Exploiting thiol-functionalized benzosiloxaboroles for achieving diverse substitution patterns - synthesis, characterization and biological evaluation of promising antibacterial agents.

Benzosiloxaboroles are an emerging class of medicinal agents possessing promising antimicrobial activity. Herein, the expedient synthesis of two novel thiol-functionalized benzosiloxaboroles 1e and 2e is reported. The presence of the SH group allowed for diverse structural modifications involving the thiol-Michael addition, oxidation, as well as nucleophilic substitution giving rise to a series of 27 new benzosiloxaboroles containing various polar functional groups, e.g., carbonyl, ester, amide, imide, nitrile, sulfonyl and sulfonamide, and pendant heterocyclic rings. The activity of the obtained compounds against selected bacterial and yeast strains, including multidrug-resistant clinical strains, was investigated. Compounds 6, 12, 20 and 22-24 show high activity against Staphylococcus aureus, including both methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) strains, with MIC values in the range of 1.56-12.5 µg mL-1, while their cytotoxicity is relatively low. The in vitro assay performed with 2-(phenylsulfonyl)ethylthio derivative 20 revealed that, in contrast to the majority of known antibacterial oxaboroles, the plausible mechanism of antibacterial action, involving inhibition of the leucyl-tRNA synthetase enzyme, is not responsible for the antibacterial activity. Structural bioinformatic analysis involving molecular dynamics simulations provided a possible explanation for this finding.

Synthesis and Anticancer Activity of Novel Dual Inhibitors of Human Protein Kinases CK2 and PIM-1.

CK2 and PIM-1 are serine/threonine kinases involved in the regulation of many essential processes, such as proliferation, differentiation, and apoptosis. Inhibition of CK2 and PIM-1 kinase activity has been shown to significantly reduce the viability of cancer cells by inducing apoptosis. A series of novel amino alcohol derivatives of parental DMAT were designed and synthesized as potent dual CK2/PIM-1 inhibitors. Concomitantly with the inhibition studies toward recombinant CK2 and PIM-1, the influence of the obtained compounds on the viability of three human carcinoma cell lines, i.e., acute lymphoblastic leukemia (CCRF-CEM), human chronic myelogenous leukemia (K-562), and breast cancer (MCF-7), as well as non-cancerous cells (Vero), was evaluated using an MTT assay. Induction of apoptosis and cell cycle progression after treatment with the most active compound and a lead compound were studied by flow-cytometry-based assay. Additionally, autophagy induction in K-562 cells and intracellular inhibition of CK2 and PIM-1 in all the tested cell lines were evaluated by qualitative/quantitative fluorescence-based assay and Western blot method, respectively. Among the newly developed inhibitors, 1,1,1-trifluoro-3-[(4,5,6,7-tetrabromo-1H-benzimidazol-2-yl)amino]propan-2-ol demonstrates the highest selectivity and the most prominent proapoptotic properties towards the studied cancer cells, especially towards acute lymphoblastic leukemia, in addition to inducing autophagy in K-562 cells.

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.

New insight into nucleo α-amino acids - Synthesis and SAR studies on cytotoxic activity of ß-pyrimidine alanines.

Three series of the ß-pyrimidine alanines, including willardiine - a naturally occurring amino acid, were prepared from the l-serine-derived sulfamidates. Compounds 3b, 4a and 4b demonstrated antiproliferative activity toward the studied cancer cell lines, albeit the effect of these compounds on human brain astrocytoma MOG-G-CCM cells was more significant than on human neuroblastoma SK-N-AS cells. The cytosine analog of willardiine, compound 4b, reduced viability of MOG-G-CCM cells with EC50 = 36 ± 2 µM, more effectively than AMPA antagonist GYKI 52466. Willardiine showed possible capability of affecting invasiveness of glioblastoma U251 MG cells with no effect on their viability and morphology. Compound 3d, the ethyl ester of willardiine, featured activity toward binding domain hHS1S2I of the GluR2 receptor. Docking analysis revealed that the location mode of compound 3d at the S1S2 domain of hGluR2 (PDB ID: 3R7X) might differ from that of willardiine.

Human dihydrofolate reductase is a substrate of protein kinase CK2α.

Dihydrofolate reductase (DHFR) is a prominent molecular target in antitumor, antibacterial, antiprotozoan, and immunosuppressive chemotherapies, and CK2 protein kinase is an ubiquitous enzyme involved in many processes, such as tRNA and rRNA synthesis, apoptosis, cell cycle or oncogenic transformation. We show for the first time that CK2α subunit strongly interacted with and phosphorylated DHFR in vitro. Using quartz crystal microbalance with dissipation monitoring (QCM-D) we determined DHFR-CK2α binding kinetic parameters (Kd below 0.5 µM, kon = 10.31 × 104 M-1s-1 and koff = 1.40 × 10-3s-1) and calculated Gibbs free energy (-36.4 kJ/mol). In order to identify phosphorylation site(s) we used site-directed mutagenesis to obtain several DHFR mutants with predicted CK2-phosphorylable serine or threonine residues substituted with alanines. All enzyme forms were subjected to CK2α subunit catalytic activity and the results pointed to serine 168 as a phosphorylation site. Mass spectrometry analyses confirmed the presence of phosphoserine 168 and revealed additionally the presence of phosphoserine 145, although the latter phosphorylation was on a very low level.

Potential bioisosteres of ß-uracilalanines derived from 1H-1,2,3-triazole-C-carboxylic acids.

The 1H-1,2,3-triazole-originated derivatives of willardiine were obtained by: (i) construction of the 1H-1,2,3-triazole ring in 1,3-dipolar cycloaddition of the uracil-derived azides and the carboxylate-bearing alkynes or α-acylphosphorus ylide, or (ii) N-alkylation of the uracil derivative with the 1H-1,2,3-triazole-4-carboxylate-derived mesylate. The latter method offered: (i) reproducible results, (ii) a significant reduction of amounts of auxiliary materials, (iii) reduction in wastes and (iv) reduction in a number of manual operations required for obtaining the reaction product. Compound 6a exhibited significant binding affinity to hHS1S2I ligand-binding domain of GluR2 receptor (EC50 = 2.90 µM) and decreased viability of human astrocytoma MOG-G-CCM cells in higher extent than known AMPA antagonist GYKI 52466.

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.

Synthesis of polybrominated benzimidazole and benzotriazole derivatives containing a tetrazole ring and their cytotoxic activity.

ABSTRACT: A series of new benzimidazole and benzotriazole derivatives containing a tetrazole moiety was synthesized by N-alkylation of 5-aryltetrazole with 4,5,6,7-tetrabromo-1-(3-chloropropyl)-1H-benzimidazole and 4,5,6,7-tetrabromo-2-(3-chloropropyl)-2H-benzotriazole. The reaction was regioselective and mostly 2,5-disubstituted tetrazole derivatives were obtained. The effect of all synthesized compounds on human recombinant casein kinase 2alpha subunit (rhCK2α) and cytotoxicity against human T-cell lymphoblast (CCRF-CEM) and breast adenocarcinoma (MCF-7) cell lines were evaluated. The results have shown that many of the synthesized compounds exhibit significant cytotoxicity at micromolar concentration.

Synthesis of novel polybrominated benzimidazole derivatives-potential CK2 inhibitors with anticancer and proapoptotic activity.

The efficient method for the synthesis of novel cell permeable inhibitors of protein kinase CK2 with anticancer and proapoptotic activity has been developed. A series of polybrominated benzimiadazole derivatives substituted by various cyanoalkyl groups have been synthesized. Cyanoethyl derivatives were obtained by Michael type addition of 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi) and 4,5,6,7-tetrabromo-2-methyl-1H-benzimidazole to acrylonitrile, whilst cyanomethyl, cyanopropyl and cyanobutyl analogs by N-alkylation of 4,5,6,7-tetrabromo-1H-benzimidazole and 4,5,6,7-tetrabromo-2-methyl-1H-benzimidazole with appropriate cyanoalkyl halides. The inhibitory activity against protein kinase rhCK2α catalytic subunit and cytotoxicity against two human cancer cell lines: acute lymphocytic leukemia (CCRF-CEM) and breast (MCF-7) were evaluated for all newly synthesized compounds. Additionally, the proapoptotic activity toward leukemia cells and intracellular inhibition of CK2 for the most cytotoxic derivatives have been performed, demonstrating 4,5,6,7-tetrabromo-2-methyl-1H-benzimidazole as a new selective inhibitor of rhCK2 with twenty-fold better proapoptotic activity than parental compound (TBBi).

[1,4]-sigmatropic rearrangement of chiral nitrones and their utilization in the synthesis of new iminosugars.

Reflection on the epimerization of the α-stereocenter of sugar nitrones leads to the conclusion that the process may occur through [1,4]-sigmatropic rearrangement. Participation of an ionic mechanism was excluded by a deuterium labeling experiment, and DFT calculations showed a reasonable energy barrier for the proposed [1,4]-shift. Products of the intramolecular 1,3-dipolar cycloaddition of the studied nitrones were utilized in the diversity-oriented synthesis of polyhydroxy derivatives of piperidine, indolizidine and quinolizidine. Minimal activity against the screened glucosidases and human melanoma cell lines was observed for some of the obtained compounds.

Thermodynamic parameters for binding of some halogenated inhibitors of human protein kinase CK2.

The interaction of human CK2α with a series of tetrabromobenzotriazole (TBBt) and tetrabromobenzimidazole (TBBz) analogs, in which one of the bromine atoms proximal to the triazole/imidazole ring is replaced by a methyl group, was studied by biochemical (IC50) and biophysical methods (thermal stability of protein-ligand complex monitored by DSC and fluorescence). Two newly synthesized tri-bromo derivatives display inhibitory activity comparable to that of the reference compounds, TBBt and TBBz, respectively. DSC analysis of the stability of protein-ligand complexes shows that the heat of ligand binding (Hbind) is driven by intermolecular electrostatic interactions involving the triazole/imidazole ring, as indicated by a strong correlation between Hbind and ligand pKa. Screening, based on fluorescence-monitored thermal unfolding of protein-ligand complexes, gave comparable results, clearly identifying ligands that most strongly bind to the protein. Overall results, additionally supported by molecular modeling, confirm that a balance of hydrophobic and electrostatic interactions contribute predominantly, relative to possible intermolecular halogen bonding, in binding of the ligands to the CK2α ATP-binding site.

Synthesis of novel chiral TBBt derivatives with hydroxyl moiety. Studies on inhibition of human protein kinase CK2α and cytotoxicity properties.

The efficient method for the synthesis of novel 4,5,6,7-tetrabromo-1H-benzotriazole (TBBt) derivatives bearing a single stereogenic center has been developed. New compounds with a variety of substituents at the meta- and para-position of the phenyl ring are reported. All of the presented compounds were obtained using classical synthetic methods, such as bromination of benzotriazole, and its subsequent alkylation by monotosylated arylpropane-1,3-diols, which in turn have been synthesized through reduction of the corresponding prochiral ß-keto esters, and the selective monotosylation of the primary hydroxyl group. The influence of the new and previously reported N-hydroxyalkyl TBBt derivatives on the activity of human protein kinase CK2α catalytic subunit was examined. The most active were derivatives with N-hydroxyalkyl substituents (IC50 in 0.80-7.35 µM range). A binding mode of (R)-1-(4,5,6,7-tetrabromo-2H-benzotriazol-2-yl)butan-3-ol 7b to hCK2α has been proposed based on in silico docking studies. Additionally, MTT-based cytotoxicity tests demonstrated high activities of novel 1-aryl-3-TBBt-propan-1-ol and 3-TBBt-propan-1,2-diol derivatives against human peripheral blood T lymphoblast (CCRF-CEM), and moderate anti-tumor activities against human breast adenocarcinoma (MCF7) cell lines.