Discovery of S-217622, a Noncovalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19

Abstract

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and threatens public health and safety. Despite the rapid global spread of COVID-19 vaccines, effective oral antiviral drugs are urgently needed. Here, we describe the discovery of S-217622, the first oral noncovalent, nonpeptidic SARS-CoV-2 3CL protease inhibitor clinical candidate. S-217622 was discovered via virtual screening followed by biological screening of an in-house compound library, and optimization of the hit compound using a structure-based drug design strategy. S-217622 exhibited antiviral activity in vitro against current outbreaking SARS-CoV-2 variants and showed favorable pharmacokinetic profiles in vivo for once-daily oral dosing. Furthermore, S-217622 dose-dependently inhibited intrapulmonary replication of SARS-CoV-2 in mice, indicating that this novel noncovalent inhibitor could be a potential oral agent for treating COVID-19.

Figure 1

Schematic flow of the screening campaign.

Figure 2

Binding modes of 3CL^pro inhibitors, their interactions, and defined pharmacophore filters for virtual screening. (a) Crystal structures of GC376 (PDB code: 6WTT), (b) 3-aminopyridine-like compound of the Postera COVID moonshot project (PDB code: 5RH2), and (c) ML188 (PDB code: 7L0D). The common H-bond acceptors are circled in red; the common hydrophobic features are circled in blue. (d) Common pharmacophore shared with inhibitors A–C. Red and green spheres represent H-bond acceptors and lipophilic features, respectively.

Figure 3

Structure-based optimization of the hit compound 1 and the profile of compounds. (a) Cytopathic effect (CPE) inhibition assay with Vero E6 cells expressing human transmembrane protease serine2 (VeroE6/TMPRSS2). (b) Percentage remaining in human liver microsomes (HLM) after 30 min. (c) Percentage remaining in rat liver microsomes (RLM) after 30 min. (d) Total clearance, (e) intravenously administered, 0.5 μmol/mL/kg (n = 2) in the nonfasted condition, (f) intravenously administered, 0.1 mg/0.2 mL/kg (n = 2) in the nonfasted condition, and (g) oral bioavailabiity. (h) Oral administration was carried out at 1 μmol/5 mL/kg (n = 2) under the nonfasted condition. (i) Oral administration was carried out at 3 mg/2 mL/kg (n = 3) under the nonfasted condition. (j) Evaluated as S-217622 fumaric acid. (k) Not tested.

Figure 4

X-ray costructure of hit compound 1 and 3CL^pro (PDB code: 7VTH). (a) Close-up view of 1 (cyan) in the binding pocket. Water molecules are shown as red spheres. Hydrogen bonds are indicated as yellow dashed lines; π–π stacking is indicated as a cyan dashed line. (b–d) Comparison of a docking pose and X-ray crystal structure of 1. Near the S2 pocket, the side chain of His41 was rotated to form a face-to-face π interaction with the 3,4,5-trifluorobenzene moiety of 1. Docked structure is in lime green, and the X-ray structure is in cyan (1) and orange (protein residues).

Figure 5

X-ray costructure of S-217622 (3) and 3CL^pro (PDB code: 7VU6). 3 is colored in orange and the protein is colored in gray. Water molecules are shown as red spheres. Hydrogen bonds are indicated as yellow dashed lines; π–π stacking is indicated as a cyan dashed line.

Figure 6

In vitro cellular activity of S-217622. Antiviral activity of S-217622 against (a) various SARS-CoV-2 strains, (b) SARS-CoV, and (c) MERS-CoV in a cytopathic effect (CPE) inhibition assay using VeroE6/TMPRSS2 cells. Antiviral activity of S-217622 against (d) HCoV-229E (Alphacoronavirus) in a CPE inhibition assay with MRC-5 cells and (e) HCoV-OC43 (Betacoronavirus) in a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay with MRC-5 cells. Data are the means ± standard deviation; n = 3 biological replicates for SARS-CoV-2 strains, MERS-CoV, HCoV-229E, and HCoV-OC43 and n = 4 for SARS-CoV.

Figure 7

Dose-dependent in vivo antiviral efficacy of S-217622 in mice infected with SARS-CoV-2. (a) Protocol for the in vivo study. bid = twice a day. (b) Effect of S-217622 (administered as S-217622 fumaric acid) treatment on lung viral titers in SARS-CoV-2 gamma strain (hCoV-19/Japan/TY7-501/2021)-infected mice. TCID₅₀ = 50% tissue culture infectious dose; each point represents an individual viral titer (n = 5–10). The broken line represents the lower limit of quantification (1.80 log₁₀ TCID₅₀/mL). The following p-values were calculated using Dunnett’s test: *p < 0.05 and **p < 0.0001 vs vehicle. (c) S-217622 plasma concentration in the infected mice (n = 4). (d) Simulated S-217622 plasma concentrations after repeated oral administration of S-217622 (administered as S-217622 fumaric acid) twice daily in infected mice as per nonparametric superposition. PA-EC₅₀ = protein-adjusted EC₅₀ extrapolated to 100% mouse serum.

Scheme 1. Synthesis of Compound 1

Reagents and conditions: (a) Ethyl isocyanato-acetate, DBU, CDI, DMA, −10 °C to rt, 90%; (b) 5-(bromomethyl)-1,2,3-trifluorobenzene, N,N-diisopropylethylamine, DMA, 60 °C; (c) 4-difluoromethoxy-2-methylaniline, tert-butanol, 100 °C, 40% in two steps; (d) (i) NaOH aq., THF/MeOH, rt; (ii) methylamine, HATU, N,N-diisopropylethylamine, THF, rt, 58% in two steps.

Scheme 2. Synthesis of Compound 2

Reagents and conditions: (a) 5-(Bromomethyl)-1,2,3-trifluorobenzene, N,N-diisopropylethylamine, DMA, 60 °C; (b) 6-chloro-2-methyl-2H-indazol-5-amine,tert-amyl alcohol, 100 °C, 44% in two steps from 5; (c) (i) NaOH aq., THF/MeOH, rt; (ii) methylamine, HATU, N,N-diisopropylethylamine, THF, rt, 29% in two steps.

Scheme 3. Synthesis of Compound 3 (S-217622)

Reagents and conditions: (a) 1-(Bromomethyl)-2,4,5-trifluorobenzene, K₂CO₃, MeCN, 80 °C, 93%; (b) TFA, rt, 97%; (c) 3-(chloromethyl)-1-methyl-1H-1,2,4-triazole hydrochloride, K₂CO₃, DMF, 60 °C, 45%; (d) 6-chloro-2-methyl-2H-indazol-5-amine, LHMDS, THF, 0 °C to rt, 25%.