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.

The Antifungal Action Mode of N-Phenacyldibromobenzimidazoles.

Our study aimed to characterise the action mode of N-phenacyldibromobenzimidazoles against C. albicans and C. neoformans. Firstly, we selected the non-cytotoxic most active benzimidazoles based on the structure-activity relationships showing that the group of 5,6-dibromobenzimidazole derivatives are less active against C. albicans vs. 4,6-dibromobenzimidazole analogues (5e-f and 5h). The substitution of chlorine atoms to the benzene ring of the N-phenacyl substituent extended the anti-C. albicans action (5e with 2,4-Cl2 or 5f with 3,4-Cl2). The excellent results for N-phenacyldibromobenzimidazole 5h against the C. albicans reference and clinical isolate showed IC50 = 8 µg/mL and %I = 100 ± 3, respectively. Compound 5h was fungicidal against the C. neoformans isolate. Compound 5h at 160-4 µg/mL caused irreversible damage of the fungal cell membrane and accidental cell death (ACD). We reported on chitinolytic activity of 5h, in accordance with the patterns observed for the following substrates: 4-nitrophenyl-N-acetyl-ß-d-glucosaminide and 4-nitrophenyl-ß-d-N,N',N″-triacetylchitothiose. Derivative 5h at 16 µg/mL: (1) it affected cell wall by inducing ß-d-glucanase, (2) it caused morphological distortions and (3) osmotic instability in the C. albicans biofilm-treated. Compound 5h exerted Candida-dependent inhibition of virulence factors.

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.

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.

Domino Reaction of Pyrrolidinium Ylides: Michael Addition/[1,2]-Stevens Rearrangement.

A novel domino reaction featuring a Michael addition/[1,2]-Stevens rearrangement reaction of pyrrolidinium ylides with electrophilic alkenes is described. Ylides generated under mild conditions from 2-aryl- N-cyanomethyl- N-methylpyrrolidinium salts entered the Michael addition, followed by a [1,3]-hydrogen shift and finally the [1,2]-Stevens rearrangement to give 3-aryl-2-cyano-2-(2-EWG-ethyl)-1-methylpiperidines.

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).

Human dihydrofolate reductase and thymidylate synthase form a complex in vitro and co-localize in normal and cancer cells.

Enzymes involved in thymidylate biosynthesis, thymidylate synthase (TS), and dihydrofolate reductase (DHFR) are well-known targets in cancer chemotherapy. In this study, we demonstrated for the first time, that human TS and DHFR form a strong complex in vitro and co-localize in human normal and colon cancer cell cytoplasm and nucleus. Treatment of cancer cells with methotrexate or 5-fluorouracil did not affect the distribution of either enzyme within the cells. However, 5-FU, but not MTX, lowered the presence of DHFR-TS complex in the nucleus by 2.5-fold. The results may suggest the sequestering of TS by FdUMP in the cytoplasm and thereby affecting the translocation of DHFR-TS complex to the nucleus. Providing a strong likelihood of DHFR-TS complex formation in vivo, the latter complex is a potential new drug target in cancer therapy. In this paper, known 3D structures of human TS and human DHFR, and some protozoan bifunctional DHFR-TS structures as templates, are used to build an in silico model of human DHFR-TS complex structure, consisting of one TS dimer and two DHFR monomers. This complex structure may serve as an initial 3D drug target model for prospective inhibitors targeting interfaces between the DHFR and TS enzymes.