Potent and specific inhibitors of trypanothione reductase from Trypanosoma cruzi: Bis(2-aminodiphenylsulfides) for fluorescent labeling studies

Article abstract of DOI:10.1016/S0968-0896(00)00312-6

In order to optimise the activity of bis 2-aminodiphenylsulfides) upon trypanothione reductase TR) from Trypanosoma cruzi, a new series of bis 2-aminodiphenylsulfides) possessing three side chains was synthesized. Various moieties were introduced at the end of the third side chain, including acridinyl or biotinyl moieties for fluorescent labeling studies. TR inhibition was improved; the most potent inhibitor IC₅₀=200 nM) was selective towards TR versus human glutathione reductase and corresponded to a single myristyl group. Compounds were also tested in vitro upon Trypanosoma cruzi and Leishmania infantum amastigotes, upon-Trypanosoma brucei trypomastigotes, and for their cytotoxicity upon human MRC-5 cells. In the presence of serum, acridine derivative was no longer detectable in mass spectrometry and its antitrypanosomal activity no longer observed. This transformation might explain the absence of correlation between the potent TR inhibition and the in vitro antiparasitic activity with both of the first generation of 2-aminodiphenylsulfides. Copyright

Full text of DOI:10.1016/S0968-0896(00)00312-6

Bioorganic & Medicinal Chemistry 9 (2001) 837±846
Potent and Speci®c Inhibitors of Trypanothione
Reductase from Trypanosoma cruzi:
Bis(2-aminodiphenylsul®des) for Fluorescent Labeling Studies
Sophie Girault,a,y Elisabeth Davioud-Charvet,a,y Louis Maes,b Jean-Francois Dubremetz
,c Marie-Ange Debreu,a Valerie Landrya and Christian Sergheraerta,*
a
Â
UMR 8525 CNRS - Universite de Lille II - Institut de Biologie et Institut Pasteur de Lille, 1 rue du Professeur Calmette, B.P. 447,
59021 Lille, France
bTibotec, Instituut voor Antiviral Onderzoek, B-32800 Mechelen, Belgium
IFR 17 - Departement 4 - Institut de Biologie et Institut Pasteur de Lille, 1 rue du Professeur Calmette, B.P. 447,
59021 Lille, France
c
Â
Received 17 July 2000; accepted 24 October 2000
AbstractÐIn order to optimise the activity of bis(2-aminodiphenylsul®des) upon trypanothione reductase (TR) from Trypanosoma
cruzi, a new series of bis(2-aminodiphenylsul®des) possessingthree side chains was synthesized. Various moieties were introduced at
the end of the third side chain, includingacridinyl or biotinyl moieties for ¯uorescent labelingstudies. TR inhibition was improved;
the most potent inhibitor (IC50=200 nM) was selective towards TR versus human glutathione reductase and corresponded to a
single myristyl group. Compounds were also tested in vitro upon Trypanosoma cruzi and Leishmania infantum amastigotes, upon-
Trypanosoma brucei trypomastigotes, and for their cytotoxicity upon human MRC-5 cells. In the presence of serum, acridine deri-
vative was no longer detectable in mass spectrometry and its antitrypanosomal activity no longer observed. This transformation
might explain the absence of correlation between the potent TR inhibition and the in vitro and in vivo antiparasitic activity with
both of the ®rst generation of 2-aminodiphenylsul®des. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
within activated macrophages.5,6 These results render
TR a potential rational drug design target against try-
panosomiasis.
Drugs presently being used for trypanosomiasis treatment
are non-speci®c and consequently fairly toxic. Investigation
of glutathione metabolism in trypanosomatids has revealed
that these organisms possess an original redox system
based upon the couple trypanothione/trypanothione
reductase (TR). TR, an NADPH-dependent ¯avoprotein,
regenerates trypanothione under its active form T(SH)2
(Fig. 1).1À3
7
Usinga microplate assay to screen TR inhibitors, we
discovered, amongother potential leads, some 2-amino-
diphenylsul®des structurally related to phenothiazines
but presentingthe advantage of a residual neuroleptic
activity.8 The position and the conformation of the
best inhibitor of the series (A series, compound 1,
Ki=25 mM, Figure 2) in the catalytic site of TR were
studied by molecular dynamics simulation. Molecular
modellingclearly indicated interactions of the amino
side-chain with acidic residues absent in GR, and of its
aromatic rings with a broad hydrophobic pocket.9
These two features of recognition inspired the synthesis
of bis(2-aminodiphenylsul®des) which were found to be
more potent and speci®c TR inhibitors (B series, Figure
2).10 In this series, compound 2 was the most potent
inhibitor, with an IC50 of 0.55 mM in the presence of
57 mM T(S)2.
Despite 40% identity in their primary sequences, TR
and human glutathione reductase (GR) are exclusive
towards their respective substrate trypanothione and
glutathione.4 Recently, genetic approaches using TR
mutants of Leishmania donovani and L. major have
shown the vital role of TR in the survival of the parasites
*Correspondingauthor. Tel.: +33-3-20-87-1211; fax: +33-3-20-87-1233;
e-mail: christian.sergheraert@pasteur-lille.fr
yThe two ®rst authors contributed equally to the results.
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00312-6

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S. Girault et al. / Bioorg. Med. Chem. 9 (2001) 837±846
Figure 1. Trypanothione and trypanothione reductase.
Figure 2. A and B series of 2-aminodiphenylsul®des and compounds 1 and 2.
With the aim of further improvingTR inhibition, we
elected to design and synthesize a new generation of
bis(2-aminodiphenylsul®des) (C series, Figure 3), corre-
spondingto the attachment of an additional side chain
to an analogue of compound 2, di€erent by the presence
of a secondary amino group within the spacer joining
the two aromatic moieties. The large volume of the
hydrophobic pocket in the TR active site justi®ed the
introduction of a hydrophobic moiety to the end of the
side chain and, given the narrower catalytic site of GR,
the greater bulk of the new molecules should favour the
TR/GR speci®city. Moreover, this side chain could be
functionalized with moieties enabling¯uorescent labeling
studies to monitor the localisation of the compounds
within the parasite. Indeed, the previous generation of
bis(2-aminodiphenylsul®des) (B series) did not reveal a
tangible correlation between TR inhibition and in vitro
trypanocidal e€ect.
In accordance with the distances evaluated by molecular
modelling, a constant length of four carbons was selected
for the additional side chain. Four terminal groups
likely to generate hydrophobic interactions were
chosen: ¯uorenylmethyloxycarbonyl (Fmoc), tert-butoxy-
carbonyl (Boc), naphtyl, myristyl as well as acridinyl
and biotinyl moieties, the latter two for ¯uorescent labeling
studies within the parasite. Amine or a carboxylic groups
capable of generating polar interactions were also used
as controls.
Figure 3. C series of bis(2-aminodiphenylsul®des).

S. Girault et al. / Bioorg. Med. Chem. 9 (2001) 837±846
839
Chemistry
The excess of amine permitted the simultaneous elim-
ination of the Fmoc group.
The di€erent compounds of the C series were deriva-
tives of the bis(2-aminodiphenylsul®de) 11 which was
synthesized as described by Scheme 1. The starting
material was iminodiacetic acid 3 whose amino was
protected by a Boc group before its transformation into
anhydride 5. This latter was opened by reaction with [3-
(4-bromo-2-nitro)phenylsulfanyl]phenylamine to give
carboxylic acid 6 which was coupled with the same
aromatic amine to produce bis(2-aminodiphenylsul®de)
7.11,12 In the ®rst step, the Boc group was preferred to
the Fmoc group since the Fmoc analogue of compound
6 did not react with the aromatic amine to form the
Fmoc analogue of compound 7.13 Since the reduction of
nitro groups was carried out under acidic conditions to
which the Boc group is sensitive, it should be preceded
by a deprotection of the central, secondary amino and
its re-protection with a Fmoc group. Nitro groups of
compound 9 were then reduced by iron in the presence
of concentrated HCl. Attachment of the lateral amino
side chains was accomplished in two steps: the reaction
of compound 10 with 3-chloropropionyl chloride, using
1-ethylpiperidine as a base, followed by the substitution
of the remainingchloro groups by 1-methylpiperazine.
1-ethylpiperidine was preferred as a tertiary amine over
pyridine to avoid formation of a pyridinium salt which
cannot be converted into the methylpiperazine derivative.10
The additional side chain was attached to compound 11
by reaction with the central, secondary amino group.
Compounds 12±17 were synthesized, as described in
Scheme 2, by couplingbis(2-aminodiphenylsul®de) 11
with N-t-Boc-g-aminobutyric acid, subsequent depro-
tection of the terminal amino group, followed by either
®xation of a Fmoc group or coupling of the terminal
amino group with myristic, 2-naphtoic or 9-acridine-
14
carboxylic acid usingPyBroP reagent.
Compound 11 also led to bis(2-aminodiphenylsul®des)
18 and 19 via a couplingof the central, secondary amino
group with biotin and glutaric anhydride respectively as
shown in Scheme 3.
Results and Discussion
The inhibitingpotency of the di€erent compounds of
the C series was evaluated by measuringthe IC
50
towards TR in the presence of 57 mM of T(S)2 and
increasingconcentrations of inhibitor (0±60 mM) (Table
1).10 Compounds were also tested upon human GR,
upon Trypanosoma cruzi and Leishmania infantum
amastigotes, upon Trypanosoma brucei trypomastigotes,
Scheme 1. Synthesis of bis(2-aminodiphenylsul®de) 11. Reagents and conditions: (a) (Boc)2O, NaOH, dioxane, H2O, rt, 18 h; (b) DCC, dry THF, rt,
24 h; (c) [3-(4-bromo-2-nitro)phenylsulfanyl]phenylamine, dry THF, re¯ux, 24 h; (d) cyanuric ¯uoride, pyridine, dry CH2Cl2, rt, 5 h then [3-(4-
bromo-2-nitro)phenylsulfanyl]phenylamine, pyridine, dry CH2Cl2, rt, 12 h; (e) TFA:CH2Cl2 1:1, rt, 4 h; (f) 9-¯uorenylmethylchloroformate,
Na2CO3, dioxane, H2O, 0 ꢀC, 4 h then rt, 12 h; (g) iron, HCl 12 N, EtOH, re¯ux, 1 h; (h) 3-chloropropionyl chloride, 1-ethylpiperidine, dry THF,
0 ꢀC, 3 h then 1-methylpiperazine, THF, re¯ux, 4 h.

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S. Girault et al. / Bioorg. Med. Chem. 9 (2001) 837±846
Scheme 2. Synthesis of compounds 12±17. Reagents and conditions: (a) N-t-Boc-g-aminobutyric acid, PyBroP, DIEA, dry DMF, rt, 2 h; (b)
TFA:CH2Cl2 1:1, rt, 4 h; (c) 9-¯uorenylmethylchloroformate, Na2CO3, dioxane, H2O, 0 ꢀC, 4 h then rt, 12 h; (d) myristic acid, PyBroP, DIEA, dry
DMF, rt, 2 h; (e) 2-naphthoic acid, PyBroP, DIEA, dry DMF, rt, 2 h; (f) 9-acridinecarboxylic acid, PyBroP, DIEA, dry DMF, rt, 2 h.
and for their in vitro cytotoxicity upon human MRC-5
cells.
compared with the correspondingmethylene rgoup
(compound 2, IC50=0.55 mM).10 The central side chain
possessinga terminal hydrophobic moiety (naphtyl,
Boc, myristyl, acridinyl, biotinyl) led in general to an
improvement of the activity (IC50 values between
0.2 and 0.3 mM, Table 1) while a terminal Fmoc group
or a free amino group o€ered no real in¯uence. In the
case of the carboxylic group (19), solubility problems
prevented IC50 measurement. Parallel enzymic studies
upon human GR revealed that the di€erent inhibitors
were speci®c towards TR from T. cruzi versus human
GR.
The presence of a protonable site within the spacer of
bis(2-aminodiphenylsul®de), caused a decrease of TR
inhibition (compound 11, IC50=1.5 mM, Table 1) when
As presented in Table 2, in vitro activities upon para-
sites were found in the low micromolar range with the
exception of Leishmania. No correlation between TR
inhibition and the in vitro trypanocidal e€ect was
apparent, as had been observed with the previous series
of 2-aminodiphenylsul®des (A and B series). Amongst
the most active compounds against T. cruzi, 12±14,
compounds 12 and 14 were found to be toxic at 12.5 mM
upon mouse peritoneal macrophages used in the Leish-
mania test, while compound 13 displayed a similar
cytotoxicity upon human MRC-5 cells. Acridinic
compound 17 was devoid of cytotoxicity and exhibited
the greatest activity against T. brucei while those of
compounds 12 and 14, given the sensitivity of this
parasite to toxic compounds, suggested a non-speci®c
e€ect.
Scheme 3. Synthesis of compound 18 and 19. Reagents and condi-
tions: (a) biotin, PyBroP, DIEA, dry DMF, rt, 2 h; (b) glutaric anhy-
dride, dry pyridine, rt, 12 h.

S. Girault et al. / Bioorg. Med. Chem. 9 (2001) 837±846
841
Table 1. Inhibitory activities of compounds 2 and 11±19 towards TR
from T. cruzi
displayed the signal corresponding to compound 17 (Fig.
4A). For the same length of incubation in HW, the mass
spectrum expressed the persistent presence of compound
17 (Fig. 4B) and all epimastigotes were dead within 1 h.
After a few minutes, three kinds of parasite could be
observed: (i) live parasites showingactive motility and
no ¯uorescence; (ii) non-motile parasites with a stumpy
shape showingseveral spots of the yellow ¯uorescence
characteristic for compound 17 (Fig. 5, parasite at right
side); (iii) remnants of burst parasites with one or two
large spot(s) of blue ¯uorescence characteristic for
Hoechst 33258 likely to correspond to remnants of the
nucleus and the kinetoplast (Fig. 5, parasite at left side).
Compound
ÀR
IC50 (mM)
2
No central amino group
No central side chain
ÀNHBoc
0.55
1.5
0.25
0.75
0.6
11
12
13
14
ÀNH2
ÀNHFmoc
15
0.2
16
0.3
The absence of ¯uorescence inside livingcells could be
related to changes in ¯uorescence polarization as the
¯uorochrome compound interacts with the TR enzyme.
Intense ¯uorescence was only associated with dying
parasites in which membrane integrity had been altered
and internal structures impaired. Blue and yellow ¯uo-
rescence were not seen together in the parasites since, as
previously reported, Hoechst 33258 stainingis associated
with a€ected cells only as an indicator of loss of membrane
integrity.15 No labelingwas observed with the biotinyl
derivative 18 followingstreptavidine interaction.
17
0.25
0.3
18
19
a
ÀCOOH
*
a*: precipitate in the enzymic bu€er at 14.25 mM (2% DMSO).
Conclusion
The construction of this new series of potent TR inhi-
bitors assumes the importance of their bioavailability to
parasites in order to obtain trypanocidal activity. Rapid
transformation of these compounds in the presence of
serum rather than absorption to serum proteins might
explain the absence of straightforward correlation
between the potent TR inhibition and the in vitro and in
vivo antitrypanosomal activity with both of the ®rst
generations of 2-aminodiphenylsul®des.8À10,16 The amino-
diphenylsul®de moiety is probably responsible for this
behaviour since structurally related phenothiazines,
used as antipsychotic or antiemetic drugs, display a high
bioavailability despite their beinghighly membrane- or
protein-bound. Therefore, structural modi®cations such
as the replacement of the sulfur atom or substitution at
the ortho position on rings will be introduced to stabilize
the aminodiphenylsul®de moiety.
Table 2. In vitro activities toward amastigote forms of T. cruzi and
of L. infantum and trypomastigote forms of T. brucei and in vitro
cytotoxicity upon human MRC-5 cells of compounds 11±19
Compound ED50 (mM) ED50 (mM) ED50 (mM)a
CC50 (mM)
Cytotoxicity
upon
T. cruzi
upon
T. brucei
upon
L. infantum
11
12
13
14
15
16
17
18
19
12
7.5
7.5
5.2
>12.5
10.5
17
>12.5
>12.5
5.75
5
4.5
4
>6.25
4.75
1.8
>6.25
>6.25
>12.5
T
>12.5
T
>12.5
>12.5
>12.5
>12.5
10
21
10
10
9.5
>25
20.5
>25
>25
>25
aT: toxic at 12.5 mM upon mouse peritoneal macrophages used in the
test.
Acridine compound 17 was found to be highly ¯uor-
escent and was therefore used for studyinguptake and
cellular localization into T. cruzi epimastigotes. Max-
imum excitation occurred at 494 nm and maximum
emission at 500 nm (30 mM, 1% DMSO, in bu€er pH 7).
When epimastigotes from the Y strain of T. cruzi were
incubated in the presence of 30 mM compound 17, con-
trastinge€ects were observed which were dependent
upon the nature of the medium, whether GLSH sup-
plemented with 10% of decomplemented foetal calf
serum (SVF), or Hanks-Wallace (HW) bu€er (Fig. 4).
Experimental
Chemistry
All reactions were monitored by thin-layer chromato-
graphy performed on 0.2 mm E. Merck silica gel plates
(60F-254) using UV light as a visualizing agent and
10% ninhydrin in acetone or Reindel Hope (R.H.)17 as
developing agents. Chromatography was carried out
usingsilica egl 60 (230±400 mesh ASTM) from
Macherey-Nagel. Thick-layer chromatography (TLC)
was performed usingsilica gel from Merck, the com-
pounds were extracted from the silica gel by the following
solvent system: acetone:NH4OH, 80:20. All meltingpoints
were determined on a Buchi meltingpoint apparatus and
In GLSH-SVF, motility of the parasites was not perturbed,
no parasitic ¯uorescence was observed and following1 h
of incubation the mass spectrum of the medium no longer

842
S. Girault et al. / Bioorg. Med. Chem. 9 (2001) 837±846
Figure 4. Time of ¯ight mass spectra of compound 17 in the di€erent media (in spectrum b, M+: 1285.9 and M++Na+: 1307.2).
were uncorrected. 1H NMR spectra were obtained using
a Brucker 300 MHz spectrometer, chemical shifts were
expressed in ppm relative to TMS used as an internal
standard. Mass spectra were recorded on a time-of-
¯ight (TOF) plasma desorption spectrometer using a
Californium source.
Procedure for oxalate salts
To a saturated solution of amine in ethyl acetate
(AcOEt) was added dropwise a saturated solution of
oxalic acid in AcOEt. The mixture was maintained at
4 ꢀC for 3 h. The salt was isolated by ®ltration, succes-
sively washed with ice cold AcOEt and ether, and dried
under vacuum.
Method A: General procedure for preparation of
compounds 8 and 13
A solution of N-Boc-amino compound (5.9 mmol, 1
equiv), in 87 mL of a 1:1 TFA:CH2Cl2 mixture was
stirred for 4 h at room temperature. The solvents were
evaporated and the oily residue treated with an AcOEt/
NaOH 2.5 N mixture. The organic layer was separated,
washed with a saturated solution of NaCl, dried over
MgSO4 and the solvent evaporated to yield the desired
product.
Method B: General procedure for preparation of
compounds 9 and 14
To a cooled solution of Na2CO3 (276 mg, 2.6 mmol, 10
equiv), in 1.6 mL of water was added a solution of
appropriate compound (0.26 mmol, 1 equiv), in 6 mL of
dioxane, then 9-¯uorenylmethylchloroformate (68 mg,
0.26 mmol, 1 equiv). After stirringthe mixture for 4 h at
0 ꢀC then for 12 h at room temperature, 5.2 mL of HCl 1
N (5.2 mmol, 20 equiv) were introduced. The solvents
Figure 5. Trypanosoma cruzi epimastigotes have been treated with
compound 17 for 1 h and observed in the presence of Hoechst 33258
under epi¯uorescence (a) and phase contrast (b). One stumpy dying
parasite (right) shows yellow dots of ¯uorescence due to compound 17,
were evaporated, and the solid residue was treated with
a CH2Cl2/HCl 1 N mixture. The organic layer was
whereas a dead burst parasite (left) shows one large spot of blue ¯uo-
rescence due to Hoechst 33258.

S. Girault et al. / Bioorg. Med. Chem. 9 (2001) 837±846
843
separated, dried over MgSO4 and the solvent evapo-
rated to yield the desired product.
was evaporated to give crude acyl ¯uoride. Then to a
cooled solution of [3-(4-bromo-2-nitro)phenylsulfanyl]-
phenylamine (6.5 g, 20 mmol, 2 equiv), and pyridine
(2.4 mL, 30 mmol, 3 equiv), in 120 mL of dry CH2Cl2
was added a solution of the latter crude acyl ¯uoride
(10 mmol, 1 equiv), in 35 mL of dry CH2Cl2. After stir-
Method C: General procedure for preparation of
compounds 12, 15±18
ꢀ
To a cooled solution of the appropriate acid (0.5 mmol,
1 equiv), and DIEA (270 mL, 1.5 mmol, 3 equiv), in
10 mL of dry DMF, PyBroP (234 mg, 0.5 mmol, 1 equiv)
ringthe mixture for 10 min at 0 C then for 12 h at room
temperature, the solvent was evaporated and the oily
residue treated with a CH2Cl2/H2O mixture. The
organic layer was separated and dried over MgSO4, the
solvent evaporated and the oily residue puri®ed by
chromatography (CH2Cl2:MeOH, 95:5), to yield com-
pound 7 as a yellow solid (5 g, 58% yield): Rf 0.5
(CH2Cl2:MeOH, 9.5:0.5); mp 115 ꢀC; 1H NMR
(CD2Cl2) d 10.04 (bs, 1H, NHCO, exchanged in D2O),
9.11 (bs, 1H, NHCO, exchanged in D2O), 8.39±8.38 (m,
2H, Ar-H), 8.04±8.03 (m, 1H, Ar-H), 7.90±7.89 (m, 1 H,
Ar-H), 7.81±7.78 (m, 2H, Ar-H), 7.51±7.42 (m, 4H, Ar-H),
7.38±7.33 (m, 2H, Ar-H), 6.88±6.83 (m, 2H, Ar-H), 4.15 (s,
2H, CH2), 4.06 (s, 2H, CH2), 1.41 (s, 9H, CH3);
TOFMS m/z 847 (M+).
ꢀ
was added. After stirringthe mixture for 10 min at 0 C,
the appropriate amino derivative (0.6mmol, 1.2 equiv)
was introduced. Followingfurther stirringof the mixture
for 10min at 0 ꢀC then for 2 h at room temperature, the
solvent was evaporated and the oily residue treated with a
CH2Cl2/Na2CO3 0.5 M mixture. The organic layer was
separated and dried over MgSO4, the solvent evaporated
and the oily residue puri®ed by TLC (acetone:NH4OH,
95:5), to yield the desired product.
N-(tert-Butoxycarbonyl)iminodiacetic acid (4). To a
cooled solution of iminodiacetic acid (10 g, 75.13 mmol,
1 equiv), in 145 mL of dioxane were added H2O
(50 mL), aqueous NaOH 2 N (50 mL, 100 mmol, 1.3
equiv), and (Boc)2O (18 g, 82.6 mmol, 1.1 equiv). After
stirringthe mixture for 30 min at 0 ꢀC then for 18 h at
room temperature, the solvents were evaporated and the
oily residue was treated with an AcOEt/HCl 1 N mix-
ture. The organic layer was separated, dried over
MgSO4 and the solvent evaporated to yield compound 4
as a white solid (11.3 g, 65% yield): Rf 0.3 (CH2Cl2); mp
2-Imino-N,N0-[3-(4-bromo-2-nitro)phenylsulfanyl]phenyl-
diacetylamide (8). Compound 8 was prepared from
compound 7 by method A and was obtained as a yellow
solid (4 g, 100% yield): Rf 0.5 (CH2Cl2:MeOH, 9:1); mp
75 ꢀC; 1H NMR (CD2Cl2) d 8.71 (bs, 2H, NHCO,
exchanged in D2O), 8.20±8.19 (m, 2H, Ar-H), 7.72±7.70
(m, 2H, Ar-H), 7.65±7.61 (m, 2H, Ar-H), 7.33±7.27 (m,
4H, Ar-H), 7.17±7.13 (m, 2H, Ar-H), 6.68±6.63 (m, 2H,
Ar-H), 3.39 (s, 4H, CH2), 1.75 (bs, 1H, NH, exchanged
in D2O); TOFMS m/z 747 (M+).
125 ꢀC; H NMR (DMSO-d6) d 12.50 (bs, 2H, COOH,
1
exchanged in D2O), 3.93±3.89 (d, J=9.9 Hz, 4H, CH2),
1.37 (s, 9H, CH3); TOFMS m/z 233 (M+).
2-(9-Fluorenylmethyloxycarbonyl)imino-N,N0-[3-(4-bromo-
2-nitro)phenylsulfanyl]phenyldiacetylamide (9). Compound
9 was prepared from compound 8 by method B and was
obtained as a yellow solid (5 g, 90% yield): Rf 0.55
N-(tert-Butoxycarbonyl)-N-{N0-[3-(4-bromo-2-nitro)phe-
nylsulfanyl]phenylacetamido}aminoacetic acid (6). To a
cooled solution of compound 4 (1.48 g, 6.35 mmol, 1
equiv), in 50 mL of dry THF was added dicyclohex-
ylcarbodiimide (1.3 g, 6.35 mmol, 1 equiv). After stirring
the mixture for 24 h at room temperature, [3-(4-bromo-
2-nitro)phenylsulfanyl]phenylamine (2.07 g, 6.35 mmol,
1 equiv) was added. Followingre¯ux of the mixture for
24 h, the solvent was evaporated and the oily residue
treated with a CH2Cl2/citric acid 20% mixture. The
organic layer was separated and dried over MgSO4, the
solvent evaporated and the oily residue puri®ed by
chromatography (CH2Cl2:MeOH, 90:10), to yield com-
pound 6 as an orange solid (1.96 g, 57% yield): Rf 0.3
(CH2Cl2:MeOH, 9:1); mp 65 ꢀC; H NMR (CD2Cl2) d
1
10.17 (bs, 1H, NHCO, exchanged in D2O), 9.67 (bs, 1H,
NHCO, exchanged in D2O), 8.37±8.34 (m, 2H, Ar-H),
8.07±8.03 (m, 1H, Ar-H), 7.92±7.88 (m, 1H, Ar-H),
7.86±7.80 (m, 2H, Ar-H), 7.73±7.70 (m, 2H, Ar-H), 7.67±
7.64 (m, 1H, Ar-H), 7.56±7.53 (m, 2H, Ar-H), 7.49±7.30
(m, 7H, Ar-H), 7.18±7.12 (m, 2H, Ar-H), 6.81±6.77 (m,
2H, Ar-H), 4.47±4.42 (m, 2H, CH2), 4.23±4.07 (m, 5H, 1
CH and 2 CH2); TOFMS m/z 970 (M+).
2-(9-Fluorenylmethyloxycarbonyl)imino-N,N0-[3-(2-amino-
4-bromo)phenylsulfanyl]phenyldiacetylamide (10). To a
solution of compound 9 (4.3 g, 4.4 mmol, 1 equiv), and
iron (1.5 g, 26.4 mmol, 6 equiv), in 15 mL of EtOH was
added HCl 12 N (915 mL, 11 mmol, 2.5 equiv). Follow-
ingre¯ux for 1 h, the mixture was ®ltrated on Celite and
the solvent evaporated to yield compound 10 as a
brown oil (4 g, 100% yield): Rf 0.6 (CH2Cl2:MeOH,
9:1); 1H NMR (CD3COCD3) d 7.90±7.50 (m, 8H, Ar-H),
7.55±7.25 (m, 10 H, Ar-H), 7.20±7.10 (m, 2H, Ar-H),
7.03±6.90 (m, 2H, Ar-H), 4.45±4.17 (m, 4H, CH2), 4.07±
3.95 (m, 3H, 1 CH and 1 CH2); TOFMS m/z 910 (M+).
ꢀ
1
(CH2Cl2:MeOH, 9:1); mp 180 C; H NMR (CD2Cl2) d
12.28 (bs, 1H, COOH, exchanged in D2O), 8.38±8.37
(m, 1H, Ar-H), 7.94±7.93 (m, 1H, Ar-H), 7.89±7.88 (m,
1H, Ar-H), 7.70 (bs, 1H, NHCO, exchanged in D2O),
7.48±7.41 (m, 2H, Ar-H), 7.29±7.25 (m, 1H, Ar-H),
6.83±6.80 (m, 2H, Ar-H), 3.94 (s, 2H, CH2), 3.83 (s, 2H,
CH2), 1.38 (s, 9H, CH3); TOFMS m/z 540 (M+).
2-(tert-Butoxycarbonyl)imino-N,N0-[3-(4-bromo-2-nitro)-
phenylsulfanyl]phenyldiacetylamide (7). At À10 ꢀC, to a
solution of compound 6 (5.9 g, 10 mmol, 1 equiv) and
pyridine (800 mL, 10 mmol, 1 equiv), in 120 mL of dry
CH2Cl2 was added, dropwise, cyanuric ¯uoride (2.7 g,
20 mmol, 2 equiv). After stirringthe mixture for 10 min
at 0 ꢀC then for 5 h at room temperature, the solvent
2-Imino-N,N0-(3-{2-[3-(4-methylpiperazin-1-yl)propanoyl-
amino-4-bromo]phenylsulfanyl}phenyl)diacetylamide (11).
Under N2, 3-chloropropionyl chloride (904 mL,

844
S. Girault et al. / Bioorg. Med. Chem. 9 (2001) 837±846
9.5 mmol, 5 equiv) was added, dropwise, into a cooled
solution of compound 10 (1 g, 1.9 mmol, 1 equiv), in
35 mL of dry THF. After stirringthe mixture for 30 min
at 0 ꢀC, three additions of 1-ethylpiperidine (781 mL,
5.7 mmol, 3 equiv), were made at intervals of 30 min.
Followingfurther stirringof the mixture for 1.5 h at
0 ꢀC, a large excess of 1-methylpiperazine (4.2 mL,
37.9 mmol, 20 equiv), was added at room temperature
and the mixture heated and held at re¯ux for 4 h. The
solvent was removed and the oily residue treated with a
CH2Cl2/water mixture. The organic layer was separated
and dried over MgSO4, the solvent evaporated and the
oily residue puri®ed by TLC (CH2Cl2:MeOH, 80:20),
to yield compound 11 as a colourless solid (945 mg,
50% yield): Rf 0.05 (CH2Cl2:MeOH, 8:2); mp 60 ꢀC;
1H NMR (CD2Cl2) d 10.66 (bs, 2H, NHCO, exchanged
in D2O), 9.08 (bs, 2H, NHCO, exchanged in D2O),
8.41±8.40 (m, 2H, Ar-H), 7.58±7.55 (m, 2H, Ar-H),
7.43±7.38 (m, 2H, Ar-H), 7.29±7.20 (m, 6H, Ar-H),
6.91±6.87 (m, 2H, Ar-H), 3.39 (s, 4H, CH2), 2.59±2.47
(m, 24 H, CH2), 2.22 (s, 6H, CH3); TOFMS m/z 995
(M+).
(203 mg, 60% yield): Rf 0.55 (acetone:NH4OH, 9.5:0.5);
1H NMR (CD2Cl2) d 10.57 (bs, 1H, NHCO, exchanged
in D2O), 10.51 (bs, 1H, NHCO, exchanged in D2O),
10.45 (bs, 1H, NHCO, exchanged in D2O), 9.43 (bs, 1H,
NHCO, exchanged in D2O), 8.42±8.33 (m, 3H, Ar-H),
7.67±7.62 (m, 2H, Ar-H), 7.52 (bs, 1H, NHC(O)O,
exchanged in D2O), 7.45±7.42 (m, 2H, Ar-H), 7.31±7.23
(m, 5H, Ar-H), 7.17±7.09 (m, 7H, Ar-H), 6.73±6.69 (m,
3H, Ar-H), 4.33 (s, 2H, CH2), 4.02 (m, 3H, 1 CH and 1
CH2), 2.49±2.25 (m, 24 H, CH2), 2.11±2.05 (m, 6H,
CH2), 2.04 (s, 6H, CH3), 1.60±1.50 (m, 2H, CH2);
TOFMS m/z 1302 (M+).
2-{N-[4-(N-Tetradecanoyl)aminobutanoyl]imino}-N,N0 -
(3-{2-[3-(4-methylpiperazin-1-yl)propanoylamino-4-bromo]
phenylsulfanyl}phenyl)diacetylamide (15). Compound 15
was prepared from myristic acid and compound 13 by
method C and was obtained as a colourless oil (200 mg,
1
31% yield): Rf 0.3 (acetone:NH4OH, 9.5:0.5); H NMR
(CD2Cl2) d 10.93 (bs, 1H, NHCO, exchanged in D2O),
10.32 (bs, 1H, NHCO, exchanged in D2O), 10.21 (bs,
1H, NHCO, exchanged in D2O), 9.99 (bs, 1H, NHCO,
exchanged in D2O), 8.59±8.56 (m, 2H, Ar-H), 7.71±7.67
(m, 2H, Ar-H), 7.54±7.41 (m, 4H, Ar-H), 7.30±7.21 (m,
4H, Ar-H), 6.82±6.72 (m, 2H, Ar-H), 6.56 (bs, 1H,
NHCO, exchanged in D2O), 4.31 (s, 2H, CH2), 4.17 (s,
2H, CH2), 3.20±3.17 (m, 2H, CH2), 2.76±2.45 (m, 32 H,
CH2), 2.44 (s, 3H, CH3), 2.36±2.32 (m, 7H, 2 CH2 and 1
CH3), 1.85±1.70 (m, 4H, CH2), 1.64±1.57 (m, 2H, CH2),
1.46±1.41 (m, 2H, CH2), 1.28 (s, 3H, CH3), 1.24±1.20
(m, 4H, CH2), 1.12±1.11 (m, 4H, CH2); TOFMS m/z
1290 (M+).
2-(N-{4-[N0-(tert-Butoxycarbonyl)amino]butanoyl}imino)-
N,N-(3-{2-[3-(4-methylpiperazin-1-yl)propanoylamino-4-
bromo]phenylsulfanyl}phenyl)diacetylamide (12). Com-
pound 12 was prepared from N-t-Boc-g-aminobutyric
acid and compound 11 by method C and was obtained
as a colourless solid (472 mg, 80% yield): Rf 0.4 (acetone:
NH4OH, 9.5:0.5); mp 60 ꢀC; 1H NMR (CD2Cl2) d 10.75
(bs, 1H, NHCO, exchanged in D2O), 10.65 (bs, 1H,
NHCO, exchanged in D2O), 10.57 (bs, 1H, NHCO,
exchanged in D2O), 9.63 (bs, 1H, NHCO, exchanged in
D2O), 8.58±8.49 (m, 2H, Ar-H), 7.68 (bs, 1H,
NHC(O)O, exchanged in D2O), 7.57±7.53 (m, 2H, Ar-
H), 7.42±7.35 (m, 2H, Ar-H), 7.30±7.21 (m, 6H, Ar-H),
6.86±6.83 (m, 2H, Ar-H), 4.20 (s, 2H, CH2), 4.08 (s, 2H,
CH2), 2.54±2.30 (m, 24 H, CH2), 2.26±2.17 (m, 4H,
CH2), 2.15 (s, 6H, CH3), 1.88±1.84 (m, 2H, CH2), 1.24
(s, 9H, CH3); TOFMS m/z 1180 (M+).
2-(N-{4-[N-(Napht-2-oyl)]aminobutanoyl}imino)-N,N0 -
(3-{2-[3-(4-methylpiperazin-1-yl)propanoylamino-4-bromo]
phenylsulfanyl}phenyl)diacetylamide (16). Compound 16
was prepared from 2-naphtoic acid and compound 13
by method C and was obtained as a yellow oil (247 mg,
40% yield): Rf 0.25 (acetone:NH4OH, 9.5:0.5); 1H
NMR (CD2Cl2) d 10.64 (bs, 1H, NHCO, exchanged in
D2O), 10.26 (bs, 1H, NHCO, exchanged in D2O), 10.04
(bs, 1H, NHCO, exchanged in D2O), 9.81 (bs, 1H,
NHCO, exchanged in D2O), 8.45±8.41 (m, 2H, Ar-H),
7.79±7.71 (m, 3H, Ar-H), 7.57 (bs, 1H, NHCO,
exchanged in D2O), 7.49±7.25 (m, 10 H, Ar-H), 7.15±
7.07 (m, 4H, Ar-H), 6.70±6.60 (m, 2H, Ar-H), 4.23 (s,
2H, CH2), 4.07 (s, 2H, CH2), 2.45±2.35 (m, 24 H, CH2),
2.05±2.03 (m, 10 H, 2 CH2 and 2 CH3), 1.74±1.69 (m,
2H, CH2); TOFMS m/z 1234 (M+).
2-[N-(4-Aminobutanoyl)imino]-N,N0-(3-{2-[3-(4-methyl-
piperazin-1-yl)propanoylamino-4-bromo]phenylsulfanyl}-
phenyl)diacetylamide (13). Compound 13 was prepared
from compound 12 by method A and was obtained as a
colourless oil (780 mg, 90% yield): Rf 0.5 (acetone:
NH4OH, 9:1); 1H NMR (CD2Cl2) d 10.65 (bs, 1H,
NHCO, exchanged in D2O), 10.58 (bs, 1H, NHCO,
exchanged in D2O), 10.55 (bs, 1H, NHCO, exchanged
in D2O), 8.90 (bs, 1H, NHCO, exchanged in D2O),
8.40±8.27 (m, 2H, Ar-H), 7.48±7.37 (m, 2H, Ar-H),
7.33±7.25 (m, 2H, Ar-H), 7.16±7.09 (m, 6H, Ar-H),
6.76±6.73 (m, 2H, Ar-H), 4.12 (s, 2H, CH2), 3.96 (s, 2H,
CH2), 3.75 (bs, 2H, NH2, exchanged in D2O), 2.45±2.25
(m, 24 H, CH2), 2.11±2.06 (m, 4H, CH2), 2.03 (s, 6H,
CH3), 1.75±1.72 (m, 2H, CH2); TOFMS m/z 1080
(M+).
2-(N-{4-[N-(Acridin-9-oyl)]aminobutanoyl}imino)-N,N0-
(3-{2-[3-(4-methylpiperazin-1-yl)propanoylamino-4-bromo]
phenylsulfanyl}phenyl)diacetylamide (17). Compound 17
was prepared from 9-acridinecarboxylic acid and com-
pound 13 by method C and was obtained as a yellow oil
(257 mg, 40% yield): Rf 0.25 (acetone:NH4OH, 9.5:0.5);
1H NMR (CD2Cl2) d 10.75 (bs, 1H, NHCO, exchanged
in D2O), 10.28 (bs, 1H, NHCO, exchanged in D2O),
10.11 (bs, 1H, NHCO, exchanged in D2O), 9.76 (bs,
1H, NHCO, exchanged in D2O), 8.39±8.34 (m, 2H, Ar-
H), 8.06±8.02 (m, 1H, Ar-H), 7.89±7.86 (m, 2H, Ar-H),
7.80 (s, 1H, NHCO, exchanged in D2O), 7.67±7.61 (m,
2H, Ar-H), 7.52±7.25 (m, 8H, Ar-H), 7.14±7.06 (m, 4H,
2-(N-{4-[N0-(9-Fluorenylmethyloxycarbonyl)amino]butan-
oyl}imino)-N,N-(3-{2-[3-(4-methylpiperazin-1-yl)propan-
oylamino - 4 - bromo]phenylsulfanyl}phenyl)diacetylamide
(14). Compound 14 was prepared from compound 13
by method B and was obtained as a colourless oil

S. Girault et al. / Bioorg. Med. Chem. 9 (2001) 837±846
845
Ar-H), 6.68±6.60 (m, 3H, Ar-H), 4.19 (s, 2H, CH2), 4.00
(s, 2H, CH2), 2.52±2.20 (m, 24 H, CH2), 2.09±2.01 (m,
10 H, 2 CH2 and 2 CH3), 1.83±1.74 (m, 2H, CH2);
TOFMS m/z 1285 (M+).
was evaluated in the presence of 57 mM of T(S)2 and 2%
DMSO.
Assays for GR inhibition
2-[N-(Biotinoyl)imino]-N,N'-(3-{2-[3-(4-methylpiperazin-
1-yl)propanoylamino-4-bromo]phenylsulfanyl}phenyl)di-
acetylamide (18). Compound 18 was prepared from
biotin and compound 11 by method C and was obtained
as a colourless oil (305 mg, 50% yield): Rf 0.15 (acetone:
Inhibitory potencies of the compounds at four con-
centrations (from 0.3 mM to 10 mM) were also deter-
mined with regard to human glutathione reductase, in
presence of 44 mM of GSSG, in 40 mM Hepes, 50 mM
KCl and 1 mM EDTA, pH 7.4, and 180 mM of
NADPH.
1
NH4OH, 9.5:0.5); H NMR (CD2Cl2) d 11.04 (bs, 1H,
NHCO, exchanged in D2O), 10.52 (bs, 1H, NHCO,
exchanged in D2O), 10.46 (bs, 1H, NHCO, exchanged
in D2O), 10.34 (bs, 1H, NHCO, exchanged in D2O),
8.47±8.40 (m, 2H, Ar-H), 7.74±7.72 (m, 1H, Ar-H),
7.60±7.53 (m, 1H, Ar-H), 7.49 (bs, 1H, NHCO,
exchanged in D2O), 7.46±7.42 (m, 2H, Ar-H), 7.31±7.25
(m, 2H, Ar-H), 7.18±7.10 (m, 4H, Ar-H), 6.72±6.69 (m,
2H, Ar-H), 6.27 (bs, 1H, NHCO, exchanged in D2O),
4.26 (s, 2H, CH2), 4.21 (s, 2H, CH2), 3.50±3.45 (m, 2H,
CH2), 3.38±3.34 (m, 2H, CH2), 2.60±2.38 (m, 27 H, 3
CH and 12 CH2), 2.33±2.22 (m, 2H, CH2), 2.18 (s, 3H,
CH3), 2.17 (s, 3H, CH3), 1.84±1.79 (m, 2H, CH2), 1.50±
1.44 (m, 2H, CH2); TOFMS m/z 1221 (M+).
In vitro activity against intracellular Trypanosoma cruzi
amastigotes
Primary mouse peritoneal macrophages were seeded in
96-well microplates at 30,000 cells per well. After 24 h,
about 100,000 trypomastigotes of T. cruzi were added
per well together with twofold dilutions of the drug. The
cultures were incubated at 37 ꢀC in 5% CO2±95% air
for 4 days. Following®xation in methanol and Giemsa
staining, the drug activity was semi-quantitatively
scored as % reduction of the total parasite load (free
trypomastigotes and intracellular amastigotes) com-
pared with untreated control cultures. Scoringwas per-
formed microscopically and ED50-values were then
extrapolated.
4-{N,N-[N0,N00-(3-{2-[4-Bromo-3-(4-methylpiperazin-1-yl)
propanoylamino]phenylsulfanyl}phenyl)acetylamido]amido}
butanoic acid (19). To a cooled solution of compound
11 (127 mg, 0.13 mmol, 1 equiv), in 1 mL of dry pyridine
was added glutaric anhydride (14.6 mg, 0.13 mmol, 1
equiv). After stirringthe mixture for 12 h at room tem-
perature, the solvent was evaporated and the oily resi-
due treated with a CH2Cl2/cooled water mixture. The
organic layer was separated and dried over MgSO4, the
solvent evaporated and the oily residue puri®ed by TLC
(acetone:NH4OH, 100:1), to yield compound 19 as a
colourless oil (100 mg, 70% yield): Rf 0.25 (acetone:
In vitro activity against Trypanosoma brucei
trypomastigotes
Bloodstream forms of T. brucei were cultivated in HMI-9
medium.19 In a 96-well microplate, 10,000 haemo¯agellates
were incubated at di€erent drugconcentrations (12.5,
6.25, 3.13 and 1.56mM) for 4 days. Parasite multiplication
was measured colorimetrically (490 nm) followingaddi-
tion of MTT tetrazolium which converts to an aqueous
soluble, formazan product.20 ED50-values were then
extrapolated.
1
NH4OH, 10:0.1); H NMR (CD2Cl2) d 11.04 (bs, 1H,
NHCO, exchanged in D2O), 10.27 (bs, 1H, NHCO,
exchanged in D2O), 10.04 (bs, 1H, NHCO, exchanged
in D2O), 10.02 (bs, 1H, NHCO, exchanged in D2O),
8.60±8.56 (m, 2H, Ar-H), 7.86±7.83 (m, 1H, Ar-H),
7.71±7.68 (m, 1H, Ar-H), 7.52±7.41 (m, 4H, Ar-H),
7.29±7.19 (m, 4H, Ar-H), 6.72±6.68 (m, 2H, Ar-H), 4.34
(s, 2H, CH2), 4.17 (s, 2H, CH2), 2.71±2.42 (m, 24 H,
CH2), 2.34 (s, 6H, CH3), 2.28±2.23 (m, 4H, CH2), 1.89±
1.86 (m, 2H, CH2); TOFMS m/z 1109 (M+).
In vitro activity against intracellular Leishamnia
infantum amastigotes
Primary mouse peritoneal macrophages were seeded in
16-well LABTEK culture slides at 30,000 cells per well.
After 24 h, amastigotes of L. infantum (derived from the
spleen of an infected donor animal) were added at an
infection ratio of 10/1 together with a twofold dilution
of the drug. The cultures were incubated at 37 ꢀC in 5%
CO2±95% air for 7 days. Treatment of uninfected con-
trol cultures was also included to determine a selective
index. Drugactivity was semi-quantitatively scored as%
reduction of the total parasite load or the number of
infected macrophages in Wright stained preparations.
Biological evaluation
All compounds were evaluated under oxalate form.
Assays for TR inhibition
Recombinant T. cruzi trypanothione reductase was
produced from the SG5 Escherichia coli strain with the
Scoringwas performed microscopically and ED
values were extrapolated.
-
50
18
overproducingexpression vector pIBITczTR.
TR
activity was measured at 21 ꢀC in a 0.02 M Hepes bu€er,
pH 7.25 containing0.15 M KCl, 1 mM EDTA and
0.2 mM NADPH with an enzyme concentration of
0.02U mLÀ1. The reaction was promoted by the addition
of the enzyme and the subsequent NADPH oxidation was
followed at 340 nm. IC50 of the di€erent compounds
Cytotoxicity test upon human MRC-5 cells
A human diploid embryonic lungcell line (MRC-5, Bio-
Whittaker 72211D) and primary peritoneal mouse
macrophages were used to assess the cytotoxicity for
host cells. The peritoneal macrophages were collected

846
S. Girault et al. / Bioorg. Med. Chem. 9 (2001) 837±846
from the peritoneal cavity 48 h after stimulation with
potato starch and seeded in 96-well microplates at
30,000 cells per well. MRC-5 cells were seeded at 5000
cells per well. After 24h, the cells were washed and a
twofold dilution of the drugwas added in 200 mL standard
culture medium (RPMI+5% FCS). The ®nal DMSO
concentration in the culture remained below 0.5%. The
cultures were incubated with four concentrations of
compounds (25, 12.5, 6.25 and 3.13 mM) at 37 ꢀC in 5%
CO2±95% air for 7 days. Untreated cultures were included
(Special Program for Research and Training in Tropical
Diseases). Finally we acknowledge Gerard Montagne
for NMR experiments, Christian Slomiany for pre-
liminary ¯uorescence experiments and Dr. Steve Brooks
for proof reading.
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20
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The authors are extremely grateful to Dr. C. Walsh and
Dr. K. Nadeau (Department of Biological Chemistry
and Molecular Pharmacology, Harvard Medical School),
and Dr. M. Bradley (Department of Chemistry, University
of Southampton, UK), for providingthe SG5 Escherichia
coli strain with the overproducingexpression vector
pIBITczTR. We express our thanks equally to Dr. De
Cha€oy (Janssen Pharmaceutica) for GR studies. Studies
on TR and GR inhibitions were supported by WHO
19. Hirumi, H.; Hirumi, K. Parasitology 1989, 75, 985.
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