Recent Advances in the Application of Nitro(het)aromatic Compounds for Treating and/or Fluorescent Imaging of Tumor Hypoxia.

This review delves into recent advancements in the field of nitro(het)aromatic bioreductive agents tailored for hypoxic environments. These compounds are designed to exploit the low-oxygen conditions typically found in solid tumors, making them promising candidates for targeted cancer therapies. Initially, this review focused on their role as gene-directed enzyme prodrugs, which are inert until activated by specific enzymes within tumor cells. Upon activation, these prodrugs undergo chemical transformations that convert them into potent cytotoxic agents, selectively targeting cancerous tissue while sparing healthy cells. Additionally, this review discusses recent developments in prodrug conjugates containing nitro(het)aromatic moieties, designed to activate under low-oxygen conditions within tumors. This approach enhances their efficacy and specificity in cancer treatment. Furthermore, this review covers innovative research on using nitro(het)aromatic compounds as fluorescent probes for imaging hypoxic tumors. These probes enable non-invasive visualization of low-oxygen regions within tumors, providing valuable insights for the diagnosis, treatment planning, and monitoring of therapeutic responses. We hope this review will inspire researchers to design and synthesize improved compounds for selective cancer treatment and early diagnostics.

Bis(benzimidazol-2-yl)amine-Based DPP-4 Inhibitors Potentially Suitable for Combating Diabetes and Associated Nervous System Alterations.

Bis(benzimidazol-2-yl)amine scaffold is not present in dipeptidyl peptidase-4 (DPP-4) inhibitors published so far. Herein, the inhibitory potential of bis(benzimidazol-2-yl)amine derivatives against DPP-4 was evaluated. In non-competitive inhibition mode, three representatives 5, 6, and 7 inhibited DPP-4 in vitro with IC50 values below 50 µM. The assessed binding pocket of DPP-4 for these benzimidazoles includes the S2 extensive subsite's residues Phe357 and Arg358. None of the lead compounds showed cytotoxicity to human neuroblastoma SH-SY5Y cells at concentrations lower than 10 µM. None showed significant binding affinity at dopamine D2, D3, and histamine H1, H3 receptors, at concentrations lower than 10 µM, leading to preferable outcomes due to mutually opposite effects of these neurotransmitters on each other. The potential beneficial effects on dopamine synthesis and the survival of dopaminergic neurons could be mediated by DPP-4 inhibition. These effective noncompetitive DPP-4 inhibitors, with inhibitory potential better than reference diprotin A (relative inhibitory potency compared to diprotin A is 3.39 and 1.54 for compounds 7 and 5, respectively), with the absence of cytotoxicity to SH-SY5Y cells, are valuable candidates for further evaluation for the treatment of diabetes and associated disruption of neuronal homeostasis.

Antiproliferative and Pro-Apoptotic Activity and Tubulin Dynamics Modulation of 1H-Benzimidazol-2-yl Hydrazones in Human Breast Cancer Cell Line MDA-MB-231.

(1) Background: The aim of the work is the evaluation of in vitro antiproliferative and pro-apoptotic activity of four benzimidazole derivatives containing colchicine-like and catechol-like moieties with methyl group substitution in the benzimidazole ring against highly invasive breast cancer cell line MDA-MB-231 and their related impairment of tubulin dynamics. (2) Methods: The antiproliferative activity was assessed with the MTT assay. Alterations in tubulin polymerization were evaluated with an in vitro tubulin polymerization assay and a docking analysis. (3) Results: All derivatives showed time-dependent cytotoxicity with IC50 varying from 40 to 60 µM after 48 h and between 13 and 20 µM after 72 h. Immunofluorescent and DAPI staining revealed the pro-apoptotic potential of benzimidazole derivatives and their effect on tubulin dynamics in living cells. Compound 5d prevented tubulin aggregation and blocked mitosis, highlighting the importance of the methyl group and the colchicine-like fragment. (4) Conclusions: The benzimidazole derivatives demonstrated moderate cytotoxicity towards MDA-MB-231 by retarding the initial phase of tubulin polymerization. The derivative 5d containing a colchicine-like moiety and methyl group substitution in the benzimidazole ring showed potential as an antiproliferative agent and microtubule destabilizer by facilitating faster microtubule aggregation and disrupting cellular and nuclear integrity.

Self-Assembled Molecular Complexes of 1,10-Phenanthroline and 2-Aminobenzimidazoles: Synthesis, Structure Investigations, and Cytotoxic Properties.

Three new molecular complexes (phen)3(2-amino-Bz)2(H+)(BF4-)·3H2O 5, (phen)3(2-amino-5(6)-methyl-Bz)2(H+)(BF4-)·H2O 6, and (phen)(1-methyl-2-amino-Bz)(H+)(BF4-) 7, were prepared by self-assembly of 1,10-phenanthroline (phen) and various substituted 2-aminobenzimidazoles. Confirmation of their structures was established through spectroscopic methods and elemental analysis. The X-ray diffraction analysis revealed that the crystal structure of 7 is stabilized by the formation of hydrogen bonds and short contacts. In addition, the molecular geometry and electron structure of molecules 5 and 6 were theoretically evaluated using density functional theory (DFT) methods. According to the DFT B3LYP/6-311+G* calculations, the protonated benzimidazole (Bz) units act as NH hydrogen bond donors, binding two phenanthrolines and a BF4- ion. Non-protonated Bz unit form hydrogen bonds with the N-atoms of a third molecule phen. The molecular assembly is held together by π-π stacking between benzimidazole and phenanthroline rings, allowing for N-atoms to associate with water molecules. The complexes were tested in vitro for their tumor cell growth inhibitory effects on prostate (PC3), breast (MDA-MB-231 and MCF-7), and cervical (HeLa) cancer cell lines using MTT-dye reduction assay. The in vitro cytotoxicity analysis and spectrophotometric investigation in the presence of ct-DNA, showed that self-assembled molecules 5-7 are promising DNA-binding anticancer agents warranting further in-depth exploration.

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.

Radical Scavenging Mechanisms of 1-Arylhydrazone Benzimidazole Hybrids with Neuroprotective Activity.

Benzimidazole-arylhydrazone hybrids showed promising potential as multifunctional drugs for the treatment of neurodegenerative disorders. The neuroprotection studies conducted using an in vitro model of H2O2-induced oxidative stress on the SH-SY5Y cell line revealed a remarkable activity of the compound possessing a vanilloid structural fragment. The cell viability was preserved up to 84% and this effect was significantly higher than the one exerted by the reference compounds melatonin and rasagiline. Another compound with a catecholic moiety demonstrated the second-best neuroprotective activity. Computational studies were further conducted to characterize in depth the antioxidant properties of both compounds. The possible radical scavenging mechanisms were estimated as well as the most reactive sites through which the compounds may deactivate a variety of free radicals. Both of the compounds are able to deactivate not only the highly reactive hydroxyl radicals but also alkoxyl and hydroperoxyl radicals, following hydrogen atom transfer or radical adduct formation mechanism. In nonpolar medium, 3e is predicted to react slightly faster than 3a with alkoxyl radicals and around two orders of magnitude faster than 3a with hydroperoxyl radicals. The most reactive sites for formal hydrogen atom transfer in 3a are the meta-hydroxy group in the phenyl ring in water and the amide N-H group in benzene; in 3e, the amide N-H group is more reactive in both solvents. The radical adduct formation can occur at several positions in 3a and 3e, the most active being C4, C6, and C14. The stability of the formed radicals was estimated by NBO calculations. The NBO calculations indicated that the spin density in the radicals formed by the abstraction of a hydrogen atom from the amide groups of both compounds is delocalized over the phenyl ring and the hydrazone chain. The obtained theoretical data for the better radical scavenging ability of the vanilloid hybrid corroborate its experimentally established better neuroprotective activity.

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.

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.

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.

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.

Evaluation of the combined activity of benzimidazole arylhydrazones as new anti-Parkinsonian agents: monoamine oxidase-B inhibition, neuroprotection and oxidative stress modulation.

Neuroprotective drugs and selective monoamine oxidase inhibitors can slow down the progression and improve symptoms of Parkinson's disease (PD). Since there is an implication of oxidative stress in the pathophysiological mechanisms of the disease, the compounds possessing an ability to reduce the oxidative stress are prime candidates for neuroprotection. Thereby our current study is focused on the development of new multi-target PD drugs capable of inhibiting the activity of monoamine oxidase-B while exerting neuroprotective and antioxidant properties. A small series of benzimidazole derivatives containing hydroxy and methoxy arylhydrazone fragments has been synthesized and the neurotoxicity of the compounds has been evaluated in vitro on neuroblastoma SH-SY5Y cells and on isolated rat brain synaptosomes by measuring the cell viability and the levels of reduced glutathione and a good safety profile has been shown. The 2-hydroxy-4-methoxy substituted arylhydrazone 7 was the least toxic on neuronal SH-SY5Y cells and showed the lowest neurotoxicity in rat brain synaptosomes. The neuroprotective properties of the test compounds were further assessed using two models: H2O2-induced oxidative stress on SH-SY5Y cells and 6-hydroxydopamine-induced neurotoxicity in rat brain synaptosomes. Compound 7 showed more pronounced neuroprotective activity on SH-SY5Y cells, compared to the referent melatonin and rasagiline. It also preserved the synaptosomal viability and the reduced glutathione levels; the effects were stronger than those of rasagiline and comparable to melatonin. All the tested compounds were capable to inhibit human monoamine oxidase-B enzyme to a significant extent, however, compound 7 exerted the most prominent inhibitory activity, similar to selegiline and rasagiline. The carried out molecular docking studies revealed that the activity is related to the appropriate molecular structure enabling the ligand to enter deeper in the narrow and highly lipophylic active site pocket of the human monoamine oxidase-B and has a favoring interaction with the key amino acid residues Tyr326 and Cys172. Since much scientific evidence points out the implication of iron dyshomeostasis in PD, the compounds were tested to reduce the ferrous iron induced oxidative molecular damage on biologically important molecules in an in vitro lecithin containing model system. All the investigated compounds denoted protection effect, stronger than the one of the referent melatonin. In order to support the assignments of the significant neuroprotective and antioxidant pharmacological activities, the radical-scavenging mechanisms of the most promising compound 7 were evaluated using DFT methods. It was found that the most probable free radicals scavenging mechanism in nonpolar phase is the hydrogen atom transfer from the amide group of compound 7, while in polar medium the process is expected to occur by a proton transfer. The current study outlines a perspective leading structure, bearing the potential for a new anti-PD drug. All performed procedures were approved by the Institutional Animal Care Committee of the Medical University of Sofia (Bulgarian Agency for Food Safety with Permission № 190, approved on February 6, 2020).

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.

New benzimidazole-aldehyde hybrids as neuroprotectors with hypochlorite and superoxide radical-scavenging activity.

BACKGROUND: Many neurodegenerative disorders include oxidative stress-mediated pathology. Melatonin and its metabolites act as endogenous reactive oxygen species (ROS) scavengers and antioxidants. N,N'-Disubstituted benzimidazole-2-thiones with extended side chains could exert antioxidant and neuroprotective properties due to structural similarities to melatonin. METHODS: The toxicological potential of the compounds was evaluated by monitoring the synaptosomal viability and the levels of reduced glutathione (GSH) in isolated rat brain synaptosomes. The neuroprotective effects were assessed in vitro in a model of 6-hydroxydopamine (6-OHDA)-induced neurotoxicity. The capability to decrease superoxide anion radical and hypochlorite was evaluated by luminol-dependent chemiluminescent assays. RESULTS: Compounds 5-7 containing residues of veratraldehyde, vanillin, and syringaldehyde at concentration 250 µM, preserved at the highest degree the synaptosomal viability and GSH levels. Further screening of compounds 5-7 at lower concentrations of 100 µM, 10 µM, and 1 µM, respectively, demonstrated that 6 and 7 do not show any toxicity within this concentration range. In the model of 6-OHDA-induced oxidative stress, 6 revealed concentration-dependent, neuroprotective, and antioxidant activities similar to melatonin. All the three compounds demonstrated ability to decrease the chemiluminescent scavenging index (CL-SI) in the hypochlorite containing system. In the superoxide system, the hydrazones exhibited different effects on the signal. CONCLUSIONS: Our studies suggest that the benzimidazole-aldehyde hybrids act as direct ROS scavengers and membranes' stabilizers against free radicals. Thus, they play a role in the antioxidative defense system and have a promising potential as therapeutic neuroprotective agents for the treatment of neurodegenerative disorders.

Folate-conjugated Helix lucorum hemocyanin - preparation, stability, and cytotoxicity.

This is the first report on the modification of a hemocyanin from Helix lucorum (HlH), a large molluscan respiratory protein, with folic acid (FA). In a two-step synthetic reaction, we prepared samples of HlH conjugated with 20 and 50 FA residues denoted as FA-HlH-1 and FA-HlH-2, respectively. Comparison of the attenuated total reflectance-Fourier transform infrared spectra in the amide I band region showed a structural rearrangement in the HlH that is due to FA conjugation. The changes in the secondary structure were more noticeable for FA-HlH-2. The thermal stability of HlH was not significantly affected by the FA modification, which is consistent with the observed structural similarities with the native protein. Preliminary cytotoxicity assays showed that FA-HlH-1 and FA-HlH-2 stimulate fibroblast proliferation when applied in concentrations of 50 and 100 µg/well. A negligible reduction of fibroblast growth was observed only for FA-HlH-1 and FA-HlH-2, exposed to 200 µg/well for 48 h. We found that FA-HlH-2 exhibits a low to moderate cytotoxic effect on two breast cancer cell lines, which express folate receptors, a hormone-dependent (MCF-7) and a hormone-independent (MDA-MB-231). FA-HlH-2 protects nontransformed cells and affects only neoplastic cells, which could be an advantage, and the protein could have potential in combination with other chemotherapeutics.

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.

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.

Biophysical Properties and Cytotoxicity of Feruloylated Helix Lucorum Hemocyanin.

For the first time Helix lucorum hemocyanin (HlH) has been feruloylated. Two HlH conjugates with 40- and 120- ferulic acid residues were prepared, denoted as FA-HlH-1 and FA-HlH-2. Expectedly, the feruloylation of HlH induced a rearrangement of the protein molecule, a decrease in the ?-helical structure at the expense of ß-structures was observed. Besides, the FA-HlH conjugates were more prone to aggregation, which is probably due to the stabilization of the partially unfolded protein molecules by non-covalent bonding. Interestingly, the thermal stability of HlH was not affected by the modification. The native and feruloylated HlH were not toxic to normal fibroblasts (BJ cells). We observed a decrease in cell viability of breast cancer MCF-7 cells to about 66% after a 48h exposure to 70 µg/well of FA-HlH-2.

Ketoprofen-Based Ionic Liquids: Synthesis and Interactions with Bovine Serum Albumin.

The development of ionic liquids based on active pharmaceutical ingredients (API-ILs) is a possible solution to some of the problems of solid and/or hydrophobic drugs such as low solubility and bioavailability, polymorphism and an alternative route of administration could be suggested as compared to the classical drug. Here, we report for the first time the synthesis and detailed characterization of a series of ILs containing a cation amino acid esters and anion ketoprofen (KETO-ILs). The affinity and the binding mode of the KETO-ILs to bovine serum albumin (BSA) were assessed using fluorescence spectroscopy. All compounds bind in a distance not longer than 6.14 nm to the BSA fluorophores. The estimated binding constants (KA) are in order of 105 L mol-1, which is indicative of strong drug or IL-BSA interactions. With respect to the ketoprofen-BSA system, a stronger affinity of the ILs containing l-LeuOEt, l-ValOBu, and l-ValOEt cation towards BSA is clearly seen. Fourier transformed infrared spectroscopy experiments have shown that all studied compounds induced a rearrangement of the protein molecule upon binding, which is consistent with the suggested static mechanism of BSA fluorescence quenching and formation of complexes between BSA and the drugs. All tested compounds were safe for macrophages.

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.