Amorphous forms of ketoamide derivatives and methods for their preparation

Abstract

The present invention relates to amorphous forms of ketoamide derivatives and methods for their preparation, which have good stability and further possess promising druggability, providing a viable drug substance option for subsequent pharmaceutical development.

Description

[Technical Field]

[0001] The present invention relates to amorphous forms of ketoamide derivatives and methods for their preparation, as well as drug substances and pharmaceutical compositions comprising the amorphous forms. [Background technology]

[0002] Before 2002, coronaviruses were considered minor human pathogens, accounting for approximately 15–25% of common cold cases. However, the severe SARS epidemic in 2002, caused by the novel coronavirus SARS-CoV, raised public awareness of coronavirus-caused diseases. To date, seven zoonotic coronaviruses causing human disease are known, including MERS-CoV, SARS-CoV, and SARS-CoV-2, which have been identified as the causative agents of severe acute respiratory syndrome (SAS). The etiological agent of COVID-19, SARS-CoV-2 (acute respiratory syndrome coronavirus 2), belongs to the Coronaviridae family and causes respiratory, hepatic, intestinal, and neurological diseases in mammals. Symptoms of infection range from asymptomatic illness to moderate-to-severe pneumonia, and even life-threatening complications such as hypoxemic respiratory failure, acute respiratory distress syndrome, and multiple organ failure, ultimately resulting in death. More seriously, this virus is not only highly contagious, but can also be transmitted through asymptomatic, symptomatic, and pre-symptomatic carriers.

[0003] 3CL protease (3C-like protease, 3CLPro) is the primary protease responsible for cleaving and processing the viral self-encoding RNA. Most of the functional (nonstructural) proteins of coronaviruses are encoded by the ORF1ab gene and are first translated into a polyprotein (7096 aa), which is then cleaved by 3CLPro into multiple active proteins, including the viral replication protein RdRp. Furthermore, this protein cleaves the intracellular protein NEMO, thereby inhibiting the activation of the interferon signaling pathway. Therefore, inhibition of 3CLPro can effectively inhibit virus infection and replication. Furthermore, studies have shown that 3CLPro also lyses host immune-related proteins, such as the human innate immune molecule STING. 3CLPro not only maintains viral replication but also destroys the host cellular immune system and inhibits anti-infection immune responses, leading to immune escape. Inhibition of 3CLPro not only effectively kills coronaviruses but also reduces immune imbalances within infected host cells. Given the potent inhibitory or killing effects of 3CLPro inhibitors against coronaviruses and their important role in the coronavirus gene replication process, 3CLPro inhibitors have become attractive targets in the field of antiviral chemotherapy.

[0004] The following compounds are reported 3CLPro inhibitor compounds: TIFF2025533223000001.tif48170

[0005] A series of ketoamide derivatives has been reported in patent PCT / CN2022 / 117124, and in vitro activity data show that some of the compounds have good in vitro anti-coronavirus activity at the cellular level without cytotoxicity, significantly higher exposure, slower clearance rate, longer half-life, and better pharmacokinetic properties. Compound 1 (Example 1, formula (I)) has relatively good overall performance and is believed to have good potential for drug discovery. TIFF2025533223000002.tif45170

[0006] Crystal form screening is one of the most important aspects in drug development. For a particular compound, the physicochemical properties of its free form, various salt forms, and corresponding crystalline forms are unknown. Therefore, it is of great significance for drug development to search for suitable crystalline or amorphous forms that further consider its drug potential and provide multiple options for intermediate products and / or drug substances for subsequent drug development. Summary of the Invention [Problem to be solved by the invention]

[0007] The first object of the present invention is to provide an amorphous form of the compound of formula (I) to overcome the shortcomings of the prior art, which has good stability and good in vitro activity against the Mpro protease of the novel coronavirus, thereby having certain drug discovery potential and providing a viable drug substance option for developing the compound of formula (I) into a clinical drug. TIFF2025533223000003.tif44170 [Means for solving the problem]

[0008] The above object of the present invention is achieved by the following technical solutions. An amorphous form of the compound of formula (I), wherein the X-ray powder diffraction pattern (XRPD) of said amorphous form is characterized by the absence of sharp diffraction peaks.

[0009] It is well known to those skilled in the art that the solid form of a drug can exist in an amorphous form in addition to a crystalline form, and the amorphous form of a drug, as a special form of a solid substance, has important applications in the preparation of drugs. Generally, in a crystalline substance, the molecules are arranged in an orderly and periodic manner, so the energy of intermolecular interaction is reduced and low, while the molecules in an amorphous form are in a highly disordered state, the surface free energy of the substance is larger, and the molecules in the solid substance have higher energy than the molecules in a crystalline solid substance, so they are easier to disperse, the dissolution rate is increased, and the bioavailability of the drug is improved. An amorphous form is considered to belong to a thermodynamically high-energy state and to have a thermodynamically metastable structure, in which the basic particles constituting the compound are arranged disorderly in three-dimensional space. Powder X-ray diffraction patterns are one of the most intuitive ways to identify amorphous forms. Specifically, when a compound exists in an amorphous form, its powder X-ray diffraction pattern generally lacks sharp diffraction peaks, i.e., the XRPD pattern generally lacks any diffraction peaks, or has one or more broad diffraction peaks. As will be understood by those skilled in the art, the broad diffraction peaks in the XRPD pattern of the amorphous form are in contrast to the narrow, sharp diffraction peaks in the XRPD pattern of a crystalline form. Generally, the broad diffraction peaks in the XRPD pattern of the amorphous form span a 2θ angle of up to 5° or more.

[0010] Specifically, the powder X-ray diffraction pattern of the amorphous compound represented by the above formula (I) has two broad diffraction peaks at 2θ angles between 5° and 15° and between 15° and 25°.

[0011] In a particular embodiment of the present invention, the amorphous form of the compound of formula (I) above has an X-ray powder diffraction pattern essentially as shown in FIG.

[0012] In some embodiments of the present invention, the amorphous form has a differential scanning calorimetry (DSC) curve with an endothermic peak onset at 71.35±3°C.

[0013] In a particular embodiment of the present invention, the amorphous form has the DSC thermogram shown in FIG.

[0014] In some embodiments of the present invention, the amorphous form has a thermogravimetric analysis curve (TGA) of 2.209% weight loss at 100.00±3° C.

[0015] In a particular embodiment of the present invention, the amorphous form has the TGA thermogram shown in FIG.

[0016] In another particular embodiment of the present invention, the amorphous form of the compound represented by formula (I) above has the powder X-ray diffraction pattern shown in FIG.

[0017] In another particular embodiment of the present invention, the amorphous form of the compound represented by formula (I) above has the powder X-ray diffraction pattern shown in FIG.

[0018] A second object of the present invention is to provide a method for preparing an amorphous form of the compound of Formula (I). The method involves suspending the compound of Formula (I) in a solvent to form a slurry, centrifuging the resulting solid, and then vacuum-drying it at room temperature. The solvent is selected from n-hexane, n-heptane, cyclohexane, water, petroleum ether, and ethyl formate, stirring the slurry at a temperature of 25-45°C, stirring for 48-72 hours, and using a compound-to-solvent mass / volume ratio of 1:0.5-2.5 g / mL. This method is suitable for large-scale industrial production of the amorphous form of the compound of Formula (I), due to its stable process, mild reaction conditions, and readily available raw materials.

[0019] The present invention further provides a drug substance comprising an amorphous form of the compound of formula (I) according to the present invention.

[0020] The present invention further provides a pharmaceutical composition comprising an amorphous form of the compound of Formula (I) and at least one pharmaceutically acceptable carrier. The pharmaceutical composition may be in the form of an oral or injectable preparation, preferably a powder, granules, minipill, capsule, tablet, solution, or lozenge. The pharmaceutical composition comprises the active ingredient and a pharmaceutically acceptable adjuvant, including, but not limited to, at least one filler, binder, disintegrant, lubricant, etc. Specifically, the beneficial effects of the amorphous form of the compound of Formula (I) of the present invention are ultimately reflected in the pharmaceutical composition. More specifically, the pharmaceutical composition may contain the active ingredient in a mass percentage of 1.00 to 99.00%, or may contain the active ingredient in a mass percentage of 5.00 to 95.00%, or may contain the active ingredient in a mass percentage of 10.00 to 90.00%.

[0021] The present invention further provides use of a pharmaceutical composition comprising an amorphous form of compound of Formula (I) in the preparation of a product for preventing, alleviating, or treating an infection or disease caused by a novel coronavirus, wherein the novel coronavirus includes a non-mutant novel coronavirus or a mutant novel coronavirus, preferably the mutant novel coronavirus includes one selected from a novel coronavirus Alpha mutant strain, a novel coronavirus Beta mutant strain, a novel coronavirus Gamma mutant strain, a novel coronavirus Delta mutant strain, a novel coronavirus Lambda mutant strain, and / or a novel coronavirus Omicron mutant strain. The infection includes fever, cough, sore throat, pneumonia, acute respiratory infection, severe acute respiratory infection, hypoxemic respiratory failure, acute respiratory distress syndrome, sepsis, or septic shock, preferably the disease includes novel coronavirus pneumonia.

[0022] Thus, since the amorphous form of the compound of formula (I) of the present invention has a certain potential for drug discovery, when the presence of the amorphous form of the compound of formula (I) in the above-mentioned drug substance and / or pharmaceutical composition is proven by a detection means, the amorphous form of the compound of formula (I) provided by the present invention is deemed to have been used. In addition to the powder X-ray diffraction as described above, the detection means may further include methods such as differential scanning calorimetry (DSC), infrared spectroscopy (IR), Raman spectroscopy (Raman), solid-state nuclear magnetic resonance (SSNMR), and all other detection methods that can be used individually or in combination to demonstrate the use of the amorphous form of the compound of formula (I) of the present invention, and the influences caused by pharmaceutical adjuvants, etc. are removed using methods commonly used by those skilled in the art, such as subtractive panning.

[0023] The present invention has the following advantages and beneficial effects over the prior art:

[0024] 1. The amorphous form of compound of formula (I) and its preparation method are disclosed for the first time. Unlike conventional compounds existing in amorphous form, which have disadvantages such as poor stability and poor efficacy, the amorphous form of compound of formula (I) of the present invention exhibits high stability, even under high temperature and high humidity conditions, as exemplified by its good in vitro activity against the novel coronavirus Mpro protease. Based on existing stability data, it can be concluded that the amorphous form of compound of formula (I) has considerable potential for drug discovery.

[0025] 2. A drug substance comprising an amorphous form of the compound of formula (I) of the present invention is provided.

[0026] 3. Provided is a pharmaceutical composition comprising an amorphous form of the compound of formula (I) and at least one pharmaceutically acceptable carrier.

[0027] 4. Use of a pharmaceutical composition comprising an amorphous form of the compound of formula (I) in the preparation of a product for preventing, alleviating, or treating an infection or disease caused by a novel coronavirus is provided. The amorphous form of the compound of formula (I) according to the present invention has a significant inhibitory effect on Mpro protease of the novel coronavirus in vitro, and a mouse pharmacokinetic model has shown that the compound of formula (I) has a lower clearance rate, a longer half-life, and better pharmacokinetic properties.

[0028] 5. By providing an amorphous form of the compound of formula (I) and a method for preparing the same, various intermediate and / or raw material options have been provided for large-scale production of drug substances and downstream processes of pharmaceuticals (e.g., formulation processes).

[0029] 6. A pharmaceutical composition comprising the above-mentioned drug substance of the present invention and a pharmaceutically acceptable adjuvant, and having beneficial effects consistent with those of the amorphous form of the above-mentioned compound of formula (I) of the present invention, has been provided. Definitions and Description

[0030] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings: A particular word or term should not be considered unclear or ambiguous unless specifically defined, and should be understood according to its ordinary meaning. Where a product name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0031] The intermediate compounds of the present invention can be prepared by various synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitution forms known to those skilled in the art, and preferred embodiments include, but are not limited to, the embodiments of the present invention.

[0032] The chemical reactions of certain embodiments of the present invention are carried out in suitable solvents, and the solvents must be suitable for the chemical transformations of the present invention and the reagents and materials required therefor. To obtain the compounds of the present invention, those skilled in the art may need to modify or select synthetic or reaction steps based on existing embodiments.

[0033] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0034] All solvents used in this invention are commercially available and can be used as is without further purification.

[0035] The solvents used in the present invention are commercially available. The following abbreviations are used in the present invention: NaCl represents sodium chloride, DCM represents dichloromethane, DMF represents N,N-dimethylformamide, DMSO represents dimethyl sulfoxide, MeOH represents methanol, TFA represents trifluoroacetic acid, EDTA represents ethylenediaminetetraacetic acid, and mp represents melting point.

[0036] 1.1 X-ray powder diffraction (XRPD) Instrument model: Bruker D8 advance (Bruker D8 Advance) X-ray powder diffractometer Test method: Use approximately 10-20 mg of sample for XRPD detection. The detailed XRPD parameters are as follows: Tube: Cu, kα, (λ=1.54056Å) Tube voltage: 40 kV, tube current: 40 mA Divergence slit: 0.60 mm Detector slit: 10.50mm Anti-scatter slit: 7.10 mm Scan range: 4~40° Step width: 0.02 deg Step length: 0.12 seconds Sample plate rotation speed: 15 rpm

[0037] 1.2 Differential Scanning Calorimeter (DSC) Instrument model: TA Q2000 differential scanning calorimeter Test method: A sample (approximately 1 mg) was placed in a DSC aluminum pot and heated from 25°C to 350°C at a rate of 10°C / min under N2 conditions of 50 mL / min.

[0038] 1.3 Thermal Gravimetric Analyzer (TGA) Instrument model: TA Q5000IR Thermogravimetric Analyzer Test method: The sample (2-5 mg) was placed in a TGA platinum pot and heated from room temperature to 350°C at a rate of 10°C / min under N2 conditions of 25 mL / min. [Brief explanation of the drawings]

[0039] [Figure 1] 1 is an XRPD pattern of Cu-Kα radiation of the amorphous form of the compound of formula (I) of Example 2. [Figure 2] 1 is a DSC thermogram of the amorphous form of the compound of formula (I). [Figure 3] 1 is a TGA thermogram of an amorphous form of the compound of formula (I). [Figure 4] 1 is an XRPD pattern of Cu-Kα radiation of the amorphous form of the compound of formula (I) of Example 3. [Figure 5] 1 is an XRPD pattern of Cu-Kα radiation of the amorphous form of the compound of formula (I) of Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention will be described in detail below with reference to examples, but this does not mean any adverse limitations of the present invention. The present invention has been described in detail herein, and specific examples thereof have been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0041] Example 1 Preparation of Compounds of Formula (I) TIFF2025533223000004.tif137170

[0042] Step 1: Synthesis of the hydrochloride salt of compound 1-2

[0043] Compound 1-1 (500 mg, 1.75 mmol) was dissolved in ethyl acetate (5 mL), and a solution of hydrogen chloride in ethyl acetate (10 mL, 4N) was added. The mixture was stirred at 20° C. for 2 hours. The mixture was concentrated under reduced pressure to give the hydrochloride salt of compound 1-2 without further purification. 1 H NMR(400 MHz,CD3OD)δ=4.28-4.20(m,1H),3.91-3.81(m,3H),3.45-3.35(m,2H),2.86-2.74( m, 1H), 2.48-2.36 (m, 1H), 2.29-2.19 (m, 1H), 2.02-1.94 (m, 1H), 1.93-1.80 (m, 1H).

[0044] Step 2: Synthesis of compounds 1-4

[0045] Compound Boc-L-cyclohexylglycine (1 g, 3.89 mmol) was added to N,N-dimethylformamide (10 mL), and 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (1.77 g, 4.66 mmol) was added thereto and stirred for 0.5 hours. Diisopropylethylamine (1.26 g, 9.72 mmol) and the hydrochloride salt of compound 1-3 (1.02 g, 4.66 mmol) were added thereto and stirred for 16 hours at 20 °C. Methyl tert-butyl ether (50 mL) was added to the reaction mixture, which was then washed with water (20 mL), 3% citric acid (20 mL × 2), and saturated aqueous sodium chloride (20 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by silica gel column chromatography (petroleum ether: ethyl acetate = 3:1) gave compound 1-4. 1 H NMR(400MHz,CDCl3)δ=5.22-5.11(m,1H),4.36(d,J=3.9Hz,1H),4.27(dd,J=6.9,9.3Hz,1H),4.21-4.12(m,2H),3.83(dd,J=7.8,10.4Hz,1H),3.70(br dd,J=3.6,10.4Hz,1H),2.81-2.61(m,2H),1.82-1.70(m,6H),1.68-1.61(m,4H ), 1.56-1.48 (m, 2H), 1.46-1.38 (m, 9H), 1.29-1.22 (m, 4H), 1.21-0.98 (m, 4H).

[0046] Step 3: Synthesis of compounds 1-5

[0047] Compound 1-4 (1.41 g, 3.34 mmol) was added to tetrahydrofuran (14 mL), and a solution of lithium hydroxide monohydrate (LiOH·HO) (280.03 mg, 6.67 mmol) in water (5 mL) was added. The mixture was stirred at 20°C for 16 hours. The crude product was neutralized with 3% citric acid solution (50 mL) and extracted with ethyl acetate (50 mL). The organic phase was washed with saturated aqueous sodium chloride solution (30 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Compound 1-5 was obtained without further purification.1 H NMR(400MHz,DMSO‐d6)δ=12.58‐12.23(m,1H),6.92‐6.82(m,1H),4.11‐3.94(m,2H),3.82‐3.76(m,1H),3.72‐3.62(m,1 H), 2.73-2.64(m, 1H), 2.62-2.55(m, 1H), 1.92-1.42(m, 12H), 1.40-1.32(m, 9H), 1.18-1.06(m, 3H), 1.00-0.81(m, 2H).

[0048] Step 4: Synthesis of compounds 1-6

[0049] Compound 1-5 (650 mg, 1.65 mmol) was added to 2-butanone (7 mL), followed by the addition of 1-hydroxybenzotriazole (222.63 mg, 1.65 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (379.03 mg, 1.98 mmol), and diisopropylethylamine (638.84 mg, 4.94 mmol). The mixture was stirred at 20 °C for 0.5 hours, and then the hydrochloride salt of compound 1-2 (366.88 mg, 1.65 mmol) was added. The mixture was stirred at 20 °C for 16 hours. Water (20 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane:methanol (30 mL x 2, 10:1). The combined organic phases were washed with 3% citric acid (20 mL x 2) and saturated aqueous sodium chloride (20 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The product was purified by silica gel column chromatography (dichloromethane:methanol=20:1) to obtain compound 1-6. 1 H NMR(400 MHz, CDCl3)δ=7.49-7.42(m,1H),6.23-6.05(m,1H),5.28-5.17(m,1H),4.64-4.51(m,1H),4.43-4.24(m,2H),3.92-3.81(m,1H),3.78-3.70( m,3H),3.39-3.27(m,2H),2.94-2.75(m,2H),2.57-2.36(m,2H),2.24- 2.07(m, 1H), 1.94-1.50(m, 14H), 1.49-1.41(m, 9H), 1.27-0.95(m, 6H).

[0050] Step 5: Synthesis of Compounds 1-7

[0051] Compound 1-6 (3.10 g, 5.51 mmol) was dissolved in tetrahydrofuran (31 mL), and lithium borohydride (240.02 mg, 11.02 mmol) was added at 0 °C. The mixture was then slowly heated to 20 °C and reacted for 2 h. Water (10 mL) and ethyl acetate (20 mL) were added to the reaction mixture, and the mixture was stirred for 10 min. A white solid precipitated and was filtered to obtain the crude filter cake, i.e., the target product 1-7. [M+1]+ = 535.4. Step 6: Synthesis of Compounds 1-8

[0052] Compound 1-7 (0.5 g, 935.13 μmol) was dissolved in dichloromethane (10 mL). Dess-Martin oxidant (594.94 mg, 1.40 mmol) was then added to the reaction mixture and stirred at 25 °C for 16 h. Saturated sodium thiosulfate (15 mL) and saturated sodium bicarbonate solution (15 mL) were added to the reaction mixture, and the mixture was stirred for 10 min. The mixture was then extracted with dichloromethane (50 mL × 2). The organic phase was washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give crude compound 1-8. [M+1]+ = 533.4.

[0053] Step 7: Synthesis of Compounds 1-9

[0054] Compound 1-8 (436 mg, 818.52 μmol) was dissolved in dichloromethane (5 mL), and glacial acetic acid (58.98 mg, 982.22 mmol) and cyclopentyl isocyanate (94.44 mg, 982.22 μmol) were added to the reaction mixture. The mixture was stirred at 25°C for 2 h. Saturated ammonium chloride solution (10 mL) was added to the reaction mixture, and the mixture was stirred for 10 min. The mixture was then extracted with dichloromethane (20 mL). The organic phase was washed with water (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Compound 1-9 was obtained by silica gel column chromatography (dichloromethane:methanol = 10:1). [M+1]+ = 688.4. Step 8: Synthesis of Compounds 1-10

[0055] Compound 1-9 (190 mg, 276.22 μmol) was dissolved in methanol (3 mL), followed by the addition of a solution of potassium carbonate (95.44 mg, 690.54 μmol) in water (2 mL). The mixture was stirred at 20°C for 16 hours. 3% citric acid (20 mL) was added to the reaction mixture, which was then extracted three times with dichloromethane (40 mL). The organic phase was washed with saturated aqueous sodium chloride (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude product 1-10. [M+1] + =646.5.

[0056] Step 9: Synthesis of Compounds 1-11

[0057] Compound 1-10 (238.00 mg, 368.52 μmol) was dissolved in dichloromethane (24 mL) and then Dess-Martin oxidant (203.19 mg, 479.08 μmol) was added. The reaction was stirred at 20 °C for 18 h. Sodium thiosulfate (15 mL) and sodium bicarbonate solution (15 mL) were added to the reaction mixture, which was stirred for 10 min. The mixture was then extracted with dichloromethane (50 mL x 2). The organic phase was washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The product 1-11 was obtained by silica gel column chromatography (dichloromethane:methanol = 20:1). [M+1] + =644.5.

[0058] Step 10: Synthesis of Compounds 1-12

[0059] Compound 1-11 (125 mg, 194.16 μmol) was dissolved in tetrahydrofuran (3 mL), and then ethyl acetate hydrochloride (4 M, 2.91 mL) was added. The reaction was stirred at 20 °C for 1 h. The reaction solution was directly rotary evaporated using an oil pump, and rotary evaporated repeatedly with a small amount of dichloromethane to give compound 1-12. [M+1] + =544.4.

[0060] Step 11: Synthesis of Compound 1

[0061] Compound 1-12 (125 mg, 229.91 μmol) was dissolved in tetrahydrofuran (2.5 mL) and added with trifluoroacetic anhydride (193.15 mg, 919.63 μmol) and pyridine (127.30 mg, 1.61 mmol) at 0°C. The mixture was stirred at 20°C for 16 hours. Water (20 mL) was added to the reaction mixture, which was then extracted with dichloromethane (40 mL x 2). The organic phase was washed with 3% citric acid (40 mL) and saturated aqueous sodium chloride (40 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by preparative HPLC to obtain compound 1. [M+1] + =640.0, 1 H NMR(400 MHz,CD3OD)δ ppm 0.94-1.10(m,2H),1.13-1.32(m,3H)1.32-1.46(m,1H),1.47-1.57(m,3H),1.59-1.68(m,4H),1.69-1.8 1(m,6H),1.83-2.00(m,5H),2.01-2.17(m,1H),2.19-2.38(m,1H),2.49-2.57(m,1H),2.58-2.70(m,1H), 2.73-2.89(m,1H),3.20-3.26(m,1H),3.37-3.45(m,1H),3.73-3.86(m,1H),3.88-3.97(m,1H),4.03-4.1 0(m,1H),4.11-4.18(m,1H),4.19-4.29(m,1H),4.29-4.37(m,1H),4.39-4.47(m,1H),4.57-4.60(m,2H).

[0062] Example 2 Preparation of an amorphous form of the compound of formula (I)

[0063] 20.3 mg of the compound of formula (I) was weighed out, 10.0 mL of n-heptane was added dropwise at room temperature, and the mixture was suspended and slurried at 25°C for 72 hours. After centrifugation, the mixture was dried under vacuum at room temperature, and the form of the resulting final product was detected by XRPD. The form of the product was found to be amorphous.

[0064] The final product obtained has an XRPD pattern of Cu-Kα radiation shown in FIG. 1, a DSC thermogram shown in FIG. 2 and a TGA thermogram shown in FIG.

[0065] Example 3 Preparation of an amorphous form of the compound of formula (I)

[0066] 19.8 mg of the compound of formula (I) was weighed out, 10.0 mL of n-hexane was added dropwise at room temperature, and the mixture was suspended and slurried at 25°C for 72 hours. After centrifugation, the mixture was dried under vacuum at room temperature, and the form of the resulting final product was detected by XRPD. The resulting final product was found to be amorphous. The resulting final product was the same amorphous form as that in Example 2. The XRPD pattern of Cu-Kα radiation of the resulting final product is shown in Figure 4.

[0067] Example 4 Preparation of an amorphous form of the compound of formula (I)

[0068] 20.3 mg of the compound of formula (I) was weighed out, 10.0 mL of water was added dropwise at room temperature, and the mixture was suspended and slurried at 25°C for 72 hours. After centrifugation, the mixture was dried under vacuum at room temperature, and the form of the resulting final product was detected by XRPD. The resulting final product was found to be amorphous. The resulting final product was the same amorphous form as that of Example 2. The XRPD pattern of Cu-Kα radiation of the resulting final product is shown in Figure 5.

[0069] Example 5: Solid-state stability testing of the amorphous form of the compound of formula (I) under high temperature and humidity conditions

[0070] Two samples of the amorphous form of the compound of formula (I) were weighed in parallel, each weighing approximately 100 mg, and placed in a glass sample vial and spread into a thin layer. The mouth of the sample vial was sealed with aluminum foil, and several small holes were made in the aluminum foil to allow sufficient contact with the outside air. The sample was then placed in a temperature- and humidity-controlled box at 40°C and 75% humidity. The samples stored under the above conditions were sampled and detected on days 0, 12, and 30. The detection results were compared with the initial detection results on day 0. The test results are shown in Table 1 below. Table 1. Solid state stability studies of amorphous forms of the compound of formula (I) TIFF2025533223000005.tif123170

[0071] The detected items of this product were compared with those on day 0.

[0072] When kept at a high temperature of 60°C for 30 days, there was no difference in the crystal form, no difference in properties, a slight increase in total impurities, a significant decrease in moisture, and a slight decrease in content.

[0073] When kept under high humidity conditions of 92.5%RH for 30 days, there was no difference in the crystal form, no difference in properties, a slight increase in total impurities, a significant increase in moisture, and a slight decrease in content. When the sample was kept in an open environment under high humidity conditions for 30 days, the weight after moisture absorption increased by 5.0%, indicating that it was hygroscopic.

[0074] When kept at room temperature of 25°C and 60% RH for 30 days, the moisture content increased slightly, and there were no significant differences in the remaining data for each detection item.

[0075] When kept under high temperature and humidity conditions of 40°C and 75%RH for 30 days, there was no difference in the crystal form, no difference in properties, no obvious difference in total impurities, a slight increase in moisture content, and a slight decrease in content.

[0076] The amorphous form of the compound of formula (I) was relatively stable under high temperature, high temperature and high humidity, and high humidity conditions, and was even more stable under room temperature conditions, compared to the compound of formula (I) prepared in Example 1.

[0077] As described above, when the amorphous form of the compound of formula (I) was kept in an open environment, it was relatively stable under high temperature, high temperature and high humidity, and high humidity conditions, and was stable under room temperature conditions.

[0078] Example 6: Stability study of the amorphous form of the compound of formula (I) in different solvents

[0079] Approximately 20 mg of amorphous compound of Formula (I) was taken and each sample was mixed with 1.0–2.5 mL of the solvent listed in the table below and stirred at 40°C. After stirring for two days, if the sample was in a solution or near-solution state, it was filtered and the solvent was removed by natural evaporation. If the sample remained in a suspension, the sample was centrifuged to collect the precipitate. The supernatant was placed in a fume hood and evaporated until the solvent had evaporated to dryness. The resulting precipitate and the solid remaining after drying the solvent were dried overnight in a vacuum oven at 40°C. All solids in the sample were collected and their state was detected by XRPD. The results are shown in Table 2. Table 2. Stability studies of the amorphous form of compound of formula (I) in different solvents TIFF2025533223000006.tif69170

[0080] The above experimental data indicate that the amorphous form of the compound of formula (I) according to the present invention is highly stable without morphological changes that occur in conventional solvents, and can provide various intermediate and / or raw material options for large-scale production of drug substances and downstream processes of pharmaceuticals (e.g., formulation processes).

[0081] Example 7 In vitro testing 1. Experimental materials: 1.1 Reagents, consumables and sources: Tris:Sigma, EDTA: Sigma; NaCl: Sigma; 384 well plate: Perkin Elmer, Dimethyl sulfoxide (DMSO): Sigma, Substrate(Dabcyl‐KTSAVLQSGFRKM‐(Edans)):GenScript, SARS‐CoV‐2 Mpro:WuXi AppTec, GC376:TargetMol. 1.2 Equipment and Sources: SpectraMax M2e Microplate Reader: Molecular Devices; Echo 655 Liquid Handling Workstation: Labcyte; Tabletop high-speed centrifuge: Eppendorf.

[0082] 2. Experimental Method:

[0083] Compounds of formula (I) were dissolved in DMSO and diluted three-fold with Echo 655 according to the concentration to be measured. Ten concentration points, each with duplicate wells, were added to a 384-well plate. Mpro protein and substrate were diluted in test buffer (100 mM NaCl, 20 mM Tris-HCl, 1 mM EDTA). Mpro protein was added to the 384-well plate and incubated with the compound at room temperature for 30 min. Substrate was then added to the plate to achieve a test concentration of 25 nM Mpro protein and 25 μM substrate. The plates were then incubated in a 30°C incubator for 60 min. The fluorescence signal at Ex / Em = 340 nm / 490 nm was then detected using a microplate reader. Simultaneously, background wells containing the substrate and compound but no Mpro protein were used as controls.

[0084] 3. Data Analysis: 1) The inhibition rate was calculated using the following formula. Inhibition rate % = [(compound - BG 化合物 )-(ZPE-BG ZPE )] / [(HPE‐BG HPE )-(ZPE-BG ZPE )] × 100% # HPE: 100% inhibition control containing 25 nM Mpro protein + 25 μM substrate + 1 μM GC376 ZPE: No-inhibition control containing 25 nM Mpro protein + 25 μM substrate and no compound Compound: Test compound wells containing 25 nM Mpro protein + 25 μM substrate + compound BG: background control wells containing 25 μM substrate + compound and no Mpro protein 2) The compound inhibition rate data (inhibition rate %) was analyzed using GraphPad Prism software by nonlinear fitting analysis using log(agonist) vs. response-variable slope to obtain the IC of the compound. 50 The values ​​were calculated and the experimental results are shown in Table 3. Table 3. In vitro activity of test compounds against the Mpro protease of the novel coronavirus TIFF2025533223000007.tif24170

[0085] Conclusion: The compounds of the present invention have good in vitro activity against the Mpro protease of the novel coronavirus.

[0086] Based on the above experiments for evaluating the stability and activity of the amorphous form of the compound of formula (I), it was found that, unlike the general perception of amorphous forms of pharmaceuticals in the art, the amorphous form of the compound of formula (I) provided in the present invention exhibits higher stability in chemical properties and physical form, and has significant inhibitory effect on the Mpro protease of the novel coronavirus, and therefore has good potential for drug discovery.

[0087] The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention are equivalent exchanges and shall fall within the protection scope of the present invention.

Claims

1. An amorphous form of the compound of formula (I), An amorphous form of the compound of formula (I), characterized in that the powder X-ray diffraction pattern of said amorphous form is free of sharp diffraction peaks.

2. 2. The amorphous form of the compound of formula (I) according to claim 1, wherein the powder X-ray diffraction pattern of the amorphous form is characterized by having two characteristic peaks between 2θ angles of 5° to 15° and 15° to 25°.

3. 2. The amorphous form of the compound of formula (I) according to claim 1, characterized in that the powder X-ray diffraction pattern of said amorphous form is essentially as shown in Figure 1.

4. 2. The amorphous form of the compound of formula (I) according to claim 1, wherein the differential scanning calorimetry curve of the amorphous form is characterized by an endothermic peak onset at 71.35±3°C.

5. 5. The amorphous form of the compound of formula (I) according to claim 4, characterized in that the DSC thermogram of said amorphous form is shown in Figure 2.

6. 2. The amorphous form of the compound of formula (I) according to claim 1, characterized in that the thermogravimetric analysis curve of the amorphous form is 100.00±3°C with a weight loss of 2.209%.

7. 7. The amorphous form of the compound of formula (I) according to claim 6, characterized in that the TGA thermogram of said amorphous form is shown in Figure 3.

8. 1. A process for preparing an amorphous form of a compound of formula (I), comprising: The preparation method includes suspending each compound of formula (I) in a solvent to form a slurry, centrifuging the system from which a solid precipitates, and then drying the system in vacuum at room temperature; A method for preparing an amorphous form of the compound of formula (I), characterized in that the solvent is selected from n-heptane, cyclohexane, water, petroleum ether, and ethyl formate, the stirring temperature for slurrying is 25-45°C, the slurrying time is 48-72 hours, and the mass / volume ratio of the compound to the solvent in the preparation method is 1:0.5-2.5 g / mL.

9. A pharmaceutical composition comprising an amorphous form of the compound of formula (I) according to any one of claims 1 to 7 and at least one pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, wherein the dosage form of the pharmaceutical composition is an oral dosage form or an injection dosage form, and preferably the oral dosage form is a powder, granules, mini-pills, capsules, tablets, solution or lozenges.

11. Use of the pharmaceutical composition of claim 9 or 10 in the preparation of a product for the prevention, alleviation or treatment of infection or disease caused by a novel coronavirus.

12. The use according to claim 11, characterized in that the novel coronavirus includes a non-mutant novel coronavirus or a mutant novel coronavirus.

13. The use described in claim 12, characterized in that the novel coronavirus mutant includes those selected from novel coronavirus Alpha mutant strains, novel coronavirus Beta mutant strains, novel coronavirus Gamma mutant strains, novel coronavirus Delta mutant strains, novel coronavirus Lambda mutant strains and / or novel coronavirus Omicron mutant strains.

14. The use according to any one of claims 11 to 13, characterized in that the infectious diseases include fever, cough, sore throat, pneumonia, acute respiratory infections, severe acute respiratory infections, hypoxemic respiratory failure, acute respiratory distress syndrome, sepsis or septic shock, preferably the diseases include novel coronavirus pneumonia.

15. A drug substance of the compound of formula (I), characterized in that it comprises an amorphous form of the compound of formula (I) according to any one of claims 1 to 7.

16. 16. A pharmaceutical composition comprising the drug substance of claim 15 and a pharmaceutically acceptable adjuvant, wherein the pharmaceutically acceptable adjuvant comprises at least one of a filler, a binder, a disintegrant, and a lubricant.

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