Imidazolidines as new anti-Trypanosoma cruzi agents: Biological evaluation and structure-activity relationships

Article abstract of DOI:10.1016/j.bmc.2007.11.077

Imidazolidine derivatives were studied as anti-Trypanosoma cruzi agents. Imidazolines can be considered as ethylenediamine/carbonyl precursors and therefore interfere with the biosynthesis of polyamines into the parasite. Some of the derivatives were found to have high and selective activity against the proliferative stages of the parasite, with IC₅₀ values against the epimastigote form in the low micromolar range as the reference drug Nifurtimox. The imidazolidines demonstrated to be stable after five days of incubation in buffer glucose, pH 7, indicating that diamines were not obtained in these conditions. But it was found that two of the studied diamine precursors were as active as the parent compounds. Probably, the imidazolidines affect the mitochondrial integrity according to the excreted end-products found in the NMR studies. The QSAR studies indicated that the bioactivities are correlated with the lipophilicities. In conclusion, we have described a new and relevant bioactivity for imidazolidines. The results support further in vivo studies of some of these imidazolidine derivatives.

Full text of DOI:10.1016/j.bmc.2007.11.077

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2226–2234
Imidazolidines as new anti-Trypanosoma cruzi agents:
Biological evaluation and structure–activity relationships
b
c
M. Cristina Caterina,a Isabel A. Perillo,a, Lucıa Boiani, Horacio Pezaroglo,
*
´
*
b
b,
a
´
Hugo Cerecetto, Mercedes Gonzalez and Alejandra Salerno
aDepartamento de Quı´mica Orga´nica, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Junı´n 956, 1113,
Buenos Aires, Argentina
bLaboratorio de Quı´mica Orga´nica, Facultad de Quı´mica-Facultad de Ciencias, Universidad de la Repu´blica,
11400 Montevideo, Uruguay
cLaboratorio de Resonancia Magne´tica Nuclear, Facultad de Quı´mica-Facultad de Ciencias, Universidad de la Repu´blica,
11400 Montevideo, Uruguay
Received 27 September 2007; revised 22 November 2007; accepted 28 November 2007
Available online 4 December 2007
Abstract—Imidazolidine derivatives were studied as anti-Trypanosoma cruzi agents. Imidazolines can be considered as ethylenedi-
amine/carbonyl precursors and therefore interfere with the biosynthesis of polyamines into the parasite. Some of the derivatives were
found to have high and selective activity against the proliferative stages of the parasite, with IC50 values against the epimastigote
form in the low micromolar range as the reference drug Nifurtimox. The imidazolidines demonstrated to be stable after five days
of incubation in buffer glucose, pH 7, indicating that diamines were not obtained in these conditions. But it was found that two of
the studied diamine precursors were as active as the parent compounds. Probably, the imidazolidines affect the mitochondrial integ-
rity according to the excreted end-products found in the NMR studies. The QSAR studies indicated that the bioactivities are cor-
related with the lipophilicities. In conclusion, we have described a new and relevant bioactivity for imidazolidines. The results
support further in vivo studies of some of these imidazolidine derivatives.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
clinical treatment of this disease, 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 lower
efficacy in long-term chronic infections.5,6
Chagas disease remains the major parasitic disease bur-
den in Latin America, despite recent advances in the
control of its vectorial and transfusional transmis-
sion.1–3 Chemotherapy to control this parasitic infection
remains unsatisfactory. Despite significant progress in
our understanding of the biochemistry and physiology
of its etiological agent, Trypanosoma cruzi (T. cruzi),4,5
which has resulted in the validation of several metabolic
steps essential for parasite survival as potential chemo-
therapeutic targets,5,6 current specific treatments are
based on old and quite unspecific drugs, associated with
long-term treatments that may give rise to severe side ef-
fects. In fact, although nifurtimox (Nfx, Fig. 1) and ben-
znidazole (Bnz, Fig. 1), the only two drugs available for
Both drugs act via the reduction of the nitro group. In
the case of Nfx, reduction generates an unstable nitro
anion radical which produces highly toxic reduced oxy-
gen species, whereas Bnz involves covalent modification
of macromolecules by nitro reduction intermediates.7
The side effects of these drugs result from the oxidative
or reductive damage in the host’s tissues and are thus
inextricably linked to its anti-parasitic activity.
The search of new anti-T. cruzi agents has been based on
different strategies,4 that is, inhibition of specific parasite
enzymes, actions on parasite DNA, and oxidative stress
damage. Other possibility has been the blockage/modifi-
cation of some biochemical pathways, like polyamine
syntheses. Some essential polyamines, like spermidine
the precursor for the synthesis of trypanothione, are
Keywords: Imidazolidines; Anti-Trypanosoma cruzi compounds;
QSAR.
*
Corresponding authors. Fax: +54 11 49648250 (I.A.P.); tel./fax: +598
525 07 49 (M.G.); e-mail addresses: iperillo@ffyb.uba.ar;
megonzal@fq.edu.uy
2
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.077

M. C. Caterina et al. / Bioorg. Med. Chem. 16 (2008) 2226–2234
2227
Taking into account that imidazolidines could act as
ethylenediamines/carbonyl precursors we describe sta-
bility in buffer glucose, pH 7, of some imidazolidines
and the activity of some ethylenediamine reactants,
54–56 (Table 3). Mammal cytotoxicities of the most ac-
tive imidazolines and changes in the T. cruzi excreted
metabolites by one of the most active imidazolidine
derivatives are described. A structure–activity relation-
ship analysis is also discussed in order to identify the
structural requirements for optimum activity. The re-
sults indicate that such derivatives are good candidates
for in vivo chemotherapeutic studies in appropriate ani-
mal models for Chagas’ disease.
O
N
N
N
H
N
N
O2N
SO2
O
NO2
Nfx
Bnz
N
N
N
N
1, -R = -OMe
2, -R = -Me
R
R
Figure 1. Nifurtimox, benznidazole, and bisimidazolidines with bac-
tericidal activity.
not supplied by a uniform mechanism in T. cruzi.
T. cruzi epimastigotes are unable to synthesize de novo
significant amounts of putrescine and cadaverine, but
take up polyamines from the medium. After conversion
into spermidine and aminopropylcadaverine, these
polyamines are used to synthesize trypanothione.8
Consequently, inhibitors/modifiers of polyamine bio-
chemistry could be a promising approach for the devel-
opment of new anti-T. cruzi drugs.9
2. Methods and results
2.1. Synthesis
Bisimidazolidines (bis(3-arylimidazolidinyl-1)methanes)
1–8 (Table 1 and Scheme 1) were synthesized by a con-
densation reaction between N-arylethylenediamines with
an excess of formaldehyde.12 Imidazolidines 9–53 were
synthesized by the classical method involving condensa-
tion between N,N0-disubstituted ethylenediamines with
a variety of aldehydes in equimolecular conditions or
an excess of aqueous formaldehyde (37%) (Table 1
and Scheme 1). N,N0-Disubstituted ethylenediamine
precursors were prepared following literature proce-
dures, those having benzyl substituents were synthesized
by condensation of ethylenediamine with aromatic alde-
hydes employing a microwave-assisted solvent-free reac-
tion developed previously28 and further reduction of the
generated Schiff bases with sodium borohydride. All
new compounds, derivatives, were characterized by
NMR (1H, NOESY, and COSY) and mass spectrome-
try. Purity was established by microanalysis (C, H, N).
1,2,3-Trisubstituted compounds display a preferential
conformation with a transoid orientation of the N-1,
N-3, and C-2 substituents.19
On the other hand, imidazolidines (tetrahydroimidazol
derivatives) are cyclic aminals of pharmacological interest
due to the bioactivity shown by some members, which is
closely related to the substitution patterns. For example,
N,N0-dibenzyl-2-arylimidazolidines showed antibacterial
and antiamebic activity.10 Fungicide, bactericide, and
antiviral activities had also been reported for N,N0-bis-
aminoalkylimidazolidines and N,N0-dihydroxyphenylim-
idazolidines.11 Recently, we have described some
bisimidazolidines (bis(3-arylimidazolidinyl-1)methanes)
with interesting bactericidal activity (i.e., compounds 1
and 2, Fig. 1).12 On the other hand, due to the hydropho-
bic nature of imidazolidines they can be used to increase
the bioavailability of biologically active precursors. Thus,
they had been employed as carriers of ethylenediamines13
or carbonyl compounds.14 They are also used as model of
the coenzyme N,N-methylenetetrahydrofolic acid, which
participates in single carbon transfer at the oxidation level
of formaldehyde.15
2.2. Biological characterization
2.2.1. Trypanosoma cruzi growth inhibition experiments.
Some of the studied compounds had a dose-dependent
effect on the growth of extracellular T. cruzi epimastig-
otes (Tulahuen 2 strain) in BHI-Tryptose medium at
28 ꢁC. The percentage of growth inhibition (PGI) at
25 lM is gathered in Table 1. For the most active deriv-
In this work, we describe the synthesis and in vitro anti-
T. cruzi proliferative activity of extracellular epimastig-
otes of a series of imidazolidines which includes terms
such as bisimidazolidines (1–8), symmetrical 1,3-diaryl
substituted (9, 16, 20), 1-aryl-3-methylimidazolidines
(18, 19), 1-aryl-3-benzylimidazolidines (10–12, 21–23),
and 1,3-dibenzylimidazolidines (13–15, 17, 24–53) (Ta-
ble 1). The use of the epimastigote form of T. cruzi as
biological model is done in view of: the existence of
the epimastigote form as an obligate mammalian intra-
cellular stage has been revisited and confirmed re-
cently.16 Moreover, we have recently demonstrated
that there is an excellent relationship between anti-pro-
liferative activity against epimastigotes and in vivo ac-
tive anti-Chagas agents. We found that different
families of compounds with excellent in vitro anti-T.
cruzi epimastigote activity have displayed very good
in vivo anti-T. cruzi activities.17
atives IC50 were evaluated (Table 2).20 The selectivity in-
21
dex (IC50(T. cruzi)/IC50(macrophages)
)
was also studied for
derivatives 25, 28, 30–32, 35–37, and 45 and it was >10
being the IC50(macrophages) > 50 lM.
2.2.2. Ethylenediamines as bioactive entities. In order to
study if the ethylenediamines are the bioactive entities
after a potential biological hydrolysis of the imidazoli-
dine carriers,13,14 two different studies were performed.
In one hand, we have studied the biological activities of
three ethylenediamines potentially produced by hydroly-
sis from the parent imidazolines (Table 3). We selected
them as examples of ethylenediamines coming from imi-

2228
M. C. Caterina et al. / Bioorg. Med. Chem. 16 (2008) 2226–2234
Table 1. Anti-proliferative activities of imidazolines against T. cruzi
Compound
–R1a
–R2a
–R3a
PGIb
1
4-CH3OC6H4
4-CH3C6H4
C6H5
—
—
—
—
0.0
0.0
2
3
—
—
—
—
0.0
0.0
4
4-ClC6H4
3-ClC6H4
5
—
—
—
—
8.0
0.0
6
4-BrC6H4
3-BrC6H4
7
—
—
—
—
10.8
0.0
8
4-NO2C6H4
C6H5
C6H5
9
H
C6H5
C6H5CH2
12.3
17.2
44.2
64.7
100.0
100.0
100.0
12.4
40.0
6.6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
Nfx
H
4-CH3C6H4
4-ClC6H4
H
C6H5CH2
C6H5CH2
H
4-ClC6H4CH2
3-ClC6H4CH2
3,4-Cl2C6H3CH2
C6H5
H
4-ClC6H4CH2
3-ClC6H4CH2
3,4-Cl2C6H3CH2
C6H5
H
H
CH3
4-CH3OC6H4CH2
C6H5
CH3
C6H5
4-CH3OC6H4CH2
CH3
CH3
4-CH3C6H4
C6H5
C6H5
C6H5
C6H5
8.9
C6H5
C6H5CH2
39.6
45.1
46.0
70.4
63.0
75.3
4.8
C6H5
C6H5
4-CH3C6H4
4-ClC6H4
C6H5CH2
C6H5CH2
C6H5CH2
C6H5CH2
C6H5
C6H5
4-CH3OC6H4CH2
2-OHC6H4CH2
4-CH3C6H4CH2
4-ClC6H4CH2
3-ClC6H4CH2
3,4-Cl2C6H3CH2
4-ClC6H4CH2
3,4-Cl2C6H3CH2
4-CH3OC6H4CH2
4-ClC6H4CH2
4-ClC6H4CH2
4-ClC6H4CH2
4-ClC6H4CH2
4-CH3OC6H4CH2
4-CH3C6H4CH2
C6H5CH2
C6H5
C6H5
4-CH3OC6H4CH2
2-OHC6H4CH2
4-CH3C6H4CH2
4-ClC6H4CH2
3-ClC6H4CH2
3,4-Cl2C6H3CH2
4-ClC6H4CH2
3,4-Cl2C6H3CH2
4-CH3OC6H4CH2
4-ClC6H4CH2
4-ClC6H4CH2
4-ClC6H4CH2
4-ClC6H4CH2
4-CH3OC6H4CH2
4-CH3C6H4CH2
C6H5CH2
C6H5
C6H5
100.0
94.5
100.0
100.0
89.4
93.9
98.6
100.0
100.0
97.4
86.8
100.0
100.0
60.0
100.0
50.0
95.0
99.0
100.0
49.0
100.0
91.0
92.0
30.0
83.0
100.0
93.0
100.0
C6H5
C6H5
2-Furyl
2-Furyl
–CH@CHC6H5
–CH@CHC6H5
4-CH3OC6H4
3-OHC6H4
2-OHC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
3-ClC6H4
3-ClC6H4
3-ClC6H4
2-ClC6H4
2-ClC6H4
3,4-Cl2C6H3
3,4-Cl2C6H3
3,4-Cl C6H3
4-NO2C6H5
4-NO2C6H5
3-NO2C6H5
—
4-ClC6H4CH2
3,4-Cl2C6H3CH2
4-CH3OC6H4CH2
4-CH3C6H4CH2
4-ClC6H4CH2
4-CH3OC6H4CH2
4-ClC6H4CH2
4-CH3OC6H4CH2
4-ClC6H4CH2
3,4-Cl2C6H3CH2
4-CH3OC6H4CH2
4-ClC6H4CH2
4-CH3OC6H4CH2
—
4-ClC6H4CH2
3,4-Cl2C6H3CH2
4-CH3OC6H4CH2
4-CH3C6H4CH2
4-ClC6H4CH2
4-CH3OC6H4CH2
4-ClC6H4CH2
4-CH3OC6H4CH2
4-ClC6H4CH2
3,4-Cl2C6H3CH2
4-CH3OC6H4CH2
4-ClC6H4CH2
4-CH3OC6H4CH2
—
a According to Scheme 1.
b PGI: percentage of parasite growth inhibition (%) at 25 lM. The results are means of three different experiments with a SD less than 10% in all
cases.
dazolines with varied bioactivities. Ethylenediamines 55
and 54 could be obtained from two of the most active 2-
phenylimidazolines, 28 (IC50 = 5 lM) and 25
(IC50 = 1.1 lM), respectively, and ethylenediamine 56
could be obtained from one of the inactive 2-phenylim-
idazolines (26, IC50 > 50 lM). Clearly, two of the dia-
mines, 54 and 55, were as active as the corresponding
imidazolines, that is, 13, 17, 25, 28, 31, 33–38, 41, 43,
45–49, and 51–53, while the ethylenediamine 56 was
inactive in the assayed conditions like the corresponding

M. C. Caterina et al. / Bioorg. Med. Chem. 16 (2008) 2226–2234
2229
Table 4. Increment in the amount of the end-products excreted to the
medium with respect to the untreated control
CH2O
NH2
R1HN
R1HN
R1
R1
N
N
N
N
Compound
Percentages (%) of amount of increment of the
excreted metabolitesa
1-8
Lac
Ala
Ace
Gly
Other metabolitesa
Pyr, Sucb
Pyr, Suc, EtOHc
R2CHO
NHR3
32
36
10
30
30
40
10
60
15
60
R1
R3
N
N
a With respect to untreated cells.
b Excreted in lower amount than 9% with respect to untreated cells.
R2
9-53
Suc, succinate.
c EtOH/ethanol.
Scheme 1. Synthetic procedures used for the development of deriva-
tives 1–53.
diamines are the biological active entities this result indi-
cates that the imidazolines-diamines transformations oc-
cur into the parasite probably by specific enzymes.
Table 2. IC50 values of the most active imidazoline derivatives
a
Compound
IC50 (lM)
Compound
IC50 (lM)
2.2.3. Changes in the excreted metabolites’ profile. In or-
der to study the changes in the biochemical pathways
promoted by the active imidazolines, we have studied
the modifications in the excreted metabolites by 1H
NMR spectroscopy. This kind of studies has probed
to be a useful tool in the mechanism of action elucida-
tion.22 For that, we have selected two of the most active
13
14
15
17
25
27
28
29
30
31
32
33
34
35
36
37
10.3
9.7
38
39
40
41
42
43
44
45
46
47
48
49
51
52
53
Nfx
14.9
9.0
15.1
37.0
1.1
23.5
8.9
25.0
14.0
13.4
4.7
12.5
5.0
14.7
6.4
1
imidazolines, namely 32 and 36, and using H NMR
25.3
11.0
16.0
9.0
spectroscopy we compared the spectra of the cell-free
medium of imidazoline-treated parasites with those of
the untreated T. cruzi-free medium as control. We have
focused mainly in the changes of the excreted salts of the
carboxylic acids, lactate (Lac) and acetate (Ace), and the
aminoacids, alanine (Ala) and glycine (Gly), being them
the most relevant modified metabolites. The studied het-
erocycles produced an increment of the excretion of Lac,
Ala, Ace, and Gly (Table 4) with respect to these metab-
olites in the medium with the parasite without treatment
(Table 4). These results could indicate some changes in
the mitochondrion integrity23 being the mitochondrion
partly oxidized end-product Ace and the metabolic
products from pyruvate (Pyr), Ala and Lac, increased.
Normally, a part of Pyr enters to the mitochondrion
and participates in the T. cruzi Krebs cycle and other
part is transaminated to Ala24 or transformed into the
secondary metabolite Lac. The presence of increasing
amount, with respect to control, of Ala and Lac could
indicate that Pyr is not involved in the mitochondrion
metabolism maybe the function of this organ is affected
by the studied compounds. Also, the increased presence
of Gly could be the result of an Ace transamination
increment due to a higher concentration of this end-
product in the cytosol as the result of a higher release
from the mitochondrion.
4.3
4.1
10.0
10.2
5.0
16.5
14.5
23.0
7.7
4.6
4.3
a IC50: concentration that causes a 50% reduction in parasite growth.
The results are means of three different experiments with a SD less
than 10% in all cases.
Table 3. IC50 values for three selected diamines
NHR1
R1HN
Compound
–R1
IC50 (lM)
a
54
55
56
4-CH3OC6H4CH2
4-ClC6H4CH2
2-OHC6H4CH2
16.2
2.0
Inactiveb
a IC50: concentration that causes a 50% reduction in parasite growth.
The results are means of three different experiments with a SD less
than 10% in all cases.
b Percentage of growth inhibition, at 25 lM, was 0.
2.3. Structure–activity relationships
heterocyclic derivative, namely 26. These results showed
that the heterocyclic activities could be the result of the
corresponding diamine activities.
Molecular modeling studies were performed on the
developed imidazoline derivatives by calculating some
stereoelectronic properties in order to understand the
mechanism of action. These properties were determined
using semiempirical AM1 calculations.25,26 A detailed
conformational search for each of the molecules was
performed, using MM methods, to find the minimum
energy and highest abundance conformer. The geometry
of this conformer was fully optimized by applying AM1
On the other hand, we have studied the stability of some
of the most relevant imidazolines, 35, 38, and 43, in the
biological conditions. In order to probe a heterocyclic
masking role, we have analyzed the presence of the ethy-
lenediamines after different treatment time in buffer glu-
cose, pH = 7.0, at 28 ꢁC. Accordingly, after five days
treatment no diamines’ presences were observed. If the

2230
M. C. Caterina et al. / Bioorg. Med. Chem. 16 (2008) 2226–2234
in the gas phase. Then, single point AM1 calculation
was performed. The properties determined and exam-
ined in this study were imidazoline carbon-2 Mulliken
charge (Q2), magnitude of dipolar moment (l),
HOMO’s and LUMO’s energies, gap (ELU-
MO ꢀ EHOMO), and the logarithm of the partition coeffi-
cient of the non-ionized molecules (logP). Theoretical
logP (clogP) was calculated using Villar method, imple-
mented in PC SPARTAN 04 package26 at the AM1 semi-
empirical level. In the equations and models, n
represents the number of data points, r2 is the correla-
tion coefficient, s is the standard deviation of the regres-
sion equation, the F value is related to the F-statistic
analysis (Fischer test), and r2 adj defines the cross-vali-
dated correlation coefficient. Activity used in the struc-
ture–activity relationship studies was the inhibitory
effect (compounds 3–5, 9–53) on the growth of T. cruzi
expressed as percentage of growth inhibition at day 5
and 25 lM, PGI. First, one-variable and multivariable
regressions between both activities and the physico-
chemical properties were studied. Only structure–activ-
ity models having a value of r2 adj above 0.5 were
considered. One of the best equations was obtained
when we analyzed the correlation between activity and
the independent clogP, (clogP)2 and the magnitude of
dipolar moment, l (Eq. 1, Table 5 and Fig. 2). The best
statistical correlation was obtained when l was omitted
in Eq. 1 (Eq. 2, Fig. 2). Some outliers compounds that
not adjust in these equations were identified, that is,
derivatives 14, 15, 19, 20, 26, 42, and 50. Probably, for
these compounds other physicochemical properties
modulate its anti-T. cruzi activities. Important orthogo-
nality was observed between the other analyzed descrip-
tors, that is, ELUMO with clogP and with l, or Q2 with
clogP, so they were not included together in the
analysis.27 No correlations were observed when gap or
EHOMO was included in the analysis. Besides, the corre-
lation matrix for the used physicochemical descriptors
was performed and cross-correlations between the
descriptors used in each equation were not obtained.27
These parameters are therefore orthogonal, a fact that
affords their use in the multilinear regression
procedure.28
3. Discussion
The results indicate that the in vitro activity of the
studied imidazolidines against T. cruzi epimastigotes
is superior in most cases to that of Nfx, the drug used
for the treatment of this disease. In particular, deriva-
tives 25, 28, 31, 32, 35–37, and 45 display low micro-
molar IC50 values (see Table 2), with appreciable
selectivity indexes. Except derivatives 25 and 32 the
most active imidazolidines have in common the struc-
tural motif 4-chlorobenzyl 1,3-bissubstitution. The eth-
ylenediamine 55, which potentially could be generated
from these imidazolidines into the parasite, was also
the most active among the evaluated diamines (see Ta-
ble 3). The rest of the imidazolidines did not display
relevant bioactivities, specially the bisimidazolidines
(1–8) with antibacterial activities12 were not active
against the protozoan T. cruzi. According to QSAR
equations the studied compounds’ lipophilicities play
a relevant role in the bioactivity being the most hydro-
phobic compounds the most active against T. cruzi. If
these derivatives are precursors of the active diamines
the hydrophobic characters are related to its capability
Table 5. Summary of QSAR results
Eq.
Statistical parameters
Expressions
r2 adj
r2
s
p
F value
n
1
2
0.8308
0.8300
0.8167
0.8208
14.80
14.63
<0.0001
<0.0001
58.91
90.34
40
40
PGI = ꢀ41.6( 16.4) + 0.7( 1.7)l + 29.6( 9.1)clogP ꢀ 1.0 ( 1.2)(clogP)2
PGI = ꢀ41.1( 16.2) + 29.8( 8.9)clogP ꢀ 1.0( 1.1)(clogP)2
100
80
60
40
20
0
100
80
60
40
20
r
= 0.9115,
s
= 12.99,
n
= 40,
p
< 0.0001
r
= 0.9110,
s
= 12.99,
n
= 40,
p
< 0.0001
0
0
20
40
60
80
100
0
20
40
60
80
100
Experimental activity (PGI)
Figure 2. Plot of PGI at 25 lM experimental versus calculated values from Eqs. 1 and 2.
Experimental activity (PGI)

M. C. Caterina et al. / Bioorg. Med. Chem. 16 (2008) 2226–2234
2231
to cross throughout the biological membranes. The ef-
fects on the metabolic pathways, studied by H NMR,
Calcd for C16H18N2: C, 80.67; H, 7.56; N, 11.76. Found:
C, 80.45; H, 7.62; N, 11.61.
1
showed that these compounds could act in some man-
ner on the mitochondrion however more studies would
be performed.
5.2.2. 1,3-Di-(3-chlorobenzyl)imidazolidine (14). This
compound was obtained as oil; 78%. H NMR d ppm:
1
7.65 (m, 2Harom.), 7.30–7.50 (m, 6Harom.), 3.81 (s, 4H,
CH2Ar), 3.50 (s, 2H, NCH2N), 2.85 (s, 2H CH2N).
MS: m/z 320 (M+Å), 322 (M+Å+2). Anal. Calcd for
C17H18Cl2N2: C, 63.56; H, 5.65; N, 8.72. Found: C,
63.62; H, 5.59; N, 8.70.
4. Conclusions
In conclusion, we have studied a new scaffold, imidazoli-
dine, as anti-T. cruzi entity. These compounds may serve
as leads for further exploration of the structural require-
ments of these anti-parasitic activities and the promising
in vitro results warrant further in vivo studies in animal
models of Chagas disease.
5.2.3. 1-Benzyl-2,3-diphenylimidazolidine (21). Mp
1
113.0–114.0 ꢁC; 70%. H NMR d ppm: 7.20–7.40 (m,
10Harom.), 7.10–7.20 (dd, 2Harom.), 6.60 (t, 1Harom.),
6.50 (d, 2Harom.), 5.05 (s, 1H, NCHN), 3.70 (d, 1H,
CH2Ar), 3.50 (d, 1H, CH2Ar), 3.20 (m, 2H, CH2N),
2.90 (m, 2H, CH2N). MS: m/z 314 (M+Å). Anal. Calcd
for C22H22N2: C, 84.08; H, 7.01; N, 8.92. Found: C,
84.16; H, 7.12; N, 8.82.
5. Experimental
5.1. Chemistry
5.2.4. 1,3-Di-(4-methylbenzyl)-2-phenylimidazolidine (27).
Mp 82.0–83.0 ꢁC; 82%. 1H NMR d ppm: 7.70 (d, 2Harom.),
7.30–7.50 (m, 3Harom.), 7.15 (dd, 4Harom.), 7.05 (dd,
4Harom.), 3.85 (s, 1H, NCHN), 3.72 (d, 2H, CH2Ar),
3.10–3.20 (m, 4H, CH2Ar and CH2N), 2.49 (m, 2H,
CH2N), 2.30 (s, 6H, CH3). MS: m/z 356 (M+Å). Anal.
Calcd for C25H28N2: C, 84.23; H, 7.92; N, 7.86. Found:
C, 84.30; H, 7.87; N, 7.93.
All starting materials were commercially available re-
search-grade chemicals and used without further purifi-
cation. All solvents were dried and distilled prior to use.
All the reactions were carried out in a nitrogen atmo-
sphere. Imidazolidines 1–8, 9, 11–13, 15–20, 22, 23–26,
28, 30, 31, 33–35, 37, 38, 40, 41, 43, 45–49, 51, and 53
were prepared applying previously described methodol-
ogy.12,18,19a,29–38 Melting points were determined with a
Buchi capillary apparatus and are uncorrected. NMR
¨
5.2.5. 1,3-Di-(3-chlorobenzyl)-2-phenylimidazolidine (29).
Mp 74.0–75.0 ꢁC; 88%. 1H NMR d ppm: 7.62 (d, 2Harom.),
7.30–7.50 (m, 3Harom.), 7.21 (s, 2Harom.), 7.00–7.15 (m,
6Harom.), 3.81 (s, 1H, NCHN), 3.69 (d, 2H, CH2Ar),
3.10–3.25 (m, 4H, CH2Ar and CH2N), 2.50 (m, 2H,
CH2N). MS: m/z 396 (M+Å), 398 (M+Å+2). Anal. Calcd
for C23H22Cl2N2: C, 69.52; H, 5.58; N, 7.05. Found:
C, 69.59; H, 5.50; N, 7.13.
spectra were recorded on a Bruker MSL 300 MHz spec-
trometer using deuteriochloroform as the solvent.
Chemical shifts are reported in ppm relative to TMS
as an internal standard. Splitting multiplicities are re-
ported as singlet (s), doublet (d), double doublet (dd),
triplet (t), and multiplet (m). Mass spectra (EI) were re-
corded with a GC-MS Shimadzu QP-1000 spectrometer
operating at 20 eV. Column chromatography was per-
formed on silica gel 60 (0.063–0.200 mesh) with typically
30–50 g of stationary phase per gram substance. TLC
analyses were carried out on aluminum sheets coated
5.2.6. 1,3-Di-(3,4-dichlorobenzyl)-2-(2-furyl)imidazolidine
(32). Mp 87.0–89.0 ꢁC; 79%. 1H NMR d ppm: 7.20–
7.50 (m, 5Harom.), 7.10 (d, 2Harom.), 6.35 (dd, 2Harom.),
4.15 (s, 1H, NCHN), 3.71 (d, 2H, CH2Ar), 3.35 (d,
2H, CH2Ar), 3.10 (m, 2H, CH2N), 2.60 (m, 2H,
CH2N). MS: m/z 454 (M+Å), 456 (M+Å+2), 458
(M+Å+4). Anal. Calcd for C21H18Cl4N2O: C, 55.29; H,
3.98; N, 6.14. Found: C, 55.32; H, 4.10; N, 6.20.
with silica gel 60 F254
.
5.2. General procedure for the synthesis of imidazolidines
10, 14, 21, 27, 29, 32, 36, 39, 42, 44, 50, and 52
Imidazolidine derivatives were obtained by reaction of
the corresponding N,N0-disubstituted ethylenediamines
(0.01 mol) and aldehydes (0.01 mol) or aqueous form-
aldehyde (37%, excess) in ethanol (20 mL) under re-
flux during 1.5 h. Compounds 10, 21, 27, 29, 32, 36,
39, 42, 44, 50, and 52 were precipitated by cooling
the mixture and were recrystallized from ethanol.
Compound 14 was extracted with chloroform and
purified by column chromatography (benzene/metha-
nol, 9:1). Melting points, yields, spectroscopic data,
and elemental analysis of the new compounds are gi-
ven below.
5.2.7. 1,3-Di-(4-chlorobenzyl)-2-(3-hydroxyphenyl)imi-
dazolidine (36). Mp 110.0–112.0 ꢁC; 70%. H NMR d
1
ppm: 10.81 (br s, 1H, OH), 7.20–7.30 (m, 9Harom.),
7.15 (d, 1Harom.), 7.00 (d, 1Harom.), 6.80 (t, 1Harom.),
4.05 (s, 1H, NCHN), 3.90 (d, 2H CH2Ar), 3.20 (d, 2H,
CH2Ar), 3.12 (m, 2H, CH2N), 2.49 (m, 2H, CH2N).
MS: m/z 412 (M+Å), 414 (M+Å+2). Anal. Calcd for
C23H22Cl2N2O: C, 66.83; H, 5.36; N, 6.78. Found: C,
66.76; H, 5.43; N, 6.84.
5.2.8. 1,3-Di-(4-methylbenzyl)-2-(4-chlorophenyl)imidaz-
1
olidine (39). Mp 108.0–109.0 ꢁC; 75%. H NMR d ppm:
5.2.1. 1-Benzyl-3-phenylimidazolidine (10). Mp 68.0–
70.0 ꢁC; 72%. 1H NMR d ppm: 6.80–7.45 (m, 10Harom.),
4.00 (s, 2H, NCH2N), 3.75 (s, 2H, CH2Ar), 3.45 (t, 2H,
CH2N), 3.01 (t, 2H, CH2N). MS: m/z 238 (M+Å). Anal.
7.60 (d, 2Harom.), 7.35 (d, 2Harom.), 7.00–7.20 (m, 8Harom.),
3.82 (s, 1H, NCHN), 3.70 (d, 2H, CH2Ar), 3.10–3.20 (m,
4H, CH2Ar and CH2N), 2.50 (m, 2H, CH2N), 2.30 (s,
6H, CH3). MS: m/z 390 (M+Å), 392 (M+Å+2). Anal. Calcd

2232
M. C. Caterina et al. / Bioorg. Med. Chem. 16 (2008) 2226–2234
for C25H27ClN2: C, 76.81; H, 6.96; N, 7.17. Found: C,
76.75; H, 6.99; N, 7.25.
containing 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 IC50 values, 50% inhibitory concen-
trations, parasite growth was followed in the absence
(control) and presence of increasing concentrations of
the corresponding drug. At day 5, the absorbance of the
culture was measured and related to the control. The
IC50 value was taken as the concentration of drug needed
to reduce the absorbance ratio to 50%.
5.2.9. 1,3-Di-(3,4-dichlorobenzyl)-2-(4-chlorophenyl)imi-
1
dazolidine (42). Mp 114.0–115.0 ꢁC; 85%. H NMR d
ppm: 7.50 (d, 2Harom.), 7.20–7.40 (m, 6Harom.), 7.05 (d,
2Harom.), 3.80 (s, 1H, NCHN), 3.65 (d, 2H, CH2Ar),
3.05–3.20 (m, 4H, CH2Ar and CH2N), 2.45 (m, 2H
CH2N). MS: m/z 498 (M+Å), 500 (M+Å+2), 502
(M+Å+4). Anal. Calcd for C23H19Cl5N2: C, 55.18; H,
3.82; N, 5.60. Found: C, 55.23; H, 3.78; N, 5.55.
5.3.2. Cytotoxicity to human macrophages. THP-1 hu-
man macrophages were seeded (100,000 cells/well) in
96-well flat bottom microplates (Nunclon) with 200 lL
of RPMI 1640 medium 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. Then, cells were exposed to the compounds (25–
1000 lM) for 48 h. Afterward, the cells were washed
with PBS and incubated (37 ꢁC) with 0.4 mg/mL
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro-
mide (MTT; Sigma) for 3 h. Then, formazan was dis-
solved with DMSO (180 lL), and optical densities
were measured. Each concentration was assayed three
times, and six growth controls were used in each test.
Cytotoxicity percentages (%C) were determined
5.2.10.
1,3-Di-(4-methylbenzyl)-2-(3-chlorophenyl)imi-
dazolidine (44). Mp 97.0–98.0 ꢁC; 79%. 1H NMR d
ppm: 7.65 (s, 1Harom.), 7.50 (m, 1Harom.), 7.28–7.30 (m,
2Harom.), 7.06 (d, 4Harom.), 7.13 (d, 4Harom.), 3.81 (s,
1H, NCHN), 3.71 (d, 2H, CH2Ar), 3.21 (d, 2H, CH2Ar),
3.15 (m, 2H, CH2N), 2.50 (m, 2H, CH2N), 2.31 (s, 6H,
CH3). MS: m/z 390 (M+Å), 392 (M+Å+2). Anal. Calcd
for C25H27ClN2: C, 76.81; H, 6.96; N, 7.17. Found: C,
76.86; H, 7.02; N, 7.25.
5.2.11. 1,3-Di-(3,4-dichlorobenzyl)-2-(3,4-dichlorophenyl)-
imidazolidine (50). Mp 64.0–65.0 ꢁC; 73%. H NMR d
1
ppm: 7.62 (s, 1Harom.), 7.40–7.50 (m, 2Harom.), 7.20–
7.38 (m, 4Harom.), 7.00–7.08 (m, 2Harom.), 3.79 (s, 1H,
NCHN), 3.60 (d, 2H, CH2Ar), 3.10–3.30 (m, 4H,
CH2Ar and CH2N), 2.50 (m, 2H, CH 2N). MS: m/z
532 (M+Å), 534 (M+Å+2), 536 (M+Å+4). Anal. Calcd for
C23H18Cl6N2: C, 51.62; H, 3.39; N, 5.23. Found: C,
51.55; H, 3.45; N, 5.17.
as
follows:
%C = [100 ꢀ (ODd ꢀ ODdm)/(ODc ꢀ
ODcm)]_100, where ODd is the mean of OD595 of wells
with macrophages and different concentrations of the
compounds, ODdm is the mean of OD595 of wells with
different compound concentrations in the medium,
ODc is the growth control, and ODcm is the mean of
OD595 of wells with medium only.
5.2.12. 1,3-Di-(4-chlorobenzyl)-2-(4-nitrophenyl)imidaz-
olidine (52). Mp 122.0–124.0 ꢁC; 90%. 1H NMR d
ppm: 8.31 (d, 2Harom.), 7.72 (d, 2Harom.), 7.20 (dd, 4Harom.),
7.10 (dd, 4Harom.), 3.95 (s, 1H, NCHN), 3.62 (d, 2H,
CH2Ar), 3.29 ( 2H, CH2Ar), 3.12 (m, 2H, CH2N), 2.51
(m, 2H, CH2N). MS: m/z 441 (M+Å), 443 (M+Å+2). Anal.
Calcd for C23H21Cl2N3O2: C, 62.45; H, 4.79; N, 9.50.
Found: C, 62.70; H, 4.72; N, 9.63.
5.3.3. 1H NMR study of the excreted metabolites. For the
spectroscopic studies, 5 mL of a 2-day-treated T. cruzi (Y
strain) with each studied imidazolidine was centrifuged
at 1500g for 10 min at 4 ꢁC. The pellet was discarded,
and the parasite-free supernatant was stored at ꢀ20 ꢁC
until used. The chemical displacements used to identify
the respective metabolites were confirmed by both add-
ing each analyzed metabolite to the studied supernatant
and by the study of a control solution with 4 lg/mL of
each metabolites in buffer phosphate, pH = 7.4.
5.3. Biology
5.3.1. Anti-T. cruzi in vitro evaluation
5.3.1.1. Trypanocidal (epimastigotes) in vitro test. Try-
panosoma cruzi epimastigotes (Tulahuen 2 strain) were
grown at 28 ꢁC in an axenic medium (BHI-Tryptose) as
previously described,39 complemented with 5% fetal bo-
vine serum (FBS). Cells from a 10-day-old culture (sta-
tionary phase) were inoculated into 50 mL of fresh
culture medium to give an initial concentration of
1 · 106 cells/mL. Cell growth was followed by measuring
everyday the absorbance of the culture at 600 nm. Before
inoculation, the media were supplemented with the indi-
cated amount of the drug from a stock solution in DMSO.
The final concentration of DMSO in the culture media
never exceeded 0.4% and the control was run in the pres-
ence of 0.4% DMSO and in the absence of any drug. No
effect on epimastigote growth was observed by the pres-
ence of up to 1% DMSO in the culture media. The percent-
ageofinhibition was calculated as follows: % = {1 ꢀ [(Ap ꢀ
A0p)/(Ac ꢀ A0c)]} · 100, where Ap = A600 of the culture
containing the drug at day 5; A0p = A600 of the culture
5.4. QSAR studies
The compounds were built with standard bond lengths
and angles using the Spartan’04, 1.0.1 version,24 suite
of programs and the geometry of each molecule was fully
optimized by applying semiempirical AM1 method in
gas phase from the most stable conformer obtained using
molecular mechanics (MMFF) methods. Then, a single
point calculation using AM1 was used. Lipophilic prop-
erties of the compounds were included into the analyses,
as clogP (logP calculated by Villar method, AM1).
Acknowledgments
This work received financial support from PEDECIBA
and University of Buenos Aires. L.B. acknowledges
the PEDECIBA fellowship.

M. C. Caterina et al. / Bioorg. Med. Chem. 16 (2008) 2226–2234
2233
Olea-Azar, C.; Rigol, C.; Maya, J. D.; Morello, A.; Piro,
References and notes
´
´
´
O. E.; Aran, V. J.; Azqueta, A.; Lopez de Cerain, A.;
Monge, A.; Rojas, M. A.; Yaluff, G. Bioorg. Med. Chem.
2006, 14, 3467.
1. Dias, J. C.; Silveira, A. C.; Schofield, C. J. Mem. Inst.
Oswaldo Cruz 2002, 97, 603.
´
21. (a) Porcal, W.; Hernandez, P.; Aguirre, G.; Boiani, L.;
Boiani, M.; Merlino, A.; Ferreira, A.; Di Maio, R.;
2. Schofield, C. J.; Jannin, J.; Salvatella, R. Trends Parasitol.
2006, 22, 583.
´
Castro, A.; Gonzalez, M.; Cerecetto, H. Bioorg. Med.
Chem. 2007, 15, 2768; (b) Perez-Rebolledo, A.; Teixeira,
3. W.H.O. Control of Chagas Disease. Technical Reports
Series, 2002; Vol. 905, pp 1–109.
´
L. R.; Batista, A. A.; Mangrich, A. S.; Aguirre, G.;
´
4. Cerecetto, H.; Gonzalez, M. Curr. Top. Med. Chem. 2002,
2, 1185.
5. Urbina, J. A.; Docampo, R. Trends Parasitol. 2003, 19,
495.
´
´
Cerecetto, H.; Gonzalez, M.; Hernandez, P.; Ferreira, A.
M.; Speziali, N. L.; Heraldo, H. Eur. J. Med. Chem. in
press, doi:10.1016/j.ejmech.2007.06.020.
22. (a) Mesa-Valle, C. M.; Castilla-Calvente, J.; Sanchez-
Moreno, M.; Moraleda-Lindez, V.; Barbe, J.; Osuna, A.
6. Urbina, J. A. Curr. Pharm. Des. 2002, 8, 287.
7. Docampo, R.; Moreno, S. N. J. Free Radical Intermedi-
ates in the Trypanocidal Action of Drugs and Phagocytic
Cells. In Free Radicals in Biology; Pryor, W. A., Ed.;
Academic Press: New York, 1984; pp 243–288.
8. Hunter, K. J.; Le Quesne, S. A.; Fairlamb, A. H. Eur. J.
Biochem. 1994, 226, 1019.
´
Antimicrob. Agents Chemother. 1996, 40, 684; (b) Sanchez-
Moreno, M.; Fernandez-Becerra, C.; Castilla, J.; Osuna,
´
A. FEMS Microbiol. Lett. 1995, 133, 119; (c) Fernandez-
´
Ramos, C.; Luque, F.; Fernandez-Becerra, C.; Osuna, A.;
Jankevicius, S. I.; Jankevicius, V.; Rosales, M. J.; Sanchez-
´
Moreno, M. FEMS Microbiol. Lett. 1999, 170, 343.
23. (a) Tielens, A. G. M.; Van Hellemond, J. J. Parasitol.
9. Khabnadideh, S.; Tan, C. L.; Croft, S. L.; Kendrick, H.;
Yardley, V.; Gilbert, I. A. Bioorg. Med. Chem. Lett. 2000,
10, 1237.
10. (a) Sharma, V.; Khan, M. S. Y. Eur. J. Med. Chem. 2001,
36, 651; (b) Kalyaham, N.; Sparthasarathy, P. C.; Anan-
than, L.; Manjunatha, S. G.; Likhate, M. A. Indian J.
Chem. Sec. B 1992, 31, 243.
11. (a) Van Hook, J. O.; Craig, W. E. U.S. 2,675,387, 1955;
Chem. Abstr. 1955, 49, 4729; (b) Craig, W. E.; Van Hook,
J. O. U.S. 2,675,381, 1956; Chem. Abstr. 1956, 50, 411.
12. Perillo, I. A.; Repetto, E.; Caterina, M. C.; Massa, R.;
Gutkind, G.; Salerno, A. Eur. J. Med. Chem. 2005, 40,
811.
13. (a) Nieper, H. A. Arzn. Forsch. 1966, 20, 18; (b) Schoe-
nenberger, H.; Adam, A.; Adam, D. Arzn. Forsch. 1966,
16, 734.
14. (a) Crank, G.; Harding, D. R. K.; Szinai, S. S. J. Med.
Chem. 1970, 13, 1212; (b) Crank, G.; Harding, D. R. K.;
Szinai, S. S. J. Med. Chem. 1970, 13, 1215.
15. (a) Biera¨ugel, H.; Plemp, R.; Pandit, U. K. Tetrahedron
1983, 39, 3987; (b) Van Der Meij, P. F. C.; Chen, T. B. R.
A.; Hilhorst, E.; De Waard, E. R.; Pandit, U. K.
Tetrahedron 1987, 43, 4015; (c) Stoit, A. R.; Pandit, U.
K. Tetrahedron 1988, 44, 6187; (d) Stoit, A. R.; Pandit, U.
K. Tetrahedron 1989, 45, 849; (e) Pandit, U. K. Pure Appl.
Chem. 1994, 66, 759.
16. (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; (c)
Tyler, K. M.; Engman, D. M. Int. J. Parasitol. 2001, 31,
472.
17. (a) Boiani, M.; Boiani, L.; Denicola, A.; Torres de Ortiz,
S.; Serna, E.; Vera de Bilbao, N.; Sanabria, L.; Yaluff, G.;
Nakayama, H.; Rojas de Arias, A.; Vega, C.; Rolan, M.;
´
Today 1998, 14, 265; (b) Miralles, D. M.; Marın, C.;
Magan, R.; Fernandez-Ramos, C.; Entrala, E.; Cordova,
´
´
´
O.; Vargas, F.; Sanchez-Moreno, M. Exp. Parasitol. 2002,
102, 23.
24. Blum, J. J. Parasitol. Today 1993, 9, 118.
25. (a) Hehre, W. J.; Shusterman, A. J.; Huang, W. W. A
Laboratory Book of Computational Organic Chemistry;
Wavefunction Inc.: California, 1996; (b) Hehre, W. J. A
Guide to Molecular Mechanics and Quantum Chemical
Calculations; Wavefunction Inc.: California, 2003.
26. (a) Wavefunction, Inc. 18401 Von Karman Avenue, Suite
370, Irvine, California 92612, USA; (b) PC Spartan Pro
User’s Guide, Wavefunction Inc., California, 1999.
27. Squared correlation matrix of descriptors used in the
QSAR study.
ELUMO
clogP
1
ꢀ0.4773
ꢀ0.1330
ꢀ0.7413
ELUMO
1
Q2
l
r
0.5803
0.3123
clogP
1
0.1189
Q2
1
l
28. (a) Myers, R. H. Classical and Modern Regression with
Application; P.W.S. Publishers: Boston, 1986; (b) Daniel,
C.; Wood, F. S. Fitting Equations to Data, 2nd ed.; Wiley:
New York, 1980; (c) Draper, N. R.; Smith, H. Applied
Regression Analysis, 2nd ed.; Wiley: New York, 1981.
29. Jaenicke, J.; Brode, E. Liebig Ann. Chem. 1959, 624, 120.
30. Caterina, M. C.; Figueroa, M. A.; Perillo, I. A.; Salerno,
A. Heterocycles 2006, 68, 701.
31. Salerno, A.; Hedrera, M. E.; D’Accorso, N. B.; Martins
Alho, M.; Perillo, I. J. Heterocyclic Chem. 2000, 37, 57.
32. Billman, J. H.; Ho, J. Y. C.; Caswell, L. R. J. Org. Chem.
1952, 17, 1375.
33. Salerno, A.; Ceriani, V.; Perillo, I. J. Heterocyclic Chem.
1992, 29, 1725.
34. Wanzlick, H. W.; Schikora, E. Chem. Ber. 1961, 94, 2389.
35. Wilson, W. L.; Le Belle, M. J. J. Pharm. Sci. 1979, 68,
1322.
´
´
Gomez-Barrio, A.; Cerecetto, H.; Gonzalez, M. J. Med.
Chem. 2006, 49, 3215; (b) Porcal, W.; Hernandez, 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.; Basombrio, M. A.;
Gonzalez, M.; Cerecetto, H. J. Med. Chem. 2007.
doi:10.1021/jm070604e.
36. Billman, J. H.; Dorman, L. C. J. Med. Chem. 1963, 6, 701.
37. Billman, J. H.; Ho, J. Y. C.; Caswell, L. R. J. Org. Chem.
1957, 22, 538.
18. Salerno, A.; Figueroa, M. A.; Perillo, I. A. Synth.
Commun. 2003, 33, 3193, and references therein.
19. (a) Salerno, A.; Buldain, G.; Perillo, I. J. Heterocyclic.
Chem. 2001, 38, 849; (b) Perillo, I.; de los Santos, C.;
Salerno, A. Heterocycles 2003, 60, 89.
ˇ
38. Jurcik, V.; Wilhelm, R. Tetrahedron 2004, 60, 3205.
39. (a) Cerecetto, H.; Di Maio, R.; Gonzalez, M.; Risso, M.;
´
Saenz, P.; Seoane, G.; Denicola, A.; Peluffo, G.; Quijano,
C.; Olea-Azar, C. J. Med. Chem. 1999, 42, 1941; (b)
´
Aguirre, G.; Cerecetto, H.; Di Maio, R.; Gonzalez, M.;
Seoane, G.; Denicola, A.; Ortega, M. A.; Aldana, I.;
20. (a) Aguirre, G.; Boiani, L.; Cerecetto, H.; Di Maio, R.;
´
Gonzalez, M.; Denicola, A.; Mo¨ller, M.; Thomson, L.;
Tortora, V. Bioorg. Med. Chem. 2005, 13, 6324; (b) Gerpe,
´
´
A.; Aguirre, G.; Boiani, L.; Cerecetto, H.; Gonzalez, M.;

2234
M. C. Caterina et al. / Bioorg. Med. Chem. 16 (2008) 2226–2234
Monge, A. Arch. Pharm. 2002, 335, 15; (c) Aguirre, G.; 2004, 337, 259; (d) Aguirre, G.; Boiani, L.; Boiani, M.;
´
Boiani, M.; Cerecetto, H.; Gerpe, A.; Gonzalez, M.;
Fernandez, Y.; Denicola, A.; Ochoa, C.; Nogal, J. J.;
´
Cerecetto, H.; Di Maio, R.; Gonzalez, M.; Porcal, W.;
Denicola, A.; Piro, O. E.; Castellano, E. E.; Sant’Anna,
´
Montero, D.; Escario, J. A. Arch. Pharm. Med. Chem.
M.; Barreiro, E. J. Bioorg. Med. Chem. 2005, 13, 6336.