Nucleoside prodrug and application thereof in treating feline infectious peritonitis

Abstract

The invention discloses a nucleoside prodrug and application thereof in treating feline infectious peritonitis, and belongs to the field of medicinal chemistry. The invention provides a compound as shown in the specification or pharmaceutically acceptable salt thereof, the compound provided by the invention can effectively inhibit feline infectious peritonitis virus (FIPV), the antiviral activity of the compound obviously exceeds that of GS-441524, and meanwhile, the compound has good pharmacokinetic properties, high bioavailability and good solubility, and can be applied to preparation of drugs for treating feline infectious peritonitis virus (FIPV) or other drugs for treating feline infectious peritonitis virus (FIPV) or other drugs for treating feline infectious peritonitis virus (FIPV). The preparation is simple, the synthesis is easy, and large-scale industrial production can be realized.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to a nucleoside prodrug and its application in the treatment of feline infectious peritonitis. Background Technology

[0002] Feline infectious peritonitis (FIP) is a highly fatal disease caused by the feline infectious peritonitis virus (FIPV). Clinically, FIP is mainly divided into two types: wet FIP ​​and dry FIP. Wet FIP ​​is characterized by abdominal swelling, while dry FIP is characterized by abscesses. Some infected cats may also experience eye and nervous system damage. Currently, there are no approved drugs for treating FIP. Clinically, treatment mainly involves antiviral, anti-inflammatory, immunosuppressive, and immunomodulatory drugs. However, these treatments cannot significantly reduce the high mortality rate of FIP; they can only slow the progression of the disease.

[0003] GS-441524 is a drug that currently has a certain therapeutic effect on feline infectious peritonitis, exhibiting inhibitory effects against various RNA viruses. GS-441524 acts on the RNA polymerase (RdRp) of RNA viruses, inhibiting the viral RNA transcription and replication process.

[0004] Current reports indicate that GS-441524 itself has poor oral bioavailability and can only be administered via injection. However, the treatment process for feline infectious peritonitis (FIP) can last up to 12 weeks; insufficient treatment time can easily lead to relapse, and prolonged injection can cause non-compliance and pain in infected cats. Therefore, finding an orally bioavailable nucleoside derivative of GS-441524 and improving its bioavailability and therapeutic efficacy has become an urgent problem to be solved. Summary of the Invention

[0005] Purpose of the invention: In view of the low oral bioavailability and inhibitory activity of the existing GS-441524, this invention designs a class of nucleoside compounds with novel structure, high bioavailability, good metabolism and low toxicity, and has excellent inhibitory activity against feline infectious peritonitis virus (FIPV).

[0006] The present invention also provides pharmaceutical uses of the said compound.

[0007] Technical solution: To solve the above-mentioned technical problems, the present invention provides a compound as shown in Formula I or a pharmaceutically acceptable salt thereof:

[0008]

[0009] in:

[0010] n is a positive integer that is 0 or greater than 1;

[0011] R 1 R 2 R 3 Each is independently selected from hydrogen, deuterium, halogen, cyano, nitro, -NR a R b -SR a Or C1-C6 alkyl;

[0012] The structure (2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-nitrile can be attached to any site on adamantane;

[0013] R a Or R b Each is independently selected from hydrogen, halogen, cyano, nitro, or C1-C6 alkyl; when n is 0, R 1 R 2 R 3 At least one of them is not hydrogen.

[0014] Furthermore, the compound or a pharmaceutically acceptable salt thereof is represented by the following formula II:

[0015]

[0016] in:

[0017] n is a positive integer greater than 0 or greater than 1;

[0018] R 1 R 2 R 3 Each is independently selected from hydrogen, deuterium, halogen, cyano, nitro, -NR a R b -SR a Or C1-C6 alkyl;

[0019] R a Or R b Each is independently selected from hydrogen, halogen, cyano, nitro, or C1-C6 alkyl; when n is 0, R 1 R 2 R 3 At least one of them is not hydrogen.

[0020] Preferably, n = 2 or 3; R 1 R 2 R 3 Each is independently selected from hydrogen, halogen, or C1-C6 alkyl.

[0021] Furthermore, n = 2; R 1 R 2 R 3 Each is independently selected from hydrogen, halogen, or C1-C6 alkyl.

[0022] The compound or its pharmaceutically acceptable salt is selected from any one of the following:

[0023]

[0024] The use of the compounds described in this invention or pharmaceutically acceptable salts thereof in the preparation of medicaments for treating feline infectious peritonitis.

[0025] The feline infectious peritonitis is a disease caused by infection with the feline infectious peritonitis virus, which is an infectious peritonitis coronavirus.

[0026] The use of the compound cpd6 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and treatment of feline infectious peritonitis, wherein the structure of the compound cpd6 is shown below:

[0027]

[0028] The pharmaceutical composition for treating feline infectious peritonitis according to the present invention comprises a therapeutically effective amount of the compound or a pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier or diluent.

[0029] The dosage form of the pharmaceutical composition is selected from capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalers, ointments, solutions, creams, gels, powders, lotions, tinctures, suppositories, or patches.

[0030] The application of the compound described in this invention, or a pharmaceutically acceptable salt thereof, or compound cpd6 or a pharmaceutically acceptable salt thereof, in the preparation of inhibitors of feline calicivirus and porcine epidemic diarrhea virus, wherein the structure of compound cpd6 is shown below:

[0031]

[0032] General schemes for the synthesis of compounds:

[0033] The following describes in detail the synthetic method of the compound represented by general formula (I), which is prepared using the following general reaction procedure:

[0034]

[0035] The compound shown in formula (I-1) is reacted with the compound shown in formula (I-2) to obtain the compound shown in formula (I). Existing methods can be used to perform the condensation reaction between the compound shown in formula (I-1) and the compound shown in formula (I-2), and no particular limitation is made herein; all such reactions are understood to be within the scope of protection of this invention.

[0036] Furthermore, before the condensation reaction proceeds, the two ortho-hydroxyl groups on the furan ring are protected with a protecting group. After the reaction with the compound shown in formula (I-2) is completed, the protecting group is removed.

[0037] Furthermore, it includes the following steps:

[0038]

[0039] The compound shown in formula (I-1) was dissolved in acetone, and the compound shown in formula (I-3) and concentrated sulfuric acid were added. The mixture was stirred for 20 to 40 minutes. After the reaction was complete, the reaction solution was poured into a sodium carbonate solution. The aqueous layer was extracted with ethyl acetate. The organic phases were combined and washed with saturated brine. After concentration, a white foamy solid was obtained. After purification by column chromatography, a white solid was obtained, which was the compound shown in formula (I-4).

[0040] The compound shown in formula (I-4) undergoes a condensation reaction with the compound shown in formula (1-2) to prepare the compound shown in formula (I-5):

[0041]

[0042] The compound shown in formula (I-2) was added to dichloromethane along with 1-ethyl-3-(3-dimethylpropylamine)carbodiimide (EDCI), 4-dimethylaminopyridine (DMAP), and the compound shown in formula (I-4). The mixture was stirred at room temperature for 24 hours. After the reactants had reacted completely, a small amount of water was added, and the mixture was extracted with dichloromethane until the aqueous layer was almost free of product. The organic phases were combined and washed with saturated brine. After drying with anhydrous sodium sulfate, the mixture was concentrated and purified by column chromatography (DCM:MeOH = 100:1). The resulting white solid was the compound shown in formula (I-5).

[0043]

[0044] The compound shown in formula (I-5) was dissolved in THF, and HCl was added under ice bath conditions. After the addition was complete, the reaction system was moved to room temperature. After the reactants disappeared completely and the product was formed, the reaction solution was poured into sodium bicarbonate solution, and the aqueous phase was extracted with ethyl acetate until the aqueous layer was almost devoid of product. The organic phases were combined and washed with saturated brine. After drying and concentration, a white solid crude product was obtained. After purification by preparative high-performance liquid chromatography, the compound shown in formula (I) was obtained.

[0045] The compounds of this invention exhibit potent inhibitory activity against feline infectious peritonitis virus (FIPV). Systematic experiments revealed that the design of the functional groups and their linkage in the specific structure of this invention significantly affects the activity, particularly the value of n, which has a significant impact on FIPV inhibition. Based on compound cpd6, the compounds of this invention exhibit excellent FIPV inhibitory activity when n is 0 (compound cpd6) and 2 (compound cpd5), with the effect being even more pronounced when n is 2, reaching levels below 50 nM. However, when n is 1 (compound cpd7), there is no inhibitory activity against FIPV. This demonstrates the importance of the choice of n in this invention, resulting in unexpected technical effects. In particular, compound cpd5, with n of 2, exhibits significantly higher inhibitory activity than existing compounds.

[0046] Furthermore, the design of the adamantane grafting site and substituents on the adamantane in this invention significantly affects the activity. For example, compared to compound cpd6, compound cpd1 shows almost no inhibitory activity against feline infectious peritonitis virus (FIPV), while attaching a bromine group (cpd4) to the adamantane also results in better activity. However, attaching dimethyl and trifluoro groups to the adamantane significantly reduces the activity, further demonstrating the uniqueness of the structure and efficacy of this invention. Therefore, the compounds of this invention or their pharmaceutically acceptable salts can be used to prepare drugs for treating feline infectious peritonitis.

[0047] Furthermore, unlike the existing use of nucleoside compounds in the preparation of products related to respiratory syncytial virus infection, this invention is the first to propose a series of compounds for the application of inhibiting feline infectious peritonitis virus (FIPV) and treating feline infectious peritonitis, with significant effects. At the same time, by expanding its substrate viruses, it was found that it also has inhibitory effects on feline calicivirus and porcine epidemic diarrhea virus.

[0048] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0049] (1) The compounds or pharmaceutically acceptable salts provided by the present invention can effectively inhibit the reproduction and replication of feline infectious peritonitis virus in host cells, and have high antiviral activity and low toxicity.

[0050] (2) The compound of the present invention has good antiviral activity against feline infectious peritonitis virus, and its antiviral activity significantly exceeds that of GS-441524 and is also significantly superior to existing compounds.

[0051] (3) The compound of the present invention has good pharmacokinetic properties and a bioavailability of up to 81% in rats, which is significantly better than GS-441524. It is expected to solve the defect of poor oral bioavailability of GS-441524, and at the same time has excellent solubility.

[0052] (4) The compounds provided by this invention or their pharmaceutically acceptable salts are simple to prepare, easy to synthesize, and can be mass-produced industrially. Attached Figure Description

[0053] Figure 1 Results of GS-441524's resistance to feline infectious peritonitis virus (scale bar 500 μm) determined by indirect immunofluorescence assay.

[0054] Figure 2 Results of the determination of cpd 6 against feline infectious peritonitis virus by indirect immunofluorescence assay (scale bar is 500 μm).

[0055] Figure 3 This is a graph showing the change in body weight of rats during the acute toxicity test of CPD-6.

[0056] Figure 4 The image shows the results of routine blood tests in rats during the acute toxicity test of CPD-6, including WBC (white blood cell count), PLT (platelet count), RBC (red blood cell count), HGB (hemoglobin count), HCT (hematocrit), MCV (mean corpuscular volume), MCHC (mean corpuscular hemoglobin concentration), and MCH (mean corpuscular hemoglobin content).

[0057] Figure 5 The image shows the blood biochemistry results of rats in the acute toxicity test of CDP-6, including the Monocyte Count (MON), Lymphocyte Count (LYC), and Granulocyte Count (GNC).

[0058] Figure 6 The image shows the liver markers detected in rats during the acute toxicity test of CPD-6, including ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), and TBIL (total bilirubin).

[0059] Figure 7 The image shows the results of kidney marker tests in rats during the acute toxicity test of CPD-6, including CREA (creatinine) and UEA (urea).

[0060] Figure 8 The image shows the results of blood glucose (GLU) and creatine kinase (CK) levels in rats during the acute toxicity test of cpd 6.

[0061] Figure 9 The image shows the results of blood lipid levels in rats during the acute toxicity test of CPD-6.

[0062] Figure 10 This is a pathological section of the rat myocardium during an acute toxicity test of cPD6.

[0063] Figure 11 This is a pathological section of the lungs of rats during an acute toxicity test of cPD6.

[0064] Figure 12 This is a pathological section of the kidney of a rat during an acute toxicity test of cPD6.

[0065] Figure 13 This is a pathological section of the spleen from a rat during an acute toxicity test of cPD6.

[0066] Figure 14 This is a pathological section of the liver of a rat during an acute toxicity test of cPD6. Detailed Implementation

[0067] The present invention will be specifically illustrated below through examples. These examples are provided to better illustrate the invention and are not intended to limit its scope. Various changes and modifications can be made to the invention without departing from its spirit and scope.

[0068] Unless otherwise specified, the starting materials used in the embodiments of the present invention are all known products, and the reagents and raw materials used in the present invention are all commercially available.

[0069] Among them, compound GS-441524 is positive control compound 1, CAS: 1191237-69-0.

[0070] Positive drug compound 2 is: See CN119306725A for details.

[0071] Example 1

[0072] Synthesis of cpd6

[0073]

[0074] Compound 1 (GS-441524, CAS: 1191237-69-0) (1 g, 3.4 mmol) and 2,2-dimethoxypropane (0.54 g, 5.2 mmol) were added to acetone (10 ml). 98% concentrated H₂SO₄ (0.44 g, 4.5 mmol) was added dropwise at room temperature. After stirring at room temperature for one hour, the reaction was monitored by thin-layer chromatography (DCM:MeOH = 20:1) until complete. The reaction solution was then poured into 50 ml of saturated sodium bicarbonate solution and extracted with ethyl acetate (20 ml * 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a white crude solid. The crude solid was purified by column chromatography (DCM:MeOH = 30:1) to obtain compound 2 (1.03 g, 3.1 mmol, yield: 91.2%).

[0075] 1 H NMR (300MHz, CDCl3) δ7.93(s,1H),7.08(d,J=4.5Hz,1H),6.66(d,J=4.6Hz,1H),5.96(s,1H),5.42(d,J=6.6Hz,1H),5.23(dd, J=6.6,2.4Hz,1H),4.66(d,J=2.4Hz,1H),4.03–3.93(m,1H),3.84(dd,J=12.5,1.8Hz,1H),1.80(s,3H),1.39(s,3H).MS[M+H] + =332.1375.

[0076]

[0077] Compound 2 (1 g, 3.01 mmol), 1-adamantanecarboxylic acid (0.40 g, 3.61 mmol), 4-dimethylaminopyridine (DMAP) (0.74 g, 6.02 mmol), and 1-ethyl-3-(3-dimethylpropylamine)carbodiimide (EDCI) (1.19 g, 6.02 mmol) were added to 10 mL of dichloromethane (DCM). The mixture was stirred at room temperature for 24 h. After the reaction was complete, it was concentrated under reduced pressure and purified by column chromatography (DCM:MeOH = 100:1) to give compound 3 (1.15 g, 2.3 mmol, yield: 77.8%) as a white solid.

[0078]

[0079] Compound 3 (200 mg, 0.50 mmol) was dissolved in THF (5 ml) solution, and HCl (2N, 1 ml) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 1 hour. After thin-layer chromatography showed that the reaction was complete, the reaction mixture was concentrated under reduced pressure and purified by preparative high-performance liquid chromatography to obtain cpd6 (96 mg, 0.27 mmol, yield: 54%).

[0080] 1 H NMR (300MHz, DMSO-d6) δ7.93(s,3H),6.92(d,J=4.5Hz,1H),6.81(d,J=4.5Hz,1H),6.37(d,J=5.9Hz,1H),5.37(d,J=5.9Hz ,1H),4.69(t,J=5.3Hz,1H),4.32–4.10(m,3H),3.98(q,J=5.9Hz,1H),1.92(s,3H),1.67(dd,J=19.5,4.7Hz,12H).MS[M+H] + =454.2118.

[0081] Example 2

[0082] Synthesis of cpd1

[0083]

[0084] Compound 2 (1 g), adamantane-2-carboxylic acid (0.40 g), 4-dimethylaminopyridine (DMAP) (0.74 g), and 1-ethyl-3-(3-dimethylpropylamine)carbodiimide (EDCI) (1.19 g) were added to 10 mL of dichloromethane (DCM). The mixture was stirred at room temperature for 24 h. After the reaction was complete, it was concentrated under reduced pressure and purified by column chromatography (DCM:MeOH = 20:1) to give compound 4 (1.2 g, yield: 92%) as a white solid.

[0085] Compound 4 (200 mg) was dissolved in THF (5 ml) solution, and HCl (2 N, 1 ml) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 1 hour. After thin-layer chromatography showed that the reaction was complete, the reaction mixture was concentrated under reduced pressure and purified by preparative high-performance liquid chromatography to obtain cpd1 (81 mg, yield: 43%). 1H NMR(300MHz,DMSO-d6)δ7.91(s,3H),6.91(d,J=4.5Hz,1H),6.79(d,J=4.5Hz ,1H),6.33(d,J=6.0Hz,1H),5.37(d,J=5.9Hz,1H),4.69(t,J=5.4Hz,1H),4. 32(d,J=9.7Hz,1H),4.28–4.14(m,2H),3.97(t,J=5.7Hz,1H),2.58(s,1H),2 .14(s,2H),1.81(d,J=11.0Hz,1H),1.77–1.62(m,9H),1.50(s,2H).MS[M+H] + =454.2084.

[0086] Example 3

[0087] Synthesis of cpd2

[0088]

[0089] Compound 2 (1 g), 3,5,7-trifluoroadamantane-1-carboxylic acid (0.40 g), 4-dimethylaminopyridine (DMAP) (0.74 g), and 1-ethyl-3-(3-dimethylpropylamine)carbodiimide (EDCI) (1.19 g) were added to 10 mL of dichloromethane (DCM). The mixture was stirred at room temperature for 24 h. After the reaction was complete, it was concentrated under reduced pressure and purified by column chromatography (DCM:MeOH = 100:1) to give compound 5 (1.06 g, yield: 80%) as a white solid.

[0090] Compound 5 (200 mg) was dissolved in THF (5 ml) solution, and HCl (2 N, 1 ml) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 1 hour. After thin-layer chromatography showed that the reaction was complete, the reaction mixture was concentrated under reduced pressure and purified by preparative high-performance liquid chromatography to obtain cpd2 (79 mg, yield: 40%). 1 H NMR (300MHz, DMSO-d6) δ7.92(s,3H),6.92(d,J=4.5Hz,1H),6.81(d,J=4.5Hz,1H),6.38(d,J=6.0Hz,1H),5.40(d,J=5.8Hz,1H),4.71(t,J= 5.4Hz,1H),4.34(d,J=10.0Hz,1H),4.26(d,J=4.3Hz,2H),3.99(d,J=5.9Hz,1H),2.18(s,3H),2.06(d,J=9.0Hz,3H),1.88(s,6H).MS[M+H]+ =508.1803.

[0091] Example 4

[0092] Synthesis of cpd3

[0093]

[0094] Compound 2 (1 g), 3,5-dimethyladamantane-1-carboxylic acid (0.40 g), 4-dimethylaminopyridine (DMAP) (0.74 g), and 1-ethyl-3-(3-dimethylpropylamine)carbodiimide (EDCI) (1.19 g) were added to 10 mL of dichloromethane (DCM). The mixture was stirred at room temperature for 24 h. After the reaction was complete, it was concentrated under reduced pressure and purified by column chromatography (DCM:MeOH = 100:1) to give compound 6 (1.25 g, yield: 92%) as a white solid.

[0095] Compound 6 (200 mg) was dissolved in THF (5 ml) solution, and HCl (2N, 1 ml) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 1 hour. After thin-layer chromatography showed that the reaction was complete, the reaction mixture was concentrated under reduced pressure and purified by preparative high-performance liquid chromatography to obtain cpd3 (91 mg, yield: 48%). MS [M+H] + =482.2363.

[0096] Example 5

[0097] Synthesis of cpd4

[0098]

[0099] Compound 2 (1 g), 3-bromo-1-adamantanecarboxylic acid (0.40 g), 4-dimethylaminopyridine (DMAP) (0.74 g), and 1-ethyl-3-(3-dimethylpropylamine)carbodiimide (EDCI) (1.19 g) were added to 10 mL of dichloromethane (DCM). The mixture was stirred at room temperature for 24 h. After the reaction was complete, it was concentrated under reduced pressure and purified by column chromatography (DCM:MeOH = 20:1) to give compound 7 (1.11 g, yield: 87%) as a white solid.

[0100] Compound 7 (200 mg) was dissolved in THF (5 ml) solution, and HCl (2 N, 1 ml) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 1 hour. After thin-layer chromatography showed that the reaction was complete, the reaction mixture was concentrated under reduced pressure and purified by preparative high-performance liquid chromatography to obtain cpd4 (83 mg, yield: 45%). 1H NMR (300MHz, DMSO-d6) δ7.93(s,3H),6.93(d,J=4.5Hz,1H),6.81(d,J=4.5Hz,1H),6.36(d,J=5.9Hz,1H),5.38(d,J=5.9Hz,1H),4.70(t,J=5.4Hz,1H) ,4.34–4.13(m,3H),3.98(q,J=5.8Hz,1H),2.31(d,J=10.6Hz,2H),2.24(s ,4H),2.10(s,2H),1.67(d,J=15.3Hz,4H),1.58(d,J=12.8Hz,2H).MS[M+H] + =534.1174.

[0101] Example 6

[0102] Synthesis of cpd5

[0103]

[0104] Compound 2 (1 g), 3-(adamantane-1-yl)propionic acid (0.40 g), 4-dimethylaminopyridine (DMAP) (0.74 g), and 1-ethyl-3-(3-dimethylpropylamine)carbodiimide (EDCI) (1.19 g) were added to 10 mL of dichloromethane (DCM). The mixture was stirred at room temperature for 24 h. After the reaction was complete, it was concentrated under reduced pressure and purified by column chromatography (DCM:MeOH = 100:1) to give compound 8 (0.67 g, yield: 67%) as a white solid.

[0105] Compound 8 (200 mg) was dissolved in THF (5 ml) solution, and HCl (2N, 1 ml) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 1 hour. After thin-layer chromatography showed that the reaction was complete, the reaction mixture was concentrated under reduced pressure and purified by preparative high-performance liquid chromatography to obtain cpd5 (50 mg, yield: 35%). 1H NMR(300MHz,DMSO-d6)δ7.92(s,2H),6.91(d,J=4.5Hz,1H),6.80(d,J=4.5Hz ,1H),6.33(d,J=6.0Hz,1H),5.37(d,J=5.8Hz,1H),4.67(t,J=5.4Hz,1H),4. 36–4.26(m,1H),4.26–4.08(m,2H),4.00–3.88(m,1H),2.26–2.15(m,2H),1. 90(s,2H),1.70–1.52(m,5H),1.43–1.35(m,5H),1.34–1.20(m,2H).MS[M+H] + =482.2404.

[0106] Comparative Example 1

[0107] Another synthesis The synthetic route is as follows, used for activity evaluation and comparison:

[0108]

[0109] Compound 2 (1 g), 3-(adamantane-1-yl)acetic acid (0.40 g), 4-dimethylaminopyridine (DMAP) (0.74 g), and 1-ethyl-3-(3-dimethylpropylamine)carbodiimide (EDCI) (1.19 g) were added to 10 mL of dichloromethane (DCM). The mixture was stirred at room temperature for 24 h. After the reaction was complete, it was concentrated under reduced pressure and purified by column chromatography (DCM:MeOH = 100:1) to give compound 9 (0.74 g, yield: 70%) as a white solid.

[0110] Compound 9 (200 mg) was dissolved in THF (5 ml) solution, and HCl (2 N, 1 ml) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 1 hour. After thin-layer chromatography showed that the reaction was complete, the reaction mixture was concentrated under reduced pressure and purified by preparative high-performance liquid chromatography to obtain cpd7 (87 mg, yield: 41%). 1H NMR (300MHz, DMSO-d6) δ7.91(s,3H),6.91(d,J=4.5Hz,1H),6.81(d,J=4.5Hz,1H),6.31(d,J=6.0Hz,1H),5.38(d,J=5.7Hz,1H) ,4.72(t,J=5.5Hz,1H),4.30–4.12(m,3H),3.95(d,J=5.5Hz,1H),2.03–1.97(m,2H),1.86(s,3H),1.74–1.40(m,12H).MS[M+H] + =468.2241.

[0111] Example 7

[0112] Nucleoside prodrug antiviral activity assay protocol

[0113] Cell preparation: Resuspend cultured CRFK cells in DMEM medium containing 10% FBS and incubate at 37°C for 18-20 hours until 90% confluence. Drug preparation: Dissolve and dilute the test drug to a concentration of 15 mM using DMSO. Drug dilution: Using DMEM medium containing 2% FBS, dilute the test drug stock solution 1000-fold at a 10-fold serial dilution. Then dilute the test drug to six concentration gradients: 3 μM, 1 μM, 333 nM, 111 nM, 37 nM, and 12.33 nM. Add 50 μL of the diluted test drug to each well, with three replicates per group. Also set up cell controls (CC) and virus controls (VC): add 50 μL of DMEM medium containing 2% FBS to each control. The cells were cultured in a 5% CO2, 37°C cell culture incubator for 4 hours. Drug dilution: Using DMEM medium containing 2% FBS, the stock solution of the test drug was diluted 1000-fold at a 10-fold ratio. The test drug was then diluted to six concentration gradients: 6 μM, 2 μM, 666 nM, 222 nM, 74 nM, and 24.66 nM. After mixing with an equal volume of virus solution, the working concentrations of the drug were 3 μM, 1 μM, 333 nM, 111 nM, 37 nM, and 12.33 nM.

[0114] Virus dilution: The virus used for assay was feline infectious peritonitis virus (FIP) FIPV-79-1146 (commercially available from the American Type Culture Collection (ATCC)). Virus was grown in CRFK cells to produce a stockpile of viruses. The FIPV-79-1146 virus stock solution was diluted to 100 TCID using DMEM medium containing 2% FBS. 50 / 50μL. Viral infection and addition of test drug: Diluted test drug and diluted virus solution were added to each well at a volume of 50μL, with 3 replicates per group. Cell control (CC) and virus control (VC) were also established: 100μL of 2% FBS DMEM medium was added to CC wells, and 50μL of 2% FBS DMEM medium and 50μL of diluted virus solution were added to VC wells. Cells were incubated at 37℃ with 5% CO2 for 72 hours, and cell status and CPE were observed daily under a microscope. Result detection: For EC 50 Cell viability was measured using the CellCounting-Lite 2.0 Luminescent Cell Viability Assay kit 72 h after drug treatment. Results analysis: The inhibition rate of different drug concentrations against the virus was calculated, and curve fitting analysis was performed using GraphPad Prism 9.5 to calculate the drug EC50. 50 The antiviral effects of the drugs were analyzed, and the results are shown in Table 1.

[0115] Table 1 Results of anti-FIPV activity test

[0116] Compound numbering <![CDATA[EC 50 (nM)]]> Compound numbering <![CDATA[EC 50 (nM)]]> cpd 1 NA cpd 2 E cpd 3 D cpd 4 B cpd 5 A cpd 6 C cpd 7 NA GS-441524 E Positive drug compound 2 C

[0117] Note: "A" represents compound EC. 50 <50 nM; "B" represents 50 nM < compound EC 50 <300nM; "C" represents 300nM < compound EC 50 <500nM; "D" represents 500nM < compound EC 50 <1000nM; "E" represents compound EC 50 >1000 nM. “NA” indicates that the compounds of this invention have no inhibitory activity.

[0118] As shown in Table 1, the compounds of this invention exhibit excellent inhibitory activity against feline infectious peritonitis virus (FIPV), and are significantly stronger than the positive control compound GS-441524. The inhibitory activity against cpd6 is comparable to that of the positive control compound.

[0119] More than ten times that of GS-441524. Furthermore, cpd7, which has only one more methylene group than cpd6, loses its inhibitory activity against feline infectious peritonitis virus (FIP), while cpd5, which has two more methylene groups than cpd6, further enhances its inhibitory activity against FIP, reaching 20 nM. This activity significantly surpasses that of cpd6 and other previously reported compounds, with effects exceeding orders of magnitude. This demonstrates that the specific structure of the specific compound of this invention has unexpected effects in inhibiting FIP.

[0120] Simultaneously, the antiviral activity of cpd6 against feline calicivirus (FCV) and porcine epidemic diarrhea virus (PEDV) was determined using the same method described above, and the results are shown in Table 2.

[0121] Table 2 Results of anti-FCV and PEDV activity tests

[0122] FCV PEDV Compound Name <![CDATA[EC 50 (μM)]]> <![CDATA[EC 50 (μM)]]> GS-441524 64 0.80 cpd 6 6.6 0.49

[0123] As shown in Table 2, the compounds of this invention also have certain antiviral activity against feline calicivirus (FCV) and porcine epidemic diarrhea virus (PEDV), and are significantly superior to the positive control drug GS-441524, proving that they may have broad-spectrum antiviral function.

[0124] Example 8

[0125] Cytotoxicity assay (CC) 50 (Measurement) Experimental Protocol

[0126] Experimental materials:

[0127] CRFK cells susceptible to FCoV were selected and cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 100 μg / ml penicillin-streptomycin to maintain the cells in the logarithmic growth phase. High-concentration stock solutions of the test compounds were prepared by dissolving them in dimethyl sulfoxide (DMSO). Before use, the solutions were diluted with culture medium to the required concentration, ensuring the final DMSO concentration was ≤1% (an equal volume of DMSO was added to the control group simultaneously). Reagents: CellTiter-Glo luminescent assay kit, 96-well cell culture plate, microplate reader.

[0128] Cell seeding and drug treatment

[0129] CRFK cells were seeded at a density of 10,000 cells / well in 96-well plates, with 100 μl of DMEM medium containing 10% FBS added to each well. The plates were incubated at 37°C in a 5% CO2 incubator for 24 hours until cell adhesion was achieved. The stock solution of the test drug was serially diluted 10-fold with culture medium, with three replicates per concentration. The control group received only medium containing 1% DMSO. The original culture medium in the 96-well plates was discarded, and 100 μl of medium containing different concentrations of the test drug was added to each well. The plates were then incubated for another 48 hours.

[0130] Cell viability assay (CellTiter-Glo method)

[0131] According to the instructions, bring the CellTiter-Glo reagent to room temperature and dilute it 1:1 with serum-free medium. For luminescence detection: Remove the 96-well plate from the incubator and equilibrate to room temperature (approximately 30 minutes). Add 100 μl of diluted CellTiter-Glo reagent to each well and gently shake for 2 minutes to lyse cells and release ATP. Incubate at room temperature for 10 minutes to stabilize the luminescence signal. Measure the luminescence intensity (RLU) of each well using a microplate reader and record the data. Set up wells containing no cells, only medium and drug, to subtract background signals. Set up wells containing cells and 1% DMSO (drug-free) as a reference for 100% cell viability. Subtract the blank control value from the luminescence value of each well, and then calculate the cell viability percentage based on the luminescence value of the solvent control group: Cell viability (%) = (Lack control group luminescence value - Blank luminescence value) / (Experimental group luminescence value - Blank luminescence value) × 100%. Plotting the drug concentration on the x-axis (logarithmic scale) and cell viability percentage on the y-axis, a nonlinear regression was performed using GraphPad Prism software. The results are shown in Table 3. Among them, CC... 50 The half-maximal toxicity concentration (WMC) refers to the drug concentration at which 50% of the host cells are destroyed in an in vitro test.

[0132] Table 3. Cytotoxicity test results

[0133] Compound numbering <![CDATA[CC 50 (μM)]]> Compound numbering <![CDATA[CC 50 (μM)]]> cpd6 A GS-441524 A

[0134] Note: "A" represents compound CC. 50 >100μM; "B" represents 5μM < compound CC 50 <100μM;

[0135] “C” represents 1 μM < compound CC 50 <5μM; "D" represents compound CC 50 <1μM.

[0136] As shown in Table 3, the compounds of the present invention have low cytotoxicity and good safety.

[0137] Example 9

[0138] Solubility test plan

[0139] Accurately weigh the analyte and dissolve it in DMSO. Prepare standard solutions of different concentration gradients using methanol dilution. Analyze the solutions by HPLC and plot a standard curve of concentration versus peak area. Subsequently, add 1–5 mg of the compound to 1 mL of water or PBS (pH = 7.4) buffer, sonicate for 1 h, then centrifuge at 12000 rpm for 10 min. Collect the supernatant for HPLC analysis, and quantify and correct the solubility of the compound using the standard curve.

[0140] Accurately weigh 1–5 mg of the compound to be tested and add it to a mixture containing 500 μL of n-octanol and 500 μL of water. Shake at room temperature for 24 h. Centrifuge the aqueous phase and organic phase separately (12000 rpm, 10 min) and analyze the sample by HPLC. Record the peak areas A (aqueous phase) and A (organic phase) respectively. The LogP value is calculated by the formula LogP = log(A (organic phase) / A (aqueous phase). The results are shown in Table 4.

[0141] Table 4 Solubility Test Results

[0142] Compound numbering solubility Compound numbering solubility cpd6 +++ GS-441524 +

[0143] Note: "+" indicates that the compound solubility is <0.05 mg / mL; "++" indicates that 0.05 mg / mL < compound solubility is <0.5 mg / mL; "+++" indicates that 0.5 mg / mL < compound solubility is <5 mg / mL. As shown in Table 4, the compound cpd 6 of this invention exhibits excellent solubility.

[0144] Example 10

[0145] Pharmacokinetic experiments

[0146] This experiment investigated the pharmacokinetic characteristics and oral bioavailability of cpd 6 and its metabolite GS-441524 in SD rats after intravenous injection and gavage administration.

[0147] Dosage and administration method for each group:

[0148] CPD 6 intravenous injection group: 2 mg of CPD 6 was injected intravenously per kg of rat body weight.

[0149] CPD 6 oral administration group: 10 mg of CPD 6 per kg of rat body weight was administered by gavage.

[0150] GS-441524 intravenous injection group: 5 mg of GS-441524 was injected intravenously per kg of rat body weight.

[0151] GS-441524 oral administration group: 25 mg of GS-441524 per kg of rat body weight was administered by gavage.

[0152] Twelve male SD rats weighing approximately 250g were divided into four groups: an intravenous injection group of CPD 6, an oral administration group of CPD 6, an intravenous injection group of GS-441524, and an oral administration group of GS-441524, with three rats in each group. The intravenous injection group received 2mg of CPD 6 per kg of rat body weight via intravenous injection; the oral administration group received 10mg of CPD 6 per kg of rat body weight via gavage; the intravenous injection group received 5mg of GS-441524 per kg of rat body weight via intravenous injection; and the oral administration group received 25mg of GS-441524 per kg of rat body weight via gavage. Blood was collected from the jugular vein at 0.03h (not collected in the oral group), 0.08h, 0.17h, 0.33h, 0.50h, 1.0h, 2.0h, 4.0h, 6.0h, 8.0h, and 12.0h, approximately 0.2ml of blood was collected and placed into heparin tubes. All samples were centrifuged at 4000rpm for 10 minutes at 4°C. Plasma samples were collected and stored at -65°C for future analysis. Each 50μl aliquot of plasma sample was treated with 250μl of acetonitrile. The samples were centrifuged at 4000rpm for 10 minutes and filtered through a 0.2μm membrane filter. The drug concentration in each sample was determined by high-performance liquid chromatography (HPLC) / mass spectrometry (MS). Analytes were separated using a SHIMADZU C18 column (2.0μm, 4.1*50mm). Plasma concentration data were statistically analyzed using WinNonlin 7.0, a metabolic data analysis software, and pharmacokinetic parameters were calculated using the non-compartmental model (NCA) method. The results are shown in Tables 5 and 6.

[0153] Table 5 Pharmacokinetic parameters of SD rats after administration of cpd 6 (measured by GS-441524, mean ± SD)

[0154]

[0155]

[0156] Table 6. Pharmacokinetic parameters in SD rats after intravenous and gavage administration of GS-414254.

[0157]

[0158] Conclusion: Intravenous injection of 2 mg / kg cpd 6 in SD rats resulted in plasma GS-441524 AUC. (0-t) The mean value was 962.17 h·μg / L; rat plasma GS-441524 AUC after oral administration of 10 mg / kg cpd 6. (0-t)The mean value was 3928.61 h·μg / L. As shown in Tables 5 and 6 above, when the compound cpd 6 of this invention enters the rat body, it rapidly breaks the ester bond to form the metabolite GS-441524. Simultaneously, its oral bioavailability is significantly improved, increasing from 21.3% to 81.6%, resulting in better oral drug-like properties.

[0159] Example 11

[0160] Immunofluorescence assay

[0161] This experiment visually demonstrates the inhibitory effect of cpd 6 on feline infectious peritonitis virus.

[0162] Cell preparation: CRFK cells were seeded into 48-well plates, with an appropriate amount of cells per well, and cultured in an incubator containing 5% CO2 at 37°C. Virus infection and treatment: After cell adhesion, FIPV-79-1146 was added to the wells, with the multiplicity of infection (MOI) set to 0.1. A control group (containing 10 μM GS-441524 dissolved in DMSO) and an experimental group (containing 10 μM cpd 6 dissolved in DMSO) were also set up. One hour after infection, the virus-containing supernatant was discarded, and the cells were gently washed 2-3 times with PBS to remove unadsorbed virus. Fresh culture medium was then added, and the cells were cultured for another 24 hours. Fixation and permeabilization: After 24 hours of culture, the 48-well plates were removed, the culture medium was discarded, and the cells were fixed with pre-cooled 100% methanol at room temperature for 10 minutes. After fixation, the methanol was discarded, and the cells were washed three times with PBS for 5 minutes each time. Next, cells were permeabilized with PBS containing 0.1% Triton X-100 at room temperature for 10 min to increase cell permeability. After permeabilization, cells were washed three times with PBS for 5 min each time. Blocking incubation: Cells were blocked with PBS containing 1% bovine serum albumin (BSA) at room temperature for 30 min to reduce nonspecific binding. After blocking, the blocking solution was discarded, and an appropriate amount of diluted FIPV monoclonal antibody (1:1000 diluted in PBS containing 1% BSA) was added to each well, and incubated at room temperature for 1.5 h. After incubation, cells were washed three times with PBS for 5 min each time. Secondary antibody incubation: FITC-labeled goat anti-mouse IgG (1:500-1:1000 diluted in PBS containing 1% BSA) was added to each well, and incubated at room temperature in the dark for 1 h. After incubation, cells were washed three times with PBS for 5 min each time. After staining, the cells were observed under a fluorescence microscope. The FITC-labeled viral proteins (green fluorescence) were observed using appropriate filters, and the results were photographed and recorded.

[0163] Experimental results are as follows Figure 1 , Figure 2As shown, with increasing drug concentration, the FITC-labeled viral protein (green fluorescence) decreased significantly, indicating that the drug has a significant inhibitory effect on feline infectious peritonitis virus (FIP). Furthermore, cpd 6 showed better anti-FIP effect than GS-441524 at the same concentration.

[0164] Example 12

[0165] In vivo acute toxicity test

[0166] This experiment investigated the safety of GS-441524 and different doses of cpd 6 in SD rats after gavage administration.

[0167] The experimental design is shown in Table 7:

[0168] Table 7. Test drugs, dosages, and administration methods for each group.

[0169]

[0170]

[0171] Note: The dosage for rats is generally 2.5 ml / 250 g;

[0172] Dissolving method: CPD 6: 10% DMSO + 35% physiological saline + 55% PEG 400

[0173] Positive control drug GS-441524: 20% DMSO + 5% saline + 75% PEG 400

[0174] Fifty SD rats weighing approximately 200g (31 males and 19 females) were divided into 5 groups: a blank solvent group (n=6), a GS-441524 (100mg / kg) group (n=11), a CPD 6 (50mg / kg) group (n=11), a CPD 6 (100mg / kg) group (n=11), and a CPD 6 (200mg / kg) group (n=11). Rats were fasted for 12 hours prior to the first administration. The rats were administered the dose daily via gavage, and changes in body weight, food intake, water intake, excrement, coat condition, and mental status were recorded. Rats were also fasted for 12 hours prior to the last administration. Four hours after drug administration, blood was collected from the abdominal aorta of 3 male and 3 female rats in each group for routine blood tests and serum biochemical index detection. The major organs of the animals in each drug administration group and control group were observed for abnormalities in color, size, and morphology. Abnormal organs were photographed promptly. The spleen and thymus were weighed and organ indices were recorded. The heart, liver, spleen, lung, and kidney were fixed in paraformaldehyde. Blood was collected from the orbital sinus of three male rats in each of the GS-441524 (100 mg / kg), cpd6 (50 mg / kg), cpd6 (100 mg / kg), and cpd6 (200 mg / kg) groups. The heart was perfused, and the heart, liver, spleen, lung, and kidney were collected for tissue distribution experiments. One male and one female mouse were kept in each of the GS-441524 (100mg / kg), cpd6 (50mg / kg), cpd6 (100mg / kg), and cpd6 (200mg / kg) groups for observation and recording of weight changes, and attention was paid to food intake, water intake, excrement, fur and mental state.

[0175] Experimental results are as follows Figures 3-14 As shown, the results indicated that SD rats tolerated GS-441524 (100 mg / kg) orally and by gavage once daily for 7 days, and all rats survived. The body weight of rats in all groups showed an increasing trend, with no significant weight loss. No rats exhibited significant central nervous system dysfunction during the intensive observation period of 0-72 hours after administration. The experimental animals remained conscious, with brisk pupillary light reflexes, normal pain response, and good voluntary motor coordination. No signs of neurological excitation or inhibition, such as tremors or tonic convulsions, were observed, indicating that cPD 6 did not induce observable acute neurotoxicity. Blood routine and biochemical data showed no statistically significant differences between the control and experimental groups, indicating good safety. Liver and kidney indicators, blood glucose, and blood lipid indicators showed no significant abnormalities. Creatine kinase levels were slightly elevated. Pathological sections of the myocardium, liver, kidney, spleen, and lungs show that the overall tissue structure of all organs is well preserved, with clear features and no obvious signs of lesions, indicating that the compound cpd 6 of this invention has good safety.

Claims

1. A compound as shown in Formula I or a pharmaceutically acceptable salt thereof: in: n is a positive integer that is 0 or greater than 1; R 1 R 2 R 3 Each is independently selected from hydrogen, deuterium, halogen, cyano, nitro, -NR a R b -SR a Or C1-C6 alkyl; The structure (2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-nitrile is attached to any site on adamantane; R a Or R b Each is independently selected from hydrogen, halogen, cyano, nitro, or C1-C6 alkyl; when n is 0, R 1 R 2 R 3 At least one of them is not hydrogen.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The compound or a pharmaceutically acceptable salt thereof is shown in Formula II: in: n is a positive integer greater than 0 or greater than 1; R 1 R 2 R 3 Each is independently selected from hydrogen, deuterium, halogen, cyano, nitro, -NR a R b -SR a Or C1-C6 alkyl; R a Or R b Each is independently selected from hydrogen, halogen, cyano, nitro, or C1-C6 alkyl; when n is 0, R 1 R 2 R 3 At least one of them is not hydrogen.

3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, characterized in that, Preferably, n = 2 or 3; R 1 R 2 R 3 Each is independently selected from hydrogen, halogen, or C1-C6 alkyl.

4. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, characterized in that, The compound is selected from any one of the following:

5. Use of a compound according to any one of claims 1-4 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating feline infectious peritonitis.

6. The application according to claim 5, characterized in that, The feline infectious peritonitis is a disease caused by infection with the feline infectious peritonitis virus, which is an infectious peritonitis coronavirus.

7. The use of compound cpd6 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating feline infectious peritonitis, wherein the structure of compound cpd6 is shown below:

8. A pharmaceutical composition for treating feline infectious peritonitis, characterized in that, It includes a therapeutically effective amount of the compound or pharmaceutically acceptable salt of any one of claims 1-4, and a pharmaceutically acceptable carrier or diluent.

9. The pharmaceutical composition according to claim 8, characterized in that, The dosage form of the pharmaceutical composition is selected from capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalers, ointments, solutions, creams, gels, powders, lotions, tinctures, suppositories, or patches.

10. The use of a compound according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, or compound cpd6 or a pharmaceutically acceptable salt thereof, in the preparation of inhibitors of feline calicivirus and porcine epidemic diarrhea virus, wherein the structure of compound cpd6 is shown below:

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