Antiviral pharmaceutical composition and method for producing the same

Abstract

Provided are oral pharmaceutical compositions comprising a spiro compound of formula (I) or a pharmaceutically acceptable salt thereof, methods for preparing the oral pharmaceutical compositions, combination products comprising the oral pharmaceutical compositions and another antiviral agent, and uses of the oral pharmaceutical compositions and combination products in the treatment or prevention of antiviral infections and related diseases caused by viral infections. TIFF2026502601000023.tif43170

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to Chinese Patent Application No. 202310081244.6, filed with the State Intellectual Property Office of China on January 16, 2023, the entire contents of which are hereby incorporated by reference herein.

[0002] The present disclosure is in the field of drug formulations, and specifically relates to oral pharmaceutical compositions, methods for their preparation and their use in antiviral infections. [Background technology]

[0003] Coronaviruses are single-stranded, sense-RNA viruses, some of which can spread widely within populations and cause severe symptoms. Currently, there are seven known coronaviruses capable of infecting 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 single polyprotein and then cleaved into multiple active proteins by 3CL proteases and PL proteases. Therefore, inhibiting the activity of 3CL proteases can effectively inhibit viral replication. Different coronavirus 3CL proteases share a high degree of structural homology; therefore, 3CL protease inhibitors typically possess broad-spectrum anticoronavirus activity.

[0004] In addition to coronaviruses, 3CL protease also plays an important role in hydrolyzing polyproteins encoded by small RNA viruses, and 3CL protease inhibitors can effectively inhibit the replication of small RNA viruses. Enterovirus 71 is a small RNA virus that is one of the common viruses that causes hand, foot, and mouth disease and can also cause various diseases such as meningitis, brainstem encephalitis, and myocarditis. In recent years, enterovirus 71 has caused multiple outbreaks in infants and young children, and clinically, there is still a lack of effective therapeutic agents.

[0005] Therefore, there remains a clinical need for antiviral drug formulations to treat viruses including coronavirus, enterovirus 71, and the like. Summary of the Invention

[0006] In one aspect, the present disclosure provides an oral pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof. [ka]

[0007] In another aspect, the present disclosure provides a method for producing the oral pharmaceutical composition, comprising: (1) mixing a compound of formula (I) or a pharmaceutically acceptable salt thereof with one or more of a disintegrant, a filler, a glidant, and a lubricant; and (2) Optionally, a method is provided that includes a step of coating the admixture obtained in step (1) with a coating agent.

[0008] In another aspect, the present disclosure further provides a combination product comprising: (1) the oral pharmaceutical composition described above; and (2) another antiviral agent.

[0009] In another aspect, the present disclosure further provides the use of the oral pharmaceutical composition or the combination product in the manufacture of a medicament for the prevention or treatment of an associated disease caused by coronavirus and / or small RNA virus infection.

[0010] In another aspect, the present disclosure further provides the use of said oral pharmaceutical composition or said combination product in the prevention or treatment of related diseases caused by coronavirus and / or small RNA virus infection.

[0011] In another aspect, the present disclosure further provides an oral pharmaceutical composition as described above or a combination product as described above for preventing or treating related diseases caused by coronavirus and / or small RNA virus infection.

[0012] In another aspect, the present disclosure provides a method for treating a coronavirus and / or related disease caused by a small RNA virus infection, comprising administering to a mammal, preferably a human, in need of such treatment a therapeutically effective amount of the oral pharmaceutical composition or the combination product.

[0013] Detailed Description In one aspect, the present disclosure provides an oral pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof. [ka]

[0014] In some embodiments, the oral pharmaceutical composition is in the form of a tablet, capsule, pill, granule, powder, emulsion, solution, or suspension.

[0015] In some embodiments, the oral pharmaceutical composition is in the form of a tablet, capsule, granule, powder, or suspension.

[0016] In some embodiments, the oral pharmaceutical composition is a tablet.

[0017] In some embodiments, the oral pharmaceutical composition further comprises a disintegrant.

[0018] In some embodiments, the disintegrant is one or more selected from low-substituted hydroxypropyl cellulose, carboxymethyl cellulose calcium, crospovidone, dry starch, sodium carboxymethyl starch, and cross-linked sodium carboxymethyl cellulose.

[0019] In some embodiments, the disintegrant is one or more selected from low-substituted hydroxypropyl cellulose, crospovidone, and cross-linked sodium carboxymethyl cellulose.

[0020] In some embodiments, the disintegrant is cross-linked sodium carboxymethylcellulose.

[0021] In some embodiments, the oral pharmaceutical composition further comprises a filler.

[0022] In some embodiments, the filler is one or more selected from microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, calcium sulfate dihydrate, lactose, sucrose, dextrin, sorbitol, starch or its derivatives, mannitol, xylitol, and fructose.

[0023] In some embodiments, the filler is one or more selected from microcrystalline cellulose, lactose, dextrin, and starch.

[0024] In some embodiments, the filler is one or more selected from microcrystalline cellulose and lactose.

[0025] In some embodiments, the filler is microcrystalline cellulose and lactose.

[0026] In some embodiments, the lactose is lactose monohydrate.

[0027] In some embodiments, the oral pharmaceutical composition further comprises a glidant.

[0028] In some embodiments, the glidant is one or more selected from colloidal silicon dioxide, talc powder, and wheat starch.

[0029] In some embodiments, the glidant is one or more selected from colloidal silicon dioxide and talc powder.

[0030] In some embodiments, the glidant is colloidal silicon dioxide.

[0031] In some embodiments, the oral pharmaceutical composition further comprises a lubricant.

[0032] In some embodiments, the lubricant is one or more selected from magnesium stearate, stearic acid, calcium stearate, zinc stearate, sodium stearyl fumarate, liquid paraffin, polyethylene glycol, sodium lauryl sulfate, and hydrogenated vegetable oil.

[0033] In some embodiments, the lubricant is one or more selected from magnesium stearate, stearic acid, calcium stearate, and sodium stearyl fumarate.

[0034] In some embodiments, the lubricant is sodium stearyl fumarate.

[0035] In some embodiments, the present disclosure provides an oral pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a disintegrant, a filler, a glidant, and a lubricant.

[0036] In some embodiments, the present disclosure provides an oral pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a disintegrant, a filler, a glidant, and a lubricant, wherein the disintegrant is cross-linked sodium carboxymethylcellulose, the fillers are microcrystalline cellulose and lactose monohydrate, the glidant is colloidal silicon dioxide, and the lubricant is sodium stearyl fumarate.

[0037] In some embodiments, the oral pharmaceutical composition comprises the compound of formula (I) or a pharmaceutically acceptable salt thereof, a disintegrant, a filler, a glidant, and a lubricant, wherein, calculated in parts by weight, the content of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the oral pharmaceutical composition is 20 to 80 parts, the content of the disintegrant is 0.5 to 20 parts, the content of the filler is 10 to 70 parts, the content of the glidant is 0.1 to 20 parts, and the content of the lubricant is 0.1 to 20 parts.

[0038] In some embodiments, the oral pharmaceutical composition contains, in parts by weight, 20 to 80 parts of the compound of formula (I) or a pharmaceutically acceptable salt thereof, 0.5 to 20 parts of cross-linked sodium carboxymethylcellulose, 10 to 70 parts of the total of microcrystalline cellulose and lactose monohydrate, 0.1 to 20 parts of colloidal silicon dioxide, and 0.1 to 20 parts of sodium stearyl fumarate.

[0039] In some embodiments, the content of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the oral pharmaceutical composition is 45 to 55 parts by weight.

[0040] In some embodiments, the content of the filler in the oral pharmaceutical composition is 30 to 45 parts by weight.

[0041] In some embodiments, the content of the disintegrant in the oral pharmaceutical composition is 2 to 10 parts by weight.

[0042] In some embodiments, the content of the glidant in the oral pharmaceutical composition is 1 to 3 parts by weight.

[0043] In some embodiments, the content of the lubricant in the oral pharmaceutical composition is 1 to 3 parts by weight.

[0044] In some embodiments, the oral pharmaceutical composition contains, in parts by weight, 45-55 parts of the compound of formula (I) or a pharmaceutically acceptable salt thereof, 2-10 parts of the disintegrant, 30-45 parts of the filler, 1-3 parts of the glidant, and 1-3 parts of the lubricant.

[0045] In some embodiments, the oral pharmaceutical composition contains, in parts by weight, 45 to 55 parts of the compound of formula (I) or a pharmaceutically acceptable salt thereof, 2 to 10 parts of cross-linked sodium carboxymethylcellulose, 30 to 45 parts of the total of microcrystalline cellulose and lactose monohydrate, 1 to 3 parts of colloidal silicon dioxide, and 1 to 3 parts of sodium stearyl fumarate.

[0046] In another aspect, the present disclosure provides an oral pharmaceutical composition comprising a compound of formula (I) above or a pharmaceutically acceptable salt thereof, a disintegrant, a filler, a glidant, a lubricant, and a coating agent.

[0047] In some embodiments, the coating is a gastric film coating.

[0048] In some embodiments, the coating comprises hydroxypropyl methylcellulose, iron oxide red, polyethylene glycol, and titanium dioxide.

[0049] In some embodiments, the present disclosure provides an oral pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, a disintegrant, a filler, a glidant, a lubricant, and a coating, wherein the disintegrant is cross-linked sodium carboxymethylcellulose, the filler is microcrystalline cellulose and lactose monohydrate, the glidant is colloidal silicon dioxide, the lubricant is sodium stearyl fumarate, and the coating comprises hydroxypropyl methylcellulose, iron oxide red, polyethylene glycol, and titanium dioxide.

[0050] In some embodiments, the content of the coating agent in the oral pharmaceutical composition is 0.5 to 20 parts by weight.

[0051] In some embodiments, the content of the coating agent in the oral pharmaceutical composition is 2 to 6 parts by weight.

[0052] In some embodiments, the oral pharmaceutical composition contains, in parts by weight, 20 to 80 parts of the compound of formula (I) or a pharmaceutically acceptable salt thereof, 0.5 to 20 parts of the disintegrant, 10 to 70 parts of the filler, 0.1 to 20 parts of the glidant, 0.1 to 20 parts of the lubricant, and 0.5 to 20 parts of the coating agent.

[0053] In some embodiments, the oral pharmaceutical composition contains, in parts by weight, 45 to 55 parts of the compound of formula (I) or a pharmaceutically acceptable salt thereof, 2 to 10 parts of the disintegrant, 30 to 45 parts of the filler, 1 to 3 parts of the glidant, 1 to 3 parts of the lubricant, and 2 to 6 parts of the coating agent.

[0054] In some embodiments, in the oral pharmaceutical composition, the content of the compound of formula (I) or a pharmaceutically acceptable salt thereof is 20 to 80 parts, the content of cross-linked sodium carboxymethylcellulose is 0.5 to 20 parts, the total content of microcrystalline cellulose and lactose monohydrate is 10 to 70 parts, the content of colloidal silicon dioxide is 0.1 to 20 parts, the content of sodium stearyl fumarate is 0.1 to 20 parts, and the content of the coating agent comprising hydroxypropyl methylcellulose, iron oxide red, polyethylene glycol, and titanium dioxide is 0.5 to 20 parts, calculated in parts by weight.

[0055] In some embodiments, in the oral pharmaceutical composition, the content of the compound of formula (I) or a pharmaceutically acceptable salt thereof is 45 to 55 parts, the content of cross-linked sodium carboxymethylcellulose is 2 to 10 parts, the total content of microcrystalline cellulose and lactose monohydrate is 30 to 45 parts, the content of colloidal silicon dioxide is 1 to 3 parts, the content of sodium stearyl fumarate is 1 to 3 parts, and the content of the coating agent including hydroxypropyl methylcellulose, iron oxide red, polyethylene glycol, and titanium dioxide is 2 to 6 parts, calculated in parts by weight.

[0056] In some embodiments, the oral pharmaceutical composition contains the compound of formula (I) or a pharmaceutically acceptable salt thereof in an amount of 10 mg to 1000 mg, for example, 10 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 375 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, or 1000 mg.

[0057] In some embodiments, when the oral pharmaceutical composition is in the form of a tablet, the content of the compound of formula (I) or a pharmaceutically acceptable salt thereof is 50 mg / tablet, 150 mg / tablet, 375 mg / tablet, or 500 mg / tablet.

[0058] In another aspect, the present disclosure provides a method for producing the oral pharmaceutical composition, comprising: (1) mixing a compound of formula (I) or a pharmaceutically acceptable salt thereof with one or more of a disintegrant, a filler, a glidant, and a lubricant; and (2) Optionally, a method is provided that includes a step of coating the admixture obtained in step (1) with a coating agent.

[0059] In another aspect, the present disclosure further provides a combination product comprising: (1) the oral pharmaceutical composition described above; and (2) another antiviral agent.

[0060] In some embodiments, the other antiviral agent is an anti-coronavirus and / or a small RNA virus agent. In some embodiments, the other antiviral agent is ritonavir.

[0061] In another aspect, the present disclosure further provides the use of the oral pharmaceutical composition or the combination product in the manufacture of a medicament for the prevention or treatment of an associated disease caused by coronavirus and / or small RNA virus infection.

[0062] In another aspect, the present disclosure further provides the use of said oral pharmaceutical composition or said combination product in the prevention or treatment of related diseases caused by coronavirus and / or small RNA virus infection.

[0063] In another aspect, the present disclosure further provides an oral pharmaceutical composition as described above or a combination product as described above for preventing or treating related diseases caused by coronavirus and / or small RNA virus infection.

[0064] In another aspect, the present disclosure provides a method for treating a coronavirus and / or related disease caused by a small RNA virus infection, comprising administering to a mammal, preferably a human, in need of such treatment a therapeutically effective amount of the oral pharmaceutical composition or the combination product.

[0065] In some embodiments, related diseases caused by coronavirus and / or small RNA virus infection described in the present disclosure include, but are not limited to, respiratory infections, pneumonia, or complications thereof.

[0066] In some embodiments, the coronavirus described herein is selected from SARS-CoV, MERS-CoV, H229E-CoV, HKU1-CoV, NL63-CoV, OC43-CoV, or SARS-CoV-2.

[0067] In some embodiments, examples of small RNA viruses described in this disclosure include, but are not limited to, Enterovirus 71. [Brief explanation of the drawings]

[0068] [Figure 1] 1A and 1B are graphs showing the inhibitory effect of the compound of formula (I) on the virus titer in mouse lungs 2 days (FIG. A) and 4 days (FIG. B) after infection in Test Example 4. FIG. [Figure 2] FIG. 1 is a graph showing changes in mouse body weight in Test Example 4. [Figure 3] 1 is a graph showing the inhibitory effect of the compound of formula (I) on the viral titer in mouse brain 4 days after infection in Test Example 4. FIG. [Figure 4] FIG. 1 shows the in vitro dissolution-release curves of coated tablets in Test Example 11. DETAILED DESCRIPTION OF THE INVENTION

[0069] Example The present invention will be described in detail below with reference to examples, but these examples are not intended to limit the present invention in any way. Although the present disclosure has already described the present invention in detail and disclosed specific embodiments thereof, it will be apparent to those skilled in the art that various modifications and improvements can be made to the specific embodiments of the present disclosure 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.

[0070] Unless otherwise specified, the ratio of the mixed solvent is a volumetric mixing ratio. Unless otherwise specified, % refers to wt%.

[0071] Compounds are named artificially or with ChemDraw® software; commercially available compounds use names from the manufacturer's catalog.

[0072] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts are in units of 10 -6 The solvents used for NMR measurements were deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard was tetramethylsilane (TMS).

[0073] Example 1: Preparation of compounds of formula (I) 1.1. Preparation of Compound 1-1: [ka] Step 1: The starting material SMA (2.74 g, 11.85 mmol), 35 mL of dichloromethane, and 35 mL of DMF were added to a reaction flask and cooled to 0 °C. The starting materials 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) were added sequentially. The temperature was raised to room temperature and the reaction was allowed to proceed for 10 h. After completion of the reaction, an appropriate amount of dichloromethane was added, and the organic phase was washed sequentially with 1 N aqueous hydrochloric acid and saturated brine. After washing, the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness, and subjected to column chromatography to obtain 3.71 g of INT-1. ESI-MS: m / z 433.2 [M+H] + ; 1 H NMR (400 MHz, DMSO-d):δ H: 6.75 (d, J = 9.2 Hz, 1H), 4.38 (t, J = 8.2 Hz, 1H), 4.25 (d, J = 10.9 Hz, 1H), 4.11 (d, J = 9.3 Hz, 1H), 3.93 (t, J = 9.3 Hz, 1H), 3.62 (s, 3H), 3.40-3.31 (m, 4H), 2.70 (dd, J = 13.1, 7.9 Hz, 1H), 2.37 (dd, J = 13.2, 8.4 Hz, 1H), 1.37 (s, 9H), 0.94 (s, 9H).

[0074] 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 was completed, 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-d):δ H :12.68 (s, 1H), 6.71 (d, J = 9.4 Hz, 1H), 4.38-4.19 (m, 2H), 4.11 (d, J = 9.4 Hz, 1H), 3.88 (d, J = 10.9 Hz, 1H), 3.41-3.29(m, 4H), 2.69 (dd, J = 13.1, 7.9 Hz, 1H), 2.34 (dd, J = 13.2, 8.9 Hz, 1H), 1.38 (s, 9H), 0.94 (s, 9H).

[0075] 1.2. Preparation of Compound 1-2: [ka] Ammonia gas-methanol solution (700 mL, 7 mol / L) and the starting material SMD (100 g, 0.349 mol) were added to the reaction flask, stirred to dissolve, and then incubated at 25±5°C for 36 hours. After the reaction was complete, the reaction mixture was concentrated to approximately 250 mL. 300 mL of isopropyl alcohol was added, and the mixture was concentrated under reduced pressure (repeated three times) until the remaining reaction mixture was approximately 250 mL. The mixture was then purged with nitrogen gas and cooled to 10±5°C. 500 mL of hydrogen chloride-isopropyl alcohol solution (4 mol / L) was added to the reaction vessel. After the addition, the mixture was heated to 25±5°C, maintained at 25±5°C, and incubated for 9 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure until the volume of the remaining reaction solution was approximately 250 mL, 300 mL of isopropyl alcohol was added, and the concentration under reduced pressure was continued until the volume of the remaining reaction solution was approximately 250 mL (repeated twice), 100 mL of isopropyl alcohol was added, and the mixture was stirred for 30±5 minutes, filtered, and the filter cake was rinsed with 50 mL of isopropyl alcohol to obtain a wet product, which was dried under vacuum at 45±5°C to obtain 66.7 g of compound 1-2. 1 H NMR (400 MHz, DMSO-d):δ H :8.45 (d, J = 5.1 Hz, 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: 172.1 m / z [M+H] + .

[0076] 1.3. Preparation of compounds of formula (I): [ka] Step 1: Compound 1-1 (419 mg, 1 mmol) was placed in a two-neck flask and, under nitrogen gas protection, 5 mL of dichloromethane was added, 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 in an ice-water bath. The ice-water bath was removed, and the system was stirred at room temperature overnight. After workup, 50 mL of dichloromethane was added, followed by washing three times with 1 M aqueous hydrochloric acid, three times with saturated aqueous sodium bicarbonate, and the organic phase with saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and rotary evaporated to give compound 1-3 (469 mg). ESI-MS: m / z 572.3 [M+H] + .

[0077] 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 thin-layer chromatography showed that the reaction was essentially complete, the solvent was thoroughly spun off. The resulting crude product was dissolved in 2 mL of dichloromethane and protected with nitrogen gas. Triethylamine (3 mmol) was added, and the system was placed in an ice-water bath. Trifluoroacetic anhydride (1.2 mmol) was added dropwise. After thin-layer chromatography showed that the reaction was essentially complete, 50 mL of dichloromethane was added, followed by washing three times with 1 M aqueous hydrochloric acid, three times with saturated aqueous sodium bicarbonate, and the organic phase with saturated brine. The organic phase was then dried over anhydrous sodium sulfate and purified by column chromatography to obtain compound 1-4 (265 mg). ESI-MS: m / z 568.3 [M+H] + .

[0078] Step 3: Compound 1-4 (113 mg, 0.2 mmol) and Burgess reagent (1.5 eq) were added to a two-neck flask, and the flask was filled and vented with nitrogen three times. Dichloromethane dried over molecular sieves was added and the mixture was stirred overnight at room temperature. Thin layer chromatography showed that the reaction was essentially complete. After workup, compound of formula (I) (41 mg) was obtained by column chromatography. 1 H NMR (400 MHz, DMSO-d6) δ 9.46 (d, J = 8.7 Hz, 1H), 9.05 (d, J = 8.6 Hz, 1H), 7.67 (s, 1H), 4.97 (ddd, J = 11.0, 8.5, 5.0 Hz, 1H), 4.53 (d, J = 8.7 Hz, 1H), 4.34 (dd, J = 9.9, 7.1 Hz, 1H), 4.26-4.14 (m, 1H), 3.92 (d, J = 10.9 Hz, 1H), 3.50-3.34 (m, 4H), 3.22-3.11 (m, 1H), 3.06 (td, J = 9.3, 7.1 Hz, 1H), 2.68-2.58 (m, 1H), 2.50-2.43 (m, 1H), 2.31 (dd, J = 13.0, 10.0 Hz, 1H), 2.23-2.07 (m, 2H), 1.71 (tdd, J = 14.9, 10.3, 7.4 Hz, 2H), 0.99 (s, 9H). ESI-MS: 550.3 m / z [M+H] + .

[0079] Example 2: Preparation of coated tablets (1) Approximately 50 g of the compound of formula (I) was weighed and pulverized using a jet mill to obtain a particle size standard: d(0.9) of 5 to 20 μm. The corresponding auxiliary materials were weighed according to the formulations in Table 1.

[0080] (2) Weighed microcrystalline cellulose (internal addition), cross-linked sodium carboxymethylcellulose (internal addition), colloidal silicon dioxide (internal addition), pulverized compound of formula (I) (internal addition), and lactose monohydrate (internal addition) were transferred in this order to a mixing drum and mixed (rotation speed: 10 rpm, mixing for 10 minutes) to obtain a premix 1.

[0081] (3) Sodium stearyl fumarate (internal addition) was added to premix 1 and continued mixing (rotation speed 10 rpm, mixing for 3 minutes) to obtain premix 2.

[0082] (4) The premix 2 was sieved using a sieving machine (hole diameter φ0.8 mm), and after sieving, the materials were further added to the mixing drum and mixed (rotation speed 10 rpm, mixing for 5 minutes) to obtain the premix 3.

[0083] (5) Premix 3 was dry granulated to obtain granules.

[0084] (6) The produced granules, colloidal silicon dioxide (externally added), and cross-linked sodium carboxymethylcellulose (externally added) were added to a mixing drum and mixed (rotation speed: 10 rpm, mixing time: 5 min) to obtain a total mixture 1.

[0085] (7) Sodium stearyl fumarate (externally added) was added to the total mixture 1 and continued to mix (rotation speed: 10 rpm, mixing for 3 minutes) to obtain total mixture 2.

[0086] (8) Total mixture 2 was compressed into tablets, and the theoretical tablet weights were 0.1 g, 0.3 g, 0.75 g, and 1 g as shown in Table 1, respectively, and the hardness was 110 to 280 N.

[0087] (9) Preparation of a coating solution with a concentration of approximately 12% (w / w): Add purified water, and when the purified water is stirred to form a swirl, add a gastric-soluble film coating premix (Colorcon, containing the following ingredients: hydroxypropyl methylcellulose, iron oxide red, polyethylene glycol, and titanium dioxide) to the top of the swirl. Stir for 1 hour until a uniform suspension is formed, then stop stirring. Next, pass the coating solution through a 60-mesh sieve. When it is confirmed that there is no residue on the sieve, the preparation of the coating solution is complete.

[0088] (10) Coating: The compressed tablets obtained in step (8) were coated with the coating solution. The entire coating process included three steps: de-dusting, pre-heating, coating, and cooling. The weight gain of the coating was controlled to 4%.

[0089] TIFF2026502601000007.tif101170

[0090] Biological activity and related property test examples Test Example 1-1: Inhibitory activity test of the compound of formula (I) against SARS-CoV-2 3CLpro Using fluorescence resonance energy transfer (FER) to detect SARS-CoV-2 3CL pro The inhibitory activity of the compound of formula (I) against enzyme activity was evaluated. The total volume of the enzyme reaction system was 120 μL, the final concentration of protease was 30 nM, and the final concentration of substrate was 20 μM. The reaction buffer contained 50 mM Tris pH 7.3 and 1 mM EDTA. SARS-CoV-2 3CL was placed in a 96-well plate. pro Protease and different concentrations of compounds were added, incubated at 30°C for 10 min, and then substrate was added and the plate was quickly read in a microplate reader. The excitation and emission light were 320 nM and 405 nM, respectively. The test time was 3.5 min, and fluorescence readings were taken every 35 s. The final results were fitted to the reaction rate using the readings from the first 2 min and compared with the control (DMSO) to calculate the inhibition rate. Fitting was performed using the software GraphPad Prism 8, and the IC50 The values ​​and inhibition rate curves were obtained.

[0091] Experimental results show that the compound of formula (I) inhibits SARS-CoV-2 3CL pro IC for 50 The value was <0.1 μM, indicating a strong inhibitory effect.

[0092] Test Example 1-2: Inhibitory activity test of the compound of formula (I) against SARS-CoV-2 Omicron strain mutant 3CL protease Experimental principle: The inhibitory effect of the compounds of the present invention on the activity of Omicron strain mutant 3CL protease (P132H) was studied using a method in which fluorescence resonance energy transfer (FRET) occurs by reacting an enzyme with a substrate.

[0093] The experimental materials are shown in the table below.

[0094] TIFF2026502601000008.tif62170

[0095] Laboratory equipment and devices: TIFF2026502601000009.tif34170

[0096] Testing Procedure: A reaction buffer containing 20 mM Tris-HCl, 1 mM EDTA, 0.01% BSA, 1 mM DTT, and 100 mM NaCl was prepared. Using the Echo pipette system, test compounds were diluted to different concentrations in dimethyl sulfoxide (DMSO) and transferred to a 384-well plate. Mutant 3CL protease was diluted in the reaction buffer and added to the 384-well plate at 10 μL / well. The mixture was centrifuged at 1000 rpm for 1 min and then incubated at room temperature for 30 min. Next, 10 μL / well of substrate was added and the mixture was centrifuged at 1000 rpm for 30 s to initiate the enzyme reaction. The final enzyme concentration in the reaction system was 50 nM, the final substrate concentration was 20 μM, and the compound concentrations ranged from 10,000 nM to 0.51 nM. Next, the Flexstation 3 microplate reader was set to Kinetic Reduction Vmax mode, and the fluorescence intensity at 490 nm was read every 75 seconds for a total of 35 consecutive readings. The reaction velocity (V) was obtained, and the inhibition rate was calculated. The XLfit software was used to perform a four-parameter fitting to determine the half-maximal inhibitory concentration (IC). 50 The inhibition rate was calculated as follows: Inhibition rate=(V max -V Compound ) / (V max -V min )×100% where V max is the reaction rate value for wells containing only enzyme and substrate, and V min is the reaction rate value for wells containing substrate only, and V Compound was the reaction rate value for the wells containing the test compound, enzyme and substrate.

[0097] Experimental results: The compound of formula (I) still retained significant inhibitory activity against the 3CL protease harboring the P132H mutation in the SARS-CoV-2 Omicron strain.

[0098] TIFF2026502601000010.tif29170

[0099] Test Example 2: Inhibitory activity test of compounds of formula (I) against coronavirus 3CL proteases of different origins Experimental Objective: To study the inhibitory effect of the compound of formula (I) on the activity of 3CL proteases from six other coronaviruses that can infect humans, namely SARS-CoV, MERS-CoV, H229E-CoV, HKU1-CoV, NL63-CoV and OC43-CoV.

[0100] Test materials: 3CL protease: Recombinant full-length coronavirus 3CL proteases were produced in-house according to the coronavirus genome sequences. 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 protein expression of the six coronavirus 3CL proteases were purchased from Nanjing Jinsirui Biotechnology Co., Ltd.

[0101] 3CL protease substrate was purchased from Nanjing Jinsirui Biotechnology Co., Ltd.

[0102] Chymotrypsin substrate was purchased from Jier Biochemical Co., Ltd.

[0103] Other reagents are listed in the table below.

[0104] TIFF2026502601000011.tif27170

[0105] Testing Procedure: A reaction buffer (containing 50 mM Tris and 1 mM EDTA) was prepared. Test compounds were dissolved in DMSO to a 100 mM stock solution and further diluted two-fold with the reaction buffer to a total of 11 concentrations. 3CL protease and different concentrations of compounds were added to a 96-well plate and incubated at room temperature for 10 minutes. Substrate was then added and the plate was quickly read in a microplate reader. The total volume of the enzyme reaction system was 120 μL. The final concentrations of SARS-CoV, MERS-CoV, H229E-CoV, HKU1-CoV, NL63-CoV, and OC43-CoV proteases were 30 nM, 80 nM, 30 nM, 20 nM, 30 nM, and 10 nM, respectively, and the final substrate concentration was 10 μM. The excitation and emission wavelengths during reading were 340 nm and 490 nm, respectively. The test lasted for 10 minutes, with fluorescence readings taken every minute. The final results were calculated by fitting the reaction rate using the readings from the first 5 minutes to calculate the inhibition rate, which was calculated using the formula: inhibition rate = 1 - (reaction rate of the test group / reaction rate of the control group).

[0106] Experimental Results: As shown in Table 3, the compound of formula (I) exhibited relatively good inhibitory effects against 3CL proteases derived from six other coronaviruses, suggesting that the compound of formula (I) may have broad-spectrum anti-coronavirus activity.

[0107] TIFF2026502601000012.tif59170

[0108] 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 Experimental Objective: This study investigated the inhibitory effect of the compound of formula (I) on the replication of SARS-CoV-2 original strain (WIV04 strain), Delta strain (B.1.617.2), and Omicron strain (B.1.1.529) in Vero E6 cells by detecting viral copy numbers in the culture supernatant using real-time fluorescent quantitative PCR. Because Vero E6 cells highly express the efflux transporter protein P-gp, the compounds were co-incubated with 0.5 μM of the P-gp inhibitor CP-100356.

[0109] Test materials: Vero E6 was purchased from ATCC (product 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 derived from the Microbial (Virulent) Seed Deposit Center of the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0110] Other reagents are listed in the table below.

[0111] TIFF2026502601000013.tif103170

[0112] Experimental equipment: Biosafety cabinet (AC2-3S1, ESCO, Singapore) Carbon dioxide incubator (Thermo Scientific HERAcell 150i, Thermo Scientific, USA) Pure water equipment (Yuyuan SYS ultrapure water machine, Chengdu) StepOne Plus Real-time PCR system (4376600, ABI, USA) TC20™ Automated Cell Counter (1450102, BIO-RAD, USA) T100™ Thermal Cycler (1861096, BIO-RAD, USA) Centrifuge (Micro21 / 21R Thermo Fisher, Thermo Scientific, USA)

[0113] Testing Procedure: Vero E6 cells were digested with trypsin, placed in 90% DMEM, 10% fetal bovine serum, and seeded at 50,000 cells / well in a 48-well plate for overnight incubation. Test compounds were dissolved in DMSO to a 40 mM stock solution, which was then diluted with 0.5 μM Pgp inhibitor-containing medium to obtain the required concentration. The final test compound concentrations ranged from 1 μM to 0.004 μM. The cell supernatant was removed, and diluted compounds (containing 0.5 μM Pgp inhibitors) were added to each well and incubated for 1 hour. Different strains of SARS-CoV-2 were added at a multiplicity of infection (MOI) of 0.01 or 0.001 in a Biosafety Level 3 (BSL-3) laboratory. After 1 hour of incubation, the supernatant was removed and washed with PBS. 200 μL of diluted compounds (containing 0.5 μM Pgp inhibitors) were added per well. The supernatant was collected at 24 or 72 hours post-infection. Supernatant viral RNA was extracted, and the viral copy number in the supernatant was detected using real-time fluorescent quantitative PCR. The compound inhibition rate was calculated based on the viral copy number, and the compound's IC was calculated using GraphPad Prism 8. 50 was calculated.

[0114] For the cytotoxicity test, Vero E6 cells were digested and placed in medium (90% DMEM, 10% fetal bovine serum), seeded at 20,000 cells / well in a 96-well plate, and cultured overnight. The test compound was dissolved in DMSO to prepare a 40 mM stock solution, which was then gradient-diluted with medium or medium containing 0.5 μM Pgp inhibitor to obtain the required concentration for the test. The final concentration range of the test compound in the experiment was 500 μM to 1.95 μM. The cell supernatant was removed from the 96-well plate, and 100 μL / well of medium containing the test compound (single agent or 0.5 μM Pgp inhibitor) was added. After 24 hours of incubation, the cell activity was detected using a CCK8 detection kit, and the inhibition rate and median cytotoxic concentration (CCT) were measured. 50 ) was calculated.

[0115] Test results: As shown in Table 4, after combined use with the P-gp inhibitor CP-100356, the compound of formula (I) can dose-dependently inhibit the replication of Delta strain in Vero E6 cells, with an IC 50 The IC value was 0.040 μM. In the original strain, the compound of formula (I) in combination with a P-gp inhibitor also exerted a relatively strong inhibitory effect, with an IC value of 0.040 μM. 50 Furthermore, the combination of the compound of formula (I) with a P-gp inhibitor was able to significantly inhibit the replication of Omicron strains in Vero E6 cells, with an IC 50 The compound of formula (I) alone and in combination with a P-gp inhibitor did not show any significant cytotoxicity against the proliferation of Vero E6 cells, and CC 50 >500 μM.

[0116] TIFF2026502601000014.tif62170TIFF2026502601000015.tif18170

[0117] Test Example 4: In vivo antiviral activity of the compound of formula (I) against SARS-CoV-2 Delta strain in hACE2-K18 transgenic mice Experimental Objective: This study evaluated the antiviral activity of compound of formula (I) against SARS-CoV-2 Delta strain in K18 transgenic mice stably expressing human angiotensin-converting enzyme 2 (ACE2) (K18-hACE2).

[0118] Test materials: Seven- to eight-week-old K18-hACE2 transgenic mice were purchased from Jiangsu Jixi Yaokang Biotechnology Co., Ltd. SARS-CoV-2 Delta strain virus was derived from the Microbial (Virulent) Species Depository Center of the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0119] Ritonavir was purchased from Shanghai Disinfectant Chemical Pharmaceutical Co., Ltd.

[0120] Vero E6 cells were purchased from ATCC (product number CRL-1586).

[0121] Other reagents are listed in the table below.

[0122] TIFF2026502601000016.tif80170

[0123] Experimental equipment: Biosafety cabinet (AC2-3S1, ESCO, Singapore) Carbon dioxide incubator (Thermo Scientific HERAcell 150i, Thermo Scientific, USA) Pure water equipment (Yuyuan SYS ultrapure water machine, Chengdu) StepOne Plus Real-time PCR system (4376600, ABI, USA) TC20™ Automated Cell Counter (1450102, BIO-RAD, USA) T100™ Thermal Cycler (1861096, BIO-RAD, USA) Centrifuge (Micro21 / 21R Thermo Fisher, Thermo Scientific, USA) Tissue polishing machine (JXFSTPRP-CL, Shanghai Jingxin, China)

[0124] Experimental Procedure: K18-hACE2 transgenic mice were infected intranasally with the SARS-CoV-2 delta strain (day 0). Two hours after infection, mice were intragastrically administered 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) twice daily for 2 days (once on day 0, twice on day 1, and once on day 2) or 4 days (once on day 0, twice on days 1, 2, and 3). Mouse weight changes were recorded, and lung and brain tissues were collected at the end of the study. The left lung was fixed with formaldehyde, embedded, sectioned, and stained with H&E for histopathological examination. The right lung and brain tissues were each divided into two portions. One portion was polished, homogenized, and RNA was extracted and reverse-transcribed for viral copy number detection by real-time fluorescent quantitative PCR. The other portion was polished, homogenized, and viral titer detection by plaque assay. Plaque assay method: Vero E6 cells were seeded at 12,000 cells / well in a 24-well plate and cultured overnight. The stock tissue homogenate was diluted 10-fold with DMEM medium for use. The cell 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.

[0125] Test results: As shown in Table 5, two days after infection, compared with the model group (mean viral copy number was 9.19±0.30 log10 copies / g), when combined with ritonavir, both 50 mg / kg and 200 mg / kg of compound of formula (I) significantly reduced the viral load in the lungs, with mean copy numbers of 7.66±0.27 log10 copies / g and 6.79±0.30 log10 copies / g, respectively, with the 200 mg / kg dose reducing the viral copy number by 2.4 log10 copies / g. Four days after infection, a sustained inhibitory effect of compound of formula (I) on viral copy number was observed.

[0126] Regarding viral titer, as shown in Figure 1, a significant inhibitory effect of the compound of formula (I) was observed. Two days after infection, a dose of 200 mg / kg completely inhibited viral replication, and no titer was measured. At 50 mg / kg, the viral titer was reduced by more than 3 log10 PFU / g compared to the model group. Four days after infection, the compound of formula (I) demonstrated a sustained inhibitory effect on viral titer. Regarding body weight, as shown in Figure 2, four days after infection, mice in the model group lost approximately 10% of their body weight, while the group administered the compound of formula (I) showed no significant weight loss, indicating that sustained administration of the compound of formula (I) did not result in any significant toxicity. Viral load in the mouse brain was further detected, and no significant infection was observed in either group two days after infection. Four days after infection, compared with the model group, compound of formula (I) at both 50 mg / kg and 200 mg / kg doses significantly reduced the viral copy number in the brains of mice. At 200 mg / kg, the viral copy number in the brains was comparable to that of the uninfected control group. Four days after infection, viral titers were also measured in the brains. As shown in Figure 3, compared with the model group, no viral titers were measured at either dose of compound of formula (I), demonstrating the potent inhibitory effect of compound of formula (I). Furthermore, histopathological analysis of the lungs showed that compound of formula (I) at a dose of 200 mg / kg significantly improved lung damage, including reducing the degree of alveolar atrophy or dilation and the degree of alveolar membrane thickening, compared with the model group.

[0127] TIFF2026502601000017.tif73170

[0128] Test Example 5: Selectivity of compounds of formula (I) against kinases Experimental Objective: To detect the inhibitory activity of the compound of formula (I) against 413 kinases and to study the selectivity of the compound of formula (I) against kinases using the KinaseProfile experimental platform.

[0129] Test materials: The Full Human Panel [10 uM ATP] KinaseProfiler is a test product provided by Eurofins, product number 50-005KP10, which contains 413 kinases.

[0130] Testing Procedure: Compounds were tested against each selected kinase using the Eurofins standard KinaseProfiler assay, following the relevant standard operating procedures. Protein kinases were detected by radioactivity, while lipid kinases were detected by HTRF. The ATP concentration during the experiments was 10 μM. Detailed information about each kinase is available on the Eurofins website: https: / / www.eurofinsdiscoveryservices.com / catalogmanagement / viewItem / Full-Human-Panel-10-uM-ATP-KinaseProfiler / 50-005KP10.

[0131] Experimental results: The compound of formula (I) showed less than 30% inhibition against 413 kinases at a concentration of 10 μM, indicating no obvious inhibitory effect, suggesting that the compound of formula (I) has excellent selectivity.

[0132] Test Example 6: Selectivity of compounds of formula (I) for safety targets Experimental Objective: To detect the effects of compound of formula (I) on 47 safety-related targets using Safetyscan experimental platform.

[0133] Test materials: The Safety47 Panel Dose Response SAFETYscan was a test product provided by Eurofins with product number 87-1003DR. This product included 78 tests related to 47 safety targets.

[0134] Testing Procedure: For the 78 tests related to the 47 safety targets, the experimental methods used included cAMP experiments, calcium flux experiments, hormone nuclear receptor experiments, kinase binding experiments, enzyme activity experiments, neurotransmitter transporter experiments, ion channel experiments, and transporter experiments. The specific methods for each experiment were available on the eurofins website, which can be found at: https: / / www.eurofinsdiscoveryservices.com / catalogmanagement / viewItem / Safety47-Panel-Dose-Response-SAFETYscan-DiscoverX / 87-1003DR.

[0135] Experimental results: The compound of formula (I) had no significant inhibitory or activating effect on any of the 47 safety-related targets at a concentration of 100 μM (EC 50 and β-glucan were all greater than 100 μM), suggesting that the compound of formula (I) has excellent selectivity.

[0136] Test Example 7: Human plasma protein binding test of the compound of formula (I) Experimental materials Human plasma was purchased from BioIVT, anticoagulated with EDTA K2, and stored at −80° C. 96-well balance dialysis plates were purchased from HTDialysis LLC. Balance dialysis membranes were purchased from Gales Ferry.

[0137] Experimental procedure A basic solution of 14.2 g / L disodium hydrogen phosphate and 8.77 g / L sodium chloride was prepared using ultrapure water, and the basic solution could be stored for 7 days at 4°C. An acidic solution of 12.0 g / L sodium dihydrogen phosphate and 8.77 g / L sodium chloride was prepared using ultrapure water, and the acidic solution could be stored for 7 days at 4°C. The basic solution was titrated with the acidic solution to a pH of 7.4, and the buffer solution could be stored for 7 days at 4°C. On the day of the experiment, the pH of the buffer solution was measured, and if it was outside the range of 7.4 ± 0.1, the pH was adjusted.

[0138] To separate the membrane into two pieces, the dialysis membrane was immersed in ultrapure water for 60 minutes, then in 20% ethanol for 20 minutes, and finally in dialysis buffer for 20 minutes.

[0139] Frozen plasma was rapidly thawed at room temperature.

[0140] The plasma was centrifuged at 3220 g for 10 minutes at 4°C to remove clots, and the supernatant was collected in a new centrifuge tube. The pH of the plasma was measured and recorded.

[0141] A 10 mM DMSO stock solution of the test substance was prepared. 2 μL of the stock solution (10 mM) was diluted with 98 μL of DMSO to obtain a working solution (200 μM). 3 μL of the working solution was taken and 597 μL of human plasma was added to give a final concentration of 1 μM (0.5% DMSO). The mixture was vortexed thoroughly and uniformly.

[0142] A 120 μL sample of drug-containing plasma was added to one side of the dialysis membrane, and an equal volume of dialysate (phosphate buffer) was added to the other side. Experiments were performed in duplicate. The dialysis plate was sealed and placed in an incubator and incubated at 37°C, 5% CO2, and a rotation speed of approximately 100 rpm for 6 hours. After incubation, the sealing membrane was removed, and 50 μL of each well was aspirated from the buffer and plasma side and transferred to a different well of a new plate.

[0143] 50 μL of blank plasma was added to the phosphate buffer sample, and an equal volume of blank phosphate buffer was added to the plasma sample. 300 μL of room-temperature quencher (internal standard acetonitrile (IS), containing 500 nM labetalol, 100 nM alprazolam, and 2 μM ketoprofen)) was added to precipitate proteins. The mixture was vortexed for 5 minutes and centrifuged at 3220 g for 30 minutes at 4°C. 100 μL of the supernatant was transferred to a new plate. Depending on the LC / MS response signal and peak shape of the analyte, the supernatant was diluted with 100 μL or 200 μL of water. After uniform mixing, the sample was analyzed by LC / MS.

[0144] All calculations were performed using Microsoft Excel. The peak areas of the test compounds on the buffer and plasma sides were determined. The calculation formulas for the plasma protein binding rates of the test compounds and control drugs were as follows: release rate = (ratio of sample peak area to internal standard peak area on the buffer side / ratio of sample peak area to internal standard peak area on the plasma side) × 100%, binding rate = 1 − release rate, recovery rate = (ratio of sample peak area to internal standard peak area on the buffer side + ratio of sample peak area to internal standard peak area on the plasma side) / (ratio of sample peak area to internal standard peak area on the 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 on the initial plasma sample represents the total concentration of the compound at the start of sample incubation.

[0145] Test results: The results are shown in Table 6. When 1 μM of the compound of formula (I) was incubated at 37° C. for 6 hours, the average release rate was 46.63%, the binding rate was 53.37%, and the recovery rate was 88.02%.

[0146] TIFF2026502601000018.tif23170

[0147] Test Example 8: Tissue distribution test by single intragastric administration of the compound of formula (I) Test materials: Balb / c mice (purchased from Shanghai Minchang Biotechnology Co., Ltd.) were used, half male and half female, a total of 60 mice, weighing 18-25 g.

[0148] Testing Procedure: The compound of formula (I) was administered intragastrically to Balb / c mice in a single dose of 100 mg / kg in a volume of 10 mL / kg.

[0149] Before administration and at 5 min, 0.25, 1.0, 2.0, 3.0, 5.0, 7.0, and 10 h post-administration (six mice, half male and half female per time point), 0.2 mL of blood was collected from the retrobulbar venous plexus in EDTA-K2 tubes and centrifuged at 11,000 rpm for 5 min to separate the plasma, which was then frozen in a -70°C refrigerator. Immediately after the whole blood collection at 0.25, 1.0, 3.0, and 7.0 h, lung tissues were dissected and washed with cold saline to remove any remaining blood and contents from the tissue surface. After drying, the tissues were labeled and stored at -70°C for further analysis. The content of compound of formula (I) in plasma and lung tissues was measured by LC / MS-MS, and the lung-blood ratio was calculated.

[0150] Test results: After a single intragastric administration of the compound of formula (I) to Balb / c mice, the ratio of lung tissue exposure to plasma exposure was 0.62, with the compound of formula (I) showing higher lung tissue exposure.

[0151] Test Example 9: Safety pharmacology test of the effect of intragastric administration of the compound of formula (I) on the cardiovascular system of cynomolgus monkeys The effects of compounds of formula (I) on the cardiovascular system were investigated together in a two-week repeated dose toxicity study in cynomolgus monkeys.

[0152] Test materials: Thirty-two cynomolgus monkeys, half male and half female, were aged 2.5 to 5 years at the time of administration.

[0153] Animal origin: Yunnan Yingmao Biological Technology Co., Ltd., Guangxi Yusen Primate Experimental Animal Culture Development Co., Ltd., Zhongke Lingrui (Zhanjiang) Biotechnology Co., Ltd.

[0154] Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MBP) were measured in all conscious animals using a smart noninvasive sphygmomanometer BP-98E with a Provantis / v10.2.3.1 electronic data acquisition system (PV-02).

[0155] Experimental Procedure: Thirty-two cynomolgus monkeys (five animals / sex / group in Groups 1 and 4, and three animals / sex / group in Groups 2 and 3, for a total of four groups) were randomly assigned to receive either the compound of Formula (I) (40, 160, or 600 mg / kg / day) or a control formulation (98.9% vehicle formulation + 1.1% MTBE, 0 mg / kg / day) by nasal gavage twice daily for a total of 14 days, followed by a 14-day recovery period. All animals were enrolled in this study to evaluate the effects of treatment on ECG parameters (including heart rate, PR interval, QRS duration, QT interval, and QTcF) and blood pressure during the pre-treatment, treatment, and recovery periods.

[0156] Test results: In this test condition, cynomolgus monkeys were administered the compound of formula (I) (40, 160 and 600 mg / kg / day) twice daily by nasogastric gavage for 14 days, and no test-related cardiovascular changes were observed, no test-related arrhythmias were observed, and no test-related changes in ECG parameters or blood pressure were observed throughout the test process.

[0157] Test Example 10: Detection of coated tablet-related substances Formulation F3 of Example 2 was subjected to related substances determination.

[0158] The measurement method for related substances was performed according to high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Four Parts General Rule 0512), and the specific results were as follows:

[0159] Sample solution: Take 4 tablets of this product, place them in a 500 mL measuring flask, add an appropriate amount of water and disintegrate them by ultrasonic shaking, add the same volume of acetonitrile and ultrasonicate for 15 minutes, then dilute to the mark with a solvent (water:acetonitrile = 1:1 v / v), shake evenly, and centrifuge. An appropriate amount of the supernatant liquid was precisely measured and diluted with the solvent to prepare a solution containing approximately 1 mg of the compound of formula (I) per mL.

[0160] Control solution: An appropriate amount of the sample solution was precisely weighed and diluted with a solvent (water:acetonitrile=1:1 v / v) to prepare a solution containing approximately 10 μg per mL.

[0161] Chromatography conditions: An octadecylsilane-bonded silica gel chromatography column (Waters Atlantis T3, 4.6 mm × 150 mm, 3 μm) was used. Mobile phase A was 0.01 mol / L ammonium perchlorate buffer (1.17 g of ammonium perchlorate was dissolved in 1 L of water, and the pH was adjusted to 2.5 with perchloric acid and mixed evenly). Mobile phase B was acetonitrile. Linear gradient elution was performed according to the table below. The detection wavelength was 210 nm, the column temperature was 35°C, the flow rate was 1.0 mL / min, and the injection volume was 10 μL.

[0162] TIFF2026502601000019.tif40170

[0163] Measurement method: The sample solution and the control solution were precisely weighed and injected into a liquid chromatograph, and the chromatograms were recorded.

[0164] TIFF2026502601000020.tif17170

[0165] The results of the detection of coated tablet-related substances showed that the coated tablets obtained in this disclosure were of good quality and had low contents of single impurities and total impurities. Furthermore, the coated tablets of this disclosure showed no increase in the maximum single impurity or total impurities compared to the drug substance.

[0166] Test Example 11: Measurement of dissolution rate of coated tablets The dissolution and release rates were measured according to the method for measuring dissolution rate and release rate (Chinese Pharmacopoeia 2020, Part 4 General Rules 0931, Method 2).

[0167] The dissolution medium was 900 mL of 0.1 mol / L hydrochloric acid solution containing 0.5% sodium lauryl sulfate, and the rotation speed was 75 rpm. The operation was performed according to the method, with sampling times of 10, 15, 20, 30, and 45 min. After the 45-min sampling, the rotation speed was adjusted to 200 rpm and sampling was continued for 60 min.

[0168] The dissolution medium was 900 mL of pH 6.8 phosphate buffer solution, the rotation speed was 75 rpm, and the operation was performed according to the method. The sampling times were 5, 10, 15, 20, 30, 45, 60, 75, and 90 min. After the 90-min sampling, the rotation speed was adjusted to 200 rpm and sampling was continued for 120 min.

[0169] The dissolution medium was 900 mL of acetate buffer solution at pH 4.5, the rotation speed was 75 rpm, and the operation was performed according to the method. The sampling times were 5, 10, 15, 20, 30, 45, 60, 75, and 90 min. After the 90-min sampling, the rotation speed was adjusted to 200 rpm and sampling was continued for 120 min.

[0170] The dissolution measurement results are shown in Table 8 and FIG.

[0171] TIFF2026502601000021.tif71170

[0172] The dissolution measurement results in Table 8 and Figure 4 showed that the coated tablets of the present disclosure had good dissolution properties in 0.1 N hydrochloric acid + 0.5% SDS medium, pH 4.5 acetate buffer, and pH 6.8 phosphate buffer.

Claims

1. An oral pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof. 【Chemistry 1】

2. 10. The oral pharmaceutical composition of claim 1, wherein the oral pharmaceutical composition is in the form of a tablet, capsule, pill, granule, powder, emulsion, solution or suspension.

3. The oral pharmaceutical composition according to any one of claims 1 to 2, further comprising a disintegrant.

4. 4. The oral pharmaceutical composition according to claim 3, wherein the disintegrant is one or more selected from low-substituted hydroxypropyl cellulose, carboxymethylcellulose calcium, crospovidone, dry starch, carboxymethyl starch sodium, and cross-linked carboxymethylcellulose sodium, or the disintegrant is one or more selected from low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, and cross-linked carboxymethylcellulose sodium, or the disintegrant is cross-linked carboxymethylcellulose sodium.

5. The oral pharmaceutical composition according to any one of claims 1 to 4, further comprising a filler.

6. 6. The oral pharmaceutical composition of claim 5, wherein the filler is one or more selected from microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, calcium sulfate dihydrate, lactose, sucrose, dextrin, sorbitol, starch or a derivative thereof, mannitol, xylitol, and fructose; or the filler is one or more selected from microcrystalline cellulose, lactose, dextrin, and starch; or the filler is one or more selected from microcrystalline cellulose and lactose; or the filler is microcrystalline cellulose and lactose.

7. The oral pharmaceutical composition according to any one of claims 1 to 6, further comprising a glidant.

8. 8. The oral pharmaceutical composition of claim 7, wherein the glidant is one or more selected from colloidal silicon dioxide, talc powder, and wheat starch; or the glidant is one or more selected from colloidal silicon dioxide, talc powder, and wheat starch; or the glidant is one or more selected from colloidal silicon dioxide and talc powder; or the glidant is colloidal silicon dioxide.

9. The oral pharmaceutical composition according to any one of claims 1 to 8, further comprising a lubricant.

10. 10. The oral pharmaceutical composition according to claim 9, wherein the lubricant is one or more selected from magnesium stearate, stearic acid, calcium stearate, zinc stearate, sodium stearyl fumarate, liquid paraffin, polyethylene glycol, sodium lauryl sulfate, and hydrogenated vegetable oil; or the lubricant is one or more selected from magnesium stearate, stearic acid, calcium stearate, and sodium stearyl fumarate; or the lubricant is sodium stearyl fumarate.

11. The oral pharmaceutical composition according to any one of claims 1 to 10, comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof, a disintegrant, a filler, a glidant and a lubricant.

12. 12. The oral pharmaceutical composition of claim 11, wherein the disintegrant is cross-linked sodium carboxymethylcellulose, the fillers are microcrystalline cellulose and lactose monohydrate, the glidant is colloidal silicon dioxide, and the lubricant is sodium stearyl fumarate.

13. The oral pharmaceutical composition according to any one of claims 1 to 12, wherein the oral pharmaceutical composition further comprises a coating agent, or the oral pharmaceutical composition further comprises a gastric soluble film coating agent, preferably the coating agent comprises hydroxypropyl methylcellulose, iron oxide red, polyethylene glycol and titanium dioxide.

14. (1) An oral pharmaceutical composition according to any one of claims 1 to 13, and (2) A combination product that includes another antiviral drug, or another anti-coronavirus and / or small RNA virus drug, or ritonavir.

15. Use of an oral pharmaceutical composition according to any one of claims 1 to 13 or a combination product according to claim 14 in the manufacture of a medicament for the prevention or treatment of related diseases caused by coronavirus and / or small RNA virus infection.

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