Trypanothione reductase inhibition/trypanocidal activity relationships in a 1,4-bis(3-aminopropyl)piperazine series

Article abstract of DOI:10.1016/S0968-0896(99)00268-0

A series of symmetrically substituted 1,4-bis(3-aminopropyl)piperazines was synthesized and tested towards trypanothione reductase and for its in vitro trypanocidal potency. The most trypanocidal amongst them was found to be totally inactive towards the enzyme and thus constitutes a lead structure for the identification of new potential Trypanosoma cruzi target(s). (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00268-0

Bioorganic & Medicinal Chemistry 8 (2000) 95±103
Trypanothione Reductase Inhibition/Trypanocidal Activity
Relationships in a 1,4-Bis(3-aminopropyl)piperazine Series
Beatrice Bonnet, a David Soullez, a Sophie Girault, a Louis Maes, b,y Valerie Landry, a
Elisabeth Davioud-Charvet a and Christian Sergheraert a,*
a
 Â
Institut de Biologie et Institut Pasteur de Lille, UMR CNRS 8525, Faculte de Pharmacie, Universite de Lille II,
1 rue du Professeur Calmette, BP 447, 59021 Lille, France
bJanssen Research Foundation, Turnhoutseweg 30, B-2340 Beerse, Belgium
Received 13 November 1998; accepted 30 August 1999
AbstractÐA series of symmetrically substituted 1,4-bis(3-aminopropyl)piperazines was synthesized and tested towards trypa-
nothione reductase and for its in vitro trypanocidal potency. The most trypanocidal amongst them was found to be totally inactive
towards the enzyme and thus constitutes a lead structure for the identi®cation of new potential Trypanosoma cruzi target(s). # 2000
Elsevier Science Ltd. All rights reserved.
Introduction
null mutant, the couple trypanothione/trypanothione
reductase has been demonstrated to be essential for the
survival of the parasites Leishmania donovani and Try-
panosoma brucei within the oxidative environment of
the host.6±8 For these reasons and facing the urgency to
®nd new treatments for trypanosome infections, TR has
been described as one of the most promising targets for
the design of new trypanocidal drugs.9
The antiparasitic activity of piperazine and its deriva-
tives has been known for several decades. In the treat-
ment of Chagas disease, compounds illustrated by
Figure 1, in which R1 is a heterocyclic moiety and R2 a
group consisting of a C1±C12 alkyl chain, have been
found to be active against experimental infections of
Trypanosoma cruzi in mice.1,2
Numerous molecules corresponding to protonated
amines linked to hydrophobic moities: imipramines,4
phenothiazines,10 substituted polyamines,11±13 2-amino-
diphenylsul®des14±17 have been described as TR inhibi-
tors. This inhibition is thought to occur through the
simultaneous interaction with a hydrophobic pocket
and a glutamic residue, via one molecule of water, all
within the TR active site.18 From the results of a high-
throughput assay set up to screen new TR inhibitors,19
we selected a series of compounds 1 possessing a piper-
azine group linked to a 2-aminodiphenylsul®de moiety
(Fig. 3).20 In this series, when the hydrophobic character
was enhanced by the association of two aminodiphe-
nylsul®des via an amide ¯anked spacer linked to the
phenyl moiety (compounds 2, Fig. 3), an increase of in
vitro trypanocidal activity and TR inhibition was
observed.16,21 Nevertheless, the association of 2-amino-
diphenylsul®des via the 2-amino group by an amino-
side chain (compounds 3, Fig. 3), led to a weak TR
inhibition particularly when the spacer was short.16 In
addition, the presence of a piperazine in the linker
(compound 4, Fig. 3) cancelled inhibitory potency
The target(s) of these molecules have never been identi-
®ed but the simultaneous presence of a hydrophobic
moiety and of protonated amines, suggests that they
could act as inhibitors of trypanothione reductase
(TR).3,4 TR is an NADPH-dependent ¯avoenzyme
which regenerates a glutathione-spermidine conjugate,
N1,N8-bis(glutathionyl)spermidine, named trypano-
thione (T(SH)2), from its oxidized form (Fig. 2).3,5 The
enzyme is responsible for the maintenance of the redox
balance in trypanosomes, which do not possess the
classic redox system based upon the couple glutathione/
glutathione reductase (GR). Despite 41% of homology,
TR and GR show almost total discrimination toward
their respective substrates. Recently, in studies with TR
Keywords: chemotherapy; antiparasitics; enzyme inhibitors; polycyclic
heterocyclic compounds.
*Corresponding author. Tel.: +33-3-2087-1211; fax: +33-3-2087-
1233; e-mail: christian.sergheraert@pasteur-lille.fr
yPresent address: Tibotec, Instituut voor Antiviral Onderzoek, B-
32800 Mechelen, Belgium.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00268-0

96
B. Bonnet et al. / Bioorg. Med. Chem. 8 (2000) 95±103
Figure 4. Symmetrical 1,4-bis(3-aminopropyl)piperazine derivatives.
Figure 1. Piperazine derivatives active in vivo against Trypanosoma
cruzi.
displace the equilibrium towards diimine intermediates
which were directly reduced by sodium borohydride.
towards TR while displaying potent trypanocidal
e€ects, suggesting that compound 4 could interact with
other target(s).22
When the required aldehyde 5 was not commercially
available for reductive amination, it was synthesized
from the corresponding carboxylic acid 7 (Scheme 2).
Reduction of carboxylic acid 7 by the borane/tetra-
hydrofuran complex led to the alcohol intermediate 8.
Oxidation of 8 was carried out in mild conditions using
pyridinium chlorochromate (PCC),23 to avoid the over-
oxidation of the sensitive aromatic aldehydes 5.
In order to establish structure±activity relationships
between TR inhibition and trypanocidal activity from
analogues of compound 4 and taking into account that
many structurally diverse aldehydes are commercially
available, we used a reductive amination reaction to pre-
pare a variety of symmetrical amine derivatives from the
1,4-bis(3-aminopropyl)piperazine. In this paper, we report
the synthesis and biological activity of these symmetrically
substituted 1,4-bis(3-aminopropyl)piperazines (Fig. 4).
The two commercially unavailable 2-(phenylsulfanyl)-
benzoic acid derivatives 7i and 7j were prepared in 60%
yield by re¯uxing the appropriate 2-iodobenzoic acid
(X=H or Cl) with thiophenol and aqueous potassium
hydroxide (5 equiv), in the presence of a catalytic
amount (0.1 equiv) of copper (Scheme 3).24
Chemistry
1,4-Bis(3-aminopropyl)piperazine derivatives 6a±y were
obtained according to Scheme 1 by condensation of 1,4-
bis(3-aminopropyl)piperazine with aldehydes 5a±y. The
reaction was carried out in absolute ethanol, in the
presence of molecular sieves to remove water and to
Most of the piperazine derivatives 6a±y were isolated
after a simple acid-base extraction with purities >98%
(assessed by HPLC). Compounds that needed further
Figure 2. Trypanothione and trypanothione reductase.
Figure 3. Compounds 1, 2, 3 and 4.

B. Bonnet et al. / Bioorg. Med. Chem. 8 (2000) 95±103
97
Scheme 1. Synthesis of 1,4-bis(3-aminopropyl)piperazine derivatives 6a±y.
Scheme 2. Synthesis of commercially unavailable aldehydes 5i, 5j and 5q.
Scheme 3. Synthesis of 2-(phenylsulfanyl)benzoic acid derivatives 7i and 7j.
puri®cation were puri®ed by ¯ash chromatography on
neutral aluminum oxide.
polyphenyl derivatives and benzofuran derivative 6q
alone displayed an IC50 inferior to 60 mM. In parallel
enzymic studies also revealed that the di€erent inhi-
bitors found with IC50 below 30 mM, were speci®c
towards TR versus human glutathion reductase.
Results and Discussion
All compounds were tested for their inhibitory
potency towards trypanothione reductase from T.
cruzi and their trypanocidal e€ects upon T. cruzi try-
pomastigote stage (Tables 1 and 2) as well as for their
trypanocidal e€ect upon T. brucei trypomastigote
stage and their cytotoxicity toward human MRC-5
cells (diploid embryonic lung cell line) (Table 3). The
solubility of compounds within the enzymic bu€er
did not permit the testing of concentrations superior
to 60 mM.
In the alicyclic series (Table 2), no TR inhibition was
observed below 60 mM proving that an aromatic moiety
is really needed for recognition.
A fairly good yet surprising correlation was observed
between compounds found to be active upon TR from
T. cruzi and their cytotoxicity upon mouse peritoneal
macrophages used in the test (Table 1). This cytotoxicity
was con®rmed by studies upon human MRC 5 cells and
by activity studies upon T. brucei, known for its sensi-
tivity to toxic compounds (Table 3).
In the aromatic series (Table 1), the most potent TR
inhibition was measured with polyphenyl derivatives 6e
and 6g±j. Only one monophenyl derivative 6k displayed
an IC50 inferior to 60 mM. The two most potent TR
inhibitors 6i and 6j (IC50 of 0.6 and 4 mM, respectively),
are diphenylsul®des, as the lead structure 4, inactive
upon TR, yet they di€er from compound 4 structurally
by the presence of a benzylamine moiety. The additional
methylene group in 6i and 6j is likely to be responsible
for the TR inhibition since it increases the length of the
spacer between the two aromatic moieties and/or intro-
ducing an additional site of basicity. The two inhibitors
6g and 6h (IC50 of 32 and 28 mM, respectively) are tri-
cyclic aromatic compounds, Benson et al. have pre-
viously described tricyclic compounds that inhibit TR.4
While the benzyloxybenzene derivative 6e constitutes a
new lead structure for TR inhibitors, 6f and 6k (IC50 of
60 and 30 mM, respectively) are structurally close to
the N4,N8-bis(3-phenylpropyl)spermine, N4,N8-bis(2-naph-
tylmethyl)spermine and N1,N8-bis(2-naphtylmethyl)
spermidine, already reported as TR inhibitors.11,12
Heterocyclic compounds were generally less potent than
Only two compounds 6a and 6u, were found to be non-
cytotoxic yet active upon T. cruzi trypomastigotes in the
low micromolar range. The absence of a potent inhibi-
tion by these compounds upon TR from T. cruzi sug-
gested that the trypanocidal activities were due to these
compounds acting like the lead compound 4, at an uni-
denti®ed site or sites. The general absence of a potent
TR inhibition has led us to prefer the search for new
targets rather than to perform QSAR studies. Since 6u
di€ers from 6a and 4 by the absence of aromatic moi-
eties sensitive to stacking interactions, we selected 6u as
a ligand for the preparation of anity chromatography
columns in order to isolate and identify the potential
target(s) of these 1,4-bis(3-aminopropyl)piperazine
derivatives. Three parasitic proteins were thus isolated
and the major of them, possessing an apparent mole-
cular weight of 52 kDa, was identi®ed as a thiol-dis-
ul®de oxidoreductase. Work describing the preparation
of anity chromatography columns, isolation and the
identi®cation of this protein has very recently been
reported.25

98
B. Bonnet et al. / Bioorg. Med. Chem. 8 (2000) 95±103
Table 1. Biological activities of compounds 6a±r prepared from aromatic aldehydes
Compounds
Aldehydes
IC50 on TR (mM)a
% Inhibition on Trypanosoma cruzi (mM)b
12.5
80
6.3
70
3.1
60
1.6
20
0.8
0
6a
6b
6c
6d
6e
>60
>6 0
>60
>60
28.5
T
T
0
T
90
0
9 8
70
0
9 5
20
0
2 0
0
0
T
T
T
99
80
6f
60
32
T
T
T
T
T
T
T
T
95
T
6g
6h
6i
28
T
T
T
T
T
T
T
T
T
T
0.6
6j
4
T
T
T
98
90
6k
6l
30
>60
>60
T
T
0
T
98
0
40
80
0
0
20
0
0
0
0
6m
6n
6o
>60
>60
89
0
40
0
0
0
0
0
0
0
6p
6q
>60
42
0
0
0
0
0
T
T
90
60
20
6r
>60
40
20
20
0
0
aClomipramine: IC50=12.5 mM.
bT indicates that compound is cytotoxic towards macrophages used in the test.

B. Bonnet et al. / Bioorg. Med. Chem. 8 (2000) 95±103
99
Table 2. Biological activities of compounds 6s±y prepared from nonaromatic aldehydes
Compound
Aldehyde
IC50 on TR (mM)a
% Inhibition on Trypanosoma cruzi (mM)b
12.5
40
6.3
0
3.1
0
1.6
0
0.8
0
6s
6t
>60
>60
0
0
0
0
0
6u
6v
>60
>60
>60
100
40
0
95
0
80
0
60
0
20
0
6w
0
0
0
0
6x
6y
>60
>60
T
T
40
T
0
0
0
0
T
70
aClomipramine: IC50=12.5 mM.
bT indicates that compound is cytotoxic towards macrophages used in the test.
Table 3. Cytotoxicity and activity upon Trypanosoma brucei of compounds 6a±y
Compound
% Cytotoxicity (mM)
% Inhibition on Trypanosoma brucei (mM)
25
12.5
6.3
3.1
6.3
3.1
1.6
6a
6b
6c
6d
6e
6f
6g
6h
6i
100
100
100
0
100
100
100
100
100
100
100
100
0
100
0
0
100
0
24
17
100
100
0
100
100
100
100
100
100
100
100
0
0
0
0
100
0
0
0
13
0
0
30
79
0
13
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
100
40
0
100
100
100
100
100
100
100
100
0
80
0
0
100
0
0
0
100
0
0
0
0
0
0
100
0
100
100
0
0
0
0
0
0
0
0
0
0
0
100
100
100
100
100
100
0
0
0
0
0
0
0
0
0
0
25
0
0
0
80
100
80
100
100
80
0
0
0
0
0
0
0
0
0
0
0
0
0
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
6u
6v
6w
6x
6y
0
0
0
0
0
0
0
0
95
0
0
100
100
0
100
0
0
40
100
0
0
0
0
0
Experimental
visualizing agent and 10% ninhydrin in acetone as
developing agent; purity of ®nal compounds was
checked by HPLC (Nucleosil cyanopropyl), before pre-
Chemistry
1
All melting points were determined on a melting
point Buchi apparatus and were uncorrected. All reac-
tions were monitored by thin-layer chromatography
(acetone:NH4OH 28%, 9:1), carried out on 0.2 mm
E. Merck silica gel plates (60F-254), using UV light as
paring oxalate or chlorhydrate salts. H and 13C NMR
spectra were obtained using a Brucker 300 MHz spec-
trometer; mass spectra were recorded on a time of ¯ight
plasma desorption spectrometer using a Californium
source.

100
B. Bonnet et al. / Bioorg. Med. Chem. 8 (2000) 95±103
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 kept at 4 ꢀC for 3 h; the salt was isolated by ®ltra-
tion, successively washed with ice cold AcOEt and
ether, and dried on vacuum.
2-(Phenylsulfanyl)phenylcarbaldehyde (5i). Yellow solid
(72%); mp 46±48 ꢀC.
5-Chloro-2-(phenylsulfanyl)phenylcarbaldehyde
Yellow oil (57%).
(5j).
Benzofur-3-ylcarbaldehyde (5q). Yellow oil (49%).
Procedure for chlorhydrate salts. To a saturated solution
of amine in methanol (MeOH), was added an excess of
an aqueous HCl 1 M solution (3 equiv). The mixture
was stirred at room temperature for 1 h. Then the sol-
vent was evaporated and the resulting salt was washed
with ether and dried on vacuum.
General procedure for the preparation of piperazine deri-
vatives 6a±y. To a solution of 1,4-bis(3-aminopropyl)-
piperazine (513 mL, 2.49 mmol, 1 equiv), and adequate
aldehyde 5a±y (5.24 mmol, 2.1 equiv), in 20 mL of
absolute ethanol was added 3 A molecular sieves (5 g).
After stirring the mixture at room temperature for 12 h,
sodium borohydride (1.9 g, 49.92 mmol, 20 equiv) was
added and the mixture was stirred for 12 h at room
temperature or under re¯ux (for compound 6c). Then
the reaction mixture was quenched by dropwise addi-
tion of water (20 mL) and ethanol was removed under
reduced pressure. The aqueous residue was extracted
with CHCl3 (3Â30 mL), then the combined organic
layers were extracted with HCl 1 N (2Â50 mL). The
combined aqueous layers were neutralized with NaOH 1
N (50 mL) and extracted with CHCl3 (3Â30 mL). The
combined organic layers were dried over MgSO4, eva-
porated to dryness and the oily residue puri®ed by ¯ash
chromatography (neutral aluminium oxide, CH2Cl2/
MeOH), to yield the desired compound 6a±y.
General procedure for the preparation of 2-(phenylsulfanyl)-
benzoic acid derivatives 7i and 7j. To a solution of
KOH (11.2 g, 0.2 mol, 5 equiv), in 75 mL of water were
added 2-iodobenzoic acid (9.9 g, 0.04 mol, 1 equiv), or
2-iodo-5-chlorobenzoic acid (11.3 g, 0.04 mol, 1 equiv),
copper in powder (254 mg, 4 mol, 0.1 equiv), and thio-
phenol (4.4 g, 0.04 mol, 1 equiv). After re¯uxing the
reaction mixture for 12 h, the resulting solution was
cooled to room temperature, acidi®ed with HCl 4N.
The resulting precipitate was isolated by ®ltration,
washed with water and dried on vacuum to yield the
desired compound 7.
2-(Phenylsulfanyl)benzoic acid (7i). White solid (60%);
mp 166±168 ꢀC.
1,4-Bis{3-[N-(benzyl)amino]propyl}piperazine (6a). White
1
5-Chloro-2-(phenylsulfanyl)benzoic acid (7j). White solid
(60%); mp 219±221 ꢀC.
solid (87%); mp 80±82 ꢀC; H NMR (CD2Cl2) d 7.40±
7.34 (m, 8H, aromatic H), 7.31±7.27 (m, 2H, aromatic
H), 3.81 (s, 4H, CH2), 2.73±2.68 (m, 4H, CH2), 2.45±
2.39 (m, 12H, CH2), 2.29 (s, 2H, NH), 1.75±1.66 (m,
4H, CH2); 13C NMR (CD2Cl2) d 128.66, 127.12, 57.35,
54.27, 53.79, 48.53, 27.37; m/z 380; biological assays
were performed using the oxalate salt of this compound.
General procedure for the preparation of alcohols 8i, 8j
and 8q. At 0 ꢀC and under nitrogen atmosphere, a
solution of borane/THF complex 1 M in THF
(50 mL, 50 mmol, 5 equiv), was added, dropwise, to a
solution of acid 7i, acid 7j or benzofur-3-ylcarboxilic
acid (10 mmol, 1 equiv), in 10 mL of THF. The reaction
mixture was stirred for 12 h at room temperature and
then hydrolysed with 1 M aq sodium carbonate
followed by evaporation under reduced pressure. The
residue was taken up in CH2Cl2, washed successively
with water and NaOH 1 M. The organic layer was dried
over MgSO4, ®ltered and concentrated to yield the
crude alcohol 8, which was used directly in the next
step.
1,4-Bis{3-[N-(4-chlorobenzyl)amino]propyl}piperazine (6b).
White solid (66%); mp 77±78 ꢀC; H NMR (CD2Cl2) d
1
7.32 (s, 8H, aromatic H), 3.76 (s, 4H, CH2), 2.68±2.62
(m, 4H, CH2), 2.40±2.35 (m, 12H, CH2), 1.69±1.64 (m,
6H, NH and CH2); 13C NMR (CD2Cl2) d 129.79,
129.28, 128.62, 57.30, 53.83, 48.46, 27.36; m/z 449; bio-
logical assays were performed using the hydrochloride
salt of this compound.
1,4-Bis{3-[N-(4-nitrobenzyl)amino]propyl}piperazine (6c).
ꢀ
1
2-(Phenylsulfanyl)benzyl alcohol (8i). Yellow oil (59%).
Yellow solid (24%); mp 58±60 C; H NMR (CD2Cl2)
d 8.21±8.17 (m, 4H, aromatic H), 7.58±7.54 (m, 4H,
aromatic H), 3.91 (s, 4H, CH2), 2.70±2.65 (m, 4H, CH2),
2.42±2.37 (m, 12H, CH2), 1.74±1.64 (m, 6H, NH and
CH2); 13C NMR (CD2Cl2) d 128.89, 123.75, 57.28,
53.82, 48.61, 27.36; m/z 470; biological assays were per-
formed using the oxalate salt of this compound.
5-Chloro-2-(phenylsulfanyl)benzyl alcohol (8j). Yellow
solid (95%); mp 42±44 ꢀC.
Benzofur-3-ylmethyl alcohol (8q). Yellow oil (100%).
General procedure for the preparation of aldehydes 5i, 5j
and 5q. To a solution of alcohol 8 (2 mmol, 1 equiv), in
30 mL of CH2Cl2 was added pyridium chlorochromate
(647 mg, 3 mmol, 1.5 equiv). After stirring for 4 h at
room temperature, the reaction mixture was ®ltered on
Celite and concentrated. The residue was puri®ed by
thick-layer chromatography (SiO2, petroleum ether:
ether, 90:10), to yield the desired compound 5.
1,4-Bis(3-{N-[4-(trimethylsilyloxy)benzyl]amino}propyl)-
piperazine (6d). White solid (27%); mp 92 ꢀC; H NMR
1
(CD3OD) d 7.21±7.18 (m, 4H, aromatic H), 6.81±6.76
(m, 4H, aromatic H), 3.20 (s, 9H, CH3), 2.90±2.69 (m,
4H, CH2), 2.60±2.37 (m, 4H, CH2), 2.32±2.00 (m, 8H,
CH2), 1.85±1.65 (m, 4H, CH2), 1.62Ð1.50 (m, 4H,
CH2); 13C NMR (CD3OD) d 162.73, 131.40, 115.41,

B. Bonnet et al. / Bioorg. Med. Chem. 8 (2000) 95±103
101
53.00, 48.55, 25.13, 21.68; m/z 556; biological assays
were performed using the hydrochloride salt of this
compound.
NMR (CD2Cl2) d 134.88, 130.01, 129.85, 129.67,
127.90, 127.22, 58.98, 53.86, 51.83, 48.51, 30.31, 27.30;
m/z 665; biological assays were performed using the hy-
drochloride salt of this compound.
1,4-Bis(3-{N-[4-(benzyloxy)benzyl]amino}propyl)piper-
ꢀ
1
azine (6e). White solid (40%); mp 86±88 C; H NMR
(CD2Cl2) d 7.46±7.37 (m, 10H, aromatic H), 7.30±7.26
(m, 4H, aromatic H), 6.97±6.94 (m, 4H, aromatic H),
5.08 (s, 4H, CH2), 3.73 (s, 4H, CH2), 2.71±2.66 (m, 4H,
CH2), 2.41±2.35 (m, 12H, CH2), 1.80 (s, 2H, NH), 1.71±
1.68 (m, 4H, CH2); 13C NMR (CD2Cl2) d 130.89,
129.98, 129.38, 129.01, 70.31, 57.39, 53.70, 53.11, 48.48,
26.99; m/z 592; biological assays were performed using
the oxalate salt of this compound.
1,4-Bis{3-[N-(3-phenylpropyl)amino]propyl}piperazine (6k).
1
Colourless oil (33%); H NMR (D2O) d 7.39±7.24 (m,
10H, aromatic H), 3.59±3.52 (m, 8H, CH2), 3.30±3.24
(m, 4H, CH2), 3.09±2.99 (m, 8H, CH2), 2.71±2.66 (m,
4H, CH2), 2.17±2.08 (m, 4H, CH2), 1.99±1.94 (m, 4H,
CH2); 13C NMR (D2O) d 128.89, 126.93, 53.91, 49.48,
47.66, 44.55, 32.17, 27.54, 22.43, 21.23; m/z 436; biolo-
gical assays were performed using the hydrochloride salt
of this compound.
1,4-Bis{3-[N-(napht-2-ylmethyl)amino]propyl}piperazine
(6f). White solid (97%); mp 50±52 ꢀC; 1H NMR
(CD2Cl2) d 7.87±7.80 (m, 8H, aromatic H), 7.54±7.46
(m, 6H, aromatic H), 3.97 (s, 4H, CH2), 2.76±2.71 (m,
4H, CH2), 2.42±2.34 (m, 12H, CH2), 1.79 (s, 2H, NH),
1.75±1.67 (m, 4H, CH2); 13C NMR (CD2Cl2) d 128.24,
127.99, 127.93, 127.04, 126.68, 126.34, 125.87, 57.39,
54.19, 53.68, 48.62, 27.01; m/z 480; biological assays
were performed using the hydrochloride salt of this
compound.
1,4±Bis{3-[N-(cinnamyl)amino]propyl}piperazine (6l).
1
Yellow oil (96%); H NMR (CD2Cl2) d 7.43±7.23 (m,
10H, aromatic H), 6.58±6.53 (m, 1H, CH), 6.38±6.30
(m, 1H, CH), 3.42±3.39 (m, 4H, CH2), 2.71±2.66 (m,
4H, CH2), 2.45±2.36 (m, 12H, CH2), 1.72±1.62 (m, 6H,
NH and CH2); 13C NMR (CD2Cl2) d 130.81, 129.62,
128.87, 127.56, 126.53, 57.32, 53.82, 52.17, 48.48, 27.54;
m/z 432; biological assays were performed using the hy-
drochloride salt of this compound.
1,4-Bis{3-[N-(pyrid-3-ylmethyl)amino]propyl}piperazine
1
1,4-Bis{3-[N-(anthr-9-ylmethyl)amino]propyl}piperazine
(6g). Yellow solid (25%); mp 65±67 ꢀC; 1H NMR
(CD2Cl2) d 8.44±8.38 (m, 6H, aromatic H), 8.06±8.02
(m, 4H, aromatic H), 7.60±7.49 (m, 8H, aromatic H),
4.72 (s, 4H, CH2), 2.97±2.92 (m, 4H, CH2), 2.36±2.31
(m, 14H, NH and CH2), 1.77±1.67 (m, 4H, CH2); 13C
NMR (CD2Cl2) d 131.94, 130.62, 129.37, 127.28,
126.29, 125.32, 124.80, 59.00, 53.84, 49.77, 46.30, 27.29;
m/z 580; biological assays were performed using the hy-
drochloride salt of this compound.
(6m). Yellow oil (91%); H NMR (CD2Cl2) d 8.56±8.54
(m, 2H, aromatic H), 8.49±8.46 (m, 2H, aromatic H),
7.77±7.68 (m, 2H, aromatic H), 7.29±7.24 (m, 2H, aro-
matic H), 3.79 (s, 4H, CH2), 2.69±2.64 (m, 4H, CH2),
2.40±2.35 (m, 12H, CH2), 1.71±1.62 (m, 6H, NH and
CH2); 13C NMR (CD2Cl2) d 150.06, 148.58, 135.85,
123.57, 57.26, 54.20, 53.78, 51.64, 48.52, 27.36; m/z 382;
biological assays were performed using the hydrochlo-
ride salt of this compound.
1,4-Bis{3-[N-(quinol-2-ylmethyl)amino]propyl}piperazine
1
1,4-Bis{3-[N-(¯uoren-2-ylmethyl)amino]propyl}piperazine
(6h). White solid (70%); mp 112±114 ꢀC; 1H NMR
(CD2Cl2) d 7.82±7.75 (m, 4H, aromatic H), 7.59±7.55
(m, 4H, aromatic H), 7.40±7.31 (m, 6H, aromatic H),
3.93 (s, 4H, CH2), 3.85 (s, 4H, CH2), 2.72±2.67 (m, 4H,
CH2), 2.42±2.36 (m, 12H, CH2), 1.71±1.64 (m, 6H, NH
and CH2); 13C NMR (CD2Cl2) d 127.08, 127.02, 126.80,
125.39, 125.31, 119.99, 57.35, 54.39, 53.83, 48.57, 37.19,
27.53; m/z 556; biological assays were performed using
the hydrochloride salt of this compound
(6n). Orange oil (99%); H NMR (CD2Cl2) d 8.87±8.85
(m, 2H, aromatic H), 8.16±8.09 (m, 4H, aromatic H),
7.74±7.70 (m, 2H, aromatic H), 7.63±7.57 (m, 2H, aro-
matic H), 7.51±7.49 (m, 2H, aromatic H), 4.28 (s, 4H,
CH2), 2.83±2.78 (m, 4H, CH2), 2.44±2.39 (m, 12H,
CH2), 1.87 (s, 2H, NH), 1.78±1.69 (m, 4H, CH2); 13C
NMR (CD2Cl2) d 150.74, 130.43, 129.24, 126.64,
123.87, 120.11, 57.29, 53.83, 50.52, 49.12, 27.28; m/z
482; biological assays were performed using the hydro-
chloride salt of this compound.
1,4-Bis(3-{N-[2-(phenylsulfanyl)benzyl]amino}propyl)-
piperazine (6i). Colourless oil (68%); 1H NMR
(CD2Cl2) d 7.40±7.20 (m, 16H, aromatic H), 3.90 (s, 4H,
CH2), 2.71±2.64 (m, 4H, CH2), 2.45±2.36 (m, 12H,
CH2), 1.82 (s, 2H, NH), 1.69±1.62 (m, 4H, CH2); 13C
NMR (CD2Cl2) d 136.15, 135.13, 134.80, 132.08, 131.11,
130.53, 130.27, 129.78, 127.97, 59.04, 53.86, 51.83, 48.55,
30.50, 28.50; m/z 596; biological assays were performed
using the hydrochloride salt of this compound.
1,4-Bis{3-[N-(indol-3-ylmethyl)amino]propyl}piperazine
(6o). White solid (31%); mp 130±132 ꢀC; 1H NMR
(CD3OD) d 7.74±7.70 (m, 2H, aromatic H), 7.49 (s, 2H,
aromatic H), 7.45±7.41 (m, 2H, aromatic H), 7.21±7.11
(m, 4H, aromatic H), 4.36 (m, 4H, CH2), 3.18±3.13 (m,
4H, CH2), 2.34±2.29 (m, 12H, CH2), 1.83±1.73 (m, 6H,
NH and CH2); 13C NMR (CD3OD) d 126.47, 122.37,
120.03, 118.01, 111.88, 56.23, 52.25, 47.99, 42.53, 22.29;
m/z 458; biological assays were performed using the hy-
drochloride salt of this compound.
1,4-Bis(3-{N-[5-chloro-2-(phenylsulfanyl)benzyl]amino}-
1
propyl)piperazine (6j). Colourless oil (33%); H NMR
1,4-Bis{3-[N-(fur-2-ylmethyl)amino]propyl}piperazine (6p).
1
(CD2Cl2) d 7.32±7.21 (m, 16H, aromatic H), 3.87 (s, 4H,
CH2), 2.66±2.61 (m, 4H, CH2), 2.44±2.34 (m, 12H,
CH2), 1.79 (s, 2H, NH), 1.66±1.61 (m, 4H, CH2); 13C
Yellow oil (78%); H NMR (CD2Cl2) d 7.38 (s, 2H,
aromatic H), 6.35±6.34 (m, 2H, aromatic H), 6.20±6.19
(m, 2H, aromatic H), 3.76 (s, 4H, CH2), 2.67±2.62

102
B. Bonnet et al. / Bioorg. Med. Chem. 8 (2000) 95±103
(m, 4H, CH2), 2.46±2.34 (m, 14H, NH and CH2), 1.69±
1.62 (m, 4H, CH2); 13C NMR (CD2Cl2) d 143.27,
141.87, 110.98, 106.76, 59.32, 53.84, 48.26, 46.63, 27.40;
m/z 360; biological assays were performed using the hy-
drochloride salt of this compound.
1,4-Bis{3-[N-(5-norbornen-2-ylmethyl)amino]propyl}piper-
1
azine (6w). Colourless oil (49%); H NMR (CD2Cl2) d
6.16±6.11 (m, 2H, CH of double bond), 5.96±5.93 (m,
2H, CH of double bond), 2.90±2.75 (m, 4H, CH2), 2.66±
2.57 (m, 4H, CH2), 2.50±2.24 (m, 18H, NH and CH2),
1.95±1.80 (m, 2H, CH), 1.75±1.60 (m, 6H, CH2 and
CH), 1.47±1.20 (m, 6H, CH2 and CH); 13C NMR
(CD2Cl2) d 137.42, 132.52, 57.48, 54.80, 53.48, 49.81,
49.34, 45.50, 42.80, 39.69, 31.78, 30.99, 27.53; m/z 412;
biological assays were performed using the hydrochlo-
ride salt of this compound.
1,4-Bis{3-[N-(benzofur-3-ylmethyl)amino]propyl}piperazine
1
(6q). Colourless oil (32%); H NMR (CD2Cl2) d 7.57±
7.54 (m, 2H, aromatic H), 7.47±7.44 (m, 2H, aromatic
H), 7.27±7.21 (m, 4H, aromatic H), 6.61±6.60 (m, 2H,
aromatic H), 3.92 (s, 4H, CH2), 2.74±2.69 (m, 4H, CH2),
2.43±2.36 (m, 12H, CH2), 1.78 (s, 2H, NH), 1.73±1.65
(m, 4H, CH2); 13C NMR (CD2Cl2) d 128.97, 123.98,
122.93, 120.99, 111.19, 103.68, 27.24, 53.83, 48.38,
47.01, 27.21; m/z 460; biological assays were performed
using the hydrochloride salt of this compound.
1,4-Bis(3-{N-[3-(cyclopentyl)propyl]amino}propyl)piper-
1
azine (6x). Colourless oil (60%); H NMR (CD2Cl2) d
2.74±2.53 (m, 8H, CH2), 2.50±2.34 (m, 12H, CH2), 1.85±
1.40 (m, 22H, CH2 and CH), 1.38±1.32 (m, 4H, CH2),
1.25±1.02 (m, 4H, CH2); 13C NMR (CD2Cl2) d 57.45,
56.76, 53.81, 50.59, 49.08, 41.13, 40.54, 34.27, 33.07,
29.63, 27.38, 25.54; m/z 420; biological assays were per-
formed using the hydrochloride salt of this compound.
1,4-Bis{3-[N-(thien-2-ylmethyl)amino]propyl}piperazine
1
(6r). Yellow oil (90%); H NMR (CD2Cl2) d 7.24±7.21
(m, 2H, aromatic H), 6.95±6.93 (m, 4H, aromatic H),
3.98 (s, 4H, CH2), 2.72±2.67 (m, 4H, CH2), 2.42±2.35
(m, 12H, CH2), 1.72 (s, 2H, NH), 1.70±1.62 (m, 4H,
CH2); 13C NMR (CD2Cl2) d 126.84, 124.78, 124.37,
57.26, 53.79, 48.85, 48.36, 27.38; m/z 388; biological
assays were performed using the hydrochloride salt of
this compound.
1,4-Bis(3-{N-[3-(cyclohexyl)propyl]amino}propyl)piper-
1
azine (6y). Colourless oil (62%); H NMR (CD2Cl2) d
2.74±2.64 (m, 4H, CH2), 2.61±2.55 (m, 4H, CH2), 2.52±
2.14 (m, 18H, NH and CH2), 1.76±1.59 (m, 14H, CH2
and CH), 1.57±1.48 (m, 4H, CH2), 1.29±1.17 (m, 8H,
CH2), 0.96±0.85 (m, 4H, CH2); 13C NMR (CD2Cl2) d
59.08, 58.29, 55.37, 52.04, 50.71, 39.56, 37.06, 35.34,
29.01, 28.66, 28.36; m/z 448; biological assays were per-
formed using the hydrochloride salt of this compound.
1,4-Bis{3-[N-(cyclopropylmethyl)amino]propyl}piperazine
1
(6s). Yellow oil (44%); H NMR (CD2Cl2) d 2.72±2.68
(m, 4H, CH2), 2.49±2.39 (m, 18H, NH and CH2), 1.74±
1.57 (m, 4H, CH2), 1.03±0.94 (m, 2H, CH), 0.54±0.47
(m, 4H, CH2), 0.18±0.13 (m, 4H, CH2); 13C NMR
(CD2Cl2) d 59.56, 54.95, 54.56, 49.03, 26.61, 11.09, 3.66;
m/z 308; biological assays were performed using the hy-
drochloride salt of this compound.
Biological evaluation
Assays for TR inhibition. Recombinant T. cruzi trypa-
nothione reductase was produced from the SG5
Escherichia coli strain with the overproducing expres-
sion vector pIBITczTR.26 TR activity was measured at
21 ꢀC in a 0.02 M Hepes bu€er, pH 7.25 containing 0.15
M KCl, 1 mM EDTA and 0.2 mM NADPH with an
enzyme concentration of 0.02 U mL 1. The reaction
began with the addition of the enzyme and the sub-
sequent decrease in absorbance was followed at 340 nm.
IC50 of the di€erent compounds was evaluated in the
presence of 57 mM of T(S)2, and 2% DMSO.
1,4-Bis{3-[N-(cyclobutylmethyl)amino]propyl}piperazine
1
(6t). Colourless oil (50%); H NMR (CD2Cl2) d 2.69±
2.64 (m, 8H, CH2), 2.54±2.38 (m, 16H, NH, CH2 and
CH), 2.10±2.07 (m, 4H, CH2), 1.95±1.88 (m, 4H, CH2),
1.72±1.67 (m, 8H, CH2); 13C NMR (CD2Cl2) d 57.61,
56.12, 53.70, 49.33, 41.11, 35.68, 26.81, 26.53, 18.89; m/z
336; biological assays were performed using the hydro-
chloride salt of this compound
1,4-Bis{3-[N-(cyclohexylmethyl)amino]propyl}piperazine
1
In vitro activity upon trypomastigotes (T. cruzi). Primary
mouse peritoneal macrophages were seeded in 96-well
microplates at 30,000 cells/well. After 24 h, about
100,000 trypomastigotes of T. cruzi were added per
well together with 2-fold dilutions of the drug. The cul-
tures were incubated at 37 ꢀC in 5% CO2 for 4 days.
After ®xation in methanol and Giemsa staining, the
drug activity was semi-quantitatively scored as %
reduction of the total parasite load (free trypomasti-
gotes and intracellular amastigotes) and compared with
untreated control cultures. Scoring was performed
microscopically.
(6u). Yellow oil (68%); H NMR (CD3OD) d 2.80±2.70
(m, 4H, CH2), 2.68±2.39 (m, 16H, CH2), 1.86±1.71 (m,
12H, CH2), 1.63±1.53 (m, 2H, CH), 1.40±1.20 (m, 8H,
CH2), 1.04±0.89 (m, 4H, CH2); 13C NMR (CD3OD) d
56.99, 56.26, 48.00, 37.51, 31.46, 26.63, 26.07, 25.50; m/z
392; biological assays were performed using the hydro-
chloride salt of this compound.
1,4-Bis{3-[N-(cyclohexen-4-ylmethyl)amino]propyl}piper-
1
azine (6v). Colourless oil (84%); H NMR (CD2Cl2) d
5.73±5.65 (m, 4H, CH of double bond), 2.66±2.61 (m,
4H, CH2), 2.52±2.35 (m, 12H, CH2), 2.20±2.00 (m, 6H,
CH2 and CH), 1.90±1.60 (m, 14H, NH and CH2), 1.30±
1.22 (m, 4H, CH2); 13C NMR (CD2Cl2) d 127.41,
126.64, 57.50, 56.36, 53.83, 49.34, 34.32, 30.47, 27.41,
25.32; m/z 388; biological assays were performed using
the hydrochloride salt of this compound.
Cytotoxicity test upon MRC-5 cells. A human diploid
embryonic lung cell line (MRC-5, Bio-Whittaker
72211D) was used to assess the cytotoxicity for host
cells. MRC-5 cells were seeded in 96-well microplates at
5,000 cells per well. After 24 h, the cells were washed

B. Bonnet et al. / Bioorg. Med. Chem. 8 (2000) 95±103
103
and 2-fold dilutions of the drug were added to 200 mL
standard culture medium (RPMI +5% FCS). The ®nal
DMSO concentration in the culture remained below
0.5%. The cultures were incubated at 37 ꢀC in 5% CO2
for 7 days. Untreated cultures were employed as con-
trols. The cytotoxicity was determined using the colori-
metric MTT assay27 and scored as % reduction of
absorption at 540 nm of treated cultures versus
untreated control cultures.
Fairlamb, A. H.; Tamar, S.; Olivier, M.; Papadopoulou, B.
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7. Tovar, J.; Cunningham, M. L.; Smith, A. C.; Croft, S. L.;
Fairlamb, A. H. Proc. Natl. Acad. Sci. USA 1998, 95, 5311.
8. Krieger, S.; Schwarz, W.; Krauth-Siegel, R. L.; Clayton, C.
1st COST B9, Sierra Nevada, Granada 31 May±3 June, 1998.
9. Schirmer, R. H.; Muller, J. G.; Krauth-Siegel, R. L. Angew.
Chem., Int. Ed. Engl. 1995, 34, 141.
10. Chan, C.; Yin, H.; Garforth, J.; McKie, J. H.; Jaouari, R.;
Speers, P.; Douglas, K. T.; Rock, P. J.; Yardley, V.; Croft, S.
L.; Fairlamb, A. H. J. Med. Chem. 1998, 41, 148.
11. O'Sullivan, M. C.; Zhou, Q. Bioorg. Med. Chem. Lett.
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In vitro activity upon trypomastigotes (T. brucei). Blood
stream forms of T. brucei were cultivated in HMI-9
medium as described by Hirumi et al.28 In a 96-well
microplate, 10,000 haemo¯agellates were incubated at
di€erent drug concentrations for 4 days. Parasite multi-
plication was measured colorimetrically (490 nm) fol-
lowing addition of MTS tetrazolium which converts to
an aqueous soluble formazam product.29
Acknowledgements
16. Girault, S.; Baillet, S.; Horvath, D.; Lucas, V.; Davioud-
Charvet, E.; Tartar, A.; Sergheraert, C. Eur. J. Med. Chem.
1997, 32, 39.
17. Girault, S.; Davioud-Charvet, E.; Salmon, L.; Berecibar,
A.; Debreu, M. A.; Sergheraert, C. Biorg. Med. Chem. Lett.
1998, 8, 1175.
We 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, Uni-
versity of Southampton, UK), for providing the SG5
Escherichia coli strain with the overproducing expres-
sion vector pIBITczTR. Studies upon trypanocidal
activities were supported by WHO (L.M., Special
Programme for Research and Training in Tropical
Diseases). We express our thanks equally to Gerard
Montagne for NMR experiments and to Dr. Steve
Brooks for the proof reading.
18. Jacoby, E. M.; Schlichting, I.; Lantwin, C. B.; Kabsch, W.;
Krauth-Siegel, R. L. Proteins 1996, 24, 73.
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21. Girault, S. Ph.D. thesis, Universite des Sciences et Tech-
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22. Sergheraert, C.; Maes, L., unpublished results.
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