Amorphous form of ketone amide derivative and preparation method thereof

Abstract

The invention relates to an amorphous form of a ketoamide derivative and a preparation method thereof, the amorphous form has considerable stability and further has a certain medicinal prospect, and a feasible bulk drug choice is provided for subsequent drug development.

Description

Amorphous ketoamide derivative and preparation method thereof Technical Field

[0001] The present invention relates to an amorphous ketoamide derivative and a preparation method thereof, as well as a raw material medicine and a pharmaceutical composition containing the amorphous ketoamide derivative. Background Art

[0002] Before 2002, coronaviruses were known only as minor pathogens in humans, accounting for approximately 15-25% of common colds. However, the emergence of the severe SARS outbreak in 2002, caused by the novel coronavirus SARS-CoV, heightened public awareness of diseases caused by coronaviruses. To date, there are seven known zoonotic coronaviruses that can cause human disease, with MERS-CoV, SARS-CoV, and SARS-CoV-2 being identified as the cause of severe acute respiratory syndrome (SARS). The causative agent of COVID-19 is SARS-CoV-2 (acute respiratory syndrome coronavirus 2), a member of the Coronaviridae family of viruses that causes respiratory, hepatic, intestinal, and neurological diseases in mammals. Symptoms of infection range from asymptomatic illness to moderate and severe pneumonia, as well as life-threatening complications, including hypoxemic respiratory failure, acute respiratory distress syndrome, multisystem organ failure, and ultimately death. Furthermore, this virus is not only highly contagious but can also be transmitted by asymptomatic individuals and those in symptomatic and pre-symptomatic stages.

[0003] 3CL protease (3C-like protease, 3CLPro) is the primary protease within the virus's own RNA that cleaves and processes. Most of the functional proteins (non-structural proteins) of coronaviruses are encoded by the ORF1ab gene, which is first translated into a polyprotein (7096 aa) and then cleaved by 3CLPro into multiple active proteins, such as the viral replication protein RdRp. Furthermore, this protein may cleave the intracellular protein NEMO, thereby inhibiting the activation of the interferon signaling pathway. Therefore, inhibiting 3CLPro can effectively inhibit viral infection and replication. Furthermore, studies have shown that 3CLPro can also cleave host immune-related proteins, such as the human innate immunity molecule STING. 3CLPro is not only essential for maintaining viral replication itself, but can also disrupt the host cell's immune system, suppress anti-infective immune responses, and cause immune escape. Inhibiting 3CLPro can not only effectively kill coronaviruses but also reduce immune imbalances within infected host cells. Given the potent inhibitory or killing effects of 3CLPro inhibitors on coronaviruses and their crucial role in the genetic replication process of coronaviruses, 3CLPro inhibitors have become attractive targets in the field of antiviral chemotherapy.

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

[0005] Patent PCT / CN2022 / 117124 reports on a series of ketoamide derivatives. In vitro activity data show that some compounds have good in vitro anti-coronavirus activity at the cellular level, are non-cytotoxic, and exhibit significantly increased exposure, slower clearance rates, longer half-lives, and better pharmacokinetic properties. Among them, Compound 1 (Example 1, Formula (I)) has relatively outstanding overall performance and is considered to have good drug development prospects.

[0006] Crystal form screening is an important step in drug development. For a specific compound, the physical and chemical properties of its free form, various salt forms, and corresponding crystal forms are unknown. Based on further consideration of its drugability, finding a suitable crystal form or amorphous form to provide a variety of intermediates and / or APIs for subsequent drug development is of great significance to drug development.

[0007] Summary of the Invention

[0008] The first object of the present invention is to solve the deficiencies of the prior art and provide an amorphous form of the compound represented by formula (I). The amorphous form has considerable stability and good in vitro anti-novel coronavirus Mpro protease activity, and thus has certain pharmaceutical prospects, providing a feasible raw material drug option for the development of the compound represented by formula (I) into a clinical drug.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The amorphous form of the compound represented by formula (I) is characterized in that the amorphous form has no sharp diffraction peaks in its X-ray powder diffraction pattern (XRPD).

[0011] It is well known to those skilled in the art that, in addition to the crystalline state, drugs also exist in an amorphous state. As a special form of solid matter, the amorphous state of drugs plays an important role in drug preparation. Generally, the ordered and periodic arrangement of molecules in crystalline substances reduces the energy of intermolecular interactions, resulting in lower energy. The highly disordered state of molecules in the amorphous state leads to a higher surface free energy. Molecules in solid substances have higher energy than those in crystalline solid substances, making them easier to disperse, increasing their solubility and improving the bioavailability of drugs. Amorphous is a thermodynamically high-energy state and a thermodynamically metastable structure. The basic particles that constitute its compound are disordered in three-dimensional space. X-ray powder diffraction spectrum is one of the most intuitive ways to judge the amorphous form. Specifically, when the compound exists in an amorphous form, its X-ray powder diffraction spectrum usually shows no sharp diffraction peaks, that is, there are no diffraction peaks in the XRPD spectrum, or there are one or several broad diffraction peaks. Those skilled in the art will understand that the broad diffraction peaks in the amorphous XRPD spectrum are relative to the narrow and sharp diffraction peaks in the crystalline XRPD spectrum. Generally speaking, the broad diffraction peaks in the amorphous XRPD spectrum have a 2θ angle span of up to 5° or even greater.

[0012] Specifically, the amorphous X-ray powder diffraction pattern of the compound represented by the aforementioned formula (I) has two broad diffraction peaks between 2θ angles of 5°-15° and 15°-25°.

[0013] In a specific embodiment of the present invention, the X-ray powder diffraction pattern of the amorphous form of the compound represented by formula (I) is substantially as shown in FIG1 .

[0014] In some embodiments of the present invention, the amorphous phase has a differential scanning calorimetry (DSC) curve with an endothermic peak starting point at 71.35±3°C.

[0015] In a specific embodiment of the present invention, the DSC spectrum of the amorphous phase is shown in FIG2 .

[0016] In some embodiments of the present invention, the amorphous phase has a thermogravimetric analysis (TGA) curve at 100.00±3° C. with a weight loss of 2.209%.

[0017] In a specific embodiment of the present invention, the TGA spectrum of the amorphous phase is shown in FIG3 .

[0018] In another specific embodiment of the present invention, the X-ray powder diffraction pattern of the amorphous form of the compound represented by formula (I) is shown in FIG4 .

[0019] In another specific embodiment of the present invention, the X-ray powder diffraction pattern of the amorphous form of the compound represented by formula (I) is shown in FIG5 .

[0020] The second object of the present invention is to provide a method for preparing an amorphous compound of formula (I), wherein the compound of formula (I) is suspended and slurried in a solvent, the system with solid precipitation is centrifuged and then dried in vacuum at room temperature, the solvent is selected from n-hexane, n-heptane, cyclohexane, water, petroleum ether, ethyl formate, the slurry stirring temperature 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. The method has stable process, mild reaction conditions, readily available raw materials, and can be used for large-scale industrial production of amorphous compound of formula (I).

[0021] The present invention also provides a bulk drug containing the amorphous form of the compound of formula (I) of the present invention.

[0022] The present invention also provides a pharmaceutical composition containing an amorphous compound of formula (I) and at least one pharmaceutically acceptable carrier. The pharmaceutical composition is an oral preparation or an injection. Preferably, the oral preparation is a powder, granules, pellets, capsules, tablets, solutions or lozenges. The pharmaceutical composition is composed of the aforementioned raw materials and pharmaceutically acceptable excipients, and the pharmaceutically acceptable excipients include but are not limited to at least one of fillers, binders, disintegrants, lubricants, etc.; specifically, based on the beneficial effects of the amorphous compound of formula (I) described in the present invention, its beneficial effects are ultimately reflected in the pharmaceutical composition; more specifically, the mass percentage of the aforementioned raw materials contained in the pharmaceutical composition is any value of 1.00 to 99.00%, further, the mass percentage of the aforementioned raw materials contained in the pharmaceutical composition is any value of 5.00 to 95.00%, and further, the mass percentage of the aforementioned raw materials contained in the pharmaceutical composition is any value of 10.00 to 90.00%.

[0023] The present invention also provides a pharmaceutical composition containing an amorphous form of a compound of formula (I) for use 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 an unmutated form of the novel coronavirus or a variant form of the novel coronavirus, preferably, the variant form of the novel coronavirus includes a novel coronavirus Alpha variant, a novel coronavirus Beta variant, a novel coronavirus Gamma variant, a novel coronavirus Delta variant, a novel coronavirus Lambda variant and / or a novel coronavirus Omicron variant. The infection includes fever, cough, sore throat, pneumonia, acute respiratory infection, severe acute respiratory infection, hypoxic respiratory failure and acute respiratory distress syndrome, sepsis or septic shock, preferably, the disease includes novel coronavirus pneumonia.

[0024] In summary, the amorphous form of the compound of formula (I) of the present invention has certain drug development prospects. Therefore, if the presence of the amorphous form of the compound of formula (I) in the aforementioned bulk drug and / or pharmaceutical composition is confirmed by a detection method, it should be deemed that the amorphous form of the compound of formula (I) provided by the present invention has been used. In addition to the aforementioned X-ray powder diffraction, the detection means may further include differential scanning calorimetry (DSC), infrared spectroscopy (IR), Raman spectroscopy (Raman), solid-state nuclear magnetic resonance (SSNMR) and other methods that can be used alone or in combination to prove the use of the amorphous form of the compound of formula (I) of the present invention, and the effects of pharmaceutical excipients, such as subtraction spectroscopy, can be removed by commonly used methods of those skilled in the art.

[0025] The present invention has the following advantages and beneficial effects compared to the prior art:

[0026] 1. Disclosed for the first time is an amorphous form of a compound of formula (I) and a method for preparing the same. Unlike conventional amorphous compounds, which suffer from poor stability and pharmaceutical performance, the amorphous form of the compound of formula (I) mentioned in the present invention has higher stability, as shown in the fact that the amorphous form of the compound of formula (I) has higher stability under conditions of high temperature and high humidity, and better in vitro anti-novel coronavirus Mpro protease activity. Based on the existing stability data, it can be judged that the amorphous form of the compound of formula (I) has certain pharmaceutical prospects;

[0027] 2. Provided is a bulk drug comprising an amorphous form of the compound of formula (I) of the present invention;

[0028] 3. Provided is a pharmaceutical composition comprising an amorphous compound of formula (I) and at least one pharmaceutically acceptable carrier;

[0029] 4. Provided is a use of a pharmaceutical composition containing an amorphous form of a compound of formula (I) for preparing a product for preventing, alleviating, or treating infection or disease caused by the novel coronavirus. The amorphous form of the compound of formula (I) mentioned in the present invention has a significant inhibitory effect on the Mpro protease of the novel coronavirus in vitro, and a mouse pharmacokinetic model found that the compound of formula (I) has a slower clearance rate, a longer half-life, and better pharmacokinetic properties;

[0030] 5. Provided are an amorphous form of a compound of formula (I) and a method for preparing the same, providing a variety of intermediates and / or APIs for the large-scale production of APIs and downstream processes of pharmaceutical products (such as formulation processes);

[0031] 6. Provided is a pharmaceutical composition, which is composed of the raw material of the present invention and pharmaceutically acceptable excipients, and has the same beneficial effects as the amorphous form of the compound of formula (I) of the present invention.

[0032] Definition and Description

[0033] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be construed as ambiguous or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0034] The intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0035] The chemical reactions of the present invention are carried out in suitable solvents that are compatible with the chemical transformations of the present invention and the reagents and materials required. To obtain the compounds of the present invention, it may sometimes be necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0036] The present invention will be described in detail below through examples, which are not intended to limit the present invention in any way.

[0037] All solvents used in the present invention were commercially available and used without further purification.

[0038] The solvents used in the present invention are commercially available. The present invention uses the following abbreviations: 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.

[0039] 1.1 X-ray powder diffractometer (XRPD)

[0040] Instrument model: Bruker D8Advance X-ray diffractometer

[0041] Test method: Approximately 10-20 mg of sample was used for XRPD analysis.

[0042] The detailed XRPD parameters are as follows:

[0043] Light pipe: Cu, kα,

[0044] Light tube voltage: 40kV, light tube current: 40mA

[0045] Divergence slit: 0.60mm

[0046] Detector slit: 10.50mm

[0047] Anti-scatter slit: 7.10mm

[0048] Scanning range: 4-40 degrees

[0049] Step diameter: 0.02deg

[0050] Step size: 0.12 seconds

[0051] Sample plate speed: 15rpm

[0052] 1.2 Differential Scanning Calorimeter (DSC)

[0053] Instrument model: TA Q2000 differential scanning calorimeter

[0054] Test method: Take a sample (-1 mg) and place it in a DSC aluminum pan for testing. The method is: under 50 mL / min N2 conditions, heat the sample from 25℃-350℃ at a heating rate of 10℃ / min.

[0055] 1.3 Thermogravimetric Analysis (TGA)

[0056] Instrument model: TA Q5000IR thermogravimetric analyzer

[0057] Test method: Take a sample (2-5 mg) and place it in a TGA platinum pot for testing. The method is: under 25 mL / min N2 conditions, heat the sample from room temperature to 350°C at a heating rate of 10°C / min. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIG1 is an XRPD spectrum of the amorphous compound of formula (I) in Example 2 under Cu-Kα radiation.

[0059] FIG2 is a DSC spectrum of the amorphous form of the compound of formula (I).

[0060] FIG3 is a TGA spectrum of the amorphous form of the compound of formula (I).

[0061] FIG4 is an XRPD spectrum of the amorphous compound of formula (I) in Example 3 under Cu-Kα radiation.

[0062] FIG5 is an XRPD spectrum of the amorphous compound of formula (I) in Example 4 under Cu-Kα radiation. DETAILED DESCRIPTION

[0063] The present invention is described in detail below by way of examples, but is not intended to limit the present invention in any way. While the present invention has been described in detail herein, and specific embodiments 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.

[0064] Example 1 Preparation of the compound of formula (I)

[0065] Step 1: Synthesis of the hydrochloride salt of compound 1-2

[0066] 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, 4 N) was added. The reaction was stirred at 20° C. for 2 hours and concentrated under reduced pressure without purification to obtain the hydrochloride salt of compound 1-2. 1 H NMR (400MHz, 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).

[0067] Step 2: Synthesis of Compounds 1-4

[0068] Compound Boc-L-cyclohexylglycine (1 g, 3.89 mmol) was added to N,N-dimethylformamide (10 mL), followed by 2-(7-azobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate (1.77 g, 4.66 mmol). The reaction was stirred for 0.5 hr. Diisopropylethylamine (1.26 g, 9.72 mmol) and the hydrochloride salt of compound 1-3 (1.02 g, 4.66 mmol) were then added. The reaction was stirred at 20°C for 16 hr. Methyl tert-butyl ether (50 mL) was added to the reaction solution, which was then washed with water (20 mL), 3% citric acid (20 mL x 2), and saturated sodium chloride solution (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 (petroleum ether:ethyl acetate = 3:1) to obtain 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).

[0069] Step 3: Synthesis of Compounds 1-5

[0070] Compound 1-4 (1.41 g, 3.34 mmol) was added to tetrahydrofuran (14 mL), followed by a solution of lithium hydroxide monohydrate (LiOH·H2O) (280.03 mg, 6.67 mmol) in water (5 mL). The reaction 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 sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Compound 1-5 was obtained without further purification. 1H 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).

[0071] Step 4: Synthesis of Compounds 1-6

[0072] Compound 1-5 (650 mg, 1.65 mmol) was added to 2-butanone (7 mL), followed by 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 reaction was stirred at 20°C for 0.5 hr. Compound 1-2 hydrochloride (366.88 mg, 1.65 mmol) was then added, and the reaction was stirred at 20°C for 16 hours. Water (20 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane:methanol (30 mL × 2, 10:1). The organic phases were combined, washed with 3% citric acid (20 mL × 2) and saturated sodium chloride solution (20 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by silica gel column chromatography (dichloromethane:methanol=20:1) gave compound 1-6. 1 H NMR(400MHz, 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).

[0073] Step 5: Synthesis of Compounds 1-7

[0074] Compound 1-6 (3.10 g, 5.51 mmol) was dissolved in tetrahydrofuran (31 mL). Lithium borohydride (240.02 mg, 11.02 mmol) was added at 0°C and the temperature was slowly raised to 20°C for 2 h. Water (10 mL) and ethyl acetate (20 mL) were added to the reaction solution and stirred for 10 min. A white solid precipitated and was filtered to obtain a filter cake, which was the crude target product 1-7. [M+1]+ = 535.4.

[0075] Step 6: Synthesis of Compounds 1-8

[0076] Compound 1-7 (0.5 g, 935.13 μmol) was dissolved in dichloromethane (10 mL), and Dess-Martin periodinane (594.94 mg, 1.40 mmol) was added to the reaction system. The reaction was stirred at 25°C for 16 h. Saturated sodium thiosulfate (15 mL) and saturated sodium bicarbonate solution (15 mL) were added to the reaction system and 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 to afford crude compound 1-8. [M+1] = 533.4.

[0077] Step 7: Synthesis of Compound 1-9

[0078] 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 isocyanide (94.44 mg, 982.22 μmol) were added to the reaction system. The reaction was stirred at 25°C for 2 h. Saturated ammonium chloride solution (10 mL) was added to the reaction system and stirred for 10 min. Dichloromethane (20 mL) was then added for extraction. The organic phase was washed with water (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by silica gel column chromatography (dichloromethane:methanol = 10:1) afforded compound 1-9. [M+1]+ = 688.4.

[0079] Step 8: Synthesis of Compounds 1-10

[0080] Compound 1-9 (190 mg, 276.22 μmol) was dissolved in methanol (3 mL), followed by a solution of potassium carbonate (95.44 mg, 690.54 μmol) in water (2 mL). The reaction was stirred at 20°C for 16 h. 3% citric acid (20 mL) was added to the reaction system, and the mixture was extracted three times with dichloromethane (40 mL). The organic phase was washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product of compound 1-10. [M+1] + =646.5.

[0081] Step 9: Synthesis of Compound 1-11

[0082] Compound 1-10 (238.00 mg, 368.52 μmol) was dissolved in dichloromethane (24 mL), and Dess-Martin periodinane (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 system and stirred for 10 min. The mixture was 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. Purification by silica gel column chromatography (dichloromethane:methanol = 20:1) gave product 1-11. [M+1] + =644.5.

[0083] Step 10: Synthesis of Compounds 1-12

[0084] 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 the rotary evaporation was repeated with a small amount of dichloromethane to obtain compound 1-12. [M+1] + =544.4.

[0085] Step 11: Synthesis of Compound 1

[0086] Compound 1-12 (125 mg, 229.91 μmol) was dissolved in tetrahydrofuran (2.5 mL) and trifluoroacetic anhydride (193.15 mg, 919.63 μmol) and pyridine (127.30 mg, 1.61 mmol) were added at 0°C. The reaction was stirred at 20°C for 16 h. Water (20 mL) was added to the reaction system, and the mixture was extracted with dichloromethane (40 mL x 2). The organic phase was washed sequentially with 3% citric acid (40 mL) and saturated sodium chloride solution (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, 1H NMR(400MHz,CD3OD)δppm 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).

[0087] Example 2 Preparation of amorphous compound of formula (I)

[0088] 20.3 mg of the compound of formula (I) was weighed, 10.0 mL of n-heptane was added dropwise at room temperature, and the mixture was suspended and slurried at 25° C. for 72 h. After centrifugation, the mixture was vacuum-dried at room temperature. The morphology of the final product was detected by XRPD, and the morphology was amorphous.

[0089] The Cu-Kα radiation XRPD spectrum of the final product is shown in FIG1 ; the DSC spectrum is shown in FIG2 ; and the TGA spectrum is shown in FIG3 .

[0090] Example 3 Preparation of amorphous form of compound of formula (I)

[0091] 19.8 mg of the compound of formula (I) was weighed, 10.0 mL of n-hexane was added dropwise at room temperature, and the mixture was suspended and slurried at 25°C for 72 h. After centrifugation, the mixture was dried under vacuum at room temperature. XRPD analysis revealed that the final product was amorphous. The final product was the same amorphous form as in Example 2. The Cu-Kα radiation XRPD spectrum of the final product is shown in Figure 4.

[0092] Example 4 Preparation of amorphous form of compound of formula (I)

[0093] 20.3 mg of the compound of formula (I) was weighed, 10.0 mL of water was added dropwise at room temperature, and the mixture was suspended and slurried at 25°C for 72 h. After centrifugation, the mixture was dried under vacuum at room temperature. XRPD analysis revealed that the final product was amorphous. The final product was the same amorphous form as in Example 2. The Cu-Kα radiation XRPD spectrum of the final product is shown in Figure 5.

[0094] Example 5: Solid Stability Test of Amorphous Compound of Formula (I) under High Temperature and High Humidity Conditions

[0095] Two amorphous samples of the compound of formula (I), approximately 100 mg each, were weighed and placed in parallel at the bottom of a glass sample bottle, spreading them into a thin layer. The bottles were sealed with aluminum foil, which was perforated with small holes to ensure adequate contact with ambient air. The samples were then placed in a constant temperature and humidity chamber at 40°C and 75% humidity. Samples were collected and tested on days 0, 12, and 30 under these conditions. The test results were compared with the initial test results on day 0. The test results are shown in Table 1 below:

[0096] Table 1: Stability test of amorphous solid of compound of formula (I)

[0097] The test items of this product are compared with those on day 0.

[0098] After being placed at high temperature of 60℃ for 30 days, there is no difference in crystal form and properties, the total impurities increase slightly, the moisture content decreases significantly, and the content decreases slightly.

[0099] When placed under high humidity conditions of 92.5% RH for 30 days, there is no difference in crystal form and properties, the total impurities increase slightly, the moisture content increases significantly, and the content decreases slightly. When the sample is placed open under high humidity conditions for 30 days, it absorbs moisture and increases in weight by 5.0%, indicating that it is hygroscopic.

[0100] After being placed at room temperature of 25℃ and 60% RH for 30 days, the moisture content increased slightly, and there was no significant difference in the data of other test items.

[0101] After being placed under high temperature and high humidity conditions of 40°C and 75% RH for 30 days, there was no difference in crystal form, properties, or total impurities. The moisture content increased slightly and the content decreased slightly.

[0102] Compared with the compound of formula (I) prepared in Example 1, the amorphous compound of formula (I) is relatively stable under high temperature, high temperature and high humidity, and high humidity conditions, and is more stable under normal temperature conditions.

[0103] In summary, the compound of formula (I) is amorphous and is relatively stable under conditions of high temperature, high temperature and high humidity, and high humidity, and is stable under normal temperature conditions.

[0104] Example 6: Stability test of the amorphous form of the compound of formula (I) in different solvents

[0105] Take approximately 20 mg of the amorphous compound of formula (I) and add 1.0-2.5 mL of the solvent listed in the table below to each aliquot, stirring at 40°C. After stirring for 2 days, if the sample is in solution or near solution, filter and remove the solvent by natural evaporation. If the sample is still a suspension, centrifuge the sample, collect the precipitate, and evaporate the supernatant in a fume hood until the solvent is completely evaporated. Then, dry the precipitate and the solvent-free solid in a vacuum drying oven at 40°C overnight. Collect the solid from all samples and analyze their state by XRPD. The results are shown in Table 2.

[0106] Table 2 Stability test of amorphous form of compound of formula (I) in different solvents

[0107] The above experimental data show that the amorphous compound of formula (I) provided by the present invention does not undergo morphological changes in conventional solvents and has high stability. It can provide a variety of intermediates and / or raw materials for the large-scale production of raw materials and downstream processes of pharmaceutical products (such as formulation processes).

[0108] Example 7 In vitro testing

[0109] 1. Experimental Materials:

[0110] 1.1 Reagents, consumables and sources:

[0111] Tris: Sigma;

[0112] EDTA: Sigma;

[0113] NaCl: Sigma;

[0114] 384 well Plate: Perkin Elmer;

[0115] Dimethyl sulfoxide (DMSO): Sigma;

[0116] Substrate(Dabcyl-KTSAVLQSGFRKM-(Edans)):GenScript;

[0117] SARS-CoV-2Mpro: WuXi AppTec;

[0118] GC376:TargetMol.

[0119] 1.2 Instruments and sources:

[0120] SpectraMax M2e microplate reader: Molecular Devices;

[0121] Echo 655 liquid handling system: Labcyte;

[0122] Benchtop high-speed centrifuge: Eppendorf.

[0123] 2. Experimental methods:

[0124] The compound of formula (I) was dissolved in DMSO and diluted 3-fold using Echo655 according to the concentration requirements to be tested, with 10 concentration points and duplicate wells for each concentration, and added to a 384-well plate. Mpro protein and substrate were diluted with test buffer (100mM NaCl, 20mM Tris-HCL, 1mM EDTA), and Mpro protein was added to a 384-well test plate and incubated with the compound at room temperature for 30min. The substrate was then added, and the test concentration of Mpro protein was 25nM and the test concentration of substrate was 25μM. Incubated in a 30°C constant temperature incubator for 60 minutes. The fluorescence signal value of Ex / Em=340nm / 490nm was then detected using a microplate reader. The background wells containing substrate and compound but not Mpro protein were also detected as controls.

[0125] 3. Data Analysis:

[0126] 1) Calculate the inhibition rate using the following formula:

[0127] Inhibition rate % = [(Compound-BG 化合物 )-(ZPE-BG ZPE )] / [(HPE-BG HPE )-(ZPE-BG ZPE )]×100%

[0128] # HPE: 100% inhibition control, containing 25nM Mpro protein + 25μM substrate + 1μM GC376

[0129] ZPE: non-inhibitory control, containing 25 nM Mpro protein + 25 μM substrate, no compound

[0130] Compound: Test compound wells, containing 25nM Mpro protein + 25μM substrate + compound

[0131] BG: Background control wells, containing 25 μM substrate + compound, without Mpro protein

[0132] 2) GraphPad Prism software was used to perform log(agonist) vs. response--Variable slope nonlinear fitting analysis on the inhibition rate data of the compound (inhibition rate%) to obtain the IC 50 The experimental results are shown in Table 3.

[0133] Table 3 In vitro anti-novel coronavirus Mpro protease activity of the test compounds

[0134] Conclusion: The compounds of the present invention have good in vitro anti-novel coronavirus Mpro protease activity.

[0135] Based on the above evaluation experiments on the stability and activity of the amorphous form of the compound of formula (I), it can be seen that: unlike the general understanding of the amorphous form of drugs in the field, the amorphous form of the compound of formula (I) provided by the present invention has higher chemical properties and physical form stability, and the amorphous form of the compound of formula (I) has a significant inhibitory effect on the new coronavirus Mpro protease. It can be seen that the amorphous form of the compound of formula (I) has good pharmaceutical prospects.

[0136] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. an amorphous form of the compound represented by formula (I), It is characterized in that The amorphous form has no sharp diffraction peaks in its X-ray powder diffraction pattern.

2. The amorphous form of the compound of formula (I) according to claim 1, characterized in that The X-ray powder diffraction pattern of the amorphous form has two characteristic peaks at 2θ angles of 5°-15° and 15°-25°.

3. The amorphous form of the compound of formula (I) according to claim 1, characterized in that The X-ray powder diffraction pattern of the amorphous phase is substantially as shown in FIG1 .

4. The amorphous form of the compound of formula (I) according to claim 1, characterized in that The differential scanning calorimetry curve of the amorphous form has an endothermic peak starting point at 71.35±3°C.

5. The amorphous form of the compound of formula (I) according to claim 4, characterized in that The DSC spectrum of the amorphous phase is shown in FIG2 .

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

7. The amorphous form of the compound of formula (I) according to claim 6, characterized in that The TGA spectrum of the amorphous phase is shown in FIG3 .

8. A method for preparing an amorphous form of a compound of formula (I), comprising suspending and slurrying the compound of formula (I) in a solvent, centrifuging the system with solid precipitation, and then vacuum drying at room temperature, wherein the solvent is selected from n-heptane, cyclohexane, water, petroleum ether, and ethyl formate, the slurrying stirring temperature 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, characterized in that The invention comprises 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 pharmaceutical composition is in the form of an oral preparation or an injection, preferably, the oral preparation is a powder, granules, pellets, capsules, tablets, solutions or lozenges.

11. Use of the pharmaceutical composition according to claim 9 or 10 in the preparation of a product for preventing, alleviating or treating infection or disease caused by the new coronavirus.

12. The use according to claim 11, wherein the novel coronavirus comprises an unmutated form of the novel coronavirus or a mutated form of the novel coronavirus.

13. The use according to claim 12, wherein the variant of the new coronavirus is selected from the coronavirus Alpha variant, coronavirus Beta variant, coronavirus Gamma variant, coronavirus Delta variant, coronavirus Lambda variant and / or coronavirus Omicron variant.

14. The use according to any one of claims 11 to 13, wherein the infection comprises fever, cough, sore throat, pneumonia, acute respiratory tract infection, severe acute respiratory tract infection, hypoxic respiratory failure and acute respiratory distress syndrome, sepsis or septic shock, and preferably, the disease comprises novel coronavirus pneumonia.

15. A bulk drug of a compound of formula (I), characterized in that The raw material medicine contains the amorphous form of the compound of formula (I) according to any one of claims 1 to 7.

16. A pharmaceutical composition, characterized in that The pharmaceutical composition is composed of the raw material drug according to claim 15 and pharmaceutically acceptable excipients, and the pharmaceutically acceptable excipients include at least one of a filler, a binder, a disintegrant, and a lubricant.

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