Application of leritegravir in preparation of anti-enterovirus drugs

Abstract

The invention relates to the technical field of medicinal chemistry, in particular to application of a compound (leritevir) shown in a formula I in preparation of an anti-enterovirus medicine. Research finds that the compound (leritevir) shown in the formula I has an efficient antiviral effect on various common and high-incidence enteroviruses such as EVA71, CVA16, CVB4 and HRV, the antiviral effect is remarkably superior to that of a control group medicine, and the market blank that no specific antiviral medicine for the enteroviruses exists at present is filled. Besides, the compound (leritevir) as shown in the formula I is a marketed anti-SARS-CoV-2 medicine, and the safety and the like of the compound are clinically verified, so that clinical application can be promoted more quickly, and the compound has the advantages of low cost and high safety.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical chemistry, and particularly relates to application of letermovir in preparation of enterovirus-resistant drugs. BACKGROUND

[0002] Enterovirus (EV) belongs to the family of small RNA viruses, is a kind of small non-enveloped single-stranded RNA virus with icosahedral structure and a diameter of about 30 nm. The traditional enterovirus can be divided into the following four types: (1) poliovirus (nearly eradicated); (2) coxsackievirus (CV), divided into group A and group B; (3) echovirus; (4) newly discovered or unable to be classified into the above three types by traditional serological methods, numbered by numbers, including enterovirus A71 type, enterovirus D68 type, etc. Human rhinovirus (HRV) is the main pathogen causing common cold (about 50% of cold cases), which was once independently classified into the genus of human rhinovirus. Modern genetic classification studies have found that the genomic structure of HRV is highly similar to that of EV, and human rhinovirus has been incorporated into the genus of enterovirus and classified into group A (HRV-A), group B (HRV-B) and group C (HRV-C).

[0003] The pathogen infecting humans mainly includes human rhinovirus group A (HRV-A), human rhinovirus group C (HRV-C), coxsackievirus group A 16 type (CVA16), group B 4 type (CVB4), enterovirus A71 type (EVA71), etc. Among them, HRV-A mainly causes upper respiratory tract infection, HRV-C mainly causes lower respiratory tract infection, CVA16 mainly causes mild hand-foot-mouth disease, CVB4 mainly causes myocarditis, meningitis, etc., and enterovirus A71 type mainly causes severe hand-foot-mouth disease and encephalitis. If the patients infected by the above pathogens do not take treatment in time, further neural system damage and myocardial damage can be caused, leading to aggravation of the disease and even death. Therefore, it is of great significance to develop enterovirus-resistant drugs.

[0004] However, so far, there is no specific antiviral drug for enterovirus infection, and general symptomatic treatment is mainly experimental drugs including ribavirin, fluoxetine, etc., but the effect is limited, and high-efficiency and safe specific drugs are urgently needed to be developed.

[0005] Leritrelvir (RAY1216) is a specific drug for treating mild to moderate SARS-CoV-2 virus infection, which has been approved for listing, and is a peptidomimetic inhibitor of 3CL protease of SARS-CoV-2. The mechanism of action is to inhibit 3CL protease, so that it cannot process the polyprotein precursor of the virus, thereby preventing viral replication. The safety of leritrelvir has been verified through clinical trials, and the development of other uses of leritrelvir can save drug development costs and speed up the clinical process. SUMMARY

[0006] In the process of studying leritrelvir, it is found that leritrelvir (structure as shown in formula I) has excellent inhibitory effect on a variety of enteroviruses, and the selection coefficient is high, which is obviously better than the positive control drug.

[0007] Based on this, the application provides a use of a compound shown in formula I in the preparation of an anti-enterovirus drug.

[0008]

[0009] Further, the enterovirus includes at least one of coxsackievirus A16, coxsackievirus B4, enterovirus A71, and human rhinovirus type 2.

[0010] Further, the compound shown in formula I or a pharmaceutically acceptable salt thereof has a drug selection coefficient SI of 192.55-85400 on a cell level for each of the enteroviruses.

[0011] Further, the compound shown in formula I or a pharmaceutically acceptable salt thereof has a selection coefficient SI of 192.55 on a cell level for the coxsackievirus A16; and / or,

[0012] a selection coefficient SI of 265.25 for the enterovirus A71; and / or,

[0013] a selection coefficient SI of 22100 for the human rhinovirus type 2; and / or,

[0014] a selection coefficient SI of 85400 for the coxsackievirus B4.

[0015] In some embodiments, the drug includes the compound shown in formula I and a pharmaceutically acceptable carrier and / or adjuvant.

[0016] Further, the dosage form of the drug includes an oral preparation or an injection preparation.

[0017] Further, the oral preparation is a solid dosage form or a liquid dosage form, and the injection preparation is a liquid dosage form.

[0018] Further, the oral preparation comprises at least one of the following: binder, lubricant, disintegrant, excipient, solubilizer, dispersant, stabilizer, suspending agent, colorant and fragrance in the carrier and / or excipient.

[0019] The injection preparation comprises at least one of the following: preservative, analgesic, solubilizer, isotonic agent and stabilizer in the carrier and / or excipient.

[0020] In some embodiments, the subject of the drug is a mammal.

[0021] Further, the subject of the drug is a human.

[0022] The present application found that the compound (Leritrelvir) shown in formula I has high efficient antiviral effect on a variety of common enteroviruses such as EVA71, CVA16, CVB4, HRV2, etc., and the antiviral effect is significantly better than that of the control drug, filling the market gap of no specific antiviral drug for enterovirus. In addition, the compound (Leritrelvir) shown in formula I used in the present application is an anti-SARS-CoV-2 drug that has been marketed, and its safety has been verified by clinical trials, so it can promote clinical application faster, with the advantages of low cost and high safety. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the description of the specific embodiments or the prior art. Obviously, the drawings described below are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0024] Figure 1 The experimental results of the anti-Coxsackie virus B4 (CVB4) of Leritrelvir of Example 1 of the present application are shown in the following table, wherein A, B are the CPE conditions of HeLa cells and virus fluorescent spot photos observed and recorded after 48h of adding different concentration gradients of control drug Fluoxetine and experimental drug Leritrelvir in the report virus rCVB4-EGFP infected HeLa cells, the scale is 100μm; C, D are the curves corresponding to the half effective concentration (EC 50 ) of the control drug and Leritrelvir against CVB4 calculated and analyzed according to the change of the number of virus fluorescent spots under different drug concentrations; E, F are the effects of different concentration gradients of Leritrelvir and control drug on cell activity detected by CCK8 method after 24h of inoculating cells, and the half cytotoxic concentration (CC 50) corresponding curves, the data is shown as mean ± SEM (n = 3).

[0025] Figure 2 The experimental results of the anti-human rhinovirus type 2 (HRV2) of Leritrelvir of Example 2 of the present application, wherein A and B are the CPE of HeLa cells after adding different concentrations of control drug Ribavirin and experimental drug Leritrelvir in HRV-infected HeLa cells, and the scale is 100 μm; C and D are the half effective concentration (EC 50 ) corresponding curves, the data is shown as mean ± SEM (n = 3); E is the effect of different concentration gradients of Ribavirin on cell activity after inoculating cells for 24 h by CCK8 method, and the half cytotoxic concentration (CC 50 ) corresponding curves, the data is shown as mean ± SEM (n = 3).

[0026] Figure 3 The experimental results of the anti-enterovirus A71 type (EVA71) of Leritrelvir of Example 3 of the present application, wherein A and B are the CPE of Vero cells after adding different concentrations of control drug Ribavirin and experimental drug Leritrelvir in EVA71-infected Vero cells, and the scale is 100 μm; C and D are the half effective concentration (EC 50 ) corresponding curves, the data is shown as mean ± SEM (n = 3); E and F are the effect of different concentration gradients of Ribavirin and Leritrelvir on cell activity after inoculating cells for 24 h by CCK8 method, and the half cytotoxic concentration (CC 50 ) in Vero cells. The data is shown as mean ± SEM (n = 3).

[0027] Figure 4 The experimental results of the anti-cosavirus A16 type (CVA16) of Leritrelvir of Example 4 of the present application, wherein A and B are the CPE of Vero cells after adding different concentrations of control drug Ribavirin and experimental drug Leritrelvir in CVA16-infected Vero, and the scale is 100 μm; C and D are the half effective concentration (EC 50) corresponding curves, data shown as mean ± SEM (n=3).

[0028] Figure 5 Figure 6 shows the results of the in vivo anti-CVB4 experiment of rintatresvir of Example 5 of the present application, in which the abscissa represents the number of days of infection and the ordinate represents the survival rate.

[0029] Figure 6 Figure 7 shows the results of the in vivo anti-EV71 experiment of rintatresvir of Example 6 of the present application, in which the abscissa represents the number of days of infection and the ordinate represents the survival rate. DETAILED DESCRIPTION

[0030] The present application is further described in connection with the following examples and embodiments. It should be understood that these examples are intended to illustrate the present application and are not intended to limit the scope of the present application. Furthermore, it should be understood that various modifications and changes can be made to the present application by those skilled in the art upon reading the contents of the present application. Such equivalent forms are intended to fall within the scope of the appended claims.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0032] Terminology

[0033] Unless otherwise indicated, or unless the context of use indicates otherwise, the terms or phrases used in this specification are intended to have the meanings presented herein. The following definitions are intended to apply uniformly unless a contrary intent is otherwise apparent from the context of use.

[0034] As used herein, the term "and / or", where used herein, is intended to encompass one or more of items listed, individually or in combination. It is noted that when at least two conjunctions selected from "and / or", "or / and", "and / or", are combined to connect at least three items, it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0035] In this document, terms such as "preferred," "better," and "more preferred" are merely descriptions of implementation methods or examples that achieve better results, and should be understood as not constituting a limitation on the scope of protection of this application.

[0036] In this application, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0037] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0038] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, optional numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0039] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0040] In this application, weight can be a well-known unit of mass in the pharmaceutical field, such as μg, mg, g, or kg.

[0041] In this application, unless otherwise specified, the terms "size", "particle size", and "diameter" generally refer to average values.

[0042] This application provides the use of the compound shown in Formula I in the preparation of an anti-enterovirus drug;

[0043]

[0044] Furthermore, the enterovirus includes at least one of Coxsackievirus A16, Coxsackievirus B4, Enterovirus A71, and Human Rhinovirus 2.

[0045] Furthermore, the selectivity SI of the compounds shown in Formula I or their pharmaceutically acceptable salts at the cellular level for each of the said enteroviruses is between 192.55 and 8.54 × 10⁻⁶. 4between.

[0046] Furthermore, the compound of Formula I or a pharmaceutically acceptable salt thereof has a selectivity SI of 192.55 for the Coxsackievirus A16 type at the cellular level; and / or,

[0047] The selectivity coefficient SI for the enterovirus A71 is 265.25; and / or,

[0048] The selectivity SI for the human rhinovirus type 2 is 22100; and / or,

[0049] The selectivity SI for the Coxsackievirus B4 type is 85400.

[0050] In some embodiments, the compound represented by Formula I or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier and / or excipient, is prepared as a formulation for use.

[0051] Furthermore, the types of the formulations include oral formulations and injectable formulations.

[0052] Furthermore, when the preparation is an oral preparation, the dosage form is a solid dosage form or a liquid dosage form; when the preparation is an injectable preparation, the dosage form is a liquid dosage form.

[0053] Furthermore, the oral formulation includes at least one of the following carriers and / or excipients: binder, lubricant, disintegrant, excipient, solubilizer, dispersant, stabilizer, suspending agent, colorant, and flavoring agent;

[0054] The injectable formulation includes at least one of the following carriers and / or excipients: preservatives, analgesics, solubilizers, isotonic agents, and stabilizers.

[0055] In some embodiments, the subject of the drug is a mammal.

[0056] Furthermore, the subjects of the drug are humans.

[0057] Application method

[0058] There are no particular limitations on the dosage form and administration method of the compound (active compound) shown in Formula I of this application.

[0059] Representative administration methods include, but are not limited to: oral, rectal, parenteral (intravenous, intramuscular, or subcutaneous) injection.

[0060] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain a buffer. Solid dosage forms such as tablets, sugar pills, capsules, pills, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound in such compositions may be delayed in a portion of the digestive tract. Examples of encapsulating components that may be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0061] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers, and emulsifiers, specifically, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. Besides these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances. For example, suspensions may contain suspending agents, specifically, for example, ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitan esters, microcrystalline cellulose, aluminum methoxide, and agar, or mixtures thereof.

[0062] Liquid dosage forms for injection include solutions or suspensions. In addition to the active compound, liquid dosage forms may contain physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous or non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0063] The following are some specific examples.

[0064] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this application document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.

[0065] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art using known methods.

[0066] Example 1

[0067] This embodiment illustrates the application of the compound represented by Formula I in the preparation of an anti-Coxsackievirus B4 (CVB4) drug.

[0068] 1.1 Experimental Procedure

[0069] 1.1.1 Cell Culture

[0070] HeLa cells were spaced at 1.5 × 10⁶ cells per well. 4 Cells were seeded at a density in 96-well plates and cultured overnight in DMEM + 10% FBS in a 37°C, 5% CO2 incubator.

[0071] 1.1.2 Infection with a virus

[0072] In the previous phase, the project team constructed a CVB4 reporter virus rCVB4-EGFP containing the reporter gene EGFP using the CVB4 clinical strain GZ-HFM01 (Genbank no. MZ540957) as the backbone (reference: Liu Q, et al. Construction and InVitro and InVivo Analysis of Coxsackievirus B4 Reporter Viruses: Attenuated Virulence but Highly Efficient for Antiviral Drug Screening and Evaluation. Journal of Medical Virology 2025, 97:e70433.). Using rCVB4-EGFP, cells were inoculated with the virus at a multiplicity of infection (MOI) of 0.01. After 2 hours, unbound viruses were removed by washing three times with PBS, and the cells were cultured in DMEM + 2% FBS at 37°C and 5% CO2.

[0073] 1.1.3 Adding drugs

[0074] Fluoxetine (Sigma-Aldrich, USA) (control drug) and leritrelvir (Zhong Sheng Rui Chuang, China) were dissolved in DMSO to prepare a 20 mM stock solution. The stock solution was serially diluted in virus culture medium, and working solutions of fluoxetine (0.625 μM–20 μM) and leritrelvir (2 nM–64 nM) were added to virus-infected cells.

[0075] 1.1.4 Observe the antiviral effect of the drug and its improvement on cell CPE.

[0076] Microscopic observation was conducted to assess the improvement of cell cytotoxicity (CPE) in virus-infected cells after treatment with different concentrations of drugs for 48 hours.

[0077] The number of EGFP fluorescent spots formed by rCVB4-EGFP in cells was observed using a fluorescence microscope. Three fields of view were photographed for each replicate well under a fluorescence microscope, and the fluorescent spots were counted using ImageJ software (National Institutes of Health, USA). The average number of spots represents the number of fluorescent spots in that well.

[0078] The median effective concentration (EC50) of the drug was calculated based on changes in the number of viral fluorescent spots. 50 Each group will conduct 3 parallel experiments.

[0079] 1.1.5 Cytotoxicity analysis of the drug

[0080] HeLa cells were spaced at 1.5 × 10⁶ cells per well. 4 Cells were seeded at a density in 96-well plates and cultured overnight at 37°C and 5% CO2. Fluoxetine working solutions were added to the cells at serial dilutions ranging from 7.8125 μM to 1000 μM and Leritrelvir working solutions at serial dilutions ranging from 56.25 μM to 3600 μM. Cell viability was assessed using the CCK-8 assay (Dojindo, Japan) after 24 hours of incubation, following the manufacturer's instructions. Absorbance at 450 nm was measured using a spectrophotometer, and the half-maximal cytotoxic concentration (MCC) of each drug was calculated. 50 Each group will conduct 3 parallel experiments.

[0081] 1.1.6 Selection Coefficient Analysis

[0082] By calculating the drug selectivity coefficient SI (SI = CC) 50 / EC 50 To evaluate the superiority of leretevir against enteroviruses.

[0083] 1.2 Experimental Results

[0084] Experimental results are as follows Figure 1 ,according to Figure 1 As can be seen, with the increase of the concentration of the control drug and Leritrelvir, the fluorescent spot signal produced by CVB4 fluorescent reporter virus decreased rapidly, both demonstrating significant antiviral ability and effectively improving cellular CPE caused by viral infection. Figure 1 (A, B); Based on the analysis of changes in viral fluorescence signals, control drugs Fluoxetine and Leritrelvir were used to evaluate the EC50 of CVB4. 50 The concentrations were 2.531 μM and 6.706 nM, respectively. Figure 1 (C, D) The effective inhibitory concentration of Leritrelvir was much lower than that of the control group.

[0085] The cytotoxicity markers CC of the control drugs Fluoxetine and Leritrelvir 50 The concentrations were 37.15 μM and 572.7 μM, respectively. Figure 1 From E and F, it can be seen that Leritrelvir is less toxic than Fluoxetine.

[0086] Analysis of the drug selectivity coefficient (SI) of leritrelvir and the control drug fluoxetine against CVB4. 50 / EC 50 The values ​​are 8.54 × 10⁻⁶. 4 The results showed that leritrelvir was significantly more selective than fluoxetine, with a selectivity of 14.68.

[0087] Example 2

[0088] This embodiment illustrates the application of the compound represented by Formula I in the preparation of an anti-human rhinovirus (HRV) drug.

[0089] 2.1 Experimental Procedure

[0090] 2.1.1 Cell Culture

[0091] HeLa cells were spaced at 1.5 × 10⁶ cells per well. 4 Cells were seeded at a density in 96-well plates and cultured overnight in DMEM + 10% FBS in a 37°C, 5% CO2 incubator.

[0092] 2.1.2 Infection with a virus

[0093] Cells were inoculated using HRV2 (ATCC VR-482) with a multiplicity of infection (MOI) of 0.01. After 2 hours, the cells were washed three times with PBS to remove unbound viruses and cultured in DMEM + 2% FBS at 37°C and 5% CO2.

[0094] 2.1.3 Adding drugs

[0095] The control drug Ribavirin (Jizhi Biochemical, China) and the experimental drug Leritrelvir (Zhong Sheng Rui Chuang, China) were dissolved in DMSO to prepare a 20 mM stock solution. The stock solution was serially diluted in virus culture medium, and working solutions of Ribavirin (12.5 μM–1600 μM) and Leritrelvir (9.5 nM–1250 nM) were added to virus-infected cells.

[0096] 2.1.4 Observe the antiviral effect of the drug and its improvement on cell CPE.

[0097] Microscopic observation was conducted to assess the improvement of cell cytotoxicity (CPE) in virus-infected cells after treatment with different concentrations of drugs for 48 hours.

[0098] The viral genome copy number change was detected using HRV real-time PCR assay kits, and the median effective concentration (EC50) of the drug was calculated accordingly. 50 Each group will conduct 3 parallel experiments.

[0099] 2.1.5 Cytotoxicity analysis of the drug

[0100] HeLa cells were spaced at 1.5 × 10⁶ cells per well. 4 Cells were seeded at a density in 96-well plates and cultured overnight at 37°C and 5% CO2. Ribavirin working solutions were added to the cells at serial dilutions between 56.25 μM and 7200 μM, and Leritrelvir working solutions were added at serial dilutions between 56.25 μM and 3600 μM. Cell viability was assessed using the CCK-8 assay (Dojindo, Japan) after 24 h of incubation, following the manufacturer's instructions. Absorbance at 450 nm was measured using a spectrophotometer, and the half-maximal cytotoxic concentration (MCC) of each drug was calculated. 50 Each group will conduct 3 parallel experiments.

[0101] 2.1.6 Selection Coefficient Analysis

[0102] By calculating the drug selectivity coefficient SI (SI = CC) 50 / EC 50 To evaluate the superiority of leretevir against enteroviruses.

[0103] 2.2 Experimental Results

[0104] Experimental results are as follows Figure 2 , Figure 2 The results showed that with increasing concentrations of the control drug Ribavirin and the experimental drug Leritrelvir, cellular cytotoxicity (CPE) induced by HRV infection was effectively improved.Figure 2 (A, B); ECG of control drug and Leritrelvir against HRV 50 The values ​​were 77.34 μM and 25.89 nM, respectively. Figure 2 (C, D) The effective inhibitory concentration of Leritrelvir was much lower than that of the control group.

[0105] The toxicity index CC of the control drug Ribavirin on HeLa cells 50 2701 μM ( Figure 2 (E).

[0106] The SI (similarity) for Leritrelvir and the control drug Ribavirin against HRV were 2.21 × 10⁻⁶. 4 At 34.92, Leritrelvir was significantly superior to Ribavirin. (Leritrelvir vs. HeLa CC) 50 See Figure 1 (F).

[0107] Example 3

[0108] This embodiment illustrates the application of the compound shown in Formula I of this application in the preparation of an anti-enterovirus A71 (EVA71) drug.

[0109] 3.1 Experimental Procedure

[0110] 3.1.1 Cell Culture

[0111] Vero cells were planted at a density of 1.5 × 10⁶ cells per well. 4 Cells were seeded at a density in 96-well plates and cultured overnight in DMEM + 10% FBS in a 37°C, 5% CO2 incubator.

[0112] 3.1.2 Viral Infection

[0113] The clinical isolate EVA71 GZ08 (Genbank no. FJ360545) was used for cell inoculation with a multiplicity of infection (MOI) of 0.01. After 2 hours, the cells were washed three times with PBS to remove unbound viruses and cultured in DMEM + 2% FBS at 37°C and 5% CO2.

[0114] 3.1.3 Adding drugs

[0115] The control drug Ribavirin (Jizhi Biochemical, China) and the experimental drug Leritrelvir (Zhong Sheng Rui Chuang, China) were dissolved in DMSO to prepare a 20 mM stock solution. The stock solution was serially diluted in virus culture medium, and Ribavirin working solutions were added to virus-infected cells using serial dilutions between 12.5 μM and 1600 μM and serial dilutions of Leritrelvir between 0.75 μM and 100 μM.

[0116] 3.1.4 Observe the antiviral effect of the drug and its improvement on cell CPE.

[0117] Microscopic observation was conducted to assess the improvement of cell cytotoxicity (CPE) in virus-infected cells after treatment with different concentrations of drugs for 48 hours.

[0118] The viral genome copy number change was detected using the EVA71 real-time PCR assay kit, and the median effective concentration (EC50) of the drug was calculated accordingly. 50 Each group will conduct 3 parallel experiments.

[0119] 3.1.5 Cytotoxicity analysis of the drug

[0120] Vero cells were planted at a density of 1.5 × 10⁶ cells per well. 4 Cells were seeded at a density in 96-well plates and cultured overnight at 37°C and 5% CO2.

[0121] Ribavirin working solutions were added to cells at serial dilutions ranging from 56.25 μM to 3600 μM and from 56.25 μM to 7200 μM, respectively. Cell viability was assessed using the CCK-8 assay (Dojindo, Japan) after 24 h of incubation, following the manufacturer's instructions. Absorbance at 450 nm was measured using a spectrophotometer, and the half-maximal cytotoxic concentration (MCC) of each drug was calculated. 50 Each group will conduct 3 parallel experiments.

[0122] 3.1.6 Selection Coefficient Analysis

[0123] By calculating the drug selectivity coefficient SI (SI = CC) 50 / EC 50 To evaluate the superiority of leretevir against enteroviruses.

[0124] 3.2 Experimental Results

[0125] Experimental results are as follows Figure 3 , Figure 3 The results showed that with increasing concentrations of the control drug Ribavirin and the experimental drug Leritrelvir, cellular cytotoxicity (CPE) induced by EVA71 infection was effectively improved. Figure 3(A, B); Control drug and Leritrelvir on ECG of EVA71 50 The values ​​were 332.2 μM and 2.466 μM, respectively. Figure 3 (C, D) The effective inhibitory concentration of Leritrelvir was much lower than that of the control group.

[0126] The toxicity markers of the control drugs Ribavirin and Leritrelvir on Vero cells (CC) 50 The values ​​were 3848 μM and 654.1 μM, respectively. Figure 3 (E, F)

[0127] The SI values ​​for Leritrelvir and the control drug Ribavirin against EVA71 were 265.25 and 11.58, respectively, with Leritrelvir being significantly superior to Ribavirin.

[0128] Example 4

[0129] This embodiment illustrates the application of the compound shown in Formula I of this application in the preparation of an anti-Coxsackievirus A16 (CVA16) drug.

[0130] 4.1 Experimental Procedure

[0131] 4.1.1 Cell Culture

[0132] Vero cells were planted at a density of 1.5 × 10⁶ cells per well. 4 Cells were seeded at a density in 96-well plates and cultured overnight in DMEM + 10% FBS in a 37°C, 5% CO2 incubator.

[0133] 4.1.2 Viral Infection

[0134] The clinical isolate CVA16 GZ08 (Genbank no. FJ198212) was used for cell inoculation with a multiplicity of infection (MOI) of 0.01. After 2 hours, the cells were washed three times with PBS to remove unbound viruses and cultured in DMEM + 2% FBS at 37°C and 5% CO2.

[0135] 4.1.3 Adding drugs

[0136] The control drug Ribavirin (Jizhi Biochemical, China) and the experimental drug Leritrelvir (Zhong Sheng Rui Chuang, China) were dissolved in DMSO to prepare a 20 mM stock solution. The stock solution was serially diluted in virus culture medium, and working solutions of Ribavirin (6.25 μM–800 μM) and Leritrelvir (1.5 μM–200 μM) were added to virus-infected cells.

[0137] 4.1.4 Observe the antiviral effect of the drug and its improvement on cell CPE.

[0138] Microscopic observation was conducted to assess the improvement of cell cytotoxicity (CPE) in virus-infected cells after treatment with different concentrations of drugs for 48 hours.

[0139] The viral genome copy number change was detected using the CVA16 real-time PCR assay kit, and the median effective concentration (EC50) of the drug was calculated accordingly. 50 Each group will conduct 3 parallel experiments.

[0140] 4.1.5 Cytotoxicity analysis of the drug

[0141] The half-cell cytotoxic concentrations (CC) of Ribavirin and Leritrelvir in Vero cells 50 See Example 3 for drug cytotoxicity analysis; the values ​​were 3848 μM and 654.1 μM, respectively. Figure 3 E and F in the middle.

[0142] 4.1.6 Selection Coefficient Analysis

[0143] By calculating the drug selectivity coefficient SI (SI = CC) 50 / EC 50 To evaluate the superiority of leretevir against enteroviruses.

[0144] 4.2 Experimental Results

[0145] Experimental results are as follows Figure 4 , Figure 4 The results showed that with increasing concentrations of the control drug Ribavirin and the experimental drug Leritrelvir, cellular chemoembolism (CPE) induced by CVA16 infection was effectively improved. Figure 4 (A, B); the EC50 values ​​of the control drug and Leritrelvir against CVA16 were 258 μM and 3.397 μM, respectively. Figure 4 (C, D). The effective inhibitory concentration of Leritrelvir was much lower than that of the control group. Since the cells used in the anti-EVA71 experiment in Example 3 and the anti-CVA16 experiment in this example were Vero cells, and the experimental drug used was Leritrelvir, the half-maximal cytotoxic concentration (CMC) of the control drugs Ribavirin and Leritrelvir on Vero cells in this example was significantly lower. 50 See Example 3. Figure 3 E and F in the middle.

[0146] The SI scores for Leritrelvir and the control drug Ribavirin against CVA16 were 192.55 and 14.91, respectively, indicating that Leritrelvir was significantly superior to Ribavirin.

[0147] The effects of the above embodiments against various enteroviruses are summarized, and the results are shown in Table 1.

[0148] Table 1. Summary of the antiviral effects of Leritrelvir on enteroviruses

[0149] Virus (host cell) Drug EC 50 ]]> CC 50 ]]> SI(CC 50 / EC 50 )]]> CVB4 (HeLa) Leritrelvir 6.706 nM 572.7 μM 8.54 x 10 4 ]] Fluoxetine 2.531 μM 37.15 μM 14.68 EVA71 (Vero) Leritrelvir 2.466 μM 654.1 μM 265.25 Ribavirin 332.2 μM 3848 μM 11.58 CVA16 (Vero) Leritrelvir 3.397 μM 654.1 μM 192.55 Ribavirin 258 μM 3848 μM 14.91 HRV (HeLa) Leritrelvir 25.89 nM 572.7 μM 2.21 x 10 4 ]]> Ribavirin 77.34 μM 2701 μM 34.92

[0150] As shown in Table 1, Leritrelvir exhibits highly effective inhibition against various common and prevalent enterovirus subtypes. The results are summarized in Table 1. The SI values ​​of Leritrelvir against each enterovirus subtype ranged from 192.55 to 85400, while the SI values ​​of the control group ranged from 11.58 to 34.92, demonstrating a significant advantage for Leritrelvir.

[0151] Example 5

[0152] This embodiment is an analysis of the in vivo anti-CVB4 effect of the compound shown in Formula I of this application.

[0153] 5.1 Experimental Procedure

[0154] 5.1.1 Animal Grouping

[0155] Two-day-old ICR suckling mice, which were used as a CVB4 in vivo infection model, were purchased from Spiefol (Beijing) Biotechnology Co., Ltd. Under specific pathogen-free (SPF) conditions, they were housed in individually ventilated cages for one day and then divided into groups (Mock group, CVB4 group, Leritrelvir group, and CVB4+Leritrelvir group) for infection experiments.

[0156] 5.1.2 Administration

[0157] (1) Mice in the CVB4 group and the CVB4+Leritrelvir group were intraperitoneally injected with CVB4 (5×10⁻⁶). 5 PFU / animal, resuspended in 20 μl PBS), infected for 2 h; the CVB4 strain used was the GZ-HFM01 strain isolated in our laboratory (Genbank no. MZ540957).

[0158] (2) Mice in the Leritrelvir group and Mock group were injected intraperitoneally with an equal volume of cell culture supernatant and waited for 2 hours.

[0159] (3) Mice in the uninfected Leritrelvir group in step (2) and mice in the infected CVB4+Leritrelvir group in step (1) were given intraperitoneal injection of leritrelvir at a dose of 12 mg / kg / day (leritrelvir was purchased from Zhong Sheng Ruichuang Biotechnology Co., Ltd., dissolved in DMSO to prepare a stock solution of 40 mg / ml, and the stock solution was diluted with PBS to the working solution, 20 μl / mouse), and were given continuously until day 5 after infection; the mortality of mice was counted and survival curves were plotted.

[0160] 5.2 Experimental Results

[0161] Experimental results are as follows Figure 5 As shown, 12 mg / kg / day of leretexvir itself did not affect the survival rate of mice. No mice in the CVB4-infected group died after administration of the drug. Leretexvir significantly improved the survival rate of mice against CVB4 in vivo (p<0.001).

[0162] Example 6

[0163] This embodiment is an analysis of the in vivo anti-EVA71 effect of the compound shown in Formula I of this application.

[0164] 6.1 Experimental Procedure

[0165] 6.1.1 Animal Grouping

[0166] Four-day-old BALB / c suckling mice with EVA71 in vivo infection model were purchased from Spiefol (Beijing) Biotechnology Co., Ltd. Under specific pathogen-free (SPF) conditions, they were housed in individually ventilated cages for one day and then divided into groups (Mock group, EVA71 group, Leritrelvir group, and EVA71+Leritrelvir group) for infection experiments.

[0167] 6.1.2 Administration

[0168] (1) Mice in the EVA71 group and the EVA71+Leritrelvir group were intraperitoneally injected with EVA71 (5×10⁻⁶). 6PFU / mouse, resuspended in 20 μl PBS, infected for 2 h; the EVA71 strain used was a mouse-adapted strain prepared in our laboratory (reference: Xu Y, et al. Clinically isolated enterovirus A71 subgenogroup C4 strain with lethalpathogenicity in 14-day-old mice and the application as an EV-A71 mouse infection model. Antiviral Research 2017, 137:67-75.).

[0169] (2) Mice in the Leritrelvir group and Mock group were injected intraperitoneally with an equal volume of cell culture supernatant and waited for 2 hours.

[0170] (3) The mice in the uninfected Leritrelvir group in step (2) and the mice in the infected EVA71+Leritrelvir group in step (1) were given intraperitoneal injection of 20 mg / kg / day of Leritrelvir (Leritrelvir was purchased from Zhong Sheng Ruichuang Biotechnology Co., Ltd., dissolved in DMSO to prepare a 40 mg / ml stock solution, and the stock solution was diluted with PBS to the working solution, 20 μl / mouse) until day 13 after infection; the mortality of mice in each group was counted and survival curves were plotted.

[0171] 6.2 Experimental Results

[0172] Experimental results are as follows Figure 6 As shown, 20 mg / kg / day of leretvir itself did not affect the survival rate of mice. No mice in the EVA71-infected group died after administration of the drug. Leretvir significantly improved the survival rate of mice by inhibiting EVA71 in vivo (p<0.0001).

[0173] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0174] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0175] The embodiments described above merely illustrate several implementation methods of this application and should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Furthermore, it should be understood that after reading the above teachings, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the protection scope of the appended claims. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. The use of a compound of Formula I in the preparation of an anti-enterovirus drug; 2. The application according to claim 1, characterized in that, The enteroviruses include at least one of Coxsackievirus A16, Coxsackievirus B4, Enterovirus A71, and Human Rhinovirus 2.

3. The application according to claim 1 or 2, characterized in that, The drug selectivity coefficients (SI) for each of the said enteroviruses at the cellular level for the compounds represented by Formula I or their pharmaceutically acceptable salts are 192.55-85400.

4. The application according to claim 3, characterized in that, The compound of Formula I or a pharmaceutically acceptable salt thereof has a selectivity SI of 192.55 for Coxsackievirus A16 at the cellular level; and / or, The selectivity coefficient SI for the enterovirus A71 is 265.25; and / or, The selectivity SI for the human rhinovirus type 2 is 22100; and / or, The selectivity SI for the Coxsackievirus B4 type is 85400.

5. The application according to claim 1 or 2, characterized in that, The drug comprises the compound represented by Formula I, as well as a pharmaceutically acceptable carrier and / or excipient.

6. The application according to claim 5, characterized in that, The dosage forms of the drug include oral formulations or injectable formulations.

7. The application according to claim 6, characterized in that, The oral formulation is in solid or liquid form, and the injectable formulation is in liquid form.

8. The application according to claim 5 or 6, characterized in that, The oral formulation includes at least one of the following carriers and / or excipients: binder, lubricant, disintegrant, excipient, solubilizer, dispersant, stabilizer, suspending agent, colorant, and flavoring agent; The injectable formulation includes at least one of the following carriers and / or excipients: preservatives, analgesics, solubilizers, isotonic agents, and stabilizers.

9. The application according to claim 1 or 2, characterized in that, The subjects of the drug are mammals.

10. The application according to claim 9, characterized in that, The subjects of the drug are humans.

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