Design, synthesis, and biological investigation of oxadiazolyl, thiadiazolyl, and pyrimidinyl linked antipyrine derivatives as potential non-acidic anti-inflammatory agents

Figures & data

Figure 1. Chemical structures of certain reported antipyrine, oxadiazolyl, thiadiazolyl, pyrimidinyl analogues and our designed compounds (4,6,8).

Scheme 1. Synthetic pathway of 1,3,4-oxadiazolyl antipyrine derivatives (4a-d).

Scheme 2. Synthetic pathway of 1,3,4-thiadiazoles derivatives (6a-d).

Scheme 3. Synthetic pathway of pyrimidines derivatives 8a-d.

Table 1. IC₅₀ values of the tested compounds as COX inhibitors.

	Comp. no.	COX-2 (IC50 nM)^a ± SD	COX-1 (IC50 nM)^a ± SD	(SI)^b
1	4a	86.92 ± 2.19	178.5 ± 2.15	2.05
2	4b	53.76 ± 2.05	238.5 ± 2.08	4.44
3	4c	69.79 ± 2.14	129.5 ± 2.30	1.86
4	4d	65.54 ± 2.23	50.76 ± 2.18	0.77
5	6a	154.80 ± 2.07	131.60 ± 2.36	0.85
6	6b	103.70 ± 2.25	82.27 ± 2.28	0.79
7	6c	86.10 ± 2.26	282.10 ± 2.34	3.28
8	6d	169.00 ± 2.34	106.00 ± 2.16	0.63
9	8a	88.62 ± 2.19	138.80 ± 2.31	1.57
10	8b	119.80 ± 2.06	209.00 ± 2.21	1.74
11	8c	129.80 ± 2.10	163.60 ± 2.39	1.26
12	8d	68.74 ± 2.19	91.59 ± 2.15	1.33
Celecoxib		45.93 ± 2.19	309.60 ± 2.16	6.74

^aData are presented as IC₅₀ values of three different experiments using the human COXs (1 and 2) assay kit.

^bIn vitro COX-2 selectivity index = (COX-1 IC₅₀/COX-2 IC₅₀).

Figure 2. (A) Proposed binding interactions of compound 4b into COX-1 enzyme (PDB ID 6Y3C). (B) Proposed binding interactions 4b into COX-2 enzyme (PDB ID 5KIR). Important residues in the active site are indicated by lines, while the gray dotted lines are used for representing H-bonds. For the sake of clarity, nonpolar Hs were removed. In the ligand-interaction diagrams the magenta arrow represents the H-bond, the green line the π–π stacking, the red line the cation-π stacking.

Figure 3. (A) Proposed binding interactions 4c into COX-1 enzyme (PDB ID 6Y3C). (B) Proposed binding interactions 4c into COX-2 enzyme. In the ligand-interaction diagrams the magenta arrow represents the H-bond, the green line the π–π stacking, the red line the cation-π stacking.

Figure 4. (A) Proposed binding interactions 6c into COX-1 enzyme (PDB ID 6Y3C). (B) Proposed binding interactions 6c into COX-2 enzyme. In the ligand-interaction diagrams the magenta arrow represents the H-bond, the green line the π–π stacking, the red line the cation-π stacking.

Figure 5. (A) Proposed binding interactions 8d into COX-1 enzyme (PDB ID 6Y3C). (B) Proposed binding interactions 8d into COX-2 enzyme. In the ligand-interaction diagrams the magenta arrow represents the H-bond, the green line the π–π stacking, the red line the cation-π stacking.

Figure 6. Effects of celecoxib (CLX), 4 b, and 4c on carrageenan-induced paw edoema. *** p < 0.001 compared to the carrageenan group.

Figure 7. (A) Representative images of hematoxylin-eosin-stained paw sections showing areas of inflammation (arrows). (B) Semi-quantitative scoring of inflammation. *** p < 0.001 compared to the control group; # p < 0.05 compared to the carrageenan group.

Figure 8. (A) Representative images of immunostained paw sections showing NF-κB positive expression (arrows). (B) Semi-quantitative scoring of NF-κB positive expression. *** p < 0.001 in comparison to the control group; # p < 0.05 compared to the carrageenan group.

Figure 9. (A) Representative images of immunostained paw sections showing COX-2 positive expression (arrows). (B) Semi-quantitative scoring of COX-2 positive expression. *** p < 0.001 compared to the control group; # p < 0.05, ## P < 0.01 compared to the carrageenan group.

Figure 10. Effects of Celecoxib (CLX), 4 b, and 4c on carrageenan-induced elevation of (A) Prostaglandin E2 (PGE2) and (B) Tumour necrosis factor-alpha (TNF-α) paw tissue levels. *** p < 0.001 compared to the control group; # p < 0.05, ### P < 0.001 compared to the carrageenan group; $$p < 0.01, $$$P < 0.001 compared to the CLX group; + p < 0.05 compared to the 4 b group.

Figure 11. Effects of Celecoxib (CLX), 4 b, and 4c on carrageenan-induced elevation of (A) malondialdehyde (MDA) and (B) Nitric oxide (NO) paw tissue contents. ** p < 0.01, *** p < 0.001 compared to the control group; # p < 0.05, ### P < 0.001 compared to the carrageenan group; $$p < 0.01 compared to the CLX group; + p < 0.05 compared to the 4 b group.