Synthesis and Antifungal Activity of Perylene Bisimide Derivatives

In this study, we have synthesized several perylene bisimide derivatives and evaluated the antifungal activity against human pathogenic yeasts (Candida spp.) and filamentous fungi (Aspergillus spp., Fusarium spp., and Trichophyton spp.). Three compounds (2-4) from the nine compounds tested presented relevant activity against Candida albicans, C. parapsilosis and C. tropicalis with minimum inhibitory concentrations (MIC90) values of 3.1-25.0 μg/mL. These three compounds also showed activity against filamentous fungi, F. oxysporum, T. rubrum and T. mentagrophytes at a concentration of 50 μg/mL. The resultant data indicates that perylene bisimides (2-4) display wide spectrum and relevant antifungal activity. Compound 4 represents a new scaffold for the development of antifungal agents and warrant further structure activity relationship studies.


Introduction
The frequency of opportunistic fungal infection has increased drastically, mainly in patients who are immunocompromised due to organ transplant, leukaemia or HIV infection. 1Candida albicans is responsible for most infections caused by fungi; however, the incidence of non-albicans species that are resistant, less susceptible or potentially resistant to currently antifungal drugs, such as Candida parapsilosis, Candida krusei, Candida tropicalis, appears to be increasing. 2Filamentous fungi infections are less frequent than Candida species infection, but are associated with high mortality rates. 3espite the addition of new classes of antifungals, the number of currently available drugs for the treatment of fungal infections remains limited.Many of them are associated with a substantial toxicity and display very complex structures.Therefore, there is a continuing need to develop novel and simpler antifungal agents being more effective and less toxic.respectively, while compound 4 showed a MIC 90 value below 3,1 µg/mL, being this compound the most potent.These three compounds also showed activity against filamentous fungi, F. oxysporum, T. rubrum and T. mentagrophytes at a concentration of 50 µg/mL.However, we didn't find any activity of perylene derivatives against Aspergillus spp at the tested concentrations.The most active compounds, 3 and 4, are the result of condensation of perylene dianhydride and the polyamine spermine.Both compounds are ammonium derivatives, compound 3 results from acidification with HCl and compound 4 from acidification with trifluroacetic acid.From the observed results, it seems that the trifluoroacetate anion in the ammonium derivatives is important for enhanced biological activity.

Conclusions
The resultant data indicates that perylene bisimides (2-4) display wide spectrum and relevant antifungal activity.Compound 4 represents a new scaffold for the development of antifungal agents and warrant further structure activity relationship studies.

General Experimental Procedures
Reactions were monitored by thin-layer chromatography (TLC) using Merck silica gel 60 F-254 in 0.25 mm-thick plates.Purifications were performed by flash chromatography on Merck silica gel (230-400 mesh).Commercial reagent grade solvents and chemicals were used as received unless otherwise noted.Combined organic extracts were washed with brine, dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure.

Antifungal activity
The antifungal activity of perylene bisimide derivatives was evaluated following the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing (AFST-EUCAST) 11  Briefly, duplicate samples (100 μL) of five serial dilutions of compounds were dispensed into 96-well microtitration plates (Becton Dickinson, New Jersey, USA) at final concentrations between 50-3.125 μg/mL.As positive controls, we included itraconazole and amphotericin B (Sigma-Aldrich, Co, MO, USA) at final concentrations of 0.031-16 μg/mL.Moreover, terbinafine (Recalcine Laboratories, Santiago de Chile, Chile) was included at final concentrations of 0.0078-4 μg/mL .One hundred microlitres of the fungal inoculum of 1-5 x 10 5 CFU/mL and 0.2-2.5 x 105 CFU/mL for yeast and filamentous fungi, respectively, were added.For the AFST-EUCAST method, the minimum inhibitory concentration (MIC 90 ) was determined after 24 h of incubation at 35 °C and was defined as the lowest concentration that resulted in 90% of growth reduction.For the CLSI M38-A method, MIC were determined after 48 h of incubation at 35 °C (F.oxysporum and Aspergillus spp) and six days at 28°C (Trichophyton spp).In this assay, MIC 50 was defined as the lowest compound dilution that resulted in total inhibition of visible growth.Compounds were considered active when presented MIC values ≤ 50 μg/mL.The MIC values were expressed as geometric means (GM-MIC) of tests performed by duplicate in the three different assays against each fungi species.
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