Penicillin Derivatives Inhibit the SARS-CoV-2 Main Protease by Reaction with Its Nucleophilic Cysteine

Abstract

The SARS-CoV-2 main protease (M^pro) is a medicinal chemistry target for COVID-19 treatment. Given the clinical efficacy of β-lactams as inhibitors of bacterial nucleophilic enzymes, they are of interest as inhibitors of viral nucleophilic serine and cysteine proteases. We describe the synthesis of penicillin derivatives which are potent M^pro inhibitors and investigate their mechanism of inhibition using mass spectrometric and crystallographic analyses. The results suggest that β-lactams have considerable potential as M^pro inhibitors via a mechanism involving reaction with the nucleophilic cysteine to form a stable acyl-enzyme complex as shown by crystallographic analysis. The results highlight the potential for inhibition of viral proteases employing nucleophilic catalysis by β-lactams and related acylating agents.

Figure 1

Examples of reported SARS-CoV-2 M^pro small-molecule inhibitors. (A) Boceprevir (1);^, (B) SDZ-224015 (2); (C) GC-376 (3);^,, (D) PF-00835231 (4) and its prodrug PF-07304814 (5);^, (E) N3 (6); (F) α-ketoamide 7; (G) PF-07321332 (8, nirmatrelvir); (H) MI-09 (9); and (I) penicillin V and G sulfone benzyl esters 10 and 11.

Scheme 1. Synthesis of Penicillin V Ester Derivatives

Reagents and conditions: (a) alkylhalide (1.2 equiv), DMF, rt, 59–93%; (b) mCPBA, CH₂Cl₂, 0 °C to rt, 89–94%. Note that the sulfoxide stereochemistry was tentatively assigned the (S)-configuration based on reported mCPBA-mediated penicillin ester oxidations to sulfoxides.^,,

Scheme 2. Synthesis of Penicillin Sulfone Benzyl Esters from (+)-6-Aminopenicillanic Acid (6-APA, 22)

Reagents and conditions: (a) benzylbromide, triethyl amine, CH₂Cl₂, 0 °C; (b) para-toluenesulfonic acid, acetone, rt; (c) NaHCO₃, ethyl acetate/H₂O, rt, 33% over three steps; (d) carboxylic acid, COMU, DMF, 0 °C to rt, 44–82%; (e) mCPBA, CH₂Cl₂, 0 °C to rt, 11–71%. Note that the C6 NHCbz penicillin benzyl ester 24a was synthesized by a different sequence, as described in the Supporting Information.

Figure 2

Evidence that penicillin V sulfone ester derivatives inhibit M^pro by selective active site cysteine covalent modification. M^pro assays with penicillin sulfone ester derivatives 10 (A, D), 25a (B, E), and 20q (C, F) were performed in the absence (A–C) or presence (D–F) of TPCK using SPE-MS as described in the Experimental Section employing SARS-CoV-2 M^pro (2.0 μM), penicillin sulfone ester derivatives (11 μM for b, and 33 μM for c and e), and TPCK (10 μM for d and e). The reactions were incubated for either 45 min (a–c) or 180 min (with TPCK, d and e) followed by additional 60 min (with a penicillin sulfone ester derivative, e) prior to analysis by SPE-MS. The reactions were performed in technical duplicates (Supporting Information Figure S5). Note (i) the clear evidence for the covalent reaction of 10 and 20q but not 25a, and (ii) that reaction is ablated by pretreatment of M^pro with the active site binding inhibitor TPCK.

Figure 3

Crystallographic evidence that penicillin V sulfone ester derivatives inhibit M^pro by active site cysteine covalent modification. Color code: M^pro: gray (protomer 1) and cyan (protomer 2); carbon-backbone of the 20e-derived complex is in orange, with the β-lactam ring-derived carbon-backbone in magenta; oxygen: red; nitrogen: blue; sulfur: yellow; and fluorine: light blue. (A) Reaction of penicillin sulfone 20e with SARS-CoV-2 M^pro. (B, C) Representative OMIT electron density map (mF_o-DF_c) contoured to 2.5σ around Cys145 and the 20e-derived complex showing clear evidence for (B) β-lactam ring opening by the active site Cys145 leading to thioester formation and (C) positioning of the SO₂H group of the 20e-derived complex formed by opening of the thiazolidine sulfone ring to enable interactions with the main chain amino group of Ser1 of the second M^pro protomer. (D) Phe140, Glu166, and the SO₂H group of the 20e-derived complex are positioned to interact with Ser1 of the second M^pro protomer. (E) C6 amido penicillin-derived side chain of the 20e-derived complex binds in the hydrophobic S2 M^pro binding site. (F) Superimposition of active sites’ views of the M^pro:20e-derived complex and the M^pro:PF-07321332 (slate blue: carbon-backbone of 8, nirmatrelvir; PDB ID: 7VH8(68)) structures.