Synthesis and anti-SARS-CoV-2 evaluation of lipid prodrugs of β-D-N4-hydroxycytidine (NHC) and a 3'-fluoro-substituted analogue of NHC

Abstract

β-D-N⁴-hydroxycytidine (NHC, EIDD-1931) is a nucleoside analogue that exhibits broad spectrum antiviral activity against a variety of RNA viruses. Herein, we report the synthesis of a series of lipid prodrugs of NHC and a novel 3'-fluoro modified NHC analogue, and evaluation of their antiviral activity against five variants of SARS-CoV-2. All lipid prodrugs showed potent antiviral activity against the tested SARS-CoV-2 variants with EC₅₀ values in the range of 0.31-3.51 μM, which were comparable to those of NHC or higher than those of remdesivir and molnupiravir. An increase in the cytostatic activity of the lipid prodrugs was found, but prodrug 2d proved equally selective as molnupinavir. The 3'-F analogue of NHC (6) only displayed minor antiviral activity against the SARS-CoV-2 Omicron variant (EC₅₀ = 29.91 μM), while no activity was found for other variants at the highest concentration tested. The promising antiviral data of the lipid prodrugs of NHC suggest that they deserve further investigation as new anti-SARS-CoV-2 drugs.

Keywords: COVID-19; EIDD-1931; Lipid prodrugs; Nucleoside analogue; SARS-CoV-2.

Graphical abstract

Fig. 1

Selected examples of biologically active nucleoside/tide anti-SARS-CoV-2 agents.

Scheme 1

Synthesis of lipid prodrugs of NHC. Reagents and conditions: (a) 1a-d, pyridine, rt, overnight, 62–82%.

Fig. 2

Variable temperature (VT) NMR spectra of 2a in DMSO‑d₆. The arrow in spectra points to minor isomer peaks.

Fig. 3

¹H NMR spectra of 2a in DMSO‑d₆ and CD₃OD. The arrow in spectra points to minor isomer peaks.

Scheme 2

Synthesis of 3′-fluoro-NHC. Reagents and conditions: (a) BSA, TMSOTf, MeCN, 80 °C, 6 h, 43%; (b) NH₃ in MeOH, rt, overnight, 77%; (c) (NH₂OH)₂·H₂SO₄, 85 °C, 5 h, 62%.

Fig. 4

Human plasma stability of 2a-d and molnupiravir.