5-Nitrofuranes and 5-nitrothiophenes with anti-trypanosoma cruzi activity and ability to accumulate squalene

Article abstract of DOI:10.1016/j.bmc.2009.09.013

Chagas disease represents a serious public health problem in South America. The first line of treatment is Nifurtimox and Benznidazole which generate toxic effects in treated patients. We have recently shown that a number of 5-nitrofuranes possess activity against Trypanosoma cruzi through oxidative stress and inhibition of parasite ergosterol biosynthesis, specifically at the level of squalene epoxidase. Here, we identify new 5-nitrofuranes and the thia-analogues with excellent effects on the viability of T. cruzi and adequate parasite/mammal selectivity indexes. Analysis of the free sterols from parasite incubated, during 120 h, with the compounds showed that some of them accumulated squalene suggesting the squalene epoxidase activity inhibition of the parasite. Nifurtimox was able to accumulate squalene only at lower incubation times. Due to this fact some derivatives were also tested as antifungal agents. Quantitative structure-activity relationship studies were also performed showing relevant features for further new derivatives design. Taken together, the results obtained in the present work point to a more general effect of 5-nitrofuranes and 5-nitrothiophenes in trypanosomatids, opening potential therapeutic possibilities of them for these infectious diseases.

Full text of DOI:10.1016/j.bmc.2009.09.013

Bioorganic & Medicinal Chemistry 17 (2009) 7500–7509
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
5-Nitrofuranes and 5-nitrothiophenes with anti-Trypanosoma cruzi activity
and ability to accumulate squalene
Alejandra Gerpe a, Guzmán Álvarez a, Diego Benítez a, Lucía Boiani a, Martín Quiroga b, Paola Hernández a,
a,
Maximiliano Sortino c, Susana Zacchino c, Mercedes González a, , Hugo Cerecetto
*
*
a Departamento de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay
b Liceo N° 4, Avda. Solari 2050, Salto, Uruguay
c Cátedra de Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 July 2009
Revised 8 September 2009
Accepted 10 September 2009
Available online 15 September 2009
Chagas disease represents a serious public health problem in South America. The first line of treatment is
Nifurtimox and Benznidazole which generate toxic effects in treated patients. We have recently shown
that a number of 5-nitrofuranes possess activity against Trypanosoma cruzi through oxidative stress
and inhibition of parasite ergosterol biosynthesis, specifically at the level of squalene epoxidase. Here,
we identify new 5-nitrofuranes and the thia-analogues with excellent effects on the viability of T. cruzi
and adequate parasite/mammal selectivity indexes. Analysis of the free sterols from parasite incubated,
during 120 h, with the compounds showed that some of them accumulated squalene suggesting the
squalene epoxidase activity inhibition of the parasite. Nifurtimox was able to accumulate squalene only
at lower incubation times. Due to this fact some derivatives were also tested as antifungal agents. Quan-
titative structure–activity relationship studies were also performed showing relevant features for further
new derivatives design. Taken together, the results obtained in the present work point to a more general
effect of 5-nitrofuranes and 5-nitrothiophenes in trypanosomatids, opening potential therapeutic possi-
bilities of them for these infectious diseases.
Keywords:
5-Nitrofuran
5-Nitrothiophene
Sterol biosynthesis inhibitors
Squalene epoxidase
Anti-Trypanosoma cruzi compounds
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Both drugs act via the reduction of the nitro group. In the case of
Nfx, reduction generates an unstable nitro anion radical which pro-
Chagas disease, or American Trypanosomiasis, remains the major
parasitic disease burden in Latin America, despite recent advances in
the control of its vectorial and transfusional transmission.1–4 Even
though the complete genome of Trypanosoma cruzi (T. cruzi) CL
Brener clone5 has been sequenced, the chemotherapy to control this
parasitic infection remains unsatisfactory.3,6 However, significant
progress in understanding the biochemistry and physiology of
T. cruzi has resulted in the validation of several metabolic steps
essential for parasite survival and for potential use as chemothera-
peutic targets.7–9 Current specific treatments are based on old and
quite unspecific drugs, Nifurtimox (Nfx, Fig. 1) and Benznidazole
(Bnz) associated with long-term treatments that may give rise to
severe side effects. In fact, although Nfx and Bnz are able to eliminate
patent parasitemia and to reduce serological titers in acute and early
chronic infections, they are not active against all T. cruzi strains and
have significantly low efficacy in long-term chronic infections.5,6
duces highly toxic reduced oxygen species.9 The side effects of this
drug results from the oxidative damage in the host’s tissues and is
thus inextricably linked to its anti-parasitic activity. Despite these
limitations, we have recently shown that novel 5-nitrofuryl deriv-
atives posses high and selective anti-T. cruzi activity in vitro and
in vivo.10–18 Moreover, we have demonstrated that one of the
mechanism of action of this family of compounds is the induction
of oxidative stress in the parasite.19–22
Regarding to the validated metabolic steps used to generate
new chemotherapeutic alternatives, membrane sterol biosynthesis
is one of the most promising targets. T. cruzi like most pathogenic
fungi and yeasts, requires specific 24-alkyl sterols for cell viability
and proliferation in all stages of its life cycle and cannot use the
abundant supply of cholesterol present in its mammalian hosts.23
The T. cruzi ergosterol biosynthesis pathway has been chemically
validated as a chemotherapeutic target at several steps,24 including
squalene epoxidase (SE; EC 1.14.99.7) a microsomal mono-oxygen-
ase that catalyzes the conversion of squalene to 2,3-oxidosqualene
using molecular oxygen.23 This conversion is the first oxygen-
dependent step in the middle stage of the sterol biosynthetic path-
way. SE employees NADPH, and FAD to catalyze the conversion of
* Corresponding authors. Tel.: +598 2 5258618; fax: +598 2 5250749 (H.C.).
E-mail addresses: megonzal@fq.edu.uy (M. González), hcerecet@fq.edu.uy (H.
Cerecetto).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.013

A. Gerpe et al. / Bioorg. Med. Chem. 17 (2009) 7500–7509
7501
N
N
N
N
O2N
N
O
SO2
Nfx
Tbf
Ntf
Bppa
Br
First generation of nitrofuranes
squalene accumulators
1
2
3
4
, n= 1, -X-= -O-, -R= -CH2CH=CHPh
, n= 1, -X-= -O-, -R= -Ph
O
O
excellent accumulators
good accumulators
N
R
N
O2N
N
H
X
O2N
N
O
O
O
, n= 0, -X-= -O-, -R= -Ph
H
n
n
, n= 0, -X-= NH-, -R= -CH(CH3)CH=CH2
5
, n= 1
, n= 0
poor accumulators
6
Second generation design
O
N
1
O2N
N
H
O
O
7-14
5-nitro and unsubstitution
phenyl and unsubstitution
propenylamide and
thiosemicarbazone/semicarbazone
furan and thiophene
Figure 1. Well-known anti-T. cruzi agents, first generation of squalene accumulator-nitrofuranes and design of the second generation.
squalene to 2,3-oxidosqualene, receiving electrons from NADPH
via NADPH-cytochrome P450 reductase (Fig. S1, Supplementary
data). SE is essential for the synthesis of cholesterol in mammals
and ergosterol in fungi and is potently inhibited by allylamines,
which have been successfully used as antifungal agents.25,26 Allyl-
amine derivatives have also shown to be potent in vitro and in vivo
T. cruzi growth inhibitors, acting by a selective reduction of the par-
asite’s endogenous membrane sterol levels.27,28 Specifically, the
antifungals terbinafine and naftifine (Tbf and Ntf, respectively,
Fig. 1)29–32 proved to be promising anti-T. cruzi agents. Also, the
synthetic compound N,N-dimethyl-3-[4-(4-bromophenyl)phenyl]-
3-phenyl-2-propenamine (Bppa, Fig. 1) showed excellent in vitro
anti-T. cruzi profile33 and in vivo for some assayed Bppa deriva-
tives.33–35 In this sense, recently our group have described a series
of 5-nitrofuranes able to accumulate squalene, and potentially in-
hibit SE, in T. cruzi parasite.18 These nitrofuranes (1–4, Fig. 1) have
an additional mechanism of action, they are able to produce oxida-
tive stress and consequently, they are dual agents.18 From a chem-
ical point of view besides the 5-nitrofuran moiety, these
compounds were designed as heteroallyl-derivatives in order to
simulate the framework allylamine present in Tbf. From a biologi-
cal point of view, and according to our results,18 this framework is
not responsible for the squalene accumulation because some of the
previously evaluated derivatives, parent compounds 2 and 3
(Fig. 1), were as good as squalene-accumulators as the heteroal-
lyl-derivatives, that is, 1 and 4 (Fig. 1), or others heteroallyl-con-
and unsubstituted ones (Fig. 1). Also, the heteroallyl-system was
maintained but in two different stereo-electronic forms, as allyla-
mide and (3-phenyl)allylamine moieties (like Ntf, Fig. 1), preserv-
ing the heterocycle ring using as the connection via
thiosemicarbazone, semicarbazone, or amide linkers (Fig. 1). Addi-
tionally, the biological effects of all the new compounds was ana-
lyzed, 7–14 and the secondary product 15 (Scheme 1), using T.
cruzi Tulahuen 2 strain as parasitic model, and for two compounds
the CL Brener clone. The selectivity towards the parasite was stud-
ied by determining the toxicity of the most active anti-T. cruzi com-
pounds against mammal-J-774 cells. The sterols level-changes
produced by the nitrocompounds into the parasite were also stud-
ied using TLC and HPLC methodologies. Furthermore, being some
antifungals active against T. cruzi,27,28 we proposed to evaluate
the designed anti-T. cruzi agents for its in vitro antifungal proper-
ties. Quantitative structure–activity relationship studies allowed
identifying some of the structural requirements for squalene
accumulation.
2. Methods and results
2.1. Chemistry
A simple and efficient procedure to generate thiosemicarbazone
derivatives, 7–12, was employed (Scheme 1a). Commercially avail-
able N4-allylthiosemicarbazide was reacted with different alde-
hydes in toluene as solvent, generating the desired compounds,
7–12, in high yields.12 Previously developed methodology36 used
in our group (carbamates synthesis followed by hydrazinoly-
sis),10,11,18 was attempted to prepare semicarbazone derivatives
containing (3-phenyl)allylamino framework (structures (II),
Scheme 1b). However, in the hydrazinolysis of the intermediate
taining nitrofuranes (i.e.,
5 and 6, Fig. 1) were enable to
accumulate this biomolecule.18 In order to complete the struc-
ture–activity information new SE inhibitors were designed, deriv-
atives 7–14 (Fig. 1). The synthetic efforts were focus on three
different structural modifications. Modifications on the 5-nitrofu-
ran system were analysed studying 5-nitrothiophene-derivatives

7502
A. Gerpe et al. / Bioorg. Med. Chem. 17 (2009) 7500–7509
a
S
S
N
H2NHN
N
H
Y
CHO
n
Y
N
H
N
H
Z
Z
p-TsOH / toluene / r.t.
n
7
,
,
,
-Y= -NO2, -Z-= -O-, n= 0
-Y= -NO2, -Z-= -O-, n= 1
-Y= -H, -Z-= -O-, n= 0
8
9
10, -Y= -H, -Z-= -O-, n= 1
11, -Y= -NO2, -Z-= -S-, n= 0
12, -Y= -H, -Z-= -S-, n= 0
b
Ph
Ph
NH2NH2.H2O
Ph
O
ClCO2Ph / Et3N
N
N
N
N
Y
N
H
N
Z
n
N
Ph
HN
N
O
N
O
toluene / 0 °C
O
H2NHN
(II)
(I)
Ph
Ph
N
t-BuOK
toluene
reflux
NNH2
O2N
O
(I) +
N
O
O
Ph
Ph
N
O2N
N
O
O
HN
N
c
N
Ph
1) SOCl2 / n-hexane / reflux
13, -Y= -NO2
14, -Y= -H
OH
N
O
Y
Y
O
O
N
Ph
O
2) Et3N /
HN
oC to r.t.
0
N
Ph
-Y= -NO2
H
N
H
N
HN
O2N
Cl
O2N
O2N
N
O
O
O
K2CO3 / acetone / reflux
Ref. [11]
O
O
N
Ph
H
N
(III)
(IV)
OH
15
O
Scheme 1. Synthetic procedures used to prepare the new studied derivatives.
(I), reduction of the allylic-double bond took place as the main pro-
cess without generation of the desired semicarbazide (see Supple-
mentary data), being in our hands impossible to separate the
desired alkene reactant from the mixture of alkene and alkane. An-
other effort to generate the desired semicarbazone derivatives was
performed using the carbamate intermediate (I) and the hydrazone
of 5-nitrofurfural as reactant (see Scheme 1b), but this procedure
was unsuccessful in our hands. Finally, the amide-linked deriva-
tives, 13 and 14 (Scheme 1c), were obtained following previously
described methodologies with adequate yield.10,11 The generation
of the 5-nitrofuryl derivative IV was endeavoured using chloroeth-
ylamide III11 and commercially available substituted-piperazine
(Scheme 1c). In this process we were enabled to obtain the desired
product IV, generating in this condition the alcohol 15 as the main
product. All the proposed structures were established by 1H, 13C
NMR (HMQC, HMBC) spectroscopy and MS. The purity was ana-
lyzed and established by TLC and microanalysis, respectively.
pounds were incorporated into the media at 25 lM and their abil-
ity to inhibit the parasite growth was evaluated in comparison to
the control (no drug added to the media) at day 5. The ID50 doses
(50% inhibitory dose) were determined for all of them (Table 1).
Nfx, Bnz, Tbf and ketoconazole (Ktz) were used as the reference
trypanosomicidal agents. The thiosemicarbazones 7 and 10 were
also studied against the high virulent39,40 CL Brener clone (Table 1).
5-Nitrofuranes 8 and 13 were the most active anti-T. cruzi deriv-
atives, with ID50 = 1.9 and 1.0 lM, near to 4 and 7 times more
active than the reference drugs Nfx and Bnz. While the 5-nitrothi-
ophene 11, the 5-unsubstituted analogue 12 and the secondary
product 15 displayed ID50 values between 4.2 and 6.4 lM, which
also resulted more active than the references (Nfx, Bnz, Tbf and
Ktz) (Table 1). The other 5-unsubstituted derivatives, 9, 10 and
14, were less or equally active than the reference drugs. No suscep-
tibility differences between both strains, with the studied com-
pounds, were observed.
2.2. Biological characterization
2.2.2. In vitro unspecific cytotoxicity
Mammal cytotoxicity of some of the new compounds, 7, 8, 10–
12, was studied in vitro using J-774 mouse macrophages as the cel-
2.2.1. In vitro anti-T. cruzi activity
The new derivatives, 7–15, were initially tested in vitro against
the epimastigote form of T. cruzi, Tulahuen 2 strain. The existence
of the epimastigote form of T. cruzi as an obligate mammalian
intracellular stage has been revisited and confirmed.37,38 The com-
lular model with doses (100.0–400.0
than the doses used for T. cruzi (25
tives were selected regarding its in vitro anti-T. cruzi activities
l
l
M) at least four times higher
M) (Table 2).41 These deriva-
and its widespread structural motives at the heterocycle level

A. Gerpe et al. / Bioorg. Med. Chem. 17 (2009) 7500–7509
7503
Table 1
microorganisms from the American Type Culture Collection (ATCC)
or clinical isolates provided by the Centro de Referencia Mic-
obiológica (CEREMIC) from Facultad de Ciencias Bioquímicas y
Farmacéuticas (Argentina) or Control Lab from Río de Janeiro (Bra-
zil) were used (Table 3). Minimal inhibitory concentration (MIC) of
each compound was determined by using broth microdilution
techniques for yeasts44 and filamentous fungi.45 For this study
we selected derivatives 7–12 with structural modifications at the
heterocycle level and with widespread squalene-accumulation
profiles (see below, Table 4). As antifungal reference compounds
Tbf, Ktz and amphotericin B (AnfB) were used. Although the stud-
ied compounds, 5-nitrofuranes, 5-nitrothiophenes and the 5-
unsubstituted analogues, were less active than the reference com-
pounds derivative 10 showed moderate activities against Crypto-
coccus neoformans, Microsporum gypseum, Trichophyton rubrum
and Trichophyton mentagrophytes.
Biological characterization of new derivatives against T. cruzi tulahuen 2 strain
a
Compd
ID50
(lM)
7
8
9
8.2 (6.4)b
1.9
>25.0
16.0 (9.6)
4.2
10
11
12
13
14
15
Nfx
Bnz
Tbf
Ktz
6.4
1.0
>25.0
5.0
7.7 (8.5)c
7.4 (4.5)c
17.0 (42.0)c
10.0 (5.0)c
a
The results are the means of three independent experiments with a SD less than
10% in all cases.
b
Values in parenthesis correspond to CL Brener clone activity.
From Ref. 41.
c
2.3. Squalene and ergosterol accumulation analysis
Since the steroids from the lipid fractions of T. cruzi epimastig-
otes has been previously described,33 this form was selected
(Tulahuen 2 strain) to study the effects on new derivatives acting
(Scheme 1). The selectivity indexes, SI, were expressed as the ratio
between ID50 in macrophages and ID50 in T. cruzi. Derivatives 7, 10
and 12 have better SI values than the biosynthesis-membrane-ste-
rol inhibitors Tbf and Ktz, having thiophene-derivative 12 1.5-fold
the selectivity of Nfx (Table 2). No structural exigencies could be
observed regarding the toxic effects.
on ergosterol biosynthesis. After
a pre-established protocol
(1 ꢀ ID50 per 8 ꢀ 106 cells/mL, 120 h of incubation),46 the controls
(untreated, Tbf- and Nfx-treated) and derivative-treated parasites
were collected and the total lipids were extracted and analysed
as described previously.33,47,24b Qualitative analyses of neutral lipid
fractions were carried out using TLC (see Supplementary data).
Then, quantitative analyses of ergosterol and squalene from sterol
fractions were done by HPLC. Derivatives 7, 8 and 11 were able to
accumulate squalene in different degrees with the concomitant de-
crease of ergosterol (Table 4, Fig. 2). The most squalene-accumula-
tor was the 5-nitrofuran derivative 7 with similar capability to Tbf
while the thia-analogue 11 showed good squalene-accumulation
level. The 5-nitrofuran homologue, derivative 8, was discrete accu-
mulator. In the three cases ergosterol concentrations were lower
than untreated-parasite values however the ergosterol depletion
promoted by Tbf was the best. This fact could be indicating some
differences on the mechanism of action between Tbf and the stud-
ied furyl/thienyl derivatives. For derivates 10, 12–15 and Nfx we
were unable to detect squalene accumulation, using HPLC analysis,
after 120 h of incubation (Table 4). However, at lower incubation
times (72 h) derivatives 13, 15 and Nfx showed, using TLC analysis,
some degree of squalene accumulation however lesser than Tbf or
derivative 7 (Fig. 2c). We were able to detect squalene accumula-
tion for 5-unsubstituted derivative 14 at 4 ꢀ ID50 doses in the same
experimental conditions.
2.2.3. In vitro antifungal activity42,43
Due to some antifungals are actives against T. cruzi27,28 we
decided to evaluate the designed anti-T. cruzi agents for its
in vitro antifungal properties. To determine the antifungal activity,
Table 2
Biological characterization of new derivatives against mammal macrophages
a
Compd
D50
J-774 macrophages
(lM)
SIb
7
200.0
24.4
8
<100.0
>400.0
<100.0
>400.0
316.0
<52.6
>25.0
<23.8
>62.5
41.0
10
11
12
Nfx
Tbf
Ktz
339.0
<100.0
19.9
<10.0
a
The results are the means of three independent experiments with a SD less than
10% in all cases.
b
SI: selectivity index, ID50,macrophage/ID50,T.
.
cruzi
Table 3
Antifungal activity of new compounds against filamentous and yeast, expressed as minimal inhibitory concentration and minimal fungicidal concentration
Compd
MIC/MFCa,b
Afu
(lg/mL)
Ca
Ct
Sc
Cn
Afl
Ani
Mg
Tr
Tm
7
8
9
>250
>250
>250
>250
>250
>250
0.78
1.56
0.5
>250
>250
>250
>250
>250
>250
1.56
>250
>250
>250
>250
>250
>250
0.50
3.12
0.5
>250
>250
250/>250
125/>250
>250
>250
0.25
0.39
0.25
>250
>250
>250
>250
>250
>250
0.50
>250
>250
>250
>250
>250
>250
0.50
>250
>250
>250
>250
>250
>250
0.50
>250
250/250
>250
125/250
>250
>250
0.125
0.04
0.05
>250
250/250
>250
125/250
>250
>250
0.075
0.01
0.025
>250
250/250
>250
125/250
>250
>250
0.075
0.025
0.025
10
11
12
AnfBc
Tbf
Ktz
1.56
0.50
0.78
0.125
0.78
0.50
1.56
0.25
a
MIC = minimal inhibitory concentration, MFC = minimal fungicidal concentration.
Ca: Candida albicans ATCC 10231, Ct: Candida tropicalis C 131 2000, Sc: Saccharomyces cerevisiae ATCC 9763, Cn: Cryptococcus neoformans ATCC 32264; An: Aspergillus niger
b
ATCC 9029, Afu: Aspergillus fumigatus ATCC 26934; Afl: Aspergillus flavus ATCC 9170, Mg: Microsporum gypseum C 115; Tr: Trichophyton rubrum C113, Tm: Trichophyton
mentagrophytes ATCC 9972.
c
AnfB: anphotericin B.

7504
A. Gerpe et al. / Bioorg. Med. Chem. 17 (2009) 7500–7509
Table 4
level comparing to the untreated control), derivatives 1, and 2,
the activity was categorized as 2; when the compounds are
good/discrete squalene accumulators (2–8-fold of squalene level
comparing to the untreated control), derivatives 3, 4, 7, 8, 11, Bppa
and Tbf, the activity was categorized as 1; and when the com-
pounds are poor or not squalene accumulators (same squalene
level that the untreated control), derivatives 5, 6, 10, 12–15 and
Nfx, the activity was categorized as 0.
Squalene and ergosterol composition of T. cruzi epimastigotes treated with com-
pounds 7, 8; 10–15; Nfx and Tbf after 120 h
Compd
Csqualene
(
l
g/mL)
Cergosterol
(lg/mL)
Ratioa
7
8
10
11
15.9 0.9
4.6 0.9
0.6b–2.1c
7.4 0.4
0.6–2.1
5.5 1.9
4.5 0.9
—
5.0 0.9
—
2.9 1.0
1.0 0.4
—
1.5 0.3
—
12
As the independent variables were employed physicochemical
parameters extracted from molecular modelling calculations.
Molecular modelling studies were performed by calculating com-
13
14
15
Nfx
Tbf
Untreated
0.6–2.1
0.6–2.1
0.6–2.1
0.6–2.1
12.0 1.0
0.6–2.1
—
—
—
—
—
—
—
—
pounds stereoelectronic properties, using DFT calculations.48,49
A
2.7 0.5
6.8 0.5
4.4 1.0
0.1–0.3
detailed conformational search for each of the molecules was per-
formed, using MM methods, to find the minimum energy and high-
est abundance conformer. The molecules with amine-protonable
group were studied as cationic forms, derivatives 13, 14, Tbf and
Bppa. The geometry of this conformer was fully optimized by
applying B3LYP/6-31G*//PM3 in the gas phase that allows to obtain
acute results with low time of computational calculi. Then, single
point B3LYP/6-31G* calculation was performed.50–52 The proper-
ties determined and examined in this study were total energy,
magnitude of dipolar moment, HOMO’s and LUMO’s energies, gap
a
b
c
Ratio: Csqualene/Cergosterol
Limit of detection 0.6 g/mL.
Limit of quantification 2.1 g/mL.
.
l
l
2.4. Structure–activity relationship studies
Initially 17 compounds were used in the structure–activity rela-
tionship studies, the parent compounds 1–6, the new derivatives 7,
8, 10–15 and the references compounds Nfx, Tbf and Bppa. The
ability to accumulate squalene (aasq) into the parasite was used
as the biological activity. Due to the different bibliographic ori-
gin18,33 of the dependent variable, squalene accumulation value,
this was used as categorical values. When the compounds are
excellent squalene accumulators (more than 8-fold of squalene
(E_LUMO–E_HOMO), hardness (
tronegativity (Elect) [(-E_LUMO–E_HOMO)/2], electrophilic index
(EI) [(Elect)2/(2
)], volume, area, and the logarithm of the parti-
g) [(E_LUMO–E_HOMO)/2], elec-
g
tion coefficient of the non-ionized molecules (Log P) (see Supple-
mentary data). Theoretical Log P (cLog P) was calculated using
the Villar method, implemented in PC SPARTAN 04 package53 at
Figure 2. (a) HPLC-lipid extracts profiles from untreated epimastigote T. cruzi using two different UV lambda detection (210, black, and 250 nm, red), acetonitrile/water (9:1),
flux rate: 0.8 mL/min. (b) HPLC-chromatograms obtained from the analysis of the lipid extracts from epimastigotes, untreated (left), Tbf-treated (centre) and 7-treated (right)
(acetonitrile, flux rate: 0.8 mL/min, detection 210 nm). (c) TLC-analysis (see Section 5 for details) of lipid extracts from epimastigote T. cruzi treated with Tbf, 7, 13, 15 and Nfx
and with 14 (at 4 ꢀ ID50 doses per 8 ꢀ 106 cells/mL) during 72 h.

A. Gerpe et al. / Bioorg. Med. Chem. 17 (2009) 7500–7509
7505
substituted series, the 5-positions of the furanes are the most con-
tributor centre.
the AM1 semiempirical level. Besides, the correlation matrix for
the used physicochemical descriptors was performed and cross-
correlations between the descriptors were not obtained (see Sup-
plementary data). Thus, these parameters are orthogonal, allowing
its safely use in the multilinear regression relationship.54,55
Non-statistical significant correlations were obtained when the
different theoretical descriptors were studied as independent var-
iable, but it was observed a clear tendency between activities and
cLog P, as it could expect the greatest squalene accumulators are
positioned in the maximum of cLog P. Consequently, multivariable
regressions between the aasq and the physicochemical descriptors
were studied. When the complete compound-population was stud-
ied adequate correlations were found between aasq and EI, cLog P,
and (cLog P)2 (r2 = 0.7802) or aasq and E_LUMO, cLog P, and
(cLog P)2 (r2 = 0.7486). When Bppa or Tbf were excluded from the
analyses the statistics of these correlations were improved
(r2 = 0.8402 and r2 = 0.7790, or r2 = 0.7872 and r2 = 0.8023, respec-
tively). When the homogeneous population of furyl/thienyl deriv-
atives were analyzed, compounds 1–8, and 10–15, the best
statistic results, Eqs. 1 and 2, were obtained. These equations show
that aasq is correlated with E_LUMO, or EI, and, in a quadratic man-
ner, with the cLog P.
Taking this in mind, we re-studied new correlations that in-
volved the atoms near to 5-position of the furan ring, specifically
the nitro nitrogen and 5-furan carbon Mulliken atomic charges
(qN and qC, respectively, Eqs. 3 and 4). Clearly, in the 5-nitrofuran
series the ability to accumulate squalene is correlated to less posi-
tive Mulliken charges of the nitro nitrogen and 5-furan carbon
atoms being Eqs. 3 and 4 completely in agreement with the
descriptors resulted in QSARs equations 1 and 2.
aasq ¼ 46 ðꢂ14Þ ꢁ 141 ðꢂ43Þ qN þ 1:8 ðꢂ0:4Þ cLogP
2
ꢁ 0:19 ðꢂ0:04Þ ðcLogPÞ
n ¼ 11; r2 ¼ 0:7790; ra2dj ¼ 0:6843; F ¼ 8:23;
p ¼ 0:0107
ð3Þ
aasq ¼ 70 ðꢂ20Þ ꢁ 151 ðꢂ41Þ qC þ 1:4 ðꢂ0:4Þ cLogP
2
ꢁ 0:15 ðꢂ0:04Þ ðcLogPÞ
n ¼ 11; r2 ¼ 0:8058; ra2dj ¼ 0:7226; F ¼ 9:7;
p ¼ 0:0069
ð4Þ
These equations also indicate that the electronic properties of the
studied compounds could play a role in the ability to accumulate
squalene. Probably, the compounds could act through their redox-
nitro moiety ability (see discussion below).
aasq ¼ ꢁ 0:4 ðꢂ0:3Þ þ 0:9 ðꢂ0:1ÞE LUMO þ 2:1 ðꢂ0:2Þ cLogP
2
ꢁ 0:21 ðꢂ0:03Þ ðcLogPÞ
n ¼ 15; r2 ¼ 0:8711; ra2dj ¼ 0:8360; F ¼ 24:8;
p < 0:0001
ð1Þ
3. Discussion
aasq ¼ ꢁ 1:3 ðꢂ0:3Þ ꢁ 0:22 ðꢂ0:03Þ EI þ 2:1 ðꢂ0:2Þ cLogP
We report the development of a second generation of 5-nitro-
furanes and their thia-analogues with anti-T. cruzi activity and
squalene accumulation capability.
2
ꢁ 0:21 ðꢂ0:03Þ ðcLogPÞ
n ¼ 15; r2 ¼ 0:8667; ra2dj ¼ 0:8304; F ¼ 23:8;
p < 0:0001
In the assay against Tulahuen 2 strain, 5-nitrofuran derivatives
8 and 13 were the most active while 7, 15 and the thiophenes 11,
and 12 were equal or more active than Nfx, Bnz, Tbf and Ktz (Table
1). Regarding the cytotoxicity against macrophages and comparing
to reference compounds (Nfx, Tbf, Ktz, Table 2) we found 5-nitro-
furan 7 and thiophene 12 with good SI being derivative 12 the best
one. The nitro-substituted derivatives are more toxic than the
unsubstituted analogues (compare cytotoxicities of 8 and 10, or
of 11 and 12) this in accordance with the well known characteristic
of nitrocompounds. However, it must be pointed out the nitro-
moiety is relevant for the squalene-accumulation properties (see
below). The 5-unsubstituted furan 10 possessed similar ID50
ð2Þ
These equations show the relevance of the compounds lipophilicity
in the ability to accumulate squalene, showing that the typical qua-
dratic behaviour corresponding to an enzymatic inhibition process
could be involved. On the other hand, according to the obtained
QSARs the electronic properties of compounds, expressed by LUMO
energy or electrophilic capability, also govern the bio-activity. Less
negative E_LUMO or less electrophile-capability of compounds pro-
mote better ability to accumulate squalene. In order to identify the
LUMO most contributor atomic centre we mapped the LUMO orbi-
tals and the electronic density maps (Fig. 3). Between the 5-nitro-
Figure 3. Electron density (solid white, isovalue 0.02) and LUMO (solid black, isovalue 0.018) isosurfaces for good (left), medium (centre), and bad (right) 5-nitrofuran-
squalene accumulators. The narrows indicate the E_LUMO map.

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A. Gerpe et al. / Bioorg. Med. Chem. 17 (2009) 7500–7509
against T. cruzi-Tulahuen 2 strain to Tbf (16.0 and 17.0
tively) however derivative 10 has better activity against CL Brener
clone (ID50s of 9.6 and 42.0 M, respectively) and SI (>25.0 and
l
M, respec-
manner of action for some of them, we could propose them as
molecular lead for further structural modifications and further bio-
logical studies, especially in vivo evaluations.
l
19.9, respectively) than the antifungal reference Tbf. On the other
hand, furan 10 was the only studied compound with some level
of activities against the studied fungus (Table 3). These results
open the therapeutic potential of 5-unsubstituted furanes and
thiophenes, that is, 10 and 12, that in general are badly considered
as pharmacophore in the neglected diseases, like trypanosomatids-
associated illness.
5. Experimental
All starting materials were commercially available research-
grade chemicals and used without further purification. All solvents
were dried and distilled prior to use. All the reactions were carried
out in a nitrogen atmosphere. 3-(5-Nitrofuryl)propenoic acid,
3-furylpropenoic acid, and intermediate III were prepared follow-
ing synthetic procedures previously reported.10,11 Melting points
were determined with an electrothermal melting point apparatus
(Electrothermal 9100) and were uncorrected. Proton and carbon
NMR spectra were recorded on a Bruker DPX-400 spectrometer.
The chemical shifts values are expressed in ppm relative to tetra-
methylsilane as internal standard. Mass spectra were determined
on a MSD 5973 Hewlett–Packard spectrometer using electronic
impact at 70 eV (EI). Infrared spectra were recorded on a Perkin–El-
mer 1310 apparatus, using potassium bromide tablets the frequen-
cies were expressed in cmꢁ1. Microanalyses were performed on a
Fisons EA 1108 CHNS-O instrument and were within 0.4% of the
calculated compositions. Column chromatography was carried
out using Merck silica gel (60–230 mesh).
In the squalene accumulation changes studies, some relevant
features could be observed. On the one hand, unlike Nfx, 5-nitro-
furanes 7, 8, and the 5-nitrothiophene 11 were able to accumulate
squalene in the studied experimental conditions (Table 4) resulting
derivatives 7 and 11 as squalene-accumulators as Tbf. In these
cases ergosterol was concomitantly depleted however the ergos-
terol-depletions for these were lower than the observed value for
Tbf showing an alternative ergosterol biosynthetic pathway prob-
ably operating for these derivatives. On the other hand, chemi-
cal-structural exigencies could be extracted from this study, for
example the necessity of an allyl-moiety is not essential for the
accumulation of squalene, like it was observed with the parent
compounds 1 and 2 (Fig. 1), being allyl-derivatives 10, and 12–14
unable to accumulate squalene when were tested at 120 h of incu-
bation. Moreover, due to 5-unsubstituted derivatives 10, 12 and 14
are not squalene-accumulators it seems to indicate the presence of
the 5-nitro-moiety is relevant for the ability to accumulate this
biological precursor.
5.1. General procedure for the preparation of
thiosemicarbazones 7–12
These structural questions could be answered analyzing the
QSARs results. On the one hand, as it is discussed above the com-
pounds lipophilic property clearly affect the compounds ability to
accumulate squalene in a quadratic manner characteristic of en-
zyme–drug interaction validating the hypothesis of an enzymatic
inhibition. On the other hand, in the cases of 5-nitro-substituted fur-
anes the QSARs seem to show the nitro-nitrogen should exhibit a
particular electrophile character to accumulate squalene. Taking
into account SE starts its catalytic cycle, terminating with the pro-
duction of 2,3-oxidosqualene, as the enzyme-reduced form56 react-
ing with molecular oxygen (Fig. S1, Supplementary data) it could be
speculated that the studied nitro-derivatives disturb this process as
electron-scavenger entities. Atleasttwodifferentpathwayscouldbe
affected by the nitro-derivatives. Firstly, as NADPH-cytochrome
P450 reductase electron-acceptor, being this enzyme the responsi-
ble of maintaining adequate levels of squalene epoxidase in its re-
duced form, the nitro-derivatives could be interfering in this
process as redox partner of NADPH or redox substrate of
NADPH-cytochrome P450 reductase (pathways (a) or (b) Fig. S2a,
Supplementary data). As it was previously probed,18–22 the nitro-
derivatives studied here are able to suffer reduction by one electron,
generating concomitantly the nitro-anion free radical (Fig. S2b, Sup-
plementary data). Secondly, as molecular oxygen scavenger, being
the squalene epoxidation an oxygen-dependent pathway the ni-
tro-derivatives could be reacting with oxygen promoting the de-
crease of this entity (pathway (c) Fig. S2a, Supplementary data).
This kind of process has been previously used as a manner to probe
the oxidative stress mechanism of action of anti-trypanosomatids
5-nitrofuranes,18–20,57 known as redox cycling studies. However,
being this process operative in most of the 5-nitrofuranes in the case
study herein the speculated consumption of molecular oxygen
seems to need a special redox potential.
The corresponding aldehyde (5-nitrofurfural, 3-(5-nitrofu-
ryl)acroleine, furfural, 5-nitrothiophene-2-carbaldehyde, or thio-
phene-2-carbaldehyde,
1.0 mmol),
4-allylthiosemicarbazone
(1.0 mmol), p-TsOH (catalytic amount) and toluene (10.0 mL) were
stirred at room temperature until the aldehyde was not present.
The solid was collected by filtration, crystallized from toluene,
washed with n-hexane and dried.
5.1.1. 4-Allyl-1-(5-nitrofurfurilydene)thiosemicarbazide (7)
Yellow solid, 221 mg (87%); mp 179.0–181.0 °C. 1H NMR
(DMSO-d6) dH: 4.24 (2H, t, J = 5.7 Hz), 5.13 (2H, m), 5.91 (1H, m),
7.37 (1H, d, J = 4.0 Hz), 7.81 (1H, d, J = 4.0 Hz), 8.00 (1H, s), 8.70
(1H, t, J = 5.7 Hz), 11.94 (1H, s). 13C NMR (DMSO-d6) dC: 46.8,
114.5, 116.0, 116.7, 130.6, 135.4, 153.3, 156.0, 178.3. EI-MS, m/z
(abundance, %): 254 (M+ꢀ, 1), 237 (M+ꢀꢁ17, 8), 209 (M+ꢀꢁNO2, 4),
197 (7), 141 (100), 115 (35), 81 (24), 56 (32). IR,
m: 619, 806,
1096, 1215, 1279, 1356, 1509, 1551, 3343. Anal. Calcd for
C9H10N4O3S.
5.1.2. 4-Allyl-1-[3-(5-nitro-2-furyl)-2-propenylidene]
thiosemicarbazide (8)
Yellow solid, 188 mg (67%); mp 175.4–178.0 °C. 1H NMR
(DMSO-d6) dH: 4.19 (2H, t, J = 5.6 Hz), 5.11 (2H, m), 5.89 (1H, m),
7.03 (3H, m), 7.75 (1H, d, J = 4.0 Hz), 7.90 (1H, d, J = 8.0 Hz), 8.61
(1H, t, J = 5.6 Hz), 11.70 (1H, s). 13C NMR (DMSO-d6) dC: 46.6,
114.5, 116.2, 116.6, 123.6, 131.0, 135.5, 142.6, 153.0, 155.6,
178.1. EI-MS, m/z (abundance, %): 280 (M+ꢀ, 6), 263 (M+ꢀꢁ17, 17),
234 (M+ꢀꢁNO2, 14), 167 (61), 115 (100), 92 (27), 81 (21), 56 (64).
IR, m: 799, 1098, 1219, 1356, 1462, 1526, 3300. Anal. Calcd for
C11H12N4O3S.
5.1.3. 4-Allyl-1-furfurylidenethiosemicarbazide (9)
Yellow solid, 127 mg (61%); mp 128.0–129.0 °C. 1H NMR
(CDCl3) dH: 4.39 (2H, m), 5.30 (2H, m), 5.98 (1H, m), 6.54 (1H, dd,
J = 1.6, 3.2 Hz), 6.74 (1H, d, J = 3.2 Hz), 7.54 (1H, s), 7.68 (1H, br
s), 7.77 (1H, br s), 9.85 (1H, br s). 13C NMR (CDCl3) dC: 47.3,
112.5, 114.5, 117.5, 132.4, 133.7, 145.2, 149.1, 177.2. EI-MS, m/z
4. Conclusions
We have developed and identified new furanes and thiophenes
as anti-T. cruzi agents. Observing high selectivity indexes and new

A. Gerpe et al. / Bioorg. Med. Chem. 17 (2009) 7500–7509
7507
(abundance, %): 209 (M+ꢀ+H, 2), 182 (4), 115 (100), 109 (9), 96 (63),
81 (19), 56 (27). IR, : 586, 745, 920, 1011, 1221, 1287, 1526, 3154.
Anal. Calcd for C9H11N3OS.
7.46 (2H, d, J = 7.8 Hz), 7.66 (1H, d, J = 1.5 Hz). 13C NMR (CDCl3)
dC: 44.0, 53.0, 61.0, 112.0, 114.0, 116.0, 126.0, 127.0, 129.0, 129.5,
133.0, 144.0, 152.0, 164.0. EI-MS, m/z (abundance, %): 322 (M+ꢀ,
20), 172 (52), 117 (100). UV (acetonitrile, 0.01 mg/mL), kmax = 255
m
5.1.4. 4-Allyl-1-(3-furyl-2-propenylidene)thiosemicarbazide
(10)
and 303 (e
= 143 mL mgꢁ1 cmꢁ1) nm. Anal. Calcd for C20H22N2O2.
Orange solid, 181 (77%); mp 130.0–132.0 °C. 1H NMR (CDCl3)
dH: 4.38 (2H, m), 5.30 (2H, m), 5.96 (1H, m), 6.47 (2H, m), 6.74
(2H, m), 7.47 (2H, br s), 7.56 (1H, d, J = 8.0 Hz), 9.29 (1H, br s).
13C NMR (CDCl3) dC: 47.3, 112.2, 112.6, 117.6, 122.7, 127.2, 133.7,
144.1, 144.2, 152.4, 177.7. EI-MS, m/z (abundance, %): 235 (M+ꢀ,
5), 135 (16), 121 (63), 115 (100), 93 (79), 80 (23), 67 (37), 56
5.1.9. N-(2-Hydroxyethyl)-3-(5-nitro-2-furyl)propenamide (15)
A mixture of intermediate III (1.0 mmol), 4-(3-phenyl-2-prope-
nyl)piperazine (1.0 mmol), K2CO3 (2.0 mmol), and acetone
(10.0 mL) was heated at reflux during 3.0 h. The acetone was evap-
orated in vacuo and the crude of reaction was treated EtOAc
(30.0 mL) and washed successively with aqueous saturated solu-
tion of NaHCO3 (10.0 mL), aqueous solution of HCl (1 M,
10.0 mL). After dried with Na2SO4 the organic layer was evaporated
in vacuo and the crude was purified by column chromatography
(Al2O3, petroleum ether/EtOAc (0–60%)). Brown oil (10%). 1H
NMR (CDCl3) dH: 2.19 (1H, m), 4.05 (2H, d, J = 9.4 Hz), 4.38 (2H,
d, J = 9.4 Hz), 6.69 (1H, d, J = 3.8 Hz), 6.86 (1H, d, J = 15.9 Hz), 7.12
(1H, d, J = 15.9 Hz), 7.35 (1H, t, J = 3.8 Hz). 13C NMR (CDCl3) dC:
55.0, 68.0, 113.0, 114.0, 120.0, 124.0, 154.0, 163.0. EI-MS, m/z
(abundance, %): 226 (M+ꢀ, 3), 208 (M+ꢀꢁH2O, 42), 162 (M+ꢀꢁNO2,
100). Anal. Calcd for C9H10N2O5.
(35). IR,
m: 583, 623, 739, 920, 957, 1007, 1075, 1136, 1221,
1285, 1545, 3152. Anal. Calcd for C11H13N3OS.
5.1.5. 4-Allyl-1-(5-nitrothenylidene)thiosemicarbazide (11)
Orange solid, 267 mg (99%); mp 201.7–202.6 °C. 1H NMR
(DMSO-d6) dH: 4.23 (2H, m), 5.14 (2H, m), 5.90 (1H, m), 7.53 (1H,
d, J = 4.4 Hz), 8.08 (1H, d, J = 4.4 Hz), 8.23 (1H, s), 8.73 (1H, t,
J = 5.6 Hz), 11.89 (1H, br s). 13C NMR (DMSO-d6) dC: 46.7, 116.6,
130.0, 131.3, 135.4, 136.2, 147.4, 151.5, 178.0. EI-MS, m/z (abun-
dance, %): 270 (M+ꢀ, 2), 244 (6), 157 (81), 115 (100), 95 (25), 81
(39), 56 (63). IR, m: 617, 733, 814, 930, 1107, 1235, 1298, 1356,
1439, 1491, 1539, 3135. Anal. Calcd for C9H10N4O2S2.
5.2. Biology
5.1.6. 4-Allyl-1-thenylidenethiosemicarbazide (12)
5.2.1. Anti-T. cruzi in vitro test using epimastigotes of Tulahuen
2 strain or CL Brener clon
White solid, 198 mg (88%); mp 161.3–162.8 °C. 1H NMR (CDCl3)
dH: 4.40 (2H, br s), 5.28 (2H, dd, J = 10.4, 17.2 Hz), 5.99 (1H, m), 7.08
(1H, dd, J = 4.0, 5.0 Hz), 7.30 (1H, d, J = 4.0 Hz), 7.40 (1H, d,
J = 5.0 Hz), 7.45 (1H, br s), 8.08 (1H, s), 9.90 (1H, br s). 13C NMR
(CDCl3) dC: 47.2, 117.4, 128.2, 128.9, 131.2, 133.7, 137.7, 138.4,
177.6. EI-MS, m/z (abundance, %): 225 (M+ꢀ, 3), 125 (9), 115
Trypanosoma cruzi epimastigotes (Tulahuen 2 strain or CL Bren-
er clon) were grown at 28 °C in an axenic medium (BHI-Tryptose)
as previously described,10–12 supplemented with 5% fetal bovine
serum (FBS). Cells from a 10-day-old culture (stationary phase)
were inoculated into 50 mL of fresh culture medium to give an ini-
tial concentration of 8 ꢀ 106 cells/mL. Cell growth was followed by
measuring everyday the absorbance of the culture at 600 nm. Be-
fore inoculation, the media were supplemented with the indicated
amount of the drug from a stock solution in DMSO. The final con-
centration of DMSO in the culture media never exceeded 0.4% and
the control was run in the presence of 0.4% DMSO and in the ab-
sence of any drug. No effect on epimastigote growth was observed
by the presence of up to 1% DMSO in the culture media. The per-
centage of growth inhibition (PGI) was calculated as follows: PGI
(%) = {1 ꢁ [(Ap ꢁ A0p)/(Ac ꢁ A0c)]} ꢀ 100, where Ap = A600 of the
culture containing the drug at day 5; A0p = A600 of the culture con-
taining the drug just after addition of the inocula (day 0); Ac = A600
of the culture in the absence of any drug (control) at day 5;
A0c = A600 in the absence of the drug at day 0. To determine ID50
values, 50% inhibitory concentrations, parasite growth was fol-
lowed in the absence (control) and presence of increasing concen-
trations of the corresponding drug. At day 5, the absorbance of the
culture was measured and related to the control. The ID50 value
was taken as the concentration of drug needed to reduce the absor-
bance ratio to 50%.
(100), 56 (40). IR, m: 594, 700, 914, 1036, 1221, 1287, 1552, 3146.
Anal. Calcd for C9H11N3S2.
5.1.7. 4-(3-Phenyl-2-propenyl)piperazine-1-ylamide of 3-(5-
nitro2-furyl)propenoic acid (13)
A mixture of 3-(5-nitrofuryl)propenoic acid (1.0 mmol), SOCl2
(2.0 mmol), and dry n-hexane (25.0 mL) was heated at reflux for
1.5 h. Then
a mixture of 4-(3-phenyl-2-propenyl)piperazine
(1.2 mmol) and Et3N (2.5 mmol) was added at 0 °C and the mixture
was stirred for 1.5 h at room temperature. The organic mixture was
treated EtOAc (30.0 mL) and washed successively with aqueous
saturated solution of NaHCO3 (10.0 mL), aqueous solution of HCl
(1 M, 10.0 mL). After dried with Na2SO4 the organic layer was evap-
orated in vacuo and the crude was purified by column chromatog-
raphy (Al2O3, petroleum ether/EtOAc (0–60%)). Brown-orange solid
(15%); mp 136.0–139.0 °C. 1H NMR (CDCl3) dH: 2.53 (4H, m), 3.23
(2H, d, J = 6.8 Hz), 3.73 (4H, m), 6.29 (1H, d, J = 15.4 Hz), 6.57 (1H,
d, J = 15.4 Hz), 6.71 (1H, d, J = 3.3 Hz), 7.17 (1H, d, J = 15.3 Hz),
7.27 (1H, t, J = 7.7 Hz), 7.36 (2H, m), 7.37 (1H, d, J = 3.3 Hz), 7.40
(2H, m), 7.47 (1H, d, J = 15.3 Hz). 13C NMR (CDCl3) dC: 45.0, 53.0,
61.0, 113.0, 115.0, 122.0, 126.0, 126.5, 128.0, 128.5, 134.0, 153.0,
164.0. EI-MS, m/z (abundance, %): 367 (M+ꢀ, 1), 350 (M+ꢀꢁ17, 13),
172 (17), 117 (100). UV (acetonitrile, 0.01 mg/mL), kmax = 246
5.2.2. Unspecific mammalian cytotoxicity41
J-774 murine macrophage-like cells (ATCC, USA) were main-
tained by passage in Dulbecco’s modified Eagle’s medium (DMEM)
containing 4 mM L-glutamine, and supplemented with 10% heat-
inactivated fetal calf serum. J-774 cells were seeded (1 ꢀ 105
(e
= 99 mL mgꢁ1 cmꢁ1) and 360 nm. Anal. Calcd for C20H21N3O4.
5.1.8. 4-(3-Phenyl-2-propenyl)piperazine-1-ylamide of 3-(2-
furyl)propenoic acid (14)
cells/well) in 96 well microplates with 200 lL of RPMI 1640 med-
Following the procedure described for derivative 13. Column
chromatography (Al2O3, petroleum ether/EtOAc (0–60%)). Yellow
solid (25%); mp 111.0–113.0 °C. 1H NMR (CDCl3) dH: 2.51 (4H, br
s), 3.19 (2H, dd, J = 6.6, 1.5 Hz), 3.70 (4H, br s), 6.34 (1H, dd,
J = 15.8, 6.9 Hz), 6.56 (1H, dd, J = 3.4, 1.9 Hz), 6.61 (1H, dd,
J = 15.8 Hz), 6.76 (1H, d, J = 3.4 Hz), 6.93 (1H, d, J = 15.2 Hz), 7.25
(1H, t, J = 7.3 Hz), 7.34 (2H, t, J = 7.6 Hz), 7.41 (1H, d, J = 15.2 Hz),
ium supplemented with 20% heat inactivated fetal calf serum. Cells
were allowed to attach for 48 h in a humidified 5% CO2/95% air
atmosphere at 37 °C and, then, exposed to compounds (100.0–
400.0 lM) for 48 h. Afterwards, cell viability was assessed by mea-
suring the mitochondrial-dependent reduction of MTT (Sigma) to
formazan. For that purpose, MTT was added to cells to a final con-
centration 0.4 mg/mL and cells were incubated at 37 °C for 3 h.

7508
A. Gerpe et al. / Bioorg. Med. Chem. 17 (2009) 7500–7509
After removing the media, formazan crystals were dissolved in
DMSO (180 L), and the absorbance at 595 nm was read using a
microplate spectrophotometer. Results are expressed as ID50 (com-
pound concentration that reduce 50% control absorbance at
595 nm). Every ID50 is the average of three different experiments.
izing the plates with iodine and heating them at 100 °C. Quantita-
tive analyses of squalene from sterol fractions was done by HPLC
using a C-18 Chromosorb column (25 cm ꢀ 0.4 cm internal diame-
l
ter, 10 lm particle size) in a Perkin–Elmer LC-135C/LC-235C Diode
Array Detector, Series 410 LC BIO PUMP, with the UV detector set
at 210 nm. The mobile phase consisted of 100% AcCN and was kept
constant at a flow-rate of 0.8 mL/min. The calibration curve of
squalene was constructed (using cholesterol as an internal stan-
dard) for quantification of this sterol in the lipid extracts from T.
5.2.3. Antifungal in vitro test
5.2.3.1. Microorganisms and media. For the antifungal evalua-
tion, strains from the American Type Culture Collection (ATCC),
Rockville, MD, USA and CEREMIC (C), Centro de Referencia Mic-
ológica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Suipa-
cha 531-(2000)-Rosario (Argentina) were used: Microsporum
canis C 112, Epidermophyton floccosum C 114, T. rubrum C 110, T.
mentagrophytes ATCC 9972 and M. gypseum C 115. Strains were
grown on Sabouraud-chloramphenicol agar slants for 48 h at
30 °C, maintained on slopes of Sabouraud-dextrose agar (SDA, Ox-
oid) and subcultured every 15 days to prevent pleomorphic trans-
formations. Inoculate of cell or spore suspensions were obtained
according to reported procedures and adjusted to 103 cells/spores
with colony forming units (CFU) /mL.
cruzi and it is the following: Csqualene
(
l
g/mL) = 4.42 ꢀ 10ꢁ5
( 0.16 ꢀ 10ꢁ5) A ꢁ 2.77 ( 2.03), where A corresponds to the area
of the peak at 5.70–5.85 min. Quantitative analyses of ergosterol
from sterol fractions was done using the same column and equip-
ment measuring at 250 nm. The mobile phase consisted of AcCN/
water (9:1) and was kept constant at a flow-rate of 0.8 mL/min.
The calibration curve of ergosterol was constructed (using choles-
terol as an internal standard) for quantification of this sterol in the
lipid extracts from T. cruzi and it is the following: Cergosterol
(
l
g/mL) = 1.52 ꢀ 10ꢁ4 ( 0.14 ꢀ 10ꢁ4) A0 ꢁ 2.02 ( 2.30), where A0
corresponds to the area of the peak at 8.50–8.75 min.
5.2.3.2. Antifungal susceptibility testing. Minimal Inhibitory
Concentration (MIC) of each studied compound was determined
by using broth microdilution techniques according to the guide-
lines of the Clinical and Laboratory Standards Institute (CLSI)
formely National Committee for Clinical Laboratory Standards for
yeasts (M27-A2) and for filamentous fungi (M 38 A). MIC values
were determined in RPMI 1640 buffered to pH 7.0 with MOPS.
The starting inocules were 1 ꢀ 105–5 ꢀ 105 CFU/mL. Microtiter
trays were incubated at 35 °C for yeasts and hialohyphomycetes
and at 28–30 °C for dermatophyte strains in a moist, dark chamber,
and MICs were visually recorded at 48 h for yeasts, and at a time
according to the control fungus growth, for the rest of fungi. For
the assay, stock solutions of compounds were twofold diluted with
5.4. Quantitative structure–activity relationship studies
The molecular structures of the studied compounds, 1–8, 10–
15, Nfx, Tbf and Bppa were subjected to complete geometry opti-
mization, in gas phase, using PC SPARTAN 04 package53 as follows:
conformational search using MMFF conformer module, after that
from the most stable conformer fully optimization by applying
*
B3LYP/6-31G //PM3. Structural, geometrical, electronic and hydro-
phobic descriptors were extracted from the calculus. Theoretical
Log P was calculated using the Villar method at the AM1 semiem-
pirical level, using the structures generated in the fully geometrical
optimization. The relationships between the selected descriptors
and the ability to accumulate squalene (aasq) were quantified by
the use of multiple linear regressions. In the equations n represents
the number of data points, r2 is the correlation coefficient, r2adj is the
adjusted correlation coefficient, and F value is related to the F-sta-
tistic analysis (Fischer test).
RPMI 1000-1
l
g/mL (final volume = 100
lL) and a final DMSO con-
centration 6 1%. A volume of 100
l
L of inocule suspension was
added to each well with the exception of the sterility control where
sterile water was added to the well instead. Pure compounds were
tested from 100 to 1 lg/mL. MIC was defined as the minimum
inhibitory concentration of compound which resulted in total inhi-
bition of the fungal growth. Ktz, Tbf and AnfB were used as positive
controls.
Acknowledgements
A.G. and L.B. thank to PEDECIBA (Uruguay) and A.G., G.A., D.B.
and P.H, thank ANII (Uruguay) for their scholarships. We thank
CSIC-UdelaR (Uruguay, Project 341) for the economical support.
5.3. Squalene and ergosterol level analysis
Epimastigote forms of T. cruzi (Tulahuen 2 strain) (16 ꢀ 106
cells/mL) were maintained in an axenic medium (BHI-Tryptose)
for analysis of the effects of the studied compounds on sterol bio-
synthesis modifications. The experiments were carried out in cul-
tures at 28 °C and with strong aeration. The studied compound
was added at a concentration equivalent of ID50 per 8 ꢀ 106 cells/
mL as DMSO solution. Control samples received only the vehicle.
Parasites were exposed to the treatment by 120 h. They were
counted by optical microscopy using a Neubauer chamber. The
control and drug-treated parasites were centrifugated at
3000 rpm during 15 min, and then the pellets were collected and
washed with buffer phosphate (10.0 mL, 0.05 M, pH 7.4) and cen-
trifugated at 3000 rpm during 15 min. The pellets were treated
with chloroform/methanol (2:1) during 12 h at 4 °C. Then the or-
ganic phases were evaporated with nitrogen and the residues were
Supplementary data
Supplementary data (synthesis, lipid analyses and QSAR data)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2009.09.013.
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