Effects of chirality on the antifungal potency of methylated succinimides obtained by Aspergillus fumigatus biotransformations. Comparison with racemic ones

Article abstract of DOI:10.3390/molecules18055669

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 w

Full text of DOI:10.3390/molecules18055669

Molecules 2013, 18, 5669-5683; doi:10.3390/molecules18055669
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Effects of Chirality on the Antifungal Potency of Methylated
Succinimides Obtained by Aspergillus fumigatus
Biotransformations. Comparison with Racemic Ones
Maximiliano Sortino, Agustina Postigo and Susana Zacchino *
Pharmacognosy Area, Faculty of Biochemical and Pharmaceutical Sciences,
National University of Rosario, Suipacha 531, 2000-Rosario, Argentina;
E-Mails: msortino@fbioyf.unr.edu.ar (M.S.); aguspostigo@hotmail.com (A.P.)
* Author to whom correspondence should be addressed; E-Mail: szaabgil@citynet.net.ar;
Tel./Fax: +54-341-437-5315.
Received: 24 April 2013; in revised form: 13 May 2013 / Accepted: 13 May 2013 /
Published: 15 May 2013
Abstract: 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.
Keywords: biotransformation; Aspergillus fumigatus; enantioselective reduction; enhanced
antifungal activity; chiral succinimides; methylated succinimides

Molecules 2013, 18
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1. 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 [1–6].
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 [9–12].
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 = (CH2)n (n = 1–4)] (Figure 1)
with excellent enantioselectivities (>99% ee) from 3-methyl- and 3,4-dimethyl-N-phenyl-
alkylmaleimides, with the same A. fumigatus strain [14].
Figure 1. Structures of (3R)-(+)-methyl-N-phenylsuccinimide 1a, (3R,4R)-(+)-dimethyl-N-
phenylsuccinimide 2a, (3R)-(+)-methyl-N-phenylalkylsuccinimides 1b–e and (3R,4R)-(+)-
dimethyl-N-phenylalkylsuccinimides 2b–e [alkyl chain = (CH2)n (n = 1–4)].
O
O
O
O
H3C
H3C
H3C
H3C
H3C
H3C
N
N
N
(CH2)n
N
(CH2)n
O
O
O
O
1a
2a
1b-e n=1-4
2b-e n=1-4
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.

Molecules 2013, 18
5671
Scheme 1. Biotransformation of 3-methyl-N-arylmaleimides 11–18 to (3R)-(+)-methyl-N-
arylsuccinimide 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].
2. Results and Discussion
For the sake of simplicity, the thirty six compounds: racemic (rac)- and chiral 1a–e, 2a–e, 3–10,
were grouped into three classes: (A) (rac)- and (3R)-methyl-N-phenyl- and N-phenylalkyl-succinimides
1a–1e; (B) (rac)- and (3R,4R)-dimethyl-N-phenyl- and N-phenylalkyl- succinimides 2a–2e; and (C)
(rac)- and (3R)-methyl-N-arylsuccinimides 3–10.
As stated above, (3R)- and (3R,4R)-succinimides of groups (A) and (B) (compounds 1a–e and 2a–e)
were previously obtained with >99% ee by fungal biotransformation of the respective maleimides with
A. fumigatus ATCC 26934 [13,14,16]. In turn, racemic ones were obtained by catalytic hydrogenation
of the respective maleimides, as previously reported [14].
Chiral compounds of group C [(3R)-3–10)], on the other hand, were obtained by submitting the
respective maleimides 11–18 to biotransformation with the same strain of A. fumigatus used for
obtaining chiral succinimides of groups (A) and (B) (Scheme 1). (Rac)-3–10 were obtained by
catalytic hydrogenation with H2 (Pd/C). In turn, maleimides 11–18 were obtained following reported
procedures [14].
The structures of 3–10 were corroborated by MS and 1H- and 13C-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.
Chiral GC of rac- and (3R)-(3–10) allowed us to determine that (3R)-enantiomers 3–10 eluted
before the S-ones. These data were used to determine the ee of each biotransformation mixture and to
confirm its absolute configuration. Results showed that 11–18 were converted into (3R)-(+)-3–10 with
ee ≥ 98%. Table 1 shows % conversion; % ee and absolute configurations of 3–10 [data of succinimide
R-1a, obtained from 3-methyl-N-phenylmaleimide (28) [13], were also included].

Molecules 2013, 18
Scheme 2. Synthesis of (3R)-methyl-N-aryl-succinimides 3–10 from (2R)-methylsuccinic
5672
acid 19 and respective anilines 20–27.
Table 1. Biotransformation of 3-methyl-N-arylmaleimides 11–18 to (3R)-(+)-methyl-N-
arylsuccinimides 3–10.
Substrate
R1
CH3
OCH3
NO2
F
R2
H
Product
(R)-(+)-3
(R)-(+)-4
(R)-(+)-5
(R)-(+)-6
(R)-(+)-7
(R)-(+)-8
(R)-(+)-9
(R)-(+)-10
(R)-(+)-1a
% ee
97
% Conv.
11
12
93
97
95
99
91
81
65
55
99
H
>99
98
13
H
14
H
98
15
Cl
H
97
16
Br
H
98
17
H
CH3
F
98
18
F
99
28 *
H
H
>99
* Results previously reported [13]; % ee: % enantiomeric excess (calculated by chiral GC); % Conv:
Conversion percentages (determined by GC analysis by using the TIC (total ion current) with the following
equation: % conversion: product TIC/(product TIC + substrate TIC) × 100.
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

Molecules 2013, 18
5673
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.
Table 2. Antifungal activity (MICs in μg·mL−1) of 3-methyl-N-phenyl- or N-
phenylalkylsuccinimides 1a–e (A); 3,4-dimethyl-N-phenyl- or phenylalkylsuccinimides
2a–e (B); and 3-methyl-N-arylsuccinimides 3–10 (C), in their racemic and enantiomeric
forms against a panel of yeasts and dermatophytes.
O
C
A
B
O
O
H3C
H3C
H3C
H3C
N
N
N
(CH2)n
(CH2)n
R
O
O
O
Type
n
Conf.
Ca 1 Ct 2 Ck 3 Cg 4 Cp 5 Cl 6 Sc 7 Cn 8 Mg 9 Tr 10 Tm 11
R-
62.5 62.5 62.5 62.5 62.5 62.5 62.5 62.5 62.5 31.3 62.5
1a
A
0
-
-
-
-
-
rac-
ER
R-
125
2
125 125 125 125 125 125 125 62.5 125 62.5
2
2
2
2
2
2
2
-
4
-
15.6 31.3 62.5 62.5 62.5 31.3 62.5 15.6 125 62.5 62.5
62.5 125 125 125 125 125 125 62.5 250 125 250
1b
1c
1d
1e
A
A
A
A
1
2
3
4
rac-
ER
R-
4
4
2
2
2
4
2
4
2
2
4
31.3 62.5 125 62.5 31.3 62.5 62.5 62.5 125 62.5 31.3
1
rac-
ER
R-
125
4
125 125 125 125 125 125 125 125 62.5 62.5
2
-
2
4
2
2
2
-
-
2
62.5 125 125 62.5 125 125 125 62.5 62.5 125 62.5
rac-
ER
R-
125
2
125 125 125 125 125 125 125 125 250 250
-
-
-
-
-
-
2
2
2
4
31.3 31.3 62.5 125 62.5 125 62.5 31.3 125 62.5 31.3
62.5 125 125 125 125 125 125 125 250 125 125
rac-
ER
2
4
2
-
2
-
2
4
2
2
4

Molecules 2013, 18
5674
Table 2. Cont.
Type
n
Conf.
3R,4R- 31.3 31.3 62.5 62.5 31.3 62.5 62.5 62.5 125 125 62.5
Ca 1 Ct 2 Ck 3 Cg 4 Cp 5 Cl 6 Sc 7 Cn 8 Mg 9 Tr 10 Tm 11
2a
2b
2c
2d
2e
B
0
-
rac-
ER
125 62.5 62.5 125 62.5 62.5 125 125 125 125 125
4
2
-
2
2
-
2
2
-
-
2
3R,4R- 31.3 62.5 62.5 125 62.5 62.5 125 62.5 62.5 62.5 31.3
rac-
ER
B
B
B
B
C
C
C
C
C
C
C
C
1
2
3
4
0
-
125
4
125 125 125 125 125 125 125 62.5 62.5 125
2
2
-
2
2
-
2
-
-
4
3R,4R- 15.6 125 125 62.5 62.5 62.5 125 62.5 125 125 62.5
rac-
ER
2
-
125
8
125 250 125 250 125 125 125 250 250 62.5
-
2
2
4
2
-
2
2
2
-
3R,4R- 31.3 62.5 62.5 62.5 62.5 62.5 62.5 62.5 62.5 31.3 31.3
rac-
ER
-
-
62.5 125 125 125 125 250 125 125 125 62.5 125
2
2
2
2
2
4
2
2
2
2
4
3R,4R- 62.5 62.5 62.5 62.5 62.5 62.5 62.5 31.3 62.5 62.5 62.5
rac-
ER
R-
125
2
125 62.5 125 125 125 125 125 125 125 125
2
-
2
2
2
2
4
2
2
2
62.5 62.5 62.5 62.5 62.5 62.5 62.5 62.5 125 125 125
3
4
4'-CH3
rac-
ER
R-
250
4
250 125 125 250 250 125 62.5 250 250 250
4
2
2
4
4
2
-
2
2
2
125 62.5 62.5 62.5 62.5 62.5 125 62.5 125 125 125
0 4'-OMe
rac-
ER
R-
250
2
125 125 125 125 125 125 62.5 250 250 250
2
2
2
2
2
-
-
2
2
2
62.5 125 62.5 125 62.5 125 125 62.5 125 125 125
5
0
0
0
0
0
0
4'-NO2
4'-F
rac-
ER
R-
250
4
125 125 125 125 125 125 125 250 250 250
-
2
-
2
-
-
2
2
2
2
62.5 62.5 31.3 62.5 31.3 62.5 62.5 62.5 62.5 62.5 62.5
6
rac-
ER
R-
250
4
250 125 125 250 250 125 62.5 125 125 125
4
4
2
8
4
2
-
2
2
2
125 62.5 125 62.5 125 62.5 62.5 31.3 125 125 125
7
4'-Cl
rac-
ER
R-
250
2
250 125 250 250 250 125 125 250 250 250
4
-
4
2
4
2
4
2
2
2
125 62.5 62.5 62.5 62.5 62.5 62.5 62.5 62.5 125 125
8
4'-Br
rac-
ER
R-
250
2
250 125 125 250 250 125 125 125 250 125
4
2
2
4
4
2
2
2
2
-
125
250
2
125 62.5 125 125 125 125 62.5 125 125 125
125 125 125 250 125 125 125 250 250 250
9
2'-CH3
2',4'-F2
rac-
ER
R-
-
2
-
2
-
-
2
2
2
2
62.5 62.5 31.3 62.5 31.3 125 62.5 62.5 62.5 62.5 125
10
rac-
ER
250
4
250 125 125 250 250 125 125 125 250 250
4
4
2
8
2
2
2
2
4
2
AmphotericinB
Ketoconazole
Terbinafine
0.97 0.48 0.49 0.49 0.98 0.98 0.48 0.24 0.12 0.06 0.06
0.48 0.12 62.5 1.95 0.98 0.98 0.48 0.24 0.06 0.03 0.03
-
-
-
-
-
-
-
-
0.03 0.01 0.03
1: Candida albicans ATCC 10231, 2: C. tropicalis CCC 131-2000: C. krusei CCC 117-2000, 4: C. glabrata
CCC 115-2000, 5: C. parapsilosis CCC 124-2000, 6: C. lusitaniae CCC 134-2000, 7: Saccharomyces cerevisiae
ATCC 9763, 8: Cryptococcus neoformans ATCC 32264, 9: Microsporum gypseum CCC 115-2000, 10:
Trichophyton rubrum CCC 113 2000, 11: T. mentagrophytes ATCC 9972. CCC = Colección de Cultivos del
CEREMIC (Centro de Referencia en Micología). ATCC: American Type Culture Collection. Conf:
configuration; ER = MIC (rac)/MIC (R form).

Molecules 2013, 18
5675
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) (Figure 2). Each graph shows the percentage of MICs
of each value, calculated as follows: [number of MICs of each value (250, 125, 62.5, 31.3 and
15.6 µg·mL−1) × 100/total number of MICs] displayed by enantiomeric forms and by racemic mixtures
within each group of compounds. The associations between MIC values and enantiomeric or racemic
forms within each group and type of fungi for the different groups were established with the Score’s
test (p < 0.05) [25].
Figure 2. Percentages of the different MIC values (number of MICs at each concentration/total
number of MICs) × 100, for the enantiomeric (3R) or (3R,4R) and racemic forms of
succinimides of either group (A), (B), or (C) acting against yeasts and dermatophytes.
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
Group A
Group B
Group C
15.6
31.3
62.5
125
250
15.6
31.3
62.5
125
250
15.6
31.3
62.5
125
250
MIC value
MIC value
MIC value
Enantiomer
Racemic
Enantiomer
Racemic
Enantiomer
Racemic
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
Group A
Group B
Group C
15.6
31.3
62.5
125
250
15.6
31.3
62.5
125
250
15.6
31.3
62.5
125
250
MIC value
MIC value
MIC value
Enantiomer
Racemic
Enantiomer
Racemic
Enantiomer Racemic
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.

Molecules 2013, 18
Figure 3. Comparative values of the inhibitory activities of antifungal succinimides in their
5676
enantiomeric and racemic forms (at 125 μg/mL) expressed as percentage of growth against
Candida albicans ATCC 10231. * p < 0.05.
a)
b)
100
100
80
60
40
20
0
Group B
enantioner
racemic
Group A
enantioner
racemic
80
60
40
20
0
*
*
*
*
*
*
*
*
*
*
2a
2b
2c
2d
2e
1a
1b
1c
1d
1e
100
c)
Group C
enantioner
racemic
*
80
60
40
20
0
*
*
*
*
*
*
*
*
1a
3
4
5
6
7
8
9
10
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.
3. Experimental
3.1. General
Solvents and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were purified
1
13
in the usual manner. H- and 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 (CHCl3 in CDCl3 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
(Southend-on-Sea, Essex, UK) and are uncorrected. Optical rotations were measured with a Jasco
DIB-1000 (Easton, USA) at room temperature. GC analyses were performed on a CG-MS Turbo Mass
Perkin Elmer (Waltman, MA, USA), equipped with a fused silica gel column (SE-30 25 m × 0.22 mm
I.D.) with He as a carrier gas in a column PE1 30 m × 0.25 mm (I.D.), film 0.1 μm, ionization energy
70 eV with a temperature program of 70–200 °C at 10 °C min−1; total time 30 min. Chiral gas
chromatograms were obtained with a CG-MS QP2010-Plus (Shimadzu, Kioto, Japan), with He as a

Molecules 2013, 18
5677
carrier gas on a Beta Dex-325 column (30 m × 0.25 mm I.D.), ionization energy 70 eV, with a
temperature program of 60–200 °C at 2 °C·min−1; total time 80 min.
3.2. Synthesis
3-Methyl-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 CHCl3 (1 mL), and maleic
anhydride 29 in CHCl3 (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
Et2O, dried over Na2SO4, 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]. Compounds 11, 13–18 have
been previously reported by Chemical Abstract Service (CAS) (11: CAS 3120-12-5, 13: CAS 10490-21-8,
14: CAS 883033-95-2, 15: CAS 59648-09-8, 16: CAS 134939-24-5, 17: CAS 131406-19-4, 18: CAS
124704-71-8) but, to the best of our knowledge, complete NMR spectral data were not available in the
literature, so below, the complete spectroscopic data of these compounds are described.
1
3-Methyl-N-(4'-methylphenyl)maleimide (11). White solid. Mp: 92–95 °C. Yield: 73%. H-NMR
(CDCl3): 2.17 (3H, d, J = 1.8 Hz, CH3); 2.37 (3H, s, CH3); 6.46 (1H, q, J = 1.8 Hz, H-3); 7.13–7.33
13
(4H, m, HAr) ppm. C-NMR (CDCl3): 11.2; 21.1; 125.9; 127.4; 128.9; 129.7; 137.8; 145.7; 169.8;
170.8 ppm. MS: m/z 201 (M+, 100%), 202 (M++1, 13%).
1
3-Methyl-N-(4'-methoxyphenyl)maleimide (12). White solid. Mp: 99–101 °C. Yield: 62%. H-NMR
(CDCl3): δ 2.17 (3H, d, J = 1.8 Hz, CH3); 3.83 (3H, s, OCH3); 6.46 (1H, q, J = 1.8 Hz, H-3); 6.97 (2H, d,
J = 8.9 Hz, H-3',5'); 7.23 (2H, d, J = 8.9 Hz, H-2';6') ppm. 13C-NMR (CDCl3): 11.2; 55.5; 114.4;
124.3; 127.4; 127.5; 145.7; 159.0; 169.9; 170.9 ppm. MS: m/z 217 (M+, 100%), 218 (M++1, 13%).
1
3-Methyl-N-(4'-nitrophenyl)maleimide (13). White solid. Mp: 126–128 °C. Yield: 51%. H-NMR
(CDCl3): 2.11 (3H, d, J = 1.8 Hz, CH3); 6.48 (1H, q, J = 1.8 Hz, H-3); 7.62 (2H, d, J = 9.3 Hz, H-2',6');
8.22 (2H, d, J = 9.3 Hz, H-3',5') ppm. 13C-NMR (CDCl3): 11.2; 124.4; 125.2; 127.9; 137.5; 146.0;
146.4; 168.5; 169.7 ppm. MS: m/z 232 (M+, 100%), 233 (M++1, 13%).
1
3-Methyl-N-(4'-fluorophenyl)maleimide (14). White solid. Mp: 122–125 °C. Yield: 72%. H-NMR
(CDCl3): 2.18 (3H, d, J = 1.8 Hz; CH3); 6.48 (1H, q, J = 1.8 Hz, H-3); 7.09–7.19 (2H, m, H-2';6');
7.28–7.36 (2H, m, H-3';5') ppm. 13C-NMR (CDCl3): 11.0; 115.8 (d, J = 22.7 Hz); 127.1 (d, J = 8.9 Hz);
136.6; 139.8; 145.8; 161.4 (d, J = 248.5 Hz); 168.6; 169.5 ppm. MS: m/z 205 (M+, 100%),
206 (M++1, 12%).
1
3-Methyl-N-(4'-chlorophenyl)maleimide (15). White solid. Mp: 127–130 °C. Yield: 46%. H-NMR
(CDCl3): 2.15 (3H, d, J = 1.3 Hz; CH3); 7.01 (1H, d, J = 1.6 Hz, H-3); 7.26 (2H, d, J = 8.8 Hz, H-3',5');
7.44 (2H, d, J = 8.7 Hz, H-2',6') ppm. 13C-NMR (CDCl3): 11.0; 126.2; 129.1; 136.4; 139.6; 142.3;
150.0; 168.4; 180.7 ppm. MS: m/z 221 (M+, 100%); 223 (M++2, 32%); 222 (M++1, 12%).

Molecules 2013, 18
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1
3-Methyl-N-(4'-bromophenyl)maleimide (16). White solid. Mp: 142–144 °C. Yield: 56%. H-NMR
(CDCl3): 2.17 (3H, d, J = 1.8 Hz, CH3); 6.48 (1H, q, J = 1.8Hz, H-3); 7.23–7.29 (2H, d, J = 8.8 Hz,
13
3',5'-H); 7.53–7.61 (2H, d, J = 8.8 Hz, 2'-6'-H) ppm. C-NMR (CDCl3): 11.2; 121.2; 127.2; 127.6;
130.4; 132.2; 145.9; 169.1; 170.6 ppm. MS: m/z 265 (M+, 100%), 267 (M++2, 97%), 265 (M++1,
12%), 268 (M++3, 11%).
1
3-Methyl-N-(2'-methylphenyl)maleimide (17). White solid. Mp: 55–57 °C. Yield: 52%. H-NMR
(CDCl3): 2.15 (3H, d, J = 1.8Hz, CH3); 2.16 (3H, s, CH3); 6.47 (1H, q, J = 1.8 Hz, H-3); 7.10 (1H, d,
13
J = 7.3, H-3'); 7.23–7.35 (3H, m, H-4'-6') ppm. C-NMR (CDCl3): 11.1; 17.9; 126.8; 127.7; 128.7;
129.5; 130.6; 131.1; 136.6; 146.2; 169.7; 170.7 ppm. MS: m/z 203 (M+, 100%), 208 (M++1, 13%).
1
3-Methyl-N-(2',4'-difluorophenyl)maleimide (18). White solid. Mp: 80–82 °C. Yield: 85%. H-NMR
(CDCl3): 2.01 (3H, d, J = 1.8Hz, CH3); 6.40 (1H, d, J = 1.8Hz, H-3); 6.89 (2H, q, J = 9.3 Hz; 5',6'-H);
7.15 (1H, dt, J = 8.6Hz; 3'-H) ppm. 13C-NMR (CDCl3): 10.8; 104.93 (dd, J = 26.38, 23.90 Hz); 111.78
(dd, J = 22.62, 3.66 Hz); 115.66 (dd, J = 13.43, 3.89 Hz); 127.8; 130.8 (dd, J = 10.09, 1.35 Hz); 146.4;
158.1 (dd, J = 254.42, 12.75 Hz); 162.5 (dd, J = 250.8, 11.3 Hz); 168.5; 169.7 ppm. MS: m/z 223
(M+, 100%), 224 (M++1, 12%).
Rac-3-methyl-N-arylsuccinimides 3–10. Each maleimide 11–18 (2 mmol) was dissolved in CH2Cl2
(2 mL) to which a catalytic amount of 5% Pd/C was added. Then, the mixture was exposed to a H2
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.
(3R)-(+)-Methyl-N-arylsuccinimides 3–10. A mixture of (R)-(+)-methylsuccinic acid 19 (5.0 mmol in
water) and substituted anilines 20–27 were maintained at 170 °C for 2 h and then cooled to 20 °C. The
aqueous solution was extracted with Et2O, dried over Na2SO4, filtered, and the solvent evaporated. The
mixtures were subjected to silica gel column chromatography using a mixture of hexane and ethyl
acetate (9:1) as eluent. Compounds 6, 9 and 10 (rac or R) are not described in the literature.
Spectroscopic NMR data of compound 4 was identical to that previously reported [18]. Compounds
rac-3, rac-5, rac-7, rac-8 have been previously reported by Chemical Abstract Service (CAS) (3: CAS
105909-89-5, 5: CAS 33624-30-5, 7: CAS 25998-50-9, 8: CAS 134939-24-5) but, to the best of our
knowledge, complete NMR spectral data were not available in the literature, so the complete
spectroscopic data of these compounds are described below.
(3R)-(+)-Methyl-N-(4'-methylphenyl)succinimide (3), White solid. Mp: 113–115 °C. Yield: 58%. 1H-NMR
(CDCl3): 1.45 (3H, d, J = 7.2 Hz, CH3); 2.38 (3H, s, CH3); 2.49 (1H, dd, J = 17.1; 3.8 Hz, H-3b);
2.94–3.16 (2H, m, H-2;3a); 7.15 (2H, d, J = 8.4Hz, H-2';6'); 7.27 (2H, d, J = 8.5 Hz, H-3';5') ppm.
13C-NMR (CDCl3): 16.9; 21.2; 126.2; 129.3; 129.8; 138.7; 175.6; 179.7 ppm. MS: m/z 203 (M+,
100%), 204 (M++1, 13%). [α]D27 +5.2 ± 1.2 (c 0.76, CHCl3).
(3R)-(+)-Methyl -N-(4'-methoxyphenyl)succinimide (4). White solid. Mp: 110–112 °C. Yield: 65%.
1H-NMR (CDCl3): 1.44 (3H, d, J = 7.2 Hz, CH3); 2.48 (1H, dd, J = 17.1; 3.8 Hz, H-3b); 2.94–3.15

Molecules 2013, 18
5679
(2H, m, H-2 and H-3a); 3.82 (3H, s, OCH3); 6.98 (2H, d, J = 8.4 Hz, H-2';6'); 7.19 (2H, d, J = 8.5 Hz,
H-3';5') ppm. 13C-NMR (CDCl3): 16.9; 34.8; 36.6; 55.5; 114.5; 124.6; 127.6; 159.5;175.7; 179.8 ppm.
MS: m/z 219 (M+, 100%), 220 (M++1, 13%). [α]D27 +4.2 ± 1.1 (c 0.96, CHCl3)
(3R)-(+)-Methyl-N-(4'-nitrophenyl)succinimide (5. White solid. Mp: 111–112 °C. Yield: 72%. 1H-NMR
(CDCl3): 1.46 (3H, d, J = 7.2 Hz, CH3); 2.56 (1H, dd, J = 17.1; 3.9 Hz, H-3b); 2.98–3.18 (2H, m, H-2;
13
3a); 7.61 (2H, d, J = 9.3 Hz, H-2',6'); 8.34 (2H, d, J = 9.4 Hz, H-3',5') ppm. C-NMR (CDCl3): 16.9;
34.9; 36.6; 123.1; 124.4; 126.8; 137.5; 174.5; 178.6; ppm. MS: m/z 234 (M+, 100%), 235 (M++1,
12%). [α]2D7 +6.1 ± 0.9 (c 0.86, CHCl3)
(3R)-(+)-Methyl-N-(4'-fluorophenyl)succinimide (6). White solid. Mp: 105–107 °C. Yield: 76%. 1H-NMR
(CDCl3): 1.45 (3H, d, J = 7.2 Hz, CH3); 2.50 (1H, dd, 17.1; 3.8 Hz, H-3b); 2.98–3.19 (2H, m, H-2 and
H-3a); 7.15 (2H, t, J = 8.7 Hz, H-2',6'); 7.28 (2H, dd, J = 8.0, 5.8 Hz, H-3',5') ppm. 13C-NMR (CDCl3):
16.9; 34.8; 36.6; 116.2 (d, JC-F = 22.9 Hz); 128.2 (d, JC-F = 8.8 Hz); 148.5; 162.3 (d, JC-F = 248.7 Hz);
175.3; 179.4 ppm. MS: m/z 207 (M+, 100%), 250 (M++1, 12%). [α]D27 +4.9 ± 0.8 (c 0.72, CHCl3).
(3R)-(+)-Methyl-N-(4'-chlorophenyl)succinimide (7). White solid. Mp: 112–113 °C. Yield: 68%. 1H-NMR
(CDCl3): 1.46 (3H, d, J = 7.2 Hz, CH3); 2.51 (1H, dd, J = 17.1; 3.8 Hz, H-3b); 2.96–3.16 (2H, m, H-2
and H-3a); 7.26 (2H, d, J = 8.8 Hz, H-3',5'); 7.44 (2H, d, J = 8.7 Hz, H-2',6') ppm. 13C-NMR (CDCl3):
16.9; 34.8; 36.6; 126.2; 129.1; 136.4; 142.3; 178.4; 178.7 ppm. MS: m/z 223 (M+, 100%), 225 (M++2,
32%), 224 (M++1, 12%). [α]D27 +4.5 ± 1.1 (c 0.66, CHCl3). [α]D27 +4.5 ± 1.1 (c 0.66, CHCl3).
(3R)-(+)-Methyl-N-(4'-bromophenyl)succinimide (8). White solid. Mp: 134–136 °C. Yield: 49%. 1H-NMR
(CDCl3): 1.46 (3H, d, J = 7.2 Hz, CH3); 2.51 (1H, dd, 17.1; 3.8 Hz, H-3b); 2.96–3.16 (2H, m, H-2 and
13
H-3a); 7.19 (2H, d, J = 8.8 Hz, H-3',5'); 7.56 (2H, d, J = 8.7 Hz, H-2',6') ppm. C-NMR (CDCl3):
16.8; 34.9; 36.6; 122.4; 127.9;130.9;132.3; 174.8; 179.2 ppm. MS: m/z 267 (M+, 100%), 269 (M++2,
98%), 268 (M++1, 12%). [α]D27 +5.6 ± 1.3 (c 0.58, CHCl3).
(3R)-(+)-Methyl-(2'-methylphenyl)succinimide (9). White solid. Mp: 102–105 °C. Yield: 74%. 1H-NMR
(CDCl3): 1.46 (3H, dd, J = 7.15, 1.46 Hz, CH3); 2.13 (3H, d, J = 1.87 Hz, CH3); 2.52 (1H, ddd,
J = 16.7, 3.4, 1.6 Hz, H-3b); 2.96–3.17 (2H, m, H- 2;3a); 7.05 (1H, dd, J = 7.2, 1.1 Hz; H-4');
7.24–7.38 (3H, m, H-3';5';6') ppm. 13C-NMR (CDCl3): 16.9; 17.5; 35.2; 36.8; 126.9; 127.9; 129.5;
131.0; 131.2; 135.5; 175.4; i179.6 ppm. MS: m/z 201 (M+, 100%), 202 (M++1, 13%). [α]D27 +7.3 ± 1.7
(c 0.47, CHCl3)
(3R)-(+)-Methyl-N-(2',4'-difluorophenyl)succinimide (10). White solid. Mp: 102–104 °C. Yield: 49%.
1H-NMR (CDCl3): 1.45 (3H, dd, J = 7.15, 1.46 Hz, CH3); 2.52 (1H, dd, J = 3.4, 1.6 Hz, H-3b);
2.95–3.16 (2H, m, H-2;3a); 6.79 (2H, q, J = 9.3 Hz; 5',6'-H); 7.15 (1H, dt, J = 8.6 Hz; 3'-H) ppm. 13C-NMR
(CDCl3): 16.8; 35.2; 36.7; 105.3 (dd, J = 26.4, 23.5 Hz), 112.0 (dd, J = 22.6, 3.6 Hz), 116.08 (dd,
J = 13.4, 3.9 Hz), 130.24 (dd, J = 10.2, 1.7 Hz), 157.7 (dd, J = 255.1, 12.8 Hz), 163.1 (dd, J = 251.9,
11.4 Hz), 168.7; 169.2 ppm. MS: m/z 225 (M+, 100%), 226 (M++1, 12%). [α]D27 +5.3 ± 1.4 (c 0.81, CHCl3).

Molecules 2013, 18
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3.3. Biotransformations
A. fumigatus ATCC 26934 was grown on a plate with agarized Czapek culture medium for 3 days
at 30 °C until well sporulated. 2-L Erlenmeyer flasks containing Czapek broth medium (1 L) were
inoculated with suspensions of conidia (2–5 × 106 CFU mL−1) and incubated at 30 °C for 72 h at
150 rpm on an orbital shaker (Innova 4000, New Brunswick Scientific Inc., Edison, NJ, USA).
Substrates (125 mg) in DMSO (5 mL) were poured into flasks containing the fungal biomass and
the reaction mixtures were incubated at 30 °C for 72 h on an orbital shaker (150 rpm). Culture controls
consisted of microorganism blank in which A. fumigatus ATCC 26934 was grown without substrate
but fed with the same amount of DMSO. Substrate controls contained the sterile medium with the
same amount of substrate and were incubated under the above conditions.
After incubation the mixtures were filtered, the aqueous phases were combined and extracted with
ethyl acetate (3 × 250 mL) and the organic phases were dried over Na2SO4. Conversion percentages
were determined by GC analysis of the crude extracts and determined by using the TIC (total ion
current) with the following equation: % conversion: product TIC/(product TIC + substrate TIC) × 100.
Compounds were purified by silica gel column chromatography using a mixture of hexane and ethyl
acetate (9:1) as eluent.
3.4. Antifungal Susceptibility Testing
The test fungi belonged either to the American Type Culture Collection (ATCC, Rockville, MD,
USA), or the Culture Collection of CEREMIC (Centro de Referencia en Micología-CCC, Facultad de
Ciencias Bioquímicas y Farmacéuticas, Rosario, Argentina). Species of Candida genus: C. albicans
ATCC 10231, C. tropicalis CCC 131-2000, C. krusei CCC 117-2000, C. glabrata CCC 115-2000,
C. parapsilosis CCC 124-2000, C. lusitaniae CCC 134-2000; S. cerevisiae ATCC 9763; C. neoformans
ATCC 32264, M. gypseum CCC 115-2000, T. rubrum CCC 113-2000 and T. mentagrophytes ATCC
9972 were used.
Strains were grown on Sabouraud-chloramphenicol agar slants at 30 °C, maintained on slopes of
Sabouraud-dextrose agar (SDA, Oxoid), and sub-cultured every 15 days to prevent pleomorphic
transformations. Inocula were obtained according to reported procedures [23,24] and adjusted to 1–5 × 103
colony forming units (CFU) mL−1.
The antifungal activity was determined by using broth microdilution techniques following the
guidelines of the CLSI for yeasts and for filamentous fungi [23,24]. Compounds stock solutions were
two-fold diluted from 250 to 0.24 μg ml−1 (final volume = 100 μL) and a final DMSO (Sigma)
concentration <1% in the culture media RPMI-1640 (Sigma) buffered to pH 7.0 with MOPS (Sigma).
The test was performed in 96 wells-microplates and included: Test wells (TW), containing the
compound solution and the inoculum of the fungus; Growth Control Well (GCW), that contains
compound-free medium and inoculum; Blank Test Wells (BTW), with the compound, culture medium
and sterile water instead of inoculum; Sterility Control Well (SCW) containing the compound diluted
in culture medium and sterile water. Ketoconazole (Sigma), terbinafine (Sigma) and amphotericin B
(Sigma), were used as standard controls.

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Plates were incubated in a moist, dark chamber 24 h for yeasts and 5 d for dermatophytes and
the MIC (Minimum Inhibitory Concentration) was determined. For yeasts, microplates turbidity
due to fungal growth were determined at 405 nm in a VERSA Max microplate reader (Molecular
Devices, Sunnyvale, CA, USA) and the % of growth were calculated as follows: % of growth =
[(ODTW − ODBTW)/(ODGCW − ODSCW) ×100.
3.5. Statistical Tests
Multiple comparisons were performed by the non-parametric ANOVA, Kruskal-Wallis test
followed by Dunn's multiple comparisons and Wilcoxon’s signed rank test. The associations between
MIC for the different groups were corroborated with the Score’s test [25]. Individual comparisons
between groups were performed with Student’s t test. A value of p < 0.05 was considered significant.
4. Conclusions
The antifungal behavior of eighteen enantiomerically pure (3R)-methyl- and (3R,4R)-dimethyl-N-
phenyl-, N-phenylalkyl- or N-arylsuccinimides, obtained by a low-cost and environmentally friendly
A. fumigatus-catalyzed reduction of maleimides, were compared to that of the corresponding racemic
ones. Results showed that the biotransformation products displayed statistically significant higher
antifungal activity against a panel of human opportunistic pathogenic fungi including yeasts as well as
dermatophytes than racemic ones as demonstrated by different approaches. These findings can be
highly useful for the development of chiral methylated succinimides as antifungal drugs by the
pharmaceutical industry and in a broader sense, they open new avenues for the development of highly
active drugs containing other 3- and 3,4-substituted chiral succinimides [26].
Acknowledgments
MS and SZ acknowledge the Agencia Nacional de Promoción Científica y Tecnológica de
Argentina (ANPCyT, PICT 0049-2010 and PICT 0608-2010) and the National University of Rosario
(BIO 258) for funds. We gratefully acknowledge Prof. Liliana Racca from Statistical Department of
the National University of Rosario for the analysis of results.
Conflict of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of compounds 11–18 are available from the authors.
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