Discovery of 2-(furan-2-ylmethylene)hydrazine-1-carbothioamide derivatives as novel inhibitors of SARS-CoV-2 main protease

Abstract

The COVID-19 posed a serious threat to human life and health, and SARS-CoV-2 M^pro has been considered as an attractive drug target for the treatment of COVID-19. Herein, we report 2-(furan-2-ylmethylene)hydrazine-1-carbothioamide derivatives as novel inhibitors of SARS-CoV-2 M^pro developed by in-house library screening and biological evaluation. Similarity search led to the identification of compound F8-S43 with the enzymatic IC₅₀ value of 10.76 μM. Further structure-based drug design and synthetic optimization uncovered compounds F8-B6 and F8-B22 as novel non-peptidomimetic inhibitors of M^pro with IC₅₀ values of 1.57 μM and 1.55 μM, respectively. Moreover, enzymatic kinetic assay and mass spectrometry demonstrated that F8-B6 was a reversible covalent inhibitor of M^pro. Besides, F8-B6 showed low cytotoxicity with CC₅₀ values of more than 100 μM in Vero and MDCK cells. Overall, these novel SARS-CoV-2 M^pro non-peptidomimetic inhibitors provide a useful starting point for further structural optimization.

Keywords: 2-(furan-2-ylmethylene)hydrazine-1-carbothioamide derivatives; Main protease; Non-peptidomimetic inhibitors; SARS-CoV-2.

Graphical abstract

Fig. 1

Chemical structures of represented inhibitors of SARS-CoV-2 M^pro.

Fig. 2

The discovery of SARS-CoV-2 M^pro inhibitors. (A) Screen the in-house library through SPR assay. (B) The chemical structure and K_D value of compound F8. (C) The dose-dependent curve of F8 against SARS-CoV-2 M^pro. (D) The similarity analysis of compound F8 with known SARS-CoV-2 M^pro inhibitors.

Fig. 3

Predicted binding mode of compound F8–S43. (A) The binding pattern of compound F8–S43 with SARS-CoV-2 M^pro in surface and 2D diagram (B). Hydrogen bonds are represented by yellow lines. Images depicting the proposed binding modes were generated using PyMOL software. (C) The design of newly identified SARS-CoV-2 M^pro inhibitors.

Scheme 1

Synthetic Route of Compounds F8-A1 to F8-A9. Reagents and conditions: (a) 5-bromofuran-2-carbaldehyde, Pd(PPh₃)₄, K₂CO₃, PhMe/EtOH/H₂O, 90 °C, overnight; (b) R¹NH₂CSNHNH₂, MeOH, 50 °C, 4 h.

Scheme 2

Synthetic Route of Compounds F8–B1 to F8–B13. Reagents and conditions: (a) (4-carbamoylphenyl)boronic acid, Pd(PPh₃)₄, K₂CO₃, PhMe/EtOH/H₂O, 90 °C, overnight; (b) Thiosemicarbazide, MeOH, 50 °C, 4 h; (c) Substituted bromobenzene, Pd(PPh₃)₂Cl₂, Na₂CO₃, MeCN/H₂O, 90 °C, overnight; or substituted bromobenzene, Pd(PPh₃)₄, K₂CO₃, PhMe/EtOH/H₂O, 90 °C, overnight; or Substituted bromobenzene, Pd(PPh₃)₂Cl₂, 2 M Na₂CO₃, DME/EtOH, 60 °C, overnight.

Scheme 3

Synthetic Route of Compounds F8–B14 to F8–B22. Reagents and conditions: (a) Corresponding aryl boronic acid, Pd(PPh₃)₄, K₂CO₃, PhMe/EtOH/H₂O, 90 °C, overnight; (b) Thiosemicarbazide, MeOH, 50 °C, 4 h; (c) benzene, trifluoromethanesulfonic acid, r.t.

Scheme 4

Synthetic Route of Compounds F8–C1 to F8–C4. Reagents and conditions: (a) (4-carbamoylphenyl)boronic acid, Pd(PPh₃)₄, K₂CO₃, PhMe/EtOH/H₂O, 90 °C, overnight; (b) Thiosemicarbazide, MeOH, 50 °C, 4 h.

Fig. 4

Michaelis-Menten kinetics analysis (A) and Lineweaver-Burk plot (B) of SARS-CoV-2 M^pro in presence or absence of F8–B6. (C) The liquid chromatograph-mass spectrometer of SARS-CoV-2 M^pro with F8–B6. (D) Reversibility assay of the inhibition ability of F8–B6 against SARS-CoV-2 M^pro.

Fig. 5

The cytotoxicity of compounds F8–B6 and F8–B22 in Vero (A) and MDCK cells (B).