Synthesis and Potent Antimicrobial Activity of Some Novel N-(Alkyl)-2-Phenyl-1H-Benzimidazole-5-Carboxamidines

A series of 22 novel 1,2-disubstituted-1H-benzimidazole-N-alkylated-5-carboxamidine derivatives were synthesized and evaluated for in vitro antibacterial activity against S. aureus and methicillin resistant S. aureus (MRSA), E. coli, E. faecalis and for antifungal activity against C. albicans. Compound 59 [1-(2,4-dichlorobenzyl)-N-(2-diethylaminoethyl)-1H-benzimidazole-5-carboxamidine], with a 3,4-dichlorophenyl group at the C-2 position, displayed the greatest activity (MIC = 3.12 μg/mL against both some bacteria and the fungus C. albicans).


Introduction
We have already reported the synthesis of a series of 1,2-disubstituted-1H-benzimidazole-Nalkylated-5-carboxamidine derivatives and their very potent antibacterial activities against S. aureus and methicillin resistant S. aureus [1]. The study revealed that compounds I-IV (Figure 1) exhibited the best activity, with MIC values of 0.78 -0.39 µg/mL against these species. As part of a continuing program focused on development of new antimicrobial benzimidazole carboxamidines, we planned to modify the structure of compounds I-IV.

Chemistry
Syntheses of the target benzimidazoles (Tables 1, 2) were achieved by two different methods, as shown in Scheme 1.

III IV
Method A involved nucleophilic displacement in DMF of the chloro group of 4-chloro-3-nitrobenzonitrile by reaction with several amines to give 1-8 (Table 3). The cyano group was then converted into the imidate ester, using a modified Pinner method [1], and the imidate esters were used directly to make the corresponding benzamidines 9-18 (Table 4). Their reduction with H 2 , Pd/C produced 19-28 (Table 5). Condensation of these derivatives with the Na 2 S 2 O 5 adducts of several benzaldehydes afforded the corresponding benzimidazoles 45-52, 55 and 59-61 [2]. Method B involved cyclization of 29-32 with the Na 2 S 2 O 5 adducts of various benzaldehydes to afford 5-cyanobenzimidazoles 33-39 (Table 6), following this, the cyano groups were converted into the imidate esters, as in method A, and these were used to prepared the corresponding amidine compounds 40-44, 53, 54 and 56-58. For its practical advantages this method was used in particular for cyanobenzimidazoles, which have better solubility in EtOH, although the yields were low.

Antimicrobial Activity
The benzimidazoles 40-61 were tested by the macro-broth dilution [4] assay for in vitro antibacterial activity against Gram positive Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA, a clinical isolate from a wound), Enterococcus faecalis and Gram negative Escherichia coli and for antifungal activity against Candida albicans. The MIC values are listed in Table 1. The synthesized compounds and reference drugs were dissolved in water or DMSOwater (40 %) at a concentration of 400 µg/mL. The concentration was adjusted to 100 µg/mL by fourfold dilution with media culture and bacteria solution at the first tube. Data was not taken for the initial solution because of the high DMSO concentration (10 %). We have already reported that 3,4-dichloro substitution on the 2-phenyl group of amidinobenzimidazoles plays an important role in their antibacterial activity [1]. Thus, the most active compound, 59, and less active compounds 45, and 55-57 all have a 3,4-dichlorophenyl group at the C-2 position. Replacement of 3,4-dichloro substitution with other functions such as fluoro, cyano, methoxy, carboxyl or methyl ester caused a reduction in inhibitory activities, and only compound 50, having a methyl ester group, exhibited moderate activity against MRSA with a MIC of 3.12 µg/mL. More lipophilic substituents on the benzimidazole N-atom such as phenyl, benzyl and 2,4-dichlorobenzyl do lead to quite active compounds (55-59), however substitution with methyl, butyl and isopropyl (cf. 46, 47, 52-54) gave no significant activity. Introduction of N,N-diethylaminoethyl substitution on the cationic amidine 59 led to inhibitory activity against E. coli and C. albicans. This is a very important result, as it represents the first example to date of inhibitory activity against E. coli with these amidinobenzimidazoles. Except for compound 59, none of the compounds showed important inhibitory activity against E. faecalis and C. albicans.

Conclusions
Introduction of aromatic amidine groups into the benzimidazole system gives a good profile of Gram-positive antibacterial activity. In particular, 1-(2,4-dichlorobenzyl)-N-(2-diethylaminoethyl)-1Hbenzimidazole-5-carboxamidine (59), having a 3,4-dichlorophenyl at the C-2 position, exhibited the greatest activity, with a MIC value of 3.12 µg/mL against S. aureus and MRSA. Detailed mechanistic studies are required to understand the potent activity of this compound.

Acknowledgments
This work was supported by Ankara University Research Fund (Project No: 0000018-2003). The Central Lab. of the Faculty of Pharmacy of Ankara University provided support for acquisition of the NMR, mass spectrometer and elemental analyser used in this work.

General
Uncorrected melting points were measured on an Electrothermal 9100 capillary melting point apparatus. 1 H-NMR spectra were recorded employing a Varian Mercury 400 MHz FT spectrometer, chemical shifts (δ) are in ppm relative to TMS, and coupling constants (J) are reported in Hertz. Mass spectra were taken on a Waters Micromass ZQ using the ESI(+) method. Microanalyses were performed by Leco CHNS-932. Some HCl salts of compounds 40-61 were prepared by using dry HCl gas in EtOH or isopropanol. All chemicals and solvents were purchased from Aldrich Chemical Co. or Fischer Scientific. Compounds 29-32 were synthesized as described in our previous study [2]

4-(2,4-Dichlorobenzyl)amino-3-nitrobenzonitrile (7)
A mixture of 2,4-dichlorobenzylamine (3.52 g, 20 mmol) and 4-chloro-3-nitrobenzonitrile (2 g, 10.95 mmol) in DMF (3 mL) was heated under reflux for 4h at 120°C. The mixture was allowed to cool and EtOH was added. The resultant yellow precipitate was filtered, washed with water and crystallised from EtOAc-n-hexane; yield 57 %; mp: 192 o C; 1 H-NMR (CDCl 3 ): 4.66 (d, J=5.6, 2H), 6.8  1-8 (4.5 mmol) and 33-39 (Table 6, 1 mmol) were suspended in absolute EtOH, cooled in a icesalt bath, and dry HCl gas was passed through the solution for 40 min. The solution was stirred in a stoppered flask at room temperature for 3 days and then diluted with dry ether. The imidate esters precipitated as yellow solids, which were washed with ether then dried under vacuum at room temperature. All imidate esters were used directly without characterisation. A suspension of imidate ester HCl in absolute EtOH was stirred with corresponding the amines (1.5 -2 fold excess) overnight at 25-30 o C. The reaction mixture was evaporated and diluted with ether, the precipitate was filtered, washed with ether, then dried. Compounds 9-18 (Table 4) were used without purification as HCl salts for the next steps since they were prepared completely pure. In contrast, crude products 40-44, 53, 54, 56-58 were treated with dilute Na 2 CO 3 solution, then water. Further purification methods are given in Table 2.    General Procedure for Synthesis of 19 -28 Compound 9-18 (3.5 mmol) in EtOH (75 mL) was subjected to hydrogenation using 40 psi of H 2 and 10 % Pd-C (40mg) until uptake of H 2 ceased. The catalyst was filtered on a bed of Celite, washed with EtOH, and the filtrate was concentrated in vacuo. The crude o-phenylenediamines (grey-purpleblack in colour) were used for the subsequent steps without crystallisation (Table 5). In order to prevent halogen reduction of compound 28, 15 psi of H 2 pressure was employed.