Orthogonal Inverse-Electron-Demand Cycloaddition Reactions Controlled by Frontier Molecular Orbital Interactions.

Chemoselective pairs of bioorthogonal reactants enable the simultaneous labeling of several biomolecules. Here, we access orthogonal click reactions by exploiting differences in frontier molecular orbital interaction energies in transition states. We establish that five-membered cyclic dienes are inert to isonitriles but readily react with strained alkynes, while tetrazines with bulky substituents readily react with isonitriles. Strained alkynes show an opposite reactivity pattern. The approach was demonstrated by orthogonally labeling two proteins with different fluorophores.

N-Phenacyldibromobenzimidazoles-Synthesis Optimization and Evaluation of Their Cytotoxic Activity.

Antifungal N-phenacyl derivatives of 4,6- and 5,6-dibromobenzimidazoles are interesting substrates in the synthesis of new antimycotics. Unfortunately, their application is limited by the low synthesis yields and time-consuming separation procedure. In this paper, we present the optimization of the synthesis conditions and purification methods of N-phenacyldibromobenzimidazoles. The reactions were carried out in various base solvent-systems including K2CO3, NaH, KOH, t-BuOK, MeONa, NaHCO3, Et3N, Cs2CO3, DBU, DIPEA, or DABCO as a base, and MeCN, DMF, THF, DMSO, or dioxane as a solvent. The progress of the reaction was monitored using HPLC analysis. The best results were reached when the reactions were carried out in an NaHCO3-MeCN system at reflux for 24 h. Additionally, the cytotoxic activity of the synthesized compounds against MCF-7 (breast adenocarcinoma), A-549 (lung adenocarcinoma), CCRF-CEM (acute lymphoblastic leukemia), and MRC-5 (normal lung fibroblasts) was evaluated. We observed that the studied cell lines differed in sensitivity to the tested compounds with MCF-7 cells being the most sensitive, while A-549 cells were the least sensitive. Moreover, the cytotoxicity of the tested derivatives towards CCRF-CEM cells increased with the number of chlorine or fluorine substituents. Furthermore, some of the active compounds, i.e., 2-(5,6-dibromo-1H-benzimidazol-1-yl)-1-(3,4-dichlorophenyl)ethanone (4f), 2-(4,6-dibromo-1H-benzimidazol-1-yl)-1-(2,4,6-trichlorophenyl)ethanone (5g), and 2-(4,6-dibromo-1H-benzimidazol-1-yl)-1-(2,4,6-trifluorophenyl)ethanone (5j) demonstrated pro-apoptotic properties against leukemic cells with derivative 5g being the most effective.

Synthesis of Novel Acyl Derivatives of 3-(4,5,6,7-Tetrabromo-1H-benzimidazol-1-yl)propan-1-ols-Intracellular TBBi-Based CK2 Inhibitors with Proapoptotic Properties.

Protein kinase CK2 has been considered as an attractive drug target for anti-cancer therapy. The synthesis of N-hydroxypropyl TBBi and 2MeTBBi derivatives as well as their respective esters was carried out by using chemoenzymatic methods. Concomitantly with kinetic studies toward recombinant CK2, the influence of the obtained compounds on the viability of two human breast carcinoma cell lines (MCF-7 and MDA-MB-231) was evaluated using MTT assay. Additionally, an intracellular inhibition of CK2 as well as an induction of apoptosis in the examined cells after the treatment with the most active compounds were studied by Western blot analysis, phase-contrast microscopy and flow cytometry method. The results of the MTT test revealed potent cytotoxic activities for most of the newly synthesized compounds (EC50 4.90 to 32.77 µM), corresponding to their solubility in biological media. We concluded that derivatives with the methyl group decrease the viability of both cell lines more efficiently than their non-methylated analogs. Furthermore, inhibition of CK2 in breast cancer cells treated with the tested compounds at the concentrations equal to their EC50 values correlates well with their lipophilicity since derivatives with higher values of logP are more potent intracellular inhibitors of CK2 with better proapoptotic properties than their parental hydroxyl compounds.

Synergistic Interactions of 5-Fluorouracil with Inhibitors of Protein Kinase CK2 Correlate with p38 MAPK Activation and FAK Inhibition in the Triple-Negative Breast Cancer Cell Line.

BACKGROUND: The combination effect of 5-fluorouracil (5-FU) with either CX-4945 or a new inhibitor of protein kinase CK2, namely 14B (4,5,6,7-tetrabromo-1-(3-bromopropyl)-2-methyl-1H-benzimidazole), on the viability of MCF-7 and triple-negative MDA-MB-231 breast cancer cell lines was studied. METHODS: Combination index (CI) values were determined using an MTT-based assay and the Chou-Talalay model. The effect of the tested drug combinations on pro-apoptotic properties and cell cycle progression was examined using flow cytometry. The activation of FAK, p38 MAPK, and ERK1/2 kinases and the expression of selected pro-apoptotic markers in MDA-MB-231 cell line after the combined treatment were evaluated by the western blot method. Confocal microscopy was used to examine actin network in MDA-MB-231. RESULTS: Our results showed that a synergistic effect (CI < 1) occurred in MDA-MB-231 after treatment with both combinations of 5-FU with 14B or CX-4945, whereas the combination of 5-FU and 14B evoked an antagonistic effect in MCF-7. We conclude that the synergistic interactions (CI < 1) observed for both the combinations of 5-FU and 14B or CX-4945 in MDA-MB-231 correlated with an activation of p38 MAPK, inhibition of FAK, increased expression of apoptogenic markers, prolongation of S-phase of cell cycle, and destabilization of actin network. CONCLUSIONS: The obtained results support the recent observation that CK2 inhibitors can improve 5-FU-based anticancer therapy and FAK kinase can be an attractive molecular target in breast cancer therapy.

Improved protein-crystal identification by using 2,2,2-trichloroethanol as a fluorescence enhancer.

The identification of initial lead conditions for successful protein crystallization is crucial for structural studies using X-ray crystallography. In order to reduce the number of false-negative conditions, an emerging number of fluorescence-based methods have been developed which allow more efficient identification of protein crystals and help to distinguish them from salt crystals. Detection of the native tryptophan fluorescence of protein crystals is one of the most widely used methods. However, this method can fail owing to the properties of the crystallized protein or the chemical composition of the crystallization trials. Here, a simple, fast and cost-efficient method employing 2,2,2-trichloroethanol (TCE) has been developed. It can be performed with a standard UV-light microscope and can be applied to cases in which detection of native tryptophan fluorescence fails. In four test cases this method had no effect on the diffraction properties of the crystals and no structural changes were observed. Further evidence is provided that TCE can be added to crystallization trials during their preparation, making this method compatible with high-throughput approaches.

Synthesis, in vitro antiproliferative activity and kinase profile of new benzimidazole and benzotriazole derivatives.

Protein kinase 2 (CK2), a member of the serine/threonine kinase family, has been established as a promising target in anticancer therapy. New derivatives of known CK2 inhibitors 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi) and 4,5,6,7-tetrabromo-1H-benzotriazole (TBBt) bearing azide or substituted triazole groups were synthesized. Their influence on the activity of human recombinant CK2α and cytotoxicity against normal and cancer cell lines were evaluated. TBBi derivatives with triazole substituted with carboxyl substituent (7 and 10) exhibited the most potent inhibitory activity against CK2 with Ki value in the range of 1.96-0.91µM, respectively. New TBBi derivatives 2, 3, 5 and 9 have demonstrated the EC50, in the range of 12-25µM and 13-29µM respectively towards CCRF-CEM and MCF-7 cells. Derivatives TBBi decreased viability of cancer cells more efficiently than BALB cells and the biggest differences were observed for the azide substituted compounds 3 and 5. The effect of the most active compounds on the activity of eight off-target kinases was evaluated. Inhibitory efficiency of CK2-mediated p65 phosphorylation was demonstrated for the TBBi and compound 12.