Use of a small molecule compound in the preparation of antiviral drugs

Abstract

This invention discloses the use of a small molecule compound with the structure shown in formula (Ⅰ) in the preparation of antiviral drugs, belonging to the field of biomedicine. Studies have found that this compound has a half-maximal effective concentration (EC50) for PRRSV and ASFV. 50 The concentrations of C14 at 30 µM were 0.34 µM and <0.1 µM, respectively. C14 significantly inhibited FIPV at 30 µM. This compound showed no significant cytotoxicity and good drug-like properties, demonstrating potential for development into a clinical therapeutic. Formula (I)

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the use of a small molecule compound in the preparation of antiviral drugs. Background Technology

[0002] Pigs are an economically vital livestock, accounting for over 30% of global meat production. The productivity of the global pig farming industry is impacted by infectious diseases, with porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV) being two of the most destructive pathogens. These viral diseases lead to high mortality rates, decreased productivity, increased disease control costs, and trade disruptions in pigs, resulting in significant economic losses. Feline infectious peritonitis virus (FIPV) primarily infects cats. This virus triggers an immune response in cats, causing excessive inflammation that affects multiple organs, such as the liver, kidneys, and lungs. Infected cats exhibit symptoms such as anorexia, fever, and weight loss, ultimately leading to organ failure. Once feline infectious peritonitis (FIP) develops, the mortality rate is almost 100%.

[0003] Antiviral compounds can provide immediate protection for animals to curb viral transmission, offering a viable alternative to vaccines. Treatments for these viruses include viral inhibitors and immunomodulators, but no specific drugs are currently widely used. Therefore, further screening and research are needed to find more effective preventative and therapeutic drugs, providing better disease control solutions for livestock farming. Summary of the Invention

[0004] The purpose of this invention is to provide a novel use of small molecule compounds in the preparation of antiviral drugs.

[0005] The small molecule compound involved in this invention has the following chemical structural formula (Ⅰ). This compound is a known compound (Specs ID: AN-465 / 41850772) with the chemical name N-{[2-(benzyloxy)-1-naphthyl]methyl}-N-[3-(4-morpholino)propyl]amine, which is abbreviated as C14 in this invention.

[0006]

[0007] Equation (I)

[0008] This study found that the small molecule compound C14 possesses activity against both PRRSV and ASFV, exhibiting significant inhibitory effects against both viruses, with a half-maximal effective concentration (EC50) of [missing value]. 50The concentrations were 0.34 µM and <0.1 µM, respectively. Subsequent studies found that the compound also exhibited good inhibitory effects on FIPV at a concentration of 30 µM. Furthermore, toxicity studies on African green monkey embryonic kidney cells (Marc-145) and porcine alveolar macrophages (PAM) after 48 h of treatment revealed that C14 had a median toxic concentration (C50) in both cell types. 50 The concentrations were approximately 80 µM and 60 µM, respectively. In CRFK cells, 30 µM C14 did not show toxic effects.

[0009] A comprehensive analysis of the absorption, distribution, metabolism, excretion, and toxicity (ADMET) of C14 revealed that, except for LogP (3.872) and LogD (3.379), which were slightly above the optimal range (1–3), all other indicators were within the range suitable for drug-like properties. Although LogP and LogD were not at their optimal values, they met the Lipinski Rule (LogP ≤ 5) and the Golden Triangle (LogD ≤ 5), indicating that C14 has good drug-like properties and the potential to be further developed into a clinical drug.

[0010] In summary, C14 has anti-PRRSV, ASFV and FIPV effects and has the potential to develop related clinical treatment drugs. This invention provides an alternative treatment for the pig farming and pet industries.

[0011] Products derived from the small molecule compound C14, including pharmaceutically acceptable prodrugs, isomers, salts, and crystal forms, are also protected within the scope of this invention in the preparation of antiviral drugs. Attached Figure Description

[0012] Figure 1 The toxicity of different concentrations of C14 in Marc-145 cells after 48 hours.

[0013] Figure 2 The toxicity of PAM cells treated with different concentrations of C14 for 48 hours.

[0014] Figure 3 The toxicity of CRFK cells treated with C14 at a concentration of 30 µM for 36 h.

[0015] Figure 4 The inhibitory effect of different concentrations of C14 on PRRSV copy number.

[0016] Figure 5 The inhibitory effect of different concentrations of C14 on ASFV copy number.

[0017] Figure 6Immunofluorescence analysis of the inhibitory effect of 30µM C14 on FIPV infection in CRFK cells.

[0018] Figure 7 Drug class analysis of C14 was performed using ADMET prediction. Detailed Implementation

[0019] To more clearly illustrate the technical solution of the present invention, the present invention will be described in detail below with reference to specific embodiments. The specific embodiments described are only some embodiments of the present invention. For those skilled in the art, other embodiments can be obtained based on these specific embodiments without creative effort.

[0020] Unless otherwise specified, all reagents or materials used in the following examples are commercially available.

[0021] Example 1: Detection of C14 cytotoxicity

[0022] 1. Test Methods

[0023] 1) Seeding cells in 96-well plates

[0024] Cells that had grown to the bottom of the flask were first digested with trypsin containing EDTA, and then resuspended in complete culture medium to prepare a single-cell suspension. Next, the suspension was seeded into 96-well plates at a density of 5000 cells per well. The culture plates were then incubated at 37°C and 5% CO2 for 24 hours.

[0025] 2) C14 treatment of cells

[0026] After aspirating the culture medium, wash three times with sterile PBS solution. Then, remove the PBS solution and add different concentrations of C14 (diluted with maintenance medium), with six replicates for each concentration. Simultaneously, wells containing the same volume of maintenance medium serve as a control group.

[0027] 3) Absorbance detection

[0028] After incubating cells in an incubator for 36 or 48 hours, the culture medium was first aspirated, and then the cells were washed three times with sterile PBS. Next, basal culture medium containing 10 µL of CCK-8 solution was added to each well, with wells without cells serving as blank controls. After incubating the cells in an incubator for 1.5 hours, the absorbance at 450 nm was measured using a microplate reader, and the values ​​were recorded to calculate cell viability.

[0029] 2. Test Results

[0030] like Figure 1As shown, the results indicate that the toxicity of different concentrations of C14 in Marc-145 cells (African green monkey embryonic kidney cells) was dose-dependent. At 5–50 µM, cell viability was approximately 100%, with virtually no cytotoxicity; at 80 µM, cell viability was close to 50%, indicating that C14 treatment was effective. 50 The concentration is approximately 80 µM; at a concentration of 240 µM C14, cell viability is below 40%.

[0031] like Figure 2 As shown, the results indicate that the cytotoxicity of PAM cells (porcine alveolar macrophages) treated with different concentrations of C14 was dose-dependent. At 0–20 µM, cell viability was approximately 100%, with virtually no cytotoxicity; at 60 µM, cell viability was close to 50%, indicating that C14 treatment was effective. 50 The concentration is approximately 60 µM; at a concentration of 200 µM C14, cell viability is below 40%.

[0032] In addition, after treating CRFK cells (cat kidney cells) with a concentration of 30 µM C14 for 36 h, the cell viability was normal and there were no obvious lesions, indicating that C14 at this concentration is basically non-cytotoxic.

[0033] Example 2: Detection of C14 anti-PRRSV and ASFV viral activity

[0034] 1. Test Methods

[0035] 1) Cellular attack

[0036] Marc-145 and PAMs cells were cultured to 80% confluence, then basal medium containing PRRSV WUH3 strain (MOI=0.1) or ASFV-Luc-EGFP (MOI=0.1) was added, and the cells were incubated at 37°C and 5% CO2. The culture plates were incubated for 1 hour to ensure complete infection, after which the inoculum was removed. C14 was dissolved in DMSO to prepare the corresponding stock solution concentration. Cells were then covered with medium containing 2% serum of the corresponding drug. A blank control group and a control group were also included in the experiment. Cytopathic effects were observed 48 hours post-infection.

[0037] 2) qPCR method to measure the efficacy of drugs against PRRSV and ASFV

[0038] Cell supernatant was collected, and viral RNA was extracted from the supernatant using FastPure Viral DNA / RNA Mini Kit (RC311, Novizan, China). The extracted viral RNA was reverse transcribed using HiScript II Q Select RT SuperMix for qPCR (R233, Novizan, China), and qPCR was performed using 2X Universal SYBR Green Fast qPCR Mix (RK21203, ABclonal, China) (Table 1).

[0039] Table 1 qPCR reaction system

[0040]

[0041] PRRSV primers: Upstream primer (NSP9): 5′-ACCCTAGGACCTGTGAAC-3′; Downstream primer (NSP9): 5′-GGCGAGTAACTTAGGAGATG-3′.

[0042] ASFV primers: Upstream primer (B646L): 5′-GATACCACAAGATCGCCG T-3′; Downstream primer (B646L): 5′-FAM-CCACGGGAGGAATACCAACCCAGTG-3′-TAMRA.

[0043] Results were monitored using the CFX96 real-time qPCR detection system (Bio-Rad, USA). The program was set to perform one 3-minute cycle at 95°C, followed by 40 cycles, with each cycle consisting of 5 seconds at 95°C and 30 seconds at 60°C. The Ct values ​​of the results were substituted into the standard curve to calculate the viral copy number.

[0044] 2. Test Results

[0045] like Figure 4 As shown, C14 inhibits PRRSV copy number in a dose-dependent manner; 10–30 µM of C14 can reduce viral copy number by more than two orders of magnitude. Analysis of the data yielded the EC50 of C14. 50 The value is approximately 0.34 µM.

[0046] like Figure 5 As shown, C14 inhibits ASFV copy number in a dose-dependent manner; 10–40 µM of C14 can reduce viral copy number by more than two orders of magnitude. Analysis of the data yielded the EC50 of C14. 50 Value <0.1µM.

[0047] Example 3: Detection of C14 anti-FIPV virus activity

[0048] 1. Test Methods

[0049] 1) Cellular attack

[0050] CRFK cells were cultured to 80% confluence, then basal medium containing cell-adapted serotype II FIPV 79-1146 (MOI=0.1) was added, and the cells were incubated at 37°C and 5% CO2. The culture plates were incubated for 1 hour to ensure complete infection, after which the inoculum was removed. C14 was dissolved in DMSO to prepare the appropriate stock solution concentration. The cells were then covered with medium containing 2% serum of the corresponding drug. A blank control group and a control group were also included in the experiment. Cytopathic effects were observed 36 hours post-infection.

[0051] 2) Immunofluorescence analysis to detect the anti-FIPV effect of drugs

[0052] CRFK cells were fixed with 4% neutral formaldehyde fixative at 4°C for 15 min, rinsed three times, completely covered with 5% BSA, and blocked at 37°C for 30 min. Since FIPV is a recombinant virus carrying a fluorescent plasmid, no primary or secondary antibody incubation was performed. Nuclear staining was performed using 4',6-diamidinyl-2-phenylindole (DAPI), incubated at room temperature in the dark for 10 min, and observed under a fluorescence inverted microscope (Olympus IX83, Olympus Co., Tokyo, Japan). 2. Experimental Results

[0053] like Figure 6 As shown, compared with the DMSO treatment group, the green fluorescence of CRFK cells was significantly reduced after treatment with 30 µM C14, indicating that C14 can significantly inhibit FIPV at this concentration.

[0054] Drug-likeness analysis of Example 4C14

[0055] This study used the ADMETlab 2.0 server (https: / / admetmesh.scbdd.com) to predict the characteristics of C14. First, the molecular structure of C14 was drawn using Chemdraw, then converted to SMILES format, copied and pasted into the ADMETlab 2.0 server, and drug-likeness analysis was performed using the default parameters.

[0056] The results are as follows Figure 7As shown, C14 exhibits good drug-like properties, with most of its predicted values ​​falling within the optimal range. The LogP and LogD values ​​of C14 are slightly above the upper limit (>3), suggesting that the solubility of these compounds may be lower. However, the molecule meets the Lipinski rule (LogP≤5) and the Golden Triangle (LogD≤5) criteria, indicating its potential for further development into clinical applications.

[0057] In summary, this study found that the small molecule compound C14 can significantly inhibit PRRSV, ASFV, and FIPV, and has no significant cytotoxic effect at therapeutic concentrations. The half-maximal effective concentration (EC50) of C14 for PRRSV and ASFV is [not specified in the original text]. 50 The concentrations of C14 at 30 µM were 0.34 µM and <0.1 µM, respectively, and C14 significantly inhibited FIPV. These results indicate that the present invention provides a new method for the drug treatment of these three viruses in clinical production.

[0058] ADMET analysis showed that C14 exhibits good drug-like properties, with its absorption, distribution, metabolism, excretion, and toxicity indicators generally within their optimal ranges. This invention provides a novel drug option for the prevention and control of PRRSV, ASFV, and FIPV. C14 is a small molecule compound with a molecular weight less than 400, and its antiviral activity has not yet been reported. This study has significant reference value for the research and application of this drug.

[0059] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of a small molecule compound with the structure shown in formula (Ⅰ) in the preparation of antiviral drugs, characterized in that: The viruses mentioned are porcine reproductive and respiratory syndrome virus (PRRSV), African swine fever virus (ASV), and feline infectious peritonitis virus (FIPV). Equation (Ⅰ).

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