Bisubstrate Inhibitors of Severe Acute Respiratory Syndrome Coronavirus-2 Nsp14 Methyltransferase

Abstract

Taking advantage of the uniquely constricted active site of SARS-CoV-2 Nsp14 methyltransferase, we have designed bisubstrate inhibitors interacting with the SAM and RNA substrate binding pockets. Our efforts have led to nanomolar inhibitors including compounds 3 and 10. As a prototypic inhibitor, compound 3 also has an excellent selectivity profile over a panel of human methyltransferases. Remarkably, C-nucleoside 10 exhibits high antiviral activity and low cytotoxicity, leading to a therapeutic index (CC₅₀/EC₅₀) greater than 139. Furthermore, a brief metabolic profiling of these two compounds suggests that they are less likely to suffer from major metabolic liabilities. Moreover, computational docking studies point to protein-ligand interactions that can be exploited to enhance inhibitory activity. In short, discovery of inhibitor 10 clearly demonstrates that potent and selective anti-SARS-CoV-2 activity can be achieved by targeting the Nsp14 methyltransferase. Therefore, the current work strongly supports the continued pursuit of Nsp14 methyltransferase inhibitors as COVID-19 therapeutics.

Figure 1

Design of bisubstrate inhibitors of Nsp14 MTase.

Figure 2

Docking of nucleosides into the active site of SARS-CoV-2 Nsp14 MTase (PDB 7EGQ). (A) Proposed binding modes of compound 3 (green) and 10 (cyan). (B) Protein–ligand interaction diagram of compound 3. (C) Protein–ligand interaction diagram of compound 10.

Scheme 1. Synthesis of Adenosine Derivatives

Reagents and conditions: (a) i. DPPA, DBU, dioxane; ii. NaN₃, TBAI, 15-crown-5, dioxane, reflux; (b) H₂, Pd/C, MeOH; (c) for 15, 2-naphthaldehyde, NaBH₄, MeOH, 5 h, 88%; for 16, 2-naphthoyl chloride, NEt₃, CH₂Cl₂, rt, 18 h, 93%; for 17–19, sulfonyl chloride, NEt₃, CH₂Cl₂, rt, 18 h, 33–93%; (d) TFA/H₂O (4:1), rt, 3 h, 38–92%; (e) Ac₂O, pyridine, rt, 52–67%; (f) H₂, Pd/C, EtOH, 18 h, 69%; (g) 2-naphthalenesulfonyl chloride, NEt₃, CH₂Cl₂, rt, 18 h, 41–44%; (h) isobutyric anhydride, pyridine, rt, 2 h, 29%; (i) H₂, Pd/C, dioxane, 18 h.

Scheme 2. Synthesis of C-Nucleosides

Reagents and conditions: (a) TMSCl, PhMgCl, ⁱPrMgCl·LiCl, THF, 0 °C, 4 h, 42%; (b) BF₃·OEt₂, Et₃SiH, CH₂Cl₂, 0 °C, 4 h, 87%; (c) BBr₃, CH₂Cl₂, – 78 °C, 2 h, 91%; (d) 2,2-dimethoxypropane, H₂SO₄, acetone, rt, 87%; (e) i. DPPA, DBU, dioxane, rt, 16 h; ii. NaN₃,15-crown-5, 110 °C, 18 h, 82% over two steps; (f) H₂, Pd/C, EtOH, 18 h, 95%; (g) 2-naphthalenesulfonyl chloride, NEt₃, CH₂Cl₂, rt, 18 h, 61%; (h) TFA/H₂O (4:1), rt, 3 h, 59%; (i) Ac₂O, pyridine, rt, 2 h, 35%.