The Use of Α-Enone Derivative in the Preparation of Some New Heterocyclic Compounds with Expected Biological and Antitumor Activities

1049 Advances in Environmental Biology, 7(6): 1049-1057, 2013 ISSN 1995-0756

This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE

The use of α-enone derivative in the preparation of some new heterocyclic compounds with expected biological and antitumor activities

Zeinab H. Ismail

Department of Chemistry, Faculty of Science, Al-Azhar University (Girls), Nasr City, Cairo, Egypt

Zeinab H. Ismail: The use of α-enone derivative in the preparation of some new heterocyclic compounds with expected biological and antitumor activities

ABSTRACT

α-Enone 1, reacts with hydrazines, hydroxylamine hydrochloride, thiourea, diethylmalonate, malononitrile ethyl cyanoacetate to give the corresponding indazole, oxime, isoxazole, quinazoline, chromene and quinoline derivatives. The structures of all the synthesized compounds were confirmed by micro analytical and spectral data. The antimicrobial and the antitumor activities of some of the synthesized compounds were tested.

Key words: α-enone, indazole, isoxazole, quinazoline, chromene and quinoline derivatives.

Introduction hydrate in DMF to obtain the formohydrazide 4 has failed instead the parent indazole 2a was obtained. Indazole derivatives are reported to exhibit However reaction of 1 with hydroxylamine antibacterial [1,2], antifungal[1.2], antiproliferative hydrochloride in ethanol or pyridine gave the [3], antigiogenic [3], antiarrhythmic and analgesic corresponding oxime and isoxazole 5 and 6, [4] activities and antihypertensive properties [5], respectively. while quinazolines exhibit antitumor [6], Compound 1, was reduced and acetylated by antimicrobial [7] antitubercular [8], antiagonists [9], zinc dust in boiling acetic anhydride solution to give anticonvulsant [10] and analgesic[11] activities. compound (7). On the other hand quinolines exhibit antimalarial Reaction of 1 with indole gave compound 8, [12], antiplasmodial [13], antiproliferative [14], through condensation of 2 moles of indole with the antibacterial [15], antifungal [15]. ketonic group in compound 1. The absence of γC=O Chromene derivatives exhibiting antimicrobial in the infrared spectrum of compound 8, support the [16], antihypertensive and antioxidant [17] activities proposed structure. have also been reported.…. Refluxing of compound 1 and thiourea in The prompted the author to use 2,6-bis(3,4- absolute ethanol with a catalytic amount of dimethoxybenzylidene) cyclohexanone (1) as key potassium hydroxide furnished quinazoline thione intermediate in the synthesis of series of heterocyclic derivative 9 in a good yield. compounds. The new derivatives were prepared The condensation of 1 with diethylmalonate in following the reaction sequences depicted in boiling benzene in the presence of diethyl sodio- Schemes (1-2). malonate as basic condensing agent afforded the Treatment of compound 1 with hydrazine chromene derivative (10). hydrate or phenyl hydrazine in absolute ethanol Enone 1 reacts under Michael conditions with afforded indazole derivatives 2a-b in a one-pot malononitrile to give the quinoline derivative (11). reaction. The olefinic double bond of 1 is activated Reaction of 1 with ethyl cyanoacetate in the by the Ketone group therefore the β-carbon atom will presence of catalytic amount of piperidine furnished accepted nucleophiles. The formation of indazole the chromene derivative (12), while its reaction with derivatives 2a-b can be visualized on the basis of ethyl cyanoacetate in the presence of ammonium cyclocondensation of hydrazines with enone 1. acetate gave the quinoline derivative (13). Treatment of compound 2a with Structure of compound 12, was further benzenesulfonylchloride, phenylisothiocyanate or established by its reaction with o-aminophenol bromine gave derivatives 2c-d and 3, respectively. and/or p-aminopyridine in absolute ethanol to give The formation of compound 3 proceeds through the corresponding amide derivatives 14a-b, however dehydrogenation and addition of bromine to the its reaction with hydrazine hydrate gave the exocyclic double bond. Reaction of 1 with hydrazine corresponding chromeno-pyrazole derivative (15)

Corresponding Author Zeinab H. Ismail, Department of Chemistry, Faculty of Science, Al-Azhar University (Girls), Nasr City, Cairo, Egypt 1050 Adv. Environ. Biol., 7(6): 1049-1057, 2013 through elimination of one molecule of ethanol and ammonia. Yield 75%; m.p. 198-200oC; IR (KBr) γ: 1598 -1 1 Compound 13 exists in a lactam-lactim (C=N) cm ; HNMR (DMSO-d6) δ: 1.25-2.26 (m, tautomerism, the existence of the lactim form was 6H, 3xCH2), 2.76 (d, 1H, CH pyrazole), 3.71 (d, 1H, proved by its reaction with POCl3 to give the chloro CH pyrazole), 3.74-3.81 (s, 12H, 4xOCH3), 6.03 (s, derivative (16). The chloro compound (16) has been 1H, CH- olefinic), 6.64-7.77 (m, 11H, Ar-H). MS used as starting material for the preparation of new (70ev) m/z%: 484 (M+) (4.52). Anal. Calcd. for compounds. Thus its reaction with sodium methoxide C30H32N2O4 (484.24); C, 74.36; H, 6.66; N, 5.78 gave the corresponding methoxy derivative (17), Found: C, 74.40; H, 6.61; N, 5.90%. however its reaction with hydrazine hydrate gave the corresponding pyrazoloquinoline derivative (18). 2,6-Bis (3,4-dimethoxybenzylidene) cyclohexanone The structure of compounds 2-19 was apparent oxime (5): from the mass spectra. The 1HNMR spectroscopic data as well as IR spectra, were also in agreement Yield 62% m.p. 160-162oC; IR (KBr) γ: 1596 -1 1 with the proposed structures. (C=N) cm ; HNMR (DMSO-d6) δ: 1.23-2.17 (m, 6H, 3xCH2), 3.76 (s, 12H, 4xOCH3), 6.75-8.03 (m, Experimental: 8H, AR-H and 1H, CH-olefinic) 8.8 (s, 1H, OH). MS (70ev) m/z%: 409 (M+) (25.88). Anal. Calcd. for Melting points were determined in open C24H27NO5 (409.19); C, 70.40; H, 6.65; N, 3.42 capillary tubes on a Gallen Kamp melting point Found: C, 70.45; H, 6.35; N, 3.45%. apparatus and are uncorrected. The elemental analysis were carried out at the Micro Analytical 8-(3,4-Dimethoxybenzylidene)-4-(3,4- Unit, Faculty of Science, Cairo University by using dimethoxyphenyl)-3a,4,5,6,7,8- Perkin-Elmer 2400 CHN elemental analysis. The IR hexahydrochromeno[2,3-c]pyrazol-3-ol (15): spectra recorded on a Shimadzu-440 IR spectrophotometer using KBr technique. The Yield 63%; m.p. 145-147oC; IR (KBr) γ: 1593 1HNMR spectra were measured on a Bruker Proton (C=N), 3424 (OH, Br), 3299 (NH) cm-1; 1HNMR NMR-Avance 300 (300 MHz, spectrometer) with (DMSO-d6) δ: 1.23-2.39 (m, 6H, 3xCH2), 3.74-3.8 (s, chemical shift (δ) expressed in ppm downfield from 12H, 4xOCH3), 4.00 (s, 1H, CH pyran), 4.32 (s, 1H, TMS as internal standard in DMSO-d6. Mass spectra OH), 6.68-7.5 (m, 6H, AR-H and 1H, CH-olefinic), were recorded on Hewlett Packard model MS-5988 9.84 (s, 1H, NH). MS (70ev) m/z%: 476 (M+) (0.24). spectrometer. Anal. Calcd. for C27H28N2O6 (476.19); C, 68.05; H, 5.92; N, 5.88 Found: C; 68.10; H, 5.95; N, 5.44%. Reaction of 1, 12 and 16 with hydrazines and NH2OH.HCl: 8-(3,4-Dimethoxybenzylidene)-4-(3,4- dimethoxyphenyl)-4,5,6,7,8,9-hexahydro-3aH- To a solution of 1, 12 and/or 16 (0.01 mole) in pyrazolo[3,4-b]quinolin-3-amine(18): ethanol or DMF (20ml) and equimolar amounts of hydrazine hydrate, phenylhydrazine and/or Yield 64%; m.p. 135-137oC; IR (KBr) γ: 3327- -1 1 NH2OH.HCl was added and the reaction mixture was 3183 (NH-NH2), 1604 (C=N) cm ; HNMR refluxed for 2-15 hours (TLC). The crude material (DMSO-d6) δ: 1.09-2.2 (m, 6H, 3xCH2), 2.7 (d, 1H, was filtered off and recrystallized from ethanol to CH pyridine), 3.34 (d, 1H, CH pyridine), 3.73 (s, give compounds 2a,b,5,15 and 18. 12H, 4xOCH3), 4.6-5.1 (s, 3H, NH-NH2), 6.86-8.6 (m, 7H, AR-H and 1H, CH-olefinic). MS (70ev) 7-(3,4-Dimethoxybenzylidene)-3-(3,4- m/z%: 474 (M+) (53.49). Anal. Calcd. for dimethoxyphenyl)-3,3a,4,5,6,7-hexahydro-2H- C27H30N4O4 (474.23); C; 68.34; H, 6.37; N, 11.81 indazole (2a): Found: C, 68.50; H, 6.40; N, 11.90%.

Yield 82%; m.p. 172-174oC; IR (KBr) γ: 1593 7-(3,4-Dimethoxybenzylidene)-3-(3,4- -1 1 (C=N), 3298 (NH) cm ; HNMR (DMSO-d6) δ: dimethoxyphenyl)-2-(phenyl-sulfonyl)-3,3a,4,5,6,7- 1.34-2.40 (m, 6H, 3xCH2), 2.8 (d, 1H, CH pyrazole), hexahydro-2H-indazole (2c): 3.7 (s, 12H, 4xOCH3), 4.26 (d, 1H, CH pyrazole), 6.68-7.25 (m, 6H, ArH), 7.4 (s, 1H, CH-olefinic), 9.8 A solution of 2a (0.01 mole) in pyridine (10ml), (s, 1H, NH). MS (70ev) m/z%: 411 (M++3) (0.97). and benzenesulfonyl chloride (0.01 mole) was heated Anal. Calcd. for C24H28N2O4 (408.2); C, 70.57; H, on water bath for 3 hours. Then, the reaction mixture 6.91; N, 6.86 Found: C, 70.64; H, 6.68; N, 6.9%. was poured into ice/HCl mixture. The crude material was filtered off and recrystallized from pet. ether 7-(3,4-Dimethoxybenzylidene)-3-(3,4- (60-80oC): Yield 82%; m.p. 120-122oC; IR (KBr) γ: dimethoxyphenyl)-2-phenyl-3,3a,4,5,6,7-hexahydro- 1597 (C=N), 1264 (O=S=O) cm-1; MS (70ev) m/z%: + 2H-indazole (2b): no parent ion; 141 (C6H5SO2 ) (63.64) Anal. Calcd.

1051 Adv. Environ. Biol., 7(6): 1049-1057, 2013 for C30H32N2O6S (548.20); C; 65.67; H, 5.88; N, (100ml) was refluxed for 1 hour, and treated with 5.11, S, 5.84 Found: C, 65.66; H, 5.78; N, 5.20; S, more zinc dust (5 g) and refluxed for 1 hour, the 5.70%. solid obtained after filtration while hot and removal of the solvent under reduced pressure, was washed 7-(3,4-Dimethoxybenzylidene)-3-(3,4- with water and crystallized from pet. ether (60-80oC) dimethoxyphenyl)-N-phenyl-3,3a,4,5,6,7- (30% yield); m.p. 70-72oC; IR (KBr) γ: 1707 (C=O) -1 1 hexahydroindazole-2-carbothioamide (2d): cm . HNMR 0.79-2.3 (m, 6H, 3xCH2), 3.21 (s, 6H, 2xCH3), 3.68-3.71 (s, 24H, 8xOCH3), 6.65-6.83 (m, A solution of 2a (0.01 mole) and phenyl 16H, Ar-H and H-olefinic), MS (70ev) m/z%, 874 + isothiocyanate (0.01 mole) in dry diethyl ether (M +1) (2.08). Anal. Calcd. for C52H58O12 (874.39); (30ml) was stirred at room temperature for 10 hours. C, 71.38; H, 6.68 Found: C, 71.40; H, 6.71%. The solid that separated was collected and recrystallized from ethanol (73% yield) m.p. 138- 2,6-Bis (3,4-dimethoxybenzylidene) cyclohexane - 140oC, IR (KBr) γ: 1596 (C=N), 1417 (C=S), 3331 1,1-diyl) bis (1H-indole) (8): (NH) cm-1. MS (70ev) m/z%: 544 (M++1) (9.81). Anal. Calcd. for C31H33N3O4S (543.22); C, 68.48; H, A mixture of 1 (0.01 mole), indole (0.01 mole) 6.12; N, 7.73; S, 5.90 Found: C, 68.50; H, 6.13; N, and sodium ethoxide [0.5 g Na + 50ml ethanol] was 7.74; S, 6.00%. refluxed on water bath for 30 hours. After cooling, the solid that separated was collected and re- 7-Bromo-7-(bromo(3,4-dimethoxyphenyl)methyl)-3- crystallized from diethyl ether. (59% yield); m.p. o 1 (3,4-dimethoxyphenyl)-4,5,6,7-tetrahydro-3aH- >300 C, HNMR (DMSO-d6) δ; 0.86-1.40 (m, 6H, indazole (3): 3xCH2), 4.64 (s, 12H, 4xOCH3), 6.74-7.71 (m, 18H, Ar-H and 2H, 2H-olefinic), m/z%, 610 (M+) (15). A solution of 2a (0.01 mole) in chloroform Anal. Calcd. for C40H38N2O4 (610.28); C, 78.66; H, (50ml), and bromine (0.01 mole) was stirred at room 6.27; N, 4.59 Found: C, 78.80; H, 6.42; N, 4.37%. temperature for 3 hours. Evaporating the solvent gave an oily product, triturated with Pet. ether (60- Reaction of enone 1 with the different nucleophiles: 80°C), the solid product obtained was crystallized from pet. ether (60-80oC). (69% yield); mp 88-90oC; General procedure: -1 1 IR (KBr) γ: 1599 (C=N) cm , HNMR (DMSO-d6) δ: 1.05-2.06 (m, 6H, 3xCH2), 3.82-4.10 (s, 12H, To a solution of 1 (0.01 mole) in ethanol (20ml) 4xOCH3), 1.90 (s, 1H, CH pyrazole), 6.30 (s, 1H, an equimolar amounts of thiourea, malononitrile or CHBr), 6.93-7.51 (m, 6H, Ar-H). MS (70ev) m/z%, ethyl cyanoacetate was added and the reaction + 566 (M +2) (0.01). Anal. Calcd. for C24H26Br2N2O4 mixture was refluxed for 4-10 hours (TLC). A (566.02); C, 50.90; H, 4.63; N, 4.95; Br, 28.22 solution of KOH (2 g) in 2ml water was added in Found: C, 51.00; H, 4.53; N, 4.94; Br, 28.21%. case of reaction with thiourea, ammonium acetate (0.4 mole) in case of reaction with malononitrile and 7-(3,4-Dimethoxybenzylidene)-3-(3,4- few drops of piperidine in case of ethyl cyanoacetate. dimethoxyphenyl)-3,3a,4,5,6,7-hexahydrobenzo(c) The crude material was filtered off, washed with H2O isoxazole (6): and recrystallized.

To a solution of 1 (0.01 mole) in pyridine 8-(3,4-Dimethoxybenzylidine)-4-(3,4- (10ml), NH2OH.HCl (0.01 mole) was added and the dimethoxyphenyl)-4,4a-5,6,7,8- reaction mixture was refluxed on a water bath for 10 hexahydroquinazoline-2(3H)-thione (9): hours. Then, the reaction mixture was poured into ice/HCl mixture. The crude material was filtered off (83% Yield); m.p. 82-84oC, IR (KBr) γ: 1611 and crystallized from ethanol. (56% yield) m.p. 108- (C=N), 1414 (C=S), 3277 (NH) cm-1; 1HNMR o -1 1 110 C, IR (KBr) γ: 1597 (C=N) cm . HNMR (DMSO-d6) δ 1.30-2.10 (m, 6H, 3xCH2), 2.67 (d, 1H, (DMSO-d6) δ: 1.20-1.77 (m, 6H, 3xCH2), (t, 4H, CH-pyrimidine), 3.72 (s, 12H, 4xOCH3), 2.11 (d, 1H, 2xCH2), 2.77 (m, 1H, CH), 3.76 (s, 12H, 4xOCH3), CH pyrimidine), 6.45 (s, 1H, CH-olefinic), 6.70-7.1 3.86 (d, 1H-CH-oxazole), 6.68 (s, 1H, CH), 7.02-7.5 (m, 6H, Ar-H), 9.8 (s, 1H, NH). MS (70ev) m/z%, + + (m, 6H, Ar-H). MS (70ev) m/z%, 409 (M ) (1.99). (452) (M ) (0.13). Anal. Calcd. for C25H28N2O4S Anal. Calcd. for C24H27NO5 (409.19); C, 70.40; H, (452.18); C, 66.35; H, 6.24; N, 6.19 Found: C, 66.31; 6.65; N, 3.42 Found: C, 70.38; H, 6.63; N, 3.47%. H, 6.18; N, 6.25%.

2,2',6,6'-Tetrakis(3,4-dimethoxybenzylidene)-[1,1'- 2-Amino-8-(3,4-dimethoxybenzylidene)-4-(3,4- bi(cyclohexane] 1,1'-diyl diacetate (7): dimethoxyphenyl)-3,4,5,6,7,8-hexahydroquinoline-3- carbonitrile (11): A mixture of 1 (0.01 mole), zinc dust (5 gm), anhydrous sod. acetate (2.5 gm) and acetic anhydride

1052 Adv. Environ. Biol., 7(6): 1049-1057, 2013

Yield 82%, m.p. 108-110oC; IR (KBr) γ:1599 To a solution of 12 (0.01 mole) in ethanol -1 1 (C=N), 2191 (C≡N) cm ; HNMR (DMSO-d6) δ: 1- (20ml) and equimolar amounts of o-aminophenol or 1.83 (m, 6H, 3xCH2), 2.08 (m, 2H, CH2), 3.78 (s, p-aminopyridine was added and the reaction mixture 12H, 4xOCH3), 7.12-7.6 (m, 7H, Ar-H and 1H, CH- was refluxed for 15 hours. The solid product olefinic), 9.68 (s, 2H, NH2). MS (70ev) m/z% 459 obtained after concentration and cooling was + (0.20) (M ). Anal. Calcd. for C27H29N3O4 (459.22); collected and re-crystallized. C, 70.57; H, 6.36; N, 9.14 Found: C, 70.89; H, 6.44; N, 9.20%. 8-(3,4-Dimethoxybenzylidene)-2-amino-4-(3,4- dimethoxyphenyl)-N-(2-hydroxyphenyl)-5,6,7,8- Ethyl-2-amino-8-(3,4-dimethoxybenzylidene)-4-(3,4- tetrahydro-4H-chromene-3-carboxamide (14a): dimethoxyphenyl)-5,6,7,8-tetrahydro-4H-chromene-3- carboxylate (12): Yield 84%, m.p. 145-147oC (ethanol); IR (KBr) γ: 1659 (C=O, amide), MS (70ev) m/z% 570 (M+) o Yield 55%, m.p. 120-122 C; IR (KBr) γ:1736 (5.00). Anal. Calcd. for C33H34N2O7 (570.24); C, 1 (C=O, ester); HNMR (DMSO-d6) δ: 0.85-1.12 (t, 69.46; H, 6.01; N, 4.91 Found: C, 69.53; H, 6.20; N, 3H, CH3CH2), 1.2-1.74 (m, 6H, 3xCH2), 2.06-2.40 4.76%. (q, 2H, CH3CH2) 2.45 (s, 1H, CH pyran), 3.73 (s, 12H, 4xOCH3), 6.64 (s, 2H, NH2), 6.83-7.59 (m, 7H, 8-(3,4-Dimethoxybenzylidene)-2-amino-4-(3,4- Ar-H and –CH olefinic). MS (70ev) m/z% 510 dimethoxyphenyl)-N-(pyridine-4-yl)-5,6,7,8- + (M +3) (22.2). Anal. Calcd. for C29H33NO7 (507.23); tetrahydro-4H-chromene-3-carboxamide (14b): C, 68.62; H, 6.55; N, 2.76 Found: C, 68.50; H, 6.76; N, 2.82%. Yield 63%, m.p. 110-112oC (diethylether); IR (KBr) γ: 1658 (C=O, amide), 1597(C=N); 1HNMR 8-(3,4-Dimethoxybenzylidene)-4-(3,4- (DMSO-d6) δ: 1.00-1.84 (m, 6H, 3xCH2), 2.08 (s, dimethoxyphenyl)-2-oxo-1,2,3,4,5,6,7,8- 2H, NH2), 3.75 (s, 12H, 4xOCH3), 4.04 (s, 1H, CH) octahydroquinoline-3-carbonitrile (13): 5.83-7.88 (m, 10H, Ar-H and 1H, CH-olefinic), 7.59 (s, 1H, NH), MS (70ev) m/z% 555 (M+) (1.7). Anal. A mixture of 1 (0.01 mole), ethyl cyanoacetate Calcd. for C32H33N3O6 (555.24); C, 69.17; H, 5.99; (0.01 mole) and ammonium acetate (2 g) was fused N, 7.56 Found: C, 69.32; H, 6.20; N, 7.82%. at 140-150oC for 15 hours. The obtained solid was washed with water and crystallized from ethanol. 8-(3,4-Dimethoxybenzylidene)-2-chloro-4-(3,4- Yield 73%, m.p. 108-110oC; IR (KBr) γ: 1682 dimethoxyphenyl)-3,4,5,6,7,8-hexahydroquinoline-3- (C=O), 2215 (C≡N), 3192 (NH); 1HNMR (DMSO- carbonitrile (16): d6) δ: 1.23-2.08 (m, 6H, 3xCH2), 3.71 (s, 12H, 4xOCH3), 3.83 (d, 1H, CH pyridine) 4.7 (d, 1H, CH A mixture of 13 (0.01 mole) and POCl3 (10ml) pyridine), 6.66-7.99 (m, 7H, Ar-H and CH olefinic), was gently refluxed for 3 hours, cooled, treated with 9.74 (s, 1H, NH). MS (70ev) m/z% 460 (M+) (72.19). crushed ice and the precipitated solid was collected Anal. Calcd. for C27H28N2 O5 (460.20); C, 70.42; H, and crystallized from ethanol: Yield 56%, m.p. 118- 6.13; N, 6.08 Found: C, 70.50; H, 6.15; N, 6.10%. 120oC; IR (KBr) γ: no γ C=O, 1600 (C=N), 2221 1 (C≡N); HNMR (DMSO-d6) δ: 1.17-1.8 (m, 6H, Ethyl-8-(3,4-dimethoxybenzylidene)-4-(3,4- 3xCH2), 2.89 (d, 1H, CH), 3.51 (d, 1H, CH), 371 (s, dimethoxyphenyl)-2-oxo-3,4,5,6,7,8-hexahydro-2H- 12H, 4xOCH3), 6.7-7.32 (m, 6H, Ar-H and 1H, CH- chromene -3-carboxylate (10): olefinic). MS (70ev) m/z% 478 (M+) (11.83). Anal. Calcd. for C27H27ClN2O4 (478.17); C, 67.71; H, 5.68; A solution of enone 1 (0.01 mole) in dry N, 5.85 Found: C, 67.40; H, 5.70; N, 5.96%. benzene (10ml) was added to suspension of ethyl sodiomalonate (0.4 g Na + 15ml dry benzene + 8-(3,4-Dimethoxybenzylidene)-4-(3,4- diethyl malonate (0.01 mole)); The mixture was dimethoxyphenyl)-2-methoxy-3,4,5,6,7,8- refluxed for 2 hours. After cooling, shake with ice- hexahydroquinoline-3-carbonitrile (17): cool dil. H2SO4, sodium carbonate solution and water successively. Extract with ether. The solid obtained A mixture of 16 (0.01 mole) and sodium after removing the solvent was crystallized from pet. methoxide [Na (0.1 mole) + absolute methanol 50ml] ether (60-80oC): Yield 55%, m.p. 112-114oC; IR was refluxed for 10 hours. The solid obtained after (KBr) γ: 1766 (C=O, ester), 1730 (C=O) cm-1; MS evaporation of the solvent was washed with water, (70ev) m/z% 508 (M+) (57.96). Anal. Calcd. for then crystallized from ethanol: Yield 72%, m.p. 130- o C29H32O8 (508.21); C, 68.49; H, 6.34; Found: C, 132 C; IR (KBr) γ: 1601 (C=N), 2219≡N); (C 1 68.50; H, 6.50%. HNMR (DMSO-d6) δ: 1.23-1.90 (m, 6H, 3xCH2), 2.78 (d, 1H, CH pyridine), 3.2 (d, 1H, CH pyridine), Reaction of 12 with aromatic amines: 3.32 (s, 3H, OCH3), 3.63 (s, 12H, 4xOCH3), 6.63- 7.82 (m, 6H, Ar-H and 1H, CH olefinic). MS (70ev)

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+ m/z% 474 (M ) (3.18). Anal. Calcd. for C28H30N2O5 (474.22); C, 70.87; H, 6.37; N, 5.90 Found: C, 70.62; Testing for anti-fungal activity: H, 6.51; N, 5.76%. Active inoculum for experiments were prepared Biological activity: by transferring many loopfuls of spores from the stock cultures to test tubes of sterile distilled water The antimicrobial screening of some of the (SDW) that were agitated and diluted with sterile synthesized compounds was done using the distilled water to achieve optical density standardized disc-agar diffusion method18. The corresponding to 2.0x105 spore/ml. Inoculum of possible antimicrobial activities of compounds 2a- 0.1% suspension was swabbed uniformly and the b,3,5-7,9-13,14b,16,18 were investigated to use inoculum was allowed to dry for 5 minutes then the standard organisms including the Gram-positive same procedure was followed as described above. bacteria: Staphylococcus aureus (ATCC 25923) and Bacillus subtilis (ATCC 6635), Gram-negative Measurement of minimal inhibition concentration bacteria: Escherichia coli (ATCC 25922) and (MIC): Salmonella typhimuruim (ATCC 14028), Yeast: Candida albicans (ATCC 10231) and Fungus MIC values of the synthesized compounds were funigatus. The obtained results are presented in Table determined using agar dilution technique [19]. Each 1. compound with high or intermediate antimicrobial Measurement of minimal inhibition effect shown in the disk diffusion test was further concentration (MIC) in Table 2. diluted with DMF to 25.6, 12.8, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, and 0.1 mg/ml respectively. Then 100μl of Screening for the anti-microbial potential: each diluted compound was mixed with 10ml of cooled (50oC) melted Mueller-Hinton agar and then Preparation of tested compound: plated into 6cm sterile Petri dish. Each dilution was prepared in duplication. The concentrations of the The tested compounds were dissolved in compounds became 256, 128, 64, 32, 16, 8, 4, 2, and dimethyl formamide (DMF) solvent and prepared in 1μg/ml respectively. Each concentration was two concentrations; 100 and 50 mg/ml and then 10μl prepared for 2 dishes. All plates were incubated at of each preparation was dropped on disk of 6 mm in 33oC for 24 hours. MIC of each compound was diameter and the concentrations became 1 and 0.5 measured from the plate with the lowest mg/disk respectively. In the case of insoluble concentration with no visible growth of specific compounds, the compounds were suspended in DMF organism. and vortexed then processed. Data in Table 1 emphasized the fact that the chemical agents symbolized 2a,b,3,5,9-12,16,18 Testing for anti-bacterial and yeasts activity: exhibit various degree of activities against Gram positive, Gram negative bacteria and fungi. Against Bacterial cultures were grown in nutrient broth Bacillus subtilis compound 16 shows high activity at medium at 30oC. After 16 h of growth, each high and low concentrations (1mg ml–1 and 0.5 mg microorganism, at a concentration of 108 cells/ml, ml-1), compounds 3, 18 show intermediate activity at was inoculated on the surface of Mueller-Hinton agar high and low concentrations, while compounds 5, 11 plates using sterile cotton swab. Subsequently, show low activity at high and low concentrations. uniform size filter paper disks (6 mm in diameter) Against Escherichia coli compound 3 shows were impregnated by equal volume (10μl) from the intermediate activity at high and low concentrations, specific concentration of dissolved compounds and while compound 18 shows intermediate and low carefully placed on surface of each inoculated plate. activities at high and low concentration respectively. The plates were incubated in the upright position at Against Candida albicans at high concentration, 36oC for 24 hours. Three replicates were carried out compounds 3, 9, 16 show high activity, compounds for each extract against each of the test organism. 2a, 2b, 10, 11, 12, 18 show intermediate activity. Simultaneously, addition of the respective solvent While at low concentration compounds 3, 9, 16 show instead of dissolved compound was carried out as high activity, compounds 2a, 11, 18 show negative controls. After incubation, the diameters of intermediate activity, while compounds 2b, 10, 12 the growth inhibition zones formed around the disc show low activity….. were measured with transparent ruler in millimeter, averaged and the mean values were tabulated.

1054 Adv. Environ. Biol., 7(6): 1049-1057, 2013

Table 1:

Mean* of Zone diameter, nearest whole mm

Gram - positive bacteria Gram - negative bacteria Yeasts and Fungi**

Organism Staphylococcm Salmonella Candida Bacillus subtilis Escherichia coli Aspergittus aureus typhimurium albicans (ATCC 6635). (ATCC 25922) fumigatus (ATCC 25923) (ATCC 14028) (ATCC 10231)

Conc. 1 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 0.5 Sample mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml

2a – – – – – – – – 20 I 15 I – – 2b – – – – – – – – 15 I 9L – – 3 – – 15 I 12 I – – 19 I 17 I 48H 45H – – 5 – – 10L 8L – – – – – – – – 6 – – – – – – – – – – – – 7 – – – – – – – – – – – – 9 – – – – – – – – 27H 24H – – 10 – – – – – – – – 12 I 7L – – 11 – – 11L 7L – – – – 21 I 18 I – – 12 – – – – – – – – 13 I 9L – – 13 – – – – – – – – – – – – 14b – – – – – – – – – – – – 16 – – 28H 22H – – – – 28H 23H – – 18 – – 22 I 16 I – – 24 I 17L 21 I 15 I – – Control # 35 26 35 25 36 28 38 27 35 28 37 26 * = Calculate from 3 values. ** = identified on the basis of routine cultural, morphological and microspical characteristics. - = No effect. L: Low activity = Mean of zone diameter ≤ ⅓ of mean zone diameter of control. I: Intermediate activity = Mean of zone diameter ≤ ⅔ of mean zone diameter of control. H: High activity = Mean of zone diameter > ⅔ of mean zone diameter of control. #: Chloramphencol in the case of Gram-positive bacteria, Cephalothin in the case of Gram-negative bacteria and cycloheximide in the case of fungi.

Table 2: MIC µg/ml Sample 9 3 16 Canadian albicans ≤64 ≤16 ≤4 (ATCC 10231)

Evaluation of Cytotoxic Effects of some new prepared compounds: for Cytotoxicity assay, the cells were seeded in 96-well plate at a cell concentration of 1x104 cells per Cell line propagation: well in 100 µl of growth medium. Fresh medium containing different concentration of the test sample The cells where propagated in Dulbecco’s was added after 24 h of seeding. Serial two-fold modified Eagle’s medium (DMEM) supplemented dilutions of the tested chemical compound were with 10% heat-inactivated fetal bovine serum, 1% l- added to confluent cell monolayers dispensed into glutamine, HEPES buffer and 50 µg/ml gentamycin. 96-well, flat-bottomed microtiter plates (Falcon, NJ, All cells where maintained at 37oC in a humidified USA) using a multichannel pipette. The microtiter o atmosphere with 5% CO2 and were subcultured two plates were incubated at 37 C in a humidified times a week. incubator with 5% CO2 for a period of 48 h. Three Cell toxicity was monitored by determining the wells were used for each concentration of the test effect of the test samples on cell morphology and cell sample. Control cells were incubated without test viability. sample and with or without DMSO. The little percentage of DMSO present in the wells (maximal Cytotoxicity evaluation using viability assay: 0.1%) was found not to affect the experiment. After

1055 Adv. Environ. Biol., 7(6): 1049-1057, 2013 incubation of the cells for 24 h at 37oC, various and mixed thoroughly, and then the absorbance of concentration of sample (50, 25, 12.5, 6.25. 3.125 the plates were measured after gently shaken on and 1.56 µg) were added and the incubation was Microplate reader (TECAN, Inc.), using a test continued for 48 h and viable cells yield was wavelength of 490 nm. All results were corrected for determined by a colorimetric method. background absorbance detected in wells without In brief, after the end of the incubation period, added stain. Treated samples were compared with media were aspirated and the crystal violet solution the cell control in the absence of the tested (1%) was added to each well for at least 30 minutes. compounds. All experiments were carried out in The stain was removed and the plates were rinsed triplicate. The cell cytotoxic effect of each tested using tap water until all excess stain is removed. compound was calculated [20-21]. Glacial acetic acid (30%) was then added to all wells

Table 3: effect of some new prepared compounds on different types of tumor cells as cytotoxic drug. a b.c Cell lines IC50 (µg/ml) 1 3 10 11 16 Doxrubcin MCF-7 46.5 19.7 30.8 24.3 11.3 0.426 HCT 31 22.3 16.8 12.0 9.8 0.469 a Cancer cell lines where breast carcinoma cells (MCF-7), colon carcinoma cells (HCT) b The assays where performed in triplicate c Doxrubcin was used as reference drug.

Scheme (1)

Cytotoxic activity: against MCF-7 and HCT cell lines with the IC50 as shown. Compound (16) selectively exhibited The cytotoxic activity of some new prepared cytotoxic activity against MCF-7 and HCT with the compounds (1,3,10,11 and 16) were performed IC50 of 11.3 and 9.8 µg/ml respectively. Compound against two cancer cell lines using a modified (11) exerted activity against HCT cell line with the method [20-21]. The results in (table 3) showed that IC50 of 12.0 µg/ml while its activity against breast all the tested compounds exhibited cytotoxic activity carcinoma cells with the IC50 of 24.3 µg/ml. It is not

1056 Adv. Environ. Biol., 7(6): 1049-1057, 2013 able that compound (1) was the most potent compound (16) which is higher than compound (3) cytotoxic agent against MCF-7 and HCT with the and subsequently compound (1) IC50 of 46.6 and 31 µg/ml respectively where the IC50 2- Regarding the presence of a dibromo- of the control Doxrubcin was 0.426 and .469 µg/ml. moiety provide a higher activity as in compound (3) compound (3) displayed activity against MCF-7 and with respect to compound (1) which didn’t contain it. HCT of 19.7 and 22.3 µg/ml, while compound (10) 3- The presence of the 2-chloro-substituent in exerted cytotoxic activity against breast cancer and the quinolin-3-carbonitrile in compound (16) has colon cancer cell lines with IC50 of 30.8 and 16.8 increased its reactivity against cancer cells more than µg/ml. thus, compounds (1, 3, 10, 11 and 16) are the presence of the 2-amino-at the same position in new series of cytotoxic agent. quinolin-3-carbonitrile as in compound (11). This is presumably due to a high lipophilicity of compound Structural activity relationship (SAR): (16) which enhances its absorption to the cancer cells. 1- As the degree of conjugated double bonds increase the activity increases, its apparent in

O S NH2 COOC H CN O 2 5 N NH N ArHC ArHC ArHC Ar Ar Ar

(9) (11) (10) S ) 2 C CN H ( e 2 (C NH -CNH H 2 tat O NH OO 2 2 C ce 2 C A 2 H m. CN 5 ) m COOC H 2 A HN O 2 5 ArHC ArHC CNCH2COOEt CNCH2COOEt Ar Ar (1) Piperidine Amm. Acetate

NH2 (13) (12) CONHAr' O POCl3 ArHC Ar Cl N2H4.H2O CN N (14) N N ArHC a; Ar' = C H OH-O Ar OH 6 4 O b; Ar' = C5H4N-p O ArHC H 2 H 4. (16) Ar N 2 N N

NH CH3ONa HN 2 (15) ArHC Ar OCH3 CN (18) N ArHC Ar = C6H3(CH3)2-3,4- Ar

(17) Scheme (2)

1057 Adv. Environ. Biol., 7(6): 1049-1057, 2013

References 12. Kgokong, J.L., G.M. Matsabisa and J.C. Breytenbach, 2001. Arznein Forsch, 51(2): 163- 1. Parikh, K.S. and S.P. Vyas, 2013. Journal of 168. chemical and Pharmaceutical Research, 5(2): 13. Egan, T., R. Hunter, C.H. Kaschula, H.M. 216-219. Marques, A. Misplon and J. Walden, 2000. J. 2. Parekh, C., A. Modi, J. Pillai and B. Patel, 2012. Med. Chem, 43(2): 283-291. Int.J. Drug Res. Tech., 2(3): 279-288. 14. Beneteau, V., A. Pierre, B. Pfeiffer, P. Renard 3. Tzanetou, E., S. Liekens, K.M. Kasiotis, N. and T. Besson, 2000. Bioorg Med Chem. Lett Fokialakis and S.A. Haroutounian, 2012. Arch 10(19): 2231-2234. Pharm Chem, 345: 804-811. 15. Renanasiddappa, B.C., E.V.S. Subrahmayan, D. 4. Mosti, L., G. Menozzi, P. Fossa, W. Filippelli, Satyanarayana and J. Thomas, 2009. Inter J. S. Gessi, B. Rinalde and G. Falcone, 2000. Chem. Tech. Research, 1(4): 1100-1104. Arzneimittelforschung, 50: 963-972. 16. El-Gabry, M.S.A., M.A. Zahran, M.M.F. Ismail 5. Goodman, K.B., H. Cui, S.E. Dowdell, D.E. and Y.A. Ammar, 2000. Farmaco, 55(3): 227- gaitanopoulos, R.L. Ivy, C.A. Sehon, R.A. 232. Stavenger, G.Z. Wang, A.Q. Viet, W. Xu et al. 17. Tsuda, H., Y. Ishitani, Y. Takemura, Y. Suzuki 2007. J. Med. Chem., 50: 6-9. and T. Kato, 1997. Heterocycles, 44(1): 139- 6. El-Azab, A.S., M.A. Al-Omar, A.A. Abdel Aziz, 142. N.I. Abdel-Aziz, M.A.A. El-Sayed, A.M. Aleisa, 18. Bauer, A.W., W.W.M. Kirby, J.C. Sherrio and M.M. Sayed Ahmed and S.G. Abdel Hamide, M. Turck, 1966. Antibiotic susceptibility testing 2010. Eur. J. Med. Chem., 45: 4188-4198. by a standardized single disc method American 7. El-Gaby, M.S.A., 2000. Phosphorus, Sulfur, Journal of Clinical Pathology, 45: 493-496. Silicon Relat Elem., 156: 157-172. 19. Andrews, J.M., Determination of minimum 8. Kunes, J., J. Bazant, M. Pour, K. Waisser, M. inhibitory concentration Journal of Slosarek and J. Farmaco, 2000. 55(11-12): 725- Antimicrobial Chemotherapy, 48(1): 5-16. 729. 20. Mosmann, T., 1983. Rapid colorimetric assay 9. Muijbwijk Koezen, J.E., H. Van Timmerman, H. for cellular growth and survival application to Goot, W.M.P.B. Menge, J. Kuenzel, D. Frytag, proliferation and cytotoxicity assays J. Immunol M. Groote and A.P. Ijzerman, 2000. J. Med. Methods, 65: 55-63. Chem., 43(11): 2227-2238. 21. Vijayan, P., C. Raghu, G. Ashok, S.A. Dhanaraj 10. Usifoh, C.O. and G.K.E. Scriba, 2000. Arch and B. Suresh, 2004. Antiviral activity of Pharm, 333(8): 261-266. medicinal plants of Nilgiris, Indian J. Med Res., 11. Abdel-Gawad, S.M., M.S.A. El-Gaby and M.M. 120: 24-29. Ghorab Farmaco, 2000. 55(4): 287-292.