Synthesis and antimicrobial activities of N-substituted imides

Article abstract of DOI:10.1016/S0014-827X(02)01217-X

In the field of our research programs concerning novel antimicrobial agents, a series of N-substituted imides was synthesized. These compounds were obtained by cyclization of amido-acids in acetic anhydride/sodium acetate or hexamethyldisilazane/zinc bromide for the hydroxy-aromatic derivatives. The hydroxy-alkyl maleimides were directly prepared by condensation of the corresponding amino-alcohol with maleic anhydride in boiling toluene. Most of N-substituted maleimides showed an interesting antimicrobial activity towards bacteria from the ATCC collection (Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) but the MIC values for P. aeruginosa were always high (128 μg/ml). The imides with alkyl substituents showed higher activities than aromatic analogues with MIC values in the range of 8-32 μg/ml. Comparatively, succinimides were practically inactive.

Full text of DOI:10.1016/S0014-827X(02)01217-X

Il Farmaco 57 (2002) 421–426
www.elsevier.com/locate/farmac
Note
Synthesis and antimicrobial activities of N-substituted imides
Frederic Zentz a, Alain Valla a, Regis Le Guillou a, Roger Labia a,*,
Anne-Gabrielle Mathot b, Danielle Sirot c
a F.R.E. 2125 C.N.R.S., 6, rue de l’Uni6ersite´ 29000 Quimper, France
b L.U.M.A.Q. E.A. 2651, 6, rue de l’Uni6ersite´, 29000 Quimper, France
c Faculte´ de Me´decine, Ser6ice de Bacte´riologie-Virologie, 28, place Henri Dunant, B.P.38, 63001 Clermont-Ferrand, France
Received 30 November 2001; accepted 12 January 2002
Abstract
In the field of our research programs concerning novel antimicrobial agents, a series of N-substituted imides was synthesized.
These compounds were obtained by cyclization of amido-acids in acetic anhydride/sodium acetate or hexamethyldisilazane/zinc
bromide for the hydroxy-aromatic derivatives. The hydroxy-alkyl maleimides were directly prepared by condensation of the
corresponding amino-alcohol with maleic anhydride in boiling toluene. Most of N-substituted maleimides showed an interesting
antimicrobial activity towards bacteria from the ATCC collection (Staphylococcus aureus ATCC 25923, Enterococcus faecalis
ATCC 29212, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) but the MIC values for P. aeruginosa were
always high (128 mg/ml). The imides with alkyl substituents showed higher activities than aromatic analogues with MIC values in
´
the range of 8–32 mg/ml. Comparatively, succinimides were practically inactive. © 2002 Published by Editions scientifiques et
me´dicales Elsevier SAS.
Keywords: Antimicrobial activities; N-Substituted imides; Maleimides
sis [11], whose structures are reported in Fig. 1. Early
reports [12–14] concerning maleimides concluded that
they interact preferably with the hydrophobic domains
of enzymes, based on the fact that the inactivation of
sulfhydryl groups is greatly affected by the side-chain
length of the derivatives.
In the present paper, we studied the antibacterial
activities of a series of maleimides and succinimides
against few reference bacteria from the ATCC collec-
tion. Some of these compounds have been previously
described, but only a very few of them were tested for
their antibacterial activities [1].
1. Introduction
Bacterial resistance to antibiotics is an increasing
problem that concerns clinicians, the pharmaceutical
industry and chemists. The multidrug-resistant bacteria
are the major cause of failure in the treatment of
infectious diseases. Thus the need for novel antibiotics
is more and more important.
Thiol-reactive compounds are often inhibitors of cys-
teine proteinases or other proteins with an essential
cysteine. Important biological properties concerning
bactericidal, fungicidal and anticancer were reported
for N-substituted imides such as maleimides [1–6] and
related compounds: acrylamides such as isohematinic
acid [7], a natural antibiotic produced by Actinoplanes
philippinensis, some vinyl ketones [8] with activities
against yeast and fungi (including Aspergillus species),
vinyl sulfones [9] with antimalarial activities, buteno-
lides [10] and thalidomide, an old drug reinvestigated
mostly as anticancer agent and in the therapy of lepro-
2. Materials and methods
2.1. Chemical preparations
The most suitable starting point for the synthesis of
maleimides is an amido-acid, obtained by acylation of
the amine by the corresponding anhydride. The subse-
quent cyclization into N-substituted imide is performed
by (CH3CO)2O/CH3COONa [15] (path a) or HMDS/
* Corresponding author.
E-mail address: roger.labia@univ-brest.fr (R. Labia).
´
0014-827X/02/$ - see front matter © 2002 Published by Editions scientifiques et me´dicales Elsevier SAS.
PII: S0014-827X(02)01217-X

422
F. Zentz et al. / Il Farmaco 57 (2002) 421–426
ZnBr2 for the hydroxy-aromatic derivative [16] (path
b). The hydroxy-alkyl maleimides were directly pre-
pared by condensation of the corresponding amino-al-
cohol with maleic anhydride in boiling toluene (path c).
2.2. Bacteriological assays
In vitro antimicrobial activities of the compounds
were determined by the two-fold broth dilution method
in Mueller Hinton nutrient broth. The concentration of
mother solutions were 1024 mg/ml (50/50 water–
dimethyl sulfoxide). Minimal inhibitory concentration
(MIC) is defined as the lowest drug concentration re-
1
All products were characterized by H NMR (Bruker
Avance DPX 400), IR (Bruker IRS 55) and the micro-
analyses were in agreement with the calculated values:
C90.25%, H90.20%, N90.20%.
Fig. 1. Chemical structures of maleimides A (1–6), isohematinic acid B (7), butenolides C (10), vinyl ketones D (8) and vinyl sulfones E (9),
succinimides F, phyllanthimide G (1), thalidomide H (11).

F. Zentz et al. / Il Farmaco 57 (2002) 421–426
423
sulting in complete inhibition of growth after 18 h of
incubation at 37 °C. Dimethylsulfoxide has no antibac-
terial activity at a concentration up to 20% in water.
The tested organisms were Staphylococcus aureus
ATCC 25923, Enterococcus faecalis ATCC 29212, Es-
cherichia coli ATCC 25922 and Pseudomonas aerugi-
nosa ATCC 27853.
21: R=Phe CO2Me: Colorless oil. IR: 1747; 1713
cm−1 1H NMR (CDCl3): 3.48 (m, ABX, 2H, CH,
.
J=11.7 Hz, J%=5.1 Hz); 3.8 (s, 3H, OMe); 5.0 (m,
ABX, 1H, CH, J=11.7 Hz, J%=5.1 Hz); 6.61 (s, 2H,
CHꢀ); 7.2 (m, 5H, Ar).
22: R=Lys CO2Me: Yellow oil. IR: 1744, 1706
1
cm−1. H NMR (CDCl3): 1.27 (m, 2H, CH2); 1.63 (m,
2H, CH2); 2,18 (m, 2H, CH2); 3.5 (m, 2H, CH2); 3.7 (s,
3H, OMe); 4.63 (t, 1H, CH); 6.7 (s, 2H, CHꢀ); 6.77 (s,
2H, CHꢀ).
3. Results
23: R=CꢀCH2 CO2Me: Oil. IR: 1722, 1640 cm−1
.
1H NMR (CDCl3): 3.83 (s, 3H, OMe); 5.93 (s, 1H,
ꢀCH); 6.63 (s, 1H, ꢀCH); 6,84 (s, 2H, CHꢀ).
3.1. Chemistry
N-Substituted maleimides and succinimides were pre-
pared by one of three available procedures (paths a, b
and c) and identified by the usual methods (IR, NMR
and microanalysis).
3.1.2. General procedure (path b) for the synthesis of
imide deri6ati6es 11 and 15
Maleamic acids are synthesized as described previ-
ously (path a).
The maleamic acid (10 mmol) in dry toluene(50 ml)
and ZnBr2 (1 equiv.) was heated to 80 °C then a
solution of HMDS (1.5 equiv.) in dry toluene (10 ml)
was slowly added, in 30 min. The reaction mixture was
refluxed for an additional 6 h and concentrated under
reduced pressure to led a crude product, which was
dissolved in EtOAc (60 ml), washed twice 15 min with
0.1 M HCl (2×60 ml). The organic phase was then
washed with 2×30 ml of saturated NaHCO3, dried
(MgSO4) and concentrated under reduced pressure. The
oily product was purified by column chromatography
on silica gel. The yields are close to 85%.
3.1.1. General procedure (path a) for the synthesis of
imide deri6ati6es 1–10, 12–14 and 18–31
First, under argon, to a stirred solution of maleic or
succinic anhydride (100 mmol) in ethyl ether or CH2Cl2
(100 ml) was added dropwise at 5–10 °C, 105 mmol of
amine or amino acid, methyl ester, in ethyl ether or
CH2Cl2 (20 ml). The resulting suspension was refluxed
for 2 h and then cooled at room temperature. The
crystallized acid was filtered off and washed with dry
ethyl ether. Yields are close to 95%.
Then, under argon, 50 mmol of the maleamic or
succinamic acid was added to a mixture of 20 ml of
acetic anhydride and 2 g of anhydrous sodium acetate.
The resulting suspension was heating on a steam bath
for 8 h. The mixture was cooled and evaporated to
dryness under reduced pressure. The residue was dis-
solved in ethyl ether or CH2Cl2, washed with 2×30 ml
of cold saturated NaHCO3 and dried (MgSO4). The
solvent, distillated under reduced pressure, gave a
brown oil. This crude product was purified by column
chromatography on silica gel. Most of the products
were obtained with good yields (\80%) and some of
them crystallize spontaneously.
Products 11 and 15 have been described, respectively,
by Beyer [18] and Choi [20].
3.1.3. General procedure (path c) for the synthesis of
imide deri6ati6es 16 and 17
The hydroxy-alkyl maleimides were synthesized di-
rectly by condensation of the maleic anhydride (50
mmol) with corresponding amino-alcohol (1.05 equiv.)
in toluene (60 ml). The resulting suspension was heated
2 h at 80 °C. The imide was removed under reduced
pressure from the organic layer, to give a oily product,
which was purified by crystallization (yields 25%).
Note that compounds 16 and 17 have been described
respectively by Beyer [18] and Miyadera [21].
Note that compounds 1, 2, 3, 4, 5, 6, have been
described by Takatori [4], 7, 8, 10, 13, 14, 24 by
Trujillo-Ferrara [16], 9 by Tome [17], 12 by Razna [19],
19 by Biagini [22], 20 by Yoon [23], 25 by Allen [24], 26
by Gesson [25], 27 by Puertas [26], 28 by Houlihan [27],
29 by Schreiber [28], 30 by Kraus [29] and 31 by
Neuberger [30].
3.2. Microbiological e6aluation
The results of the screening tests of N-maleimides
and N-succinimides derivatives are reported in Tables 1
and 2. These tables show the MIC values of imides
against two Gram-positive bacteria (S. aureus ATCC
25923, E. faecalis ATCC 29212) and two Gram-nega-
tive bacteria (E. coli ATCC 25922, P. aeruginosa ATCC
27853). Dimethyl sulfoxide has no antibacterial effect at
a concentration up to 512 mg/ml.
Products 18, 21, 22 and 23 are new ones:
18: R=Ser(OAc) CO2Me: Colorless oil. IR: 1749,
1
1714 cm−1. H NMR (CDCl3): 2.0 (s, 3H, CH3COO);
3.8 (s, 3H, OMe); 4.5 (dd, ABX, 1H, J=11.6 Hz;
J%=10.0 Hz); 4.8 (dd, ABX, 1H, J=11.7 Hz, J%=4.3
Hz); 5.0 (m, ABX, 1H, J=10.0 Hz, J%=4.3 Hz); 6.8 (s,
2H, CHꢀ).

424
F. Zentz et al. / Il Farmaco 57 (2002) 421–426
Table 1
In vitro antimicrobial activities of maleimide compounds (MIC values, mg/ml)
N-(R) maleimides
No. S. aureus ATCC 25923 E. faecalis ATCC 29212
E. coli ATCC 25922
P. aeruginosa ATCC 27853
C6H5
C6H5CH2
Cyclohexyl
n-C4H9
n-C3H7
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
64
8
16
8
8
128
8
32
16
16
32
32
64
16
16
256
256
128
256
256
128
]512
256
128
256
256
]512
256
256
256
128
128
256
256
256
256
256
256
iso-C3H7
8
8
16
o-C6H4OMe
o-C6H4OAc
m-C6H4OMe
m-C6H4OAc
m-C6H4OH
p-C6H4NO2
p-C6H4OMe
p-C6H4OAc
p-C6H4OH
(CH2)2OH
(CH2)3OH
Ser(OAc) CO2Me
Ala CO2Me
Gly CO2Me
Phe CO2Me
Lys CO2Me
CꢀCH2 CO2Me
]512
64
]512
64
]512
128
64
128
32
32
64
32
32
64
64
]512
32
]512
128
64
64
16
16
128
64
64
16
64
256
]512
64
32
32
16
16
128
32
64
16
64
64
32
16
8
64
32
32
256
256
64
128
Table 2
In vitro antimicrobial activities of succinimide compounds (MIC values, mg/ml)
N-(R) succinimides
No. S. aureus ATCC 25923 E. faecalis ATCC 29212
E. coli ATCC 25922
P. aeruginosa ATCC 27853
C6H5
24
25
26
27
28
29
30
31
128
512
512
128
512
512
512
512
128
256
256
256
256
256
256
256
128
256
256
256
256
256
256
256
128
256
256
256
256
256
256
256
C6H5CH2
Cyclohexyl
n-C4H9
n-C3H7
iso-C3H7
p-C6H4OMe
Gly CO2Me
4. Discussion
maleimides (23 compounds) and succinimides (8 com-
pounds). All succinimides (compounds 24–31) showed
practically no antibacterial activity towards the differ-
ent bacteria strains tested. During a previous work, we
observed that some succinimides displayed a noticeable
antibacterial activity against bacteria isolated from the
marine environment. Maleimides (compounds 1–23)
showed more or less interesting antibacterial activities.
Globally, within all products, and the organisms used
for tests, P. aeruginosa is clearly the less susceptible
organism and all tested compounds are poorly active
with MIC values of 128 mg/ml or higher.
The other organisms are much more susceptible and
the most active compounds are: 4, 5, 6, 16 and 17. They
are maleimides substituted by a short alkyl side-chain:
propyl 5, isopropyl 6, butyl 4, hydroxy-ethyl 16 or
hydroxy-propyl 17. Related compounds, such as benzyl
A lot of bioactive compounds including many antibi-
otics, are enzyme inhibitors. In the present work con-
cerning novel antibacterial agents, we looked for
thiol-reactive compounds, which are often inhibitors of
cysteine proteinases or other proteins with an essential
cysteine. It is known that maleimides are inhibitors of
cysteine proteinases and N-ethylmaleimide (NEM) re-
acts very rapidly with sulfhydryl groups. NEM is cur-
rently used for the titration of cysteines in proteins [31].
This compound, which is too reactive, has a poor
specificity but was proposed as antimitotic [32]. In the
present work we explored the incidence of N-sub-
stituents of maleimides on their antibacterial activities.
31 compounds were synthesized as possible antibac-
terial agents. These compounds belong to two families:

F. Zentz et al. / Il Farmaco 57 (2002) 421–426
425
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most of maleimides, the lowest MIC values being 8
mg/ml. Pseudomonas aeruginosa is the less susceptible
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