Synthesis and SAR Study of Novel Amidino 2-substituted Benzimidazoles as Potential Antibacterial Agents

A series of novel 2-substituted benzimidazole derivatives were synthesized and their antibacterial activity was assessed against selected Gram-positive and Gram-negative bacteria. The specific moiety at the 2-position of the benzimidazole was extensively modified with several fused heterocyclic functional groups containing nitrogen and sulfur heteroatoms. In addition, the influence of different amidino groups at the position 5 of benzimidazole scaffold was evaluated. The values of clogP (a partition coefficient) and clogD7.5 (calculated distribution coefficient, pH 7.5) were determined and the lipophilic character of compounds has been found to be important parameter for the observed activity of the tested benzimidazole derivatives against Moraxella catarrhalis. The indolo 2-substituted benzimidazole 13a demonstrated solid activity against Staphylococcus aureus (MICs 16 μg/mL) and Moraxella catarralis (MICs 2 μg/mL) and represents promising lead molecule for further optimization.


INTRODUCTION
ACTERIAL organisms causing infectious diseases represent an increasing public health problem regardless of the current availability of numerous antimicrobial agents.Not only has the incidence of infections caused by resistant bacterial pathogens been continuously increasing during the last decades, but even the formerly commensal species have raised serious health concerns because of dramatic changes in their genetic makeup.This is the case with Moraxella catarrhalis. [1]In recent years, Moraxella catarrhalis has established its position as an important human mucosal pathogen, no longer being regarded as just a commensal bacterium.It has been associated with childhood otitis media (OM) and lower respiratory tract infections in adults with chronic pulmonary disease as a third common bacterial cause after Haemophilus influenzae and Streptococcus pneumoniae. [2]Even though Moraxella catharralis has not been identified as an important human pathogen, it is obvious that it has a significant financial impact on global health care systems. [3]enzimidazoles displayed a variety of biological effects, such as antihelmintic, [4] antiviral, [5,6] anticancer, [7] anti-inflammatory [8] or antioxidant [9] activities.Consequently, benzimidazole core received attention as an important pharmacophore for the development of novel B antimicrobial agents in inhibiting synthesis of microbial nucleic acids and proteins and preventing the growth of various microorganisms (Figure 1). [10][13] The most used heterocycles containing one or two nitrogen atoms have been imidazole, pyridine and pyrrole, and fused scaffolds like benzothiophens, indols, benzothiazoles and quinolines. [14]urthermore, the amidine moiety is found in a variety of biologically active molecules. [15]The amidine derivatives generally possess broad spectrum of biological activities: anticancer, [16] antimicrobial, [17] anti-HIV [18] and anti-inflammatory. [19,20]The positively charged amidine groups are attracted by electrostatic interactions to negatively charged DNA thus enabling selective binding. [21]n view of the afore-mentioned findings and in a continuation of our research on biologically active heterocycles, [22][23][24][25] we hereby describe the synthesis of a new 2,5(6)-disubstituted benzimidazole derivatives and their evaluation as antibacterial agents.The structure activity relationship of their antibacterial activities was also examinate.

EXPERIMENTAL PART
Chemistry GENERAL Melting points were determined by SMP10 Bibby apparatus and are uncorrected.IR spectra were recorded on FTIR-ATR spectrophotometer. 1 H and 13 C NMR spectra were recorded on Bruker AV 300 and Bruker AV 600 spectrometers using TMS as an internal standard in DMSO-d6.Chemical shifts are reported in ppm (d) relative to TMS.Elemental analyses for carbon, hydrogen, and nitrogen were performed on a Perkin-Elmer 2400 elemental analyzer and a Perkin-Elmer, Series II, CHNS analyzer 2400.All compounds were routinely checked by TLC with Merck silica gel 60F-254 glass plates.The spectroscopic data regarding 13 C NMR spectra and elemental analysis are reported in the Supporting Materials.

General method for preparation of amidino substituted benzimidazoles 3a,b-14a,b
A mixture of equivalent amounts of corresponding 4-Nsubstituted-1,2-phenylenediamine 2a-2b, aromatic aldehyde 1a-1l and p-benzoquinone in absolute ethanol (10-15 mL) was stirred at reflux for 4 hours under nitrogen atmosphere.The reaction mixture was cooled to room temperature and resulting product was filtered off and washed with diethylether (20 mL).After recrystallization from ethanol/diethylether or ethanol, light powders were obtained.

Antibacterial Activity
Determination of minimal inhibitory concentrations (MICs) was performed according to guidelines of the Clinical Laboratory Standards Institute. [26]Testing was performed by the standard broth microdilution method with azithromycin as the reference antibiotic.Bacterial strains used as the primary screening panel included two strains of Gram-negative species, Esherichia coli (TolC-) (efflux pump deficient strain ECM1556 tolC:Tn10) [27] and Moraxella catarrhalis (ATCC 23246), and two fully sensitive strains of Gram-positive species Staphylococcus aureus (ATCC 29213) and Enterococcus faecalis (ATCC 29212).Bacteria were grown on appropriate agar plates: Mueller-Hinton agar with 5 % sheep blood for enterococci and Moraxella catarrhalis and Mueller Hinton agar for staphylococci and E. coli.Compounds were tested as double dilutions (concentration range 128-0.25 μg/ml) in 96-well microtitre plates.Bacterial inocula were prepared by direct colony suspension method and plates inoculated with 5×10 4 cfu/well.Results were determined by visual read-out after overnight incubation at 37 °C in ambient air.
In Silico Predictions of plausible biological targets and pharmacological activities were made by web-service PASS (http://www.pharmaexpert.ru/passonline/index.php) which is based on the identification of substructure features typical for active molecules. [28,29]Lipophilicity parameters clogP and clogD at pH 7.5 for synthesized compounds were calculated using Volsurf+ approach. [30]Classification models were built using decision tree algorithm See5, release 2.07. [31]
The structures of all newly prepared derivatives were determined by using 1 H and 13 C NMR spectroscopy and elemental analysis.NMR analysis was based on the values of H-H coupling constants and chemical shifts in the 1 H and 13 C NMR spectra.

In Silico Analysis, Antibacterial Activity and Structure Activity Relationship (SAR)
Biological potential of synthesized compounds (Scheme 1 and Figure 2) were estimated using Prediction of Activity Spectra for Substances (PASS) via web service.PASS is widely utilized for prediction of pharmacotherapeutic effects and biological targets of drug-like compounds. [30,31]he estimates are based on the SAR analysis of known compounds with experimentally established biological activity spectra.][29][30]32] The probability, Pa, reflects the similarity of a molecule under prediction with the structures of molecules, which are the most typical in a sub-set of 'actives' in the structure-activity relationship (SAR) PASS database. Accrding to the PASS output the synthetized compounds could potentially act as omptin inhibitors (Pa ≥ 0.5).Omptins represent a unique group of integral outer membrane (OM) proteases implicated in pathogenicity and are present in a number of gram-negative pathogens.Because of their direct involvement in pathogenesis, omptins are prime candidates for therapeutic targeting.[33] According to these predicted results we tested the in vitro antibacterial activity of novel 2,5( 6 were determined and are presented in Table 1.Structural modifications at position 2 of benzimidazole have included various aromatic fragments (Ar: pyrrole, pyridine, naphthalene, quinoline, benzothiazole, benzofurane, benzothiophene and indole) and at position 5 two types of end groups (R: un-substituted amidine and cyclic amidines) (Scheme 1).The antibacterial testing demonstrated that variation of the fused heterocycles nuclei at position 2 of benzimidazole had significant impact on selectivity and activity towards different bacterial species (Table 1).In general, all tested compounds showed weak to moderate activity towards tested bacteria with the exception of activity against Moraxella catarrhalis against which tested compounds showed the highest potency.The most active compounds (5a-b, 8a, 10a-b, 11a, 13a-b, 14a-b) exhibited activity against Moraxella catarrhalis in the range of 2-4 µg/mL.These compounds have a substituent containing fused benzene ring and six of them have indole ring bonded to benzimidazole at position 3.The activities of molecules 10a-b, 13a-b and 14a-b, were not affected by substituents at N-1 of indole or the type of amidino/imidazolinyl group at C-5 of benzimidazole.The compound 13a also displayed acceptable antibacterial activity towards Staphylococcus aureus (MIC value 16 µg/mL), indicating its potential for further development as a broad-spectrum antibacterial.Among indole substituted benzimidazoles, the best activity was observed for derivatives with indole bonded at their position 3 to benzimidazole.
In addition to activity against Moraxella catarralis, the benzothiophene derivative (8a) also showed moderate activity towards gram-positive bacterial strains, Staphylococcus aureus and and Enterococcus faecalis, with MICs values 16 and 32 µg/mL, respectively (Table 1).Similarly, benzothiazole derivatives (9a-b) were also active against Enterococcus faecalis (MIC 32 and 64µg/mL, respectively) and Escherichia coli (MIC of 8 µg/mL).These three indole derivatives have shown the broadest spectrum of activity against the tested bacterial strains.In comparison to molecules with heterocycles containing fused benzene ring, the com-pounds 15ab, 16a-b and 17a-b, were generally less active against Moraxella catarrhalis as well as other three tested strains (Table 1).The substitution of pyrrole fragment with the indole moiety, lead to 2-8 fold increase of the antibacterial activity against Moraxella catarrhalis.Moreover, the activity towards gram positive bacteria, especially against Staphylococcus aureus was also increased by this structural modification.In contrast to the compounds comprising indole and heterocycles with sulphur, derivatives containing other heterocycles with fused benzene ring such as na-phthalene, (3a-b), quinoline (4a-b), bezofuran (7a-b) had moderate activity against Moraxella catarrhalis (MICs 8-16 µg/mL).
Additionally, our compound design also included structural modifications of the amidine moiety as our previous experience with un-substituted amidines and cyclic amidines revealed pharmacologically relevant analogues with prominent biological activity. [22,33]urthermore, it was previously shown that positively charged terminal groups are responsible for accumulation into cells. [34,35]Here presented pairs of acyclic and cyclic amidine derivatives have shown almost identical activities against all examined bacterial species.The lipophilic character of compounds has been found to be important parameter for the observed activity of the tested benzoimidazole derivatives against Moraxella catarrhalis.In general, the compounds with clogP > 1.56 and logD7.5 > 0.68 showed noteworthy antibacterial activity (≤16 µg/mL) against Moraxella catarrhalis (Table 2).These results are in agreement with previous findings which suggested increased susceptibility of Moraxella catarrhalis to hydrophobic agents. [36]The observed relationship is probably associated with more efficient intracellular partitioning of active compounds.
The outer membrane of gram-negative bacteria is composed of lipopolysaccharide molecules (LPS) that form a hydrophilic environment providing protection against hydrophobic molecules. [37]Additionally, cells of Moraxella catarrhalis have on their surface low molecular weight lipooligosaccharides (LOS) instead of LPS which contribute to the increased hydrophobicity of its outer membrane.Consequently, more lipophilic molecules may show distinct antimicrobial activity against Moraxella catarrhalis as a result of higher cellular uptake.

CONCLUSION
In conclusion, a series of novel amidino substituted benzimidazole derivatives were designed and synthesized in good yields.A convenient and efficient synthetic route was applied starting from commercially available different heterocyclic aldehydes and all new compounds were characterized by 1 H NMR, 13 C NMR and elemental analysis.The in vitro antimicrobial activities of newly prepared benzimidazole derivatives were evaluated against four bacterial strains.
All tested compounds except compounds 17a and 17b inhibited the growth of human respiratory tract pathogen Moraxella catarrhalis.The lipophilic character of compounds has been found to be related to the observed activity of the tested benzimidazole derivatives against Moraxella catarrhalis.The compounds with clogP > 1.56 and clogD7.5 > 0.68 showed antibacterial activity against Moraxella catarrhalis.The analysis of antibacterial, clogP and logD results revealed that the most promising compound is the one with the fused heterocyclic group at 2-position of amidino substituted 1-methylindol-2-yl derivative 13a.Moreover compound 13a exhibited potent antibacterial activity towards Staphylococcus aureus and Moraxella catarralis.Furthermore, the SAR results obtained in this study will be applied for the further optimization of this heteroaromatic core and for the design of novel derivatives in order to improve initially observed antibacterial activity.

Table 1 .
cLogP/logD Values and minimal inhibitory concentrations (MICs) determined against four bacterial strains

Table 2 .
Classification models for two definitions of active (YES) and inactive (NO) compounds, against M. catarrhalis, built in terms of calculated lipophilicity parameters a) (Total number of compounds in a given class/ number of misclassified compounds) For example, of 6 inactive compounds, five are correctly predicted as inactive and one is a false positive. (