Antiviral pharmaceutical composition and preparation method thereof

Abstract

Provided are an oral pharmaceutical composition comprising a spiro compound represented by formula (I) or a pharmaceutically acceptable salt thereof, a preparation method of the oral pharmaceutical composition, and a combined product comprising the oral pharmaceutical composition and another antiviral drug, the invention also discloses application of the oral pharmaceutical composition and the combination product in resisting virus infection and treating or preventing related diseases caused by virus infection. # imgabs0 #

Description

Antiviral drug composition and preparation method thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefits and priority of 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 into this document in its entirety. Technical Field

[0003] The present disclosure belongs to the field of pharmaceutical preparations, and in particular, relates to an oral pharmaceutical composition, a preparation method thereof, and use thereof in antiviral infection. Background Art

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

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

[0006] Therefore, there is still a clinical need for antiviral drug preparations for the treatment of coronaviruses, enterovirus 71, etc.

[0007] SUMMARY OF THE INVENTION

[0008] In one aspect, the present disclosure provides an oral pharmaceutical composition, wherein the oral pharmaceutical composition comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof

[0009] On the other hand, the present disclosure provides a method for preparing 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, coating the mixture obtained in step (1) with a coating agent.

[0010] In another aspect, the present disclosure also provides a combination product comprising: (1) the oral pharmaceutical composition, and (2) other antiviral drugs.

[0011] On the other hand, the present disclosure also provides use of the oral pharmaceutical composition or the combination product in preparing a drug for preventing or treating related diseases caused by coronavirus and / or picornavirus infection.

[0012] On the other hand, the present disclosure also provides use of the oral pharmaceutical composition or the combination product in preventing or treating diseases related to coronavirus and / or picornavirus infection.

[0013] On the other hand, the present disclosure also provides the oral pharmaceutical composition or the combination product for preventing or treating related diseases caused by coronavirus and / or picornavirus infection.

[0014] In another aspect, the present disclosure provides a method for treating diseases caused by coronavirus and / or picornavirus infection, comprising administering a therapeutically effective amount of the oral pharmaceutical composition or the combination product to a mammal, preferably a human, in need of such treatment.

[0015] Detailed description

[0016] In one aspect, the present disclosure provides an oral pharmaceutical composition, wherein the oral pharmaceutical composition comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof

[0017] In some embodiments, the oral pharmaceutical composition is in the form of a tablet, capsule, pill, granule, powder, emulsion, solution, or suspension.

[0018] In some embodiments, the oral pharmaceutical composition is in the form of a tablet, capsule, granule, powder or suspension.

[0019] In some embodiments, the oral pharmaceutical composition is a tablet.

[0020] In some embodiments, the oral pharmaceutical composition further comprises a disintegrant.

[0021] In some embodiments, the disintegrant is selected from one or more of low-substituted hydroxypropyl cellulose, carboxymethyl cellulose calcium, crospovidone, dry starch, sodium carboxymethyl starch, and croscarmellose sodium.

[0022] In some embodiments, the disintegrant is selected from one or more of low-substituted hydroxypropyl cellulose, crospovidone, and croscarmellose sodium.

[0023] In some embodiments, the disintegrant is croscarmellose sodium.

[0024] In some embodiments, the oral pharmaceutical composition further comprises a filler.

[0025] In some embodiments, the filler is selected from one or more of microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, calcium sulfate dihydrate, lactose, sucrose, dextrin, sorbitol, starch or its derivatives, mannitol, xylitol and fructose.

[0026] In some embodiments, the filler is selected from one or more of microcrystalline cellulose, lactose, dextrin, and starch.

[0027] In some embodiments, the filler is selected from one or more of microcrystalline cellulose and lactose.

[0028] In some embodiments, the fillers are microcrystalline cellulose and lactose.

[0029] In some embodiments, the lactose is lactose monohydrate.

[0030] In some embodiments, the oral pharmaceutical composition further comprises a glidant.

[0031] In some embodiments, the glidant is selected from one or more of colloidal silicon dioxide, talc, and wheat starch.

[0032] In some embodiments, the glidant is selected from one or more of colloidal silicon dioxide and talc.

[0033] In some embodiments, the glidant is colloidal silicon dioxide.

[0034] In some embodiments, the oral pharmaceutical composition further comprises a lubricant.

[0035] In some embodiments, the lubricant is selected from one or more of magnesium stearate, stearic acid, calcium stearate, zinc stearate, sodium stearyl fumarate, liquid paraffin, polyethylene glycol, sodium lauryl sulfate, and hydrogenated vegetable oil.

[0036] In some embodiments, the lubricant is selected from one or more of magnesium stearate, stearic acid, calcium stearate, and sodium stearyl fumarate.

[0037] In some embodiments, the lubricant is sodium stearyl fumarate.

[0038] In some embodiments, the present disclosure provides an oral pharmaceutical composition, wherein the oral pharmaceutical composition comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof, a disintegrant, a filler, a glidant, and a lubricant.

[0039] In some embodiments, the present disclosure provides an oral pharmaceutical composition, wherein the oral pharmaceutical composition comprises 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 carboxymethyl cellulose, the filler is microcrystalline cellulose and lactose monohydrate, the glidant is colloidal silicon dioxide, and the lubricant is sodium stearyl fumarate.

[0040] In some embodiments, the oral pharmaceutical composition comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof, a disintegrant, a filler, a glidant and a lubricant, wherein, in parts by weight, the content of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the oral pharmaceutical composition is 20-80 parts, the content of the disintegrant is 0.5-20 parts, the content of the filler is 10-70 parts, the content of the glidant is 0.1-20 parts and the content of the lubricant is 0.1-20 parts.

[0041] In some embodiments, in the oral pharmaceutical composition, the content of the compound of formula (I) or a pharmaceutically acceptable salt thereof is 20-80 parts, the content of cross-linked carboxymethyl cellulose sodium is 0.5-20 parts, the total content of microcrystalline cellulose and lactose monohydrate is 10-70 parts, the content of colloidal silicon dioxide is 0.1-20 parts, and the content of sodium stearyl fumarate is 0.1-20 parts, based on parts by weight.

[0042] In some embodiments, the content of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the oral pharmaceutical composition is 45-55 parts by weight.

[0043] In some embodiments, the content of the filler in the oral pharmaceutical composition is 30-45 parts by weight.

[0044] In some embodiments, the content of the disintegrant in the oral pharmaceutical composition is 2-10 parts by weight.

[0045] In some embodiments, the content of the glidant in the oral pharmaceutical composition is 1-3 parts by weight.

[0046] In some embodiments, the content of the lubricant in the oral pharmaceutical composition is 1-3 parts by weight.

[0047] In some embodiments, in the oral pharmaceutical composition, the content of the compound of formula (I) or a pharmaceutically acceptable salt thereof is 45-55 parts, the content of the disintegrant is 2-10 parts, the content of the filler is 30-45 parts, the content of the glidant is 1-3 parts, and the content of the lubricant is 1-3 parts, based on parts by weight.

[0048] In some embodiments, in the oral pharmaceutical composition, the content of the compound of formula (I) or a pharmaceutically acceptable salt thereof is 45-55 parts, the content of cross-linked carboxymethyl cellulose sodium is 2-10 parts, the total content of microcrystalline cellulose and lactose monohydrate is 30-45 parts, the content of colloidal silicon dioxide is 1-3 parts, and the content of sodium stearyl fumarate is 1-3 parts, based on parts by weight.

[0049] In another aspect, the present disclosure provides an oral pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof, a disintegrant, a filler, a glidant, a lubricant, and a coating agent.

[0050] In some embodiments, the coating is a gastric soluble film coating.

[0051] In some embodiments, the coating comprises hypromellose, red iron oxide, polyethylene glycol, and titanium dioxide.

[0052] In some embodiments, the present disclosure provides an oral pharmaceutical composition, wherein the oral pharmaceutical composition comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof, a disintegrant, a filler, a glidant, a lubricant and a coating agent, wherein the disintegrant is cross-linked sodium carboxymethyl cellulose, the filler is microcrystalline cellulose and lactose monohydrate, the glidant is colloidal silicon dioxide, the lubricant is sodium stearyl fumarate, and the coating agent comprises hypromellose, red iron oxide, polyethylene glycol and titanium dioxide.

[0053] In some embodiments, the content of the coating agent in the oral pharmaceutical composition is 0.5-20 parts by weight.

[0054] In some embodiments, the content of the coating agent in the oral pharmaceutical composition is 2-6 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 20-80 parts, the content of the disintegrant is 0.5-20 parts, the content of the filler is 10-70 parts, the content of the glidant is 0.1-20 parts, the content of the lubricant is 0.1-20 parts and the content of the coating agent is 0.5-20 parts, based on parts by weight.

[0056] In some embodiments, in the oral pharmaceutical composition, the content of the compound of formula (I) or a pharmaceutically acceptable salt thereof is 45-55 parts, the content of the disintegrant is 2-10 parts, the content of the filler is 30-45 parts, the content of the glidant is 1-3 parts, the content of the lubricant is 1-3 parts, and the content of the coating agent is 2-6 parts, based on parts by weight.

[0057] In some embodiments, in the oral pharmaceutical composition, the content of the compound of formula (I) or a pharmaceutically acceptable salt thereof is 20-80 parts, the content of cross-linked carboxymethyl cellulose sodium is 0.5-20 parts, the total content of microcrystalline cellulose and lactose monohydrate is 10-70 parts, the content of colloidal silicon dioxide is 0.1-20 parts, the content of sodium stearyl fumarate is 0.1-20 parts, and the content of the coating agent comprising hypromellose, red iron oxide, polyethylene glycol and titanium dioxide is 0.5-20 parts.

[0058] In some embodiments, in the oral pharmaceutical composition, the content of the compound of formula (I) or a pharmaceutically acceptable salt thereof is 45-55 parts, the content of cross-linked carboxymethyl cellulose sodium is 2-10 parts, the total content of microcrystalline cellulose and lactose monohydrate is 30-45 parts, the content of colloidal silicon dioxide is 1-3 parts, the content of sodium stearyl fumarate is 1-3 parts, and the content of the coating agent comprising hypromellose, red iron oxide, polyethylene glycol and titanium dioxide is 2-6 parts.

[0059] In some embodiments, the content of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the oral pharmaceutical composition is 10 mg-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.

[0060] In some embodiments, when the oral pharmaceutical composition is 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.

[0061] On the other hand, the present disclosure provides a method for preparing 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, coating the mixture obtained in step (1) with a coating agent.

[0062] In another aspect, the present disclosure also provides a combination product comprising: (1) the oral pharmaceutical composition, and (2) other antiviral drugs.

[0063] In some embodiments, the other antiviral drug is a drug against coronavirus and / or small RNA virus. In some embodiments, the other antiviral drug is ritonavir.

[0064] On the other hand, the present disclosure also provides use of the oral pharmaceutical composition or the combination product in preparing a drug for preventing or treating related diseases caused by coronavirus and / or picornavirus infection.

[0065] On the other hand, the present disclosure also provides use of the oral pharmaceutical composition or the combination product in preventing or treating diseases related to coronavirus and / or picornavirus infection.

[0066] On the other hand, the present disclosure also provides the oral pharmaceutical composition or the combination product for preventing or treating related diseases caused by coronavirus and / or picornavirus infection.

[0067] In another aspect, the present disclosure provides a method for treating diseases caused by coronavirus and / or picornavirus infection, comprising administering a therapeutically effective amount of the oral pharmaceutical composition or the combination product to a mammal, preferably a human, in need of such treatment.

[0068] In some embodiments, the diseases caused by coronavirus and / or picornavirus infection described in the present disclosure include but are not limited to respiratory tract infection, pneumonia or its complications.

[0069] In some embodiments, the coronavirus described in the present disclosure is selected from SARS-CoV, MERS-CoV, H229E-CoV, HKU1-CoV, NL63-CoV, OC43-CoV or SARS-CoV-2.

[0070] In some embodiments, examples of picornaviruses described herein include, but are not limited to, enterovirus 71.

[0071] BRIEF DESCRIPTION OF THE DRAWINGS

[0072] FIG1 is a graph showing the inhibitory effect of the compound of formula (I) on the viral titer in the lungs of mice 2 days after infection (Figure A) and 4 days after infection (Figure B) in Test Example 4.

[0073] FIG2 is a graph showing changes in mouse body weight in Test Example 4.

[0074] FIG3 is a graph showing the inhibitory effect of the compound of formula (I) on the viral titer in the mouse brain 4 days after infection in Test Example 4.

[0075] FIG4 is an in vitro dissolution and release curve of the coated tablets in Test Example 11. Example

[0076] The invention is described in detail below by way of examples, but this is not intended to limit the invention in any way. While the present invention has been described in detail herein, including specific embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments without departing from the spirit and scope of the present disclosure. All reagents used in the present disclosure are commercially available and can be used without further purification.

[0077] Unless otherwise specified, the ratios expressed for mixed solvents are volume ratios. Unless otherwise specified, % refers to wt%.

[0078] Compounds are manually or Software naming, commercially available compounds use supplier catalog names.

[0079] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The units of NMR shifts are 10 -6 The solvents for NMR measurements are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and tetramethylsilane (TMS) is the internal standard.

[0080] Example 1: Preparation of compound of formula (I)

[0081] 1.1. Preparation of compound 1-1:

[0082] Step 1: Add the starting material SMA (2.74 g, 11.85 mmol), 35 ml of dichloromethane, and 35 ml of DMF to a reaction flask, cool to 0°C, and sequentially add the starting material SMB (3.56 g, 11.86 mmol), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP, 6.29 g, 14.22 mmol), and N-methylmorpholine (NMM, 3.91 ml, 35.56 mmol). Warm to room temperature and react for 10 h. After the reaction, add an appropriate amount of dichloromethane, and wash the organic phase with 1N aqueous hydrochloric acid and saturated brine. Dry the organic phase over anhydrous sodium sulfate, concentrate to dryness, and column chromatography to obtain 3.71 g of INT-1; ESI-MS: 433.2 m / z [M+H] + ; 1 H NMR (400 MHz, DMSO-d6): δ H :6.75(d,J=9.2Hz,1H),4.38(t,J=8.2Hz,1H),4.25(d,J=10.9Hz,1H),4.11(d,J=9.3Hz,1H),3.93(t,J=9.3Hz,1H),3 .62(s,3H),3.40-3.31(m,4H),2.70(dd,J=13.1,7.9Hz,1H),2.37(dd,J=13.2,8.4Hz,1H),1.37(s,9H),0.94(s,9H).

[0083] Step 2: INT-1 (3.71 g, 8.58 mmol), 37 ml of THF, 37 ml of purified water, and lithium hydroxide monohydrate (0.72 g, 17.16 mmol) were added to a reaction flask and reacted at room temperature for 2 h. After the reaction, the pH was adjusted to 4 with concentrated hydrochloric acid and filtered to obtain 3.4 g of compound 1-1; ESI-MS: 419.2 m / z [M+H] + ; 1 H NMR (400 MHz, DMSO-d6): δ H :12.68(s,1H),6.71(d,J=9.4Hz,1H),4.38–4.19(m,2H),4.11(d,J=9.4Hz,1H),3.88(d,J=10.9Hz,1H), 3.41-3.29(m,4H),2.69(dd,J=13.1,7.9Hz,1H),2.34(dd,J=13.2,8.9Hz,1H),1.38(s,9H),0.94(s,9H).

[0084] 1.2. Preparation of compound 1-2:

[0085] Add 700 ml of ammonia-methanol solution (7 mol / L) and the starting material SMD (100 g, 0.349 mol) to the reaction flask, stir to dissolve, and maintain at 25±5°C for 36 hours. After the reaction is completed, concentrate the reaction solution until the remaining reaction solution is approximately 250 ml. Add 300 ml of isopropanol and continue to concentrate under reduced pressure until the remaining reaction solution is approximately 250 ml (repeat three times). Replace the atmosphere with nitrogen, cool the temperature to 10±5°C, and add 500 ml of hydrogen chloride-isopropanol solution (4 mol / L) to the reactor. After the addition is complete, heat the mixture to 25±5°C and maintain at 25±5°C for 9 hours. After the reaction, the reaction solution was concentrated under reduced pressure to a residual volume of about 250 ml. 300 ml of isopropanol was added and the reaction solution was further concentrated under reduced pressure to a residual volume of about 250 ml (repeated twice). 100 ml of isopropanol was added and stirred for 30 ± 5 min. The mixture was filtered and the filter cake was rinsed with 50 ml of isopropanol to obtain a wet product. The mixture was then dried under vacuum at 45 ± 5 °C to obtain 66.7 g of compound 1-2. 1 H NMR (400 MHz, DMSO-d6): δ H :8.45(d,J=5.1Hz,3H),8.25-8.04(m,1H),7.95(s,1H),7.67-7.49(m,1H),3.85-3.80(m,1H),3.19-3.13( m,2H),2.59-2.51(m,1H),2.32-2.27(m,1H),2.05-1.98(m,1H),1.82-1.66(m,2H); ESI-MS:172.1m / z[M+H] + .

[0086] 1.3. Preparation of compound of formula (I):

[0087] Step 1: Compound 1-1 (419 mg, 1 mmol) was placed in a two-necked flask. 5 mL of dichloromethane was added under nitrogen, followed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (400 mg, 1.1 mmol). The reaction mixture was stirred at room temperature for 1 h. Compound 1-2 (1 mmol) was dissolved in 1 mL of dichloromethane and added to the above system. N,N-diisopropylethylamine (2 mmol) was then added under an ice-water bath. The ice-water bath was removed and the system was stirred at room temperature overnight. After post-treatment, 50 mL of dichloromethane was added, followed by washing with 1 M aqueous hydrochloric acid three times, saturated aqueous sodium bicarbonate three times, and saturated brine. The organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and dried to obtain compound 1-3 (469 mg). ESI-MS: m / z 572.3 [M+H] + .

[0088] Step 2: Compound 1-3 (572 mg, 1 mmol) was dissolved in 3 mL of 4 M hydrogen chloride / 1,4-dioxane solution and stirred at ambient temperature. After TLC analysis indicated that the reaction of the starting material was essentially complete, the solvent was fully dried. The crude product was dissolved in 2 mL of dichloromethane under nitrogen protection. After adding triethylamine (3 mmol), the system was placed in an ice-water bath and trifluoroacetic anhydride (1.2 mmol) was added dropwise. After TLC analysis indicated that the reaction of the starting material was essentially complete, 50 mL of dichloromethane was added and the mixture was washed three times with a 1 M aqueous hydrochloric acid solution, three times with a saturated aqueous sodium bicarbonate solution, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and subjected to column chromatography to obtain compound 1-4 (265 mg). ESI-MS: m / z 568.3 [M+H] + .

[0089] Step 3: Compound 1-4 (113 mg, 0.2 mmol) and Burgess reagent (1.5 eq) were added to a two-necked flask. The mixture was flushed and released with nitrogen three times, followed by the addition of molecular sieve-dried dichloromethane. The mixture was stirred overnight at room temperature. Thin-layer chromatography indicated that the reaction was essentially complete. Post-treatment and column chromatography yielded the compound of formula (I) (41 mg). 1 H NMR(400MHz, DMSO-d6)δ9.46(d,J=8.7Hz,1H),9.05(d,J=8.6Hz,1H),7.67(s,1H),4.97(ddd,J=11.0,8.5,5.0 Hz,1H),4.53(d,J=8.7Hz,1H),4.34(dd,J=9.9,7.1Hz,1H),4.26–4.14(m,1H),3.92(d,J=10.9Hz,1H),3.50–3. 34(m,4H),3.22–3.11(m,1H),3.06(td,J=9.3,7.1Hz,1H),2.68–2.58(m,1H),2.50–2.43(m,1H),2.31(dd,J=1 3.0,10.0Hz,1H),2.23–2.07(m,2H),1.71(tdd,J=14.9,10.3,7.4Hz,2H),0.99(s,9H).ESI-MS:550.3m / z[M+H] + .

[0090] Example 2: Preparation of coated tablets

[0091] (1) Weigh about 50 g of the compound of formula (I) and grind it using a jet mill. The particle size standard is: d(0.9) is 5-20 μm. Weigh the corresponding excipients according to the prescription amount in Table 1.

[0092] (2) The weighed microcrystalline cellulose (added internally), croscarmellose sodium (added internally), colloidal silicon dioxide (added internally), the crushed compound of formula (I) (added internally), and lactose monohydrate (added internally) were transferred to a mixing barrel and mixed (rotating speed 10 rpm, mixing for 10 min) to obtain premix 1.

[0093] (3) Sodium stearyl fumarate (internal addition) was added to premix 1 and the mixture was mixed continuously (rotating speed 10 rpm, mixing for 3 min) to obtain premix 2.

[0094] (4) Use a granulator (aperture) ) Premix 2 was sieved and then added to the above mixing barrel for mixing (rotation speed 10 rpm, mixing for 5 min) to obtain premix 3.

[0095] (5) Dry granulate the premix 3 to obtain granules.

[0096] (6) The prepared particles, colloidal silicon dioxide (externally added) and cross-linked sodium carboxymethyl cellulose (externally added) were added to a mixing barrel and mixed (rotation speed 10 rpm, mixing time 5 min) to obtain a total mixture 1.

[0097] (7) Sodium stearyl fumarate (external addition) was added to the above-mentioned total mixture 1 and continued to mix (rotation speed 10 rpm, mixing for 3 minutes) to obtain total mixture 2.

[0098] (8) The total mixture 2 was compressed into tablets. The theoretical tablet weights were 0.1 g, 0.3 g, 0.75 g, and 1 g, respectively, as shown in Table 1. The hardness ranged from 110 to 280 N.

[0099] (9) Prepare a coating solution with a concentration of about 12% (w / w): Add purified water. When the purified water is stirred into a vortex, add a gastric-soluble film coating premix (Colorcon, ingredients include: hydropropyl methylcellulose, red iron oxide, polyethylene glycol and titanium dioxide) to the shoulder of the vortex. Stir for 1 hour to form a uniform suspension. Then stop stirring. Then pass the coating solution through a 60-mesh sieve and confirm that there is no residue on the sieve. The preparation of the coating solution is completed.

[0100] (10) Coating: The compressed tablets obtained in step (8) are coated with a coating liquid. The entire coating process includes three steps: dust removal and preheating, coating, and cooling. The coating weight gain is controlled at 4%.

[0101] Table 1 Tablet prescription composition

[0102] Biological activity and related properties test examples

[0103] Test Example 1-1: Test on the activity of the compound of formula (I) in inhibiting SARS-CoV-2 3CLpro

[0104] Evaluation of the effect of compound of formula (I) on SARS-CoV-2 3CL using fluorescence resonance energy transfer method pro The volume of the entire enzymatic reaction system was 120 μL, the final concentration of the protease was 30 nM, and the final concentration of the substrate was 20 μM. The reaction system buffer included 50 mM Tris pH 7.3 and 1 mM EDTA. SARS-CoV-2 3CL was added to a 96-well plate. pro The protease and compound at different concentrations were incubated at 30°C for 10 minutes. The substrate was added and the plate was quickly placed in a microplate reader for reading. The excitation and emission values ​​were 320nM and 405nM, respectively. The test time was 3.5 minutes, and the fluorescence value was read every 35 seconds. The final result was the reaction rate fitted with the readings of the first 2 minutes, and the inhibition rate was calculated by comparing with the control group (DMSO). The IC was fitted using GraphPad Prism 8 software. 50 values ​​and inhibition rate curves.

[0105] The experimental results show that the compound of formula (I) has an anti-SARS-CoV-2 3CL pro IC 50 The value was <0.1 μM, indicating a strong inhibitory effect.

[0106] Test Example 1-2: Inhibitory activity test of the compound of formula (I) against the mutant 3CL protease of SARS-CoV-2 Omicron strain

[0107] Experimental principle: The method of fluorescence resonance energy transfer (FRET) produced by the reaction between enzyme and substrate was used to study the inhibitory effect of the compound of the present invention on the activity of the mutant 3CL protease (P132H) of the Omicron strain.

[0108] The experimental materials are shown in the following table:

[0109] Experimental instruments and equipment:

[0110] Experimental steps:

[0111] Prepare a reaction buffer containing 20mM Tris-HCl, 1mM EDTA, 0.01% BSA, 1mM DTT, and 100mM NaCl. Use an Echo pipetting system to dilute the test compound to different concentrations in dimethyl sulfoxide (DMSO) and transfer it to a 384-well plate. Dilute the mutant 3CL protease with reaction buffer and add 10μL / well to a 384-well plate, centrifuge at 1000rpm for 1min, and then incubate at room temperature for 30 minutes. Then add 10μL / well of substrate and centrifuge at 1000rpm for 30s to start the enzyme reaction. In the reaction system, the final concentration of the enzyme is 50nM, the final concentration of the substrate is 20μM, and the concentration range of the compound is 10000nM to 0.51nM. Kinetic Reduction Vmax mode was then selected on the Flexstation 3 microplate reader. The fluorescence value at a wavelength of 490 nm was read continuously every 75 seconds for a total of 35 times to obtain the reaction rate value (V). The inhibition rate was calculated and the half-maximal inhibitory concentration (IC50) was obtained by four-parameter fitting using XLfit software. 50 The inhibition rate is calculated as follows: Inhibition rate = (V max -V compound ) / (V max -V min )*100%

[0112] Among them, V max is the reaction rate value of the well containing only enzyme and substrate, V min is the reaction rate value of the well containing only substrate, V Compound is the reaction rate value for the wells containing the test compound, enzyme, and substrate.

[0113] Experimental results: The compound of formula (I) still maintains significant inhibitory activity against the 3CL protease with the P132H mutation in the SARS-CoV-2 Omicron strain.

[0114] Table 2 Inhibitory effect of the compound of formula (I) on the 3CL protease activity of SARS-CoV-2 Omicron strain

[0115] *Indicates three independent repeated experiments.

[0116] Test Example 2: Inhibitory activity test of the compound of formula (I) against coronavirus 3CL protease from different sources

[0117] Experimental purpose: To study the inhibitory effect of the compound of formula (I) on the 3CL protease activity of six other coronaviruses that can infect humans, namely SARS-CoV, MERS-CoV, H229E-CoV, HKU1-CoV, NL63-CoV and OC43-CoV.

[0118] Experimental Materials:

[0119] 3CL protease: Recombinant full-length coronavirus 3CL protease was prepared based on the coronavirus genome sequence. The GenBank numbers of the SARS-CoV, MERS-CoV, H229E-CoV, HKU1-CoV, NL63-CoV, and OC43-CoV genomes used were AAP13442.1, MT387202.1, AF304460.1, AY597011.2, AY567487.2, and AY903459.1, respectively. The DNA sequences required for the expression of the six coronavirus 3CL protease proteins were purchased from Nanjing GenScript Biotechnology Co., Ltd.

[0120] 3CL protease substrate was purchased from Nanjing GenScript Biotechnology Co., Ltd.

[0121] Chymotrypsin substrate was purchased from Gill Biochemical Co., Ltd.

[0122] Other reagents are shown in the following table:

[0123] Experimental steps:

[0124] Prepare reaction buffer (containing 50mM Tris and 1mM EDTA). Dissolve the test compound in DMSO to a 100mM stock solution and further perform a 2-fold serial dilution in reaction buffer for a total of 11 concentrations. Add 3CL protease and different concentrations of the compound to a 96-well plate, incubate at room temperature for 10 minutes, add substrate, and quickly place in a microplate reader for reading. The volume of the entire enzymatic reaction system is 120μL. The final concentrations of SARS-CoV, MERS-CoV, H229E-CoV, HKU1-CoV, NL63-CoV, and OC43-CoV proteases are 30nM, 80nM, 30nM, 20nM, 30nM, and 10nM, respectively, and the final concentration of substrate is 10μM. The excitation and emission wavelengths for reading are 340nm and 490nm, respectively. The test time was 10 minutes, and the fluorescence value was read every 1 minute. The final result was the reaction rate obtained by fitting the readings of the first 5 minutes, and the inhibition rate was calculated using the following formula: inhibition rate = 1 – (reaction rate of the test group / reaction rate of the control group).

[0125] Experimental results: As shown in Table 3, the compound of formula (I) exhibited a good inhibitory effect on 3CL proteases from six other coronaviruses, suggesting that the compound of formula (I) may have broad-spectrum anti-coronavirus activity.

[0126] Table 3 Inhibitory effect of compounds of formula (I) on 3CL proteases from other coronaviruses

[0127] 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

[0128] Objective: This study investigated the inhibitory effect of the compound of formula (I) on the replication of SARS-CoV-2 (WIV04), Delta (B.1.617.2), and Omicron (B.1.1.529) in Vero E6 cells by detecting viral copy number in the culture supernatant using real-time fluorescence quantitative PCR. Since Vero E6 cells highly express the efflux transporter P-gp, 0.5 μM of the P-gp inhibitor CP-100356 was added to the cells for co-incubation.

[0129] Experimental Materials:

[0130] Vero E6 was purchased from ATCC (catalog number CRL-1586), and the SARS-CoV-2 original strain (SARS-CoV-2-WIV04), Delta strain (B.1.617.2), and Omicron strain (B.1.1.529) were obtained from the Microbial Culture Collection Center of the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0131] Other reagents are shown in the following table:

[0132] Experimental instruments:

[0133] Biological safety cabinet (AC2-3S1, ESCO, Singapore)

[0134] CO2 incubator (Thermo Scientific HERAcell 150i, Thermo Scientific, USA )

[0135] Pure water machine (SYS ultrapure water machine, Chengdu)

[0136] StepOne Plus Real-time PCR system (4376600, ABI, USA)

[0137] TC20 TM Automatic cell counter (1450102, BIO-RAD, USA)

[0138] T100 TM Thermal Cycler (1861096, BIO-RAD, USA)

[0139] Centrifuge (Micro21 / 21R Thermo Scientific, USA)

[0140] Experimental steps:

[0141] Vero E6 cells were trypsinized and plated in culture medium (90% DMEM, 10% fetal bovine serum) in 48-well plates at 50,000 cells per well and cultured overnight. Test compounds were dissolved in DMSO to a 40 mM stock solution and serially diluted in culture medium containing 0.5 μM Pgp inhibitor to obtain the desired concentrations. Final test compound concentrations ranged from 1 μM to 0.004 μM. The supernatant was removed, and the diluted compound (containing 0.5 μM Pgp inhibitor) was added to each well and incubated for 1 hour. In a biosafety level 3 (BSL-3) laboratory, different strains of SARS-CoV-2 were added at a multiplicity of infection (MOI) of 0.01 or 0.001. After a 1-hour incubation, the supernatant was removed, the cells were washed with PBS, and 200 μL / well of the diluted compound (containing 0.5 μM Pgp inhibitor) was added. The supernatant was collected 24 or 72 hours after infection. The supernatant viral RNA was extracted and the supernatant viral copy number was detected by real-time fluorescence quantitative PCR. The compound inhibition rate was calculated based on the viral copy number, and the IC of the compound was calculated using GraphPad Prism 8. 50 .

[0142] In the cytotoxicity test, Vero E6 cells were digested and placed in culture medium (90% DMEM, 10% fetal bovine serum), seeded into 96-well plates, 20,000 cells per well, and cultured overnight. The test compound was dissolved in DMSO to prepare a 40mM stock solution, and further diluted with culture medium or culture medium containing 0.5μM Pgp inhibitor to obtain the required concentration for the test. The final concentration range of the test compound in the experiment was 500μM to 1.95μM. The cell supernatant in the 96-well plate was removed, and 100μL / well of the test compound (single drug or containing 0.5μM Pgp inhibitor) culture medium was added. After incubation for 24 hours, the cell viability was detected using the CCK8 detection kit, and the inhibition rate and half cytotoxic concentration (CC) were calculated. 50 ).

[0143] Test results: As shown in Table 4, when combined with the P-gp inhibitor CP-100356, the compound of formula (I) can inhibit the replication of the Delta strain in Vero E6 cells in a dose-dependent manner, IC 50 The compound of formula (I) combined with P-gp inhibitors also exerted a strong inhibitory effect in the original strain, IC 50In addition, the compound of formula (I) combined with P-gp inhibitors can significantly inhibit the replication of Omicron strain in Vero E6 cells, IC 50 The compound of formula (I) alone or in combination with P-gp inhibitors had no significant cytotoxicity to the proliferation of Vero E6 cells. 50 >500μM.

[0144] Table 4 Inhibitory effect of compound of formula (I) combined with P-gp inhibitor on SARS-CoV-2 in Vero E6 cells

[0145] Test Example 4: In vivo antiviral effect of the compound of formula (I) against SARS-CoV-2 Delta strain in hACE2-K18 transgenic mice

[0146] Objective: This study evaluated the antiviral activity of the compound of formula (I) against SARS-CoV-2 delta strain in K18 transgenic mice stably expressing human angiotensin-converting enzyme 2 (ACE2) (K18-hACE2).

[0147] Experimental Materials:

[0148] 7-8 week old K18-hACE2 transgenic mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. SARS-CoV-2 Delta strain virus was obtained from the Microbial Culture Collection Center of Wuhan Institute of Virology, Chinese Academy of Sciences.

[0149] Ritonavir was purchased from Shanghai Desano Chemical Pharmaceutical Co., Ltd.

[0150] Vero E6 cells were purchased from ATCC (Cat. No. CRL-1586).

[0151] Other reagents are shown in the following table:

[0152] Experimental instruments:

[0153] Biological safety cabinet (AC2-3S1, ESCO, Singapore)

[0154] CO2 incubator (Thermo Scientific HERAcell 150i, Thermo Scientific, USA )

[0155] Pure water machine (SYS ultrapure water machine, Chengdu)

[0156] StepOne Plus Real-time PCR system (4376600, ABI, USA)

[0157] TC20 TM Automatic cell counter (1450102, BIO-RAD, USA)

[0158] T100 TM Thermal Cycler (1861096, BIO-RAD, USA)

[0159] Centrifuge (Micro21 / 21R Thermo Scientific, USA)

[0160] Tissue grinder (JXFSTPRP-CL, Shanghai Jingxin, China )

[0161] Experimental procedures: K18-hACE2 transgenic mice were infected with the SARS-CoV-2 Delta strain via intranasal drops, with this day being designated as day 0. Two hours after infection, vehicle, 50 mg / kg, or 200 mg / kg of the compound of formula (I) (combined with 50 mg / kg of the cytochrome P450 inhibitor ritonavir) were administered orally, with dosing occurring twice daily for 2 days (one dose on day 0, two doses on day 1, and one dose on day 2) or 4 days (one dose on day 0, and two doses on days 1, 2, and 3, respectively). Mouse body weight changes were recorded, and lung and brain tissues were collected at the endpoint. The left lung was formaldehyde-fixed, embedded, sectioned, and stained with H&E for histopathological examination. The right lung and brain tissue were each divided into two aliquots. One aliquot was ground and homogenized to extract RNA, which was then reverse transcribed, and viral copy number was determined by real-time fluorescence quantitative PCR. The other aliquot was ground and homogenized to determine viral titer using a plaque assay. The plaque assay was performed as follows: Vero E6 cells were seeded in a 24-well plate at 12,000 cells per well and cultured overnight. A stock tissue homogenate was serially diluted 10-fold in DMEM medium. The supernatant was removed, and the diluted tissue homogenate was added and incubated for 1 hour. The supernatant was then removed, and medium containing 1% sodium methylcellulose and 2% FBS was added and cultured for 4 days. The medium was then removed, and the cells were fixed with paraformaldehyde and stained with 1% (w / v) crystal violet. The number of plaques in each well was counted.

[0162] Test results: As shown in Table 5, two days after infection, compared to the model group (mean viral copy number of 9.19±0.30log10copies / g), the compound of formula (I) at 50 mg / kg and 200 mg / kg, combined with ritonavir, significantly reduced the viral load in the lungs, with mean copy numbers of 7.66±0.27log10copies / g and 6.79±0.30log10copies / g, respectively. At a dose of 200 mg / kg, the viral copy number decreased by 2.4log10copies / g. A sustained inhibitory effect of the compound of formula (I) on viral copy number was observed four days after infection.

[0163] In terms of viral titer, as shown in Figure 1, a significant inhibitory effect of the compound of formula (I) was observed. Two days after infection, the 200 mg / kg dose completely inhibited viral replication, and no titer was measured. At 50 mg / kg, the viral titer decreased by more than 3 log10 PFU / g compared to the model group. After four days of infection, the compound of formula (I) demonstrated a sustained inhibitory effect on viral titer. As shown in Figure 2, after four days of infection, the model group mice lost approximately 10% of their body weight, while the body weight of the group receiving the compound of formula (I) did not significantly decrease, indicating that the compound of formula (I) did not show significant toxicity under continuous administration. We further tested the viral load in the mouse brain. After two days of infection, no obvious infection was observed in any group. Four days after infection, compared to the model group, the compound of formula (I) at both 50 mg / kg and 200 mg / kg doses significantly reduced the number of viral copies in the mouse brain. In particular, at the 200 mg / kg dose, the number of viral copies in the brain was comparable to that of the uninfected normal group. We further tested viral titers in the brain 4 days after infection. The results, shown in Figure 3, showed no detectable viral titers at both doses compared to the model group, demonstrating the potent inhibitory effect of the compound (I). Furthermore, lung histopathological analysis showed that the compound (I) significantly improved lung damage at a dose of 200 mg / kg compared to the model group, including reduced alveolar atrophy or dilation and alveolar membrane thickening.

[0164] Table 5 Viral loads in the lungs and brains of mice 2 and 4 days after infection (mean ± SD)

[0165] Test Example 5: Selectivity of the compound of formula (I) for kinases

[0166] Experimental purpose: The inhibitory activity of the compound of formula (I) against 413 kinases was detected on the KinaseProfile experimental platform to study the selectivity of the compound of formula (I) against kinases.

[0167] Experimental Materials:

[0168] Full Human Panel [10uM ATP] Kinase Profiler is a test product provided by Eurofins, product number: 50-005KP10, this product contains 413 kinases.

[0169] Experimental steps:

[0170] Compounds were tested for each selected kinase using the standard Eurofins KinaseProfiler assay, following the relevant standard operating procedures. Protein kinases were detected radiometrically, while lipid kinases were detected using HTRF. The ATP concentration used in the assays was 10 μM. Detailed information for each kinase is available on the Eurofins website at: https: / / www.eurofinsdiscoveryservices.com / catalogmanagement / viewItem / Full-Human-Panel-10-uM-ATP-KinaseProfiler / 50-005KP10.

[0171] Experimental results: For 413 kinases, the inhibition rate of the compound of formula (I) at a concentration of 10 μM was less than 30%, with no obvious inhibitory effect, suggesting that the compound of formula (I) has excellent selectivity.

[0172] Test Example 6: Selectivity of the compound of formula (I) for safety targets

[0173] Experimental purpose: The effects of the compound of formula (I) on 47 safety-related targets were detected on the Safetyscan experimental platform.

[0174] Experimental Materials:

[0175] Safety47 Panel Dose Response SAFETYscan is a test product provided by Eurofins, product number: 87-1003DR. This product contains 78 tests related to 47 safety targets.

[0176] Experimental steps:

[0177] The 78 tests for 47 safety targets utilize assays including cAMP assays, calcium flux assays, hormone nuclear receptor assays, kinase binding assays, enzyme activity assays, neurotransmitter transporter assays, ion channel assays, and transporter assays. Detailed protocols for each assay are available on the eurofins website at: https: / / www.eurofinsdiscoveryservices.com / catalogmanagement / viewItem / Safety47-Panel-Dose-Response-SAFETYscan-DiscoverX / 87-1003DR.

[0178] Experimental results: For 47 safety-related targets, the compound of formula (I) had no significant inhibitory or activating effect at a concentration of 100 μM (EC 50 were all greater than 100 μM), suggesting that the compound of formula (I) has excellent selectivity.

[0179] Test Example 7: Human plasma protein binding test of the compound of formula (I)

[0180] Experimental Materials

[0181] Human plasma was purchased from BioIVT, anticoagulated with EDTA K2, and stored at −80° C. 96-well equilibrium dialysis plates were purchased from HTDialysis LLC. Equilibrium dialysis membranes were purchased from Gales Ferry.

[0182] Experimental procedures

[0183] Prepare an alkaline solution with ultrapure water containing 14.2 g / L sodium dihydrogen phosphate and 8.77 g / L sodium chloride. This alkaline solution can be stored at 4°C for 7 days. Prepare an acidic solution with ultrapure water containing 12.0 g / L sodium dihydrogen phosphate and 8.77 g / L sodium chloride. This acidic solution can be stored at 4°C for 7 days. Titrate the alkaline solution with the acidic solution to a pH of 7.4. This buffer can be stored at 4°C for 7 days. Test the buffer pH on the day of the experiment and adjust the pH if it is outside the range of 7.4 ± 0.1.

[0184] The dialysis membrane was soaked in ultrapure water for 60 minutes to separate the membrane into two pieces, then soaked in 20% ethanol for 20 minutes, and finally soaked in the dialysis buffer for 20 minutes.

[0185] Frozen plasma was rapidly thawed at room temperature.

[0186] The plasma was centrifuged at 3,220 g for 10 minutes at 4°C to remove clots, and the supernatant was collected into a new centrifuge tube. The pH of the plasma was measured and recorded.

[0187] Prepare a 10 mM DMSO stock solution of the test substance. Dilute 2 μL of the 10 mM stock solution with 98 μL of DMSO to obtain a 200 μM working solution. Take 3 μL of the working solution and add 597 μL of human plasma to a final concentration of 1 μM (0.5% DMSO). Vortex thoroughly.

[0188] Add 120 μL of drug-spiked plasma sample to one side of the dialysis membrane and an equal volume of dialysate (phosphate buffered saline) to the other side. Perform the experiment in duplicate. Seal the dialysis plate and place it in an incubator. Incubate for 6 hours at 37°C, 5% CO2, and approximately 100 rpm. After incubation, remove the seal and pipette 50 μL from the buffer and plasma sides of each well into separate wells of a new plate.

[0189] Add 50 μL of blank plasma to the phosphate buffer sample, and add an equal volume of blank phosphate buffer to the plasma sample. Precipitate the protein by adding 300 μL of room temperature quencher (containing the internal standard acetonitrile (IS), 500 nM labetalol, 100 nM alprazolam, and 2 μM ketoprofen). Vortex for 5 minutes. Centrifuge at 3220 g for 30 minutes at 4°C. Transfer 100 μL of the supernatant to a new plate. Dilute the supernatant with 100 μL or 200 μL of water, depending on the HPLC-MS response and peak shape of the analyte. Mix thoroughly, and analyze the sample by HPLC-MS.

[0190] All calculations were performed using Microsoft Excel. The peak areas of the test substance on the buffer and plasma sides were determined. The plasma protein binding rates of the test substance and control drug were calculated using the following formulas: Free rate = (ratio of sample peak area to internal standard peak area, buffer side) / ratio of sample peak area to internal standard peak area, plasma side) * 100%, Binding rate = 1 - Free rate, Recovery rate = (ratio of sample peak area to internal standard peak area, buffer side + ratio of sample peak area to internal standard peak area, plasma side) / (ratio of sample peak area to internal standard peak area, initial plasma sample) * 100%. The ratio of sample peak area to internal standard peak area on the buffer side represents the free concentration of the compound, the ratio of sample peak area to internal standard peak area on the plasma side represents the sum of the free and bound concentrations of the compound, and the ratio of sample peak area to internal standard peak area in the initial plasma sample represents the total concentration of the compound at the start of sample incubation.

[0191] Test results:

[0192] See Table 6. When 1 μM of the compound of formula (I) was incubated at 37° C. for 6 hours, the average free rate was 46.63%, the binding rate was 53.37%, and the recovery rate was 88.02%.

[0193] Table 6 Human plasma protein binding test results of the compound of formula (I)

[0194] Test Example 8: Tissue distribution test of a single oral gavage of a compound of formula (I)

[0195] Experimental Materials:

[0196] A total of 60 Balb / c mice (purchased from Shanghai Minchang Biotechnology Co., Ltd.) were used, half male and half female, weighing 18-25 g.

[0197] Experimental steps:

[0198] Balb / c mice were given a single oral administration of the compound of formula (I) at a dose of 100 mg / kg in a volume of 10 mL / kg.

[0199] Before administration and at 5 minutes, 0.25, 1.0, 2.0, 3.0, 5.0, 7.0, and 10 hours after administration (six mice, half male and half female, were included at each time point). At these time points, 0.2 ml of blood was collected from the retroorbital venous plexus, placed in an EDTA-K2 tube, centrifuged at 11,000 rpm for 5 minutes, and plasma was separated and frozen at -70°C. Lung tissue was immediately dissected and collected at 0.25, 1.0, 3.0, and 7.0 hours after whole blood collection. The tissue was rinsed with cold saline to remove residual blood and contents, blotted dry, labeled, and stored at -70°C until testing. The content of the compound of formula (I) in plasma and lung tissue was determined by LC / MS-MS, and the lung-to-blood ratio was calculated.

[0200] Test results:

[0201] After a single oral administration of the compound of formula (I) to Balb / c mice, the ratio of lung tissue exposure to plasma exposure was 0.62, indicating that the exposure of the compound of formula (I) in the lung tissue was high.

[0202] Test Example 9: Safety pharmacology study on the effects of oral administration of the compound of formula (I) on the cardiovascular system of cynomolgus monkeys

[0203] In a 2-week repeated-dose toxicity study in cynomolgus monkeys, the effects of the compound of formula (I) on the cardiovascular system were also investigated.

[0204] Experimental Materials:

[0205] Thirty-two cynomolgus monkeys, half male and half female, aged 2.5-5 years at the time of administration were used.

[0206] Source of animals: Yunnan Yingmao Biotechnology Co., Ltd.; Guangxi Xiongsen Primate Experimental Animal Breeding and Development Co., Ltd.; Zhongke Lingrui (Zhanjiang) Biotechnology Co., Ltd.

[0207] Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MBP) of all awake animals were measured using an intelligent non-invasive sphygmomanometer BP-98E, and the Provantis / v10.2.3.1 electronic data acquisition system (PV-02) was used.

[0208] Experimental procedures: 32 cynomolgus monkeys (5 animals / sex / group in Group 1 and Group 4, 3 animals / sex / group in Group 2 and Group 3, a total of 4 groups) were randomly divided into groups and administered by nasogastric administration of the compound of formula (I) (40, 160 and 600 mg / kg / day) or the control formulation (98.9% vehicle formulation + 1.1% MTBE, 0 mg / kg / day) twice a day for a total of 14 days, followed by a 14-day recovery period. All animals were included in this study to evaluate the effects of drug administration on ECG parameters (including heart rate, PR interval, QRS duration, QT interval and QTcF) and blood pressure during the pre-dose period, drug administration period and recovery period.

[0209] Test results: Under the conditions of this test, crab-eating monkeys were given the compound of formula (I) (40, 160 and 600 mg / kg / day) by nasogastric gavage twice a day for 14 days. No test article-related changes in the cardiovascular system were observed; no test article-related arrhythmias were observed; and no test article-related changes in ECG parameters or blood pressure were observed throughout the entire test.

[0210] Test Example 10: Detection of Related Substances in Coated Tablets

[0211] The related substances of the formulation F3 in Example 2 were determined.

[0212] The determination method of relevant substances is in accordance with the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Chapter 0512), as follows:

[0213] Test solution: Take 4 tablets of this product and place them in a 500ml volumetric flask. Add an appropriate amount of water and shake ultrasonically to disintegrate them. Then add the same volume of acetonitrile and sonicate for 15 minutes. Then dilute to the scale with solvent (water:acetonitrile = 1:1 v / v), shake well, centrifuge, accurately measure an appropriate amount of supernatant, and dilute with solvent to make a solution containing approximately 1 mg of the compound of formula (I) per 1ml.

[0214] Control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with solvent (water: acetonitrile = 1:1 v / v) to make a solution containing approximately 10 μg per 1 ml.

[0215] Chromatographic conditions: An octadecylsilane bonded silica gel column (Waters Atlantis T3, 4.6 mm × 150 mm, 3 μm) was used with 0.01 mol / L ammonium perchlorate buffer (1.17 g of ammonium perchlorate was dissolved in 1 L of water, the pH was adjusted to 2.5 with perchloric acid, and the mixture was mixed) as mobile phase A and acetonitrile as mobile phase B. Linear gradient elution was performed as shown in 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.

[0216] Determination method: Accurately measure the test solution and control solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0217] Table 7 Determination results of related substances in coated tablets

[0218] The results of related substance testing of the coated tablets showed that the coated tablets obtained in the present disclosure were of good quality, with low levels of single and total impurities. In addition, the maximum single and total impurities of the coated tablets in the present disclosure were not increased compared with the API.

[0219] Test Example 11: Dissolution Determination of Coated Tablets

[0220] It was determined according to the dissolution and release test method (Method 2, Part 4, General Chapter 0931, Chinese Pharmacopoeia 2020 Edition).

[0221] Use 900 ml of 0.1 mol / L hydrochloric acid solution containing 0.5% sodium lauryl sulfate as the dissolution medium, the rotation speed is 75 revolutions per minute, and the operation is carried out according to the method. The sampling time is 10, 15, 20, 30, and 45 minutes. After the 45-minute sampling is completed, adjust the rotation speed to 200 revolutions per minute and sample for 60 minutes.

[0222] Use 900 ml of pH 6.8 phosphate buffer as the dissolution medium, the rotation speed is 75 revolutions per minute, operate according to the method, and the sampling time is 5, 10, 15, 20, 30, 45, 60, 75, and 90 minutes. After the 90-minute sampling, adjust the rotation speed to 200 revolutions per minute and sample for 120 minutes.

[0223] Use 900 ml of pH 4.5 acetate buffer as the dissolution medium, the rotation speed is 75 revolutions per minute, operate according to the method, and the sampling time is 5, 10, 15, 20, 30, 45, 60, 75, and 90 minutes. After the 90-minute sampling, adjust the rotation speed to 200 revolutions per minute and sample for 120 minutes.

[0224] The dissolution test results are shown in Table 8 and Figure 4.

[0225] Table 8 Dissolution test results of coated tablets

[0226] The dissolution test results in Table 8 and FIG4 show that the coated tablets of the present disclosure have good dissolution characteristics in 0.1N 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, 2. The oral pharmaceutical composition according to claim 1, wherein the oral pharmaceutical composition is in the form of tablets, capsules, pills, granules, powders, emulsions, solutions or suspensions.

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

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

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

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

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

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

10. The oral pharmaceutical composition according to claim 9, wherein the lubricant is selected from one or more of 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 selected from one or more of 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, wherein the oral pharmaceutical composition comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof, a disintegrant, a filler, a glidant and a lubricant.

12. The oral pharmaceutical composition of claim 11, wherein the disintegrant is croscarmellose sodium, the filler is 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 hypromellose, red iron oxide, polyethylene glycol and titanium dioxide.

14. A combination product comprising: (1) an oral pharmaceutical composition according to any one of claims 1 to 13, and (2) another antiviral drug or another anti-coronavirus and / or anti-piconavirus drug or ritonavir.

15. Use of the oral pharmaceutical composition according to any one of claims 1 to 13 or the combination product according to claim 14 in the preparation of a medicament for preventing or treating related diseases caused by coronavirus and / or picornavirus infection.

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