FIELD OF THE INVENTION

[001] This invention relates to dendritic polyglycerol (dPG) compounds having alkyl- carboxylate functional groups that irreversibly inhibit viruses and are useful in the treatment of viral infections such as COVID-19.

BACKGROUND OF THE INVENTION

[002] The recent emergence of SARS-CoV-2 resulted in a global pandemic (COVID- 19), threatening the health of the world’s population and causing dramatic socio-economic damage. It is known that new viruses can emerge or re-emerge every 3-4 years, as previously shown by H1N1, Ebola, H5N1, Zika, etc., all episodes that revealed how our society is unprepared to respond to novel viruses. Indeed, even the percentages of people infected by known viruses such as HSV, HIV, and influenza evidence the urgency of developing novel strategies in fighting viral diseases.

[003] At present, there are two primary weapons against viruses: vaccines and antivirals. Vaccines are preventive drugs composed of modified or attenuated pathogens that are meant to stimulate an immunological bio-response prior to exposure to a live virus. At the moment, vaccines represent the most effective approach to preventing viral infections. However, the durability of protection following vaccination is not 100%. Vaccines are not always available, particularly in underdeveloped countries, and existing vaccines are highly unlikely to be effective against a virus that has not yet emerged. Thus, there remains a large unmet medical need for therapeutic interventions that can help at-risk and infected individuals. Antivirals are drugs designed to fight against viruses and viral infections directly.

[004] The life cycle of a virus is composed of multiple steps: 1) attachment, 2) entry, 3) uncoating, 4) biosynthesis, and 5) assembly and release. The typical mechanism of action of existing antivirals involves inhibiting a step of the viral life cycle, thereby stopping replication. Most antivirals target one or more of steps 2-5, requiring each antiviral to be specific for the manner in which such step is carried out by a particular virus. Given the error-prone nature of viral replication, viruses are often known to mutate and develop resistance to antivirals.

[005] The first step of the viral life cycle is attachment. In this step the virus recognizes a host cell using receptors on viral attachment ligands (VALs) that recognize and bind to specific proteins present on host cell membranes. It is known that the VALs of a significant percentage of all viruses target either heparane sulfate proteoglycans (HSPG) or sialic acid (SA) terminal moieties of proteins present on cell membranes. This facilitates a different approach to designing antivirals by mimicking HSPG or SA with a molecule (ranging from polymers to dendrimers, oligomers, nanoparticles, liposomes, monoclonal antibodies, and small molecules) that will bind to a virus and block viral entry. Many of these compounds have shown broad-spectrum activity and limited toxicity, yet none has been translated into a successful drug. The main limitation of such binding inhibitors lies in their mechanism itself. Binding is a reversible event, particularly when the environment (e.g., the bloodstream) surrounding a compound that is bound to a virus causes dissociation of the virus-compound complex, separating the virus from the compound that prevented binding and leaving the virus free to bind again. Unfortunately, dilution is a common event, especially in vivo. Such temporary blocking of viral attachment and/or replication is referred to as virustatic.

[006] The irreversible inhibition of the infectivity of a virus following interaction with an antiviral compound or composition is referred to as virucidal. Many known compounds, ranging from strong surfactants to alcohol, can irreversibly inhibit the infectivity of viruses. Most of these compounds, however, have not translated into acceptable drugs due to issues such as toxicity. Viruses are made of components similkar to thosze of the host, so a drug that damages or interferes with such common components in a virus or an infected cell will also damage (i.e. , be toxic to) the host. Only a few compounds have demonstrated virucidal properties together with low toxicity, such as certain reported peptides, but these have been virus-specific, not broad spectrum.

[007] Therefore, there is still a need for virucidal agents that have low toxicity, excellent virucidal potency and broad-spectrum action.

SUMMARY OF THE INVENTION

[008] In accordance with the objects outlined above, the present invention provides compositions and methods that can be used to treat viral diseases, e.g., COVID-19.

[009] In one aspect, the invention provides a compound, pharmaceutically acceptable salt or pharmaceutically acceptable ester of Formula I:

Formula I wherein: dPGC is dendritic polyglycerol core having an average molecular weight from 5 to 100 kDa as measured by GPC;

R can be the same or different and is selected from the group comprising: -H, -COOH, optionally substituted Cs to C ₃₀ alkyl, C ₃ to Csoalkene, -(optionally substituted C5 to C ₃₀ alkyl)-COOH, -(C ₅ to C ₃₀ alkene)-COOH, -(CH ₂ )z-0-(CH ₂ ) _y -C00H, and -(CH ₂ ) _z -S-(CH ₂ ) _y -COOH; y is an integer from about 4 to about 30; z is an integer from 1 to about 20; y + z is an integer from about 5 to about 30; and having a DF of at least about 30% as measured by ¹ HNMR.

[010] In Formula I, "OR" represents the free OH groups within and at the periphery of the core.

[011] The compounds of Formula I are useful as active agents in practice of the methods of treatment and in manufacture of the pharmaceutical formulations of the invention, and as intermediates in the synthesis of such active agents.

[012] Another aspect of the present invention provides a pharmaceutical composition comprising an effective amount of the one or more compounds of the invention and at least one pharmaceutically acceptable excipient, carrier and/or diluent.

[013] Another aspect of the present invention provides the compounds of the invention for use in treating and/or preventing viral infections and/or diseases associated with viruses, particularly SARS-CoV-2.

[014] Another aspect of the present invention provides a virucidal composition comprising an effective amount of one or more compounds of the invention and at least one suitable carrier or aerosol carrier.

[015] Another aspect of the present invention provides a device comprising the virucidal composition of the invention or one or more compounds of the invention and means for applying and/or dispensing thereof.

[016] Another aspect of the present invention provides a method of disinfection and/or sterilization of non-living surfaces using one or more compounds of the invention or the virucidal composition of the invention.

[017] Another aspect of the present invention provides a use of one or more compounds of the invention or the virucidal composition of the invention for sterilization and/or for disinfection of human or animal skin and/or hair.

[018] Another aspect of the present invention provides a use of one or more compounds of the invention or the virucidal composition of the invention for manufacturing virucidal surfaces.

[019] Another aspect of the present invention provides a device comprising a surface coated with one or more compounds of the invention or with the virucidal composition of the invention.

BRIEF DESCRIPTION OF THE FIGURES

[020] Figure 1 shows a dendritic polyglycerol core and a route for its synthesis by anionic ring opening of glycidol.

[021] Figure 2 illustrates another dendritic polyglycerol core.

[022] Figure 3 shows the hydrodynamic diameter measured by DLS (dynamic light scattering) of the non-functionalized core dPG (C1) in phosphate buffer at concentration 1mg/ml_. The results are for three measurements.

[023] Figure 4 shows gel permeation chromatography (GPC) diagram of the non- functionalized core dPG (C1). (Mn:7.2 kDa, Mw: 10 kDa, PDI: 1.4)

[024] Figure 5 shows the ¹ HNMR of dPGiokDa-C3SCio-C001\la ⁺ ioo% in D2O.

[025] Figure 6 shows the hydrodynamic diameter measured by DLS (dynamic light scattering) of dPGio _kDa -C3SCio-COO-Na ⁺ _95% in water.

[026] Figure 7 shows the ¹ HNMR and DLS characterizing data for dPGskDa-

C ₃ SCio-COO Na ⁺ ioo _% ("EM 124").

[027] Figure 8 shows the ¹ HNMR and DLS characterizing data for dPG25 _kDa -

C ₃ SCio-COO Na ⁺ ioo _% ("EM 126").

[028] Figure 9 shows the ¹ HNMR and DLS characterizing data for dPGioo _kDa -

C ₃ SCio-COO Na ⁺ ioo _% ("EM 131").

[029] Figure 10 shows the ¹ HNMR and DLS characterizing data for dPGskDa-

C ₃ SCii-C001\la ⁺ ioo _% ("EM 125").

[030] Figure 11 shows the SARS-CoV-2 antiviral activity and virucidal activity of dPGio _kDa -C ₃ SCio-COO-Na ⁺ ioo _% , along with the cytotoxicity data for the test compound.

[031] Figure 12 shows the SARS-CoV-2 antiviral and virucidal activity of dPGs _kDa -

C ₃ SCio-COO Na ⁺ ioo _% ("EM 124").

[032] Figure 13 shows the SARS-CoV-2 antiviral and virucidal activity of dPGs _kDa -

C ₃ SCii-C001\la ⁺ ioo _% ("EM 125").

[033] Figure 14 shows the SARS-CoV-2 antiviral and virucidal activity of dPG25 _kDa -

C ₃ SCio-COO Na ⁺ ioo _% ("EM 126").

[034] Figure 15 shows the SARS-CoV-2 antiviral and virucidal activity of dPGioo _kDa -C ₃ SCio-COO Na ⁺ ioo _% ("EM 131").

[035] Figure 16 shows the HSV-2 antiviral activity and virucidal activitry of d PG 1 _0k Da-CsSC _{l 0} -COO N a ⁺ _95% . [036] Figure 17 shows the the SARS-CoV-2 in vivo antiviral activity of dPGio _k Da-

C3SCio-COONa ⁺ ioo _% as tested in the Syrian hamster model. The figure shows the change in percentage of body weight relative to each half day (each point on x-axys is 12h). End point day 14.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[037] As used in the present specification, the following words and phrases are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise. In the case of conflict, the present specification, including definitions, will control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following abbreviations and definitions are supplied in order to facilitate the understanding of the present invention.

[038] As used in the specification and claims, the singular form "a", "an" and "the" includes plural references unless the context clearly dictates otherwise.

[039] The term “about” as used in conjunction with a number or a range of numbers, indicates that such number/range will be understood to be approximate. Thus, “about 2” encompasses the integers 1, 2, 3 and 4. The term “about 5 to 30” should be read as "about 5 to about 30" and encompasses, e.g., ranges from 4 to 32, 4 to 28, 6 to 33 and 3 to 27.

[040] As used herein the term "alkene" or “alkenyl” refers to a monoradical branched or unbranched, unsaturated or polyunsaturated hydrocarbon chain, having from about 2 to 30 carbon atoms, more preferably about 5 to 30 carbon atoms and still more preferably about 7 to about 15 carbon atoms. This term is exemplified by groups such as ethenyl, but- 2-enyl, hex-2, 5-dienyl, (2E,6£)-5-methyl^ ³ -nona-2, 6-diene and the like. The term “alkenyl” when recited to specify a group linking to another moiety [such as carboxyalkenyl or -(C5 to C30 alkene)-COOH] refers to a diradical branched or unbranched, unsaturated or polyunsaturated hydrocarbon chain consisting of an alkenyl monoradical, a terminal hydrogen of which is substituted by such other moiety.

[041] As used herein, the term “alkyl” refers to a monoradical branched or unbranched saturated hydrocarbon chain containing from 1 to 50 carbon atoms, preferably 5 to 30 carbon atoms. Representative examples of alkyl include, but are not limited to methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, n-pentyl, n-hexyl, n- heptyl, n-octyl, n-nonyl, n-decyl and 6-ί3or ^I-3-GhbΐI^I-10l ³ ^bq3hb. The term "alkyl" when recited to specify a group linking to another moiety (such as carboxyalkyl) refers to a diradical branched or unbranched saturated hydrocarbon chain derived from an alkyl monoradical, a terminal hydrogen of which is substituted by such other moiety; exemplified by groups such as methylene (-CH2-), ethylene (-CH2CH2-), propylene isomers [e.g., -CH2CH2CH2- and -CH(CH3)-CH2-] and the like. The term “substituted alkyl” refers to an alkyl group in which 1 or more (up to about 5, preferably up to about 3) hydrogen atoms is/are independently replaced by a substituent selected from the group comprising: alkenyl, alkenylthio, alkenyloxy, alkoxy, alkoxyalkoxy, alkoxyalkoxyalkoxy, alkoxyalkoxyalkyl, alkoxyalkyl, alkoxycarbonyl, alkoxycarbonylalkoxy, alkoxycarbonylalkyl, alkoxysulfonyl, alkylamidoalkyl, alkylcarbonyl, alkylcarbonylalkoxy, alkylcarbonylalkyl, alkylcarbonylalkylthio, alkylcarbonyloxy, alkylcarbonylthio, alkylsulfinyl, alkylsulfinylalkyl, alkyl sulfonyl, alkylsulfonylalkyl, alkylthio, alkylthio alkyl, alkylthioalkoxy, alkynyl, alkynyloxy, alkynylthio, aryl, arylcarbonyl, aryloxy, arylsulfonyl, carboxy, carboxyalkyl, carboxyalkoxy, cyano, cyanoalkoxy, cyanoalkyl, cyanoalkylthio, 1,3-dioxolanyl, dioxanyl, dithianyl, ethylenedioxy, formyl, formylalkoxy, formylalkyl, haloalkenyl, haloalkenyloxy, haloalkoxy, haloalkyl, haloalkynyl, haloalkynyloxy, halogen, heterocycle, heterocyclocarbonyl, heterocycloxy, heterocyclosulfonyl, hydroxy, hydroxyalkoxy, hydroxyalkyl, mercapto, mercapto alkoxy, mercapto alkyl, methylenedioxy, nitro, sulfinyl and sulfonyl. Preferred substiituents for "substituted alkyl" are selected from the group comprising: alkenyl, alkenylthio, alkoxysulfonyl, alkylcarbonylthio, alkylsulfinyl, alkylsulfinylalkyl, alkyl sulfonyl, alkylsulfonylalkyl, alkylthio, alkylthioalkoxy, alkynylthio, aryl, arylcarbonyl, aryloxy, arylsulfonyl, cyanoalkylthio, dithianyl, heterocyclosulfonyl, hydroxy, hydroxyalkoxy, hydroxyalkyl, mercapto, mercapto alkoxy, and mercapto alkyl.

[042] As used herein, the term “and/or” used in a phrase such as “A and/or B” herein is intended to include “A and B”, “A or B”, “A”, and “B”.

[043] As used in the specification and claims, the term “at least one” used in a phrase such as “at least one C atom” can mean “one C atom” or “two C atoms” or more than two C atoms.

[044] As used herein, the terms "carboxylate", "carboxy", "carboxyl" and "carboxylic" refer to the moiety "-C(0)OH", which can be written, interchangeably, as "-COOH" and "-COO", and should be read to include pharmaceutically acceptable salts, such as "-COONa ⁺ ". The term also applies to such moieties attached to a hydrocarbon linker, for example a C1-50 alkyl group as defined herein, to form a carboxyalkyl moiety.

[045] The term “comprise” is generally used in the sense of include, that is to say permitting the presence of one or more features or components. Also as used in the specification and claims, the language “comprising” can include analogous embodiments, as contrasted with the terms “consisting of” which includes only the embodiment recited and “consisting essentially of” which includes analogous embodiments to the extent that they do not materially affect the basic and novel characteristics of the claimed invention. [046] As used herein, the term “degree of functionalization” can be abbreviated as “DF”. The term refers to the number of R functional groups bearing a moiety selected from the group: -(optionally substituted Cs to C ₃₀ alkyl)-COOH, -(C ₅ to C ₃₀ alkene)-COOH, -(CH ₂ ) _z -0-(CH ₂ ) _y -C00H, and -(CH ₂ ) _z -S-(CH ₂ ) _y -COOH, determined as a percentage of all of the free hydroxyl groups in a given dendritic polyglycerol. The DF for the dendritic polyglycerols of the invention is at least 30% (i.e. , between 30% and 100%) or preferably between about 30% and 90%, more preferably between about 40% and 75%, or about 50%. Degree of functionalization can be measured by ¹ HNMR, as described in greater detail below. It will be appreciated by those skilled in the art that some, but not all of the free hydroxyl groups within the dPGC will be functionalized in the syntheses described below and bear the functional moiety represented by R and that it is not feasible to assign the specific location of a particular functional group as existing at one or the other of the two groups shown as R in Formula I. Such functionalization within the dPGC will diminish proceeding inward from the more accessible periphery to the less accessible center of the core, but will be inherrently counted in the degree of functionlization measurement by ¹ HNMR.

[047] As used herein, the term “dendrimer” refers to nano-sized, synthetic, highly branched polymers and oligomers having a well-defined chemical structure that that radially, symmetrically, identically branches from an initial monomeric unit, typically forming spherical (e.g., ovoid, ellipsoid, etc...) macromolecules.

[048] As used herein, the term “dendritic” refers to dendrimer-like highly branched polymers, copolymers or oligomers having a chemical structure resembling that of a dendrimer. Dendritic compounds have a core including a given number of generations of branches or arms, and a plurality of end groups. The branches start from an initial monomeric unit (e.g., trimethylolpropane) but are not identical, typically as the result of incomplete bonding in the early steps of polymerization. The generations of arms consist of structural monomeric units; these can be identical or incomplete for a given generation of arms (or non-identical in the case of dendritic copolymers), and can be the same as the first generation or can branch differently for subsequent generations of arms. (These monomeric units are glycerol in a dendritic polyglycerol.) The generations of arms extend radially in a geometrical progression from the initial monomeric unit until the end (or N ^th ) generation (also described as the periphery). Dendritic (in the sense of dendrimer-like) includes molecules containing non-symmetrical branching. Dense star polymers, starburst polymers and rod-shaped dendrimers can be considered dendritic. [049] As used herein, the term "dendritic polyglycerol" or "dPG" refers to a glycerol polymer having a plurality of branch points and multifunctional branches that lead to further branching with polymer growth. Dendritic polymers can be obtained by a one-step polymerization process and form a polydisperse system with varying degrees of branching. Figure 1 illustrates the structure and a method for synthesizing a dPG. Methods of making a variety of such polymers are known in the art and further described herein.

[050] As used herein, the term “dendritic polyglycerol core” or “dPGC” refers to an entire dendritic polyglycerol serving as a substrate for functionalization.

[051] As used herein, the term "functionalized" means having chemically bound substituent groups, also referred to as functional groups, functional moieties or functional units, such as bioactive ligands. The dendritic polyglycerols useful for the present invention can contain only a single functional unit per branch or can contain two of the same or different functional units per branch.

[052] As used herein, the terms “glycerol” and “glycerine” and “glycerin” all refer to the monomeric unit propane-1, 2, 3- triol.

[053] As used herein, the term "hyperbranched" as in "hyperbranched polymer" or "HBP" or “hBP” is used synonymously with "dendritic) when it refers to a polymer or oligomer that branches radially from a central core incorporating plural copies of at least one branching monomer unit. This term is not synonymous with "dendritic" in the case of linear polymers that branch following a cylindrical symmetry or any other branching macromolecule that does not follow a radial branching symmetry. In contrast, hyperbranched polymers (HBPs) incorporate monomers that have three or more reacting groups and thus result in branched polymers. HBPs can be homopolymers composed of a hyperbranched single monomer, or can be copolymers of branching monomers (those able to react at three or more positions) with other branching monomers or with linear monomers (those able to react at only two positions). The HBP compounds employed herein typically are considered to be biocompatible or pharmaceutically acceptable polymers, such that they are suitable for administration to human and/or veterinary subjects. Certain disclosed embodiments of the HBP, e.g., the hyperbranched polyglycerol (hPG) polymer are homopolymers that contain only repeating glycerol subunits. In another example, the HBP can be a heteropolymer that includes one, two or more other polymer subunits. HBPs are well known in the art (see, e.g., Gao and Yan, Prog. Polym. Sci. 29 (2004) 183-275). Examples of HBP compounds, methods of synthesizing them using, for example, a single monomer methodology and double-monomer methodology, modifying, and functionalizing the compounds are disclosed herein and in Macromolecules 1999, 32, 4240-4246 (polyglycerol) and in Biomaterials 2006, 27:5471-5479, and Gao and Yan, Prog. Polym. Sci. 29 (2004) 183-275. [054] As used herein, the term “mammal” (for purposes of treatment) refers to any animal classified as a mammal, including humans, domestic and farm animals or pet animals, such as dogs, horses, cats, cows, monkeys etc. Preferably, the mammal is human.

[055] The term “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, “optionally substituted alkyl” means either “alkyl” or “substituted alkyl,” as defined. It will be understood by those skilled in the art with respect to any group containing one or more substituents that such groups are not intended to introduce any substitution or substitution patterns (e.g., substituted alkyl including optionally substituted cycloalkyl groups, which in turn are defined as including optionally substituted alkyl groups, potentially ad infinitum) that are sterically impractical and/or synthetically non-feasible.

[056] The term "pharmaceutically acceptable ester" refers to esters of the compounds of the present invention, which hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety advantageously has not more than 6 carbon atoms. Examples of particular esters include, but are not limited to, formates, acetates, propionates, butyrates, acrylates, ethylsuccinates, morpholinoethyl esters and the like.

[057] The term "pharmaceutically acceptable salts" as used herein refers to salts that retain the desired biological activity of the compounds the invention and includes pharmaceutically acceptable acid addition salts and base addition salts. Suitable pharmaceutically acceptable acid addition salts of the compounds of Formula I may be prepared from an inorganic acid or from an organic acid, or can be prepared in situ during the final isolation and purification of the compounds of the invention. Examples of such inorganic acids are hydrochloric, sulfuric, and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, fumaric, maleic, alkyl sulfonic, arylsulfonic. Suitable pharmaceutically acceptable base addition salts of the compounds of Formula I include metallic salts made from lithium, sodium, potassium, magnesium, calcium, aluminium, and zinc, and organic salts made from organic bases such as choline, diethanolamine, morpholine. Other examples of organic salts are: ammonium salts, quaternary salts such as tetramethylammonium salt; amino acid addition salts such as salts with glycine and arginine. Additional information on pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Co., Easton, PA 1995. In an embodiment, the pharmaceutically acceptable salt of the compounds of the invention is a sodium salt.

[058] As used herein the terms "subject" or "patient" are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human, and other, e.g., avian animals, such as a chicken. In preferred embodiments, the terms "subject" or "patient" refer to a human and animals, such as dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, chicken. In some embodiments, the subject is a subject in need of treatment, or a subject being infected by a virus. In other embodiment, a subject can be an animal infected by a virus, such as a chicken. However, in other embodiments, the subject can be a healthy subject or a subject who has already undergone treatment. The term does not denote a particular age or sex. Thus, adult, children and newborn subjects, whether male or female, are intended to be covered.

[059] As used herein, the term “therapeutically effective amount” refers to an amount of a compound of the invention effective to alter a virus, and to render it inert, in a recipient subject, and/or if its presence results in a detectable change in the physiology of a recipient subject, for example ameliorates at least one symptom associated with a viral infection, prevents or reduces the rate transmission of at least one viral agent.

[060] As used herein, the term “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already being infected by a virus, as well as those in which the viral infection is to be prevented or those who are likely to come into contact with a virus. Hence, the mammal, preferably human, to be treated herein may have been diagnosed as being infected by a virus, or may be predisposed or susceptible to be infected by a virus. Treatment includes ameliorating at least one symptom of, curing and/or preventing the development of a disease or condition due to the viral infection. Preventing is meant attenuating or reducing the ability of a virus to cause infection or disease, for example by affecting a post-entry viral event.

[061] As used herein, the term “virucidal” refers to a characterization of antiviral efficacy determined by in vitro testing demonstrating irreversible inhibition of the infectivity of a virus following interaction with an antiviral compound or composition. Even following termination of the interaction (for example, by dilution) and absent any added materials or conditions promoting viral reconstitution, it is essentially impossible for the virus to resume infectivity. Interaction with antiviral compound or composition alters the virus, rendering it inert, and thereby prevents further infections. [062] As used herein, the term “virustatic” refers to a characterization of antiviral efficacy determined by in vitro testing demonstrating reversible inhibition of the infectivity of a virus following interaction with an antiviral compound or composition. Once the interaction terminates (for example, by dilution) and absent any added materials or conditions promoting viral reconstitution, it is possible for the virus to resume infectivity.

Compounds of the Present Invention

[063] The present invention provides certain dendritic polyglycerol compounds. The compositions are antivirals, have proved to be virucidal inhibitors of SARS-CoV-2, and can be used to treat COVID-19 and other viral diseases.

[064] The context in which the compounds of the invention were discovered is significant, in that the first carboxyalkyl functionalized dendritic polyglycerol was synthesized to be employed as a negative control in testing the utility of other compounds for inhibition of SARS-CoV-2. It was believed that the cell surface receptors sought by the virus did not comprise -COO- groups. When tested together with other promising anti- SARS-CoV-2 candidate molecules, it was surprisingly discovered that the carboxyalkyl dendritic polyglycerol indeed has virucidal activity.

[065] Accordingly, the present invention relates to the compounds, pharmaceutical formulations, methods of treatment employing such compositions, as represented by Formula I:

Formula I wherein: dPGC is dendritic polyglycerol core in which OR represents the free OH groups within and at the peripheryu of the core, having an average molecular weight from 5 to 100 kDa as measured by GPC;

R can be the same or different and is selected from the group comprising: -H, -COOH, optionally substituted Cs to C ₃₀ alkyl, C ₃ to Csoalkene, -(optionally substituted C ₅ to C ₃₀ alkyl)-COOH, -(C ₅ to C ₃₀ alkene)-COOH, -(CH ₂ ) _z -0-(CH ₂ ) _y -C00H, and -(CH ₂ ) _z -S-(CH ₂ ) _y -COOH; y is an integer from about 4 to about 30; z is an integer from 1 to about 20; y + z is an integer from about 5 to about 30; and having a DF of at least about 30%, or a pharmaceutically acceptable salt or pharmaceutically acceptable ester. [066] R is preferably functionalized with a carboxylic acid bearing aliphatic chain, more preferably having from about 6 to about 20 carbon atoms, and still more preferably from about 8 to about 15 carbon atoms. The variable "y" is preferably an integer from about 4 to about 20, more preferably from about 8 to about 13 and still more preferably about 11. The variable "z" is preferably an integer from about 2 to about 10, more preferably from about 2 to about 5, and still more preferably about 3. The sum "y + z" is preferably an integer from about 10 to about 16, more preferably about 14.

[067] The dendritic polyglycerol core (dPGC) according to the invention has a size from about 4 to 15 nm and a molecular weight from about 5 to 100 kDa. It is composed of repeated units of glycerine with the formula (RO-CH ₂ )2CH-OR wherein R = H or an adjacent glycerine unit on a multifunctional polyhydroxy starter molecule having a plurality of OH groups, for example 2 to 4 OH groups, such as 2-ethyl-2-(hydroxymethyl)propane- 1,3-diol. Figure 1 shows a representative example of a dPG formed from 2-ethyl-2- (hydroxymethyl)propane-1,3-diol polymerized with glycerol monomers. One particular dPGC identified as "(C1)" has a hydrodynamic diameter (size) of 5.34 ± 0.293 nm and molecular weight => GPC (H2O) M _n = 7.2 kDa, M _w = 10.4 kDa. Figures 3 and 4 show the results of dynamic light scattering (DLS) characterizing the size and gel permeation chromatography (GPC) characterizing the molecular weight of dendritic polyglycerol core (C1). It should be noted that the weight and size of functionalized dPG products of Formula I will be greater than the weight and size of the core (dPGC) that was functionalized.

[068] In one aspect of the invention, the R substituents that contribute to DF are selected from the group comprising: -(optionally substituted C5 to C30 alkyl)-COOH, -(C5 to C30 alkene)-COOH, -(CH ₂ )z-0-(CH ₂ ) _y -C00H , and -(CH ₂ ) _z -S-(CH ₂ ) _y -COOH. In another aspect, the R substituents that contribute to DF are selected from the group comprising: -(optionally substituted C5 to C30 alkyl)-COOH, -(CH ₂ ) _z -0-(CH ₂ ) _y -C00H, and -(CH ₂ ) _z -S-(CH ₂ ) _y -COOH.

[069] According to an embodiment, the present invention provides a compound of Formula I comprising a dendric polyglycerol core as defined above functionalized with a plurality of same or different R substituents as defined above that are bound to the core, provided however, that not each of said R substituents necessarily comprises a COOH group, and wherein the degree of functionalization is as defined above. R will comprise a hydrogen or an incompletely reacted precursor group when not fully functionalized with a COOH-bearing aliphatic chain. Incompletely reacted precursor groups for R can include, without limitation: H, -optionally substituted C5 to C30 hydroxyalkyl, or -C3 to C30 alkenyl (particularly -CH ₂ -CH=CH ₂ ). Nomenclature

[070] In the present specification, the dendritic polyglycerols are named using the following format: The subscript following “dPG” indicates the weight of the dendritic polyglycerol being named, e.g., dPGio _kDa names a dendritic polyglycerol having a weight of 10 kDa. The text following such weight indication identifies the functional group(s) and the subscript that follows indicates the degree of functionalization. Thus, dPGio _kDa -CsSCio- COO Na ⁺ ioo _% names a dendritic polyglycerol having the structure shown below in Formula dPG1:

Formula dPG1 which is a compound of Formula 1 where the dPG core has a weight of 10 kDa, R is -(CH ₂ ) _z -S-(CH ₂ ) _y -COO-Na ⁺ where z is 3 and y is 10, with a DF of 100%.

Synthesis of the Compounds of Formula I

[071] Syntheses of the compounds of Formula I are described below with reference to the Reaction Schemes.

Synthetic Reaction Parameters

[072] The terms "solvent", "inert organic solvent" or "inert solvent" mean a solvent inert under the conditions of the reaction being described in conjunction therewith [including, for example, benzene, toluene, acetonitrile, tetrahydrofuran ("THF"), dimethylformamide ("DMF"), chloroform, methylene chloride (or dichloromethane), diethyl ether, methanol, pyridine and the like]. Unless specified to the contrary the solvents used in the reactions of the present invention are inert organic solvents. Reactions take place at room temperature and 1 atmosphere of pressure unless otherwise indicated.

[073] Isolation and purification of the compounds and intermediates described herein can be effected, if desired, by any suitable separation or purification procedure such as, for example, filtration, extraction, crystallization, column chromatography, thick-layer chromatography, or a combination of these procedures. Specific illustrations of suitable separation and isolation procedures can be had by reference to the examples hereinbelow. However, other equivalent separation or isolation procedures can, of course, also be used. Starting Materials

[074] The starting materials, such as the glycerol (1) are commercially available or can be readily prepared by those skilled in the art using commonly employed synthetic methodology.

[075] Reaction Scheme 1

[076] Preparation of dendritic polyqlvcerol: The dPG cores used in the invention can be synthesized according to procedures described in the literature, e.g., Sunder, A.; Frey, H.; Muelhaupt, R. Macromol Symp 2000, 153, 187.

[077] As illustrated above in Reaction Scheme 1, (a.) a glycerol such as trimethylolpropane ("TMP") (1) is deprotonated with about 0.4 molar equivalents ("eq.") of potassium methoxide solution (e.g., in methanol). The methanol is evaporated at about 60 °C under vacuum (about 3 mbar). (b.) The synthesis reactor is heated to about 100 °C and an excess (e.g., number of branches desired x 3 eq.) of (oxiran-2-yl)methanol ("glycidol") (2) is added slowly, e.g., over a period of about 24 hours, providing ring opening multi branching polymerization conditions, to afford a dendritic polyglycerol (3), such as C1, the dPG illustrated in Figure 1, or the dPG illustrated in Figure 2, which can be conventionally isolated and purified. The molecular weight of the resulting dPG can be controlled by adjusting the molar ratio of glycidol to TMP and the ring-opening polymerization reaction time accordingly. It will be appreciated by those skilled in the art that the resulting product will be a mixture of dPGs falling within a narrow range of molecular weights, such as 8 kDa to 12 kDa, and will have an average molecular weight such as 10 kDa.

[078] Reaction Scheme 2 [079] Preparation of dPGC-allyl (5): As illustrated above in Reaction Scheme 2, a dPG core (3) is functionalized in preparation for a subsequent thiol-ene click reaction, by converting the free hydroxyl groups to allyl groups through reaction with an allyl halide (e.g., bromide) (4) where z can be 0 to about 18. The dPG is dissolved in a suitable solvent (e.g., DMF) and the reaction takes place over about 12 hours; it is performed in dry condition in presence of NaH as base for deprotonation of hydroxyl groups. The resulting allyl-functionalized product (5) is conventionally isolated and purified (e.g., solvent removed under vacuum and purification by dialysis in MeOH for 2 days). The degree of functionalization (DF) can be controlled by adjusting the ratio of allyl halide to dPG, the amount of NaH, the reaction time and/or conditions, and is confirmed by 1H NMR. For example, by limiting the equivalents of allyl halide and NaH, the product corresponding to (5) where more of the groups corresponding to R remain hydrogen can be obtained.

[080] Reaction Scheme 3

[081] Preparation of Formulae la and lb: As illustrated above in Reaction Scheme

3, dPG-di-allyl (5) (DF=100) (where z can be 0 to about 18) and about 2 eq. of an co- mercaptoalkyl carboxylic acid sodium salt (6) (where y is an integer from about 4 to about 30) are dissolved in suitable solvent (e.g., methanol). To this solution are added 2,2- dimethoxy-2-phenylacetophenone (DM PA) as radical initiator and a catalytic amount of tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCI) (to avoid oxidation of the thiol intermediate). A few drops of water can be added to dissolve any precipitation and obtain a clear solution. The solution is degassed by flushing argon through the reaction mixture for about 10 minutes. The reaction mixture is then stirred and irradiated with UV light using a high pressure UV lamp at room temperature for about 5 hours. The product(s) from the reaction mixture can be conventionally isolated and purified, for example being dialyzed (MWCO 2 kDa), e.g., against methanol/water mixture, to remove the TCEP.HCI, DMPA and any excess of unreacted thiol compound. The product(s) will have a DF of about 85-95%. Most of the product will be Formula lb. The products can be conventionally separated (e.g., by gel permeation chromatography or size exsclusion chromatography). The successful formation of Formula la and/or Formula lb can be confirmed by ¹ HNMR of pure product by correlating the aliphatic protons of ligands at 1.5-1.00 ppm with the polyglycerol backbone protons at 3.7-3.2 ppm. In addition, elemental analysis can be used for the sulfur content measurement confirming the click reaction.

[082] Reaction Scheme 4

Formula Id

[083] Synthesis of dPGC-carboxyalkyl: As illustrated in Reaction Scheme 4, dPG (3) is reacted with an o-halo-alkan-1-olic acid salt (7) (about 1.5 eq.) (where p is an integer from 5 to 30) in the presence of NaH (about 2 eq.) (as a base for deprotonation of the dPG hydroxyl groups). The reaction mixture is allowed to stir for about 24 hours at about 40 °C and is then quenched by adding methanol and conventionally isolated and purified (e.g., by dialysis against methanol) to afford the corresponding dPGC-alkyl carboxylate of Formula lc (with a DF of about 50%) or Formula Id (with a DF of about 100%) The remaining hydroxyl groups of Formula lc can be further functionalized, for example, by sulfation. [084] Reaction Scheme 5

[085] Synthesis of dPGC-R-carboxyalkene/carboxyalkene or H: As illustrated in Reaction Scheme 5, dPG (3) is reacted with an co-bromo-alkenyl-1-carboxylic acid sodium salt (8) (where p is an integer from 1 to 28, q is an integer from 1 to 26, and p+q is an integer from 4 to 30) in the presence of NaH. The reaction mixture is allowed to stir for about 24 hours at about 60 °C to afford one or both of the corresponding alkenyl sulfonate products of Formula le and Formula If, which are conventionally separated (e.g., by gel permeation chromatography or size exclusion chromatography), isolated and purified.

[086] Reaction Scheme 6 [087] Synthesis of hvdroxyalkyl dPG: As illustrated in Reaction Scheme 6, Step 1, dPG (3) is reacted with an co-halo-alkan-1-ol (9) in the presence of NaH. The alkanol (9) is preferably added slowly, dropwise, to minimize the formation of dimers. The reaction mixture is allowed to stir for about 24 hours at about 60 °C to afford one or both of the corresponding hydroxyalkyl products (10) and/or (11), which can be carried forward together or conventionally separated (e.g., by gel permeation chromatography or size exsclusion chromatography) and carried forward independently. The products can be conventionally isolated and purified.

[088] Synthesis of carboxyalkoxyalkyl dPGs: As illustrated in Reaction Scheme 6, Step 2, hydroxyalkyl (10) and/or (11) is/are reacted with ano-halo-alkan-1-olic acid salt (7) in the presence of NaH. The reaction mixture is allowed to stir for about 24 hours at about 60 °C to afford one or all of the corresponding cartboxyalkyl / carboxyalkoxy products of Formula Ig, Formula Ih and/or Formula li, which are conventionally separated (e.g., by gel permeation chromatography or size exsclusion chromatography), isolated and purified.

Preferred Processes and Last Steps

[089] A compound of Formula I is prepared by a thiol-ene click reaction between a dPG-allyl and an co-mercaptoalkyl carboxylic acid.

[090] A compound of Formula I is contacted with a pharmaceutically acceptable acid to form the corresponding acid addition salt.

[091] A pharmaceutically acceptable acid addition salt of Formula I is contacted with a base to form the corresponding free base of Formula I.

Preferred Compounds

[092] Preferred for the compounds, pharmaceutical formulations, methods of manufacture and use of the present invention are the following combinations and permutations of substituent groups of Formula I (sub-grouped, respectively, in increasing order of preference):

• R contributing to DF is selected from the group comprising: -(optionally substituted C ₅ to C ₃₀ alkyl)-COOH, -(C ₅ to C ₃₀ alkene)-COOH, -(CH ₂ )z-0-(CH ₂ ) _y -C00H, and -(CH ₂ ) _z -S-(CH ₂ ) _y -COOH. o Especially where R contributing to DF is selected from the group comprising: -(optionally substituted Cs to C ₁₅ alkyl)-COOH, -( Cs to C ₁₅ alkene)-COOH, -(CH ₂ ) _z -0-(CH ₂ ) _y -C00H, and -(CH ₂ ) _z -S-(CH ₂ ) _y -COOH.

^■ Particularly where y is from about 8 to 13, z is from about 2 to 5, and y+z is from about 10 to 16.

^■ Particularly where R not contributing to DF is selected from the group comprising -H, -COOH and -CH2-CH=CH2.

• Preferably where y is from about 8 to 13, z is from about 2 to 5, and y+z is from about 10 to 16. o More preferably where dPGC has an average molecular weight of 10 kDa.

^■ Most preferably where COOH is COO Na ⁺ .

^■ Most preferably where DF is at least about 50%.

^■ Most preferably where DF is at least about 85- 95%.

• Preferably where y is from about 9 to 11 and z is 3. o More preferably where dPGC has an average molecular weight of 10 kDa.

^■ Most preferably where COOH is COO Na ⁺ .

^■ Most preferably where DF is at least about 50%.

^■ Most preferably where DF is at least about 85- 95%.

^■ Particularly where y is from about 9 to 11 and z is 3.

• Preferably where dPGC has an average molecular weight of 10 kDa. o More preferably where COOH is COO Na ⁺ . o More preferably where DF is at least about 50%. o More preferably where DF is at least about 85-95%. Especially where R contributing to DF is selected from the group comprising: -(optionally substituted Cs to C15 alkyl)-COOH, -(CH ₂ ) _z -0-(CH ₂ ) _y -C00H, and -(CH ₂ ) _z -S-(CH ₂ ) _y -COOH.

^■ Particularly where y is from about 8 to 13, z is from about 2 to 5, and y+z is from about 10 to 16.

• Preferably where y is from about 9 to 11 and z is 3.

^■ Particularly where R not contributing to DF is selected from the group comprising -H, -COOH and -CH2-CH=CH2.

• Preferably where y is from about 8 to 13, z is from about 2 to 5, and y+z is from about 10 to 16. o More preferably where dPGC has an average molecular weight of 10 kDa.

^■ Most preferably where COOH is COO Na ⁺ . ^■ Most preferably where DF is at least about 50%.

^■ Most preferably where DF is at least about 85- 95%.

The above-described groups and sub-groups are individually preferred and can be combined to describe further preferred aspects of the invention.

[093] Particularly preferred for the compounds, pharmaceutical formulations, methods of manufacture and use of the present invention are the following:

• dPGiokDa-C ₃ SCio-COO-Na ⁺ ioo%,

• dPGiokDa-C3SCio-COO Na ⁺ 95%/allyl5%, and

• d PG 1 okDa-C ₃ SCi o-COO ^' N a ⁺ 7o%/ally o% .

[094] More preferred for the compounds, pharmaceutical formulations, methods of manufacture and use of the present invention is the following:

• dPGiokDa-C ₃ SCio-COO-Na ⁺ ioo%.

Utility, Testing, Administration and Formulation

General Utility

[095] The compositions of the invention find use in a variety of applications. As will be appreciated by those in the art, the compositions are antiviral and have demonstrated virucidal activity against HSV-2 and surprisingly against SARS-CoV-2.

[096] An aspect of the invention provides a method of treating and/or preventing COVID-19 and other respiratory diseases caused by coronaviruses, influenza virus infections and/or diseases associated therewith, comprising administering to a subject in need thereof, a therapeutically effective amount of one or more virucidal compositions of the invention. These treatable viruses and diseases are discussed in greater detail below. [097] Coronaviruses (CoVs) are abundant and tend to cause mild to serious upper- respiratory tract syndromes, like the common cold or lower respiratory diseases like wheezy bronchitis and other affections of the lower respiratory tract. Coronaviruses tend to reinfect the same human hosts (Archives of Disease in Childhood, 1983, 58, 500-503). Coronaviruses are zoonotic and circulate among pigs, horses, cats, bats, camels, among other species. When a coronavirus jumps from an animal to humans, they can cause the mild to moderate diseases associated to coronaviruses such as HCV229E (alpha CoV), HCVOC43 (beta CoV), HCVNL63 (alpha CoV), HCVOC43 (beta coronavirus), HCVHKU1 (beta coronavirus), all of which do not have a distinct pathognomonic syndrome named after the individual virus. In the last two decades, three CoVs have caused serious respiratory (upper and lower) syndromes with dedicated syndromes being named to describe their infections: MERS-CoV (beta CoV that causes Middle East Respiratory Syndrome, or MERS); SARS-CoV (the beta CoV that causes severe acute respiratory syndrome, or SARS) and SARS-CoV-2 (the novel CoV that causes COVID-19). Like all viruses, CoVs use either Sialic Acids (SAs) and/or Heparan Sulfate Proteoglycans (HSPGs), among other cell surface receptors such as the Angiotensin Converting Enzyme to infect the host cell. HCVNL63 and SARS-CoV use primarily SAs to dock onto host cells while the docking used by other variants is still under investigation (Microorganisms 2020, 8, 1894; doi:10.3390/microorganisms8121894).

[098] CoVs are also of great importance in the veterinary and livestock industries because they cause diseases to animals. The Equine Coronavirus (ECoV), a beta-CoV causes enteric inflammation on horses and is closely related to the bovine CoV (BCoV), also a beta-CoV that causes enzootic pneumonia complex and dysentery in calves and has been reported to cause winter dysentery in adult cattle. Both ECoV and BCoV infect the host cells via the N-acetyl-9-O-acetylneuraminic acid receptor, also referred to as Sialic acid. In pigs, the Porcine Respiratory Coronavirus (PRCv) causes a respiratory disease to which the only treatment is isolation of the contaminated animal. Other CoVs affect pigs, such as Transmissible Gastroenteritis Virus (TGEV), Porcine epidemic diarrhoea virus (PEDV), and porcine haemagglutinating encephalomyelitis virus (PHEV). PDCoV (porcine deltacoronavirus) TGEV and PRCV are alpha CoVs and closely associated to the CoVs that affect cats and dogs, and to PEDV and human CoVs HCV229E and HCVNL63. PHEV and PDCoV are the beta CoVs. Poultry and many avian species also develop diseases caused by CoVs such as coronaviruses of the domestic fowl - infectious bronchitis virus IBV, that causes respiratory illness to chicken (Gallus gallus), turkey (Meleagris gallopavo) and pheasant (Phasianus colchicus). Improvements in testing and detection will likely increase the list of coronaviruses that affect animals. The fear of new outbreaks of CoVs relevant to human health may also increase this list as the source of new outbreaks lies predominantly in livestock.

[099] The compositions and methods provided herein are particularly deemed useful for the treatment of COVID-19.

[0100] Another aspect of the invention provides a method of disinfection and/or sterilization of surfaces using one or more compounds of the invention or the virucidal composition of the invention or the pharmaceutical composition of the invention. The disinfection and/or sterilization is preferably done on living surfaces or non-living surfaces. The living surfaces are human or animal skin and/or hair. The non-living surface are, but not limited to, medical equipment, touch screens, textile, clothing, masks, gloves, furniture, and any other surfaces present in rooms, transport means, public spaces such as schools, airports, public transportation and cinemas. In some other embodiments, the non-living surfaces are fabric surfaces (masks, gloves, doctor coats, curtains, bed sheet), metal surfaces (lifts, door handle, nobs, railings, medical equipment and instruments, public transport and places), wood material surfaces (furniture, floors, partition panels), concrete surfaces (hospitals, clinics and isolation wards and walls), and plastic surfaces (medical equipment and instruments, touch screens, switches, kitchen and home appliances).

[0101] In a preferred embodiment, the method of disinfection and/or sterilization of surfaces comprises the steps of (i) providing at least one compound of the invention or a virucidal composition of the invention, or pharmaceutical composition of the invention, (ii) contacting a virus-contaminated surface or a surface suspected to be contaminated by a virus with the at least one compound of the invention or a virucidal composition of the invention or pharmaceutical composition of the invention for a time sufficient to obtain virucidal effect. In some embodiments, the virus-contaminated surface is human or animal skin and/or hair. In other embodiments, the virus-contaminated surface is a non-living surface. The non-living surface is, but not limited to, medical equipment, touch screens, textile, clothing, masks, gloves, furniture, and any other surfaces present in rooms, transport means, public spaces such as schools, airports, public transportation and cinemas. In some other embodiments, the non-living surfaces are fabric surfaces (masks, gloves, doctor coats, curtains, bed sheet), metal surfaces (lifts, door handle, nobs, railings, medical equipment and instruments, public transport and places), wood material surfaces (furniture, floors, partition panels), concrete surfaces (hospitals, clinics and isolation wards and walls), and plastic surfaces (medical equipment and instruments, touch screens, switches, kitchen and home appliances).

[0102] Another aspect of the invention provides a use of a compound of the invention or a virucidal composition of the invention or a pharmaceutical composition of the invention for sterilization and/or for disinfection. In some embodiments, sterilization and disinfection is for virus-contaminated surfaces or surfaces suspected to be contaminated by a virus. In some preferred embodiments, the surfaces are human or animal skin and/or hair. Thus in some embodiments, the invention provides a use of a compound of the invention or a virucidal composition of the invention or a pharmaceutical composition of the invention for sterilization and/or for disinfection of human or animal skin and/or hair. In other preferred embodiments, the surfaces are non-living surfaces. The non-living surfaces are, but not limited to, medical equipment, touch screens, textile, clothing, masks, gloves, furniture, and any other surfaces present in rooms, transport means, public spaces such as schools, airports, public transportation and cinemas. In some other embodiments, the non-living surfaces are fabric surfaces (masks, gloves, doctor coats, curtains, bed sheet), metal surfaces (lifts, door handle, nobs, railings, medical equipment and instruments, public transport and places), wood material surfaces (furniture, floors, partition panels), concrete surfaces (hospitals, clinics and isolation wards and walls), and plastic surfaces (medical equipment and instruments, touch screens, switches, kitchen and home appliances). In an embodiment, the virucidal composition of the invention or the pharmaceutical composition of the invention is used as virucidal hand disinfectant for frequent use. In another embodiment, the virucidal composition of the invention or the pharmaceutical composition of the invention is applied by spraying. In a further embodiment, the virucidal composition of the invention of the pharmaceutical composition of the invention is applied on a protective mask.

[0103] Another aspect of the invention provides a use of the compounds of the invention or the virucidal composition of the invention for manufacturing (producing) virucidal surfaces (i.e. able to inactivate viruses). Such surfaces are, but not limited to, textile, clothing, masks, touch screens, medical equipment, furniture. In some other embodiments, the surfaces are fabric surfaces (masks, gloves, doctor coats, curtains, bed sheet), metal surfaces (lifts, door handle, nobs, railings, medical equipment and instruments, public transport and places), wood material surfaces (furniture, floors, partition panels), concrete surfaces (hospitals, clinics and isolation wards and walls), and plastic surfaces (medical equipment and instruments, touch screens, switches, kitchen and home appliances). In some embodiments, the surfaces can be modified with the one or more compounds of the invention either through chemical modification or physical coating known in the art. Examples of physical coating are spraying or dipping the surface in a solution comprising the one or more compounds of the invention.

[0104] Another aspect of the invention provides a method for manufacturing (producing) a virucidal surface, wherein the method comprises coating the surface with the one or more compounds of the invention or the virucidal composition of the invention. The surface is, but not limited to, textile, clothing, masks, touch screens, medical equipment, furniture. In some other embodiments, the surface is fabric surface (masks, gloves, doctor coats, curtains, bed sheet), metal surface (lifts, door handle, nobs, railings, medical equipment and instruments, public transport and places), wood material surface (furniture, floors, partition panels), concrete surface (hospitals, clinics and isolation wards and walls), and plastic surface (medical equipment and instruments, touch screens, switches, kitchen and home appliances). The coating can be done either through chemical modification or physical coating known in the art.

[0105] Another aspect of the invention provides a virucidal surface coating composition comprising the one or more compounds of the invention or the virucidal composition of the invention. The virucidal surface coating composition of the invention can be sprayed or painted on surfaces. The surfaces are, but not limited to, medical equipment, touch screens, textile, clothing, masks, gloves, furniture, and any other surfaces present in rooms, transport means, public spaces such as schools, airports, public transportation and cinemas. In some other embodiments, the surfaces are fabric surfaces (masks, gloves, doctor coats, curtains, bed sheet), metal surfaces (lifts, door handle, nobs, railings, medical equipment and instruments, public transport and places), wood material surfaces (furniture, floors, partition panels), concrete surfaces (hospitals, clinics and isolation wards and walls), and plastic surfaces (medical equipment and instruments, touch screens, switches, kitchen and home appliances).

[0106] Another aspect of the invention provides a device comprising a surface coated with one or more compounds of the invention or with the virucidal composition of the invention. Such an antiviral coated device can be, but is not limited to, clothing, a mask, a glove, a touch screen, medical equipment, furniture, etc.... In one preferred embodiment, the device is a mask, clothing or medical equipment. In another preferred embodiment, the device is a medical device.

Testing

[0107] The weight, size, and degree of functionalization of the compounds of the invention can be determined, for example, by gel permeation chromatography, by dynamic light scattering and by nuclear magnetic resonance, as described in Bhatia, et al. , "Linear polysialoside outperforms dendritic analogs for inhibition of influenza virus infection in vitro and in vivo", Biomaterials, Vol. 138, 22-34 (September 2017) ((DOI: 10.1039/c7py01470h) (https://doi.Org/10.1016/j.biomaterials.2017.05.028)

[0108] Degree of branching can be calculated using inverse gated (IG) ¹³ C NMR, for example, as described in Haag, R.; Sunder, A.; Stumbe, J.-F. J Am Chem Soc 2000, 122, 2954.

[0109] In vitro activity for SARS-CoV-2 inhibition is determined, for example, as described in Gasbarri et al., Microorganisms 2020, 8, 1894 (2020).

[0110] In vivo activity for SARS-CoV-2 inhibition is determined, for example, as described in Kaptein et al., "Favipiravir at high doses has potent antiviral activity in SARS- CoV-2-infected hamsters, whereas hydroxychloroquine lacks activity," Proc Natl Acad Sci U S A, 2020 Oct 27; 117(43): 26955-26965, or in Imai et al., "Syrian hamsters as a small animal model for SARS-CoV-2 infection and countermeasure development," https://www.pnas.org/content/117/28/16587, or in Rosenke et al., "Orally delivered MK- 4482 inhibits SARS-CoV-2 replication in the Syrian hamster model," https://www.nature.com/articles/s41467-021-22580-8.

[0111] Cytotoxicity is determined by exposing Vero cells to varying concentrations of test drug and measuring the percentage of cells surviving such exposure. Antiviral activity is determined by plaque reduction assays on infected Vero cells, measuring the number of plaques that form in wells exposed to a mixture of a fixed concentration of the virus and varying concentrations of test drug. Virucidal activity is determined by exposing Vero cells to different dilutions of a prep-incubated mixture of virus and an effective amount of test drug. After incubation, the solution is removed and the cells are incubated again, measuring the plaques that form, evaluating the viral titer. The decrease of viral titer with respect to an untreated control is an indication of virucidal activity. These determinations can be carried out, for example, as described in Cagno, et al., "Broad-spectrum non-toxic antiviral nanoparticles with a virucidal inhibition mechanism," Nature Materials 17, 195-203 (2018).

Administration

[0112] The compounds of Formula I are administered at a therapeutically effective dosage, e.g., a dosage sufficient to provide treatment for the disease states previously described. Administration of the compounds of the invention or the pharmaceutically acceptable salts thereof can be via any of the accepted modes of administration for agents that serve similar utilities.

[0113] While human dosage levels have yet to be optimized for the compounds of the invention, generally, a daily dose is from about 0.001 to 2.0 mg/kg of body weight/day, preferably about 0.005 to 0.75 mg/kg of body weight/day, and most preferably about 0.01 to 0.5 mg/kg of body weight. Thus, for administration to a 70 kg person, the dosage range would be about 0.07 to 140 mg per day, preferably about 0.35 to 52.5 mg per day, and most preferably about 0.7 to 35 mg per day. Administration can be as a single daily dose or divided into 2 or more doses per day, over a period of treatment lasting from about 1 to about 7 days. The amount of active compound administered will, of course, be dependent on the subject and disease state being treated, the severity of the affliction, the manner and schedule of administration and the judgment of the prescribing physician.

Formulation

[0114] The compounds of the invention that are used in the methods of the present invention can be incorporated into a variety of formulations and medicaments for therapeutic administration. More particularly, the compounds as provided herein can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers, excipients and/or diluents, and can be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, pills, powders, granules, dragees, gels, slurries, ointments, solutions, suppositories, injections, inhalants and aerosols. As such, administration of the compounds can be achieved in various ways, including oral, buccal, inhalation (pulmonary, nasal), rectal, parenteral, intraperitoneal, intradermal, topical, transdermal, intracranial and/or intratracheal administration. Moreover, the compounds can be administered in a local rather than systemic manner, e.g., in a topical cream or gel, a depot or a sustained release formulation. The compounds can be formulated with common excipients, diluents or carriers, and compressed into tablets, or formulated as elixirs or solutions for convenient oral administration, or administered by the intramuscular or intravenous routes. The compounds can be administered alone, in combination with each other, or they can be used in combination with other known compounds including oither antiviral agents. Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences (Mack Publishing Company (1985) Philadelphia, PA, 17th ed.), which is incorporated herein by reference. Moreover, for a brief review of methods for drug delivery, see, Langer, Science (1990) 249:1527-1533, which is incorporated herein by reference. [0115] As to the appropriate excipients, carriers and diluents, reference may be made to the standard literature describing these, e.g. to chapter 25.2 of Vol. 5 of "Comprehensive Medicinal Chemistry", Pergamon Press 1990, and to "Lexikon der Hilfsstoffe fur Pharmazie, Kosmetik und angrenzende Gebiete", by H.P. Fiedler, Editio Cantor, 2002. The term "pharmaceutically acceptable carrier, excipient and/or diluent" means a carrier, excipient or diluent that is useful in preparing a pharmaceutical composition that is generally safe, and possesses acceptable toxicities. Acceptable carriers, excipients or diluents include those that are acceptable for veterinary use as well as human pharmaceutical use. A "pharmaceutically acceptable carrier, excipient and/or diluent" as used in the specification and claims includes both one and more than one such carrier, excipient and/or diluent. [0116] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the compounds of the invention, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and [gamma] ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT(TM) (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3- hydroxybutyric acid.

[0117] The compounds of the invention can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0118] The pharmaceutical compositions described herein can be manufactured in a manner that is known to those of skill in the art, i.e. , by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. The following methods and excipients are merely exemplary and are in no way limiting. For injection, a compound of the invention (and optionally another active agent) can be formulated into preparations by dissolving, suspending, or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers, and preservatives. Preferably, the compounds of the invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0119] Preferably, pharmaceutical formulations for parenteral administration include aqueous solutions of the compounds of the invention in water-soluble form. Additionally, suspensions of the compounds of the invention can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compounds of the invention to allow for the preparation of highly concentrated solutions. [0120] The amount of a compound of the invention that can be combined with a carrier material to produce a single dosage form will vary depending upon the viral disease treated, the mammalian species, and the particular mode of administration. It will be also understood, that the specific dose level for any particular patient will depend on a variety of factors including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the individual being treated; the time and route of administration; the rate of excretion; other drugs that have previously been administered; and the severity of the particular viral disease undergoing therapy, as is well understood by those of skill in the area.

[0121] Another aspect of the invention provides a virucidal composition comprising an effective amount of one or more compounds of the invention and optionally at least one suitable carrier or aerosol carrier. “An effective amount” refers to the amount sufficient for irreversibly inhibiting viruses; i.e. sufficient for obtaining virucidal effect. In an embodiment, the suitable carrier is selected from the group comprising stabilisers, fragrance, colorants, emulsifiers, thickeners, wetting agents, or mixtures thereof. In another embodiment, the virucidal composition can be in the form of a liquid, a gel, a foam, a spray or an emulsion. In a further embodiment, the virucidal composition can be an air freshener, a sterilizing solution or a disinfecting solution.

[0122] Another aspect of the invention provides a device (or a product) comprising the virucidal composition of the invention or one or more compounds of the invention and means for applying and/or dispensing thereof (i.e. the compounds of the invention or the virucidal composition). In another embodiment, the means comprise a dispenser, a spray applicator or a solid support soaked with the compounds of the invention. In another embodiment, the support is a woven or non-woven fabric, a textile, a paper towel, cotton wool, an absorbent polymer sheet, or a sponge.

[0123] Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients can also be administered.

[0124] Formulations of the active compound or a salt may also be administered to the respiratory tract as an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the formulation have diameters of less than 50 microns, preferably less than 10 microns.

[0125] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

EXAMPLES

[0126] The following examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. All references cited herein are incorporated by reference in their entirety. Example 1 dPG-Carboxylate

[0127] 1A. Preparation of Trimethylolpropane (TMP) (25 mmol) was deprotonated by potassium methoxide solution (10 mmol KOH in 10 mL methanol). The resulting methanol was evaporated at 60 °C under vacuum (3 mbar). The synthesis- reactor was then heated to 100 °C and glycidol was added slowly over a period of 24h. The resulting dPG, having an average molecular weight of 10 kDa (“dPGio _kDa ” or “C1”), was used for functionalization. The product was characterized by NMR and GPC (H ₂ 0) for the determination of absolute molecular weights and polydispersity index [GPC (H ₂ 0): M _n = 9.6 kDa, M _w = 12.6 kDa, D = 1.31] The degree of branching (DB) was calculated to be 59%, using inverse gated (IG) ¹³ C NMR, as reported in literature.

[0128] By varying the amount of glycidol and adjusting the period of addition, the corresponding dPGs, having average molecular weights of 5 kDa ("dPGs _k Da"), 25 kDa ("dPG ₂ 5kDa"), and 100 kDa ("dPGiookDa") were similarly obtained.

[0129] 1B. Preparation of dPGio _k r> _a -allyl (5): Using the product obtained, e g., as described in Example 1A, dPGio _kDa (200 mg, 2.69 mmol OH to be functionalized) was dried at 60 °C overnight under high vacuum. The dried dPG was dissolved in dry DMF (20 mL) and cooled to 0 °C in an ice bath. To the stirred solution of dPG in dry DMF at 0 °C, NaH (129.12 mg, 5.38 mmol, 2 eq.) was added. After the NaH addition, the ice bath was removed and the temperature of the reaction mixture was allowed to reach room temperature. The reaction mixture was allowed to stir for 1 hour at room temperature, then stirred for 1 hour at 40 °C and then cooled down again using an ice bath. The allyl bromide (3-bromoprop-1-ene) (465 pL, 5.38 mmol, 2.0 eq.) in dry DMF (1 mL) was added dropwise to the reaction mixture using a syringe. The ice bath was removed and after stirring for 24 hours at 40 °C the reaction was quenched by addition of methanol and the resulting mixture was dialyzed in MeOH to afford dPG-allyl (DF = 100). Degree of allylation was quantified by ¹ H NMR in CD ₃ OD.

[0130] 1C. Preparation of Formula I: In dPG-allyl (DF=100) (50 mg, 0.67 mmol of allyl group) and 11-mercaptoundecanoic acid (1.35 mmol, 295 mg) were dissolved in methanol (5 mL). 2,2-Dimethoxy-2-phenylacetophenone (DMPA) as radical initiator (50 mg, 0.19 mmol) and a catalytic amount of tris(2-carboxyethyl)phosphine hydrochloride (TCEP- HCI) were added to the reaction to avoid oxidation of the thiol intermediate. Upon observing precipitation, a few drops of water were added to dissolve the precipitate and obtain a clear solution. The solution was degassed by flushing argon through the reaction mixture for 10 minutes. The reaction mixture was stirred and irradiated with UV light using a high pressure UV lamp at room temperature for 5 hours. The reaction mixture was dialyzed (MWCO 2 kDa) against methanol/water mixture to remove the TCEP.HCI, DMPA and any excess unreacted thiol. The successful formation of product, dPGio _kD a-C ₃ SCio-COO Na ⁺ ioo _% , was confirmed by ¹ HNMR of pure product by correlating the aliphatic protons of ligands at 1.5- 1.00 ppm with the polyglycerol backbone protons at 3.7-3.2 ppm. In addition, elemental analysis was used for the sulfur content measurement confirming the click reaction.

[0131] 1D. Other Compounds of Formula I: By following the procedures of

Examples 1 B and 1C and substituting dPGio _kDa with the following compounds as obtained in Example 1A, dPGs _kDa , dPG ₂ 5 _kDa and dPGioo _kDa , there were obtained the following:

• dPG _5kD a-C ₃ SCio-COO Na ⁺ ioo% ("EM124"),

• dPG _25kD a-C ₃ SCio-COO-Na ⁺ ioo% ("EM 126") and

• d PG 1 oo _kDa -C ₃ SCi o-COO N a ⁺ i oo _% ("EM 131"), the characterization data for which are shown in Figures 7, 8 and 9.

[0132] 1E. Other Compounds of Formula I: By following the procedures of

Examples 1B and 1C and substituting dPGio _kDa with dPGs _kDa , as obtained in Example 1A, and substituting 11-mercaptoundecanoic acid with 12-mercaptododecanoic acid, there was obtained the following:

• dPG _5kD a-C ₃ SCii-COO Na ⁺ ioo% ("EM125"), the characterization data for which is shown in Figure 10.

Example 2

Other Compounds of Formula I

[0133] 2A. Formula 5: By following the procedures of Example 1 B and substituting allyl bromide with: a) 4-bromobut-1-ene, b) 5-bromopent-1-ene, c) 7-bromo-5-methylhept-1-ene, and d) 10-iodo-5-isopropyl-6-methyldec-1-ene; there are obtained the following compounds, respectively: a) dPGiokDa-but-1-eneioo%, b) dPGiokDa-pent-1-eneioo%, c) dPGiokDa-5-methylhept-1-eneioo%, and d) dPGiokDa-5-isopropyldec-1-eneioo%.

[0134] 2B. Formula I: By following the procedure described in Example 1C and substituting dPGio _kDa -allyhoo _% with compounds obtained in Example 2A, there are obtained the following respective compounds: a) d PG 1 o _kDa -C4SCi o-COO N a ⁺ i oo _% , b) d PG 10 _kDa -CsSC _l 0-COO N a ⁺ _l oo _% , c) dPGiokDa-C7(5-Me)SCio-COO Na ⁺ ioo%, and d) dPGiokDa-Cio(5-iPr)SCio-COO Na ⁺ ioo%-

[0135] 2C. Formula I: By following the procedure described in Example 1C and substituting 11-mercaptoundecanoic acid with: a) 8-mercaptooctanoic acid, b) 3-(6-merdcaptohexyl)pentanedioic acid, c) 10-mercaptodecanoic acid, d) 12-mercaptododecanoic acid, and e) 13-mercaptotridecanoic acid; there are obtained the following compounds, respectively: a) dPGiokDa-C ₃ SC7-COO-Na ⁺ ioo%, b) dPGiokDa-C3SC7-(di-CH2-COO Na ⁺ )ioo%, c) dPGiokDa-C ₃ SC ₉ -COO-Na ⁺ ioo%, d) dPGiokDa-C ₃ SCn-COO-Na ⁺ ioo%, and e) d PG 1 o _kDa -C ₃ SCi 2-COO N a ⁺ i oo _%

[0136] 2D. Formula I: By following the procedures of Example 1B and substituting allyl bromide with: a) 8-bromooctanoic acid, b) 9-bromononanoic acid, c) 10-bromodecanoic, and d) 11-iodoundecanoic acid; there are obtained the following compounds, respectively: a) dPGl0kDa-C7-COOHl00%, b) dPGl0kDa-C ₈ -COOHl00%, c) dPGiokDa-C ₉ -COOHioo%, and d) dPGl0kDa-Cl0-COOHl00%.

[0137] 2E. Formula 11: By following the procedures of Example 1B and substituting allyl bromide with: a) 3-bromopropanol, and b) 4-brombutanol; there are obtained the following compounds, respectively: a) dPGiokDa-C3-OHioo%, and b) dPGl0kDa-C4-OHi00%-

[0138] 2F. Formula I: By following the procedures of Example 1C and substituting dPGiokDa-allyhoo% with compounds obtained in Example 2E, there are obtained the following respective compounds: c) dPGiokDa-C ₃ OCio-COO-Na ⁺ ioo%, and d) dPGiokDa-C4 OCio-COO Na ⁺ ioo%.

[0139] Example 3 Inhibition of SARS-CoV-2

[0140] A. Cells and Virus: Vero C1008 (clone E6) (ATCC CRL-1586) cells are propagated in DMEM High Glucose + Glutamax supplemented with 10% fetal bovine serum (FBS) and 1% pen ici 11 i n/streptavi d i n (pen/strep). SARS-

CoV2/Switzerland/GE9586/2020 is isolated from a clinical specimen in Vero-E6 and passaged twice before the experiments. Cells are infected with the virus and the supernatant is collected 3 days post infection, clarified, aliquoted, and frozen at -80 °C and subsequently titrated by plaque assay in Vero-E6.

[0141] B. Inhibition assay against SARS-CoV-2: The antiviral effect of dendritic polyglycerol against SARS-CoV-2 is tested by plaque reduction assays on Vero-E6 cells. Vero-E6 cells are plated 24h before the experiment in 24-well plates at a density of 10 ⁵ cells. A fixed amount of virus (MOI = 0.0005) is pre-incubated for 1 hour at 37°C with serial dilutions of the compound of interest. The solution is then transferred onto the cells and incubated for 1 hour. Afterwards, the solution is removed and the cells incubated for 24h in DMEM w5% FBS with 0.4w% Avicel. The cells are then stained with crystal violet and the plaques counted. The inhibition at each concentration is then compared to an untreated control and percentage of inhibiton calculated.

[0142] C. Virucidal Assay: Viruses (10 ⁵ pfu of SARS-CoV-2) and test compounds are incubated for 1 h at 37°C, and then the virucidal effect is investigated by adding serial dilutions of the mixtures on Vero-E6 for 1 h, followed by addition of medium containing avicel. Viral titers are determined at dilutions at which the material is not effective.

[0143] D. Results: When tested as described above in Example 3B, dPGio _kDa -C3-S- Cio-COONa ⁺ ioo _% inhibited SarsCoV-2 as shown in Figure 11. When tested as described above in Example 3C, the composition dPGiokDa-C3-S-Cio-C001\la ⁺ ioo% shows virucidal efficacy as shown in Figure 11.

[0144] D. Results: When tested as described above in Example 3B and Example 3C, the compounds dPG5kDa-C3SCio-C001\la ⁺ ioo% ("EM124"), dPG5kDa-C3SCii-C001\la ⁺ ioo% ("EM125"), d PG ₂ 5 _k Da-C ₃ SCi o-COO N a ⁺ i _00% ("EM126") and dPGioo _kDa -C ₃ SCio-COO-Na ⁺ ₁₀ o _% ("EM131") inhibited SarsCoV-2 and had virucidal activity as shown in Figures 12, 13, 14 and 15, respectively.

[0145] Example 4 Inhibition of HSV-2

[0146] A. Toxicity assay on Vero Cells: Cytotoxicity of the dendritic polyglycerol is tested on mammalian cells. Vero cells are plated 24h before the experiment in 96-well plates in order to have a confluent layer. Cells are then incubated with different concentrations of the compound being tested at 37°C for 24 h in DMEM w2% FBS. The solution is then removed and the cells washed with DMEM w2%FBS. 100 ul of DMEM w2% FBS is added in each well with 20 ul of MTS (CellTiter 96® AQueous One Solution Cell Proliferation Assay). After 4 hours of incubation at 37°C, the absorbance of each well is measured through a plate-reader (l=490 nm). A percentage of cytotoxicity was then calculated comparing the absorbance with a reference, in which cells were incubated with just DMEM w2% FBS.

[0147] B. Inhibition assay against HSV-2: The antiviral effect of dendritic polyglycerol against HSV-2 is tested by plaque reduction assays on Vero cells. Vero cells are plated 24h before the experiment in 24-well plates at a density of 10 ⁵ cells. A fixed amount of virus (MOI = 0.0005) is pre-incubated for 1 hour with serial dilutions of the compound of interest. The solution is then transferred onto the cells and incubated for 1 hour. Afterwards, the solution is removed and the cells incubated for 24h in DMEM w2% FBS with 0.45w% Methyl-Cellulose. The cells are then stained with crystal violet and the plaques counted. The inhibition at each concentration is then compared to an untreated control and percentage of inhibition calculated.

[0148] C. Virucidal assay against HSV-2: The virucidal activity of the dendritic polyglycerol against HSV-2 is tested by virucidal assay. Vero cells are plated 24h before the experiment in 96-well plates in order to have a confluent layer. An effective amount of dendritic polyglycerol (100-300-500 mg/ml) is incubated with a fixed amount of viruses (10 ⁵ - 10 ⁶ pfu/ml) for 1 hour at 37°C in DMEM - 2%FBS. A serial dilution of this solution is added in each well and incubated for 1 hour at 37°C. Afterwards, the solution is removed and the cells incubated for 24 h in DMEM w2% FBS with 0.45w% Methyl-Cellulose. The cells are then stained with crystal violet and the plaques counted. The viral titer is evaluated and compared against a reference with no compound.

[0149] D. Data Analysis: The EC50 values for inhibition curves (dose-response assay) are calculated using GraphPad Prism 8.0 using a 4-parameter.

[0150] E. Results: The compound of the invention, dPGiokDa-C3-S-Cio-COONa ⁺ ioo%, when tested, for example as described above, was found to have an IC50 of about 79 mg/ml and to have virucidal activity against HSV-2, as shown in Figure 16.

[0151] Example 5

In Vivo Testing in the Syrian Hamster Model [0152] Hamsters 6 wk to 10 wk old are anesthetized with ketamine/xylazine/atropine and inoculated intranasally with 50 pl_ containing 2 ^c 106 TCID50. Test compound is administered daily for 4 days intranasally (100mI in PBS). Two different doses are tested: 1.5 mg/kg/day and 4.5 mg/kg/day. A placebo is used (100 mI of PBS) as a negative control. MK-4482 [an orally administered bioavailable prodrug (5'-isobutyric ester form) of the cytidine nucleoside analog EIDD-1931 ^-D-N4-hydroxycytidine; NHC)] is used as a positive control. MK-4482 is administered twice a day via oral gavage (187mI) in formulation (MK-4482 at 100mg/ml in 85:10:2.5 water: PEG400:Chremophor) as reported in Rosenke et al. Hamsters are monitored daily for appearance, behavior, and weight. At day 14 hamsters are euthanized and tissues [lungs, small intestine (ileum)] are collected. Viral RNA and infectious virus are quantified by RT-qPCR and end-point virus titration, respectively.

[0153] When tested as described above, compounds of Formula I are effective in treating hamsters infected with SARS-CoV-2, including inhibition of weight loss. Figure 17 shows the results of testing dPGio _kDa -C ₃ -S-Cio-C001\la ⁺ ioo _% , where the control is indicated by a grey circle, placebo is indicated by a solid square, Di-Low represents 1.5 mg/kg/day of dPGio _kDa -C ₃ -S-Cio-COO-Na ⁺ ioo _% and is indicated by an upward-pointing triangle, D1-Med represents 4.5 mg/kg/day of dPGiokDa-C3-S-Cio-C001\la ⁺ ioo% and is indicated by a downward-pointing triangle, and MK-4482 is indicated by a solid black circle. Example 6

Lip Balm Formulation

As disclosed in US 2013/0150312, polyethylene 1450 and 300 are melted at 50° C with stirring. The dPGio _kDa -C ₃ SCio-COO Na ⁺ ioo _% , 2-deoxy-D-glucose, silica gel and stevioside are triturated together. The triturated powders are slowly sifted into the melted PEGs with stirring. The flavoring is added, followed by thorough mixing. The mixture is poured into applicator tubes and allowed to cool to room temperature.

Example 7

Aqueous Cream Formulation

As disclosed in US 2013/0150312, a part of the dPGio _kDa -C ₃ SCio-COO Na ⁺ ioo _% is dissolved in water with the 2-deoxy-D-glucose and propylene glycol at ambient temperature to produce an aqueous solution. The paraffins and emulsifiers (cetostearyl alcohol and sodium lauryl sulphate) are mixed together, heated to 60° C, and emulsified with the aqueous solution, also at 60° C. The remaining dPGio _kDa -C3SCio-COO Na ⁺ ioo _% is added, the mixture dispersed, allowed to cool, and filled into lacquered aluminum tubes.

Example 8

Nebulizer Formulation

A nebuliser, for example, as disclosed in US 9,364,618 B2 or EP 3,517,117 A1 is provided comprising the following pharmaceutical composition in its fluid reservoir: 14.0 mg dPGio _kDa -C ₃ SCio-COO-Na ⁺ ₁₀ o _% , 0.9% w/v NaCI dissolved in sterile deionised water. The nebuliser is used to deliver the composition by inhalation as an aerosol to the lower respiratory tract of a patient suffering from influenza.

[0154] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. All patents and publications cited above are hereby incorporated by reference.