Synthesis and biological evaluation of some novel sulfamoylphenyl-pyridazinone as anti-inflammatory agents (Part-II)

Abstract

Seven novel 6-aryl-2-(p-sulfamoylphenyl)-4,5-dihydropyridazin-3(2H)-ones (2a-g) were synthesized by the condensation of appropriate aroylpropionic acid and 4-hydrazinobenzenesulfonamide hydrochloride in ethanol. Structure of all compounds have been elucidated by elemental analysis, IR, ¹H NMR, ¹³C NMR, DEPT and MS spectrscopy. These compounds were tested for their anti-inflammatory activity in carrageenan-induced rat paw edema model. Compound 2b exhibited anti-inflammatory activity comparable to that of celecoxib (at 5 h). Two other compounds 2d and 2g showed promising anti-inflammatory activity (edema reduction more than 80% at 5 h). These compounds (2b, 2d and 2g) did not produce any ulceration in gastric region.

Keywords::

• Pyridazinone
• benzenesulfonamide
• anti-inflammatory

Introduction

Pyridazin-3(2H)-one derivatives represent one of the most active class of compounds possessing a wide spectrum of pharmacological activities ranging from cardiovascular properties, anti-inflammatory, antidiabetic, analgesic, anti-AIDs, anticancer and anticonvulsant activities^1,2. Pyridazinone derivatives also possess affinity for benzodiazepine receptors³ and the ability to inhibit the human matrix metalloproteinase⁴ and aldose reductase⁵ enzymes. Recently, these derivatives have been reported as potent hepatoprotective agents⁶, antibacterial and antifungal agents⁷, COX-2 inhibitors⁸, platelet aggregation inhibitor^9–12, phosphodiesterase^13,14. They also act as cytotoxic (cell-killing) agents and anti-hormonal drugs, which reduce the proliferation of the tumors^15–16.

The sulfonamides constitute an important class of drugs, with several types of pharmacological agents possessing antibacterial¹⁷, anti-carbonic anhydrase^18,19, diuretic^18,20, hypoglycemic²¹, antithyroid²², and antiprotons activities^23–25. A large number of structurally novel sulfonamide derivatives have recently been reported to show substantial antitumor activity, both in vitro and/or in vivo. Although they have a common chemical motif of aromatic/heterocyclic sulfonamide, there are a variety of mechanisms of their antitumor action, most of them poorly understood at this moment. Some of these derivatives are currently being evaluated in clinical trials, and there is much optimism that they may lead to novel alternative anticancer drugs, devoid of the side effects of the presently available pharmacological agents²⁶. Anti-inflammatory activity of celecoxib (celebrex) which was used clinically in the 1990s is attributed to the presence of SO₂NH₂ substituted at the para position of one aryl groups^27,28.

Therefore, it was thought worthwhile to synthesize novel pyridazinone derivatives bearing sulphonamide moiety and screen them for the anti-inflammatory activity.

Results and discussion

Synthesis of compounds

The synthetic route used to synthesize title compounds (2a-j) is outlined in Scheme 1. Various intermediates 1,4-ketoacids (β-aroylpropionic acids) required for the synthesis of pyridazinones were prepared by Friedel-Crafts succinoylation of substituted aromatic compounds²⁹. The cyclization to pyridazinone derivatives bearing a benzene sulfonamide moiety was afforded by the condensation of appropriate aroylpropionic acid and 4-hydrazinobenzenesulfonamide hydrochloride in ethanol in 60–75% yield. The structures of pyridazinone derivatives (2a-g) were determined by elemental analysis, IR, ¹H NMR, ¹³C NMR, DEPT and MS.

Scheme 1.  Reagents and conditions: EtOH, reflux 18–24 h.

The structure was supported by the evidence of presence of prominent bands in IR spectra for NH₂ (3284–3449, 3019–3298 and 2916–2954), cyclic carbonyl (1637–1657), C=N (1462–1595) and SO₂N< (1310–1377 and 1151–1189) groups. The structure was further established by ¹HNMR spectral data. The signal for SO₂NH₂ was observed as two-proton singlet at δ 7.10–7.98. Two triplets each integrating for two protons observed at δ 2.74–2.87 and δ 3.03–3.21 indicate the presence of−CH₂–CH₂– system of dihydropyridazinone ring. DEPT spectra indicate the presence of CH₃, CH₂ and CH carbons. Elemental analysis (C, H, N and S) data were within ±0.4 of the theoretical values.

Biological activity

All compounds (2a-g) where tested for anti-inflammatory activity by using carrageenan-induced rat hind paw edema method³⁰. All the pyridazinone derivatives (2a-g) exhibited varying degree of anti-inflammatory activity (23.3–78.9% at 3 h and 52.4–88.1% at 5 h) (Table 1). Compound 2b has shown maximum activity at 5h (comparable to that of celecoxib at 5 h). Compound 2d and 2g showed promising activity.

Table 1.  Effect of pyridazinone derivatives on carrageenan induced hind paw edema in rats.

Treatment	Dose per kg b.w.	Increase in paw volume (mL ± SEM) after carrageenan administration
		3 h	5 h
Vehicle	10 ml/kg	0.38 ± 0.0307	0.42 ± 0.0401
Celecoxib	20 mg	0.066 ± 0.0210* (82.6%)	0.06 ± 0.02* (85.7%)
2a	20 mg	0.2 ± 0.000* (47.4%)	0.13 ± 0.0210* (69%)
2b	20 mg	0.15 ± 0.02236* (60.5%)	0.05 ± 0.03416* (88.1%)
2c	20 mg	0.28 ± 0.01667* (23.3%)	0.2 ± 0.0258* (52.4%)
2d	20 mg	0.08 ± 0.01667* (78.9%)	0.08 ± 0.01667* (81%)
2e	20 mg	0.23 ± 0.0333* (39.4%)	0.13 ± 0.0333* (69%)
2f	20 mg	0.2 ± 0.02582* (47.3%)	0.12 ± 0.03073* (71.4%)
2g	20 mg	0.1 ± 0.02582* (73.6%)	0.08 ± 0.03073* (80.9%)

*P < 0.05 compared to control (one-way analysis of variance followed by Dunnett’s test). Values are presented as mean ± S.E.M. (n = 6). Values in parentheses represent percent inhibitions.

No specific structural activity relationship was established from the data shown in Table 1.

Acute gastric ulcerogenic effect of the compound 2b, 2d and 2g at 60 mg/kg (three times) was evaluated in Wistar rats³¹. No ulceration in gastric region was observed.

Experimental

Melting points were determined by open capillary tubes and are uncorrected. All the Fourier Transform Infra Red spectra were recorded on a Brukers Vector 22 spectrophotometer in film; υ_max values are given in cm⁻¹. ¹H NMR spectra were recorded on a Bruker Spectrospin DPX 300-MHz spectrometer using deuterated DMSO as a solvent and tetramethyl silane (TMS) as an internal standard. Chemical shifts are given in δ (ppm) scale and coupling constants (J values) are expressed in Hz. Mass spectra were scanned by affecting FAB ionization JEOL-JMS-DX 303 system, equipped with direct inlet probe system and ESI Bruker Esquire 3000. The m/z values of the more intense peaks are mentioned. ¹³C NMR and DEPT spectra were recorded on Bruker spectrospin DPX 400 MHz and 500 MHz using deuterated DMSO as a solvent and TMS as internal standard. Purity of the compounds was checked on TLC plates (silica gel G) which were visualized by exposing to iodine vapors. Elemental analysis was carried out on CHNS Elementar (Vario EL III).

Procedure for the synthesis of Aroylpropionic acids (1a-g)

To liquid aromatic hydrocarbon (30 mL), anhydrous aluminium chloride (16.6 g, 0.125 mole) was added. It was stirred on magnetic stirrer at room temperature for 30 min. To it succinic anhydride (5 g, 0.05 mol) was added in five portions with continuous stirring. Vigorous reaction started with evolution of HCl gas. Stirring was continued for another 6 h at room temperature. It was left at room temperature for 48 h and then decomposed by adding ice-cold hydrochloric acid (50%, 100 mL). The excess solvent was removed by steam distillation. The solid precipitated out was treated with aqueous saturated sodium bicarbonate solution and filtered. Filtrate was acidified with dilute HCl (4% v/v) to give a precipitate. It was filtered and residue was washed with cold water, dried and crystallized from the appropriate solvent to give (1a-g)²⁹.

General Procedure for the synthesis of pyridazinones (2a-g)

A mixture of appropriate aroylpropionic acid (1a-g) (0.001 mol) and 4-hydrazinobenzenesulfonamide hydrochloride (0.001 mol) in absolute ethanol (20–30 mL) was refluxed for 18–24 h. The reaction mixture was concentrated to one-third of its volume and left at room temperature when a solid separated out. Crude product was filtered off, washed with little volume of alcohol. The residue thus obtained was stirred with 5% sodium bicarbonate solution (25 mL) for 1h. It was filtered, washed with 2% acetic acid and then with water. It was dried and crystallized from methanol to give (2a-g).

6-(2′,4′-Dimethylphenyl)-2-(4-sulfamoylphenyl)-4,5-dihydropyridazin-3(2H)-one (2a)

Yield = 61%, m.p. 156–1580C, Rf = 0.61 (toluene: ethyl acetate: formic acid, 5: 4: 1). IR υ_max (Solvent, in cm⁻¹): 3304, 3232 and 2920 (NH₂), 1649 (C=O), 1310 and 1185 cm⁻¹ (SO₂N). ¹H NMR (300 MHz, DMSO, δ): 2.30 (3H, s, CH₃), 2.41 (3H, s, CH₃), 2.78 & 3.03 (each t, 2x-CH₂-), 7.11 (2H, s, SO₂NH₂), 7.38 (3H, m, H-2′, H-5′, H-6′), 7.72 (2H, d, J = 8.59 Hz, H-3′′, H-5′′), 7.85 (2H, d, J = 8.59 Hz, H-2′′, H-6′′). ESI⁺ (m/z): 356 (M⁺-1), 380 (M⁺+Na). ¹³C NMR (DMSO, δ): 20.92 (-CH₃, C-3′), 22.19 (-CH₃, C-4′), 27.42 (-CH₂ of pyridazinone), 46.16 (-CH₂ of pyridazinone), 165.73 (C=N), 177.95 (C=O). DEPT (DMSO, δ): 19.036 (2x-CH₃, C-3′, C-4′), 27.875 (-CH₂ of pyridazinone), 46.680 (-CH₂ of pyridazinone). Anal. Calcd. For C₁₈H₁₉N₃O₃S, Calculated: C = 60.49, H = 5.36, N = 11.76, S = 8.97, Found: C = 60.87, H = 4.98, N = 12.02, S = 9.13.

6-(4′-Phenylphenyl)- 2-(4-sulfamoylphenyl)-4,5-dihydropyridazin-3(2H)-one (2b)

Yield = 55%, m.p. >260°C, Rf = 0.70 (toluene: ethyl acetate: formic acid, 5: 4: 1). IR υ_max (Solvent, in cm⁻¹): 3317, 1657 (C=O), 1595 (C=N), 1326 and 1151 cm⁻¹ (SO₂N). ¹H NMR (300 MHz, DMSO, δ): 2.80 (2H, t, CH₂ of pyridazinone), 3.21 (2H, t, CH₂ of pyridazinone), 7.40-7.98 (15H, m, N-phenyl protons, biphenyl protons, SO₂NH₂). ESI⁺ (m/z): 405 (M⁺), 406 (M⁺ +1), 407 (M⁺ +2). ¹³C NMR (DMSO, δ): 24.4 (-CH₃), 32.4 (-CH₂ of pyridazinone), δ 32.4 (-CH₂ of pyridazinone), 152.50 (C=N), 165.70 (C=O). DEPT (DMSO, δ): 22.20 (-CH₂ of pyridazinone), 27.44 (-CH₂ of pyridazinone). Anal. Calcd. For C₂₂H₁₉N₃O₃S, Calculated: C = 65.17, H = 4.72, N = 10.36, S = 7.91, Found: C = 65.12, H = 4.69, N = 10.36, S = 7.89.

6-(4′-phenoxyphenyl)- 2-(4-sulfamoylphenyl)-4,5-dihydropyridazin-3(2H)-one (2c)

Yield = 65%, m.p. 235–2360C, Rf = 0.57 (toluene: ethyl acetate: formic acid, 5: 4: 1). IR υ_max (Solvent, in cm⁻¹): 3449 (NH₂), 1637 (C=O), 1329 and 1153 cm⁻¹ (SO₂N). ¹H NMR (300 MHz, DMSO,d): 2.80 (2H, t, -CH₂ of pyridazinone), 3.18 (2H, t, -CH₂ of pyridazinone), 7.10- 7.94 (15H, m, SO₂NH₂, phenyl phenoxy & N-phenyl). ESI⁺ (m/z): 421 (M⁺), 422 (M⁺ +1), 423 (M⁺ +2), 299. ¹³C NMR (DMSO, δ): 22.47 (-CH₂ of pyridazinone), 27.59 (-CH₂ of pyridazinone), 152.33 (C=N), 165.56 (C=O). DEPT (DMSO, δ): 22.23 (-CH₂ of pyridazinone), 27.46 (-CH₂ of pyridazinone). Anal. Calcd. for C₂₂H₁₉N₃O₄S, Calculated: C = 62.69, H = 4.54, N = 9.97, S = 7.61, Found: C = 62.63, H = 4.51, N = 9.96, S = 7.60.

6-(β-napthyl)-2-(4-sulfamoylphenyl)-4,5-dihydropyridazin-3(2H)-one(2d)

Yield = 65%, m.p. 252–2540C, Rf = 0.57 (toluene: ethyl acetate: formic acid, 5: 4: 1). IR υ_max (Solvent, in cm⁻¹): 3298 (NH₂), 1657 (C=O), 1590 (C=N), 1377 and 1189 cm⁻¹ (SO₂N). ¹H NMR (300 MHz, DMSO, δ): 2.87 (2H, t, -CH₂ of pyridazinone), 7.60-8.21 (10H, m, N-phenyl and napthelene protons except H-α), 7.43 (2H, s, SO₂NH₂), 8.44 (1H, s, H-α). ESI⁺ (m/z): 379 (M⁺), 337, 300. ¹³C NMR (DMSO, δ): 22.82 (-CH₂ of pyridazinone), 28.07 (-CH₂ of pyridazinone), 153.19 (C=N), 166.20 (C=O). DEPT (DMSO, δ): 20.76 (-CH₂ of pyridazinone), 27.47 (-CH₂ of pyridazinone). Anal. Calcd. For C₂₀H₁₇N₃O₃S, Calculated: C = 63.31, H = 4.52, N = 11.07, S = 8.45, Found: C = 63.25, H = 4.48, N = 11.07, S = 8.43.

6-(4′-bromophenyl)-2-(4-sulfamoylphenyl)-4,5-dihydropyridazin-3(2H)-one (2e)

Yield = 55%, m.p. 241–2420C, Rf = 0.59 (toluene: ethyl acetate: formic acid, 5: 4: 1). IR υ_max (Solvent, in cm⁻¹): 3312, 3019 and 2954 (NH₂), 1651 (C=O), 1462 (C=N), 1338 and 1154 cm⁻¹ (SO₂N). ¹H NMR (300 MHz, DMSO, δ): 2.76 (2H, t, -CH₂ of pyridazinone), 3.14 (2H, t, -CH₂ of pyridazinone), 7.37 (2H, s, SO₂NH₂), 7.64− 7.87 (8H, m, N-phenyl protons & bromophenyl protons). ESI⁺ (m/z): 409 (M⁺+1), 431 (M⁺+Na), 407 (M⁺-1), 405, 378, 338. ¹³C NMR (DMSO, δ): 22.11 (-CH₂ of pyridazinone), 27.28 (-CH₂ of pyridazinone), 151.79 (C=N), 165.57 (C=O). DEPT (DMSO, δ): 22.18 (-CH₂ of pyridazinone), 27.35 (-CH₂ of pyridazinone). Anal. Calcd. For C₁₆H₁₄BrN₃O₃S, Calculated: C = 47.07, H = 3.46, N = 10.29, S = 7.85, Found: C = 47.03, H = 3.43, N = 10.29, S = 7.84.

6-(3′,4′-dimethoxyphenyl)-2-(4-sulfamoylphenyl)-4,5-dihydropyridazin-3(2H)-one (2f)

Yield = 65%, m.p. 203–2040C, Rf = 0.5 (toluene: ethyl formate: formic acid, 7.5: 2: 0.5). ¹H NMR (300 MHz, DMSO, δ): 2.74 (2H, t, -CH₂ of pyridazinone), 3.13 (2H, t, -CH₂ of pyridazinone), 3.79 (6H, s, 2 × OCH₃), 7.02 (1H, d, J= 8.4 Hz, H-5′), 7.39 (4H, m, H-2′, H-6′, SO₂NH₂), 7.82 (4H, m, N-phenyl protons). ESI⁺ (m/z): 389 (M⁺), 388 (M⁺-1), 412 (M⁺+Na), 325, 311, 255. ¹³C NMR (DMSO, δ): 22.16 (-CH₂ of pyridazinone), 27.6 (-CH₂ of pyridazinone), 55.55 (OCH₃,C-3′ and C-4′), 148.68 (C=N), 165.74 (C=O). DEPT (DMSO, δ): 22.23 (-CH₂ of pyridazinone), 27.63 (-CH₂ of pyridazinone), δ 55.615 (2x OCH₃, C-3′, C-4′). Anal. Calcd. for C₁₈H₁₉N₃O₅S, Calculated: C = 55.52, H = 4.92, N = 10.79, S = 8.23, Found: C = 55.47, H = 4.88, N = 10.78, S = 8.22.

6-(3′,4′-dichlorophenyl)-2-(4-sulfamoylphenyl)-4,5-dihydropyridazin-3(2H)-one (2g)

Yield = 70%, m.p. 260–2610C, Rf = 0.70 (toluene: ethyl acetate: formic acid, 5: 4: 1). IR υ_max (Solvent, in cm⁻¹): 3284, 3218 and 2916 (NH₂), 1643 (C=O), 1468 (C=N), 1336 and 1151 cm⁻¹ (SO₂N). ¹H NMR (300 MHz, DMSO, δ): 2.74 (2H, t, -CH₂ of pyridazinone), 3.15 (2H, t, -CH₂ of pyridazinone), 7.37 (2H, s, SO₂NH₂), 7.71-7.87 (6H, m, N-phenyl protons, H-5′, H-6′), 8.05 (1H, s, H-2′). ES⁺-MS (m/z): 397 (M⁺), 399 (M+2), 365, 348. ¹³C NMR (DMSO, δ): 22.78 (-CH₂ of pyridazinone), 28.07 (-CH₂ of pyridazinone), 153.19 (C=N), 166.20 (C=O). DEPT (DMSO, δ): 26.394 (-CH₂ of pyridazinone), 28.225 (-CH₂ of pyridazinone). Anal. Calcd. for C₁₆H₁₃Cl₂N₃O₃S, Calculated: C = 48.25, H = 3.29, N = 10.55, S = 8.05, Found: C = 48.21, H = 3.26, N = 10.55, S = 8.04.

Anti-inflammatory activity

Carrageenan-induced hind paw edema method was used for evaluating anti-inflammatory activity³⁰. Wistar rats (either sex) weighing 140–180 g were procured from Central Animal House facility of Jamia Hamdard, New Delhi (Registration no. 173/CPCSEA). The experiments were performed in accordance with the guidelines for the care and use of laboratory animals, laid down by the Committee for the Purpose of Control and Supervision of Experiments in Animals (CPCSEA), Ministry of Social Justice and Empowerment, Govternment of India, January 2000. Overnight fasted rats (16 h) were divided into groups of six animals each. One group of rats, which served as control was given vehicle (1% Carboxymethyl cellulose (CMC) in water in a volume of 10 mL/kg p.o.) only. Test compounds (20 mg/kg body weight.) and celecoxib (20 mg/kg b.w.) suspended in vehicle (10 mL/kg) were administered orally to respective groups. After 30 min, all animals were injected with 0.1 mL of 1% carrageenan solution (prepared in normal saline) in the subplantar aponeurosis of left hind paw to induce inflammation and the volume of injected paw was measured by using plethysmometer immediately (at 0 h). The paw volume was again measured after 3 h and 5 h. The average paw volume in a group of treated rats was compared with vehicle (control group) and the percentage inhibition of edema was calculated by using the formula:

Where Vt is the mean paw volume of the treated rats and Vc is the mean paw volume of the control.

Ulcerogenic activity

Acute gastric ulcerogenic effect of the compound 2b, 2d and 2g was evaluated in Wistar rats³¹. Albino rats of Wistar strain (150–200 g) fasted over 24 h were randomly allotted into three groups of six animals each. The animals of one group were given vehicle 10 mL/kg (CMC 1% w/v in distilled water) orally. The compound 2b, 2d and 2g standard drug (60 mg/kg) suspended in vehicle was administered orally in a volume of 10 mL/kg to the animals. They were scarified under deep ether anesthesia after 6 h of the oral treatment at a dose of 60 mg/kg. Their stomach were removed and opened through greater curvature for examining lesions or bleedings.

Conclusions

The structures proposed to the synthesized compounds (2a-g) are well supported by spectroscopic data and elemental analysis. Among the synthesized compounds 2b exhibited potent anti-inflammatory activity which is comparable to that of celecoxib (at 5 h). Other two compounds (2d and 2g) were found to have promising anti-inflammatory activity (edema reduction more than 80% at 5 h). These compounds (2b, 2d and 2g) did not produce any ulceration in gastric region.

Acknowledgments

This work was supported by Grant No. 32–228/2006 (SR) from the University Grants Commission, New Delhi, India. One of the authors, Rafia Bashir is thankful to UGC for fellowship. Thanks are due to SAIF, CDRI, Lucknow, for providing Mass spectra.

Declaration of interest

The authors report no conflict of interest. The authors alone are responsible for the content and writing of the article.