2-Phenyl substituted Benzimidazole derivatives: Design, synthesis, and evaluation of their antiproliferative and antimicrobial activities

The inability to meet the desired outcomes of anticancer treatment and decrease in treatment success of bacterial and fungal infections accelerated research in these areas. Our research group has conducted numerous studies, especially on benzimidazole ring systems’ antiproliferative and antimicrobial activities. In this study, the antiproliferative activity of benzimidazole compounds was tested against A549, A498, HeLa, A375, and HepG2 cancer cell lines by MTT assay. All compounds exhibited good to potent antiproliferative activity against all tested cancer cell lines. Compounds 6-chloro-2-(4-fluorobenzyl)-1H-benzo[d]imidazole (30) and 6-chloro-2-phenethyl-1H-benzo[d]imidazole (46) were especially active against HeLa and A375 cancer cell lines with IC50 values in the range of 0.02–0.04 µM. In contrast, compounds 6-chloro-2-((p-tolyloxy)methyl)-1H-benzo[d]imidazole (67) and 5(6)-chloro-2-((4-hydroxyphenoxy)methyl)-1H-benzimidazole (68) were active against A549 and A498 cancer cell lines with an IC50 value of 0.08 µM. These compounds (30, 46, 67, and 68) were less toxic to normal human cells than the positive control compound methotrexate, which was screened to determine its toxicity against normal cell lines (HEK293). In the second part of the study, all compounds were tested to demonstrate their antimicrobial properties. All compounds exhibited moderate activity against all tested bacteria and fungi. However, some phenoxy methyl derivatives 5-chloro-2-((4-chlorophenoxy)methyl)-1H-benzo[d]imidazole (69) and 5,6-dichloro-2-((4-chlorophenoxy)methyl)-1H-benzo[d]imidazole and (74) were most active against Candida (<3.90 µg/mL). Molecular docking studies were carried out against certain proteins in order to identify potential targets of the antiproliferative effects of the synthesized compounds. The docking scores of the compounds were found to be significantly compatible with the antiproliferative activity results. Graphical abstract Graphical abstract


Introduction
Nitrogen-containing heterocycles present a tremendous research topic for being bioactive natural compounds. This reputation of N-heterocycles makes these compounds vital for investigating biological activities [1]. Among the nitrogencontaining compounds, benzazoles (benzimidazole, benzoxazole, and benzothiazole, etc.) are a subject of great interest to our research group for investigating drug development due to their known pharmacological and biological activities [2][3][4][5][6][7][8][9][10][11]. Benzimidazole is primarily defined as a privileged substructure in terms of purine-like scaffold feature and its ability to interact with various unrelated molecular targets [12,13]. Many drugs are commercially available with benzimidazole as their basic core structure, including antihelmintic drugs, such as albendazole and cyclobendazole [14]; antiulcer drugs, such as omeprazole [15]; and antipsychotic drugs, such as droperidol [16]. These drugs have a benzimidazole structure with various pharmacological activities ( Fig. 1) [17].
The literature survey shows that 2-substituted benzimidazole is an essential pharmacophore in drug discovery [18][19][20][21], making it vital for future planning [22]. Synthesis and biological investigations of various 2-substituted benzimidazoles have resulted in the discovery of gastric antacid, rabeprazole, and pantoprazole. The synthesis of benzimidazoles continues to be the focus of clinical research in recent years. The easy synthesis of benzimidazole structures has also made these compounds an interesting research topic for many researchers [23][24][25].
The anticancer effects of benzimidazole structures have been widely reported in the literature [12,13,26].
All active benzimidazole core structures have been attached to an aromatic ring by aliphatic or heteroaliphatic linkers with binding aromatic substituents, such as CH 3 , OCH 3 , or OH, as shown in Fig. 2.
On the contrary, some microorganisms that have become resistant to conventional antibiotics are often overused against microbial pathogens. Therefore, various classes of compounds with antibiotic properties are synthesized and used to treat and prevent microbial infections. The benzimidazole structure is a versatile precursor molecule used in antimicrobial drug development [17]. Antimicrobial drug development studies on compounds containing benzimidazole core have gained momentum in the last two decades [34]. Iwahi and Satoh reported that compound 9 exhibited antibacterial properties against Campylobacter pylori [35]. Compound 10 has been reported to have antibacterial activities equivalent to omeprazole against C. pylori [36]. 2,5-substituted benzimidazole derivatives, compounds 11 and 12, have been reported to exhibit moderate to good antibacterial activities against Gram-positive and Gram-negative bacteria as well as methicillin-resistant Staphylococcus aureus (MRSA) [37,38]. Bansal et al. reported that a similar derivative they synthesized (DMA) inhibited bacterial topoisomerase I (Fig. 3) [39,40].
In modern drug discovery studies, there is also a goal of identifying potential compounds for multiple targets from different compounds. Multi-target drugs have raised considerable interest in the last two decades due to their advantages in treating complex diseases and health conditions linked to drug resistance issues. Benzimidazole structure, which has a broad spectrum of action, can be a good starting point for such a study. In this study, we designed and synthesized 45 different 2-substituted (benzyl/phenylethyl/ phenoxymetyl) and 5,6-mono/di-substituted (chloro) benzimidazoles derivatives to increase antiproliferative and antimicrobial activities against five different cell lines (A549, A498, HeLa, A375, HepG2) and Gram (+) and Gram (−) bacteria and two fungi (C. albicans and C. parapsilosis), respectively. The general structure of synthesized compounds is shown in Fig. 4. We also evaluated the role of linker groups in benzimidazole and phenyl moieties for molecular flexibility, markedly affecting, positively or negatively, the activity and selectivity in antiproliferative [41,42], antibacterial and antifungal activities.

Chemistry
The title 2-substituted benzimidazoles are prepared by a highly efficient one-pot procedure, cyclodehydration of the corresponding accessible carboxylic acids and 1,2-phenylenediamine derivatives, using 5 N hydrochloric acid as the catalyst and solvent according to Phillips method as shown in Fig. 4 [43].
In synthesizing some compounds containing -CH 2 -linker in their structures, some problems were faced regarding the reaction time and solubility of the compounds. We solved these problems using microwave irradiation.  For this, an appropriate 1,2-phenylenediamine derivative (1 eqiv.) and appropriate carboxylic acid derivative (1,1 eqiv.) were dissolved in 5 N HCl and introduced in a microwave tube. The mixture was irradiated in a household microwave oven for three minutes. After irradiation, the mixture was poured into cold water and neutralized by mixing with NaHCO 3 . The resulting precipitate was filtered off, washed with cold water, and crystallized with a suitable solvent. The resulting crystalline compounds were filtered, and the vacuumed product was dried. The structures of all synthesized compounds are shown in Fig. 5.

Antiproliferative evaluation
The antiproliferative effects of the synthesized benzimidazole derivatives were tested against five different cell lines (A549, A498, HeLa, A375, HepG2). The derivatives were also evaluated to study their cytotoxic nature against HEK293 (normal human cell), which helped determine the selectivity of compounds toward cancerous cells. If the compound is more selective toward cancerous cells, it must be less toxic to the normal cells. The synthesized compounds were classified under three groups based on their linkers (-CH 2 -, -CH 2 -CH 2 -and -CH 2 -O-) (Tables 1-3) to determine the linkers' role in antiproliferative effects between benzimidazole and phenyl ring.
In the first group, 2-benzylbenzimidazole derivatives (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35), as presented in Table 1, were screened for their activity. Compound 30 is the most active compound in the series with IC 50 values of 0.04-0.4 µM. It was determined that the compound had higher activity (IC 50 : 0.02 and 0.04 µM, respectively) against HeLa and A375 cells in particular. The selectivity of the compound against these cells was found to be 50 and 25 times more selective for cancer cells HeLa and A375, respectively, compared to the normal healthy cell line HEK293. Among this series, the second most active compound 33 has very high antiproliferative activity with IC 50 values of 0.04-0.16 µM. Compound 33 was determined to be 25 times more selective especially against A375 and HepG2 cells. Among this series, compounds generally appear to be more susceptible to HeLa and A375 cancer cell lines. Considering the structure-activity relationships of the active compounds in the series, the presence of -Cl substituent in the 6-position  of the benzimidazole ring increases the activity. Similar activity can be seen in 5,6-dichloro benzimidazole derivatives, but the activity is highest in the presence of mono-Cl substituent. In the R 3 position, the substitution of the -F and -OH groups play a positive role in the activity. Among the second group, although compounds 49 and 54 are the highest antiproliferative compounds (IC 50 : 0.02-0.08 µM), they show toxic effect against healthy cell HEK293 with IC 50 : 0.2 µM. Following this, compound 46 is the most active with IC 50 values of 0.04-0.4 µM and it targets HeLa and A375 cancer cells with 25-fold higher selectivity compared to the healthy cell line. The second most active compound among this series is compound 44 and it was observed as the most potent antiproliferative agent against A549 with an IC 50 value of 0.08 µM and possessed high selectivity against HEK293 with an IC 50 value of 1.00 μM which means that it shows sixteen times more selectivity toward A549 cancer cell lines. In addition, it can be said that in general the second group compounds target HeLa and A375 cancer cells.
In the evaluation of the antiproliferative activities of the second group compounds by structure-activity relationships, the -Cl substituent at the 6 th position of the benzimidazole ring increases the antiproliferative activity. Similar to the first group, the activity of dichloro compounds decreases partially. In the ethyl benzene group, the p-OH substituent plays a positive role in the activity (Table 2).
In the third group, 2-(phenoxymethyl)benzimidazole derivatives (61-75), presented in Table 3, were screened. All the compounds of third group were generally more effective than the other derivatives (first and second groups) against the tested cell lines. From this group, compounds 67 and 68 showed 12 times more selectivity toward A549 and A498 cell lines with IC 50 values of 1.00 μM against HEK293 and 0.08 μM against A549 and A498 cell lines. Also, compound 65 showed ten times more selectivity toward the A375 cell line (Table 3). In general, the compounds were found to be more sensitive to the A498 cancer cell line. In the structural activity examination, the presence of the -Cl substituent in the 6-position of the benzimidazole ring and the presence of the p-CH 3 (67) or p-OH (68) substituent of the phenyl ring were determined to increase the activity.
Generally, potent antiproliferative activity was obtained using third group compounds (61-75). Those compounds were ten times more potent than first and second group compounds and were more active against A549 and A498 cancer cell lines, but the selectivity slightly decreased with these compounds. The antiproliferative activity study concluded that the 2-(aryl)benzimidazole derivatives would significantly lead to further investigation of antiproliferative activity by changing the substituted positions and designing new linkers with different heteroatoms.

Antimicrobial evaluation
In this study, the antimicrobial properties of all the tested three series of benzimidazole scaffold derivatives were assessed. A series of 2-(benzyl/phenylethyl/phenoxymethyl) benzimidazole derivatives generally exhibited potent antibacterial activities against Gram-positive bacteria and fungi and relatively low to moderate activity against Gramnegative bacteria. The benzimidazole derivatives 43 and 54 showed sensitivity against E. coli and Pseudomonas aeruginosa. Also, they were active against Enterococcus faecalis, Staphylococcus aureus, Streptococcus pneumoniae, whereas compounds 54, 65, 69, and 73 showed activity against Bacillus subtilis (Table 4). Compounds 69 and 74 were determined to be the most active compounds with the same MIC value (<3.90 µM) as the reference compound fluconazole possess against Candida albicans and Candida parapsilosis. In this study, among the synthesized benzimidazole derivatives, especially compounds 54 and 74 containing R 1 = R 2 = R 3 = -Cl substituents showed higher biological effects on bacteria and fungi.
The best antiproliferative activity results were obtained from the third group of compounds and phenoxymethyl derivatives for Candida.

Molecular Docking
It is known that benzimidazole derivatives have various antiproliferative effects. Antiproliferative effects of substituted benzimidazole derivatives have been reported in A549, HepG2, A498, HeLa, A375 cell lines. Various macromolecules thought to be associated with these cell lines were identified and in silico studies were performed. The targets thought to be related the antiproliferative effects were selected from the proteins where the benzimidazole ring-bearing compounds are effective. Tubulin-Colchicine: Stathmin-Like Domain Complex (PDB ID:1SA0) for A549 cell line [44], Sphingosine kinase 1 complex (PDB ID: 4V24) for A498 cell line [45], HER2 Kinase Domain Complexed (PDB ID: 3RCD) for HeLa cell line [46], and Human indoleamine 2,3dioxygenase (PDB ID:2D0T) was selected as a potential target for the A375 cell line [47] and molecular docking studies were performed. As a result of molecular docking studies, the docking scores of the compounds are given in Table 5

ADMET properties
Compounds must reach their sites of action in order to show their activity. The physicochemical properties of the    compounds play an important role in their transport to the target site. Many compounds could not be used as drugs due to their unsuitable properties. Lack of efficacy and safety are the two major causes leading to drug failure, which means the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of chemicals play vital roles in every stage of drug discovery and development. Therefore, it is necessary to find efficacious molecules with better ADMET properties. In this study, various physicochemical properties and estimated toxic properties of the synthesized compounds were calculated ( Table 6). The "Rule of Five", the original and best known rulebased drug-like filter to distinguish whether a molecule is well absorbed orally: molecular weight (MW) ≤ 500, octanol/water partition coefficient (A log P) ≤ 5, hydrogen bond donor number (HBDs) ≤ 5 and the number of hydrogen bond acceptors (HBAs) ≤ 10.
The properties of all synthesized compounds comply with the rule of five (Lipisnki's five rule). It can be seen that the volume and PSA values of the compounds are close to each other, and it can be seen that among the most active compounds, compounds 30, 46 and 67 have these values in the average range. Caco-2 permeability and percent human oral absorption, which are the absorption parameter, were calculated. The obtained results showed that all synthesized molecules have proper Caco-2 permeability. percent human oral absorption, which is desired to be high, was also calculated as 100%.
The compounds have very high Percent Human Oral Absorption values and their Qplog HERG values are below −5. In addition, the drug-likeness scores of the compounds range from −2,369 to 0.100. The compounds do not have mutagenic, tumorigenic and irritant effect (except for compounds 62, 67, and 72) according to the predicted results. It can be said that these scores are appropriate in terms of drug feasibility. These data indicate that substituted benzimidazole derivatives can be potential drug candidates.

Conclusion
A series of 2-(benzyl/phenylethyl/phenoxymethyl) benzimidazole derivatives with various substituents were designed and synthesized. In silico studies show that the physicochemical properties of the compounds are suitable for drug feasibility and do not have significant toxicity. The derivatives showed significant antiproliferative activities against five human cancer cell lines by comparing the results with the standard antiproliferative drug, methotrexate. Antiproliferative activity results indicated that substituted benzimidazole derivatives positively affect the activity. Result analysis may conclude that compounds with aliphatic linkers (21-35, 41-55) have good to potent antiproliferative activity with high selectivity. Interestingly, the antiproliferative activity study concluded that the 2-(aryl)benzimidazole derivatives would significantly lead to further investigation of antiproliferative activity by changing the substituted positions and designing new linkers with different heteroatoms. All compounds exhibited potent antimicrobial activity against Gram-positive bacteria and fungi. As a result of antimicrobial activity studies, higher effects on bacteria and fungi were observed among the synthesized benzimidazole derivatives, especially with -Cl substituent. Among all the groups of compounds tested, the antimicrobial effect appears to be highest when R 1 = R 2 = R 3 : -Cl (compounds 34, 54 and 74, respectively). In addition, it was determined that compound 54 carrying -CH 2 -CH 2 backbone was highly effective against E. faecalis and S. aureus bacteria, and compound 74 carrying -CH 2 -O backbone was highly effective against fungal microorganisms. Interestingly, among all cancer cell lines, compound 54 was determined to have the highest activity, and compound 74 to be the second highest active compound. These results may lead to the development of new compounds with both antimicrobial and anticancer activity. Even more potent compounds can be designed by extending the linker in the compounds or substituting the R 1 , R 2 and R 3 groups with groups such as -F, -Br. In silico studies of the designed compounds and synthesizing and testing of more effective compounds are among our ongoing studies.  phenylenediamine derivatives (1 eq) and the corresponding carboxylic acid derivaties (1.1 eq) was refluxed for a period of 15-27 h in 5 M hydrochloric acid. The reaction mixture was poured onto ice water and neutralized by mixing with NaHCO 3 till slightly basic pH (8)(9) to get the precipitate. The resulting precipitate was filtered off and washed with cold water. Recrystallized with a suitable solvent. The resulting crystalline compounds were filtered and the vacuumed product was dried (Fig. 8).   Table 6 Some predicted ADME, toxicological, and drug-like properties of all compounds , and human colon cancer cell line (A375) were performed using MTT assay [48]. The obtained results of in vitro antiproliferative activities are summarized in Table 1. The selectivity of these compounds toward cancerous cells is evaluated against HEK293 (normal human cell) to determine the non-toxic concentrations of the compounds on relatively healthy cells. All cell lines were obtained from the cell culture collections of Mersin University. All cells were incubated at 37°C in 5% (v/v) CO 2 .

Antiproliferative activity
The cytotoxic activity of the compounds was determined using an MTT assay [48,49]  violations of these rules are more likely to be orally available prepared from a stock culture grown in tryptic soy agar (TSA) at 28°C for 24 h and in Mueller-Hinton agar (MHA) at 37°C for 24 h, respectively. The microorganism suspension concentrations were adjusted to 0.5 McFarland turbidity tubes using sterilized saline. Stock solutions of the title compounds were prepared in DMSO at 1000 µg/mL. A modified microdilution test was performed for antimicrobial activity, and the experiments were run in duplicate independently. For the antifungal activity testing, a 100 µL Tryptic Soy Broth (TSB) was added to each of the 11 wells.
A 100 µL aliquot of the tested chemical solution was added to the first well, and twofold dilutions were prepared. Then, 5 µL of fungal suspension was added to each tube except the last one, which acted as the control well. For the antibacterial activity testing, a 100 µL Mueller-Hinton broth (MHB) was added to each of the 11 wells. A 100 µL aliquot of the chemical derivative solution was added to the first tube, and twofold dilutions were prepared. Then, 5 µL of the bacterial suspension was added to each tube, except the last control well. A control tube containing 5 µL of the fungal and bacterial suspensions alone without the tested compounds was also prepared. All plates were incubated at 28°C (for fungi) and 37°C (for bacteria) for 24 h. After incubation, the MICs (Table 1) were obtained by noting the growth inhibitions. The concentration resulting in a 50% reduction in the optical density (OD) values was compared to a reproduction control at 450 nm by spectrophotometric evaluation and defined as the MIC value. Fluconazole and ampicillin were used as reference drugs. The results were read visually and by measuring the OD for 24 h.

Molecular docking
Maestro 12.8 (Schrodinger) was used in all stages of molecular modeling studies. The crystal structures of the target proteins (1SA0, 4V24, 3RCD, 2D0T) [44][45][46][47] downloaded from www.rcsb.org were prepared using the standard protocol with the Protein Preparation module. While preparing the compounds, LipPrep module and OPLS3 force filed were used. The possible ionization states were generated at the target site at pH 7.0 ± 2.0. Ligands were docked to the active sites of target proteins 100 times with standard precision (SP). For the validation of the docking studies, the native ligands in the crystal structure of the proteins were extracted, minimized and redocked [52,53]. RMSD values were calculated as 0.162, 0.091, 0.120, and 0.192 Å. The interaction of ligands with proteins was visualized by Schrodinger's XP visualize.

ADMET prediction
The structures of the compounds were drawn using the 2D Sketcher module of the Maestro program. The molecular weights, logP, volume, PSA, Qplog HERG, QPP Caco, Qplog Khsa, Percent Human Oral Absorption, Rule of Five and Rule of Three values of the compounds were calculated with the QikProp software [3]. The estimated toxic effects of molecules (Mutagenic, Tumorigenic, Irritant) and Druglikeness scores were also evaluated with the DataWarrior 4.07.02 software [54].

Data availability
NMR and MS spectra are depicted in the Supplementary Material.