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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 5–7containing 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 5–7at lower concentrations of 100 μM, 10 μM, and 1 μM, respectively, demonstrated that 6and 7do not show any toxicity within this concentration range. In the model of 6-OHDA-induced oxidative stress, 6revealed 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.

Aims: The aim of the current study was to develop and explore a series of new cytotoxic agents based on theconjugation 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 thienopyrimidinederivatives were synthesized by modifying the structure of the clinically used anticancer quinazoline EGFRinhibitors of the first generation - gefitinib, and second-generation - dacomitinib and canertinib. It was reported thatsome thieno[2,3-d]pyrimidine derivatives showed improved EGFR inhibitory activity. On the other hand, the benzimidazoleheterocycle 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 benzimidazolederivative 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 thecompounds and investigation of their effects towards human cancer HT-29, MDA-MB-231, HeLa, HepG2 and to normalhuman 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 toestablish the cytotoxicity of the tested compounds. SAR analysis and in silico assessment of the inhibitory potential towardshuman 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 highinhibitory 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 a1,3-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 activityagainst MDA-MB-231 and HepG2, with IC50 1.44 nM and 1.11 nM respectively. To outline the possible biological target ofthe studied coumpounds, their potential to interact with human oncogenic V599EB-Raf was explored by a docking study. Asa result, it was suggested that the benzimidazolyl and glycyl fragments could enhance the binding ability of the new compoundsby 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 normalhuman Lep3 cell lines demonstrated cytotoxicity in the nanomolar range. It was established that compounds 9, 10 and21 showed many times lower toxicity against normal Lep3 cells that can provide a high selectivity towards all fourcancer cell lines at small concentrations. Based on the analysis of the structure-activity relationship, the observedtrends 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-Rafshowed that the compounds might be able to stabilize the enzyme in its inactive form.

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.

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.

Background: One of the successful approaches for rational design of bioactive compounds is the bioisosterism strategy. Thus, the bioisosteric relation of thieno[2,3-d]pyrimidin-4- ones with quinazolines, cytosine and uracil resulted in the generation of compounds with variety of biological properties including antiparasitic activity. In contrast to mammals, all parasites are unable to synthesize purines de novo and rely instead on the Purine Salvage Pathway (PSP) to obtain purines, which are essential for their survival. Having in view the above mentioned facts we designed and synthesized some thieno[2,3-d]pyrimidines as bioisosters in order to evaluate their antitrichinellosis efficacy. Methods: The target compounds were synthesized by one-pot cyclocondensation reaction passing dry hydrogen chloride gas through a solution of 2-aminothiophene-carboxylate and a series of alkyl respectively aryl nitriles as precursors. The parasitological screening in vitro was carried out by using encapsulated infective larvae of Trichinella spiralis, 100 speciments per 1 mL physiological solution, released in advance from the muscle capsules by digestion with acid pepsin solution. Results: Fourteen 2-substituted-thieno[2,3-d]pyrimidin-4-ones were synthesized and their structure was identified. The data obtained by the antitrichinellosis screening showed that compounds 2, 8 and 15 exhibit higher activity than the reference drugs albendazole and ivermectin at concentrations of 0.37, 0.35 and 0.48 μM resp. 0.92, 0.88 and 1.2 μM after 24-hour incubation of the samples. The highest efficacy at both incubations was revealed by 2-benzyl-5,6,7,8- tetrahydrothieno[2,3-d]pyrimidin-4(3H)-one 2 - 97.94% and 100%, respectively. Compound 15 demonstrated 97.5% antiparasitic activity after 48h at concentration of 250 μg/ml. The theoretically calculated intestinal absorption (%ABS) of the tested thieno[2,3-d]pyrimidines displayed higher values than that of albendazole. The structure-activity relationship (SAR) data of the tested compounds 2, 4-8 and 15 complies with the Lipinski's rule. It was assumed that the low molecular volume (Vol.) and logP of compound 15 led to enhancement of the compound activity by increasing the penetration through the ion channels of the parasitic cells as it was observed in some albendazole derivatives. Conclusion: The tested thienopyrimidin-4-ones possess higher anthelminthic effect than albendazole against Trichinella spiralis. Compounds 2, 4-8 and 15 demonstrated higher absorption %ABS than albendazole. According to the SAR analysis, the partition coefficient (logP) is essential for parasite drug penetration due to diffusion through the cell membrane.

Benzimidazole-arylhydrazone hybrids showed promising potential as multifunctional drugs for the treatment of neurodegenerative disorders. The neuroprotection studies conducted using an in vitromodel 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, 3eis predicted to react slightly faster than 3awith alkoxyl radicals and around two orders of magnitude faster than 3awith hydroperoxyl radicals. The most reactive sites for formal hydrogen atom transfer in 3aare 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 3aand 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.

Recently, we found that two cyclodidepsipeptides, 3,6-di-(propan-2-yl)-4-methyl-morpholine-2,5-dione (1)and 3-(2-methylpropyl)-6-(propan-2-yl)-4-methyl-morpholine-2,5-dione (2), are excellent inhibitors of xanthine oxidase. In order to obtain more information about the toxicological potential of compounds 1and 2on bone cells, the current study was designed to evaluate the effect of these compounds on viability and proliferation of MC3T3-E1 cells. Compound 1showed neither cytotoxic nor stimulatory effect on cell viability, while compound 2showed a slight stimulatory effect on cell viability. Both studied compounds showed slight stimulatory effects on proliferation of MC3T3-E1 cells, in a dose dependent manner. Additionally, an in silicotoxicological study of compounds 1and 2was performed, and the results indicate that they have a good probability of safe biological intake.