New potent imidazoisoquinolinone derivatives as anti-Trypanosoma cruzi agents: Biological evaluation and structure-activity relationships

Article abstract of DOI:10.1016/j.bmc.2009.01.011

A series of novel benzoimidazo and N-aryl-5-oxo-imidazo[1,2-b]isoquinoline-10-carbothioamides was developed. All the compounds were evaluated for their in vitro action against the epimastigote form of Trypanosoma cruzi. Four of them showed higher activity than Nifurtimox. Their unspecific cytotoxicity was evaluated using HeLa and L6 cells, being non-toxic at concentrations at least 15 and 200 times higher than that of T. cruzi IC_50. To gain insight into the mechanism of action, their DNA binding properties and reactivity with glutathione were studied, and QSAR study was performed.

Full text of DOI:10.1016/j.bmc.2009.01.011

Bioorganic & Medicinal Chemistry 17 (2009) 1437–1444
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New potent imidazoisoquinolinone derivatives as anti-Trypanosoma cruzi
agents: Biological evaluation and structure–activity relationships
Mariela Bollini a, Juan José Casal a, Diego E. Alvarez a, Lucía Boiani b, Mercedes González b,
Hugo Cerecetto b, Ana María Bruno a,
*
a Departamento de Química Orgánica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina
b Departamento de Química Orgánica, Facultad de Ciencias y Facultad de Química, Universidad de la República, Montevideo, Uruguay
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel benzoimidazo and N-aryl-5-oxo-imidazo[1,2-b]isoquinoline-10-carbothioamides was
developed. All the compounds were evaluated for their in vitro action against the epimastigote form of
Trypanosoma cruzi. Four of them showed higher activity than Nifurtimox. Their unspecific cytotoxicity
was evaluated using HeLa and L6 cells, being non-toxic at concentrations at least 15 and 200 times higher
than that of T. cruzi IC50. To gain insight into the mechanism of action, their DNA binding properties and
reactivity with glutathione were studied, and QSAR study was performed.
Received 25 November 2008
Revised 9 January 2009
Accepted 10 January 2009
Available online 15 January 2009
Keywords:
Imidazoisoquinolinones
QSAR
Ó 2009 Elsevier Ltd. All rights reserved.
anti-Trypanosoma cruzi agents
1. Introduction
In this regard, agents based on different strategies, such as inhi-
bition of specific parasite enzymes, actions on parasite DNA and
Chagas’ disease or American Trypanosomiasis is a parasitic dis-
ease that constitutes an important health problem in Central and
South America and affects around 20 million people living in
poorly constructed homes in rural areas. It is estimated that
50,000 infected people die each year.1 The causative agent of this
disease is a haemoflagellate protozoan, Trypanosoma cruzi (T. cruzi),
which enters the human body through damaged skin.
Despite significant progress in the understanding of the bio-
chemistry and physiology of its etiological agent, current specific
treatments are usually effective only in the acute stage. Once the
disease has progressed to the later stages, there is no effective cure
and severe side effects arise in association with long-term
treatments.
There are two drugs available for the etiological treatment of
Chagas’ disease: Nifurtimox (Nfx) and benznidazole (Bz). Nfx is a
nitrofurane and Bz is a nitroimidazole compound.2 The use of these
drugsto treattheacutephaseofthediseaseis widelyaccepted. How-
ever, their use in the treatment of the chronic phase is controversial.
The undesirable side effects of both drugs are a major drawback in
their use, frequently forcing the physician to stop the treatment.
The development of safer and more efficient therapeutic
anti-Trypanosoma cruzi compounds continues to be a major goal
in trypanocidal chemotherapy.
oxidative stress damage, have been described.3 A variety of lead
compounds, including acridines, phenothiazines, benzoazepines
and pyridoquinolines show trypanocidal activities.4
Isoquinolines generally constitute an important branch of het-
erocyclic compounds and are currently used against parasitic infec-
tions.5 Imidazoisoquinolinones are valuable substrates for the
synthesis of potentially biologically active compounds with struc-
tural features different from those of the existing drugs. Recently,
we have described the synthesis of a series of imidazoisoquinolinon-
es C-10 and N-1 substituted,6 which possess a planar structure with
an heterocyclic scaffold present in antiprotozoal agents.
In this work, we describe the synthesis and in vitro anti-T. cruzi
proliferative activity of a series of imidazoisoquinolinones aryl-
thioamides. A structure-activity relationship analysis is also dis-
cussed in order to identify the structural requirements for
optimum activity.
In an attempt to gain better insight into the trypanocidal mech-
anism of action of these compounds, DNA affinity and redox
metabolism were evaluated.
2. Methods and results
2.1. Synthesis
Derivatives 2–9 were prepared by a reaction between arylisoth-
iocyanates and 2,3-dihydroimidazo[1,2-b]isoquinoline-5 (1H)-one
* Corresponding author. Tel.: +54 1149648251.
E-mail address: anabruno@ffyb.uba.ar (A.M. Bruno).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.011

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M. Bollini et al. / Bioorg. Med. Chem. 17 (2009) 1437–1444
1.6 The arylisothiocyanates were prepared according to the proce-
dures described in the literature in two steps7 with minor modifi-
cations. Initially, we obtained the different 1,3-diarylthioureas
from the corresponding anilines substituted and carbon disulfide
and assayed different basic catalysts (potassium hydroxide, pyri-
dine, 4-(dimethylamino) pyridine (DMAP)) in order to diminish
the time of the reaction. DMAP was effective in 18 h as compared
to other catalysts, which were effective in 2 days.8 In the second
step, by reaction of 1,3-diarylthioureas with sulfuric acid and ace-
tic anhydride, we obtained the corresponding isothiocyanates9
(Scheme 1) by isolating them by steam distillation. All the precur-
sor compounds were identified by spectroscopy methods and are
in agreement with those described in the literature.
after varying time and power irradiation, products of decomposition
were obtained. Then, the synthesis was carried out using equimolar
concentrations of reagentsand drops of DMFas solvent. Thisallowed
a greater heating in the reaction medium and thus, the possibility to
obtainthe product in much reduced reaction times, in a cleaner reac-
tion and with excellent yields.
Finally, coupling of compound 12 with phenylisothiocyanate
with DMAP as catalyst afforded the C-10 substituted final com-
pound 13 (Scheme 2).
All the products were fully characterized by 1H and 13C NMR,
MS and IR (see Section 5).
2.2. Biological characterization
Therefore, coupling of compound 1 with isothiocyanates affor-
ded the C-10 substituted final compounds 2–9 (Scheme 1). Reac-
tion time varied from 6 to 72 h depending on the substitution of
the isothiocyanates used and the yields were poor. Thus, to obtain
better yields, we used DMAP in concentrations equimolar. In the
case of 3-trifluormethylphenylisothiocyanate no reaction was
observed.
2.2.1. Antitrypanosomal activities
The existence of the epimastigote form of T. cruzi as an obligate
intracellular stage in mammals has been revised and confirmed re-
cently.10 Therefore, compounds were tested in vitro against the
epimastigote form of the parasite.11 As a first screening, the ability
of developed derivatives to inhibit the growth of the epimastigote
Compound 6 was treated with hydrogen peroxide at 0–5 °C to
obtain compound 10 to compare its biological activity to that of
sulfur compounds. Furthermore, we synthesized derivative 11 by
acylation with acetyl chloride and DMAP as catalyst to determine
whether the substitution in N-1 was necessary to show activity.
Compound 12 was synthesized in order to introduce a new aro-
matic moiety to enlarge compound 1 scaffold. For this, we used an
efficient and environmentally friendly synthetic method for the syn-
thesis under microwave irradiation, in whichhomophthalic acid and
2-aminoaniline directly reacted on a silica gel support under sol-
vent-free conditions. Experiments were carried out using a domestic
microwave oven modified with a reflux condenser. Nevertheless,
form of T. cruzi (Tulahuen 2 strain) was evaluated at 25 lM. Be-
sides, the IC50 concentrations (50% inhibitory dose) were deter-
mined for the derivatives 1–13 (Table 1). Parasites were grown
in the presence of the compound for 5 days, and the percentage
of growth inhibition was determined against control (no drug
added to the medium). Nifurtimox (Nfx) was used as the reference
trypanocidal drug, as explained in Section 5. Imidazoisoquinoli-
nones 2, 3, 6, 7 and 13 showed either good or excellent activity
against the epimastigote form. Compounds 2 and 3 exhibited
remarkable trypanocidal activity and were 15- and 8-fold more po-
tent than Nfx, respectively. Differently, compounds 1 and 12 did
not show antichagasic activity.
S
(AcO)2O
Ethanol
R
NH2
+
R
NCS
CS2
R
NHCNH
R
H2S
+
Reflux, 1h
Reflux, 18h
DMAP
O
R: 4-ClPh; 4-CH3Ph; 1-Naph; 3-CH3Ph; 3-CF3Ph; Ph; 4-OCH3Ph; 2-Py; 4-ClBn
N
O
N
H
H2O2 30%
HN
KOH 2%
0º
O
10
O
DMAP
N
N
S
NCS
R
+
O
N
H
N
H
CH2Cl2
Reflux
1
N
HN
R
N
CH3COCl
O
DMAP/CH2Cl2
0-5 °C
HN
S
R: 4-ClPh
4-CH3Ph
1-Naph
3-CH3Ph
Ph
2
3
4
5
6
7
8
9
11
4-OCH3Ph
2-Py
4-ClBn
Scheme 1. (a) Synthesis of isothiocyanates and (b) synthesis of compounds 2–11.

M. Bollini et al. / Bioorg. Med. Chem. 17 (2009) 1437–1444
1439
O
O
COOH
COOH
NH2
NH2
MW
N
N
PhNC S
DMAP
N
H
N
H
DMF
12
HN
S
13
Scheme 2. Synthesis of compounds 12 and 13.
Table 1
Table 3
In vitro activity of imidazo and benzoimidazoisoquinolinones derivatives and Nfx on
Antiprotozoal activity (Onchocerca gutturosa) of compound 1
T. cruzi
Compound
Mot Redna (%)
MTT Rednb (%)
Activity score
Compound
IC50 epimastigotea
(lM)
IC50 HeLa (
l
M)
SIb
Amocarzine (positive control)
1
85.71
14.29
48.57
0.00
2
1
1
2
3
4
5
6
7
8
>25
0.5
1
25
20
3
12.3
30
35
30
43
>25
6.4
7.7
>100
>90
>100
Ntc
>52.5
>100
22
>100
>90
>100
Nt
>100
98
—
>180
>100
—
a
Motility score (mean % reduction at 120 h).
MTT colorimetry (mean inhibition of formazan formation).
b
—
>33
1.7
—
—
—
—
—
15
1.5
tured human cervix adenocarcinoma (HeLa) cells. Nfx was
included as trypanocidal reference. We found that none of the
compounds evaluated was cytotoxic at any of the concentrations
assayed, and that derivative 7 was slightly toxic (Table 1).
9
10
11
12
13
Nfx
2.3. Study of anti-T. cruzi mechanism of action
12
a
IC50: concentration that produces 50% inhibitory effect.
2.3.1. DNA binding properties
b
c
SI: selectivity index = % IC50Hela/% IC50epimastigote
Not tested.
.
To gain insight into the trypanocidal mechanism of action of
these compounds, DNA affinity was evaluated.12
All the final products were tested for their ability to bind DNA.
The binding capacity of these compounds was evaluated by mea-
suring the hypochromic and bathochromic effects of their absor-
bance in the UV spectra.13 The standard experiment was
enhanced by means of a slow rotation of DNA-drug mixture stir-
ring, in a 5:1 ratio for 24 h. The procedure was validated by repeat-
ing assays with well-known intercalating agents (m-AMSA and
mitoxantrone) and a compound which binds closely in the minor
groove (bis-benzimide, Hoechst No. 33258). The degree of interac-
tion was expressed by the ratio between the final absorbance area
after 24 h (a24) and that of the compound at the same concentra-
tion (a0), centred at maximal absorbance. Values of 1 indicated a
total lack of affinity and value of 0 showed that the whole com-
pound was bound to DNA. The a24/a0 coefficient values of com-
pounds 1–13 are between 0.80 and 0.95. The DNA binding assay
showed that the compounds tested are poor DNA ligands with
low affinity, thus suggesting that DNA interaction is probably not
the mechanism of action of the imidazoisoquinolinone derivatives.
2.2.2. Antiparasitic activity
Four of the synthesized products were selected for a primary
in vitro screening at Tropical Disease Research (TDR) Program,
World Health Organization (WHO, Switzerland). Compounds 1, 2,
10 and 13 were tested in vitro against Plasmodium falciparum,
Leishmania donovani, Leishmania infantum and Trypanosoma b. rho-
desiense, causative agents of malaria, leishmaniasis and sleeping
sickness, respectively, and their cytotoxicity was tested on rat L6
myoblasts. The activity data are given as lg/mL concentration that
produces 50% inhibition (IC50) in the assays used and are shown in
Table 2.
Compound 1 presented selective and good activity against T. b.
rhodesiense. The remaining compounds did not exhibit antipara-
sitic activity on the protozoa evaluated. In addition, compound 1
was evaluated against Onchocerca gutturosa, the causative agent
of onchocercasis, and found to be inactive (Table 3).
2.2.3. Unspecific cytotoxicity
2.3.2. Reactivity with glutathione
Cytotoxicity of the best trypanocidal compounds against mam-
In order to confirm the capacity of the new imidazoisoquinoli-
nones to react with thiols in the reductive metabolism of the par-
asite, Glutathione (GSH) was used as a model assay.14 The reaction
between compound 2 and GSH was followed spectroscopically in
the UV–visible region. The results are shown in Figure 1. Clearly,
it was confirmed that the thioamide derivative 2 reacted with this
biological thiol. This compound was able to react with GSH at 28 °C
and after 2 min of incubation showed hypochromic effect. After 2 h
of incubation, it exhibited bathochromic and hyperchromic effects.
Taking into account the UV-signal shifts, the GSH reacted mainly
via a redox pathway, producing Glutathione disulfide (GSSG) and
the corresponding reduced derivative from compound 2, 10-((4-
malian cells was evaluated in vitro at 4, 20, and 100 lM, using cul-
Table 2
Antiprotozoal activity of compounds 1, 2, 10 and 13 (IC50 values are given in
Compound P. falciparum L. infantum T. b rhodesiense Cytotoxicity L-6
Ref. drugs Chloroquina 0.026 Pentostam 2.4 Suramin 0.13
1
2
l
g/mL)a
Tamoxifen 4.9
>100
>10
>20.7
>22.7
>19.5
>19.5
1.20
>22.7
>19.5
>19.5
>22.7
>19.5
>22.7
>22.8
>19.6
>22.8
10
13
a
IC50 = 50% growth inhibition.

1440
M. Bollini et al. / Bioorg. Med. Chem. 17 (2009) 1437–1444
Figure 1. Studies of the reaction between compound 2 and GSH using UV–vis spectroscopy.
Chlorophenylamino)methyl)-2,3-dihydroimidazo[1,2-b]isoquino-
lin-5(1H)-one (2a).
tory effect (compounds 1–13) on the growth of T. cruzi (Tulahuen
strains) expressed as 50% growth inhibition on day 5 (% IC50). We
used log10 (IC50) values as the dependent variables in the lineariza-
tion procedure. First, one-variable and multivariable regressions
between these activities and the physicochemical properties (cal-
culated descriptors) were studied. Only structure–activity models
having a value of r2 adj above 0.5 were considered. The models
are shown in Table 4 and indicate that Eq. 2 is the best one-variable
relationship obtained where activity is correlated with heat forma-
tion (HF).17 This result could be modestly improved by including a
lipophilic descriptor in the analysis (Eq. 3), however, this showed
that the clogP term did not lead to significant increase in the r2 cor-
relation coefficient. The best equation was obtained when we ana-
lysed the correlation between activity, the independent heat
2.4. Structure–activity relationship
Molecular modelling studies were performed on the developed
imidazoisoquinolinones derivatives by calculating the stereoelec-
tronic properties in order to understand the mechanism of action.
These properties were determined using DFT/B3LYP calcula-
tions.15,16 A detailed conformational search for each of the mole-
cules was performed, using MM methods, to find the minimum
energy and highest abundance conformer. The geometry of this
*
conformer was fully optimized by applying B3LYP/6-31G , and
density functional calculation was performed in the gas phase.
The properties determined and examined in this study were vol-
ume, total energy, solvation (water), heat formation energy, mag-
nitude of dipolar moment, HOMO’s and LUMO’s energies, gap
(ELUMO–EHOMO) and the logarithm of the partition coefficient of
the non-ionized molecules (logP). Theoretical logP (clogP) was cal-
formation (HF) the magnitude of dipolar moment
l (Table 4, Eq.
4). Besides, the correlation matrix for the physicochemical descrip-
tors used was performed and no cross-relations between the
descriptors used in each equation were obtained.18 These parame-
ters are therefore orthogonal, a fact that affords their use in the
multilinear regression procedure.19
´
culated using the Villar method, implemented in PC SPARTAN 02
*
package at the B3LYP/6-31G density functional. In the equations
and models, n represents the number of data points, r2 the correla-
tion coefficient, s the standard deviation of the regression equation,
the F value is related to the F-statistic analysis (Fischer test) and r2
adj defines the cross-validated correlation coefficient. The activity
used in the structure–activity relationship studies was the inhibi-
3. Discussion
New imidazoisoquinolinone derivatives were synthesized by a
simple methodology and in good yields. Compound 12 was synthe-
sized under microwave irradiation and was obtained in higher
Table 4
QSAR study
Eq
Statistic parameter
Expression
r2 adj
r2
s
p
F value
n
1
2
3
4
0.680
0.801
0.771
0.899
0.655
0.849
0.8627
0.921
0.4120
0.2873
0.2928
0.2212
0.002
79.72
44.98
22.00
11
11
11
11
Log(1/IC50) = 2.26 ( 0.32) À 0.38( 0.09)l
0.000001
0.0001
0.00001
Log(1/IC50) = 1.44( 0.11) À 0.19( 0.02)HF
Log(1/IC50) = 1.75 ( 0.38) À 0.17( 0.02)HF À 0.10 ( 0.12)logP
120.34
Log(1/IC50) = 1.82 ( 0.17) À 0.14( 0.02)HF À 0.41 ( 0.05)l
n, represents the number of data points; r2, the correlation coefficient; s, the standard deviation of the regression equation; F value is related to the F-statistic analysis (Fischer
test) and r2 adj defines the cross-validated correlation coefficient.

M. Bollini et al. / Bioorg. Med. Chem. 17 (2009) 1437–1444
1441
Table 5
Assessment of Lipinski’s rules and TPSA values for imidazoisoquinolinones derivatives
Compound
Lipinski rules
TPSAb,c
clogPa
No. of H-bond donorb
No. of H-bond acceptorsa
Mol. Wt.
No. of criteria met
Regla
2
3
6
7
<5
65
2
2
2
2
610
5
5
5
6
<500
At least 3
3.77
3.44
3.13
2.89
4.11
8.51
355.9
335.4
321.4
351.4
369.4
287.0
All
All
All
All
All
3
50.6
50.6
50.6
64.7
50.6
108.7
13
Nfx
2
0
4
8
Nfx: nifurtimox.
a
´
Theorical LogP (clogP) was calculated using Villar method, at AM1 semiempirical. method, implemented in Spartan02, 1.0.1 version, suite of programs.
http://www.molinspiration.com/cgi-bin/properties.
TPSA, topological polar surface area.
b
c
yield (85%) and a cleaner reaction than conventional heating. In
situation aggravates the precarious defence against oxidative
stress.20 Accordingly, in mammalian cells, the effects of derivative
2 could be precluded by the enzyme Glutathione reductase (GR)
maintaining GSH levels. On the contrary, in the parasitic cells, com-
pound 2 may decrease GSH levels irreversibly because TR is unable
to reduce GSSG effectively, although another possibility is that
GSSG might deplete T(SH)2, Tryparedoxin (Tpx), and/or TR as a re-
sult of chemical21 or biochemical pathways.22,23
addition, the reaction time was reduced from 4 h to 3 min.
The results indicate that the in vitro activity of the imidazoiso-
quinolinones studied against T. cruzi epimastigotes is superior, in
most cases, to Nfx, the drug used for treatment of this disease. In
particular, derivatives 2, 3, 6, 7 and 13 displayed low micromolar
IC50 values (Table 1), with an adequate selective index.
In order to establish the key structural scaffold for the activity
of these compounds, we selected compound 6 and carried out a
variation of the individual substituents in this lead structure. The
structure–activity relationship (SAR) studies indicated that func-
tionality at position 4 of the phenyl group was not essential for
good activity. However, the 4-chloro (2) and 4-methyl (3) group
yielded the most potent compounds in this series. Replacement
of the 4-methyl group with a 4-methoxy group resulted in a de-
crease in the inhibitory activity. Introduction of a methyl substitu-
ent at position 3 of the phenyl group (5) resulted in a significant
decrease in potency. Interestingly, acylation of N-1 in compound
11 completely abolished the activity. This result suggests that NH
should be free for binding through a hydrogen bond to a target
receptor.
On the other hand, if we consider the potential use of imidazo-
isoquinolinone derivatives as drugs, an adequate in vivo behaviour
according to Lipinski’s rules24 and their topological polar surface
area (TPSA) should be present.25 Lipinski has described the desired
ranges for certain properties thought to be important for drug bio-
availability and absorption. These are: clogP <5, number of hydro-
gen bond donor 65, number of hydrogen bond acceptor 65 and
molecular weight <500. A compound that fulfils at least three out
´
of the four criteria is said to adhere to Lipinskis ‘rule of 5’. Besides,
TPSA is correlated with various types of drug transport properties
including intestinal absorption, blood-brain barrier penetration,
and Caco-2 cell permeability. Table 5 lists such physicochemical
properties for the trypanocidal imidazoisoquinolinone derivatives.
All of the potent antiparasitic imidazoisoquinolinones presented
In addition, only the change of sulfur atom (6) by oxygen atom
(10) abolished the antichagasic activity.
´
herein are fully compatible with Lipinskis rule and possess better
Furthermore, the insertion of a methylene group (9) resulted in
a significant decrease in potency. No further improvement was ob-
served when the phenyl group was replaced by a pyridine or naph-
thyl group. This result shows that phenylthioamide moiety is
essential for antichagasic activity.
When we included a benzene ring at position 2, 3 in compound
6 the activity was decreased 2-fold; however, derivative 13 exhib-
ited better activity than Nifurtimox.
QSAR studies demonstrated the relevance of the dipolar mo-
ment module and the heat formation for trypanocidal activity on
the Tulahuen strain. The relationship between activity and dipolar
moment could indicate the electronic requirements of the deriva-
tives to have optimal interaction with target biomolecules. In this
study, it was observed that the activity of the compounds increases
as heat formation increases.
From the DNA binding study, it is possible to conclude that the
imidazoisoquinolinone derivatives are not an appropriate ligand
(in structure and function) for interaction with DNA. On the other
hand, the reactivity with the glutathione assay showed that com-
pound 2 can react with GSH in a redox process.
TPSA than Nifurtimox.
4. Conclusion
The new imidazoisoquinolinone derivatives developed in this
study are in vitro active against T. cruzi epimastigotes. In general,
the toxic effects of the parasite are not associated with mammal
cytotoxicity in HeLa cells. Derivative 2, with
phenylthioamide substituent, possesses the best activity (IC50
0.5 M). These results provide supporting evidence to stimulate
a 4-chloro-
l
further in vivo studies of these compounds in appropriate animal
models of Chagas’ disease.
5. Experimental
5.1. Chemistry
Melting points were determined in a capillary with an Electro-
thermal 9100 SERIES-Digital apparatus and are uncorrected. IR
spectra were recorded with a FT Perkin Elmer Spectrum One from
KBr discs. UV spectra were measured with a Jasco V-570 UV/vis/
NIR spectrophotometer. 1H NMR (300 MHz) spectra were obtained
with a Bruker spectrometer at room temperature with tetrameth-
ylsilane as internal standard. Chemical shifts (d) are reported in
ppm and coupling constants (J) in Hertz. Elemental analysis was
carried out in our laboratories with a Coleman Analyser.
It is well-known that GSH is one of the essential precursors of
Trypanothione (T(SH)2) synthesis in the parasite cells. In addition,
T. cruzi has much lower GSH levels than those of the mammalian
host. Therefore, in parasites, the depletion of GSH levels and conse-
quently of T(SH)2 levels, could be more dangerous than the GSH
decrease in mammals. While in mammals GSH synthesis can be
inhibited up to 80–90% without evidence of toxicity, in T. cruzi this

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M. Bollini et al. / Bioorg. Med. Chem. 17 (2009) 1437–1444
Microwave-assisted reaction carried out in a household MW
oven (BGH-QUICK chef 15240). The apparatus was modified for
laboratory application with an external reflux condenser.
5.1.7. N-(3-Methylphenyl)-5-oxo-1,2,3,5-tetrahydroimidazo[1,
2-b]isoquinolin-10-carbothioamide (5)
Reaction time: 5 h, yield: 0.65 g, 72%, mp: 246–248 °C. 1H NMR
(DMSO-d6) d (ppm): 10.45 (s, 1H, NH (thioamide)), 8.03 (d, 1H, H-6,
J = 6.0 Hz), 7.60 (s, 1H, NH, exchangeable with D2O), 7.48–7.54 (m,
2H, H-Ar), 7.08–7.28 (m, 5H, H-Ar), 4.22 (t, 2H, H-3, J = 8.8 Hz), 3.89
5.1.1. General procedure for the preparation of 1,
3-diarylthioureas
(t, 2H, H-2, J = 8.8 Hz), 2.32 (s, 3H, CH3). IRm(KBr): 3377 (N–Hthioam-
A mixture of corresponding aniline (25 mmol), CS2 (2.4 mL,
40 mmol) and DMAP (0.3 g, 2.5 mmol) in ethanol absolute was
heated at reflux for 18 h. Then the mixture was allowed to cool
to room temperature and the product was collected and washed
with ethanol. Physical properties of the product obtained were
according to literature data.26
ide), 3240 (N–H), 1698 (C@O), 1560 (N–CO), 1540, 1299 and 1044
(C@S), 890, 805 and 765 (C–H Ar) cmÀ1. Anal. Calcd for C19H17N3OS:
C, 68.03; H, 5.11; N, 12.53. Found: C, 68.29; H, 5.31; N, 12.33.
5.1.8. N-Phenyl-5-oxo-1,2,3,5-tetrahydroimidazo[1,2-b]
isoquinolin-10-carbothioamide (6)
Reaction time: 3 h, yield: 0.39 g, 45%, mp: 228–230 °C (mp lit.27
221–222 °C). 1H NMR (DMSO-d6) d (ppm): 11.75 (s, 1H, NH (thioam-
ide)), 8.02 (d, 1H, H-6, J = 7.7 Hz), 7.89 (s, 1H, NH), 7.39–7.5 (m, 7 H,
H-Ar), 7.08–7.13 (m, 1H, H-Ar), 4.14 (t, 2H, H-3, J = 8.5 Hz), 3.66
(t, 2H, H-3, J = 8.5 Hz). 13C NMR (DMSO-d6) d (ppm): 210.5 (C@S),
159.1 (C@O), 149.2 (C-10a), 144.0 (C-Ar), 133.3 (C-Ar), 132.4 (C-Ar),
128.4 (C-Ar), 126.7 (C-Ar), 122.9 (C-Ar), 121.0 (C-Ar), 119.7
5.1.2. General procedure for the preparation of aryl, naphthyl,
pyrimidylisothiocyanates
The corresponding 1,3-diarylthioureas (2.4 g, 8 mmol) was dis-
solved in anhydride acetic (5 mL) and was heated at reflux for 1 h.
The product was isolated by steam distillation, the organic layer
was separated, dried (MgSO4), filtered and purified by simple dis-
tillation. Physical properties of the product obtained were accord-
ing to literature data.9
(C-Ar), 110.6 (C-10), 50.0 (C-3), 43.9 (C-2). IR m (KBr): 3375 (N–H thi-
oamide), 3219(N–H),1644(C@O), 1568(N–CO),1519, 1293and1086
(C@S), 760 and 668 (C–H Ar) cmÀ1. m/z (%): 322.05 ([M+1]+, 9.96),
321.05 (M+, 47.58), 288.10 (M+-SH, 100), 211.15 (M+À33ÀC6H5,
17.62), 186.15 (M+ÀCSNHC6H5, 71.77), 135 (C6H5NHCS, 73.22),
77.04 ([C6H5]+, 74.90). Anal. Calcd for C18H15N3OS: C, 67.27; H, 4.70;
N, 13.07. Found: C, 67.81; H, 5.05l; N, 13.44.
5.1.3. General procedure for the synthesis of compounds 2–9
and 13
A mixture of compound 16 (0.5 g, 2.7 mmol), the corresponding
isothiocyanate (2.7 mmol), DMAP (0.6 g, 4.9 mmol) and CH2Cl2
(10 mL) was stirred at reflux for 6–24 h. Then the mixture was al-
lowed to room temperature and the crystalline solid was collected,
washed with ethanol and dried in vacuum.
5.1.9. N-(4-Methoxyphenyl)-5-oxo-1,2,3,5-tetrahydroimidazo
[1,2-b]isoquinolin-10-carbothioamide (7)
Reaction time: 9 h, yield: 0.57 g, 60%, mp: 182–184 °C. 1H NMR
(DMSO-d6) d (ppm): 11.60 (s, 1H, NH (thioamide)), 8.03 (d, 1H, H-6,
J = 6.0 Hz), 7.80 (s, 1H, NH, exchangeable with D2O), 7.08–7.28 (m,
7H, H-Ar), 4.24 (t, 2H, H-3, J = 8.4 Hz), 3.60 (t, 2H, H-2, J = 8.2 Hz),
3.45 (s, 3H, OCH3). Anal. Calcd for C19H17N3O2S: C, 64.94; H, 4.88;
N, 11.96. Found: C, 65.12; H, 5.16; N, 11.74.
5.1.4. N-(4-Chlorophenyl)-5-oxo-1,2,3,5-tetrahydroimidazo[1,
2-b]isoquinolin-10-carbothioamide (2)
Reaction time: 6 h, yield: 0.52 g, 55%, mp 207–209 °C. 1H NMR
(DMSO-d6) d (ppm): 11.74 (s, 1H, NH (thioamide)), 8.02 (d, 1H,
H-6, J = 7.6 Hz), 7.86 (s, 1H, NH, exchangeable with D2O), 7.43–
7.50 (m, 6H, H-Ar), 7.19–7.30 (m, 1H, H-Ar), 4.17 (t, 2H, H-3,
J = 8.8 Hz), 3.98 (t, 2H, H-2, J = 8.7 Hz). IR
amide), 3240 (N–H), 1699 (C@O), 1548 (N–CO), 1520, 1297 and
1080 (C@S), 886 and 767 (C–H Ar) cmÀ1
Anal. Calcd for
C18H14ClN3OS: C, 60.76; H, 3.97; N, 11.81. Found: C, 60.41; H,
3.59; N, 11.96.
m (KBr): 3274 (N–H thio-
5.1.10. 5-Oxo-N-(2-pyridinyl)-1,2,3,5-tetrahydroimidazo[1,
2-b]isoquinolin-10-carbothioamide (8)
Reaction time: 4 h, yield: 0.28 g, 32%, mp: 209–210 °C. 1H NMR
.
(DMSO-d6) d (ppm): 11.60 (s, 1H, NH (thioamide), 8.43 (d, 1H, a-H-
pyridin, J = 5.7 Hz), 8.07 (d, 1H, H-6, J = 7.1 Hz), 7.82 (s, 1H, NH,
exchangeable with D2O), 7.20–7.38 (m, 2H, H-Ar), 7.08–7.15 (m,
4H, H-Ar), 4.12 (t, 2H, H-3, J = 8.9 Hz), 3.90 (t, 2H, H-2, J = 8.8 Hz).
Anal. Calcd for C17H14N4OS: C, 63.33; H, 4.38; N, 17.38. Found: C,
63.47; H, 3.99; N, 17.19.
5.1.5. N-(4-Methylphenyl)-5-oxo-1,2,3,5-tetrahydroimidazo[1,
2-b]isoquinolin-10-carbothioamide (3)
Reaction time: 6 h, yield: 0.46 g, 51%, mp 210–212 °C. 1H NMR
(DMSO-d6) d (ppm): 11.64 (s, 1H, NH (thioamide)), 8.03 (d, 1H,
H-6, J = 6.0 Hz), 7.74 (s, 1H, NH, exchangeable with D2O), 7.08–
7.28 (m, 7H, H-Ar), 4.17 (t, 2H, H-3, J = 8.4 Hz), 3.66 (t, 2H, H-
5.1.11. N-(4-Chlorobenzyl)-5-oxo-1,2,3,5-tetrahydroimidazo
[1,2-b]isoquinolin-10-carbothioamide (9)
Reaction time: 4 h, yield: 0.29 g, 29%, mp: 267–269 °C (d). 1H NMR
(DMSO-d6) d (ppm): 11.20 (s, 1H, NH (thioamide)), 8.04 (d, 1H, H-6,
J = 6.9 Hz), 7.59–7.68 (m, 4H, H-Ar), 7.20–7.38 (m, 3H, H-Ar), 7.02 (s,
1H, NH, exchangeable with D2O), 4.15 (t, 2H, H-3, J = 9.1 Hz), 3.91 (s,
2H, CH2), 3.88 (t, 2H, H-2 J = 9.0 Hz). Anal. Calcd for C19H16ClN3OS:
C, 61.70; H, 4.36; N, 11.36. Found: C, 61.35; H, 4.18; N, 11.55.
2 J = 8.3 Hz), 2.32 (s, 3H, CH3). IR
m (KBr): 3370 (N–H thioamide),
3240 (N–H), 1701 (C@O), 1568 (N–CO), 1519, 1297 and 1044
(C@S), 882 and 768 (C–H Ar) cmÀ1. Anal. Calcd for C19H17N3OS:
C, 68.03; H, 5.11; N, 12.53. Found: C, 68.42; H, 4.71; N, 12.13.
5.1.6. N-(1-Naphtalenyl)-5-oxo-1,2,3,5-tetrahydroimidazo[1,
2-b]isoquinolin-10-carbothioamide (4)
Reaction time: 3 h, yield: 0.49 g, 49%, mp: 189–191 °C. 1H NMR
(DMSO-d6) d (ppm): 10.29 (s, 1H, NH thioamide), 8.12 (d, 1H, H-6,
J = 8.2 Hz), 7.89–7.99 (m, 3H, H-Ar), 7.64 (s, 1H, NH, exchangeable
with D2O), 7.52–7.59 (m, 5H, H-Ar), 7.43 (dd, 1H, H-Ar, J = 7.3 Hz,
J = 1.2 Hz), 7.35 (dt, 1H, H-Ar, J = 6.4 Hz, J = 1.4 Hz), 4.52 (t, 2H, H-
5.1.12. 5-Oxo-N-phenyl-1,2,3,5-tetrahydroimidazo[1,2-b]
isoquinolin-10-carboxamide (10)
To a stirred solution of compound 6 (0.15 g, 0.46 mmol) and
KOH (2%) (1.5 mL), in ethanol (3 mL), 0.11 mL H2O2 (30%) was
slowly added. The temperature was maintained at 0 °C for
15 min. and then warmed to room temperature for 30 min. The
yellow crystalline solid was collected, washed with benzene and
dried in vacuum. Yield 63 mg, 45%, mp >320 °C. 1H NMR (DMSO-
d6) d (ppm): 10.1 (s, 1H, NH (amide)), 8.0 (d, 1H, H-6, J = 8.2 Hz),
7.69 (d, 2H, H-Ar, J = 7.7 Hz), 7.47 (s, 1H, NH, exchangeable with
3, J = 8.2 Hz), 4.20 (t, 2H, H-2, J = 8.5 Hz). IR
thioamide), 3229 (N–H), 1700 (C@O), 1599 (N–CO), 1519, 1297
and 1044 (C@S), 882, 768 (C–H Ar) cmÀ1
Anal. Calcd for
m (KBr): 3372 (N–H
.
C22H17N3OS: C, 71.14; H, 4.61; N, 11.31. Found: C, 70.88; H,
5.01; N, 11.11.

M. Bollini et al. / Bioorg. Med. Chem. 17 (2009) 1437–1444
1443
D2O), 7.28 (t, 1H, H-Ar, J = 7.9 Hz), 6.9–7.07 (m, 5H, H-Ar), 4.09 (t,
2H, H-3, J = 8.4 Hz), 3.70 (t, 2H, H-2, J = 8.4 Hz). 13C NMR (DMSO-
d6) d (ppm): 162.3 (C@O), 159.1 (C@O), 150.0 (C-10a), 140.8
(C-Ar), 133.1 (C-Ar), 131.1 (C-Ar), 128.9 (C-Ar), 127.0 (C-Ar),
122.5 (C-Ar), 119.7 (C-Ar), 118.0 (C-Ar), 111.0 (C-10), 51.4 (C-3),
lowed by measuring the absorbance of the culture at 600 nm every
day. Before inoculation, the medium was supplemented with the
indicated amount of the studied compound from a stock solution
in DMSO. The final concentration of DMSO in the culture medium
never exceeded 0.4%, and the control was run in the presence of
0.4% DMSO and in the absence of compound. No effect on epima-
tigotes growth was observed by the presence of up to 1% DMSO
in the culture medium. The percentage of inhibition was calculated
as follows: I% = {1 À [(Ap À A0p)/(Ac À A0c)]} Â 100, where Ap = A600
of the culture containing the drug at day 5; A0p = A600 of the culture
containing the compound just after the addition of the inocula
(day 0); Ac = A600 of the culture in the absence of any compound
(control) at day 5; A0c = A600 in the absence of the compound at
day 0. To determine IC50 values, 50% inhibitory concentrations, par-
asite growth was followed in the absence (control) and presence of
increasing concentrations of the corresponding compound. At day
5, the absorbance of the culture was measured and related to the
control. The IC50 value was taken as the concentration of com-
pound needed to reduce the absorbance ratio to 50%.
44.1 (C-2). IR
m (KBr): 3393 (N–H), 1658 (C@O), 1616 (C@O),
1598 (N–CO), 756 and 617 (C–H Ar) cmÀ1. EM m/z (%): 306.10
([M+1]+, 10.61), 305.00 (M+, 31.60), 212.45 (M+-NH2C6H5, 100),
186.10 (M+-CONHC6H5, 31.89), 93.10 (C6H5NH2+, 30.96), 77.05
([C6H5]+, 20.51). Anal. Calcd for C18H15N3O2: C, 70.81; H, 4.95; N,
13.76. Found: C, 70.44; H, 4.73; N, 14.00.
5.1.13. 1-Acetyl-5-oxo-N-phenyl-1,2,3,5-tetrahydroimidazo[1,
2-b]isoquinolin-10-carbothioamide (11)
A mixture of compound 6 (0.15 g, 0.46 mmol), DMAP (56 mg,
0.46 mmol) and CH2Cl2 as a solvent was stirred. After 5 min, the
acetyl chloride (0.15 mL, 3.5 mmol) was added over a period of
several minutes and the mixture was stirred at 0–5 °C for 2 h.
The organic layer was washed with H2O (20 mL) and then with
K2CO3 (10%) (3 Â 20 mL). The organic layer was dried (MgSO4), fil-
tered and evaporated under pressure, the residue was recrystal-
lized with ethanol. Yield 0.23 g, 56%, mp 94–96 °C. 1H NMR
(DMSO-d6) d (ppm): 9.98 (s, 1H, NH (amide)), 8.51 (dd, 1H, H-6,
J = 7.9 Hz, J = 1.3 Hz), 8.03 (dt, 1H, H-Ar, J = 7.2 Hz, J = 1.3 Hz),
7.83–7.92 (m, 1H, H-Ar), 7.63–7.71 (m, 4H, H-Ar), 7.40 (d, 2H, H-
Ar, J = 7.2 Hz), 4.93 (m, 2H, H-3), 4.68 (m, 2H, H-2), 2.60 (s, 3H,
CH3). Anal. Calcd for C20H17N3O2S: C, 66.10; H, 4.71; N, 11.76.
Found: C, 66.18; H, 4.58; N, 11.22.
5.3. Antiparasitic activity29
The evaluation of antiparasitic29 activity was performed at Trop-
ical Disease Research (TDR) Program, World Health Organization
(WHO, Switzerland)1, following the well-known in vitro disease-ori-
ented primary antiparasitc screening against P. falciparum, L. dono-
vani, L. infantum, T. b. rhodesiense, and O. gutturosa. For antimalarial
activity, K1 strain was used. If the IC50 is >5
is classified as inactive. If the IC50 is 0.5–5 g/mL, the compound is
classified as moderately active. If the IC50 is <0.5 g/mL, the com-
pound is classified as active. For sleeping sickness, if the IC50 is
>16 g/mL, the compound is classified as inactive. If the IC50 is
between 1 and 16 g/mL, the compound is classified as moderately
active. If the IC50 is <1 g/mL, the compound is classified as active.
For Leishmania activity, if the IC50 is >16 g/mL, the compound is
classified as inactive. If the IC50 is between 1 and 16 g/mL, the com-
pound is classified as moderately active. If the IC50 is <1 g/mL, the
lg/mL, the compound
l
5.1.14. Benzo [4,5] imidazo [1,2-b]isoquinolin-5(1H)-11-one (12)
A mixture of homophthalic acid (5 g, 24 mmol), o-phenylendi-
amine (2.5 g, 24 mmol), and 0.1 mL of DMF was introduced into
bottom flask and subjected to microwave irradiation at 480 W
for 10 min. The reaction was monitored by thin layer chromatogra-
phy. After complete conversion the reaction mixture the product
was recrystallized with ethanol to yield 2 (5.96 g, 85%) mp
>330 °C (mp lit.:28 324–326 °C). 1H NMR (DMSO-d6) d (ppm):
11.87 (s, 1H, N–H, exchangeable with D2O), 8.62 (d, 1H, H-10,
J = 7.4 Hz), 8.26 (dt, 1H, H-Ar, J = 7.4 Hz, J = 1.2 Hz, J = 0.5 Hz),
7.58–7.62 (m, 2H, H-Ar), 7.32–7.42 (m, 2H, H-Ar), 7.16–7.25 (m,
2H, H-Ar), 6.35 (s, 1H, H-6). 13C NMR (DMSO-d6) d (ppm): 159.03
(C-11), 141.43 (C-5), 138.69 (C-Ar), 133.19 (C-Ar), 131.9 (C-Ar),
127.89 (C-Ar), 126.9 (C-Ar), 126.05 (C-Ar), 124.74 (C-Ar), 121.43
(C-Ar), 120.03 (C-Ar), 117.42 (C-Ar), 115.79(C-Ar), 109.22 (C-Ar),
l
l
l
l
l
l
l
compound is classified as active. For O. gutturosa a test compound
is considered active if there is 50% or greater reduction in motility
score and/or 50% or greater inhibition of formazan formation com-
pared to untreated controls. Compounds are classified as moder-
ately active if there is a 50–75% reduction in motility and/or
inhibition of formazan, or highly active at 76–100%.
78.61 (C-6). IR
m
(KBr): 3135 (N–H), 1679 (C@O), 1512 (N–CO),
5.4. Reactivity with glutathione14
780 and 680 cmÀ1. Anal. Calcd for C15H10N2O: C, 76.91; H, 4.30;
N, 11.96. Found: C, 76.56; H, 4.69; N, 12.07.
Solution A: GSH (15.6 mM) in phosphate buffer (0.1 M Na2HPO4,
1.5 mM EDTA, pH 7.4). Solution B: studied compound 2 (3 mM) in
anhydrous MeOH. The reaction was started by mixing solution A
5.1.15. N-Phenyl-11-oxo-benzo[4,5]imidazo[1,2-b]isoquinolin-
5(1H)-6-carbothioamide (13)
(500
(studied compoundfinal concentration 500
(1000 L). The reaction mixture was maintained at 28 °C. At variable
lL) (GSH final concentration 1 mM) with solution B (500
lL)
Reaction time: 6 h, yield: 0.59 g, 60%, mp >300 °C. 1H NMR
(CDCl3) (ppm): 11.80 (s, 1H, NH thioamide), 8.60 (d, 1H, H-10,
J = 7.9 Hz), 8.18–8.28 (m, 5H, H-Ar), 7.59–7.73 (m, 3H, H-Ar),
7.32–7.39 (m, 2H, H-Ar), 7.19–7.25 (m, 3H, 2H-Ar, NH, exchange-
lM) in phosphatebuffer
l
times (0, 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h and 6 h) the reac-
tions were quenched by cooling, at 0 °C, and the UV–vis spectra were
acquired between 200 and 600 nm. As negative controls, GSH and
each compound were incubated at 28 °C in phosphate buffer.
Compound 10-((4-Chlorophenylamino)methyl)-2,3-dihydroim-
idazo[1,2-b]isoquinolin-5(1H)-one (2a) was isolated from the reac-
tion mixture after 6 h and purified by column chromatography
(SiO2, petroleum ether/EtOAc (7:3)). 1H NMR (DMSO-d6) d (ppm):
8.02 (dd, 1H, H-Ar, J = 7.92, 0.93 Hz), 7.59–7.62 (dd, 2H, H-Ar,
J = 6.75 Hz), 7.45–7.47 (m, 1H, H-Ar), 7.31–7.28 (m, 2H, H-Ar),
7.03–7.05 (m, 1H, H-Ar), 6.98 (s, 1H, N–H, exchangeable with
D2O), 5.20 (s, 1H, NH, exchangeable with D2O), 4.11 (t, 2H, H-3,
J = 8.3 Hz), 3.62 (m, 4H, H-2, CH2). Anal. Calcd for C18H16ClN3O: C,
66.36; H, 4.95; N, 12.90. Found: C, 66.21; H, 5.01; N, 12.85.
able with D2O). IR
m (KBr): 3375 (N–H thioamide), 3258 (N–H),
1699 (C@O), 1559 (N–CO), 1520, 1213 and 1080 (C@S), 890, 760
and 668 (C–H Ar) cmÀ1. Anal. Calcd for C22H15N3OS: C, 71.52; H,
4.09; N, 11.37. Found: C, 71.49; H, 3.78; N, 11.41.
5.2. In vitro anti-T. cruzi activity (Epimastigote Form)
Trypanosoma cruzi epimastigotes (Tulahuen 2 strain) were
grown at 28 °C in an axenic medium (BHI-tryptose) complemented
with 5% fetal calf serum. Cells from 5-day-old culture (stationary
phase) were inoculated to 50 mL of fresh culture medium to give
an initial concentration of 1 Â 106 cells/mL. Cell growth was fol-

1444
M. Bollini et al. / Bioorg. Med. Chem. 17 (2009) 1437–1444
5.5. DNA affinity assay
and Tropical Disease Research (TDR) Program-World Health Orga-
nization (WHO, Switzerland) for primary antiparasitic assays. This
work was supported by a grant from University of Buenos Aires
UBACyT B020 M.B. was funded by PROSUL-CNPq (Programa Sul-
Americano de Apoio às Atividades de Cooperação em Ciência e Tec-
nología- Conselho Nacional de Desenvolvimento Científico e Tec-
nológico-Brasil).
DNA solution: Calf thymus DNA (12.5 mg) was slowly magnet-
ically stirred in Tris–HCl buffer 10 mM, pH 7.4 (5 mL), for 24 h at
4 °C. 0.6 mL was taken from this solution and diluted to 25 mL with
the same buffer.
The test compound solution was prepared at 10À4 M concentra-
tion using a minimal volume of ethanol and then diluted adding
water to a concentration of 2 Â 10À5 M. 3 mL sample of this solu-
tion was mixed with 3 mL of the DNA solution. The mixture was
slowly rotated during 24 h and, then, its UV spectra were recorded
at 20 °C using a 1 cm cell.
References and notes
1. www.who.int.
2. Moreno, S. N.; Docampo, R.; Mason, R. P.; Leon, W.; Stoppani, A. O. Arch.
Biochem. Biophys. 1982, 218, 585.
3. Cerecetto, H.; Gonzalez, M. Curr. Trop. Med. Chem. 2002, 2, 1185.
4. Duschak, G.; Couto, A. S. Recent Patents on anti-Infective Drug Discovery 2007, 2,
19.
5.6. Cytotoxic activity
5. Bringman, G.; Hamm, A.; Gunther, C.; Michael, M.; Brun, R.; Mudogo, V. J. Nat.
Prod. 2000, 63, 1465.
6. Bollini, M.; Asís, S. E.; Bruno, A. M. Synthesis 2006, 2, 237.
7. Vogel, A. Practical Organic Chemistry, 3rd ed.; Longmans, 1956.
8. Asís, S. E.; Bruno, A. M.; Gaozza, C. H. Acta Farm. Bonaerense 1997, 16, 209.
9. Werner, E. J. Chem. Soc. 1891, 59, 396.
10. (a) Faucher, J. F.; Baltz, T.; Petry, K. G. Parasitol. Res. 1995, 81, 441; (b) Almeida-
de-Faria, M.; Freymuller, E.; Colli, W.; Alves, M. J. Exp. Parasitol. 1999, 92, 263.
11. Tyler, K. M.; Engman, D. M. Int. J. Parasitol. 2001, 31, 472.
12. Bodley, A. L.; Shapiro, T. A. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 3726.
13. Bollini, M.; Casal, J. J.; Bruno, A. M. Bioorg. Med. Chem. 2008, 16, 8003.
14. Porcal, W.; Hernández, P.; Boiani, M.; Aguirre, G.; Boiani, L.; Chidichimo, A.;
Cazzulo, J. J.; Campillo, N. E.; Paez, J. A.; Castro, A.; Krauth-Siegel, R. L.; Davies,
C.; Miguel; Basombrío, A.; González, M.; Cerecetto, H. J. Med. Chem. 2007, 50,
6004.
The HeLa cells were cultured under standard culture conditions
at 37 °C and 5% CO2 atmosphere in a humidified incubator in Dul-
becco’s modified Eagle’s medium (DMEM) supplemented with
10% fetal bovine serum, 100 UI/mL penicillin and 100 mg/L strepto-
mycin. Cells 9 Â 10À5 cell/mL were seeded in 96 multiwell plates
(Falcon) and after 48 h different concentrations of compound 2 (4,
20 and 100 lM) were added. After 24 h of incubation, cells were
washed twice with PBS and 3-(4,5-dimethylthiazol-2yl)-2,5-diphe-
nyltetrazolium bromide (MTT) from a stock solution (5 mg/mL) was
added at a final concentration of 0.5 mg/mL. Plates were incubated
for 4 h at 37 °C. Finally, blue precipitates were dissolved in 0.1 mL
isopropanol/HCl (300:1) and cells were incubated 1 h a 37 °C. Sam-
ples were read on a plate reader (Spectra CountTM BS 10001) at
570 nm. The values of absorbance showed a good correlation with
viable cell counts using trypan blue. Values from blank plates con-
taining only medium and reagents were subtracted from the values
of the samples. Cell survival percentage (%SP) was expressed as per-
centage of the control cells. All MTT assays were repeated at least
three times by using four samples per assay.
15. (a) Hehre, W. J.; Radom, L.; Schleyer, P. V. R.; Pople, J. A. In Ab Initio Molecular
Orbital Theory; Wiley: New York, 1986; (b) Hehre, W. J.; Shusterman, A. J.;
Huang, W. W.
Wavefunction: California, 1996; (c) Hehre, W. J.
A
Laboratory Book of Computational Organic Chemistry;
Guide to Molecular
A
Mechanics and Quantum Chemical Calculations; Wavefunction: California, 2003.
16. (a) Wavefunction, Inc. 18401 Von Karman Avenue, Suite 370, Irvine, California
92612, USA.; (b) PC Spartan Pro User’s Guide, Wavefunction Inc., California,
1999.
17. (a) Gupta, S. P. Chem. Rev. 1994, 94, 1507; (b) Gandhi, N. Arkivoc, 2006, XVI, 109.
18. Squared correlation matrix of descriptors used in the QSAR study
The evaluation of cytotoxicity activity with L-6 cells was per-
formed at Tropical Disease Research (TDR) Program, World Health
Organization (WHO, Switzerland).1
1
µ
Hf
0.04
0.13
µ
1
clogP
r2
0.06
Hf
1
5.7. QSAR studies
clogP
Quantum-chemical semiempirical AM1 calculations were per-
formed for the lowest energy conformation of the compounds using
the Spartan’02 suite of programs. The compounds were built with
standard bond lengths and angles using the Spartan’02 1.0.1 ver-
sion, and the geometry of each molecule was fully optimized by
applying the semiempirical AM1 method in gas phase from the most
stable conformer obtained using molecular mechanics (MMFF)
methods. Then, a single point calculation using DFT/6-31G* was
used. Lipophilic properties of the compounds were included into
the analyses, as clogP (logP calculated by Villar method, AM1).
.
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Acknowledgements
We thank Dr. Andrea Gamarnik from Leloir Institute, Buenos
Aires—Argentina, for in vitro evaluation of cytotoxicity activity
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