Syntheses, in vitro antibacterial and cytotoxic activities of a series of 3-substituted succinimides

Article abstract of DOI:10.1016/j.farmac.2004.07.007

We have synthesized a series of 3-substituted succinimides and their in vitro antibacterial activities have been tested towards Gram-positive and Gram-negative bacteria from the ATCC collection. Some of them possess significant antibacterial activity against Gram-positive organisms (Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212) but all are poorly active or inactive against Gram-negative organisms (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853). The compounds with the lowest minimal inhibitory concentrations (esters of 3-hydroxy succinimides) are also the most cytotoxic against green monkey Vero cell line (ATCC CCL-81) and could explain that perhaps apoptosis should be implicated in eukaryotic cell cytotoxicity of succinimides.

Full text of DOI:10.1016/j.farmac.2004.07.007

IL FARMACO 59 (2004) 879–886
http://france.elsevier.com/direct/FARMAC/
Syntheses, in vitro antibacterial and cytotoxic activities of a series
of 3-substituted succinimides
Frédéric Zentz a,*, Régis Le Guillou a, Roger Labia a, Danielle Sirot b,
Boris Linard c, Alain Valla a
a C.N.R.S., Chimie et Biologie des Substances Naturelles, 6, rue de l’Université, 29000 Quimper, France
b Faculté de Médecine, Service de Bactériologie-Virologie, 28, place Henri Dunant, BP 38, 63001 Clermont-Ferrand, France
c LUMAQ EA 265, 6, rue de l’Université, 29000 Quimper, France
Received 29 November 2003; received in revised form 17 June 2004; accepted 2 July 2004
Available online 21 September 2004
Abstract
We have synthesized a series of 3-substituted succinimides and their in vitro antibacterial activities have been tested towards Gram-positive
and Gram-negative bacteria from the ATCC collection. Some of them possess significant antibacterial activity against Gram-positive
organisms (Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212) but all are poorly active or inactive against
Gram-negative organisms (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853). The compounds with the lowest
minimal inhibitory concentrations (esters of 3-hydroxy succinimides) are also the most cytotoxic against green monkey Vero cell line (ATCC
CCL-81) and could explain that perhaps apoptosis should be implicated in eukaryotic cell cytotoxicity of succinimides.
© 2004 Elsevier SAS. All rights reserved.
1. Introduction
bitory concentration (MIC) was defined as the lowest drug
concentration resulting in complete inhibition of growth after
18 h of incubation at 37 °C. Dimethyl sulfoxide (DMSO) had
no antibacterial activity at a concentration up to 20% in
water. The tested organisms were Staphylococcus aureus
ATCC 25923, Enterococcus faecalis ATCC 29212, Escheri-
chia coli ATCC 25922 and Pseudomonas aeruginosa ATCC
27853.
We have recently reported on the synthesis and antimicro-
bial activities of N-substituted maleimides [1]. We described
herein the antibacterial and the cytotoxicity of a series of
3-substituted succinimides which biological properties were
poorly documented. It appeared that only a patent in date of
1982 described a synthesis of 3-substituted succinimides and
their use as fungicides [2] and a paper in date of 1997
reported their analgesic actions [3].
2.2. Cytotoxicity assays
2.2.1. Cells
African green monkey (Cercopithecus aethiops) kidney
cells (Vero cell line no. ATCC CCL-81) was grown in RPMI
1640 (BioWest, Nuaille France) containing 100 UI/ml peni-
cillin, 100 µg/ml streptomycin and 2 mM L-glutamine
(BioWest, Nuaille France), supplemented with 10% fetal calf
serum (FCS). Cells were grown at 37 °C under 5% CO2 and
routinely passed before they reached confluence every
3 days. Cells were harvested using trypsin EDTA solution
(Bio-Whittaker Europe, Belgium) and washed. Cellular sus-
pension was incubated (4500 cells per well in 150 µl of
media) with various concentrations of imides (0–2000 µg/ml,
four wells per concentration) in 96-wells plates for 2 days.
2. Materials and methods
2.1. Bacteriological assays
In vitro antimicrobial activities were determined by the
twofold broth dilution method in Mueller Hinton nutrient
broth. The concentration of mother solutions was 1024 µg/ml
(in 50/50 water–dimethyl sulfoxide solution). Minimal inhi-
* Corresponding author. Tel. and Fax: +33 (0)2 98 90 02 27
E-mail address: zentz@iutquimp.univ-brest.fr (F. Zentz).
0014-827X/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.07.007

880
F. Zentz et al. / IL FARMACO 59 (2004) 879–886
1
Daily cell examinations were performed under a phase-
contrast microscope to determine the minimum concentra-
tion that induced cell morphology alterations such as swel-
ling, shrinkage, granularity and floating. Cytotoxicity of
imides was assessed using determination of cell viability
performed by neutral red dye method as previously described
by McLaren et al. [4] and Langlois et al. [5]. Briefly, 50 µl of
media containing neutral red dye (0.02%) was added by well.
After 4 h incubation at 37 °C, the media was removed and the
cells were washed four times. Lysis buffer (50 µl 1% acetate
50% ethanol per well) before reading DO absorbance at
540 nm. A second method was the reduction of tetrazolium
salt by viable cells. Cell titer 96 (Promega, France, Charbon-
nière) (20 µl per well) was added and the plates incubated 4 h
incubation at 37 °C before reading DO absorbance at 492 nm.
17: H RMN: 0.91 (t, 3H, J = 7.34, CH3); 1.3 (m, 2H,
CH2); 1.56 (m, 2H, CH2); 2.15 (s, 3H, CH3); 2.64 (dd, 1H,
J = 18.3, J = 4.8, CH); 3.14 (dd, 1H, J = 18.3, J = 8.7, CH);
3.52 (t, 2H, J = 7.41, CH2); 5.41 (dd, 1H, J = 8.7, J = 4.8,
CH). IR: 1751, 1714 cm–1.
1
18: H RMN: 1.42 (d, 6H, J = 18.3, CH3); 2.18 (s, 3H,
CH3); 2.64 (dd, 1H, J = 18.3, J = 4.9, CH); 3.12 (dd, 1H,
J = 18.3, J = J = 8.9, CH); 4.42 (m, 1H, CH); 5.37 (dd, 1H,
J = 8.9, J = 4.9, CH). IR: 1750, 1709 cm–1.
19: 1H RMN: 0.93 (m, 3H, CH3); 1.64 (m, 2H, CH2); 2.18
(s, 3H, CH3); 2.69 (dd, 1H, J = 18.3, J = 4.8, CH); 3.16 (dd,
1H, J = 18.3, J = 8.7, CH); 3.53 (t, 2H, J = 7.46, CH2); 5.43
(dd, 1H, J = 8.7, J = 4.8, CH). IR: 1750, 1712 cm–1.
20: 1H RMN: 2.18 (s, 3H, CH3); 2.69 (dd, 1H, J = 18.4,
J = 4.9, CH); 3.19 (dd, 1H, J = 18.4, J = 8.7, CH); 4.72 (m,
2H, CH2); 5.47 (dd, 1H, J = 4.9, J = 8.7, CH); 7.37 (m, 5H,
Ar). IR: 1749, 1706 cm–1.
21: 1H RMN: 2.04 (s, 3H, CH3); 2.19 (s, 3H, CH3); 2.71
(dd, 1H, J = 4.9, J =18.3, CH); 3.2(dd, 1H, J = 18.3, J = 8.8,
CH); 3.84 (t, 2H, J = 5.2, CH2); 4.27 (m, 2H, CH2); 5.46 (dd,
1H, J = 4.9, J = 8.8, CH). IR: 1789, 1744, 1717 cm–1.
3. Results
3.1. Chemistry
1
22: H RMN: 2.07(s, 6H, CH3); 2.18 (s, 3H, CH3); 2.73
The syntheses of maleimides 1–10 and succinimide 11
have been previously reported by our group [1]. The synthe-
ses of 3-acetoxysuccinimides 12–26 have been synthesized,
starting with malic acid, using the method that we described
in reference [6].
(dd, 1H, J = 5.1, J = 18.3, CH); 3.18 (dd, 1H, J = 18.3, J = 8.9,
CH); 4.48 (m, 4H, CH2); 4.60 (m, 1H, CH); 5.41(dd, 1H,
J = 5.1, J = 8.9, CH). IR: 1790, 1744, 1718 cm–1
23: 1H RMN: 1.62 (s, 6H, CH3); 2.06 (s, 3H, CH3); 2.18
(s, 3H, CH3); 2.64 (dd, 1H, J = 5.6, J = 18.1, CH); 3.08 (dd,
1H, J = 18.1, J = 9.1, CH); 4.44 (m, 2H, CH2); 5.36 (dd, 1H,
J = 5.6, J = 9.1, CH). IR: 1790, 1745, 1715 cm–1.
3.1.1. Physicochemical properties of derivatives 12–26
1
24: 1H RMN: 1.61 (s, 9H, CH3); 2.19 (s, 3H, CH3); 2.61
(dd, 1H, J = 5.5, J = 18.1); 3.06 (dd, 1H, J = 18.1, J = 8.1,
CH); 5.35 (dd, 1H, J = 5.5, J = 8.1, CH). IR: 1750, 1713 cm–1.
25: 1H RMN: 2.23 (s, 3H, CH3); 2.34 (s, 3H, CH3); 2.90
(dd, 1H, J = 5.1, J = 18.5, CH); 3.36(dd, 1H, J = 18.5, J = 9.0,
CH); 5.58 (dd, 1H, J = 5.1, J = 9.0, CH); 7.25 (m, 2H, Ar);
7.38 (m, 2H, Ar). IR: 1789, 1747, 1714 cm–1.
12: H RMN: 1.62 (d, 3H, J = 7.3, CH3); 2.20 (s, 3H,
CH3); 2.74 (dd, 1H, J = 18.4, J = 4.9, CH); 3.23 (m, 1H, CH);
3.77 (s, 3H, CH3); 4.87 (m, 1H, CH); 5.5 (m, 1H, CH). IR:
1750, 1719 cm-1.
1
13: H RMN: 2.2 (s, 3H, CH3); 2.78 (dd, 1H, J = 18.4,
J = 4.9, CH); 3.29 (dd, 1H, J = 18.4, J = 8.7, CH); 3.79 (s, 3H,
CH3); 4.3 (s, 2H, CH2); 5.57 (dd, 1H, J = 8.7, J = 4.9, CH).
IR: 1746, 1720 cm–1.
26: 1H RMN: 1.98 (m, 2H, CH2); 2.09 (s, 3H, CH3); 2.19
(s, 3H, CH3); 2.7 (dd, 1H, J = 4.8, J = 18.4, CH); 3.18(dd, 1H,
J = 18.4, J = 8.8, CH); 3.68(t, 2H, J = 7.0, CH2); 4.1(t, 2H,
J = 6.1, CH2); 5.4(dd, 1H, J = 4.8, J = 8.8, CH). IR: 1789,
1745, 1717 cm–1.
The 3-methylsuccinimides 27–29 were obtained by
condensation of 2-methylsuccinic acid with the appropriate
amine and further cyclisation, according to the method des-
cribed in reference [6], Fig. 1.
1
14: H RMN: (two diastereoisomers) 2.12–2.16 (2s, 3H,
CH3); 2.45–2.58 (2m, 1H, CH); 2.97–3.10 (2m, H, CH);
3.43–3.51 (2m, 2H, CH2); 3.80 (2s, 3H, CH3); 5.05 (2m, 1H,
CH); 5.25–5.36 (2m, 1H, CH2); 7.14–7.31 (2m, 5H, Ar). IR:
1749, 1721 cm–1.
15: 1H RMN: 2.19 (s, 3H, CH3); 2.82 (dd, 1H, J = 18.4,
J = 5.1, CH); 3.31 (dd, 1H, J = 18.4, J = 9.0, CH); 3.83 (s, 3H,
CH3); 5.59 (m, 1H, J = 9.0, J = 5.1, CH); 5.98 (s, 1H, CH);
6.70 (s, 1 H, CH). IR: 1729 cm–1 (broad).
1
16: H RMN: 1.22–2.17 (m, 10H, CH2); 2.18 (s, 3H,
3.1.2. Physicochemical properties of derivatives 27–29
27: 1H NMR (MeOD) 1.26 (d, 3H, CH3, J = 6.0); 2.36 (m,
1H, CH); 2.93 (m, 2H, CH2); 4.6 (s, 2H, CH2); 7.29 (m, 5H,
Ar). IR: 1701 cm–1.
CH3); 2.64 (dd, 1H, J = 18.3, J = 4.9, CH); 3.12 (dd, 1H,
J = 18.3, J = 8.8, CH); 4.02 (m, 1H, CH); 5.38 (dd, 1H,
J = 8.8, J = 4.9, CH). IR: 1747, 1708 cm–1.
Fig. 1. Syntheses of 3-methylsuccinimides 27–29.

F. Zentz et al. / IL FARMACO 59 (2004) 879–886
881
1
28: H NMR: 1.30 (d, 3H, J = 7.0, CH3); 1.36 (dd, 6H,
diethyl ether–pentane (50–50%) as eluant, to give 0.56 g of
the product 36 (71%).
CH3, J = 7.0, J = 1.1); 2.24 (dd, 1H, CH, J = 4.2, J = 17.6); 2.7
(m, 1H, CH); 2.8 (dd, H, J = 9; J = 17.6, CH); 4.35 (d, 1H,
J = 6.9, CH). IR: 1713 cm–1.
3.1.4. Physicochemical properties of derivatives 35–39
35: 1H NMR: 0.93(t, 3H, J = 7.5, CH3); 1.64(m, 2H, CH2);
2.64(dd, 1H, J = 4.8, J = 18.3, CH); 3.15(dd, 1H, J = 18.3,
J = 8.7, CH); 3.53(t, 2H, J = 7.5, CH2); 3.75(s, 2H, CH2);
5.46(dd, 1H, J = 4.8, J = 8.7, CH); 7.33(m, 5H,Ar). IR: 1750,
1701 cm–1.
1
29: H NMR: 1.30 (d, 3H, J = 7.2, CH3); 2.08 (m, 2H,
CH2); 2.34 (m, 1H, CH); 2.91 (m, 2H, CH2); 3.55 (t, 2H,
CH2, J = 6.9) 1.3 (t, 2H, J = 5.96, CH2). IR: 1700 cm–1.
The corresponding 3-hydroxysuccinimides 30–34 were
acquired by transesterification of the above described ace-
toxysuccinimides, using acetyl chloride in hot ethanol.
In a typical example, the synthesis of the compound 34
was realized according to the following protocol: Under
argon, 2.2 ml (3 eq.) of acetyl chloride was added to a stirred
solution of 2.01 g (10.1 mmol, 1 eq.) of the compound 19 in
60 ml of anhydrous ethanol. The resulting mixture was hea-
ted to 50 °C for 3 h. The mixture was cooled and distilled to
dryness under reduced pressure. The crude product was puri-
fied by column chromatography on silica gel. The product
34 was obtained with a good yield (81%, 1.28 g) and crystal-
lizes spontaneously in pentane (m.p. 68 °C).
36: 1H NMR: 2.64 (dd, 1H, J = 4.8, J = 18.3, CH); 3.16
(dd, 1H, J = 18.3, J = 8.7, CH); 3.75 (dd, 2H, J = 19.9,
J = 14.8, CH2); 4.71 (dd, 2H, J = 19.9, J = 14.8, CH2); 5.49
(dd, 1H, J = 4.8, J = 8.7, CH); 7.36 (m, 10H, Ar). IR:
1715 cm–1.
37: 1H NMR: 2.58(dd, 1H, J = 4.9, J = 18.3, CH); 3.06(dd,
1H, J = 18.3, J = 8.8, CH); 3.59(s, 2H, CH2); 3.75(s, 2H,
CH2); 3.85(t, 2H, J = 5.2, CH2); 4.30(m, 2H, CH2); 5.34(dd,
1H, J = 4.9, J = 8.8, CH); 7.32(m, 10H, Ar). IR: 1737,
1716 cm–1.
38: 1H NMR: 0.96(t, 3H, J = 7.4, CH3); 1.68(m, 2H, CH2);
2.78(dd, 1H, J = 4.8, J = 18.3, CH); 3.25(dd, 1H, J = 18.3,
J = 8.7, CH); 3.58(m, 2H, CH2); 5.56(dd, 1H, J = 4.8, J = 8.7,
CH); 6.5(d, 1H, J = 16.0, CH); 7.43(m, 3H, Ar); 7.56(m, 2H,
Ar); 7.8(d, 1H, J = 16.0 CH). IR: 1712 cm–1.
3.1.3. Physicochemical properties of derivatives 30–34
30: 1H NMR: 2.70 (dd, 1H, J = 4.8, J = 18.2, CH); 3.08
(dd, 1H, J = 18.2, J = 8.4, CH); 4.63 (dd, 1H, J = 4.8, J = 8.4,
CH); 4.68 (d, 2H, J = 3.0, CH2); 7.35 (m, 5H, Ar). IR:
1704 cm–1.
39: 1H NMR: 0.9 (t, 3H, J = 7.4, CH3); 0.94(t, 3H, J = 7.0,
CH3); 1.3(m, 12H, CH2); 1.66(m, 4H, CH2); 2.4(m, 2H,
CH2); 2.67(dd, 1H, J = 4.8, J = 18.3, CH); 3.17(dd, 1H,
J = 18.3, J = 8.7, CH); 3.54(t, 2H, J = 7.2, CH2); 5.44(dd, 1H,
J = 4.8, J = 8.7, CH). IR: 1708, 1739 cm–1.
31: 1H NMR (CH3COCH3) 2.51(dd, 1H, J = 4.6, J = 17.8);
3.05(dd, 1H, J = 17.8, J = 8.4); 3.57(m, 2H, CH2); 3.64(m,
2H, CH2); 3.87(s, 1H, OH); 4.66(dd, 1H, J = 4.6, J = 8.4,
CH); 5.21(s, 1H, OH). IR: 1703 cm–1.
Compounds 40–42 were synthesized from itaconic acid,
using the method previously reported by Stratford and Cur-
ley [7].
1
32: H NMR: 2.04 (s, 3H, CH3); 2.72(dd, 1H, J = 4.8,
J = 18.2, CH); 3.11(dd, 1H, J = 18.2, J = 8.5, CH); 3.2(s, 1H,
OH); 3.82(t, 2H, J = 5.2, CH2); 4.27(m, 2H, CH2); 4.67(m,
1H, CH). IR: 1707 cm–1.
Other derivatives: 43 were obtained by reacting benzoyl
chloride with the free base of compound 41. From the free
amine of compound 42, the amino-methyl N-benzyl succini-
mides 44–49 were produced, using the appropriate alkanoyl
chloride (44: benzoyl; 45: cinnamoyl; 46: crotonyl; 47:
acetyl; 48: hexanoyl; 49: trimethylacetyl).
In a typical example, the synthesis of the compound 44
was realized according to the following protocol: Under
argon, 0.26 ml (1.05 eq.) of triethylamine in 5 ml of tetrahy-
drofurane (THF) was added to a stirred solution of 0.45 g
(1.76 mmol, 1 eq.) of the compound 42 in 15 ml of THF. The
resulting suspension was agitated for 18 h at room tempera-
ture. Then, 0.26 ml of triethylamine and 0.30 ml (1.25 eq.) of
benzoyl chloride was added and the mixture was stirred for
24 h at room temperature. After filtration of the triethylamine
hydrochloride, the mixture was distilled to dryness under
reduced pressure. The crude oily product crystallizes sponta-
neously in acetone (m.p. 114 °C). 0.38 g of the product 44
was obtained (64%).
33: 1H NMR (DMSO): 2.6(dd, 1H, J = 4.7, J = 17.6, CH);
3.09(dd, 1H, J = 17.6, J = 8.5, CH); 4.6(dd, 1H, J = 4.7,
J = 8.5, CH); 6.2(m, 1H, OH); 6.8(m, 2H, Ar); 7.1(m, 2H,
Ar); 9.8(s, 1H, OH). IR: 1705 cm–1.
34: 1H NMR 0.93(t, 3H, J = 7.5, CH3); 1.64(m, 2H, CH2);
2.7(dd, 1H, J = 4.8, J = 18.2, CH); 2.94(s, 1H, OH); 3.09(dd,
1H, J = 18.2, J = 8.4, CH); 3.51 (t, 2H, CH2); 4.65(dd, 1H,
J = 4.8, J = 8.4, CH). IR: 1701 cm–1.
The esters of 3-hydroxy succinimides were produced by
esterification of 3-hydroxysuccinimides, using phenylacetyl
chloride (35–37), cinnamoyl chloride (38) or decanoyl chlo-
ride (39).
In a typical example, the synthesis of the compound 36
was realized according to the following protocol: under ar-
gon, at 5–10 °C, 0.5 ml (1.5 eq.) of benzoyl chloride in 10 ml
of dry toluene was added drop wise to a stirred solution of
0.5 g (2.44 mmol, 1 eq.)) of the compound 30 in 10 ml of dry
toluene. The resulting solution was heated to 75 °C for 4 h
and then cooled at room temperature. The mixture was dis-
tilled to dryness under reduced pressure. The crude product
was purified by column chromatography on silica gel, using
3.1.5. Physicochemical properties of derivatives 43–49
43: RMN 1H NMR: 2.49–2.76 (m, 2H, CH2), 2.98
(m, 1H, CH), 3.49–3.97 (m, 4H, CH2), 4.61 (s, 2H, CH2),
4.72 (s, 2H, CH2), 7.12–7.41 (m, 15H, Ar). IR: 1774, 1703,
1648 cm–1.

882
F. Zentz et al. / IL FARMACO 59 (2004) 879–886
44: 1H NMR: 2.47–2.80 (m, 2H, CH2); 2.91 (m, 1H, CH),
room temperature. The triethylamine hydrochloride was re-
moved by vacuum filtration. The filtrate was distillated under
reduced pressure and the crude product was purified by
column chromatography on silica gel, using cyclohexane–
ethyl acetate as eluant (50–50%), to afford 0.12 g (10%) of
52, which crystallize spontaneously (m.p. 129 °C).
3.44–3.65 (m, 4H, CH2), 4.58 (s, 2H, CH2), 6.13(t, 1H,
J = 5.9, NH), 7.18–7.36(m, 10H, Ar). IR: 1773, 1701,
1650 cm–1.
1
45: H NMR: 2.63–3.0(m, 2H, CH2), 3.10 (m, 1H, CH),
3.59–3.93 (m, 2H, CH2), 4.67(s, 2H, CH2), 6.12(s, 1H, NH),
6.28 (d, 1H, J = 15.6, CH), 7.29–7.52 (m, 10H, Ar) 7.63 (d,
1H, CH); IR: 1773, 1701, 1659 cm–1.
3.1.6. Physicochemical properties of derivatives 50–52
50: 1H NMR: 2.63 (dd, 1H, J = 18.3, J = 5.2, CH); 2.9 (dd,
1H, J = 18.3, J = 9.3, CH); 3.05 (m, 1H, CH); 3.54 (m, 1H,
CH); 3.72 (m, 1H, CH); 4.68 (s, 2H, CH2); 5.50 (s, 1H, NH);
7.08–7.39 (m, 10H, Ar). IR: 1778, 1735, 1701 cm–1.
51: 1H NMR: 2.21 (s, 3H, CH3); 2.25 (m, 4H, CH2) 2.52
(dd, 1H, J = 18.4, J = 4.6, CH); 2.74 (dd, 1H, J = 18.4, J = 9.1,
CH); 2.9 (m, 1H, CH); 3.21 (m, 4H, CH2); 3.40 (m, 1H, CH);
3.59 (m, 1H, CH); 4.55 (s, 2H, CH2); 5.21 (s, 1H, NH);
7.18–7.29 (m, 5H, Ar). IR: 1779, 1703, 1630 cm–1.
52: 1H NMR: 2.41 (s, 3H, CH3); 2.65 (dd, 1H, J = 18.5,
J = 5.2, CH); 2.9 (dd, 1H, J = 18.5, J = 9.2, CH); 3.06 (m, 1H,
CH); 3.59 (m, 1H, CH); 3.74 (m, 1H, CH);4.64 (s, 2H, CH2);
5.70 (s, 1H, NH); 6.97 (s, 1H, NH); 7.18–7.68 (m, 9H, Ar).
IR: 1770, 1722, 1702, 1620 cm–1.
46: 1H NMR: 1.86 (dd, 3H, J = 6.8, J = 1.2, CH), 2.6–2.9
(m, 2H, CH2), 3.0 (m, 1H, CH), 3.55–3.93 (m, 2, CH2), 4.64
(s, 2H, CH2) 5.69 (dd, 1H, J = 15.2, J = 1.4, CH) 5.9 (s, 1H,
NH), 6.81(m, 1H, CH); 7.31–7.39 (m, 5H, Ar). IR: 1771,
1699, 1665 cm–1.
1
47: H NMR: 1.93 (s, 3H, CH3) 2.54–2.9(m, 2H, CH2),
3.0 (m, 1H, CH), 3.44–3.75 (m, 2H, CH2), 4.66 (s, 2H, CH2)
6.0 (s, 1H, NH); 7.3–7.38(m, 5H, C6H5). IR: 1773, 1696,
1649 cm–1.
1
48: H NMR: 0.90 (t, 3H, J = 7.0, CH3), 1.29(m, 4H,
CH2); 1.57 (m, 2H, CH2); 2.10 (m, 2H, CH2); 2.55–2.90 (m,
2H, CH2); 2.95 (m, 1H, CH); 3.51–3.72 (m, 2H, CH2); 4.66
(s, 2H, CH2); 6.0 (s, 1H, NH); 7.3–7.4 (m, 5H, Ar). IR: 1773,
1702, 1646 cm–1.
49: 1H NMR: 1.1(s, 9H, CH3); 2.51–2.9(m, 2H, CH2); 3.0
(m, 1H, CH); 3.48–3.72 (m, 2H, CH2); 4.65 (s, 2H, CH2);
6.21 (s, 1H, NH); 7.3–7.38 (m, 5H,Ar). IR: 1773, 1705 cm–1.
From the free amine, derived from salt 42, the carbamate
50 was produced, by reaction with phenyl chloroformate.
From this latter, the urea 51 was acquired with N-methyl
piperazine and the urea 52 with 4-amino acetophenone [8].
The synthesis compounds 50, 51 and 52 were realized
according to the following procedures:
50: Under argon, 0.27 ml (1.05 eq.) of triethylamine in
5 ml of tetrahydrofurane (THF) was added to a stirred solu-
tion of 0.47 g (1.85 mmol, 1 eq.) of 42 in 15 ml of THF. The
resulting suspension was agitated for 10 h at room tempera-
ture. Then, 0.27 ml of triethylamine and 0.25 ml (1.07 eq.) of
phenyl chloroformate was added and the mixture was stirred
for another 70 h at room temperature. After filtration of the
triethylamine hydrochloride, the mixture was distilled to
dryness under reduced pressure. The crude oily product crys-
tallizes in acetone after 12 h at –18 °C. 0.38 g of white
crystals (m.p. 141–142 °C) were obtained (61%).
51: Under argon, 2.19 ml (6 eq.) of N-methylpiperazine in
5 ml of THF were added to a stirred solution of 1.09 g
(3.22 mmol, 1 eq.) of 50 in 15 ml of THF. The resulting
solution was stirred for 66 h at room temperature. The
triethylamine hydrochloride was removed by vacuum filtra-
tion and the filtrate was distillated to dryness under reduced
pressure. The crude oil was purified by column chromatogra-
phy on silica gel, using dichloromethane-methanol as eluant
(90–10%), to give 0.56 g of yellow oil in a nearly quantitative
yield.
From the free amine derived from salt 42, the oxamide 53
was produced by reaction with oxalyl chloride.
The synthesis of 53 was realized according to the fol-
lowing procedure: Under argon, 0.47 ml (1.05 eq.) of triethy-
lamine in 5 ml of THF was added to a stirred solution of
0.81 g (3.18 mmol, 1 eq.) of 42 in 15 ml of THF. The resulting
suspension was stirred for 18 h at room temperature. Then,
0.47 ml of triethylamine and 0.147 ml (0.525 eq.) of oxalyl
chloride was added and the mixture was stirred for another
24 h at room temperature. After filtration of the triethylamine
hydrochloride, the volatile fractions were removed under
reduced pressure. The concentrated solution was dissolved in
acetone and the compound 53 (0.48 g, 62%) crystallizes
(m.p. 195 °C) with addition of a small amount of diethyl
ether.
3.1.7. Physicochemical properties of derivative 53
53: 1H NMR: 2.53 (dd, 2H, J = 18.1, J = 3.85, CH); 2.88
(dd, 2H, J = 18.4, J = 9.3, CH); 3.06 (m, 2H, CH); 3.63 (m,
2H, CH); 3.76 (m, 2H, CH); 4.65 (s, 4H, CH2); 7.27–7.39 (m,
10H, Ar); 7.84 (s, 2H, NH); IR: 1701, 1777.
3.2. Microbiological evaluation
The results of the screening tests of succinimides were
reported in Tables 1–3. These tables showed the MICs of
succinimides against two Gram-positive bacteria (S. aureus
ATCC 25923, E. faecalis ATCC 29212) and two Gram-
negative bacteria (E. coli ATCC 25922, P. aeruginosa ATCC
27853). DMSO had no antibacterial effect at a concentration
up to 512 µg/ml. These strains have also been tested with
reference antibiotics: ampicillin, kanamycin, erythromycin
and ofloxacin. The results are given at the end of Table 1.
52: Under argon, 1.69 g (4 eq.) of 4-aminoacetophenone
and 0.9 ml (2 eq.) of triethylamine in 5 ml of THF was added
to a stirred solution of 1.06 g (3.13 mmol, 1 eq.) of 50 in
15 ml of THF. The resulting solution was agitated for 70 h at

F. Zentz et al. / IL FARMACO 59 (2004) 879–886
883
Table 1
In vitro antimicrobial activities of N-benzyl succinimide, 3-methyl succinimides and 3-hydroxy succinimides (see Fig. 2) and reference compounds (MICs,
µg/ml)
No.
11
R1
R2
S. aureus ATCC 25923 E. faecalis ATCC 29212
E. coli ATCC 25922
P. aeruginosa ATCC 27853
C6H5CH2–
C6H5CH2–
IsoC3H7–
H–
512
512
512
512
512
512
512
512
512
256
256
256
256
256
256
512
512
512
256
256
256
256
256
256
512
512
512
256
256
256
256
256
256
512
512
512
27
28
29
30
31
32
33
34
CH3–
CH3–
CH3–
HO–
HO–
HO–
HO–
HO–
HO(CH2)3–
C6H5CH2–
HO(CH2)2–
CH3CO2 (CH2)2–
p OHC6H4–
NC3H7–
Reference compounds
Ampicillin
0.5
2
1
32
2
4
>16
>16
>4
Kanamycin
2
Erythromycin
Ofloxacin
0.25
0.5
>4
0.03
2
2
Table 2
In vitro antimicrobial activities of esters of 3-hydroxy succinimides (MICs, µg/ml) (see Fig. 3). In the presence of cysteine (1 mg/ml) all MICs are >512 µg/ml
No
12
13
14
15
16
17
18
19
35
38
39
20
36
37
21
22
23
24
25
26
R1
R2
S. aureus ATCC 25923 E. faecalis ATCC 29212 E. coli ATCC 25922
P. aeruginosa ATCC 27853
CH3OCOAla–
CH3OCOGly–
CH3OCOPhe–
CH3OCO(CH2=C)–
Cyclohexyl–
n C4H9–
CH3–
CH3–
CH3–
CH3–
CH3–
CH3–
CH3–
CH3–
C6H5CH2–
32
32
128
128
256
128
256
256
256
128
128
256
128
256
256
128
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
256
128
256
256
256
256
256
256
128
16
64
16
128
32
256
64
16
32
IsoC3H7–
32
64
n C3H7–
16
32
n C3H7–
64
64
n C3H7–
C6H5CH=CH– 256
128
128
32
n C3H7–
n C9H19
–
128
64
C6H5CH2–
C6H5CH2–
CH3–
C6H5CH2–
64
64
C6H5CH2CO2(CH2)2– C6H5CH2–
128
128
128
64
64
CH3CO2(CH2)2–
(CH3CO2CH2)2CH–
CH3CO2C(CH3)2–
C(CH3)3–
CH3–
CH3–
CH3–
CH3–
CH3–
CH3–
128
128
128
256
256
128
128
256
128
pAc O C6H4–
CH3CO2(CH2)3–
Table 3
In vitro antimicrobial activities of 3-amino-methyl N-benzyl succinimides (MICs, µg/ml) (see Fig. 4)
No.
41
42
40
44
43
45
46
47
48
49
50
51
52
53
R3
H
R4
S. aureus ATCC 25923 E. faecalis ATCC 29212
E. coli ATCC 25922
P. aeruginosa ATCC 27853
C6H5CH2
H
256
512
512
512
>512
512
512
512
512
512
256
256
>512
>512
64
256
256
256
256
>512
256
256
256
256
256
256
256
>512
256
256
256
256
256
>512
256
256
256
256
256
256
256
>512
128
H
256
256
256
>512
256
256
256
256
256
256
256
>512
256
C6H5CH2– CH3CO–
H– C6H5CO–
C6H5CH2– C6H5CO–
H–
H–
H–
H–
H–
H–
H–
H–
H–
C6H5CH=CHCO–
CH3CH=CHCO–
CH3CO–
n C5H11CO–
(CH3)3CCO–
C6H5OCO–
See Fig. 5A
See Fig. 5B
See Fig. 5C

884
F. Zentz et al. / IL FARMACO 59 (2004) 879–886
Table 7
3.3. Cytotoxic evaluation (determination of 50% cytotoxic
concentration (CC50))
In vitro cytotoxic activities of 3-amino-methyl N-benzyl succinimides
(CC50, µg/ml) (see Fig. 4)
No.
R3
R4
CC50 (µg/ml)
(with cysteine
1 mg/ml)
The results of the screening tests of imides are reported in
Tables 4–7. Cells were incubated with various concentrations
of imide molecules (0–2000 µg/ml, four wells per concentra-
tion). The effect of cysteine on imide cytotoxicity was as-
sessed at the concentrate of 1 mg/ml in the media. Correla-
tion curve was obtained between pinocytosis of neutral red
(assessed by DO absorbance at 540 nm) and Neperian loga-
rithm of imide concentrations. The CC50 of the tested mole-
cules was defined as the concentration that reduced the absor-
bance to 50% of that of controls. Negatives controls showed
41
42
43
45
46
47
48
49
50
51
52
53
H–
C6H5CH2–
H–
6.05 (67)
H–
8.17 (>2000)
33
C6H5CH2–
H–
C6H5CO–
C6H5CH=CHCO– 110
H–
CH3CH=CHCO–
CH3CO–
601
H–
365
H–
N C5H11CO–
(CH3)3CCO–
C6H5OCO–
See Fig. 5A
See Fig. 5B
See Fig. 5C
284
H–
572
H–
299
H–
1096
<31.25 a
200
Table 4
H–
In vitro cytotoxic activities of maleimides 1–10 (CC50, µg/ml) (see Fig. 6)
H–
No
1
R1
CC50 (µg/ml) (with cysteine 1 mg/ml)
a For compound 52, the lowest concentration tested was 31.25 µg/ml.
C6H5CH2
2.23 (735)
2
3
5
6
7
8
9
10
Cyclohexyl
n-C4H9–
9
that DMSO had no cytotoxic effect at concentration up to
20 mg/ml.
2.59
p-C6H4OMe
(CH2)2OH–
Ser(OAc) CO2Me
Ala CO2Me
Gly CO2Me
Phe CO2Me
<31.25 a
4.06
27
4. Discussion
4.95
15.6
<31.25 a (493)
More than 40 succinimides have been synthesized. They
were substituted at the nitrogen atom (position 1) and at
position 3 (see Figs. 2–5). Ten maleimides (see Fig. 6) were
added, in order to compare their cytotoxycities (products
1–10). Antibacterial activities of these latter have been repor-
ted in Ref. [1].
a For compounds 5 and 10, the lowest concentration tested was
31.25 µg/ml.
Table 5
In vitro cytotoxic activities of N-benzyl succinimide, 3 methyl N-benzyl
If R2 is a hydrogen atom, a methyl or a hydroxyl group, the
compounds have no noticeable antibacterial activity (see
Table 1). Three of them (with R2 = H: 11, CH3: 27 or OH: 28)
were selected for toxicity studies: they demonstrated poor
toxicity towards Vero cells, with CC50 close to 1 mg/ml.
Twenty molecules with an ester sub-unit display some
moderate activities against bacteria, with the lowest MICs in
succinimide and 3 hydroxy N-benzyl succinimide compounds (CC50
µg/ml) (see Fig. 2)
,
No
11
27
28
R1
R2
CC50 (µg/ml)
C6H5CH2–
C6H5CH2–
C6H5CH2–
H–
898
735
972
CH3–
HO–
Table 6
In vitro cytotoxic activities of esters of 3-hydroxy succinimides (CC50
µg/ml) (see Fig. 3)
,
No.
R1
R2
CC50 (µg/ml)
(with cysteine
1 mg/ml)
10
Fig. 2. 3-Methyl succinimides and 3-hydroxy succinimides.
12
13
14
19
35
38
39
20
36
21
22
23
24
25
26
CH3OCOAla–
CH3OCOGly–
CH3OCOPhe–
nC3H7–
CH3–
CH3–
40
CH3–
<31.25 a (>2000)
4.05 (105)
4.95 (110)
89
CH3–
nC3H7–
C6H5CH2–
C6H5CH=CH–
nC3H7–
nC3H7–
NC9H19
–
11.2 (181)
9.97 (1339)
20.9 (>2000)
31.25
C6H5CH2–
C6H5CH2–
CH3CO2(CH2)2–
CH3–
Fig. 3. Esters of 3-hydroxy succinimides.
C6H5CH2–
CH3–
(CH3CO2CH2)2CH– CH3–
CH3CO2C(CH3)2– CH3–
121.5
8.58
C(CH3)3–
CH3–
CH3–
CH3–
4.05
pAc O C6H4–
225
CH3CO2(CH2)3–
31.25
a For compound 14, the lowest concentration tested was 31.25 µg/ml.
Fig. 4. 3-Amino-methyl N-benzyl succinimides.

F. Zentz et al. / IL FARMACO 59 (2004) 879–886
885
Fig. 5. Nature of substituents R4.
the range of 16–32 µg/ml towards Gram-positive strains (see
Table 2). We could notice that the antibacterial activities
against Gram-negative bacteria (E. coli, P. aeruginosa) re-
mained very poor. The toxicity against Vero cells was also
higher as demonstrated for 15 molecules, but large variations
were observed (see Table 6). The most toxic compounds are
19 and 24 with CC50 = 4.05 µg/ml and the less toxic is 25
with CC50 = 225 µg/ml (55 times less toxic). The most toxic
molecules bear a short alkyl chain at position 1. For molecu-
les with R2 being CH3, alkyl, benzyl or styryl (see Table 6),
the antibacterial activity is generally lower but the toxicity
against Vero cells is not changed.
Molecules 51 and 52 (ureas) and 53 (oxamide) were also
prepared, as they are related to publish substances that pos-
sess antibacterial activities [9].
In a first step, we could suspect that the activity of
3-acetoxysuccinimides is due to in vivo elimination of the
acetoxy group, leading to maleimide compounds that are
known to be inhibitors of SH-enzymes. In a second step, in
order to clarify this point, several tests were performed in the
presence of 1 mg/ml of cysteine.
Concerning the antibacterial activities and toxicity tests,
all combinations were inactive and non-toxic in the presence
of cysteine, as it occurs for maleimides (see Table 4).
For molecule 42, a 3-amino-methyl succinimide taken as
an example, formation of a maleimide is strictly impossible
in biological conditions. Surprisingly CC50 toxicity values
shift from 8 to >2000 µg/ml in the presence of cysteine. Thus,
the mechanism of action of these molecules remains to be
clarified.
Observation of cell morphology alterations during the first
part of the tests showed that the cells begin to shrink around
the CC50 (Fig. 7), although membrane permeability is not yet
affected, as described when a cell is undergoing an apoptosis
process [10]. Moreover, a previous paper showed that a
conjugated vinyl-ketone (ethacrynic acid, a diuretic, inhibi-
tor of some SH-enzymes) which had some similarities with
these maleimides induce apoptosis in cell line [11] and it was
Fourteen amino-methyl succinimides were synthesized
(Fig. 4). Only two of them display a moderate antibacterial
activity: compound 41 versus E. faecalis (MIC: 64 µg/ml)
and 53 versus P. aeruginosa (128 µg/ml) (see Table 3). With
few exceptions, these compounds were weakly toxic (see
Table 7).
Fig. 6. Maleimides.
Fig. 7
. For an example, cytotoxic effects of succinimide 19 (CC50 = 4.05 µg/ml) onVero cells were grown for 2 days in absence (A) or in presence of succinimide
19 (B) at 31.25 µg/ml, and colored with neutral red dye.

886
F. Zentz et al. / IL FARMACO 59 (2004) 879–886
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