Severe acute respiratory syndrome coronavirus papain-like novel protease inhibitors: design, synthesis, protein-ligand X-ray structure and biological evaluation

Abstract

The design, synthesis, X-ray crystal structure, molecular modeling, and biological evaluation of a series of new generation SARS-CoV PLpro inhibitors are described. A new lead compound 3 (6577871) was identified via high-throughput screening of a diverse chemical library. Subsequently, we carried out lead optimization and structure-activity studies to provide a series of improved inhibitors that show potent PLpro inhibition and antiviral activity against SARS-CoV infected Vero E6 cells. Interestingly, the (S)-Me inhibitor 15 h (enzyme IC(50) = 0.56 microM; antiviral EC(50) = 9.1 microM) and the corresponding (R)-Me 15 g (IC(50) = 0.32 microM; antiviral EC(50) = 9.1 microM) are the most potent compounds in this series, with nearly equivalent enzymatic inhibition and antiviral activity. A protein-ligand X-ray structure of 15 g-bound SARS-CoV PLpro and a corresponding model of 15 h docked to PLpro provide intriguing molecular insight into the ligand-binding site interactions.

Figure 1

Structures of PLpro inhibitors 1-3 and 15g

Figure 2

Stereo representation of 15g bound to PLpro, including the conserved waters adjacent to the binding site that may influence the binding configuration, as described in the text.

Figure 3

The X-ray structure of inhibitor 15g-bound (yellow) PLpro (grey) (PDB id: WXYZ) superimposed on the X-ray structure of inhibitor 2-bound (cyan) PLpro (pink) (PDB id: 3E9S).

Figure 4

(A) Superposition of enantiomer 15h (blue) with the crystal structure of 15g-bound (yellow) PLpro. (B) Docked alignment of the gem-dimethyl substituted compound in the 15g-ligand removed PLpro crystal structure. The bumping collision of one of the methyl groups of the gem-dimethyl (magenta) 15k with the Asp-165 carboxylate is noted.

Scheme 1

Reagents and conditions: (a) 5a or 5b, EDCI, HOBT, NMM, CH₂Cl₂, 23 °C, 5 h; (b) TFA, 0 °C to 23 °C, 6 h; (c) 1-naphthaldehyde, Na(OAc)₃BH, AcOH, CH₂Cl₂, 23 °C, 12 h.

Scheme 2

Reagents and conditions: (a) KOt-Bu, DMSO, 23 °C, 48 h (b) 10% HCl, THF, 23 °C, 18 h; (c) NaHCO₃, 23 °C, 16 h; (c) H₂, PtO₂, EtOAc, 23 °C, 2 h.

Scheme 3

Reagents and conditions: (a) NaCN, DMF, reflux, 16 h; (b) LiOH·H₂O, THF/MeOH/H₂O (3:1:1), 23 °C, 16 h; (c) 5a-d, EDCI, HOBT, DIPEA, CH₂Cl₂/DMF (9:1), 23 °C, 15 h.

Scheme 4

Reagents and conditions: (a) NaBH₃CN, MeOH/AcOH (50:1), 23 °C, 48 h; (b) TFA, CH₂Cl₂, 23 °C, 2 h; (c) 5c, N,N′-carbonyldiimidazole, CH₂Cl₂, 23 °C, 4 h.