Structure-Based Optimization of Pyridone α-Ketoamides as Inhibitors of the SARS-CoV-2 Main Protease

Abstract

The main protease M^pro is a clinically validated target to treat infections by the coronavirus SARS-CoV-2. Among the first reported M^pro inhibitors was the peptidomimetic α-ketoamide 13b, whose cocrystal structure with M^pro paved the way for multiple lead-finding studies. We established structure-activity relationships for the 13b series by modifying residues at the P1', P3, and P4 sites. Guided by cocrystal structures, we reduced the P1' substituent size to better fill the pocket and added a fluorine substituent to the pyridone ring, enabling a new hydrogen bond with Gln189 in P3. Among 22 novel analogues, 6d and 12d inhibited M^pro with IC₅₀s of 110 nM and 40 nM, improving the potency of 13b by up to 9.5-fold. Compound 6d had pronounced antiviral activity with an EC₅₀ of 1.6 μM and was stable in plasma and microsomes. The study illustrates the potential of structure-based design to systematically improve peptidomimetic α-ketoamides.

Figure 1

Structures of selected, previously reported M^pro inhibitors and design considerations for this study. (A) Structures of selected M^pro inhibitors with covalent warheads. (B) Rationale for modifying P1′, P3, and P4 residues based on the cocrystal structure of 13b-K with M^pro (PDB Code: 6Y2G). S1′: reduce the site of P1′ rest to improve pocket fit, S3: add a substituent to enable H-bonding to Gln189, S4: modify P4 rest to benefit from the spacious S4 pocket. 13b-K is represented as sticks in pink, and the M^pro surface is shown in light blue.

Scheme 1. Improved Synthesis of Aldehyde 4a

Reagents and conditions: (a) (i) KBr, H₂SO₄, NaNO₂, 0 °C—rt, 16 h; (ii) SOCl₂/CH₃OH, 0 °C—rt, 16 h; 90% over two steps; (b) Cs₂CO₃/CH₃CN, 16 h, 50 °C, 70%; (c) (i) LiOH·H₂O, aq.CH₃OH, 0 °C—rt, 1.5 h; (ii) methyl (S)-2-amino-3-((S)-2-oxopyrrolidin-3-yl)propanoate hydrochloride, HATU/TEA, DMF, 0 °C—rt, 10 h, 92% over two steps; (d) (i) LiBH₄, CH₂Cl₂, 2 h; (ii) DMP, CH₂Cl₂, 2 h, 96% over two steps.

Scheme 2. Synthesis of α-Ketoamides 6a–d

Reagents and conditions: (a) alkyl isocyanide, CH₃COOH, CH₂Cl₂, 0 °C—rt, 22 h, 85–92%; (b) (i) LiOH·H₂O, MeOH, H₂O, 0 °C—rt, 1.5 h; (ii) DMP, CH₂Cl₂, rt, 2 h, 49–63% over two steps.

Scheme 3. Synthesis of α-Ketoamides (9a–d)

Reagents and conditions: (a) acetone cyanohydrin, TEA, DCM, 0 °C—rt, 3 h, 82%; (b) NaCN, sodium bisulfite, MeOH, H₂O, 0 °C, 88–92%; (c) 30% H₂O₂ (aq), LiOH·H₂O, MeOH, 0 °C—rt, 1 h, 33–68%; (d) for 9a: DMP, DMF, rt, 3 h, 31%; (e) for 9b–d: IBX, acetone, reflux, 5 h, 58–69%.

Figure 2

Overall cocrystal structure of the M^pro in complex with 6d (PDB: 9F3A). Compound 6d is colored in pink, and all H-bonds between the inhibitor and the corresponding M^pro residues are colored in red.

Figure 3

Overall crystal structure of M^pro in complex with 13b-K (PDB: 6Y2E), 9a (9GMQ), 6c (8AIU), and 6a (8AIV). The overall pattern of interactions between inhibitors and M^pro in the S1 pocket is conserved.

Figure 4

Crystal structures of M^pro in complex with 6a (A, PDB code: 8AIV) and 6c (B, PDB code: 8AIU). The P1′ cyclopropyl derivative 6c shows close contact with Thr26. Crystal structure of M^pro in complex with 13b-K (C, PDB code: 6Y2G) and 9b (D, PDB code: 9F2V).

Scheme 4. Synthesis of Acylated α-Ketoamides 12a–h

Reagents and conditions: (a) 4 M HCl, CH₂Cl₂, 0 °C—rt, 6 h, 84–98%; (b) R₂-COOH, HATU, TEA, DMF, 0 °C, 10 h, 65–95%; (c) for 11e: PhCH₂OCOCl, NaHCO₃, aq. THF, 0 °C—rt, 16 h, 83%; (d) (i) LiOH·H₂O, MeOH, H₂O, 0 °C—rt, 1.5 h; (ii) DMP, CH₂Cl₂, rt, 2 h, 47–68%.

Scheme 5. Synthesis of Urea α-Ketoamides 16a–e and 17

Reagents and Conditions: (a) alkyl/aryl isocyanates, CH₂Cl₂, DMF, 0 °C—rt, 24 h, 38–68%; (b) (i) LiOH·H₂O, MeOH, H₂O, 0 °C—rt, 1.5 h; (ii) DMP, CH₂Cl₂, rt, 2 h, 36–68% over two steps.

Figure 5

Structural model of cathepsin L in complex with 9b. Model constructed using electron density map of cathepsin L in complex with 13b (PDB code: 8PRX): Strong H-bonds are formed between Gln-19 Nε2 and P1́O (2.7 Å) and between Cys25 N and P1́O (2.8 Å); H-bonds are colored in red.

Figure 6

Summary of the structure–activity relationship of pyridone α-ketoamides derived in this study.

Figure 7

Pharmacokinetic studies of pyridone α-ketoamides in mice. Plasma concentrations are displayed after cassette dosing of 1 mg/kg IV for 6a, 6c, 6d, 9a, and 9b (upper panel) and for 12b, 12c, 12d, 16c, and 16e (lower panel). n = 2 animals were used per study.