Novel 1,3,4-thiadiazolium-2-phenylamine chlorides derived from natural piperine as trypanocidal agents: Chemical and biological studies

Article abstract of DOI:10.1016/j.bmc.2007.12.049

We herein describe the synthesis and characterization of nine new 1,3,4-thiadiazolium-2-phenylamine chlorides derived from natural piperine. We evaluate their toxic effects against the different evolutive forms of Trypanosoma cruzi, and the host cell (murine macrophages). The results obtained show that mesoionic hydrochloride MI possesses the best activity profile. Compound MI may be a prototype for use in the development of a new chemotherapeutic agent with high efficiency, which may be employed in the treatment of Chagas' disease.

Full text of DOI:10.1016/j.bmc.2007.12.049

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2984–2991
Novel 1,3,4-thiadiazolium-2-phenylamine chlorides derived
from natural piperine as trypanocidal agents:
Chemical and biological studies
a
b
b
´
Welisson da Silva Ferreira, Leonardo Freire-de-Lima, Vıctor Barbosa Saraiva,
b
b
b
´
Frederico Alisson-Silva, Lucia Mendonc¸a-Previato, Jose Osvaldo Previato,
Aurea Echevarriaa and Marco Edilson Freire de Limaa,*
a
ˆ
Universidade Federal Rural do Rio de Janeiro, Instituto de Ciencias Exatas, Departamento de Quımica,
´
Rodovia BR 465, Km 7, CEP 23.890-000 Serope´dica, RJ, Brazil
ˆ
b
Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude, Instituto de Biofısica Carlos Chagas Filho-Ilha da Cidade
Universita´ria, CEP 21.990-400 Rio de Janeiro, RJ, Brazil
´
´
Received 29 August 2007; revised 15 December 2007; accepted 20 December 2007
Available online 25 December 2007
Abstract—We herein describe the synthesis and characterization of nine new 1,3,4-thiadiazolium-2-phenylamine chlorides derived
from natural piperine. We evaluate their toxic effects against the different evolutive forms of Trypanosoma cruzi, and the host cell
(murine macrophages). The results obtained show that mesoionic hydrochloride MI possesses the best activity profile. Compound
MI may be a prototype for use in the development of a new chemotherapeutic agent with high efficiency, which may be employed in
the treatment of Chagas’ disease.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
of insect feces with eyes, mouth, or open skin lesions.
The disease has two phases: acute and chronic. After
the initial acute phase, which is asymptomatic in most
cases, a chronic condition establishes which can lead in
ꢀ40% of cases to irreversible lesions in gastrointestinal
tract and heart.3 The efforts in insect control and in the
quality of blood for transfusion have reduced the impact
of this parasitic illness, but the treatment of people al-
ready infected, most of them in the chronic phase of the
disease, is still a challenge.1–4 Currently, the chemothera-
peutic agents available are unsatisfactory for chronic
cases and there is neither a vaccine nor other preventive
treatments. There are only two drugs available, the nitro-
heterocyclic compounds nifurtimox and benznidazole (1
and 2, Fig. 1).5 Both compounds have low efficacy and
produce severe side effects. Chagas’ disease is now consid-
ered a neglected disease, among other parasitic illnesses,
due to the fact that the pharmaceutical industry does not
regard as cost effective the investment on drugs to fight
these illnesses. Therefore, it is necessary to find new ther-
apeutic alternatives to treat chagasic patients.6
Chagas’ disease, or American trypanosomiasis, is an en-
demic parasitic illness widespread in Latin America,
from northern Mexico to Argentina.1,2 Together with
malaria, leishmaniasis, and African trypanosomiasis,
Chagas’ disease is a major parasitic cause of death and
hardship, especially in developing countries.3 According
to the World Health Organization, there are 16–18 mil-
lion people already infected and some 120 million at risk
of becoming infected, with ꢀ200,000 new cases and
more than 14,000 deaths every year.1,2
The etiologic agent of Chagas’ disease is the flagellated
protozoan Trypanosoma cruzi, which is transmitted to
humans either by transfusion of infected blood, from
an infected mother to her child, or by hematophagous
Reduviid vectors.3 Contagion usually occurs by contact
Keywords: Chagas’ disease; Mesoionic compounds; 1,3,4-Thiadiazoli-
um-2-phenylamine; Piperine; Trypanosoma cruzi.
*
Kapil described the results of an investigation of the
activity of natural piperine 3 (Fig. 1) against promasti-
Corresponding author. Tel.: +55 2126821872; fax: +55
2126822807; e-mail: marco@ufrrj.br
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.049

W. da Silva Ferreira et al. / Bioorg. Med. Chem. 16 (2008) 2984–2991
2985
acyl chlorides26,27 or aromatic aldehydes28 as synthetic
precursors.
O
CH3
N
N
N
O2N
N
N
H
O
SO2
1
Scheme 1 shows the synthesis of precursors obtained di-
rectly from natural product 3. Piperic acid 4 was ob-
2
NO2
O
tained from
3 in excellent yield through basic
hydrolysis.9,11 The saturated derivative 5 was prepared
from 4 by catalytic hydrogenation of the conjugated
double bonds9 while the nitro-derivative 6 was prepared
from 5 by nitration under acidic condition.29
O
O
N
3
Figure 1. Structures of nifurtimox 1, benznidazole 2, and piperine 3.
The derivatives of the cinnamic series (Scheme 2) were
obtained employing piperonal 7 as starting material,
through classical reactions such as Knoevenagel’s meth-
odology (8 and 12),18 catalytic hydrogenation (9),9 and
nitration (10 and 11).29
gote forms of Leishmania donovani.7 More recently,
Raay and co-workers described encouraging data ob-
tained for the activity of piperine intercalated into
mannose-coated liposomes with hamsters infected with
L. donovani.8 Based on these results, we investigated
the activity of this natural amide and of a series of deriv-
atives and analogues on T. cruzi.9 The natural amide 3 is
the main secondary metabolite in Piper nigrum, occur-
ring mainly in the fruits. P. nigrum (popularly known
as black pepper) is the most common spice worldwide.
It is a perennial vine, widely used in folk medicine in In-
dia, where it originates. Piperine 3 is very abundant in
the plant, being extracted from the dry fruits with a yield
of 3–7%.10,11 Various biological activities have been
attributed to piperine including insecticidal,12–14 inhibi-
tion of liver metabolism,15 stimulation of melanocyte
proliferation,16 and anti-tumoral activity.17
The main reagent for the synthesis of all these mesoionic
hydrochlorides, 1,4-diphenylthiosemicarbazide (C6H5N
HNHCSNHC6H5), is commercially available or can be
easily prepared by a known method.30 Scheme 3 shows
the synthesis of mesoionic hydrochlorides (MI–IX)
employing the respective acyl chlorides and 1,4-diphe-
nylthiosemicarbazide in dry 1,4-dioxane,18,27 affording
the mesoionic compounds (MI–VII) in moderate to high
yields (34–83%, Entries A and B). The aromatic alde-
hydes 7 and 11 (Scheme 3, Entry C) reacted with 1,4-
diphenylthiosemicarbazide, in the presence of trimethyl-
silyl chloride in dry dimethylformamide,28 generating
the mesoionic salts MVIII (68%) and MIX (40%). Thus,
we were able to prepare three series of compounds
(Scheme 3, Entries A, B, and C) having a saturated or
unsaturated side chain with none, two or four carbon
atoms as spacers between the methylenedioxyphenyl
and mesoionic moieties.
Recently we described the synthesis and evaluation of
toxicity of a series of 1,3,4-thiadiazolium-2-phenylamine
salts derived from different substituted cinnamic acids
against promastigote and amastigote forms of Leish-
mania amazonensis, which afforded very promising re-
sults.18 Since protozoa from genera Leishmania and
Trypanosoma belong to the same family (Trypanoso-
matidae), we devised the preparation of new mesoionic
1,3,4-thiadiazolium-2-phenylamine chlorides derived
from piperine, thus combining the mesoionic and the
natural amide moieties, having in mind the concept of
molecular hybridization, which has proven to be a very
useful tool in the design of new bioactive compounds.19
We planned the preparation of nitro derivatives of the
three series of mesoionic salts (Scheme 3, Entries A–
C), since the nitro group present in the structures of nif-
urtimox (1) and benznidazole (2) is pharmacophoric to
the trypanocidal activity showed by these compounds.
The toxic effects of nitro derivatives are due to the for-
mation of various free radical intermediates and/or elec-
trophilic metabolites from the nitroreductases, that act
on the nitro group of R-NO2-type molecules.5
Mesoionic systems are dipolar five-membered heterocy-
clic rings in which both the negative and positive charges
are delocalized throughout endo- and exocyclic
atoms.20,21 Mesoionic rings are present in the structure
of numerous compounds with useful and wide-ranging
biological activities, including anti-bacterial,22,23 inhibi-
tion of platelet aggregation,24 and anti-tumoral
effects.25,26
The structures of all mesoionic salts MI–IX (Scheme 3),
derivatives and analogues of the natural piperine, were
1
fully characterized by IR, MS, H and 13C NMR spec-
tral data, and microanalyses. The spectral assignments
were based on literature data9,18,27 and are consistent
with the structures described.
2.2. Biological activity
2. Results and discussion
2.1. Chemistry
The toxic effects of mesoionic derivatives were evalu-
ated against the different evolutive forms of T. cruzi
(Y strain) and also against the host cell (murine mac-
rophages). The results obtained are depicted in Table
1. Firstly, we evaluated the anti-proliferative effects
of nine derivatives on epimastigotes of T. cruzi obtain-
ing IC50 values ranging from 0.64 to 113.06 lM. The
four more active derivatives (MI, MII, MIII, and
Nine new mesoionic 1,3,4-thiadiazolium-2-phenylamine
chlorides (MI–MIX), derivatives and analogues of natu-
ral piperine 3, were synthesized following two experi-
mental procedures found in the literature, employing

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W. da Silva Ferreira et al. / Bioorg. Med. Chem. 16 (2008) 2984–2991
O
COOH
O
O
O
a
N
O
4
3
b
COOH
COOH
O
c
O
O
O
6
NO2
5
Scheme 1. Preparation of acid precursors from piperine 3. Reagents and conditions: (a) KOH, ethanol, reflux, 24 h, then HCl (aq), 0 °C (93%); (b)
ethyl acetate, Pd/C, H2, 3 h (76%); (c) CH3COOH (glacial), HNO3 (concd) 3 h (77%).
COOH
COOH
CHO
b
O
O
O
O
O
O
a
7
9
8
c
c
COOH
CHO
NO2
COOH
O
O
O
O
a
NO2
O
O
NO2
12
10
11
Scheme 2. Preparation of the precursors of the cinnamic series (8, 9, 10, and 12). Reagents and conditions: (a) malonic acid, pyridine, piperidine,
reflux, 8 h, then HCl (aq), 0 °C (75–93%); (b) ethyl acetate, Pd/C, H2, 3 h (80%); (c) CH3COOH (glacial), HNO3 (concd) 3 h (62–82%).
MIX), which showed the lowest IC50 values on epim-
astigotes, were evaluated against murine macrophages
and the results show a better selectivity profile for
MI (Table 1). The higher toxicity on epimastigotes
shown by MI, MII, and MIII derivatives suggests that
the conjugation between the mesoionic and methylene-
dioxyphenyl rings is an important structural feature
for the activity of these compounds. Derivatives MII,
MIII, and MIX showed significant toxic effects on
epimastigotes of T. cruzi, but they were also found
to be toxic for host cells (murine macrophages). Fur-
thermore, MI was evaluated against trypomastigote
and amastigote forms of T. cruzi, showing an interest-
ing activity profile, better than its precursor, the natu-
ral amide piperine (3), and being more active against
the intracellular amastigote form than the reference
drug benznidazole (2, Table 1).
4. Experimental protocols
4.1. Chemistry
Melting points were determined in a capillary with a
Melt-Temp. H and 13C NMR were recorded at room
1
temperature using Bruker-AC 200 MHz, Bruker-DRX
600 MHz, and Bruker-DRX 400 MHz spectrometers,
employing tetramethylsilane as internal reference. The
chemical shifts (d) are given in ppm and coupling con-
stants (J) in Hertz. Mass spectra were obtained at
70 eV on a Shimadzu-CG/MS QP 2000A equipped with
a solid probe. Fragments were described as m/z ratio. IR
spectra were recorded on a Perkin-Elmer 5987A spec-
trometer in KBr pellets. The microanalyses were carried
out with elemental apparatus CHNS-O EA-1110 of Car-
lo Erba Instruments. Thin layer chromatography (TLC)
was carried out on silica gel plates with a fluorescence
indicator F254 (0.2 mm, E. Merck); the spots were visu-
alized in UV light (254 nm). Column chromatography
was performed on silica using Kieselgel 60 (230–400
mesh, E. Merck). All solvents and reagents used in the
present study were of analytical grade.
3. Conclusions
We were able to prepare a new series of mesoionic 1,3,4-
thiadiazolium-2-phenylamine chlorides which are deriv-
atives or analogues of the natural amide piperine. The
results obtained in the synthesis and biological evalua-
tion of the new compounds highlight the potential use
of natural piperine as a precursor for new molecules
which may be employed in the treatment of Chagas’ dis-
ease. Compound MI showed the best activity profile.
4.1.1. General procedure for the preparation of derivatives
1,3,4-thiadiazolium-2-phenylamine chlorides (MI–MVII).
These compounds were synthesized by the coupling of
the corresponding acyl chlorides (1.37 mmol) which
were added to a stirred solution of 1,4-diphenylthiose-

W. da Silva Ferreira et al. / Bioorg. Med. Chem. 16 (2008) 2984–2991
2987
Entry A (unsaturated series):
COCl
n
COOH
O
O
O
O
a
b
n
X
X
(4a) n = 2; X = H
(8a) n = 1; X = H
(12a) n = 1; X = NO2
(4) n = 2; X = H
(8) n = 1; X = H
(12) n = 1; X = NO2
Cl
N
N
N
H
O
O
S
n
X
(MI) n = 2; X = H
(MII) n = 1; X = H
(MIII) n = 1; X = NO2
Entry B (saturated series):
X
O
COCl
Cl
O
N
a
n
COOH
n
b
n
N
O
S
X
O
X
O
(5a) n = 4; X = H
(6a) n = 4; X = NO2
(9a) n = 2; X = H
(10a) n = 2; X = NO2
N
O
(5) n = 4; X = H
(6) n = 4; X = NO2
(9) n = 2; X = H
(10) n = 2; X = NO2
H
(MIV) n = 4; X = H
(MV) n = 4; X = NO2
(MVI) n = 2; X = H
(MVII) n = 2; X = NO2
Entry C (from aromatic aldehydes):
Cl
N
N
N
CHO
X
O
O
O
O
S
c
H
X
(MVIII) X = H
(MIX) X = NO2
(7) X = H
(11) X = NO2
Scheme 3. Preparation of mesoionic hydrochlorides (MI–MIX). Reagents and conditions: (a) (COCl)2, 25 °C, 2 h (100%); (b) 1,4-diphenylthiose-
micarbazide, 1,4-dioxane, 25 °C, 24–48 h (34–83%); (c) 1,4-diphenylthiosemicarbazide, TMSCl, DMF, 25 °C, 24 h (40–68%).
1
micarbazide (1.37 mmol) in dry 1,4-dioxane (2 mL) at
room temperature.22,23 After 48 h standing, the products
were separated by vacuum filtration and washed with a
sequence of solvents (1,4-dioxane, toluene, and ethyl
ether, respectively). In this way, the mesoionic com-
pounds MI–MVII were prepared.
692; H NMR (DMSO-d6, 400 MHz) d 12.39 (s, NH),
7.78 (m, H200), 7.73 (m, H300 and 400), 7.54 (m, Hb),
7.46 (s, H20), 7.38 (t, H30, J = 7.5 Hz), 7.22 (s, H2000),
7.18 (m, Hc and d), 7.09 (t, H40, J = 7.3 Hz), 7.04 (d,
H6000, J = 8.0 Hz), 6.94 (d, H5000, J = 8.0 Hz), 6.52 (d,
Ha, J = 14.8 Hz) 6.06 (s, OCH2O); 13C NMR
(DMSO-d6, 400 MHz) 159.80 (C2), 149,01 (C3000 and
C4000), 138,00 (Cd), 132.52 (C400), 131.50 (C1000), 131.20
(C30), 130.61 (C300), 127.33 (C6000 and 200), 125.61 (Cc),
124.70 (C40), 120.30 (C20), 114.52 (Ca), 109.91 (C5000),
107.01 (C2000), 102.83 (OCH2O); MS (70 eV, m/z) (%)
426 (5), 284 (15), 177 (100), 161 (65), 149 (31), 127
(22), 77 (10). Anal. Calcd for C25H20N3SO2Cl: C,
65.00; H, 4.36; N, 9.10. Found: C, 65.08; H, 4.27; N,
9.16.
In order to facilitate the assignment of NMR data, we
have opted for numbering the structures according to
the example given below (Fig. 2).
4.1.1.1. 4-Phenyl-5-[4-(3,4-methylenedioxyphenyl)-1(E)-
3(E)-butadienyl]-1,3,4-thiadiazolium-2-phenylamine chlo-
ride (MI). Yield 57%; mp 155 °C (decomposed); IR
(KBr, cmꢁ1) 3449, 2926, 1600, 1565, 1361, 1256, 758,

2988
W. da Silva Ferreira et al. / Bioorg. Med. Chem. 16 (2008) 2984–2991
Table 1. In vitro activities of mesoionic compounds MI–MIX against three evolutive forms of Trypanosoma cruzi and its cytotoxicity on murine
macrophages
Compound
IC50 (lM)
LD50 (lM)
Epimastigotes
Trypomastigotes
Amastigotes
Cytotoxicitya
b
Piperine
MI
MII
7.31 1.5
10.83 2.2
4.13 1.2
>[ ]max
4.91 1.1
1.35 0.95
NTc
20.01 3.35
38.56 4.6
1.95 0.5
1.08 0.23
NT
6.70 1.7
>[ ]max
>[ ]max
NT
MIII
MIV
0.64 0.14
31.50 2.0
45.24 5.5
83.42 6.6
40.84 4.1
NT
NT
MV
MVI
NT
NT
NT
NT
NT
>[ ]max
NT
NT
MVII
MVIII
MIX
Benznidazoled
NT
NT
NT
NT
113.06
10.09
13.42 3.0
2.21 0.85
>[ ]max
6.61 2.4
NT
2.51 0.7
6.62 2.3
—
a Against murine macrophages.
b IC50 values highest than the maximum concentration allowed.
c Not tested.
d Reference drug.
and 4000), 143.87 (Cb), 138.77 (C100), 137.77 (C10),
132.72 (C400), 131.10 (C30), 130.44 (C300), 126.90 (C200),
125.46 (C40), 119.97 (C20), 115.49 (Ca), 108.57 (C2000),
106.52 (C5000), 105.11 (OCH2O); MS (70 eV, m/z) (%)
444 (11), 368 (10), 269 (77), 208 (25), 135 (95), 118
(35), 91 (65), 77 (100). Anal. Calcd for C23H17N4SO4Cl:
C, 57.44; H, 3.56; N, 11.65. Found: C, 57.49; H, 3.63; N,
11.58.
3´´
4´´
2´´
Cl
4´
3´
3
1´´
N
N
4
δ
β
2
2´´´
5´´´
1´
3´´´
4´´´
N
H
2´
O
O
S
1
5
1´´´
6´´´
γ
α
MI
Figure 2. Chemical structure of the mesoionic salt MI. The skeleton
numbering is only for NMR assignments.
4.1.1.4. 4-Phenyl-5-[4-(3,4-methylenedioxyphenyl)-
butyl]-1,3,4-thiadiazolium-2-phenylamine chloride (MIV).
Yield 51%; mp 234–236 °C; IR (KBr, cmꢁ1) 3443, 3026,
2924, 1600, 1567, 1320, 1247, 759, 693; 1H NMR
(CDCl3, 200 MHz) d 12.61 (s, NH), 7.78 (m, H200),
7.64 (m, H300 and 400), 7.52 (d, H20, J = 7.8 Hz), 7.14 (t,
H30, J = 7.8 Hz), 6.96 (t, H40, J = 7.4 Hz), 6.67 (d,
H5000, J = 7.8 Hz), 6.50 (dd, H6000, J = 6.0 and 1.6 Hz),
6.45 (d, H2000, J = 1.6 Hz), 5.90 (s, OCH2O), 3.19 (t,
Hd, J = 7.2 Hz), 2.39 (t, Ha, J = 7.2 Hz), 1.72 (m, Hb),
1.56 (m, Hc); 13C NMR (CDCl3, 200 MHz) d 169.70
(C5), 159.71 (C2), 147.41 (C3000), 145.50 (C4000), 138.32
(C100), 136.90 (C10), 131.40 (C400), 129.81 (C30), 128.60
(C300), 125.51 (C40), 123.42 (C200), 120.81 (C6000), 118.50
(C20), 108.40 (C5000), 107.90 (C2000), 100.61 (OCH2O),
34.41 (Ca), 30.63 (Cd), 28.51 (Cb), 24.80 (Cc); MS
(70 eV, m/z) (%) 429 (35), 280 (100), 135 (23), 77 (15).
Anal. Calcd for C25H24N3SO2Cl: C, 69.74; H, 5.62; N,
9.76. Found: C, 69.68; H, 5.59; N, 9.81.
4.1.1.2. 4-Phenyl-5-[2-(3,4-methylenedioxyphenyl)-(E)-
ethenyl]-1,3,4-thiadiazolium-2-phenylamine chloride (MII).
Yield 34%; mp 294–296 °C; IR (KBr, cmꢁ1) 3425, 2900,
2674, 1600, 1567, 1362, 1259, 754, 690; 1H NMR
(DMSO-d6, 400 MHz) d 12.24 (s, NH), 7.87 (d, Hb,
J = 15.5 Hz), 7.81 (m, H200) 7.74 (s, H300 and 400), 7.56
(d, H20, J = 7,6 Hz), 7.41 (t, H30, J = 7.5 Hz), 7.41 (s,
H2000), 7.32 (d, H6000, J = 8.0 Hz), 7.14 (t, H40,
J = 7.1 Hz), 7.01 (d, H5000, J = 7.8 Hz), 6.95 (d, Ha,
J = 15.9 Hz), 6.10 (s, OCH2O); 13C NMR (DMSO-d6,
400 MHz) d 166.03 (C5), 161.71 (C2), 152.98 (Cb),
149.53 (C4000 and C3000), 132.71 (C400), 131.35 (C200),
130.68 (C300), 128.13 (C5000), 127.31 (C30), 125.28 (C40),
119.98 (C20), 110.58 (Ca), 110.06 (C2000), 103.31
(OCH2O); MS (70 eV, m/z) (%) 399 (100), 283 (45),
250 (20), 191 (65), 161 (46), 118 (32), 77 (25). Anal.
Calcd for C23H18N3SO2Cl: C, 63.37; H, 4.16; N, 9.64.
Found: C, 63.25; H, 4.27; N, 9.72.
4.1.1.5. 4-Phenyl-5-[4-(6-nitro-3,4-methylenedioxy-
phenyl)-butyl]-1,3,4-thiadiazolium-2-phenylamine chloride
(MV). Yield 50%; mp 226–228 °C; IR (KBr, cmꢁ1
)
4.1.1.3. 4-Phenyl-5-[2-(6-nitro-3,4-methylenedioxyphe-
nyl)-(E)-ethenyl]-1,3,4-thiadiazolium-2-phenylamine chlo-
3444, 3052, 2923, 2774, 1609, 1567, 1380, 1330, 1252,
758, 694; 1H NMR (CDCl3, 600 MHz) d 12.69 (s,
NH), 7.71 (m, H200), 7.66 (m, H300 and 400), 7.59 (d,
H20, J = 7.9 Hz), 7.21 (t, H30, J = 7.6 Hz), 7.01 (t, H40,
J = 7.2 Hz), 6.64 (s, H2000), 5.90 (s, OCH2O), 3.19 (t,
Hd, J = 7.6 Hz), 2,74 (t, Ha, J = 7.6 Hz), 1.85 (qui,
Hb, J = 7.6 Hz), 1.64 (qui, Hc, J = 7.5 Hz); 13C NMR
(DMSO-d6, 200 MHz) d 170.30 (C5), 160.31 (C2),
151.70 (C6000), 146.13 (C4000), 000142.20 (C3000), 138.65
(C100), 137.04 (C10), 133.25 (C1 ), 131.63 (C400), 130.07
(C30), 129.31 (C300), 125.70 (C200), 123.70 (C40), 118.10
ride (MIII). Yield 37%; mp 274–276 °C; IR (KBr, cmꢁ1
3426, 3052, 2922, 2736, 1615, 1565, 1385, 1330, 1260,
)
1
757, 692; H NMR (DMSO-d6, 400 MHz) d 12.69 (s,
NH), 8.09 (d, Hb, J = 15.8 Hz), 7.83 (m, H200), 7.74
(m, H300 and 400), 7.71 (s, H5000), 7.59 (d, H20,
J = 7.5 Hz), 7.43 (t, H30, J = 7.9 Hz), 7.43 (s, H2000),
7.18 (t, H40, J = 7.4 Hz), 7.07 (d, Ha, J = 15.6 Hz),
6.27 (s, OCH2O); 13C NMR (DMSO-d6, 400 MHz) d
168.52 (C5), 162.20 (C2), 150.87 (C6000), 149.13 (C3000

W. da Silva Ferreira et al. / Bioorg. Med. Chem. 16 (2008) 2984–2991
2989
(C20), 110.30 (C5000), 104.92 (C2000), 103.10 (OCH2O),
31.73 (Cd), 29.21 (Cc), 28.43 (Cb), 28.09 (Ca); MS
(70 eV, m/z) (%) 474 (15), 228 (12), 280 (75), 135 (100),
118 (64), 91 (48), 77 (85). Anal. Calcd for
C25H23N4SO4Cl: C, 58.76; H, 4.54; N, 10.96. Found:
C, 58.83; H, 4.78; N, 11.05.
H6000, J = 8.1 and 1.7 Hz), 7.06 (d, H5000, J = 8.2 Hz),
6.94 (d, H2000, J = 1.6 Hz), 6.13 (s, OCH2O); 13C NMR
(CD3OD, 400 MHz) d 165.13 (C5), 161.47 (C2), 152.72
(C3000), 149.05 (C4000), 139.68 (C100), 139.04 (C10), 132.60
(C400), 131.18 (C300), 130.70 (C300), 127.31 (C6000), 127.27
(C200), 125.30 (C4 ), 119.70 (C20), 117.28 (C1000), 110.50
(C2000), 110.38 (C5000), 103.91 (OCH2O); MS (70 eV, m/
z) 373 (82), 257 (24), 224 (27), 165 (100), 118 (18), 91
(10), 77 (14). Anal. Calcd for C21H16N3SO2Cl: C,
61.54; H, 3.93; N, 10.25. Found: C, 61.47; H, 4.02; N,
10.37.
4.1.1.6. 4-Phenyl-5-[2-(3,4-methylenedioxyphenyl)-
ethyl]-1,3,4-thiadiazolium-2-phenylamine chloride (MVI).
Yield 83%; mp 272–274 °C; IR (KBr, cmꢁ1) 3423, 3023,
1
2897, 2769, 1600, 1570, 1321, 1242, 752, 690; H NMR
(CDCl3, 400 MHz) d 12.26 (s, NH), 7.70 (m, H200, 300
and 400), 7.56 (d, H20, J = 8.5 Hz), 7.36 (t, H30,
J = 7.9 Hz), 7.09 (t, H40, J = 7.3 Hz), 6.78 (d, H5000,
J = 8.0 Hz), 6.75 (d, H2000, J = 1.5 Hz), 6.5 (dd, H6000,
J = 7.9 and 1.7 Hz), 5.98 (s, OCH2O), 3.38 (t, Ha,
J = 7.3 Hz), 2.94 (m, Hb); 13C NMR (DMSO-d6,
400 MHz) d 149.70 (C3000), 148.60 (C4000), 139.72 (C100),
138.41 (C10), 133.10 (C400), 132.52 (C1000), 131.34 (C30),
130.53 (C300), 126.61 (C200), 126.03 (C40), 123.02 (C6000),
120.20 (C20), 109.91 (C5000), 109.53 (C2000), 102.50
(OCH2O), 34.91 (Ca), 34.91 (Cb); MS (70 eV, m/z) (%)
401 (100), 280 (15), 211 (93), 135 (50), 118 (20), 91
(10), 77 (23). Anal. Calcd for C23H20N3SO2Cl: C,
63.08; H, 4.60; N, 9.59. Found: C, 63.15; H, 4.48; N, 9.64.
4.1.2.2. 4-Phenyl-5-(6-nitro-3,4-methylenedioxyphenyl)-
1,3,4-thiadiazolium-2-phenylamine chloride (MIX). Yield
40%; mp 264–266 °C; IR (KBr, cmꢁ1) 3437, 3010, 2777,
1
1610, 1566, 1329, 1267, 754, 690; H NMR (DMSO-d6,
200 MHz) d 12.58 (s, NH), 7.92 (s, H5000), 7.63 (m, H200),
7.57 (m, H300 and 400), 7.55 (m, H20), 7.45 (t, H30,
J = 7.5 Hz), 7.45 (sl, H2000), 7.18 (t, H40, J = 7.4 Hz),
6.36 (s, OCH2O); 13C NMR (DMSO-d6, 400 MHz) d
162.7 (C5), 152.4 (C2), 151.2 (C6000), 146.4 (C4000), 142.3
(C3000), 138.4 (C100), 137.0 (C10), 134.3 (C1000), 131.7
(C400), 130.0 (C30), 129.5 (C300), 125.1 (C200), 124.2
(C40), 118.6 (C20), 110.8 (C5000), 106.0 (C2000), 105.1
(OCH2O); MS (70 eV, m/z) (%) 418 (20), 298 (52), 206
(28), 178 (64), 135 (75), 91 (18), 77 (100). Anal. Calcd
for C21H15N4SO4Cl: C, 55.45; H, 3.32; N, 12.32. Found:
C, 55.57; H, 3.28; N, 12.57.
4.1.1.7. 4-Phenyl-5-[2-(6-nitro-3,4-methylenedioxy-
phenyl)-ethyl]-1,3,4-thiadiazolium-2-phenylamine chloride
(MVII). Yield 42%; mp 248–250 °C; IR (KBr, cmꢁ1
3426, 3052, 2932, 2732, 1615, 1565, 1385, 1330, 757,
)
4.2. Biological assays
1
692; H NMR (DMSO-d6, 200 MHz) d 12.34 (s, NH),
7.70 (m, H200, 300 and 400), 7.57 (m, H20), 7.52 (s, H5000),
7.40 (t, H30, J = 7.8 Hz), 7.09 (m, H40), 7.09 (s, H2000),
6.21 (s, OCH2O), 3.47 (m, Ha), 3.15 (m, Hb); 13C
NMR (DMSO-d6, 200 MHz) d 170.32 (C5), 160.31
(C2), 151.72 (C6000), 146.12 (C4000), 142.24 (C3000), 138.60
(C100), 137.03 (C10), 133.21 (C1000), 131.61 (C400), 130.01
(C30), 129.33 (C300), 125.71 (C200), 123.70 (C40), 118.12
(C20), 110.31 (C5000), 104.91 (C2000), 103.10 (OCH2O),
28.40 (Cb), 28.02 (Ca), MS (70 eV, m/z) (%) 446 (5),
282 (12), 269 (100), 208 (28), 177 (40), 136 (32), 91
(43), 77 (46). Anal. Calcd for C23H19N4SO4Cl: C,
57.20; H, 3.97; N, 11.60. Found: C, 57.39; H, 3.82; N,
11.42.
4.2.1. Parasites. Trypanosoma cruzi (Y strain) was ob-
tained from the Fundac¸a˜o Oswaldo Cruz (Rio de Ja-
neiro, Brazil) culture collection. The epimastigote
forms were cultured in brain heart infusion (BHI) sup-
plemented with 10 mg of hemin and 20 mg of folic acid
literꢁ1 and 5% heat-inactivated fetal calf serum (FCS)
(BHI-FCS medium) at 28 °C.31 Tissue culture-derived
trypomastigotes were obtained after infection of conflu-
ent monolayers of Vero cells with blood trypomastigotes
(Y strain) to establish the intracellular cycle and main-
tained in RPMI 1640 medium containing 10% FCS un-
der an atmosphere of 5% CO2 at 37 °C.32 These tissue
culture-derived trypomastigotes were used to infect mur-
ine peritoneal macrophages in vitro to evaluate the toxic
effect of the drugs.
4.1.2. General procedure for the preparation of derivatives
1,3,4-thiadiazolium-2-phenylamine chlorides (MVIII) and
(MIX). To a solution of 1,4-diphenylthiosemicarbazide
(0.25 mmol) in dry DMF (1 mL) was added TMS-Cl
(0.8 mmol), followed by aldehydes (0.62 mmol). The
mixture was stirred for 24 h at room temperature,28
the products were separated by vacuum filtration and
washed with a sequence of solvents (1,4-dioxane, tolu-
ene, and ether ethylic, respectively). In this way, the fol-
lowing compounds (MVIII–MIX) were prepared.
4.2.2. Anti-epimastigote effect. The toxic effect of the
drugs on epimastigotes was evaluated as previously de-
scribed.9 The drugs were stored as 1.0 mg mLꢁ1 stock
solution in DMSO (Sigma) and were used in serial dilu-
tion (1:2) in BHI-FCS medium before use. Drug-free
control medium contained comparable final concentra-
tion of DMSO (0.05%). Epimastigotes (1.105 cells) were
incubated in BHI-FCS medium with or without drugs in
a final volume of 1.0 mL in 24-well plates (TPP). After 7
days of treatment, the toxic effect of the drugs was quan-
tified by the direct count of the live epimastigotes in a
Neubauer chamber.
4.1.2.1. 4-Phenyl-5-(3,4-methylenedioxyphenyl)-1,3,4-
thiadiazolium-2-phenylamine chloride (MVIII). Yield
68%; mp 286–287 °C; IR (KBr, cmꢁ1) 3455, 3054,
1
2993, 2645, 1600, 1565, 1314, 1245, 761, 693; H NMR
(CD3OD, 400 MHz) d 12.37 (s, NH), 7.70 (m, H200),
7.62 (m, H300 and 400), 7.59 (d, H20, J = 7.8 Hz), 7.43 (t,
H30, J = 7.7 Hz), 7.15 (t, H40, J= 7.6 Hz), 7.11 (dd,
4.2.3. Cytotoxicity to macrophages. The evaluation of
the toxic effects of the compounds was carried out as
previously described.33 Murine peritoneal macrophages

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W. da Silva Ferreira et al. / Bioorg. Med. Chem. 16 (2008) 2984–2991
were seeded (1.106 cells/well) in 24-well plates (TPP)
with 1 mL of RPMI medium containing 10% FCS.
The cells were allowed to attach for 24 h at 37 °C
and then exposed to the active compounds dissolved
in DMSO against epimastigotes (maximum final con-
centration of solvent was 0.05%) for 72 h. Afterwards,
the cells were washed with PBS, and RPMI was added
to the culture before the addition of vital dye trypan
blue in a final concentration of 0.01%. The toxic effect
of the drugs was monitored by the count of 200 cells in
a Neubauer chamber where plasma membrane perme-
ability was evaluated.
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(American Trypanosomiasis) [http://www.paho.org/english/
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4.2.4. Anti-trypomastigote activity. The anti-trypomasti-
gote activity of the drugs was monitored at concentra-
tions which were found non-toxic to macrophages.
The parasites were harvested from infected cultures as
described above,33,34 and the motility of the trypom-
astigotes was quantified 24 h after the treatment, by
the direct count of the live trypomastigotes in a Neu-
bauer chamber.
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4.2.5. Anti-amastigote effect. Resident peritoneal cells
from non-infected BALB/c mice were harvested and cul-
tured as previously described.34 Adhered macrophages
were infected with T. cruzi metacyclic trypomastigotes
at a 10:1 parasite/macrophage ratio and incubated at
37 °C under 5% CO2 for 1 h. Subsequently, non-infec-
tive cells were removed by extensive washing with
PBS, and the infected macrophage cultures were treated
with increasing concentrations (0.5, 1.0, 2.5, 5.0, and
10 lg/mL) of MI per 72 h. After that, monolayers were
washed with PBS at 37 °C, fixed in methanol, and
stained with Giemsa. The amastigote survival was deter-
mined by the counting of 200 cells in triplicate, where
the percentage of infected cells was analyzed, as well
as the number of amastigotes per macrophage and the
endocytic index of these infected cells.34
4.2.6. Statistical analysis. The 50% inhibitory concentra-
tions (IC50) values shown in the Table 1 represent the
mean of experiments carried out in triplicate. The IC50
of all compounds were determined by linear regression
analysis using the program IGOR Pro 2.03 (Lake Oswe-
go, Oregon - USA).
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Stuttgart 2000, 11, 1565.
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Acknowledgments
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Authors acknowledge financial supports from FAPERJ,
CNPq, and CAPES (PROCAD). Fellowships from
CNPq to Welisson da Silva Ferreira and V.B. Saraiva
and from Capes to L. Freire-de-Lima are gratefully
acknowledged. Authors are grateful to the National
Center for Nuclear Magnetic Resonance of Macromol-
ecules (UFRJ) for acquisition of NMR spectra at 400
and 600 MHz.
28. Kappel, J. C.; Yokum, T. S.; Barany, G. J. Comb. Chem.
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