Antiviral prodrugs, pharmaceutical formulations, and methods

Abstract

Compounds, including antiviral prodrugs, and pharmaceutical formulations containing the compounds, which may be orally bioavailable or formulated for intramuscular injection. Methods for producing compounds, such as antiviral prodrugs. Methods for treating coronavirus and other RNA virus infections in mammals. Methods for producing drug triphosphates.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 145,698, filed February 4, 2021, U.S. Provisional Patent Application No. 63 / 110,596, filed November 6, 2020, U.S. Provisional Patent Application No. 63 / 078,427, filed September 15, 2020, U.S. Provisional Patent Application No. 63 / 070,695, filed August 26, 2020, and U.S. Provisional Patent Application No. 63 / 055,944, filed July 24, 2020, which are incorporated herein by reference.

[0002] STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant No. AI131424 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] The present invention relates to antiviral prodrugs, methods for making antiviral prodrugs, and methods of use for the treatment of coronavirus infections in mammals. [Background technology]

[0004] Over the past two decades, spillover events have introduced the highly contagious β-coronavirus strains SARS-CoV, MERS-CoV, and SARS-CoV-2 into the human population. Although case fatality rates vary, each has demonstrated the ability to induce substantial morbidity and mortality, particularly in patients over 55 years of age and / or with underlying comorbid medical conditions. While SARS-CoV and MERS-CoV have been largely contained through epidemiological interventions, SARS-CoV-2 has evolved into a global pandemic.

[0005] Efforts to develop a SARS-CoV-2 vaccine have been challenged by strain diversity, the possibility that vaccine-induced immunity may be short-lived, potential diminished immune recognition in individuals as young as 30 years old, and the possibility that antibody-dependent enhancement may be observed. Reported cases of reinfection have raised substantial new concerns regarding long-lasting immunity, even after recovery from natural infection. While SARS-CoV-2 vaccine efforts are expected to be successful, unfortunately, after a third of a century, no AIDS vaccine has been developed. Highly successful drug development efforts have transformed the face of HIV by providing highly effective, affordable, and scalable preventative and therapeutic tools. During coronavirus vaccine efforts, it would be desirable to prepare for equally potent therapeutic efforts.

[0006] Remdesivir nucleoside triphosphate (RVn triphosphate) potently inhibits the enzymatic activity of the polymerase of all coronaviruses tested to date, including SARS CoV-2 (see, e.g., Yan, VC et al. ACS Med. Chem. Lett. 2020;11(7):1361-1366).

[0007] This broad range of activity may reflect the relative molecular conservation of coronavirus RNA-dependent RNA polymerase (RdRp). Remdesivir (RDV) is an aryloxyphosphoramidate triester prodrug that must be converted through a series of reactions to the active antiviral metabolite, RVn triphosphate. RVn triphosphate is a potent inhibitor of viral RdRp (see, e.g., Gordon, CJ et al. J. Biol. Chem. 2020;295:4773-4779). However, the antiviral activity of RDV is highly variable in different cell types, which may be due to variable expression of the four enzymes required for conversion to RVn-P (Yan, VC et al. ACS Med. Chem. Lett. 2020;11(7):1361-1366). The base of RDV is a 1'-cyano-substituted adenine C nucleoside (GS-441524, RVn), which is thought to be underphosphorylated. To circumvent the recognized slow first phosphorylation, developers have relied on an aryloxyphosphoramidate triester prodrug that is converted through a complex series of four reactions to remdesivir nucleoside monophosphate (RVn-P), which is then efficiently converted to the active metabolite, RVn triphosphate. RDV may be more active in some SARS-CoV-2-infected tissues than others, which may explain its incomplete clinical impact against SARS-CoV-2.

[0008] Remdesivir has beneficial antiviral and clinical effects in animal models of coronavirus infection (see, e.g., de Wit, E. et al. Proc. Natl. Acad. Sci. USA, 2020;115:6771-6776). These effects are primarily demonstrable when administered before or immediately after viral challenge. RDV is not highly bioavailable after oral administration and must be administered intravenously, functionally limiting its clinical application to hospitalized patients with relatively advanced disease. Furthermore, the persistence of RDV in plasma is known to be very short.

[0009] Specifically, RDV is a prodrug designed to bypass the first phosphorylation step of remdesivir nucleoside (RVn), which may be rate-limiting in the synthesis of the active metabolite, RVn-triphosphate. However, this approach does not appear to offer any benefit, as results have shown that the antiviral activity of RVn is greater than that of RDV in Vero E6 cells, a monkey kidney cell line (see, for example, Pruijssers, AJ et al., Cell Rep. 2020 Jul 21;32(3):107940). Other recognized drawbacks of RDV include lack of oral bioavailability, difficult synthesis, instability in plasma, poor delivery to the lungs, and / or hepatotoxicity. In patients with Covid-19 and in Syrian hamster models of SARS-CoV-2 disease, in addition to high viral loads in the nasal turbinates, trachea, and lungs, many other tissues become infected with SARS-CoV-2 as the infection progresses, including the intestine, heart, liver, spleen, kidneys, brain, lymph nodes, and vascular endothelium. However, RDV antiviral activity was limited to an EC of 1.65 μM in E6 cells, 0.28 μM in Calu3 2B4, and 0.010 μM in human alveolar epithelial cells (HAE). 50 values, appearing to vary widely in lung and kidney cell lines with a 165-fold difference (see, e.g., Pruijssers, AJ et al., Cell Rep. 2020 Jul 21;32(3):107940). It has been suggested that this may be due to variable amounts of the enzyme that converts RDV to RVn-P (see, e.g., Yan, VC et al. ACS Med. Chem. Lett. 2020;11(7):1361-1366).

[0010] There remains a need for highly active and / or orally bioavailable analogs of RVn that can provide sustained levels of intact antiviral in plasma, including those that provide increased oral bioavailability by improving pulmonary exposure to the active antiviral. Summary of the Invention

[0011] Provided herein are compounds, such as antiviral prodrugs, and pharmaceutical formulations that overcome one or more drawbacks of currently used drugs. For example, embodiments of the compounds and pharmaceutical formulations provided herein include orally effective antiviral prodrugs that can specifically target organs where viral replication is maximal and can be conveniently administered on a large scale at any stage of disease. For oral use and enhanced pulmonary exposure, novel prodrug embodiments of RVn provided herein can achieve one or more of the following three steps: 1) kinase bypass of the first nucleoside phosphorylation, 2) providing increased oral bioavailability, and 3) delivering antivirally significant concentrations to the lungs and gastrointestinal tract. Also provided herein are methods for the synthesis and antiviral evaluation of compounds, including novel lipophilic prodrugs of RVn-monophosphate that are substantially more active than remdesivir in Vero E6 cells infected with SARS-CoV-2. Without wishing to be bound by any particular theory, embodiments of the compounds herein are prodrugs that may enable earlier and / or more effective treatment upon diagnosis of SARS-CoV-2 infection. The prodrugs herein may represent a potential approach that may target antivirals to the lungs and away from the liver, where the primary dose limitation of remdesivir is directed.

[0012] In one aspect, provided herein are compounds comprising antiviral prodrugs. In some embodiments, the compounds have the formula (I): [ka] wherein Nuc is selected from the group consisting of antiviral nucleosides and antiviral nucleoside analogs, and Y is independently hydrogen, C1-C 30 and a covalent bond to a carbon atom of a 5-carbon sugar moiety of an antiviral nucleoside or antiviral nucleoside analog, wherein x is 0 or 1, L is a C1-C6 hydrocarbyl, and R is independently selected from the group consisting of C10 ~C 30 is selected from the group consisting of hydrocarbyl and a substituent of formula (A): [ka] In the formula, R 1 and R 2 are independently hydrogen and C1 to C 30 is selected from the group consisting of hydrocarbyl.

[0013] In another aspect, a pharmaceutical preparation is provided.In some embodiments, the pharmaceutical preparation comprises one or more compounds as described herein.The pharmaceutical preparation can be formulated for intramuscular injection.The pharmaceutical preparation can be orally bioavailable.

[0014] In a further aspect, therapeutic methods are provided, such as methods for treating viruses (e.g., coronaviruses), including viral infections in mammals. In some embodiments, the methods comprise administering an effective amount of a compound described herein, or a pharmaceutical formulation described herein.

[0015] In yet a further aspect, methods for preparing compounds, such as prodrugs, are provided. In some embodiments, the methods include: (i) providing a compound of formula (a); [ka] (ii) providing a compound of formula (b); [ka] (iii) contacting a compound of formula (a) with a compound of formula (b) to form a compound of formula (c); [ka] (iv) contacting a compound of formula (c) with an acid to form a compound of formula (d); [ka] where Het is a C1-C aryl group containing at least one heteroatom. 30 hydrocarbyl, and Y is hydrogen, C1-C 30 and a pharmaceutically acceptable cation, x is 0 or 1, L is a C1-C6 hydrocarbyl, and R is C 10 ~C 30 is selected from the group consisting of hydrocarbyl and a substituent of formula (A): [ka] In the formula, R 1 and R 2 are independently hydrogen and C1 to C 30 The method may include carrying out an intramolecular esterification reaction of a product such as a phosphodiester to form a cyclic phosphate, such as a 3',5' cyclic phosphate.

[0016] In yet another aspect, methods for producing a drug triphosphate are also provided. In some embodiments, the methods include providing a plurality of cells, contacting the plurality of cells with an amount of a drug, and incubating the plurality of cells and the amount of drug for a period of time effective to form a drug triphosphate.

[0017] Additional aspects will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the aspects described herein. The advantages described herein may be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive. [Brief explanation of the drawings]

[0018] [Figure 1A]1 shows concentration-response curves for embodiments of compounds described herein for SARS-CoV-2 infection in Vero E6 cells for two separate experiments performed in duplicate. [Figure 1B] 1 shows concentration-response curves for embodiments of compounds described herein for SARS-CoV-2 infection in Vero E6 cells for two separate experiments performed in duplicate. [Figure 1C] 1 shows concentration-response curves for embodiments of compounds described herein for SARS-CoV-2 infection in Vero E6 cells for two separate experiments performed in duplicate. [Figure 1D] 1 shows the concentration-response curve for remdesivir for SARS-CoV-2 infection in Vero E6 cells from two separate experiments performed in duplicate. [Figure 1E] Figure 1 shows the concentration-response curve for remdesivir nucleoside for SARS-CoV-2 infection in Vero E6 cells in two separate experiments performed in duplicate. [Figure 1F] The concentration-response curves are shown in Figures 1A to 1E. [Figure 2] 1 shows a plot of relative viability for several embodiments of compounds described herein, remdesivir, and remdesivir nucleosides. [Figure 3] 1 shows the results of an embodiment in which the synthesis of remdesivir triphosphate was carried out in Vero E6 cells. [Figure 4A] 1 shows antiviral dose-response curves for remdesivir (GS-5734) and compounds herein against human coronavirus 229E in MRC-5 cells. [Figure 4B] 1 shows cytotoxicity in MRC-5 cells incubated for 72 hours in the presence of the indicated drugs and compound embodiments herein at the indicated concentrations. [Figure 5A] 1 shows 7-day oral pharmacokinetics in Syrian hamsters for embodiments of the compounds herein. [Figure 5B]1 shows the 7-day oral pharmacokinetics of remdesivir in Syrian hamsters. [Figure 6A] 1 shows the stability of ODE-P-RVn and ODBG-P-RVn in human plasma using K2EDTA as an anticoagulant. [Figure 6B] 1 shows the stability of ODE-P-RVn and ODBG-P-RVn in human plasma using sodium heparin as an anticoagulant. DETAILED DESCRIPTION OF THE INVENTION

[0019] In one aspect, provided herein are compounds comprising a compound of formula (I): [ka]

[0020] "Nuc" in formula (I) can be any suitable nucleoside. The nucleoside can be linked to the compound in any manner. For example, the 5'-hydroxyl of the nucleoside can be linked to the phosphate moiety as an ester bond.

[0021] The nucleoside, in some embodiments, is an antiviral nucleoside. The antiviral nucleoside may be an antiviral ribonucleoside. The nucleoside, in some embodiments, is an antiviral nucleoside analog. The antiviral nucleoside analog may be an antiviral ribonucleoside analog.

[0022] In some embodiments, Nuc is RVn (GS-441524), β-D-N4-hydroxycytidine (NHC), or (2'R)-2-amino-2'-deoxy-2'-fluoro-N,2'-dimethyladenosine (CAS number: 1998705-62-6). In some embodiments, Nuc is GS-441524 and the compound of formula (I) has the following structure: [ka]

[0023] Other antiviral agents for coronavirus infections can also be modified using the methods provided herein. For example, N4-hydroxycytidine (NHC) is an antiviral candidate undergoing clinical Phase I evaluation. Other nucleoside analogs known to inhibit RNA viruses are also suitable for modification using the present disclosure.

[0024] "Y" in formula (I) may be any of the substituents described herein. In some embodiments, Y is hydrogen, C1-C 30 A hydrocarbyl, a pharmaceutically acceptable cation, or a covalent bond to a carbon atom of the 5-carbon sugar moiety of the antiviral nucleoside or antiviral nucleoside analog.

[0025] When Y is a covalent bond to a carbon atom of the 5-carbon sugar moiety of the antiviral nucleoside or antiviral nucleoside analog, the covalent bond can be to any carbon atom (e.g., the 1', 2', 3', or 4' carbon) of the 5-carbon sugar moiety of the antiviral nucleoside or antiviral nucleoside analog. In other words, the covalent bond can be between (i) the oxygen to which Y is attached in Formula (I) and (ii) any carbon atom (e.g., the 1', 2', 3', or 4' carbon) of the 5-carbon sugar moiety of the antiviral nucleoside or antiviral nucleoside analog. For example, Nuc can be GS-441524, and the covalent bond can be between the oxygen to which Y is attached in Formula (I) and the 3' carbon of the 5-carbon sugar moiety of GS-441524, and the compound of Formula (I) has the following structure: [ka]

[0026] When Y is a pharmaceutically acceptable cation, the pharmaceutically acceptable cation may be Na+.

[0027] In some embodiments, Y is C1-C20 Hydrocarbyl, C1-C 10 In some embodiments, Y is a C1-C6 hydrocarbyl, or a C1-C6 hydrocarbyl. In some embodiments, Y is a C1-C6 alkyl, which may be unsubstituted. In some embodiments, Y comprises at least one cyclic moiety. The at least one cyclic moiety may be a monocyclic moiety or a polycyclic moiety, such as a bicyclic moiety, a spiro moiety, etc. In some embodiments, Y is an aryl, arylalkyl, heteroaryl, heteroarylalkyl, or heterocycloalkyl, each of which may be unsubstituted or substituted. In some embodiments, Y is an unsubstituted or substituted pyridinyl. In some embodiments, Y is an unsubstituted or substituted benzyl. The unsubstituted or substituted benzyl may have a structure according to formula (B): [ka] In the formula, R 3 , R 4 , R 5 , R 6 , and R 7 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, aryl(alkyl), heteroaryl(alkyl), (heterocyclyl)alkyl, hydroxy, alkoxy, acyl, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, nitro, azido, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanomethanesulfonamido, amino, monosubstituted amino, and disubstituted amino. 3 , R 4 , R 5 , R 6 , and R 7 is hydrogen. In some embodiments, R 3, R 4 , R 5 , R 6 , and R 7 At least two of R are hydrogen. 3 , R 4 , R 5 , R 6 , and R 7 At least three of R 3 , R 4 , R 5 , R 6 , and R 7 At least four of the are hydrogen.

[0028] When Y is unsubstituted or substituted benzyl of formula (B), the compound of formula (I) has the following structure: [ka]

[0029] In formula (I), x can be 1 or 0. When X is 1, the "-OL-" moiety is present in the compound of formula (I). When X is 0, R is attached to and points towards the oxygen of the phosphonate moiety, as shown in the following structure: [ka]

[0030] When "L" is present in the compounds of Formula (I), "L" may be selected from any of the substituents described herein. In some embodiments, L is a C1-C 30 Hydrocarbyl, C1-C 20 Hydrocarbyl, C1-C 10In some embodiments, L is optionally unsubstituted ethyl. In some embodiments, L is optionally unsubstituted methyl. In some embodiments, L is optionally unsubstituted propyl.

[0031] "R" in formula (I) may be selected from any of the substituents described herein. In some embodiments, R is a C1-C 30 Hydrocarbyl, C5-C 30 Hydrocarbyl, C 10 ~C 30 Hydrocarbyl, C 12 ~C 24 Hydrocarbyl, C 13 ~C 29 Hydrocarbyl, C 15 ~C 24 Hydrocarbyl, or C 20 ~C 24 R is a hydrocarbyl. In some embodiments, R is a heteroalkyl. R can contain 0 to 6 unsaturated bonds, 1 to 6 unsaturated bonds, 2 to 6 unsaturated bonds, 3 to 6 unsaturated bonds, or 4 to 6 unsaturated bonds. The "unsaturated bond" described herein can include any non-single bond, and when more than one unsaturated bond is present, two or more unsaturated bonds can be independently selected from double bonds or triple bonds. When one or more double bonds are present, the one or more double bonds can be cis bonds, trans bonds, or a combination thereof. R can include a cyclopropyl moiety, such as a terminal cyclopropyl moiety.

[0032] In some embodiments, R is [ka] wherein a is 1 to 29. In some embodiments, a is 15 to 25. In some embodiments, a is 18 to 22. In some embodiments, a is 19. In some embodiments, a is 6 to 10. In some embodiments, a is 8.

[0033] In some embodiments, R is [ka] wherein b is 1 to 29, c is 0 to 28, and the sum of b and c is 29 or less. In some embodiments, b is 1 to 4, and c is 15 to 20. In some embodiments, b is 3, and c is 15. In some embodiments, b is 2, and c is 17.

[0034] In some embodiments, R is a group of formula (A): [ka] where R 1 and R 2 is hydrogen or C1-C 30 Hydrocarbyl, for example, C 10 ~C 30 Hydrocarbyl, or C 12 ~C 24 It is a hydrocarbyl. 1 , R 2 , or R 1 and R 2 Both of R may contain at least one cyclic moiety, which may be a monocyclic moiety or a polycyclic moiety, e.g., a bicyclic moiety, a spiro moiety, etc. 1 , R 2 , or R 1 and R 2 Both of R may contain 0 to 6 unsaturated bonds, 1 to 6 unsaturated bonds, 2 to 6 unsaturated bonds, 3 to 6 unsaturated bonds, or 4 to 6 unsaturated bonds. When one or more double bonds are present, the one or more double bonds may be cis bonds, trans bonds, or a combination thereof. 1, R 2 , or R 1 and R 2 may include a branched hydrocarbyl, such as a penultimate branched hydrocarbyl. 1 and R 2 At least one of R is hydrogen. 1 and R 2 Both of these are independent, C1 to C 30 The alkyl group is selected from the group consisting of alkyl, alkoxy, alkoxysilyl ...

[0035] In some embodiments, R 1 , R 2 , or R 1 and R 2 are both independently selected from the group consisting of aryl, arylalkyl, heteroaryl, heteroarylalkyl, and heterocycloalkyl, each of which may be unsubstituted or substituted. The arylalkyl may be unsubstituted or substituted benzyl. The unsubstituted or substituted benzyl may have a structure according to formula (C): [ka] In the formula, R 8 , R 9 , R 10 , R 11 , and R 12are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, aryl(alkyl), heteroaryl(alkyl), (heterocyclyl)alkyl, hydroxy, alkoxy, acyl, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, nitro, azido, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanomethanesulfonamido, amino, monosubstituted amino, and disubstituted amino. 8 , R 9 , R 10 , R 11 , and R 12 Each of R is hydrogen. 8 , R 9 , R 10 , R 11 , and R 12 At least two of R are hydrogen. 8 , R 9 , R 10 , R 11 , and R 12 At least three of R 8 , R 9 , R 10 , R 11 , and R 12 At least four of R 8 , R 9 , R 10 , R 11 , and R 12 At least five of the are hydrogen.

[0036] In some embodiments, R 1 teeth, [ka] wherein d is 1 to 29. In some embodiments, d is 5 to 29, 10 to 29, 15 to 29, 20 to 29, 25 to 29, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5.

[0037] In some embodiments, R 1 teeth, [ka] In the formula, e is 1 to 27, f is 0 to 26, and the sum of e and f is 27 or less.

[0038] In some embodiments, R2 is [ka] wherein g is 1 to 29. In some embodiments, g is 5 to 10. In some embodiments, g is 7.

[0039] The substituents of formula (A) can be racemic, Sn-1 stereoisomers, or Sn-3 stereoisomers. Throughout this disclosure, when a formula such as formula (A) is shown without an indication of spatial orientation, the formula reads with respect to all isomers, e.g., stereoisomers, of the compound of the formula. For example, in some embodiments, the compound can have a structure as set forth in formula (I), where x is 0 and R is a substituent of formula (A). [ka]

[0040] This formula lacks any indication of spatial orientation and therefore reads in terms of its sn-3 isomer, its sn-1 isomer, and mixtures of the sn-3 and sn-1 isomers, including racemic mixtures of the formula below. [ka]

[0041] Further non-limiting embodiments of compounds of Formula (I) are provided in the table below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0042] As used herein with respect to the selection of substituents, the term "independently" indicates that (i) the substituents at a particular position may be the same or different for each molecule of the formula (e.g., (i) a compound of formula (I) may comprise two molecules of formula (I), each molecule containing the same or different C-C groups selected for R). 30 and / or (ii) two different labeled substituents selected from the same pool of substituents may be the same or different (e.g., when R and Y in a molecule of a compound of formula (I) are both "C-C 30 and R and Y are selected from the group consisting of C-C 30 The hydrocarbyls may be the same or different).

[0043] Phrase “C1~C 30 Hydrocarbyl," "C 10 ~C 30"Hydrocarbyl" and the like, as used herein, generally refer to an aliphatic, aryl, or arylalkyl group containing 1 to 30 carbon atoms, or 10 to 30 carbon atoms, respectively, including substituted derivatives thereof, which may include, but are not limited to, heteroaryl, heteroarylalkyl, heterocycloalkyl, and the like. Examples of aliphatic groups include, but are not limited to, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkadienyl, cyclic, and the like, in each instance, including all substituted, unsubstituted, branched, and / or straight-chain analogs or derivatives having 1 to 30 total carbon atoms, or 10 to 30 total carbon atoms, for "C1-C30 hydrocarbyl" and "C10-C30 hydrocarbyl," respectively. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, and dodecyl. Cycloalkyl moieties can be monocyclic or polycyclic, and examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and adamantyl (including any heteroatom-substituted derivatives thereof). Further examples of alkyl moieties have straight-chain, branched-chain, and / or cyclic moieties (e.g., 1-ethyl-4-methyl-cyclohexyl). Representative alkenyl moieties include vinyl, allyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2-dimethyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 2-decenyl, and 3-decenyl.Representative alkynyl moieties include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl, 6-heptynyl, 1-octynyl, 2-octynyl, 7-octynyl, 1-nonynyl, 2-nonynyl, 8-nonynyl, 1-decynyl, 2-decynyl, and 9-decynyl. Examples of aryl or arylalkyl moieties include, but are not limited to, anthracenyl, azulenyl, biphenyl, fluorenyl, indan, indenyl, naphthyl, phenanthrenyl, phenyl, 1,2,3,4-tetrahydro-naphthalene, anthracenyl, tolyl, xylyl, mesityl, benzyl, and the like.

[0044] Unless otherwise indicated, the term "substituted," when used to describe a chemical structure or moiety, means (i) that a polyvalent non-carbon atom (e.g., oxygen, nitrogen, sulfur, phosphorus, etc.) is bonded to one or more carbon atoms of the chemical structure or moiety (e.g., a "substituted" C hydrocarbyl can include, but is not limited to, a pyrimidinyl moiety, a pyridinyl moiety, a dioxanyl moiety, a diethyl ether moiety, a methyl propionate moiety, a N,N-dimethylacetamide moiety, a butoxy moiety, etc., a "substituted" aryl C 12Hydrocarbyls can include, but are not limited to, an oxydibenzene moiety, a benzophenone moiety, and the like), or (ii) one or more of its hydrogen atoms (e.g., chlorobenzene can be generally characterized as an aryl C hydrocarbyl "substituted" with a chlorine atom) can be substituted with an acyl, alcohol, alkoxy, alkanoyloxy, alkoxycarbonyl, alkenyl, alkyl (e.g., methyl, ethyl, propyl, t-butyl), alkynyl, alkylcarbonyloxy (-OC(O)alkyl), amido (-C(O)NH-alkyl- or -alkylNHC(O)alkyl), primary, secondary, and tertiary amino (e.g., alkylamino, arylamino, arylalkylamino), aryl, arylalkyl, aryloxy, azo, azido, carbamoyl (-NHC(O)Oalkyl- or -OC(O)NH-alkyl), carbamyl (e.g., CONH and CONH-alkyl, CONH-aryl). "NHCONH" refers to derivatives of that structure or moiety substituted with a chemical moiety or functional group such as -NHCONH-alkyl, -NHCONH-aryl ...

[0045] Pharmaceutical preparations Pharmaceutical formulations are also provided herein. The pharmaceutical formulations may contain compounds described herein, such as compounds of formula (I). In some embodiments, the pharmaceutical formulations are orally bioavailable. In some embodiments, the pharmaceutical formulations are formulated for intramuscular injection.

[0046] A pharmaceutical formulation may include one compound described herein or more than one (eg, two, three, etc.) compound described herein.

[0047] The pharmaceutical formulation may optionally contain one or more pharmaceutically acceptable excipients.

[0048] Treatment method Also provided herein are therapeutic methods, including methods of treating a viral infection, such as a coronavirus infection. The viral infection may be an infection in a mammal.

[0049] In some embodiments, the method comprises administering to the mammal an effective amount of a compound described herein or a pharmaceutical formulation described herein.

[0050] The viral infection may be an RNA viral infection, hi some embodiments, the RNA viral infection is caused by an RNA virus of a viral family selected from the group consisting of Filoviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, Fenuviridae, Nairoviridae, Arenaviridae, Flaviviridae, and Coronaviridae.

[0051] Compounds, including prodrugs, provided herein can be screened for inhibitory activity against SARS-CoV-2 and related coronaviruses (or other viruses) using conventional techniques for assessing anti-coronavirus activity and cytotoxicity. Typically, compounds are first screened in vitro for inhibition of coronaviruses, and those that show significant antiviral activity are then screened in vivo for efficacy.

[0052] Non-limiting examples of potentially useful in vitro assays include: a) using the OC43β-coronavirus strain (ATCC1558) in the human adenocarcinoma cell line HCT-8 (ATCC CCL-244) or coronavirus 229E in MRC-5 human lung fibroblasts. Endpoints may include semi-quantitative RT-PCR and pfu determined by triplicate serial dilution. b) Compound activity may be studied using laboratory and clinical isolates of SARS-CoV-2 in Vero E6 cells, Caco-2, Calu-3, HPSC human lung cells, or Huh7.5 cells. An initial SARS-CoV-2 growth inhibition assay may quantify plaque reduction in Vero cells grown in 12-well plates using a commercially available mouse anti-SARS-CoV-2 spike protein detection antibody (item 40021-MM07 on SinoBiological.com). Virus can also be quantified in culture supernatants by serial dilution in VERO cell lawns and by RT-PCR. For example, laboratory strains available from the BEI resource (strains NR52281 and NR522282) and clinical strains isolated from patients participating in clinical trials can be used. Cytotoxicity can be measured by commercially available MTT or Cell Titer Glo assays. The compound with the lowest 90% inhibitory concentration and requiring the highest concentration to induce cellular cytotoxicity can be selected for further evaluation. Anticoronavirus compounds can also be evaluated in a lung explant model of SARS-CoV infection. Candidate molecules with the highest therapeutic index in VERO E6 cells can be advanced to studies in human lung explants to determine activity in primary cells derived from the organ most clinically affected by the virus.

[0053] Methods for producing compounds Also provided herein are methods for making compounds, such as those described herein, which can be prodrugs.

[0054] The compounds provided herein may be prepared by various processes, including those described herein. In some embodiments, a protected analog of remdesivir nucleoside, RVn,2, is prepared and then coupled to a suitable alkoxyalkyl phosphate to form a phosphodiester. Removal of the protecting group can result in a compound of formula (I). [ka]

[0055] In some embodiments, 2-C-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2,5-anhydro-D-altrononitrile (RVn, 2) is first converted to its 2',3'-isopropylidene derivative. The mixture of alkoxyalkylphosphate and protected RVn may then be treated with N,N-dicyclohexylcarbodiimide (DCC) and N,N-dimethylaminopyridine (DMAP) under conditions suitable for preparing the phosphodiester. Removal of the isopropylidene protecting group by treatment with dilute HCl or other suitable acid can provide compounds of Formula (I) in suitable yield and purity.

[0056] In some embodiments, the method includes providing a compound of formula (a): [ka] wherein x, R, L, and Y are as described herein.

[0057] In some embodiments, the method includes providing a compound of formula (b): [ka] wherein Het is as described herein. In some embodiments, Het is [ka] is selected from the group consisting of:

[0058] Also, as described herein, formula (b) does not include any stereochemical designation and therefore reads for at least the following stereoisomers of formula (b). [ka]

[0059] In some embodiments, the method comprises contacting a compound of Formula (a) and a compound of Formula (b) to form a compound of Formula (c). [ka]

[0060] Contacting the compound of formula (a) and the compound of formula (b) may occur at any temperature or pressure and in the presence of any suitable liquid, such as a C1-C2 compound containing at least one cyclic moiety, at least one heteroatom such as nitrogen, or a combination thereof. 30 C1-C such as hydrocarbyl 30 In some embodiments, the liquid is N,N-dicyclohexylcarbodiimide, 4-dimethylaminopyridine, or a combination thereof.

[0061] In some embodiments, the method comprises contacting a compound of formula (c) with an acid to form a compound of formula (d). [ka]

[0062] The acid may include any acid capable of promoting the formation of the compound of formula (d). The acid may be an organic acid or an inorganic acid. The acid may include a hydrogen halide, such as hydrogen chloride. Contacting the compound of formula (c) with the acid may occur in the presence of any suitable liquid. The liquid may be a C1-C cyclic ring containing at least one cyclic moiety, at least one heteroatom, or a combination thereof. 30C1-C such as hydrocarbyl 30 It may be a hydrocarbyl, hi some embodiments, the liquid is tetrahydrofuran.

[0063] In some embodiments, the method comprises carrying out an intramolecular esterification reaction of a compound of formula (d) to form a cyclic phosphate, such as a 3',5' cyclic phosphate.

[0064] Method for preparing drug triphosphates Also provided herein are methods for producing a drug triphosphate. In some embodiments, the method includes providing a plurality of cells, contacting the plurality of cells with an amount of a drug, and incubating the plurality of cells and the amount of drug for a period of time effective to form the drug triphosphate. The plurality of cells can include any suitable cells. In some embodiments, the plurality of cells includes Vero E6 cells, Calu-2 cells, Caco-2 cells, MRC5 human lung fibroblast cells, Huh7.5 cells, and PSC human lung cells. In some embodiments, the drug includes remdesivir or remdesivir nucleoside (GS441524).

[0065] All referenced publications are incorporated herein by reference in their entirety. Furthermore, if a definition or use of a term in a reference incorporated herein by reference contradicts or is contrary to the definition of that term provided herein, the definition of that term provided herein shall apply and the definition of that term in the reference shall not apply.

[0066] Unless otherwise defined, all technical and scientific terms and any acronyms used herein have the same meaning as commonly understood by one of ordinary skill in the art of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, exemplary methods, devices, and materials are described herein.

[0067] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are fully explained in such publications as Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F.M. Usubel et al., eds., 1987, and periodic updates); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Remington, The Science and Practice of Pharmacy, 20th ed., (Lippincott, Williams & Wilkins 2003), and Remington, The Science and Practice of Pharmacy, 22nd ed., (Pharmaceutical Press and Philadelphia College of Pharmacy at University of the Sciences 2012).

[0068] Although certain aspects of the prior art have been discussed to facilitate disclosure of various embodiments, applicants have not in any way abandoned these technical aspects, and it is contemplated that the present disclosure may encompass one or more of the conventional technical aspects discussed herein.

[0069] The present disclosure may address one or more of the problems and deficiencies of known methods and processes. However, it is believed that various embodiments may prove useful in addressing other problems and deficiencies in certain technology areas. Thus, the present disclosure should not necessarily be construed as limited to addressing any of the specific problems or deficiencies discussed herein.

[0070] Where any document, act, or item of knowledge is referenced or discussed in this specification, such reference or discussion is not an admission that the document, act, or item of knowledge, or any combination thereof, was at the priority date, was published, was generally known, was part of the common general knowledge, or otherwise constitutes prior art under applicable statutory provisions, or is known to be relevant to any attempt to solve any problem to which this specification pertains.

[0071] As used herein, the terms "comprises," "comprising," "including," "having," "contains," "containing," "characterized by," or any other variation thereof, are intended to encompass the non-exclusive inclusion of the listed elements, subject to any limitations not expressly stated. For example, a fusion protein, pharmaceutical composition, and / or method "comprising" a list of elements (e.g., components, features, or steps) is not necessarily limited to only those elements (or components, or steps), but may include other elements (or components, or steps) not expressly listed or inherent in the fusion protein, pharmaceutical composition, and / or method.

[0072] As used herein, the transitional phrases "consists of" and "consisting of" exclude any unspecified element, step, or component. For example, "consists of" or "consisting of" used in a claim limits the claim to the components, materials, or steps specifically recited in the claim, excluding impurities normally associated therewith (i.e., impurities within a given component). When the phrase "consists of" or "consisting of" appears in a clause in the body of a claim rather than immediately following a preamble, the phrase "consists of" or "consisting of" limits only the element (or component or step) recited in that clause and does not exclude other elements (or components) from the claim as a whole.

[0073] As used herein, the transitional phrases "consists essentially of" and "consisting essentially of" are used to define fusion proteins, pharmaceutical compositions, and / or methods that include materials, steps, features, components, or elements in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristics of the claimed invention. The term "consisting essentially of" occupies a middle ground between "comprising" and "consisting."

[0074] It will be understood that aspects and embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" aspects and embodiments.

[0075] The terms "a," "an," and "the" are intended to include plural alternatives, e.g., at least one. For example, disclosure of "a compound," "a pharmaceutical agent," "an acid," etc., is meant to encompass one or more mixtures or combinations of one or more compounds, pharmaceutical agents, acids, etc., unless otherwise specified.

[0076] The term "and / or," when used in a list of two or more items, means that any one of the listed items may be used by itself or in combination with any one or more of the listed items. For example, the phrase "A and / or B" is intended to mean either or both of A and B, i.e., A alone, B alone, or a combination of A and B. The phrase "A, B, and / or C" is intended to mean A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0077] Various numerical ranges may be disclosed herein. When applicants disclose or claim any type of range, applicants' intent is to separately disclose or claim each possible number that such range may reasonably encompass, including the endpoints of the range, and any subranges and combinations of subranges encompassed within that range, unless otherwise specified. Moreover, all numerical endpoints of ranges disclosed herein are approximate. As a representative example, applicants disclose that in some embodiments, "a is 15 to 25." This range should be interpreted as including 15 and 25, and further includes each of 16, 17, 18, 19, 20, 21, 22, 23, and 24, including any ranges and subranges between any of these values.

[0078] When such values ​​or ranges are expressed, other disclosed embodiments include the specific values ​​recited from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that there are a number of values ​​disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. In embodiments, "about" can be used to mean, for example, within 10% of the recited value, within 5% of the recited value, or within 2% of the recited value.

[0079] As used herein, the term "pharmaceutical composition" refers to a pharmaceutically acceptable composition that comprises a pharmaceutically active agent and, in some embodiments, further comprises a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition may be a combination of a pharmaceutically active agent and a carrier.

[0080] The term "combination" refers to either a fixed combination in a single dosage unit form or a kit of parts for combined administration, where one or more active compounds and combination partners (e.g., another drug described below, also referred to as a "therapeutic agent" or "co-agent") can be administered independently, simultaneously, or separately at time intervals. In some situations, the combination partners exhibit a synergistic effect, e.g., a synergistic effect. As used herein, terms such as "co-administration" or "co-administration" are intended to encompass the administration of selected combination partners to a single subject (e.g., patient) in need of the selected combination, and are intended to include therapeutic regimens in which the agents are not necessarily administered by the same route of administration or at the same time. As used herein, the term "pharmaceutical combination" refers to a product resulting from the mixing or combining of two or more active ingredients, and includes both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that both active ingredients, e.g., a compound and a combination partner, are administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient as separate entities simultaneously or sequentially without any specific time limit, such administration providing therapeutically effective levels of the two compounds in the patient's body. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0081] As used herein, the term "pharmaceutically acceptable" means approved by a federal or state government regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopeia, as well as other formulations that are safe for use in animals, particularly in humans and / or non-human mammals.

[0082] As used herein, the term "pharmaceutically acceptable carrier" refers to an excipient, diluent, preservative, solubilizer, emulsifier, adjuvant, and / or vehicle into which the demethylated compound is administered. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like, as well as oils containing polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents. Antibacterial agents such as benzyl alcohol or methylparabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and agents for isotonicity adjustment such as sodium chloride or dextrose may also be carriers. Methods for preparing compositions in combination with carriers are known to those skilled in the art. In some embodiments, the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. See, for example, Remington, The Science and Practice of Pharmacy, 20th ed., (Lippincott, Williams & Wilkins 2003). Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated.

[0083] As used herein, a "therapeutically effective amount" refers to a sufficient amount of a pharmaceutically active compound to treat or ameliorate, or in some way alleviate, symptoms associated with a disease or medical condition. When used in reference to a method, the method is sufficiently effective to treat or ameliorate, or in some way reduce, symptoms associated with the disease or condition. For example, an effective amount in the context of a disease is an amount sufficient to block or prevent onset, or where disease pathology has been initiated, to alleviate, ameliorate, stabilize, reverse, or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. In either case, the effective amount can be given in a single dose or in divided doses.

[0084] As used herein, the terms "treat," "treatment," or "treating" encompass at least an improvement in symptoms associated with a disease in a patient, where improvement is used broadly to refer to at least a reduction in the magnitude of a parameter, e.g., a symptom associated with the disease or condition being treated. Thus, "treatment" also includes situations in which a disease, disorder, or pathological condition, or at least the symptoms associated therewith, are completely inhibited (e.g., prevented from occurring) or arrested (e.g., terminated) so that the patient is no longer afflicted with the condition, or at least the symptoms that characterize the condition.

[0085] As used herein, and unless otherwise specified, the terms "prevent," "preventing," and "prevention" refer to the prevention of the onset, recurrence, or spread of a disease or disorder, or one or more symptoms thereof. In certain embodiments, the terms refer to treatment or administration of a compound or dosage form provided herein, with or without one or more other additional active agents, before the onset of symptoms, particularly to a subject at risk for a disease or disorder provided herein. The terms encompass the inhibition or reduction of symptoms of a particular disease. In certain embodiments, subjects with a family history of a disease are likely candidates for a preventative regimen. In certain embodiments, subjects with a history of recurrent symptoms are also likely candidates for prevention. In this regard, the term "prevention" can be used interchangeably with the term "prophylactic treatment."

[0086] As used herein, and unless otherwise specified, a "prophylactically effective amount" of a compound is an amount sufficient to prevent a disease or disorder or prevent its recurrence. A prophylactically effective amount of a compound refers to the amount of a therapeutic agent, alone or in combination with one or more other agents, that provides a prophylactic benefit in the prevention of disease. The term "prophylactically effective amount" can encompass an amount that improves overall prophylaxis or enhances the prophylactic effectiveness of another prophylactic agent. As used herein, and unless otherwise specified, the term "subject" is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In certain embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably herein to refer to a mammalian subject, e.g., a human.

[0087] As used herein, and unless otherwise specified, compounds described herein are intended to encompass all possible stereoisomers unless a particular stereochemistry is specified. Where structural isomers of a compound are interconvertible via a low energy barrier, the compound may exist as a single tautomer or a mixture of tautomers. This can take the form of proton tautomerism, or, for example, so-called valence tautomerism in compounds containing aromatic moieties.

[0088] "Nucleic acid" or "nucleic acid molecule" refers to a polymeric compound containing two or more covalently linked nucleosides or nucleoside analogs with nitrogenous heterocyclic bases or base analogs, where the nucleosides are linked together by phosphodiester or other linkages to form a polynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNA polymers, or oligonucleotides, and their analogs. The nucleic acid backbone can be composed of various linkages, including one or more of sugar phosphodiester linkages, peptide-nucleic acid linkages, phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar portion of a nucleic acid can be ribose, deoxyribose, or similar compounds with known substitutions (e.g., 2'-methoxy and 2'-halide substitutions). The nitrogenous bases can be conventional bases (A, G, C, T, U) or their analogs (e.g., inosine, 5-methylisocytosine, isoguanine). Nucleic acids can contain only conventional sugars, bases, and linkages, as found in RNA and DNA, or they can contain conventional building blocks and substitutions (e.g., conventional bases linked by a 2'-methoxy backbone, or nucleic acids containing a mixture of conventional bases and one or more base analogs). Nucleic acids can include "locked nucleic acids" (LNAs), in which one or more nucleotide monomers have a bicyclic furanose unit locked into an RNA-mimicking sugar conformation, enhancing hybridization affinity to complementary sequences in single-stranded RNA (ssRNA), single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA). Nucleic acids can also contain modified bases that alter the function or behavior of the nucleic acid (e.g., adding a 3'-terminal dideoxynucleotide to block additional nucleotides from being added to the nucleic acid). Synthetic methods for making nucleic acids in vitro are well known in the art, but nucleic acids can also be purified from natural sources using routine techniques. Nucleic acids can be single-stranded or double-stranded.

[0089] Nucleic acids are usually single- or double-stranded and generally contain phosphodiester bonds, although in some cases may have alternative backbones, including, but not limited to, phosphoramides, including nucleic acid analogs as outlined herein (Beaucage et al. (1993) Tetrahedron 49(10):1925 and references therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl et al. (1977) Eur. J. Biochem. 81:579; Letsinger et al. (1986) Nucl. Acids Res. 14:3487; Sawai et al. (1984) Chem. Lett. 805; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470; and Pauwels et al. al. (1986) Chemica Scripta 26:1419 (each incorporated by reference), phosphorothioates (Mag et al. (1991) Nucleic Acids Res. 19:1437, and U.S. Patent Application No. 5,644,048 (both incorporated by reference), phosphorodithioates (Briu et al. (1989) J. Am. Chem. Soc. 111:2321 (incorporated by reference), O-methyl phosphoramidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press (1992) (incorporated by reference), and peptide nucleic acid backbones and linkages (Egholm (1992) J. Am. Chem. Soc. 114:1895, Meier et al. al. (1992) Chem. Int. Ed. Engl. 31:1008; Nielsen (1993) Nature 365:566, and Carlsson et al. (1996) Nature 380:207, each of which is incorporated by reference.Other analog nucleic acids include those with positively charged backbones (Denpcy et al. (1995) Proc. Natl. Acad. Sci. USA 92:6097, which is incorporated by reference), non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141, and 4,469,863; Angew (1991) Chem. Intl. Ed. English 30:423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470; Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597, Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense"). Non-ribose backbones, including those described in "Carbohydrate Modifications in Antisense Research," Ed. Y.S. Sanghvi and P. Dan Cook, Mesmaeker et al. (1994) Bioorganic and Medicinal Chem: Lett. 4:395, Jeffs et al. (1994) J. Biomolecular NMR 34:17, and Tetrahedron Lett. 37:743 (1996) (each of which is incorporated by reference), and U.S. Pat. Nos. 5,235,033 and 5,034,506, Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Ed. Y.S. Sanghvi and P. Dan Cook (each of which references is incorporated by reference). Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (Jenkins et al. al. (1995) Chem. Soc. Rev. pp 169-176, which is incorporated by reference. Some nucleic acid analogs are also described, for example, in Rawls, C & E News Jun. 2, 1997 page 35, which is incorporated by reference.These modifications of the ribose-phosphate backbone can be made to facilitate the addition of additional moieties, such as labels, or to alter the stability and half-life of such molecules in physiological environments.

[0090] In addition to the naturally occurring heterocyclic bases typically found in nucleic acids (e.g., adenine, guanine, thymine, cytosine, and uracil), nucleic acid analogs also include those containing non-naturally occurring heterocyclic or modified bases, many of which are described herein or otherwise referenced. In particular, for many non-naturally occurring bases, see, for example, Seela et al. (1991) Helv. Chim. Acta 74:1790, Grein et al. (1994) Bioorg. Med. Chem. Lett. 4:971-976, and Seela et al. (1999) Helv. Chim. Acta 82:1640, each of which is incorporated by reference. As a further example, certain bases used in nucleotides that act as melting temperature (TO) modifiers are optionally included. For example, some of these include 7-deazapurines (e.g., 7-deazaguanine, 7-deazaadenine, etc.), pyrazolo[3,4-d]pyrimidines, propynyl-dN (e.g., propynyl-dU, propynyl-dC, etc.), etc. See, e.g., U.S. Patent No. 5,990,303, entitled "SYNTHESIS OF 7-DEAZA-2'-DEOXYGUANOSINE NUCLEOTIDES," issued November 23, 1999 to Seela, which is incorporated by reference. Other representative heterocyclic bases include, for example, hypoxanthine, inosine, xanthine; 2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, 8-aza derivatives of hypoxanthine, inosine, and xanthine; 7-deaza-8-aza derivatives of adenine, guanine, 2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine, and xanthine; 6 5-azacytosine; 5-fluorocytosine; 5-chlorocytosine; 5-iodocytosine; 5-bromocytosine; 5-methylcytosine; 5-propynylcytosine; 5-bromovinyluracil; 5-fluorouracil; 5-chlorouracil; 5-iodouracil; 5-bromouracil; 5-trifluoromethyluracil; 5-methoxymethyluracil; 5-ethynyluracil; 5-propynyluracil, and the like.

[0091] Examples of modified bases and nucleotides are described, for example, in U.S. Pat. No. 5,484,908, entitled "OLIGONUCLEOTIDES CONTAINING 5-PROPYNYL PYRIMIDINES," issued Jan. 16, 1996 to Froehler et al.; U.S. Pat. No. 5,645,985, entitled "ENHANCED TRIPLE-HELIX AND DOUBLE-HELIX FORMATION WITH OLIGOMERS CONTAINING MODIFIED PYRIMIDINES," issued Jul. 8, 1997 to Froehler et al.; U.S. Pat. No. 5,830,653, entitled "METHODS OF USING OLIGOMERS CONTAINING MODIFIED PYRIMIDINES," issued Nov. 3, 1998 to Froehler et al.; U.S. Pat. No. 5,830,653, entitled "METHODS OF USING OLIGOMERS CONTAINING MODIFIED PYRIMIDINES," issued Oct. 28, 2003 to Kochkine et al.; No. 6,639,059 entitled "ONE STEP SAMPLE PREPARATION AND DETECTION OF NUCLEIC ACIDS IN COMPLEX BIOLOGICAL SAMPLES," issued Oct. 16, 2001 to Skouv; U.S. Patent Application Publication No. 2003 / 0092905 entitled "SYNTHESIS OF [2.2.1] BICYCLO NUCLEOSIDES," published May 15, 2003 by Kochkine et al., each of which is incorporated by reference.

[0092] "Oligonucleotide" or "oligomer" refers to a nucleic acid comprising at least two nucleic acid monomer units (e.g., nucleotides), usually more than three monomer units, and more usually more than ten monomer units. The exact size of an oligonucleotide generally depends on various factors, including the ultimate function or use of the oligonucleotide. Oligonucleotides are optionally prepared by any suitable method, including, but not limited to, isolation of existing or naturally occurring sequences, DNA replication or amplification, reverse transcription, cloning and restriction digestion of appropriate sequences, or direct chemical synthesis by methods such as the phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68:90-99, the phosphotriester method of Brown et al. (1979) Meth. Enzymol. 68:109-151, the diethyl phosphoramidate method of Beaucage et al. (1981) Tetrahedron Lett. 22:1859-1862, the triester method of Matteucci et al. (1981) J. Am. Chem. Soc. 103:3185-3191, or the solid support method described in U.S. Pat. No. 4,458,066, or other methods known in the art. All of these references are incorporated by reference.

[0093] The compounds of the invention and methods of use for inhibiting RNA viruses include the following viral families: Filoviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, Fenuiviridae, Nairoviridae, Arenaviridae, Flaviviridae, and Coronaviridae. Exemplary virus names within each family are included in the table below. [Table 2-1] [Table 2-2]

[0094] Embodiment Embodiments of the compounds, pharmaceutical formulations, and methods described herein are provided in the following list: Embodiment 1. A compound of formula (I): [ka] wherein Nuc is selected from the group consisting of antiviral nucleosides and antiviral nucleoside analogs; and Y is independently hydrogen, C1-C 30 a hydrocarbyl, a pharmaceutically acceptable cation, and a covalent bond to a carbon atom of a 5-carbon sugar moiety of an antiviral nucleoside or antiviral nucleoside analog; x is 0 or 1; L is a C1-C6 hydrocarbyl; and R is independently selected from the group consisting of C 10 ~C 30 hydrocarbyl, and the substituents of formula (A) are selected from the group consisting of: [ka] In the formula, R 1 and R 2 are independently hydrogen and C1 to C 30 The compound of formula (I) is selected from the group consisting of hydrocarbyl. Embodiment 2. The compound of embodiment 1, wherein the antiviral nucleoside or antiviral nucleoside analog is an antiviral ribonucleoside or antiviral ribonucleoside analog, respectively. Embodiment 3. The compound of any one of the preceding embodiments, wherein Nuc is selected from the group consisting of GS-441524, β-D-N4-hydroxycytidine (NHC), and (2'R)-2-amino-2'-deoxy-2'-fluoro-N,2'-dimethyladenosine. Embodiment 4. The compound of any one of the preceding embodiments, wherein Nuc is GS-441524. [ka] Embodiment 5. Y is an unsubstituted C1-C6 alkyl, C1-C 20 Hydrocarbyl, C1-C 10Hydrocarbyl, C1-C6 hydrocarbyl, or Na + The compound of any one of the preceding embodiments, Embodiment 6. The compound of any one of the preceding embodiments, wherein Y comprises at least one cyclic moiety. Embodiment 7. A compound of any one of the preceding embodiments wherein Y is selected from the group consisting of aryl, arylalkyl, heteroaryl, heteroarylakyl, and heterocycloalkyl, each of which is unsubstituted or substituted. Embodiment 8. A compound of any one of the preceding embodiments, wherein heteroaryl is unsubstituted or substituted pyridinyl. Embodiment 9. A compound of any one of the preceding embodiments, wherein arylalkyl is unsubstituted or substituted benzyl. Embodiment 10. The unsubstituted or substituted benzyl has the structure according to formula (B): [ka] In the formula, R 3 , R 4 , R 5 , R 6 , and R 7 are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, aryl(alkyl), heteroaryl(alkyl), (heterocyclyl)alkyl, hydroxy, alkoxy, acyl, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, azido, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanomethanesulfonamido, amino, monosubstituted amino, and disubstituted amino. Embodiment 11.R 3 , R 4 , R 5, R 6 , and R 7

[0023] The compound of any one of the preceding embodiments, wherein at least two of are hydrogen. Embodiment 12. R is (i) unsubstituted or substituted C 12 ~C 24 The compound of any one of the preceding embodiments, which is hydrocarbyl, (ii) contains 0 to 6 unsaturated bonds, (iii) contains a cyclopropyl moiety, or (iv) a combination thereof. Embodiment 13. R is (i) unsubstituted or substituted C 13 ~C 29 The compound of any one of the preceding embodiments, wherein the compound is heteroalkyl, (ii) contains 0-6 unsaturations, or (iii) a combination thereof. Embodiment 14. R is (i) [ka] (wherein a is 1 to 29), and (ii) [ka] wherein b is 1 to 29, c is 0 to 28, and the sum of b and c is 29 or less. Embodiment 15. The compound of any one of the preceding embodiments, wherein (i) a is 15-25, or (ii) b is 1-4 and c is 15-20. Embodiment 16. The compound of any one of the preceding embodiments, wherein (i) a is 19; (ii) b is 3 and c is 15; or (iii) b is 2 and c is 17. Embodiment 17. The compound of any one of the preceding embodiments, wherein a is 8. Embodiment 18.R 1 (i) unsubstituted or substituted C 12 ~C 24The compound of any one of the preceding embodiments, wherein the compound is hydrocarbyl, (ii) contains 0 to 6 unsaturated bonds, or (iii) a combination thereof. Embodiment 19.(i)R 1 , (ii) R 2 , or (iii) R 1 and R 2 and each independently contain at least one cyclic moiety. 30 The compound of any one of the preceding embodiments, wherein the compound is selected from hydrocarbyl. Embodiment 20.(i)R 1 , (ii) R 2 , or (iii) R 1 and R 2 and R are independently selected from the group consisting of aryl, arylalkyl, heteroaryl, heteroarylakyl, and heterocycloalkyl, each of which is unsubstituted or substituted. Embodiment 21. A compound of any one of the preceding embodiments, wherein arylalkyl is unsubstituted or substituted benzyl. Embodiment 22. The unsubstituted or substituted benzyl has the structure according to formula (C): [ka] In the formula, R 8 , R 9 , R 10 , R 11 , and R 12are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, aryl(alkyl), heteroaryl(alkyl), (heterocyclyl)alkyl, hydroxy, alkoxy, acyl, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, azido, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanomethanesulfonamido, amino, monosubstituted amino, and disubstituted amino. Embodiment 23.R 8 , R 9 , R 10 , R 11 , and R 12

[0023] The compound of any one of the preceding embodiments, wherein at least two of are hydrogen. Embodiment 24. A compound of any one of the preceding embodiments, wherein the substituent of Formula (A) is a racemate, an sn-1 stereoisomer, or an sn-3 stereoisomer. Embodiment 25. (i)R 1 but, (a) [ka] (wherein d is 1 to 29), and (b) [ka] wherein e is 1 to 27, f is 0 to 26, and the sum of e and f is 27 or less; (ii)R 2 but, [ka] wherein g is 1 to 29; or (iii) A compound according to any one of the preceding embodiments, which is a combination thereof. Embodiment 26. The compound of any one of the preceding embodiments, wherein g is 5-10. Embodiment 27. The compound of any one of the preceding embodiments, wherein g is 7. Embodiment 28. A compound of any one of the preceding embodiments wherein x is 1 and L is unsubstituted or substituted C1-C3 hydrocarbyl. Embodiment 29. A compound of any one of the preceding embodiments wherein L is selected from the group consisting of unsubstituted methyl, unsubstituted ethyl, and unsubstituted propyl. Embodiment 30. A pharmaceutical formulation comprising a compound according to any one of embodiments 1 to 29. Embodiment 31. The pharmaceutical formulation of embodiment 30, wherein the pharmaceutical formulation is orally bioavailable. Embodiment 32. The pharmaceutical formulation of embodiment 30, wherein the pharmaceutical formulation is formulated for intramuscular injection. Embodiment 33. A method for treating a coronavirus infection in a mammal, comprising administering to the mammal an effective amount of a compound of any one of embodiments 1 to 29, or a pharmaceutical formulation of any one of embodiments 30 to 32. Embodiment 34. A method for treating a viral infection in a mammal, comprising administering to the mammal an effective amount of a compound of any one of embodiments 1 to 29 or a pharmaceutical formulation of any one of embodiments 30 to 32, wherein the virus is an RNA virus of a viral family selected from the group consisting of Filoviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, Fenuviridae, Nairoviridae, Arenaviridae, Flaviviridae, and Coronaviridae. Embodiment 35. A method for producing a prodrug, the method comprising: (i) providing a compound of formula (a); [ka] (ii) providing a compound of formula (b); [ka] (iii) contacting a compound of formula (a) with a compound of formula (b) to form a compound of formula (c); [ka] (iv) contacting a compound of formula (c) with an acid to form a compound of formula (d); [ka] where Het is a C1-C aryl group containing at least one heteroatom. 30 hydrocarbyl, and Y is hydrogen, C1-C 30 and a pharmaceutically acceptable cation, x is 0 or 1, L is a C1-C6 hydrocarbyl, and R is C 10 ~C 30 is selected from the group consisting of hydrocarbyl and a substituent of formula (A): [ka] In the formula, R 1 and R 2 are independently hydrogen and C1 to C 30 A method for producing a prodrug, wherein the prodrug is selected from the group consisting of hydrocarbyl. Embodiment 36. The method for producing a prodrug of any one of the preceding embodiments, wherein the contacting of the compound of Formula (a) with the compound of Formula (b) occurs in the presence of N,N-dicyclohexylcarbodiimide, 4-dimethylaminopyridine, or a combination thereof. Embodiment 37. A method for producing a prodrug according to any one of the preceding embodiments, wherein the acid comprises HCl. Embodiment 38. A method for producing a prodrug according to any one of the preceding embodiments, wherein the contacting with the acid of Formula (c) occurs in the presence of tetrahydrofuran (THF). Embodiment 39. Het [ka] 10. A method for producing a prodrug according to any one of the preceding embodiments, selected from the group consisting of: Embodiment 40. A method for producing a prodrug of any one of the preceding embodiments, further comprising carrying out an intramolecular esterification reaction of a compound of formula (d) to form a cyclic phosphate, such as a 3',5' cyclic phosphate. Embodiment 41. A method for producing a drug triphosphate, comprising providing a plurality of cells; contacting the plurality of cells with an amount of a drug; and incubating the plurality of cells and the amount of drug for a period of time effective to form the drug triphosphate. Embodiment 42. The method of embodiment 41, wherein the plurality of cells comprises Vero E6 cells. Embodiment 43. The method of embodiment 41 or 42, wherein the drug comprises remdesivir. [Example]

[0095] The present invention is further illustrated by the following examples, which should not be construed as imposing limitations on the scope of the invention in any way. On the contrary, it is to be clearly understood that the present invention is contemplated to have various other aspects, embodiments, modifications, and equivalents thereof, which may suggest themselves to those skilled in the art after reading the description herein, without departing from the spirit of the invention or the scope of the appended claims. Accordingly, other aspects of the present invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein.

[0096] Example 1 - Preparation of Compounds In the examples, several general methods were used to generate various products and / or intermediates, although other known synthetic techniques may also be used.

[0097] A.GS-441524 Synthesis of alkyl and alkoxyalkyl esters of 5'-monophosphate [ka] Synthesis of Alkyl and Alkoxyalkyl Phosphates (Schemes 1, 2a-c) General method A. Long-chain alcohols 1a–c were phosphorylated to give phosphates 2a–c as previously described (Ruiz, J., Beadle, JR, Aldern, KA, Keith, K., Hartline, C., Kern, E., Hostetler, KY (2007). Synthesis and antiviral evaluation of alkoxyalkyl-phosphate conjugates of cidofovir and adefovir. Antiviral Res., 75, 87–90). Briefly, a solution of the long-chain alcohol (1 equivalent) and triethylamine (2 equivalents) in anhydrous tetrahydrofuran (THF) was added dropwise to a solution of phosphorus oxychloride (1.5 equivalents) in THF with stirring, maintaining the temperature below 20°C. Stirring was continued for an additional hour at 0°C, followed by the addition of water and overnight stirring, followed by extraction with ethyl ether. The crude solid from the ether layer was recrystallized from hexane to give phosphates 2a–c.

[0098] 2a eicosyl dihydrogen phosphate 1 H NMR (400 MHz, chloroform-d) δ 8.30 (s, 1H), 3.98 (t, 2H), 1.61 (m, 1H), 1.26 (br s, 16H), 0.86 (t, 1H). ESI-MS 650.38 [M−H] -

[0099] 2b 3-Dihydrogen(hexadecyloxy)propyl phosphate 1 H NMR (400MHz, Chloroform-d) δ 4.03 (dt, 2H), 3.49 (t, 2H), 3.40 (t, 2H), 1.94 (p, 2H), 1.59-1.55 (m, 2H), 1.26 (br s, 18H), 0.86 (t, 3H).

[0100] 2c 2-Dihydrogen(octadecyloxy)ethyl phosphate 1 H NMR (400MHz, Chloroform-d) δ 4.12 (dt, 2H), 3.77 (t, 2H), 3.42 (t, 2H), 1.29 (br s, 20H), 0.94-0.85 (t, 3H).

[0101] Coupling of phosphates 2a-c to GS-441524 acetonide (remdesivir nucleoside, RVn acetonide) General method B. N,N-Dicyclohexylcarbodiimide (DCC, 1.5 equiv.) was added to a mixture of GS-441524 acetonide (1 equiv., CAS# 1191237-80-5, purchased from Ontario Chemicals), long-chain dihydrogen phosphate (1.0 equiv.), and 4-dimethylaminopyridine (DMAP, 1.0 equiv.) in dry pyridine. The mixture was then heated to 90 °C and stirred for 24 h. Water was added to quench the reaction, and the pyridine was evaporated under vacuum. The residue was adsorbed onto silica gel and purified by flash column chromatography on silica gel 60. Gradient elution (CHCl / methanol 10–20%) afforded the protected phosphodiester compound.

[0102] General method C. N,N-Diisopropylcarbodiimide (DIC, 3.3 mmol) was added to a mixture of GS-441524 acetonide (1.65 mmol), lipid phosphate (1.65 mmol), and 1-methylimidazole (NMI, 406 mg, 4.95 mmol) in dry pyridine (30 mL), and the mixture was stirred at room temperature for 48 h. Analysis of the reaction mixture by TLC indicated substantial formation of the coupled product. Water (5 mL) was then added and concentrated on a rotary evaporator. The residue was adsorbed onto silica gel and purified by flash column chromatography on silica gel 60. Gradient elution (100% CHCl to CHCl / 20% methanol) afforded the protected phosphodiester analog.

[0103] 3a Eicosyl-phospho-RVn acetonide. GS-441524 acetonide was coupled to 2a according to general procedure C. The structure was shown in ESI-MS 690.50 [MH]. - was confirmed by.

[0104] 3b 3-(Hexadecyloxy)propyl-phospho-RVn acetonide. GS-441524 acetonide was coupled to 2b according to general procedure B. N-N-dicyclohexylcarbodiimide (DCC, 619 mg, 3 mmol) was added to a mixture of GS-441524 acetonide (300 mg, 0.91 mmol), 3-hexadecylpropyl phosphate (2b, 414 mg, 1.10 mmol), and 4-dimethylaminopyridine (DMAP, 122 mg, 1.0 mmol) in 25 mL of dry pyridine. The mixture was then heated to 90 °C and stirred for 24 h. Pyridine was then evaporated, and the residue was purified by flash column chromatography on silica gel 60. Gradient elution (CHCl / methanol 10–20%) afforded 423 mg (67% yield) of compound 3b. 1 H NMR(500MHz,chloroform-d)δ 8.42(s,1H),7.98(s,1H),7.70(s,2H),6.22(d,J=6.0Hz,1H),5.68(d,J=6.2Hz,1H),5.15(d,J=1.0H z,1H),4.70(dd,J=3.8,0.9Hz,1H),4.48-4.42(m,1H),4.26(ddd,J=11.2,8.5,2.6Hz,1H),4.15(ddd, J=11.1,8.5,2.6Hz,1H),4.02(dt,J=8.5,6.3Hz,2H),3.49(t,J=6.1Hz,2H),3.40(t,J=6.1Hz,2H),1. 95(p,J=6.2Hz,2H),1.54(tt,J=7.4,6.1Hz,2H),1.31(s,3H),1.32-1.24(m,26H),0.94-0.85(m,3H). ESI-MS 691.6[MH] - .

[0105] 3c, 2-(octadecyloxy)ethyl-phospho-RVn acetonide. GS-441524 acetonide was coupled to 2c according to general procedure B. N-N-dicyclohexylcarbodiimide (DCC, 0.3 g, 1.4 mmol) was added to a mixture of GS-441524 acetonide (0.23 g, 0.7 mmol), phosphate 2c (0.27 g, 0.68 mmol), and 4-dimethylaminopyridine (DMAP, 0.07 g, 0.6 mmol) in 10 mL of dry pyridine, which was then heated to 90 °C and stirred for 3 days. The pyridine was then evaporated, and the residue was purified by flash column chromatography on silica gel 60. Gradient elution (CHCl / methanol 10–20%) afforded 0.22 g (45% yield) of phosphodiester 3c.

[0106] Synthesis of 4a-c: Removal of the acetonide protecting group General Method D (HCl / THF) Concentrated HCl (0.1 mL) in tetrahydrofuran (THF, 1 mL) was added to a stirred solution of acetonide-protected (2',3'-isopropylidene)phosphodiester (0.25 mmol) in THF (10 mL) at room temperature. The mixture was stirred for 3 h, and then sodium bicarbonate (50 mg) and water (2 mL) were added. After stirring for an additional 15 min, the solvent was evaporated and cold water (10 mL) was added to the residue. The crude product was collected by vacuum filtration and dried under vacuum. Purification by flash column chromatography (100% CHCl to CHCl / 35% methanol) afforded the pure phosphodiester analog.

[0107] General Method E The acetonide analog (1 mmol) was added to formic acid (25 mL) at room temperature and stirred. The reaction was monitored by TLC until deprotection was complete in approximately 4 hours. The formic acid was removed by rotary evaporation, and the residue was co-evaporated with EtOH (2 x 25 mL), then adsorbed onto silica gel and purified by flash column chromatography. Gradient elution (100% CHCl) 2~ CH2Cl2 / 35% methanol) to give the product.

[0108] 4a Eicosyl-phospho-RVn - prepared from 3a according to general method E. The structure was confirmed by ESI-MS 650.38 [MH]-.

[0109] 4b 3-(Hexadecyloxy)propyl-phospho-RVn. Prepared from 3b according to general procedure D. Concentrated HCl (0.1 mL) in tetrahydrofuran (THF, 1 mL) was added to a stirred solution of 3b (100 mg, 0.14 mmol) in THF (10 mL) at room temperature. The mixture was stirred for 3 h, and then sodium bicarbonate (50 mg) and water (2 mL) were added. After stirring for an additional 15 min, the solvent was evaporated, and cold water (10 mL) was added to the residue. The solid product was collected by vacuum filtration and dried under vacuum to give 4b (79 mg, 87% yield) as an off-white solid. 1 H NMR(500MHz, CDCl3-methanol-d4)δ 8.42(s,1H),7.98(s,1H),7.70(s,1H),6.22(d,J=6.0Hz,1H),5.70(d,J=6.0Hz,1H),5.12(d,J=4.2Hz,1H ),4.55(ddd,J=5.5,2.7,0.9Hz,1H),4.40(dtd,J=6.8,2.6,0.8Hz,1H),4.33-4.27(m,2H),4.25(ddd,J=11 .1,8.4,2.6Hz,1H),4.16(ddd,J=11.3,8.5,2.6Hz,1H),4.02(dt,J=8.5,6.3Hz,2H),3.49(t,J=6.1Hz,2H ),3.40(t,J=6.1Hz,2H),1.95(p,J=6.2Hz,2H),1.59-1.50(m,1H),1.34-1.24(m,23H),0.94-0.85(m,3H). ESI MS:652.39[MH] - Purity by HPLC: 99.7%

[0110] 4c, 2-(octadecyloxy)ethyl-phospho-RVn, was prepared from 3c according to general procedure D. Concentrated HCl (0.3 mL) was slowly added to a stirred solution of 3c (0.2 g, 0.28 mmol) in THF (10 mL) at 0 °C. The mixture was allowed to warm to room temperature overnight, then diluted with water (2 mL) and adjusted to pH = 8 by adding saturated sodium bicarbonate. The product was extracted with chloroform (3 × 30 mL), and the organic layer was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel. Elution with 20% MeOH / CHCl gave 0.10 g (55% yield) of compound 4c. 1 H NMR (400 MHz, CDCl3-methanol-d4) δ ppm 7.89(s,1H),6.94(d,J=4.65Hz,1H),6.89(d,J=4.65Hz,1H),4.40(d,J=4.65Hz ,2H),4.21-4.28(m,1H),4.12-4.20(m,1H),4.04-4.12(m,1H),3.91(d,J=4.89 Hz,2H),3.46-3.57(m,2H),3.42(td,J=6.85,1.96Hz,2H),3.34(dt,J=3.18,1. 59Hz, 2H), 1.53 (d, J = 6.85Hz, 2H), 1.20-1.37 (m, 30H), 0.89 (t, J = 6.97Hz, 3H). ESI MS:666.43[MH] - Purity by HPLC 98.4%.

[0111] B. Synthesis of 2-(octadecyloxy)ethylbenzylphospho-RVn (long-acting formulation) (Scheme 1, 6c) Compound 3c (160 mg, 0.22 mmol), benzyl alcohol (48 mg, 0.45 mmol), diisopropylethylamine (DIEA, 58 mg, 0.45 mmol), and (1H-benzotriazol-1-yloxy)-tripyrrolidinophosphonium hexafluorophosphate (PyBOP, 230 mg, 0.45 mmol) in dry DMF (5 mL) were stirred at room temperature for 3 h. The DMF was then evaporated, and the residue was dissolved in ethyl acetate (50 mL) and washed with saturated NaHCO (3 × 10 mL). The organic layer was dried over MgSO and concentrated. The residue was purified by column chromatography on silica gel eluting with chloroform / methanol (0–15%) to give 5c (60 mg, 35% yield). 1 H NMR(400MHz,CDCl3-CD3OD)δ ppm 7.87(d,J=4.03Hz,1H),7.27-7.37(m,5H),6.91-6.95(m,1H),6.83-6.89 (m,1H),5.41(d,J=6.97Hz,1H),4.92-5.06(m,3H),4.54-4.60(m,1H),4.2 4-4.31(m,2H),4.07-4.15(m,2H),3.53-3.60(m,2H),3.38-3.51(m,2H),3 .32-3.37(m,2H),1.78-1.96(m,2H),1.75(s,3H),1.50-1.60(m,2H),1.42 (s,3H),1.15-1.38(m,30H),0.89(t,J=6.54Hz,3H).ESI MS:798.51[M+H] + , 820.56[M+Na] + .

[0112] To a solution of 5c (60 mg, 0.075 mmol) in THF (2 mL) was added concentrated HCl (0.1 mL) at 0 °C. After 20 min, the ice bath was removed and the reaction was monitored by TLC. After 3 h, the ice bath was replaced and the mixture was neutralized with saturated NaHCO. The mixture was concentrated in vacuo, and the residue was purified by column chromatography (silica gel, dichloromethane / methanol 10-20%) to give 35 mg (62% yield) of 6c. 1H NMR(400MHz,CDCl3+methanol d4)δ ppm 7.84-7.90(m,1H),7.29-7.38(m,5H),6.89-6.93(m,1H),6.82-6.86(m,1H),5.03(d,J=1 1.36Hz,2H),4.76-4.81(m,1H),4.40-4.45(m,1H),4.304.37(m,1H),4.17-4.31(m,2H), 4.06-4.14(m,2H),3.54-3.60(m,2H),3.39-3.47(m,2H),3.33-3.37(m,2H),3.12-3.18( m,2H),1.82-1.91(m,2H),1.49-1.59(m,2H),1.20-1.37(m,30H),0.89(t,J=6.60Hz,3H). ESI MS:758.32[M+H] + ,780.43[M+Na] + .

[0113] C.GS-441524 Synthesis of 1-O-Alkyl-2-O-Substituted-sn-Glyceryl Esters of 5'-Monophosphate The following scheme (Scheme 2) illustrates an embodiment of the synthetic method used to generate the following embodiments of 1-O-alkyl-2-O-substituted-sn-glyceryl esters of GS-441524 5'-monophosphate. [ka]

[0114] Synthesis of 1-O-alkyl-sn-glycerols (Scheme 2, 10a-d) General Method F. In this embodiment, alkylation of 2,3-isopropylideneglycerol with alkyl methanesulfonates was carried out as described in the literature (Fernandez, DM; Contreras, LJ; Moreno, BM; Silva, EG; Mayorga, HG. Enantiomeric synthesis of natural alkylglycerols and their antibacterial and antibiotic activities. Nat. Prod. Res. 2019, 1-7.). Briefly, sodium hydride and DMF were stirred in a flask. Isopropylideneglycerol was added slowly (hydrogen evolution!) and cooled, if necessary, to keep the temperature below 35°C. Stirring continued for an additional 30 minutes. The alkyl methanesulfonate was added all at once and stirred vigorously for 5 hours. The reaction mixture was poured onto crushed ice and gently stirred. The solid was collected on a fritted funnel and washed with water. Deprotection: The filter cake was added to 80% acetic acid and heated at 80°C for 1 hour. The flask was cooled, the product was allowed to crystallize, and the vacuum filtrate was collected and dried. The crude product was either recrystallized in hexane or purified by flash column chromatography on silica gel 60.

[0115] General Method G (Alkylation using 1-bromoalkanes / alkenes as described in the literature: (Halldorsson, A., et al. Tetrahedron: Asymmetry, 2004, 15, 2893-2899)). Briefly, isopropylideneglycerol (1 equiv.), 1-bromoalkane / alkene (1 equiv.), and tetrabutylammonium bromide (0.2 equiv.) were vigorously stirred in a round-bottom flask. Crushed potassium hydroxide (2 equiv.) was slowly added, and the mixture was stirred in an oil bath at 35–40°C for approximately 15 hours. The alkylated product was extracted into hexane, and the organic phase was washed with HO and then evaporated to give 1-O-alkyl-2,3-isopropylidene-sn-glycerol. Deprotection: The product was refluxed overnight with p-toluenesulfonic acid (10 mol%) in THF / water. After concentration in vacuo, the residue was dissolved in diethyl ether, washed with water and brine solution, dried over anhydrous magnesium sulfate, and the solvent was removed in vacuo on a rotary evaporator to give 1-O-alkyl-sn-glycerol.

[0116] 10a. 1-O-Tetradecyl-sn-glycerol. Synthesized according to general method F. Analytical data were consistent with literature values ​​(Barragan, CA; Silva, EG; Moreno, BM; Mayorga, HW. Inhibition of quorum sensing by compounds from two Eunicea species and synthetic saturated alkylglycerols. Vitae 2018, 25, 92-103).

[0117] 10b. 1-O-Hexadecyl-sn-glycerol was purchased from Bachem America.

[0118] 10c. 1-O-Octadecyl-sn-glycerol was purchased from Bachem America.

[0119] 10d. 1-O-oleyl-sn-glycerol. Synthesized according to general procedure G. A mixture of oleyl bromide (541 mg, 1.63 mmol), Bu₄NBr (0.2 equiv.), 2,3-isopropylidene-sn-glycerol (1 equiv.), and KOH (powder, 2.5 equiv.) was stirred at 40 °C overnight. Workup afforded 583 mg of crude 9d as an oil. Crude 9d was treated with p-TsOH.HO (0.15 equiv.) in THF (6 mL) and HO (2.5 mL) at reflux overnight. Purification of the crude oil (540 mg) by flash column chromatography (MeOH in DCM 0-8%) afforded 420 mg of 1-O-oleyl-sn-glycerol 10d as an oil. Yield: 75% (2 steps). 1H NMR(CDCl3)δ 5.36-5.33(m,2H),3.86-3.85(m,1H),3.72(dd,1H),3.62(dd,1H),3.52(dd,1H),3.46(dd,1H),3.50-3. 42(m,2H),2.02-1.99(m,4H),1.59-1.55(quintet,2H),1.35-1.26(m,22H),0.88(t,3H)ESI-MS:343.67[M+H] + ,365.61[M+Na] + .

[0120] Synthesis of 1-O-alkyl-2-O-substituted-sn-glycerols (Scheme 2, 12a-m) General Method H Protection of the 3-hydroxy group of 1-O-substituted sn-glycerol was carried out as described in the literature (Kini, GD, Hostetler, SE, Beadle, JR, Aldern, K.A. Synthesis and antiviral activity of 1-O-octadecyl-2-O-alkyl-sn-glycero-3-foscarnet conjugates in human cytomegalovirus-infected cells, Antiviral Research, 1997, 36, 115; and Huang, Z., Szoka, Z. (2008). Sterol-Modified Phospholipids: Cholesterol and Phospholipid Chimeras with Improved Biomembrane Properties. J. Am. Chem. Soc., 130, 15702-15712). Briefly, triethylamine (1.5 equiv.) was added to a solution of 1-O-alkyl-sn-glycerol (1 equiv.), N,N-dimethylaminopyridine (DMAP, 0.1 equiv.), and triphenylchloride (TrCl, 1.5 equiv.), and the mixture was stirred for 18 h. The reaction mixture was then quenched with water, evaporated, adsorbed onto silica gel, and purified by flash column chromatography on silica gel. An increasing gradient of ethyl acetate in hexane (0-20%) eluted the appropriate fractions.

[0121] 11a 1-O-tetradecyl-3-O-trityl-sn-glycerol - prepared as described in Huang, Z., Szoka, Z. (2008). Sterol-Modified Phospholipids: Cholesterol and Phospholipid Chimeras with Improved Biomembrane Properties. J. Am. Chem. Soc., 130, 15702-15712.

[0122] 11b 1-O-Hexadecyl-3-O-trityl-sn-glycerol - prepared as described in Huang, Z., Szoka, Z. (2008). Sterol-Modified Phospholipids: Cholesterol and Phospholipid Chimeras with Improved Biomembrane Properties. J. Am. Chem. Soc., 130, 15702-15712.

[0123] 11c 1-O-Octadecyl-3-O-trityl-sn-glycerol. Prepared from 10c according to general method H. Yield 87%. 1 H NMR(CDCl3):δ 0.9(t,3H),1.3(bs,30H),1.55(m,4H)3.2(m,2H),3.4-3.6(m,3H),3.95(m,1H)7.2-7.5(m,15H).

[0124] 11d 1-O-oleyl-3-O-trityl-sn-glycerol. Prepared from 10d according to general method H. Yield 77%. 1 H NMR(CDCl3),δ 0.88(t,J=7.2,3H);1.27(br,22H);1.55(m,2H);2.0(m,4H);2.40(br,1H);3.20(m, 2H);3.41-3.56(m,4H);3.95(m,1H);5.35(m,2H);7.25(m,9H);7.45(m,6H).ESI-MS 607.75[M+Na] +

[0125] General Method I Alkylation and deprotection of 1-O-alkyl-3-O-trityl-sn-glycerol were carried out as previously described (Kini, GD, Hostetler, SE, Beadle, JR, Aldern, K.A. Synthesis and antiviral activity of 1-O-octadecyl-2-O-alkyl-sn-glycero-3-foscarnet conjugates in human cytomegalovirus-infected cells, Antiviral Research, 1997, 36, 115). Briefly, sodium hydride (2.5 equivalents) was added to a stirred solution of 1-O-alkyl-3-O-trityl-sn-glycerol (1 equivalent) in DMF at 0°C. After 20 minutes, the bromo or methanesulfonate derivative of R2- (1.8 equivalents) was added. The reaction mixture was then stirred at room temperature for 5 hours or until the reaction was substantially complete by TLC. Workup and column chromatography gave the acid-detritylated 1-O-alkyl-2-O-substituted-3-O-trityl-sn-glycerols, which were then purified by workup and column chromatography to give the 1-O-alkyl-2-O-substituted-sn-glycerols.

[0126] 12a 1-O-tetradecyl-2-O-benzyl-sn-glycerol - prepared from 11a and benzyl bromide according to general method I. Structure: ESI-MS: 401.51 [M+Na] + was confirmed by.

[0127] 12b 1-O-Hexadecyl-2-O-benzyl-sn-glycerol - Prepared from 11b and benzyl bromide according to general method I. 1 H NMR (300MHz, CDCl3): δ 7.36-7.27(m,5H),4.65(m,2H),3.79-3.59(m,5H),3.55(t,2H),1.57-1.51(m,2H),1.29(br s,26H),0.89(t,3H).

[0128] 12c 1-O-Hexadecyl-2-O-(3-fluoro-4-methoxybenzyl)-sn-glycerol was prepared from 11b and 3-fluoro-4-methoxybenzyl bromide according to general method I.

[0129] 12d 1-O-octadecyl-2-O-benzyl-sn-glycerol, 12e 1-O-octadecyl-2-O-benzyl-rac-glycerol, 12f 1-O-octadecyl-2-O-octyl-sn-glycerol, 12g 1-O-octadecyl-2-O-(cyclohexylmethyl)-sn-glycerol, 12h 1-O-octadecyl-2-O-(3-fluorobenzyl)-sn-glycerol, 12i 1-O-octadecyl-2-O-(4-methoxybenzyl)-sn-glycerol, 12j 1-O-octadecyl-2-O-(3-fluoro-4-methoxybenzyl)-sn-glycerol, and 12k 1-O-octadecyl-2-O-(pyridin-3-yl-methyl)-sn-glycerol was prepared from 11c and the appropriate bromide according to general method I.

[0130] 12l 1-O-oleyl-2-O-benzyl-sn-glycerol-. Sodium hydride (1.3 equiv.) was added to 11d (531 mg, 0.91 mmol) in DMF (4 mL) at 0 °C. The resulting mixture was stirred at room temperature for 1 h before benzyl bromide (1.3 equiv.) was added. The reaction mixture was stirred at room temperature overnight. Workup and column chromatography afforded 354 mg of crude product, and 200 mg of 11d was also recovered. Deprotection afforded 12l. 1H NMR(300MHz,chloroform-d)δ 7.35-7.26(m,4H),5.36-5.32(m,2H),3.84-3.62(m,5H),3.54-3.46(m,2H),3.44(t,2H),2.88-2.75(m,2H),2.02(m,4H),1.54-1.50(pentet) 2H), 1.29(br s, 22H), 0.88(t, 3H). 455.73[M+Na] +

[0131] 12m 1-O-oleyl-2-O-(3-fluoro-4-methoxybenzyl)-sn-glycerol. Prepared from 11d and 3-fluoro-4-methoxybenzyl bromide according to general method I. 1H NMR (300 MHz, chloroform-d) δ 7.12 (m, 2H), 6.95 (t, 1H), 5.36-5.32 (m, 2H), 4.65-4.52 (dd, 2H), 3.75-3.70 (m, 2H), 3.67-3.60 (m, 2H), 3.57-3.55 (m, 2H), 3.45 (t, 2H), 2.01-1.97 (m, 2H), 1.28 (br s, 16H), 0.87 (t, 3H). ESI-MS: 503.79 [M+Na] +

[0132] Synthesis of 1-O-alkyl-2-O-substituted-sn-glyceryl esters of GS-441524 5'-monophosphate (Scheme 3, 15a-m) The following scheme illustrates an embodiment of the synthetic steps used to generate 1-O-alkyl-2-O-substituted-sn-glyceryl esters of GS-441524 5'-monophosphate. [ka]

[0133] General Method J. Phosphorylation of 1-O-alkyl-2-O-substituted-sn-glycerols was accomplished as described in Kates, M., Adams, GA, Blank, ML, Snyder, FM (1991). Chemical synthesis and physiological activity of sulfonium analogues of platelet activating factor. Lipids, 26, 1095-1101. Briefly, 1-O-alkyl-2-O-substituted-sn-glycerol (11.5 mmol) and 1-methylimidazole (14.4 mmol) were dissolved in dry pyridine (100 mL) and stirred at room temperature. A solution of bis(trichloroethyl)chlorophosphate (5.5 g, 14.4 mmol) in diethyl ether (20 mL) was added dropwise over 10 min, and the mixture was then stirred overnight. Analysis by TLC indicated complete phosphorylation. Water (10 mL) was added to quench the excess reagent, and the mixture was then concentrated by rotary evaporation and coevaporated with toluene to remove pyridine. The residue was adsorbed onto silica gel 60 (approximately 30 g) and purified by flash column chromatography. The protected phosphorylated product was isolated using a gradient elution from 100% hexane to 25% EtOAc / hexane.

[0134] The product (9.65 mmol) was dissolved in a mixture of chloroform (50 mL) and glacial acetic acid (90 mL), then vigorously stirred and cooled in an ice-water bath. Zinc powder (5 g) was added to the mixture and stirred for 1 h. The ice-water bath was then removed and stirring continued for an additional 2 h. The remaining zinc was removed by vacuum filtration, and the clear filtrate was concentrated by rotary evaporation. The residue was taken up in 20% MeOH / CHCl (250 mL) and extracted with 1 M HCl (3 × 50 mL). The organic layer was then concentrated and coevaporated with ethanol (2 × 50 mL). The waxy residue was dissolved in 1,4-dioxane, frozen, and then lyophilized in vacuo (18 h) to give glyceryl phosphate.

[0135] Compounds 13a, 13b, 13c, 13d, 13e, 13f, 13g, 13h, 13i, 13j, and 13k were prepared according to general method J.

[0136] 13L 1-O-oleyl-2-O-benzyl-sn-glyceryl-3-phosphate. Prepared according to general procedure A. 1 H NMR(300MHz,chloroform-d)δ 7.36-7.23(m,5H),5.36-5.32(m,2H),4.69(d,J=11.9Hz,1H),4.62(d,J=11.8Hz,1H),4.11-4.09(m,2H),3.80-3 .77(m,2H),3.76-3.69(m,1H),3.53-3.47(m,1H),3.42(t,2H),2.00(tq,J=7.1,3.7Hz,4H),1.50(m,2H),1.26(br s,22H),0.87(t,3H). ESI-MS:513.72[M+1] +

[0137] 13m 1-O-oleyl-2-O-(3-fluoro-4-methoxybenzyl)-sn-glyceryl-3-phosphate. Prepared according to general method A.

[0138] Coupling of phosphates 13a-m to GS-441524 acetonide (Remdesivir nucleoside, RVn acetonide) 14a 1-O-tetradecyl-2-O-benzyl-sn-glyceryl-phospho-RVn acetonide - prepared from GS-441524 acetonide and 13a according to general procedure C. Structure: ESI-MS 770.50 [M−H] - was confirmed by.

[0139] 14b 1-O-Hexadecyl-2-O-benzyl-sn-glyceryl-phospho-RVn acetonide - Prepared from GS-441524 acetonide and 13b according to general procedure C.

[0140] 14c 1-O-Hexadecyl-2-O-(3-fluoro-4-methoxy-benzyl)-sn-glyceryl-phospho-RVn acetonide - Prepared from GS-441524 acetonide and 13c according to general procedure C.

[0141] 14d 1-O-Octadecyl-2-O-benzyl-sn-glyceryl-phospho-RVn acetonide—Prepared from GS-441524 acetonide and 13d according to general procedure B. N,N-Dicyclohexylcarbodiimide (DCC, 310 mg, 1.5 mmol) was added to a mixture of acetonide (300 mg, 0.91 mmol), phosphate 13d (515 mg, 1.0 mmol), and 4-dimethylaminopyridine (DMAP, 122 mg, 1.0 mmol) in 25 mL of dry pyridine, and the mixture was then heated to 90 °C and stirred for 24 h. The pyridine was then evaporated, and the residue was purified by flash column chromatography on silica gel 60. Gradient elution (CHCl / methanol 10–20%) afforded 210 mg (28% yield) of compound 14d. ESI MS 826.58[MH] - .

[0142] 14e 1-O-Octadecyl-2-O-benzyl-rac-glyceryl-phospho-RVn acetonide - prepared from GS-441524 acetonide and 13e according to general method C.

[0143] 14f 1-O-Octadecyi-2-O-octyl-sn-glyceryl-phospho-RVn acetonide - Prepared from GS-441524 acetonide and 13f according to general method C.

[0144] 14 g 1-O-Octadecyl-2-O-(cyclohexylmethyl)-sn-glyceryl-phospho-RVn acetonide - can be prepared from GS-441524 acetonide and 13 g according to general method C.

[0145] 14h 1-O-Octadecyl-2-O-(3-fluoro-benzyl)-sn-glyceryl-phospho-RVn acetonide - Prepared from GS-441524 acetonide and 13h according to general procedure C.

[0146] 14i 1-O-Octadecyl-2-O-(4-methoxy-benzyl)-sn-glyceryl-phospho-RVn acetonide - can be prepared from GS-441524 acetonide and 13i according to general method C.

[0147] 14j 1-O-Octadecyl-2-O-(3-fluoro-4-methoxy-benzyl)-sn-glyceryl-phospho-RVn acetonide - Prepared from GS-441524 acetonide and 13j according to general procedure C.

[0148] 14k 1-O-Octadecyl-2-O-(pyridin-3-yl-methyl)-sn-glyceryl-phospho-RVn acetonide - can be prepared from GS-441524 acetonide and 13k according to general procedure C.

[0149] 14l 1-O-oleyl-2-O-benzyl-sn-glyceryl-phospho-RVn acetonide - prepared from GS-441524 acetonide and 13l according to general procedure B.

[0150] 14m 1-O-oleyl-2-O-(3-fluoro-4-methoxy-benzyl)-sn-glyceryl-phospho-RVn acetonide. Prepared from GS-441524 acetonide and 13m according to general method B.

[0151] Removal of the acetonide protecting group 15a 1-O-tetradecyl-2-O-benzyl-sn-glyceryl-phospho-RVn - Prepared from compound 14a according to general method E and isolated as an off-white powder. The structure was confirmed by ESI-MS [M−H]. - =730.41.

[0152] 15b 1-O-Hexadecyl-2-O-benzyl-sn-glyceryl-phospho-RVn. Prepared from compound 14b according to general method E. 1 H NMR(500MHz,DMSO-d6)δ 7.94(s,1H),7.89(s,1H),7.81(s,1H),7.32-7.25(m,3H),7.22(ddd,J=8.7,5.4,2.6Hz,1H),6.88(d,J=4.5Hz,1H), 6.80(d,J=4.5Hz,1H),6.24(s,1H),5.95(d,J=4.0Hz,1H),4.55(q,J=12.1,12.1,12.1Hz,3H),4.09(dt,J=6.7,4.3, 4.3Hz,1H),3.92(d,J=4.5Hz,1H),3.78(dtt,J=24.4,7.8,7.8,4.4,4.4Hz,2H),3.66-3.55(m,3H),3.43(dd,J=10.6 ,3.5Hz,1H),3.32-3.28(m,2H),1.42(q,J=6.5,6.5,6.0Hz,2H),1.20(d,J=7.7Hz,24H),0.83(t,J=7.0,7.0Hz,3H). LC / MS purity=99.8%;[M+H] + 760.6.

[0153] 15c 1-O-Hexadecyl-2-O-(3-fluoro,4-methoxybenzyl)-sn-glyceryl-phospho-RVn. Prepared from compound 14c according to general method E. 1H NMR(500MHz,DMSO-d6)δ 7.89(s,1H),7.80(s,1H),7.11(d,J=12.3Hz,1H),7.04(d,J=6.1Hz,2H),6.87(d,J=4.6Hz,1H) ,6.81(d,J=4.5Hz,1H),6.17(s,1H),4.56(d,J=5.0Hz,1H),4.52-4.41(m,2H),4.10(s,1H),3.9 3q,J=5.4,5.2,5.2Hz,1H),3.78(s,3H),3.63(s,2H),3.57(d,J=4.6Hz,1H),3.41(dd,J=10.4, 3.5Hz, 1H), 3.29 (s, 3H), 1.41 (d, J = 6.5Hz, 2H), 1.25-1.17 (m, 24H), 0.83 (t, J = 7.0, 7.0Hz, 3H). LC / MS purity=99.8%;[M+H] + 808.9.

[0154] 15f 1-O-Octadecyi-2-O-octyl-sn-glyceryl-phospho-RVn - Prepared from compound 14f according to general method E. 1 H NMR(500MHz,DMSO-d6)δ 7.89(s,1H),7.79(s,1H),6.88(d,J=4.5Hz,1H),6.80(d,J=4.5Hz,1H),6.10(s,1H),5.92(s, 1H),4.56(t,J=5.2,5.2Hz,1H),4.08(t,J=5.7,5.7Hz,1H),3.91(q,J=5.0,4.9,4.9Hz,1H),3. 79(d,J=18.1Hz,3H),3.54(d,J=19.3Hz,3H),3.46-3.35(m,5H),3.33(s,2H),3.27-3.23(m,1H) ),1.41(dt,J=16.0,7.4,7.4Hz,4H),1.21(d,J=4.5Hz,36H),0.83(td,J=7.1,7.0,5.7Hz,6H). LC / MS purity 99.7%; [M+H] + 810.7.

[0155] 15 g 1-O-Octadecyl-2-O-(ethylcyclohexyl)-sn-glyceryl-phospho-RVn--can be prepared from compound 14 g according to general method E.

[0156] 15h 1-O-Octadecyl-2-O-(3-fluoro-benzyl)-sn-glyceryl-phospho-RVn--Prepared from compound 14h according to general method E. 1 H NMR(500MHz,DMSO-d6)δ 8.47(s,2H),7.88(s,2H),7.36-7.26(m,1H),7.12(q,J=8.4,6.9,6.9Hz,2H),7.03(td,J=8.5,8.4,2.9Hz,1H),6.8 8(d,J=4.5Hz,1H),6.80(d,J=4.5Hz,1H),6.30(s,1H),5.97(s,1H),4.64-4.50(m,3H),4.13-4.07(m,1H),3.92(t, J=5.8,5.8Hz,1H),3.79(dddd,J=33.7,12.0,7.6,4.3Hz,2H),3.63(dtt,J=14.0,10.1,10.1,5.7,5.7Hz,3H),3.43 (dd,J=10.7,3.4Hz,2H),1.43(p,J=6.5,6.5,6.5,6.5Hz,2H),1.20(d,J=11.1Hz,30H),0.83(t,J=6.9,6.9Hz,3H). LC / MS purity 98.7%; [M+H] + 806.8.

[0157] 15i 1-O-Octadecyl-2-O-(4-methoxybenzyl)-sn-glyceryl-phospho-RVn--can be prepared from compound 14i according to general method E.

[0158] 15j 1-O-Octadecyl-2-O-(3-fluoro-4-methoxy-benzyl)-sn-glyceryl-phospho-RVn--Prepared from compound 14j according to general method E. 1H NMR(500MHz,DMSO-d6)δ 8.43(d,J=6.7Hz,2H),7.99-7.73(m,2H),7.04(d,J=5.5Hz,1H),6.87(q,J=3.7,3.7,3.2Hz,1H),6.82(dd,J=7.3,4.3Hz,1H), 6.17-5.74(m,1H),4.59(t,J=4.9,4.9Hz,1H),4.53-4.41(m,1H),4.11(q,J=4.9,4.9,4.9Hz,1H),3.93(q,J=5.4,5.4,5.4Hz, 1H),3.84(dq,J=11.3,6.5,5.1,5.1Hz,1H),3.80-3.70(m,3H),3.61(ddd,J=27.7,10.9,5.2Hz,3H),3.30(dd,J=6.6,3.0Hz,3 H),3.21(dq,J=9.8,5.1,5.1,4.9Hz,1H),1.43(p,J=6.6,6.6,6.5,6.5Hz,2H),1.27-1.16(m,30H),0.83(t,J=6.8,6.8Hz,3H). LC / MS purity 94.7%; [M+H] + 836.8.

[0159] 15k 1-O-Octadecyl-2-O-(pyridin-3-yl-methyl)-sn-glyceryl-phospho-RVn - can be prepared from compound 14k according to general method E.

[0160] 15l 1-O-oleyl-2-O-benzyl-sn-glyceryl-phospho-RVn - Prepared from compound 14l according to general method D and isolated as an off-white solid in 84% yield. 1H NMR(300MHz,CDCl3+CD3OD)δ 7.79(s,1H),7.38(s,2H),7.27-7.21(m,5H),6.92(d,J=6.0 Hz,1H),6.90(d,J=6.0Hz,1H),5.30(t,J=6.0Hz,2H),4.70(d,J=11Hz,1H),4.62 (d,J=5Hz,1H),4.34-4.49(m,1H),4.20(m,1H),3.90-3.87(m,2H),4.18-4.06(m, 2H),3.71-3.69(m,2H),3.52(ddd,J=11.7,3.1,1.3Hz,1H),3.35(t,2H),1.96-1. 93(m,4H)1.49-1.47(m,2H),1.33-1.23(m,20H),0.83(t,2H).LC / MS purity 99%;[M+H] + 786.78.

[0161] 15m 1-O-oleyl-2-O-(3-fluoro,4-methoxybenzyl)-sn-glyceryl-phospho-RVn - Prepared from compound 14m according to general method D and isolated as an off-white solid. Yield was 92%. 1 H NMR(300MHz,CD3OD)δ 7.74(s,1H),7.32(s,2H),6.99(d,J=6.0Hz,1H),6.92-6.77(m,3H),5.23(t,J=6.0Hz,2H),4.67(d,J=11Hz,1H),4.49(d,J=5Hz,2 H),4.30(m,1H),4.20(m,1H),3.83-3.81(m,2H),3.75(s,3H),3.63(m,1H),3.31(t,2H),1.91-1.89(m,4H),1.44(m,2H),1.18(br s,22H),0.77(t,2H).LC / MS purity 99%;[M+H] + 834.87.

[0162] D. Synthesis of 1-O-octadecyl-2-O-benzyl-sn-glyceryl-benzyl-phospho-RVn [ka]

[0163] Compound 14d (160 mg, 0.22 mmol), benzyl alcohol (48 mg, 0.45 mmol), diisopropylethylamine (DIEA, 58 mg, 0.45 mmol), and (1H-benzotriazol-1-yloxy)-tripyrrolidinophosphonium hexafluorophosphate (PyBOP, 230 mg, 0.45 mmol) in dry DMF (5 mL) were stirred at room temperature for 3 h. The DMF was then evaporated, and the residue was dissolved in ethyl acetate (50 mL) and washed with saturated NaHCO (3 × 10 mL). The organic layer was dried over MgSO and concentrated. The residue was purified by column chromatography on silica gel eluting with chloroform / methanol (0–15%) to give 16. ESI-MS 918.33 [MH] - .

[0164] Compound 16 was added to formic acid and the deprotection was monitored by TLC. The mixture was concentrated in vacuo and the residue was purified by column chromatography (silica gel, dichloromethane / methanol 10-20%) to give compound 17. The structure was confirmed by ESI-MS: 878.35 [M+H]. + , 900.43[M+Na] + I confirmed it.

[0165] E. Synthesis of GS-441524 - 3',5'-cyclic monophosphate, 1-O-octadecyl-2-O-benzyl-sn-glyceryl ester In another embodiment, the compound of the present invention is a 3',5'-cyclic monophosphate. The 3',5'-cyclic monophosphate can be prepared from known or readily prepared starting materials according to methods known to those skilled in the art of organic synthesis. As an example, the 3',5'-cyclic monophosphate 18 can be prepared from the phosphodiester 15d by an intramolecular esterification reaction.

[0166] A solution of 1-O-octadecyl-2-O-benzyl-sn-glyceryl-phospho-RVn (1 mmol) in dry pyridine (25 mL) was added dropwise to a solution of triisopropylbenzenesulfonyl chloride (3 mmol) and 1-methylimidazole (1 mmol) in dry pyridine (100 mL). The mixture was stirred at room temperature for 2 days or until TLC showed substantial conversion to the 3',5'-cyclic phosphate. The solvent was evaporated under vacuum, and the residue was purified by column chromatography on silica gel to give compound 18 as a mixture of equatorial and axial isomers. The isomers can be separated using either preparative HPLC or preparative chiral HPLC prior to antiviral testing. [ka]

[0167] To measure the cell-based anti-SARS CoV-2 activity of RVn 3',5'-cyclic phosphate prodrugs, assays such as those described in Examples 2-11 below can be performed. The data obtained can indicate that the compounds have significant antiviral activity.

[0168] Example 2 - Assay of anti-coronavirus activity in Vero E6 cells This example refers to the following compounds, including remdesivir nucleoside analogs and related intermediates: [ka]

[0169] The compounds of this example were assayed for anti-coronavirus activity in Vero E6 cells in comparison with remdesivir (RDV) and remdesivir nucleoside (RVn). 10,000 Vero E6 cells were seeded in 100 microliters of culture medium in a 96-well plate. The following day, serial two-fold dilutions of antiviral compounds or DMSO-containing vehicle were added to each well. The USA WA-01 strain of SARS-CoV-2 was added to each well 30 minutes later at a multiplicity of infection of 0.1. Cells were incubated for 48 hours, washed twice in PBS, and lysed with TRIzol. RNA was extracted using Directzol microRNA columns. RNA was generated into cDNA and assayed for SARS-CoV-2 spike protein and housekeeping gene (RPLPO) RNA by qPCR. Data represent the average of duplicate wells. Cell cytotoxicity was also measured in Vero E6 cells. As shown below, each of the synthesized compounds exhibited enhanced anti-SARS CoV-2 activity compared to remdesivir or remdesivir nucleosides with selectivity indices ranging from 22.8 to >227. 50 ) was assessed using a commercially available MTT assay.

[0170] As shown in the table below, ODE-P-RVn (4c) and ODBG-P-RVn (15d) were 9-15 times more active against the USA WA-1 strain of SARS-CoV-2019 in Vero E6 cells. Similarly, HDP-P-RVn (4b) was 3.3 times more active than remdesivir. [Table 3]

[0171] Example 3. Additional synthesis and testing of RVn monophosphate prodrugs. Antiviral Activity: Concentration-response curves for ODBG-P-RVn (15d), ODE-P-RVn (4c), and HDP-P-RVn (4b), remdesivir (RDV), and remdesivir nucleoside (RVn) against SARS-CoV-2 infection in Vero E6 cells were also generated in two separate experiments performed in duplicate (Figures 1A-1F). Dose-response curves for three remdesivir analogs (Figures 1A, 1B, and 1C), remdesivir (GS-5734) (Figure 1D), and remdesivir nucleoside (GS-441524) (Figure 1E) against SARS-CoV-2 infection in Vero E6 cells. Vero E6 cells were pretreated with the indicated drug at the indicated dose for 30 minutes and then infected with SARS-CoV-2 isolate USA-WA1 / 2020 for 48 hours. Relative SARS-CoV-2 spike RNA expression was determined by qRT-PCR. Each dose-response comparison was performed simultaneously for all drugs on two separate occasions. Data from both experiments are shown in Figure 1A-1F. Data points represent the average relative expression from duplicate wells. Error bars indicate standard deviation (SD). The vertical black dashed line indicates where there is 50% inhibition (EC 50 ) concentrations are shown (Figure 1F). Combined inhibition curves for all five compounds and DMSO on a single chart. DMSO was the vehicle for all compounds and had no effect on SARS-CoV-2 replication at the concentrations used. All three lipid esters of RVn-monophosphate were substantially more active than RDV and RVn.

[0172] The table below shows the effective concentration (EC 50 , E.C. 90 ), 50% cytotoxic concentration (CC 50 ), and selection index, mean ± SD are shown. Cytotoxicity (CC 50 ) was assessed using Cell Titer Glo. EC of RDV and RVn 50 The values ​​were 4.6 and 1.7 μM, respectively. The lipid prodrugs had EC values ​​ranging from 0.19 ± 0.023 to 0.96 ± 0.17. 50ODBG-P-RVn and ODE-P-RVn were the most active and selective compounds. EC 50 Based on the values, the most active compound, ODBG-P-RVn, was 24-fold more active than RDV and 8.9-fold more active than RVn (p<0.001 and 0.005), with a selectivity index of 240. [Table 4]

[0173] Of all the recognized drawbacks of RVn, this example chose to design a prodrug of RVn that could provide oral bioavailability, because an effective oral drug would allow much earlier treatment of individuals diagnosed with SARS-CoV-2 infection. As shown in this example, this was achieved by constructing a liponucleotide of RVn that resembles the lysophospholipids normally absorbed in the GI tract. RVn liponucleotides are not rapidly metabolized in plasma and gain rapid entry into cells, often demonstrating significantly increased antiviral activity.

[0174] In contrast to RDV activation, which requires four transformations, intracellular kinase bypass by this type of compound generated nucleoside monophosphates when the lipid ester moiety was cleaved in a single reaction catalyzed by acid phospholipase C or acid sphingomyelinase (sphingomyelin phosphodiesterase I).

[0175] One of the compounds, ODBG-P-RVn(15d), was likely delivered relatively more to the lungs and less to the liver, as previously shown in lethal varicella infection of mice (Hostetler KY, Beadle JR, Trahan J, Aldern KA, Owens G, Schriewer J, Melman L, Buller RM. Oral 1-O-octadecyl-2-O-benzyl-sn-glycero-3-cidofovir targets the lung and is effective against a lethal respiratory challenge with ectromelia virus in mice. Antiviral Res. 2007 Mar;73(3):212-8. doi:10.1016 / j.antiviral.2006.10.009. Epub 2006 Nov 9. PMID:17123638; PMCID:PMC1859865).

[0176] The synthesis of the lipid prodrugs in this example was much simpler than that of RDV and was readily scalable.

[0177] In this example, we synthesized three lipid prodrugs of RVn that were substantially more active than RDV or RVn in Vero E6 cells. The two most active compounds, ODBG-P-RVn and ODE-P-RVn, were 24- and 9.8-fold more active than RDV. These compounds were orally bioavailable, stable in plasma, and predicted to provide significant exposure and antiviral activity in all tissues infected with SARS-CoV-2.

[0178] Compounds: Remdesivir (GS-5734) and remdesivir nucleoside (GS-441524) were purchased from AA Block (San Diego, CA) and Mason-Chem (Palo Alto, CA), respectively.

[0179] Cells: VERO E6 were obtained from ATCC and grown in DMEM (Corning) with 10% FBS and penicillin-streptomycin (Gibco).

[0180] SARS-CoV-2 infection: SARS-CoV-2 isolate USA-WA1 / 2020 (BEI Resources) was propagated and infectious units were quantified by plaque assay using Vero E6 (ATCC) cells. Approximately 10 per well. 4 Vero E6 cells were seeded into 96-well plates and incubated overnight. Compounds or controls were added at the indicated concentrations 30 min prior to infection, followed by SARS-CoV-2 at a multiplicity of infection equal to 0.01. After 48 h of incubation at 37°C and 5% CO2, cells were washed twice with PBS and lysed in 200 μl of TRIzol (ThermoFisher).

[0181] RNA extraction, cDNA synthesis, and qPCR: RNA was purified from TRIzol lysates using the Direct-zol RNA Microprep Kit (Zymo Research) according to the manufacturer's recommendations, including DNase treatment. RNA was converted to cDNA using the iScript cDNA Synthesis Kit (BioRad). qPCR was performed using the iTaq Universal SYBR Green Supermix (BioRad) and an ABI 7300 real-time PCR system. cDNA was amplified using the following primers: RPLP0 F-GTGTTCGACAATGGCAGCAT; RPLP0 R-GACACCCTCCAGGAAGCGA; SARS-CoV-2 Spike F-CCTACTAAATTAAATGATCTCTGCTTTACT; SARS-CoV-2 Spike R-CAAGCTATAACGCAGCCTGTA. The relative expression of SARS-CoV-2 spike RNA was calculated by delta-delta-Ct by first normalizing to the housekeeping gene RPLP0 and then comparing with untreated SARS-CoV-2 infected Vero E6 cells (reference control). Curves were fitted and the 50 and 90% effective concentrations EC50 and EC 90 Values ​​were calculated using Prism8.

[0182] CellTiter-glo luminescent cell viability assay: approximately 10 per well 4 Vero E6 cells were seeded into opaque-walled 96-well cell culture plates and incubated overnight. Compounds or controls were added at the indicated concentrations. After 48.5 hours of incubation at 37°C and 5% CO2, an equal volume of CellTiter-Glo reagent (Cat. #G7570, Promega, Madison, WI) was added, and luminescence was recorded on an EnSpire Multimode Plate Reader (PerkinElmer) according to the manufacturer's recommendations. Viability was calculated relative to untreated controls and CC values ​​were calculated. 50 Values ​​were calculated using Prism8 (Table S10).

[0183] Determination of cytotoxicity: 50% cytotoxic concentration (CC 50 ) was determined according to the manufacturer's recommendations (Cat.#G7570, Promega, Madison, WI). Calculated CC 50 The values ​​are shown in the table above.

[0184] Vero E6 cells were treated with increasing concentrations of remdesivir analogs, remdesivir (GS-5734), remdesivir nucleoside (GS441524), or DMSO vehicle (control) for 48.5 hours. Relative viability was measured by the CellTiter-Glo luminescent cell viability assay, as shown in Figure 2.

[0185] Example 4 - Production of remdesivir triphosphate in Vero E6 cells In this example, Vero E6 cells were grown at approximately 3.4 x 10 cells per well in 2 mL of medium (DMEM, 10% FBS). 5 Cells were seeded in a 6-well plate.

[0186] The cells were then incubated at 37°C for 24 hours. The medium was then aspirated and replaced with 2 mL of control medium (fresh Dulbecco's Modified Eagle Medium (DMEM), 10% FBS) or 2 mL of medium with drug at a concentration of 1 μM. The cells were incubated with various drugs for 48 hours. The medium was aspirated, and the cells were rinsed twice with phosphate-buffered saline (PBS), trypsinized with 1 mL of ATV for 5 minutes, triturated, removed to a 15 mL centrifuge tube, rinsed with 1 mL of PBS, which was then also removed to a 15 mL centrifuge tube, and triturated again. The cells were counted using a Reichert hemocytometer using two 10 μL samples to determine the number of cells in each sample.

[0187] The cells were centrifuged at 1200 rpm for 10 min, the diluent was aspirated, and the pellet was resuspended in 250 μL of methanol / distilled water (70 / 30) and analyzed by LC / MS / MS. The results, shown in Figure 3, were obtained from pmol / 10 6 Cells and are the average of two or three measurements. Abbreviations for Figure 3: RDV, remdesivir; RVn, remdesivir nucleoside (GS-441524); ODE-P-RVn, octadecyloxyethyl-phospho-RVn (4c); ODBG-P-RVn, 1-O-octadecyl-2-O-benzyl-glyceryl-sn-3-phospho-RVn (15d).

[0188] As shown in Figure 3, in Vero E6 cells, remdesivir triphosphate (RVn-TP) synthesis progressively increased up to 48 hours after exposure to 1 micromolar ODE-P-RVn and OBDG-P-RVn. RVn-induced RVn-TP levels peaked at 8 hours and then decreased. RDV-induced RVn-TP levels were below quantifiable levels at 8 and 24 hours.

[0189] Example 5 - Human coronavirus 229E infection In this example, human coronavirus 229E (ATCC) was propagated and infectious units were determined using MRC-5 cells at TCID 50For antiviral testing, approximately 104 MRC-5 cells were seeded per well in a 96-well plate in EMEM (10% FCS) overnight at 37°C. The medium was removed from each well, and the cells were incubated with 100 TCID in 100 μL of medium. 50 The virus was then allowed to infect the mice for 2 hours.

[0190] The cells were washed once with medium, and then compounds or controls were added at the indicated concentrations. After 3 days, CPE was observed under a microscope and quantified using an MTT cell proliferation assay kit (Abcam) read on an ELx800 Universal Microplate Reader (BIO-TEK Instruments, INC.). [Table 5]

[0191] The % inhibition was calculated as (Atv-Acv) / (Acd-Acv) x 100%, where Atv represents the absorbance of the test compound with virus-infected cells, and Acv and Acd represent the absorbance of the virus control and the cell control, respectively. The mean 50% maximal effective concentration (EC 50 ) was defined as the concentration that achieved 50% inhibition of the virus-induced cytopathic effect.

[0192] Example 6 - SARS-CoV-2 infection assay Approximately 12e3 TMPRSS2-Vero cells or 20e3 Huh7.5 cells were seeded per black well of a clear, flat-bottom 96-well plate and incubated overnight. Compounds or controls were added at the indicated concentrations, followed by the addition of SARS-CoV-2 at a multiplicity of infection (FFU / cell) equal to 0.01 for TMPRSS2-Vero and 0.1 for Huh7.5, approximately 30 to 60 minutes prior to infection.

[0193] After 32 hours of incubation for TMPRSS2-Vero or 48 hours for Huh7.5 at 37°C and 5% CO2, the medium was removed and cells were incubated with 4% formaldehyde for 30 minutes at room temperature. Formaldehyde-fixed cells were washed with PBS, permeabilized for immunofluorescence in 0.1% Triton-X100 in PBS with 1% bovine serum albumin (BSA) fraction V (Millipore-Sigma), and stained for SARS-CoV-2 with a primary anti-nucleocapsid antibody (GeneTex GTX135357) followed by an AlexaFluor 594 secondary antibody (Thermo Fisher Scientific A-11012) with the nuclear counterstain Sytox Green (Thermo Fisher Scientific).

[0194] Five images per well were acquired at 10x magnification using an Incucyte S3 (Sartorius). The percentage of infected cells and nuclei number were calculated using the built-in image analysis tools for the Incucyte S3. Calculations of EC50, EC90, and CC50 were performed using nonlinear regression analysis in GraphPad Prism9 with bottom and top parameters constrained to 0 and 100, respectively. [Table 6-1] [Table 6-2]

[0195] Example 7 - Antiviral activity in various cell types infected with SARS-CoV-2 Vero E6, Caco-2, and Calu-3 cell lines were obtained from ATCC. Huh7.5 cells were obtained from Apath LLC. Calu-3 and Caco-2 cells were grown in DMEM (Corning), 10% FBS, and penicillin-streptomycin (Gibco). Vero E6 and Huh7.5 cells were grown in DMEM (Corning) with 10% FBS and penicillin-streptomycin (Gibco). Generation of human PSC-lung cells. Human lung organoids were generated as previously described (Leibel SL, McVicar RN, Winquist AM, Niles WD, Snyder EY. Generation of complete multi-cell type lung organoids from human embryonic and patient-specific induced pluripotent stem cells for infectious disease modeling and therapeutics validation. Curr. Protoc. Stem Cell Biol., 54(1) (2020 Sep), Article e118). H9 embryonic stem cells (WiCell) were cultured under feeder-free conditions on Matrigel (Corning #354230)-coated plates in mTeSR medium (StemCellTech #85850). The medium was changed daily, and stem cells were subcultured using the enzyme-free dissociation reagent ReLeSR™ (Stem Cell Tech #05872). Cultures were maintained in an undifferentiated state at 37°C in a 5% CO2 incubator.

[0196] For proximal lung organoid generation, human PSCs were dissociated into single cells and then cultured at 5.3 x 10 in definitive endoderm (DE) induction medium (RPMI 1640, 2% B27 supplement, 1% HEPES, 1% Glutamax, 50 U / mL penicillin / streptomycin). 4 cells / cm 2Cells were seeded onto Matrigel-coated plates (BD Biosciences) at a density of 100 ng / mL human activin A (R&D), 5 μM CHIR99021 (Stemgent), and 10 μM ROCK inhibitor, Y-27632 (R&D Systems) on day 1. On days 2 and 3, cells were cultured in DE induction medium with 100 ng / mL human activin A alone. Anterior foregut endoderm (AFE) was generated on days 4–6 by supplementing serum-free basal medium (3 parts IMDM: 1 part F12, B27+N2 supplement, 50 U / mL penicillin / streptomycin, 0.25% BSA, 0.05 mg / mL L-ascorbic acid, 0.4 mM monothioglycerol) with 10 μM SB431542 (R&D) and 2 μM dorsomorphin (StemGent). On day 7, AFE medium was changed to lung progenitor cell (LPC) induction medium containing serum-free basal medium supplemented with 10 ng / mL human recombinant BMP4 (R&D), 0.1 μM all-trans retinoic acid (Sigma-Aldrich), and 3 μM CHIR99021. Medium was changed every other day for days 9–11. To generate 3D human proximal lung organoids, a previously published protocol (KB McCauley, F. Hawkins, M. Serra, DC Thomas, A. Jacob, and DN Kotton. (2017) Efficient Derivation of Functional Human Airway Epithelium from Pluripotent Stem Cells via Temporal Regulation of Wnt Signaling. Cell Stem Cell;20(6):844-857) was modified.

[0197] LPCs were dissociated in Accutase for 10 min and plated onto Matrigel in 12-well 0.4 μm pore size Transwell (Corning) culture inserts at 5.0 × 10 4Cells were resuspended in 200 μl of Matrigel. Cells were cultured in proximal lung organoid maturation medium using serum-free basal medium supplemented with 250 ng / mL FGF2, 100 ng / mL rhFGF10, 50 nM dexamethasone (Dex), 100 μM 8-bromoadenosine 3',5'-cyclic monophosphate sodium salt (Br-cAMP), 100 μM 3-isobutyl-1-methylxanthine (IBMX), and 10 μM ROCK inhibitor (Y-27632). The proximal lung organoid medium was changed every other day for 3 weeks. Human PSC-derived lung organoids were dissociated into single cells and seeded at 20,000 cells per well on Matrigel-coated 96-well plates one day before transfection. Transwells containing proximal organoids in Matrigel were incubated in 2 U / mL dispase for 30 minutes at 37° C. Cold PBS was added to the mixture, which was then centrifuged at 400×g for 5 minutes.

[0198] The supernatant was carefully removed and the cells were resuspended in 2-3 ml of TrypLE Express (Gibco #12605010) for 20 minutes at 37°C. The reaction was quenched with 2% FBS in DMEM / F12 and then centrifuged at 400 x g for 5 minutes. The supernatant was aspirated, and the cell pellet was resuspended in 1 ml of quenching medium supplemented with 10 μM Rock inhibitor (Y-27632). A cell count was performed, and the respective volume of cells was transferred into a reagent reservoir trough, resuspended in proximal lung organoid maturation medium, and seeded as a monolayer into a 96-well plate at 100 μl per well via a multichannel pipette.

[0199] SARS-CoV-2 infection: SARS-CoV-2 isolate USA-WA1 / 2020 (BEI Resources) was propagated, and infectious units were quantified by plaque assay using Vero E6 (ATCC) cells. Approximately 12,000 cells from each cell line were seeded per well in a 96-well plate. Vero E6 and Huh7.5 were seeded approximately 24 hours before treatment / infection. Calu-3 and Caco-2 were seeded approximately 48 hours before treatment / infection. Human PSC lung cell infection and cytotoxicity experiments were performed when cells reached 100% confluency. Compounds or controls were added at the indicated concentrations 30 minutes before infection, followed by SARS-CoV-2 at a multiplicity of infection equal to 0.01. After 48 h of incubation at 37°C and 5% CO2, cells were washed twice with PBS and lysed in 200 μl of TRIzol (ThermoFisher). All work with SARS-CoV-2 was performed under biosafety level 3 conditions at the University of California, San Diego, with approval from the Institutional Biosafety Committee.

[0200] RNA extraction, cDNA synthesis, and qPCR: RNA was purified from TRIzol lysates using the Direct-zol RNA Microprep Kit (Zymo Research) according to the manufacturer's recommendations, including DNase treatment. RNA was converted to cDNA using the iScript cDNA Synthesis Kit (BioRad). qPCR was performed using the iTaq Universal SYBR Green Supermix (BioRad) and an ABI 7300 real-time PCR system. cDNA was amplified using the following primers: RPLP0 F-GTGTTCGACAATGGCAGCAT; RPLP0 R-GACACCCTCCAGGAAGCGA; SARS-CoV-2 Spike F-CCTACTAAATTAAATGATCTCTGCTTTACT; SARS-CoV-2 Spike R-CAAGCTATAACGCAGCCTGTA. The relative expression of SARS-CoV-2 spike RNA was calculated by delta-delta-Ct by first normalizing to the housekeeping gene RPLP0 and then comparing with untreated SARS-CoV-2 infected Vero E6 cells (reference control). Curves were fitted using a nonlinear regression-logarithmic (inhibitor) vs. response (four parameters) model using Prism 9. Effective concentration EC 50 and EC 90 To calculate values, qRT-PCR values ​​were normalized to percent inhibition and curve-fit using a nonlinear regression-log (agonist) vs. response (four parameters) model with base and top constrained to 0 and 100, respectively, using Prism9.

[0201] Cell viability assay: Cell types were seeded according to the SARS-CoV-2 infection assay in opaque-walled 96-well cell culture plates or the 229E infection assay in clear 96-well cell culture plates and incubated overnight. Compounds or controls were added at the indicated concentrations. For SARS-CoV-2-related assays, cells were incubated at 37°C and 5% CO2 for 72 hours. An equal volume of CellTiter-Glo reagent (Cat. #G7570, Promega, Madison, WI) was added according to the manufacturer's recommendations, and luminescence was recorded on a Veritas Microplate Luminometer (Turner BioSystems). For the related 229E assays, cells were incubated at 37°C and 5% CO2 for 72 hours. The supernatant was removed, and 50 μL of serum-free medium and 50 μL of MTT reagent (Abcam ab211091) were added to each well and incubated at 37°C for 3 hours. Absorbance was measured with an ELx800 Universal Microplate Reader (BIO-TEK Instruments, INC.) according to the manufacturer's recommendations. Viability was calculated relative to untreated controls and CC 50 Values ​​were calculated using Prism9. [Table 7]

[0202] In all cell lines, there was dose-dependent inhibition of viral RNA by ODBG-P-RVn, ODE-P-RVn, HDP-P-RVn, remdesivir (RDV), and remdesivir nucleoside (RVn). In Vero E6 cells, the mean 50% maximal effective concentration (EC 50 ) and mean 90% effective concentration (EC 90 ) were 0.14 μM and 0.16 μM, respectively. The EC 50 ODE-P-RVn and HDP-P-RVn also showed EC values ​​of 0.3 μM and 0.63 μM in Vero E6. 50The EC values ​​of ODBG-P-RVn and ODE-P-RVn were strong antiviral activity. 50 The antiviral activity of ODBG-P-RVn and ODE-P-RVn was significantly better than that of RVn in PSC-Lung cells. ODBG-P-RVn, ODE-P-RVn, and HDP-P-RVn inhibited EC 50 In the Caco-2 cell line, ODBG-P-RVn showed potent antiviral activity in Huh7.5 cells where the EC value was less than 0.2 μM, with no significant difference from that of RDV or RVn. 50 The EC of ODE-P-RVn was 0.3 μM, significantly lower than that of RVn but similar to that of RDV. 50 was 0.77 μM, significantly higher than RDV.

[0203] The cytotoxicity of each compound was determined by incubating each of these cell lines with serial dilutions of each compound from 1.23 μM to 100 μM for 48 hours. The mean 50% cytotoxic concentration (CC) of all compounds was calculated. 50 ) had a CC of 32.7 μM in PSC-lung cells and 15.2 μM in the human liver cell line Huh7.5. 50 The selectivity index of ODBG-P-RV was greater than 60 μM in all cell lines except for RDV, which has ODBG-P-RVn (EC). In the five cell types tested in this example, the selectivity index of ODBG-P-RV ranged from 295 to 699. 50 0.14μM~0.30μM and CC 50 The range of antiviral activity and cytotoxicity of RDV (EC 61.5μM-98.2μM) was 50 0.06μM~1.13μM and CC 50 The potency of RVn monophosphate prodrugs was more consistent across cell types than that of RVn monophosphate prodrugs (15.2 μM to >100 μM) (Table 1). Collectively, these data demonstrate that lipid RVn monophosphate prodrugs are potent antiviral agents against SARS-CoV-2 in vitro with low toxicity and excellent selectivity indices.

[0204] Example 8 - Effect of antiviral drugs on human coronavirus 229E-infected cells Human coronavirus 229E (ATCC) was propagated and TCID was measured using MRC-5 cells. 50 For antiviral testing, infectious units were quantified by approximately 10 4 MRC-5 cells were seeded per well in EMEM (10% FCS) in a 96-well plate overnight at 37°C. The medium was removed from each well and the cells were incubated with 100 TCID in 100 μL medium. 50 The virus was then allowed to infect the mice for 2 hours.

[0205] The cells were washed once with medium, and then compounds or controls were added at the indicated concentrations. After 3 days, CPE was observed under a microscope and quantified using an MTT cell proliferation assay kit (Abcam) read on an ELx800 Universal Microplate Reader (BIO-TEK Instruments, INC.). % inhibition was calculated as (A tv -A cv ) / (A cd -A cv ) × 100%, where A tv indicates the absorbance of the test compound with virus-infected cells, and A cv and A cd indicates the absorbance of the virus control and the absorbance of the cell control, respectively. 50 ) was defined as the concentration that achieved 50% inhibition of the virus-induced cytopathic effect.

[0206] Figures 4A and 4B: ODBG-P-RVn (15d) inhibits human alphacoronavirus 229E. Figure 4A shows the antiviral dose-response curves for remdesivir (GS-5734) and ODBG-P-RVn against human coronavirus 229E in MRC-5 cells. Cells were infected with 229E for 2 hours and subsequently treated with the indicated drug at the indicated dose for 72 hours. Relative CPE was determined by measuring cell viability using an MTT assay.

[0207] Figure 4B shows cytotoxicity in MRC-5 cells incubated for 72 hours in the presence of the indicated drugs at the indicated concentrations, after which cell viability was measured by the CellTiter-Glo assay. Data points represent the mean from three independent experiments performed in duplicate. Error bars represent the standard error of the mean (SEM).

[0208] Both ODBG-P-RVn and RDV showed dose-dependent inhibition of cytopathic effect (CPE). 50 The values ​​were 0.15 μM and 0.04 μM, and the EC 90 The CC of ODBG-P-RVn and RDV was 0.54 mM and 0.26 mM, respectively. 50 was greater than 50 μM in MRC-5 cells, which, together with the antiviral data for SARS-CoV-2, indicates that ODBG-P-RVn has antiviral activity against two genetically distinct human pathogenic coronaviruses.

[0209] Example 9 - Orally administered ODBG-P-RVn(15d) achieves therapeutic plasma levels in Syrian hamsters. ODBG-P-RVn in 0.1 M sodium carbonate / bicarbonate buffer (pH 9.0) was administered to Syrian hamsters by oral irrigation every 12 hours for 7 days. ODBG-P-RVn was present as the sodium salt. It was well tolerated, and no adverse clinical signs were observed. Peak plasma levels of ODBG-P-RVn were observed at 1 hour and declined by 50% by approximately 5 hours.

[0210] Plasma curves were generally similar on days 1 and 7, except for 16.9 mg / kg, where the values ​​on day 7 were slightly higher than the levels on day 1. At 12 hours, ODBG-P-RVn levels were above the EC 0.01 for ODBG-P-RVn on both days 1 and 7 in all cell lines tested, including Vero E6 cells and PSC lung cells. 90The levels of RVn, a nucleoside metabolite of ODBG-P-RVn, peaked 3 hours after administration and then decreased. Plasma levels of RVn were higher than the EC 0.01 for RVn in both PSC lung cells and Vero E6 cells. 90 The observed low levels of RVn suggest that the antiviral activity attributable to this metabolite is minimal and is consistent with findings of OBDG-P-RVn stability in human plasma. Collectively, these results suggest that OBDG-P-RVn is effective in suppressing viral replication in various tissue types in vivo.

[0211] Figures 5A and 5B show 7-day oral pharmacokinetics in Syrian hamsters. Syrian hamsters received vehicle or ODBG-P-RVn by oral irrigation every 12 hours for 7 days. Groups of three animals received vehicle or drug at doses of 16.9 and 13.2 mg / kg. Animals were weighed daily and monitored for clinical signs. Plasma samples were obtained at 1, 3, 6, and 12 hours on days 1 and 7 and frozen for analysis by LC / MS / MS for (Figure 5A) ODBG-P-RVn and (Figure 5B) RVn.

[0212] Analytical Method: ODBG-P-RVn: A hamster plasma sample (10 μL) containing ODBG-P-RVn and K2EDTA as anticoagulants was added to a polypropylene tube containing 100 μL of water, 100 μL of internal standard solution (1,000 ng / mL ODE-P-RVn in ACN:DMF (1:1, v / v)), and ACN:DMF (1:1, v / v). The solution was mixed, then acidified with phosphoric acid, 85% w / v:water (1:19, v / v; 10 μL), mixed, then diluted with 200 μL of IPA, mixed, then diluted with 500 μL of water, and mixed. The sample was extracted with a Sep-Pak® tC18 96-well solid-phase extraction plate (25 mg; water, Milford, MA). Extraction was performed under positive pressure using nitrogen. Samples were washed consecutively with 1 mL of water:acetonitrile:formic acid (475:25:0.5, v / v) and 0.4 mL of water:acetonitrile:formic acid (350:150:0.5, v / v), followed by sequential elution with 100 μL and 150 μL of a 2% w / v solution of water:{acetonitrile:isopropyl alcohol (1:1, v / v)}:formic acid:ammonium formate:citric acid (15:85:0.1:0.1:0.1, v / v / v). The citric acid solution was prepared as water:citric acid monohydrate (20:0.4, v / w). After elution, 100 μL of water was added to each sample. The ODBG-P-RVn extract was analyzed using an Agilent 1200 HPLC system (Agilent, Santa Clara, CA) coupled to an API5500 mass spectrometer (SCIEX, Foster City, CA). Analytes were chromatographically separated using a Dacapo DX-C18 MF column (100 × 2 mm, 2.5 μm; ImtaktUSA, Portland, OR) with a mobile phase system consisting of mobile phase A (water:formic acid:[water:ammonium formate:citric acid (25:5:0.5, v / w)] (1,000:1:1:1, v / v / v)) and mobile phase B (acetonitrile:isopropyl alcohol:formic acid:[water:ammonium formate:citric acid (25:5:0.5, v / w)] (800:200:1:1, v / v / v)). The total analytical run time was 4.5 min.The mobile phase was nebulized using heated nitrogen in a Turbo-V source / interface set in electrospray positive ionization mode. Ionized compounds were detected using multiple reaction monitoring via the m / z transitions 788.4 > 229 (V2043) and 668.4 > 467.2 (V2041). This method was applied to measure ODBG-P-RVn concentrations ranging from 6.25 to 3,000 ng / mL using 10.0 μL of plasma for extraction. Peak areas of ODBG-P-RVn and RVn were acquired using Analyst v.1.6.2 (SCIEX, Framingham, MA). A calibration curve was obtained using Analyst by fitting the analyte / IS peak area ratio and standard concentration to a linear equation with a 1 / x2 weighting. The peak area ratio was then used to interpolate the analyte concentration in the sample using the equation for the calibration curve. The peak areas used in the calculations were not rounded.

[0213] Analytical Method: RVn (GS-441524): Hamster plasma samples (20 μL) containing GS-441524 and K2EDTA as anticoagulants were added to an Eppendorf LoBind microcentrifuge tube containing 300 μL of acetonitrile and 60 μL of water:acetonitrile (2:8, v / v). The solution was mixed and centrifuged at 16,000 g for 5 minutes. The supernatant (300 μL) was then filtered through an Ostro protein precipitation and phospholipid removal plate (25 mg, Waters, Milford, MA). Filtration was performed under positive pressure using nitrogen. The collected filtered sample was capped, mixed, and stored at 10°C awaiting analysis. GS-441524 extracts were analyzed using an Acquity UPLC system (Waters, Milford, MA) coupled to a G2-SQTof mass spectrometer (Waters, Milford, MA). Analytes were chromatographically separated using a Unison-UK Amino HT column (100 x 2 mm, 3 μm; ImtaktUSA, Portland, OR) with a mobile phase system consisting of mobile phase A (0.008% ammonium hydroxide, 0.012% acetic acid in water, v / v / v) and mobile phase B (0.008% ammonium hydroxide, 0.012% acetic acid in acetonitrile, v / v / v). The total analytical run time was 12.5 min. The mobile phase was nebulized using heated nitrogen with a Z-spray source / interface set in electrospray positive ionization mode. Ionized compounds were detected using Time of Flight (Tof) MS scan monitoring in sensitivity mode scanning from 50.0 to 700 m / z. This method is applicable to measure GS-441524 concentrations ranging from 1.00 to 1,000 ng / mL using 20.0 μL of plasma for extraction. The peak area of ​​GS-441524 was obtained using MassLynx V4.2 (Waters, Milford, MA). A calibration curve was generated using MassLynx by calculating the peak area ratio of the analyte and the standard concentration as 1 / x. 2The peak areas were then used to interpolate the concentration of the analyte in the sample using the equation of the calibration curve. The peak areas used in the calculation were not rounded.

[0214] Example 10 - Stability of ODE-P-RVn (4c) and ODBG-P-RVn (15d) in human plasma One drawback of remdesivir is its plasma instability, which has been reported to persist at virologically significant levels for less than 2 hours after intravenous infusion. (1,2) Remdesivir also has a reported T of 69 minutes. 1 / 2 (Siegel D, Hui HC, Doerffler E, Clarke MO, Chun K, Zhang L, Neville S, Carra E, Lew W, Ross B, Wang Q, Wolfe L, Jordan R, Soloveva V, Knox J, Perry J, Perron M, Stray KM, Barauskas O, Feng JY, Xu Y,Lee G,Rheingold AL,Ray AS,Bannister R,Strickley R,Swaminathan S,Lee WA,Bavari S,Cihlar T,Lo MK,Warren TK,Mackman RL.Discovery and Synthesis of a Phosphoramidate Prodrug of a Pyrrolo[2,1-f][triazin-4-amino]Adenine C-Nucleoside (GS-5734) for the Treatment of Ebola and Emerging Viruses.J Med Chem.2017 Mar 9;60(5):1648-1661).

[0215] The stability of ODE-P-RVn and ODBG-P-RVn in human plasma was evaluated with either K2EDTA or sodium heparin as anticoagulants.

[0216] Plasma was spiked with ODE-P-RVn or ODBG-P-RVn at a concentration of 2 μg / mL and incubated at 37°C. Samples were collected at 0.5, 1, 2, 4, 8, and 24 hours and frozen for subsequent analysis by LC / MS / MS according to the method described in Example C. Figures 6A and 6B show that both ODE-P-RVn and ODBG-P-RVn were stable for at least 24 hours in human plasma with either KEDTA (Figure 6A) or sodium heparin (Figure 6B) as the anticoagulant. (See, e.g., Warren T. et al. Nature. 2016 Mar. 17;531(7594):381-5; and Tempestilli, M. et al. J. Antimicrob Chemother. 2020 Oct. 1;75(10):2977-2980.)

Claims

1. A compound of formula (I) 【Chemistry 1】 During the ceremony, Nuc is GS-441524 【Chemistry 2】 and Y is independently selected from the group consisting of hydrogen and a pharmaceutically acceptable cation; x is 0, R is independently (a) CH 3 (CH 2 ) 15 O(CH 2 ) 3 −, (b) CH 3 (CH 2 ) 17 O(CH 2 ) 2 —, or (c) 【Transformation 3】 A compound selected from:

2. The compound of claim 1, wherein Y is hydrogen.

3. The compound of claim 1, wherein Y is a pharmaceutically acceptable cation.

4. The compound of claim 1, wherein R is CH 3 (CH 2 ) 15 O(CH 2 ) 3 —.

5. The compound of claim 1, wherein R is CH 3 (CH 2 ) 17 O(CH 2 ) 2 —.

6. R is 【Chemistry 4】 2. The compound of claim 1, wherein:

7. The compound according to claim 6, wherein the compound is hexadecyloxypropyl-phospho-RVn. 【Transformation 5】 or a pharmaceutically acceptable salt thereof.

8. The compound according to claim 7, wherein the compound is octadecyloxyethyl-phospho-RVn 【Transformation 6】 or a pharmaceutically acceptable salt thereof.

9. The compound according to claim 8, wherein the compound is 1-O-octadecyl-2-O-benzyl-sn-glyceryl-phospho-RVn 【Transformation 7】 or a pharmaceutically acceptable salt thereof.

10. A compound having one of the following structures or a pharmaceutically acceptable salt thereof: Table 1-1 Table 1-2

11. A pharmaceutical formulation comprising a compound according to any one of claims 1 to 10.

12. 12. The pharmaceutical formulation of claim 11, wherein the pharmaceutical formulation is orally bioavailable.

13. 12. The pharmaceutical formulation of claim 11, wherein the pharmaceutical formulation is formulated for intramuscular injection.

14. The pharmaceutical formulation of claim 11, wherein the pharmaceutical formulation is formulated for intravenous administration.

15. A pharmaceutical preparation according to any one of claims 11 to 14 for treating coronavirus infections in mammals.

16. A pharmaceutical formulation according to any one of claims 11 to 15 for treating a viral infection in a mammal, comprising:

1. The pharmaceutical formulation, wherein the virus is an RNA virus of a viral family selected from the group consisting of Filoviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, Phenuiviridae, Nairoviridae, Arenaviridae, Flaviviridae, and Coronaviridae.

17. 1. A method for producing a prodrug, the method comprising: (i) providing a compound of formula (a); 【Transformation 8】 (ii) providing a compound of formula (b); 【Chemistry 9】 (iii) contacting the compound of formula (a) with the compound of formula (b) to form a compound of formula (c); 【Chemistry 10】 (iv) contacting the compound of formula (c) with an acid to form a compound of formula (d); 【Chemistry 11】 During the ceremony, Het, 【Chemistry 12】 and Y is selected from the group consisting of hydrogen and a pharmaceutically acceptable cation; x is 0, R is, (a) CH 3 (CH 2 ) 15 O(CH 2 ) 3 −, (b) CH 3 (CH 2 ) 17 O(CH 2 ) 2 -, or (c) 【Chemistry 13】 A method selected from the following.

18. 18. The method of claim 17, wherein the contacting of the compound of formula (a) with the compound of formula (b) occurs in the presence of N,N-dicyclohexylcarbodiimide, 4-dimethylaminopyridine, or a combination thereof.

19. 18. The method of claim 17, wherein the acid comprises HCl.

20. 18. The method of claim 17, wherein said contacting with said acid of formula (c) occurs in the presence of tetrahydrofuran (THF).

21. 18. The method of claim 17, further comprising carrying out an intramolecular esterification reaction of the compound of formula (d) to form a cyclic phosphate.

22. 1. A method for producing a drug triphosphate, comprising: providing a plurality of cells; contacting the plurality of cells with an amount of a drug; and incubating the plurality of cells and the amount of drug for a period of time effective to form the drug triphosphate, wherein the drug is GS-441524. 【Chemistry 14】 That's the method.

23. 23. The method of claim 22, wherein the plurality of cells comprises Vero E6 cells, Caco-2 cells, Calu-3 cells, HPSC (human pluripotent stem cell) lung cells, Huh7.5 cells, or a combination thereof.

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