Synthesis and antiviral activity of fatty acyl conjugates of remdesivir against severe acute respiratory syndrome coronavirus 2 and Ebola virus

Abstract

We report here the synthesis, purification, and characterization of mono- and di-fatty acyl conjugates of remdesivir (RDV) and their in vitro antiviral activity against SAR-CoV-2, an Ebola virus transcription- and replication-competent virus-like particle (trVLP) system, and infectious Ebola virus. The most potent monofatty acyl conjugate was 4b, containing a 4-oxatetradecanolyl at the 3′ position. Monofatty acyl conjugates, 3′- O -tetradecanoyl ( 4a ) (IC 50(VeroE6) = 2.3 μM; IC 50(Calu3) = 0.24 μM), 3′- O -4-oxatetradodecanoyl ( 4b ) (IC 50(VeroE6) = 2.0 μM; IC 50(Calu3) = 0.18 μM), and 3′- O -(12-ethylthiododecanoyl) ( 4e ) (IC 50(VeroE6) = 2.4 μM; IC 50(Calu3) = 0.25 μM) derivatives exhibited less activity than RDV (IC 50(VeroE6) = 0.85 μM; IC 50(Calu3) = 0.06 μM) in both VeroE6 and Calu3 cells. Difatty acylation led to a significant reduction in the antiviral activity of RDV (as shown in conjugates 5a and 5b ) against SARS-CoV-2 when compared with monofatty acylation ( 3a-e and 4a-e ). About 77.9% of 4c remained intact after 4 h incubation with human plasma while only 47% of parent RDV was observed at the 2 h time point. The results clearly indicate the effectiveness of fatty acylation to improve the half-life of RDV. The antiviral activities of a number of monofatty acyl conjugates of RDV, such as 3b , 3e , and 4b , were comparable with RDV against the Ebola trVLP system. Meanwhile, the corresponding physical mixtures of RDV and fatty acids 6a and 6b showed 1.6 to 2.2 times less antiviral activity than the corresponding conjugates, 4a and 4c , respectively, against SARS-CoV-2 in VeroE6 cells. A significant reduction in viral RNA synthesis was observed for selected compounds 3a and 4b consistent with the IC 50 results. These studies indicate the potential of these compounds as long-acting antiviral agents or prodrugs of RDV.

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

“…The enhanced lipophilicity of these lipid prodrugs relative to NHC and its isopropyl prodrug molnupiravir was expected to increase their permeability properties to achieve a higher concentration of the prodrugs in the infected cells [22] , [23] , [24] , [25] . This conjugation may result in the development of new prodrug forms of NHC having a longer duration of action by higher uptake into infected cells and sustained intracellular release of active substrates.…”

“…5′- O -myristoyl derivative of FTC exhibited 10–24-times higher anti-HIV activity than FTC alone [24] . Recently, EI-Sayed et al reported that remdesivir can be modified with fatty acids such as myristic acid, 12-azidododecanoic, and palmitic acid without demonstrating a significant loss of antiviral activity in cell culture against SARS-CoV-2 and EBOV while generating long-acting effect in vitro [25] . Furthermore, the fatty acyl conjugates and other lipid prodrugs have also been reported to exhibit reduced toxicity, enhanced cellular uptake, improved lung exposure/selectivity and oral bioavailability compared to their parent nucleoside analogues [26] , [27] , [28] .…”

“…Four fatty acids, tetradecyl carbonate, palmitic acid, myristic acid, and lauric acid were conjugated to NHC at its N 4 -hydroxy position. The fatty acid selection was based on the antiviral activity for the previously reported lipid prodrugs of NAs [22] , [23] , [24] , [25] . It was expected that the lipid prodrugs of NHC may have different physicochemical properties from molnupiravir, leading to improved antiviral activities and biological profiles [25] , [26] , [27] , [28] , [29] .…”

“…The fatty acid selection was based on the antiviral activity for the previously reported lipid prodrugs of NAs [22] , [23] , [24] , [25] . It was expected that the lipid prodrugs of NHC may have different physicochemical properties from molnupiravir, leading to improved antiviral activities and biological profiles [25] , [26] , [27] , [28] , [29] . In addition, in view of the proofreading exonuclease of coronaviruses can excise incorporated nucleotide analogues and diminish the inhibitory effects, to investigate the influence of 3′-hydroxy of NHC on anti-SARS-CoV-2 activity, a previously unknown 3′-deoxy-3′-fluoro modified NHC analogue was also prepared and evaluated.…”

Ebolavirus Species-Specific Interferon Antagonism Mediated by VP24

“…TransIt-LT1 transfection reagent (Mirus Bio) was used at a 3:1 ratio of reagent to DNA to transfect Huh7 cells with L at 166.7 ng, VP35 at 20.8 ng, NP at 20.8 ng, VP30 at 12.5 ng, T7 at 41.7 ng, and the EBOV tetracistronic minigenome at 41.7 ng per well. The EBOV tetracistronic minigenome encodes for Renilla luciferase, VP40, GP, and VP24 under a T7 promoter for initial transcription of the minigenome (28). As a transfection control, 0.8 ng per well of firefly luciferase was included.…”