Chemical reactivity and antimicrobial activity of N-substituted maleimides

Article abstract of DOI:10.3109/14756366.2011.580455

Several N-substituted maleimides containing substituents of varying bulkiness and polarity were synthesised and tested for antimicrobial and cytostatic activity. Neutral maleimides displayed relatively strong antifungal effect minimum inhibitory concentrations (MICs in the 0.54 g ml^-1 range); their antibacterial activity was structure dependent and all were highly cytostatic, with IC₅₀ values below 0.1 g ml^-1. Low antimicrobial but high cytostatic activity was noted for basic maleimides containing tertiary aminoalkyl substituents. Chemical reactivity and lipophilicity influenced antibacterial activity of neutral maleimides but had little if any effect on their antifungal and cytostatic action. N-substituted maleimides affected biosynthesis of chitin and β(1,3)glucan, components of the fungal cell wall. The membrane enzyme, β(1,3)glucan synthase has been proposed as a putative primary target of N-ethylmaleimide and some of its analogues in Candida albicans cells.

Full text of DOI:10.3109/14756366.2011.580455

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(1): 117–124
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.580455
RESEARCH ARTICLE
Chemical reactivity and antimicrobial activity of
N-substituted maleimides
Natalia Salewska1, Joanna Boros-Majewska2, Izabela Łącka2, Katarzyna Chylińska1, Michał Sabisz2,
Sławomir Milewski2, and Maria J. Milewska1
1Department of Organic Chemistry, Gdansk University of Technology, Organic Chemistry, Gdansk, Poland, and
2Department of Pharmaceutical Technology & Biochemistry, Gdansk University of Technology, Gdansk, Poland
Abstract
Several N-substituted maleimides containing substituents of varying bulkiness and polarity were synthesised and
tested for antimicrobial and cytostatic activity. Neutral maleimides displayed relatively strong antifungal effect
minimum inhibitory concentrations (MICs in the 0.5–4 µg ml−1 range); their antibacterial activity was structure
dependent and all were highly cytostatic, with IC50 values below 0.1 µg ml−1. Low antimicrobial but high cytostatic
activity was noted for basic maleimides containing tertiary aminoalkyl substituents. Chemical reactivity and
lipophilicity influenced antibacterial activity of neutral maleimides but had little if any effect on their antifungal and
cytostatic action. N-substituted maleimides affected biosynthesis of chitin and β(1,3)glucan, components of the
fungal cell wall. The membrane enzyme, β(1,3)glucan synthase has been proposed as a putative primary target of
N-ethylmaleimide and some of its analogues in Candida albicans cells.
Keywords: Antimicrobial action, fungicidal activity, inhibitors, glucan synthase
Introduction
enzymes containing reactive cysteinyl residues, essen-
Microbial resistance to antimicrobials is an emerging
challenge for clinicians and pharmaceutical indus-
try. e multi-drug-resistant bacteria and fungi are
the major cause of failure in chemotherapy of infec-
tious diseases. us, the need for novel antimicrobial
agents is especially urgent. Among different strate-
gies of searching for new potential drugs, identifica-
tion of novel, unexploited molecular targets and their
inhibitors seems one of the most promising. ese days
many researchers concentrate on bacterial and fungal
enzymes that catalyse important biochemical reaction
in microbial cells. Bioactive compounds that selectively
inactivate such proteins and block metabolic pathways
in human pathogenic bacteria and fungi could be
potential antimicrobial drugs.
tial for their catalytic activity2–4. Because many of
these enzymes are important for growth and survival
of micro-organisms, antimicrobial activity of NEM is
not surprising. Antimicrobial properties of NEM and
other N-substituted maleimides have been already
reported5–9; however, none of those studies related anti-
microbial activity of substituted maleimides to their
physicochemical properties, including chemical reac-
tivity and lipophilicity. In the present work, such corre-
lation has been attempted for 12 various N-substituted
maleimides. Moreover, novel information on molecular
mechanism of antifungal action of compounds studied
has been provided.
Materials and methods
Although N-ethyl maleimide (NEM) may react with
amines and imidazole derivatives to form N-acylation
products, it is widely known as a thiol-reacting com-
pound1 and as such is an effective inhibitor of several
Chemistry
Solvents and reagents were purchased from Sigma-
Aldrich. Melting points are uncorrected. 1H NMR
Address for Correspondence: Slawomir Milewski, Department of Pharmaceutical Technology & Biochemistry, Gdansk University of
Technology, 11/12 NarutoWicza St., 80-233, Gdansk, Poland. Tel: +48 583472107. Fax: +48 583471144. E-mail: slamilew@pg.gda.pl
(Received 14 February 2011; revised 21 March 2011; accepted 08 April 2011)
117

118 N. Salewska et al.
spectra were recorded on the Varian Gemini 200 and
500 MHz spectrometers. UV–vis spectra were recorded
in an UV–vis Lambda 45, Perkin Elmer spectrophotom-
eter. in layer chromatography (TLC) was performed
on silica gel (Merck) in a solvent system benzene:ethyl
acetate:ethanol (50:15:5). Elementary analysis (EA) was
run on the Carlo Erba, type EA 1108 analyser.
N-(2-tert-butylphenyl)maleimide (7)
Yield 85% and m.p. 90°C. 1H NMR (CDCl3) δ [ppm]:
7.2 (d, 1H, J= 8 Hz, C6H5); 7.1 (t, 1H, J= 7 Hz, C6H5); 7.9
(s, 1H, = CH); 6.93 (d, 1H, C6H5); 6.2 (s,1H, CH=); 1.1 (s,
9H, (CH3)3C). Anal. found: C 73.11, H 6.54, N 6.07%.
Calculated for C14H15NO2: C 73.34, H 6.59, N 6.11%.
N-(4-bromophenyl)maleimide (8)
Yield 53% and m.p. 118–119°C. 1H NMR (CDCl3) δ [ppm]:
7.73 (m, 2H, C6H5); 7.71 (m, 2H, C6H5); 6.7 (s, 2H, CH=CH).
Anal. found: C 47.66, H 2.51, N 5.32%. Calculated for
C10H6NO2Br: C 47.65, H 2.40, N 5.36%.
General procedure for the synthesis of
maleimides 3–10
Maleic anhydride (20 mmol) was dissolved in 20 ml of
CH2Cl2. e stirred solution was cooled to 0–5°C and
equimolar amount of an appropriate amine dissolved
in 15 ml of CH2Cl2 was added to the mixture. e result-
ing suspension was refluxed for 2 h and then cooled to
room temperature. e precipitated solids were filtered
off and washed twice with ethyl ether. e maleamic
acids formed in the first step (15 mmol) were dissolved in
24 ml of acetic anhydride and anhydrous sodium acetate
(10 mmol) was added to the mixture. e resulting sus-
pension was refluxed and reaction progress was followed
by TLC. When the reaction was completed, the mixture
was cooled and 100 ml of water was added. e result-
ing solution of maleimide in water was extracted with
ethyl acetate (3 × 50 ml), extracts were combined, dried
with MgSO4, filtered and the solvent was evaporated.
Crude products were re-crystallised from the solvent or
distilled.
N-(4-methoxyphenyl)maleimide (9)
Yield 58% and m.p. 150–151°C. 1H NMR (CDCl3) δ [ppm]:
7.5 (d, 2H, J= 8.8 Hz, C6H5); 7.2 (d, 2H, J= 8.8 Hz, C6H5); 6.5
(s, 2H, CH=CH); 3.4 (s, 3H,−OCH3). Anal. found: C 64.98,
H 4.48, N 6.79%. Calculated for C11H9NO3: C 65.02, H 4.46,
N 6.89%.
N-(4-nitrophenyl)maleimide (10)
Yield 70% and m.p. 161°C. 1H NMR (CDCl3) δ [ppm]: 8.33
(d, 2H, J= 2 Hz, C6H5); 7,.8 (d, 2H, J= 2 Hz, C6H5); 6.3 (s,
2H, CH=CH). Anal. found: C 55.04, H 2.75, N 12.77%.
Calculated for C10H6N2O4: C 55.05, H 2.77, N 12.84%.
N-[2-(N,N-dimethylamino)ethyl]maleimide (11)
Yield 52% and m.p. 175–176°C. 1H NMR (CDCl3): δ [ppm]:
6.67 (s, 2H, CH=CH), 3.61 (t, J= 6.3 Hz, 2H, CH2), 2.47 (t,
J= 6.4 Hz, 2H, CH2), 2.23 (s, 6H, CH3). Anal. found: C 56.99,
H 7.09, N 16.51%. Calculated for C8H12N2O2: C 57.14, H,
7.14, N 16.67%.
Basic maleimides 11 and 12 were synthesised accord-
ing to the described procedures10,11
.
N-cyclohexylmaleimide (3)
Yield 70% and m.p. 84°C. 1H NMR (CDCl3): δ [ppm]:
7.29 (s, 1H, = CH); 6.65 (s, 1H, HC=); 3.93 (t, 1H, J= 3.9
Hz, CH); 2.08 (qd, 2H, J= 12.9 Hz and 3.9 Hz, CH2); 1.86
(d, 2H, J= 13.2 Hz, CH2); 1.68 (m, 3H, CH2), 1.3 (m, 3H,
CH2). Analytically found (Anal. Found): C 66.93, H 7.27,
N 7.78%. Calculated for C10H13NO2: C 67.02, H 7.31, N
7.82%.
N-[2-(N-morpholinoethyl]maleimide (12)
Yield 33% and m.p. 132°C. 1H NMR (CDCl3): δ [ppm]: 6.65
(s, 2H, CH=CH), 3.55 (t, J= 6.8 Hz, 2H, CH2), 2.49 (t, J= 6.8
Hz, 2 H, CH2), 2.42 (m, 8H, CH2). Anal. found: C 56.99,
H, 6.48, N 13.17%. Calculated for C10H14N2O3: C 57.14, H
6.67, N 13.33%.
Determination of the model reaction rate
N-benzylmaleimide (4)
Yield 58% and m.p. 69°C. H NMR (CDCl3) δ [ppm]: 7.35
(m, 5H, C6H5); 6.72 (s, 2H, CH=CH); 4.69 (s, 2H, CH2).
Anal. found: C 71.48, H 5.01, N 7.42%. Calculated for
C11H9NO2: C 71.48, H 4.85, N 7.48%.
Progress of the reaction between maleimides and
N-acetyl methyl cysteinate (ACME) was determined
spectrophotometrically, measuring decrease in the
absorption at 300 nm (A300). Stock solutions of maleim-
ides (50 µmol ml−1 in dimethyl sulfoxide) and ACME
(50 µmol ml−1 in water) were prepared. Sample of the
maleimide solution (50 µl) was added to 1400 µl of the
sodium phosphate buffer (pH 5.0 or 7.0) and A300 of the
resulting solution was recorded. Reaction was initiated
by addition of 50 µl of the ACME solution and mea-
surement of A300 was continued at room temperature.
Concentration of a particular maleimide in the reac-
tion mixture at the given moment was calculated from
the standard curves A300 = f(c) determined separately
for each compound from the 1–12 series. Data were
plotted as 1/c versus time, to give the second order rate
constants k as slopes of the linear plots.
1
N-(2,6-dimethylphenyl)maleimide (5)
Yield 51% and m.p. 105–107°C. 1H NMR (CDCl3) δ [ppm]:
7.1 (m, 3H, C6H5); 6.8 (s, 2H, CH=CH); 2.2 (s, 6H, CH3).
Anal. found: C 69.34, H 5.58, N 6.83%. Calculated for
C12H11NO2: C 69.63, H 5.51, N 6.95%.
N-(2,4,6-trimethylphenyl)maleimide (6)
Yield 64% and m.p. 99–100°C. H NMR (CDCl3) δ [ppm]:
6.7 (m, 5H, C6H5); 6.6 (s, 2H, CH=CH); 2.1 (s, 3H, CH3);
2.7 (s, 6H, CH3). Anal. found: C 72.21, H 6.11, N 6.45%.
Calculated for C13H13NO2: C 72.54, H 6.09, N 6.51%.
1
Journal of Enzyme Inhibition and Medicinal Chemistry

Antimicrobial properties of N-substituted maleimides 119
Calculation of logP
Preparation of the mixed membrane fraction and
glucan synthase activity assay
Values of the theoretical 1−octanol/water partition coef-
ficients (logP) were calculated for all compounds using
the ALOGPS 2.1 software12.
e mixed membrane fractions (MMFs) of C. albicans
cells were prepared using the method of Cabib and
Kang15. Activity of β(1,3)-glucan synthase present in MMF
was measured with uridine-5′-diphospho-d-glucose
as a substrate, using the microtitre plate fluorescence
method described by Shedletzky et al.16, based on the
quantification of the aniline blue–glucan complex. e
fluorescence was measured with the microplate reader
at the excitation wavelength of 400 nm and the emission
wavelength of 460 nm.
Determination of antimicrobial activity
Antifungal activity was determined by the M27-A2
method specified by CLSI (formerly NCCLS) in RPMI-
1640 medium buffered to pH 7.013. e test micro-or-
ganisms were Candida albicans ATCC 10231, Candida
glabrata DSM 11226, Candida tropicalis KKP 334 and
Saccharomyces cerevisiae ATCC 9763. Antibacterial
activity was determined by the microtitre plate method
in Tryptic Soy Broth medium, pH 7.0 (GibcoBRL) against
Gram-negative Escherichia coli ATCC 10536 and Gram-
positive Staphylococcus aureus ATCC 6538 and Bacillus
subtilis ATCC 6633. Wells containing serially diluted
examined compounds and compound-free control
were inoculated with 12-h cultures of tested strains to
the final concentration of 104 colony-forming units (cfu)
ml−1. Plates were incubated for 24 h at 37°C and growth
was then quantified by measuring an optical density at
531 nm, using the microplate reader (Victor3V; Perkin
Elmer, Centre of Excellence ChemBioFarm). e MIC
was defined as the lowest drug concentration at which
at least 80% decrease in turbidity, in comparison to the
drug-free control, was observed.
Determination of chitin synthase activity and
measurement of chitin content
Chitin synthase activity was determined in MMF of C.
albicans cells by the method of Munro et al.17 e MMFs
were activated by 5-min incubation with 100 ng trypsin
μl−1 MMF at 30°C and the reactions were stopped by
addition of 150 ng soybean trypsin inhibitor μl−1 MMF.
Standard reactions for measuring chitin synthase
activity were carried out in a 50 μl volume and were
composed of: 50 μg MMF protein, 25 mM N-acetyl-D-
glucosamine, 1 mM UDP-N-acetyl-D-glucosamine that
included 25 nCi UDP-[U-14C] N-acetyl-D-glucosamine,
50 mM Tris–HCl pH 7.5, 10 mM MgCl2 and an inhibi-
tor tested at appropriate concentration (0–200 μg ml−1
range). Incubations were carried out at 30°C for 30 min
and the reaction was stopped by addition of 1 ml of 66%
(v/v) ethanol. e reaction mixture was then filtered
through GF/C filter discs (Whatman), which had been
pre-soaked in 10% (v/v) trichloroacetic acid. e reac-
tions tubes were rinsed out with 2 × 1 ml of 1% (v/v)
Triton X-100 and each filter was then washed with
4 × 2 ml of 66% (v/v) ethanol. e radio-labelled chitin
synthesised in the reaction was trapped on the filters
and unincorporated substrate was removed by wash-
ing. Filters were dried at 80°C and their radioactivity
counted in a scintillation counter.
e number of viable yeast cells was determined by
subculturing diluted cell suspensions on agar plates.
Samples of 0.05 ml were collected from microtitre
plate wells, appropriately diluted with sterile water
and aliquots of 0.1 ml were spread onto the surface of
Sabouraud agar plates. Plates were incubated at 30°C
for 24 h and colonies were counted to give the cfu
values.
Morphological observations
C. albicans cells from the overnight cultures in Sabouraud
medium were harvested, washed with sterile saline and
suspended in RPMI-1640 medium to the final cell density
of 105 cfu ml−1. Tested compounds were added at appro-
priate concentrations and cultures were incubated for 6h
at 30°C, with shaking. At time intervals, samples of 0.1ml
were collected and cells were stained for chitin or glucan.
For chitin staining, the culture sample was combined
with 0.1ml of the Calcofluor White solution (0.3mg ml−1
in 25mM phosphate-buffered saline (PBS), pH 7.0) and
incubated for 5min at 25°C. e cells were then harvested,
washed four times with PBS, re-suspended in 0.1ml of the
fresh PBS and transferred to a microscopic slide. Staining
for glucan was performed using an aniline blue dye, as
described by Kippert and Lloyd14. Aliquots of the cell sus-
pension (0.1ml) were harvested, washed four times with
PBS (pH 7.5), mounted in the aniline blue solution (0.5mg
ml−1 in PBS) and transferred to a microscopic slide. e
stained cells were examined with the Olympus BX 60 F5
fluorescence microscope and photographs were taken
using the Studio Lite software (Pixera Corporation).
For the quantitative determination of chitin content
in cells cultivated for 24 h in the presence of maleimides,
the amount of N-acetyl-D-glucosamine released by chi-
tinase/cytohelicase was measured by the colorimetric
method described previously18. Reaction mixtures con-
tained 0.1 U ml−1 of chitinase from Streptomyces griseus
and 1 mg ml−1 of cytohelicase from Helix pomatia.
Determination of haemolytic effect
Human red cells were re-suspended in cold saline to the
final number of 2 × 107 cells ml−1. Compounds were added
to the cell suspensions, samples were incubated at 37°C
for 30 min and then centrifuged (1700g, 10 min, 4°C).
Supernatants were collected and absorbance at 540 nm
was measured to determine haemoglobin concentra-
tion. e maximal possible haemolysis level (positive
control) was determined in supernatants obtained from
cell suspensions pre-treated with 0.1 % Triton X-100.
Values of drug concentration causing 50% haemolysis in
© 2012 Informa UK, Ltd.

120 N. Salewska et al.
comparison with the positive control (EH50) were read
from the A540 = f(concentration) plots.
N-benzylmaleimide 4 afforded product with the follow-
ing spectral data: 1H NMR (CDCl3) δ [ppm]: 8.19 (m, 1H,
NH); 7.35 (m, 5H, C6H5); 4.71 (s, 2H, CH2Ph); 4.38 (m, 1H,
CHC(O)); 3.74 (t, 1H, CHS); 3.58 (s, 3H, CH3O); 3.01 (m,
2H, CH2C(O)); 2.84 and 2.74 (AB, 2H, CH2S); 1.92 (s, 3H,
CH3C(O)). Kinetics of reactions of ACME with maleim-
ides was also studied. e reaction progress was moni-
tored spectrophotometrically at 300 nm, by measuring
decrease of absorption deriving from the maleimide
chromophore. e determined k values are summarised
in Table 1. As expected for the nucleophilic 1,4-addition
of thiols to the conjugated double bond system, reactions
of ACME with maleimides were faster at higher pH, due
to the enhanced thiol nucleophilicity. is finding con-
firms the previous observations concerning reaction of
NEM with reduced glutathione19. Differences in relative
reactivity of maleimides at the given pH were noted. e
most reactive compounds exhibited velocity constants
more than an order of magnitude higher than the least
reactive ones. Relatively high reactivity was observed
for NEM 1 and maleimides with benzyl or phenylalkyl
substituents at the nitrogen atom (4–7), while lower for
compounds containing polar fragments in their N-aryl
or N-alkyl substituents (8–12) and for bulky N-tert-butyl
maleimide 2 and N-cyclohexyl maleimide 3.
Table 1 also contains values of logP parameters, cal-
culated for compounds 1–12, using the ALOGPS 2.1
software. e maleimides under study differed markedly
in terms of their lipophilicity, reflected by the calculated
values of logP. Although NEM 1 was only about two times
better soluble in octanol than in water, solubility of the
most hydrophobic N-(2-tert-butylphenyl)maleimide 7 in
octanol was almost 500 times higher than that in water.
Generally, compounds 1–10 should be considered mod-
erately or highly hydrophobic, whereas 11 and 12 are
moderately hydrophilic.
Cytotoxicity assay
Human cervix carcinoma HeLa S3 cells were main-
tained in high glucose Dulbecco’s modified Eagle’s
medium (pH 7.0) supplemented with 10% foetal bovine
serum, 2 mM l-glutamine and antibiotics (100 units
ml−1 penicillin and 100 mg ml−1 streptomycin) at 37°C in
10% CO2/air atmosphere. Cells were screened routinely
for Mycoplasma by the PCR method with Mycoplasma
Plus PCR Primer Set (Stratagene, La Jolla, CA). e
cytotoxicity was determined by the MTT assay. Briefly,
exponentially growing cells were attached at 3 × 104
cells per well in a 24-well multi-well plates and the cel-
lular viability was determined after 120 h of continuous
exposure to different drug concentrations. Cells were
incubated with the MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium tetrazolium salt) for 4 h
at 37°C, and formazan formation was measured by
a microplate reader. e concentrations required to
inhibit cell growth by 50% compared to untreated con-
trols (IC50) were determined from the curves plotting
survival as a function of dose by use of the Slide Write
program. All values are averages of at least two inde-
pendent experiments, each done in duplicate.
Results
Synthesis of N-substituted maleimides
N-Ethylmaleimide 1 (NEM) and N-tert-butylmaleimide
2 were purchased from Sigma-Aldrich. N-substituted
maleimides 3–10 were synthesised in a two-step pro-
cedure (Scheme 1). In the first step, N-substituted
maleamic acid derivatives were formed upon reac-
tion of maleic anhydride and an appropriate amine.
Cyclisation to N-substituted maleimides 3–10 was
achieved by heating of maleamic acid derivatives in
the presence of acetic anhydride containing catalytic
amounts of sodium acetate. N-[2-(N,N-dimethylamino)
ethyl]maleimide 11 and N-[2-(N-morpholinoethyl]
maleimide 12 were synthesised according to the litera-
ture procedures.
e maleimide ring is known to undergo hydrolysis
to maleamic acid in a pH-dependent manner, especially
quickly in alkaline media19, 20. HPLC analysis of buffered
aqueous solutions of maleimides 1–12 revealed that
Chemical reactivity and lipophilicity of maleimides
Chemical reactivity of N-substituted maleimides 1–12
was determined in the model reaction with ACME, mim-
icking cysteinyl residues present at the enzyme active
centres. Reactions were carried out at room temperature,
in buffered aqueous media, at pH 5.0 and 7.0. Reagents
were present in equimolar amounts. All reactions pro-
ceeded according to Scheme 2, leading to the formation
of respective products of nucleophilic 1,4 addition, as was
confirmed by high-performance liquid chromatography
Scheme 1. Synthesis of neutral maleimides.
1
(HPLC) and H NMR analysis. e NMR data of prod-
ucts formed in the reactions of ACME with maleimides
1–12 were consistent with structures of general formula
shown in Scheme 2. For example, reaction of ACME with
Scheme 2. Reaction of maleimides with ACME.
Journal of Enzyme Inhibition and Medicinal Chemistry

Antimicrobial properties of N-substituted maleimides 121
these compounds were completely stable at pH 4–6.5
for at least 24 h and only a very slow decomposition to
maleamic acid derivatives was observed at pH 6.5–7.0,
with 5–11% of the decomposition products observed at
pH 7.0 after 24 h. erefore, a possibility of substantial
decomposition of maleimides under conditions of deter-
mination of their antimicrobial, haemolytic and cyto-
static activity (pH ≤ 7.0) should be excluded.
by the microplate serial dilution method. Data shown
in Table 2 indicate that all compounds but 11 and 12
exhibited strong antifungal activity, with MICs ranging
between 0.5 and 4 µg ml−1. is activity was comparable
to that of known antifungals, Nystatin and Fluconazole.
Compounds 11 and 12 were notable exceptions, demon-
strating much lower antifungal activity. On the other hand,
antibacterial activity of 1–12 was differentiated but gener-
ally low, with MICs between 1 and 128 µg ml−1. Especially
high MIC values were noted against Gram-negative E. coli.
N-(4-bromophenyl)maleimide 8, N-(4-methoxyphenyl)
maleimide 9, N-(4-nitrophenyl)maleimide 10 and two
basic maleimides 11 and 12 showed the lowest antibacte-
rial activity among the all compounds tested, whereas the
highest was observed for 1.
Antimicrobial activity
e N-substituted maleimides 1–12were tested for growth
inhibitory activity against four yeast and three bacterial
strains. MICs of the studied compounds were determined
Table 1. Lipophilicity and chemical reactivity of maleimides.
k (M−1 s−1)
Effect of maleimides action on C. albicans viability and
morphology of cells
Compound
logP
0.29
0.87
1.58
1.35
1.88
2.26
2.67
1.91
1.13
1.11
−0.11
−0.46
pH 5.0
80.3
3.23
4.74
47.2
39.7
58.3
85.0
1.11
1.82
0.82
2.28
2.11
pH 7.0
404
1
C. albicans cells grown in RPMI-1640 medium were
treated with malemides 1–10, 8 µg ml−1, i.e., concentra-
tion exceeding their MIC values. Cells were counted
and changes in cell morphology were monitored by
fluorescence microscopy after staining of fungal cell
wall components with fluorescent dyes. Action of all
compounds resulted in a substantial reduction of the
cell number, reflected by the cfu ml−1 value, dropped
from 105 cfu ml−1 to 1–1.4 × 104 cfu ml−1. e most striking
changes in cell morphology shown in Figure 1 included
apparent “chaining” of the cells, most likely resulting
from cell agglutination or formation of pseudohyphal
forms and in some cases evident shrinking and distor-
tion of dye-stained cell wall constituents, especially
2
40.9
69.8
202
3
4
5
195
6
221
7
364
8
14.1
17.1
10.0
25.6
16.7
9
10
11
12
Data include calculated 1-octanol/water partition coefficients
(logP) and determined apparent second order velocity constants
(k) for the reaction of maleimides with ACME.
Table 2. Antibacterial and antifungal in vitro activity of maleimide compounds and reference antifungals.
MIC (μg ml−1)
Compd.
R
C. a.
2
C. g.
4
C. t.
1
S. c.
2
E. c.
8
B. s.
1
S. a.
2
1
Ethyl
2
tert-Butyl
2
4
0.5
1
2
32
2
2
3
Cyclohexyl
1
1
1
64
16
2
32
2
4
Benzyl
2
2
1
2
32
5
2,6-Dimethylphenyl
2,4,6-Trimethylphenyl
2-tert-Butylphenyl
4-Bromophenyl
4-Methoxyphenyl
4-Nitrophenyl
N-dimethylaminoethyl
N-morpholinoethyl
Nystatin
2
4
2
2
64
8
8
6
2
1
0.5
1
1
64
4
2
7
4
2
2
64
16
64
64
64
128
128
NT
NT
8
8
1
2
2
1
128
128
128
128
128
NT
NT
128
128
128
128
128
NT
NT
9
2
4
2
2
10
11
12
2
2
1
1
128
128
2
128
128
2
64
64
2
64
64
2
Fluconazole
1
2
2
1
C. a., Candida albicans; C. g., Candida glabrata; C. t., Candida tropicalis; S. c., Saccharomyces cerevisiae; E. c., Escherichia coli; B. s.,
Bacillus subtilis; S. a., Staphylococcus aureus; NT, not tested.
© 2012 Informa UK, Ltd.

122 N. Salewska et al.
those stained with the β(1,3)-glucan-specific aniline
blue, was observed.
Haemolytic and cytostatic activity
N-substituted maleimides 1–12 were tested for hae-
molytic activity against human erythrocytes. None of
the studied compounds at concentrations <300 µg ml−1
induced haemoglobin release from red blood cells at a
measurable rate. Higher concentrations could not be
tested because of the solubility problems. In the paral-
lel determination, a well-known membrane-active anti-
fungal antibiotic Nystatin caused rapid haemolysis and
disruption of 50% of erythrocytes was noted at 80 µg ml−1
of this compound. It may be therefore concluded that
N-substituted maleimides are not haemolytic and do not
disturb membrane function in erythrocytes.
e maleimide derivatives were also tested for cyto-
static activity in tissue culture against HeLa S3 cells. All
compounds appeared highly cytostatic. e ED50 val-
ues for 1–12 were lower than 0.1 µg ml−1, for example:
0.068 0.007 µg ml−1 for 1, 0.045 0.008 µg ml−1 for 4, and
0.05 0.01 µg ml−1 for 9, while for the reference, known
cytostatic agent doxorubicin, the respective value was
0.011 0.009 µg ml−1 −
Influence of maleimides on chitin content of
C. albicans cells and activity of chitin synthase and β(1,
3)-glucan synthase
Action of maleimides on C. albicans slightly affected
chitin content in the yeast cells. Compared to the
control untreated cells, cell treated with maleimides
1–10 at 5 µg ml−1 contained 10–12% less chitin, while
the treatment with maleimides at 50 µg ml−1 reduced
the chitin content by 20–25%. No reduction of chitin
content was observed for cells treated with 11 or 12 at
50 µg ml−1. Slight inhibition of chitin synthase activity
present in the MMF of C. albicans cells by maleimides
tested was noted. Enzyme activity was 14–21% lower in
samples containing malemides 1–12 at 50 µg ml−1 but
none of the compounds studied reached 50% inhibi-
tion of chitin synthase activity at concentration <200
µg ml−1. In the parallel determination, IC50 = 9.5 0.5 µg
ml−1 was determined for nikkomycin Z, a well-known
inhibitor of chitin synthase21.
On the other hand, a notable in vitro inhibition by
maleimides 1–10 of β(1,3)-glucan synthase activity
present in MMF was noted. Exemplary data shown in
Figure 2 clearly indicate that at 5 µg ml−1, i.e., concentra-
tion slightly higher than the MICs of these maleimides,
enzyme activity was inhibited in about 35%, while at 50
µg ml−1 the extent of inhibition was enhanced to 60–65%.
Concentrations causing 50% inhibition of the enzyme
activity (IC50) were: 8.5 1.1 µg ml−1 for 1, 13.7 2.3 µg
ml−1 for 2 and 21.2 2.5 µg ml−1 for 8, respectively. Similar
enzyme inhibitory potency, with IC50 values within the
8–25 µg ml−1 range, was noted for other maleimides,
except for 11 and 12, where only 20–24% inhibition was
observed at 100 µg ml−1. e known glucan synthase
inhibitor, trans-cinnamaldehyde22, inhibited the enzyme
with IC50 = 140 8 µg ml−1.
Discussion
A series of neutral N-substituted maleimides (1–10)
studied in this work displayed reasonably high antifungal
activity, with very little if any structure–antifungal activity
relationship. On the other hand, the antibacterial activ-
ity of NEM 1 and its derivatives 2–12 was generally lower
and structure dependent. e especially low activity of all
compounds studied but NEM against Gram-negative E.
coli seems to be related to their lipophilicity and bulki-
ness, as high MICs (<32 µg ml−1) were found for the com-
pounds 2–10 exhibiting logP values higher than 0.8 and
containing relatively large substituents, while the lowest
for the least lipophilic and the smallest NEM 1. Biological
basis of this difference is not known but one may assume
that the lipophilic, relatively large maleimide derivatives
Figure 1. Visualisation of the effect of maleimides on morphology of Candida albicans yeast cells. (A–C) Cells stained for chitin with
Calcofluor White. (A) Control, (B) cells treated with 1, 5 µg ml−1; (C) cells treated with 7, 5 µg ml−1. (D–F) Cells stained for glucan with aniline
blue. (D) Control, (E) cells treated with 1, 5 µg ml−1; (F) cells treated with 7, 5 µg ml−1.
Journal of Enzyme Inhibition and Medicinal Chemistry

Antimicrobial properties of N-substituted maleimides 123
inhibition of β(1,3)-glucan synthase was noted. All neu-
tral maleimides at concentrations slightly exceeding their
MIC values inhibited enzyme activity by more than 50%.
In this respect, it is worth mentioning that fungal β(1,3)-
glucan synthases contain sulfhydryl groups, important for
their activity, localised most probably in the GTP-ase part
of the enzyme complex23. Our present finding confirms
previous reports on inhibition of β(1,3)glucan synthase
activity by other sulfhydryl-acting compounds24. e pre-
viously reported inhibition of S. cerevisiae β(1,3)-glucan
synthase by N-phenylpropyl-3,4-dichloromaleimide9
also supports our findings, as that compound should be
considered a good electrophilic acceptor for nucleophilic
1,4-addition of reactive sulfhydryl group(s) present in
glucan synthase.
Figure 2. In vitro inhibition of β(1→3)-glucan synthase activity by
maleimides. e assay was performed using the microtitre-based
fluorescence method. Values are the means of three independent
experiments. Bars represent SD.
e facts that maleimides tested strongly inhibited
β(1,3)-glucan synthase activity at concentrations close to
those inhibiting fungal growth, they were fungicidal and
induced morphological alterations characteristic for the
consequences of cell wall inhibition, suggested that the
enzyme might be a primary molecular target of these com-
pounds in yeast cells. Because glucan synthase, responsible
for the formation of the main component of C. albicans cell
wall and thus essential for cell viability, is a cytoplasmic
membrane-localised protein, it is supposed to be espe-
cially exposed to the action of hydrophobic inhibitors that
may accumulate and concentrate in the membrane lipidic
environment. It is worth mentioning, therefore, that this
enzyme is inhibited by many structurally unrelated but
generally hydrophobic compounds, like glycolipidic papu-
lacandins, acid terpenoids and N-acylated cyclic hexapep-
tides25, but also by the trans-cinnamaldehyde as well as
by chalcone derivatives, including those developed in our
laboratory22,26,27, both containing the conjugated double
bond systems, that could serve as electrophilic acceptors in
nucleophilic 1,4-addition. Many of the above-mentioned
compounds targeting glucan synthase inhibit enzyme
activity in vitro by more than 50%, at concentrations
slightly higher than their MICs27,28. e same phenomenon
was found by us for maleimides 1–10 what additionally
points at glucan synthase as the primary target of these
compounds in C. albicans. Much lower antifungal activ-
ity of moderately hydrophilic basic maleimides 11 and 12
and low inhibitory potential of these compounds against
β(1,3)-glucan synthase present in the membrane fraction
could be explained by their hydrophilicity and possible low
accumulation in the membrane, although the presence of a
positively charged aminoalkyl substituent may also directly
affect affinity to the binding site at the enzyme molecule.
e maleimides tested appeared strongly cytostatic
against mammalian cells in tissue culture, despite the
fact that they did not induce lysis of human erythrocytes.
However, the latter can be easily explained by the fact that
erythrocytes are unusual cells, rather “biological sacs”
filled with haemoglobin, so that one can hardly find there
any potential target for maleimides. Lack of the haemo-
lytic effect thus only indicates that maleimides are not
membrane-disrupting compounds. On the other hand,
do not cross the bacterial outer membrane and remain
captured there, unable to reach any intracellular targets,
whereas the smallest and less hydrophobic NEM easily
penetrates the outer barrier and reaches an unidentified
target in the periplasmic space, cytoplasmic membrane
or cytosol. e low activity of moderately hydrophilic
basic maleimides 11 and 12 against E. coli seems to be an
exception to this rule; however, their relative bulkiness
and presence of several hydrophobic regions in these
molecules may be a reason for the strongly reduced activ-
ity against Gram-negative bacteria. In the case of Gram-
positive bacteria, three compounds, namely 8, 9 and 10,
bearing more or less polar substituents in the phenyl
ring, as well as the basic maleimides 11 and 12, were
apparently less active than the other ones. Reason for this
difference is not known, although it does not seem to be
directly related to the general lipophilicity. Because com-
pounds 8–12, demonstrating the lowest activity against
Gram-positive bacteria, were found to be the least reac-
tive in the model reaction with ACME, it is possible that
their low growth inhibitory potency against B. subtilis and
S. aureus results from poor reactivity against an unidenti-
fied target containing essential cysteinyl residue.
Undoubtedly, differences in chemical reactivity and
lipophilicity did not influence the antifungal activity of
the maleimide derivatives tested, except for the two com-
pounds containing N-aminoalkyl substituents 11 and 12,
demonstratingmuchlowerantifungalefficacy.Reduction
of the cell number and changes in morphology of C. albi-
cans cells treated with highly active compounds at con-
centrations exceeding their MIC values indicated the cell
lysis and suggested a possible effect of these maleimides
on formation of cell wall components. is was checked
in two ways: (1) quantification of possible influence on
chitin biosynthesis by monitoring the total chitin con-
tent in C. albicans cells treated with maleimides and (2)
in vitro effect of maleimides on activity of C. albicans
chitin synthase and β(1,3)-glucan synthase in MMF.
Although the compounds studied only slightly affected
chitin content and chitin synthase activity, a substantial
© 2012 Informa UK, Ltd.

124 N. Salewska et al.
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Declaration of interest
e authors report no conflict of interests.
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