Towards safer anti-inflammatory therapy: synthesis of new thymol–pyrazole hybrids as dual COX-2/5-LOX inhibitors

Figures & data

Figure 1. Structures of compounds having COX-2 or 5-LOX inhibitory activities.

Figure 2. Design of dual COX-2/5-LOX inhibitors.

Scheme 1. Synthesis of the target thymol–1,5-disubstitutedpyrazole hybrids.

Table 1. In vitro COX-1, COX-2, and 5-LOX enzyme inhibitory activities, ^aIC50 values, and ^bselectivity indices (SI) of the tested compounds.

Code	COX-1 µM IC50	COX-2 µM IC50	Selectivity index	5-LOX µM IC50
Celecoxib	14.7 ± 0.07	0.045 ± 0.007	327	–
Diclofenac sodium	3.9 ± 0.04	0.8 ± 0.009	4.87	–
Quercetin	–	–	–	3.34 ± 0.05
4a	10.3 ± 0.1	0.068 ± 0.01	151.5	3.05 ± 0.067
4b	11.81 ± 0.15	0.17 ± 0.01	69.5	1.84 ± 0.06
4c	10.22 ± 0.046	0.19 ± 0.007	53.8	2.91 ± 0.068
8a	11.21 ± 0.15	0.12 ± 0.007	93.4	1.96 ± 0.036
8b	13.61 ± 0.096	0.043 ± 0.001	316.5	1.58 ± 0.026
8c	12.87 ± 0.097	0.063 ± 0.001	204	1.91 ± 0.053
8d	10.19 ± 0.036	0.11 ± 0.007	92.6	3.11 ± 0.046
8e	11.24 ± 0.075	0.12 ± 0.007	93.6	2.56 ± 0.025
8f	11.18 ± 0.57	0.14 ± 0.01	79.8	2.91 ± 0.064
8g	12.1 ± 0.16	0.045 ± 0.01	268.8	1.60 ± 0.042
8h	12.61 ± 1.36	0.16 ± 0.01	78.8	1.97 ± 0.026
8i	12.58 ± 0.46	0.17 ± 0.012	74	2.78 ± 0.016

^aIC₅₀ is the concentration (µM) needed to cause 50% inhibition of COX-1 and COX-2 enzymatic activity. All values are expressed as a mean of three replicates with standard deviation less than 10% of the mean.

^bSelectivity Index (SI) = (COX-1 IC₅₀/COX-2 IC₅₀).

Figure 3. Comparison between different groups according to % inhibition of paw oedema volume.

Figure 4. Comparison between the different groups according to % relative potency (4h) celecoxib.

Figure 5. Comparison between the different groups according to %relative potency (4h) diclofenac.

Table 2. In vivo anti-inflammatory activities of selected compounds in formalin-induced rat paw oedema bioassay (acute inflammation model).

Code		Volume of oedema (ml)		Abs diff.	% Inhibition of oedema volume	% Relative potency (4h) celecoxib	% Relative potency (4h) diclofenac
		0	4h
Vehicle DMSO (control)	1	4.25	6.47	2.22
	2	4.20	6.37	2.17
	3	4.18	6.31	2.13
Mean ± SD		4.21 ± 0.04	6.38 ± 0.08	2.17 ± 0.05
Reference standard (celecoxib)	1	4.25	5.69	1.44	35.10
	2	4.34	5.67	1.33	38.70
	3	4.32	5.70	1.38	35.20
Mean ± SD		4.30 ± 0.05	5.69^a ± 0.02	1.38^a ± 0.06	36.33 ± 2.05
Reference standard (diclofenac)	1	4.20	5.37	1.17	47.30	134.60
	2	4.34	5.33	0.99	54.40	140.50
	3	4.25	5.19	0.94	55.90	158.70
Mean ± SD		4.26 ± 0.07	5.30^ab ± 0.09	1.03^ab ± 0.12	52.53^b ± 4.59	144.6 ± 12.56
4a	1	5.15	5.50	0.35	84.20	239.70	178.10
	2	5.10	5.65	0.55	74.70	192.90	137.30
	3	5.25	5.55	0.30	85.90	244.0	153.80
Mean ± SD		5.17^abc ± 0.08	5.57^ac ± 0.08	0.40^abc ± 0.13	81.60^bc ± 6.04	225.5^c ± 28.34	156.4 ± 20.52
4b	1	5.20	6.18	0.98	55.90	159.0	118.10
	2	5.15	6.17	1.02	53.0	136.90	97.50
	3	5.15	6.15	1.0	53.10	150.70	95.0
Mean ± SD		5.17^abc ± 0.03	6.17^abc ± 0.02	1.0^ab ± 0.02	54.0^b ± 1.65	148.9 ± 11.16	103.5 ± 12.68
4c	1	5.35	6.10	0.75	66.20	188.50	140.0
	2	5.30	6.20	0.90	58.50	151.20	107.60
	3	5.25	6.20	0.95	55.40	157.30	99.20
Mean ± SD		5.30^abc ± 0.05	6.17^abc ± 0.06	0.87^ab ± 0.10	60.03^b ± 5.56	165.7 ± 20.01	115.6 ± 21.54
8a	1	5.02	6.35	1.33	40.10	114.10	84.80
	2	4.96	6.35	1.39	35.90	92.90	66.10
	3	5.02	6.30	1.28	39.90	113.30	71.40
Mean ± SD		5.0^abc ± 0.03	6.33^bc ± 0.03	1.33^ac ± 0.06	38.63^c ± 2.37	106.8 ± 12.02	74.10 ± 9.64
8b	1	5.02	6.35	1.33	40.10	114.10	84.80
	2	4.96	6.40	1.44	33.60	86.90	61.90
	3	5.02	6.45	1.43	32.90	93.30	58.80
Mean ± SD		5.0^abc ± 0.03	6.40^bc ± 0.05	1.40^ac ± 0.06	35.53^c ± 3.97	98.10^c ± 14.22	68.50 ± 14.20
8c	1	5.02	5.90	0.88	60.40	171.80	127.60
	2	4.97	5.80	0.83	61.80	159.50	113.60
	3	5.01	5.80	0.79	62.90	178.70	112.60
Mean ± SD		5.0^abc ± 0.03	5.83^ac ± 0.06	0.83^ab ± 0.05	61.70^b ± 1.25	170.0 ± 9.73	117.9 ± 8.39
8d	1	5.02	5.95	0.93	58.10	165.40	122.90
	2	4.96	6.03	1.07	50.70	131.0	93.20
	3	5.02	6.02	1.0	53.10	150.70	95.0
Mean ± SD		5.0^abc ± 0.03	6.0^abc ± 0.04	1.0^ab ± 0.07	53.97^b ± 3.78	149.0 ± 17.26	103.7 ± 16.65
8e	1	5.35	6.25	0.90	59.50	169.20	125.70
	2	5.25	6.35	1.10	49.30	127.40	90.70
	3	5.40	6.40	1.0	53.10	150.70	95.0
Mean ± SD		5.33^abc ± 0.08	6.33^bc ± 0.08	1.0^ab ± 0.10	53.97^b ± 5.15	149.1 ± 20.95	103.8 ± 19.09
8f	1	5.15	6.25	1.10	50.50	143.60	106.70
	2	5.15	6.35	1.20	44.70	115.50	82.20
	3	5.20	6.40	1.20	43.70	124.0	78.20
Mean ± SD		5.17^abc ± 0.03	6.33^bc ± 0.08	1.17^a ± 0.06	46.30 ± 3.67	127.7 ± 14.41	89.03 ± 15.43
8g	1	5.10	6.15	1.05	52.70	150.0	111.40
	2	5.15	6.10	0.95	56.20	145.20	103.40
	3	5.25	6.25	1.0	53.10	150.70	95.0
Mean ± SD		5.17^abc ± 0.08	6.17^abc ± 0.08	1.0^ab ± 0.05	54.0^b ± 1.92	148.6 ± 2.99	103.3 ± 8.20
8h	1	5.02	6.05	1.03	53.60	152.60	113.30
	2	4.96	6.15	1.19	45.20	116.70	83.10
	3	5.02	6.0	0.98	54.0	153.30	96.60
Mean ± SD		5.0^abc ± 0.03	6.07^abc ± 0.08	1.07^ab ± 0.11	50.93^b ± 4.97	140.9 ± 20.93	97.67 ± 15.13
8i	1	5.30	6.02	0.72	67.60	192.30	142.90
	2	5.30	5.93	0.63	71.0	183.30	130.50
	3	5.40	5.95	0.55	74.20	210.70	132.80
Mean ± SD		5.33^abc ± 0.06	5.97^abc ± 0.05	0.63^abc ± 0.09	70.93^bc ± 3.30	195.4^c ± 13.97	135.4 ± 6.60

^aStatistically significant difference in comparison with control group.

^bStatistically significant difference in comparison with celecoxib reference group.

^cStatistically significant difference in comparison with diclofenac reference group.

Figure 6. Gross appearance of gastric mucosa.

Figure 7. Mode of binding (2D) of celecoxib (A), 4a (B), 8b (C), and 8g (D) inside the active site of COX-2.

Figure 8. Overlay of celecoxib of crystallised (yellow) and docked (cyan) with RMSD = 0.422.

Figure 9. Overlay of compounds 4a (green), 8b (yellow), 8g (pink), and celecoxib (cyan) inside the active site of COX-2.

Figure 10. Mode of binding (3D) of celecoxib (A), 4a (B), 8b (C), and 8g (D) into COX-2 active site.

Figure 11. Mode of binding (2D) of Arachidonic acid (A), 4a (B), 8b (C), and 8g (D) inside the active site of 5-LOX.

Figure 12. Overlay of compounds 4a (green), 8b (yellow), 8g (pink), and arachidonic acid (cyan) inside the active site of 5-LOX.

Figure 13. Mode of binding (3D) of arachidonic acid (A), 4a (B), 8b (C), and 8g (D) inside the active site of 5-LOX.

Table 3. Docking results of the active compounds in COX-2 active site.

Code	Score in Kcal/mol	Hydrogen bonds interaction amino acids	Hydrophobic interaction amino acids	Polar interaction amino acids
Celecoxib (Figures 7–10)	−9.32	Four bonds: two bonds between N of SO2NH2 and Ser339, Leu338; each O of SO2 with Arg499 and Ile503	Val335, Leu370, Phe367, Leu517, Leu345, Trp373, Val102, Met508, Ala502, Phe504, Val509	His75, Tyr341, Ser516, Tyr371, Gly340, Arg106, Gln178, Gly512, arene-H between phenyl of methylphenyl and Ala513
4a (Figures 7–9)	−7.79	One bond: between NH of CONH and *Leu338	Pro177, Val335, Ala513, Ala513, Phe504, Ala502, Pro500, Val509	Asp501, Thr79, His75, Arg499, Gln178, Gly340, Tyr341, arene-H between pyrazole and Ser339
8b (Figures 7–9)	−10.40	One bond: between NH of CONH and Leu338	Val335, Leu517, Trp373, Met508, Val509, Ala513, Phe367, Phe504, Ala502, Pro500, Leu345, Val102	Gly512, Asp501, Thr79, His80, Arg499, Tyr341, His75, Gln178, Gly340, Ser516, Arg106, Tyr371, arene-H between pyrazole and Ser339
8g (Figures 7–9)	−8.76	One bond: between NH of CONH and Leu338	Pro177, Val335, Leu517, Met508, Ala513, Phe504, Ala502, Pro500, Val509	Gly512, Asp501, Thr79, His337, Arg499, Tyr341, His75, Gln178, Gly340, Arg106, Ser339

*Amino acids interacted with the co-crystallised ligand (celecoxib) were marked in bold format.

Table 4. Docking results of the active compounds in 5-LOX active site.

Code	Score in Kcal/mol	Hydrogen bonds interaction amino acids	Hydrophobic interaction amino acids	Polar interaction amino acids
Co-crystallised ligand (arachidonic acid)	−9.42	No hydrogen bonds	Ile406, Phe177, Val175, Leu607.	Gln557, Gly174, Asn554, His367, Asp176, Gln363, Lys173, Ser171, Glu172
4a (Figures 10–12)	−7.19	One hydrogen bond between N1 of pyrazole and *Asp176	Ala410, Phe555, Ala672, Phe177, Phe610, Leu607, Ile406	Asn554, His372, Gly174, His367, Gln557, FE701, Glu614, Lys173, Gln413
8b (Figures 10–12)	−8.38	One hydrogen bond between N of sulphonamide and Ala672	Val604, Leu368, Phe177, Val671, Ala672, Phe610, Leu607, Phe555, Pro668.	Tyr558, Gln557, Ser608, His367, His550, Gln363, Glu614, Gly174, Asn554, Lys173, FE701, Thr364.
8g (Figures 10–12)	−7.78	Coordinate bond between carbonyl oxygen and FE701.	Leu607, Val604, Phe555, Phe610, Val671, Phe177, Ala672, Ala410, Leu368.	Glu614, Gly174, Gln363, Tyr558, His367, Gln557, Asn554, His550, His372.

*Amino acids interacted with the co-crystallised ligand (Arachidonic acid) were marked in bold format.