Exploring the dual effect of novel 1,4-diarylpyranopyrazoles as antiviral and anti-inflammatory for the management of SARS-CoV-2 and associated inflammatory symptoms

Abstract

COVID-19 and associated substantial inflammations continue to threaten humankind triggering death worldwide. So, the development of new effective antiviral and anti-inflammatory medications is a major scientific goal. Pyranopyrazoles have occupied a crucial position in medicinal chemistry because of their biological importance. Here, we report the design and synthesis of a series of sixteen pyranopyrazole derivatives substituted with two aryl groups at N-1 and C-4. The designed compounds are suggested to show dual activity to combat the emerging Coronaviruses and associated substantial inflammations. All compounds were evaluated for their in vitro antiviral activity and cytotoxicity against SARS-CoV infected Vero cells. As well, the in vitro assay of all derivatives against the SARS-CoV M^pro target was performed. Results revealed the potential of three pyranopyrazoles (22, 27, and 31) to potently inhibit the viral main protease with IC₅₀ values of 2.01, 1.83, and 4.60 μM respectively compared with 12.85 and 82.17 μM for GC-376 and lopinavir. Additionally, in vivo anti-inflammatory testing for the most active compound 27 proved its ability to reduce levels of two cytokines (TNF-α and IL-6). Molecular docking and dynamics simulation revealed consistent results with the in vitro enzymatic assay and indicated the stability of the putative complex of 27 with SARS-CoV-2 M^pro. The assessment of metabolic stability and physicochemical properties of 27 have also been conducted. This investigation identified a set of metabolically stable pyranopyrazoles as effective anti-SARS-CoV-2 M^pro and suppressors of host cell cytokine release. We believe that the new compounds deserve further chemical optimization and evaluation for COVID-19 treatment.

Keywords: Antiviral; COVID-19; Molecular docking; Molecular dynamics; Physicochemical properties; SARS-CoV-2 M(pro).

Graphical abstract

Fig. 1

Prominent SARS-CoV-2 M^pro inhibitors reported in the literature.

Fig. 2

Structures of pyrazoles, pyranopyrazoles and bioisosteric antiviral and anti-inflammatory molecules.

Fig. 3

Molecular design of new pyranopyrazoles as suggested SARS-CoV-2 M^pro inhibitors and anti-inflammatory molecules.

Scheme 1

Synthetic strategy of new pyranopyrazoles.

Scheme 2

Suggested mechanism for formation of 1,4-dihydropyranopyrazoles; B^●● = Piperidine.

Fig. 4

SAR analysis of target pyranopyrazoles correlated with M^pro inhibition activity.

Fig. 5

Dose-response curve and IC₅₀ value of compounds 22 (upper panel) and 27 (lower panel) against SARS CoV-2-M^Pro.

Fig. 6

Dose-response curve of compound 27 report with IC₅₀ values at different time intervals.

Fig. 7

Heatmap correlation between the cytotoxicity effect, cellular antiviral effect (% inhibition), and SARS-CoV-2 M^pro inhibition of tested pyranopyrazoles and reference standards.

Fig. 8

Effect of compound 27 in a dose of 100 mg/kg (triplicate ± SD) on serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), in male rats administrated Carrageenan compared to negative control and diclofenac-treated groups (25 mg/kg) in pg/mL.

Fig. 9

2D and 3D binding interactions of the co-crystalized ligand (upper row) compared to the active analog 27 (lower row) in the active site of SARS-CoV-2 M^pro.

Fig. 10

Results of MD of SARS-CoV-2 M^pro (PDB ID: 7L11) complex. RMSD of complex with reference compound (upper panel) and 27 (lower panel). The RMSDs were stable after 100 ns.

Fig. 11

Results of MD of SARS-CoV-2 M^pro (PDB ID: 7L11) complexes. (A) Ligand Root Mean Square Fluctuation (l-RMSF) for reference complexed with SARS-CoV-2 M^pro (left) and for 27 complexed with SARS-CoV-2 M^pro (right). (B) Secondary structure elements (SSE) of the complex SARS-CoV-2 M^pro with reference compound (left) and with 27 (right). (C) % SSE of the two complexes of SARS-CoV-2 M^pro with reference and 27 compounds. No significant change in the percentage of SSE between the apo and bound proteins indicates conformational stability.

Fig. 12

Results of MD of SARS-CoV 2 M^pro (PDB ID: 7L11) complexes. (A) Timeline of the total contacts of the complex SARS-CoV 2 M^pro with reference compound (right) and 27 (left). (B) Stacked bar chart represents the normalized interactions throughout the trajectory. Y-axis represents the % of simulation time the specific interaction was maintained of the complex SARS-CoV–2 M^pro with reference (right) and 27 (left). (C) Ligand-Protein Contacts of the two complexes of SARS-CoV–2 M^pro with reference compound and 27.