Synthesis, trypanocidal activity and molecular modeling studies of 2-alkylaminomethylquinoline derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.05.035

Research and development of new drugs effective in the treatment of Trypanosoma cruzi infections are a real need for the 16 million people infected in the Americas. In a previous work, a quinoline derivative substituted by a 2-piperidylmethyl moiety showe

Full text of DOI:10.1016/j.ejmech.2011.05.035

European Journal of Medicinal Chemistry 46 (2011) 3696e3703
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journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, trypanocidal activity and molecular modeling studies
of 2-alkylaminomethylquinoline derivatives
,
,
c
,
,
Gisela C. Muscia a 1, Silvia I. Cazorla b 1, Fernanda M. Frank b c, Gabriela L. Borosky d, Graciela Y. Buldain a,
Silvia E. Asís a 1, Emilio L. Malchiodi b
, ,
,
c
,
,1
**
*
a Departamento de Química Orgánica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina
b Cátedra de Inmunología e Instituto de Estudios de la Inmunidad Humoral (IDEHU), CONICET-UBA, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956,
1113 Buenos Aires, Argentina
c Departamento de Microbiología, Parasitología e Inmunología, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, 1121 Buenos Aires, Argentina
d Departamento de Matemática y Física, INFIQC, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, 5000 Córdoba, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
Research and development of new drugs effective in the treatment of Trypanosoma cruzi infections are
a real need for the 16 million people infected in the Americas. In a previous work, a quinoline derivative
substituted by a 2-piperidylmethyl moiety showed to be active against Chagas disease and was
considered a lead compound for further optimization. A series of ten analogous derivatives were tested
against epimastigotes as a first approach. In view of their promising results, six of them were evaluated
against the blood and intracellular replicative forms of the parasite in humans. Among them, compound
12 which possesses a 6-acetamidohexylamino substituent showed remarkable improvement in activity
against epimastigotes, trypomastigotes and amastigotes compared with the structure lead, as well as
a good selectivity index for the two parasite stages present in humans. In addition, treatment of infected
mice with compound 12 induced a significant reduction in parasitemia compared with non-treated mice.
Molecular modeling studies were performed by computational methods in order to elucidate the factors
determining these experimental bioactivities.
Received 11 February 2011
Received in revised form
19 April 2011
Accepted 13 May 2011
Available online 20 May 2011
Keywords:
Chagas disease
Trypanosoma cruzi
Quinoline derivatives
Trypanocidal activity
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
parasitemia, followed by an indeterminate stage that can last for
years without signs or symptoms. Major complications, such as
Chagas disease, also known as American trypanosomiasis, is
caused by the flagellate protozoan Trypanosoma cruzi. One century
after its discovery, Chagas disease still remains a major health
problem in Latin America [1]. In the past two decades, migration of
thousands of paucisymptomatic infected persons from rural to
urban areas in South America and from endemic regions to
developed countries has changed the epidemiology of the disease
[2,3]. Nowadays, it is an important public health issue in South
America and an emerging disease in Europe and North America
[4,5]. Chagas disease is characterized by an acute phase with high
disability due to chronic cardiomyopathy and stroke, occur in
20e30% of patients in the chronic phase of disease [6].
Prophylactic and therapeutic vaccines have been pursued for
decades but sterilizing immunity has not been achieved yet [7e10].
Therefore, chemotherapy remains the only treatment option for
controlling infection once acquired, but none of the different
chemotherapeutic strategies used in the past has proven consis-
tently successful. Although other chemicals have been recently
analyzed in the therapy of trypanosomiasis and several natural
products such as quinone and terpene derivatives have also shown
trypanocidal activity [11e13], the treatment modalities for trypa-
nosomatid infections mostly rely on drugs that date back over 50
years and suffer from poor efficacy, high toxicity, and increasing
resistance [14]. Accordingly, research and development of new
drugs effective in the treatment of T. cruzi infections are a real need
and novel strategies for drug design are required [15,16].
* Corresponding author. Tel.: þ54 11 4964 8251; fax: þ54 11 4964 8252.
** Corresponding author. Cátedra de Inmunología e Instituto de Estudios de la
Inmunidad Humoral (IDEHU), CONICET-UBA, Facultad de Farmacia y Bioquímica,
Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina. Tel./fax: þ54
11 4964 8259.
To develop other alternative drugs, new compounds have been
searched based on empirical screening or ethnopharmacological
studies. Thus, 2-substituted quinolines isolated from a Bolivian
E-mail addresses: elizabet@ffyb.uba.ar (S.E. Asís), emalchio@ffyb.uba.ar
(E.L. Malchiodi).
1
These authors contributed equally.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.035

G.C. Muscia et al. / European Journal of Medicinal Chemistry 46 (2011) 3696e3703
3697
compound 2 was considered a new lead compound for further
optimization of trypanocidal activity.
R1
R1
R4
R3
R2
R2
R4
R3
MW
O
In this work, the preparation of 2-alkylaminomethylquinoline-
3-carboxylic acid derivatives (3e12) in good yields is described
(Scheme 2 and Table 1). These syntheses were carried out in
refluxing methylene chloride for conventional heating whereas
under MW irradiation the reactions were neat.
The final products were evaluated in vitro against different
developmental stages of T. cruzi (epimastigotes, trypomastigotes
and amastigotes). With the aim of establishing preliminary struc-
tureeactivity relationships (SAR) studies for these compounds,
density functional theory (DFT) methods were employed to eval-
uate electronic properties such as molecular electrostatic potentials
(MEPs), charge densities, dipole moments, and HOMOeLUMO
energy values.
+
HCl cat
O
NH2
N
1
R1 = Ph, R2 = Cl,
R3 = CH2Cl, R4 = COOEt
Scheme 1. Synthesis of quinolines via the Friedländer reaction.
medicinal plant, Galipea longiflora Kr (Rutaceae), have shown effi-
cacy in the experimental treatment of cutaneous leishmaniasis as
well as in Balb/c mice chronically infected with T. cruzi [17]. Later
on, the synthesis of 2-substituted quinolines and their in vitro
biological evaluation against the causal agent of Chagas disease
among other parasites was reported [18]. Another series of quino-
lines were also synthesized and tested and one of them exhibited
promising anti-trypanosomal activity [19]. Recently, a quinoline
derivative having anticancer activity (tipifarnib) and synthesized
analogs have shown strong activity against T. cruzi [20e22]. We
have previously reported a series of polysubstituted quinolines
synthesized via the Friedländer reaction employing microwave
irradiation (MW) and a catalytic amount of hydrochloric acid
(Scheme 1) [23]. These products were obtained in good yields at
short times and were tested in vitro against the parasites causing
malaria, leishmaniasis, sleeping sickness and Chagas disease at the
Tropical Disease Research Laboratory, World Health Organization
(WHO). This kind of structures had been designed considering the
antiparasitic activity of 2-substituted quinoline alkaloids, many of
which were isolated from medicinal plants [24,25].
2. Results and discussion
2.1. Synthesis and anti-epimastigote activity
A series of ten 2-alkylaminomethylquinoline derivatives of the
structure lead 2 (Table 1) were designed and synthesized by taking
into account principles of medicinal chemistry such as isosterism,
ring transformations, open-chain analogs and the inclusion of
a suitable side chain containing a second protected amino group
(compound 12). This moiety was chosen because it is a recognized
structure feature necessary for antimalarial activity of quinoline
derivatives [26] and it is known that many compounds are active
against both parasites. In addition, synthetic derivatives of the
natural alkaloid piperine possessing different alkyl chain length
with an amido group at the end, showed trypanocidal activity [27].
The acetamido group was selected just to protect the primary
amine and could be assumed that it cleaves under test conditions to
give the more reactive free primary amine.
The series of 2-alkylaminomethylquinoline derivatives were
obtained by the nucleophilic substitution reaction of a variety of
alkylamines and 2-chloromethyl-quinoline 1 under MW irradiation
or conventional heating. Although the product yields were quite
similar for both reaction-promoting methods, the reactions pro-
ceeded to completion in between 2 and 15 min when they were
MW-assisted whereas otherwise 3e6 h were needed.
Moreover, the 2-piperidylmethyl derivative 2 was prepared in
good yields from the corresponding 2-chloromethylquinoline 1
under MW (Scheme 2) and was achieved in only 6 min instead of
3 h with conventional heating. This compound was moderately
active against Plasmodium falciparum and active against T. cruzi
epimastigotes [23]. Then, it was selected for a secondary in vivo
screening at WHO and this assay was later repeated at higher doses
(results not shown). In view of its remarkable performance,
As first screening, the starting material 1, the lead compound 2
and its ten analogs 3e12 were assayed in vitro against T. cruzi epi-
mastigotes, using benznidazole as reference drug. Compounds 4, 5,
6 and 9 exhibited IC50 values against epimastigotes quite close to the
Ph
Ph
NH
Cl
COOEt
CH2N
Cl
COOEt
CH2Cl
DMF, K2CO3
MW
N
2
N
1
lead compound (8.2
possesses a 6-acetamidohexylamino substituent, showed a remark-
able improvement in activity (3.4 M, Table 1). The replacement of
the piperidyl moiety by N-methylpiperazinyl (4) caused a slight
decrease in activity (IC50 11.8 M), as well as the ring contraction in
the pyrrolidinyl derivative 5 (IC50 10.0 M) and its open-chain
analog 6 (IC50 11.6 M). When the group benzylmethylamine was
introduced (9), the resulting activity was also slightly decreased
(IC50 9.7 M). The isosteric replacement of the piperidyl ring by
morpholinyl (3) markedly decreased the activity (IC50 85.3 M). The
mM), whereas only compound 12, which
H2NR1 or HNR1R2
DMF, K2CO3
MW
m
m
m
Ph
N
Ph
N
m
Cl
COOR
Cl
or
COOR
m
CH2NR1R2
CH2NHR1
m
introduction of benzyl and 2-chlorobenzyl moieties (7 and 8) led to
products which could not be tested because of their low solubility.
The presence of the iso-propyl group (10) as well as the
2-hydroxypropyl chain (11) decreased the activity against epi-
7
8
9
R = H, R1 =
CH2C6H5
3
4
R = Et, NR R =
1 2
N
N
N
O
N
R = H, R =
CH2(2-ClC6H4)
1
R = Et, NR R =
CH3
1
2
R = Et, R1 = CH3 R =
CH2C6H5
2
mastigotes (IC50 89.6 and 50.4 mM, respectively). It is worthy to
10 R = H, R1 = CH(CH3)2
note that the ethyl ester and the carboxylic acid groups attached at
position 3 do not change the electronic properties of the whole
quinoline nucleus and they do not influence the parameters for the
nitrogen atom at position 2, according to our computational
studies in this work and trypanocidal activity in previous results
5
6
R = Et, NR R =
1 2
11 R = H, R1 = CH2CHOHCH3
12 R = H, R1 = (CH2)6NHCOCH3
R = Et, R1 = R2 = CH2CH3
Scheme 2. Synthesis of compound 2 and derivatives 3e12 (see Table 1)

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G.C. Muscia et al. / European Journal of Medicinal Chemistry 46 (2011) 3696e3703
Table 1
In vitro anti-trypanosomal and cytotoxic activity of compounds 1e12.
Compd
Structure
IC50
(m
M) Epia
IC50
nd
(m
M) Trypob
IC50
nd
(
m
M) Amasc
CC50
nd
(m
M)
SI Trypo
nd
SI Amas
nd
Ph
Cl
COOEt
CH2Cl
1
31.4
N
Ph
Cl
Cl
COOEt
CH2N
2
3
8.2
51.3
37.9
310.6
6.05
8.2
N
Ph
COOEt
CH2N
85.3
nd
nd
nd
nd
nd
N
O
N
Ph
N
Cl
COOEt
CH2N
4
11.8
10.0
11.6
nd
6.4
62.4
42.6
10.4
40.3
33.0
nd
35.9
490.0
479.9
nd
5.6
7.8
3.5
12.1
14.5
nd
CH3
Ph
Cl
Cl
COOEt
CH2N
5
N
Ph
COOEt
CH2N
6
11.3
CH2CH3
CH2CH3
N
Ph
N
Cl
COOH
7
nd
nd
CH2NHCH2
Ph
Cl
Cl
COOH
Cl
8
nd
nd
nd
nd
nd
nd
N
CH2NHCH2
Ph
COOEt
9
9.7
127.5
61.0
315.4
2.5
5.2
N
CH2N CH2
CH3
Ph
N
Cl
COOH
CH3
CH2NHCH
CH3
10
89.6
nd
nd
nd
nd
nd

G.C. Muscia et al. / European Journal of Medicinal Chemistry 46 (2011) 3696e3703
3699
Table 1 (continued )
Compd
Structure
IC50
(
m
M) Epia
IC50
nd
(m
M) Trypob
IC50
nd
(
m
M) Amasc
CC50
nd
(m
M)
SI Trypo
nd
SI Amas
Ph
N
Cl
COOH
11
OH
50.4
nd
CH2NHCH2CH
CH3
Ph
Cl
COOH
12
3.4
5.8
3.1
12.8
770.9
706.0
248.7
60.2
O
N
CH2NH (CH2)6 NHCCH3
N
NO2
N
Bzd
34.1
3.6
20.70
196.1
H
N
O
nd: not determined.
a
Epi: epimastigotes.
b
Trypo: trypomastigotes.
Amas: amastigotes.
Bz: benznidazole.
c
d
[23]. Moreover, both groups can be found in active natural
compounds [24].
since a SI > 50 is considered adequate for trypanocidal drugs under
development [28].
2.2. In vitro activity against the blood and intracellular replicative
forms
2.3. In vivo assay
In view of the promising results obtained in vitro with
compound 12, we analyzed the in vivo effect by treating infected
mice for 10 days starting in day 5 post-infection. Untreated mice
infected with T. cruzi trypomastigotes displayed high levels of
parasitemia compared with those treated with compound 12 or
benznidazole (Fig. 1). Considering the parasitemia curve
throughout the acute phase of infection, calculated as the area
under the parasitemia curve (AUC) [7], compound 12-treated
animals presented a threefold reduction in the number of parasites
when compared to untreated ones (1.5 and 4.5 ꢀ 106, respectively).
More importantly, at the peak of parasitemia (day 14), compound
12-treated mice presented a significant reduction in parasitemia
The promising finding of the antiparasitic activity of compounds
2, 4, 5, 6, 9 and 12 against T. cruzi epimastigotes prompted us to
analyze their activity against the main stages found in humans:
bloodstream trypomastigotes and the intracellular replicative
amastigotes. We did not observe any significant differences
between the activity against trypomastigotes and amastigotes
when the piperidyl ring 2 (IC50 51.3 and 37.9
replaced by the pyrrolidinyl ring 5 (IC50 62.4 and 40.3
open-chain analog 6 (IC50 42.6 and 33.0 M). By contrast, the
mM, respectively) was
mM), or by its
m
structural modification resulting from the introduction of N-
methyl-N-benzyl 9 was not successful as an active compound
against T. cruzi (IC50 127.5 and 61.0 mM).
The substitution of piperidyl (2) by piperazinyl group (4)
resulted in an incremental activity against trypomastigotes and
with respect to untreated ones (6.2
ꢁ
1.6
ꢀ
104 versus
5.4 ꢁ 0.2 ꢀ 105 parasites/mL). Interestingly, the number of
amastigotes (IC50 6.4 and 10.4
lead compound (51.3 and 37.9
m
M, respectively) compared to the
mM). It is possible that the N-meth-
ylpiperazinyl group could make compound 4 to reach easily the site
of action of trypomastigotes and amastigotes compared with 2
because of the additional basic nitrogen atom and also its larger
size owing to the methyl group. However, when the cytotoxic
activity against the COS-7 cell line was analyzed, compound 4 was
found to be toxic, having a CC50 value of 35.9 mM. A second amino
group seems to be very important for the differential effect on
amastigotes and trypomastigotes compared with epimastigotes,
since compound 12 behaves similarly to compound 4 in this
respect. Thus, an important decrease in the IC50 value was observed
for compound 12 against trypomastigotes (3.1
mM) and amastigotes
(12.8 M). More importantly, the selectivity index (SI) used to
m
compare the toxicity for mammalian cells and the activity against
the parasites for compound 4 was 5.6 against trypomastigotes and
3.5 against amastigotes, while for the most active compound 12 it
was 248.7 and 60.2, respectively, showing good selectivity for both
parasite stages present in humans. This is particularly significant
Fig. 1. Parasitemia levels during the acute infection period. C3H mice were infected
with 5 ꢀ 103 T. cruzi bloodstream trypomastigotes and treated with compound 12,
benznidazole or PBS. Data represent the mean ꢁ SEM. Results presented are repre-
sentative of three independent experiments.

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G.C. Muscia et al. / European Journal of Medicinal Chemistry 46 (2011) 3696e3703
circulating parasites of benznidazole-treated and compound 12-
treated animals was not significantly different (AUC: 1.3 ꢀ 106 and
1.5 ꢀ 106, respectively).
electronic factors. Instead, the determined IC50 values could be gov-
erned by steric effects.
The structures of the active compounds illustrate the degree of
steric hindrance exerted by the different substituents on the
nitrogen atom of the 2-alkylaminomethyl group in common. The
reduction in activity of derivative 6, brought about by the highly
rotational nature of its open-chain substituent should be noticed.
The lack of activity shown by derivative 3 could be attributed to
the electron density of the oxygen atom in the morpholinyl
substituent, being more available for electrophilic attack than the
nitrogen atom mentioned above. This assumption would also apply
to the hydroxyl group in compound 11. This would also be the case
for N-methylpiperazinyl derivative 4, which showed a decrease in
activity in relation to piperidyl analog 2. The HOMO coefficient for
the N atom under consideration was actually the lowest of the
series (0.236), while the corresponding coefficient for the N-methyl
atom was 0.293. On the other hand, the carbonyl group in the most
active compound 12 is very distant from the proposed reactive
nitrogen to influence the activity.
2.4. Molecular modeling studies
In order to elucidate the factors determining these experimental
bioactivities, molecular modeling studies were performed by
computational methods [29]. DFT geometry optimizations at the
B3LYP/6-31G* level were carried out for all the structures. For the
obtained potential energy minima, several physicochemical prop-
erties were analyzed in order to establish SAR: Mulliken charge
densities, molecular electrostatic potentials (MEPs), dipole moments
(m), frontier molecular orbital energies (3HOMO, 3LUMO), and chemical
hardness ( 3HOMO)) [30,31]. Results are displayed in
h
¼ (3LUMO ꢂ
Table 2. HOMO and LUMO surfaces were also examined for derivative
12 (see supplementary material).
The molecular electrostatic potential (MEP) is the potential
generated by the charge distribution of a molecule and denotes
chemical reactivity, showing nucleophilic and electrophilic sites
indicated by MEP contour maps [32]. Negative electrostatic
potential corresponds to the attraction of a proton by the concen-
trated electron density in the molecule (lone pairs, pi-bonds); thus,
revealing sites for electrophilic attack (colored in shades of red in
standard contour diagrams). Positive electrostatic potential corre-
sponds to the repulsion of a proton by the atomic nuclei in regions
where low electron density exists and the nuclear charge is
incompletely shielded (colored in shades of blue in standard
contour diagrams).
The values in Table 2 did not reveal any correlation between the
computed parameters and the assessed bioactivity. Neither did
molecular orbital surfaces provide any further indication, as the
surface shapes of the frontier orbitals were very similar in all the
compounds. However, inspection of the MEP diagrams (see supple-
mentary material) led to a plausible clarification of the activity data.
All the studied compounds only differ in the substituent attached to
the nitrogen atom of the 2-alkylaminomethyl group. According to the
electrostaticpotentialmaps, these substituentsdifferently modifythe
degree of exposure of the nitrogen nonbonding electron pair and,
consequently affect the accessibility to the electron density at this
point. Therefore, this nitrogen atom was proposed as the reactive
center for the studied compounds. In this way, the HOMO would be
a proper descriptor of the activity. Nevertheless, the coefficient of the
nitrogen considered in this molecular orbital was comparable for
almost every derivative (Table 2). These observations suggest that
relative activities of these series of compounds are not determined by
3. Conclusions
We herein show the syntheses of new 2-alkylaminomethylquino
-line derivatives which exhibited a more selective anti-trypano-
somal activity than the original lead compound (2). They could be
obtained in good yields in a two-pathway simple reaction with
affordable starting materials. The molecular modeling studies
propose that the determined IC50 values could be governed by steric
effects. Consequently, the trypanocidal activity could be improved if
the nitrogen atom is linked to appropriate chains, allowing its
nonbonding electron pair to be exposed.
Activity experiments showed that compound 12 is not only
active against epimastigotes but also against amastigote and try-
pomastigote forms, which are the mammal stages of the parasite.
Compound 12 differentially affects the parasite and the mammalian
cell, with a significant selectivity index and is able to reduce par-
asitemia in infected mice. These characteristics make compound 12
an excellent candidate for extensive studies of in vivo activity and
toxicity in murine models of acute and chronic T. cruzi infection.
4. Experimental methods
4.1. Chemistry
The structures of the synthesized compounds were established
through their 1H and 13C NMR, MS and IR spectra. Melting points
Table 2
Bioactivities and computed molecular parameters for the compounds.
MEPN (a.u.)b
HOMO (a.u.)
NHOMO
LUMO (a.u.)
h (a.u.)
a
c
Compd
IC50
(m
M)
m
(Debye)
qN
12
2
9
5
6
4
1
11
3
10
7
3.4
8.2
9.7
4.1734
2.7074
3.4927
3.7357
2.3105
3.5735
3.1998
3.0390
4.3046
3.5826
3.2841
3.1617
ꢂ0.565
ꢂ0.407
ꢂ0.363
ꢂ0.367
ꢂ0.351
ꢂ0.418
nd
ꢂ0.557
ꢂ0.392
ꢂ0.566
ꢂ0.548
ꢂ0.548
ꢂ0.053
ꢂ0.008
ꢂ0.063
ꢂ0.067
ꢂ0.056
ꢂ0.036
nd
ꢂ0.053
ꢂ0.032
ꢂ0.054
ꢂ0.053
ꢂ0.051
ꢂ0.2031
ꢂ0.2170
ꢂ0.1992
ꢂ0.2026
ꢂ0.1887
ꢂ0.1820
ꢂ0.2411
ꢂ0.2053
ꢂ0.1986
ꢂ0.2136
ꢂ0.2183
ꢂ0.2146
0.528
0.496
0.491
0.532
0.513
0.236
nd
0.515
0.464
0.544
0.518
0.522
ꢂ0.0664
ꢂ0.0616
ꢂ0.0668
ꢂ0.0660
ꢂ0.0652
ꢂ0.0662
ꢂ0.0742
ꢂ0.0643
ꢂ0.0670
ꢂ0.0652
ꢂ0.0725
ꢂ0.0691
0.1368
0.1554
0.1324
0.1366
0.1235
0.1158
0.1669
0.1410
0.1316
0.1484
0.1458
0.1456
10.0
11.6
11.8
31.4
50.4
85.3
89.6
nd
8
nd
nd: not determined.
a
Mulliken charge density at the nitrogen atom of interest (see text).
MEP value at the nitrogen atom of interest (see text).
HOMO coefficient for the nitrogen atom of interest (see text).
b
c

G.C. Muscia et al. / European Journal of Medicinal Chemistry 46 (2011) 3696e3703
3701
were determined in a capillary Electrothermal 9100 SERIES-Digital
apparatus and are given uncorrected. 1H and 13C-NMR spectra were
recorded at rt using a Bruker 300 MHz spectrometer. The operating
frequency for protons and carbons was 300.13 and 75.46 MHz,
CH2CH3), 2.16e2.30 (m, 4H, CH2CH2), 3.20e3.40 (br, 2H, NeCH2),
3.98 (q, J ¼ 7.0 Hz, 2H, CH2CH3), 4.15e4.40 (br, 2H, NeCH2), 4.70 (s,
2H, Het-CH2), 7.29e7.31 (m, 2H, Ph-H), 7.52e7.55 (m, 3H, Ph-H),
7.59 (d, J ¼ 2.1 Hz, 1H, Het-H), 7.75 (dd, J ¼ 9.1 Hz and 2.1 Hz, 1H,
respectively. The chemical shifts (
d) are given in ppm. IR spectra
Het-H), 8.25 (d, J ¼ 9.1 Hz, 1H, Het-H); 13C NMR (CDCl3):
d 13.30,
were recorded on a FT Perkin Elmer Spectrum One from KBr discs.
Elemental analysis (C, H and N) was performed on an Exeter CE 440
and the results were within ꢁ0.4% of the calculated values.
Analytical TLCs were performed on DC-Alufolien Kieselgel 60 F254
Merck. Microwave-assisted reactions were carried out in a CEM
Discover oven. All the alkyl (or heterocyclic) amines were
commercially purchased, except N-(6-aminohexyl) acetamide
24.10, 54.27, 56.20, 62.17, 125.45, 125.72, 126.83, 128.60, 128.97,
129.06, 131.50, 132.37, 134.42, 134.82, 145.55, 147.32, 148.14, 166.86;
IR (cmꢂ1):
y 2997, 1718, 1619, 1221, 833, 703, 637; Anal. Calcd. for
C23H23ClN2O2: C, 69.95; H, 5.87; N, 7.09; found: C, 69.91; H, 5.90; N,
7.12.
4.1.5. Ethyl 6-chloro-2-(diethylaminomethyl)-4-phenylquinoline-3-
carboxylate (6)
which was prepared from
a mixture of 1,6-diaminohexane
(0.16 mol) and AcOEt (0.07 mol) stirred at rt for a week. The
Yield 65%; mp 177e179 ꢃC; reaction time 14 min; crystallized
product was purified by fractionating distillation yield 76%; b.p.
from cyclohexane; 1H NMR (CDCl3):
d
0.90 (t, J ¼ 7.0 Hz, 3H,
162e164 ꢃC (3 mmHg); 1H NMR (CDCl3):
d
1.27e1.32 (m, 4H),
CH2CH3), 1.56 (t, J ¼ 7.0 Hz, 6H, NeCH2CH3), 3.55 (br, 2H,
NeCH2CH3), 3.73 (br, 2H, NeCH2CH3), 4.07 (q, J ¼ 7.1 Hz, 2H,
CH2CH3), 4.63 (s, 2H, Het-CH2), 7.32e7.34 (m, 2H, Ph-H), 7.53e7.55
(m, 3H, Ph-H), 7.62 (d, J ¼ 2.2 Hz,1H, Het-H), 7.77 (dd, J ¼ 8.9 Hz and
2.1 Hz, 1H, Het-H), 8.20 (d, J ¼ 8.9 Hz, 1H, Het-H); 13C NMR (CDCl3):
1.39e1.49 (m, 4H), 1.66 (br, 1H), 1.93 (s, 3H), 2.62e2.67 (m, 3H),
3.16e3.22 (m, 2H), 5.90 (br,1H); 13C NMR (CDCl3):
d
23.19, 26.60,
y 3418, 3067,
29.44, 33.39, 39.44, 41.90, 42.00, 170.11; IR (cmꢂ1):
2927, 2848, 1646, 1377, 1300.
d
9.98, 13.42, 49.03, 50.55, 62.44, 125.54, 126.58, 126.80, 128.67,
4.1.1. General procedure for compounds 3e12
129.05, 129.24, 131.30, 132.41, 134.53, 134.55, 145.42, 147.24, 148.03,
A mixture of 1 (0.40 g, 1.10 mmol), alkyl (or heterocyclic) amine
(1.50 mmol), anhydrous K2CO3 (0.15 g, 1.10 mmol) was subjected to
microwave irradiation, at 400 W. In the case of solid alkylamines,
0.3 mL DMF was added. After reaction completion (TLC), the reac-
tion mixture was diluted with CH2Cl2 and washed with water, HCl
5% (10 mL) and brine, which was then dried (Na2SO4) and
concentrated under reduced pressure to give a solid product which
was triturated or crystallized from the proper solvent. Compound
10 could only be achieved under conventional heating, in refluxing
CH2Cl2 as solvent, employing the same work-up.
166.71; IR (cmꢂ1):
y
2977, 1713, 1603, 1222, 838, 761, 708; Anal.
Calcd. for C23H25ClN2O2: C, 69.60; H, 6.35; N, 7.06; found: C, 69.57;
H, 6.31; N, 7.02.
4.1.6. 2-(Benzylaminomethyl)-6-chloro-4-phenylquinoline-3-carb-
oxylic acid (7)
Yield 55%; mp 206e208 ꢃC, reaction time 7 min; crystallized
from EtOH; 1H NMR (CDCl3):
d 4.46 (s, 2H, NHeCH2ePh), 4.81 (s,
2H, Het-CH2), 7.33e7.36 (m, 5H, Ph-H), 7.46e7.49 (m, 2H, Ph-H),
7.60e7.62 (m, 3H, Ph-H), 7.74 (dd, J ¼ 9.0 Hz and 2.3 Hz, 1H, Het-
H), 7.82 (d, J ¼ 2.3 Hz, 1H, Het-H), 8.09 (d, J ¼ 9.0 Hz, 1H, Het-H);
4.1.2. Ethyl 6-chloro-2-(morpholin-4-ylmethyl)-4-phenylquinoline-
3-carboxylate (3)
13C NMR (CDCl3):
d 46.43, 50.30, 130.70, 126.24, 127.92, 128.04,
128.18, 128.45, 128.89, 129.17, 129.87, 130.69, 131.94, 132.87, 136.28,
Yield 50%; mp 222e223 ꢃC; reaction time 8 min; triturated with
147.08, 148.08, 160.81, 165.49; IR (cmꢂ1):
y
3436, 3067, 3033, 1698,
cyclohexane; 1H NMR (CDCl3):
d
0.82 (t, J ¼ 7.2 Hz, 3H, CH2CH3), 3.59
1613, 1227, 831, 758,700. Anal. Calcd. for C24H19ClN2O2: C, 71.55; H,
4.75; N, 6.95; found: C, 71.59; H, 4.71; N, 6.92.
(br, 4H, NeCH2), 4.02 (q, J ¼ 7.2 Hz 2H, CH2CH3), 4.08 (br, 4H, OeCH2),
4.59(s, 2H, Het-CH2), 7.29e7.32 (m, 2H, Ph-H), 7.52 (m, 3H, Ph-H), 7.61
(d, J ¼ 2.1 Hz, 1H, Het-H), 7.77 (dd, J ¼ 9.0 Hz and 2.3 Hz, 1H, Het-H),
4.1.7. 2-(2-Chlorobenzylaminomethyl)-6-chloro-4-
phenylquinoline-3-carboxylic acid (8)
8.20 (d, J ¼ 9.0 Hz, 1H, Het-H). 13C NMR (CDCl3):
d 13.33, 51.99,
58.25, 62.38, 63.91, 125.50, 126.90, 127.14, 128.63, 128.99, 129.18,
Yield 77%; mp 223e225 ꢃC; reaction time 10 min; triturated
131.32,132.54,134.60,134.77,145.49,146.16,148.27,166.86;IR(cmꢂ1):
with EtOH; 1H NMR (CDCl3):
d 4.56 (s, 2H, NHeCH2ePh), 4.98 (s,
y
3063,1712,1484, 1277, 835, 711, 610; Anal. Calcd. for C23H23ClN2O3:
2H, Het-CH2), 7.24e7.61 (m, 9H, Ph-H), 7.76 (dd, J ¼ 9.0 and 2.3 Hz,
C, 67.23; H, 5.64; N, 6.82; found: C, 67.27; H, 5.59; N, 6.87.
1H, Het-H), 7.84 (d, J ¼ 2.1 Hz, 1H, Het-H), 8.1 (d, J ¼ 9.1 Hz, 1H, Het-
H); 13C NMR (CDCl3):
d 43.69, 50.77, 120.43, 126.24, 127.41, 128.02,
4.1.3. Ethyl 6-chloro-2-(4-methylpiperazin-1-ylmethyl)-4-
phenylquinoline-3-carboxylate (4)
128.16, 129.17, 129.33, 129.72, 129.86, 130.64, 130.71, 131.93, 131.97,
132.89, 133.83, 133.91, 147.12, 148.12, 160.81, 165.62; IR (cmꢂ1):
y
Yield 68%; mp 237e239 ꢃC; reaction time 5 min; crystallized
3392, 3067, 1705, 1652, 1227, 832, 758, 702. Anal. Calcd. for
C24H18Cl2N2O2: C, 65.91; H, 4.15; N, 6.41; found: C, 65.94; H, 4.13; N,
6.44.
from cyclohexane; 1H NMR (CDCl3):
d
0.93 (t, J ¼ 7.1 Hz, 3H,
CH2CH3), 2.74 (s, 3H, NeCH3), 2.81e2.92 (m, 4H, NeCH2), 3.08e3.12
(m, 2H, NeCH2), 3.38e3.42 (m, 2H, NeCH2), 4.01 (q, J ¼ 7.1 Hz, 2H,
CH2CH3), 4.09 (s, 2H, Het-CH2), 7.31e7.34 (m, 2H, Ph-H), 7.51e7.53
(m, 3H, Ph-H), 7.57 (d, J ¼ 2.1 Hz, 1H, Het-H), 7.69 (dd, J ¼ 8.9 Hz and
2.3 Hz, 1H, Het-H), 8.07 (d, J ¼ 9.1 Hz, 1H, Het-H); 13C NMR (CDCl3):
4.1.8. Ethyl 2-[(benzyl-methyl-amino)-methyl]-6-chloro-4-phenyl-
quinoline-3-carboxylate (9)
Yield 77%; mp 142e145 ꢃC; reaction time 10 min; triturated with
d
13.68, 43.42, 49.01, 53.56, 61.13, 62.02, 125.27, 126.89, 127.14,
EtOH; 1H NMR (CDCl3):
d
0.89 (t, J ¼ 7.1 Hz, 3H, CH2CH3), 2.15 (s, 3H,
128.45, 128.85, 129.15, 131.07, 131.41, 133.31, 134.84, 145.59, 146.75,
NeCH3), 3.55 (s, 2H, NeCH2ePh), 3.96 (q, J ¼ 7.0 Hz, 2H, CH2CH3),
4.07 (s, 2H, Het-CH2), 7.20e7.25 (m, 5H, Ph-H), 7.35e7.38 (m, 2H,
Ph-H), 7.51e7.53 (m, 3H, Ph-H), 7.57 (d, J ¼ 2.3 Hz, 1H, Het-H), 7.67
(dd, J ¼ 8.8 Hz and 2.3 Hz,1H, Het-H), 8.06 (d, J ¼ 8.8 Hz,1H, Het-H);
154.52,167.82; IR (cmꢂ1):
y 2980,1719,1604,1226, 831, 761, 700; EI-
MS m/z: 424 [M þ H]þ; Anal. Calcd. for C24H26ClN3O2: C, 68.00; H,
6.18; N, 9.91; found: C, 68.05; H, 6.15; N, 9.87.
13C NMR (CDCl3):
d 13.47, 41.78, 60.95, 61.66, 63.46, 125.35, 126.87,
4.1.4. Ethyl 6-chloro-4-phenyl-2-(pyrrolidin-1-ylmethyl) quinoline-
3-carboxylate (5)
127.02, 127.95, 128.33, 128.57, 129.11, 129.27, 130.87, 130.97, 132.76,
135.32, 138.38, 145.41, 146.25, 157.71, 167.86; IR (cmꢂ1):
y
2980,
Yield 70%; mp 199e201 ꢃC, reaction time 2 min; crystallized
1725, 1652, 1278, 836, 736, 703. Anal. Calcd. for C27H25ClN2O2: C,
72.88; H, 5.66; N, 6.30; found: C, 72.84; H, 5.68; N, 6.27.
from cyclohexane; 1H NMR (CDCl3):
d
0.82 (t, J ¼ 7.0 Hz, 3H,

3702
G.C. Muscia et al. / European Journal of Medicinal Chemistry 46 (2011) 3696e3703
4.1.9. 6-Chloro-2-(isopropylaminomethyl)-4-phenylquinoline-3-
carboxylic acid (10)
The trypanocidal effect of the compound was also tested on
bloodstream trypomastigotes as previously described in Ref. [12].
Briefly, mouse blood containing trypomastigotes was diluted in
complete RPMI medium to adjust parasite concentration to
Yield 70%; mp 230e232 ꢃC; conventional heating 4 h; triturated
with cyclohexane; 1H NMR (CDCl3):
d
1.31 (d, J ¼ 6.9 Hz, 6H, CH3), 4.53
(s, 2H, Het-CH2), 4.61e4.72 (m, 1H, CH), 7.45e7.50 (m, 2H, Ph-H),
7.58e7.60 (m, 3H, Ph-H), 7.74e7.76 (m, 1H, Het-H), 7.77 (dd,
J ¼ 8.7 Hz and 2.3 Hz, 1H, Het-H), 8.12 (d, J ¼ 8.7 Hz, 1H, Het-H); 13C
1.5 ꢀ 106/mL. Parasites were seeded (150
mL/well) in duplicate in
a 96-well microplate and 2 mL of the compound/well (0.5e100 mg/
mL final concentration), or control drug (benznidazole) were
added. Plates were incubated for 24 h at 4 ꢃC and the remaining live
parasites were counted in a Neubauer chamber. The percentage of
inhibition was calculated as 100 ꢂ {[(live parasites in wells after
compound treatment)/(live parasites in untreated wells)] ꢀ 100}.
NMR (CDCl3):
d 20.48, 42.57, 45.85, 121.41, 126.20, 128.08, 129.02,
129.79, 130.56, 131.79, 132.10, 132.79, 146.75, 147.86, 161.01, 164.86; IR
(cmꢂ1):
y
3043, 2970, 1689, 1607, 1225, 841, 763, 701. Anal. Calcd. for
C20H19ClN2O2: C, 67.70; H, 5.40; N, 7.89; found: C, 67.75; H, 5.36; N, 7.87.
4.1.10. 2-[(2-Hydroxypropylamino)methyl]-6-chloro-4-phenylquin-
oline-3 carboxylic acid (11)
4.3.2. Cytotoxicity assay
African green monkey kidney (COS-7) cell lines were employed
for the determination of cell viability by the MTT assay. Cells
(6 ꢀ 104) were settled in a 96-well flat-bottom microtiter plate in
the absence and presence of increasing concentrations of the
Yield 51%; mp 197e199 ꢃC; reaction time 6 min; crystallized
from EtOH; 1H NMR (CDCl3):
d
1.25 (d, J ¼ 6.2 Hz, 3H, CH3), 1.62 (br,
1H, OH), 2.53 (br, 1H, NH), 3.57 (dd, J ¼ 14.4 Hz and 7.9 Hz, 1H, CH2),
3.70 (dd, J ¼ 14.4 Hz and 3.3 Hz, 1H, CH2), 4.17 (m, 1H, CH), 4.69 (d,
J ¼ 17.4 Hz, 1H, Het-CH2), 4.77 (d, J ¼ 17.4 Hz, 1H, Het-CH2),
7.39e7.43 (m, 2H, Ph-H), 7.54e7.58 (m, 3H, Ph-H), 7.74 (d, J ¼ 2.4 Hz,
1H, Het-H), 7.78 (dd, J ¼ 8.9 Hz and 2.4 Hz, 1H, Het-H), 8.10 (d,
compounds (10e500
For the last 4 h, 20 L of MTT solution (1.5 mg/mL in complete RPMI
m
g/mL) and incubated at 37 ꢃC 5% CO2 for 24 h.
m
medium without phenol red (Gibco) containing 10% fetal bovine
serum) was added into each well. The purple formazan crystals
were completely dissolved by adding 150 mL of ethanol and
J ¼ 9.1 Hz, 1H, Het-H); 13C NMR (CDCl3):
d
21.43, 50.76, 52.90, 67.37,
126.23, 128.11, 129.15, 129.76, 130.63, 132.01, 147.99, 160.99; IR
absorbance was detected at 570 nm in a microplate reader. The
results were calculated as the relationship between viable cells in
the presence and absence of the compound ꢀ 100. The selectivity
(cmꢂ1):
y 3415, 2977,1669,1608,1230, 838, 761, 702. Anal. Calcd. for
C20H19ClN2O3: C, 64.78; H, 5.16; N, 7.55; found: C, 64.75; H, 5.19; N,
7.52.
index (SI) was calculated as the 50% cytotoxic concentration (CC50
)
divided by the 50% inhibitory concentration (IC50) of the compound
4.1.11. 2-[6-(Acetamidohexylamino)methyl]-6-chloro-4-
phenylquinoline-3-carboxylic acid (12)
for T. cruzi trypomastigotes and amastigotes.
Yield 56%; mp 136e139 ꢃC; reaction time 7.5 min; triturated
4.3.3. Amastigote growth inhibition assay
with cyclohexane; 1H NMR (CDCl3):
d
1.36e1.43 (m, 4H, CH2),
Ninety-six well tissue culture plates (Sarstedt Inc., Newton, NC)
1.59e1.69 (m, 5H, CH2, NH), 1.80 (s, 3H, COCH3), 3.08e3.14 (m, 2H,
CH2NHCO), 3.58 (t, J ¼ 7.1 Hz, 2H, NHCH2), 4.50 (s, 2H, Het-CH2),
7.34e7.36 (m, 2H, Ph-H), 7.49e7.51 (m, 3H, Ph-H), 7.68e7.73 (m, 2H,
were seeded with a murine macrophage cell line, J774, at 5 ꢀ 103
per well in 100 m
L volumes and incubated 2 h at 37 ꢃC 5% CO2. Cells
were infected with transfected trypomastigotes expressing
b-
d, Het-H), 8.06 (d, J ¼ 8.9 Hz, 1H, Het-H); IR (cmꢂ1):
y
3343, 2929,
galactosidase [36], at a parasite cell ratio 10:1, kindly provided by
Dr. Buckner. After 24 h of co-culture, plates were washed twice with
PBS to remove unbound parasites and drug compounds were added
1689, 1641, 1239, 833, 758, 699. Anal. Calcd. for C25H28ClN3O3: C,
66.14; H, 6.22; N, 9.26; found: C, 66.17; H, 6.25; N, 9.24.
at 0.5e100 mg/mL per well in 200 mL fresh complete RPMI medium
4.2. Computational procedures
without phenol red. Each concentration was tested in duplicate.
Controls included uninfected JTT4 cells with RPMI alone (0%
infection control) and cell monolayers infected with trypomasti-
gotes (100% infection control). On day 7, the assays were developed
DFT full geometry optimizations were performed with the
Gaussian 03 program [29] using the B3LYP functional [33e35] and the
6-31G* basis set. Molecularparameters of the optimized minimawere
evaluated at the same level of theory. Distribution of the electrostatic
potential derived from the electron density of the compounds was
estimated by energy calculations at the optimized structures.
by addition of CPRG (100 mM final concentration) and Nonidet P-40
(1% final concentration). Plates were incubated for 4e6 h at 37 ꢃC.
Wells with galactosidase activity turned the media from yellow to
red, and this was quantitated using a microplate reader (A570 nm,
Bio-Rad Laboratories). The percentage of inhibition was calculated
as 100 ꢂ {[(Absorbance of treated infected cells)/(Absorbance of
untreated infected cells)] ꢀ 100}, and the IC50 was estimated.
4.3. Biology
4.3.1. In vitro trypanocidal activity assay
T. cruzi epimastigotes (RA strain) were grown in biphasic
medium and maintained by weekly passages at 26 ꢃC. T. cruzi
bloodstream trypomastigotes were obtained from infected CF1
mice by cardiac puncture at the peak of parasitemia on day 15 post-
infection. Trypomastigotes were routinely maintained by infecting
21-day-old CF1 mice.
4.3.4. Animals
Female C3H/HeN mice were nursed at the Departamento de
Microbiología, Facultad de Medicina, Universidad de Buenos Aires.
Mice were housed in groups of 5 per cage. They were kept in
a conventional room at 24 ꢁ 1 ꢃC with free access to a standard
commercial diet and water ad libitum under a 12 h light/12 h dark
cycle. All procedures were approved by the Ethics Review Board of
the Instituto de Estudios de la Inmunidad Humoral (IDEHU-CONI-
CET) and conducted in accordance with the guidelines established
by the National Research Council, Guide for the Care and Use of
Laboratory Animals [37].
Growth inhibition of T. cruzi (RA) epimastigotes was evaluated
by a [3H] thymidine uptake assay as previously described in Ref.
[11]. Compounds were tested at 100, 50, 10, 5, 1 and 0.5 mg/mL (final
concentration). Briefly, parasites were adjusted to a cell density of
1.5 ꢀ 106/mL and cultured in the presence of compounds for 72 h.
Benznidazole (5e20 mM; Roche) was used as positive control. The
percentage of inhibition was calculated as 100 ꢂ {[(cpm of treated
parasites)/(cpm of untreated parasites)] ꢀ 100}, and the IC50 was
estimated.
4.3.5. In vivo assays
Groups of five female C3H/HeN mice (6e8 week-old,
23.8 ꢁ 2.6 g), were infected with 5 ꢀ 103 bloodstream T. cruzi

G.C. Muscia et al. / European Journal of Medicinal Chemistry 46 (2011) 3696e3703
3703
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Financial support was received from: Consejo Nacional de
Investigaciones Científicas y Técnicas PIP 441 (to GYB), University of
Buenos Aires B027 (to ELM); Agencia Nacional de Investigaciones
Científicas y Técnicas PICT 608 (to ELM) and SECyT-UNC (to GLB),
Argentina. ELM is also supported by the Fogarty International
Center (TW007972), USA; and the International Center for Genetic
Engineering and Biotechnology (CRP/ARG09-02), Italy.
Appendix. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.05.035
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