Design, Synthesis and Evaluation of New 2, 5-Di Substituted Benzimidazole Derivatives

Antimicrobial resistance is a persistent and growing threat to global public health, significantly lowering the efficacy of many standard antibacterial and antifungal treatments and emphasizing the need for novel bioactive chemical scaffolds. Along with the rapid rise of multidrug-resistant bacterial strains, invasive and opportunistic fungal infections have become more clinically important. Microbial pathogens, which include Gram-positive and Gram-negative bacteria, as well as pathogenic fungi, cause a wide range of infections, from moderate and localized to severe, systemic, and possibly lethal. These infections disproportionately affect immune-compromised people, critically ill patients, and those undergoing lengthy hospital stays or invasive medical procedures. The increasing prevalence of drug-resistant bacterial and fungal species, combined with the limited effectiveness and safety concerns of current antimicrobial agents, highlights the critical need for the development of new broad-spectrum antimicrobial compounds with improved efficacy and resilience to resistance-modulating capabilities.

Within this context, benzimidazole has emerged as a highly important and adaptable scaffold in medicinal chemistry, providing a foundation for the design and development of a wide range of therapeutic medicines. Because of their structural closeness to purine nucleobases, benzimidazole motifs can interact successfully with a wide range of biological targets, which contributes to the diverse spectrum of biological activity documented for this family of molecules. Benzimidazole-containing compounds have been extensively studied and developed as antibacterial, antifungal, antiviral, and anticancer drugs. Furthermore, the benzimidazole core's ease of chemical modification makes it ideal for the rational creation of novel bioactive compounds. A recent study found that benzimidazole compounds have notable antibacterial activity against both fungal and bacterial infections.

MATERIALS AND METHODS:

Insilico screening:

DESIGNING OF MOLECULES

     Chem Draw version 12.0 was used to design the molecules, various substitutions were done using structure mode in the softwares.

Table:1- Designed  Molecules

Lipinski’s rule of 5 filtration:

The files were inserted in *.pdb, *.mol, *.mol2, *.xyz, *.sdf, or .smile formats. Care was taken to avoid whitespace(s) in the input file name. The window opened and the files were uploaded in the above mentioned formats. pH was adjusted from 0-14 as required. Upon submission, results were obtained.

OSIRIS property explorer (version 2):

OSIRIS property explore version 2 (which requires JAVA platform to run) was used in the present study. The structure of designed molecule when drawn in or when pasted in smiles format will show the results at right side with colour coding. A green colour indicates non-toxic and red indicates toxicity.

Prediction of activity spectra for substances (PASS):

Molecules which have been filtered through Lipinski rule were subjected to online PASS software to predict their biological activities. The Pa and Pi values from 0.000 to 1.000. To define the threshold for selecting type of activity to be predicted.

Molsoft property explorer (version v.3.7-2):

Molsoft property explorer version v.3.7-2 was used in the present study. The structure when drawn directly on the window or when inserted in mol, Inch, smiles formats will calculate properties like MlogP, MlodS.

Docking (version 4.2):

AutoDock is a molecule modeling simulation software. It is especially effective for protein ligand docking. It has two versions Auto Dock 4.2, vina. Vina is a advanced version

Docking is used to find the exact binding conformation and orientation of the ligand molecule into the active site of the protein. The synthesized five benzimidazole compounds and standard (ciprofloxacin) were docked against beta tubulin using Auto-Dock Tool 4.0, an automated docking tool.

The docking process involves four main steps,

(i) Protein preparation

(ii) Ligand preparation

(iii) Grid preparation and

(iv) Docking.

The Lamarckian genetic algorithm has been used as the search algorithm to search for the best conformers. The initial population size was set randomly as 150 individuals and ten generations was set for each genetic algorithm run and the maximum number of energy evaluations was set to 2,500,000. The grid box size was set as to include all the active site residues present in rigid macromolecules. The grid box was centered at 8.671 Å x -8.036 Å x 0.67 Å and the dimensions of the grid box have been set as 40, 40, 40 (X,Y,Z co-ordinates) so as to include all the active site residues.

SCHEME :

A= Glycine  , HCl reflux for 12hrs

B= Different aldehydes with ethanol reflux for 14hrs

General procedure for the synthesis of 2,5-Di substituted benzimidazole:

Place 5gms of 4-chloro Phenylenediamine (OPDA)  in a 250ml round bottomed flask add 19.7gm of glycine and add hydrochloric acid(4N). Heat the mixture on a water bath at 100⁰C for 12 hrs. The progess of the reaction was monitored by TLC(chloroform : ethanol in the ratio of 1:1). Cool and add sodium hydroxide (10%)solution slowly with constant stirring of the solution until the mixture is just alkaline to litmus. Filter off the crude product, wash with ice-cold water, drain well and again wash with 20ml of cold water. The obtained solid was recrystallized from water to give a pure colour (brown,2,5-disubstitued bezimidazole)compound.

Synthesis of N-[(6-chloro-1H-benzimidazol-2-yl)methyl]-1- substituted phenylmethanimine (3a,3b,3e):

A mixture of compound 2 (0.01mol )and 3a, 3b, 3e  with aromatic benzaldehyde(substituted 4-chloro benzaldehyde and 2-chloro benzaldehyde) (0.01 mol) in ethanol (30 ml) was refluxed under stirring for 5-14 hours and monitered by TLC (chloroform : ethanol in the ratio of 0.2:1.8) after that the mixture cooled at room temperature, then poured on crushed ice. The precipitate was collected by filtration and gives orange to brown crystals.

EVALUTION OF ANTIMICROBIAL ACTIVITY

Microbes are living organisams that multiply frequently and spread rapidly. An antimicrobial is an agent that kills microorganisms or inhibits their growth. Antimicrobial medicines can be grouped according to the microorganisms against which they are primarily effective. Antibacterials (commonly known as antibiotics) are used against bacteria and fungals are used against fungi. Drugs have been developed to achieve better pharmacokinetic and pharmacodynamic properties. In addition antimicrobial agents that are associated with serious side effects have been replaced by other safe drugs.

ANTIBACTERIAL ACTIVITY

The antibacterial activity of the test compounds was assayed systematically against four non-pathogenic strains of bacteria i.e. S.aureus ,S.typhi, E.coli  and  M.luteus          The antibacterial activity of a compound is its ability to inhibit the growth of bacteria in nutrient broth or agar. The method used in this present investigation was diffusion method.

NUTRIENT AGAR COMPOSITION

Beef extract  : 10g

Peptone  : 10g

Sodium chloride  : 5g

Distilled water  : 100ml

Media was prepared  by dissolving required quantity of nutrient agar (28g for 1Lit) in distilled water by heating water bath if required and pH was adjusted to 7.0-7.2. The prepared media was sterilized by autoclaving at 121⁰C (151b\in²) for  about 15 min and transferred to each petri plate.

CUP PLATE METHOD

The test compounds in the concentration of  75, 100µg/mL were prepared by dissolving in DMSO. Sterilized media was cooled to 40⁰C, inoculated with respective bacteria and poured into petri plates. After solidification of the medium at room temperature, cups of 4mm diameter were made in each plate with sterile with borer. Accurately 0.01mL of test solution was transferred to cups and labelled accordingly. The plates were kept undisturbed for at least two hours at room temperature to allow diffusion properly. Incubation of the petri plates was done at 37±1⁰C for 24h. The growth/inhibition of bacteria was observed the solvent effects. The diameter of zone of inhibition was read and results were calculated. Ciprofloxacin and Streptomycin were chosen as standards.

MINIMUM INHIBITORY CONCENTRATION-BROTH DILUTION METHOD

PRINCIPLE

The minimum inhibitory concentration  Assay is a technique used to determine the lowest concentration of a particular antibiotic needed to kill bacteria. This assay is typically performed on planktonic (free floating) bacterial cells.

PROCEDURE

• Take  clean and dry test tubes, sterilize and label them.
• Prepare dilutions for different concentrations ( 25, 50, 100µg/mL) of test compounds
• Then nutrient broth was prepared, sterilized and allowed to cool to room temperature, it was transferred to test tubes, 10-15ml in each tube
• The bacterial culture was inoculated into the test tubes in laminar chamber at aseptic conditions to avoid contamination. The test tubes were shaken to allow proper mixing.
• Various concentrations of test compounds were added to the test tubes.
• The test tubes were shaken well and tightly closed with cotton  plug.
• The tubes were allowed to incubate overnight (18-24hrs).
• Broth tubes were appeared turbid are indicative of bacterial growth while tubes that remained clear indicate no growth.
• MIC was calculated by using following formula.

MIC=  Highest conc. that inhibit growth + Lowest conc. that  allow growth of microorganism

RESULTS AND DISCUSSION:

Table-2: Physical data of synthesized compounds:

Table-3: Spectral Data:

Insilico screening:

Table-4: Results of  lipinski’s rule of 5 filtration:

Based on the results of Lipinski rule of 5 out of 70 compounds 67 compounds were obeyed Lipinski rule of 5.

Those 67 compounds are subjected to OSIRIS to know the toxicity
 

Table-5:  Results of OSIRIS property explorer (version 2):