Pegylated phosphoric acid prodrug and preparation therefor and use thereof

Abstract

A compound of formula I or a pharmaceutically acceptable salt, wherein R1 and R2 are independently selected from H, or a C1-C6 alkyl, a 3-8-membered cycloalkyl or a 6-20-membered aryl, and the alkyl, cycloalkyl and aryl can be further substituted with one or more mutually independent substituents; m is an integer greater than 1; and n is an integer greater than or equal to 0. Further disclosed are a method for preparing the above-mentioned compound or the pharmaceutically acceptable salt, and the use of the compound or the pharmaceutically acceptable salt. A new pegylated phosphoric acid prodrug compound molecule is synthesized, and the synthesis route has the characteristics of readily available raw materials, mild reaction conditions, simple operation and low costs, and is suitable for subsequent research and production. The compound and a composition thereof can be used for treating or preventing viral infections in humans or animals. The compound and the composition thereof can be used to effectively inhibit feline infectious peritonitis virus (FIPV) infections and treat feline infectious peritonitis.

Description

A PEGylated phosphoric acid prodrug and its preparation and application Technical Field

[0001] The present invention belongs to the technical field of antiviral drug research and development, and specifically relates to a PEGylated phosphate prodrug and its preparation and application. Background Art

[0002] Remdesivir is an FDA-approved antiviral drug used to treat severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) infection. The drug is an arylphosphoamidate triester prodrug that utilizes ProTide nucleotide prodrug technology to deliver the remdesivir nucleoside (RVn) candidate molecule. After administration, remdesivir releases RVn monophosphate, a precursor to the active RVn triphosphate, which effectively inhibits the enzymatic activity of the polymerase of every coronavirus tested to date, including SARS CoV-2. However, due to the instability of remdesivir in plasma, it must be administered intravenously. Researchers are currently studying various prodrug approaches to improve its oral bioavailability, plasma instability, insufficient pulmonary delivery, and liver toxicity.

[0003] Summary of the Invention

[0004] Provided herein are PEGylated phosphate prodrugs of remdesivir nucleoside monophosphate that can be used in pharmaceutical compositions for treating or preventing viral infections in humans or animals.

[0005] In one aspect, the present invention provides a compound of formula I:

[0006] or a pharmaceutically acceptable salt, wherein:

[0007] Each R1 and R2 is independently selected from H, or C1-C6 alkyl, 3-8 membered cycloalkyl or 6-20 membered aryl, or an optional substituent thereof; the alkyl, cycloalkyl, aryl may be further substituted by one or more independent substituents;

[0008] m is an integer greater than 1; n is an integer greater than or equal to 0.

[0009] As an embodiment, m is 1, 2, 3, 4, 5, 6, 7, or 8.

[0010] As an embodiment, n is 0, 1, 2, 3, 4, 5, 6, 7, 8.

[0011] The substituent group can be a variety of groups, such as alkyl (preferably C1-C6 alkyl), alkoxy (preferably C1-C6 alkoxy), carboxyl, ester (preferably containing 1-6 carbon atoms), hydroxyl, aldehyde, etc. For example, the optional substituent of phenyl can be phenyl, phenyl substituted with a single alkyl group, phenyl substituted with multiple alkyl groups, phenyl substituted with oxygen-containing groups (such as methoxy, carboxyl, ester, hydroxyl, aldehyde, etc.), phenyl substituted with multiple groups, etc. In another aspect, the present application provides a pharmaceutical preparation comprising a pharmaceutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

[0012] In another aspect, the present application provides the use of a compound of formula I or a pharmaceutically acceptable salt thereof for treating or preventing viral infections in humans or animals.

[0013] In another aspect, the present application provides a method for treating or preventing a viral infection in a human, wherein the method comprises administering a compound of Formula I or a pharmaceutically acceptable salt thereof to the human.

[0014] In another aspect, the present application provides methods for preparing and producing pharmaceutical compositions characterized by using a compound of Formula I or a pharmaceutically acceptable salt thereof.

[0015] The foregoing summary is illustrative only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will be apparent by reference to the drawings and the following detailed description.

[0016] The present disclosure relates to PEGylated phosphate prodrugs, and pharmaceutical compositions thereof can be used to treat or prevent viral infections in humans or animals.

[0017] definition

[0018] Unless otherwise indicated, the following terms and phrases used in this Agreement are intended to have the following meanings:

[0019] As used herein, "a compound of the present disclosure" or "a compound of Formula I" refers to a compound of Formula I or a pharmaceutically acceptable salt thereof. Similarly, the phrase "a compound of Formula (number)" refers to a compound of the formula and a pharmaceutically acceptable salt thereof.

[0020] As used herein, "salt" refers to a solid containing equal amounts of anions and cations. A salt can be a "single-component" solid. A salt can also be a "multi-component" solid containing one or more additional substances, such as nonionic molecules, eutectic molecules, and / or solvent molecules.

[0021] "Alkyl" refers to an unbranched or branched saturated hydrocarbon chain. For example, an alkyl group can have 1 to 6 carbon atoms (i.e., a C1-C6 alkyl group). Examples of suitable alkyl groups include, but are not limited to, methyl (Me, CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s- Bu,s-butyl,-CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu,t-butyl,-C(CH3)3), 1-pentyl (n-pentyl,-CH2CH2CH2CH2CH3), 2-pentyl (CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH (CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl -2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2) and 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3).

[0022] "Cycloalkyl" generally refers to a cycloalkyl group containing 3 to 8 carbon atoms, including cyclopropyl (-C3H7), cyclobutyl (-C4H9), cyclopentyl (-C5H11), cyclohexyl (-C6H11) or their "optional substituents".

[0023] "Aryl" refers to an aromatic hydrocarbon radical derived by removing a single hydrogen atom from a single carbon atom of a parent aromatic ring system. For example, an aryl group can have 6 to 20 carbon atoms, 6 to 14 carbon atoms, or 6 to 10 carbon atoms. Typical aryl groups include, but are not limited to, radicals derived from benzene (e.g., phenyl), as well as phenyl, naphthyl, anthracenyl, biphenyl, and the like.

[0024] The term "optionally substituent(s)" when referring to a particular moiety of a compound of Formula I (e.g., optionally substituted phenyl) refers to a moiety wherein all substituents are hydrogen or wherein one or more hydrogens of the moiety may be replaced with a recited substituent.

[0025] Certain commonly used alternative chemical names may or may not be used. For example, a divalent group such as a divalent "alkyl" group, a divalent "aryl" group, a divalent "cycloalkyl" group, etc., may also be referred to as an "alkylene" group or an "alkenyl" group or an alkyl group; an "arylene" group or an "alkenyl" group or an aryl group; a "cycloalkylene" group or a "cycloalkylenyl" group or a cycloalkyl group.

[0026] As used herein, the term "treating," unless otherwise indicated, means reversing, alleviating, inhibiting, or preventing the progression of the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. As used herein, the term "treating" means the act of treating, as "treating" is specifically defined above.

[0027] As used herein, the term "therapeutically effective amount" (or "pharmaceutically effective amount") is the amount of a compound of Formula I present in a composition described herein that, when such composition is administered by a selected route of administration, is required to provide the desired levels of drug in the secretions and tissues of the airways and lungs, or in the blood of the subject to be treated to impart the desired physiological response or desired biological effect. The precise amount will depend on many factors, such as the specific compound of Formula I, the specific activity of the composition, the delivery device employed, the physical properties of the composition, its intended use, and patient considerations such as the severity of the disease state, patient cooperation, and can be readily determined by one skilled in the art based on the information provided herein.

[0028] Compound

[0029] Provided herein are compounds of Formula I:

[0030] or a pharmaceutically acceptable salt, wherein:

[0031] Each R1 and R2 is independently H, or C1-C6 alkyl, 3-8 membered cycloalkyl or aryl, or an optional substituent thereof;

[0032] m is an integer greater than 1; n is an integer greater than or equal to 0.

[0033] In some embodiments, wherein R1 and m are defined as in Formula I, R2 = H, and n = 0; that is, the compound of Formula I is a compound having Formula Ia:

[0034] In some embodiments, wherein R1=CH3, m=2, R2=H, n=0 in Formula I, that is, the compound of Formula I has the following structure:

[0035] In some embodiments, in Formula I, R1=CH3, m=4, R2=H, and n=0; that is, the structure of the compound of Formula I is as follows:

[0036] In some embodiments, in Formula I, R1=R2, m=n; the compound of Formula I is a compound having Formula Ib:

[0037] In some embodiments, in Formula I, R1=R2=CH3, m=n=2, that is, Formula I has the following structure:

[0038] A pharmaceutical preparation comprising a pharmaceutically effective amount of a compound or a pharmaceutically acceptable salt thereof according to any of the above technical solutions. In some embodiments, the pharmaceutical preparation is for subcutaneous administration, intravenous administration, oral administration, or inhalation administration.

[0039] In other embodiments, the pharmaceutical preparation is a granule, capsule, tablet, pill, injection preparation, suspension preparation, solution preparation, inhalant such as aerosol, powder spray, spray, etc.

[0040] Use of a compound described in any one of the above technical solutions or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or preventing viral infection in humans or animals.

[0041] The compound is administered to humans or animals orally, intravenously, subcutaneously or by inhalation.

[0042] The viral infection is a coronavirus infection.

[0043] Furthermore, the animal is a feline. Furthermore, the animal is a cat.

[0044] Furthermore, the viral infection is feline infectious peritonitis virus (FIPV) infection. The compound of the present invention can effectively inhibit feline infectious peritonitis virus (FIPV) infection and treat feline infectious peritonitis.

[0045] A method for preparing the compound described in any of the above technical solutions, comprising:

[0046] (1) Prepare the intermediate int-1 with the following structure:

[0047] (2) Synthesizing PEGylated phosphoric acid or its corresponding salt having the following structure:

[0048] (3) Reaction of the intermediate with PEGylated phosphoric acid or its corresponding salt to obtain the following compound:

[0049] (IV) Removing the protecting group to obtain the following compound:

[0050] Taking PEGylated phosphoric acid as an example, the reaction equation is as follows:

[0051] In some embodiments, the preparation method of compound int-1 is shown in the following formula:

[0052] The steps include:

[0053] (1) Compound 1 is mixed with 2,2-dimethoxypropane and TsOH in an organic solvent I (e.g., acetone), stirred for 20-60 minutes, and isopropyl acetate is slowly added; the resulting mixture is filtered, washed with 2:1 heptane / isopropyl acetate, and dried under vacuum (30-50°C) to obtain compound 2;

[0054] (2) Compound 2 was mixed with a K2CO3 solution and an organic solvent II (such as ethyl acetate), and the layers were separated; the organic layer was washed with water and concentrated to a predetermined volume; isopropyl acetate was added again, and the mixture was concentrated; the flask was refilled with isopropyl acetate and the mixture was concentrated; heptane was slowly added to the viscous mixture; the mixture was filtered and washed with 2:1 heptane / isopropyl acetate; and the mixture was dried under vacuum (30-50°C) to obtain compound 3; compound 3 was named compound Int-1.

[0055] Furthermore, a method for synthesizing the PEGylated phosphate prodrug compound (single substitution, n=0) of the above structure is shown in the following formula:

[0056] POCl3 was added to anhydrous MeCN; the atmosphere was replaced with nitrogen, and compound 101-1 was added dropwise at 0°C; the reaction mixture was raised to room temperature and stirred to complete the reaction; water was added to the solution, and the mixture was heated at 50-60°C while protecting with N2 for 1.5-3 hours; the mixture was dried and purified in a high vacuum to obtain compound 101-2;

[0057] Compound Int-1, DCC, and DMAP were added to a solution of compound 101-2 in anhydrous pyridine (9.5 mL); the reaction mixture was stirred at room temperature for 10 to 20 hours, and post-treated to obtain compound 101-3;

[0058] HCl was added to a THF solution of compound 101-3; the reaction mixture was stirred at room temperature for 1 to 3 hours, and post-treated to obtain compound JWY-101.

[0059] Furthermore, a method for synthesizing the PEGylated phosphate prodrug compound of the above structure (disubstituted, m≠0, n≠0) is shown in the following formula:

[0060] The steps include:

[0061] Compound 102-1 was added to a THF solution containing NaH at -5 to 5°C; a THF solution containing POCl3 was added at 0°C and stirred at room temperature for 20 to 60 minutes; the reaction mixture was then stirred at room temperature for 10 to 20 hours and post-treated to obtain compound 102-2;

[0062] A solution of DMAP and compound 102-2 was added to a DCM solution of Int-1 at 0°C; the reaction mixture was stirred at room temperature for 10 to 20 hours, and post-treated to give compound 102-3;

[0063] At -5 to 5°C, HCl was added to a THF (0.8 mL) solution of compound 102-3; the reaction mixture was stirred at room temperature for 1 to 3 hours, and post-treated to obtain JWY-102.

[0064] The embodiments of the present application are not limited to any compositions and methods, which can change and be understood by technicians. In order to make it easier to understand the present disclosure, some terms are first defined. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the embodiments of the present disclosure relate. Many similar, modified or equivalent methods and materials described herein can be used in the practice of the embodiments of the present disclosure without the need for unnecessary experiments. Preferred materials and methods are described herein. When describing and claiming embodiments of the present disclosure, the following terms will be used according to the definitions listed below.

[0065] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0066] This invention innovatively synthesizes novel PEGylated phosphate prodrug compounds. The synthetic route utilizes readily available raw materials, mild reaction conditions, simple operation, and low cost, making it suitable for subsequent research and production. The innovative compounds and compositions of the present invention can be used to treat or prevent viral infections in humans or animals. These compounds and compositions can effectively inhibit feline infectious peritonitis virus (FIPV) infection and treat feline infectious peritonitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 shows the results of CPE (cytopathic effect assay);

[0068] Figure 2 shows the results of cell pathological changes after FIPV infection;

[0069] Figure 3 shows the cytotoxicity assay (CC 50 Determination) results;

[0070] Figure 4 shows EC 50 Measurement results;

[0071] FIG5 is a graph showing the average concentration-time curve of JWY-101 in plasma of SD rats after subcutaneous administration of JWY-101.

[0072] FIG6 is a graph showing the average concentration-time curve of JWY-103 in plasma of SD rats after subcutaneous administration of JWY-101.

[0073] FIG7 is a graph showing the mean concentration-time curve of JWY-101 in plasma after subcutaneous administration of JWY-101 to kittens.

[0074] FIG8 is a graph showing the mean concentration-time curve of JWY-101 in plasma after subcutaneous administration of 5 mg / kg JWY-101 to adult cats.

[0075] FIG9 is a graph showing the mean concentration-time curve of JWY-101 in plasma of adult cats after oral administration of 10 mg / kg JWY-101.

[0076] FIG10 is a graph showing the white-ball ratio of cats in the medium-dose group, i.e., after subcutaneous administration of 5 mg / kg JWY-101.

[0077] FIG11 is a graph showing the white-ball ratio of cats in the high-dose group, i.e., after subcutaneous administration of 7 mg / kg JWY-101. DETAILED DESCRIPTION

[0078] The present invention is further illustrated by the following examples, which should not be considered as limiting in any way. Embodiments of the present invention are further defined in the following non-limiting examples. It should be understood that these examples, although representing certain embodiments of the present invention, are given by way of example only. From the above discussion and these examples, those skilled in the art can ascertain the essential characteristics of the present invention, and, without departing from its characteristics and scope, various changes and modifications can be made to the embodiments of the present invention to adapt them to various usages and conditions. Therefore, in addition to the embodiments shown and described herein, various modifications of the embodiments of the present invention will be feasible for those skilled in the art from the above description. Such modifications also fall within the scope of the appended claims.

[0079] Chemical substances

[0080] All compounds were purchased from Sigma-Aldrich without further purification.

[0081] Analytical methods

[0082] Nuclear magnetic resonance spectroscopy

[0083] In general, the structure of the final salt product was confirmed by nuclear magnetic resonance (NMR) spectroscopy. Proton NMR spectroscopy was performed using a Bruker Advance 500 (500 MHz), and NMR chemical shift values ​​are reported in ppm. Unless otherwise stated, proton NMR measurements were performed at ambient temperature; the following abbreviations have been used to characterize NMR peaks: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublet; ddd, doublet of doublet; dt, doublet of triplet; bs, broad signal. Chemical shifts (δ) of NMR peaks are reported in parts per million (ppm) relative to tetramethylsilane (TMS) and are referenced to the respective residual undeuterated solvent peaks: CDCl3 = 7.26 ppm, MeOH-d4 = 3.31 ppm (1H-NMR). Coupling constants (J) are expressed in Hz.

[0084] Example 1: Synthesis of intermediate compound (Compound Int-1)

[0085] 1.1 Preparation of Compound 2

[0086] A mixture of compound 1 (1 g, 3.43 mmol, 1.0 equiv.) and 2,2-dimethoxypropane (2.1 mL, 17.2 mmol, 5.0 equiv.) in acetone (5 mL) was added TsOH (649 mg, 3.77 mmol, 1.1 equiv.) at room temperature. After stirring for 30 minutes, isopropyl acetate (5 mL) was slowly added. The resulting mixture was filtered, and the filtrate was washed with 2:1 heptane / isopropyl acetate (5 mL). The mixture was dried under vacuum at 40°C to yield compound 2 (800 mg, 46.3%).

[0087] 1.2 Preparation of compound 3

[0088] Compound 2 (800 mg, 1.59 mmol, 1.0 equiv.) was added to a flask, and 5% aqueous K2CO3 solution (10 ml) and ethyl acetate (10 ml) were added. The layers were separated, and the aqueous layer was extracted with ethyl acetate. The organic layer was washed with water and concentrated to approximately 5 ml. Isopropyl acetate (5 ml) was added back to the flask and concentrated to approximately 5 ml. The flask was then refilled with isopropyl acetate (5 ml) and concentrated to 5 ml. Heptane (5 ml) was slowly added to the viscous mixture. The resulting mixture was filtered and washed with 2:1 heptane / isopropyl acetate (5 ml). Compound 3 (650 mg) was obtained by vacuum drying at 40°C. Compound 3 was designated Compound Int-1, and JWY-101 and JWY-102 were synthesized by the following steps.

[0089] Example 2: Synthesis of JWY-101

[0090] 1.1 Preparation of Compound 101-2

[0091] Anhydrous MeCN (40 mL) was added to a dry flask, and POCl₃ (0.38 mL, 4.16 mmol, 0.5 equiv.) was added via syringe. The flask was purged with nitrogen. Compound 101-1 (1.0 g, 8.32 mmol, 1.0 equiv.) was added dropwise to the solution at 0°C. The reaction mixture was allowed to warm to room temperature and stirred overnight. Water (20 mL) was added to the solution, and the mixture was heated at 55°C while protected by N₂ for 2 h. The remaining residue was dried under high vacuum overnight. Purification by column chromatography (DCM:ethanol:water = 60:30:5) afforded compound 101-2 (300 mg, 11.9%) as a colorless oil. (LCMS(ESI)[M+1] + =201.3)

[0092] 1.2 Preparation of Compound 101-3

[0093] Compound Int-1 (1.2 g, 3.6 mmol, 1.0 equiv), DCC (760 mg, 3.6 mmol, 1 equiv) and DMAP (44 mg, 0.36 mmol, 0.1 equiv) were added to a solution of compound 101-2 (736 mg, 3.6 mmol, 1.0 equiv) in anhydrous pyridine (9.5 mL). The reaction mixture was stirred at room temperature for 16 hours. LCMS showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure to obtain a crude product, which was purified by preparative HPLC to obtain compound 101-3 (71 mg, 3.76%). (LCMS (ESI) [M+1] + =514.3)

[0094] 1.3 Preparation of JWY-101

[0095] To a solution of compound 101-3 (71 mg, 0.14 mmol) in THF (3 ml) was added HCl (1 N, 1 ml). The reaction mixture was stirred at room temperature for 2 hours. LCMS indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure to obtain the crude product, which was purified by preparative HPLC to afford JWY-101 (17.8 mg, 26.9%).

[0096] LCMS (ESI) [M+1] + =474.3. 1 H NMR (400MHz, MeOD) δ8.06(s,1H),7.30(d,J=4.8Hz,1H),7.13(d,J=4.8Hz,1H),4.77(d,J=5.3Hz,1H),4.40–4.32(m ,1H),4.28–4.19(m,2H),4.17–4.09(m,1H),4.03–3.94(m,2H),3.67–3.58(m,4H),3.54–3.48(m,2H),3.32(s,3H).

[0097] Example 3: Synthesis of JWY-102

[0098] 1.1 Preparation of Compound 102-2

[0099] Compound 102-1 (1.14 g, 9.5 mmol, 1.0 equiv) was added to a solution of NaH (380 mg, 9.5 mmol, 1.0 equiv) in THF (14 mL) at 0°C. A solution of POCl (1.5 g, 9.8 mmol, 1.0 equiv) in THF (10 mL) was added at 0°C and stirred at room temperature for 0.5 h. The reaction mixture was then stirred at room temperature for 3 h. The solvent was removed in vacuo to afford compound 102-2, which was used in the next step.

[0100] 1.2 Preparation of compound 102-3

[0101] DMAP (24 mg, 0.2 mmol, 0.1 equiv) and a solution of compound 102-2 (0.25 M, DCM, 8 mL, 2 mmol, 1.0 equiv) were added to a DCM solution (6 mL) of compound Int-1 (660 mg, 2 mmol, 1.0 equiv) at 0°C. The reaction mixture was stirred at room temperature for 16 hours. LCMS showed that the reaction was complete. The solvent was removed in vacuo and purified by preparative HPLC to give compound 102-3 (48 mg, 3.9%). (LCMS (ESI) [M+1] + =616.4)

[0102] 1.3 Preparation of JWY-102

[0103] To a solution of compound 102-3 (48 mg, 0.078 mmol, 1.0 equiv) in THF (0.8 mL) was added HCl (1 N, 0.8 mL) at 0°C. The reaction mixture was stirred at room temperature for 2 hours. LCMS indicated the reaction was complete. The solvent was removed in vacuo, and the product was purified by preparative HPLC to afford JWY-102 (10.3 mg, 23.1%).

[0104] LCMS (ESI) [M+1] + =576.4. 1 H NMR(400MHz,MeOD)δ7.89(s,1H),6.95(d,J=4.6Hz,1H),6.91(d,J=4.6Hz,1H),4.97–4.87(m,1H),4.43–4.35(m,2H),4. 34–4.26(m,1H),4.21(t,J=5.6Hz,1H),4.18–4.11(m,4H),3.66–3.57(m,8H),3.54–3.48(m,4H),3.33(d,J=3.4Hz,6H).

[0105] Example 4: Inhibition of Feline Infectious Peritonitis Virus (FIPV) Infection

[0106] CRFK (Crandell-Rees feline kidney) cells were pre-incubated with the test compound and then infected with FIPV at an MOI of 0.01. The culture plates were incubated for 24 h, and each well was assayed for infection-induced phenotypic changes.

[0107] The results of the CPE (cytopathic effect) assay are shown in Figure 1. The cell images in Figure 1 are all magnified 100x. The results show that at a drug concentration of 0 μM, i.e., when the drug is absent, most cells in the wells exhibit pathological changes after FIPV infection, including shrinkage, stacking, cell death, and detachment, with large blank areas appearing at the bottom of the wells. The positive control compound GS-441524, at 10 μM, completely prevented FIPV-induced pathological changes, resulting in cells in the wells that were morphologically unchanged from normal cells. Compounds JWY-101 and JWY-102 exhibited similar activity to GS-441524.

[0108] We used plate imaging-based cell analysis software to quantitatively evaluate the morphological pathological changes in FIPV-infected cells (GS-441524, JWY-101, and untreated controls) and healthy cells. Six parallel experiments were performed simultaneously. Figure 2 shows the percentage of cells with pathological changes in each group. Among them, cells in the untreated negative control group underwent significant pathological changes after FIPV infection, with 80-100% of cells showing pathological changes; 0-20% of cells in the JWY-101 and GS-441524 treatment groups showed pathological changes; and 0% of healthy cells showed pathological changes. The results showed that JWY-101 can effectively inhibit the pathological changes of FIPV-infected cells, and the effect is comparable to that of GS-441524.

[0109] Example 5: Cytotoxicity and in vitro anti-FIPV activity assay of compounds JWY-101 and JWY-102

[0110] 1. Drug dilution method

[0111] 1) Dissolution: Add DMSO, pipette to dissolve and mix thoroughly, see Table 1:

[0112] Table 1

[0113] 2) Filter using a 0.22 μm filter

[0114] 2 CC 50 Determination

[0115] 1) Cell plating: CRFK cells were seeded in 96-well plates and incubated at 37°C for 24 h. The cell number was approximately 2 × 10 4 Pieces / hole.

[0116] 2) Drug dilution: Take 10 sterilized 1.5mL centrifuge tubes and press 1 # ~10 # Numbered in sequence, to 1 # Add 990 μL of culture medium containing 3% FBS to the tube from 2 # Add 900 μL of culture medium containing 3% FBS to each tube. Add 10 μL of 100 mM JWY101 / 102 solution to tube 1, vortex for 15 seconds, then aspirate 100 μL and add 2 # Tube, dilute the drug to 9 times # Tube #1 was added with culture medium containing 1% DMSO and 3% FBS as solvent control.

[0117] 3) Cell washing: Use an 8-channel pipette to remove the culture medium in the 96-well plate and wash twice with PBS.

[0118] 4) Add Drug: 100 μL of the diluted drug solution was added to each well of the plate, starting from the lowest concentration and proceeding from right to left, to columns 9 to 1. 100 μL of culture medium containing 1% DMSO and 3% FBS was added to column 10, and 100 μL of culture medium containing 3% FBS was added to column 11. The 96-well plate was placed in an incubator (37°C, 5% CO2) and incubated for 24 hours. The cells were then observed.

[0119] 5) Cell viability assay: 10 μL of CCK8 was added to each well. After incubation for 1 h, the absorbance at 450 nm was read using a microplate reader. The results were entered into Excel and then imported into GraphPad Prime 9 for analysis.

[0120] 6) Cell activity calculation formula: A is the absorbance of the sample at 450 nm.

[0121] 3 EC 50 Determination

[0122] 1) Cell plating: CRFK cells were seeded in 96-well plates and incubated at 37°C for 24 h. The cell number was approximately 2 × 10 4 Pieces / hole.

[0123] 2) Sample preparation

[0124] a) Drug dilution: culture medium containing 3% FBS and 10, 6, 5, 2, 1, 0.75, and 0.5 μM JWY-101 and JWY-102 was prepared respectively.

[0125] b) Virus dilution: dilute the virus to 10 3.0 TCID 50 / mL.

[0126] 3) Virus inoculation: Add 100 μL of serum-free culture medium to each well in column 10 of a 96-well plate, and add 100 μL of virus dilution solution to each well in columns 1-9. Incubate at 37°C for 1 h.

[0127] 4) Medium change: Aspirate the liquid in each well and replace it with culture medium containing 3% FBS and different concentrations of drugs. Incubate at 37°C for 24 hours and observe the cytopathic effect.

[0128] 5) Result determination: Collect the supernatant of each well, mix the wells with the same drug concentration in the same group and determine the TCID 50 The data were imported into GraphPad Prime 9 to obtain the results.

[0129] 4The results are as follows:

[0130] 1) Cytotoxicity assay (CC) 50 The results of the assay are shown in Figure 3.

[0131] CC 50 The half toxic concentration refers to the drug concentration at which 50% of the host cells are destroyed in the in vitro test. As shown in Figure 3, the CC of JWY-101 and JWY-102 50 The measured values ​​were all above 100 μM, indicating low cytotoxicity.

[0132] 2) In vitro anti-FIPV activity assay (EC 50 The measurement results are shown in Figure 4.

[0133] EC 50 The EC50 is the effective concentration (EC50), which refers to the drug concentration at which 50% of virus-infected cells are inhibited by the drug in in vitro experiments. 50 The measured values ​​were 0.6653μM, 0.8315μM and 0.6384μM respectively. Both JWY-101 and JWY-102 can achieve effective inhibition at low drug concentrations, and the inhibitory effect of JWY-101 is comparable to that of GS-441524.

[0134] Example 6: The compound JWY-101 synthesized in Example 2 was subjected to the following pharmacokinetic test

[0135] (I) This study investigated the pharmacokinetic characteristics of JWY-101 and its metabolite JWY-103 in SD rats under subcutaneous administration of JWY-101.

[0136] 1. Sample configuration and preparation

[0137] (1) Synthesis of intermediate metabolite JWY-103

[0138] Due to the requirements of the experimental analysis method, the intermediate metabolite JWY-103 was also synthesized.

[0139] The method for synthesizing the structure is shown below:

[0140] The synthesis steps are as follows: Compound 103-1 was weighed into a Schlenk flask. Under a nitrogen atmosphere, ultra-dry THF, triethylamine, and POCl3 were added. The mixture was allowed to react at room temperature for 3 hours. Compound 2 was then isolated by column chromatography. The clean compound 103-2 was transferred to a round-bottom flask, and THF and 4N HCl were added. The reaction was continued at room temperature. The reaction was completed by TLC monitoring until the starting material spot of compound 2 disappeared. After post-treatment, compound JWY-103 was obtained.

[0141] (2) Preparation of test samples

[0142] The test article was prepared to a final concentration of 2 mg / mL for subcutaneous administration.

[0143] The test sample was prepared in DMSO and diluted to the final concentration with PBS. After preparation, JWY-101 was a colorless and transparent liquid.

[0144] 2. Experimental system

[0145] (1) Experimental animals

[0146] Species & strain: SD rat

[0147] Source of experimental animals: Spirochete (Beijing) Biotechnology Co., Ltd.

[0148] Planned age at dosing: 6-8 weeks at the start of dosing.

[0149] Planned body weight at the time of dosing: 180-250 g at the start of dosing.

[0150] Number of animals and sex: 4 male rats, 1 of which was a spare rat.

[0151] (2) Feeding and management

[0152] The animals were housed in transparent resin plastic cages (530 mm x 400 mm x 200 mm).

[0153] Fluorescent lighting was used, with 12 hours of light per day (08:00-20:00) and 12 hours of darkness. Dark periods could be interrupted intermittently as needed for research-related activities. The animal room ambient temperature and relative humidity were maintained within the ranges of 20-26°C and 40-70%, respectively, and monitored and recorded daily.

[0154] The diet is a certified rodent diet. The batch number of the diet will be recorded in the study records.

[0155] Drinking water was filtered and sterilized by an ultrapure water machine. All animals had free access to water during the experiment.

[0156] (3) Animal marking

[0157] Use a high-quality marker to write the number directly on the rat's tail and mark the back with basic fuchsin. Attach a label to the outside of the animal cage with information such as the study number, animal number, group number, and sex.

[0158] 3. Brief description of experimental methods

[0159] (1) Groups and dosages

[0160] No random grouping was performed. Animal body weights were measured before administration, and healthy animals with similar body weights were selected for inclusion in the experiment (see Table 2).

[0161] Table 2

[0162] *During the experiment, if any unexpected situation occurs during the administration, the blank group animals can be promptly replaced. After the experiment, the blank group animals will be used to collect blank samples.

[0163] Dosage frequency: Single dose.

[0164] (2) Sample collection

[0165] After ether anesthesia, at least 0.2 mL of blood was collected from the jugular vein and an anticoagulant, sodium heparin, was added.

[0166] Collection time: 30 minutes before administration, and 5 minutes, 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours after administration.

[0167] (3) Sample processing

[0168] After blood sample collection, place it in a labeled centrifuge tube and quickly centrifuge to separate the plasma. The centrifugation conditions are: 3000 rpm, 15 minutes, 4°C. The plasma is stored below -80°C until testing.

[0169] 4. Standard curve and quality control samples

[0170] (1) Preparation of JWY-101, JWY-103 standard curve and quality control samples: Prepare standard curve working solution and quality control working solution using 50% methanol water as diluent. Take 5 μL of each working solution and mix with 45 μL of blank plasma to prepare plasma standard curve and quality control samples. Add 250 μL of internal standard working solution (200 g / mL, methanol, methyldopa) to the prepared standard curve and quality control samples, vortex mix for 1 min, and centrifuge at 4°C, 15400g for 10 min. Take the supernatant for sample injection and analysis.

[0171] (2) Sample pretreatment

[0172] Plasma sample pretreatment: After thawing at room temperature, 40 μL of plasma samples were taken, 200 μL of internal standard (200 ng / mL, methanol, methyldopa) was added, vortexed for about 1 min, centrifuged at 4°C, 15400g for 10 min, and the supernatant was taken for sampling and analysis.

[0173] 5. Data collection and statistical analysis

[0174] Analyst 1.6.3 software outputs original spectra, concentration, accuracy and other data.

[0175] Microsoft Excel 2007 software was used to calculate the mean, standard deviation, and coefficient of variation.

[0176] WinNonlin software non-compartmental model method (NCA) was used to perform AUC and C max , t 1 / 2 Calculation of main pharmacokinetic parameters.

[0177] CT plots were performed using GraphPad Prism 9.

[0178] Specific steps:

[0179] Four SD rats of similar weight were used in the experiment. Rats 1-3 were subcutaneously administered JWY-101 at a dose of 4 mg / kg, a single dose. Blood was collected 30 minutes before administration and at 5 minutes, 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours after administration. The remaining rats served as a backup group for backup and blank matrix collection. The LC-MS / MS analysis method was used to detect the levels of JWY-101 and JWY-103 in plasma, with a limit of quantification of 4 ng / mL. The plasma concentration data were statistically analyzed using the metabolic kinetic data analysis software WinNonlin 7.0, and the pharmacokinetic parameters were calculated using the non-compartmental model (NCA) method.

[0180] The main pharmacokinetic parameters of JWY-101 in SD rats after administration are shown in Table 3:

[0181] Table 3 Main pharmacokinetic parameters of JWY-101 in SD rats after administration

[0182] The average concentration-time curves of JWY-101 and JWY-103 in the plasma of SD rats after subcutaneous administration of JWY-101 are shown in Figures 5 and 6.

[0183] Conclusion: Subcutaneous administration of 4 mg / kg JWY-101, SD rat plasma JWY-101, t 1 / 2 The average value is 0.265h, C max The mean value was 3740 ng / mL, AUC 0-t The mean value was 1522 g·h / mL; rat plasma JWY-103, t 1 / 2 The average value is 0.241h, C max The mean value was 80.7 ng / mL, AUC 0-t The mean value was 32.6 g·h / mL. The data in the table above and the results in Figures 5 and 6 indicate that in the JWY-101-administered group, a large amount of the prodrug JWY-101 prototype component and a certain amount of the ester-cleaved metabolite JWY-103 were simultaneously detected in the rat plasma. The identical peak times for JWY-101 and JWY-103 indicate that the prodrug's ester bond cleaves rapidly in the systemic circulation. The data demonstrate that JWY-101 effectively enters the rat systemic circulation after administration, and ester bond cleavage occurs within the systemic circulation within a short period of time to produce the metabolite JWY-103.

[0184] (2) This study investigated the pharmacokinetic characteristics of JWY-101 in cats (kittens) under the condition of subcutaneous administration of JWY-101.

[0185] 1. Preparation of test samples

[0186] The test sample was prepared to a final concentration of 5 mg / mL for the preparation of working solution.

[0187] The test sample was prepared in DMSO, which was diluted to the final concentration by 50% methanol in water. It was a colorless and transparent liquid after preparation.

[0188] 2. Experimental Animals

[0189] Species & Strain: Cat (approximately 600g).

[0190] 3. Brief description of experimental methods

[0191] (1) Groups and dosages

[0192] No random grouping was performed. The body weight of the animals was measured before administration, and healthy animals with similar body weight were selected for inclusion in the experiment (see Table 5).

[0193] Table 4 Experimental animal grouping

[0194] *During the experiment, if any unexpected situation occurs during the administration, the blank group animals can be promptly replaced. After the experiment, the blank group animals will be used to collect blank samples.

[0195] Dosage frequency: Single dose.

[0196] (2) Sample collection

[0197] After ether anesthesia, at least 0.2 mL of blood was collected from the jugular vein and an anticoagulant, sodium heparin, was added.

[0198] Collection time: 30 minutes before administration, and 5 minutes, 15 minutes, 30 minutes, 1 hour, 1.5 hours, and 2 hours after administration.

[0199] (3) Sample processing

[0200] After blood sample collection, place it in a labeled centrifuge tube and quickly centrifuge to separate the plasma. The centrifugation conditions are: 3000 rpm, 15 minutes, 4°C. The plasma is stored below -80°C until testing.

[0201] 4. Standard curve and quality control samples

[0202] (1) Preparation of JWY-101 standard curve and quality control samples: Prepare the standard curve working solution and quality control working solution using 50% methanol water as the diluent. Take 5 μL of each working solution and mix it with 45 μL of blank plasma to prepare the plasma standard curve and quality control samples. Add 250 μL of internal standard working solution (200 g / mL, methanol, methyldopa) to the prepared standard curve and quality control samples, vortex mix for 1 minute, and centrifuge at 4°C, 15400g for 10 minutes. Take the supernatant for sampling and analysis.

[0203] (2) Sample pretreatment

[0204] Plasma sample pretreatment: After thawing at room temperature, 40 μL of plasma samples were taken, 200 μL of internal standard (200 ng / mL, methanol, methyldopa) was added, vortexed for about 1 min, centrifuged at 4°C, 15400g for 10 min, and the supernatant was taken for sampling and analysis.

[0205] 5. Data collection and statistical analysis

[0206] Analyst 1.6.3 software outputs original spectra, concentration, accuracy and other data.

[0207] Microsoft Excel 2007 software was used to calculate the mean, standard deviation, and coefficient of variation.

[0208] WinNonlin software non-compartmental model method (NCA) was used to perform AUC and C max , t 1 / 2 Calculation of main pharmacokinetic parameters.

[0209] CT plots were performed using GraphPad Prism 9.

[0210] 6. Experimental steps

[0211] Four cats of similar weight were divided into two groups. Cats 1-3 received a single subcutaneous dose of 5 mg / kg of JWY-101. Blood was collected 30 minutes before, and 5 minutes, 15 minutes, 30 minutes, 1 hour, 1.5 hours, and 2 hours after administration. The remaining cats were assigned to Group 2, a reserve group for backup and blank matrix collection. LC-MS / MS analysis was used to determine the levels of JWY-101 and GS441524 in plasma, with a limit of quantification of 4 ng / mL. The plasma concentration data were analyzed using WinNonlin 7.0, a metabolic kinetic data analysis software, and pharmacokinetic parameters were calculated using the non-compartmental analysis method (NCA).

[0212] The main pharmacokinetic parameters of JWY-101 in cats after administration are shown in Table 5:

[0213] Table 5 Main pharmacokinetic parameters of JWY-101 in cats after administration

[0214] The mean concentration-time curve of JWY-101 in the plasma of cats after subcutaneous administration of 5 mg / kg JWY-101 is shown in Figure 7 .

[0215] 7. Experimental Summary

[0216] In the JWY-101-treated group, significant amounts of the prodrug, JWY-101, were detected in the cats' plasma. These data demonstrate that JWY-101 effectively enters the systemic circulation of the experimental kittens following administration.

[0217] (3) This study investigated the pharmacokinetic characteristics of JWY-101 in adult cats under subcutaneous administration of JWY-101.

[0218] 1. Preparation of test samples

[0219] The test sample was prepared to a final concentration of 5 mg / mL for the preparation of working solution.

[0220] The test sample was prepared in DMSO, which was diluted to the final concentration by 50% methanol in water. It was a colorless and transparent liquid after preparation.

[0221] 2. Experimental Animals

[0222] Species & Strain: Cat (approximately 2.5 kg).

[0223] 3. Brief description of experimental methods

[0224] (1) Groups and dosages

[0225] No random grouping was performed. The body weight of the animals was measured before administration, and healthy animals with similar body weight were selected for inclusion in the experiment (see Table 8).

[0226] Table 6 Experimental animal grouping

[0227] *During the experiment, if any unexpected situation occurs during the administration, the blank group animals can be promptly replaced. After the experiment, the blank group animals will be used to collect blank samples.

[0228] Dosage frequency: Single dose.

[0229] (2) Sample collection

[0230] After ether anesthesia, at least 0.2 mL of blood was collected from the jugular vein and an anticoagulant, sodium heparin, was added.

[0231] Collection time: 30 minutes before administration, 5 minutes, 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, and 24 hours after administration.

[0232] (3) Sample processing

[0233] After blood sample collection, place it in a labeled centrifuge tube and quickly centrifuge to separate the plasma. The centrifugation conditions are: 3000 rpm, 15 minutes, 4°C. The plasma is stored below -80°C until testing.

[0234] 4. Standard curve and quality control samples

[0235] (1) Preparation of JWY-101 standard curve and quality control samples: Prepare the standard curve working solution and quality control working solution using 50% methanol water as the diluent. Take 5 μL of each working solution and mix it with 45 μL of blank plasma to prepare the plasma standard curve and quality control samples. Add 250 μL of internal standard working solution (200 g / mL, methanol, methyldopa) to the prepared standard curve and quality control samples, vortex mix for 1 minute, and centrifuge at 4°C, 15400g for 10 minutes. Take the supernatant for sampling and analysis.

[0236] (2) Sample pretreatment

[0237] Plasma sample pretreatment: After thawing at room temperature, 40 μL of plasma samples were taken, 200 μL of internal standard (200 ng / mL, methanol, methyldopa) was added, vortexed for about 1 min, centrifuged at 4°C, 15400g for 10 min, and the supernatant was taken for sampling and analysis.

[0238] 5. Data collection and statistical analysis

[0239] Analyst 1.6.3 software outputs original spectra, concentration, accuracy and other data.

[0240] Microsoft Excel 2007 software was used to calculate the mean, standard deviation, and coefficient of variation.

[0241] WinNonlin software non-compartmental model method (NCA) was used to perform AUC and C max , t 1 / 2 Calculation of main pharmacokinetic parameters.

[0242] CT plots were performed using GraphPad Prism 9.

[0243] 6. Experimental steps

[0244] Four cats of similar weight were divided into two groups. Cats 1-3 received a single subcutaneous dose of 5 mg / kg of JWY-101. Blood was collected 30 minutes before dosing and at 5, 15, 30 minutes, 1 hour, 1.5 hours, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, and 24 hours after dosing. The remaining cats were assigned to Group 2, a reserve group for use as a backup and for blank matrix collection. LC-MS / MS analysis was used to determine the levels of JWY-101 and GS441524 in plasma, with a limit of quantification of 4 ng / mL. The plasma concentration data were analyzed using WinNonlin 7.0, a metabolic kinetic data analysis software, and pharmacokinetic parameters were calculated using the non-compartmental (NCA) method.

[0245] The main pharmacokinetic parameters of JWY-101 in cats after administration are shown in Table 7:

[0246] Table 7 Main pharmacokinetic parameters of JWY-101 in cats after administration

[0247] The mean concentration-time curve of JWY-101 in the plasma of cats after subcutaneous administration of 5 mg / kg JWY-101 is shown in Figure 8 .

[0248] 7. Experimental Summary

[0249] In the JWY-101-treated group, significant amounts of prodrug JWY-101 were detected in the plasma of cats. These data demonstrate that JWY-101 effectively enters the systemic circulation of adult cats following administration.

[0250] (IV) This study investigated the pharmacokinetic behavior of JWY-101 in adult cats after oral administration of JWY-101.

[0251] 1. Preparation of test samples

[0252] The test sample was prepared to a final concentration of 5 mg / mL for the preparation of working solution.

[0253] The test sample was prepared in DMSO, which was diluted to the final concentration by 50% methanol in water. It was a colorless and transparent liquid after preparation.

[0254] 2. Experimental Animals

[0255] Species & Strain: Cat (approximately 3.5 kg).

[0256] 3. Brief description of experimental methods

[0257] (1) Groups and dosages

[0258] No random grouping was performed. The body weight of the animals was measured before administration, and healthy animals with similar body weight were selected for inclusion in the experiment (see Table 8).

[0259] Table 8 Experimental animal grouping

[0260] Dosage frequency: Single dose.

[0261] (2) Sample collection

[0262] After ether anesthesia, at least 0.2 mL of blood was collected from the jugular vein and an anticoagulant, sodium heparin, was added.

[0263] Collection time: 30 minutes before administration, 5 minutes, 10 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, and 24 hours after administration.

[0264] (3) Sample processing

[0265] After blood sample collection, place it in a labeled centrifuge tube and quickly centrifuge to separate the plasma. The centrifugation conditions are: 3000 rpm, 15 minutes, 4°C. The plasma is stored below -80°C until testing.

[0266] 4. Standard curve and quality control samples

[0267] (1) Preparation of JWY-101 standard curve and quality control samples: Prepare the standard curve working solution and quality control working solution using 50% methanol water as the diluent. Take 5 μL of each working solution and mix it with 45 μL of blank plasma to prepare the plasma standard curve and quality control samples. Add 250 μL of internal standard working solution (200 g / mL, methanol, methyldopa) to the prepared standard curve and quality control samples, vortex mix for 1 minute, and centrifuge at 4°C, 15400g for 10 minutes. Take the supernatant for sampling and analysis.

[0268] (2) Sample pretreatment

[0269] Plasma sample pretreatment: After thawing at room temperature, 40 μL of plasma samples were taken, 200 μL of internal standard (200 ng / mL, methanol, methyldopa) was added, vortexed for about 1 min, centrifuged at 4°C, 15400g for 10 min, and the supernatant was taken for sampling and analysis.

[0270] 5. Data collection and statistical analysis

[0271] Analyst 1.6.3 software outputs original spectra, concentration, accuracy and other data.

[0272] Microsoft Excel 2007 software was used to calculate the mean, standard deviation, and coefficient of variation.

[0273] WinNonlin software non-compartmental model method (NCA) was used to perform AUC and C max , t 1 / 2 Calculation of main pharmacokinetic parameters.

[0274] CT plots were performed using GraphPad Prism 9.

[0275] 6. Experimental steps

[0276] Four cats of similar weight were selected for the experiment and divided into cats 1-4. Cats 1-3 were orally administered JWY-101 at a dose of 10 mg / kg, a single dose. Blood was collected 30 minutes before administration and 5 minutes, 10 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, and 24 hours after administration. Cat 4 was used as a backup group for backup and blank matrix collection. The LC-MS / MS analysis method was used to detect the content of JWY-101 and GS441524 in plasma, and the lower limit of quantification of the method was 4 ng / mL. The plasma concentration data were statistically analyzed using the metabolic kinetic data analysis software WinNonlin 7.0, and the pharmacokinetic parameters were calculated using the non-compartmental model method (NCA).

[0277] The main pharmacokinetic parameters of JWY-101 in cats after administration are shown in Table 9:

[0278] Table 9 Main pharmacokinetic parameters of JWY-101 in cats after administration

[0279] The mean concentration-time curve of JWY-101 in the plasma of cats after oral administration of 10 mg / kg JWY-101 is shown in Figure 9.

[0280] 7. Experimental Summary

[0281] In this example, JWY-101 was administered, and a small amount of prodrug JWY-101 was detected in cat plasma. This result shows that after oral administration of JWY-101 to cats, JWY-101 can effectively and rapidly enter the systemic circulation.

[0282] Example 7: The compound JWY-101 synthesized in Example 2 was subjected to the following animal experiments

[0283] 1. Experimental subjects

[0284] A total of 8 sick cats diagnosed with feline infectious peritonitis were recruited from the pet hospital.

[0285] 2. Experimental plan

[0286] JWY-101 was prepared into a solution with normal saline and injected subcutaneously once daily.

[0287] JWY-101 was administered subcutaneously to the confirmed sick cats. The experimental cats were divided into a high-dose medication group and a medium-dose medication group. The high-dose medication group received a dose of 7 mg / kg, and the medium-dose medication group received a dose of 5 mg / kg.

[0288] Discharge criteria: Mental state and appetite return to normal, pleural / peritoneal effusion decreases, other clinical symptoms are relieved or disappear, and the white-to-globulin ratio recovers and reaches around 0.4 or above 0.4.

[0289] 3. Detection indicators

[0290] Blood biochemical indicators

[0291] 4. Experimental results

[0292] Regular check-ups of sick cats were conducted during and after the medication.

[0293] The blood white blood cell ratio of cats diagnosed with feline infectious peritonitis after administration of JWY-101 is shown in Table 10:

[0294] Table 10 Record of blood leukocyte ratio of sick cats after administration of JWY-101

[0295] Figures 10 and 11 show the blood leukocyte counts of cats diagnosed with feline infectious peritonitis after subcutaneous administration of 5 mg / kg and 7 mg / kg JWY-101, respectively.

[0296] Cats diagnosed with feline infectious peritonitis all showed recovery of appetite and activity within three days of treatment; all met discharge criteria and were discharged after one week of treatment. With the exception of Fa Cai, whose data could not be followed up after discharge, all other cats recovered after treatment (mental state and appetite returned to normal, pleural and abdominal effusions disappeared, intestinal lymph nodes significantly shrank, and white-globulin ratio returned to normal). As shown in Table 10, Figures 10, and 11, regardless of whether the cats were diagnosed with dry or wet infectious peritonitis, the white-globulin ratio of the cats recovered to varying degrees within a short period of time after JWY-101 treatment. During maintenance treatment until recovery, the white-globulin ratio of the cats gradually returned to a healthy value above 0.6. This result shows that subcutaneous administration of JWY-101 can rapidly improve the mental state and appetite of cats, increase the white-globulin ratio, and effectively cure cats diagnosed with infectious peritonitis.

[0297] 5. Experimental Conclusion

[0298] Our experimental results show that when JWY-101 is administered subcutaneously to cats diagnosed with infectious peritonitis (regardless of dry or wet), it can quickly improve the cats' spirits and appetite, increase the blood white blood cell ratio, and effectively cure infectious peritonitis in cats.

Claims

1. The compound shown in formula I: or a pharmaceutically acceptable salt; in: R1 and R2 are independently selected from H, or C1-C6 alkyl, 3-8 membered cycloalkyl or 6-20 membered aryl, and the alkyl, cycloalkyl, aryl may be further substituted by one or more independent substituents; m is an integer greater than 1; n is an integer greater than or equal to 0.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R1 and m are as defined in formula I, and R2=H and n=0; having the structure shown in the following chemical formula:

3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, wherein For Formula I, R1=CH3, m=2, R2=H and n=0; having the structure shown in the following chemical formula: Alternatively, for Formula I: R1=CH3, m=4, R2=H and n=0; having the structure shown in the following chemical formula:

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein For Formula I, R1=R2 and m=n; having the structure shown in the following chemical formula:

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein For formula I: R1=R2=CH3, m=n=2; its structure is shown below:

6. A pharmaceutical preparation comprising a pharmaceutically effective amount of the compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof.

7. The pharmaceutical preparation according to claim 7, wherein The pharmaceutical preparation is a granule, capsule, tablet, pill, injection, suspension, solution, inhalant, powder aerosol or spray.

8. Use of a compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing viral infection in humans or animals.

9. The use according to claim 8, wherein the viral infection is a coronavirus infection.

10. The use according to claim 8, wherein the viral infection is feline FIPV virus infection.

11. A method for preparing the compound according to claim 1, wherein: include: (I) Prepare the intermediate int-1 with the following structure: (ii) synthesizing PEGylated phosphoric acid or its corresponding salt having the following structure: (III) The intermediate int-1 reacts with pegylated phosphoric acid or its corresponding salt to obtain the following compound: (IV) Removal of protecting groups:

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