Patent Description:
Coronaviruses are a group of RNA viruses that cause diseases in mammals and birds. Typically, in humans these viruses cause respiratory tract infections. In humans, some coronaviruses can be responsible for the common cold as well as more serious diseases such as Severe acute respiratory syndrome (SARS) and Coronavirus disease <NUM> (COVID-<NUM>).

In <NUM>, a novel coronavirus known as severe acute respiratory syndrome coronavirus <NUM> (SARS-CoV-<NUM>) emerged causing coronavirus disease <NUM> (COVID-<NUM>). Covid-<NUM> quickly spread across the planet causing a global pandemic. The severity of COVID-<NUM> varies from mild through to lethal. Risk factors include underlying health conditions, old age and obesity. Cases of symptoms lingering for many months after initial recovery from the disease (so called long Covid-<NUM>) have also been reported. The virus is particularly contagious and rapidly spread throughout the world during late <NUM> and <NUM> causing significant morbidity and economic disruption.

Given the severity of Covid-<NUM> and the ease of transmission of SARS-CoV-<NUM>, there is a need for new antiviral treatments for Covid-<NUM>.

African Swine Fever is a disease that causes a high mortality rate in domestic pigs. The disease is caused by the African Swine Fever virus (ASFV) which is a large double stranded DNA virus in the Asfarviridae family. The disease can be devastating to domestic pig populations. There is a need for new treatments to address this disease.

<CIT> relates to compounds for use to treat Herpesviradae Human Papilloma Virus, bacterial infections and fungal infections.

GUMENIUK et al. (<NUM>)<NUM>, relates to the antiviral activity of decamethoxin regardless of antigenetic structure, giving grounds for use of decamethoxin in respiratory tract infections, including coronavirus infection.

In a first aspect of the present invention, there is provided a compound, or a composition comprising the compound, for use in the treatment of an infection caused by a coronavirus or African swine fever virus; wherein the compound is a compound of formula I:
<CHM>.

In the case that the compound (or the composition comprising the compound) is for use in treating an infection caused by a coronavirus, the compound or composition may be for use in treating an infection caused by a severe acute respiratory syndrome coronavirus, for example severe acute respiratory syndrome coronavirus <NUM> (SARS-CoV-<NUM>).

The compound (or the composition comprising the compound) may be for use in treating a coronavirus disease, for example coronavirus disease <NUM> (COVID-<NUM>).

The compound (or the composition comprising the compound) may be for use in treating an infection caused by African swine fever virus, for example African swine fever.

In the compound of formula (I) R may be, for example, a linear or branched saturated or unsaturated alkylene chain having between <NUM> and <NUM> carbon atoms, for example <NUM> and <NUM> carbon atoms, for example <NUM> and <NUM> carbon atoms, for example <NUM> and <NUM> carbon atoms, for example <NUM> carbon atoms. Preferably, R is a linear saturated alkylene chain having between <NUM> and <NUM> carbons, for example <NUM> carbon atoms. Herein alkylene means an alkane based di-radical, so has two points of attachment to the rest of the molecule.

R may be, for example, a group according to formula (la), in which Ra and Rb are each a linear or branched saturated or unsaturated alkylene chain having between <NUM> and <NUM> carbon atoms, for example <NUM> and <NUM> carbon atoms, for example <NUM> and <NUM> carbon atoms, for example <NUM> and <NUM> carbon atoms, for example <NUM> carbon atoms. Ra and Rb may each be a linear or branched saturated alkylene chain having a different number of carbon atoms or the same number of carbon atoms.

Preferably, Ra and Rb are each a linear saturated alkylene chain having between <NUM> and <NUM> carbon atoms, for example <NUM> carbon atoms.

A saturated linear alkylene chain may be represented by the following formula:
<CHM>
wherein n is the number of repeat units, that is the number of carbon atoms in the linear alkylene chain. Thus, in the case of R being an alkylene chain having between <NUM> and <NUM> carbon atoms, it is preferred that R is
<CHM>
wherein n is between <NUM> and <NUM>, for example <NUM>. In the case of R being an amine having the formula (la) as set out above, it is preferred that Ra and Rb are each
<CHM>
wherein n is between <NUM> and <NUM>, for example <NUM>.

In the case of, for example, R being an alkylene chain having <NUM> carbons, R may be represented by the following formula
<CHM>.

A may, for example, comprise halide ions, such as chloride (CI) ions, bromide (Br-) ions, iodide ions (I-) and/or fluoride ions (F-). A may, for example, comprise ions of other organic and non-organic acids, such as sulphate (SO<NUM><NUM>-), carbonate (CO<NUM><NUM>-), hydrogen carbonate (HCO<NUM>-), hydrogen sulphate (HSO<NUM>-), acetate ions (CH<NUM>COO-), and/or formate ions (HCOO-). In the case of R being an alkylene chain having between <NUM> and <NUM> carbon atoms, preferably A comprises two halide ions, for example two chloride ions, thus having a total charge of -<NUM>. In the case of R being a group having formula (la) as set out above, preferably A comprises two chloride ions and a bromide ion, thus having a total charge of -<NUM>.

R<NUM>, R<NUM>, R<NUM>, R<NUM>, R<NUM> and R<NUM> may each independently be selected from C<NUM>-<NUM> alkyl and H, for example R<NUM>, R<NUM>, R<NUM>, R<NUM>, R<NUM> and R<NUM> may each be methyl. RE may be chloride, bromide, iodide, or fluoride. In the case of the ester oxygen being bonded to the aromatic ring of E at position <NUM> with respect to the methyl group, RE may be bonded to the aromatic ring at position <NUM>, <NUM> or <NUM> with respect to the methyl group. In the case of the ester oxygen being bonded to the aromatic group of E at position <NUM> with respect to the methyl group, RE may be bonded to the aromatic ring at position <NUM>, <NUM> or <NUM> with respect to the methyl group.

Preferably, the compound of formula I above has the following formula:
<CHM>
or
<CHM>
in which R, R<NUM>, R<NUM>, R<NUM>, R<NUM> and A are as defined above for formula I.

Preferably, R<NUM>, R<NUM>, R<NUM>, R<NUM>, R<NUM> and R<NUM> are each methyl.

The compound of formula I may have the following formula:
<CHM>
or
<CHM>
<CHM>
or
<CHM>.

Preferably, the compound of formula I has the following formula:
<CHM>
or
<CHM>.

In formula IV and V, it will be appreciated that R is a linear saturated alkylene chain having <NUM> carbon atoms, and A is two chloride ions. In formula VI and VII, it will be appreciated that R is a quaternary amine, in which Ra and Rb are each saturated linear alkylene chains having <NUM> carbon atoms, and A is two chloride ions and one bromide ion. It will be appreciated that in formula IV and V, R<NUM>, R<NUM>, R<NUM> and R<NUM> are each methyl. It will be appreciated that in formula VI and VII, R<NUM>, R<NUM>, R<NUM>, R<NUM>, R<NUM> and R<NUM> are each methyl.

In the case in which the compound of formula I (or the composition comprising the compound of formula I) is for use in treating an infection caused by a severe acute respiratory syndrome coronavirus, for example caused by Severe acute respiratory syndrome coronavirus <NUM> (SARS-CoV-<NUM>), or in the case in which the compound of formula I (or the composition comprising the compound of formula I) may be for use in treating a coronavirus disease, for example coronavirus disease <NUM> (COVID-<NUM>), then preferably the compound of formula I has the following formula:
<CHM>
wherein R is an alkylene chain having between <NUM> and <NUM> carbon atoms, for example R may be a saturated linear alkylene chain having between <NUM> and <NUM> carbon atoms, for example <NUM> carbon atoms.

The compound of formula II may have the following formula:
<CHM>.

In the case in which the compound of formula I (or the composition comprising the compound of formula I) is for use in treating an infection caused by African Swine Fever virus, for example for use in treating African Swine Fever, then preferably the compound of formula I has the following formula:
<CHM>
or
<CHM>
wherein R is an alkylene chain having between <NUM> and <NUM> carbon atoms, for example R may be a saturated linear alkylene chain having between <NUM> and <NUM> carbon atoms, for example <NUM> carbon atoms.

The compound of formula III may have the following formula:
<CHM>.

In other embodiments, the compound may be a compound of formula VIII
<CHM>.

Each of R, R<NUM>, R<NUM>, R<NUM>, R<NUM> and R<NUM>, Ra, Rb, and A may be as defined according to any statement above.

Preferably, the compound of formula VIII above has the following formula:
<CHM>
or
<CHM>
wherein R, R<NUM>, R<NUM>, R<NUM>, R<NUM>, X and A are as defined above for formula (IX).

Preferably, the compound of formula (VIII) has the following formula:
<CHM>
or
<CHM>
or
<CHM>
or
<CHM>
wherein X is <NUM> or <NUM>.

In formula XI and XII, it will be appreciated that R is a linear saturated alkylene chain having <NUM> carbon atoms; and A is two chloride ions.

In formula XIII and XIV, it will be appreciated that R is a quaternary ammonium in which Ra and Rb are each saturated linear alkylene chains having <NUM> carbon atoms, and A is two chloride ions and one bromide ion. It will be appreciated that in formula XI and XII, R<NUM>, R<NUM>, R<NUM> and R<NUM> are each methyl. It will be appreciated that in formula XIII and XIV, R<NUM>, R<NUM>, R<NUM>, R<NUM>, R<NUM> and R<NUM> are each methyl.

The composition may be a pharmaceutical composition, for example a human pharmaceutical composition and/or a veterinary pharmaceutical composition.

The pharmaceutical composition may be in a form suitable for one or more of oral, rectal, parenteral, transdermal, intravenous, intra-arterial, intraosseous infusion, intracerebral, intracerebroventricular, intrathecal, intramuscular, subcutaneous, intravaginal, intraperitoneal, epidural, intracerebral, intraosseous infusion, intravitreal, transmucosal, buccal, or nasal administration. Preferably, the pharmaceutical composition is in the form suitable for oral or intravenous administration.

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, such as aqueous solution, non-toxic excipients, including salts and preservatives, or buffers.

Examples of suitable aqueous and non-aqueous pharmaceutical carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate.

Examples of pharmaceutically acceptable excipients include antiadherents, binders, coatings, colourings, disintegrant, flavourings, glidants, lubricants, preservatives, sorbents and sweeteners.

The pharmaceutical composition may also contain additives such as but not limited to preservatives, wetting agents, emulsifying agents, surfactants and dispersing agents. Antibacterial and antifungal agents can be included to prevent growth of microbes and includes, for example, m-cresol, benzyl alcohol, paraben, chlorobutanol, phenol, sorbic acid, and the like. If a preservative is included, benzyl alcohol, phenol and/or m-cresol are preferred; however, the preservative is by no means limited to these examples. Furthermore, it may be desirable to include isotonic agents such as sugars, or sodium chloride.

A pharmaceutical composition suitable for oral administration may be in the form of, for example, a tablet, a pill, a sugar coated agent, a powder, a capsule, a liquid, a gel, a syrup, a slurry, a suspension, a cachet and the like. The composition may comprise a pharmaceutically acceptable carrier, for example liposomes, lactose, trehalose, sucrose, mannitol, xylitol, crystalline cellulose, chitosan, calcium carbonate, talc, titanium oxide, or silica.

The pharmaceutical composition may be obtained, for example, by combining the compounds of the invention with a solid excipient, pulverizing the mixture (if necessary) and inserting into a capsule, for example, a soft sealed capsule consisting of a gelatin capsule, gelatin and coating (e.g., glycerol or sorbitol) or a capsule composition suitable for vegetarians. In the soft capsule, the composition may be dissolved or suspended in an appropriate liquid, such as a fatty oil, liquid paraffin or liquid polyethylene glycol, with or without a stabilizer.

The compound or composition may be administered according to one or more of the following administration routes: oral, rectal, parenteral, transdermal, intravenous, intra-arterial, intraosseous infusion, intracerebral, intracerebroventricular, intrathecal, intramuscular, subcutaneous, intravaginal, intraperitoneal, epidural, intracerebral, intraosseous infusion, intravitreal, transmucosal, buccal, or nasal. Preferably, the compound or composition is administered orally or intravenously.

The compound may be administered in a dose of from <NUM> to <NUM>µg per kg of bodyweight.

The present invention will now be described in more detail with reference to the following examples and the accompanying drawings in which:.

In a first step, <NUM>,<NUM>-dibromodecane is reacted with <NUM> molar equivalents of dimethylamine to form <NUM>,<NUM>-Bis(dimethylamino)decane. The reaction takes place at <NUM>-<NUM>° C in a suitable solvent such as benzene, diethyl ether or dimethylamine and is followed by a step of acid extraction followed by alkaline treatment and extraction with diethyl ether. The extracted fractions are dried over magnesium sulphate and then purified by vacuum distillation.

In a second step, Carvacrol (<NUM>-Methyl-<NUM>-(<NUM>-methylethyl)-phenol) is reacted with chloroacetyl chloride. The reaction is carried out at -<NUM>° C for <NUM> hour and then stirred at room temperature for <NUM> hours. The reaction mixture is then washed with acid, followed by treatment with sodium bicarbonate and then water. The organic layer is dried over sodium sulphate, filtered and the solvent is removed under vacuum.

In a third step, <NUM> molar equivalents of the compound formed in the second step is reacted with one molar equivalent of <NUM>,<NUM>-Bis(dimethylamino)decane to form the compound of formula (IV). The reaction in the third step is carried out using an appropriate solvent such as acetonitrile and the reaction mixture is stirred for <NUM> hours.

It will be appreciated that further purification and separation steps may also be included in the process, for example between each of the above steps and also after the process is complete to purify the final compound having formula (IV).

Separation steps may include steps of performing column chromatography, low pressure liquid chromatography, high performance liquid chromatography and the like. Purification steps may include standard purification processes known in the art, for example, filtration, evaporation, liquid-liquid extraction, crystallisation, adsorption, recrystallization, chromatography, distillation and the like.

The compound of formula (IV) has the following systematic name, formula and molecular weight:.

In a first step, <NUM>,<NUM>-dibromodecane is reacted with <NUM> molar equivalents of dimethylamine to form <NUM>,<NUM>-Bis(dimethylamino)decane. The reaction takes place at <NUM>-<NUM>° C in benzene in a suitable solvent such as benzene, diethyl ether or dimethylamine and is followed by a step of acid extraction followed by alkaline treatment and extraction with diethyl ether. The extracted fractions are dried over magnesium sulphate and then purified by vacuum distillation.

In a second step, thymol (<NUM>-isopropyl-<NUM>-methylphenol) is reacted with chloroacetyl chloride. The reaction is carried out at -<NUM>° C for <NUM> hour and then stirred at room temperature for <NUM> hours. The reaction mixture is then washed with acid, followed by treatment with sodium bicarbonate and then water. The organic layer is dried over sodium sulphate, filtered and the solvent is removed under vacuum.

In a third step, <NUM> molar equivalents of the compound formed in the second step is reacted with one molar equivalent of <NUM>,<NUM>-Bis(dimethylamino)decane to form the compound of formula (V). The reaction in the third step is carried out using an appropriate solvent such as acetonitrile and the reaction mixture is stirred for <NUM> hours.

It will be appreciated that further purification and separation steps may also be included in the process, for example between each of the above steps and also after the process is complete to purify the final compound having formula (V).

In a first step, <NUM> molar equivalents of <NUM>,<NUM>-Dibromodecane are reacted with <NUM> molar equivalents of dimethylamine. The reaction takes place at <NUM>-<NUM>° C in a suitable solvent such as benzene or diethyl ether and is followed by a step of acid extraction followed by alkaline treatment and extraction with diethyl ether. The extracted fractions are dried over magnesium sulphate and then purified by vacuum distillation.

In a second step, carvacrol (<NUM>-Methyl-<NUM>-(<NUM>-methylethyl)-phenol) is reacted with chloroacetyl chloride. The reaction is carried out at -<NUM> C for <NUM> hour and then stirred at room temperature for <NUM> hours. The reaction mixture is then washed with acid, followed by treatment with sodium bicarbonate and then water. The organic layer is dried over sodium sulphate, filtered and the solvent is removed under vacuum.

In a third step, one molar equivalent of the compound formed in step <NUM> is reacted with the <NUM> molar equivalents of the compound formed in step <NUM> to form the compound having formula (VI). The reaction in the third step is carried out using an appropriate solvent such as acetonitrile and the reaction mixture is stirred for <NUM> hours.

It will be appreciated that further purification and separation steps may also be included in the process, for example between each of the above steps and also after the process is complete to purify the final compound having formula (VI).

In a first step, <NUM> molar equivalents of <NUM>,<NUM>-Dibromodecane are reacted with <NUM> molar equivalents of dimethylamine The reaction takes place at <NUM>-<NUM>° C in a suitable solvent such as benzene or diethyl ether and is followed by a step of acid extraction followed by alkaline treatment and extraction with diethyl ether. The extracted fractions are dried over magnesium sulphate and then purified by vacuum distillation.

In a second step, thymol (<NUM>-isopropyl-<NUM>-methylphenol) is reacted with chloroacetyl chloride. The reaction is carried out at -<NUM> C for <NUM> hour and then stirred at room temperature for <NUM> hours. The reaction mixture is then washed with acid, followed by treatment with sodium bicarbonate and then water. The organic layer is dried over sodium sulphate, filtered and the solvent is removed under vacuum.

In a third step, one molar equivalent of the compound formed in step <NUM> is reacted with the <NUM> molar equivalents of the compound formed in step <NUM> to form the compound having formula (VII). The reaction in the third step is carried out using an appropriate solvent such as acetonitrile and the reaction mixture is stirred for <NUM> hours.

It will be appreciated that further purification and separation steps may also be included in the process, for example between each of the above steps and also after the process is complete to purify the final compound having formula (VII).

It will be appreciated that to synthesise the bromine complexes defined by formula XI to XIV, the compounds of formula IV to VII are treated with bromine to form the complexes of formulas XI to XIV. It will be appreciated that further purification and separation steps may also be included in this process.

Compound IV was evaluated using a SARS-COV-<NUM> reporter gene assay which uses a reporter gene tagged infectious SARS-CoV-<NUM> virus. The assay is described in the following article: <NPL>.

The following SARS-Cov-<NUM> nanoluciferase (SARS-Cov-<NUM> Nluc) antiviral assay protocol was used.

A549-hACE2 cells (<NUM>,<NUM> cells per well in phenol-red free medium containing <NUM>% fetal bovine serum (FBS)) were plated into a white opaque <NUM>-well plate (Corning).

<NUM>-fold serial dilutions from <NUM> (final assay concentration) of compound IV were prepared in water in <NUM> well format and further diluted <NUM>-fold in the phenol-red free culture medium containing <NUM>% FBS.

Cell culture medium was removed and incubated with <NUM>µL of the diluted compound solutions and <NUM>µL of SARS-CoV2-Nluc viruses (at Multiplicity of Infection (MOI) of <NUM>).

At <NUM> post-infection, <NUM>µL Nano luciferase substrates (Promega) were added to each well and Luciferase signals measured using a Synergy™ Neo2 microplate reader.

Relative luciferase signals were calculated by normalizing the luciferase signals of compound-treated groups to that of the water-treated groups (set as <NUM>%).

The EC50 (compound concentration for reducing <NUM>% of luciferase signal) data were calculated from technical repeats using a nonlinear regression model.

The results of the assay are illustrated in <FIG>.

<FIG> shows the results of the assay and indicates that compound IV inhibits the SARS-CoV-<NUM> reporter gene in a dose dependent manner.

The effect of compound IV on cell viability (CC50 value) was determined using the Cell TiterGlo (CTG) assay protocol (Promega) in a similar fashion except that the virus infection was omitted.

Compound IV was tested using the CellTiter-Glo® Luminescent Cell Viability Assay.

The assay principle of the homogeneous CellTiter-Glo® Luminescent Cell Viability Assay is based on determining the number of viable cells in tissue culture and dependent on the quantification of the ATP content correlating with the number of metabolically active cells (www. Following the instructions of the manufacturer, the assay is used in multi-well plate formats for determination of EC50 values of test compounds in standard readouts such as cell proliferation and cytotoxicity. For the evaluation of Compound IV, A549 NSCLC cells were seeded at <NUM>,<NUM> cells per well in <NUM> well plate format. <NUM> hours after seeding, the test compound was added to the cells and treatment continued for <NUM>. In order to evaluate Compound IV over a broad concentration range starting from <NUM> as the highest concentration, a <NUM>-fold serial dilution was conducted with a total of <NUM> data points. At the end of the treatment, cells were processed according to the manufacturer's instructions and luminescence was measured using a Tecan plate reader M1000. The EC50 value of Compound IV was determined using the GraphPad Prism software.

The results of this assay are illustrated in <FIG>, which indicates that compound IV does not affect cell viability.

RPMI media was prepared at 37ºC and glutamine and HEPES (<NUM>-(<NUM>-hydroxyethyl)-<NUM>-piperazineethanesulfonic acid) buffer were added to the media.

<NUM> grams of each of the tested compounds (compounds IV and V) was dissolved separately in <NUM> distilled water (pH <NUM>).

<NUM> of media was transferred to a first tube and <NUM> of the test compound (Compound IV or V) solution was added to the tube. <NUM> of the mixture was then transferred to a second tube and <NUM> of RPMI media was added to the second tube to form a diluted mixture. This <NUM> fold dilution step was repeated sequentially until <NUM> tubes were filled.

<NUM>µL of each diluted sample (<NUM>-<NUM>) was transferred from each tube and placed in a <NUM>-well microtiter plate and mixed with <NUM>µL of log <NUM> pure African swine fever virus solution to form a mixture. Each mixture was incubated for <NUM> hours.

For the negative control samples, <NUM>µL of RPMI media was placed in the wells. For the positive control group, <NUM>µL of RPMI media was mixed with <NUM>µL of the virus solution and transferred to the wells.

Each incubated mixture was then transferred to the primary cells (PAM cells) and viral multiplication and cell toxicity were observed at <NUM>, <NUM>, <NUM>, <NUM> and <NUM> hours post infection. Viral multiplication was determined by the observation of rosetta on the surface of the cell plates.

The results for both compounds IV and V are described in table <NUM> below.

As demonstrated in Table <NUM>, Compounds IV and V were both effective at inhibiting African swine fever virus.

Claim 1:
A compound for use in the treatment of an infection caused by a coronavirus or African Swine fever virus, wherein the compound has formula I:
<CHM>
wherein R is an alkylene chain having between <NUM> and <NUM> carbon atoms, and A is one or more anions having a total charge of -<NUM>,
or R is a group having the following formula:
<CHM>
wherein Ra and Rb are each an alkylene chain having between <NUM> and <NUM> carbon atoms, and A is one or more anions having a total charge of -<NUM>;
R<NUM>, R<NUM>, R<NUM>, R<NUM>, R<NUM> and R<NUM> are each independently selected from C<NUM>-<NUM> alkyl and H;
E has the following formula
<CHM>
wherein the ester oxygen is bonded to the aromatic ring of each E at the same position, the position being position <NUM> or <NUM>; and wherein RE is H or a halide.