Design, Synthesis and Antibacterial Activity Evaluation of 4,5-Diphenyl-1 H -Imidazoles Derivatives

Due to the continuous emergence and rapid spread of drug-resistant strains of bacteria, there is an urgent need for the development of novel antimicrobials. Along this line, the synthesis and antibacterial activity of 4,5-diphenyl-imidazol-2-thiol derivatives 2a-g and 6a-e are reported. The structures of the synthesized compounds were confirmed by Nuclear Magnetic Resonance (NMR) and High Resolution Mass Spectrometry (HRMS). All compounds were screened in vitro for their antibacterial activity against Pseudomonas aeruginosa and Escherichia coli (Gram-negative bacteria) and also against Staphyloccocus aureus and Enterococcus faecalis (Gram-positive bacteria). The results showed most of the synthesized compounds have no antibacterial activity. However compound 6d was two-fold potent than ciprofloxacin against Staphylococcus aureus with Minimum Inhibitory Concentration (MIC) of 4 µg/mL and 6c showed moderate biological activity against Staphylococcus aureus (16 µg/mL) and Enterococcus faecalis (16 µg/mL).


Introduction
Infectious diseases caused by bacterial pathogens represent a major public health issue in recent years due to the emergence and spread of new strains of bacteria [1] and the widespread occurrence of drug resistance [2]. Thus, the development of new types of antibacterial drugs, especially those with a new drug target and/or with the ability to overcome drug resistance [3] is an urgent need. Structural modification of antimicrobial drugs to which resistance has developed, has been shown to be an effective way to prolong the lifespan of antifungal agents such as azoles [4], antiviral agents such as nonnucleoside reverse transcriptase inhibitors [5], and various antibacterial agents including imidazole and benzimidazole [6] [7] [8] [9]. Currently, some of the various marketed drugs available ( Figure 1) have imidazole or benzimidazole in their structures.
Because the heterocyclic ring includes the core of the active moiety or pharmacophores, we have been interested in working on structural modification of imidazole and benzimidazole. Besides this, these two heterocyclics containing nitrogens can be substituted in different positions to give several compounds with antibacterial activity [10] [11].
Considering the importance of both moieties imidazole and benzimidazole, we hypothesize their presence in the same structure would lead to compounds with higher potent. In the present study, we report the design, synthesis and antibacterial activities of some compounds containing both imidazole and benzimidazole motifs (Scheme 1 and Scheme 2), modified from 4,5-diphenyl-imidazol -2-thiol. All the structures of the synthesized compounds were confirmed by NMR ( 1 H and 13 C) and HRMS spectra and their activities were illustrated against some gram-positive and gram-negative bacteria.

Chemistry
All chemical reagents and solvents used were of reagent grade or purified using standard methods. NMR spectra were recorded at 1 H (400 MHz or 600 MHz) and 13 C (101 MHz or 400 MHz) on a Bruker instrument. Coupling constants (J) and chemical shifts (δ) are given in hertz and ppm respectively, using TMS ( 1 H NMR) and solvents ( 13 C NMR) as internal standards.
High resolution mass spectrometry (HRMS) analyses are performed on a hybrid quadrupole time-of-flight mass spectrometer (Micromass-Waters Q-TOF Ultima global), equipped with an electrospray ionization source and a MALDI source. All these analyses are carried out at the Laboratory of Glycochemistry, Antimicrobials and Agroresources (LG2A) of the University of Picardie Jules Verne, Amiens. The melting points were measured using a KOFLER bench.

Chemistry
The synthetic route of the compounds 2a-g and 6a-e is outlined in Scheme 1 and Scheme 2 respectively. The synthesis of 2-(benzylthio)-4,5-diphenyl-1Himidazoles 2a-g was carried out in two steps from benzoin. First, 4,5-diphenyl-1H-imidazol-2-thiol 1 was prepared by the condensation of benzoin with thiourea using the reported procedure [12].  carbons) of the (SCH 2 ) groups as well as the increase of the signals of the aromatic protons and carbons confirm the formation of compounds 2a-g. The structures of synthesized compounds 2a-g and their physicochemical properties are given in Table 1.
The 2-[(benzimidazol-2-yl)methylthio]-4,5-diphenyl-1H-imidazoles 6a-e were obtained after purification by silica gel chromatography in good yields, from 66 to 77%. Compounds 6a-e were characterized by 1 H NMR, 13 C NMR and HRMS. Their 1 H NMR spectra indicate the disappearance of the signal at 3.5 ppm corresponding to the thiol proton (SH) in compound 1 and the appearance of new signals attributed to the protons of the (SCH 2 ) group at 4.58 -4.66 ppm. Chemical structures of synthesized compounds 6a-e and their physicochemical properties are given in Table 2.

Antibacterial Activity
The synthesized compounds 2a-e and 6a-e were evaluated for their in-vitro an-tibacterial activity against the gram positive Staphylococcus aureus and Enterococcus faecalis bacteria and the gram negative Pseudomonas aeruginosa and Escherichia coli bacteria. The MIC values were determined by using liquid medium micromethod performed in a 96-well plate. Ciprofloxacin was used as the reference antibacterial agents. All the biological results of the tested compounds are shown in Table 3. It was found that compounds 2a-e and 6a,b,e were not active against all bacteria tested at used concentrations. However two compounds 6c and 6d showed MIC values respectively at 16 µg/mL against gram positive bacteria (Staphylococcus aureus and Enterococcus faecalis) and at 4 µg/mL against Staphylococcus aureus. Thus, the investigation of antibacterial screening revealed that most of the synthesized compounds were inactive but 6c has shown half antibacterial activity against gram positive while 6d has shown two fold antibacterial activity against Staphylococcus aureus compared to the reference ciprofloxacin. From the results, it is evident that the presence of chloride or trifluoromethyl at 5-position of benzimidazole leads to the appearance of  Overall, the results indicate very useful information for researchers interested in designing antibacterial agents containing imidazole and benzimidazole moities in their structure for both improved potency and for avoiding the chemical space that would not be productive.

Conclusion
We have synthesized a series of heterocyclic compounds 2a-g and 6a-e from 4,5-diphenylimidazol-2-thiol 1. The antibacterial activity of compounds 2a-e and 6a-e was evaluated against gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli) and gram-positive bacteria (Staphyloccocus aureus and Enterococcus faecalis) in a 96-well plate using the liquid dilution method (liquid micromethod) and ciprofloxacin as a reference. Most of the synthesized compounds were inactive but 6c has shown half antibacterial activity against gram-positive while 6d has shown two-fold antibacterial activity against Staphylococcus aureus compared to the reference ciprofloxacin.