Synthesis and evaluation of anticancer activity of new 4,5,6,7-tetrabromo-1H-benzimidazole derivatives.

An efficient method for the synthesis of new 4,5,6,7-tetrabromo-1H-benzimidazole derivatives has been developed. New ketones were obtained by N-alkylation of TBBi or 2-Me-TBBi with various phenacyl halides and then reduced to the corresponding alcohols. All compounds were obtained with satisfactory yields in the range of 40-91 %. The synthesized compounds appeared a weak CK2 and PIM-1 inhibitors but exhibit an interesting cytotoxic activity against cancer cell lines, i.e. MCF-7, PC-3, CCRF-CEM, K-562. 1-Phenyl-2-(4,5,6,7-tetrabromo-1H-benzimidazol-1-yl)ethanone 3aA exhibits the highest cytotoxic activity with IC50 value of 5.30 µM for MCF-7 and 6.80 µM for CCRF-CEM. Moreover, this compound shows the highest selectivity against both MCF-7 and CCRF-CEM with SI selectivity coefficients (against MRC-5 and Vero cells) equal 5.45 and 4.30 for MCF-7 and 4.25 and 3.35 for CCRF-CEM, respectively. Furthermore, it was shown that compound 3aA exhibits very good pro-apoptotic properties, through induction of the mitochondrial apoptotic pathway in CCRF-CEM cells. These results correlate with data showing the effect of 3aA on intracellular level of CK2α protein and CK2-mediated phosphorylation of Ser529 in NF-κBp65. Study of the effect of compound 3aA on mRNA levels of CK2α and CK2α' showed no significant differences in gene expression levels in control CCRF-CEM and cells treated with 3aA, indicating 3aA action at the protein level.

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.

In Vitro Anti-Candida Activity and Action Mode of Benzoxazole Derivatives.

A newly synthetized series of N-phenacyl derivatives of 2-mercaptobenzoxazole, including analogues of 5-bromo- and 5,7-dibromobenzoxazole, were screened against Candida strains and the action mechanism was evaluated. 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-bromophenyl)ethanone (5d), 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,3,4-trichloro-phenyl)ethanone (5i), 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,4,6-trichlorophenyl)ethanone (5k) and 2-[(5-bromo-1,3-benzoxazol-2-yl)sulfanyl]-1-phenylethanone (6a) showed anti-C. albicans SC5314 activity, where 5d displayed MICT = 16 µg/mL (%R = 100) and a weak anti-proliferative activity against the clinical strains: C. albicans resistant to azoles (Itr and Flu) and C. glabrata. Derivatives 5k and 6a displayed MICP = 16 µg/mL and %R = 64.2 ± 10.6, %R = 88.0 ± 9.7, respectively, against the C. albicans isolate. Derivative 5i was the most active against C. glabrata (%R = 53.0 ± 3.5 at 16 µg/mL). Benzoxazoles displayed no MIC against C. glabrata. Benzoxazoles showed a pleiotropic action mode: (1) the total sterols content was perturbed; (2) 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(3,4-dichlorophenyl)ethanol and 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,3,4-trichlorophenyl)ethanol (8h-i) at the lowest fungistatic conc. inhibited the efflux of the Rho123 tracker during the membrane transport process; (3) mitochondrial respiration was affected/inhibited by the benzoxazoles: 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-chlorophenyl)ethanol and 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-bromophenyl)ethanol 8c-d and 8i. Benzoxazoles showed comparable activity to commercially available azoles due to (1) the interaction with exogenous ergosterol, (2) endogenous ergosterol synthesis blocking as well as (3) membrane permeabilizing properties typical of AmB. Benzoxazoles display a broad spectrum of anti-Candida activity and action mode towards the membrane without cross-resistance with AmB; furthermore, they are safe to mammals.

Lipase-catalyzed kinetic resolution of novel antitubercular benzoxazole derivatives.

Novel benzoxazole derivatives were synthesized, and their antitubercular activity against sensitive and drug-resistant Mycobacterium tuberculosis strains (M. tuberculosis H37 Rv, M. tuberculosis sp. 210, M. tuberculosis sp. 192, Mycobacterium scrofulaceum, Mycobacterium intracellulare, Mycobacterium fortuitum, Mycobacterium avium, and Mycobacterium kansasii) was evaluated. The chemical step included preparation of ketones, alcohols, and esters bearing benzoxazole moiety. All racemic mixtures of alcohols and esters were separated in Novozyme SP 435-catalyzed transesterification and hydrolysis, respectively. The transesterification reactions were carried out in various organic solvents (tert-butyl methyl ether, toluene, diethyl ether, and diisopropyl ether), and depending on the solvent, the enantioselectivity of the reactions ranged from 4 to >100. The enzymatic hydrolysis of esters was performed in 2 phase tert-butyl methyl ether/phosphate buffer (pH = 7.2) system and provided also enantiomerically enriched products (ee 88-99%). The antitubercular activity assay has shown that synthesized compounds exhibit an interesting antitubercular activity. Racemic mixtures of alcohols, (±)-4-(1,3-benzoxazol-2-ylsulfanyl)butan-2-ol ((±)-3a), (±)-4-[(5-bromo-1,3-benzoxazol-2-yl)sulfanyl]butan-2-ol ((±)-3b), and (±)-4-[(5,7-dibromo-1,3-benzoxazol-2-yl)sulfanyl]butan-2-ol ((±)-3c), displayed as high activity against M. scrofulaceum, M. intracellulare, M. fortuitum, and M. kansasii as commercially available antituberculosis drug-Isoniazid. Moreover, these compounds exhibited twice higher activity toward M. avium (MIC 12.5) compared with Isoniazid (MIC 50).

Synthesis of novel tetrazole derivatives and evaluation of their antifungal activity.

With the appearance of the antifungal resistance, novel antifungal agents need to be identified. In this context new 2,5-disubstituted tetrazole derivatives containing benzothiazole, benzoxazole or phenylsulfonyl moiety were synthesized by N-alkylation of aryltetrazole with 2-[(3-chloropropyl)sulfanyl]-1,3-benzothiazole or 2-[(3-chloropropyl)sulfanyl]-1,3-benzoxazole and Michael-type addition of aryltetrazole to phenyl vinyl sulfone. The chemical structures of the synthesized compounds were confirmed by means of 1H NMR, 13C NMR, IR and HRMS spectral data. The compounds were tested against the moulds: Fusarium sambucinum, Fusarium oxysporum, Colletotrichum coccodes, Aspergillus niger, and the yeast Candida albicans. The results showed that among the moulds only C. coccodes was significantly sensitive to all the structures examined. All the tetrazole derivatives acted at the same level against C. albicans and demonstrated a high cell growth inhibition (97-99%) at the concentrations ranging from 16 to 0.0313µg/mL. The mode of action of 2-({3-[5-(4-chlorophenyl)-2H-tetrazol-2-yl]propyl}sulfanyl)-1,3-benzoxazole (5c) and 2-({3-[5-(2-chlorophenyl)-2H-tetrazol-2-yl]propyl}sulfanyl)-1,3-benzoxazole (5d) was established by verifying fungal growth in the presence of osmotic protector-sorbitol. The effect of compound 5c or 5d combined with Fluconazole was determined using the checkerboard method. The calculated fractional inhibitory concentration index (FIC) indicated antagonism (FIC >1). Additionally, survival experiments with lepidopteran Galleria mellonella treated with compounds 5c and 5d were performed and demonstrated the lack of toxicity of these compounds.

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).

Lipase-Catalyzed Kinetic Resolution of Novel Antifungal N-Substituted Benzimidazole Derivatives.

A series of new N-substituted benzimidazole derivatives was synthesized and their antifungal activity against Candida albicans was evaluated. The chemical step included synthesis of appropriate ketones containing benzimidazole ring, reduction of ketones to the racemic alcohols, and acetylation of alcohols to the esters. All benzimidazole derivatives were obtained with satisfactory yields and in relatively short times. All synthesized compounds exhibit significant antifungal activity against Candida albicans 900028 ATCC (% cell inhibition at 0.25 µg concentration > 98%). Additionally, racemic mixtures of alcohols were separated by lipase-catalyzed kinetic resolution. In the enzymatic step a transesterification reaction was applied and the influence of a lipase type and solvent on the enantioselectivity of the reaction was studied. The most selective enzymes were Novozyme SP 435 and lipase Amano AK from Pseudomonas fluorescens (E > 100).

Tetrazole activity against Candida albicans. The role of KEX2 mutations in the sensitivity to (±)-1-[5-(2-chlorophenyl)-2H-tetrazol-2-yl]propan-2-yl acetate.

Series of 4-(5-aryl-2H-tetrazol-2-yl)butan-2-ol, 1-(5-aryl-2H-tetrazol-2-yl)propan-2-ol and their acetates have been screened against Candida albicans. Among the tested compounds, (±)-1-[5-(2-chlorophenyl)-2H-tetrazol-2-yl]propan-2-yl acetate (E5) proved to be the most effective inhibitor of fungal growth and was further evaluated against young (adhesion phase) and mature biofilm in vitro. The activity exhibited by the tested tetrazole derivatives against C. albicans associated with minor cytotoxicity towards Vero epithelial cells make us suggest that E5 could be a promising structure in the development of new antifungals. Serine protease Kex2 appeared essential for the resistance mechanism. Further investigations of in vivo activity, drug interactions, and E5 structure optimization are needed.

Enzymatic hydrolysis of esters containing a tetrazole ring.

The lipase-catalyzed enantioselective hydrolysis of acetates containing tetrazole moiety was studied. Among all tested lipases, Novozyme SP 435 allowed to obtain optically active 4-(5-aryl-2H-tetrazol-2yl)butan-2-ol and 1-(5-aryl-2H-tetrazol-2yl)-propan-2-ol and their acetates with the highest optical purities (ee = 95%-99%) and excellent enantioselectivity (E>100). Some of the synthesized tetrazole derivatives were screened for their antifungal activity. Racemic mixtures of 4-[5-(4-chlorophenyl)-2H-tetrazol-2-yl)butan-2-ol as well as pure enantiomers of this compound showed promising antifungal activity against F. sambucinum, F. oxysporum, C. coccodes, and A. niger.

Has a High Dose of Vitamin D3 Impacted Health Conditions in Older Adults?-A Systematic Review and Meta-Analysis Focusing on Dose 100,000 IU.

BACKGROUND: Older adults are prone to vitamin D3 (VD3) deficiency, which may impair their health. A high dose of VD3 (HDVD3 = 100,000 IU) could improve their 25-hydroxyvitamin D3 [25(OH)D] level and health outcomes. However, evidence for such a beneficial effect of HDVD3 in older adults coming from clinical trials is mixed. OBJECTIVE: To review the literature on the efficacy of a single dose of 100,000 IU of VD3 in older people. METHODS: We searched PubMed/Medline, Science Direct, and NIH's clinical trials registry for clinical studies on the effect of a single high dose of VD3 on various health outcomes in older people. We also performed a meta-analysis using the standardized mean difference to assess the effect of VD3 on its blood level. Due to expected high heterogeneity, its amount (i.e., tau2) was estimated using the DerSimonian-Laird estimator. To estimate tau2, the Q-test for heterogeneity and the I2 statistic were calculated. RESULTS: Search results identify 13 studies that reported diverse health outcomes, such as lung and cardiovascular function, skin cancer progression, intensive care unit mortality, immune system response, and bone density. The meta-analysis showed a significant increase in 25(OH)D blood levels after treatment in 10 studies, with an average standardized mean difference of 2.60 ng/mL (95% CI: 2.07 to 3.13). Their results suggested that a single high dose of VD3 may benefit intensive care unit patients and skin cancer patients in remission. However, evidence for other beneficial health effects of HDVD3 was mixed due to high heterogeneity among studies. CONCLUSIONS: A single high dose of VD3 may positively affect some health outcomes in older people, possibly due to its pleiotropic and immunomodulatory effects. However, the evidence needs to be more extensive and consistent, and more rigorous studies are required to confirm the benefits and safety of VD3 high doses in older patients.

Design and synthesis of CK2 inhibitors.

A series of new polybrominated benzimidazoles and benzotriazoles has been synthesized and their influence on the activity of protein kinase CK2 was evaluated. It was revealed that the most active inhibitors are those with methyl or ethyl substituent at benzene ring, namely 5,6,7-tribromo-4-methyl-1H-benzotriazole (38, IC(50) 0.51 µM) and 5,6,7-tribromo-4-ethyl-1H-benzotriazole (40, IC(50) 0.16 µM). The derivatives with large aromatic or heterocyclic substituents connected to benzimidazole or benzotriazole scaffold appeared to be less potent inhibitors.

Synthesis of tetrazole derivatives bearing pyrrolidine scaffold and evaluation of their antifungal activity against Candida albicans.

The increase of opportunistic fungal infections raises the need for design and synthesis of new antifungal agents. Taking into account that tetrazole derivatives exhibit antifungal activity, and some of them are in the phase of clinical trials, new tetrazole derivatives bearing pyrrolidine moiety were synthesized in order to present their action mode against C. albicans. The target compounds were obtained by N-alkylation of various 2-arylpyrrolidines with several 1-(3-chloropropyl)-5-aryl-2H-tetrazoles. Regardless of the substituents at tetrazole or pyrrolidine rings reactions took place in 48 h and with satisfactory yields ranging from 53 to 70%. We performed screen of the synthesized compounds to identify these nontoxic inhibiting the C. albicans planktonic and sessile cells, and conducted a series of follow up studies to examine the in vitro and in vivo activity of the most potent antifungals. The leading antifungal inhibitor: 2-{3-[2-(3-Methylphenyl)pyrrolidin-1-yl]propyl}-5-phenyl-2H-tetrazole (3aC) and the randomly selected ones: 5-phenyl-2-[3-(2-phenylpyrrolidin-1-yl)propyl]-2H-tetrazole (3aA), 5-(4-chlorophenyl)-2-{3-[2-(4-fluorophenyl)pyrrolidin-1-yl]propyl}-2H-tetrazole (3cD), and 5-(4-chlorophenyl)-2-{3-[2-(4-chlorophenyl)pyrrolidin-1-yl]propyl}-2H-tetrazole (3cE) showed little to no toxicity against the Vero cell line and Galleria mellonella. 3aC and 3aD, the most active against biofilm in vitro, demonstrated in vivo activity in the invertebrate model of disseminated candidiasis. Flow cytometry analysis showed that necrotic cell death was generated under 3aC due to its interactions with the fungal membrane; this confirmed by the mitochondrial damage (XTT assay) and reduced adhesion to the TR-146 cell line at 46.05 µM. Flow cytometry was used to directly measure the redox state of the treated cells with the fluorescent DCFH probe. Pro-necrotic tetrazole derivatives (3aA, 3aC, 3cD) are unable to induce ROS production in the C. albicans cells. Moreover, CLSM analyses revealed that the tetrazole derivatives (principally 3aC, 3aD, and 3aE) inhibit C. albicans' ability to neutralize macrophages; a more effective phagosomes organisation was observed. 3aC's and 3aD's activity reflected in an attenuation of virulence in disseminated candidiasis in vivo.

Effect of Simultaneous Inhibition of Protein Kinase CK2 and Thymidylate Synthase in Leukemia and Breast Cancer Cells.

BACKGROUND/AIM: Protein kinase CK2 was recently identified as a promising therapeutic target for combination therapy. Our study aims to investigate the anticancer effect of a simultaneous inhibition of thymidylate synthase (TS) and CK2 in MCF-7 breast cancer and CCRF-CEM leukemia cells. MATERIALS AND METHODS: The type of interaction between CK2 inhibitors: CX-4945, 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi), or recently obtained 4,5,6,7-tetrabromo-2-methyl-1H-benzimidazol-1-yl)acetonitrile (2b) and TS-directed anticancer drug, 5-fluorouracil (5-FU) was determined using the MTT assay and a combination index method. The influence of the combined treatment on apoptosis in leukemia cells, as well as on cell-cycle progression and the levels of TS, CK2α and P-Ser529-p65 were determined in both cell lines, using flow cytometry and western blot analysis, respectively. RESULTS: The best synergistic effect was observed in CCRF-CEM cell line with the combination of 5-FU and 2b which correlated with a decrease in the endocellular CK2 activity and enhancement of the pro-apoptotic effect. CONCLUSION: The obtained results demonstrate the ability of CK2 inhibitors to enhance the efficacy of 5-FU in anticancer treatment, indicating a different molecular mechanism of the studied CK2 inhibitors interaction with 5-FU.

New 1,5 and 2,5-disubstituted tetrazoles-dependent activity towards surface barrier of Candida albicans.

A series of novel tetrazole derivatives was synthetized using N-alkylation or Michael-type addition reactions, and screened for their fungistatic potential against Candida albicans (the lack of endpoint = 100%). Among them, the selected compounds 2d, 4b, and 6a differing in substituents at the tetrazole ring were non-toxic to Galleria mellonella larvae in vivo and exerted slight toxicity against Caco-2 in vitro (CC50 at 256 µg/mL). An antagonistic effect of tetrazole derivatives 2d, 4b, and 6a respectively in combination with Fluconazole was shown using the checker board and colorimetric methods (fractional inhibitory concentration indexes FICIs >1). The most active 2d and 6a displayed an inverse relation between MICs in the presence of exogenous ergosterol, the effect was opposite to Itraconazole and Amphotericin B. The differences between 6a's and 2d's action mode were noted. Combining both flow cytometry and fluorescence image analyses respectively showed the complexity of planktonic and biofilm cell demise mode under the tetrazole derivatives tested. The following evidences for 6a's interaction with fungal membrane were noted: necrosis-like programmed cell death (97.03 ±â€¯0.88), DNA denaturation (no laddering), mitochondrial damage (XTT assay), reduced adhesion to human epithelium (>50% at 0.0313 µg/mL, p ≤ .05), irregular deposit of chitin, and attenuated morphogenesis in mature biofilm. The treatment with 6a reduced pathogenicity of C. albicans during infection in G. mellonella. Contrariwise, 2d enhancing fungal adhesion displayed mechanism targeted to the cell wall (due to the presence of 3-chloropropyl clubbed with aryltetrazole) in the presence of osmotic protector. Under 2d, the accidental cell death (88.60% ±â€¯4.81) was observed. In conclusion, all tetrazole derivatives were obtained in satisfactory yields (60-95%) using efficient, simple and not expensive methods. Fungistatic and slightly anticancer tetrazole derivatives with the novel action mode can circumvent an appearance of antifungal-resistant strains. These results indicate that they are worthy of further studies.

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.