Antimicrobial and Pharmacological Studies of some Newly Synthesized Aryl Sulfonamide Derivatives

Introduction

Sulfonamide and their derivatives are used in medicine due to their pharmacological properties such as antibacterial activity^(1,2). Oxadiazole derivatives belong to an important group of heterocyclic compounds that have  a vital role in medicinal chemistry such as antibacterial^(3,4), antifungal^(4,5), anti- inflammatory⁽⁶⁾ and analgesic^(7,8). Moreover pyrrole and pyrazole derivatives bearing biologically active sulfonamide moiety were reported to exhibit antibacterial and antifungal activities⁽⁹⁾. Also pyrazole derivatives have been reported as analgesic and ant-inflammatory⁽¹⁰⁾. Quinoline and its derivatrives have attracted much more attention as an important class of heterocyclic compounds in the field of medicine and pharmaceutical as antibacterial and  antifungal⁽¹¹⁾, analgesic⁽¹²⁾ and anti-inflammatory^(13,14). In this work novel aryl sulfonamide derivatives containing different heterocyclic moieties as pyrrole, pyrazole, 1,3,4-oxadiazole and quinoline derivatives were synthesized hopping to produce potent antibacterial, antifungal ,analgesic and anti-inflammatory with no or less tendency to evoke gastric ulceration.

Results and Discussion

Chemistry

The preparation of the new compounds is outlined in schemes 1&2. The starting compound 4-tosylaminobenzohydrazide 4 was prepared via reaction of ethyl4-tosylaminobenzoate with hydrazine hydrate 99% under reflux as reported method ^(15). The hydrazide 4 was reacted with 2,5-hexanedione in glacial acetic acid to give compound 5.  Refluxing the hydrazide 4 with acetylacetone, benzoyl acetone and ethyl acetoacetate in ethanol containing drops of acetic acid gave compounds 6a, b and 7, respectively. Hydrazone 8 was prepared via refluxing the hydrazide 4 with 3,4,5-trimethoxybenzaldehyde in ethanol. Moreover stirring the hydrazide 4 with p-chlorobenzoyl chloride in glacial acetic acid yielded compound 9.

Compounds 10a-c were prepared by refluxing hydrazide 4 with maleic anhydride, dichloromaleic anhydride and phthalic anhydride in glacial acetic acid, respectively.

Scheme 1 Click here to View scheme

 

Scheme 2 Click here to View scheme

 

Table 1: Antimicrobial activity of synthesized compounds by cup plate diffusion method

Samples	Diameter (mm) of inhibition zones against the corresponding standard strains of different microorganisms
	Gram-positive bacteria			Gram-negative bacteria				Fungi
	Staphylococcus aureus ATCC6538	Staphylococcus epidermidiss ATCC12228	Micrococcus spp. ATCC10240	Pseudomonas aeruginosae ATCC9027	Klebsiella pneumoniae ATCC27736	Salmonella typhimurium ATCC14028	Escherichia coli ATCC10536	Aspragillus niger ATCC16404	Candida albicans ATCC10231
5	15	14	19	17	18	18	19	24	22
6a	15	14	17	18	19	19	20	22	21
6b	17	16	19	17	17	17	18	25	24
7	14	14	16	17	18	18	19	18	16
8	22	21	23	18	19	19	20	22	22
9	18	17	20	18	19	19	20	22	22
10a	17	16	19	18	18	18	19	22	21
10b	21	20	23	17	18	18	19	24	23
11	20	19	22	18	19	19	20	23	22
12a	18	17	20	17	18	18	19	23	22
12b	15	15	17	18	19	19	20	19	17
12c	20	19	22	17	17	17	17	21	19
12d	19	18	20	17	18	18	19	22	21
12f	17	16	19	18	18	18	19	22	21
12g	17	16	19	18	18	18	20	26	25
12h	18	18	20	18	19	19	20	24	23
13	19	19	20	20	21	21	22	23	22
14a	15	14	17	19	19	19	20	20	18
14b	16	15	17	18	18	18	20	21	19
15a	15	15	17	18	19	19	20	18	16
Cefotaxime (Refrence)	30	28	35	30	32	33	35	–	–
Sulphamethoxazole (Refrence)	25	23	30	25	27	28	30	–	–
Nuystatin (Refrence)	–	–	–	–	–	–	–	25	20
DMF (control)	–	–	–	–	–	–	–	–	–

• · The method used is cup plate diffusion method                               · Each cup is filled with 100 micro liter from each sample
• · Conc. Of each sample is 50 mg/ml                                       · Conc. of antimicrobial agents is 5 mg/ml

Table 2: Analgesic activity evaluation. of compounds 5, 6a,b, 7, 8, 10a, 11, 12b-f, 13, 14a,b and 15a.

Group	Reaction time in seconds after
Mean±SEM	10 min.	20 min.	30 min	60 min.	90 min.	120 min.
Control	13.4±0.2	13.8±0.3	15.4±0.4*	15.2±0.3*	15.8±0.3*	12.8±2.9
Celecoxibe	28.8±1.2#	30.4±1.4#	32.6±1.6#	33.4±1.5*#	34±1.1*#	33.8±0.8*#
Diclofenac (Na.) Voltarin	31.2±1.4#	32±1.3#	33.2±1.4#	34.2±1.3#	34.2±0.8#	34.4±0.8#
5	13.6±0.5	13.8±0.5	12.8±0.5#	13.8±0.4	13.8±0.7#	14.4±0.4
6 a	12.4±0.2#	13±0.3	13.8±0.3*#	13.8±0.3*#	12.8±0.5#	13.4±0.6
6 b	14±0.7	12.2±1.9	13.6±0.4#	14±0.3#	13.4±0.6#	14±0.6
7	12.6±0.2#	14.2±0.3*	13.2±0.5#	13.8±0.5	13.4±0.6#	14.2±0.755
8	18±0.5*#	19.6±0.4*#	21.8±0.3*#	23±0.5*#	23.6±0.4*#	24.4±0.4*#
10 a	18.6±0.6*#	23.4±1.1*#	23.8±0.9*#	24.8±1*#	24.8±0.8*#	25.8±0.5*#
11	37±0.8#	37.4±0.7#	36.2±0.5#	36.6±0.6#	36±0.4#	36.2±1.4#
12 b	11.8±0.3#	12.2±0.7	13.8±0.5	14±0.4*	13.4±0.87#	14±0.7*
12 c	12.8±0.3	13.8±0.5	14±0.6	13.6±0.6	13.8±0.5#	13.6±0.6
12 d	13.8±0.5	15.2±1.5	14±0.4#	15.4±0.2*	15±0.3	15.2±0.6
12 f	13.8±0.5	13.8±0.5	13.8±0.5	13±0.5#	14±0.6#	16±0.4*
12 g	13.8±0.6	14±0.5	14.8±0.4	14±0.7	13±0.7	12.4±0.7
13	18.2±1.3#	20.8±0.7#	22±0.5*#	23.8±0.3*#	23.8±0.4*#	21.4±0.2*#
14 a	16±0.5#	15±0.8	15.6±0.5	17.4±0.5#	14.2±0.4#	14.6±0.4
14 b	19.8±0.8#	21±0.7#	22±0.8#	23±0.8*#	23.6±0.6*#	20.6±0.4#
15 a	16±0.6#	15.4±0.2#	15.4±0.5	14.4±0.2*	14±0.6#	14±0.4*

*Significantly different from 10 min.time point in the same group P<0.05

# Significantly different from the same time point in control group<0.05.

Figure 1 Click here to View figure

 

Table 3: Anti-inflammatory activity of compounds 5, 6a, b, 7, 8, 10a, 11, 12b-f, 13, 14a,b and 15a.

Time	Thickness
Mean±SEM	Zero time	1st hr	2nd hr	3rd hr	4th hr
Control	0.31±0.01	0.78±0.04*	0.89±0.03*	1.16±0.02*	1.4±0.04*
Celecoxib	0.31±0.01	0.38±0.01*#	0.47±0.01*#	0.53±0.03*#	0.63±0.02*#
Volatrin	0.31±0.01	0.43±0.03*#	0.54±0.03*#	0.57±0.02*#	0.71±0.03*#
5	0.31±0.01	0.57±0.02*#	0.65±0.02*#	0.69±0.03*#	0.77±0.03*#
6a	0.31±0.01	0.71±0.04*	0.85±0.02*	1.14±0.02*	1.24±0.02*#
6b	0.31±0.01	0.75±0.02*	0.80±0.03*	0.98±0.06*#	1.24±0.02*#
7	0.31±0.01	0.82±0.06*	0.90±0.04*	1.16±0.02*	1.26±0.06*#
8	0.31±0.01	0.86±0.02*	1.03±0.06*	1.10±0.05*	1.20±0.03*
10a	0.31±0.01	0.62±0.02*#	0.82±0.02*	0.85±0.02*#	0.92±0.02*#
11	0.31±0.01	0.48±0.03*#	0.47±0.01*#	0.53±0.03*#	0.64±0.02*#
12b	0.31±0.01	0.80±0.04*	0.88±0.02*	1.1±0.05*	1.3±0.04*
12c	0.31±0.01	0.75±0.02*	0.94±0.02*	1.16±0.02*	1.4±0.03*
12d	0.31±0.01	0.60±0.03*#	0.63±0.03*#	0.72±0.03*#	0.79±0.01*#
12f	0.31±0.01	0.55±0.02*#	0.63±0.02*#	0.71±0.04*#	0.72±0.03*#
12g	0.31±0.01	0.55±0.03*#	0.61±0.01*#	0.65±0.03*#	0.72±0.02*#
13	0.31±0.01	0.78±0.02*	0.94±0.02*#	1.16±0.02*	1.36±0.04*
14a	0.31±0.01	0.61±0.02*#	0.70±0.02*#	0.77±0.01*#	0.86±0.02*#
14b	0.31±0.01	0.84±0.04*	0.88±0.02*	1.20±0.03*	1.34±0.04*
15a	0.31±0.02	0.47±0.03*#	0.55±0.04*#	0.61±0.05*#	0.85±0.02*#

*significantly different from zero time point in the same group P<0.05

# significantly different from the same time point in control group P<0.05.

 

Figure 2 Click here to View figure

 

Table 4: Ulcerogenic activity of compounds 5, ,8,10a,11,12d-g,13,14a,b and 15a.

Group	score
Mean ± SEM
Control	0.00 ± 0.00
Celecoxib	0.00 ± 0.00
Diclofenac	4.60 ± 0.24 #
5	0.20 ± 0.20
8	0.00 ± 0.00
10a	0.20 ± 0.20
11	0.20 ± 0.20
12d	2.20 ± 0.37 #
12f	0.20 ± 0.20
12g	0.40 ± 0.24
13	0.20 ± 0.20
14a	0.40 ± 0.24
14b	0.40 ± 0.24
15a	0.40 ± 0.24

# significantly different from the control group p< 0.05.

 

Figure 3 Click here to View figure

A- Chemistry

Melting points were determined with a Gallen Kamp melting point apparatus and are uncorrected. IR spectra (KBr, cm^-1) were recorded on Bruker or Testscan Shimadzu FT 8000 spectrometers. ¹HNMR ,C¹³NMR spectra were recorded on Varian Gemini 200, 200 MH_z, Varian Mercury (300 MH_z) using DMSO-d₆ or CDCl₃ as solvent and (TMS) as internal stander (chemical shift in d, ppm). Electron impact mass spectra were determined using a GC/MS Mat112 S at 70 eV spectrometer. Elemental analysis were determined using the Elemental analyzer, Heraeus and Automatic Elemental analyzer, Model 2400 Perkin Elemer at Microanalytical Center, Faculty of Science, University of Cairo, Egypt. Thin layer chromatography (TLC) was performed on Silica gel G for TLC (Merck) and spots were visualized by iodine vapors or by irradiation with ulteraviolet light (uv; 254 nm). Compounds 3, 4 were prepared according to reported procedures. ⁽¹⁵⁾ Compound (3) m.p    198-200C^o,    yield 95% as reported IR (KBr, cm^-1): 3216 (NH), 3062 (CH, aromatic), 2987, 2937 (CH, aliphatic), 1691 (C=O ester), 1604 (C=N), 1508 (C=C) and 1303 (SO₂). Compound (4) m.p. 279-280°C, yield 90% as reported. Microanalysis for C₁₄H₁₅N₃O₃S (305) Calcd%, C, 55.08; H, 4.9; N, 13.77, Found % C, 54.93; H, 4.59; N, 13.76. IR (KBr, cm^-1): 3322 (NH₂), 3149 (NH), 3048 (CH, aromatic), 2940 (CH, aliphatic), 1650 (C=O), 1608 (C=N), 1579 (C=C), 1342 (SO_2­). Ms: m/z (%) 305 (5.21) M⁺, 290 (0.98), 274 (100), 182 (2.39), 155 (12), 139 (9.20), 119 (33.96), 91 (75.80), 65 (22.98).

N-(2, 5-Dimethyl-1H-pyrrol-1-yl) 4-tosylaminobenzamide(5)

A mixture of the hydrazide 4 (0.610 gm, 0.002 mol) and 2,5-hexanedione (0.228 gm, 0.002 mol) in glacial acetic acid (10 ml) was  stirred at room temperature overnight.  Dilution with water gave reddish  solid  was filtered, crystallized from aqueous ethanol yield, 95% ,m.p.148-150C^o.Microanalysis for C₂₀H₂₁N₃O₃S (383.46) Calcd% C, 62.64; H, 5.52; N, 10.96 Found% C, 62.76; H, 5.30; N, 10.76. IR(KBr, cm^-1): 3320 (NH), 3193 (NH), 3051 (CH, aromatic), 2922 (CH, aliphatic), 1760 (C=O), 1605 (C=N), 1487 (C=C), 1337(SO₂). ¹HNMR (DMSO-d6) d ppm: 2.05 (s, 6H, 2CH₃), 2.4 (s, 3H, CH₃), 5.74 (s, 2H, CH pyrrol) 7.257 (d, 2H J=8.1Hz ArH),7.387(d,2H, J=8.1Hz ArH)7.754(d,2H, J=7.8Hz ArH),7.848(d,2H, J=8.7Hz ArH) 10.80 (s, 1H, NH), 11.13 (s, 1H, NH). ¹³C-NMR (DMSO-d6) d ppm 11.012, 20.979, 103.067, 118.14, 126.56, 126.72, 128.86, 129.85, 137.6, 141, 143.6, 161.11.

General procedure for the preparation of 6a, b&7: To a solution of the acid hydrazide 4 (10 mmol) in ethanol (30 ml) containing  glacial acetic acid( 2 ml )an equimolar amount of acetyl acetone or benzoyl acetone or ethyl acetoacetate was added. The reaction mixture was heated under reflux for 12 hrs, concentrated to a small volume, cooled, and added to ice-cold water(30 ml). The separated product was filtered, washed with H₂O and recrystalized from an appropriate solvent.

[(3,5-Dimethyl-1H-pyrazol-1-yl) 4-(tosylamino)] phenyl methanone (6a):

Crystallized from EtOH  Yield  75% ,m.p. 238-240°C, Microanalysis for C₁₉H₁₉N₃O₃S (369.44) Calcd% C, 61.77; H, 5.18; N, 11.37 Found% C, 61.37; H, 5.59%; N, 11.46. IR (KBr, cm^-1): 3258 (NH), 3055 (CH, aromatic), 2979, 2930 (CH, aliphatic), 1690 (C=O), 1603 (C=N), 1505 (C=C),and 1336 (SO₂). ¹HNMR (DMSO-d6). dppm: 1.28 (s, 3H, CH₃), 2.16 (s, 3H, CH₃), 2.36 (s, 3H, CH₃), 6.24 (s, 1H, CH of pyrazol), 7.26-7.82 (m, 8H, ArH), 10.81 (s, 1H, NH).

[(3-Methyl-5-phenyl-1H-pyrazol-1-yl)-4-(tosylamino)] phenyl methamone (6b):

Crystallized from EtOH  yield 70%; m.p. 285-290°C . Microanalysis for C₂₄H₂₁N₃O₃S (431.5) Calcd% C, 66.80; H, 4.91; N, 9.74 Found% C, 66.97; H, 5.48%; N, 9.28. IR (KBr, cm^-1): 3195 (NH), 3058 (CH, aromatic), 2930 (CH, aliphatic), 1620 (C=O), 1509 (C=N), 1444 (C=C), 1338 (SO₂). ¹HNMR (DMSO-d6) dppm 2.03 (s, 3H, CH₃), 2.30 (s, 3H, CH₃), 5.80 (s, 1H, CH of pyrazol), 7.20-7.95 (m, 13H, ArH), 10.71 (s, 1H, NH).

[(3-Methyl-5-oxo-1H-pyrazolin-1-yl) 4-(tosylamino)] phenyl methanone (7):

Crystallized from EtOH  yield 65%; m.p. 195-198°C . Microanalysis for C₁₈H₁₇N₃O₄S (371.4) Calcd% C, 58.21; H, 4.61; N, 11.31 Found% C, 58.41; H, 4.96%; N, 11.00. IR (KBr, cm^-1): 3210 (NH), 3060 (CH, aromatic), 2980, 2937(CH, aliphatic), 1688 (C=O), 1605 (C=N), 1506 (C=C), 1335 (SO₂). ¹HNMR (DMSO-d6) dppm: 1.28 (s, 3H, CH₃), 2.32 (s, 3H, CH₃), 4.20 (d,1H, J= 6.6Hz CH₂) ,4.24(d,1H, J= 6.6Hz,CH₂)7.19(d,2H,J=8.1Hz, ArH), 7.34(d,2H, J=7.8Hz ArH)7.714(d,2H ,J= 7.8Hz,ArH)7,79(d,2H,J= 8.1Hz,ArH) 11.00 (s, 1H, NH).

N-[3, 4, 5-Trimethoxybenzylidene]-4-tosylamino benzohydrazide (8)

A mixture of the hydrazide 4 (0.610gm,0.002mol) and 3,4,5-trimethoxybenzaldehyde (0.392 gm, 0.002 mol) was dissolved in ethanol (15 ml) and heated under reflux for 3 hrs. Cool the mixture the separated solid was filtered washed with water and crystallized from absolute EtOH, yield 90%; m.p. 259-260°C. Microanalysis for C₂₄H₂₅N₃O₆S (484) Calcd% C, 59.61; H, 5.21; N, 8.69 Found% C, 59.15; H, 5.71%; N, 8.59. IR (KBr, cm^-1): 3474 (NH), 3173 (NH), 3050 (CH, aromatic), 2937 (CH, aliphatic), 1645 (C=O), 1607 (C=N), 1574 (C=C), 1329 (SO₂). ¹HNMR (DMSO-d6) dppm: 2.33 (s, 3H, CH₃), 3.69 (s, 3H, OCH₃), 3.82 (s,6H,2OCH₃),6.99(s,2H,ArH),7.218(d,2H,J=8.7Hz,ArH),7.381(d,2H,J=8.4Hz,ArH)7.70-7.77 (m,4H,ArH) 8.3 (s, 1H, HC=N), 10.69 (s, 1H, NH), 11.69 (s, 1H, NH).

N-(4-Chlorobenzoyl)-4-tosylaminobenzohydrazide (9):

A mixture of the hydrazide 4(0.610 gm, 0.002 mol)  and p-chlorobenzoyl chloride(0.348gm,0.002mol)  in glacial acetic acid,(10ml)  stirred  at  room temperature for 3 hrs, the separated solid was filtered, washed with water and crystallized from absolute EtOH, yield 80%; m.p. 210-212°C. Microanalysis for C₂₁H₁₈ClN₃O₄S (444) Calcd% C, 56.82; H, 4.09; N, 9.47 Found% C, 57.50; H, 4.45%; N, 9.56. IR (KBr, cm^-1): 3236 (NH), 3007(CH, aromatic), 2843 (CH, aliphatic), 1685 (C=O), 1610 (C=N), 1569 (C=C), 1331 (SO₂). ¹HNMR (DMSO-d6) dppm: 2.32 (s, 3H, CH₃), 7.19-7.97 (m, 12H, ArH), 10.38 (s, 1H, NH), 10.54 (s, 1H, NH), 10.68 (s, 1H, NH),. Ms. m/z (%) 444 (0.27) , 443 (1.13) M⁺-1, 313 (0.56), 274 (100), 139 (24.2), 119 (16.31), 111 (12.05), 91 (35.47).

General procedure for the preparation of compounds 10a-c. A mixture of acid hydrazide 4 (0.610g, 0.002 mol)and equimolar amount of maleic anhydride or dichloromaleic anhydride or phthalic anhydride in glacial acetic acid(20ml) was heated under reflux for 8 hrs. The product  was cooled and then poured into ice water 30 ml. The separated solid was filtered and crystallized from EtOH.

N-[(2, 5-Dioxo-2, 5-dihydropyrrol-1-yl)-4-(tosylamino)] benzamide (10 a):

Yield 75%; m.p. 178-180°C. Microanalysis for C₁₈H₁₅N₃O₅S (385) Calcd% C, 56.10; H, 3.92; N, 10.90 Found% C, 55.65; H, 4.40; N, 10.68. IR (KBr, cm^-1): 3256 (NH), 3005 (CH, aromatic), 2928 (CH, aliphatic), 1735, 1672 (2C=O), 1605 (C=N), 1496. (C=C), 1336. (SO₂). ¹HNMR (DMSO-d6) dppm: 2.32 (s, 3H, CH₃), 7.19 (s, 2H, HC=CH), 7.36-7.70 (m, 8H, ArH), 10.18 (s, 2H, 2NH). Ms. m/z(%) 385 (0.26), 329 (0.61), 305 (1.05), 290 (18.20), 274 (100), 155 (20.35), 119 (33.65), 107 (11.25), 91 (91.14), 65 (20.65).

N-[(3,4-Dichloro-2,5-dioxo-2,5-dihydropyrrol-1-yl)-4-(tosylamino)]benzamide(10b):

Yield 65%; m.p. 168-170°C. Microanalysis for C₁₈H₁₃Cl₂N₃O₅S (454) Calcd% C, 47.59; H, 2.88; N, 9.25 Found% C, 47.40; H, 3.00; N, 8.9. IR (KBr, cm^-1): 3243. (NH), 3050 (CH, aromatic), 2926.(CH, aliphatic), 1667 (2C=O), 1606 (C=N), 1498 (C=C), 1335(SO₂). Ms. m/z (%) 454 (0.63) , 453 (2.85), 435 (4.78), 426 (2.71), 367 (8.60), 327 (5.41), 191 (26.95), 149 (100), 125 (32.9), 111 (43.81), 85 (35.73), 69 (99.85), 55 (35.14).

N-[(1,3-Dioxoisoindolin-2-yl)-4-(tosylamino)]benzamide(10c):

Yield 85%; m.p. 198-200°C. Microanalysis for C₂₂H₁₇N₃O₅S (435.5) Calcd% C, 60.68; H, 4.93; N, 9.95 Found% C, 60.23; H, 5.00; N, 10.04. IR (KBr, cm^-1): 3424(NH), 3220 (NH), 3060 (CH, aromatic), 2930 (CH, aliphatic), 1737(C=O), 1686 (C=O), 1608 (C=N), 1497 (C=C), 1335 (SO₂). Ms. m/z (%) 435 (0.7) , 434 (1.1), 274 (100), 250 (7.6), 120 (13.5), 91 (65.8), 77 (6.9), 51 (9.7).

5-(4-Tosylaminophenyl) 1,3,4-oxadiazole-2-thiol(11)

To a solution of acid hydrazide 4 (0.610gm,0.002 mol) in EtOH(50 ml) (95%) containing KOH (0.130gm,0.0022 mol), CS₂ (10 ml )was added. The reaction mixture was heated under reflux for 12 hrs while stirring, then concentrated, cooled and acidified with dilute HCl. The separated product was filtered, washed with H₂O and crystalized from aqueous EtOH. yield 95%; m.p. 229-230°C. Microanalysis for C₁₅H₁₃N₃O₃S₂ (347) Calcd% C, 51.86; H, 3.77; N, 12.10 Found% C, 51.74; H, 3.70; N, 12.49. IR (KBr, cm^-1): 3278 (2NH), 3056 (CH, aromatic), 2924 (CH, aliphatic), 1508 (C=N), 1470 (C=C), 1344 (SO₂). ¹HNMR (DMSO-d6) dppm 2.33 (s, 3H, CH₃), 7.24-7.73 (m, 8H, ArH), 10.50 (s, 1H, NH), 14.62 (s, 1H, NH). Ms. m/z (%) 347 (63.66), 287 (13.74), 274 (12.99), 192 (32.49), 155 (20.80), 132 (95.80), 91 (100), 78 (10.46), 65 (30.53).

2-(4-Tosylaminophenyl)-5-(alkylthio)-1,3,4-oxadiazoles(12a-d)2-(5-(4-tosylaminophenyl)-1,3,4-oxadiazol-2-ylthio)-N-4(substituted phenyl) acetamides (12e-i)

A mixture of 11 (0.347gm,0.001 mol) and appropriate alkyl or aryl halid or chloroacetamide derivatives(0.001 mol) in ethanol(30ml) containing KOH (0.1gm,0.0017 mol) was stirred at room temperature overnight. The reaction mixture was poured into ice water(50ml). The separated solid was filtered and crystallized from appropriate solvent   (Table 5).

Table 5:  2-(4-(Tosylaminophenyl-5-alkylthio)1,3,4-oxadiazoles(12a-d),2-(5-(4-tosylamino phenyl)-1,3,4-oxadiazol-2-yl thio-N-4-(substituted phenyl) acetamides (12e-i).

Comp. No	R2	M.P.°C	Yield %	Crystalization solvent	M.F (M.W)	Analysis (%) calcd/found
						C	H	N
12a	CH3–	122-125	85	a	C16H15N3O3S2 (361.44)	53.17 53.31	4.18 3.89	11.63 11.91
12b	C2H5–	198-200	75	a	C17H17N3O3S2 (375)	54.38 54.23	4.56 4.33	11.19 10.90
12c	(CH3)2CH-	138-140	80	a	C18H19N3O3S2 (389.49)	55.51 55.23	4.92 4.61	10.79 10.49
12d	C6H5-CH2–	188-190	85	a	C22H19N3O3S2 (438)	60.39 60.16	4.38 4.20	9.60 9.29
12e	C6H5-NH-CO-CH2–	205-207	80	b	C23H20N4O4S2 (480.56)	57.48 57.63	4.19 4.45	11.66 11.73
12f	4-Cl(C6H4)NH-CO-CH2–	218-220	80	b	C23H19ClN4O4S2 (515)	53.64 53.35	3.72 4.33	10.88 11.28
12g	4-OCH3(C6H4)NH-CO-CH2–	208-210	85	b	C24H22N4O5S2 (511)	56.46 56.51	4.34 4.52	10.97 11.12
12h	4-CH3(C6H4)NH-CO-CH2–	204-206	75	b	C24H22N4O4S2 (494.5)	58.28 58.55	4.94 4.59	11.33 11.71
12i	2-CH3(C6H4)NH-CO-CH2–	199-201	70	b	C24H22N4O4S2 (494.5)	58.28 58.38	4.94 4.91	11.33 11.55

a = CHCl_3/ pet. ether 60-80C^o

b= Ethanol

Compound 12a

IR (KBr, cm^-1): 3103 (NH), 3008 (CH, aromatic), 2926 (CH, aliphatic), 1608 (C=N), 1469 (C=C), 1340 (SO₂), ¹HNMR (CDCl₃) dppm 2.03 (s, 3H, CH₃), 2.73 (s, 3H, CH₃, S-CH₃), 6.87-7.94 (m, 8H, ArH),10.79(s,1H,NH)

Compound 12b

IR (KBr, cm^-1): 3198 (NH), 3058 (CH, aromatic), 2930 (CH, aliphatic), 1477 (C=N), 1404 (C=C), 1335 (SO₂), ¹HNMR (CDCl₃) dppm 1.50 (t, 3H, CH₃), 2.38 (s, 3H, CH₃), 3.35 (q, 2H, CH₂), 7.26-7.73 (m, 8H, ArH),10,97(s,1H,NH).

Compound 12c

IR (KBr, cm^-1): 3100 (NH), 3072 (CH, aromatic), 2965, 2921 (CH, aliphatic), 1609 (C=N), 1503 (C=C), 1338 (SO₂), ¹HNMR (DMSO-d6) dppm  1.438 (d, 6H,J= 6.9Hz, 2CH₃), 2.31 (s, 3H, CH₃), 3.86-3.88 (m, 1H, CH), 7.301 (d,2H,J=7.2Hz, ArH), 7.371(d,2H,J=7.8Hz,ArH)7.729(d,2H,J= 8.1Hz,ArH)7.845(d,2H,J=8.7Hz,ArH)10.78 (s, 1H, NH). ¹³CNMR (DMSO-d6) dppm: 20.943, 23.049, 38.39, 127.37, 128.51, 136.16, 137.18, 139.28, 145.8, 150.7, 153.09, 171.9, 174.18.

Compound 12d

IR (KBr, cm^-1): 3198 (NH), 3058 (CH, aromatic), 2930 (CH, aliphatic), 1607 (C=N), 1477 (C=C), 1338 (SO₂), ¹HNMR (CDCl₃) dppm: 2.33 (s, 3H, CH₃), 4.51 (s, 2H, CH₂), 7.26-7.84 (m, 13H, ArH).10.79 (s,1H,NH)

Compound 12e

IR (KBr, cm^-1): 3208 (NH), 3073 (CH, aromatic), 2929 (CH, aliphatic), 1665 (C=O), 1608 (C=N), 1337 (SO₂), ¹HNMR (DMSO-d6) dppm 2.22 (s, 3H, CH₃), 4.23(s, 2H, CH₂), 7.02-8.23 (m, 13H, ArH), 10.41 (s, 1H, NH), 10.70 (s, 1H, NH).

Compound 12f

IR (KBr, cm^-1): 3211 (NH), 3072 (CH, aromatic), 2925 (CH, aliphatic), 1665 (C=O), 1605 (C=N), 1337 (SO₂). M.s m/z (%) 515 (0.81) , 498 (0.76), 481 (0.76), 462 (1.21), 452 (1.03), 440 (14.81), 422 (2.19), 367 (5.06), 361 (1.65), 348 (100), 330 (1.47), 3.17 (0.55), 2.87 (39.46).

Compound 12g: IR (KBr, cm^-1): 3210 (NH), 3050 (CH, aromatic), 2926 (CH, aliphatic), 1663 (C=O), 1607 (C=N), 1576 (C=C), 1337 (SO₂), ¹HNMR (DMSO-d6) dppm: 2.33 (s, 3H, CH₃),3.73(s,3H,OCH₃₎ 4.26 (s,2H,CH₂),7.258(d,2H,J=7.8Hz, ArH),7.374(d,2H,J=7.8Hz,ArH)

7.723(d,4H,J=8.1Hz,ArH) 7.799(d,2H,J=8.1Hz,ArH),7.957(d,2H,J=8.1Hz,ArH),

10.27 (s, 1H, NH), 10.79 (s, 1H, NH).

Compound 12h

IR (KBr, cm^-1): 3210 (NH), 3071(CH, aromatic), 2924 (CH, aliphatic), 1665 (C=O), 1605 (C=N), 1545 (C=C), 1336 (SO₂), ¹HNMR (DMSO-d6) dppm: 2.32 (s, 3H, CH₃), 2.42(s,3H,CH₃),4.33(s,2H,CH₂),7.30-7.76(m,12H,ArH), 10.28 (s, 1H, NH), 10.76(s, 1H, NH).

Compound 12i

IR (KBr, cm^-1): 3257 (NH), 3072, 3004 (CH, aromatic), 2928 (CH, aliphatic), 1653 (C=O), 1606 (C=N), 1545 (C=C), 1331 (SO₂). ¹HNMR (DMSO-d6) dppm: 2.20 (s, 3H, CH₃), 2.33 (s, 3H, CH₃), 4.23 (s, 2H, CH₂), 6.74-7.86 (m, 12H, ArH), 10.69 (s, 1H, NH), 11.21 (s, 1H, NH).

 N-[(5,5-Dimethyl-3-oxocyclohex-1-enyl)4-tosylamino] benzohydrazide (13)

A mixture of acid hydrazid 4 (1.2 gm, 0.004 mol) and equimolar amount of 5,5-dimethyl-1,3-cyclohexanedione (dimedone) (0.56 gm, 0.004 mol) in  toluene(20 ml)  was heated under reflux for 15 hrs. The reaction mixture allow to cool to room temperature. The obtained crystalline product was filtered and recrystallized from ethanol as yellow crystal, yield 75%, m.p. 237-238°C. Microanalysis for C₂₂H₂₅N₃O₄S (427) Calcd % C, 61.81; H, 5.89; N, 9.83 Found % C, 61.84; H, 5.94; N,. 10.17, IR (KBr, cm^-1): 3237 (3NH), 3013 (CH, aromatic), 2951 (CH, aliphatic), 1671 (2C=O), 1549 (C=C), 1331 (SO₂). ¹HNMR (DMSO-d6) dppm: 0.78 (s, 3H, CH₃), 1.00 (s, 3H, CH₃), 2.01 (s, 2H, CH₂), 2.30 (s, 5H, CH₃+ CH₂), 4.89 (s, 1H, =CH), 7.14-7.68 (m, 8H, ArH), 9.58 (s, 1H, NH), 10.60 (s, 1H, NH), 11.20 (s, 1H, NH).

N-(2-Amino-3-cyano-7,7-dimethyl-4-(4-substitutedphenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinolin-1-yl-4-tosylaminobenzamides 14(a-c)

A mixture of equimolar amounts of enaminone 13 (0.626 gm, 0.001 mol) and the appropriate 2-(4-substituted benzylidene) malonodinitrile (0.001 mol) in absolute ethanol (15 ml) containing TEA (3 drops) was heated under reflux for 12-20 hrs. The reaction mixture was cooled and the separated solid product was filtered, washed with water and crystallized from ethanol (Table 6).

Table 6:  N-(2-Amino-3-cyano-7,7-dimethyl-4-(4-substitutedphenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinolin-1-yl-4-tosylaminobenzamides 14(a-c).

Comp No.	X	Time reflux (hrs)	m.p. °C	Yield %	MF (M.W)	Analysis% Calcd/found
						C	H	N
14a	Cl	15	180-182	65	C32H30ClN5O4S (616.13)	62.38 62.24	5.75 5.38	11.37 11.00
14b	NO2	12	218-220	70	C32H30N6O6S (626.68)	61.33 61.43	4.83 5.43	13.41 13.38
14c	OCH­3	20	205-208	60	C33H33N5O5S (611.7)	64.79 64.71	5.44 5.88	11.45 11.32

Compound (14a)

IR (KBr, cm^-1): 3457, 3349 (NH₂), 3215, (NH), 3055 (CH, aromatic), 2957 (CH, aliphatic), 2183 (CN), 1647 (C=O), 1486 ( (C=C), 1336 (SO₂). Ms: m/z (%) 616 (4.02), 615 (0.15), 597 (50.59), 442 (75.84), 290 (35.32), 274 (48.69), 91 (73.57), 64 (100).

Compound (14b)

IR (KBr, cm^-1): 3457, 3351 (NH₂), 3205 (NH), 3051 (CH, aromatic), 2952 (CH, aliphatic), 2183(CN), 1632 (C=O), 1512 (C=N), 1459.8 ,1379.8(NO₂), 1341 (SO₂). ¹HNMR (DMSO-d6) d ppm: 0.80 (s, 3H, CH₃), 0.85 (s, 3H, CH₃), 2.00-2.20 (m, 4H, 2CH₂),2.30(s,3H,CH₃)4.40(s,1H,CH),6.58(s,2H,NH_2,exchangeable),7.0267(d,2H,J=8.4Hz,ArH)),7.0389(d,2H,J=7.8Hz,ArH),7.0661-7.0776(m,4H,ArH)7.0874(d,2H,J=8.4Hz,ArH), 8.0152(d,2H,J=8.7Hz,ArH), 11.00 (broad 2H, 2NH, exchangeable).  ¹³CNMR (DMSO-d6) dppm: 21.02, 27.6, 28.11, 37.1, 49.2, 57.3, 110.9, 117.77, 121.2, 123.4, 126.8, 129.7, 136.6, 142.2, 143.7, 146, 165.7, 194.64.

Compound (14c)

IR (KBr, cm^-1): 3374 (NH₂), 3238 (NH), 3050 (CH, aromatic), 2952 (CH, aliphatic), 2184 (CN), 1645 (C=O), 1606.(C=N), 1506 (C=C), 1337 (SO₂). ¹HNMR (DMSO-d6) d ppm: 0.83 (s, 3H, CH₃), 0.98 (s, 3H, CH₃), 2.00-2.20 (m, 4H, 2CH₂), 2.30  (s, 3H, CH₃), 3.80 (s, 3H, OCH₃), 4.85 (s, 1H, CH), 6.58 (s, 2H, NH₂), 7.24-7.76 (m, 12H, ArH), 10.40 (s, 1H, NH), 10.85 (s, 1H, NH).

N-(Ethyl-2-amino-7,7-dimethyl-4-(4-substitutedphenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinolin-1-yl-3-carboxylate-4-tosylaminobenzamides (15a-b)

The title compound were prepared as described under 14a-c using   the enaminone 13 and ethyl 3-(4-substituted phenyl) 2-cyanoacrylate  then heating under reflux for 10-12 hrs, crystallization from ethanol(Table 7).

 Table 7:  N-(Ethyl-2-amino-7,7-dimethyl-4-(4-substitutedphenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinolin-1-yl-3-carboxylate-4-tosylaminobenzamides (15a-b).

Comp No.	X	Time reflux (hrs)	m.p. °C	Yield %	MF (M.W)	Analysis% Calcd/found
						C	H	N
15a	Cl	12	270-272	65	C34H33ClN4O6S (363.18)	61.58 61.96	5.32 5.31	8.45 8.80
15b	NO­2	10	282-285	70	C34H35N5O8S (673.22)	60.61 60.55	5.24 5.11	10.39 10.54

Compound (15a) IR (KBr, cm^-1): 3436, 3233 (NH₂), 3149 (NH), 3045 (CH, aromatic), 2932 (CH, aliphatic), 1692 (C=O), 1604 (C=N), 1503 (C=C), 1333 (SO₂). M.S: m/z (%) 663 (5.62) , 662 (8.08), 597 (15.20), 436 (22.74), 422 (26.11), 369 (50.78), 257 (20.44),. 208 (23.86), 185 (21.08), 173 (18.41), 147 (35.42), 127 (28.52), 96 (53.50), 73 (70.3).

Compound (15b) IR (KBr, cm^-1): 3492, 3322 (NH₂), 3181 (NH), 3050 (CH, aromatic), 1925 (CH, aliphatic), 1664(C=O), 1621 (C=O), 1507,1379.8 (NO₂), 1339 (SO₂).  ¹HNMR (DMSO-d6) d ppm: 0.76 (s, 3H, CH₃), 0.95 (s, 3H, CH₃), 1.08 (t, 3H, CH₃), 1.9-2.1 (m, 4H, 2CH₂), 2.34 (s, 3H, CH₃)3.91 (d,  2H,J=6.9Hz,CH₂), 4.86(s,1H,CH), 7.24 (d,2H,J=8.7Hz,ArH), 7.37 (d,2H,J=7.8Hz,ArH),7.45(brs,2H,NH₂), 7.74 (d,2H,J=8.1Hz,ArH) ,7.91 (d,2H,J=8.1Hz,ArH), 8.03 (d,2H,J=8.4Hz,ArH), 8.09 (d,2H,J=9Hz,ArH)10.80(s, 1H, NH), 11.06(s, 1H, NH).

B- Biological studies

1- Antimicrobial activity

Different bacteria and fungi were subjected to susceptibility test on Muller-Hinton agar medium by cup plate  method^(16-17). The antimicrobial activity of some of the newly synthesized compounds was tested against each of the mentioned strains using Cefotaxime, Sulphamethoxazole and Nystatin as reference (table 1). Control testing using DMF was used to determine the solvent effect.

Overnight culture was streaked on the cups of Muller-Hinton agar plate with 50 mg/ml concentration of each sample under investigation in DMF solvent the plate incubated at 37°C for 24 hrs and examined for inhibition zones to determine the activity of tested compounds

2- Pharmacological activities:

Analgesic activity evaluation

The hot plate method of Jacob and Bosovski⁽¹⁸⁾was used to evaluate the  analgesic activity. Mature albino mice of both sex weighing 20-25 gm were    classified into 19 groups (each of 5). The first group was left as control and injected i.p. with the solvent(DMSO).While the second  Group was injected with Celecoxib and Voltarin at a dose of 1.7 mg/kg. Each of the remaining groups was Injected i.p with a test compound at adose of 1.7mg/kg.Ten minutes latter, each mouse was  placed in two liter-beaker immersed in water bath thermostatically controlled at 56C^o .The time elapsed till the mouse licks its paw or jumps was considered the reaction time and was taken as a measure of the analgesic effect. Reading  were taken at 10,20,30,60,90 and 120 minutes post treatment (Table2)

Anti-inflammatory activity evaluation

The rat hind paw odema method^(19-21)was applied to determined the Anti-inflammatory activity of the test compounds using Celecoxib ,Voltarin as standerd.Mature albino rats of both sex weighing 200-250gm were used the animal were divided into 19 equal groups (each of 5). The first group was left as control, while the second and third groups was injected(i.p) with Celecoxib and Voltarin at a dose 18  mg/kg- one hour later odema in the right hind paw was induced  by injection of 0.1cm³ of 10% carrageenin. The thickness of the paw was measured 60,120,180 and 240 minutes after carrageenin injection to determine  the  anti-inflammatory activity of the test compounds(Table3)

Ulcerogenic activity⁽²²⁾

Compounds 5,6a ,8,10a,11,12d,12f,12g,14a,14b and15a were tested for their ulcerogenic activity  using Celecoxib and Indomethacin as refrence drug. Male albino rats weighing 150-200gm were fasted for 12hrs prior to drug     administration. Water was given ad-libitum.The animal were divided into 15 equal groups(each4).The first group received1% gum acacia(suspending vehicle) orally once a day and was left as control. While the second and third groups received Indomethacin and Celecoxib at a dose 18 mg /kg/day orally.the remaining groups received the test compounds at a dose of 18 mg /kg/day orally. The drugs were administrated once a  day for successive days .The animal were killed by an over dosage of ether 6hrs after the last dose .The stomach was removed .Open along the greater curvature and examined ulceration. The number and the severity of discrete areas of damage in the glandular mucosa were scored (Table 4)

Acknowledgement

I am heart fully thankful to extent my profound gratitude to Ass. Lecture Nader Shawky Mohammed  ,Microbiology department, Faculty of pharmacy, Zagazig university for his kind support for performing antimicrobial activity.

My extreme thanks and deep gratitude to Prof. Dr. Saed  Abdel Aziz, Professor of pharmacology ,Faculty of Veterinary Medicine,Zagazig university for his valuable efforts in carrying the analgesic, anti-inflammatory and ulcerogenic testing.

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