Fluconazole-COX Inhibitor Hybrids: A Dual-Acting Class of Antifungal Azoles.

When used in combination with azole antifungal drugs, cyclooxygenase (COX) inhibitors such as ibuprofen improve antifungal efficacy. We report the conjugation of a chiral antifungal azole pharmacophore to COX inhibitors and the evaluation of activity of 24 hybrids. Hybrids derived from ibuprofen and flurbiprofen were considerably more potent than fluconazole and comparable to voriconazole against a panel of Candida species. The potencies of hybrids composed of an S-configured azole pharmacophore were higher than those with an R-configured pharmacophore. Tolerance, defined as the ability of a subpopulation of cells to grow in the presence of the drug, to the hybrids was lower than to fluconazole and voriconazole. The hybrids were active against a mutant lacking CYP51, the target of azole drugs, indicating that these agents act via a dual mode of action. This study established that azole-COX inhibitor hybrids are a novel class of potent antifungals with clinical potential.

Antibiotic-Based Conjugates Containing Antimicrobial HLopt2 Peptide: Design, Synthesis, Antimicrobial and Cytotoxic Activities.

Recent studies have shown that modified human lactoferrin 20-31 fragment, named HLopt2, possesses antibacterial and antifungal activity. Thus, we decided to synthesize and evaluate the biological activity of a series of conjugates based on this peptide and one of the antimicrobials with proven antibacterial (ciprofloxacin, CIP, and levofloxacin, LVX) or antifungal (fluconazole, FLC) activity. The drugs were covalently connected to the peptide via amide, methylenecarbonyl moieties, or a disulfide bridge. The antibacterial and antifungal activities were evaluated under Clinical and Laboratory Standard Institute (CLSI) recommended conditions or in a low-salt brain-heart infusion diluted medium (BHI1/100). Results showed that conjugation of the peptide with the drug increased its antimicrobial activity up to 4-fold. Under CLSI-recommended conditions, all the compounds revealed rather low efficiency. Among conjugates, the highest antibacterial activity was recorded for the CIP-Cys-S-S-HLopt2-NH2 (III). In BHI1/100, which had lower differentiating properties, all of the conjugates revealed low MIC and MMC (minimum inhibitory and microbicidal concentrations) values. The disulfide bridge used as a linker in the most active conjugate (III) upon incubation with S. aureus cells is reduced, releasing constituent peptide and CIP-Cys. In addition, we showed that its fluorescently labeled analogue and constituent peptide are able to be internalized into both C. albicans and S. aureus cells. Moreover, the invaluable advantage of the presented conjugates was their low toxicity to mammalian cells and very low hemolytic activity. The current research can form a solid basis for further in vivo studies and drug development.

Fluorescent Tracking of the Endoplasmic Reticulum in Live Pathogenic Fungal Cells.

In fungal cells, the endoplasmic reticulum (ER) harbors several of the enzymes involved in the biosynthesis of ergosterol, an essential membrane component, making this organelle the site of action of antifungal azole drugs, used as a first-line treatment for fungal infections. This highlights the need for specific fluorescent labeling of this organelle in cells of pathogenic fungi. Here we report on the development and evaluation of a collection of fluorescent ER trackers in a panel of Candida, considered the most frequently encountered pathogen in fungal infections. These trackers enabled imaging of the ER in live fungal cells. Organelle specificity was associated with the expression of the target enzyme of antifungal azoles that resides in the ER; specific ER labeling was not observed in mutant cells lacking this enzyme. Labeling of live Candida cells with a combination of a mitotracker and one of the novel fungal ER trackers revealed sites of contact between the ER and mitochondria. These fungal ER trackers therefore offer unique molecular tools for the study of the ER and its interactions with other organelles in live cells of pathogenic fungi.

Benzofuro[3,2-d]pyrimidines inspired from cercosporamide CaPkc1 inhibitor: Synthesis and evaluation of fluconazole susceptibility restoration.

In a context of growing resistance to classical antifungal therapy, the design of new drugs targeting alternative pathways is highly expected. Benzofuro[3,2-d]pyrimidine derivatives, derived from (-)-cercosporamide, were synthesized and evaluated as potential Candida albicans PKC inhibitors in the aim of restoring susceptibility to azole treatment. Co-administration assay of benzofuropyrimidinedione 23 and fluconazole highlighted a synergistic effect on inhibition of cell growth of a Candida albicans resistant strain.

Design, Synthesis, and Biological Activity of New Triazole and Nitro-Triazole Derivatives as Antifungal Agents.

In this study two series of fluconazole derivatives bearing nitrotriazole (series A) or piperazine ethanol (series B) side chain were designed and synthesized and then docked in the active site of lanosterol 14α-demethylase enzyme (1EA1) using the Autodock 4.2 program (The scripps research institute, La Jolla, CA, USA). The structures of synthesized compound were confirmed by various methods including elemental and spectral (NMR, CHN, and Mass) analyses. Then antifungal activities of the synthesized compound were tested against several natural and clinical strains of fungi using a broth microdilution assay against several standard and clinical fungi. Nitrotriazole derivatives showed excellent and desirable antifungal activity against most of the tested fungi. Among the synthesized compounds, 5a-d and 5g, possessing nitrotriazole moiety, showed maximum antifungal activity, in particular against several fluconazole-resistant fungi.

Potent Synergy between Spirocyclic Pyrrolidinoindolinones and Fluconazole against Candida albicans.

A spiroindolinone, (1S,3R,3aR,6aS)-1-benzyl-6'-chloro-5-(4-fluorophenyl)-7'-methylspiro[1,2,3a,6a-tetrahydropyrrolo[3,4-c]pyrrole-3,3'-1H-indole]-2',4,6-trione, was previously reported to enhance the antifungal effect of fluconazole against Candida albicans. A diastereomer of this compound was synthesized, along with various analogues. Many of the compounds were shown to enhance the antifungal effect of fluconazole against C. albicans, some with exquisite potency. One spirocyclic piperazine derivative, which we have named synazo-1, was found to enhance the effect of fluconazole with an EC50 value of 300 pM against a susceptible strain of C. albicans and going as low as 2 nM against some resistant strains. Synazo-1 exhibits true synergy with fluconazole, with an FIC index below 0.5 in the strains tested. Synazo-1 exhibited low toxicity in mammalian cells relative to the concentrations required for antifungal synergy.

Synthesis and biological evaluation of fluconazole analogs with triazole-modified scaffold as potent antifungal agents.

In order to find new azole antifungals, we have recently designed a series of triazole alcohols in which one of the 1,2,4-triazol-1-yl group in fluconazole structure has been replaced with 4-amino-5-aryl-3-mercapto-1,2,4-triazole motif. In this paper, we focused on the structural refinement of the primary lead, by removing the amino group from the structure to achieve 5-aryl-3-mercapto-1,2,4-triazole derivatives 10a-i and 11a-i. The in vitro antifungal susceptibility testing of title compounds demonstrated that most compounds had potent inhibitory activity against Candida species. Among them, 5-(2,4-dichlorophenyl)triazole analogs 10h and 11h with MIC values of <0.01 to 0.5µg/mL were 4-256 times more potent than fluconazole against Candida species.

Synthesis and biological evaluation of novel fluconazole analogues bearing 1,3,4-oxadiazole moiety as potent antifungal agents.

A novel series of fluconazole based mimics incorporating 1,3,4-oxadiazole moiety were designed and synthesized. All the title compounds were characterized by (1)H-NMR, (13)C-NMR, and Q-TOF-MS. Preliminary results revealed that most of analogues exhibited significant antifungal activity against seven pathogenic fungi. Compounds 9g and 9k (MIC80 ≤ 0.125 µg/mL, respectively) were found more potent than the positive controls itraconazole and fluconazole as broad-spectrum antifungal agents. The observed docking results showed that the 1,3,4-oxadiazole moiety enhanced the affinity binding to the cytochrome P450 14α-demethylase (CYP51).

Efeitos de compostos naturais, sintéticos e da fototerapia antifúngica sobre Candida tropicalis resistente ao fluconazol / Effects of natural compounds, synthetic and herbal medicine on Candida tropicalis antifungal fluconazole-resistant

A candidíase é uma infecção oportunista provocada por diversas espécies de fungos do gênero Candida, frequentemente encontrados integrando a microbiota, da superfície cutânea, no trato gastrointestinal e cavidades mucosas do ser humano desde o seu nascimento. A incidência das infecções fúngicas sistêmicas têm aumentado consideravelmente nas últimas décadas em função do grande número de pacientes com SIDA, a grande quantidade de transplantes e condições crônicas como o câncer, a terapia prolongada com imunossupressores e o uso de agentes corticosteroides. Além disso, a exposição prolongada aos antifúngicos azólicos promove a seleção de patógenos resistentes. No presente estudo avaliou-se a atividade antifúngica do complexo Rutênio-pirocatecol (RPC) frente a um isolado clinico de Candida tropicalis resistente ao fluconazol. A metodologia empregada para os testes de susceptibilidade foi de acordo com o documento M27-A3 do National Committee for Clinical Laboratory Standards (NCCLS, 2008). Esplenócitos de camundongos Balb/c foram obtidos de forma asséptica para avaliar a citotoxicidade do composto para células de mamíferos. O estresse oxidativo promovido pelo composto foi avaliado através da reação ao ácido tiobarbitúrico (TBARS) e ensaios de fluorescência com a sonda diclorodihidrofluoresceína diacetato (DCFH2DA). O Calcofluor White foi empregado para avaliar a integridade da parede celular. A análise ultraestrutural foi realizada através da microscopia eletrônica de varredura e transmissão. Os resultados encontrados para os testes de atividade antifúngica foram analisados através do teste estatístico ANOVA e pós-teste Dunnett. Os resultados encontrados para os testes de atividade antifúngica do RPC mostraram uma Concentração Inibitória de 50% (IC50) de 20,3 μM, enquanto em esplesnócitos a concentração efetiva de 50% foi de 325 μM mostrando um índice de seletividade igual a 16...

Candidiasis is an opportunistic infection caused by several species of fungi of the genus Candida, often found is the microbiota, on the skin, gastrointestinal tract and mucous cavities of the human beings birth. The incidence of systemic fungal infections have increased considerably in recent decades due to the large number of AIDS patients, the large number of transplants and chronic conditions such as cancer, prolonged therapy promotes the selection of resistant pathogen with immunosuppressant and corticosteroid agents. Also prolonged exposure azole antifungals to make them strong candidates for patients resistance. In the present study we evaluated the antifungal activity of Ruthenium-pyrocatechol complex (RPC) against a clinical isolate of Candida tropicalis resistant to fluconazole. The methodology for susceptibility testing was in accordance with the M27-A3 document of there National Committee for Clinical Laboratory Standards (NCCLS, 2008). Splenocytes from Balb/c mice were obtained aseptically to evaluate the cytotoxicity of the compound to mammalian cells. Oxidative stress caused by the compound was assessed by reaction to thiobarbituric acid (TBARS) and fluorescence assays with the probe diclorodihidrofluoresceína diacetate (DCFH2DA). The Calcofluor White was used to evaluate the integrity of the cell wall. The ultrastructural analysis was performed by scanning and transmission electron microscopy. The results for the antifungal activity tests were analyzed using ANOVA and pos-test Dunnett test statistic. The results for the tests of antifungal activity of the RPC showed a 50% inhibitory concentration (IC50) of 20.3 μM while in splenocytes the 50% effective concentration was 325 μM showing a selectivity index of 16...

Synthesis and antifungal activity of the novel triazole derivatives containing 1,2,3-triazole fragment.

A series of fluconazole analogues containing 1,2,3-triazole fragment have been designed and synthesized on the basis of the active site of the cytochrome P450 14α-demethylase (CYP51). Their structures were characterized by (1)H NMR, (13)C NMR and LC-MS. The MIC80 values indicate that the target compounds 1a-r showed higher activities against nearly all the fungi tested to some extent except against Aspergillus fumigatus. Compounds 1c, e, f, l and p showed 128 times higher activity (with the MIC80 value of 0.0039 mg/mL) than that of fluconazole against Candida albicans and also showed higher activity than that of the other positive controls.

Design, synthesis and antimicrobial evaluation of novel benzimidazole type of Fluconazole analogues and their synergistic effects with Chloromycin, Norfloxacin and Fluconazole.

A novel series of benzimidazole type of Fluconazole analogues were synthesized and characterized by (1)H NMR, (13)C NMR, IR, MS and HRMS spectra. All the new compounds were screened for their antimicrobial activities in vitro by two-fold serial dilution technique. The bioactive evaluation showed that 3,5-bis(trifluoromethyl)phenyl benzimidazoles gave comparable or even stronger antibacterial and antifungal efficiency in comparison with reference drugs Chloromycin, Norfloxacin and Fluconazole. The combination of 2,4-difluorobenzyl benzimidazole derivative 5m and its hydrochloride 7 respectively with antibacterial Chloromycin, Norfloxacin or antifungal Fluconazole showed better antimicrobial efficiency with less dosage and broader antimicrobial spectrum than the separated use of them alone. Notably, these combined systems were more sensitive to Fluconazole-insensitive Aspergillus flavus and methicillin-resistant MRSA.

Synthesis of novel fluconazoliums and their evaluation for antibacterial and antifungal activities.

A series of novel fluconazoliums were synthesized and their bioactive evaluation as potential antibacterial and antifungal agents were described. Some target compounds displayed good and broad-spectrum antimicrobial activities with low MIC values ranging from 0.25 to 64 µg/mL against all the tested strains, including three Gram-positive bacteria (Staphylococcus aureus, MRSA and Bacillus subtilis), three Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Bacillus proteus) as well as two fungi (Candida albicans and Aspergillus fumigatus). Among all tested title compounds, the octyl, dichlorobenzyl, naphthyl and naphthalimino derivatives gave comparable or even better antibacterial and antifungal efficiency in comparison with the reference drugs Fluconazole, Chloromycin and Norfloxacin.

Design, synthesis and antifungal evaluation of 1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-1H-1,2,4-triazol-5(4H)-one.

Based on the structure of the active site of cytochrome P450 14α-demethylase (CYP51) and the conclusions of the structure-activity relationships of azole antifungals, a series of 1-(2-(2,4-difluoro-phenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-1H-1,2,4-triazol-5(4H)-one of fluconazole analogs was synthesized. All compounds were characterized by IR, HRMS, (1)HNMR and (13)C NMR spectroscopic analysis. Results of preliminary antifungal in vitro test using eight human pathogenic species showed that some compounds displayed comparable or even better antifungal activities than reference drug fluconazole and that compound 3i exhibited significant activity against Candida albicans being worthy of further optimization.

Fluconazole analogues containing 2H-1,4-benzothiazin-3(4H)-one or 2H-1,4-benzoxazin-3(4H)-one moieties, a novel class of anti-Candida agents.

As a part of our program to develop new antifungal agents, a series of fluconazole analogues was designed and synthesized wherein one of the triazole moieties in fluconazole was replaced with 2H-1,4-benzothiazin-3(4H)-one or 2H-1,4-benzoxazin-3(4H)-one moiety. The new chemical entities thus synthesized were screened against various fungi and it was observed that the compounds 4a and 4i are potent inhibitors of Candida strains. The structure-activity relationship for these compounds is discussed.

Design, synthesis, and biological evaluation of novel 1-(1H-1,2,4-triazole-1-yl)-2-(2,4-difluorophenyl)-3-substituted benzylamino-2-propanols.

Based on the results of computational docking to the active site of the cytochrome P450 14alpha-demethylase (CYP51), a series of 1-(1H-1,2,4-triazole-1-yl)-2-(2,4-difluorophenyl)-3-substituted benzylamino-2-propanols as analogs of fluconazole were designed, synthesized, and evaluated as antifungal agents. Results of preliminary antifungal tests against eight human pathogenic fungi in vitro showed that all the title compounds exhibited excellent activities with broad spectrum.

Synthesis and antifungal activity of 1,2,3-triazole containing fluconazole analogues.

Fluconazole based novel mimics containing 1,2,3-triazole were designed and synthesized as antifungal agents. Their antifungal activities were evaluated in vitro by measuring the minimal inhibitory concentrations (MICs). Compounds 12, 15, and 16 were found to be more potent against Candida fungal pathogens than control drugs fluconazole and amphotericin B. The studies presented here provide structural modification of fluconazole to give 1,2,3-trazole containing molecules. Furthermore, these molecules were evaluated in vivo against Candida albicans intravenous challenge in Swiss mice and antiproliferative activities were tested against human hepatocellular carcinoma Hep3B and human epithelial carcinoma A431. It was found that compound 12 resulted in 97.4% reduction in fungal load in mice and did not show any profound proliferative effect at lower dose (0.001 mg/ml).

Carbon analogs of antifungal dioxane-triazole derivatives: synthesis and in vitro activities.

A new series of triazole compounds possessing a carbon atom in place of a sulfur atom were efficiently synthesized and their in vitro antifungal activities were investigated. The carbon analogs showed excellent in vitro activity against Candida, Cryptococcus, and Aspergillus species. The MICs of compound 1c against C. albicans ATCC24433, C. neoformans TIMM1855, and A. fumigatus ATCC26430 were 0.016, 0.016, and 0.125 microg/mL, respectively (MICs of fluconazole: 0.5, >4, and >4 microg/mL; MICs of itraconazole: 0.125, 0.25, and 0.25 microg/mL).

Synthesis and antifungal activities of new fluconazole analogues with azaheterocycle moiety.

A series of fluconazole analogues 5-20 incorporating azaindole and indole moieties were prepared using oxirane intermediates synthesized under microwave irradiation. All of the compounds were evaluated in vitro against two clinically important fungi, Candida albicans and Aspergillus fumigatus. Four derivatives 6, 13, 14 and 18 exerted high antifungal activity against C. albicans with MIC(80) values 3- to 28-fold lower than that of fluconazole.

Carboxylic acid and phosphate ester derivatives of fluconazole: synthesis and antifungal activities.

Two classes of fluconazole derivatives, (a) carboxylic acid esters and (b) fatty alcohol and carbohydrate phosphate esters, were synthesized and evaluated in vitro against Cryptococcus neoformans, Candida albicans, and Aspergillus niger. All carboxylic acid ester derivatives of fluconazole (1a-l), such as O-2-bromooctanoylfluconazole (1g, MIC=111 microg/mL) and O-11-bromoundecanoylfluconazole (1j, MIC=198 microg/mL), exhibited higher antifungal activity than fluconazole (MIC > or = 4444 microg/mL) against C. albicans ATCC 14053 in SDB medium. Several fatty alcohol phosphate triester derivatives of fluconazole, such as 2a, 2b, 2f, 2g, and 2h, exhibited enhanced antifungal activities against C. albicans and/or A. niger compared to fluconazole in SDB medium. For example, 2-cyanoethyl-omega-undecylenyl fluconazole phosphate (2b) with MIC value of 122 microg/mL had at least 36 times greater antifungal activity than fluconazole against C. albicans in SDB medium. Methyl-undecanyl fluconazole phosphate (2f) with a MIC value of 190 microg/mL was at least 3-fold more potent than fluconazole against A. niger ATCC 16404. All compounds had higher estimated lipophilicity and dermal permeability than those for fluconazole. These results demonstrate the potential of these antifungal agents for further development as sustained-release topical antifungal chemotherapeutic agents.

Synthesis of 18F-labeled fluconazole and positron emission tomography studies in rabbits.

[4-18F] 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-l-yl)-2-propanol [( 4-18F] fluconazole) was synthesized from its amino precursor. Fieldel-Crafts acylation of 3-fluoroacetanilide with chloroacetyl chloride produced 2'-fluoro-4'-acteamido-2-(1H-1,2,4-triazole-1-yl) acetophenone in 12% yield. Sequential reaction with (1) dimethylsulphoxonium methylide and (2) 1,2,4-triazole followed by in situ hydrolysis resulted in 2-(2-fluoro-4-aminophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)-2-propan ol in 19% yield. A modified Schiemann reaction on this product resulted in [4-18F]fluconazole with a radiochemical yield of 1.0-2.0% (EOS) within 2 h. [4-18F]Fluconazole was used to measure the pharmacokinetics of fluconazole in rats by measurement of radioactivity in excised tissues and in rabbits by PET. In both species, there was rapid equilibration of [4-18F]fluconazole to a relatively uniform distribution of radioactivity in most organs.