Synthesis and Antioxidant Activity of a Variety of Sulfonamidomethane Linked 1,3,4-Oxadiazoles and Thiadiazoles

Abstract

A variety of sulfonamidomethane linked 1,3,4-oxadiazoles and 1,3,4-thiadiazoles were prepared and tested for antioxidant activity. The methyl substituted arylsulfonylaminomethyl-1,3,4-oxadiazole 9b showed excellent antioxidant activity.

Among different five membered heteroarenes, 1,3,4-oxadiazoles and 1,3,4-thiadiazoles have gained importance in medicinal chemistry and in materials science as they constitute the structural features of many bioactive compounds. Oxadiazole derivatives possess a wide range of biological properties such as antimicrobial, anticonvulsant etc.^1,2) They are known to work as ester and amide bioisosteres.^3,4) It has been shown that 1,3,4-thiadiazole derivatives have multiple biological activities including anticancer,^5–7) antibacterial⁸⁾ and antifungal.⁹⁾ In fact the structure of ‘N–C–S’ in 1,3,4-thiadiazole derivatives work as active centre. Besides, sulfonamides constitute an important class of drugs with several types of pharmacological activities such as antibacterial,¹⁰⁾ hypoglycemic,¹¹⁾ antithyroid,¹²⁾ antiviral^13,14)etc. The aryl/heteroaryl sulfonamides act as antitumor agents.^15–19) Motivated by these findings it is proposed to synthesize and investigate the antioxidant activity of a new series of molecules having sulfonamide and amide backbone to diazole nucleus which will definitely contribute substantially in increasing pharmacological activity.

Results and Discussion

Chemistry

The synthetic routes adopted to prepare the target molecules are illustrated and outlined in scheme. The precursors arylsulfonylaminoacetic acid hydrazide (1), phenylsulfonylacetic acid (3), benzoylaminoacetic acid (4) and phenylsulfonylaminoacetic acid (5) were prepared according to the literature methods^20–22) and commercially available phenylacetic acid (2) was used as such. The cyclocondensation of 1 with 2 in the presence of POCl₃ led to 2-arylsulfonylaminomethyl-5-benzyl-1,3,4-oxadiazole (6). The compound 2-arylsulfonylaminomethyl-5-phenylsulfonylmethyl-1,3,4-oxadiazole (7) was prepared by the reaction of 1 with 3. Similarly 2-arylsulfonylaminomethyl-5-benzoylaminomethyl-1,3,4-oxadiazole (8) was obtained by the reaction between 1 and 4. Likewise, the compound 2-arylsulfonylaminomethyl-5-phenylsulfonylaminomethyl-1,3,4-oxadiazole (9) was obtained by the treatment of 1 with 5 (Chart 1).

Chart 1

The compounds 6–9 in their IR spectra displayed absorption bands in the regions 1130–1144 and 1318–1336 for SO₂, 1580–1608 for C=N and 3334–3347 cm⁻¹ for NH. Besides, the compound 8 presented another absorption band ≈1700 cm⁻¹ for CO–NH. The ¹H-NMR spectra of 6a exhibited two singlets at δ 4.86, 4.42, 7a at 4.55, 4.89 and 8a at 4.39, 4.87 ppm for methylene protons attached to C-2 and C-5, respectively. However, compound 9a displayed a singlet at 4.48 ppm for methylene protons which indicated that it is a symmetric molecule. Besides, all the compounds showed a broad singlet in the region ≈8.40 ppm due to SO₂–NH. The compound 8a exhibited another broad singlet at 10.15 ppm due to CO–NH. The signals of highly acidic protons disappeared when D₂O was added. The ¹³C-NMR spectra of 6a displayed signals at δ 188.2, 168.4, 41.6, 31.9, 7a at 165.3, 158.2, 44.6, 59.9 and 8a at 176.6, 158.6, 40.1, 47.2 ppm which were assigned to C-2, C-5, CH₂-(C-2), CH₂-(C-5), whereas the signals observed at 177.9, 158.0, 47.1 ppm in 9a were attributed to C-2, C-5, CH₂-(C-2, C-5), respectively. Besides, 8a showed a signal at 169.8 ppm due to CO. Interconversion of oxadiazoles 6–9 to the respective thiadiazoles was effected by treatment with thiourea in THF. Thus the compounds 2-arylsulfonylaminomethyl-5-benzyl-1,3,4-thiadiazole (10), 2-arylsulfonylaminomethyl-5-phenylsulfonylmethyl-1,3,4-thiadiazole (11), 2-arylsulfonylaminomethyl-5-benzoylaminomethyl-1,3,4-thiadiazole (12) and 2-arylsulfonylaminomethyl-5-phenylsulfonylaminomethyl-1,3,4-thiadiazole (13) were prepared. This transformation of oxadiazole to respective thiadiazole was supported from ¹H-NMR data which showed singlets for methylene protons attached to C-2 and C-5 at slightly downfield region. The compound 10a displayed two singlets at δ 5.07, 4.63, 11a at 4.75, 5.10 and 12a at 4.59, 5.08 ppm due to methylene protons attached to C-2 and C-5, respectively. However compound 13a displayed a singlet at 4.69 ppm for methylene protons. The downfield shift of C-2, C-5 and methylene carbons was also observed in the ¹³C-NMR spectra of these compounds. The signals observed at δ 190.1, 170.2, 43.9, 33.7 in 10a, at 167.1, 160.0, 46.4, 61.7 in 11a, at 178.8, 160.3, 41.9, 49.2 ppm in 12a were attributed to C-2, C-5, CH₂-(C-2), CH₂-(C-5), respectively. The compound 13a displayed signals at 179.6, 160.1, 49.5 ppm due to C-2, C-5, CH₂-(C-2 & C-5).

Antioxidant Activity

The compounds 6–13 were tested for antioxidant property by 2,2-diphenyl-1-picrylhydrazyl (DPPH),^23,24) nitric oxide (NO)^25,26) and hydrogen peroxide (H₂O₂)²⁷⁾ methods at three different concentrations 50, 75 and 100 µg/mL. The observed data on the antioxidant activity of the compounds and controlled drug were shown in Tables 1–3 and Figs. 1–3. Amongst oxadiazole and thiadiazole containing compounds, the oxadiazole derivatives showed good radical scavenging activity than the corresponding thiadiazoles. In fact, the compound 9b, methyl substituted bis(arylsulfonamidomethyl)oxadiazole displayed excellent antioxidant activity in all the three methods greater than the standard drug ascorbic acid. It was observed that there is not much difference in the activity of the compounds having sulfonylmethyl (7) and amidomethyl (8) groups attached to oxadiazole ring. On the other hand, the compound 6 displayed least activity when compared with the other oxadiazole derivatives. Amongst compounds having thiadiazole ring, the one with bis(arylsulfonamidomethyl) moiety 13 showed good activity. Further it was observed that in each set of compounds, those having electron donating methyl substituent on the aromatic ring displayed slightly high activity.

Fig. 1. Free Radical Scavenging Activity of Compounds 6–13 by DPPH Method

Fig. 2. Free Radical Scavenging Activity of Compounds 6–13 by Nitric Oxide Method

Fig. 3. Free Radical Scavenging Activity of Compounds 6–13 in H₂O₂ Method

Table 1. Antioxidant Activities of the Test Compounds 6–13 and Standard Using DPPH Scavenging Method—% DPPH Radical Scavenging Activity

Compound	Concentration
	50 µg/mL (%)	75 µg/mL (%)	100 µg/mL (%)
6a	43.85±0.86	46.12±0.37	48.85±1.22
6b	45.45±1.43	48.20±1.22	50.45±0.69
6c	42.20±0.93	47.75±0.15	49.05±1.32
7a	63.90±0.28	67.75±0.63	68.46±1.12
7b	65.14±0.69	68.55±1.45	70.25±1.06
7c	62.71±0.29	65.36±0.72	67.58±1.28
8a	64.00±0.35	67.15±0.73	69.58±1.08
8b	65.94±0.88	68.92±1.54	70.78±1.04
8c	62.82±0.36	65.73±0.65	68.10±1.24
9a	75.64±1.40	79.34±2.41	83.42±1.02
9b	79.17±1.20	83.66±1.23	86.83±1.57
9c	74.26±1.02	78.73±1.08	82.28±1.10
10a	42.13±1.32	44.95±1.31	46.85±1.85
10b	43.70±0.71	45.50±0.87	47.38±1.55
10c	41.78±1.30	44.14±0.96	46.25±1.65
11a	49.12±0.26	50.95±0.16	53.08±1.10
11b	49.54±0.63	51.34±0.52	53.54±1.05
11c	48.73±0.08	50.06±1.26	52.45±1.32
12a	58.15±0.15	60.10±1.87	61.85±0.95
12b	58.82±0.45	60.42±1.81	62.75±1.12
12c	57.50±1.28	59.55±1.42	61.10±1.31
13a	63.84±0.25	66.45±1.38	68.54±0.90
13b	64.15±1.48	67.38±1.40	69.66±1.52
13c	62.50±1.50	65.75±1.75	67.24±1.28
Ascorbic acid	77.15±0.45	80.95±0.39	83.82±0.81
Blank	—	—	—

(—) Showed no scavenging activity. Values were the means of three replicates±S.D.

Table 2. Antioxidant Activities of the Test Compounds 6–13 and Standard Using NO Scavenging Method—% NO Radical Scavenging Activity

Compound	Concentration
	50 µg/mL (%)	75 µg/mL (%)	100 µg/mL (%)
6a	62.54±0.89	63.74±1.20	65.83±0.25
6b	62.75±1.24	64.10±1.10	66.25±0.54
6c	62.16±0.53	63.50±1.25	64.12±0.63
7a	66.45±0.12	68.54±1.24	71.00±1.30
7b	66.90±1.63	69.97±1.55	72.98±1.45
7c	65.10±1.45	67.85±0.65	70.20±0.75
8a	66.83±0.15	69.15±1.34	71.12±1.28
8b	67.20±1.64	70.43±1.60	72.68±1.42
8c	65.83±1.43	66.78±0.70	71.50±0.74
9a	78.12±0.90	79.74±0.80	80.16±0.83
9b	80.43±0.82	82.25±1.22	83.85±1.54
9c	77.75±0.72	78.08±1.75	79.65±1.83
10a	45.20±1.55	47.12±1.95	49.26±1.42
10b	45.70±1.32	47.35±1.83	49.84±1.35
10c	44.70±1.27	46.54±1.08	48.44±1.20
11a	58.16±0.86	59.54±1.66	62.10±1.86
11b	58.63±1.34	60.34±1.09	62.38±1.63
11c	57.54±1.32	59.13±1.38	61.70±1.07
12a	64.10±1.26	66.50±0.67	70.15±1.52
12b	66.75±1.35	69.82±1.50	71.64±0.96
12c	63.43±0.97	65.15±0.23	70.05±0.54
13a	74.60±0.15	76.68±1.70	79.48±0.84
13b	75.45±0.26	77.54±0.54	80.55±1.60
13c	74.15±0.55	76.05±0.54	78.15±1.45
Ascorbic acid	78.23±0.17	81.46±1.37	82.79±0.80
Blank	—	—	—

(—) Showed no scavenging activity. Values were the means of three replicates±S.D.

Table 3. Antioxidant Activities of the Test Compounds 6–13 and Standard Using H₂O₂ Scavenging Method—% H₂O₂ Radical Scavenging Activity

Compound	Concentration
	50 µg/mL (%)	75 µg/mL (%)	100 µg/mL (%)
6a	60.44±1.57	61.66±1.54	64.63±1.54
6b	60.80±1.63	62.13±1.58	64.80±1.17
6c	60.08±1.67	61.23±1.78	64.34±1.01
7a	66.05±1.56	68.01±1.53	70.13±1.38
7b	67.43±1.48	69.15±1.26	73.24±1.22
7c	65.93±1.67	68.42±1.48	72.93±1.11
8a	66.20±1.59	69.14±1.43	70.63±1.42
8b	67.75±1.57	69.54±1.24	70.74±1.25
8c	66.65±1.78	68.62±1.36	69.14±1.15
9a	77.12±1.02	78.75±1.92	80.15±1.23
9b	79.54±1.43	80.45±1.38	84.53±1.42
9c	76.70±1.57	77.34±1.45	79.75±1.25
10a	43.12±1.95	45.54±1.24	46.62±1.40
10b	43.54±1.35	45.82±1.32	46.84±1.14
10c	43.07±0.85	45.13±1.26	46.25±1.20
11a	54.10±1.10	56.20±1.06	58.20±1.02
11b	54.34±1.17	56.70±1.52	58.84±1.70
11c	53.80±1.45	55.82±1.10	58.05±1.28
12a	66.00±1.42	69.21±1.72	70.53±0.82
12b	67.54±0.87	69.72±1.13	70.70±1.54
12c	66.08±1.67	69.03±1.21	70.14±0.75
13a	73.10±1.54	75.10±1.42	76.43±1.26
13b	74.58±1.25	75.84±1.73	77.38±1.48
13c	72.60±1.32	74.75±1.33	76.05±1.36
Ascorbic acid	77.68±0.51	79.27±1.29	83.16±0.44
Blank	—	—	—

(—) Showed no scavenging activity. Values were the means of three replicates±S.D.

Conclusion

In conclusion we have prepared a new class of sulfonamidomethane linked 1,3,4-oxadiazoles and 1,3,4-thiadiazoles and studied their antioxidant activity. The oxadiazole derivatives exhibited greater activity than the corresponding thiadiazoles. The presence of electron donating methyl substituent on the aromatic ring enhanced the activity. Thus the results exemplified that the methyl substituted bis(arylsulfonamidomethyl)oxadiazole 9b is the most powerful antioxidant agent.

Experimental

General

Melting points were determined in open capillaries on a Mel-Temp apparatus and are uncorrected. The purity of the compounds was checked by TLC (silica gel H, BDH, hexane–ethyl acetate, 3 : 1). The IR spectra were recorded on a Thermo Nicolet IR200 FT-IR spectrometer as KBr pellets and the wave numbers were given in cm⁻¹. The ¹H-NMR spectra were recorded in CDCl₃–DMSO-d₆ on a Brucker-400 spectrometer (400 MHz). The ¹³C-NMR spectra were recorded in CDCl₃–DMSO-d₆ on a Brucker spectrometer operating at 100 MHz. All chemical shifts are reported in δ (ppm) using tetramethylsilane as an internal standard. The microanalyses were performed on a Perkin-Elmer 240C elemental analyzer.

General Procedure for the Synthesis of 2-Arylsulfonylaminomethyl-5-benzyl-1,3,4-oxadiazole (6a–c)/2-Arylsulfonylaminomethyl-5-phenylsulfonylmethyl-1,3,4-oxadiazole (7a–c)/2-Arylsulfonylaminomethyl-5-benzoylaminomethyl-1,3,4-oxadiazole (8a–c)/2-Arylsulfonylaminomethyl-5-phenylsulfonylaminomethyl-1,3,4-oxadiazole (9a–c)

A mixture of arylsulfonylaminoacetic acid hydrazide (1) (10 mmol), phenylacetic acid (2)/phenylsulfonylacetic acid (3)/benzoylaminoacetic acid (4)/phenylsulfonylaminoacetic acid (5) (10 mmol) and POCl₃ (7 mL) was heated under reflux for 5–8 h. The excess POCl₃ was removed under reduced pressure and the residue was poured onto crushed ice. The resulting precipitate was filtered, washed with saturated sodium bicarbonate solution and then with water. It was dried and recrystallized from ethanol.

2-Phenylsulfonylaminomethyl-5-benzyl-1,3,4-oxadiazole (6a): Pale yellow solid, yield 72%, mp 112–144°C. IR (KBr) cm⁻¹: 1135, 1329 (SO₂), 1589 (C=N), 3334 (NH). ¹H-NMR (CDCl₃) δ: 4.42 (s, 2H, CH₂-(C-5)), 4.86 (s, 2H, CH₂-(C-2)), 7.40–8.07 (m, 10H, Ar-H), 8.41 (br s, 1H, SO₂-NH). ¹³C-NMR (CDCl₃) δ: 31.9 (CH₂-(C-5)), 41.6 (CH₂-(C-2)), 168.4 (C-5), 188.2 (C-2), 124.8, 126.9, 128.5, 129.2, 137.2, 138.0, 143.7, 145.9 (aromatic carbons). MS m/z: 329.37 (M⁺). Anal. Calcd for C₁₆H₁₅N₃O₃S: C, 58.34; H, 4.59; N, 12.76. Found: C, 58.39; H, 4.62; N, 12.86.

2-(p-Methylphenylsulfonylaminomethyl)-5-benzyl-1,3,4-oxadiazole (6b): Pale yellow solid, yield 68%, mp 105–107°C. IR (KBr) cm⁻¹: 1138, 1318 (SO₂), 1593 (C=N), 3341 (NH). ¹H-NMR (CDCl₃) δ: 2.19 (s, 3H, Ar-CH₃), 4.36 (s, 2H, CH₂-(C-5)), 4.82 (s, 2H, CH₂-(C-2)), 7.31–8.05 (m, 9H, Ar-H), 8.37 (br s, 1H, SO₂-NH). ¹³C-NMR (CDCl₃) δ: 21.0 (Ar-CH₃), 31.1 (CH₂-(C-5)), 41.0 (CH₂-(C-2)), 167.8 (C-5), 187.0 (C-2), 124.4, 126.7, 128.4, 129.1, 136.7, 137.4, 143.3, 145.5 (aromatic carbons). MS m/z: 343.41 (M⁺). Anal. Calcd for C₁₇H₁₇N₃O₃S: C, 59.46; H, 4.99; N, 12.24. Found: C, 59.54; H, 5.04; N, 12.31.

2-(p-Chlorophenylsulfonylaminomethyl)-5-benzyl-1,3,4-oxadiazole (6c): Pale yellow solid, yield 73%, mp 127–129°C. IR (KBr) cm⁻¹: 1143, 1320 (SO₂), 1608 (C=N), 3343 (NH). ¹H-NMR (CDCl₃) δ: 4.51 (s, 2H, CH₂-(C-5)), 4.91 (s, 2H, CH₂-(C-2)), 7.41–8.11 (m, 9H, Ar-H), 8.47 (br s, 1H, SO₂-NH). ¹³C-NMR (CDCl₃) δ: 32.5 (CH₂-(C-5)), 42.1 (CH₂-(C-2)), 168.9 (C-5), 188.8 (C-2), 124.9, 127.2, 128.7, 129.8, 137.5, 139.2, 144.8, 146.1 (aromatic carbons). MS m/z: 363.82 (M⁺). Anal. Calcd for C₁₆H₁₄ClN₃O₃S: C, 52.82; H, 3.88; N, 11.55. Found: C, 52.89; H, 3.90; N, 11.68.

2-Phenylsulfonylaminomethyl-5-phenylsulfonylmethyl-1,3,4-oxadiazole (7a): Pale yellow solid, yield 75%, mp 151–153°C. IR (KBr) cm⁻¹: 1140, 1324 (SO₂), 1580 (C=N), 3339 (NH). ¹H-NMR (DMSO-d₆) δ: 4.55 (s, 2H, CH₂-(C-2)), 4.89 (s, 2H, CH₂-(C-5)), 6.90–7.81 (m, 10H, Ar-H), 8.46 (br s, 1H, SO₂-NH). ¹³C-NMR (DMSO-d₆) δ: 44.6 (CH₂-(C-2)), 59.9 (CH₂-(C-5)), 158.2 (C-5), 165.3 (C-2), 126.8, 128.3, 129.1, 129.9, 131.2, 133.8, 136.8, 144.3 (aromatic carbons). MS m/z: 393.44 (M⁺). Anal. Calcd for C₁₆H₁₅N₃O₅S₂: C, 48.84; H, 3.84; N, 10.68. Found: C, 48.80; H, 3.86; N, 10.60.

2-(p-Methylphenylsulfonylaminomethyl)-5-phenylsulfonylmethyl-1,3,4-oxadiazole (7b): Pale yellow solid, yield 70%, mp 140–142°C. IR (KBr) cm⁻¹: 1142, 1322 (SO₂), 1594 (C=N), 3346 (NH). ¹H-NMR (DMSO-d₆) δ: 2.23 (s, 3H, Ar-CH₃), 4.50 (s, 2H, CH₂-(C-2)), 4.80 (s, 2H, CH₂-(C-5)), 6.89–7.78 (m, 9H, Ar-H), 8.40 (br s, 1H, SO₂-NH). ¹³C-NMR (DMSO-d₆) δ: 21.2 (Ar-CH3), 44.2 (CH₂-(C-2)), 59.7 (CH₂-(C-5)), 157.9 (C-5), 164.9 (C-2), 126.7, 128.1, 128.8, 129.4, 130.6, 133.7, 136.2, 143.2 (aromatic carbons). MS m/z: 407.46 (M⁺). Anal. Calcd for C₁₇H₁₇N₃O₅S₂: C, 50.11; H, 4.21; N, 10.31. Found: C, 50.19; H, 4.24; N, 10.42.

2-(p-Chlorophenylsulfonylaminomethyl)-5-phenylsulfonylmethyl-1,3,4-oxadiazole (7c): Pale yellow solid, yield 66%, mp 169–170°C. IR (KBr) cm⁻¹: 1144, 1327 (SO₂), 1603 (C=N), 3344 (NH). ¹H-NMR (DMSO-d₆) δ: 4.61 (s, 2H, CH₂-(C-2)), 4.94 (s, 2H, CH₂-(C-5)), 6.93–7.84 (m, 9H, Ar-H), 8.51 (br s, 1H, SO₂-NH). ¹³C-NMR (DMSO-d₆) δ: 45.0 (CH₂-(C-2)), 60.2 (CH₂-(C-5)), 158.8 (C-5), 165.6 (C-2), 126.9, 128.6, 129.2, 130.1, 132.5, 134.6, 137.5, 144.5 (aromatic carbons). MS m/z: 427.88 (M⁺). Anal. Calcd for C₁₆H₁₄ClN₃O₅S₂: C, 44.91; H, 3.30; N, 9.82. Found: C, 44.87; H, 3.29; N, 9.89.

2-Phenylsulfonylaminomethyl-5-benzoylaminomethyl-1,3,4-oxadiazole (8a): Pale brown solid, yield 76%, mp 122–124°C. IR (KBr) cm⁻¹: 1132, 1334 (SO₂), 1582 (C=N), 1708 (CO), 3347 (NH). ¹H-NMR (DMSO-d₆) δ: 4.39 (s, 2H, CH₂-(C-2)), 4.87 (s, 2H, CH₂-(C-5)), 6.61–8.12 (m, 10H, Ar-H), 8.43 (br s, 1H, SO₂-NH), 10.15 (br s, 1H, CO–NH). ¹³C-NMR (DMSO-d₆) δ: 40.1 (CH₂-(C-2)), 47.2 (CH₂-(C-5)), 158.6 (C-5), 176.6 (C-2), 169.8 (CO), 127.6, 130.7, 133.5, 133.8, 134.8, 136.0, 136.9, 143.5 (aromatic carbons). MS m/z: 372.41 (M⁺). Anal. Calcd for C₁₇H₁₆N₄O₄S: C, 54.83; H, 4.33; N, 15.04. Found: C, 54.89; H, 4.35; N, 15.15.

2-(p-Methylphenylsulfonylaminomethyl)-5-benzoylaminomethyl-1,3,4-oxadiazole (8b): Pale brown solid, yield 69%, mp 101–103°C. IR (KBr) cm⁻¹: 1130, 1332 (SO₂), 1592 (C=N), 1710 (CO), 3342 (NH). ¹H-NMR (DMSO-d₆) δ: 2.19 (s, 3H, Ar-CH₃), 4.36 (s, 2H, CH₂-(C-2)), 4.82 (s, 2H, CH₂-(C-5)), 6.53–8.05 (m, 9H, Ar-H), 8.40 (br s, 1H, SO₂–NH), 10.10 (br s, 1H, CO–NH). ¹³C-NMR (DMSO-d₆) δ: 21.4 (Ar-CH₃), 39.9 (CH₂-(C-2)), 46.4 (CH₂-(C-5)), 158.4 (C-5), 176.4 (C-2), 169.2 (CO), 127.2, 130.0, 133.1, 133.6, 134.0, 135.3, 135.8, 142.9 (aromatic carbons). MS m/z: 386.42 (M⁺). Anal. Calcd for C₁₈H₁₈N₄O₄S: C, 55.95; H, 4.70; N, 14.50. Found: C, 56.14; H, 4.67; N, 14.42.

2-(p-Chlorophenylsulfonylaminomethyl)-5-benzoylaminomethyl-1,3,4-oxadiazole (8c): Pale brown solid, yield 68%, mp 140-142°C. IR (KBr) cm⁻¹: 1134, 1336 (SO₂), 1606 (C=N), 1714 (CO), 3334 (NH). ¹H-NMR (DMSO-d₆) δ: 4.42 (s, 2H, CH₂-(C-2)), 4.93 (s, 2H, CH₂-(C-5)), 6.65–8.16 (m, 9H, Ar-H), 8.46 (br s, 1H, SO₂–NH), 10.21 (br s, 1H, CO–NH). ¹³C-NMR (DMSO-d₆) δ: 40.6 (CH₂-(C-2)), 47.7 (CH₂-(C-5)), 159.2 (C-5), 177.0 (C-2), 170.2 (CO), 127.8, 131.1, 133.9, 134.5, 135.4, 137.7, 138.2, 145.2 (aromatic carbons). MS m/z: 406.84 (M⁺). Anal. Calcd for C₁₇H₁₅ClN₄O₄S: C, 50.19; H, 3.72; N, 13.77. Found: C, 50.26; H, 3.73; N, 13.71.

2-Phenylsulfonylaminomethyl-5-phenylsulfonylaminomethyl-1,3,4-oxadiazole (9a): Pale yellow solid, yield 74%, mp 172–174°C. IR (KBr) cm⁻¹: 1138, 1321 (SO₂), 1585 (C=N), 3343 (NH). ¹H-NMR (CDCl₃) δ: 4.48 (s, 4H, CH₂), 7.33–7.99 (m, 10H, Ar-H), 8.48 (br s, 2H, SO₂–NH). ¹³C-NMR (CDCl₃) δ: 47.1 (CH₂), 158.0 (C-5), 177.9 (C-2), 126.6, 128.1, 130.3, 132.7, 134.5, 136.0, 137.9, 139.5 (aromatic carbons). MS m/z: 408.46 (M⁺). Anal. Calcd for C₁₆H₁₆N₄O₅S₂: C, 47.05; H, 3.95; N, 13.72. Found: C, 47.09; H, 3.92; N, 13.84.

2-(p-Methylphenylsulfonylaminomethyl)-5-phenylsulfonylaminomethyl-1,3,4-oxadiazole (9b): Pale yellow solid, yield 77%, mp 196–198°C. IR (KBr) cm⁻¹: 1133, 1319 (SO₂), 1591 (C=N), 3340 (NH). ¹H-NMR (CDCl₃) δ: 2.35 (s, 3H, Ar-CH₃), 4.42 (s, 4H, CH₂), 7.30–7.94 (m, 9H, Ar-H), 8.44 (br s, 2H, SO₂–NH). ¹³C-NMR (CDCl₃) δ: 21.9 (Ar-CH₃), 46.6 (CH₂), 157.4 (C-5), 177.4 (C-2), 126.2, 128.0, 130.1, 132.6, 133.0, 133.8, 134.8, 139.1 (aromatic carbons). MS m/z: 422.48 (M⁺). Anal. Calcd for C₁₇H₁₈N₄O₅S₂: C, 48.33; H, 4.29; N, 13.26. Found: C, 48.28; H, 4.31; N, 13.36.

2-(p-Chlorophenylsulfonylaminomethyl)-5-phenylsulfonylaminomethyl-1,3,4-oxadiazole (9c): Pale yellow solid, yield 73%, mp 221–223°C. IR (KBr) cm⁻¹: 1140, 1326 (SO₂), 1605 (C=N), 3346 (NH). ¹H-NMR (CDCl₃) δ: 4.51 (s, 4H, CH₂), 7.35–8.00 (m, 9H, Ar-H), 8.52 (br s, 2H, SO₂–NH). ¹³C-NMR (CDCl₃) δ: 47.6 (CH₂), 158.6 (C-5), 178.3 (C-2), 126.8, 128.4, 130.6, 133.2, 134.9, 136.7, 138.0, 139.8 (aromatic carbons). MS m/z: 442.90 (M⁺). Anal. Calcd for C₁₆H₁₅ClN₄O₅S₂: C, 43.39; H, 3.41; N, 12.65. Found: C, 43.48; H, 3.46; N, 12.76.

General Procedure for the Synthesis of 2-Arylsulfonylaminomethyl-5-benzyl-1,3,4-thiadiazole (10a–c)/2-Arylsulfonylaminomethyl-5-phenylsulfonylmethyl-1,3,4-thiadiazole (11a–c)/2-Arylsulfonylaminomethyl-5-benzoylaminomethyl-1,3,4-thiadiazole (12a–c)/2-Arylsulfonylaminomethyl-5-phenylsulfonylaminomethyl-1,3,4-thiadiazole (13a–c)

In a sealed test tube, a mixture of compounds 6–9 (5 mmol), thiourea (20 mmol) and tetrahydrofuran (5 mL) were taken and heated at 120–150°C in an oil bath for 20–26 h. After the reaction was completed, it was extracted with dichloromethane. The organic layer was washed with water, brine solution and dried over anhydrous Na₂SO_4. The solvent was removed under reduced pressure and the resultant solid was recrystallized from methanol.

2-Phenylsulfonylaminomethyl-5-benzyl-1,3,4-thiadiazole (10a): Pale yellow solid, yield 72%, mp 133–135°C. IR (KBr) cm⁻¹: 1133, 1325 (SO₂), 1585 (C=N), 3337 (NH). ¹H-NMR (CDCl₃) δ: 4.63 (s, 2H, CH₂-(C-5)), 5.07 (s, 2H, CH₂-(C-2)), 7.60–8.27 (m, 10H, Ar-H), 8.61 (br s, 1H, SO₂–NH). ¹³C-NMR (CDCl₃) δ: 33.7 (CH₂-(C-5)), 43.9 (CH₂-(C-2)), 170.2 (C-5), 190.1 (C-2), 126.8, 128.7, 130.5, 131.0, 139.2, 140.2, 145.5, 147.9 (aromatic carbons). MS m/z: 345.44 (M⁺). Anal. Calcd for C₁₆H₁₅N₃O₂S₂: C, 55.63; H, 4.38; N, 12.16. Found: C, 55.73; H, 4.36; N, 12.24.

2-(p-Methylphenylsulfonylaminomethyl)-5-benzyl-1,3,4-thiadiazole (10b): Pale yellow solid, yield 68%, mp 147–149°C. IR (KBr) cm⁻¹: 1136, 1315 (SO₂), 1587 (C=N), 3342 (NH). ¹H-NMR (CDCl₃) δ: 2.40 (s, 3H, Ar-CH₃), 4.57 (s, 2H, CH₂-(C-5)), 5.02 (s, 2H, CH₂-(C-2)), 7.51–8.19 (m, 9H, Ar-H), 8.57 (br s, 1H, SO₂–NH). ¹³C-NMR (CDCl₃) δ: 22.8 (Ar-CH₃), 33.5 (CH₂-(C-5)), 43.8 (CH₂-(C-2)), 169.7 (C-5), 188.8 (C-2), 126.2, 128.6, 130.4, 131.2, 138.9, 139.7, 145.7, 147.3 (aromatic carbons). MS m/z: 359.47 (M⁺). Anal. Calcd for C₁₇H₁₇N₃O₂S₂: C, 56.80; H, 4.77; N, 11.69. Found: C, 56.86; H, 4.72; N, 11.79.

2-(p-Chlorophenylsulfonylaminomethyl)-5-benzyl-1,3,4-thiadiazole (10c): Pale yellow solid, yield 75%, mp 155–157°C. IR (KBr) cm⁻¹: 1141, 1319 (SO₂), 1604 (C=N), 3345 (NH). ¹H-NMR (CDCl₃) δ: 4.72 (s, 2H, CH₂-(C-5)), 5.13 (s, 2H, CH₂-(C-2)), 7.62–8.31 (m, 9H, Ar-H), 8.68 (br s, 1H, SO₂–NH). ¹³C-NMR (CDCl₃) δ: 34.5 (CH₂-(C-5)), 44.0 (CH₂-(C-2)), 170.7 (C-5), 190.7 (C-2), 126.7, 129.1, 130.7, 131.9, 139.7, 141.5, 147.2, 147.9 (aromatic carbons). MS m/z: 379.88 (M⁺). Anal. Calcd for C₁₆H₁₄ClN₃O₂S₂: C, 50.59; H, 3.71; N, 11.06. Found: C, 50.70; H, 3.74; N, 11.20.

2-Phenylsulfonylaminomethyl-5-phenylsulfonylmethyl-1,3,4-thiadiazole (11a): Pale yellow solid, yield 69%, mp 165–167°C. IR (KBr) cm⁻¹: 1143, 1320 (SO₂), 1570 (C=N), 3332 (NH). ¹H-NMR (DMSO-d₆) δ: 4.75 (s, 2H, CH₂-(C-2)), 5.10 (s, 2H, CH₂-(C-5)), 7.10–7.91 (m, 10H, Ar-H), 8.67 (br s, 1H, SO₂–NH). ¹³C-NMR (DMSO-d₆) δ: 46.4 (CH₂-(C-2)), 61.7 (CH₂-(C-5)), 160.0 (C-5), 167.1 (C-2), 128.6, 130.2, 131.1, 132.0, 133.4, 136.1, 139.2, 146.1 (aromatic carbons). MS m/z: 409.51 (M⁺). Anal. Calcd for C₁₆H₁₅N₃O₄S₃: C, 46.93; H, 3.69; N, 10.26. Found: C, 47.06; H, 3.68; N, 10.37.

2-(p-Methylphenylsulfonylaminomethyl)-5-phenylsulfonylmethyl-1,3,4-thiadiazole (11b): Pale yellow solid, yield 67%, mp 157–159°C. IR (KBr) cm⁻¹: 1128, 1316 (SO₂), 1575 (C=N), 3335 (NH). ¹H-NMR (DMSO-d₆) δ: 2.44 (s, 3H, Ar-CH₃), 4.72 (s, 2H, CH₂-(C-2)), 5.01 (s, 2H, CH₂-(C-5)), 7.05–7.98 (m, 9H, Ar-H), 8.60 (br s, 1H, SO₂–NH). ¹³C-NMR (DMSO-d₆) δ: 23.6 (Ar-CH₃), 46.0 (CH₂-(C-2)), 61.5 (CH₂-(C-5)), 159.7 (C-5), 166.7 (C-2), 128.5, 130.0, 130.8, 131.5, 132.8, 136.0, 138.6, 145.0 (aromatic carbons). MS m/z: 423.53 (M⁺). Anal. Calcd for C₁₇H₁₇N₃O₄S₃: C, 48.21; H, 4.05; N, 9.92. Found: C, 48.30; H, 4.09; N, 9.86.

2-(p-Chlorophenylsulfonylaminomethyl)-5-phenylsulfonylmethyl-1,3,4-thiadiazole (11c): Pale yellow solid, yield 76%, mp 187–189°C. IR (KBr) cm⁻¹: 1135, 1323 (SO₂), 1589 (C=N), 3338 (NH). ¹H-NMR (DMSO-d₆) δ: 4.82 (s, 2H, CH₂-(C-2)), 5.14 (s, 2H, CH₂-(C-5)), 7.15–7.93 (m, 9H, Ar-H), 8.72 (br s, 1H, SO₂-NH). ¹³C-NMR (DMSO-d₆) δ: 46.8 (CH₂-(C-2)), 62.0 (CH₂-(C-5)), 160.9 (C-5), 167.4 (C-2), 128.7, 130.5, 131.2, 132.2, 134.7, 136.9, 139.9, 146.3 (aromatic carbons). MS m/z: 443.95 (M⁺). Anal. Calcd for C₁₆H₁₄ClN₃O₄S₃: C, 43.29; H, 3.18; N, 9.47. Found: C, 43.25; H, 3.15; N, 9.56.

2-Phenylsulfonylaminomethyl-5-benzoylaminomethyl-1,3,4-thiadiazole (12a): Pale brown solid, yield 78%, mp 153–155°C. IR (KBr) cm⁻¹: 1137, 1316 (SO₂), 1593 (C=N), 1705 (CO), 3341(NH). ¹H-NMR (DMSO-d₆) δ: 4.59 (s, 2H, CH₂-(C-2)), 5.08 (s, 2H, CH₂-(C-5)), 6.81–8.32 (m, 10H, Ar-H), 8.62 (br s, 1H, SO₂-NH), 10.25 (br s, 1H, CO-NH). ¹³C-NMR (DMSO-d₆) δ: 41.9 (CH₂-(C-2)), 49.2 (CH₂-(C-5)), 160.3 (C-5), 178.8 (C-2), 171.6 (CO), 130.0, 132.4, 135.5, 136.0, 136.8, 137.9, 138.7, 145.3 (aromatic carbons). MS m/z: 388.46 (M⁺). Anal. Calcd for C₁₇H₁₆N₄O₃S₂: C, 52.56; H, 4.15; N, 14.42. Found: C, 52.63; H, 4.16; N, 14.49.

2-(p-Methylphenylsulfonylaminomethyl)-5-benzoylaminomethyl-1,3,4-thiadiazole (12b): Pale brown solid, yield 75%, mp 166–168°C. IR (KBr) cm⁻¹: 1140, 1318 (SO₂), 1608 (C=N), 1715 (CO), 3343 (NH). ¹H-NMR (DMSO-d₆) δ: 2.40 (s, 3H, Ar-CH₃), 4.56 (s, 2H, CH₂-(C-2)), 5.02 (s, 2H, CH₂-(C-5)), 6.73–8.25 (m, 9H, Ar-H), 8.59 (br s, 1H, SO₂–NH), 10.20 (br s, 1H, CO–NH). ¹³C-NMR (DMSO-d₆) δ: 23.2 (Ar-CH₃), 41.7 (CH₂-(C-2)), 48.4 (CH₂-(C-5)), 160.2 (C-5), 178.2 (C-2), 171.1 (CO), 129.6, 132.3, 135.3, 135.7, 136.0, 137.2, 137.6, 144.7 (aromatic carbons). MS m/z: 402.49 (M⁺). Anal. Calcd for C₁₈H₁₈N₄O₃S₂: C, 53.71; H, 4.51; N, 13.92. Found: C, 53.83; H, 4.49; N, 14.03.

2-(p-Chlorophenylsulfonylaminomethyl)-5-benzoylaminomethyl-1,3,4-thiadiazole (12c): Pale brown solid, yield 77%, mp 172–174°C. IR (KBr) cm⁻¹: 1143, 1320 (SO₂), 1580 (C=N), 1720 (CO), 3339 (NH). ¹H-NMR (DMSO-d₆) δ: 4.64 (s, 2H, CH₂-(C-2)), 5.15 (s, 2H, CH₂-(C-5)), 6.89–8.36 (m, 9H, Ar-H), 8.67 (br s, 1H, SO₂–NH), 10.27 (br s, 1H, CO–NH). ¹³C-NMR (DMSO-d₆) δ: 42.8 (CH₂-(C-2)), 49.9 (CH₂-(C-5)), 161.2 (C-5), 179.2 (C-2), 172.5 (CO), 130.2, 133.4, 135.8, 136.6, 137.4, 139.6, 140.0, 147.1 (aromatic carbons). MS m/z: 422.91 (M⁺). Anal. Calcd for C₁₇H₁₅ClN₄O₃S₂: C, 48.28; H, 3.58; N, 13.25. Found: C, 48.18; H, 3.63; N, 13.33.

2-Phenylsulfonylaminomethyl-5-phenylsulfonylaminomethyl-1,3,4-thiadiazole (13a): Pale yellow solid, yield 72%, mp 185–187°C. IR (KBr) cm⁻¹: 1136, 1317 (SO₂), 1586 (C=N), 3342 (NH). ¹H-NMR (CDCl₃) δ: 4.69 (s, 4H, CH₂), 7.53–8.20 (m, 10H, Ar-H), 8.69 (br s, 2H, SO₂–NH). ¹³C-NMR (CDCl₃) δ: 49.5 (CH₂), 160.1 (C-5), 179.6 (C-2), 128.4, 130.0, 132.3, 134.8, 136.7, 138.3, 140.3, 141.3 (aromatic carbons). MS m/z: 424.52 (M⁺). Anal. Calcd for C₁₆H₁₆N₄O₄S₃: C, 45.27; H, 3.80; N, 13.20. Found: C, 45.34; H, 3.77; N, 13.30.

2-(p-Methylphenylsulfonylaminomethyl)-5-phenylsulfonylaminomethyl-1,3,4-thiadiazole (13b): Pale yellow solid, yield 75%, mp 216–218°C. IR (KBr) cm⁻¹: 1142, 1319 (SO₂), 1595 (C=N), 3340 (NH). ¹H-NMR (CDCl₃) δ: 2.57 (s, 3H, Ar-CH₃), 4.63 (s, 4H, CH₂), 7.50–8.14 (m, 9H, Ar-H), 8.64 (br s, 2H, SO₂–NH). ¹³C-NMR (CDCl₃) δ: 24.1 (Ar-CH₃), 48.7 (CH₂), 159.5 (C-5), 179.6 (C-2), 128.0, 129.9, 132.1, 134.7, 135.2, 136.1, 137.2, 140.9 (aromatic carbons). MS m/z: 438.55 (M⁺). Anal. Calcd for C₁₇H₁₈N₄O₄S₃: C, 46.56; H, 4.14; N, 12.78. Found: C, 46.50; H, 4.13; N, 12.86.

2-(p-Chlorophenylsulfonylaminomethyl)-5-phenylsulfonylaminomethyl-1,3,4-thiadiazole (13c): Pale yellow solid, yield 78%, mp 238–240°C. IR (KBr) cm⁻¹: 1145, 1322 (SO₂), 1602 (C=N), 3345 (NH). ¹H-NMR (CDCl₃) δ: 4.76 (s, 4H, CH₂), 7.57–8.24 (m, 9H, Ar-H), 8.74 (br s, 2H, SO₂–NH). ¹³C-NMR (CDCl₃) δ: 49.6 (CH₂), 160.4 (C-5), 180.4 (C-2), 128.6, 130.3, 132.6, 135.3, 137.1, 139.0, 140.4, 141.6 (aromatic carbons). MS m/z: 458.97 (M⁺). Anal. Calcd for C₁₆H₁₅ClN₄O₄S₃: C, 41.87; H, 3.29; N, 12.21. Found: C, 41.95; H, 3.23; N, 12.34.

Antioxidant Activity

The compounds 6–13 were tested for antioxidant property by 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO) and hydrogen peroxide (H₂O₂) methods at three different concentrations 50, 75 and 100 µg/mL.

DPPH Radical Scavenging Activity

The hydrogen atom or electron donation ability of the compounds was measured from the bleaching of the purple colored methanol solution of DPPH radical. The spectrophotometric assay uses the stable radical DPPH as a reagent. To 4 mL of 0.004% (w/v) methanol solution of DPPH, 1 mL of various concentrations of the test compounds (50, 75, 100 µg/mL) in methanol were added. After a 30 min incubation period at room temperature, the absorbance was read against blank at 517 nm. Ascorbic acid was used as the standard. The percent of inhibition (I %) of free radical production from DPPH was calculated by the following equation

  

where A_control is the absorbance of the control reaction (containing methanolic DPPH and ascorbic acid), A_sample is the absorbance of the test compound (containing methanolic DPPH and test compound) and A_blank is the absorbance of the blank (containing only methanolic DPPH). Tests were carried out in triplicate.

NO Scavenging Activity

Nitric oxide scavenging activity was measured by slightly modified methods of Green and Marcocci et al. NO radicals were generated from sodium nitroprusside. One mL of sodium nitroprusside (10 mm) and 1.5 mL of phosphate buffer saline (0.2 m, pH 7.4) were added to different concentrations (50, 75, 100 µg/mL) of the test compounds and incubated for 150 min at 25°C. After incubation 1 mL of the reaction mixture was treated with 1 mL of Griess reagent (1% sulfanilamide, 2% H₃PO₄ sand 0.1% naphthylethylenediamine dihydrochloride). The absorbance of the chromatophore was measured at 546 nm. Ascorbic acid was used as standard. NO scavenging activity was calculated by the following equation

  

where A_control is the absorbance of the control reaction (containing all reagents and Ascorbic acid), A_sample is the absorbance of the test compound (containing all reagents and test compound) and A_blank is the absorbance of the blank (containing only reagents). Tests were carried out in triplicate.

H₂O₂ Scavenging Activity

The H₂O₂ scavenging ability of the test compound was determined according to the method of Ruch et al. A solution of H₂O₂ (40 mm) was prepared in phosphate buffer (pH 7.4). 50, 75 and 100 µg/mL concentrations of the test compounds in 3.4 mL phosphate buffer were added to H₂O₂ solution (0.6 mL, 40 mm). The absorbance value of the reaction mixture was recorded at 230 nm. The percent of scavenging of H₂O₂ was calculated by the following equation

  

where A_control is the absorbance of the control reaction (containing all reagents and ascorbic acid), A_sample is the absorbance of the test compound (containing all reagents and test compound) and A_blank is the absorbance of the blank (containing only reagents). Tests were carried out in triplicate.

Acknowledgment

The authors are grateful to DST, New Delhi, for financial assistance under major research project.

References

• 1)  Jha K. K., Samad A., Kumar Y., Shaharyar M., Khosa R. L., Jain J., Kumar V., Singh P., Eur. J. Med. Chem., 45, 4963–4967 (2010).
• 2)  Singh P., Jangra P. K., Der Chemica Sinica, 1, 118–123 (2010).
• 3)  Omar F. A., Mahfouz N. M., Rahman M. A., Eur. J. Med. Chem., 31, 819–825 (1996).
• 4)  Leung D., Du W., Hardouin C., Cheng H., Hwang I., Cravatt B. F., Boger D. L., Bioorg. Med. Chem. Lett., 15, 1423–1428 (2005).
• 5)  Elson P. J., Kvols L. K., Vogl S. E., Glover D. J., Hahn R. G., Trump D. L., Carbone P. P., Earle J. D., Davis T. E., Invest. New Drugs, 6, 97–103 (1988).
• 6)  Matysiak J., Eur. J. Med. Chem., 42, 940–947 (2007).
• 7)  Zou X. J., Zhang S. W., Liu Y., Liu Z. M., Gao J. W., Song Q. L., Pan Y., Zhang J. Z., Lix J., Chin. J. Struct. Chem., 30, 1001–1005 (2011).
• 8)  Du H. T., Du H. J., Chin. J. Org. Chem., 30, 137–141 (2010).
• 9)  Liu F., Luo X. Q., Song B. A., Bhadury P. S., Yang S., Jin L. H., Xue W., Hu D. Y., Bioorg. Med. Chem., 16, 3632–3640 (2008).
• 10)  Drews J., Science, 287, 1960–1964 (2000).
• 11)  Boyd A. E. 3rd, Diabetes, 37, 847–850 (1988).
• 12)  Thornber C. W., Chem. Soc. Rev., 8, 563–580 (1979).
• 13)  Scozzafava A., Owa T., Mastrolorenzo A., Supuran C. T., Curr. Med. Chem., 10, 925–953 (2003).
• 14)  Supuran C. T., Scozzafava A., Casini A., Med. Res. Rev., 23, 146–189 (2003).
• 15)  Casini A., Scozzafava A., Mastrolorenzo A., Supuran L. T., Curr. Cancer Drug Targets, 2, 55–75 (2002).
• 16)  Supuran C. T., Scozzafava A., Casini A., Med. Res. Rev., 23, 146–189 (2003).
• 17)  Kivelä A. J., Kivelä J., Saarnio J., Parkkila S., World J. Gastroenterol., 11, 155–163 (2005).
• 18)  Supuran C. T., Scozzafava A., Bioorg. Med. Chem., 15, 4336–4350 (2007).
• 19)  Supuran C. T., Nat. Rev. Drug Discov., 7, 168–181 (2008).
• 20)  Kenney W. J., Walsh J. A., Davenport D. A., J. Am. Chem. Soc., 83, 4019–4022 (1961).
• 21)  Tian B., He M., Tan Z., Tang S., Hewlett I., Chen S., Jin Y., Yang M., Chem. Biol. Drug Des., 77, 189–198 (2011).
• 22)  Wang P., Naduthambi D., Mosley R. T., Niu C., Furman P. A., Otto M. J., Sofia M. J., Bioorg. Med. Chem. Lett., 21, 4642–4647 (2011).
• 23)  Cuendet M., Hostettmann K., Potterat O., Dyatmiko W., Helv. Chim. Acta, 80, 1144–1152 (1997).
• 24)  Burits M., Bucar F., Phytother. Res., 14, 323–328 (2000).
• 25)  Green L. C., Wagner D. A., Glogowski J., Skipper P. L., Wishnok J. S., Tannenbaum S. R., Anal. Biochem., 126, 131–138 (1982).
• 26)  Marcocci L., Maguire J. J., Droy-Lefaix M. T., Packer L., Biochem. Biophys. Res. Commun., 201, 748–755 (1994).
• 27)  Ruch R. J., Cheng S. J., Klaunig J. E., Carcinogenesis, 10, 1003–1008 (1989).