N-Phenyl and N-phenylalkyl-maleimides acting against Candida spp.: Time-to-kill, stability, interaction with maleamic acids

Article abstract of DOI:10.1016/j.bmc.2007.08.030

N-Phenyl and N-phenylalkyl maleimides (alkyl chain = (CH₂)n; n = 0-4) and their respective open derivatives (maleamic acids) were evaluated against Candida spp. with the microbroth dilution method following the guidelines of CLSI (formely NCCLS). MIC values of maleimides without pre-incubation and submitted to different pre-incubation times into the growth media, time-to-kill studies as well as a time-dependent UV-spectroscopy study of the maleimides in water, led to determine that maleimides display antifungal activities with their intact maleimide ring, being in addition their activities not dependent on the length of the alkyl chain. They are not only fungistatic but fungicidal with very low MICs and MFCs, displaying strong fungicide activities not only against standardized but also clinical isolates of Candida albicans and non-albicans Candida spp.

Full text of DOI:10.1016/j.bmc.2007.08.030

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 560–568
N-Phenyl and N-phenylalkyl-maleimides
acting against Candida spp.: Time-to-kill, stability,
interaction with maleamic acids
a
b
b
a,
*
´
ˆ
Maximiliano Sortino, Valdir Cechinel Filho, Rogerio Correa and Susana Zacchino
aFarmacognosia, Facultad de Ciencias Bioquı´micas y Farmace´uticas, Universidad Nacional de Rosario,
Suipacha 531, 2000 Rosario, Argentina
b
ˆ
´
Nu´cleo de Investigac¸o˜es Quı´mico-Farmaceuticas (NIQFAR), Universidade do Vale do Itajaı (UNIVALI),
88.302-202, Itajaı´ (SC), Brazil
Received 4 June 2007; revised 14 August 2007; accepted 20 August 2007
Available online 24 August 2007
Abstract—N-Phenyl and N-phenylalkyl maleimides (alkyl chain = (CH2)n; n = 0–4) and their respective open derivatives (maleamic
acids) were evaluated against Candida spp. with the microbroth dilution method following the guidelines of CLSI (formely NCCLS).
MIC values of maleimides without pre-incubation and submitted to different pre-incubation times into the growth media, time-to-
kill studies as well as a time-dependent UV-spectroscopy study of the maleimides in water, led to determine that maleimides display
antifungal activities with their intact maleimide ring, being in addition their activities not dependent on the length of the alkyl chain.
They are not only fungistatic but fungicidal with very low MICs and MFCs, displaying strong fungicide activities not only against
standardized but also clinical isolates of Candida albicans and non-albicans Candida spp.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Although amphotericin B, fluocytosine and azoles such
as ketoconazole, fluconazole or itraconazole have been
considered efficient for the treatment of candidosis,
some of these drugs show toxicity, produce recurrence
because they are fungistatic and not fungicide, or lead
to development of resistance due in part to the intensive
prophylactic use of antifungal drugs.6
Candida spp. are usually present in skin, mucous mem-
branes, gastrointestinal tract, and genitourinary organs
of healthy human beings. Nevertheless, they produce seri-
ous infections in patients with immunodeficiencies such as
individuals submitted to organ transplantations or anti-
neoplastic chemotherapy, those suffering the acquired
immunodeficiency syndrome (AIDS), extremely aged per-
sons and patients in intensive care units, among others.1
There is, therefore, a clear need for the discovery of new
structures with anti-Candida activity, which could con-
stitute alternatives for the management of candidosis
mainly in immunocompromised hosts.7
Candidosis has been shown to be the fourth most com-
mon nosocomial blood stream infection, Candida albi-
cans representing more than 60% of all isolates from
clinical infections.2,3 In the last years, there has been
an increase in the percentage of Candida infections
caused by non-albicans Candida spp. such as C. glabrata,
C. tropicalis and C. parapsilosis, and in a lower percent-
age C. guillermondii, C. lusitaneae, C. krusei, C. dublini-
ensis, and C. rugosa, among others.1,4,5
As part of our ongoing project on the detection of anti-
fungal compounds,8–12 we recently reported that N-phe-
nyl and N-phenylalkylmaleimides (Fig. 1) possess strong
antifungal activities when tested in agar dilution assays
against a panel of 11 strains of clinically important fungi
including Candida spp.13
In contrast to results obtained with N-phenyl and
N-phenylalkyl-3,4-dichloromaleimides, the antifungal
activity of N-phenyl and N-phenylalkylmaleimides ap-
peared not to be dependent on the presence or on the
length of the alkyl chain. Nevertheless, the fact that they
Keywords: Maleimides; Antifungal; Stability; Candida.
*
Corresponding author. Fax: +54 341 4375315; e-mail: szaabgil@
citynet.net.ar
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.030

M. Sortino et al. / Bioorg. Med. Chem. 16 (2008) 560–568
561
showed Minimum Inhibitory Concentrations (MICs)
lower than 12.5 lg/mL, 6.25 lg/mL or 3.25 lg/mL in
40, 30 or 18 tests, respectively, out of the 55 agar dilu-
tion tests performed13 prompted us to continue with
the study of the antifungal behavior of N-phenyl and
N-phenylalkylmaleimides, results that are presented
here. The MICs and Minimum Fungicidal Concentra-
tions (MFCs) of maleimides along with time-to-kill val-
ues were determined at first against C. albicans ATCC
10231, by using the Clinical and Laboratory Standards
Institute (CLSI; formerly National Committee for Clin-
ical and Laboratory Standards, NCCLS) approved ref-
erence method for antifungal susceptibility of yeasts.14
At last, to gain insight into a future clinical application
of these structures, MICs and MFCs of maleimides
against an extended panel of clinical isolates of C. albi-
cans and non-albicans Candida spp. were determined.
2. Results and discussion
2.1. Chemistry
The syntheses of N-substituted maleamic acids and
maleimides were achieved by a two steps procedure pre-
viously reported.17 Reactions of maleic anhydride 1 with
the appropriate amine (Ph-(CH2)n-NH2; n = 0–4) in
CHCl3 at room temperature (rt) generated the corre-
sponding N-substituted maleamic acids 2–6 in near
quantitative yields. Compounds 5 and 6 have not been
reported up to date in the literature. Maleamic acids
were subsequently cyclized to the corresponding N-phe-
nyl and N-phenylalkylmaleimides 7–11 by heating 2–6 in
the presence of acetic anhydride containing catalytic
amounts of sodium acetate (Scheme 1).
Then, considering the possibility that in the aqueous cul-
ture media, the maleimide ring could undergo hydrolysis
to maleamic acids as it was demonstrated for non N-
substituted maleimide in alkaline medium and under cer-
tain conditions15 (Fig. 2), we performed a study on the
time-dependant stability of each compound in the growth
media to determine if the maleimides under study (with
the intact ring) are the true responsible for the antifungal
activity or instead, their open analogues contribute to
their antifungal behavior. In addition, the type of interac-
tion (synergistic, antagonistic or additive)16 that could ex-
ist between maleamic acids and the respective maleimide,
and a time-dependent UV spectroscopy study of the five
maleimides in water were performed too.
1H and 13C NMR spectra were coincident with the ex-
pected structures. A variation of the NMR signals was
observed in the first step due to the lost of symmetry
in the reaction. Cyclization in the second step was pro-
ven by NMR signals in agreement with the symmetrical
structures of N-arylmaleimides obtained.
2.2. MIC and MFC determinations
O
The MICs of maleamic acids 2–6 and maleimides 7–11
were determined against C. albicans ATCC 10231 by
using the standardized microbroth dilution method M-
27 A2 for yeasts according to the guidelines of the CLSI
(formerly NCCLS)14 which assures confident and repro-
ducible results.18
(CH2)n
N
O
n = 0-4
Figure 1. General structure of N-phenyl and N-phenylalkylmaleimides.
Results showed that maleimides 7–11 possessed strong
antifungal activities against C. albicans (all
MICs = 3.9 lg/mL; Table 1, first row), also corroborat-
ing our previous findings that the activity did not de-
pend on the length of the alkyl chain. Regarding
maleamic acids 2–6, they did not possess any antifungal
activity at concentrations up to 250 lg/mL. MFCs of
maleimides 7–11 were accomplished afterwards by
O
O
-
OH
NH2
O-
NH
O
O
Figure 2. Opening of maleimide to maleamic acid under basic
conditions.
Scheme 1.

Table 1. Minimum inhibitory concentration (MIC) and Minimum fungicidal concentration (MFC) values (lg/mL) of N-phenyl- and N-phenylalkyl-maleamic acids and maleimides acting against Candida
spp.
O
O
(CH2)n
N
H
(CH2)n
N
OH
O
O
A
B
Candida spp.
Voucher number
Maleamic acids (type A)
Maleimides (type B)
St. drug
n = 0
2
n = 1
3
n = 2
4
n = 3
5
n = 4
6
n = 0
7
n = 1
8
n = 2
9
n = 3
10
n = 4
11
Amp B
Ket
MIC
MIC
MIC
MIC
MIC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MIC
C. albicans 1
C. albicans 2
C. albicans 3
C. albicans 4
C. albicans 5
C. albicans 6
C. albicans 7
ATCC 10231
C 125-2000
C 126-2000
C 127-2000
C 128-2000
C 129-2000
C 130-2000
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
3.9
7.8
3.9
7.8
3.9
7.8
3.9
7.8
3.9
7.8
1.0
0.5
0.48
0.97
0.97
0.97
0.48
0.48
1.95
3.9
0.97
0.97
1.95
1.95
1.95
0.97
1.95
3.9
1.95
3.9
1.95
7.81
3.9
0.97
0.97
1.95
3.9
1.95
1.95
3.9
0.97
0.97
1.95
0.97
0.97
1.95
1.95
1.95
3.9
0.78
1.56
0.78
1.56
0.78
0.39
6.25
1.56
6.25
6.25
12.5
6.25
1.95
3.9
3.9
3.9
1.95
3.9
7.81
1.95
1.95
7.81
3.9
1.95
1.95
3.9
1.95
1.95
3.9
1.95
1.95
1.95
1.95
0.97
1.95
C. glabrata
C. parapsilosis
C. lusitaniae
C. colliculosa
C. krusei
C 115-2000
C 124-2000
C 131-2000
C 122-2000
C 117-2000
C 123-2000
C 137-1997
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
1.95
1.95
3.9
3.9
3.9
1.95
1.95
1.95
1.95
3.9
1.95
3.9
1.95
3.9
3.9
3.9
1.95
3.9
3.9
3.9
1.95
1.95
3.9
3.9
7.8
0.39
0.78
0.39
0.39
0.39
0.78
0.12
1.56
0.78
25
7.8
3.9
3.9
3.9
7.8
1.95
3.9
7.8
32.25
7.8
3.9
3.9
1.95
1.95
1.95
7.81
7.81
3.9
1.95
3.9
7.81
7.8
3.9
7.8
0.78
50
1.95
3.9
3.9
15.75
15.75
15.75
7.81
32.25
C. kefyr
3.9
15.75
3.9
32.25
3.9
15.75
3.9
32.25
7.81
32.25
1.95
15.75
0.78
0.12
C. tropicalis
15.75
´
ATCC, American Type Culture Collection (Rockville, MD, USA); C, Centro de Referencia en Micologıa (CEREMIC, Rosario, Argentina); Amp B, amphotericin B; Ket, ketoconazole.

M. Sortino et al. / Bioorg. Med. Chem. 16 (2008) 560–568
563
sub-culturing an aliquot from MIC wells showing no
growth, onto drug-free agar plates. MFCs for all tested
maleimides were 7.8 lg/mL (Table 1, first row).
mining the MICs of maleimide 7 at different pre-incuba-
tion times. Each line, which contains a series of twofold
dilutions (1000–0.98 lg/mL) of the tested maleimide,
was prepared in 7 consecutive days beginning for bot-
tom to top. At the end of the seventh day, plates were
inoculated with C. albicans ATCC 10231 and incubated
24 h at 37 ꢁC. Results showed that the MIC of malei-
mide 7 was 3.9 lg/mL up to 2 days of contact with the
growth media, suggesting that during this period the
maleimide would remain intact. The MIC gently raised
to a value of 500 lg/mL after 7 days of pre-incubation
suggesting a gradual opening of the ring. Similar results
were obtained for all maleimides tested (Table 2).
2.3. Time-to-kill studies
The times needed for compounds 7–11 to kill C. albicans
(time-to-kill studies), were determined by enumerating
viable cells of fungal suspensions exposed to 1· MFC
of each compound at different exposition times (0–
24 h) as explained in the Experimental section. Plots of
UFC/mL versus time of incubation for maleimides 7–
11 are shown in Figure 3 (times longer than 120 min
are not shown). Results revealed that the fungus was
killed by all compounds within 90 min of incubation.
2.5. Interaction of maleimides with their corresponding
maleamic acids
2.4. MICs of maleimides submitted to different
pre-incubation times
For a correct interpretation of the above results, it
seemed in order to determine if the interaction of malei-
mides and their respective maleamic acids produce syn-
ergism, antagonism, or no effect in the case of acting
together as antifungal agents.
Each maleimide was pre-incubated at 37 ꢁC into growth
media during 0–7 days prior to inoculation. Figure 4
shows the 96-wells microplate that was used for deter-
Chequerboard experiments were used to determine the
Fractional Inhibitory Concentration (FIC, in lg/mL)
of the combination of each maleimide with its corre-
spondent maleamic acid for C. albicans ATCC 10231
and to calculate the Fractional Inhibitory Concentration
Index (FICI). FICI value for each maleimide/maleamic
acid was interpreted as follows: FICI 6 0.5, synergistic;
0.5 < FICI 6 4, indifferent; FICI > 4, antagonistic.19
30000
25000
7
8
20000
9
15000
10
11
10000
Results showed that each maleimide and its maleamic
acid do not interact with each other and therefore only
an additive effect can be expected (Table 3).
5000
0
0
10
20
30
40
50
60
70
80
90
100 110 120
Table 2. MIC values (lg/mL) against Candida albicans ATCC 10231
of maleimides 7–11, without pre-incubation (0 days) and with different
times (1–7 days) of pre-incubation at 37 ꢁC in the growth media, using
the CLSI (formerly NCCLS) M27 A2 method
time (min)
Figure 3. Time-to-kill curves of maleimides 7–11 at 1 · MFC against
C. albicans ATCC 10231.
T.P.
7
8
9
10
11
0
1
2
3
4
5
6
7
3.90
3.90
3.90
7.80
15.75
62.50
125
3.90
3.90
7.80
7.80
15.75
125
3.90
3.90
7.80
7.80
31.25
125
3.90
3.90
3.90
15.75
15.75
62.5
125
3.90
3.90
3.90
15.75
31.25
125
Concentration (μg/ml)
1000 500 250 125 62.5 31.25 15.75 7.80 3.90 1.95 0.98
0
0
1
2
3
4
5
6
7
250
500
250
500
250
1000
500
1000
T.P., Time of pre-incubation of maleimides in the growth media.
Table 3. Fractional Inhibitory Concentration Indexes (FICI) of
maleimides 7–11 in combination with maleamic acids 2–6 against
Candida albicans ATCC 10231
Combination
of Compounds
FICI
Result
7/2
8/3
0.80
1.125
0.75
1.00
0.75
Indifferent
Indifferent
Indifferent
Indifferent
Indifferent
Figure 4. Microtiter plate showing MIC values in lg/mL (white line)
of N-phenylmaleimide 7 without pre-incubation (0 days) and with
different times (1–7 days) of pre-incubation into the growth media,
against Candida albicans ATCC 10231 using the CLSI (formerly
NCCLS) M27 A2 method.
9/4
10/5
11/6

564
M. Sortino et al. / Bioorg. Med. Chem. 16 (2008) 560–568
Considering that each maleimide and its open derivative
are indifferent when co-existing in a solution, mixtures
of maleimides/maleamic acids at different concentrations
were prepared and their MICs were determined and
compared with those of Table 2, in order to estimate
the % of hydrolysis that would have been produced at
the different pre-incubation times. Results showed
(Table 4) that for all pairs of compounds tested,
maleimides with up to 48 h of pre-incubation (Table 2)
could have suffered a maximum of 10–20% of hydrolysis
(see Table 4). According to these results, the hydrolysis
of maleimides 7–11 at the time required to kill the fungi
(90 min) would have been negligible.
antifungal activity, prompted us to test compounds 7–11
in a second panel conformed by thirteen clinical isolates
of the Candida genus. Six of them were C. albicans and
seven were non-albicans Candida spp., including C. trop-
icalis, C. glabrata, C. parapsilopsis, C. krusei, C. kefyr,
C. collicullosa and C. lusitaneae. The inclusion of non-
albicans Candida spp. was due to the high incidence of
infections by non-albicans Candida spp. observed in re-
cent years in immunocompromised patients as it was
stated in the Introduction.1
Results showed (Table 1) that maleimides 7–11 pos-
sessed strong antifungal activities against all the tested
Candida strains with MICs between 0.48 and 3.90 lg/
mL, values that are similar to those of amphotericin B
(0.12—1.56 lg/mL) and in some cases better that those
of ketoconazole (0.12–6.25 lg/mL). In addition, these
maleimides killed C. albicans and most non-albicans
Candida strains with MFCs between 0.97 and 7.8 lg/
mL and 1.95–7.8 lg/mL, respectively. The maximum
MFC value observed was 32.25 lg/mL.
2.6. Stability of maleimides 7–11 in water
Considering that the culture media is an aqueous solu-
tion, we corroborated the above results by analyzing
the stability of maleimides 7–11 in water by spectropho-
tometric measurements. The UV spectra of maleimides
dissolved in water was plotted (250–350 nm) at intervals
from 2 h to 3 days taking into account that a typical
absorption wavelength for a cyclic imide group is
300 nm, while an amide group does not absorb here.
So, a decrease in the absorption peak at 300 nm should
demonstrate the cleavage of the imide bond.15
3. Conclusion
These studies deepen the knowledge of the behavior of
N-phenyl- and N-phenylalkylmaleimides 7–11 as anti-
candidal agents demonstrating that (a) they display anti-
fungal activities with their intact maleimide ring; (b) the
opening of the maleimide ring would lead to a loss of
antifungal activity; (c) they are not only fungistatic but
fungicidal with very low MICs and MFCs, killing the
fungi in about 90 min; (d) they display strong fungicidal
activities not only against standardized but also clinical
isolates of C. albicans and non-albicans Candida spp.,
enhancing the importance of their antifungal behavior.
Spectra of pure maleimides 7–11 and pure maleamic
acids 2–6 at time = 0 h are presented in Figure 5a–e. Fig-
ure 5a0–e0 shows the spectra of the aqueous solutions of
maleimides after 0, 2, 24, 48, and 72 h. In these figures, it
is observed that all maleimides spectra were almost iden-
tical at the beginning of the experiment (0 h) as well as
after 2 h, suggesting that at the time needed for killing
the fungus (90 min), the hydrolysis would be almost neg-
ligible for N-phenylmaleimides and N-phenylalkylmalei-
mides 7–11, confirming that maleimides 7–11 act with
their intact maleimide ring against C. albicans.
Taking into account that fungicidal compounds, mainly
acting against Candida spp., are highly needed, these re-
sults open an important possibility for the development
of new promising anticandidal drugs.
2.7. Antifungal activity of maleimides against clinical
isolates of the Candida genus
The high activity shown by maleimides against the
ATCC standardized C. albicans strain and the demon-
stration that they are truly responsible for the observed
4. Experimental
4.1. Chemistry
Table 4. MIC values (lg/mL) of combinations of maleimides 7–11
(100% ! 0%) with maleamic acids 2–6 (0% ! 100%)
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
%
Maleimide/Maleamic acid
13C NMR spectra were recorded on
a Bruker
Maleimide Maleamic 7/2
Acid
8/3
9/4
10/5
11/6
200 MHz (Bruker, Karlsruhe, Germany). Compounds
were dissolved in deuterated solvents from commercial
sources (Sigma) with tetramethylsilane (TMS) as the
internal standard. Chemical shifts are reported in ppm
(d) relative to the solvent peak (CHCl3 in CDCl3 at
7.26 ppm for protons and at 77.0 ppm for carbons). Sig-
nals are designated as follows: s, singlet; d, doublet; dd,
doublets of doublets; t, triplet; dt: doublets of triplets;
m, multiplet; q, quartet; quint, quintuplet; b, broad.
All gas chromatograms were obtained in a CG-MS Tur-
bo Mass Perkin Elmer, column PE1 30m · 0.25 mm of
inner diameter, film 0.1 lm, ionization energy 70 eV.
100
90
80
70
60
50
40
30
20
10
0
0
10
20
30
40
50
60
70
80
90
100
3.90
3.90
3.90
7.80
7.80
7.80
7.80
3.90
3.90
3.90
7.80
7.80
7.80
7.80
3.90
3.90
3.90
7.80
7.80
7.80
3.90
3.90
7.80
7.80
7.80
7.80
3.90
3.90
7.80
7.80
7.80
15.75
15.75 15.75 15.75
15.75 15.75 15.75 15.75 15.75
31.25 31.25 31.25 31.25 31.25
62.5
750
62.5
750
31.25 62.5
1000 1000
62.5
1000

M. Sortino et al. / Bioorg. Med. Chem. 16 (2008) 560–568
565
0.5
a
a'
1
0.8
0.6
0.4
0.2
0
0.45
0.4
0 h
2 h
24 h
48 h
72 h
2
7
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
250
250 260 270 280 290 300 310 320 330 340 350
270
290
310
330
350
λ(nm)
λ (nm)
1.2
1
1.2
b'
c'
b
c
1
0.8
0.6
0.4
0.2
0
0 h
2h
24 h
48 h
72 h
3
8
0.8
0.6
0.4
0.2
0
250
270
290
310
330
350
250
270
290
310
330
350
λ(nm)
λ (nm)
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
4
9
0 h
2 h
24 h
48 h
72 h
250
270
290
310
330
350
250
270
290
310
330
350
λ(nm)
λ (nm)
0.6
0.6
d'
e'
d
e
0.5
0.4
0.3
0.2
0.1
0
0.5
0.4
0.3
0.2
0.1
0
0 h
2 h
24 h
48 h
72 h
5
10
250
270
290
310
330
350
250
270
290
310
330
350
(nm)
λ
λ (nm)
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0 h
2 h
24 h
48 h
72 h
6
11
250
270
290
310
330
350
250
270
290
310
330
350
λ(nm)
λ (nm)
Figure 5. (a–e) UV spectra of each maleimide and its respective maleamic acid in recently prepared water solutions. In detail: (a) N-
phenylamaleimide 7 and N-phenylmaleamic acid 2. (b) N-bencylmaleimide 8 and N-bencylmaleamic acid 3. (c) N-phenethylmaleimide 9 and
N-phenethylmaleamic acid 4. (d) N-phenylpropylmaleimide 10 and N-phenylpropylmaleamic acid 5. (e) N-phenylbutylmaleimide 11 and N-
phenylbutylmaleamic acid 6. (a0–e0) UV spectra of each maleimide (250 lg/mL) 7–11 in aqueous solutions, after different times of incubation
(0 to 72 h).

566
M. Sortino et al. / Bioorg. Med. Chem. 16 (2008) 560–568
UV–vis spectra were recorded in a Beckman DU-640
spectrophotometer (USA). Elemental analyses were per-
formed on a Perkin-Elmer 2400 series II analyzer (Nor-
walk, CT, USA). 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 apparatus (U.K.) and were uncorrected.
4.2.4. N-Phenylpropyl maleamic acid (5). White crystals;
yield 90%; mp: 101.3–101.6 ꢁC. UV (MeOH): not maxi-
mum appears from 350 to 250 nm; at k < 310 the absor-
bance increases rapidly up to 250 nm. 1H RMN
(200 MHz) d 1.95 (2H, dt (appearing as a quint),
J = 7.4 Hz, H-20), 2.69 (2H, t, J = 7.2 Hz, H-30), 3.41
(2H, dt (appearing as a q) J = 7.0 Hz, H-10), 6.28 (1H,
d, J = 12.8, H-2), 6.41 (1H, d, J = 12.8 Hz, H-3), 7.20
(5H, m, HAr), 7.81 (1H, bs, NH); 13C RMN (50 MHz)
d 29.9 (C-30), 33.0 (C-20), 40.1 (C-10), 126.0 (C-400),
128.3 (C-200,600), 128.4 (C-300,500), 132.7 (C-2), 135.3 (C-
3), 140.9 (C-10), 166.3 (CONHR), 166.7 (COOH); GC:
tR = 12.36 min. EIMS m/z 215 (M+-H2O) (25), 117
(100), 111 (45), 91 (38), 82 (25). Anal for C13H15NO3.
Calcd (%): C, 66.94; H, 6.48; N, 6.02. Found (%): C,
66.74; H, 6.45; N, 6.04.
4.2. General procedure for the synthesis of maleamic acids
2–6
The compounds were obtained according to published
methodologies17 with yields ranging from 90 to 98%.
Reaction mixtures containing maleic anhydride (1)
(500 mg, 5.1 mmol in CHCl3 (5 mL)) and equimolar
amounts of the appropriate amine (Ph-(CH2)n-NH2;
n = 0-4) dissolved in CHCl3 (1 mL) were stirred during
1–2 h. The solid which precipitated out of the reaction
mixture was filtered and washed thoroughly with water.
N-phenyl maleamic acid 2 and N-phenylalkylmaleamic
acids 3 and 4 have been previously reported20,21. To
our knowledge, their spectroscopic data were not de-
scribed previously. For the numbering of maleamic
acids, see Scheme 1.
4.2.5. N-Phenylbutyl maleamic acid (6). White crystals;
yield 92%; mp: 96.7–97.3 ꢁC. UV (MeOH): not maxi-
mum appears from 350 to 250 nm; at k < 310 the absor-
bance increases rapidly up to 250 nm. 1H RMN
(200 MHz) d 1.67 (4H, m, H-20,30), 2.65 (2H, t,
J = 7.2 Hz, H-40), 3.39 (2H, dt (appearing as a q)
J = 6.8 Hz, H-10), 6.28 (1H, d, J = 12.8 Hz, H-2), 6.43
(1H, d, J = 12.8 Hz, H-3), 7.24 (5H, m, HAr), 7.76
13
4.2.1. N-Phenyl maleamic acid (2). White crystals; yield
98%; mp: 203.0–203.6 ꢁC. UV (MeOH) kmax (log e): 265
(2.9).1H RMN (200 MHz) d 6.33 (1H, d, J = 12.3, H-2),
6.51 (1H, d, J = 12.3 Hz, H-3), 7.45 (5H, m, HAr); 13C
RMN (50 MHz) d 119.4 (C-200,600), 123.8 (C-400), 128.7
(C-300,500), 130.3 (C-100), 131.4 (C-2), 138.5 (C-3), 163.2
(CONHR), 163.2 (COOH); GC: tR = 9.18 min. EIMS
m/z 173 (M+-H2O) (100), 129 (28), 93 (27), 54 (52). Anal
for C13H15NO3. Calcd (%): C, 62.82; H, 4.74; N, 7.33.
Found (%): C, 62.93; H, 4.75; N, 7.34.
(1H, bs, NH); C RMN (50 MHz) d 28.1 (C-30), 28.5
(C-20), 35.3 (C-40), 40.3 (C-10), 125.8 (C-400), 128.2 (C-
200,600), 128.3 (C-300,500), 135.2 (C-2), 135.2 (C-3), 141.8
(C-100), 166.2 (C-4), 166.3 (C-1); GC: tR = 13.55 min.
EIMS m/z 229 (M+-H2O; 47%), 131 (35), 110 (30), 91
(100), 82 (24). Anal for C14H17NO3. Calcd (%): C,
68.00; H, 6.93; N, 5.66. Found (%): C, 68.16; H, 6.94;
N, 5.68.
4.3. General procedure for the synthesis of maleimides 7–
11
4.2.2. N-Benzyl maleamic acid (3). White crystals; yield
96%; mp: 142.2–142.9 ꢁC. UV (MeOH): not maximum
appears from 350 to 250 nm; at k < 310 the absorbance
A reaction mixture containing the appropriated malea-
mic acid in 5 mL of acetic anhydride and 100 mg of so-
dium acetate was heated for 2 h under reflux, according
to previous reports.17,22 The reaction was cooled and
quenched with water. The aqueous solution was ex-
tracted with Et2O, dried with Na2SO4, filtered, and the
solvent was evaporated. N-phenylmaleimide 7 and N-
phenylalkylmaleimides 8–11 were obtained with yields
ranging from 45% to 98%. Their spectroscopic data were
identical to reported data.23
1
increases rapidly up to 250 nm. H RMN (200 MHz) d
4.47 (2H, s, H-10), 6.26 (1H, d, J = 12.6, H-2), 6.41
(1H, d, J = 12.6 Hz, H-3), 7.45 (5H, m, HAr); 13C
RMN (50 MHz) d 44.7 (C-10), 128.6 (C-400), 129.0 (C-
200,600), 129.7 (C-300,500), 130.9 (C-2), 133.4 (C-3), 134.2
(C-100), 167.7 (CONHR), 176.8 (COOH); GC:
tR = 19.0 min. EIMS m/z 106 (100), 91 (65). Anal for
C13H15NO3. Calcd (%): C, 64.38; H, 5.40; N, 6.83.
Found (%): C, 64.47; H, 5.41; N, 6.85.
4.4. Antifungal evaluation
4.2.3. N-Phenylethyl maleamic acid (4). White crystals;
yield 96%; mp: 138.0–138.7 ꢁC. UV (MeOH): not maxi-
mum appears from 350 to 250 nm; at k < 310 the absor-
bance increases rapidly up to 250 nm. 1H RMN
(200 MHz) d 2.92 (2H, t, J = 7.2 Hz, H-20), 3.68 (2H,
dt (appearing as a q), J = 7.0 Hz, H-10), 6.10 (1H, d,
J = 12.8, H-2), 6.36 (1H, d, J = 12.8 Hz, H-3), 7.20
4.4.1. Microorganisms and media. Fungal species from
the American Type Culture Collection (ATCC, Rock-
ville, MD, USA), and clinical isolates from the Centro
´
de Referencia en Micologıa (C, CEREMIC, Rosario,
Argentina) were used. The panel included 14 strains of
Candida spp. (seven of them were C. albicans and seven
were non-albicans Candida spp.); voucher specimens are
detailed in Table 1. Strains were grown on Sabouraud-
chloramphenicol agar slants for 24 h at 37 ꢁC, and main-
tained on slopes of Sabouraud-dextrose agar (SDA,
Oxoid). Inocula of cell suspensions were obtained
according to reported procedures and adjusted to 1–
5 · 104 colony forming units (CFU)/mL.14
13
(5H, m, HAr); C RMN (50 MHz) d 34.7 (C-20), 41.7
(C-10), 126.7 (C-400), 128.6 (C-200,600), 128.7 (C-300,500),
132.5 (C-2), 135.4 (C-3), 138.0 (C-100), 166.3 (CONHR),
166.7 (COOH); GC: tR = 10.71 min. EIMS m/z 201
(M+-H2O) (25), 110 (34), 104 (100), 91 (40). Anal for
C13H15NO3. Calcd (%): C, 65.74; H, 5.98; N, 6.39.
Found (%): C, 65.87; H, 6.99; N, 6.40.

M. Sortino et al. / Bioorg. Med. Chem. 16 (2008) 560–568
567
4.4.2. MIC and MFC determinations of maleimides and
maleamic acids. Stock solutions of compounds (100 lL)
were diluted twofold with RPMI-1640 (Sigma, St. Louis,
MO, USA) (final concentrations 250–0.98 lg/mL and a
final DMSO concentration 62%) according to the
M27-A2 method recommended by CLSI (formerly
NCCLS),14 with the modification that the inoculum
used was 1–5 · 104 instead of 1–5 · 103 CFU/mL.
About 100 lL of the inoculum suspension was added
to each well. Ketoconazole (Janssen Pharmaceutical,
Titusville, NJ, USA) and amphotericin B (Sigma) were
used as positive controls. MIC was defined as the lowest
concentration of a compound that induced total inhibi-
tion of growth as read visually. MFC was determined by
plating in duplicate 5 lL from each clear well of MIC
determinations, onto a 150-mm SDA plate. After 48 h
at 37 ꢁC, MFCs were determined as the lowest concen-
tration of each compound showing no growth.
with C. albicans (1–5 · 104 CFU/mL) in a 200 lL final
volume.
The FICI was calculated by adding the FIC of each
maleimide (M) to the FIC of maleamic acid (MA) for
each combination using the following formula:28
FICI ¼ FICM þ FICMA
¼ ðMICM combination=MICM alone
Þ
þ ðMICMA combination=MICMA aloneÞ:
The experiments were performed by duplicate and re-
sults are the mean of both values.
4.4.6. MIC determinations of different mixtures of
maleimide/maleamic acids. Stock solutions of mixtures
at different ratios (0–100%/100–0%) of each pair of
maleimide/maleamic acid, namely 7/2, 8/3, 9/4, 10/5,
and 11/6 (100 lL), were twofold diluted with RPMI-
1640 to obtain a series of concentrations (250–0.98 lg/
mL and a final DMSO concentration 62%). About
100 lL of inoculum suspension was added to each well
and MIC was determined according to CLSI guidelines
(formerly NCCLS) as explained above.
4.4.3. MIC determinations of maleimides pre-incubated in
the growth media. Five empty 96-microwell plates were
used for this assay. Each row of the plates was filled-in
during 7 consecutive days (see Fig. 4). (a) Day 0: two-
fold diluted series (1000–0.98 lg/mL) of each maleimide
(with all ingredients as to determine MIC except the
inoculum) were put into the first row of each plate,
therefore having the five plates with only the first row
filled-in. (b) Day 1: a second series of dilutions of each
maleimide was prepared and put into the second row
of the plates. (c) Days 2–7: The same procedure was per-
formed during six more consecutive days at the same
time of the day on the six following rows.
4.4.7. Spectrophotometric experiments. The absorption
spectra were obtained scanning the samples between
250 and 350 nm with a Beckman DU-640 spectropho-
tometer. Each experiment was started with a solution
of each maleimide in water at 250 lg/mL (with the addi-
tion of minimal quantities of DMSO to assure their sol-
ubility). Beer’s law was confirmed for each maleimide in
a range between 0.10 and 1000 lg/mL.
At the end of the seventh day, all wells of each plate
were inoculated with C. albicans ATCC 10231 (inocula
size 1–5 · 104 CFU/mL) and put at 37 ꢁC (Dalvo Incu-
bator, Buenos Aires) for 24 h. A control of growth
was added to each row. The MIC of each maleimide
tested with 0–7 days pre-incubation times was recorded.
The experiment was performed in duplicate.
An aliquot was quickly transferred to a quartz cell kept
pre-incubated (37 ꢁC) in the thermostatic cell-holder of
the spectrophotometer. The progress of hydrolysis was
followed by monitoring the variability of the peak at
300 nm at regular intervals (0, 2, 24, 48, and 72 h). UV
spectra of maleamic acids were obtained under the same
conditions as maleimides without incubation.
4.4.4. Time-to-kill assays. The time-course of fungicidal
activity of each maleimide was determined by enumerat-
ing viable cells in 5 mL of RPMI-1640 medium contain-
ing an inoculum of C. albicans ATCC 10231 (2–2.5 · 104
CFU/mL) and 1· MFC of the tested compound. The
culture vials were incubated with agitation at
37 ꢁC.24,25 Samples of 10 lL were removed from each
vial at fixed times (0, 30, 60, 90, and 120 min, 4, 8, 12,
and 24 h), diluted appropriately if necessary, and plated
onto drug-free SDA plates. Colony were counted after
incubation of the plates at 37 ꢁC for 24–48 h. All time-
to kill experiments were conducted in duplicate.
Acknowledgments
S.Z. and M.S. are grateful to ANPCyT (PICTR 260)
and CONICET, CYTED (RIBIOFAR network/RT
0284) and UNR (1BIO133). V.C.F. thanks CNPq/
Brazil.
References and notes
4.4.5. Chequerboard method. Interactions of maleimides
7–11 with maleamic acids 2–6 when acting against C.
albicans ATCC 10231 were determined by using the
microbroth dilution chequerboard method with incuba-
tions at 37 ꢁC for 24 h.26,27 Seven twofold dilutions of
each maleimide and seven twofold dilutions of maleamic
acids at such a range of drug concentrations that encom-
passed the MIC of each compound used were tested in
combination. Microbroth dilution wells were inoculated
1. Pfaller, M.; Diekema, D. Clin. Microb. Rev. 2007, 20, 133.
´
2. Beck-Sague, C. M.; Jarvis, W. R. The National Nosoco-
mial Infections Surveillance System. J. Infect. Dis. 1993,
167, 1247.
3. Edmond, M. B.; Wallace, S. E.; McClish, D. K.; Pfaller,
M. A.; Jones, R. N.; Wenzel, R. P. Clin. Infect. Dis. 1999,
29, 239.
4. Pfaller, M.; Diekema, D. J. Clin. Microb. 2004, 42, 4419.
5. Nucci, M.; Marr, K. Clin. Infect. Dis. 2005, 41, 521.

568
M. Sortino et al. / Bioorg. Med. Chem. 16 (2008) 560–568
6. Cuenca-Estrella, M.; Rodriguez, D.; Almirante, B.; Mor-
gan, J.; Planes, A. M.; Almela, M.; Mensa, J.; Sanchez, F.;
17. Kalgutkar, A.; Crews, B. C.; Marnett, L. J. J. Med. Chem.
1996, 39, 1692.
´
´
Ayats, J.; Gimenez, M.; Salvado, M.; Warnock, D.;
18. Odds, F.; Motyl, M.; Andrade, R.; Bille, J.; Canton, E.;
Cuenca-Estrella, M.; Davidson, A.; Durussel, C.; Ellis, D.;
Foraker, E.; Fothergill, A.; Ghannoum, M.; Giacobbe, R.;
Pahissa, A.; Rodriguez-Tudela, J. L. On behalf of the
Barcelona Candidemia Project Study Group. J. Antimic-
rob. Chemother. 2005, 55, 194.
`
Gobernado, M.; Hadke, R.; Laverdiere, M.; Yang, W.;
´
7. Vicente, M. F.; Basilio, A.; Cabello, A.; Pelaez, F. Clin.
Microbiol. Infect. 2003, 9, 15.
Merz, W.; Ostrosky, L.; Peman, J.; Perea, S.; Perfect, J.;
Pfaller, M.; Proia, L.; Rex, J.; Rinaldi, M.; Rodriguez, J.;
Schell, W.; Shields, C.; Sutton, D.; Verjweij, P.; Warnock,
D. J. Clin. Microb. 2004, 42, 3475.
´
´
8. Lopez, S.; Ramallo, I.; Gonzalez Sierra, M.; Zacchino, S.;
´
Furlan, R. PNAS 2007, 104, 441.
´
´
9. Sortino, M.; Delgado, P.; Juarez, S.; Quiroga, J.; Abonıa,
R.; Insuasty, B.; Nogueras, M.; Rodero, L.; Garibotto, F.;
Enriz, R.; Zacchino, S. Bioorg. Med. Chem. 2007, 15, 484.
19. Quan, H.; Cao, Y.; Xu, Z.; Zhao, J.; Gao, P.; Qin, X.;
Jiang, Y. Antimicrob. Agents Chemother. 2006, 50, 1096.
20. Metha, N.; Phillips, A.; Fu, F.; Brooks, R. J. Org. Chem.
1960, 25, 1012.
´
10. Massman, M.; Somlai, C.; Rodrıguez, A.; Sortino, M.;
Zacchino, S.; Enriz, R. Bioorg. Med. Chem. 2006, 14, 7604.
11. Suvire, F.; Sortino, M.; Kouznetsov, V.; Vargas, L.;
Zacchino, S.; Enriz, R. Bioorg. Med. Chem. 2006, 14,
1851.
21. Frankel, M.; Liwschitz, Y.; Amiel, Y. J. Am. Chem. Soc.
1953, 75, 330.
22. Cava, M.; Deana, A.; Muth, K.; Mitchell, M. Org. Synth.
1973, 5, 944.
12. Vargas, L.; Castelli, M.; Kouznetsov, V.; Urbina, J.;
23. Cechinel Filho, V.; Queiroz, E.; Lima, E.; Pinheiro,
T.; Nunes, R.; Yunes, R. Quim Nova. 1996, 19,
590.
´
Lopez, S.; Sortino, M.; Enriz, R.; Ribas, J. C.; Zacchino,
S. Bioorg. Med. Chem. 2003, 11, 1531.
´
ˆ
13. Lopez, S.; Castelli, M. V.; de Campos, F.; Correa, R.;
Cechinel Filho, V.; Yunes, R.; Zamora, M.; Enriz, R.;
24. Klepser, M.; Wolfe, E.; Jones, R.; Hightingale, C.;
Pfaller, M. Antimicrob. Agents Chemother. 1997, 41,
1392.
25. Klepser, M.; Ernst, E.; Lewis, R.; Ernst, M.; Pfaller, M.
Antimicrob. Agents Chemother. 1998, 42, 1207.
26. Rand, K.; Houck, H.; Brown, P.; Bennett, D. Antimicrob.
Agents Chemother. 1993, 37, 613.
´
Ribas, J. C.; Furlan, R.; Zacchino, S. Arzn Forsch (Drug
Res). 2005, 55, 123.
14. NCCLS, National Committee for Clinical and Laboratory
Standards, Method M27-A2, 2nd ed., Wayne Ed.; 2002,
Vol. 22 (15), pp 1–29.
15. Khan, M. J. Pharm. Sci. 1984, 73, 1767.
16. Johnson, M.; McDougall, C.; Ostrosky-Zeichner, L.;
Perfect, J.; Rex, J. Antimicrob. Agents Chemother. 2004,
48, 693.
27. Lewis, R.; Diekema, D.; Messer, S.; Pfaller, M.; Klebser,
M. J. Antimicrob. Chemother. 2002, 49, 345.
28. Arikan, S.; Lozano-Chiu, M.; Paetznick, V.; Rex, J.
Antimicrob. Agents Chemother. 2002, 46, 245.