Benzimidazoles Containing Piperazine Skeleton at C-2 Position as Promising Tubulin Modulators with Anthelmintic and Antineoplastic Activity.

Benzimidazole anthelmintic drugs hold promise for repurposing as cancer treatments due to their interference with tubulin polymerization and depolymerization, manifesting anticancer properties. We explored the potential of benzimidazole compounds with a piperazine fragment at C-2 as tubulin-targeting agents. In particular, we assessed their anthelmintic activity against isolated Trichinella spiralis muscle larvae and their effects on glioblastoma (U-87 MG) and breast cancer (MDA-MB-231) cell lines. Compound 7c demonstrated exceptional anthelmintic efficacy, achieving a 92.7% reduction in parasite activity at 100 µg/mL after 48 hours. In vitro cytotoxicity analysis of MDA-MB 231 and U87 MG cell lines showed that derivatives 7b, 7d, and 7c displayed lower IC50 values compared to albendazole (ABZ), the control. These piperazine benzimidazoles effectively reduced cell migration in both cell lines, with compound 7c exhibiting the most significant reduction, making it a promising candidate for further study. The binding mode of the most promising compound 7c, was determined using the induced fit docking-molecular dynamics (IFD-MD) approach. Regular docking and IFD were also employed for comparison. The IFD-MD analysis revealed that 7c binds to tubulin in a unique binding cavity near that of ABZ, but the benzimidazole ring was fitted much deeper into the binding pocket. Finally, the absolute free energy of perturbation technique was applied to evaluate the 7c binding affinity, further confirming the observed binding mode.

2-Alkyl-Substituted-4-Amino-Thieno[2,3-d]Pyrimidines: Anti-Proliferative Properties to In Vitro Breast Cancer Models.

Thienopyrimidines are structural analogs of quinazolines, and the creation of new 2-alkyl derivatives of ethyl 4-aminothienopyrimidine-6-carboxylates for the study of their anti-proliferative properties is of great pharmacological interest. Some 2-alkyl-4-amino-thieno[2,3-d]pyrimidines 2-5 were synthesized, and their cyto- and phototoxicity against BALB 3T3 cells were established by an in vitro 3T3 NRU test. The obtained results indicate that the tested compounds are not cytotoxic or phototoxic, and that they are appropriate to be studied for their anti-proliferative and anti-tumor properties. The anti-proliferative potential of the compounds was investigated on MCF-7 and MDA-MB-231 cancer cells, as well as a MCF-10A cell line (normal human mammary epithelial cells). The most toxic to MCF-7 was thienopyrimidine 3 with IC50 13.42 µg/mL (IC50 0.045 µM), followed by compound 4 (IC50 28.89 µg/mL or IC50 0.11 µM). The thienopyrimidine 4 revealed higher selectivity to MCF-7 and lower activity (IC50 367 µg/mL i.e., 1.4 µM) than compound 3 with MCF-10A cells. With respect to MDA-MB-231 cells, ester 2 manifested the highest effect with IC50 52.56 µg/mL (IC50 0.16 µM), and 2-ethyl derivative 4 revealed IC50 62.86 µg/mL (IC50 0.24 µM). It was estimated that the effect of the substances on the cell cycle progression was due to cell cycle arrest in the G2 stage for MDA-MB-231, while arrest in G1 was detected for the estrogen (ER)-positive MCF-7 cell line. The tested compound's effects on the change of the zeta potential in the tumorigenic cells utilized in this study were determined. The calculation which we performed of the physicochemical properties and pharmacokinetic parameters influencing the biological activity suggested high intestinal absorption, as well as drug-likeness.

Fused Triazinobenzimidazoles Bearing Heterocyclic Moiety: Synthesis, Structure Investigations, and In Silico and In Vitro Biological Activity.

[1,3,5]Triazino[1,2-a]benzimidazole-2-amines bearing heterocyclic moiety in 4-position were synthesized. The compounds were characterized by elemental analysis, IR, 1H-NMR, 13C-NMR, and HRMS spectroscopy. The molecular geometry and electron structure of these molecules were theoretically studied using density functional theory (DFT) methods. The molecular structure of the synthesized fused triazinobenzimidazole was confirmed to correspond to the 3,4-dihydrotriazinobenzimidazole structure through the analysis of spectroscopic NMR data and DFT calculations. The antinematodic activity was evaluated in vitro on isolated encapsulated muscle larvae (ML) of Trichinella spiralis. The results showed that the tested triazinobenzimidazoles exhibit significantly higher efficiency than the conventional drug used to treat trichinosis, albendazole, at a concentration of 50 µg/mL. The compound 3c substituted with a thiophen-2-yl moiety exhibited the highest anthelmintic activity, with a larvicidal effect of 58.41% at a concentration of 50 µg/mL after 24 h of incubation. Following closely behind, the pyrrole analog 3f demonstrated 49.90% effectiveness at the same concentration. The preliminary structure-anti-T. spiralis activity relationship (SAR) of the analogues in the series was discussed. The cytotoxicity of the benzimidazole derivatives against two normal fibroblast cells (3T3 and CCL-1) and two cancer human cell lines (MCF-7 breast cancer cells and chronic myeloid leukemia cells AR-230) was evaluated using the MTT-dye reduction assay. The screening results indicated that the compounds showed no cytotoxicity against the tested cell lines. An in silico study of the physicochemical and pharmacokinetic characteristics of the novel synthesized fused triazinobenzimidazoles showed that they were characterized by a significant degree of drug-likeness and optimal properties for anthelmintic agents.

Design, Cytotoxicity and Antiproliferative Activity of 4-Amino-5-methyl-thieno[2,3-d]pyrimidine-6-carboxylates against MFC-7 and MDA-MB-231 Breast Cancer Cell Lines.

Novel 4-amino-thieno[2,3-d]pyrimidine-6-carboxylates substituted at the second position were prepared by cyclocondensation of 2-amino-3-cyano-thiophene and aryl nitriles in an acidic medium. The design of the target compounds was based on structural optimization. The derivatives thus obtained were tested in vitro against human and mouse cell lines. The examination of the compound effects on BLAB 3T3 and MFC-10A cells showed that they are safe, making them suitable for subsequent experiments to establish their antitumor activity. The photoirritancy factor of the compounds was calculated. Using the MTT test, the antiproliferative activity to MCF-10A, MCF-7 and MDA-MB-231 cell lines was estimated. The best antiproliferative effect in respect to the MCF-7 cell line revealed compound 2 with IC50 4.3 ± 0.11 µg/mL (0.013 µM). The highest selective index with respect to MCF-7 cells was shown by compound 3 (SI = 19.3), and to MDA-MB-231 cells by compound 2 (SI = 3.7). Based on energy analysis, the most stable conformers were selected and optimized by means of density functional theory (DFT). Ligand efficiency, ligand lipophilicity efficiency and the physicochemical parameters of the target 4-amino-thienopyrimidines were determined. The data obtained indicated that the lead compound among the tested substances is compound 2.

New C2- and N3-Modified Thieno[2,3-d]Pyrimidine Conjugates with Cytotoxicity in the Nanomolar Range.

AIMS: The aim of the current study was to develop and explore a series of new cytotoxic agents based on the conjugation between the thieno[2,3-d]pyrimidine moiety and a second pharmacophore at the C2 or N3 position. BACKGROUND: As the thieno[2,3-d]pyrimidine core is a bioisostere of the 4-anilinoquinazoline, various new thienopyrimidine derivatives were synthesized by modifying the structure of the clinically used anticancer quinazoline EGFR inhibitors of the first generation - gefitinib, and second-generation - dacomitinib and canertinib. It was reported that some thieno[2,3-d]pyrimidine derivatives showed improved EGFR inhibitory activity. On the other hand, the benzimidazole heterocycle is present as a pharmacophore unit in the structure of many clinically used chemotherapeutic agents. Some 2-aminobenzimidazole derivatives, possessing anticancer activity, demonstrated EGFR inhibition and the benzimidazole derivative EGF816 is currently in the second phase of clinical trials. OBJECTIVE: The objectives of the study were the design of a novel series thieno[2,3-d]pyrimidines, synthesis of the compounds and investigation of their effects towards human cancer HT-29, MDA-MB-231, HeLa, HepG2 and to normal human Lep3 cell lines. (American Type Culture Collection, ATCC, Rockville, MD, USA). METHODS: The synthetic protocol implemented cyclocondensation of 2-amino-thiophenes and nitriles in an inert medium, aza- Michael addition to benzimidazole derivatives and nucleophylic substitution at the N3 place. MTS test was used in order to establish the cytotoxicity of the tested compounds. SAR analysis and in silico assessment of the inhibitory potential towards human oncogenic V599EB-Raf were performed using Molinspiration tool and Molecular Operating environment software. RESULTS: The MTS test data showed that almost all studied thieno[2,3-d]pyirimidines (9-13, 21-22 and 25) manifest high inhibitory effect on cell proliferation at nanomolar concentrations, whereas compounds 9 (IC50 = 130 nM) and 10 (IC50 = 261 nM) containing amino acid moiety, and 21 (IC50 = 108 nM) possessing two thienopyrimidine moieties attached to a 1,3-disubstituted benzimidazole linker, revealed many times lower toxicity against Lep3 cells compared to the cancer cells. Thienopyrimidines 11-13 possessed high selectivity against HeLa cells. Compound 13 showed high inhibitory activity against MDA-MB-231 and HepG2, with IC50 1.44 nM and 1.11 nM respectively. To outline the possible biological target of the studied coumpounds, their potential to interact with human oncogenic V599EB-Raf was explored by a docking study. As a result, it was suggested that the benzimidazolyl and glycyl fragments could enhance the binding ability of the new compounds by increasing the number of hydrogen bond acceptors and by stabilizing the inactive form of the enzyme. CONCLUSION: The thienopyrimidines tested in vitro for human cancer HT-29, MDA-MB-231, HeLa, HepG2 and normal human Lep3 cell lines demonstrated cytotoxicity in the nanomolar range. It was established that compounds 9, 10 and 21 showed many times lower toxicity against normal Lep3 cells that can provide a high selectivity towards all four cancer cell lines at small concentrations. Based on the analysis of the structure-activity relationship, the observed trends in the cytotoxicity could be related to the lipophilicity and the topological polar surface area of the tested compounds. The docking study on the potential of the new thieno[2,3-d]pyrimidine-4-ones to interact with mutant V599EB-Raf showed that the compounds might be able to stabilize the enzyme in its inactive form.

Modulation Effect on Tubulin Polymerization, Cytotoxicity and Antioxidant Activity of 1H-Benzimidazole-2-Yl Hydrazones.

1H-benzimidazol-2-yl hydrazones with varying hydroxy and methoxy phenyl moieties were designed. Their effect on tubulin polymerization was evaluated in vitro on porcine tubulin. The compounds elongated the nucleation phase and slowed down the tubulin polymerization comparably to nocodazole. The possible binding modes of the hydrazones with tubulin were explored by molecular docking at the colchicine binding site. The anticancer activity was evaluated against human malignant cell lines MCF-7 and AR-230, as well as against normal fibroblast cells 3T3 and CCL-1. The compounds demonstrated a marked antineoplastic activity in low micromolar concentrations in both screened in vitro tumor models. The most active were the trimethoxy substituted derivative 1i and the positional isomers 1j and 1k, containing hydroxy and methoxy substituents: they showed IC50 similar to the reference podophyllotoxin in both tumor cell lines, accompanied with high selectivity towards the malignantly transformed cells. The compounds exerted moderate to high ability to scavenge peroxyl radicals and certain derivatives-1l containing metha-hydroxy and para-methoxy group, and 1b-e with di/trihydroxy phenyl moiety, revealed HORAC values high or comparable to those of well-known phenolic antioxidants. Thus the 1H-benisimidazol-2-yl hydrazones with hydroxy/methoxy phenyl fragments were recognized as new agents exhibiting promising combined antioxidant and antineoplastic action.

1H-benzimidazole-2-yl hydrazones as tubulin-targeting agents: Synthesis, structural characterization, anthelmintic activity and antiproliferative activity against MCF-7 breast carcinoma cells and molecular docking studies.

In the present study, fifteen benzimidazolyl-2-hydrazones 7a-7o of fluoro-, hydroxy- and methoxy-substituted benzaldehydes and 1,3-benzodioxole-5-carbaldehyde were synthesized and their structure was identified by IR, NMR, and elemental analysis. The compounds 7j 2-(3-hydroxybenzylidene)-1-(5(6)-methyl-1H-benzimidazol-2-yl)hydrazone and 7i 2-(3-hydroxybenzylidene)-1-(1H-benzimidazol-2-yl)hydrazone have exerted the strongest anthelmintic activity (100% after 24 h incubation period at 37 °C) against isolated muscle larvae of Trichinella spiralis in an in vitro experiment. The in vitro cytotoxicity assay towards MCF-7 breast cancer cells and mouse embryo fibroblasts 3T3 showed that the studied benzimidazolyl-2-hydrazones exhibit low to moderate cytotoxic effects. The ability of the studied benzimidazolyl-2-hydrazones to modulate microtubule polymerization was confirmed and suggested that their anthelmintic action is mediated through inhibition of the tubulin polymerization likewise the other known benzimidazole anthelmitics. It was also shown that the four most promising benzimidazolyl-2-hydrazones do not affect significantly the AChE activity even at high tested concentration, thus indicating that they do not have the potential for neurotoxic effects. The binding mode of compounds 7j and 7n in the colchicine-binding site of tubulin were clarified by molecular docking simulations. Taken together, these results demonstrate that for the synthesized benzimidazole derivatives the anthelmintic activity against T. spiralis and the inhibition of tubulin polymerization are closely related.

Thieno[2,3-d]pyrimidin-4(3H)-one Derivatives of Benzimidazole as Potential Anti- Breast Cancer (MDA-MB-231, MCF-7) Agents.

AIMS: The purpose of this study was the synthesis of some new thienopyrimidine derivatives of 1,3-disubstituted benzimidazoles and the evaluation of their cytotoxicity against MDA-MB-231, MCF-7, and 3T3 cells lines. BACKGROUND: An overexpression or mutational activation of TK receptors EGFR and HER2/neu is characteristic of tumors. It has been found that some thieno[2,3-d]pyrimidines exhibited better inhibitory activity against Epidermal Growth Factor Receptor (EGFR/ErbB-2) tyrosine kinase in comparison to aminoquinazolines. Breast cancer activity towards MDA-MB-231 and MCF-7 cell lines by inhibiting EGFR was revealed by a novel 2-arylbenzimidazole. This motivated the synthesis of new thienopyrimidines possessing benzimidazole fragments in order to evaluate their cytotoxicity to the above-mentioned cell lines. OBJECTIVE: The objectives of the study were to design and synthesize a novel series of thieno[2,3-d]pyrimidines bearing biologically active moieties, such as 1,3-disubstituted-benzimidazole heterocycle, structurally similar to diaryl ureas in order to evaluate their cytotoxicity against MDA-MB-231, and MCF-7 breast cancer cell lines. METHODS: N,N-disubstituted benzimidazole-2-one carbonitriles were synthesized by Aza-Michael addition and used as precursors to generate some of the new thieno[2,3-d]pyrimidines in acidic medium The interaction of chloroethyl-2-thienopyrimidines, 2-amino-benzimidazole and benzimidazol-2-one nitriles under solid-liquid transfer catalysis conditions led to new thienopyrimidines. MTT assay for cell survival was performed in order to evaluate the cytotoxicity of the tested compounds. A fluorescence study was conducted to elucidate some aspects of the mechanism of action. RESULTS: The effects of nine synthesized compounds were investigated towards MDA-MB-231, MCF-7 and 3T3 cell lines. Thieno[2,3-d]pyirimidine-4-one 16 (IC50 - 0.058µM) and 21 (IC50 - 0.029µM) possess high cytotoxicity against MDA-MB-231 cells after 24h. The most cytotoxic compounds against breast cancer MCF-7 cells was compound 21 (IC50 - 0.074µM), revealing lower cytotoxicity against mouse fibroblast 3T3 cells with IC50 - 0.20µM. SAR analysis was performed. Fluorescence study of the treatment of MDA-MB cells with compound 21 was carried out in order to clarify some aspects of the mechanism of action. CONCLUSION: The relationship between cytotoxicity of compounds 14 and 20 against MCF-7 and 3T3 cells can suggest a similar mechanism of action. The antitumor potential of the tested compounds proves the necessity for further investigation to estimate the exact inhibition pathway in the cellular processes. The fluorescence study of the treatment of MDA-MB cells with compound 21 showed a rapid process of apoptosis.

Benzimidazole-based dual dipeptidyl peptidase-4 and xanthine oxidase inhibitors.

Multiple-targeting compounds might reduce complex polypharmacy of multifactorial diseases, such as diabetes, and contribute to the greater therapeutic success. Targeting reactive oxygen species-producing enzymes, as xanthine oxidase (XO), might suppress progression of diabetes-associated vascular complications. In this study a small series of benzimidazole derivatives (1-9) was evaluated for inhibitory activity against dipeptidyl peptidase-4 (DPP-4) and XO. One 1,3-disubstituted-benzimidazole-2-imine (5) and 1,3-thiazolo[3,2-a]benzimidazolone derivative (8) were shown as effective dual DPP-4 and XO inhibitors, with IC50 values lower than 200 µM, and predicted binding modes with both target enzymes. Both selected dual inhibitors (compounds 5 and 8) did not show cytotoxicity to a greater extent on Caco-2 cells even at concentration of 250 µM. These structures represent new non-purine scaffolds bearing two therapeutic functionalities, being DPP-4 and XO inhibitors, more favorable in comparison to DPP-4 inhibitors with DPP-4 as a single target due to pleiotropic effects of XO inhibition.

Benzo[4,5]thieno[2,3-d]pyrimidine phthalimide derivative, one of the rare noncompetitive inhibitors of dipeptidyl peptidase-4.

A small library of benzo[4,5]thieno[2,3-d]pyrimidine phthalimide and amine derivatives was evaluated for inhibitory activity against dipeptidyl peptidase-4 (DPP-4). The phthalimide derivatives exhibited better activity than the amine precursors, with 2-(2-(3-chlorobenzyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)isoindoline-1,3-dione (compound 14) as the most effective inhibitor (IC50 = 34.17 ± 5.11 µM). The five most potent selected inhibitors did not show cytotoxicity to a greater extent on Caco-2 cells, even at a concentration of 250 µM. Compound 14 is considered as a novel representative of the rare noncompetitive DPP-4 inhibitors. Molecular docking and dynamics simulation indicated the importance of the Tyr547, Lys554, and Trp629 residues of DPP-4 in the formation of the enzyme-inhibitor complex. These observations could be potentially utilized for the rational design and optimization of novel (structurally similar, with phthalimide moiety, or different) noncompetitive DPP-4 inhibitors, which are anyway rare, but favorable in terms of the saturation of substrate competition.

Synthesis and DNase I inhibitory properties of some 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidines.

A series of six novel and six known thieno[2,3-d]pyrimidin-4-amines 2-13 were synthesized, and further were used as a starting material for preparation of a small series of eight novel thieno[2,3-d]pyrimidin-4-phthalimides 14-21. Eight compounds, five amine and three phthalimide derivatives, inhibited bovine pancreatic DNase I with an IC50 below 200 µM, being more effective than referent inhibitor crystal violet. Phthalimide derivatives 16, 18 and 19 exhibited higher DNase I inhibitory activity compared to their amine precursors 7, 10 and 11. Compound 19, as the most potent (IC50 = 106 ±â€¯16 µM), offers a good starting point for a design of new DNase I inhibitors. The Pharma RQSAR model showed a significant enhancement of thieno[2,3-d]pyrimidines activity using aryl substituents at R1 position. The E-State RQSAR model clarified the most important structural fragments relevant for DNase I inhibition. Molecular docking and Site Finder module defined the thieno[2,3-d]pyrimidines interactions with the most important catalytic residues of DNase I, including Glu 39, His 134, Asp 168 and His 252. We also found that steric effects and increase of molecular volume play a vital role in DNase I inhibition.

Benzimidazoles as novel deoxyribonuclease I inhibitors.

Inhibitory potential of 19 benzimidazoles against bovine pancreatic deoxyribonuclease I (DNase I) was investigated in vitro. Three compounds inhibited DNase I with IC50 below 100 µM and proved to be more potent DNase I inhibitors than crystal violet (IC50 = 351.82 ± 29.41 µM), used as a positive control. Compound 9 showed the most potent DNase I inhibition with an IC 50 value of 79.46 ± 11.75 µM. To further explore the relationship between inhibitory activity and 2D pharmacophore features, Pharma/E-State R-group quantitative structure-activity relationship (RQSAR) models were generated and validated using Schrödinger Suite. RQSAR models showed a significant enhancement of benzimidazoles activity using hydrogen-bond acceptor substituents at the R2, R3, and R4 positions, or aryl substituents at the R4 position. The Site Finder module and molecular docking defined the benzimidazoles interactions with the most important catalytic residues of DNase I, including H-acceptor interaction with residue His 134 and His 252 and/or H-donor interaction with residue Glu 39. We also found a positive correlation between IC50 inhibition values and relative binding free energies of the most active benzimidazoles. In addition, a molecular dynamics simulation was performed for DNase I-compound 9 docking complex in Desmond. Trajectory analysis showed that docking complex and intermolecular interactions were stable throughout the entire production part of simulations. Furthermore, the results of protein structure alignment module suggested the potential translational impact of benzimidazoles against human DNase I. Due to the significant involvement of DNase I in the pathophysiology of many disease conditions, benzimidazoles as DNase I inhibitors could have potential therapeutic applications.

Synthesis, fluorescence-sensing and molecular logic of two water-soluble 1,8-naphthalimides.

Two novel highly water-soluble fluorescence sensing 1,8-naphthalimides are synthesized and investigated. The novel compounds are designed on the "fluorophore-receptor1-spacer-receptor2" model as a molecular fluorescence probe for determination of cations and anions in 100% aqueous media. The novel probes comprising N-imide and N-phenylpiperazine or morpholine substituents are capable to operate simultaneously via ICT and PET signaling mechanism as a function of pH and to recognize selectively Cu2+ and Hg2+ over the other representative metal ions. Due to the remarkable fluorescence changes in the presence of protons, hydroxyl anions, Hg2+ and Cu2+, INH and doubly disabled INH logic gates are executed and the systems are able to act as a single output combinatorial logic circuit with four chemical inputs.

Synthesis, anticancer activity and photostability of novel 3-ethyl-2-mercapto-thieno[2,3-d]pyrimidin-4(3H)-ones.

Some derivatives of 3-ethyl-2-mercapto-thieno[2,3-d]pyrimidin-4(3H)-ones were synthesized using ethyl 2-aminothiophene-3-carboxylates as precursors in order to estimate their cytotoxicity, respectively proliferative activity. Thienopyrimidinones containing thiosemicarbazide as well as 1,3,4-thiadiazole moieties were evaluated for their cytotoxical effect on four cancer cell lines: HT-29, breast cancer cells MDA-MB-231, HeLa, HepG2 as well as human diploid cell line Lep-3. Compounds 5b, 6a and 6b revealed cytotoxicity to the four studied cancer cell lines. The highst cytotoxicity against MDA-MB-31 exhibited the thiosemicarbazide 5b with IC50 2.31.10(-4) µM, but most active towards HT-29 cell lines was thienopyrimidine 6c with IC50 0.001 µM. Compound 6a showed the highest inhibitory activity with IC50 - 0.99 µM to human liver carcinoma HepG2 cells and low cytotoxicity towards Lep3 (IC50 = 191 µM). The thienopyrimidine derivative linked to thiadiazole 6b was toxic to the four studied cancer cell lines, especially to HeLa (IC50-0.83 µM), and besides that the compound demonstrated toxicity to Lep 3 cells at very high concentration 89 × 10(3) µM. The solid-state photostability of the derivatives 5a-c and 6a-c was tested by irradiation with UV light. All of the studied compounds show solid-state photostability in 240 min of irradiation. Using MOE software molecular docking of the three ligands 5b, 6b and 7 was accomplished into an internal pocket formed by the activation segment and the P-loop of (V599E)B-Raf. It was established that the binding of the ligands to (V599E)B-Raf promotes an inactive conformation of the enzyme.

Synthesis, electronic properties, antioxidant and antibacterial activity of some new benzimidazoles.

Two groups of benzimidazole derivatives were synthesized using as precursors 5(6)-substituted 2-mercapto-benzimidazol-thiols and their antioxidant activity was investigated using TBA-MDA test. In the group of 1,3-disubstituted-benzimidazol-2-imines the highest lipid peroxidation inhibition effect 74.04% (IC50=141.89 µg/mL) revealed ethyl [3-(2-ethoxy-2-oxoethyl)-2-imino-5-benzoyl-2,3-dihydro-1H-benzimdazol-1-yl]acetate 12 while in the group of 2-substituted-1,3-thiazolo[3,2-a]benzimidazolones the highest inhibition effect showed 2-(4-fluorobenzylidene)-7-(phenylcarbonyl)[1,3]thiazolo[3,2-a]benzimidazol-3(2H)-one 17 90.76% (IC50=53.70 µg/mL). In order to estimate the capability of the studied benzimidazoles to act as radical scavengers the structure of the most active derivative within the both subseries was optimized at B3LYP/6-311++G(∗∗) level and the respective bond dissociation enthalpies were calculated. The appropriate models for the HAT and SET-mechanism of the antioxidant activity were proposed. The antibacterial activity of the compounds was evaluated against two Gram-positive bacteria (Bacillus subtilis ATCC 6633 and Staphylococcus aureus ATCC 6538) and three Gram-negative bacteria (Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027 and Salmonella abony NCTC 6017). 1,3-Diphenylpropyl-5-methyl-1,3-dihydro-2H-benzimidazol-2-imine 14 exhibited significant activity against B. subtilis, S. aureus, S. abony and E. coli (with MIC values of 0.125, 0.016, 0.50 and 0.50mg/mL, respectively). The group of thiazolobenzimidazolones did not reveal antibacterial activity against the tested strains.

Synthesis and antiproliferative activity of some new thieno[2,3-d]pyrimidin-4(3H)-ones containing 1,2,4-triazole and 1,3,4-thiadiazole moiety.

Some new thieno[2,3-d]pyrimidin-4(3H)-ones containing 1,2,4-triazole and 1,3,4-thiadiazole moiety were synthesized using thieno[2,3-d]pyrimidin-3(4H)-yl)acetohydrazides as precursors in order to determine their cytotoxicity. Compounds 5, 7-8 and 10-18 were evaluated for their cytotoxical effect on four cancer cell lines: human colorectal cancer cell line HT-29, breast cancer cells MDA-MB-231, cervical cancer cells HeLa, human liver carcinoma HepG2 and human normal diploid cell line Lep3. Exclusively high cytotoxic activity of compounds 8, 16 and 17 against MDA-MB-231 cells was ascertained and the calculated IC50 values were 3.91·10(-2), 1.2·10(-3) and 3.74·10(-2) µM respectively. Thienopyrimidinones 10, 15 and 17 exhibited high cytotoxicity against HT-29 cell and the IC50 values were in the range 1.56·10(-3) µM- 0.13 µM. To HeLa cell lines cytotoxicity demonstrated compounds 8, 10, 11, 13 and 15-18 but the substance 13 was the most toxic with IC50 - 9.5·10(-4) µM. Distinctly high antiproliferative activity of derivatives 10, 14-15 and 17-18 was estimated against Hep G2, compound 15 showed IC50 - 0.21 µM. Proliferative effects to Lep 3 demonstrated compounds 5, 7-8, 11-14, 16, 18 whose EC50 values were from 0.12 to 2.21 µM. The biological data highlighted that the nature and the position of the substituents influence both the cytotoxicity to the cancer cells and the proliferation properties to Lep3 of the tested compounds.

Design, synthesis and antiproliferative properties of some new 5-substituted-2-iminobenzimidazole derivatives.

Some new 1,3,5-substituted-2,3-dihydro-2-imino-benzimidazoles were synthesized under solid-liquid phase transfer catalysis conditions using 5-substituted-2-aminobenzimidazoles as precursors in order to assess their cytotoxicity respectively proliferative activity. The structures of the compounds were confirmed by IR, (1)H NMR, (13)C NMR and elemental analysis. Compounds 9-10, 12 and 16-17 were evaluated for their cytotoxical effect on four cancer cell lines: HT-29, breast cancer cells MDA-MB-231, HeLa, HepG2 and as well as human diploid cell line Lep-3. Significant cytotoxicity of hydrazone 16 against MDA-MB-231 was established by biologically study, the IC50 was 6.2 nM while the EC50 value to Lep 3 is 0.21 nM. Relative high antiproliferative effects of the acetate 12 and compound 16 against HT-29 were ascertained and the calculated IC50 values were IC50 - 0.85 nM and IC50 - 2.83 nM respectively. Cytotoxic activity against HeLa and HepG2 cells was demonstrated by hydrazone 17, IC50 was 7.2 nM and 117 nM respectively. All tested compounds revealed proliferative activities to human diploid cell line Lep-3. The EC50 values were in the range from 0.05 to 16.91 nM. The obtained results prove the selective cytotoxicity of the tested compounds and are promising for further evaluation of the investigated compounds in vivo experiments using experimentally induced tumors in laboratory animals.

Synthesis, characterization and cytotoxicity of some novel 1,3-disubstituted-2,3-dihydro-2-iminobenzimidazoles.

Some new 1,3-disubstituted-2,3-dihydro-2-iminobenzimidazoles were synthesized using 1-(un)substituted-2-aminobenzimidazoles as precursors in order to determine their cytotoxicity. The structures of the compounds were confirmed by IR, (1)H NMR, (13)C NMR and elemental analysis. Compounds 4, 7-11 and 13-14 were evaluated for their cytotoxical effect on two cancer cell lines: human colorectal cancer cell line HT-29, breast cancer cells MDA-MB-231 and as well as normal spleen cells. The distinctly marked antiproliferative activity of 1,3-bis(3-phenylpropyl-1)-1,3-dihydro-2H-benzimidazol-2-imine hydro bromide 7, N-(aminopropyl)-2-(3-{2-[(aminopropyl)-amino]-2-oxoethyl}-2-imino-2,3-dihydro-1H-benzimidazol-1-yl)acetamide 9 and 1,3-bis[2-(4-methylpiperazin-1-yl)-2-oxoethyl]-2,3-dihydro-2H-benzimidazol-2-imine 11 against human colorectal cancer cell line HT-29 was ascertained and the calculated IC(50) were 9.26, 0.56 and 0.013 nM respectively. Compounds 4, 9, 10 and 13 exhibited relative high cytotoxic activity against MDA-MB-231 cells. The calculated IC(50) values were in the range 0.123-1.65 nM. All tested compounds excluding compound 1,3-bis[2-(4-methylpiperazin-1-yl)-2-oxoethyl]-2,3-dihydro-2H-benzimidazol-2-imine (11) revealed proliferative activities to normal spleen cells. The computed EC(50) values varied from 0.05 to 16.91 nM.

Synthesis, antitrichinnellosis and antiprotozoal activity of some novel thieno[2,3-d]pyrimidin-4(3H)-ones containing benzimidazole ring.

Some novel thieno[2,3-d]pyrimidin-4(3H)-ones containing benzimidazol-2-yl-thioethyl- and benzimidazol-2-yl-methanethioethyl moiety in second position of the pyrimidine ring were synthesized in order to determine their antitrichinellosis and antiprotozoal effects. The structures of the compounds were confirmed by IR, (1)H NMR and elemental analysis. The antiparasitic screening showed that the benzimidazole derivatives of thieno[2,3-d]pyrimidin-4(3H)-ones exhibited higher activity against Trichinella spiralis in vitro in comparison albendazole. The most active compound, 2-[2-(5-nitro-1H-benzimidazol-1-yl)ethyl]-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one 22 revealed 95% activity at a dosage of 5 mg/kg mw after 24 h, while compounds 8 and 10 applied at the same dose showed efficacy of 90% after 48 h. The compound 2-{2-[(5(6)-nitro-1H-benzimidazol-2-yl)thio]ethyl}-5,6,7,8-tetrahydro[1]-benzothieno[2,3-d]pyrimidin-4(3H)-one 11 exhibited 90% efficacy after 24 h. The pharmaco-therapeutic study in vivo on invaded with Lamblia muris white mice showed 100% effectiveness of the compounds 8, 10, 11, 13-15 and 22, 23 after five-days-treatment course.

Synthesis, cytotoxicity and effects of some 1,2,4-triazole and 1,3,4-thiadiazole derivatives on immunocompetent cells.

Novel derivatives of 4,5-substituted-1,2,4-triazole-thiones and 2,5-substituted-1,3,4-thiadiazoles were synthesized and evaluated for their cytotoxicity. The biological study indicated that compounds 4-ethyl-5-(4,5,6,7-tetrahydro-1-benzothien-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 13, N-ethyl-5-(4,5,6,7-tetrahydro-1-benzothien-2-yl)-1,3,4-thiadiazol-2-amine 16, 4-amino-5-(4,5,6,7-tetrahydro-1-benzothien-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 20 and 4-amino-5-(5-phenylthien-2-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 21 possessed high cytotoxicity in vitro against thymocytes. The corresponding IC(50) values were 0.46 microM, 5.2 x 10(-6)microM, 0.012 microM and 1.0 x 10(-6)microM. Most toxic against lymphocytes was compound 21, IC(50) - 0.012 microM. The tested compounds showed a general stimulation effect on B-cells' response.