Marcela Raimondi

Antifungal Activity of Eugenol Analogues. Influence of Different Substituents and Studies on Mechanism of Action

Twenty one phenylpropanoids (including eugenol and safrole) and synthetic analogues, thirteen of them new compounds, were evaluated for antifungal properties, first with non-targeted assays against a panel of human opportunistic pathogenic fungi. Some structure-activity relationships could be observed, mainly related to the influence of an allyl substituent at C-4, an OH group at C-1 and an OCH3 at C-2 or the presence of one or two NO2 groups in different positions of the benzene ring. All active compounds were tested in a second panel of clinical isolates of C. albicans and non-albicans Candida spp., Cryptococcus neoformans and dermatophytes. The eugenol derivative 4-allyl-2-methoxy-5-nitrophenol (2) was the most active structure against all strains tested, and therefore it was submitted to targeted assays. These studies showed that the antifungal activity of 2 was not reversed in the presence of an osmotic support such as sorbitol, suggesting that it does not act by inhibiting the fungal cell wall synthesis or assembly. On the other hand, the Ergosterol Assay showed that 2 did not bind to the main sterol of the fungal membrane up to 250 µg mL−1. In contrast, a 22% of fungal membrane damage was observed at concentrations = 1 × MIC and 71% at 4× MIC, when 2 was tested in the Cellular Leakage assay. The comparison of log P and MICs for all compounds revealed that the antifungal activity of the eugenol analogues would not to be related to lipophilicity.

New small-size peptides possessing antifungal activity

The synthesis, in vitro evaluation, and conformational study of a new series of small-size peptides acting as antifungal agents are reported. In a first step of our study we performed a conformational analysis using Molecular Mechanics calculations. The electronic study was carried out using Molecular electrostatic potentials (MEPs) obtained from RHF/6-31G calculations. On the basis of the theoretical predictions three small-size peptides, RQWKKWWQWRR-NH(2), RQIRRWWQWRR-NH(2), and RQIRRWWQW-NH(2) were synthesized and tested. These peptides displayed a significant antifungal activity against human pathogenic strains including Candida albicans and Cryptococcus neoformans. Our experimental and theoretical results allow the identification of a topographical template which can serve as a guide for the design of new compounds with antifungal properties for potential therapeutic applications against these pathogenic fungi.

Penetratin and derivatives acting as antifungal agents

The synthesis, in vitro evaluation, and conformational study of RQIKIWFQNRRMKWKK-NH(2) (penetratin) and related derivatives acting as antifungal agents are reported. Penetratin and some of its derivatives displayed antifungal activity against the human opportunistic pathogenic standardized ATCC strains Candida albicans and Cryptococcus neoformans as well as clinical isolates of C. neoformans. Among the compounds tested, penetratin along with the nonapeptide RKWRRKWKK-NH(2) and the tetrapeptide RQKK-NH(2) exhibited significant antifungal activities against the Cryptococcus species. A comprehensive conformational analysis on the peptides reported here using three different approaches, molecular mechanics, simulated annealing and molecular dynamics simulations, was carried out. The experimental and theoretical results allow us to identify a topographical template which may provide a guide for the design of new compounds with antifungal characteristics against C. neoformans.

Synthesis, antifungal and antitumor activity of novel (Z)-5-hetarylmethylidene-1,3-thiazol-4-ones and (z)-5-ethylidene-1,3-thiazol-4-ones.

New hetaryl- and alkylidenerhodanine derivatives 3a–d, 3e, and 4a–d were prepared from heterocyclic aldehydes 1a–d or acetaldehyde 1e. The treatment of several rhodanine derivatives 3a–d and 3e with piperidine or morpholine in THF under reflux, afforded (Z)-5-(hetarylmethylidene)-2-(piperidin-1-yl)thiazol-4(5H)-ones and 2-morpholinothiazol-4(5H)-ones 5a–d, 6a–d, and (Z)-5-ethylidene-2-morpholinothiazol-4(5H)-one (5e), respectively, in good yields. Structures of all compounds were determined by IR, 1D and 2D NMR and mass spectrometry. Several of these compounds were screened by the U.S. National Cancer Institute (NCI) to assess their antitumor activity against 60 different human tumor cell lines. Compound 3c showed high activity against HOP-92 (Non-Small Cell Lung Cancer), which was the most sensitive cell line, with GI50 = 0.62 μM and LC50 > 100 μM from the in vitro assays. In vitro antifungal activity of these compounds was also determined against 10 fungal strains. Compound 3e showed activity against all fungal strains tested, but showed high activity against Saccharomyces cerevisiae (MIC 3.9 μg/mL).

Synthesis of novel thiazole-based 8,9-dihydro-7H-pyrimido[4,5-b][1,4]diazepines as potential antitumor and antifungal agents.

A new series of novel thiazole-based 8,9-dihydro-7H-pyrimido[4,5-b][1,4]diazepines 6a-g and 7a-g were obtained with high regioselectivity from the reaction of triamino- or tetraaminopyrimidines 4 and 5 with α,β-unsaturated carbonyl compounds 3a-g based on 2,4-dichlorothiazol-5-carbaldehyde 1. Twelve of the synthesized compounds were selected and tested by US National Cancer Institute (NCI) for their antitumor activity against 60 different human tumor cell lines. Compounds 7d and 7g showed important GI50 ranges of 1.28-2.98 μM and 0.35-2.78 μM respectively under in vitro assays. In addition, 6a-g and 7a-g were tested for antifungal properties against the clinical important fungi Candida albicans and Cryptococcus neoformans. Although these compounds showed moderate activities against C. albicans, the 2-amino derivatives 7a-g and mainly 7a and 7b, showed high activity against standardized and clinical isolates of C. neoformans with MIC50 = 7.8-31.2 μg/mL, MIC80 = 15.6-31.2 μg/mL and MIC100 = 15.6-62.5 μg/mL. In addition, since both compounds were fungicide rather than fungistatic these thiazole-based 8,9-dihydro-7H-pyrimido[4,5-b][1,4]diazepines appear as good candidates for further development not only as antifungal but also as antitumor drugs.

Penetratin analogues acting as antifungal agents

The synthesis, in vitro evaluation, and conformational study of penetratin analogues acting as antifungal agents are reported. Different peptides structurally related with penetratin were evaluated. Analogues of penetratin rich in Arg, Lys and Trp amino acids were tested. In addition, HFRWRQIKIWFQNRRM[O]KWKK-NH(2), a synthetic 20 amino acid peptide was also evaluated. These penetratin analogues displayed antifungal activity against human pathogenic strains including Candida albicans and Cryptococcus neoformans. In contrast, Tat peptide, a well-known cell penetrating peptide, did not show a significant antifungal activity against fungus tested here. We also performed a conformational study by means experimental and theoretical approaches (CD spectroscopic measurements and MD simulations). The electronic structure analysis was carried out from Molecular Electrostatic Potentials (MEP) obtained by using RHF/6-31G ab initio calculations. Our experimental and theoretical results permitted us to identify a topographical template which may provide a guide for the design of new peptides with antifungal effects.

Structure–activity relationship study of nitrosopyrimidines acting as antifungal agents

The design, synthesis, in vitro evaluation, and conformational study of nitrosopyrimidine derivatives acting as antifungal agents are reported. Different compounds structurally related with 4,6-bis(alkyl or arylamino)-5-nitrosopyrimidines were evaluated. Some of these nitrosopyrimidines have displayed a significant antifungal activity against human pathogenic strains. In this paper, we report a new group of nitrosopyrimidines acting as antifungal agents. Among them, compounds 2a, 2b and 15, the latter obtained from a molecular modeling study, exhibited antifungal activity against Candida albicans, Candida tropicalis and Cryptococcus neoformans. We have performed a conformational and electronic analysis on these compounds by using quantum mechanics calculations in conjunction with Molecular Electrostatic Potentials (MEP) obtained from B3LYP/6-31G(d) calculations. Our experimental and theoretical results have led us to identify a topographical template which may provide a guide for the design of new nitrosopyrimidines with antifungal effects.

Fungicide Activity of 5-(4-Chlorobenzylidene)-(Z)-2-dimethylamino-1,3-thiazol-4-one against Cryptococcus Neoformans

The present work describes the synthesis and antifungal evaluation of new 5‐arylidene‐(Z)‐2‐dimethylamino‐1,3‐thiazol‐4‐ones 4a–f, obtained by the reaction of aromatic aldehydes 1 and rhodanine 2 followed by treatment with DMF. All compounds were tested against a panel of yeasts, hialohyphomycetes, and dermatophytes using the microbroth dilution method. Compounds 4a and 4c showed antifungal activity, with compound 4a being the most active one. Compound 4a demonstrated to be fungicidal rather than fungistatic and selective activity against Cryptococcus neoformans and dermatophytes. MIC100, MIC80, and MIC50 of 4a were determined against a panel of clinical isolates of C. neoformans showing ranges of MICs between 2 and 16 μg/mL.

Detection of synergistic combinations of Baccharis extracts with Terbinafine against Trichophyton rubrum with high throughput screening synergy assay (HTSS) followed by 3D graphs. Behavior of some of their components

Forty four extracts from nine Baccharis spp. from the Caulopterae section were tested in combination with terbinafine against Trichophyton rubrum with the HTSS assay at six different ratios with the aim of detecting those mixtures that produced a ≥50% statistically significant enhancement of growth inhibition. Since an enhanced effect of a combination respective of its components, does not necessarily indicate synergism, three-dimensional (3D) dose-response surfaces were constructed for each selected pair of extract/antifungal drug with the aid of CombiTool software. Ten extracts showed synergistic or additive combinations which constitutes a 22% hit rate of the extracts submitted to evaluation. Four flavonoids and three ent-clerodanes were detected in the active Baccharis extracts with HPLC/UV/ESI-MS methodology, all of which were tested in combination with terbinafine. Results showed that ent-clerodanes but not flavonoids showed synergistic or additive effects. Among them, bacchotricuneatin A followed by bacrispine showed synergistic effects while hawtriwaic acid showed additive effects.

Antifungal Activity of a Library of Cyclitols and Related Compounds

Affiliations: Roy and Diana Vagelos Laboratories, Penn/Merck Laboratory for High-Throughput Experimentation, Department of Chemistry, University of Pennsylvania, United States. Depto. de Quimica Organica, Facultad. de Quimica, Universidad de la Republica, UdelaR, Montevideo, Uruguay. Farmacognosia, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Rosario, Argentina. Departamento de Farmacia, Faculdade de Ciencias Farmaceuticas, Universidade de Sao Paulo, Sao Paulo, SP, Brazil.