Biological activity and computational analysis of novel acrylonitrile derived benzazoles as potent antiproliferative agents for pancreatic adenocarcinoma with antioxidative properties.

Continuing our research into the anticancer properties of acrylonitriles, we present a study involving the design, synthesis, computational analysis, and biological assessment of novel acrylonitriles derived from methoxy, hydroxy, and N-substituted benzazole. Our aim was to examine how varying the number of methoxy and hydroxy groups, as well as the N-substituents on the benzimidazole core, influences their biological activity. The newly synthesized acrylonitriles exhibited strong and selective antiproliferative effects against the Capan-1 pancreatic adenocarcinoma cell line, with IC50 values ranging from 1.2 to 5.3 µM. Consequently, these compounds were further evaluated in three other pancreatic adenocarcinoma cell lines, while their impact on normal PBMC cells was also investigated to determine selectivity. Among these compounds, the monohydroxy-substituted benzimidazole derivative 27 emerged with the most profound and broad-spectrum anticancer antiproliferative activity being emerged as a promising lead candidate. Moreover, a majority of the acrylonitriles in this series exhibited significant antioxidative activity, surpassing that of the reference molecule BHT, as demonstrated by the FRAP assay (ranging from 3200 to 5235 mmolFe2+/mmolC). Computational analysis highlighted the prevalence of electron ionization in conferring antioxidant properties, with computed ionization energies correlating well with observed activities.

Synthesis and Biological Evaluation of Novel Amino and Amido Substituted Pentacyclic Benzimidazole Derivatives as Antiproliferative Agents.

Newly designed pentacyclic benzimidazole derivatives featuring amino or amido side chains were synthesized to assess their in vitro antiproliferative activity. Additionally, we investigated their direct interaction with nucleic acids, aiming to uncover potential mechanisms of biological action. These compounds were prepared using conventional organic synthesis methodologies alongside photochemical and microwave-assisted reactions. Upon synthesis, the newly derived compounds underwent in vitro testing for their antiproliferative effects on various human cancer cell lines. Notably, derivatives 6 and 9 exhibited significant antiproliferative activity within the submicromolar concentration range. The biological activity was strongly influenced by the N atom's position on the quinoline moiety and the position and nature of the side chain on the pentacyclic skeleton. Findings from fluorescence, circular dichroism spectroscopy, and thermal melting assays pointed toward a mixed binding mode-comprising intercalation and the binding of aggregated compounds along the polynucleotide backbone-of these pentacyclic benzimidazoles with DNA and RNA.

Design, Synthesis and Biological Activity of Novel Methoxy- and Hydroxy-Substituted N-Benzimidazole-Derived Carboxamides.

This work presents the design, synthesis and biological activity of novel N-substituted benzimidazole carboxamides bearing either a variable number of methoxy and/or hydroxy groups. The targeted carboxamides were designed to investigate the influence of the number of methoxy and/or hydroxy groups, the type of substituent placed on the N atom of the benzimidazole core and the type of substituent placed on the benzimidazole core on biological activity. The most promising derivatives with pronounced antiproliferative activity proved to be N-methyl-substituted derivatives with hydroxyl and methoxy groups at the phenyl ring and cyano groups on the benzimidazole nuclei with selective activity against the MCF-7 cell line (IC50 = 3.1 µM). In addition, the cyano-substituted derivatives 10 and 11 showed strong antiproliferative activity against the tested cells (IC50 = 1.2-5.3 µM). Several tested compounds showed significantly improved antioxidative activity in all three methods compared to standard BHT. In addition, the antioxidative activity of 9, 10, 32 and 36 in the cells generally confirmed their antioxidant ability demonstrated in vitro. However, their antiproliferative activity was not related to their ability to inhibit oxidative stress nor to their ability to induce it. Compound 8 with two hydroxy and one methoxy group on the phenyl ring showed the strongest antibacterial activity against the Gram-positive strain E. faecalis (MIC = 8 µM).

Synthesis and Antiproliferative Activity of 2,6-Disubstituted Imidazo[4,5-b]pyridines Prepared by Suzuki Cross Coupling.

A series of novel 2,6-diphenyl substituted imidazo[4,5-b]pyridines was designed and synthesized using optimized Suzuki cross coupling to evaluate their biological activity in vitro. The conditions of the Suzuki coupling were evaluated and optimized using a model reaction. To study the influence of the substituents on the biological activity, we prepared N-unsubstituted and N-methyl substituted imidazo[4,5-b]pyridines with different substituents at the para position on the phenyl ring placed at position 6 on the heterocyclic scaffold. Antiproliferative activity was determined on diverse human cancer cell lines, and the selectivity of compounds with promising antiproliferative activity was determined on normal peripheral blood mononuclear cells (PBMC). Pronounced antiproliferative activity was observed for p-hydroxy substituted derivatives 13 and 19, both displaying strong activity against most of the tested cell lines (IC50 1.45-4.25 µM). The unsubstituted N-methyl derivative 19 proved to be the most active derivative. There was a dose-dependent accumulation of G2/M arrested cells in several cancer cell lines after exposure to compound 19, implying a cell cycle-phase-specific mechanism of action. Additionally, the novel series of derivatives was evaluated for antiviral activity against a broad panel of viruses, yet the majority of tested compounds did not show antiviral activity.

Novel Biologically Active N-Substituted Benzimidazole Derived Schiff Bases: Design, Synthesis, and Biological Evaluation.

Herein, we present the design and synthesis of novel N-substituted benzimidazole-derived Schiff bases, and the evaluation of their antiviral, antibacterial, and antiproliferative activity. The impact on the biological activity of substituents placed at the N atom of the benzimidazole nuclei and the type of substituents attached at the phenyl ring were examined. All of the synthesized Schiff bases were evaluated in vitro for their antiviral activity against different viruses, antibacterial activity against a panel of bacterial strains, and antiproliferative activity on several human cancer cell lines, thus enabling the study of the structure-activity relationships. Some mild antiviral effects were noted, although at higher concentrations in comparison with the included reference drugs. Additionally, some derivatives showed a moderate antibacterial activity, with precursor 23 being broadly active against most of the tested bacterial strains. Lastly, Schiff base 40, a 4-N,N-diethylamino-2-hydroxy-substituted derivative bearing a phenyl ring at the N atom on the benzimidazole nuclei, displayed a strong antiproliferative activity against several cancer cell lines (IC50 1.1-4.4 µM). The strongest antitumoral effect was observed towards acute myeloid leukemia (HL-60).

Imidazo[4,5-b]pyridine derived tubulin polymerization inhibitors: Design, synthesis, biological activity in vitro and computational analysis.

Imidazo[4,5-b]pyridine derived acrylonitriles were synthesized and explored for their in vitro antiproliferative effect on a diverse human cancer cell line panel. Three compounds, 20, 21 and 33, showed strong activity in the submicromolar range (IC50 0.2-0.6 µM), and were chosen for further biological experiments. Immunofluorescence staining and tubulin polymerization assays confirmed tubulin as the main target, but excluded its colchicine-binding site as a potential interacting unit. This was supported by the computational analysis, which revealed that the most potent ligands act on the extended colchicine site on the surface between interacting tubulin subunits, where they interfere with their polymerization and reveal pronounced antitumor properties. In addition, lead molecule 21 potently inhibited cancer cell migration, while it did not affect the viability of normal cells even at the highest concentration tested (100 µM).

Design, synthesis, biological evaluation and QSAR analysis of novel N-substituted benzimidazole derived carboxamides.

As a result of our previous research focussed on benzimidazoles, herein we present design, synthesis, QSAR analysis and biological activity of novel N-substituted benzimidazole derived carboxamides. Carboxamides were designed to study the influence of the number of methoxy groups, the type of the substituent placed at the benzimidazole core on biological activity. Pronounced antioxidative activity displayed unsubstituted 28 (IC50 ≈ 3.78 mM, 538.81 mmolFe2+/mmolC) and dimethoxy substituted derivative 34 (IC50 ≈ 5.68 mM, 618.10 mmolFe2+/mmolC). Trimethoxy substituted 43 and unsubstituted compound 40 with isobutyl side chain at N atom showed strong activity against HCT116 (IC50 ≈ 0.6 µM, both) and H 460 cells (IC50 ≈ 2.5 µM; 0.4 µM), being less cytotoxic towards non-tumour cell. Antioxidative activity in cell generally confirmed relatively modest antioxidant capacity obtained in DPPH/FRAP assays of derivatives 34 and 40. The 3D-QSAR models were generated to explore molecular properties that have the highest influence on antioxidative activity.

Biological Activity of Amidino-Substituted Imidazo [4,5-b]pyridines.

A series of cyano- and amidino-substituted imidazo[4,5-b]pyridines were synthesized using standard methods of organic synthesis, and their biological activity was evaluated. Biological evaluation included in vitro assessment of antiproliferative effects on a diverse selection of human cancer cell lines, antibacterial activity against chosen Gram-positive and Gram-negative bacterial strains, and antiviral activity on a broad panel of DNA and RNA viruses. The most pronounced antiproliferative activity was observed for compound 10, which contained an unsubstituted amidino group, and compound 14, which contained a 2-imidazolinyl amidino group; both displayed selective and strong activity in sub-micromolar inhibitory concentration range against colon carcinoma (IC50 0.4 and 0.7 µM, respectively). All tested compounds lacked antibacterial activity, with the exception of compound 14, which showed moderate activity against E. coli (MIC 32 µM). Bromo-substituted derivative 7, which contained an unsubstituted phenyl ring (EC50 21 µM), and para-cyano-substituted derivative 17 (EC50 58 µM) showed selective but moderate activity against respiratory syncytial virus (RSV).

Preclinical in vitro screening of newly synthesised amidino substituted benzimidazoles and benzothiazoles.

Newly synthesised benzimidazole/benzotiazole derivatives bearing amidino, namely 3,4,5,6-tetrahydropyrimidin-1-ium chloride, substituents have been evaluated for their potential antitumor activity in vitro. Compounds and standard drugs (doxorubicin, staurosporine and vandetanib) were tested on three human lung cancer cell lines A549, HCC827 and NCI-H358. We tested compounds in MTS citotoxicity assay and in BrdU proliferative assay performed on 2 D and 3 D assay format. Because benzmidazole scaffold is similar to natural purines, we tested the most active compounds for ability to induce cell apoptosis of A549 by binding to DNA in comparison with doxorubicin and saturosporine. Additionally, the ADME properties of the most active benzothiazole/benzimidazole and non-active compounds were determined to see if the different ADME properties are the cause of different activity in 2 D and 3 D assays, as well as to see if the tested active compounds have drug like properties and potency for further profilation. ADME characterisation included solubility, lipophilicity, permeability, metabolic stability and binding to plasma proteins. In general, the benzothiazole derivatives were more active in comparison to their benzimidazole analogues. The exception was 2-phenyl substituted benzimidazole 6a being active with very pronounced activity especially towards HCC827 cells. All active compounds have similar mode of action on A549 cell line as standard compound doxorubicin, which binds to nucleic acids with the DNA double helix. Tested active benzothiazole compounds were characterised by moderate to good solubility, good metabolic stability, low permeability and high binding to plasma proteins. One tested active benzimidazole derivative showed ADME properties, but lower lipophilicity resulted in low PPB and higher metabolic instability. In addition, no significant difference was observed in ADME profile between active and non-active compounds.

Biological Activity of Newly Synthesized Benzimidazole and Benzothizole 2,5-Disubstituted Furane Derivatives.

Newly designed and synthesized cyano, amidino and acrylonitrile 2,5-disubstituted furane derivatives with either benzimidazole/benzothiazole nuclei have been evaluated for antitumor and antimicrobial activity. For potential antitumor activity, the compounds were tested in 2D and 3D cell culture methods on three human lung cancer cell lines, A549, HCC827 and NCI-H358, with MTS cytotoxicity and BrdU proliferation assays in vitro. Compounds 5, 6, 8, 9 and 15 have been proven to be compounds with potential antitumor activity with high potential to stop the proliferation of cells. In general, benzothiazole derivatives were more active in comparison to benzimidazole derivatives. Antimicrobial activity was evaluated with Broth microdilution testing (according to CLSI (Clinical Laboratory Standards Institute) guidelines) on Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Additionally, Saccharomyces cerevisiae was included in testing as a eukaryotic model organism. Compounds 5, 6, 8, 9 and 15 showed the most promising antibacterial activity. In general, the compounds showed antitumor activity, higher in 2D assays in comparison with 3D assays, on all three cell lines in both assays. In natural conditions, compounds with such an activity profile (less toxic but still effective against tumor growth) could be promising new antitumor drugs. Some of the tested compounds showed antimicrobial activity. In contrast to ctDNA, the presence of nitro group or chlorine in selected furane-benzothiazole structures did not influence the binding mode with AT-DNA. All compounds dominantly bound inside the minor groove of AT-DNA either in form of monomers or dimer and higher-order aggregates.

Biological Potential of Novel Methoxy and Hydroxy Substituted Heteroaromatic Amides Designed as Promising Antioxidative Agents: Synthesis, 3D-QSAR Analysis, and Biological Activity.

This paper discusses antioxidative and biological activities of 25 novel amidino substituted benzamides with a variety of heteroaromatic nuclei attached to the benzamide moiety and with a variable number of methoxy or hydroxy substituents. Targeted compounds, bearing either amidino or 2-imidazolinyl substituent, were obtained in the Pinner reaction from cyano precursors. 3D-QSAR models were generated to predict antioxidative activity of the 25 novel aromatic and heteroaromatic benzamide derivatives. The compounds were tested for antioxidative activity using in vitro spectrophotometric assays. Direct validation of 3D-QSAR approach for predicting activities of novel benzamide derivatives was carried out by comparing experimental and computationally predicted antioxidative activity. Experimentally determined activities for all novel compounds were found to be within a standard deviation of error of the models. Following this, structure-activity relationships among the synthesized compounds are discussed. Furthermore, antiproliferative activity in vitro against HeLa cells as well as antibacterial and antifungal activity was tested to confirm the other biological activities of the prepared compounds.

Amino-Substituted Benzamide Derivatives as Promising Antioxidant Agents: A Combined Experimental and Computational Study.

We prepared a range of N-arylbenzamides with a variable number of methoxy and hydroxy groups, bearing either amino or amino-protonated moieties, and used DPPH and FRAP assays to evaluate their antioxidant capacity. Most of the systems exhibit improved antioxidative properties relative to the reference BHT molecule in both assays. Combining results from both sets of experiments, the most promising antioxidative potential was displayed by the trihydroxy derivative 26, which we propose as a lead compound for a further optimization of the benzamide scaffold. Computational analysis helped in interpreting the observed trends and demonstrated that protonated systems are better antioxidants than their neutral counterparts, while underlying the positive influence of the electron-donating methoxy group on the antioxidant properties, thus confirming the experiments. It also revealed that the introduction of the hydroxy groups shifts the reactivity from both amide and amine groups toward this moiety and additionally enhances antioxidative features. This is particularly evident in 26H•+, which owes its pronounced reactivity to the stabilizing [O•···H-O] hydrogen bonding between the created phenoxyl radical and the two neighboring hydroxy groups. We demonstrated that its antioxidative activities are more favorable than those for analogous trihydroxy derivatives without the N-phenyl group or without the amide moiety, which strongly justifies the employed strategy in utilizing bisphenylamides in designing potent antioxidants.

Antibacterial and antiproliferative activity of novel 2-benzimidazolyl- and 2-benzothiazolyl-substituted benzo[b]thieno-2-carboxamides.

Novel nitro (3a-3f)- and amino (4a-4f and 5a-5f)-substituted 2-benzimidazolyl and 2-benzothiazolyl benzo[b]thieno-2-carboxamides were designed and synthesized as potential antibacterial agents. The antibacterial activity of these compounds has been evaluated against Gram-positive (Staphylococcus aureus and Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli and Moraxella catarrhalis). The most promising antibacterial activity was observed for the nitro- and amino-substituted benzimidazole derivatives 3a, 4a, 5a and 5b with MICs 2-8 [Formula: see text]. Additionally, compounds with inferior antibacterial activity were further tested for their antiproliferative activity in vitro against three human cancer cell lines. Amino-substituted benzothiazole hydrochloride salt 5d displayed the most pronounced and selective activity against the MCF-7 cell line with an [Formula: see text] of 40 nM. Furthermore, DNA binding experiments of selected derivatives indicated that DNA cannot be considered as a primary biological target for this type of compounds.

Antioxidative and antiproliferative activities of novel pyrido[1,2-a]benzimidazoles.

A series of pyrido[1,2-a]benzimidazoles has been designed, and novel examples are synthesized and evaluated for their potential antiproliferative activity against four human tumour cell lines-cervical (HeLa), colorectal (SW620), breast (MCF-7) and hepatocellular carcinoma (HepG2). In addition, their antioxidative potency has been evaluated by in vitro spectrophotometric assays. Preliminary structure-activity relationships among the synthesized compounds are discussed. Evaluation of their antioxidative capacity has shown that two compounds (25 and 26) possess promising reducing characteristics and free radical scavenging activity. Selective antiproliferative effect in the single-digit micromolar range was observed for compound 25 on MCF-7 [Formula: see text] and HeLa [Formula: see text] cell lines, comparable to the standards 5-fluorouracil and cisplatin. The combination of the radical scavenging activity and antiproliferative activity of compound 25 positions this compound as a potential lead candidate for further optimization.

Synthesis and antiproliferative activity of amino-substituted benzimidazo[1,2-α]quinolines as mesylate salts designed by 3D-QSAR analysis.

An experimental search for new benzimidazole derivatives with enhanced antiproliferative activity was successfully guided by QSAR modelling. Robust 3D-QSAR models were derived on an available database of compounds with previously measured activities. Our QSAR analysis revealed that an increase of the antiproliferative activities towards H460, HCT 116, MCF-7 and SW 620 cells will be obtained if new compounds are charged at a pH range from 5 to 7 and if their hydrophobicity is increased compared to the dataset compounds. Novel benzimidazo[1,2-a]quinolines bearing quarternary amino groups with corresponding aliphatic chains were designed, and their antiproliferative activities were computationally predicted. Using uncatalysed microwave-assisted amination reactions, 14 novel compounds were obtained to assess their antiproliferative activities towards H460, HCT 116, MCF-7, and SW 620 tumour cell lines in vitro. Novel compounds showed antiproliferative activities at micromolar and submicromolar inhibition concentrations. Experimental measurements of antiproliferative activities validation the QSAR models showing very good agreement between experimentally measured activities and computational predictions. In an attempt to elucidate the mode of action through which benzimidazole derivatives accomplish their antiproliferative activities, thermal denaturation experiments were performed to test their DNA-binding properties.

Antitumor activity of amidino-substituted benzimidazole and benzimidazo[1,2-a]quinoline derivatives tested in 2D and 3D cell culture systems.

Due to a poor clinical predictive power of 2D cell cultures, standard tool for in vitro assays in drug discovery process, there is increasing interest in developing 3D in vitro cell cultures, biologically relevant assay feasible for the development of robust preclinical anti-cancer drug screening platforms. Herein, we tested amidino-substituted benzimidazoles and benzimidazo[1,2-a]quinolines as a small platform for comparison of antitumor activity in 2D and 3D cell culture systems and correlation with structure-activity relationship. 3D cell culture method was applied on a human cancer breast (SK-BR-3, MDA-MB-231, T-47D) and pancreatic cancer cells (MIA PaCa-2, PANC-1). Results obtained in 2D and 3D models were highly comparable, but in some cases we have observed significant disagreement indicating that some prominent compounds can be discarded in early phase of researching because of compounds with false positive result. To confirm which of cell culture systems is more accurate, in vivo profiling is needed.

Synthesis, QSAR, and Molecular Dynamics Simulation of Amidino-substituted Benzimidazoles as Dipeptidyl Peptidase III Inhibitors.

A molecular modeling study is performed on series of benzimidazol-based inhibitors of human dipeptidyl peptidase III (DPP III). An eight novel compounds were synthesized in excellent yields using green chemistry approach. This study is aimed to elucidate the structural features of benzimidazole derivatives required for antagonism of human DPP III activity using Quantitative Structure-Activity Relationship (QSAR) analysis, and to understand the mechanism of one of the most potent inhibitor binding into the active site of this enzyme, by molecular dynamics (MD) simulations. The best model obtained includes S3K and RDF045m descriptors which have explained 89.4 % of inhibitory activity. Depicted moiety for strong inhibition activity matches to the structure of most potent compound. MD simulation has revealed importance of imidazolinyl and phenyl groups in the mechanism of binding into the active site of human DPP III.

Novel acrylonitrile derived imidazo[4,5-b]pyridines as antioxidants and potent antiproliferative agents for pancreatic adenocarcinoma.

We present the design, synthesis, computational analysis, and biological assessment of several acrylonitrile derived imidazo[4,5-b]pyridines, which were evaluated for their anticancer and antioxidant properties. Our aim was to explore how the number of hydroxy groups and the nature of nitrogen substituents influence their biological activity. The prepared derivatives exhibited robust and selective antiproliferative effects against several pancreatic adenocarcinoma cells, most markedly targeting Capan-1 cells (IC50 1.2-5.3 µM), while their selectivity was probed relative to normal PBMC cells. Notably, compound 55, featuring dihydroxy and bromo substituents, emerged as a promising lead molecule. It displayed the most prominent antiproliferative activity without any adverse impact on the viability of normal cells. Furthermore, the majority of studied derivatives also exhibited significant antioxidative activity within the FRAP assay, even surpassing the reference molecule BHT. Computational analysis rationalized the results by highlighting the dominance of the electron ionization for the antioxidant features with the trend in the computed ionization energies well matching the observed activities. Still, in trihydroxy derivatives, their ability to release hydrogen atoms and form a stable O-H⋯O•⋯H-O fragment upon the H• abstraction prevails, promoting them as excellent antioxidants in DPPH• assays as well.

Design, synthesis, antitumor activity and NMR-based metabolomics of novel amino substituted tetracyclic imidazo[4,5-b]pyridine derivatives.

Newly prepared tetracyclic imidazo[4,5-b]pyridine derivatives were synthesized to study their antiproliferative activity against human cancer cells. Additionally, the structure-activity was studied to confirm the impact of the N atom position in pyridine nuclei as well as the chosen amino side chains on antiproliferative activity. Targeted amino substituted regioisomers were prepared by using uncatalyzed amination from corresponding chloro substituted precursors. The most active compounds 6a, 8 and 10 showed improved activity in comparison to standard drug etoposide with IC50 values in a nanomolar range of concentration (0.2 - 0.9 µM). NMR-based metabolomics is a powerful instrument to elucidate activity mechanism of new chemotherapeutics. Multivariate and univariate statistical analysis of metabolic profiles of non-small cell lung cancer cells before and after exposure to 6a revealed significant changes in metabolism of essential amino acids, glycerophospholipids and oxidative defense. Insight into the changes of metabolic pathways that are heavily involved in cell proliferation and survival provide valuable guidelines for more detailed analysis of activity metabolism and possible targets of this class of bioactive compounds.

Novel hydroxy- and amidino-substituted benzimidazoles and benzothiazoles as antibacterial and antiproliferative agents.

Aim: The aim was synthesis of novel benzazoles bearing amidino and 2-hydroxyphenyl substituents to explore their biological activity. Methods: Condensation of 5-substituted salicylaldehydes and intermediates gave new benzazoles by previously published and developed procedures, which were tested for antibacterial and antiproliferative activity in vitro. Results: The best antibacterial activity showed benzimidazole with 2-imidazolinyl group 27 and benzothiazole with an unsubstituted amidine 48 (minimum inhibitory concentration 8 µg/ml). Benzothiazole 53 proved most potent at inhibiting proliferation of all cancer cells (IC50: 1.2-2.0 µM). Conclusion: Most active compounds have been recognized as lead compounds for additional optimization to improve their biological activity. The type of amidine moiety markedly influenced the biological activity. Benzothiazoles showed improved antiproliferative activity in comparison to benzimidazoles.