Synthesis, cyclooxygenase inhibition, anti-inflammatory evaluation and ulcerogenic liability of new 1,5-diarylpyrazole derivatives

Abstract

A new series of 1,5-diarylpyrazoles 10a–l was designed and synthesized for evaluation as COX inhibitors and as anti-inflammatory agents. All compounds were more selective for COX-2 isozyme and showed good in vivo anti-inflammatory activity. Compound 10e was the most COX-2 selective compound (S.I. = 10.67) and the most potent anti-inflammatory derivative (ED₅₀ = 46 μmol/kg) which is approximately 11-folds more potent than ibuprofen (ED₅₀ = 499 μmol/kg) and had 2/3 potency of celecoxib (ED₅₀ = 31 μmol/kg). All compounds were less ulcerogenic (ulcer indexes = 1.20–4.61) than ibuprofen (ulcer index = 20.25) and comparable to celecoxib (ulcer index = 2.90).

Keywords:

• Anti-inflammatory
• cyclooxygenase-2 inhibitors
• 1,5-diarylpyrazole

Introduction

Inflammation is a multi-staged process whose critical phase is thought to be driven by acutely released arachidonic acid and its metabolites including prostaglandins1. Two cyclooxygenase (COX) isozymes, COX-1 and COX-2 are known to catalyze the rate limiting step of prostaglandin synthesis2. Non-steroidal anti-inflammatory drugs (NSAIDs) have become the most widely used pharmaceuticals for treatment of inflammation and pain by blocking the action of both COX isoforms3. COX-2 is upregulated by inflammatory mediators and forms prostaglandins which intensify the inflammatory response while COX-1 is the house keeping isozyme making prostaglandins which are important for maintaining physiological functions in the body4. Traditional NSAIDs as aspirin, ibuprofen and indomethacin exert their anti-inflammatory (AI) effect through inhibition of both COX-1 and COX-2 and in turn their use was accompanied with adverse effects including gastric bleeding and ulceration5. Thus, it was thought that selective COX-2 inhibitors would have reduced side effects with improved gastric safety profile. Most of the selective COX-2 inhibitors (Figure 1) (coxibs) are diarylheterocycles in which, two vicinal (adjacent) aryl moieties are attached to a five-membered ring as pyrazole in celecoxib (1)6, furanone in rofecoxib (2)7 or isoxazole in valdecoxib (3)8. Also, one of the two aryl rings is substituted at para position with one COX-2 pharmacophoric moiety either sulfamoyl (SO₂NH₂) or methanesulfonyl (SO₂CH₃) moiety9,10. Realization about the importance of COX-2 selective inhibitors for decreasing side effects associated with nonselective COX inhibitors has stimulated our group for designing and synthesis of new series of diarylpyrazolines 4, 5 and diarylpyrazoles 6, 7 of moderate COX-2 selectivity and good safety profile11–13.

Figure 1. Chemical structures of the selective cyclooxygenase-2 inhibitors celecoxib (1), rofecoxib (2), valdecoxib (3), diarylpyrazoline derivatives (4, 5) and the diarylpyrazoles (6, 7).

Based on the aforementioned information, we now describe the synthesis, in vitro evaluation as COX-1/COX-2 inhibitors, in vivo AI activity and ulcerogenic liability for a new series of diarylpyrazoles 10a–l which maintains the vicinal diarylheterocycle scaffold and is closely related to the structure of celecoxib (1) but with three modifications: (i) the tolyl moiety of celecoxib was replaced with electron withdrawing moieties as (4-bromophenyl in 10a–c, 4-nitrophenyl in 10d–f or 4-chlorophenyl in 10g–i) or replaced with another electron donating moiety (2-thienyl in 10j–l) to check the effect of these substituents on the biological activity, (ii) trifluoromethyl moiety at C-3 of the central five-membered pyrazole ring was removed since it was reported that the substituent at C-3 of the central ring has very few steric restrictions with respect to COX-2 binding14 and (iii) The COX-2 pharmacophore SO₂NH₂ was maintained (10a, 10d, 10g and 10j), replaced with another COX-2 pharmacophore SO₂CH₃ (10b, 10e, 10h and 10k) or replaced with COOH (10c, 10f, 10i and 10l) (Figure 2).

Figure 2. Chemical structures of the selective cyclooxygenase-2 inhibitor celecoxib (1) and the designed dihydropyrazoles 10a–l.

Results and discussion

Chemistry

The enaminone derivatives (8a–d) were prepared via condensation of the appropriate aryl or heteroaryl methyl ketone with dimethylformamide dimethylacetal (DMFDMA) in xylene following procedures applied in literatures15–21. Cyclocondensation of the appropriate enaminone (8a–d) with 4-hydrazinylbenzenesulfonamide hydrochloride (9a)22, methanesulfonylphenylhydrazine hydrochloride (9b)23,24 or 4-hydrazinobenzoic acid (9c)25 in aqueous ethanol afforded the respective 1,5-diarylpyrazoles (10a–l) in good yields (56–88%) (Scheme 1).

Scheme 1. Reagents and conditions: (a) EtOH (95%), reflux, 36 h.

Biological evaluation

In vitro cyclooxygenase inhibition assay

The in vitro COX-1/COX-2 isozyme inhibition studies measure the ability of tested compounds to inhibit ovine COX-1 and human recombinant COX-2 using an enzyme immunoassay (EIA)26. The results (Table 1) showed that all the diarylpyrazoles (10a–l) are week inhibitors for COX-1 isozyme (IC₅₀ = 2.91–10.21 μM range) and exhibited moderate COX-2 isozyme inhibitory activities (IC₅₀ = 0.33–3.41 μM range) with COX-2 selectivity indexes in the range of 2.79–10.67. While the 4-nitrophenyl analogs (10d–f) had the highest COX-2 potency (IC₅₀ = 0.33–3.41 μM range) and the highest COX-2 selectivity indexes (S.I. = 8.56–10.67), the other analogs (4-bromophenyl analogs 10a–c, 4-chlorophenyl analogs 10g–i and 2-thienyl analogs 10j–l) had lower COX-2 potency (IC₅₀ = 1.43–2.89, 2.24–2.98 and 1.52–3.41 μM ranges respectively) and lower COX-2 selectivity indexes (S.I. = 2.91–4.80, 2.79–3.42 and 2.84–3.93 ranges, respectively). Within the all derivatives (10a–l), the 4-nitrophenylmethanesulphonylphenyl derivative (10e) had the highest COX-2 selectivity index (S.I. = 10.67) which is also higher than that of the COX-2 selective reference drug celecoxib (S.I. = 9.29). Also, for each group of compounds; while the COOH derivative (10c) was the most COX-2 selective (S.I. = 4.80) for the 4-bromophenyl analogs (10a–c) and the SO₂CH₃ derivative (10e) was the most COX-2 selective (S.I. = 10.67) for the 4-nitrophenyl analogs (10d–f), the SO₂NH₂ derivatives (10g, S.I. = 3.42, 10j, S.I. = 3.93) were the most COX-2 selective derivatives for 4-chlorophenyl analogs 10g–i and 2-thienyl analogs 10j–l, respectively.

Table 1. In vitro COX-1, COX-2 inhibition, anti-inflammatory activity of diarylpyrazoles (10a–l), and reference drugs celecoxib and ibuprofen.

Compound	R	R^1	Ar	COX-1 IC50 (μM)^a	COX-2 IC50 (μM)^a	COX-2 S.I.^b	ED50 (μmol/kg)^c
10a	SO2NH2	H	4-BrC6H4	7.52	2.33	3.22	465
10b	SO2CH3	H	4-BrC6H4	8.42	2.89	2.91	604
10c	COOH	H	4-BrC6H4	6.87	1.43	4.80	758
10d	SO2NH2	H	4-NO2C6H4	3.51	0.41	8.56	145
10e	SO2CH3	H	4-NO2C6H4	5.87	0.55	10.67	46
10f	COOH	H	4-NO2C6H4	2.91	0.33	8.81	148
10g	SO2NH2	H	4-ClC6H4	7.67	2.24	3.42	525
10h	SO2CH3	H	4-ClC6H4	8.11	2.63	3.08	737
10i	COOH	H	4-ClC6H4	8.34	2.98	2.79	503
10j	SO2NH2	H	2-thienyl	5.98	1.52	3.93	878
10k	SO2CH3	H	2-thienyl	9.23	3.25	2.84	305
10l	COOH	H	2-thienyl	10.21	3.41	2.99	537
Celecoxib	SO2NH2	CF3	4-CH3C6H4	7.34	0.79	9.29	31
Ibuprofen^d	–	–	–	2.9	1.1	2.64	499

^aThe concentration of test compound produce 50% inhibition of COX-1, COX-2 enzyme, the result is the mean of two value obtained by assay of enzyme kits obtained from (Cayman Chemicals Inc., Ann Arbor, MI).

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

^c Inhibitory activity in a carrageenan induced rat paw edema assay. The results are expressed as the ED₅₀ value (μmol/kg) at 3 h after oral administration of the test compound.

^d Data quoted from literature28.

In vivo anti-inflammatory activity

The AI activities exhibited by the synthesized compounds were determined using a carrageenan-induced rat paw edema model and the dose causing 50% edema inhibition (ED₅₀) was determined in comparison to the reference drugs celecoxib and ibuprofen28. The tested diarylpyrazoles (10a–l) exhibited a broad AI activity range (ED₅₀ = 46–878 μmol/kg) in comparison with reference drugs celecoxib (ED₅₀ = 31 μmol/kg) and ibuprofen (ED₅₀ = 499 μmol/kg). Similar to the in vitro results, while the 4-nitrophenyl analogs (10d–f) had the highest AI activities (ED₅₀ = 46–148 μmol/kg range), the other analogs (4-bromophenyl analogs 10a–c, 4-chlorophenyl analogs 10g–i and 2-thienyl analogs 10j–l) had lower AI activities (ED₅₀ = 465–758, 503–737 and 305–878 μmol/kg ranges respectively). Also, within the all derivatives (10a–l), the 4-nitrophenyl-methanesulphonylphenyl derivative (10e), the most COX-2 selective derivative, was the most potent derivative (ED₅₀ = 46 μmol/kg) which is approximately 11-folds more potent than ibuprofen (ED₅₀ = 499 μmol/kg) and 2/3 potency of celecoxib (ED₅₀ = 31 μmol/kg).

Ulcerogenic liability

The target compounds 10a–l were subjected to further study to determine their ulcerogenic effect (ulcer index) using ED₅₀ dose in comparison with celecoxib (ED₅₀ dose) and small dose of Ibuprofen (120 μmol/kg)29. The results revealed that all compounds 10a–l were less ulcerogenic (ulcer indexes = 1.20–4.61) than ibuprofen (ulcer index = 20.25) and comparable to celecoxib (ulcer index = 2.90) (Table 2). Also, it was clear that, within each group of analogs, the carboxylic derivative was more ulcerogenic than the other derivatives which could be correlated to local acidity effect of their carboxylic group. For 4-bromophenyl analogs 10a–c, the carboxylic derivative 10c (ulcer index = 4.61) while the sulphamoyl derivative 10a and the methanesulphonyl derivative 10b had ulcer indexes = 2.92 and 2.68, respectively. Similarly, within 4-nitrophenyl analogs 10d–f, 4-chlorophenyl analogs 10g–i and 2-thienyl analogs 10j–l the carboxylic derivatives 10f, 10i and 10l (ulcer indexes = 4.32, 4.36 and 3.89 respectively) while the sulphamoyl derivatives 10d, 10g and 10j (ulcer indexes = 1.20, 3.03 and 2.63, respectively) and the methanesulphonyl derivatives 10e, 10h and 10k (ulcer indexes = 2.47, 3.02 and 2.66 respectively). The sulphamoyl derivative 10d was the most safe derivative (ulcer index = 1.20) with relative ulcerogenicities to the reference drugs celecoxib and ibuprofen 0.41 and 0.06, respectively.

Table 2. Ulcerogenic liability for diarylpyrazoles (10a–l) and reference drugs celecoxib and ibuprofen.

Compound	Average severity	Average no. of ulcer^a	% incidence/10	Ulcer index
10a	0.73 ± 0.017***^,^b	0.2 ± 0.004***^,^b	2	2.92
10b	0.38 ± 0.016***^,^b	0.3 ± 0.001***^,^b	2	2.68
10c	1.2 ± 0.037***^,^b	0.4 ± 0.016***^,^b	3	4.61
10d	0.1 ± 0.002***^,^b	0.1 ± 0.003***^,^b	1	1.2
10e	0.28 ± 0.015***^,^b	0.2 ± 0.007^b	2	2.47
10f	1.00 ± 0.040^b	0.3 ± 0.008***^,^b	3	4.32
10g	0.83 ± 0.030***	0.2 ± 0.007***^,^b	2	3.03
10h	0.62 ± 0.026***^,^b	0.4 ± 0.015***^,^b	2	3.02
10i	0.85 ± 0.035***^,^b	0.5 ± 0.012,b	3	4.36
10j	0.25 ± 0.009***^,^b	0.4 ± 0.006,b	2	2.63
10k	0.17 ± 0.004***^,^b	0.5 ± 0.012***^,^b	2	2.66
10l	0.57 ± 0.013***^,^b	0.3 ± 0.004***^,^b	3	3.89
Celecoxib	0.5 ± 0.013^b	0.4 ± 0.006^b	2	2.9
Ibuprofen	2.25 ± 0.13	8	10	20.25

***Significant difference with celecoxib at p < 0.001.

^a Values represent means ± SEM of 10 animals for each group.

^b Significant difference with ibuprofen at p < 0.001.

Conclusion

A new series of 1,5-diarylpyrazoles 10a–l was synthesized for evaluation as selective COX-2 inhibitors, AI agents and ulcerogenic liability. Structure-activity data acquired and biological studies showed that (i) all compounds were more COX-2 inhibitors than COX-1, (ii) the 4-nitrophenyl analogs (10d–f) had higher AI activities than the other analogs (4-bromophenyl analogs 10a–c, 4-chlorophenyl analogs 10g–i and 2-thienyl analogs 10j–l), (iii) the 4-nitrophenyl-methanesulphonylphenyl derivative (10e), the most COX-2 selective derivative, was the most potent derivative which is approximately 11-folds more potent than ibuprofen (ED₅₀ = 499 μmol/kg) and 2/3 potency of celecoxib (ED₅₀ = 31 μmol/kg), and (iv) all compounds were less ulcerogenic than ibuprofen and showed ulceration effect comparable to that of celecoxib.

Experimental

Chemistry

Melting points were determined on a Thomas-Hoover capillary apparatus and are uncorrected. Infrared (IR) spectra were recorded as films on KBr plates using a Nicolet 550 Series II Magna FT-IR spectrometer. ¹H NMR and ¹³C NMR spectra were measured on a Bruker 400 MHz NMR Spectrophotometer, Faculty of Pharmacy, Beni-Suef University, Egypt in CDCl₃ or DMSO-d₆ with TMS as the internal standard, where J (coupling constant) values are estimated in Hertz (Hz). Mass spectra (MS) were recorded on a Water’s Micromass ZQ 4000 mass spectrometer using the electro-spray (ES) ionization mode. Microanalyses were performed for C, H and N were carried out on Perkin-Elmer 2400 analyzer (Perkin-Elmer, Norwalk, CT) at the micro analytical unit of Cairo University, Egypt. All compounds were within ±0.4% of the theoretical values. Silica gel column chromatography was performed using Merck silica gel 60 ASTM (70–230 mesh). Compounds 8a–d15–21, 9a–c22–25 were prepared according to the reported procedures.

General method for preparation of diarylpyrazoles (10a–l)

A solution of the appropriate enaminone (8a–d, 0.1 mol) in ethanol (50 mL) was heated under reflux with a mixture of p-substituted-phenylhydrazine hydrochloride (9a–c, 0.1 mol) for 36 h, cooled and diluted with cold water. The precipitated crude product was filtered and recrystallized from ethanol to give the respective pyrazoles (10a–l). Physical and spectral data are listed below.

4-[5-(4-Bromophenyl)-pyrazol-1-yl]benzenesulfonamide (10a)

68% yield; white solid; m.p. 269–271 °C; IR (KBr disk) 3375, 3202 (NH₂), 1336, 1162 (SO₂); ¹H NMR (DMSO-d₆) δ 6.55 (d, J = 1.6 Hz, 1H, pyrazole H-3), 7.12 (d, J = 8.0 Hz, 2H, bromophenyl H-3, H-5), 7.28 (s, 2H, NH₂, D₂O exchangeable) 7.45 (d, J = 8.2 Hz, 2H, aminosulfonylphenyl H-3, H-5), 7.51 (d, J = 8.0 Hz, 2H, bromophenyl H-2, H-6), 7.78 (d, J = 1.6 Hz, 1H, pyrazole H-4), 7.91 (d, J = 8.2 Hz, 2H, aminosulfonylphenyl H-2, H-6); ¹³C NMR (DMSO-d₆) δ 109.33, 123.24, 124.57, 124.99, 127.54, 127.62, 128.96, 130.30, 131.25, 132.14, 143.17; MS (m/z, relative abundance %): 378.24 (M^+., 1.17); Anal. Calcd for C₁₅H₁₂BrN₃O₂S: C, 47.63; H, 3.20; N, 11.11; Found: C, 47.35; H, 3.35; N, 11.35.

5-(4-Bromophenyl)-1-(4-methanesulfonyl-phenyl)-1H-pyrazole (10b)

72% yield; white solid; m.p. 241–243 °C; IR (KBr disk) 1308, 1151 (SO₂); ¹H NMR (DMSO-d₆) δ 3.05 (s, 1H, SO₂CH₃), 6.56 (d, J = 1.6 Hz, 1H, pyrazole H-3), 7.12 (d, J = 8.0 Hz, 2H, bromophenyl H-3, H-5), 7.51 (m, 4H, methanesulfonylphenyl H-3, H-5 and bromophenyl H-3, H-5), 7.79 (d, J = 1.6 Hz, 1H, pyrazole H-4), 7.93 (d, J = 8.2 Hz, 2H, methanesulfonylphenyl H-2, H-6); ¹³C NMR (DMSO-d₆) δ 44.49, 109.53, 124.68, 125.11, 128.50, 128.58, 129.43, 130.05, 131.02, 135.16, 140.36, 141.60; MS (m/z, relative abundance %): 377.26 (M^+., 0.92); Anal. Calcd for C₁₆H₁₃BrN₂O₂S: C, 50.94; H, 3.47; N, 7.43; Found: C, 50.59; H, 3.35; N, 7.65.

4-[5-(4-Bromophenyl)pyrazol-1-yl]benzoic acid (10c)

56% yield; white solid; m.p. 205–207 °C; IR (KBr disk) 3430 (OH), 1683 (C=O) 1381, 1172 (SO₂); ¹H NMR (DMSO-d₆) δ 6.55 (d, J = 1.6 Hz, 1H, pyrazole H-3), 7.19 (d, J = 7.8 Hz, 2H, bromophenyl H-3, H-5), 7.35 (d, J = 7.8 Hz, 2H, benzoic H-3, H-5), 7.42 (d, J = 8.0 Hz, 2H, bromophenyl H-2, H-6), 7.80 (d, J = 1.6 Hz, 1H, pyrazole H-4), 8.10 (d, J = 7.8 Hz, 2H, benzoic H-2, H-6), 13.11 (s, 1H, COOH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ 109.07, 124.57, 128.05, 128.72, 128.98, 129.04, 129.80, 130.05, 130.47, 134.86, 141.07, 170.22; MS (m/z, relative abundance %): 343.17 (M^+., 3.12); Anal. Calcd for C₁₆H₁₁BrN₂O₂: C, 56.00; H, 3.23; N, 8.16; Found: C, 56.35; H, 3.35; N, 7.95.

4-[5-(4-Nitrophenyl)pyrazol-1-yl]benzenesulfonamide (10d)

88% yield; yellow solid; m.p. 255–257 °C; IR (KBr disk) 3423, 3338 (NH₂), 1339, 1165 (SO₂); ¹H NMR (DMSO-d₆) δ 6.87 (d, J = 1.6 Hz, 1H, pyrazole H-3), 7.50 (m, 2H, NH₂, D₂O exchangeable and 2H, nitrophenyl H-3, H-5), 7.54 (d, J = 8.0 Hz, 2H, aminosulfonylphenyl H-3, H-5), 7.79 (d, J = 8.2 Hz, 2H, nitrophenyl H-2, H-6), 7.92 (d, J = 1.6 Hz, 1H, pyrazole H-4), 8.24 (d, J = 8.0 Hz, 2H, aminosulfonylphenyl H-2, H-6); ¹³C NMR (DMSO-d₆) δ 110.78, 124.40, 125.77, 127.37, 130.24, 136.53, 141.27, 141.85, 142.06, 143.55, 147.56; MS (m/z, relative abundance %): 344.35 (M^+., 9.34); Anal. Calcd for C₁₅H₁₂N₄O₄S: C, 52.32; H, 3.51; N, 16.27; Found: C, 52.44; H, 3.35; N, 15.95.

1-(4-Methanesulfonylphenyl)-5-(4-nitrophenyl)-1H-pyrazole (10e)

74% yield; yellow solid; m.p. 222–224 °C; IR (KBr disk) 1301, 1149 (SO₂); ¹H NMR (DMSO-d₆) δ 3.10 (s, 1H, SO₂CH₃), 6.59 (d, J = 1.6 Hz, 1H, pyrazole H-3), 7.44 (d, J = 8.2 Hz, 2H, nitrophenyl H-3, H-5), 7.50 (d, J = 8.2 Hz, 2H, methanesulfonylphenyl H-3, H-5), 7.84 (d, J = 1.6 Hz,1H, pyrazole H-4), 7.96 (d, J = 8.2 Hz, 2H, nitrophenyl H-3, H-5), 8.24 (d, J = 8.2 Hz, 2H, methanesulfonylphenyl H-2, H-6); ¹³C NMR (DMSO-d₆) δ 44.50, 110.57, 124.19, 125.31, 128.74, 129.48, 136.19, 139.54, 141.08, 141.87, 143.68, 144.14; MS (m/z, relative abundance %): 343.36 (M^+., 35.57); Anal. Calcd for C₁₆H₁₃N₃O₄S: C, 55.97; H, 3.82; N, 12.24; Found: C, 55.61; H, 3.45; N, 11.95.

4-[5-(4-Nitrophenyl)pyrazol-1-yl]benzoic acid (10f)

69% yield; yellowish white solid; m.p. 256–258 °C; IR (KBr disk) 3426 (OH), 1685 (C=O) 1413, 1152 (SO₂); ¹H NMR (DMSO-d₆) δ 6.92 (d, J = 1.6 Hz, 1H pyrazole H-3), 7.41 (d, J = 7.8 Hz, 2H, nitrophenyl H-3, H-5), 7.52 (d, J = 7.8 Hz, 2H, benzoic H-3, H-5), 7.91 (d, J = 1.6 Hz, 1H, pyrazole H-4), 7.98 (d, J = 7.8 Hz, 2H, bromophenyl H-2, H-6), 8.12 (d, J = 7.8 Hz, 2H, benzoic H-2, H-6), 13.13 (s, 1H, COOH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ 110.66, 124.36, 125.44, 125.52, 129.42, 130.14, 130.39, 130.77, 130.94, 136.54, 141.22, 167.00; MS (m/z, relative abundance %): 309.01 (M^+., 6.22); Anal. Calcd for C₁₆H₁₁N₃O₄: C, 62.14; H, 3.58; N, 13.59; Found: C, 62.35; H, 3.35; N, 13.95.

4-[5-(4-Chlorophenyl)pyrazol-1-yl]benzenesulfonamide (10g)

62% yield; white solid; m.p. 229–231 °C; IR (KBr disk) 3346, 3194 (NH₂), 1338, 1162 (SO₂); ¹H NMR (DMSO-d₆) δ 6.55 (d, J = 1.6 Hz, 1H, pyrazole H-3), 7.19 (d, J = 8.2 Hz, 2H, chlorophenyl H-3, H-5), 7.28 (s, 2H, NH₂, D₂O exchangeable), 7.32 (d, J = 8.0 Hz, 2H, aminosulfonylphenyl H-3, H-5), 7.41 (d, J = 8.2 Hz, 2H, chlorophenyl H-2, H-6), 7.79 (d, J = 1.6 Hz, 1H, pyrazole H-4), 8.09 (d, J = 8.0 Hz, 2H, aminosulfonylphenyl H-2, H-6); ¹³C NMR (DMSO-d₆) δ 109.34, 124.56, 125.01, 127.55, 127.62, 129.20, 129.37, 130.06, 131.04, 140.18, 141.39; MS (m/z, relative abundance %): 333.03 (M^+., 70.42); Anal. Calcd for C₁₅H₁₂ClN₃O₂S: C, 53.97; H, 3.62; N, 12.59; Found: C, 54.35; H, 3.35; N, 12.75.

5-(4-Chlorophenyl)-1-(4-methanesulfonylphenyl)-1H-pyrazole (10h)

79% yield; yellow solid; m.p. 236–238 °C; IR (KBr disk) 1305, 1150 (SO₂); ¹H NMR (DMSO-d₆) δ 3.09 (s, 1H, SO₂CH₃), 6.56 (d, J = 1.6 Hz, 1H, pyrazole H-3), 7.19 (d, J = 7.8 Hz, 2H, chlorophenyl H-3, H-5), 7.36 (d, J = 7.8 Hz, 2H, methanesulfonylphenyl H-3, H-5), 7.51 (d, J = 7.8 Hz, 2H, chlorophenyl H-3, H-5), 7.79 (d, J = 1.6 Hz, 1H, pyrazole H-4), 7.93 (d, J = 7.8 Hz, 2H, methanesulfonylphenyl H-2, H-6); ¹³C NMR (DMSO-d₆) δ 44.53, 109.52, 123.32, 125.11, 128.50, 128.91, 130.29, 132.19, 138.94, 141.66, 142.32, 144.02; MS (m/z, relative abundance %): 332.04 (M^+., 71.47); Anal. Calcd for C₁₆H₁₃ClN₂O₂S: C, 57.74; H, 3.94; N, 8.42; Found: C, 57.39; H, 3.75; N, 8.15.

4-[5-(4-Chlorophenyl)pyrazol-1-yl]benzoic acid (10i)

77% yield; yellowish white solid; m.p. 244–246 °C; IR (KBr disk) 3431 (OH), 1684 (C=O) 1381, 1174 (SO₂); ¹H NMR (DMSO-d₆) δ 6.55 (d, J = 1.6 Hz, 1H pyrazole H-3), 7.18 (d, J = 8.0 Hz, 2H, chlorophenyl H-3, H-5), 7.35 (d, J = 7.8 Hz, 2H, benzoic H-3, H-5), 7.45 (d, J = 8.0 Hz, 2H, chlorophenyl H-2, H-6), 7.78 (d, J = 1.6 Hz, 1H, pyrazole H-4), 7.91 (d, J = 8.0 Hz, 2H, benzoic H-2, H-6), 13.11 (s, 1H, COOH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ 109.08, 124.53, 124.57, 127.90, 128.71, 128.98, 129.04, 130.05, 134.87, 141.04, 143.94, 169.38; MS (m/z, relative abundance %): 298.06 (M^+., 100.00); Anal. Calcd for C₁₆H₁₁ClN₂O₂: C, 64.33; H, 3.71; N, 9.38; Found: C, 64.35; H, 3.35; N, 8.95.

4-(5-Thiophen-2-yl-pyrazol-1-yl)benzenesulfonamide (10j)

77% yield; brown solid; m.p. 258–260 °C; IR (KBr disk) 3427, 3302 (NH₂), 1341, 1160 (SO₂); ¹H NMR (DMSO-d₆) δ 6.61 (d, J = 1.6 Hz, 1H, pyrazole H-3), 6.90 (s, 1H, thienyl H-4), 7.03 (s, 1H, thienyl H-5), 7.28 (s, 2H, NH₂, D₂O exchangeable), 7.38 (d, J = 4.4 Hz, 1H, thienyl H-3), 7.56 (d, J = 8.0 Hz, 2H, aminosulfonylphenyl H-3, H-5), 7.76 (d, J = 1.6 Hz, 1H, pyrazole H-4), 7.95 (d, J = 8.0 Hz, 2H, aminosulfonylphenyl H-2, H-6); ¹³C NMR (DMSO-d₆) δ 109.71, 125.49, 127.37, 127.42, 127.71, 128.11, 141.28, 141.63, 145.00, 145.22, 145.41; MS (m/z, relative abundance %): 305.07 (M^+., 6.32); Anal. Calcd for C₁₃H₁₁N₃O₂S₂: C, 51.13; H, 3.63; N, 13.76; Found: C, 51.35; H, 3.35; N, 13.95.

1-(4-Methanesulfonylphenyl)-5-thiophen-2-yl-1H-pyrazole (10k)

58% yield; orange solid; m.p. 233–235 °C; IR (KBr disk) 1384, 1152 (SO₂); ¹H NMR (DMSO-d₆) δ 3.07 (s, 3H, SO₂CH₃), 6.61 (d, J = 1.2 Hz, 1H, pyrazole H-3), 6.89 (d, J = 3.6 Hz, thienyl H-4), 7.02 (dd, J = 4.0, 8.8 Hz, thienyl H-4), 7.39 (d, J = 5.2 Hz, 1H, thienyl H-5), 7.60 (d, J = 8.4 Hz, 2H, methanesulfonylphenyl H-3, H-5), 7.76 (d, J = 1.2 Hz, 1H, pyrazole H-4), 7.96 (d, J = 8.4 Hz, 2H, methanesulfonylphenyl H-2, H-6); ¹³C NMR (DMSO-d₆) δ 44.56, 109.91, 125.10, 125.61, 127.53, 127.77, 128.22, 129.00, 130.11, 130.36, 141.38, 143.97; MS (m/z, relative abundance %): 304.09 (M^+., 21.83); Anal. Calcd for C₁₄H₁₂N₂O₂S₂: C, 55.24; H, 3.97; N, 9.20; Found: C, 55.00; H, 3.71; N, 8.88.

4-(5-Thiophen-2-yl-pyrazol-1-yl)benzoic acid (10l)

72% yield; brown solid; m.p. 297–299 °C; IR (KBr disk) 3428 (OH), 1693 (C=O) 1347, 1173 (SO₂); ¹H NMR (DMSO-d₆) δ 6.67 (d, J = 1.6 Hz, 1H, pyrazole H-3), 6.02 (s, 1H, thienyl H-4), 7.07 (s, 1H, thienyl H-5), 7.48 (d, J = 7.6 Hz, 2H, benzoic H-3, H-5), 7.62 (d, J = 4.4 Hz, 1H, thienyl H-3), 7.81 (d, J = 1.6 Hz, 1H, pyrazole H-4), 8.05 (d, J = 7.6 Hz, 2H, benzoic H-2, H-6), 13.19 (s, 1H, COOH, D₂O exchangeable); ¹³C NMR (DMSO-d₆) δ 109.71, 125.49, 127.37, 127.42, 127.71, 128.11, 141.05, 141.28, 141.63, 145.00, 145.22, 169.81; MS (m/z, relative abundance %): 270.05 (M^+., 3.18); Anal. Calcd for C₁₄H₁₀N₂O₂S: C, 62.21; H, 3.73; N, 10.36; Found: C, 62.35; H, 3.35; N, 9.95.

Biological evaluation

COX-1/COX-2 inhibition colorimetric assay

The ability of the test compounds listed in Table 1 to inhibit ovine COX-1 and human recombinant COX-2 (IC₅₀ value, μM) was determined using an enzyme immuno assay (EIA) kit (catalog no. 560131, Cayman Chemical, Ann Arbor, MI) according to the previously reported method26.

In vivo anti-inflammatory activity

Animals

Adult male Wistar Albino rats (100–150 g) were used in the pharmacological studies. The animals (five per cage) were maintained under standard laboratory conditions (light period of 12 h/day and temperature 27 ± 2 °C), with access to food and water. The experimental procedures were carried out in strict compliance with the Institutional Animal Ethics Committee regulations. All experiments were performed in the morning according to the guidelines for the care of laboratory animals.

The dose causing 50% edema inhibition (ED₅₀) for test compounds 10a–l and reference drugs celecoxib and ibuprofen were determined using the in vivo carrageenan-induced rat paw edema model and the measurement of paw thickness was done at 3 h after oral administration of the test compound as reported previously27.

Ulcerogenic liability study

Ulcerogenic liability of the target compounds 10a–l were determined using ED₅₀ dose in comparison with celecoxib (ED₅₀ dose) and small dose of Ibuprofen (120 μmol/kg) according to the previously reported method29.

Declaration of interest

The authors have declared no conflict of interest.