Biphenylpyrans with broad-spectrum methyltransferase (2'-o-mtase) inhibition against coronaviruses

Abstract

Provided are antiviral compounds, pharmaceutical compositions and methods for treating coronavirus infections.

Description

[0001] PCT APPLICATION 112746-110233 (P24015)

[0002] BIPHENYLPYRANS WITH BROAD-SPECTRUM METHYLTRANSFERASE (2 -0- MTASE) INHIBITION AGAINST CORONAVIRUSES

[0003] FIELD OF THE INVENTION

[0004] The invention relates to inhibitors of the methyltransferase (2'-O-MTase) of coronaviruses, a highly conserved non-structural protein (NsplO-16).

[0005] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes and to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.

[0006] BACKGROUND OF THE INVENTION

[0007] The COVID- 19 pandemic sparked a global effort to rapidly develop antiviral therapeutics for those infected by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As a result, novel messenger RNA (mRNA) vaccines from Pfizer and Modema, as well as a traditional viral vector vaccine from Johnson and Johnson (J&J), were developed in recordbreaking time - largely thanks to years of prior research, modem biotechnology, and Operation Warp Speed.

[0008] The protein in SARS and MERS coronaviruses responsible for mRNA maturation and host innate immune response evasion is the 2'-( -methyltransferase (2’-O-MTase; NsplO-16; PDB ID: 6W4H. 3R24, and 5YNB). which is an Nsp heterodimer complex highly conserved among all Betacoronaviruses. The 2'-O-MTases of SARS coronaviruses share primary amino acid sequences up to 95% and 99% identical to Nsp 16 and Nsp 10, respectively. A recent study suggests that substrate selectivity is broader in SARS-CoV-2 and determined by the Nsp 10 cofactor ofthe NsplO-16 complex. This highly conserved nature of amino acids and substratebinding structures suggests that 2'-O-MTase is a potential broad-spectrum target amongst coronaviruses. Targeting the 2'-O-MTase would prevent the virus’s mRNA from maturing before translation, which is required to mimic eukaryotic mRNA and avoid detection and degradation by cytosolic ribonucleases (RNases). The 2'-O-MTase uses S-adenosylmethionine

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[0011] (SAM) to methylate Cap-O-RNA (m7GpppA2'-OH-RNA) into Cap-1 -RNA (m7GpppAm2'-O- RNA). Small molecule antivirals that inhibit SAM’s natural role as an active methylator of adenosine may prevent coronaviruses from subverting the induction of interferons (IFNs) and translating their viral RNA.

[0012] Unlike current vaccines, which immunize the body by inducing the generation of antibodies for future protection, antiviral agents that directly target viral nonstructural proteins (Nsps) are potential alternative treatments for current and future pandemics. A need exists in the art for such alternative antiviral agents.

[0013] SUMMARY OF THE INVENTION

[0014] Provided are antiviral compounds, pharmaceutical compositions and methods for treating a coronavirus infections.

[0015] In one embodiment of the invention, a compound is provided from the biphenylpyran (BPP) scaffold: or pharmaceutically acceptable salt thereof.

[0016] In another embodiment of the invention, provided is a pharmaceutical composition, comprising a therapeutically effective amount of a compound above, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0017] In a further embodiment of the invention, provided is a method for treating a coronavirus infection comprising the step of administering a therapeutically effective of a compound above, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

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[0020] DETAILED DESCRIPTION OF THE INVENTION

[0021] Currently, there are only preclinical and clinical candidates aimed at targeting the NsplO-16 complex. These include sinefungin, a SAM nucleoside analog: 3-Deazaneplanocin A (DZNep), a SAM cycle inhibitor; and other small molecule inhibitors. Broad affinity to multiple Nsps is the major reoccurring issue for many of these experimental therapeutics, thus emphasizing the need for a selective inhibitor with reduced chances of off-target effects that may result in cytotoxicity and unintended mechanisms of action (MoA). As a result, a candidate compound, CPM-1, was sourced from a unique in-house pool of nearly 500 compounds (SI-2 Docking List) composed of natural product scaffolds, SAM analogs, and repurposed FDA-approved antivirals used to treat SARS-CoV-2 infections.

[0022] In certain embodiments of the invention, cannabinoid-inspired synthetic antivirals were designed, screened, and synthesized to target the 2'-<9-MTase in Coronaviridae, such as SARS- CoV-2. These desired antivirals were chosen based on unique natural product-inspired scaffolds, synthetic feasibility, and high computational binding affinity and selectivity to NsplO-16. Embodiments of the antiviral compounds of the invention include:

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[0024] 3257680.1 PCT APPLICATION 112746-110233 (P24015)

[0025] TPMs CPMs

[0026] TPM-3 CPM-3

[0027] Nomenclature was assigned following the system guidelines set by the International Union of Pure and Applied Chemistry (IUPAC) for naming and classifying chemical compounds as follows:

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[0029] 3257680.1 PCT APPLICATION 112746-110233 (P24015)

[0030] Cannabinoid-like synthetics, such as CPM-1 and analogs, may be potential antiviral Nsp inhibitors due to high computational in silico binding affinities to coronavirus Nsps, as well as in vitro activity, as demonstrated in this study. Additionally, CPM-1 may also be non-cytotoxic, non-mutagenic, and non-psychoactive due to a unique CBN-like BCP core motif that posses an aromatic ring rather than an alkene. For instance, there are no medically reported fatal overdoses from cannabidiol (CBD) or related cannabinoids, which are recreationally used by 14% or nearly 50 million Americans. Some during the 2020 COVID-19 pandemic claimed that the use of CBD alleviated symptoms. Interestingly, some studies demonstrate that CBD potently inhibited SARS-CoV-2 replication in lung epithelial cells while reducing the expression of the ACE2 receptor and pro-inflammatory cytokines. One of the studies indicated that the MoA of cannabinoid ligands is the formation of stable conformations with Nsp binding pockets responsible for the viral RTC. In another, CBD was found to be associated with preventing viral gene expression by up-regulating the host’s IFN signaling pathways that target viral RNA, thus preventing translation. Similar studies have shown potency for both A9-THC (IC50 = 10.3 pM) and CBD (IC50 = 7.9 pM) against SARS-CoV-2 using Vero cells, which also demonstrated higher molar concentration doses as potentially safe and non-cytotoxic.

[0031] Logistical and natural product sourcing challenges resulted in synthetic alternatives to natural product scaffolds for analog production. As a result, CPM-1, a cannabinoid-like synthetic compound, was selected due to computational broad-spectrum coronavirus 2'-( -MTase selectivity, but with the most promising binding affinity to SARS-CoV-2 of -9.8 kcal / mol (Table 1 and S3). The synthesis of CPM-1 resulted in the generation of four novel molecules of two novel classes: tetrahydrophenylmethanones (TPMs)- TPM-1 and 2, and chromenephenylmethanones (CPMs)- CPM-1 and 2. These all demonstrated high

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[0033] 3257680.1 PCT APPLICATION 112746-110233 (P24015) computational docking scores in comparison to SAM and cannabinoids sharing core motifs with the synthetic cannabinoid-like molecules.

[0034] Table 1. In silico binding affinities (kcal / mol) to SARS-CoV-2 Nsps.

[0035] Computational Docking to SARS-CoV-2 Nsps

[0036] Compound 2'-(9-MTase 3CLpro RdRp (Nspl2- PLpro (Nsp3)

[0037] (NsplO-16) (Nsp5) 7-8)

[0038] SAM -7.8 -7.9 -7.1 -9.5

[0039] Remdesivir -8.9 -7.4 -7.1 -7.2

[0040] TPM-1 -9.7 -7.9 -6.7 -7.4

[0041] CFMA -9.8 -8.1 -7.1 -8.2

[0042] TPM-2 -9.6 -9.5 -7.1 -8.8

[0043] CPM-2 -9.5 -8.7 -6.8 -8.1

[0044] CBN -7.8 -7.3 -5.6 -7.1

[0045] A8-THC -8.1 -7.2 -5.6 -6.9

[0046] A9-THC -8.1 -7.3 -5.4 -8.0

[0047] PET -8.9 -7.6 -6.7 -8.0

[0048] The compounds of the invention can be synthesized via the general synthetic schemes as shown below :

[0049] Friedel-Crafts Alkylation Reaction

[0050] Scheme SI. First step of the BPP synthesis of TPM-1 and TPM-2 via the Friedel-Crafts alkylation chemical reaction.

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[0053] Terpenoid Aromatization Reaction

[0054] Scheme S2. Second step of the synthesis of a) CPM-1 and b) CPM-2 via the terpenoid aromatization chemical reaction.

[0055] Reaction mechanisms for the formation of TPMs and CPMs are as follows:

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[0058] To produce CPM-1, a synthetic chemical reaction between fS')-c7.s-\ erbenol and a resorcinol moiety was determined as the most straightforward and cost-effective option for TBP motif synthesis. This would be followed by an aromatization reaction to yield a BCP motif. (S)-cis- verbenol is naturally found in the feverfew flowering plant (Tanacetum parthenium) and a few other organisms, whereas many types of resorcinols are synthetics or semi-synthetics produced from plant resins. Resorcinols with biphenyl core motifs (e g., benzophenones and phenyl stilbenes) react with verbenol to produce biphenylpyrans (BPPs). The two-step synthesis of cannabinoid derivatives used in this study to produce a TBP core motif (found in A8-THC) and a BCP core motif (found in CBN) is outlined in Scheme 1.

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[0061] The first step in the synthesis as shown above involves a Friedel-Crafts alk lation reaction to form a TBP motif, which is the core ring system of A8-THC and other cannabinoids. The second step involves an aromatization reaction of the terpenoid cyclohexane from the TBP motif to form a BCP motif, which is the core ring system of CBN and other cannabinoids. The previously synthesized TPM regioisomers were used to produce CPM regioisomers via the terpenoid aromatization reaction. A total of four BPP products were synthesized: two TPMs (TPM-1 and TPM-2) and two CPMs (CPM-1 and CPM-2).

[0062] Anti-SARS-CoV-2 activity was assessed by measuring percent inhibition at 10 pM in Vero cells, which ranged from 98. 1 to 99.3% inhibition of SARS-CoV-2 in vitro, as seen in Table 2. In addition, ECso data were also obtained via antiviral dose-response assays for the four synthesized BPPs, which ranged from 4.3 to 8.6 pM. The four synthetics were nearly as potent as the control, remdesivir, which is a repurposed drug that became the first FDA-approved antiviral medication for COVID-19.

[0063] Table 2. Assessment of anti-SARS-CoV-2 activity via a virus yield reduction assay on Vero cells and antiviral dose-response assay for 50% effective concentration (EC50) of maximal response against Vero cells.

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[0066] Anti-SARS-CoV-2 Activity

[0067] Compound % Inhibition at 10 pM EC50 (pM)

[0068] Vero cells Vero cells

[0069] Remdesivir 99.9 2.3

[0070] TPM-1 99.3 4.3

[0071] CPM-1 98.9 7.65

[0072] TPM-2 98.9 7.8

[0073] CPM-2 98.1 8.6

[0074] A cytotoxicity assessment of the four BPP synthetics was carried out on PBM and Vero cells, as seen in Table 3. CPM-1 was the only synthesized BPP with an acceptable ‘safe’ cytotoxicity of CC50 of >100 pM for both PBM and Vero cells. Interestingly, remdesivir portrayed high cytotoxicity (2.0 pM) in PBM cells. Although Vero cells are non-human, they’re regularly utilized for initial toxicity screening. Additional antiviral and cytotoxicity testing is planned via HepG2, a hepatic cell line, and A559, the human lung cell line, for future synthetic cannabinoid-inspired analogs.

[0075] Table 3. The cytotoxic concentration of synthesized compounds that inhibited cell proliferation by 50% (CC50) on PBM and Vero cells.

[0076] Cytotoxicity

[0077] Compound CCso (pM)

[0078] PBM cells Vero cells

[0079] Remdesivir 2.0 >100

[0080] TPM-1 59.2 13.2

[0081] CPM-1 >100 >100

[0082] TPM-2 34.9 45.2

[0083] CPM-2 41.2 55.4

[0084] The ability of the four BPPs to inhibit the purified SARS-CoV-2 2'-O-MTase was determined using an established commercially available kinetic assay. The SARS-CoV-2 2'-O-MTase is composed of two proteins, NsplO and Nspl6, both of which were expressed and purified according to previous work. Enzyme activity was measured under linear conditions with Capfl (m7GpppAUUAA) RNA as a substrate, as described in the supporting information. All the

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[0086] 3257680.1 PCT APPLICATION 112746-110233 (P24015) synthesized compounds demonstrated inhibition of 2'-O-MTase, ranging from 1.5 - 6.7 pM (Table 4). For comparison, a control nucleoside-based methyltransferase inhibitor, sinefungin, has an IC50 of 3.4 ± 0.4 pM. These data reveal that the synthesized molecules, TPM-1 and CPM-1, comparably inhibit the SARS-CoV-2 2'-(9-MTase as well or better than the nucleoside-based sinefungin.

[0087] Table 4. Inhibitory activity of the TPM and CPM compounds against the SARS-CoV-2 1-0- MTase.

[0088] Compound 2'-O-MTase Inhibition

[0089] ICso (pM)

[0090] Sinefungin 3.4 ± 0.4

[0091] TPM-1 2.8 ± 0.5

[0092] CPM-1 1.5 ± 0.2

[0093] TPM-2 6.4 ± 1.5

[0094] CPM-2 6.7 ± 0.5

[0095] Synthesized compound CPM-1 was selected to be assayed for mutagenicity due to its minimal cytotoxicity in vitro and potent inhibition of SARS-CoV-2 2'-O-MTase. As a result, the Ames fluctuation test for CPM-1 at different concentrations was utilized to induce a mutagenic index (MI) in two different strains of Salmonella. A compound is considered mutagenic if the peak of the average number of reverse mutant colonies of strains TA98 and TAI 00 is greater than twice the average number of the negative control (DMSO). None of the six concentrations of CPM-1 tested with Salmonella TA98 or TA100 induced a reverse mutation.

[0096] Possible Inhibition Mechanisms

[0097] A previous study identified the catalytic KDKE tetrad motif (Lys6844-Asp6928-Lys6968- Glu7001) in SARS-CoV-2 2’-(9-MTase as essential for methyltransferase activity. Asp6928 initiates cap formation and facilitates methyl transfer from SAM to RNA, while the lysine residues stabilize the RNA, and glutamic acid ensures structural integrity. Analysis of the 2’- (9-MTase-SAM binding domain revealed that Asp6928 forms a hydrogen bond with the N- atom of SAM’s methionine unit. For TPMs and CPMs, the binding interactions involve distinct amino acid residues from that of SAM. However, their location within the narrow binding domain of SAM, in close proximity to the Asp6928 residue, suggests that CPMs and TPMs

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[0099] 3257680.1 PCT APPLICATION 112746-110233 (P24015) may inhibit methyltransferase activity by obstructing access to the catalytic KDKE tetrad \. This finding aligns with an earlier study on Machaeriols RS-1 and RS-2. The highly conserved nature of the SAM-dependent 2'-( -MTase protein across Betacoronaviruses , including SARS- CoV-2. SARS, and MERS, is well established. A previous study demonstrated that superposition and sequence alignment of the 2'-( -MTase protein structures of SARS-CoV-2 (PDB ID: 6W4H), SARS (PDB ID: 3R24), and MERS (PDB ID: 5YNB) revealed similar SAM-binding domains, all of which share the catalytic KDKE tetrad. Thus, TPMs and CPMs may serve as potential broad-spectrum inhibitors of 2'-O-MTase in Betacoronaviruses.

[0100] Definitions

[0101] A “subject'’ is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey, chimpanzee, baboon or rhesus, and the terms “subject” and “patient” are used interchangeably herein.

[0102] The invention also includes pharmaceutical compositions comprising an effective amount of the antiviral of the invention and a pharmaceutically acceptable carrier. The invention includes antiviral of the invention provided as a pharmaceutically acceptable prodrug, hydrate, salt, such as a pharmaceutically acceptable salt, enantiomers, stereoisomers, or mixtures thereof.

[0103] Representative “pharmaceutically acceptable salts” include, e.g., water-soluble and waterinsoluble salts, such as the acetate, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzonate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fiunarate, gluceptate, gluconate, glutamate, glycollylarsanilate. hexafluorophosphate, hexylresorcinate. hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, magnesium, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, A-methylglucamine ammonium salt, 3-hydroxy-2- naphthoate, oleate, oxalate, palmitate, pamoate (l.l-methene-bis-2-hydroxy-3-naphthoate, einbonate), pantothenate, phosphate / diphosphate, picrate, poly galacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosalicylate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate salts.

[0104] The term “carrier,” as used in this disclosure, encompasses carriers, excipients, and diluents and means a material, composition or vehicle, such as a liquid or solid filler, diluent, excipient,

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[0106] 3257680.1 PCT APPLICATION 112746-110233 (P24015) solvent or encapsulating material, involved in carrying or transporting a pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body.

[0107] The term '“treating,” with regard to a subject, refers to improving at least one symptom of the subject's disorder. Treating can be curing, improving, or at least partially ameliorating the disorder.

[0108] The term ““disorder” is used in this disclosure to mean, and is used interchangeably with, the terms disease, condition, or illness, unless otherwise indicated.

[0109] The term “administer,” “administering,” or “administration” as used in this disclosure refers to either directly administering a compound or pharmaceutically acceptable salt of the compound or a composition to a subject, or administering a prodrug derivative or analog of the compound or pharmaceutically acceptable salt of the compound or composition to the subject, which can form an equivalent amount of active compound within the subject’s body.

[0110] In one embodiment, the antivirals of the invention can each be administered in amounts that are sufficient to treat ALS. Administration of the antivirals of the invention can be accomplished via any mode of administration for therapeutic agents. These modes include systemic or local administration such as oral, nasal, parenteral (intravenous), intramuscular, intrathecal, intra-vitreal, transdermal, subcutaneous, vaginal, buccal, rectal, topical administration modes or as a drug-eluting stent.

[0111] Depending on the intended mode of administration, the compositions can be in solid, semisolid or liquid dosage form, such as, by way of non-limiting examples, injectables, tablets, suppositories, pills, time-release capsules, elixirs, tinctures, emulsions, syrups, powders, liquids, suspensions, nanoparticles or the like, sometimes in unit dosages and consistent with conventional pharmaceutical practices. Likewise, they can also be administered in intravenous (non-limiting examples include bolus and infusion), intraperitoneal, intrathecal, intra-vitreal injection, subcutaneous or intramuscular form, all using forms well known to those skilled in the pharmaceutical arts.

[0112] Non-limiting illustrative pharmaceutical compositions are tablets and gelatin capsules comprising the antivirals of the invention and a pharmaceutically acceptable carrier, such as: a) a diluent, e.g., purified water, triglyceride oils, such as hydrogenated or partially

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[0114] 3257680.1 PCT APPLICATION

[0115] 112746-110233 (P24015) hydrogenated vegetable oil, or mixtures thereof, com oil, olive oil, sunflower oil, safflower oil, fish oils, such as EPA or DHA, or their esters or triglycerides or mixtures thereof, omega-3 fatty acids or derivatives thereof, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, sodium, saccharin, glucose and / or glycine; b) a lubricant, e.g., silica, talcum, stearic acid, its magnesium or calcium salt, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and / or polyethylene glycol; for tablets also; c) a binder, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, magnesium carbonate, natural sugars such as glucose or betalactose, com sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, waxes and / or polyvinylpyrrolidone, if desired; d) a disintegrant, e.g., starches, agar, methyl cellulose, bentonite, xanthan gum, alginic acid or its sodium salt, or effervescent mixtures: e) absorbent, colorant, flavorant and sweetener; f) an emulsifier or dispersing agent, such as Tween 80, Labrasol. HPMC, DOSS, caproyl 909. labrafac, labrafil. peceol. transcutol, capmul MCM, capmul PG-12, captex 355, gelucire, vitamin E TGPS or other acceptable emulsifier; and / or g) an agent that enhances absorption of the compound such as cyclodextrin, hydroxypropyl-cyclodextrin, PEG400, PEG200.

[0116] Liquid, particularly injectable compositions can, for example, be prepared by dissolution, dispersion, etc. For example, the antivirals of the invention are dissolved in or mixed with a pharmaceutically acceptable solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form an injectable isotonic solution or suspension. Proteins such as albumin, chylomicron particles, or serum proteins can be used to solubilize the antivirals of the invention.

[0117] The antivirals of the invention can be also formulated as a suppository that can be prepared from fatty emulsions or suspensions; using polyalkylene glycols such as propylene glycol, as the carrier.

[0118] In further embodiments, the pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations

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[0121] The antivirals of the invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, containing cholesterol, stearylamine or phosphatidylcholines. In some embodiments, a film of lipid components is hydrated with an aqueous solution of drug to a form lipid layer encapsulating the drug, as described in United States Patent No. 5,262,564, the contents of which are herein incorporated by reference in their entirety.

[0122] Parenteral injectable administration is generally used for subcutaneous, intramuscular or intravenous injections and infusions. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions or solid forms suitable for dissolving in liquid prior to injection.

[0123] Compositions can be prepared according to conventional mixing, granulating or coating methods, respectively, and the present pharmaceutical compositions can contain from about 0. 1 % to about 80 %, from about 5 % to about 60 %, or from about 1 % to about 20 % of the antivirals of the invention by weight or volume.

[0124] A “therapeutically effective amount” when used in connection with the antivirals of the invention is an amount effective for treating or preventing, for example, Covid. The dosage regimen utilizing the antivirals of the invention is selected in accordance with a van etv of factors including type, species, age, weight, sex, race, diet, concomitant medications, and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal or hepatic function of the patient; and the particular the antivirals of the invention employed. A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.

[0125] Therapeutically effective amounts of the present invention, when used for the indicated effects, range from about 0. 1 mg to about 5000 mg of the active ingredient per unit dose which could be administered. In one embodiment, the compositions are in the form of a tablet that can be scored. Appropriate dosages of the antivirals of the invention can be determined as set forth in Goodman, L. S.; Gilman, A. The Pharmacological Basis of Therapeutics, 5th ed.; MacMillan: New York, 1975, pp. 201-226, the contents of which are hereby incorporated by reference.

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[0128] The antivirals of the invention can also be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times daily. Furthermore, the antivirals of the invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of atransdermal delivery system, the dosage administration can be continuous rather than intermittent throughout the dosage regimen. Other illustrative topical preparations include creams, ointments, lotions, aerosol sprays and gels, wherein the concentration of the antivirals of the invention ranges from about 0. 1 % to about 15 %, w / w or w / v.

[0129] EXAMPLES

[0130] The disclosure is further illustrated by the following examples, which are not to be construed as limiting this disclosure in scope or spirit to the specific procedures herein described. It is to be understood that the examples are provided to illustrate certain embodiments and that no limitation to the scope of the disclosure is intended thereby. It is to be further understood that resort may be had to various other embodiments, modifications, and equivalents thereof which may suggest themselves to those skilled in the art without departing from the spirit of the present disclosure and / or scope of the appended claims.

[0131] Example 1

[0132] Synthesis of Certain Compounds of the Invention

[0133] ACS and HPLC grade chemicals, reagents, and solvents were purchased through VWR, ThermoFisher Scientific, and Sigma Aldrich. Starting reagents for producing TPMs were (5)- cA-verbenol (95%) and 2,2’,4,4’-tetrahydroxybenzophenone (TBP) (97%), along with tetrafluoroboric acid-diethyl ether (HBF4 OEt2), used as a reaction catalyst. (,S')-c7.s-\ erbenol is naturally found in the feverfew flowering plant (Tanacetum parthenium) and a few other organisms, whereas many types of resorcinols, such as TBP, are synthetics produced from plant resins. (A)- / .s-\ erbenol has the “5” configuration and has the following specific rotation: [cr]o° = -9° in chloroform. A vanillin stain solution (15 g 1250 mL EtOH + 2.5 mL H2SO4), in conjunction with a heat gun, was utilized to observe and monitor the reaction and final products. Iodine (I2, >99%) was utilized as a reagent for the aromatization reaction. A Fast

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[0136] 112746-110233 (P24015)

[0137] Blue B Salt solution (0. 1 g / 250 mL DI H2O) was utilized to stain CPM compounds possessing aromatic rings. Plasmids encoding the NsplO and Nspl6 subunits were obtained from BEI Resources (NR-52425 and NR-52427). Cap-0 (m7GpppAUUAA) mRNA was synthesized by BioSynthesis.

[0138] All solvents and chemicals were used as purchased without further purification. All moisture and air-sensitive reactions were performed under an inert atmosphere via argon gas in oven-dried or flame-dried glassware. For this experiment, dried Schlenk tubes were utilized as a reaction vessel. Reactions that required heating were carried with a stir-hot plate using a heated external oil bath. The progress of all reactions was monitored on Merck precoated silica gel plates (with fluorescence indicator UV254) using ethyl acetate / n-hexane as a solvent system. Column chromatography was performed with SiliaFlash silica gel P60 (230-400 mesh) with the solvent mixtures specified in the corresponding experiment. Spots were visualized by irradiation with ultraviolet light (254 nm). The synthesized compounds were isolated by high- performance liquid chromatography (HPLC). The instrument was a Waters 486 Tunable Absorbance Detector and Automated Gradient Controller, and the column was a Kinetex® LC C18 100 A Column (250 x 21.2 mm). A program gradient, with a run time of 45 mins and a flow rate of 7 mL / min, consisted of an initial gradient of 75% H2O (DI H2O + 0.1% formic acid) and 25% MeOH (HPLC-grade), flowing to 0% H2O and 100% MeOH. All compounds are > 95% pure by HPLC analysis. Proton (' H) and carbon (13C) NMR spectra were recorded on a Bruker Avance II 600 MHz with UltraShield Plus magnet technology and a probe temperature of 307K. Samples were dissolved using deuterated chloroform (CDCh) as a solvent. Chemical shifts are given in parts per million (ppm) (8 relative to residual solvent peak for ' H and13C). For 'H NMR spectra, the proton signal (ppm) was at 7.28. For13C NMR spectra, the carbon signals were at 76.8, 77.0, and 77.20. One-dimensional (ID) 'H NMR data was collected via the ' PROTON parameter and "zg30" pulse sequence, and13C NMR data was collected via ‘C13CPD” parameter and “zgpg30’’ pulse sequence. Mass spectrometry (MS) data was collected via Impact II Elute QTOF UPLC (Bruker Daltonics, Bremen, Germany). Data were analyzed using DataAnalysis (Bruker Daltonics).

[0139] Synthesis of TPM- 1 and TPM-2

[0140] To a stirred solution of 2.2’,4,4’-tetrahydrobenzophenone resorcinol (246 mg, 1.0 mmol. 1.0 equiv.) in dry acetone (2 mL) at -78 °C was added HBF4-OEt2 (0.3 mL) dropwise. A

[0141] 17

[0142] 3257680.1 PCT APPLICATION

[0143] 112746-110233 (P24015) solution of fS'J-c / .s-verbenol (183 mg, 1.2 mmol, 1.2 equiv.) in DCM (4 rnL) was added dropwise to the reaction mixture. The reaction mixture was allowed to stay at this temperature for 2 hrs. The reaction mixture was removed from the cold bath and stirred at room temperature for 1 hr. The reaction was monitored for completion by TLC (254 nm, vanillin stain). The reaction was quenched by the addition of sat. aq. NaHCOs. The phases were separated, and the organics were washed with NaHCCh (x3). The aqueous phase was extracted with dry DCM (x3). The combined organic extracts were dried over anhydrous Na2SO4. After fdtration, the solvent was removed in vacuo, and the products were isolated from the crude reaction mixture by normal phase silica gel gradient column chromatography (5%, 20%, 50%, and 100% ethyl acetate in hexanes as eluent) as yellow oils. The 20% ethyl acetate fraction was then further fractionated via an isocratic elution using silica gel. Further purification was achieved by reverse phase HPLC [gradient- 75% H2O / 25% MeOH to 0% H2O / 100% MeOH. The final yields of TPM-1 and TPM-2 were 15.2% (65.0 mg) and 10.6% (45.5 mg), respectively. Prior to HPLC, the fraction was injected into a small disposable cartridge: a C18-E SPE sorbent (attached to a 13mm syringe filter with a 0.2 pM PTFE membrane).

[0144] TPM-1 (2, 4-dihydroxyphenyl)((6aR,10a / ?)-l -hydroxy-6, 6, 9-trimethyl-6a, 7,10,10a- tetrahydi o-6 / / -benzo[c|chromen-2-yl)methanone.

[0145] Off-white solid; yield 15.2%; 'H NMR (600 MHz, CDCh 7.28 ppm): 6 = 12.04 (s, 1H. OH), 11.13 (s, 1H, OH), 7.53 (d, J = 8.7 Hz, 1H, Ar-H), 7.39 (d, J = 8.9 Hz, 1H, Ar-H), 6.47 (d, J= 2.5 Hz, 1H, Ar-H), 6.40 (dd, J= 8.7, 2.5 Hz, 1H, Ar-H), 6.37 (d, J= 8.9 Hz, 1H, Ar-H), 5.54 (brs, 1H, OH), 5.46 - 5.43 (m, 1H, CH=), 3.36 (dd, J= 17.1, 4.1 Hz, 1H, CH2). 2.80 (td, .7= 10.9, 4.8 Hz. 1H, CH). 2.20 - 2.14 (m, 1H, CH2), 1.91 - 1.77 (m, 3H, CH2), 1.72 (s, 3H, CH3), 1.43 (s, 3H, CH3), 1.15 ppm (s, 3H, CH3).13C NMR (150 MHz, CDC13): 5 = 199.5, 164.1, 163.9, 161.6, 160.6, 135.2, 134.9, 132.3, 118.9, 114.2, 114.1, 112.6, 109.4, 107.2, 104.0, 78.6, 44.6, 35.5, 31.5, 27.8, 27.4, 23.4, 18.8 ppm. HPLC analysis: TPM-1 was collected at 4% H2O, 96% MeOH on the 43rd minute of the gradient run. MS: 381.208 m / z [M + H]+for C23H240S.

[0146] TPM-2 (2,4-dihydroxyphenyl)((6aR,10a / ?)-3-hydi oxy-6,6,9-tiimethyl-6a,7,10,10a- tetrahydro-6 / 7-benzo[c|chromen-2-yl)methaiione.

[0147] Pale yellow solid; yield 10.6%;JH NMR (600 MHz, CDC137.28 ppm): 5 = 11.30 (s, 1H, OH), 10.88 (s, 1H. OH). 7.52 (d, J = 8.7 Hz, 1H, Ar-H), 7.45 (d. J = 1.3 Hz. 1H, Ar-H), 6.49

[0148] 18

[0149] 3257680.1 PCT APPLICATION 112746-110233 (P24015)

[0150] (d, J= 2.5 Hz, 1H, Ar-H), 6.43 (dd J= 8.7, 2.5 Hz, 1H, Ar-H), 5.53 (brs, 1H, OH), 5.47 - 5.45 (m, 1H, CH=), 2.69 (td, J = 11.4, 5.5 Hz, 1H, CH), 2.49 - 2.46 (dd, 1H, CH), 2.47 (m, 1H, CH2), 2.20 - 2.14 (m, 1H, CH), 1.94 - 1.88 (m, 1H, CH2), 1.87 - 1.80 (m, 2H, CH2), 1.71 (s, 3H. CH3), 1.42 (s, 3H, CH3). 1.21 ppm (s. 3H. CH3).13C NMR (150 MHz, CDCh): 5 = 199.3, 164.5, 162.1, 161.7, 160.2, 135.0, 132.9, 132.2, 119.9, 117.8, 114.2, 113.7, 107.2, 105.2, 104.1, 78.9, 42.6, 36.6, 31.5, 27.5, 27.3, 23.4, 19.7 ppm. HPLC analysis: TPM-2 was collected at 7% H2O, 93% MeOH on the 41stminute of the gradient run. MS: 381.212 m / z [M + H]+for C33H24O5.

[0151] Synthesis of CPM-1

[0152] To a stirred solution of TPM-1 (52.5 mg, 0.138 mmol) in toluene (5.25 mL, 1 mL / mmol) at 90 °C was added iodine (I2) (52.5 mg, 1.0 equiv., 0.207 mmol). After 14.5 hrs, additional I2 (52.5 mg, 1.0. equiv.) was added. The reaction was monitored for completion by TLC (254 nm UV and Fast Blue staining). After 18 hrs., the reaction mixture was allowed to cool to room temperature and was quenched by the addition of sat. aq. NaHCO?,. The phases were separated, and the organics were washed with NaHCO3(x3), Na2O3S2 (x3), and brine sat. aq. solutions. The aqueous phase was extracted with hexane (x3). The combined organic extracts were dried over anhydrous N zSC - After filtration, the solvent was removed in vacuo. Purification was achieved by reverse phase HPLC gradient- 75% H2O / 25% MeOH to 0% H2O / 100% MeOH. The final yield of CPM-1 was (6.2 mg, yield 11.8%). Prior to HPLC, the fraction was injected into a small disposable cartridge: a C18-E SPE sorbent (attached to a 13mm syringe filter with a 0.2 pM PTFE membrane.

[0153] CPM-1 (2,4-dihydroxyphenyl)(l-hydroxy-6,6,9-trimethyl-6 / / -benzo[rjchi omen-2- yl)methanone).

[0154] Yellow solid; yield 11.8%; 'H NMR (600 MHz, CDC137.28 ppm): 5 = 12.45 (s, 1H, OH), 11.13 (s, 1H, OH), 8.47 (s, 1H, Ar-H), 7.55 (d, J= 8.7 Hz, 1H, Ar-H), 7.49 d, J= 8.7 Hz, 1H, Ar-H), 7.16 (d. J = 7.9 Hz, 1H, Ar-H), 7.13 (dd, J = 7.9, 1.6 Hz, 1H, Ar-H), 6.54 (d, J = 8.7 Hz. 1H, Ar-H), 6.49 (d, J = 2.5 Hz, 1H. Ar-H). 6.43 (dd. J = 8.7, 2.5 Hz, 1H, Ar-H), 2.41 (s, 3H, CH3), 1.65 ppm (s, 3H, CH3), 1.65 (s, 3H, CH3) ppm.13C NMR (150 MHz, CDC13): 8 = 199.95, 164.4, 162.4, 161.7, 160.2, 137.2, 136.0, 135.3, 133.9, 128.5, 127.5, 126.4, 122.4, 122.4, 114.1, 113.9, 109.5, 107.5, 104.0, 78.9, 27.5, 27.5, 21.6 ppm. HPLC analysis: CPM-1

[0155] 19

[0156] 3257680.1 PCT APPLICATION 112746-110233 (P24015) was collected at 6% H2O, 96% MeOH on the 42ndminute of the gradient run. MS: 377.269 m / z [M + H]+for C23H20O5.

[0157] Synthesis of CPM-2

[0158] To a stirred solution under reflux of TPM-2 (20 mg, 0.0526 mmol) in toluene (2.7 mL, 1 mL / mmol) at 80 °C was added iodine (I2) (20 mg, 1.0 equiv., 0.0788 mmol). After 1 hr. and 30 mins., additional I2 (20 mg, 1.0. equiv.) was added. The reaction was monitored for completion by TLC (254 nm UV and Fast Blue staining). After 3 hrs. and 30 mins., heat was removed, and the reaction product was allowed to reach room temperature. The reaction was quenched by the addition of sat. aq. NaHCCh. The phases were separated, and the organics were washed with NaHCCh (x3), Na20sS2 (x3), and NaCl salt brine (x3) sat. aq. solutions. The aqueous layer was separated from the organic layer during the washing steps and further extracted with hexane to collect the remaining organic product. No normal phase column chromatography was required. Further purification could be achieved by reverse phase HPLC gradient- 75% H2O I 25% MeOH to 0% H2O / 100% MeOH. The final yield of CPM-2 was (19.0%, 3.8 mg). Prior to HPLC, the fraction was injected into a small disposable cartridge: a C 18-E SPE sorbent (attached to a 13 mm syringe filter with a 0.2 pM PTFE membrane).

[0159] CPM-2 (2,4-dihydroxyphenyl)(3-hydroxy-6,6,9-trimethyl-6 / 7-benzo[cjchromen-2- yljinethanone.

[0160] Yellow solid; yield 19.0%; 'H NMR (600 MHz, CDCI3 7.28 ppm): 5 = 11.31 (s, 1H, OH), 11.06 (s, 1H, OH), 7.96 (d, J= 1.5 Hz, 1H, Ar-H), 7.63 (d, J= 8.7 Hz, 1H, Ar-H), 7.31 (s, 1H, Ar-H), 7.14 (d. J = 8.7 Hz, 1H, Ar-H), 7.10 (dd, J = 8.7, 1.5 Hz. 1H, Ar-H), 6.60 (s, 1H, Ar- H), 6.53 (d, J = 2.6 Hz, 1H, Ar-H), 6.47 (dd, J = 8.7, 2.6 Hz, 1H, Ar-H), 5.54 (brs, 1H, OH), 2.37 (s, 3H, CH3), 1.67 (s, 3H, CH3), 1.67 (s, 3H, CH3) ppm.13C NMR (150 MHz, CDC13): 5 = 199.4, 164.8, 163.9, 162.0, 159.8, 137.7, 135.5, 135.1, 128.5, 127.6, 127.2, 123.4, 121.9, 114.6, 114.1, 107.5, 106.3, 104.2, 79.3, 28.2, 28.2, 21.3 ppm. HPLC analysis: CPM-2 was collected at 10% H2O, 90% MeOH on the 41st minute of the gradient run. MS: 377.273 m'z [M + H]+for C23H24O5.

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[0162] 3257680.1 PCT APPLICATION 112746-110233 (P24015)

[0163] Example 2

[0164] Biological Examples

[0165] In Silico Molecular Binding Assay

[0166] The compound structures were improved using MM2 in Chem3D Ultra version 16.0. The crystal structures of the 2'-O-MTase (PDB ID: 6W4H (x-center = 83.181, y-center = 16.183, z-center = 28. 120), 3R24 (x-center = 57.272, y-center = 62.272, z-center = 68.032), and 5YNB (x-center = 61.628, y-center = 87.066, z-center = 148.084)) were obtained from Protein Data Bank. AutoDockTools version 1.5.6 was used to prepare the receptor proteins and ligands for the molecular docking experiment. The grid box parameters were: grid box spacing = 1.0 A; x-dimension = y-dimension = z-dimension = 20 A. AutoDock Vina program was used to perform the docking and calculate the binding affinity. Lastly, the results were processed and analyzed using the BIOVIA Discover}' Studio Visualizer v21. 1.0.20298.

[0167] ICso Determination Using MTaseGlo Coupling Assay

[0168] RNA methyltransferase activity of the Nspl0-16 complex was measured using the commercially available coupled assay MTaseGlo (Promega). The MTaseGlo kit measures the byproduct of methyltransferase reactions, .S'-adenosyl homocysteine (SAH), using luminescence. NsplO-16 methyltransferase activity was assessed with 25 nM heterodimer (NsplO-16), 1 pM excess Nsp 10. 20 mM Tns-HCl pH 8.0, 1 mM EDTA. 2 mM MgCl2, 300 nM Cap-0 (m7GpppAUUAA) RNA, and 5 pM .S’-adenosy I methionine and varying amounts of inhibitor solubilized in DMSO. The control reaction (no inhibitor) also contained 1% DMSO. Reactions were initiated with 2'-( -MTase and terminated at 15 min, as described in the manufacturer's instructions. Previous studies showed that under these conditions, the rate of methyltransferase activity was linear for at least 20 minutes. The amount of SAH in each reaction was determined using an SAH standard curve and the same coupled assay. Measurements were corrected by subtracting luminescence associated with a response that lacked RNA substrate. Reactions were done in at least duplicate. In order to avoid false positives from compounds that interfere with the MTaseGlo coupling system, each inhibitor was evaluated for its ability to alter the SAH standard curve. At 10 pM, none of the inhibitors affected the standard curve. The percent activity at all inhibitor concentrations was determined by taking the rate of NsplO-16 in the presence of the inhibitor divided by the rate of NsplO- 16 in the absence of the inhibitor and multiplied by 100. Measurements were taken in at least

[0169] 21

[0170] 3257680.1 PCT APPLICATION 112746-110233 (P24015) duplicate, and the average for each measurement was plotted as a function of inhibitor concentration. Data were fitted to the Hill Equation using guidelines outlined previously.

[0171] In Vitro Cytotoxicity Assay

[0172] An MTS (3-(4.5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2 / 7- tetrazol i um) assay was performed on PBM cells and Vero cells using the CellTiter 96® Non-Radioactive Cell Proliferation (Promega) kit as previously described. Briefly, cell proliferation was measured with or without test compounds after two to four days of incubation. Cytotoxicity was expressed as the concentration of test compounds that inhibited cell proliferation by 50% (CC50) and calculated using the Chou and Talalay method. The protocol for this assay followed the methods utilized by Zandi et al. in “Repurposing nucleoside analogs for human coronaviruses”.

[0173] In Vitro Antiviral Assay

[0174] An antiviral evaluation assay was conducted to determine the potential antiviral effects of the natural products and synthetics against in vitro replication of SARS-CoV-2 in cell culture. A confluent monolayer of Vero cells in a 96-well cell culture microplate was treated with 10 pM of compounds, followed by inoculation with 0. 1 MOI of the virus. To assess the antiviral activity, a vims yield reduction assay using specific qRT-PCR for each virus was performed. A dose-dependent antiviral assay was conducted for BPPs: TPM-1, CPM-1. TPM-2, and CPM- 2. Antiviral activity was further confirmed by virus yield reduction assay using specific qRT- PCR for SARS-CoV-2 by measuring the RNA copy number of the vims after 2-days posttreatment for Vero cells in supernatant of treated-infected cells in a dose-response manner. One step qRT-PCR was carried out in a final volume of 10 LIL containing extracted viral RNA, specific probe / primer mix, and qScript-Tough master mix (Quantibio, USA). Quantitative PCR measurement was performed using LightCycler 480 PCR system (Roche, Germany) according to the manufacturer’s protocol. The protocol for this assay followed the methods utilized by Zandi et al..

[0175] In Vitro Ames Assay

[0176] The Ames-MOD ISO™ test protocol was performed according to the method’s supplier protocol (Environmental Bio-Detection Products Inc). Sodium aside (NaN , 5 pg / mL) and 2-

[0177] 22

[0178] 3257680.1 PCT APPLICATION

[0179] 112746-110233 (P24015) nitrofluorene (2-NF, 300pg / mL) were used as positive controls for TAI 00 and TA98, respectively, while dimethyl sulfoxide (DMSO) was used as the negative control. The overnight cultures were diluted to OD600 = 0.05 for TAI 00 and = 0.1 for TA98 with exposure medium. Then. 200 pL of diluted bacterial culture and 200 pL of exposure medium were added to each well. After 100 min of incubation at 37 °C, 1.6 mL bacterial culture from 24 well plates was mixed with 8.71 mL reversion medium and then transferred into twelve 96- well plates for each strain evaluated. Plates were sealed into the Ziploc bags and incubated for 4 days at 37 °C. The plates were scored visually: yellow and partial yellow wells were scored as positive; purple wells were scored as negative.

[0180] DP4+ Calculations

[0181] The 2D structures of all plausible regioisomers of TPM and CPM compounds (TPM-1 (6a / ?, 10a / ?), TPM-2 (6a / ?, 10a / ?), TPM-3 (6a / ?, 10a / ?), TPM-1 (6a / ?,10a ), TPM-2 (6a / ?,10aS), TPM-3 (6a / ?,10a5), CPM-1, CPM-2 and CPM-3) were sketched in Maestro [Maestro, Schrodinger, LLC, New York, NY, 2020] and 3D-energy minimized at physiological pH 7.4 using the Limper [Schrodinger software Release 2020-4 Limper, Schrodinger, LLC, New York, NY, 2020] module of the Schrodinger software. The conformational searches of each Regio isomer were performed using MacroModel, considering mixed torsional / low-mode sampling. The energy window cutoff was set to 10 kcal / mol to cover all possible lowest energy conformers. Redundant conformers were eliminated using RMSD cutoff = 0.5 A. The conformations that showed >1% Boltzmann population from molecular mechanics calculations were further geometry optimized using DFT with mPWlPW91 / 6-311+G(d,p), using Gaussian 16 Rev. B.01 software [Gaussian, Inc., Wallingford, CT, 2016], CHCh was used as a solvent with the PCM solvation model. All the geometry optimizations included subsequent frequency calculations to verify that true minima on the potential energy surface were obtained. The Boltzmann-weighted optimized low-energy conformers in CHCh were used in the chemical shift calculations using GIAO NMR at the DFT mPWlPW91 / 6-311+G(d,p) level. The DP4+ probability analysis was performed to predict the correct regioisomers of TPM and CPM compounds. The DP4+ probability analysis revealed that TPM-1 (6a / ?, 10a / ?), TPM-2 (6a / ?, 10a / ?), CPM-1, and CPM-2 showed excellent agreement (100% probability) with the experimental NMR data of compounds TPM-1, TPM-2, CPM-1 and CPM-2, respectively. In addition, the standard statistical parameters of carbon and proton data, such as mean absolute error (MAE) and corrected MAE, also match with DP4+ probability data with some exceptions.

[0182] 23

[0183] 3257680.1 PCT APPLICATION 112746-110233 (P24015)

[0184] The table below portrays the DP4+ outcome for CPM-1 compared to the other regioisomers,

[0185] CPM-2 and CPM-3.

[0186] The half maximal effective concentration (EC50) for antiviral activity was calculated using GraphPad PRISM for Windows, version 9 (GraphPad Software Inc., San Diego, CA, 2005). In addition, for the Ames test, a sample is considered mutagenic when there is a significant increase in the number of positive wells in treated plates over those found in the negative control plates (i.e., mutagenic index (MI)). The results were expressed as Mutagenicity Ratio (MR = number of positive wells in treated plates / number of positive wells in control plates).

[0187] Supplemental Information

[0188] The following supplemental information amplifies the protocols used and the results obtained in Example 2 above:

[0189] Antiviral Test

[0190] Cell Lines

[0191] Vero (African Green Monkey Kidney) and Peripheral Blood Mononuclear (PBM) cell lines were used in this study. Cells were maintained and cultured in MEM containing 10% heat inactivated fetal bovine serum (FBS). The cells were incubated at 37 °C in the presence of 5% CO2. At the time of virus inoculation and antiviral assays, the concentration of FBS was reduced to 2%. SARS-CoV-2 (Isolate USA-WA1 / 2020) was provided by BEI Resources PCT APPLICATION 112746-110233 (P24015)

[0192] (Manassas, VA). SARS-CoV-2 has been propagated in each cell line and titrated by TCID50 method followed by storage of aliquots at - 80 °C until further use in the experiments.

[0193] Virus Kinetic Replication Assays

[0194] To determine the kinetic replication of SARS-CoV-2 in each cell line, a confluent monolayer of Vero cells in a 96-well cell culture microplate were inoculated at an MOI of 0.1 and the yield of progeny virus production was assessed in different time points using a specific qRT- PCR for SARS-CoV-2 for each cell line. Briefly, a one-step qRT-PCR was conducted in a final volume of 10 pL containing extracted viral RNA, probe / primer mix (Table 2), and qScript- Tough master mix (Quantibio, USA). Quantitative PCR measurement was performed using LightCycler 480 PCR system (Roche, Germany) according to manufacturer’s protocol. The protocol for this assay followed the methods utilized by Zandi et al. in ‘'Repurposing Nucleoside Analogs for Human Coronaviruses."’

[0195] Forward and reverse primers isolated from SARS-CoV-2 are listed in the table as their 5’ to 3" RNA genetic sequence. qRT-PCR was conducted to amplify viral RNA for a kinetic replication assay.

[0196] Ames Test

[0197] Compound Preparation

[0198] CPM-2 was prepared for incubation via 6 series dilution: 10 pM (1 / 1), 5 pM (1 / 2), 2.5 pM (1 / 4). 1.25 (1 / 8). 0.625 (1 / 16), and 0.3125 pM (1 / 32). A 10 mM stock solution of CPM-2 in DMSO was diluted (1 / 1000) in culture medium to get 10 pM. Diluted concentrations were

[0199] 25

[0200] 3257680.1 PCT APPLICATION 112746-110233 (P24015) performed in triplicates in 24-well plates and 1.6 mL of the test substance or the controls were used per well.

[0201] Cell Lines

[0202] The Ames mutagenicity assays were conducted via Salmonella TA98 and TA100 strains. The two strains were cultured in Dr. Zhiwei Ye’s laboratory at the Medical University of South Carolina, Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Charleston, South Carolina. TA98 strain was utilized to detect potential frameshift mutations, whereas TAI 00 strain was utilized to detect potential base-pair substitutions.

[0203] Biochemical Target-Validation Test

[0204] Expression and Purification of the SARS-CoV-22'-O-MTase

[0205] The SARS-CoV-2 RNA 2'-O-MTase is composed of two subunits, NsplO andNspl6. Plasmids encoding the NsplO and Nspl6 subunits of the 2'-(?-MTase were obtained from BEI Resources (NR-52425 and NR-52427) and were used to transform NiCo21(DE3) cells. Expression and purification of NsplO and Nspl6 proteins followed the previously described protocol with the following modifications. Cell cultures were induced with 0.5 mM IPTG at an OD 0.8 for Nsp 10 and an OD 1.8 for Nsp 16. Cell growths were harvested and resuspended in lysis buffer (50 mM Tris-HCl pH 8.3, 500 mM NaCl, 10% glycerol, 0.1% IGEPAL) and sonicated. Lysate was clarified through centrifugation and the resulting supernatant was applied to Gold Bio Nickel resin and incubated at 4 °C for 2 hours. The nickel resin was washed with 10 mM Tris-HCl pH 8.3, 500 mM NaCl, and 25 mM imidazole, and protein eluted off the resin in 10 mM Tris-HCl pH 8.3, 500 mM NaCl, 1 M imidazole. Nsp 10 was further purified by gel filtration in 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCh, 0.5 mM DTT, and 5% glycerol. Proteins were dialyzed into 10 mM Tns-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 0.5 mM DTT, and 5% glycerol. Proteins were concentrated, flash frozen in liquid nitrogen, and stored at -80 °C.

[0206] 26

[0207] 3257680.1 PCT APPLICATION 112746-110233 (P24015)

[0208] In Silico Assays

[0209] Binding Affinity

[0210] The RCSB PDB was utilized to obtain high resolution angstrom (A°) 3D crystal structures for Coronaviridae Nsps used for in silico computational analyses. The Nsps from SARS-CoV-2 and other coronaviruses derived from X-ray crystallography or NMR spectroscopy from the literature. The data for Nspl0-Nspl6 (6w4h and 3r24) was obtained from “High-resolution structures of the SARS-CoV-2 2’-O-methyltransferase reveal strategies for structure-based inhibitor design” and “Biochemical and Structural Insights into the Mechanisms of SARS Coronavirus RNA Ribose 2-O-Methylation by nspl6 / nspl0 Protein Complex. Drug binding affinities and protein-ligand interaction visuals were obtained via AutoDock Vina.

[0211] The table below shows the binding affinities (kcal / mol) of synthesized BPPs and major cannabinoids to SARS and MERS coronavirus protein targets: methyltransferase (2’-<9-MTase. NsplO-16), 3C-like proteinase protease (3CLpro, Nsp5), RNA-dependent RNA polymerase (RdRp, Nsp 12-7-8), and papain-like protease (PLpro, Nsp3). Binding affinity threshold of less than -8.5 kcal / mol is highlighted: Light blue- 2'-O-MTase, Peach- 3CLpro, and Light green- PLpro.

[0212] 27

[0213] 3257680.1 PCT APPLICATION 112746-110233 (P24015)

[0214] The figure below shows a two-dimensional (2D) in silico simulation of the potential inhibition mechanism of SAM, TPM-1, TPM-2, CPM-1, and CPM-2 between the amino acid residues of the SARS-CoV-2 2'-O-MTase.

[0215] EQUIVALENTS

[0216] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

[0217] 28

[0218] 3257680.1

Claims

PCT APPLICATION 112746-110233 (P24015)CLAIMS;1. A compound, selected from the group consisting of:or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition, comprising a therapeutically effective amount of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

3. A method for treating a coronavirus infection, comprising the step of administering a therapeutically effective amount of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.293257680.1

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