Synthesis and Antimicrobial Activity of 2-[2-(2,6-dichloro phenyl)amino]benzyl-3-(5-substituted phenyl-4,5-dihydro-1H-pyrazol-3-yl-amino)-6,8-dibromoquinazolin-4(3H)ones

A series of 2-[2-(2,6-dichlorophenyl)amino]benzyl-3-(5-substituted phenyl-4,5-dihydro-1H-pyrazol-3-yl-amino)-6,8-dibromoquinazolin-4(3H) ones 6a-m have been synthesized by the reaction of 2-[2-(2,6-dichlorophenyl)amino]benzyl-3-substituted phenylacrylamido-6,8-dibromoquinazolin-4(3H) ones 5a-m with hydrazine hydrate in the presence of glacial acetic acid. The chalcones 5a-m were prepared by the condensation of 2-[2-(2,6-dichlorophenyl)amino]benzyl-3-acetamido-6,8-dibromoquinazolin-4(3H)one 4 with different substituted aromatic aldehyde. The benzoxazinone 2 was synthesized from 2-[2-(2,6-dichlorophenyl)amino]phenyl acetyl chloride 1 on treatment with 3,5-dibromoanthranilic acid in pyridine, which on reaction with hydrazine hydrate and then on acetylation reaction yielded 4. The structures of these compounds have been elucidated by elemental analyses, IR, and NMR spectral data. The title compounds pyrazolyl-quinazolin-4(3H)ones 6a-m were evaluated for their antibacterial and antifungal activities in vitro.


Antimicrobial activity
The in vitro antimicrobial activities of compounds 6a-m were carried out by the cup-plate method. [27] Antibacterial activity was screened against two gram-positive bacteria S. aureus (ATCC 12228) and B. subtilis (ATCC 11778), and two gram-negative bacteria E. coli (ATCC 8739) and Certium (ATCC 27957), by measuring the zone of inhibition on agar plates at two different concentrations 100 µg/ml and 50 µg/ml. While antifungal activity was tested by measuring the zone of inhibition on agar plates with two fungal species C. albicans (ATCC 10231) and A. niger (ATCC 16404) at two different concentrations 20 µg/ml and 10 µg/ml. Penicillin-G was used as a standard antibacterial agent, whereas fluconazole was used as a standard antifungal agent.

RESULTS AND DISCUSSION
The title compounds 2-[2-(2,6-dichlorophenyl)amino] benzyl-3-(5-substituted phenyl-4,5-dihydro -1H-pyrazol-3-yl-amino)-6,8-dibromoquinazolin-4(3H)ones 6a-m were synthesized according to described process in Scheme 1. The structures of all the synthesized compounds were elucidated by the elemental analysis as well as IR and NMR spectral data. IR spectra showed strong C=O and C=N stretching of quinazolinone at around 1720 cm -1 and 1610 cm -1 . The 1 H NMR spectra of compounds 6a-m indicated that the -CH 2 protons of the pyrazoline ring resonated as a pair of doublet of doublets (Ha and Hb) due to geminal and vicinal coupling. The CH proton appeared as a doublet of doublet (Hx) due to vicinal coupling with the two magnetically nonequivalent protons of methylene group at position 4 of pyrazoline ring. The Ha proton which is cis to Hx resonates upfield in the range δ 3.02-3.07 as a doublet of doublet while Hb, the other proton which is trans to Hx resonates downfield in the range δ 3.45-3.49 as a doublet of doublet. The Hx proton which is vicinal to two methylene protons (Ha and Hb) resonates as a doublet of doublet in the range δ 5.46-5.51. In 13 C NMR spectra, signals at around δ 36.5, δ 55.5, and δ 161 confirms the presence of CH 2 , CH, and C=N of pyrazoline ring, respectively, whereas C=O and C=N signals of quinazolinone ring are appear at around δ 162 and δ 168, respectively.
The results of antibacterial activity are shown in Table  1. Compounds 6a (R = H) and 6h (R = 2-NO 2 ) showed good activities against gram-positive bacteria (70.87% and 63.39% against S. aureus respectively; 73.97% and 69.56% against B. subtilis, respectively). The remaining compounds showed moderate activities (44.19-55.29%) against grampositive bacteria as compared to penicillin-G. Compounds containing para-substituted hydroxyl, chloro and methoxy groups were found active than orthoand meta-substituted compounds while ortho substituted nitro compound showed good activity than metaand para-substituted nitro compounds against gram-positive bacteria. Compounds 6h (R = 2-NO 2 ) and 6i (R = 3-NO 2 ) exhibited good activities against gram-negative bacteria (65.04% and 74% against E. coli, respectively; 68.97% and 78.63% against Certium, respectively). The remaining compounds showed moderate activities (37.98-58.63%) against gram-negative bacteria as compared to penicillin-G. Meta-substituted hydroxyl and nitro compounds possessed good activity against gram-negative bacteria as compared to orthoand para-substituted compounds. Whereas ortho-substituted chloro compound was found good as compared to meta and para, and methoxy group containing compounds share an equal activity against gram-negative bacteria E. coli. On the other hand, ortho-substituted methoxy compound was found good than para-substituted compound and metasubstituted chloro compound showed lower activity as compared to orthoand para-substituted chloro compound against gram-negative bacteria Certium. Furthermore, compound 6h (R = 2-NO 2 ) was active against all gram positive as well as gram-negative bacteria, while compound 6a (R = H) was active against both grampositive bacteria and compound 6i (R = 3-NO 2 ) was active against both gram-negative bacteria. Also, compound 6l (R = 2-OCH 3 ) displayed good activity (61.15 %) against gram-negative bacteria Certium. In addition compounds containing dimethylamino group exhibited quite low activity than others against gram-positive as well as gramnegative bacteria.
The results of antifungal activity are shown in Table   2   C H = zone of inhibition at 20 µg/ml; C L = zone of inhibition at 10 µg/ml; Pot. = potency in % albicans and A. niger. On the other hand, para-substituted hydroxyl compound was found good against C. albicans as compared to ortho and meta, while meta-substituted hydroxyl compound possessed higher activity against A. niger than orthoand para-substituted compounds. Both ortho and para-substituted methoxy group containing compounds exhibited same activity against C. albicans and A. niger.

CONCLUSIONS
All the compounds showed satisfactory elemental as well as IR and NMR spectral results. Compounds bearing 2-nitro group showed promising activity against all bacterial species while chloro group-containing compounds were found active against both fungal species. All this findings give ideas to improve antimicrobial activity for further studies.