Synthesis and antimicrobial activity evaluation of some benzimidazole and indole derivatives

In this study, 16 benzimidazole and indole derivatives were synthesized and tested for in vitro antimicrobial activity by tube dilution and disc diffusion susceptibility test methods. The in vitro antimicrobial activity of the compounds was carried out by determining minimum inhibitory concentration value against various Gram-positive, Gram-negative bacteria and candida species fungi. The most active compound was 4-(5-methyl-3-phenyl-1H-indole) phenol (Compound 15) as shown by most inhibitory effect on Candida albicans, Candida glabrata, Staphylococcus aureus and Bacillus subtilis. Compound 15 showed better activity against yeast and Gram-positive bacteria than Gramnegative bacteria.


INTRODUCTION
The incidence of morbidity and mortality by bacterial and fungal infections has increased globally mostly in developing countries due to the growing of antibacterial and antifungal drug resistance.It comprises serious problems such as increased resistance of microorganisms to a number of antimicrobial agents such as β-lactam antibiotics, macrolides, quinolones and vancomycin (Ozkay et al., 2010).Candida species and Staphylococcus aureus are the most known multi-drug resistant microbial pathogens.Candida species are the fourth leading cause of health care-associated bloodstream infections and are associated with significant morbidity and mortality (Rentz et al., 1998).Candida albicans and Candida glabrata can cause serious mucosal or systemic infection (Wingard, 1995).C. albicans is the most common cause of candidaemia.C. glabrata also causes approximately 5 to 15% of non-albicans Candida infections (Fidel, et al., 1999).
The emergence of fluconazole-resistant C. glabrata bloodstream infections has had important implications because therapy requires higher doses of fluconazole or the use of other antifungal agents such as echinocandins or polyenes (Pappas et al., 2004).
S. aureus is currently the most common cause of various infections such as nosocomial infections.Currently, the most important problem is methicillin-resistant Staphylococcus aureus (MRSA).Since MRSA strains are resistant to all β-lactam antibiotics, the therapeutic options are limited significantly.
Successful treatment of infectious diseases requires the availability of more effective new antimicrobial compounds.As pathogens mutate, continued success in treating infectious diseases requires a steady stream of new antimicrobial agents to which existing bacteria and fungi have not developed resistance.*Corresponding author.E-mail: oztekinalgul@mersin.edu.tr.Tel: +90 532 4140199.Fax: 0 324 3413022.Extensive biochemical and pharmacological studies of benzimidazole and indole derivatives have confirmed their highly effectiveness against various strains of microorganisms (Sharma et al., 2009;Al-Qawasmeh et al., 2010;Göker et al., 2002;Ayhan-Kılcıgil and Altanlar, 2003;Pawar et al., 2004;Mahboobi et al., 2008;Ansari and Lal, 2009;Ryu et al., 2009), as well as have various therapeutic effects.Benzimidazole and indole cores were recognized by organism, because they are placed in the natural structure of Vitamin B 12 (Cyanocobalamine), tryptophane and serotonin (O'Niel et al., 2001;Fernandes et al., 2004).Although vitamin B 12 is capable of inducing the growth of bacteria, some of the benzimidazole derivatives such as Astemizol, Mebendazole, Enviroxim, Carbendazim and Benomyl repress the bacterial growth and have been used in clinics widely (The Merck Index, 1996).On the other hand, compounds which have indole moeity also act as anti-inflammatory drug, such as indometazin, etodolac and acemetacin.
Prompted by these observations, in the present study, some substituted benzimidazoles and indoles were synthesized as the lead compounds in order to examine their in vitro antimicrobial activities against different Grampositive, Gram-negative bacteria and fungi in comparison with control drugs.
In the present work, some 2-mono or 1,2-/2,5-disubstituted benzimidazoles (Compound 1-14) and 1,2 and 5trisubstituted indoles (Compound 15 and 16) were synthesized (Figure 1) as the lead compounds in order to examine their in vitro antimicrobial activities against different Gram-positive, Gram-negative bacteria and the yeasts Candida species in comparison with two control drugs.

MATERIALS AND METHODS
In this study, we established two assays for testing and identifying new antimicrobial agents from a series of benzimidazole and indole derivatives.Some of the compounds have been synthesized before either by our group or by other scientist (Al-Awadi et al., 2005, Kabeer et al., 2012;Algul et al., 2008;Navarrete-Vazquez et al., 2006;Lee et al., 2007;Iyengar et al., 1997;Kassim et al., 2012).

Chemistry
The structures of the synthesized compounds (Figure 1) were characterized by FT-IR and 1 H-NMR, as well as MS spectra.FT-IR spectra were recorded on Varian FTS 1000 spectrophotometer and -The newly synthesized molecules.
1 H-NMR spectra on a Varian Mercury 400 MHz FT-NMR spectrometer using tetramethylsilane (TMS) as an internal reference (chemical shift in δ ppm).Mass spectra were taken on a Agilent 6460 Triple Quadropol LC-MS instrument.

General method for the synthesis of 2-(p-subtitutedphenyl)-1Hbenzimidazoles derivatives
Method A: The 4-substituted benzoic acid (1.5 mmol), 1,2phenylendiamine (1.0 mmol) and PPA (5 mL) were placed in a round bottomed flask.The mixture was heated and stirred at 180-200°C for 3-7 h.After the reaction was complete, the mixture was allowed to cool to room temperature and poured into cold water (50 mL).The mixture was neutralized with NaHCO3.The resulting precipitate was filtered off, washed several times with water and purified by recrystallization.

Microbiology
Antimicrobial susceptibility testing was performed by modification of the following literature methods (National Committee for Clinical Laboratory Standarts, 2002;Alexpoulos and Mims, 1979;Ghannoum and Rice, 1999).We used microbial strains such as S. aureus ATCC 25925, B. subtilis ATCC 6633, E. coli ATCC 25923, Aeromonas hydrophlia ATCC 95080 Acinetobacter baumanii ATCC 02026, C. albicans (clinical isolate strain), Candida glabrata ATCC 4322, Candida parapsilosis ATCC 22019 and Candida glabrata (clinical isolate strains).The yeast and bacterial cell inoculum were prepared from the stock culture grown in Tryptic Soy Broth (TSB) at 28°C for 24 h and Mueller-Hinton Broth (MHB) 37°C for 24 h, respecti el .he microorganisms concentrations ere ad usted according to McFarland 0. turbidit tubes ( 10 5 cells per mL) using sterilized TSB and MHB.Stock solutions of chemical derivatives Yuksek et al. 1711 were prepared in DMSO at 1000 μg/mL.A modified macrodilution test was applied for antimicrobial activity and the experiments were run in duplicates independently.
For antifungal activity testing, 1 mL TSB was added to each of 10 sterilized test tubes. 1 mL of chemical derivative solution was added to the first tube and 2-fold dilutions of stock solution were of the east cell (McFarland 0. ) as prepared.hen, 10 μL of this stock solution was added to each tube except the last one which acted as control tube.
For antibacterial activity testing, 1 mL MHB was added to each of ten sterilized tubes. 1 mL of chemical derivative solution was added to the first tube and 2-fold dilutions of stock solution were prepared and the bacterial cell (McFarland 0. ) as prepared.hen, 10 μL of this stock solution was added to each tube except the last control tube.Only 2 ml of yeast and bacterial cell were added in to control tube without chemical and used as control for growing.All tubes were incubated at 28°C (for fungi) and at 37°C (for bacteria) for 24 h.After the incubation, the minimal inhibitory concentrations (MIC) (Tables 1 and 2) were noted by controlling the growth inhibition for each chemical compound.Fluconazole and Ampicillin were used as reference drug.The results of modified macrodilution test, some chemical compounds were determined active for C. glabrata ATCC 4322.Only active compounds were selected and tested for C. glabrata, b "Disc diffusion method".Dimeth l sulfoxide as used as the solvent to prepare desired solution (1 mg/mL) of the selected compounds initially.Different dilutions of isolated compound that is 31.2, 62. , 12 , 2 0 and 00 μg/mL for each, were employed.Fluconazole was used as reference antifungal drug and DMSO was also tested for a possible antifungal activity.In this method, the filter paper discs (6 mm in diameter) were individually absorbed with 30 µL each of all concentrations (31.25, 62.5, 125, 2 0 and 00 μg/mL) and placed on Potato Dextrose Agar (PDA) plates, which had been previously inoculated with the tested microorganism, and was adjusted according to McFarland 0.5 using sterilized TSB.The Petri-plates were incubated at 28±1°C for 24 h.The diameters of the inhibition zone were measured in mm.

Chemical assays
The procedures for synthesis used in this study are illustrated in Figures 2 to 5. All benzimidazole derivatives were prepared according to a modified one-step reaction sequence in analogy to a method described (Bahrami et al., 2007;Navarrete-Vazquez et al., 2006).
We also determined the antimicrobial activity of some indole derivatives as shown in Figure 5.The compounds were synthesized according to modified indole synthesized method from p-tolylhydrazine HCl and the corresponding benzyl phenyl ketone in ethanol (Robinson, 1963).

Antimicrobial assay
We observed that substitution at the second position of the benzimidazole and indole rings played an important role on antimicrobial activity.These compounds also showed potent antimicrobial activity; however they have not been introduced into the pharmaceutical market yet.
In recent years, antimicrobial compounds, which target  directly bacterial DNA and therefore exhibit more selective effect, are beginning to investigate.Benzimidazole and indole rings are isosteres of basic structures of DNA bases (purine and pyrimidine moieties), which raises the possibility that these compounds could be antimetabolite of purine.Therefore, we also focused on investigation of antimicrobial activities of some new benzimidazole and indole derivatives.
The most active compound was 4-(5-methyl-3-phenyl-1H-indole) phenol (Compound 15) as shown by the most inhibitory effect on C. albicans, C.glabrata, S. aureus and B. subtilis.Additionally, we found that Compound 3 to Candida parapsilosis, Compound 6 to Aeromonas hydrophlia and Compounds 1 and 14 to Acinetobacter baumanii were more effective than other compounds tested.
First, antibacterial and antifungal activities of all  compounds were determined by tube dilution method; Ampicillin and Fluconazole were used as standard drugs, respectively.Results were also confirmed with the disc diffusion method.All compounds showed low activity against S. aureus and B. subtilis, except Compound 15, which had considerable activity against these microorganisms.Different activity profiles of Compound 15 and 16 show that activity is not just determined by main structure.
All compounds were also examined for antifungal activity.It was found that Compounds 1-3, 5, 6, 9, and 14-16 were fully susceptible to C. glabrata and Compound 3 and 14 had a significant activity against C. albicans and C. parapsilosis, respectively.Compounds 1-3, 5, 6, 9, and 14-16 showed activity against C. glabrata in all tested dilutions and these results were also confirmed with disc diffusion method.Generally, the compounds were active against C. glabrata.
As a result of this study, Compound 15 is a promising compound which is active in both methods, the other compound 3 showed promising activities in the medium level.Also, Compound 15 showed considerable activity against 10 different clinical strain of C. Glabrata in disc diffusion method with inhibition zone (18 mm) compared with Fluconazole inhibition zone (15 mm).

Conclusion
Nowadays, growing resistance of pathogens against antibacterial compounds used is a common and important problem, which shows clearly that research on new compounds against these pathogens is needed.We synthesized 16 novel substituted benzimidazole and indole derivatives and screened their antimicrobial activities.In future studies, novel derivatives of Compound 15 should be designed to reveal a new antifungal compound group.Among the investigated compounds, indole derivatives showed better antibacterial activity than benzimidazole derivatives.It was observed that substitution at the second position of the benzimidazole and indole rings played an important role on antimicrobial activity and increased activity when 4-OH substituted phenyl at the second position of the indole.Additionally, aromatic ring substitution at first position of benzimidazole ring decreased antimicrobial activity.
We need to study the in vivo and mode of action of these compounds to determine the potential of their antibacterial and antifungal activities.

Figure 1 .
Figure 1.Chemical structures of the synthesized compounds.