Synthesis and Antimicrobial Activities of a New Class of 6-Arylbenzimidazo [ 1 , 2-c ] quinazolines

Uma série de compostos 6-arilbenzimidazo[1,2-c]quinazolina (11-20) foram sintetizados pela condensação do 2-(o-aminofenil)benzimidazol com diferentes arilaldeídos seguida pela ciclização oxidativa dos 2-o-arilidenoaminofenilbenzimidazol (1-10). Todos os produtos foram caracterizados por espectroscopia no infravermelho (IR), ressonância magnética nuclear de prótons e de carbono, (RMN H e RNN C), de massas (MS) e análise elementar. A atividade antimicrobial de todos os 6-arilbenzimidazo[1,2-c]quinazolina foi testada com três linhagens de bactérias Gram-positivas (S. aureus, B. subtilis e S. pyogenes), três Gram-negativas (S. typhimurium, E. coli e K. pneumonia) e de três linhagens de fungos (A. niger, C. albicans e T. viridae). Dos compostos testados, o 13, o 19 e o 20 mostraram-se ser os mais potentes inibidores contra os organismos testados.


Introduction
Heterocycles have a central position in medicinal as well as in organic chemistry 1-3 and considerable attention has been focused on their syntheses.Nitrogen heterocycles in particular exhibit diverse biological and pharmacological activities [4][5][6] due in part to the similarities with many natural and synthetic molecules with known biological activity. 71][12] In general, quinazoline compounds have been well-recognized for their pharmacological properties, such as anti-inflammatory, 13,14 antihypertensive, 15 anti-HIV, 16 bronco-dilatory, 17 antiallergic, 18 anti-cancer, [19][20][21] anticonvulsant, 22,23 antihelmintic, 24 analgesic, 25 antimalarial 26 and antimicrobial 27 activities.Literature survey reveals that, benzimidazo [1,2-c] quinazoline derivatives also show various biological activities, 23,[28][29][30] such as anticancer, antiviral, antimicrobial, anti-inflammatory and anticonvulsants.However, search is continuously on to identify a more potent lead molecule as these molecules are developing resistance over a period.Based on the importance of these molecules, our attention was attracted towards synthesis of novel quinazoline derivatives in order to find more potent biologically active molecules.Hence, we report here the synthesis and characterization of new 6-arylbenzimidazo[1,2-c]quinazoline compounds (11-20).In addition, the antimicrobial activities of all synthesized quinazolines against different bacteria and fungi were evaluated.Among the compounds tested some of quinazolines were found to be superior in inhibiting all the bacterial and fungal strains.

Chemistry
The condensation of o-phenylenediamine with anthranilic acid was carried out in presence of polyphosphoric acid at 250 °C for 4 h to obtain the known 2-(o-aminophenyl) benzimidazole (A) (mp 214 o C). 31 The target compounds were prepared by using the reaction sequence in Scheme 1.Initially, we synthesised ten IR spectra of compound A showed absorption band at 3350 cm -1 assigned to aminophenyl ring and in the case of compounds A and 1-10 two absorption bands in the region 3150-3300 cm -1 and 1420 cm -1 were assigned to -NH stretching and bending vibrations of imidazolyl ring, respectively. 33,34The absence of aminophenyl ring absorption at 3350 cm -1 and appearance of a strong intensity band in the IR spectra of compounds (1-10) in the range of 1610-1650 cm -1 attributable to nC=N provides a strong evidence for the condensation and also confirms the formation of the azomethines 1-10. 35The oxidative cyclisation of compounds 1-10 to 11-20 were accompanied by the disappearance of the absorption maxima at 3150-3300 cm -1 , which are ascribed to the vibrations of NH group of the benzimidazole ring; at the same time the appearance of a new maximum at 1360-1388 cm --1 , which is characteristic for benzimidazoquinazoline ring with a tertiary nitrogen atom, appears. 23,36he 1 H NMR spectra of the compound A as well as its derivatives have been recorded in CDCl 3 /DMSO-d 6 using TMS as internal standard.In the spectra of 2-(o-aminophenyl) benzimidazole (A), signals at d 6.4 and d 8.25 ppm were appeared corresponding to free amino and imidazolyl protons respectively. 33,35The aromatic protons of various environments present in all compounds appeared as multiplets in the range of d 6.84-8.81ppm. 35,37 1  NMR spectra of compounds 1-10 contain signals corresponding to CH=N protons in the range of d 8.07-8.29 ppm suggesting the condensation of aromatic aldehydes with compound A. However, in the spectra of 1-10 compounds the disappearance of signals at d 6.4 ppm which is due to NH 2 protons of compound A supports the involvement of amino group in condensation and confirms the formation of azomethines.Finally, in the spectra of 11-20 compounds disappearance of signals corresponding to -NH of imidazolyl ring and CH=N protons supports the benzimidazoquinazoline ring structure.23,36 Scheme 1. Synthesis of 6-arylbenzimidazo[1,2-c]quinazolines (11-20).signals in the range of d 141.1-171.2ppm confirming the presence of carbon, which is doubly bonded to nitrogen.The aromatic carbons of various environments present in all the compounds appeared as signals in the range of d 103.2-156.2ppm.37 The 1 H and 13 C NMR spectra (Figure S2 and Figure S3) of compound 20 are given in supplementary material.All the compounds (except 2 and 12) showed a single peak in ESI-MS suggesting the molecular formulae of the azomethines and quinazolines.Compounds, 2 and 12 exhibited molecular ion peaks (M + ) at m/z 391, (M +2 ) at m/z 393 and (M + ) at m/z 389, (M +2 ) at m/z 391 respectively, which is due to the isotopic abundance of bromine.The FAB mass spectrum of compound 20 shows a molecular ion peak (M + ) at m/z 296 (65.1%), which confirms the proposed formula (C 19 H 12 N 4 ).The peaks have been observed at m/z values 218 (40.3%), 193 (52.2%), 192 (100%), 180 (74.8%) and 76 (24.4%), which indicate the fragmentation pattern and their intensity gives an idea about the abundance and stability of the fragments.The mass fragmentation pattern (Scheme S1) of compound 20 as assigned on the basis of mass spectra (Figure S3) is presented in supplementary material.

Antimicrobial activity
The in vitro antimicrobial activity was carried out by cupplate method. 38All the synthesized quinazolines were screened for antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Streptococcus pyogenes (Gram positive) and Salmonella typhimurium, Escherichia coli, Klebsiella pneumonia (Gram negative) bacterial strains using ampicillin as standard.The antifungal activity was investigated against Aspergillus niger, Candida albicans, Trichoderma viridae fungal strains using ketoconazole as standard.Preliminary screening of target compounds and standard drugs were performed at fixed concentrations of 1000 µg mL -1 .Inhibition was recorded by measuring the diameter of the inhibition zone at the end of 48 h for bacteria and 72 h for fungi.Each experiment was repeated thrice and the average of the three independent determinations was recorded.Screening results are summarized in Table 1.Among the compounds tested 13 was found to exhibit good activity against all bacterial and fungal strains.Furthermore, the most potent activity was observed in 19 and 20 against all bacterial and fungal strains when compared to respective standard drugs ampicillin and ketoconazole.The minimum inhibitory concentration 39 of these quinazolines (13, 19 and  20) was also verified by the liquid dilution method in which the effectiveness was observed at lower concentrations.The detailed antibacterial and antifungal preliminary screening methods are given in supplementary material.

Conclusions
Quinazolines and related derivatives are classes of heterocyclic compounds contain the pyrimidine nucleus in their structures.Literature survey reveals that a variety of antimicrobial agents contains quinazoline moiety and also fused quinazoline ring systems such as benzimidazoloquinazolines, indoloquinazolines and many other.In a search for new fused quinazoline systems with potential biological activities, we planned to prepare new 6-arylbenzimidazo[1,2-c]quinazolines, from 2-(o-aminophenyl) benzimidazole and various aldehydes as starting compounds.We have chosen six bacterial and three fungal strains for microbial studies of these entire quinazoline compounds.From this study it is evident that all quinazoline compounds are showing moderate to good activity towards inhibiting all tested bacterial   a Ampicillin; b Ketoconazole and fungal strains.Furthermore, the most potent activity was observed in (13, 19 and 20) against all bacterial and fungal strains when compared to respective standard drugs ampicillin and ketoconazole.A possible explanation for this result is that the biological activity of compounds may be depending on the basic skeleton of molecule as well as on the nature of substituents.Hence we conclude that the most potent activity of 13, 19 and 20 compounds may be due to the presence of heterocyclic isoquinolyl, pyridyl and nitro substituted aryl groups at C 6 position of the benzimidazo[1,2-c]quinazoline moiety.

Experimental
Analar grade reagents and freshly distilled solvents were used throughout the investigations.Compounds were checked for their purity by TLC using Merck 60 F 254 silica gel plates and spots were located by iodine vapors.Micro analytical (C, N, H) data was obtained by using a Perkin-Elmer 2400 CHN elemental analyzer.The IR spectra were recorded in KBr pellets on Perkin-Elmer-283 spectrophotometer. 1 H NMR spectra were acquired at 400 MHz and 13

Procedure for preparation of mono-2-o-(arylideneamino) phenyl benzimidazoles (1-10)
To a hot stirred solution of 2-(o-aminophenyl) benzimidazole (A) (2 mmol) in ethanol (20 mL) were added the appropriate aldehyde (2 mmol) and 2-3 drops of acetic acid.The resultant mixture was refluxed under heat (40-55 °C) for 2 h after which the compound was obtained.The product was filtered and recrystalized from a dichloromethane-methanol mixture.

4-Benzo
new 2-o-arylideneaminophenylbenzimidazoles (1-10) via the condensation between 2-(o-aminophenyl b e n z i m i d a z o l e(A ) a n d va r i o u s a r y l al d e hy d e s .At last, we prepared 6-arylbenzimidazo [1,2-c] quinazolines (11-20) through oxidative cyclisation 32 of 2-o-arylideneaminophenylbenzimidazoles (1-10).

Figure 1 .
Figure 1.Comparison of MIC values (in µg mL -1 ) of quinazolines and standard drugs against different bacteria and fungi.
C NMR was acquired at 67.93 MHz on a Bruker NMR spectrometer.FAB mass spectra were recorded on a Finnigan-MAT 1020 instrument.An ion trap mass spectrometer (Agilent Series LC/MSD Trap SL) equipped with an electrospray ionization (ESI) source was used for MS analyses.Hot air oven (Instrument and Equipment Pvt. Ltd., Mumbai), incubator (Instrument and Equipment Pvt. Ltd., Mumbai), laminar airflow unit (Clas laminar technologies Pvt. Ltd.Secunderabad), autoclave (Medica Instrument Mfg.Co., Mumbai) were used in the present investigations.Organisms like Staphylococcus aureus, Bacillus subtilis, Streptococcus pyogenes (Gram positive), Salmonella typhimurium, Escherichia coli, Klebsiella pneumonia (Gram negative) bacteria and Aspergillus niger, Candida albicans, Trichoderma viridae fungi were used in the present investigations.