Synthesis, Characterization and in Vitro Antimicrobial

Journal of Saudi Chemical Society (2011) xxx, xxx–xxx

King Saud University Journal of Saudi Chemical Society

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ORIGINAL ARTICLE Synthesis, characterization and in vitro antimicrobial activity of some 1-(substitutedbenzylidene) -4-(4-(2-(methyl/phenyl)-4-oxoquinazolin-3(4H)- yl)phenyl)semicarbazide derivatives

Govindaraj Saravanan a,*, Veerachamy Alagarsamy b, Chinnasamy Rajaram Prakash c a Medicinal Chemistry Research Laboratory, Bapatla College of Pharmacy, Jawaharlal Nehru Technological University, Hyderabad, Andhra Pradesh, India b Medicinal Chemistry Research Laboratory, M.N.R. College of Pharmacy, Sangareddy, Andhra Pradesh, India c Department of Medicinal Chemistry, DCRM Pharmacy College, Inkollu, Andhra Pradesh, India

Received 19 October 2011; accepted 6 December 2011

KEYWORDS Abstract A series of 1-(substitutedbenzylidene)-4-(4-(2-(methyl/phenyl)-4-oxoquinazolin-3(4H)-yl) Quinazolin-4(3H)-one; phenyl)semicarbazide derivatives were synthesized with the aim of developing potential antimicro- Semicarbazide; bials. It was characterized by FT-IR, 1H NMR, Mass spectroscopy and elemental analysis. In addi- Schiff base; tion, the in vitro antibacterial and antifungal properties were tested against some human pathogenic Antibacterial activity; microorganisms by employing the disc diffusion technique and agar streak dilution method. All title Antifungal activity compounds showed activity against the entire strain of microorganisms. The relationship between the functional group variation and the biological activity of the evaluated compounds were well dis- cussed. Based on the results obtained, compound 5j was found to be very active compared to the rest of the compounds which were subjected to antimicrobial assay. ª 2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

* Corresponding author. Address: Dept. of Medicinal Chemistry, Bapatla College of Pharmacy, JNT University, Hyderabad, Andhra Pradesh, India. Mobile: +91 9963023257. E-mail address: [email protected] (G. Saravanan).

Peer review under responsibility of King Saud University. doi:10.1016/j.jscs.2011.12.010

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Please cite this article in press as: Saravanan, G. et al., Synthesis, characterization and in vitro antimicrobial activity of some 1- (substitutedbenzylidene) 2 G. Saravanan et al.

1. Introduction broad spectrum of in vitro and in vivo chemotherapeutic activities (El-Gazzar et al., 2009). Diverse examples of few antimi- Microbial infections are a growing problem in contemporary crobial quinazolinone are indicated in Fig. 1. Quinazolinone medicine, and the use of antibiotics is inevitable. The global derivatives such as 2-((2,6-dichlorophenylamino)methyl)qui- sales of antibiotics are generally higher when compared to nazolin-4(3H)-one I exerted antibacterial effect against Staphy- other drugs which are prescribed. Antibiotic resistance is a lococcus aureus at a very low concentration (Jantova et al., major problem in hospitals as well as in community settings. 2004); while 2-(6,8-dichloro-4-oxo-2-phenylquinazolin-3(4H)- Morbidity and mortality due to enteric bacterial infection is yl)cyclopent-4-ene-1,3-dione II and 3-(benzylideneamino)-6,8- most common around the world and in specific regions such dibromo-2-phenylquinazolin-4(3H)-one III exhibited potent as the Indian subcontinent, part of South America and tropical antimicrobial activities (Zhou et al., 2004; Panneerselvam part of Africa are worst affected (Qadri et al., 2005; Devasia et et al., 2009). A significant in vivo activity, low toxicity and good al., 2006). A continuous increase in the number of infections pharmacokinetic profile were exhibited by 7-chloro-3-(3-(2,4- caused by bacterial resistance to one or multiple class of anti- difluorophenyl)-4-(1H-1,2,4-triazol-1-yl)butan-2-yl)qui nazo- biotics poses a significant threat as it may lead to treatment lin-4(3H)-one IV (Bartroli et al., 1998; Chandrika et al., failures and associated complications (Beekmann et al., 2005; 2008). Quinazolin-4(3H)-ones with substitution such as substi- Spellberg et al., 2008). Thus the treatment of bacterial infec- tuted phenyl ring moieties (Abdel-Rahman, 1997), bridged tions remains a challenging therapeutic problem. Despite the phenyl rings (Kumar et al., 1982; Hassan et al., 1991), hetero- many antibiotics and chemotherapeutics available, the emer- cyclic rings (Pandey et al., 2009) and aliphatic systems (El- gence of old and new antibiotic resistant bacterial strains in Sharief et al., 2001; Ouyang et al., 2006) at position 3, were re- the last decades constitutes a substantial need for new classes ported to be associated with antimicrobial properties (Moham- of antibacterial agents (Chopra et al., 2008). Moreover a ed et al., 2010). spread of resistance among the common respiratory pathogens On the other hand, Schiff bases have gained importance be- was recognized as one of the three major areas of concern by cause of the physiological and pharmacological activities the Infectious Diseases Society of America which creates a associated with them. Compounds containing azomethine need for the development of newer antibiotics (Spellberg group (–C‚N–) in the structure are known as Schiff bases, et al., 2008). which are usually synthesized by the condensation of primary The importance of heterocyclic compounds has long been amines and active carbonyl groups. Schiff bases are well known recognized in the field of synthetic organic chemistry and has for their pharmacological properties like antibacterial, anti- been extensively studied due to their important properties fungal, anticancer and antiviral agents (Wang et al., 2001). and applications at present. It is well known that a number Since the quinazolinone moiety seems to be a possible of heterocyclic compounds containing nitrogen exhibited a pharmacophore in various pharmacologically active agents, we wide variety of biological activity. Among these compounds, decided to synthesize compounds with this functionality quinazolinone derivatives have become especially noteworthy coupled with Schiff base as possible antimicrobial agents which in recent years. The quinazolinone derivatives have emerged could furnish better therapeutic results. In view of the facts as antimicrobial agents of an immense interest because of their mentioned above and as part of our efforts to discover

O O O Cl H Cl N N H N O N N

Cl Cl 2-((2,6-dichlorophenylamino)methyl) quinazolin-4(3H)-one (I) 2-(6,8-dichloro-4-oxo-2-phenylquinazolin- 3(4H)-yl)cyclopent-4-ene-1,3-dione (II)

O CH3 O

N Br N C H N N N

N Cl N N F

3-(benzylideneamino)-6,8-dibromo- F 2-phenylquinazolin-4(3H)-one (III) 7-chloro-3-(3-(2,4-difluorophenyl)-4-(1H-1,2,4- triazol-1-yl)butan-2-yl)quinazolin-4(3H)-one (IV)

Figure 1 Diverse examples of antimicrobial quinazolinone.

Please cite this article in press as: Saravanan, G. et al., Synthesis, characterization and in vitro antimicrobial activity of some 1- (substitutedbenzylidene) Synthesis, characterization and in vitro antimicrobial activity 3 potentially active new agents, various quinazolin-4(3H)-ones obtained was filtered, dried and recrystallized from ethanol derivatives 5a–n were synthesized and evaluated for their antimi- (Alagarsamy et al., 2002). crobial activity. 2.3.1.1. 2-Methyl-4H-benzo-(1,3)-oxazin-4-one (1a). Yield: 2. Materials and methods 71% m.p. 182 C. IR: (KBr, cmÀ1) 3096 (Ar C–H), 2882 (CH3 C–H), 1712 (C‚O), 1636 (C‚N), 1600 (C‚C), 1055 1 2.1. General (C–O–C). H NMR (CDCl3, 300 MHz) d ppm: 2.38 (3H, s, CH3), 6.92–7.40 (4H, m, Ar–CH). ESI–MS: MS: m/z 161 + All solvents used were of laboratory grade and were obtained (M ). Anal. Cald for C9H7NO2: C, 67.07; H, 4.38; N, 8.69. from SD fine chemicals (Mumbai, India), and Merck (Mumbai, Found: C, 67.16; H, 4.40; N, 8.66. India). Melting points were determined in open glass capillary tubes and were uncorrected. Compounds were routinely 2.3.1.2. 2-Phenyl-4H-benzo-(1,3)-oxazin-4-one (1b). Yield: À1 checked for their purity on Silica gel G (Merck) thin layer chro- 80% m.p. 120 C. IR: (KBr, cm ) 3077 (Ar C–H), 1751 1 matography (TLC) plates. Iodine chamber and UV lamp were (C‚O), 1625 (C‚N), 1616 (C‚C), 1038 (C–O–C). H used for visualization of TLC spots. The IR spectra were re- NMR (CDCl3, 300 MHz) d ppm: 6.95–7.78 (9H, m, Ar–CH). + corded in KBr pellets on (BIO-RAD FTS) FT-IR spectropho- ESI–MS: MS: m/z 223 (M ). Anal. Cald for C14H9NO2:C, tometer. 1H NMR spectra were recorded on Bruker DPX-300 75.33; H, 4.06; N, 6.27. Found: C, 75.42; H, 4.05; N, 6.29. NMR spectrometer in CDCl3 using tetramethylsilane (TMS) as an internal standard. The chemical shifts were reported in 2.3.2. Synthesis of 3-(4-aminophenyl)-2-(methyl/phenyl)- ppm scale. Mass spectra were obtained on a JEOL-SX-102 quinazolin-4(3H)-one (2a–2b) instrument using electron impact ionization. Elemental analyses 4H-benzo-(1,3)-oxazin-4-one 1a/1b (1.61/2.23 g, 0.01 mol) and for C, H, and N were performed on a Perkin Elmer model 240C p-phenylenediamine (1.08 g, 0.01 mol) were dissolved in 50 ml analyzer and were within ±0.4% of the theoretical values. of anhydrous pyridine and heated on sand bath for 6 h. The resulting solution was cooled in ice bath and treated with 2.2. Test microorganisms and medium 100 ml of dilute hydrochloric acid. The products thus separated 2a/2b were filtered, washed with water, and recrystallized All the microorganisms used in this study were purchased from from ethanol. CL laboratories, Chennai, India. All the synthesized compounds were screened for antimicrobial activities by paper disc 2.3.2.1. 3-(4-Aminophenyl)-2-methylquinazolin-4(3H)-one À1 diffusion technique. The antibacterial activity of the (2a). Yield: 73% m.p. 227 C. IR: (KBr, cm ) 3380 (NH), ‚ compounds were evaluated against four Gram-positive bacte- 3068 (Ar C–H), 2954 (CH3 C–H), 1712 (C O), 1641 ‚ ‚ 1 ria (S. aureus ATCC 9144, Staphylococcus epidermidis ATCC (C N), 1609 (C C). H NMR (CDCl3, 300 MHz) d ppm: 155, Micrococcus luteus ATCC 4698, and Bacillus cereus 2.63 (3H, s, CH3), 3.76 (2H, s, NH2), 6.92–7.97 (8H, m, Ar– + ATCC 11778) and three Gram-negative bacteria (Escherichia CH). ESI–MS: MS: m/z 251 (M ). Anal. Cald for coli ATCC 25922, Pseudomonas aeruginosa ATCC 2853, and C15H13N3O: C, 71.70; H, 5.21; N, 16.72. Found: C, 71.92; Klebsiella pneumoniae ATCC 11298). The antifungal activities H, 5.19; N, 16.67. of the synthesized compounds were evaluated against two fungi (Aspergillus niger ATCC 9029 and Aspergillus fumigatus 2.3.2.2. 3-(4-Aminophenyl)-2-phenylquinazolin-4(3H)-one À1 ATCC 46645). The minimum inhibitory concentrations (2b). Yield: 77% m.p. 287 C. IR: (KBr, cm ) 3395 (NH), (MIC) of the compounds were also determined by agar streak 3091 (Ar C–H), 1735 (C‚O), 1650 (C‚N), 1617 (C‚C). 1 dilution method. Bacterial strains were cultured over night at H NMR (CDCl3, 300 MHz) d ppm: 3.88 (2H, s, NH2), + 37 C in Mueller–Hinton broth and the yeast was cultured 6.76–8.09 (13H, m, Ar–CH). ESI–MS: MS: m/z 313 (M ). overnight at 30 C in YEPDE agar for antibacterial and anti- Anal. Cald for C20H15N3O: C, 76.66; H, 4.82; N, 13.41. fungal activity tests. Test strains were suspended in nutrient Found: C, 76.42; H, 4.83; N, 13.45. agar to give a final density of 5 · 10À5 cfu/ml. 2.3.3. Synthesis of 1-(4-(2-(methyl/phenyl)-4-oxoquinazolin- 2.3. Chemistry 3(4H)-yl)phenyl)urea (3a–3b) Compound 2a/2b (2.51/3.13 g, 0.01 mol) was dissolved in 2.3.1. Synthesis of 2-(methyl/phenyl)-4H-benzo-(1,3)-oxazin- 10 ml glacial acetic acid and the volume was diluted to 4-one (1a–1b) 100 ml with the same in a conical flask. It was added with a For the synthesis of 2-methyl derivative a mixture of anthranilic solution of sodium cyanate (0.65 g, 0.01 mol) and 50 ml of acid (1.37 g, 0.01 mol) and acetic anhydride (10.2 ml, 0.1 mol) warm water slowly with continuous stirring. Then the reaction was refluxed at 50 C for 1 h. The excess of acetic anhydride mixture was allowed to stand for 30 minutes at room temper- was distilled off under reduced pressure and the residue was dis- ature, and then cooled in crushed ice, allowed to stand for a solved in petroleum ether and was kept aside for 1 h. The light further period of 30 minutes in ice bath. The product 3a/3b brown solid 1a was obtained. It was further filtered, dried and thus obtained was filtered and washed with cold water, finally immediately used for the next step (Alagarsamy et al., 2003). dried at 100 C. The product was recrystallized at least once To a solution of anthranilic acid (13.7 g, 0.1 mol) dissolved from ethanol to give the pure form. in pyridine (60 ml), benzoyl chloride (28 g, 0.2 mol) was added fraction wise slowly with stirring to synthesize 2-phenyl deriv- 2.3.3.1. 1-(4-(2-Methyl-4-oxoquinazolin-3(4H)-yl)phenyl)urea ative. The mixture was stirred for 30 min at room temperature (3a). Yield: 76% m.p. 218 C. IR: (KBr, cmÀ1) 3362 (NH), ‚ followed by treatment with 5% NaHCO3 (15 ml). The solid 1b 3077 (Ar C–H), 2948 (CH3 C–H), 1736 (C O), 1630

Please cite this article in press as: Saravanan, G. et al., Synthesis, characterization and in vitro antimicrobial activity of some 1- (substitutedbenzylidene) 4 G. Saravanan et al.

1 (C‚N), 1605 (C‚C). H NMR (CDCl3, 300 MHz) d ppm: 2.3.5. General procedure for the synthesis of 5a–5n 2.40 (3H, s, CH3), 3.64 (2H, s, NH2), 7.06–8.12 (8H, m, Ar– Title compounds 5a–5n were synthesized by adding 4-(4-(2- CH), 8.49 (1H, s, urea-NH). ESI–MS: MS: m/z 294 (M+). (methyl/phenyl)-4-oxoquinazolin-3(4H)-yl)phenyl)semicarbaz- Anal. Cald for C16H14N4O2: C, 65.30; H, 4.79; N, 19.04. ide 4a/4b (3.09/3.71 g, 0.01 mol) as fraction portions with the Found: C, 65.51; H, 4.78; N, 18.98. well stirred mixture of different aromatic aldehydes (0.01 mol) in ethanol (50 ml) and 5 ml of glacial acetic acid. 2.3.3.2. 1-(4-(4-Oxo-2-phenylquinazolin-3(4H)-yl)phenyl)urea Then this mixture was refluxed at 100 C for 6–8 h and kept (3b). Yield: 72% m.p. 246 C. IR: (KBr, cmÀ1) 3347 (NH), aside for 24 h. The product that separated out was filtered, 3072 (Ar C‚H), 1748 (C‚O), 1635 (C‚N), 1603 (C‚C). washed with water, dried, and recrystallized from ethanol. 1 H NMR (CDCl3, 300 MHz) d ppm: 3.79 (2H, s, NH2), The method used for the preparation and isolation of the com- 6.95–7.92 (13H, m, Ar–CH), 8.54 (1H, s, urea-NH). pounds gave materials of good purity, as evidenced by their + ESI–MS: MS: m/z 356 (M ). Anal. Cald for C21H16N4O2: spectral analyses and by thin layer chromatography. The title C, 70.77; H, 4.53; N, 15.72. Found: C, 71.01; H, 4.52; compounds are found to be soluble in chloroform, dimethyl N, 15.75. sulfoxide, and dimethylformamide. The physicochemical properties of the synthesized compounds are given in Table 1. 2.3.4. Synthesis of 4-(4-(2-(methyl/phenyl)-4-oxoquinazolin- 3(4H)-yl)phenyl)semicarbazide (4a–4b) 2.3.6. Spectral data of synthesized compounds 5a–5n The compounds 3a/3b (2.94/3.56 g, 0.01 mol) were dissolved in 2.3.6.1. 1-Benzylidene-4-(4-(2-methyl-4-oxoquinazolin-3(4H)- 30 ml of absolute ethanol. To this 2.5 ml of hydrazine hydrate yl)phenyl)semicarbazide (5a). IR: (KBr, cmÀ1) 3348 (NH), was added fraction wise. After complete addition of hydrazine 3042 (Ar C–H), 2956 (CH3 C–H), 1729 (C‚O), 1635 1 hydrate the entire mixture was refluxed at 100 C for 10–12 h (C‚N), 1623 (C‚C). H NMR (CDCl3, 300 MHz) d ppm: with stirring. The contents were concentrated to half of its vol- 2.54 (3H, s, CH3), 6.98–7.81 (13H, m, Ar–CH), 8.17 (1H, s, ume and poured onto crushed ice. The resultant precipitate urea-NH), 8.45 (1H, s, N‚CH), 9.82 (1H, s, CONH). ESI– was filtered, thoroughly washed with water, dried, and recrys- MS: MS: m/z 397 (M+). tallized from ethanol. 2.3.6.2. 1-(4-Nitrobenzylidene)-4-(4-(2-methyl-4-oxoquinazo- 2.3.4.1. 4-(4-(2-Methyl-4-oxoquinazolin-3(4H)-yl)phenyl)semi lin-3(4H)-yl)phenyl)semicarbazide (5b). IR: (KBr, cmÀ1) À1 carbazide (4a). Yield: 70% m.p. 283 C. IR: (KBr, cm ) 3383 3384 (NH), 3097 (Ar C–H), 2942 (CH3 C–H), 1733 (C‚O), ‚ 1 (NH), 3050 (Ar C–H), 2927 (CH3 C–H), 1745 (C O), 1649 1628 (C‚N), 1606 (C‚C), 1546 & 1315 (NO2). H NMR ‚ ‚ 1 (C N), 1617 (C C). H NMR (CDCl3, 300 MHz) d ppm: (CDCl3, 300 MHz) d ppm: 2.38 (3H, s, CH3), 7.13–8.07 2.54 (3H, s, CH3), 5.78 (2H, s, NH2), 6.92–8.06 (8H, m, (12H, m, Ar–CH), 8.35 (1H, s, urea-NH), 8.76 (1H, s, Ar–CH), 8.31 (1H, s, urea-NH), 9.86 (1H, s, CONH). N‚CH), 9.99 (1H, s, CONH). ESI–MS: MS: m/z 442 (M+). + ESI–MS: MS: m/z 309 (M ). Anal. Cald for C16H15N5O2: C, 62.13; H, 4.89; N, 22.64. Found: C, 62.32; H, 4.88; 2.3.6.3. 1-(4-Hydroxybenzylidene)-4-(4-(2-methyl-4-oxoqui- N, 22.57. nazolin-3(4H)-yl)phenyl)semicarbazide (5c). IR: (KBr, cmÀ1) 3512 (OH), 3367 (NH), 3045 (Ar C–H), 2931 (CH3 C–H), 1725 1 2.3.4.2. 4-(4-(4-Oxo-2-phenylquinazolin-3(4H)-yl)phenyl)semi (C‚O), 1644 (C‚N), 1618 (C‚C). HNMR(CDCl3, À1 carbazide (4b). Yield: 79% m.p. 255 C. IR: (KBr, cm ) 3389 300 MHz) d ppm: 2.41 (3H, s, CH3), 5.87 (1H, s, OH), 6.86–7.99 (NH), 3025 (Ar C–H), 1744 (C‚O), 1652 (C‚N), 1618 (12H,m,Ar–CH), 8.23 (1H, s, urea-NH), 8.52 (1H, s, N‚CH), 1 + (C‚C). H NMR (CDCl3, 300 MHz) d ppm: 5.85 (2H, s, 9.85 (1H, s, CONH). ESI–MS: MS: m/z 413 (M ). NH2), 7.15–8.11 (13H, m, Ar–CH), 8.42 (1H, s, urea-NH), 9.97 (1H, s, CONH). ESI–MS: MS: m/z 371 (M+). Anal. Cald 2.3.6.4. 1-(4-(Dimethylamino)benzylidene)-4-(4-(2-methyl-4- for C21H17N5O2: C, 67.91; H, 4.61; N, 18.86. Found: C, 67.65; oxoquinazolin-3(4H)-yl)phenyl) semicarbazide (5d). IR: À1 H, 4.63; N, 18.92. (KBr, cm ) 3355 (NH), 2989 (Ar C–H), 2950 (CH3 C–H),

Table 1 Physicochemical data of all synthesized test compounds (5a–5n).

Compound R R1 M.p. (C) Yield (%) Molecular formula Analysis (%), found (calc.): C; H; N

5a CH3 H 194–196 72 C23H19N5O2 69.33 (69.51); 4.84 (4.82); 17.67 (17.62) 5b CH3 4-NO2 201–204 79 C23H18N6O4 62.60 (62.44); 4.11 (4.10); 18.95 (19.00) 5c CH3 4-OH 232–234 77 C23H19N5O3 66.99 (66.82); 4.61 (4.63); 16.88 (16.94) 5d CH3 4-N(CH3)2 265–267 75 C25H24N6O2 68.36 (68.17); 5.47 (5.49); 19.12 (19.08) 5e CH3 3-NO2 219–222 70 C23H18N6O4 62.21 (62.44); 4.11 (4.10); 18.94 (19.00) 5f CH3 4-Cl 246–248 79 C23H18ClN5O2 64.18 (63.96); 4.21 (4.20); 16.17 (16.22) 5g CH3 4-OH-3-OCH3 228–230 74 C24H21N5O4 64.79 (65.00); 4.79 (4.77); 15.73 (15.79) 5h C6H5 H 169–172 78 C28H21N5O2 73.42 (73.19); 4.62 (4.61); 15.19 (15.24) 5i C6H5 4-NO2 241–243 71 C28H20N6O4 66.43 (66.66); 4.01 (4.00); 16.72 (16.66) 5j C6H5 4-OH 182–184 76 C28H21N5O3 70.98 (70.73); 4.44 (4.45); 14.69 (14.73) 5k C6H5 4-N(CH3)2 253–255 70 C30H26N6O2 71.92 (71.70); 5.19 (5.21); 16.67 (16.72) 5l C6H5 3-NO2 177–179 74 C28H20N6O4 66.54 (66.66); 3.99 (4.00); 16.61 (16.66) 5m C6H5 4-Cl 260–262 81 C28H20ClN5O2 68.27 (68.08); 4.07 (4.08); 14.14 (14.18) 5n C6H5 4-OH-3-OCH3 212–214 73 C29H23N5O4 68.67 (68.90); 4.61 (4.59); 13.89 (13.85)

1 1721 (C‚O), 1612 (C‚N), 1606 (C‚C). H NMR (CDCl3, 2.3.6.12. 1-(3-Nitrobenzylidene)-4-(4-(4-oxo-2-phenylquinazo- À1 300 MHz) d ppm: 2.19 (3H, s, CH3), 2.63 (6H, s, N(CH3)2), lin-3(4H)-yl)phenyl)semicarbazide (5l). IR: (KBr, cm ) 3361 7.01–8.15 (12H, m, Ar–CH), 8.31 (1H, s, urea-NH), 8.54 (NH), 2994 (Ar C–H), 1727 (C‚O), 1625 (C‚N), 1612 1 (1H, s, N‚CH), 9.73 (1H, s, CONH). ESI–MS: MS: m/z (C‚C), 1535 & 1328 (NO2). H NMR (CDCl3, 300 MHz) d 440 (M+) ppm: 7.00–8.17 (17H, m, Ar–CH), 8.39 (1H, s, urea-NH), 8.74 (1H, s, N‚CH), 10.02 (1H, s, CONH). ESI–MS: MS: 2.3.6.5. 1-(3-Nitrobenzylidene)-4-(4-(2-methyl-4-oxoquinazo- m/z 504 (M+). lin-3(4H)-yl)phenyl)semicarbazide (5e). IR: (KBr, cmÀ1) 3350 (NH), 3038 (Ar C–H), 2934 (CH3 C–H), 1737 (C‚O), 2.3.6.13. 1-(4-Chlorobenzylidene)-4-(4-(4-oxo-2-phenylquinaz- 1 À1 1639 (C‚N), 1622 (C‚C), 1531 & 1323 (NO2). H NMR olin-3(4H)-yl)phenyl)semicarbazide (5m). IR: (KBr, cm ) ‚ ‚ (CDCl3, 300 MHz) d ppm: 2.36 (3H, s, CH3), 7.22–8.19 3354 (NH), 3048 (Ar C–H), 1732 (C O), 1637 (C N), 1629 1 (12H, m, Ar–CH), 8.40 (1H, s, urea-NH), 8.79 (1H, s, (C‚C), 765 (C–Cl). H NMR (CDCl3, 300 MHz) d ppm: N‚CH), 10.01 (1H, s, CONH). ESI–MS: MS: m/z 442 (M+). 7.19–8.22 (17H, m, Ar–CH), 8.24 (1H, s, urea-NH), 8.47 (1H, s, N‚CH), 9.95 (1H, s, CONH). ESI–MS: MS: m/z 2.3.6.6. 1-(4-Chlorobenzylidene)-4-(4-(2-methyl-4-oxoquinazo- 495 (M+2). lin-3(4H)-yl)phenyl)semicarbazide (5f). IR: (KBr, cmÀ1) 3372 (NH), 3026 (Ar C–H), 2949 (CH3 C–H), 1722 (C‚O), 1623 2.3.6.14. 1-(4-Hydroxy-3-methoxybenzylidene)-4-(4-(4-oxo-2- 1 (C‚N), 1615 (C‚C), 758 (C–Cl). H NMR (CDCl3, phenylquinazolin-3(4H)-yl)phenyl) semicarbazide (5n). IR: À1 300 MHz) d ppm: 2.25 (3H, s, CH3), 6.90–7.93 (12H, m, Ar– (KBr, cm ) 3515 (OH), 3373 (NH), 3026 (Ar C–H), 1729 1 CH), 8.19 (1H, s, urea-NH), 8.42 (1H, s, N‚CH), 9.98 (1H, (C‚O), 1614 (C‚N), 1607 (C‚C). H NMR (CDCl3, s, CONH). ESI–MS: MS: m/z 433 (M+2). 300 MHz) d ppm: 3.72 (3H, s, OCH3), 5.69 (1H, s, OH), 7.06–8.14 (16H, m, Ar–CH), 8.27 (1H, s, urea-NH), 8.51 (1H, 2.3.6.7. 1-(4-Hydroxy-3-methoxybenzylidene)-4-(4-(2-methyl- s, N‚CH), 9.74 (1H, s, CONH). ESI–MS: MS: m/z 505 (M+). 4-oxoquinazolin-3(4H)-yl)phenyl) semicarbazide (5g). IR: (KBr, cmÀ1) 3520 (OH), 3359 (NH), 2981 (Ar C–H), 2937 1 3. Biological activities (CH3 C–H), 1735 (C‚O), 1626 (C‚N), 1608 (C‚C). H NMR (CDCl3, 300 MHz) d ppm: 2.30 (3H, s, CH3), 3.77 3.1. Paper disc diffusion technique (3H, s, OCH3), 5.64 (1H, s, OH), 7.17–8.06 (11H, m, Ar– CH), 8.37 (1H, s, urea-NH), 8.80 (1H, s, N‚CH), 10.05 (1H, s, CONH). ESI–MS: MS: m/z 443 (M+). The sterilized (Gillespie, 1994) (autoclaved at 120 C for 30 min) medium (40–50 C) was inoculated (1 ml/100 ml of À5 2.3.6.8. 1-Benzylidene-4-(4-(4-oxo-2-phenylquinazolin-3(4H)- medium) with the suspension (5 · 10 cfu/ml) of the microor- yl)phenyl)semicarbazide (5h). IR: (KBr, cmÀ1) 3363 (NH), ganism (matched to McFarland barium sulfate standard) and 3075 (Ar C–H), 1738 (C‚O), 1642 (C‚N), 1629 (C‚C). poured into a petridish to give a depth of 3–4 mm. The paper 1 impregnated with the test compounds (50, 100 and 150 lg/ml H NMR (CDCl3, 300 MHz) d ppm: 6.89–7.92 (18H, m, Ar–CH), 8.14 (1H, s, urea-NH), 8.38 (1H, s, N‚CH), 9.87 in dimethyl formamide) was placed on the solidified medium. (1H, s, CONH). ESI–MS: MS: m/z 459 (M+). The plates were pre-incubated for 1 h at room temperature and incubated at 37 C for 24 and 48 h for antibacterial and 2.3.6.9. 1-(4-Nitrobenzylidene)-4-(4-(4-oxo-2-phenylquinazo- antifungal activities, respectively. Ciprofloxacin (100 lg/ml/ lin-3(4H)-yl)phenyl)semicarbazide (5i). IR: (KBr, cmÀ1) disc) and Ketoconazole (100 lg/ml/disc) were used as standard 3387 (NH), 3052 (Ar C–H), 1724 (C‚O), 1620 (C‚N), 1601 for antibacterial and antifungal activities respectively. All the 1 observed zone of inhibition is presented in Table 2. (C‚C), 1522 & 1319 (NO2). H NMR (CDCl3, 300 MHz) d ppm: 6.94–8.08 (17H, m, Ar–CH), 8.32 (1H, s, urea-NH), 8.87 (1H, s, N‚CH), 9.71 (1H, s, CONH). ESI–MS: MS: m/ 3.2. Minimum inhibitory concentration (MIC) z 504 (M+). MIC of the compound was determined by agar streak dilution method (Hawkey and Lewis, 1994). A stock solution of the syn- 2.3.6.10. 1-(4-Hydroxybenzylidene)-4-(4-(4-oxo-2-phenylqui- thesized compound (100 lg/ml) in dimethyl formamide was pre- nazolin-3(4H)-yl)phenyl)semicarbazide (5j). IR: (KBr, cmÀ1) pared and graded quantities of the test compounds were 3528 (OH), 3365 (NH), 3081 (Ar C–H), 1726 (C‚O), 1632 incorporated in a specified quantity of molten sterile agar (C‚N), 1614 (C‚C). 1HNMR(CDCl, 300 MHz) d ppm: 5.72 3 (nutrient agar for antibacterial activity and Sabouraud’s dex- (1H, s, OH), 7.25–8.14 (17H, m, Ar–CH), 8.28 (1H, s, urea-NH), trose agar for antifungal activity) medium. A specified quantity 8.63 (1H, s, N‚CH), 9.96 (1H, s, CONH). ESI–MS: MS: m/z of the medium (40–50 C) containing the compound was poured 475 (M+). into a petridish to give a depth of 3–4 mm and allowed to solid- ify. Suspension of the microorganism was prepared to contain 2.3.6.11. 1-(4-(Dimethylamino)benzylidene)-4-(4-(4-oxo-2- approximately 5 · 10À5 cfu/ml and applied to plates with seri- phenylquinazolin-3(4H)-yl)phenyl) semicarbazide (5k). IR: ally diluted compounds in dimethyl formamide to be tested À1 (KBr, cm ) 3346 (NH), 3012 (Ar C–H), 2923 (CH3 C–H), and incubated at 37 C for 24 and 48 h for bacteria and fungi, 1 1734 (C‚O), 1647 (C‚N), 1625 (C‚C). H NMR (CDCl3, respectively. The MIC was considered to be the lowest concen- 300 MHz) d ppm: 2.71 (6H, s, N(CH3)2), 6.97–8.01 (17H, m, tration of the test substance exhibiting no visible growth of bac- Ar–CH), 8.16 (1H, s, urea-NH), 8.59 (1H, s, N‚CH), 9.81 teria or fungi on the plate. The observed MIC is presented in (1H, s, CONH). ESI–MS: MS: m/z 502 (M+) Table 3.

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Table 2 Disc diffusion method results of the synthesized compounds and standard reagents (diameter of the zone of inhibition (mm)) at 50, 100 and 150 lg/ml. Compounds S. aureus S. epidermidis M. luteus B. cereus E. coli P. aeruginosa K. pneumoniae A. niger A. fumigatus 50 100 150 50 100 150 50 100 150 50 100 150 50 100 150 50 100 150 50 100 150 50 100 150 50 100 150 5a 9 16 19 11 16 20 8 14 17 7 12 16 8 15 18 7 11 15 7 12 16 7 13 15 8 15 17 5b 3 8 12 6 11 13 5 11 15 – 5 8 6 10 13 3 7 10 – 5 9 3 9 14 5 11 14 5c 13 22 25 13 23 27 14 21 23 11 20 24 12 20 25 13 22 26 15 21 25 12 19 21 10 20 23 5d 12 20 24 10 19 21 11 16 20 9 18 22 12 18 21 11 15 18 13 17 19 11 16 20 9 19 23 5e 6 10 13 5 12 15 – 7 12 5 11 14 4 8 13 3 6 10 – 7 10 6 10 14 – 6 10 5f 4 11 15 7 13 18 5 10 13 – 6 11 7 11 14 – 4 9 5 10 14 – 5 11 – 8 14 5g 12 19 24 12 21 24 11 18 22 10 17 21 11 16 20 13 17 22 10 15 18 13 18 21 9 16 20 5h 10 18 20 11 17 21 9 17 19 8 15 18 10 15 19 9 14 16 8 13 17 9 12 15 10 15 18 5i 8 12 15 9 14 16 8 13 16 6 10 12 7 12 16 4 8 10 8 11 14 6 10 13 8 13 15 5j 18 25 28 13 24 27 17 27 30 12 21 25 13 21 26 16 24 28 14 21 26 12 20 23 11 22 25 5k 15 22 26 17 29 31 14 23 25 10 20 23 16 25 29 14 20 24 13 20 23 15 22 25 14 26 29 5l 7 11 14 8 13 17 7 11 14 7 12 17 5 10 13 5 7 11 6 11 15 10 13 17 8 11 14 5m 8 13 17 10 15 18 6 12 16 5 10 14 9 13 17 4 9 12 7 12 14 4 8 11 6 10 15 5n 15 23 26 12 21 25 12 20 24 13 22 27 14 22 26 15 21 23 13 19 24 16 23 27 13 25 29 Ciproﬂoxacin (100 lg/ml) 27 28 25 24 27 22 23 – – Ketoconazole (100 lg/ml) – – – – – – – 28 30 Control – – – – – – – – –

Table 3 MIC (minimum inhibitory concentration in lg/ml) of synthesized compounds (5a–5n). Compounds S. aureus S. epidermidis M. luteus B. cereus E. coli P. aeruginosa K. pneumoniae A. niger A. fumigatus 5a 31.25 15.62 15.62 31.25 31.25 15.62 31.25 31.25 15.62 5b 31.25 31.25 31.25 62.5 31.25 31.25 62.5 31.25 31.25 5c 15.62 7.81 15.62 15.62 15.62 15.62 7.81 31.25 15.62 5d 31.25 15.62 15.62 31.25 31.25 15.62 7.81 31.25 15.62 5e 31.25 31.25 62.5 31.25 31.25 31.25 62.5 31.25 62.5 5f 31.25 31.25 31.25 62.5 31.25 62.5 31.25 62.5 62.5 5g 31.25 15.62 15.62 31.25 31.25 7.81 15.62 31.25 31.25 5h 31.25 15.62 15.62 31.25 31.25 15.62 15.62 31.25 15.62 5i 31.25 31.25 31.25 31.25 31.25 31.25 15.62 31.25 31.25 5j 7.81 15.62 15.62 7.81 31.25 7.81 3.9 31.25 7.81 5k 15.62 7.81 15.62 15.62 15.62 7.81 3.9 15.62 15.62 5l 31.25 31.25 15.62 31.25 31.25 31.25 15.62 31.25 31.25 5m 31.25 15.62 31.25 31.25 31.25 31.25 31.25 31.25 31.25 5n 15.62 15.62 7.81 7.81 31.25 15.62 7.81 31.25 15.62 Ciproﬂoxacin 15.62 7.81 7.81 7.81 15.62 7.81 3.9 – – Ketoconazole – – – – – – – 15.62 7.81

4. Results and discussion hydrate to get respective semicarbazide derivatives 4a/4b. In the last step, the compounds 5a–5n were synthesized by a Schiff base 4.1. Chemistry reaction, in which a different aromatic aldehydes (carbonyl compound) and amino derivative (quinazolinone analog) un- In this study, a series of novel quinazolin-4(3H)-one derivative dergo nucleophilic addition, forming a hemiaminal. This reac- was synthesized by substituting different 1-(substitutedbenzy- tion followed by dehydration results in title compounds 5a–5n lidene)-4-phenylsemicarbazide at third position and methyl/ by forming a stable imine according to the synthetic Scheme 1. 1 phenyl group at second position. Initially, anthranilic acid and IR, H NMR, mass spectra, and elemental analyses of the acetic anhydride/benzoyl chloride were used as starting materi- synthesized compounds are in accordance with the assigned als to synthesize 2-(methyl/phenyl)-4H-benzo-[1,3]-oxazin-4- structures. The IR spectra of all synthesized compounds one 1a/1b by a simple acetylation/benzoylation followed by ring showed some characteristic peaks indicating the presence of closure reaction. In the subsequent step, 3-(4-aminophenyl)-2- particular groups. Formation of the benzo(1,3)-oxazin-4-one (methyl/phenyl)-quinazolin-4(3H)-one 2a/2b was synthesized ring in compound 1a/1b was confirmed by the presence of À1 through simple reaction by treating compound 1a/1b with p- absorption peak at 1712–1751 and 1038–1055 cm in IR phenylenediamine with the elimination of water molecule. On due to presence of C‚O and C–O–C stretching respectively. stirring with sodium cyanate and glacial acetic acid, compounds The formations of compounds 2a/2b were confirmed by the ab- À1 get converted to their respective urea derivatives 3a/3b. Be- sence of absorption bands around 1050 cm corresponds to fore the final step, the compounds 3a/3b treated with hydrazine C–O–C stretching and appearance of peak around 3400 cm

NH O 2 O

COOH O p-NH C H NH (CH3CO)2O / 2 6 4 2 N Pyridine; 6 h C6H5COCl

NH2 N R N R (1a-1b) Anthranilic acid (2a-2b)

Glacial CH3COOH NaOCN Ethanol

H N NH2 O C

O N

1a, 2a, 3a, 4a: R= CH3 N R (3a-3b) 1b, 2b, 3b, 4b: R= C6H5

NH2NH2.H2O 5a: R= CH3, R1= H 10-12 h Ethanol 5b: R= CH3, R1= 4-NO2 Reflux 5c: R= CH3, R1= 4-OH H H 5d: R= CH3, R1= 4-N(CH3)2 N N

5e: R= CH3, R1= 3-NO2 O C NH2

5f: R= CH3, R1= 4-Cl O 5g: R= CH , R = 4-OH-3OCH 3 1 3 N 5h: R= C6H5, R1= H 5i: R= C6H5, R1= 4-NO2

5j: R= C6H5, R1= 4-OH N R (4a-4b) 5k: R= C6H5, R1= 4-N(CH3)2 5l: R= C H , R = 3-NO R 6 5 1 2 1 Ethanol 5m: R= C6H5, R1= 4-Cl CHO Glacial CH3COOH 5n: R= C H , R = 4-OH-3OCH 6 5 1 3 6-8 h

H H N N O C N

R1 O N

N R Title compounds (5a-5n)

Scheme 1 Synthetic protocols of intermediates and title compounds.

À1 in IR corresponds to N–H stretching of NH2. Appearance tra of synthesized compounds were recorded in CDCl3. The of a singlet around d 3.85 ppm for two protons in its 1H following conclusions can be derived by comparing the 1H NMR spectra which might be assigned to NH2 group also con- NMR spectra of title compounds: firms the formation of 2a/2b.In1H NMR spectra appearance of a singlet for single proton at d 8.49–8.54 ppm which might A singlet at d 2.19–2.54 ppm for 2-CH3 (only for 2-methyl be assigned to NH group of compound 3a/3b. Further pres- quinazolinone derivative), ence of a single proton in CONH of compound 4a/4b was con- A multiplet at d 6.86–8.22 ppm for Ar–CH, firmed by the appearance of a singlet at d 9.85–9.97 ppm. The A singlet at d 8.14–8.40 ppm for urea-NH, IR spectrum of title compound shows absorption bands at A singlet at d 8.38–8.87 ppm for N‚CH, 3346–3387 cmÀ1, 1721–1738 cmÀ1, and weak band at 1612– A singlet at d 9.71–10.05 ppm for CONH. 1647 cmÀ1, which can be assignable to NH, C‚O, and C‚N vibrations respectively. IR spectrum of compounds 5c, 4.2. Antimicrobial activity 5g, 5j and 5n showed an absorption band in the region of 3512–3528 cmÀ1 which may be assigned to O–H stretching. All the synthesized compounds were evaluated for their in vitro Compounds 5b, 5e, 5i, and 5l showed absorption bands at antibacterial and antifungal activities. A comparison of anti- 1522–1546 and 1315–1328 cmÀ1 due to the presence of nitro microbial activity of the synthesized compounds with that of group. In addition the presence of chlorine in compounds 5f standard drugs is effectively presented in Table 2. All com- and 5 m was confirmed by the appearance of absorption bands pounds were found to exhibit significant activity against all at 758–765 cmÀ1 in IR spectrum and appearance of M+2 the tested bacteria and fungi. Compounds 5c, 5j, 5k, and 5n peaks in mass spectrum. The proton magnetic resonance spec- showed excellent antimicrobial properties against the entire

Please cite this article in press as: Saravanan, G. et al., Synthesis, characterization and in vitro antimicrobial activity of some 1- (substitutedbenzylidene) 8 G. Saravanan et al. microorganisms that were included in the present evaluation. biological activity, it has been observed that the most potent When compared to Ciprofloxacin, compound 5j showed antibacterial and antifungal activities exhibited by com- remarkable activity against M. luteus ATCC 4698 and P. aeru- pounds 5j, 5k, and 5n might be due to the presence of elec- ginosa ATCC 2853. Moreover, compound 5k exhibited more tron donating substituents like hydroxy, N-dimethyl amino activity against S. epidermidis ATCC 155 when compared with and 4-hydroxy-3-methoxy group respectively on the phenyl the reference drug Ciprofloxacin. ring of benzylidene. Similarly compounds 5c, 5d, and 5g also Among the tested compounds against S. aureus ATCC 9144, exhibited significant antimicrobial activity due to the above M. luteus ATCC 4698, P. aeruginosa ATCC 2853, and K. pneu- said reason. The role of electron donating group in improv- moniae ATCC 11298, compound 5j showed better activity. Fur- ing antimicrobial activities was supported by the previous ther out of several tested title compounds, compound 5k was studies (Jie et al., 2010). While other compounds containing found to be more potent against S. epidermidis ATCC 155, electron withdrawing substituents such as nitro and chloro E. coli ATCC 25922, and A. fumigatus ATCC 46645; Whereas groups (5b, 5e, 5f, 5i, 5l and 5m) do not exhibit significant compound 5n exhibited better activity than the other tested in vitro antimicrobial activity. The unsubstituted derivatives compounds against B. cereus ATCC 11778 and A. niger ATCC 5a and 5h showed moderate activity. The MIC values were 9029. Compounds 5a, 5d, 5g and 5h showed moderate to aver- determined as the lowest concentration that totally inhibited age antibacterial and antifungal activities. Rest of the com- the visible growth of microorganisms. The chemical structure pounds 5b, 5e, 5f, 5i, 5l and 5m showed weaker antimicrobial and antimicrobial activity relationship of the synthesized activity. Entire series of title compounds demonstrated signifi- compounds revealed that the compounds having electron cant levels of activity against fungus strain A. niger ATCC withdrawing moiety exhibited least activity when compared 9029 and A. fumigatus ATCC 46645. with compounds having electron releasing moieties In addi- All the synthesized compounds were subjected to MIC tion it was observed that 2-phenyl quinazolinone analog (minimum inhibitory concentration) studies against all exhibited better activity than corresponding 2-methyl quinaz- microorganisms. The MICs of Ciprofloxacin and Ketocona- olinone analog. zole were determined in parallel experiments in order to control the sensitivity of the test organisms. MIC values of the 5. Conclusions compounds and the standards are presented in Table 3.As seen in Table 3 While all compounds showed lower activities In summary, a series of 1-(substitutedbenzylidene)-4-(4-(2- than the standard against S. aureus, compounds 5c, 5k, and (methyl/phenyl)-4-oxoquinazolin-3(4H)-yl)phenyl)semicarbaz- 5n showed the same activity (MIC: 15.62 lg/ml). Moreover ide derivatives were synthesized and characterized by FT-IR, 5j demonstrated better activity (MIC: 7.81 lg/ml) than Cipro- 1H NMR, mass spectroscopy, and elemental analysis. These floxacin against S. aureus. Compounds 5c and 5k showed the derivatives were evaluated for their in vitro antimicrobial activ- same activity (MIC: 7.81 lg/ml) against S. epidermidis, while ity against seven bacteria (including four gram positive and other compounds exhibited lower activity (MIC: 15.62– three gram negative) and two fungi strains. In general, electron 31.25 lg/ml) than standard. Compound 5n showed the same donating group substituted derivatives exhibited better antimi- activity (MIC: 7.81 lg/ml) as Ciprofloxacin against M. luteus, crobial properties than electron withdrawing compounds. while others demonstrated lower activity than standard. Com- Moreover, 2-phenyl quinazolinone analog showed better activ- pound 5e showed very least activity (MIC: 62.5 lg/ml) against ity than corresponding 2-methyl quinazolinone analog. M. luteus. Compounds 5j and 5n exhibited the same activity Among the tested compounds5c, 5j, 5k, and 5n exhibited more (MIC: 7.81 lg/ml) against B. cereus, while other compounds antimicrobial activity than rest of the compounds. However, exhibited lower activity (MIC: 15.62–62.5 lg/ml) than stan- the electron withdrawing groups like nitro and chloro substi- dard. Compounds 5b and 5f showed least activity (MIC: tuted derivatives showed least activity toward tested microor- 62.5 lg/ml) against B. cereus among the tested compounds. ganisms. The above results revealed that electron donating All the compounds showed lower activity except 5c and 5k group substituted analogs may act as useful leads for antimi- (showed the same activity with MIC: 15.62 lg/ml) against crobial drug development in the future. E. coli. Compounds 5 g, 5j, and 5k showed the same activity (MIC: 7.81 lg/ml) as Ciprofloxacin against P. aeruginosa. While the rest of compounds showed the lower activity Declaration of interest (MIC: 7.81–62.5 lg/ml) than standard against K. pneumoniae compounds 5j and 5k showed the same activity (MIC: The authors report no conflicts of interest. The authors 3.9 lg/ml) as standard. Only compound 5k demonstrated the alone are responsible for the content and writing of the same activity (MIC: 15.62 lg/ml) like standard, while com- paper. pound 5f showed lower activity (MIC: 62.5 lg/ml) against A. niger. Compound 5j showed equal activity (MIC: 7.81 lg/ml) against A. fumigatus while the others have lower activity Acknowledgement (MIC: 15.62–62.5 lg/ml) than standard (MIC: 7.81 lg/ml). The authors gratefully acknowledge the Central Instrumenta- 4.3. Structure Activity Relationships (SAR) tion Facility, IIT Chennai, India for the spectral analysis of the compounds used in this study. The authors are also wish The current results revealed that most of the synthesized to thank the management of Bapatla College of Pharmacy derivatives exhibited significant antimicrobial activity. On for providing infrastructure facilities to carry out this research correlating the structures of the sample candidate with their work.

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