CN116528849A - Antiviral compounds and methods of use thereof - Google Patents

Abstract

Antiviral compounds and methods of using antiviral compounds are described. The compounds may be used in methods of reducing the rate of viral infection in a subject in need thereof and in methods of treating viral infection. The virus may be a coronavirus, such as SARS-CoV-2.

Description

Antiviral compounds and methods of use thereof

Cross Reference to Related Applications

The application claims the following benefits: U.S. provisional application No. 63/043,024, filed on 6/23 in 2020; U.S. provisional application No. 63/043,048, filed on even 23 in 6/2020; U.S. provisional application No. 63/043,054, filed on even 23 in 6/2020; U.S. provisional application No. 63/043,059, filed on even 23 in 6/2020; and U.S. provisional application No. 63/043,065 filed on day 23 of 6/6 in 2020. The entire teachings of the above application are incorporated herein by reference.

Background

Coronaviruses are a large family of viruses commonly found in humans and in animals of many different species. Some coronaviruses (e.g., SARS-CoV-2) can cause serious diseases in humans, particularly respiratory diseases.

Disclosure of Invention

The present invention relates to antiviral compounds and pharmaceutical compositions comprising such compounds; methods of reducing viral infection, replication, or viruses, for example, in a subject in need thereof using these compounds and compositions; and methods of treating viral infections in subjects in need thereof with these compounds and compositions. The compounds described herein can inhibit viral proteases and are useful, for example, as antiviral agents against: coronavirus infection that results in Severe Acute Respiratory Syndrome (SARS) (e.g., SARS coronavirus (SARS-CoV) infection); MERS coronavirus (MERS-CoV) that causes Middle East Respiratory Syndrome (MERS); and SARS-CoV-2 (also known as "2019 novel coronavirus" or "2019-nCoV") that causes the coronavirus disease 2019 (COVID-19).

In one aspect, the invention provides a pharmaceutical composition comprising a compound selected from the group consisting of: the compound of table 1 or table 3, or a pharmaceutically acceptable salt thereof, and a pharmaceutically suitable carrier.

In another aspect, the invention provides a method of reducing viral infection, replication and/or viruses in a subject, the method comprising administering to the subject a pharmaceutical composition comprising a compound selected from the group consisting of: the compound of table 1 or table 3, or a pharmaceutically acceptable salt thereof, and a pharmaceutically suitable carrier.

In another aspect, the invention provides a method of treating a subject diagnosed with a coronavirus-related disease (e.g., SARS, MERS, or covd-19), the method comprising administering to the subject a pharmaceutical composition comprising a compound selected from the group consisting of: the compound of table 1 or table 3, or a pharmaceutically acceptable salt thereof, and a pharmaceutically suitable carrier.

The compounds described herein are suitable for use in monotherapy or in combination therapy. For example, the methods described herein can include administering a plurality of compounds described herein, e.g., a plurality of compounds listed in table 1 or table 3. In some embodiments, a compound described herein is administered to a subject in combination with a second agent (e.g., a second antiviral compound, an anti-inflammatory agent, an anticoagulant, or an analgesic). In some embodiments, a plurality of compounds described herein (e.g., a plurality of compounds listed in table 1 or table 3) is administered to a subject in combination with another agent (e.g., another antiviral compound, an anti-inflammatory agent, an anticoagulant, or an analgesic).

In embodiments, the other agent or second agent is, for example, famprivir, gab Li Xiwei, adefovir, ifenprodil, lopinavir, ritonavir, BPI-002; steroids, e.g., dexamethasone; anticoagulants, such as heparin or enoxaparin; antibodies, e.g., antibodies to human granulocyte macrophage colony-stimulating factor (GM-CSF), e.g., TJM2, melitumumab; antibodies to human interleukin-6 (IL-6) or IL-6R, e.g., tobrazumab or AT-100 (rhSP-D); antibodies to CCR5, e.g., leno mab or combinations thereof. In embodiments, the other agent or the second agent is derived from convalescent plasma of a human infected with a coronavirus as described herein. In embodiments, the second agent is selected from the compounds of table 2 or a pharmaceutically acceptable salt thereof.

The compounds described herein (e.g., the compounds of table 1 or table 3) can be administered separately from the second agent (e.g., the compound of table 2). The compounds of table 1 or table 3 may be formulated as separate pharmaceutical compositions from the compounds of table 2. When formulated separately, the pharmaceutical compositions may be administered simultaneously or sequentially in any manner that achieves their intended purpose.

In other embodiments, a single pharmaceutical composition may include one or more compounds described herein (e.g., table 1 or table 3) and one or more of the second agents (e.g., table 2).

Drawings

The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

FIGS. 1A-B are cell viability curves of Vero E6 cells treated with various compounds (0.001-1000. Mu.M or vehicle control (DMSO)) for 48 hours, as assessed by CellTiterGlo. Data were normalized to untreated controls.

FIGS. 2A-O show the effect of pretreatment with various concentrations of food-derived compounds on the relative levels of SARS-CoV-2 viral load in Vero E6 cells and host cell numbers. Viral load is indicated by the relative level of immunofluorescent staining of the SARS-CoV-2N protein. Host cell numbers are indicated by the relative levels of DAPI positive immunofluorescent staining.

FIGS. 3A-G are dose response curves for several food-derived compounds against SARS-CoV-2 major protease (Mpro).

FIGS. 4A-G are dose response curves for food-derived compounds against SARS-CoV-2 papain-like protease (PLpro).

FIGS. 5A-F are graphs showing the effect of food-derived compounds on the relative host cell viability of SARS-CoV-2 infected Vero E6 cells with Sweden Wei Zuge.

Definition of the definition

As used herein, a "pharmaceutical composition" or "pharmaceutical formulation" is a composition or formulation, and/or its final dosage form or formulation, that has pharmacological activity or other direct impact on alleviating, treating, or preventing a disease, and is indicated for human use.

As used herein, the term "pharmaceutically acceptable carrier or excipient" means a formulation aid suitable for human use and generally is not toxic or inert. The pharmaceutically acceptable carrier or excipient may be a liquid, solid, or semi-solid filler, diluent, or encapsulating material. Some examples of materials that may serve as pharmaceutically acceptable carriers are: sugars such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol and phosphate buffer; other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate; colorants, release agents, coating agents, sweeteners, flavoring agents, and fragrances; preservatives and antioxidants can also be present in the composition at the discretion of the formulator.

As used herein, the term "pharmaceutically acceptable salts" refers to those salts that are, within the sound medical judgment, suitable for use in contact with the tissues of humans and other mammals, and commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are known in the art and are described, for example, in Gupta et al, 2018,Salts of Therapeutic Agents:Chemical,Physicochemical,and Biological Considerations [ salts of therapeutic agents: chemical, physicochemical and biological considerations.

As used herein, the term "subject" means an animal, preferably a mammal, e.g., a human, or a veterinary animal or agricultural animal. In embodiments, the subject is a non-human mammal, such as a non-human primate (e.g., monkey, ape), ungulate (e.g., cow, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horse, donkey), carnivorous (e.g., dog, cat), rodent (e.g., rat, mouse), or lagomorph (e.g., rabbit).

As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of the compositions described herein refer to the amounts as follows: when administered to a subject, including a mammal (e.g., a human), is sufficient to produce a beneficial or desired result, including an effect on cellular level, tissue level, or clinical outcome, and thus "effective" or synonyms thereof, depend on the context in which it is used. The amount of a given composition described herein corresponding to that amount will vary depending upon a variety of factors, such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, the identity of the subject (e.g., age, sex, weight) or host being treated, etc., but can still be routinely determined by one of skill in the art.

As used herein, "treatment" refers to the medical management of a subject that aims to improve, alleviate, stabilize (i.e., not worsen), prevent or cure a disease, pathological condition, or disorder. The term includes active therapy (therapy aimed at ameliorating a disease, pathological condition or disorder), causal therapy (therapy aimed at the cause of the associated disease, pathological condition or disorder), palliative therapy (therapy aimed at alleviating symptoms), prophylactic therapy (therapy aimed at minimizing or partially or completely inhibiting the development of the associated disease, pathological condition or disorder); and supportive treatment (treatment for supplementing another therapy). Treatment also includes reducing the extent of the disease or disorder (e.g., reducing viral infection, replication, and/or viruses); preventing the spread of a disease or disorder; delay or slow the progression of the disease or disorder; improving or alleviating a disease or condition; and mitigation (whether partial or complete), whether detectable or undetectable. By ameliorating or reducing a disease or disorder is meant that the extent of the disease, disorder or disorder and/or the time course of the reduction and/or progression of adverse clinical manifestations is slowed or prolonged as compared to the extent or time course in the absence of treatment. "treatment" may also mean prolonged survival compared to the expected survival without treatment. Those in need of treatment include those already with the condition or disorder, as well as those at risk of having the condition or disorder, or those in which the condition or disorder is to be prevented.

As used herein, "combination therapy" or "combined administration" means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or disorder. The treatment regimen defines the dose and period of administration of each agent such that the effects of the individual agents on the subject coincide. In some embodiments, two or more agents are delivered simultaneously or in parallel, and these agents may be co-formulated. In other embodiments, the two or more agents are not co-formulated, but are administered sequentially as part of a prescribed regimen. In some embodiments, two or more agents or treatments are administered in combination such that the decrease in symptoms or other parameters associated with the disorder is greater than the result that would be observed by delivering one agent or treatment alone or without the other. The effects of the two treatments may be partially additive, fully additive, or greater than additive (e.g., synergistic). Sequential or substantially simultaneous administration of the therapeutic agents may be accomplished by any suitable route including, but not limited to, oral route, intravenous route, intramuscular route, and direct absorption through mucosal tissue. The therapeutic agents may be administered by the same route or by different routes. For example, a first therapeutic agent of the combination may be administered by intravenous injection, while a second therapeutic agent of the combination may be administered orally.

Detailed Description

The description of the example embodiments is as follows.

Summary of the method

Extensive computer screening involving Artificial Intelligence (AI) algorithms was performed to identify compounds that might inhibit viral proteases. Approximately 40,000 compounds were screened for their ability to reduce viral replication of the novel SARS-CoV-2 coronavirus, and the compounds described herein were selected for further study.

Only a portion of those compounds selected for further investigation exhibited antiviral activity. Additional tests in the direct enzyme assay showed that these compounds inhibited the activity of two isolated SARS CoV-2 proteases, the main protease (Mpro) and papain-like protease (PLpro). Major proteases (Mpro) are required to cleave viral polyproteins, including those requiring viral replication. Inhibition of this protease prevents viral replication, and is marked as a possible therapeutic target for the prevention or treatment of SARS-CoV-2 infection (Sacco et al (2020); coelho et al (2020)). Similarly, papain-like protease (PLpro) is required for processing of multimeric proteins and represents an alternative therapeutic target for the treatment of SARS CoV-2 infection (Klemm et al (2020)).

Computer screening and laboratory experiments have highlighted the challenge of identifying compounds that are effective in inhibiting viral replication. About 40,000 compounds were screened in silico to identify compounds for further study. However, of those tested, only a few reduced viral load and inhibited viral proteases, mpro and/or PLpro.

Coronavirus

Coronaviruses are a large family of viruses commonly found in humans and in animals of many different species. Some coronaviruses may cause serious diseases in humans. Some significant coronaviruses include the Severe Acute Respiratory Syndrome (SARS) coronavirus (SARS-CoV), which causes SARS; MERS coronavirus (MERS-CoV) that causes Middle East Respiratory Syndrome (MERS); and SARS-CoV-2, which causes coronavirus disease 2019 (COVID-19).

Infection with coronavirus can lead to fever, cough and shortness of breath. Infections can be particularly dangerous in elderly people, people with weakened immune systems, and people with potentially healthy conditions (e.g., cardiovascular disease, diabetes, and chronic lung disease, etc.).

Antiviral compounds

Based on the in silico screening, the compounds of table 1 were identified as compounds that were effective in inhibiting viral proteases of coronaviruses. In one embodiment of any aspect of the invention, the compound is a compound listed in table 1 or a pharmaceutically acceptable salt thereof.

Table 1: antiviral compounds

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

/>

Examples of pharmaceutically acceptable salts

Examples of pharmaceutically acceptable salts of the compounds of table 1 and (where appropriate) table 2 include salts derived from: suitable inorganic and organic acids, and suitable inorganic and organic bases. Examples of pharmaceutically acceptable acid addition salts are salts of amino groups with inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid) or with organic acids (such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid), or by using other methods used in the art (such as ion exchange). Other pharmaceutically acceptable acid addition salts include adipates, alginates, ascorbates, aspartate, benzenesulfonates, benzoates, bisulfate, borates, butyrates, camphorates, camphorsulfonates, cinnamates, citrates, cyclopentanepropionates, digluconates, dodecylsulfate, ethanesulfonates, formates, fumarates, glucoheptonates, glycerophosphate, gluconate, glutarates, glycolates, hemisulfates, heptanoates, caprates, hydroiodides, hydroxybenzoates, 2-hydroxy-ethanesulfonates, hydroxymaleates, lactonates, lactates, laurates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmates, pamonates, pectinates, persulfates, 2-phenoxybenzoates, phenylacetates, 3-phenylpropionates, phosphates, pivalates, propionates, pyruvates, salicylates, stearates, succinates, sulfates, tartaric acid salts, thiocyanates, p-toluenesulfonates, undecanoates, valerates, and the like.

Mono-, di-or tri-acid salts may be formed and such salts may exist in hydrated, solvated or substantially anhydrous form.

Salts derived from suitable bases include salts derived from inorganic bases (e.g., alkali metals, alkaline earth metals, and ammonium bases), salts derived from aliphatic, alicyclic, or aromatic organic amines (e.g., methylamine, trimethylamine, and picoline), or n+ ((C1-C4) alkyl) 4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, barium, and the like. Additional pharmaceutically acceptable salts include nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions (e.g., halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate) as appropriate.

Pharmaceutical composition

The pharmaceutical compositions described herein comprise a therapeutically effective amount of a compound of table 1, or a pharmaceutically acceptable salt thereof, formulated with one or more pharmaceutically acceptable carriers or excipients. The pharmaceutical composition may be administered orally, parenterally, by inhalation spray, topically, or via an implanted reservoir.

The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. Liquid formulations for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable formulations, such as sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Acceptable vehicles and solvents that may be employed are water, ringer's solution, u.s.p. And isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids (such as oleic acid) are useful in the preparation of injectables.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound may be admixed with at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and/or: a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and silicic acid, b) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia, c) wetting agents, such as glycerol, d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, e) solution retarding agents, such as paraffin, f) absorption accelerators, such as quaternary ammonium compounds, g) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents, such as kaolin and bentonite clays, and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like, can also be used, as are solid compositions of similar type for use as fillers in soft and hard-filled gelatin capsules. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents, and may also be compositions such that they release one or more active ingredients in a delayed manner, optionally only in or preferably in a certain part of the intestinal tract. Examples of embedding compositions that can be used include polymeric substances and waxes.

Compositions suitable for buccal or sublingual administration include tablets, troches and lozenges wherein the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth, or gelatin and glycerin.

Dosage forms for topical or transdermal administration of the compounds of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier and any required preservatives or buffers as required. Ophthalmic formulations, ear drops, ophthalmic ointments, powders and solutions are also contemplated as falling within the scope of the present invention. Ointments, pastes, creams and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

In addition to the compounds of the invention, powders and sprays can contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder or mixtures of these substances. The spray may additionally contain conventional propellants such as chlorofluorohydrocarbons. Transdermal patches have the additional advantage of providing controlled delivery of compounds to the body. Such dosage forms may be prepared by dissolving or dispersing the compound in an appropriate medium. Absorption enhancers may also be used to increase the transdermal flux of a compound. This rate may be controlled by providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

For pulmonary delivery, the therapeutic composition is formulated in solid or liquid particulate form and administered to the subject by direct administration (e.g., inhalation into the respiratory system). Solid or liquid particulate forms of the active compounds prepared for the practice of the present invention include inhalable sized particles: i.e. particles of a size small enough to pass through the mouth and throat into the bronchi and alveoli of the lung upon inhalation. Delivery of aerosolized therapeutic agents, particularly aerosolized antibiotics, is known in the art (see, e.g., van Devanter et al, U.S. Pat. No. 5,767,068, smith et al, U.S. Pat. No. 5,508,269 to Montgomery, WO 98/43650 to Germinario et al, U.S. Pat. No. 9,956,360 to Germinario et al, U.S. patent publication No. 2020/0170301A1, and PCT publication No. WO 2020/072478 A1, all of which are incorporated herein by reference in their entirety).

Therapeutic method

In the methods described herein, a viral infection or disorder in a subject (e.g., a human or another animal) is treated by administering to the subject a therapeutically effective amount of a compound or composition described herein in an amount and for a time necessary to achieve the desired result. An effective amount of a compound described herein may range from about 0.01mg/kg to about 500mg/kg, for example, from about 0.01 to about 50mg/kg, from 0.1 to 50mg/kg, or from 0.1 to 25mg/kg of body weight. The effective amount or dose will also vary depending on the route of administration and the possibility of co-use with other agents.

The total daily dose of the compound administered to a human or other animal in a single dose or divided doses may be divided amounts. A single dose composition may include this amount or an approximate amount (sub-multiple) thereof to make up a daily dose. Treatment regimens for the compounds and methods described herein may comprise administration of about 10mg to about 1000mg, or in some cases, more than 1000mg of one or more compounds per day, in single or multiple doses, to a patient in need of such treatment.

Dosages below or above those described above may be required. The particular dosage and treatment regimen for any particular patient will depend upon a variety of factors including the activity of the particular compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, the condition or symptoms, the patient's disposition to the disease, condition or symptoms, and the discretion of the treating physician.

Combination therapy

In certain embodiments, the methods described herein comprise administering a combination of a compound described herein and one or more additional therapeutic or prophylactic agents. In some embodiments, the compound and the additional agent are co-formulated. In another embodiment, the compound and the additional therapeutic agent are co-administered, but are administered in different formulations.

In combination therapy, both the compound and the additional agent should generally be present at a dosage level of between about 1% and 100%, and more preferably between about 5% and 95% of the normal administered dosage in a monotherapy regimen. The additional agents may be administered separately from the compounds of the invention as part of a multi-dose regimen. Alternatively, those agents may be part of a single dosage form, mixed together with the compounds of the invention in a single composition. The "additional therapeutic or prophylactic agent" may include, but is not limited to, immunotherapy (e.g., interferon), therapeutic vaccine, anti-inflammatory agents (e.g., angiotensin-converting enzyme 2 (ACE 2) inhibitors, corticosteroids or NSAIDs), bronchodilators (e.g., beta-2 adrenergic agonists and xanthines (e.g., theophylline)), mucolytics, antimuscarinics, anti-leukotrienes, cell adhesion inhibitors (e.g., ICAM antagonists), cytokine antagonists, pulmonary surfactants, and/or antimicrobial and antiviral agents.

Table 2: additional (second) therapeutic agent

/>

/>

/>

/>

/>

Where appropriate, the agents of table 2 may be formulated as pharmaceutically acceptable salts as described herein.

Synthesis method

Methods of synthesizing the compounds herein will be apparent to those of ordinary skill in the art. Synthetic chemical transformations and protecting group methods for synthesizing the compounds described herein are known in the art and include, for example, methods as described in the following documents: larock (eds.), comprehensive Organic Transformations [ comprehensive organic transformations ], volume 4: AGuide to Functional Group Preparations [ guidelines for the preparation of functional groups ], wiley [ Weily Verlag ]. 3 rd edition (2018); wuts, greene's Protective Groups in Organic Synthesis [ protecting group in green organic synthesis ], wiley [ weili press ] 5 th edition (2014); ho, fiesers' Reagents for Organic Synthesis [ Fei Saier ] organic synthetic reagent (book 29), wiley [ Weily Verlag ] (2019); paquette (editions), encyclopedia of Reagents for Organic Synthesis [ encyclopedia of organic synthetic reagents ], wiley [ Weily Verlag ] (2009). The compounds of the invention may be modified by the addition of suitable functional groups to enhance selective biological properties. Such modifications are known in the art and may include those that increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility in order to allow administration by injection, alter metabolism, and alter excretion rates.

Cell and vesicle based vectors

The compounds or compositions described herein may be administered into vesicle-or other membrane-based carriers.

In embodiments, the compounds or compositions described herein are administered in or via a cell-based, vesicle, or other membrane carrier. In one embodiment, the compound or composition may be formulated in a liposome or other similar vesicle. Liposomes are spherical vesicle structures composed of a lipid bilayer of one or more layers surrounding an inner aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be anionic, neutral or cationic. Liposomes are biocompatible, non-toxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasmatic enzymes, and load transport across the biological membrane and the Blood Brain Barrier (BBB) (for reviews see, e.g., sphch and Navarro, journal of Drug Delivery [ journal of drug delivery ], volume 2011, article ID 469679, page 12, 2011.doi:10.1155/2011/469679).

Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to form liposomes as drug carriers. Methods for preparing multilamellar vesicle lipids are known in the art (see, e.g., U.S. patent No. 6,693,086, the teachings of which are incorporated herein by reference for the preparation of multilamellar vesicle lipids). Although vesicle formation is spontaneous when lipid membranes are mixed with aqueous solutions, vesicle formation can also be accelerated by applying force in the form of oscillation using a homogenizer, sonicator or squeeze device (for reviews see, e.g., sphch and Navarro, journal of Drug Delivery [ journal of drug delivery ], volume 2011, article ID 469679, page 12, 2011.doi:10.1155/2011/469679). The extruded lipids may be prepared by extrusion through a filter having a reduced size, as described in Templeton et al, nature Biotech [ Nature Biotech ],15:647-652,1997, the teachings of which are incorporated herein by reference for the preparation of extruded lipids.

Lipid nanoparticles are another example of a carrier that provides a biocompatible and biodegradable delivery system for the compounds or compositions described herein. Nanostructured Lipid Carriers (NLCs) are modified Solid Lipid Nanoparticles (SLNs) that retain the characteristics of SLNs, improve drug stability and loading capacity, and prevent drug leakage. Polymeric Nanoparticles (PNPs) are an important component of drug delivery. These nanoparticles can effectively direct drug delivery to specific targets and improve drug stability and controlled drug release. Lipid polymer nanoparticles (PLN), a novel carrier combining liposomes and polymers, can also be used. These nanoparticles have the complementary advantage of PNP and liposomes. PLN is composed of a core-shell structure; the polymer core provides a stable structure and the phospholipid shell provides good biocompatibility. Thus, the two components increase the drug encapsulation efficiency, promote surface modification, and prevent leakage of the water-soluble drug. For reviews, see, for example, li et al 2017, nanomaterials [ nanomaterials ]7,122; doi 10.3390/nano7060122.

Additional non-limiting examples of carriers include carbohydrate carriers (e.g., anhydride modified phytoglycogen or glycogen type materials), protein carriers (e.g., proteins covalently linked to cyclic polyribonucleotides), or cationic carriers (e.g., cationic lipopolymers or transfection reagents). Non-limiting examples of carbohydrate carriers include phyto-octenyl succinate, phyto-glycogen beta-dextrin, and anhydride modified phyto-glycogen beta-dextrin. Non-limiting examples of cationic carriers include lipofectamine (lipofectamine), polyethylenimine, poly (trimethyl imine), poly (tetramethyl imine), polypropylenimine, aminoglycoside-polyamine, dideoxy-diamino-B-cyclodextrin, spermine, spermidine, poly (2-dimethylamino) ethyl methacrylate, poly (lysine), poly (histidine), poly (arginine), cationic gelatin, dendrimers, chitosan, 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP), N- [1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTMA), 1- [2- (oleoyloxy) ethyl ] -2-oleyl-3- (2-hydroxyethyl) imidazolium chloride (DOTIM), 2, 3-dioleoyloxy-N- [2 (spermimido) ethyl ] -N, N-dimethyl-1-trifluoroammonium acetate (DOSPA), 3B- [ N- (N, N' -dimethylaminoethane) -carbamoyl ] cholesterol hydrochloride (DC-cholesterol HCl), di-heptadecylaminocardamine (DOGS), N-distearyl-N, N-dimethyl ammonium bromide (DDAB), N- (1, 2-dimyristoxyprop-3-yl) -N, N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE) and N, N-dioleyl-N, N-dimethyl ammonium chloride (DODAC). Non-limiting examples of protein carriers include Human Serum Albumin (HSA), low Density Lipoprotein (LDL), high Density Lipoprotein (HDL), or globulin.

Exosomes may also be used as drug delivery vehicles for the compounds or compositions described herein. For review, see Ha et al, 2016, 7, acta Pharmaceutica SinicaB, journal of pharmacy, volume 6, stage 4, pages 287-296; https:// doi.org/10.1016/j.apsb.2016.02.001.

Ex vivo differentiated erythrocytes can also be used as carriers for the compounds or compositions described herein. See, for example, WO 2015073587; WO 2017123646; WO 2017123644; WO 2018102740; wO 2016183482; WO 2015153102; WO 2018151829; WO 2018009838; shi et al 2014.Proc Natl Acad Sci USA [ Proc. Natl. Acad. Sci. USA ].111 (28): 10131-10136; us patent 9,644,180; huang et al 2017.Nature Communications [ Nature communication ]8:423; shi et al 2014.Proc Natl Acad Sci USA [ Proc. Natl. Acad. Sci. USA ].111 (28): 10131-10136.

For example, fusion compositions as described in WO 2018208728 may also be used as a carrier for delivering a compound or composition described herein.

Virosomes and virus-like particles (VLPs) may also be used as vectors for delivering the compounds or compositions described herein to targeted cells.

For example, plant nanovesicles and Plant Messenger Packages (PMPs) as described in WO 2011097480, WO 2013070324, WO 2017004526, or WO 2020041784 may also be used as carriers for delivering the compounds or compositions described herein.

Illustrative examples

Example 1: preparation of the Compounds

This example describes the preparation of the compounds described herein.

Example 1.1: hypericin

Hypericin was purchased from Sigma Aldrich (SKU 56690). Hypericin can be extracted from Hypericum perforatum using ethanol and high pressure according to the protocol in [ Cossua et al (2011) Journal of Food Process Engineering [ journal of food processing engineering ], volume 35, pages 222-235, phase 2 DOI:10.1111/j.1745-4530.2010.00583.X ].

Example 1.2: wheat nutrient

The extraction of hordeins from dry buckwheat herbs was performed according to the protocol in [ Hinneburg and Neubert (2005) J.Agric.food Chem. [ J.agricultural and food chemistry ], vol.53, 1 st, pages 3-7 https:// doi.org/10.1021/jf049118f ].

Example 1.3: original hypericin

The procyanidins are obtained by subsequent oxidation of emodin dianthrone with oxygen in methanol containing triethylamine according to the protocol in [ Barnard, d.l. et al (1992) Antiviral Research [ antiviral study ]. 17 volume: pages 63-77. PMID:1310583 ].

Example 1.4: ficus simplicissima extract

Pseudohypericin was purchased from sigma aldrich (CAS No. 55954-61-5). Briefly, the isolation of pseudohypericin from Hypericum perforatum by purification using hydroalcoholic dry extracts and column chromatography can be confirmed using liquid chromatography-mass spectrometry according to the protocol in [ Karioti et al, (2009), J.Sep.science [ journal of separation science ], vol.32:1374-1382, https:// doi.org/10.1002/jssc.200800700 ].

Example 1.5: artemisinin A

Artemisinin A was extracted from dry root bark or pineapple in n-hexane, benzene and acetone, followed by column chromatography and filtration according to the protocol in [ Hano Y et al, (1989) Heterocycles [ heterocycle ] volume 29:1447-1453, DOI:10.3987/COM-89-5019 ].

Example 1.6: juglone (Sanjuglone)

Juglone was extracted from the dry bark of walnuts (common walnut tree) in hexane, chloroform, ethyl acetate and methanol, concentrated and separated by silica gel column chromatography according to the protocol in [ Strugstad, m., and desmotovski, s. (2013). Journal of Ecosystems and Management [ society for ecosystem and management ], volume 13 (3) pages 1-16 ].

Example 1.7: mahonia stem tanning booth

The equisetum was purchased from Nacalai USA. According to [ Rodrigues et al, mar Drugs [ marine pharmaceuticals ] (2019) for 7 months; the protocol in volume 17 (7): page 403 ] also isolated vain from lilac or mangrove.

Example 1.8: new suona extract I

New suona extract I was purchased from Nacali corporation of America.

Example 1.9: lignin

The lignin was isolated from Alnus alnus according to the protocol in Hirokane et al A unified strategy for the synthesis of highly oxygenated diaryl ethers featured in ellagitannins [ unified strategy for high oxygen containing diaryl ethers in synthetic ellagitannins ] [ Hirokane et al (2014) Nature Communications [ Nature communication ] Vol. 5:3478. DOI: 10.1038/ncoms 4478 ].

Example 1.10: chestnut tree ellagic agent

Chestnut ellagic agent is available from sigma aldrich.

Example 1.11: punicalagin

Punicalagin was purchased from sigma aldrich.

Example 1.12: theaflavin

Theaflavins are purchased from sigma aldrich. Described in US 2008/0254190 A1, urea is typically extracted from tea and purified by HPLC.

Example 1.13: theaflavin-3-gallate

Theaflavin-3-gallate was purchased from sigma aldrich. Described in US 2008/0254190 A1, urea is typically extracted from tea and purified by HPLC.

Example 1.14: hinokitiol

Hinokitiol was synthesized according to the protocol [ Koichi Nakazawa (1967), tetrahedron Letters [ tetrahedral communication ], volume 8, stage 51, pages 5223-5225, https:// doi. Org/10.1016/S0040-4039 (01) 89648-9 ].

Example 1.15: ginkgo yellow extract

Ginkgo biloba extract was purchased from sigma aldrich.

Example 1.16: arhat pine biflavone A

Arhat pine biflavone A was purchased from Lifetein.

Example 1.17: sequoia biflavone

The sequoyibiflavones were extracted from Ouretea ferruginea according to the protocol in [ Fidelis QC et al, (2012) molecular [ molecule ]. 17 (7): pages 7989-8000. Doi: 10.3390/molecular 17077989 ]. Briefly, the ground leaves were extracted with methanol and purified by column chromatography.

Example 1.18: su Tehuang element

Su Tehuang is available from MedChemExpress.

Example 1.19: taiwan high flavone A

Taiwan homoflavone a was purchased from baiaocrick corporation (BioCrick).

Example 1.20: amentoflavone from amentoflavone

Amentoflavone was purchased from sigma aldrich.

Example 1.21: ginkgo extract

Ginkgo biloba extract was purchased from sigma aldrich.

Example 1.22: glabrous greenbrier rhizome extract

Glabrous greenbrier rhizome element was synthesized according to the protocol in US 6,706,865 B2. Briefly, protected catechins are reacted with sugar derivatives.

Example 1.23: caryophyllin F

The carnosine F is synthesized by cyclic peptide synthesis.

Example 1.24: 2-Phlaroeckol

2-Phlotoeckol was extracted from E.stonifera according to the protocol in [ Yoon et al, (2008) Fisher Sci ] [ fishery science ] volume 74, page 200, https:// doi.org/10.1111/j.1444-2906.2007.01511.X ]. Briefly, e.stolonifera was triturated and extracted with ethanol, then isolated by hexane-ethyl acetate extraction. The ethyl acetate fraction was dried and purified by HPLC to give 2-phlotoneckol.

Example 1.25: ergotamine

Ergotamine was purchased from sigma aldrich.

Example 1.26: bismahanine

Bismahanine was extracted from Murray Ke Niji leaves according to the protocol in [ Tachibana et al, (2003) J.Agilc Food Chem [ journal of agricultural and Food chemistry ], vol.51, pages 6461-6467, https:// doi.org/10.1021/jf034700+ ]; briefly, the ground leaves were extracted with dichloromethane, partitioned with ethyl acetate, and run on a silica gel column to yield Bismahanine.

Example 1.27: lactose C

Lactose C was extracted from pterygium samarangense according to Hou et al, J.Nat Prod [ journal of Natural products ], method 2003. Briefly, plants were extracted with acetone, dried, solvent extracted with n-butanol, and purified by HPLC.

Example 1.28: walnut phthalide C

According to Sun et al, chem Pharm Bull [ chemical and pharmaceutical bulletins ],2012, walnut phthalide C was extracted from wild walnut. Briefly, root bark was extracted with ethanol, then redistributed in n-butanol and purified by HPLC to produce walnut phthalide C.

Example 1.29: dongfuma Qian Ci base

Dongfuma Qian Ci base was extracted from blue fruits according to the method in [ Frederich M et al, (1999), antimicrob Agents Chemother [ antimicrobial chemotherapy ] volume 43 (9), pages 2328-31, doi:10.1128/AAC.43.9.2328 ]. Briefly, root bark was pulverized, extracted with ethanol and purified by HPLC.

Example 1.30: grandione

Grandione was extracted from Arecae semen according to the protocol in Kusumoto et al, phytopherer.Res. [ phytotheraphy study ], (1995), volume 9, pages 180-184, https:// doi.org/10.1002/ptr.2650090305. Briefly, ground betel nuts were extracted with acetone, followed by extraction in hexane and ethyl acetate. The ethyl acetate fraction was purified by HPLC to yield Arecatann.

Example 1.31: neoacrimarimine H

Neoacrimine H was extracted from grapefruit root according to the method in Takemura et al, chem Pharm Bullet [ chemical and pharmaceutical bulletins ], (1998), volume 46, pages 1518-1521, https:// doi.org/10.1248/cpb.46.1518. Briefly, roots were ground and extracted with acetone and purified by HPLC to produce neoacrrimaine H.

Example 2: antiviral Activity assay

Vero E6 cells were obtained from ATCC and plated according to manufacturer's instructions. The grown Vero E6 cells were plated into 96-well plates and pre-treated with 0, 0.01, 0.1, 1,10,100, 1000 and 10000nmol of the compound prepared as described in example 1 for 1-24 hours and dissolved in DMSO, water or PBS. 6. Cytotoxicity of individual compounds was assessed using cell titer Glo after 12, 24, 48 and 72 hours.

After verifying the optimal concentration range for compound pretreatment, SARS-CoV-2 was then applied to pretreated Vero E6 cells at a multiplicity of infection of 0.01, 0.05, 0.1, 0.5, 1,10 plus no virus control. Cells were fixed with 10% formalin 2 days after infection. Fixed cells were immunofluorescent stained with a primary antibody directed against SARS-CoV-2 nucleoprotein. Nuclei were stained with DAPI. The infection rate was determined by quantifying SARS-CoV-2 positive cells. Cell viability was determined by counting DAPI positive cells and compared to control.

Example 3: treatment of SARS-CoV-2 infection with food-derived compounds

Many food-derived compounds were screened to determine their ability to reduce viral replication of the novel SARS-CoV-2 coronavirus. Disclosed herein are certain compounds that exhibit antiviral activity.

Example 3 demonstrates the ability of the compounds disclosed herein (table 3) to reduce viral replication of the novel SARS-CoV-2 coronavirus in infected Vero E6 primate cells and inhibit viral proteases in a direct enzyme assay.

TABLE 3 list of food source compounds

Names of Compounds	Suppliers (suppliers)	Catalog numbering
			Amentoflavone from amentoflavone	Sigma Aldrich Co Ltd	PHL80351-10MG
Ginkgo extract	Sigma Aldrich Co Ltd	PHL83840-5MG
			Delphinidin-3, 5-glucoside (delphinidin)	Sigma Aldrich Co Ltd	PHL89626-5MG
Dioscin	Sigma Aldrich Co Ltd	SMB00576-25MG
			Ergot isoamine	Sigma Aldrich Co Ltd	1241550-100MG
Ginkgo yellow extract	Sigma Aldrich Co Ltd	PHL83501-10MG
			Hypericin	Sigma Aldrich Co Ltd	PHL89226-10MG
Galactoside/quercetin 3-glucuronide	Sigma Aldrich Co Ltd	PHL80349-10MG
			Procyanidin B2	Sigma Aldrich Co Ltd	PHL89552-10MG
Punicalagin	Sigma Aldrich Co Ltd	P0023-10MG
			Robinia pseudoacacia element	Sigma Aldrich Co Ltd	PHL83246-10MG
Rutin	Sigma Aldrich Co Ltd	PHL89270-50MG
			Theaflavin	Sigma Aldrich Co Ltd	PHL83341-10MG
Theaflavin 3-gallate	Sigma Aldrich Co Ltd	PHL83342-10MG
			Tilia Miqueliana Maxim glycoside	Sigma Aldrich Co Ltd	PHL89809-10MG

a) Effect of food-derived Compounds on Vero E6 cell viability

Dosage is as follows:

all compounds were dissolved in DMSO to reach a stock concentration of 10 mM. Vero E6 cells were treated with compounds at final concentrations of 1nM, 10nM, 100nM, 1 μm, 10 μm, 100 μm and 1mM in cell culture medium, or vehicle control (DMSO).

Experimental design and results:

from ATCC (VERO C1008[ Vero 76, clone E6, vero E6 ]](CRL-1586 ^TM ) Obtaining Vero E6 cellsAnd grown and maintained according to the supplier's instructions. Vero E6 cell line derived from Vero kidney epithelial cells was used as an in vitro model of SARS-CoV-2 infection. Cells at 70% -80% confluency were harvested, counted and plated in cell culture medium into 96 well culture treated well plates at an plating density of 10,000 cells per well. 24 hours after inoculation, cells were incubated in cell culture medium with 1nM, 10nM, 100nM, 1. Mu.M, 10. Mu.M, 100. Mu.M and 1mM of compound from Table 3 or vehicle-only control (DMSO, sigma, D2650) for 48 hours at 37 ℃. These experiments were performed in triplicate.

To determine the effect of these compounds on Vero E6 cell viability, celltiter glo (Promega) luminescent cell viability assays were performed according to the manufacturer's instructions and luminescence intensities were measured on a SpectraMax microplate reader. Luminescence data were normalized to DMSO control samples and percent viability was plotted against compound concentration (fig. 1A-B).

For amentoflavone, ginkgetin, delphinidin, ergotamine, procyanidin B2, locust in, rutin, theaflavin and theaflavin-3-gallate, concentrations of less than or equal to 10 μm did not significantly affect Vero E6 cell viability compared to DMSO-treated controls, with toxicity observed at 100 μm and above.

For tiliroside, concentrations less than or equal to 10 μm only slightly affected Vero E6 cell viability compared to DMSO-treated controls, with toxicity observed at 100 μm and above.

For dioscin, concentrations less than or equal to 0.1 μm did not significantly affect Vero E6 cell viability compared to DMSO-treated controls, with toxicity observed at 1 μm and above.

For ginkgetin, hypericin, punicalagin, concentrations of less than or equal to 1 μm did not significantly affect Vero E6 cell viability compared to DMSO-treated controls, with toxicity observed at 10 μm and above.

For galactosides, concentrations less than or equal to 100 μm did not significantly affect Vero E6 cell viability compared to DMSO-treated controls, with toxicity observed at 1000 μm.

b) Effect of Compound pretreatment on viral load of SARS-CoV-2 infected Vero E6 cells

To next determine the effect of compound pretreatment on SARS-CoV-2 virus replication, vero E6 cells were seeded into 96-well plates at 10,000 cells/well and pretreated in triplicate with the corresponding compounds or vehicle-only controls at final concentrations of 1nM, 10nM, 100nM, 1. Mu.M, 10. Mu.M, and 100. Mu.M for 1 hour.

SARS-CoV-2 virus (isolate USA_WA1/2020, offered by the major researchers in CDC Natalie Thornburg and the world's emerging and arbovirus reference center (World Reference Center for Emerging Viruses and Arboviruses (WRCEVA)) were then added directly to the wells at a multiplicity of infection (MOI) of 0.05 and incubated in the BSL-4 laboratory at 37℃for 48 hours in the presence of the compound. After 48 hours, cells were fixed using 10% formalin. Immobilized cells were immunofluorescent with a primary antibody against SARS-CoV-2 nucleoprotein (Luo Kelan; 200-401-A50; 1:2000), and a GFP-labeled secondary antibody (goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, alexa Fluor 488 (ThermoFisher; A-11034; 1:200)). The resulting fluorescent signal is used as a representation of the viral load. Nuclei were stained with DAPI (Sigma; D9542; 1:5000) to quantify total cell numbers.

Images were processed using CellProfiler 3.1.9 to identify the nuclei (DAPI) and the presence of SARS-CoV-2 infection (GFP intensity). Each D API positive cell was then classified as "GFP positive" (SARS-CoV-2 infected) or "GFP negative" (non-SARS-CoV-2 infected), with the threshold set based on images of cells not exposed to any SARS-CoV-2 (negative control) and images of virus-infected cells not treated with any compound (positive control). If both DAPI-positive and GFP-positive staining were observed, the cells were considered infected. If the cell is only DAPI-positive and GFP-negative, the cell is considered to be uninfected by the SARS-CoV-2 virus. The infection rate was determined by normalizing SARS-CoV-2 infected cells to the total cell number of DAPI-positive cells. The relative levels of infection ("viral load") were further calculated by normalizing the infection rate for vehicle-treated controls (DMSO) at each concentration. The number of DAPI positive cells at each treatment condition was used as an indicator of cell number ("host cell number"). A standard curve of 3 parameters was fitted, with concentration as x value. Control GFP positive cells (vehicle control DMSO treated, virus infected) were scaled to 100%. EC50 is defined as the concentration of inhibitor that reaches 50% response according to the standard curve.

The effect of increasing compound concentration on viral load and host cell number can be found in fig. 2A-O. Delphinidin, galactoside, procyanidin B2, locust bean gum, theaflavin, rutin, or tilianin are examples of compounds that do not show an effect on viral load or cell viability, as expressed by unchanged viral load and host cell number levels at the concentrations tested (fig. 2C, H, I, K, L, M, O). Dioscin showed a decrease in viral load levels, and a similar decrease in host cell numbers, indicating toxicity of the compound (figure 2D). Pretreatment with amentoflavone, ginkgetin, ergotamine, ginkgetin, hypericin, punicalagin, or theaflavin-3-gallate, but at concentrations inconsistent with loss of host cell numbers, viral load levels decreased with therapeutic indices greater than 2 (figure 2A, B, E, F, G, J, N). These results indicate that these compounds can reduce SARS-CoV-2 virus replication in Vero E6 primate cells.

c) Effect of Compounds on SARS-CoV-2 Master protease (M) and papain-like (PL) protease in direct enzyme assay

In view of the in vitro inhibition of SARS-CoV-2 viral load, several compounds (amentoflavone, theaflavin-3-gallate, punicalagin) and some metabolites of punicalagin (urolithin A (Sigma Aldrich), urolithin B (Sigma Aldrich), ellagic acid (Sigma Aldrich), and chestnut ellagic acid (Sigma Aldrich)) were tested in a direct enzyme assay for their ability to inhibit the activity of two isolated SARS CoV-2 proteases. Major proteases (Mpro) are required to cleave viral polyproteins, including those requiring viral replication. Inhibition of this protease prevents viral replication, and is marked as a possible therapeutic target for the prevention or treatment of SARS-CoV-2 infection (Sacco et al (2020); coelho et al (2020)). Similarly, papain-like protease (PLpro) is required for processing of multimeric proteins and represents an alternative therapeutic target for the treatment of SARS CoV-2 infection (Klemm et al (2020)).

Mpro assay: the total reaction volume was 50. Mu.L. Compounds were pre-dispensed into black 384 well plates (Corning) using an Echo 550 acoustic dispenser (Labcyte). All compounds were dissolved in DMSO. The compound volumes varied depending on the final concentration, the wells were topped up with DMSO using a Multidrop Combi nL reagent dispenser (sameidie company) to provide a final concentration of 2% (total volume 1 μl). A Tempest dispenser (formula Co.) was used to add 30. Mu.L of the reaction mixture to give a final concentration of 50mM HEPES pH7.5,5mM L-reduced glutathione, 0.1mg/mL BSA and 0.0125. Mu.M Mpro. The compound solution was incubated at room temperature for 10 minutes, and then the reaction was started by adding 19 μl of 25 μM fluorogenic peptide substrate using a Multidrop Combi nL reagent dispenser (sameidie). The plate was placed in a vacuum chamber for 1.5 minutes to remove bubbles and fluorescence was read every 65 seconds for 14 minutes in a Synergy Neo2 plate reader (Biotek) with excitation wavelength of 360nm and emission wavelength of 460 nm.

PLpro assay: the total reaction volume was 50. Mu.L. Compounds were pre-dispensed into black 384 well plates (Corning) using an Echo 550 acoustic dispenser (Labcyte). All compounds were dissolved in DMSO. The compound volumes varied depending on the final concentration, the wells were topped up with DMSO using a Multidrop Combi nL reagent dispenser (sameidie company) to provide a final concentration of 2% (total volume 1 μl). A Tempest dispenser (formula Co.) was used to add 30. Mu.L of the reaction mixture to give a final concentration of 50mM HEPES pH7.5,5mM L-reduced glutathione, 0.1mg/mL BSA and 0.1. Mu.M PLpro. The compound solution was incubated at room temperature for 10 minutes, and then the reaction was started by adding 19 μl of 325 μΜ fluorogenic peptide substrate using Multidrop Combi nL reagent dispenser (sameidie company). The plate was placed in a vacuum chamber for 1.5 minutes to remove bubbles and fluorescence was read every 65 seconds in a Synergy Neo2 plate reader (berteng instruments ltd) with an excitation wavelength of 320nm and an emission wavelength of 405nm for up to 14 minutes.

The screening data consisted of 14 minute kinetic readings. The slope was calculated using data from 0 to 14:05 minutes for PLpro and from 0 to 9:45 minutes for Mpro. Slope on each plate was normalized using a control-based normalization, where:

where S = sample slope

Slope of L = low activity control

Slope of H = high activity control

Dose response data was fitted using a four parameter logistic (4 PL) nonlinear regression model, limiting the maximum response to 1 and the minimum response to 0. The equation for the 4PL curve is:

where y=sample response in relative luminescence units, x=drug concentration, a=maximum response of infinite standard concentration, b= -hill slope, c=inflection point, d=response at standard concentration of 0.

Using these equations, the drug concentration that resulted in a 50% decrease in enzyme activity (IC 50) was calculated. Dose response curves were generated for the various compounds of the M protease (FIGS. 3A-G). Dose response curves were generated for various compounds of PL protease (fig. 4A-G).

d) Combined effect of food-derived compound and adefovir on host cell viability of SARS-CoV-2 infected Vero E6 cells

The combination of food-derived compounds with the current antiviral agent, adefovir, was tested against SARS-CoV-2 infection. Vero E6 cells were seeded in 96-well plates at 25,000 cells/well and SARS-CoV-2 was added to the wells at a MOI of 0.01. After 1 hour, final concentrations were 0.0316. Mu.M, 0.1. Mu.M, 0.316. Mu.M, 1. Mu.M, 3.16. Mu.M, 10. Mu.M and 31.6 mu.M of compound or vehicle-only control (DMSO) was added to the wells, while adefovir or vehicle-only control (DMSO) was added to the wells at final concentrations of 0.15. Mu.M, 0.31. Mu.M, 0.62. Mu.M, 1.25. Mu.M, and 2.5. Mu.M, and incubated at 37 ℃. After 72 hours incubation, the use was according to manufacturer's instructionsLuminescent cell viability assay (Promega Corp.) host cell viability was assessed. Results were normalized to the highest dose of adefovir alone (2.5 μm).

As expected, redeSivir alone improved host cell viability of SARS-CoV-2 infected Vero E6 cells (FIGS. 5A-F). Surprisingly, the known metabolites of punicalagin, ellagic acid and urolithin a exhibit the ability to act synergistically with adefovir to further increase host cell viability of SARS-CoV-2 infected Vero E6 cells. In particular, ellagic acid (10 and 31.6. Mu.M) synergistically acts with adefovir (0.625. Mu.M and below). Urolithin A (10 and 31.6. Mu.M) synergistically acts with Rede-ciclovir (1.25. Mu.M and below).

Incorporated by reference; equivalent forms

The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

While the exemplary embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

Claims (58)

1. A method of reducing the rate of viral infection in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising (a) a compound selected from the group consisting of: amentoflavone, ginkgetin, ergotamine, ginkgetin, hypericin, punicalagin, theaflavin-3-gallate, urolithin a, urolithin B, ellagic acid and castanogin, or any pharmaceutically acceptable salt thereof, and (B) a pharmaceutically acceptable carrier or excipient.

2. The method of claim 1, wherein the method reduces the rate of coronavirus infection by one or more of nasal tissue, bronchi, lung, kidney, esophagus, ileum, colon, rectum, heart, thymus, liver, and blood.

3. The method of claim 1, wherein the method reduces the rate of coronavirus infection by one or more of epithelial cells, decidua cells, parenchymal cells, and immune cells.

4. The method of any one of claims 1-3, further comprising administering to the subject at least one additional therapeutic agent selected from the group consisting of: a second antiviral agent, an anti-inflammatory agent, an anticoagulant, and an analgesic.

5. The method of any one of claims 1-3, further comprising administering to the subject at least one additional therapeutic agent selected from table 2.

6. The method of any one of claims 1-3, wherein the subject has, or is at risk of, a coronavirus infection.

7. The method of claim 6, wherein the coronavirus infection is a Severe Acute Respiratory Syndrome (SARS) infection, a Middle East Respiratory Syndrome (MERS) infection, or a coronavirus 2019 (covd-19) infection.

8. The method of any one of claims 1-3, wherein the virus is SARS-CoV-2.

9. A method according to any one of claims 1 to 3 wherein (a) is amentoflavone.

10. A method according to any one of claims 1 to 3 wherein (a) is a ginkgetin.

11. A process according to any one of claims 1 to 3, wherein (a) is ergotamine.

12. A method according to any one of claims 1 to 3 wherein (a) is ginkgetin.

13. A method according to any one of claims 1 to 3 wherein (a) is hypericin.

14. A method according to any one of claims 1-3, wherein (a) is punicalagin.

15. A method as claimed in any one of claims 1 to 3 wherein (a) is theaflavin-3-gallate.

16. A method according to any one of claims 1 to 3, wherein (a) is urolithin a.

17. The method of claim 16, further comprising administering adefovir.

18. A method according to any one of claims 1 to 3, wherein (a) is urolithin B.

19. A method according to any one of claims 1 to 3, wherein (a) is ellagic acid.

20. The method of claim 19, further comprising administering adefovir.

21. A method as claimed in any one of claims 1 to 3, wherein (a) is chestnut ellagic acid.

22. A method of treating a viral infection in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising (a) a compound selected from the group consisting of: amentoflavone, ginkgetin, ergotamine, ginkgetin, hypericin, punicalagin, theaflavin-3-gallate, urolithin a, urolithin B, ellagic acid and castanogin, or any pharmaceutically acceptable salt thereof, and (B) a pharmaceutically acceptable carrier or excipient.

23. The method of claim 22, further comprising administering to the subject at least one additional therapeutic agent selected from the group consisting of: a second antiviral agent, an anti-inflammatory agent, an anticoagulant, and an analgesic.

24. The method of claim 22, further comprising administering to the subject at least one additional therapeutic agent selected from table 2.

25. The method of any one of claims 22-24, wherein the viral infection is a coronavirus infection.

26. The method of claim 25, wherein the coronavirus infection is a Severe Acute Respiratory Syndrome (SARS) infection, a Middle East Respiratory Syndrome (MERS) infection, or a coronavirus 2019 (covd-19) infection.

27. The method of any one of claims 22-24, wherein the virus is SARS-CoV-2.

28. The method of any one of claims 22-24, wherein (a) is amentoflavone.

29. The method of any one of claims 22-24, wherein (a) is a ginkgetin.

30. The method of any one of claims 22-24, wherein (a) is ergotamine.

31. The method of any one of claims 22-24, wherein (a) is ginkgetin.

32. The method of any one of claims 22-24, wherein (a) is hypericin.

33. The method of any one of claims 22-24, wherein (a) is punicalagin.

34. The method of any one of claims 22-24, wherein (a) is theaflavin-3-gallate.

35. The method of any one of claims 22-24, wherein (a) is urolithin a.

36. The method of claim 35, further comprising administering adefovir.

37. The method of any one of claims 22-24, wherein (a) is urolithin B.

38. The method of any one of claims 22-24, wherein (a) is ellagic acid.

39. The method of claim 38, further comprising administering adefovir.

40. The method of any one of claims 22-24, wherein (a) is chestnut ellagic acid.

41. A pharmaceutical composition comprising between 0.01mg/kg and 500mg/kg of a unit dose of (a) a compound selected from the group consisting of: amentoflavone, ginkgetin, ergotamine, ginkgetin, hypericin, punicalagin, theaflavin-3-gallate, urolithin a, urolithin B, ellagic acid and castanogin, or any pharmaceutically acceptable salt thereof, and (B) a pharmaceutically acceptable carrier or excipient.

42. The pharmaceutical composition of claim 41, wherein the pharmaceutical composition comprises at least one additional therapeutic agent selected from the group consisting of: a second antiviral agent, an anti-inflammatory agent, an anticoagulant, and an analgesic.

43. The pharmaceutical composition of claim 41, wherein the pharmaceutical composition comprises at least one additional therapeutic agent selected from table 2.

44. The pharmaceutical composition of any one of claims 41-43, wherein (a) is amentoflavone.

45. The pharmaceutical composition of any one of claims 41-43, wherein (a) is a ginkgetin.

46. The pharmaceutical composition of any one of claims 41-43, wherein (a) is ergotamine.

47. The pharmaceutical composition of any one of claims 41-43, wherein (a) is ginkgetin.

48. The pharmaceutical composition of any one of claims 41-43, wherein (a) is hypericin.

49. The pharmaceutical composition of any one of claims 41-43, wherein (a) is punicalagin.

50. The pharmaceutical composition of any one of claims 41-43, wherein (a) is theaflavin-3-gallate.

51. The pharmaceutical composition of any one of claims 41-43, wherein (a) is urolithin A.

52. The pharmaceutical composition of claim 51, further comprising adefovir.

53. The pharmaceutical composition of any one of claims 41-43, wherein (a) is urolithin B.

54. The pharmaceutical composition of any one of claims 41-43, wherein (a) is ellagic acid.

55. The pharmaceutical composition of claim 54, further comprising adefovir.

56. The pharmaceutical composition of any one of claims 41-43, wherein (a) is chestnut ellagic acid.

57. A method of reducing viral infection-induced decrease in cell viability in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising (a) a compound selected from the group consisting of: amentoflavone, ginkgetin, ergotamine, ginkgetin, hypericin, punicalagin, theaflavin-3-gallate, urolithin a, urolithin B, ellagic acid and castanogin, or any pharmaceutically acceptable salt thereof, and (B) a pharmaceutically acceptable carrier or excipient.

58. A method of preventing viral infection-induced cell death in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising (a) a compound selected from the group consisting of: amentoflavone, ginkgetin, ergotamine, ginkgetin, hypericin, punicalagin, theaflavin-3-gallate, urolithin a, urolithin B, ellagic acid and castanogin, or any pharmaceutically acceptable salt thereof, and (B) a pharmaceutically acceptable carrier or excipient.