CN117222406A - Compounds for the treatment and prophylaxis of covd-19 - Google Patents

Abstract

The present invention discloses compounds of formula (I)Wherein R is ₁ To R ₆ Identical OR different and is H, OH-OR OR ₇ Wherein R is ₇ Is C ₁ To C ₃ Alkyl or C ₁ To C ₄ Acyl, provided that R ₁ To R ₆ Is not H, is useful for the treatment and prophylaxis of covd-19, particularly for inhibiting SARS-CoV-2, in a human subject.

Description

Compounds for the treatment and prophylaxis of covd-19

Technical Field

The present invention relates to the treatment and prevention of covd-19.

Background

In month 12 2019, a new type of coronavirus (SARS-CoV-2) began to induce a pandemic that would continue until an effective and safe vaccine could be provided or a causal medication for the disease could help avoid serious and fatal cases. Millions of people infected worldwide are waiting for effective treatment of this disease and this number is increasing.

To date, specific drugs for controlling this disease have not been found. Development of new therapeutic approaches is often very time consuming, so the use of a broad-spectrum antiviral drug for diversion (repurposing) is generally considered an effective strategy for immediate response. Thus, intensive studies have been made on the actions of known antiviral compounds.

In particular, "diverting" naturally occurring or previously developed synthetic antiviral compounds with a broad spectrum of biochemical mechanisms for covd-19 is considered a promising strategy against this pandemic.

Substances such as glifexine, nafamostat (targeted cell entry), disulfiram, lopinavir/ritonavir, darunavir, nelfinavir (for SARS-CoV-2 protease), fepra5-Weili-bavirin, penciclovir, adefovir, gan Li Xiwei (targeted RNA-dependent RNA polymerase (RdRp)) have been proposed as promising candidates, but have not provided specific effective treatments for SARS-CoV-2 for patients infected with covd-19 (Ghanbari et al, fut. Microbiol.15 (2020), 1747-1758; harrison, nat. Biotechnol.38 (2020), 379-381).

Phenolic compounds are homogeneously dispersed phytochemicals found in any tissue of most plant families throughout the world and are abundant, especially in fruits and vegetables that are part of the plant. Phenolic compounds are classified according to their chemical structure into phenolic acids (phenolic acids), flavonoids (flavonoids), tannins (tannins), coumarins (coumarins), lignans (lignans), quinones (quinines), stilbenes (stilbenes) and curcuminoids (curcuminoids). Phenolic compounds are synthesized by the shikimate pathway in plants, as secondary metabolites are usually involved in the adaptation of plants to environmental stress conditions. Phenols and flavonoids are secondary metabolites of plants with aromatic rings having at least one hydroxyl group. Among the chemically diverse natural therapeutic agents, flavonoids and phenolic compounds are considered to be the most promising anti-SARS-CoV-2 active compounds due to their excellent pharmacokinetic properties.

Phenolic related compounds have been reported to possess a variety of biological activities such as antioxidant, anti-cancer, anti-inflammatory, antibacterial, cardioprotective and immune system promoting activities. Several studies have shown that phenol and flavonoid related compounds in medicinal plants can improve human health and enhance human immunity. The natural polyphenol compounds are mainly derived from plants. These phenolic and flavonoid compounds have antiviral activity against a variety of viruses, such as rhinoviruses, hepatitis c viruses, HIV, yellow fever, herpes simplex viruses, and influenza viruses. Today, pharmacological companies produce potential drug molecules in a short time by means of bioinformatic tools and applications.

Many natural compounds (which have previously shown inhibitory effects on other pathogens, such as polyphenols) have been suggested for the treatment of new coronaries by in silico prediction and biochemical in vitro binding assays of SARS-CoV-2 component, also based on silico screening (representative of Rathinvel et al, bioint.Res. Appl. Chem.11 (2021), 10161-10173; mhatre et al, phytomed (2020), 153286; chojnaka et al, J.Funct. Food73 (2020), 104146; ghosh et al, J.Biom. St. Dyn. (2020), 1-13-doi.org-10.1080-07391102.2020; islam et al, phytopher. Res.3 (2020), 2471-2492; WO 2020/037095A 1). The advantage of such compounds is that they are "generally considered safe" ("GRAS"; e.g., U.S. Federal food, drug and cosmetic Act 201(s) and 409) and therefore can be administered without preclinical investigation (and also as a food supplement) and can be used immediately to treat patients with COVID-19 and prevent them from suffering from serious disease.

Wahedi et al (J.Biomol. Structure. Dyn.39 (2021): 3225-3234) suggested mainly resveratrol and as a representative 3',4',2,4 tetrahydroxy (-trans-) stilbene as a candidate drug against COVID-19. Zhang et al (Cell Discovery 6 (2020), 80) reported that heparan sulfate could assist SARS-CoV-2 in entering cells and could be targeted ex vivo by the approved drugs, with mitoxantrone (as a potent heparan sulfate inhibitor), sunitinib and BNTX being possible candidates for this approach. Piceatannol was further reported to bind heparin. CN 111 228,343A appears to disclose that an extract containing piceatannol can be used to treat "2019-nCoV" infections. Han et al (J.Med. Virol.87 (2015): 2054-2060) reported HIV inhibiting activity of 3,3', 4', 5' -hexahydroxy-trans-stilbene.

However, these substances have not been shown to have suitable antiviral activity against SARS-CoV-2 in order to provide effective therapeutic or prophylactic options for patients infected with SARS-CoV-2.

Disclosure of Invention

It is therefore an object of the present invention to provide a realistic and effective transition substitute for the prevention or treatment of covd-19. These alternatives should be effective in preventing or inhibiting SARS-CoV-2 from becoming pathogenic to human patients. Thus, the agent should be capable of inhibiting SARS-CoV-2 and/or preventing or impeding the entry of SARS-CoV-2 into human cells, at least to some extent, thereby providing a significant benefit to the patient in order to reduce the risk of developing COVID-19 or to reduce the risk of being severely affected by COVID-19. Another object is that these substances are easy to obtain, well tolerated and can be successfully applied to humans without invasive methods, preferably by inhalation, aerosol delivery, etc.

Accordingly, the present invention provides a compound of formula I

Wherein R is ₁ To R ₆ Identical OR different and is H, OH-OR OR ₇ Wherein R is ₇ Is C ₁ To C ₃ Alkyl or C ₁ To C ₄ Acyl, provided that R ₁ To R ₆ At least four, preferably at least 5, especially at least six, are other than H, for use in the treatment and prophylaxis of covd-19, particularly inhibition of SARS-CoV-2, in a human subject.

The compounds according to the invention were identified in the course of the present invention as exhibiting a practical and potent inhibitory activity against SARS-CoV-2. Thus, the compounds according to the present invention are effective in preventing or inhibiting SARS-CoV-2 from causing disease in human patients. Accordingly, the compounds according to the present invention are capable of inhibiting SARS-CoV-2 and/or preventing or impeding the entry of SARS-CoV-2 into human cells, at least to some extent, thereby providing a significant benefit to the patient in order to reduce the risk of developing COVID-19 or to reduce the risk of being severely affected by COVID-19. The compounds of the present invention are readily available, well tolerated and can be successfully applied to humans without the need for invasive methods. Furthermore, the compounds of the present invention are considered "GRAS", i.e., they are generally considered safe (according to federal food, drug and cosmetic act 201(s) and 409).

Preferably, the compounds used according to the invention are selected from 3,3', 5' -tetramethoxystilbene (stillbene), 3, 4', 5-tetramethoxystilbene, 3',4,5 '-tetramethoxystilbene, 3',4,5 '-pentamethoxyl stilbene, 3',5 '-tetrahydroxystilbene, 3, 4', 5-tetrahydroxystilbene, 3',4,5' -tetrahydroxy stilbene, 3',4', 5-pentahydroxy stilbene, 3',4,5' -pentahydroxy stilbene, 3, 4',5,5' -pentahydroxystilbene, 3',4',5 '-pentahydroxystilbene, 3',4', 5' -hexahydroxystilbene, preferably 3,3',4,5' -pentamethoxystilbene, 3',4', 5-pentahydroxystilbene, 3', preferably 3,3',4,5 '-pentamethoxystilbene, 3',4,4', 5-pentahydroxystilbene, 3'.

Surprisingly, the compounds of the present invention have significantly higher activity in inhibiting SARS-CoV-2 than similar polyphenol substances believed to have viral inhibitory effects. Although (-) epigallocatechin gallate or (-) -gallocatechin gallate also showed more pronounced inhibition of SARS-CoV-2 than other polyphenols, particularly other compounds from green tea and black tea, the inhibition of compounds according to the present invention was significantly enhanced over those substances.

The compounds according to the invention preferably inhibit SARS-CoV-2 replication and/or are useful for preventing the cytopathic effect of active viral replication of SARS-CoV-2 and/or for preventing SARS-CoV-2 viral infection by air transmission.

In the course of the present invention, nine polyphenol plant components and one synthetic polyphenol compound 3,3', 4', 5' -hexahydroxy trans-stilbene (synthesized as an analogue of resveratrol as a wine component) were tested for antiviral effects in vitro. Nine natural compounds, gallic acid, (-) -catechin gallate, (-) -epigallocatechin gallate, (-) -epicatechin gallate, (-) -epigallocatechin, (-) -gallocatechin gallate, ellagic acid (which has been suggested in the prior art as a potential antiviral compound), and hexahydroxy trans-stilbene (as representative of synthetic resveratrol (trans-3, 5,4' -trihydroxy stilbene) analogues) were studied for their ability to inhibit viral replication in vitro.

The stilbene derivatives according to the invention have been previously disclosed, for example, in WO 02/50007A2, WO 02/057219A1, WO 2005/016860 A1 and WO 2007/002973 A2.

These (poly) hydroxy phenols have stronger anticancer and anti-inflammatory effects than resveratrol because of the increased number of para-hydroxy groups of the polyhydroxylated stilbenes. Hexahydroxystilbene shows the most potent anti-inflammatory and anti-cancer activity in vitro, an anti-cancer effect in animal studies, and an inhibition of HIV infection in very early stages in vitro. These substances are also disclosed as modulators of T cells, neutrophils, macrophages and corresponding cytokines.

In CN 1736986A, stilbene derivatives are proposed as antiviral substances for SARS-CoV-1, wherein the compounds disclosed as having antiviral activity are compounds containing pyridine groups and 2,2' -OH or CH ₃ O-substituted compounds and 3,3' methyl-butenyl substituted compounds. Furthermore, the nature of these compounds with true anti-SARS activity is not disclosed.

All compounds tested are excellent free radical scavengers and exhibit a broad range of biochemical actions, such as inhibiting key enzymes of DNA synthesis or inflammation. Furthermore, some of them have been shown to inhibit different viral infections by nonspecifically inhibiting viral entry or replication. These compounds are chosen for their chemical structure and availability in natural sources such as tea or fruit.

Inhibition of viral infection by all compounds was studied using an in vitro cell culture model.

The receptor for SARS-CoV-2 to interface with cells is ACE 2 receptor. Binding studies were performed on the three compounds that showed the most potent antiviral effects.

In contrast to other suggestions in the prior art describing the potential anti-SARS-CoV-2 properties of several polyphenols from green and black tea, the present invention demonstrates that polyphenols vary greatly in their anti-SARS-CoV-2 effects, with only a few of these being effective.

These experiments conducted during the course of the present invention demonstrate that the synthetic resveratrol analogues according to the invention, preferably in the form of tetrahydroxy, pentahydroxy and hexahydroxy, especially hexahydroxystilbene, exhibit significantly better inhibitory activity against SARS-CoV-2 than other natural polyphenolic compounds, even a small number of natural polyphenolic compounds which proved to have real world antiviral activity against SARS-CoV-2 (as opposed to optimistic predictions based on analogy or computer predictions with other viruses).

The compounds of the invention are in principle referred to as pharmaceutical substances. Thus, formulations known and proven to be effective for delivering these compounds to human subjects are also suitable for use in the present invention. The route of administration may be divided into enteral or parenteral, such as oral, sublingual, intramuscular, subcutaneous, nasal, oral mucosal, dermal, peritoneal or rectal, etc., preferably oral, sublingual or nasal, in particular as inhalable and/or aerosol compositions. The compounds of the present invention or pharmaceutical compositions comprising the same may be administered in unit dosage form. The dosage form may be a liquid dosage form or a solid dosage form. For example, the liquid dosage form may be a true solution, gel, particulate, emulsion, or suspension. Examples of dosage forms include tablets; caplets (caplets); capsules, such as hard gelatin capsules and soft elastic gelatin capsules; cachets (cachets); round lozenges (troches); lozenges (lozenges); a dispersing agent; a suppository; ointments (oils); wet cloth agent (cataplasm/poulted); paste (paste); a powder; dressing; cream (streams); plaster (plasters); a solution; patches (patches); aerosols (e.g. nasal sprays or inhalers); sublingual film or tablet; lollipop; gel; liquid dosage forms suitable for oral or mucosal administration to a patient include suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil liquid emulsions), solutions, and elixirs. The compound of the invention can be prepared into common preparations, sustained release agents, controlled release agents, targeted preparations and various particle administration systems.

The present invention therefore relates to the use of a pharmaceutical formulation comprising a compound of the invention and a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be any excipient known to be suitable and used in the compounds of the present invention, especially a carrier or diluent.

For preparing unit dosage forms, various carriers known in the art can be widely used.

Examples of carriers or diluents are, for example, absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders such as water, glycerol, polyethylene glycol, ethanol, propanol, starch syrup, dextrin, syrup, honey, dextrose solution, acacia syrup, gelatin syrup, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, and the like; disintegrants such as dry starch, alginate, agar powder, alginate, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oils and the like; absorption promoters such as quaternary ammonium salts, sodium lauryl sulfate, and the like; lubricants such as talc, silica, corn starch, stearic acid, boric acid, liquid paraffin, polyethylene glycol, and the like. Other carriers such as polyacrylic resins, liposomes, water soluble carriers such as PEG4000 and PEG6000, PVP and the like. The tablet may be further processed into coated tablet such as sugar coated tablet, film coated tablet, enteric coated tablet, or double-layer tablet and multilayer tablet. For example, in order to make the administration unit into a pill, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, kaolin, talc, etc.; binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter (batter) and the like; disintegrants, for example, agar powder, dry starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, and the like. For example, in order to capsule the administration unit, the compound of the present invention is mixed as an active ingredient with the above-mentioned various carriers, and the resulting mixture is placed in a hard gelatin capsule or a soft capsule. The active ingredients of the compounds of the present invention may also be formulated as microcapsules, suspended in an aqueous medium to form a suspension, or filled into hard capsules or formulated for injection, inhalant or aerosol administration. For example, the compounds of the invention are formulated as injection, inhalant or aerosol formulations, such as solutions, suspensions, emulsions, freeze-dried powders, which may be aqueous or non-aqueous.

The pharmaceutical formulations of the present invention may contain one and/or more pharmaceutically acceptable excipients, such as carriers, diluents, binders, lubricants, preservatives, surfactants or dispersants. For example, the diluent may be selected from water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, and the like. In addition, for the preparation of isotonic inhalation/suspension, aerosol, injection, an appropriate amount of sodium chloride, glucose or glycerin may be added to the injectable preparation. In addition, conventional solubilizers, buffers, pH adjusters, and the like may be added. These adjuvants are usual in the art. In addition, coloring agents, preservatives, flavors, sweeteners or other materials may be added to the pharmaceutical formulation, if desired.

The medicament or pharmaceutical composition of the present invention may be administered by any known administration method for achieving the purpose of administration and enhancing the therapeutic effect. The dosage of the extract or compound or pharmaceutical composition of the present invention depends on various factors such as the nature and severity of the disease to be prevented or treated, sex, age, weight, character and individual response of the patient or animal, administration route, the number of administrations and the purpose of treatment, and thus the therapeutic dosage of the present invention may vary widely. In general, the dosage of the pharmaceutically effective ingredients of the present invention is well known to those skilled in the art. Depending on the actual amount of drug contained in the final formulation of the compound composition of the present invention, the dosage of the compound of the present invention may be adjusted appropriately to meet the requirements of a therapeutically effective dosage, and the dosage of the compound of the present invention may be accomplished, preferably, from 0.1 to 20mg/Kg body weight.

The above-described dosages may be administered in a single dosage form or divided into several dosage forms, e.g., two, three or four dosage forms, which are limited by the clinical experience of the administering physician and the dosage regimen (including the use of other therapeutic means).

According to a preferred embodiment, the polyphenol compounds of the invention are formulated for delivery to the upper respiratory system. Exemplary formulations include nasal, bronchial, oral, and pulmonary formulations. However, the compounds according to the invention may also be formulated for topical application, including liquids, gels, waxes or pastes. It is particularly preferred to formulate the compositions of the present invention as aerosols. The aerosol may be a liquid or powder aerosol. In some embodiments, the composition contains one or more pharmaceutically acceptable excipients, such as glycerol. The composition may contain 0.01% -20% w/v of the active ingredient of the present invention and 10% -20% glycerol.

The pharmaceutical compositions and unit dosage forms disclosed herein also typically include one or more pharmaceutically acceptable excipients, particularly carriers or diluents. The particular compounds disclosed herein provide advantages such as increased solubility and/or enhanced flowability, purity, or stability (e.g., hygroscopicity) characteristics that may make them more suitable for pharmaceutical formulation and/or administration to patients than the prior art.

Suitable excipients are well known to those skilled in the pharmaceutical or pharmacy arts. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art, including the manner in which the dosage form is administered to a patient. For example, oral dosage forms such as tablets or capsules may contain excipients that are not suitable for use in parenteral dosage forms. The suitability of a particular excipient may also depend on the particular active ingredient in the dosage form. For example, the breakdown of some active ingredients may be accelerated by some excipients (e.g., lactose) or when exposed to water. Active ingredients comprising primary or secondary amines are particularly susceptible to such accelerated decomposition.

Particularly preferred pharmaceutically acceptable excipients are antioxidants (reducing agents). Antioxidants commonly used in pharmaceutical compositions include citric acid and its salts (E330-E333), tartaric acid and its salts (E334-E337), phosphoric acid and its salts (E338-E343), and ethylenediamine tetraacetic acid (EDTA) and its salts (disodium calcium EDTA, E385), vitamins C, E, and the like.

The present disclosure further encompasses pharmaceutical compositions and dosage forms comprising one or more compounds that reduce the rate of decomposition of the active ingredient. Examples of such compounds are stabilizers, such as antioxidants (e.g. ascorbic acid), pH buffers or salt buffers. In addition, the pharmaceutical compositions or dosage forms of the present disclosure may contain one or more solubility modifiers, such as sodium chloride, sodium sulfate, sodium or potassium phosphate, or organic acids. One particular solubility modifier is tartaric acid.

As with the amount and type of excipients, the amount and specific type of compound of the invention in the dosage form may depend on factors such as the route of administration to the patient. Typical dosage forms of the compounds of the present invention contain the active ingredient of the present invention in an amount of about 100 μg to about 10g, preferably in an amount of about 1mg to about 1g, more preferably in an amount of 10mg to 500mg, even more preferably in an amount of about 20mg to about 300 mg. Preferred dosage forms contain the compound of the invention in an amount of from about 10mg to about 1000mg, preferably in an amount of from about 25mg to about 750mg, more preferably in an amount of from 50mg to 500mg, even more preferably in an amount of from about 30mg to about 100 mg.

Preferably, the pharmaceutical composition may also include a carrier, such as a sugar alcohol, for example, but not limited to, glycerol, mannitol, sorbitol, xylitol, and erythritol. In a specific embodiment, the sugar alcohol is glycerol.

Typical topical dosage forms include liquids, creams, lotions, ointments, gel waxes, pastes, sprays, aerosols, solutions, emulsions and other forms known to those skilled in the art. In a preferred embodiment, the compounds of the invention are delivered to the oral, nasal or bronchial tissues in a suitable topical dosage form.

For non-sprayable topical dosage forms, viscous to semi-solid or solid forms are typically employed which comprise a carrier or one or more excipients compatible with topical application and have a dynamic viscosity preferably greater than water. Suitable formulations include solutions, suspensions, emulsions, creams, ointments, powders, gels, waxes, pastes, liniments, ointments (salves) and the like, which are sterilized or otherwise combined with adjuvants (e.g., preservatives, stabilizers, wetting agents, buffers or salts) as necessary to affect various properties such as osmotic pressure.

Nasal spray pharmaceutical products contain a therapeutically active ingredient in a solution or excipient mixture dissolved or suspended in a non-pressurized dispenser that delivers a metered dose of spray containing a dose of the active ingredient. The dose may be metered by a spray pump or may be pre-metered during manufacture. Nasal spray devices may be designed for unit dose administration, or may spray up to several hundred metered doses of a formulation containing a drug substance. Nasal sprays are applied to the nasal cavity to produce local and/or systemic effects.

According to another preferred embodiment, the compounds of the present invention are provided in the form of inhalation solutions and suspension pharmaceutical products. Such products are typically water-based formulations, which contain the therapeutically active ingredient and may also contain additional excipients. Aqueous-based oral inhalation solutions and suspensions must be sterile. Inhalation solutions and suspensions are intended for delivery to the lungs by oral inhalation to produce local and/or systemic effects, and are used with specific nebulizers. Inhalation spray pharmaceutical products consist of a formulation and a container closure system. These formulations are generally water-based and must be sterile. Inhalation sprays are intended to be delivered to the lungs by oral inhalation to produce local and/or systemic effects. Other suitable topical dosage forms include sprayable aerosol formulations wherein the active ingredient (preferably in combination with a solid or liquid inert carrier) is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant such as freon) or in a squeeze bottle. Examples of sprayable aerosol formulations include metered dose inhalers, dry powder inhalers, and nebulizers. If desired, humectants (moistuzers or humectants) may also be added to the pharmaceutical compositions and dosage forms.

The pharmaceutical compositions according to the invention may also advantageously be provided in transdermal and mucosal dosage forms, such as eye drops, patches, sprays, aerosols, creams, lotions, suppositories, ointments, gels, solutions, emulsions, suspensions or other forms known to the person skilled in the art. Dosage forms suitable for the treatment of mucosal tissue in the oral cavity may be formulated as mouthwashes, oral gels or buccal patches. Other transdermal dosage forms include reservoir or matrix patches that can be applied to the skin and worn for a specific period of time to allow the desired amount of active ingredient to penetrate. Suitable excipients (e.g., carriers and diluents) and other materials useful in providing transdermal and mucosal dosage forms are well known to those skilled in the pharmaceutical arts and depend on the particular tissue or organ to which a given pharmaceutical composition or dosage form is to be administered. In view of this fact, typical excipients include water, acetone, ethanol, ethylene glycol, propylene glycol, 1, 3-butylene glycol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof, to form non-toxic and pharmaceutically acceptable dosage forms. Depending on the particular tissue to be treated, additional components may be used prior to, concurrently with, or after treatment with the compounds of the present invention. For example, penetration enhancers may be used to aid in the delivery of the active ingredient to or through the tissue. Suitable permeation enhancers include acetone; alcohols such as ethanol, oleyl alcohol, and tetrahydrofuran; alkyl sulfoxides such as dimethyl sulfoxide; dimethylacetamide; dimethylformamide; polyethylene glycol; pyrrolidones, such as polyvinylpyrrolidone; kollidon grade (Povidone); urea; and various water-soluble or insoluble sugar esters such as TWEEN 80 (polysorbate 80) and SPAN 60 (sorbitan monostearate).

The pH of the pharmaceutical composition or dosage form, or the tissue to which the pharmaceutical composition or dosage form is applied, may also be adjusted to improve the delivery of the active ingredient. Similarly, the polarity of the solvent carrier, its ionic strength, or tonicity can be adjusted to improve delivery. Compounds such as stearates may also be added to pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or lipophilicity of the active ingredient, thereby improving delivery. In this regard, the stearate may be used as a lipid carrier for the formulation, as an emulsifier or surfactant, and as a delivery or penetration enhancer.

The compounds of the present invention may also be formulated as extended or delayed release formulations. Prolonged and delayed release formulations of the various active ingredients are known in the art, for example by encapsulation.

The compounds of the present invention are present in the pharmaceutical composition from about 0.001% to about 50% w/v, typically from about 0.01% to about 0.1% w/v, more typically from about 1% to about 20% w/v. In a preferred embodiment, the compounds of the present invention are present at about 0.01% to about 20% w/v. The pharmaceutically acceptable excipients and the final other compounds or agents present in the pharmaceutical composition then make up 100%.

The compounds and compositions of the present invention are useful for treating one or more symptoms of SARS-CoV-2 viral infection. Preferably, the pharmaceutical composition according to the invention is formulated for nasal or oral administration, such as drops, applicators (applicators) or sprays. One embodiment provides a composition according to the invention for use in the prophylactic or therapeutic treatment of a patient infected with SARS-CoV-2 or a person at risk of being infected with SARS-CoV-2, in particular for use in the prevention of viral infection via the airborne route.

According to a further aspect, the present invention relates to the use of a compound according to the invention for the preparation of a pharmaceutical formulation according to the invention for the treatment and prophylaxis of covd-19, in particular for inhibiting SARS-CoV-2, in a human subject. Preferably, the use is for inhibiting the replication of SARS-CoV-2 and/or for preventing cytopathic effects of active viral replication of SARS-CoV-2 and/or for preventing infection by SARS-CoV-2 virus through an air-borne channel.

According to another aspect, the present invention relates to a method for the treatment and prophylaxis of covd-19, in particular for inhibiting SARS-CoV-2, in a human subject, wherein an effective amount of a compound of the invention or a pharmaceutical formulation of the invention is administered to a subject infected with or at risk of infection by SARS-CoV-2. The method is particularly suitable for inhibiting replication of SARS-CoV-2 and/or preventing cytopathic effects of active viral replication of SARS-CoV-2 and/or preventing viral infection of SARS-CoV-2 by the airborne route in a human subject.

The invention is further illustrated by the following examples and figures, but is not limited thereto.

Drawings

FIG. 1 shows the compounds tested and their chemical structures; compound I: gallic acid (gallic acid), compound II: (-) -catechin, compound III: (-) -catechin gallate, compound IV: (-) epigallocatechin gallate (epigallocatechin gallate), compound V: (-) -epicatechin, compound VI: (-) -epicatechin gallate (epicatechin gallate), compound VII: (-) -epigallocatechin (epigallocatechin), compound VIII: (-) -gallocatechin gallate (gallocatechin gallate), compound IX: ellagic acid (ellagic acid), and compound X: hexahydroxy-trans-stilbene (hexahydroxy-stilbene);

FIG. 2 shows the effect of pre-incubation with a specified substance on TCID50 of SARS-CoV-2

Figures 3, 4 and 5 show the behavior of compounds IV, VIII and X in Vero cell culture assays with SARS-CoV-2 infected cells, where the cells were infected after incubation and the viral load was determined after 48 hours incubation with different concentrations of compound. Quantifying viral infection in the supernatant using an RT PCR assay;

FIGS. 6 and 7 show RT PCR assays in supernatants to quantify viral infections of compounds I to X ("s 1" to "s 10") and Remdesivir (remdesivir);

FIG. 8 shows two rounds of viral inhibition;

FIG. 9 shows the structures of resveratrol (resveratrol), piceatannol (piceatannol), and hexahydroxystilbene (Compound X);

FIG. 10 shows viral copy number at various concentrations of compounds (dots: resveratrol, squares: piceatannol, upward triangle: hexahydroxystilbene (compound X); downward triangle: positive control);

FIG. 11 shows viral copy numbers at different concentrations of compounds, wherein RT PCR assays in supernatants were used to quantify viral infection; right column positive control: (a) resveratrol, (B) piceatannol, (C) hexahydroxy stilbene (compound X);

figures 12 to 14 show the performance of hexahydroxystilbene (compound X), resveratrol and piceatannol in Vero cell culture assays with SARS-CoV-2 infected cells, which were infected after incubation and viral load was determined after 48 hours incubation with different concentrations of compound.

Examples

In these examples, the inhibition of SARS-CoV-2 by compounds I through X was studied, including TCID50 of SARS-CoV-2 and Vero cell culture assays with SARS-CoV-2 infected cells.

Materials and methods

Cells were infected and incubated with all compounds at different concentrations.

Median tissue culture infection dose (TCID 50) assay

Vero cells were grown at 1.5-2X 10 at night prior to the experiment ⁴ Density of wells was plated into 96-well plates of growth medium (DMEM containing 10% FCS, 100. Mu.M of nonessential amino acids, 1mM sodium pyruvate and penicillin/streptomycin). The next morning, the growth medium was removed, and 95% confluent Vero cells were pre-incubated with DMSO or with 50 μm doses of substance I, IV, VII, VIII and X for 45 minutes and infected with 100 μl serial dilutions of SARS-CoV-2 virus stock. Preincubation and virus infection were performed in medium containing only 2% fcs instead of 10% fcs. After 5 to 7 days, all wells were examined for cell viability and TCID calculated by the methods of Reed and Munch (Reed et al, am. J. Epidemic. 27 (1938), 493-497) in the presence or absence of the indicated substance ₅₀ Dosage. The wells with living and infected cells were identified by a backlight microscope and showed clear and intact Vero cell monolayer (living cells), or wells with large numbers of CPE (cytopathic effect), characterized by a large number of dead cells and the absence of monolayer (infected cells).

After 5 to 7 days, all wells were examined for cytopathic effect (CPE) and TCID in the presence or absence of the indicated material was calculated by Reed et al ₅₀ Dosage.

Cell culture

VeroE6 (obtained from biomedica) was maintained in Gibco minimal essential medium supplemented with Earle's salt and L-glutamine (Gibco 11095-080, 500 mL), 5% FCS and 1% PenStrep, hereinafter MEM 5%. If not otherwise stated, 37 ℃ and 5% CO ₂ And (5) preserving heat.

Virus: stock preparation and titer determination

SARS-CoV-2 strain: human 2019-nCoV isolate

Product description Ref-SKU:026V-03883, human 2019-nCoV infected cell culture supernatant

Product risk grouping: RG3 ICTV

Classification: ssRNA (+)/Neuroviridae/Coronaviridae/Corona/Beta coronavirus

Virus name: human 2019-nCoV (except China) strain: bavPat1/2020

Separating: germany (except China)

Based on the 2.2E+06PFU/mL human 2019-nCoV isolate stock, virus working stock (VPN 3) was cultured in EMEM (Gibco) +10% FCS. For working stock, TCID ₅₀ Both titer and plaque assays were repeated in duplicate, and the results showed a concentration of 1.30E+06 copies of viral RNA per μL of stock. Aliquots were stored at-80 ℃. For infection assays, the working stock was thawed and diluted to MOI 0.002 in MEM 2%.

The European Commission ranks SARS-CoV-2 as a risk class 3 pathogen. Experiments with live virus and high concentration virus stock were performed under biosafety grade 3 conditions (BSL-3). At the university of medical science, pathology institute of Graz, all working steps with live virus were performed under BSL-3 conditions and equipped with an enhanced personal safety device to avoid transmission of the virus through aerosols.

Substance (B)

The material was dissolved in DMSO (Sigma) to give a material stock concentration of 5mM or 1mM, respectively. For some materials, a further 1:2 dilution was performed to obtain 2.5mM and 0.5mM stock solutions. The final experimental concentration was 1:100 dilution of stock solution of the substance in MEM 2% per well. Stock solutions of the materials were freshly prepared prior to each assay.

Cytotoxicity assays

For cytotoxicity assays VeroE6 cells were seeded into 96-well plates using MEM supplemented with 2% FCS, 8500 to 10000 cells per well, 24 hours prior to substance treatment. 37 ℃ and 5% CO ₂ And (5) heat preservation.

After incubation, the cells were exposed to a stock solution of material diluted 1:100 in MEM+2% FCS (as described above). Each material and concentration was tested in triplicate. Immediately after the addition of the substances, 24 hours, and 48 hours. Subsequently, metabolic activity was measured with an alamar blue (resazurin) based assay. After addition of alamar blue (10 μm concentration), the increase in Relative Fluorescence Units (RFU) was measured continuously for 3 hours and subjected to linear regression analysis. The slope of the material-treated cells was normalized to the slope of untreated cells (untreated cells = cells + corresponding amount of medium) to calculate relative metabolic activity.

Infection assay

Unless otherwise stated, veroE6 cells used for infection assays were 24 hours ahead of time at 2.5X10 per well (300. Mu.L) ⁴ Up to 3.0x10 ⁴ Density of individual cells was seeded in mem+2% fcs in 48 well plates (Corning Costar, cell culture media treated).

On the day of infection, the seeding medium was removed and the cells were treated with medium (MEM 2% FCS) containing 1% dmso+dissolved to the indicated concentration, with a final volume of 198 μl. Subsequently, 2. Mu.L of diluted viral stock containing SARS-CoV-2 (calculated as MOI 0.002) was added. Cells at 5% CO ₂ And infection with virus at 37℃for 1 hour. After the heat preservation step, the heat preservation device,the infection medium was removed and the cells were washed 2 times with MEM without FCS. Cells were supplemented with 440. Mu.l fresh MEM 2% FCS (with or without material). After 10 minutes, the supernatant from time point 0 (140. Mu.L) was collected, frozen at 80℃or inactivated with 560. Mu.L of AVL buffer.

Cells at 5% CO ₂ And incubation at 37℃for a further 48 hours, until time point 48 the supernatant was harvested and inactivated with 560. Mu.l of AVL buffer. In addition, either cells were lysed at Kong Zhonglie for intracellular virus detection (RTX buffer of Qiagen RNeasy kit, then RNA was prepared according to the protocol of the kit), or plates were fixed in 4% formalin for SARS-specific immunohistochemical staining (IHC).

RNA isolation and RT-qPCR

After inactivation of the virus-containing supernatant samples with AVL buffer (Qiagen), viral RNA was isolated using QIamp viral RNA mini kit (QiAmp Viral RNAMini kit, qiagen) according to the CDC recommended manufacturer protocol. RNA at 40. Mu.L ultra-pure H ₂ Eluted in O and stored at-80 ℃.

RT-qPCR for detecting viral load in samples was performed as suggested by CDC using QuantiTect multiplex RT-PCR kit and Rotor Gene Q cycler:

2019-nCoV_N1-F2019-nCoV_N1 Forward primer 5'-GAC CCC AAA ATC AGC GAA AT-3'

2019-nCoV_N1-R2019-nCoV_N1 reverse primer 5'-TCT GGT TAC TGC CAG TTG AAT CTG-3'

2019-nCoV_N1-P2019-nCoV_N1 Probe 5'-FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1-3' FAM, BHQ-1

Rotorgene

qRT-PCR analysis was performed with Rotorgene and following manufacturer's protocol.

Immunohistochemistry

Cells were fixed with 4% formalin and washed with PBS and then with 0.1% Triton X100Is permeabilized for 10 minutes (200. Mu.l per well) and then washed 3 times with 200. Mu.l PBS. 3%H for endogenous peroxidases ₂ O ₂ Is blocked for 30 minutes. Thereafter, the cells were incubated with 100. Mu.L/well of primary antibody (SARS-CoV-2 nucleocapsid; 1:1000 dilution in antibody dilution) for 1 hour by washing 3 times with 200. Mu.L of PBS. Subsequently, the cells were washed 3 times with 200 μl PBS and, in a further incubation step, treated with secondary antibody (EnVision) for at least 30 minutes (protected from light).

After washing (PBS 3 x), substrate AEC (2 drops) was dropped onto the cells and incubated until virus-infected cells were stained red (observed under a microscope), but no later than 3 minutes. The reaction was quenched by washing with PBS (3X). Wells were kept in PBS until photographic archiving.

Molecular interaction assay to detect inhibition of ACE2 receptor binding by RBD

Molecular interaction assays to detect inhibition of ACE2 receptor binding by RBD were performed as described in the literature (Gattinger et al, allergy,76 (2021), 878-883), with the following modifications: 200ng His-tagged RBD were incubated with different doses (100, 50, 25, 12.2 and 6 mM) of substance X for 3 hours at room temperature, followed by 3 hours of overlay on ACE2 (2. Mu.g/ml) conjugated plates. The bound RBD was then labeled with mouse monoclonal anti-His antibody, followed by HRP-labeled anti-mouse IgG ₁ Antibodies were detected and ABTS was used. All measurements were performed in duplicate, changes<5％。

Results

In the first set of experiments, SARS-CoV-2 TCID was analyzed with Vero cells after preincubation with DMSO or with a specified substance (I-X) dissolved in DMSO ₅₀ . As expected, DMSO caused TCID of SARS-CoV-2 ₅₀ 1Log is reduced. Cells were seeded, then infected for 1 hour, and then compounds at different concentrations were added for 48 hours. All substances showed cytoprotective effects, TCID50 was reduced by at least 1Log scale. TCID (TCID) ₅₀ Is reduced by 50. Mu.M of substance IV (99.7-fold and 802-fold reduction) The marked decrease in VIII (99.7-fold and 5.14-fold decreases) and X (99.7-fold and 2107-fold decreases) indicates inhibition of viral replication (table 1 and fig. 2).

Table 1:

in vitro inhibition of viral proliferation was repeated in another laboratory:

vero cells were seeded into 48-well plates as described in the methods section, then infected and incubated with all 10 compounds at different concentrations for 48 hours. Compounds IV, VIII and X showed inhibitory effect on viral proliferation, which was comparable to the first set of experiments (TCID ₅₀ Measurement), the results observed were consistent. qPCR on SARS-CoV-2 was then performed in the supernatant after RNA isolation. Compound IV resulted in an increase in ct value above 30 at a concentration of 50 μm in one of the two replicates. An increase in ct value above 30 was also observed after incubation of 50. Mu.M of substance VIII. Compound X resulted in an increase in ct values (above 30) after 48 hours of treatment at 25 and 50 μm, showing inhibition of viral proliferation (see also figures 3, 4 and 5).

To determine if inhibition of viral proliferation is due to alteration of viral uptake into the host cells, the cells were further incubated for one hour with compounds IV, VIII and X (compounds that proved effective in previous experiments) while cell infection was performed, and then the compounds were washed away. Cell supernatants were then tested by qPCR as described above. Interestingly, compounds IV and VIII did not have any effect on virus proliferation compared to untreated controls, whereas compound X was able to inhibit virus proliferation in a concentration dependent manner after a short incubation period of 1 hour. The results are shown in FIG. 7; after incubation for one hour with 25 or 50 μm, the ct value increases significantly.

The results indicate that viral uptake into host cells can be inhibited by compound X. Thus, it was investigated whether compound X could block ACE2 receptors which are said to be the entry of viruses into cells.

Compounds IV, VIII and X were then tested for their effect on ACE2 receptors. In this test, compounds IV and VIII did not show any inhibition of ACE2 receptor binding. Compound X inhibits ACE2 binding in a concentration dependent manner. At a concentration of 100. Mu.M, it inhibited ACE2 binding by 21-31% (see FIG. 8). This suggests that compound X inhibits viral uptake into cells at a very early stage, due at least in part to the binding of the compound to ACE2 receptor.

Comparison of Trihydroxystilbene (resveratrol), tetrahydroxy stilbene (piceatannol) and hexahydroxy stilbene (Compound X of the invention) Examples

Comparative tests of resveratrol, piceatannol and hexahydroxy stilbene (compound X of the invention) were performed as described above. Vero E6 cells were used as the cell system. Cells were infected with virus for 1 hour, then the test compound was added. Then washed and incubated with the compound under test for 48 hours. Results/reads were obtained by supernatant qPCR and IHC.

Results

The results of these comparative tests are shown in FIGS. 9-14. Hexahydroxystilbene is capable of completely inhibiting viral replication (100. Mu.M and 80. Mu.M); trihydroxystilbene showed low inhibition of viral replication (100. Mu.M, showing concentration dependence); tetrahydroxy stilbene did not show an inhibitory effect on viral replication.

In view of the present disclosure and examples, the present specification discloses the following preferred embodiments:

1. compounds of formula I

Wherein R is ₁ To R ₆ Identical OR different and is H, OH-OR OR ₇ Wherein R is ₇ Is C ₁ To C ₃ Alkyl or C ₁ To C ₄ Acyl, provided that R ₁ To R ₆ Is not H, for use in the treatment and prophylaxis of covd-19, particularly inhibition of SARS-CoV-2, in a human subject.

2. A compound for use according to embodiment 1 wherein the compound is selected from 3,5,4 '-trimethoxystilbene, 3',5 '-tetramethoxystilbene, 3, 4', 5-tetramethoxystilbene, 3',4,5' -tetramethoxystilbene, 3',4,5' -pentamethoxystilbene, 3', 5' -tetrahydroxystilbene, 3, 4', 5-tetrahydroxy stilbene, 3',4,5 '-tetrahydroxy stilbene, 3',4', 5-pentahydroxy stilbene, 3',4,5 '-pentahydroxystilbene, 3, 4',5 '-pentahydroxystilbene, 3',4,4', 5' -hexahydroxy stilbene, preferably 3,3',4,5' -pentamethoxy stilbene, 3',4', 4,4', 5' -hexahydroxystilbene, preferably 3,3',4,5' -pentamethoxystilbene, 3', 4'.

3. The compound for use according to embodiment 1 or 2, wherein inhibiting SARS-CoV-2 is inhibiting the cytopathic effect of replication of SARS-CoV-2 and/or preventing active viral replication of SARS-CoV-2 and/or preventing infection by the SARS-CoV-2 virus by the airborne route.

4. A pharmaceutical formulation comprising, or consisting of, a compound according to any one of embodiments 1 to 3 as an active ingredient and a pharmaceutically acceptable excipient.

5. The pharmaceutical formulation for use according to embodiment 4, wherein the formulation is a formulation for oral, sublingual, intramuscular, subcutaneous, intranasal, oral mucosal, bronchial, pulmonary, dermal, peritoneal or rectal administration, preferably a formulation for oral, sublingual or intranasal administration, in particular the formulation is provided as an inhalable and/or aerosol composition, or as a nasal or oral spray, and/or as a sublingual film or tablet, and/or in the form of a lollipop.

6. The pharmaceutical formulation for use according to embodiment 4 or 5, wherein the pharmaceutically acceptable excipient is selected from diluents such as water, acetone, ethanol, polyethylene glycol, polypropylene glycol, 1, 3-propanediol, l, 3-butanediol, isopropyl myristate, isopropyl palmitate, mineral oil, ethoxylated isostearyl alcohol, polyoxyisostearyl alcohol, polyoxyethylene sorbitol fatty acid esters; carriers such as glycerol, mannitol, sorbitol, xylitol and erythritol; absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, kaolin, microcrystalline cellulose, aluminum silicate; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch syrup, dextrin, syrup, honey, dextrose solution, acacia syrup, gelatin syrup, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone; disintegrants, for example, dry starch, alginates, agar powder, alginates, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, ethylcellulose; disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oils; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate; lubricants, for example talc, silica, corn starch, stearic acid, boric acid, liquid paraffin, polyethylene glycol; polyacrylic resins, liposomes, water soluble carriers such as PEG4000 and PEG6000, PVP; an antioxidant; pH buffers and/or salt buffers; a solubility modifier; a permeation enhancer; antioxidants such as citric acid and its salts, tartaric acid and its salts, phosphoric acid and its salts, and ethylenediamine tetraacetic acid (EDTA) and its salts, vitamins C, E; or mixtures thereof.

7. The pharmaceutical formulation for use according to any one of embodiments 4 to 6, wherein the formulation is an inhalant or aerosol formulation, in particular an isotonic inhalation solution or suspension or a liquid or powder aerosol.

8. The pharmaceutical formulation for use according to any one of embodiments 4 to 7, which comprises the compound in an amount of about 100 μg to about 10g, preferably about 1mg to about 1g, more preferably about 10mg to 500mg, even more preferably about 20mg to about 300mg.

9. The pharmaceutical formulation for use according to any one of embodiments 4 to 8, which comprises the compound in an amount of about 10mg to about 1000mg, preferably about 25mg to about 750mg, more preferably 50mg to 500mg, even more preferably about 30mg to about 100mg.

10. The pharmaceutical formulation for use according to any one of embodiments 4 to 9, wherein the formulation is contained in a metered dose inhaler, a dry powder inhaler or a nebulizer.

11. A pharmaceutical formulation for use according to any one of embodiments 4 to 9, comprising 0.001% to 50% w/v, preferably 0.01% to 20% w/v, more preferably 0.1% w/v to 20% w/v, in particular 1% to 20% w/v of the compound.

12. Use of a compound according to embodiments 1 to 3 for the preparation of a pharmaceutical formulation according to any one of embodiments 4 to 11 for the treatment and prevention of covd-19, in particular for inhibiting SARS-CoV-2, in a human subject.

13. The use according to embodiment 12, for inhibiting the replication of SARS-CoV-2, and/or for preventing the cytopathic effect of active viral replication of SARS-CoV-2, and/or for preventing infection by the SARS-CoV-2 virus by the airborne route.

14. A method for the treatment and prophylaxis of covd-19, in particular for inhibiting SARS-CoV-2, in a human subject, wherein an effective amount of a compound according to embodiments 1 to 3 or a pharmaceutical formulation according to any one of embodiments 4 to 11 is administered to a subject that has been infected with SARS-CoV-2 or is at risk of being infected with SARS-CoV-2.

15. A method of inhibiting SARS-CoV-2 replication and/or preventing cytopathic effects of SARS-CoV-2 active viral replication and/or preventing infection by SARS-CoV-2 virus by the airborne route in a human subject, wherein an effective amount of a compound according to embodiments 1 to 3 or a pharmaceutical formulation according to any one of embodiments 4 to 11 is administered to a subject infected with SARS-CoV-2 or at risk of infection by SARS-CoV-2.

1. A compound selected from (-) -epigallocatechin gallate (epigallocatechin gallate), (-) -gallocatechin gallate (gallocatechin gallate) or a mixture thereof, preferably (-) -gallocatechin gallate or a mixture thereof, for use in the treatment and prevention of covd-19, in particular for inhibiting SARS-CoV-2, in a human subject.

2. A compound for use according to embodiment 1, wherein the compound is (-) -gallocatechin gallate ("substance number VIII").

3. The compound for use according to embodiment 1 or 2, wherein inhibiting SARS-CoV-2 is inhibiting the cytopathic effect of replication of SARS-CoV-2 and/or preventing active viral replication of SARS-CoV-2 and/or preventing infection by the SARS-CoV-2 virus by the airborne route.

4. A pharmaceutical formulation comprising, or consisting of, a compound according to any one of embodiments 1 to 3 as an active ingredient and a pharmaceutically acceptable excipient.

5. A pharmaceutical formulation for use according to embodiment 4, wherein the formulation is for oral, sublingual, intramuscular, subcutaneous, nasal, oral mucosal, bronchial, pulmonary, skin, peritoneal or rectal administration, preferably for oral, sublingual or nasal administration, in particular the formulation is provided as an inhalable and/or aerosol composition or as a nasal or oral spray and/or as a sublingual film or tablet and/or as a lollipop.

6. The pharmaceutical formulation for use according to embodiment 4 or 5, wherein the pharmaceutically acceptable excipient is selected from diluents such as water, acetone, ethanol, polyethylene glycol, polypropylene glycol, 1, 3-propanediol, l, 3-butanediol, isopropyl myristate, isopropyl palmitate, mineral oil, ethoxylated isostearyl alcohol, polyoxyisostearyl alcohol, polyoxyethylene sorbitol fatty acid esters; carriers such as glycerol, mannitol, sorbitol, xylitol and erythritol; absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, kaolin, microcrystalline cellulose, aluminum silicate; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch syrup, dextrin, syrup, honey, dextrose solution, acacia syrup, gelatin syrup, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone; disintegrants, for example, dry starch, alginates, agar powder, alginates, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, ethylcellulose; disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oils; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate; lubricants, for example talc, silica, corn starch, stearic acid, boric acid, liquid paraffin, polyethylene glycol; polyacrylic resins, liposomes, water soluble carriers such as PEG4000 and PEG6000, PVP; an antioxidant; pH buffers and/or salt buffers; a solubility modifier; a permeation enhancer; antioxidants such as citric acid and its salts, tartaric acid and its salts, phosphoric acid and its salts, and ethylenediamine tetraacetic acid (EDTA) and its salts, vitamins C, E; or mixtures thereof.

7. The pharmaceutical formulation for use according to any one of embodiments 4 to 6, wherein the formulation is an inhalation or aerosol formulation, in particular an isotonic inhalation solution or suspension or a liquid or powder aerosol.

8. The pharmaceutical formulation for use according to any one of embodiments 4 to 7, wherein the formulation contains about 100 μg to about 10g, preferably about 1mg to about 1g, more preferably 10mg to 500mg, even more preferably about 20mg to about 300mg of the compound.

9. The pharmaceutical formulation for use according to any one of embodiments 4 to 8, wherein the formulation contains an amount of about 10mg to about 1000mg, preferably about 25mg to about 750mg, more preferably 50mg to 500mg, even more preferably about 30mg to about 100mg of the compound.

10. The pharmaceutical formulation for use according to any one of embodiments 4 to 9, wherein the formulation is contained in a metered dose inhaler, a dry powder inhaler or a nebulizer.

11. The pharmaceutical formulation for use according to any one of embodiments 4 to 9, wherein the formulation contains 0.001% to 50% w/v, preferably 0.01% to 20% w/v, more preferably 0.1% to 20% w/v, in particular 1% to 20% w/v of the compound.

12. Use of a compound according to any one of embodiments 1 to 3 for the preparation of a pharmaceutical formulation according to any one of embodiments 4 to 11 for the treatment and prevention of covd-19, in particular for inhibiting SARS-CoV-2, in a human subject.

13. The use according to embodiment 12 for inhibiting replication of SARS-CoV-2 and/or for preventing cytopathic effects of active viral replication of SARS-CoV-2 and/or for preventing infection by an airborne pathway of SARS-CoV-2 virus.

14. A method for the treatment and prophylaxis of covd-19, in particular for inhibiting SARS-CoV-2, in a human subject, wherein an effective amount of a compound according to any one of embodiments 1 to 3 or a pharmaceutical formulation according to any one of embodiments 4 to 11 is administered to a subject infected with SARS-CoV-2 or at risk of being infected with SARS-CoV-2.

15. A method of inhibiting the replication of SARS-CoV-2 and/or preventing the cytopathic effect of SARS-CoV-2 active viral replication and/or preventing viral infection of SARS-CoV-2 through an air-borne channel in a human subject, wherein an effective amount of a compound according to any one of embodiments 1 to 3 or a pharmaceutical formulation according to any one of embodiments 4 to 11 is administered to a subject infected with SARS-CoV-2 or at risk of being infected with SARS-CoV-2.

Sequence listing

<110> T.Seiki Lai Shen (Szekers, thomas)

W-elegance grid (Jager, walter)

<120> Compounds for the treatment and prevention of COVID-19

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<150> EP21164852

<151> 2021-03-25

<150> EP21164854

<151> 2021-03-25

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<170> PatentIn version 3.5

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Claims (15)

1. The use of a compound of the formula I,

wherein R is ₁ To R ₆ Identical OR different and is H, OH-OR OR ₇ Wherein R is ₇ Is C ₁ To C ₃ Alkyl or C ₁ To C ₄ Acyl, provided that R ₁ To R ₆ At least four, preferably at least 5, especially at least six, other than H, for use in the treatment and prophylaxis of covd-19, particularly inhibition of SARS-CoV-2, in a human subject; wherein the compound is selected from the group consisting of 3,3', 5' -tetramethoxystilbene, 3, 4', 5-tetramethoxystilbene, 3',4,5 '-tetramethoxystilbene, 3',4,5 '-pentamethoxystilbene, 3',4,5 '-tetramethoxystilbene, 3',4,5 '-pentamethoxystilbene, 3',4', 5' -pentahydroxystilbene, 3',4',5 '-hexahydroxystilbene, preferably 3,3',4,5 '-pentamethoxyl stilbene, 3',4', 5-pentahydroxystilbene, 3',4,5 '-pentamethoxystilbene, 3',4,4', 5-pentahydroxystilbene, 3'.

2. Use of a compound according to claim 1, wherein inhibiting SARS-CoV-2 is inhibiting the cytopathic effect of replication of SARS-CoV-2 and/or preventing active viral replication of SARS-CoV-2 and/or preventing infection by the airborne route of SARS-CoV-2 virus.

3. A pharmaceutical formulation comprising, or consisting of, a compound for use according to claim 1 or 2 as active ingredient and a pharmaceutically acceptable excipient.

4. A pharmaceutical formulation for use according to claim 3, wherein the formulation is a formulation for oral, sublingual, intramuscular, subcutaneous, intranasal, oral mucosal, bronchial, pulmonary, dermal, peritoneal or rectal administration, preferably a formulation for oral, sublingual or intranasal administration.

5. Pharmaceutical formulation for use according to claim 3 or 4, wherein the formulation is provided as an inhalable and/or aerosol composition, or as a nasal or oral spray, and/or as a sublingual film or tablet, and/or in the form of a lollipop.

6. The pharmaceutical formulation for use according to any one of claims 3 to 5, wherein the pharmaceutically acceptable excipient is selected from diluents such as water, acetone, ethanol, polyethylene glycol, polypropylene glycol, 1, 3-propanediol, l, 3-butanediol, isopropyl myristate, isopropyl palmitate, mineral oil, ethoxylated isostearyl alcohol, polyoxyisostearyl alcohol, polyoxyethylene sorbitol fatty acid esters; carriers such as glycerol, mannitol, sorbitol, xylitol and erythritol; absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, kaolin, microcrystalline cellulose, aluminum silicate; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch syrup, dextrin, syrup, honey, dextrose solution, acacia syrup, gelatin syrup, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone; disintegrants, for example, dry starch, alginates, agar powder, alginates, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, ethylcellulose; disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oils; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate; lubricants, for example talc, silica, corn starch, stearic acid, boric acid, liquid paraffin, polyethylene glycol; polyacrylic resins, liposomes, water soluble carriers such as PEG4000 and PEG6000, PVP; an antioxidant; pH buffers and/or salt buffers; a solubility modifier; a permeation enhancer; antioxidants such as citric acid and its salts, tartaric acid and its salts, phosphoric acid and its salts, and ethylenediamine tetraacetic acid (EDTA) and its salts, vitamins C, E; or mixtures thereof.

7. A pharmaceutical formulation for use according to any one of claims 3 to 6, wherein the formulation is an inhalant or aerosol formulation, in particular an isotonic inhalation solution or suspension or a liquid or powder aerosol.

8. The pharmaceutical formulation for use according to any one of claims 3 to 7, which comprises said compound in an amount of about 100 μg to about 10g, preferably about 1mg to about 1g, more preferably about 10mg to 500mg, even more preferably about 20mg to about 300mg.

9. The pharmaceutical formulation for use according to any one of claims 3 to 8, which comprises said compound in an amount of about 10mg to about 1000mg, preferably about 25mg to about 750mg, more preferably 50mg to 500mg, even more preferably about 30mg to about 100mg.

10. The pharmaceutical formulation for use according to any one of claims 3 to 9, wherein the formulation is contained in a metered dose inhaler, a dry powder inhaler or a nebulizer.

11. Pharmaceutical formulation for use according to any one of claims 3 to 9, comprising 0.001% to 50% w/v, preferably 0.01% to 20% w/v, more preferably 0.1% w/v to 20% w/v, in particular 1% to 20% w/v of the compound.

12. Use of a compound according to claim 1 or 2 for the preparation of a pharmaceutical formulation according to any one of claims 3 to 11 for the treatment and prophylaxis of covd-19, in particular for inhibiting SARS-CoV-2, in a human subject.

13. Use according to claim 12 for inhibiting the replication of SARS-CoV-2 and/or for preventing cytopathic effects of active viral replication of SARS-CoV-2 and/or for preventing infection by the SARS-CoV-2 virus by the airborne route.

14. A method for the treatment and prophylaxis of covd-19, in particular for inhibiting SARS-CoV-2, in a human subject, wherein an effective amount of a compound according to claim 1 or 2 or a pharmaceutical formulation according to any one of claims 3 to 11 is administered to a subject who has been infected with SARS-CoV-2 or is at risk of being infected with SARS-CoV-2.

15. A method of inhibiting the replication of SARS-CoV-2 and/or preventing the cytopathic effect of SARS-CoV-2 active viral replication and/or preventing viral infection of SARS-CoV-2 through an air-borne channel in a human subject, wherein an effective amount of a compound according to claim 1 or 2 or a pharmaceutical formulation according to any one of claims 3 to 11 is administered to a subject infected with SARS-CoV-2 or at risk of being infected with SARS-CoV-2.