Inhibition of the RNA-Dependent RNA-Polymerase from SARS-CoV-2 by 6-Chloropurine Isoxazoline-Carbocyclic Monophosphate Nucleotides

Figures

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  Scheme 1. Chemical Design of Carbocyclic Nor-Nucleotides

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  Scheme 2. General Synthetic Strategy toward Brominated/Phosphorylated Nor-Nucleotides of Type 4

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  Chart 1. Chemical Structure of Designed Compounds 4a and 4b, Couples of Enantiomers

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  Figure 1. Docking poses (A1,2, B1,2) of enantiomeric compounds 4a and 4b in the triphosphate forms in the precatalytic structure.

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  Figure 2. Docking poses (A1,2, B1,2) of enantiomeric compounds 4a and 4b in the monophosphate forms in the postcatalytic structure.

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  Figure 3. Closure of the binding pocket observed in the MD simulations. Scatter plot of the average distances between Asp 671 and Lys 798 and the distance between Asp 761 and Ala 550 in (a) complexes obtained with the 7CTT structure and (b) 7BV2 (refer to Chart 1 for structures). The label ‘Control’ indicate average for MD simulations of the X-ray complex structures of Remdesivir and/or Favipiravir bound RdRp. These are (a) FTP bound complex with C or U as complementary base; RTP bound complex with U as complementary base. (b) RMP bound complex; RMP bound complex after incorporation into the chain. The pdb code label indicates crystal structure values as further reference. (c) RdRp in complex with FTP in 7CTT.

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  Scheme 3. Syntheses of Regioisomeric Nor-Nucleotides of Type 4a,b

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  Figure 4. Inhibition of cell viability assessed by the trypan blue assay (a, b) and of metabolic activity assessed by MTS assay (c, d) in HEp-2 cells (a, c) and HepG2 cells (b, d) treated with nucleotides 4a and 4b for 24 h (experiments in triplicate). Cytotoxicity, calculated as concentration of compounds needed to reduce the viability or metabolic activity by 50% with respect to control cells. CC50 values (expressed as mean ± SD: (a) 4a = 46.79 ± 1.18, 4b = 22.36 ± 1.04; (b) 4a = 79.67 ± 1.10, 4b = 22.85 ± 1.19; (c) 4a = 30.96 ± 1.2, 4b = 23.64 ± 1.3; (d) 4a = 28.14 ± 1.3, 4b = 32.37 ± 1.4.

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  Figure 5. Inhibition of SARS-CoV-2 RdRp activity by nucleotides (a) 4a and (b) 4b, assessed by a cell/virus-free fluorometric RdRp activity assay. Data presented as the mean ± SD. Statistical significance analyzed using the 1 way ANOVA Bonferroni’s multiple comparisons test: *p < 0.001 vs all (experiments in triplicate). Inhibitory activity was calculated as the concentration of compounds needed to reduce RdRp activity by 50%. IC50 values, expressed as mean ± SD, were: 4a = 43.83 ± 1.07; 4b = 29.31 ± 1.05.