Ronak Muhammedsadek Ersan

Ph.D.

Medicinal Chemistry

M.S.

Medicinal Chemistry

B.A.

Pharmacy

Bisbenzoxazole Derivatives: Design, Synthesis, in Vitro Antimicrobial, Antiproliferative Activity, and Molecular Docking Studies

Four series of bisbenzoxazole derivatives were designed, synthesized, and screened for antiproliferative and antimicrobial activities. Generally, all synthesized bisbenzoxazoles (9–24) displayed significant antiproliferative activity; these effects were shown to be related to oxazole rings and substituents in bisbenzoxazole compounds. Especially, the series bearing chloro-substituent (9–12) exhibited better antiproliferative activity with higher selectivity than the other series (13–24); the IC50 values were observed in the range of 0.045–0.342 µM. Interestingly, only the compound with a nitro substituent (22) showed maximum potency with an IC50 value of 0.011 µM, which is two-fold more active than the standard drug methotrexate, with moderate selectivity. The compounds bearing fluoro-substituent (14–16) were found to exhibit potent antibacterial activity against the Gram-positive Enterococcus faecalis, with a MIC value of 62.5 μg/mL, and moderate activity against Gram-negative bacteria and fungi. Only the compound 23 showed potent activity against Escherichia coli, with a MIC value of 62.5 μg/mL. In order to better evaluate the activity results, crystal structures of five different proteins Human Anaplastic Lymphoma Kinase (PDB ID: 2XP2), CYP2C8dH complexed (PDB ID: 2NNI), factor-human kinase-β enzyme IKK-β enzyme (PDB ID: 4KIK), a tubulin heterodimer complex containing α and β sub-units (PDB ID: 1Z2B) and penicillin-binding protein 4 (PBP4) from Enterococcus faecalis (PDB ID: 6MKI) were used in the docking study to examine antiproliferative and antimicrobial activity. Finally, an ADMET screening test was applied to determine the drug-like, toxicological, and optimum physicochemical properties for all of the synthesized compounds. The strategy applied in this research may act as a perspective for the rational design of potential anticancer drugs.

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.

Cytotoxic activity evaluation of naphthalene substituted benzimidazole derivatives

Compounds bearing naphthalene and benzimidazole pharmacophores have been reported to possess excellent anticancer activity. In view of this, we designed, synthesized and characterized a series of naphthalene substituted benzimidazole derivatives (11–19), and further evaluated them for antiproliferative activities by employing MTT method. Among the nine investigated molecules, compounds (11 and 13) showed good antiproliferation of the tested cancer cell lines with IC50 values ranging from 0.078 to 0.625 µM. In addition, compound (18) exhibited selective cytotoxicity against HepG2 cell lines with high safety to normal cell (HEK293). Furthermore, cytotoxicity studies of these compounds against normal Human embryonic kidney cells (HEK293) revealed that the target molecules were less selective against HEK293 as compared to methotrexate (positive control). The high potency and selective cytotoxicity suggested that compound 18 could be a starting point for further optimization to develop novel antitumor agents towards liver cancer.

Design, synthesis, and computational studies of benzimidazole derivatives as new antitubercular agents

The increase in the drug-resistant strains of Mycobacterium tuberculosis has led researchers to new drug targets. The development of new compounds that have effective inhibitory properties with the selective vital structure of Mycobacterium tuberculosis is required in new scientific approaches. The most important of these approaches is the development of inhibitor molecules for Mycobacterium cell wall targets. In this study, first of all, the antitubercular activity of 23 benzimidazole derivatives was experimentally determined. And then molecular docking studies were carried out with 4 different targets: Arabinosyltransferase C (EmbC), Filamentous Temperature Sensitive Mutant Z (FtsZ), Protein Tyrosine Phosphatase B (PtpB), and Decaprenylphosphoryl-β-D-ribose-2′-oxidase (DprE1). It has been determined that benzimidazole derivatives show activity through the DprE1 enzyme. It is known that DprE1, which has an important role in the synthesis of the cell envelope from Arabinogalactan, is also effective in the formation of drug resistance. Due to this feature, the DprE1 enzyme has become an important target for drug development studies. Also, it was chosen as a target for this study. This study aims to identify molecules that inhibit DprE1 for the development of more potent and selective antitubercular drugs. For this purpose, molecular docking studies by AutoDock Vina, and CDOCKER and molecular dynamics (MD) simulations and in silico ADME/Tox analysis were implemented for 23 molecules. The molecules exhibited binding affinity values of less than −8.0 kcal/mol. After determining the compound’s anti-TB activities by a screening test, the best-docked results were detected using compounds 20, 21, and 30. It was found that 21, was the best molecule with its binding affinity value, which was supported by MD simulations and in silico ADME modeling results.

In vitro antiproliferative activity of some fluoro-substituted benzimidazole-based scaffolds

In the present work, a series of fluoro-substituted benzimidazole derivatives were designed and synthesized as antiproliferative agents. The antiproliferative activity of these compounds was investigated using MTT assay. Fluoro-substituted benzimidazole derivatives showed significant antiproliferative activity against all the tested cancer cell lines. All the derivatives were found to be less toxic as compared to methotrexate (positive control) in human cells, indicating selective and efficient antiproliferative activity of these benzimidazole derivatives. These findings suggest that compounds ORT14 and ORT15 among this series are most effective and have potential for detailed investigations

Bisbenzimidazole derivatives as potential antimicrobial agents: Design, synthesis, biological evaluation and pharmacophore analysis

In an attempt to design and synthesize a potent class of antimicrobials, 1,2-phenylenediamine derivatives were reacted with various aliphatic and heteroaliphatic dicarboxylic acids to generate a small library of 26 head-to-head bisbenzimidazole compounds (16 – 42) using the polyphosphoric acid method. These compounds were screened for their antibacterial activity and their antifungal activity. Compound 25 showed maximum potency against both Gram-positive and Gram-negative bacterial strains with minimum inhibitory concentration (MIC) values in the range of 7.81 – 31.25 μg/mL. In particular, it showed the maximum MIC values of 7.81 μg/mL against Gram-negative bacteria, which was four-fold more active than the standard drug ampicillin (MIC = 32.25 μg/mL). Compound 19 was found to be the most active against S. aureus with a MIC value of < 3.90 μg/mL, whereas the remaining compounds showed only low-to-moderate activity. Furthermore, all compounds exhibited low activity against all fungal strains in comparison to the standard drug fluconazole. I addition, pharmacophore hypotheses were generated to analyze structure–activity relationships between the molecular structures and antimicrobial activities on E. coli. This pharmacophore model can be useful in order to design new antimicrobial drugs. It can be suggested that the substitution of a phenyl ring at the 5/6 and 5′/6′ positions in symmetric bisbenzimidazole derivatives produces compounds with promising antimicrobial activity.

An efficient synthesis of novel di-heterocyclic benzazole derivatives and evaluation of their antiproliferative activities

A series of unsymmetrical nine di-heterocyclic compounds of benzazole derivatives were synthesized at one step via cyclization reaction. The compounds evaluated for in vitro cytotoxic activity against A549, A498, HeLa, and HepG2 cancer cell lines. The biological evaluation results show that 23, 26 and 29 exhibit better activity against HepG2 and HeLa cancer cell lines. Compound 23 also showed good activity against A549, and A498 cancer cell lines. The analogs were further performed molecular docking studies against human cytochrome P450 2C8 monooxygenase enzyme, calculated some theoretical quantum parameters, ADMET descriptor and molecular electrostatic potential analysis. The strategy applied in this research work may act as a perspective for the rational design of potential anticancer drugs.

One‐pot synthesis of novel benzimidazoles with a naphthalene moiety as antimicrobial agents and molecular docking studies

In an attempt to design a greener approach for the synthesis of a potent class of antimicrobials, 1,2-phenylenediamine derivatives were reacted with various 1/2-carboxylic acid-substituted naphthalene derivatives to generate a series of naphthyl-substituted benzimidazole derivatives (11–19) using polyphosphoric acid as catalyst under microwave irradiation and conventional synthesis method. This is an eco-friendly and swift reaction method for a synthetic approach to diverse benzimidazoles. Structures of the synthesized compounds were established on the basis of spectral data and they were screened for their antimicrobial activity. Compound 18 showed maximum potency against all Gram-positive and Gram-negative bacterial strains with a minimum inhibitory concentration (MIC) value in the range of 7.81–62.50 μg/ml. Only compound 17 was found to be the most active against all fungal strains with a MIC value of 15.62 μg/ml. In this study, we performed molecular docking experiments to understand the interactions between compounds 17 and 18 and E. coli topoisomerase I, and we compared the results obtained with that of 2-(3,4-dimethoxyphenyl)-5-[5-(4-methylpiperazin−1-yl)-1H-benzimidazol-2-yl]-1H-benzimidazole (DMA). Compounds 17 and 18 demonstrated strong interactions with important active site residues, similar to DMA. As a result, the compounds obtained from this study can be used in designing new potent inhibitors of E. coli topoisomerase I.

Head-to-head bisbenzazole derivatives as antiproliferative agents: design, synthesis, in vitro activity, and SAR analysis

In the present work, a series of bisbenzazole derivatives were designed and synthesized as antiproliferative agents. The antiproliferative activity of these compounds was investigated using MTT assay. Bisbenzazole derivatives showed significant antiproliferative activity against all the four tested cancer cell lines. Among the various bisbenzazole derivatives, bisbenzoxazole derivatives exhibited the most promising anticancer activity followed by bisbenzimidazole and bisbenzothiazole derivatives. All the derivatives were found to be less toxic as compared to methotrexate (positive control) in normal human cells, indicating selective and efficient antiproliferative activity of these bisbenzazole derivatives. The structure–activity relationships of heteroaromatic systems and linkers present in bisbenzazole derivatives were analyzed in detail. In silico ADMET prediction revealed that bisbenzazole is a drug-like small molecule with a favorable safety profile. Compound 31 is a potential antiproliferative hit compound that exhibits unique cytotoxic activity distinct from methotrexate.

New-generation benzimidazole-based plasmid delivery reagents with high transfection efficiencies on the mammalian cells

Gene transfer and gene therapy studies require high-efficiency gene delivery reagents. By transferring the piece of DNA that we are interested in, we can alter the expression of certain gene or genes to further characterize its role in the cell function or in the organism’s development, metabolism, immune system, etc. Transfection reagents that enable efficient delivery of the DNA to the cells are important tools in the molecular and cellular biology studies. There are chemical products and tools that have been used for transfection of the cells but they are not as efficient as desired or they can induce cytotoxicity. It is crucial to design and generate new transfection reagents to further support the field of biotechnology, molecular studies, cellular biology, and in vitro studies relying on them. The more efficient and the less cytotoxic compounds will be especially useful for the field. We synthesized a new set of benzimidazole-based transfection reagents that have higher efficiency to carry GFP expressing plasmid in to the mammalian cells compared with the commercially available ones with low cytotoxicity. GFP expression levels were tracked by flow cytometry to determine the transfection efficiencies. Benzimidazole-based transfection reagents can be safely used for transfection studies in tissue culture as well as in gene therapy applications due to their high efficiency in the gene transfer to the mammalian cells.

Bisbenzoxazole derivatives had anti-proliferative effect on human cancer cells

A series of symmetric bis-benzoxazole derivatives were synthesized using one-pot cyclisation reaction between 4-fluoro substituted 2-aminophenol and suitable carboxylic acids. Synthesized compounds’ anticancer activities were tested by using MTT assay on human prostate (DU145) and breast (MCF7) cancer cells. Screening results revealed that all compounds possessed a high level anti-cancer potential by significantly decreasing the cell proliferation in prostate and breast cancer cell lines. Our compounds exerted their anti-proliferative effects in a dose and time dependent manner. Our results suggest that they can be highly potent since they were biologically active even at low concentration ranges. Our study presents a series of new bis-benzoxazole based compounds with potential therapeutic effects against tumor cells. Therefore, characterization of new generation bis-benzoxazole derivatives will have a significant contribution on the development of new era anti-cancer drug candidates.

Immunomodulatory and immunostimulatory effects of some bisbenzoxazole derivatives on lipopolysaccharide stimulated mammalian macrophages

Benzoxazoles and their derivatives have exerted anti-cancer and anti-inflammatory (immunomodulatory) potential due to their anti-proliferative effect on the cells. These molecules are DNA basebioisosteres, therefore, their mechanism of action could be by mimicking the structures of the DNA bases and halting the DNA polymerization processes. Based on their anti-proliferative effect, in our study we aimed to decipher the potential anti-inflammatory activities of unique bisbenzoxazole derivatives in vitro on mammalian macrophages. Being able to manipulate the inflammatory function of macrophages would enable the regulation of the immune response against danger stimuli. This would enable us better prognosis against different types of the diseases ranging from autoimmune disorders to cancer. Our results support the stark anti-inflammatory potential of bisbenzoxazole derivatives RHE 241 and RHE 248 in vitro on the LPS activated mammalian macrophages. After further delineation of their mechanism of action in vitro and their in vivo potency, these molecules could be utilized as potent anti-inflammatory medicines.

Symmetric bis-benzoxazole-based chemicals exerted anti-inflammatory effect on danger signal LPS-stimulated macrophages

Inflammatory responses are generated against the danger molecules under normal conditions but excessive or chronic inflammation leads to tissue degeneration and loss of function. To prevent the disease symptoms associated with the inflammatory disorders or the growth of tumor types that require inflammatory environment, generation of immunomodulatory drugs with suppressive functions have great potentials. In this study, we synthesized new generation of anti-proliferative bis-benzoxazole derivatives and tested their anti-inflammatory and anti-cancer potencies. For this purpose, we used a well-characterized mouse macrophage cell line (RAW 264.7). Furthermore, anti-cancer activity of these compounds were tested using MTT assay on prostate (DU145) and breast (MCF7) cancer cells. The screening results revealed that all compounds possessed a high-level anti-inflammatory potential by reducing the expression of inflammatory cytokines in LPS-stimulated macrophages. There were significant and substantial reductions in the secreted TNFα, IL6, and IL1β levels by chemically treated LPS-induced macrophages compared to non-treated induced ones. Our compounds exerted their anti-inflammatory effect in a dose-dependent manner and they were biologically active even in low nanomolar range concentrations. Bis-benzoxazole derivatives had anti-proliferative effect on MCF-7 and DU-145 cancer cells. Together, our results present a series of new bis-benzoxazole-based compounds with potential therapeutic effects in inflammatory diseases and on tumor cells.

Bisbenzoxazole derivatives had an antiinflammatory effect on in vitro stimulated macrophages

Benzoxazoles are DNA base bioisosteres and studies suggest that their derivatives have antiproliferative activities. Based on their antiproliferative activities they have been mostly studied as new generation anticancer drugs. In our study we exploited their antiproliferative effect, aiming to delineate bisbenzoxazole derivatives'(RHE 231 and RHE 238) potential antiinflammatory effect on mouse macrophages that are activated in vitro through danger signal LPS stimulation. RAW 267.4 mammalian macrophages were activated in the presence of our derivatives with or without danger mimic E. coli derived LPS. We present data that support the strong antiinflammatory activity of the bisbenzoxazole derivatives RHE 231 and RHE 238 on stimulated mammalian macrophages. There was a significant and substantial decrease in the production levels of TNF-, IL-1, and IL-6 proinflammatory cytokines in the presence of RHE 231 and RHE 238. These molecules had an antiproliferative effect on the macrophages and, probably, this was their mechanism of action on the cells to alter their inflammatory functions. Our results show that bisbenzoxazole structures RHE 231 and RHE 238 have potential to be used as antiinflammatory drug agents.

DNA base bioisosteres, bis-benzoxazoles, exert anti-proliferative effect on human prostate and breast cancer cells

A series of symmetric bis-benzoxazole derivatives were synthesized using one-pot cyclisation reaction between 4-chloro substituted 2-aminophenol and suitable dicarboxylic acids. Synthesized compounds’ anticancer activities were tested by using MTT assay on human prostate (DU145) and breast (MCF7) cancer cells. Screening results revealed that all compounds possessed a high level anti-cancer potential by significantly decreasing the cell proliferation in prostate and breast cancer cell lines. Our compounds exerted their anti-proliferative effects in a dose and time dependent manner. Our results suggest that they can be highly potent since they were biologically active even at low concentrations. Our study presents a series of new bis-benzoxazole based compounds with potential therapeutic effects against tumor cells. Therefore, characterization of new generation bis-benzoxazole derivatives will have a significant contribution on the development of new era anti-cancer drug candidates.

Synthesis and anti-proliferative activity of fluoro-substituted chalcones

A series of novel fluoro-substituted chalcone derivatives have been synthesized. All synthesized compounds were characterized by 1H nuclear magnetic resonance (NMR), 13C NMR, and elemental analysis. Their anti-proliferative activities were evaluated against five cancer cells lines, namely, A549, A498, HeLa, A375, and HepG2 using the MTT method. Most of the compounds showed moderate to high activity with IC50 values in the range of 0.029–0.729 μM. Of all the synthesized compounds, 10 and 19 exhibited the most potent anti-proliferative activities against cancer cells, and 10 was identified as the most promising compound