Synthesis, anti-inflammatory, analgesic, COX1/2-inhibitory activity, and molecular docking studies of hybrid pyrazole analogues

Figures & data

Figure 1 Chemical structure of some pyrazole-containing anti-inflammatory drugs.

Figure 2 Strategy for design of target compound with structural resemblance of reference ligand.

Abbreviation: IC₅₀, half-maximal inhibitory concentration.

Table 1 In vivo results of N-((3-(4-(benzyloxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methyl) aniline derivatives (5a–5u)

Compound	Ar	R	Anti-inflammatory activity (% inhibition ± SEM)		Analgesic activity (% inhibition ± SEM)	Ulcerogenic activity (severity index ± SEM)
			3 hours	4 hours
5a	Phenyl-	H	50.77±4.28c	47.91±4.39c	ND	ND
5b	4-Chlorophenyl-	H	73.42±2.1a	72.37±2.4a	71.89±2.33c	1.07±0.27b
5c	3-Chlorophenyl-	H	56.32±1.07c	38.31±4.11c	ND	ND
5d	2-Chlorophenyl-	H	71.45±2.04a	69.36±4.32a	53.45±2.26d	ND
5e	2,5-Dichlorophenyl-	H	59.77±1.31c	53.77±2.35c	ND	ND
5f	2-Fluorophenyl-	H	61.08±2.04c	54.72±2.28c	33.25±2.37c	ND
5g	4-Fluorophenyl-	H	71.08±2.04a	61.72±2.28c	49.21±1.87c	1.03±0.13b
5h	4-Bromophenyl-	H	70.91±2.5a	67.43±4.02a	29.56±1.85c	ND
5i	3-Chloro-4-fluorophenyl-	H	76.34±2.6a	75.55±2.3a	72.50±1.22	0.82±0.35b
5j	3-Methyl-4-bromophenyl-	H	41.25±1.32c	33.7±2.2c	ND	ND
5k	2,4-Nitrophenyl-	H	42.95±2.5c	20.43±1.93c	ND	ND
5l	3-Nitrophenyl-	H	57.27±2.82c	41.27±3.82c	ND	ND
5m	4-Hydroxyphenyl-	H	63.43±1.22c	58.71±2.54a	ND	ND
5n	3-Methoxyphenyl-	H	71.06±4.23a	59.23±5.21a	67.89±2.33c	0.76±0.36b
5o	3,4-Dimethoxyphenyl-	H	52.47±3.41c	30.91±5.16c	ND	ND
5p	4-Methylphenyl-	H	52.42±3.1c	43.06±3.78c	ND	ND
5q	Phenyl-	SO2NH2	56.72±3.24c	60.63±3.39a	57.33±1.87c	ND
5r	4-Chlorophenyl-	SO2NH2	79.63±0.49a	80.01± a 2.23	57.24±1.55c	0.93±0.12a
5s	2-Chlorophenyl-	SO2NH2	80.87±2.67a	76.56± a 2.34	73.56±1.25c	0.64±0.43b
5t	4-Fluorophenyl-	SO2NH2	77.85±2.23a	68.74± a 3.2	65.12±1.2c	0.85±0.36b
5u	4-Methylphenyl-	SO2NH2	80.63±0.53c	78.09± c 2.45	73.72±1.87c	0.81±0.3b
Ibuprofen	–	–	81.32±0.59a	79.23±0.4a	74.12±1.2c	1.16±0.4
Control	–	–	–	–	–	–

Notes:

a P<0.05;

b P<0.01;

c P<0.001;

d P<0.005.

Abbreviations: SEM, standard error of the mean; ND, not determined; ANOVA, analysis of variance.

Figure 3 Histopathological study of stomachs of rats treated with standard drug (ibuprofen) and active compounds in comparison to healthy controls.

Notes: Green arrow shows a normal mucosal cell. Blue arrow shows mucosal cell damage when treated with Ibuprofen. Black arrow represents the mucosal cell damage (ulcer) in high resolution (×40).

Table 2 Results of in vitro COX1/COX2 enzyme inhibition, NO-inhibition assay, and cytotoxicity study

Compound	IC50 (µM)a		SIb	NO (nitrite/mg, mmol/mg) 20 µM	Cytotoxicity (% cell viability 20 µM)
	COX1	COX2
5b	204.51	4.31	47.45	12.16±0.7	84±2.41
5d	98.62	5.03	19.6	11.93±0.57	71±2.35
5f	95.25	9.45	10.07	17.71±0.35	65±2.45
5g	139.23	4.12	33.79	10.06±0.48	73±1.18
5i	137.45	4.25	32.34	7.89±0.52	92±2.59
5n	103.52	4.71	21.97	11.43±0.26	70±2.37
5h	125.12	9.63	12.99	18.26±0.55	41±2.84
5q	45.23	3.04	14.87	8.47±0.21	59±2.84
5r	192.58	2.99	64.4	9.50±0.21	92.5±2.41
5s	183.11	2.51	72.95	7.08±0.51	94±2.12
5t	63.76	2.87	22.21	10.96±0.51	90±1.33
5u	134.07	1.79	74.92	7.97±0.79	91±1.96
Celecoxib	24.2	0.31	78.06	7.53±0.23	93±1.44
LPS	ND	ND	ND	20.46±1.03	ND
Control	ND	ND	ND	–	100

Notes:

a For means of three determinations where deviation from the mean is <10% of the mean value;

b COX1 IC₅₀/COX2 IC₅₀.

Abbreviations: IC₅₀, half-maximal inhibitory concentration; SI, selectivity index; ND, not determined; NO, nitric oxide; LPS, lipopolysaccharide.

Figure 4 In vitro TNFα assay of synthesized compound.

Notes: Data analyzed by one-way analysis of variance followed by Dunnett’s t-test and expressed as mean ± standard error of the mean from six observations.

Table 3 In silico docking score of N-((3-(4-(benzyloxy)phenyl)-1-phenyl-1H-pyrazol-4-yl)methyl) aniline derivatives (5a–5u)

Compound	Ar	R	Docking score
			COX1	COX2
5a	Phenyl-	H	−12.128	−11.412
5b	4-Chlorophenyl-	H	−12.777	−11.072
5c	3-Chlorophenyl-	H	−12.482	−11.345
5d	2-Chlorophenyl-	H	−12.307	−11.245
5e	2,5-Dichlorophenyl-	H	−12.404	−12.18
5f	2-Fluorophenyl-	H	−12.822	−11.311
5g	4-Fluorophenyl-	H	−11.88	−11.123
5h	4-Bromophenyl-	H	−12.728	−11.414
5i	3-Chloro-4-fluorophenyl-	H	−12.423	−11.701
5j	3-Methyl-4-bromophenyl-	H	−10.132	−10.625
5k	2,4-Nitrophenyl-	H	−10.059	−10.185
5l	3-Nitrophenyl-	H	−11.707	−11.481
5m	4-Hydroxyphenyl-	H	−11.314	−10.293
5n	3-Methoxyphenyl-	H	−11.576	−11.668
5o	3,4-Dimethoxyphenyl-	H	−11.344	−10.023
5p	4-Methylphenyl-	H	−12.774	−11.055
5q	Phenyl-	SO2NH2	#	−11.842
5r	4-Chlorophenyl-	SO2NH2	#	−11.978
5s	2-Chlorophenyl-	SO2NH2	#	−12.240
5t	4-Fluorophenyl-	SO2NH2	#	−12.123
5u	4-Methylphenyl-	SO2NH2	#	−12.907
Celecoxib	#	#	#	−9.924

Note: “#” Compounds do not give any docking score for COX1.

Figure 5 2-D LigPlot interaction diagrams.

Notes: (A) Compound 5u; (B) compound 5s.
Abbreviation: 2-D, two-dimensional.

Figure 6 Docked pose of 5s and 5u.

Notes: (A) Best docked pose of compound 5u (green), represented as ball-and-stick model in the binding site of COX2, showing hydrogen-bond interaction (yellow dashed lines) with Ser530, Arg120, His90, Arg513, Phe518, Ser353, Gln192, and Ile517; (B) zooming in on the docked-pose sulfonamide structure of compound 5u (green), showing hydrogen-bond interaction; (C) best docked pose of compound 5s (turquoise), represented as ball-and-stick model in the binding site of COX2, showing hydrogen-bond interaction (yellow dashed lines) with Ser530, Arg120, His90, Arg513, Phe518, Ser353, and Gln192; (D) zooming in on the docked-pose sulfonamide structure of compound 5s (turquoise), showing hydrogen-bond interaction.

Figure 7 Superimposed and hydrophobic interactions of 5u.

Notes: (A) Superimposed docked pose of celecoxib (pink), with compound 5u (green) represented as stick model at the binding site of COX2, showing alignment and orientation with interacting amino acid residues; (B) hydrophobic enclosures of compound 5u: hydrophobic atoms on the ligand are represented as green ball-and-stick model, and hydrophobic amino acids are displayed in gray CPK representation.

Figure 8 Receptor-surface model.

Notes: Receptor-surface model of compound 5u (green stick) bound to COX2, showing hydrophobic pocket 1 (p-methylaniline), hydrophobic pocket 2 (benzyloxyphenyl), and selectivity pocket (SO₂NH₂).

Scheme 1 Protocol for the synthesis of pyrazole analogues (5a–5u).

Abbreviations: DMF, dimethylformamide; EtOH, ethanol; GAA, glacial acetic acid.