Phenotypic Test of Benzo[4,5]imidazo[1,2-c]pyrimidinone-Based Nucleoside and Non-Nucleoside Derivatives against DNA and RNA Viruses, Including Coronaviruses

Figures

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  Figure 1. Examples of nucleobase modifications in various antivirals.

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  Figure 2. Examples of LLPS modulators.

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  Scheme 1. The preparation of the benzo[4,5]imidazo[1,2-c]pyrimidin-1(2H)-one derivatives. Reagents and conditions: (a) 1,4-benzoquinone, sodium acetate buffer (pH 4.5), 37 °C; (b) DMTr-Cl, Py, rt; (c) CnH2n+1OH (n = 1 (7a,b), 4 (4a and 5a), 6 (4b and 5b), 8 (4c and 5c), 10 (4d and 5d), 12 (4e and 5e)), PPh3, DIAD, CH2Cl2, 0 °C→rt; (d) CH3COOH, H2O, 50 °C; and (e) 12 N HCl, CH3OH, 50 °C.

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  Figure 3. The effects of compounds 3a and 3b on the activity of RdRp in vitro. The RNA substrate, consisting of an RNA primer 32P-labeled at the 5′-end (red) and the template strand (black), is shown on the top. The direction of primer extension by RdRp is indicated by the red arrow. (A,B) Analysis of RNA products at various concentrations of 3a (A) and 3b (B). Positions of the RNA primer and the extended full-length RNA product are indicated by black arrows. The length of RNA is indicated. (C) Quantitative analysis of the inhibition of RdRp activity by 3a and 3b. Means and standard deviations from three independent measurements are shown. The data were fitted to a hyperbolic equation (see Section 3.3.2 for details).

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  Figure 4. The effects of the antiviral compounds on biocondensate formation in vitro: cell-free LLPS assays. (A) Fluorescence microscopy images of the condensates assembled from the labeled SARS-CoV-2 N-protein (3 µM) and the gRNA fragment (6 µM) in the absence (wt) and in the presence of the compounds (20 µM). Scale bar: 10 µm. Additional representative large-field images are provided in Supplementary Information. (B) Size distribution of condensates obtained in the absence (wt) and in the presence of the compounds (20 µM). (C) Summary of the compounds’ effects on condensate formation estimated as the average number of droplets per slide (D) Antivirals’ effects on the partitioning coefficient of the N-protein. (E) Dose-dependence of the effects of compound 3a. (F) Representative fluorescence microscopy images illustrating the dose-dependent effects of 3a. Scale bar: 10 µm. Error bars in C-E represent the SD of six repeats. * p < 0.05; ** p < 0.001; *** p < 0.0001 (two-tailed Student’s t-test).