Design, Synthesis, and Biological Evaluation of Novel Phenoxy Acetic Acid Derivatives as Selective COX-2 Inhibitors Coupled with Comprehensive Bio-Pharmacological Inquiry, Histopathological Profiling, and Toxicological Scrutiny

Abstract

COX-2 plays a key role in converting arachidonic acid into prostaglandins. This makes it a significant target for treating inflammation. Selective COX-2 inhibitors have marked a new phase in inflammatory treatment, providing significant effectiveness while reducing negative side effects. Herein, we aimed at the design and synthesis of new anti-inflammatory agents 5a-f, 7a-b, 10a-f, and 13a-b with expected selective inhibition for COX-2. Compounds 5d-f, 7b, and 10c-f showed significant COX-2 inhibition with IC₅₀ in the range of 0.06-0.09 μM, indicating powerful pharmacological potential. In light of this, eight compounds were selected for further testing in vivo to assess their selectivity toward COX-1/COX-2 enzymes with the ability to reduce paw thickness. Compounds 5f and 7b showed significant anti-inflammatory effects without causing stomach ulcers, as they showed significant in vivo inhibition for paw thickness at 63.35% and 46.51%, as well as paw weight at 68.26% and 64.84%. Additionally, the tested compounds lowered TNF-α by 61.04% and 64.88%, as well as PGE-2 by 60.58% and 57.07%, respectively. Furthermore, these potent compounds were thoroughly analyzed for their pain-relieving effects, histological changes, and toxicological properties. Assessing renal and stomach function, as well as measuring liver enzymes AST and ALT, together with kidney indicators creatinine and urea, offered valuable information on their safety profiles. Molecular modeling studies explain the complex ways in which the strong interacts with the COX-2 enzyme. This comprehensive strategy emphasizes the therapeutic potential and safety profiling of these new analogues for managing inflammation.

Keywords: COX-1/COX-2; PGE-2; anti-inflammatory; paw edema model; ulcerogenic effects.

Figure 1

Schematic review of commercial COX-1 with marketed and reported selective COX-2 inhibitors, including the rationale for the design of new selective COX-2 inhibitors.

Scheme 1

(i) DMF/K₂CO₃, stirring 12 h; (ii) NaOH/MeOH, reflux 12 h; (iii) EtOH/AcOH, reflux 6 h.

Scheme 2

(i) Ar-NH₂/DMF/K₂CO₃, stirring 12 h; (ii) NHNH₂.H₂O/MeOH, reflux 4 h; (iii) PhCOCl/NaOH, stirring at 0 °C 2 h; (iv) MeOH/H₂SO₄, reflux 6 h; (v) NHNH₂/ MeOH, reflux 3 h; (vi) EtOH/AcOH, reflux 6 h.

Figure 2

SAR analysis of most designed compounds 5a–f, 7a, b, 10a–f, and 13a, b through in vitro study of COX-2 isozyme.

Figure 3

A heat map represents the paw thickness difference at hourly intervals. The darkest green color represents the lowest values, while the yellow color signifies the highest values. The intensity of colors highlights the efficacy of test compounds 5f and 7b (indicated with a downward arrow), with the deepest green indicating them as the most effective test compounds for inhibiting paw thickness in the carrageenan paw edema inflammation model. Con: control; Car: carrageenan; Cel: celecoxib; Mef: mefenamic acid. The data are presented in individual cells as mean values.

Figure 4

Impact of test compounds on the exudate content of TNF-α (A) PGE2 (B) in a carrageenan-induced paw edema inflammation model. The data are presented as mean ± SD and were analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons test; n = 6. ^a Significantly different from the control group at p < 0.05, ^b Significantly different from the carrageenan group at p < 0.05, ^c Significantly different from the celecoxib group at p < 0.05, ^d Significantly different from the mefenamic acid group at p < 0.05. TNF-α: tumor necrosis factor-alpha; PGE2: prostaglandin E2; Con: control; Car: carrageenan; Cel: celecoxib; Mef: mefenamic acid.

Figure 5

Histopathological photomicrographs illustrate the evident characteristics of an inflammatory model of paw edema across multiple experimental groups. (A) Normal control group demonstrates the normal histological structure of the epidermis and dermis; (B) carrageenan group demonstrates dermal edema (star) with infiltration by inflammatory cells mainly lymphocytes and eosinophils (arrow); (C) celecoxib group demonstrates dermal infiltration by high number of inflammatory cells mainly neutrophils and lymphocytes (arrows); (D) mefenamic acid group demonstrates infiltration of dermis by high number of inflammatory cells mainly lymphocytes and eosinophils (arrow); (E) 5f group demonstrates dermal edema (star) with infiltration by a few numbers of inflammatory cells mainly eosinophils and lymphocytes (arrow); (F) 7b group demonstrates dermal infiltration by a moderate number of mononuclear inflammatory cells (arrow) (Hematoxylin and Eosin staining).

Figure 6

Inflammatory cell infiltration score in a carrageenan-induced paw edema inflammation model. The data are presented as mean ± SD and were analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons test; n = 6. ^a Significantly different from the control group at p < 0.05, ^b Significantly different from the carrageenan group at p < 0.05, ^d Significantly different from the mefenamic acid group at p < 0.05. Con: control; Car: carrageenan; Cel: celecoxib; Mef: mefenamic acid.

Figure 7

Histopathological photomicrographs of the gastric mucosa of the following: (A) Control group demonstrates normal histological structure of gastric mucosa; (B) carrageenan group demonstrates small ulcer formation at superficial part of gastric mucosa (arrow); (C) celecoxib group demonstrates ulcer formation with eroded gastric mucosa (arrow); (D) mefenamic acid group demonstrates ulcer formation with desquamation of gastric mucosal epithelium (arrow); (E) 5f group demonstrates normal histological structure of gastric mucosa; (F) 7b group demonstrates infiltration of gastric mucosa by a few numbers of mononuclear inflammatory cells (arrow) (Hematoxylin and Eosin staining).

Figure 8

Two-dimensional schematic diagram of potent compounds 5f (left) and 7b (right) on the active site of COX-2 enzyme.