CN118994243A - Pegylated phosphoric acid prodrug and preparation and application thereof - Google Patents

Abstract

The present invention discloses a compound of formula I or a pharmaceutically acceptable salt; wherein: _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, and aryl may be further substituted by one or more independent substituent groups; m is an integer greater than 1; n is an integer greater than or equal to 0. The present invention also discloses a preparation method and application of the above-mentioned compound or pharmaceutically acceptable salt. The present invention innovatively synthesizes a new PEGylated phosphoric acid prodrug compound molecule, and the raw materials of the synthesis route are easy to obtain, the reaction conditions are mild, the operation is simple, the cost is low, and it is suitable for subsequent research and production. The innovative compound of the present invention and its composition can be used to treat or prevent viral infections in humans or animals. The use of the compound and its composition can effectively inhibit feline infectious peritonitis virus (FIPV) infection and treat feline infectious peritonitis.

Description

Pegylated phosphoric acid prodrug and preparation and application thereof

Technical Field

The invention belongs to the technical field of antiviral drug research and development, and particularly relates to a polyethylene glycol phosphate prodrug, and preparation and application thereof.

Background

REMDESIVIR (radevir) is an FDA approved antiviral drug for the treatment of severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) infection. The drug is an aryl phosphoramide triester prodrug for delivery of a radexine Wei Hegan (RVn) candidate molecule using the ProTide nucleotide prodrug technique. After administration, the Rede Wei Shifang RVn monophosphate, a precursor of the active RVn triphosphate, is effective in inhibiting the enzymatic activity of each of the coronavirus (including SARS CoV-2) polymerases tested to date. However, because of the instability of adefovir in plasma, it must be injected intravenously. Various prodrug approaches are currently being investigated by researchers to improve their oral bioavailability, plasma instability, pulmonary inadequate delivery, and hepatotoxicity.

Disclosure of Invention

Provided herein are pegylated phosphate prodrugs of ryposide Wei Hegan monophosphate, which are useful in pharmaceutical compositions for treating or preventing viral infections in humans or animals.

In one aspect, the invention provides a compound of formula I:

or a pharmaceutically acceptable salt, wherein:

Each R ₁ and R ₂ 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 groups may be further substituted with one or more substituents independently of each other;

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

As an alternative to the use of a single-phase polymer, m is 1,2,3,4,5,6,7,8.

As one embodiment, n is 0,1,2,3,4,5,6,7,8.

The substituent may be various groups such as alkyl (preferably C1-C6 alkyl), alkoxy (preferably C1-C6 alkoxy), carboxyl, ester (preferably having 1-6 carbon atoms), hydroxyl, aldehyde, etc. For example, the optional substituents of the phenyl group may be phenyl, mono-alkyl substituted phenyl, polyalkyl substituted phenyl, oxy group (e.g., methoxy, carboxyl, ester, hydroxy, aldehyde, etc.) substituted phenyl, multi-group mono-or multi-substituted phenyl, and the like. In another aspect, the application provides a pharmaceutical formulation comprising a pharmaceutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or adjuvant.

In another aspect, the application provides the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, for the treatment or prophylaxis of a viral infection in a human or animal.

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

In another aspect, the present application provides methods of preparing and producing pharmaceutical compositions characterized by the use of a compound of formula I or a pharmaceutically acceptable salt thereof.

The foregoing abstract 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 presented by reference to the drawings and the following detailed description.

The present disclosure relates to pegylated phosphate prodrugs, pharmaceutical compositions thereof, useful for treating or preventing viral infections in humans or animals.

Definition of the definition

Unless otherwise indicated, the following terms and phrases used in the present protocol are intended to have the following meanings:

As used herein, "compounds of the present disclosure" or "compounds of formula I" refer to compounds of formula I or pharmaceutically acceptable salts thereof. Similarly, the phrase "compound of formula (x)" refers to a compound of formula (x) and pharmaceutically acceptable salts thereof.

As used herein, "salt" refers to a solid that contains equal amounts of anions and cations. The salt may be a "one-component" solid. Salts may also be "multicomponent" solids containing one or more additional substances, such as nonionic molecules, eutectic molecules, and/or solvent molecules.

"Alkyl" refers to an unbranched or branched saturated hydrocarbon chain. For example, an alkyl group may 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, CH ₃), ethyl (Et, -CH ₂CH₃), 1-propyl (n-Pr, n-propyl, -CH ₂CH₂CH₃), 2-propyl (i-Pr, i-propyl, -CH (CH ₃)₂), 1-butyl (n-Bu, n-butyl, -CH ₂CH₂CH₂CH₃), 2-methyl-1-propyl (i-Bu, i-butyl, -CH ₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl, -CH (CH ₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl, -C (CH ₃)₃), 1-pentyl (n-pentyl, -CH ₂CH₂CH₂CH₂CH₃), 2-pentyl (CH (CH ₃)CH₂CH₂CH₃), 3-pentyl (-CH (CH ₂CH₃)₂), 2-methyl-2-butyl (-C (CH ₃)₂CH₂CH₃)), 3-methyl-2-butyl (-CH (CH ₃)CH(CH₃)₂), 3-methyl-1-butyl (-CH ₂CH₂CH(CH₃)₂), 2-methyl-1-butyl (-CH ₂CH(CH₃)CH₂CH₃), 1-hexyl (-CH ₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl (-CH (CH ₃)CH₂CH₂CH₂CH₃), 3-hexyl (-CH (CH ₂CH₃)(CH₂CH₂CH₃)), 2-methyl-2-pentyl (-C (CH ₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl (-CH (CH ₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (-CH (CH ₃)CH₂CH(CH₃)₂), 3-methyl-3-pentyl (-C (CH ₃)(CH₂CH₃)₂), 2-methyl-3-pentyl (-CH (CH ₂CH₃)CH(CH₃)₂), 2, 3-dimethyl-2-butyl (-C (CH ₃)₂CH(CH₃)₂) and 3, 3-dimethyl-2-butyl (-CH (CH ₃)C(CH₃)₃)).

"Cycloalkyl" refers generally to cycloalkyl groups containing 3 to 8 carbon atoms, including cyclopropyl (-C3H 7), cyclobutyl (-C4H 9), cyclopentyl (-C5H 11), cyclohexyl (-C6H 11) or their "optional substituents".

"Aryl" refers to an aromatic radical derived by removing one 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.

The term "optional substituent" when referring to a particular moiety of a compound of formula I (e.g., an optionally substituted phenyl group) refers to a moiety wherein all substituents are hydrogen or wherein one or more hydrogens of the moiety may be replaced with a listed substituent.

Some common 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 "arylalkenyl" group or an aryl group; "cycloalkylene" or "cycloalkylalkenyl" or cycloalkyl.

The term "treating" as used herein, unless otherwise indicated, refers to reversing, alleviating, inhibiting or preventing the progression of a disorder or condition to which the term applies, or one or more symptoms of the disorder or condition. The term "treatment" as used herein refers to the act of treatment, "treatment" is defined explicitly above.

The term "therapeutically effective amount" (or "pharmaceutically effective amount") as used herein is the amount of a compound of formula I present in a composition described herein that, when administered by a selected route of administration, is required to provide a desired level of drug in secretions and tissues of the airways and lungs, or in the blood of a subject to be treated, to administer a desired physiological response or a desired biological effect. The exact amount will depend on many factors, such as the particular compound of formula I, the particular activity of the composition, the delivery device employed, the physical characteristics of the composition, its intended use, and patient considerations such as the severity of the disease state, patient compliance, and the like, and can be readily determined by one of skill in the art based on the information provided herein.

Compounds of formula (I)

Provided herein are compounds of formula I:

or a pharmaceutically acceptable salt, wherein:

Each of R ₁ and R ₂ is independently H, or C ₁-C₆ alkyl, 3-8 membered cycloalkyl or aryl, or an optional substituent thereof;

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

In some embodiments, wherein R ₁ and m are as defined in formula I, R ₂ =h, n=0; i.e. the compound of formula I is a compound of formula Ia:

In some embodiments, wherein R ₁＝CH₃,m＝2,R₂ = H, n = 0 in formula I, i.e., the compound of formula I has the following structure:

In some embodiments, R ₁＝CH₃,m＝4,R₂ = H, n = 0 in formula I; i.e. the structure of the compound of formula I is as follows:

In some embodiments, in formula I, R ₁＝R₂, m=n; the compound of formula I is a compound having formula Ib:

in some embodiments, R ₁＝R₂＝CH₃, m=n=2 in formula I, i.e., formula I has the structure:

a pharmaceutical formulation comprising a pharmaceutically effective amount of a compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof. In some embodiments, wherein the pharmaceutical formulation is for subcutaneous administration, intravenous administration, oral administration, or inhalation administration.

In other embodiments, the pharmaceutical formulation is a granule, capsule, tablet, pill, injection, suspension, solution, inhalant such as aerosol, powder spray, or the like.

Use of a compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment or prophylaxis of a viral infection in a human or animal.

Wherein the compound is administered to the human or animal by oral, intravenous, subcutaneous, or inhalation.

Wherein the viral infection is a coronavirus infection.

Further, the animal is a feline. Further, the animal is a cat.

Further, the viral infection is a Feline Infectious Peritonitis Virus (FIPV) infection. The compounds of the invention may be effective in inhibiting infection by Feline Infectious Peritonitis Virus (FIPV) and treating infectious peritonitis in cats.

A method of preparing a compound according to any one of the preceding claims, comprising:

(one) preparing an intermediate int-1 with the following structure:

(II) synthesizing PEGylated phosphoric acid or corresponding salt thereof with the following structure:

(III) reacting the intermediate with polyethylene glycol phosphoric acid or corresponding salt thereof to obtain the following compound:

(IV) removing the protecting group to obtain the following compound:

taking polyethylene glycol phosphoric acid as an example, the reaction equation is as follows:

in some embodiments, a method for preparing compound int-1 is represented by the formula:

The method comprises the following steps:

(1) Mixing the compound 1 with 2, 2-dimethoxy propane and TsOH in an organic solvent I (such as acetone can be selected), stirring for 20-60 minutes, and slowly adding isopropyl acetate; filtering the obtained mixed solution, and washing with 2:1 heptane/isopropyl acetate; vacuum drying at 30-50 deg.c to obtain compound 2;

(2) Compound 2 was mixed with a K ₂CO₃ solution and an organic solvent II (such as ethyl acetate) and the layers separated; the organic layer is concentrated to a set volume after being washed by water; adding isopropyl acetate again, concentrating; recharging the flask with isopropyl acetate, and concentrating; adding heptane slowly into the viscous mixed solution; the obtained mixture is filtered and washed with 2:1 heptane/isopropyl acetate; vacuum drying at 30-50 deg.c to obtain compound 3; compound 3 is designated compound Int-1.

Further, a method of synthesizing the pegylated phosphate prodrug compound of the above structure (monosubstituted, n=0) is shown as the following formula:

Adding POCl ₃ into anhydrous MeCN; nitrogen replacement, namely dropwise adding the compound 101-1 at the temperature of 0 ℃; the reaction mixture was warmed to room temperature and stirred to finish the reaction; adding water into the solution, heating at 50-60 ℃, protecting with N ₂, and reacting for 1.5-3 h; drying and purifying in high vacuum to obtain a compound 101-2;

To a solution of compound 101-2 in anhydrous pyridine (9.5 mL) were added compound Int-1, DCC and DMAP; stirring the reaction mixture at room temperature for 10-20 hours, and performing post-treatment to obtain a compound 101-3;

Adding HCl to a solution of compound 101-3 in THF; the reaction mixture was stirred at room temperature for 1 to 3 hours and worked up to give compound JWY-101.

Further, a method of synthesizing the pegylated phosphate prodrug compound of the above structure (disubstituted, m.noteq.0, n.noteq.0) is shown as the following formula:

The method comprises the following steps:

Adding the compound 102-1 into a THF solution containing NaH at the temperature of-5 to 5 ℃; adding a THF solution containing POCl ₃ at 0 ℃, and stirring at room temperature for 20-60 min; then stirring the reaction mixture at room temperature for 10-20 hours, and carrying out post-treatment to obtain a compound 102-2;

DMAP and a solution of compound 102-2 were added to Int-1 in DCM at 0deg.C; stirring the reaction mixture at room temperature for 10-20 hours, and performing post-treatment to obtain a compound 102-3;

HCl is added into a THF (0.8 mL) solution of the compound 102-3 at the temperature of minus 5 to 5 ℃; the reaction mixture was stirred at room temperature for 1 to 3 hours and worked up to give JWY-102.

Embodiments of the application are not limited to any compositions and methods, which may vary and are understood by the skilled artisan. For easier understanding of the present disclosure, certain terms are first defined. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of this disclosure relate. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the present disclosure without undue experimentation. Preferred materials and methods are described herein. In describing and claiming embodiments of the present disclosure, the following terminology will be used in accordance with the definitions set out below.

Compared with the prior art, the invention has the beneficial effects that:

The invention innovatively synthesizes new polyethylene glycol phosphate prodrug compound molecules, and the synthetic route has the advantages of easily available raw materials, mild reaction conditions, simple and convenient operation and lower cost, and is suitable for subsequent research and production. The novel compounds of the present invention and compositions thereof are useful for the treatment or prevention of viral infections in humans or animals. The compound and the composition thereof can effectively inhibit infection of infectious peritonitis virus (FIPV) of cats and treat infectious peritonitis of cats.

Drawings

FIG. 1 shows CPE (cytopathic effect assay) results;

FIG. 2 is a graph showing the results of pathological changes in cells after FIPV infection;

FIG. 3 shows cytotoxicity detection (CC ₅₀ assay) results;

FIG. 4 shows the results of EC ₅₀ assay;

FIG. 5 is a graph of average drug JWY-101 in plasma after subcutaneous administration JWY-101 in SD rats.

FIG. 6 is a graph of average drug JWY-103 in plasma after subcutaneous administration JWY-101 in SD rats.

FIG. 7 is a graph of average drug time JWY-101 in plasma after subcutaneous administration JWY-101 in kittens.

FIG. 8 is a graph of average drug JWY-101 in plasma after subcutaneous administration of 5mg/kg JWY-101 to adult cats.

FIG. 9 is a graph of average drug JWY-101 in plasma after oral administration of 10mg/kg JWY-101 to adult cats.

FIG. 10 is a graph of white ball ratio after subcutaneous administration of 5mg/kg JWY-101 to a middle dose group, i.e., a sick cat.

FIG. 11 is a graph of white ball ratio after subcutaneous administration of 7mg/kg JWY-101 to a high dose group, i.e., a sick cat.

Detailed Description

The invention is further illustrated by the following examples, which should not be construed 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 embodiments, while indicating certain embodiments of the invention, are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the embodiments of the invention to adapt it to various usages and conditions. Accordingly, various modifications of the embodiments of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Chemical substances

All compounds were purchased from Sigma-Aldrich and were not further purified.

Analysis method

Nuclear magnetic resonance spectroscopy

In general, the structure of the final product of the salt is determined by Nuclear Magnetic Resonance (NMR) spectroscopy. Proton magnetic resonance spectroscopy used Bruker Advance 500 (500 MHz) and nuclear magnetic resonance chemical shift values were in ppm. Proton nuclear magnetic resonance measurements are performed at ambient temperature unless otherwise indicated; the following abbreviations have been used to characterize nuclear magnetic resonance peaks: s, single peak: d, double peaks; t, triplet; q, quartet; m, multiple peaks; dd, doublet; ddd, double doublet peak; dt, double triplet; bs, wide signal. Chemical shift (δ) of nuclear magnetic resonance peak is in parts per million (ppm) of Tetramethylsilane (TMS) and refers to the respective residual non-deuterated solvent peak: CDCl ₃ =7.26 ppm, meoh-d4=3.31 ppm (1H-NMR). The coupling constant (J) is expressed in Hz.

EXAMPLE 1 Synthesis of intermediate Compound (Compound Int-1)

1.1 Preparation of Compound 2

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

1.2 Preparation of Compound 3

Compound 2 (800 mg,1.59mmol,1.0 equiv.) is added to the flask, and 5% K ₂CO₃ in water (10 ml) and ethyl acetate (10 ml) are added. The layers were separated and the aqueous layer was extracted with ethyl acetate. The organic layer was washed with water and concentrated to about 5ml. The flask was again charged with isopropyl acetate (5 ml) and concentrated to about 5ml. The flask was again charged with isopropyl acetate (5 ml) and concentrated to 5ml. To the viscous mixture was slowly added heptane (5 ml). The resulting mixture was filtered and washed with 2:1 heptane/isopropyl acetate (5 ml). Vacuum drying at 40℃afforded Compound 3 (650 mg). Compound 3 was designated compound Int-1 and JWY-101 and JWY-102 were synthesized by the following steps.

EXAMPLE 2 Synthesis of JWY-101

1.1 Preparation of Compound 101-2

In a dry flask was added anhydrous MeCN (40 mL) and POCl ₃ (0.38 mL,4.16mmol,0.5 equiv.) was added via syringe. Nitrogen was purged in the flask. Compound 101-1 (1.0 g,8.32mmol,1.0 equiv.) was added dropwise to the solution at 0 ℃. The reaction mixture was allowed to warm to room temperature and stirred overnight. Water (20 ml) was added to the solution, which was heated at 55℃while being protected with N ₂, and reacted for 2 hours. 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 oily liquid. (LCMS (ESI) [ m+1] ⁺ =201.3)

1.2 Preparation of Compound 101-3

To a solution of compound 101-2 (356 mg,3.6mmol,1.0 equiv) in anhydrous pyridine (9.5 mL) were added compound Int-1 (1.2 g,3.6mmol,1.0 equiv), DCC (760 mg,3.6mmol,1 equiv) and DMAP (44 mg,0.36mmol,0.1 equiv). The reaction mixture was stirred at room temperature for 16 hours. LCMS showed the reaction was complete. The reaction mixture was concentrated under reduced pressure to give a crude product, which was purified by preparative high performance liquid chromatography to give compound 101-3 (71 mg, 3.76%). (LCMS (ESI) [ m+1] ⁺ =514.3)

1.3 Preparation of JWY-101

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

LCMS(ESI)[M+1]⁺＝474.3.¹H 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).

EXAMPLE 3 Synthesis of JWY-102

1.1 Preparation of Compound 102-2

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

1.2 Preparation of Compound 102-3

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

1.3 Preparation of JWY-102

To a solution of compound 102-3 (48 mg,0.078mmol,1.0 equiv.) in THF (0.8 mL) at 0deg.C was added HCl (1N, 0.8 mL). The reaction mixture was stirred at room temperature for 2 hours. LCMS showed the reaction was complete. The solvent was removed in vacuo and purified by preparative high performance liquid chromatography to give JWY-102 (10.3 mg, 23.1%).

LCMS(ESI)[M+1]⁺＝576.4.¹H 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).

Example 4 inhibition of feline-infected peritoneal inflammatory Virus (FIPV) infection

CRFK (Crandell-REES FELINE KIDNEY) cells were pre-incubated with test compounds and then infected with FIPV at an MOI of 0.01. The dishes were incubated for 24h and each well was examined for infection-induced phenotypic changes.

CPE (cytopathic effect assay) results are shown in figure 1. The cell images in FIG. 1 are all at 100 x magnification. From the results, when the drug concentration is 0 mu M, namely no drug acts, most cells in the hole are pathologically changed after being infected by FIPV, the cells shrink, stack, die and fall off, and a large blank area appears at the bottom of the hole; the positive control compound GS-441524 completely prevents the FIPV from inducing pathological changes at 10 mu M, and the cell morphology in the hole is not different from that of normal cells; compounds JWY-101 and JWY-102 showed similar activity to GS-441524.

We quantitatively assessed the pathological changes in cell morphology of FIPV infected cells (GS-441524, jwy-101, untreated control) and healthy cells using well-plate imaging based cell analysis software, while 6 parallel experiments were performed and the results of figure 2 show the percentage of cells in each group that developed the pathological changes. Wherein, after FIPV infection of cells of the non-drug negative control group, obvious pathological changes are generated, and 80-100% of cells show pathological changes; JWY-101 and GS-441524 showed pathological changes in 0-20% of cells; healthy cells 0% of the cells show pathological changes. The results show that JWY-101 can effectively inhibit the cytopathy of FIPV infection, and the effect is equivalent to that of GS-441524.

Example 5: cytotoxicity and in vitro anti-FIPV Activity assay of Compounds JWY-101, JWY-102

1 Method of diluting a drug

1) Dissolving: adding DMSO, blowing for dissolution and mixing well, see Table 1:

TABLE 1

Medicament	Molecular weight	Mass/mg	DMSO/μL	Final concentration/mM
					JWY-101	473.38	11.7	247.16	100
JWY-102	575.51	11.2	194.61	100

2) Filtration was performed using a 0.22 μm filter

2CC ₅₀ assay

1) Cell plating: CRFK cells were seeded in 96-well plates and incubated at 37 ℃ for 24h with cell numbers of approximately 2 x 10 ⁴ cells/well.

2) Drug dilution: 10 sterilized 1.5mL centrifuge tubes were sequentially numbered 1 ^#～10^#, 990. Mu.L of 3% FBS-containing culture medium was added to 1 ^# tubes, and 900. Mu.L of 3% FBS-containing culture medium was added to each tube from 2 ^#. mu.L of 100mM JWY101/102 solution was added to the 1# tube, and after vortexing for 15s, 100. Mu.L of the solution was aspirated and added to the 2 ^# tube, the drug was diluted in the order of fold to 9 ^# tube, and the 10# tube was added with a culture medium containing 1% DMSO and 3% FBS as a solvent control.

3) Cell washing: the culture broth in the 96-well plate was aspirated with an 8-channel pipette and washed twice with PBS.

4) Adding the medicine: the diluted drug solution was sequentially applied to columns 9 to 1 from right to left in this order from low to high in 100. Mu.L of a culture solution containing 1% DMSO and 3% FBS per well, and to column 10, 100. Mu.L of a culture solution containing 3% FBS was added to column 11. The 96-well plate was placed in an incubator (37 ℃ C., 5% CO 2) and incubated for 24 hours to observe the cell condition.

5) Cell activity detection: 10 mu L of CCK8 is added to each well, after incubation for 1 hour, absorbance at 450nm is read by using a microplate reader, excel is input, and GRAPHPAD PRIME is introduced after analysis to obtain a result.

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

3EC ₅₀ assay

1) Cell plating: CRFK cells were seeded in 96-well plates and incubated at 37 ℃ for 24h with cell numbers of approximately 2 x 10 ⁴ cells/well.

2) Sample preparation

A) Drug dilution: culture solutions containing 3% FBS and 10, 6, 5, 2,1, 0.75 and 0.5 mu M JWY-101 and JWY-102 were prepared, respectively.

B) Dilution of virus liquid: the virus was diluted to 10 ^3.0TCID₅₀/mL.

3) Virus inoculation: 100. Mu.L of serum-free medium was added to each well of column 10 of the 96-well plate, 100. Mu.L of virus dilution was added to each well of columns 1 to 9, and incubated at 37℃for 1 hour.

4) Liquid replacement: the liquid in each well was aspirated, replaced with a culture medium containing 3% fbs and different concentrations of drug, incubated at 37 ℃ for 24h, and cytopathic effect was observed.

5) Results determination: the supernatants from each well were collected, the same set of drug concentration wells were mixed and then assayed for TCID ₅₀ (Reed-Muench method), and the data was imported GRAPHPAD PRIME to yield the results.

The results were as follows:

1) The cytotoxicity test (CC ₅₀ assay) results are shown in FIG. 3.

CC ₅₀ is the median toxic concentration, which refers to the concentration of drug at which 50% of the host cells are destroyed in an in vitro assay. As can be seen from the results of FIG. 3, the measured values of CC ₅₀ of JWY-101 and JWY-102 were 100. Mu.M or more, and the cytotoxicity was low.

2) The results of the in vitro anti-FIPV activity assay (EC ₅₀ assay) are shown in figure 4.

EC ₅₀ is the median effective concentration, which refers to the concentration of drug in an in vitro assay when 50% of virus-infected cells are inhibited by the drug. As can be seen from the results of FIG. 4, the EC ₅₀ measurements for JWY-101, JWY-102, and GS-441524 are 0.6653. Mu.M, 0.8315. Mu.M, and 0.6384. Mu.M, respectively. JWY-101 and JWY-102 can achieve effective inhibition at low drug concentrations, and the inhibition effect of JWY-101 is equivalent to that of GS-441524.

Example 6: the following pharmacokinetic test was performed on the compound JWY-101 synthesized in example 2 (one) to examine the pharmacokinetic behavior characteristics of JWY-101 and the metabolite JWY-103 in rats under the condition of JWY-101 given subcutaneously to SD rats

1. Sample configuration and preparation

(1) Synthesis of intermediate metabolite JWY-103

As required by the experimental analysis method, the intermediate metabolites JWY-103 were additionally synthesized.

The method for synthesizing the structure is shown as the following formula:

The synthesis steps are as follows: weighing the compound 103-1 in a Schlenk bottle, adding ultra-dry THF, triethylamine and POCl ₃ under the nitrogen atmosphere, reacting for 3 hours at normal temperature, and separating by column chromatography to obtain the compound 2. Transferring the clean compound 103-2 into a round-bottom flask, adding THF and 4N HCl for reaction at normal temperature, monitoring the disappearance of the starting point of the compound 2 by TLC, ending the reaction, and carrying out post treatment to obtain the compound JWY-103.

(2) Test sample preparation

The final concentration of the test sample is 2mg/mL, and the test sample is used for subcutaneous administration.

The test sample is diluted with DMSO to a final concentration, and JWY-101 is colorless transparent liquid.

2. Experimental system

(1) Experimental animal

Species & strain: SD rat

Experimental animal sources: sibefu (Beijing) Biotechnology Co., ltd.

Week of planned dosing: the administration is started for 6-8 weeks.

Body weight at planned dosing: 180-250g at the beginning of administration.

Number and sex of animals: 4 males, 1 of which were spare rats.

(2) Feeding and management

Animals were kept in clear resin plastic squirrel cages (530mm x 400mm x 200mm).

Fluorescent lighting, 12 hours of lighting (08:00-20:00) per day and no lighting for 12 hours. The dark time may be intermittently interrupted due to the need to study related activities. The ambient temperature and relative humidity of the animal house are controlled to be in the range of 20-26 ℃ and 40-70% respectively, and are monitored and recorded every day.

The feed is a well-tested rodent feed. The lot number of the feed will be recorded in the study record.

The drinking water is filtered and sterilized by an ultrapure water machine. All animals were free to drink water during the experiment.

(3) Animal marking

The tail was directly numbered using a premium Mark pen and the basic fuchsin was marked on the back. The animal cage is externally provided with a label, and information such as study number, animal number, group number, sex and the like is marked.

3. Brief description of the experimental methods

(1) Group and dose

No random grouping is performed. Animal body weight was measured prior to dosing and healthy animals with similar body weights were selected for inclusion in the experiment, see table 2.

TABLE 2

* In the experimental process, if accidents occur in the administration process, animals in a blank group can be replaced in time. At the end of the experiment, the animals of the blank group will be used to collect the blank samples.

Frequency of administration: single administration.

(2) Sample collection

After anesthesia with diethyl ether, the jugular vein was sampled at least 0.2mL, and anticoagulant was added: heparin sodium.

Acquisition time: 30min before administration, 5min, 15min, 30min, 1h, 1.5h, 2h, 4h, 6h, 8h, 12h, 24h after administration.

(3) Sample processing

After blood sample collection, placing the blood sample in a marked centrifuge tube, and rapidly centrifuging to separate out plasma, wherein the centrifugation conditions are as follows: 3000rpm, 15 min, 4 deg.C, and plasma at-80 deg.C.

4. Standard curve and quality control sample

(1) JWY-101, preparation of JWY-103 standard curve and quality control sample: and preparing a standard curve working solution and a quality control working solution by taking 50% methanol water as a diluting solvent. 5 mu L of each working solution is mixed with 45 mu L of blank plasma to prepare a plasma standard curve and a quality control sample. 250 mu L of internal standard working solution (200 g/mL, methanol and methyldopa) is added into the standard yeast and quality control sample after the preparation, and after vortex mixing for 1min, centrifugation is carried out for 10min at 4 ℃ and 15400 g. Taking supernatant and analyzing by sample injection.

(2) Sample pretreatment

Pretreatment of plasma samples: after thawing the plasma samples at room temperature, 40. Mu.L of each of the samples was taken, 200. Mu.L of an internal standard (200 ng/mL, methanol, methyldopa) was added, and after vortexing for about 1min, centrifugation was performed at 4℃for 10min under 15400g conditions, and the supernatant was sampled for analysis.

5. Data acquisition and statistical analysis

The analysis 1.6.3 software outputs data such as original map, concentration, accuracy and the like.

Microsoft Excel 2007 software calculates the mean, standard deviation, coefficient of variation, etc.

The WinNonlin software non-atrioventricular model method (NCA) performs calculation of main pharmacokinetic parameters such as AUC, C _max、t_1/2 and the like.

GRAPHPAD PRISM 9C-T plots were made.

The method comprises the following specific steps:

the experiment selects 4 SD rats with similar weight, and the subcutaneous administration of JWY-101 to rats 1-3 is carried out at a dosage of 4mg/kg, and the single administration is carried out. Blood was collected at time points of 30min before administration, 5min, 15min, 30min, 1h, 1.5h, 2h, 4h, 6h, 8h, 12h, 24h after administration. The remaining rats were used as a spare group for spare and empty matrix collection. The LC-MS/MS analysis method is adopted to detect the JWY-101 and JWY-103 content in the blood plasma, and the lower limit of the quantification of the method is 4ng/mL. Plasma concentration data were counted using the pharmacokinetic data analysis software WinNonlin 7.0 and pharmacokinetic parameters were calculated using the non-compartmental model method (NCA).

The main pharmacokinetic parameters in SD rats after JWY-101 dosing are shown in Table 3:

Tables 3 JWY-101 Main pharmacokinetic parameters in SD rats after administration

The average drug of JWY-101 and JWY-103 in plasma after subcutaneous administration of JWY-101 in SD rats is shown in FIG. 5 and FIG. 6.

Conclusion: the subcutaneous 4mg/kg administration is JWY-101, SD rat plasma JWY-101, t _1/2 mean is 0.265 h, C _max mean is 3740 ng/mL, AUC _0-t mean is 1522 g-h/mL; rat plasma JWY-103, t _1/2 mean 0.241 h, C _max mean 80.7 ng/mL, AUC _0-t mean 32.6 g h/mL. From the above table data results and the results of fig. 5 to 6, it is understood that a large amount of pro-drug JWY-101 prototype component and a certain amount of ester bond cleavage metabolite JWY-103 were simultaneously detected in rat plasma in JWY-101 administration group. The same peak arrival times of JWY-101 and JWY-103 indicate rapid cleavage of the ester linkage of the prodrug in the systemic circulation. The data show that JWY-101 can effectively enter the systemic circulation of rats after administration, and that ester bond cleavage occurs in the systemic circulation in a short period of time to produce metabolites JWY-103.

(II) this experiment examined pharmacokinetic behavior profile of JWY-101 in cats under conditions of subcutaneous administration JWY-101 in experimental cats (kittens).

1. Test sample preparation

The final concentration of the sample preparation is 5mg/mL, and the sample preparation is used for preparing working solution.

The test sample is prepared by diluting DMSO with 50% methanol water to final concentration, and preparing into colorless transparent liquid.

2. Experimental animal

Species & strain: cats (about 600 g).

3. Brief description of the experimental methods

(1) Group and dose

No random grouping is performed. Animal body weight was measured prior to dosing and healthy animals with similar body weights were selected for inclusion in the experiment, see table 5.

Table 4 experimental animal grouping table

* In the experimental process, if accidents occur in the administration process, animals in a blank group can be replaced in time. At the end of the experiment, the animals of the blank group will be used to collect the blank samples.

Frequency of administration: single administration.

(2) Sample collection

After anesthesia with diethyl ether, the jugular vein was sampled at least 0.2mL, and anticoagulant was added: heparin sodium.

Acquisition time: 30min before administration, 5min, 15min, 30min, 1h, 1.5h, and 2h after administration.

(3) Sample processing

After blood sample collection, placing the blood sample in a marked centrifuge tube, and rapidly centrifuging to separate out plasma, wherein the centrifugation conditions are as follows: 3000rpm, 15 min, 4 deg.C, and plasma at-80 deg.C.

4. Standard curve and quality control sample

(1) JWY-101 standard curve and preparation of quality control samples: and preparing a standard curve working solution and a quality control working solution by taking 50% methanol water as a diluting solvent. 5 mu L of each working solution is mixed with 45 mu L of blank plasma to prepare a plasma standard curve and a quality control sample. 250 mu L of internal standard working solution (200 g/mL, methanol and methyldopa) is added into the standard yeast and quality control sample after the preparation, and after vortex mixing for 1min, centrifugation is carried out for 10min at 4 ℃ and 15400 g. Taking supernatant and analyzing by sample injection.

(2) Sample pretreatment

Pretreatment of plasma samples: after thawing the plasma samples at room temperature, 40. Mu.L of each of the samples was taken, 200. Mu.L of an internal standard (200 ng/mL, methanol, methyldopa) was added, and after vortexing for about 1min, centrifugation was performed at 4℃for 10min under 15400g conditions, and the supernatant was sampled for analysis.

5. Data acquisition and statistical analysis

The analysis 1.6.3 software outputs data such as original map, concentration, accuracy and the like.

Microsoft Excel 2007 software calculates the mean, standard deviation, coefficient of variation, etc.

The WinNonlin software non-atrioventricular model method (NCA) performs calculation of main pharmacokinetic parameters such as AUC, C _max、t_1/2 and the like.

GRAPHPAD PRISM 9C-T plots were made.

6. Experimental procedure

The experiment selects 4 cats with similar weight, and the cats are divided into 2 groups, and the subcutaneous administration of JWY-101 is carried out on the cats 1-3, wherein the administration dosage is 5mg/kg, and the single administration is carried out. Blood was collected at time points of 30min before administration, 5min, 15min, 30min, 1h, 1.5h, and 2h after administration. The remaining cats were organized into group 2 as a spare group for spare and empty matrix collection. The LC-MS/MS analysis method is adopted to detect the JWY-101 content of the GS441524 in the blood plasma, and the lower limit of the quantification of the method is 4ng/mL. Plasma concentration data were counted using the pharmacokinetic data analysis software WinNonlin 7.0 and pharmacokinetic parameters were calculated using the non-compartmental model method (NCA).

The main pharmacokinetic parameters in cats following JWY-101 administration are shown in table 5:

tables 5 JWY-101 Main pharmacokinetic parameter tables in cats following administration

The graph of the average drug JWY-101 in plasma after subcutaneous administration of 5mg/kg JWY-101 in cats is shown in FIG. 7.

7. Summary of the experiment

JWY-101 dosing groups, large amounts of prodrug JWY-101 were detectable in cat plasma. The data show that JWY-101 can effectively enter the systemic circulation of experimental kittens after administration.

(III) this experiment examined pharmacokinetic behavior profile of JWY-101 in cats under conditions of subcutaneous administration JWY-101 in experimental cats (adult cats).

1. Test sample preparation

The final concentration of the sample preparation is 5 mg/mL, and the sample preparation is used for preparing working solution.

The test sample is prepared by diluting DMSO with 50% methanol water to final concentration, and preparing into colorless transparent liquid.

2. Experimental animal

Species & strain: cats (about 2.5 kg).

3. Brief description of the experimental methods

(1) Group and dose

No random grouping is performed. Animal body weight was measured prior to dosing and healthy animals with similar body weights were selected for inclusion in the experiment, see table 8.

TABLE 6 grouping table of experimental animals

* In the experimental process, if accidents occur in the administration process, animals in a blank group can be replaced in time. At the end of the experiment, the animals of the blank group will be used to collect the blank samples.

Frequency of administration: single administration.

(2) Sample collection

After anesthesia with diethyl ether, the jugular vein was sampled at least 0.2mL, and anticoagulant was added: heparin sodium.

Acquisition time: the time period is 30 minutes before the administration, 5min,15min,30min,1h,1.5h,2h,4h,6h,8h,

10h，12h，24h。

(3) Sample processing

After blood sample collection, placing the blood sample in a marked centrifuge tube, and rapidly centrifuging to separate out plasma, wherein the centrifugation conditions are as follows: 3000rpm, 15 min, 4 deg.C, and plasma at-80 deg.C.

4. Standard curve and quality control sample

(1) JWY-101 standard curve and preparation of quality control samples: and preparing a standard curve working solution and a quality control working solution by taking 50% methanol water as a diluting solvent. 5 mu L of each working solution is mixed with 45 mu L of blank plasma to prepare a plasma standard curve and a quality control sample. 250 mu L of internal standard working solution (200 g/mL, methanol and methyldopa) is added into the standard yeast and quality control sample after the preparation, and after vortex mixing for 1min, centrifugation is carried out for 10min at 4 ℃ and 15400 g. Taking supernatant and analyzing by sample injection.

(2) Sample pretreatment

Pretreatment of plasma samples: after thawing the plasma samples at room temperature, 40. Mu.L of each of the samples was taken, 200. Mu.L of an internal standard (200 ng/mL, methanol, methyldopa) was added, and after vortexing for about 1min, centrifugation was performed at 4℃for 10min under 15400g conditions, and the supernatant was sampled for analysis.

5. Data acquisition and statistical analysis

The analysis 1.6.3 software outputs data such as original map, concentration, accuracy and the like.

Microsoft Excel 2007 software calculates the mean, standard deviation, coefficient of variation, etc.

The WinNonlin software non-atrioventricular model method (NCA) performs calculation of main pharmacokinetic parameters such as AUC, C _max、t_1/2 and the like.

GRAPHPAD PRISM 9C-T plots were made.

6. Experimental procedure

The experiment selects 4 cats with similar weight, and the cats are divided into 2 groups, and the subcutaneous administration of JWY-101 is carried out on the cats 1-3, wherein the administration dosage is 5mg/kg, and the single administration is carried out. The time period is 30 minutes before the administration, 5min,15min,30min,1h,1.5h,2h,4h,6h,8h,10h,12h,24h after administration blood was collected at time points. The remaining cats were organized into group 2 as a spare group for spare and empty matrix collection. The LC-MS/MS analysis method is adopted to detect the JWY-101 content of the GS441524 in the blood plasma, and the lower limit of the quantification of the method is 4ng/mL. Plasma concentration data were counted using the pharmacokinetic data analysis software WinNonlin 7.0 and pharmacokinetic parameters were calculated using the non-compartmental model method (NCA).

The main pharmacokinetic parameters in cats following JWY-101 administration are shown in Table 7:

tables 7 JWY-101 Main pharmacokinetic parameter Table in cats following administration

The graph of the average drug JWY-101 in plasma after subcutaneous administration of 5mg/kg JWY-101 in cats is shown in FIG. 8.

7. Summary of the experiment

JWY-101 dosing groups, large amounts of prodrug JWY-101 were detectable in cat plasma. The data show that JWY-101 can effectively enter the systemic circulation of experimental adult cats after administration.

(IV) the experiment examined pharmacokinetic behavior profile of experimental cats (adult cats) JWY-101 in cats given JWY-101 orally.

1. Test sample preparation

The final concentration of the sample preparation is 5 mg/mL, and the sample preparation is used for preparing working solution.

The test sample is prepared by diluting DMSO with 50% methanol water to final concentration, and preparing into colorless transparent liquid.

2. Experimental animal

Species & strain: cats (about 3.5 kg).

3. Brief description of the experimental methods

(1) Group and dose

No random grouping is performed. Animal body weight was measured prior to dosing and healthy animals with similar body weights were selected for inclusion in the experiment, see table 8.

Table 8 experimental animal grouping table

Frequency of administration: single administration.

(2) Sample collection

After anesthesia with diethyl ether, the jugular vein was sampled at least 0.2mL, and anticoagulant was added: heparin sodium.

Acquisition time: the time period is 30 minutes before the administration, 5min,10min,30min,1h,1.5h,2h,4h,6h,8h,10h,12h,24h after administration.

(3) Sample processing

After blood sample collection, placing the blood sample in a marked centrifuge tube, and rapidly centrifuging to separate out plasma, wherein the centrifugation conditions are as follows: 3000rpm, 15 min, 4 deg.C, and plasma at-80 deg.C.

4. Standard curve and quality control sample

(1) JWY-101 standard curve and preparation of quality control samples: and preparing a standard curve working solution and a quality control working solution by taking 50% methanol water as a diluting solvent. 5 mu L of each working solution is mixed with 45 mu L of blank plasma to prepare a plasma standard curve and a quality control sample. 250 mu L of internal standard working solution (200 g/mL, methanol and methyldopa) is added into the standard yeast and quality control sample after the preparation, and after vortex mixing for 1min, the mixture is centrifuged at 15400g at 4 ℃ for 10 min. Taking supernatant and analyzing by sample injection.

(2) Sample pretreatment

Pretreatment of plasma samples: after thawing the plasma samples at room temperature, 40. Mu.L of each of the samples was taken, 200. Mu.L of an internal standard (200 ng/mL, methanol, methyldopa) was added, and after vortexing for about 1min, centrifugation was performed at 4℃for 10min under 15400g conditions, and the supernatant was sampled for analysis.

5. Data acquisition and statistical analysis

The analysis 1.6.3 software outputs data such as original map, concentration, accuracy and the like.

Microsoft Excel 2007 software calculates the mean, standard deviation, coefficient of variation, etc.

The WinNonlin software non-atrioventricular model method (NCA) performs calculation of main pharmacokinetic parameters such as AUC, C _max、t_1/2 and the like.

GRAPHPAD PRISM 9C-T plots were made.

6. Experimental procedure

The experiment selects 4 cats with similar weights, and the cats are divided into 1-4. The cat No. 1-3 is orally administered JWY-101 at a dose of 10mg/kg in a single dose. The time period is 30 minutes before the administration, 5min,10min,30min,1h,1.5h,2h,4h,6h,8h,10h,12h,24h after administration blood was collected at time points. Cat No. 4, as a spare group, was used for spare and empty matrix collection. The LC-MS/MS analysis method is adopted to detect the JWY-101 content of the GS441524 in the blood plasma, and the lower limit of the quantification of the method is 4ng/mL. Plasma concentration data were counted using the pharmacokinetic data analysis software WinNonlin 7.0 and pharmacokinetic parameters were calculated using the non-compartmental model method (NCA).

The main pharmacokinetic parameters in cats following JWY-101 administration are shown in Table 9:

tables 9 JWY-101 Main pharmacokinetic parameter Table in cats following administration

The graph of JWY-101 mean drug in plasma after oral administration of 10mg/kg JWY-101 in cats is shown in FIG. 9.

7. Summary of the experiment

In this example, JWY-101 doses were administered and small amounts of prodrug JWY-101 were detected in cat plasma. The results show that JWY-101 is able to enter the systemic circulation effectively and rapidly after oral administration of JWY-101 to cats.

Example 7: the following animal experiments were conducted using the compound JWY-101 synthesized in example 2

1. Experimental objects

The definitive diagnosis of the hospital recruitment of pets is 8 cats with infectious peritonitis.

2. Experimental protocol

JWY-101 was prepared as a solution in physiological saline, subcutaneously injected, once daily.

The method comprises the steps of performing JWY-101 subcutaneous injections on the diagnosed cat, wherein the experimental cat is divided into a high-dose administration group and a medium-dose administration group, the high-dose administration group is 7mg/kg, and the medium-dose administration group is 5mg/kg.

Discharge standard: the spirit and appetite are recovered to be normal, the hydrothorax/abdominal cavity is reduced, other clinical symptoms are relieved or disappeared, and the white ball ratio is recovered to be about 0.4 or more than 0.4.

3. Detection index

Biochemical index of blood

4. Experimental results

And (5) periodically rechecking the treated cats after the administration is finished.

The white blood ball ratios of JWY-101 fed cats from the abdominal cavity to the diagnosed cats are shown in Table 10:

tables 10JWY-101 blood white ball ratio record table of sick cats after administration

The ratio of white blood balls of cats with abdominal pain syndrome is shown in FIG. 10 and FIG. 11 after subcutaneous administration of 5mg/kg and 7mg/kg JWY-101, respectively.

The cat transmits abdominal pain to diagnose the sick cat, and the appetite and the activity are all observed to be recovered within 3 days of medication; discharge standard is basically reached and discharged after one week of medication. Except that the wealth cannot be continuously tracked after treatment and discharge, other sick cats are healed after treatment (spirit and appetite are recovered to be normal, hydrothorax and abdominal cavity liquid disappear, intestinal lymph nodes are obviously reduced, and the white ball ratio is recovered to be normal). As shown in Table 10, FIG. 10 and FIG. 11, regardless of whether the sick cat is diagnosed with dry or wet infectious peritonitis, the white ball ratio of the sick cat is restored to a different degree within a short time after JWY-101 dosing treatment; the white ball ratio of the sick cat gradually returns to a healthy value above 0.6 during the course of maintenance treatment to recovery. The result shows that the subcutaneous administration JWY-101 can quickly improve the spirit and appetite of the sick cat, improve the white ball ratio and effectively cure the infectious peritonitis confirmed-diagnosed sick cat.

5. Conclusion of the experiment

The experimental result shows that under the condition of subcutaneously administrating JWY-101, JWY-101 can quickly improve the spirit and appetite of the sick cat, improve the blood white ball ratio and effectively cure the infectious peritonitis of the cat.

Claims (11)

1.A compound of formula I:

or a pharmaceutically acceptable salt;

Wherein:

R ₁ and R ₂ are independently selected from H, or C1-C6 alkyl, 3-8 membered cycloalkyl or 6-20 membered aryl, which alkyl, cycloalkyl, aryl may be further substituted with one or more independent substituents;

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

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

3. the compound according to claim 2, or a pharmaceutically acceptable salt thereof, wherein, for formula I, R ₁＝CH₃,m＝2,R₂ =h and n=0; has a structure shown in the following chemical formula:

Or for formula I: r ₁＝CH₃,m＝4,R₂ =h and n=0; has a structure shown in the following chemical formula:

4. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein, for formula I, R ₁＝R₂ and m = n; has a structure shown in the following chemical formula:

5. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein for formula I: r ₁＝R₂＝CH₃, m=n=2; the structure is shown as the following formula:

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

7. The pharmaceutical formulation of claim 7, wherein the pharmaceutical formulation is a granule, capsule, tablet, pill, injection, suspension, solution, inhalant, powder spray or spray.

8. Use of a compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prophylaxis of a viral infection in a human or animal.

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

10. The use of claim 8, wherein the viral infection is a feline FIPV viral infection.

11. A method of preparing the compound of claim 1, comprising:

(one) preparing an intermediate int-1 with the following structure:

(II) synthesizing PEGylated phosphoric acid or corresponding salt thereof with the following structure:

(III) reacting the intermediate int-1 with polyethylene glycol phosphoric acid or corresponding salt thereof to obtain the following compound:

(IV) removing the protecting group: