Application of bufalin and derivatives thereof in preparation of medicines for preventing and / or treating various virus infections

Abstract

The invention provides application of bufotalin or a derivative thereof or a pharmaceutically acceptable salt thereof or a substance taking the bufotalin or the derivative thereof or the pharmaceutically acceptable salt thereof or a prodrug thereof as an active ingredient in preparation of at least one of the following products: A1) a product for preventing and / or treating various virus infections; the bufotalin and the derivatives thereof have the advantages that the bufotalin and the derivatives thereof can be used for preparing the bufotalin and the derivatives thereof, the bufotalin and the derivatives thereof can be used for preparing the bufotalin and the derivatives thereof, the products can be used for inhibiting various viruses, the products can be used for preventing and / or treating diseases caused by various virus infections, and the bufotalin and the derivatives thereof can be used for improving symptoms caused by various virus infections.

Description

Technical Field

[0001] This invention relates to the field of new uses of pharmaceuticals, and in particular to the use of bufotalin and its derivatives in the preparation of drugs for the prevention and / or treatment of various viral infections. Background Technology

[0002] Since 1940, the number of emerging infectious diseases worldwide has been increasing, peaking in the 1980s. In recent years, the emergence of new and highly pathogenic infectious diseases such as SARS-CoV-2, MERS-CoV, SARS-CoV, highly pathogenic avian influenza (H5N1, H7N9), influenza A (H1N1), Ebola (EBOV), and Zika fever has seriously endangered human security and tested the ability of countries around the world to prevent and control emerging and highly pathogenic infectious diseases.

[0003] Emerging and highly contagious infectious diseases are characterized by their high transmissibility, rapid spread, and wide reach. Furthermore, since most emerging and highly contagious diseases are zoonotic, the viruses mutate rapidly within their natural hosts, and the population generally lacks immunity to these viruses. The development of modern biological technologies makes these emerging and highly contagious viruses potentially deadly biological weapons; therefore, the prevention and control of emerging and highly contagious infectious diseases is a crucial component of national biosecurity.

[0004] Vaccines and drugs are effective means of combating emerging and highly infectious diseases. Vaccines for infectious diseases include different types such as inactivated vaccines, live attenuated vaccines, and genetically engineered vaccines, while drugs mainly consist of small molecule compounds, antibodies, and antisera. However, due to the unpredictable nature of emerging and highly infectious diseases in their early stages, most emerging and highly infectious diseases lack effective vaccine and drug reserves, leaving the country in a very passive position when dealing with disease outbreaks. Developing vaccines and drugs is not only the most urgent problem to be solved in the field of emerging and highly infectious diseases, but also a crucial means of combating bioterrorism in the field of biosecurity.

[0005] Viruses are simple microorganisms that must parasitize host cells to replicate and multiply. Currently, antiviral drugs are mainly categorized by their targets. For example, drugs that directly target the virus can bind to viral proteins or nucleic acids to affect viral entry, transcription, replication, assembly, and release during viral adsorption, internalization, transport, membrane fusion, and genome release. Drugs that directly target the host can affect viral entry, transcription, replication, processing, transport, and the activity of signaling pathways. The main molecular forms of antiviral drugs include small molecules, peptides, neutralizing antibodies, interferons, CRISPR-Cas systems, si / shRNA, and other nucleic acid polymers. However, compared to the conservation of host cells, viruses, especially RNA viruses and small DNA viruses, have a much higher rate of nucleotide substitution during replication. This is because viruses are more prone to genetic mutations during self-replication, causing the original drug targets to become ineffective. Furthermore, there are currently no drugs on the market targeting broad-spectrum viruses; only drugs targeting single (or similar) viruses exist, and these single (or similar) virus-targeting drugs often face the challenge of drug resistance. Therefore, in-depth research into the common mechanisms of interaction between viruses and host cells, and the discovery of new antiviral targets, are key issues that need to be addressed in the prevention and control of viral infectious diseases.

[0006] Bufalin and its derivatives (such as bufalin and bufalin) are extracted from toad venom (secretions of the Chinese giant toad), and belong to the steroidal class of compounds. 24 Characteristic structures include a hydroxyl group at C3 and an acetoxy group at C3. Bufotalin or its derivatives possess anticancer properties and can inhibit tumor cell proliferation, regulate the NF-κB pathway, and inhibit Na+. + / K + -ATPase activity and other functions, but there are no reports of bufotalin and its derivatives inhibiting multiple viruses. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the present invention provides the application of bufotalin and its derivatives in the preparation of drugs for the prevention and / or treatment of various viral infections.

[0008] In one aspect, the present invention provides the use of a drug in the preparation of at least one of the following products: A1) Products for the prevention and / or treatment of multiple viral infections; A2) Products that inhibit multiple viruses; A3) Products for the prevention and / or treatment of diseases caused by various viral infections; A4) Products that improve symptoms caused by various viral infections; The drug includes bufotalin or its derivatives or pharmaceutically acceptable salts thereof, or a substance with bufotalin or its derivatives or pharmaceutically acceptable salts thereof or a prodrug as its active ingredient; The drug can inhibit the replication of various viruses at the systemic or cellular level.

[0009] According to an embodiment of the present invention, the inhibition of multiple viruses works throughout the entire process of viral entry into the host or host cell (i.e., the entire infection cycle) and during the genome replication stage after entry into the cell.

[0010] According to embodiments of the present invention, bufotalin and its derivatives are used to prepare products for treating diseases caused by poxvirus, herpes simplex virus, coronavirus, enterovirus, flavivirus, respiratory syncytial virus, influenza virus, adenovirus, fever with viral infection, and / or for preparing products for preventing various viruses.

[0011] According to an embodiment of the present invention, the poxvirus is an orthopoxvirus, preferably the vaccinia virus Tian Tan strain (VACV_TT); the herpes simplex virus is herpes simplex virus type 1 (HSV-1); the enterovirus is a coxsackievirus, preferably coxsackievirus A9 (CV-A9); and the coronavirus is a porcine epidemic diarrhea virus, preferably porcine epidemic diarrhea virus strain CV777 (PEDV), pangolin coronavirus, or porcine severe acute diarrhea syndrome coronavirus, preferably porcine severe acute diarrhea syndrome coronavirus CN / GDWT / 2017 (SADS). -COV); flaviviruses include encephalitis virus and Zika virus, with Japanese encephalitis virus SA14 strain (JEV) and Zika virus FSS13025 strain (ZIKV) being preferred; fever with virus is Bunyavirus, with Bunyavirus SDTA-1 strain (SFTSV) being preferred; respiratory syncytial virus is respiratory syncytial virus type A2 (RSV-A); adenovirus is adenovirus type C5 (AdV-5); influenza virus is influenza A virus, with influenza A virus A / PR / 8 / 34 strain (PR8) being preferred.

[0012] According to an embodiment of the present invention, the bufotalin and its derivatives comprise the structure shown in the following formula:

[0013] R1 is selected from hydroxyl, hydroxyl C1-C3 alkyl, ester; R2 is H, hydroxyl; R3 is C1-C6 alkyl, hydroxyl, hydroxyl C1-C3 alkyl, acyl; R4 is H, ketone (oxo) or β-hydroxy; R5 is H, ketone (oxo) or β-hydroxy; R6 is C1-C6 alkyl; R7 is hydroxyl, C 14 -C 15 Epoxy group; R8 is hydrogen, hydroxyl, ester or acyl.

[0014] According to an embodiment of the present invention, the acyl group is a formyl group or an acetyl group.

[0015] According to an embodiment of the present invention, the C1-C6 alkyl group is methyl.

[0016] According to an embodiment of the present invention, the ester group is a formate group or an acetate group.

[0017] According to an embodiment of the present invention, the hydroxyalkyl group is hydroxymethyl.

[0018] According to embodiments of the present invention, the bufotaline and its derivatives comprise at least one of the following compounds: bufotaline (CAS: 471-95-4), cinobufotalin (CAS: 1108-68-5), telocinobufagin (CAS: 472-26-4), gamabufotalin (CAS: 465-11-2), areenobufagin (CAS: 464-74-4), cinobufagin (CAS: 470-37-1), and bufalin (CAS: 465-21-4). ), Resibufogenin (CAS:465-39-4), Acetylarenobufagin (CAS:184673-79-8), Pseudobufarenogin (CAS:17008-69-4), Deacetylcinobufotalin (CAS:4099-30-3), Resibufagin (CAS:20987-24-0), Cinobufaginol (CAS:6691-83-4), and Bufotalidin (CAS:465-90-7).

[0019] According to an embodiment of the present invention, the bufotalin exhibits extremely high sensitivity to adenovirus, respiratory syncytial virus, and porcine severe acute diarrhea syndrome coronavirus (SADS-CoV), and its EC50... 50 <10 nM. According to an embodiment of the present invention, the bufotalin exhibits extremely high sensitivity to poxvirus, enterovirus, pangolin coronavirus (GX_P2V), herpes simplex virus, Japanese encephalitis virus (JEV), and Zika virus (ZIKV), and its EC50... 50 <50 nM. According to an embodiment of the present invention, the bufotalin is highly sensitive to porcine epidemic diarrhea virus (PEDV) and Zika virus (ZIKV), and its EC50 value is <50 nM. 50 <100nM.

[0020] According to an embodiment of the present invention, the bufotalin exhibits extremely high sensitivity to fever accompanied by virus, and its EC50... 50 <10 nM. According to an embodiment of the present invention, the bufotalin exhibits extremely high sensitivity to adenovirus, enterovirus, herpes simplex virus, and Zika virus (ZIKV), and its EC50... 50 <50 nM. According to an embodiment of the present invention, the bufotalin exhibits high sensitivity to pangolin coronavirus (GX_P2V), and its EC50... 50 <100nM.

[0021] According to an embodiment of the present invention, the far-infrared bufotoxin exhibits extremely high sensitivity to poxvirus, adenovirus, respiratory syncytial virus, fever-associated virus, and Zika virus (ZIKV), and its EC50... 50 <10 nM. According to an embodiment of the present invention, the bufotoxin has extremely high sensitivity to enteroviruses, pangolin coronaviruses (GX_P2V), herpes simplex virus, Japanese encephalitis virus (JEV), and porcine severe acute diarrhea syndrome coronavirus (SADS-CoV), and its EC50... 50 <50 nM. According to an embodiment of the present invention, the far-infrared bufotoxin exhibits high sensitivity to porcine epidemic diarrhea virus (PEDV) and influenza virus, and its EC50... 50 <100nM.

[0022] According to embodiments of the present invention, the bufotalin exhibits extremely high sensitivity to poxvirus, adenovirus, enterovirus, herpes simplex virus, respiratory syncytial virus, swine severe acute diarrhea syndrome coronavirus (SADS-CoV), and fever-associated virus, and its EC50... 50 <10 nM. According to an embodiment of the present invention, the bufotalin exhibits extremely high sensitivity to pangolin coronavirus (GX_P2V), Japanese encephalitis virus (JEV), influenza virus, and Zika virus (ZIKV), and its EC50... 50 <50 nM. According to an embodiment of the invention, the bufotalin is highly sensitive to porcine epidemic diarrhea virus (PEDV), and its EC50... 50 <100nM.

[0023] According to an embodiment of the present invention, the sand toad extract exhibits extremely high sensitivity to poxvirus, adenovirus, enterovirus, herpes simplex virus, influenza virus, respiratory syncytial virus, swine severe acute diarrhea syndrome coronavirus (SADS-CoV), and fever with virus, and its EC 50 <10 nM. According to an embodiment of the present invention, the sand toad extract exhibits extremely high sensitivity to pangolin coronavirus (GX_P2V), Japanese encephalitis virus (JEV), porcine epidemic diarrhea virus (PEDV), and Zika virus (ZIKV), and its EC50 is <10 nM. 50<50 nM.

[0024] According to an embodiment of the present invention, the bufotoxin base exhibits extremely high sensitivity to enteroviruses, Japanese encephalitis virus (JEV), and respiratory syncytial virus, and its EC50... 50 <10nM. According to embodiments of the present invention, the bufotoxin base exhibits extremely high sensitivity to poxvirus, adenovirus, pangolin coronavirus (GX_P2V), herpes simplex virus, porcine epidemic diarrhea virus (PEDV), influenza virus, swine severe acute diarrhea syndrome coronavirus (SADS-CoV), and fever-associated virus, and its EC50... 50 <50 nM. According to an embodiment of the present invention, the bufotoxin gene is highly sensitive to Zika virus (ZIKV), and its EC50... 50 <100nM.

[0025] According to an embodiment of the present invention, the bufotoxin exhibits extremely high sensitivity to poxvirus, enterovirus, herpes simplex virus, influenza virus, respiratory syncytial virus, and swine severe acute diarrhea syndrome coronavirus (SADS-CoV), and its EC50... 50 <10 nM. According to an embodiment of the present invention, the bufotoxin exhibits extremely high sensitivity to adenovirus, pyrethroid virus, and Zika virus (ZIKV), and its EC50... 50 <50 nM. According to an embodiment of the present invention, the bufotoxin exhibits high sensitivity to pangolin coronavirus (GX_P2V), Japanese encephalitis virus (JEV), and porcine epidemic diarrhea virus (PEDV), and its EC50 value is <50 nM. 50 <100nM.

[0026] According to an embodiment of the present invention, the toad-like substance exhibits extremely high sensitivity to porcine epidemic diarrhea virus (PEDV), with an EC50 < 50 nM. According to an embodiment of the present invention, the toad-like substance exhibits high sensitivity to influenza virus, with an EC50 < 50 nM. 50 <100nM.

[0027] According to an embodiment of the present invention, the bufotoxin is highly sensitive to porcine epidemic diarrhea virus (PEDV), and its EC50... 50 <100nM.

[0028] According to embodiments of the present invention, the bufotoxin alcohol exhibits extremely high sensitivity to poxvirus, adenovirus, enterovirus, herpes simplex virus, Japanese encephalitis virus (JEV), influenza virus, respiratory syncytial virus, swine severe acute diarrhea syndrome coronavirus (SADS-CoV), fever-associated virus, and Zika virus (ZIKV), and its EC50... 50 <50 nM. According to an embodiment of the present invention, the bufotalin alcohol exhibits high sensitivity to pangolin coronavirus (GX_P2V), and its EC50...50 <100nM.

[0029] According to an embodiment of the present invention, the acetylasin exhibits extremely high sensitivity to poxvirus, adenovirus, enterovirus, herpes simplex virus, Japanese encephalitis virus (JEV), respiratory syncytial virus, and porcine severe acute diarrhea syndrome coronavirus (SADS-CoV), and its EC50... 50 <10 nM. According to an embodiment of the present invention, the acetylasin toxin exhibits extremely high sensitivity to pangolin coronavirus (GX_P2V), symptomatic virus, and Zika virus (ZIKV), and its EC50... 50 <50 nM. According to an embodiment of the present invention, the acetylastin exhibits high sensitivity to porcine epidemic diarrhea virus (PEDV) and influenza virus, and its EC50 content is <50 nM. 50 <100nM.

[0030] According to an embodiment of the present invention, the toad tadalafil exhibits extremely high sensitivity to poxvirus, adenovirus, enterovirus, herpes simplex virus, Japanese encephalitis virus (JEV), respiratory syncytial virus, swine severe acute diarrhea syndrome coronavirus (SADS-CoV), pangolin coronavirus (GX_P2V), septicemia with virus, Zika virus (ZIKV), swine epidemic diarrhea virus (PEDV), and influenza virus, and its EC50... 50 <10nM.

[0031] According to embodiments of the present invention, bufotalin and its derivatives exhibit good dose-dependent antiviral effects and low cytotoxicity against a variety of viral infections.

[0032] According to an embodiment of the present invention, bufotalin and its derivatives are used to inhibit viral nucleic acid expression, viral protein expression, and infectious particle formation after the virus enters the cell, thereby achieving an antiviral effect. According to an embodiment of the present invention, bufotalin and its derivatives are used to inhibit viral mRNA expression.

[0033] According to an embodiment of the present invention, the bufotalin and its derivatives are administered after the virus enters the cell and inhibit the nucleic acid expression, protein expression and infectious viral particle formation of orthopoxvirus in a dose-dependent manner.

[0034] According to an embodiment of the present invention, the dose-dependent effect is manifested as follows when the concentration of Bufo bufo increases: (1) the Virus / GAPDH mRNA expression ratio decreases; (2) the viral antigen expression level detected by IFA decreases; and (3) the number of infectious viral plaques decreases.

[0035] According to an embodiment of the present invention, when the bufotalin and its derivatives are applied after orchiopeptidosis virus enters the cell, they can still significantly inhibit viral protein expression (IFA) and infectious particle formation (PFU), and the effect is similar to that of the Full-Time group and significantly better than that of the Entry group.

[0036] According to an embodiment of the present invention, the viral infection level was detected by IFA. The Post-Entry administration group was similar to the Full-Time group, both significantly lower than the Entry group and the virus control group.

[0037] According to an embodiment of the present invention, viral infectivity is evaluated by plaque forming units (PFU). The number of plaques in the Post-Entry group is similar to that in the Full-Time group, but significantly lower than that in the Entry group and the virus control group.

[0038] According to an embodiment of the present invention, the bufotalin and its derivatives mainly exert antiviral effects in the post-viral entry stage, possibly by inhibiting viral gene expression or particle assembly, and have the potential for therapeutic use.

[0039] A second aspect of the present invention provides the use of a composition in the preparation of at least one of the following products: A1) Products for the prevention and / or treatment of multiple viral infections; A2) Products that inhibit multiple viruses; A3) Products for the prevention and / or treatment of diseases caused by various viral infections; A4) Products that improve symptoms caused by various viral infections; The inhibition of multiple viruses can be achieved by inhibiting the replication of multiple viruses at the organismal or cellular level. The composition comprises at least one of the following: bufotalin or its derivatives or pharmaceutically acceptable salts thereof, or a substance having bufotalin or its derivatives or pharmaceutically acceptable salts thereof or a prodrug as the active ingredient.

[0040] According to an embodiment of the present invention, the bufotalin and its derivatives are as described above.

[0041] According to an embodiment of the present invention, the composition further includes at least one other antiviral drug.

[0042] According to embodiments of the present invention, the other antiviral drugs include, but are not limited to: tecovirimat and cidofovir against poxviruses; acyclovir, valacyclovir, and famciclovir against herpes simplex virus; sofosbuvir, ribavirin, and daclatasvir against flaviviruses; and palilizumab against respiratory syncytial virus. For example, ribavirin and mab; for adenovirus, cidofovir and brincidofovir; for coronavirus, remdesivir, nimatrelvir / ritonavir and molnupiravir; for influenza virus, oseltamivir, zanamivir and baloxavir; and for fever with virus, ribavirin and favipiravir.

[0043] According to embodiments of the present invention, the composition can be administered in various forms such as tablets, capsules, injections, or inhalers, depending on the type of virus, the site of infection, and the patient's condition.

[0044] In a second aspect, the present invention provides a composition comprising the above-mentioned bufotalin or its derivatives or its pharmaceutically acceptable salts or prodrugs.

[0045] According to an embodiment of the present invention, the composition further includes at least one other antiviral drug.

[0046] According to an embodiment of the present invention, the other antiviral drugs are as described above.

[0047] According to embodiments of the invention, the composition further includes a pharmaceutically acceptable carrier or excipient. Examples include fillers, diluents, lubricants, surfactants, flow aids, binders, dispersants, suspending agents, disintegrants, penetrants, tableting aids, chemical enhancers (cell membrane disordered compounds and solvents or binary systems containing cell membrane disordered compounds and solvents), water-soluble polymers, water-swellable polymers (penetrating polymers or hydrogels), copolymers (plasticizers and thickeners), homopolymers, matrix materials, pH adjusters, pigments, antioxidants, thickeners, film-forming agents, granulation aids, flavoring agents (flavoring agents), soluble polymer matrices, sweeteners, coating agents, solubilizers, and combinations thereof. In another embodiment, depending on the desired release profile, the oral solid dosage form of the invention may contain appropriate amounts of controlled-release agents, extended-release agents, or modulating-release agents.

[0048] Compared with the prior art, the present invention has the following beneficial effects: This invention screens bufotalin and its derivatives, which have broad-spectrum antiviral activity, from antiviral drugs based on multiple viral drug screening models, and has important clinical application value.

[0049] The bufotalin and its derivatives of this invention exhibit good inhibitory activity against various viruses, such as poxvirus, herpes simplex virus, flavivirus, respiratory syncytial virus, adenovirus, coronavirus, influenza virus, and febrile virus. Therefore, the bufotalin and its derivatives of this invention have important clinical application value in the treatment of diseases caused by various viruses, such as poxvirus, herpes simplex virus, flavivirus, respiratory syncytial virus, adenovirus, coronavirus, influenza virus, and febrile virus.

[0050] In this invention, bufotalin and its derivatives exhibit good inhibitory activity against a variety of viruses. At the cellular level, most bufotalin and its derivatives show excellent antiviral efficacy (90%~99%) at the nanomolar (nM) level. These results indicate that bufotalin and its derivatives have significant potential clinical application value in the pre-remission and / or treatment of viral infections. Attached Figure Description

[0051] Figure 1 This is a graph showing the initial screening results of the drug library.

[0052] Figure 2 bufotalin is an EC2 inhibitor for multiple viruses. 50 CC 50 And SI.

[0053] Figure 3 Huachanotoxin is an EC2 inhibitor for multiple viruses. 50 CC 50 And SI.

[0054] Figure 4 For the EC of Yuanhua Bufotoxin against multiple viruses 50 CC 50 And SI.

[0055] Figure 5 EC for bufotalin against multiple viruses 50 CC 50 And SI.

[0056] Figure 6 EC for sand toad essence against multiple viruses 50 CC 50 And SI.

[0057] Figure 7 EC2 of bufotoxin against multiple viruses 50 CC 50 And SI.

[0058] Figure 8 EC50 for Bufotoxin against multiple viruses 50 CC 50 And SI.

[0059] Figure 9 EC for Toad Power Su against multiple viruses 50 CC 50 And SI.

[0060] Figure 10 EC50 of deacetylated bufotenoids against multiple viruses 50 CC 50 And SI.

[0061] Figure 11 EC50 of bufotalin against multiple viruses 50 CC 50 And SI.

[0062] Figure 12 EC50 of bufotalin against multiple viruses 50 CC 50 And SI.

[0063] Figure 13 EC50 of acetylsalicylic acid against multiple viruses 50 CC 50 And SI.

[0064] Figure 14 EC50 of pseudobufotoxin against multiple viruses 50 CC 50 And SI.

[0065] Figure 15 EC2 inhibitors for toads against multiple viruses 50 CC 50And SI.

[0066] Figure 16 The results are from the gradient effectiveness experiment of bufotoxin.

[0067] Figure 17 The TOA test results are for bufotoxin. Detailed Implementation

[0068] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0069] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions.

[0070] The term "acyl" refers to a group containing a carbonyl group bonded to a radical. These radicals may include, but are not limited to, groups selected from: hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, alkoxyalkyl, haloalkoxy, aryl, heterocyclic, heteroaryl, alkylsulfinylalkyl, alkylsulfonylalkyl, aralkyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, alkylthio, arylthio, amino, alkylamino, dialkylamino, arylalkoxy, arylthio, and alkylthioalkyl. Non-limiting examples of "acyl" include formyl, acetyl, benzoyl, trifluoroacetyl, phthalyl, malonyl, nicotinyl, etc.

[0071] The term "ester group" refers to a functional group having the general formula –COOR (where R represents a straight-chain or branched alkyl, aryl, or heterocyclic group, etc.). "Ester group" includes, but is not limited to, structures such as methyl ester, ethyl ester, propyl ester, benzoyl ester, and alkoxycarbonyl. Unless otherwise specified, the R group in this invention can be a substituted or unsubstituted hydrocarbon group. As a structural modification unit, the ester group can be used to adjust properties such as the polarity, lipophilicity, bioavailability, or enzymatic stability of the target compound. For example, "ester group" refers to -CO-O-(C1-C5 alkyl) or -O-CO-(C1-C5 alkyl).

[0072] The term "hydroxyalkyl" refers to an alkyl group substituted with one or more (e.g., one, two, or three) hydroxyl groups. In one embodiment, the hydroxyalkyl group is a monohydroxyalkyl group, i.e., substituted with one hydroxyl group. In another embodiment, the hydroxyalkyl group is a dihydroxyalkyl group, i.e., substituted with two hydroxyl groups. In yet another embodiment, the hydroxyalkyl group is selected from C1-C4 hydroxyalkyl groups. Non-limiting examples of hydroxyalkyl groups include hydroxymethyl, hydroxyethyl, hydroxypropyl, and hydroxybutyl, such as 1-hydroxyethyl, 2-hydroxyethyl, 1,2-dihydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 3-hydroxybutyl, 4-hydroxybutyl, 2-hydroxy-1-methylpropyl, and 1,3-dihydroxypropyl-2-yl.

[0073] The term "alkoxy" refers to an optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, or optionally substituted alkynyl group attached to a terminal oxygen atom. In one embodiment, the alkoxy group is selected from C1-C4 alkoxy groups. In another embodiment, the alkoxy group is selected from C1-C4 alkyl groups attached to a terminal oxygen atom, such as methoxy, ethoxy, and terbutoxy.

[0074] The viral strains used in the embodiments of this invention include: CV-A9, which is Coxsackievirus A9 strain BUCT01, with its accession number at the China General Microbiological Culture Collection Center (CGMCC No.). 20091; RSV-A is respiratory syncytial virus type A2, with accession number VR1540 at the American Type Culture Collection (ATCC); SFTSV is Bunyavirus SDTA-1 strain for fever with thrombocytopenia syndrome; SADS-CoV is porcine severe acute diarrhea syndrome coronavirus CN / GDWT / 2017, with GenBank accession number MG557844; GX_P2V is pangolin coronavirus, with GenBank accession number MT072864.1; PEDV is porcine epidemic diarrhea virus CV777 strain, with GenBank accession number AF353511.1; ZIKV is Zika virus FSS13025 strain, with GenBank accession number KU955593.1; JEV is Japanese encephalitis virus SA14 strain, with GenBank accession number KY683775.1.

[0075] The virus titration assay (TCID) used in the embodiments of this invention 50 Experiment): 2.5 × 10 4 Cells were seeded into 96-well plates and incubated at 37°C with 5% CO2 for 24 h. The viral stock solution was then serially diluted 10-fold to a final concentration of 10-1. -9 Discard the complete culture medium from the 96-well plate and add 10... -2 ~10 -91 ml of virus dilution was inoculated into each well of a 96-well plate at a rate of 100 μL (each dilution was inoculated in parallel with a total volume of 1 mL). After 72 h of incubation, CPE characterization of each well was observed and statistically analyzed. The results were then calculated using Reed-Muench to obtain the virus titer.

[0076] The following examples use GraphPad statistical software to process the data. The experimental results are expressed as mean ± standard deviation, and a t-test is used. This indicates a significant difference (P < 0.1). This indicates a significant difference (P < 0.01). This indicates a highly significant difference (P < 0.001). This indicates a highly significant difference (P < 0.0001).

[0077] The compound structures used in the following examples are shown in the table below.

[0078]

[0079]

[0080]

[0081]

[0082] Example 1: Cell Culture and Virus Culture 1. Cell Culture African green monkey kidney cell line Vero E6, human rhabdomyosarcoma cell line RD, human liver cancer cell line Huh7, human laryngeal epidermoid carcinoma cell line Hep-2, and canine kidney cell line MDCK were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM medium (Gibco) containing 10% fetal bovine serum (FBS; Gibco Invitrogen) at 37°C in a 5% CO2 incubator.

[0083] 2. Virus culture The Coxsackievirus A9 (CV-A9) strain BUCT01 was amplified and its titer was determined in RD cells. Vaccine virus Tiantan strain (VACV_TT), adenovirus C type 5 strain (AdV-5), herpes simplex virus type 1 (HSV-1), porcine epidemic diarrhea virus CV777 strain (PEDV), pangolin coronavirus (GX_P2V), fever with thrombocytopenia syndrome Bunyavirus SDTA-1 strain (SFTSV), Zika virus FSS13025 strain (ZIKV), and Japanese encephalitis virus SA14 strain (JEV) were amplified and titers determined in Vero E6 cells; porcine severe acute diarrhea syndrome coronavirus (SADS-CoV) CN / GDWT / 2017 strain was amplified and titers determined in Huh 7 cells; respiratory syncytial virus type A 2 (RSV-A) was amplified and titers determined in Hep-2 cells; and influenza A virus A / PR / 8 / 34 strain (PR8) was amplified and titers determined in MDCK cells. All infection experiments were performed in a biosafety level 2 (BSL-2) laboratory.

[0084] Example 2: Initial drug screening in FDA-approved drug libraries and natural product libraries In 96-well cell culture plates pre-seeded with Vero E6 cells, monomeric drugs (at a concentration of 20 μM) from the FDA-approved drug library and natural product library corresponding to the MOI, VAV_TT and GLPBIO, were added. After 24 h of culture, the relative expression of Virus / GAPDH mRNA was detected by nucleic acid extraction, reverse transcription, and RT-qPCR. Based on the results, the antiviral efficacy of the monomeric components was evaluated. Active components with an inhibition rate greater than 90% were considered potential antiviral components for further antiviral activity verification and preliminary mechanism studies. The initial screening results of the drug library are shown in the figure below. Figure 1 As shown.

[0085] Figure 1 The results showed that, through systematic screening, arenobufagin, cinobufagin, bufalin, and resibufogenin, among the active compounds, exhibited significant inhibitory effects on VCV_TT infection at a concentration of 10 μM, with inhibition rates all greater than 95%. After reviewing the literature, nine other bufalin-based compounds, including bufalin, were purchased, and their broad-spectrum antiviral activities were tested.

[0086] Example 3: Bufotalin and its derivatives inhibit the EC of various viruses 50 Measurement 1. 2.5 × 10 4Cells were seeded into 96-well plates and cultured at 37°C with 5% CO2 for 24 h. Then, serial dilutions of bufotalin or its derivatives were added to the cell culture wells at final concentrations of 5.00 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, 0.15625 μM, 0.78125 μM, 0.040 μM, 0.020 μM, 0.010 μM, 0.005 μM, 0.0025 μM, 0.00125 μM, and 0 μM, respectively. Cytopathic effects were observed under a microscope after infection with different viruses (multiple-infection number and time are shown in the table below; MOI was calculated from the virus titer determined in Example 1). Table 1. Multiples of Infection (MOI) and Time for Different Viruses

[0087] 2. Real-time quantitative PCR was used to quantitatively detect the expression of viral RNA and the intracellular reference gene GAPDH in cells. GraphPad-Prism 8 software was used for data analysis and EC50 calculation. 50 The result is as follows Figures 2-15 As shown.

[0088] The primers used are shown in Table 2, the SYBR-Green amplification procedure is shown in Table 3, and the reaction system is shown in Table 4.

[0089] Table 2 Primer sequences used in the study

[0090] Table 3. Dye-based qPCR amplification program

[0091] Table 4 Dye reaction system

[0092] Example 4: Bufotalin and its derivatives inhibit the C-cell activity of various viruses. 50 Measurement CC was performed using the CellTiter-Blue method. 50 The detection. 2.5 × 10 4Cells were seeded into 96-well plates, and experiments were conducted when the cell density reached 60%-80%. After changing the medium, diluted drug was added to final concentrations of 5.00 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, 0.15625 μM, 0.78125 μM, 0.040 μM, 0.020 μM, 0.010 μM, 0.005 μM, 0.0025 μM, 0.00125 μM, and 0 μM. Cells were incubated at 37℃ with 5% CO2 for 48 h. The luminescence intensity at 593 nm was detected using CellTiter-Blue reagent, and CC was calculated using GraphPad-Prism 8 software. 50 .

[0093] EC 50 This refers to the drug concentration that can effectively inhibit viral infection in 50% of cells; the lower the value, the better the inhibitory effect on the virus. 50 This is the drug concentration at which 50% of cells become diseased. A higher value indicates lower cytotoxicity. The selectivity index (SI) is measured in CC. 50 With EC 50 The higher the ratio, the higher the likelihood of it becoming a drug.

[0094] Bufotalin and its derivatives inhibit the EC of various viruses 50 The measurement results are as follows Figures 2-15 As shown. Bufotalin and its derivatives have CC effects on various viruses. 50 The measurement results are as follows Figures 2-15 As shown in the figure. The left vertical axis represents the inhibition rate of bufotalin and its derivatives against viruses, the right vertical axis represents the toxicity of bufotalin and its derivatives to cells, and the horizontal axis represents the concentration of bufotalin and its derivatives.

[0095] Figure 2 The results showed that bufotalin was effective against the EC50 of different viruses. 50 =3.03~245.60 nM. Figure 3 The results showed that bufotalin was effective against the EC50 of different viruses. 50 =3.02~564.60 nM. Figure 4 The results showed that bufotalin was effective against the EC5 of different viruses. 50 =2.80~73.18 nM. Figure 5 The results showed that bufotalin was effective against different viruses and EC2. 50 =1.30~61.62 nM. Figure 6 The results showed that sand toad essence has an effect on the EC of different viruses. 50 =0.50~39.06 nM. Figure 7The results showed that the bufotoxin gene was effective against the EC of different viruses. 50 =1.56~76.86 nM. Figure 8 The results showed that bufotalin was effective against the EC50 of different viruses. 50 =1.12~78.50 nM. Figure 9 The results showed that Toad-like sclerotherapy is effective against the EC50 of different viruses. 50 =35.00~893.00 nM. Figure 10 The results showed that deacetylated toad venom is effective against the EC5 of different viruses. 50 =1.67~18.43 μM. Figure 12 The results showed that bufotalin is effective against the EC5 of different viruses. 50 =0.05~2.14 μM. Figure 13 The results showed that bufotalin has an effect on the EC50 of different viruses. 50 =11.30~150.30 nM. Figure 14 The results showed that acetylsalicylic acid bufotoxin is effective against the EC5 of different viruses. 50 =1.13~94.33 nM. Figure 14 The results showed that pseudobufotoxin has an effect on the EC50 response of different viruses. 50 =0.32~3.16 μM. Figure 15 The results showed that toad talidone is effective against the EC of different viruses. 50 =0.33~9.94nM. For a variety of viruses, most drugs at the nanomolar level have good antiviral efficacy (90%~99%), suggesting that bufotalin and its derivatives are potential broad-spectrum antiviral cell inhibitors.

[0096] Example 5: Verification of the effectiveness of Bufotatoxin in inhibiting multiple viruses and preliminary investigation of its mechanism. The efficacy of bufotoxin and orthopoxvirus was verified and the preliminary mechanism was investigated. Gradient effectiveness experiment: 2.5 × 10 4 Cells were seeded into 48-well plates, and gradient efficacy assays were performed when the cell density reached 70%–80%. 125 μL of bufotoxin dilution buffer (final concentrations of 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM) and 125 μL of VAV_TT with MOI=0.01 or MOI=0.1 were added to the cell culture wells, respectively. Three replicates were set up for qPCR, immunofluorescence (IFA), and plaque assays. Cytopathic effects were observed under a microscope after VAV_TT infection, and the expression of viral DNA and the internal reference gene GAPDH was quantitatively detected by qRT-PCR. Viral DNA expression levels were calculated using GraphPad-Prism 8 software.

[0097] Time of Addition (TOA): 2.5 × 10⁻⁶ mg / L of the drug was added at different time points. 4 Cells were seeded into 48-well plates, and TOA (Total Activity Acid) experiments were performed when the cell density reached 70%–80%. Four groups were set up: Full-Time, Entry, Post-Entry, and Vitamin C (VC) group. After aspirating the culture medium, 125 μL of VCV_TT (MOI = 0.1) was added to each well. Three replicates were set up for qPCR, immunofluorescence (IFA), and plaque assays, respectively. At 0 h, 125 μL of a 25 nM dilution of bufotoxin was added to the Entry and Full-Time groups, and 250 μL was added to the wells of the other groups. 250 μL of maintenance medium was also added to the CC group. The cells were then incubated in a CO2 cell culture incubator for 2 h; after 2 h, the cells were washed three times with PBS. 125 μL of a 25 nM dilution of bufotoxin was added to both the Post-Entry and Full-Time groups, and the volume of each group was brought up to 250 μL with maintenance medium. The cells were then incubated in a CO2 cell culture incubator for 12 h. The expression levels of viral DNA and the intracellular reference gene GAPDH were quantitatively detected by qRT-PCR. Viral DNA expression levels were calculated using GraphPad-Prism 8 software for data analysis.

[0098] Immunofluorescence (IFA): 2.5 × 10⁻⁶ 4 Cells were seeded into 48-well plates, and IFA experiments were performed when the cell density reached 70%–80%. After the preceding gradient effectiveness and TOA experiments, upon collection, the liquid in the 48-well plates was aspirated, and the cells were washed with PBS for 3 min each time, for a total of 3 times. 150 μL of ice-cold methanol was added to each well, and the plates were fixed at room temperature for 20 min. The ice-cold methanol was aspirated, and the cells were washed with PBS for 3 min each time, for a total of 3 times. 150 μL of diluted primary antibody was added to each well, and the plates were incubated at room temperature for 1 h or overnight at 4°C. The primary antibody was recovered, and the cells were washed with PBS for 3 min each time. 150 μL of diluted secondary antibody was added to each well, and the plates were incubated at room temperature in the dark for 1 h. The secondary antibody was recovered, and the cells were washed with PBS for 3 min each time. 150 μL of diluted DAPI (1:10000) was added to each well, and the plates were incubated at room temperature in the dark for 5 min. DAPI was recovered, and the cells were washed with PBS for 3 min each time. Finally, 150 μL of PBS was added to each well, and the plates were developed in the dark.

[0099] Plaque assay: Cells were seeded into four 6-well plates and the assay was performed when the cell density reached 40%–50%. Plaque samples from the gradient effectiveness assay or TOA assay were removed from the cryogenic freezer, thawed at room temperature, and mixed thoroughly by pipetting in a clean bench. The virus was diluted 10-fold, resulting in a sample dilution buffer of 10. -1 ~10 -6 Discard the complete culture medium from the 6-well plate and add 1 mL of sample diluent to each well. After 2 hours, discard the sample diluent, wash three times with PBS to thoroughly remove any virus particles not adsorbed to the cell surface, and then quickly add 2 mL of pre-prepared maintenance medium containing 2% methylcellulose to each well. Incubate in a CO2 cell culture incubator for 3 days. When empty plaques appear in the wells, add 1 mL of 4% paraformaldehyde fixative to each well and fix for at least 15 minutes. Discard the fixative, add an appropriate amount of crystal violet solution for staining, wash away excess dye after 10 minutes, and photograph and save the experimental data.

[0100] The gradient efficacy and TOA test results of bufotoxin are as follows: Figure 16-17 As shown.

[0101] The results of the gradient efficacy experiment of farhua bufotoxin against VACV_TT in Vero E6 cells are shown in [the table below]. Figure 16 .Depend on Figure 16 It was found that the inhibitory effect of bufotalin on orthopoxvirus was dose-dependent. Analysis of qPCR, IFA, and the number of infectious viral particles showed that the viral load increased with decreasing concentration of the bufotalin dilution, demonstrating from multiple perspectives that the inhibitory effect of bufotalin on orthopoxvirus was dose-dependent.

[0102] The TOA assay results of bufotoxin against VACV_TT in Vero E6 cells are shown below. Figure 17 .Depend on Figure 17 It was found that adding bufotalin dilution only in the post-invasion stage significantly reduced the relative expression level of orthopoxvirus nucleic acid, suggesting that bufotalin may play an inhibitory role in the post-invasion stage of orthopoxvirus. In the IFA imaging results, the Post-Entry group was similar to the Full-Time group, and the viral load was significantly lower than that of the Entry group and the virus control group. Regarding the number of infectious viral particles, the Post-Entry group and the Full-Time group showed almost the same number of plaques, while the number of plaques in the Entry group was on the same order of magnitude as that in the virus control group, suggesting that bufotalin plays a decisive role in the post-invasion stage. Both sets of data are consistent with the qPCR results, further verifying that bufotalin mainly functions in the post-invasion stage of the virus.

[0103] In summary, at the cellular level, bufotalin and its derivatives exhibit good dose-dependent antiviral efficacy and low cytotoxicity against various viral infections, and their effect against orthopoxvirus primarily occurs after cell entry. Currently, there are no specific broad-spectrum antiviral drugs; therefore, this invention has significant reference value for future clinical application of broad-spectrum antiviral drugs.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The use of a drug in the preparation of at least one of the following products, A1) Products for the prevention and / or treatment of multiple viral infections; A2) Products that inhibit multiple viruses; A3) Products for the prevention and / or treatment of diseases caused by various viral infections; A4) Products that improve symptoms caused by various viral infections; in, The drug includes bufotalin or its derivatives or pharmaceutically acceptable salts thereof, or substances with bufotalin or its derivatives or pharmaceutically acceptable salts thereof or prodrugs as active ingredients; The drug can inhibit the replication of various viruses at the systemic or cellular level; Optionally, the drug exerts an inhibitory effect throughout the entire process of viral infection, including before the virus enters the host cell, after entry into the cell, and during the viral genome replication stage.

2. The application as described in claim 1, characterized in that; The viruses mentioned include poxvirus, herpes simplex virus, coronavirus, enterovirus, flavivirus, respiratory syncytial virus, influenza virus, adenovirus, and fever with virus; Optionally, the viruses include poxvirus, herpes simplex virus type 1, Coxsackievirus, porcine epidemic diarrhea virus, pangolin coronavirus, porcine severe acute diarrhea syndrome coronavirus, encephalitis virus, Zika virus, Bunyavirus, respiratory syncytial virus, adenovirus, and influenza A virus.

3. The application as described in claim 1, characterized in that; The bufotalin and its derivatives include the structures shown in the following formula: R1 is selected from hydroxyl, hydroxyl C1-C3 alkyl, ester; R2 is H, hydroxyl; R3 is C1-C6 alkyl, hydroxyl, hydroxyl C1-C3 alkyl, acyl; R4 is H, ketone (oxo) or β-hydroxy; R5 is H, ketone (oxo) or β-hydroxy; R6 is C1-C6 alkyl; R7 is hydroxyl, C 14 -C 15 Epoxy group; R8 is hydrogen, hydroxyl, ester or acyl group; Optionally, the acyl group is a formyl group or an acetyl group; Optionally, the C1-C6 alkyl group is methyl; Optionally, the ester group is a formate group or an acetate group; Optionally, the hydroxyalkyl group is hydroxymethyl.

4. The application as described in claim 3, characterized in that; The bufotaline and its derivatives include at least one of the following compounds: bufotaline (CAS: 471-95-4), cinobufotalin (CAS: 1108-68-5), telocinobufagin (CAS: 472-26-4), gamabufotalin (CAS: 465-11-2), areenobufagin (CAS: 464-74-4), cinobufagin (CAS: 470-37-1), bufalin (CAS: 465-21-4), and bufotaline. (Resibufogenin, CAS:465-39-4), Acetylarenobufagin, Pseudobufarenogin, Deacetylcinobufotalin, CAS:4099-30-3, Resibufagin, CAS:20987-24-0, Cinobufaginol, 6691-83-4, and Bufotalidin, CAS:465-90-7.

5. The application as described in claim 1, characterized in that; The bufotalin and its derivatives have good dose-dependent antiviral effects and low cytotoxicity against a variety of viral infections. Optionally, the bufotalin and its derivatives exert antiviral effects during the post-viral entry stage; Optionally, the bufotalin and its derivatives are used to inhibit viral nucleic acid expression, viral protein expression and infectious particle formation after the virus enters the cell to achieve antiviral effects; Optionally, the bufotalin and its derivatives are used to inhibit viral nucleic acid expression, protein expression, and infectious virus particle formation in a dose-dependent manner after the virus enters the cell. Optionally, the dose-dependent effect is manifested as follows when the concentration of Bufo bufo increases: (1) the Virus / GAPDH mRNA expression ratio decreases; (2) the viral antigen expression level detected by IFA decreases; and (3) the number of infectious viral plaques decreases.

6. The use of a composition in the preparation of at least one of the following products: A1) Products for the prevention and / or treatment of multiple viral infections; A2) Products that inhibit multiple viruses; A3) Products for the prevention and / or treatment of diseases caused by various viral infections; A4) Products that improve symptoms caused by various viral infections; in, The composition can inhibit the replication of a variety of viruses at the organismal or cellular level; The composition comprises at least one of the following: bufotalin or its derivatives or pharmaceutically acceptable salts thereof, or a substance having bufotalin or its derivatives or pharmaceutically acceptable salts or prodrugs as active ingredients, as described in any one of claims 1-5.

7. The application as described in claim 6, characterized in that: The composition also includes at least one other antiviral drug; Optionally, the composition may further include a pharmaceutically acceptable carrier or excipient; Optionally, the composition can be administered in various forms such as tablets, capsules, injections, or inhalers.

8. A composition, characterized in that: The composition comprises bufotalin or its derivatives or its pharmaceutically acceptable salts or prodrugs as described in any one of claims 1-5.

9. The composition according to claim 8, characterized in that, The composition also includes at least one other antiviral drug.

10. The composition according to claim 8, characterized in that, The composition also includes a pharmaceutically acceptable carrier or excipient.

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