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 for 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 the cytotoxicity to SH-SY5Y cells, are valuable candidates for further evaluation for the treatment of diabetes and associated disruption of neuronal homeostasis.

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.

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.

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 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.