Miniaturized Modular Click Chemistry-enabled Rapid Discovery of Unique SARS-CoV-2 Mpro Inhibitors With Robust Potency and Drug-like Profile

Abstract

The COVID-19 pandemic has required an expeditious advancement of innovative antiviral drugs. In this study, focused compound libraries are synthesized in 96- well plates utilizing modular click chemistry to rapidly discover potent inhibitors targeting the main protease (M^pro) of SARS-CoV-2. Subsequent direct biological screening identifies novel 1,2,3-triazole derivatives as robust M^pro inhibitors with high anti-SARS-CoV-2 activity. Notably, C5N17B demonstrates sub-micromolar M^pro inhibitory potency (IC₅₀ = 0.12 µM) and excellent antiviral activity in Calu-3 cells determined in an immunofluorescence-based antiviral assay (EC₅₀ = 0.078 µM, no cytotoxicity: CC₅₀ > 100 µM). C5N17B shows superior potency to nirmatrelvir (EC₅₀ = 1.95 µM) and similar efficacy to ensitrelvir (EC₅₀ = 0.11 µM). Importantly, this compound displays high antiviral activities against several SARS-CoV-2 variants (Gamma, Delta, and Omicron, EC₅₀ = 0.13 - 0.26 µM) and HCoV-OC43, indicating its broad-spectrum antiviral activity. It is worthy that C5N17B retains antiviral activity against nirmatrelvir-resistant strains with T21I/E166V and L50F/E166V mutations in M^pro (EC₅₀ = 0.26 and 0.15 µM, respectively). Furthermore, C5N17B displays favorable pharmacokinetic properties. Crystallography studies reveal a unique, non-covalent multi-site binding mode. In conclusion, these findings substantiate the potential of C5N17B as an up-and-coming drug candidate targeting SARS-CoV-2 M^pro for clinical therapy.

Keywords: SARS‐CoV‐2; click chemistry; direct screening; main protease; miniaturized synthesis; non‐covalent inhibitors.

Figure 1

A) Structures of the marketed drugs, including Nirmatrelvir, Simnotrelvir, Leritrelvir, and Ensitrelvir, and the preclinical compounds GC‐14 and AA‐625. EC₅₀ values and CC₅₀ values correspond to antiviral effects and cytotoxicity, respectively. B) Illustration of the general design concept of focused compound libraries.

Scheme 1

Synthetic route to the intermediates and alkyne fragments C4, C5, and C6 ^a.^aReagents and conditions: i) THF, −20 °C, N₂; ii) dichlorosulfoxide, dichloromethane (DCM), ice bath; iii) 3‐(ethyliminomethylideneamino)‐N,N‐dimethylpropan‐1‐amine, hydrochloride (EDCI), 1‐Hydroxybenzotriazole (HOBt), N‐methylmorpholine (NMM), DCM, r.t.; iv) hydrogen chloride dioxane solution (4 m), DCM, r.t.; v) CH₃COOK, methanol (MeOH), 70 °C; vi) O‐(7‐azabenzotriazol‐1‐yl)‐N,N,N″,N″‐tetramethyluronium hexafluorophosphate (HATU), N,N‐diisopropylethylamine (DIPEA), DCM, r.t.

Scheme 2

Synthetic Route to the Intermediates and Alkyne fragment C3 ^a; ^aReagents and conditions: i) HATU, DIPEA, niacin, DCM, r.t.; ii) hydrogen chloride dioxane solution (4 m), DCM, r.t.; iii) 3‐fluorophenylboronic acid, Cu(OAc)₂, pyridine, O₂, DCM,r.t.; iv) 3‐bromopropyne, t‐BuOK, ethanol, 60 °C.

Figure 2

Structures of azides (N1 to N69) employed as starting materials.

Figure 3

Effects of the racemate C5N17 and its two enantiomers (C5N17A, C5N17B) on M^pro inhibition and antiviral efficacy determined in a cellular assay. A) M^pro inhibitory potency of C5N17, C5N17A, C5N17B, and Nirmatrelvir. B) Anti‐SARS‐CoV‐2 activity of C5N17, C5N17A, C5N17B, and Nirmatrelvir in Calu‐3 cells. C) Toxicity of C5N17, C5N17A, C5N17B, and Nirmatrelvir on Calu‐3 cells. D) Western blot analysis for detecting viral S and N proteins in Calu‐3 cells in the presence of C5N17B or Nirmatrelvir on day 2 post‐infection. E) Quantitative RT‐PCR for measuring SARS‐CoV‐2 viral RNA amounts accumulated in Calu‐3 cell culture supernatants in the presence of increasing concentrations of C5N17B or Nirmatrelvir on day 2 post‐infection. n.t., not tested. In all experiments, Nirmatrelvir was used as a positive control. In panels (A–C) and (E), all values are represented as means ± SD from three independent experiments.

Figure 4

X‐ray co‐crystal structures of the (R)‐configurated C5N17B (PDB ID: 9G0I) and its (S)‐configurated enantiomer C5N17A (PDB ID: 9G0H) in complex with M^pro. A) View of C5N17B (green) in the binding pocket. Hydrogen bonds are shown as magenta‐colored dashed lines; π–π stacking is indicated as green dashed lines. B) View of the interactions of C5N17B (green) with His41. C) View of C5N17A (red) in the binding pocket. D) Binding pose comparison of C5N17B (green) and C5N17A (red).

Figure 5

Comparison of the M^pro binding modes of C5N17B and selected other inhibitors with piperazine scaffolds. A) Binding pose comparison of C5N17B (green), GC‐14 ^{[ 13 ]} (yellow, PDB ID: 8ACL), and JZD‐07 ^{[ 30 ]} (cyan, PDB ID: 8GTV). B) Superposition of the binding modes of C5N17B (green), nirmatrelvir (orange, PDB ID: 7VH8), and ensitrelvir (blue, PDB ID: 7VU6).

Figure 6

A) The comparison of C5N17B and nirmatrelvir B) against SARS‐CoV‐2 nirmatrelvir‐resistant mutants.

Figure 7

A–C) Antiviral activity of C5N17B against SARS‐CoV‐2 variants and D) HCoV‐OC43.

Figure 8

Comparison of the location of C5N17B and C5N17A in the substrate envelope of SARS‐CoV‐2 M^pro. A) The 3D shape of the substrate envelope of SARS‐CoV‐2 M^pro. B) The fitting of C5N17B (green) and C5N17A (brick red) within the substrate envelope.

Figure 9

A) Plasma concentration−time curve of C5N17B following oral administration (p.o., 10 mg kg⁻¹ in combination with 20 mg kg⁻¹ Ritonavir) in ICR mice. B) Plasma concentration−time curve of Nirmatrelvir following oral administration (p.o., 10 mg kg⁻¹ in combination with 20 mg kg⁻¹ Ritonavir) in ICR mice.

Figure 10

Visual presentation of in vivo toxicity experiment results for C5N17B. A) Time courses of body weight in the 7‐day acute toxicity experiment. B) Time courses of body weight in the 15‐day subacute toxicity experiment. C) Microscopic images of organ slices from mice treated in subacute toxicity study. The heart, liver, spleen, lung, and kidney were sectioned and stained with hematoxylin and eosin.