A crystal form of a cyano-substituted polypeptide compound and a preparation method thereof

Abstract

The present invention discloses a crystal form of a polypeptide compound containing cyano substitution and a preparation method thereof, specifically discloses a crystal form of a compound of formula (I) and a preparation method thereof.

Description

[0001] This application claims the priority of Chinese Patent Application No. 2022112639720 filed on October 14, 2022. This application cites the entire text of the above Chinese Patent Application. Technical Field

[0002] The present invention relates to a crystal form of a cyano-substituted polypeptide compound and a preparation method thereof, and in particular to a crystal form of a compound of formula (I) and a preparation method thereof. Background Art

[0003] In December 2019, the novel coronavirus disease 2019 (CoVID-19) broke out and quickly evolved into a pandemic with significant impacts on global public health systems and the global economy. The pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has a very high RNA genome (~80%) similarity to SARS-CoV-1, which caused the SARS outbreak in 2003. In response to the prevalence and severity of SARS-CoV-2 infection, many countries and institutions have made great efforts to develop prevention and treatment methods, such as the development and large-scale vaccination of vaccines, as well as the development and application of some therapeutic drugs.

[0004] Coronavirus is an enveloped, single-stranded, positive-sense RNA virus that encodes structural and nonstructural proteins to facilitate viral entry and replication in the host. In the viral replication cycle, the nonstructural protein 3CL (3-chymotrypsin-like protease) protease plays a vital role, and its main function is to hydrolyze the two polyproteins expressed by the virus. Sequence analysis shows that 3CL protease may become one of the key targets for drug design.

[0005] The 3CL proteases currently on the market include Pfizer's Namatevir tablets, which reduce hospitalization or mortality by 89% and 70% in both high-risk and low-risk populations, respectively. They have been approved for marketing or emergency authorization use in the United States and many other countries. There are currently many 3CL proteases in various stages of clinical trials at home and abroad. Therefore, in order to treat 2019 novel coronavirus or other coronavirus infections, the development of 3CL protease inhibitors is particularly important. Summary of the invention

[0006] The present invention provides a crystalline form A of a compound of formula (I),

[0007]

[0008] The invention is characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 10.655±0.200°, 11.988±0.200°, 16.055±0.200°, 18.356±0.200°, 20.083±0.200°.

[0009] In some embodiments of the present invention, the X-ray powder diffraction pattern of the crystal form A of the compound of formula (I) above, expressed by 2θ angle, comprises at least 6, 7, or 8 diffraction peaks selected from the following: 6.917±0.200°, 10.655±0.200°, 11.988±0.200°, 14.481±0.200°, 16.055±0.200°, 17.653±0.200°, 18.356±0.200°, 20.083±0.200°.

[0010] In some embodiments of the present invention, the A form of the compound of formula (I) has an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2θ angles: 6.917±0.200°, 10.655±0.200°, 11.988±0.200°, 14.481±0.200°, 16.055±0.200°, 17.653±0.200°, 18.356±0.200°, 20.083±0.200°.

[0011] In some embodiments of the present invention, the A crystal form of the compound of formula (I) has an X-ray powder diffraction pattern, represented by 2θ angle, comprising at least 12, 13, 14, 15, or 16 diffraction peaks selected from the following: 6.917±0.200°, 10.655±0.200°, 11.988±0.200°, 13.837±0.200°, 14.481±0.200°, 16.05 5±0.200°, 17.653±0.200°, 18.356±0.200°, 20.083±0.200°, 20.839±0.200°, 21.388±0.200°, 22.379±0.200°, 24.577±0.200°, 25.104±0.200°, 26.402±0.200°, 31.540±0.200°.

[0012] In some embodiments of the present invention, the X-ray powder diffraction pattern of the A crystal form of the compound of formula (I) has characteristic diffraction peaks at the following 2θ angles: 6.917±0.200°, 10.655±0.200°, 11.988±0.200°, 13.837±0.200°, 14.481±0.200°, 16.055±0.200°, 17.6 53±0.200°, 18.356±0.200°, 20.083±0.200°, 20.839±0.200°, 21.388±0.200°, 22.379±0.200°, 24.577±0.200°, 25.104±0.200°, 26.402±0.200°, 31.540±0.200°.

[0013] In some embodiments of the present invention, the crystal form A of the compound of formula (I) has an X-ray powder diffraction pattern as shown in Figure 1 shown.

[0014] In some embodiments of the present invention, the peak position, interplanar spacing and relative intensity of the diffraction peaks in the X-ray powder diffraction (XRPD) spectrum of the above-mentioned crystal form A of the compound of formula (I) are shown in Table 1:

[0015] Table 1 Peak positions, interplanar spacings and relative intensities of diffraction peaks in the XRPD spectrum of Form A

[0016]

[0017] In some embodiments of the present invention, the differential scanning calorimetry (DSC) curve of the above-mentioned crystalline form A of the compound of formula (I) has an endothermic peak at 173.5°C±3°C.

[0018] In some embodiments of the present invention, the above-mentioned compound of formula (I) A crystal form has a differential scanning calorimetry curve as shown in Figure 2 shown.

[0019] In some embodiments of the present invention, the thermogravimetric analysis curve (TGA) of the above-mentioned crystalline form A of the compound of formula (I) shows a weight loss of 0.00% at 150.0°C±3°C.

[0020] In some embodiments of the present invention, the above-mentioned compound of formula (I) A crystal form has a thermogravimetric analysis curve as shown in Figure 3 shown.

[0021] The present invention also provides a method for preparing the above-mentioned crystal form A of the compound of formula (I), comprising the step of stirring the compound of formula (I) in water, and further comprising the step of separation.

[0022] In some embodiments of the present invention, the crystal form A of the compound of formula (I) of the present invention is prepared by the following method:

[0023] (1) adding water to the compound of formula (I) and stirring;

[0024] (2) Filter and concentrate under reduced pressure to remove water.

[0025] In some embodiments of the present invention, the stirring in step (1) is performed under heating conditions; preferably, the stirring temperature is 50°C.

[0026] The present invention also provides a crystal form B of the compound of formula (I),

[0027]

[0028] The invention is characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 9.202±0.200°, 13.394±0.200°, 14.212±0.200°, 15.516±0.200°, 19.917±0.200°.

[0029] In some embodiments of the present invention, the X-ray powder diffraction pattern of the B form of the compound of formula (I) above, expressed by 2θ angle, comprises at least 6, 7, or 8 diffraction peaks selected from the following: 9.202±0.200°, 10.968±0.200°, 13.394±0.200°, 14.212±0.200°, 15.516±0.200°, 16.483±0.200°, 18.306±0.200°, and 19.917±0.200°.

[0030] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystalline form B of compound of formula (I) has characteristic diffraction peaks at the following 2θ angles: 9.202±0.200°, 10.968±0.200°, 13.394±0.200°, 14.212±0.200°, 15.516±0.200°, 16.483±0.200°, 18.306±0.200°, 19.917±0.200°.

[0031] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystalline form B of the compound of formula (I) comprises at least 12, 13, 14, 15, or 16 diffraction peaks selected from the following, expressed as 2θ angles: 5.459±0.200°, 7.096±0.200°, 9.202±0.200°, 10.968±0.200°, 13.394±0.200°, 14.212 ±0.200°, 15.516±0.200°, 16.483±0.200°, 18.306±0.200°, 19.488±0.200°, 19.917±0.200°, 20.681±0.200°, 21.526±0.200°, 22.409±0.200°, 24.622±0.200°, 25.661±0.200°.

[0032] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form B of the compound of formula (I) has characteristic diffraction peaks at the following 2θ angles: 5.459±0.200°, 7.096±0.200°, 9.202±0.200°, 10.968±0.200°, 13.394±0.200°, 14.212±0.200°, 15.51 6±0.200°, 16.483±0.200°, 18.306±0.200°, 19.488±0.200°, 19.917±0.200°, 20.681±0.200°, 21.526±0.200°, 22.409±0.200°, 24.622±0.200°, 25.661±0.200°.

[0033] In some embodiments of the present invention, the B crystal form of the compound of formula (I) has an X-ray powder diffraction pattern (XRPD) as shown in Figure 4 shown.

[0034] In some embodiments of the present invention, the peak position, interplanar spacing and relative intensity of the diffraction peaks in the X-ray powder diffraction pattern of the above-mentioned crystal form B of the compound of formula (I) are shown in Table 2:

[0035] Table 2 Peak positions, interplanar spacings and relative intensities of diffraction peaks in the XRPD spectrum of Form B

[0036]

[0037] In some embodiments of the present invention, the crystalline form B of the compound of formula (I) has a differential scanning calorimetry (DSC) curve with an endothermic peak at 128.4°C±3°C.

[0038] In some embodiments of the present invention, the above-mentioned compound of formula (I) crystalline form B has a differential scanning calorimetry curve as shown in Figure 5 shown.

[0039] In some embodiments of the present invention, the thermogravimetric analysis (TGA) curve of the above-mentioned crystalline form B of the compound of formula (I) shows a weight loss of 9.77% at 150.0°C±3°C.

[0040] In some embodiments of the present invention, the above-mentioned compound of formula (I) B crystal form has a thermogravimetric analysis curve as shown in Figure 6 shown.

[0041] In some embodiments of the present invention, the above-mentioned crystal form B of the compound of formula (I) may exist in the form of solvate crystals.

[0042] The present invention also provides a method for preparing the above-mentioned crystal form B of the compound of formula (I), comprising the step of stirring the compound of formula (I) in an alcohol / n-heptane mixed solvent at room temperature, and further comprising a separation step.

[0043] In some embodiments of the present invention, the crystal form B of the compound of formula (I) of the present invention is prepared by the following method:

[0044] (1) adding an alcohol / n-heptane mixed solvent to the compound of formula (I) and stirring at room temperature;

[0045] (2) Evaporate at low temperature and concentrate under reduced pressure.

[0046] In some embodiments of the present invention, the alcohol / n-heptane mixed solvent in step (1) is preferably ethanol / n-heptane.

[0047] In some embodiments of the present invention, the solvent ratio of ethanol / n-heptane is preferably 1 / 4 (volume ratio).

[0048] In some embodiments of the present invention, the low temperature in the above step (2) is preferably 5°C.

[0049] The present invention also provides a crystal form C of the compound of formula (I),

[0050]

[0051] The invention is characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 6.334±0.200°, 8.399±0.200°, 9.435±0.200°, 12.060±0.200°, and 18.126±0.200°.

[0052] In some embodiments of the present invention, the X-ray powder diffraction pattern of the C form of the compound of formula (I) above, expressed by 2θ angle, comprises at least 6, 7, or 8 diffraction peaks selected from the following: 4.883±0.200°, 6.334±0.200°, 8.399±0.200°, 8.928±0.200°, 9.435±0.200°, 11.168±0.200°, 12.060±0.200°, and 18.126±0.200°.

[0053] In some embodiments of the present invention, the X-ray powder diffraction pattern of the C form of the compound of formula (I) above has characteristic diffraction peaks at the following 2θ angles: 4.883±0.200°, 6.334±0.200°, 8.399±0.200°, 8.928±0.200°, 9.435±0.200°, 11.168±0.200°, 12.060±0.200°, 18.126±0.200°.

[0054] In some embodiments of the present invention, the X-ray powder diffraction pattern of the crystalline form C of the compound of formula (I) above, expressed as 2θ angle, comprises at least 12, 13, 14, 15, or 16 diffraction peaks selected from the following: 4.883±0.200°, 6.334±0.200°, 8.399±0.200°, 8.928±0.200°, 9.435±0.200°, 11.168±0.200°, 12.880±0.200°, 13.600±0.200°, 14.700±0.200°, 15.900±0.200°, 16.600±0.200°, 17.600±0.200°, 18.600±0.200°, 19.700±0.200°, 20.600±0.200°, 21.400±0.200°, 23.400±0.200°, 24.400±0.200°, 25. 0.200°, 12.060±0.200°, 12.988±0.200°, 14.821±0.200°, 16.182±0.200°, 18.126±0.200°, 18.545±0.200°, 19.474±0.200°, 20.199±0.200°, 24.294±0.200°, 25.697±0.200°.

[0055] In some embodiments of the present invention, the X-ray powder diffraction pattern of the C crystal form of the compound of formula (I) has characteristic diffraction peaks at the following 2θ angles: 4.883±0.200°, 6.334±0.200°, 8.399±0.200°, 8.928±0.200°, 9.435±0.200°, 11.168±0.200°, 12.060 ±0.200°, 12.988±0.200°, 14.821±0.200°, 16.182±0.200°, 18.126±0.200°, 18.545±0.200°, 19.474±0.200°, 20.199±0.200°, 24.294±0.200°, 25.697±0.200°.

[0056] In some embodiments of the present invention, the crystal form C of the compound of formula (I) is characterized in that the X-ray powder diffraction pattern of the crystal form C is as follows: Figure 7 shown.

[0057] In some embodiments of the present invention, the peak position, interplanar spacing and relative intensity of the diffraction peaks in the X-ray powder diffraction pattern of the above-mentioned crystal form C of the compound of formula (I) are shown in Table 3:

[0058] Table 3 Peak positions, interplanar spacings and relative intensities of diffraction peaks in the XRPD spectrum of Form C

[0059]

[0060]

[0061] In some embodiments of the present invention, the differential scanning calorimetry curve of the Form C of the compound of formula (I) above has an endothermic peak at 113.5°C±3°C.

[0062] In some embodiments of the present invention, the DSC spectrum of the crystal form C of the compound of formula (I) is as follows: Figure 8 shown.

[0063] In some embodiments of the present invention, the thermogravimetric analysis curve of the crystal form C of the compound of formula (I) above shows a weight loss of 3.52% at 110.0°C±3°C and a weight loss of 10.58% at 150.0°C±3°C.

[0064] In some embodiments of the present invention, the TGA spectrum of the crystal form C of the compound of formula (I) is as follows: Fig. 9 shown.

[0065] In some embodiments of the present invention, the crystal form C of the compound of formula (I) may exist in the form of a solvate crystal.

[0066] The present invention also provides a method for preparing the above-mentioned crystal form C of the compound of formula (I), comprising the step of stirring the compound of formula (I) in an ethyl acetate / n-heptane mixed solvent at room temperature, and further comprising a separation step.

[0067] In some embodiments of the present invention, the crystal form C of the compound of formula (I) of the present invention is prepared by the following method:

[0068] (1) adding ethyl acetate / n-heptane mixed solvent to the compound of formula (I) and stirring at room temperature;

[0069] (2) Evaporate at low temperature and concentrate under reduced pressure.

[0070] In some embodiments of the present invention, the solvent ratio of ethyl acetate / n-heptane in the above step (1) is preferably 1 / 4 (volume ratio).

[0071] In some embodiments of the present invention, the low temperature in the above step (2) is preferably -20°C.

[0072] The present invention also provides a crystal form D of the compound of formula (I),

[0073]

[0074] The invention is characterized in that its X-ray powder diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 9.136±0.200°, 9.908±0.200°, 17.787±0.200°, 18.329±0.200°, and 24.298±0.200°.

[0075] In some embodiments of the present invention, the X-ray powder diffraction pattern of the D crystal form of the compound of formula (I) above has characteristic diffraction peaks at the following 2θ angles: 9.136±0.200°, 9.908±0.200°, 17.787±0.200°, 18.329±0.200°, 24.298±0.200°.

[0076] In some embodiments of the present invention, the XRPD pattern of the D crystal form of the compound of formula (I) is as follows: Fig.10 shown.

[0077] In some embodiments of the present invention, the peak position, interplanar spacing and relative intensity of the diffraction peaks in the X-ray powder diffraction pattern of the D crystal form of the compound of formula (I) are shown in Table 4:

[0078] Table 4 Peak positions, interplanar spacings and relative intensities of diffraction peaks in the XRPD spectrum of Form D

[0079]

[0080] In some embodiments of the present invention, the differential scanning calorimetry curve of the D-form of the compound of formula (I) above has an endothermic peak at 113.3°C±3°C.

[0081] In some embodiments of the present invention, the DSC spectrum of the crystal form D of the compound of formula (I) is as follows: Fig.11 shown.

[0082] In some embodiments of the present invention, the thermogravimetric analysis curve of the D crystal form of the compound of formula (I) above shows a weight loss of 6.59% at 90.0°C±3°C and a weight loss of 15.60% at 150.0°C±3°C.

[0083] In some embodiments of the present invention, the TGA spectrum of the D crystal form of the compound of formula (I) is as follows: Fig.12 shown.

[0084] In some embodiments of the present invention, the D crystal form of the compound of formula (I) may exist in the form of a solvate crystal.

[0085] The present invention also provides a method for preparing the D crystal form of the compound of formula (I), comprising the step of stirring the compound of formula (I) in a dichloromethane / n-heptane mixed solvent at room temperature, and further comprising a separation step.

[0086] In some embodiments of the present invention, the D crystal form of the compound of formula (I) of the present invention is prepared by the following method:

[0087] (1) adding a dichloromethane / n-heptane mixed solvent to the compound of formula (I) and stirring at room temperature;

[0088] (2) Evaporate at low temperature and concentrate under reduced pressure.

[0089] In some embodiments of the present invention, the solvent ratio of dichloromethane / n-heptane in the above step (1) is preferably 1 / 4 (volume ratio).

[0090] In some embodiments of the present invention, the low temperature in the above step (2) is preferably -20°C.

[0091] The present invention also provides a crystalline form E of the compound of formula (I),

[0092]

[0093] The invention is characterized in that its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 5.987±0.200°, 9.928±0.200°, 11.998±0.200°, 13.499±0.200°, and 18.044±0.200°.

[0094] In some embodiments of the present invention, the X-ray powder diffraction pattern of the E crystal form of the compound of formula (I) above has characteristic diffraction peaks at the following 2θ angles: 5.987±0.200°, 9.928±0.200°, 11.998±0.200°, 13.499±0.200°, 18.044±0.200°, 19.492±0.200°, 25.173±0.200°.

[0095] In some embodiments of the present invention, the XRPD pattern of the E crystal form of the compound of formula (I) is as follows: Fig.13 shown.

[0096] In some embodiments of the present invention, the peak position, interplanar spacing and relative intensity of the diffraction peaks in the X-ray powder diffraction pattern of the E crystal form of the compound of formula (I) are shown in Table 5:

[0097] Table 5 Peak positions, interplanar spacings and relative intensities of diffraction peaks in the XRPD spectrum of Form E

[0098]

[0099] In some embodiments of the present invention, the differential scanning calorimetry curve of the E-form of the compound of formula (I) has an endothermic peak at 113.7°C±3°C.

[0100] In some embodiments of the present invention, the DSC spectrum of the E crystal form of the compound of formula (I) is as follows: Fig.14 shown.

[0101] In some embodiments of the present invention, the thermogravimetric analysis curve of the E crystal form of the compound of formula (I) above shows a weight loss of 13.97% at 150.0°C±3°C.

[0102] In some embodiments of the present invention, the TGA spectrum of the E crystal form of the compound of formula (I) is as follows: Fig.15 shown.

[0103] In some embodiments of the present invention, the E crystal form of the compound of formula (I) may exist in the form of a solvate crystal.

[0104] The present invention also provides a method for preparing the E crystal form of the compound of formula (I), comprising the step of stirring the compound of formula (I) in toluene at room temperature, and further comprising the step of separation.

[0105] In some embodiments of the present invention, the E crystal form of the compound of formula (I) of the present invention is prepared by the following method:

[0106] (1) adding toluene to the compound of formula (I) and stirring at room temperature;

[0107] (2) Evaporate and concentrate under reduced pressure.

[0108] In some embodiments of the present invention, the volatilization temperature in the above step (2) is preferably room temperature.

[0109] The present invention also provides a crystalline composition comprising Form A, Form B, Form C, Form D, or Form E of the compound of formula (I), wherein the form accounts for more than 50% by weight of the crystalline composition, preferably more than 80%, more preferably more than 90%, and most preferably more than 95%.

[0110] The present invention also provides a pharmaceutical composition, which contains a therapeutically effective amount of the A crystal form, B crystal form, C crystal form, D crystal form, or E crystal form of the compound of formula (I), or the above-mentioned crystalline composition. The pharmaceutical composition of the present invention may contain or not contain pharmaceutically acceptable excipients. In addition, the pharmaceutical composition of the present invention may further include one or more other therapeutic agents.

[0111] The present invention also provides a method for treating coronavirus infection, comprising administering to an individual in need thereof a therapeutically effective amount of the crystal form A, crystal form B, crystal form C, crystal form D, or crystal form E of the compound of formula (I) of the present invention, or the above-mentioned crystalline composition, or the above-mentioned pharmaceutical composition.

[0112] The present invention also provides the use of the above-mentioned crystal form A, crystal form B, crystal form C, crystal form D, or crystal form E of the compound of formula (I), or the above-mentioned crystalline composition, or the above-mentioned pharmaceutical composition in the preparation of drugs for treating diseases related to coronavirus infection.

[0113] In some embodiments of the present invention, the above-mentioned disease related to coronavirus infection is SARS-CoV-2 virus infection.

[0114] Technical Effects

[0115] The crystal form of the compound of the present invention has good PK properties and the effect of treating SARS-CoV-2 virus. The crystal form is stable, has good hygroscopicity, and is less affected by light and heat.

[0116] Definition and Description

[0117] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

[0118] For any given crystalline form, the relative intensity of the diffraction peaks can change due to preferred orientations caused by factors such as crystal morphology, which is well known in the art of crystallography. Where there is a preferred orientation effect, the peak intensity changes, but the position of the diffraction peaks of the crystal form cannot be changed. In addition, for any given crystal form, there may be slight errors in the position of the peaks, which is also well known in the art of crystallography. For example, due to changes in temperature when analyzing a sample, movement of the sample, or calibration of the instrument, the position of the peak can move, and the measurement error of the 2θ value is sometimes about ± 0.2 degrees. Therefore, it is well known to those skilled in the art that this error should be taken into account when determining each crystalline structure.

[0119] DSC measures the transition temperature when a crystal absorbs or releases heat due to changes in its crystalline structure or melting of the crystal. For the same type of crystal form of the same compound, in consecutive analyses, the error of thermal transition temperature and melting point is typically within about 5°C or 3°C. When we say that a compound has a given DSC peak or melting point, this refers to the DSC peak or melting point ±5°C or ±3°C. DSC provides an auxiliary method for distinguishing different crystal forms. Different crystalline forms can be identified based on their different transition temperature characteristics. It should be pointed out that for mixtures, their DSC peaks or melting points may vary over a larger range. In addition, since decomposition is accompanied by the melting of the substance, the melting temperature is related to the heating rate.

[0120] For the same crystal form, the TGA weight loss temperature may vary due to factors such as the measuring instrument, measuring method / conditions, etc. For any specific crystal form, the weight loss temperature may have an error of about ±5°C or about ±3°C.

[0121] It should be noted that when preparing drug crystal forms, during the contact between drug molecules and solvent molecules, it is difficult to avoid the situation that the solvent molecules and compound molecules form a co-crystal and remain in the solid material due to external conditions and internal factors, thereby forming a solvate, specifically including a stoichiometric solvate and a non-stoichiometric solvate. The solvates are all included in the scope of the present invention.

[0122] The "pharmaceutically acceptable excipients" refer to inert substances that are administered together with the active ingredients and are conducive to the administration of the active ingredients, including but not limited to any glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, disintegrants, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers that are acceptable for use in humans or animals (such as livestock) and approved by the State Food and Drug Administration.

[0123] The term "crystalline composition" refers to a mixture of a crystalline form of the compound of formula (I) of the present invention and other crystalline forms or amorphous substances or other impurities of the compound. For example, a crystalline composition of crystalline form A of the compound of formula (I) contains other crystalline forms or amorphous substances or other impurities of the compound of formula (I) in addition to crystalline form A of the compound of formula (I).

[0124] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present invention or their salts and pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present invention to an organism.

[0125] The therapeutic dosage of the compounds of the invention may be determined, for example, based on the specific use of the treatment, the manner in which the compound is administered, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of the compounds of the invention in the pharmaceutical composition may not be fixed, depending on a variety of factors, including dosage, chemical properties (e.g., hydrophobicity), and route of administration.

[0126] The term "treatment" means administering the compound or formulation of the present invention to improve or eliminate a disease or one or more symptoms associated with the disease, and includes:

[0127] (i) inhibiting a disease or disease state, i.e. arresting its development;

[0128] (ii) ameliorating a disease or condition, even if causing regression of the disease or condition.

[0129] The term "therapeutically effective amount" means an amount of a compound of the present invention that (i) treats a particular disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of a compound of the present invention that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art based on their own knowledge and this disclosure.

[0130] Unless otherwise required in the present invention, throughout the specification and the claims that follow, the word "comprise" and its English variations such as "comprises" and "comprising" should be interpreted as having an open and inclusive meaning, that is, "including but not limited to".

[0131] References to "one embodiment" or "an embodiment" or "in another embodiment" or "in certain embodiments" throughout this specification are intended to include in at least one embodiment the specific referenced elements, structures or features described in connection with that embodiment. Thus, the phrases "in one embodiment" or "in an embodiment" or "in another embodiment" or "in certain embodiments" appearing in different places throughout the specification do not necessarily all refer to the same embodiment. Furthermore, the specific elements, structures or features may be combined in any appropriate manner in one or more embodiments.

[0132] It should be understood that the singular article "a", "an" and "the" used in the present specification and the appended claims includes plural objects unless the context clearly dictates otherwise. Thus, for example, a reference to a reaction including "a catalyst" includes one catalyst, or two or more catalysts. It should also be understood that the term "or" is generally used in its sense including "and / or" unless the context clearly dictates otherwise.

[0133] The intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention.

[0134] The chemical reactions of the specific embodiments of the present invention are carried out in a suitable solvent, which must be suitable for the chemical changes of the present invention and the reagents and materials required. In order to obtain the compounds of the present invention, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction processes based on the existing embodiments.

[0135] The present invention will be described in detail below by way of examples, which are not intended to limit the present invention in any way.

[0136] All solvents used in the present invention were commercially available and used without further purification.

[0137] Compounds are named according to the conventional nomenclature in the art or using The software names were used, and commercially available compounds were named using the supplier's catalog names.

[0138] Instruments and analytical methods

[0139] 1.1 X-ray powder diffractometer (XRPD) method of the present invention

[0140] Instrument model: PANalytacal X-ray diffractometer

[0141] Test method: About 1-2 mg of sample was used for XRPD detection.

[0142] The detailed XRPD parameters are as follows:

[0143] Light pipe: Cu, kα, Kα1 1.540598, Kα2 1.544426, Kα2 / Kα1intensity ratio: 0.50

[0144] Light tube voltage: 45kV, light tube current: 40mA

[0145] Divergence slit: 1 / 8°

[0146] Scan mode: Continuous

[0147] Scanning range (°2Theta): 3-40

[0148] Scan time per step (s): 46.7

[0149] Scan step size (°2Theta): 0.0263

[0150] Test time: ~5min

[0151] 1.2 Differential Scanning Calorimeter (DSC) method of the present invention

[0152] Instrument model: TA 2500 differential scanning calorimeter

[0153] Test method: Take a sample (~1 mg) and place it in a DSC aluminum pan for testing. Under 50 mL / min N2 conditions, heat the sample from 25°C (room temperature) to the set temperature at a heating rate of 10°C / min.

[0154] 1.3 Thermogravimetric analysis (TGA) method of the present invention

[0155] Instrument model: TA 5500 Thermogravimetric Analyzer

[0156] Test method: Take a sample (~1 mg) and place it in a TGA platinum pan for testing. Under 25 mL / min N2 conditions, heat the sample from room temperature to the set temperature at a heating rate of 10°C / min.

[0157] or:

[0158] Thermogravimetric Analyzer TGA550

[0159] Test method: Take a sample (5-10 mg) and place it in the aluminum pan inside the TGA platinum pot for testing. Under 60 mL / min N2 conditions, heat the sample from room temperature to 300°C at a heating rate of 10°C / min.

[0160] 1.4 Dynamic Vapor Sorption (DVS) Method of the Present Invention

[0161] Instrument model: SMS DVS Advantage Dynamic Vapor Sorption Instrument

[0162] Test conditions: Take a sample (10-30 mg) and place it in the DVS sample tray for testing.

[0163] The detailed DVS parameters are as follows:

[0164] Temperature: 25℃

[0165] Balance: dm / dt = 0.002% / min (minimum: 10min, maximum: 180min)

[0166] RH(%) range: 70%RH-95%RH-0%RH-95%RH

[0167] RH (%) gradient: 10% (90%RH-0%RH-90%RH), 5% (95%RH-90%RH and 90%RH-95%RH). BRIEF DESCRIPTION OF THE DRAWINGS

[0168] Figure 1 is the Cu-Kα radiation XRPD spectrum of Form A of the compound of formula (I);

[0169] Figure 2 is the DSC spectrum of Form A of the compound of formula (I);

[0170] Figure 3 is the TGA spectrum of Form A of the compound of formula (I);

[0171] Figure 4 is a Cu-Kα radiation XRPD spectrum of Form B of the compound of formula (I);

[0172] Figure 5 is the DSC spectrum of Form B of the compound of formula (I);

[0173] Figure 6 is the TGA spectrum of the B crystal form of the compound of formula (I);

[0174] Figure 7 is a Cu-Kα radiation XRPD spectrum of Form C of the compound of formula (I);

[0175] Figure 8 is the DSC spectrum of Form C of the compound of formula (I);

[0176] Fig. 9 is the TGA spectrum of Form C of the compound of formula (I);

[0177] Fig.10 is the Cu-Kα radiation XRPD spectrum of the D crystal form of the compound of formula (I);

[0178] Fig.11is the DSC spectrum of the D crystal form of the compound of formula (I);

[0179] Fig.12 is the TGA spectrum of the D crystal form of the compound of formula (I);

[0180] Fig.13 is a Cu-Kα radiation XRPD spectrum of Form E of the compound of formula (I);

[0181] Fig.14 is the DSC spectrum of the E crystal form of the compound of formula (I);

[0182] Fig.15 is the TGA spectrum of the E crystal form of the compound of formula (I);

[0183] Fig.16 It is the DVS spectrum of Form A of the compound of formula (I). DETAILED DESCRIPTION

[0184] The present invention is described in detail below by way of examples, but it is not intended to impose any adverse limitations on the present invention. The present invention has been described in detail herein, and specific embodiments thereof are also disclosed therein. It will be apparent to those skilled in the art that various changes and modifications may be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0185] Example 1: Preparation of Compound 1

[0186]

[0187] Step 1: Synthesis of compound 1-2

[0188] Compound 1-1 (5 g, 54.32 mmol) was dissolved in methanol (50 mL) and refluxed at 70° C. for 48 h. The reaction system was concentrated under reduced pressure to obtain a crude target product. The crude product had a high purity and was directly used in the next step to obtain compound 1-2.

[0189] 1 H NMR (400MHz, CDCl3) δ=4.81 (s, 1H), 3.77 (s, 3H), 3.43 (s, 3H).

[0190] Step 2: Synthesis of Compound 1-3

[0191] Dissolve compound 1-2 in toluene (3 mL), cool to 0°C, slowly drop compound (R)-(+)phenylethylamine (1.5 g, 12.38 mmol, 1.60 mL), and stir at 20°C for 1 h. Add ethyl acetate (60 mL) and saturated brine (30 mL) to the reaction system to extract and separate the organic phase, dry over anhydrous sodium sulfate, and spin dry to obtain a crude product. Purify by silica gel column chromatography (petroleum ether: ethyl acetate = 1:0-5:1) to obtain the target compound 1-3.

[0192] 1 H NMR (400MHz, CDCl3) δ = 7.95-7.56 (m, 1H), 7.31-7.17 (m, 5H), 4.71-4.40 (m, 1H), 3.95-3.71 (m, 3H), 1.67-1.51 (m, 3H).

[0193] Step 3: Synthesis of Compound 1-4

[0194] Compound 1-3 (0.5 g, 2.61 mmol) was dissolved in 2,2,2-trifluoroethanol (5 mL), trifluoroacetic acid (313.04 mg, 2.75 mmol, 203.28 μL) was added, the temperature was cooled to -10 °C, and the mixture was stirred for 1 h. The temperature was controlled at -10 °C, and cyclopentadiene (207.40 mg, 3.14 mmol) was slowly added dropwise, and the mixture was stirred for 0.5 h. The reaction system was concentrated under reduced pressure, methyl tert-butyl ether (60 mL) and saturated sodium bicarbonate solution (30 mL×2) were added, and the mixture was stirred for 10 min. The organic phase was extracted and separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Compound 1-4 was obtained by purification by silica gel column chromatography (petroleum ether: ethyl acetate = 1:0-5:1), and the configuration was confirmed by two-dimensional NMR.

[0195] 1 H NMR (400MHz, CDCl3) δ = 7.34-7.18 (m, 5H), 6.59-6.41 (m, 1H), 6.31 (dd, J = 1.6, 5.6Hz, 1H), 4.35 (br d, J=1.3Hz, 1H), 3.39 (s, 3H), 3.18-3.03 (m, 1H), 2.95 (br s, 1H), 2.33-2.22 (m, 1H), 2.14 (br d, J=8.4Hz, 1H), 1.54-1.41 (m, 4H).

[0196] MS m / z(ESI): [M+H] + =258.2.

[0197] Step 4: Synthesis of Compound 1-5

[0198] Compound 1-4 (100.00 mg, 388.61 μmol) was dissolved in tetrahydrofuran (1.25 mL), cooled to -70 °C, and borane tetrahydrofuran complex (1M, 427.47 μL) was slowly added dropwise, and the temperature was slowly raised to 20 °C and stirred for 1 h. The temperature was lowered to 0 °C, and a 10% aqueous sodium hydroxide solution (0.55 mL) and a 30% hydrogen peroxide solution (220.28 mg, 1.94 mmol, 186.68 μL) were added, and the temperature was slowly raised to 20 °C and stirred for 1 h. A saturated sodium thiosulfate solution (10 mL) was added to the reaction system and stirred for 10 min to quench the reaction, followed by extraction with saturated saline (20 mL) and ethyl acetate (60 mL × 2) to separate the organic phase. A small amount of sample solution was taken, and the pH was adjusted to less than 8 with 3% citric acid. After the starch potassium iodide test paper was negative, it was dried over anhydrous sodium sulfate and concentrated under reduced pressure at 30 °C. Compound 1-5 was obtained by purification through silica gel column chromatography (petroleum ether: ethyl acetate = 1:0-5:1).

[0199] 1 H NMR (400MHz, CDCl3) δ = 7.30-7.13 (m, 5H), 3.93 (br d, J=6.5Hz, 1H), 3.78 (brs, 1H), 3.70-3.54 (m, 1H), 3.39-3.32 (m, 1H), 3.3 1-3.24(m, 3H), 2.49-2.40(m, 1H), 2.26(s, 1H), 2.09-2.00(m, 1H), 1.72(br d, J=10.1Hz, 1H), 1.46 (br d, J=6.5Hz, 1H), 1.41-1.33 (m, 3H).

[0200] MS m / z(ESI): [M+H] + =276.1.

[0201] Step 5: Synthesis of the hydrochloride salt of compound 1-6

[0202] Compound 1-5 (3 g, 10.90 mmol) was dissolved in ethanol (80 mL), and hydrochloric acid (1.19 g, 32.69 mmol) and wet palladium carbon (15 g, 10.68 mmol) were added. The reaction was stirred at 20° C. for 16 h. The reaction solution was filtered through diatomaceous earth and then directly spin-dried to obtain the hydrochloride of crude compound 1-6.

[0203] 1H NMR (400MHz, DMSO-d6) δ = 10.32-9.78 (m, 1H), 8.94-8.43 (m, 1H), 5.51-5.12 (m, 1H), 4.05-3.98 (m, 1H), 3.96 -3.86(m, 2H), 3.83-3.71(m, 3H), 2.70-2.60(m, 1H), 2.36-2.20(m, 1H), 1.92-1.81(m, 1H), 1.51-1.31(m, 2H)

[0204] MS m / z(ESI): [M+H] + =172.0.

[0205] Step 6: Synthesis of Compound 1-8

[0206] Compound 1-7 (1.87 g, 10.90 mmol) was dissolved in N, N-dimethylformamide (20 mL), O-(7-azabenzotriazole-1-yl)-N, N, N, N-tetramethyluronium hexafluorophosphate (4.78 g, 12.58 mmol) and diisopropylethylamine (4.34 g, 33.55 mmol) were added, and after stirring for 30 min, the hydrochloride of compound 1-6 (190 mg, 1.12 mmol) was added. The reaction was stirred at 20 ° C for 16 h. Water (15 mL) was added to the reaction solution, and ethyl acetate (60 mL) was extracted twice. The organic phases were combined, and the organic phases were washed twice with 5% citric acid (30 mL), washed four times with brine (20 mL), dried over anhydrous sodium sulfate, filtered, and spin-dried. Compound 1-8 was obtained by column chromatography (petroleum ether: ethyl acetate = 3: 1).

[0207] 1 H NMR (400MHz, CDCl3) δ = 5.28-5.16 (m, 1H), 4.50 (br s, 1H), 4.28 (d, J=9.8Hz, 1H), 3.92 (s, 1H), 3.74 (s, 3H), 2.81 (s, 1H), 2.67 (s, 1H ), 2.17(brdd, J=6.1, 12.7Hz, 1H), 1.99-1.93(m, 1H), 1.90-1.84(m, 1H), 1.59(br d, J=13.3Hz, 2H), 1.43 (s, 9H), 1.04 (s, 9H).

[0208] MS m / z(ESI): [M+H] + =385.2.

[0209] Step 7: Synthesis of Compound 1-9

[0210] Compound 1-8 (500 mg, 1.30 mmol) was dissolved in acetonitrile (7.5 mL), 2-iodoacylbenzoic acid (976.31 mg, 3.49 mmol) was added, and stirred at 60°C for 16 h. The reaction solution was directly filtered through diatomaceous earth and dried by spin drying. Compound 1-9 was obtained without purification.

[0211] MS m / z(ESI): [M-55] + =327.1.

[0212] Step 8: Synthesis of Compound 1-10

[0213] Compound 1-9 (0.7 g, 1.83 mmol) was dissolved in tetrahydrofuran (14 mL), and TEBBE (μ-chloro-μ-methylene[di(cyclopentadienyl)titanium]-dimethylaluminum) reagent (0.5 M, 14.64 mL) was added at 0°C. Stirred at 0°C for 1 h, then heated to 15°C and continued to stir for 3 h. Slowly poured into saturated sodium bicarbonate solution (50 mL) into the reaction mixture, filtered with diatomaceous earth, extracted with ethyl acetate (30 mL×3), and washed with saturated brine (30 mL×2). Purified by column chromatography (petroleum ether: ethyl acetate = 5:1) to obtain compound 1-10.

[0214] 1H NMR (400MHz, DMSO-d6) δ = 6.63-6.54 (m, 1H), 5.21-5.15 (m, 1H), 4.88-4.82 (m, 1H), 4.76-4.68 (m, 1H), 4.25-4.19 (m, 1H), 3.90-3.85 (m , 1H), 3.66-3.61(m, 3H), 3.19-3.11(m, 1H), 2.42-2.28(m, 2H), 1.98-1.92(m, 1H), 1.61-1.53(m, 1H), 1.38(s, 9H), 1.00-0.93(m, 9H).

[0215] MS m / z(ESI): [M+H] + =381.1.

[0216] Step 9: Synthesis of Compound 1-11

[0217] Under nitrogen protection, diethylzinc (1M, 13.14mL) was slowly added to 1,2-dichloroethane (80mL) at 0°C. Stir for 0.25h, diiodomethane (7.04g, 26.28mmol, 2.12mL) was slowly added to the reaction solution at 0°C, and stirred for 0.25h. Trifluoroacetic acid (149.84mg, 1.31mmol, 97.30μL) was slowly added to the reaction system, and stirring continued for 0.5h. Compound 1-10 (0.5g, 1.31mmol) in 1,2-dichloroethane (5mL) was added to the reaction system, and the temperature was raised to 20°C and stirring continued for 12h. The reaction was quenched with saturated sodium bicarbonate solution (200mL), extracted with dichloromethane (100mL), dried over anhydrous sodium sulfate, and filtered. Concentrated. The crude product was purified by preparative HPLC (column type: Phenomenex luna C18 80*40mm*3μm; mobile phase: [H2O(HCl)-acetonitrile]; acetonitrile%: 1%-30%, 7min) to obtain compound 1-11.

[0218] MS m / z(ESI): [M+H] + =295.2.

[0219] Step 10: Synthesis of Compound 1-12

[0220] Compound 1-11 (0.1 g, 339.69 μmol) was dissolved in 1,4-dioxane (3 mL), and potassium carbonate (187.79 mg, 1.36 mmol) and di-tert-butyl dicarbonate (111.20 mg, 509.53 μmol, 117.06 μL) in water (1 mL) were added, and the reaction was stirred at 15°C for 12 h. The reaction solution was poured into water (30 mL), extracted with ethyl acetate (20 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated. Compound 1-12 was obtained by column chromatography (petroleum ether: ethyl acetate = 5: 1).

[0221] 1H NMR (400MHz, DMSO-d6) δ = 12.52-12.16 (m, 1H), 6.56-6.31 (m, 1H), 4.66-4.58 (m, 1H), 4.24-4.18 (m, 1H), 4.02 (s, 1H), 3.36-3.28 (m, 1H), 1.97 -1.91(m, 2H), 1.81-1.73(m, 2H), 1.66-1.59(m, 1H), 1.36(s, 9H), 0.99 -0.93(m,9H),0.80-0.70(m,1H),0.64-0.53(m,1H),0.49-0.33(m,2H)

[0222] MS m / z(ESI): [M+H]+ =395.2.

[0223] Step 11: Synthesis of Compound 1-13

[0224] Compound 1-12 (88.13 mg, 223.40 μmol) was dissolved in tetrahydrofuran (2 mL) and methanol (0.6 mL), and lithium hydroxide monohydrate (28.12 mg, 670.21 μmol) was dissolved in water (0.6 mL) and added. The reaction was stirred at 15° C. for 2 h. The reaction was adjusted to pH about 5 with 3% citric acid, extracted with ethyl acetate (20 mL×3), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 1-13.

[0225] MS m / z(ESI): [M+H] + =381.3.

[0226] Step 12: Synthesis of Compound 1-15

[0227] Compound 1-13 (0.056 g, 148.79 μmol) was dissolved in N, N-dimethylformamide (2 mL), O-(7-azabenzotriazole-1-yl)-N, N, N, N-tetramethyluronium hexafluorophosphate (84.86 mg, 223.18 μmol) was added to the reaction system, and the reaction was stirred at 15° C. for 0.5 h. Diisopropylethylamine (76.92 mg, 595.16 μmol, 103.67 μL) was then added to the reaction solution, and a solution of the hydrochloride of compound 1-14 (43.26 mg, 208.31 μmol) dissolved in N, N-dimethylformamide (0.5 mL) was added to the reaction system, and the reaction was stirred at 15° C. for 12 h. The reaction was diluted with water (20 mL), extracted with ethyl acetate (20 mL×3), and the organic phase was washed with 3% citric acid (20 mL), washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 1-15.

[0228] 1H NMR (400MHz, DMSO-d6) δ = 8.22-8.08 (m, 1H), 7.56 (s, 1H), 7.32-7.16 (m, 1H), 7.01 (s, 1H), 6.51 (br d, J=9.4Hz, 1H), 4.60-4.49 (m, 1H), 4.28-4.17 (m, 2H), 4.14 (s, 1H), 3.18-2. 98(m, 2H), 2.47-2.35(m, 1H), 2.17-2.09(m, 2H), 1.96-1.84(m, 2H), 1.76(br d, J=11.0Hz, 1H), 1.71-1.42 (m, 4H), 1.39-1.34 (m, 9H), 0.95 (s, 8H), 0.84-0.77 (m, 1H), 0.73-0.65 (m, 1H), 0.39 (br s, 2H)

[0229] MS m / z(ESI): [M+H] + =534.4.

[0230] Step 13: Synthesis of Compound 1-16

[0231] Compound 1-15 (0.02 g, 37.48 μmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (141.02 mg, 1.24 mmol, 91.57 μL) was added to the reaction system. Stir at 15 °C for 1 h. The reaction was directly quenched with sodium bicarbonate solution (10 mL), extracted with dichloromethane (5 mL×5), and the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 1-16.

[0232] 1H NMR (400MHz, DMSO-d6) δ = 8.37-8.26 (m, 1H), 8.14 (br d, J=3.6Hz, 3H), 7.59-7.53(m, 1H), 7.40-7.32(m, 1H), 7.05-6.92(m, 1H), 4.33-4.21(m, 2H), 3.93(br d, J=5.0Hz, 1H), 3.17-3.08(m, 1H), 3.06-2.97(m, 1H), 2.44-2.32(m, 1H), 2.18-2.08(m, 2H), 1.96-1.86(m, 2H ), 1.82-1.57(m, 4H), 1.51-1.40(m, 1H), 1.04(s, 9H), 0.95-0.85(m, 1H), 0.76-0.67(m, 1H), 0.54-0.34(m, 2H)

[0233] MS m / z(ESI): [M+H] +=434.2.

[0234] Step 14: Synthesis of Compound 1

[0235] Compound 1-16 (0.03 g, 69.20 μmol) was dissolved in dichloromethane (1 mL), and trifluoroacetic anhydride (58.13 mg, 276.79 μmol, 38.50 μL) was added to the reaction system. Stir at 15°C for 1 h. The reaction was directly quenched with sodium bicarbonate solution (10 mL), extracted with dichloromethane (5 mL×5), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by preparative HPLC (column type: Waters Xbridge BEH C18 100*30mm*10μm; mobile phase: [H2O(NH4HCO3)-acetonitrile]; acetonitrile%: 10%-50%, 8 min) to obtain compound 1, i.e., compound of formula (I).

[0236] 1H NMR (400MHz, DMSO-d6) δ = 9.31 (br d, J=8.0Hz, 1H), 8.87 (d, J=8.6Hz, 1H), 7.66 (s, 1H), 5.01-4.87 (m, 1H), 4.72-4.59 (m, 2H), 4.07 (s, 1H), 3.17-3.10(m, 1H), 3.09-2.98(m, 1H), 2.45-2.33(m, 1H), 2.19-2.05(m, 3H), 1.85-1.64(m, 5H), 1.56(br d, J=12.0Hz, 1H), 1.20-1.13 (m, 1H), 1.01 (s, 8H), 0.82-0.67 (m, 2H), 0.54-0.46 (m, 1H), 0.45-0.34 (m, 1H)

[0237] MS m / z(ESI): [M+H] + =512.2.

[0238] Example 2: Preparation of Crystalline Form A of Compound (I)

[0239] The compound of formula (I) (9.4 g, 18.38 mmol) was dissolved in H2O (235 mL) and stirred at 50°C for 48 hr. Filtered to obtain a white solid, which was concentrated under reduced pressure to remove water to obtain Form A, which was detected by XRPD.

[0240] Example 3: Preparation of Crystalline Form B of Compound (I)

[0241] The compound of formula (I) (15 mg, 29.32 μmol) was added to a glass vial, and 0.5 mL of ethanol / n-heptane (1:4) solvent was added. The sample was placed at room temperature with magnetic stirring (1000 rpm) for about 5 days, then lowered to a low temperature (5° C.) and stirred for volatilization. The solid was collected by centrifugation and concentrated under reduced pressure to obtain Form B, which was detected by XRPD.

[0242] Example 4: Preparation of Crystalline Form C of Compound (I)

[0243] The compound of formula (I) (15 mg, 29.32 μmol) was added to a glass vial, and 0.5 mL of ethyl acetate / n-heptane (1:4) solvent was added. The sample was placed at room temperature with magnetic stirring (1000 rpm) for about 5 days, then lowered to a low temperature (-20°C) and stirred for volatilization. The solid was collected by centrifugation and concentrated under reduced pressure to obtain Form C, which was detected by XRPD.

[0244] Example 5: Preparation of Crystalline Form D of Compound (I)

[0245] The compound of formula (I) (15 mg, 29.32 μmol) was added to a glass vial, and 0.5 mL of dichloromethane / n-heptane (1:4) solvent was added. The sample was placed at room temperature with magnetic stirring (1000 rpm) for about 5 days, then lowered to a low temperature (-20°C) and stirred for volatilization. The solid was collected by centrifugation and concentrated under reduced pressure to obtain Form D, which was detected by XRPD.

[0246] Example 6: Preparation of Crystalline Form E of Compound of Formula (I)

[0247] The compound of formula (I) (15 mg, 29.32 μmol) was added to a glass vial, and 0.5 mL of toluene solvent was added. The sample was placed at room temperature with magnetic stirring (1000 rpm) for about 5 days to a clear state. The temperature was then lowered to a low temperature (-20°C) and stirred. No precipitation was observed. The temperature was then raised to room temperature and evaporated to obtain a white solid. The mixture was concentrated under reduced pressure to obtain Form E, which was detected by XRPD.

[0248] Example 7: Hygroscopicity study of the crystalline form of compound A of formula (I)

[0249] Experimental Materials:

[0250] SMS DVS Advantage Dynamic Vapor Sorption Tester

[0251] Experimental methods:

[0252] 10-30 mg of the crystalline form A of the compound of formula (I) was placed in a DVS sample tray for testing.

[0253] Experimental results:

[0254] The DVS spectrum of the crystal form A of the compound of formula (I) is as follows Fig.16 As shown, ΔW = 0.114%.

[0255] Experimental conclusion:

[0256] The weight gain of the crystal form A of the compound of formula (I) at 25° C. and 80% RH is 0.114%, and it has no or almost no hygroscopicity.

[0257] Example 8: Stability study of the crystalline form of compound A of formula (I)

[0258] The stability test results of Form A under different conditions are shown in Table 6.

[0259] Table 6 Stability test under different conditions

[0260]

[0261] Experimental conclusion: The crystalline form A of the compound of the present invention has good stability under light, high temperature and high humidity conditions.

[0262] Biological test data:

[0263] Experimental Example 1: Evaluation of the in vitro anti-novel coronavirus Mpro protease activity of the test compound

[0264] 1. Experimental materials:

[0265] 1.1 Reagents and consumables are shown in Table 7.

[0266] Table 7

[0267]

[0268] 1.2 Instruments and brands are shown in Table 8.

[0269] Table 8

[0270] instrument brand 1 SpectraMax M2e Microplate Reader Molecular Devices 2 Echo 655 Liquid Workstation Labcyte 3 Desktop high speed centrifuge Eppendorf

[0271] 2. Experimental methods:

[0272] The compound was dissolved in DMSO, and Echo655 was used to dilute the compound in a 3-fold gradient according to the concentration to be tested, with 10 concentration points and double wells for each concentration, and added to a 384-well plate. Mpro protein and substrate were diluted with test buffer (100mM NaCl, 20mM Tris-HCL, 1mMEDTA), and Mpro protein was added to a 384-well test plate, incubated with the compound at room temperature for 30 minutes, and then the substrate was added. The test concentration of Mpro protein was 25nM and the test concentration of substrate was 25μM. Incubate in a 30°C constant temperature incubator for 60 minutes. Then use an enzyme marker to detect the Ex / Em=340nm / 490nm fluorescence signal value. At the same time, the background wells containing substrate and compound but not Mpro protein were detected as controls.

[0273] 3. Data Analysis:

[0274] 1) Calculate the inhibition rate using the following formula:

[0275] Inhibition rate % = [(compound-BG 化合物 )-(ZPE-BG ZPE )] / [(HPE-BG HPE )-(ZPE-BG ZPE )]*100%

[0276] # HPE: 100% inhibition control, containing 25nM Mpro protein + 25μM substrate + 1μM GC376

[0277] ZPE: non-inhibitory control, containing 25 nM Mpro protein + 25 μM substrate, no compound

[0278] Compound: Test compound wells. Contains 25nM Mpro protein + 25μM substrate + compound

[0279] BG: Background control wells. Contains 25 μM substrate + compound, without Mpro protein

[0280] 2) Use GraphPad Prism software to perform log(agonist) vs. response--Variable slope nonlinear fitting analysis on the inhibition rate data of the compound (inhibition rate %) to obtain the IC of the compound. 50 value.

[0281] Table 9: In vitro anti-novel coronavirus Mpro protease activity of the tested compounds

[0282] Compound No. <![CDATA[IC 50 (nM)]]> 1 1.8

[0283] Conclusion: The compounds of the present invention have good in vitro anti-novel coronavirus Mpro protease activity.

[0284] Example 2: Evaluation of the in vitro anti-coronavirus activity of compounds using a cytopathic model

[0285] 1. Experimental materials are shown in Tables 10 and 11.

[0286] Table 10 Reagents and consumables

[0287]

[0288]

[0289] Table 11 Instruments

[0290] instrument brand 1 ELISA reader BioTek 2 Cell Counter Beckman 3 <![CDATA[CO2 Incubator]]> Thermo

[0291] 1.1 Cells and viruses

[0292] MRC5 cells and coronavirus HCoV OC43 were purchased from ATCC.

[0293] MRC5 cells were cultured in MEM (Sigma) supplemented with 10% fetal bovine serum (Excell), 1% double antibody (Hyclone), 1% L-glutamine (Gibco) and 1% non-essential amino acids (Gibco). MEM (Sigma) supplemented with 5% fetal bovine serum (Excell), 1% double antibody (Hyclone), 1% L-glutamine (Gibco) and 1% non-essential amino acids (Gibco) was used as the experimental culture medium.

[0294] 2. Experimental Methods

[0295] Table 12 Virus test methods used in this study

[0296]

[0297] The cells were inoculated into a 96-well microplate at a certain density (Table 12) and cultured overnight in a 5% CO2, 37°C incubator. The next day, the compound after multiple dilution (8 concentration points, duplicate wells) was added, 50 μL per well. The diluted virus was then added at 100 TCID 50Add cells, 50 μL per well. Set up cell controls (cells, no compound treatment or virus infection), virus controls (cells infected with virus, no compound treatment) and culture medium controls (culture medium only). The final volume of the experimental culture medium is 200 μL, and the final concentration of DMSO in the culture medium is 0.5%. The cells are cultured in a 5% CO2, 33°C incubator for 5 days. Use the cell viability detection kit CellTiter Glo (Promega) to detect cell viability. The cytotoxicity experiment has the same conditions as the antiviral experiment, but without virus infection.

[0298] 3. Data Analysis

[0299] The antiviral activity and cytotoxicity of the compound are respectively expressed by the inhibition rate (%) and cell viability (%) of the compound at different concentrations on the virus-induced cytopathic effect. The calculation formula is as follows:

[0300] Inhibition rate (%) = (test well reading - virus control average) / (cell control average - virus control average) × 100

[0301] Cell viability (%) = (test well reading - average value of culture medium control) / (average value of cell control - average value of culture medium control) × 100

[0302] GraphPad Prism was used to perform nonlinear fitting analysis on the inhibition rate and cell viability of the compounds and calculate the half effective concentration (EC 50 ) and the half cytotoxic concentration (CC 50 )value.

[0303] Table 13: Evaluation of the anti-coronavirus activity of compounds in vitro using cytopathic model

[0304] Compound No. <![CDATA[EC 50 (nM)]]> <![CDATA[EC 90 (nM)]]> <![CDATA[CC 50 (nM)]]> 1 5.1 7.9 ＞10000

[0305] Conclusion: The compounds of the present invention have good in vitro anti-coronavirus activity at the cellular level and are non-cytotoxic.

[0306] Experimental Example 3: Pharmacokinetics test in rats

[0307] In this study, male and female SD rats were selected as test animals. The LC / MS / MS method was used to quantitatively determine the plasma drug concentration of the test compound at different time points after a single intravenous injection of 2 mg / kg and a single oral gavage of 30 mg / kg of compound 1 A crystal form. The pharmacokinetic characteristics of the test drug in rats were evaluated.

[0308] The animals were divided into groups of 3 / group / sex, and the animals were given the A-form of compound 1, and plasma samples were collected at 0.083 (only intravenous injection group), 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after administration, and the blood drug concentration was determined by LC-MS / MS. The experimental results are shown in Table 14.

[0309] Table 14 Pharmacokinetic parameters in rats

[0310]

[0311] a: Bioavailability is calculated from AUC0-1ast and theoretical dose. ND: Not determined. --: Not applicable.

[0312] Experimental conclusion: The compound of the present invention has a higher exposure in plasma and a higher bioavailability.

[0313] Experimental Example 4: Evaluation of the in vivo antiviral efficacy of the test compound using the coronavirus OC43 strain suckling mouse infection model

[0314] C57BL / 6J suckling mice were infected with a lethal dose of coronavirus via intranasal drops, and 2 hours before infection, the suckling mice were treated with a solvent (5% DMSO + 40% PEG400 + 55% water) and compound 1. During the experiment, the weight, health status and survival of the suckling mice were monitored daily to evaluate the protective effect of compound 1 on suckling mice at different doses.

[0315] The body weight of the suckling mice in the vehicle group continued to decrease on the 6th day after virus inoculation, and the terminal survival rate was 0%. Compound 1 (12.5, 25, 50 mpk) was first administered 2 hours before infection, and the terminal survival rates were 87.5%, 100% and 100%, respectively. The experimental results are shown in Table 15.

[0316] Table 15. Mouse survival analysis

[0317]

[0318]

[0319] *Compared with vehicle group

[0320] Experimental conclusion: Compound 1 has good anti-coronavirus efficacy in vivo and shows a good dose-effect relationship.

Claims

1. Crystal form A of the compound of formula (I), It is characterized in that Its X-ray powder diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 10.655±0.200°, 11.988±0.200°，16.055±0.200°，18.356±0.200°，20.083±0.200°。 2. According to claim 1, the crystal form A of the compound of formula (I) is characterized in that: The X-ray powder diffraction pattern of the A crystal form, expressed by 2θ angle, also contains at least 1, 2, or 3 diffraction peaks selected from the following: 6.917±0.200°, 14.481±0.200°, 17.653±0.200°.

3. According to claim 1, the crystal form A of the compound of formula (I) has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 6.917±0.200°, 10.655±0.200°, 11.988±0.200°, 14.481±0.200°, 16.055±0.200°, 17.653±0.200°, 18.356±0.200°, 20.083±0.200°.

4. The crystal form A of the compound of formula (I) according to claim 1, characterized in that: The X-ray powder diffraction pattern of the A crystal form, expressed by the 2θ angle, also includes at least 7, 8, 9, 10, or 11 diffraction peaks selected from the following: 6.917±0.200°, 13.837±0.200°, 14.481±0.200°, 17.653±0.200°, 20.839±0.200°, 21.388±0.200°, 22.379±0.200°, 24.577±0.200°, 25.104±0.200°, 26.402±0.200°, and 31.540±0.200°.

5. According to claim 1, the crystal form A of the compound of formula (I) has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 6.917±0.200°, 10.655±0.200°, 11.988±0.200°, 13.837±0.200°, 14.481±0.200°, 16.055±0.200°, 17.6 53±0.200°, 18.356±0.200°, 20.083±0.200°, 20.839±0.200°, 21.388±0.200°, 22.379±0.200°, 24.577±0.200°, 25.104±0.200°, 26.402±0.200°, 31.540±0.200°.

6. The crystal form A of the compound of formula (I) according to claim 1, characterized in that: The X-ray powder diffraction pattern of the A crystal form is shown in FIG1 .

7. The crystal form A of the compound of formula (I) according to claim 1, characterized in that: The differential scanning calorimetry curve of the A crystal form has an endothermic peak at 173.5°C±3°C.

8. The crystal form A of the compound of formula (I) according to claim 1, whose differential scanning calorimetry curve is shown in Figure 2.

9. The crystal form A of the compound of formula (I) according to claim 1, characterized in that: The thermogravimetric analysis curve of the A crystal form shows a weight loss of 0.00% at 150.0°C±3°C.

10. The crystal form A of the compound of formula (I) according to claim 1, whose reanalysis curve is shown in Figure 3.

11. Crystal form B of the compound of formula (I), It is characterized in that Its X-ray powder diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 9.202±0.200°, 13.394±0.200°, 14.212±0.200°, 15.516±0.200°, 19.917±0.200°.

12. The crystal form B of the compound of formula (I) according to claim 11, characterized in that: The X-ray powder diffraction pattern of the B crystal form, expressed by 2θ angle, also includes at least 1, 2, or 3 diffraction peaks selected from the following: 10.968±0.200°, 16.483±0.200°, 18.306±0.200°.

13. According to claim 11, the crystal form B of the compound of formula (I) has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 9.202±0.200°, 10.968±0.200°, 13.394±0.200°, 14.212±0.200°, 15.516±0.200°, 16.483±0.200°, 18.306±0.200°, 19.917±0.200°.

14. The crystal form B of the compound of formula (I) according to claim 11, characterized in that: The X-ray powder diffraction pattern of the B crystal form, expressed by 2θ angle, also includes at least 7, 8, 9, 10, or 11 diffraction peaks selected from the following: 5.459±0.200°, 7.096±0.200°, 10.968±0.200°, 16.483±0.200°, 18.306±0.200°, 19.488±0.200°, 20.681±0.200°, 21.526±0.200°, 22.409±0.200°, 24.622±0.200°, 25.661±0.200°.

15. The crystal form B of the compound of formula (I) according to claim 11, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 5.459±0.200°, 7.096±0.200°, 9.202±0.200°, 10.968±0.200°, 13.394±0.200°, 14.212±0.200°, 15.5 16±0.200°, 16.483±0.200°, 18.306±0.200°, 19.488±0.200°, 19.917±0.200°, 20.681±0.200°, 21.526±0.200°, 22.409±0.200°, 24.622±0.200°, 25.661±0.200°.

16. The crystal form B of the compound of formula (I) according to claim 11, characterized in that: The X-ray powder diffraction pattern of the B crystal form is shown in FIG4 .

17. The crystal form B of the compound of formula (I) according to claim 11, characterized in that: The differential scanning calorimetry curve of the B crystal form has an endothermic peak at 128.4°C±3°C.

18. The crystal form B of the compound of formula (I) according to claim 11, whose differential scanning calorimetry curve is shown in Figure 5.

19. The crystal form B of the compound of formula (I) according to claim 11, characterized in that: The thermogravimetric analysis curve of the B crystal form shows a weight loss of 9.77% at 150.0°C±3°C.

20. The crystal form B of the compound of formula (I) according to claim 11, whose thermogravimetric analysis curve is shown in Figure 6.

21. The crystal form B of the compound of formula (I) according to claim 11, characterized in that: The B crystal form may exist in the form of a solvate crystal.

22. Crystal form C of the compound of formula (I), It is characterized in that Its X-ray powder diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 6.040±0.200°, 6.334±0.200°, 8.399±0.200°, 9.435±0.200°, 12.060±0.200°, and 18.126±0.200°.

23. The crystal form C of the compound of formula (I) according to claim 22, characterized in that: The X-ray powder diffraction pattern of the C crystal form, expressed by 2θ angle, also contains at least 1, 2, or 3 diffraction peaks selected from the following: 4.883±0.200°, 8.928±0.200°, 11.168±0.200°.

24. According to claim 22, the crystal form C of the compound of formula (I) has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 4.883±0.200°, 6.040±0.200°, 6.334±0.200°, 8.399±0.200°, 8.928±0.200°, 9.435±0.200°, 11.168±0.200°, 12.060±0.200°, and 18.126±0.200°.

25. The crystal form C of the compound of formula (I) according to claim 22, characterized in that: The X-ray powder diffraction pattern of the C crystal form, expressed by 2θ angle, also includes at least 7, 8, 9, 10, or 11 diffraction peaks selected from the following: 4.883±0.200°, 8.928±0.200°, 11.168±0.200°, 12.988±0.200°, 14.821±0.200°, 16.182±0.200°, 18.545±0.200°, 19.474±0.200°, 20.199±0.200°, 24.294±0.200°, 25.697±0.200°.

26. The crystal form C of the compound of formula (I) according to claim 22, wherein its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 4.883±0.200°, 6.040±0.200°, 6.334±0.200°, 8.399±0.200°, 8.928±0.200°, 9.435±0.200°, 11.168±0.200° , 12.060±0.200°, 12.988±0.200°, 14.821±0.200°, 16.182±0.200°, 18.126±0.200°, 18.545±0.200°, 19.474±0.200°, 20.199±0.200°, 24.294±0.200°, 25.697±0.200°.

27. The crystal form C of the compound of formula (I) according to claim 22, characterized in that: The X-ray powder diffraction pattern of the C crystal form is shown in FIG7 .

28. The crystal form C of the compound of formula (I) according to claim 22, characterized in that: The differential scanning calorimetry curve of the C crystal form has an endothermic peak at 113.3°C±3°C.

29. The crystal form C of the compound of formula (I) according to claim 22, whose differential scanning calorimetry curve is shown in Figure 8.

30. The crystal form C of the compound of formula (I) according to claim 22, characterized in that: The thermogravimetric analysis curve of the C crystal form shows a weight loss of 3.52% at 110.0°C±3°C and a weight loss of 10.58% at 150.0°C±3°C.

31. The crystal form C of the compound of formula (I) according to claim 22, whose thermogravimetric analysis curve is shown in Figure 9.

32. The crystal form C of the compound of formula (I) according to claim 22, characterized in that: The C crystal form may exist in the form of a solvate crystal.

33. Crystalline Form D of the compound of formula (I), It is characterized in that Its X-ray powder diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 9.136±0.200°, 9.908±0.200°, 17.787±0.200°, 18.329±0.200°, and 24.298±0.200°.

34. The crystal form D of the compound of formula (I) according to claim 33, characterized in that: The X-ray powder diffraction pattern of the D crystal form is shown in FIG10 .

35. The crystal form D of the compound of formula (I) according to claim 33, characterized in that: The differential scanning calorimetry curve of the D crystal form has an endothermic peak at 113.3°C±3°C.

36. The D crystal form of the compound of formula (I) according to claim 33, whose differential scanning calorimetry curve is shown in Figure 11.

37. The crystal form D of the compound of formula (I) according to claim 33, characterized in that: The thermogravimetric analysis curve of the D crystal form shows a weight loss of 6.59% at 90.0°C±3°C and a weight loss of 15.60% at 150.0°C±3°C.

38. The D crystal form of the compound of formula (I) according to claim 33, whose thermogravimetric analysis curve is shown in Figure 12.

39. The crystal form D of the compound of formula (I) according to claim 33, characterized in that: The D crystal form may exist in the form of solvate crystals.

40. Crystal form E of the compound of formula (I), It is characterized in that Its X-ray powder diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 5.987±0.200°, 9.928±0.200°, 11.998±0.200°, 13.499±0.200°, and 18.044±0.200°.

41. According to claim 40, the E crystal form of the compound of formula (I) has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ angles: 5.987±0.200°, 9.928±0.200°, 11.998±0.200°, 13.499±0.200°, 18.044±0.200°, 19.492±0.200°, 25.173±0.200°.

42. According to the E crystal form of the compound of formula (I) according to claim 40, its XRPD pattern is shown in Figure 13.

43. The E crystal form of the compound of formula (I) according to claim 40, characterized in that The differential scanning calorimetry curve of the E crystal form has an endothermic peak at 113.7°C±3°C.

44. The E crystal form of the compound of formula (I) according to claim 40, whose DSC spectrum is shown in Figure 14.

45. The E crystal form of the compound of formula (I) according to claim 40, characterized in that The thermogravimetric analysis curve of the E crystal form shows a weight loss of 13.97% at 150.0°C±3°C.

46. ​​The E crystal form of the compound of formula (I) according to claim 40, whose thermogravimetric analysis curve is shown in Figure 15.

47. The E crystal form of the compound of formula (I) according to claim 40, characterized in that The E crystal form may exist in the form of solvate crystals.

48. A crystalline composition comprising the crystal form A according to any one of claims 1 to 10, the crystal form B according to any one of claims 11 to 21, the crystal form C according to any one of claims 22 to 32, the crystal form D according to any one of claims 33 to 39, or the crystal form E according to any one of claims 40 to 47.

49. A pharmaceutical composition comprising the crystal form A according to any one of claims 1 to 10, the crystal form B according to any one of claims 11 to 21, the crystal form C according to any one of claims 22 to 32, the crystal form D according to any one of claims 33 to 39, or the crystal form E according to any one of claims 40 to 47.

50. Use of the crystal form A according to any one of claims 1 to 10, the crystal form B according to any one of claims 11 to 21, the crystal form C according to any one of claims 22 to 32, the crystal form D according to any one of claims 33 to 39, the crystal form E according to any one of claims 40 to 47, the crystalline composition according to claim 48, or the pharmaceutical composition according to claim 49 in the preparation of a medicament for treating diseases related to coronavirus infection.

51. The use according to claim 50, wherein: Diseases associated with coronavirus infection are SARS-CoV-2 virus infection.

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