Discovery of the Clinical Candidate S-892216: A Second-Generation of SARS-CoV-2 3CL Protease Inhibitor for Treating COVID-19

Abstract

The coronavirus disease 2019 (COVID-19) pandemic crisis has been mitigated by worldwide efforts to develop vaccines and therapeutic drugs. However, there remains concern regarding public health and an unmet need for therapeutic options. Herein, we report the discovery of S-892216, a second-generation SARS-CoV-2 3C-like protease (3CL^pro) inhibitor, to treat COVID-19. S-892216 is a reversible covalent 3CL^pro inhibitor with highly potent antiviral activity and an EC₅₀ value of 2.48 nM against SARS-CoV-2 infected cells. Structure-based design of a covalent modifier for compound 1 revealed that introducing a nitrile warhead increased 3CL^pro inhibition activity by 180-fold. Subsequent optimization efforts yielded S-892216, which combined a favorable pharmacokinetic profile and high off-target selectivity. S-892216 exhibited antiviral activity against diverse SARS-CoV-2 variants, including major mutations reducing antiviral activities of nirmatrelvir and ensitrelvir. In SARS-CoV-2-infected mice, S-892216 inhibited viral replication in the lungs similar to ensitrelvir, although at a 30-fold lower dose.

1

X-ray crystal structures of SARS-CoV2 3CL^pro complexed with (A) ensitrelvir (PDB: 7VU6), (B) nirmatrelvir (PDB: 7RFS), (C) compound 1 (PDB: 9LVR) and (D) compound 4 (PDB: 9LVT), respectively. The inhibitors are shown as cyan sticks. The backbone of 3CL^pro is shown as a white ribbon, the amino acids interacting to inhibitors are highlighted as gray sticks, and hydrogen bonds are indicated as yellow dashed lines. The structures of inhibitors are also depicted at the bottom.

2

X-ray crystal structure of S-892216 complexed with SARS-CoV-2 3CL^pro (PDB: 9LVV). S-892216 is shown as a cyan stick, and the protein backbone is shown as a gray ribbon. Residues interacting with S-892216 are indicated as gray and orange sticks. Hydrogen bonds are indicated as yellow dashed lines, and π–π stacking is indicated as a cyan dashed line.

3

Dissociation kinetics of S-892216 from 3CL^pro measured using a SPA-based assay. (A) Schematic illustration of S-892216 dissociation kinetics study. Sample 1: [¹⁴C]-S-892216 alone; Sample 2: excess amount of nonlabeled S-892216 was added immediately before measurement; Sample 3 excess amount of nonlabeled S-892216 was added before preincubation. [¹⁴C]-S-892216: red circle, Nonlabeled S-892216: blue circle, buffer: white circle. (B) Dissociation kinetics of [¹⁴C]-S-892216 was monitored using scintillation signals. Data fitted in GraphPad Prism 9 using one-phase exponential decay.

4

In vitro antiviral activity of S-892216. (A) Antiviral activity of S-892216 against various SARS-CoV-2 strains in a CPE inhibition assay using VeroE6/TMPRSS2 cells. (B) Antiviral activity of S-892216 against Omicron strains in suppressing virus production using human airway epithelial cells (hAECs). Data are the means ± SD; n = 3 biological replicates.

5

Dose-dependent in vivo antiviral efficacy of S-892216 in mice infected with SARS-CoV-2. (A) Schematic diagram illustrating the in vivo study protocol. (B) Effect of S-892216 (0.1, 0.3, 1, 3, and 10 mg/kg bid) and ETV (ensitrelvir fumaric acid, 32 mg/kg [as a free form] bid) treatment on lung virus titers in SARS-CoV-2 Gamma strain (hCoV-19/Japan/TY7-501/2021)-infected mice. Each point represents an individual viral titer (n = 5). The broken line represents the LLOQ (1.80 log₁₀ TCID₅₀/mL). The following p-values were calculated using Dunnett’s test: **p < 0.01 and **p < 0.001 vs vehicle. (C) S-892216 plasma concentration in the infected mice after a single oral administration (n = 4). (D) Simulated plasma concentrations for S-892216 via oral administration twice daily in infected mice as per nonparametric superposition. PA-EC₉₀ = protein-adjusted EC₉₀ extrapolated to 100% mouse serum.

1. Synthetic Scheme of Benzyl Type Pyrimidine-Dione Derivatives 2–12

2. Synthetic Scheme of Aryl-type Pyrimidine-Dione Derivatives 13–18