Characterization of the Effect of N-(2-Methoxyphenyl)-1-methyl-1H-benzimidazol-2-amine, Compound 8, against Leishmania mexicana and Its In Vivo Leishmanicidal Activity.

Chemotherapy currently available for leishmaniasis treatment has many adverse side effects and drug resistance. Therefore, the identification of new targets and the development of new drugs are urgently needed. Previously, we reported the synthesis of a N-(2-methoxyphenyl)-1-methyl-1H-benzimidazol-2-amine, named compound 8, with an IC50 value in the micromolar range against L. mexicana, it also inhibited 68.27% the activity of recombinant L. mexicana arginase. Herein, we report studies carried out to characterize the mechanism of action of compound 8, as well as its in vivo leishmanicidal activity. It was shown in our ultrastructural studies that compound 8 induces several changes, such as membrane blebbing, the presence of autophagosomes, membrane detachment and mitochondrial and kinetoplast disorganization, among others. Compound 8 triggers the production of ROS and parasite apoptosis. It reduced 71% of the parasite load of L. mexicana in an experimental model of cutaneous leishmaniasis in comparison with a control. Altogether, the data obtained suggest the potential use of compound 8 in the treatment of cutaneous leishmaniasis.

Sex-divergent expression of cytochrome P450 and SIRTUIN 1-7 proteins in toxicity evaluation of a benzimidazole-derived epigenetic modulator in mice.

Efforts in precision medicine to combat aberrant epigenome have led to the development of epigenetic targeting drugs. We have previously reported the capability of the BZD9L1 epigenetic modulator to impede colorectal tumour growth in vitro and in vivo through sirtuin (SIRT) inhibition. Although most benzimidazole derivatives are commonly less toxic, their effects on SIRTs and cytochrome P450 (CYP) regulations have not been explored alongside toxicity assessments. SIRTs are histone deacetylases that are crucial in maintaining metabolic homeostasis, whereas CYP is essential in drug metabolism. This study aims to determine the toxicology profile of BZD9L1 through oral acute and repeated dose toxicity evaluations, along with molecular analyses of SIRT, CYP and relevant toxicity markers through western blot and quantitative polymerase chain reaction (qPCR). BZD9L1 demonstrated no sign of acute toxicity at the limit dose (2000 mg/kg). The 28-day toxicity study highlighted the tolerability of repeated dose administration without adverse effects. BZD9L1 showed a sex-divergent regulation of hepatic SIRT1-7, CYP2A5 and CYP2D proteins. Furthermore, BZD9L1 did not induce the expression of organ injury proteins or alter the gene expression of cellular function indicators in mouse liver and kidneys, hence demonstrating, at least in part, the safety of BZD9L1 in short-term evaluations. The present study cautions for personalised strategies when employing benzimidazole-derived epigenetic therapeutics.

Effects of a New Benzimidazole Derivative with Actoprotective Properties on Spermatogenic Epithelium of the Testes and Fertility of Male White Rats.

The effects of a new derivative of benzimidazole (K-134) in doses of 5 and 50 mg/kg on the spermatogenesis and fertilizing ability of spermatozoa were studied on male rats. It was found that 2-month course treatment with the studied substance enhances the producing ability of the spermatogenic epithelium and improves fertilizing ability of spermatozoa.

Evaluation of New Benzimidazole Derivatives as Cysticidal Agents: In Vitro, in Vivo and Docking Studies.

Based on our previous research on cysticidal drugs, we report the synthesis and evaluation of three new benzimidazole derivatives. In these compounds, the amido group was used as a bioisosteric replacement of the ester group. The molecular docking on ß-tubulin revealed that the derivatives interacted through hydrogen bonding with N165, E198 and V236. All compounds showed in vitro activity against Taenia crassiceps cysts. Among them, benzimidazole 3 was found to be the most potent of the series (EC50 0.86 µM). This compound also exhibited the highest probability of binding and the lowest binding free energy score and was therefore selected for in vivo evaluation. Results indicated that the efficacy of compound 3 was comparable to that of the reference drug, albendazole (50.39 vs. 47.16% parasite reduction). Animals treated with compound 3 seemed to tolerate this benzimidazole well, with no changes in behavior, or food and water consumption. These findings are consistent with the in silico prediction results, which indicated low toxicity risks. The pharmacokinetic study showed that the half-life and mean residence time (6.06 and 11.9 h, respectively) were long after oral administration. Together, these results indicate that this new benzimidazole derivative represents a promising structure with cysticidal activity.

Benzimidazole derivative M084 extends the lifespan of Caenorhabditis elegans in a DAF-16/FOXO-dependent way.

With the growth of aging population, there is increasing demand to develop strategy to improve the aging process and aging-related diseases. Benzimidazole and its derivatives are crucial heterocyclic backbone of many drugs and compounds with diverse therapeutic applications, including alleviation of aging-related diseases. Here, we investigate if the benzimidazole derivative n-butyl-[1H]-benzimidazol-2-amine (M084), a novel inhibitor of TRPC4 and TRPC5 channels and antidepressant, could affect the lifespan of Caenorhabditis elegans (C. elegans). Our results showed that M084 could extend the lifespan of C. elegans, delay age-related decline of phenotypes, and improve stress resistance. M084 could not extend the lifespan of the loss-of-function mutants of daf-16, daf-2, pdk-1, aak-2, clk-1, isp-1, sir-2.1, and skn-1. M084 could decrease the ATP level and increase the gene expression of mitochondrial unfolded protein response factors. Thus, M084 might inhibit the mitochondrial respiration, activate mitochondrial unfolded protein response and AMPK, recruite SIR-2.1 and SKN-1, and finally through the transcription factor DAF-16, delay the aging process of C. elegans. Our findings reveal the new pharmaceutical potential of benzimidazole derivatives and provide clue for developing novel anti-aging agents.

Alternative energy production pathways in Taenia crassiceps cysticerci in vitro exposed to a benzimidazole derivative (RCB20).

Biochemical studies of benzimidazole derivatives are important to determine their mode of action and activity against parasites. The lack of antihelminthic alternatives to treat parasitic infections and albendazole resistance cases make the search for new antiparasitary drugs of utmost importance. The 6-chloro-5-(1-naphthyloxy)-2-(trifluoromethyl)-1H-benzimidazole (RCB20) is a benzimidazole derivative with promising effect. This study evaluated the effect of different concentrations of RCB20 in the alternative energetic pathway of in vitro Taenia crassiceps cysticerci. The parasites were in vitro exposed to 6.5 and 13 µM of RCB20 and albendazole sulfoxide (ABZSO). The quantification of acetate, acetoacetate, ß-hydroxybutyrate, fumarate and propionate was performed by high-performance liquid chromatography. The quantification of urea, creatinine and total proteins was performed by spectrophotometry. The increase in ß-hydroxybutyrate reflects the enhancement of the fatty acid oxidation in the treated groups. Volatile fatty acids secretion, acetate and propionate, was increased in the treated groups. The secretion mechanisms of the treated parasites were impaired due to organic acids increased concentrations in the cysticerci. It is possible to conclude that the metabolic effect on alternative energetic pathways is slightly increased in the parasites treated with RCB20 than the ones treated with ABZSO.

Bactericidal and biofilm eradication efficacy of a fluorinated benzimidazole derivative, TFBZ, against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major inducement of nosocomial infections and its biofilm formation render the high tolerance to conventional antibiotics, which highlights the requirement to develop new antimicrobial agents urgently. In this study, we identified a fluorinated benzimidazole derivative, TFBZ, with potent antibacterial efficacy toward planktonic MRSA (MIC = 4 µg/mL, MBC = 8 µg/mL) and its persistent biofilms (≥99%, MBEC = 8 µg/mL). TFBZ manifested significant irreversible time-dependent killing against MRSA as characterized by diminished cell viability, bacterial morphological change and protein leakage. Furthermore, the results from CBD devices, crystal violet assay in conjunction with live/dead staining and scanning electron microscopy confirmed that TFBZ was capable of eradicating preformed MRSA biofilms with high efficiency. Simultaneously, TFBZ reduced the bacterial invasiveness and exerted negligible hemolysis and cytotoxicity toward mammalian cells, which ensuring the robust therapeutic effect on mouse skin abscess model. The transcriptome profiling and quantitative RT-PCR revealed that a set of encoding genes associated with cell adhesion, biofilm formation, translation process, cell wall biosynthesis was consistently downregulated in MRSA biofilms upon exposure to TFBZ. In conclusion, TFBZ holds promise as a valuable candidate for therapeutic applications against MRSA chronic infections.

Synthesis and characterization of a novel Mannich base benzimidazole derivative to explore interaction with human serum albumin and antimicrobial property: experimental and theoretical approach.

The novel Mannich base benzimidazole derivative (CB-1), 1-((1H-benzo[d]imidazol-1-yl)(3-chlorophenyl)methyl)-3-phenylurea) has been designed and synthesized by reacting benzimidazole, 3-chloro benzaldehyde, and N-Phenyl urea. CB-1 has been characterized by UV- Visible, FTIR, and 1H NMR. CB-1 was explored to study the interaction with the most abundant blood protein which involved in the role of transport of molecules (drugs), human serum albumin (HSA). Fluorescence results are evident for the presence of both dynamic and static quenching mechanisms in the binding of CB-1 to HSA. Antimicrobial screening were carried out against three bacteria and three fungi pathogens employing disc diffusion method. Molecular docking using AutoDock Vina tool further confirms the experimental binding interactions obtained from fluorescence. Density functional theory (DFT) with B3LYP/6-311G++ basis set was used for correlating theoretical data and obtaining optimized structures of CB-1 along with reactants with molecular electrostatic potential (MEP) map and HOMO→LUMO energy gap calculation. HIGHLIGHTSThe novel Mannich base benzimidazole derivative (CB-1) has been designed and synthesized by Mannich reaction.CB-1 has been characterized by UV- Visible, FTIR, and 1H NMR.Fluorescence quenching reveals that HSA binds to CB-1 via aromatic residues, which is corroborated by molecular docking.Antifungal and antibacterial activity was evaluated in comparison to Nystatin and Gentamicin standard drugs, respectively.DFT calculations support experimental data and provide HOMO-LUMO energy gap.Communicated by Ramaswamy H. Sarma.

Investigation of anti-cancer effects of new pyrazino[1,2-a]benzimidazole derivatives on human glioblastoma cells through 2D in vitro model and 3D-printed microfluidic device.

AIMS: Recent studies show targeted therapy of new pyrazino[1,2-a]benzimidazole derivatives with COX-II inhibitory effects on different cancer cells. This study aimed to investigate 2D cell culture and 3D spheroid formation of glioblastoma multiforme (GBM) cells using a microfluidic device after exposure to these compounds. MAIN METHODS: After isolating astrocytes from human GBM samples, IC50 of 2,6-dimethyl pyrazino[1,2-a]benzimidazole (L1) and 3,4,5-trimethoxy pyrazino[1,2-a]benzimidazole (L2) were determined as 13 µM and 85 µM, respectively. Then, in all experiments, cells were exposed to subtoxic concentrations of L1 (6.5 µM) and L2 (42.5 µM), which were ½IC50. In the following, in two phases, cell cycle, migration, and gene expression through 2D cell culture and tumor spheroid formation ability using a 3D-printed microfluidic chip were assessed. KEY FINDINGS: The obtained results showed that both compounds have positive effects in reducing G2/M cell population and GBM cell migration. Furthermore, real-time gene expression data showed that L1 and L2 significantly impact the upregulation of P21 and P53 and down-regulation of cyclin D1, MMP2, and MMP9. On the other hand, GBM spheroids exposed to L1 and L2 become smaller with fewer live cells. SIGNIFICANCE: Our data on human isolated astrocyte cells in 2D and 3D cell culture conditions showed that L1 and L2 compounds could reduce GBM cells' invasion by controlling gene expressions associated with migration and proliferation. Moreover, designing microfluidic platform and related cell culture protocols facilitates the broad screening of 3D multicellular tumor spheroids derived from GBM tumor biopsies and provides effective drug development for brain gliomas.

Benzimidazole Derivative Ameliorates Opioid-Mediated Tolerance during Anticancer- Induced Neuropathic Pain in Mice.

BACKGROUND: Cancer is known to be the second significant cause of death worldwide. Chemotherapeutic agents such as platinum-based compounds are frequently used single-handedly or accompanied by additional chemotherapies to treat cancer patients. Chemotherapy-induced peripheral painful neuropathy is seen in around 40% of patients who are treated with platinum-based compounds, including cisplatin. This not only decreases the quality of life of patients but also patients' compliance with cisplatin. OBJECTIVES: Nalbuphine, an opioid, is frequently used to treat acute and chronic pain, coupled with cisplatin in cancer patients. However, long term use of nalbuphine induces tolerance to its analgesic effects. We employed the same strategy to induce tolerance in mice. METHODS: Here, we investigated analgesic effects of 2-[(pyrrolidin-1-yl) methyl]-1H-benzimidazole (BNZ), a benzimidazole derivative, on nalbuphine-induced tolerance during cisplatin-induced neuropathic pain using hot plate test, tail-flick tests and von Frey filament in mouse models. Furthermore, we investigated the effects of BNZ on the expression of Tumor Necrosis Factor-alpha (TNF-α) in the spinal cord. RESULTS: The results showed that BNZ reduced tolerance to analgesic effects of nalbuphine and TNF-α expression in mice. CONCLUSION: BNZ could be a potential drug candidate for the management of nalbuphine-induced tolerance in cisplatin-induced neuropathic pain.

Assessment of cytotoxic effects of new derivatives of pyrazino[1,2-a] benzimidazole on isolated human glioblastoma cells and mitochondria.

AIMS: Glioblastoma multiforme (GBM) is a highly devastating malignant brain tumor with poor pharmacotherapy. Based on COX-2 inhibitory effects in preventing cancer progression, new pyrazino[1,2-a]benzimidazole derivatives were assessed on isolated human GBM cells. MAIN METHODS: In this study, firstly, primary culture of astrocytes from human GBM samples was prepared and exposed to 2,6-dimethyl pyrazino[1,2-a]benzimidazole (L1) and 3,4,5-trimethoxy pyrazino[1,2-a]benzimidazole (L2) for finding their half-maximal inhibitory concentration (IC50). In the following, in two phases, cell apoptosis pathway and mitochondrial markers were investigated on GBM and also HEK293 cells (as non-cancerous normal cells). KEY FINDINGS: The MTT results represented a remarkable selective cytotoxic effect of both L1 and L2 on GBM cells, and interestingly not on normal cells. After 48 h, IC50 of L1 and L2 were calculated as 13 µM and 85 µM, respectively. Annexin/PI staining showed that L1 and L2 induce apoptosis in GBM cells, and caspase measurement showed that apoptosis occurs through mitochondrial signaling. In the clonogenic assay, GBM cells formed more paraclones and fewer holoclones after treating with L1 and L2. L1 and L2 also selectively enhanced mitochondrial damaged markers, including reactive oxygen species (ROS) formation, and mitochondrial swelling, decreased mitochondrial membrane potential (MMP) and cytochrome c release in isolated cancerous GBM mitochondria. SIGNIFICANCE: Our findings on human primary astrocyte cells illustrated that L1 and L2 compounds, with COX-2 inhibitory effect, through the intrinsic pathway of apoptosis concerning mitochondrial damage enhancement have therapeutic potentials on GBM.

Partial inhibition of the tricarboxylic acid cycle in Taenia crassiceps cysticerci after the in vitro exposure to a benzimidazole derivative (RCB15).

The benzimidazole derivative, 6-chloro-5-(2,3-dichlorophenoxy)-2-(trifluoromethyl)-1H-benzimidazole (RCB15), has a similar mode of action and efficacy as albendazole, a commonly used anthelminthic drugs. The aim of this study was to evaluate its influence on the tricarboxylic acid cycle in Taenia crassiceps cysticerci. The parasites were cultured in supplemented RPMI medium containing albendazole sulfoxide (ABZSO) or RCB15, for 24 h. Then, frozen in liquid nitrogen for organic metabolites extraction. Samples were analyzed by high performance liquid chromatography and organic acids of the tricarboxylic acid cycle were detected. It was possible to observe changes in the concentrations of all acids involved in this metabolic pathway, with the exception of α-ketoglutarate, which was not detected in the control group neither in most of the treated groups. It indicates that the parasite presented a partial inhibition of the tricarboxylic acid cycle. The significant increase in the concentration of citrate, oxaloacetate and succinate in the RCB15 treated groups may indicate an activation of the fumarate reductase pathway, leading to metabolic distress. Therefore RCB15 may be considered an alternative for the treatment of tissue parasitic diseases, since it induced changes in the main metabolic pathway of the parasite.

Reinvestigating the synthesis and efficacy of small benzimidazole derivatives as presequence protease enhancers.

Presequence protease (PreP) is a proteostatic enzyme that plays a key role in the maintenance of mitochondrial health. Defects in PreP stability are associated with neurological disorders in humans, and altered activity of this enzyme modulates the progress of Alzheimer's disease-like pathology in mice. As agonists that boost PreP proteolytic activity represent a promising therapeutic avenue, we sought to determine the structural basis for the action of benzimidazole derivatives (3c and 4c), first reported by Vangavaragu et al. (Eur. J. Med. Chem. 76 (2014) 506-516) that enhance the activity of PreP. However, we found the published procedure for the synthesis of 3c yielded aldimine A instead. We then developed an alternative synthesis and obtained 3c, termed compound C, and an alternative benzimidazole derivative, termed compound B. We tested compounds A, B and C for their ability to enhance the activities of human PreP. In contrast to the previous report, we observed that none of the compounds A, B, or C (3c) modulated the catalytic activity of human PreP. Here we report our findings on the mis-identification of the reported benzimidazoles and the lack of biological activity of such compounds on human PreP. Thus, PreP modulators for PreP-based therapies remain to be discovered.

A benzimidazole derivative (RCB15) in vitro induces the alternative energetic metabolism and glycolysis in Taenia crassiceps cysticerci.

The emergence of resistance to albendazole has encouraged the search for effective alternatives for cysticercosis and other parasitosis treatment. RCB15 is a benzimidazole derivative that may be used against such diseases. The aim of this study was to determine the in vitro effect of RCB15 on the alternative energetic pathways of Taenia crassiceps cysticerci. The cysticerci were in vitro exposed to albendazole sulphoxide (ABZSO) or RCB15 at different concentrations during 24h. The cysticerci extract and the culture medium were analyzed through spectrophotometry and high performance liquid chromatography as to detect glucose, urea, creatinine and organic acids of the energetic metabolism. The drugs did not influence the protein catabolism. Fatty acids oxidation was enhanced through significantly higher acetate concentrations in the groups treated with RCB15 and ABZSO. Beta-hydroxybutyrate concentrations were decreased which indicates the use of fatty acids towards acetyl-CoA synthesis. There was a decrease in glucose uptake and pyruvate concentrations. The absence of lactate indicates the use of pyruvate in gluconeogenesis. Therefore it is possible to conclude that RCB15 enhanced the alternative energetic pathways of cysticerci in vitro exposed to different concentration, with emphasis on the fatty acids catabolism.

Crystal structure of 3-(2-hy-droxy-eth-yl)-2-methyl-sulfanyl-6-nitro-3H-benzimidazol-1-ium chloride monohydrate.

In the cation of the title hydrated molecular salt, C10H12N3O3S+·Cl-·H2O, the benzimidazolium ring system is almost planar (r.m.s. deviation = 0.006 Å) and the nitro group is inclined at an angle of 4.86 (9)° to this plane. In the crystal, C-H⋯O hydrogen bonds form centrosymmetric R22(20) dimers and these are further aggregated through N-H⋯O and O-H⋯Cl hydrogen bonds involving the water mol-ecules and chloride anions. Aromatic π-π stacking inter-actions are also found between two parallel benzene rings or the benzene and imidazolium rings, with centroid-centroid distances of 3.5246 (9) and 3.7756 (9) Å, respectively. Analysis of the bond lengths and comparison with related compounds show that the nitro substituent is not involved in conjugation with the adjacent π-system and hence has no effect on the charge distribution of the heterocyclic ring.

Crystal structure of catena-poly[1,3-di-benzyl-benzimidazolium [[chlorido-mercurate(II)]-di-µ-chlorido]].

The asymmetric unit of the polymeric title compound, {(C21H19N2)[HgCl3]} n , comprises one-half of the cationic mol-ecule, the other half being generated by application of twofold rotation symmetry, one Hg and two Cl atoms. The Hg(II) atom, lying on a twofold rotation axis, exhibits a distorted triangular coordination environment and is surrounded by three Cl atoms with Hg-Cl distances in the range 2.359 (2)-2.4754 (13) Å. Two additional longer distances [Hg⋯Cl = 3.104 (14) Å] lead to the formation of polymeric [HgCl1/1Cl4/2](-) chains extending along [001]. The crystal packing can be described by cationic layers alternating parallel to (-110) with the anionic chains located between the layers. The packing is consolidated by π-π stacking inter-actions between the benzene rings of the central benzimidazole entities, with centroid-to-centroid distances of 3.643 (3) Å.

Benzimidazole derivative, BM601, a novel inhibitor of hepatitis B virus and HBsAg secretion.

Hepatitis B virus (HBV) belongs to the Hepadnaviridae family. HBsAg, greatly outnumbered mature virion, has been mysterious since the discovery of HBV. A novel benzimidazole derivative, BM601, is identified inhibiting the secretion of HBV virions and HBsAg, with 50% effective concentration of 0.6µM and 1.5µM, as well as 50% cytotoxicity concentration of 24.5µM. It has no effect on transcription, protein production, nucleocapsid formation or intracellular HBV DNA synthesis. Immunofluorescence analysis suggests that BM601 might inhibit virion and HBsAg secretion by interfering surface protein aggregation in trans Golgi apparatus. Furthermore, BM601 does not trigger cellular stress response or affect HBeAg or host protein secretion. We hypothesize that BM601 is a secretion inhibitor functioning at the level of virion and HBsAg secretion pathway.