Antitumor activity of the protein kinase inhibitor 1-(ß-D-2'-deoxyribofuranosyl)-4,5,6,7-tetrabromo- 1H-benzimidazole in breast cancer cell lines.

BACKGROUND: The protein kinases CK2 and PIM-1 are involved in cell proliferation and survival, the cell cycle, and drug resistance, and they are found overexpressed in virtually all types of human cancer, including breast cancer. In this study, we investigated the antitumor activity of a deoxynucleoside derivative, the protein kinase inhibitor compound 1-(ß-D-2'-deoxyribofuranosyl)-4,5,6,7-tetrabromo-1H-benzimidazole (K164, also termed TDB), inter alia CK2 and PIM-1, on breast cancer cell lines (MDA-MB-231, MCF-7, and SK-BR-3). METHODS: An evaluation of the cytotoxic and proapoptotic effects, mitochondrial membrane potential (ΔΨm), and cell cycle progression was performed using an MTT assay, flow cytometry, and microscopic analysis. The Western blotting method was used to analyze the level of proteins important for the survival of breast cancer cells and proteins phosphorylated by the CK2 and PIM-1 kinases. RESULTS: The examined compound demonstrated the inhibition of cell viability in all the tested cell lines and apoptotic activity, especially in the MCF-7 and SK-BR-3 cells. Changes in the mitochondrial membrane potential (ΔΨm), cell cycle progression, and the level of the proteins studied were also observed. CONCLUSIONS: The investigated CK2 and PIM-1 kinase inhibitor K164 is a promising compound that can be considered a potential agent in targeted therapy in selected types of breast cancer; therefore, further research is necessary.

Synthesis and Crystal Structure of Adamantylated 4,5,6,7-Tetrahalogeno-1H-benzimidazoles Novel Multi-Target Ligands (Potential CK2, M2 and SARS-CoV-2 Inhibitors); X-ray/DFT/QTAIM/Hirshfeld Surfaces/Molecular Docking Study.

A series of new congeners, 1-[2-(1-adamantyl)ethyl]-1H-benzimidazole (AB) and 1-[2-(1-adamantyl)ethyl]-4,5,6,7-tetrahalogeno-1H-benzimidazole (Hal=Cl, Br, I; tClAB, tBrAB, tIAB), have been synthesized and studied. These novel multi-target ligands combine a benzimidazole ring known to show antitumor activity and an adamantyl moiety showing anti-influenza activity. Their crystal structures were determined by X-ray, while intermolecular interactions were studied using topological Bader's Quantum Theory of Atoms in Molecules, Hirshfeld Surfaces, CLP and PIXEL approaches. The newly synthesized compounds crystallize within two different space groups, P-1 (AB and tIAB) and P21/c (tClAB and tBrAB). A number of intramolecular hydrogen bonds, C-H⋯Hal (Hal=Cl, Br, I), were found in all halogen-containing congeners studied, but the intermolecular C-H⋯N hydrogen bond was detected only in AB and tIAB, while C-Hal⋯π only in tClAB and tBrAB. The interplay between C-H⋯N and C-H⋯Hal hydrogen bonds and a shift from the strong (C-H⋯Cl) to the very weak (C-H⋯I) attractive interactions upon Hal exchange, supplemented with Hal⋯Hal overlapping, determines the differences in the symmetry of crystalline packing and is crucial from the biological point of view. The hypothesis about the potential dual inhibitor role of the newly synthesized congeners was verified using molecular docking and the congeners were found to be pharmaceutically attractive as Human Casein Kinase 2, CK2, inhibitors, Membrane Matrix 2 Protein, M2, blockers and Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2, inhibitors. The addition of adamantyl moiety seems to broaden and modify the therapeutic indices of the 4,5,6,7-tetrahalogeno-1H-benzimidazoles.

Benzimidazole inhibitors of protein kinase CK2 potently inhibit the activity of atypical protein kinase Rio1.

Benzimidazole derivatives of 5,6-dichlorobenzimidazole 1-ß-D-ribofuranoside (DRB) comprise the important class of protein kinase CK2 inhibitors. Depending on the structure, benzimidazoles inhibit CK2 with different selectivity and potency. Besides CK2, the compounds can inhibit, with similar activity, other classical eukaryotic protein kinases (e.g. PIM, DYRK, and PKD). The present results show that a majority of the most common CK2 inhibitors can affect the atypical kinase Rio1 in a nanomolar range. Kinetic data confirmed the mode of action of benzimidazoles as typical ATP-competitive inhibitors. In contrast to toyocamycin-the first discovered small-molecule inhibitor of Rio1-the most potent representative of benzimidazoles TIBI (IC50 = 0.09 µM, K i  = 0.05 µM) does not influence the oligomeric state of the Rio1 kinase. Docking studies revealed that TIBI can occupy the ATP-binding site of Rio1 in a manner similar to toyocamycin, and enhances the thermostability of the enzyme.

In Search of the Antimicrobial Potential of Benzimidazole Derivatives.

A broad series of 4,5,6,7-tetrahalogenated benzimidazoles and 4-(1H-benzimidazol-2-yl)-benzene-1,3-diol derivatives was tested against selected bacteria and fungi. For this study three plant pathogens Colletotrichum sp., Fusarium sp., and Sclerotinia sp., as well as Staphylococcus sp., Enterococcus sp., Escherichia sp., Enterobacter sp., Klebsiella spp. , and Candida spp. as human pathogens were used. MIC values and/or area of growth reduction method were applied in order to compare the activity of the synthesized compounds. From the presented set of 22 compounds, only 8, 16, 18 and 19 showed moderate to good inhibition against bacterial strains. Against Candida strains only compound 19 with three hydroxyl substituted benzene moiety presented high inhibition at nystatin level or lower.

Cell-permeable dual inhibitors of protein kinases CK2 and PIM-1: structural features and pharmacological potential.

It has been proposed that dual inhibitors of protein kinases CK2 and PIM-1 are tools particularly valuable to induce apoptosis of cancer cells, a property, however, implying cell permeability, which is lacking in the case of selective CK2/PIM-1 inhibitors developed so far. To fill this gap, we have derivatized the scaffold of the promiscuous CK2 inhibitor TBI with a deoxyribose moiety, generating TDB, a selective, cell-permeable inhibitor of CK2 and PIM-1. Here, we shed light on the structural features underlying the potency and narrow selectivity of TDB by exploiting a number of TDB analogs and by solving the 3D structure of the TDB/CK2 complex at 1.25 Å resolution, one of the highest reported so far for this kinase. We also show that the cytotoxic efficacy of TDB is almost entirely due to apoptosis, is accompanied by parallel inhibition of cellular CK2 and PIM-1, and is superior to both those observed combining individual inhibitors of CK2 and PIM-1 and by treating cells with the CK2 inhibitor CX4945. These data, in conjunction with the observations that cancer cells are more susceptible than non-cancer cells to TDB and that such a sensitivity is maintained in a multi-drug resistance background, highlight the pharmacological potential of this compound.

Exploiting the repertoire of CK2 inhibitors to target DYRK and PIM kinases.

Advantage has been taken of the relative promiscuity of commonly used inhibitors of protein kinase CK2 to develop compounds that can be exploited for the selective inhibition of druggable kinases other than CK2 itself. Here we summarize data obtained by altering the scaffold of CK2 inhibitors to give rise to novel selective inhibitors of DYRK1A and to a powerful cell permeable dual inhibitor of PIM1 and CK2. In the former case one of the new compounds, C624 (naphto [1,2-b]benzofuran-5,9-diol) displays a potency comparable to that of the first-in-class DYRK1A inhibitor, harmine, lacking however the drawback of drastically inhibiting monoamine oxidase-A (MAO-A) as harmine does. On the other hand the promiscuous CK2 inhibitor 4,5,6,7-tetrabromo-1H-benzimidazole (TBI,TBBz) has been derivatized with a sugar moiety to generate a 1-(ß-D-2'-deoxyribofuranosyl)-4,5,6,7-tetrabromo-1H-benzimidazole (TDB) compound which inhibits PIM1 and CK2 with comparably high efficacy (IC50 values<100nM) and remarkable selectivity. TDB, unlike other dual PIM1/CK2 inhibitors described in the literature is readily cell permeable and displays a cytotoxic effect on cancer cells consistent with concomitant inhibition of both its onco-kinase targets. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

Quantum-chemical insight into structure-reactivity relationship in 4,5,6,7-tetrahalogeno-1H-benzimidazoles: a combined X-ray, DSC, DFT/QTAIM, Hirshfeld surface-based, and molecular docking approach.

The weak interaction patterns in 4,5,6,7-tetrahalogeno-1H-benzimidazoles, protein kinase CK2 inhibitors, in solid state are studied by the X-ray method and quantum chemistry calculations. The crystal structures of 4,5,6,7-tetrachloro- and 4,5,6,7-tetrabromo-1H-benzimidazole are determined by X-ray diffraction and refined to a final R-factor of 3.07 and 3.03%, respectively, at room temperature. The compound 4,5,6,7-tetrabromo-1H-benzimidazole, which crystallizes in the I41/a space group, is found to be isostructural with previously studied 4,5,6,7-tetraiodo-1H-benzimidazole in contrast to 4,5,6,7-tetrachloro-1H-benzimidazole, which crystallizes as triclinic P1̅ with 4 molecules in elementary unit. For 4,5,6,7-tetrachloro-1H-benzimidazole, differential scanning calorimetry (DSC) revealed a second order glassy phase transition at Tg = 95°/106° (heating/cooling), an indication of frozen disorder. The lack of 3D isostructurality found in all 4,5,6,7-tetrahalogeno-1H-benzimidazoles is elucidated on the basis of the intra- and intermolecular interactions (hydrogen bonding, van der Waals contacts, and C-H···π interactions). The topological Bader's Quantum Theory of Atoms in Molecules (QTAIM) and Spackman's Hirshfeld surface-based approaches reveal equilibration of electrostatic matching and dispersion van der Waals interactions between molecules consistent with the crystal site-symmetry. The weakening of van der Waals forces accompanied by increasing strength of the hydrogen bond (N-H···N) result in a decrease in the crystal site-symmetry and a change in molecular packing in the crystalline state. Crystal packing motifs were investigated with the aid of Hirshfeld surface fingerprint plots. The ordering 4,5,6,7-tetraiodo > 4,5,6,7-tetrabromo > 4,5,6,7-tetrachloro > 4,5,6,7-tetrafluoro reflects not only a decrease in crystal symmetry but also increase in chemical reactivity (electronic activation), which could explain some changes in biological activity of compounds from the 4,5,6,7-tetrahalogeno-1H-benzimidazole series. The ability of formation of a given type of bonds by 4,5,6,7-tetrahalogeno-1H-benzimidazole molecules is the same in the crystal and in CK2. Analysis of the interactions in the crystal permits drawing conclusions on the character (the way) of connections between a given 4,5,6,7-tetrahalogeno-1H-benzimidazole as a ligand with CK2 protein to make a protein-ligand complex.

Modified tetrahalogenated benzimidazoles with CK2 inhibitory activity are active against human prostate cancer cells LNCaP in vitro.

A series of novel CK2 inhibitors, tetrahalogenated benzimidazoles carrying an aminoalkylamino group at position 2, has been prepared by nucleophilic substitution of the respective 2,4,5,6,7-pentabromobenzimidazoles and 2-bromo-4,5,6,7-tetraiodobenzimidazoles. The new derivatives as well as some previously obtained tetrahalogenobenzimidazoles, including 4,5,6,7-tetrabromobenzimidazole (TBI) and 4,5,6,7-tetraiodobenzimidazole (TIBI), were evaluated for activity against the hormone-sensitive human prostate cancer cell line LNCaP. The activity of 2-aminoalkylamino derivatives was notably higher (LD(50) 4.75-9.37 µM) than that of TBI and TIBI (LD(50) ≈ 20 µM). The determination of the LD(50) value identified the 2-aminoethylamino-4,5,6,7-tetraiodobenzimidazole with an additional methyl group at position 1 (6) as the most efficient compound (LD(50): 4.75 ± 1.02 µM). Interestingly, there was no clear correlation between cell viability and apoptosis induction indicating additional cell death mechanisms.

Synthesis and proapoptotic properties of new casein kinase II inhibitors.

Casein kinase II (CK2) is the most pleiotropic of all protein kinases with more than 300 substrates implicated in a wide variety of cellular functions as signal transduction, proliferation and cell survival. Increased levels of CK2 has been demonstrated in a number of cancers, where it regulates the activity of various oncoproteins and tumor suppressor proteins. Therefore, CK2 inhibitors could be considered as potential anticancer drugs in monotherapy or in combination with known cytostatics. In this study, we examined proapoptotic activity of new strong CK2 inhibitor - 4,5,6,7-tetraiodobenzimidazole (TIBI) (IC50 = 38 nM) as well as new derivatives of 4,5,6,7-tetrabromobenzimidazole and 4,5,6,7-tetraiodobenzimidazole. All the tested compounds induced apoptosis and cytostatic effects in the promyelocytic leukemia cell line (HL-60). The proapoptotic effect was concentration and time dependent. The changes of the mitochondrial membrane potential and cell cycle progression were also observed.

Tetraiodobenzimidazoles are potent inhibitors of protein kinase CK2.

A series of novel iodinated benzimidazoles have been prepared by iodination of respective benzimidazole with iodine and periodic acid in sulfuric acid solution. Additionally several 2-substituted- and N-1-carboxymethyl-substituted derivatives of 4,5,6,7-tetraiodobenzimidazole (TIBI) were obtained. For sake of comparison, some new 4,5,6,7-tetrabromobenzimidazoles were also synthesized. The ability of the new compounds to inhibit protein kinase CK2 has been evaluated. The results show that 4,5,6,7-tetraiodobenzimidazoles are more powerful inhibitors of CK2 than their tetrabrominated analogs. Molecular modeling supports the experimental data showing that tetraiodobenzimidazole moiety fills better the binding pocket than respective tetrabromo and tetrachlorocompounds. To note that 4,5,6,7-tetraiodobenzimidazole (TIBI) is one of the most efficient CK2 inhibitors (K(i)=23 nM) described to date.

Synthesis of new analogs of benzotriazole, benzimidazole and phthalimide--potential inhibitors of human protein kinase CK2.

New derivatives of 4,5,6,7-tetrabromo-1H-1,2,3-benzotriazole (TBBt), 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi), and N-substituted tetrabromophthalimides were synthesized and their effect on the activity of human protein kinase CK2 was examined. The most active were derivatives with N-hydroxypropyl substituents (IC(50) in 0.32-0.54 microM range) whereas derivatives of phthalimide were almost ineffective.

The selectivity of inhibitors of protein kinase CK2: an update.

CK2 (casein kinase 2) is a very pleiotropic serine/threonine protein kinase whose abnormally high constitutive activity has often been correlated to pathological conditions with special reference to neoplasia. The two most widely used cell permeable CK2 inhibitors, TBB (4,5,6,7-tetrabromo-1H-benzotriazole) and DMAT (2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole), are marketed as quite specific CK2 blockers. In the present study we show, by using a panel of approx. 80 protein kinases, that DMAT and its parent compound TBI (or TBBz; 4,5,6,7-tetrabromo-1H-benzimidazole) are potent inhibitors of several other kinases, with special reference to PIM (provirus integration site for Moloney murine leukaemia virus)1, PIM2, PIM3, PKD1 (protein kinase D1), HIPK2 (homeodomain-interacting protein kinase 2) and DYRK1a (dual-specificity tyrosine-phosphorylated and -regulated kinase 1a). In contrast, TBB is significantly more selective toward CK2, although it also inhibits PIM1 and PIM3. In an attempt to improve selectivity towards CK2 a library of 68 TBB/TBI-related compounds have been tested for their ability to discriminate between CK2, PIM1, HIPK2 and DYRK1a, ending up with seven compounds whose efficacy toward CK2 is markedly higher than that toward the second most inhibited kinase. Two of these, K64 (3,4,5,6,7-pentabromo-1H-indazole) and K66 (1-carboxymethyl-2-dimethylamino-4,5,6,7-tetrabromo-benzimidazole), display an overall selectivity much higher than TBB and DMAT when tested on a panel of 80 kinases and display similar efficacy as inducers of apoptosis.

Synthesis and antimycobacterial activity of selected nitrobenzyloxylated benzotriazoles.

A series of 1-nitrobenzyloxybenzotriazoles was prepared by the benzylation of the respective halogenosubstituted 1-hydroxybenzotriazoles. The newly obtained compounds were tested against four Mycobacterium strains. Particularly high antimycobacterial activity, comparable with that of isoniazide, was found for 5,6-dichloro- 1-(3,5-dinitrobenzyloxy)- 1H-benzotriazole.

Deoxynucleosides with benzimidazoles as aglycone moiety are potent anticancer agents.

Abnormally high levels of CK2 and PIM-1 serine/threonine kinases have been documented in many cases of cancer. The elevation of CK2 and PIM-1 in cells entails suppression of apoptosis and implies a protective role for the kinases against cell death. Downregulation of these enzymes by chemical methods promotes apoptosis in cells. The aim of the present study was to explore the anticancer activity of inhibitors of protein kinases CK2 and PIM-1 on neoplastic cell lines in vitro. We studied a series of deoxynucleosides with various tetrahalobenzimidazoles as aglycone moiety. Cytotoxicity, induction of apoptosis by the tested inhibitors, mitochondrial membrane potential, activity of caspases, changes in cell cycle progression, as well as a mechanism of action were determined by flow cytometry and other methods. The results indicate that the studied compounds, e.g., 1-(ß-D-2'-deoxyribofuranosyl)-4,5,6,7-tetrabromo-1H-benzimidazole called K164 (also termed TDB), showed diverse cytotoxicity and proapoptotic efficacy in cell lines. Our results showed that the tested compounds are potential anticancer agents for targeted therapy, particularly in the treatment of myeloid leukaemia and androgen-responsive prostate cancer.

Synthesis and anti-HIV studies of 2-adamantyl-substituted thiazolidin-4-ones.

A series of novel thiazolidin-4-ones bearing a lipophilic adamantyl substituent at position 2, and versatile substituents on the nitrogen atom of the thiazolidine ring, were synthesized whereas several compounds exhibited a modest anti-HIV-1 activity, (+/-)-2-adamantan-1-yl-3-(4,6-dimethyl-pyridin-2-yl)-thiazolidin-4-one 22 was endowed with a remarkable antiviral potency (EC(50)=0.35 microM). The adamantane moiety played an important role in the eventual antiviral activity of the compound. This compound behaved as a typical non-nucleoside reverse transcriptase (RT) inhibitor (NNRTI) with non-competitive inhibition against RT with respect to the substrate (K(i)=12 microM). Separation of the enantiomers via diastereoisomeric salts was performed for 22. X-ray studies enabled us to ascribe an S configuration to (-)-2-adamantan-1-yl-3-(4,6-dimethyl-pyridin-2-yl)-thiazolidin-4-one (-)-22. Furthermore, it was found that the (+)-22 isomer was predominantly responsible for the potent anti-HIV-1 activity (EC(50) value of 0.178 microM), while the levo isomer was more than 60-fold less active.

Synthesis and antibacterial activity of 5-adamantan-1-yl-methyl analogues of trimethoprim.

A series of new trimethoprim [5-(3,4,5-trimethoxy-benzyl)-pyrimidine-2,4- diamine] analogues were prepared by condensation of adamantane-1-carbaldehyde with 3-methoxypropionitrile, followed by reaction of resulting mixture of 2-adamantan-1-ylmethyl-3-methoxy-acrylonitrile and 3-adamantan-1-yl-2-methoxymethyl-acrylonitrile with guanidine, acetamidine and thiourea, respectively. The activity of compounds obtained and sulfamethoxazole, alone and in combination, against several bacterial strains, as well as fungi was investigated.

Synthesis and antiviral activity of some 5'-N-phthaloyl-3'-azido-2',3'-dideoxythymidine analogues.

A variety of substituted 5'-N-phthaloyl-3'-azido-2',3'-dideoxythymidine derivatives has been evaluated for their activity against HIV-1, HIV-2 and Moloney murine sarcoma virus (MSV) in cell culture. Most of the 3'-azido-2',3'-dideoxythymidine (AZT, zidovudine) derivatives showed antiviral activity in the lower micromolar concentration range and there was a close correlation between their anti-HIV and anti-MSV activity (r = 0.99). The adamantyl phthaloyl derivative was active at submicromolar concentrations. None of the compounds showed marked cytostatic activity. They did not inhibit recombinant HIV-1 reverse transcriptase. All compounds were inactive against HIV in thymidine kinase-deficient cells, pointing to the compounds' requirement to release free AZT to afford antiviral efficacy.

Synthesis and antimicrobial activity of new adamantane derivatives III.

Some novel ester imides synthesised from trimellitic acid anhydride and 1-adamantanol or 2-adamantanol, were tested as antimicrobial compounds. Unfortunately, these agents showed a modest antibacterial activity (MIC > 6 microg/ml). However, a comparison of these N-substituted adamantylester imides with the series published previously, indicated that the incorporation of L-alanine and L-phenylalanine into the phthalimide moiety was the best choice regardless of the series and leads to antimicrobial activity against Staphylococcus aureus strains.

Tumor necrosis factor-alpha production-regulating activity of phthalimide derivatives in genetically modified murine melanoma cells B78H1.

The effect of imides, monothioimides, trimellitimides, as well as 5'-deoxy-5'-phthaloylamino-derivatives of azidothymidine on tumor necrosis factor-alpha (TNF-alpha) production by genetically modified murine B78H1 melanoma cells transduced with the gene for human TNF-alpha (B78/TNF) was investigated. It was found that N-(adamant-1-yl)monothiophthalimide (1e) and N-(adamant-2-yl)-monothiophthalimide (1f) showed over 200% enhancing of TNF-alpha production while some of imides were inhibitors.

Novel adamantylated pyrimidines and their preliminary biological evaluations.

The synthesis of adamantylated pyrimidines was based on the reaction of 3-(adamantan-1-yl)-3-oxopropionic acid ethyl ester with urea, thiourea, guanidine as well as acetamidine, respectively. Then the compounds obtained were converted into respective bromo-, thio- and S-alkyl derivatives. The molecular structures for some compounds were studied by X-ray methods. The significant anticancer and antimicrobial properties of [2-(6-adamantan-1-yl-2-methylpyrimidin-4-ylthio)ethyl]dimethylamine were found.