ONE-POT SYNTHESIS AND ANTIMICROBIAL SCREENING OF NEW [ 1 , 5 ] – BENZODIAZEPINE DERIVATIVES CATALYZED BY TBAB

A new [1,5]-benzodiazepines (VI) were synthesized by the reaction of ophenylenediamine with 2-chloro-3-acetylquinoline in the presence of tetrabutyl ammonium bromide (TBAB) in short reaction time with excellent yield.


INTRODUCTION
Benzodiazepines are one of the most important class that have various therapeutic applications.Many members of the benzodiazepine family have a wide range of biological activity as anticonvulsants, anti-anxiety, analgesic, sedative, anti-depressant, hypnotic (Randall, 1974) anti-inflammatory (Roma, et al., 1991), anti-viral (Kavali and Badami, 2000), anti-HIV (Di Braccio, et al., 2001), antimicrobial, (Kumar and Joshi, 2007) and anti-tumor (Kamala, et al., 2008) ctivities.Accordingly 1, 5-Benzodiazepine and its derivatives attract our attention to synthesize new members depending on wide range of pharmacological activity and industrial applications, the development of mild and efficient protocols for their preparation continues to be a challenging endeavor in the synthetic organic chemistry (Herbert, et al., 1974).The common procedure for the synthesis of these compounds is a one pot condensation between o-phenelendiamine and newly synthesized carbonyl compounds .However, from the modified literature survey a variety of catalysts such as BF 3 O, NaBH 4 , PPA-SiO 2 , MgO-POCl 3 , Yb(OTf) 3 , amberlyst-15, Ag 3 PW12O40, solid super acid sulphated zirconia, acetic acidunder MWI, AgNO3, zinc montmorilonite as catalyst at room temperature were used.These reactions also occur with various catalysts under solvent free conditions (Chen and Lu, 2005;Kumar, et al., 2006;Li, et al., 2007;An, et al., 2008;Mohamed and Asgar, 2012;Heravi, et al., 1985).Indeed these methodologies have several disadvantages such as harsh reaction condition, long reaction time, a large amount of catalysts, expensive reagents, unsatisfactory product yields, critical product isolation procedures, tedious workup and formation of side products.Keeping in mind in the present study we have developed one pot synthesis of [1,5]-benzodiazepines by condensation of o-phenelendiamine with ketones catalyzed by tetrabutylammonium bromide (Scheme 1).

General
Melting points were measured in capillary tube on a Graffin melting point apparatus and are uncorrected.The IR spectra were recorded on Pye Unicam SP 1000 IR spectrophotometer using KBr discs (λmax in cm -1 ). 1 HNMR spectra were performed either on a Jeol ECA (500 MHz) or Gemini 300BB (300MHz) spectrometer, using TMS as internal standard and DMSO-d6 as solvent; the chemical shifts are reported in ppm (δ) and coupling constant (J) values are given in Hertz (Hz).Signal multiplicities are represented by s (singlet), d (doublet), t (triplet), q (quadruplet), and m (multiplet).All of the new compounds were analyzed for C, H and N and agreed with the proposed structures within ±0.4% of the theoretical values by the automated CHN analyzer at the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt.Mass spectra were recorded on Hewlett Packard 5988 spectrometer at the RCMB.The purity of the compounds was checked by thin layer chromatography (TLC) on Merck silica gel 60 F254 precoated sheets.The starting Compounds( I-III ) were prepared according to reported method (Wright, 1985;Neelima, et al., 1986;Cyrous, et al., 2006;Monir, et al., 2004;Hang and Browne, 2010).

General procedure for the synthesis of 6-substituted-2-Chloro-N-methoxy-Nmethylquinoline-3-carboxamide (IVa-e)
To a solution of )III) in dry THF (10 mL), a solution of N,O-dimethyl hydroxylamine in THF (prepared from 2.2 mmol of N,O-dimethyl hydroxylamine hydrochloride, and 2.2 mmol of triethylamine in 5 mL of dry THF was added at 20 o C over 5min.The reaction mixture was stirred at r.t. for 24 h.The solvent was removed in vacuo and the residue was dissolved in EtOAc.The organic layer was washed sequentially with saturated citric acid, satd.Na 2 CO 3 , H 2 O, and dried over MgSO 4 .Evaporation in vacuo to dryness gave the desired products (IVa-e), which were recrystallized from the appropriate solvent.In a typical flask experiment's 0.02 mol of Grignard reagent was added at once to stirred solution of (IV) in 100 cc of dry ether that previously cooled by ice / acetone mixture ,the added Grignard reagent were cooled also previously .After stirring for 2-3h the cool bath was removed and the mixture treated with ammonium chloride solution to get the crud product of compounds (Va-e) at room temperature.11.18. Found C,53.07;H,3.14;N,11.02.

RESULT AND DISCUSSION
Tetrabutylammonium bromide is a cheap and readily available reagent it efficiently catalyze the condensation of ketones with o-phenelendiamine at 60 o C under mild conditions in short reaction time (50 min) with excellent yield of the product.In our work a series of [1,5]-Benzodiazepine derivatives were prepared from starting intermediate 2-oxo-1,2dihydroquinoline-3-carboxylic acid derivatives which prepared according to the reported procedure 14-18 .The structures of compound (II) were confirmed by 1 HNMR, 13 C-NMR, In the 1 H-NMR spectra, where two sharp proton signal observed at the δ 5.4 and 11.68 ppm, one belong OH at C-2 and one belong COOH respectively, and due to resonance isomerism the NH(secondry amine) it appear at higher chemical shift at 8.9 as abroad signal more than expected due to neighboring carbonyl group, In the 13 C-NMR spectra of these compounds one C=O peak at about 178 ppm and one around 166 ppm corresponded to the signals of the 3-carboxylic acid and carbonyl groups in the quinoline rings.On contrary the IR spectra of compound (III) showed the disappearance of OH streatching band and this explaine the conversion of acid into acid chloride and the mass spectra of compound (III) confirm the conversion of carbonyl at C-2 into chloride moiety , so when the compound (III) react N,Odimethyl hydroxylamine it produce the compound (IV) which IR spectra confirm the presence of these compound because it showed clearly decreasing of stretching absorption band of C=O from 1812 cm -1 and appearance of sharp medium stretching absorption bands at range of 1636 cm -1 which belong to (C=O) for amide, when compound (IV) react with alkyl Grignard reagent it produce compound (V), which upon cyclocondensation with ophenelendiamine it afford new 1,5-benzodiazepine derivatives (VI), where the elemental analysis and spectral data confirm the existence of this cyclocondensation in which the 1 HNMR of new 1,5-benzodiazepines contain singlet signal due to CH 3 at C-2, in addition the presence of NH singlet signal at 4.17-4.25,furthermore the presence of singlet signal due to the two hydrogen at C-3 of benzodiazepine ring and all this fact will prove this cyclocondensation reaction and formation of 1,5-benzodiazepine ring .

Antimicrobial activity:
The newly synthesized of [1,5]-benzodiazepine compounds were screened for their in-vitro antibacterial activity against Staphylococcus aureus , Bacillus subtilius , Pseudomonas aeruginosa , Escherichia coli and candida albicans bacterial stains by serial plate dilution method (Barry, 1991), Serial dilutions of the drug in Muller Hinton broth were taken in tubes and their pH was adjusted to 5.0 using phosphate buffer.A standardized suspension of the test bacterium was inocubated for 16 to 18 hours at 37 0 C. The minimum inhibitory concentration (MIC) was noted by observing the lowest concentration of the drug at which there was no visible growth.Numbers of antibacterial discs were placed on the agar for the sole purpose of producing zones of inhibition in the bacterial lawn.Twenty milliliters of agar media was poured into each petri dish.Excess of suspension was decanted and plates were dried by placing in an incubator at 37 0 C for an hour.Using a punch, wells were made on these seeds agar plates and minimum inhibitory concentrations of the test compounds in