Antifungal, cytotoxic and SAR studies of a series of N-alkyl, N-aryl and N-alkylphenyl-1,4-pyrrolediones and related compounds

Article abstract of DOI:10.1016/j.bmc.2011.03.038

The synthesis, in vitro evaluation and SAR studies of 67 maleimides and derivatives acting as antifungal agents are reported. A detailed SAR study supported by theoretical calculations led us to determine that: an intact maleimido ring appears to be necessary for a strong antifungal activity, dissimilarly affected by the substituents in positions 2 and 3. The best activities were shown by 2,3-nonsubstituted followed by 2,3 dichloro- and 2-methyl-substituted maleimides. They all were fungicide rather than fungistatic enhancing the importance of their antifungal activity. 2,3-Dimethyl and 2,3-diphenyl-maleimides possessed marginal or null activity. The presence of a flexible connecting chain in N-phenylalkyl maleimides appears not to be essential for antifungal activity, although its length shows a correlation with the antifungal behavior, displaying maleimides with alkyl chains of n = 3 and n = 4 the best antifungal activities in most fungi. Different substituents on the benzene ring did not have a clear influence on the activity. Values of chemical potential properties as well as of energy do not sufficiently discriminate between active and inactive compounds. Nevertheless, it was found that, although log P alone is not strong enough to properly predict the antifungal activity, the comparison of its values for compounds within the same sub-type, showed an enhancement of antifungal activity along with an increment of lipophilicity. In addition, the LUMO's electronic clouds of the highly active compounds showed to be concentrated on the imido ring, indicating that their carbon atoms are potential sites for nucleophilic attack. Same results were obtained from MEPs. Most of the active compounds did not show cytotoxic activity against human cancer cell lines and no one possessed hemolytic activity, indicating that their activity is selective to pathogenic fungi and that they are not toxic at MIC concentrations.

Full text of DOI:10.1016/j.bmc.2011.03.038

Bioorganic & Medicinal Chemistry 19 (2011) 2823–2834
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antifungal, cytotoxic and SAR studies of a series of N-alkyl, N-aryl
and N-alkylphenyl-1,4-pyrrolediones and related compounds
M. Sortino a,b, F. Garibotto a,c, V. Cechinel Filho d, M. Gupta e, R. Enriz b,c, , S. Zacchino a,
⇑
,
a Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000-Rosario, Argentina
b IMIBIO-CONICET, Universidad Nacional de San Luis, Chacabuco 915, 5700-San Luis, Argentina
c Química General, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Chacabuco 915, 5700-San Luis, Argentina
d Núcleo de Investigacões Químico-Farmacèuticas (NIQFAR), Universidade do Vale do Itajaí (UNIVALI), 88.302-202, Itajaí (SC), Brazil
e Centro de Investigaciones Farmacognósticas de la Flora Panameña (CIFLORPAN), Facultad de Farmacia, Universidad de Panamá, Panama
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis, in vitro evaluation and SAR studies of 67 maleimides and derivatives acting as antifungal
agents are reported. A detailed SAR study supported by theoretical calculations led us to determine that:
an intact maleimido ring appears to be necessary for a strong antifungal activity, dissimilarly affected by
the substituents in positions 2 and 3. The best activities were shown by 2,3-nonsubstituted followed by
2,3 dichloro- and 2-methyl-substituted maleimides. They all were fungicide rather than fungistatic
enhancing the importance of their antifungal activity. 2,3-Dimethyl and 2,3-diphenyl-maleimides pos-
sessed marginal or null activity. The presence of a flexible connecting chain in N-phenylalkyl maleimides
appears not to be essential for antifungal activity, although its length shows a correlation with the anti-
fungal behavior, displaying maleimides with alkyl chains of n = 3 and n = 4 the best antifungal activities
in most fungi. Different substituents on the benzene ring did not have a clear influence on the activity.
Values of chemical potential properties as well as of energy do not sufficiently discriminate between
active and inactive compounds. Nevertheless, it was found that, although log P alone is not strong enough
to properly predict the antifungal activity, the comparison of its values for compounds within the same
sub-type, showed an enhancement of antifungal activity along with an increment of lipophilicity. In addi-
tion, the LUMO’s electronic clouds of the highly active compounds showed to be concentrated on the
imido ring, indicating that their carbon atoms are potential sites for nucleophilic attack. Same results
were obtained from MEPs. Most of the active compounds did not show cytotoxic activity against human
cancer cell lines and no one possessed hemolytic activity, indicating that their activity is selective to path-
ogenic fungi and that they are not toxic at MIC concentrations.
Received 3 February 2011
Revised 11 March 2011
Accepted 18 March 2011
Available online 23 March 2011
Keywords:
Maleimides
Antifungal
SAR (structure–activity relationships)
DFT calculations
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
and N-phenylalkyl-1,4-pyrrolediones (common name: malei-
mides) 1–5 and 2,3-dichloro-derivatives 6–10, with an alkyl chain
Fungal infections have emerged as a major cause of morbidity
and often of mortality in immunocompromised and debilitated
patients over the past two decades.1 A matter of concern in the
treatment of fungal infections is the limited number of efficacious
antifungal drugs. Many of the currently available drugs are toxic,
produce recurrence because they are fungistatic and not fungicide
or lead to the development of resistance due in part to the pro-
longed periods of administration of the available antifungal drugs.2
There is a clear need for the discovery of new structures which
could be hits for the development of new antifungal drugs.
length from n = 0 to 4 (Fig. 1 A and B), possess strong antifungal
activities against a panel of clinically important fungi including
yeasts, Aspergillus species and dermatophytes with minimum
inhibitory concentrations (MICs) similar than some of the standard
antifungal drugs.
Results obtained in that previous work showed that the
antifungal activities of 1–5 were not dependent on the length of
the connecting alkyl chain. In contrast, the antifungal behavior of
6–10 appeared to depend on the alkyl chain’s length. From a con-
formational and electronic study of the last compounds, it was cal-
culated that the optimum distance for the alkyl connecting chain
was in the range 3.2–4.9 Å that corresponds to an alkyl chain of
3 carbon atoms.3
As part of our ongoing project on the detection of antifungal
compounds, we recently reported3 that non-substituted N-phenyl
Unfortunately, this study3 was performed with the agar dilution
method which, as it is known, is a quantitative non-standardized
test which could have generated not reproducible MICs.4 In a re-
cent paper, Cos et al.5 stated that the use of a primary standardized
⇑
Corresponding author. Tel./fax: +54 341 4375315.
E-mail addresses: denriz@unsl.edu.ar (R. Enriz), szaabgil@citynet.net.ar (S.
Zacchino).
This work was co-directed by both authors.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.038

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M. Sortino et al. / Bioorg. Med. Chem. 19 (2011) 2823–2834
O
O
O
: n=0
: n=0
6
11: n=0
12
: n=1
1
Cl
Cl
2: n=1
7: n=1
: n=2
4: n=3
5: n=4
: n=2
13: n=2
N
(CH2)n
3
N
(CH2)n
8
HN (CH2)n
OH
14
: n=3
15
: n=4
9: n=3
10: n=4
O
O
O
A
B
C
Figure 1. Structure of (A) N-phenyl and N-phenylalkyl-maleimides, (B) 2,3-dichloro-N-phenyl and N-phenylalkyl-maleimides and (C) N-phenyl and N-phenylalkyl-maleamic
acids.
validated primary screening assay that generates confident results
is essential to guarantee the reproducibility of the results. In this
regard, the Clinical and Laboratory Standards Institute [CLSI, for-
merly National Committee for Clinical and Laboratory Standards
(NCCLS)] established consensus’ procedures to facilitate the agree-
ment among laboratories in the measuring the antifungal suscep-
tibility of yeasts6 and filamentous fungi.7 Parameters such as
growth media, preparation and size of the inoculum, reference
compounds, preparation of antifungal stock solutions, dilutions
for testing, temperature and duration of incubation, end-point def-
initions, reference MIC ranges and quality controls were standard-
ized in the CLSI documents.
ylalkylmaleimides and change of a phenylalkyl to an alkyl group;
(II) compounds with a modified maleimido ring, in which breaking
of the integrity of the maleimido ring, elimination of the double
bond or reduction of one carbonyl group, were performed.
The 67 synthesized compounds include maleimides 1–10,
maleamic acids 11–15, (of which 1–5 and 11–15 were tested pre-
viously with the CLSI methodology only against Candida spp. and
6–10 were tested only in agar dilution assays)3,8 (Fig. 1) and other
52 derivatives 16–67 not tested previously as antifungal com-
pounds, of which 17 are new ones. They were evaluated against
a panel of 10 clinically important human pathogenic fungi includ-
ing yeasts, Aspergillus spp. as well as dermatophytes. In addition
we report a structure–activity relationship (SAR) study, including
computational analyses which aid to determine the minimal struc-
tural requirements to produce the antifungal effect. The evaluation
of the cytotoxic properties of the active compounds against a panel
of three cellular lines was included in this study, too.
So, in a further paper,8 we re-evaluated maleimides 1–5 with
the CLSI method only against Candida species and we corroborated
that these compounds possess strong anticandidal activity, which
was not dependent on the length of the alkyl chain. An important
concern was clarified in that paper related to the stability of malei-
mides in the culture medium. Considering that in aqueous culture
media, maleimide rings could undergo hydrolysis to the inactive
maleamic acids 11–15 (Fig. 1C), we could demonstrate that 1–5
display antifungal activities with their intact maleimido ring and
that they are not only fungistatic but fungicide with very low MICs
and MFCs, killing the fungi in 90 min.
The study of the antifungal behavior of this ample series of
maleimides and structural analogues against yeast and filamen-
tous fungi seems appropriate for a more precise comprehension
of the importance of maleimides and related compounds as anti-
fungal hits.
Taking into account the previous works,3,8 maleimides ap-
peared as excellent candidates for the development of antifungal
drugs since they possess strong antifungal activities against
important human opportunistic pathogenic fungi. Nevertheless,
according to Barrett,9 besides a strong activity, some other require-
ments are necessary to develop an antifungal agent. Among them,
the possibility of performing chemical modifications on the struc-
ture is an important advantage to take into account when selecting
an antifungal hit for developing a lead.
In that regard, we propose herein the syntheses and antifungal
evaluation with a more ample fungal panel, with the CLSI method-
ologies, of an ample series of 67 maleimides and related com-
pounds grouped in two main classes (Fig. 2): (I) compounds with
an intact maleimido ring, in which the following variation of the
substituents on positions 2,3 or modification of the N-substituents
were performed: variation of the alkyl chain length in phenylalkyl
derivatives, introduction of substituents on benzene ring of phen-
2. Results and discussion
2.1. Chemistry
For the sake of clarity, the synthesized compounds were
grouped according to their structural features (Fig. 3). Among com-
pounds with an intact maleimido ring (I) the following structural
types were synthesized, Type IA: 2,3-non-substituted N-phenyl
and N-phenylalkyl-maleimides with a variable alkyl chain (n = 0–
4) 1–5; Type IB: 2,3-substituted N-phenyl and N-phenylalkyl-
maleimides (6–10, 16–30) (n = 0–4); Type IC: N-aryl-maleimides
31–43; Type ID: N-alkyl-maleimides 44–47 (alkyl chain of 1 to 4
carbons).
Among compounds with modifications in the maleimido ring
(II), Type IIA: N-phenyl and N-phenylalkyl-maleamic acids 11–
15; Type IIB: N-phenyl and N-phenylalkyl-succinimides 48–52
and Type IIC: N-phenyl and N-phenylalkyl-4-hydroxy-2,3-dehy-
dro-c-lactames 53–67 were prepared. Scheme 1 shows reactions
and conditions for the obtainment of all synthesized compounds
(1–67).8,10
I. COMPOUNDS WITH AN INTACT MALEIMIDO RING
2.1.1. Synthesis of type I-compounds
Modification of the N-
substituent
Variation of substituent
on position 2,3
Reaction of maleic anhydrides 68–72 with aniline 73 or the
appropriate phenylalkylamines 74–77 in CHCl3 at room tempera-
ture generated the corresponding maleimides 1–10 (type IA) and
16–30 (type IB) via the corresponding maleamic acids (Scheme 1,
plain arrows). Maleic anhydride 68 and substituted anilines 78–
90 (Scheme 1, wavy arrows) or alkyamines 91–94 (Scheme 1,
dashed arrows), produced N-aryl-maleimides 31–43 (type IC) and
N-alkyl-maleimides 44–47 (type ID), respectively.
O
R1
R2
N
R0
O
Elimination of
the double bond
Reduction of
carbonyl group
Integrity of the
ring
2.1.2. Synthesis of type II-compounds
II. COMPOUNDS WITH MODIFICATIONS IN THE MALEIMIDO RING
N-Phenyl- and N-phenylalkyl-maleamic acids 11–15 (type IIA,
Fig. 1) were obtained as intermediates of the synthesis of 1–5. N-
Figure 2. Types of synthesized compounds.

M. Sortino et al. / Bioorg. Med. Chem. 19 (2011) 2823–2834
2825
I. COMPOUNDS WITH AN INTACT MALEIMIDO RING
Type 1B: 2,3-substituted N-
Type IA: N-phenyl and
N-phenylalkyl- maleimides
Type ID: N-alkylmaleimides
Type IC: N-arylmaleimides
phenyl and N-phenylalkyl-
maleimides
R3
R4
O
O
O
O
R1
R2
(CH2)n
R5
N CnH2n+1
N
N
(CH2)n
N
O
O
O
O
1 5
-
6 10, 16 30
-
31 43
44 47
-
-
-
II. COMPOUNDS WITH MODIFICATIONS IN THE MALEIMIDO RING
Type IIA
N-phenylalkyl maleamic acids
Type IIB
N-phenylalkylsuccinimides
Type IIC
: N-phenyl and N-phenylalkyl-4-
: N-phenyl and
: N-phenyl and
γ
OH-2,3-dehydro- -lactames
O
O
O
HN (CH2)n
OH
(CH2)n
(CH2)n
N
N
O
11-15
48-52
Figure 3. Structures of synthesized compounds.
53-67
O
OH
Phenyl and N-phenylalkyl-succinimides 48–52 (type IIB) were syn-
thesized utilizing the same procedure used for maleimides but
with succinic anhydride 95 and aniline 73 (or phenylalkylamines
74–77) as the starting material.
those displayed by 1–5. Regarding methylated derivatives, mono-
methylated maleimides 16–20 show a significant loss of activity
respective the non-substituted analogues 1–5. They possess mod-
erate antifungal activity against yeasts and Aspergillus spp.
4-Hydroxy-2,3-dehydro-
c
-lactames 53–57 (type IIC) were pre-
(MICs = 15.63–125
lg/mL) although they maintain strong capacity
pared from 1–5 by chemo-selective reduction with NaBH4 in the
presence of CeCl3ꢀ7H2O, according to reported procedures (Scheme
1, bold arrows).11 In turn, 4-hydroxy-3-methyl- and 4-hydroxy-2-
for inhibiting dermatophytes (MICs = 1.95–31.25
l
g/mL). Dimethyl
derivatives 21–25 were almost inactive against yeasts and Aspergil-
lus spp. and showed moderate to low activity against dermato-
phytes. In turn, diphenyl derivatives 26–30 were all inactive up
methyl-2,3-dehydro-c-lactames (58–62 and 63–67, respectively)
were prepared by chemo- and regio-selective reduction of 16–20
with both, NaBH4 and NaBH4 plus CeCl3ꢀ7H2O, respectively, accord-
ing to reported procedures (Scheme 1, bold arrows).11
to 250 lg/mL.
(b) Influence of the modification of the N-substituents.
(b1) Influence of the variation of the length of the alkyl chain.
The presence of a flexible alkyl chain in compounds 1–10, 16–
47 would not be essential for the antifungal effect, as it can be
clearly seen in the activities displayed by compounds 1, 16 and
31–43, which do not posses it.
In spite of this, it is observed that within the most active series,
when the N-substituent did possess an alkyl chain (compounds 2–
5, 7–10, 17–20, 44–47), its length appears to play a role in the
activity, being three or four the optimum number of carbons, in
most cases. So, among compounds 2–5, the best activities were
shown by compounds 4 (n = 3) and 5 (n = 4), although this differ-
ence was not observed against C. albicans.
2.2. Biological activity
MICs were determined with the microbroth dilution methods
M27-A3 and M38-A2 of CLSI,6,7 against a panel of 10 fungal species
comprising four yeasts (Candida albicans, Candida tropicalis, Crypto-
coccus neoformans and Saccharomyces cerevisiae), three Aspergillus
spp. (Aspergillus niger, Aspergillus fumigatus and Aspergillus flavus)
and three dermatophytes (Trichophyton rubrum, Trichophyton ment-
agrophytes and Microsporum gypseum. Compounds with MICs
>250
tive; between 62.5 and 125
MICs 6 31.25 g/mL, highly active. Particularly, structures display-
ing MICs 6 10 g/mL were considered of great interest for further
l
g/mL were considered inactive; MICs = 250
lg/mL, low ac-
l
g/mL, moderately active and with
Among the 2,3-dichloro derivatives 7–10, maleimide 9 (n = 3)
was the most active compound followed by 10, coincident with
the previously reported results.3
l
l
development. Minimum fungicidal concentrations (MFCs) were
accomplished by sub-culturing an aliquot from MIC wells showing
no growth onto drug-free agar plates. Results are shown in Table 1.
A similar relationship between length of the alkyl chain and
antifungal activity was observed for N-butylmaleimide 47 (n = 4)
that showed the lowest values of MICs among N-alkylmaleimides
44–47.
2.3. Structure–activity relationships
(b2) Influence of the introduction of substituents on the ben-
zene ring.
(a) Influence of variation of the substituents on positions 2,3
(compounds 1–10, 16–30).
Results showed (Table 1) that compounds 1–5 (IA) displayed
high antifungal activities against all yeasts (MICs = 0.97–15.63
The comparison of the activities displayed by 1 with those of
31–43 showed that the presence of electron-withdrawing or elec-
tron-donor substituents, at any position of the phenyl ring does not
exert a clear influence on the activity. The most striking result was
the higher activity of 43 against C. albicans but this did not consti-
tute a clear SAR neither.
(b3) Influence of the change of a phenylalkyl to an alkyl group
(compounds 1D).
The activities displayed by these compounds demonstrated that
the presence of a phenyl or an aryl moiety in the N-substituent was
not essential to produce the biological effect.
!
l
g/mL) and dermatophytes (MICs = 0.48–7.81
ent strengths of activities against all Aspergillus spp. (MICs = 7.81–
250
g/mL).8 The introduction of substituents on positions 2,3 (IB,
lg/mL) and differ-
l
compounds 6–10, 16–30) produced a dissimilar influence on the
antifungal activities, which appears to depend on the type of sub-
stituents introduced. In fact, the 2,3-dichloro maleimides 7–10 dis-
play strong antifungal effects against the whole panel, similar than

2826
M. Sortino et al. / Bioorg. Med. Chem. 19 (2011) 2823–2834
O
a
a
b
H2N CnH2n+1
N
CnH2n+1
91: n=1
92
: n=2
O
93: n=3
94: n=4
O
R
R
b
O
H2N
N
78: R=4-OCH3
: R=3-OCH3
O
O
79
80: R=3,4-(OCH3)2
81: R=2,4-(OCH3)2
82: R=2-CH3
O
OH
N
O
O
O
c
83: R=4-CH3
b
N
N
84: R=3,4-OCH2O-
85: R=4-F
H
OH
O
86
: R=4-Cl
Cl
Cl
O
O
87: R=4-Br
88: R=4-I
O
O
89: R=2,4-F2
Cl
Cl
90
: R=2-t-Bu
O
O
b
b
N
OH
N
H3C
H3C
O
d
O
O
a
O
H3C
O
O
(CH2)n
H2N
N
73
74
: n=0
: n=1
O
H3C
H3C
H3C
O
c
75: n=2
76: n=3
77: n=4
O
N
O
H3C
H3C
O
OH
b
N
O
C6H5
C6H5
O
O
O
C6H5
C6H5
O
b
b
N
O
O
O
O
O
N
O
Scheme 1. Reagents and conditions: (a) CHCl3, 1 h, rt; (b) (CH3CO)2O, NaOCOCH3, 1 h, reflux; (c) NaBH4, CeCl3ꢀ7H2O, CH3OH, rt, 1 h; (d) NaBH4, CH3OH, rt, 1 h.
(II) Influence of the modification of the imido ring.
1–5 gives 4-hydroxy-2,3-dehydro-
with MIC values from 31.25 to 250
The methylation of 53–57 (type IIC) rendered two groups of
regioisomers, the 3-methyl- and the 2-methyl-4-hydroxy-2,3-
c
-lactames 53–57 (type IIC)
The antifungal activities of maleimides 1–5 (type IA) were com-
pared to their open analogues (maleamic acids 11–15, type IIA)
and to their reduced analogues [succinimides 48–52 (type IIB)
l
g/mL.
and 4-hydroxy-2,3-dehydro-c-lactames 53–57 (type IIC)].
dehydro-c-lactames, 58–62 and 63–68, respectively which dis-
In contrast to type IA compounds 1–5, type IIA maleamic acids
11–15 were inactive against Candida and Aspergillus spp. and C.
neoformans and some of them displayed moderate to low activity
against S. cerevisiae and dermatophytes. These results suggest that
the intact imido ring would be necessary for the antifungal activity
against the whole panel, in concordance with our previous findings
for the same compounds against Candida spp.8 In addition, the
complete lack of activity of type IIB succinimides 48–52 (MIC
>250 lg/mL) adds the data that a 2,3-unsaturated structure ap-
pears to be necessary for the activity.
The presence of both carbonyl groups appears to be important
too, since the replacement of a carbonyl by a hydroxyl group in
played marginal or null antifungal activity without differences be-
tween regioisomers, but with a clear decrease of activity respective
the non-methylated analogues 53–57.
At this stage of our SAR study, some general trends can be
established:
(i) An intact maleimido ring appears to be necessary for a
strong antifungal activity, dissimilarly affected by the sub-
stituents in positions 2 and 3.
(ii) Although the presence of a flexible connecting chain in N-
phenylalkyl maleimides 1–10, 16–20, 44–47 appears not to
be essential for antifungal activity, its length shows a

M. Sortino et al. / Bioorg. Med. Chem. 19 (2011) 2823–2834
2827
Table 1
Minimum inhibitory concentrations (MIC, in
l
g/mL)/minimum fungicidal concentrations (MFC, in
R3 R4
lg/mL) of synthesized compounds
O
O
O
O
O
O
O
R1
R2
R1
R2
(CH2)n
(CH2)n
CnH2n+1
N
N
R5
N
HN (CH2)n
OH
(CH2)n
(CH2)n
N
N
N
O
O
O
O
O
OH
O
IA
IB
IC
ID
IIC
IIA
IIB
Type R1
R2
R3
R4
R5
—
n
Ca
Ct
Sc
Cn
Afu
Afl
An
Mg
Tr
Tm
1
2
3
4
5
IA
IA
IA
IA
IA
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
0
1
2
3
4
3.90/
7.81a
3.90/
7.81a
3.90/
7.81a
3.90/
7.81a
3.90/
7.81a
7.81/
3.90/
7.81
7.81/
15.63
3.90/
7.81
0.97/
1.95
0.97/
1.95
15.63/
62.5
250/
>250
125/
62.5/
125
62.5/
125
31.25/
31.25
31.25/
62.5
7.81/
15.63
31.25/
62.5
31.25/
62.5
15.63/
15.63
15.63/
15.63
7.81/
7.81
7.81/
15.63
3.90/
7.81
3.90/
7.81
0.97/
1.95
0.97/
1.95
3.90/
7.81
3.90/
7.81
1.95/
3.90
1.95/
1.95
0.48/
0.97
7.81/
7.81
3.90/
7.81
7.81/
15.63
3.90/
7.81
3.90/
7.81
15.63a
15.63/
32.25a
15.63/
32.25a
3.90/
—
—
—
—
7.81/
15.63
7.81/
15.63
15.63/
31.25
15.63/
31.25
250
31.25/
31.25
15.63/
31.25
15.63/
31.25
15.63a
7.81/
15.63a
6
7
IB
IB
Cl
Cl
Cl
Cl
—
—
—
—
—
—
0
1
i
i
i
i
i
i
i
i
i
i
15.63/
31.25
7.81/
15.63
7.81/
15.63
7.81/
15.63
31.25/
31.25
15.63/
31.25
7.81/
15.63
7.81/
15.63
7.81/
15.63
3.90/
7.81
3.90/
3.90
3.90/
7.81
7.81/
15.63
7.81/
7.81
3.90/
3.90
3.90/
3.90
15.63/
31.25
15.63/
15.63
15.63/
31.25
31.25/
62.5
31.25/
31.25
15.63/
15.63
15.63/
31.25
15.63/
31.25
31.25/
62.5
15.63/
31.25
15.63/
31.25
15.63/
15.63
15.63/
31.25
7.81/
15.63
3.90/
7.81
3.90/
7.81
3.90/
7.81
7.81/
15.63
7.81/
15.63
3.90/
7.81
3.90/
7.81
8
9
IB
IB
IB
Cl
Cl
Cl
Cl
Cl
Cl
—
—
—
—
—
—
—
—
—
2
3
4
15.63/
31.25
15.63/
15.63
15.63/
15.63
10
16
17
18
19
20
IB
IB
IB
IB
IB
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
0
1
2
3
4
62.5/
125
62.5/
125
62.5/
125
62.5/
125
62.5/
125
125/250
31.25/
125
31.25/
62.5
31.25/
62.5
15.63/
62.5
31.25/
62.5
15.63/
31.25
31.25/
62.5
15.63/
31.25
31.25/
62.5
31.25/
62.5
31.25/
62.5
31.25/
31.25
15.63/
31.25
31.25/
62.5
62.5/
125
62.5/
125
62.5/
250
31.25/
31.25
62.5/
125
125/
250
62.5/
125
62.5/
250
31.25/
31.25
62.5/
125
7.81/
15.63
3.90/
7.81
3.90/
7.81
3.90/
7.81
3.90/
15.63
7.81/
15.63
3.90/
7.81
7.81/
15.63
1.95/
3.90
31.25/
62.5
31.25/
31.25
31.25/
62.5
15.63/
31.25
31.25/
31.25
125/250
125/125
62.5/
125
62.5/
125
31.25/
62.5
3.90/
15.63
21
22
23
24
25
IB
IB
IB
IB
IB
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
0
1
2
3
4
i
i
i
125/
250
125/
250
250/
>250
125/
250
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
250/
>250
125/
250
250/
>250
125/
250
62.5/
125
62.5/
125
62.5/
250
62.5/
250
125/
250
125/
250
250/
>250
250/
>250
125/
>250
125/
>250
i
125/
>250
i
250/
>250
i
i
i
i
i
i
i
250/
>250
125/
250
26
27
28
29
30
IB
IB
IB
IB
IB
C6H5 C6H5
C6H5 C6H5
C6H5 C6H5
C6H5 C6H5
C6H5 C6H5
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
0
1
2
3
4
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
31
32
33
34
35
IC
IC
IC
IC
IC
—
—
—
—
—
—
—
—
—
—
H
H
OCH3
0
0
0
0
0
62.5/
125
15.63/
62.5
31.25/
62.5
15.63/
15.63
15.63/
15.63
62.5/
125
15.63/
62.5
15.63/
15.63
15.63/
15.63
15.63/
31.25
31.25/
62.5
3.90/
7.81
7.81/
15.63
7.81/
7.81
7.81/
7.81
15.63/
15.63
3.90/
7.81
7.81/
15.63
7.81/
7.81
62.5/
125
31.25/
62.5
62.5/
125
31.25/
62.5
62.5/
125
31.25/
62.5
62.5/
125
62.5/
125
62.5/
62.5
31.25/
62.5
31.25/
62.5
62.5/
125
15.63/
62.5
3.90/
7.81
3.90/
7.81
3.90/
7.81
3.90/
7.81
3.90/
7.81
3.90/
3.90
1.95/
3.90
3.90/
7.81
3.90/
7.81
1.95/
3.90
3.90/
7.81
1.95/
3.90
3.90/
7.81
3.90/
3.90
1.95/
3.90
H
OCH3
H
H
OCH3 OCH3
OCH3
CH3
H
H
OCH3
H
7.81/
7.81
62.5/
125
15.63/
62.5
36
37
38
39
40
IC
IC
IC
IC
IC
—
—
—
—
—
—
—
—
—
—
H
H
H
H
H
H
CH3
0
0
0
0
0
15.63/
31.25
31.25/
62.5
15.63/
15.63
62.5/
62.5
15.63/
31.25
31.25/
62.5
15.63/
15.63
7.811/
15.63
15.63/
62.5
3.90/
7.81
7.81/
15.63
3.90/
7.81
3.90/
7.81
7.81/
15.63
3.90/
7.81
31.25/
62.5
62.5/
125
15.63/
31.25
62.5/
125
31.25/
62.5
15.63/
15.63
62.5/
125
15.63/
15.63
62.5/
125
15.63/
31.25
62.5/
125
7.81/
7.81
31.25/
62.5
31.25/
62.5
1.95/
3.90
1.95/
1.95
3.90/
7.81
3.90/
7.81
3.90/
3.90
1.95/
3.90
1.95/
3.90
3.90/
7.81
3.90/
7.81
1.95/
3.90
1.95/
7.81
1.95/
3.90
3.90/
7.81
3.90/
7.81
1.95/
3.90
OCH2O
7.81/
15.63
7.81/
15.63
3.90/
7.81
H
H
H
F
Cl
Br
15.63/
31.25
7.81/
15.63
31.25/
62.5
(continued on next page)

2828
M. Sortino et al. / Bioorg. Med. Chem. 19 (2011) 2823–2834
Table 1 (continued)
Type R1
R2
—
R3
H
R4
H
R5
I
n
Ca
Ct
Sc
Cn
Afu
Afl
An
Mg
Tr
Tm
41
42
43
IC
IC
IC
—
—
—
0
31.25/
62.5
15.63/
31.25
3.90/
3.90
31.25/
62.5
15.63/
31.25
3.90/
3.90
7.81/
7.81
7.81/
15.63
3.90/
7.81
7.81/
15.63
7.81/
7.81
3.90/
7.81
62.5/
125
31.25/
31.25
15.63/
31.25
62.5/
125
62.5/
62.5
62.5/
62.5
62.5/
125
15.63/
31.25
62.5/
125
3.90/
7.81
3.90/
7.81
1.95/
3.90
3.90/
7.81
3.90/
7.81
1.95/
3.90
3.90/
7.81
3.90/
7.81
1.95/
3.90
—
—
F
H
H
F
0
0
t-Bu
H
44
45
46
47
ID
ID
ID
ID
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
1
2
3
4
62.5/
125
62.5/
250
62.5/
125
3.90/
15.63
31.25/
62.5
62.5/
125
62.5/
125
7.81/
15.63
7.81/
15.63
15.63/
31.25
1.95/
1.95
15.63/
62.5
15.63/
62.5
15.63/
31.25
15.63/
15.63
15.63/
15.63
1.95/
7.81/
15.63
7.81/
15.63
15.63/
15.63
3.90/
3.90
7.81/
7.81
7.81/
7.81
3.90/
7.81
3.90/
3.90
3.90/
15.63
3.90/
3.90
3.90/
7.81
7.81/
15.63
7.81/
15.63
7.81/
7.81
15.63/
31.25
7.81/
15.63
3.90/
7.81
15.63/
31.25
15.63/
31.25
1.95/
1.95
3.90/
3.90
1.95/
1.95
1.95/
3.90
7.81
11
12
13
14
15
IIA
IIA
IIA
IIA
IIA
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
0
1
2
3
4
ia
ia
ia
ia
ia
ia
ia
ia
ia
ia
125/
>250
250/
>250
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
125/
>250
i
62.5/
250
i
250/
>250
i
250/
>250
250/
>250
i
250/
>250
250/
>250
i
i
i
i
i
i
48
49
50
51
52
IIB
IIB
IIB
IIB
IIB
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
0
1
2
3
4
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
53
54
55
56
57
IIC
IIC
IIC
IIC
IIC
H
H
H
H
H
H
H
H
H
H
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
0
1
2
3
4
i
i
i
250/
>250
250/
>250
62.5/
>250
62.5/
>250
250/
>250
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
62.5/
125
62.5/
>250
i
250/
>250
125/250
250/
>250
62.5/
125
i
62.5/
125
62.5/
125
125/
250
62.5/
125
62.5/
250
62.5/
250
31.25/
62.5
31.52/
62.5
62.5/
125
125/
250
i
250/
>250
i
i
i
58
IIC
H
CH3
—
—
—
0
i
i
250/
i
i
i
i
i
i
>250
59
60
61
IIC
IIC
IIC
H
H
H
CH3
CH3
CH3
—
—
—
—
—
—
—
—
—
1
2
3
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
250/
>250
i
250/
>250
i
250/
>250
i
250/
>250
i
62
IIC
H
CH3
—
—
—
4
i
i
i
i
i
i
63
64
65
66
67
IIC
IIC
IIC
IIC
IIC
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
0
1
2
3
4
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
i
AMP
KTZ
TBF
0.97/
1.95
0.48/
1.95
—
0.48/
0.97
0.12/
0.97
—
0.48/
1.95
0.48/
0.97
—
0.24/
0.97
0.24/
1.95
—
0.48/
0.97
0.12/
3.90
—
0.48/
1.95
0.48/
3.90
—
0.48/
3.90
0.24/
1.95
—
0.12/
0.48
0.06/
0.24
0.03/
0.06
0.06/
0.24
0.03/
0.12
0.01/
0.03
0.06/
0.24
0.03/
0.12
0.03/
0.06
Ca: Candida albicans ATCC 10213, Ct: C. tropicalis C 131 2000, Sc: Saccharomyces cerevisiae ATCC 9763, Cn: Cryptococcus neoformans ATCC 32264, Afu: Aspergillus fumigatus ATCC
26934, Afl: A. flavus ATCC 9170, An: A. niger ATCC 9029, Mg: Microsporum gypseum C 115 2000, Tr: Trichophyton rubrum C 113 2000, Tm: T. mentagrophytes ATCC 9972.
C = CEREMIC (Centro de Referencia en Micología). ATCC: American Type Culture Collection AMP: Amphothericine B, KTZ: Ketoconazole, TBF: Terbinafine. i: inactive (>250 lg/
mL).
a
Results published in Ref.8.
correlation with the antifungal behavior, showing alkyl
chains of n = 3 and n = 4 the best antifungal activities in most
fungi.
representing more than 60% of isolates from clinical infec-
tions,13 it is important to highlight that out of the 67 com-
pounds tested, 2,3-non-substituted maleimides 1–5, 43
and 47 showed the best antifungal activities against this
clinically important fungus.
(iii) Results obtained for compounds possessing different sub-
stituents on the benzene ring showed that they would not
have a clear influence in the activity. Nevertheless, 43
showed the highest antifungal activity of N-aryl-maleimides.
(iv) Considering that C. albicans produces the fourth most preva-
lent blood stream infection in immunocompromised hosts,
As an additional information to the SAR studies, it is important
to take into account that MFC values of all active compounds were
one or two dilutions higher than their respective MICs. MFC has

M. Sortino et al. / Bioorg. Med. Chem. 19 (2011) 2823–2834
2829
been pointed out as being more relevant than MIC to therapeutic
outcome.12
Results shown in Table 2 indicate that values of CPP as well as
energy values do not sufficiently discriminate between active and
inactive compounds.
So, other parameters such as dipolar moment and log P were
calculated for all representative compounds. Results are showed
in Table 3.
2.4. Computational studies
To better understand the above experimental results, we per-
formed both a conformational and an electronic study on one rep-
resentative compound of each type reported here, which could
allow discriminating between active and inactive structures. The
purpose was to obtain more precise information on how closely
these compounds resemble each other in terms of the spatial ori-
entations of the essential moieties to produce the antifungal
activity.
Regarding conformational studies, it was necessary at first to
define the three-dimensional geometry of the relevant conforma-
tion of each molecule, which could be either that of the lowest-en-
ergy or the topographically congruent with any other active
compound.
Once obtained the relevant conformations for the different
compounds, we evaluated the electronic and hydrophobic aspects
of the molecules, by using different electronic parameters as well
as molecular electrostatic potentials (MEPs).
Theoretical calculations obtained for the representative com-
pounds 1, 2, 5, 7, 12, 17, 22, 27, 31, 36, 38, 42–47, 49, 54, 59 and
64 are considered descriptive of the whole series and are shown
in Table 2.
In general, the low-active or inactive compounds 12, 22, 27, 49,
54, 59 and 64 showed relatively higher values of
l (range 1.90–
4.75 D) than active compounds (1, 2, 5, 7, 17, 31, 36, 38, 42–47)
(0.90–1.72 D) (table 4). The active compound 42 with
l
= 2.11 D
is an exception.
Table 3
Dipolar moments and log P for representative compounds
Compound
l
(dipolar moment) (Debye)
log P
1
2
5
1.26
1.15
1.13
1.25
2.88
1.72
1.90
2.24
1.14
0.90
1.03
2.11
0.90
1.12
1.03
1.02
0.91
1.13
1.96
4.16
4.06
4.75
1.61
2.04
3.22
3.21
1.39
2.53
3.04
6.01
1.63
0.89
1.30
1.46
3.25
ꢁ0.52
ꢁ0.18
0.30
1.31
1.31
2.04
2.04
1.61
1.79
7
12
17
22
27
31
36
38
42
43
44
45
46
47a
47b
49
54
59
64
Thus, the first objective of the calculations was to study the
chemical potential properties (CPP) of each selected compound
that are defined by different variables tightly related among them:
electron affinity (EA), ionization potential (IP), hardness (
g), elec-
tronegativity ( ) and electrophilicity ( ). Such variables have dif-
v
x
ferent meanings but nevertheless, as a whole, they measure the
tendency of a group to release or capture electrons; in fact, they
constitute an index of potential chemical reactivity.14 In addition,
the energy of neutral and charged molecules (+1 and ꢁ1) are re-
corded in Table 2.
a
Conformation extended.
Conformation folded.
b
Table 2
Chemical potential properties and energy properties for representative compounds
Compd Electro affinity
EA = E(0) ꢁ E(ꢁ1) potential
(eV)
Ionization
Hardness
Electro
Electro-
philicity
x = l2/2g
Energy of neutral
molecule (0)
(Hartree)
Energy of charged
molecule (+1)
(Hartree)
Energy of charged
molecule (ꢁ1)
(Hartree)
g
= (I ꢁ A)/2 negativity
IP = E(+1) ꢁ E(0)
(eV)
v
= (I + A)/2
(eV)
(eV)
(eV)
1
2
5
0.61
8.37
8.77
8.23
8.66
8.43
8.51
8.49
7.68
8.46
7.52
8.61
8.64
8.50
9.80
9.62
9.50
9.39
9.45
8.61
8.28
6.18
5.90
3.88
4.15
3.92
3.82
4.20
4.10
4.15
3.73
3.96
3.24
4.03
4.00
3.97
4.74
4.64
4.57
4.52
4.52
4.96
4.54
2.85
2.82
4.49
4.61
4.32
4.84
4.23
4.41
4.34
3.95
4.49
4.28
4.58
4.64
4.52
5.06
4.98
4.93
4.93
4.87
3.65
3.74
3.33
3.08
0.20
0.16
0.16
0.20
0.99
0.36
0.44
0.11
0.16
0.76
0.13
0.56
0.10
0.13
0.11
0.11
0.14
0.09
0.25
1.90
2.90
4.00
ꢁ590.48
ꢁ629.80
ꢁ747.74
ꢁ1548.98
ꢁ706.22
ꢁ669.12
ꢁ708.44
ꢁ819.52
ꢁ629.80
ꢁ1091.92
ꢁ689.71
ꢁ788.94
ꢁ747.72
ꢁ398.74
ꢁ438.06
ꢁ477.37
ꢁ516.69
ꢁ516.69
ꢁ631.04
ꢁ630.98
ꢁ669.66
ꢁ669.65
ꢁ590.17
ꢁ629.47
ꢁ747.44
ꢁ1548.66
ꢁ705.91
ꢁ668.81
ꢁ708.13
ꢁ819.24
ꢁ629.49
ꢁ1091.64
ꢁ689.39
ꢁ788.62
ꢁ747.41
ꢁ398.38
ꢁ437.71
ꢁ477.03
ꢁ516.34
ꢁ516.34
ꢁ630.72
ꢁ630.67
ꢁ669.43
ꢁ669.43
ꢁ590.50
ꢁ629.81
ꢁ747.75
ꢁ1549.02
ꢁ706.22
ꢁ669.13
ꢁ708.45
ꢁ819.53
ꢁ629.82
ꢁ1091.95
ꢁ689.73
ꢁ788.96
ꢁ747.74
ꢁ398.76
ꢁ438.07
ꢁ477.39
ꢁ516.70
ꢁ516.70
ꢁ630.99
ꢁ630.95
ꢁ669.67
ꢁ669.66
0.46
0.40
1.02
0.03
0.32
0.19
0.22
0.54
1.03
0.56
0.65
0.55
0.32
0.34
0.36
0.35
0.41
ꢁ1.31
ꢁ0.79
0.49
0.27
7
12
17
22
27
31
36
38
42
43
44
45
46
47a
47b
49
54
59
64
a
Conformation extended.
Conformation folded.
b

2830
M. Sortino et al. / Bioorg. Med. Chem. 19 (2011) 2823–2834
Table 4
Cytotoxic and hemolytic activity of active antifungal compounds
Compd
Cytotoxic activity
GI50
Hemolytic activity
H-460
MCF-7
SF268
HC50
1
2
3
4
5
>10.0
>10.0
4.7
>10.0
>10.0
>10.0
>10.0
3.6
5.2
>10.0
>10.0
>10.0
4.1
5.8
>10.0
>250
>250
>250
>250
>250
7
8
9
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>250
>250
>250
>250
10
16
17
18
19
20
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>250
>250
>250
>250
>250
Figure 4. Spatial representation of the frontier molecular orbital LUMO of the
compounds 5 (a), 43 (b) and 47 (c) at the ground state using B3LYP/6-31G(d)
calculations.
31
32
33
34
35
36
37
38
39
40
41
42
43
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
44
45
46
47
>10.0
6.7
>10.0
>10.0
>10.0
9.9
>10.0
>10.0
>10.0
6.5
>10.0
>10.0
>250
>250
>250
>250
GI50 (growth inhibition 50, in
mL).
lg/mL) and HC50 (hemolytic concentration 50, in lg/
Regarding log P, it was found that this descriptor alone is not
strongly enough to properly predict the antifungal activity. The ac-
tive compounds 5, 43 and 47 displayed values of 3.22, 3.23 and
1.31, respectively, whereas the low-active or inactive compounds
22, 49, 54, 59 and 64 showed values of 3.04, 2.04, 2.04, 1.61 and
1.79, respectively (Table 3). However, within compounds of the
same type, the comparison of the values of log P shows that an
enhancement of antifungal activity is directly related to the incre-
ment of lipophilicity. For instance, within compounds of each IA
(1–5), IC (31–43) or ID (44–47) types, maleimides 5, 43 and 47,
possess the highest log P values and, concomitantly, displayed
the best antifungal activities.
In addition, it is known that the relative ordering of HOMO
and/or LUMO contour plots provides a reasonable indication of
the excitation properties of a compound.15–17 Figure 4 gives a
detailed description of LUMO orbitals, including spatial character-
istics, nodal patterns, and individual atom contributions of 5, 43
and 47. It is interesting to note that the LUMO contour plot
obtained for 5 (Fig. 1a) is closely related to those obtained from
compounds 43 and 47 ( Fig. 1b and c). We can observe that the
LUMO’s electronic clouds of the three compounds are concentrated
Figure 5. Electrostatic potential-encoded electron density surfaces of compounds 5
(a), 43 (b) and 47 (c). The surfaces were generated with GAUSSIAN 03 after B3LYP
minimization with
a 6-31G(d) basis set. The coloring represents electrostatic
potential with red indicating the strongest attraction to a positive point charge and
blue indicating the strongest repulsion. The electrostatic potential is the energy of
interaction of the positive point charge with the nuclei and electrons of a molecule.
sites.18–20 Figure 5 gives the MEPs obtained for compounds 5, 43
and 47 which exhibit two clear minimum values (deep red zones)
in the maleimido ring. This region is symmetrical with respect to
the carbonyl groups and displays a deep and extensive negative
potential zone with values of about ꢁ0.070 kcal/mol. The other rel-
evant characteristic of these MEPs is the presence of a relatively
extended hydrophobic zone (light blue and green zones).
2.5. Cytotoxic and hemolytic activity
In addition to the antifungal evaluation, cytotoxic activities
against breast (MCF-7), lung (H-460) and central nervous system
(SF-268) human cancer cell lines of the most active compounds
(those whit MICs 6 10 lg/mL in at least one fungus) (1–5, 7–10,
16–20, 31–47) were evaluated in order to determine the selectivity
of maleimides towards the most sensitive fungi. Results showed
(Table 4) that the cytotoxic activities (GI50) were >10 lg/mL for
all selected compounds, except for 3, 4 and 45, whose GI50 values
were similar or higher than the antifungal ones (Table 4).
on the imido ring, which is mostly composed by
p
⁄ orbitals (inter-
ring antibonding character). This indicates that the carbon atoms
in the imido ring are potential sites for nucleophilic attack, thus
indicating that its contribution is crucial.
The same results can also be appreciated by observing the MEPs
obtained from DFT calculations. MEPs have shown to provide reli-
able information, both on the interaction sites of molecules with
point charges and on the comparative reactivities of these

M. Sortino et al. / Bioorg. Med. Chem. 19 (2011) 2823–2834
2831
Then, considering the possibility that active compounds might
have a simple lytic mode of action and be broadly cytotoxic against
human cells, we determined the hemolytic activity of these com-
pounds against human erythrocytes. Results indicate that no com-
pounds induced hemoglobin release from red blood cells at
concentrations up to 250 lg/mL, indicating that these compounds
are not hemolytic and do not disturb membrane functions in eryth-
rocytes at antifungal MICs (Table 4).
shifts are reported in ppm (d) relative to the solvent peak (CHCl3
in CDCl3 al 7.26 ppm for protons and at 77.0 for carbons). Signals
are designated as follows: s, singlet; d, doublet; dd, doublets of
doublets; t, triplet; dt; doublets of triplets; m, multiplet; quint,
quintuplet. Mass spectra were obtained in a Turbo Mass Perkin El-
mer, ionization energy 70 eV. Elemental analyses were performed
on a Carlo Erba EA1108 analyzer. Percentages of C, H and N were
in agreement with the product formula (within 0.4% of theoretical
values). Melting points were obtained in a Electrothermal appara-
tus (UK) and were uncorrected.
3. Conclusions
4.1.1. Synthesis of maleimides
The synthesis, in vitro evaluation and SAR studies of 67 malei-
mides and derivatives acting as antifungal agents are reported. A
detailed SAR study supported by theoretical calculations led us to
determine the minimal structural requirements of this series to
produce the antifungal effect: (i) an intact maleimido ring appears
to be necessary for a strong antifungal activity; (ii) the substituents
in positions 2 and 3 dissimilarly affected the antifungal activity.
The best activities were shown by 2,3-nonsubstituted 1–5, 31–
47, followed by 2,3 dichloro- 6–10 and by 2-methyl-substituted
maleimides 16–20. 2,3-Dimethyl- and 2,3-diphenyl-maleimides
21–30 possessed marginal or null activity; (iii) the presence of a
flexible connecting chain in N-phenylalkyl maleimides appears
not to be essential for antifungal activity, although its length shows
a correlation with the antifungal behavior, displaying maleimides
with alkyl chains of n = 3 and n = 4 the best antifungal activities
in most fungi; (iv) different substituents on the benzene ring did
not have a clear influence on the activity.
Values of CPP as well as of energy do not sufficiently discrimi-
nate between active and inactive compounds. Nevertheless, it
was found that log P, although alone is not strong enough to prop-
erly predict the antifungal activity, the comparison of its values for
compounds within the same type, showed an enhancement of
antifungal activity along with an increment of lipophilicity.
In addition, the LUMO’s electronic clouds of the highly active
compounds 5, 43 and 47 showed to be concentrated on the imido
ring, indicating that their carbon atoms are potential sites for
nucleophilic attack. Same results were obtained from MEPs.
Most active compounds did not show cytotoxic activity against
human cancer cell lines and no one possessed hemolytic activity,
indicating that their activity is selective to pathogen fungi and that
they are not toxic at MIC concentrations.
The synthesis of maleimides 1–47 was performed by mixing
an equimolar amount of the appropriate maleic anhydrides 68–
72 in 5 mL of CHCl3 and anilines 73, 78–90, amines 91–94 or
phenylalkylamines 74–77 (5 mmol) dissolved in 1 mL of CHCl3
and stirred during 1 h. The solid (maleamic acid) which precipi-
tated out of the reaction mixture was filtered off. The whole
amount of maleamic acid was dissolved in 5 mL of acetic anhy-
dride and 100 mg of sodium acetate was added. The mixture
was heated for 2 h under reflux. The reaction was cooled and
quenched with water; then, the aqueous solution was extracted
with Et2O, dried with Na2SO4, filtered, and the solvent was evap-
orated. The product was purified by silica gel column chromatog-
raphy using a mixture of hexane and ethyl acetate (9:1) as eluent.
Compounds 1–10, 16–26, 31–34, 36, 38–42 and 43–47 were
previously reported.3,8,21–28
4.1.1.1. 2,3-Diphenyl-N-benzylmaleimide 27. Yellow crystals.
Mp 99–100 °C. Yield: 42%. 1H NMR (CDCl3, 300 MHz): d 3.98 (2H,
s, CH2); 7.22–7.58 (15H, m, HAr) ppm. 13C NMR (CDCl3, 75 MHz):
d 46.5 (CH2); 126.8; 127.1; 128.6; 128.7; 128.8; 129.8; 136.2;
136.5; 143.2; 170.5 ppm. EM: m/z = 339 (M+). Anal. Calcd for
C23H17NO2: C, 81.3; H, 5.0; N, 4.1. Found: C, 81.2; H, 5.0; N, 4.1.
4.1.1.2. 2,3-Diphenyl-N-phenethylmaleimide 28. Yellow crys-
tals. Mp 79–81 °C. Yield: 36%. 1H NMR (CDCl3, 300 MHz): d 3.77
(2H, t, J = 7.2, ArCH2); 3.92 (2H, t, J = 7.2, NCH2); 7.18–7.52 (15H,
m, HAr) ppm. 13C NMR (CDCl3, 75 MHz): d 34.6 (ArCH2); 40.0;
126.2; 128.8; 128.9; 129.8; 136.2; 138.1; 129.8; 170.6 ppm. EM:
m/z = 353 (M+). Anal. Calcd for C24H19NO2: C, 81.5; H, 5.4; N, 4.0.
Found: C, 81.6; H, 5.4; N, 4.0.
Considering the high antifungal activities of types IA and IB
compounds, a study of their mode of action constitutes a necessary
next step for future research. In this regard, it is important to take
into account that compound 9 showed, in a previous work,3 to be
inhibitory of the fungal cell-wall. Since fungal but not mammalian
cells possess a wall, its inhibition is highly appreciated for the pos-
sible development of safe antifungal drugs.
In addition, a rational design that lead to further modifications
of the unsaturated, and diketo ring system will allow to find new
structure–activity relationships and will help to have a more pro-
found and comprehensive knowledge of the potentiality of malei-
mide-related compounds as antifungal compounds.
4.1.1.3. 2,3-Diphenyl-N-propylphenylmaleimide 29. Yellow
crystals. Mp 82–84 °C. Yield: 35%. 1H NMR (CDCl3, 300 MHz): d
1.79 (2H, quint, J = 7.5, CH2CH2CH2); 2.66 (2H, t, J = 7.5, ArCH2);
2.74 (2H, t, J = 7.5, NCH2); 7.15–7.52 (15H, m, HAr) ppm. 13C NMR
(CDCl3, 75 MHz): d 33.2; 35.0; 41.6; 125.8; 125.9; 128.2; 128.4;
129.5; 135.4; 142.0; 142.0; 155.9; 170.3 ppm. EM: m/z = 367
(M+). Anal. Calcd for C25H21NO2: C, 81.6; H, 5.7; N, 3.8. Found: C,
81.4; H, 5.7; N, 3.8.
4.1.1.4.
2,3-Diphenyl-N-butylphenylmaleimide 30. Yellow
crystals. Mp 150–152 °C. Yield: 41%. 1H NMR (CDCl3, 300 MHz): d
1.28 (4H, m, CH2(CH2)2CH2); 3.01 (2H, t, J = 7.5, ArCH2); 3.86 (2H,
t, J = 7.5, NCH2); 7.12–7.45 (15H, m, HAr) ppm. 13C NMR (CDCl3,
75 MHz): d 22.4; 26.4; 34.6; 39.6; 125.3; 126.7; 128.8; 128.9;
129.4; 129.8; 136.2; 136.4; 138.1; 170.6 ppm. EM: m/z = 381
(M+). Anal. Calcd for C26H23NO2: C, 81.8; H, 6.0; N, 3.7. Found: C,
82.1; H, 6.0; N, 3.7.
4. Experimental
4.1. Chemistry
Solvents and reagents were purchased from Sigma (St. Louis,
MO, USA) and Aldrich (Steinheim, Germany) and were purified in
the usual manner. The 1H and 13C NMR spectra were recorded on
a Bruker 300 MHz (Bruker, Karlsruhe, Germany). Compounds were
dissolved in deuterated solvents from commercial sources (Sigma)
with tetramethylsilane (TMS) as the internal standard. Chemical
4.1.1.5. N-(30,40-Dimethoxylphenyl) maleimide 33. Yellow
crystals. Mp 92–94 °C Yield 85%. 1H NMR (CDCl3, 300 MHz): d
3.90 (3H, s, 30-OCH3); 3.92 (3H, s, 40-OCH3); 6.85 (2H, s, H-2,3);
6.82–7.98 (3H, m, HAr) ppm. 13C NMR (CDCl3, 75 MHz): d 55.0;
56.1; 110.0; 111.2; 119.0; 124.0; 134.2; 148.9; 149.3; 169.8 ppm.

2832
M. Sortino et al. / Bioorg. Med. Chem. 19 (2011) 2823–2834
Anal. Calcd for C12H11NO4: C, 61.7; H, 4.7; N, 6.0. Found: C, 62.0; H,
4.7; N, 6.0.
Anal. Calcd for C12H13NO2: C, 70.9; H, 6.4; N, 6.9. Found: C, 70.7;
H, 6.4; N, 6.9.
4.1.1.6. N-(30,40-Methylendioxyphenyl)-maleimide 37. White
crystals. Mp 147–148 °C Yield 74%. 1H NMR (CDCl3, 300 MHz): d
6.03 (2H, s, OCH2O); 6.79 (1H, dd, J = 9.0; 2.0, H-60); 6.84 (s, 2H, H-
2,3); 6.80–6.77 (2H, m, H-20,50) ppm. 13C NMR (CDCl3, 75 MHz): d
101.8; 107.7; 108.4; 120.2; 124.6; 134.2; 147.4; 148.1; 169.6 ppm.
Anal. Calcd for C11H7NO4: C, 60.8; H, 3.2; N, 6.4. Found: C, 61.0; H,
3.2; N, 6.5.
4.1.3.3.
N-Propylphenyl-4-hydroxy-2,3-dehydro-c-lactame
56. White crystals. Mp 125–127 °C. Yield: 76%. 1H NMR (CDCl3,
300 MHz): d 1.92 (2H, m, CH2CH2CH2); 2.39 (2H, m, ArCH2); 3.22
(1H, dt, J = 14.1; 7.2, CHaHb); 3.60 (1H, dt, J = 14.1; 7.2, CHaHb);
5.38 (1H, d, J = 10.8, H-4); 6.08 (1H, d, J = 6.0, H-2); 6.91 (1H, dd,
J = 6.0; 1.6, H-3); 7.11–7.37 (5H, m, HAr) ppm. 13C NMR (CDCl3,
75 MHz): d 30.1; 33.3; 39.0; 83.4; 125.9; 128.3; 128.4; 128.4;
141.4; 145.7; 169.6 ppm. Anal. Calcd for C13H15NO2: C, 71.8; H,
6.9; N, 6.4. Found: C, 71.7; H, 6.9; N, 6.5.
4.1.2. Synthesis of succinimides
4.1.2.1. N-Phenyl- and N-phenylalkylsuccinimides 48–
52. They were prepared following the same procedure used for
maleimides 1–47 utilizing succinic anhydride 95 as the reactive in-
stead of maleic anhydrides. Compounds 48–50 were previously
reported.28
4.1.3.4.
N-Butylphenyl-4-hydroxy-2,3-dehydro-c-lactame
57. White crystals. Mp 132–134 °C. Yield: 74%. 1H NMR (CDCl3,
300 MHz): d 1.58–1.77 (4H, m, CH2(CH2)2CH2); 2.64 (2H, t, J = 7.0,
ArCH2); 3.01 (1H, d, J = 11.4, OH); 3.34 (1H, dt, NCHaHb); 3.60
(1H, dt, NCHaHb); 5.19 (1H, d, J = 11.4, H-4); 7.09–7.35 (7H, m,
H-2; H-3 and HAr) ppm. 13C NMR (CDCl3, 75 MHz): d 27.7; 28.4;
35.3; 40.3; 82.1; 125.9; 126.9; 128.4; 128.4; 141.8; 142.8;
162.6 ppm. Anal. Calcd for C14H17NO2: C, 71.8; H, 6.9; N, 6.4.
Found: C, 72.0; H, 6.9; N, 6.5.
4.1.2.2. N-Propylphenylsuccinimide 51. White crystals. Mp
110–113 °C Yield 71%. 1H NMR (CDCl3, 300 MHz): d 1.96 (2H, quint,
J = 7.8, CH2CH2CH2); 2.57 (4H, s, H-2,3); 2.68 (2H, t, J = 7.8, ArCH2);
3.78 (2H, t, J = 7.8, NCH2); 7.13–7.38 (5H, m, HAr) ppm. 13C NMR
(CDCl3, 75 MHz): d 28.1; 28.4; 33.2; 38.7; 126.0; 128.2; 128.4;
141.0; 177.3 ppm. Anal. Calcd for C13H15NO2: C, 71.8; H, 6.9; N,
6.4. Found: C, 72.0; H, 6.9; N, 6.5.
4.1.3.5.
3-Methyl-N-phenethyl-4-hydroxy-2,3-dehydro-c-lac-
tame 60. White crystals. Mp 143–145 °C. Yield: 69%. 1H NMR
(CDCl3, 300 MHz): d 2,00 (3H, s, CH3); 2,89 (2H, t, J = 7,2, ArCH2);
3,47 (1H, dt, J = 14,2; 7,2, NCHaHb); 3,74 (1H, dt, J = 14,2; 7,2,
NCHaHb); 4,99 (1H, d, J = 10,9, H-4); 5,68 (1H, s, H-2); 7,12–7,43
(5H, m, HAr) ppm. 13C NMR (CDCl3, 75 MHz): d 13.5; 34.7; 40.8;
85.3; 122.3; 126.5; 128.6; 128.7; 138.9; 158.1; 170.4 ppm. Anal.
Calcd for C13H15NO2: C, 71.8; H, 6.9; N, 6.4. Found: C, 71.6; H,
6.9; N, 6.5.
4.1.2.3. N-Butylphenylsuccinimide 52. White crystals. Mp 103–
104 °C Yield 69%. 1H NMR (CDCl3, 300 MHz): d 1.63 (4H, m,
CH2(CH2)2CH2); 2.24 (2H, t, J = 7.2, ArCH2); 2.70 (4H, s, H-2,3);
3.55 (2H, t, J = 6.9, NCH2); 7.12–7.33 (5H, m, HAr) ppm. 13C NMR
(CDCl3, 75 MHz): d 27.3; 28.1; 28.6; 35.3; 38.6; 125.8; 128.3;
128.4; 141.9; 177.2 ppm. Anal. Calcd for C14H17NO2: C, 72.6; H,
7.4; N, 6.1. Found: C, 72.5; H, 7.4; N, 6.0.
4.1.3.6. 3-Methyl-N-propylphenyl-4-hydroxy-2,3-dehydro-c-lac-
tame 61. White crystals. Mp 112–114 °C. Yield: 71%. 1H NMR
(CDCl3, 300 MHz): d 1.89 (2H, m, CH2CH2CH2); 2.04 (3H, s, CH3);
3.63 (1H, d, J = 11.4, OH); 3.63 (2H, t, J = 7.8, ArCH2); 3.26 (1H, dt,
J = 14.2; 7,2, NCHaHb); 3.54 (1H, dt, J = 14.2; 7.2, NCHaHb); 5.08
(1H, d, J = 11.4, H-4); 5.68 (1H, s, H-2); 7.15–7.32 (5H, m, HAr) ppm.
13C NMR (CDCl3, 75 MHz): d 13.5; 30.1; 33.1; 38.9; 84.9; 122.4;
125.9; 128.3; 128.4; 157.8; 141.4; 170.4 ppm. Anal. Calcd for
4.1.3. Synthesis of 4-hydroxy-2,3-dehydro-
c-lactames
4-Hydroxy-2,3-dehydro- -lactames 53–57 were prepared by
c
dissolving maleimides 1–5 (1 mmol) in CH3OH (2 mL) added with
2 mmol of CeCl3ꢀ7H2O and stirring (5 min); then NaBH4 (2 mmol)
was added portionwise and stirred for 1 h. The same procedure
was used for obtaining 63–67 from 16–20. 3-Methyl-4-hydroxy-
2,3-dehydro-c-lactames 58–62 were prepared similarly but with-
C14H17NO2: C, 72.6; H, 7.4; N, 6.1. Found: C, 72.4; H, 7.3; N, 6.0.
out the addition of CeCl3. The reaction mixture was quenched with
ice water, CH3OH was removed under reduced pressure and the
solution was extracted with EtOAc, dried with Na2SO4, filtered,
and the solvent was evaporated. The mixtures were subjected to
silica gel column chromatography using a mixture of hexane and
ethyl acetate (9:1) as eluent. Compounds 54, 58–59, 63–64 were
previously reported.11
4.1.3.7. 3-Methyl-N-butylphenyl-4-hydroxy-2,3-dehydro- -lac-
c
tame 62. White crystals. Mp 123–125 °C. Yield: 66%. 1H NMR
(CDCl3, 300 MHz): d 1,62–164 (4H, m, CH2(CH2)2CH2); 1,88 (3H,
s, CH3); 2,65 (2H, t, J = 7,0 ArCH2); 3,28 (1H, dt, NCHaHb); 3,56
(1H, dt, NCHaHb); 5,24 (1H, s, H-4); 6,53 (1H, s, H-2); 7,19–7,34
(5H, m, HAr) ppm. 13C NMR (CDCl3, 75 MHz): d 13.5; 28.0; 28.6;
35.4; 38.8; 84.8; 122.4; 125.8; 128.3; 128.4; 142.1; 157.7;
170.3 ppm. Anal. Calcd for C15H19NO2: C, 73.4; H, 7.7; N, 5.7.
Found: C, 73.2; H, 7.8; N, 5.7.
4.1.3.1. N-Phenyl-4-hydroxy-2,3-dehydro-c-lactame 53. White
crystals. Mp 117–120 °C. Yield: 88%. 1H NMR (CDCl3, 300 MHz): d
6.00 (1H, d, H-4); 6.24 (1H, d, J = 6.0, H-2); 7.06 (1H, dd, J = 6,0;
1,7, H-3); 7.21 (1H, t, J = 7.9, H-40); 7.42 (2H, t, J = 7.9, 30,50-H);
7.60–7.74 (2H, d, J = 7.9, H-20,60) ppm. 13C NMR (CDCl3, 75 MHz): d
84.1; 121.2; 125.1; 129.1; 129.2; 136.7; 144.9; 168.4 ppm. Anal.
Calcd for C10H9NO2: C, 68.5; H, 5.1; N, 8.0. Found: C, 68.6; H, 5.1;
N, 8.0.
4.1.3.8.
2-Methyl-N-phenethyl-4-hydroxy-2,3-dehydro-c-lac-
tame 65. White crystals. Mp 128–130 °C. Yield: 64%. 1H NMR
(CDCl3, 300 MHz): d 1.89 (3H, s, CH3); 2.28 (1H, d, J = 11.1, OH);
2.93 (2H, t, J = 7.2, ArCH2); 3.55 (1H, dt, J = 14.2; 7.2, NCHaHb);
3.79 (1H, dt, J = 14.2; 7.2, NCHaHb); 5.04 (1H, d, J = 11.1, H-4);
6.49 (1H, d, J = 1.5, H-3); 7.17–7.35 (5H, m, HAr) ppm. 13C NMR
(CDCl3, 75 MHz): d 10.9; 34.7; 41.1; 82.1; 126.5; 128.6; 128.8;
137.2; 138.2; 139.0; 170.3 ppm. Anal. Calcd for C13H15NO2: C,
71.8; H, 6.9; N, 6.4. Found: C, 71.6; H, 6.9; N, 6.4.
4.1.3.2.
N-Phenethyl-4-hydroxy-2,3-dehydro-c-lactame
55. White crystals. Mp 124–126 °C. Yield: 59%. 1H NMR (CDCl3,
300 MHz): d 2,71 (2H, t, J = 7,2, ArCH2); 3,60 (1H, dt, J = 14,1; 7,2,
CHaHb); 3.85 (1H, dt, J = 14.1; 7.2, CHaHb); 5.48 (1H, d, J = 10.7,
H-4); 6.28 (1H, d, J = 5.1, H-2); 6.41 (1H, dd, J = 5.1; 1.6, H-3);
7.21–7.39 (5H, m, HAr) ppm. 13C NMR (CDCl3, 75 MHz): d 33.7;
41.0; 85.4; 126.0; 128.4; 128.3; 128.4; 142.4; 146.7; 170.6 ppm.
4.1.3.9. 2-Methyl-N-propylphenyl-4-hydroxy-2,3-dehydro-c-lac-
tame 66. White crystals. Mp 115–117 °C. Yield: 70%. 1H NMR
(CDCl3, 300 MHz): d 1.87 (3H, s, CH3); 1.91 (2H, m, CH2CH2CH2);

M. Sortino et al. / Bioorg. Med. Chem. 19 (2011) 2823–2834
2833
2.85 (1H, d, J = 10.9, OH); 3.65 (2H, t, J = 7.8, ArCH2); 3.33 (1H, dt,
J = 14.2; 7.2, NCHaHb); 3.58 (1H, dt, J = 14,2; 7,2, NCHaHb); 5.25
(1H, d, J = 10.9, H-4); 6.53 (1H, d, J = 1.5, H-3); 7.21–7.32 (5H, m,
counted, diluted with fresh medium, and added to 96-well micro-
titer plates (100 L/well) containing test materials (1 mg in
100 L in DMSO). Test plates were incubated for 2 days at 37 °C
in a 5% CO2 incubator. All treatments were performed in duplicate.
After incubation periods, cells were fixed by addition of 50 L of
l
l
H
Ar) ppm. 13C NMR (CDCl3, 75 MHz): d 10.9; 30.1; 33.3; 39.3;
81.7; 125.9; 128.3; 128.4; 137.2; 138.2; 141.4; 170.4 ppm. Anal.
Calcd for C14H17NO2: C, 72.6; H, 7.4; N, 6.1. Found: C, 72.7; H,
7.3; N, 6.0.
l
cold 50% aqueous TCA solution (4 °C for 60 min), washed 4–5
times with tap water, and air-dried. The fixed cells were stained
with 100 lL sulforhodamine B (SRB) (0.4% wt/vol in 1% acetic
4.1.3.10. 2-Methyl-N-butylphenyl-4-hydroxy-2,3-dehydro-
c-lac-
acid) for 15 min. Free SRB solution was then removed by rinsing
tame 67. White crystals. Mp 122–124 °C. Yield: 71%. 1H NMR
(CDCl3, 300 MHz): d 1.52–1.72 (4H, m, CH2(CH2)2CH2); 2.04 (3H,
s, CH3); 2.45 (2H, t; J = 6.6, ArCH2); 3.48 (1H, dt, NCHaHb); 3.53
(1H, dt, NCHaHb); 5.06 (1H, d, J = 1.5, H-4); 5,69 (1H, d, J = 1.5, H-
3); 7.15–7.35 (5H, m, HAr) ppm. 13C NMR (CDCl3, 75 MHz): d
10.9; 28.0; 28.7; 35.5; 39.2; 81.6; 125.8; 128.3; 128.4; 138.0;
138.0; 142.1; 170.3 ppm. Anal. Calcd for C15H19NO2: C, 73.4; H,
7.7; N, 5.7. Found: C, 73.6; H, 7.8; N, 5.7.
with 1% acetic acid (5ꢂ). The plates were then air-dried, the
bound dye was solubilized with 100 lL of 10 mM tris-base, and
the absorbance was determined at 515 nm using an ELISA plate
reader (Bio-Tek Instruments, Inc. Model ELX-800). Finally, the
absorbance values obtained with each of the treatment proce-
dures were averaged, and the average value obtained with the
zero day control was subtracted measuring in this way the rela-
tive cell growth or inviability in treated and untreated cells. From
the curves, growth inhibition and 50% inhibition of growth (GI50
)
4.2. Biological evaluation
were calculated.30
4.2.1. Microorganisms and media
4.2.4. Hemolysis assay
For the antifungal evaluation, reference strains from the Amer-
ican Type Culture Collection (ATCC), Rockville, MD, USA, and Cul-
ture Collection of Centro de Referencia en Micología-CEREMIC
(CCC), Facultad de Ciencias Bioquímicas y Farmacéuticas, Suipacha
531-(2000)-Rosario, Argentina, were used: C. albicans ATCC 10231,
C. tropicalis CCC 191, S. cerevisiae ATCC 9763, C. neoformans ATCC
32264, A. flavus ATCC 9170, A. fumigatus ATTC 26934, A. niger ATCC
9029, M. gypseum CCC 115, T. rubrum CCC 110, T. mentagrophytes
ATCC 9972. Strains were grown on Sabouraud-chloramphenicol
agar slants at 30 °C, maintained on slopes of Sabouraud-dextrose
agar (SDA, Oxoid), and subcultured every 15 days to prevent pleo-
morphic transformations. Inocula were obtained according to re-
ported procedures and adjusted to 1–5 ꢂ 103 colony forming
units (CFU)/mL.6,7
Stock solutions of compounds solubilized in DMSO were diluted
with sterile 5% glucose to yield final test concentrations ranging
from 250 to 0.24
lg/ml and added to 96-well microtiter plates
(100 L/well). Freshly obtained heparinized human red blood cells
l
(RBC) were washed three times by centrifugation (2500 rpm for
10 min) in isotonic PBS, pH 7.0 at rt. Two milliliters of RBC were
added to 50 mL of sterile 5% glucose to obtain a 4% suspension.
One-hundred microliters of RBC suspension was added to each
well, mixed, and plates incubated at 37 °C. The absorbance of the
liberated hemoglobin was measured spectrophotometrically at
540 nm in a Versa Max spectrophotometer. HC50 was defined as
the concentration at which there was 50% lysis in comparison with
drug-free value.31 The same amount of DMSO alone causes no lysis.
4.3. Computational methods
4.2.2. Antifungal susceptibility testing
MIC of each compound was determined by using broth microdi-
lution techniques following the guidelines of the CLSI for yeasts6
and for filamentous fungi.7 MIC values were determined in RPMI-
1640 (Sigma) buffered to pH 7.0 with MOPS (Sigma). Microtiter
trays were incubated at 35 °C for yeasts and hyalohyphomycetes
and at 28 °C for dermatophyte strains in a moist, dark chamber;
MICs were recorded at 48 h for yeasts, and at a time according to
the control fungus growth, for the rest of fungi. The susceptibilities
of the standard drugs Ketoconazole, Terbinafine, and Amphotericin
B were defined as the lowest concentration of drug which resulted
in total inhibition of fungal growth. For the assay, compound stock
solutions were twofold diluted with RPMI-1640 from 250 to
All the computational studies were carried out using density
functional theory (DFT) methods implemented in the GAUSSIAN 03
suite of programs.32 Correlation effects were included in the pres-
ent work using DFT with the Becke3-Lee-Yang-Parr (B3LYP)33–35
functional and the 6-31G(d) basis set. Thus, molecular geometry
optimizations were performed at the DFT (B3LYP/6-31G(d) level
of theory, using the GAUSSIAN 03 program employing standard basis
set with no modifications. Convergence criteria were according to
the limits imposed internally by GAUSSIAN 03. Low energy conforma-
tions were confirmed from a vibrational analysis using B3LYP/6-
31G(d) calculations.
Molecular electrostatic potentials (MEPs) were calculated by
using RB3LYP/6-31G(d) wave functions. MEP graphical presenta-
tions were created using the MOLEKEL program.36 The calculated
electronic properties for maleimides were: chemical potential
properties (electron affinity, ionization potential, hardness, elec-
tronegativity, electrophilicity and dipolar moments. These proper-
ties were calculated from B3LYP/6-31G(d) calculations. All the
calculations were performed in gas phase with the purpose of
obtaining the intrinsic properties of the maleimides studied, free
of any interaction. Calculations of the partition coefficient (log P)
were performed from CHEM OFFICE 10.0 program.37
0.24
l
g/ml (final volume = 100
l
L) and a final DMSO (Sigma) con-
L of inoculum suspension was
centration <1%. A volume of 100
l
added to each well with the exception of the sterility control where
sterile water was added to the well instead. MIC was defined as the
minimum inhibitory concentration of the compound, which re-
sulted in total inhibition of the fungal growth. MFC was deter-
mined by plating in duplicate 5 lL from each clear well of MIC
determinations, onto a 150-mm SDA plate and incubated at the
same conditions used previously. MFCs were determined as the
lowest concentration of each compound showing no growth on
the plates.
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