Graphical abstract
Keywords: β-D-N4-Hydroxycytidine, RNA-Polymerase, DENV-2, Influenza viruses, SARS-CoV-2, β-D-N4-O-Isobutyrylcytidine, Nucleosides
Abstract
Drug repurposing approach was applied to find a potent antiviral agent against RNA viruses such as SARS-CoV-2, influenza viruses and dengue virus with a concise strategy of small change in parent molecular structure. For this purpose, β-D-N4-hydroxycytidine (NHC, 1) with a broad spectrum of antiviral activity was chosen as the parent molecule. Among the prepared NHC analogs (8a-g, and 9) from uridine, β-D-N4-O-isobutyrylcytidine (8a) showed potent activity against SARS-CoV-2 (EC50 3.50 μM), Flu A (H1N1) (EC50 5.80 μM), Flu A (H3N2) (EC50 7.30 μM), Flu B (EC50 3.40 μM) and DENV-2 (EC50 3.95 μM) in vitro. Furthermore, its potency against SARS-CoV-2 was >5-fold, 3.4-fold, and 3-fold compared to that of NHC (1), MK-4482 (2), and remdesivir (RDV) in vitro, respectively. Ultimately, compound 8a was expected to be a potent inhibitor toward RNA viruses as a viral mutagenic agent like MK-4482.
Over the last half a century, >50 nucleoside analogs have been developed as the potent chemotherapeutic agents, and many analogs are ongoing clinical or preclinical trials1, 2, 3, 4, 5, 6, 7 since 5-iodo-2′-deoxyuridine (IDU) was approved for treatment of herpes simplex keratitis in 1962.8, 9 Most recently, at the end of 2019, coronavirus disease (COVID-19) broke out by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and then rapidly spread around the world to be over 611 million infections and 6.51 million deaths as of September 2022.10 When it was in a public health emergency in the early stage, unfortunately, the development of vaccine and chemotherapy to treat COVID-19 was insufficient. To date, there are three efficient therapies11 and mainly-eight types of vaccines12 to overcome pandemic crisis. Still, more efficient chemotherapy is strongly needed to restrain new infections by mutated viruses. Thus, a variety of investigations were performed to find therapeutic agents from a pool of approved drugs or candidates for viral infections. Indeed, β-D-N 4-hydroxycytidine (NHC, EIDD-1931)13, 14 and its prodrug analog, or MK-4482 (EIDD-2801)15 were suitable target molecules utilizing a drug repurposing strategy.16 As the promising antiviral agent, NHC has exhibited a broad-spectrum potency against many RNA viruses including hepatitis C virus (HCV),17 norovirus (NV),18 Ebola virus19, chikungunya virus (CHIKV),20 Venezuelan equine encephalitis virus (VEEV),21 influenza and respiratory syndrome viruses (RSV),22 β-coronavirus (β-CoVs: murine hepatitis virus (MHV) and Middle East respiratory syndrome coronavirus (MERS-CoV)),23 and Mayaro virus (MAYV).24 Its derivative, MK-4482 (EIDD-2801), was first developed as a polymerase inhibitor of influenza viruses25 and then repurposed for cure against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection26, 27, 28 and is currently used in an orally treatment of pandemic SARS-CoV-2 infection as a viral mutagenic agent.29, 30 Additionally, NHC ester prodrugs have exhibited a potent antiviral activity against some DNA viruses such as vaccinia, monkeypox and cowpox viruses.31
The antiviral potency of NHC and its analogs prompts us to modify NHC to find more potent its analog against RNA viruses with the strategy of a local transformation or exchange of a functional group in the parent molecular structure. In this article, we wish to report synthesis of eight β-D-N 4-hydroxycytidine (NHC) analogs (8a-g and 9) from uridine. Among the prepared NHC derivatives, the antiviral potency of β-D-N 4-isobutyryloxycytidine (8a) against DENV-2, influenza viruses, and SARS-CoV-2 was provided as our preliminary result in vitro, respectively (Fig 1 ).
Fig. 1.
Structures of β-D-N4-hydroxycytidine (NHC), MK-4482 and targeting molecules.
Initially the β-D-N 4-hydroxycytidine (1) as the parent moiety was synthesized from uridine (3) according to a previously reported synthetic method (Patent).14 Three hydroxyl groups of 3 was protected with t-butyldimethylsilyl chloride (TBDMSCl) in the presence of imidazole in N,N-dimethylforamide (DMF) to obtain tri-TBDMS-O-d-uridine derivative (4) in 79 % yield. The reaction of 4 with 2,4,6‑triisopropylbenzenesulfonyl chloride, of which a bulky sulfonyl group was needed to induce a substitution reaction at 4-carbon position of pyrimidine base instead of sulfur position on sulfonyl group, successfully provided a desired intermediate 5 in situ. The reaction of 5 with hydroxyl amine hydrochloride (NH2OH-HCl) in the presence of excess of diisopropylethylamine (DIEA) afforded silyl protected N 4-hydroxycytidine (6a) in 78 % yield. The treatment of 6a with ammonium fluoride (NH4F) in MeOH gave N 4-hydroxycytidine (1) in 45 % yield. In the next, the novel N 4-O-isobutyryl cytidine (8a) was synthesized from an intermediate 6a in two steps. The protection reaction of 6a with isobutyryl chloride and triethylamine (Et3N) in CH2Cl2 afforded N 4-O-isobutyryl cytidine (7a) in 66 % yield along with and N 4-O-diisobutyryl cytidine (7b) in 34 % yield. Subsequently, the treatment of both 7a and 7b with Et3N-3HF in THF interestingly delivered N 4-O-isobutyryl cytidine (8a) in 75 % and 70 % yield with the same 1H NMR spectrum, respectively. Furthermore, a couple of NHC analogs (8b-g) were prepared utilizing the in-situ intermediate 5 with the similar synthetic procedure.
The sulfonyl group of 5 was substituted with tert-butyl hydrazinecarboxylate (NH2NHBoc), 2-ethanolamine (HOCH2CH2NH2), and hydrochloric l-amino acid ethyl esters (GlyOEt, AlaOEt, LeuOEt and PheOEt)32 as isosteric analogs of NHO-isobutylryl moiety to afford their corresponding N 4-substituted cytidine derivatives (6b-g) in 29–80 % yields. With the same removal condition of 8a above, compounds 6b-g were converted to their corresponding products (8b-g) such as C4-Boc-hyrazinyl cytidine (8b), C4-2‑hydroxylethyl cytidine (8c), C4-GlyOEt-cytidine (8d), C4-AlaOEt-cytidine (8e), C4-LeuOEt-cytidine (8f) and C4-PheOEt-cytidine (8 g) in 41–82 % yields summarized in scheme 1 , respectively. Additionally, the tert-butoxycarbonyl (Boc) group of 8b was removed with hydrogen chloride in methanol to afford N 4-aminocytidine (9) in 67 % yield.13.
Scheme 1.
Reagents and reaction conditions: a) TBDMSCl, imidazole, DMF, 0 °C → rt, 12 h; b) 2,4,6‑Triisopropylbenzenesulfonyl chloride, DIEA, DMAP, CH3CN, 0 °C → rt, 15 h; c) HCl·NH2OH for 6a; NH2NHBoc for 6b; NH2CH2CH2OH for 6c; HCl·GlyOEt for 6d; HCl·AlaOEt for 6e; HCl·LeuOEt for 6f; HCl·PheOEt for 6 g, DIEA, 0 °C → rt, 12 h for 6a-g; d) Isobutyryl chloride, Et3N, CH2Cl2, 0 °C, 12 h; e) Et3N-3HF, THF, rt, 48 h; f) HCl, MeOH, rt, 4 h; g) NH4F, MeOH, 50 °C, 12 h.
Finally, MK-4482 (2) was prepared as a reference material of biological activities according to the previous reported method from compound 4.15 The treatment of 4 with Et3N-3HF followed by reaction with isobutyryl chloride in presence of Et3N gave the compound 11 in 14 % yield in two steps. The reaction of 11 with 2,4,6‑triisopropylbenzenesulfonyl chloride, and then treated with hydroxyl amine successfully afforded its corresponding N 4-hydroxylcytidine derivative (12) in 54 % yield. Subsequently, the removal of silyl group of 12 with Et3N-3HF in THF delivered N 4-hydroxyl-5′-O-isobutyryl-cytidine (2) in 40 % yield depicted in scheme 2 . Totally, eight new compounds (8a-g, and 9) as the novel NHC analogs and NHC (1) and MK-4482 as the positive control compounds were prepared from uridine (3) in moderate to good yields.
Scheme 2.
Reagents and reaction conditions: a) Et3N-3HF, THF, 0 °C → rt, 24 h; b) Isobutyryl chloride, Et3N, CH2Cl2, 0 °C; 18 h; c) i. 2,4,6‑Triisopropylbenzenesulfonyl chloride, DIEA, DMAP, CH3CN, 0 °C → rt, 15 h; ii. NH2OH-HCl, DIEA, 0 °C → rt, 12 h; d) Et3N-3HF, THF, 0 °C → rt, 24 h.
With the new synthesized NHC analogs in hands, we investigated antiviral activity against SARS-CoV-2 (hCoV-19/Korea/KCDC-06/2020) in Vero cells (Vero CCL-81 cells), DENV-2 replicon BHK-21 (DENV2-BHK) cells, and Flu A/Puerto Rico/8/34 (PR8; H1N1), Flu A/Hong Kong/8/68 (HK; H3N2), and Flu B/Lee/40 (Lee) in MDCK cells (CCL-34) according to the previously reported methods.33, 34, 35 Cell viability of non-infected or infected cells was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) to determine the half-maximal cytotoxic concentration (CC50). For dengue virus, the half-maximal effective concentration (EC50) was determined by using Renilla Luciferase assay system.36 The EC50 values for SARS-CoV-2 and influenza viruses were determined with cell-based antiviral assays by utilizing an anti-spike antibody and MTT, respectively. EC50 and CC50 values were calculated from a nonlinear regression equation.36 The preliminary antiviral results of the prepared analogs (8a-g, and 9) were summarized in Table 1 . Novel N 4-O-isobutyryl cytidine (8a) showed most potent activity against SARS-CoV-2 in vitro. The value of EC50 of 8a was 3.50 µM along with >100 µM of CC50. The potency of 8a was improved by >4.7-fold and 3.4-fold compared with that of NHC (1, EC50 > 16.5 µM) and MK4482 (2, EC50 11.8 µM), respectively. Furthermore, activity of 8a against SARS-CoV-2 was superior to that of remdesivir (RDV, EC50 11.1 µM) in vitro. Although activities of 8a against Flu A (EC50 5.8 µM) and B (EC50 7.3 µM) had similar values of 1 and 2, the potency of anti-Flu B (EC50 3.4 µM) of 8a was enhanced by 7-fold compared with that of 2 (EC50 23.8 µM). The efficiency of 8a (EC50 3.95 µM) along with CC50 10.85 µM against DENV-2 was similar to that of 1 (EC50 2.84 µM, CC50 5.75 µM) but was meaningfully improved by 5.9-folds compared with that of 2 (EC50 23.38 µM, CC50 42.25 µM). When the value of 8a was compared with that of ribavirin (RBV) as a control reagent, the activity of 8a was more potent around by 3-folds for SARS-CoV-2, 6-folds for Flu A (H1N1), 5-fold for Flu A (H3N2), 10-folds for Flu B, but was reduced by 3-fold for DENV-2. Also, when the potency of 8a was compared with that of T-705 (favipiravir, 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) and amantadine (AMT), its values of EC50 against Flu B and DENV-2 displayed about 2-fold but showed less activity than T-705 against Flu A (H3N2). Additionally, activity of 8a against Flu B was similar to that of AMT.
Table 1.
Antiviral activity of NHC analogs.
| Ent | Comp | Antiviral activity (µM)a | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| SARS-CoV-2 | Flu A (H1N1) | Flu A (H3N2) | Flu B | DENV-2 | |||||||
| EC50 | CC50 | EC50 | CC50 | EC50 | CC50 | EC50 | CC50 | EC50 | CC50 | ||
| 1 | 1 | >16.5 | 16.5 | 1.7 | >100 | 2.3 | >100 | <1.2 | >100 | 2.84 | 5.75 |
| 2 | 2 | 11.8 | >100 | 6.6 | >100 | 6.0 | >100 | 23.8 | >100 | 23.38 | 42.25 |
| 3 | 8a | 3.5 | >100 | 5.8 | >100 | 7.3 | >100 | 3.4 | >100 | 3.95 | 10.85 |
| 4 | 8b | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | 100 | 100 |
| 5 | 8c | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | 100 | 100 |
| 6 | 8d | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | 100 | 100 |
| 7 | 8e | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | 100 | 100 |
| 8 | 8f | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | 100 | 100 |
| 9 | 8 g | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | 100 | 100 |
| 10 | 9 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | 100 | 100 |
| 11 | RDVb | 11.1 | >100 | ndf | nd | nd | nd | nd | nd | nd | nd |
| 12 | RBVc | nd | nd | 37.7 | >100 | 38.7 | >100 | 33.3 | >100 | 1.16 | 5.46 |
| 13 | T-705d | nd | nd | 3.2 | >100 | 8.5 | >100 | 7.6 | >100 | nd | nd |
| 14 | AMTd | nd | nd | >100 | >100 | 6.4 | >100 | >100 | >100 | nd | nd |
EC50: 50% effective concentration; CC50: 50% cytotoxic concentration; bRDV: remdesivir; cRBV: ribavirin; dT-705: favipiravir; eAMT: amantadine; NHC (1), MK-4482 (2), RBV, T-705 and AMT were used as control; fnd: no determination.
Unfortunately, none of the other prepared compounds including N 4-NHBoc (8b), N 4-2‑hydroxylethyl (8c) and C4-amino acid derivatives (8d-g) and N 4-aminocytidine (9) exhibited significant antiviral activities against SARS-CoV-2, Flu A/B and DENV-2 with no cell cytotoxicity (>100 µM) in vitro.
In conclusion, we synthesized the eight novel NHC analogs (8a-g and 9) from uridine in mild to good yield. Among prepared them, β-D-N 4-O-isobutyrylcytidine (8a) showed only significant antiviral activity against SARS-CoV-2 (EC50 3.50 µM), Flu A (H1N1) (EC50 5.80 µM), Flu A (H3N2) (EC50 7.30 µM), Flu B (EC50 3.40 µM) and DENV-2 (EC50 3.95 µM) with no significant cytotoxicity in vitro, respectively. The potency of 8a against SARS-CoV-2 was improved by >5-fold for NHC (1), 3-fold for MK-4482 (2) and 3-fold for remdesivir (RDV), respectively. Ultimately, novel β-D-N 4-O-isobutyrylcytidine (8a) is a promising candidate for developing RNA-virus polymerase inhibitor via a mechanism of RNA mutation by the polymerases of RNA-SARS-CoV-2, Flu A and B, and DENV-2.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported in part by BB 21 Plus Project and supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (MIST) (Nos. 2020R1F1A1077099 to J.H.C. and 2018M3A9H4089601 to M.K.). We thank Dr. E. H. Lee for mass spectrometric assistance. The SARS-CoV-2 resource (NCCP No., 43328) was provided by the National Culture Collection for Pathogens, Republic of Korea.
Footnotes
Supplementary data to this article can be found online at https://doi.org/10.1016/j.bmcl.2023.129174.
Appendix A. Supplementary data
The following are the Supplementary data to this article:
Data availability
Data will be made available on request.
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Supplementary Materials
Data Availability Statement
Data will be made available on request.