Effects of Chirality on the Antifungal Potency of Methylated Succinimides Obtained by Aspergillus fumigatus Biotransformations. Comparison with Racemic Ones

Eighteen (3R) and (3R,4R)-N-phenyl-, N-phenylalkyl and N-arylsuccinimides were prepared with high enantioselectivity by biotransformation of maleimides with A. fumigatus. This environmentally friendly, clean and economical procedure was performed by the whole-cell fungal bioconversion methodology. Their corresponding eighteen racemic succinimides were prepared instead by synthetic methods. Both, the racemic and the chiral succinimides were tested simultaneously by the microbroth dilution method of CLSI against a panel of human opportunistic pathogenic fungi of clinical importance. Chiral succinimides showed higher antifungal activity than the corresponding racemic ones and the differences in activity were established by statistical methods. The bottlenecks for developing chiral drugs are how to obtain them through a low-cost procedure and with high enantiomeric excess. Results presented here accomplish both these objectives, opening an avenue for the development of asymmetric succinimides as new antifungal drugs for pharmaceutical use.


Introduction
Chiral succinimides, containing asymmetric carbons (in position 3-or 3,4-of the imido ring) have demonstrated to be core structural units with interesting biological activities. They have shown anxiolytic, antidepressant effects, and the ability to inhibit protein synthesis and human enzymes, such as leucocyte elastase, cathepsin G, proteinase 3 and glycosidase, among others. As a consequence, they have become good clinical drug candidates for several diseases [1][2][3][4][5][6].
Regarding antimicrobial activity, the chiral succinimides andrimid and moraimide B showed potent in vitro antibacterial activity against antibiotic-resistant human pathogens as methicillin-resistant Staphylococcus aureus [7]. In turn, hirsutellones inhibited the growth of Mycobacterium tuberculosis [4]. These findings have led to an increased interest in the clinical use of these asymmetric structures as a class of potential antimicrobial agents.
The bottleneck for developing chiral drugs for pharmaceutical use is to obtain them in a cheap and friendly procedure with high enantiomeric excesses (ees). Asymmetric synthesis by chemical procedures typically requires the use of expensive catalysts containing transition metal ions [8], which often prevents its commercial development. In contrast, the application of biocatalysts using whole cells in their native forms in aqueous/organic media has shown to be a highly selective, environmentally safe and cost effective method of producing enantiomeric compounds [9][10][11][12].
In the course of our project aimed at generating new chiral compounds through fungal biotransformations, we previously reported the preparation of (3R)-(+)-methyl-N-phenylsuccinimide (1a) and (3R,4R)-(+)-dimethyl-N-phenylsuccinimide (2a). These compounds were obtained in >99% ee by bioconversion of 3-methyl-N-phenyl-and 3,4-dimethyl-N-phenylmaleimide with Aspergillus fumigatus ATCC 26934, which proved to be the most effective catalyst among the fifteen strains tested [13]. In a subsequent paper, we reported the production of (3R)-(+)-methyl-N-phenylalkylsuccinimides 1b-e and (3R,4R)-(+)-dimethyl-N-phenylalkylsuccinimides 2b-e [alkyl chain = (CH 2 ) n (n = 1-4)] ( Figure 1) with excellent enantioselectivities (>99% ee) from 3-methyl-and 3,4-dimethyl-N-phenylalkylmaleimides, with the same A. fumigatus strain [14]. This prompted us to expand the knowledge on the ability of A. fumigatus to stereoselectively hydrogenate eight related 3-methyl-N-arylmaleimides to produce asymmetric succinimides 3-10 (Scheme 1). These compounds possess a substituted benzene ring with either electron-withdrawing or electron-donor groups on its p-or o-positions. It is known that the application of the same microorganism on different substrates does not always result in similar transformations or enantioselectivities of catalyzed reactions. Scheme 1. Biotransformation of 3-methyl-N-arylmaleimides 11-18 to (3R)-(+)-methyl-Narylsuccinimide 3-10 with Aspergillus fumigatus ATCC 26934. Chiral compounds 3-10, along with the previously obtained chiral 1a-e and 2a-e, were tested here for antifungal properties against a panel of human opportunistic pathogenic fungi using standardized procedures. In addition, racemic succinimides (±)-1a-e; -2a-e; -3-10 were synthesized and tested simultaneously against the same panel of fungi in order to compare their antifungal activities. It is well known that isomers can differ in their biological activities, thus the knowledge of the properties of both, racemic and enantiomeric forms of a compound is of great significance in the pharmaceutical field, leading to a better understanding of the concentration-effect relationships, adverse effects, activity or toxicity [15].
The structures of 3-10 were corroborated by MS and 1 H-and 13 C-NMR. To assist in the assignment of both the absolute configuration and the ee of each chiral succinimide, R-enantiomers of 3-10 were also synthesized from (R)-2-methylsuccinic acid 19 and the respective anilines 20-27 (Scheme 2) [17]. Synthetic (3R)-3-10 were all dextrorotatory, therefore indicating that the biotransformation products (+)-3-10 had the R-configuration.
The above results expand the knowledge on the ability of A. fumigatus ATCC 26934 to enantioselectively hydrogenate eight related prochiral 3-methyl-N-arylmaleimides 11-18 to produce chiral succinimides (3R)-3-10 with high enantioselectivity and in high yields. The fungus showed the same enantioface preference, irrespective of the substituent on the benzene ring which was the same results that has been observed with their analogues 1a-e and 2a-e [13,14]. To our knowledge, there are no previous reports on chiral synthesis of (3R)-methyl-N-arylsuccinimides 3, 5-10 by any of the chemical or enzymatic methods. Instead, (3R)-(+)-4 has been previously obtained by biotransformation with the plant Marchantia polymorpha [18].
Compounds (3R)-(3-10), along with the previously obtained chiral compounds 1a-e and 2a-e, were tested for antifungal properties against a panel of eleven human opportunistic pathogenic fungi comprising yeasts (Candida spp., Cryptococcus neoformans, Saccharomyces cerevisiae and the dermatophytes Microsporum gypseum, Trichophyton rubrum and Trichophyton mentagrophytes). The selection of these species was due to their high clinical incidence mainly among immunocompromised patients. Thus, species of the genus Candida are among the leading causes of nosocomial, blood stream infections worldwide and, although C. albicans was in the past the usual species associated with invasive infections, at present non-albicans Candida spp. (C. tropicalis, C. glabrata, C. parapsilopsis, C. krusei and C. lusitaneae) comprise more than half of human candidiasis isolates [19].
In turn, C. neoformans was selected because it remains an important life-threatening species for immunocompromised hosts, particularly for patients infected with HIV and therefore, new compounds that act against this fungus are highly welcome [20,21]. Regarding dermatophytes of the genus Microsporum and Trichophyton, they were selected because both genera are the cause of approximately 80-93% of chronic and recurrent human superficial infections which, although not life-threatening, diminish the quality of life of patients because they are difficult to eradicate [22].
To determine the Minimum Inhibitory Concentration (MIC), amounts of compounds from 250 μg·mL −1 were incorporated into growth media according to the CLSI standardized procedures [23,24]. Amphotericin B, terbinafine, and ketoconazole were used as positive controls. The Enhancement Ratio (ER), which is a measure of how many-fold the MIC was reduced in each enantiomeric compound compared to its corresponding racemic one, was calculated as the ratio between MIC (rac)/MIC (enantiomer). Results are shown in Table 2.    The comparison of the 198 MICs of racemic mixtures (18 compounds x 11 fungi) with the same number of MICs of enantiomers, allowed us to detect that 158 MICs of chiral forms (80%) were statistically significantly lower (p < 0.05) than the corresponding racemic form ones. Of them, 117 MICs were two orders of magnitude lower (ER = 2); 38 MICs were four-fold lower (ER = 4), and three MICs were eight-fold lower (ER = 8). C. albicans was shown to be the most sensitive species to chirality, showing enhancements in all pairs of compounds tested. The statistical analyses were performed by the non-parametric ANOVA, Kruskal-Wallis test followed by Dunn's multiple comparisons and Wilcoxon's signed rank test (p < 0.05).
This overall trend of a better antifungal behavior of (3R)-and (3R,4R)-forms, with respect to racemic ones against the whole panel of fungi, was analyzed within each group of compounds for each type of fungus (yeasts and dermatophytes) (   To corroborate the above findings from another point of view, we compared the percentages of fungal growth inhibition for the enantiomeric form and the racemic mixture of each compound, at a fixed concentration. Figure 3 shows the comparative antifungal inhibitory activities of chiral vs racemic forms of each of the eighteen structures tested at 125 µg·mL −1 as a measure of % of growth of C. albicans. It can be observed in the three groups, that each enantiomer showed a significantly lower percent of growth that its respective racemic form, confirming the previous analyses. These comparisons between groups were performed with Student's t test.
It is worth noting that the overall antifungal behavior of chiral forms was better than that of racemic ones, irrespective of the succinimides' N-substituents, since chiral succinimides with or without an alkyl chain between the N atom and the phenyl group, or with or without substituents in the 2' and 4'-positions of the benzene ring, showed better antifungal activity than their corresponding racemic forms.

General
Solvents and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were purified in the usual manner. 1 H-and 13 C-NMR spectra were recorded on a Bruker (Karlsruhe, Germany) 300 MHz NMR spectrometer. Compounds were dissolved in deuterated solvents from commercial sources (Sigma-Aldrich) with tetramethylsilane (TMS) as the internal standard. Chemical shifts (δ) are reported in ppm relative to the solvent peak (CHCl 3 in CDCl 3 at 7.26 ppm for protons and at 77.0 ppm for carbons). Signals are designated as follows: s, singlet; d, doublet; dd, doublets of doublets; t, triplet; m, multiplet; q, quartet. Melting points were obtained on or using an Electrothermal apparatus  -N-arylmaleimides 11-18. The synthesis of maleimides 11-18 was performed by mixing an equimolecular amount of substituted anilines 20-27 (5 mmol), dissolved in CHCl 3 (1 mL), and maleic anhydride 29 in CHCl 3 (5 mL) and stirring during 1 h. The solid which precipitated out of the reaction mixture (maleamic acid) was filtered off. The whole amount of maleamic acid was dissolved in acetic anhydride (5 mL) and sodium acetate (100 mg) was added. The mixture was heated for 2 h under reflux. The reaction was cooled, quenched with water and the aqueous solution was extracted with Et 2 O, dried over Na 2 SO 4 , filtered, and the solvent evaporated. The product was purified by silica gel column chromatography using a mixture of hexane and ethyl acetate (9:1) as eluent. Spectroscopic NMR data of compound 12 was identical to that previously reported [18].   (16  Rac-3-methyl-N-arylsuccinimides 3-10. Each maleimide 11-18 (2 mmol) was dissolved in CH 2 Cl 2 (2 mL) to which a catalytic amount of 5% Pd/C was added. Then, the mixture was exposed to a H 2 atmosphere at room temperature for 2 h. The crude mixture was filtered and the solvent was evaporated. The resulting product was purified by silica gel column chromatography using a mixture of hexane and ethyl acetate (9:1) as eluent.