Lowering of 5-nitroimidazole's mutagenicity: Towards optimal antiparasitic pharmacophore

Article abstract of DOI:10.1016/j.ejmech.2008.05.015

To improve the antiparasitic pharmacophore, 20 5-nitroimidazoles bearing an arylsulfonylmethyl group were prepared from commercial imidazoles. The antiparasitic activity of these molecules was assessed against Trichomonas vaginalis, the in vitro cytotoxicity was evaluated on human monocytes and the mutagenicity was determined by the Salmonella mutagenicity assay. All IC₅₀ on T. vaginalis were below the one of metronidazole. The determination of the specificity indexes (SIs), defined as the ratios of the cytotoxic activity and the antitrichomonas activity, indicated that 11 derivatives had a SI over the one of metronidazole. Molecules, bearing an additional methyl group on the 2-position, showed a lower mutagenicity than metronidazole. Moreover, three derivatives were characterized by a low mutagenicity and an efficient antitrichomonas activity.

Full text of DOI:10.1016/j.ejmech.2008.05.015

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 653e659
http://www.elsevier.com/locate/ejmech
Original article
Lowering of 5-nitroimidazole’s mutagenicity: Towards optimal
antiparasitic pharmacophore
a
Maxime D. Crozet , Celine Botta , Monique Gasquet , Christophe Curti , Vincent Remusat ,
c
b
a
a
´
Sebastien Hutter , Olivier Chapelle , Nadine Azas , Michel De Meo , Patrice Vanelle
´
b
b
b
c
a,
*
´
´
a Laboratoire de Pharmaco-Chimie Radicalaire, Faculte´ de Pharmacie, Universite´ d’Aix-Marseille I, II et III - CNRS, UMR 6264:
Laboratoire Chimie Provence, 27 Bd Jean Moulin, 13385 Marseille Cedex 05, France
b Laboratoire de Bioge´notoxicologie et Mutagene`se Environnementale, EA 1784, IFR PMSE 112, Universite´ de la Me´diterrane´e,
Faculte´ de Pharmacie, 27 Bd Jean Moulin, 13385 Marseille Cedex 05, France
c Laboratoire de Parasitologie, Mycologie me´dicale, Hygie`ne et Zoologie, UMR MD3, Universite´ de la Me´diterrane´e, Faculte´ de Pharmacie,
27 Bd Jean Moulin, 13385 Marseille Cedex 05, France
Received 7 November 2007; received in revised form 7 May 2008; accepted 19 May 2008
Available online 27 May 2008
Abstract
To improve the antiparasitic pharmacophore, 20 5-nitroimidazoles bearing an arylsulfonylmethyl group were prepared from commercial
imidazoles. The antiparasitic activity of these molecules was assessed against Trichomonas vaginalis, the in vitro cytotoxicity was evaluated
on human monocytes and the mutagenicity was determined by the Salmonella mutagenicity assay. All IC50 on T. vaginalis were below the
one of metronidazole. The determination of the specificity indexes (SIs), defined as the ratios of the cytotoxic activity and the antitrichomonas
activity, indicated that 11 derivatives had a SI over the one of metronidazole. Molecules, bearing an additional methyl group on the 2-position,
showed a lower mutagenicity than metronidazole. Moreover, three derivatives were characterized by a low mutagenicity and an efficient
antitrichomonas activity.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: 5-Nitroimidazoles; Arylsulfonylmethyl group; Antiparasitic activity; Mutagenicity
1. Introduction
and therapeutic activities of these compounds in eukaryotic
cells yet. A body of indirect evidence suggests that the nitro
group reduction, directly involved in the mechanism of action
of the 5-nitroimidazoles is also responsible for the expression
of mutagenicity and drug residue formation in mammalian
cells [2]. Effectively, the metabolic way of the 5-nitroimidazoles
(inhibition of the ferredoxine reductase) has been showed to
produce free radicals and cause DNA damage [3]. Further-
more, it has been assumed, that the separation of antiprotozoal
and genotoxic activities is not feasible, both of theses proper-
ties being mediated through a common metabolic intermedi-
ate. A nitroimidazole possessing good pharmacological
activities with no mutagenicity would, therefore, be of great
interest not only from a safety point of view but would also
provide a basis for further investigations of the mode of action
and mechanism of expression of mutagenicity. Moreover,
emergence of metronidazole-resistant T. vaginalis results in
5-Nitroimidazoles are a well-established group of pharma-
ceutical drugs. These chemotherapeutic agents inhibit the
growth of both anaerobic bacteria and some anaerobic proto-
zoa [1]. Nowadays, among the nitroimidazole family, the
leader drug is metronidazole and it uses extensively for the
treatment of both infections caused by protozoa such as
Trichomonas vaginalis, Entamœba histolytica, Giardia intesti-
nalis and infections induced by anaerobic bacteria. However,
the 5-nitroimidazoles have been found to possess a high muta-
genic activity in prokaryotic microorganisms. Although a few
hypotheses have been published, active research in this area
has not led to a comprehensive mechanism for the mutagenic
* Corresponding author. Tel.: þ33 4 91 83 55 80; fax: þ33 4 91 79 46 77.
E-mail address: patrice.vanelle@pharmacie.univ-mrs.fr (P. Vanelle).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.05.015

654
M.D. Crozet et al. / European Journal of Medicinal Chemistry 44 (2009) 653e659
O
S
O
S
O
+
Na
Cl
-
DMSO
58 °C
O
BrCH2Cl
+
X
X
2a X = H
63%
X = H, CH3, Cl
2b X = CH3 53%
2c X = Cl
47%
Scheme 1.
decreased success of current therapies [4]. These refractory
cases are usually treated with higher doses of metronidazole,
which leads to an increase in the occurrence of side effects.
So, alternative curative therapies are needed.
recrystallization, the desired 1-bromo-4-(chloromethylsulfonyl)-
benzene (2d) and 1-fluoro-4-(chloromethylsulfonyl)benzene (2e)
were obtained, respectively, in 67% and 65% yield.
The 1,2-dialkyl-4-arylsulfonylmethyl-5-nitro-1H-imidazoles
Our previous study was based on the evaluation of mutagenic
and the genotoxic activities of 48 imidazoles derived from dime-
tridazole and metronidazole [5]. Although substituents at the 1-
and 2-positions were found to modulate these activities, all the
tested compounds induced mutagenicity in the Salmonella muta-
genicity assay and genotoxicity as assessed by the SOS chromot-
est. In order to enlarge the pharmacological potential of the
nitroimidazole ring, we have decided, in this present study, to
modulate the 4-position of easily obtainable 1-methyl-5-nitro-
1H-imidazole [6] or commercial dimetridazole, metronidazole
and secnidazole. We focused our workonthe synthesis oforiginal
compounds from 1,2-dialkyl-5-nitro-1H-imidazoles or 1-methyl-
5-nitro-1H-imidazole bearing an arylsulfonylmethyl group in the
4-position by vicarious nucleophilic substitution of hydrogen
(VNS) of Makosza followed by the evaluations of antitrichomo-
nas activity (TV87), THP1 celltoxicity and mutagenicity (Salmo-
nella mutagenicity assay). The arylsulfonylmethyl group in the
4-position was never investigated in these areas.
and
4-arylsulfonylmethyl-1-methyl-5-nitro-1H-imidazoles
(Table 1) were prepared according to our published procedure
by vicarious nucleophilic substitution of hydrogen from chloro-
methylaryl sulfones with 1-methyl-5-nitro-1H-imidazole (3)
(easily obtainable) [6] or commercial dimetridazole (4), metro-
nidazole (5), secnidazole (6) (Scheme 3) [9].
3. Results and discussion
The activities of 20 nitroimidazole derivatives were com-
pared with that of precursors, 1-methyl-5-nitro-1H-imidazole
(3), dimetridazole (4), metronidazole (5) and secnidazole (6)
in four series of molecules. For the purpose of screening these
new compounds, we have directly compared their IC50 and SI
with those of precursors (Table 2). The 24-nitroimidazole
derivatives showed antitrichomonas activity with IC50 ranging
from 0.044 mM (9c) to 2.746 mM (5). The reference standards
dimetridazole, metronidazole and secnidazole exhibited IC50
of 0.708 mM, 2.746 mM and 0.810 mM, respectively. In the
1-methyl-5-nitro-1H-imidazole and metronidazole series of
molecules, all compounds were more effective against T.
vaginalis than precursors.
Among the 24 tested molecules, 21 showed stronger activ-
ity against T. vaginalis than metronidazole and two had similar
activity. The highest activity was obtained with 2-[4-(4-chlor-
ophenylsulfonylmethyl)-2-methyl-5-nitro-1H-imidazol-1yl]
ethanol (9c) with a specificity index 13136. Fifteen molecules
did not have any toxicity on THP1 cells. Among remaining
molecules, the cell toxicity (IC50) ranged from 59 mM to
578 mM. The present study demonstrated the positive influ-
ence of the phenylsulfonylmethyl moiety in 4-position. The
influence of the sulfonyl group was already observed in 2-
substituted-5-nitroimidazole series [10]. The compounds
2. Chemistry
Chloromethylaryl sulfones (2aec) were prepared by alkyl-
ation of the corresponding sulfinate salts with bromochlorome-
thane in DMSO at 58 ꢀC (Scheme 1) [7].
Non-commercially available sulfinate salts could be prepared
from sodium sulfite-mediated reduction of the corresponding sul-
fonyl chlorides in water (Scheme 2). In order to optimize the pro-
cedure described by Antane et al. [8], DMSO was added in the
aqueous reaction mixture to facilitate the solubilizations of the
4-bromobenzene-1-sulfonyl chloride and 4-fluorobenzene-1-
sulfonyl chloride. The reduced sulfonyl chlorides were treated
with a phase transfer catalyst (PTC) and an excess of bromo-
chloromethane at 75 ꢀC during 20 h. After work-up and
O
S
O
S Cl
O
O
S
O
+
Na
-
Cl
Na2SO3 / NaHCO3
H2O / DMSO
BrCH2Cl
TBABr
O
X
X
X
2d X = Br 67%
2e X = F 65%
X = Br, F
Scheme 2.

M.D. Crozet et al. / European Journal of Medicinal Chemistry 44 (2009) 653e659
655
Table 1
Preparation of 4-arylsulfonylmethyl-1,2-dialkyl-5-nitro-1H-imidazoles and 4-
arylsulfonylmethyl-1-methyl-5-nitro-1H-imidazoles
4. The Salmonella mutagenicity assay
The 24-nitroimidazole derivatives were mutagenic on
TA100 and YG1042. The calculated mutagenic powers (MP)
are included in Table 3. The MP ranged from 0.56 rev/nmol
(9a) to 44.03 rev/nmol (7c) for TA100 and from 1.29 rev/
nmol (10d) to 222.71 rev/nmol (4) for YG1042. The mutage-
nicity could be linked to the NO2 reduction by nitroreductase
activity for 19/24 molecules as assessed by comparison of the
MPTA100 and the MPYG1042. Addition of the phenylsulfonyl-
methyl, (4-methylphenylsulfonyl)methyl, (4-chlorophenyl
sulfonyl)methyl and (4-bromophenylsulfonyl)methyl on the
4-position resulted in significant decreases of MPTA100 for
the 4e6 series of molecules. These additions either did not
modify or increased the MPTA100 for the 3 series. These results
underlined the critical role of substituents in the 2-position as
the only difference between the four series was the methyl
group in the 2-position of the nitroimidazole ring.
Entry
Sulfone derivatives
Compound
number
Yield
(%)
X
R1
R2
1
H
CH3
CH3
H
7a
55
29
42
57
54
28
20
49
53
40
24
24
41
51
20
37
37
51
69
17
2
CH3
Cl
Br
F
H
7b
7c
3
CH3
CH3
H
4
H
7d
7e
5
CH3
CH3
H
6
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
8a
7
CH3
Cl
Br
F
CH3
CH3
8b
8c
8
9
CH3
CH3
8d
8e
10
11
12
13
14
15
16
17
18
19
20
H
(CH2)2OH
(CH2)2OH
(CH2)2OH
(CH2)2OH
(CH2)2OH
CH2CH(CH3)OH
CH2CH(CH3)OH
CH2CH(CH3)OH
CH2CH(CH3)OH
CH2CH(CH3)OH
9a
CH3
Cl
Br
F
9b
9c
9d
9e
H
10a
10b
10c
10d
10e
Different correlations between MP and IC50 were attemp-
ted. The only statistical significant correlation was found be-
CH3
Cl
Br
F
tween MPTA100 and IC50
(Fig. 1). However, this
T. vaginalis
correlation was limited to 21 molecules. The molecules 9b,
9c and 10b, characterized by a low MPTA100 and an efficient
IC50 T. vaginalis, were excluded from the non-linear regression.
All the tested molecules were found to be mutagenic in the
Salmonella mutagenicity assay using the most sensitive strain
TA100 [12,13]. It has been shown that mono- or bicyclic nitro-
aromatics revert the base substitution strains (such as TA100)
more efficiently and nitro compounds with three or more
aromatic rings induce mainly mutations in frameshift tester
strains (such as TA98). Comparison of MP between TA100
and YG1042 showed that the nitro group reduction played
a key role in the mutagenic activity for 19 molecules (Table 3).
For the five remaining molecules, the nitroreduction could
not be statistically demonstrated although the MPYG1042 were
always above the MPTA100. The nitroaromatics are generally
reduced to hydroxylamines that are subsequently converted to
electrophilic nitrogen species. The nitronium ions react with
the nucleophilic sites of cellular macromolecules (such as
DNA). Furthermore, the methyl group at the 2-position and
the arylsulfonylmethyl group at the 4-position modulated the
nitroreduction at the 5-position. We have shown that the
Conditions: KOH (5 eq.), 1,2-dialkyl-5-nitro-1H-imidazole (1 eq.) or 1-
methyl-5-nitro-1H-imidazole (1 eq.), chloromethylaryl sulfone (1 eq.),
DMSO.
have side chains that are more hydrophobic than those of met-
ronidazole, and this may reflect the increased activities
because the site of metronidazole activation in Trichomonas
is the membrane-localized electron transport pathway. The
mechanism of action of metronidazole in Mz-susceptible
parasites requires reduction of the critical nitro group to toxic
radicals by ferrodoxin, which is reduced by the membrane-lo-
calized enzyme pyruvate: ferrodoxin oxido-reductase (PFOR)
[11]. However, compounds 8e and 10e were less effective than
their precursors dimetridazole and secnidazole, respectively,
and 9e exhibited the lowest antitrichomonas activity for the
metronidazole series of molecules, showing that the substitu-
tion on the phenylsulfonyl group by a fluorine atom resulted
in a decrease of IC50 T. vaginalis. These encouraging results
had to be completed with genotoxicity evaluation by Salmo-
nella mutagenicity assays.
X
O
O
S
O
S
7a-e, 8a-e,
N
N
Cl
9a-e, 10a-e
O
KOH
DMSO
+
O2N
O2N
R2
R2
N
R1
N
X
R1
a X = H
b X = CH3
c X = Cl
d X = Br
e X = F
7 R1 = CH3 R2 = H
3 R1 = CH3 R2 = H
X = H, CH3, Cl, Br, F
8 R1 = CH3 R2 = CH3
4 R1 = CH3 R2 = CH3
9 R1 = (CH2)2OH R2 = CH3
10 R1 = CH2CH(CH3)OH R2 = CH3
5 R1 = (CH2)2OH R2 = CH3
6 R1 = CH2CH(CH3)OH R2 = CH3
Scheme 3.

656
M.D. Crozet et al. / European Journal of Medicinal Chemistry 44 (2009) 653e659
Table 2
Antitrichomonas activity and cell toxicity
in regard to other studies [4,18] which explain that metronida-
zole therapeutic use is done in microaerophilic conditions, it
could appear interesting to continue our work by testing aero-
bic conditions. Furthermore, it could be valuable to use not
only a metronidazole sensitive reference strain, but also a clin-
ical strain with elevated minimum lethal concentration (MLC).
These complementary studies could allow the selection of new
interesting molecules.
Mol.
IC50
IC50 THP 1
(mM)
SITrichomonas
(mM)
Trichomonas
3
0.629
0.199
0.155
0.158
0.394
0.334
>1966a
>888a
>846a
174
>694a
>835a
>3125a
>4462a
>5458a
1101
>1761a
>2500a
7a
7b
7c
7d
7e
5. Experimental section
4
0.708
0.240
0.355
0.918
0.660
2.553
>1771a
>846a
242
>2501a
>3525a
684
8a
8b
8c
8d
8e
5.1. Synthetic methodology
68
326
>797a
74
493
>312a
Yields refer to purified products and are not optimized.
Melting points were determined on Buchi melting point
¨
5
2.746
1.536
0.067
0.044
1.635
1.747
>1460a
>768a
59
>531a
>500a
880
B-540 apparatus and are uncorrected. NMR experiments
were recorded on a Bruker Avance 200 spectrometer (operat-
ing at 200 MHz for 1H and 50 MHz for 13C). 1H and 13C NMR
shifts (d) were reported in parts per millions (ppm) with re-
9a
9b
9c
9d
9e
578
124
>728a
13136
75
>416a
spect to CDCl3 7.26 ppm for H and 77.00 ppm for 13C and
1
1
DMSO-d6 2.50 for H and 39.70 ppm for 13C. Multiplicities
6
0.810
0.615
0.124
0.732
0.418
2.378
>1350a
418
>1666a
679
10a
10b
10c
10d
10e
are represented by s (singlet), d (doublet), t (triplet), q (quar-
tet) and m (multiplet). Coupling constant (J ) are in Hertz
(Hz). Elemental analyses were performed on a Thermo
Finnigan EA 1112 at the Spectropole of the University of
Aix-Marseille III. Thin-layer chromatography (TLC) was per-
formed on 5 cm ꢁ 10 cm aluminium plates coated with silica
gel 60F-254 (Merck) in an appropriate solvent. Column chro-
matography was performed over silica gel 60 (70e230 mesh).
Procedures for the synthesis of compounds 2aed, 7aed, 8ae
d, 9aed and 10aed have been previously described [8]. Anal-
yses indicated by the symbols of the elements or functions
were within ꢂ0.4% of the theoretical values.
>707a
>668a
126
>5701a
>912a
301
>699a
>293a
Mol: molecules and IC50: mean of three independent experiments.
a
No toxicity at the highest tested concentration related to the solubility of
the molecule.
replacement of the methyl group at the 2-position by a lactam
group could double the mutagenicity of metronidazole [5]. In
the present study, the methyl group at the 2-position combined
with the arylsulfonylmethyl group at the 4-position lowered the
MPTA100 of dimetridazole, metronidazole and secnidazole
series. The arylsulfonylmethyl group alone did not modify
the MPTA100 of the 1-methyl-5-nitro-1H-imidazole series.
In eukaryotic cells, 5-nitroimidazoles are primarily metab-
olized by the oxidative P450 enzymes (IA, IIB, IIC and IIIA)
to generate hydroxyl and acetic acid metabolites. The hy-
droxyl metabolite has been shown to be more mutagenic
than the original compound [14]. Another hypothesis is the
production of radical oxygen species through the futile cycle
of incomplete nitroreduction in mammalian cells [15].
In conclusion, a good correlation was demonstrated be-
tween the antiprotozoal activity and the mutagenicity for 21
molecules which reflects their ability to damage DNA through
covalent binding and induction of DNA breaks [16]. However,
three molecules were excluded from this correlation. They
were characterized by a low mutagenicity and high antipara-
sitic activity. Although the mechanism of action explaining
their biological activity remains to be elucidated, these newly
synthesized compounds allow the disconnection between
mutagenicity and biological activity for the first time. This
approach offers the possibility of synthesizing new and poten-
tially safer 5-nitroimidazoles.
5.2. 1-Fluoro-4-(chloromethylsulfonyl)benzene (2e) [19]
A stirred mixture of 4-fluorobenzene-1-sulfonyl chloride
(3.00 g, 15.4 mmol), sodium sulfite (3.30 g, 26.2 mmol), and
sodium bicarbonate (2.20 g, 26.3 mmol) in water (20 mL)
was heated at 80 ꢀC for 6 h. The crude sodium sulfinate
solution was allowed to cool for 30 min, and then treated
with bromochloromethane (15 mL, 230 mmol) and tetra-N-
butylammonium bromide (1.00 g, 3.10 mmol). The resultant
mixture was heated at 75 ꢀC for 10 h and poured into an
iceewater mixture. The residue was extracted with chloroform
(3 ꢁ 50 mL). The combined organic layers were washed with
water (3 ꢁ 30 mL), brine, dried over MgSO4 and removed un-
der reduced pressure. The crude solid residue was purified by
column chromatography eluting with chloroform/petroleum
ether (7:3) and recrystallized from propan-2-ol giving a white
solid (2.07 g, 65%): mp 88 ꢀC (propan-2-ol) (Ref. [19] mp
88e89 ꢀC); 1H NMR (CDCl3, 200 MHz) d 4.53 (s, 2H,
CH2), 7.25e7.34 (m, 2H, CH ꢁ 2), 7.97e8.04 (m, 2H,
CH ꢁ 2); 13C NMR (CDCl3, 50 MHz) d 58.5 (CH2), 116.8
(d, J ¼ 23 Hz, CH ꢁ 2), 131.6 (C), 132.4 (d, J ¼ 10 Hz,
CH ꢁ 2), 166.6 (d, J ¼ 258 Hz, C). Anal. C7H6ClFO2S: (C,
H, S).
T. vaginalis tests are not standardized [17], but under anaer-
obic conditions, they are well documented [10]. Nevertheless,

M.D. Crozet et al. / European Journal of Medicinal Chemistry 44 (2009) 653e659
657
Table 3
Mutagenic activities (MA) and mutagenic powers (MP) of the 24-nitroimidazole derivatives
Mol.
Mutagenic activities
TA100
YG1042
r2
P
PE
MA
MP
Ratio
r2
P
PE
MA
MP
Ratio
Comment
3
0.99
0.99
0.99
0.99
0.99
0.99
<10ꢃ5
<10ꢃ5
<10ꢃ5
<10ꢃ5
<10ꢃ5
<10ꢃ5
0.08
0.05
0.52
0.13
0.94
0.53
121.79
50.91
69.71
139.44
41.41
35.17
15.48
14.32
20.59
44.03
14.92
10.53
1.85
1.71
2.46
5.27
1.78
1.26
0.98
0.99
0.99
0.96
0.98
0.99
<10ꢃ5
<10ꢃ5
<10ꢃ5
<10ꢃ5
<10ꢃ5
<10ꢃ5
0.05
0.08
0.99
0.23
0.32
0.85
1551.06
176.64
185.37
167.38
164.42
157.34
197.14
49.69
54.74
52.85
59.22
47.09
2.34
0.59
0.65
0.63
0.70
0.56
NO2 (12.7)
NO2 (3.5)
NO2 (2.7)
No NO2 (1.2)
NO2 (4.0)
NO2 (4.5)
7a
7b
7c
7d
7e
4
0.98
0.99
0.98
0.96
0.98
0.97
<10ꢃ5
<10ꢃ5
<10ꢃ5
<10ꢃ5
<10ꢃ5
<10ꢃ5
0.54
0.05
0.98
0.16
0.06
0.57
153.80
27.91
15.03
7.15
21.71
8.24
4.65
2.36
6.59
1.48
2.60
0.99
0.56
0.28
0.79
0.18
0.99
0.98
0.99
0.98
0.98
0.97
<10ꢃ5
<10ꢃ5
<10ꢃ5
<10ꢃ5
<10ꢃ5
<10ꢃ5
0.53
0.62
0.09
0.74
0.06
0.07
1578.04
74.18
54.97
25.21
32.91
24.26
222.71
21.91
17.00
8.31
2.64
0.26
0.20
0.10
0.15
0.09
NO2 (10.3)
NO2 (2.7)
NO2 (3.7)
NO2 (3.5)
NO2 (1.9)
NO2 (5.1)
8a
8b
8c
8d
8e
17.61
4.72
12.32
7.60
5
0.96
0.74
0.77
0.94
0.84
0.80
<10ꢃ5
2Eꢃ3
0.09
0.82
0.20
0.14
0.74
0.47
48.85
1.72
4.62
6.22
2.73
1.91
8.36
0.56
1.57
2.24
1.10
0.66
1.00
0.07
0.19
0.27
0.13
0.08
0.95
0.95
0.89
0.96
0.95
0.90
<10ꢃ5
<10ꢃ5
<10ꢃ5
<10ꢃ5
<10ꢃ5
<10ꢃ5
0.46
0.92
0.29
0.54
0.06
0.67
469.79
8.36
6.14
84.34
2.72
2.08
5.30
3.73
3.46
1.00
0.03
0.02
0.06
0.04
0.04
NO2 (10.1)
NO2 (4.9)
No NO2 (1.3)
NO2 (2.4)
NO2 (3.4)
NO2 (5.3)
9a
9b
9c
9d
9e
7Eꢃ5
<10ꢃ5
1Eꢃ5
1Eꢃ5
14.73
9.123
10.09
6
0.97
0.69
0.87
0.93
0.97
<10ꢃ5
5Eꢃ4
<10ꢃ5
<10ꢃ5
<10ꢃ5
0.53
0.15
0.55
0.28
0.75
25.74
2.58
2.07
2.18
3.36
4.77
0.88
0.73
0.81
1.41
0.57
0.10
0.09
0.10
0.17
0.81
0.68
0.81
0.58
0.67
2Eꢃ5
6Eꢃ4
2Eꢃ5
4Eꢃ3
7Eꢃ6
0.24
0.50
0.06
0.13
0.66
128.44
10.76
6.76
23.78
3.65
2.39
1.39
1.29
0.28
0.04
0.03
0.02
0.02
NO2 (5.0)
NO2 (4.2)
10a
10b
10c
10d
NO2 (3.3)
No NO2 (1.7)
No NO2 (0.9)
3.72
3.08
Mol.: molecule; Mod.: mathematical model selected for the regression (Mar-1, Mar-2, Mar-3 and Mar-4 take into account the mutagenic and toxic effects); r2:
square of the correlation coefficient; P: model probability (should be <0.05); PE: error probability of the model (should be >0.05); MA: mutagenic activities (num-
ber of revertants/mg); MP: mutagenic powers (number of revertants/nmol); Ratio: ratio of the MP of the molecule and the MP of metronidazole for each strain;
Comment NO2: mutagenicity that is linked to the reduction of the NO2 substituent by comparison of the MP of TA100 and YG1042; and Comment no NO2:
mutagenicity that is not linked to the reduction of the NO2 substituent by comparison of the MP of TA100 and YG1042. In brackets the value of the ratio
MPYG1042/MPTA100 is indicated. Ratios ꢄ 2 reflected MP linked to NO2 group reduction.
5.3. General procedure for the synthesis of compounds
7ee10e
1H NMR (CDCl3, 200 MHz) d 3.97 (s, 3H, CH3), 4.87 (s, 2H,
CH2), 7.16e7.25 (m, 2H, CH ꢁ 2), 7.51 (s, 1H, CH), 7.82e
7.89 (m, 2H, CH ꢁ 2); 13C NMR (CDCl3, 50 MHz) d 36.1
(CH3), 56.1 (CH2), 116.5 (d, J ¼ 23 Hz, CH ꢁ 2), 131.3 (d,
To a stirred suspension of powdered KOH (21.90 mmol) in
DMSO (20 mL) were added a solution of corresponding 5-
nitroimidazole (4.40 mmol) and 1-fluoro-4-(chloromethylsul-
fonyl)benzene (2e) (4.40 mmol) in DMSO (15 mL) and the
reaction was carried out at room temperature for 15 min.
The mixture was poured into an iceewater mixture, acidified
with 1 N HCl until pH ¼ 1e2 and extracted with chloroform
(3 ꢁ 50 mL). The combined organic layers were washed
with water (3 ꢁ 30 mL), dried over MgSO4 and removed un-
der reduced pressure. After purification by chromatography
over silica gel, washing with acetone and recrystallization
Correlation
30
MP100 = -0.07 + (4.08/CI50
)
r2 = 0.55, P = 3*10-5, n = 21
4
7b
20
10
0
3
7d
7a
7e
from
arylsulfonylmethyl-1-alkyl-5-nitro-1H-imidazole
appropriate
solvent,
corresponding
4-
(7ee10e)
5
8a
8d
6
8b
10d
was obtained.
9c
9b
10b
8c
8e
9d
10a
10c
10e
9e
9a
5.4. 4-(4-Fluorobenzenesulfonylmethyl)-1-methyl-5-
nitro-1H-imidazole (7e)
0.0
0.5
1.0
1.5
IC50 Tricho (µM)
2.0
2.5
3.0
Fig. 1. Correlation between the mutagenic powers determined with TA100 and
the IC50 on Trichomonas vaginalis. Circle: the molecules 9b, 9c and 10b were
excluded from the non-linear regression analysis. The dashed lines indicate the
95% confidence levels.
Following general procedure, compound 7e was purified by
column chromatography eluting with ethyl acetate and recrys-
tallized in butan-1-ol giving a white solid (54%): mp 206 ꢀC;

658
M.D. Crozet et al. / European Journal of Medicinal Chemistry 44 (2009) 653e659
J ¼ 10 Hz, CH ꢁ 2), 133.5 (C), 135.1 (C), 139.9 (CH), 142.8
(C), 166.1 (d, J ¼ 257 Hz, C). Anal. C11H10FN3O4S: (C, H,
N, S).
one day following regular subculturing and were in the log
phase of growth.
In vitro drug susceptibility assays were carried out using
standard procedure [20]. Test compounds were dissolved in
DMSO (analytical grade, Sigma). The compounds were tested
in the concentration range of 0.01e5 mg/mL.
5.5. 4-(4-Fluorobenzenesulfonylmethyl)-1,2-dimethyl-5-
nitro-1H-imidazole (8e)
Working concentrations of 5 ꢁ 103 trophozoites/mL were
selected for the assay. Triplicate culture of the T. vaginalis
was incubated anaerobically at 37 ꢀC in the presence of a range
of concentration of nitroimidazole derivatives. A second cul-
ture containing DMSO (final concentration of 0.1%) served
as the negative control. After a 48 h-incubation period, antitri-
chomonas activity was assessed by trypan blue staining to de-
termine the number of viable cells. The IC values were defined
as the concentration of drug inducing a mortality of 50% of
parasites compared to the control (IC50 Trichomonas). They
were calculated by non-linear regression analysis processed
on doseeresponse curves, using the Table curve program (Jan-
del scientific).
Following general procedure, compound 8e was purified by
column chromatography eluting with ethyl acetate and recrys-
tallized in propan-1-ol giving a white solid (40%): mp 183 ꢀC;
1H NMR (CDCl3, 200 MHz) d 2.48 (s, 3H, CH3), 3.87 (s, 3H,
CH3), 4.84 (s, 2H, CH2), 7.17e7.26 (m, 2H, CH ꢁ 2), 7.85e
7.92 (m, 2H, CH ꢁ 2); 13C NMR (CDCl3, 50 MHz) d 14.1
(CH3), 34.1 (CH3), 56.1 (CH2), 116.5 (d, J ¼ 23 Hz,
CH ꢁ 2), 131.3 (d, J ¼ 10 Hz, CH ꢁ 2), 132.5 (C), 135.2
(C), 137.1 (C), 148.7 (C), 166.0 (d, J ¼ 257 Hz, C). Anal.
C12H12FN3O4S: (C, H, N, S).
5.6. 2-[4-(4-Fluorobenzenesulfonylmethyl)-2-methyl-5-
nitroimidazol-1-yl]ethanol (9e)
7. Cells toxicity assays
Following general procedure, compound 9e was purified by
column chromatography eluting with chloroform/acetone (6:4)
and recrystallized in propan-1-ol giving a beige solid (20%):
In vitro toxicity of nitroimidazoles was assessed on human
monocytes (THP1 cells) maintained at 37 ꢀC in 6% CO2, in
a medium of RPMI (Eurobio, Paris, France) supplemented
with 10% foetal calf serum (Eurobio, Paris, France), 25 mM
of HEPES, 25 mM of NaHCO3 (Gibco-BRL, Paisley, Scot-
land), and 1% of L-glutamine/penicillinestreptomycin mix.
Cells were replicated every 7 days [21].
THP1 cell (105 cells/mL) were incubated with different
concentrations of tested products dissolved in DMSO. A
viability control free of drug and a control containing
DMSO (final concentration of 0.5%) were performed in paral-
lel. After a 72 h-incubation at 37 ꢀC and 6% CO2 in complete
RPMI medium, cell growth and viability were measured by
flow cytometry after staining the monocytes by 5 mL of propi-
dium iodide (1 mg/mL) (Sigma) [22].
1
mp 189 ꢀC; H NMR (DMSO-d6, 200 MHz) d 2.41 (s, 3H,
CH3), 3.57e3.65 (m, 2H, CH2), 4.31 (t, J ¼ 5.2 Hz, 2H,
CH2), 4.91 (s, 2H, CH2), 5.04 (t, J ¼ 5.3 Hz, 1H, OH),
7.39e7.48 (m, 2H, CH ꢁ 2); 7.72e7.79 (m, 2H, CH ꢁ 2);
13C NMR (DMSO-d6, 50 MHz) d 14.1 (CH3), 48.9 (CH2),
55.7 (CH2), 59.8 (CH2), 116.6 (d, J ¼ 23 Hz, CH ꢁ 2), 131.4
(d, J ¼ 10 Hz, CH ꢁ 2), 133.0 (C), 135.1 (C), 136.6 (C),
150.1 (C), 166.4 (d, J ¼ 253 Hz, C). Anal. C13H14FN3O4S:
(C, H, N, S).
5.7. 1-[4-(4-Fluorobenzenesulfonylmethyl)-2-methyl-5-
nitroimidazol-1-yl]propan-2-ol (10e)
Antiproliferative activity was evaluated by counting the
number of living cells on a 100 mL-sample. Inhibitory concen-
tration 50 (IC50 human) was defined as the concentration of drug
inducing a reduction of 50% in the THP1 cell proliferation
compared to the control [23].
Following general procedure, compound 10e was purified
by column chromatography eluting with chloroform/acetone
(7:3), washed with petroleum ether and recrystallized in
propan-2-ol giving a yellow solid (17%): mp 128 ꢀC; 1H
NMR (CDCl3, 200 MHz) d 1.30 (d, J ¼ 5.6 Hz, 3H, CH3),
1.87 (broad s, 1H, OH), 2.52 (s, 3H, CH3), 3.96e4.46 (m,
3H, CH2 and CH), 4.83 (s, 2H, CH2), 715e7.88 (m, 4H,
CH ꢁ 4); 13C NMR (CDCl3, 50 MHz) d 14.7 (CH3), 21.3
(CH3), 53.4 (CH2), 56.3 (CH2), 67.3 (CH), 116.4 (d,
J ¼ 23 Hz, CH ꢁ 2), 131.4 (d, J ¼ 10 Hz, CH ꢁ 2), 133.1
(C), 135.1 (C), 136.6 (C), 150.2 (C), 166.0 (d, J ¼ 257 Hz,
C). Anal. C14H16FN3O5S: (C, H, N, S).
A specificity index (SI) corresponding to the ratio of
toxicities on human cells and on parasite was calculated
according to the following formula: SI
¼ IC50 human
/
T. vaginalis
IC50 T. vaginalis
8. The Salmonella mutagenicity assay
The Salmonella mutagenicity [24] test was carried out ac-
.
´
cording to De Meo et al. [25]. Salmonella typhimurium strain
6. Antitrichomonas activity
TA100 was a generous gift of B.N. Ames (Berkeley, CA,
USA). This strain has been shown to be the most sensitive
to the mutagenic effect of nitroimidazole derivatives [5,26].
S. typhimurium YG1042 strain was obtained from T. Nohmi
(National Institute of Hygienic Sciences, Tokyo, Japan). This
strain is a derivative of TA100 and includes an additional
T. vaginalis parasites (TVR87) were grown in Trichomonas
medium TM 161 medium (Oxoid) supplemented with 10%
heat-inactivated horse serum (Eurobio, Paris, France). Para-
sites to be used in drug susceptibility assays were grown for

M.D. Crozet et al. / European Journal of Medicinal Chemistry 44 (2009) 653e659
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