Use of adefovir dipivoxil and its structural analogues to treat pseudorabies virus - Patents.com

Abstract

The present invention relates to the medical field, in particular to the use of adefovir dipivoxil and structural analogues thereof for treating pseudorabies virus. In particular, the present invention relates to the use of a compound of formula (I), a stereoisomer thereof, a solvate thereof, a pharmaceutically acceptable salt thereof or a solvate of a pharmaceutically acceptable salt thereof for inhibiting pseudorabies virus, treating pseudorabies virus infection or treating a disease associated with pseudorabies virus infection. [Formula 1] JPEG2023508919000006.jpg5632

Description

[Technical field]

[0001] The present invention relates to the pharmaceutical field, in particular to the use of adefovir dipivoxil and its structural analogues for treating pseudorabies virus. [Background technology]

[0002] Pseudorabies virus (PRV), also known as porcine herpesvirus type I, infectious bulbar paralysis virus, scrapie virus and Aujeszky's disease virus, is a member of the α-herpesvirus genus, whose genome consists of a DNA double strand of approximately 150 kb encoding at least 72 proteins. PRV can easily infect and cause death in a variety of mammals. Pigs are the natural host of PRV. PRV infects pigs and causes a severe infectious disease in the host, which causes huge economic losses to the pig industry worldwide. Although PRV vaccines have been widely used for a long time and outbreaks of the disease have been well controlled, in recent years pseudorabies suddenly broke out in vaccinated pig farms in China and spread throughout the country, becoming one of the biggest hazards in the pig industry in China.

[0003] Currently, there is no specific treatment for PRV on the market, so it is of great importance to develop an effective specific treatment for PRV.

[0004] Adefovir dipivoxil is the chemical name for 9-[2-[bis(pivaloyloxy)methyl]phosphinyl]methoxy]ethyl]adenine (molecular formula: C 20 H 32 N5O8P) and its structural formula is shown below:

[0005] [ka]

[0006] Adefovir dipivoxil is primarily used to treat adult patients with chronic hepatitis B who have compensated liver function and active hepatitis B virus replication and persistent elevations in serum amino acid transferases (ALT or AST) or histological evidence of active lesions in the liver. After oral absorption, adefovir dipivoxil is rapidly converted to the active structure (see structural formula below) of adefovir in the body and exerts its therapeutic effects.

[0007] [ka]

[0008] To date, the antiviral activity of adefovir dipivoxil or its structural analogues against PRV has not been reported. Summary of the Invention

[0009] The present inventors have unexpectedly discovered that adefovir dipivoxil and its structural analogues have strong antiviral activity against pseudorabies virus (PRV), and provide the following invention.

[0010] In one aspect, the present application provides a compound of formula (I):

[0011] [ka]

[0012] wherein R1 and R2 are each independently selected from H, C1-C4 alkyl, haloC1-C4 alkyl, -CHOCO-(C1-C4 alkyl) and -CHOCO-(haloC1-C4 alkyl), and wherein R1 and R2 are the same or different; or a stereoisomer, solvate or pharma- ceutically acceptable salt of the compound; or a solvate of a pharma-ceutically acceptable salt of the compound.

[0013] In some embodiments, the C1-C4 alkyl can be independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, and isobutyl.

[0014] In some embodiments, the halo C1-C4 alkyl can be independently selected from fluoro C1-C4 alkyl (eg, trifluoromethyl, trifluoroethyl, etc.), chloro C1-C4 alkyl, bromo C1-C4 alkyl, and iodo C1-C4 alkyl.

[0015] In some embodiments, one of R1 and R2 is H.

[0016] In some embodiments, one of R1 and R2 is -CH2OCO-(C1-C4 alkyl), such as -CH2OCO-tert-butyl and -CH2OCO-isopropyl.

[0017] In some embodiments, one of R1 and R2 is -CH2OCO-(haloC1-C4 alkyl), such as -CH2OCO-trifluoromethyl.

[0018] In some embodiments, both R1 and R2 are H.

[0019] In some embodiments, both R1 and R2 are -CH2OCO-(C1-C4 alkyl), such as -CH2OCO-tert-butyl and -CH2OCO-isopropyl.

[0020] In some embodiments, both R1 and R2 are -CH2OCO-(haloC1-C4 alkyl), such as -CH2OCO-trifluoromethyl.

[0021] In some embodiments, the compound is adefovir or adefovir dipivoxil.

[0022] In some embodiments, the pharma- ceutically acceptable salt of the compound is selected from hydrochloride, aspartate, taurate, gluconate, fructonate, salicylate, and maleate, hi some embodiments, the pharma- ceutically acceptable salt of the compound is adefovir dipivoxil hydrochloride.

[0023] In some embodiments, the solvate is a hydrate. In some embodiments, the solvate of the compound is adefovir dipivoxil hydrate.

[0024] In some embodiments, the solvate of the pharma- ceutically acceptable salt of the compound is a hydrate of the hydrochloride salt.In some embodiments, the solvate of the pharma- ceutically acceptable salt of the compound is a hydrate of adefovir dipivoxil hydrochloride.

[0025] In one aspect, the application relates to the use of a pharmaceutical composition in the preparation of a medicament for treating pseudorabies virus infection or a disease associated with pseudorabies virus infection, the pharmaceutical composition comprising a compound of formula (I), or a stereoisomer, solvate or pharmaceutically acceptable salt of the compound, or a solvate of a pharmaceutically acceptable salt of the compound, and one or more pharmaceutically acceptable carriers or excipients.

[0026] Pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human albumin, buffer substances such as phosphates, glycerol, sorbic acid, potassium sorbate, some mixtures of glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycols, sodium carboxymethylcellulose, polyacrylates, beeswax and lanolin.

[0027] An excipient refers to an additive other than the main drug in a pharmaceutical preparation. The excipient has stable properties, is not incompatible with the main drug, does not cause side effects, does not affect the therapeutic effect, and is not easily deformed, cracked, moldy, or eaten by insects at room temperature. The excipient is harmless, has no physiological effect on animals and humans, does not react chemically or physically with the main drug, and does not affect the determination of the content of the main drug. The excipient can be a binder, a filler, a disintegrant, and a lubricant in a tablet, and a preservative, an antioxidant, a flavoring, a fragrance, a cosolvent, an emulsifier, a solubilizer, an osmotic regulator, a coloring agent, etc. in an oral liquid preparation.

[0028] The pharmaceutical composition of the present invention can be formed into any pharma- ceutical acceptable dosage form, such as tablets, capsules, pills, oral liquids, granules, powders, or injections, etc. The pharmaceutical composition of the present invention can be administered orally, by injection, implantation, external application, spraying, or inhalation.

[0029] The compounds of the invention, their stereoisomers, solvates or pharma- ceutically acceptable salts, or solvates of their pharma- ceutically acceptable salts, when used to treat pseudorabies virus infections, can be used as anti-infective therapeutic agents, optionally in combination with another therapeutic agent, and thus in some embodiments the pharmaceutical composition further comprises a second therapeutic agent.

[0030] In yet another aspect, the application provides the use of a compound of formula (I), a stereoisomer, solvate or pharma- ceutically acceptable salt of the compound, or a solvate of a pharma- ceutically acceptable salt of the compound, in the preparation of a medicament for use in the treatment of pseudorabies virus infection or a disease associated with pseudorabies virus infection, in combination with a second therapeutic agent.

[0031] In some embodiments, the second therapeutic agent for use in the present invention is selected from interferon drugs, broad-spectrum antiviral drugs, other herpes virus treatments, and anti-inflammatory drugs.

[0032] In some embodiments, interferon drugs include, but are not limited to, alpha interferon, beta interferon, gamma interferon, and the like.

[0033] In some embodiments, broad spectrum antivirals include, but are not limited to, ribavirin.

[0034] In some embodiments, other herpes virus treatments include, but are not limited to, acyclovir, ganciclovir, valacyclovir, and the like.

[0035] In some embodiments, anti-inflammatory drugs include, but are not limited to, ibuprofen, aspirin, indomethacin, and the like.

[0036] In some embodiments of the combination dosing, the compound, a stereoisomer, solvate or pharma- ceutically acceptable salt of the compound, or a solvate of a pharma- ceutically acceptable salt of the compound and the second therapeutic agent can be in the same formulation unit or in different formulation units.

[0037] In some embodiments of the combined administration, the compound, a stereoisomer, solvate or pharma- ceutically acceptable salt of the compound, or a solvate of a pharma- ceutically acceptable salt of the compound and a second therapeutic agent can be administered simultaneously or separately to a subject in need of treatment. The compound, a stereoisomer, solvate or pharma- ceutically acceptable salt of the compound, or a solvate of a pharma- ceutically acceptable salt of the compound can be administered first, and then after a certain period of time, the second therapeutic agent can be administered. Alternatively, the second therapeutic agent can be administered first, and then after a certain period of time, the compound, a stereoisomer, solvate or pharma- ceutically acceptable salt of the compound, or a solvate of a pharma- ceutically acceptable salt of the compound can be administered.

[0038] In one aspect, the present application provides a compound of formula (I), a stereoisomer, solvate or pharma- ceutically acceptable salt of the compound, or a solvate of the pharma- ceutically acceptable salt of the compound, for use in the treatment of pseudorabies virus infection or a disease associated with pseudorabies virus infection.

[0039] In one aspect, the application provides a method of treating pseudorabies virus infection or a disease associated with pseudorabies virus infection, comprising administering to a subject in need of treatment a therapeutically effective dose of a compound of formula (I), a stereoisomer, solvate or a pharmaceutically acceptable salt of said compound, or a solvate of a pharmaceutically acceptable salt of said compound, or a pharmaceutical composition as described above.

[0040] In some embodiments, the subject is a mammal, including but not limited to, pigs, cows, dogs, mice, rabbits, sheep, and humans. In some embodiments, the subject is a domestic or wild animal (such as, for example, cows, sheep, pigs, dogs, cats, and raccoons), or a laboratory animal (such as, for example, rabbits, guinea pigs, and mice), or a human.

[0041] In the present invention, diseases associated with pseudorabies virus infection include, but are not limited to, pseudorabies (e.g., pseudorabies occurring in animals such as pigs, cattle and sheep), and encephalitis, retinitis or endophthalmitis associated with pseudorabies virus infection.

[0042] The present application provides a method of inhibiting pseudorabies virus (e.g., inhibiting the replication or growth of pseudorabies virus), comprising providing to the pseudorabies virus a compound of formula (I), a stereoisomer, solvate or a pharma- ceutically acceptable salt of the compound, or a solvate of a pharma- ceutically acceptable salt of the compound. In some embodiments, the pseudorabies virus is present in a subject and the method is performed in vivo. In some embodiments, the pseudorabies virus is present in vitro and the method is performed in vitro. The method may be used for therapeutic or non-therapeutic purposes (e.g., scientific research).

[0043] In some embodiments, the method is performed in vitro and uses a compound, a stereoisomer, a solvate or a pharma- ceutically acceptable salt of the compound, or a solvate of a pharma- ceutically acceptable salt of the compound at a concentration of 0.05 μmol / L to 5 μmol / L (e.g., 5 μmol / L, 2.5 μmol / L, 1.25 μmol / L, 0.625 μmol / L, 0.313 μmol / L, 0.156 μmol / L, or 0.078 μmol / L).

[0044] The present application further provides the use of a compound of formula (I), or a stereoisomer, solvate or pharma- ceutically acceptable salt of the compound, or a solvate of a pharma- ceutically acceptable salt of the compound, for the preparation of a formulation that inhibits pseudorabies virus (e.g., inhibits the replication or proliferation of pseudorabies virus).

[0045] The application further provides a compound of formula (I), or a stereoisomer, solvate or pharma- ceutically acceptable salt of the compound, or a solvate of a pharma- ceutically acceptable salt of the compound, for use in inhibiting pseudorabies virus (e.g., inhibiting the replication or proliferation of pseudorabies virus).

[0046] Definition of Terms In this application, unless otherwise specified, the scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art, but for a better understanding of the present invention, the definitions and explanations of some relevant terms are provided below. In addition, if the definitions and explanations of terms provided in this application conflict with the meanings that are commonly understood by those skilled in the art, the definitions and explanations of terms provided in this application shall prevail.

[0047] In the present application, "C1-C4 alkyl" refers to straight-chain or branched alkyl having 1 to 4 carbon atoms, and includes, for example, "C1-C3 alkyl", "C1-C2 alkyl", etc., such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, and isobutyl.

[0048] In this application, "halo" means substitution with one or more (eg, 1, 2, 3, or 4) halogen atoms (eg, fluorine, chlorine, bromine, or iodine atoms).

[0049] In this application, "halo C1-C4 alkyl" refers to a group derived by replacing one or more hydrogen atoms on a C1-C4 alkyl with one or more halogen atoms. "Halogen atom" and "C1-C4 alkyl" are as defined above.

[0050] In this application, the "stereoisomerism" of a compound is divided into conformational isomerism and configurational isomerism, which is further divided into cis-trans isomerism and optical isomerism. Configurational isomerism refers to a stereoisomerism in which an atom or group of atoms in an organic molecule with a certain configuration has various arrangements in space due to rotation or twisting of a carbon-carbon single bond. Common configurational isomerism includes alkane and cycloalkane structures, such as the chair and boat shapes of cyclohexane. "Stereoisomer" means that when a compound of the present invention contains one or more asymmetric centers, the compound may be a racemate, a racemic mixture, a single enantiomer, a diastereomeric mixture, or a single diastereomer. The compounds of the present invention may have asymmetric centers, which each independently give rise to two optical isomers, and all possible optical isomers, diastereomeric mixtures, and pure or partially pure compounds are included within the scope of the present invention. The compounds of the present invention may exist in tautomeric forms in which the position of hydrogen is bonded differs due to the rearrangement of one or more double bonds. For example, a ketone and its enol form are keto-enol tautomers. Each tautomer and mixtures thereof are included in the compounds of the present invention. All enantiomers, diastereomers, racemates, cis-trans isomers, tautomers, geometric isomers and epimers of the compounds of formula (I), and mixtures thereof, are included within the scope of the present invention.

[0051] In this application, "solvate" refers to a substance formed by association of a compound of the present invention or a salt thereof with a solvent molecule. The solvent can be an organic solvent (e.g., methanol, ethanol, propanol, acetonitrile, etc.), water, etc. For example, the compound of the present invention or a salt thereof can form a hydrate with water.

[0052] In this application, a "pharmaceutically acceptable salt" of a compound includes a salt of an acidic functional group present in the compound with a suitable inorganic or organic cation (base), including salts of the acidic functional group with alkali metals or alkaline earth metals, ammonium salts, and salts of the acidic functional group with nitrogen-containing organic bases. "Pharmaceutically acceptable salt" also includes salts of basic functional groups present in the compounds (e.g., purine groups, etc.) with suitable inorganic or organic anions (acids), including salts of basic functional groups with inorganic or organic acids, such as salts of basic functional groups with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, perchloric acid, fumaric acid, acetic acid, propionic acid, succinic acid, glycolic acid, formic acid, lactic acid, maleic acid, tartaric acid, citric acid, pamoic acid, malonic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, hydroxynaphthoic acid, hydroiodic acid, aspartic acid, fructonic acid, gluconic acid, tauric acid, malic acid, tannic acid, and the like.

[0053] In this application, "effective dosage" refers to a dosage sufficient to achieve or at least partially achieve a desired effect. For example, a therapeutically effective amount refers to a dosage sufficient to treat or at least partially halt a disease and its complications in a patient already suffering from the disease. Determination of such effective dosages is well within the capabilities of one of ordinary skill in the art. For example, a dosage effective for therapeutic use will depend on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's overall condition, such as age, weight, and sex, the method of administration of the drug, other treatments being administered at the same time, and the like.

[0054] The dosage of the compound of formula (I), its stereoisomer, solvate or pharma- ceutically acceptable salt, or solvate of its pharma- ceutically acceptable salt, or the second therapeutic agent depends on the type and severity of the disease or condition, and the characteristics of the subject, such as general health, age, sex, body weight, and tolerance to the drug. The dosage also depends on factors such as the duration or time interval of administration, as well as the type of formulation and the method of administration of the drug. Those skilled in the art will be able to determine the appropriate dosage based on these and other factors. Typically, the daily dosage of the compound of formula (I), its stereoisomer, solvate or pharma- ceutically acceptable salt, or solvate of its pharma- ceutically acceptable salt, or the second therapeutic agent for treatment can be about 0.0001 mg / kg (body weight) / day to 1000 mg / kg (body weight) / day, and the daily dosage can be administered in one or more doses as needed. Effect of the Invention

[0055] Adefovir dipivoxil and its structural analogs have strong therapeutic activity against PRV infection and can be used as a therapeutic agent for PRV. In addition, adefovir dipivoxil and its structural analogs have the advantages of being easily accepted by patients, low-cost, and easy to obtain. Adefovir dipivoxil and its structural analogs can be taken for a long period of time, can effectively inhibit pseudorabies virus infection and pseudorabies virus epidemic, and can be expected to become a class of clinical drugs that can treat pseudorabies virus infection. [Brief description of the drawings]

[0056] [Figure 1] FIG. 1 shows the inhibitory effect of adefovir dipivoxil on pseudorabies virus replication in Example 1. [Diagram 2] 1 is a graph showing the results of an EC50 assay for the inhibition of adefovir dipivoxil against pseudorabies virus in Example 1. [Diagram 3]1 is a graph showing in Example 1 that adefovir dipivoxil has no inhibitory activity against EB virus. [Figure 4] 1 is a graph showing the inhibitory effect of adefovir dipivoxil on the release of pseudorabies virus from cells in Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] Hereinafter, the technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the drawings of the embodiments of the present invention. It is clear that the embodiments described in this specification are only a part, and are not all of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention fall within the scope of the present invention.

[0058] In the following examples, the test materials used and their suppliers are as follows:

[0059] Adefovir dipivoxil was purchased from MCE. High glucose DMEM medium, fetal bovine serum FBS (Gibco) and 96-well plates (Nunc brand) were purchased from Thermo Company.

[0060] In the following Examples 1 and 2, adefovir dipivoxil was diluted in culture medium to a stock solution at a concentration of 10 mmol / L, and then diluted with culture medium during use to use various gradient concentrations.

[0061] The cell line chosen in the following examples is porcine kidney cell PK-15 (culture medium is high glucose DMEM medium + 10% FBS, CCL-33 purchased from ATCC™).

[0062] Example 1 Inhibitory effect of adefovir dipivoxil on pseudorabies virus replication The experimental method was immunofluorescence. The principle of this method is to first label a known antigen or antibody with a fluorescent group according to the principle of antigen-antibody reaction, and then use the fluorescent antibody (or antigen) as a probe to examine the corresponding antigen (or antibody) in cells or tissues. A fluorescent microscope can be used to visualize the cells or tissues where the fluorescence is located, to determine the nature and localization of the antigen or antibody, and a quantitative method such as flow cytometry can be used to determine its amount.

[0063] The specific experimental steps were as follows: Cells with a density of about 90%-95% were digested with pre-heated trypsin, then resuspended in complete medium, and thoroughly pipetted to obtain a single cell suspension, after which counting was performed. A 12-well plate for cell culture was prepared, and first, a few drops of culture medium were added to each well at the position where the slide was to be placed according to the size of the slide. Then, the slide was placed on the drop and pressed firmly so that the slide and the culture dish would adhere to each other by the tension of the medium, so that the slide would not float when the cell suspension was added, and no double-layer cell patches would occur. Cells with an appropriate density were seeded in the 12-well plate. After 24 or 48 hours, the cell density was observed and determined according to the rate of cell proliferation. After the 12-well plate was filled with cells, various concentrations of adefovir dipivoxil and 100 pfu of pseudorabies virus were added in sequence, and then immunofluorescence detection was performed 24 hours after infection. During cell fixation, immunofluorescence and medium removal, PBS was usually added and the cells were washed three times for 5 min each time. For cell fixation, 1 ml of 4% paraformaldehyde was added to the wells and the cells were fixed for 20 min at room temperature. Paraformaldehyde was removed by absorption and rinsed three times with PBS for 5 min each time. To permeabilize the cells, 0.5% Triton X-100 (prepared in PBS) was added to the wells and permeabilized for 20 min at room temperature. Then, Triton X-100 was removed and rinsed three times with PBS for 5 min each time. Blocking with 10% goat serum (prepared in PBS) or 5% BSA homologous to the secondary antibody was performed for 2 h (the blocking solution selected was the same as the antibody dilution used in the subsequent procedures). Rinsing with PBS was not necessary after blocking. The blocking solution was removed by absorption, and a sufficient amount of primary antibody with appropriate concentration was dropped into each well (the antibody was initially used at the recommended concentration according to the antibody's instruction manual, and the appropriate concentration of the antibody could be examined for the subsequent experimental steps), followed by overnight incubation in a humidified box at 4°C. The primary antibody was removed by absorption, and rinsed with PBS three times for 5 minutes each time. A sufficient amount of secondary antibody with appropriate concentration was dropped into the well at 37°C, followed by 1 hour of incubation at room temperature in the dark.It should be noted that the operation should be performed in the dark because the secondary antibody was labeled with fluorescein. The secondary antibody was removed by absorption and rinsed with PBS three times for 5 min each. Nuclei were counterstained with blue fluorescence, usually by adding a drop of DAPI to the glass slide or using Hoechst, and then incubated in the dark for 5 to 10 min. The cells were gently rinsed with PBS three times for 5 min each to remove excess DAPI. Since the cell slide was tightly connected to the bottom of the petri dish and had a large tension, to take the cell slide, the needle tip of the syringe was bent backwards to make a small hook, and the cell slide could be gently hooked and taken out with small tweezers. The liquid on the cell slide was dried with absorbent paper, and then the cell slide was blocked with a blocking solution containing a non-fluorescent quencher (it should be noted that the cell slide needs to be mounted inversely on the polylysine slide). Then, observation and image acquisition were performed under a fluorescent microscope. It should be noted that the excitation light source corresponding to the antibody should be selected.

[0064] The inhibitory effect of adefovir dipivoxil on pseudorabies virus replication is shown in Figure 1. The concentrations of adefovir dipivoxil used were 5μmol / L, 2.5μmol / L, 1.25μmol / L, 0.625μmol / L, 0.313μmol / L, 0.156μmol / L and 0.078μmol / L. As can be seen from Figure 1, a high concentration of adefovir dipivoxil can significantly inhibit the replication and proliferation of pseudorabies virus, which confirms that adefovir dipivoxil has a significant proliferation inhibitory effect on pseudorabies virus.

[0065] EC for inhibition of adefovir dipivoxil against pseudorabies virus 50 The results of the assay are shown in Figure 2. The concentrations of adefovir dipivoxil used were 5 μmol / L, 2.5 μmol / L, 1.25 μmol / L, 0.625 μmol / L, 0.313 μmol / L, 0.156 μmol / L and 0.078 μmol / L. As can be seen from Figure 2, the EC50 reaches nanomolar levels, confirming that adefovir dipivoxil has good inhibitory effect on pseudorabies virus.

[0066] For comparison, the inhibitory activity of adefovir dipivoxil against EBV replication was tested, and the results are shown in Figure 3. The concentrations of adefovir dipivoxil used were 5 μmol / L, 2.5 μmol / L, 1.25 μmol / L, 0.625 μmol / L, 0.313 μmol / L, 0.156 μmol / L, and 0.078 μmol / L. As can be seen from Figure 3, adefovir dipivoxil has no inhibitory activity against EBV (EBV is also a herpes virus).

[0067] Example 2 Inhibitory effect of adefovir dipivoxil on the release of pseudorabies virus from cells The method used was virus plaque titration. According to the principle of this method, the virus solution of each dilution is inoculated into a monolayer cell culture environment, and after 2 hours of adsorption, the monolayer cells are covered with agarose, and the virus is allowed to infect the cells and grow inside the cells. This causes the cells to be destroyed and killed. Due to the limitations of the solid medium, the released virus can only spread around the initially infected cells. After several growth cycles, a localized diseased cell area, that is, a virus plaque, is formed. After staining with the dye, the live cells turn red, but the cells in the plaque area are not stained, forming an unstained area.

[0068] The corresponding virus-host cells were prepared, then homogenously digested, and then the cell concentration was adjusted. The host cells were inoculated into 6-well plates at appropriate concentrations. After the cells grew into a monolayer, the culture medium was removed by absorption. The virus solution was prepared and diluted 10-fold gradient to obtain five virus solutions with different concentrations. An appropriate amount of virus solution was added dropwise to each well, then absorbed at 37°C for 2 hours, and the excess virus solution was removed. A low-melting 2% agarose solution was prepared and placed in a water bath at a temperature of 40°C to 50°C for later use. The above-mentioned agarose and 2× cell maintenance solution were mixed in a 1:1 ratio. The mixture was then added to the wells at a dosage of 2mL / well, and then cooled and solidified into a coating layer. The culture plate was placed upside down in a carbon dioxide incubator at 37°C. After 48 hours, the cells were fixed with 10% formaldehyde for 30 minutes. The coating gel was then removed, and staining with 1% crystal violet was performed for 15 minutes. After removing the dye solution, viral plaques were counted.

[0069] Various concentrations of adefovir dipivoxil and 100 pfu of pseudorabies virus were added to PK15 cells. After 24 hours of culture, the culture supernatant of PK15 cells was collected for virus plaque titration test. The results are shown in Figure 4. The concentrations of the drugs used were 5 μmol / L, 2.5 μmol / L, 1.25 μmol / L, 0.625 μmol / L, 0.313 μmol / L, 0.156 μmol / L and 0.078 μmol / L. As can be seen from Figure 4, adefovir dipivoxil can significantly inhibit the release of pseudorabies virus from cells.

[0070] The above-mentioned embodiments are merely preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. This specification discloses the following inventions. (1) In the preparation of a medicament for treating a pseudorabies virus infection or a disease associated with a pseudorabies virus infection, [ka] (In the formula, R 1 and R 2 are each independently H, C 1 ~C 4 Alkyl, haloC 1 ~C 4 Alkyl, -CH 2 OCO-(C 1 ~C 4 Alkyl) and -CH 2 OCO-(HaroC 1 ~C 4 alkyl), where R 1 and R 2 is the same as or different from Preferably, said C 1 ~C 4 alkyl is independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, and isobutyl; Preferably, the halo C 1 ~C 4 The alkyl is independently fluoroC 1 ~C 4 Alkyl, Chloro C 1 ~C 4 Alkyl, bromo C 1 ~C 4 Alkyl and iodoalkyl C 1 ~C 4 alkyl, Preferably, R 1 and R 2 One of them is H, Preferably, R 1 and R 2 One of them is -CH 2 OCO-(C 1 ~C 4 alkyl), Preferably, R 1 and R 2 One of them is -CH 2 OCO-(HaroC 1 ~C 4 alkyl), Preferably, R 1 and R 2 are both H, Preferably, R 1 and R 2 Both -CH 2 OCO-(C 1 ~C 4 alkyl), Preferably, R 1 and R 2 Both -CH 2 OCO-(HaroC 1 ~C 4 5. The use of a compound of formula (I) (I is an alkyl group), a stereoisomer, solvate or a pharma- ceutically acceptable salt of said compound, or a solvate of a pharma- ceutically acceptable salt of said compound, preferably wherein said compound is adefovir or adefovir dipivoxil. (2) the pharma- ceutically acceptable salt is selected from hydrochloride, aspartate, taurate, gluconate, fructonate, salicylate and maleate; Preferably, the pharma- ceutically acceptable salt of the compound is adefovir dipivoxil hydrochloride. (3) the solvate is a hydrate, Preferably, the solvate of the compound is adefovir dipivoxil hydrate; Preferably, the solvate of the pharma- ceutically acceptable salt of the compound is a hydrate of the hydrochloride salt; Preferably, the solvate of the pharma- ceutically acceptable salt of the compound is a hydrate of adefovir dipivoxil hydrochloride. (4) Use of a pharmaceutical composition in the preparation of a medicament for treating pseudorabies virus infection or a disease associated with pseudorabies virus infection, the pharmaceutical composition comprising the compound according to any one of (1) to (3), a stereoisomer, solvate or pharmaceutically acceptable salt of the compound, or a solvate of the pharmaceutically acceptable salt of the compound, and one or more pharmaceutically acceptable carriers or excipients; Preferably, the pharmaceutical composition is in the form of a tablet, a capsule, a pill, an oral liquid, a granule, a powder, or an injection; Preferably, the pharmaceutical composition is administered orally, injected, implanted, applied topically, sprayed or inhaled. (5) the pharmaceutical composition further comprises a second therapeutic agent; Preferably, the second therapeutic agent is selected from interferon drugs such as alpha interferon, beta interferon and gamma interferon, broad spectrum antiviral drugs such as ribavirin, other herpes virus treatment drugs such as acyclovir, ganciclovir and valacyclovir, anti-inflammatory drugs such as ibuprofen, aspirin and indomethacin; Preferably, the compound, a stereoisomer, a solvate or a pharma- ceutically acceptable salt of the compound, or a solvate of a pharma- ceutically acceptable salt of the compound and the second therapeutic agent are in the same formulation unit or in different formulation units. The use according to (4). (6) Use of a compound according to any one of (1) to (3), a stereoisomer, a solvate or a pharma- ceutically acceptable salt of the compound, or a solvate of the pharma- ceutically acceptable salt of the compound, in the preparation of a medicament for use in the treatment of a pseudorabies virus infection or a disease associated with a pseudorabies virus infection, in combination with a second therapeutic agent, Preferably, the second therapeutic agent is selected from interferon drugs such as alpha interferon, beta interferon and gamma interferon, broad spectrum antiviral drugs such as ribavirin, other herpes virus treatment drugs such as acyclovir, ganciclovir and valacyclovir, anti-inflammatory drugs such as ibuprofen, aspirin and indomethacin; Preferably, the compound, a stereoisomer, a solvate or a pharma- ceutically acceptable salt of the compound, or a solvate of a pharma- ceutically acceptable salt of the compound and the second therapeutic agent are in the same formulation unit or in different formulation units. (7) A method for inhibiting pseudorabies virus, comprising administering to a pseudorabies virus a compound according to any one of (1) to (3), a stereoisomer, a solvate or a pharmaceutically acceptable salt of the compound, or a solvate of the pharmaceutically acceptable salt of the compound; A method performed in vitro and for non-therapeutic purposes. (8) Use of the compound according to any one of (1) to (3), a stereoisomer, a solvate or a pharma- ceutically acceptable salt of the compound, or a solvate of the pharma-ceutically acceptable salt of the compound, for preparing a formulation that inhibits pseudorabies virus.

Claims

1. 2. Use of a compound, a stereoisomer, solvate or a pharma- ceutically acceptable salt of the compound, or a solvate of a pharma- ceutically acceptable salt of the compound, in the preparation of a medicament for treating a pseudorabies virus infection or a disease associated with a pseudorabies virus infection, wherein the compound is adefovir or adefovir dipivoxil, and the adefovir has the chemical structure: 【Chemistry 1】 The adefovir dipivoxil has the following chemical structure: 【Chemistry 2】 use.

2. The use according to claim 1, wherein the pharma- ceutically acceptable salt is selected from hydrochloride, aspartate, taurate, gluconate, fructonate, salicylate and maleate.

3. The use described in claim 1, wherein the pharma- ceutically acceptable salt of the compound is adefovir dipivoxil hydrochloride.

4. The use according to claim 1 , wherein the solvate is a hydrate.

5. The use described in claim 1, wherein the solvate of the compound is adefovir dipivoxil hydrate.

6. The use according to claim 1, wherein the solvate of a pharma- ceutically acceptable salt of the compound is a hydrate of the hydrochloride salt.

7. The use described in claim 1, wherein the solvate of a pharma- ceutically acceptable salt of the compound is a hydrate of adefovir dipivoxil hydrochloride.

8. 8. Use of a pharmaceutical composition in the preparation of a medicament for treating pseudorabies virus infection or a disease associated with pseudorabies virus infection, the pharmaceutical composition comprising a compound as defined in any one of claims 1 to 7, a stereoisomer, solvate or pharma- ceutically acceptable salt of the compound, or a solvate of a pharma-ceutically acceptable salt of the compound, and one or more pharma-ceutically acceptable carriers or excipients.

9. The use according to claim 8, wherein the pharmaceutical composition is formulated into a dosage form of a tablet, capsule, pill, oral liquid, granule, powder or injection.

10. The use of claim 8, wherein the pharmaceutical composition is administered orally, injected, implanted, applied topically, sprayed or inhaled.

11. The use of claim 8, wherein the pharmaceutical composition further comprises a second therapeutic agent.

12. The use described in claim 11, wherein the compound, a stereoisomer, solvate or pharma- ceutically acceptable salt of the compound, or a solvate of a pharma- ceutically acceptable salt of the compound and the second therapeutic agent are in the same formulation unit or in different formulation units.

13. 10. Use of a compound, a stereoisomer, solvate or pharma- ceutically acceptable salt of said compound, or a solvate of a pharma- ceutically acceptable salt of said compound, as defined in any one of claims 1 to 7, in the preparation of a medicament for use in the treatment of pseudorabies virus infection or a disease associated with pseudorabies virus infection, in combination with a second therapeutic agent.

14. The use of claim 13, wherein the second therapeutic agent is selected from interferon drugs, broad-spectrum antiviral drugs, other herpes virus treatment drugs, and anti-inflammatory drugs.

15. The use of claim 14, wherein the interferon drug is selected from alpha interferon, beta interferon and gamma interferon, the broad-spectrum antiviral drug is ribavirin, the other herpes virus treatment drug is selected from acyclovir, ganciclovir and valacyclovir, and the anti-inflammatory drug is selected from ibuprofen, aspirin and indomethacin.

16. The use of claim 13, wherein the compound, a stereoisomer, solvate or pharma- ceutically acceptable salt of the compound, or a solvate of a pharma- ceutically acceptable salt of the compound and the second therapeutic agent are in the same formulation unit or in different formulation units.

17. A method for inhibiting the replication or proliferation of pseudorabies virus, comprising administering to the pseudorabies virus a compound as defined in any one of claims 1 to 7, a stereoisomer, solvate or pharma- ceutically acceptable salt of said compound, or a solvate of a pharma-ceutically acceptable salt of said compound, A method performed in vitro and for non-therapeutic purposes.

18. Use of a compound as defined in any one of claims 1 to 7, a stereoisomer, solvate or a pharma- ceutically acceptable salt of said compound, or a solvate of a pharma-ceutically acceptable salt of said compound, for the preparation of a formulation for inhibiting the replication or proliferation of pseudorabies virus.

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