstituted benzene ring compound as shown in formula I is 15-40 ml / g; And / or, in method 2, the stirring is magnetic stirring; And / or, in method 2, the stirring temperature is 0-50°C; And / or, in method 2, the volume-to-mass ratio of solvent B to the crystal form B of the polysubstituted benzene ring compound maleate as shown in formula II is 20-50 ml / g; And / or, in method 3, the volume ratio of dimethyl sulfoxide to ethyl acetate in the mixed solvent is 1:(2-5); And / or, in method 3, the volume-to-mass ratio of the mixed solvent to the crystal form B of the polysubstituted benzene ring compound maleate as shown in formula II is 20-50 ml / g; And / or, in method 4, the solvent D is added dropwise; And / or, in method 4, the volume ratio of solvent D to solvent C is (7-15):1; And / or, in method 5, the temperature of the gas-liquid permeation crystallization experiment is room temperature; And / or, in method 5, the volume ratio of the antisolvent to the normal solvent is (7-15):1; And / or, in method 6, the temperature of the gas-solid permeation experiment is room temperature; And / or, in method 6, the gas-solid permeation experiment lasts for 20-50 days; And / or, in method 6, the volume-to-mass ratio of solvent E to the crystal form B of the polysubstituted benzene ring compound maleate as shown in formula II is 100-400 ml / g; And / or, in method 3, the crystallization is carried out by slow cooling; And / or, in method 4, if a solid precipitates after adding solvent D, and no solid precipitates, the temperature is lowered to 5°C. If no solid still precipitates, the temperature is then increased to room temperature for evaporation or vacuum dried at 50°C. And / or, in method 5, the crystallization method is room temperature crystallization or vacuum drying at 50°C; And / or, methods 1-6 include the following post-processing steps: separation and drying, wherein the separation is performed by centrifugation or filtration, and the drying is performed by vacuum drying.

8. The method for preparing crystal form A of the polysubstituted benzene ring compound maleate as shown in Formula II according to claim 7, characterized in that, In Method 1, the molar ratio of the polysubstituted benzene ring compound as shown in Formula I to the maleic acid is 1:1.07; And / or, in method 1, the volume-to-mass ratio of solvent A to the polysubstituted benzene ring compound as shown in formula I is 17.6 ml / g; And / or, in method 2, the stirring temperature is room temperature, 50°C, or temperature cycling is performed at 50°C-5°C and 0.1°C / min. When the stirring temperature is 50°C, the stirring time is 5-8 days; when the stirring temperature is room temperature, the stirring time is 25-35 days; when the stirring temperature is temperature cycling, the temperature cycling conditions are 50°C-5°C, 0.1°C / min, 2-4 cycles. And / or, in method 2, the volume-to-mass ratio of solvent B to the crystal form B of the polysubstituted benzene ring compound maleate as shown in formula II is 25 ml / g; And / or, in method 3, the volume ratio of dimethyl sulfoxide to ethyl acetate in the mixed solvent is 1:3; And / or, in method 3, the volume-to-mass ratio of the mixed solvent to the crystal form B of the polysubstituted benzene ring compound maleate as shown in formula II is 25 ml / g; And / or, in method 4, the volume ratio of solvent D to solvent C is 9:1; And / or, in method 5, the volume ratio of the antisolvent to the normal solvent is 8:1; And / or, in method 6, the gas-solid permeation experiment lasts for 30 days; And / or, in method 6, the volume-to-mass ratio of solvent E to the crystal form B of the polysubstituted benzene ring compound maleate as shown in formula II is 200 ml / g; And / or, in method 3, the saturated solution is filtered; And / or, in method 3, the slow cooling method is to cool from 50°C to 5°C, and the slow cooling rate is 0.1-0.5°C / min.

9. The method for preparing crystal form A of the polysubstituted benzene ring compound maleate as shown in Formula II according to claim 7, characterized in that, In method 3, the saturated solution is filtered through a filter membrane; And / or, in method 3, the slow cooling rate is 0.1°C / min, and if the solution remains clear, it is transferred to room temperature for evaporation.

10. The method for preparing crystal form A of the maleate salt of the polysubstituted benzene ring compound as shown in Formula II according to any one of claims 6-9, characterized in that, The method for preparing crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II includes the following steps: dissolving the polysubstituted benzene ring compound as shown in Formula I with maleic acid in ethanol, cooling to crystallize, and adding methyl tert-butyl ether.

11. The method for preparing crystal form A of the maleate salt of the polysubstituted benzene ring compound as shown in Formula II as described in claim 10, characterized in that, In the method for preparing crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II, the molar ratio of the polysubstituted benzene ring compound as shown in Formula I to the maleic acid is 1:(0.95-0.98). And / or, in the method for preparing crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II, the mass ratio of ethanol to the polysubstituted benzene ring compound as shown in Formula I is (7-9):1; And / or, in the method for preparing crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II, the cooling crystallization temperature is 15-25°C; And / or, in the method for preparing crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II, the mass ratio of the methyl tert-butyl ether to the polysubstituted benzene ring compound as shown in Formula I is (12-15):1; And / or, the method for preparing crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II includes the following post-processing steps: filtration and drying.

12. The method for preparing crystal form A of the maleate salt of the polysubstituted benzene ring compound as shown in Formula II as described in claim 11, characterized in that, In the method for preparing crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II, the molar ratio of the polysubstituted benzene ring compound as shown in Formula I to the maleic acid is 1:0.

978. And / or, in the method for preparing crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II, the mass ratio of ethanol to the polysubstituted benzene ring compound as shown in Formula I is 6.9:1; And / or, in the method for preparing crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II, the cooling crystallization temperature is 20°C; And / or, in the method for preparing crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II, the mass ratio of the methyl tert-butyl ether to the polysubstituted benzene ring compound as shown in Formula I is 13:1; And / or, the method for preparing crystal form B of the polysubstituted benzene ring compound maleate as shown in Formula II includes the following post-processing steps: the filter cake obtained after filtration is further washed with methyl tert-butyl ether.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises crystal form A of a maleate salt of a polysubstituted benzene ring compound as shown in Formula II as described in any one of claims 1-5, and a pharmaceutically acceptable carrier.

14. Use of the polysubstituted benzene ring compound maleate of formula II as described in any one of claims 1-5, or the pharmaceutical composition of claim 13, in the preparation of a medicament for the prevention or treatment of EZH2-mediated diseases.

15. The use as described in claim 14, characterized in that, The EZH2-mediated disease is cancer.

16. The use as described in claim 15, characterized in that, The cancers mentioned include brain cancer, thyroid cancer, cardiac sarcoma, lung cancer, oral cancer, stomach cancer, liver cancer, kidney cancer, pancreatic cancer, esophageal cancer, nasopharyngeal cancer, laryngeal cancer, colorectal cancer, breast cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, lymphoma, leukemia, adrenal neuroblastoma, or skin cancer.

17. The use as described in claim 15, characterized in that, The cancer in question is melanoma, glioblastoma, or astrocytoma.

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