Alkyne Derivatives of SARS-CoV-2 Main Protease Inhibitors Including Nirmatrelvir Inhibit by Reacting Covalently with the Nucleophilic Cysteine

Abstract

Nirmatrelvir (PF-07321332) is a nitrile-bearing small-molecule inhibitor that, in combination with ritonavir, is used to treat infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Nirmatrelvir interrupts the viral life cycle by inhibiting the SARS-CoV-2 main protease (M^pro), which is essential for processing viral polyproteins into functional nonstructural proteins. We report studies which reveal that derivatives of nirmatrelvir and other M^pro inhibitors with a nonactivated terminal alkyne group positioned similarly to the electrophilic nitrile of nirmatrelvir can efficiently inhibit isolated M^pro and SARS-CoV-2 replication in cells. Mass spectrometric and crystallographic evidence shows that the alkyne derivatives inhibit M^pro by apparent irreversible covalent reactions with the active site cysteine (Cys145), while the analogous nitriles react reversibly. The results highlight the potential for irreversible covalent inhibition of M^pro and other nucleophilic cysteine proteases by alkynes, which, in contrast to nitriles, can be functionalized at their terminal position to optimize inhibition and selectivity, as well as pharmacodynamic and pharmacokinetic properties.

Figure 1

Nirmatrelvir inhibits SARS-CoV-2 M^pro by reversible covalent reaction with the nucleophilic Cys145. (a) Reversible covalent reaction of nirmatrelvir (PF-07321332, 1) with the nucleophilic thiolate of SARS-CoV-2 M^pro Cys145 (deprotonated by His41). (b) View from a crystal structure of SARS-CoV-2 M^pro (gray cartoon) in complex with nirmatrelvir (1; green carbon backbone) (PDB ID: 7TE0(22)). The thioimidate is located in an oxyanion hole (involving the main chain amide NHs of Cys145 and Gly143).

Figure 2

SARS-CoV-2 M^pro and DPP4 inhibitors. (a) Ensitrelvir (S-217622, 2), (b) GC376 (3),^,, (c) MI-09 (4), (d) vildagliptin (5), and saxagliptin (6), and (e) selected reported nirmatrelvir derivatives (7a–d).

Scheme 1. Synthesis of Alkyne-Bearing Nirmatrelvir Derivative 13

Reagents and conditions: (a) LiOH, THF/H₂O, 0 °C to room temperature (rt), 83%; (b) Me(OMe)NH·HCl, 1,1′-carbonyldiimidazole (CDI), ⁱPr₂NEt, CH₂Cl₂, rt, 58%; (c) LiAlH₄, THF/Et₂O, −78 °C, 93%; (d) 10,^, K₂CO₃, MeOH, rt, 43%; (e) HCl (4 M in dioxane), CH₂Cl₂, rt, app. quant.; (f) 12, COMU,N-methylmorpholine (NMM), DMF/CH₂Cl₂, 0 °C to rt, 17%.

Figure 3

Dose–responses observed for nitrile- or alkyne-bearing SARS-CoV-2 M^pro inhibitors. Representative dose–response curves of M^pro inhibitors shown in (a) Table 1, (b) Table 2, and (c) Table 3. High Z′-factors (>0.5 for each inhibition plate) indicate excellent solid-phase extraction coupled to mass spectrometry (SPE-MS) assay quality (Supporting Figure S5).

Scheme 2. Synthesis of Alkyne Derivatives of the Investigational COVID-19 Therapeutics GC376 (3) and MI-09 (4)

Reagents and conditions: (a) 16(27) or 17, COMU, NMM, DMF/CH₂Cl₂, 0 °C to rt, 14 and 13%, respectively; (b) COMU, NMM, DMF/CH₂Cl₂, 0 °C to rt, 33%.

Scheme 3. Synthesis of Nirmatrelvir Derivatives Bearing an Internal Alkyne

Reagents and conditions: (a) aryl iodide, CuI (5 mol %), PdCl₂(PPh₃)₂ (2.5 mol %), NEt₃/THF, 80 °C, 46% (25a) or 44% (25b); (b) HCl (4 M in dioxane), CH₂Cl₂, rt, app. quant.; (c) 12, COMU, NMM, DMF/CH₂Cl₂, 0 °C to rt, 17% (27a) or 11% (27b); (d) TMSCF₃, CuI, K₂CO₃, N,N,N′,N′-tetramethylethylenediamine (TMEDA), DMF, rt, air, 26%.

Figure 4

Crystallographic evidence that the alkyne of the nirmatrelvir derivative 13 reacts covalently with the nucleophilic thiolate of M^pro Cys145. Color code: SARS-CoV-2 M^pro, gray; carbon backbone of 13 in complex with M^pro is in orange; oxygen, red; nitrogen, blue; sulfur, yellow; and fluorine, light blue. (a) Reaction of M^pro with alkyne 13; (b) representative OMIT electron density map (mF_o–DF_c) contoured to 3σ around Cys145 and 13 in complex with M^pro (PDB ID: 8B2T); (c) superimposition of a view from the M^pro:13 complex structure (PDB ID: 8B2T) with the reported M^pro:nirmatrelvir (1) complex structure (pale yellow: M^pro; green: carbon backbone of 1 in complex with M^pro; PDB ID: 7TE0(22)) showing the interaction of 1 but not 13 with residues forming the oxyanion hole, i.e., Cys145 and Gly143.

Figure 5

Nirmatrelvir alkyne derivatives inhibit SARS-CoV-2 M^pro via covalent reaction with Cys145. (a) Reaction of M^pro (bottom) with nirmatrelvir (1, center) and its alkyne derivative 13 (top); SPE-MS analysis indicates near-quantitative reaction of 1 or 13 with M^pro (∼500 Da mass shifts). (b) Addition of a 10-fold excess of alkyne 28 to the covalent complexes of M^pro with 1 or 13, followed by 30 min incubation; SPE-MS analysis of the mixtures reveals formation of a covalent complexes of M^pro with 28 only for the M^pro complex with 1 (∼66 Da mass shift, center), but not with 13 (top), as compared with unreacted M^pro (bottom). (c) SPE-MS analysis of the mixtures of the covalent complexes of M^pro with 1 or 13 with 28 indicates that 28 reacts slowly with the M^pro complex with 1 (∼66 Da mass shift, center), but not with 13 (top), as compared with unreacted M^pro (bottom). M^pro assays were performed using SPE-MS as described in the Experimental Section employing SARS-CoV-2 M^pro (2.0 μM) in buffer (20 mM HEPES, pH 7.5).^,

Figure 6

Alkynes are important functional groups in human therapeutics. (a) Representative human therapeutics that contain alkynes (in green); (b) covalent reaction of acalabrutinib (36) with Btk Cys481.