Structure-based design, synthesis, and biological evaluation of a series of novel and reversible inhibitors for the severe acute respiratory syndrome-coronavirus papain-like protease

Abstract

We describe here the design, synthesis, molecular modeling, and biological evaluation of a series of small molecule, nonpeptide inhibitors of SARS-CoV PLpro. Our initial lead compound was identified via high-throughput screening of a diverse chemical library. We subsequently carried out structure-activity relationship studies and optimized the lead structure to potent inhibitors that have shown antiviral activity against SARS-CoV infected Vero E6 cells. Upon the basis of the X-ray crystal structure of inhibitor 24-bound to SARS-CoV PLpro, a drug design template was created. Our structure-based modification led to the design of a more potent inhibitor, 2 (enzyme IC(50) = 0.46 microM; antiviral EC(50) = 6 microM). Interestingly, its methylamine derivative, 49, displayed good enzyme inhibitory potency (IC(50) = 1.3 microM) and the most potent SARS antiviral activity (EC(50) = 5.2 microM) in the series. We have carried out computational docking studies and generated a predictive 3D-QSAR model for SARS-CoV PLpro inhibitors.

Figure 1

Structure of inhibitors 1, 2, 24 and 4

Figure 2

Docking in the presence of conserved water molecules is critical for replicating the binding conformation of inhibitors in the active site of the bound form of SARS-CoV PLpro. (A) Docking of compound 2 in the absence of water molecules in the active site of the inhibitor-bound form of SARS-CoV PLpro causes the naphthyl rings to flip down into a pocket, as shown by the docked conformation of compound 2 (in green) when compared to the crystal structure conformation of compound 24 (in cyan). In the x-ray structure of compound 24, the naphthyl rings are flipped up in the opposite direction, holding the flexible loop in place. The yellow dotted lines show the possible interactions of the docked compound 2 with residues Tyr269, Gln270 and Asp165 (catalytic domain residue numbering). (B) The three conserved water molecules are marked; two of them are buried deep in the pocket (P5) whereas the third one lies in a groove between residues Lys158 and Glu168. The position of these water molecules is integral to structure-based inhibitor design efforts. The crystal structure conformation of compound 24 is shown in cyan whereas the docked conformation of compound 2 in the presence of water molecules (red dots) is shown in green.

Figure 3

Correlation plot between the predicted percent inhibition of 41 compounds and the actual experimental percent inhibition values obtained at 100 μM.

Figure 4

Compound 24 is shown in cyan in its bound conformation as in the crystal structure aligned with the docked conformation of the most active compound 2, in white, when docked in the presence of 3 conserved water molecules. A) The electrostatic contour map for this series of SARS-CoV PLpro inhibitors. Blue is the region of unfavorable negative charge and red is of favorable negative charge. B) The hydrophobic contour map, where white is the region of unfavorable lipophilic interactions and magenta is of favorable lipophilicity. C) A steric contour map, with green denoting the region of favorable steric interactions and yellow denoting unfavorable regions.

Figure 4

Compound 24 is shown in cyan in its bound conformation as in the crystal structure aligned with the docked conformation of the most active compound 2, in white, when docked in the presence of 3 conserved water molecules. A) The electrostatic contour map for this series of SARS-CoV PLpro inhibitors. Blue is the region of unfavorable negative charge and red is of favorable negative charge. B) The hydrophobic contour map, where white is the region of unfavorable lipophilic interactions and magenta is of favorable lipophilicity. C) A steric contour map, with green denoting the region of favorable steric interactions and yellow denoting unfavorable regions.

Scheme 1

Reagents and conditions: (a) EDCI, HOBT, DIPEA, 23 ºC, 16 h. [Table: see text]

Scheme 2

Reagents and conditions: (a) Boc₂O, Et₃N, dioxane/H₂O (2:1), 23 ºC, 16 h; (b) (R)-(+)-1-(2-naphthyl)ethylamine, EDCI, HOBT, DIPEA, CH₂Cl₂, 23 ºC, 16 h; (c) TFA, CH₂Cl₂, 23 ºC, 2 h.

Scheme 3

Reagent and conditions: (a) AlCl₃, 1,2-dichloroethane, 35 ºC, 4 h; (b) NH₄OAc, NaBH₃CN, MeOH, 23 ºC, 24 h; (c) o-toluic acid, EDCI, HOBT, DIPEA, DMF, 23 ºC, 16 h; (d) o-toluic acid, EDCI, HOBT, DIPEA, CH₂Cl₂, 23 ºC, 16 h.

Scheme 4

Reagents and conditions: (a) ClCO₂Me, K₂CO₃, dioxane/H₂O (1:1), 0 ºC, 1 h; (b) LiAlH₄, THF, reflux, 1 h; (c) o-toluic acid, EDCI, HOBT, DIPEA, DMF, 23 ºC, 16 h; (d) 7, EDCI, HOBT, DIPEA, DMF, 23 ºC, 16 h; (e) TFA, CH₂Cl₂, 23 ºC, 2 h.

Scheme 5

Reagents and conditions: (a) H₂, Pd-C, EtOAc/MeOH (1:1), 23 ºC, 15 h; (b) Ac₂O, Et₃N, CH₂Cl₂, 23 ºC, 18 h; (c) MeLi, CeCl₃, THF, 23 ºC, 2 h; (d) 2-Methyl-5-nitrobenzoic acid, EDCI, HOBT, DIPEA, CH₂Cl₂, 23 ºC, 16 h; (e) H₂, Pd-C, EtOAc/MeOH (1:1), 23 ºC, 15 h.

Scheme 6

Reagents and conditions: (a) KI, NaIO₄, conc. H₂SO₄, 25–30 ºC, 2 h; (b) (R)-(+)-1-(1-naphthyl)ethylamine 18, EDCI, HOBT, DIPEA, DMF/CH₂Cl₂ (1:1), 23 ºC, 48 h; (c) CuCN, KCN, DMF, 130 ºC, 16 h; (d) SOCl₂, MeOH, reflux, 4 h; (e) NBS, Bz₂O₂, CCl₄, reflux, 24 h; (f) NaH, NaOMe, MeOH, 50 ºC, 4 h; (g) LiOH·H₂O, THF/H₂O (5:1), 23 ºC, 1.5 h; (h) (R)-(+)-1-(1-naphthyl)ethylamine 18, EDCI, HOBT, DIPEA, DMF/CH₂Cl₂ (1:1), 23 ºC, 16 h; (i) H₂, Pd-C, EtOAc, 23 ºC, 10 h.

Scheme 7

Reagents and conditions: (a) H₂, Pd-C, EtOAc, 23 ºC, 16 h; (b) NaNO₂, conc. HCl, CuCN, NaCN, H₂O, 23 ºC, 3 h; (c) Boc₂O, NiCl₂·6H₂O, NaBH₄, MeOH, 23 ºC, 2 h; (d) MeI, KHMDS, THF, 23 ºC, 16 h; (e) LiOH·H₂O, THF/H₂O (9:1), 23 ºC, 16 h; (f) (R)-(+)-1-(1-naphthyl)ethylamine 18, EDCI, HOBT, DIPEA, CH₂Cl₂, 23 ºC, 16 h; (g) TFA, CH₂Cl₂, 23 ºC, 2 h.