Antiviral prodrugs, intermediate and long acting formulations, and methods

Abstract

The compounds and pharmaceutical formulations comprising the compounds and oils, which may be formulated for intermediate or long acting intramuscular injection.Methods for treating respiratory syncytial virus (RSV), human immunodeficiency virus (HIV), coronavirus, SARS CoV-2, and other RNA virus infections in mammals.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 303,376, filed January 26, 2022, which is incorporated herein by reference.

[0002] government funding This invention was made with Government support under Grant No. AI131424 and Grant No. AI1161348 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] The present disclosure relates to antiviral prodrugs, methods for making antiviral prodrugs, methods of treatment, and pharmaceutical formulations, such as long- or intermediate-acting formulations, of antiviral prodrugs for the treatment of various diseases, such as SARS CoV-2 infection or human immunodeficiency infection in mammals. [Background technology]

[0004] Over the past 18 years, spillover events have introduced the highly contagious β-coronavirus strains SARS CoV, MERS CoV, and SARS CoV-2 into human populations (see, e.g., Zhong NS, et.al. Lancet 2003;362:1353-1358; Zaki, AM, et.al., N Engl J Med 2012;367:1814-1820; and Zhu N., et.al. N Engl J Med. 2020;382:727-733). Although case fatality rates vary, each has demonstrated the ability to induce substantial morbidity and mortality, especially in patients over 55 years of age and / or with underlying comorbid medical conditions (see, e.g., Wu C., et.al., JAMA Intern Med 2020;180:934-943, and Zhou F. et.al. Lancet 2020;395:1054-1062). While SARS CoV and MERS CoV were largely contained by epidemiological interventions, the current outbreak has evolved into a global pandemic responsible for hundreds of millions of infections and over 5 million deaths (see, e.g., Johns Hopkins Coronavirus Resource Center https: / / coronavirus.jhu.edu / map.html (accessed January 16, 2022)). The intensive effort to develop a COVID vaccine has resulted in the development of several highly effective vaccines that provide substantial levels of protection from severe disease and death (see, e.g., Polack FP et.al. N Engl J Med 2021;383;2603-2615, and Baden LRN Engl J Med. 2021;384:403-416). Even with the most effective vaccines, immunity begins to wane within 5 months (see, e.g., Keehner J. et.al, N Engl J Med. 2021;384:1774-1775).Despite their efficacy, vaccine hesitancy is widespread, with up to 50% of people in many parts of the world refusing to be vaccinated (see, e.g., Daniel CNet.al.Vaccine.2022:S0264-410X(21)01667-4). A substantial subset of the population is immunocompromised and / or has underlying conditions that limit vaccine efficacy. Thus, despite significant successes in vaccine development, SARS CoV-2 infection rates remain high, and COVID-related morbidity and mortality are expected to persist in the coming years without effective antiviral therapeutics.

[0005] Antiviral therapeutic efforts include both monoclonal antibodies directed at neutralizing epitopes on the viral spike protein and small molecules directed at the viral RNA-dependent RNA polymerase or the viral 3-CL-like protease enzyme. Remdesivir nucleoside triphosphate (RVn triphosphate) potently inhibits the enzymatic activity of the polymerase of all coronaviruses tested to date, including SARS CoV-2. Remdesivir also inhibits the polymerases of several other pathogenic RNA viruses, including Ebola virus, Nipah virus, and respiratory syncytial virus (see, e.g., Wang M., et.al., Cell Res 2020;30:269-271; Yan VC,eg, ACS Med Chem Lett. 2020;11:1361-1366; and Mulangu S.et.al, N Engl J Med 2019;381:2293-2303).

[0006] Clinical trials of antivirals for COVID-19 (including remdesivir) have shown the greatest benefit when used prior to hospitalization (see, for example, Gotttlieb RL New Engl J Med, 2021 Dec 22: NEJMoa2116846). Remdesivir can reduce hospitalization or death by 85-90% when administered as an intravenous infusion three times daily prior to hospitalization. This approach, while highly effective, is extremely difficult to scale because it requires three consecutive days of intravenous infusion in SARS CoV-2 infected patients. Nevertheless, the approach is proof of concept that early remdesivir, if used early enough in the disease, can have a substantial impact on the disease. The availability of a remdesivir prodrug that can be administered orally over a week or intramuscularly on a single occasion to outpatients with SARS CoV-2 at risk for severe COVID-19 disease would greatly expand the clinical scope of this agent in the treatment of COVID-19 and potentially for the treatment of Ebola, Nipah, and RSV infections. A sustained-release injectable formulation of remdesivir prodrug would be of interest because RDV is not highly bioavailable following oral administration and must be administered intravenously, thereby severely complicating its administration to pre-hospitalized, high-risk patients, often with relatively advanced disease. A long-acting formulation of remdesivir nucleoside monophosphate that could enable plasma antiviral activity in excess of 90% effective concentrations for at least about 5 to about 10 days following a single intramuscular or subcutaneous dose would be particularly useful.

[0007] Antiretroviral therapy (ART) has resulted in substantial reductions in HIV-1-related morbidity and mortality in the United States and worldwide (see, e.g., Walensky RPet. al., J Infect Dis 2006;194:11-19, and April MD, et. al. J Infect Dis. 2014;209:491-9). By 2021, 28 million of the 38 million people with HIV infection were receiving antiretroviral therapy (see, e.g., https: / / www.unaids.org / en / resources / fact-sheet (accessed January 19, 2022)). Of those receiving antiretroviral therapy, it is estimated that as many as 90% can be fully virally suppressed in resource-rich settings. Because plasma HIV-1 RNA levels are not routinely measured in sub-Saharan Africa, the proportion of the HIV-1-infected population currently receiving fully effective therapy is unknown but is likely substantially lower. Preventing transmission of HIV-1 is also an urgent global priority. The likelihood of HIV transmission has been shown to be directly related to plasma (and therefore genital) levels of HIV-1 (see, e.g., Quinn TC, et.al., N Engl J Med 2000;342:921-929), and there is growing optimism that the epidemic may be ended by aggressive global expansion of antiretroviral therapy used for both treatment and prevention (see, e.g., Granich RM, et.al., N Engl J Med 2000;342:921-929). Indeed, reductions in incident infections associated with treatment of HIV-1 infected individuals have been shown both in clinical trials and at the population level (see, e.g., Cohen MS, et.al., N Engl J Med. 2011;365:493-505; Montaner JS, et.al., Lancet 2010;376:532-539; Montaner JS, et.al., PLoS One 2014;9:e87872; and Tanser JF, et.al., Science 2013.339:966-971). 1.5 million new infections occurred in 2020.Despite early optimism about the development of an effective AIDS vaccine, there is little actual insight into how to elicit protective responses in a scalable commercial vaccine product. Treatment of high-risk, HIV-1 uninfected individuals (called pre-exposure prophylaxis or "PrEP") has been effective in some populations but not others, with protection rates ranging from 0 to 62% (see, e.g., Grant RMN Engl J Med 2010;363:2587-2599; Baeten JM,et.al.,N Engl J Med 2012;367:399-410; Thigpen MCN Engl J Med.2012;367:423-434; Choopanya K,et.al.,Lancet 2013;381:2083-90; Van Damme L,et.al.,N Engl J Med.2012;367:411-22; and Marrazzo JM,et.al.,N Engl J Med 2015;372:509-18). PrEP is effective when adherence is high, but those at highest risk for HIV-1 infection are often those who face the greatest barriers to adherence. The failure of the large FEM PrEP and VOICE trials to show any degree of protection, despite simultaneous assessment of 88-90% adherence by self-report and number of products returned, only highlights the adherence challenge imposed by oral PrEP (see, for example, Van Damme L,et.al.,N Engl J Med.2012;367:411-22, and Marrazzo JM,et.al.,N Engl J Med 2015;372:509-18). Nevertheless, results from oral and topical PrEP trials involving TFV have been more consistent than perhaps one might think: high levels of protection can be achieved when active concentrations of the drug are present at the time of exposure.The dual benefits of ART, including improving the health of HIV-1-infected individuals receiving therapy and reducing the likelihood of viral transmission, have made the rapid expansion of antiretroviral therapy a major global priority (see, for example, UNAIDS. 90-90-90 An Ambitious Treatment Target to help end the AIDS epidemic. Geneva, Switzerland 2014. Available from: http: / / www.unaids.org / sites / default / files / media_asset / 90-90-90_en_0.pdf).

[0008] With the mitigation of many of the acute and chronic toxicities of first-generation ARVs with several currently available once-daily single-pill regimens (STRs), treatment adherence has emerged as the greatest barrier to successful long-term treatment. While most patients are able to comply with daily regimens, many of the most vulnerable populations (for prevention and treatment) include those whose adherence is complicated by mental health problems, substance use, and stigma. The development of an effective, inexpensive, well-tolerated, and convenient modality that maintains active concentrations at sites of viral replication and exposure for extended periods of time would represent a major advance in ART for both treatment and prevention. Long-acting injectable products such as cabotegravir, rilpivarine, and islatravir provide proof of concept for this approach (see, e.g., Margolis DA, et.al., Lancet Infect Dis. 2015;15:1145-55; Verloes R, et.al., HIV Med. 2015;16:477-84; and Matthers RP, et.al., Nature Med. 2021;27,1712-1717). The FDA's recent decision to place islatravir on clinical hold due to leukopenia in a subset of patients highlights the need to develop agents within multiple classes if long-acting ART is to be used widely in patient populations vulnerable to different toxicities and with viruses of different drug susceptibility.

[0009] There remains a need for pharmaceutical formulations that can be delivered in a variety of ways, such as intramuscularly. There also remains a need for pharmaceutical formulations that are long-acting, e.g., formulations that can provide effective plasma levels for extended periods of time after infrequent administration, such as once a month. Summary of the Invention

[0010] Provided herein are compounds, such as antiviral prodrugs, and pharmaceutical formulations that overcome one or more of the shortcomings of currently used drugs and / or formulations. For example, embodiments of the compounds and pharmaceutical formulations provided herein include orally useful antiviral prodrugs that can specifically target organs where viral replication is greatest and can be conveniently administered on a large scale at any stage of disease. For oral use and enhanced pulmonary exposure, some embodiments of prodrugs or formulations of RVn provided herein can achieve one or more of the following: 1) kinase bypass of the first nucleoside phosphorylation, 2) providing increased oral bioavailability, and 3) delivery of antivirally significant concentrations to the lungs and gastrointestinal tract and formulations that can provide sustained levels in plasma for 5-30 days after a single injection. 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 an approach that may be able to target the antiviral to the lungs and away from the liver, where the main dose limitations of remdesivir are directed.

[0011] In one aspect, provided herein are compounds that comprise antiviral prodrugs. In some embodiments, the compounds have a structure according to formula (I): [ka] wherein Nuc is selected from the group consisting of antiviral nucleosides and antiviral nucleoside analogs; 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, x is 0 or 1, and L is independently selected from the group consisting of C1-C 30 hydrocarbyl (such as C1-C6 hydrocarbyl), and R is independently 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 is selected from the group consisting of hydrocarbyl.

[0012] In another aspect, a pharmaceutical formulation is provided. In some embodiments, the pharmaceutical formulation comprises an oil and one or more compounds described herein. The pharmaceutical formulation can be formulated for injection, such as intramuscular or subcutaneous injection. The pharmaceutical formulation can be orally bioavailable.

[0013] In a further aspect, a therapeutic method is provided, such as a method for treating a virus (e.g., a coronavirus), including a viral infection in a mammal. In some embodiments, the method comprises administering an effective amount of a compound described herein, or a pharmaceutical formulation described herein.

[0014] 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, Y is hydrogen, C1-C 30 hydrocarbyl, and a pharma- ceutically acceptable cation, x is 0 or 1, and L is C1-C 30 Hydrocarbyl (e.g., C1-C6 hydrocarbyl), 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.

[0015] In yet another aspect, methods of 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.

[0016] 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 description of the drawings]

[0017] [Fig. 1A-1F] Concentration-response curves of ODBG-P-RVn, ODE-P-RVn, and HDP-P-RVn (4b), remdesivir (RDV), and remdesivir nucleoside (RVn) for SARS-CoV-2 infection in Vero E6 cells in two separate experiments performed in duplicate. [Diagram 2] 1 shows the relative viability of an embodiment of treated Vero E6 cells as measured by the CellTiter-Glo luminescent cell viability assay. [Diagram 3] LC / MS / MS analysis of cells centrifuged at 1200 rpm for 10 min, the diluent aspirated, and resuspended in 250 μL of methanol / distilled water (70 / 30) is shown. [Figure 4A-4B] Plasma levels of ODBG-P-RVn (FIG. 4A) and RVn (FIG. 4B) in a 7-day oral pharmacokinetic study in Syrian hamsters are shown. [Figure 5A-5B] The stability of ODE-P-RVn and ODBG-P-RVn in human plasma with either K2EDTA (Figure 5A) or sodium heparin (Figure 5B) as the anticoagulant is shown. [Figure 6A-6B] 1 shows the pharmacokinetics of ODE-Bn-TFV and ODE-TFV, respectively, following intramuscular administration of 100 mg / kg in formulation F3 to rats. [Figure 7A-7B]13 is a plot of the effect of four different formulations on the pharmacokinetics of ODE-Bn-TFV and ODE-TFV, respectively, following intramuscular administration of 100 mg / kg to rats. [Figure 8] 1 shows a comparison of ODE-Bn-TFV pharmacokinetics at 96 mg / kg in rats using formulations F3 vs. F8. [Figure 9] 1 shows rat plasma levels of ODE-Bn-TFV and ODE-TFV during a 3-month exposure to monthly intramuscular doses of ODE-Bn-TFV in formulation F3. [Figure 10] 1 shows plasma levels of ODE-Bn-TFV and ODE-TFV in beagle dogs treated with ODE-Bn-TFV in formulation F3 at 100 mg / kg. [Figure 11A-11B] Figure 11A shows TFVpp persistence in HFF cells for ODE-TFV (Figure 11A) and ODE-Et-TFV (Figure 11B). [Figure 12A-12B] 12A and 12B show plasma concentrations of embodiments of formulations containing ODE-Bn-TFV (FIG. 12A) or ODE-TFV (FIG. 12B) (ODE-Bn-TFV ED90 10.9 ng / mL, ODE-TFV ED90 6.3 nm / mL). [Fig. 12C-12D] 12C and 12D show plasma concentrations of embodiments of formulations containing ODE-Bn-TFV (FIG. 12C) and ODE-TFV (FIG. 12D). [Fig. 12E-12F] 12E and 12F show plasma concentrations of embodiments of formulations containing ODE-Bn-TFV (FIG. 12E) and ODE-TFV (FIG. 12F). [Fig. 12G-12H] 12G and 12H show plasma concentrations of embodiments of formulations containing ODE-Bn-TFV (FIG. 12G) and ODE-TFV (FIG. 12H). [Figure 12I] 1 shows the mean plasma concentrations of embodiments of the formulation. [Figure 13] 1 shows the nanograms / mL of active ingredient present in rat plasma following administration of an embodiment of a formulation herein. [Figure 14] 1 shows the amount of an embodiment of a diphosphate in PBMCs following administration of an embodiment of a formulation herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] In one aspect, a pharmaceutical formulation is provided herein. The pharmaceutical formulation may include (i) an oil and (ii) a compound of formula (I) as described herein. In some embodiments, the pharmaceutical formulation also includes benzyl alcohol, benzyl benzoate, ethyl alcohol, or a combination thereof.

[0019] As used herein, the term "oil" refers to a non-polar compound or a mixture of two or more non-polar compounds that are insoluble in water (e.g., solubility of less than 1 g per liter of water), such as non-polar compounds that contain at least partially saturated carbon chains (e.g., hydrocarbons, fatty acids, etc.).

[0020] Any known oil may be used in the pharmaceutical formulations provided herein. In some embodiments, the oil is selected from the group consisting of sesame oil, triglycerides (e.g., medium chain triglycerides), and combinations thereof.

[0021] The oil may be present in the pharmaceutical formulations provided herein in any effective concentration. For example, the oil may be present in the pharmaceutical formulation in an amount of about 60% to about 95% by weight, about 65% to about 95% by weight, about 70% to about 95% by weight, about 75% to about 95% by weight, about 80% to about 95% by weight, or about 85% to about 95% by weight, based on the weight of the pharmaceutical formulation.

[0022] In some embodiments, the pharmaceutical formulation also includes benzyl alcohol, benzyl benzoate, ethyl alcohol, or a combination thereof. When present, the benzyl alcohol, ethyl alcohol, benzyl benzoate, or a combination thereof may be present in a total amount of about 0.01% to about 25% by weight, about 0.01% to about 20% by weight, about 0.01% to about 15% by weight, about 0.01% to about 10% by weight, or about 5% to about 10% by weight, based on the weight of the pharmaceutical formulation.

[0023] The compound of formula (I) may be present in any effective concentration in the pharmaceutical formulation provided herein. For example, the compound of formula (I) may be present in an amount of about 0.01% by weight to about 40% by weight, about 0.01% by weight to about 30% by weight, about 0.01% by weight to about 20% by weight, about 0.01% by weight to about 10% by weight, or about 0.01% by weight to about 5% by weight based on the weight of the pharmaceutical formulation.

[0024] The pharmaceutical formulations provided herein can be configured for any route of administration. In some embodiments, the pharmaceutical formulations are configured for oral administration. In some embodiments, the pharmaceutical formulations are configured for injection, such as intramuscular or subcutaneous injection.

[0025] The pharmaceutical formulations provided herein may be configured to be administered as a single dose. For example, the pharmaceutical formulations may be configured to be administered as a single, long-acting therapeutic agent, as described herein. The single dose may provide an effective amount of the pharmaceutical formulation, such as the compound of formula (I), present in the pharmaceutical formulation. The pharmaceutical formulations provided herein may be configured to be administered as a series of two or more doses, each dose being administered at least 7 days, at least 14 days, at least 21 days, or at least 28 days after the preceding dose. For example, each of the two or more doses may be a long-acting therapeutic agent, as described herein.

[0026] In some embodiments, the pharmaceutical formulation comprises one or more of the components of any of formulations F3, F4, F7, or F8: [Table 1]

[0027] In some embodiments, the pharmaceutical formulation is a long-acting therapeutic. As used herein, the phrases "long-acting therapeutic," "long-acting dose," and the like refer to a therapeutic agent or dose that promotes plasma antiviral activity greater than 90% effective concentration for about 5 to about 30 days, about 5 to about 28 days, about 5 to about 21 days, about 5 to about 14 days, or about 5 to about 7 days, respectively, after administration of a single dose.

[0028] The pharmaceutical formulations described herein may include one or more additives that do not undesirably affect one or more characteristics of the pharmaceutical formulation, such as the activity of the compound of formula (I), bioavailability, etc. Non-limiting examples of additives include excipients, colorants, flavoring agents, buffers, preservatives, surfactants, other active ingredients, such as anti-inflammatory agents, pain-reducing agents, etc.

[0029] In one aspect, provided herein are compounds comprising a compound of formula (I): [ka]

[0030] "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.

[0031] 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.

[0032] In some embodiments, Nuc is RVn (GS-441524), β-dN 4 -hydroxycytidine (NHC), or (2'R)-2-amino-2'-deoxy-2'-fluoro-N,2'-dimethyladenosine (CAS number is 1998705-62-6). In some embodiments, Nuc is GS-441524 and the compound of formula (I) has the following structure: [ka]

[0033] Other antiviral agents for coronavirus infections may also be modified by the methods provided herein. For example, N 4 -Hydroxy-cytidine (NHC) is an antiviral candidate entering clinical Phase I evaluation. Other nucleoside analogues known to inhibit RNA viruses are also suitable for modification according to the present disclosure.

[0034] "Y" in formula (I) may be any of the substituents described herein. In some embodiments, Y is hydrogen, C1-C 30 It is a hydrocarbyl, a pharma- ceutically acceptable cation, or a covalent bond to a carbon atom of the 5-carbon sugar moiety of the antiviral nucleoside or antiviral nucleoside analog.

[0035] 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 a covalent bond to any carbon atom (e.g., the 1' carbon, the 2' carbon, the 3' carbon, or the 4' carbon) of the 5-carbon sugar moiety of the antiviral nucleoside or antiviral nucleoside analog. In other words, the covalent bond can be a covalent bond between (i) the oxygen to which Y is attached in formula (I) and (ii) any carbon atom (e.g., the 1' carbon, the 2' carbon, the 3' carbon, or the 4' carbon) of the 5-carbon sugar moiety of the antiviral nucleoside or antiviral nucleoside analog. For example, Nuc can be GS-441524, 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]

[0036] When Y is a pharma- ceutically acceptable cation, the pharma- ceutically acceptable cation is Na + It may be.

[0037] In some embodiments, Y is C1-C 20 Hydrocarbyl, C1-C 10In 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, and the like. 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 is 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, trihalomethanesulfonamido, 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 , R6 , and R 7 At least two of R 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.

[0038] When Y is unsubstituted or substituted benzyl of formula (B), the compound of formula (I) has the structure: [ka]

[0039] In formula (I), x can be 1 or 0. When x is 1, an "-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]

[0040] When "L" is present in the compound 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 10 In some embodiments, L is an optionally unsubstituted ethyl group. In some embodiments, L is an optionally unsubstituted methyl group. In some embodiments, L is an optionally unsubstituted propyl ...

[0041] "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. R, in some embodiments, is a heteroalkyl. R can include 0-6 unsaturated bonds, 1-6 unsaturated bonds, 2-6 unsaturated bonds, 3-6 unsaturated bonds, or 4-6 unsaturated bonds. An "unsaturated bond" as described herein may include any non-single bond, and when more than one unsaturated bond is present, the two or more unsaturated bonds may be independently selected from double bonds or triple bonds. When one or more double bonds are present, the one or more double bonds may be cis, trans, or a combination thereof. R can include a cyclopropyl moiety, such as a terminal cyclopropyl moiety.

[0042] 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.

[0043] 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.

[0044] In some embodiments, R is a substituent of formula (A): [ka] In the formula, 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 can contain at least one cyclic moiety, which can 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 0-6 unsaturated bonds, 1-6 unsaturated bonds, 2-6 unsaturated bonds, 3-6 unsaturated bonds, or 4-6 unsaturated bonds. When one or more double bonds are present, the one or more double bonds may be cis, trans, or a combination thereof. 1 , R 2 , or R 1 and R 2 may include a branched hydrocarbyl, such as a penultimate branched hydrocarbyl. In some embodiments, R 1 and R 2 At least one of R is hydrogen. 1 and R 2 Both are independent, C1~C 30 is selected from hydrocarbyl.

[0045] 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 the structure according to formula (C): [ka] In the formula, R 8 , R 9 , R 10 , R 11 , and R 12 is 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, trihalomethanesulfonamido, 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 8 , R9 , 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.

[0046] In some embodiments, R 1 is [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.

[0047] In some embodiments, R 1 is [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.

[0048] In some embodiments, R 2 teeth, [ka] wherein g is 1 to 29.

[0049] In some embodiments, g is 5 to 10. In some embodiments, g is 7.

[0050] The substituent of formula (A) can be racemic, sn-1 stereoisomer, or sn-3 stereoisomer. Throughout this disclosure, when a formula such as formula (A) is shown without 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 the structure described in formula (I), where x is 0 and R is a substituent of formula (A). [ka]

[0051] This formula lacks any indication of spatial orientation and therefore reads with respect to mixtures of the sn-3 and sn-1 isomers, including the sn-3 isomer, the sn-1 isomer, and racemic mixtures of the formula below. [ka]

[0052] Further non-limiting embodiments of compounds of formula (I) are provided in the table below. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

[0053] 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 having the same or different C1-C substituted groups selected for R). 30 and / or (ii) the two differently labeled substituents selected from the same pool of substituents may be the same or different (e.g., R and Y of the molecule of the compound of formula (I) are both “C1-C 30 hydrocarbyl" and C1 to C4 selected for R and Y. 30 The hydrocarbyls may be the same or different).

[0054] 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 unsubstituted and substituted derivatives thereof, which may include, but are not limited to, heteroaryl, heteroarylalkyl, heterocycloalkyl, and the like, as described herein. Thus, it is understood that a substituent herein may be, for example, "C1-C 30 When characterized as "hydrocarbyl," "C1-C 30 The term "hydrocarbyl" includes, unless otherwise specified, unsubstituted and substituted C1-C 30 Examples of aliphatic groups include, but are not limited to, alkyl groups, cycloalkyl groups, alkenyl groups, cycloalkenyl groups, alkynyl groups, alkadienyl groups, and cyclic groups, each of which is defined as "C1 to C 30 Hydrocarbyl" and "C 10 ~C 30"Hydrocarbyl" includes, in each instance, all substituted, unsubstituted, branched, and / or straight chain analogs or derivatives thereof having 1 to 30 total carbon atoms or 10 to 30 total carbon atoms. 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 may be monocyclic or polycyclic, examples of which 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, 2,3-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, including any heteroatom substituted derivatives thereof.

[0055] Unless otherwise indicated, the term “substituted,” when used to describe a chemical structure or moiety, means (i) 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) is selected from the group consisting of 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, and CONH-aryl). "NH-arylalkyl", carboxyl, carboxylic acid, cyano, cycloalkyl, cycloalkenyl, ester, ether (e.g., methoxy, ethoxy), halo, haloalkyl (e.g., -CCl3, -CF3, -C(CF3)3), haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamide, heteroalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl, isocyanate, isothiocyanate, nitrile, nitro, oxo, phosphodiester, silyl, sulfide, sulfonamide (e.g., SO2NH2), sulfone, sulfenyl, sulfinyl, sulfonyl (including alkylsulfonyl, arylsulfonyl, and arylalkylsulfonyl), sulfoxide, thiocarbonyl, thiocarbamyl, thiocyanato, thiol (e.g., sulfhydryl, thioether), or urea (-NHCONH-alkyl-).

[0056] 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.

[0057] In some embodiments, the methods include administering to the mammal an effective amount of a compound described herein or a pharmaceutical formulation described herein.

[0058] The viral infection may be an RNA viral infection. In 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, Fenuiviridae, Nairoviridae, Arenaviridae, Flaviviridae, and Coronaviridae.

[0059] 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 evaluating anti-coronavirus activity and cytotoxicity. Typically, compounds are first screened in vitro for inhibition of coronaviruses, and then those that show significant antiviral activity are screened in vivo for efficacy.

[0060] 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 three-way serial dilution. b) Compound activity may be tested 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). The virus can also be quantified in culture supernatants, by serial dilution in Vero cell lawns and by RT-PCR. For example, laboratory strains that can be obtained from the BEI resource (strains NR52281 and NR522282) can be used, as well as clinical strains that can be isolated from patients participating in clinical trials. Cytotoxicity can be measured by commercially available MTT or Cell Titer Glo. The compound with the lowest 90% inhibitory concentration and that requires the highest concentration to induce cellular cytotoxicity can be selected for further evaluation. Anti-coronavirus compounds can also be evaluated in a lung explant model for 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 from the organ most clinically affected by the virus.

[0061] How to make a compound Also provided herein are methods for making compounds, such as those described herein, which may be prodrugs.

[0062] 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]

[0063] 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 phosphodiesters. Removal of the isopropylidene protecting group by treatment with dilute HCl or other suitable acid may provide compounds of formula (I) in suitable yield and purity.

[0064] In some embodiments, the method includes providing a compound of formula (a): [ka] wherein x, R, L, and Y are as described herein.

[0065] 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:

[0066] Also, as described herein, formula (b) does not include any stereochemical indication and therefore reads at least as follows for stereoisomers of formula (b). [ka]

[0067] In some embodiments, the method includes contacting a compound of formula (a) and a compound of formula (b) to form a compound of formula (c). [ka]

[0068] Contacting the compound of formula (a) and the compound of formula (b) may occur at any temperature or pressure and may occur in the presence of any suitable liquid. The liquid may be a C1-C cyclic compound having at least one cyclic moiety. 30 Hydrocarbyl, C1-C 30 It may include at least one heteroatom, such as hydrocarbyl, nitrogen, or a combination thereof. In some embodiments, the liquid is N,N-dicyclohexylcarbodiimide, 4-dimethylaminopyridine, or a combination thereof.

[0069] In some embodiments, the method comprises contacting a compound of formula (c) with an acid to form a compound of formula (d). [ka]

[0070] The acid may include any acid capable of promoting the formation of a 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 system having at least one cyclic moiety.30 Hydrocarbyl, C1-C 30 It may be a hydrocarbyl, at least one heteroatom, or a combination thereof, in some embodiments, the liquid is tetrahydrofuran.

[0071] In some embodiments, the method includes carrying out an intramolecular esterification reaction of a compound of formula (d) to form a cyclic phosphate, such as a 3',5'-cyclic phosphate.

[0072] Methods for Producing Drug Triphosphates Also provided herein is a method of 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 a drug triphosphate. The plurality of cells may 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).

[0073] 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.

[0074] 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 invention, exemplary methods, devices, and materials are described herein.

[0075] The practice of the present invention employs, 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 described in Molecular Cloning: A Laboratory Manual, 2 nd ed. (Sambrook et al., 1989), Oligonucleotide Synthesis (MJGait, ed., 1984), Animal Cell Culture (RIFreshney, ed., 1987), Methods in Enzymology (Academic Press, Inc.), Current Protocols in Molecular Biology (FMAusubel et al., eds., 1987 and regular updates), PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994), Remington, The Science and Practice of Pharmacy, 20 th ed., (Lippincott, Williams & Wilkins 2003), and Remington, The Science and Practice of Pharmacy, 22 th ed., (Pharmaceutical Press and Philadelphia College of Pharmacy at University of the Sciences 2012).

[0076] 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 include one or more of the prior art aspects discussed herein.

[0077] 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 some technology areas. Thus, the present disclosure should not necessarily be construed as being limited to addressing any of the specific problems or deficiencies discussed herein.

[0078] 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 in any attempt to solve any problem to which this specification pertains.

[0079] As used herein, the terms "comprises," "comprising," "including," "having," "contains," "containing," "characterized by," or any other variation thereof, are intended to encompass a non-exclusive inclusion of the recited elements, subject to any limitations not expressly stated. For example, a fusion protein, pharmaceutical composition, and / or method that "comprises" a recitation 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 recited or inherent to the fusion protein, pharmaceutical composition, and / or method.

[0080] As used herein, the transitional phrases "consists of" and "consisting of" exclude any element, step, or component not specified. 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 elements (or components or steps) recited in that clause and does not exclude other elements (or components) from the claim as a whole.

[0081] As used herein, the transitional phrases "consists essentially of" and "consisting essentially of" are used to define compounds, 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 of."

[0082] It is understood that aspects and embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" aspects and embodiments.

[0083] The terms "a," "an," and "the" are intended to include multiple 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.

[0084] 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.

[0085] Various numerical ranges may be disclosed herein. When the applicant discloses or claims any type of range, the applicant's intention is to disclose or claim individually each possible number that such range may reasonably encompass, including the endpoints of the range, and any subranges and combinations of subranges encompassed within the range, unless otherwise specified. Moreover, all numerical endpoints of ranges disclosed herein are approximate. As a representative example, the applicant discloses 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.

[0086] When such values ​​or ranges are expressed, other embodiments disclosed 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 disclosed herein 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.

[0087] As used herein, the term "pharmaceutical composition" refers to a pharma- ceutically acceptable composition that includes a pharma- ceutically active agent and, in some embodiments, further includes a pharma- ceutically acceptable carrier. In some embodiments, a pharmaceutical composition may be a combination of a pharma- ceutically active agent and a carrier.

[0088] The term "combination" refers to either a fixed combination in one 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 circumstances, the combination partners exhibit a cooperative effect, e.g., a synergistic effect. Terms such as "co-administration" or "co-administration" as used herein are meant to encompass 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. The term "pharmaceutical combination" as used herein refers to a product resulting from the mixing or combination of two or more active ingredients, and including both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient at the same time in the form of a single entity or dosage. The term "non-fixed combination" means that both active ingredients, e.g., a compound and a combination partner, are administered to a patient as separate entities simultaneously or sequentially without specific time limitations, 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.

[0089] As used herein, the term "pharmaceutical acceptable" means approved by a regulatory agency of a federal or state government, or listed in the United States Pharmacopeia, or other generally recognized pharmacopoeias, in addition to other formulations that are safe for use in animals, and particularly in humans and / or non-human mammals.

[0090] As used herein, the term "pharmaceutical acceptable carrier" refers to an excipient, diluent, preservative, solubilizer, emulsifier, adjuvant, and / or vehicle in which the demethylated compound is administered. Such carriers may 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, oils including polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents. Antibacterial agents, such as benzyl alcohol or methyl parabens, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, and agents for isotonicity, such as sodium chloride or dextrose, may also be carriers. Methods of preparing compositions in combination with carriers are known to those skilled in the art. In some embodiments, the language "pharmaceutical acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharma- ceutical 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.

[0091] As used herein, "therapeutically effective amount" refers to an amount of a pharmacoactive compound sufficient to treat or improve, 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 improve, or in some way reduce, symptoms associated with a disease or condition. For example, an effective amount in relation to a disease is an amount sufficient to block or prevent onset, or where disease pathology has been initiated to alleviate, improve, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. In either case, the effective amount may be given in a single dose or in divided doses.

[0092] As used herein, the terms "treat," "treatment," or "treating" encompass at least an amelioration of 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., in symptoms associated with the disease or condition being treated. Thus, "treatment" also includes situations in which a disease, disorder, or pathological condition, or at least symptoms associated therewith, are completely inhibited (e.g., prevented from occurring) or halted (e.g., terminated) such that the patient is no longer afflicted with the condition, or at least the symptoms that characterize the condition.

[0093] 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 additional active agents, before the onset of symptoms, particularly to subjects at risk of 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 preventive regimen. In certain embodiments, subjects with a history of recurrent symptoms are also likely candidates for prevention. In this regard, the term "prevention" may be used interchangeably with the term "prophylactic treatment".

[0094] 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 an amount of a therapeutic agent, alone or in combination with one or more other agents, that provides a prophylactic benefit in the prevention of a disease. The term "prophylactically effective amount" may include an amount that improves overall prophylaxis or enhances the prophylactic efficacy 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, and the like. In certain embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably herein to refer to a mammalian subject, such as, for example, a human.

[0095] As used herein, and unless otherwise specified, the compounds described herein are intended to encompass all possible stereoisomers, unless a specific 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 may take the form of proton tautomerism, or, for example, so-called valence tautomerism in compounds containing aromatic moieties.

[0096] "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 bonds or other linkages to form a polynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNA polymers, or oligonucleotides, and analogs thereof. The nucleic acid backbone may be composed of a variety of linkages, including one or more of sugar phosphodiester linkages, peptide-nucleic acid linkages, phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar portion of the nucleic acid may be ribose, deoxyribose, or similar compounds with known substitutions (e.g., 2'-methoxy and 2'-halide substitutions). The nitrogenous bases may be conventional bases (A, G, C, T, U) or their analogs (e.g., inosine, 5-methylisocytosine, isoguanine). Nucleic acids may contain only conventional sugars, bases, and linkages as found in RNA and DNA, or may 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 may contain "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 may 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 may be purified from natural sources using routine techniques. Nucleic acids may be single-stranded or double-stranded.

[0097] Nucleic acids are typically single- or double-stranded and will generally contain phosphodiester bonds, although in some cases, as outlined herein, may contain, for example and without limitation, phosphoramide bonds (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. (1986) Chemica Scripta 26:1419), phosphorothioates (Mag et al. (1991) Nucleic Acids Res. 19:1437, and U.S. Pat. No. 5,644,048, both of which are incorporated by reference), phosphorodithioates (Briu et al. (1989) J. Am. Chem. Soc. 111:2321, both of which are incorporated by reference), O-methyl phosphoramidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press (1992), each of which is incorporated by reference), and peptide nucleic acid backbones and linkages (Egholm (1992) J. Am. Chem. Soc. 114:1895, Meier et al. (1992) J. Am. Chem. Soc. 114:1895, each of which is incorporated by reference). al. (1992) Chem. Int. Ed. Engl. 31:1008, Nielsen (1993) Nature 365:566, and Carlsson et al. (1996) Nature 380:207).Other analog nucleic acids include those with positively charged backbones (Denpcy et al. (1995) Proc. Natl. Acad. Sci. USA 92:6097, which are incorporated by reference), non-ionic backbones (see U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141, and 4,469,863, each of which is incorporated by reference; 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, 1999). 580, "Carbohydrate Modifications in Antisense Research", Ed. YS Sanghvi and P. Dan Cook, Mesmaeker et al. (1994) Bioorganic & Medicinal Chem:Lett. 4:395, Jeffs et al. (1994) J. Biomolecular NMR 34:17, and Tetrahedron Lett. 37:743 (1996)), and U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Ed. YS Sanghvi and P. Dan Cook, each of which is incorporated by reference. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al. (1995) Chem. Soc. Rev. pp 169-176, incorporated by reference). Some nucleic acid analogs are also described, for example, in Rawls, C&E News Jun. 2, 1997 page 35, 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.

[0098] In addition to these naturally occurring heterocyclic bases commonly found in nucleic acids (e.g., adenine, guanine, thymine, cytosine, and uracil), nucleic acid analogs also include those with non-naturally occurring heterocyclic or modified bases, many of which are described herein or otherwise referenced. In particular, many non-naturally occurring bases are further described, for example, in 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, for example, U.S. Patent No. 5,990,303, entitled "SYNTHESIS OF 7-DEAZA-2'-DEOXYGUANOSINE NUCLEOTIDES," issued Nov. 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 -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.

[0099] Examples of modified bases and nucleotides are described, for example, in U.S. Pat. No. 5,484,908, issued Jan. 16, 1996 to Froehler et al., entitled "OLIGONUCLEOTIDES CONTAINING 5-PROPYNYL PYRIMIDINES," U.S. Pat. No. 5,645,985, issued Jul. 8, 1997 to Froehler et al., entitled "ENHANCED TRIPLE-HELIX AND DOUBLE-HELIX FORMATION WITH OLIGOMERS CONTAINING MODIFIED PYRIMIDINES," U.S. Pat. No. 5,830,653, issued Nov. 3, 1998 to Froehler et al., entitled "METHODS OF USING OLIGOMERS CONTAINING MODIFIED PYRIMIDINES," U.S. Pat. No. 5,830,653, issued Oct. 28, 2003 to Kochkine et al., entitled "SYNTHESIS OF No. 6,639,059, entitled "ONE STEP SAMPLE PREPARATION AND DETECTION OF NUCLEIC ACIDS IN COMPLEX BIOLOGICAL SAMPLES," issued Oct. 16, 2001 to Skouv; and 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.

[0100] "Oligonucleotide" or "oligomer" refers to a nucleic acid that contains 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 phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68:109-151, the diethyl phosphoramidite 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.

[0101] The disclosed compounds 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 in each family are included in the table below. [Table 3-1] [Table 3-2]

[0102] Embodiment Embodiments of the compounds, pharmaceutical formulations, and methods described herein are provided in the following list: Embodiment 1. A compound of formula (I) or a pharma- ceutically acceptable salt thereof, [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, x is 0 or 1, and L is independently selected from the group consisting of C1-C 30 R is independently selected from the group consisting of: 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 is selected from the group consisting of hydrocarbyl, Optionally, the compound of formula (I) (i) achieves kinase bypass of the first nucleoside phosphorylation, (ii) provides increased oral bioavailability, (iii) delivers antivirally significant concentrations to the lungs and gastrointestinal tract and formulations, (iv) provides sustained levels in plasma for at least about 5 to about 30 days after administration of a single dose, such as intravenously, or (v) a combination thereof, or a pharma- ceutically acceptable salt thereof.

[0103] Embodiment 2. The compound of embodiment 1, wherein the antiviral nucleoside or antiviral nucleoside analog is an antiviral ribonucleoside or antiviral ribonucleoside analog, respectively.

[0104] Embodiment 3. Nuc is selected from the group consisting of GS-441524, β-dN 4 The compound of any one of the preceding embodiments, wherein the compound is selected from the group consisting of -hydroxycytidine (NHC), and (2'R)-2-amino-2'-deoxy-2'-fluoro-N,2'-dimethyladenosine.

[0105] Embodiment 4. Nuc comprises (i)GS-441524: [ka] The compound of any one of the preceding embodiments,

[0106] Embodiment 5. Y is an unsubstituted C1-C6 alkyl, C1-C 20 Hydrocarbyl, C1-C 10 Hydrocarbyl, C1-C6 hydrocarbyl, or Na + The compound of any one of the preceding embodiments,

[0107] Embodiment 6. A compound according to any one of the preceding embodiments, wherein Y comprises at least one cyclic moiety.

[0108] Embodiment 7. A compound according to any one of the preceding embodiments, wherein Y is selected from the group consisting of aryl, arylalkyl, heteroaryl, heteroarylalkyl, and heterocycloalkyl, each of which is unsubstituted or substituted.

[0109] Embodiment 8. A compound according to any one of the preceding embodiments, wherein heteroaryl is unsubstituted or substituted pyridinyl.

[0110] Embodiment 9. A compound of any one of the preceding embodiments, wherein arylalkyl is unsubstituted or substituted benzyl.

[0111] 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 is 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, trihalomethanesulfonamido, amino, monosubstituted amino, and disubstituted amino.

[0112] Embodiment 11.R 3 , R 4 , R 5 , R 6 , and R 7 The compound of any one of the preceding embodiments, wherein at least two of are hydrogen.

[0113] 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 0-6 unsaturated bonds; (iii) contains a cyclopropyl moiety; or (iv) a combination thereof.

[0114] Embodiment 13. R is (i) unsubstituted or substituted C 13 ~C 29The compound of any one of the preceding embodiments, wherein the compound is heteroalkyl, (ii) contains 0-6 unsaturation, or (iii) a combination thereof.

[0115] 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.

[0116] Embodiment 15. The compound of any one of the preceding embodiments, wherein (i) a is 15 to 25, or (ii) b is 1 to 4 and c is 15 to 20.

[0117] 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.

[0118] The compound of any one of the preceding embodiments, wherein embodiment 17.a is 8.

[0119] Embodiment 18.R 1 and / or R 2 is independently: (i) unsubstituted or substituted C 12 ~C 24 The compound of any one of the preceding embodiments, wherein the compound is hydrocarbyl, (ii) contains 0-6 unsaturated bonds, or (iii) a combination thereof.

[0120] Embodiment 19.(i)R 1 , (ii) R2 or (iii) R 1 and R 2 each independently comprises at least one cyclic moiety. 30 The compound of any one of the preceding embodiments, wherein the compound is selected from hydrocarbyl.

[0121] 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, heteroarylalkyl, and heterocycloalkyl, each of which is unsubstituted or substituted.

[0122] Embodiment 21. A compound according to any one of the preceding embodiments, wherein arylalkyl is unsubstituted or substituted benzyl.

[0123] 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 12is 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, trihalomethanesulfonamido, amino, monosubstituted amino, and disubstituted amino.

[0124] Embodiment 23.R 8 , R 9 , R 10 , R 11 , and R 12 The compound of any one of the preceding embodiments, wherein at least two of are hydrogen.

[0125] Embodiment 24. A compound according to any one of the preceding embodiments, wherein the substituent of formula (A) is a racemate, an sn-1 stereoisomer (e.g., glyceryl-sn-1-phospho), or an sn-3 stereoisomer (e.g., glyceryl-sn-3-phospho).

[0126] EMBODIMENT 25. (i)R 1 and / or R 2 But independently, (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 1 and / or R 2 But independently, [ka] [ka] or (iii) a combination thereof.

[0127] The compound of any one of the preceding embodiments, wherein embodiment 26.g is 5-10.

[0128] Embodiment 27. The compound of any one of the preceding embodiments, wherein g is 7.

[0129] Embodiment 28. A compound according to any one of the preceding embodiments, wherein x is 1 and L is unsubstituted or substituted C1-C3 hydrocarbyl.

[0130] Embodiment 29. A compound according to any one of the preceding embodiments, wherein L is selected from the group consisting of unsubstituted methyl, unsubstituted ethyl, and unsubstituted propyl.

[0131] Embodiment 30. The compound of any one of the preceding embodiments, wherein the compound of formula (I) is at least one of the following compounds or a pharma- ceutically acceptable salt thereof: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0132] EMBODIMENT 31. (i) oil; (ii) A compound according to any one of embodiments 1 to 30.

[0133] Embodiment 32. A pharmaceutical formulation according to embodiment 31, wherein the oil is selected from the group consisting of sesame oil, triglycerides (e.g., medium chain triglycerides), and combinations thereof.

[0134] Embodiment 33. The pharmaceutical formulation of embodiment 31 or 32, wherein (A) the oil is present in the pharmaceutical formulation in an amount of about 60% to about 95% by weight, about 65% to about 95% by weight, about 70% to about 95% by weight, about 75% to about 95% by weight, about 80% to about 95% by weight, or about 85% to about 95% by weight, based on the weight of the pharmaceutical formulation; (B) the compound of formula (I) is present in an amount of about 0.01% to about 40% by weight, about 0.01% to about 30% by weight, about 0.01% to about 20% by weight, about 0.01% to about 10% by weight, about 0.01% to about 5% by weight, based on the weight of the pharmaceutical formulation; or (C) a combination thereof.

[0135] Embodiment 34. The pharmaceutical formulation of any one of embodiments 31 to 33, wherein the pharmaceutical formulation further comprises benzyl alcohol, benzyl benzoate, ethyl alcohol, or a combination thereof, optionally wherein the benzyl alcohol, ethyl alcohol, benzyl benzoate, or a combination thereof is present in a total amount of about 0.01% to about 25% by weight, about 0.01% to about 20% by weight, about 0.01% to about 15% by weight, about 0.01% to about 10% by weight, or about 5% to about 10% by weight, based on the weight of the pharmaceutical formulation.

[0136] Embodiment 35. The pharmaceutical formulation of any one of embodiments 31-34, wherein the pharmaceutical formulation (A) is formulated for injection, such as intramuscular or subcutaneous injection, (B) is configured to achieve kinase bypass of the first nucleoside phosphorylation, (C) delivers antivirally significant concentrations to the lungs and / or gastrointestinal tract, (D) provides sustained levels of the active ingredient in plasma for about 5 to about 30 days, about 5 to about 28 days, about 5 to about 21 days, about 5 to about 14 days, or about 5 to about 7 days after administration of a single dose, such as by intramuscular injection, or (E) is a combination thereof.

[0137] Embodiment 36. A method for treating a coronavirus infection in a mammal (e.g., a human), comprising administering to the mammal an effective amount of a pharmaceutical formulation according to any one of embodiments 31 to 35.

[0138] Embodiment 37. The method of embodiment 36, wherein the effective amount of the pharmaceutical formulation is administered as a single dose.

[0139] Embodiment 38. The method of embodiment 36 or 37, wherein the pharmaceutical formulation comprises an intermediate acting oil formulation, such as formulation F8 or formulation F7, as described herein.

[0140] Embodiment 39. A method for treating HIV infection in a mammal, comprising administering to the mammal an effective amount of a pharmaceutical preparation according to any one of embodiments 31 to 35.

[0141] Embodiment 40. The method of embodiment 39, wherein the pharmaceutical formulation is a long-acting therapeutic agent.

[0142] Embodiment 41 The method of embodiment 39 or 40, wherein the pharmaceutical formulation comprises formulation F3 or formulation F4, as described herein.

[0143] Embodiment 42. A method for treating and / or inhibiting the replication of respiratory syncytial virus (RSV), comprising administering to a mammal an effective amount of a pharmaceutical formulation according to any one of embodiments 31 to 35.

[0144] Embodiment 43. A method for treating a viral infection in a mammal, comprising administering to the mammal an effective amount of a pharmaceutical preparation according to any one of embodiments 31 to 35, wherein the virus is an RNA virus of a viral family selected from the group consisting of Filoviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, Fenuiviridae, Nairoviridae, Arenaviridae, Flaviviridae, and Coronaviridae.

[0145] Embodiment 44. A method for producing a prodrug, 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, Y is hydrogen, C1-C 30 hydrocarbyl, and a pharma- ceutically acceptable cation; x is 0 or 1; and L is independently selected from the group consisting of C1-C 30 Hydrocarbyl, 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 of any one of claims 1 to 5, wherein the alkyl group is selected from the group consisting of hydrocarbyl.

[0146] Embodiment 45. A method for producing a prodrug according to 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.

[0147] Embodiment 46 A method for producing a prodrug according to any one of the preceding embodiments, wherein the acid comprises HCl.

[0148] Embodiment 47. 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). EXAMPLES

[0149] The present disclosure is further illustrated by the following examples, which should not be interpreted as imposing limitations on the scope of the present invention in any way. On the contrary, it is clearly understood that the present disclosure must 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 present disclosure or the scope of the appended claims. Thus, other aspects of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the present disclosure disclosed herein.

[0150] Example 1 - Preparation of Compounds In the examples, several general methods were used to generate the various products and / or intermediates, although other known synthetic techniques may also be used.

[0151] A.GS-441524 Synthesis of alkyl and alkoxyalkyl esters of 5'-monophosphate [ka] Scheme 1. Synthesis of alkyl and alkoxyalkyl esters of GS-441524 5'-monophosphate. Reagents: a) POCl3, TEA, THF; b) GS-441524 acetonide, DCC / DMAP or DIC / NMI, Pyridine; c) formic acid, room temperature or concentrated HCl / THF; d) PyBOP, DIEA, DMF.

[0152] 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 eq.) and triethylamine (2 eq.) in anhydrous tetrahydrofuran (THF) was added dropwise to a solution of phosphorus oxychloride (1.5 eq.) in THF with stirring while maintaining the temperature below 20° C. Stirring was continued for an additional hour at 0° C., then water was added and stirring was continued overnight, followed by extraction with ethyl ether. The crude solids from the ether layer were recrystallized from hexane to give phosphates 2a-c.

[0153] 2a Eicosyl dihydrogen phosphate 1 H (400MHz, 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[MH] -

[0154] 2b 3-(Hexadecyloxy)propyl dihydrogen phosphate 1 H NMR (400 MHz, 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).

[0155] 2c 2-(Octadecyloxy)ethyl dihydrogen phosphate 1H NMR (400 MHz, 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).

[0156] Coupling of phosphates 2a-c to GS-441524 acetonide (remdesivir nucleoside, RVn acetonide) General Method BN,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, then the mixture was 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 (CH2Cl2 / methanol 10-20%) afforded the protected phosphodiester compound.

[0157] General MethodsCN,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 then stirred at room temperature for 48 h with analysis of the reaction mixture by TLC indicating 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% CH2Cl2 to CH2Cl2 / 20% methanol) afforded the protected phosphodiester analog.

[0158] 3a Eicosyl-phospho-RVn acetonide. GS-441524 acetonide was coupled to 2a following general procedure C. The structure was confirmed by ESI-MS 690.50 [MH]- Confirmed by.

[0159] 3b 3-(hexadecyloxy)propyl-phospho-RVn acetonide. GS-441524 acetonide was coupled to 2b according to general method B. N,N-dicyclohexylcarbodiimide (DCC, 619 mg, 3 mmol) was added to a mixture of GS-441524 acetonide (300 mg, 0.91 mmol), 3-(hexadecyloxy)propyl phosphate (2b, 414 mg, 1.10 mmol), 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. 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=1.0Hz, 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=1 1.1,8.5,2.6Hz,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] - .

[0160] 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, then heated to 90 °C and stirred for 3 days. 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.

[0161] 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, 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.

[0162] General Method E. 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 about 4 h. Formic acid was removed by rotary evaporation and the residue was coevaporated with EtOH (2×25 mL) and then adsorbed onto silica gel and purified by flash column chromatography. Gradient elution (100% CH2Cl2 to CH2Cl2 / 35% methanol) afforded the product.

[0163] 4a Eicosyl-phospho-RVn - prepared from 3a according to general method E. The structure was confirmed by ESI-MS 650.38 [MH] - Confirmed by.

[0164] 4b 3-(Hexadecyloxy)propyl-phospho-RVn. Prepared from 3b according to general method 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, 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%

[0165] 4c 2-(Octadecyloxy)ethyl-phospho-RVn Prepared from 3c according to general method D. Concentrated HCl (0.3 mL) was added slowly 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 / CH2Cl2 gave 0.10 g (55% yield) of compound 4c. 1 H NMR (400MHz, 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=4.89H z,2H),3.46-3.57(m,2H),3.42(td,J=6.85,1.96Hz,2H),3.34(dt,J=3.18,1.59 Hz,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%.

[0166] 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. Then, DMF was evaporated and the residue was dissolved in ethyl acetate (50 mL) and washed with saturated NaHCO3 (3 x 10 mL). The organic layer was dried over MgSO4 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),4.24-4.31(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] + .

[0167] 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 NaHCO3. 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=11 .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,2 H),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] + .

[0168] 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 methodology used to generate the following embodiments of 1-O-alkyl-2-O-substituted-sn-glyceryl esters of GS-441524 5'-monophosphate. [ka] Scheme 2. Synthesis of 1-O-alkyl-2-O-substituted-sn-glycerols. Reagents: a) R1 bromide or methanesulfonate, NaH, DMF; b) 80% CH3COOH, reflux; c) trityl chloride, TEA, DMAP, CH2Cl2; d) R2 bromide or methanesulfonate, NaH, DMFe) acidic deprotection.

[0169] Synthesis of 1-O-alkyl-sn-glycerols (Scheme 2, 10a-d) General Method F. In this embodiment, alkylation of 2,3-isopropylideneglycerol using alkyl methanesulfonates was carried out as described in the literature (Fernandez, DM; Contreras, LJ; Moreno, BM; Silva, EG; Mayorga, WH Enantiomeric synthesis of natural alkylglycerols and their antibacterial and antibiotic activities. Nat. Prod. Res. 2019, 1-7.).

[0170] Sodium hydride and DMF were stirred in the flask. Isopropylidene glycerol was added slowly for expected hydrogen evolution, with cooling as necessary to keep the temperature below 35°C. After stirring 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 solids were collected on a fritted funnel and then washed with water. To achieve deprotection, the filter cake was added to 80% acetic acid and heated at 80°C for 1 hour. The flask was cooled and the product crystallized, collected via vacuum filtration and dried. The crude product was recrystallized in hexane or purified by flash column chromatography on silica gel 60.

[0171] General method G. (Alkylation with 1-bromoalkanes / alkenes as described in the literature: (Halldorsson, A., et al. Tetrahedron: Asymmetry, 2004, 15, 2893-2899)).

[0172] 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 stirred in an oil bath at 35-40°C for approximately 15 hours. The alkylation 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.

[0173] Deprotection: The product was refluxed with p-toluenesulfonic acid (10 mol%) in THF / water overnight. After concentration in vacuum, the residue was dissolved in diethyl ether, washed with water and brine solution, dried over anhydrous magnesium sulfate, and solvent removal was achieved in vacuum on a rotary evaporator to give 1-O-alkyl-sn-glycerols.

[0174] 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.)

[0175] 10b. 1-O-Hexadecyl-sn-glycerol was purchased from Bachem America.

[0176] 10c. 1-O-Octadecyl-sn-glycerol was purchased from Bachem America.

[0177] 10d. 1-O-oleyl-sn-glycerol. Synthesized according to general method 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. Subsequent workup afforded 583 mg of crude 9d as an oil. Crude 9d was treated with p-TsOH.H2O (0.15 equiv.) in THF (6 mL) and H2O (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). 1 H 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] + .

[0178] Synthesis of 1-O-alkyl-2-O-substituted-sn-glycerols (Scheme 2, 12a-p) General Method H. Protection of the 3-hydroxy group of 1-O-substituted-sn-glycerols was performed as described in the literature (e.g., Kini, GD, Hostetler, SE, Beadle, JR, Aldern, KA 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). 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.) in anhydrous dichloromethane, 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 hexanes (0-20%) eluted the appropriate fractions.

[0179] 11a 1-O-Tetradecyl-3-O-trityl-sn-glycerol - prepared as described in the literature (Huang, Z., Szoka, Z. (2008). Sterol-Modified Phospholipids: Cholesterol and Phospholipid Chimeras with Improved Biomembrane Properties. J. Am. Chem. Soc., 130, 15702-15712).

[0180] 11b 1-O-Hexadecyl-3-O-trityl-sn-glycerol - prepared as described in the literature (Huang, Z., Szoka, Z. (2008). Sterol-Modified Phospholipids: Cholesterol and Phospholipid Chimeras with Improved Biomembrane Properties. J. Am. Chem. Soc., 130, 15702-15712).

[0181] 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).

[0182] 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] +

[0183] General Method I. Alkylation and deprotection of 1-O-alkyl-3-O-trityl-sn-glycerol was carried out as previously described (Kini, GD, Hostetler, SE, Beadle, JR, Aldern, KA 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 equiv.) was added to a stirred solution of 1-O-alkyl-3-O-trityl-sn-glycerol (1 equiv.) in DMF at 0° C. After 20 min, the bromo or methanesulfonate derivative of R2- (1.8 equiv.) was added. The reaction mixture was then stirred at room temperature for 5 h or until the reaction was essentially complete by TLC. Workup and column chromatography gave the 1-O-alkyl-2-O-substituted-3-O-trityl-sn-glycerols detritylated with acid. Workup and column chromatography gave the 1-O-alkyl-2-O-substituted-sn-glycerols.

[0184] 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] + Confirmed by.

[0185] 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).

[0186] 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.

[0187] 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.

[0188] 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. Work-up and column chromatography gave 354 mg of crude product and 200 mg of 11d was also recovered. Deprotection gave 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] +

[0189] 12m 1-O-oleyl-2-O-(3-fluoro-4-methoxybenzyl)-sn-glycerol. Prepared according to general method I from 11d and 3-fluoro-4-methoxybenzyl bromide. 1 H NMR(300MHz,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,2 H),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] +

[0190] 12n 3-O-Octadecyl-2-O-benzyl-sn-glycerol.

[0191] 12o 1-O-Octadecyl-2-O-(3-trifluoromethyl)benzyl)-sn-glycerol ESI-MS 525.47[M+Na] + .

[0192] 12p 1-O-Octadecyl-2-O-(4-trifluoromethyl)benzyl)-sn-glycerol. ESI-MS 503.35 [M+H] + .

[0193] Synthesis of 1-O-alkyl-2-O-substituted-sn-glyceryl esters of GS-441524 5'-monophosphate (Scheme 3, 15a-p) 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] Scheme 3. Synthesis of 1-O-alkyl-2-O-substituted-sn-glyceryl esters of GS-441524 5'-monophosphate. Reagents: a) POCl3 or bis(trichloroethyl)chlorophosphate / zinc powder, b) DCC / DMAP or DIC / NMI, pyridine, c) formic acid or concentrated HCl / THF.

[0194] General Method J. Phosphorylation of 1-O-alkyl-2-O-substituted-sn-glycerols was accomplished as described in the literature (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). 1-O-alkyl-2-O-substituted-sn-glycerols (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 showed complete phosphorylation. Water (10 mL) was added to quench excess reagent, then the mixture was 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. Gradient elution from 100% hexane to 25% EtOAc / hexane was used to isolate the protected phosphorylated product.

[0195] The product (9.65 mmol) was dissolved in a mixture of chloroform (50 mL) and glacial acetic acid (90 mL), then stirred vigorously and cooled in an ice-water bath. Zinc powder (5 g) was added to the mixture and stirred for 1 h, then the ice-water bath was removed and stirring was continued for another 2 h. Residual 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), then the organic layer was 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.

[0196] Compounds 13a, 13b, 13c, 13d, 13e, 13f, 13g, 13h, 13i, 13j, and 13k were prepared according to general method J.

[0197] 13l 1-O-oleyl-2-O-benzyl-sn-glyceryl-3-phosphate. Prepared according to general method 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] +

[0198] 13m 1-O-oleyl-2-O-(3-fluoro-4-methoxybenzyl)-sn-glyceryl-3-phosphate. Prepared according to general method A.

[0199] 13n 3-O-Octadecyl-2-O-benzyl-sn-glyceryl-1-phosphate. Prepared according to general method A.

[0200] 13o 1-O-Octadecyl-2-O-(3-trifluoromethyl)benzyl-sn-glyceryl-3-phosphate. Prepared according to general method A. ESI-MS 581.49 [MH] - .

[0201] 13p 1-O-Octadecyl-2-O-(4-trifluoromethyl)benzyl-sn-glyceryl-3-phosphate. Prepared according to general method A. ESI-MS 581.47 [MH] - .

[0202] Coupling of phosphates 13a-p 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 method C. The structure was confirmed by ESI-MS 770.50 [MH] - Confirmed by.

[0203] 14b 1-O-Hexadecyl-2-O-benzyl-sn-glyceryl-phospho-RVn acetonide - prepared from GS-441524 acetonide and 13b according to general procedure C.

[0204] 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.

[0205] 14d 1-O-Octadecyl-2-O-benzyl-sn-glyceryl-phospho-RVn acetonide - prepared from GS-441524 acetonide and 13d according to general method 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. 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] - .

[0206] 14e 1-O-Octadecyl-2-O-benzyl-rac-glyceryl-phospho-RVn acetonide. - Prepared from GS-441524 acetonide and 13e according to general method C.

[0207] 14f 1-O-Octadecy-2-O-octyl-sn-glyceryl-phospho-RVn acetonide - Prepared from GS-441524 acetonide and 13f according to general procedure C.

[0208] 14g 1-O-Octadecyl-2-O-(cyclohexylmethyl)-sn-glyceryl-phospho-RVn acetonide - can be prepared from GS-441524 acetonide and 13g according to general method C.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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 method C.

[0213] 14l 1-O-oleyl-2-O-benzyl-sn-glyceryl-phospho-RVn acetonide - prepared from GS-441524 acetonide and 13l according to general method B.

[0214] 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.

[0215] 14n 3-O-Octadecyl-2-O-benzyl-sn-glyceryl-1-phospho-RVn acetonide Prepared from GS-441524 acetonide and 13n according to general method B.

[0216] 14o 1-O-Octadecyl-2-O-(3-trifluoromethyl)benzyl-sn-glyceryl-phospho-RVn acetonide. Prepared from GS-441524 acetonide and 13o according to general method B. ESI-MS 894.66 [MH] - .

[0217] 14p 1-O-Octadecyl-2-O-(4-trifluoromethyl)benzyl-sn-glyceryl-phospho-RVn acetonide. Prepared from GS-441524 acetonide and 13p according to general method B. ESI-MS 894.53 [MH] - .

[0218] 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 [MH] - =730.41.

[0219] 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),3.3 2-3.28(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.

[0220] 15c 1-O-Hexadecyl-2-O-(3-fluoro,4-methoxybenzyl)-sn-glyceryl-phospho-RVn. Prepared from compound 14c according to general method E. 1 H 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),3.93(q,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),3.2 9(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.

[0221] 15f 1-O-Octadecy-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=1 6.0,7.4,7.4Hz,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.

[0222] 15g 1-O-Octadecyl-2-O-(ethylcyclohexyl)-sn-glyceryl-phospho-RVn--can be prepared from compound 14g according to general method E.

[0223] 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),6.88(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=5.8,5.8 Hz,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.

[0224] 15i 1-O-Octadecyl-2-O-(4-methoxybenzyl)-sn-glyceryl-phospho-RVn--can be prepared from compound 14i according to general method E.

[0225] 15j 1-O-Octadecyl-2-O-(3-fluoro-4-methyl-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,3H),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.

[0226] 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.

[0227] 15l 1-O-oleyl-2-O-benzyl-sn-glyceryl-phospho-RVn - Prepared from compound 14l according to general method D and isolated in 84% yield as an off-white solid. 1H NMR(300MHz,CDCl3+CD3OD) δ 7.79(s,1H),7.38(s,2H),7.27-7.21(m,5H),6.92(d,J=6.0Hz,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),3.9 0-3.87(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.

[0228] 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.

[0229] 15n 3-O-Octadecyl-2-O-benzyl-sn-glyceryl-1-phospho-RVn Prepared from compound 14n following general method D and isolated as an off-white solid.

[0230] 15o 1-O-Octadecyl-2-O-(3-trifluoromethyl)benzyl-sn-glyceryl-phospho-RVn Prepared from compound 14o according to general method D and isolated as an off-white solid. ESI-MS 854.48 [MH] - .

[0231] 15p 1-O-Octadecyl-2-O-(4-trifluoromethyl)benzyl-sn-glyceryl-phospho-RVn Prepared from compound 14p according to general method D and isolated as an off-white solid. ESI-MS 854.46 [MH] - .

[0232] D. Synthesis of 1-O-octadecyl-2-O-benzyl-sn-glyceryl-benzyl-phospho-RVn [ka] Scheme 4. Reagents: a) benzyl alcohol, PyBOP, DIEA, DMF; b) formic acid, room temperature 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. Then, DMF was evaporated and the residue was dissolved in ethyl acetate (50 mL) and washed with saturated NaHCO3 (3 × 10 mL). The organic layer was dried over MgSO4 and concentrated. The residue was purified by column chromatography on silica gel, eluted with chloroform / methanol (0-15%) to give 16. ESI-MS 918.33 [MH] - .

[0233] Compound 16 was added to formic acid and the deprotection was monitored by TLC. The mixture was concentrated under vacuum 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] + Confirmed by.

[0234] E. GS-441524 - Synthesis of 3',5'-cyclic monophosphate, octadecyloxyethyl, and 1-O-octadecyl-2-O-benzyl-sn-glyceryl ester In another embodiment, the compounds of the present disclosure are 3',5'-cyclic phosphates. The 3',5'-cyclic phosphates are prepared from known or easily prepared starting materials according to methods known to those skilled in the art of organic synthesis. As an example, the 3',5'-cyclic phosphates 18-eq, 18-ax, 19-eq, and 19-ax are prepared from the phosphodiesters 15d and 4c by an intramolecular esterification reaction.

[0235] A solution of 1-O-octadecyl-2-O-benzyl-sn-glyceryl-phospho-RVn (15d, 1 mmol) in dry DMF (5 mL) was added to a solution of 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) and stirred at room temperature for 3 h until TLC showed substantial conversion to the 3',5'-cyclic phosphate. Water (mL) was added and the solvent was evaporated under vacuum. The residue was purified by column chromatography on silica gel to give compound 18-eq (ESI-MS: 770.18 [M+H] + ) and 18-ax(ESI-MS:770.29[M+H] + ) was obtained.

[0236] Similarly, a solution of octadecyloxyethyl-phospho-RVn (4c, 1 mmol) in dry DMF (5 mL) was added to a solution of diisopropylethylamine (DIEA, 160 mg, 1.5 mmol) and (1H-benzotriazol-1-yloxy)-tripyrrolidinophosphonium hexafluorophosphate (PyBOP, 780 mg, 1.5 mmol) in dry DMF (5 mL) and stirred at room temperature for 3 h until TLC showed substantial conversion to the 3',5'-cyclic phosphate. Water (1 mL) was added and the solvent was evaporated under vacuum. The residue was purified by column chromatography on silica gel to give compound 19-eq (ESI-MS: 650.41 [M+H] + ) and 19-ax(ESI-MS:650.42[M+H] + ) was obtained. [ka] F. Synthesis of 1-O-Octadecyl-2-O-(4-cyano)benzyl-sn-glyceryl-phospho-RVn. OD-4-CN-Bn-P-RVn was prepared using the method outlined above in Section C. 1-O-Octadecyl-3-O-trityl-sn-glycerol was alkylated with 4-cyanobenzyl chloride and the trityl was removed (Method I) to provide 1-O-Octadecyl-2-O-(4-cyano)benzyl)-sn-glycerol. The alcohol was then phosphorylated (Method A), coupled to GS-441524 acetonide (Method C) and deprotected (Method E) to give the title compound. [ka]

[0237] Example 2 - Anti-coronavirus activity assay in Vero E6 cells In this example, reference is made to the following compounds, including remdesivir nucleoside analogs and related intermediates: [ka]

[0238] The compounds of this example were assayed for anti-coronavirus activity in Vero E6 cells in comparison to 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 next day, serial 2-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 made 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 nucleoside with selectivity indices ranging from 22.8 to >227. Cytotoxicity (CC 50 ) was assessed using a commercially available MTT assay.

[0239] 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 4]

[0240] Example 3. Further 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) for SARS-CoV-2 infection in Vero E6 cells were also generated in two separate experiments performed in duplicate (Figure 1A-Figure 1F). Dose-response curves for three remdesivir analogs (Figure 1A, Figure 1B, and Figure 1C), remdesivir (GS-5734) (Figure 1D), and remdesivir nucleoside (GS-441524) (Figure 1E) for SARS-CoV-2 infection in Vero E6 cells. Vero E6 cells were pretreated with the indicated drug at the indicated doses for 30 min and then infected with SARS-CoV-2 isolate USA-WA1 / 2020 for 48 h. 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-F. Data points represent the average relative expression from duplicate wells. Error bars indicate standard deviation (SD). The black vertical 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.

[0241] The following table shows the effective concentration (EC 50 , E.C. 90 ), 50% cytotoxic concentration (CC 50 ), and selectivity index, mean ± SD. 50 ) was evaluated 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 5]

[0242] Of all the recognized shortcomings of RVn, it was chosen in this example to design a prodrug of RVn that could provide oral bioavailability, since 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 that often show a significant increase in antiviral activity.

[0243] In contrast to activation of RDV, which requires four transformations, intracellular kinase bypass by this class of compounds 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).

[0244] 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 mouse chickenpox infections (Hostetler KY, Beadle JR, et al. 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).

[0245] The synthesis of the lipid prodrugs of this example was much simpler than that of RDV and was readily scalable.

[0246] In this example, three lipid prodrugs of RVn were synthesized 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.

[0247] Compounds: Remdesivir (GS-5734) and remdesivir nucleoside (GS-441524) were purchased from AA Block (San Diego, CA) and Mason-Chem (Palo Alto, CA), respectively.

[0248] Cells: Vero E6 were obtained from ATCC and grown in DMEM (Corning) with 10% FBS and penicillin-streptomycin (Gibco).

[0249] SARS-CoV-2 infection: SARS-CoV-2 isolate USA-WA1 / 2020 (BEI Resources) was grown and infectious units were quantified by plaque assay using Vero E6 (ATCC) cells. Approximately 10 per well 4 Vero E6 cells were seeded in 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 ul of TRIzol (ThermoFisher).

[0250] 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) and qPCR was performed using an iTaq Universal

[0251] The PCR was performed using SYBR Green Supermix (BioRad) and ABI 7300 real-time PCR system. cDNA was amplified using the following primers RPLP0 F-GTGTTCGACAATGGCAGCAT (SEQ ID NO: 1), RPLP0 R-GACACCCTCCAGGAAGCGA (SEQ ID NO: 2), SARS-CoV-2 Spike F-CCTACTAAATTAAATGATCTCTGCTTTACT (SEQ ID NO: 3), SARS-CoV-2 Spike R-CAAGCTATAACGCAGCCTGTA (SEQ ID NO: 4). 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 to untreated SARS-CoV-2 infected Vero E6 cells (reference control). Curves were fitted and 50 and 90% effective concentration EC50 and EC90 values ​​were calculated using Prism8.

[0252] CellTiter-glo Luminescent Cell Viability Assay: Approximately 10 per well 4 Vero E6 cells were seeded in opaque walled 96-well cell culture plates and incubated overnight. Compounds or controls were added at the indicated concentrations. After 48.5 h of incubation at 37° C. and 5% CO2, an equal volume of CellTiter-Glo reagent (catalog no. G7570, Promega, Madison, WI) was added, mixed, 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 50 Values ​​were calculated using Prism8.

[0253] Determination of cytotoxicity: 50% cytotoxic concentration (CC 50 ) was determined with Cell Titer Glo (catalog no. G7570, Promega, Madison, WI) according to the manufacturer's instructions. Calculated CC 50 The values ​​are given in the table above.

[0254] 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.

[0255] Example 4 - Production of Remdesivir Triphosphate in Vero E6 Cells In this example, Vero E6 cells were grown at approximately 3.4×10 per well in 2 mL of medium (DMEM, 10% FBS). 5 Cells were seeded in 6-well plates.

[0256] 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 drugs at a concentration of 1 μM. The cells were incubated with the 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 the cells triturated again. The cells were counted in a Reichert hemocytometer using two 10 μL samples to determine the number of cells in each sample.

[0257] 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 pmol / 10 6 Cells and were the average of two or three determinations. 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).

[0258] As shown in Figure 3, in Vero E6 cells, synthesis of remdesivir triphosphate (RVn-TP) was progressively increased up to 48 hours after exposure to 1 micromolar ODE-P-RVn and OBDG-P-RVn. With RVn, the level of RVn-TP peaked at 8 hours and then decreased. With RDV, the level of RVn-TP was below quantifiable levels at 8 and 24 hours.

[0259] 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 96-well plates in EMEM (10% FCS) overnight at 37°C. The medium was removed from each well and the cells were incubated with 100 TCID 50 The virus was then allowed to infect for 2 hours.

[0260] 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 the MTT cell proliferation assay kit (Abcam) read on an ELx800, Universal Microplate Reader (BIO-TEK Instruments, INC). [Table 6-1] [Table 6-2]

[0261] 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 half-maximal effective concentration (EC 50 ) was defined as the concentration that achieved 50% inhibition of virus-induced cytopathic effect.

[0262] Example 6 - SARS-CoV-2 Infection Assay Approximately 12e3 TMPRSS2-Vero cells or 20e3 Huh7.5 cells were seeded per well in a black with clear flat-bottom 96-well plate and incubated overnight. Compounds or controls were added at the indicated concentrations approximately 30 to 60 minutes prior to infection with SARS-CoV-2 at a multiplicity of infection (FFU / cell) equal to 0.01 for TMPRSS2-Vero and 0.1 for Huh7.5.

[0263] 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 nuclear counterstain Sytox Green (Thermo Fisher Scientific).

[0264] Five images per well were acquired at 10x magnification using an Incucyte S3 (Sartorius). The percentage of infected cells and the number of nuclei were calculated using the built-in image analysis tools for the Incucyte S3. EC 50 , E.C. 90 , and C.C. 50 Calculations of were performed using nonlinear regression analysis in GraphPad Prism 9 with bottom and top parameters constrained to 0 and 100, respectively. [Table 7-1] [Table 7-2] [ka] [Table 8-1] [Table 8-2]

[0265] SARS-CoV-2 infection assay: Approximately 20k Calu-3 cells were seeded per well in black 96-well plates with clear flat bottom (Corning #3904) and incubated for 48-72 h. Compounds or controls were added at the indicated concentrations 30-60 min prior to infection with addition of SARS-CoV-2 at a multiplicity of infection (FFU / cell) equal to 0.01. After 44 h of incubation at 37 °C and 5% CO2, the medium was removed and cells were incubated with 4% formaldehyde for 30 min 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 nuclear counterstain Sytox Green (Thermo Fisher Scientific). Five images per well were obtained at 10x magnification using an Incucyte S3 (Sartorius).

[0266] The percentage of infected cells and the number of nuclei were calculated using the built-in image analysis tools for the Incucyte S3. 50 , E.C. 90 , and C.C. 50 Calculations of were performed using nonlinear regression analysis in GraphPad Prism9 with bottom and top parameters constrained to 0 and 100, respectively. All work on authentic SARS-CoV-2 was performed under biosafety level 3 conditions at the University of California, San Diego.

[0267] Cell viability assay: For selected compounds, CC50 was calculated using CellTiter-Glo up to a maximum of 100 μM. For these experiments, approximately 20k Calu-3 cells were seeded per well in opaque white 96-well plates (catalog no. 655073, Greiner Bio-One, Monroe, North Carolina) and incubated for 48-72 hours. Compounds or controls were added and incubated for 44 hours at 37°C and 5% CO2. An equal volume of CellTiter-Glo reagent (catalog no. G7570, Promega, Madison, WI) was then added, mixed, and luminescence was recorded on a Veritas Microplate Luminometer (Turner BioSystems) according to the manufacturer's recommendations.

[0268] Example 7 - Antiviral activity in different 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 MEM (Corning), 10% FBS, 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 enzyme-free dissociation reagent ReLeSR™ (Stem Cell Tech #05872). Cultures were maintained in an undifferentiated state at 37°C in a 5% CO2 incubator.

[0269] For proximal lung organoid generation, human PSCs were dissociated into single cells and then cultured at 5.3 × 10 in definitive endoderm (DE) induction medium (RPMI1640, 2% B27 supplement, 1% HEPES, 1% Glutamax, 50 U / mL penicillin / streptomycin). 4 cells / cm 2Cells were seeded at a density of 100 ng / mL onto Matrigel-coated plates (BD Biosciences) and supplemented with 100 ng / mL human activin A (R&D), 5 μM CHIR99021 (Stemgent), and 10 μM of the ROCK inhibitor, Y-27632 (R&D Systems) on day 1. On days 2 and 3, cells were cultured in DE induction medium with only 100 ng / mL human activin A. Anterior foregut endoderm (AFE) was generated 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 days 4–6. 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 9–11 days. To generate 3D human proximal lung organoids, we modified 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).

[0270] LPCs were dissociated in Accutase for 10 min and plated at 5.0 × 10 cells on Matrigel in 12-well 0.4-μm pore-size Transwell (Corning) culture inserts. 4Cells were resuspended in Matrigel at 200 ul / cell. 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). 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 of Matrigel-coated 96-well plates 1 day prior to 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.

[0271] The supernatant was carefully removed and resuspended in 2-3 ml of TrypLE Express (Gibco #12605010) for 20 min at 37°C. The reaction was quenched with 2% FBS in DMEM / F12 and then centrifuged at 400 x g for 5 min. 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 the reagent reservoir trough, resuspended in proximal lung organoid maturation medium and seeded into 96-well plates as a monolayer at 100 ul per well via a multichannel pipette.

[0272] SARS-CoV-2 infection: SARS-CoV-2 isolate USA-WA1 / 2020 (BEI Resources) was grown 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 prior to treatment / infection. Calu-3 and Caco-2 were seeded approximately 48 hours prior to 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 prior to infection, followed by SARS-CoV-2 at a multiplicity of infection equal to 0.01. After 48 hours of incubation at 37°C and 5% CO2, cells were washed twice with PBS and lysed in 200 ul of TRIzol (ThermoFisher). All SARS-CoV-2 work was conducted under biosafety level 3 conditions at the University of California, San Diego, with approval from the Institutional Biosafety Committee.

[0273] RNA extraction, cDNA synthesis and qPCR: RNA was purified from TRIzol lysates using the Direct-zol RNA Microprep kit (Zymo Research) following the manufacturer's recommendations, including Dnase treatment. RNA was converted to cDNA using the iScript cDNA synthesis kit (BioRad) and 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 (SEQ ID NO: 1), RPLP0 R-GACACCCTCCAGGAAGCGA (SEQ ID NO: 2), SARS-CoV-2 Spike F-CCTACTAAATTAAATGATCTCTGCTTTACT (SEQ ID NO: 3), SARS-CoV-2 Spike R-CAAGCTATAACGCAGCCTGTA (SEQ ID NO: 4). 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 to untreated SARS-CoV-2 infected Vero E6 cells (reference control). Curves were fitted using a nonlinear regression-log(inhibitor) vs response (four parameters) model using Prism 9. To calculate effective concentration EC50 and EC90 values, qRT-PCR values ​​were normalized to percent inhibition and curve fit using a 20 nonlinear regression-log(agonist) vs response (four parameters) model with bottom and top constrained to 0 and 100, respectively, using Prism 9.

[0274] Cell viability assay: Cell types were seeded according to the SARS-CoV-2 infection test in opaque walled 96-well cell culture plates or the 229E infection test in clear 96-well cell culture plates and incubated overnight. Compounds or controls were added at the indicated concentrations. For SARS-CoV-2 related tests, cells were incubated for 48.5 h at 37 °C and 5% CO2, an equal volume of CellTiter-Glo reagent (catalog no. G7570, Promega, Madison, WI) was added, mixed, and luminescence was recorded on a Veritas Microplate Luminometer (Turner BioSystems) according to the manufacturer's recommendations. For related 229E, cells were incubated for 72 h at 37 °C and 5% CO2, 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 for 3 h at 37 °C. Absorbance was measured with an ELx800, Universal Microplate Reader, (BIO-TEK Instruments, INC.) according to the manufacturer's recommendations. Percent viability was calculated relative to untreated controls and CC 50 Values ​​were calculated using Prism9. [Table 9]

[0275] 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 half-maximal effective concentration (EC 50 ) and the 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 was less than 0.35 μM in PSC-Lung and Calu-3, both models of human lung infection. The antiviral activities of ODBG-P-RVn and ODE-P-RVn were significantly better than 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, and there was no significant difference with 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.

[0276] 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 hepatic cell line Huh7.5. 50 In all cell lines except for RDV, which has ODBG-P-RVn (EC 50 0.14μM~0.30μM and CC 50 The range of antiviral and cytotoxicity of RDV (EC 50 0.06μM~1.13μM and CC 50 15.2 μM to >100 μM) was more consistent across cell types (see table above). Collectively, these data indicate that lipid RVn monophosphate prodrugs are potent antiviral agents against SARS-CoV-2 in vitro with low toxicity and excellent selectivity indices.

[0277] Example 8 - Effect of antiviral drugs on human coronavirus 229E-infected cells Human coronavirus 229E (ATCC) was propagated and infectious units were determined using TCID 2016 using MRC-5 cells. 50 For antiviral testing, 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 50 The virus was then allowed to infect for 2 hours.

[0278] 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). The % 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 cell control, respectively. The mean half-maximal effective concentration (EC 50 ) was defined as the concentration that achieved 50% inhibition of virus-induced cytopathic effect.

[0279] As described herein, various compounds of the present disclosure inhibit human alphacoronavirus 229E. Cells were infected with 229E for 2 hours, followed by treatment with the indicated drug at the indicated dose for 72 hours. Relative CPE was determined by measuring cell viability using an MTT assay. Cytotoxicity in MRC-5 cells incubated for 72 hours in the presence of the indicated drug at the indicated concentration, after which cell viability was measured by CellTiter-Glo assay. Data points represent the mean from three independent experiments performed in duplicate. Error bars represent standard error of the mean (SEM). Both ODBG-P-RVn and RDV showed dose-dependent inhibition of cytopathic effect (CPE). EC of ODBG-P-RVn and RDV 50 The values ​​are 0.15 μM and 0.04 μM, and the EC 90 The CC of ODBG-P-RVn and RDV were 0.54 mM and 0.26 mM, respectively. 50 was >50 μM in MRC-5 cells. The most active compounds were those with 3-fluoro,4-methoxy substitution. This, together with the antiviral data for SARS-CoV-2, indicates that ODBG-P-RVn-related analogs have antiviral activity against two genetically distinct human pathogenic coronaviruses.

[0280] Example 9 - Orally administered ODBG-P-RVn(15d) achieved 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 noted at 1 hour and declined by 50% in approximately 5 hours.

[0281] Plasma curves were generally similar on days 1 and 7, except for 16.9 mg / kg, where the 7d values ​​were slightly higher than the levels on day 1. At 12 h, ODBG-P-RVn levels were above the EC50 for ODBG-P-RVn on both days 1 and 7 in all cell lines tested, including Vero E6 and PSC lung cells. 90 The levels of RVn, a nucleoside metabolite of ODBG-P-RVn, peaked 3 hours after administration and then decreased. Plasma levels of RVn were greater than the EC 90 The observed low levels of RVn suggest that the antiviral activity attributable to this metabolite is minimal and is also consistent with findings of OBDG-P-RVn stability in human plasma. Collectively, these results suggest that ODBG-P-RVn is effective in suppressing viral replication in various tissue types in vivo.

[0282] Figures 4A and 4B show 7-day oral pharmacokinetics in Syrian hamsters. Syrian hamsters were given vehicle or ODBG-P-RVn by oral irrigation every 12 hours for 7 days. Groups of 3 animals were administered 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 ODBG-P-RVn (Figure 4A) and RVn (Figure 4B).

[0283] Analytical method: ODBG-P-RVn: Hamster plasma samples (10 μL) containing ODBG-P-RVn and K2EDTA as anticoagulants were added to polypropylene tubes containing water (100 μL), internal standard solution (10 μL; 1,000 ng / mL ODE-P-RVn in ACN:DMF (1:1, v / v)), and 10 μL 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 IPA, mixed, then diluted with 500 μL water, and mixed. Samples were extracted with Sep-Pak® tC18 96-well solid phase extraction plates (25 mg; water, Milford, MA). Extraction was performed under positive pressure conditions using nitrogen. Samples were washed successively with 1 mL water:acetonitrile:formic acid (475:25:0.5, v / v / v) and 0.4 mL water:acetonitrile:formic acid (350:150:0.5, v / v / v) and then successively eluted with 100 μL and 150 μL water:{acetonitrile:isopropyl alcohol (1:1, v / v)}:formic acid:ammonium formate:citric acid solution, 2% w / v (15:85:0.1:0.1:0.1, v / v / v / w / v). Citric acid solution was prepared as water:citric acid monohydrate (20:0.4, v / w). After elution, 100 μL 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) using a mobile phase system consisting of mobile phase A (water:formic acid:[water:ammonium formate:citric acid (25:5:0.5, v / w / w)] (1,000:1:1, v / v / v) and mobile phase B (acetonitrile:isopropyl alcohol:formic acid:[water:ammonium formate:citric acid (25:5:0.5, v / w / w)] (800:200:1:1, v / 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 with transitions m / z 788.4>229 (V2043) and 668.4>467.2 (V2041). The method is 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 by fitting the analyte / IS peak area ratios and standard concentrations to a linear equation with 1 / x2 weighting using Analyst. The peak area ratios were then used to interpolate the concentrations of the analytes in the samples using the equation of the calibration curve. The peak areas used in the calculations were not rounded.

[0284] Analytical Methods: RVn (GS-441524): Hamster plasma samples (20 μL) containing GS-441524 and K2EDTA as anticoagulants were added to Eppendorf LoBind microcentrifuge tubes containing acetonitrile (300 μL) and water:acetonitrile (2:8, v / v; 60 μL). The solutions were mixed and centrifuged at 16,000 g for 5 min. 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 samples were capped, mixed, and stored at 10°C pending 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 × 2 mm, 3 μm; ImtaktUSA, Portland, OR) using 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 Tof MS scan monitoring in sensitivity mode scanning from 50.0 to 700 m / z. The method is applied 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). MassLynx was used to calculate the peak area ratio of the analytes and the standard concentration as 1 / x 2 A calibration curve was obtained by fitting the peak areas to a linear equation with weighting: The peak areas were then used to interpolate the concentrations of the analytes in the samples using the equation of the calibration curve. The peak areas used in the calculations were not rounded.

[0285] 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 two 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 MedChem.2017 Mar 9;60(5):1648-1661).

[0286] The stability of ODE-P-RVn and ODBG-P-RVn in human plasma was evaluated with either K2EDTA or sodium heparin as anticoagulants.

[0287] Plasma was spiked with ODE-P-RVn or ODBG-P-RVn at a concentration of 2 micrograms / ml and incubated at 37°C. Samples were taken at 0.5, 1, 2, 4, 8, and 24 hours and frozen for later analysis by LC / MS / MS according to the method shown in Example C. Figures 5A and 5B show that both ODE-P-RVn and ODBG-P-RVn were stable for at least 24 hours in human plasma with either K2EDTA (Figure 5A) or sodium heparin (Figure 5B) as the anticoagulant. (See, e.g., Warren TK 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).

[0288] Example 11 - Effect of antiviral agents on respiratory syncytial virus replication Methods: The SRV A2 strain of RSV was obtained from ATCC. 10,000 Hep-2 cells were seeded into each well of a 96-well plate in EMEM containing 10% fetal bovine serum and 1% penicillin and streptomycin. Each well was dosed with 100 TCID 50 After 2 hours, wells were washed once with medium and serial 4-fold dilutions of candidate antiviral drugs were added. Cell cultures were observed for 72-96 hours for cytopathic effects. The concentrations of drugs that reduced CPE by 50 or 90%, respectively, were calculated using Prism 7 software and were given as EC 50 and E.C. 90 It is expressed as:

[0289] Cell viability assay: For selected compounds, CC50 was calculated using CellTiter-Glo up to a maximum of 100 μM. For these experiments, approximately 20k Calu-3 cells were seeded per well in opaque white 96-well plates (catalog no. 655073, Greiner Bio-One, Monroe, North Carolina) and incubated for 48-72 hours. Compounds or controls were added and incubated for 44 hours at 37°C and 5% CO2. An equal volume of CellTiter-Glo reagent (catalog no. G7570, Promega, Madison, WI) was then added, mixed, and luminescence was recorded on a Veritas Microplate Luminometer (Turner BioSystems) according to the manufacturer's recommendations. [Table 10]

[0290] Without wishing to be bound by any particular theory, it is believed that compounds 15j, 15m, and 15c may be at least 2-3 times more active than compound 15d. For example, compound 15m may be more active than remdesivir.

[0291] Compounds of the present disclosure had highly significant antiviral activity in Hep-2 cells infected with RSV A2. Cytotoxicity was moderate, and selectivity (CC50 / EC50) ranged from 605 to 2,500.

[0292] Example 12 - Preparation of Formulations Synthesis of HIV antiviral agents: Synthesis of ((2S,5R)-5-(4-amino-5-fluoro-2-oxo-3,4-dihydropyrimidin-1(2H)-yl)-1,3-oxathiolan-2-yl)methylbenzyl(2-(octadecyloxy)ethyl)phosphate (ODE-benzylphospho-emtricitabine) [ka] Scheme 5. Exemplary process for the synthesis of emtricitabine 5'-monophosphate analogs of the present disclosure. Reagents and conditions: a) emtricitabine, DCC, DMAP, pyridine, 80°C, 72 hours; b) benzyl alcohol, PyBOP, DIEA, DMF, room temperature, 3 hours.

[0293] N,N-Dicyclohexylcarbodiimide (DCC, 410 mg, 2.0 mmol) was added to a solution of emtricitabine (230 mg, 0.9 mmol), octadecyloxyethyl phosphate (320 mg, 0.74 mmol), and 4-dimethylaminopyridine (DMAP, 110 mg, 0.9 mmol) in 10 mL of dry pyridine, and the mixture was then heated to 80 °C and stirred for 3 days. Pyridine was then evaporated and the residue was purified by flash column chromatography on silica gel 60. Gradient elution (CHCl / methanol 10-20%) afforded 220 mg (47% yield) of octadecyloxyethyl-phospho-emtricitabine: ESI MS: 622.29 [MH] - ;624.10[M+H] + ,646.30[M+Na] + ,662.26[M+K] + .

[0294] Octadecyloxyethyl-phospho-emtricitabine (220 mg, 0.35 mmol), benzyl alcohol (76 mg, 0.70 mmol), diisopropylethylamine (DIEA, 0.12 ml, 0.70 mmol), and (1H-benzotriazol-1-yloxy)-tripyrrolidinophosphonium hexafluorophosphate (PyBOP, 360 mg, 0.70 mmol) in dry DMF (10 mL) were stirred at room temperature for 3 h. DMF was evaporated under vacuum. The residue was dissolved in ethyl acetate (50 mL) and washed with saturated NaHCO3 (3 x 10 mL). The organic layer was dried over MgSO4 and the ethyl acetate was evaporated. Purification of the residue by silica gel column chromatography using ethyl acetate and ethanol (0-4%) yielded 130 mg (52% yield) of octadecyloxyethyl benzylphospho-emtricitabine.1 H NMR(400MHz,CDCl3-CD3OD) δ ppm 7.91(d,J=5.87Hz,1H),7.40-7.48(m,4H),6.27-6.34(m,1H),5.35-5.4 1(m,1H),5.19(d,J=8.43Hz,2H),4.33-4.48(m,2H),4.21-4.28(m,2H),3 .65-3.71(m,2H),3.48-3.61(m,3H),3.37-3.42(m,2H),3.06-3.18(m,1 H),1.53-1.64(m,2H),1.25-1.40(m,30H),0.93(t,sCJ=6.23Hz,3H).ESI MS:714.32[M+H] + ,736.43[M+Na] + .

[0295] Synthesis of ((2S,5R)-5-(4-amino-5-fluoro-2-oxo-3,4-dihydropyrimidin-1(2H)-yl)-1,3-oxathiolan-2-yl)methylbenzyl ((R)-2-(benzyloxy)-3-(octadecyloxy)propyl)phosphate (ODBG-benzyl-phospho-emtricitabine) [ka] 1-O-Octadecyl-2-O-benzyl-sn-glyceryl phosphate (ODBG-P-) was coupled to emtricitabine in pyridine using DCC / DMAP as described in Scheme 5 to give ODBG-phospho-emtricitabine. Esterification of the phosphate diester with benzyl alcohol was achieved using PyBOP / DIEA as in Scheme 5 to give the phosphotriester, ODBG-benzyl-phospho-emtricitabine.

[0296] Synthesis of benzyl (2-(octadecyloxy)ethyl)((((R)-1-(2,6-diamino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphonate (ODE-benzyl 9-(R)-[2-(phosphonomethoxy)-propyl]2,6-diaminopurine) [ka] Scheme 6. Exemplary process for the synthesis of 9-(R)-[2-(phosphonomethoxy)propyl]2,6-diaminopurine) analogs of the present disclosure. Reagents and conditions: a) DCC, DMAP, pyridine, 80° C., 72 hours; b) benzyl alcohol, PyBOP, DIEA, DMF, room temperature, 3 hours. 9-[2-(R)-(Phosphonomethoxy)propyl]-2,6-diaminopurine(R)-PMPDAP and its prodrugs: Optimized preparation, including identification of by-products formed, and antiviral evaluation in vitro, Bioorganic & Medicinal. Chemistry, 2013, 21:1199-1208), octadecyloxyethan-1-ol (1 eq.), and 4-dimethylaminopyridine (DMAP, 1 eq.) are added and the mixture is heated to 80° C. until conversion to the esterified product is complete. The monoester (ODE-(R)-PMPDAP) is isolated after column chromatography. To a mixture of ODE-(R)-PMPDAP (1 eq.), benzyl alcohol (1.5 eq.), and diisopropylethylamine (DIEA, 1.5 eq.) in dry DMF, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP, 1.5 eq.) is added and the mixture is stirred at room temperature until formation of the diester is complete. The pure product, ODE-benzyl-(R)-PMPDAP, is isolated by column chromatography.

[0297] Synthesis of benzyl(2-(octadec-9-en-1-yloxy)ethyl)((((R)-1-(2,6-diamino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphate (OLE-benzyl 9-(R)-[2-(phosphonomethoxy)-propyl]2,6-diaminopurine)L [ka]

[0298] A mixture of (R)-PMPDAP and oleyloxyethan-1-ol in pyridine was treated with DCC / DMAP as in Scheme 6 to give the monoester, OLE-(R)-PMPDAP.

[0299] OLE-(R)-PMPDAP was further esterified using benzyl alcohol, PyBOP, and DIEA, and OLE-benzyl-(R)-PMPDAP was isolated after column chromatography.

[0300] Long-Acting HIV Antivirals: For use in the long-acting intramuscular treatment of infection with the Human Immunodeficiency Virus, antiviral compounds suitable for use are described in this application and in U.S. Pat. No. 8,835,630 and include: Compounds useful as long-acting HIV antiviral agents [ka] [Table 11]

[0301] The compounds of this example have an EC in the range of 0.09 to 18 nanomolar. 50 It was active at these values.

[0302] Pharmaceutical formulations: Pharmaceutical formulations are also provided herein. The pharmaceutical formulations comprise a compound described herein, such as a compound of formula (I). In some embodiments, the pharmaceutical formulations are orally bioavailable. In some embodiments, the pharmaceutical formulations are formulated for injection, such as intramuscular injection.

[0303] The pharmaceutical formulations included one compound described herein or more than one (e.g., two, three, etc.) compound described herein. The pharmaceutical formulations included any one or more pharma- ceutically acceptable excipients. The oil formulations for the compounds of the present disclosure have the following composition by weight (w / w%): [Table 12]

[0304] The following materials were used in the formulations in the table above: [Table 13]

[0305] In some embodiments, one or more of the compounds described herein, such as the compound of formula (I), were soluble in oil, such as one of formulations F3, F4, F7, and F8. When a quantity of one or more compounds described herein, such as the compound of formula (I), was soluble in oil, the resulting pharmaceutical formulation can be used in any of the methods herein, including the method of treating coronavirus with a single dose of the pharmaceutical formulation.

[0306] The formulations in the above table were prepared using compounds of the present disclosure. Antiviral prodrugs of the present disclosure using HIV antiviral agents or RNA virus antiviral compounds described in PCT / US2021 / 043094 can be used in the formulation. As a non-limiting example, the following pharmacokinetic experiments were performed using ODE-Bn-tenofovir as the antiviral prodrug.

[0307] Comparison of formulations F3, F4, F7, and F8 after a single IM dose in rats - Groups: vehicle control, formulations 3, 4, 7, 8 at 10 mg / kg, 30 mg / kg, and 100 mg / kg.

[0308] Procedure: At time zero, rats were injected intramuscularly with 100 microliters of vehicle and the formulations described. Animals were observed for general health and body weights were obtained at baseline and twice weekly thereafter. Blood was obtained at 6, 24, and 48 hours, and at 7, 14, 28, 35, and 42 days, and plasma levels of ODE-Bn-TFV and ODE-TFV (the first metabolites, also the active anti-HIV compounds) were determined by LC / MS / MS.

[0309] Figures 6A and 6B show the pharmacokinetics of ODE-Bn-TFV and ODE-TFV, respectively, following intramuscular administration of 100 mg / kg in formulation F3 to rats.

[0310] Results: Rats were injected IM on day 0 and body weights and clinical observations were obtained twice weekly for 42 days. Body weight gain in vehicle and drug treated rats was identical and no clinical signs were observed. A comprehensive metabolic panel at the end of the study did not show any abnormalities in liver or kidney function tests. CBC was normal control and all drug treatment groups. After administration, ODE-Bn-TFV was metabolized to the more active metabolite, ODE-TFV. As can be seen in the ODE-Bn-TFV curve (left) and the curve for the active first metabolite, ODE-TFV (right), clearly the 10 and 30 mg / kg (groups 2 and 3) doses were not sufficient to provide antivirally significant levels of the compound for over 30 days. Only the 100 mg / kg dose (group 4) provided virologically significant levels of ODE-Bn-TFV and ODE-TFV for 28 days.

[0311] The following two logs 10The panels show the various formulations at a dose of 100 mg / kg (formulation drug concentration of 200 mg / ml). The left panel is ODE-Bn-TFV and the right panel shows the plasma levels (nM / L) of ODE-TFV. All four formulations given at 100 mg / kg allow maximum plasma levels of ODE-Bn-TFV at 6-48 hours, followed by a gradual decline from 2.87 ng / ml (F8) to 6.95 (F3). The highest values ​​on day 28 were seen for F3, ODE-Bn-TFV 6.95 ng / ml (10.3 nM) and ODE-TFV 1.78 ng / ml (3.05 nM). These values ​​are the EC values ​​for these two compounds. 50 (1.7 nM and 1.1 nM).

[0312] 7A and 7B are plots of the effect of four different formulations on the pharmacokinetics of ODE-Bn-TFV and ODE-TFV, respectively, following intramuscular administration of 100 mg / kg to rats. [Table 14]

[0313] The figure below shows a comparison between intramuscular ODE-Bn-TFV in formulation F3 versus formulation F8 in rats. ODE-Bn-TFV in F3 provided lower initial levels of drug compared to F8, but levels were longer sustained and increased HIV EC5A levels over 28 days. 90 The F8 formulation provided higher drug levels through day 15 and was better suited for a 5-14 day treatment course.

[0314] FIG. 8 shows a comparison of ODE-Bn-TFV pharmacokinetics at 96 mg / kg in rats using formulations F3 vs. F8.

[0315] The calculated area under the curve (AUC) percentages during the first and second 14 days showing the release profile are shown in the table below. [Table 15]

[0316] Formulation F3 provided the greatest exposure during the second 14 days. Formulations F8 and F7 showed the highest exposure percentages, 89 and 83%, during the first 14 days.

[0317] A 3-month study of formulation F3 in rats measuring plasma levels at 15 and 30 days after monthly IM injections of ODE-Bn-TFV - since initial plasma values ​​were very high (approximately 100 ng / ml) and progressively decreased to slightly suboptimal values ​​at day 28, it was deemed important to know PBMC TFVpp levels in a longer, multi-dose study. The 3-month study was designed to examine mid- and trough plasma levels of ODE-Bn-TFV and ODE-TFV in male Sprague Dawley rats after monthly IM injections of F3 containing ODE-Bn-TFV.

[0318] A maximum volume IM injection of 100 microliters was given to groups of three rats on days 0, 30, and 60. There were three groups of control animals that received monthly injections of F3 vehicle. Plasma drug levels were analyzed by LC / MS / MS. Body weight was assessed over a three month period. Rats gained weight from 355 gm to 705 mg at the end of the study at 105 days. A constant (maximum) IM volume of 100 microliters was given so the initial dose was 90 mg / kg, and the two subsequent injections on days 30 and 60 were lower, at 62 mg / kg, due to weight gain in the rats. There were no significant clinical observations during the three month study, and metabolic panel and CBC showed no abnormalities at day 105.

[0319] FIG. 9 shows rat plasma levels of ODE-Bn-TFV and ODE-TFV during a 3-month exposure to monthly intramuscular doses of ODE-Bn-TFV in formulation F3.

[0320] Plasma drug levels: 15 days after the first dose, ODE-Bn-TFV levels were high and the first metabolite, ODE-TFV, was approximately 30% of the parent compound. However, as the 3-month study progressed, ODE-TFV levels gradually became the predominant active compound in plasma. After the final dose at 60 days, ODE-TFV levels remained substantial until day 105, when they were 11 nmol. EC 90 is 10 nM (Beadle JR, Aldern KA, Zhang XQ, Valiaeva N, Hostetler KY, Schooley RT. Octadecyloxyethyl benzyl tenofovir: a novel tenofovir diester provided sustained intracellular levels of tenofovir diphosphate. (Antiviral Res. 2019 Nov;171:104614). Therapeutic levels of ODE-Bn-TFV and ODE-TFV were maintained out to 105 days after three monthly IM doses at 0, 30, and 60 days. Rats received a maximum (volume) dose of 100 microliters. The second and third doses were only 62 mg / kg because body weight doubled during treatment. Even with the lower second and third doses, therapeutic levels of the drug were maintained for 105 days. Therapeutic levels of ODE-TFV metabolites were sustained for 105 days.

[0321] Dog pharmacokinetics of ODE-Bn-TFV in Formulation F3 - A 30-day study in beagle dogs was conducted at a 30 mg / kg IM dose of ODE-Bn-TFV in Formulation F3. Figure 10 shows the levels of ODE-Bn-TFV (circles) and ODE-TFV (squares).

[0322] EC 90 Values ​​are indicated with triangles to indicate when plasma drug levels fall below the 90% effective level. Intramuscular ODE-Bn-TFV in formulation F3 at 30 mg / kg had an EC 90 It provided 30-34 days above the value.

[0323] FIG. 10 shows plasma levels of ODE-Bn-TFV and ODE-TFV in beagle dogs treated with ODE-Bn-TFV in formulation F3 at 100 mg / kg.

[0324] There were no adverse effects, except for some transient swelling at the injection site. Pre- and post-CBC and metabolic panel before and after testing did not show any abnormalities. Notably, no abnormal liver or kidney function tests were observed. Other compounds of the present disclosure may be used in formulation F3 to provide long-term protection from HIV infection.

[0325] Antiviral Compounds and Formulations for Intramuscular Treatment of Coronavirus Infections - In SARS-CoV-2 infections, active viral replication typically persists for 5-10 days. To treat with an intramuscular dose, an antiviral prodrug in formulation F8 at 200-400 mg / ml was expected to provide effective drug levels for at least 7 days. Non-limiting examples of effective antiviral prodrugs of remdesivir nucleoside monophosphate may be selected from the structures shown below. [ka] [ka] [ka]

[0326] Antiviral Compounds and Formulations for Intramuscular Treatment of Human Immunodeficiency Infections - For use in the long-acting intramuscular treatment of infections by the human immunodeficiency virus, antiviral compounds suitable for use are described in U.S. Patent No. 8,835,630. These compounds may be used in formulations F3 or F4.

[0327] Non-limiting examples of compounds that may be used in the formulations described herein include the following: [ka] [Table 16]

[0328] As described herein and in this Example, 22 analogs of ODE-TFV were synthesized to determine the effective diester substituent compared to the benzyl in ODE-Bn-TFV. Antiviral activity was determined in HIV-infected human PBMCs. Pharmacokinetics of TFVpp was determined in HFF cells over a 28-day period.

[0329] Variation of the diester substituent on the anti-HIV activity of ODE-TFV is shown in the table below. [Table 17]

[0330] Figures 11A and 11B show TFVpp persistence in HFF cells of ODE-TFV (Figure 11A) and ODE-Et-TFV (Figure 11B).

[0331] The persistence of TFVpp in HFF cells after 3 days of exposure is shown in the table below. HFF cells were exposed to 1 micromolar drug for 3 days. On days 0, 1, 2, 4, 7, 14, 21, and 28, cells were trypsinized, counted, and processed for LC / MS measurement of TFVpp. Data are expressed as ftg / cell.nm=not significant; ftg-gemtogram. [Table 18]

[0332] In this example, six oil-based formulations for ODE-Bn-TFV were prepared. Accelerated stability of the 20 mg / mL formulations was determined. API loading was evaluated.

[0333] In this example, a formulation of ODE-Bn-TFV for IM use (20 mg / mL formulation) was prepared and included the components (w / w%) in the table below. [Table 19]

[0334] In this example, a two month accelerated stability study was performed, and the results and observations of this study are provided in the table below. [Table 20] [Table 21] [Table 22]

[0335] Additional formulations were prepared to be tested as provided in the table below (w / w%). [Table 23]

[0336] A 42-day pharmacokinetic study of formulations 3, 4, 7, and 8 of this example in rats was conducted. In the study, 100, 30, and 10 mg / kg of each formulation were administered to male Sprague Dawley rats (100 microliters) by intramuscular (IM) injection. Blood was collected 0.25, 1, 2, 7, 14, 21, 28, 35, and 42 days after injection to determine plasma levels of ODE-Bn-TFV and ODE-TFV. Rats were subjected to clinical observations twice weekly and body weights were collected.

[0337] 12A and 12B show the ODE-Bn-TFV (FIG. 12A) and ODE-TFV (FIG. 12B) (ODE-Bn-TFV ED 90 10.9ng / mL, ODE-TFV ED 90 6 shows the plasma concentration of formulation F3V containing 6.3 nm / mL of riboflavin.

[0338] Figures 12C and 12D show the plasma concentrations of formulations F4V containing ODE-Bn-TFV (Figure 12C) and ODE-TFV (Figure 12D).

[0339] Figures 12E and 12F show the plasma concentrations of formulations F7V containing ODE-Bn-TFV (Figure 12E) and ODE-TFV (Figure 12F).

[0340] Figures 12G and 12H show the plasma concentrations of formulations F8V containing ODE-Bn-TFV (Figure 12G) and ODE-TFV (Figure 12H).

[0341] FIG. 12I shows the mean plasma concentrations of formulations F3V, F4V, F7V, and F8V.

[0342] The API pharmacokinetic parameters for this example are provided in the table below. [Table 24]

[0343] The provided ODE-BN-TFV release observed in this example is summarized, at least in part, in the table below. [Table 25]

[0344] Through chemical and formulation modifications, TFV was converted to a long-acting anti-HIV state, as a result of this example. Formulations F3 and F4 of this example, for example, released 69% and 79% of the lead compound in the first 14 days, but this release profile can be modified as described herein. Using formulation F3, a 3-month study was conducted in rats with monthly IM injections of 100 microliters, and plasma drug levels and PBMC levels of TFVpp were measured.

[0345] 105-day pharmacokinetic study (monthly IM injections of F3 in rats): 100 microliters of Formulation 3 API was administered monthly by IM injection to male Sprague Dawley rats. Due to the fact that the rats gained significant weight over the 105-day period, the actual doses based on body weight were as follows: 90, 62, and 60 mg / kg. Blood was collected on days 15, 30, 45, 60, 75, 90, 98, and 105. Plasma levels of ODE-Bn-TFV and ODE-TFV were measured. PBMCs were also obtained the day before and f mo l / 10 6 The cells were determined.

[0346] Figure 13 shows nanograms / mL of ODE-Bn-TFV and ODE-TFV in plasma. Figure 14 shows TFV diphosphate in PBMCs.

[0347] The rats in this example gained weight over the 105 day study; from about 350 grams to about 670 grams. There were no differences in growth curves between control and treated rats. No clinical observations were reported in either group. CBCs in treated and control animals were normal. Complete metabolic panels were normal in both groups. Of importance was the fact that no changes were noted in BUN or creatinine.

[0348] Example 13 - Synthesis of 2, 3, and 4-Cyanobenzyl Analogs Below are schemes showing embodiments of the synthesis of 2-, 3-, and 4-cyanobenzyl analogs. [ka] Scheme 7. Synthesis of 2, 3, and 4-cyanobenzyl analogues. Reagents: a) sodium hydride, 2, 3, or 4-(bromomethylbenzonitrile, tetrabutylammonium iodide, THF; b) p-TsOH (cat.), CH2Cl2, MeOH; c) i) POCl3, triethylamine, CH2Cl2, ii) acetone / ice water; d) RVn (GS-441524) acetonide, DIC / NMI, pyridine, 35 °C; e) formic acid, room temperature.

[0349] Example 13a. Synthesis of 1-O-Octadecyl-2-O-(4-cyanobenzyl)-sn-glyceryl-phospho-RVn (OD(4-CN-Bn)GP-RVn) [ka] Sodium hydride (440 mg, 11 mmol) was added to a cooled (0° C.) solution of (R)-1-((4-methoxyphenyl)diphenylmethoxy)-3-(octadecyloxy)propan-2-ol (3.39 g, 5.5 mmol) and tetrabutylammonium iodide (600 mg, 1.65 mmol) in dry THF (30 mL). After vigorously stirring for 20 min, 4-(bromomethyl)benzonitrile (1.61 g, 8.2 mmol) was added and the mixture was allowed to warm to ambient temperature and stirred overnight. The reaction was then quenched with ice (25 mL), diluted with ethyl ether (75 mL), washed with H2O (2×25 mL), and dried over anhydrous MgSO4. The residue was adsorbed onto silica gel and purified by flash column chromatography (gradient: 0-50% EtOAc- in hexanes) to give (R)-4-(((1-((4-methoxyphenyl)diphenylmethoxy)-3-(octadecyloxy)propan-2-yl)oxy)methyl)benzonitrile (610 mg, 15%) as a white solid. MS (ESI) m / z [M+Na] + 754.54.

[0350] To a solution of (R)-4-(((1-((4-methoxyphenyl)diphenylmethoxy)-3-(octadecyloxy)propan-2-yl)oxy)methyl)benzonitrile (400 mg, 0.54 mmol) in 1:1 CHCl / MeOH (30 mL) was added p-toluenesulfonate monohydrate (5 mg, 0.03 mmol) and the mixture was stirred at room temperature until deprotection was complete according to TLC analysis (approximately 3 h). Saturated aqueous NaHCO (200 mg) was added and the solvent was evaporated under vacuum. The residue was adsorbed onto silica gel and purified by flash column chromatography (gradient: 0-10% EtOAc in hexanes) to give (S)-4-(((1-hydroxy-3-(octadecyloxy)propan-2-yl)oxy)methyl)benzonitrile as a clear oil (80 mg, 32%). MS (ESI) m / z [M+Na] + 482.55.

[0351] A solution of (S)-4-(((1-hydroxy-3-(octadecyloxy)propan-2-yl)oxy)methyl)benzonitrile (80 mg, 0.17 mmol) and triethylamine (0.08 mg, 0.8 mmol) was added to a cooled (0 °C) solution of POCl3 (30 mg, 0.20 mmol) in CHCl2 (3 mL) and stirred for 3 h. The mixture was added to acetone / ice water and stirred for 1 h before being extracted with CHCl2. (R)-2-((4-cyanobenzyl)oxy)-3-(octadecyloxy)propyl dihydrogen phosphate was isolated (80 mg, 87%) and used without further purification. MS (ESI) m / z [MH] - 538.47.

[0352] The phosphate (200 mg, 0.37 mmol) was coupled to RVn (GS-441524)-acetonide (132 mg, 0.40 mmol) using diisopropylcabodiimide (DIC, 100 mg, 0.80 mmol), N-methylimidazole (NMI, 98 mg, 1.2 mmol) in pyridine (10 mL) to give ((3aR,4R,6R,6aR)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-6-cyano-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl((R)-2-((4-cyanobenzyl)oxy)-3-(octadecyloxy)propyl)hydrogen phosphate (60 mg, 19%, MS m / z [MH]-851.55), which was then treated with formic acid (1 mL) and stirred for 6 h. Evaporation of the solvent and purification by flash column chromatography afforded ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl((R)-2-((4-cyanobenzyl)oxy)-3-(octadecyloxy)propyl)hydrogen phosphate (20 mg, 35%) as an off-white solid. 1 H NMR(400MHz,CD3OD + CDCl3) δ 7.76(s,1H,H2),7.60(d,J=8.0Hz,2H,-CH2-aryl-H3+H5),7.49(d,J=7.9Hz,2H,-CH2-aryl-H2+H6),6.9 6(d,J=4.4Hz,1H,H9),6.86(d,J=4.5Hz,1H,H8),4.80(d,J=5.3Hz,1H,H2´),4.77-4.63(m,2H,-CH2-aryl ),4.37(d,J=4.7Hz,1H,H4´),4.25(t,J=5.4Hz,1H,H3´),4.11(d,J=18.7Hz,2H,H5´),3.90(s,2H,H3´´) ,3.80-3.68(m,1H,H2´´),3.49(qd,J=10.6,4.9Hz,2H,H1´´),3.39(tt,J=6.0,3.0Hz,2H,-OCH2CH2(CH2) 15 -),1.52(p,J=6.9Hz,2H,-OCH2CH2(CH2)15 -),1.26(br s,J=5.7Hz,30H,-OCH2CH2(CH2) 15 -), 0.89(t,J=6.8Hz,3H). 13 C NMR (101MHz, CD3OD+CDCl3) δ 156.63(C6), 145.57(C2), 132.62, (-CH2-aryl-C1), 128.47(-CH2-aryl-C3+C5), 125.14(-CH2-aryl-C2+C6), 119.38(-CN), 117.29(C7), 111.95(C5), 111.28(-CN´), 84.60(C4´), 80.18(C1´), 75.38(C2´), 72.24(C2´´), 71.57(-CH2-aryl), 71.40(-OCH2CH2(CH2) 14 -),71.12(C1´´),65.75(C3´´),65.20(C5´),32.57(-OCH2CH2(CH2) 14 -),30.89-29.57(m)(m,-OCH2CH2(CH2) 14 -),26.75(-CH2CH2CH3),23.26(-CH2CH2CH3),14.21(-CH3). HRMS(ESI)m / z[MH] - C 41 H 60 N6O9P calculated value 811.4165, found value 811.4178. HPLC purity 98.6%.

[0353] Example 13b. Synthesis of 1-O-Octadecyl-2-O-(3-cyanobenzyl)-sn-glyceryl-phospho-RVn (OD(3-CN-Bn)GP-RVn) [ka] Sodium hydride (60 mg, 2.5 mmol) was added to a cooled (0° C.) solution of (R)-1-(octadecyloxy)-3-(trityloxy)propan-2-ol (540 mg, 0.92 mmol) and tetrabutylammonium iodide (180 mg, 0.5 mmol) in dry THF (30 mL). After the mixture was stirred vigorously for 20 min, 3-(bromomethyl)benzonitrile (480 mg, 2.6 mmol) was added and the mixture was then allowed to warm to ambient temperature and stirred overnight. The reaction was then quenched with ice (25 mL), diluted with ethyl ether (75 mL), washed with H2O (2×25 mL), and dried over anhydrous MgSO4. The residue was adsorbed onto silica gel and purified by flash column chromatography (gradient: 0-50% EtOAc- in hexanes) to give (R)-3-(((1-(octadecyloxy)-3-(trityloxy)propan-2-yl)oxy)methyl)benzonitrile (600 mg, 93%) as a white solid. MS (ESI) m / z [M+Na] + 724.57.

[0354] To a solution of (R)-3-(((1-(octadecyloxy)-3-(trityloxy)propan-2-yl)oxy)methyl)benzonitrile (600 mg, 0.85 mmol) in 1:1 CHCl / MeOH (30 mL) was added p-toluenesulfonate monohydrate (8 mg, 0.04 mmol) and the mixture was stirred at room temperature until deprotection was complete according to TLC analysis (ca. 3 h). Saturated aqueous NaHCO (200 mg) was added and the solvent was evaporated under vacuum. The residue was adsorbed onto silica gel and purified by flash column chromatography (gradient: 0-10% EtOAc in hexanes) to give (S)-3-(((1-hydroxy-3-(octadecyloxy)propan-2-yl)oxy)methyl)benzonitrile as a clear oil (380 mg, 97%). MS (ESI) m / z [M+H] + 460.57, [M+Na] + 482.52.

[0355] A solution of (S)-3-(((1-hydroxy-3-(octadecyloxy)propan-2-yl)oxy)methyl)benzonitrile (360 mg, 0.78 mmol) and triethylamine (340 mg, 3.0 mmol) was added to a cooled (0 °C) solution of POCl3 (140 mg, 2.0 mmol) in CHCl2 (5 mL) and stirred for 3 h. The mixture was added to acetone / ice water and stirred for 1 h before being extracted with CHCl2. Evaporation of the organic phase gave (R)-2-((3-cyanobenzyl)oxy)-3-(octadecyloxy)propyl dihydrogen phosphate (850 mg, 1.57 mmol, MS (ESI) m / z [MH] - 538.46), which was then coupled to RVn (GS-441524)-acetonide (600 mg, 1.8 mmol) using DIC (450 mg, 3.6 mmol), NMI (440 mg, 5.4 mmol) in pyridine (30 mL). Overnight reaction at 35° C., followed by chromatographic purification, gave ((3aR,4R,6R,6aR)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-6-cyano-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl((R)-2-((3-cyanobenzyl)oxy)-3-(octadecyloxy)propyl)hydrogen phosphate (360 mg, 26%) MS (ESI) m / z [M+Na] + 875.49.

[0356] The acetonide (340 mg, 0.39 mmol) was added to formic acid (7 mL) at room temperature and stirred for 3 h. Evaporation of the solvent and purification by flash column chromatography afforded ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl((R)-2-((3-cyanobenzyl)oxy)-3-(octadecyloxy)propyl)hydrogen phosphate (290 mg, 90%) as an off-white solid. 1H NMR(400MHz,CD3OD+CDCl3) δ 7.71(s,1H,H2),7.70(s,1H,-CH2-aryl-H2),7.62(d,J=7.8Hz,1H,-CH2-aryl-H4),7.56(d,J=7.8Hz,1H,-CH2-aryl-H5),7.44(t,J=7. 6Hz,1H,-CH2-aryl-H6),6.96(d,J=4.5Hz,1H,H9),6.86(d,J=4.5Hz,1H,H8),4.72(d,J=5.3Hz,1H,H2´),4.72(d,J=12.4Hz,1H,-CH2-ar yl),4.65(d,J=12.4Hz,1H,-CH2-aryl),4.39(q,J=4.4Hz,1H,H4´),4.27(t,J=5.5Hz,1H,H3´),4.10(d,J=11.5Hz,1H,H5´),3.89(d,J= 11.2Hz,1H,H5´),3.78(m,2H,H3´´),3.71(q,J=5.0Hz,1H,H2´´),),3.51-3.40(m,2H,H1´´),3.41(tt,J=5.5,2.7Hz,2H,-OCH2CH2(CH2) 15 -),1.54(p,J=6.6Hz,2H,-OCH2CH2(CH2) 15 -),1.26(br s,30H,-OCH2CH2(CH2) 15 ), 0.89(t,3H,-CH3). 13 C NMR (101MHz, CD3OD+CDCl3) δ 156.64(C6), 147.68(C2), 141.50(-CH2-aryl-C1), 132.77(-CH2-aryl-C2), 131.73(-CH2-aryl-C4), 131.57(-CH2-aryl-C6), 129.88(-CH2-aryl-C5), 125.25(C7), 119.50(C5), 117.41(-CN), 112.62(-CN´), 111.98(C8), 102.47(C9), 84.91(C4´), 80.07(C1´), 75.57(C2´´), 72.40(-CH2-aryl), 71.58(-OCH2CH2(CH2) 14 -),71.44(C1´´),71.23(C3´),65.73(C3´´),65.21(C5´),32.67(-OCH2CH2(CH2) 14-),31.02-29.33(m,-OCH2CH2(CH2) 14 -),26.85(-CH2CH2CH3),23.37(-CH2CH2CH3),14.39(-CH3). HRMS(ESI)m / z[MH] - C 41 H 60 N6O9P calculated value 811.4165, found value 811.4178. HPLC purity 98.6%.

[0357] Example 13c. Synthesis of 1-O-Octadecyl-2-O-(2-cyanobenzyl)-sn-glyceryl-phospho-RVn (OD(2-CN-Bn)GP-RVn) [ka] Sodium hydride (60 mg, 2.5 mmol) was added to a cooled (0° C.) solution of (R)-1-(octadecyloxy)-3-(trityloxy)propan-2-ol (587 mg, 1.0 mmol) and tetrabutylammonium iodide (180 mg, 0.5 mmol) in dry THF (30 mL). After the mixture was stirred vigorously for 20 min, 2-(bromomethyl)benzonitrile (455 mg, 2.5 mmol) was added and the solution was then allowed to warm to ambient temperature overnight. The reaction was then quenched with ice (25 mL), diluted with ethyl ether (75 mL), washed with H2O (2×25 mL), and dried over anhydrous MgSO4. The residue was adsorbed onto silica gel and purified by flash column chromatography (gradient: 0-50% EtOAc- in hexanes) to give (R)-2-(((1-(octadecyloxy)-3-(trityloxy)propan-2-yl)oxy)methyl)benzonitrile (580 mg, 83%) as a white solid. MS (ESI) m / z [M+Na] + 724.62.

[0358] To a solution of (R)-2-(((1-(octadecyloxy)-3-(trityloxy)propan-2-yl)oxy)methyl)benzonitrile (580 mg, 0.83 mmol) in 1:1 CHCl / MeOH (15 mL) was added p-toluenesulfonate monohydrate (8 mg, 0.04 mmol) and the mixture was stirred at room temperature until deprotection was complete according to TLC analysis (approximately 3 h). Saturated aqueous NaHCO (200 mg) was added and the solvent was evaporated under vacuum. The residue was adsorbed onto silica gel and purified by flash column chromatography (gradient: 0-10% EtOAc in hexanes) to give the compound (S)-2-(((1-hydroxy-3-(octadecyloxy)propan-2-yl)oxy)methyl)benzonitrile as a clear oil (380 mg, 97%). MS (ESI) m / z [M+H] + 460.57, [M+Na] + 482.52.

[0359] A solution of (S)-2-(((1-hydroxy-3-(octadecyloxy)propan-2-yl)oxy)methyl)benzonitrile (360 mg, 0.78 mmol) and triethylamine (340 mg, 3.0 mmol) was added to a cooled (0 °C) solution of POCl3 (140 mg, 2.0 mmol) in CHCl2 (5 mL) and stirred for 3 h. The mixture was added to ice water and stirred for 1 h before being extracted with CHCl2. Evaporation of the organic phase gave the phosphate (R)-2-((2-cyanobenzyl)oxy)-3-(octadecyloxy)propyl dihydrogen phosphate (850 mg), which was used without further purification. MS (ESI) m / z [MH] - 538.46.

[0360] The phosphate (460 mg, 0.85 mmol) was coupled to RVn (GS-441524)-acetonide (280 mg, 0.85 mmol) using DIC (210 mg, 1.7 mmol), NMI (140 mg, 1.7 mmol) in dry pyridine (15 mL). Reaction at 35 °C for 23 h, followed by chromatographic purification, yielded ((3aR,4R,6R,6aR)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-6-cyano-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl((R)-2-((2-cyanobenzyl)oxy)-3-(octadecyloxy)propyl)hydrogen phosphate (190 mg, 22%) MS (ESI) m / z [M+H] + 851.47.

[0361] The coupled acetonide (190 mg, 0.22 mmol) was added to formic acid (5 mL) and stirred for 3 h. The formic acid was evaporated and the residue was purified by flash column chromatography to give ((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl((R)-2-((2-cyanobenzyl)oxy)-3-(octadecyloxy)propyl)hydrogen phosphate as an off-white solid (120 mg, 67%). 1<h2 style=";text-align:left;direction:ltr">H NMR(400MHz,CD3OD+CDCl3) δ 7.73(s,1H,H2),7.65(d,J=7.6,Hz,2H,-CH2-aryl-H3+H5),7.59(td,J=7.6,1.3Hz,1H,H4),7.39(td,J=7 .6,1.3Hz,1H,H6),6.97(d,J=4.6Hz,1H,H9),6.86(d,J=4.5Hz,1H,H8),4.81(d,J=5.7Hz,1H,H2´),4.38(q ,J=4.2Hz,1H,H4´),4.28(t,J=5.3Hz,1H,H3´),4.19-3.84(m,2H,H5´),3.93(tp,J=10.7,4.7Hz,2H,H3´´),3.85-3.70(m,1H,H2´´),3.54(qd,J=10.6,5.0Hz,2H,H1´´),3.41(td,J=6.6,2.7Hz,2H,-OCH2CH2(CH2)<h2 style=";text-align:left;direction:ltr"> 15 <h2 style=";text-align:left;direction:ltr"> -),1.52(p,J=6.6Hz,2H,-OCH2CH2(CH2)<h2 style=";text-align:left;direction:ltr"> 15 <h2 style=";text-align:left;direction:ltr"> -),1.26(m,J=7.2Hz,30H,-OCH2CH2(CH2)<h2 style=";text-align:left;direction:ltr"> 15 <h2 style=";text-align:left;direction:ltr"> -),0.89(t,3H,-CH3).<h2 style=";text-align:left;direction:ltr"> 13 <h2 style=";text-align:left;direction:ltr"> C NMR(101MHz,CD3OD+CDCl3)δ156.53(C6),147.49(C2),142.96(-CH2-アリール-C1),133.62,(-C H2-アリール-C3),133.23,(-CH2-アリール-C6),129.66,(-CH2-アリール-C5),128.68,(-CH2-アリール-C4), 125.15,(C7),117.85,(C5),117.26,(-CN),111.84,(-CN´),111.58,(C8),102.35,(C9),84. 75,(C4´),79.89(C1´),78.99(C2´),75.45(C2´´),72.21(-CH2-アリール),71.30(OCH2CH2(CH2)<h2 style=";text-align:left;direction:ltr"> 14 <h2 style=";text-align:left;direction:ltr"> -),71.09(C1´´),70.45(C3´),65.73(C3´´),65.03(C5´),32.51(-OCH2CH2(CH2)<h2 style=";text-align:left;direction:ltr"> 14 <h2 style=";text-align:left;direction:ltr"> -),31.09-29.39(m,-OCH2CH2(CH2)<h2 style=";text-align:left;direction:ltr"> 14-),26.67,(-CH2CH2CH3),23.21(-CH2CH2CH3),14.19(-CH3). HRMS(ESI)m / z[M+H] + C 41 H 62 N6O9P calculated value 813.4310, found value 8813.4302. HPLC purity 99.4%. [Table 26]

Claims

1. The following compounds or pharmaceutically acceptable salts thereof. 【Chemistry 1】

2. Use of the compound described in Claim 1 or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical formulation for use in a method for treating coronavirus infection in mammals, The method includes administering an effective amount of the pharmaceutical preparation to the mammal, The aforementioned pharmaceutical preparation contains oil, and is used.

3. The use according to claim 2, wherein the effective amount of the pharmaceutical preparation is administered as a single dose.

4. The use according to claim 2, wherein the pharmaceutical preparation comprises preparation F8 or preparation F7 as described herein.

5. Use of the compound described in Claim 1 or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical formulation for use in a method for treating HIV infection in mammals, The method includes administering an effective amount of the pharmaceutical preparation to the mammal, The aforementioned pharmaceutical preparation contains oil, and is used.

6. The use according to claim 5, wherein the pharmaceutical preparation is a long-acting therapeutic agent.

7. The use according to claim 5, wherein the pharmaceutical preparation comprises preparation F3 or preparation F4 as described herein.

8. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical formulation for use in a method for treating and / or inhibiting the replication of respiratory syncytial virus (RSV) in mammals, The method includes administering an effective amount of the pharmaceutical preparation to the mammal, The aforementioned pharmaceutical preparation contains oil, and is used.

9. Use of the compound described in Claim 1 or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical formulation for use in a method for treating viral infections in mammals, The method includes administering an effective amount of the pharmaceutical preparation to the mammal, The aforementioned virus is an RNA virus belonging to a viridae family selected from the group consisting of Filoviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, Phenuiviridae, Nairoviridae, Arenaviridae, Flaviviridae, and Coronaviridae. The aforementioned pharmaceutical preparation contains oil, and is used.

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