Nucleoside and nucleotide analogs as antiviral agents and uses therof

Abstract

The present invention provides a compound having the structure: wherein Base, R1-R5, n, m, X, Y and Z are disclosed herein.

Description

Docket: 92532-A-PCT / GJG / YXNUCLEOSIDE AND NUCLEOTIDE ANALOGS AS ANTIVIRAL AGENTS AND USES THEROF

[0001] Throughout this application, various publications are referenced, including referenced in parenthesis. The disclosures of all publications mentioned in this application in their entireties are hereby incorporated by reference into this application in order to provide additional description of the art to which this invention pertains and of the features in the art which can be employed with this invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U. S. Provisional Application No. 63 / 759,950 filed February 18, 2025, the contents of which is hereby incorporated by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under AI171401 awarded by National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0004] Positive-stranded RNA (+ssRNA) viruses are a large and diverse group belonging to three broad phyla, whose genomes act as mRNAs for direct translation by host ribosomes. They utilize RNA-dependent RNA polymerases (RdRps) to replicate their genomes.

[0005] RNA-dependent RNA polymerase (RdRp), the enzyme responsible for RNA synthesis in positive-sense RNA viruses, is one of the most conserved nonstructural proteins in these viruses (Venkataraman 2018; Gao 2020; Jia 2019; Kandwal 2023; and te Velthuis 2014). It is a low fidelity enzyme with no counterparts in human cells, making it an ideal target for antiviral therapeutics. Nucleoside / nucleotide analogs are among the most important RdRp inhibitors. Their active triphosphate forms compete with natural nucleoside triphosphates for RdRp incorporation during viral RNA synthesis. Once incorporated, nucleotide analogs can lead to chain termination or reduction of viral RNA synthesis, as well as lethal mutations, thus hindering viral replication in the host. Because these nucleoside / nucleotide analogs interact with the enzyme’s highly conserved active site, they have broad-spectrum antiviral potential (Jockusch 2020; Shannon 2023; Kabinger 2021; Barik 2022; Hillen 2020; Chien 2020; Ju 2020; and Jiang 2021).

[0006] The SARS-CoV-2 RdRp complex, the fastest viral RdRp characterized to date, consists of the main functional nonstructural protein 12 (Nspl2) along with Nsp7 and two Nsp8 proteins acting as cofactors (Hillen 2020 and Seifert 2021). Due to the large viral genome (~ 30kb) and the low fidelity RdRp, SARS-CoV-2 also possesses a proofreading exonuclease complex (ExoN, consisting of Nspl4 and NsplO)to remove mis-incorporated nucleotides as well as nucleotide analogs in the replicating RNA to maintain its genome integrity (Moeller 2022). This poses a major challenge to nucleotide analog-based drug development for coronaviruses. For example, studies have revealed that Remdesivir, the first FDA-approved nucleotide analog drug against COVID- 19, has no ExoN resistance, partially undermining its antiviral activity and clinical efficacy (Gordon 2020; Kokic 2020; and Jockusch 2020).

[0007] Azvudine is a cytidine analog carrying a 2'-deoxy-2'-p-fluoro-4'-azido modified ribose that targets viral polymerases and has demonstrated broad-spectrum antiviral activity against human immunodeficiency virus, hepatitis C virus and hepatitis B virus (Wang 2021; Wang 2014: Ren 2020). Recent clinical trials suggest that Azvudine exhibits anti-SARS-CoV-2 activity with efficacy comparable to the FDA-approved drug Paxlovid, leading to its conditional authorization for COVID- 19 treatment in China (Sun 2023; Wu 2021; and Yu 2020). An early study revealed that Azvudine homes to the thymus and functions as an effective COVID-19 antiviral drug (Wu 2021). Azvudine has also been shown to have an anti-inflammatory activity in SARS-CoV-2 infected rhesus macaques (Li 2024). However, the detailed enzymatic mechanisms of Azvudine inhibition of SARS-CoV-2 have not been determined.

[0008] The coronaviruses, such as SARS-CoV-2, due to their large genome and error-prone RdRp also possesses a 3 ’-5’ exonuclease (ExoN) proofreading function to remove mis-incorporated nucleotides from the replicating RNA thereby maintaining their genome integrity (Zhu 2020; V’kovski P 2021; and Shannon 2020). To serve as potential coronavirus therapeutics, nucleotide / nucleoside inhibitors must be able to compete sufficiently with natural nucleoside triphosphates in vivo and either (1) resist removal by ExoN, (2) be combined with ExoN inhibitors, or (3) result in lethal viral mutagenesis.

[0009] The replication complex of coronaviruses consists of several viral proteins, including the RdRp (Nspl2) and its two accessory proteins (Nsp7 and Nsp8), and the exonuclease (Nspl4) with its accessory protein (NsplO) (Kirchdoerfer RN 2019; Gao Y 2020: Hillen 2020; and Bouvet M 2012).

[0010] Unlike the coronaviruses, the +ssRNA flaviviruses and alphaviruses, such as dengue vims (DENV) and Chikungunya vims (CHIKV), respectively, have much smaller genomes with fewer viral proteins, and lack the exonuclease activity. This would reduce the challenge of developing nucleotide-based drugs for these viruses.

[0011] DENV and CHIKV, growing threats to global human health, utilize their RdRps (NS5 in DENV and nsP4 in CHIKV) to synthesize the viral genomes (te Velthuis AJ 2014 and Radoshitzky SR 2023). While a vaccine has been developed for DENV, it cannot be used in a large percent of the population. There is a concerted effort to repurpose or develop dmgs for DENV and other flaviviruses. While several of these have some antiviral activity in vitro, there are no approved dmgs for these diseases. Among thosestudied, several are nucleotide inhibitors of RdRp including RDV, Balapiravir, Sofosbuvir and AT-752, while others are non-nucleoside / non -nucleotide small molecules that target the NS5 or host functions (Radoshitzky SR 2023; Gordon CJ 2021; Nguyen NM 2013; Xu HT 2017; Good SS 2021; Shimizu H 2019; Lim SP 2016; Barbosa-Lima G 2020 and Lu G 2017). There are also reports on molecules that inhibit the NS5 RdRp of other flaviviruses such as the Zika virus (ZIKV) and the yellow fever virus (YFV) (Bluemling GR 2017 and LeCher J. C 2022). CHIKV belongs to the +ssRNA alphavirus genus and causes the disease Chikungunya that can often lead to severe chronic arthralgia and occasionally to neurologic disorders (Skidmore AM 2023). There is no effective vaccine or drug treatment for CHIKV, so development of therapeutics is a high priority.

[0012] DENV and CHIKV have small genomes compared to SARS-CoV-2 and no exonuclease proofreading function. Moreover, DENV and CHIKV have similar RdRp active sites as SARS-CoV-2 (Radoshitzky 2023, Pathania S 2022 and Jia 2019). Since Remdesivir (RDV) has broad spectrum antiviral activity in in vitro assays against many +ssRNA viruses, the modifications of nucleotide prodrugs of RDV analogs, with rational design, might lead to ideal inhibitors of the flaviviruses (e.g., DENV, YFV, ZIKV) and alphaviruses (e g., CHIKV, EEEV) RdRps (Radoshitzky SR 2023: Gordon CJ 2021; Pathania S 2022: Jia H 2019; Noble CG 2022, and Hucke FI 2020).BRIEF SUMMARY OF THE INVENTION[00131 The present disclosure provides a compound having the structure:whereinBASE is adenine, guanine, cytosine, thymine, uracil or derivatives thereof;[A]R₁, R₂, R₃, R₄, R₅ and R₆ are each independently -H, halogen, -CN, -N₃, -NH₂, -OH, -CHF₂, -CH₂F, -CF₃, -OCHF₂, -OCF₃, -OCH₂F, -NO₂, -OAc, -COOH, alkyl, alkenyl, alkynyl, -OR₁₃, -COR₁₃, -SH, -SR₁₃, -SO₂R₁₃, -NHR₁₃, -NR₁₄R₁₅, -NHCOR₁₃, or -CONR₁₄R₁₅,wherein R₁₃, R₁₄, and R₁₅ are each independently -H, alkyl, alkenyl, alkynyl, aldehyde, ketone, ester, ether, carboxylate, aryl, or heteroaryl;n is 0-10;Z is -O-, -CH₂-, -NH-, or -S-; and[B](a) R₁ is -CN, R₂ is -OH, R₄ is -NH₂, and R₃, R₅ and R₆ are each independently -H, halogen, -CN, -N₃, -NH₂, -OH, -CHF₂, -CH₂F, -CF₃, -OCHF₂, -OCF₃, -OCH₂F, -NO₂, -OAc, -COOH, alkyl, alkenyl, alkynyl, -OR₁₃, -COR₁₃, -SH, -SR₁₃, -SO₂R₁₃, -NHR₁₃, -NR₁₄R₁₅, -NHCOR₁₃, or -CONR₁₄R₁₅,(b) R₁ is -CN, R₃ is -F, R₄ is -OH, R₆ is -N₃, and R₂ and R₅ are each independently -H, halogen, -CN, -N₃, -NH₂, -OH, -CHF₂, -CH₂F, -CF₃, -OCHF₂, -OCF₃, -OCH₂F, -NO₂, -OAc, -COOH, alkyl, alkenyl, alkynyl, -OR₁₃, -COR₁₃, -SH, -SR₁₃, -SO₂R₁₃, -NHR₁₃, -NR₁₄R₁₅, -NHCOR₁₃, or -CONR₁₄R₁₅, or(c) R₁ is -CN, R₂ is -OH, R₄ is -F, and R₃, R₅ and R₆ are each independently -H, halogen, -CN, -N₃, -NH₂, -OH, -CHF₂, -CH₂F, -CF₃, -OCHF₂, -OCF₃, -OCH₂F, -NO₂, -OAc, -COOH, alkyl,alkenyl, alkynyl, -OR₁₃, -COR₁₃, -SH, -SR₁₃, -SO₂R₁₃, -NHR₁₃, -NR₁₄R₁₅, -NHCOR₁₃, or -CONR₁₄R₁₅,n is 0-10;Z is -O-, -CH₂-, -NH-, or -S-; andY is -OH, R?, or;wherein R₇, R₈, R₉, R₁₀ are each independently halogen, -CN, -N₃, -NH₂, -OH, -CHF₂, -CH₂F, -CF₃, -OCHF₂, -OCF₃, -OCH₂F, -NO₂, -OAc, -COOH, alkyl, alkenyl, alkynyl, -OR₁₃, -COR₁₃, -SH, -SR₁₃, -SO₂R₁₃, -NHR₁₃, -NR₁₄R₁₅, -NHCOR₁₃, -CONR₁₄R₁₅, or HO-P(O)-POH OH wherein R₁₃, R₁₄, and R₁₅ are each independently -H, alkyl, alkenyl, alkynyl, aldehyde, ketone, ester, ether, carboxylate, aryl, or heteroaryl; and t is 0-10; andn is 0-10;or a pharmaceutically acceptable salt or ester thereof.

[0014] Hie present disclosure provides a compound having the structure:whereinBASE is adenine, guanine, cytosine, thymine, uracil or derivatives thereof;(a) R₁ is -CN, R₂ is -OH, R₄ is -NH₂, and R₃, R₅ and R₆ are each independently -H, halogen, -CN, -N₃, -NH₂, -OH, -CHF₂, -CH₂F, -CF₃, -OCHF₂, -OCF₃, -OCH₂F, -NO₂, -OAc, -COOH, alkyl, alkenyl, alkynyl, -OR₁₃, -COR₁₃, -SH, -SR₁₃, -SO₂R₁₃, -NHR₁₃, -NR₁₄R₁₅, -NHCOR₁₃, or -CONR₁₄R₁₅,(b) R₁ is -CN, R₃ is -F, R₄ is -OH, R₆ is -N₃, and R₂ and R₅ are each independently -H, halogen, -CN, -N₃, -NH₂, -OH, -CHF₂, -CH₂F, -CF₃, -OCHF₂, -OCF₃, -OCH₂F, -NO₂, -OAc, -COOH, alkyl, alkenyl, alkynyl, -OR₁₃, -COR₁₃, -SH, -SR₁₃, -SO₂R₁₃, -NHR₁₃, -NR₁₄R₁₅, -NHCOR₁₃, or -CONR₁₄R₁₅,(c) R₁ is -CN, R₂ is -OH, R₄ is -F, and R₃, R₅ and R₆ are each independently -H, halogen, -CN, -N₃, -NH₂, -OH, -CHF₂, -CH₂F, -CF₃, -OCHF₂, -OCF₃, -OCH₂F, -NO₂, -OAc, -COOH, alkyl, alkenyl, alkynyl, -OR₁₃, -COR₁₃, -SH, -SR₁₃, -SO₂R₁₃, -NHR₁₃, -NR₁₄R₁₅, -NHCOR₁₃, or -CONR₁₄R₁₅,(d) R₃ is -F, R₄ is -OH, R₆ is -N₃, and R₁, R₂ and R₅ are each independently -H, halogen, -CN, -N₃, -NH₂, -OH, -CHF₂, -CH₂F, -CF₃, -OCHF₂, -OCF₃, -OCH₂F, -NO₂, -OAc, -COOH, alkyl, alkenyl, alkynyl, -OR₁₃, -COR₁₃, -SH, -SR₁₃, -SO₂R₁₃, -NHR₁₃, -NR₁₄R₁₅, -NHCOR₁₃, or -CONR₁₄R₁₅,NH2(e) BASE is [structure], R₂ is -OH, and R₁, R₃, R₄, R₅, and R₆ are each independently -H, halogen, -CN, -N₃, -NH₂, -OH, -CHF₂, -CH₂F, -CF₃, -OCHF₂, -OCF₃, -OCH₂F, -NO₂, -OAc, -COOH, alkyl, alkenyl, alkynyl, -OR₁₃, -COR₁₃, -SH, -SR₁₃, -SO₂R₁₃, -NHR₁₃, -NR₁₄R₁₅, -NHCOR₁₃, or -CONR₁₄R₁₅, or(f) BASE is [structure], R₁ is -CN, R₂ is -OH, R₄ is -OH, and R₃, R₅ and R₆ are each independently -H, halogen, -CN, -N₃, -NH₂, -OH, -CHF₂, -CH₂F, -CF₃, -OCHF₂, -OCF₃, -OCH₂F, -NO₂, -OAc, -COOH, alkyl, alkenyl, alkynyl, -OR₁₃, -COR₁₃, -SH, -SR₁₃, -SO₂R₁₃, -NHR₁₃, -NR₁₄R₁₅, -NHCOR₁₃, or -CONR₁₄R₁₅,wherein R₁₃, R₁₄, and R₁₅ are each independently -H, alkyl, alkenyl, alkynyl, aldehyde, ketone, ester, ether, carboxylate, aryl, or heteroaryl;n is 0-10;Z is -O-, -CH₂-, -NH-, or -S-; andO11 HO- P- Yis -OH, OHwherein when Y is -OH,(i) then BASE is [structure], R₁ is -CN; R₂ is -OH, R₄ is -NH₂, R₃, R₅, R₆ are each -H, n is 0 and Z is -CH₂-, or(ii) then BASE is [structure], R₁ is -CN; R₃ is -F, R₄ is -OH, R₆ is -N₃, R₂ and R₅ are each -H, n is 0 and Z is -CH₂-; andherein when Y is OH(iii) then BASE is [structure], R₁ is -CN; R₂ is -OH, R₄ is -NH₂, R₃, R₅, R₆ are each -H, n is 1 and Z is -CH₂-;H, NL2 N- N(iv) then BASE is [structure], R₁ is -CN; R₂ is -OH, R₃, R₄, R₅, R₆ are each -H, n is 1 and Z is -CH2-;'2(v) then BASE is [structure], R₂ is -OH, R₁, R₃, R₄, R₅, R₆ are each -H, n is 1 and Z is -CH₂-;NH2(vi) then BASE is [structure], R₃ is -F; R₄ is -OH, R₆ is -N₃, R₁, R₂, R₅ are each -H, n is 1 andZ is -CH2-; or a pharmaceutically acceptable salt or ester thereof.

[0015] The present disclosure provides a compound having structure:BASE is adenine, guanine, cytosine, uracil, thymine or derivatives thereof;m is 0-10: andX is halogen, -CN, -N₃, -NH₂, -OH, -CHF₂, -CH₂F, -CF₃, -OCHF₂, -OCF₃, -OCH₂F, -NO₂, -OAc, -COOH, alkyl, alkenyl, alkynyl, -OR₁₃, -COR₁₃, -SH, -SR₁₃, -SO₂R₁₃, -NHR₁₃, -NR₁₄R₁₅, -NHCOR₁₃, or -CONR₁₄R₁₅,wherein R₁₃, R₁₄, and R₁₅ are each independently -H, alkyl, alkenyl, alkynyl, aldehyde, ketone, ester, ether, carboxylate, aryl, or heteroaryl,or a pharmaceutically acceptable salt or ester thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Fig 1. Models of ATP (A) and Tenofovir derivatives (B-E) in the pre-incorporation state of the SARS-CoV-2 RdRp. Molecular modeling of (A) ATP and (B) TFV-DP was reported

[0036] , Structures of each compound shown on the left. This study indicates that removing the -CH, group or replacing it with a more polar -CH2X (e.g. -CH2F) in a configuration avoids clashes with key amino acids in the active sites, making it a better polymerase substrate.

[0017] Fig. 2 (A) Structures of A and.8 isomers of F-TFV-DP; (B) Synthesis of TFV analogs (X = F, CN, Ns, NH2, OH or other related groups).

[0018] Fig. 3 The R isomer of F-TFV-DP (b) is incorporated > 10-fold better than the 5 isomer (c) by SARS-CoV-2 RdRp. The RNA substrate (sequence shown at the top of the figure) was mixed with SARS-CoV-2 RdRp and incubated with three different nucleotides [a, ATP; b. (R) F-TFV-DP: c, (S) F-TFV-DP] to produce extension products. The results were analyzed by MALDI-TOF MS. When the RNA template-loop-primer was incubated with 0.5 pM ATP, an extension product at 8200 Da was observed (a), indicating single base extension. (R) F-Tenofovir-DP is readily incorporated into the primer at a concentration of 64 pM (b) while (S) F-Tenofovir-DP is barely incorporated into the primer at 256 pM (c). This result clearly shows that the (R) F-Tenofovir-DP is incorporated much more efficiently than the corresponding S isomer.

[0019] Fig. 4 Three RNAs (a, A-RNA; b, (R) F-TFV-RNA; c, TFV-RNA) were produced by polymerase incorporation of ATP, (R) F-TFV-DP and TFV-DP at the 3‘ ends of the RNA substrates, respectively. (R) F-TFV-RNA (b) and TFV-RNA (c) display ~3x better protection against SARS-CoV-2 ExoN excision than A-RNA (a). The intact RNAs and the products of the ExoN reactions were analyzed by MALDI-TOF MS. The cleavage products are shown in the lower panels, represented by peaks with lower MWs. Less cleavage was observed for (R) F-TFV (e) and TFV (f) terminated RNAs relative to the natural RNA strand (d).

[0020] Fig.5 Example synthesis of disoproxil and alafenamide prodrugs of (R) X-TFV or (S) X-TFV, (X = F, CN, Ns, NH2, OH or other related groups)

[0021] Fig.6 Azvudine-TP is incorporated by SARS-CoV-2 RdRp ~4 times less efficiently than CTP. The reaction w as performed using similar conditions as in Fig.3. When the RNA template-loop-primer w as incubated with 0.8 pM CTP. an extension product at 8191 Da was observed (a). At the same concentration, the Azvudine incorporated peak (8236 Da) (b) was ~ 4 times lower than that of CTP.

[0022] Fig.7 RNA extended by Azvudine-TP (Azv-RNA) significantly inhibits subsequent nucleotide incorporation by RdRp. RNA substrates w'ith C or Azv at the 3’ ends (C-RNA and Azv-RNA) (position z) were incubated with SARS-CoV-2 RdRp and ATP. ATP incorporation to position z+7 on C-RNA (a, b) ismore efficient than its incorporation on Azv-RNA (c, d) at both 0.4 pM and 1.6 pM based on MALDI-TOF MS analysis.

[0023] Fig. 8 Azvudine (Azv) terminated RNA (Azv-RNA) has no resistance to SARS-CoV-2 ExoN, similar to natural RNA (C-RNA) (a, b). A mixture of RNAs extended by CTP and Azv-TP at the same concentration (a) were incubated with ExoN, yielding the cleavage profile in (b). The similar decrease of both RNA substrate peaks indicates the absence of ExoN excision protection.

[0024] Fig.93 ’-deoxy ATP is incorporated by SARS-CoV-2 RdRp ~8 times less efficiently than ATP based on MALDI-TOF MS analysis. When the RNA template-loop-primer was incubated with 0.5 pM ATP, an extension product at 8201 Da was observed (a). 4 pM 3’-dATP is needed to achieve a similar incorporation percentage as 0.5 pM ATP (b).

[0025] Fig. 10 Synthesis of isobutyric ester and alafenamide prodrugs, and triphosphate of I’-CN-Azvudine (A) and synthesis of isobutyric ester and alafenamide prodrugs, and triphosphate of 3’-deoxy-RDV (B)

[0026] Fig. 11 Synthesis of l’-cyano-3’-deoxy-5’-CH2-4-aza-7,9-dideaza-adenosine C-nucleotide (5’-CH2-3’ -deoxy RDV) phosphoramidate prodrug (A) and disoproxil prodrug (B).

[0027] Fig. 12 Synthesis of disoproxil (A) and alafenamide (B) prodrugs of 5’-CH2-RDV and triphosphate of 5’-CH2-RDV (C).

[0028] Fig. 13 Synthesis of isobutyric ester (A) and ProTide (B) prodrugs of 3’-NH2-RDV and triphosphate of 3’-NH2-RDV (C).

[0029] Fig. 14 Delayed chain termination of SARS-CoV-2 RdRp reaction by 5’-CH2-RDV-TP. The SARS-CoV-2 RdRp reaction results indicate that 5’-CH2-RDV-TP acts as a delayed chain terminator. Three RNA substrates extended by (a) ATP (A-RNA), (b) 5’-CH2-RDV-TP (CFL-RDV-RNA) and (c) RDV-TP (RDV-RNA) at initial position (z) were produced as the starting RNA substrates. Their relative abilities to be extended by RdRp were evaluated by mixing them with SARS-CoV-2 RdRp and UTP / GTP / ATP. and the final RNA products were analyzed by MALDI-TOF MS. (d) For A-RNA, the major product is an extended RNA with 5 nucleotides incorporated (z+5). (e) The major extension product for 5’-CH2-RDV-RNA is an extended RNA with 3 nucleotides incorporated (z— 3). Thus, fewer nucleotides were incorporated by RdRp compared to the natural A-RNA, indicating that 5’-CH2-RDV-TP has delayed RNA chain termination activity, (f) RDV-RNA showed similar delayed chain termination activity with the major extension product at z+3 followed by z+4.

[0030] Fig. 15 Incorporation of active triphosphate form of 3 -NH -RDV into RNA by SARS-CoV-2 RdRp. A reaction consisting of SARS-CoV-2 RdRp, RNA substrate, and 3’-NH2-RDV-TP resulted in extension of the RNA by the 3’-NH2-Remdesivir-TP. The starting RNA (MW 7873 Da) and the extended RNA (8226 Da) are shown in the MALDI-TOF mass spectrum.

[0031] Fig. 16 Synthesis of disoproxil (A) and alafenamide (B) prodrugs of 3’-NH2-5’-CH2-RDV and triphosphate of 3’-NH2-5’-CH2-RDV (C).

[0032] Fig. 17 Synthesis of disoproxil (A) and alafenamide (B) prodrugs of 3’-F-5’-CH2-RDV and triphosphate of 3’-F-5’-CH2-RDV (C).

[0033] Fig 18. 3’-dATP has a similar DENV-2 RdRp incorporation ability as ATP and better than that of RDV. Hie RNA substrate (sequence shown at the top of the figure) was mixed with DENV-2 RdRp and incubated with three different nucleotides [a. ATP: b, RDV-TP; c, 3’-dATP] to produce extension products. The results were analyzed by MALDI-TOF MS. When the RNA template-loop-primer was incubated with 1 pM ATP, an extension product at 8197 Da was observed (a). At the same concentration, 3’-dATP was incorporated into the RNA strand as efficiently as ATP and better than RDV-TP (b, c).

[0034] Fig. 19 Synthesis of disoproxil and alafenamide prodrugs (A) and triphosphate of 5’-CH2-3’dA (B).

[0035] Fig. 20 Synthesis of isobutyryl ester and alafenamide prodrugs of 3'-deoxyA

[0036] Fig. 21 Synthesis of disoproxil and alafenamide prodrugs of 5’-CH2-Azvudine.

[0037] Fig.22 The active form of 3’-NH2-RDV prodrug (3’-NH2-RDV-TP) is aDENV RdRp reaction terminator. The present disclosure cloned the DENV-2 RdRp and found that 3’-NH2-RDV-TP terminates the RdRp reaction using MALDI-TOF-MS assay.

[0038] Fig 23 ExoN resistance of Azv-RNA+1 is not sequence specific, (a) Annealed RNA template-loop-primer substrate (top) and RNA products after RdRp reaction (bottom), (b) MS analysis of time-dependent CTP and Azv-TP incorporation by RdRp. Incubation time is indicated to the left of each figure. Azv-TP can be incorporated by RdRp in the presence of CTP with 2- to 3-fold lower incorporation when the ratio between CTP and Azv-TP is 1:40.

[0039] Fig. 24 Azv hinders RNA extension by SARS-CoV-2 RdRp. (a) Annealed RNA template-loop-primer substrates (top) and RNA products (bottom) after RdRp reaction. MS analysis of ATP incorporation by RdRp into C-RNA (b) or Azv-RNA (c) at different time points noted on the left of each figure. This result shows that Azv hinders next base incorporation 2-3-fold at all time points.

[0040] Fig. 25 The incorporated Azv reduces RNA synthesis, (a) Annealed Azv- or C-extended RNA substrate (top) and the fully extended RNA products (bottom) after RdRp reaction. MS analysis of continuous nucleotide incorporation of natural RNA (b) or Azv-RNA (c) by RdRp with an excess of NTPs. Incorporation of Azv reduces further RNA elongation when compared to natural RNA elongation, shown by the remaining 20% Azv-RNA (-8233 Da) after the RdRp reaction. Percentage of remaining starting material is shown by the corresponding peaks of each spectrum.

[0041] Fig. 26 ExoN excision of Azv-RNA and fully extended Azv-embedded RNA. a Azv- or C-RNA substrate. ExoN excision direction is indicated by the arrow, b MS analysis of the RNA mixture before (-ExoN) and after ExoN reaction. Azv-RNA (-8234 Da) showed no ExoN resistance, c frilly extended natural RNA substrate, d MS analysis of natural RNA ExoN excision as a control showed no ExoN resistance, e fully extended Azv-embedded RNA substrate, f ExoN excision of fully extended Azv-embedded RNA stops at Azv+1 position generating Azv-RNA+A (-8572 Da) which almost completely resists ExoN. Percentage of ExoN resistance of Azv-RNA and Azv-RNA+A are shown by the corresponding peaks in the MS figure.

[0042] Fig. 27 Strong ExoN resistance of Azv-RNA+1 with A at the i+1 position, a Natural C-RNA+A used as a control, b Azv-RNA+A substrate. ExoN excision direction is indicated by the arrow, c MS analysis of natural C-RNA+A (-8523 Da) before (-ExoN) and after ExoN reaction indicating no ExoN resistance, d MS analysis of Azv-RNA+A before (-ExoN) and after ExoN reaction showing that Azv-RNA+A (~8568Da) has strong ExoN resistance.

[0043] Fig. 28 ExoN resistance of Azv-RNA+1 is not sequence specific, a Natural C-RNA+A used as control, b Azv-RNA+C substrate, c Azv-RNA+U substrate, d Azv-RNA+GG substrate, e MS analysis of natural C-RNA+A (-8522 Da) before (-ExoN) and after ExoN reaction, f MS analysis of Azv-RNA+C before (-ExoN) and after ExoN reaction, g MS analysis of Azv-RNA+U before (-ExoN) and after ExoN reaction, h MS analysis of Azv-RNA+GG before (-ExoN) and after ExoN reaction. All three Azv-RNA+1 showed strong ExoN resistance.

[0044] Fig.29 Phosphonate Modification of Nucleotide Analogs to Enhance Phosphorylation to Form Active Drugs and Resist ExoN Excision

[0045] Fig. 30 RdRp Incorporation and ExoN Resistance of Tenofovir-DP and its Analogues.

[0046] Fig. 31 Synthesis of Tenofovir-based SARS-CoV-2 RdRp Inhibitors with Different Substituents at the R Position.

[0047] Fig. 32 SARS-CoV-2 ExoN Active Site (DEEDh)..DETAILED DESCRIPTION OF THE INVENTION

[0048] The present disclosure provides a compound having the structure:whereinBASE is adenine, guanine, cytosine, thymine, uracil or derivatives thereof;[A]R₁, R₂, R₃, R₄, R₅ and R₆ are each independently -H, halogen, -CN, -N₃, -NH₂, -OH, -CHF₂, -CH₂F, -CF₃, -OCHF₂, -OCF₃, -OCH₂F, -NO₂, -OAc, -COOH, alkyl, alkenyl, alkynyl, -OR₁₃, -COR₁₃, -SH, -SR₁₃, -SO₂R₁₃, -NHR₁₃, -NR₁₄R₁₅, -NHCOR₁₃, or -CONR₁₄R₁₅,wherein R, R14, and R15 are each independently -H, alkyl, alkenyl, alkynyl, aldehyde, ketone, ester, ether, carboxylate, aryl, or heteroaryl;n is 0-10;Z is -O-, -CH₂-, -NH-, or -S-; and(a) Ri is -CN, R2is -OH, R4 is -NH2, and R3, Rs and Re are each independently -H, halogen, - CN, -Ns, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAC, -COOH, alkyl, alkenyl, alkynyl, -ORI3. -COR, -SH, -SR, -SO2R, -NHRB, -NRMRIS. - NHCORB, or -CONR14R15,(b) R₁ is -CN, R₃ is -F, R₄ is -OH, R₆ is -N₃, and R₂ and R₅ are each independently -H, halogen, -CN, -N₃, -NH₂, -OH, -CHF₂, -CH₂F, -CF₃, -OCHF₂, -OCF₃, -OCH₂F, -NO₂, -OAc, -COOH, alkyl, alkenyl, alkynyl, -OR₁₃, -COR₁₃, -SH, -SR₁₃, -SO₂R₁₃, -NHR₁₃, -NR₁₄R₁₅, -NHCOR₁₃, or -CONR₁₄R₁₅, or(c) R₁ is -CN, R₂ is -OH, R₄ is -F, and R₃, R₅ and R₆ are each independently -H, halogen, -CN, -N₃, -NH₂, -OH, -CHF₂, -CH₂F, -CF₃, -OCHF₂, -OCF₃, -OCH₂F, -NO₂, -OAc, -COOH, alkyl,alkenyl, alkynyl, -ORB, -CORB, -SH, -SRB, -SO2RB, -NHRB, -NR4RB, -NHCOR, or - CONR14R15.n is 0-10;Z is -O-, -CH₂-, -NH-, or -S-; andY is -OH, R?, or wherein R7, Rs, R% Rio are each independently halogen, -CN, -Ns, -NH2, -OH, -CHF2, - CH2F, -CFS, -OCHF2, -OCFs, -OCH2F, -NO2, -OAC, -COOH, alkyl, alkenyl, alkynyl, - ORB, -CORB, -SH, -SRis, -SO2RB, -NHRB. -NR14R15, -NHCORB, -CONR14R15, or O O HO-pfo-P^OH OH 5wherein RB, RW, and RB are each independently -H, alkyl, alkenyl, alkynyl, aldehyde, ketone, ester, ether, carboxylate, aryl, or heteroaryl; and t is 0-10; andn is 0-10;or a pharmaceutically acceptable salt or ester thereof.

[0049] In some embodiments, in [A], Ri. R2, Rs, R4, R5 and Rs are each independently -H, halogen, -CN. -Ns, -NH2, -OH, -CHF2, -CH2F. -CFS. -OCHF2. -OCFS, -OCH2F, -NO2. -OAC, or -COOH.

[0050] In some embodiments, in [B](a), R3, Re and Re are each independently -H, halogen, -CN, -Ns, -NH2, -OH, -CHF2, -CH2F, -CFS, -OCHF2. -OCFS. -OCH2F. -NO2. -OAC, or -COOH.

[0051] In some embodiments, in [B](b), R2and Re are each independently -H, halogen, -CN. -Ns, -NH2, -OH, -CHF2. -CH2F. -CFS. -OCHF2, -OCFS, -OCH2F, -NO2. -OAC. or -COOH.

[0052] In some embodiments, in [B](c), Rs, Re and Re are each independently -H, halogen. -CN. -Ns, -NH2, -OH, -CHF2, -CH2F, -CFS, -OCHF2, -OCFS, -OCH2F, -NO2, -OAC, or -COOH.

[0053] In some embodiments, in [A], Ri, R2. R3. R4. Re and Re are each independently -H, -F, -Cl, -CN, -Ns, -NH2, -OH, -CHF2, -CH2F, or -CFs.

[0054] In some embodiments, in [B](a), Rs, Rs and Re are each independently -H, -F, -Cl, -CN, -Ns, -NH2, -OH, -CHF2, -CH2F, or -CF3.

[0055] In some embodiments, in [B](b), R2and Rs are each independently -H, -F, -Cl, -CN, -Ns, -NH2, -OH, -CHF2, -CH2F, or -CF3.

[0056] In some embodiments, in [B](c), Rs, Rs and Re are each independently -H, -F, -Cl, -CN, -Ns, -NH2, -OH, -CHF2, -CH2F, or -CF3.

[0057] In some embodiments, in [A], Ri, R2, Rs, R4, Rs and Re are each independently -H, -F, -CN, -Ns, -NH2, or -OH.

[0058] In some embodiments, in [B](a), Rs, Rs and Re are each independently -H, -F, -CN, -Ns, -NH2, or -OH.

[0059] In some embodiments, in [B](b), R2and Rs are each independently -H, -F, -CN, -N3, -NH2, or -OH.

[0060] In some embodiments, in [B](c), Rs, Rs and Re are each independently -H, -F, -CN, -Ns, -NH2, or -OH.

[0061] In some embodiments, wherein in [B], Ri, R2, R3are each independently -H, -F, -CN, or -OH; and R4, Rs and Re are each independently -H, -F, -CN, -N3, -NH2, or -OH.

[0062] In some embodiments, wherein in [B], n is 0-1.

[0063] In some embodiments, wherein in [B], t is 0-2.O

[0064] In some embodiments, wherein in [B], Y is -OH,, or,wherein R7, Rs Rg, Rio are each independently -OH, -NHRis. C1-C3 alkyl, or -ORis.

[0065] In some embodiments, n is 0-5.

[0066] In some embodiments, t is 0-5.

[0067] In some embodiments, Z is -O- or -CH2-.

[0068] In some embodiments, n is 0-5.

[0069] In some embodiments, n is 0-2.

[0070] In some embodiments, t is 0-3.

[0071] In some embodiments, t is 0-2.oo

[0072] In some embodiments, Yis -OH,, orKl)wherein R7, Rs, Rg. Rio are each independently halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAc, -COOH, alkyl, alkenyl, alkynyl, -NHR, -NR14R15, or -OR13.oO11 ■R8-P-!

[0073] In some embodiments, Yis -OH, R7R9wherein R7, R: Rg, Rio are each independently halogen, -CN, -Ns, -NH2, -OH, -CHF2, - CH2F, -CFS, -OCHF2, -OCF3, -OCH2F, -NO2, -OAc, -COOH, alkyl, -NHR13, -NR14R15, or -OR13.O

[0074] In some embodiments, Yis -OH,, orwherein R7, Rg, R9, Rio are each independently halogen, -CN, -N3, -NH2, -OH, -CHF2, - CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -NHRB, C1-C6alkyl, -NR14R15, or -ORB, wherein R, R14, and R15 are each independently aldehyde, ketone, ester, ether, carboxylate, or aryl.

[0075] Hie present disclosure provides a compound having the structure:whereinBASE is adenine, guanine, cytosine, thymine, uracil or derivatives thereof;(a) Ri is -CN, R2is -OH, R4 is -NH2, and R3, R5 and Re are each independently -H. halogen, - CN, -Ns, -NH2, -OH, -CHF2. -CH2F, -CF3, -OCHF2, -0CF3, -OCH2F, -NO2, -OAc, -COOH, alkyl, alkenyl, alkynyl, -ORB, -COR, -SH, -SR, -SO2RB, -NHR, -NR14R15, - NHCORB, or -CONRI4RI5.(b) Ri is -CN, R3 is -F, R4 is -OH, Re is -Ns, and R2and R5 are each independently -H, halogen, -CN, -Ns, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAc, - COOH, alkyl, alkenyl, alkynyl, -ORB, -CORB, -SH, -SR. -SO2RB, -NHR. -NR]4Ri5. - NHCORB, or -CONR14R15,(c) Ri is -CN, R2is -OH, R4 is -F, and R3, Rs and s are each independently -H, halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAC. -COOH, alkyl, alkenyl, alkynyl, -ORJ3, -CORJ3, -SH, -SRi3, -SO2RB, -NHRB, -NR14R15, -NHCOR13, or -CONRuRis,(d) R3is -F. R4 is -OH, s is -N3, and Ri, R2and R5 are each independently -H, halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAC, -COOH, alkyl, alkenyl, alkynyl, -OR]3, -CORI3, -SH, -SRB, -SO2RI3, -NHRB, -NR14R15, -NHCORI3, or -CONR14R15.(e) BASE is [structure], R₂ is -OH, and R₁, R₃, R₄, R₅, and R₆ are each independently -H, halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAc, -COOH, alkyl, alkenyl, alkynyl, -ORB, -CORn, -SH, -SRI3, -SO2RI3, -NHRB, -NR14R15. -NHCORB, or -CONR14R15, orH2N ~ / =N(f) BASE is '~iAZ, Ri is -CN, R2is -OH, R4 is -OH. and R3, Rs and Rs are each independently -H, halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAc, -COOH, alkyl, alkenyl, alkynyl, -ORJ3, -CORis, -SH, -SR]3, -SO2RI3, -NHR13, -NR14R15, -NHCOR]3, or -CONR14R15.wherein RB, RH, and Ru are each independently -H, alkyl, alkenyl, alkynyl, aldehyde, ketone, ester, ether, carboxylate, aryl, or heteroaryl;n is 0-10;Z is -O-, -CH₂-, -NH-, or -S-; andwherein when Y is -OH,(i) then BASE is [structure], R₁ is -CN; R₂ is -OH, R₄ is -NH₂, R₃, R₅, R₆ are each -H, n is 0 and Z is -CH₂-, or(ii) then BASE is [structure], R₁ is -CN; R₃ is -F, R₄ is -OH, R₆ is -N₃, R₂ and R₅ are each -H, n is 0 and Z is -CH₂-; andherein when Y is OH(iii) then BASE is [structure], R₁ is -CN; R₂ is -OH, R₄ is -NH₂, R₃, R₅, R₆ are each -H, n is 1 and Z is -CH₂-;H, NL2 N- N(iv)(iv) then BASE is [structure], R₁ is -CN; R₂ is -OH, R₃, R₄, R₅, R₆ are each -H, n is 1 and Z is -CH2-;'2then BASE is, R2is -OH, Ri, Rs, R4, Rs, Rs are each -H, n is 1 and Z is - CH2-;NH2(vi) then BASE is [structure], R₃ is -F; R₄ is -OH, R₆ is -N₃, R₁, R₂, R₅ are each -H, n is 1 andZ is -CH2-; or a pharmaceutically acceptable salt or ester thereof.

[0076] In some embodiments, wherein in (a), Rs, Rs and R, are each independently -H, halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCFS, -OCH2F, -NO2, -OAc, or -COOH.

[0077] In some embodiments, wherein in (b), R2and Rs are each independently -H, halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAC, or -COOH.

[0078] In some embodiments, wherein in (c), R3, Rs and R. are each independently -H, halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAC, or -COOH.

[0079] In some embodiments, wherein in (d), Ri, R2and Rs are each independently-H, halogen, -CN, -Ns, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCFS, -OCH2F, -NO2, -OAC, or -COOH.

[0080] In some embodiments, wherein in (e), Ri, R3, R4, Rs, and Re are each independently-H, halogen, -CN, -Ns, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHFZ, -OCFS, -OCH2F, -NO2, -OAC, or -COOH.

[0081] In some embodiments, wherein in (f), R3, Rs and s are each independently-H, halogen, -CN, -Ns, -NH2, -OH, -CHF2, -CH2F, -CFS, -OCHF2, -OCFS, -OCH2F, -NO2, -OAC, or -COOH.

[0082] In some embodiments, wherein in (a), R3, Rs and Re are each independently -H, -F, -Cl, -CN, -Ns, -NH2, -OH, -CHF2, -CH2F, or -CF3.

[0083] In some embodiments, wherein in (b), R2and Rs are each independently -H, -F, -Cl, -CN, -Ns, -NH2, -OH, -CHF2, -CH2F, or -CF3.

[0084] In some embodiments, wherein in (c), R3, Rs and Re are each independently -H, -F, -Cl, -CN, -Ns, -NH2, -OH, -CHF2, -CH2F, or -CF3.

[0085] In some embodiments, wherein in (d), Ri, R2and Rs are each independently-H, -F, -Cl, -CN, -Ns, -NH2, -OH, -CHF2, -CH2F, or -CF3.

[0086] In some embodiments, wherein in (e), Ri, R3, i, Rs, and Re are each independently-H, -F, -Cl, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, or -CF3.

[0087] In some embodiments, wherein in (f), R3, Rs and Re are each independently-H, -F, -Cl, -CN, -Ns, -NH2, -OH, -CHF2, -CH2F, or -CF3.

[0088] In some embodiments, wherein in (a), Rs, Rs and Re are each independently -H, -F, -CN, -Ns, -NH2, or -OH.

[0089] In some embodiments, wherein in (b), R2and Rs are each independently -H, -F, -CN, -Ns, -NH2, or -OH.

[0090] In some embodiments, wherein in (c), Rs, Rs and Re are each independently -H, -F, -CN, -Ns, -NH2, or -OH.

[0091] In some embodiments, wherein in (d), Ri, R2 and Rs are each independently— H, -F, -CN, -N3, -NHs, or -OH.

[0092] In some embodiments, wherein in (e), Ri, R3, R4, Rs, and R., are each independently-H, -F, -CN, -N3, -NH2, or -OH.

[0093] In some embodiments, wherein in (f), R3, Rs and 5 are each independently— H, -F, -CN, -N3, -NH2, or -OH.

[0094] In some embodiments, n is 0-2.

[0095] In some embodiments, Z is -O- or -CH2-.

[0097]

[0098] In some embodiments, wherein the derivative of adenine, guanine, cytosine, thymine, and uracil have the structures:whereinRis, RIQ, R20, R21, and R22 are each independently -H, halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAc, -COOH, alkyl, alkenyl, alkyny l, -ORI3, -COR]3, -SH, -SR13, -SO2RI3, -NHR13, -NR14R15, -NHCORB, or -CONR14R15wherein R|3. Ru, and Ris are each independently -H, alkyl, alkenyl, alkynyl, aldehyde, ketone, ester, ether, carboxylate, ar l, or heteroaryl.

[0099] In some embodiments, Ris, R19. R20, R21, and R22 are each independently -H, halogen, -CN, -N3, -NH2. -OH. -CHF2. -CH2F. -CF3, -OCHF2, -OCF3, -OCH2F, -NO2. -OAc, -COOH, alkyl, alkenyl, alkynyl, -ORI3, -CORI3, -SH, or -SRI3.

[0100] In some embodiments, Ris, R19. R20, R21, and R22 are each independently -H. halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, or -NO2.

[0101] In some embodiments, Ris, R19, R20, R21, and R22 are each independently -H, or -NH2.

[0102] In some embodiments, the derivative is

[0103] Tire present disclosure provides a compound having structure:wherein BASE is adenine, guanine, cytosine, uracil, thymine or derivatives thereof, and R is -OH, -F, -N3, or -NH2.

[0104] Tire present disclosure provides a compound having structure:wherein BASE is adenine, guanine, cytosine, uracil, thymine or derivatives thereof.

[0105] The present disclosure provides a compound having structure:wherein BASE is adenine, guanine, cytosine, uracil, thymine or derivatives thereof, and R is -F, -Ns, or -NH2.

[0106] The present disclosure provides a compound having structure:wherein BASE is adenine, guanine, cytosine, uracil, thymine or derivatives thereof, and R is -OH, -F, -Ns, or -NH2.

[0107] Tire present disclosure provides a compound having structure:wherein R is -OH. -F, -Ns or -NH2.

[0108] The present disclosure provides a compound having structure:HO

[0109] The present disclosure provides a compound having structure:

[0110] The present disclosure provides a compound having structure:BASE is adenine, guanine, cytosine, uracil, thymine or derivatives thereof;m is 0-10; andX is halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3. -OCH2F, -NO2. -OAc, - COOH, alkyl, alkenyl, alkynyl, -OR]3, -COR]3, -SH, -SRI3, -SO2RI3, -NHRI3, -NRuRis. - NHCORis, or -CONR14R15,wherein RI3, U. and Ris are each independently -H, alkyl, alkenyl, alkynyl, aldehyde, ketone, ester, ether, carboxylate, aryl, or heteroaryl,or a pharmaceutically acceptable salt or ester thereof.

[0111] In some embodiments, m is 0-5; more preferably m is 0-3; more preferably m is 2.

[0112] In some embodiments, the compound has the structure:

[0113] In some embodiments, the compound has the structure:

[0114] In some embodiments, X is halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, - OCF3, -OCH2F, -NO2, -OAc, or -COOH.

[0115] In some embodiments, X is -F, -Cl, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, or -CF3.

[0116] In some embodiments, X is -F, -CN, -N3, -NH2, or -OH.

[0117] In some embodiments, the derivative of adenine, guanine, cytosine, thymine, and uracil has the structure:whereinRis, RIQ, R20, R21, and R22 are each independently -H, halogen, -CN, -Ns, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAc, -COOH, alkyl, alkenyl, alkynyl, -ORI3, -COR]3, -SH, -SR13, -SO2RI3, -NHRB, -NR14R15, -NHCORis, or -CONR14R15,wherein R|3. R14. and R15 are each independently -H, alkyl, alkenyl, alkynyl, aldehyde, ketone, ester, ether, carboxylate, aryl, or heteroaryl;preferably, Ris, R19, R20, R21, and R22 are each independently -H, halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2. -OCF3, -OCH2F, -NO2, -OAc, -COOH, alkyl, alkenyl, alkynyl, -ORis, -CORI3, -SH, or -SRu.more preferably, Ris, R19, R20, R21, and R22are each independently -H, halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHFZ, -OCF3, -OCH2F, or -NO2;more preferably, Ris, R19, R2o, R2I, and R22are each independently -H, or -NH2;more preferably, the derivative is

[0118] Tire present disclosure provides a compound having structure:

[0119] Tire present disclosure provides a composition comprising the compound disclosed herein or a pharmacally acceptable salt thereof.

[0120] The present disclosure provides a composition comprising at least one of tire following compounds or a pharmaceutically acceptable salt thereof for the treatment of viral infections caused by one or more viruses selected from the group comprising coronaviruses, flaviviruses and alphaviruses:wherein BASE is adenine, guanine, cytosine, uracil, thymine or derivatives thereof,X is -F, -CN, -N₃, -NH₂ or -OH, and X has either an R or S configuration or a mixture of both stereoisomers.

[0121] Tire present disclosure provides a composition comprising at least one of the following compounds or a pharmaceutically acceptable salt thereof for the treatment of viral infection caused by one or more viruses selected from the group comprising coronaviruses, flaviviruses and alphaviruses:wherein X is -F, -CN, -N₃, -NH₂, -OH and X has either an R or S configuration or a mixture of both stereoisomers.

[0122] In some embodiments, coronavirus infections were caused by viruses comprising SARS-CoV2, SARS-CoV, and MERS-CoV, and wherein the X is in the R configuration.

[0123] The present disclosure provides a composition comprising at least one of the following compounds or a pharmaceutically acceptable salt thereof for the treatment of viral infection caused by one or more viruses selected from the group comprising coronaviruses, flaviviruses and alphaviruses:

[0124] The present disclosure provides a composition comprising at least one of the following compounds or a pharmaceutically acceptable salt thereof for the treatment of viral infection caused by one or more viruses selected from the group comprising coronaviruses, flaviviruses and alphaviruses,R is -OH, -F, -N3, or -NH2; andwherein BASE is adenine, guanine, cytosine, uracil, thymine or derivatives thereof.

[0125] In some embodiments, wherein a flaviviruses comprise Dengue virus (DENV), Zika virus (ZIKV) and yellow fever virus (YFV); and / or alphaviruscs comprise Chikungunya virus (CHIKV) and Eastern equine encephalitis virus (EEEV).

[0126] Tire present disclosure provides a method of treating viral infection in a subject comprising administering one or more compound disclosed herein and composition disclosed herein to the subject.

[0127] In some embodiments, the subject is a mammal, preferably, the subject is a human.

[0128] In some embodiments, the viral infection is caused by one or more viruses selected from the group comprising coronaviruses, flaviviruses and alphaviruses.

[0129] In some embodiments, the coronavirus infection caused by viruses comprising SARS-CoV2, SARS-CoV, and MERS-CoV.

[0130] In some embodiments, the flaviviruse is Dengue virus (DENV), Zika virus (ZIKV) and yellow fever virus (YFV).

[0131] In some embodiments, the alphaviruse is Chikungunya virus (CHIKV) and Eastern equine encephalitis virus (EEEV).

[0132] In some embodiments, the method further comprises administering one or more anti-viral medication.

[0133] In some embodiments, the compound disclosed herein is incorporated by viral RNA-dependent RNA polymerase (RDRP) and is resistant to excision by viral 3 ’-5’ exonuclease (ExoN).

[0134] The present disclosure provides a process of producing tire compound disclosed herein. wherein the process comprises:(a) reacting a nitrogenous basewith X to obtain a compound of formula I,BASE HOx(i);TsO O^P-OEti(b) reacting the compound of formula I with OEt to obtain a compound of formula II BASEF.tCk / / °OEtdX (II);(c) reacting the compound of formula II with an acid to produce a compound of formula III O BASEOik / / ,0OHX (III); and(d) reacting the compound having formula III with phosphorylating reagent.

[0135] In some embodiments,(a) step (a) is conducted in the presence of a base, preferably, an inorganic base, more preferably, KOH, NaOH Ca(OH)2;(b) step (b) is conducted in the presence of MeMgCl, t-BuOH and cyclohexane;(c) the acid in step (c) is HBr / AcOH;(d) step (d) is conducted in the presence of CDI / DMF, MeOH, TEAB buffer, and THP: and / or (e) the phosphory lating reagent is tributylammonium pyrophosphate.

[0136] In some embodiments, whereinO(a)has the structure ofxBASE o BASEF.tO^ / / EtO„ / / P P^Ol t() EtC)(b)has the structure of; andBASE BASE OH.p / z OI K / PP\ / OOH OH(c)has the structure of

[0137] In some embodiments, the process for producing the compound disclosed herein comprises:NHBzwith a desilylation agent; or(c) reacting the compound of formula (III) with phenol to produce a compound of formula (IV)(d) reacting the compound of formula (IV) with SOCk a compound of formula (V)NH->O X)(e) reacting the compound of formula Vwith; orNHBzto produceNHBz(1) reactingwith a desilylation agent.

[0138] The present disclosure provides a process of producing the compound disclosed herein comprising:HO(a) reactingTBSO (VI) or OBnwith a phosphorylating reagent: orHO- °\ <.0O^H Cl(b) reactingTBSO (VI) with O to produce a compound of(c) reacting compound of formula (VII) with a desilylation agent; or(d) reacting tire compound of formula (VI) withto produce a compound of formula (VIII)TBSO (VIII),(e) reacting compound of formula (VIII) with a desilylation agent.

[0139] In some embodiments, wherein the desilylation agent is BCE, or TBAF / THF.

[0140] The present disclosure provides a composition comprising at least one of the following compounds or a pharmaceutically acceptable salt thereof for the treatment of viral infections caused by one or more viruses selected from the group comprising coronaviruses, flaviviruses and alphaviruses:wherein BASE is Adenine, Guanine, Cytosine, Thymine, Uracil or derivatives thereof,X is F, CN, N3, NH2or OH, or related groups and X may have either an R or S configuration or a mixture of both stereoisomers;orwherein X is F, CN, N₃, NH₂, OH or related groups and X may have either an R or S configuration or a mixture of both stereoisomers.

[0141] In some embodiments, the coronavirus infections are caused by viruses comprising SARS-CoV2, SARS-CoV, and MERS-CoV, and wherein tire X substitution is in the R configuration.

[0142] The present disclosure provides a composition comprising at least one of the following 5’-methylene nucleotide compounds or a pharmaceutically acceptable salt thereof for the treatment of viral infection caused by one or more viruses selected from the group comprising coronaviruses, flaviviruses and alphaviruses:HO ora composition comprising at least one of the following l’-CN, 2’-F, 4 ’-Ns- 1 -Deazacytidine nucleoside or a pharmaceutically acceptable salt of thereof for the treatment of viral infection caused by one or more viruses selected from the group comprising coronaviruses, flaviviruses and alphaviruses:ora composition comprising at least one of the following 3 ’-deoxyadenosine nucleoside prodrugs or a pharmaceutically acceptable salt thereof for the treatment of viral infection caused by one or more viruses selected from the group comprising coronaviruses, flaviviruses and alphaviruses:OH OH

[0143] The present disclosure provides a composition comprising at least one of the following compounds or a pharmaceutically acceptable salt thereof for the treatment of viral infection caused by one or more viruses selected from the group comprising coronaviruses, flaviviruses and alphaviruses.NH₂OH OHwherein BASE is adenine, guanine, cytosine, uracil. Thymine or derivatives thereof: orwherein BASE is adenine, guanine, cytosine, uracil. Thymine or derivatives thereof, and R is F, Ns, NH2or related groups; orwherein R is F, N?, or NH2or related groups.

[0144] The present disclosure provides a composition comprising at least one of the following compounds or a pharmaceutically acceptable salt thereof for the treatment of viral infection caused by one or more viruses selected from the group comprising flaviviruses and alphaviruses.wherein BASE is adenine, guanine, cytosine, uracil. Thymine or derivatives thereof, and R is OH, F, N3, NH2or related groups.

[0145] In some embodiments, wherein flaviviruses comprise Dengue virus (DENV), Zika virus (ZIKV) and yellow fever virus (YFV) or wherein alphaviruses is a Chikungunya virus (CHIKV) and Eastern equine encephalitis virus (EEEV).Definitions

[0146] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in tire practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. Incase of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0147] In the discussion unless otherwise stated, adjectives such as ‘'substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to + / - 10% of the specified value. In embodiments, about includes the specified value. Unless otherwise indicated, the word “or” in the specification and claims is considered to be the inclusive '‘or” rather than the exclusive or, and indicates at least one of and any combination of items it conjoins.

[0148] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a,” “an” and “at least one” are used interchangeably in this application.

[0149] “And / or” as used herein, for example with option A and / or option B, encompasses the separate embodiments of (i) option A, (ii) option B, and (iii) option A plus option B.

[0150] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0151] In the description and claims of the present application, each of the verbs, “comprise,” “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb. Other terms as used herein are meant to be defined by their well-known meanings in the art.

[0152] The compounds of the subject invention may have spontaneous tautomeric forms. In cases wherein compounds may exist in tautomeric forms, such as keto-enol tautomers, each tautomeric form iscontemplated as being included within this invention whether existing in equilibrium or predominantly in one form.

[0153] In the compound structures depicted herein, hydrogen atoms are not shown for carbon atoms having less than four bonds to non-hydrogen atoms. However, it is understood that enough hydrogen atoms exist on said carbon atoms to satisfy the octet rule.

[0154] This invention also provides isotopic variants of the compounds disclosed herein, including wherein the isotopic atom is2H and / or wherein the isotopic atom13C. Accordingly, in the compounds provided herein hydrogen can be enriched in the deuterium isotope. It is to be understood that the invention encompasses all such isotopic forms.

[0155] It is understood that the structures described in the embodiments of the methods hereinabove can be the same as the structures of the compounds described hereinabove.

[0156] It is understood that where a numerical range is recited herein, the present invention contemplates each integer between, and including, the upper and lower limits, unless otherwise stated.

[0157] Except where otherwise specified, if the structure of a compound of this invention includes an asymmetric carbon atom, it is understood that tire compound occurs as a racemate, racemic mixture, and isolated single enantiomer. All such isomeric forms of these compounds are expressly included in this invention. Except where otherwise specified, each stereogenic carbon may be of the R or S configuration. It is to be understood accordingly that the isomers arising from such asymmetry' (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure fonn by classical separation techniques and by stereochemically controlled synthesis, such as those described in " Enantiomers, Racemates and Resolutions" by J. Jacques, A. Collet and S. Wilen, Pub. John Wiley & Sons, NY, 1981. For example, the resolution may be carried out by preparative chromatography on a chiral column.

[0158] Except where otherwise specified, the subject invention is also intended to include all isotopes of atoms occurring on the compounds disclosed herein. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.

[0159] It will be noted that any notation of a carbon in structures throughout this application, when used without further notation, are intended to represent all isotopes of carbon, such as12C,13C, or14C. Furthermore, any compounds containing13C or14C may specifically have the structure of any of the compounds disclosed herein.

[0160] It will also be noted that any notation of a hydrogen in structures throughout this application, when used without further notation, are intended to represent all isotopes of hydrogen, such as 'H,2H, or3H. Furthermore, any compounds containing2H or3H may specifically have the structure of any of the compounds disclosed herein.

[0161] Isotopically -labeled compounds can generally be prepared by conventional techniques known to those skilled in the art using appropriate isotopically-labeled reagents in place of the non-labeled reagents employed.

[0162] In tire compounds used in the method of the present invention, the substituents may be substituted or unsubstituted, unless specifically defined otherwise.

[0163] In the compounds used in the method of the present invention, alkyl, heteroalkyl, monocycle, bicycle, aryl, heteroaryl and heterocycle groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano, carbamoyl and aminocarbonyl and aminothiocarbonyl.

[0164] In choosing the compounds used in the method of the present invention, one of ordinary skill in the art will recognize that the various substituents, i.e. Ri, R2, etc. are to be chosen in conformity with well-known principles of chemical structure connectivity.

[0165] The present invention provides a use of the compound or the composition disclosed herein in inhibiting the growth of cancer cells or treating cancer.

[0166] The present invention provides the compound or the composition disclosed herein for use in inhibiting the growth of cancer cells or treating cancer.

[0167] In some embodiments, the present invention includes a pharmaceutically acceptable salt of any of the above compounds of the present invention.

[0168] In some embodiments, a salt of the compound of the present invention is used in any of the above methods, uses, packages or compositions.

[0169] In some embodiments, a pharmaceutically acceptable salt of the compound of the present invention is used in any of tire above methods, uses, packages or compositions.

[0170] In some embodiments, an ester of tire compound of the present invention is used in any of the above methods, uses, packages or compositions.

[0171] Any of the above compounds may be used in any of the disclosed methods, uses, packages or pharmaceutical compositions.

[0172] Any of the compounds used in the disclosed methods, uses, packages or pharmaceutical compositions may be replaced with any other compound disclosed in the present invention.

[0173] Any of the above generic compounds may be used in any of the disclosed methods, uses, packages or compositions.

[0174] A person skilled in the art may use the techniques disclosed therein to prepare compounds which are not enriched in deuterium and thereafter use the techniques disclosed herein to prepare deuterium analogs thereof.

[0175] Except where otherwise specified, the structure of a compound of this invention includes an asymmetric carbon atom, it is understood that the compound occurs as a racemate, racemic mixture, scalemic mixtures and isolated single enantiomers. All such isomeric forms of these compounds are expressly included in this invention. Except where otherwise specified, each stereogenic carbon may be of the R or S configuration. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stcrcochcmically controlled synthesis, such as those described in " Enantiomers, Racemates and Resolutions" by J. Jacques, A. Collet and S. Wilen, Pub. John Wiley & Sons, NY, 1981. For example, the resolution may be carried out by preparative chromatography on a chiral column.

[0176] Deuterium (2H or D) is a stable, non-radioactive isotope of hydrogen and has an atomic weight of 2.0144. Hydrogen atom in a compound naturally occurs as a mixture of the isotopes 'H (hydrogen or protium), D (2H or deuterium), and T (3H or tritium). The natural abundance of deuterium is 0.0156%. Thus, in a composition comprising molecules of a naturally occurring compound, the level of deuterium at a particular hydrogen atom site in that compound is expected to be 0.0156%. Thus, a composition comprising a compound with a level of deuterium at any site of hydrogen atom in the compound that has been enriched to be greater than its natural abundance of 0.0156% is novel over its naturally occurring counterpart.

[0177] In the compounds used in the method of the present invention, the substituents may be substituted or unsubstituted, unless specifically defined otherwise.

[0178] This invention also provides isotopic variants of the compounds disclosed herein, including wherein the isotopic atom is2H and / or wherein the isotopic atom13C. Accordingly, in the compounds provided herein hydrogen can be enriched in the deuterium isotope. It is to be understood that the invention encompasses all such isotopic forms.

[0179] It is understood that the structures described in the embodiments of the methods hereinabove can be the same as the structures of the compounds described hereinabove.

[0180] It is understood that where a numerical range is recited herein, the present invention contemplates each integer between, and including, the upper and lower limits, unless otherwise stated.

[0181] Except where otherwise specified, if the structure of a compound of this invention includes an asymmetric carbon atom, it is understood that the compound occurs as a racemate, racemic mixture, and isolated single enantiomer. All such isomeric forms of these compounds are expressly included in this invention. Except where otherwise specified, each stereogenic carbon may be of tire R or S configuration. It is to be understood accordingly that the isomers arising from such asymmetry (e.g.. all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis, such as those described in " Enantiomers, Racemates and Resolutions" by J. Jacques, A. Collet and S. Wilen, Pub. John Wiley & Sons, NY, 1981. For example, the resolution may be carried out by preparative chromatography on a chiral column.

[0182] In tire compounds used in tire method of the present invention, alkyl, heteroalkyl, monocycle, bicycle, aryl, heteroaryl and heterocycle groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.

[0183] As used herein, "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. Thus, Ci -Cnas in “Ci-Cnalkyl" is defined to include groups having 1, 2, n-1 or n carbons in a linear or branched arrangement, and specifically includes methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, isopropyl, isobutyl, sec-butyl and so on. An embodiment can be C1-C12 alkyl, C2-C12 alkyl, C3-C12 alkyl, C4-C12 alkyl and so on. ’’Alkoxy" represents an alkyl group as described above attached through an oxygen bridge.

[0184] Tlie term "alkenyl" refers to a non -aromatic hydrocarbon radical, straight or branched, containing at least 1 carbon to carbon double bond, and up to the maximum possible number of nonaromatic carbon-carbon double bonds may be present. Tirus. C2-Cnalkenyl is defined to include groups having 1, 2...., n-1 or n carbons. For example, " C2-C6 alkenyl" means an alkenyl radical having 2, 3, 4, 5, or 6 carbon atoms, and at least 1 carbon-carbon double bond, and up to, for example, 3 carbon-carbon double bonds in the case of a C, alkenyl, respectively. Alkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl. As described above with respect to alkyl, the straight, branched or cyclic portion of the alkenyl group may contain double bonds and may be substituted if a substituted alkenyl group is indicated. An embodiment can be C2-C12 alkenyl, C3-C12 alkenyl, C4-C12 alkenyl and so on.

[0185] The term "alkynyl" refers to a hydrocarbon radical straight or branched, containing at least 1 carbon to carbon triple bond, and up to the maximum possible number of non-aromatic carbon-carbon triple bonds may be present. Thus, C2-Cnalkynyl is defined to include groups having 1, 2...., n-1 or n carbons. For example, " C2-C6 alkynyl" means an alkynyl radical having 2 or 3 carbon atoms, and 1 carbon-carbon triple bond, or having 4 or 5 carbon atoms, and up to 2 carbon-carbon triple bonds, or having 6 carbon atoms, and up to 3 carbon-carbon triple bonds. Alkynyl groups include ethynyl. propynyl and butynyl. As described above with respect to alkyl, the straight or branched portion of the alkynyl group may contain triple bonds and may be substituted if a substituted alkynyl group is indicated. An embodiment can be a C2-Cnalkynyl. An embodiment can be C2-C12 alkynyl, C3-C12 alkynyl, C4-C12 alkynyl and so on.

[0186] ‘‘Alkylene”, “alkenylene” and “alkynylene” shall mean, respectively, a divalent alkane, alkene and alkyne radical, respectively. It is understood that an alkylene, alkenylene, and alkynylene may be straight or branched. An alkylene, alkenylene, and alkynylene may be unsubstituted or substituted.

[0187] As used herein, "heteroalkyl" includes both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms and at least 1 heteroatom within the chain or branch.

[0188] As used herein, "heterocycle" or "heterocyclyl" as used herein is intended to mean a 5- to 10-membered nonaromatic ring containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S, and includes bicyclic groups. " Heterocyclyl" therefore includes, but is not limited to the following: imidazolyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl, dihydropiperidinyl, tetrahydrothiophenyl and the like. If the heterocycle contains a nitrogen, it is understood that the corresponding N-oxidcs thereof arc also encompassed by this definition.

[0189] As used herein, "cycloalkyl" shall mean cyclic rings of alkanes of three to eight total carbon atoms, or any number within this range (i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl).

[0190] As used herein, "monocycle" includes any stable polyatomic carbon ring of up to 10 atoms and may be unsubstituted or substituted. Examples of such non-aromatic monocycle elements include but are not limited to: cyclobutyl, cy clopentyl, cyclohexyl, and cycloheptyl. Examples of such aromatic monocycle elements include but are not limited to: phenyl.

[0191] As used herein, "bicycle" includes any stable polyatomic carbon ring of up to 10 atoms that is fused to a polyatomic carbon ring of up to 10 atoms with each ring being independently unsubstituted or substituted. Examples of such non-aromatic bicycle elements include but are not limited to:decahydronaphthalene. Examples of such aromatic bicycle elements include but are not limited to: naphthalene.

[0192] As used herein, "aryl" is intended to mean any stable monocyclic, bicyclic or polycyclic carbon ring of up to 10 atoms in each ring, wherein at least one ring is aromatic, and may be unsubstituted or substituted. Examples of such aryl elements include phenyl, p-toluenyl (4-methylphenyl), naphthyl, tetrahydro-naphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl. In cases where the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring.

[0193] As used herein, the term “polycyclic” refers to unsaturated or partially unsaturated multiple fused ring structures, which may be unsubstituted or substituted.

[0194] The term “arylalkyl” refers to alkyl groups as described above wherein one or more bonds to hydrogen contained therein are replaced by a bond to an aryl group as described above. It is understood that an “arylalkyl” group is connected to a core molecule through a bond from the alkyl group and that the aryl group acts as a substituent on the alkyl group. Examples of arylalkyl moieties include, but are not limited to, benzyl (phenylmethyl), p-trifluoromethylbenzyl (4-trifIuoromethylphenylmethyl), 1 -phenylethyl, 2-phcnylcthyl, 3-phcnylpropyl, 2-phcnylpropyl and the like.

[0195] Tire term "heteroaryl", as used herein, represents a stable monocyclic, bicyclic or polycyclic ring of up to 10 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Bicyclic aromatic heteroaryl groups include phenyl, pyridine, pyrimidine or pyridizine rings that are (a) fused to a 6-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom; (b) fused to a 5- or 6-membered aromatic (unsaturated) heterocyclic ring having two nitrogen atoms; (c) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom together with either one oxygen or one sulfur atom; or (d) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one heteroatom selected from O, N or S. Heteroaryl groups within the scope of this definition include but are not limited to: benzoimidazolyl, benzofuranyl, benzofurazanyl. benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyL furanyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyL isoindolyL isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, aziridinyl, 1,4-dioxanyl. hexahydroazepinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl. dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyL dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl,dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl, tetrahydrothienyl, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, isoxazolyl, isothiazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline. In cases where the heteroaryl substituent is bicyclic and one ring is non -aromatic or contains no heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom containing ring, respectively. If the heteroaryl contains nitrogen atoms, it is understood that the corresponding N-oxides thereof are also encompassed by this definition.

[0196] Tire term “alkylheteroaryl” refers to alkyl groups as described above wherein one or more bonds to hydrogen contained therein are replaced by a bond to an hctcroaryl group as described above. It is understood that an “alkylheteroaryl” group is connected to a core molecule through a bond from the alkyl group and that the heteroaryl group acts as a substituent on the alkyl group. Examples of alkylheteroaryl moieties include, but are not limited to, -CEb-CCsFUN), -CfE-CfE-lCMEN) and the like.

[0197] The temr "heterocycle" or “heterocyclyl” refers to a mono- or poly-cyclic ring system which can be saturated or contains one or more degrees of unsaturation and contains one or more heteroatoms. Preferred heteroatoms include N, 0, and / or S, including N-oxides, sulfur oxides, and dioxides. Preferably the ring is three to ten-membered and is either saturated or has one or more degrees of unsaturation. The heterocycle may be unsubstituted or substituted, with multiple degrees of substitution being allowed. Such rings may be optionally fused to one or more of another "heterocyclic" ring(s), heteroaryl ring(s), aryl ring(s), or cycloalkyl ring(s). Examples of heterocycles include, but are not limited to, tetrahydrofuran, pyran, 1,4-dioxane, 1,3-dioxane, piperidine, piperazine, pyrrolidine, morpholine, thiomorpholine, tetrahydrothiopyran, tetrahydrothiophene, 1,3 -oxathiolane, and the like.

[0198] The alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl substituents may be substituted or unsubstituted, unless specifically defined otherwise. In the compounds of the present invention, alkyl, alkenyl, alkynyl, and, heterocyclyl and heteroaryl groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.

[0199] As used herein, tire term "halogen" refers to F, Cl, Br, and I.

[0200] Tire terms “substitution”, “substituted” and “substituent” refer to a functional group as described above in which one or more bonds to a hy drogen atom contained therein are replaced by a bond to non-hydrogen or non-carbon atoms, provided that normal valencies are maintained and that thesubstitution results in a stable compound. Substituted groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Examples of substituent groups include the functional groups described above, and halogens (i.e., F, Cl, Br, and I); alkyl groups, such as methyl, ethyl, n-propyl, isopropry 1, n-butyl, tert-butyl, and trifluoromethyl; hydroxyl; alkoxy groups, such as methoxy, ethoxy, n-propoxy, and isopropoxy; aryloxy groups, such as phenoxy; arylalkyloxy, such as benzyloxy (phenylmethoxy) and p-trifluoromethylbenzyloxy (4-trifluoromethylphenylmethoxy); heteroaryloxy groups; sulfonyl groups, such as trifluoromethanesulfonyl, methanesulfonyl, and p-toluenesulfonyl; nitro, nitrosyl; mercapto; sulfanyl groups, such as methylsulfanyl, ethylsulfanyl and propylsulfanyl; cyano; amino groups, such as amino, methylamino, dimethylamino, ethylamino, and diethylamino; and carboxyl. Where multiple substituent moieties are disclosed or claimed, the substituted compound can be independently substituted by one or more of the disclosed or claimed substituent moieties. singly or pluraly. By independently substituted, it is meant that the (two or more) substituents can be the same or different.

[0201] It is understood that substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.

[0202] In choosing the compounds of the present invention, one of ordinary skill in the art will recognize that the various substituents, i.e. Ri, R2, etc. are to be chosen in confonnity with well-known principles of chemical structure connectivity.

[0203] The various R groups attached to the aromatic rings of the compounds disclosed herein may be added to the rings by standard procedures, for example those set forth in Advanced Organic Chemistry: Part B: Reaction and Synthesis, Francis Carey and Richard Sundberg, (Springer) 5th ed. Edition. (2007), the content of which is hereby incorporated by reference.

[0204] The compounds used in the method of the present invention may be prepared by techniques well known in organic synthesis and familiar to a practitioner ordinarily skilled in the art. However, these may not be the only means by which to synthesize or obtain the desired compounds.

[0205] Tire compounds used in the method of the present invention may be prepared by techniques described in Vogel’s Textbook of Practical Organic Chemistry, A. I. Vogel, A. R. Tatchell, B. S. Fumis, A. J. Hannaford, P. W. G. Smith, (Prentice Hall) 5thEdition (1996), March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, Michael B. Smith, Jerry March, (Wiley-Interscience) 5thEdition (2007), and references therein, which are incorporated by reference herein. However, these may not be the only means by which to synthesize or obtain the desired compounds.

[0206] Another aspect of the invention comprises a compound used in the method of the present invention as a pharmaceutical composition.

[0207] In some embodiments, a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable carrier.

[0208] As used herein, the term “pharmaceutically active agent” means any substance or compound suitable for administration to a subject and furnishes biological activity or other direct effect in the treatment, cure, mitigation, diagnosis, or prevention of disease, or affects the structure or any function of the subject. Pharmaceutically active agents include, but are not limited to, substances and compounds described in the Physicians’ Desk Reference (PDR Network, LLC; 64th edition; November 15, 2009) and “Approved Drug Products with Therapeutic Equivalence Evaluations” (U. S. Department Of Health And Human Services, 30thedition, 2010), which are hereby incorporated by reference. Pharmaceutically active agents which have pendant carboxylic acid groups may be modified in accordance with the present invention using standard esterification reactions and methods readily available and known to those having ordinary skill in the art of chemical synthesis. Where a pharmaceutically active agent does not possess a carboxylic acid group, the ordinarily skilled artisan will be able to design and incorporate a carboxylic acid group into the pharmaceutically active agent where esterification may subsequently be carried out so long as the modification does not interfere with the pharmaceutically active agent’s biological activity or effect.

[0209] The compounds used in the method of the present invention may be in a salt form. As used herein, a “salt” is a salt of the compounds disclosed herein w hich has been modified by making acid or base salts of the compounds. In tire case of compounds used to treat an infection or disease caused by a pathogen, the salt is pharmacally acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to. mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as phenols. The salts can be made using an organic or inorganic acid. Such acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like. Phenolate salts are the alkaline earth metal salts, sodium, potassium or lithium. Hie term "pharmaceutically acceptable salt" in this respect, refers to the relatively non-toxic, inorganic and organic acid or base addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base or free acid form with a suitable organic or inorganic acid or base, and isolating the salt thus formed. Representativesalts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge et al. (1977) " Pharmaceutical Salts",. Pharm. Set. 66:1-19).

[0210] Tire compounds of the present invention may also form salts with basic amino acids such a lysine, arginine, etc. and with basic sugars such as N-methylglucamine, 2-amino-2-deoxyglucose, etc. and any other physiologically non-toxic basic substance.

[0211] As used herein, “administering” an agent may be performed using any of the various methods or delivery systems well known to those skilled in the art. The administering can be performed, for example, orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery, subcutaneously, intraadiposally, intraarticularly, intrathecally, into a cerebral ventricle, intraventicularly, intratumorally, into cerebral parenchyma or intraparenchchymally.

[0212] Tire compounds used in the method of the present invention may be administered in various fomrs, including those detailed herein. Tire treatment with the compound may be a component of a combination therapy or an adjunct therapy, i.e. the subject or patient in need of the drug is treated or given another drug for the disease in conjunction with one or more of the instant compounds. This combination therapy can be sequential therapy where the patient is treated first with one drug and then the other or the two drugs are given simultaneously. These can be administered independently by the same route or by two or more different routes of administration depending on the dosage forms employed.

[0213] As used herein, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the animal or human. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Liposomes are also a pharmacally acceptable carrier as are slow-release vehicles.

[0214] Tire dosage of tire compounds administered in treatment will vary depending upon factors such as the pharmacodynamic characteristics of a specific chemotherapeutic agent and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with; and the desired therapeutic effect.

[0215] A dosage unit of the compounds used in the method of the present invention may comprise a single compound or mixtures thereof with additional antitumor agents. The compounds can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups,and emulsions. The compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by injection, topical application, or other methods, into or topically onto a site of disease or lesion, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.

[0216] Tire compounds used in the method of the present invention can be administered in admixture with suitable pharmaceutical diluents, extenders, excipients, or in carriers such as the novel programmable sustained-release multi-compartmental nanospheres (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for oral, nasal, rectal, topical, intravenous or direct injection or parenteral administration. The compounds can be administered alone or mixed with a pharmaceutically acceptable carrier. This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. Tire active agent can be co-administered in the fomr of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

[0217] Techniques and compositions for making dosage forms useful in the present invention are described in the following references: 7 Modem Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmacal Sciences Vol. 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Fonns (Drugs and the Pharmacal Sciences, Series 36 (James McGinity, Ed., 1989); Pharmacal Particulate Carriers: Therapeutic Applications: Drugs and the Phamraceutical Sciences. Vol 61 (Alain Rolland, Ed.. 1993); DrugDelivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.

[0218] Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. For instance, for oral administration in the dosage unit form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, com sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

[0219] The compounds used in tire method of the present invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids such as lecithin, sphingomyelin, proteolipids, protein-encapsulated vesicles or from cholesterol, stearylamine, or phosphatidylcholines. Tire compounds may be administered as components of tissue-targeted emulsions.

[0220] Tire compounds used in the method of the present invention may also be coupled to soluble polymers as targetable drug carriers or as a prodrug. Such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol, polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.

[0221] Gelatin capsules may contain the active ingredient compounds and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period ofhours. Compressed tablets can be sugar-coated or film-coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.

[0222] For oral administration in liquid dosage form, the oral drug components are combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.

[0223] Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance. In general, water, asuitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. Tn addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmacal Sciences, Mack Publishing Company, a standard reference text in this field.

[0224] Tire compounds used in the method of the present invention may also be administered in intranasal form via use of suitable intranasal vehicles, or via transdennal routes, using those forms of transdennal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdennal deliver}7system, the dosage administration will generally be continuous rather than intermittent throughout the dosage regimen.

[0225] Parenteral and intravenous forms may also include minerals and other materials such as solutol and / or ethanol to make them compatible with the type of injection or deliver}7system chosen.

[0226] The compounds and compositions of the present invention can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by topical administration, injection or other methods, tothe afflicted area, such as a wound, including ulcers of the skin, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.

[0227] Specific examples of pharmaceutically acceptable carriers and excipients that may be used to formulate oral dosage forms of the present invention are described in U. S. Pat. No. 3,903,297 to Robert, issued Sept. 2, 1975. Techniques and compositions for making dosage forms useful in the present invention are described-in the following references: 7 Modem Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drags and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences. Vol 61 (Alain Rolland, Ed., 1993); Drug Deliver,’ to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.

[0228] Tire active ingredient can be administered orally in solid dosage forms, such as capsules, tablets, powders, and chewing gum; or in liquid dosage forms, such as elixirs, syrups, and suspensions, including, but not limited to, mouthwash and toothpaste. It can also be administered parentally, in sterile liquid dosage forms.

[0229] Solid dosage forms, such as capsules and tablets, may be enteric-coated to prevent release of the active ingredient compounds before they reach the small intestine. Materials that may be used as enteric coatings include, but are not limited to, sugars, fatty acids, proteinaceous substances such as gelatin, waxes, shellac, cellulose acetate phthalate (CAP), methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxy propyl methyl cellulose phthalate, hydroxy propyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), and methyl methacrylatemethacrylic acid copolymers.

[0230] Tire compounds and compositions of the invention can be coated onto stents for temporary or permanent implantation into the cardiovascular system of a subject.

[0231] Variations on those general synthetic methods will be readily apparent to those of ordinary skill in the art and are deemed to be within the scope of the present invention.

[0232] In the compounds used in the method of the present invention, alkyl, heteroalkyl, monocycle, bicycle, aryl, heteroaryl and heterocycle groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.

[0233] It is understood that substituents and substitution patterns on the compounds used in the method of the present invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.

[0234] The compounds used in the method of the present invention may be prepared by techniques well known in organic synthesis and familiar to a practitioner ordinarily skilled in the art. However, these may not be the only means by which to synthesize or obtain the desired compounds.

[0235] The compounds used in the method of the present invention may be prepared by techniques described in Vogel’s Textbook of Practical Organic Chemistry, A. I. Vogel, A. R. Tatchell, B. S. Fumis, A. J. Hannaford, P. W. G. Smith, (Prentice Hall) 5thEdition (1996), March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Michael B. Smith, Jerry March, (Wiley-Interscience) 5thEdition (2007), and references therein, which are incorporated by reference herein. However, these may not be the only means by which to synthesize or obtain the desired compounds.

[0236] The various R groups attached to the aromatic rings of the compounds disclosed herein may be added to the rings by standard procedures, for example those set forth in Advanced Organic Chemistry: Part B: Reactions and Synthesis, Francis Carey and Richard Sundberg, (Springer) 5th ed. Edition. (2007), the content of which is hereby incorporated by reference.

[0237] Another aspect of the invention comprises a compound used in the method of the present invention as a pharmaceutical composition.

[0238] Tire compounds used in the method of the present invention may be in a salt form. As used herein, a “salt” is a salt of the instant compounds which has been modified by making acid or base salts of the compounds. In the case of compounds used to treat a disease or medical disorder, the salt is pharmacally acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as phenols; alkali or organic salts of acidic residues such as carboxylic acids. The salts can be made using an organic or inorganic acid. Such acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like.Phenolate salts are the sodium, potassium, or lithium salts, and the like. Carboxylate salts are the sodium, potassium, or lithium salts, and the like. The term "pharmaceutically acceptable salt" in this respect, refers to the relatively non-toxic, inorganic and organic acid or base addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base or free acid form with a suitable organic or inorganic acid or base, and isolating the salt thus fonned. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge et al. (1977) " Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).

[0239] As used herein, "treating" means preventing, slowing, halting, or reversing the progression of a disease. Treating may also mean improving one or more symptoms of a disease.General

[0240] For the foregoing embodiments, each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments.

[0241] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections. All combinations of the various elements disclosed herein are within the scope of the invention.

[0242] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in tire following examples.

[0243] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0244] Examples arc provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, thescope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only.

[0245] Various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative, and not limiting. Every embodiment and feature described in the application should be understood to be interchangeable and combinable with every embodiment contained within.EXAMPLESExample A

[0246] Incorporation of active triphosphate form of Tenofovir (TFV) analogs into RNA by SARS-CoV-2 RdRp (Fig. 3)

[0247] The detailed SARS-CoV-2 RdRp incorporation method is as follows: The RNA template-loop-primer (5'-UUUUCAUCGCGUAGUUUUCAUCGCG-3') was used for extension with F-Tenofovir-DP isomers and ATP. The RNA template-loop-primer was annealed by heating to 75 °C for 3 min and cooling to room temperature in 1 x RdRp buffer. Then, 5 pL of the appropriate annealed RNA template-loop-primer solution (4 pM) was added to 5 pL of 4.3 pM SARS-CoV-2 Nspl2 / Nsp7 / Nsp8 (RdRp complex) and incubated at room temperature for 5 min. Finally, 10 pL of 128 pM (R) F-Tenofovir-DP or 512 pM (. S') F-Tcnofovir-DP, or 10 pL of 1 pM ATP as positive control in 1 X RdRp buffer w as added into the mixture of RdRp and RNA template -loop-primer and incubated at 30 °C for 2 hr. The final extension reaction solution (20 pL) contained 1 pM RNA template-loop-primer, 1.1 pM SARS-CoV-2 RdRp complex, and 64 pM (R) F-Tenofovir-DP or 256 pM (. S’) F-Tenofovir-DP, or 0.5 pM ATP. The lx RdRp reaction buffer contains 10 mM pH 8 Tris-HCl, 10 mM KC1, 2 mM MgCL and 1 mM -mercaptoethanol. Desalting of the reaction was performed first with ZipTip and then with Oligo Clean & Concentrator kit (Zymo Research), resulting in ~10 pL purified aqueous RNA solutions. 1 pL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The remaining ~9 pL RNA products were used to test exonuclease activity.

[0248] The present disclosure showd that the R isomer of F-TFV-DP (b) is incorporated > 10-fold better than the 5 isomer (c) by SARS-CoV-2 RdRpExample B

[0249] SARS-CoV-2 exonuclease resistance of RNAs terminated by TFV analogs (Fig. 4)

[0250] Tire present disclosure examined whether RNA extended with (R) F-TFV showed resistance to excision by SARS-CoV-2 exonuclease (Nspl4) in the presence of the Nspl4 accessory’ protein Nsp 10. Thetemplate-loop-primer RNA (5’-UUUUCAUCGCGUAGUUUUCAUCGCG-3’) was modified with AMP, TFV, and (R) F-TFV at the 3' end using polymerase extension assays as follows:

[0251] The template-loop-primer RNA was purchased from Horizon Discovery (Lafayette, CO). The template-loop-primer RNA was annealed by heating to 75 °C for 3 min and cooling to room temperature in lx SuperScript IV reverse transcriptase (SSRT) buffer. Then, 10 pL of the appropriate annealed RNA template-loop-primer solution (10 pM) was added to 8 pL of the reverse transcriptase solution consisting of 200 U of SuperScript IV RT in lx SuperScript IV RT buffer. Finally. 2 pL of a solution containing 5 mM Tenofovir-DP or (R) F-Tenofovir-DP was added and incubation was carried out for 3 hr at 45 °C. The final extension reaction solution (20 pL) contained 200 U SuperScript IV RT, 5 pM RNA-template-loop-primer, and 500 pM Tenofovir-DP or Adefovir-DP. Desalting of the reaction mixture was performed with Oligo Clean & Concentrator kit (Zymo Research), resulting in ~10 pL purified aqueous RNA solutions. 1 pL of each solution was subjected to MALDI-TOF MS (Broker ultrafleXtreme) analysis. The remaining ~9 pL RNA products were used to test exonuclease activity.

[0252] The 5 pL annealed extended RNA template-loop-primer solutions (2 pM) were added to a mixture of 5 pL of exonuclease Nspl4 / Nspl0 complex (20 nM) in 1 X exonuclease buffer, 9 pL 1 X exonuclease buffer and 1 pL DMSO, and incubate at 37 °C for 15 min. The final concentration of the reagents in the 20 pL reactions were 500 nM different RNAs and 5 nMNspl4 / NsplO. The 1 x exonuclease reaction buffer contains 40 mM Tris-HCl pH 8, 1.5 mM MgCl₂, and 5 mM DTT. After incubation, each reaction was quenched by the addition of 2.2 pL EDTA (100 mM). Following desalting using an Oligo Clean & Concentrator kit (Zymo Research), 1 pL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The signal intensity was normalized to the highest peak.

[0253] (R) F-TFV-RNA (b) and TFV-RNA (c) display ~3x better protection against SARS- CoV-2 ExoN excision than A-RNA (a). (R) F-Tfv-RNA and Tfv-RNA have high resistance against SARS-CoV-2 ExoN excision than natural RNA.Example C

[0254] Incorporation of active triphosphate form of Azvudine (AZV) analogs into RNA by SARS-CoV-2 RdRp (Fig. 6)

[0255] The detailed SARS-CoV-2 RdRp incorporation method is as follows: The RNA template-loop-primer (5’-UUUUCAGCGCGUAGUUUUCAUCGCG-3’) was used for Azvudine-TP and CTP. The RNA was annealed by heating to 75 °C for 3 min and cooling to room temperature in lx RdRp buffer. Then, 5 pL of the appropriate annealed RNA template-loop-primer solution (4 pM) was added to 5 pL of 4 pM SARS-CoV-2 Nspl2 / Nsp7 / Nsp8 (RdRp) complex and incubated at room temperature for 5 min. Finally,10 pL of 1.6 pM Azvudine-TP, or 10 pL of 1.6 M CTP as positive control in lx RdRp buffer was added into the mixture of RdRp and RNA template-loop-primer and incubated at 30 °C for 2 hr. The final extension reaction solution (20 pL) contained 1 pM RNA template-loop-primer, 1 pM SARS-CoV-2 RdRp complex, and 0.8 pM Azvudine-TP or 0.8 pM CTP. The lx RdRp reaction buffer contains 10 mM pH 8 Tris-HCL 10 mM KCL 2 mM MgCL and 1 mM P-mercaptoethanol. Desalting of the reaction was performed first with ZipTip and then with Oligo Clean & Concentrator kit (Zymo Research), resulting in ~10 pL purified aqueous RNA solutions. 1 pL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The remaining ~9 pL RNA products were used to test exonuclease activity.

[0256] The present disclosure showed that Azvudine-TP is incorporated by SARS-CoV-2 RdRp ~4 times less efficiently than CTP.Example D

[0257] Evaluation of termination and further extension of AZV extended RNA using SARS-CoV-2 RdRp reactions (Fig. 7)

[0258] The present disclosure used RNA extended with AZV-TP at the 3 ’terminus to determine the termination activity, with CTP extended RNA as control. The template-loop-primer RNA extended with AZN or CMP was produced from the extension reaction as follows:

[0259] The RNA template-loop-primer (5’-UUUUCAGCGCGUAGUUUUCAUCGCG-3’) was annealed by heating to 75 °C for 3 min and cooling to room temperature in lx RdRp buffer. Then, 5 pL of the appropriate annealed RNA template-loop-primer solution (10 pM) was added to 5 pL of 4 pM SARS-CoV-2 Nspl2 / Nsp7 / Nsp8 (RdRp complex) and incubate at room temperature for 5 min. Finally, 10 pL of 22 pM CTP or 44 pM AZV-TP, was added into the mixture of RdRp and RNA template-loop-primer and incubate at 30 °C for 2 hr. The final extension reaction solution (20 pL) contained 2.5 pM RNA templateloop-primer, 1 pM SARS-CoV-2 RdRp complex, and 11 pM CTP or 22 pM AZV-TP. Desalting of the reaction was performed with Oligo Clean & Concentrator kit (Zymo Research), resulting in ~10 pL purified aqueous RNA solutions. 1 pL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The remaining ~9 pL extended RNA products were used to test termination activity.

[0260] The AZV or CMP extended RNA were further extended with ATP to evaluate tennination efficiency. A solution of 5 pL of 4 pM SARS-CoV-2 Nspl2 / Nsp7 / Nsp8 (RdRp complex) was added to 5 pL of C extended RNA (C-RNA) or 5 pL of Azvudine extended RNA (Azv-RNA) template-loop-primer solutions (4 pM) and incubated at room temperature for 5 min in 1 x RdRp buffer. Then, 10 pL of ATP (0.8 pM or 3.2 pM) in 1 x RdRp buffer was added into the mixture of RdRp with the extended RNAs and incubated at 30 °C for 2 hr. The final extension reaction solution (20 pL) contains 1 mM tenninated RNA,1 pM SARS-CoV-2 RdRp complex, and different concentrations of ATP. The 1 x RdRp reaction buffer contains 10 mM pH 8 Tris-HCl, 10 mM KC1, 2 mM MgCL and 1 mM [3-mercaptoethanol. Desalting of the reaction was performed with Oligo Clean & Concentrator kit (Zymo Research), resulting in ~10 pL purified aqueous RNA solutions. 1 pL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis.

[0261] The present disclosure showed that RNA extended by Azvudine-TP (Azv-RNA) significantly inhibits subsequent nucleotide incorporation by RdRp.Example E

[0262] Evaluation of SARS-CoV-2 exonuclease (ExoN) resistance of RNAs terminated by ZN (Fig.8)

[0263] The RNAs extended with AZV (AZV-RNA) and CMP (C-RNA) were obtained as described in the previous example. C-RNA was used as the control for the experiments. A single tube RNA excision reaction was performed to determine the exonuclease resistance of AZV as explained below:

[0264] A 10 pL mixture of the same amount of annealed AZV-RNA and C-RNA template-loop-primer solution (2 pM) was added to a mixture of 5 pL of exonuclease Nspl4 / Nspl0 complex (20 nM) in 1 Xexonuclease buffer, 9 pL lx exonuclease buffer and 1 pL DMSO, and incubate at 37 °C for 15 min. The final concentration of the reagents in the 20 pL reactions were 500 nM different RNAs and 5 nM Nspl4 / Nspl0. The lx exonuclease reaction buffer contains 40 mM Tris-HCl pH 8, 1.5 mM MgCE. and 5 mM DTT. After incubation, each reaction was quenched by the addition of 2.2 pL EDTA (100 mM). Following desalting using an Oligo Clean & Concentrator kit (Zymo Research), 1 pL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The signal intensity was normalized to tire highest peak.

[0265] The present disclosure showed that Azvudine (Azv) terminated RNA (Azv-RNA) has no resistance to SARS-CoV-2 ExoN. similar to natural RNA (C-RNA).Example F

[0266] F: Incorporation of active triphosphate form of 3 ’-deoxyadenosine into RNA by SARS-CoV-2 RdRp (Fig. 9)

[0267] The detailed SARS-CoV-2 RdRp incorporation method is as follows: The RNA template-loop-primer (5’-UUUUCAUCGCGUAGUUUUCAUCGCG-3’) for 3’-dATP and ATP was used. The RNA was annealed by heating to 75 °C for 3 min and cooling to room temperature in lx RdRp buffer. Then, 5 pL of the appropriate annealed RNA template-loop-primer solution (4 pM) was added to 5 pL of 4 pM SARS-CoV-2 Nspl2 / Nsp7 / Nsp8 (RdRp complex) and incubate at room temperature for 5 min. Finally, 10 pL of 8 pM Azvudine-TP, or 10 pL of 1 pM ATP as positive control in lx RdRp buffer was added into the mixture of RdRp and RNA template-loop-primer and incubated at 30 °C for 2 hr. The final extension reaction solution (20 pL) contained 1 pM RNA template-loop-primer, 1 pM SARS-CoV-2 RdRp complex, and 4 pM 3’-dATP or 0.5 pM ATP. The lx RdRp reaction buffer contains 10 mM pH 8 Tris-HCl, 10 mM KCl, 2 mM MgCl₂ and 1 mM β-mercaptoethanol. Desalting of the reaction was performed first with ZipTip and then with Oligo Clean & Concentrator kit (Zymo Research), resulting in ~10 pL purified aqueous RNA solutions. 1 pL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis.

[0268] Tire present disclosure showed that 3’-deoxyATP is incorporated by SARS-CoV-2 RdRp ~8 times less efficiently than ATP.Example G

[0269] Evaluation of triphosphate form of 5’-CH2-RDV as terminator of the SARS-CoV-2 RdRp reactions (Fig. 14)

[0270] The 5'-CH2-RDV, RDV and AMP extended RNAs (5’-CH2-RDV-RNA, RDV-RNA and A-RNA, respectively) were produced using our polymerase extension assay as described below:

[0271] The RNA template-loop-primer (5’-UUUUCAUCGCGUAGUUUUCAUCGCG-3’) was annealed by heating to 75 °C for 3 min and cooling to room temperature in lx RdRp buffer. Then, 5 pL of the appropriate annealed RNA template-loop-primer solution (10 pM) was added to 5 pL of 4 pM SARS-CoV-2 Nspl2 / Nsp7 / Nsp8 (RdRp complex) and incubate at room temperature for 5 min. Finally, 10 pL of 5 pM ATP, 100 pM 5’-CH2-RDV-DP or 20 pM RDV-DP, was added into the mixture of RdRp and RNA template-loop-primer and incubate at 30 °C for 2 hr. The final extension reaction solution (20 pL) contained 2.5 pM RNA template-loop-primer, 1 pM SARS-CoV-2 RdRp complex, and 2.5 pM ATP, 50 pM 5'-CH2-RDV-DP or 40 pM RDV-DP. Desalting of the reaction was performed with Oligo Clean & Concentrator kit (Zymo Research), resulting in ~10 pL purified aqueous RNA solutions. 1 pL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The remaining ~9 pL extended RNA products were used to test termination activity.

[0272] 5'-CH2-RDV-RNA, RDV-RNA and A-RNA were annealed by heating to 75 °C for 3 min and cooling to room temperature in lx RdRp buffer. Then. 5 pL of the appropriate annealed RNA template-loop-primer solution (2 pM) was added to 5 pL of 4 pM SARS-CoV-2 Nspl2 / Nsp7 / Nsp8 (RdRp complex) and incubate at room temperature for 5 min. Finally, 10 pL mixture of 1 pM UTP, 1.5 pM GTP and 1 pM ATP was added into the mixture of RdRp and RNA template-loop-primer and incubated at 30 °C for 2 h. The 20 pL extension reaction buffer contained 500 nM RNA template-loop-primer, 1 pM SARS-CoV-2RdRp complex, and a mixture of 2 pM UTP, 3 pM GTP, and 2 pM ATP. Desalting of the reaction was performed with Oligo Clean & Concentrator kit (Zymo Research), resulting in ~10 pL purified aqueous RNA solutions. 1 pL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis.

[0273] 5’-CH2-RDV-TP acted as a delayed chain terminator of the SARS-CoV-2 RdRp reaction. Example H

[0274] Incorporation of active triphosphate form of S’-NFb-RDV into RNA by SARS-CoV-2 RdRp (Fig. 15)

[0275] The detailed SARS-CoV-2 RdRp incorporation method is as follows: The RNA template-loop-primer (5’-UUUUCAUCGCGUAGUUUUCAUCGCG-3 ) for 3’-NH2-RDV was used for the extension experiment. Tire RNA was annealed by heating to 75 °C for 3 min and cooling to room temperature in lx RdRp buffer. Then, 5 pL of the appropriate annealed RNA template-loop-primer solution (4 pM) was added to 5 pL of 4 pM SARS-CoV-2 Nspl2 / Nsp7 / Nsp8 (RdRp complex) and incubate at room temperature for 5 min. Finally, 10 pL of 256 pM S’-NFF-RDV in lx RdRp buffer was added into the mixture of RdRp and RNA template-loop-primer and incubated at 30 °C for 2 hr. The extension reaction solution (20 pL) contained 1 pM RNA template-loop-primer, 1 pM SARS-CoV-2 RdRp complex, and 128 pM 3'-NH₂-RDV. The 1 x RdRp reaction buffer contains 10 mM pH 8 Tris-HCl, 10 mM KCl, 2 mM MgCl₂ and 1 mM β-mercaptoethanol. Desalting of the reaction was performed first with ZipTip and then with Oligo Clean & Concentrator kit (Zymo Research), resulting in ~10 pL purified aqueous RNA solutions. 1 pL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis.

[0276] The present disclosure showed that 3’-NH2-Remdesivir-TP was incorporated by SARS-CoV-2 RdRp as a terminator of RNA synthesis.Example I

[0277] Incorporation of active triphosphate form of 3 ’-deoxyadenosine (3’-dA) and Remdesivir into RNA by DENV RdRp (Fig. 18)

[0278] The detailed DENV-2 RdRp incorporation method is as follows: The RNA template-loop-primer (5 -UUUUCAUCGCGUAGUUUUCAUCGCG-3 ) for 3’-dATP and RDV-TP was used in this extension reaction. The RNA was annealed by heating to 75 °C for 3 min and cooling to room temperature in 1 x extension buffer. Then, 5 pL of the appropriate annealed RNA template-loop-primer solution (4 pM) was added to 5 pL of 4 pM DENV-2 and incubate at room temperature for 5 mins. Finally, 10 pL of 4 pM 3’-dATP, 10 pL of 4 pM RDV-TP or 10 pL of 2 pM ATP as positive control in lx extension buffer was added into the mixture of RdRp and RNA template-loop-primer and incubated at 30 °C for 2 hr. The extension reaction solution (20 pL) contained 1 pM RNA template-loop-primer, 1 pM DENV-2 RdRpcomplex, and 2 LIM 3’-dATP or 2 pM RDV-TP or 1 pM ATP. The lx extension reaction buffer contains 50 mM pH 7.5 Tris-HCl, 50 mM KC1, 6 mM MgCl2and 2 mM DTT. Desalting of the reaction was performed first with ZipTip and then with Oligo Clean & Concentrator kit (Zymo Research), resulting in ~10 pL purified aqueous RNA solutions. 1 pL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis.

[0279] The present disclosure showed that 3 -dATP has a similar DENV-2 RdRp incorporation ability as ATP and better than that of RDV.Example J

[0280] Evaluation of antiviral activity of nucleotides and their prodrugs against DENV-2 and CHIKV in cellular assays (Table 1 and Table 2)

[0281] Huh-7 (Table 1) or Huh-7.5 (Table 2) cells were either CHIKV or DENV infected at a MOI of 0.05 (Table 1) and MOI of 0.1 (Table 2) and treated for 2 days, after which, the supernatant was harvested, and the viral RNA levels were quantified via RT-PCR. The cytotoxicity of tire compounds was also evaluated in Huh-7 cells and Huh-7.5 cells, allowing calculation of the selectivity index (SI) as the ratio of the CCA and EC50 values.

[0282] 5'-CH2-RDV and 3’-NH2-RDV displayed ~10-fold better inhibition than the previously best-known nucleotide-based inhibitor RDV for DENV and Sofosbuvir for CHIKV. 3’-deoxyadenosine (3’-deoxyA), 3'-deoxyA isobutyryl ester prodrug, and 5’-CH2-3’-deoxyA disoproxil prodrug displayed 3~6 folds better inhibition than tire previously best-knowm nucleotide -based inhibitor (Sofosbuvir) for CHIKV.Example K

[0283] Synthesis of the triphosphate fonns of Tenofovir (TFV) analogs (X=F, CN, N3, OH or other related groups) (Fig. 2B)

[0284] Adenine is alkylated with an X (F, CN, N3, OH or other related groups) derivatized 4-(X-modified)-1.3-dioxolan-2-one (R or S) to afford 9-((l-X-modified-2-hydroxyl)propyl)adenine, which is further alkylated w ith tosylated hydroxymethylphosphonate diester (DESMP) yielding diethyl phosphonate ester. Tire diethyl phosphonate ester is then hydrolyzed affording phosphonic acid, which is further converted to X-modified Tenofovir-diphosphate by phosphorylation. For synthesis of NH2-TFV-DP, the Ns-Tenofovir-diphosphate is treated with THP yielding the expected NH2-TFV-DP.Example L

[0285] Example synthesis of disoproxil and alafenamide prodrugs of (R) X-TFV or (. S') X-TFV, (X = F, CN, N3. NH2. OH or other related groups) (Fig. 5)

[0286] X-modified Tenofovir phosphonate (see Fig. 2) is esterified with chloromethyl isopropylcarbonate in the presence of EtjN installing the isopropyl carbonate ester groups and affording the disoproxil prodrug of X-TFV. The resulting N3-TFV prodrug is treated with TPP to generate the NH2-TFV prodrug.

[0287] X-modified Tenofovir phosphonate (see Fig. 2) is treated with phenol in the presence of DCC and DMAP, generating tire phenyl ester, which is further activated by thionyl chloride forming tire chlorophosphonate derivative. Coupling of the isopropyl ester of L-alanine to the chlorophosphonate affords the phenoxy isopropyl L-alaninate phosphoramidate moiety, leading to the alafenamide prodrug of X-TFV. The resulting N3-TFV prodrug can be further treated with TPP to generate the NH3-TFV prodrug.Example M

[0288] Synthesis of isobutyric ester, alafenamide prodrugs and triphosphate of l’-CN-Azvudine (A) and Synthesis of isobutyric ester, alafenamide prodrugs and triphosphate of 3'-deoxy-RDV (B) (Fig. 10)

[0289] (A) 3’,5’-O protected 3’-deoxy-P-D-ribofuranose is converted to dihydro-2(3H)-furanon by oxidation, from which 2’-fluorinated-dihydro-furanone is generated by treatment with (C. SCfCO. followed by substitution with DAST. The resulting 2’-F-dihydro-2(3H)-furanone is converted to I’-OH-C-nucleoside by treatment with N6protected 3-bromo-3-deaza-cytosine. The resulting 1 '-OH nucleoside is converted to l’-CN by treatment with TMSCN, affording l’-cyano-2’-F-3-deaza-cytidine C-nucleoside. The deprotection of 3’,5’-O silyl affords the 5 ’-OH nucleoside, which is further iodinated and reacted with DBU to afford a 4’-ene compound via an elimination reaction. The subsequent reaction with NaNs and IC1 results in l'-cyano-2’-F-3’-OH-4'-N3-5’-iodo-3-deaza-cytidine C-nucleoside. After protection of the 3’-OH, the 5 ’-iodo group is converted the to 5 ’-OH group by treatment with benzoic acid and sliver acetate, followed by the hydrolysis of 5’-O-Bz group. Tire resulting 5 ’-OH group is masked by treatment with chlorophosphoramidate and N-methyl imidazole, yielding 5’-O-phosphoramidate, and the subsequent deprotection of 3 -0 silane results in the alafenamide prodrug of l’-cyano-2’-F-3’-OH-4’-N3-3-deaza-cytidine C-nucleoside (I'-CN-AZV). The isobutyryl ester prodrug of I'-CN-AZV is synthesized by esterification of the 5 '-OH group with isobutyric anhydride, followed by deprotection of the 3’-0 silane with TBAF. Triphosphorylation on 5’-OH and the following 3’-O-deprotection generates the expected T-CN-Azvudine-TP.

[0290] (B) Synthesis of l’-cyano-3’-deoxy-4-aza-7,9-dideaza-adenosine C-nucleotide phosphoramidate. 3’, 5 -0 protected (with Benzyl and TBDMS respectively) 3’-deoxy-P-D-ribofuranose is converted to dihydro-2(3H)-furanon by? oxidation, which is then treated with 9-bromo-4-aza-7,9-dideazaadenosine to form I’-OH-C-nucleoside. The resulting I’-OH is replaced with the cyano group by treatment with TMSCN, affording l’-cyano-3’-deoxy-4-aza-7,9-dideazaadenosine C-nucleoside. Selectivedeprotection of 5’-O- generates 5 ’-OH group, which is further masked with freshly prepared chlorophosphoramidate reagent in the presence of N-methyl imidazole, yielding the l’-CN-3’ -deoxy -nucleotide phosphoramidate. The subsequent deprotection of 2’-0 results in expected l’-cyano-3’-deoxy-4-aza-7,9-dideaza-adenosine C-nucleotide phosphoramidate prodrug (alafenamide prodrug of 3'-Deoxy-RDV). The isobutyryl ester prodrug of 3'-deoxyRDV is synthesized by esterification of 5 '-OH of the 2'-0 silyl protected 3'-deoxy RDV with isobutyric anhydride, followed by deprotection of the 2 -0 with BCls. For the synthesis of 3’-deoxy-RDV-TP, 5’-OH intermediate is treated with BChto remove the 2’-0-Bn protection, then triphosphorylation on 5 ’-OH generates the expected 3’-deoxy-RDV-triphosphate.Example N

[0291] Synthesis of l’-cyano-3’-dcoxy-5’-CH2-4-aza-7,9-didcaza-adcnosinc C-nuclcotidc (5’-CH2-3 ’-deoxy RDV) alafenamide prodrug (A) and disoproxil prodrug (B). (Fig. 11)

[0292] (A) 2'-OH and exocyclic-NH2protected 5’ -methyl enephosphonic acid derivative of 3 ’-deoxy RDV is reacted with phenol to couple to the phosphonic acid using DCC / DMAP to generate the monophenyl ester. This is further activated by thionyl chloride treatment, and coupled with isopropyl ester of L-alanine. The deprotection of 2 ’-OH results in the phenoxy isopropyl L-alaninate phosphoramidate prodrug of 5’-CH2-3’-deoxy RDV (5’-CH2-deoxy RDV alafenamide).

[0293] (B) 2’,5’-O protected 3’-deoxy-0-D-ribofuranose is converted to dihydro-2(3H)-furanone by oxidation, which is treated with 9-bromo-4-aza-7,9-dideazaadenosine to generate I’-OH-C-nucleoside. The resulting l'-OH is converted to a cyano group by treatment with TMSCN, affording l’-cyano-3’-deoxy-4-aza-7,9-dideazaadenosine C-nucleoside. The subsequent selective 5’-O-deprotection generates free 5 ’-OH group, which is further converted to an aldehyde by oxidation. The Wittig reaction using methylenetriphenylphosphorane treatment generates a 5’-ene compound, which is converted to 5’-CH2-phosphonate by addition of diethyl phosphonate to the double bond. The hydrolysis of 5’-CH2-diethyl phosphonate results in the 5’-CH2phosphonic acid. Then chloromethyl isopropyl carbonate is used to esterify the phosphonic acid, followed by removal of 2’-0 and N6 protective groups with BCI3 and TBAF resulting in the disoproxil prodrug of 5’-CH2-3’-deoxy RDV.Example O

[0294] Synthesis of disoproxil (A) and alafenamide (B) prodrugs of 5’-CH2-RDV and triphosphate of 5 ’-CH2-RDV (C). (Fig.12)

[0295] (A) 2’,3'-OH and exocyclic-NH2protected 5’-methylenephosphonic acid derivative of RDV is reacted with chloromethyl isopropyl carbonate to esterify the phosphonic acid. The subsequent deprotection of 2’,3’-OH and exocyclic-NH2under acidic conditions results in the disoproxil prodrag of 5’-CH2-RDV.

[0296] (B) For the preparation of alafenamide prodrug of 5’-CH2-RDV, the same procedure was used as shown in Fig. 11A, except for the final deprotection of 2’,3’-O and N6 protective groups with TFA.

[0297] (C) After deprotection of 2’, 3 ’-OH and exocyclic-NH2 groups, the resulting 5’-methylenephosphonic acid derivative of RDV is activated with CDI followed by treatment with pyrophosphate to yield the triphosphate of 5’-CH2-RDV (5’-CH2-RDV-TP).Example P

[0298] Synthesis of isobutyric ester (A) and ProTide (B) prodrugs of 3’-NH2-RDV and triphosphate of 3 -NH2-RDV (C). (Fig. 13)

[0299] (A) Starting from r-cyano-3’-azido-4-aza-7,9-dideaza-adenosine C-nucleoside, the ester prodrug of 3'-NH;-RDV is synthesized by esterification of the 5 ’-OH with isobutyric anhydride, followed by reductive conversion of the 3’-N?, to NH2 using triphenylphosphine.

[0300] (B) Starting from S’-Ns-RDV. chlorophosphoramidate andN-methyl imidazole is used to mask the 5 ’ -OH group, yielding 5 ’ -O-phosphoramidate, and the subsequent reductive conversion of 3 ’ -N3 to NH2 with triphenylphosphine results in the ProTide prodrug form of 3’-NH2-RDV.

[0301] (C) Triphosphorylation on the 5 ’-OH of S’-Ns-RDV and the subsequent reductive conversion of 3’-Ns to NH2 with THP yields the active triphosphate form of 3’-NH2-RDV (3’-NH2-RDV-TP).Example Q

[0302] Synthesis of disoproxil (A) and alafenamide (B) prodrugs of 3'-NH2-5'-CH2-RDV and triphosphate of 3’-NH2-5’-CH2-RDV (C). (Fig. 16)

[0303] (A) Using the 3’-azido-5'-methylenephosphonic acid derivative of RDV as starting material, the 2'-OH is protected, followed by esterification of the phosphonic acid with chloromethyl isopropyl carbonate. The subsequent reduction of 3’-N2to NH2using triphenylphosphine and the deprotection of 2’-OH yields the disoproxil prodrug of 3’-NH2-5’-CH2-RDV.

[0304] (B) Tire same starting material is used to synthesize the alafenamide prodrug of 3’-NH2-5’- CH2-RDV. After protection of the 2’-OH with silane, phenol is coupled to the phosphonic acid to generate the phenyl ester, which is further activated by thionyl chloride. Coupling of the isopropyl ester of L-alanine to the chlorophosphonate installs the phenoxy isopropyl L-alaninate phosphoramidate moiety, which results in the alafenamide prodrug of 3’-NH2-5’-CH2-RDV after reduction of 3’-N2to NH2with triphenylphosphine and 2'-OH deprotection.

[0305] (C) To investigate the mechanism of action of the above 2 prodrugs, the 3’-azido-5’-methylenephosphonic acid derivative of RDV is used to generate 3’-NH2-5’-CH2-RDV triphosphate via CDI activation and pyrophosphate coupling, followed by reduction of 3 ’-Ns to NH2with THP.Example R

[0306] Synthesis of disoproxil (A) and alafenamide (B) prodrugs of 3’-F-5'-CH2-RDV and triphosphate of 3’-F-5'-CH2-RDV (C). (Fig.17)

[0307] (A) With 3’-F-5’-methylenephosphonic acid derivative of RDV as starting material, the 2'-OH is protected, then the phosphonic acid is esterified by treatment with chloromethyl isopropyl carbonate. Subsequent deprotection of the 2’-OH yields the disoproxil prodrug of 3’-F-5’-CH2-RDV.

[0308] (B) The same starting material is used to synthesize the alafenamide prodrug of 3’-F-5'-CH2- RDV After protection of the 2 ’-OH with silane, phenol is coupled to the phosphonic acid to generate the phenyl ester, which is further activated by thionyl chloride. Coupling of the isopropyl ester of L-alanine to chlorophosphonate installs the phenoxy isopropyl L-alaninate phosphoramidate moiety, which leads to the alafenamide prodrug of 3’-F-5’-CH2-RDV after deprotection of2'-OH.

[0309] (C) Finally, the 3’-F-5’-methylenephosphonic acid derivative of RDV is used to generate the triphosphate of 3'-F-5’-CH2-RDV through CDI activation and pyrophosphate coupling for performing enzymatic assays and for structural studies.Example S

[0310] Synthesis of disoproxil and alafenamide prodrugs (A) and triphosphate of 5’-CH2-3’dA (B).(Fig. 19)

[0311] (A) Oxidation of the starting material 2’-0 and N6 protected 3’-deoxyadenosine results in the 5’-aldehyde intermediate, which is converted to the 5’-alkene product by treatment with tetraethyl methylenediphosphonate in the presence of t-ButOK. Further reduction of the alkene results in 5’-CH2-phosphonate-3 ’deoxyadenosine, which is hydrolyzed to give 5’-CH2-phosphonic acid-3’-deoxyadenosine. Chloromethyl isopropyl carbonate is used to esterify 5’-CH2-phosphonic acid-3 '-deoxyadenosine, and subsequent deprotection of 2’-0 silyl with TBAF results in the disoproxil prodrug of 5'-CH2-3'-deoxyA. For the preparation of alafenamide prodrug of 5’-CH2-3’-deoxyA, the same procedure was used as shown in Fig. 11A, except for the final deprotection of 2’-0 protective group with TBAF.

[0312] (B) Dess-Martin periodinane oxidation of the starting material, 2’-0 and N6protected 3’-deoxyadenosine, results in the 5 ’-aldehyde intermediate, which is converted to the 5 ’-alkene product by treatment with tetraethyl methylenediphosphonate in the presence of t-ButOK. Further reduction of the alkene yields 5’-ethylphosphonate-3’-deoxyadenosine, which is readily hydrolyzed to give 5’-CH2-phosphonic acid-3 ’-deoxyadenosine. Activation of the phosphonic acid with 1,1’ -carbonyldiimidazole and the subsequent coupling of the resulting tributylammonium pyrophosphate yields the triphosphate. Finally, deprotection generates tire expected product, 5’-CH2-3’-deoxyadenosine-triphosphate.Example T

[0313] Synthesis of isobutyryl ester and alafenamide prodrug of 3'-deoxyA. (Fig. 20)

[0314] Tire isobutyryl ester prodrug of 3'-dcoxyA was synthesized by esterification of 5 ’-OH of the 2’-0 silyl protected 3'-deoxyA with isobutyric anhydride, followed by deprotection of the 2’-0 with TBAF. Treatment of 3'-deoxyA with freshly prepared chlorophosphoramidate reagent in the presence of N-methyl imidazole generates alafenamide prodrug of 3 ’-deoxyadenosine after removal of 2’-0 protection.Example U

[0315] Synthesis of disoproxil and alafenamide prodrug of 5’-CH2-Azvudine. (Fig. 21)

[0316] For synthesis of 5’-CH2-AZV, the intermediate for synthesis of Azvudine 2’-F-3’-OH-4’-N3-5’-I-Nb-Bz -cytidine is used as starting material. After protection of 3 ’-OH, 5 ’-I is converted to 5 ’-OH, which is further oxidized to 5’-aldehyde. The Wittig reaction using methylenetriphenylphosphorane treatment generates a 5'-ene compound, which is converted to 5’-CH2-phosphonate by addition of diethyl phosphonate to the 5’-ene. Hydrolysis of 5’-CH2-diethyl phosphonate results in 5’-CH2 phosphonic acid of AZV. Then, chloromethyl isopropyl carbonate is used to esterify the phosphonic acid, and the removal of 3’-0 and N6 protection with NH3 / MeOH and TBAF results in the disoproxil prodrug of 5’-CH2-AZV. For the preparation of alafenamide prodrug of 5’-CH2-AZV, same procedure was used as shown in Fig. HA, except for the final deprotection of 3’-OTBS was carried out with TBAF, andNHBz with NHJMcOH. Example V

[0317] Synthesis of disoproxil and alafenamide prodrug of 5'-CH2-Azvudine. (Fig. 21)

[0318] RNA oligonucleotides (Template-Loop-Primers) were purchased from Dhannacon or IDT. Azvudine triphosphate was purchased from Arctom Scientific. NTPs were purchased from Thermo Scientific. Other chemicals were purchased from Fisher Scientific or Sigma- Aldrich. The RdRp and ExoN of SARS-CoV-2 were cloned and purified as described (Wang 2022).

[0319] Primer extension reactions with Azvudine-TP and CTP in the same tube.

[0320] The RNA template-loop-primer (sequences shown in Fig. 23a) was annealed by heating to 75 °C for 3 min and cooling to room temperature in lx RdRp buffer. Then, 5 pL of 4 pM SARS-CoV-2 Nspl2 / Nsp7 / Nsp8 (RdRp complex) was added to 5 pE annealed RNA template-loop-primer solution (4 pM) and incubated at room temperature for 5 min. Finally, 10 pL of 2 pM CTP and 80 pM Azvudine-TPin lx RdRp buffer were added to the mixture of RdRp and RNA template-loop-primer and stopped at 7.5, 15, 30, and 60 min. The 20 pL extension reaction buffer contained 1 pM RNA template-loop-primer, 1 pM SARS-CoV-2 RdRp complex, and 1 pM CTP and 40 pM Azvudine-TP. The lx RdRp reaction buffer contained 10 mM pH=8 Tris-HCl, 10 mM KC1, 2 mM MgCh and 1 mM [3-mercaptoethanol. The reactions were quenched with 2.2 pL 100 mM EDTA at 7.5, 15, 30, and 60 min. Desalting of the reaction was perfonned with an Oligo Clean & Concentrator kit (Zymo Research), resulting in ~10 pL purified aqueous RNA solutions. 1 pL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtream) analysis. The remaining ~9 pL RNA products were used to test exonuclease activity and to perform further elongation.

[0321] Production of Azvudine-terminated RNAs.

[0322] Different RNA template-loop-primers (5’-UUUUCUGCGCGUAGUUUUCAUCGCG-3’, 5’- UUUUCAGCGCGUAGUUUUCAUCGCG-3’, 5’-UUUUCCGCGCGUAGUUUUCAUCGCG-3’ or 5’-UUUUCGGCGCGUAGUUUUCAUCGCG-3’) were annealed by heating to 75 °C for 3 min and cooling to room temperature in lx RdRp buffer. Then, 5 pL of 4 pM SARS-CoV-2 Nspl2 / Nsp7 / Nsp8 (RdRp complex) was added to 5 pL annealed RNA template-loop-primer solution (10 pM) and incubated at room temperature for 5 min. Finally, 10 pL 256 pM Azvudine-TP in lx RdRp buffer were added to tire mixture of RdRp and RNA template-loop-primer and incubated at 30 °C for 2 hr. The 20 pL extension reaction buffer contained 1 pM RNA template-loop-primer, 1 pM SARS-CoV-2 RdRp complex, and 128 pM Azvudine-TP. The lx RdRp reaction buffer contained 10 mM pH=8 Tris-HCl, 10 mM KC1, 2 mM MgCL and 1 mM p-mercaptoethanol. Desalting of the reaction was performed with an Oligo Clean & Concentrator kit (Zymo Research), resulting in ~10 pL purified aqueous RNA solutions. 1 pL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtream) analysis. The remaining ~9 pL RNA products were used to test exonuclease activity or to perform further elongation.

[0323] Single base elongation of nucleotide / nucleotide analog-extended RNA.

[0324] The C-tenninated RNA or Azvudine-terminated RNA was annealed by heating to 75 °C for 3 min and cooling to room temperature in lx RdRp buffer. Then, 5 pL of 4 pM SARS-CoV-2 Nspl2 / Nsp7 / Nsp8 (RdRp complex) was added to 5 pL of C-terminated RNA (C-RNA) or Azv-terminated RNA (4 pM) and incubated at room temperature for 5 min. Finally, 10 pL of the NTP (20 pM for time dependent subsequent nucleotide hindrance shown in Fig. 24) for the next base incorporation in lx RdRp buffer was added into the mixture of RdRp and tenninated RNA and incubated at 30 °C for 7.5, 15, 30, and 60 min for time dependent reactions (Fig. 24). The 20 pL extension reaction buffer contained 1 mM terminated RNA, 1 pM SARS-CoV-2 RdRp complex, and different concentrations of the NTP (10 pM for time dependent subsequent nucleotide hindrance shown in Fig. 24) for the next base incorporation. The lxRdRp reaction buffer contains 10 mM pH=8 Tris-HCl, 10 mM KC1, 2 mM MgCl? and 1 mM 0-mercaptoethanol. The reactions were quenched with 2.2 pL 100 mM EDTA at 7.5, 15, 30, and 60 min. Desalting of the reaction was performed with Oligo Clean & Concentrator kit (Zymo Research), resulting in ~10 pL purified aqueous RNA solutions. 1 pL of each solution was subjected to MALDI-TOF MS (Broker ultrafleXtream) analysis. Tire remaining ~9 pL RNA products were used to test for exonuclease activity.

[0325] Further elongation of nucleotide / nucleotide analog-extended RNA.

[0326] The C-terminated RNA or Azvudine -terminated RNA (sequences shown in Fig. 25a) was annealed by heating to 75 °C for 3 min and cooling to room temperature in lx RdRp buffer. Then, 5 pL of 4 pM SARS-CoV-2 Nspl2 / Nsp7 / Nsp8 (RdRp) complex was added to 5 pL of C terminated RNA (C-RNA) or 5 pL of Azvudine terminated RNA (Azv-RNA) template-loop-primer solutions (4 pM) and incubate at room temperature for 5 min. Finally, 10 pL of 10 pM ATP and 10 pM GTP mixture in lx RdRp buffer was added to the mixture of RdRp and RNA substrates and incubated at 30 °C for 2 hr. The 20 pL extension reaction buffer contained 1 pM RNA substrate, 1 pM SARS-CoV-2 RdRp complex, 5 pM ATP and 5 pM GTP. The lx RdRp reaction buffer contains 10 mM pH=8 Tris-HCl. 10 mM KC1, 2 mM MgCL and 1 mM 0-mercaptoethanol. Desalting of the reaction was performed with Oligo Clean & Concentrator kit (Zymo Research), resulting in ~10 pL purified aqueous RNA solutions. 1 pL of each solution was subjected to MALDI-TOF MS (Broker ultrafleXtream) analysis. The remaining ~9 pL RNA products were used to test exonuclease activity.

[0327] Comparison of SARS-CoV-2 exonuclease reaction for Azvudine (Azv)-terminated RNA with natural RNA in the same tube.

[0328] Tire C-extended RNA (2 pM) and Azv-extended RNA (2 pM) (sequences shown in Fig. 4a) were annealed by heating to 75 °C for 3 min and cooling to room temperature in 1 * exonuclease reaction buffer. After annealing, the two RNAs were mixed in equal volumes. To a lO pL solution of lOnM exonuclease complex (Nspl4 / Nspl0) in lx exonuclease reaction buffer and 10% DMSO. 10 pL annealed RNA mixture (1 pM each) was added and incubated at 37 °C for 15 min. The final concentrations of reagents in the 20 pL reactions were 5 nM Nspl4 / Nspl0 complex, 500 nM C-terminated RNA, 500 nM Azv-terminated RNA and 5% DMSO. After incubation for 15 min, each reaction was quenched by adding 2.2 pL of an aqueous solution of EDTA (100 mM). The lx ExoN reaction buffer contains 50 mM pH=8 Tris-HCl, 2 mM MgCL and 2 mM DTT. Following desalting using an Oligo Clean & Concentrator (Zymo Research), the samples were subjected to MALDI-TOF MS (Broker ultrafleXtreme) analysis.

[0329] Comparison of SARS-CoV-2 exonuclease reaction for full length Azvudine (Azv)-embedded RNA and natural RNA.

[0330] The fully extended Azv-embedded RNA (2 pM) and natural RNA (2 pM) produced from the RdRp extension experiment (sequences shown in Fig. 26c, e) were annealed by heating to 75 °C for 3 min and cooling to room temperature in lx exonuclease reaction buffer. To a 14 pL solution of lOnM exonuclease complex (Nspl4 / Nspl0) in 1 x exonuclease reaction buffer and 1 uL DMSO, 5 pL of annealed RNA (2 pM each) was added and incubated at 37 °C for 15 min. Tire final concentrations of reagents in the 20 pL reactions were 5 nM Nspl4 / Nspl0 complex, 500 nM of fully extended Azv-embedded RNA or natural RNA, and 5% DMSO. The lx ExoN reaction buffer contained 50 mM pH=8 Tris-HCl, 2 mM MgCT and 2 mM DTT. After incubation for 15 min, each reaction was quenched by adding 2.2 pL of an aqueous solution of EDTA (100 mM). Following desalting using an Oligo Clean & Concentrator (Zymo Research), the samples were subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis.

[0331] Production of Azvudine-embedded RNAs with different subsequent nucleotides.

[0332] Different Azv terminated RNAs (5’-UUUUCUGCGCGUAGUUUUCAUCGCGAzv-3’, 5’-UUUUCAGCGCGUAGUUUUCAUCGCGAzv-3 ’, 5 -UUUUCCGCGCGUAGUUUUCAUCGCGAzv-3’ or 5’-UUUUCGGCGCGUAGUUUUCAUCGCGAzv-3’) were annealed by heating to 75 °C for 3 min and cooling to room temperature in lx RdRp buffer. Then, 5 pL of 4 pM SARS-CoV-2 Nspl2 / Nsp7 / Nsp8 (RdRp complex) was added to 5 pL annealed RNA template-loop-primer solution (10 pM) and incubated at room temperature for 5 min. Finally, 10 pL 20 pM corresponding NTP for subsequence nucleotide incorporation in lx RdRp buffer was added to the mixture of RdRp and RNA template-loop-primer and incubated at 30 °C for 2 hr. The 20 pL extension reaction buffer contained 1 pM RNA substrate, 1 pM SARS-CoV-2 RdRp complex, and 10 pM corresponding NTP. The lx RdRp reaction buffer contained 10 mM pH=8 Tris-HCl, 10 mM KC1, 2 mM MgCL and 1 mM p-mercaptoethanol. Desalting of the reaction was performed with an Oligo Clean & Concentrator kit (Zymo Research), resulting in ~10 pL purified aqueous RNA solutions. 1 pL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtream) analysis. The remaining ~9 pL RNA products were used to test exonuclease activity.

[0333] Comparison of SARS-CoV-2 exonuclease reaction for Azv+1 RNAs and natural RNA.

[0334] The Azv+1 RNA (2 pM) and same length natural RNA (2 pM) produced from RdRp extension experiments (sequences shown in Fig. 27 and 28) were annealed by heating to 75 °C for 3 min and cooling to room temperature in 1 x exonuclease reaction buffer. To a 14 pL solution of 10 nM exonuclease complex (Nspl4 / Nspl0) in lx exonuclease reaction buffer and 1 pL DMSO, 5 pL of annealed RNA (2 pM each) was added and incubated at 37 °C for 15 min. The final concentrations of reagents in the 20 pL reactionswere 5 nM Nspl4 / NsplO complex, 500 nM of Azv+1 RNA or natural RNA, and 5% DMSO. The lx ExoN reaction buffer contains 50 mM pH=8 Tris-HCl, 2 mM MgCh and 2 mM DTT. After incubation for 15 min, each reaction was quenched by adding 2.2 pL of an aqueous solution of EDTA (100 mM). Following desalting using an Oligo Clean & Concentrator (Zymo Research), the samples were subjected to MALDI-TOF MS (Broker ultrafleXtreme) analysis.DISCUSSION

[0335] SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) (Nspl2 / 7 / 8) and exonuclease (ExoN) (Nspl4 / 10) play critical roles in viral genome replication and transcription. The proofreader ExoN removes certain nucleotide inhibitors incorporated into the viral RNA, thereby decreasing the efficacy of these drugs for treating COVID-19.

[0336] Tire present disclosure designs and synthesizes novel nucleotide analogs that terminate coronavirus RdRp reaction and resist the viral ExoN excision as potent coronavirus inhibitors. Tire present disclosure uses SAR and rational design to discover nucleoside / nucleotide analogues with enhanced SARS-CoV-2 RdRp reaction tennination activity and ExoN resistance and also designs and synthesizes novel nucleoside / nucleotide analogues that potently inhibit Dengue and Chikungunya Viruses.

[0337] The present disclosure has identified a variety of nucleotide inhibitors of the SARS-CoV-2 RdRp which terminate RNA synthesis, including some that synergize with compounds identified with ExoN inhibitory activity to inhibit viral replication in cellular assays, and others that themselves resist ExoN excision (Ju J 2020; Jockusch S 2020; Chien M 2020; Ju J 2023; Sacramento CQ 2021; Jockusch S 2020; and Wang X 2022). The FDA-approved COVID- 19 drug Remdesivir (RDV) is a delayed terminator but shows minimal resistance to ExoN (Jockusch S 2020; Gordon CJ 2020 and Bravo JPK 2021).

[0338] The present disclosure has investigated a library of nucleotide analogs targeting the SARS-CoV-2 RdRp (Ju J 2020: Jockusch S 2020 and Chien M. 2020). Of particular interest are those incorporated into RNA by the viral RdRp, where they halt or slow further replication, which gives us structure-activity information to further modify these molecules. These include the FDA-approved COVID- 19 drug RDV, a delayed terminator and Tenofovir (TFV), an HIV reverse transcriptase obligate terminator (Jockusch S 2020; Chien M 2020; Gordon CJ 2020; and Bravo JPK2021). However, except for RDV, the active forms of all the other nucleotide analogs cannot compete with the corresponding natural nucleotides. While several nucleoside / nucleotide analogs show promising results against SARS-CoV-2, in vitro pharmacological parameters often exceed human plasma exposure for the clinically approved doses. The present disclosure has identified nucleotide inhibitors based on the TFV and other nucleotide scaffolds that can be incorporated as terminators and resist ExoN excision. Tire present disclosure discloses the nucleotideanalogs which can be incorporated as terminators and resist ExoN excision, and will serve as broadspectrum coronavirus therapeutics.

[0339] The present disclosure designed and synthesized ester prodrugs oftwo RDV analogs, a 5 -CH2-RDV prodrug, which is expected to enhance cellular phosphorylation to the active triphosphate and prevent reversion to the nucleoside, and a 3 ’-NH2-RDV prodrug, which was designed to be an immediate terminator of RdRp. Both prodrugs had more potent antiviral activity against DENV than RDV and Sofosbuvir in Huh7 cell in vitro assays. The present disclosure indicated that these two prodrugs had better inhibitory activity for CEIIKV than Sofosbuvir, and that RDV has no inhibitory activity against CEIIKV. This is consistent with recent reports indicating that RDV has no inhibitory activity for CHIKV (Radoshitzky SR 2023, and Garcia G Jr 2023).

[0340] The present disclosure designed and synthesized 3 ’-deoxyadenosine (3’-dA)-related prodrugs and demonstrated that they have significant anti-viral activities against DENV and CHIKV in cell culture assays. The present disclosure cloned the DENV-2 RdRp and tested the incorporation activity of 3’-dATP using our MALDI-TOF mass spectrometry-based RdRp assays. The present disclosure designed showed that 3’-dATP has similar incorporation activity as the natural nucleotide ATP.

[0341] The present disclosure provides nucleotide inhibitors as coronavirus therapeutics that compete sufficiently with natural nucleotides in vivo and resist removal by ExoN. The present disclosure has implemented two high resolution molecular assays using mass-spectrometry' detection to assess SARS-CoV-2 RdRp and SARS-CoV-2 ExoN activities for screening inhibitors of these enzymes and identifying their mechanism of action. The MS-based approach, due to its exquisite resolution (~10 Da differences), has the advantage of precisely identify ing the nucleotides that arc added or removed from the RNA, which is not feasible with gel-based assays. The present disclosure has shown that Tenofovir-diphosphate (TFV-DP) could be incorporated into RNA as an obligate terminator by SARS-CoV-2 RdRp. but with very low efficiency (Chien M 2020). As an acyclic nucleotide, TFV-DP lacks a sugar ring configuration and it is less likely to be recognized by SARS-CoV-2 ExoN. The present disclosure showed that TFV, once incorporated into RNA, was mostly resistant to excision by the SARS-CoV-2 ExoN (Wang X 2022). This result suggests that a new derivative of TFV-DP designed to have higher RdRp incorporation efficiency, while retaining its substantial resistance to ExoN, would inhibit SARS-CoV-2. Cryo-EM structures of wild-type and mutant SARS-CoV-2 ExoN in complex with an RNA substrate bearing a 3 '-end mismatched nucleotide were recently reported (Liu C 2021).

[0342] The present disclosure determined that effective cleavage by ExoN required a 2’- and 3'-OH on the RNA. Their observations are consistent with the results, where RNA terminated with the acyclic nucleotide TFV, lacking both 2 ’-OH and 3 ’-OH groups, was largely resistant to ExoN excision (Wang X,2022). The molecular modeling results of Tenofovir and ATP in the pre -incorporation state of the SARS-CoV-2 RdRp, reported by Feng et al., is consistent with the enzymatic assay result (Feng, J. Y. 2022). The SARS-CoV-2 RdRp recognizes ribose substrates through hydrogen bond interaction between a set of polar residues (D623, T680, S682, T687, and N691) and the ribose structure. Tenofovir, lacking the 2’ and 3’-OH groups, does not fonn hydrogen bond interaction with the surrounding polar residue at the preincorporation state. The presence of the non-polar methyl group clashes with N691 and makes Tenofovir more incompatible within the polar pocket. Thus, removing the methyl group of Tenofovir to avoid clashes with N691 should increase the compound’s capability to fit in the pocket (Fig. 1C). Adefovir, the Tenofovir derivative lacking the methyl group, was tested in our MS-based assay and was shown to be incorporated onto the RNA by SARS-CoV-2 RdRp while Tenofovir was poorly incorporated.

[0343] To avoid the clash between the methyl group andN691, the present disclosure also synthesized an isomer of Tenofovir, (S)-Tenofovir, which has the methyl group pointed to the opposite direction of N691 as shown in (Fig. ID). The (. S)-Tcnofovir was shown to have 3 times higher incorporation efficiency compared to its R isomer. Since the residue N691 forms H-bond interaction with 2’-OH of ATP and clashes with the nonpolar methyl group of TFV, by replacing -CH3 of Tenofovir with a polar -CH2F (F-Tenofovir) introduces H-bonding and increases the incorporation efficiency of this Tenofovir scaffold-based compound as shown in (Fig. IE). The present disclosure synthesized R and 5 isomers of the fluoro derivative of TFV-DP (F-TFV-DP) (Fig. 2A, B) and discovered that the R isomer is incorporated > 10-fold better than the S isomer by SARS-CoV-2 RdRp (Fig. 3) and displays -3 times better protection against excision from RNA by SARS-CoV-2 ExoN than ATP (Fig. 4). Under the conditions described in Fig. 3, (7?)-TFV-DP was barely incorporated by SARS-CoV-2 RdRp, while (R)-F-TFV-DP was incorporated -350 times more efficiently than TFV-DP. Thus, the present disclosure includes several analogs of Tenofovir in which the F is replaced with CN, N3, NH2, OH or other related groups (Fig.2, 5) as SARS-CoV-2 and other coronaviruses inhibitors.

[0344] Tire present disclosure has also analyzed the mechanism of action of several other nucleotides that have previously been reported to have anti-coronavirus activity using our MALDI-TOF mass spectrometry-based RdRp and ExoN assays, which allow precise determination of the extension and cleavage products of these reactions, with the goal of designing and synthesizing derivatives of these nucleotide analogs to further improve their antiviral activity (Wang X 2022). Azvudine, a cytidine nucleoside analog, has been approved as a therapeutic for COVID- 19 in China (Chen M P, 2024, Chan, 2024 and Zhang 2021). However, its mechanism of action has not been clearly determined at the enzyme level. The present disclosure showed that Azvudine-TP is incorporated by SARS-CoV-2 RdRp -4 times less efficiently than CTP (Fig. 6). Its incorporation into RNA significantly inhibits subsequent nucleotideincorporation (Fig. 7), but shows no identifiable protection against excision from RNA by SARS-CoV-2 ExoN relative to natural RNA (Fig.8). It has also been reported that 3’-deoxynucleotides display substantial resistance to SARS-CoV-2 ExoN excision from RNA than natural nucleotides and 3 ’-deoxyadenosine has been reported to inhibit SARS-CoV-2 and other viruses (Liu C, 2021, Chinthapatla R 2023, Dubhashi S, 2023). The present disclosure detennined that 3'-deoxyATP is incorporated by SARS-CoV-2 RdRp ~8 times less efficiently than ATP (Fig. 9). Thus, it is desirable to further improve the RdRp incorporation efficiency of both Azvudine-TP and 3’-deoxyATP in order to efficiently inhibit coronaviruses. Remdesivir (RDV), due to its I’-CN modification, is incorporated more efficiently than ATP by SARS-CoV-2 RdRp (Malone, 2023). Thus, the present disclosure discloses the Azvudine (Fig. 10A) and 3 ’-deoxyadenosine (Fig. 10B) with l'-CN substitution, their triphosphate and prodrug forms.

[0345] Tire present disclosure discloses nucleoside and nucleotide analogs comprising analogs based on the Remdesivir scaffold with 3' modifications (-H, -F, -Ns, -NIL or other related groups) and their prodrug fonns. This present disclosure also discloses 5’ methylene modifications of Remdesivir, 5’ methylene modifications of 3 ’-deoxyadenosine and 5’ methylene modifications of 3’ modified Remdesivir analogs (Fig. 11) as DENV and CHIKV RdRp inhibitors and effective against infections caused by these viruses.

[0346] The present disclosure described several types of nucleoside-based / nucleotide-based inhibitors that have the ability to block coronavirus replication. Tire first group of compounds are triphosphates and prodrugs of Tenofovir analogs that can be incorporated into RNA by SARS-CoV-2 RdRp where they serve as terminators of the polymerase reaction. Using MALDI-TOF MS-based detection of the SARS-CoV-2 RdRp extension and exonuclease excision products, the present disclosure showed that the (R) F-TFV triphosphate, once incorporated into RNA, shows similar resistance to excision by the SARS-CoV-2 exonuclease as TFV. The other nucleoside analogs Azvudine (AZV), approved in China to treat SARS-CoV-2 infection, and 3 ’-deoxyadenosine (3 ’-deoxy A), have been reported to have antiviral activity against SARS-CoV-2 infection. AZV acts as a non-obligate terminator, once incorporated onto the RNA strand by SARS-CoV-2 and has no resistance toward the proofreading exonuclease. Both AZV and 3'-deoxyA have lower incorporation efficiency than natural NTPs. The present disclosure designed new triphosphate and prodrug structures based on the AZV and 3’-deoxyA scaffolds by adding a l’-CN group to improve SARS-CoV-2 RdRp incorporation efficiency, thereby improving drug potency.

[0347] The present disclosure also described several types of nucleoside-based / nucleotide-based inhibitors that have the ability to block replication of other +ssRNA viruses, such as DENV and CHIKV. The first group of compounds are the triphosphates and prodrugs of RDV analogs. These prodrugs of RDV analogs were detennined to have better antiviral activity against CHIKV and DENV than the control drugsSofosbuvir and Remdesivir (CHIKV and DENV, respectively). The present disclosure designed prodrugs of 5 -CH2-RDV to enhance cellular phosphorylation to the active triphosphate and prevent reversion to the nucleoside. The present disclosure showed that the triphosphate of 5’-CH2-RDV is a delayed terminator of RNA synthesis, using the SARS-CoV-2 polymerase assay. The present disclosure also designed and synthesized triphosphate and prodrug forms of 3 -NH2-RDV. replacing tire 3’-OH of RDV with 3’-NH2, enabling the triphosphate form to act as an obligate and immediate terminator for the RNA polymerase reaction. Addition of 5’-CH2 to 3’-NH2-RDV enhances cellular phosphory lation. The present disclosure showed that 3 ’-deoxy A and its prodrugs have antiviral activity in cell based assays for both DENV and CHIKV. The present disclosure have shown that 3 ’-deoxy ATP can be incorporated better than Remdesivir-TP by DENV RdRp in our MALDI-TOF MS-based assay. The present disclosure also synthesized triphosphates and prodrugs of 5’-CH2-3'-deoxyA for enhanced phosphorylation in cells and to prevent reversion to the nucleoside.

[0348] The present disclosure designed and synthesized three Remdesivir (RDV) related prodrugs, the disoproxil prodrug of 5 -CH2-RDV and an isobutyryl ester prodrug of 3’-NH2-RDV, and demonstrated that they have significant anti-viral activities against DENV and CHIKV in cell culture assays (Table 1). In Huh-7 cells, these compounds displayed ~ 10-fold better inhibition than the previously best-known nucleotide-based inhibitors of these viruses (RDV for DENV and Sofosbuvir for CHIKV). Molnupiravir has been recently reported to have limited activity for flaviviruses such as DENV and ZIKV (Wang 2023).A direct comparison in Huh-7 cells shows that the 3’-NH2-RDV prodrug has much higher antiviral activity for DENV than RDV, Sofosbuvir and Molnupiravir. For instance, the 5’-CH2-RDV prodrug had an EC50 of 0.05 pM for DENV compared to RDV with an EC50 of 0.7 pM and Sofosbuvir with an EC50 of 3.4 pM. For CHIKV, the 5’-CH2-RDV prodrug had an EC50 of 0.1 pM, while RDV had no activity and Sofosbuvir had an EC50 of 1.2 pM. The 3’-NH2-RDV prodrug had an EC50 of 0.06 pM for DENV and 0.7 pM for CHIKV. The cytotoxicity profiles of both prodrugs (5’-CH2-RDV and 3’-NH2-RDV) are 3-4 times better than that of RDV. In a separate experiment in Huh-7 cells at an MOI at 0.5. 3'-NH2-RDV isobutyryl ester prodrug had an EC50 of 0.09 pM, compared to Remdesivir, which had an EC50 of 1.2 pM.

[0349] Table 1(A) - Antiviral activities of the compounds tested against DENV & CHIKV.DENV CHIKVCompounds ECso(pM) CCso(pM) SI ECso(pM) CCso(pM) SI Sofosbuvir 3.4 ± 0.5 320 ± 23 94 1.2 ± 0.2 320 ± 23 106 Molnupiravir >10 70 ± 15 / 8.3 ± 0.7 70 ± 15 8 Remdesivir (RDV) 0.7 ± 0.1 98 ± 32 140 No Activity 98 ± 32 / 3’-NH2-RDV Isobutyryl Ester 0.06 ±341 ± 22 5683 0.7 ± 0.1 341 ± 22 487Prodrug 0.020.05 ±5’-CH2-RDV Disop roxil Prodrug 393 ± 23 7860 0.1 ± 0.03 393 ± 23 39300.02

[0350] Table 1(B) - Prodrug of S’-NHi-RDV exhibits more potent antiviral activity than RDV for DENV-2 in vitro (Huh-7 cells).Patancya gainst DENV-2 in Drug fcM) CC«(MM)mean *SEM _ mean*SEM MO! -0.05Remdesivir (RDV) 0.71=0.1 1.2*1.3 98*32 3s-NH RDV isobury ryl 3’~NH2-RDV isobutyryl ester Prodfug 0.08*0.02 0.09=43.05 341*23 ester Prodrug

[0351] Tire present disclosure synthesized a 11 brary of RdRp inhibitors based on the RDV scaffold and discovered that an isobutyryl ester of 3’-NH2-RDV displayed ~10-fold better in vitro antiviral activity (better EC50 and higher CC50 values) for DENV-2 serotype than Remdesivir (RDV), previously known as the most potent nucleotide-based inhibitor of this virus. The 3’-NH2-RDV prodrug had an EC50 of 0.06 pM for DENV-2 at an MOI of 0.05 and 0.09 pM at an MOI of 0.5. The cytotoxicity profile of this prodrug, with an SI >1000, was ~3.5 times better than that of RDV.

[0352] Huh-7 cells were infected with CHIKV or DENV at an MOI of 0.05 and treated Ih after inoculation. 2 days after infection, the supernatant was harvested, and quantitative RT-PCR was performed to detect viral RNA levels.3 ’- lfe-RDV Isobutyryl Ester Prodrug 5 "-CPb-RDV Disoproxil Prodrug

[0353] The synthetic schemes for the different prodrug fonns and triphosphates of 5'-CH2-RDV and 3’-NH2-RDV are shown in Fig. 12 and Fig. 13, respectively.

[0354] DENV, CHIKV and SARS-CoV-2 are all +ssRNA viruses. The present disclosure has cloned and expressed large amount ts of the SARS-CoV-2 RdRp complex (Nspl2 / 7 / 8). Therefore, the present disclosure initially used this RdRp complex to evaluate how well the 5’-CH2-RDV-TP and 3’-NH2-RDV-TP are incorporated in the RdRp reaction, relative to the natural substrate ATP, to elucidate theirmechanisms of action. Fig. 14 shows the results for 5’-CH2-RDV-TP and Fig. 15 shows the results for 3’-NHz-RDV-TP.

[0355] The SARS-CoV-2 RdRp reaction results (Fig. 14) indicate that 5’-CH2-RDV-TP acts as a delayed RNA chain terminator. Three RNA substrates (Fig. 14a) A-RNA, (Fig. 14b) CH2-RDV-RNA or (Fig. 14c) RDV-RNA were produced by extension with ATP, 5’-CH2-RDV-TP or RDV-TP, respectively. These RNA substrates were then evaluated for their ability to be further extended by SARS-CoV-2 RdRp in the presence of natural nucleotides complementary to the template strand. MALDI-TOF MS results indicated that extension of the A-RNA was nearly complete, with incorporation of 5 nucleotides (as indicated by the peak at i+5) (Fig. 14d). The major extension product for 5 -CH2-RDV-RNA is an extended RNA with 3 nucleotides incorporated (i+3) (Fig. 13e). Thus, fewer nucleotides were incorporated by RdRp compared to the natural A-RNA, indicating that 5’-CH2-RDV-TP has delayed RNA chain termination activity. RDV-RNA also showed delayed chain termination activity with the dominant extension product at i+3 followed by i+4 (Fig. 14f).

[0356] Replacement of the 3 '-OH by a 3’-NH2 turns RDV into an immediate obligate terminator. As indicated in Fig. 15, the 3'-NH2-RDV-TP is a substrate for SARS-CoV-2 RdRp. where it acts as a terminator of further RNA extension. The 5’-CH2-3’-NH2-RDV prodrugs and triphosphates were synthesized based on the synthetic route in Figs. 16. The 5’-CH2-3’-F-RDV prodrugs and triphosphates were synthesized based on the synthetic route in Figs. 17.

[0357] The present disclosure designed and synthesized 3 ’-deoxyadenosine (3’-dA) related prodrugs and demonstrated that they have significant anti-viral activities against DENV and CHIKV in cell culture assays (Table 2). In Huh-7.5 cells, some of these compounds displayed better inhibition than the previously best-known nucleotide-based inhibitors of these viruses (RDV for DENV and Sofosbuvir for CHIKV). A direct comparison in Huh-7.5 cells shows that the 3’-dA and its isobutyryl ester prodrug have much higher antiviral activity for CHIKV than Sofosbuvir. The 3’-dA isobutyryl ester prodrug had an EC?o of 0.2 pM, while RDV had no activity and Sofosbuvir had an EC50 of 1.3 pM. The 3’-dA isobutyryl ester prodrug had an EC50 of 0.07 pM for DENV compared to RDV with an EC50 of 0.05 pM and Sofosbuvir with an EC50 of 2.1 pM. The nucleoside 3 '-dA had an EC50 of 0.1 pM for DENV and 0.2 pM for CHIKV. Tire cytotoxicity profiles of both compounds are 10 times better than that of RDV. In a separate experiment in Huh-7 cells, 3'-deoxyA isobutyryl ester prodrug had an EC50 of 0.2 pM (MOI = 0.05) and an EC50 of 0.7 pM (MOI = 0.5) against CHIKV, compared to Sofosbuvir, which had an EC50 of 1.2 pM (MOI = 0.05) and an EC50 of 8.9 pM (MOI = 0.5) against CHIKV.

[0358] Table 2(A) - Antiviral activities of the compounds tested against DENV & CHIKV. Prodrug of 3’-dA and 5’-CH2-3’-dA exhibit more potent antiviral activity than Sofosbuvir for CHIKV in vitro.DENV CHIKVCompounds ECso(pM) CCso(pM) SI EC5o(pM) CCso(pM) SI Sofosbuvir 2.1 ± 0.2 138 ± 2.1 66 1.3 ± 0.5 138 ± 2.1 1060.05 ±Remdesivir 0.03 33 ± 1.5 660 >10 33 ± 1.5 / 3'-deoxyA 0.1 ± 0.02 291 ± 18 2910 0.2 ± 0.05 291 ± 18 14550.07 ±3'-deoxyA Isobutyryl Ester Prodrug 289 ± 23 4129 0.2 ± 0.03 289 ± 23 14450.043’-deoxyA ProTide Prodrug >10 312 ± 2 / >10 312 ± 2 / 5’-CH2-3’-deoxyA Disoproxil Prodrug 1.9 ± 0.5 32 ± 1.5 17 0.5 ± 0.2 32 ± 1.5 64 5’-CH2-3’-deoxyA ProTide Prodrug >10 387 ± 2.7 1.05 ± 0.2 387 ± 2.7 369 /

[0359] Table 2(B) - Novel nucleotide analog prodrug 3’-deoxy-A ester displayed potent inhibition of CHIKV in vitro.CHIKV Antiviral Activity of 3'-deoxy~A Prodrug Wteacy agsiwt <11 IKV m vHm CMotaxic C&ncentratho F ECAO CP. M) W teanfoS^^Drug MOI 0.05 MOI 0.5Sfoosbuvir L2±0.2 OMJ 321M233 Meexy-A-Ester Prodmg 0J-M03 0.7-M4 280 for

[0360] In prel im in ary experiments, the present disclosure showed that an isobutyryl ester prodrug form of 3’-dA had a 10-fold lower EC50 and a 2-fold higher CC50 relative to Sofosbuvir, currently the best available drug candidate for treating CHIKV, using CHIKV-infected Huh-7 cells. Tire present disclosure designed and synthesized a disoproxil prodrug of 5'-CH2-3'-dA to improve conversion to triphosphate inside cells and showed that it also had a higher potency than Sofosbuvir.

[0361] Huh-7.5 cells were infected with CHIKV or DENV at an MOI of 0.1 and treated Ih after inoculation. 2 days after infection, the supernatant was harvested, and quantitative RT-PCR was performed to detect viral RNA levels.3'~deoxyA 3*-deoxyA Isobutyryl Ester Prodnig 3’-deoxyA ProTide Prodrug5’-CHa-3’- eoxyA Disoproxil Prodrug 5 '-CHi-d’-deoxyA ProTide Prodrug

[0362] The present disclosure cloned the DENV-2 RdRp and tested the incorporation activity of 3’-dATP using our MALDI-TOF mass spectrometry-based RdRp assays (Fig. 18). Tire present disclosure showed that 3'-dATP has similar incorporation activity as the natural nucleotide ATP and is -2 times better than Remdesivir-TP. The 3 ’-deoxy A prodrugs used for the cellular assay and triphosphates used for enzymatic assays were synthesized based on the synthetic route in Figs. 19 and 20. The synthesis of disoproxil and alafenamide prodrugs of 5’-CH2-3’-deoxyA are shown in Fig. 19A. The triphosphate of the 5’-CH2-3’-deoxyA is show n in Fig. 19B. Isobutyryl ester and alafenamide prodrugs of 5’-CH2-3’-deoxyA are shown in Fig. 20. Additional synthetic scheme for disoproxil and alafenamide prodrug of 5’-CH2-Azvudine is shown in Fig.21.

[0363] The cytidine analog Azvudine-TP (Azv-TP) is incorporated into RNA by SARS-CoV-2 RdRp less efficiently than CTP.

[0364] Azv-TP has a cytosine base with a modified ribose ring. To study the mechanism of action of Azvudine (Azv), the present disclosure first assessed whether Azv-TP can be incorporated by SARS-CoV-2 RdRp using a MALDI-TOF MS-based enzymatic assay using CTP as the control (Wang 2022).

[0365] To further investigate the relative incorporation ability of Azv-TP in the presence of CTP in RNA elongation by RdRp, a time course experiment was performed in which CTP and Azv-TP were mixed at a 1:40 ratio, and the products were analyzed by MALDI-TOF MS (Fig. 23a). Both C-RNA (-8191 Da) and Azv-RNA (-8231 Da) peaks showed a time-dependent increase with comparable decreases of the initial RNA substrate (-7885 Da) (Fig. 23b). The two peaks maintained a 2-3-fold difference at all time points, indicating that Azv-TP was incorporated -100 times less efficiently than CTP. This lower incorporationcould be ascribed to the lack of a hydroxyl group at the 2’-position and the presence of the 4’-azido group on the ribose of Azv-TP.

[0366] Azv significantly inhibits incorporation of the subsequent nucleotide.

[0367] Azv has been reported to interrupt with RNA / DNA synthesis in other viruses and Klumpp et al. demonstrated that Azv is an obligate terminator for hepatitis C virus RdRp (Wang 201; Xu 2020; Chang 2022; Klumpp 2008 and Picarazzi 2020). The present disclosure showed that Azv hindered subsequent base incorporation in a time-dependent manner (Fig. 24). Tire lower incorporation of the nucleotide following Azv suggests that Azv functions as a non-obligate tenninator in RNA elongation and can be partially overcome by high concentrations of the subsequent nucleotide.

[0368] To investigate whether Azv could hinder RNA synthesis beyond the i+1 position, an excess of 5 pM of each NTP was mixed with C-RNA or Azv -RNA to overcome the tennination effect at the i position (Fig. 25a). As shown in Fig. 25b, the natural C-RNA showed full extension with no evidence of RdRp stalling (C-RNA+6). In contrast, Azv-RNA extension resulted in a major product of 5 nucleotides being incorporated (Azv at position i, Fig. 25c), with a significant portion of Azv-RNA (—20%) remaining unrcactcd. These results showed that Azv acts as a non-obligatc terminator in RNA elongation that slows down the overall RNA synthesis.

[0369] Azv is excised by ExoN when positioned at the 3’-tenninus of the RNA but resists excision when located one nucleotide upstream of the 3 ’-terminus.

[0370] To determine whether RNA terminated with Azv can resist proofreading ExoN cleavage, the present disclosure characterized the RNA products of tire ExoN reaction using the MALDI-TOF MS-based assay. A mixture of C-RNA (-8194 Da) and Azv-RNA (-8234 Da) was incubated with ExoN. yielding the cleavage profile shown in Fig. 26a, b. The similar decrease in both RNA substrate peaks indicates that Azv, when located at the 3 ’-end of the RNA, does not resist ExoN excision.

[0371] However, the polymerase reaction results combined with the ExoN reaction results in Fig. 26b are insufficient to fully explain Azvudine’s strong antiviral activity for SARS-CoV-2 both in vitro and in vivo (Ren 2020; Sun 2023; Wu 2021 and Yu 2020). The present disclosure therefore performed the following experiments to explore whether the elongated RNA with embedded Azv displays any ExoN resistance. Fully extended natural RNA (Fig. 26c) and Azv-embedded RNA (Fig. 26e) were produced for the ExoN excision experiments and the products were analyzed by MALDI-TOF MS. As shown in Fig.26d, all natural RNAs were cleaved to the end of the double-stranded portion of the RNA substrate, whereas cleavage of the Azv-embedded RNA halted at the i+1 position where Azv is at position z, generating Azv-RNA+A (-8572 Da, Fig. 26f). To further examine tire ExoN resistance of this specific RNA, Azv-RNA+Aand C-RNA+A were produced and incubated with ExoN (Fig. 27a, b). While the natural C-RNA+A was completely cleaved by ExoN (Fig. 27c), Azv-RNA+A showed almost complete ExoN resistance as indicated by the negligible degraded RNA peaks (Fig. 27d). Finally, to investigate whether the resistance of Azv is affected by the specific nucleotide at the i+1 position, RNAs with tire other three nucleotides (C, G, U) at the i+1 position were incubated with ExoN (Fig. 28). The results indicated that RNAs with Azv incorporated at position z, followed by C, G. or U at the i+1 position, also showed high resistance to ExoN cleavage (Figs. 28). These results demonstrated that RNA with Azvudine incorporated one nucleotide upstream of 3 ’-terminus strongly resists cleavage by SARS-CoV-2 proofreading exonuclease.

[0372] In conclusion, the present disclosure revealed the detailed molecular mechanism of Azvudine’s SARS-CoV-2 antiviral activity, indicating that it targets both the virus’s RdRp and ExoN. The present invention showed that Azv-TP can be incorporated by SARS-CoV-2 RdRp into the viral RNA, even in the presence of its natural counterpart CTP. Tire Azv nucleotide acts as a non-obligate tenninator that hinders further RNA extension. Tire present disclosure demonstrated that the ExoN resistance of Azv-embedded RNA is dependent on the position of the incorporated Azv. When Azv is located at the 3 ’-terminus, the RNA has no ExoN resistance, whereas when Azv is located at the penultimate position from the 3’-terminus, the RNA exhibits near-complete resistance to ExoN cleavage. This resistance and inhibition of proofreading allows accumulation of Azv in the RNA, which would slow down viral RNA synthesis catalyzed by RdRp, ultimately reducing viral replication in host cells. Moreover, because both RdRp and ExoN active sites are highly conserved among the coronaviruses, Azvudine may serve as a broad-spectrum therapeutic for this viral family (Rona 2022: Yan 2022; Wang 2022; Xu 2020 and Chang 2022).

[0373] Positive-stranded RNA (+ssRNA) viruses are a large and diverse group belonging to three broad phyla, whose genomes act as mRNAs for direct translation to viral proteins by host ribosomes. Flaviviruses and togaviruses are +ssRNA viruses utilizing RNA-dependent RNA polymerases (RdRps) to replicate their genomes and transcribe their viral RNAs. These viruses do not possess a proofreading exonuclease, and this enhances the likelihood of developing successful oral therapeutics based on nucleotide analogs targeting the viral RdRps.

[0374] For DENV, the present disclosure has synthesized a library of RdRp inhibitors and identified a novel nucleoside ester prodrug of 3’-NH2-Remdesivir which displays ~10-fold better in vitro antiviral activity (better EC50 and higher CC50 values) for DENV-2 serotype than Remdesivir (RDV), previously known as the most potent nucleotide-based inhibitor of this virus. The prevent disclosure determined that the 3’-NH2-RDV triphosphate is an immediate DENV-2 RdRp reaction terminator.

[0375] For CHIKV, present disclosure designed and synthesized isobutyryl ester prodrug of 3’-deoxyadenosine and disoproxil prodrug of 5 ’-CH2-3’ -deoxyadenosine. In cellular assays, these compoundsdisplay ~10-fold beter inhibition than the previously best-known nucleotide-based inhibitor, Sofosbuvir, against CHIKV.

[0376] The present disclosure relates to nucleosides / nucleotides which are inhibitors or terminators of viral RNA dependent RNA polymerases (RdRp). These nucleosides / nucleotides or their prodrugs can be used to treat infections caused by coronaviruses, flaviviruses, alphaviruses and other positive strand RNA viruses.

[0377] The cytidine analog Azvudine showed promising therapeutic effects against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), comparable to Paxlovid, leading to its conditional authorization as a COVID-19 treatment in China. Once Azvudine enters infected cells, it is converted to the active form, Azvudine-triphosphate, which has been suggested to inhibit viral RNA-dependent RNA polymerase (RdRp). However, the detailed enzymatic mechanisms of Azvudine inhibition of SARS-CoV-2 have not been determined. Here the present disclosure show that when Azvudine is incorporated into RNA, it hinders incorporation of the subsequent nucleotide. More importantly, the present disclosure demonstrate that when Azvudine is incorporated at the penultimate position of the RNA strand, it prevents RNA cleavage by the viral proofreading exonuclease (ExoN). These properties of Azvudine slow viral RNA synthesis and thereby contribute to its antiviral activity. 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Claims

CLAIMSWhat is claimed is:

1. A compound having the structure:whereinBASE is adenine, guanine, cytosine, thymine, uracil or derivatives thereof;[A]Ri, R2, R3. RU Rs and Re are each independently -H, halogen, -CN. -Ns, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAc, -COOH, alkyl, alkenyl, alkynyl, -ORI3, -COR]3, -SH, -SR13, -SO2RI3, -NHRIS, -NR14R15, -NHCORis, or -CONR14R15,wherein RI3, Ru, and R15 are each independently -H, alkyl, alkenyl, alkynyl, aldehyde, ketone, ester, ether, carboxylate, aryl, or heteroaryl;11 is 0-10;Z is -O-, -CH₂-, -NH-, or -S-; and[B](a) Ri is -CN, R2 is -OH, R4 is -NH2, and R3, R5 and Re are each independently -H, halogen, - CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAC, -COOH, alkyl, alkenyl, alkynyl, -ORI3, -CORI3, -SH, -SRi3, -SO3RI3, -NHRJ3, -NR14R15, -NHCORI3, or - CONR14R15,(b) Ri is -CN, R3is -F, R4 is -OH, Re is -N3, and R2 and R5 are each independently -H, halogen, -CN. -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2. -OCF3. -OCH2F, -NO2. -OAc, -COOH, alkyl, alkenyl, alkynyl, -OR,,. -COR,,. -SH, -SRB, -SO2R3, -NHRB, -NR S, - NHCORis, or -CONR4R5, or(c) Ri is -CN, R2 is -OH, R is -F, and R3, R5 and are each independently -H, halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -0CF3, -OCH2F, -NO2, -OAc, -COOH, alkyl, alkenyl, alkynyl, -ORI3, -COR13, -SH, -SRI3, -SO2R13, -NHR13, -NR14R15, -NHCOR13, or - CONR14R15.n is 0-10;Z is -O-, -CH₂-, -NH-, or -S-; andY is -OH,R?. or ■ wherein R7, Rs, R% Rio are each independently halogen, -CN, -N3, -NH2, -OH, -CHF2, - CH2F, -CFS, -OCHF2, -OCFs, -OCH2F, -NO2, -OAc, -COOH, alkyl, alkenyl, alkynyl, - OR13, -COR13, -SH, -SR13, -SO2R3, -NHR13, -NR14R5, -NHCOR13, -CONR4R5, or O O HO-P11^ / -O-P11-jiOH OH wherein Rn. R14, and R15 are each independently -H, alkyl, alkenyl, alkynyl, aldehyde, ketone, ester, ether, carboxylate, aryl, or heteroaryl; and t is 0-10; andn is 0-10;or a pharmaceutically acceptable salt or ester thereof.

2. A compound having the structure:whereinBASE is adenine, guanine, cytosine, thymine, uracil or derivatives thereof;(a) Ri is -CN, R2 is -OH, R4 is -NH2, and R3, Rs and Re are each independently -H, halogen, - CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHFZ, -OCF3, -OCH2F, -NO2, -OAC, -COOH, alkyl, alkenyl, alkynyl, -ORB, -CORB, -SH, -SRB, -SO2R, -NHRB, -NR14R15, -NHCORB, or - CONR14R15,(b) Ri is -CN, R3is -F, R4 is -OH, Re is -N3, and R2and R5 are each independently -H, halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAc, -COOH, alkyl, alkenyl, alkynyl, -ORB, -CORB, -SH, -SR, -SO2R, -NHRB, -NRWR, -NHCORB, or -CONR14R15,(c) Ri is -CN. R2is -OH, R4 is -F, and R3, Rs and Re are each independently -H, halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAC. -COOH, alkyl, alkenyl, alkynyl, -ORB, -CORB, -SH, -SRB, -SO2RB, -NHRB, -NR14R15, -NHCOR, or -CONRuRis,(d) R3is -F, R4 is -OH, Re is -N3, and Ri, R2and R5 are each independently -H, halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAC, -COOH, alkyl, alkenyl, alkynyl, -ORB, -CORB, -SH, -SRB, -SO2RB, -NHR, -NR14R15, -NHCOR, or -CONR14R15.(e) BASE is■, R2is -OH, and Ri, R3, R4, Rs, and Re are each independently -H, halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAc, -COOH, alkyl, alkenyl, alkynyl, -OR, -CORB, -SH, -SRB, -SO2R, -NHRB, -NRUR, -NHCORB, or -CONR14R15, or(f) BASE is 1, Ri is -CN, R2is -OH, R4 is -OH, and R3, Re and Re are each independently -H, halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAC, -COOH, alkyl, alkenyl, alkynyl, -ORB, -COR, -SH. -SRB, -SO2RB, -NHRB, -NR14R15, -NHCORB, or -CONR14R15.wherein RB, R14, and Ris are each independently -H, alkyl, alkenyl, alkynyl, aldehyde, ketone, ester, ether, carboxylate, aryl, or heteroaryl;n is 0-10;Z is -0-, -CH2-, -NH-, or -S-; andwherein when Y is -OH,(i) then BASE is1, Ri is -CN; R2is -OH, R4 is -NH2, Rs, Rs Rs are each -H, n is 0 and Z is -CH2-, or(ii) then BASE is1, Ri is -CN; Rs is -F, R4 is -OH, Rs is -Ns, R2and Rs are each -H, n is 0 and Z is -CH2-; and11HO- P- lwherein when Y is OH,» / vw(iii) then BASE is1, Ri is -CN; R2is -OH, R4 is -NH2, Rs, R5 Re are each -H, n is 1 and Z is -CH2-;IEN(iv) then BASE is or1, Ri is -CN; R2is -OH, Rs, R4, Rs Rs are each - H, n is 1 and Z is -CH2-;then BASE is. R2is -OH, Ri, R3, R4, Rs, Rs are each -H, n is 1 and Z is -(vi) then BASE is ', R3 is -F: R4 is -OEI, Rs is -N3, Ri, R2, Rs are each -H, n is 1 and Z is -CH2-;or a pharmaceutically acceptable salt or ester thereof.

3. The compound of claim 1, wherein(a) in [A], Ri, R2, R3, R4, Rs and s are each independently -H, halogen, -CN, -N3, -NH2, - OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3. -OCH2F, -NO2, -OAc, or -COOH;(b) in [B](a), R3. Rs and R are each independently -H, halogen, -CN, -N3, -NH2, -OH. - CHF2, -CH2F, -CF3, -OCHF2, -0CF3, -OCH2F. -NO2, -OAc. or -COOH:(c) in [B](b), R2and Rs are each independently -H, halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CFS, -OCHF2, -OCF3, -OCH2F, -NO2, -OAC, or -COOH;(d) in [B](c), R3, Rs and Rs are each independently -H, halogen, -CN, -N3, -NH2, -OH, - CHF2, -CH2F, -CF3, -OCHF2, -0CF3, -OCH2F. -NO2, -OAc, or -COOH;(e) n is 0-5;(f) t is 0-5;(g) Z is -0- or -CH2-; and / or(h) Y is -OH,OrR9,wherein R7, Rs. R9, Rio are each independently halogen, -CN, -N3, -NH2, -OH, - CHF2, -CH2F, -CF3, -OCHF2, -OCR, -OCH2F, -NO2, -OAC, -COOH, alkyl, alkenyl, alkynyl, -NHRI3, -NR14R15, or -OR13.

4. The compound of claim 2, wherein(a) wherein in (a), Rs, Rs and Re are each independently -H, halogen, -CN, -N3, -NH2, - OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAc, or -COOH;(b) wherein in (b), R2 and Rs are each independently -H, halogen, -CN, -N3, -NH2, -OH, - CHF2, -CH2F, -CF3, -OCHF2, -OCFS, -OCH2F, -NO2, -OAC, or -COOH;(c) wherein in (c), R3, R5 and Re are each independently -H, halogen, -CN, -N3, -NH2, - OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3. -OCH2F. -NO2, -OAC, or -COOH;(d) wherein in (d), Ri, R2and Rs are each independently-H, halogen, -CN, -Ns, -NH2, - OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAc, or -COOH;(e) wherein in (e), Ri, R3, R4, Rs, and Re are each independently-H, halogen, -CN, -Ns, - NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCFS, -OCH2F, -NO2, -OAC, or -COOH; (f) wherein in (f), R3, R5and Re are each independently-H, halogen. -CN, -N3, -NH2, - OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCFS. -OCH2F. -NO2, -OAC, or -COOH;(g) n is 0-5; and / or(h) Z is -0- or -CH2-.

5. The compound of claim 1, wherein(a) in [A], Ri, R2, R3, R4, Rs and Re are each independently -H, -F. -Cl, -CN, -Ns, -NH2, - OH, -CHF2, -CH2F, or -CF3:(b) in [B](a), R3, Rs and Re are each independently -H, -F, -Cl, -CN, -N3, -NH2, -OH, - CHFs, -CH2F, or -CF3;(c) in [B](b), R2 and Rs are each independently -H, -F, -Cl, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, or -CFs;(d) in [B](c), Rs. Rs and Re are each independently -H, -F. -Cl, -CN, -Ns, -NH2, -OH, - CHF2, -CH2F, or -CFs:(e) n is 0-3;(f) t is 0-2;(g) Z is -0- or -CH2-; and / orO(h) Y is -OH,Orwherein R7, Rs. R9, Rio are each independently halogen, -CN, -N3, -NH2, -OH, -CHF2, - CH2F, -CF3, -OCHF2, -OCFs, -OCH2F, -NO2, -OAc, -COOH, alkyl, -NHRis, -NR14R15, or -ORis6. The compound of claim 2, wherein(a) wherein in (a), R3, Rs and Re are each independently -H, -F, -Cl, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, or -CF3;(b) wherein in (b), R2and R5 are each independently -H, -F, -Cl, -CN, -Ns, -NH2, -OH, - CHF2, -CH2F, or -CF3;(c) wherein in (c). R3, R5 and Re are each independently -H. -F, -Cl. -CN, -N3, -NH2, -OH, -CHF2, -CH2F, or -CF3:(d) wherein in (d), Ri, R2and R5 are each independently-H, -F, -Cl, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, or -CF3;(e) wherein in (e), Ri, R3, R4, Rs, and Re are each independently-H, -F, -Cl, -CN, -Ns, - NH2. -OH, -CHF2, -CH2F, or -CF3:(f) wherein in (f), Rs, Rs and Re are each independently-H, -F, -Cl, -CN, -Ns, -NH2. -OH, -CHF2, -CH2F, or -CFs:(g) n is 0-3;and / or(h) Z is -0- or -CH2-.

7. The compound of claim 1, wherein(a) in [A], Ri, R2, Rs, R4, Rs and Re are each independently -H, -F. -CN, -Ns, -NH2, or - OH;(b) in [B](a), R3, Rs and Re are each independently -H, -F, -CN, -Ns, -NH2, or -OH;(c) in [B](b), R2and Rs are each independently -H, -F, -CN, -Ns, -NH2, or -OH;(d) in [B](c), Rs. Rs and Rs are each independently -H, -F, -CN, -Ns, -NH2, or -OH;(e) n is 0-2;(f) t is 0-2; and / or(g) Z is -0- or -CH2-;O(h) Y is -OH,, or,wherein R7, Rs, R9, Rio are each independently halogen, -CN, -Ns, -NH2, -OH, -CHF2, - CH2F, -CFS, -OCHFS, -OCFs, -OCH2F, -NO2, -NHR13, Ci-C6alkyl, -NR14R15, or -OR13, wherein R13, Ru. and R15 are each independently aldehyde, ketone, ester, ether, carboxylate, or aryl.

8. The compound of claim 2, wherein(a) wherein in (a), Rs, Rs and Rs are each independently -H, -F, -CN, -Ns, -NH2, or -OH; (b) wherein in (b), R2 and Rs are each independently -H, -F, -CN, -Ns, -NH2, or -OH; (c) wherein in (c), Rs, Rs and Re are each independently -H, -F, -CN, -Ns, -NH2, or -OH; (d) wherein in (d), Ri, R2 and Rs are each independently— H, -F, -CN, -Ns, -NH2, or -OH; (e) wherein in (e), Ri, Rs, R4, Rs, and Re are each independently-H, -F, -CN, -Ns, -NH2, or -OH;(f) wherein in (f), Rs, Rs and Re are each independently-H, -F, -CN, -Ns, -NH2, or -OH; (g) n is 0-2; and / or(h) Z is -0- or -CH2-.

9. The compound of claim 1, wherein in [B],(a) Ri. R2. Rs are each independently -H, -F. -CN. or -OH; and R4, Rs and Rs are each independently -H, -F, -CN, -Ns, -NH2, or -OH;(b) n is 0-1;(c) t is 0-2; and / orO(d) Y is -OH,OrR9wherein R7, RS, R RIO are each independently -OH, -NHR13, C1-C3 alkyl, or -OR13.

10. Tire compound of claim 9, wherein12. The compound of any one of claims 1-11, wherein the derivative of adenine, guanine, cytosine, thymine, and uracil has the structures:oR20 whereinRis, RI. R20, R21, and R22 are each independently -H, halogen, -CN, -N3, -NH3, -OH, -CHF3, -CH3F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO3, -OAc, -COOH, alkyl, alkenyl, alkynyl, -ORI3, -COR]3, -SH, -SR13, -SO3RI3, -NHRIS, -NR14R15, -NHC0R13, or -CONR14R15,wherein R|3. R14, and R15 are each independently -H, alkyl, alkenyl, alkynyl, aldehyde, ketone, ester, ether, carboxy late, aryl, or heteroaryl;preferably, Ris, R19, R20, R21, and R22 are each independently -H, halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2. -OCF3, -OCH2F, -NO2, -OAc, -COOH, alkyl, alkenyl, alkynyl, -ORI3, -CORI3, -SH, or -SRI3,more preferably, Ri. Ri R20, R21, and R22 are each independently -H, halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCFs, -OCH2F, or -NO3;more preferably, Ris, R19, R20, R21, and R33are each independently -H, or -NH3;more preferably, the derivative is13. The compound of claim 2 having the structure:wherein BASE is adenine, guanine, cytosine, uracil, thymine or derivatives thereof, and R is -OH, -F, -N3, or -NH2.

14. The compound of claim 2 having the structure:wherein BASE is adenine, guanine, cytosine, uracil, thymine or derivatives thereof.

15. The compound of claim 10 having the structure:wherein BASE is adenine, guanine, cytosine, uracil, thymine or derivatives thereof, and R is -F. -Ns, or -NH2.

16. The compound of claim 2 having the structure:wherein BASE is adenine, guanine, cytosine, uracil, thymine or derivatives thereof, and R is -OH, -F, -N3, or -NH2.

17. The compound of claim 2 having the structure:wherein R is -OH, -F, -Ns or -NH?.

18. Tire compound of claim 2 having the structure:

19. The compound of claim 1 having the structure:NH₂ O O O / \ / H.0!i O A; \\ 'T HO P P P; K| N HO OH HO -Z,CN N£ OH20. A compound having the structure:whereinBASE is adenine, guanine, cytosine, uracil, thymine or derivatives thereof;m is 0-10; preferably, 0-5; more preferably 0-3; more preferably 2; andX is halogen, -CN, -N3, -NH2, -OH, -CHF2, -CH2F, -CF3, -OCHF2, -OCF3, -OCH2F, -NO2, -OAc, - COOH, alkyl, alkenyl, alkynyl, -ORB, -CORJ3, -SH, -SRJ3, -SO2RJ3, -NHRI3, -NRuRis, - NHCORis, or -CONR14R15,wherein R13, Ru, and R15 are each independently -H, alkyl, alkenyl, alkynyl, aldehyde, ketone, ester, ether, carboxylate, aryl, or heteroarykor a pharmaceutically acceptable salt or ester thereof.

21. The compound of claim 20 having the structure:

22. The compound of any one of claims 20-21 having the structure:

23. The compound of any one of claims 20-22, wherein X is halogen, -CN, -N3, -NH2, -OH, -CHF2, - CH2F, -CF3, -OCHF2, -0CF3, -OCH2F, -NO2, -OAc, or -COOH; preferably, X is -F, -Cl, -CN, - N3, -NH2, -OH, -CHFJ, -CH3F, or -CF3.

24. The compound of claim 23, wherein X is -F, -CN. -N3, -NH2, or -OH.

25. The compound of any one of claims 20-24, wherein the derivative of adenine, guanine, cytosine, thymine, and uracil has the structures:whereinRis, Rig, R20, R21, and R22 are each independently -H, halogen, -CN, -Ns, -NH2, -OH, -CHF2, -CH2F, -CFs, -OCHF2, -OCFs, -OCH2F, -NO2, -OAc, -COOH, alkyl, alkenyl, alkyny l, -ORis, -CORis, -SH, -SRis, -SOsRis, -NHR13, -NR14R15, -NHCORis, or -CONR14R15wherein R Ru, and Ris are each independently -H, alkyl, alkenyl, alkynyl, aldehyde, ketone, ester, ether, carboxylate, ar l, or heteroary 1;preferably, Ris, Rig, R20, R21, and R22 are each independently -H, halogen, -CN, -Ns. -NH2, -OH, -CHF2, -CH2F, -CFS, -OCHF2. -OCFs. -OCH2F, -NO2. -OAc, -COOH. alkyl, alkenyl, alky nyl, -ORis, -CORis, -SH, or -SRis,more preferably, Ris. Rig, R20, R21, and R22 are each independently -H, halogen, -CN, -Ns, -NH2, -OH, -CHFS, -CH2F, -CFS, -OCHF2, -OCFs, -OCH2F, or -NOs;more preferably, Ris, Rig, R20, R21, and R22 are each independently -H, or -NH2;more preferably, the derivative is26. Tire compound of claim 20 having the structure:NH2NH2" AN < VN9 2 9 " V 2 2 2 NVHO-P-O-P-O-P^W> HO-P-O-P-O-P^O^>OH OH OH £ OH OH OH £^NH2^FNIR NH22 2 9 NV 2 2 2 NVHO-P-O-P-O-P^.0^ HO-P-O-P-O-P^O^>OH OH OH £ OH OH OH £^CN " N3ʼnll27. A composition comprising the compound of any one of claims 1-26 or a pharmaceutically acceptable salt thereof.

28. The composition of claim 27 comprising at least one of the following compounds or a pharmaceutically acceptable salt thereof for the treatment of viral infections caused by one or more viruses selected from the group comprising coronaviruses, flaviviruses and alphaviruses:wherein BASE is adenine, guanine, cytosine, uracil, thymine or derivatives thereof,X is -F, -CN, -Ns, -NH2or -OH, and X has either an R or S configuration or a mixture of both stereoisomers.

29. Tire composition of claim 27 comprising at least one of the following compounds or a pharmacally acceptable salt thereof for the treatment of viral infection caused by one or more viruses selected from the group comprising coronaviruses, flaviviruses and alphaviruses:wherein X is -F, -CN, -N₃, -NH₂, -OH and X has either an R or S configuration or a mixture of both stereoisomers.

30. The composition of any one of claims 28-29 for the treatment of coronavirus infections caused by viruses comprising SARS-CoV2, SARS-CoV, and MERS-CoV wherein the X is in the R configuration.

31. Tire composition of claim 27 comprising at least one of tire following compounds or a pharmaceutically acceptable salt thereof for the treatment of viral infection caused by one or more viruses selected from the group comprising coronaviruses, flaviviruses and alphaviruses:wherein R is -F, -N3, or -NH2:

32. The composition of claim 27 comprising at least one of tire following compounds or a pharmacally acceptable salt thereof for the treatment of viral infection caused by one or more viruses selected from the group comprising coronaviruses, flaviviruses and alphaviruses,R is -OH, -F, -N3, or -NH2; andwherein BASE is adenine, guanine, cytosine, uracil, thymine or derivatives thereof.

33. The composition of claim 28-32, wherein flavivimses comprise Dengue virus (DENV), Zika virus (ZIKV) and yellow fever virus (YFV); and / or alphaviruses comprise Chikungunya virus (CHIKV) and Eastern equine encephalitis virus (EEEV).

34. A method of treating viral infection in a subject comprising administering one or more compound of any one of claims 1-26 and composition of claim 27 to the subject: preferably, the viral infection is caused by one or more viruses selected from the group comprising coronaviruses, flaviviruses and alphaviruses, more preferably, the coronavirus infection caused by viruses comprising SARS-CoV2, SARS-CoV, and MERS-CoV.

35. The method of claim 34, wherein(a) the flaviviruse is Dengue virus (DENV), Zika virus (ZIKV) and yellow fever virus (YFV); (b) the alphaviruse is Chikungunya virus (CHIKV) and Eastern equine encephalitis virus (EEEV);(c) the method further comprises administering one or more anti-viral medication; and / or (d) the compound is incorporated by viral RNA-dependent RNA polymerase (RDRP) and is resistant to excision by viral 3’-5’ exonuclease (ExoN).

36. A process of producing the compound of claim 1 or 2, wherein the process comprises:(a) reacting a nitrogenous basewithxto obtain a compound of formula I,BASEx(I);TsO OP— OEtI(b) reacting the compound of formula I with OEt0obtain a compound of formula II O BASEEt(X / / IEtO Ix(II);(c) reacting the compound of formula 11 with an acid to produce a compound of formula 111BASEOH OH(III): and(d) reacting the compound having formula III with phosphorylating reagent.

37. The process of claim 36, wherein(a) step (a) is conducted in the presence of a base, preferably, an inorganic base, more preferably, KOH. NaOH Ca(OH)2;(b) step (b) is conducted in the presence of MeMgCl, t-BuOH and cyclohexane:(c) the acid in step (c) is HBr / AcOH;(d) step (d) is conducted in the presence of CDI / DMF, MeOH, TEAB buffer, and THP; and / or (e) the phosphorylating reagent is tributylammonium pyrophosphate.

38. The process of any one of claims 36-37, whereinOO^XI,0(a)has the structure ofxBASE O BASE EKX EKK z / P EK) Eto'(b)has the structure of: andBASE BASE OIL - OIL zPP FL O OH OH(c)has the structure of39. Tire process for producing the compound of claim 1 or 2, comprising(a) reacting(III) withNHBzwith a desilylation agent; or (d) reacting the compound of formula (III) with phenol to produce a compound of formula (IV)(e) reacting the compound of formula (IV) with SOCk a compound of formula (V)x(V); and(f) reacting the compound of formula VwithNHBzto produceNHBz(j) reactingwith a desilylation agent.

40. A process of producing the compound of claim 1, comprising(a) reactingTBSO (VI) or OBn with a phosphorylating reagent; or(b) reactingto produce a compound of(c) reacting compound of formula (VII) with a desilylation agent; or(d) reacting tire compound of formula (VI) withto produce a compound of formula (VIII)TBSO (VIII),(e) reacting compound of formula (VIII) with a desilylation agent.

41. The process of any one of claims 30-40, wherein the desilylation agent is BCE, or TBAF / THF.

42. A composition comprising at least one of the following compounds or a pharmaceutically acceptable salt thereof for the treatment of viral infections caused by one or more viruses selected from tire group comprising coronaviruses, flaviviruses and alphaviruses:wherein BASE is Adenine. Guanine, Cytosine. Thymine, Uracil or derivatives thereof.X is F. CN, Ns, NH2 or OH, or related groups and X may have either an R or configuration or a mixture of both stereoisomers;orwherein X is F, CN, N3, NH2, OH or related groups and X may have either an R or S configuration or a mixture of both stereoisomers.

43. Tire composition of claim 42 for the treatment of coronavirus infections caused by viruses comprising SARS-CoV2, SARS-CoV, and MERS-CoV wherein tire X substitution is in the R configuration.

44. A composition comprising at least one of the following 5 ’-methylene nucleotide compounds or a pharmaceutically acceptable salt thereof for the treatment of viral infection caused by one or more viruses selected from the group comprising coronaviruses, flaviviruses and alphaviruscs:HO ora composition comprising at least one of the following l’-CN, 2’-F, 4 ’-Ns- 1 -Deazacytidine nucleoside or a pharmaceutically acceptable salt of thereof for the treatment of viral infection caused by one or more viruses selected from the group comprising coronaviruses, flaviviruses and alphaviruses:ora composition comprising at least one of the following 3 ’-deoxyadenosine nucleoside prodrugs or a pharmaceutically acceptable salt thereof for the treatment of viral infection caused by one or more viruses selected from the group comprising coronaviruses, flaviviruses and alphaviruses:OH OH45. A composition comprising at least one of the following compounds or a pharmaceutically acceptable salt thereof for the treatment of viral infection caused by one or more viruses selected from the group comprising coronaviruses, flaviviruses and alphaviruses.NH₂OHwherein BASE is adenine, guanine, cytosine, uracil. Thymine or derivatives thereof: orwherein BASE is adenine, guanine, cytosine, uracil. Thymine or derivatives thereof, and R is F, Ns, NH2or related groups; orwherein R is F, N₃ or NH₂ or related groups.

46. A composition comprising at least one of the following compounds or a pharmaceutically acceptable salt thereof for the treatment of viral infection caused by one or more viruses selected from the group comprising flaviviruses and alphaviruses.wherein BASE is adenine, guanine, cytosine, uracil. Thymine or derivatives thereof, and R is OH, F, Ns, NH2or related groups.

47. The composition of claims 42-46., wherein flaviviruses comprises Dengue virus (DENV), Zika virus (ZIKV) and yellow fever virus (YFV) or wherein alphaviruses are a Chikungunya virus (CHIKV) and Eastern equine encephalitis virus (EEEV).

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