4'-halogen containing nucleotide and nucleoside therapeutic compositions and uses related thereto

Abstract

Disclosed are halogen containing nucleotide and nucleoside therapeutic compositions and uses related thereto. In certain embodiments, the disclosure relates to the treatment or prophylaxis of viral infections. Such viral infections can include tongaviridae, bunyaviridae, arenaviridae, coronaviridae, flaviviridae, picornaviridae, Eastern, Western, and Venezuelan Equine Encephalitis (EEE, WEE and VEE, respectively), Chikungunya fever (CHIK), Ebola, Influenza, RSV, and Zika virus infections.

Description

In one embodiment, the compound is administered after the subject has been 5 exposed to the malaria parasite. In another embodiment, a compound disclosed herein is administered before the subject travels to a country where malaria is endemic. The compounds or the above-mentioned pharmaceutical compositions may also be used in combination with one or more other therapeutically useful substances selected from the group comprising antimalarials like quinolines (e.g., quinine, chloroquine, amodiaquine, 10 mefloquine, primaquine, tafenoquine); peroxide antimalarials (e.g., artemisinin, artemether, artesunate); pyrimethamine-sulfadoxine antimalarials (e.g., Fansidar); hydroxynaphtoquinones (e.g., atovaquone); acroline-type antimalarials (e.g., pyronaridine); and antiprotozoal agents such as ethylstibamine, hydroxystilbamidine, pentamidine, stilbamidine, quinapyramine, puromycine, propamidine, nifurtimox, melarsoprol, 15 nimorazole, nifuroxime, aminitrozole and the like. In an embodiment, compounds disclosed herein can be used in combination one additional drug selected from the group consisting of chloroquine, artemesin, qinghaosu, 8-aminoquinoline, amodiaquine, arteether, artemether, artemisinin, artesunate, artesunic acid, artelinic acid, atovoquone, azithromycine, biguanide, chloroquine phosphate, 20 chlorproguanil, cycloguanil, dapsone, desbutyl halofantrine, desipramine, doxycycline, dihydrofolate reductase inhibitors, dipyridamole, halofantrine, haloperidol, hydroxychloroquine sulfate, imipramine, mefloquine, penfluridol, phospholipid inhibitors, primaquine, proguanil, pyrimethamine, pyronaridine, quinine, quinidine, quinacrineartemisinin, sulfonamides, sulfones, sulfadoxine, sulfalene, tafenoquine, 25 tetracycline, tetrandine, triazine, salts or mixture thereof. Cancer In a typical embodiment, the disclosure relates to a method treating cancer comprising administering to a patient a compound disclosed herein. In some embodiments, the disclosure relates to a compound disclosed herein, or a pharmaceutically acceptable salt 30 thereof for uses in treating cancer. In some embodiments, the disclosure relates to a compound disclosed herein, or a pharmaceutically acceptable salt thereof, as defined herein for use in the treatment of cancer of the breast, colorectum, lung (including small cell lung cancer, non- small cell lung cancer and bronchioalveolar cancer) and prostate. 2020418425   14 Sep 2026 In some embodiments, the disclosure relates to a compound disclosed herein, or a pharmaceutically acceptable salt thereof, as defined herein for use in the treatment of cancer of the bile duct, bone, bladder, head and neck, kidney, liver, gastrointestinal tissue, oesophagus, ovary, endometrium, pancreas, skin, testes, thyroid, uterus, cervix and vulva, 5 and of leukaemias (including ALL and CML), multiple myeloma and lymphomas. In some embodiments, the disclosure relates to a compound disclosed herein, or a pharmaceutically acceptable salt thereof, as defined herein for use in the treatment of lung cancer, prostate cancer, melanoma, ovarian cancer, breast cancer, endometrial cancer, kidney cancer, gastric cancer, sarcomas, head and neck cancers, tumors of the central 10 nervous system and their metastases, and also for the treatment of glioblastomas. In some embodiments, compounds disclosed herein could be used in the clinic either as a single agent by itself or in combination with other clinically relevant agents. This compound could also prevent the potential cancer resistance mechanisms that may arise due to mutations in a set of genes. 15          The anti-cancer treatment defined herein may be applied as a sole therapy or may involve, in addition to the compound of the disclosure, conventional surgery or radiotherapy or chemotherapy. Such chemotherapy may include one or more of the following categories of anti-tumour agents: (i) antiproliferative / antineoplastic drugs and combinations thereof, as used in 20 medical oncology, such as alkylating agents (for example cis-platin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulfan and nitrosoureas); antimetabolites (for example antifolates such as fluoropyrimidines like 5-fluorouracil and gemcitabine, tegafur, raltitrexed, methotrexate, cytosine arabinoside and hydroxyurea); antitumour antibiotics (for example anthracyclines like adriamycin, bleomycin, 25 doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (for example vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like taxol and taxotere); and topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan and camptothecin); and proteosome inhibitors (for example bortezomib [Velcade®]); and the 30 agent anegrilide [Agrylin®]; and the agent alpha-interferon; (ii) cytostatic agents such as anti-estrogens (for example tamoxifen, toremifene, raloxifene, droloxifene and iodoxyfene), oestrogen receptor down regulators (for example fulvestrant), antiandrogens (for example bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (for example goserelin, 2020418425   14 Sep 2026 leuprorelin and buserelin), progestogens (for example megestrol acetate), aromatase inhibitors (for example as anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5a-reductase such as finasteride; (iii) agents that inhibit cancer cell invasion (for example metalloproteinase inhibitors 5 like marimastat and inhibitors of urokinase plasminogen activator receptor function); (iv) inhibitors of growth factor function, for example such inhibitors include growth factor antibodies, growth factor receptor antibodies (for example the anti-erbb2 antibody trastuzumab [Herceptin™] and the anti-erbbl antibody cetuximab) , farnesyl transferase inhibitors, tyrosine kinase inhibitors and serine / threonine kinase inhibitors, for example 10 inhibitors of the epidermal growth factor family (for example EGFR family tyrosine kinase inhibitors such as: N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3- morpholinopropoxy)quinazolin-4-a mine (gefitinib), N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib), and 6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazolin-4-amine (CI 1033), for example 15 inhibitors of the platelet-derived growth factor family and for example inhibitors of the hepatocyte growth factor family, for example inhibitors or phosphotidylinositol 3-kinase (PI3K) and for example inhibitors of mitogen activated protein kinase kinase (MEK1 / 2) and for example inhibitors of protein kinase B (PKB / Akt), for example inhibitors of Src tyrosine kinase family and / or Abelson (AbI) tyrosine kinase family such as dasatinib (BMS-354825) 20 and imatinib mesylate (Gleevec™); and any agents that modify STAT signalling; (v) antiangiogenic agents such as those which inhibit the effects of vascular endothelial growth factor, (for example the anti-vascular endothelial cell growth factor antibody bevacizumab [Avastin™]) and compounds that work by other mechanisms (for example linomide, inhibitors of integrin ocv03 function and angiostatin); 25          (vi) vascular damaging agents such as Combretastatin A4; (vii) antisense therapies, for example those which are directed to the targets listed above, such as an anti-ras antisense; (viii) gene therapy approaches, including for example approaches to replace aberrant genes such as aberrant p53 or aberrant BRCAl or BRCA2, GDEPT (gene-directed enzyme 30 pro-drug therapy) approaches such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches to increase patient tolerance to chemotherapy or radiotherapy such as multi-drug resistance gene therapy; and (ix) immunotherapy approaches, including for example ex-vivo and in-vivo approaches to increase the immunogenicity of patient tumour cells, such as transfection 2020418425   14 Sep 2026 with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, approaches to decrease T-cell anergy, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumour cell lines and approaches using anti-idiotypic antibodies, and approaches 5 using the immunomodulatory drugs thalidomide and lenalidomide [Revlimid®]. Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment. Such combination products employ the compounds of this disclosure, or pharmaceutically acceptable salts thereof, within the dosage range described hereinbefore and the other pharmaceutically-active agent 10 within its approved dosage range. Formulations Pharmaceutical compositions disclosed herein may be in the form of pharmaceutically acceptable salts, as generally described below. Some preferred, but nonlimiting examples of suitable pharmaceutically acceptable organic and / or inorganic acids 15 are hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, acetic acid and citric acid, as well as other pharmaceutically acceptable acids known per se (for which reference is made to the references referred to below). When the compounds of the disclosure contain an acidic group as well as a basic group, the compounds of the disclosure may also form internal salts, and such compounds 20 are within the scope of the disclosure. When a compound of the disclosure contains a hydrogen-donating heteroatom (e.g., NH), the disclosure also covers salts and / or isomers formed by the transfer of the hydrogen atom to a basic group or atom within the molecule. Pharmaceutically acceptable salts of the compounds include the acid addition and base salts thereof. Suitable acid addition salts are formed from acids which form non-toxic 25 salts. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulphate / sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, 30 nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts. Suitable base salts are formed from bases that form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, 2020418425   14 Sep 2026 olamine, potassium, sodium, tromethamine and zinc salts. Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts. For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002), incorporated herein by reference. 5          The compounds described herein may be administered in the form of prodrugs. A prodrug can include a covalently bonded carrier that releases the active parent drug when administered to a mammalian subject. Prodrugs can be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compounds. Prodrugs include, for example, 10 compounds wherein a hydroxyl group is bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxyl group. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol functional groups in the compounds. Methods of structuring a compound as a prodrug are known, for example, in Testa and Mayer, Hydrolysis in Drug and Prodrug Metabolism, Wiley (2006). 15 Typical prodrugs form the active metabolite by transformation of the prodrug by hydrolytic enzymes, the hydrolysis of amide, lactams, peptides, carboxylic acid esters, epoxides or the cleavage of esters of inorganic acids. It has been shown that ester prodrugs are readily degraded in the body to release the corresponding alcohol. See e.g., Imai, Drug Metab Pharmacokinet. (2006) 21(3):173-85, entitled “Human carboxylesterase isozymes: catalytic 20   properties and rational drug design.” Pharmaceutical compositions for use in the present disclosure typically comprise an effective amount of a compound and a suitable pharmaceutical acceptable carrier. The preparations may be prepared in a manner known per se, which usually involves mixing the at least one compound according to the disclosure with the one or more pharmaceutically 25 acceptable carriers, and, if desired, in combination with other pharmaceutical active compounds, when necessary under aseptic conditions. Reference is made to U.S. Pat. No. 6,372,778, U.S. Pat. No. 6,369,086, U.S. Pat. No. 6,369,087 and U.S. Pat. No. 6,372,733 and the further references mentioned above, as well as to the standard handbooks, such as the latest edition of Remington's Pharmaceutical Sciences. 30          Generally, for pharmaceutical use, the compounds may be formulated as a pharmaceutical preparation comprising at least one compound and at least one pharmaceutically acceptable carrier, diluent or excipient, and optionally one or more further pharmaceutically active compounds. 2020418425   14 Sep 2026 The pharmaceutical preparations of the disclosure are preferably in a unit dosage form, and may be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable single-dose or multi-dose holder or container (which may be properly labeled); optionally with one or more leaflets containing product information 5 and / or instructions for use. Generally, such unit dosages will contain between 1 and 1000 mg, and usually between 5 and 500 mg, of the at least one compound of the disclosure, e.g., about 10, 25, 50, 100, 200, 300 or 400 mg per unit dosage. The compounds can be administered by a variety of routes including the oral, ocular, rectal, transdermal, subcutaneous, sublingual, intravenous, intramuscular or intranasal 10 routes, depending mainly on the specific preparation used. The compound will generally be administered in an "effective amount", by which is meant any amount of a compound that, upon suitable administration, is sufficient to achieve the desired therapeutic or prophylactic effect in the subject to which it is administered. Usually, depending on the condition to be prevented or treated and the route of administration, such an effective amount will usually 15 be between 0.01 to 1000 mg per kilogram body weight of the patient per day, every other day, twice weekly, or weekly, more often between 0.1 and 500 mg, such as between 1 and 250 mg, for example about 5, 10, 20, 50, 100, 150, 200 or 250 mg, per kilogram body weight of the patient per day, every other day, twice weekly, or weekly, which may be administered as a single daily, every other day, twice weekly, or weekly dose, or divided 20 over one or more daily, every other day, twice weekly, or weekly doses. The amount(s) to be administered, the route of administration and the further treatment regimen may be determined by the treating clinician, depending on factors such as the age, gender and general condition of the patient and the nature and severity of the disease / symptoms to be treated. Reference is made to U.S. Pat. No. 6,372,778, U.S. Pat. No. 6,369,086, U.S. Pat. 25 No. 6,369,087 and U.S. Pat. No. 6,372,733 and the further references mentioned above, as well as to the standard handbooks, such as the latest edition of Remington's Pharmaceutical Sciences. For an oral administration form, the compound can be mixed with suitable additives, such as excipients, stabilizers or inert diluents, and brought by means of the customary 30 methods into the suitable administration forms, such as tablets, coated tablets, hard capsules, aqueous, alcoholic, or oily solutions. Examples of suitable inert carriers are gum arabic, magnesia, magnesium carbonate, potassium phosphate, lactose, glucose, or starch, in particular, cornstarch. In this case, the preparation can be carried out both as dry and as moist granules. Suitable oily excipients or solvents are vegetable or animal oils, such as 2020418425   14 Sep 2026 sunflower oil or cod liver oil. Suitable solvents for aqueous or alcoholic solutions are water, ethanol, sugar solutions, or mixtures thereof. Polyethylene glycols and polypropylene glycols are also useful as further auxiliaries for other administration forms. As immediate release tablets, these compositions may contain microcrystalline cellulose, 5 dicalcium phosphate, starch, magnesium stearate and lactose and / or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art. When administered by nasal aerosol or inhalation, the compositions may be prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable 10 preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art. Suitable pharmaceutical formulations for administration in the form of aerosols or sprays are, for example, solutions, suspensions or emulsions of the compounds of the disclosure or their physiologically tolerable salts in a pharmaceutically acceptable solvent, such as ethanol or water, or a mixture of such solvents. 15 If required, the formulation may additionally contain other pharmaceutical auxiliaries such as surfactants, emulsifiers and stabilizers as well as a propellant. For subcutaneous or intravenous administration, the compounds, if desired with the substances customary therefore such as solubilizers, emulsifiers or further auxiliaries are brought into solution, suspension, or emulsion. The compounds may also be lyophilized 20 and the lyophilizates obtained used, for example, for the production of injection or infusion preparations. Suitable solvents are, for example, water, physiological saline solution or alcohols, e.g. ethanol, propanol, glycerol, sugar solutions such as glucose or mannitol solutions, or mixtures of the various solvents mentioned. The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, 25 parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. When rectally administered in the form of suppositories, the formulations may be 30 prepared by mixing the compounds of formula I with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquefy and / or dissolve in the rectal cavity to release the drug. In certain embodiments, it is contemplated that these compositions can be extended release formulations. Typical extended release formations utilize an enteric coating. 2020418425   14 Sep 2026 Typically, a barrier is applied to oral medication that controls the location in the digestive system where it is absorbed. Enteric coatings prevent release of medication before it reaches the small intestine. Enteric coatings may contain polymers of polysaccharides, such as maltodextrin, xanthan, scleroglucan dextran, starch, alginates, pullulan, hyaloronic acid, 5 chitin, chitosan and the like; other natural polymers, such as proteins (albumin, gelatin etc.), poly-L-lysine; sodium poly(acrylic acid); poly(hydroxyalkylmethacrylates) (for example poly(hydroxyethylmethacrylate)); carboxypolymethylene (for example CarbopolTM); carbomer; polyvinylpyrrolidone; gums, such as guar gum, gum arabic, gum karaya, gum ghatti, locust bean gum, tamarind gum, gellan gum, gum tragacanth, agar, pectin, gluten and 10 the like; poly(vinyl alcohol); ethylene vinyl alcohol; polyethylene glycol (PEG); and cellulose ethers, such as hydroxymethylcellulose (HMC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), methylcellulose (MC), ethylcellulose (EC), carboxyethylcellulose (CEC), ethylhydroxyethylcellulose (EHEC), carboxymethylhydroxyethylcellulose (CMHEC), hydroxypropylmethyl-cellulose (HPMC), 15 hydroxypropylethylcellulose (HPEC) and sodium carboxymethylcellulose (Na-CMC); as well as copolymers and / or (simple) mixtures of any of the above polymers. Certain of the above-mentioned polymers may further be crosslinked by way of standard techniques. The choice of polymer will be determined by the nature of the active ingredient / drug that is employed in the composition of the disclosure as well as the desired rate of release. 20 In particular, it will be appreciated by the skilled person, for example in the case of HPMC, that a higher molecular weight will, in general, provide a slower rate of release of drug from the composition. Furthermore, in the case of HPMC, different degrees of substitution of methoxyl groups and hydroxypropoxyl groups will give rise to changes in the rate of release of drug from the composition. In this respect, and as stated above, it may be desirable to 25 provide compositions of the disclosure in the form of coatings in which the polymer carrier is provided by way of a blend of two or more polymers of, for example, different molecular weights in order to produce a particular required or desired release profile. Microspheres of polylactide, polyglycolide, and their copolymers poly(lactide-co-glycolide) may be used to form sustained-release protein delivery systems. Proteins can be 30 entrapped in the poly(lactide-co-glycolide) microsphere depot by a number of methods, including formation of a water-in-oil emulsion with water-borne protein and organic solvent-borne polymer (emulsion method), formation of a solid-in-oil suspension with solid protein dispersed in a solvent-based polymer solution (suspension method), or by dissolving the protein in a solvent-based polymer solution (dissolution method). One can attach 2020418425   14 Sep 2026 poly(ethylene glycol) to proteins (PEGylation) to increase the in vivo half-life of circulating therapeutic proteins and decrease the chance of an immune response. Liposomal suspensions (including liposomes targeted to viral antigens) may also be prepared by conventional methods to produce pharmaceutically acceptable carriers. This 5 may be appropriate for the delivery of free nucleosides, acyl nucleosides or phosphate ester prodrug forms of the nucleoside compounds according to the present invention. It is appreciated that nucleosides of the present invention have several chiral centers and may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses 10 any racemic, optically active, diastereomeric, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein. It is well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic 15 separation using a chiral stationary phase). Carbons of the nucleoside are chiral, their nonhydrogen substituents (the base and the CHOR groups, respectively) can be either cis (on the same side) or trans (on opposite sides) with respect to the sugar ring system. The four optical isomers therefore are represented by the following configurations (when orienting the sugar moiety in a 20 horizontal plane such that the oxygen atom is in the back): cis (with both groups "up", which corresponds to the configuration of naturally occurring P-D nucleosides), cis (with both groups "down", which is a nonnaturally occurring P-L configuration), trans (with the C2' substituent "up" and the C4' substituent "down"), and trans (with the C2' substituent "down" and the C4' substituent "up"). The "D-nucleosides" are cis nucleosides in a natural 25 configuration and the "L-nucleosides" are cis nucleosides in the nonnaturally occurring configuration. Likewise, most amino acids are chiral (designated as L or D, wherein the L enantiomer is the naturally occurring configuration) and can exist as separate enantiomers. Examples of methods to obtain optically active materials are known in the art, and 30 include at least the following. i) physical separation of crystals-a technique whereby macroscopic crystals of the individual enantiomers are manually separated. This technique can be used if crystals of the separate enantiomers exist, i.e., the material is a conglomerate, and the crystals are visually distinct; ii) simultaneous crystallization-a technique whereby the individual enantiomers are separately crystallized from a solution of the racemate, 2020418425   14 Sep 2026 possible only if the latter is a conglomerate in the solid state; iii) enzymatic resolutions-a technique whereby partial or complete separation of a racemate by virtue of differing rates of reaction for the enantiomers with an enzyme; iv) enzymatic asymmetric synthesis-a synthetic technique whereby at least one step of the synthesis uses an enzymatic reaction to 5 obtain an enantiomerically pure or enriched synthetic precursor of the desired enantiomer; v) chemical asymmetric synthesis--a synthetic technique whereby the desired enantiomer is synthesized from an achiral precursor under conditions that produce asymmetry (i.e., chirality) in the product, which may be achieved using chiral catalysts or chiral auxiliaries; vi) diastereomer separations-a technique whereby a racemic compound is reacted with an 10 enantiomerically pure reagent (the chiral auxiliary) that converts the individual enantiomers to diastereomers. The resulting diastereomers are then separated by chromatography or crystallization by virtue of their now more distinct structural differences and the chiral auxiliary later removed to obtain the desired enantiomer; vii) first- and second-order asymmetric transformations-a technique whereby diastereomers from the racemate 15 equilibrate to yield a preponderance in solution of the diastereomer from the desired enantiomer or where preferential crystallization of the diastereomer from the desired enantiomer perturbs the equilibrium such that eventually in principle all the material is converted to the crystalline diastereomer from the desired enantiomer. The desired enantiomer is then released from the diastereomer; viii) kinetic resolutions-this technique 20 refers to the achievement of partial or complete resolution of a racemate (or of a further resolution of a partially resolved compound) by virtue of unequal reaction rates of the enantiomers with a chiral, non-racemic reagent or catalyst under kinetic conditions; ix) enantiospecific synthesis from non-racemic precursors--a synthetic technique whereby the desired enantiomer is obtained from non-chiral starting materials and where the 25 stereochemical integrity is not or is only minimally compromised over the course of the synthesis; x) chiral liquid chromatography--a technique whereby the enantiomers of a racemate are separated in a liquid mobile phase by virtue of their differing interactions with a stationary phase. The stationary phase can be made of chiral material or the mobile phase can contain an additional chiral material to provoke the differing interactions; xi) chiral gas 30 chromatography-a technique whereby the racemate is volatilized and enantiomers are separated by virtue of their differing interactions in the gaseous mobile phase with a column containing a fixed non-racemic chiral adsorbent phase; xii) extraction with chiral solvents-a technique whereby the enantiomers are separated by virtue of preferential dissolution of one enantiomer into a particular chiral solvent; xiii) transport across chiral membranes-a 2020418425   14 Sep 2026 technique whereby a racemate is placed in contact with a thin membrane barrier. The barrier typically separates two miscible fluids, one containing the racemate, and a driving force such as concentration or pressure differential causes preferential transport across the membrane barrier. Separation occurs as a result of the non-racemic chiral nature of the 5 membrane that allows only one enantiomer of the racemate to pass through. Chiral chromatography, including simulated moving bed chromatography, is used in one embodiment. A wide variety of chiral stationary phases are commercially available. Some of the compounds described herein contain olefinic double bonds and unless otherwise specified, are meant to include both E and Z geometric isomers. 10          In addition, some of the nucleosides described herein, may exist as tautomers, such as, keto-enol tautomers. The individual tautomers as well as mixtures thereof are intended to be encompassed within the compounds of the present invention. Combination Therapies The compound described herein can be administered adjunctively with other active 15 compounds. These compounds include but are not limited to analgesics, anti-inflammatory drugs, antipyretics, antidepressants, antiepileptics, antihistamines, antimigraine drugs, antimuscarinics, anxioltyics, sedatives, hypnotics, antipsychotics, bronchodilators, antiasthma drugs, cardiovascular drugs, corticosteroids, dopaminergics, electrolytes, gastrointestinal drugs, muscle relaxants, nutritional agents, vitamins, parasympathomimetics, 20 stimulants, anorectics, anti-narcoleptics, and antiviral agents. In a particular embodiment, the antiviral agent is a non-CNS targeting antiviral compound. “Adjunctive administration”, as used herein, means the compound can be administered in the same dosage form or in separate dosage forms with one or more other active agents. The additional active agent(s) can be formulated for immediate release, controlled release, or 25 combinations thereof. Specific examples of compounds that can be adjunctively administered with the compounds include, but are not limited to, aceclofenac, acetaminophen, adomexetine, almotriptan, alprazolam, amantadine, amcinonide, aminocyclopropane, amitriptyline, amolodipine, amoxapine, amphetamine, aripiprazole, aspirin, atomoxetine, azasetron, 30 azatadine, beclomethasone, benactyzine, benoxaprofen, bermoprofen, betamethasone, bicifadine, bromocriptine, budesonide, buprenorphine, bupropion, buspirone, butorphanol, butriptyline, caffeine, carbamazepine, carbidopa, carisoprodol, celecoxib, chlordiazepoxide, chlorpromazine, choline salicylate, citalopram, clomipramine, clonazepam, clonidine, clonitazene, clorazepate, clotiazepam, cloxazolam, clozapine, codeine, corticosterone, 2020418425   14 Sep 2026 cortisone, cyclobenzaprine, cyproheptadine, demexiptiline, desipramine, desomorphine, dexamethasone, dexanabinol, dextroamphetamine sulfate, dextromoramide, dextropropoxyphene, dezocine, diazepam, dibenzepin, diclofenac sodium, diflunisal, dihydrocodeine, dihydroergotamine, dihydromorphine, dimetacrine, divalproxex, 5 dizatriptan, dolasetron, donepezil, dothiepin, doxepin, duloxetine, ergotamine, escitalopram, estazolam, ethosuximide, etodolac, femoxetine, fenamates, fenoprofen, fentanyl, fludiazepam, fluoxetine, fluphenazine, flurazepam, flurbiprofen, flutazolam, fluvoxamine, frovatriptan, gabapentin, galantamine, gepirone, ginko bilboa, granisetron, haloperidol, huperzine A, hydrocodone, hydrocortisone, hydromorphone, hydroxyzine, ibuprofen, 10 imipramine, indiplon, indomethacin, indoprofen, iprindole, ipsapirone, ketaserin, ketoprofen, ketorolac, lesopitron, levodopa, lipase, lofepramine, lorazepam, loxapine, maprotiline, mazindol, mefenamic acid, melatonin, melitracen, memantine, meperidine, meprobamate, mesalamine, metapramine, metaxalone, methadone, methadone, methamphetamine, methocarbamol, methyldopa, methylphenidate, methylsalicylate, 15 methysergid(e), metoclopramide, mianserin, mifepristone, milnacipran, minaprine, mirtazapine, moclobemide, modafinil (an anti-narcoleptic), molindone, morphine, morphine hydrochloride, nabumetone, nadolol, naproxen, naratriptan, nefazodone, neurontin, nomifensine, nortriptyline, olanzapine, olsalazine, ondansetron, opipramol, orphenadrine, oxaflozane, oxaprazin, oxazepam, oxitriptan, oxycodone, oxymorphone, pancrelipase, 20 parecoxib, paroxetine, pemoline, pentazocine, pepsin, perphenazine, phenacetin, phendimetrazine, phenmetrazine, phenylbutazone, phenytoin, phosphatidylserine, pimozide, pirlindole, piroxicam, pizotifen, pizotyline, pramipexole, prednisolone, prednisone, pregabalin, propanolol, propizepine, propoxyphene, protriptyline, quazepam, quinupramine, reboxitine, reserpine, risperidone, ritanserin, rivastigmine, rizatriptan, rofecoxib, ropinirole, 25 rotigotine, salsalate, sertraline, sibutramine, sildenafil, sulfasalazine, sulindac, sumatriptan, tacrine, temazepam, tetrabenozine, thiazides, thioridazine, thiothixene, tiapride, tiasipirone, tizanidine, tofenacin, tolmetin, toloxatone, topiramate, tramadol, trazodone, triazolam, trifluoperazine, trimethobenzamide, trimipramine, tropisetron, valdecoxib, valproic acid, venlafaxine, viloxazine, vitamin E, zimeldine, ziprasidone, zolmitriptan, zolpidem, 30 zopiclone and isomers, salts, and combinations thereof. In certain embodiments, the exemplary compounds and pharmaceutical compositions can be administered in combination with another antiviral agent(s) such as abacavir, acyclovir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, AT-511, AT-527, atazanavir, atripla, balapiravir, BCX4430 / Galidesivir, boceprevir, cidofovir, 2020418425   14 Sep 2026 combivir, daclatasvir, darunavir, dasabuvir, delavirdine, didanosine, ocosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir, favipiravir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, GS-441524, GS-5734 / Remdesivir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, interferon type III, 5 interferon type II, interferon type I, lamivudine, ledipasvir, lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfinavir, nevirapine, nexavir, NITD008, ombitasvir, oseltamivir, paritaprevir, peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin , raltegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, simeprevir, sofosbuvir, stavudine, telaprevir, telbivudine, tenofovir, tenofovir disoproxil, 10 Tenofovir Exalidex, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine zalcitabine, zanamivir, or zidovudine and combinations thereof. In certain embodiments, the exemplary compounds and pharmaceutical compositions can be administered in combination with another agent(s) such as 15 chloroquine, chloroquine phosphate, hydroxychloroquine, hydroxychloroquine sulfate, Ampligen, APN01, Ganovo, IFX-1, BXT-25, CYNK-001, Tocilizumab, Leronlimab, Ii-key, COVID-19 S-Trimer, Camrelizumab, thymosin, Brilacidin, INO-4800, Prezcobix, cobicistat, mRNA-1273, Arbidol, umifenovir, REGN3048, REGN3051, TNX-1800, fingolimod, methylprednisolone, nitazoxanide, benzopurpin B, C-467929, C-473872, NSC- 20   306711, N-65828, C-21, CGP-42112A, L-163491, xanthoangelol, or bevacizumab and combinations thereof. In certain embodiments, the exemplary compounds and pharmaceutical compositions disclosed herein can be administered in combination with any of the compounds disclosed in: 25  WO2003090690A2, WO2003090690A3, WO2003090691A2, WO2003090691A3, WO2004005286A2, WO2004005286A3, WO2004006843A2, WO2004006843A3, WO2004031224A2, WO2004031224A3, WO2004035576A2, WO2004035576A3, WO2004035577A2, WO2004035577A3, WO2004050613A2, WO2004050613A3, WO2004064845A1, WO2004064846A1, WO2004096286A2, WO2004096286A3, 30  WO2004096287A2, WO2004096287A3, WO2004096818A2, WO2004096818A3, WO2004100960A2, WO2005002626A2, WO2005002626A3, WO2005012324A2, WO2005012324A3, WO2005028478A1, WO2005039552A2, WO2005039552A3, WO2005042772A1, WO2005042773A1, WO2005047898A2, WO2005047898A3, WO2005063744A2, WO2005063744A3, WO2005063751A1, WO2005064008A1, 2020418425   14 Sep 2026 WO2005064008A9, WO2005066189A1, WO2005070901A2, WO2005070901A3, WO2005072748A1, WO2005117904A2, WO2005117904A3, WO2006015261A2, WO2006015261A3, 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EIDD-1931 and prodrugs thereof, e.g EIDD-2801, can be administered in combination with, or formulated with, another antiviral agent(s) such as: • Nucleoside reverse transcriptase inhibitors (NRTIs) • Non-nucleoside reverse transcriptase inhibitors (NNRTIs) 10      • Protease inhibitors (PIs) • Integrase inhibitors (INSTIs) • Fusion inhibitors (FIs) • Chemokine receptor antagonists • Entry inhibitors 15 Specific examples of agents include abacavir, acyclovir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, AT-511, AT-527, atazanavir, atripla, balapiravir, BCX4430 / Galidesivir, boceprevir, cidofovir, combivir, daclatasvir, darunavir, dasabuvir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, 20 enfuvirtide, entecavir, famciclovir, favipiravir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, GS-441524, GS-5734 / Remdesivir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, interferon type III, interferon type II, interferon type I, lamivudine, ledipasvir, lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfinavir, nevirapine, nexavir, NITD008, ombitasvir, oseltamivir, paritaprevir, peginterferon alfa-2a, 25 penciclovir, peramivir, pleconaril, podophyllotoxin , raltegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, simeprevir, sofosbuvir, stavudine, telaprevir, telbivudine, tenofovir, tenofovir disoproxil, Tenofovir Exalidex, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine zalcitabine, zanamivir, zidovudine, or chloroquine, chloroquine phosphate, 30 hydroxychloroquine, hydroxychloroquine sulfate, Ampligen, APN01, Ganovo, IFX-1, BXT-25, CYNK-001, Tocilizumab, Leronlimab, Ii-key, COVID-19 S-Trimer, Camrelizumab, thymosin, Brilacidin, INO-4800, Prezcobix, cobicistat, mRNA-1273, Arbidol, umifenovir, REGN3048, REGN3051, TNX-1800, fingolimod, methylprednisolone, nitazoxanide, benzopurpin B, C-467929, C-473872, NSC-306711, N- 2020418425   14 Sep 2026 65828, C-21, CGP-42112A, L-163491, xanthoangelol, bevacizumab, polyclonal antibodies derived from patients and monoclonal antibodies (including those antibodies from patients of COVID-19 or monoclonal or polyclonal antibodies which bind SARS-CoV-2), and combinations thereof. In addition, the compounds of this invention can be combined with 5 compounds that are favorable to preventing lung damage associated with COVID-19, including for example anti-IL-6 and TNF inhibitors, specifically including for example , tocilizumab (Actemra), siltuximab (Sylvant), Tocilizumab, Sarilumab, olokizumab (CDP6038), elsilimomab, BMS- 945429 (ALD518), sirukumab (CNTO 136), levilimab (BCD-089), and CPSI- 10   2364 and ALX-0061, ARGX-109, FE301, FM10, infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), and golimumab (Simponi), etanercept (Enbrel), Thalidomide (Immunoprin) and its derivatives lenalidomide (Revlimid) and pomalidomide (Pomalyst, Imnovid), xanthine derivatives (e.g. pentoxifylline) and bupropion and 5-HT, 15 agonist hallucinogens including (R)-DOI, TCB-2, LSD and LA-SS-Az. In exemplified embodiments, the exemplary compounds and pharmaceutical compositions can be administered in combination with y o H o l x n„^° y.N^ \__ / ''CN [ 1 HO OH . In exemplified embodiments, Va h ° k X / N" P-O- ........................ 6 0 0 f\H ctiy ° V._y    HO OH    can be administered in nh2 \__ / CN HO OH . 2020418425   14 Sep 2026 with 0 In exemplified embodiments, HO OH can be administered in comination In exemplified embodiments, HO OH can be administered in comination with 5                          EXAMPLES Example 1. Conjugate Preparation Mono and diphosphate prodrugs have been prepared by several groups. See Jessen et al., Bioreversible Protection of Nucleoside Diphosphates, Angewandte Chemie- 10 International Edition English 2008, 47 (45), 8719-8722, hereby incorporated by reference. In order to prevent rupture of the P-O-P anhydride bond, one utilizes a pendant group that fragments rapidly (e.g. bis-(4-acyloxybenzyl)-nucleoside diphosphates (BAB-NDP) that is deacylated by an endogenous esterase) to generate a negative charge on the second phosphate. See also Routledge et al., Synthesis, Bioactivation and Anti-HIV Activity of 4 15 Acyloxybenzyl-bis(nucleosid-5'-yl) Phosphates, Nucleosides & Nucleotides 1995, 14 (7), 1545-1558 and Meier et al., Comparative study of bis(benzyl)phosphate triesters of 2',3'- 2020418425   14 Sep 2026 dideoxy-2',3'-didehydrothymidine (d4T) and cycloSal-d4TMP -hydrolysis, mechanistic insights and anti-HIV activity, Antiviral Chemistry and Chemotherapy 2002, 13,101-114, both hereby incorporated by reference. Once this occurs, the P-O-P anhydride bond is less susceptible to cleavage and the remaining protecting group can then do its final unraveling 5 to produce the nucleoside diphosphate. Other methods to prepare diphosphate and monothiodiphosphate prodrugs are shown in Figure 5. Standard coupling conditions are used to prepare sphingolipidnucleoside monophosphate prodrugs. The corresponding diphosphate prodrugs may be prepared according to the protocols shown in Figure 5 and as provided in Smith et al., 10 Substituted Nucleotide Analogs. U.S. Patent Application 2012 / 0071434; Skowronska et al., Reaction of Oxophosphorane-Sulfenyl and Oxophosphorane-Selenenyl Chlorides with Dialkyl Trimethylsilyl Phosphites - Novel Synthesis of Compounds Containing a Sulfur or Selenium Bridge Between 2 15 Phosphoryl Centers, Journal of the Chemical Society-Perkin Transactions 1 1988, 8, 21972201; Dembinski et al., An Expedient Synthesis of Symmetrical Tetra-Alkyl Monothiopyrophosphates, Tetrahedron Letters 1994, 35 (34), 6331-6334; Skowronska et al., Novel Synthesis of Symmetrical Tetra-Alkyl Monothiophosphates, Tetrahedron Letters 20   1987, 28 (36), 4209-4210; and Chojnowski et al., Methods of Synthesis of O,O-Bis TrimethylSilyl Phosphorothiolates. Synthesis-Stuttgart 1977, 10, 683-686, all hereby incorporated by reference in their entirety. Example 2. General Procedure for Base Coupling 25          The persilylated nucleobase was prepared in a round bottom flask charged with dry nucleobase (15.5 mmol), chlorotrimethylsilane (12.21 mmol), and bis(trimethylsilyl)amine (222 mmol) under nitrogen. The mixture was refluxed with stirring overnight (16 h) until all solids dissolved. The mixture was cooled to room temperature and volatiles were removed by rotary evaporation followed by high vacuum to give persilylated nucleobase. 30 This compound was used immediately in the next step. The freshly prepared persilylated nucleobase (15.50 mmol) was dissolved in 1,2-dichloroethane (50 mL) or chlorobenzene (50 mL) under nitrogen with stirring at room temperature. A solution of □-D-ribofuranose 1,2,3,5-tetraacetate (7.75 mmol) in 1,2- 2020418425   14 Sep 2026 dichloroethane (50 mL) or chlorobenzene (50 mL) was added all at once to the stirred mixture. To this mixture was added SnCl4 (11.63 mmol) dropwise via syringe, and the mixture was stirred at room temperature 6 h until all starting material was consumed. The 5 mixture was cooled to 0°C and a sat. aq. NaHCO3 solution (125 mL) was added. The mixture was warmed to room temperature and stirred 30 min. The mixture was extracted with EtOAc (2 x 200 mL) and the combined organic layers were washed with brine (1 x 100 mL), dried over Na2SO4, filtered, and concentrated by rotary evaporation to give 5.5 g crude product. The crude material was taken up in dichloromethane, immobilized on Celite, and 10 subjected to flash chromatography to provide the desired acetate protected product. The ribonucleoside was deprotected using the general deprotection conditions. Example 3. General Cytosine Analog Coupling In a flask charged with N4-benzoyl protected cytosine analog (0.793 mmol) was added 15 bis(trimethylsilyl)amine (8.45 mmol) and ammonium sulfate (0.02 mmol) under N2. This was heated at reflux for 2 h, after cooling to rt, solvent was removed in vacuo and further dried under high vacuum for 1 h. The residue was dissolved in dry chlorobenzene (10 ml) and □ -D- or □-L-ribofuranose 1,2,3,5-tetraacetate (0.53 mmol) was added. Then SnCh (0.27 ml, 2.3 mmol) was added dropwise. After stirring at rt for 1 h, this was heated to 60 oC overnight. 20 After cooling to 0 oC, solid sodium bicarbonate (0.85 g) was added, followed by EtOAc (5 mL). This was allowed to stir for 15 min and then water (0.5 mL) was added slowly. The insoluble material was filtered off and washed wtih more EtOAc (2.5 mL). The filtrate was washed with water once, bine once, dried (Na2SO4) and concentrated in vacuo. The crude material was purified by SiO2 column chromatography. 25 Example 4. General Deamination Conditions A solution of benzoyl protected cytidine ribonucleoside (1.02 mmol) in 80% aqueous AcOH (30 mL) was heated under reflux for 16 h. The solvent was then removed in vacuo and dried under high vacuum. The white solid was triturated with ether, filtered off and washed 30 with more ether to obtain the desired product. Example 5. General Uracil Analog Coupling The persilylated uracil was prepared in a round bottom flask charged with uracil (15.5 mmol), chlorotrimethylsilane (12.21 mmol), and bis(trimethylsilyl)amine (222 mmol) under 2020418425   14 Sep 2026 nitrogen. The mixture was refluxed with stirring overnight (16 h) until all solids dissolved until a clear colorless solution formed. The mixture was cooled to room temperature and volatiles were removed by rotary evaporation followed by high vacuum to give persilylated uracil. This compound was used immediately in the next step. 5          The freshly prepared persilylated uracil (15.50 mmol) was dissolved in 1,2- dichloroethane (50 mL) under nitrogen with stirring at room temperature. A solution of □D- or D-L-ribofuranose 1,2,3,5-tetraacetate (7.75 mmol) in 1,2-dichloroethane (50 mL) was added all at once to the stirred mixture. To this mixture was added SnCl4 (11.63 mmol) dropwise via syringe, and the 10 mixture was stirred at room temperature 6 h until all starting material was consumed. The mixture was cooled to 0°C and a sat. aq. NaHCO3 solution (125 mL) was added. The mixture was warmed to room temperature and stirred 30 min. The mixture was extracted with EtOAc (2 x 200 mL) and the combined organic layers were washed with brine (1 x 100 mL), dried over Na2SO4, filtered, and concentrated by rotary evaporation to give 5.5 g crude 15 product. The crude material was taken up in dichloromethane, immobilized on Celite, and subjected to flash chromatography on the Combiflash (120 g column, 5 to 50% EtOAc in hexanes gradient) to provide the product. Example 6. General Acetate or Benzoyl Deprotection Conditions 20          Benzoyl protected ribonucleoside analog (0.25 mmol) was stirred with 7 N ammonia in MeOH at rt for 15.5 h. The solvent was then removed and the crude material was purified by SiO2 column chromatography to obtain the desired ribonucleoside. Example 7. Synthesis of 1’-Deuterated Nucleoside Analogs 389                       390 The lactone (0.0325 mol) was added to a dry flask under an argon atmosphere and was then dissolved in dry THF (250 mL). The solution as then cooled to -78°C and a DIBAL-D solution in toluene (0.065 mol) was dropwise. The reaction was allowed to stir at -78°C for 3-4 hours. The reaction was then quenched with the slow addition of water (3 30 mL). The reaction was then allowed to stir while warming to room temperature. The mixture was then diluted with two volumes of diethyl ether and was then poured into an 2020418425   14 Sep 2026 10 15 equal volume of saturated sodium potassium tartrate solution. The organic layer was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified on silica eluting with hexanes / ethyl acetate. The resulting lactol was then converted to an acetate or benzolyate and subjected to base coupling conditions to introduce the desired nucleobase. Example 8. A 1 L rbf was charged with uridine (36.6 g, 150 mmol) and acetone (Volume: 700 ml) with stirring under nitrogen at rt. The slurry was treated with concentrated sulfuric acid (0.800 ml, 15.00 mmol) and the mixture was stirred at rt overnight. After stirring 16 h, triethylamine (41.8 ml, 300 mmol) was added all at once, the mixture was stirred 30 min, and then concentrated by rotary evaporation to give a sticky white solid. The solid was dissolved in boiling iPrOH (~1.4 L) and allowed to cool overnight at rt. After cooling overnight, small crystals had formed. The flask was placed in the freezer for 3 h and more crystals formed. The mixture was vacuum filtered, and the solids were washed with ice-cold 2020418425   14 Sep 2026 iPrOH (2 x 200 mL) and ice-cold ether (2 x 200 mL). The solid was recovered to give compound 1 (21.75 g, 77 mmol, 51.0 % yield) as a white powdery solid. A round bottom flask was charged with compound 1 (21.75 g, 77 mmol) and DCM (219 ml) and the mixture was stirred under nitrogen. Solid 4-DMAP (23.37 g, 191 mmol) 5 was added all at once, and the mixture was stirred at rt until all solids dissolved. The mixture was cooled to 0°C, and tosyl chloride (17.50 g, 92 mmol) was added portionwise as a solid over 5 min. The mixture was stirred at rt for 1 h until all starting material was consumed. The mixture was transferred to a separatory funnel, and the organic layer was washed with 1 N HCl (2 x 200 mL), sat. aq. NaHCO3 (1 x 200 mL), and brine (1 x 200 mL), 10 then dried over Na2SO4, filtered and concentrated by rotary evaporation to give compound 2 (34.52 g, 74.8 mmol, 98 % yield) as a white solid. To a stirred solution of compound 2 (3.95 g, 9.01 mmol) in THF (30 mL) at 0°C under nitrogen. Solid potassium tert-butoxide (3.03 g, 27.0 mmol) was added all at once, the reaction mixture turned into a yellow slurry. The mixture was stirred at 0°C for 2 h. Silica 15 gel (6 g) and Celite (14 g) were added along with more THF, and the mixture was concentrated by rotary evaporation. Flash chromatography on the Isco (80 g column, 1 to 5% MeOH in DCM) gave compound 3 (2.17 g, 8.15 mmol, 90 % yield) as a white powdery solid. A round bottom flask was charged with a stir bar, compound 3 (2.17 g, 8.15 mmol), 20 silver(I) fluoride (5.17 g, 40.8 mmol), and DCM (Volume: 152 ml, Ratio: 14) at 0°C. To this vigorously stirred mixture was added a solution of iodine (4.14 g, 16.30 mmol) in THF (Volume: 10.87 ml, Ratio: 1.000) dropwise via syringe over 40 min. After addition was complete, the mixture was stirred another 15 min at 0°C, then a 1:1 mixture of sat. aq. NaHCO3:sat. aq. Na2S2O3 was added (75 mL) and the whole mixture was filtered through a 25 Celite pad, washing with DCM (2 x 50 mL). The filtrates were transferred to a separation funnel, and the organic layer was dried over Na2SO4, filtered, and concentrated by rotary evaporation to give 4 g. Flash chromatography on the Isco (120 g column, 5 to 25% EtOAc in DCM) gave compound 4 (2.06 g, 5.00 mmol, 61.3 % yield) as a pale yellow flaky solid. A round bottom flask was charged with compound 4 (10.76 g, 26.1 mmol), 30 tetrabutylammonium sulfate (8.86 g, 26.1 mmol), potassium hydrogen phosphate dibasic trihydrate (8.94 g, 39.2 mmol), DCM (Volume: 1088 ml, Ratio: 5) and water (Volume: 218 ml, Ratio: 1.000) and the biphasic mixture was stirred vigorously at rt. To this mixture was added solid mCPBA, 77% w / w (29.3 g, 131 mmol) all at once and the mixture was stirred at rt overnight. After stirring 20 h at rt, all SM had been consumed by TLC analysis. The 2020418425   14 Sep 2026 mixture was quenched by slow addition of sat. aq. Na2S2O3 (375 mL) followed by sat. aq. Na2CO3 (375 mL). The organic layer was removed, and the aqueous layer was extracted with DCM (1 x 450 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated by rotary evaporation to give 22 g crude. The crude was taken up in DCM, 5 and flash chromatography on the Isco (330 g column, 5 to 25% EtOAc in DCM) gave 10 g of semipure product. The compound was taken up in DCM, and flash chromatography on the Isco (330 g column, 5 to 70% EtOAc in hexanes) gave compound 5 (6.91 g, 15.68 mmol, 60.0 % yield) as an off-white flaky solid. A round bottom flask was charged with compound 5 (3.53 g, 8.0 mmol) and 10 ammonia in MeOH (34.3 ml, 240 mmol) at 0°C. The mixture was stirred for 5 h, at which point all starting material was consumed. The mixture was concentrated by rotary evaporation to give ~4 g crude as a yellow oil. The crude was taken up in DCM, and flash chromatography on the Isco (120 g column, 1 to 5% MeOH in DCM) gave compound 6 (2.20 g, 7.28 mmol, 91 % yield) as a white powdery solid. 15          A 1L 3-neck RBF equipped with temperature probe, overhead stirrer and additiion funnel (argon inlet) was charged with phosphorus oxychloride (15.50 ml, 166 mmol) in THF (300 ml), evacuated and purged with argon 3x, then cooled to <-70°C using dry ice / acetone. A solution of 2-(hydroxymethyl)phenol (18.77 g, 151 mmol) and triethylamine (44.3 ml, 317 mmol) in 200mL of THF was slowly added via addition funnel over 20 30minutes. The resulting light tan mixture was slowly warmed to RT and stirred for 3hrs. Cooled to 0°C using an ice bath and added triethylamine (25.3 ml, 181 mmol), then slowly added a THF (100mL) solution of 2,3,4,5,6-pentafluorophenol (25.05 g, 136 mmol) to the rapidly stirred mixture. Warmed to RT and monitored by TLC (25% EtOAc / hexanes). SM consumed in <2hrs, only product (Rf = 0.5) present. The oil was purified by SGC (glass 25 column, 10-25% EtOAc / hexanes), fractions containing product were pooled and concentrated under reduced pressure to yield compound 7 (41.2 g, 117 mmol, 77 % yield) as a white solid. To a stirred solution of compound 6 (1.95 g, 6.45 mmol) in THF (Volume: 96 ml, Ratio: 5) at 0°C under nitrogen, was added a solution of tert-butylmagnesium chloride, 1.0 30 M in THF (14.19 ml, 14.19 mmol) dropwise via syringe. A white precipitate formed; the mixture was warmed to rt and stirred for 30 min, then recooled to 0°C. A solution of compound 7 (5.68 g, 16.13 mmol) in THF (Volume: 19.20 ml, Ratio: 1.000) was added dropwise via syringe, and the mixture was warmed to rt and stirred overnight. After 18 h stirring, a little SM remained and one slightly less polar product had formed. The mixture 2020418425   14 Sep 2026 10 15 was quenched by addition of solid NH4Cl (2 g) and the mixture was immobilized on Celite. Flash chromatography on the Isco (220 g column, 1 to 5% MeOH in DCM) gave 1.94 g of a white solid that consisted of desired product and pentafluorophenol. The solid was taken up in DCM and washed with sat. aq. NaHCO3 (3 x 100 mL). The organic layer was dried over Na2SO4, filtered, and concentrated by rotary evaporation to give compound 8 (1.70 g, 3.61 mmol, 56.0 % yield) as a white powdery solid. A round bottom flask was charged with compound 8 (.250 g, 0.532 mmol) and formic acid, 80% aq. (Volume: 10 mL). The mixture was stirred at rt under nitrogen overnight. After stirring 20 h, all volatiles were removed by rotary evaporation. The residue was taken up in MeOH and immobilized on Celite. Flash chromatography on the Isco (24 g column, 1 to 15% MeOH in DCM) gave a white powdery solid, 175 mg, 90-95% pure by NMR. The white powder was taken up in a 5:1 water:MeCN mixture, and reverse phase flash chromatography on the Isco (100 g C18 column, 100% water to 100% MeCN) gave good separation of the impurity. The fractions containining desired product were concentrated, taken up in 5:1 water:MeCN, frozen in a dry ice bath, and lyophilized to provide compound 9, EIDD-02838. Example 9. 17 20 Uridine (1 mmol) was suspended in dioxane (4 mL) followed by the addition of pyridine (2 mmol), PPh3 (1.5 mmol), and iodine (1.5 mmol) under an argon atmosphere. The mixture was stirrd at room temperature overnight. The reaction mixture was quenched 2020418425   14 Sep 2026 with methanol and saturated aqueous Na2S2O3and was then evaporated to dryness to provide crude compound 10, which was used directly in the next step. Crude compound 10 was dissolved in dry DMF under an argon atmosphere followed by the addition of imidazole (5 equivalents) and TBSCl (4 equivalents) at 0°C. The mixture 5 was allowed to warm to room temperature and stir overnight. The reaction mixture was partitioned between AcOEt / H2O (3:1). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The resulting residue was purified on a silica gel column eluting with hexanes and etheyl acetate to provide compound 11. Compound 11 was dissolved in dry MeCN and treated with DBN (2.25 equivalents) 10 at 0°C under an argon atmosphere. The reaction was allowed to stir overnight. The reaction mixture was neutralized with AcOH and then was evaporated to dryness. The residue was partitioned between DCM and saturated aqueous NaHCO3. The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The resulting residue was purified on a silica gel column eluting with hexanes and etheyl acetate to 15 provide compound 12. To a solution of compound 12 in dry DCM (20 mL / mmol 12) was added DMDO (0.1M in acetone, 1.2 equivalents) at -30°C under an argon atmosphere. The reaction was allowed to stir for 1 hour and was then evaporated to dryness to afford compound 13, which was used immediately in the next step. 20         To a solution of compound 13 in dry DCM (20 mL / mmol 13) was added SnCl4 (3 equivalents) at -30°C under an argon atmosphere. The misture was allowed to stir for 1 hour and was then quenched with saturated aqueous NaHCO3. The mixture was filtered through a celite pad, and the filtrate was partitioned between DCM and saturated aqueous NaHCO3. The organic layer was dried over MgSO4, filtered, and concentrated under 25 reduced pressure. The resulting residue was purified on a silica gel column eluting with hexanes and etheyl acetate to provide compounds 14 and 15 in a 2:1 ratio. Compound 15 was treated with TBAF (2.5 equivalents) in THF. After starting material was consumed, the reaction mixture was concentrated under reduced pressure and purified by reverse phase to obtain compound 16. 30         Compound 15 was treated under the same conditions as compound 6 followed by treatment with TBAF to obtain compound 17. 2020418425   14 Sep 2026 Example 10. A round bottom flask was charged with compound 5 (.250 g, 0.567 mmol) and formic acid, 80% aq. (Volume: 10 mL). The mixture was stirred at room temperature under nitrogen 5 overnight. After stirring 20 h, all volatiles were removed by rotary evaporation. The residue was taken up in MeOH and immobilized on Celite. Flash chromatography on the Isco (24 g column, 1 to 15% MeOH in DCM) gave a white powdery solid 90-95% pure by NMR. The white powder was taken up in a 5:1 water:MeCN mixture, and reverse phase flash chromatography on the Isco (100 g C18 column, 100% water to 100% MeCN) gave good 10 separation of the impurity. The fractions containining desired product were concentrated, taken up in 5:1 water:MeCN, frozen in a dry ice bath, and lyophilized to provide compound 18. A round bottom flask was charged with compound 18 (3.53 g, 8.8 mmol) and ammonia in MeOH (34.3 ml, 240 mmol) at 0°C. The mixture was allowed to stir for 5 15 hours, at which point all starting material was consumed. The mixture was concentrated by rotary evaporation to give ~4 g crude as a yellow oil. The crude was taken up in DCM, and flash chromatography on the Isco (120 g column, 1 to 5% MeOH in DCM) gave compound 19, EIDD-02749, (2.20 g, 7.28 mmol, 91 % yield) as a white powdery solid. Example 11. 23                               24                             25 To a stirred solution of DMP (27.5g, 64.9mmol) in DCM (162mL, 0.2M) was cooled to 0°C and 23 (15g, 32.4mmol) was added. The reaction was stirred at 0°C and allowed to warm to room temperature. After stirring for18 hours the reaction mixture was 25 concentrated under reduced pressure to a past which was then slurried in 100mL ethyl ether followed by filtration through a 50g pad of sillica / mag sulfate 1:1 by mass and washed with a total of 400mL ethyl ether. The ether layer was washed with 2.5g of sodium thiosulfate in 2020418425   14 Sep 2026 15mL water then 2x30mL cooled sodium bicarbonate, and finally with 30mL brine. The filtrate was then dried over sodium sulfate, filtered, and concentrated under reduced pressure to provide a foam which was used without further purification. Before use in the next step, a solution of ketone (32.6mmol) in DCM (200mL) was prepared and stirred 5 overnight over 5g of magnesium sulfate at room temperature. After 18 hours of stirring, the solution was filtered and concentrated under reduced presure. To a -78°C solution of TMS Ethylene (11.4mL, 80mmol) in dry THF (100mL) under argon was added butyl lithium (30.5mL, 2.5M hexanes, 76mmol). After 30 minutes of stirring, lithiated alkyne was cannulated into a -78°C suspension of anhydrous CeCl3 ( 10   33.5g, 90mmol, dried overnight 150°C under high vacuum) in dry THF (130mL) with 2x15mL rinses of THF. After 90 minutes of stirring, a solution of 24 (32.4mmol) in dry THF (50mL) was added via cannula (2x10mL rinse THF). After 3 hours of stirring, the resulting solution was quenched with saturated aqueous ammonium chloride (100mL). The reaction was warmed to room temperature and filtered through a celite pad. The celite pad 15 was washed with ethyl ether (3x100mL) and with saturated aqueous ammonium chloride (100mL). The filtrate was separated and the organics were washed with saturated aqueous ammonium chloride (100mL) and brine (100mL). The filtrate was dried over sodium sulfate, filtered, and concentrated under reduced pressure to provide an oil which was purified by silica gel chromatography 10-50% ethyl acetate in hexanes to provide the 20 product as a mixture of anomers.. To a stirred 0°C solution of the above product (32.4mmol) in dry DCM (163mL, 0.2M) under argon was added sequentially triethyl amine (18mL, 130mmol) DMAP (3.98g, 32.4mmol), and benzoyl chloride (9.46mL, 82mmol). After stirring for 16 hours, the reaction was concentrated under reduced pressure and then slurried in 200mL ethyl ether 25 and filtered. The organics were concentrated under reduced pressure to provide a paste which was purified by silica gel chromatography eluting with 10-25% ethyl acetate in hexanes to provide 25 as a mixture of anomers. Compound 25 can then be subjected to general base coupling conditions followed by the appropriate deprotection conditions. Example 12. BzO OBz               BzO OBz                    BzO OBz               HO OH 26                        27                              28                        29 2020418425   14 Sep 2026 The lactone (0.0325 mol) was added to a dry flask under an argon atmosphere and was then dissolved in dry THF (250 mL). The solution was then cooled to -78°C and a DIBAL-D solution in toluene (0.065 mol) was added dropwise. The reaction was allowed to stir at -78°C for 3-4 hours. The reaction was then quenched with the slow addition of 5 water (3 mL). The reaction was then allowed to stir while warming to room temperature. The mixture was then diluted with two volumes of diethyl ether and was then poured into an equal volume of saturated sodium potassium tartrate solution. The organic layer was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified on silica eluting with hexanes / ethyl acetate. The resulting lactol, as a 10 solution in dry DCM, was then treated with benzoyl chloride, trimethylamine, and DMAP. The reaction was allowed to stir at 0°C until all the strating material was consumed. Next, the reaction mixture was washed with water and then brine. The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The product was purified on silica eluting with hexanes / ethyl acetate. 15          To a stirred suspension of uracil (3.92g, 2 eq) in HMDS (18mL) was added ammonium sulfate (230mgs, 0.1 eq). The suspension was then refluxed 18h to obtain a clear solution. The solution was cooled to room temperature and concentrated under reduced pressure to a paste. Sugar 27 was dissolved in 1,2-dichloroethane (120mL) and concentrated under reduced pressure to about 80mL. The sugar solution was then cannulated into the 20 flask containing silylated base with 2x20mL rinses of DCE. The reaction was cooled to 0°C and then tin tetrachloride was added dropwise over 5minutes. After 30 minutes of stirring, the reaction was allowed to warm to room temperature and was stirred for a further 18 hours overnight. The reaction was charged with 10g sodium bicarbonate and 10g celite. 10mL saturated aqueous sodium bicarbonate was added dropwise (gas evolution occured). After 25 the quench, the reaction was allowed to stir 30 minutes and then was filtered through a celite pad. The pad was washed with DCM (2x150mL) and the combined organics were washed with 100mL saturated aqueous sodium bicarbonate. The organics were collected, dried over sodium sulfate, filtered and concentrated under reduced pressure to provide a brown paste that was purified by sillica gel chromatography eluting with 25-100% ethyl 30 aceate in hexanes. A round bottom flask was charged with compound 28 and ammonia in MeOH at 0°C. The mixture was allowed to stir for 5 hours, at which point all starting material was consumed. The mixture was concentrated by rotary evaporation to give ~4 g crude as a 2020418425   14 Sep 2026 yellow oil. The crude was taken up in DCM, and flash chromatography on the Isco (120 g column, 1 to 5% MeOH in DCM) gave compound 29. Example 13. 37 5         A 1 L rbf was charged with compound 29 (36.6 g, 150 mmol) and acetone (Volume: 700 ml) with stirring under nitrogen at rt. The slurry was treated with concentrated sulfuric acid (0.800 ml, 15.00 mmol) and the mixture was stirred at rt overnight. After stirring 16 h, triethylamine (41.8 ml, 300 mmol) was added all at once, the mixture was stirred 30 min, and then concentrated by rotary evaporation to give a sticky white solid. The solid was 10 dissolved in boiling iPrOH (~1.4 L) and allowed to cool overnight at rt. After cooling overnight, small crystals had formed. The flask was placed in the freezer for 3 h and more crystals formed. The mixture was vacuum filtered, and the solids were washed with ice-cold iPrOH (2 x 200 mL) and ice-cold ether (2 x 200 mL). The solid was recovered to give compound 30 (21.75 g, 77 mmol, 51.0 % yield) as a white powdery solid. 15         A round bottom flask was charged with compound 30 (21.75 g, 77 mmol) and DCM (219 ml) and the mixture was stirred under nitrogen. Solid 4-DMAP (23.37 g, 191 mmol) 2020418425   14 Sep 2026 was added all at once, and the mixture was stirred at rt until all solids dissolved. The mixture was cooled to 0°C, and tosyl chloride (17.50 g, 92 mmol) was added portionwise as a solid over 5 min. The mixture was stirred at rt for 1 h until all starting material was consumed. The mixture was transferred to a separatory funnel, and the organic layer was 5 washed with 1 N HCl (2 x 200 mL), sat. aq. NaHCO3 (1 x 200 mL), and brine (1 x 200 mL), then dried over Na2SO4, filtered and concentrated by rotary evaporation to give compound 31 (34.52 g, 74.8 mmol, 98 % yield) as a white solid. To a stirred solution of compound 31 (3.95 g, 9.01 mmol) in THF (30 mL) at 0°C under nitrogen. Solid potassium tert-butoxide (3.03 g, 27.0 mmol) was added all at once, the 10 reaction mixture turned into a yellow slurry. The mixture was stirred at 0°C for 2 h. Silica gel (6 g) and Celite (14 g) were added along with more THF, and the mixture was concentrated by rotary evaporation. Flash chromatography on the Isco (80 g column, 1 to 5% MeOH in DCM) gave compound 32 (2.17 g, 8.15 mmol, 90 % yield) as a white powdery solid. 15          A round bottom flask was charged with a stir bar, compound 32 (2.17 g, 8.15 mmol), silver(I) fluoride (5.17 g, 40.8 mmol), and DCM (Volume: 152 ml, Ratio: 14) at 0°C. To this vigorously stirred mixture was added a solution of iodine (4.14 g, 16.30 mmol) in THF (Volume: 10.87 ml, Ratio: 1.000) dropwise via syringe over 40 min. After addition was complete, the mixture was stirred another 15 min at 0°C, then a 1:1 mixture of sat. aq. 20 NaHCO3:sat. aq. Na2S2O3 was added (75 mL) and the whole mixture was filtered through a Celite pad, washing with DCM (2 x 50 mL). The filtrates were transferred to a separation funnel, and the organic layer was dried over Na2SO4, filtered, and concentrated by rotary evaporation to give 4 g. Flash chromatography on the Isco (120 g column, 5 to 25% EtOAc in DCM) gave compound 33 (2.06 g, 5.00 mmol, 61.3 % yield) as a pale yellow flaky solid. 25         A round bottom flask was charged with compound 33 (10.76 g, 26.1 mmol), tetrabutylammonium sulfate (8.86 g, 26.1 mmol), potassium hydrogen phosphate dibasic trihydrate (8.94 g, 39.2 mmol), DCM (Volume: 1088 ml, Ratio: 5) and water (Volume: 218 ml, Ratio: 1.000) and the biphasic mixture was stirred vigorously at rt. To this mixture was added solid mCPBA, 77% w / w (29.3 g, 131 mmol) all at once and the mixture was stirred at 30 rt overnight. After stirring 20 h at rt, all SM had been consumed by TLC analysis. The mixture was quenched by slow addition of sat. aq. Na2S2O3 (375 mL) followed by sat. aq. Na2CO3 (375 mL). The organic layer was removed, and the aqueous layer was extracted with DCM (1 x 450 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated by rotary evaporation to give 22 g crude. The crude was taken up in DCM, 2020418425   14 Sep 2026 and flash chromatography on the Isco (330 g column, 5 to 25% EtOAc in DCM) gave 10 g of semipure product. The compound was taken up in DCM, and flash chromatography on the Isco (330 g column, 5 to 70% EtOAc in hexanes) gave compound 34 (6.91 g, 15.68 mmol, 60.0 % yield) as an off-white flaky solid. 5         A round bottom flask was charged with compound 34 (3.53 g, 8.0 mmol) and ammonia in MeOH (34.3 ml, 240 mmol) at 0°C. The mixture was stirred for 5 h, at which point all starting material was consumed. The mixture was concentrated by rotary evaporation to give ~4 g crude as a yellow oil. The crude was taken up in DCM, and flash chromatography on the Isco (120 g column, 1 to 5% MeOH in DCM) gave compound 35 10   (2.20 g, 7.28 mmol, 91 % yield) as a white powdery solid. A 1L 3-neck RBF equipped with temperature probe, overhead stirrer and additiion funnel (argon inlet) was charged with phosphorus oxychloride (15.50 ml, 166 mmol) in THF (300 ml), evacuated and purged with argon 3x, then cooled to <-70°C using dry ice / acetone. A solution of 2-(hydroxymethyl)phenol (18.77 g, 151 mmol) and triethylamine 15   (44.3 ml, 317 mmol) in 200mL of THF was slowly added via addition funnel over 30minutes. The resulting light tan mixture was slowly warmed to RT and stirred for 3hrs. Cooled to 0°C using an ice bath and added triethylamine (25.3 ml, 181 mmol), then slowly added a THF (100mL) solution of 2,3,4,5,6-pentafluorophenol (25.05 g, 136 mmol) to the rapidly stirred mixture. Warmed to RT and monitored by TLC (25% EtOAc / hexanes). SM 20 consumed in <2hrs, only product (Rf = 0.5) present. The oil was purified by SGC (glass column, 10-25% EtOAc / hexanes), fractions containing product were pooled and concentrated under reduced pressure to yield compound 7 (41.2 g, 117 mmol, 77 % yield) as a white solid. To a stirred solution of compound 35 (1.95 g, 6.45 mmol) in THF (Volume: 96 ml, 25 Ratio: 5) at 0°C under nitrogen, was added a solution of tert-butylmagnesium chloride, 1.0 M in THF (14.19 ml, 14.19 mmol) dropwise via syringe. A white precipitate formed; the mixture was warmed to rt and stirred for 30 min, then recooled to 0°C. A solution of compound 7 (5.68 g, 16.13 mmol) in THF (Volume: 19.20 ml, Ratio: 1.000) was added dropwise via syringe, and the mixture was warmed to rt and stirred overnight. After 18 h 30 stirring, a little SM remained and one slightly less polar product had formed. The mixture was quenched by addition of solid NH4Cl (2 g) and the mixture was immobilized on Celite. Flash chromatography on the Isco (220 g column, 1 to 5% MeOH in DCM) gave 1.94 g of a white solid that consisted of desired product and pentafluorophenol. The solid was taken up in DCM and washed with sat. aq. NaHCO3 (3 x 100 mL). The organic layer was dried over 2020418425   14 Sep 2026 Na2SO4, filtered, and concentrated by rotary evaporation to give compound 36 (1.70 g, 3.61 mmol, 56.0 % yield) as a white powdery solid. A round bottom flask was charged with compound 36 (.250 g, 0.532 mmol) and formic acid, 80% aq. (Volume: 10 mL). The mixture was stirred at rt under nitrogen 5 overnight. After stirring 20 h, all volatiles were removed by rotary evaporation. The residue was taken up in MeOH and immobilized on Celite. Flash chromatography on the Isco (24 g column, 1 to 15% MeOH in DCM) gave a white powdery solid, 175 mg, 90-95% pure by NMR. The white powder was taken up in a 5:1 water:MeCN mixture, and reverse phase flash chromatography on the Isco (100 g C18 column, 100% water to 100% MeCN) gave 10 good separation of the impurity. The fractions containining desired product were concentrated, taken up in 5:1 water:MeCN, frozen in a dry ice bath, and lyophilized to provide compound 37. Example 14. 6: R = H                   38: R = H                 40: R = H 35: R = D                39: R = D               41:R = D 15 Either the nucleoside 6 or 35 was suspended in methylene chloride (40 mL, partially soluble). After stirring at rt for 30 min the mixture was treated sequentially with PDC, acetic anhydride and then tert-butanol. The mixture was allowed to stir at room temperature. TLC (5% methanol in DCM) and LCMS indicated only a small amount of remaining starting material at 4 hours. The mixture was filtered through a pad of silica gel 20 that was loaded into a 150 mL fritted funnel. The silica was eluted with ethyl acetate. The collected filtrate was concentrated by under reduced pressure. The crude dark oil was purified by chromatography over silica gel (25 mm x 175 mm) with 2:1 hexanes:ethyl acetate to ethyl acetate gradient. The pure fractions were collected and concentrated under reduced pressure to give of a white gum. The material was placed under high vacuum for 2 25 days to provide either compound 38 or 39. The material was used in the next step without further purification. The 5’-protected nucleoside 38 or 39 was dissolved in 200 proof ethanol and was then treated with solid sodium borodeuteride. The mixture became homogeneous and was 2020418425   14 Sep 2026 then heated to 80°C. After 12h, a white / pale yellow precipitate formed. The mixture was allowed to cool to rt. TLC (5% methanol in methylene chloride) indicates complete conversion of starting material. The mixture was cooled to 0°C with an ice-bath and then slowly quenched with acetic acid (approximately 1 mL). The clear solution was warmed to 5 rt and then partitioned between ethyl acetate (30 mL) and brine (3 mL). The organic phase was concentrated and then purified by chromatography over silica gel (19 mm x 180 mm) using a mobile phase of 5% methanol in methylene chloride to provide compound 40 or 41. Compounds 40 and 41 can then be deprotected to obtain the unprotected ribonucleoside using 80% formic acid as decscribed previously. Additionally, Compounds 40 and 41 can 10 be conjugated to prodrug reagent 7 followed by deprotection as described previously. Example 15. Prepared according to Boumendjel, Ahcene and Miller, Stephen Journal of Lipid 15 Research 1994, 35, 2305. A mixture of sphingosine (450 mg, 1.50 mmol) and di-tert-butyl dicarbonate (0.656 g, 3.01 mmol) in methylene chloride (100 mL) at 4oC was treated dropwise with diisopropylethylamine (0.53 mL, 3.01 mmol). After gradual warming to rt, the mixture was stirred for an additional 12 h and then diluted with methylene chloride (100 mL) followed 20 by a wash with water (30 mL) and brine (30 mL). The organic phase was dried over sodium sulfate, filtered and concentrated to dryness. The crude residue was purified by flash column chromatography over silica gel (19 mm x 175 mm) using 50% ethyl acetate in hexanes to give N-tert-butyloxycarbonyl-sphingosine (540 mg, 90%) as a white solid. 1H NMR (300 MHz, Chloroform-d) 5 5.77 (dt, J = 15.4, 8.4 Hz, 1H), 5.52 (dd, J= 25    15.4, 8.4 Hz, 1H), 3.93 (dd, J = 11.4, 3.7 Hz, 1H), 3.70 (dd, J = 11.4, 3.7 Hz, 1H), 3.59 (s, 3H), 2.05 (q, J = 7.0 Hz, 2H), 1.52 (s, 9H), 1.25 (s, 22 H), 0.87 (t, J = 6.5 Hz, 3H). Example 16. 2020418425   14 Sep 2026 125 N-tert-Butyloxycarbonyl-sphingosine 124(540 mg, 1.35 mmol) was rendered anhydrous by co-evaporation with anhydrous pyridine (2 x 12 mL). The residue was then 5 dissolved in anhydrous pyridine and treated with carbon tetrabromide (622 mg, 1.88 mmol). The mixture was cooled to 0oC and treated dropwise with a solution of trimethylphosphite (0.25 mL, 2.10 mmol) in anhydrous pyridine (3 mL) over a 30 min period. After an additional 12 h at rt, both LCMS and tlc (5% methanol in methylene chloride) analysis indicated complete conversion. The mixture was quenched with water (2 mL) and then 10 concentrated to dryness. The resulting dark oil was dissolved in ethyl acetate (150 mL) and washed with 3% HCL solution ( 2 x 20 mL) followed by saturated sodium bicarbonate solution (30 mL). The organic layer was dried over sodium sulfate, filtered and concentrated. The crude residue was purified by flash column chromatography over silica gel (19 mm x 175 mm) using 2% methanol in methylene chloride to give N-tert- 15   butyloxycarbonyl-sphingosine-1-O-dimethylphosphate 125 (350 mg, 51%) as a gum. 1H NMR (400 MHz, Chloroform-d) 5 5.82 (dt, J = 15.4, 7.1 Hz, 1H), 5.48 (dd, J = 15.4, 7.1 Hz, 1H), 4.99 (d, J = 8.9 Hz, 1H), 4.32 (ddd, J = 10.7, 8.0, 4.6 Hz, 1H), 4.11 (ddt, J = 10.7, 7.4, 3.1 Hz, 2H), 3.77 (dd, J = 11.1, 2.1 Hz, 6H), 2.01 (q, J = 7.1 Hz, 2H), 1.41 (s, 9H), 1.34 (m, 2H), 1.23 (m, 20H), 0.86 (t, J = 6.4 Hz, 3H). 20         31P NMR (162 MHz, Chloroform-d) 5 2.00. MS C17H25NO4 [M+Na+]; calculated: 330.2, found: 330.2. Example 17. O ?H HO-P-O' / Y^^C13H27 OH NH2 126 A solution ofN-tert-butyloxycarbonyl-sphingosine-1-O-dimethylphosphate 125 (350 25 mg, 0.689 mmol) in anhydrous methylene chloride (8 mL) was treated dropwise with trimethylsilyl bromide (0.45 mL, 3.45 mmol) at 0oC. After warming to room temperature, the mixture was allowed to stir at rt for 6h and then concentrated to dryness. The resulting residue was co-evaporated with methylene chloride to remove excess trimethylsilyl bromide 2020418425   14 Sep 2026 and then treated with 66% aqueous THF (6 mL). The resulting precipitate was collected by filtration to give sphingosine-1-phosphate 126 (218 mg, 83%) as a white solid. 1H NMR (400 MHz, Methanol-d4+ CD3CO2D) 5 5.84 (dt, J = 15.5, 6.7 Hz, 1H), 5.46 (dd, J = 15.5, 6.7 Hz, 1H), 4.33 (t, J = 6.0 Hz, 1H), 4.13 (ddd, J = 11.8, 7.7, 3.6 Hz, 5    1H), 4.03 (dt, J = 11.8, 8.4 Hz, 1H), 3.47 (ddd, J = 8.3, 4.8, 3.2 Hz, 1H), 2.10 - 1.99 (m, 2H), 1.37 (m, 2H), 1.24 (m, 20H), 0.83 (t, J = 6.4 Hz, 3H). 31P NMR (162 MHz, Chloroform-d) 5 0.69. MS C18H38NO5P [M-H+]; calculated: 378.2, found: 378.2. Example 18. OH O 131 F3C NH OH 10 To a slurry of phytosphingosine (4 g, 12.6 mmol) and anhydrous powdered potassium carbonate (5.22 g, 37.8 mmol) in methylene chloride (85 mL) was added trifluoroacetic anhydride (1.96 mL, 13.9 mmol). The mixture was stirred at rt for 18 h and then diluted with methylene chloride (500 mL). The mixture was washed with water (100 15 mL). Methanol (60 mL) was added to break the emulsion. The organic phase was then dried over sodium sulfate, filtered and concentrated to give 131 (4.9 g, 94 %) as a white solid 1H NMR (400 MHz, DMSO-d6) 5 8.90 (s, 1H), 4.90 - 4.68 (m, 1H), 4.56 (d, J = 6.1 Hz, 1H), 4.43 (s, 1H), 3.97 (d, J = 7.6 Hz, 1H), 3.65 (d, J = 10.8 Hz, 1H), 3.46 (t, J = 10.2 20 Hz, 1H), 3.32 - 3.16 (m, 1H), 1.42 (tt, J = 15.7, 7.5 Hz, 2H), 1.20 (s, 24H), 0.83 t, J = 6.8 Hz, 3H). O 132 Example 19. 25          N-Trifluoroacetyl-phytosphingosine (131, 1.88 g, 4.5 mmol) in anhydrous pyridine (23 mL) was treated with DMAP (56 mg, 0.45 mmol) and then dropwise with tertbutyldiphenylsilyl chloride (1.38 g, 5.0 mmol). After 18 h concentrated to dryness. The 2020418425   14 Sep 2026 resulting residue was dissolved in ethyl acetate (200 mL) and washed with saturated ammonium chloride (2x 50 mL) and then brine (50 mL). The aqueous phases was back-extracted with ethyl acetate (50 mL). Combined organic phases were dried over sodium sulfate and concentrated to give crude 1-O-tert-Butyldiphenylsilyl-2-N-trifluoroacetyl- 5 phytosphingosine 132 (3g, 100%) as a gum. The material was used in the next step without further purification. 1H NMR (400 MHz, Chloroform-d) S 7.62 (m, 2H), 7.60 - 7.56 (m, 2H), 7.47 - 7.31 (m, 6H), 7.07 (d, J = 8.4 Hz, 1H), 4.23 (dd, J = 8.5, 4.1 Hz, 1H, 4.04 (dt, J = 11.0, 2.5 Hz, 1H), 3.82 (ddd, J = 11.0, 4.3, 1.8 Hz, 1H), 3.64 (dq, J = 10.6, 6.0, 4.3 Hz, 2H), 1.45 (m, 10   2H), 1.39 - 1.15 (m, 24H), 1.05 (m, 9H), 0.94 - 0.80 (t, J = 6.9 Hz 3H). Example 20. A solution of 1-O-tert-Butyldiphenylsilyl-2-N-trifluoroacetyl-phytosphingosine 132 (3g,4.5 mmol) in 1 / 1 (v / v) 2,2-dimethoxypropane / THF was treated with catalytic amount of 15 p-toluenesulfonic acid (87 mg, 0.45 mmol) and allowed to stir for 16h at rt. The mixture was quenched with saturated sodium bicarbonate (30 mL) and then excess THF / 2,2-dimethoxypropane was removed under vacuum. The mixture was extracted with ethyl acetate (200 mL). After washing with brine, the organic layer was dried over sodium sulfate, filtered and concentrated. The crude oil was purified by column chromatography 20   (25 mm x 175mm) over silica gel with a hexanes / ethyl acetate mobile phase to give 133(2.45 g, 78%). 1H NMR (400 MHz, Chloroform-d) S 7.68 - 7.63 (m, 2H), 7.63 - 7.57 (m, 2H), 7.39 (m, 6H), 6.54 (d, J = 9.4 Hz, 1H), 4.23 (dd, J = 8.2, 5.6 Hz, 1H), 4.12 (ddd, J = 13.3, 6.9, 3.8 Hz, 2H), 3.96 (dd, J = 10.5, 3.9 Hz, 1H), 3.69 (dd, J = 10.5, 2.9 Hz, 1H), 1.52 - 1.36 (m, 25   2H), 1.33 (s, 3H), 1.31 (s, 3H), 1.24 (m, 24H), 1.03 (s, 9H), 0.86 (t, J = 53.7, 6.9 Hz, 3H). Example 21. 2020418425   14 Sep 2026 A solution of 1-O-tert-Butyldiphenylsilyl-3,4-O-isopropylidene-2-N-trifluoroacetyl-phytosphingosine 133 (2.45 g, 3.54 mmol)in THF (18 mL) was treated with 5 tetrabutylammonium fluoride (4.25 mL of a 1.0 M solution in THF, 4.25 mmol) and stirred at rt for 12h. The mixture was diluted with ethyl acetate (100 mL) and saturated ammonium chloride (2 x 50 mL) and then brine (50 mL). The organic phase was dried over sodium sulfate, filtered, and concentrated to give a white solid that was further purified by column chromatography (25 mm x 175 mm) over silica gel with a 9:1 hexanes: ethyl acetate mobile 10 phase to afford 134(1.5g, 93%) as a white solid. 1H NMR (300 MHz, Chloroform-d) 5 6.92 (d, J = 8.7 Hz, 1H), 4.31 - 4.16 (m, 2H), 4.11 (dq, J = 11.7, 3.7 Hz, 1H), 4.00 (dd, J = 11.5, 2.6 Hz, 1H), 3.70 (dd, J = 11.5, 3.6 Hz, 1H), 1.48 (s, 3H), 1.35 (s, 3H), 1.25 (m, 26H), 0.88 (t, J = 6.9 Hz 3H). Example 22. O 0^0 ’ p -O' 0 Y c„h29 c p NH Y 15                                          0    135 A solution of 3,4-O-Isopropylidene-2-N-Trifluoroacetyl-phytosphingosine 134(630 mg, 1.39 mmol) was rendered anhydrous by co-evaporation with anhydrous pyridine (2 x 12 mL). The residue was then dissolved in anhydrous pyridine (12 mL) and treated with carbon tetrabromide (533 mg, 1.67 mmol). The mixture was cooled to 0oC and treated 20 dropwise with a solution of trimethylphosphite (0.23 mL, 1.95 mmol) in anhydrous pyridine (3 mL) over a 30 min period. After an additional 12 h at rt, both LCMS and tlc (5% methanol in methylene chloride) analysis indicated complete conversion. The mixture was quenched with water (2 mL) and then concentrated to dryness. The resulting dark oil was dissolved in ethyl acetate (100 mL) and washed with 3% HCL solution ( 2 x 20 mL) 25 followed by saturated sodium bicarbonate solution (30 mL). The organic layer was dried over sodium sulfate, filtered and concentrated. The crude residue was purified by flash 2020418425   14 Sep 2026 column chromatography over silica gel (19 mm x 175 mm) using 2% methanol in methylene chloride to give 135 (650 mg, 83%). 1H NMR (300 MHz, Chloroform-d) S 7.42 (d, J = 8.8 Hz, 1H), 4.36 (td, J = 10.9, 5.0 Hz, 1H), 4.25 (m, 1H), 4.19 (m, J = 6.5, 2.0 Hz, 3H), 3.77 (dd, J = 11.2, 7.5 Hz, 6H), 1.44 5   (s, 3H), 1.33 (s, 3H), 1.25 (m, 26H), 0.87 (t, J = 6.6 Hz, 3H). 31P NMR (121 MHz, Chloroform-d) S 1.69. MS C25H47F3NO7P [M-H+]; calculated: 560.3, found: 560.2. Example 23. 3,4-O-Isopropylidene-2-N-trifluoroacetyl-phytosphingosine-1-phosphate (136) 10 A solution of 3,4-O-Isopropylidene-2-N-trifluoroacetyl-phytosphingosine-1-O-dimethylphosphate 135 (650 mg, 1.16 mmol) in anhydrous methylene chloride (12 mL) was treated dropwise with trimethylsilyl bromide (0.81 mL, 6.23 mmol) at 0oC. After 12h at rt, the mixture was concentrated to dryness and the resulting residue co-evaporated with 15 methylene chloride (3 x 50 mL) to remove excess trimethylsilyl bromide. The residue then was dissolved in cold (4oC) solution of 1% NH4OH while maintaining pH 7-8. After 10 min at rt, the mixture was concentrated to dryness, and the resulting solid triturated with methanol / acetonitrile. The solid was collected by filtration, washed with acetonitrile, and dried under high vacuum to give 136 (500 mg, 75%) as a white solid. 20          1H NMR (300 MHz, Methanol-d4) S 4.31 (dd, J = 8.7, 5.4 Hz, 1H), 4.09 (m, 4H), 1.42 (s, 3H), 1.36 (s, 3H), 1.31 (m, 26H), 0.89 (t, J = 6.4 Hz, 3H). 31P NMR (121 MHz, Methanol-d4) S 1.28. 19F NMR (282 MHz, Methanol-d4) S -77.13. HRMS C23H42F3NO7P [M-H+]; calculated: 532.26565, found: 532.26630. 25 Example 24. 2020418425   14 Sep 2026 A mixture of N-trifluoroacetyl-phytosphingosine-1-phosphate 136(200mg, 0.373 mmol) and 2’,3’-dideoxy-2’-fluoro-7-deazaguanine (100 mg, 0.373 mmol) was rendered anhydrous by co-evaporation with anhydrous pyridine (3 x 10 mL). The resulting residue 5 then was dissolved in anhydrous pyridine (4 mL) and treated with diisopropylcarbodiimide (127 mg, 1.01 mmol) and HOBt (60 mg, 0.447 mmol). After 24 h at 75oC, the reaction mixture was cooled to rt and concentrated to dryness. The crude material was purified by flash column chromatography (19 mm x 170 mm) over silica gel using a solvent gradient from 5 to 7.5% methanol in chloroform with 1% (v / v) NH4OH to give 137(80 mg, 27%) as 10 a white solid. 1H NMR (300 MHz, Methanol-d4) 5 6.88 (d, J = 3.8 Hz, 1H), 6.46 (d, J = 3.8 Hz, 1H), 6.24 (d, J = 19.9 Hz, 1H), 5.34 (dd, J = 52.4, 4.6 Hz, 1H), 4.53 (s, 1H), 4.34 - 3.97 (m, 6H), 2.63 - 2.17 (m, 2H), 1.40 (s, 3H), 1.30 (s, 3H), 1.27 (m, 26H), 0.89 (t, J = 6.6 Hz, 3H). 31P NMR (121 MHz, Methanol-d4) 5 12.50. 15          19F NMR (282 MHz, Methanol-d4) 5 -77.10 , -179.69 - -180.25 (m). MS C34H522F4N5O9P [M-H+]; calculated: 781.3, found: 782.2. Example 25. Experimental procedure for synthesis of prodrugs A solution of isopropyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate (0.397 g, 20   1.300 mmol) in anhydrous THF (5 ml) was added to a -78 °C stirred solution of 2’-deoxy- 2’-fluoronucleoside (0.812 mmol) and 1-methyl-1H-imidazole (0.367 ml, 4.63 mmol) in pyridine (10.00 ml). After 15 min the reaction was allowed to warm to room temperature and was stirred for an additional 3 hours. Next, the solvent was removed under reduced pressure. The crude product was dissolved in 120 ml of DCM and was washed with 20 ml 1 25 N HCl solution followed by 10 ml water. The organic phase was dried over sodium sulfate, filtered and concentrated in vacuo. The residues were separated over silica column (neutralized by TEA) using 5% MeOH in DCM as a mobile phase to yield the respective products as diastereomers. 30 Example 26. 2020418425   14 Sep 2026 N-tert-Butyloxycarbonyl-phytosphingosine (174) OH q^-Nh oh o 174 A suspension of phytosphingosine (10.6 g, 33.5 mmol) and triethylamine (5.6 ml, 40.2 mmol) in THF (250 mL) was treated dropwise with di-tert-butyl dicarbonate (8.6 mL, 5   36.9 mmol). After 12h at rt, the mixture was concentrated to dryness and the resulting white solid was recrystallized from ethyl acetate (80 mL) and then dried under high vacuum at 35oC for 12h to give 174(10.5 g, 75%). 1H NMR (400 MHz, Chloroform-d) S 5.31 (d, J = 8.5 Hz, 1H), 3.89 (d, J = 11.1 Hz, 1H), 3.83 (s, 2H), 3.74 (dd, J = 11.1, 5.2 Hz, 1H), 3.65 (d, J = 8.3 Hz, 1H), 3.61 (d, J = 3.9 10 Hz, 1H), 1.43 (s, 9H), 1.23 (s, 27H), 0.86 (t, J = 6.4 Hz, 3H). Example 27. 2-O-tert-Butyldiphenylsilyl-1-N-tert-butyloxycarbonyl-phytosphingosine (175) A solution of N-tert-Butyloxycarbonyl-phytosphingosine 174 (9.5 g, 22.65 mmol) 15 and triethylamine (3.8 mL, 27.2 mmol) in anhydrous methylene chloride / DMF (120 mL / 10 mL) was treated dropwise with tert-butylchlorodiphenylsilane (7 mL, 27.25 mmol). After 18h at rt, the mixture was diluted with methylene chloride (200 mL) and washed with 0.2N HCl (100 mL) and then brine (100 mL). The organic phase was dried over sodium sulfate, filtered and then concentrated to give 175 (14.9 g) as an oil which was used in the next 20 reaction without further purification. 1H NMR (400 MHz, Chloroform-d) S 5.31 (d, J = 8.5 Hz, 1H), 3.89 (d, J = 11.1 Hz, 1H), 3.83 (m, 1H), 3.74 (dd, J = 11.1, 5.2 Hz, 1H), 3.65 (d, J = 8.3 Hz, 1H), 3.61 (d, J = 3.9 Hz, 1H), 1.43 (s, 9H), 1.23 (s, 27H), 0.86 (t, J = 6.4 Hz, 3H). Example 28. 25   2-O-tert-Butyldiphenylsilyl-1-N-tert-butyloxycarbonyl-3,4-O-isopropylidene- phytosphingosine (176) 2020418425   14 Sep 2026 A solution of 2-O-tert-Butyldiphenylsilyl-1-N-tert-butyloxycarbonyl- phytosphingosine (175, 14.9 g, 22.65 mmol) in 1 / 1 (v / v) THF / 2,2-dimethoxypropane was treated with catalytic para-toluenesulfonic acid (860 mg, 4.53 mmol). After 24h, the 5 mixture was quenched with saturated sodium bicarbonate solution (50 mL). The mixture was concentrated and then dissolved in ethyl acetate (200 mL) and washed with brine (2 x 50 mL). The organic phase was dried over sodium sulfate, filtered and concentrated to give 176 (15.7 g) as a gum which was used in the next step without further purification. 1H NMR (400 MHz, Chloroform-d) S 7.66 (m, 4H), 7.51 - 7.27 (m, 6H), 4.78 (d, J 10   = 10.0 Hz, 1H), 4.18 (dd, J = 9.3, 5.5 Hz, 1H), 3.89 (dd, J = 9.9, 3.3 Hz, 1H), 3.80 (d, J = 9.9 Hz, 1H), 3.72 (d, J = 9.9 Hz, 1H), 1.45 (s, 9H), 1.42 (s, 3H), 1.35 (s, 3H), 1.25 (s, 27H), 1.05 (s, 9H), 0.87 (t, J = 6.5 Hz, 3H). Example 29. 1-N-tert-butyloxycarbonyl-3,4-O-isopropylidene-phytosphingosine (177). O^O ho-^Y xc14h29 \ o NH Ay 15                                     X O 177 A solution of 2-O-tert-Butyldiphenylsilyl-1-N-tert-butyloxycarbonyl-3,4-O-isopropylidene-phytosphingosine 176 (15.7 g,22.6 mmol) in THF at 0oC was treated dropwise with a solution of tetrabutylammonium fluoride (1.0 M in THF, 24.9 mL, 24.9 mmol) over a 20 min period. After 16h at rt, tlc (3:1 hexanes:ethyl acetate) indicated 20 complete conversion. The mixture was concentrated to dryness and the resulting residue was dissolved in ethyl acetate (300 mL) and washed with water (3 x 100 mL). The organic phase was dried over sodium sulfate, filtered and concentrated. The resulting oil purified by flash column chromatography (35 mm x 180 mm) using a solvent gradient from 25 to 50% ethyl acetate in hexanes to give 177 (7.3 g, 71% over 3 steps) as a white solid. 25          1H NMR (400 MHz, Chloroform-d) S 4.93 (d, J = 9.1, 1H), 4.16 (q, J = 7.1, 6.4 Hz, 1H), 4.07 (t, J = 6.5 Hz, 1H), 3.83 (dd, J = 11.1, 2.4 Hz, 1H), 3.76 (m, 1H), 3.67 (dd, J = 2020418425   14 Sep 2026 11.2, 3.6 Hz, 1H), 1.43 (s, 3H), 1.42 (s, 9H), 1.32 (s, 3H), 1.23 (s, 27H), 0.86 (t, J = 6.9 Hz, 3H). Example 30. General Procedure for the Preparation of 5’-Phosphoramidate Prodrugs 5 Synthesis of chlorophosphoramidate: 251 Thionyl chloride (80 g, 49.2 mL, 673 mmol) was added dropwise over a 30 min period to a suspension of l-alanine (50g, 561 mmol) in isopropanol (500 mL). The mixture 10 was heated to a gentle reflux for 5h and then concentrated by rotary evaporator (bath set at 60oC). The resulting thick gum solidified upon trituration with ether (150 ml). The white powder was triturated a second time with ether (150 mL), collected by filtration while under a stream of argon, and then dried under high vacuum for 18h to give (S)-isopropyl 2-aminopropanoate hydrochloride (88 g, 94%). 15          1H NMR (400 MHz, DMSO-d6) 5 8.62 (s, 3H), 5.10 - 4.80 (m, 1H), 3.95 (q, J = 7.2 Hz, 1H), 1.38 (d, J = 7.2 Hz, 3H), 1.22 (d, J = 4.6 Hz, 3H), 1.20 (d, J = 4.6 Hz, 3H). Example 31. 252 A solution of phenyl dichlorophosphate (30.9 g, 146 mmol) in dichloromethane (450 20 mL) was cooled to 0oC then treated with (S)-isopropyl 2-aminopropanoate hydrochloride (24.5 g, 146 mmol). The mixture was further cooled to -78oC and then treated dropwise with triethylamine (29.6 g, 40.8 mL, 293 mmol) over a 30 min period. The mixture continued to stir at -78oC for an additional 2 h and then allowed to gradually warm to rt. After 18h the mixture was concentrated to dryness and the resulting gum dissolved in 25 anhydrous ether (150 mL). The slurry was filtered while under a stream of argon, and the collected solid washed with small portions of anhydrous ether (3 x 30 mL). Combined 2020418425   14 Sep 2026 filtrates were concentrated to dryness by rotary evaporator to give a 1:1 diastereomeric mixture of phosphochloridate (41.5 g, 93%) as pale yellow oil. 1H NMR (300 MHz, Chloroform-d) S 7.43 - 7.14 (m, 5H), 5.06 (m, 1H), 4.55 (dd, J = 14.9, 7.0 Hz, 1H), 4.21 - 4.01 (m, 1H), 1.48 (d, J = 7.0 Hz, 2H), 1.27 (d, J = 6.2 Hz, 3H), 5   1.26 (d, J = 5.8 Hz, 3H). 31P NMR (121 MHz, Chloroform-d) S 8.18 and 7.87. Example 32. Synthesis of 2-chloro-4-nitrophenyl phosphoramidate: NO2 10                                           253 A solution of phenyl dichlorophosphate (60 g, 42.5 mL, 284 mmol) in dichloromethane (300 mL) was cooled to 0oC and then treated with (S)-isopropyl 2-aminopropanoate hydrochloride (47.7 g, 284 mmol). The mixture was further cooled to -78oC and treated dropwise with a solution of triethylamine (57.6 g, 79 mL, 569 mmol) in 15 methylene chloride (300 mL) over a 1 h period. The reaction mixture was warmed to 0oC for 30 min and then treated with a preformed mixture of 2-chloro-4-nitrophenol (46.9 g, 270 mmol) and triethylamine (28.8 g, 39.6 mL, 284 mmol) in dichloromethane (120 mL) over a 20 min period. After 2 h at 0oC, the mixture was filtered through a fritted funnel, and the collected filtrate concentrated to dryness. The crude gum was dissolved MTBE (500 mL) 20 and washed with 0.2 M K2CO3 (2 x 100 mL) followed by 10% brine (3 x 75 mL). The organic phase was dried over sodium sulfate, filtered and concentrated to dryness by rotary evaporator to give a diastereomeric mixture (100 g, 93%) as a pale yellow oil. 1H NMR (400 MHz, Chloroform-d) S 8.33 (dd, J = 2.7, 1.1 Hz, 1H, diastereomer 1), 8.31 (dd, J = 2.7, 1.1 Hz, 1H, diastereomer 2), 8.12 (dd, J = 9.1, 2.7 Hz, 1H), 7.72 (dt, J = 25   9.1, 1.1 Hz, 1H), 7.40 - 7.31 (m, 2H), 7.28 - 7.19 (m, 6H), 5.01 (pd, J = 6.3, 5.2 Hz, 1H), 4.22 - 4.08 (m, 1H), 3.96 (td, J = 10.7, 9.1, 3.6 Hz, 1H), 1.43 (dd, J = 7.0, 0.6 Hz, 3H), 1.40 (dd, J = 7.2, 0.6 Hz, 3H, diastereomer 2), 1.25 - 1.20 (m, 9H). 2020418425   14 Sep 2026 Example 33. Separation of compound 253 diastereomers: Sp-diastereomer               Rp-diastereomer 254                        255 5          The diastereomeric mixture 253 (28 g, 63.2 mmol) was dissolved in 2:3 ethyl acetate:hexanes (100 mL) and cooled to -20oC. After 16 h, the resulting white solid was collected by filtration and dried under high vacuum to give a 16:1 Sp:Rp-diastereomeric mixture (5.5 g, 19.6%). The mother liquor was concentrated and the resulting residue dissolved in 2:3 ethyl acetate:hexanes (50 mL). After 16h at -10oC, the resulting white solid 10 was collected and dried under high vacuum to give a 1:6 Sp:Rp-diastereomeric mixture (4g, 14%). The 16:1 Sp:Rp-diastereomeric mixture (5.5 g, 12.4 mmol) was suspended in hot hexanes (50 mL) and treated slowly with ethyl acetate (approximately 10 mL) until complete dissolution. After cooling to 0oC, the resulting white solid was collected by filtration, washed with hexanes, and dried under high vacuum to give the Sp -diastereomer 15 of 254 (4.2 g, 76%) as a single isomer. 1H NMR (Sp-diastereomer, 400 MHz, Chloroform-d) 5 8.33 (dd, J = 2.7, 1.1 Hz, 1H), 8.12 (dd, J = 9.1, 2.7 Hz, 1H), 7.71 (dd, J = 9.1, 1.2 Hz, 1H), 7.41 - 7.30 (m, 2H), 7.29 - 7.11 (m, 3H), 5.00 (m, 1H), 4.25 - 4.07 (m, 1H), 3.97 (dd, J = 12.7, 9.4 Hz, 1H), 1.43 (d, J = 7.0 Hz, 3H), 1.23 (d, J = 2.2 Hz,3H), 1.21 (d, J = 2.2 Hz, 3H). 20          The 1:6 Sp:Rp-diastereomeric mixture (4 g, 12.4 mmol) was suspended in hot hexanes (50 mL) and treated slowly with ethyl acetate (approximately 5 mL) until complete dissolution. After cooling to 0oC, the resulting white solid was collected by filtration, washed with hexanes, and dried under high vacuum to give the Rp -diastereomer of 255 (3.2g, 80%) as a single isomer. Absolute stereochemistry was confirmed by X-ray analysis. 25   1H NMR (Rp-diastereomer , 400 MHz, Chloroform-d) 5 8.31 (dd, J = 2.7, 1.1 Hz, 1H), 8.11 (dd, J = 9.1, 2.7 Hz, 1H), 7.72 (dd, J = 9.1, 1.2 Hz, 1H), 7.42 - 7.30 (m, 2H), 7.31 - 7.14 (m, 3H), 5.01 (p, J = 6.3 Hz, 1H), 4.15 (tq, J = 9.0, 7.0 Hz, 1H), 4.08 - 3.94 (m, 1H), 1.40 (d, J = 7.0 Hz, 3H), 1.24 (d, J = 3.5 Hz, 3H), 1.22 (d, J = 3.5 Hz, 3H). 2020418425   14 Sep 2026 Example 34. General procedure for phosphoramidate prodrug formation: The desired nucleoside (1 equivalent) to be converted into its 5’-phosphoramidate prodrug was dried in a vaccum oven at 50°C overnight. The dry nucleoside is placed in a 5 dry flask under an inert atmosphere and suspended in either dry THF or dry DCM to achieve a 0.05M solution. The flask was then cooled to 0°C, and the chlorophosphoramidate reagent (5 equivalents) was added to the suspended nucleoside. Next, 1-methylimidazole (8 equivalents) was added to the reaction mixture dropwise. The reaction was allowed to stir at room temperature for 12-72 hours. After the reaction was 10 complete as judged by TLC, the reaction mixture was diluted with ethyl acetate. The diluted reaction mixture was then washed with saturated aqueous ammonium chloride solution. The aqueous layer was re-extracted with ethyl acetate. The combined organic layers were then washed with brine, dried over MgSO4, filtered, and concentrated. The concentrated crude product was then purified on silica eluting with a gradient of DCM to 15  5% MeOH in DCM. Example 35. General Procedure for Preparation of 5’-Triphosphates: Nucleoside analogue was dried under high vacuum at 50oC for 18h and then dissolved in anhydrous trimethylphosphate (0.3 M). After addition of proton-sponge® (1.5 20 molar equiv), the mixture was cooled to 0oC and treated dropwise with phosphoryl chloride (1.3 molar equiv) via microsyringe over a 15 min period. The mixture continued stirring at 0oC for 4 to 6 h while being monitored by tlc (7:2:1 isopropanol: conc. NH4OH: water). Once greater than 85% conversion to the monophosphate, the reaction mixture was treated with a mixture of bis(tri-n-butylammonium pyrophosphate) (3 molar equiv) and 25 tributylamine (6 molar equiv) in anhydrous DMF (1 mL). After 20 min at 0oC with monitoring by tlc (11:7:2 NH4OH: isopropanol: water), the mixture was treated with 20 mL of a 100 mM solution of triethylammonium bicarbonate (TEAB), stirred for 1h at rt and then extracted with ether (3 x 15 mL). The aqueous phase was then purified by anion-exchange chromatography over DEAE Sephadex® A-25 resin (11 x 200 mm) using a buffer 30 gradient from 50 mM (400 mL) to 600 mM (400 mL) TEAB. Fractions of 10 mL were analyzed by tlc (11:7:2 NH4OH: isopropanol: water). Triphosphate (eluted @ 500 mM TEAB) containing fractions were combined and concentrated by rotary evaporator (bath < 25oC). The resulting solid was reconstituted in DI water (10 mL) and concentrated by lyophilization. 2020418425   14 Sep 2026 Example 36. Synthesis of (R)-2,2,2-trifluoro-N-(1-hydroxyoctadecan-2-yl)acetamide OH NH2 OH 305 O EtO^CF3 ,NEt3 EtOH rt OH OH 131 TBDPSCI, DMAP, NEt3 CH2CI2:DMF 4:1 0°C to rt 80% TBDPSO OH 132 C14H29 l2, imid, PPh3 NaOH______ Toluene, reflux 54% H2, Pd(OH)2 MeOH TBAF x THF 0°C to rt 60% over two steps HO NH2 OH 305 OH C14H29 O EtO^CF3 ,NEt3 ---------------------------J EtOH rt Phytosphingosine (15.75 mmol) was dissolved in EtOH (0.5M) and ethyl trifluoroacetate (15.75 mmol) was added dropwise. NEt3 (24.41mmol) was added next the reaction mixture stirred overnight. The solvent was removed in vacuo and the residue was 10 taken up in EtOAc and brine, washed, dried and concentrated. The crude material that was a white powder was good enough to use in the next step without further purification. Characterization matched literature: Synthesis, 2011, 867. Example 37. 15 OH OH 131 TBDPSCI, DMAP, NEt3 CH2CI2:DMF 4:1 0°C to rt OH TBDPSO OH 132 C14H29 The primary alcohol (15.75 mmol), DMAP (1.575 mmol) and NEt3 (39.4 mmol) were dissolved in CH2CI2 and DMF (0.18M) mixture and cooled to 0°C. TBDPSCI (19.69 2020418425   14 Sep 2026 mmol) was added dropwise then the solution was allowed to warm to room temperature and stirred overnight. NH4Cl solution was added to quench. The reaction mixture was extracted with EtOAc and the combined organic layers were washed with water (x2) to remove DMF. It was then dried and concentrated. A column was run to purify the mixture. 10-20% EtOAc / Hex. Characterization matched literature: Synthesis, 2011, 867. Example 38. TBDPSO O. OH >\X14H29 NH OH cf3 132 l2, imid, PPh3 NaOH______ Toluene, reflux 54% TBDPSO Y 0^ ,NH c14h29 CF3 306 The diol (12.58 mmol), triphenylphosphine (50.3 mmol) and imidazole (50.03 10 mmol) were dissolved in toluene and reheated to reflux. The iodine (37.7 mmol) was then added slowly and the reaction mixture continued to be stirred at reflux. After three hours it was cooled to room temperature and 1 equivalent of iodine (12.58 mmol) was added followed by 8 equivalents of 1.5M NaOH (100.64 mmol). The reaction mixture was stirred until all the solids dissolved. The aqueous layer was removed in a separatory funnel and the 15 organic layer was washed with Na2S2O3 solution then NaHCO3 solution then brine. It was dried and concentrated. A column was run to purify the mixture 0-20% EtOAc / Hex and a mixture of cis and trans was obtained but carried on to the next step. § 1H NMR (400 MHz, Chloroform-d) 8 7.64 (ddt, J = 7.8, 3.8, 1.7 Hz, 4H), 7.51 -7.35 (m, 6H), 6.68 (dd, J = 16.0, 8.2 Hz, 1H), 5.6 - 5.40 (m, 2H), 4.57 - 4.46 (m, 1H), 3.84 20   - 3.62 (m, 2H), 2.04 (q, J = 7.0 Hz, 1H), 1.28-1.21 (m, 24H), 1.15 - 0.98 (m, 9H), 0.90 (t, J = 6.8 Hz, 3H). HRMS: 617.38759. Example 39. TBDPSO O cf3 306 Ci4H29 H2, Pd(OH)2 TBDPS0 MeOH         ° cf3 307 c14h29 25         The alkene (2.91 mmol) was dissolved in MeOH (0.1M) and Pd(OH)2 / C (0.146 mmol) was added. A Parr Hydrogenator was used at 40 psi. The palladium catalyst was carefully filtered off through celite and rinsed with EtOAc. The crude material was used in the next step and provided quantitative yield. 2020418425   14 Sep 2026 Example 40. The silyl ether was dissolved in THF and cooled to 00C then TBAF was added dropwise. After stirring for 1 hour it was warmed to room temperature. After two hours 5 NH4Cl solution was added and it was extracted with EtOAc, washed with brine and dried and concentrated. A column was run 10-50% EtOAc / Hex. 1H NMR (400 MHz, Chloroform-d) 5 7.60 (tt, J = 7.0, 1.5 Hz, 2H), 7.48 - 7.33 (m, 4H), 3.73 3.61 (m, 1H), 1.24 (d, J = 3.5 Hz, 18H), 1.05 (s, 6H), 0.86 (t, J = 6.8 Hz, 3H). HRMS : 381.28546. 10 Example 41. o o E.O^AOE. (CH2)15CH3 380 To 33.4 g sodium ethoxide solution (21% wt) in ethanol, diethyl malonate(15g) and then 1-bromohexadecane (31.5g) were added dropwise. After reflux for 8 hrs, ethanol was evaporated in vacuo. The remaining suspension was mixed with ice-water( 200 ml) and 15 extracted with diethyl ether (3 X 200ml). The combined organic layers were dried over MgSO4, filtered and the filtrate was evaporated in vacuo to yield a viscous oil residue. This residue was purified by column chromatography(silica: 500 g) using hexane / diethyl ether( 12:1) as mobile phase to yield the main compound. Example 42. HO'^Y^'OH (CH2)15CH3 20                                          381 In a 250 mL round-bottomed flask was aluminum lithium hydride (2.503 g, 66.0 mmol) in Diethyl ether (90 ml) to give a suspension. To this suspension was added diethyl 2-hexadecylmalonate (18.12 g, 47.1 mmol) dropwise and the reaction was refluxed for 6 h. The reaction was followed up by TLC using PMA and H2SO4 as drying agents. The excess 25 lithium aluminium hydride was destroyed by 200ml of ice-water. 150 ml of 10 % H2SO4 was added to dissolve aluminium hydrate. The reaction mixture was extracted by diethyl ether (100 ml X 3). The organic layer including undissolved product was filtered. The collect solids were washed with ethyl acetate. The filtrate was dried over MgSO4, filtered 2020418425   14 Sep 2026 and concentrated under reduced pressure. The product was purified on silica (100g) column eluting with Hexane:EtOAc (3:1) to (1:1). Example 43. (CH2)15CH3 382 5          To a solution of 2-hexadecylpropane-1,3-diol (7.04 g, 23.43 mmol) in 100 ml of DCM was added dropwise phosphorous trichloride (3.59 g, 23.43 mmol) dissolved in 20 ml of DCM followed by triethylamine (6.53 ml, 46.9 mmol). The reaction was refluxed for one hour. TLC analysis showed that the starting material was consumed and two new spots formed. The mixture was concentrated to dryness, dissolved in dry diethyl ether and 10 filtered. The filtrate was concentrated to yield the crude product (8.85 g) that was used in the next step without further purification. Example 44. Synthesis of 5’-Deuterated Nucleoside Analogs 386                       387 15          The nucleoside was suspended in methylene chloride (40 mL, partially soluble). After stirring at rt for 30 min the mixture was treated sequentially with PDC, acetic anhydride and then tert-butanol. The mixture continued to stir at room temperature. TLC (5% methanol in DCM) and LCMS indicated only a small amount of remaining starting material at 4 hours. The mixture was filtered through a pad of silica gel that was loaded 20 into a 150 mL fritted funnel. The silica was eluted with ethyl acetate. The collected filtrate was concentrated by under reduced pressure. The crude dark oil was purified by chromatography over silica gel (25 mm x 175 mm) with 2:1 hexanes:ethyl acetate to ethyl acetate gradient. The pure fractions were collected and concentrated to give of a white gum. The material was placed under high vacuum for 2 days and was used in the next step 25 without further purification. The 5’-protected nucleoside was dissolved in 200 proof ethanol and was then treated with solid sodium borodeuteride. The mixture became homogeneous and was then heated 2020418425   14 Sep 2026 to 80°C. After 12h, a white / pale yellow precipitate formed. The mixture was allowed to cool to rt. TLC (5% methanol in methylene chloride) indicates complete conversion of starting material. The mixture was cooled to 0°C with an ice-bath and then slowly quenched with acetic acid (approximately 1 mL). The clear solution was warmed to rt and 5 then partitioned between ethyl acetate (30 mL) and brine (3 mL). The organic phase was concentrated and then purified by chromatography over silica gel (19 mm x 180 mm) using a mobile phase of 5% methanol in methylene chloride. Example 45. 480 10 A solution of 2’-deoxy-2’-fluorouridine (6g, 24.37 mmol) and 4,4'-(chloro(phenyl) methylene)-bis(methoxybenzene) (9.91 g, 29.2 mmol) in pyridine (48.7 ml) was stirred at rt for 16 hours. The mixture was treated with MeOH (20 mL), concentrated to dryness and was partitioned between water (50 mL) and EtOAc (250 mL). The aqueous phase was back extracted with EtOAc (50 mL) and the combined organic layers were washed with water 15   (50 mL) and dried over Na2SO4. The solution was concentrated to give 2’-deoxy-2’- fluoro-5’-(4’,4’-dimethoxytrityl)uridine (14g, quant.) which was used without further purification. TBDMSO F 481 20           To a solution of 2’-deoxy-2’-fluoro-5’-(4’,4’-dimethoxytrityl)uridine (13.37 g, 24.37 mmol) in methylene chloride (30 mL) were added 1H-imidazole (2.48 g, 36.6 mmol) and tert-butylchlorodimethylsilane (5.51 g, 36.6 mmol). The reaction was stirred for 16 hours and then was diluted with EtOAc (250 mL). The mixture was washed with saturated 2020418425   14 Sep 2026 aqueous sodium bicarbonate (50 mL) and brine (50 mL), dried over Na2SO4, filtered and concentrated to give 2’-Deoxy-2’-fluoro-3’-O-(tert-butyldimethylsilyl)-5’-(4’,4’-dimethoxytrityl)uridine (16 g, 99%). This product was used in the next step without further purification. 0 TBDMSO F 482 To a solution of 2’-deoxy-2’-fluoro-3’-O-(tert-butyldimethylsilyl)-5’-(4’,4’-dimethoxytrityl) uridine (13.37 g, 20.17 mmol) in DCM (10 mL) were added acetic acid (20.19 ml, 353 mmol) and water (5 ml). The reaction was stirred at room temperature for 20 hours, diluted with EtOAc (250 mL), washed with saturated aqueous NaHCO3 (2 x 100 10 mL) and brine (100 mL), dried (sodium sulfate), filtered and concentrated. The residue was purified by column chromatography over silica gel (1% MeOH in DCM, 2% MeOH in DCM) to afford 2’-deoxy-2’-fluoro-3’-O-(tert-butyldimethylsilyl)uridine (6.73 g, 93 % yield) as a yellow solid. 0 TBDMSO F 483 15 To a suspension of PDC (14.05 g, 37.3 mmol) in anhydrous DCM (37.3 ml) / DMF (9.34 ml) were added sequentially 2-methylpropan-2-ol (35.7 ml, 373 mmol), 2’-deoxy-2’-fluoro-3’-O-(tert-butyldimethylsilyl)uridine (6.73 g, 18.67 mmol) and acetic anhydride (17.62 ml, 187 mmol). After 18 hours, the mixture was quenched with absolute EtOH (5 20 mL), diluted with EtOAc (15 mL), dried over Na2SO4, filtered through Celite and concentrated. The crude residue was purified by column chromatography over silica gel using 1% MeOH in DCM to give (2S,3R,4R,5R)-tert-butyl 3-((tert-butyldimethylsilyl)oxy)- 2020418425   14 Sep 2026 5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluorotetrahydrofuran-2-carboxylate (6.72 g, 83%) TBDMSO F 484 5           To a solution of (2S,3R,4R,5R)-tert-butyl 3-((tert-butyldimethylsilyl)oxy)-5-(2,4- dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluorotetrahydrofuran-2-carboxylate (3.29 g, 7.64 mmol) was added sodium borodeuteride (1.422 g, 30.6 mmol) in one portion. The reaction was stirred at 80°C for 20 hours in a sealed tube. The mixture was cooled to room temperture and then quenched with acetic acid (6.99 ml, 122 mmol). The mixture was 10 neutralized with saturated aqueous sodium bicarbonate and extracted with EtOAc. After concentrating, the resulting residue was purified by column chromatography over silica gel (Rf = 0.5 hexane EtOAc 1:1) to give [5’-2H2]-2’-deoxy-2’-fluoro-3’-O-(tert-butyldimethylsilyl)uridine (1g, 36%). 0 f|    NH d oh   T HO-V p HO F 15                                        485 To a solution of [5’-2H2]-2’-deoxy-2’-fluoro-3’-O-(tert-butyldimethylsilyl)uridine (200mg, 0.552 mmol) in MeOH (6 mL) was added Dowex 50WX8 (H+ form) (6 g) in one portion. The mixture was stirred for 72 h, filtered and concentrated to give [5’-2H2]-2’-deoxy-2’-fluorouridine (150 mg, quant.). 20 2020418425   14 Sep 2026 0 rf^NH o D d|I 7 HO-P-oA' T'N'X) OH Q HO F 486 To a solution of phosphoryl trichloride (1.69 mL, 18.13 mmol) in trimethyl phosphate (2 mL) at 5°C, under N2, was added [5’-2H2]-2’-deoxy-2’-fluorouridine (100 mg, 0.403 mmol) in small portions. The solution was stirred vigorously for 2h at 5°C and then 5 was quenched by dropwise addition of DI water (8 mL). The reaction mixture was extracted with chloroform (2 x 10 mL), and the aqueous phase was treated with concentrated with NH4OH to pH 6.5, while keeping the solution below 30 °C. The aqueous layer was extracted once more with chloroform (10 mL) and then concentrated to dryness. The residue was suspended in MeOH (15 mL), filtered, and concentrated. The resulting solid was 10 purified by column chromatography over silica gel (7:2:1 iPrOH / conc. NH4OH, H2O, Rf = 0.2). The product was further purified by column chromatography over DEAE using methanol followed by a mobile phase gradient from 0 to 100 mM aqueous ammonium bicarbonate. Fractions were concentrated to dryness, dissolved in water and lyophilized to give [5’-2H2]-2’-deoxy-2’-fluorouridine-5’-monophosphate (27 mg, 20%) as an amorphous 15 white solid. ,CN O C16H33 487 A suspension of 3-hexadecyloxypropan-1-ol (2.02 g, 6.72 mmol) and DIPEA (4.7 20 mL, 26.9mmol) in anhydrous methylene chloride (45 mL) was treated dropwise over a 10 minute period with 3-((chloro(diisopropylamino)phosphino)oxy)propanenitrile (3 mL, 13.45 mmol). After 18hours at room temperature, the mixture was quenched with saturated sodium bicarbonate solution (15 mL) and extracted with ethyl acetate (2 x 100 mL). Combined organic phases were concentrated to dryness, and the resulting crude residue 2020418425   14 Sep 2026 purified by chromatography over silica gel (25 mm x 140 mm) using a solvent gradient from 10 to 20% ethyl acetate in hexanes to give hexadecyloxypropyl-(2-cyanoethyl) diisopropylphosphoramidite (2.1 g, 65%) as a white solid. 1H NMR (400 MHz, Chloroform-d) S 3.89 - 3.54 (m, 6H), 3.49 (t, J = 6.3 Hz, 2H), 5   3.39 (t, J = 6.7 Hz, 2H), 2.64 (t, J = 6.6 Hz, 2H), 1.87 (p, J = 6.3 Hz, 2H), 1.57 (p, J = 6.3 Hz, 2H), 1.25 (s, 26H), 1.18 (dd, J = 6.8, 3.5 Hz, 12H), 0.87 (t, J = 6.6 Hz, 3H). 31P NMR (162 MHz, Chloroform-d) S 147.40. O 488 10 A solution of [5’-2H2]-2’-deoxy-2-fluoro-3’-O-(tert-butyldimethylsilyl)uridine (600 mg, 1.65 mmol) and hexadecyloxypropyl-(2-cyanoethyl) diisopropylphosphoramidite (1.65 g, 3.31 mmol) in anhydrous THF (22 mL) was treated dropwise with 1-H-tetrazole (14.7 15 mL of 0.45 M solution in acetonitrile, 6.62 mmol). After 16hours at room temperature, the mixture was treated dropwise with tert-butyl hydroperoxide (1.5 mL of a 5.5 M solution in nonane, 8.28 mmol) and stirred at room temperature for 1hour and then quenched with 1.0 M aqueous solution of sodium thiosulfate (40 mL). After 30 min, the mixture was extracted with ethyl acetate (2 x 80mL). Combined organic phases were washed with brine (40 mL) 20 and dried over sodium sulfate, filtered, and concentrated. The resulting residue was purified by column chromatography over silica gel (40g) with a mobile phase gradient from 1% to 5% methanol in methylene chloride to give the cyanoethyl phosphate intermediate which without further purification was dissolved in methanol (30 mL) and treated with concentrated ammonium hydroxide (5 mL, 128 mmol). After 4hours at room temperature, 25 the mixture was concentrated to dryness. The resulting residue was purified by column chromatography over silica gel using a CombiFlash instrument equipped with a 40 g silica cartridge eluting with a solvent gradient from 5 to 25% methanol in methylene chloride to give [5’-2H2]-2’-deoxy-2’-fluoro-3’-O-(tert-butyldimethylsilyl)-5’-((hexadecyloxypropyl) phospho)uridine (1 g, 82%) as a white foam. 2020418425   14 Sep 2026 ^16^33 O 0 0 zPx 1 0 I 0 OH HO F 489 A solution of [5’-2H2]-2’-deoxy-2’-fluoro-3’-O-(tert-butyldimethylsilyl)-5’-((hexadecyloxypropyl) phospho)uridine (1 g, 1.38 mmol) in THF (15 mL) was treated with acetic acid (0.5 g, 8.28 mmol) and triethylammonium fluoride (1.2 g, 5.52 mmol). After 5 36hours, the mixture was concentrated and the resulting residue eluted through a short column (11 mm x 90 mm) of Dowex 50WX8 (H+ form) using methanol (120 mL) as the mobile phase. The product was further purified by column chromatography over silica gel (24 g) using a mobile phase gradient from 0 to 25% methanol in methylene chloride with 2.5% (v / v) ammonium hydroxide. Pure fractions were pooled and concentrated. The 10 resulting solid was co-evaporated with methylene chloride (2 x 75 mL) and then dried under high vacuum for 19hours to give [5’-2H2]-2’-deoxy-2’-fluoro-5’-((hexadecyloxypropyl)phospho)- uridine (455 mg, 54%) as a white solid. 1H NMR (400 MHz, Chloroform-d4 / Methanol-d4) 5 7.75 (d, J = 8.1 Hz, 1H), 5.95 (dd, J = 17.9, 1.6 Hz, 1H), 5.70 (d, J = 8.1 Hz, 1H), 5.01 (ddd, J = 52.8, 4.6, 1.7 Hz, 1H), 15   4.30 (ddd, J = 20.7, 8.1, 4.5 Hz, 1H), 4.16 - 4.07 (m, 3H), 3.51 (t, J = 6.2 Hz, 2H), 3.41 (t, J = 6.7 Hz, 2H), 1.92 (p, J = 7.6 Hz, 2H), 1.53 (p, J = 7.6 Hz, 2H), 1.25 (s, 26H), 0.87 (d, J = 7.6 Hz, 3H). 13C NMR (101 MHz, Chloroform-d4 / Methanol-d4) 5 164.31, 150.24, 140.33, 102.11, 94.19, 92.32, 88.88, 88.53, 80.83, 80.75, 71.18, 67.62, 67.45, 66.50, 66.40, 64.83, 20   64.77, 63.81, 31.81, 30.37, 30.29, 29.59, 29.57, 29.54, 29.51, 29.47, 29.41, 29.25, 26.00, 25.96, 22.57, 13.96. 31P NMR (162 MHz, Chloroform-d4 / Methanol-d4) 5 -0.87. HRMS C28H49D2FN2O9P [M+H+]; calculated: 611.34359, found: 611.34363. Example 46. 25 Assay Protocols (1) Screening Assays for DENV, JEV, POWV, WNV, YFV, PTV, RVFV, CHIKV, EEEV, VEEV, WEEV, TCRV, PCV, JUNV, MPRLV Primary cytopathic effect (CPE) reduction assay. Four-concentration CPE inhibition assays are performed. Confluent or near-confluent cell culture monolayers in 96-well 2020418425   14 Sep 2026 disposable microplates are prepared. Cells are maintained in MEM or DMEM supplemented with FBS as required for each cell line. For antiviral assays the same medium is used but with FBS reduced to 2% or less and supplemented with 50 gg / ml gentamicin. The test compound is prepared at four logio final concentrations, usually 0.1, 1.0, 10, and 100 ugml 5 or gM. The virus control and cell control wells are on every microplate. In parallel, a known active drug is tested as a positive control drug using the same method as is applied for test compounds. The positive control is tested with each test run. The assay is set up by first removing growth media from the 96-well plates of cells. Then the test compound is applied in 0.1 ml volume to wells at 2X concentration. Virus, normally at <100 50% cell culture 10 infectious doses (CCID50) in 0.1 ml volume, is placed in those wells designated for virus infection. Medium devoid of virus is placed in toxicity control wells and cell control wells. Virus control wells are treated similarly with virus. Plates are incubated at 37oC with 5% CO2 until maximum CPE is observed in virus control wells. The plates are then stained with 0.011% neutral red for approximately two hours at 37oC in a 5% CO2 incubator. The neutral 15 red medium is removed by complete aspiration, and the cells may be rinsed 1X with phosphate buffered solution (PBS) to remove residual dye. The PBS is completely removed and the incorporated neutral red is eluted with 50% Sorensen’s citrate buffer / 50% ethanol (pH 4.2) for at least 30 minutes. Neutral red dye penetrates into living cells, thus, the more intense the red color, the larger the number of viable cells present in the wells. The dye 20 content in each well is quantified using a 96-well spectrophotometer at 540 nm wavelength. The dye content in each set of wells is converted to a percentage of dye present in untreated control wells using a Microsoft Excel computer-based spreadsheet. The 50% effective (EC50, virus-inhibitory) concentrations and 50% cytotoxic (CC50, cell-inhibitory) concentrations are then calculated by linear regression analysis. The quotient of CC50 25   divided by EC50 gives the selectivity index (SI) value. Secondary CPE / Virus yield reduction (VYR) assay. This assay involves similar methodology to what is described in the previous paragraphs using 96-well microplates of cells. The differences are noted in this section. Eight half-log10 concentrations of inhibitor are tested for antiviral activity and cytotoxicity. After sufficient virus replication occurs, a 30 sample of supernatant is taken from each infected well (three replicate wells are pooled) and held for the VYR portion of this test, if needed. Alternately, a separate plate may be prepared and the plate may be frozen for the VYR assay. After maximum CPE is observed, the viable plates are stained with neutral red dye. The incorporated dye content is quantified as described above. The data generated from this portion of the test are neutral red EC50, 2020418425   14 Sep 2026 CC50, and SI values. Compounds observed to be active above are further evaluated by VYR assay. The VYR test is a direct determination of how much the test compound inhibits virus replication. Virus that was replicated in the presence of test compound is titrated and compared to virus from untreated, infected controls. Titration of pooled viral samples 5 (collected as described above) is performed by endpoint dilution. This is accomplished by titrating log10 dilutions of virus using 3 or 4 microwells per dilution on fresh monolayers of cells by endpoint dilution. Wells are scored for presence or absence of virus after distinct CPE (measured by neutral red uptake) is observed. Plotting the log10 of the inhibitor concentration versus log10 of virus produced at each concentration allows calculation of the 10   90% (one log10) effective concentration by linear regression. Dividing EC90 by the CC50 obtained in part 1 of the assay gives the SI value for this test. Example 47. (2) Screening Assays for Lassa fever virus (LASV) Primary Lassa fever virus assay. Confluent or near-confluent cell culture 15 monolayers in 12-well disposable cell culture plates are prepared. Cells are maintained in DMEM supplemented with 10% FBS. For antiviral assays the same medium is used but with FBS reduced to 2% or less and supplemented with 1% penicillin / streptomycin. The test compound is prepared at four logio final concentrations, usually 0.1, 1.0, 10, and 100 ugml or gM. The virus control and cell control will be run in parallel with each tested compound. 20 Further, a known active drug is tested as a positive control drug using the same experimental set-up as described for the virus and cell control. The positive control is tested with each test run. The assay is set up by first removing growth media from the 12-well plates of cells, and infecting cells with 0.01 MOI of LASV strain Josiah. Cells will be incubated for 90 min: 500 gl inoculum / M12 well, at 37°C, 5% CO2 with constant gentle 25 rocking. The inoculums will be removed and cells will be washed 2X with medium. Then the test compound is applied in 1 ml of total volume of media. Tissue culture supernatant (TCS) will be collected at appropriate time points. TCS will then be used to determine the compounds inhibitory effect on virus replication. Virus that was replicated in the presence of test compound is titrated and compared to virus from untreated, infected controls. For 30 titration of TCS, serial ten-fold dilutions will be prepared and used to infect fresh monolayers of cells. Cells will be overlaid with 1% agarose mixed 1:1 with 2X MEM supplemented with 10%FBS and 1%penecillin, and the number of plaques determined. Plotting the log10 of the inhibitor concentration versus log10 of virus produced at each 2020418425   14 Sep 2026 concentration allows calculation of the 90% (one log10) effective concentration by linear regression. Secondary Lassa fever virus assay. The secondary assay involves similar methodology to what is described in the previous paragraphs using 12-well plates of cells. 5 The differences are noted in this section. Cells are being infected as described above but this time overlaid with 1% agarose diluted 1:1 with 2X MEM and supplemented with 2% FBS and 1% penicillin / streptomycin and supplemented with the corresponding drug concentration. Cells will be incubated at 37oC with 5% CO2 for 6 days. The overlay is then removed and plates stained with 0.05% crystal violet in 10% buffered formalin for 10 approximately twenty minutes at room temperature. The plates are then washed, dried and the number of plaques counted. The number of plaques is in each set of compound dilution is converted to a percentage relative to the untreated virus control. The 50% effective (EC50, virus-inhibitory) concentrations are then calculated by linear regression analysis. Example 48. 15   (3) Screening Assays for Ebola virus (EBOV) and Nipah virus (NIV) Primary Ebola / Nipah virus assay. Four-concentration plaque reduction assays are performed. Confluent or near-confluent cell culture monolayers in 12-well disposable cell culture plates are prepared. Cells are maintained in DMEM supplemented with 10% FBS. For antiviral assays the same medium is used but with FBS reduced to 2% or less and 20 supplemented with 1% penicillin / streptomycin. The test compound is prepared at four log10 final concentrations, usually 0.1, 1.0, 10, and 100 gg / ml or uM. The virus control and cell control will be run in parallel with each tested compound. Further, a known active drug is tested as a positive control drug using the same experimental set-up as described for the virus and cell control. The positive control is tested with each test run. The assay is set up 25 by first removing growth media from the 12-well plates of cells. Then the test compound is applied in 0.1 ml volume to wells at 2X concentration. Virus, normally at approximately 200 plaque-forming units in 0.1 ml volume, is placed in those wells designated for virus infection. Medium devoid of virus is placed in toxicity control wells and cell control wells. Virus control wells are treated similarly with virus. Plates are incubated at 37°C with 5% 30   CO2 for one hour. Virus-compound inoculums will be removed, cells washed and overlaid with 1.6% tragacanth diluted 1:1 with 2X MEM and supplemented with 2% FBS and 1% penicillin / streptomycin and supplemented with the corresponding drug concentration. Cells will be incubated at 37°C with 5% CO2 for 10 days. The overlay is then removed and plates stained with 0.05% crystal violet in 10% buffered formalin for approximately twenty 2020418425   14 Sep 2026 minutes at room temperature. The plates are then washed, dried and the number of plaques counted. The number of plaques is in each set of compound dilution is converted to a percentage relative to the untreated virus control. The 50% effective (EC50, virus-inhibitory) concentrations are then calculated by linear regression analysis. 5         Secondary Ebola / NIpah virus assay with VYR component. The secondary assay involves similar methodology to what is described in the previous paragraphs using 12-well plates of cells. The differences are noted in this section. Eight half-log10 concentrations of inhibitor are tested for antiviral activity. One positive control drug is tested per batch of compounds evaluated. For this assay, cells are infected with virus. Cells are being infected 10 as described above but this time incubated with DMEM supplemented with 2% FBS and 1% penicillin / streptomycin and supplemented with the corresponding drug concentration. Cells will be incubated for 10 days at 37°C with 5% CO2, daily observed under microscope for the number of green fluorescent cells. Aliquots of supernatant from infected cells will be taken daily and the three replicate wells are pooled. The pooled supernatants are then used 15 to determine the compounds inhibitory effect on virus replication. Virus that was replicated in the presence of test compound is titrated and compared to virus from untreated, infected controls. For titration of pooled viral samples, serial ten-fold dilutions will be prepared and used to infect fresh monolayers of cells. Cells are overlaid with tragacanth and the number of plaques determined. Plotting the log10 of the inhibitor concentration versus log10 of virus 20 produced at each concentration allows calculation of the 90% (one log10) effective concentration by linear regression. Example 49. Anti-Dengue Virus Cytoprotection Assay: Cell Preparation -BHK21 cells (Syrian golden hamster kidney cells, ATCC catalog # 25   CCL-I 0) , Vero cells (African green monkey kidney cells, ATCC catalog# CCL-81), or Huh-7 cells (human hepatocyte carcinoma) were passaged in DMEM supplemented with 10% FBS, 2 mM L-glutamine,100 U / mL penicillin, and 100 pg / mL streptomycin in T-75 flasks prior to use in the antiviral assay. On the day preceding the assay, the cells were split 1:2 to assure they were in an exponential growth phase at the time of infection. Total cell 30 and viability quantification was performed using a hemocytometer and Trypan Blue dye exclusion. Cell viability was greater than 95% for the cells to be utilized in the assay. The cells were resuspended at 3 x 103 (5 x 105 for Vero cells and Huh-7 cells) cells per well in tissue culture medium and added to flat bottom microtiter plates in a volume of 100 pL. The 2020418425   14 Sep 2026 plates were incubated at 37°C / 5%C02 overnight to allow for cell adherence. Monolayers were observed to be approximately 70% confluent. Virus Preparation-The Dengue virus type 2 New Guinea C strain was obtained from ATCC (catalog# VR-1584) and was grown in LLC-MK2 (Rhesus monkey kidney cells; 5 catalog #CCL-7.1) cells for the production of stock virus pools. An aliquot of virus pretitered in BHK21 cells was removed from the freezer (-80°C) and allowed to thaw slowly to room temperature in a biological safety cabinet. Virus was resuspended and diluted into assay medium (DMEM supplemented with 2% heat-inactivated FBS, 2 mM L-glutamine, 100 U / mL penicillin, and 100 ^gmL streptomycin) such that the amount of 10 virus added to each well in a volume of 100 pL was the amount determined to yield 85 to 95% cell killing at 6 days post-infection. Plate Format-Each plate contains cell control wells (cells only), virus control wells (cells plus virus), triplicate drug toxicity wells per compound (cells plus drug only), as well as triplicate experimental wells (drug plus cells plus virus). 15          Efficacy and Toxicity XTT-Following incubation at 37°C in a 5% C02 incubator, the test plates were stained with the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide). XTT-tetrazolium was metabolized by the mitochondrial enzymes of metabolically active cells to a soluble formazan product, allowing rapid quantitative analysis of the inhibition of virus-induced 20 cell killing by antiviral test substances. XTT solution was prepared daily as a stock of 1 mg / mL in RPMI 1640. Phenazine methosulfate (PMS) solution was prepared at 0.15mg / mL in PBS and stored in the dark at -20°C. XTT / PMS stock was prepared immediately before use by adding 40 pL of PMS per ml of XTT solution. Fifty microliters ofXTT / PMS was added to each well of the plate and the plate was reincubated for 4 hours at 37°C. Plates 25 were sealed with adhesive plate sealers and shaken gently or inverted several times to mix the soluble formazan product and the plate was read spectrophotometrically at 450 / 650 nm with a Molecular Devices Vmax plate reader. Data Analysis -Raw data was collected from the Softmax Pro 4.6 software and imported into a Microsoft Excel spreadsheet for analysis. The percent reduction in viral 30 cytopathic effect compared to the untreated virus controls was calculated for each compound. The percent cell control value was calculated for each compound comparing the drug treated uninfected cells to the uninfected cells in medium alone. Example 50. Anti-RSV Cytoprotection Assay: 2020418425   14 Sep 2026 Cell Preparation-HEp2 cells (human epithelial cells, A TCC catalog# CCL-23) were passaged in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U / mL penicillin, 100 pg / mL streptomycin 1 mM sodium pyruvate, and 0.1 mM NEAA, T-75 flasks prior to use in the antiviral assay. On the day preceding the assay, the cells were split 1:2 to assure 5 they were in an exponential growth phase at the time of infection. Total cell and viability quantification was performed using a hemocytometer and Trypan Blue dye exclusion. Cell viability was greater than 95% for the cells to be utilized in the assay. The cells were resuspended at 1 x 104 cells per well in tissue culture medium and added to flat bottom microtiter plates in a volume of 100 pL. The plates were incubated at 37°C / 5% CO2 10 overnight to allow for cell adherence. Virus Preparation -The RSV strain Long and RSV strain 9320 were obtained from ATCC (catalog# VR-26 and catalog #VR-955, respectively) and were grown in HEp2 cells for the production of stock virus pools. A pretitered aliquot of virus was removed from the freezer (-80°C) and allowed to thaw slowly to room temperature in a biological safety cabinet. Virus was resuspended and diluted into assay 15 medium (DMEMsupplemented with 2% heat-inactivated FBS, 2 mM L-glutamine, 100 U / mL penicillin, 100 pg / mL streptomycin, 1 mM sodium pyruvate, and 0.1 mM NEAA) such that the amount of virus added to each well in a volume of 100 pL was the amount determined to yield 85 to 95% cell killing at 6 days post-infection. Efficacy and Toxicity XTT-Plates were stained and analyzed as previously described for the Dengue 20 cytoprotection assay. Example 51. Anti-Influenza Virus Cytoprotection Assay: Cell Preparation-MOCK cells (canine kidney cells, ATCC catalog# CCL-34) were passaged in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U / mL penicillin, 25   100 pg / mL streptomycin 1 mM sodium pyruvate, and 0.1 mM NEAA, T-75 flasks prior to use in the antiviral assay. On the day preceding the assay, the cells were split 1:2 to assure they were in an exponential growth phase at the time of infection. Total cell and viability quantification was performed using a hemocytometer and Trypan Blue dye exclusion. Cell viability was greater than 95% for the cells to be utilized in the assay. The cells were 30 resuspended at 1 x 104 cells per well in tissue culture medium and added to flat bottom microtiter plates in a volume of 100 pL. The plates were incubated at 37°C / 5% C02 overnight to allow for cell adherence. Virus Preparation-The influenza A / PR / 8 / 34 (A TCC #VR-95), A / CA / 05 / 09 (CDC),A / NY / 18 / 09 (CDC) and A / NWS / 33 (ATCC #VR-219) strains were obtained from 2020418425   14 Sep 2026 ATCC or from the Center of Disease Control and were grown in MDCK cells for the production of stock virus pools. A pretitered aliquot of virus was removed from the freezer (-80°C)and allowed to thaw slowly to room temperature in a biological safety cabinet. Virus was resuspended and diluted into assay medium (DMEM supplemented with 0.5%BSA, 2 5 mM L-glutamine, 100 U / mL penicillin, 100 iigmL streptomycin, 1 mM sodium pyruvate, 0.1 mM NEAA, and 1 pg / ml TPCK-treated trypsin) such that the amount of virus added to each well in a volume of 100 pL was the amount determined to yield 85 to 95% cell killing at 4 days post-infection. Efficacy and Toxicity XTT-Plates were stained and analyzed as previously described for the Dengue cytoprotection assay. 10 Example 52. Anti-Hepatitis C Virus Assay: Cell Culture -The reporter cell line Huh-luc / neo-ET was obtained from Dr. Ralf Bartenschlager (Department of Molecular Virology, Hygiene Institute, University of Heidelberg, Germany) by ImQuest BioSciences through a specific licensing agreement. 15 This cell line harbors the persistently replicating I389luc-ubi-neo / NS3-3’ / ET replicon containing the firefly luciferase gene-ubiquitin-neomycin phosphotransferase fusion protein and EMCV IRES driven NS3-5B HCV coding sequences containing the ET tissue culture adaptive mutations (E1202G, Tl2081, and K1846T). A stock culture of the Huh-luc / neo-ET was expanded by culture in DMEM supplemented with I 0% FCS, 2mM glutamine, 20 penicillin (100 pU / mL) / streptomycin (100 pg / mL) and I X nonessential amino acids plus 1 mg / mL G418. The cells were split 1:4 and cultured for two passages in the same media plus 250 pg / mL G418. The cells were treated with trypsin and enumerated by staining with trypan blue and seeded into 96-well tissue culture plates at a cell culture density 7.5 x 103 cells per well and incubated at 37°C 5% CO2 for 24 hours. Following the 24 hour incubation, 25 media was removed and replaced with the same media minus theG418 plus the test compounds in triplicate. Six wells in each plate received media alone as a no-treatment control. The cells were incubated an additional 72 hours at 37°C 5%C02 then anti-HCV activity was measured by luciferase endpoint. Duplicate plates were treated and incubated in parallel for assessment of cellular toxicity by XTT staining. 30          Cellular Viability- The cell culture monolayers from treated cells were stained with the tetrazolium dye XTT to evaluate the cellular viability of the Huh-luc / neo-ET reporter cell line in the presence of the compounds. Measurement of Virus Replication-HCV replication from the replicon assay system was measured by luciferase activity using the britelite plus luminescence reporter gene kit 2020418425   14 Sep 2026 according to the manufacturer's instructions (Perkin Elmer, Shelton, CT). Briefly, one vial of britelite plus lyophilized substrate was solubilized in 10 mL of britelite reconstitution buffer and mixed gently by inversion. After a 5 minute incubation at room temperature, the britelite plus reagent was added to the 96 well plates at 100 pL per well. The plates were 5 sealed with adhesive film and incubated at room temperature for approximately 10 minutes to lyse the cells. The well contents were transferred to a white 96-well plate and luminescence was measured within 15 minutes using the Wallac 1450Microbeta Trilux liquid scintillation counter. The data were imported into a customized Microsoft Excel 2007 spreadsheet for determination of the 50% virus inhibition concentration (EC50). 10 Example 53. Anti-Parainfluenza-3 Cytoprotection Assay: Cell Preparation- HEp2 cells (human epithelial cells, ATCC catalog# CCL-23) were passaged in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U / mL penicillin, 100 pg / mL streptomycin 1 mM sodium pyruvate, and 0.1 mM NEAA, T-75 flasks prior to 15 use in the antiviral assay. On the day preceding the assay, the cells were split 1:2 to assure they were in an exponential growth phase at the time of infection. Total cell and viability quantification was performed using a hemocytometer and Trypan Blue dye exclusion. Cell viability was greater than 95% for the cells to be utilized in the assay. The cells were resuspended at 1 x 104cells per well in tissue culture medium and added to flat bottom 20 microtiter plates in a volume of 100 pL. The plates were incubated at 37°C / 5% C02 overnight to allow for cell adherence. Virus Preparation - The Parainfluenza virus type 3 SF4 strain was obtained from ATCC (catalog# VR-281) and was grown in HEp2 cells for the production of stock virus pools. A pretitered aliquot of virus was removed from the freezer (-80°C) and allowed to 25 thaw slowly to room temperature in a biological safety cabinet. Virus was resuspended and diluted into assay medium (DMEM supplemented with 2% heat-inactivated FBS, 2 mM L-glutamine, 100 U / mL penicillin, and 100 pg / mL streptomycin) such that the amount of virus added to each well in a volume of 100 pL was the amount determined to yield 85 to 95% cell killing at 6 days post-infection. 30           Plate Format - Each plate contains cell control wells (cells only), virus control wells (cells plus virus), triplicate drug toxicity wells per compound (cells plus drug only), as well a triplicate experimental wells (drug plus cells plus virus). Efficacy and Toxicity XTT-Following incubation at 37°C in a 5% C02 incubator, the test plates were stained with the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5- 2020418425   14 Sep 2026 [(phenylamino)carbonyl]-2H-tetrazol hydroxide). XTT-tetrazolium was metabolized by the mitochondrial enzymes of metabolically active cells to a soluble formazan product, allowing rapid quantitative analysis of the inhibition of virus-induced cell killing by antiviral test substances. XTT solution was prepared daily as a stock of 1mg / mL in 5   RPMI1640. Phenazine methosulfate (PMS) solution was prepared at 0.15mg / mL in PBS and stored in the dark at - 20°C. XTT / PMS stock was prepared immediately before use by adding 40 pL of PMS per ml of XTT solution. Fifty microliters of XTT / PMS was added to each well of the plate and the plate was reincubated for 4 hours at 3 7°C. Plates were sealed with adhesive plate sealers and shaken gently or inverted several times to mix the soluble 10 fom1azan product and the plate was read spectrophotometrically at 450 / 650 nm with a Molecular Devices Vmax plate reader. Data Analysis - Raw data was collected from the Softmax Pro 4.6 software and imported into a Microsoft Excel spreadsheet for analysis. The percent reduction in viral cytopathic effect compared to the untreated virus controls was calculated for each 15 compound. The percent cell control value was calculated for each compound comparing the drug treated uninfected cells to the uninfected cells in medium alone. Example 54. Influenza Polymerase Inhibition Assay: Virus Preparation - Purified influenza virus A / PR / 8 / 34 (1 ml) was obtained from 20 Advanced Biotechnologies, Inc. (Columbia, MD), thawed and dispensed into five aliquots for storage at -80°C until use. On the day of assay set up, 20 pL of 2.5% Triton N-101 was added to 180 pL of purified virus. The disrupted virus was diluted 1:2 in a solution containing 0.25% Triton and PBS. Disruption provided the source of influenza ribonucleoprotein (RNP) containing the influenza RNA-dependent RNA polymerase and 25 template RNA. Samples were stored on ice until use in the assay. Polymerase reaction - Each 50 pL polymerase reaction contained the following: 5 pL of the disrupted RNP, 100 mM Tris-HCl (pH 8.0), 100 mM KCl, 5 mM MgCl2. 1 mM dithiothreitol, 0.25% Triton N-101, 5 pCi of [a-32P] GTP, 100 pM ATP, 50 pM each (CTP, UTP), 1 pM GTP, and 200 pM adenyl (3'-5') guanosine. For testing the inhibitor, the 30 reactions contained the inhibitor and the same was done for reactions containing the positive control (2'-Deoxy-2'-fluoroguanosine-5'-triphosphate). Other controls included RNP +reaction mixture, and RNP + I% DMSO. The reaction mixture without the ApG primer and NTPs was incubated at 30°C for 20 minutes. Once the ApG and NTPs were added to the reaction mixture, the samples were incubated at 30°C for 1 hour then 2020418425   14 Sep 2026 immediately followed by the transfer of the reaction onto glass-fiber filter plates and subsequent precipitation with 10% trichloroacetic acid (TCA ). The plate was then washed five times with 5% TCA followed by one wash with 95% ethanol. Once the filter had dried, incorporation of [a-32P] GTP was measured using a liquid scintillation counter (Micro 5 beta). Plate Format - Each test plate contained triplicate samples of the three compounds (6 concentrations) in addition to triplicate samples of RNP + reaction mixture (RNP alone), RNP + 1% DMSO, and reaction mixture alone (no RNP). Data Analysis - Raw data was collected from the Micro Beta scintillation counter. 10 The incorporation of radioactive GTP directly correlates with the levels of polymerase activity. The "percent inhibition values" were obtained by dividing the mean value of each test compound by the RNP + 1% DMSO control. The mean obtained at each concentration of 2DFGTP was compared to the RNP + reaction control. The data was then imported into Microsoft Excel spreadsheet to calculate the IC50 values by linear regression analysis. 15 Example 55. HCV Polymerase Inhibition Assay: Activity of compounds for inhibition of HCV polymerase was evaluated using methods previously described (Lam eta!. 2010. Antimicrobial Agents and Chemotherapy 54(8):3187-3196). HCV NS5B polymerase assays were performed in 20 pL volumes in 96 20 well reaction plates. Each reaction contained 40 ng / pL purified recombinant NS5BA22 genotype-1b polymerase, 20 ng / pL of HCV genotype-1b complimentary IRES template, 1 pM of each of the four natural ribonucleotides, 1 U / mL Optizyme RNAse inhibitor (Promega, Madison, WI), 1 mM MgCl2, 0.75 mM MnCl2, and 2 mM dithiothreitol (DTT) in 50 mM HEPES buffer (pH 7.5). Reaction mixtures were assembled on ice in two steps. Step 25   1 consisted of combining all reaction components except the natural nucleotides and labeled UTP in a polymerase reaction mixture. Ten microliters (10 pL) of the polymerase mixture was dispensed into individual wells of the 96 well reaction plate on ice. Polymerase reaction mixtures without NS5B polymerase were included as no enzyme controls. Serial half-logarithmic dilutions of test and control compounds, 2'-O-Methyl-CTP and 2'-O-Methyl- 30 GTP (Trilink, San Diego, CA), were prepared in water and 5 pL of the serial diluted compounds or water alone (no compound control) were added to the wells containing the polymerase mixture. Five microliters of nucleotide mix (natural nucleotides and labeled UTP) was then added to the reaction plate wells and the plate was incubated at 27°C for 30 minutes. The reactions were quenched with the addition of 80 pL stop solution (12.5 mM 2020418425   14 Sep 2026 EDTA, 2.25 M NaCl, and 225 mM sodium citrate) and the RNA products were applied to a Hybond-N+ membrane (GE Healthcare, Piscataway, N.J) under vacuum pressure using a dot blot apparatus. The membrane was removed from the dot blot apparatus and washed four times with 4X SSC (0.6 M NaCl, and 60 mM sodium citrate), and then rinsed one time 5 with water and once with 100% ethanol. The membrane was air dried and exposed to a phosphoimaging screen and the image captured using a Typhoon 8600 Phospho imager. Following capture of the image, the membrane was placed into a Micro beta cassette along with scintillation fluid and the CPM in each reaction was counted on a Micro beta 1450. CPM data were imported into a custom Excel spreadsheet for determination of compound 10    IC50s. Example 56. NS5B RNA-dependent RNA polymerase reaction conditions Compounds were assayed for inhibition of NS5B-521 from HCV GT-1b Con-1. Reactions included purified recombinant enzyme, 1 u / pL negative-strand HCV IRES RNA 15 template, and 1pM NTP substrates including either [32P]-CTP or [32P]-UTP. Assay plates were incubated at 27°C for 1 hour before quench. [32P] incorporation into macromolecular product was assessed by filter binding. Example 57. Human DNA Polymerase Inhibition Assay: 20          The human DNA polymerase alpha (catalog# 1075), beta (catalog# 1077), and gamma (catalog# 1076) were purchased from CHIMERx (Madison, WI). Inhibition of beta and gamma DNA polymerase activity was assayed in microtiter plates in a 50 uL reaction mixture containing 50 mM Tris-HCl (pH 8.7), KCl (10 mM for beta and 100mM for gamma), 10 mM MgCl2, 0.4 mg / mL BSA, 1 mM DTT, 15% glycerol, 0.05 mM of dCTP, 25 dTTP, and dATP, 10 uCi [32P]-alpha-dGTP (800 Ci / mmol), 20 ug activated calf thymus DNA and the test compound at indicated concentrations. The alpha DNA polymerase reaction mixture was as follows in a 50 uL volume per sample: 20mM Tris-HCl (pH 8), 5 mM magnesium acetate, 0.3 mg / mL BSA, 1 mM DTT, 0.1 mM spermine, 0.05 mM of dCTP, dTTP, and dATP, 10 uCi [32P]-alpha-dGTP (800 Ci / mmol), 20 ug activated calf 30 thymus DNA and the test compound at the indicated concentrations. For each assay, the enzyme reactions were allowed to proceed for 30 minutes at 37°C followed by the transfer onto glass-fiber filter plates and subsequent precipitation with 10% trichloroacetic acid (TCA). The plate was then washed with 5% TCA followed by one wash with 95% ethanol. 2020418425   14 Sep 2026 Once the filter had dried, incorporation of radioactivity was measured using a liquid scintillation counter (Microbeta). Example 58. HIV infected PBMC assay: 5         Fresh human peripheral blood mononuclear cells (PBMCs) were obtained from a commercial source (Biological Specialty) and were determined to be seronegative for HIV and HBV. Depending on the volume of donor blood received, the leukophoresed blood cells were washed several times with PBS. After washing, the leukophoresed blood was diluted 1:1 with Dulbecco’s phosphate buffered saline (PBS) and layered over 15mL of 10 Ficoll-Hypaque density gradient in a 50ml conical centrifuge tube. These tubes were centrifuged for 30 min at 600g. Banded PBMCs were gently aspirated from the resulting interface and washed three times with PBS. After the final wash, cell number was determined by Trypan Blue dye exclusion and cells were re-suspended at 1 x 10A6 cells / mL in RPMI 1640 with 15% Fetal Bovine Serum (FBS), 2 mmol / L L-glutamine, 2 ug / mL PHA- 15 P, 100 U / mL penicillin and 100 ug / mL streptomycin and allowed to incubate for 48-72 hours at 37°C. After incubation, PBMCs were centrifuged and resuspended in tissue culture medium. The cultures were maintained until use by half-volume culture changes with fresh IL-2 containing tissue culture medium every 3 days. Assays were initiated with PBMCs at 72 hours post PHA-P stimulation. 20          To minimize effects due to donor variability, PBMCs employed in the assay were a mixture of cells derived from 3 donors. Immediately prior to use, target cells were resuspended in fresh tissue culture medium at 1 x 10A6 cells / mL and plated in the interior wells of a 96-well round bottom microtiter plate at 50 uL / well. Then, 100 uL of 2X concentrations of compound-containing medium was transferred to the 96-well plate 25 containing cells in 50 uL of the medium. AZT was employed as an internal assay standard. Following addition of test compound to the wells, 50 uL of a predetermined dilution of HIV virus (prepared from 4X of final desired in-well concentration) was added, and mixed well. For infection, 50-150 TCID50 of each virus was added per well (final MOI approximately 0.002). PBMCs were exposed in triplicate to virus and cultured in the 30 presence or absence of the test material at varying concentrations as described above in the 96-well microtiter plates. After 7 days in culture, HIV-1 replication was quantified in the tissue culture supernatant by measurement of reverse transcriptase (RT) activity. Wells with cells and virus only served as virus controls. Separate plates were identically prepared without virus for drug cytotoxicity studies. 2020418425   14 Sep 2026 Reverse Transcriptase Activity Assay - Reverse transcriptase activity was measured in cell-free supernatants using a standard radioactive incorporation polymerization assay. Tritiated thymidine triphosphate (TTP; New England Nuclear) was purchased at 1 Ci / mL and 1 uL was used per enzyme reaction. A rAdT stock solution was prepared by mixing 5 0.5mg / mL poly rAand 1.7 U / mL oligo dT in distilled water and was stored at -20°C. The RT reaction buffer was prepared fresh daily and consists of 125 uL of 1 mol / L EGTA, 125 uL of dH2O, 125 uL of 20% Triton X-100, 50 uL of 1 mol / L Tris (pH 7.4), 50 uL of 1 mol / L DTT, and 40 uL of 1 mol / L MgCl2. For each reaction, 1 uL of TTP, 4 uL of dH2O, 2.5 uL of rAdT, and 2.5 uL of reaction buffer were mixed. Ten microliters of this reaction 10 mixture was placed in a round bottom microtiter plate and 15 uL of virus-containing supernatant was added and mixed. The plate was incubated at 37°C in a humidified incubator for 90 minutes. Following incubation, 10 uL of the reaction volume was spotted onto a DEAE filter mat in the appropriate plate format, washed 5 times (5 minutes each) in a 5% sodium phosphate buffer, 2 times (1 minute each) in distilled water, 2 times (1 minute 15 each) in 70% ethanol, and then air dried. The dried filtermat was placed in a plastic sleeve and 4 mL of Opti-Fluor O was added to the sleeve. Incorporated radioactivity was quantified utilizing a Wallac 1450 Microbeta Trilux liquid scintillation counter. Example 59. HBV: 20         HepG2.2.15 cells (100pL) in RPMI1640 medium with 10% fetal bovine serum was added to all wells of a 96-well plate at a density of 1 x 104 cells per well and the plate was incubated at 37°C in an environment of 5% CO2 for 24 hours. Following incubation, six ten-fold serial dilutions of test compound prepared in RPMI1640 medium with 10% fetal bovine serum were added to individual wells of the plate in triplicate. Six wells in the plate 25 received medium alone as a virus only control. The plate was incubated for 6 days at 37°C in an environment of 5% CO2. The culture medium was changed on day 3 with medium containing the indicated concentration of each compound. One hundred microliters of supernatant was collected from each well for analysis of viral DNA by qPCR and cytotoxicity was evaluated by XTT staining of the cell culture monolayer on the sixth day. 30          Ten microliters of cell culture supernatant collected on the sixth day was diluted in qPCR dilution buffer (40pg / mL sheared salmon sperm DNA) and boiled for 15 minutes. Quantitative real time PCR was performed in 386 well plates using an Applied Biosystems 7900HT Sequence Detection System and the supporting SDS 2.4 software. Five microliters (5 pL) of boiled DNA for each sample and serial 10-fold dilutions of a quantitative DNA 2020418425   14 Sep 2026 standard were subjected to real time Q-PCR using Platinum Quantitative PCR SuperMix-UDG (Invitrogen) and specific DNA oligonucleotide primers (IDT, Coralville, ID) HBV-AD38-qF1 (5’-CCG TCT GTG CCT TCT CAT CTG-3’), HBV-AD38-qR1 (5’-AGT CCA AGA GTY CTC TTA TRY AAG ACC TT-3’), and HBV-AD38-qP1 (5’-FAM CCG TGT 5 GCA / ZEN / CTT CGC TTC ACC TCT GC-3’BHQ1) at a final concentration of 0.2 pM for each primer in a total reaction volume of15 pL. The HBV DNA copy number in each sample was interpolated from the standard curve by the SDS.24 software and the data were imported into an Excel spreadsheet for analysis. The 50% cytotoxic concentration for the test materials are derived by measuring the 10 reduction of the tetrazolium dye XTT in the treated tissue culture plates. XTT is metabolized by the mitochondrial enzyme NADPH oxidase to a soluble formazan product in metabolically active cells. XTT solution was prepared daily as a stock of 1 mg / mL in PBS. Phenazine methosulfate (PMS) stock solution was prepared at 0.15 mg / mL in PBS and stored in the dark at -20°C. XTT / PMS solution was prepared immediately before use 15 by adding 40 pL of PMS per 1 mL of XTT solution. Fifty microliters of XTT / PMS was added to each well of the plate and the plate incubated for 2-4 hours at 37°C. The 2-4 hour incubation has been empirically determined to be within linear response range for XTT dye reduction with the indicated numbers of cells for each assay.      Adhesive plate sealers were used in place of the lids, the sealed plate was inverted several times to mix the soluble 20 formazan product and the plate was read at 450 nm (650 nm reference wavelength) with a Molecular Devices SpectraMax Plus 384 spectrophotometer. Data were collected by Softmax 4.6 software and imported into an Excel spreadsheet for analysis. Example 60. Dengue RNA-dependent RNA polymerase reaction conditions 25         RNA polymerase assay was performed at 30 °C using 100pl reaction mix in 1.5ml tube. Final reaction conditions were 50mM Hepes (pH 7.0), 2mM DTT, 1mM MnCl2, 10mM KCl, 100nM UTR-Poly A (self-annealing primer), 10pM UTP, 26nM RdRp enzyme. The reaction mix with different compounds (inhibitors) was incubated at 30 °C for 1 hour. To assess amount of pyrophosphate generated during polymerase reaction, 30pl of 30 polymerase reaction mix was mixed with a luciferase coupled-enzyme reaction mix (70pl). Final reaction conditions of luciferase reaction were 5mM MgCl2, 50mM Tris-HCl (pH 7.5), 150mM NaCl, 200pU ATP sulfurylase, 5pM APS, 10nM Luciferase, 100pM D-luciferin. White plates containing the reaction samples (100pl) were immediately 2020418425   14 Sep 2026 transferred to the luminometer Veritas (Turner Biosystems, CA) for detection of the light signal. Example 61. Procedure for Cell Incubation and Analysis 5          Huh-7 cells were seeded at 0.5x10A6 cells / well in 1 mL of complete media in 12 well tissue culture treated plates. The cells were allowed to adhere overnight at 37o / 5% CO2. A 40 pM stock solution of test article was prepared in 100% DMSO. From the 40 pM stock solution, a 20 pM solution of test article in 25 ml of complete DMEM media was prepared. For compound treatment, the media was aspirated from the wells and 1 mL of the 10   20 gM solution was added in complete DMEM media to the appropriate wells. A separate plate of cells with “no” addition of the compound was also prepared. The plates were incubated at 37o / 5% CO2 for the following time points: 1, 3, 6 and 24 hours. After incubation at the desired time points, the cells were washed 2X with 1 mL of DPBS. The cells were extracted by adding 500 pl of 70% methanol / 30% water spiked with the internal 15 standard to each well treated with test article. The non-treated blank plate was extracted with 500 ul of 70% methanol / 30% water per well. Samples were centrifuged at 16,000 rpm for 10 minutes at 4oC. Samples were analyzed by LC-MS / MS using an ABSCIEX 5500 QTRAP LC-MS / MS system with a Hypercarb (PGC) column. Example 62. 20 Zika RNA-dependent RNA polymerase reaction conditions RNA polymerase assay was performed at 30 °C using 100pl reaction mix in 1.5ml tube. Final reaction conditions were 50mM Hepes (pH 7.0), 2mM DTT, 1mM MnCl2, 10mM KCl, 100nM UTR-Poly A (self-annealing primer), 10pM UTP, 26nM RdRp enzyme. The reaction mix with different compounds (inhibitors) was incubated at 30 °C for 25   1 hour. To assess amount of pyrophosphate generated during polymerase reaction, 30pl of polymerase reaction mix was mixed with a luciferase coupled-enzyme reaction mix (70pl). Final reaction conditions of luciferase reaction were 5mM MgCl2, 50mM Tris-HCl (pH 7.5), 150mM NaCl, 200pU ATP sulfurylase, 5pM APS, 10nM Luciferase, 100pM D-luciferin. White plates containing the reaction samples (100pl) were immediately 30 transferred to the luminometer Veritas (Turner Biosystems, CA) for detection of the light signal. Example 63. Zika infectious assay conditions 2020418425   14 Sep 2026 Vero cells were passaged in DMEM medium in T-75 flasks prior to use in the antiviral assay. On the day preceding the assay, the cells were split 1:2 to assure they were in exponential growth phase at the time of infection. The cells were resuspended at 5 x 103 cells per well in tissue culture medium and added to flat bottom microtiter plates in a 5 volume of 100 mL. The plates were incubated at 37°C / 5% CO2 overnight to allow for cell adherence. Separately, Zika virus was titrated in LLCMK2 cells to define the inoculum for use in the antiviral assay. Virus was diluted in DMEM medium such that the amount of virus added to each well in a volume of 100 mL was the amount determined to achieve 85 to 95% cell killing at 5 days post-infection. Following incubation test plates were stained 10 with XTT dye. XTT solution was prepared daily as a stock solution of 1 mg / mL in RPMI1640. PMS solution was prepared at 0.15 mg / mL in PBS and stored in the dark at -20°C. XTT / PMS stock was prepared immediately before use by adding 40 mL of PMS per mL of XTT solution. Fifty microliters of XTT / PMS was added to each well of the plate, and the plate was reincubated for 4 hours at 37°C. Plates were sealed with adhesive plate 15 sealers ad shaken gently to mix the soluble formazan product, and the plate was read spectrophotometrically read 450 / 650 nm with a Molecular Devices Vmax plate reader. The raw data was collected from Softmax Pro and imported into a Microsoft Excel XLfit4 spreadsheet for analysis using four parameter curve fit calculations. Example 64. 20 POLRMT methods. POLRMT enzyme purification A variant of human POLRMT coding sequence was amplified from a POLRMT cDNA plasmid (Accession: BC098387, Clone ID: 5264127, Dharmacon, CO) and cloned into a pMal-c5X vector under control of the tac promoter. For protein expression, the 25   plasmid was transformed into Stellar competent cells (Clontech). Expression vector pMal- c5X contains a lacI gene which allows inducible expression of POLRMT in Stellar cells. The transformed cells were grown in LB medium containing 100 iigmi ampicillin at 35°C to an optical density of 1 at 600 nm. Cells were cooled down in a 4°C fridge for 1 hour. MgCl2 was added to final concentration of 1 mM. Protein expression was induced at 16°C 30 overnight by the addition of 0.4 mM IPTG. Cells were harvested by centrifugation at 4000 x g for 20 min at 4°C. The cell pellet was stored at -80°C until further processed. For protein purification, the cell pellet was re-suspended in sonication buffer (20 mM Tris-HCl pH 7.5, 10% glycerol, 500 mM NaCl, 0.5% Triton X-100, 10 mM DTT, 10 mM MgCl2, 30 mM imidazole and 1X protease inhibitor cocktail). Cell disruption was performed on ice for 2020418425   14 Sep 2026 10 min using an ultrasound probe sonicator. The cell extract was clarified by centrifugation at 16,000 x g for 20 min at 4°C. The supernatant was incubated with HisPur Ni-NTA agarose resin with gentle rocking for 15 minutes at 4°C. The resin was then washed 5 times with 10 volumes of wash buffer (20 mM Tris-HCl pH 7.5, 10% glycerol, 500 mM NaCl, 5   0.1% Triton X-100, 1 mM DTT, 2 mM MgCl2) containing 30 mM imidazole and then once with the wash buffer containing 2M NaCl. The protein was eluted from the resin with 1 volume of elution buffer (20 mM Tris-HCl, pH 7.5, 10% glycerol, 50 mM NaCl, 0.5% Triton X-100, 10 mM DTT and 300 mM imidazole). The eluted enzyme was adjusted to 50% glycerol and stored at -80 °C before use. Protein identification was performed by mass 10 spectrometry. The concentration of a targeted protein was measured by SDS-PAGE using BSA (Sigma, St. Louis, MO) as a standard. Measurement of ribonucleotide analog incorporation efficiency Different templates were designed to test individual analog rNTPs, Table 1. Different concentrations of tested ribonucleotide analogs were added to reaction mixtures 15 containing 10 nM P / T and 20 nM POLRMT in a reaction buffer (5 mM Tris-HCl, pH 7.5, 10 mM DTT, 20 mM MgCl2, 0.5% X-100, 10% glycerol) to initiate the reactions. The reactions were continued at 22°C for different time and subsequently quenched with quenching buffer (8 M Urea, 90 mM Tris base, 29 mM taurine, 10 mM EDTA, 0.02% SDS and 0.1% bromophenol blue). The quenched samples were denatured at 95°C for 15 min 20 and the primer extension products were separated using 20% denaturing polyacrylamide gel electrophoresis (Urea PAGE) in 1X TTE buffer (90 mM Tris base, 29 mM Taurine and 0.5 mM EDTA). After electrophoresis, gels were scanned using an Odyssey infrared imaging system. The intensity of different RNA bands was quantified using Image Studio Software Lite version 4.0. The incorporation efficiencies of different rNTP analogs were evaluated by 25 measurement the K1 / 2 and corresponding Discrimination Values (ref. G Lu). Primer extension polymerase activity assay POLRMTs polymerase activity was determined in a primer extension reaction using a fluorescently labeled RNA primer / DNA template complex. A typical primer extension reaction was performed in a 20-pl reaction mixture containing reaction buffer (5 mM Tris- 30 HCl, pH7.5, 10 mM DTT, 20mM MgCl2, 0.1% Triton X-100, 0.01 U RNasin, 10% glycerol), 10 nM P / T complex, and 20 nM POLRMT. The reaction was initiated by the addition of rNTPs at a final concentration of 100 pM, followed by incubation for 1 h at 22 °C. The reactions were quenched by the addition of 20 pl quenching buffer (8 M Urea, 90 mM Tris base, 29 mM taurine, 10 mM EDTA, 0.02% SDS and 0.1% bromophenol blue). 2020418425   14 Sep 2026 The quenched samples were denatured at 95°C for 15 min and the primer extension products were separated using 20% denaturing polyacrylamide gel electrophoresis (Urea PAGE) in 1X TTE buffer (90 mM Tris base, 29 mM Taurine and 0.5 mM EDTA). After electrophoresis, gels were scanned using an Odyssey infrared imaging system (LI-COR 5 Biosciences, Lincoln, NE). The images were analyzed and the proper RNA bands were quantified using Image Studio software Lite version 4.0 (LI-COR Biosciences, Lincoln, NE). Example 65. EIDD-02838 Togaviridae Activity Virus Cell Line EC50 (gM) EC90 (gM) CC50 (gM) VEEV Vero76 8.28 >230 VEEV Vero76 8.51 >230 EEEV Vero76 2.76 >230 EEEV Vero76 3.22 >230 EEEV Vero 76 28.4 >230 EEEV Vero 76 12.4 >230 WEEV Vero76 5.98 >230 WEEV Vero76 7.82 >230 WEEV Vero 76 24.15 >230 WEEV Vero 76 5.75 >230 CHIKV Vero76 2.3 >230 CHIKV Vero76 3.68 >230 CHIKV Vero 76 23.92 >230 CHIKV Vero 76 3.91 >230 10 Example 66. EIDD-02838 Bunyaviridae Activity Virus Cell Line EC50 (gM) EC90 (gM) CC50 (gM) RVFV Vero76 3.91 >230 RVFV Vero76 3.68 >230 RVFV Vero 76 2.19 >230 RVFV Vero 76 4.14 >230 Heartland virus Vero 8.05 >230 La Crosse virus Vero 76 <0.23 >230 La Crosse virus Vero 76 <0.23 193 La Crosse virus Vero 76 0.12 >23 La Crosse virus Vero 76 0.69 >23 Marpol virus Vero 25.3 >230 Marpol virus Vero 19.1 >230 Punta Toro virus Vero 76 6.21 >230 Punta Toro virus Vero 76 5.75 >230 2020418425   14 Sep 2026 Severe fever thrombocytopenia syndrome virus Vero 7.82 >230 Example 67. EIDD-02838 Arenaviridae Activity Virus Cell Line EC50 (gM) EC90 (gM) CC50 (gM) Lassa fever virus Vero 0.14 >230 Lassa fever virus Vero 1.3 213 Junin virus Vero 0.37 >230 Junin virus Vero <0.23 >230 Junin virus Vero 0.023 >23 Junin virus Vero 0.023 >23 Lymphocytic choriomeningitis virus Vero 4.6 >230 Pichinde virus Vero 2.76 >230 Pichinde virus Vero 1.45 >230 Pichinde virus Vero 0.55 >23 Pichinde virus Vero 0.35 >23 Tacaribe virus Vero <0.23 >230 Tacaribe virus Vero <0.23 >230 Tacaribe virus Vero 0.046 >23 Tacaribe virus Vero 0.046 >23 5 Example 68. EIDD-02838 Influenza Activity Virus Cell Line EC50 (gM) EC90 (gM) CC50 (gM) Influenza A H1N1 MDCK 1.2 >230 Influenza A H1N1 MDCK 1.59 >230 Flu A H7N9 (High Path) MDCK 5.29 >230 Flu A H7N9 (High Path) MDCK 4.37 >230 Flu A H5N1 (High Path) MDCK 23 >230 Flu A H5N1 (High Path) MDCK 11.27 >230 Flu A H1N1 MDCK 1.98 >230 Flu A H1N1 MDCK 2.3 >230 Flu A H3N2 MDCK 0.97 >230 Flu A H3N2 MDCK 0.87 >230 Flu A H5N1 (Low Path) MDCK 0.78 >230 Flu A H5N1 (Low Path) MDCK 0.71 >230 Flu B MDCK 0.78 >230 Flu B MDCK 0.74 >230 Flu B MDCK 0.74 >230 Flu B MDCK 0.67 >230 2020418425   14 Sep 2026 Example 69. EIDD-02838 Parainfluenza and RSV Activity Virus Cell Line EC5g(^M) EC9g(^M) CC5g(^M) Parainfluenza virus 3 MA-104 73.6 >230 Parainfluenza virus 3 MA-104 78.2 >230 RSV MA-104 3.45 >230 RSV MA-104 2.76 >230 RSV MA-104 7.82 >230 RSV MA-104 6.21 >230 5 Example 70. EIDD-02838 Ebola Activity Virus Cell Line EC5g(^M) EC9g(^M) CC5g(^M) EBOV Vero 149.5 >230 Example 71. EIDD-02838 Coronaviridae Activity Virus Cell Line EC50 (gM) EC90 (gM) CC50 (gM) MERS Vero76 1.98 >230 MERS Vero E6 2.53 >230 10 Example 72. EIDD-02838 Flaviviridae Activity Virus Cell Line EC50(gM) EC90(gM) CC50(gM) POWV BHK 75.9 >230 POWV BHK 98.9 >230 ZIKV Vero 76 7.82 >230 ZIKV Vero 76 9.2 >230 DENV2 Vero 76 29.9 >230 DENV2 Vero 76 >230 >230 JEV Vero 76 3.91 >230 JEV Vero 76 78.2 >230 Usutu virus Vero 76 25.76 >230 Usutu virus Vero 76 6.9 >230 ZIKV Vero 76 43.7 156.4 ZIKV Vero 76 8.28 156.4 15 2020418425   14 Sep 2026 Example 73. EIDD-02838 Picornaviridae Activity Virus Cell Line EC5o(gM) EC9o(gM) CC5o(gM) Poliovirus Vero 76 7.36 >230 Poliovirus Vero 76 8.74 >230 Coxsackie virus B3 Vero 76 4.83 >230 Coxsackie virus B3 Vero 76 2.76 >230 Enterovirus-68 RD 7.36 >230 Enterovirus-68 RD 7.82 >230 HRV-14 HeLa-Ohio >230 >230 HRV-14 HeLa-Ohio >230 >230 Example 74. 5   EIDD-02749 Norovirus Activity Virus Cell Line EC50 (gM) EC90 (gM) CC50 (gM) Murine Norovirus RAW264.7 2.2 7.2 >100 Example 75. 2020418425   14 Sep 2026 Reagents and conditions; a) Acetone, H2SO4, 2,2-DMP, RT, 12 hr, 80-85%; b) Boc-L-Val-OH, DCC, DMAP, DCM, RT 5-6 hr; c) 1,2,4-triazole, POC13, triethylamine, MeCN; d) 50%NH2OH in water, MeCN; e) conc.HCl, MeOH, RT, 24 hr A 2L 3-neck RBF was charged with 1-[(3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]pyrimidine-2,4-dione (61.4g, 251.43 mmol) and acetone (1400 mL). The resulting slurry was stirred at RT and sulfuric acid (2 mL was 5 added. Stirring was continued overnite. The clear colorless solution was quenched / adjusted to basic pH with 100 mL of trimethylamine. The crude solution was concentrated under reduced pressure to yield a pale yellow oil. The residue was dissolved in 600 mL of EtOAc and washed with water x 2, bicarb x 2, water, brine x 2 and dried over sodium sulfate. The colorless solution was concentrated under reduced pressure to yield 1-[(3aR,6R,6aR)-6- 10 (hydroxymethyl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-4-yl]pyrimidine-2,4-dione (45 g) as a white solid. A 200 mL RBF was charged with 1-[(3aR,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-4-yl]pyrimidine-2,4-dione (2.36 g, 8.3 2020418425   14 Sep 2026 mmol) and DCM (50 mL). The reaction was stirred until a solution was formed. Next, (2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoic acid (2.16 g, 9.96 mmol) and N,N-dimethylpyridin-4-amine (0.1 g, 0.8300 mmol) were added. The reaction was cooled to 0°C with an ice bath. A DCM solution of N,N'-dicyclohexylcarbodiimide (2.06 g, 9.96 mmol) 5 was added slowly. The reaction mixture was allowed to warm to rt. Monitored by TLC (EtOAc). A precipitate (DCU) formed after about 1 hr and no starting material was detected after 3 hrs. The solids were filtered off and rinsed with EtOAc. The filtrate was washed with water, brine, dried over sodium sulfate and concentrated under reduced pressure to 10 yield white, gooey solid. The gummy solid was triturated with ether and filtered to remove the solid. The filtrate was concentrated under reduced pressure to yield about 8 g of thick viscous oil. The product was purified by SGC, pooled fractions 6-25 and concentrated under reduced pressure to yield [(3aR,6R,6aR)-4-(2,4-dioxopyrimidin-1-yl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuro[3,4-d][1,3]dioxol-6-yl]methyl (2S)-2-(tert-butoxycarbonylamino)- 15 3-methyl-butanoate (3.8 g,7.8592 mmol, 94.667% yield) as a foamy white solid after drying in vacuo. 1,2,4-triazole was taken in anhydrous acetonitrile and stirred at RT after 30 min, the reaction mixture was cooled to 0°C and POCl3 was added dropwise and continued stirring for 2 hr. After 2 hr triethylamine was added added dropwise and continue stirring for 1 hr, 20 the reaction mixture was slowly brought to RT, and the uridine derived substrate from the above reaction was added as solution in acetonitrile. The reaction mixture stirred at RT overnight. After completion of the reaction, the solvent was removed under reduced pressure and taken in DCM and extracted with water. The organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude product 25 was purified by flash column chromatography. To a solution of the substrate in acetonitrile (10 mL / gm), 50% hydroxylamine in water was added dropwise and stirred at rt for 2-3 hrs. After completion of the reaction, solvent was removed under reduced pressure and the crude product was purified by flash column chromatography using hexane and EtOAc as eluent. 30          1 g of substrate was taken in 20 mL of methanol and treated with 2 mL of conc.HCl (36%) and after 3-4 hr 30% completion was obsereved. Another 5 mL of conc.HCl was added and stirred overnight. After completion of the reaction, solvent was removed and the crude product was taken in minimum methanol and added dropwise to excess diethylether with stirring, product was crashed out of solution and allowed to settle, ether was decanted 2020418425   14 Sep 2026 and fresh ether was added, stirred, settled and decanted, the same process was repeated two times. After ether was decanted, solid was dried over a rotavap and high vaccum to get free flowing white solid. Ether was trapped in the solid and was difficult to remove. The solid was dissolved in methanol, evaporated and dried to get colorless foam, which still holds 5 methanol. The foam was taken in water and a purple solution was observed. The purple solution was purified by reverse phase ISCO column chromatography using water and acetonitrile. The fractions containing product were evaporated under reduced pressure and lyophilized to get colorless solid. Example 76. 10                                                                                                     EIDD-2800 A 3-neck 1L round bottom flask equipped with an overhead stirrer, temperature probe and addition funnel was charged with uridine (25 g, 102.38 mmol) and ethyl acetate (500 mL). The white slurry was stirred at ambient temperature while triethylamine (71.39 mL, 511.88 mmol) and DMAP (0.63 g, 5.12 mmol) were added to the mixture. The slurry 15 was cooled in an ice bath and isobutyric anhydride (56.02 mL, 337.84 mmol) was slowly added to the reaction mixture over a 5 minute period. The temperature rose 25°C during the addition. The resulting slurry was stirred at ambient temperature and monitored by TLC. After 1 hour, a clear colorless solution had formed and TLC showed no starting material. The reaction was quenched with 200mL of water, stirred at rt for 20 minutes. The layers 20 were separated, and the organics were washed with water (2 x 100 mL), saturated aqueous bicarbonate solution (100 mL x 2), 100 mL of water, brine (100 mL x 2), and then dried over sodium sulfate. The organics were filtered and the filtrate was concentrated under reduced pressure at 45°C to yield a yellow oil. The oil was used in the next step without any further purification. 25          A 2L 3-neck flask equipped with an argon inlet, overhead stirrer and temperature probe was charged with 1H-1,2,4-triazole (50.88 g, 736.68 mmol), triethylamine (114.17 mL, 818.54 mmol) and MeCN (350 mL). The reaction mixture was stirred at rt for 20 minutes. An ethyl acetate (350 mL) solution of [(2R,3R,4R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-bis(2-methylpropanoyloxy)tetrahydrofuran-2-yl]methyl 2-methylpropanoate (46.5 g, 2020418425   14 Sep 2026 102.32 mmol) was added and the mixture was cooled to <5°C using an ice bath. Stirring continued for 20 minutes. Next, phosphorous(V)oxychloride (14.35 mL, 153.48 mmol) was added slowly under argon at less than 20°C over 15 minutes. The reaction was monitored by TLC (100% EtOAc), starting material (Rf = 0.89) consumed in less than 2 hours and a 5 new spot due to product (Rf = 0.78) present. The reaction was quenched with 500 mL of water and 400 mL of EtOAc. The quenched reaction was allowed to stir at rt for 15 minutes. The layers were separated and the organic layer was washed with water (2 x 100 mL), 200mL of 0.5N HCl, and brine (2 x 100 mL). The organics were dried over sodium sulfate, filtered and concentrated under reduced pressure to yield [(2R,3R,4R)-3,4-bis(2- 10   methylpropanoyloxy)-5-[2-oxo-4-(1,2,4-triazol-1-yl)pyrimidin-1-yl]tetrahydrofuran-2- yl]methyl 2-methylpropanoate (49 g, 96.93 mmol, 94.735% yield) as a yellow oil. The crude material was used in the next step without further purification. A 500mL round bottom flask was charged with [(2R,3R,4R)-3,4-bis(2-methylpropanoyloxy)-5-[2-oxo-4-(1,2,4-triazol-1-yl)pyrimidin-1-yl]tetrahydrofuran-2- 15 yl]methyl 2-methylpropanoate (48.9 g, 96.73 mmol), ethyl acetate (400 mL), and isopropyl alcohol (100 mL). The reaction mixture was stirred at rt until all of the starting material was dissolved. The orange solution was treated with hydroxylamine (6.52 mL, 106.41 mmol), and the resulting pale yellow solution was stirred at rt and monitored by TLC (EtOAc). No starting material was observed after 1 hour. The reaction was quenched with 20   500mL of water, and the layers were separated. The organics were washed with 100mL of water, 100 mL x 2 of brine, and then dried over sodium sulfate. The organics were filtered and concentrated under reduced pressure to yield the crude product. The crude product was dissolved in 180 mL of hot MTBE and allowed to cool to rt. Seed crystals were added, and the flask was placed in the freezer. The white solid that formed was collected by filtration, 25 washed with a minimal amount of MTBE and dried in vacuo to yield the desired product. Example 77. 2020418425   14 Sep 2026 A 1L round bottom flask was charged with uridine (25 g, 102.38 mmol) and acetone (700 mL). The reaction mixture was allowed to stir at rt. The slurry was then treated with sulfuric acid (0.27 mL, 5.12 mmol). Stirring was allowed to continue at rt for 18 hours. 5 The reaction was quenched with 100 mL of trimethylamine and was used in the next step without further pruficication. A 1L round bottom flask was charged with the reaction mixture from the previous reaction. Triethylamine (71.09 mL, 510.08 mmol) and 4-dimethylaminopyridine (0.62 g, 5.1 mmol) were then added. The flask was cooled using an ice bath and then 2 10 methylpropanoyl 2-methylpropanoate (17.75 g, 112.22 mmol) was slowly added. The reaction mixture was allowed to stir at rt until the reaction was complete. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in 600 mL ethyl acetate and washed with saturated aqueous bicarbonate solution x 2, water x 2 and brine x 2. The organics were dried over sodium sulfate and concentrated under reduced 15 pressure to yield a clear colorless oil. The crude product was used in the next step without further purification. A 1L round bottom flask was charged with the crude product from above (36 g, 101.59 mmol) and MeCN (406.37 mL). The reaction mixture was allowed to stir until all the starting material was dissolved. Next, 1,2,4-triazole (50.52 g, 731.46 mmol) was added 20 followed by the addition of N,N-diethylethanamine (113.28 mL, 812.73 mmol). The reaction mixture was allowed to stir at rt until all solids dissolved. The reaction was then cooled to 0°C using an ice bath. Phosphorous oxychloride (24.44 mL, 152.39 mmol) was added slowly. The slurry that formed was allowed to stir under argon while slowly warming to rt. The reaction was then allowed to stir until complete by TLC (EtOAc). The 25 reaction was then quenched by the addition of 100mL of water. The slurry then became a dark colored solution, which was then concentrated under reduced pressure. The residue 2020418425   14 Sep 2026 was dissolved in DCM and washed with water and brine. The organics were then dried over sodium sulfate, filtered, and concentrated under reduced pressure. The product was purified by silica gel chromatography (2 x 330 g columns). All fractions containing product were collected and concentrated under reduced pressure. 5          A 500 mL round bottom flask was charged with the product from the previous step (11.8 g, 29.11 mmol) and isopropyl alcohol (150 mL). The reaction mixture was allowed to stir at rt until all solids dissolved. Next, hydroxylamine (1.34 mL, 43.66 mmol) was added and stirring continued at ambient temperature. When the reaction was complete (HPLC) some solvent was removed under high vacuum at ambient temperature. The remaining 10 solvent was removed under reduced pressure at 45°C. The resulting residue was dissolved in EtOAc and was washed with water and brine. The organics were dried over sodium sulfate, filtered, and concentrated under reduced pressure to yield oil. Crystals formed upon standing at rt. The crystals were collected by filtration, washed with ether x 3, and dried in vacuo to provide the product as a white solid. 15          A 200 mL round bottom flask was charged with the product from the previous step (6.5 g, 17.6 mmol) and formic acid (100 mL, 2085.6 mmol). The reaction mixture was allowed to stir at rt overnight. The progress of the reaction was monitored by HPLC. The reaction mixture was concentrated under reduced pressure at 42°C to yield a clear, pale pink oil. Next, 30 mL of ethanol was added. Solvent was then removed under reduced pressure. 20 MTBE (50 mL) was added to the solid and heated. Next, isopropyl alcohol was added and heating was continued until all solid material dissolved (5 mL). The solution was then allowed to cool and stand at rt. A solid started to form after about 1hr. The solids were collected by filtration, washed with MTBE, and dried in vacuo to yield the EIDD-2801 as a white solid. The filtrate was concentrated under reduced pressure to yield a sticky solid, 25 which was dissolved in a small amount of isopropyl alcohol with heating. The solution was allowed to stand at rt overnight. A solid formed in the flask, which was collected by filtration, rinsed with isopropyl alcohol and MTBE, and dried in vacuo to an additional crop of desired product. EIDD-2801 (25 g) was dissolved in 250 mL of isopropyl alcohol by heating to 30   70°C to give a clear solution. The warm solution was polish filtered and filtrate transferred to 2L three neck flask with overhead stirrer. It was warmed back to 70°C and MTBE (250 mL) was slowly added into the flask. The clear solution was seeded and allowed to cool slowly to rt with stirring for 18 hrs. The EIDD-2801 solid that formed was filtered and washed with MTBE and dried at 50°C under vacuum for 18hours. The filtrate was 2020418425   14 Sep 2026 10 concentrated, redissolved in 50 mL isopropyl alcohol and 40 mL MTBE by warming to give clear solution and allowed to stand at rt to give a second crop of EIDD-2801. Example 78. Norovirus Activity for EIDD-02749 Virus Strain Cell Line EC50 (gM) EC90 (gM) CC50 (gM) Murine Norovirus MNV-1 RAW264.7 2.2 7.2 >100 Human Norovirus G1 HG23 2.7 9.3 100 Example 79. Togaviridae Activity for EIDD-02749 Virus Strain Cell Line EC50 (gM) EC90 (gM) CC50 (gM) CHIKV S27 (VR-64) Vero76 1.98 >380 CHIKV S27 (VR-64) Vero76 2.05 >380 CHIKV S27 (VR-64) Vero 76 9.12 >380 CHIKV S27 (VR-64) Vero 76 3.42 >380 VEEV TC-83 Huh-7 1.6 >380 VEEV TC-83 Huh-7 19 >380 VEEV TC-83 Vero 76 213 >380 VEEV TC-83 Vero 76 7.6 >380 EEEV FL93-939 Vero76 380 >380 EEEV FL93-939 Vero76 >380 >380 WEEV California Vero76 9.88 >380 WEEV California Vero76 11.8 >380 Example 80. Flaviviridae Activity for EIDD-02749 Virus Strain Cell Line EC50(gM) EC90 (gM) CC50 (gM) DENV2 New Guinea C Huh-7 >380 >380 DENV2 New Guinea C Huh-7 >380 >380 ZIKV MR766 Vero 76 16.3 >380 ZIKV MR766 Vero 76 3.8 >380 ZIKV MR766 Vero 76 >380 >380 ZIKV MR766 Vero 76 28.1 >380 Example 81. Picornaviridae Activity for EIDD-02749 Virus Strain Cell Line EC50(gM) EC90 (gM) CC50 (gM) ENTV71 Tainan / 4643 / 98 Vero 76 3.27 >380 ENTV71 Tainan / 4643 / 98 Vero 76 1.33 >380 Coxsackie virus B3 HA201933 Vero 76 3.12 >380 Coxsackie virus B3 HA201933 Vero 76 3.15 >380 Coxsackie virus B3 HA201933 Vero 76 >380 >380 Coxsackie virus B3 HA201933 Vero 76 12 >380 Enterovirus-68 US / KY / 14-18953 RD 13.7 >380 Enterovirus-68 US / KY / 14-18953 RD 11.8 >380 Poliovirus Mahoney Vero 76 12.2 >380 Poliovirus Mahoney Vero 76 11.8 >380 Example 82. Respiratory Virus Activity for EIDD-02749 2020418425   14 Sep 2026 Virus Strain Cell Line EC50 (hm) CC50 (hm) Influenza A H1N1 CA / 07 / 20 / 09 MDCK <0.38 >380 Influenza A H1N1 CA / 07 / 20 / 09 MDCK <0.38 >380 Flu A H3N2 Perth / 16 / 2009 MDCK <0.38 >380 Flu A H3N2 Perth / 16 / 2009 MDCK <0.38 >380 Flu A H5N1 (Low Path) Duck / MN / 1525 / 81 MDCK <0.38 >380 Flu A H5N1 (Low Path) Duck / MN / 1525 / 81 MDCK <0.38 >380 Flu B Brisbane / 60 / 2008 MDCK <0.38 >380 Flu B Brisbane / 60 / 2008 MDCK <0.38 >380 Flu B Florida / 4 / 2006 MDCK <0.38 >380 Flu B Florida / 4 / 2006 MDCK <0.38 >380 Parainfluenza virus 3 14702 MA-104 98.8 >380 Parainfluenza virus 3 14702 MA-104 60.8 >380 HRV-14 1059 HeLa-Ohio 1.22 32.7 HRV-14 1059 HeLa-Ohio 1.14 31.9 RSV A2 MA-104 1.22 >380 RSV A2 MA-104 <0.38 >380 Example 83. Coronavirus Activity for EIDD-02749 Virus Strain Cell Line EC50 (hM) CC50 (hM) MERS EMC Vero76 >380 >380 MERS EMC Vero76 >380 >380 Example 84. Bunyaviridae Activity for EIDD-02749 Virus Strain Cell Line EC5o(hM) EC90 (hm) CC50 (hm) RVFV MP-12 Vero76 3.46 >380 RVFV MP-12 Vero76 2.96 >380 RVFV MP-12 Vero 76 23.9 >380 RVFV MP-12 Vero 76 16.3 >380 RVFV MP-12 Vero 76 4.3 >380 Heartland virus MO-4 Vero 121.6 >380 La Crosse virus Wisconsin 1960 (VR-744) Vero 76 <0.38 >380 La Crosse virus Wisconsin 1960 (VR-744) Vero 76 <0.38 >380 La Crosse virus Wisconsin 1960 (VR-744) Vero 76 0.27 >380 La Crosse virus Wisconsin 1960 (VR-744) Vero 76 0.38 >380 Marpol virus HV97021050 Vero E6 95 >380 Marpol virus HV97021050 Vero E6 57 >380 Punta Toro virus Adames Vero 76 72.2 >380 Punta Toro virus Adames Vero 76 91.2 >380 Severe fever thrombocytopenia syndrome virus HB-29 Vero >380 >380 Example 85. Arenaviridae Activity for EIDD-02749 2020418425   14 Sep 2026 Virus Strain Cell Line EC50« EC90 (pM) CC50 (pM) Tacaribe virus TRVL11573 Vero <0.38 >380 Tacaribe virus TRVL 11573 Vero <0.38 >380 Tacaribe virus TRVL11573 Vero 0.87 >380 Tacaribe virus TRVL 11573 Vero 0.34 >380 Lassa fever virus Josiah Vero 0.38 >380 Lassa fever virus Josiah Vero 9 >380 Lymphocytic choriomeningitis virus Armstrong Vero 7.22 >380 Pichinde virus An 4763 Vero 2.81 >380 Pichinde virus An 4763 Vero 1.03 >380 Pichinde virus An 4763 Vero 0.19 >380 Junin virus Candid #1 Vero 1.86 >380 Junin virus Candid #1 Vero 1.29 >380 Junin virus Candid #1 Vero 0.38 >380 Example 86. Filovirus Activity for EIDD-02749 Virus Strain Cell Line EC50(pM) EC90 (pM) CC50 (pM) EBOV Zaire Vero 1.52 >380 EBOV Zaire Vero 22 >380 Marburg virus Angola Vero >380 >380 Example 80. EIDD-02749 Cytotoxicity ID Cell Line CC50 EIDD-02749 CEM 107.3 EIDD-02749 HepG2 >400 EIDD-02749 Huh-7 >400 EIDD-02749 A-204 >400 EIDD-02749 A549 >400 EIDD-02749 BxPc3 >400 EIDD-02749 H9c-2 >400 EIDD-02749 IEC-6 371.7 EIDD-02749 Vero >400 Example 81. Synthesis of EIDD-02749-5’-Monophosphate (EIDD-02986) 5 A heavy wall 350 mL round-bottomed pressure vessel was charged with 5’-(3-chlorobenzoyloxy)-4’-fluoro-2’,3’-O-isopropylideneuridine (4.1 g, 9.3 mmol) and 7N 10 ammonia in methanol (66 mL, 462 mmol). The mixture was stirred for 6h at room temperature after which time tlc indicated complete consumption of starting material. The 2020418425   14 Sep 2026 mixture was concentrated in vacuo, and the resulting residue purified by column chromatography over silica gel (40 g) eluting with a methylene chloride / methanol gradient to give 4’-fluoro-2’,3’-O-isopropylideneuridine (2.5 g, 89%) as a white solid. 1H NMR (400 MHz, Chloroform-d) S 9.24 (s, 1H), 7.23 (d, J = 8.0 Hz, 1H), 5.77 (d, 5 J = 8.0 Hz, 1H), 5.72 (s, 1H), 5.24 (dd, J = 12.6, 6.5 Hz, 1H), 5.07 (dd, J = 6.4, 1.3 Hz, 1H), 2.50 (s, 1H), 1.59 (s, 3H), 1.38 (s, 3H). 19F NMR (376 MHz, Chloroform-d) S -115.53 (dd, J = 12.4, 8.8 Hz). A solution of tristriazolide in acetonitrile was freshly prepared by treating a mixture of 1,2,4-triazole (468.91 mg, 6.79 mmol) and triethylamine (0.95 mL, 6.79 mmol) in 10 acetonitrile (7.5 mL) dropwise with phosphorus oxychloride (0.21mL, 2.27mmol) over a 5 min period at -15°C. After stirring for an additional 20 min at -15°C, the triethylammonium precipitate was removed by centrifuge, and the solution of tristriazolide was added to an acetonitrile solution (7.5 mL) of 4’-fluoro-2’,3’-O-isopropylideneuridine (225 mg, 0.74 mmol) at -15°C. After stirring for 15 min at -15°C, the mixture was allowed to warm to rt, 15 and continued for another 1.5 hr. The mixture was quenched with 50 mM TEAB (30 mL), stirred for 1h at rt, and concentrated to dryness in vacuo. The resulting residue was coevaporated with water (2 x 20 mL) and purified by ion-exchange chromatography over DEAE-Sephadex A-25 (HCO3- form) eluting with a gradient from 0 to 0.2 M (700 mL) aqueous ammonium bicarbonate in 10% ethanol. Fractions were analyzed by tlc (7:2:1 20   iPa:NH4OH:water), and target fractions combined and concentrated. The product was further purified by reversed-phase chromatography with a CombiFlash equipped with a C-18 column (50g) eluting with 0.01M aqueous ammonium bicarbonate. Product containing fractions were pooled, frozen, and concentrated by lyophilization to give 4’-fluoro-2’,3’-O-isopropylideneuridine 5’-O-phosphate (131 mg, 46%) as a white solid. 25          1H NMR (400 MHz, D2O) S 7.64 (d, J = 8.0 Hz, 1H), 6.08 (s, 1H), 5.81 (d, J = 7.8 Hz, 1H), 5.21 (dd, J = 12.4, 6.6 Hz, 1H), 5.14 (d, J = 6.5 Hz, 1H), 4.02 - 3.73 (m, 2H), 1.54 (s, 3H), 1.36 (s, 3H). 31P NMR (162 MHz, D2O) S 3.46. 19F NMR (376 MHz, D2O) S -113.90 (q, J = 12.4, 11.9 Hz). 30 4’-Fluorouridine-5’-monophosphate (EIDD-02986) A 50 mL round-bottomed flask was charge with 4’-fluoro-2’,3’-O-isopropylideneuridine-5’-O-phosphate (171 mg, 0.43 mmol), water (0.5 mL) and acetic acid (1.5 mL). The solution was cooled to 10°C and treated with cold aqueous 90% 2020418425   14 Sep 2026 trifluoroacetic acid (3.3 mL, 43.15 mmol). After 5 min, the mixture was allowed to warm to room temperature and stirred an additional 2h. The mixture was concentrated in vacuo and the resulting gum co-evaporated with water (5 x 10 mL) followed by methanol (3 x 10 ml). The crude product as a solution in methanol (10 mL) was filtered, concentrated to 5 approximately 4mL in volume and treated with a cold solution of 1M sodium perchlorate in acetone (20 mL). After 20 min at 0°C, the white precipitate was collected by centrifuge. The white solid was washed with acetone (5 x 14 mL), dissolved in water (4 mL) and concentrated by lyophilization to give 4’-fluorouridine-5’-monophosphate (EIDD-02986) (78 mg, 45%) as the disodium form. 10          1H NMR (400 MHz, D2O) 5 7.75 (d, J = 8.1 Hz, 1H), 6.09 (s, 1H), 5.92 - 5.82 (m, 1H), 4.58 - 4.49 (m, 1H), 4.42 (dd, J = 6.4, 1.9 Hz, 1H), 4.11 (t, J = 5.2 Hz, 3H). 31P NMR (162 MHz, D2O) 5 -0.27. 19F NMR (376 MHz, D2O) 5 -121.26 (dt, J = 19.1, 5.1 Hz). LCMS Calculated for C9 H11FN2O9P [M-H+]: 341.0; found: 340.9. 15 Example 82. Synthesis of EIDD-02749-5’-triphosphate (EIDD-02991) A 2 L three-necked round-bottomed flask flushed with argon and fitted with a mechanical stirrer, thermometer was charged with 5’-deoxy-5’-iodouridine (80 g, 225.92 20 mmol) and dry methanol (500 mL). Under argon atm, the white suspension was treated with a solution of 25% (4.37 M) sodium methoxide in methanol (103.4 mL, 451.85 mmol). The resulting homogeneous solution was stirred at 60°C for 3 h. Methanol was removed in vacuo, and the resulting residue dissolved in anhydrous acetonitrile (300 mL). After addition of acetic anhydride (70.2 mL, 743 mmol), the mixture was heated to 60°C for 5 h. 25 Once cooled to room temperature, the mixture was concentrated in vacuo, and the resulting residue dissolved in ethyl acetate (500 mL) and treated with saturated sodium bicarbonate 2020418425   14 Sep 2026 (100 mL). The organic layer was separated, washed with brine (100 mL), dried and concentrated to dryness to give 2’,3’-di-O-acetyl-4’,5’-didehydro-5’-deoxyuridine (70 g, 99% yield). 1H NMR (400 MHz, DMSO-d6) 5 11.53 (d, J = 1.9 Hz, 1H), 7.75 (d, J = 8.1 Hz, 5    1H), 6.07 (d, J = 4.3 Hz, 1H), 5.92 (d, J = 6.5 Hz, 1H), 5.69 (dd, J = 8.0, 1.8 Hz, 1H), 5.63 (dd, J = 6.4, 4.3 Hz, 1H), 4.52 (t, J = 1.9 Hz, 1H), 4.28 (d, J = 2.4 Hz, 1H), 2.08 (s, 3H), 2.04 (s, 3H). In a 1 L round-bottomed flask, a solution of 2’,3’-di-O-acetyl-4’,5’-didehydro-5’-deoxyuridine (70 g, 225.6 mmol) in methanol (350 mL) was treated with 30% ammonium 10 hydroxide (85.3 mL, 2190.7 mmol). After 18 h at room temperature, the mixture was concentrated in vacuo and the resulting residue dissolved in a 65:35:5 mixture of acetonitrile:isopropanol:methanol. After 30 min, the white precipitate was collected by vacuum filtration and washed with acetonitrile and hexanes. A second crop was isolated by concentrating the filtrate and stirring the resulting solid with acetonitrile. Combined crops 15 were dried under high vacuum for 18 h to give 4’,5’-didehydro-5’-deoxyuridine (35 g. 68% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) 5 11.44 (s, 1H), 7.59 (d, J = 8.1 Hz, 1H), 5.96 (d, J = 5.4 Hz, 1H), 5.64 (d, J = 8.1 Hz, 1H), 5.60 (d, J = 5.8 Hz, 1H), 5.46 (d, J = 5.7 Hz, 1H), 4.38 (t, J = 5.5 Hz, 1H), 4.33 (s, 1H), 4.24 (q, J = 5.5 Hz, 1H), 4.17 (d, J = 1.8 Hz, 1H). 20          A 2 L three-necked round-bottomed flask was charged with 4’,5’-didehydro-5’- deoxyuridine (35 g, 154.7 mmol) and anhydrous acetonitrile (400 mL). The suspension was cooled to 0°C under argon atm and treated with triethylamine trihydrofluoride (12.6 mL, 77.4 mmol) followed by the addition of N-iodosuccinimide (45.3 g, 201.2 mmol). After 1 h at 0°C, tlc (10% methanol in methylene chloride) indicated complete conversion. While still 25 cold, the mixture was vacuum filtered. The isolated solid was washed sequentially with acetonitrile, dichloromethane, hexanes, and then dried under high vacuum for 18 h to give 5’-deoxy-4’-fluoro-5’-iodouridine (35 g, 61%). 1H NMR (400 MHz, Methanol-d4) 5 7.77 (d, J = 8.1 Hz, 1H), 6.05 (s, 1H), 5.69 (d, J = 8.1 Hz, 1H), 4.43 (dd, J = 18.2, 6.5 Hz, 1H), 4.25 (d, J = 6.6 Hz, 1H), 3.85 - 3.63 (m, 30   2H). 19F NMR (376 MHz, Methanol-d4) 5 -112.49 (ddd, J = 20.9, 18.1, 6.1 Hz). A 150 mL round-bottomed flask was charged with 5’-deoxy-5’-iodo-4’-fluorouridine (2.6 g, 6.99 mmol) and methylene chloride (35 mL). After stirring for 20 min at room temperature, the suspension was cooled to 0°C and treated with benzyl 2020418425   14 Sep 2026 chloroformate (4.49 mL, 31.44 mmol) followed by dropwise addition of 1-methylimidazole (3.34 mL, 41.93 mmol) over a 10 min period. The mixture was stirred an additional 10 min at 0°C and then allowed to slowly warm to room temperature. After 18h, the turbid mixture was diluted with methylene chloride (120 mL) and washed with 0.5M HCl solution (75 5 mL), water (50 mL), and brine (50 mL). The organic layer was separated, dried and concentrated in vacuo. The resulting residue was purified by column chromatography over silica gel (80g) eluting with a methylene chloride / methanol gradient. Pure fractions were combined and concentrated in vacuo to give 2’,3’-di-O-benzyloxycarbonyl-5’-deoxy-4’-fluoro-5’-iodouridine (4.2 g, 94% yield) as a white solid. 10          1H NMR (400 MHz, CDCI3) 5 9.02 (s, 1H), 7.44 - 7.28 (m, 10H), 7.14 (d, J = 8.0 Hz, 1H), 5.86 - 5.72 (m, 2H), 5.69 - 5.57 (m, 2H), 5.19 (d, J = 4.3 Hz, 2H), 5.09 (d, J = 3.1 Hz, 2H), 3.71 - 3.35 (m, 2H). 19F NMR (376 MHz, CDCl3) 5 -107.06 (td, J = 18.6, 7.3 Hz). In a 100 mL round-bottomed flask a 55% tetrabutylammonium hydroxide solution 15 in water (8.04mL, 9.37mmol) was adjusted to pH 3.5 by dropwise addition of trifluoroacetic acid (0.72mL, 9.37mmol) while maintaining a temperature below 25°C. The mixture was then treated with a methylene chloride (15 mL) solution of 2’,3’-di-O-benzyloxycarbonyl-5’-deoxy-4’-fluoro-5’-iodouridine (2g, 3.12 mmol) followed by addition of 3-chloroperbenzoic acid (3.6g, 15.62 mmol) in portions over a 30 min period. After one hour 20 the pH drifted to pH 1.4. The mixture was adjusted back to pH 3.5 with 1N sodium hydroxide and allowed to stir for 16 h after which time tlc (10% methanol in methylene chloride) and LCMS indicated complete conversion. The reaction mixture was quenched by addition of sodium thiosulfate (3.21g, 20.31 mmol) slowly in portions while maintaining a temperature below 25°C. After stirring for 30 min, the methylene chloride layer was 25 separated, and the aqueous layer extracted with additional methylene chloride (2 x 30 mL). Combined organic layers were dried over sodium sulfate, concentrated, and purified by column chromatography over silica gel (80 g) eluting with 60% ethyl acetate in hexanes followed by a second column of silica gel (80 g) eluting with a methylene chloride / methanol gradient to give 2’,3’-di-O-benzyloxycarbonyl-4’-fluorouridine (1.05 g , 30   63% yield) as a white solid. 1H NMR (400 MHz, CDCl3) 5 9.30 (s, 1H), 7.39 - 7.29 (m, 10H), 7.21 (d, J = 8.1 Hz, 1H), 5.83 (dd, J = 17.8, 7.0 Hz, 1H), 5.77 - 5.71 (m, 2H), 5.61 (dd, J = 7.0, 2.4 Hz, 1H), 5.17 (d, J = 4.8 Hz, 2H), 5.09 (s, 2H), 3.86 (q, J = 5.8, 4.9 Hz, 2H), 3.06 (s, 1H). 19F NMR (376 MHz, CDCl3) 5 -121.03 (dt, J = 17.7, 4.6 Hz). 2020418425   14 Sep 2026 4’-Fluorouridine 5’-O-triphosphate (EIDD-02991) A 10 mL round-bottomed flask charged with 2’,3’-di-O-benzyloxycarbonyl-4’-fluorouridine (348 mg, 0.66 mmol) and anhydrous trimethyl phosphate (3.5 mL). After stirring for 20 min at room temperature, the solution was cooled to 0°C and treated with 1 5 methyl-imidazole (115 gL, 1.44 mmol) followed by dropwise addition of phosphorus oxychloride (122 gL, 1.31 mmol) over a 40 min period. The mixture continued to stir at 0°C for 3.5h after which time tlc (10% methanol in DCM and then 7:2:1 iPa:NH4OH:water) indicated complete phosphorylation. The mixture was treated with tributylamine (0.94mL, 3.94mmol), tris(tetrabutylammonium)pyrophosphate (887 mg, 0.98 mmol), and anhydrous 10 DMF (1.5 mL). After 1h at room temperature, the reaction mixture was quenched with 100 mM TEAB (20 mL), stirred for 1h, degassed by pump-fill with argon (3x) and treated with 10% palladium on carbon (100 mg). After cooling with an ice-bath, the mixture was pump-filled with hydrogen (2x) followed by vigorous stirring under atm pressure of hydrogen for 30 min. The mixture was pump-filled with argon and then vacuum filtered through a pad of 15 Celite. The palladium was washed with water (2 x 20 mL). Combined filtrates were washed with ether (4 x 60 mL) and then concentrated in vacuo at 25°C. The residue was coevaporated with water (2 x 25 mL) and purified by column chromatography over DEAE-Sephadex GE A-25 (10 mm x 130 mm) eluting with a gradient from 100 mM to 500 mM TEAB (900 mL). Pure fractions as determined by tlc (8:1:1 NH4OH:iPrOH:water) were 20 combined and concentrated in vacuo with the bath temperature set at 25°C. The resulting solid was dissolved in methanol (1 mL) and treated with saturated solution of sodium perchlorate in acetone (10 mL). The resulting white precipitate was collected by centrifuge and washed with acetone (5 x 5 mL).The solid was dissolved in water (1 mL), frozen and lyophilized to yield 4’-fluorouridine 5’-O-triphosphate (3.14 mg, 0.81% yield) as the 25 tetrasodium form.. 1H NMR (400 MHz, D2O) 5 7.77 (d, J = 8.0 Hz, 1H), 6.15 (d, J = 1.9 Hz, 1H), 5.91 (d, J = 8.1 Hz, 1H), 4.72 - 4.57 (m, 1H), 4.41 (d, J = 6.3 Hz, 1H), 4.30 (ddd, J = 10.2, 6.3, 3.0 Hz, 1H), 4.17 (dt, J = 10.8, 5.0 Hz, 1H). 31P NMR (162 MHz, D2O) 5 -7.81(d), -11.84 (d, J = 19.2 Hz), -22.23 (t). 30         19F NMR (376 MHz, D2O) 5 -121.09 (unresolved dt, J = 19.2 Hz). LCMS Calculated for C9 H13FN2O15P3 [M-H+]: 500.9; found: 500.8. Example 83. Synthesis of EIDD-02749-5’-Isobutyl ester (EIDD-02947) 2020418425   14 Sep 2026 To a 25 mL pear-shaped flask charged with 1-[(3aS,4S)-4-fluoro-4- (hydroxymethyl)-2,2-dimethyl-6,6a-dihydro-3aH-furo[3,4-d][1,3]dioxol-6-yl]pyrimidine-2,4-dione (0.1 g, 0.33 mmol) and DMAP (2.0 mg, 0.02 mmol) was added EtOAc (1.1 mL) to give a colorless solution. The vessel was vacuumed and charged with argon. Then Et3N 5   (083.12 mL, 0.83 mmol) was added, followed by isobutyric anhydride (0.07 mL, 0.4 mmol). This reaction solution was allowed to stir at room temperature overnight. After overnight stirring, TLC showed no SM. The reaction solution was transferred into a separation funnel and water was added. The aqueous layer was separated and re-extracted with DCM once. The combined organic layers was dried (Na2SO4), filtered and 10 concentrated in vacuo. The crude material was purified by ISCO column chromatography (12 g) eluting from 100% hexanes to 80% EtOAc in hexanes to afford [(3aS,4S)-6-(2,4-dioxopyrimidin-1-yl)-4-fluoro-2,2-dimethyl-6,6a-dihydro-3aH-furo[3,4-d][1,3]dioxol-4-yl]methyl 2-methylpropanoate (0.11 g, 89%) as a white glassy solid. To a 25 mL pear-shaped flask charged with [(3aS,4S)-6-(2,4-dioxopyrimidin-1-yl)-4-fluoro-15 2,2-dimethyl-6,6a-dihydro-3aH-furo[3,4-d][1,3]dioxol-4-yl]methyl 2-methylpropanoate (0.11g, 0.3000mmol) was added 95% formic acid (12 mL, 0.3 mmol) to give a colorless solution. After stirring at room temperature for 3.5 h, solvent was removed in vacuo. Then water and Celite were added, concentrated in vacuo. The crude material was purified by ISCO column chromatography (12 g) eluting from 100% DCM to 15% MeOH in DCM to 20 afford the product with some impurity. This material was re-purified by ISCO column chromatography (12 g) eluting from 100% hexanes to 100% EtOAc to afford [(2S,3S)-5-(2,4-dioxopyrimidin-1-yl)-2-fluoro-3,4-dihydroxy-tetrahydrofuran-2-yl]methyl 2-methylpropanoate (EIDD-02947) (7.8 mg, 8% yield) after lyophilizing overnight as a white fluffy solid. 25          1H NMR (400 MHz, Methanol-d4) S 7.59 (d, J = 8.1 Hz, 1H), 5.87 (d, J = 2.1 Hz, 1H), 5.69 (d, J = 8.1 Hz, 1H), 4.51 (dd, J = 19.2, 6.9 Hz, 1H), 4.44 - 4.34 (m, 2H), 4.28 (dd, J = 11.9, 8.1 Hz, 1H), 2.72 - 2.53 (m, 1H), 1.17 (dd, J = 7.0, 4.8 Hz, 6H). 19F NMR (376 MHz, Methanol-d4) S -123.39 (dt, J = 19.1, 8.0 Hz). 2020418425   14 Sep 2026 13C NMR (101 MHz, CD3OD) 5 176.24, 164.59, 150.18, 142.88, 142.80, 116.65, 114.36, 101.82, 101.62, 95.71, 95.46, 70.98, 70.88, 70.27, 70.07, 61.44, 61.02, 33.66, 33.51, 17.90, 17.84, 17.80. Example 84. Synthesis of EIDD-02749-5’-L-Valine ester (EIDD-02971) 5 H° °H '                                                                                 EIDD-02971 To a 25 mL pear-shaped flask charged with 1-[(3aS,4S,6R,6aR)-4-fluoro-4-(hydroxymethyl)-2,2-dimethyl-6,6a-dihydro-3aH-furo[3,4-d][1,3]dioxol-6-yl]pyrimidine-2,4-dione (0.15 g, 0.5 mmol), Boc-L-Valine (0.13 g, 0.6 mmol) and DMAP (0.01g, 0.05 10 mmol) was added dry DCM (2 mL) to give a colorless solution. The reaction vessel was vacuumed and charged with argon. Then DCC (0.12 g, 0.6 mmol) was added all at once to give a white suspension. After overnight stirring, the white suspension was filtered through Celite and the solids were washed with DCM. Celite was added to the filtrate, and the filtrate was then concentrated in vacuo. The crude material was purified by ISCO column 15 chromatography (24 g) eluting from 100% hexanes to 100% EtOAc to afford [(3aS,4S,6R,6aR)-6-(2,4-dioxopyrimidin-1-yl)-4-fluoro-2,2-dimethyl-6,6a-dihydro-3aH-furo[3,4-d][1,3]dioxol-4-yl]methyl (2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoate (0.158 g, 63%). To a 10 mL pear-shaped vial charged with [(3aS,4S,6R,6aR)-6-(2,4-dioxopyrimidin- 20 1-yl)-4-fluoro-2,2-dimethyl-6,6a-dihydro-3aH-furo[3,4-d][1,3]dioxol-4-yl]methyl (2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoate (50 mg, 0.1 mmol) was added isopropyl acetate (1.3 mL) to give a colorless solution under argon. This was cooled to 0 oC and then 5-6 N HCl in IPA (0.05 mL) was added dropwisely. After 1.5 h, TLC showed mainly SM. Then more 5-6 N HCl in IPA (0.05 mL) was added and this was put in the fridge overnight. 25 The next day, some solids formed in the flask. This was filtered through a medium sintered glass frit and washed with Et2O. Since the solids were hygroscopic, it was dissolved in MeOH and concentrated in vacuo. The previous mother liquor had contained added solids, which were filtered, dissolved in MeOH, and combined with the previous solution. Concentration in vacuo gave the product with an impurity. This was re-dissolved in EtOH 30 and triturated with Et2O. After stirring for a while, this mixture was filtered and the solids were dissolved in EtOH. Then more Et2O was added and the solids were filtered after 2020418425   14 Sep 2026 stirring. Finally, the solids were dissolved in MeOH, concentrated in vacuo, dissolved in water and lyophilized overnight to afford [(1S)-1-[[(2S,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-2-fluoro-3,4-dihydroxy-tetrahydrofuran-2-yl]methoxycarbonyl]-2-methyl-propyl]ammonium chloride (EIDD-02971) (11 mg, 30%) as light yellow solids. 5 Example 85. Synthesis of EIDD-02749-2’, 3’, 5’-Isoburyl triester (EIDD-02954) o EIDD-02954 To a 50 mL rbf charged with 1-[(2R,3R,4S,5S)-5-fluoro-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]pyrimidine-2,4-dione (68 mg, 0.26 mmol) and DMAP (6.3 mg, 0.05 mmol) was added EtOAc (2.6 mL) to give a suspension. This was vacuumed 10 and charged with argon. Then Et3N (0.18 mL, 1.3 mmol) was added. The flask was cooled to 0°C and isobutyric anhydride (0.15 mL, 0.91 mmol) was added dropwisely. After 15 min, the resulting colorless solution was allowed to stir at room temperature. After 3.5 h, TLC showed no SM. Then water was added dropwisely. After stirring for 5 min, the reaction mixture was transferred into a separation funnel and more EtOAc was added. The organic 15 layer was separated, dried (Na2SO4), filtered and concentrated in vacuo with Celite. The crude material was purified by ISCO column chromatography (24 g) eluting from 100% hexanes to 100% EtOAc to afford [(2S,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-2-fluoro-3,4-bis(2-methylpropanoyloxy)tetrahydrofuran-2-yl]methyl 2-methylpropanoate (EIDD-02954) (0.1 g, 82%) aswhite solids. 20         1H NMR (400 MHz, Methanol-d4) 5 7.62 (d, J = 8.0 Hz, 1H), 5.93 - 5.78 (m, 2H), 5.69 (d, J = 7.9 Hz, 1H), 5.64 (dd, J = 7.2, 2.1 Hz, 1H), 4.35 (dd, J = 7.8, 3.5 Hz, 2H), 2.68 - 2.57 (m, 3H), 1.26 - 1.07 (m, 18H). 19F NMR (376 MHz, Methanol-d4) 5 -120.16 (dt, J = 19.6, 7.8 Hz). 25 Example 86. Synthesis of 4‘-fluoro-4-thiouridine: 2020418425   14 Sep 2026 b) Reagents and conditions; a) TBSC1, imidazole, DMF, RT, 70%; b) Lawesson reagent, K2CO3, toluene, reflux, 60%; c) TBAF, THF, 5 hr. Preparation of 2‘,3‘,5‘-tri-O-(t-butyldimethylsilyl)-4‘-fluoro-uridine To a solution of 4‘-fluoro-uridine (500 mg, 1.9 mmol) in DMF (20ml) taken in 100 ml RBF, TBDMSCl (1.2 gm, 7.6 mmol) and imidazole (650 mg, 9.5 mmol) were added 5 under inert atmosphere at 0 °C and continued stirring at room temperature. After completion, the reaction mixture was concentrated under reduced pressure and the crude product was dissolved in dichloromethane and washed with saturated aq. NaHCO3 followed by brine. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by chromatography. Product was obtained 10 as colorless foam (yield 58%). Preparation of 2‘,3‘,5‘-tri-O-(t-butyldimethylsilyl)-4‘-fluoro-4-thiouridine To a solution of 2‘,3‘,5‘-tri-O-(t-butyldimethylsilyl)-4'-fluoro-uridine (600 mg, 1 mmol) in anhydrous THF (20 ml), Lawesson’s reagent (freshly purchased) (590 mg, 1.5 mmol) and potassium carbonate (29 mg, 0.2 mmol) were added and the reaction mixture 15 was refluxed for 5 hr. After completion the reaction mixture was concentrated under reduced pressure and the crude product was purified by column chromatography. Product was obtained as colorless foam (yield 52%). Preparation of 4‘-fluoro-4-thiouridine To a solution of 2‘,3‘,5‘-tri-O-(t-butyldimethylsilyl)-4'-fluoro-4-thiouridme (250 mg, 20   0.41 mmol) in anhydrous tetrahydrofuran (5 ml), 1M solution of tetrabutylammonium fluoride (2 ml) was added and stirred at room temperature for 5 hr. After completion, the reaction mixture was concentrated under reduced pressure and the crude product was purified by silica gel column chromatography. 2020418425   14 Sep 2026 1H NMR 400 MHz, CD3OD, 5 7.77 (1H, d, J = 8 Hz), 6.06 (1H, d, J = 4 Hz), 5.69 (1H, d, J = 8 Hz), 4.42 (1H, dd, J = 6.4 Hz, 20 Hz), 4.25 (1H, dd, 6.4 Hz, 2.4 Hz), 3.73 (2H, m); 19F NMR 376 MHz 5 -123.57, (1F, dt, J = 18.8 Hz, 3.7 Hz) Example 87. Synthesis of 1‘-deutero-4‘-fluorouridine Reagents and conditions; a) NaBD4, THF, H2O, 0 °C, 3-4 hr, 65%; b) Ac2O, pyridine, RT, 70%, c) i) 80% AcOH, 50 °C, 12 hr; ii) Ac2O, pyridine, RT, 62%; d) i) uracil, HMDS, 126 °C, (NH4)2SO4; ii) DCE, SnCl4, reflux, 5 hr, 50%; e) NH3, MeOH, RT, TBAF, THF, 5 hr; ii) Iodine, PPh3, Imidazole, THF; f) NaOMe, MeOH, 65 °C; ii) Ac2O, DMAP, MeCN, RT; g) NIS, (HF)3.Et3N, MeCN, 0 °C to RT; h) mCPBA, TBAHS, K2HPO4, DCM, H20; i) NH3, MeOH, RT Preparation of 1-deutero-2,3-O-isopropylidene-D-ribofuranose To a solution of 2,3-O-isopropylidene-D-ribonolactone (3 g, 16 mmol) in 9:1 (THF: H2O) (50ml), taken in a 250 ml RBF, NaBD4 (1g, 24 mmol) was added slowly in portions at 0 °C with continued stirring. After completion, the reaction mixture was quenched with 10 acetone and stirred at room temperature for additional 30 min. The reaction mixture was diluted with excess ethyl acetate (100 ml) and washed with saturated aq. NH4Cl followed by saturated aq. NaHCO3 and brine. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by chromatography. The product was obtained as colorless oil (yield 65%). 15 2020418425   14 Sep 2026 Preparation of 1,5-di-O-acetyl-1-deutero-2,3-O-isopropylidene-D-ribofuranose To a solution of 1-deutero-2,3-O-isopropylidene-D-ribofuranose (1.9 g, 10 mmol) in DCM (50 ml), acetic anhydride (2.4 ml, 25 mmol), trimethylamine (4.2 ml, 30 mmol), and DMAP ( 195 mg,1.6 mmol) were added at 0 °C. Stirring continued at room temperature. 5 After completion, the reaction mixture was washed with saturated aq. NH4Cl followed by saturated aq. NaHCO3 (twice) and brine. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by chromatography. Product was obtained as colorless syrup (yield 70%). Preparation of 1,2,3,5-tetra-O-acetyl-1-deutero-D-ribofuranose 10           1,5-di-O-acetyl-1-deutero-2,3-O-isopropylidene-D-ribofuranose (2g, 7.2 mmol) was dissolved in 80% acetic acid (50 ml) in 100 ml RBF and stirred at 50 °C for 12 hr. After completion, the reaction mixture was concentrated under reduced and co-evaporated with toluene twice. The crude product was dissolved in pyridine (20 ml). Acetic anhydride (1.7 ml, 18 mmol) and DMAP (122 mg, 1 mmol) were added at 0 °C and stirring continued at 15 room temperature. After completion, the reaction mixture was concentrated under reduced pressure and the crude product was dissolved in dichloromethane and washed with 5% aq. HCl followed by saturated aq. NaHCO3 and brine. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by chromatography. Product was obtained as syrup which crystallizes upon standing (yield 20   62% for 2 steps). Preparation of 2’,3’,5’-tri-O-acetyl-1’-deutero-uridine To a suspension of uracil (670 mg, 6 mmol) in HMDS (10 ml) in a 100 ml RBF, catalytic ammonium sulfate was added and refluxed at 126 °C under inert atmosphere for 12 hr. The reaction mixture cooled down and concentrated under reduced pressure. The residue 25 was subjected to high vacuum and charged with anhydrous acetonitrile, compound 1,2,3,5-tetra-O-acetyl-1-deutero-D-ribofuranose (950 mg, 3 mmol) in acetonitrile and tin tetrachloride (350 ^L, 3 mmol). The reaction mixture was refluxed under inert atmosphere for 5 hr. After completion, the reaction mixture was quenched with solid NaHCO3 and Celite and stirred at room temperature for 30 min. A few drops of saturated aq. NaHCO3 30 and continued stirring for 2-3 hr. The white precipitate formed was filtered and washed with DCM, the filtrate was concentrated under reduced pressure and purified by column chromatography. Product was obtained as colorless solid (yield 50%). Preparation of 5’-deoxy-1’-deutero-5’-iodo-uridine 2020418425   14 Sep 2026 To a solution of 2’,3’,5’-tri-O-acetyl-1’-deutero-uridine (745 mg, 2 mmol) in methanol (10 ml), 7N ammonia in methanol was added and stirred at room temperature. After completion, the reaction mixture was concentrated under reduced pressure. The crude product was triturated with ethyl acetate and the resulting solid was taken in a 100 ml RBF 5 and suspended in THF. Triphenylphosphine (786 mg, 3 mmol), imidazole (200 mg, 3 mmol) and iodine (600 mg, 2.3 mmol) were added and stirred at room temperature for 8 hr. After completion, the reaction mixture was concentrated under reduced pressure and the residue was stirred with isopropanol. The colorless solid formed was filtered and dried (yield 45%). 10 Preparation of compound 2’,3’-di-O-acetyl-1’-deutero-5’-deoxy-4’,5’-didehydrouridine To a solution of 5’-deoxy-1’-deutero-5’-iodo-uridine (530 mg, 1.5 mmol) in methanol, sodium methoxide 25% by weight in methanol (325 gL) was added and stirred at 65 °C under inert atmosphere. After completion, the reaction mixture was concentrated 15 under reduced pressure. The crude product was taken in MeCN (10ml) and treated with acetic anhydride (425 gL, 4.5 mmol) and DMAP (20 mg, 0.15 mmol) and stirred at room temperature for 12 hr. After completion, the reaction mixture was quenched with saturated aq. NaHCO3, diluted with DCM, washed with saturated aq. NaHCO3 and brine. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced 20 pressure. The residue was purified by chromatography yielding product as colorless solid. g) Preparation of compound 2’,3’-di-O-acetyl-1’-deutero-5’-deoxy-5’-iodo-4’-fluorouridine To a solution of compound 2’,3’-di-O-acetyl-1’-deutero-5’-deoxy-4’,5’-didehydrouridine (460 mg, 2mmol) in anhydrous acetonitrile (5ml) in 50 ml RBF, 25 triethylamine trihydrofluoride (162 gL, 1 mmol) and N-iodosuccinimide (2.6 mmol) were added at 0 °C. After 60 min, the reaction mixture was slowly warmed to room temperature. After completion, the reaction mixture was concentrated under reduced pressure and purified by column chromatography. h) Preparation of compound 2’,3’-di-O-acetyl-1’-deutero-5’-m-chlorobenzoate-4’- 30 fluorouridine To a solution of 2’,3’-di-O-acetyl-1’-deutero-5’-deoxy-5’-fluoro-4’-iodouridine (460 mg, 1 mmol) in 5:1 (DCM:H2O) (50 ml) in a 100 ml RBF, tetrabutylammonium hydrogen sulfate (370 mg, 1.1 mmol) and potassium phosphate dibasic (260 mg, 1.5 mmol) were added, and the reaction mixture was cooled to 0 °C. meta-chloroperbenzoic acid (860mg, 4 2020418425   14 Sep 2026 mmol) was added slowly in portions and reaction mixture was allowed to warm to room temperature and vigorous stirring was continued for another 12 hr. After completion, the reaction mixture was quenched with aq. Na2SO3 and diluted with DCM (30 ml). The organic layer was separated and washed with saturated aq. NaHCO3 and brine. The 5 combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by chromatography. i) Preparation of 1’-deutero-4’-fluorouridine To a solution of 2’,3’-di-O-acetyl-1’-deutero-5’-m-chlorobenzoate-4’-fluorouridine (250 mg, 0.5 mmol) in methanol (10 ml), 7N ammonia in methanol (2 ml) was added and 10 stirred at room temperature. After completion, the reaction mixture was concentrated under reduced pressure and purified by column chromatography. Example 88. Synthesis of 4‘-fluoro-carbauridine Reagents and conditions; a) 2,2-dimethoxypropane, pTSA.H20; 8 hr; b) Iodine, PPh3, Imidazole, THF; c) NaOMe, MeOH, 65 °C; d) i) AgF, Iodine, DCM, 0 °C to RT; e) mCPBA, TBAHS, K2HPO4, DCM, H2O; f) 80% AcOH, 50 °C ii) NH3, MeOH, RT. To a suspension of compound carbauridine (2.5 gm, 10 mmol) in anhydrous acetone 15   (200 ml), 2,2-dimethoxypropane (1.2 ml, 10 mmol) and concentrated sulfuric acid (200 DL, 2 mmol) were added at 0 °C under inert atmosphere and stirring continued at room temperature. After completion, the reaction mixture was quenched with NaHCO3, stirred for 30 min, and was filtered. The filtrate was concentrated under reduced pressure and the residue was purified by column chromatography. 20 2020418425   14 Sep 2026 To a solution of the above synthesized acetonide (1.4 gm, 5 mmol) in THF (100ml) in a 250 ml RBF, triphenylphosphine (2 gm, 7.5 mmol), imidazole (500 mg, 7.5 mmol) and iodine (1.4 gm, 5.5 mmol) were added at 0 °C under inert atmosphere and stirred at room temperature for 8 hr. After completion, the reaction mixture was concentrated under 5 reduced pressure and the residue was taken up in isopropanol (100ml) and stirred at room temperature. The colorless solid formed was filtered and dried. To a solution of the iodo compound synthesized above (1 gm, 2.5 mmol) in methanol, sodium methoxide 25% by weight in methanol (1.1 ml, 5 mmol) was added and stirred at 65 °C under an inert atmosphere. After completion, the reaction mixture was 10 concentrated under reduced pressure. The crude product was dissolved in DCM (100 ml) and filtered through a Celite bed. The filtrate was concentrated under reduced pressure and the residue was purified by column chromatography. To a solution of the alkene product obtained above (800 mg, 3 mmol) in anhydrous DCM (50ml) in a 100 ml RBF, silver fluoride (950 mg, 7.5 mmol) was added followed by 15 dropwise addition of iodine (1.5 gm, 6 mmol) in THF. After addition, the reaction mixture was slowly allowed to warm to room temperature and stirred for additional 30 min at room temperature. After completion, the reaction mixture was filtered through a Celite bed and the filtrate was washed with saturated aq. Na2S2O3 followed by saturated aq. NaHCO3 and brine. The combined organic layers were dried over Na2SO4, filtered, and concentrated 20 under reduced pressure. The residue was purified by chromatography. To a solution of the above compound (610 mg, 1.5 mmol) in 5:1 (DCM:H2O) (50 ml) in a 100 ml RBF, tetrabutylammonium hydrogen sulfate (560 mg, 1.65 mmol) and potassium phosphate dibasic (400 mg, 2.3 mmol) were added. The reaction mixture was cooled to 0 °C. m-Chloroperbenzoic acid (1.0 gm, 6 mmol) was added slowly in portions, 25 and the reaction mixture was allowed to warm to room temperature. Vigorous stirring was continued for another 12 hr. After completion, the reaction mixture was quenched with aq. Na2SO3 and diluted with DCM (50ml). The organic layer was separated and washed with saturated aq. NaHCO3 and brine. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by 30 chromatography. The above compound (450 mg, 1 mmol) was taken up in 80% acetic acid (20 ml) and stirred at 50 °C for 12 hr. After completion, the reaction mixture was concentrated under reduced pressure and co-evaporated twice with anhydrous toluene. The residue was taken in methanol (20 ml) and treated with 7N ammonia in methanol (2 ml) and stirred at 2020418425   14 Sep 2026 room temperature. After completion, the reaction mixture was concentrated under reduced pressure and purified by column chromatography to provide the final desired product. Example 89. Synthesis of 4’-fluoro-2-thiouridine: TBSO' 'OTBS HO' OH Reagents and conditions; a) i) TIPSC1, DMAP, Et3N, DCM; (ii) 2,6-dimethylphenol, Et3N, DCM, RT 5-6 hr; 53% b) NH3 in MeOH, RT, 88%; c) TBSC1, imidazole, DMF, RT, 71%; d) Lawesson reagent, K2CO3, toluene, reflux, 74%; e) TMG, syn-o-nitrobenzaldoxime, MeCN; f) TBAF, THF, 2-3 hr, 74%. Preparation of 4-O-(2,6-dimethylphenyl)-2‘,3‘-di-O-acetyl-5‘-O-(4-chlorobenzoyl)-4‘-fluorouridine 2‘,3'-di-O-acetyl-5'-O-(4-chlorobenzoyl)-4‘-fluorouridine (1gm, 2mmol) was dissolved in anhydrous dicholoromethane (30 ml) in 100 ml RBF. EtsN (542 gL, 3.75 10 mmol), 2,4,6-triisopropylbenzensulfonyl chloride (690 mg, 2.26mmol), and 4-(dimethylamino)pyridine (62 mg, 0.5 mmol) were added added at 0 °C under an inert atmosphere with continued stirring at room temperature. After completion of the reaction, 2,6-dimethylphenol (300 mg, 2.45 mmol), Et3N (3.45 mL, 25 mmol), and 1,4-diazabicyclo[2,2,2]octane (23 mg, 0.2 mmol) were added at 0 °C under an inert atmosphere 15 with continued stirring at room temperature for 3-4 hr. The reaction mixture was diluted with dichloromethane (30ml) and washed once with saturated NaHCO3 (aqueous) and twice with brine. The combined organic extracts were dried over Na2SO4, filtered, and 2020418425   14 Sep 2026 concentrated under reduced pressure. The residue was purified by column chromatography. Product was obtained as colorless solid (yield 53%). Preparation of 4-O-(2,6-Dimethylphenyl)-4‘-fluorouridine To a solution of 4-O-(2,6-Dimethylphenyl)-2',3‘-di-O-acetyl-5'-O-(4- 5   chlorobenzoyl)-4'-fluorouridine (600 mg) in anhydrous methanol (6 ml) in 25 ml RBF, 1 ml of 7N ammonia in methanol was added and stirred at room temperature for 8 hr. After completion, the reaction mixture was concentrated under reduced pressure and the crude product was obtained as colorless solid (yield 88%). Preparation of 4-O-(2,6-dimethylphenyl)-2‘,3',5‘-tri-O-(t-butyldimethylsilyl)-4‘-10 fluorouridine To a solution of 4-O-(2,6-dimethylphenyl)-4‘-fluorouridine (720mg) in anhydrous DMF (10 ml) in a 50 ml RBF, tert-butyldimethylsilyl chloride (1185 mg, 7.8 mmol) and imidazole (670 mg, 9.8 mmol) were added at 0 °C under an inert atmosphere with continued stirring at room temperature for 12 hr. After completion the reaction mixture was 15 concentrated under reduced pressure and the crude product was taken up in DCM and washed with saturated aq.NaHCO3 and with brine. The combined organic extracts were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to get colorless foam (yield 71%) Preparation of 4-O-(2,6-dimethylphenyl)- 2‘,3‘,5‘-tri-O-(t-butyldimethylsilyl)-4‘-20 fluoro-2-thiouridine To a solution of 4-O-(2,6-dimethylphenyl)- 2',3‘,5'-tri-O-(t-butyldimethylsilyl)-4'-fluorouridine (750 mg, 1 mmol) in anhydrous toluene (20 ml), Lawesson’s reagent (freshly purchased) (590 mg, 1.5 mmol) and potassium carbonate (29 mg, 0.2 mmol) were added and the reaction mixture was refluxed for 8 hr. After completion, the reaction mixture was 25 concentrated under reduced pressure and the crude product was purified by column chromatography. Product was obtained as colorless foam (yield 74%). Preparation of 2‘,3‘,5‘-tri-O-(t-butyldimethylsilyl)-4‘-fluoro-2-thiouridine To a solution of 4-O-(2,6-dimethylphenyl)-2‘,3‘,5‘-tri-O-(t-butyldimethylsilyl)-4‘-fluoro-2-thiouridine (500 mg, 0.68 mmol) in acetonitrile (10 ml), 1,1,3,3 30 tetramethylguanidine (260 ^L, 2 mmol) and syn-o-nitrobenzaldoxime (343 mg, 2 mmol) were added and stirred at room temperature for 5 hr. After completion, the mixture was concentrated under reduced pressure. The crude product was dissolved in dichloromethane and washed with saturated aq. NaHCO3 and with brine. The combined organic layers were 2020418425   14 Sep 2026 dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by chromatography. Product was obtained as colorless foam (yield 67%). Preparation of 4‘-fluoro-2-thiouridine To a solution of 2‘,3‘,5‘-tri-O-(t-butyldimethylsilyl)-4‘-fluoro-2-thiouridine (270 mg, 5   0.43 mmol) in anhydrous tetrahydrofuran (5 ml), 1M solution of tetrabutylammonium fluoride (2 ml) was added and stirred at room temperature for 5 hr. After completion, the reaction mixture was concentrated under reduced pressure and the crude product was purified by silica gel column chromatography. Product was obtained as off white solid (yield 74%). 10          1H NMR 400 MHz, CD3OD, 5 8.11 (1H, d, J = 8 Hz), 6.84 (1H, s), 5.94 (1H, d, J = 8 Hz), 4.26 (2H, m), 3.78 (2H, m); 13C NMR 100 MHz 5 176.49, 159.90, 140.78, 119.46, 117.16, 107.34, 95.08, 72.83, 68.59, 59.80; 19F NMR 376 MHz 5 -122.77, (1F, d, J = 18.8 Hz); LCMS: [M+1]+ 279.0. Example 90. Protocol for Determining Plasma Stability 15          Test article was incubated in triplicate at 1.00 gM in pooled mixed gender human plasma (BioIVT, K2EDTA), in pooled male CD-1 mouse plasma (BioIVT, K2EDTA), in pooled male Sprague-Dawley rat plasma (BioIVT, lithium heparin). Incubations were performed in 13 x 100 mm glass culture tubes. Samples were placed in a water bath shaker set at 37°C and shaken at 150 rpm. Procaine, Benfluorex or Enalapril (1 gM, each) were 20 run in parallel as a positive controls for human, mouse or rat plasma activity, respectively. Aliquots of 100 gL were taken at the following time-points: 0, 5, 15, 30, 60, and 120 minutes. These aliquots were mixed with 400 gL of 100% acetonitrile in 1.7-mL conical polypropylene microcentrifuge tubes. Samples were vortexed for about 10 seconds and then clarified by centrifugation (2 minutes at 15,000 g). Supernatants were analyzed by LC-25 MS / MS. HPLC separation was performed on an Agilent 1200 system (Agilent Technologies, Santa Clara, CA, USA) equipped with a column oven, UV lamp, and binary pump. A Thermo Hypercarb PGC (150 x 4.6 mm, 5 gm) column (ThermoFisher, Waltham, MA USA) was used for the separation. Mobile Phase A consisted of 100 mM Ammonium 30 Bicarbonate buffer in HPLC grade Water (pH 10) and Mobile phase B consisted of neat acetonitrile. A gradient 0-85% of B was run for 3 minutes followed by 0% B for 4 minutes was used for the separation. Mass Spectrometry analysis was performed on a Triple Quad 5500 Mass Spectrometer (AB Sciex, Farmingham, MA, USA) using Negative Mode 2020418425   14 Sep 2026 Electrospray Ionization (ESI) in Multiple Reaction Monitoring (MRM) Mode. Data analysis was performed using Analyst Software (AB Sciex, Farmingham, MA, USA). Analyte concentrations were calculated based on standard curve. Half-lives (t1 / 2) were calculated by plotting the natural logarithm of the analyte concentration vs. time and 5 obtaining the slope of the line. Assuming first-order kinetics, the elimination rate constant, k, is the negative (-) of the slope of the plot (In [pM] vs. time). Half-life (ti / 2) (min) =-0.693 / (slope). Example 91. Protocol for Determining Liver Microsome Stability Test article was incubated in triplicate at i.00 pM in i00 mM phosphate buffer (pH i0 7.4), Phase I cofactors (NADPH Regenerating System) and 0.5 mg (total protein) from pooled gender human liver microsomes (BioIVT), pooled male CD-i mouse liver microsomes (XenoTech) or pooled male Sprague-Dawley rat liver microsomes (BioIVT). Incubations were performed in i3 x i00 mm glass culture tubes. Samples were placed in a water bath shaker set at 37°C and shaken at i50 rpm. Verapamil (i pM) was run in parallel i5 as a positive control. HPLC separation was performed on an Agilent i200 system (Agilent Technologies, Santa Clara, CA, USA) equipped with a column oven, UV lamp, and binary pump. A Thermo Hypercarb PGC (i50 x 4.6 mm, 5 pm) column (ThermoFisher, Waltham, MA USA) was used for the separation. Mobile Phase A consisted of i00 mM Ammonium 20 Bicarbonate buffer in HPLC grade Water (pH i0) and Mobile phase B consisted of neat acetonitrile. A gradient 0-85% of B was run for 3 minutes followed by 0% B for 4 minutes were used for the separation. Mass Spectrometry analysis was performed on a Triple Quad 5500 Mass Spectrometer (AB Sciex, Farmingham, MA, USA) using Negative Mode Electrospray Ionization (ESI) in Multiple Reaction Monitoring (MRM) Mode. Data analysis 25 was performed using Analyst Software (AB Sciex, Farmingham, MA, USA). Analyte concentrations were calculated based on Standard curve. Half-lives (ti / 2) were calculated by plotting the natural logarithm of the analyte concentration vs. time and obtaining the slope of the line. Assuming first-order kinetics, the elimination rate constant, k, is the negative (-) of the slope of the plot (ln [pM] vs. time). Half-life (ti / 2) (min) =-30   0.693 / (slope). Example 92. Protocol for Determining pH Stability Test article in methanol, water, 0.iN HCl, PBS or pH9 buffer were placed in the HPLC autosampler set at 25°C or 4°C. Samples were injected on the LC-MS / MS at times: 0, i, 2, 3, 4, 6 and 24 hours. HPLC separation was performed on an Agilent i200 system 2020418425   14 Sep 2026 (Agilent Technologies, Santa Clara, CA, USA) equipped with a column oven, UV lamp, and binary pump. A Thermo Hypercarb PGC (100 x 4.6 mm, 5 pm) column (ThermoFisher, Waltham, MA USA) was used for the separation. Mobile Phase A consisted of 25 mM ammonium bicarbonate buffer in HPLC grade water (pH 9.4) and 5 Mobile phase B consisted of neat acetonitrile. Initial mobile phase conditions of 5%B were held for a minute. A gradient 5-60% of B was run for next 7 minutes, followed by reequilibration of the column, was used. Mass Spectrometry analysis was performed on a QTRAP 5500 Mass Spectrometer (AB Sciex, Framingham, MA, USA) using Negative Mode Electrospray Ionization (ESI) in Multiple Reaction Monitoring (MRM) Mode and 10 UV at 260 nm. Data analysis was performed using Analyst Software (AB Sciex, Framingham, MA, USA). Stability was determined by the % UV peak area change from the time-zero samples. Example 93. Stability of EIDD-02749 in Solvents and Buffers The stability of EIDD-02749 in solvents and acidic, neutral, and basic buffers is 15 shown in Figures 1-5. 20 25 30 2020418425   14 Sep 2026 Example 94. Stability of EIDD-02749 Prodrugs species matrix drug (1.00 rM, each) half-life (min) max

[2749] , rM human plasma EIDD-2749 (nuc) 26 NA EIDD-2838 (cycloSal) 12 0.0521 EIDD-2947 (mono-ester) 2 0.653 EIDD-2954 (tri-ester) 2 0.0390 EIDD-2971 (valine) 81 0.0152 liver microsomes EIDD-2749 (nuc) 22 NA EIDD-2838 (cycloSal) 7 0.130 EIDD-2947 (mono-ester) 44 0.209 EIDD-2954 (tri-ester) <1 0.342 EIDD-2971 (valine) 38 0.179 mouse plasma EIDD-2749 (nuc) 37 NA EIDD-2838 (cycloSal) 1 0.0216 EIDD-2947 (mono-ester) 1 1.18 EIDD-2954 (tri-ester) 1 0.716 EIDD-2971 (valine) 59 0.267 liver microsomes EIDD-2749 (nuc) 27 NA EIDD-2838 (cycloSal) 7 0.0347 EIDD-2947 (mono-ester) 1 0.602 EIDD-2954 (tri-ester) <1 0.256 EIDD-2971 (valine) 11 0.305 rat plasma EIDD-2749 (nuc) 22 NA EIDD-2838 (cycloSal) <1 0.0341 EIDD-2947 (mono-ester) 9 0.518 EIDD-2954 (tri-ester) 1 0.142 EIDD-2971 (valine) 50 0.155 liver microsomes EIDD-2749 (nuc) 25 NA EIDD-2838 (cycloSal) 8 0.218 EIDD-2947 (mono-ester) 10 0.423 EIDD-2954 (tri-ester) <1 0.149 EIDD-2971 (valine) 33 0.203 Example 95. EIDD-02991 Concentrations in Huh-7 Cells EIDD-02991 concentrations in Huh-7 cells incubated with EIDD-02749, EIDD- 5   02947, EIDD-02954, or EIDD-02971 are shown in Figure 6. Example 96. EIDD-02991 Concentrations in Vero Cells EIDD-02991 concentrations in Vero cells incubated with EIDD-02749, EIDD-02947, EIDD-02954, or EIDD-02971 are shown in Figure 7. Example 83. Mouse PK protocol 10         Female ICR (CD-1) mice (from Envigo) between the ages of 7 to 8 weeks were acclimated to their environment for at least three days prior to dosing. Mice were weighed at least once before dosing to determine the dosing volume. 2020418425   14 Sep 2026 Test article was dissolved in sterile saline at 1 mg / mL for IP dosing. For oral dosing, test article was resuspended in 10 mM trisodium citrate / 0.5% Tween 80 / Water. For IP dosing mice were dosed with a 10 mL / kg dose volume and mice dosed PO were dosed with a 10 mL / kg dose volume. 5          Blood samples collected from mice dosed by oral gavage were collected pre-dose, 0.25, 0.50, 1, 2, 3, 4, 8, and 24 hours post-dose. Blood samples collected from mice dosed by intraperitoneal injection were collected pre-dose, 0.08, 0.25, 0.50, 1, 2, 3, 4, and 8 hours post-dose. Blood samples were collected by reto-orbital bleeding under isoflurane anesthesia into lithium-heparin microtainer tubes, centrifuged at 2000 x g for 10 min at 5 C, 10 and the plasmas were transferred into fresh tubes and stored at -80 C before processing for quantitation by LC-MS / MS. 50 pL aliquots of mouse plasma were extracted with 950 pL of acetonitrile that included EIDD-2216 as an Internal Standard. Samples were clarified by centrifugation at 20,000 x g at 4 °C for 10 min. The clarified supernatants were transferred to HPLC vials for 15 analysis. Samples were maintained at 4 °C in a Leap Pal Autosampler (CTC Analytics AG, Zwingen, Switzerland). HPLC separation was performed on an Agilent 1200 system (Agilent Technologies, Santa Clara, CA, USA) equipped with a column oven, UV lamp, and binary pump. An Agilent SB-Phenyl (150 x 4.6 mm, 5 pm) column (Agilent 20 technologies, Santa Clara, CA, USA) was used for the separation. Mobile Phase A consisted of 100 mM Ammonium Formate buffer in HPLC grade Water and Mobile phase B consisted of pure acetonitrile. An initial 1 minute isocratic step was used at 5% Mobile Phase B followed by a 1.5 minute gradient to 100% Mobile Phase B, which was held for 1.5 minutes before returning to starting conditions for 1.5 minutes. Mass Spectrometry analysis 25 was performed on an QTRAP 5500 Mass Spectrometer (AB Sciex, Farmingham, MA, USA) using Negative Mode Electrospray Ionization (ESI) in Multiple Reaction Monitoring (MRM) Mode. Data analysis was performed using Analyst Software (AB Sciex, Farmingham, MA, USA). PK parameters are calculated using the Phoenix WinNonLin 6.4 (Build 6.4.0.768) 30 Non-compartmental analysis tool (Certara, Princeton, NJ, USA). Bioavailability is calculated by comparing the exposure (AUCinf) after oral dosing with the exposure after intraperitoneal dosing. 2020418425   14 Sep 2026 Example 84. EIDD-02749 Mouse PK Results Graphs for EIDD-02749 CD-1 mouse PK are shown in Figure 8. The PK parameters are shown in the table below. Dose (mg / kg) ^max (h) On ax (nmol / mL) (nmol / mL) AUC-INF (h*nmol / mL) Cm„ / Dose (kg*nmol / mL / mmol) AUC-INF / Dose (h*kg*nmol / mL / mmol) 11 / 2 (h) Bioavailability* 10-IP 0.25 7.7 ±2.3 0.75 ±0.1 19.6 202.1 514.5 6.3 N / A 5O-PO 0.5 35.2 ±9.7 2.8 ±1.1 232.3 184.3 1216.0 8.3 236% 150-PO 1 43.6 ±2.8 3.4±1.3 392.3 76.2 685.8 8.5 133% 500-PO 1 76.0 ±15.3 9.8 ±4.2 755.2 39.9 396.0 12.9 77.0% 5 Example 85. Mouse Tolerability Protocol AG129 mice between the ages of 6 to 10 weeks were acclimated to their environment for at least three days prior to dosing. Mice were weighed daily as well as monitored for morbidity and mortality daily. For oral dosing, test article was resuspended in 10 mM trisodium citrate / 0.5% Tween 80 / Water. Mice dosed PO were dosed with a 10 10 mL / kg dose volume. Mice were dosed PO at 10, 30, and 100 mg / kg QD for 10 days. Example 86. Tolerability of EIDD-02749 in AG129 Mice Results for tolerability of EIDD-02749 in AG129 mice arer shown in Figure 9. Example 87. MERS Activity for EIDD-02749 Virus Strain Cell Line EC5o(gM) CC50(gM) MERS EMC Vero 76 5.7 >380 15 Example 88. Measles Activity for EIDD-02749 Virus Strain Cell Line EC50(gM) CC50(pM) Measles CC Vero 76 1.06 >380 20 Example 89. Picornaviridae Activity for EIDD-02749 Virus Strain Cell Line EC5o(^M) EC90 (pM) CC5o(pM) Coxsackie HA 201933 Vero 76 1.63 7.22 372 ENTV-68 US / KY / 14-18953 RD 0.65 1.06 228 Poliovirus Mahoney Vero 76 2.01 3.8 > 380 Example 90. Togaviridae Activity for EIDD-02749 25 2020418425   14 Sep 2026 Virus Strain Cell Line EC5o(^M) EC90 (pM) CC5o(^M) EEEV Fl39-939 Vero 76 0.65 1.79 > 380 Mayaro BEAR 505411 Vero 76 2.81 9.5 > 380 Example 91. Hantaviridae Activity for EIDD-02749 Virus Cell Line EC5o(^M) CC5o(^M) Andes Vero 0.11 > 100 Andes Vero 0.035 > 100 Andes Vero 0.028 > 100 Example 92. Arenaviridae Activity for EIDD-02749 Virus Strain Cell Line EC5o(gM) EC90 (MM) CC5o(MM) Tacribe TRVL 11573 Vero < 0.12 < 0.12 > 380 Tacribe TRVL 11573 Vero < 0.12 < 0.12 > 380 Tacribe TRVL 11573 Vero 0.13 0.33 > 380 Pichinde An 4763 Vero 3.5 0.32 > 380 Pichinde An 4763 Vero 1.9 0.36 > 380 Junin Candid #1 Vero 0.33 < 0.12 > 380 Junin Candid #1 Vero 0.35 < 0.12 > 380 Junin Romero HeLa 0.02 - 77 Junin Romero Huh-7 0.25 - > 100 Junin Romero Vero E6 0.08 - > 100 Lassa Josiah HeLa 0.45 - 77 Lassa Josiah Huh-7 0.9 - > 100 Lassa Josiah Vero E6 0.36 - > 100 Machupo Carvallo HeLa 0.07 - 77 Machupo Carvallo Huh-7 0.31 - > 100 Machupo Carvallo Vero E6 1.49 - > 100 Lujo Zambia 2008 HeLa 0.07 - 77 Lujo Zambia 2008 Huh-7 1.32 - > 100 Lujo Zambia 2008 Vero E6 0.83 - > 100 10 Example 93. Cytotoxicity for EIDD-02749 Cell Line CEM Huh7 HG23 HepG2 / Gal* BxPC-3 A204 RD HBTECs BEAS-2B BEAS-2B / Gal* A549 Species Human Human Human Human Human Human Human Human Human Human Human Cell Type Lymphoid Liver Liver Liver Pancreas Muscle Muscle Respiratory Tract Lung Lung Lung CCs0(nM) 107 >400 (n=3) 100 >400 >400 >400 >380 >500 >500 250 >400 Cell Line Hep-2 HeLa IEC-6 H9c2 RAW 264.7 Vero MA-104 MDCK MDCK / Gal* BHK-T7 Species Human Human Rat Rat Mouse Green Monkey Green Monkey Canine Canine Hamster Cell Type Larynx Cervix Small Intestine Heart Macrophage Kidney Kidney Kidney Kidney Kidney CC^nM) >500 55 ±32 (n=2) >400 >400 > 100 >380 (n=42) >380 (n=2) >380 (n=6) 444 >500 2020418425   14 Sep 2026 Example 94. Mouse PK Parameters for EIDD-02749 Graphs for EIDD-02749 CD-1 mouse PK are shown in Figure 10. The PK parameters are shown in the table below. Dosing Route Dose (mg / kg) Tmax (h) Cmax (nmol / mL) Ti / 2 (h) Auc (0-~) (h*nmol / mL) IP 10 0.25 7.7 6.3 19.6 PO 1.5 0.25 1.2 17.9 6.58 PO 5 0.25 7.1 15.2 28.6 PO 15 0.5 23.3 20.3 86.9 PO 50 0.5 35.2 8.28 232.3 PO 150 1 43.6 8.46 392.3 PO 500 1 76 12.9 755.2 Example 95. Ferret PK Parameters for EIDD-02749 10 Graphs for EIDD-02749 ferret PK are shown in Figure 11. The PK parameters are shown in the table below. Dose (mg / kg) tmax (h) Cmax {nmol / mL} AUC-INF (h*nmol / mL) CmaJ1 / Dose (kg*nmol / mL / mmol) AUC-INF / Dose (h*kg*nmol / mL / mmol) tV2 (h) 15 1.0 1 0.0 34.81 6.5 154.01 27.6 608.21 114.0 2691.21 483.7 5.9710.35 50 2.3 1 1.5 63.31 10.9 413.1178.1 332.0157.5 2166.11 409.8 4.310.80 Example 96. Guinea Pig PK Parameters for EIDD-02749 15 Graphs for EIDD-02749 Guinea pig PK are shown in Figure 12. The PK parameters are shown in the table below. Dose (mg / kg) tmax (h) r „ max (nmol / mL) AUC. . (h•nmol / mL) Cmax / Dose (kg-nmol / mL-mmol) AUC0 . / Dose (h-kg-nmol / mL-mmol) Vd (L / kg) CL (L / h-kg) t1 / 2 (h) 5 IV 0.083 143.5 ± 115.4 107.5 ± 4.5 7525.0 ± 6050.9 5639.4 ± 234.2 2.4. ± 0.4 0.18 ± 0.01 9.3 ± 1.3 5 PO 1.3 ± 0.5 20.1 ± 2.3 92.9 ± 16.1 1054.5 ± 118.8 4869.4 ± 841.9 13.0 ± 1.6 1.3 ± 0.5 20.1 ± 2.3 50 PO 1.8 ± 0.5 86.4 ± 9.8 557.5 ± 39.9 453.0 ± 51.2 2923.1 ± 209.6 14.9 ± 1.9 1.8 ± 0.5 86.4 ± 9.8 20 Example 97. Prophylactic Efficacy of EIDD-02749 in a Mouse Model of Tacaribe Infection AG129 mice, inoculated with 500 CCID50 TCRV (strain TRVL 11573) i.p., received 3, 10, 25 or 30 mg / kg / d EIDD-02749 PO for 14 days starting 2 hours before infection. Survival curves for the study are shown in Figure 15 and viral titers measured on Day 9 are shown in Figure 16. Example 98. Therapeutic Efficacy of EIDD-02749 in a Mouse Model of Tacaribe 30 Infection 2020418425   14 Sep 2026 AG129 mice, inoculated with 500 CCID50 TCRV (strain TRVL 11573) i.p., received 10 mg / kg / d EIDD-02749 PO. Treatment started 2 hours, 1 Day, and 3 Days post infection and continued for 14 days, 5 Days post infection and continued for 12 days, and 7 Days post infection and continued for 10 days. Survival curves for the study are shown in Figure 17 5 and viral titers measured on Day 9 are shown in Figure 18. Example 99: SARS / MERS Assay Calu: At 48hrs prior to infection, Calu3 2B4 cells were plated in a 96-well black walled clear 10 bottom plate at 5x104 cells / well. A 10mM stock of EIDD-2749 was serially diluted in 100% DMSO in 3-fold increments to obtain a ten-point dilution series. MERS-nLUC was diluted in DMEM 10% FBS, and 1% Antibiotic-Antimycotic to achieve a multiplicity of infection (MOI) of 0.08. Cells were infected and treated with EIDD-2749 in triplicate per drug dilution for 1hr after which, viral inoculum was aspirated, cultures were rinsed once and fresh medium 15 containing drug or vehicle was added. At 48hrs post infection, nanoluciferase expression as a surrogate for virus replication was quantitated on a Spectramax (Molecular Devices) plate reader according to the manufacturer’s instructions (Promega, NanoGlo). For our 100% inhibition control, diluted MERS-nLUC was exposed to short-wave UV light (UVP, LLC) for 6 minutes to inhibit the ability of the virus to replicate. For our 0% inhibition control, cells 20 were infected in the presence of vehicle only. DMSO was kept constant in all conditions at 0.05%. Values from triplicate wells per condition were averaged and compared to controls to generate a percent inhibition value for each drug dilution. The IC50 value was defined as the concentration at which there was a 50% decrease in luciferase expression. Data was analyzed using GraphPad Prism 8.0 (La Jolla, CA). The IC50 values were calculated by non-linear 25 regression analysis using the dose-response (variable slope) equation (four parameter logistic equation): Y = Bottom + (Top-Bottom) / (1+10A((LogIC50-X)*HillSlope)). To measure cell viability to determine if there was any NHC induced cytotoxicity, Calu3 2B4 cells were plated and treated with EIDD-2749 only as described above. Cells were exposed to the same ten-point dilution series created for the in vitro efficacy studies. As above, 0.05% DMSO-treated 30 cells served as our 0% cytotoxicity control. Wells without cells served as our 100% cytotoxic positive control. After 48hr, cell viability was measured on a Spectramax (Molecular Devices) via Cell-Titer Glo Assay (Promega) according to the manufacturer’s protocol. Similar data was obtained in three independent experiments. 35 Example 100: EIDD-2749 Coronavirus Activity Including COVID-19 (SARS-2-CoV-2) Activity 2020418425   14 Sep 2026 Virus Strain Cell Line EC5o(gM)a EC9o(gM)b CC5o(gM) MERS - Vero 76 5.7 9.5 >380 HCoV-alpha 229E Huh-7 1.67 9.5 >380 HCoV-beta OC43 RD 1.06 2.85 >380 SARS Urbani Vero 76 14.1 - >380 SARS-CoV-2 USA_WA1 / 2020 Vero 76 2.3 1.52 >380 SARS-CoV-2 USA_WA1 / 2020 Calu3 1.9 - > 50

Claims

1. A method of treating a viral infection in a subject in need thereof, the methodcomprising administering to the subject a compound having a structure represented by a formula:Formula XXVIIIor a pharmaceutical or physiological salt thereof, whereinR1 is hydrogen,Y is O;R2 is hydroxy;R3 is hydroxy;R6, R6’, R6’’, and R6’’’ are each independently selected from hydrogen, hydroxyl, amino, halogen, alkoxy, or alkyl;wherein the compound is administered to the subject greater than or equal to 3 days after the subject presents with clinical signs of disease.

2. The method of claim 1, wherein the compound is selected from the following:2020418425   14 Sep 2026and3.     The method of claim 2, wherein the compound is:

4. A method of treating a viral infection in a subject in need thereof, the methodcomprising administering to the subject a compound having a structure represented by aformula:0HO OHOHO OH2020418425   14 Sep 2026wherein the compound is administered to the subject greater than or equal to 3 days after the subject presents with clinical signs of disease.

5. The method of claim 4, wherein the compound is0HO OH .

6. The method of claim 4, wherein the compound isOHO OH7.     The method of any one of claims 1-6, wherein the viral infection is in the centralnervous system (CNS) of the subject.

8. The method of any one of claims 1-6, wherein the viral infection is encephalitic andthe compound is delivered to the brain or the central nervous system of the subject.

9. The method of any one of claims 1-6, wherein the compound is administered to thesubject greater than or equal to 5 days after the subject presents with clinical signs ofdisease.

10. The method of any one of claims 1-6, wherein the compound is administered to thesubject greater than or equal to 7 days after the subject presents with clinical signs ofdisease.

11. The method of any one of claims 1-6, wherein the compound is administered in combination with a second antiviral agent.

12. The method of claim 11, wherein the second antiviral agent is selected from abacavir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, AT-511 (Bemnifosbuvir), AT-527 (Bemnifosbuvir hemisulfate), atazanavir, atripla, balapiravir, BCX4430 (Galidesivir), boceprevir, cidofovir, combivir, daclatasvir, darunavir, dasabuvir, delavirdine, didanosine, ocosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide,2020418425   14 Sep 2026entecavir, famciclovir, favipiravir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, GS-441524 ((2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)oxolane-2-carbonitrile), GS-5734 (Remdesivir), ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, interferon type III, interferon type II, interferon type I, lamivudine, ledipasvir, lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfinavir, nevirapine, nexavir, NITD008 ((2R,3R,4R,5R)-2-(4-aminopyrrolo[2,3-d]pyrimidin-7-yl)-3-ethynyl-5-(hydroxymethyl)oxolane-3,4-diol), ombitasvir, oseltamivir, paritaprevir, peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin raltegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, simeprevir, sofosbuvir, stavudine, telaprevir, telbivudine, tenofovir, tenofovir disoproxil, Tenofovir Exalidex, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine zalcitabine, zanamivir, zidovudine, chloroquine, chloroquine phosphate, hydroxychloroquine, hydroxychloroquine sulfate, Ampligen, APN01 (Alunacedase alfa / recombinant human soluble ACE2 / rhACE2), Ganovo, IFX-1 (Vilobelimab), BXT-25 (originator BioXyTran), CYNK-001 (Taniraleucel), Tocilizumab, Leronlimab, Ii-key, COVID-19 S-Trimer, Camrelizumab, thymosin, Brilacidin, INO-4800 (Reluscovtogene Ralaplasmid), Prezcobix, cobicistat, mRNA-1273 (COVID-19 Vaccine Moderna / Elasomeran), Arbidol, umifenovir, REGN3048 (originator Regeneron Pharmaceuticals), REGN3051 (originator Regeneron Pharmaceuticals), TNX-1800 (originator Tonix Pharmaceuticals), fingolimod, methylprednisolone, nitazoxanide, benzopurpin B, C-467929 (5-[[3-carboxy-4-[(3-nitrobenzoyl)amino]phenyl]methyl]-2-[(3-nitrobenzoyl)amino]benzoic acid), C-473872 (3,3'-methylenebis[6-([1,1'-biphenyl]-4-carbonylamino)benzoic acid]), NSC-306711 (dicopper;4-amino-6-[[4-[4-[[3-methyl-5-oxido-1-(4-sulfonatophenyl)pyrazol-4-yl]diazenyl]-3-oxidophenyl]-2-oxidophenyl]diazenyl]-5-oxidonaphthalene-1,3-disulfonate;hydron), N-65828 (8-amino-5-[[4-(4-hydroxyphenyl)phenyl]diazenyl]naphthalene-2-sulfonic acid), C-21(Buloxibutid / Compound 21), CGP-42112A ((2S,3S)-2-[[(2S)-1-[(2S)-2-[[(2S)-6-[[(2S)-5-(diaminomethylideneamino)-2-(phenylmethoxycarbonylamino)pentanoyl]amino]-2-[[(2S)-3-(4-hydroxyphenyl)-2-(pyridine-3-carbonylamino)propanoyl]amino]hexanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]pyrrolidine-2-carbonyl]amino]-3-methylpentanoic acid), L-163491 (5,7-dimethyl-2-ethyl-3- [[2'- [(butyloxycarbonyl)aminosulfonyl]-5'-(3 -methoxybenzyl)-[1,1'-biphenyl]-4-yl]methyl]-3H-imidazo[4,5-b]pyridme), xanthoangelol, bevacizumab, or a combination thereof.2020418425   14 Sep 202613. Use of a compound in the manufacture of a medicament for the treatment of a viral infection in a subject in need thereof, wherein the compound is compound having a structure represented by a formula:R2 R3Formula XXVIIIor a pharmaceutical or physiological salt thereof, whereinR1 is hydrogen,Y is O;R2 is hydroxy;R3 is hydroxy;R6, R6’, R6’’, and R6’’’ are each independently selected from hydrogen, hydroxyl, amino, halogen, alkoxy, or alkyl;wherein the medicament is administered to the subject greater than or equal to 3 days after the subject presents with clinical signs of disease.

14. The use of claim 13, wherein the compound is selected from the following:2020418425   14 Sep 2026and15. The use of claim 14, wherein the compound is:

16. Use of a compound in the manufacture of a medicament for the treatment of a viral infection in a subject in need thereof, wherein the compound is a compound having astructure represented by a formula:0HO OHOHO OHwherein the medicament is administered to the subject greater than or equal to 3 days after the subject presents with clinical signs of disease.2020418425   14 Sep 202617. The use of claim 16, wherein the compound isHO OH18. The use of claim 16, wherein the compound isOHO OH19. The use of any one of claims 13-18, wherein the viral infection is in the central nervous system (CNS) of the subject.

20. The use of any one of claims 13-18, wherein the viral infection is encephalitic and the medicament is delivered to the brain or the central nervous system of the subject.

21. The use of any one of claims 13-19, wherein the medicament is administered to the subject greater than or equal to 5 days after the subject presents with clinical signs of disease.

22. The use of any one of claims 13-19, wherein the medicament is administered to the subject greater than or equal to 7 days after the subject presents with clinical signs of disease.

23. The use of any one of claims 13-18, wherein the medicament is administered in combination with a second antiviral agent.

24. The use of claim 23, wherein the second antiviral agent is selected from abacavir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, AT-511 (Bemnifosbuvir), AT-527 (Bemnifosbuvir hemisulfate), atazanavir, atripla, balapiravir, BCX4430 (Galidesivir), boceprevir, cidofovir, combivir, daclatasvir, darunavir, dasabuvir, delavirdine, didanosine, ocosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir, favipiravir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, GS-441524 ((2R,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)oxolane-2-carbonitrile), GS-5734 (Remdesivir), ibacitabine, imunovir,2020418425   14 Sep 2026idoxuridine, imiquimod, indinavir, inosine, interferon type III, interferon type II, interferon type I, lamivudine, ledipasvir, lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfinavir, nevirapine, nexavir, NITD008 ((2R,3R,4R,5R)-2-(4-aminopyrrolo[2,3-d]pyrimidin-7-yl)-3-ethynyl-5-(hydroxymethyl)oxolane-3,4-diol), ombitasvir, oseltamivir, paritaprevir, peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin raltegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, simeprevir, sofosbuvir, stavudine, telaprevir, telbivudine, tenofovir, tenofovir disoproxil, Tenofovir Exalidex, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine zalcitabine, zanamivir, zidovudine, chloroquine, chloroquine phosphate, hydroxychloroquine, hydroxychloroquine sulfate, Ampligen, APN01 (Alunacedase alfa / recombinant human soluble ACE2 / rhACE2), Ganovo, IFX-1 (Vilobelimab), BXT-25 (originator BioXyTran), CYNK-001 (Taniraleucel), Tocilizumab, Leronlimab, Ii-key, COVID-19 S-Trimer, Camrelizumab, thymosin, Brilacidin, INO-4800 (Reluscovtogene Ralaplasmid), Prezcobix, cobicistat, mRNA-1273 (COVID-19 Vaccine Moderna / Elasomeran), Arbidol, umifenovir, REGN3048 (originator Regeneron Pharmaceuticals), REGN3051 (originator Regeneron Pharmaceuticals), TNX-1800 (originator Tonix Pharmaceuticals), fingolimod, methylprednisolone, nitazoxanide, benzopurpin B, C-467929 (5-[[3-carboxy-4-[(3-nitrobenzoyl)amino]phenyl]methyl]-2-[(3-nitrobenzoyl)amino]benzoic acid), C-473872 (3,3'-methylenebis[6-([1,1'-biphenyl]-4-carbonylamino)benzoic acid]), NSC-306711 (dicopper;4-amino-6-[[4-[4-[[3-methyl-5-oxido-1-(4-sulfonatophenyl)pyrazol-4-yl]diazenyl]-3-oxidophenyl]-2-oxidophenyl]diazenyl]-5-oxidonaphthalene-1,3-disulfonate;hydron), N-65828 (8-amino-5-[[4-(4-hydroxyphenyl)phenyl]diazenyl]naphthalene-2-sulfonic acid), C-21 (Buloxibutid / Compound 21), CGP-42112A ((2S,3S)-2-[[(2S)-1-[(2S)-2-[[(2S)-6-[[(2S)-5-(diaminomethylideneamino)-2-(phenylmethoxycarbonylamino)pentanoyl]amino]-2-[[(2S)-3-(4-hydroxyphenyl)-2-(pyridine-3-carbonylamino)propanoyl]amino]hexanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]pyrrolidine-2-carbonyl]amino]-3-methylpentanoic acid), L-163491 (5,7-dimethyl-2-ethyl-3- [[2'- [(butyloxycarbonyl)aminosulfonyl]-5'-(3 -methoxybenzyl)-[1,1'-biphenyl]-4-yl]methyl]-3H-imidazo[4,5-b]pyridine), xanthoangelol, bevacizumab, or a combination thereof.

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