Structure-guided approach on the role of substitution on amide-linked bipyrazoles and its effect on their anti-inflammatory activity

Figures & data

Figure 1. Logical flow and the rational of the study.

Figure 2. (A) The docking poses of 5a-e for chloro, bromo, nitro, fluoro and methyl substituents, as cyan, pale rose, orange and purple sticks, respectively, in the binding site of COX-2. (B) Focussed view at the substituents of 5a-e. (C), (D) and (E), Focussed view at substituents isopropyl, 5-membered dummy ring and 6-membered phenyl groups, respectively. Polar and non-polar regions of the binding site were presented by red and green coloured molecular surface, respectively. Dashed lines indicate favourable interactions, while red arrows represent steric clashes and unfavourable interactions. Non-polar hydrogen atoms were omitted for clarity.

Figure 3. Synthesis of the target compounds 5a-e. Reagents and reaction conditions: i: C₆H₅NH₂, EtOH, Reflux; ii: cyanoacetic acid, acetic anhydride, 50 °C; iii: 4-R-C₆H₄NCS, KOH, DMF, 0 °C; iv: 4-aminoantipyrine, oil bath at 170 °C.

Table 1. The docking score of the active compounds in the binding site of COX-2.

Compound	Docking score^a (AutoDock VinaXB)	Rescoring via (AutoDock Vina)
Celecoxib	−10.9	−10.9
Indomethacin	−7.8	−7.8
5a	−8.0	−8.0
5b	−8.0	−8.0
5c	−8.1	−8.1
5d	−7.8	−7.8
5e	−8.0	−8.0

^aThe docking scores of AutoDock Vina and VinaXB are expressed as binding affinity (kcal/mol).

Figure 4. (A) Overlay of the docking pose of 5a (cyan), 5b (pale rose), 5d (orange) and 5e (purple) in the binding site of COX-2 (PDB: 6BL4). Non-polar hydrogen atoms were omitted for clarity. (B) Interactions of 5a docking pose with COX-2 protein in a 2 D depiction.

Figure 5. (A) Root mean square deviation (RMSD) of the protein alpha carbon atoms during the simulation for the apo and complexed forms with 5d and 5e. (B) Radius of gyration (Rg) for the COX-2 protein in the apo and complexed forms with 5d and 5e during the simulation time. The 50000 ps simulation time (x- axis) implies 50 ns time. (C) Per residue root mean square fluctuation (RMSF) for the apo and complexed forms with 5d and 5e. (D) and (E) Hydrogen bond count during the MD simulation for both 5d and 5e in the binding site, respectively.

Table 2. COX-1 and COX-2 inhibition of the tested compounds.

	COX-2 IC50 (µM)	COX-1 IC50 (µM)	Selectivity ratio COX-1/COX-2
Indomethacin	0.079 ± 0.001*	0.099 ± 0.001*	1.253
Celecoxib	0.046 ± 0.002^×	14.50 ± 0.100^×	315.217
5a	0.135 ± 0.001*^×	7.50 ± 0.200*^×	55.555
5b	0.103 ± 0.006*^×	8.5 ± 0.100*^×	82.524
5c	0.089 ± 0.001*^×	11.5 ± 0.100*^×	129.213
5d	0.060 ± 0.001*^×	10.5 ± 0.200*^×	175.000
5e	0.069 ± 0.001*^×	12.5 ± 0.100*^×	181.159

Data was reported as mean ± standard deviation.

*p < 0.05 relative to celecoxib.

^×p < 0.05 relative to indomethacin.

Figure 6. Biological results for the formalin-induced paw edoema assay.

Table 3. Real-time PCR results.

	COX-1	COX-2	LOX
5a	4.04 ± 0.61^a,b	0.32 ± 0.04^a	1.88 ± 0.39
5b	1.29 ± 0.17	0.24 ± 0.04^a	0.74 ± 0.11
5c	3.36 ± 0.56^a,b	0.28 ± 0.02^a	1.83 ± 0.31
5d	0.79 ± 0.23	0.22 ± 0.06^a	1.37 ± 0.11
5e	1.99 ± 0.18	0.40 ± 0.04^a	1.05 ± 0.19
Indomethacin	0.82 ± 0.02	0.17 ± 0.01^a	1.32 ± 0.01
Vehicle	1.45 ± 0.01	1.22 ± 0.42	0.93 ± 0.02

^aThe mean difference is significant at the 0.05 level relative to the vehicle.

^bThe mean difference is significant at the 0.05 level relative to the Indomethacin.

Table 4. Acute ulcerogenic side effect of the tested compounds.

Tested compounds	Abou Zeit Har Score
5a	0.0 ± 0.00
5b	0.0 ± 0.00
5c	0.0 ± 0.00
5d	0.2 ± 0.45
5e	4.0 ± 1.41*
Celecoxib	0.6 ± 0.58
Indomethacin	11.2 ± 1.64*
Vehicle	0.0 ± 0.00

*p < 0.01 as compared with the vehicle group.