Novel tetrazole and cyanamide derivatives as inhibitors of cyclooxygenase-2 enzyme: design, synthesis, anti-inflammatory evaluation, ulcerogenic liability and docking study

Figures & data

Figure 1. General structure of some known selective COX-2 inhibitors.

Figure 2. Design for the newly synthesised compounds 3a&b: 9a&b.

Table 1. In vitro COX-1 and COX-2 inhibition of tested compounds and reference drug, celecoxib.

	IC50^a (μM)				IC50^a (μM)
Compound no.	COX-1	COX-2	SI^b	Compound no.	COX-1	COX-2	SI^b
3a	12.54	0.57	22	6a	9.21	0.38	24.23
3b	6.74	0.24	28.08	6b	12.89	0.51	25.27
4a	6.23	0.43	14.48	7a	3.45	0.26	13.26
4b	6.11	0.27	22.62	7b	3.52	0.24	14.66
4c	15.97	0.38	42.02	8a	8.67	0.32	27.09
5a	7.09	0.18	39.38	8b	3.87	0.11	35.18
5b	12.41	1.14	10.88	9a	7.11	0.33	21.54
5c	11.23	0.71	15.81	9b	7.37	0.21	35.13
5d	11.31	0.45	25.13	Celecoxib	7.31	0.16	45.68
5e	7.14	0.19	37.57	Diclofenac	3.9	0.8	4.87
5f	6.45	0.14	46.07	Indomethacin	0.039	0.49	0.07

^a The in vitro test compound concentration required to produce 50% inhibition of COX-1 or COX-2. The result (IC₅₀, μM) is the mean of two determinations acquired using an ovine COX-1/COX-2 assay Kit (Catalog No. 560131, Cayman Chemicals Inc., Ann Arbor, MI) and the deviation from the mean is <10% of the mean value.

^b The in vitro COX-2 selectivity index (COX-1 IC₅₀/COX-2 IC₅₀).

Scheme 1. Reagent and conditions: (i) NaN₃, TEOF, gl. HAc, reflux, 12 h, (ii) ArCHO, KOH, abs. EtOH, r.t., 10–12 h, (iii) ArCHO, CN(CH₂)CN, NH₄OAc, abs. EtOH, (iv) ArCHO, CNCH₂COOEt, NH₄OAc, abs. EtOH, (v) CN(CH₂)CN, NH₄OAc, abs. EtOH, (vi) CNCH₂COOEt, NH₄OAc, abs. EtOH, (vii) p-substitutedphenylhydrazine hydrochloride, abs. EtOH, reflux, 6–8 h.

Scheme 2. Reagents and conditions: (i) thiourea or urea, KOH, abs. ethanol, reflux 10–12 h.

Scheme 3. Reagents and conditions: (i) p-methanesulphonylphenyl hydrazine hydrochloride, abs. ethanol; (ii) p-benzene sulphonamide hydrazine hydrochloride, abs. ethanol.

Table 2. Results of in vivo anti-inflammatory activities of tested compounds using carrageenan-induced rat paw oedema assay.

	Paw thickness changemean ± SEM (%Inhibition of paw oedema)
Compound	1h	3h	5h
Control	1.57 ± 0.09 (0%)	1.67 ± 0.12 (0%)	1.33 ± 0.10 (0%)
3a	1.18 ± 0.14 (25%)	1.53 ± 0.09 (9%)	1.18 ± 0.13 (2%)
3b	0.73 ± 0.08*** (54%)	1.45 ± 0.18 (13%)	1.33 ± 0.08 (0%)
4a	0.78 ± 0.08** (51%)	1.50 ± 0.19 (10%)	1.33 ± 0.05 (0%)
4b	1.28 ± 0.10 (19%)	1.53 ± 0.08 (9%)	1.28 ± 0.14 (4%)
4c	0.83 ± 0.15** (47%)	0.70 ± 0.11*** (58%)	1.23 ± 0.11 (8%)
5a	0.61 ± 0.08*** (61%)	0.63 ± 0.18*** (54%)	0.53 ± 0.09*** (61%)
5b	0.98 ± 0.18* (38%)	1.13 ± 0.15 (33%)	1.10 ± 0.15 (17%)
5c	0.68 ± 0.09*** (57%)	1.18 ± 0.11 (30%)	1.15 ± 0.05 (14%)
5d	0.45 ± 0.10*** (71%)	0.61 ± 0.12***(63%)	0.76 ± 0.15*(43%)
5e	0.45 ± 0.16*** (71%)	0.98 ± 0.03*(42%)	0.68 ± 0.08** (49%)
5f	0.33 ± 0.09*** (79%)	1.08 ± 0.10 (36%)	0.93 ± 0.15 (30%)
6a	1.10 ± 0.23 (30%)	0.98 ± 0.23*(42%)	1.28 ± 0.08 (4%)
6b	0.80 ± 0.26** (49%)	1.05 ± 0.09 (37%)	1.18 ± 0.13 (12%)
7a	1.20 ± 0.11 (24%)	1.30 ± 0.15 (22%)	1.33 ± 0.08 (0%)
7b	1.08 ± 0.11 (32%)	0.95 ± 0.17*(43%)	1.33 ± 0.08 (0%)
8a	1.40 ± 0.14*** (75%)	0.89 ± 0.19**(52%)	1.13 ± 0.11 (15%)
8b	0.45 ± 0.10*** (71%)	0.88 ± 0.09**(48%)	0.83 ± 0.17 (38%)
9a	0.40 ± 0.00*** (75%)	1.03 ± 0.09 (39%)	1.08 ± 0.20 (19%)
9b	0.40 ± 0.11*** (75%)	1.00 ± 0.16*(40%)	1.18 ± 0.20 (12%)
Celecoxib	0.38 ± 0.03*** (76%)	0.58 ± 0.11*** (66%)	1.08 ± 0.13 (19%)

Values represent mean ± SEM (n = 4). Significance levels *p > .05, **p < .01 and ***p > .001 as compared to the control group.

Table 3. Ulcerogenic liability for compounds 4c, 5a, 5d-f, 8a&b and 9a&b compared to reference drugs celecoxib and indomethacin.

Compound	Average number of ulcers	Ulcer index^a
Control	0.25	0.25
4c	1.75	1.75
5a	0.75	1.25
5d	1.25	1.50
5e	1.00	1.25
5f	1.25	1.25
8a	1.50	1.50
8b	0.75	0.75
9a	1.25	2.00
9b	0.25	0.50
Celecoxib	0.50	0.50
Indomethacin	14.25***	22.50***

^a The ulcer index is the sum of % incidence, average severity and average number of ulcers after oral administration of the tested compounds or the reference drug with dose equal 50 mg/kg.

Table 4. Molecular modelling data for best poses of the designed compounds 3a&b, 4a-c, 5a-f, 6a&b, 7a&b, 8a&b, 9a&b and SC-558 during docking in COX-2 (PDB: 1CX2) active site.

	COX-2					COX-2
Compound no.	Affinity Kcal/mol	Distance (in A°) from main residue		Functional group	Compound no.	Affinity Kcal/mol	Distance (in A°) from main residue	Functional group
SC-558	−10.0340	His90Tyr355	2.422.77	SO2NH2Pyrazole N-2	5e	−19.5404	Tyr355Ser530	2.962.96
3a	−14.0261	His90Tyr355	2.422.88	C = O3-OMe	5f	−22.4323	His90Tyr355Ser530Tyr385	2.532.992.853.32
3b	−16.2638	His90Tyr355	2.422.80	C = O3-OMe	6a	−10.6634	His90Tyr355	2.662.54
4a	−18.7919	His90	2.50	Tetrazole N-2	6b	−11.5834	His90Gly354	2.902.64
4b	−4.8888	His90	2.51	Tetrazole N-2	7a	−5.5894	His90Tyr355Ser530	2.882.473.02
4c	−18.5365	His90Tyr385Ser530	2.502.472.98	Tetrazole N-24-OMe4-OMe	7b	−5.5429	Ser530	2.73
5a	−18.3959	His90	2.51	Tetrazole N-2	8a	−12.8979	His90Tyr385	2.363.26
5b	−18.1932	His90	2.52	Tetrazole N-2	8b	−15.2648	His90	2.39
5c	−12.5497	His90Tyr355	2.543.01	Tetrazole N-2Pyridine N	9a	−15.1385	His90Tyr385Ser530	2.412.462.83
5d	−7.0670	Tyr355Tyr355	2.752.80	OHC≡N	9b	−16.9066	His90Ser353Ser530Ser530	2.382.582.862.92

Figure 3. Results of hot plate assay.

Figure 4. Results of acetic acid-induced writhing test.

Figure 5. Haematoxylin and eosin immunohistochemical staining of gastric ulcers after ulcer induction in rats for specimen intact Mucous membrane in control, indomethacin, celecoxib-treated rat and test compounds 4c, 5a, 5d and 5e.

Figure 6. Haematoxylin and eosin immunohistochemical staining of gastric ulcers after ulcer induction in rats for specimen intact mucous membrane in 5f, 8a, 8 b, 9a and 9 b-treated rats.

Figure 7. 2D (left image) and 3D (right image) interaction of ligand SC-558 in the active site of COX-2 receptor. It is possible to observe the binding using H-bond to His90 and Tyr355 amino acids.

Figure 8. 2D (left image) and 3D (right image) interaction of compound 5f in the active site of COX-2 receptor. It is possible to observe the binding using H-bond to His90, Tyr355, Tyr385 and Ser530 amino acids.