Use of Compounds for the Treatment of Viral Infections

Abstract

The present invention relates to the use of compounds of formula I for the inhibition of cellular V-ATPase activity and to methods of treating viral infections using compounds of formula I. [Formula 1] TIFF2023525103000036.tif84135

Description

[Technical Field]

[0001] The present invention relates to the use of compounds for the inhibition of cellular V-ATPase activity, in particular as inhibitors of viral V-ATPase activity, such as SARS-CoV-2 or influenza virus, for the treatment of viral infections. [Background technology]

[0002] The SARS-CoV-2 virus and COVID-19 disease have wreaked havoc worldwide, regardless of cultural, demographic, technological and religious differences.

[0003] Logical solutions such as computational and experimental drug repurposing or demonstration of second use are useful in developing treatments for COVID-19. Existing drugs can provide a starting point and significantly shorten the time required for clinical development. The literature is replete with repurposing strategies that target various events in the viral life cycle, starting with viral entry and ending with the release of mature virus particles. The use of hydroxychloroquine, chloroquine, and other antivirals as potential treatment options for COVID-19 provides evidence of the potential of this approach.

[0004] One such promising target is vacuolar ATPase (V-ATPase), inferred from the antiviral efficacy of V-ATPase inhibitors such as bafilomycin. V-ATPase has been proposed as a promising target for blocking viral entry into host cells. V-ATPase is a ubiquitous proton pump located in the endomembrane system of all eukaryotic cells, i.e., the endoplasmic reticulum (ER), Golgi apparatus, etc. Influenza virus, flavivirus, vaccinia virus, bornavirus, rhabdovirus, and coronavirus utilize V-ATPase-mediated endosomal acidification as a critical cellular process for entry into host cells.

[0005] Because V-ATPase is a host protein, V-ATPase inhibitors may be a potential intervention in viral entry that is less susceptible to the development of drug resistance.

[0006] Several V-ATPase inhibitors, such as bafilomycin (the first and most notable example), have been tested for their antiviral potential. Despite their potent antiviral efficacy, toxicity has been a major obstacle to their clinical application. In addition, the poor water solubility of V-ATPase inhibitors poses challenges for drug delivery therapy.

[0007] Given the important role of V-ATPase in coronaviruses, e.g., SARS-CoV-2 infection, and the application of its inhibitors to combat COVID-19, there is a need to develop potent and effective V-ATPase inhibitors as potential therapies to target coronavirus infections. Summary of the Invention

[0008] In one embodiment, the invention relates to the use of a compound of formula I for the inhibition of cellular V-ATPase activity. [ka] During the ceremony, E is selected from C and N; Q is O or -NH; n is 0 to 6, R 1 and R 2 is selected from -H and -C(O)O-alkyl such as -C(O)OC2H5, or R 1 and R 2 combine to form a substituted or unsubstituted 5- or 6-membered ring such as a lactone, R 3 , R 4 , R 5 and R 6 are each independently selected from —H, —OH, and alkoxy; R 7 and R8 is selected from -H, -OH, alkoxy, -X, where X can be F, Cl, Br, or R 7 and R 8 are linked together to form a five-membered ring containing one or more heteroatoms such as O, a and b indicate the presence or absence of a double bond at the corresponding position, R 11 and R 12 are each independently selected from -H, or R 11 and R 12 can be a substituted or unsubstituted 5- or 6-membered ring such as a lactone, or —C(O)O-alkyl; R is selected from the following substituted or unsubstituted phenyl rings: [ka] (In the formula, R 9 , R 10 , R 11 and R 12 are each independently selected from -H, -OH, alkoxy, and the following formula: [ka] In the formula, R 13 is H or alkyl, * represents a bond to Q or a -CH2 group.

[0009] In another embodiment, the invention relates to the use of a compound of formula E for the inhibition of cellular V-ATPase activity. [ka] [Brief explanation of the drawings]

[0010] [Figure 1] A schematic diagram of the ELISA assay is shown. [Figure 2] A standard curve for V-ATPase within the concentration range of 31.25 pg / ml to 2000 pg / ml is shown. [Figure 3]1 shows the dose-response curve of remidesivir. [Figure 4] 1 shows the dose response curve for the compound of formula E of the present invention. [Figure 5] Figure 5 shows the effect of administration of the compound of formula E on SARS-CoV-2 infection in hamsters. Figure 5A shows the percentage change in body weight of hamsters from the day of exposure to day 4 post-infection. Figure 5B shows images of whole lungs removed from euthanized animals showing inflammation and interstitial pneumonia. Figure 5C shows images of the spleen showing symptoms of splenomegaly. [Figure 6] Figure 6 shows the antiviral and immunomodulatory effects of administration of the compound of formula E on SARS-CoV-2 infection in hamsters. Figure 6A is a bar graph showing the relative lung viral load in various groups 4 days after infection, and Figure 6B is a bar graph showing the relative mRNA expression levels of splenic cytokines in various experimental groups. Each bar represents the mean + SEM. [Figure 7] 1 shows the effect of administration of the compound of formula E on lung pathology in SARS-CoV-2 infected hamsters. DETAILED DESCRIPTION OF THE INVENTION

[0011] Detailed Description of the Invention The present invention relates to the use of compounds of formula I for the inhibition of cellular V-ATPase activity. [ka] During the ceremony, E is selected from C and N; Q is O or -NH; n is 0 to 6, R 1 and R 2 is selected from -H and -C(O)O-alkyl such as -C(O)OC2H5, or R 1 and R 2 combine to form a substituted or unsubstituted 5- or 6-membered ring such as a lactone, R 3 , R 4 , R 5 and R6 are each independently selected from —H, —OH, and alkoxy; R 7 and R 8 is selected from -H, -OH, alkoxy, -X, where X can be F, Cl, Br, or R 7 and R 8 are linked together to form a five-membered ring containing one or more heteroatoms such as O, a and b indicate the presence or absence of a double bond at the corresponding position, R 11 and R 12 are each independently selected from -H, or R 11 and R 12 can be a substituted or unsubstituted 5- or 6-membered ring such as a lactone, or —C(O)O-alkyl; R is selected from the following substituted or unsubstituted phenyl rings: [ka] (In the formula, R 9 , R 10 , R 11 and R 12 are each independently selected from -H, -OH, alkoxy, and the following formula: [ka] In the formula, R 13 is H or alkyl, * represents a bond to Q or a -CH2 group.

[0012] In one embodiment, a and b in Formula I indicate the presence or absence of a double bond at the corresponding position. For example, when E is -N and R 3 When E is -N and R is -N, the double bond at the corresponding position is absent. 3 is absent, the double bond at the corresponding position is present.

[0013] In one embodiment, the invention relates to the use of a compound of formula II for the inhibition of cellular V-ATPase activity. [ka]

[0014] In one embodiment, the invention relates to the use of a compound of formula III for the inhibition of cellular V-ATPase activity. [ka] The substituents are as defined for Formula I above.

[0015] In one embodiment, the present invention relates to the use of the compounds shown in Table 1 for the inhibition of cellular V-ATPase activity, which act as antiviral agents in the treatment of SARS-CoV-2 or influenza infection.

[0016] [Table 1-1]

[0017] [Table 1-2]

[0018] [Table 1-3]

[0019] [Table 1-4]

[0020] [Table 1-5]

[0021] [Table 1-6]

[0022] In another embodiment, the present invention relates to the use of a compound of formula E for inhibiting cellular V-ATPase activity. The compound of formula E is effective in treating SARS-CoV-2 or influenza infection. [ka]

[0023] The compound of formula E has a molecular weight of about 450 and a partition coefficient (logP) value close to 4. The compound is soluble in solvents such as dimethyl sulfoxide (DMSO), methanol, and ethyl acetate.

[0024] The compounds of Formulas I to III and Formulas A to U may also be used in the form of their prodrugs, metabolites, pharmaceutically acceptable salts, solvates or polymorphs.

[0025] The compounds of Formulas I-III and A-U find use in the inhibition of viruses by inhibiting V-ATPase activity in cells infected with the virus. It is believed that inhibiting cellular V-ATPase activity inhibits the virus.

[0026] The compounds of Formulas I-III and A-U find use in inhibiting viruses, such as SARS-CoV-2 virus or influenza virus.

[0027] The compounds of formula E find use in inhibiting SARS-CoV-2 or influenza viruses.

[0028] The present invention further relates to a method for inhibiting V-ATPase activity in a cell, comprising contacting the cell with at least one of the compounds of Formulas I-III and Formulas AU.

[0029] The present invention further relates to a method for inhibiting a virus, preferably a SARS-CoV-2 virus or an influenza virus, comprising contacting at least one of the compounds of Formulae I-III and A-U with a virus-infected cell.

[0030] A method of inhibiting cellular V-ATPase activity comprises contacting a cell or a virus-infected cell with a compound of formula E.

[0031] The present invention further relates to a method for treating a viral infection, preferably for treating SARS-CoV-2 or influenza virus infection, by administering a therapeutically effective amount of at least one compound of Formulae I-III and A-U to inhibit cellular V-ATPase activity.

[0032] In one embodiment, the method of treatment comprises administering a therapeutically effective amount of a compound of Formulas I-III and Formulas AU in combination with at least one additional compound having V-ATPase inhibitory activity.

[0033] In other embodiments, the method of treatment comprises administering a compound of formula E as one of the compounds.

[0034] The compounds of the present invention are highly effective in inhibiting cellular V-ATPase activity. In both in vitro and in vivo studies, the compounds exhibited excellent antiviral effects without demonstrating cytotoxicity against normal human lymphocytes. These results suggest that the compounds can be used effectively and safely in the treatment of viral infections, particularly SARS-CoV-2 infections.

[0035] In one embodiment, compounds encompassed by the present invention are used in the manufacture of a medicament for treating a viral infection, such as a SARS-CoV-2 or influenza infection, by inhibiting cellular V-ATPase activity.

[0036] Some specific compounds falling within the scope of the present invention are disclosed herein, with the understanding that the disclosed compounds do not limit the scope of the invention. [Example]

[0037] The compounds of the present invention are prepared by known methods, as disclosed in WO2018193476 and WO2020129082, which are incorporated herein by reference.

[0038] Example 1 Determination of V-ATPase inhibitory activity in cells The vacuolar ATPase (V-ATPase) is a proton pump responsible for regulating the intracellular and extracellular pH of cells. The structure of V-ATPase is highly conserved among all eukaryotic cells and is involved in diverse functions across species. V-ATPase is most widely known for acidifying endosomes and lysosomes, but is also important for luminal acidification in specific cells.

[0039] Among host factors that can be targeted in antiviral therapy, V-ATPase is a promising target for intervening in virus entry into host cells. Against viral threats such as influenza, flaviviruses, vaccinia, bornaviruses, rhabdoviruses, and coronaviruses, V-ATPase-mediated endosomal acidification may pave the way for new antiviral therapies that are more versatile and less susceptible to drug-resistance mutations.

[0040] To determine the V-ATPase inhibitory effect of the compounds of the present invention, an ELISA test was carried out using a human V-type proton ATPase ELISA kit (My BioSource catalog number: MBS911862).

[0041] method: One million MDAMB231 cells (a V-ATPase-enriched cell line) were seeded into 60 mm plates and placed at 37°C and 5% CO2 for 24 hours. Cells were treated with test compounds (Formulas A-U) at a concentration of 100 nM (0.1 μM) / 10,000 cells and incubated for 48 hours. After incubation, the supernatant was discarded, and the cells were washed with DPBS (Dulbecco's Phosphate-Buffered Saline), collected, and stored at -80°C for 48 hours. The cells were thawed, vortexed, and homogenized for 7 minutes, followed by centrifugation at 3000 rpm for 3 minutes. The supernatant was collected for V-ATPase assay.

[0042] The assay used was a quantitative sandwich enzyme immunoassay technique as shown in FIG.

[0043] Standards and samples were pipetted into the wells. A V-ATPase-specific antibody was precoated on the microplate. Any V-ATPase present in the standards / samples would bind to the immobilized antibody. After the incubation period, any unbound material was removed by washing, and a V-ATPase-specific biotin-conjugated antibody was added to the wells. The plate was again incubated at 37°C and 5% CO2. After washing, avidin-conjugated horseradish peroxidase (HRP) was added to the wells. Following washing to remove unbound avidin-enzyme reagent, a substrate solution was added to the wells, resulting in the development of an amount of color proportional to the amount of V-ATPase bound in the initial step. Color development was stopped, and the optical density was measured spectrophotometrically at 450 nm. The V-ATPase concentration was calculated by substituting the optical density into the regression equation obtained using the standard samples. The percent (%) reduction of V-ATPase was calculated by subtracting the amount of V-ATPase obtained from the untreated sample / V-ATPase concentration in the untreated sample×100.

[0044] result A standard curve of V-ATPase concentrations ranging from 31.25 pg / ml to 2000 pg / ml was plotted as shown in Figure 2. The plot showed good linearity (R 2=0.954).

[0045] Table 2 shows the results of the V-ATPase assay for compounds of the present invention.

[0046] [Table 2]

[0047] conclusion The results showed that the compounds of formulae A-U resulted in a decrease in V-ATPase activity in cells.

[0048] Example 2 Toxicity studies of compounds of the present invention in normal cells Toxicity studies were carried out on human peripheral blood lymphocytes obtained by differential centrifugation of defibrinated blood. To determine their toxicity, MTT assays were carried out on the compounds of the present invention. MTT assays are simple and sensitive assays that measure the metabolic activity of cells. The assays were carried out as follows:

[0049] The required volume of cell suspension was prepared for each cell seeding efficiency. 200 μl of the prepared cell suspension was added to a labeled 96-well plate, and the plate was placed in an incubator at 37°C and 5% CO2 for 18–24 hours. 2 μl of each diluted compound was then added, and the plate was placed in an incubator at 37°C and 5% CO2 for 48 hours. The suspension was aspirated from the plate, and 100 μl of working MTT solution (0.5 mg / ml MTT prepared from a 5 mg / ml MTT stock solution in 1x complete medium) was added. The plate was incubated in an incubator at 37°C and 5% CO2 for 4 hours. The plate was centrifuged, the supernatant was removed, 200 μl of DMSO was added, gently mixed, and placed in an incubator at 37°C and 5% CO2 for 10 minutes. The absorbance at 570 nm was read, and the percent viability and IC of the compound were determined. 50 Values ​​were calculated by regression analysis.

[0050] Table 3 shows the results of the MTT assay performed on compounds of the present invention.

[0051] [Table 3]

[0052] conclusion From the above results, the compounds of formulas B to E, I, L, O, P, and U did not exert any toxicity on normal human lymphocytes up to 100 μM, which means that the compounds of the present invention did not exert any toxicity on normal peripheral blood lymphocytes.

[0053] Other Integrity Studies Various preclinical GLP (Good Laboratory Practice) and non-GLP studies were conducted on the compound of formula E. These studies included 7-day and 28-day studies in rats and dogs, and acute toxicity in rats, mice, and dogs. The effects of the compound of formula E on respiratory function, nervous and cardiovascular systems, bacterial mutation, metabolism, CaCO2 permeability, protein binding, and cytochrome effects were also studied. These studies showed that the compound of formula E is nontoxic, nonmutagenic, nonclastogenic, and has no effect on the respiratory, nervous, and cardiovascular systems. Furthermore, the compound of formula E did not show any toxic or adverse effects in rats, mice, and dogs, even at doses higher than 2000 mg / kg. This result indicates that the compound of formula E is safe to administer.

[0054] Example 3 In-vitro anti-SARS-CoV-2 activity of compounds of the present invention Neutral Red (cytopathic effect / toxicity assay)

[0055] method: Reduction of virus-induced cytopathic effect (early CPE assay) Confluent or near-confluent cultured T-cell monolayers of Vero76 cells were prepared in 96-well disposable microplates the day before testing. Cells were maintained in MEM (Eagle's Minimum Essential Medium) supplemented with 5% FBS (fetal bovine serum). For antiviral assays, the same medium was used, but the FBS was reduced to 2% and 50 μg / ml gentamicin was added. Compounds were dissolved in DMSO, saline, or diluent. Compounds with low solubility were subjected to vortexing, heating, and sonication, and if these did not result in a solution, they were tested as colloidal suspensions. Test compounds were diluted in a four-step logarithmic scale. 10 Compounds were prepared at concentrations of typically 0.1, 1.0, 10, and 100 μg / ml or μM. Lower concentrations were used when insufficient compound was available. Five microwells were used per dilution: three for infected cultures and two for uninfected, virulent cultures. Experimental controls consisted of six microwells per plate that were infected but untreated (virus control) and six microwells that were untreated and uninfected (cell control). A known active agent was tested in parallel as a positive control, using the same method as used for the test compounds. A positive control was also tested during each run.

[0056] Culture medium was removed from the cells and 2x concentrations of test compounds were added to the wells in a 0.1 ml volume. Typically, up to 60 CCID in a 0.1 ml volume. 50Virus (50% culture cell infectious dose) was added to the wells for virus infection. Virus-free medium was added to the toxicity control wells and cell control wells. The plates were incubated at 37°C and 5% CO2 until clear CPE (>80% CPE for most virus strains) was observed in the virus control wells. The plates were then stained with 0.011% neutral red for approximately 2 hours in a 37°C and 5% CO2 incubator. The neutral red medium was removed by thorough aspiration, and cells may be rinsed with 1x phosphate-buffered saline (PBS) to remove residual stain. The PBS was then completely removed, and the incorporated neutral red was eluted with 50% Sorensen citrate buffer / 50% ethanol for at least 30 minutes. Because neutral red stain penetrates live cells, the darker the red color, the greater the number of live cells present in the well. The stain content of each well was quantified using a spectrophotometer at a wavelength of 540 nm. The dye content of each set of wells was converted to a percentage of the dye present in untreated control wells using a Microsoft Excel computer-based spreadsheet and normalized based on the virus control. The 50% effective concentration (EC 50 , viral inhibitory concentration) and 50% cytotoxic concentration (CC 50 , cytotoxicity) was calculated by regression analysis. 50 EC 50 The quotient obtained by dividing by this is the selectivity index (SI) value.

[0057] Table 3 shows the results of the Neutral Red (cytopathic effect / toxicity assay).

[0058] [Table 4]

[0059] In the neutral red assay, the EC of the compound of formula E 50 , C.C. 50 and S.I. 50The values ​​were 0.35 μg / ml, 2.5 μg / ml and 7.1, respectively. These values ​​indicate that the compounds, particularly the compound of formula E, inhibit viral replication.

[0060] conclusion The compound of formula E is EC 50 0.35 and SI as 50 7.1 as a compound of formula F, 50 2.9 and SI 50 These compounds are believed to have good antiviral activity.

[0061] Example 4 In-vitro anti-SARS-CoV-2 activity of the compound of formula E (assay method: IC 50 (Estimate of

[0062] method: The assay was performed in a 96-well plate format with three wells per sample. 4 VeroE6 cells were seeded into each well and incubated overnight at 37°C to form a monolayer. The following day, cells were incubated with seven concentrations of test substance (TS). For remidesivir, the following concentrations were used: 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, and 0.01 μM. For compound of formula E, a 10 μM stock solution was serially diluted (2-fold) with DMSO. Two microliters from each dilution were used in the screening assay. The seven concentrations of compound of formula E were 10 μg, 3 μg, 1 μg, 0.3 μg, 0.1 μg, 0.03 μg, and 0.01 μg. Control cells were incubated with 0.5% DMSO only. Cells were infected with SARS-CoV-2 at an MOI of 0.01. After 48 hours, viral RNA was extracted from 100 μl of culture supernatant and subjected to qRT-PCR (in duplicate) to determine the Ct values ​​of the N and E gene sequences. Inhibition of viral replication was determined based on the fold change in Ct values ​​in TS-treated cells compared to the control. IC 50 The values ​​are shown in Table 4 and are based on the AAT Bioquest IC 50 It was determined using a calculator.

[0063] [Table 5]

[0064] Figure 3 shows the dose-response curve for remidesivir. Figure 4 shows the dose-response curve for the compound of formula E.

[0065] conclusion The results showed that the compound of formula E had excellent anti-SARS-CoV-2 activity (IC 50 The results show that the effect of ATP on the ATP-dependent agonist activity was <10 μM.

[0066] Example 5 In-vitro anti-SARS-CoV-2 activity of compounds of formula E Assay Method: Cytotoxicity

[0067] method The assay was performed in a 96-well plate format with three wells per sample. 4VeroE6 cells were seeded into each well and incubated overnight at 37°C to form a monolayer. The following day, cells were incubated with the indicated concentrations of test substance (TS) to a final DMSO concentration of 0.5%. Control cells were incubated with 0.5% DMSO alone. After 24 and 48 hours, cells were stained with Hoechst 33342 and Sytox orange stains. Sixteen images covering 90% of the well area at 10x magnification were taken for each well using an ImageXpress confocal microscope (Molecular Devices). Hoechst 33342 nucleic acid stain is a common cell-permanent nuclear counterstain that emits blue fluorescence upon binding to double-stranded DNA. It stains all live and dead cells. Sytox orange stains nucleic acids in cells with heteromorphic membranes. This stain is an indicator of cell death. Software first counts the total number of cells in the Hoechst image. In the Sytox image, the number of Sytox-positive cells is counted from among the Hoechst-positive cells.

[0068] Antiviral Screening method: The assay was performed in a 96-well plate format with three wells per sample. 4 VeroE6 cells were seeded into each well and incubated overnight at 37°C to form a monolayer. The following day, cells were incubated with the indicated concentrations of test substance (TS) to a final DMSO concentration of 0.5%. Control cells were incubated with 0.5% DMSO alone. Cells were infected with SARS-CoV-2 at an MOI of 0.01. After 24 and 48 hours, viral RNA was extracted from 100 μl of culture supernatant and subjected to qRT-PCR (in duplicate) to determine Ct values ​​for N and E gene sequences. Inhibition of viral replication was determined based on the fold change in Ct values ​​in TS-treated cells compared to the control. Remidesivir was used as a positive control for viral inhibition.

[0069] result Table 5 shows the results of the cytotoxicity and antiviral activity of compounds of formula E.

[0070] [Table 6]

[0071] The results above show that the compound of formula E exhibits slightly higher cell viability after 24 hours compared to remidesivir. The percentage of viral replication inhibition by the compound of formula E at 24 and 48 hours after viral infection was comparable to that of remidesivir.

[0072] conclusion Since remidesivir is effective against the SARS-CoV-2 virus, the compound of formula E is also effective in inhibiting the SARS-CoV-2 virus and can be effectively used to treat SARS-CoV-2 virus infection.

[0073] Example 6 In-Vivo Study of Anti-SARS-CoV-2 Activity of Compounds of Formula E In vivo studies were performed in golden hamsters infected with SARS-CoV-2 as described below.

[0074] methodology Animals: Male golden hamsters, 6-8 weeks old, were obtained from the Central Drug Research Institute (CDRI) and transferred to the Small Animal Facility (SAF) at the Translational Health Science and Technology Institute (THSTI), where they were quarantined for 7 days prior to the challenge study. During the pretreatment regimen, animals were housed in the Small Animal Facility (SAF) and then transferred to an Animal Biosafety Level-3 (ABSL-3) research facility for the SARS-CoV-2 challenge study. Animals were maintained under a light-dark cycle, fed a standard pellet diet, and had free access to water for less than 12 hours. All experimental protocols, including immunizations, booster doses, and animal exposures, were approved by the institutional ethics committees IAEC (IAEC / THSTI / 118), IBS, and RCGM. Virus culture and titration: SARS-associated coronavirus 2, isolate USA-WA1 / 2020 virus, was propagated and titered in VeroE6 cell line cultured in complete Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g / L D-glucose, 100,000 U / L penicillin-streptomycin, 100 mg / L sodium pyruvate, 25 mM HEPES, and 2% FBS. Virus stocks were purified by plaque purification in the THSTI IDRF facility within ABSL3, following laboratory biosafety standards.

[0075] Virus culture and titration SARS-associated coronavirus 2, isolate USA-WA1 / 2020 virus, was propagated and titered in VeroE6 cell line cultured in complete Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g / L D-glucose, 100,000 U / L penicillin-streptomycin, 100 mg / L sodium pyruvate, 25 mM HEPES, and 2% FBS. Virus stocks were purified by plaque purification in the THSTI IDRF facility within ABSL3, following laboratory biosafety standards.

[0076] SARS-CoV2 infection and medication in golden hamsters Thirty-three male golden hamsters were randomly assigned to separate groups (n = 5): 1) exposed controls (n = 5); 2) remidesivir controls (n = 5); and 3) unexposed controls (n = 3) housed in individual cages. The pretreatment group (δIII / p400) received 400 mpk of the compound of formula E (drug) by oral gavage starting on day 2 of exposure. The remaining three groups, δI / 200, δII / 800, and δIV / 400, received 200, 800, and 400 mpk of the compound of formula E, respectively, by oral gavage daily after exposure until the end point. All animals, except the unexposed controls, were anesthetized in an ABSL3 facility with an intraperitoneal injection of ketamine (150 mg / kg) and xylazine (10 mg / kg), and 50 μl of the compound was administered intranasally via a catheter into each nostril for 10 min. 5 The mice were exposed to PFU of SARS-CoV-2 (Chan et al., 2020; Rizvi et al., 2021; Sia et al., 2020). An unexposed control group received mock PBS intranasally. All experimental protocols involving viral culture and animal infection handling were approved by the RCGM, Institutional Biosafety and IAEC Animal Ethics Committee.

[0077] Large-scale clinical parameters of SARS-CoV-2 infection All infected animals were euthanized at ABSL3 four days after infection. Changes in body weight and animal activity were monitored daily after challenge. After euthanasia, the lungs and spleens of the animals were excised and imaged for gross morphological changes. The lower lobe of the right lung was fixed in 10% neutral formalin solution and used for histological analysis. For estimation of viral load, the intact left lobe of the lung was homogenized in 2 ml of Trizol solution. The spleen was homogenized in 2 ml of Trizol solution. Tissue samples in Trizol were immediately stored at -80°C until further use. Blood was withdrawn from the animals by direct cardiac puncture, and serum was isolated and stored at -80°C until further use.

[0078] result Effect of oral administration of compound of formula E on large-scale clinical parameters in SARS-CoV-2 infected hamsters To understand the protective efficacy of the compound of Formula E (according to the dosing regimen) against SARS-CoV-2 challenge in hamsters, the infected groups receiving different doses of the compound of Formula E, i.e., δI / 200, δII / 800, δIII / p400, and δIV / 400, were evaluated for large-scale clinical parameters associated with SARS-CoV-2 infection in hamsters compared to experimental controls: uninfected (UI), SARS-CoV-2 infected (I), and SC remidesivir-treated hamsters (R).

[0079] As shown in Figure 5A, the results showed that golden hamsters administered δIII / p400 exhibited the highest protection against weight loss and did not lose weight after infection. Furthermore, the δI / 200 group exhibited the second most favorable outcome in terms of weight loss, whereas the remaining two groups exhibited the least protection compared to infected controls and showed moderate weight loss after infection.

[0080] Similar to the above, lungs isolated from animals euthanized 4 days after exposure showed significantly fewer areas of interstitial pneumonia and inflammation in the δIII / p400 group, followed by the δII / 800 group, suggesting that these two groups may have less SARS-CoV2-associated lung injury compared to infected groups (Figure 5B). Splenomegaly, another clinical parameter of SARS-CoV2-associated pathology in hamsters, also showed a significant reduction in the δIII / p400 group compared to infected controls, whereas the other drug groups showed no or only a small reduction in spleen size (Figure 5C).

[0081] Antiviral and immunomodulatory properties of compound of formula E against SARS-CoV-2 infected hamsters To understand the antiviral efficiency of the compound of formula E relative to the viral load, quantification of the N2 gene of SARS-CoV2 was performed in lungs excised from hamsters.

[0082] The results in Figure 6A showed significant reductions in relative lung viral load in the δI / 200, δII / 800, and δIII / p400 groups. The greatest reduction in viral load was observed in δIII / p400, which showed a 5-fold reduction in viral load compared to the infected control group, whereas other groups, such as δI / 200 and δII / 800, showed approximately a 2- to 2.5-fold reduction in viral load. Interestingly, there was no reduction in viral load in the δIV / 400 group, and heterogeneity in lung viral load was also observed in this group.

[0083] To understand the immunomodulatory potential of compound of formula E, we performed mRNA expression profiling of IFNγ, IL4, and IL17A genes relative to the HGPRT endogenous control in splenocyte samples from various groups. The data showed a dramatic inhibition of IL4 across all groups and an inhibition of IL17A expression in δI / 200 and δIII / p400 (Figure 6B). Interestingly, δI / 200 showed the highest induction of IFNγ across all groups.

[0084] Histological study of SARS-CoV2 infection-associated lung disease in hamsters treated with compound of formula E To understand the effect of administration of the compound of formula E on lung disease during SARS-CoV2 infection, detailed histological analysis of lung samples isolated on day 4 post-infection was performed.

[0085] Histological evaluation of lung disease was performed by H&E staining of hamsters treated with compound of Formula E and compared with the control group. Figure 7 shows a 40x histological image of an H&E-stained lung, showing areas of interstitial pneumonia (blue arrows), inflammation (black arrows), lung damage (red arrows), and alveolar epithelial cells (green arrows), along with the corresponding pathology scores for each, and the overall disease score for the lung sample.

[0086] H&E-stained lung samples from all drug-treated groups showed significant reductions in interstitial pneumonia, alveolar epithelial damage, and lung inflammation scores. The δIII / p400 group showed the highest protection for the histological parameters and overall disease scores studied. Interestingly, little or no protection was seen in the δIV / 400 drug group compared to infected controls.

[0087] conclusion A SARS-CoV-2 challenge study in golden hamsters showed that of the four dosing regimens tested, pretreatment with 400 mpk of the compound of formula E provided the highest overall protective efficacy against SARS-CoV-2 infection, resulting in reduced lung viral load and interstitial pneumonia compared to remidesivir, as well as reduced lung pathology and suppression of pathogenic IL-4 and IL-17A immune responses.

[0088] The above studies show that the compounds of formula I are effective in reducing intracellular V-ATPase, thereby aiding in the treatment of viral infections by inhibiting the SARS CoV-2 virus. The compounds of formula I, especially the compound of formula E, were highly effective as antiviral drugs to treat viral infections without causing toxicity.

[0089] The above description of the present invention is intended to be illustrative and not limiting. Since modifications that incorporate the spirit and substance of the present invention into the disclosed implementations may occur to those skilled in the art, the present invention should be understood to include all modifications within the scope of the present disclosure.

Claims

1. A pharmaceutical agent for inhibiting cellular V-ATPase activity, comprising, as an active ingredient, a compound selected from the group consisting of the following formulae B to K and MU: 【Chemistry 1】 【Chemistry 2】 【Transformation 3】

2. A medicament comprising a compound of formula E for inhibiting cellular V-ATPase activity. 【Chemistry 4】

3. The method of claim 1 or 2, wherein the compound is a pharmaceutically acceptable salt, solvate, or polymorph thereof.

4. The pharmaceutical agent according to any one of claims 1 to 3, for inhibiting viruses by inhibiting cellular V-ATPase activity.

5. The medicament according to claim 4 for inhibiting SARS-CoV-2 virus or influenza virus.

6. A medicament for inhibiting SARS-CoV-2 virus or influenza virus, comprising, as an active ingredient, a compound selected from the group consisting of the following formulae B to K and MU: 【Transformation 5】 【Transformation 6】 【Transformation 7】

7. A medicament comprising a compound of formula E as an active ingredient for inhibiting SARS-CoV-2 virus or influenza virus. 【Transformation 8】

8. 1. Use of a compound selected from the group consisting of formulae BK and MU for the manufacture of a medicament for inhibiting cellular V-ATPase activity for the treatment of viral infection. 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】

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