Synthesis, anti-inflammatory, analgesic, COX1/2-inhibitory activity, and molecular docking studies of hybrid pyrazole analogues

Abstract

This article reports on the design, synthesis, and pharmacological activity of a new series of hybrid pyrazole analogues: 5a-5u. Among the series 5a-5u, the compounds 5u and 5s exhibited potent anti-inflammatory activity of 80.63% and 78.09% and inhibition of 80.87% and 76.56% compared with the standard drug ibuprofen, which showed 81.32% and 79.23% inhibition after 3 and 4 hours, respectively. On the basis of in vivo studies, 12 compounds were selected for assessment of their in vitro inhibitory action against COX1/2 and TNFα. The compounds 5u and 5s showed high COX2-inhibitory activity, with half-maximal inhibitory concentrations of 1.79 and 2.51 μM and selectivity index values of 72.73 and 65.75, respectively, comparable to celecoxib (selectivity index =78.06). These selected compounds were also tested for TNFα, cytotoxicity, and ulcerogenicity. Docking studies were also carried out to determine possible interactions of the potent compounds (5u and 5s), which also showed high docking scores of -12.907 and -12.24 compared to celecoxib, with a -9.924 docking score. These selective COX2 inhibitors were docked into the active site of COX2, and showed the same orientation and binding mode to that of celecoxib (selective COX2 inhibitor). Docking studies also showed that the SO₂NH₂ of 5u and 5s is inserted deep inside the selective pocket of the COX2-active site and formed a hydrogen-bond interaction with His90, Arg513, Phe518, Ser353, Gln192, and Ile517, which was further validated by superimposed docked pose with celecoxib.

Keywords: TNFα inhibition; analgesic activity; anti-inflammatory activity; molecular docking studies; pyrazole; selective COX2 inhibition.

Figure 1

Chemical structure of some pyrazole-containing anti-inflammatory drugs.

Figure 2

Strategy for design of target compound with structural resemblance of reference ligand. Abbreviation: IC₅₀, half-maximal inhibitory concentration.

Figure 3

Histopathological study of stomachs of rats treated with standard drug (ibuprofen) and active compounds in comparison to healthy controls. Notes: Green arrow shows a normal mucosal cell. Blue arrow shows mucosal cell damage when treated with Ibuprofen. Black arrow represents the mucosal cell damage (ulcer) in high resolution (×40).

Figure 4

In vitro TNFα assay of synthesized compound. Notes: Data analyzed by one-way analysis of variance followed by Dunnett’s t-test and expressed as mean ± standard error of the mean from six observations.

Figure 5

2-D LigPlot interaction diagrams. Notes: (A) Compound 5u; (B) compound 5s. Abbreviation: 2-D, two-dimensional.

Figure 6

Docked pose of 5s and 5u. Notes: (A) Best docked pose of compound 5u (green), represented as ball-and-stick model in the binding site of COX2, showing hydrogen-bond interaction (yellow dashed lines) with Ser530, Arg120, His90, Arg513, Phe518, Ser353, Gln192, and Ile517; (B) zooming in on the docked-pose sulfonamide structure of compound 5u (green), showing hydrogen-bond interaction; (C) best docked pose of compound 5s (turquoise), represented as ball-and-stick model in the binding site of COX2, showing hydrogen-bond interaction (yellow dashed lines) with Ser530, Arg120, His90, Arg513, Phe518, Ser353, and Gln192; (D) zooming in on the docked-pose sulfonamide structure of compound 5s (turquoise), showing hydrogen-bond interaction.

Figure 7

Superimposed and hydrophobic interactions of 5u. Notes: (A) Superimposed docked pose of celecoxib (pink), with compound 5u (green) represented as stick model at the binding site of COX2, showing alignment and orientation with interacting amino acid residues; (B) hydrophobic enclosures of compound 5u: hydrophobic atoms on the ligand are represented as green ball-and-stick model, and hydrophobic amino acids are displayed in gray CPK representation.

Figure 8

Receptor-surface model. Notes: Receptor-surface model of compound 5u (green stick) bound to COX2, showing hydrophobic pocket 1 (p-methylaniline), hydrophobic pocket 2 (benzyloxyphenyl), and selectivity pocket (SO₂NH₂).

Scheme 1

Protocol for the synthesis of pyrazole analogues (5a–5u). Abbreviations: DMF, dimethylformamide; EtOH, ethanol; GAA, glacial acetic acid.