Crystal of spiro compound and preparation method thereof

Abstract

The invention relates to a crystal form of a spiro compound as shown in formula (I), a preparation method of the crystal form and application of the crystal form to treatment of diseases caused by coronavirus infection and / or small RNA virus infection, and # imgabs0 #

Description

Crystallization of spirocyclic compound and preparation method thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority and benefits of Chinese Patent Application No. 202310064780.5 filed with the State Intellectual Property Office of China on January 16, 2023, and the contents disclosed in said application are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to the field of pharmaceutical chemistry, and in particular to a crystal form of a spirocyclic compound, a preparation method thereof, and a pharmaceutical composition and use thereof. Background Art

[0004] Coronaviruses are single-stranded, positive-sense RNA viruses, some of which can spread widely among humans and cause severe symptoms. Currently, there are seven known coronaviruses that can infect humans: HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2. Most of the functional proteins of coronaviruses are encoded by the ORF1ab gene, which is first translated into a polyprotein and then cleaved into multiple active proteins by 3CL proteases and PL proteases. Therefore, inhibiting 3CL protease activity can effectively inhibit viral replication. The 3CL proteases of different coronaviruses share a high degree of structural homology, and therefore, 3CL protease inhibitors generally have broad-spectrum anti-coronavirus activity.

[0005] In addition to coronaviruses, 3CL proteases also play a key role in the hydrolysis of polyproteins encoded by picornaviruses. 3CL protease inhibitors can effectively inhibit picornavirus replication. Enterovirus 71, a picornavirus, is a common cause of hand, foot and mouth disease (HFMD) and can also cause a variety of other illnesses, including meningitis, brainstem encephalitis, and myocarditis. In recent years, EV71 has repeatedly caused outbreaks in infants and young children, and effective treatments remain clinically lacking.

[0006] Therefore, there is still a clinical need for the development of pharmaceutically active substances that can be used to inhibit RNA / small RNA viruses including coronaviruses and enterovirus 71.

[0007] Summary of the Invention

[0008] In one aspect, the present disclosure provides a crystalline compound of formula (I)

[0009] In another aspect, the present disclosure provides a crystalline form A of a compound of formula (I),

[0010] The X-ray powder diffraction pattern of the A crystal form expressed by the diffraction angle 2θ has diffraction peaks at 10.88±0.20°, 15.09±0.20°, 17.67±0.20°, 18.28±0.20° and 20.64±0.20°.

[0011] In another aspect, the present disclosure provides a method for preparing Form A of the compound of formula (I), comprising:

[0012] The compound of formula (I) is mixed with a solvent (i) and a solvent (ii), crystallized, and a solid is separated; the solvent (i) is selected from at least one of isopropyl acetate, ethyl acetate, and isopropanol, and the solvent (ii) is selected from at least one of n-hexane and n-heptane.

[0013] In another aspect, the present disclosure provides a solvate of a compound of formula (I),

[0014] The solvate is selected from methyl tert-butyl ether solvate and 2-methyltetrahydrofuran solvate.

[0015] In another aspect, the present disclosure provides a methyl tert-butyl ether solvate of a compound of formula (I) in Form B,

[0016] The X-ray powder diffraction pattern of the B crystal form represented by the diffraction angle 2θ has diffraction peaks at 6.26±0.20°, 17.66±0.20° and 20.24±0.20°.

[0017] In another aspect, the present disclosure provides a method for preparing crystal form B, comprising: mixing the compound of formula (I) with methyl tert-butyl ether, stirring, and separating a solid.

[0018] In another aspect, the present disclosure provides a 2-methyltetrahydrofuran solvate of a compound of formula (I) in Form C,

[0019] The X-ray powder diffraction pattern of the C crystal form represented by the diffraction angle 2θ has diffraction peaks at 6.21±0.20°, 17.48±0.20° and 20.86±0.20°.

[0020] In another aspect, the present disclosure provides a method for preparing Form C, comprising: mixing the compound of formula (I) with 2-methyltetrahydrofuran, stirring, and separating a solid after crystallization.

[0021] In yet another aspect, the present disclosure provides a pharmaceutical combination comprising the compound of formula (I) described herein in crystalline form A, crystalline form B, crystalline form C, or a combination thereof, and other antiviral drugs.

[0022] In another aspect, the present disclosure provides a pharmaceutical composition comprising the compound of formula (I) described herein, crystalline form A, crystalline form B, crystalline form C, or a combination thereof, and pharmaceutically acceptable excipients.

[0023] In another aspect, the present disclosure provides the use of the crystalline form A, crystalline form B, crystalline form C of the compound of formula (I) described herein, or a combination thereof, the drug combination described herein, or the pharmaceutical composition described herein in the preparation of a medicament for preventing or treating diseases related to coronavirus and / or picornavirus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG1 is an XRPD pattern of 2-methyltetrahydrofuran solvate Form C of the compound of formula (I).

[0025] FIG2 is a DSC spectrum of 2-methyltetrahydrofuran solvate Form C of the compound of formula (I).

[0026] FIG3 is an XRPD pattern of methyl tert-butyl ether solvate Form B of the compound of formula (I).

[0027] FIG4 is a DSC spectrum of methyl tert-butyl ether solvate Form B of the compound of formula (I).

[0028] Figure 5 is the XRPD pattern of the crystalline form A of compound of formula (I).

[0029] FIG6 is a DSC spectrum of the crystalline form A of compound of formula (I).

[0030] Figure 7 is a TGA spectrum of the crystal form A of compound of formula (I).

[0031] FIG8 is a graph showing the inhibitory effect of the compound of formula (I) on the viral titer in the lungs of mice 2 days after infection (Figure A) and 4 days after infection (Figure B) in Test Example 4.

[0032] FIG9 is a graph showing changes in mouse body weight in Test Example 4.

[0033] FIG10 is a graph showing the inhibitory effect of the compound of formula (I) on the viral titer in the mouse brain 4 days after infection in Test Example 4. DETAILED DESCRIPTION

[0034] The compound of formula (I) is a novel 3CL protease inhibitor with broad-spectrum anti-coronavirus activity. The structure of the compound of formula (I) is as follows:

[0035] The present disclosure provides crystals of the compound of formula (I).

[0036] The present disclosure provides a crystalline form A of a compound of formula (I), wherein the crystalline form A has an X-ray powder diffraction pattern represented by a diffraction angle 2θ, with diffraction peaks at 10.88±0.20°, 15.09±0.20°, 17.67±0.20°, 18.28±0.20° and 20.64±0.20°.

[0037] In some embodiments, the crystal form A of the compound of formula (I) has an X-ray powder diffraction pattern represented by a diffraction angle 2θ, with diffraction peaks at 10.88±0.20°, 15.09±0.20°, 16.61±0.20°, 17.67±0.20°, 18.28±0.20°, 18.50±0.20°, 20.09±0.20° and 20.64±0.20°.

[0038] In some embodiments, the crystalline form A of the compound of formula (I) has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ, with diffraction peaks at 10.27±0.20°, 10.88±0.20°, 11.86±0.20°, 15.09±0.20°, 16.61±0.20°, 17.67±0.20°, 18.28±0.20°, 18.50±0.20°, 20.09±0.20° and 20.64±0.20°.

[0039] In some embodiments, the crystalline form A of the compound of formula (I) has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ at 9.29±0.20°, 10.27±0.20°, 10.88±0.20°, 10.97±0.20°, 11.86±0.20°, 14.27±0.20°, 14.92±0.20°, 15.09±0.20°. , 15.61±0.20°, 15.78±0.20°, 16.61±0.20°, 17.67±0.20°, 18.28±0.20°, 18.50±0.20°, 20.09±0.20°, 20.64±0.20°, 22.18±0.20°, 23.80±0.20° and 25.63±0.20°.

[0040] In some embodiments, the crystal form A of the compound of formula (I) has an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, and the diffraction peaks are shown in Table 3.

[0041] In some embodiments, the Form A of the compound of Formula (I) has an X-ray powder diffraction pattern represented by a diffraction angle 2θ substantially as shown in FIG5 .

[0042] In some embodiments, the Form A of the compound of Formula (I) has a DSC spectrum with a peak at 213.78°C±5.0°C.

[0043] In some embodiments, the DSC spectrum of Form A of the compound of Formula (I) is substantially as shown in FIG6 .

[0044] The present disclosure also provides a method for preparing a crystalline form of compound A of formula (I), comprising:

[0045] The compound of formula (I) is mixed with a solvent (i) and a solvent (ii), crystallized, and a solid is separated; the solvent (i) is selected from at least one of isopropyl acetate, ethyl acetate, and isopropyl alcohol, preferably isopropyl acetate, and the solvent (ii) is selected from at least one of n-hexane and n-heptane, preferably n-heptane.

[0046] The mixing method of the compound of formula (I) with solvent (i) and solvent (ii) includes at least the following specific methods: the compound of formula (I) is first mixed with solvent (i) and then further mixed with solvent (ii); or, the compound of formula (I) is mixed with a mixed solvent of solvent (i) and solvent (ii).

[0047] In some embodiments, in the preparation method of the crystal form A, the volume (mL) of the solvent (i) is 1 to 100 times, preferably 2 to 50 times, the mass (g) of the compound.

[0048] In some embodiments, in the preparation method of the crystal form A, the volume (mL) of the solvent (ii) is 2 to 100 times, preferably 5 to 50 times, the mass (g) of the compound.

[0049] In some embodiments, the crystallization in the preparation method of the crystal form A is carried out at 10-80°C, preferably at 15-45°C, and more preferably at 20-30°C.

[0050] In some embodiments, in the method for preparing the crystal form A, the separation step comprises filtering, collecting the solid, and drying.

[0051] The present disclosure also provides a solvate of the compound of formula (I), wherein the solvate is selected from methyl tert-butyl ether solvate and 2-methyltetrahydrofuran solvate.

[0052] In some embodiments, in the solvate of the compound of formula (I), the molar ratio of the compound of formula (I) to the solvent is about 0.5-2.

[0053] In some embodiments, in the solvate of the compound of formula (I), the molar ratio of the compound of formula (I) to the solvent is about 0.8 to 1.2.

[0054] In some embodiments, in the solvate of the compound of formula (I), the molar ratio of the compound of formula (I) to the solvent is about 1.0.

[0055] The present disclosure also provides a methyl tert-butyl ether solvate of the compound of formula (I) in Form B, wherein the Form B has an X-ray powder diffraction pattern represented by a diffraction angle 2θ, with diffraction peaks at 6.26±0.20°, 17.66±0.20°, and 20.24±0.20°.

[0056] In some embodiments, the B crystalline form has an X-ray powder diffraction pattern represented by a diffraction angle 2θ, and has diffraction peaks at 6.26±0.20°, 7.27±0.20°, 11.47±0.20°, 13.06±0.20°, 15.57±0.20°, 17.66±0.20°, 20.24±0.20° and 22.72±0.20°.

[0057] In some embodiments, the B crystalline form has an X-ray powder diffraction pattern expressed as a diffraction angle 2θ, and has diffraction peaks at 6.26±0.20°, 7.27±0.20°, 8.84±0.20°, 10.18±0.20°, 10.30±0.20°, 11.47±0.20°, 13.06±0.20°, 14.44±0.20°, 15.57±0.20°, 17.66±0.20°, 20.24±0.20°, 20.51±0.20° and 22.72±0.20°.

[0058] In some embodiments, the B crystal form has an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, and the diffraction peaks are shown in Table 2.

[0059] In some embodiments, the Form B has an X-ray powder diffraction pattern represented by a diffraction angle 2θ substantially as shown in FIG3 .

[0060] In some embodiments, the Form B has a DSC spectrum with peaks at 93.36°C±5.0°C, 170.25°C±5.0°C, and 214.18±5.0°C.

[0061] In some embodiments, the DSC spectrum of the Form B is substantially as shown in FIG4 .

[0062] The present disclosure also provides a method for preparing Form B, comprising:

[0063] The compound of formula (I) is mixed with methyl tert-butyl ether, stirred, and a solid is separated.

[0064] In some embodiments, in the preparation method of the Form B, the volume (mL) of the methyl tert-butyl ether is 2 to 100 times, preferably 5 to 50 times, and more preferably 10 to 30 times the mass (g) of the compound of formula (I).

[0065] In some embodiments, in the preparation method of the Form B, the stirring step is carried out at 15-55°C, preferably at 45-55°C, and more preferably at 50-55°C.

[0066] In some embodiments, in the preparation method of the crystal form B, the compound of formula (I) is a 2-methyltetrahydrofuran solvate of the compound of formula (I).

[0067] The present disclosure also provides Form C of a 2-methyltetrahydrofuran solvate of a compound of formula (I), wherein the Form C has an X-ray powder diffraction pattern represented by a diffraction angle 2θ, with diffraction peaks at 6.21±0.20°, 17.48±0.20°, and 20.86±0.20°.

[0068] In some embodiments, the C crystalline form has an X-ray powder diffraction pattern represented by a diffraction angle 2θ, with diffraction peaks at 6.21±0.20°, 7.04±0.20°, 11.71±0.20°, 17.48±0.20°, 20.59±0.20° and 20.86±0.20°.

[0069] In some embodiments, the C crystalline form has an X-ray powder diffraction pattern represented by a diffraction angle 2θ, and has diffraction peaks at 6.21±0.20°, 7.04±0.20°, 11.71±0.20°, 14.74±0.20°, 15.77±0.20°, 17.48±0.20°, 20.59±0.20° and 20.86±0.20°.

[0070] In some embodiments, the C crystal form has an X-ray powder diffraction pattern represented by a diffraction angle of 2θ, and the diffraction peaks are shown in Table 1.

[0071] In some embodiments, the Form C has an X-ray powder diffraction pattern represented by a diffraction angle 2θ substantially as shown in FIG1 .

[0072] In some embodiments, the Form C has a DSC spectrum with peaks at 90.39°C±5.0°C, 167.59°C±5.0°C, and 214.22±5.0°C.

[0073] In some embodiments, the DSC spectrum of the Form C is substantially as shown in FIG2 .

[0074] The present disclosure also provides a method for preparing Form C, comprising:

[0075] The compound of formula (I) is mixed with 2-methyltetrahydrofuran, stirred, and crystallized to separate the solid.

[0076] In some embodiments, in the preparation method of the crystal form C, the volume (mL) of the 2-methyltetrahydrofuran is 2 to 50 times the mass (g) of the compound of formula (I), preferably 3 to 20 times, and more preferably 5 to 15 times.

[0077] In some embodiments, in the method for preparing the Form C, the stirring step is performed at 15-80°C, preferably at 40-80°C, for example, at 50-60°C or 70-80°C.

[0078] In some embodiments, in the preparation method of the crystal form C, the crystallization is cooling crystallization.

[0079] In some embodiments, in the preparation method of the crystal form C, the cooling step is to cool the reaction system to 0-35°C, preferably to 5-30°C, and more preferably to 10-25°C.

[0080] On the other hand, the present disclosure also provides a drug combination comprising the above-mentioned compound of formula (I) in crystal form A, crystal form B, crystal form C, or a combination thereof, and other antiviral drugs.

[0081] On the other hand, the present disclosure provides a pharmaceutical composition comprising the above-mentioned compound of formula (I) in crystalline form A, crystalline form B, crystalline form C, or a combination thereof, and pharmaceutically acceptable excipients; optionally, the pharmaceutical composition further comprises other antiviral drugs.

[0082] In some embodiments, the other antiviral drug is ritonavir.

[0083] On the other hand, the present disclosure provides the use of the above-mentioned crystal form A, crystal form B, crystal form C of the compound of formula (I) or a combination thereof, or the above-mentioned drug combination or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing or treating related diseases caused by coronavirus and / or picornavirus infection.

[0084] On the other hand, the present disclosure provides the use of the above-mentioned Form A, Form B, Form C of the compound of formula (I) or a combination thereof, or the above-mentioned drug combination or the above-mentioned pharmaceutical composition in preventing or treating diseases related to coronavirus and / or picornavirus infection.

[0085] On the other hand, the present disclosure provides the above-mentioned compound of formula (I) crystal form A, crystal form B, crystal form C or a combination thereof, or the above-mentioned drug combination or the above-mentioned pharmaceutical composition for preventing or treating related diseases caused by coronavirus and / or picornavirus infection.

[0086] On the other hand, the present disclosure provides a method for treating diseases related to coronavirus and / or picornavirus infection, comprising administering a therapeutically effective amount of the above-mentioned Form A, Form B, Form C of the compound of formula (I), or a combination thereof, or the above-mentioned drug combination or the above-mentioned pharmaceutical composition to a subject in need of such treatment (e.g., a mammal, preferably a human).

[0087] The related diseases caused by coronavirus and / or picornavirus infection described in the present disclosure include but are not limited to respiratory tract infection, pneumonia or its complications.

[0088] The coronavirus of the present disclosure is selected from SARS-CoV, MERS-CoV, H229E-CoV, HKU1-CoV, NL63-CoV, OC43-CoV or SARS-CoV-2. The small RNA virus of the present disclosure is selected from enterovirus 71.

[0089] The crystal form A, crystal form B or crystal form C of the compound of formula (I) disclosed in the present invention has at least one advantage in terms of pharmacological efficacy, physicochemical properties, etc., and is suitable for preparation into the desired pharmaceutical composition.

[0090] Definitions and Explanations of Terms

[0091] Unless otherwise indicated, the terms used in this disclosure have the following meanings. The definitions of groups and terms described in this disclosure, including their definitions as examples, exemplary definitions, preferred definitions, definitions described in tables, and definitions of specific compounds in the examples, may be combined and coupled with each other in any manner. A particular term should not be considered as undefined or unclear unless specifically defined, but should be understood according to its ordinary meaning in the art. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0092] The term "solvate" refers to a complex or aggregate formed by one or more molecules of a solute and one or more molecules of a solvent. Solvate generally has a substantially fixed molar ratio of solute to solvent. This term also includes cage compounds, including cage compounds with water. Representative solvents include, for example, water, methanol, ethanol, isopropanol, acetic acid, 2-methyltetrahydrofuran, methyl tert-butyl ether, etc. When the solvent is water, the solvate formed is a hydrate.

[0093] The term "about" is used in this disclosure to mean approximately, around, roughly, or approximately. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower limits of the numerical range stated. Unless otherwise indicated, the term "about" is used herein to modify the upper and lower limits of a numerical value stated by a deviation of 10%.

[0094] Unless otherwise indicated, the terms "comprise," "comprise," or "comprises" and variations thereof, such as comprises or comprising, should be construed in an open, non-exclusive sense, ie, "including but not limited to."

[0095] References to "alternative embodiments" or "embodiments" in this disclosure mean that at least one embodiment includes the specific referenced elements, structures, or features described in connection with that embodiment. Thus, the phrases "alternative embodiments" or "embodiments" appearing in different places in this disclosure do not necessarily all refer to the same embodiment. Furthermore, the specific elements, structures, or features may be combined in any suitable manner in one or more embodiments.

[0096] The room temperature described in the present disclosure refers to 20±5.0°C.

[0097] The range "m to n" described in the present disclosure represents an abbreviation for any combination of real numbers between m and n, where m and n are both real numbers.

[0098] The “X-ray powder diffraction pattern” described in the present disclosure is measured using CuKα radiation.

[0099] The "X-ray powder diffraction pattern or XRPD pattern" described in the present disclosure refers to the Bragg formula 2d Sinθ = nλ (wherein, d is the interplanar spacing, θ is the diffraction angle, λ is the wavelength of the incident X-ray, and the diffraction order n is any positive integer, generally the first-order diffraction peak is taken, n = 1). When the X-ray is incident on an atomic plane with a lattice plane spacing d in a crystal or a partial crystal sample at a grazing angle θ (the complementary angle of the incident angle, also known as the Bragg angle), the Bragg equation is satisfied, thereby measuring this set of X-ray powder diffraction patterns.

[0100] For the same crystalline form of the same compound, the peak positions of their XRPD patterns are generally similar, but the relative intensity errors may be large. It should also be noted that in the identification of mixtures, some diffraction lines may be missing due to factors such as a decrease in content. In this case, it is not necessary to rely on all diffraction peaks observed in a high-purity sample; even a single diffraction peak may be characteristic for a given crystal.

[0101] The "2θ or 2θ angle" described in the present disclosure refers to the diffraction angle, θ is the Bragg angle, and the unit is ° or degree.

[0102] Those skilled in the art will appreciate that the molar ratios of the disclosed compounds to the acid / base molecules and the solvent molecules in the solvate often have a certain degree of error due to factors such as crystal defects and measurement errors. Generally, a ±10% ratio is considered within a reasonable error range. While there may be a certain degree of error depending on the context in which the molar ratio is used, this error should not exceed ±10%, preferably ±5%.

[0103] The term "therapeutically effective amount" refers to an amount of a compound of the present disclosure that (i) treats a specific disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a specific disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of a specific disease, condition, or disorder as described herein. The amount of a compound of the present disclosure 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 one skilled in the art based on their knowledge and this disclosure.

[0104] The term "treatment" means administering the compound or formulation described herein to improve or eliminate a disease or one or more symptoms associated with the disease, and includes:

[0105] (i) inhibiting a disease or disease state, i.e., arresting its development;

[0106] (ii) ameliorating the disease or condition, i.e., causing regression of the disease or condition.

[0107] The term "prevention" means administering a compound or formulation described herein to prevent a disease or one or more symptoms associated with the disease, and includes:

[0108] Preventing a disease or disease state from occurring in a subject (eg, a mammal) is particularly useful when such subject is susceptible to having the disease state but has not yet been diagnosed as having the disease state.

[0109] As used herein, the term "subject" or "patient" includes mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates (e.g., chimpanzees and other apes and monkeys); livestock, such as cattle, horses, sheep, goats, and pigs; domestic animals, such as rabbits, dogs, and cats; and laboratory animals, including rodents, such as rats, mice, and guinea pigs. Examples of non-human mammals include, but are not limited to, birds and fish.

[0110] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0111] The term "pharmaceutically acceptable excipient" refers to an excipient that is non-irritating to organisms and does not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art and include, for example, carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.

[0112] The term "drug combination" refers to a combination of two or more active ingredients. In some embodiments of the present disclosure, the active ingredients in the drug combination can be administered simultaneously. In some embodiments of the present disclosure, the active ingredients in the drug combination can also be administered separately or sequentially.

[0113] The pharmaceutical compositions of the present disclosure can be prepared by combining the compounds of the present disclosure or their pharmaceutically acceptable salts or solvates with suitable pharmaceutically acceptable excipients, and can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.

[0114] Typical routes of administration of the compounds of the present disclosure, or pharmaceutically acceptable salts thereof, or solvates thereof, or pharmaceutical compositions comprising the same, include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0115] The pharmaceutical composition of the present disclosure can be manufactured by methods well known in the art, such as conventional mixing methods, dissolution methods, granulation methods, emulsification methods, freeze-drying methods, and the like.

[0116] In some embodiments, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These excipients enable the compounds of the present disclosure, or pharmaceutically acceptable salts thereof, or solvates thereof, to be formulated into tablets, pills, lozenges, dragees, capsules, liquids, gels, slurries, suspensions, and the like for oral administration to a patient.

[0117] Solid oral compositions can be prepared by conventional mixing, filling or tableting methods. For example, they can be obtained by mixing the active compound with a solid excipient, optionally grinding the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into granules to obtain tablets or dragee cores.

[0118] The pharmaceutical composition may also be suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in appropriate unit dosage forms.

[0119] The pharmaceutical composition of the present disclosure contains a therapeutically effective amount of the compound of formula (I) or its solvate crystalline form selected from 0.001 mg / kg body weight to 1000 mg / kg body weight, for example 0.01 mg / kg body weight to 500 mg / kg body weight, in the form of single or divided doses.

[0120] Those skilled in the art recognize that the measured data of XRPD peak positions and / or intensities for a given crystalline form of the same compound will vary within a range of error. The 2θ values ​​in this disclosure encompass an appropriate range of error, which is typically represented by "±". For example, a 2θ value represented in this disclosure as a specific angle value of ±0.20° represents that the specific angle value has an error range of ±0.20°, i.e., 5.92±0.20°2θ represents a 2θ range of 6.12 to 5.72. Depending on the sample preparation technique, the calibration technique applied to the instrument, human operator bias, etc., those skilled in the art recognize that an appropriate error range for XRPD diffraction angles may be ±0.20°, ±0.15°, ±0.10°, ±0.05° or less, and that some variability in peak intensity is allowed. The terms "substantially the same" or "substantially as shown" when used to describe an XRPD pattern refer to a pattern that includes diffraction peaks that have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the diffraction angles within a standard deviation of ±0.2° 2θ.

[0121] As those skilled in the art recognize that the measured data of the DSC spectra for a given crystalline form of the same compound will vary within an error tolerance. A suitable error range is allowed for the single peak value (expressed in degrees Celsius). Typically, the error range is represented by "±". For the same crystal form of the same compound, in consecutive analyses, the thermal transition temperature and melting point errors are typically within ±5.0°C. For example, a peak value of "170.25±5.0" is represented as being within the range of 165.25 to 175.25. Depending on the sample preparation technique, the calibration technique applied to the instrument, human operator bias, etc., those skilled in the art recognize that the appropriate error range for the single peak value may be ±5.0, ±4.0, ±3.0, ±2.0 or less.

[0122] The salt forms and / or crystal forms of the present disclosure may also be isotopically labeled. The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but in which one or more atoms are replaced by atoms having an atomic mass or mass number different from that normally found in nature. Examples of isotopes that may be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H. 3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 123 I. 125 I and 36 Cl et al.

[0123] Certain isotopically labeled compounds of the present disclosure (e.g., 3 H and 14 C-labeled) can be used in compound and / or substrate tissue distribution assays. 3 H) and carbon-14 (i.e. 14 C) isotopes are particularly preferred due to their ease of preparation and detectability. Positron emitting isotopes, such as 15 O. 13 N. 11 C and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the disclosure can generally be prepared by following procedures analogous to those disclosed in the Schemes and / or Examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0124] In addition, the use of heavier isotopes such as deuterium (i.e. 2 H)) substitution may offer certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and may therefore be preferred in certain circumstances, wherein deuterium substitution may be partial or complete, partial deuterium substitution meaning that at least one hydrogen is replaced by deuterium.

[0125] The compounds of the present invention or their pharmaceutically acceptable salts or solvates thereof can be prepared by various 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 examples disclosed herein.

[0126] The chemical reactions described in the embodiments of the present disclosure are carried out in a suitable solvent that is compatible with the chemical transformations described herein and the reagents and materials required. To obtain the compounds of the present disclosure, or pharmaceutically acceptable salts or solvates thereof, it may sometimes be necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0127] Test conditions of the instruments used in this public experiment:

[0128] 1. X-ray powder diffraction

[0129] Instrument model: Bruker D8 Focus

[0130] X-ray source: Cu Kα

[0131] 1.54060; 1.54439; Kα2 / Kα1 intensity ratio: 0.50

[0132] wavelength 1.54060

[0133] Slit (°): 2.5

[0134] Scanning mode: θ / 2θ, scanning range: 3-40° (2θ angle)

[0135] Dwell time (seconds): 0.12

[0136] Scan step (°2θ): 0.01

[0137] Scanning flow rate: 5° / min

[0138] Voltage: 40kV

[0139] Current: 40mA

[0140] 2. Differential Scanning Calorimeter

[0141] Instrument model: Discovery DSC2500

[0142] Purge gas: nitrogen

[0143] Sample pan: aluminum pan, non-sealed cover

[0144] Method: Linear temperature ramp

[0145] Heating rate: 10℃ / min

[0146] Temperature range: 30℃~300℃; or 30℃~400℃;

[0147] 3. Thermogravimetric analyzer

[0148] Instrument model: Discovery TA 55

[0149] Purge gas: nitrogen

[0150] Sample pan: platinum, open

[0151] Method: Linear temperature ramp

[0152] Heating rate: 10℃ / min

[0153] Temperature range: 30℃~300℃

[0154] 4. Dynamic moisture adsorption instrument

[0155] Instrument model: DVS Intrinsic PLUS

[0156] DVS parameters:

[0157] Temperature: 25℃;

[0158] Balance: dm / dt = 0.002% / min

[0159] RH (%) test level: 10%

[0160] RH (%) test step range: 0%-90%-0%.

[0161] The compounds disclosed herein 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 examples disclosed herein.

[0162] The chemical reactions of the embodiments of the present disclosure are carried out in a suitable solvent that is compatible with the chemical transformations of the present disclosure and the reagents and materials required. In order to obtain the compounds of the present disclosure, it is sometimes necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0163] DETAILED DESCRIPTION

[0164] The invention is described in detail below by way of examples, but this is not intended to limit the present disclosure in any way. While the present disclosure has been described in detail herein, including specific embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments without departing from the spirit and scope of the present disclosure. All reagents used in the present disclosure are commercially available and can be used without further purification.

[0165] Unless otherwise specified, the ratios expressed for mixed solvents are volume ratios. Unless otherwise specified, % refers to wt%.

[0166] Compounds are artificially or Software naming, commercially available compounds use supplier catalog names.

[0167] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The units of NMR shifts are 10 -6 The solvents for NMR measurements are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and tetramethylsilane (TMS) is the internal standard.

[0168] Example 1: Preparation of compound of formula (I)

[0169] 1.1. Preparation of compound 1-1:

[0170] Step 1: Add the starting material SMA (2.74 g, 11.85 mmol), 35 ml of dichloromethane, and 35 ml of DMF to a reaction flask, cool to 0°C, and sequentially add the starting material SMB (3.56 g, 11.86 mmol), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP, 6.29 g, 14.22 mmol), and N-methylmorpholine (NMM, 3.91 ml, 35.56 mmol). Warm to room temperature and react for 10 h. After the reaction, add an appropriate amount of dichloromethane, and wash the organic phase with 1N aqueous hydrochloric acid and saturated brine. Dry the organic phase over anhydrous sodium sulfate, concentrate to dryness, and column chromatography to obtain 3.71 g of INT-1; ESI-MS: 433.2 m / z [M+H] + ; 1 H NMR (400 MHz, DMSO-d6): δ H:6.75(d,J=9.2Hz,1H),4.38(t,J=8.2Hz,1H),4.25(d,J=10.9Hz,1H),4.11(d,J=9.3Hz,1H),3.93(t,J=9.3Hz,1H),3 .62(s,3H),3.40-3.31(m,4H),2.70(dd,J=13.1,7.9Hz,1H),2.37(dd,J=13.2,8.4Hz,1H),1.37(s,9H),0.94(s,9H).

[0171] Step 2: INT-1 (3.71 g, 8.58 mmol), 37 ml of THF, 37 ml of purified water, and lithium hydroxide monohydrate (0.72 g, 17.16 mmol) were added to a reaction flask and reacted at room temperature for 2 h. After the reaction, the pH was adjusted to 4 with concentrated hydrochloric acid and filtered to obtain 3.4 g of compound 1-1; ESI-MS: 419.2 m / z [M+H] + ; 1 H NMR (400 MHz, DMSO-d6): δ H :12.68(s,1H),6.71(d,J=9.4Hz,1H),4.38–4.19(m,2H),4.11(d,J=9.4Hz,1H),3.88(d,J=10.9Hz,1H), 3.41-3.29(m,4H),2.69(dd,J=13.1,7.9Hz,1H),2.34(dd,J=13.2,8.9Hz,1H),1.38(s,9H),0.94(s,9H).

[0172] 1.2. Preparation of compound 1-2:

[0173] Add 700 ml of ammonia-methanol solution (7 mol / L) and the starting material SMD (100 g, 0.349 mol) to the reaction flask, stir to dissolve, and maintain at 25±5°C for 36 hours. After the reaction is completed, concentrate the reaction solution until the remaining reaction solution is approximately 250 ml. Add 300 ml of isopropanol and continue to concentrate under reduced pressure until the remaining reaction solution is approximately 250 ml (repeat three times). Replace the atmosphere with nitrogen, cool the temperature to 10±5°C, and add 500 ml of hydrogen chloride-isopropanol solution (4 mol / L) to the reactor. After the addition is complete, heat the mixture to 25±5°C and maintain at 25±5°C for 9 hours. After the reaction, the reaction solution was concentrated under reduced pressure to a residual volume of about 250 ml. 300 ml of isopropanol was added and the reaction solution was further concentrated under reduced pressure to a residual volume of about 250 ml (repeated twice). 100 ml of isopropanol was added and stirred for 30 ± 5 min. The mixture was filtered and the filter cake was rinsed with 50 ml of isopropanol to obtain a wet product. The mixture was then dried under vacuum at 45 ± 5 °C to obtain 66.7 g of compound 1-2. 1 H NMR (400 MHz, DMSO-d6): δ H :8.45(d,J=5.1Hz,3H),8.25-8.04(m,1H),7.95(s,1H),7.67-7.49(m,1H),3.85-3.80(m,1H),3.19-3.13( m,2H),2.59-2.51(m,1H),2.32-2.27(m,1H),2.05-1.98(m,1H),1.82-1.66(m,2H); ESI-MS:172.1m / z[M+H] + .

[0174] 1.3. Preparation of compound of formula (I):

[0175] Step 1: Compound 1-1 (419 mg, 1 mmol) was placed in a two-necked flask. 5 mL of dichloromethane was added under nitrogen, followed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (400 mg, 1.1 mmol). The reaction mixture was stirred at room temperature for 1 h. Compound 1-2 (1 mmol) was dissolved in 1 mL of dichloromethane and added to the above system. N,N-diisopropylethylamine (2 mmol) was then added under an ice-water bath. The ice-water bath was removed and the system was stirred at room temperature overnight. After post-treatment, 50 mL of dichloromethane was added, followed by washing with 1 M aqueous hydrochloric acid three times, saturated aqueous sodium bicarbonate three times, and saturated brine. The organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and dried to obtain compound 1-3 (469 mg). ESI-MS: m / z 572.3 [M+H] + .

[0176] Step 2: Compound 1-3 (572 mg, 1 mmol) was dissolved in 3 mL of 4 M hydrogen chloride / 1,4-dioxane solution and stirred at ambient temperature. After TLC analysis indicated that the reaction of the starting material was essentially complete, the solvent was fully dried. The crude product was dissolved in 2 mL of dichloromethane under nitrogen protection. After adding triethylamine (3 mmol), the system was placed in an ice-water bath and trifluoroacetic anhydride (1.2 mmol) was added dropwise. After TLC analysis indicated that the reaction of the starting material was essentially complete, 50 mL of dichloromethane was added and the mixture was washed three times with a 1 M aqueous hydrochloric acid solution, three times with a saturated aqueous sodium bicarbonate solution, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and subjected to column chromatography to obtain compound 1-4 (265 mg). ESI-MS: m / z 568.3 [M+H] + .

[0177] Step 3: Compound 1-4 (113 mg, 0.2 mmol) and Burgess reagent (1.5 eq) were added to a two-necked flask. The mixture was flushed and released with nitrogen three times, followed by the addition of molecular sieve-dried dichloromethane. The mixture was stirred overnight at room temperature. Thin-layer chromatography (TLC) indicated that the reaction was essentially complete. Post-treatment with column chromatography afforded the compound of formula (I) (41 mg), which was confirmed to be amorphous by XRPD. 1 H NMR(400MHz, DMSO-d6)δ9.46(d,J=8.7Hz,1H),9.05(d,J=8.6Hz,1H),7.67(s,1H),4.97(ddd,J=11.0,8.5,5.0 Hz,1H),4.53(d,J=8.7Hz,1H),4.34(dd,J=9.9,7.1Hz,1H),4.26–4.14(m,1H),3.92(d,J=10.9Hz,1H),3.50–3. 34(m,4H),3.22–3.11(m,1H),3.06(td,J=9.3,7.1Hz,1H),2.68–2.58(m,1H),2.50–2.43(m,1H),2.31(dd,J=1 3.0,10.0Hz,1H),2.23–2.07(m,2H),1.71(tdd,J=14.9,10.3,7.4Hz,2H),0.99(s,9H).ESI-MS:550.3m / z[M+H] + .

[0178] Example 2: Preparation of 2-Methyltetrahydrofuran Solvate C of Formula (I)

[0179] Compound 1-4 (10.00 g) was dissolved in 100 ml of dichloromethane, and 8.39 g of Burgess reagent was added. The mixture was allowed to react at 25-26°C for 1 h. After the reaction, the mixture was quenched with 50 ml of saturated aqueous sodium bicarbonate solution. The organic phase was washed with 50 ml of 1 M aqueous hydrochloric acid and 50 ml of saturated aqueous sodium chloride, respectively. The mixture was then concentrated under reduced pressure to yield the compound of formula (I). The resulting compound of formula (I) was added to 100 ml of 2-methyltetrahydrofuran, heated to 70-80°C, dissolved, slowly cooled to crystallize, and filtered to yield 7.92 g of a solid. The resulting solid was confirmed to be 2-methyltetrahydrofuran solvate Form C of the compound of formula (I) by NMR, TGA, and X-ray powder diffraction analysis. The XRPD pattern of Form C is shown in Figure 1, and its DSC pattern is shown in Figure 2. The positions of its XRPD diffraction peaks are shown in Table 1 below.

[0180] Table 1

[0181] 1 H NMR (400MHz, DMSO-d6) δ9.47(d,J=7.6Hz,1H),9.06(d,J=8.6Hz,1H),7.68(s,1H),4.99-4.93(m,1H),4.52(d,J=7.0Hz,1H),4 .32(dd,J=9.9Hz,7.1Hz,1H),4.19(d,J=11.0Hz,1H),3.91(d,J=10.9Hz,1H),3.79-3.86(m,1H),3.78-3.71(m,1H),3.58-3.51 (m,1H),3.47-3.35(m,4H),3.14(t,J=9.3Hz,1H),3.08-3.02(m,1H),2.61(dd,J=13.0Hz,7.1Hz,1H),2.46-2.39(m,1H),2.35 -2.25(m,1H),2.18-2.07(m,2H),2.00-1.75(m,3H),1.75-1.67(m,2H),1.35-1.27(m,1H),1.12(d,J=6.1Hz,3H),0.97(s,9H).

[0182] Example 3: Preparation of Form B of Methyl Tert-Butyl Ether Solvate of Formula (I)

[0183] 1.6 kg of 2-methyltetrahydrofuran solvate of the compound of formula (I) was dissolved in 4.5 L of dichloromethane, concentrated under reduced pressure to a dry solid, pulverized, and added portionwise to 45 L of methyl tert-butyl ether. After the addition was completed, the mixture was heated to 50-55° C. and slurried for 1 hour, then slowly cooled to 20° C., filtered, and dried to obtain 1.41 kg of a solid. The resulting solid was confirmed to be Form B of the methyl tert-butyl ether solvate of the compound of formula (I) by nuclear magnetic resonance, TGA, and X-ray powder diffraction analysis. The XRPD pattern of Form B is shown in Figure 3 , and its DSC pattern is shown in Figure 4 . The positions of its XRPD diffraction peaks are shown in Table 2 below.

[0184] Table 2

[0185] 1H NMR (400MHz, DMSO-d6): δ9.48(d,J=8.7Hz,1H),9.06(d,J=8.6Hz,1H),7.68(s,1H),5.00-4.94(m,1H ),4.53(d,J=8.7Hz,1H),4.33(dd,J=9.8Hz,7.1Hz,1H),4.19(d,J=10.7Hz,1H),3.92(d,J=10.9Hz,1H ),3.44-3.37(m,4H),3.17-3.02(m,2H),3.08(s,3H),2.61(dd,J=12.8Hz,7.4Hz,1H),2.46-2.43(m,1 H), 2.30 (dd, J = 12.9Hz, 10.1Hz, 1H), 2.18-2.08 (m, 2H), 1.75-1.65 (m, 2H), 1.11 (s, 9H), 0.98 (s, 9H).

[0186] Example 4: Preparation of Crystalline Form A of Compound (I)

[0187] Method 1:

[0188] 270 g of the 2-methyltetrahydrofuran solvate of the compound of formula (I) was added to a mixed solvent of 540 ml of isopropyl acetate and 5.4 L of n-heptane, the temperature was raised to 55-65° C., and the mixture was beaten for 12 hours. The temperature was slowly lowered to 20-30° C., the mixture was stirred at this temperature for 1 hour, filtered, and dried to obtain 215 g of a solid. The resulting solid was confirmed to be Form A of the compound of formula (I) by nuclear magnetic resonance and X-ray powder diffraction. The XRPD pattern of Form A is shown in Figure 5 , its DSC pattern is shown in Figure 6 , and its TGA pattern is shown in Figure 7 . The positions of the XRPD diffraction peaks are shown in Table 3 below.

[0189] Table 3

[0190] 1H NMR (400MHz, DMSO-d6): δ9.47(d,J=8.4Hz,1H),9.06(d,J=8.6Hz,1H),7.68(s,1H),5.00-4.94 (m,1H),4.53(d,J=8.5Hz,1H),4.33(dd,J=9.8Hz,7.1Hz,1H),4.19(d,J=10.8Hz,1H),3.92(d, J=11.0Hz,1H),3.44-3.32(m,4H),3.17-3.02(m,2H),2.61(dd,J=12.7Hz,5.6Hz,1H),2.50-2. 43(m,1H),2.30(dd,J=12.8Hz,10.0Hz,1H),2.19-2.08(m,2H),1.75-1.68(m,2H),0.98(s,9H).

[0191] Method 2:

[0192] 10 mg of methyl tert-butyl ether solvate of the compound of formula (I) was dissolved in 50 μL of ethyl acetate. After adding 50 μL of n-heptane, the solution became turbid. After evaporating the solvent, the solid obtained was confirmed to be the crystal form A of the compound of formula (I) by X-ray powder diffraction spectrum.

[0193] Method 3:

[0194] 15 mg of methyl tert-butyl ether solvate of the compound of formula (I) was dissolved in 150 μL of isopropanol, and 400 μL of n-heptane was added, resulting in a turbid solution. Stirring was continued for 2 hours, and an additional 40 μL of n-heptane was added, resulting in a turbid solution. After evaporating the solvent, the resulting solid was confirmed to be Form A of the compound of formula (I) by X-ray powder diffraction.

[0195] Biological activity and related properties test examples

[0196] Test Example 1-1: Test on the activity of the compound of formula (I) in inhibiting SARS-CoV-2 3CLpro

[0197] Evaluation of the effect of compound of formula (I) on SARS-CoV-2 3CL using fluorescence resonance energy transfer method pro The volume of the entire enzymatic reaction system was 120 μL, the final concentration of the protease was 30 nM, and the final concentration of the substrate was 20 μM. The reaction system buffer included 50 mM Tris pH 7.3 and 1 mM EDTA. SARS-CoV-2 3CL was added to a 96-well plate. proThe protease and compound at different concentrations were incubated at 30°C for 10 minutes. The substrate was added and the plate was quickly placed in a microplate reader for reading. The excitation and emission values ​​were 320nM and 405nM, respectively. The test time was 3.5 minutes, and the fluorescence value was read every 35 seconds. The final result was the reaction rate fitted with the readings of the first 2 minutes, and the inhibition rate was calculated by comparing with the control group (DMSO). The IC was fitted using GraphPad Prism 8 software. 50 values ​​and inhibition rate curves.

[0198] The experimental results show that the compound of formula (I) has an anti-SARS-CoV-2 3CL pro IC 50 The value was <0.1 μM, indicating a strong inhibitory effect.

[0199] Test Example 1-2: Inhibitory activity test of the compound of formula (I) against the mutant 3CL protease of SARS-CoV-2 Omicron strain

[0200] Experimental principle: The method of fluorescence resonance energy transfer (FRET) generated by enzyme-substrate reaction was used to study the inhibitory effect of the compound of formula (I) disclosed herein on the activity of the mutant 3CL protease (P132H) of the Omicron strain.

[0201] The experimental materials are shown in the following table:

[0202] Experimental instruments and equipment:

[0203] Experimental steps:

[0204] Prepare reaction buffer containing 20mM Tris-HCl, 1mM EDTA, 0.01% BSA, 1mM DTT and 100mM NaCl. Use the Echo pipetting system to dilute the test compound to different concentrations in dimethyl sulfoxide (DMSO) and transfer it to a 384-well plate. Dilute the mutant 3CL protease with reaction buffer and add 10μL / well to the 384-well plate, centrifuge at 1000rpm for 1min, and then incubate at room temperature for 30 minutes. Then add 10μL / well of substrate and centrifuge at 1000rpm for 30s to start the enzyme reaction. In the reaction system, the final concentration of the enzyme is 50nM, the final concentration of the substrate is 20μM, and the concentration range of the compound is 10000nM to 0.51nM. Kinetic Reduction Vmax mode was then selected on the Flexstation 3 microplate reader. The fluorescence value at a wavelength of 490 nm was read continuously every 75 seconds for a total of 35 times to obtain the reaction rate value (V). The inhibition rate was calculated and the half-maximal inhibitory concentration (IC50) was obtained by four-parameter fitting using XLfit software. 50The inhibition rate is calculated as follows: Inhibition rate = (V max -V compound ) / (V max -V min )*100%

[0205] Among them, V max is the reaction rate value of the well containing only enzyme and substrate, V min is the reaction rate value of the well containing only substrate, V Compound is the reaction rate value for the wells containing the test compound, enzyme, and substrate.

[0206] Experimental results: The compound of formula (I) still maintains significant inhibitory activity against the 3CL protease with the P132H mutation in the SARS-CoV-2 Omicron strain.

[0207] Table 4: Inhibitory effect of the compound of formula (I) on the 3CL protease activity of SARS-CoV-2 Omicron strain

[0208] *Indicates three independent repeated experiments.

[0209] Test Example 2: Inhibitory activity test of the compound of formula (I) against coronavirus 3CL protease from different sources

[0210] Experimental purpose: To study the inhibitory effect of the compound of formula (I) on the 3CL protease activity of six other coronaviruses that can infect humans, namely SARS-CoV, MERS-CoV, H229E-CoV, HKU1-CoV, NL63-CoV and OC43-CoV.

[0211] Experimental Materials:

[0212] 3CL protease: Recombinant full-length coronavirus 3CL protease was prepared based on the coronavirus genome sequence. The GenBank numbers of the SARS-CoV, MERS-CoV, H229E-CoV, HKU1-CoV, NL63-CoV, and OC43-CoV genomes used were AAP13442.1, MT387202.1, AF304460.1, AY597011.2, AY567487.2, and AY903459.1, respectively. The DNA sequences required for the expression of the six coronavirus 3CL protease proteins were purchased from Nanjing GenScript Biotechnology Co., Ltd.

[0213] 3CL protease substrate was purchased from Nanjing GenScript Biotechnology Co., Ltd.

[0214] Chymotrypsin substrate was purchased from Gill Biochemical Co., Ltd.

[0215] Other reagents are shown in the following table:

[0216] Experimental steps:

[0217] Prepare reaction buffer (containing 50mM Tris and 1mM EDTA). Dissolve the test compound in DMSO to a 100mM stock solution and further perform a 2-fold serial dilution in reaction buffer for a total of 11 concentrations. Add 3CL protease and different concentrations of the compound to a 96-well plate, incubate at room temperature for 10 minutes, add substrate, and quickly place in a microplate reader for reading. The volume of the entire enzymatic reaction system is 120μL. The final concentrations of SARS-CoV, MERS-CoV, H229E-CoV, HKU1-CoV, NL63-CoV, and OC43-CoV proteases are 30nM, 80nM, 30nM, 20nM, 30nM, and 10nM, respectively, and the final concentration of substrate is 10μM. The excitation and emission wavelengths for reading are 340nm and 490nm, respectively. The test time was 10 minutes, and the fluorescence value was read every 1 minute. The final result was the reaction rate obtained by fitting the readings of the first 5 minutes, and the inhibition rate was calculated using the following formula: inhibition rate = 1-(reaction rate of the test group / reaction rate of the control group).

[0218] Experimental results: As shown in Table 5, the compound of formula (I) exhibited a good inhibitory effect on 3CL proteases from six other coronaviruses, suggesting that the compound of formula (I) may have a broad-spectrum anti-coronavirus activity.

[0219] Table 5: Inhibitory effect of compounds of formula (I) on 3CL proteases from other coronaviruses

[0220] Test Example 3: Inhibitory effect of the compound of formula (I) on SARS-CoV-2 Vero E6 original strain (WIV04), Delta strain (B.1.617.2), and Omicron strain (B.1.1.529) at the cellular level

[0221] Objective: This study investigated the inhibitory effect of the compound of formula (I) on the replication of SARS-CoV-2 (WIV04), Delta (B.1.617.2), and Omicron (B.1.1.529) in Vero E6 cells by detecting viral copy number in the culture supernatant using real-time fluorescence quantitative PCR. Since Vero E6 cells highly express the efflux transporter P-gp, 0.5 μM of the P-gp inhibitor CP-100356 was added to the cells for co-incubation.

[0222] Experimental Materials:

[0223] Vero E6 was purchased from ATCC (catalog number CRL-1586), and the SARS-CoV-2 original strain (SARS-CoV-2-WIV04 strain), Delta strain (B.1.617.2), and Omicron strain (B.1.1.529) viruses were from the Microbial Bacteria (Virus) Collection Center of the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0224] Other reagents are shown in the following table:

[0225] Experimental instruments:

[0226] Biological safety cabinet (AC2-3S1, ESCO, Singapore)

[0227] CO2 incubator (Thermo Scientific HERAcell 150i, Thermo Scientific, USA )

[0228] Pure water machine (SYS ultrapure water machine, Chengdu)

[0229] StepOne Plus Real-time PCR system (4376600, ABI, USA)

[0230] TC20 TM Automatic cell counter (1450102, BIO-RAD, USA)

[0231] T100 TM Thermal Cycler (1861096, BIO-RAD, USA)

[0232] Centrifuge (Micro21 / 21R Thermo Scientific, USA)

[0233] Experimental steps:

[0234] Vero E6 cells were trypsinized and plated in culture medium (90% DMEM, 10% fetal bovine serum) in 48-well plates at 50,000 cells per well and cultured overnight. Test compounds were dissolved in DMSO to a 40 mM stock solution and serially diluted in culture medium containing 0.5 μM Pgp inhibitor to obtain the desired concentrations. Final test compound concentrations ranged from 1 μM to 0.004 μM. The supernatant was removed, and the diluted compound (containing 0.5 μM Pgp inhibitor) was added to each well and incubated for 1 hour. In a biosafety level 3 (BSL-3) laboratory, different strains of SARS-CoV-2 were added at a multiplicity of infection (MOI) of 0.01 or 0.001. After a 1-hour incubation, the supernatant was removed, the cells were washed with PBS, and 200 μL / well of the diluted compound (containing 0.5 μM Pgp inhibitor) was added. The supernatant was collected 24 or 72 hours after infection. The supernatant viral RNA was extracted and the supernatant viral copy number was detected by real-time fluorescence quantitative PCR. The compound inhibition rate was calculated based on the viral copy number, and the IC of the compound was calculated using GraphPad Prism 8. 50 .

[0235] In the cytotoxicity test, Vero E6 cells were digested and placed in culture medium (90% DMEM, 10% fetal bovine serum), seeded into 96-well plates, 20,000 cells per well, and cultured overnight. The test compound was dissolved in DMSO to prepare a 40mM stock solution, and further diluted with culture medium or culture medium containing 0.5μM Pgp inhibitor to obtain the required concentration for the test. The final concentration range of the test compound in the experiment was 500μM to 1.95μM. The cell supernatant in the 96-well plate was removed, and 100μL / well of the test compound (single drug or containing 0.5μM Pgp inhibitor) culture medium was added. After incubation for 24 hours, the cell viability was detected using the CCK8 detection kit, and the inhibition rate and half cytotoxic concentration (CC) were calculated. 50 ).

[0236] Test results: As shown in Table 6, when combined with the P-gp inhibitor CP-100356, the compound of formula (I) can inhibit the replication of the Delta strain in Vero E6 cells in a dose-dependent manner, IC 50 The compound of formula (I) combined with P-gp inhibitors also exerted a strong inhibitory effect in the original strain, IC 50 In addition, the compound of formula (I) combined with P-gp inhibitors can significantly inhibit the replication of Omicron strain in Vero E6 cells, IC 50 The compound of formula (I) alone or in combination with P-gp inhibitors had no significant cytotoxicity to the proliferation of Vero E6 cells. 50 >500μM.

[0237] Table 6: Inhibitory effect of compound of formula (I) combined with P-gp inhibitor on SARS-CoV-2 in Vero E6 cells

[0238] Test Example 4: In vivo antiviral effect of the compound of formula (I) against SARS-CoV-2 Delta strain in hACE2-K18 transgenic mice

[0239] Objective: This study evaluated the antiviral activity of the compound of formula (I) against SARS-CoV-2 delta strain in K18 transgenic mice stably expressing human angiotensin-converting enzyme 2 (ACE2) (K18-hACE2).

[0240] Experimental Materials:

[0241] 7-8 week old K18-hACE2 transgenic mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. SARS-CoV-2 Delta strain virus was obtained from the Microbial Culture Collection Center of Wuhan Institute of Virology, Chinese Academy of Sciences.

[0242] Ritonavir was purchased from Shanghai Desano Chemical Pharmaceutical Co., Ltd.

[0243] Vero E6 cells were purchased from ATCC (Cat. No. CRL-1586).

[0244] Other reagents are shown in the following table:

[0245] Experimental instruments:

[0246] Biological safety cabinet (AC2-3S1, ESCO, Singapore)

[0247] CO2 incubator (Thermo Scientific HERAcell 150i, Thermo Scientific, USA )

[0248] Pure water machine (SYS ultrapure water machine, Chengdu)

[0249] StepOne Plus Real-time PCR system (4376600, ABI, USA)

[0250] TC20 TM Automatic cell counter (1450102, BIO-RAD, USA)

[0251] T100 TMThermal Cycler (1861096, BIO-RAD, USA)

[0252] Centrifuge (Micro21 / 21R Thermo Scientific, USA)

[0253] Tissue grinder (JXFSTPRP-CL, Shanghai Jingxin, China )

[0254] Experimental procedures: K18-hACE2 transgenic mice were infected with the SARS-CoV-2 Delta strain via intranasal drops, with this day being designated as day 0. Two hours after infection, vehicle, 50 mg / kg, or 200 mg / kg of the compound of formula (I) (combined with 50 mg / kg of the cytochrome P450 inhibitor ritonavir) were administered orally, with dosing occurring twice daily for 2 days (one dose on day 0, two doses on day 1, and one dose on day 2) or 4 days (one dose on day 0, and two doses on days 1, 2, and 3, respectively). Mouse body weight changes were recorded, and lung and brain tissues were collected at the endpoint. The left lung was formaldehyde-fixed, embedded, sectioned, and stained with H&E for histopathological examination. The right lung and brain tissue were each divided into two aliquots. One aliquot was ground and homogenized to extract RNA, which was then reverse transcribed, and viral copy number was determined by real-time fluorescence quantitative PCR. The other aliquot was ground and homogenized to determine viral titer using a plaque assay. The plaque assay was performed as follows: Vero E6 cells were seeded in a 24-well plate at 12,000 cells per well and cultured overnight. A stock tissue homogenate was serially diluted 10-fold in DMEM medium. The supernatant was removed, and the diluted tissue homogenate was added and incubated for 1 hour. The supernatant was then removed, and medium containing 1% sodium methylcellulose and 2% FBS was added and cultured for 4 days. The medium was then removed, and the cells were fixed with paraformaldehyde and stained with 1% (w / v) crystal violet. The number of plaques in each well was counted.

[0255] Test results: As shown in Table 7, two days after infection, compared to the model group (mean viral copy number of 9.19±0.30log10copies / g), the compound of formula (I) at 50 mg / kg and 200 mg / kg, combined with ritonavir, significantly reduced the viral load in the lungs, with mean copy numbers of 7.66±0.27log10copies / g and 6.79±0.30log10copies / g, respectively. At a dose of 200 mg / kg, the viral copy number decreased by 2.4log10copies / g. A sustained inhibitory effect of the compound of formula (I) on viral copy number was observed four days after infection.

[0256] In terms of viral titer, as shown in Figure 8, a significant inhibitory effect of the compound of formula (I) was observed. After 2 days of infection, the 200 mg / kg dose completely inhibited viral replication and no titer was measured. At 50 mg / kg, the viral titer decreased by more than 3 log10 PFU / g compared to the model group. After 4 days of infection, the compound of formula (I) demonstrated a sustained inhibitory effect on viral titer. As shown in Figure 9, after 4 days of infection, the model group mice lost approximately 10% of their body weight, while the body weight of the group receiving the compound of formula (I) did not decrease significantly, indicating that the compound of formula (I) did not show significant toxicity under continuous administration. We further tested the viral load in the mouse brain. After 2 days of infection, no obvious infection was observed in any group. After 4 days of infection, compared to the model group, the compound of formula (I) at both 50 mg / kg and 200 mg / kg doses significantly reduced the number of viral copies in the mouse brain. In particular, at the 200 mg / kg dose, the number of viral copies in the brain was comparable to that of the uninfected normal group. We further tested viral titers in the brain 4 days after infection. The results are shown in Figure 10. Compared with the model group, no viral titers were detected at both doses of the compound of formula (I), demonstrating its potent inhibitory effect. In addition, lung histopathological analysis showed that the compound of formula (I) significantly improved lung damage at a dose of 200 mg / kg compared with the model group, including reducing the degree of alveolar atrophy or expansion and the degree of alveolar membrane thickening.

[0257] Table 7 Viral loads in the lungs and brains of mice 2 and 4 days after infection (mean ± SD)

[0258] Test Example 5: Selectivity of the compound of formula (I) for kinases

[0259] Experimental purpose: The inhibitory activity of the compound of formula (I) against 413 kinases was detected on the KinaseProfile experimental platform to study the selectivity of the compound of formula (I) against kinases.

[0260] Experimental Materials:

[0261] Full Human Panel [10uM ATP] Kinase Profiler is a test product provided by Eurofins, product number: 50-005KP10, this product contains 413 kinases.

[0262] Experimental steps:

[0263] Compounds were tested for each selected kinase using the standard Eurofins KinaseProfiler assay, following the relevant standard operating procedures. Protein kinases were detected radiometrically, while lipid kinases were detected using HTRF. The ATP concentration used in the assays was 10 μM. Detailed information for each kinase is available on the Eurofins website at: https: / / www.eurofinsdiscoveryservices.com / catalogmanagement / viewItem / Full-Human-Panel-10-uM-ATP-KinaseProfiler / 50-005KP10.

[0264] Experimental results: For 413 kinases, the inhibition rate of the compound of formula (I) at a concentration of 10 μM was less than 30%, with no obvious inhibitory effect, suggesting that the compound of formula (I) has excellent selectivity.

[0265] Test Example 6: Selectivity of the compound of formula (I) for safety targets

[0266] Experimental purpose: The effects of the compound of formula (I) on 47 safety-related targets were detected on the Safetyscan experimental platform.

[0267] Experimental Materials:

[0268] Safety47 Panel Dose Response SAFETYscan is a test product provided by Eurofins, product number: 87-1003DR. This product contains 78 tests related to 47 safety targets.

[0269] Experimental steps:

[0270] The 78 tests for 47 safety targets utilize assays including cAMP assays, calcium flux assays, hormone nuclear receptor assays, kinase binding assays, enzyme activity assays, neurotransmitter transporter assays, ion channel assays, and transporter assays. Detailed protocols for each assay are available on the eurofins website at: https: / / www.eurofinsdiscoveryservices.com / catalogmanagement / viewItem / Safety47-Panel-Dose-Response-SAFETYscan-DiscoverX / 87-1003DR.

[0271] Experimental results: For 47 safety-related targets, the compound of formula (I) had no significant inhibitory or activating effect at a concentration of 100 μM (EC 50were all greater than 100 μM), suggesting that the compound of formula (I) has excellent selectivity.

[0272] Test Example 7: Human plasma protein binding test of the compound of formula (I)

[0273] Experimental Materials

[0274] Human plasma was purchased from BioIVT, anticoagulated with EDTA K2, and stored at −80° C. 96-well equilibrium dialysis plates were purchased from HTDialysis LLC. Equilibrium dialysis membranes were purchased from Gales Ferry.

[0275] Experimental procedures

[0276] Prepare an alkaline solution with ultrapure water containing 14.2 g / L sodium dihydrogen phosphate and 8.77 g / L sodium chloride. This alkaline solution can be stored at 4°C for 7 days. Prepare an acidic solution with ultrapure water containing 12.0 g / L sodium dihydrogen phosphate and 8.77 g / L sodium chloride. This acidic solution can be stored at 4°C for 7 days. Titrate the alkaline solution with the acidic solution to a pH of 7.4. This buffer can be stored at 4°C for 7 days. Test the buffer pH on the day of the experiment and adjust the pH if it is outside the range of 7.4 ± 0.1.

[0277] The dialysis membrane was soaked in ultrapure water for 60 minutes to separate the membrane into two pieces, then soaked in 20% ethanol for 20 minutes, and finally soaked in the dialysis buffer for 20 minutes.

[0278] Frozen plasma was rapidly thawed at room temperature.

[0279] The plasma was centrifuged at 3,220 g for 10 minutes at 4°C to remove clots, and the supernatant was collected into a new centrifuge tube. The pH of the plasma was measured and recorded.

[0280] Prepare a 10 mM DMSO stock solution of the test substance. Dilute 2 μL of the 10 mM stock solution with 98 μL of DMSO to obtain a 200 μM working solution. Take 3 μL of the working solution and add 597 μL of human plasma to a final concentration of 1 μM (0.5% DMSO). Vortex thoroughly.

[0281] Add 120 μL of drug-spiked plasma sample to one side of the dialysis membrane and an equal volume of dialysate (phosphate buffered saline) to the other side. Perform the experiment in duplicate. Seal the dialysis plate and place it in an incubator. Incubate for 6 hours at 37°C, 5% CO2, and approximately 100 rpm. After incubation, remove the seal and pipette 50 μL from the buffer and plasma sides of each well into separate wells of a new plate.

[0282] Add 50 μL of blank plasma to the phosphate buffer sample, and add an equal volume of blank phosphate buffer to the plasma sample. Precipitate the protein by adding 300 μL of room temperature quencher (containing the internal standard acetonitrile (IS), 500 nM labetalol, 100 nM alprazolam, and 2 μM ketoprofen). Vortex for 5 minutes. Centrifuge at 3220 g for 30 minutes at 4°C. Transfer 100 μL of the supernatant to a new plate. Dilute the supernatant with 100 μL or 200 μL of water, depending on the HPLC-MS response and peak shape of the analyte. Mix thoroughly, and analyze the sample by HPLC-MS.

[0283] All calculations were performed using Microsoft Excel. The peak areas of the test substance on the buffer and plasma sides were determined. The plasma protein binding rates of the test substance and control drug were calculated using the following formulas: Free rate = (ratio of sample peak area to internal standard peak area, buffer side) / ratio of sample peak area to internal standard peak area, plasma side) * 100%, Binding rate = 1 - Free rate, Recovery rate = (ratio of sample peak area to internal standard peak area, buffer side + ratio of sample peak area to internal standard peak area, plasma side) / (ratio of sample peak area to internal standard peak area, initial plasma sample) * 100%. The ratio of sample peak area to internal standard peak area on the buffer side represents the free concentration of the compound, the ratio of sample peak area to internal standard peak area on the plasma side represents the sum of the free and bound concentrations of the compound, and the ratio of sample peak area to internal standard peak area in the initial plasma sample represents the total concentration of the compound at the start of sample incubation.

[0284] Test results:

[0285] See Table 8. When 1 μM of the compound of formula (I) was incubated at 37° C. for 6 hours, the average free rate was 46.63%, the binding rate was 53.37%, and the recovery rate was 88.02%.

[0286] Table 8 Human plasma protein binding test results of the compound of formula (I)

[0287] Test Example 8: Tissue distribution test of a single oral gavage of a compound of formula (I)

[0288] Experimental Materials:

[0289] A total of 60 Balb / c mice (purchased from Shanghai Minchang Biotechnology Co., Ltd.) were used, half male and half female, weighing 18-25 g.

[0290] Experimental steps:

[0291] Balb / c mice were given a single oral administration of the compound of formula (I) at a dose of 100 mg / kg in a volume of 10 mL / kg.

[0292] Before administration and at 5 minutes, 0.25, 1.0, 2.0, 3.0, 5.0, 7.0, and 10 hours after administration (six mice, half male and half female, were included at each time point). At these time points, 0.2 ml of blood was collected from the retroorbital venous plexus, placed in an EDTA-K2 tube, centrifuged at 11,000 rpm for 5 minutes, and plasma was separated and frozen at -70°C. Lung tissue was immediately dissected and collected at 0.25, 1.0, 3.0, and 7.0 hours after whole blood collection. The tissue was rinsed with cold saline to remove residual blood and contents, blotted dry, labeled, and stored at -70°C until testing. The content of the compound of formula (I) in plasma and lung tissue was determined by LC / MS-MS, and the lung-to-blood ratio was calculated.

[0293] Test results:

[0294] After a single oral administration of the compound of formula (I) to Balb / c mice, the ratio of lung tissue exposure to plasma exposure was 0.62, indicating that the exposure of the compound of formula (I) in the lung tissue was high.

[0295] Test Example 9: Safety pharmacology study on the effects of oral administration of the compound of formula (I) on the cardiovascular system of cynomolgus monkeys

[0296] In a 2-week repeated-dose toxicity study in cynomolgus monkeys, the effects of the compound of formula (I) on the cardiovascular system were also investigated.

[0297] Experimental Materials:

[0298] Thirty-two cynomolgus monkeys, half male and half female, aged 2.5-5 years at the time of administration were used.

[0299] Source of animals: Yunnan Yingmao Biotechnology Co., Ltd.; Guangxi Xiongsen Primate Experimental Animal Breeding and Development Co., Ltd.; Zhongke Lingrui (Zhanjiang) Biotechnology Co., Ltd.

[0300] Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MBP) of all awake animals were measured using an intelligent non-invasive sphygmomanometer BP-98E, and the Provantis / v10.2.3.1 electronic data acquisition system (PV-02) was used.

[0301] Experimental procedures: 32 cynomolgus monkeys (5 animals / sex / group in Group 1 and Group 4, 3 animals / sex / group in Group 2 and Group 3, a total of 4 groups) were randomly divided into groups and administered by nasogastric administration of the compound of formula (I) (40, 160 and 600 mg / kg / day) or the control formulation (98.9% vehicle formulation + 1.1% MTBE (methyl tert-butyl ether), 0 mg / kg / day) twice a day for a total of 14 days, followed by a 14-day recovery period. All animals were included in this study to evaluate the effects of drug administration on ECG parameters (including heart rate, PR interval, QRS duration, QT interval and QTcF) and blood pressure during the pre-dose period, drug administration period and recovery period.

[0302] Test results: Under the conditions of this test, crab-eating monkeys were given the compound of formula (I) (40, 160 and 600 mg / kg / day) by nasogastric gavage twice a day for 14 days. No test article-related changes in the cardiovascular system were observed; no test article-related arrhythmias were observed; and no test article-related changes in ECG parameters or blood pressure were observed throughout the entire test.

[0303] Test Example 10: Chemical stability test

[0304] Test method: The chemical stability of the crystalline form A of the compound of formula (I) and the methyl tert-butyl ether solvate form B of the compound of formula (I) under conditions of high temperature, light, accelerated temperature, and high humidity was investigated. Both the unpackaged and packaged (with two layers of polyethylene and one layer of pharmaceutical composite film) cases were investigated under light conditions. The results are shown in Table 9.

[0305] Test results: When crystal form B was placed under various influencing factors for 12 days, and when crystal form A was placed under various influencing factors for 12 days and 30 days, there was no significant change in the content. The chemical stability of crystal form A and crystal form B were both stable.

[0306] Table 9 Stability test results

[0307] Test Example 11: Crystal Stability Test

[0308] Test method:

[0309] The crystalline form A of compound of formula (I) was packaged and sampled according to the following scheme, and the sample was taken out for XRPD analysis on the 30th day.

[0310] The test results show that after the crystal form A of compound of formula (I) is placed under various influencing factors for 30 days, the characteristic diffraction peaks of the crystal form are consistent and there is no significant difference in crystallinity, and the crystal form is stable.

[0311] Test Example 12: Hygroscopicity test

[0312] Test method:

[0313] (1) Experimental instrument: Dynamic water adsorption instrument DVS Intrinsic PLUS;

[0314] (2) Experimental conditions: Take samples (appropriate amount) and place them in the DVS sample tray for testing.

[0315] (3)DVS parameters:

[0316] Temperature: 25℃;

[0317] Balance: dm / dt≤0.002% / min

[0318] RH (%) test level: 10%

[0319] RH (%) test step range: 0%-90%-0%.

[0320] Hygroscopicity evaluation standards (based on the description of hygroscopic characteristics and the definition of hygroscopic weight gain in the "9103 Guiding Principles for Hygroscopicity of Drugs" in Part IV of the 2020 edition of the Chinese Pharmacopoeia):

[0321] *Weight gain by moisture absorption at 25±1°C and 80±2%RH.

[0322] Test results:

[0323] The weight gain of the crystal form A of the compound of formula (I) under the conditions of 80% RH and 90% RH was only 0.5% and 0.6%, respectively.

Claims

1. Crystallization of the compound of formula (I) 2. Crystal form A of the compound of formula (I), The X-ray powder diffraction pattern of the A crystal form represented by the diffraction angle 2θ has diffraction peaks at 10.88±0.20°, 15.09±0.20°, 17.67±0.20°, 18.28±0.20° and 20.64±0.20°; or The X-ray powder diffraction pattern of the A crystal form represented by the diffraction angle 2θ has diffraction peaks at 10.88±0.20°, 15.09±0.20°, 16.61±0.20°, 17.67±0.20°, 18.28±0.20°, 18.50±0.20°, 20.09±0.20° and 20.64±0.20°; or The A crystal form has an X-ray powder diffraction pattern represented by a diffraction angle 2θ, and has diffraction peaks at 10.27±0.20°, 10.88±0.20°, 11.86±0.20°, 15.09±0.20°, 16.61±0.20°, 17.67±0.20°, 18.28±0.20°, 18.50±0.20°, 20.09±0.20° and 20.64±0.20°; or The X-ray powder diffraction pattern of the A crystal form represented by the diffraction angle 2θ is 9.29±0.20°, 10.27±0.20°, 10.88±0.20°, 10.97±0.20°, 11.86±0.20°, 14.27±0.20°, 14.92±0.20°, 15.09±0.20°, 15.61±0.2 Diffraction peaks are present at 0°, 15.78±0.20°, 16.61±0.20°, 17.67±0.20°, 18.28±0.20°, 18.50±0.20°, 20.09±0.20°, 20.64±0.20°, 22.18±0.20°, 23.80±0.20° and 25.63±0.20°; or The A crystal form has an X-ray powder diffraction pattern represented by a diffraction angle 2θ, and the diffraction peaks are shown in Table 3 Table 3 ;or The X-ray powder diffraction pattern of the A crystal form represented by the diffraction angle 2θ is basically as shown in Figure 5.

3. The crystal form A of the compound of formula (I) as claimed in claim 2, which has a DSC spectrum with a peak at 213.78°C±5.0°C; or The DSC spectrum of the A crystal form is basically shown in Figure 6.

4. A method for preparing the crystal form A of the compound of formula (I) according to claim 2 or 3, comprising: The compound of formula (I) is mixed with a solvent (i) and a solvent (ii), and crystallized to separate a solid; the solvent (i) is selected from at least one of isopropyl acetate, ethyl acetate, and isopropyl alcohol, preferably isopropyl acetate, and the solvent (ii) is selected from at least one of n-hexane and n-heptane, preferably n-heptane.

5. A solvate of a compound of formula (I), The solvate is selected from methyl tert-butyl ether solvate and 2-methyltetrahydrofuran solvate.

6. The solvate of the compound of formula (I) according to claim 5, wherein The molar ratio of the compound of formula (I) to the solvent is about 0.5 to 2, preferably about 0.8 to 1.2, and more preferably about 1.

0.

7. Form B of the methyl tert-butyl ether solvate of the compound of formula (I), The X-ray powder diffraction pattern of the B crystal form represented by the diffraction angle 2θ has diffraction peaks at 6.26±0.20°, 17.66±0.20° and 20.24±0.20°; or The X-ray powder diffraction pattern of the B crystal form represented by the diffraction angle 2θ has diffraction peaks at 6.26±0.20°, 7.27±0.20°, 11.47±0.20°, 13.06±0.20°, 15.57±0.20°, 17.66±0.20°, 20.24±0.20° and 22.72±0.20°; or The X-ray powder diffraction pattern of the B crystal form represented by the diffraction angle 2θ has diffraction peaks at 6.26±0.20°, 7.27±0.20°, 8.84±0.20°, 10.18±0.20°, 10.30±0.20°, 11.47±0.20°, 13.06±0.20°, 14.44±0.20°, 15.57±0.20°, 17.66±0.20°, 20.24±0.20°, 20.51±0.20° and 22.72±0.20°; or The X-ray powder diffraction pattern of the B crystal form represented by the diffraction angle 2θ, the diffraction peaks are shown in Table 2 Table 2 ;or The X-ray powder diffraction pattern of the B crystal form represented by the diffraction angle 2θ is basically as shown in Figure 3.

8. The crystal form B according to claim 7, which has a DSC spectrum with peaks at 93.36°C±5.0°C, 170.25°C±5.0°C and 214.18±5.0°C; or The DSC spectrum of the B crystal form is basically shown in Figure 4.

9. A method for preparing the B crystal form according to claim 7 or 8, comprising: The compound of formula (I) is mixed with methyl tert-butyl ether and stirred to separate out a solid.

10. Form C of the 2-methyltetrahydrofuran solvate of the compound of formula (I), The X-ray powder diffraction pattern of the C crystal form represented by the diffraction angle 2θ has diffraction peaks at 6.21±0.20°, 17.48±0.20° and 20.86±0.20°; or The X-ray powder diffraction pattern of the C crystal form represented by the diffraction angle 2θ has diffraction peaks at 6.21±0.20°, 7.04±0.20°, 11.71±0.20°, 17.48±0.20°, 20.59±0.20° and 20.86±0.20°; or The X-ray powder diffraction pattern of the C crystal form represented by the diffraction angle 2θ has diffraction peaks at 6.21±0.20°, 7.04±0.20°, 11.71±0.20°, 14.74±0.20°, 15.77±0.20°, 17.48±0.20°, 20.59±0.20° and 20.86±0.20°; or In the X-ray powder diffraction pattern of the C crystal form represented by the diffraction angle 2θ, the diffraction peaks are shown in Table 1 Table 1 ;or The X-ray powder diffraction pattern of the C crystal form represented by the diffraction angle 2θ is basically as shown in Figure 1.

11. The crystal form C according to claim 10, which has a DSC spectrum with peaks at 90.39°C±5.0°C, 167.59°C±5.0°C and 214.22±5.0°C; or The DSC spectrum of the C crystal form is basically shown in Figure 2.

12. A method for preparing the crystal form C according to claim 10 or 11, comprising: The compound of formula (I) is mixed with 2-methyltetrahydrofuran, stirred, and separated into a solid after crystallization.

13. A drug combination comprising the crystal form A of the compound of formula (I) according to claim 2 or 3, the crystal form B according to claim 7 or 8, the crystal form C according to claim 10 or 11, or a combination thereof, and other antiviral drugs; preferably, the other antiviral drug is ritonavir.

14. A pharmaceutical composition comprising the crystal form A of the compound of formula (I) according to claim 2 or 3, the crystal form B according to claim 7 or 8, the crystal form C according to claim 10 or 11, or a combination thereof, and a pharmaceutically acceptable excipient.

15. Use of the crystal form A of the compound of formula (I) according to claim 2 or 3, the crystal form B according to claim 7 or 8, the crystal form C according to claim 10 or 11, or a combination thereof, or the drug combination according to claim 13, or the pharmaceutical composition according to claim 14 in the preparation of a medicament for preventing or treating related diseases caused by coronavirus and / or picornavirus infection.

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