New pharmaceutical application of N4-hydroxycytidine and derivatives thereof

Abstract

The invention discloses application of N4-hydroxycytidine and a derivative thereof in preparation of a medicine for treating cat abdominal infection. The test result shows that the N4-hydroxycytidine and the derivative thereof have a very small toxic effect on normal cat cells, have an obvious inhibiting effect on cat coronavirus (FCoV), and can obviously reduce the virus titer of the virus in the cells. Therefore, the N4-hydroxycytidine and the derivative thereof can be used for preparing a feline coronavirus inhibitor for treating feline infectious peritonitis, and are good in curative effect and high in safety.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to new medicinal uses of N4-hydroxycytidine and its derivatives. Background Art

[0002] Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV), which mainly affects young cats, cats with weakened immune systems, and cats living in cat colonies. FIP occurs due to mutations in the feline coronavirus, which causes the immune system to overreact, ultimately causing systemic inflammation and organ damage. The clinical symptoms of FIP vary depending on the type, and are mainly divided into two types: wet and dry: wet (exudative): This type of FIP usually manifests as fluid accumulation in the abdominal or thoracic cavity, resulting in ascites or pleural effusion. Cats usually show rapid weight loss, loss of appetite, rapid breathing, or abdominal distension. Dry (non-exudative): The clinical manifestations of dry FIP are more diverse, and there are usually no obvious symptoms of effusion, but inflammation and damage to different organs due to immune response. Commonly affected organs include the eyes and nervous system, and symptoms may include loss of vision, neurological symptoms (such as seizures, unsteady gait), etc.

[0003] FIP was once considered an incurable and fatal disease, but in recent years, the treatment outlook for FIP has improved with the advent of antiviral drugs. GS441524 (similar to remdesivir) is an effective antiviral drug that has been shown to significantly improve survival in some cats with FIP. Other treatment options include immunosuppressive drugs and supportive care (such as fluid and nutritional support).

[0004] Although the scientific community has made some progress in treating FIP, it is still a serious health problem in cats. Therefore, the development of new antiviral drugs for the treatment and prevention of feline coronavirus infection, the treatment and prevention of feline infectious peritonitis, and the improvement of cat health are technical issues that technical personnel need to solve urgently. Summary of the invention

[0005] The purpose of the present invention is to provide the use of N4-hydroxycytidine and its derivatives in the preparation of drugs for treating feline infectious peritonitis in view of the deficiencies in the prior art.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] Provided is the use of a compound as shown in formula (I) or a derivative thereof in the preparation of a drug for treating feline infectious peritonitis, wherein the derivative is a pharmaceutically acceptable salt, prodrug, solvate or hydrate thereof;

[0008]

[0009] Among them, R 1 , R 2 , R 3 are each independently a hydrogen atom, a deuterium atom, or a fluorine atom;

[0010] R4 is a hydrogen atom, or

[0011]

[0012] wherein X is independently a hydrogen atom, a deuterium atom, or a fluorine atom.

[0013] Furthermore, the compound represented by formula (I) includes the following compounds:

[0014]

[0015]

[0016] Furthermore, the drug for treating FIP inhibits the activity of feline coronavirus (FCoV).

[0017] The drug for treating feline infectious peritonitis of the present invention can be topically administered in any acceptable dosage form.

[0018] Formulations include the following: inserts, pump systems, foams, gels, creams, lotions, solutions, suspensions, emulsions, ointments, powders, crystalline forms, liposomes, sprays, pastes, salves, sustained-release nanoparticles, dimethyl sulfoxide (DMSO)-based solution suspensions, sustained-release microparticles.

[0019] The drug for treating feline infectious peritonitis of the present invention can be optionally administered orally, intravenously or intraarterially, intrafatally or liposome-administered, spray inhaled, parenterally or intraperitoneally, rectally, subcutaneously, intrafatally or intraarticularly, nasally, via local delivery (e.g., catheter or stent), buccally, vaginally, or by implantable drug box. The preferred mode of administration is oral administration, intraperitoneal administration, or intravenous injection.

[0020] The drug for treating feline infectious peritonitis of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0021] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0022] The test results of the present invention show that N4-hydroxycytidine and its derivatives have little toxic effect on normal cat cells, but have obvious inhibitory effect on feline coronavirus (FCoV), and can significantly reduce the toxicity of the virus in cells.

[0023] Therefore, N4-hydroxycytidine and its derivatives can be used to prepare feline coronavirus inhibitors for treating feline infectious peritonitis with good efficacy and high safety. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0025] In the following examples, unless otherwise indicated, temperatures are expressed in degrees Celsius (°C); operations are carried out at room or ambient temperature, "rt" or "RT" (usually in the range of 18-25°C); evaporation of solvents is carried out using a rotary evaporator under reduced pressure (usually 4.5-30 mmHg) with a bath temperature of up to 60°C; the progress of reactions is usually followed by thin layer chromatography (TLC); melting points are uncorrected; products show satisfactory 1H-NMR and / or microanalytical data; the following conventional abbreviations are used: L (liter), mL (milliliter), mmol (millimole), g (gram), mg (milligram), min (minute) and h (hour).

[0026] Unless otherwise specified, all solvents and reagents were purchased from commercial suppliers and used without further purification. Unless otherwise specified, reactions were performed under nitrogen atmosphere. Compounds were observed under UV light (254 nm). Spectra were recorded on a 300 MHz NMR spectrometer. 1 HNMR and 13 CNMR spectrum.

[0027] Example 1 Preparation of Compound H-1

[0028]

[0029] 1) Preparation of intermediate A-2

[0030] Under argon protection, at room temperature, a 200 mL round-bottom flask was charged with 1-[(2R, 3R, 4S, 5R)-3,4-dihydroxy-5-(hydroxymethyl)oxacyclopentane-2-yl]-6-fluoro-1,2,3,4-tetrahydropyrimidine-2,4-dione (A-1, 3.0 g, 11.44 mmol) and dichloromethane (30 mL). The flask was cooled with an ice bath, and 4-DMAP (0.150 g, 1.2 mmol) and imidazole (3.3 g, 49 mmol) were slowly added. TBSCl (6.5 g, 53 mmol) was then added, the mixture was warmed to ambient temperature, and stirred overnight. The reaction was quenched with 50 mL of water and separated. The organic layer was washed with water (50 ml × 2), bicarbonate solution (50 ml × 2), and brine (100 mL), and the organic phase was washed with anhydrous Na 2 SO4 Dry, filter and concentrate. The crude product is purified by flash column chromatography to obtain intermediate A-2.

[0031] 2) Preparation of intermediate A-3

[0032] Under argon protection, at room temperature, a 200 mL round-bottom flask was charged with intermediate A-2 (1 g, 1.65 mmol) and acetonitrile (20 mL). The flask was cooled with an ice bath, and then p-toluenesulfonyl chloride (0.61 g, 3.2 mmol), 4-DMAP (0.202 g, 1.65 mmol) and triethylamine (0.90 mL, 6.39 mmol) were slowly added in sequence. The mixture was stirred for 3 h under an ice bath, and hydroxylamine hydrochloride (1.15 g, 16.5 mmol) was added. The mixture was heated at 50 ° C until the reaction was complete and cooled to room temperature. The reaction was quenched with 50 mL of water. EtOAc (100 mL) was added to dilute and separated. The organic layer was washed with water (50 ml × 2), bicarbonate solution (50 ml × 2), and brine (100 mL), and the organic phase was washed with anhydrous Na 2 SO 4 Dry, filter and concentrate. The crude product is purified by flash column chromatography to obtain intermediate A-3.

[0033] 3) Preparation of compound H-1

[0034] Under argon protection, at room temperature, intermediate A-3 (0.31 g, 0.5 mmol), tetramethylammonium fluoride (0.196 g, 2.64 mmol) and DMF (5 mL) were loaded into a 100 mL round-bottom flask. The flask was cooled using an ice bath, and acetic acid (0.157 mL, 2.75 mmol) was then slowly added. The mixture was heated at 50 ° C until the reaction was complete and cooled to room temperature. Rotary evaporation was concentrated. The crude product was purified by rapid column chromatography to obtain compound H-1 as a white solid.

[0035] LCMS (ESI+): m / z 278.07 (M+H)

[0036] 1 H NMR (400 MHz, D 2 O): δ5.87(d,J=5.5Hz,1H),5.71(d,J=8.4,1H),4.27(t,J=5.5Hz,1H),4.20(t,J=4.8Hz,1H),4.12(m,1H),3.85(m,1H),3.77(m,1H)

[0037] Example 2 Preparation of Compound H-2

[0038]

[0039] 1) Synthesis of intermediate B-2

[0040] Under nitrogen protection, at room temperature, a 200mL three-necked round-bottom flask was charged with 1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxacyclopentane-2-yl]-6-fluoro-1,2,3,4-tetrahydropyrimidine-2,4-dione (3.0 g, 11.44 mmol) and acetone (500 mL). The resulting mixture was stirred in an ice bath, and sulfuric acid (0.1 mL) was slowly added. Stirring was continued at room temperature for 16 hours. The reaction was quenched with 5 mL of trimethylamine. EtOAc (30 mL) was added. Wash with water (50 ml × 2), sodium bicarbonate solution (50 ml × 2), and brine (100 mL), and the organic phase was washed with anhydrous Na 2 SO 4 Dry, filter and concentrate to obtain crude B-2 as a white solid.

[0041] 2) Synthesis of intermediate B-3

[0042] Under nitrogen protection, at room temperature, a 200 mL three-necked round-bottom flask was charged with B-2. EtOAc (30 mL), triethylamine (7.1 mL, 51 mmol) and 4-dimethylaminopyridine (0.06 g, 0.5 mmol) were then added. An ice bath was used to cool the flask, and then acetic anhydride (1.25 g, 12.22 mmol) was slowly added. The reaction mixture was stirred at room temperature until the reaction was complete. The reaction was quenched with 5 mL of water and washed with saturated sodium bicarbonate aqueous solution (30 mL × 2), water (30 mL × 2) and brine (30 mL × 2). The organic phase was washed with anhydrous Na 2 SO 4 Drying and concentration under reduced pressure gave a clear colorless oil. The crude product was used in the next step without purification.

[0043] 3) Synthesis of intermediate B-4

[0044] Under argon protection, at room temperature, a 200mL round-bottom flask was charged with the crude product B-3 from the previous step and anhydrous acetonitrile (30mL), and stirred until all the starting materials were dissolved. Then, 1,2,4-triazole (5.0g, 73mmol) and triethylamine (11mL, 81mmol) were added, stirred at room temperature for 30 minutes, and then the reaction solution was cooled to 0°C using an ice bath, POCl3 (2.4mL, 15mmol) was added dropwise, and stirring was continued for 2 hours, and slowly heated to room temperature. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed, 30mL of water was added to quench the reaction and extracted with DCM. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography to obtain intermediate B-4.

[0045] 4) Synthesis of intermediate B-5

[0046] The product B-4 (2 g) and acetonitrile (20 mL) of the previous step are loaded into a 200 mL round-bottom flask. The reaction mixture is stirred at room temperature until all solids are dissolved, hydroxylamine (0.27 mL, 8.8 mmol) is added, and stirred at room temperature. After the reaction is complete, the solvent is removed under reduced pressure, and the obtained residue is dissolved in ethyl acetate, and washed with water and salt water. Organic matter is dried over sodium sulfate, filtered, and concentrated under reduced pressure, and the crude product is purified by rapid column chromatography to obtain white solid product B-5.

[0047] 5) Synthesis of Compound H-2

[0048] The product B-5 (1.5 g) from the previous step, 15 mL of methanol and 15 mL of formic acid were placed in a 100 mL round-bottom flask. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure at 42 °C to obtain a clear light pink oil. Compound H-2 was obtained by column chromatography.

[0049] LCMS (ESI+): m / z 320.08 (M+H)

[0050] 1 H-NMR (400MHz, CD 3 OD)δ / ppm: δ5.81(d,J=5.0Hz,1H),5.69(1H,d,J=8.4Hz),4.39(m,2H),4.27-4.13(m,3H),2.12(s,3H)

[0051] Example 3 Preparation of Compound H-3

[0052]

[0053] The target compound H-3 was prepared according to the method described in Example 2, except that the reagent acetic anhydride in Example 2 was replaced by the reagent isobutyric anhydride, and the purity of the product was >97%.

[0054] LCMS (ESI+): m / z 348.11 (M+H)

[0055] 1 H-NMR (400MHz, CD 3 OD)δ / ppm: δ5.81(d,J=5.0Hz,1H),5.69(1H,d,J=8.4Hz),4.39(m,2H),4.27-4.13(m,3H),2.74-2.69(m,1H),1.25-1.19(m,6H)

[0056] Example 4 Preparation of Compound H-6 and Compound H-7

[0057]

[0058] 1) Preparation of intermediate D-1

[0059] Under argon protection, at room temperature, a 1L round-bottom flask was charged with intermediate B-2 (10 g, 33 mmol), IBX (28 g, 100 mmol) and acetonitrile (300 ml) (1 equivalent). The resulting suspension was refluxed for 1.5 hours until the reaction was complete (TLC). The suspension was cooled to room temperature and The filter cake was washed with EtOAc and the filtrate was then concentrated in vacuo to afford crude aldehyde D-1, which was used directly in the next step without further purification.

[0060] 2) Preparation of intermediate D-2

[0061] Under argon protection, at room temperature, a 1L round-bottom flask was charged with the crude aldehyde D-1 from the previous step, and deuterated ethanol (300 mL) was added. After stirring at room temperature for 30 minutes, the reaction solution was cooled to 0°C using an ice bath, and sodium borodeuteride (1.4 g, 34 mol) was slowly added. After the addition, the temperature was raised to room temperature and reacted for 1 hour. After the reaction was completed, water (50 ml) was added to quench, and the solvent was concentrated. EtOAc (300 mL) was added, and the mixture was washed with water (50 ml × 2), bicarbonate solution (50 ml × 2), and brine (100 mL). The organic phase was washed with anhydrous Na 2 SO 4 Drying, filtration and concentration gave intermediate D-2.

[0062] 3) Synthesis of intermediate D-3

[0063] Under nitrogen protection, at room temperature, a 200 mL three-necked round-bottom flask was charged with D-2 (3 g, 9.9 mmol), followed by the addition of EtOAc (30 mL), triethylamine (7 mL) and 4-dimethylaminopyridine (0.06 g, 0.5 mmol). The flask was cooled with an ice bath, and then acetic anhydride (1.1 g, 11 mmol) was slowly added. The reaction mixture was stirred at room temperature until the reaction was complete. The reaction was quenched with 5 mL of water and washed with saturated sodium bicarbonate aqueous solution (30 mL × 2), water (30 mL × 2) and brine (30 mL × 2). The organic phase was washed with anhydrous Na 2 SO 4 Drying and concentration under reduced pressure gave a clear colorless oil. The crude product was used in the next step without purification.

[0064] 4) Synthesis of intermediate D-4

[0065] Under argon protection, at room temperature, a 200 mL round-bottom flask was charged with the crude product D-3 from the previous step and anhydrous acetonitrile (30 mL), and stirred until all the starting materials were dissolved. Then, 1,2,4-triazole (4.5 g, 66.4 mmol) and triethylamine (10 mL, 73 mmol) were added, and after stirring at room temperature for 30 minutes, the reaction solution was cooled to 0 °C using an ice bath, and POCl was added dropwise. 3 D-4-(2-[(2-nitro-1-yl)-2-nitropropene ...

[0066] 5) Synthesis of intermediate D-5

[0067] The product D-4 (2 g) and acetonitrile (20 mL) of the previous step are loaded into a 200 mL round-bottom flask, and the reaction mixture is stirred at room temperature until all solids are dissolved. Hydroxylamine (0.27 mL, 8.8 mmol) is added and stirred at room temperature. After the reaction is completed, the solvent is removed under reduced pressure, and the obtained residue is dissolved in ethyl acetate, and washed with water and salt water. Organic matter is dried over sodium sulfate, filtered, and concentrated under reduced pressure, and the crude product is purified by rapid column chromatography to obtain a white solid product D-5.

[0068] 6) Synthesis of Compound H-7

[0069] The product of the previous step (1.5 g), 15 mL of methanol and 15 mL of formic acid were placed in a 100 mL round-bottom flask. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure at 42 °C to obtain a clear light pink oil. Compound H-7 was obtained by column chromatography.

[0070] LCMS (ESI+): m / z 321.09 [M+H+]

[0071] 1 H-NMR (400MHz, CD 3 OD)δ:ppmδ5.82(d,J=4.8Hz,1H),5.62(1H,d,J=8.4Hz),4.40(m,1H),4.27-4.13(m,3H),2.12(s,3H)

[0072] 7) Synthesis of Compound H-6

[0073] Under argon protection, compound H-7 (500 mg) and saturated ammonia / methanol solution (100 mL) were added to a 250 mL round-bottom flask at room temperature and stirred for reaction. After the reaction was completed, the reaction mixture was concentrated under reduced pressure at 42 °C and purified by column chromatography to obtain compound H-6.

[0074] LCMS (ESI+): m / z 279.09 [M+H+]

[0075] Example 5 Preparation of Compound H-16

[0076]

[0077] 1) Synthesis of compound E-2

[0078] Under argon protection, at room temperature, a 200 mL round-bottom flask was charged with compound E-1 (1.0 g, 3.35 mmol), dichloromethane (20 mL), triethylamine (0.60 mL, 5 mmol) and 4-DMAP (3.4 mg, 0.028 mmol) in turn, and the mixture was stirred at room temperature for 30 minutes. The reaction solution was then cooled to 0 ° C using an ice bath, and a solution of 4,4'-dimethoxytrityl chloride (1.05 g, 3.08 mmol) in dichloromethane (16 mL) was added dropwise, and stirring was continued for 2 hours, and slowly heated to room temperature. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed, 30 mL of water was added to quench the reaction. The organic layer was washed with brine (1×30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography to obtain intermediate E-2.

[0079] 2) Synthesis of compound E-4

[0080] Under argon protection, at room temperature, a 200 mL round-bottom flask was charged with compounds, intermediate E-2 (1 g, 1.66 mmol), anhydrous magnesium chloride (1.7 g, 18.1 mmol), compound E-3 (0.7 g, 5.27 mmol) and 30 ml acetonitrile, and N, N-diisopropylethylamine (1.4 mL, 8.3 mmol) was added dropwise. The temperature was raised to 50 degrees Celsius for 2 hours. The reaction was complete when detected by TLC. The mixture was cooled to 0°C, quenched with saturated ammonium chloride, and concentrated under reduced pressure to remove acetonitrile. Water and ethyl acetate were added, the layers were separated, the aqueous phase was extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain intermediate E-4.

[0081] 3) Synthesis of compound H-16

[0082] Under argon protection, at room temperature, a 200 mL round-bottom flask was charged with compound intermediate E-4 (0.5 g), 10 ml methanol and 10 ml 80% aqueous formic acid. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed, 30 mL water and 30 mL dichloromethane were added to quench the reaction. The pH was adjusted to neutral with sodium bicarbonate solution. The organic layer was washed with brine (1 × 30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography to obtain compound H-16.

[0083] LCMS (ESI+): m / z 571.24 (M+H)

[0084] 1 HNMR (400MHz, CD3OD) δ7.69-7.13(m,6H), 6.02-5.70(m,2H), 4.90-4.08(m,7H), 3.90(m,1H), 1.44-1.26(m,8H), 1.20-0.86(m,6H)

[0085] Example 6 Preparation of Compound H-4

[0086]

[0087] The target compound H-4 was prepared by the method described in Example 5, except that the intermediate D-2 was used to replace the compound E-1 in Example 5. The purity of the product was >97%; LCMS (ESI+): m / z 589.23 (M+H).

[0088] Example 7 Preparation of Compound H-5

[0089]

[0090] The preparation was basically carried out according to the method described in Example 2, except that (2S)-2-(tert-butoxycarbonylamino)-3-propionic acid was used to replace the reagent acetic anhydride in Example 2, thereby preparing the target compound H-5. The purity of the product was detected to be >97%; LCMS (ESI+): m / z 349.13 (M+H).

[0091] Example 8 Preparation of Compound H-8

[0092]

[0093] The target compound H-8 was prepared by the method described in Example 4, except that the reagent acetic anhydride in Example 4 was replaced by the reagent isobutyric anhydride. The purity of the product was >97%; LCMS (ESI+): m / z 349.11 (M+H).

[0094] Example 9 Preparation of Compound H-9

[0095]

[0096] The target compound H-9 was prepared by the method described in Example 5, except that the compound E-1 in Example 5 was replaced by the compound H-6. The purity of the product was >97%; LCMS (ESI+): m / z 590.23 (M+H).

[0097] Example 10 Preparation of Compound H-10

[0098]

[0099] The preparation was basically carried out according to the method described in Example 5, except that the reagent acetic anhydride in Example 5 was replaced by (2S)-2-(tert-butoxycarbonylamino)-3-propionic acid, and the compound E-1 in Example 5 was replaced by compound H-1, thereby preparing the target compound H-10. The purity of the product was >97%; LCMS (ESI+): m / z 350.13 (M+H).

[0100] Example 11 Preparation of Compound H-12

[0101]

[0102] The target compound H-12 was prepared by the method described in Example 4, except that 5′-deoxy-2′,3′-O-(1-methylethylidene)-5′-oxouridine was used to replace the reagent B-2 in Example 4. The purity of the product was >97%; LCMS (ESI+): m / z 303.11 (M+H);

[0103] 1 H-NMR (400MHz, CD 3 OD)δ:ppmδ6.91(1H,d,J=8.4Hz),5.82(d,J=4.8Hz,1H),5.62(1H,d,J=8.4Hz),4.38(m,1H),4.27-4.13(m,3H),2.12(s,3H)

[0104] Example 12 Preparation of Compound H-11

[0105]

[0106] Prepared according to the method described in Example 4, except that the target compound H-12 was used to obtain the target compound H-11. The purity of the product was >97%; LCMS (ESI+): m / z 261.11 (M+H);

[0107] NMR (400MHz, CD 3 OD), δ7.16(m,1H),5.87(d,J=5.0Hz,1H),5.59(d,J=8.4

[0108] Hz,1H),4.17-4.11(m,2H),3.94(d,J=3.8Hz,1H);3.77-3.65(m,1H)

[0109] Example 13 Preparation of Compound H-13

[0110]

[0111] The target compound H-13 was prepared by the method described in Example 4, except that 5′-deoxy-2′,3′-O-(1-methylethylidene)-5′-oxouridine was used to replace the reagent B-2 in Example 4, and fluoroacetic anhydride was used to replace the reagent acetic anhydride in Example 4. The purity of the product was >97%; LCMS (ESI+): m / z 321.11 (M+H).

[0112] Example 14 Preparation of Compound H-14

[0113]

[0114] The target compound H-14 was prepared by the method described in Example 5, except that the compound E-1 in Example 5 was replaced by the compound H-11. The purity of the product was >97%; LCMS (ESI+): m / z 572.22 (M+H).

[0115] Example 15 Preparation of Compound H-15

[0116]

[0117] The preparation was basically carried out according to the method described in Example 4, except that (2S)-2-(tert-butoxycarbonylamino)-3-propionic acid was used to replace the reagent acetic anhydride in Example 4, thereby obtaining the target compound H-15. The purity of the product was detected to be >97%; LCMS (ESI+): m / z 332.13 (M+H).

[0118] Example 16 Preparation of Compound H-18

[0119]

[0120] The preparation was basically carried out according to the method described in Example 5, except that 5-fluoro-2′,3′-O-(1-methylethylidene)uridine-4-oxime was used to replace the compound E-1 in Example 5, thereby obtaining the target compound H-18. The purity of the product was detected to be >97%; LCMS (ESI+): m / z 589.20 (M+H).

[0121] Example 17 Preparation of Compound H-17

[0122]

[0123] The target compound H-8 was prepared by the method described in Example 4, except that acetic anhydride was replaced by 2-deuterioisobutyric anhydride, and reagent B-2 was replaced by 5′-deoxy-2′,3′-O-(1-methylethylidene)-5′-oxouridine. The purity of the product was >97%; LCMS (ESI+): m / z 332.13 (M+H).

[0124] Example 18 Preparation of Compound H-19

[0125]

[0126] The preparation was basically carried out according to the method described in Example 5, except that 2-deuterium isobutyric anhydride was used to replace the reagent E-3 in Example 5, thereby obtaining the target compound H-19. The purity of the product was detected to be >97%; LCMS (ESI+): m / z 331.13 (M+H).

[0127] 1H-NMR (300MHz, MeOH-d4) δ6.91(d,J=8.3Hz,1H),5.82(d,J=4.8Hz,1H),5.61(d,J=8.2Hz,1H),4.29(d,J=3.6Hz,2H),4.15-4.07(m,3H),1.18(s,6H);

[0128] Verification Example 1EC 50 In vitro experiments

[0129] 1. Experimental Materials

[0130] 1) Cell line: Crandell-Reese cat kidney (CRFK) cells.

[0131] 2) Virus: Laboratory-adapted strain of feline infectious peritonitis virus (FIPV).

[0132] 3) Test compound: the compound prepared in Examples 1-18.

[0133] 4) Culture medium: Dulbecco's modified medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics.

[0134] 5) Cell viability assay reagent: CellTiter-Glo kit.

[0135] 6) PCR reagents: used to quantify viral RNA by qRT-PCR.

[0136] 7) 96-well cell culture plate.

[0137] 2. Operation steps

[0138] 1) Cell preparation:

[0139] CRFK cells were seeded into 96-well plates at 10,000 cells per well.

[0140] At 37°C, 5% CO 2 Incubate in an incubator overnight to allow attachment.

[0141] 2) Compound dilution:

[0142] The test compounds were serially diluted in DMSO or culture medium to prepare a final concentration gradient ranging from 0.1 nM to 100 μM.

[0143] 3) Viral infection:

[0144] Cells were infected with FIPV using a multiplicity of infection (MOI) of 0.01.

[0145] Incubate at 37°C for 1 hour to promote virus adsorption; then wash with culture medium to remove unadsorbed viruses.

[0146] 4) Compound treatment:

[0147] The culture medium was replaced with fresh culture medium containing the predetermined concentration of the test compound.

[0148] A negative control group (infected with virus but without inhibitor) and a sham-infected control group (not infected with virus) were set up.

[0149] 5) Incubation:

[0150] Incubate for 48 to 72 hours, depending on the replication kinetics of FIPV.

[0151] 6) End point determination:

[0152] a. Quantification of viral replication:

[0153] The cell supernatant was collected and viral RNA was extracted.

[0154] Viral RNA levels were measured using qRT-PCR.

[0155] b. Cell viability detection:

[0156] MTT or CellTiter-Glo reagent was added directly into the wells.

[0157] The absorbance or luminescence signal was measured to evaluate cell viability.

[0158] 7) Data analysis:

[0159] Plot the relationship between inhibitor concentration and viral replication level.

[0160] Dose-response curves were fitted using GraphPad Prism software.

[0161] Calculation of EC 50 The values ​​(concentration of compound required to inhibit 50% viral replication) are shown in Table 1 below.

[0162] Table 1

[0163]

[0164]

[0165] It can be seen that N4-hydroxycytidine and its derivatives can inhibit infectious peritonitis virus in cats.

[0166] Verification Example 2 Cell toxicity test

[0167] 1. Cell Preparation

[0168] Take CCL-94 (cat kidney cells) with good growth status and digest and subculture them. Use cell growth medium to adjust the cell density to 1×10 5 / mL, inoculated into 96-well plates, 100 μL per well, and placed at 37°C, 5% CO 2 Incubate for 16 hours in an incubator. After inoculation, observe the cells evenly attached using an inverted microscope to ensure that the cells are in good condition.

[0169] 2. Cleaning and culture medium replacement

[0170] After 16 hours, the culture medium was discarded, and the cells were slowly washed three times with sterile PBS (1% FBS), and the plate was finally dried to remove the residual liquid. 100 μL of cell maintenance solution was added to the first column of each group, and 50 μL of cell maintenance solution was added to the second to sixth columns to provide a basic liquid environment for subsequent drug dilution.

[0171] 3. Drug treatment

[0172] The target compound is added to the first column of each group, and DMSO is used as the solvent. A concentration gradient (200 μM to 6.25 μM) is generated by the 2-fold dilution method, and accuracy is ensured by step-by-step dilution. A cell control without drug addition is retained, and a solvent control (such as an equal amount of DMSO) is set up to correct for the potential effects of the solvent on the cells.

[0173] 4. Duration of drug action

[0174] After drug treatment, the 96-well plate was returned to the incubator and incubated at 37°C and 5% CO 2 The cells were incubated under the same conditions and the data were recorded for 72 h and 96 h, respectively, to analyze the effect of time on cytotoxicity.

[0175] 5. Cell Viability Detection

[0176] The cell viability was determined by a reagent (luminescence kit), and the cell survival rate was calculated according to the following formula:

[0177] Cell survival rate (%)

[0178] = Average luminescence value of drug-treated cells / average luminescence value of cell control group

[0179] Record the luminescence value of each concentration group, calculate the cell survival rate, draw the concentration-effect curve and determine the half inhibitory concentration (CC 50 )

[0180] Table 2

[0181] Compound <![CDATA[CC 50 (μM)]]> Compound <![CDATA[CC 50 (μM)]]> H-1 >100 H-10 >100 H-2 >100 H-11 >100 H-3 >100 H-12 >100 H-4 >100 H-13 >100 H-5 >100 H-14 >100 H-6 >100 H-15 >100 H-7 >100 H-16 >100 H-8 >100 H-18 >100 H-9 >100 H-19 >100

[0182] The results showed that the toxicity to CCL-94 cells was small. 50 Greater than 100μM.

[0183] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a compound as represented by formula (I) or a derivative thereof in the preparation of a drug for treating feline infectious peritonitis, characterized in that: The derivative is a pharmaceutically acceptable salt, prodrug, solvate or hydrate thereof; wherein R1, R2, and R3 are each independently a hydrogen atom, a deuterium atom, or a fluorine atom; R4 is a hydrogen atom, or wherein X is independently a hydrogen atom, a deuterium atom, or a fluorine atom.

2. The use according to claim 1, characterized in that: The compound represented by formula (I) includes the following compounds:

3. The use according to claim 1, characterized in that: The drug for treating feline infectious peritonitis inhibits the activity of feline coronavirus (FCoV).

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