Antitrypanosomal activities and cytotoxicity of 5-nitro-2-furancarbohydrazides

Article abstract of DOI:10.1016/S0960-894X(02)00788-6

A series of 5-nitro-2-furancarbohydrazides were synthesized. In vitro antitrypanosomal activities of these compounds were determined against the closely related protozoa Trypanosoma cruzi Trypanosoma brucei and discussed in relation to potential targets.

Full text of DOI:10.1016/S0960-894X(02)00788-6

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3601–3604
Antitrypanosomal Activities and Cytotoxicity of
5-Nitro-2-furancarbohydrazides
Regis Millet,a,y Louis Maes,b Valerie Landry,a
Christian Sergheraerta and Elisabeth Davioud-Charveta,*,{
a
´
UMR 8525 CNRS, Universite de Lille 2, Institut de Biologie de Lille et Institut Pasteur de Lille,
1 rue du Professeur Calmette, BP 447, F-59021 Lille, France
bTibotec, L11 Gen. de Wittelaan, B-32800 Mechelen, Belgium
Received 23 May 2002; revised 27 August 2002; accepted 10 September 2002
Abstract—A series of 5-nitro-2-furancarbohydrazides were synthesized. In vitro antitrypanosomal activities of these compounds
were determined against the closely related protozoa Trypanosoma cruzi Trypanosoma brucei and discussed in relation to potential
targets.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
Trypanosomatids are parasitic hemoflagellate protozoa
which are the causative agents of tropical diseases
including human African sleeping sickness (Trypano-
soma brucei gambiense, T. brucei rhodesiense), Chagas’
disease (South American trypanosomiasis, T. cruzi) and
the visceral, cutaneous, and mucocutaneous manifes-
tations of leishmaniasis (e.g., Leishmania donovani,
Leishmania tropica, Leishmania braziliensis). Several
millions of new infections by trypanosomatid parasites
are detected annually. The current drugs1 used in the
treatment of these diseases have serious side effects,
show poor clinical efficacy or are ineffective due to an
increase in resistance. For African trypanosomiasis, the
trivalent arsenical drug melarsoprol (Fig. 1) is still used
against the second stage in which Trypanosoma brucei
rhodesiense or gambiense has invaded the central ner-
vous system. However, its use is limited by the high
toxicity of the treatment. Against the T. cruzi parasite,
nitroheterocyclic compounds2 such as benznidazole
or nifurtimox (Fig. 1) are used for the treatment of
Chagas’ disease. Benznidazole is the only drug still
available since the production of nifurtimox was
stopped. The limited available treatment accentuates the
Figure 1. Antitrypanosomal agents.
need to develop safer and more efficient new therapeutic
agents.
By random screening, we have identified another nitro-
heterocyclic derivative (compound 1, Fig. 2) as a lead
structure for a novel class of antitrypanosomal agents.
Many 5-nitrofuran derivatives2b,3 from various series
have been synthesized since their structure is associated
with antitrypanosomal, antibacterial and antifungal
activities. Nevertheless only one example of a 5-nitro-
thiophenecarbohydrazide has been described so far for
its antitrypanosomal activity.4
We have therefore undertaken the synthesis and an in
vitro antiparasitic activity study against the protozoa
*Corresponding author at present address. E-mail: elisabeth.
davioud@gmx.edu
yPresent address: Institut de Chimie Pharmaceutique Albert Lespag-
nol, 3 rue du Pr Laguesse, BP 83, F-59006 Lille, France.
{Present address: Universitat Heidelberg, Biochemie-Zentrum
Heidelberg (BZH), Im Neuenheimer Feld 504, D-69120 Heidelberg,
Germany.
Figure 2. Compound 1 and furancarbohydrazide derivatives.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00788-6

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R. Millet et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3601–3604
Biological Evaluation
In vitro activity
In vitro activity against intracellular T. cruzi amasti-
gotes. 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 2-fold dilutions of the
drug. The cultures were incubated at 37 ꢀC in 5%
CO2–95% air for 4 days. Following fixation in metha-
nol and Giemsa staining, the drug activity was semi-
quantitatively scored as % reduction of the total para-
site load (free trypomastigotes and intracellular amasti-
gotes) compared with untreated control cultures.
Scoring was performed microscopically and ED50-
values were then extrapolated.
Scheme 1. Reagents and conditions: (a) PyBOP, DIEA, tert-butyl-
carbazate, CH2Cl2, rt, 16 h, 71%; (b) MeOH, HCl, rt, 16 h, 95%; (c)
PyBOP, DIEA, CH2Cl2, appropriate acid, rt, 16 h, 25–72%; (d)
appropriate acyl chloride, NEt3, CH2Cl2, rt, 16 h, 50–70%; (e) appro-
priate sulfonyl chloride, NEt3, CH2Cl2, rt, 16 h, 40–50%; (f) 1.
Cs2CO3, MeOH, H2O, 2. CH3I, DMF, 91%; (g) hydrazine mono-
hydrate, MeOH, reflux, 24 h, 25%.
In vitro activity against T. brucei trypomastigotes.
Bloodstream forms of T. brucei were cultivated in HMI-
9 medium. In a 96-well microplate, 10,000 haemo-
flagellates were incubated at different drug concentra-
tions for 4 days. Parasite multiplication was measured
colorimetrically (490nm) following addition of MTT
tetrazolium which converts to an aqueous soluble for-
mazan product. ED50-values were then extrapolated.
Trypanosoma cruzi and Trypanosoma brucei of 5-nitro-
2-furancarbohydrazides in which we have replaced the
2-naphthylmethylcarbonyl of the lead compound by
several aromatic acyl or aromatic sulfonyl moieties (Fig.
2). In this study, the 5-nitro-2-furancarbo-hydrazide
group presented a good activity/toxicity ratio, allowing
to develop a new series of not toxic compounds.
In vitro activity against intracellular Leishmania infan-
tum amastigotes. Primary mouse peritoneal macro-
phages 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 two-fold dilution of the drug. The cul-
tures were incubated at 37 ꢀC in 5% CO2–95% air for 7
days. Treatment of uninfected control cultures was also
included to determine a selective index. Drug activity was
semi-quantitatively scored as % reduction of the total
parasite load or the number of infected macrophages in
Wright stained preparations. Scoring was performed
microscopically and ED50-values were extrapolated.
Chemistry
Compounds 1 and 3–17 were obtained as shown in
Scheme 1.
The hit 1 was obtained according to standard proce-
dures of peptidic synthesis in solution.5 Briefly, the
commercial 5-nitro-2-furoic acid 2 was reacted with
tert-butyl carbazate using PyBOP (benzotriazole-1-yl-
oxy-tris-pyrrolidino-phosphonium hexafluorophosphate)6
as coupling agent and DIEA (N,N-diisopropylethyl-
amine) as a base to give compound 4. Removal of the
Boc group by methanolic HCl 1 M solution resulted in
5-nitro-2-furancarbohydrazide hydrochloride 5.
Cytotoxicity test
A human diploid embryonic lung cell line (MRC-5, Bio-
Whittaker 72211D) and mouse primary peritoneal
macrophages were used to assess the cytotoxic effects on
host cells. The peritoneal macrophages were collected
from the peritoneal cavity 48 h after stimulation with
potato starch and they were seeded in 96-well micro-
plates at 30,000 cells per well. MRC-5 cells were seeded
at 5000 cells per well. After 24 h, the cells were washed
and two-fold dilutions of the drug were added in 200 mL
standard culture medium (RPMI+5% FCS). The final
DMSO concentration in the culture remained below
0.5%. The cultures were incubated with several con-
centrations of the compounds (between 32 and 1.6 mM)
at 37 ꢀC in 5% CO2–95% air for 7 days. Untreated cul-
tures were included as controls. For MRC-5 cells, the
cytotoxicity was determined using the colorimetric
MTT assay and scored as a % reduction of absorption
at 540nm of treated cultures versus untreated control
cultures. For macrophages, scoring was performed
microscopically.
An alternative synthetic route was also used to prepare
this latter compound. It involved the preparation of
methyl ester 3 via the alkylation of the cesium salt of
5-nitro-2-furoic acid by methyl iodide in DMF. Then,
ester 3 was refluxed with hydrazine monohydrate in
MeOH to give the desired 5-nitro-2-furancarbo-
hydrazide 5 in 25% yield. However, this method pro-
duced a lower yield than the peptidic procedure
adopted. Compound 5 was then coupled with 2-naph-
thyl acetic or another appropriate acid to produce the
diacylhydrazines 1 or 9–11, 13 respectively. For com-
pounds 6–8, the aromatic moiety was introduced by
direct acylation of hydrazide 5 by the commercially
available acyl chloride, in CH2Cl2 in the presence of
NEt3. The sulfonamide derivatives 15–17 were obtained
by reaction of hydrazide 5 with appropriate sulfonyl
chloride.

R. Millet et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3601–3604
3603
Enzymology
Table 1. In vitro activities towards trypomastigote forms of T. brucei
and amastigote forms of T. cruzi and L. infantum and in vitro cyto-
toxicity upon MRC-5 cells and MPM of compounds 1–17
When tested as potential inhibitors of trypanothione
reductase from T. cruzi (TcTR), compounds 1 and 4–6
were relatively ineffective with little TcTR inhibition
evident at concentrations up to 50 mM (IC50 >50 mM
for 4–6, and >25 mM for 1) (data not shown).7 This was
not surprising since many derivatives lack a protonable
nitrogen atom in the side-chain, the compounds 1 and
4–6 were not well-recognized by TcTR, which illustrates
the essential requirement of a positively charged amino
group for TR recognition.8
T. cruzi T. brucei L. infantum
ED50
Compda
R
ED50
(mM)
ED50
(mM)
CC50
(mM)
(mM)b
1
2
3
4
5
CO-CH2-2(Napht)
0.4
>32
>32
1
0.59
8
>32
>32
>32
>32
4
>32
>32
12
—
—
Boc
H
5
>32
2
0.5
5
9
Compounds 1 and 4–6 were also evaluated as subversive
substrates of the lipoamide dehydrogenase from T. cruzi
(TcLipDH).9 At 133 mM none of the compounds
showed any increase in the reaction rate compared to
the intrinsic oxidase activity of the enzyme. In compar-
ison, 133 mM nitrofuroxazid3a used as positive control
yielded an activity of 0.115 U/mL, suggesting another
mechanism to explain the observed potent anti-
trypanosomal action of the synthesized nitrofurans
described in this study.
6
3
3
8
4
7
8
9
10
CO-Ph
CO-Bzl
CO-2(Napht)
CO-1(Napht)
16
1
4
32
0.3
0.13
0.4
>32
17
2
2
8
8
8
3
1
11
12
4
2
0.25
2
8
6
14
8
>32
Result and Discussion
13
14
1
4
0
.5
2
Antitrypanosomal activities of the compounds towards
the intracellular amastigote stage of both T. cruzi and
L. infantum and against the T. brucei bloodstream try-
pomastigotes are given in Table 1, as ED50 values. The
standard drugs melarsoprol, benznidazole and nifurti-
mox were used as positive controls for T. brucei and
T. cruzi respectively. All compounds were tested also for
their cytotoxicity towards human MRC-5 cells (CC50
values) and mouse peritoneal macrophages (MPM).
0.64
2
28
1
0.2
8
0.77
15
16
17
Mel
Benz
Nifur
SO2-Ph
SO2-2(Napht)
SO2-1(Napht)
16
>32
0.8
—
3.13
0
>32
>32
8
—
—
4
18
0.75
12.5
>50
>50—
—
—
—
—
.4
—
aMel: melarsoprol, Benz: benznidazole, Nifur: nifurtimox.
bAll the compounds were cytotoxic only at 32 mM against MPM used in
L. infantum test.
The results show that the 5-nitro-2-furancarbo-hydrazide
structure is an excellent scaffold for the design of new
antitrypanosomal derivatives. The hydrazides shown in
Table 1 present potency profiles that are similar to those
seen with standard drugs as melarsoprol, benznidazole
and nifurtimox. This was not observed for acid 2 and
ester 3 that verified the essential role of hydrazide func-
tion. The trypanocidal effects observed in this series
suggested to us to study the inhibitory potencies or the
redox-cycling properties of several representatives
among the most active compounds (1,4–6,11) against
T. cruzi in cultures, with two flavoenzymes character-
ized in T. cruzi, the trypanothione reductase and the
lipoamide dehydrogenase, respectively.10 Both enzymes
have been validated as attractive targets for anti-
parasitic chemotherapy, by satisfactory correlations
between redox-cycling properties and antitrypanosomal
action of several nitrofurans and NQ.3a,11 The data
obtained from these experiments showed that obviously
the 5-nitro-2-furancarbohydrazides prepared in this
study do not act through interaction with TR and
LipDH activities.
and shown to display potent antitrypanosomal action
against cultured bloodstream forms of T. b. brucei.4,12
The lack of correlation between the trypanocidal activ-
ity and the inhibition of the purified T. brucei trypano-
pain by these products4 supported the suggestion that
the protease was not the major direct target of these
compounds. However, the broad antiparasitic—anti-
malarial,12 antitrypanosomal4—action of the acyl
hydrazide series suggests that these compounds could
act through a similar mechanism and merit further
investigation as potential lead structures for the search
of new treatments against trypanosomiasis.
Acknowledgements
The authors gratefully thank Professor. Luise Krauth-
Siegel for carrying out the enzymic assays with
TcLipDH and sharing her expertise, Dr. Simon Croft
and Vanessa Yardley for conducting the in vivo testing
of two compounds in mice infected with T. brucei, Ger-
ard Montagne for NMR experiments, and Dr Sophie
Girault-Mizzi for proof reading.
On the other hand, some acyl hydrazides have already
been described as reversible inhibitors of T. brucei try-
panopain the major cysteine proteinase of T. b. brucei

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R. Millet et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3601–3604
were determined in the presence of 50 mM T(S)2 and TR
(0.02 U per assay).
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