Studies on the trypanocidal activity of semi-synthetic pyran[b-4,3] naphtho[1,2-d]imidazoles from β-lapachone

Article abstract of DOI:10.1016/j.ejmech.2004.02.015

We synthesized new naphthoimidazoles from β-lapachone with an aromatic moiety linked to the imidazole ring, using phenylic and heterocyclic aldehydes. The most active compound against Trypanosoma cruzi had a p-methyl group linked to the phenyl ring, presenting an EC₅₀ value of 15.5 ± 2.9 μM. No reliable correlation could be established with the biological activity and the structure of in the phenylic series. For the heterocyclic series, activity was associated with a three bond-distance from nitrogen to the imidazole ring, in accordance with our previous work.

Full text of DOI:10.1016/j.ejmech.2004.02.015

European Journal of Medicinal Chemistry 39 (2004) 639–645
www.elsevier.com/locate/ejmech
Laboratory note
Studies on the trypanocidal activity of semi-synthetic
pyran[b-4,3]naphtho[1,2-d]imidazoles from b-lapachone
a
b
b
a
Kelly C.G. De Moura , Kelly Salomão , Rubem F.S. Menna-Barreto , Flávio S. Emery ,
a
a
b,
Maria do Carmo F.R. Pinto , Antônio V. Pinto , Solange L. de Castro *
a
Núcleo de Pesquisas em Produtos Naturais, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro 21944-970, Brazil
b
Laboratório de Biologia Celular, DUBC, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, Manguinhos, CP 926,
Rio de Janeiro 21045-900, Brazil
Received 22 September 2003; received in revised form 29 January 2004; accepted 12 February 2004
Available online 17 June 2004
Abstract
We synthesized new naphthoimidazoles from b-lapachone with an aromatic moiety linked to the imidazole ring, using phenylic and
heterocyclic aldehydes. The most active compound against Trypanosoma cruzi had a p-methyl group linked to the phenyl ring, presenting an
EC50 value of 15.5 ± 2.9 µM. No reliable correlation could be established with the biological activity and the structure of in the phenylic series.
For the heterocyclic series, activity was associated with a three bond-distance from nitrogen to the imidazole ring, in accordance with our
previous work.
©
2004 Elsevier SAS. All rights reserved.
Keywords: Trypanosoma cruzi; Chagas’ disease; Naphthoquinones; b-lapachone; Naphthoimidazoles; Chemotherapy
1
. Introduction
and safe agents for the treatment of this disease [6], since the
available nitroheterocyclic (benznidazole) presents severe
side effects, and its efficacy depends on the susceptibility of
different parasite populations. b-lapachone, among several
natural naphthoquinones, showed the highest activity against
T. cruzi, leading to the generation of free radicals and inhibi-
tion of nucleic acid and protein synthesis [7–9].
Due to the variety of microbicidal effects, the easy access
to natural sources of quinones from Brazilian flora, and the
synthetic alternative routes already developed by our group
for the reactivity of quinoidal carbonyl towards nucleophylic
agents, we took naphthoquinones, especially b-lapachone, as
starting points for medicinal chemistry studies [7,10–17]. We
have previously synthesized several derivatives from the re-
action of naphthoquinones isolated from Tabebuia (Tecoma)
spp., leading to naphthoimidazoles, naphthoxazoles, and
other groups of heterocyclics. The original quinones plus
Naphthoquinones present a broad distribution in the plant
kingdom and their structures endow them with redox proper-
ties, which could interfere in different biological oxidative
processes [1]. In folk medicine, plants containing naphtho-
quinones have been employed for the treatment of several
diseases [2]. Among naturally occurring naphthoquinones
we have lapachol and b-lapachone, from the heartwood of
trees of Bignoniaceae and Verbanaceae families. Their mi-
crobicidal activity had been described, in Brazil, by
Gonçalves Lima et al. [3], and subsequent studies described
other biological activities, such as anti-tumoural, anti-
inflammatory, bactericidal, fungicidal and virucidal [4].
The potential use of b-lapachone and semi-synthetic de-
rivatives as chemotherapeutic agent against Chagas disease
has been investigated. This disease, endemic in Latin
America, is caused by the protozoa Trypanosoma cruzi and
affects about 16–18 million people [5]. Efforts have been
addressed by several research groups to find more efficient
37 derivatives were assayed against trypomastigotes of
T. cruzi, the infective form for the mammalian host [4,18,19].
Among the 13 naphthoimidazoles and 14 naphthoxazoles
assayed, 50% of the compounds presented activity higher
than crystal violet, indicating a trend of trypanocidal activity
among these naphthalenic heterocyclics. Two naphthoimida-
*
Corresponding author. Tel.: +55-21-2598-4624;
fax: +55-21-2260-4434.
E-mail address: solange@ioc.fiocruz.br (S.L. de Castro).
©
2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.02.015

640
K.C.G. De Moura et al. / European Journal of Medicinal Chemistry 39 (2004) 639–645
NH
O
O
N
N
NH
NH
O
O
O
1
2
3
R4
R3
R5
N
R2
R6
R1
N
NH
NH
O
O
A
B
R6
4
5
6
1
1
1
1
1
1
1
2
2
,7,10 R =X
R -R =H
2 5
1
N
,8,11 R =X
R ,R -R =H
1 3 5
2
2
2
,9,12 R =X
R ,R ,R ,R =H
3
1 2 4 5
3
4
5
6
7
8
9
0
1
R =CH R -R =H
N
1
3
2
5
R =CH R ,R -R =H
23
2
3
1
3
5
R =CH R ,R ,R ,R =H
3
3
1
2
4
5
R =CF R -R =H
1
3
2
5
R =CF R ,R -R =H
2
3
1
3
5
R =CF R ,R ,R ,R =H
3
3
1
2
4
5
R =CN R -R =H
1
2
5
R =CN R ,R -R =H
2
1
3
5
R =CN R ,R ,R ,R =H
3
1
2
4
5
4-6 X=F; 7-9 X=Cl; 10-12 X=Br
Scheme 1. Structures of the naphtoimidazoles obtained from b-lapachone.
zoles derived from b-lapachone (1), with the aromatic moi-
eties phenyl (2) and 3-indolyl (3) linked to the imidazole
ring, showed the highest activity against the parasite. These
results stimulated us to continuing the screening of the naph-
thoimidazoles, synthesizing new derivatives with structures
related to 2 and 3 (Scheme 1).
heartwood of Tabebuia sp. and purified by recrystalizations
as previously described [13].
The naphtoimidazoles were synthesized by the reaction of
b-lapachone and the proper aldehyde, in the presence of
ammonium acetate [4,18,19]. The spectral data for the syn-
thesized compounds are in agreement with the structures
depicted in Scheme 1.
2. Chemistry
3
. Results and discussion
b-lapachone was obtained through cyclization in sulfuric
acid by nucleophylic attack of oxygen of the isoprenyl side
chain of lapachol, which was extracted in large scale from the
Although quinoidal substances are important sources of
heterocyclics, there are only a few reports about the reactivity

K.C.G. De Moura et al. / European Journal of Medicinal Chemistry 39 (2004) 639–645
641
Table 1
Effect of naphthoimidazoles derived from b-lapachone against T. cruzi
a
#
1
2
3
4
5
6
7
8
9
1
EC50/24 h (µM)
RA
b
b
d
d
d
c
391.5 ± 16.5
1.37
14.49
34.81
2.20
1.44
5.47
13.60
0.50
0.23
0.27
3.63
6.31
5.90
14.29
34.58
1.20
4.16
2.36
–
37.0 ± 0.7
15.4 ± 0.2
243.3 ± 24.6
372.0 ± 38.7
98.0 ± 4.8
39.4 ± 8.1
1064.2 ± 61.6
2286.3 ± 21.1
2004.0 ± 22.9
147.8 ± 12.5
84.9 ± 3.2
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
3
4
5
6
7
8
9
0
1
2
3
90.8 ± 5.8
Scheme 2. Naphthoimidazoles with pyridinyl and quinolyl rings (22 and
37.5 ± 12.8
15.5 ± 2.9
2
3), in which the nitrogen atom of the heterocyclic substituent are be located
three bonds-distant from its point of attachment to the pyran[b-
4,3]naphtho[1,2-d]imidazolic skeleton.
448.0 ± 55.7
128.7 ± 29.4
227.5 ± 58.0
>8000
non-substituted imidazole (2). Although the compounds
4–21 have substituents with different size and electronic
characteristics, no consistent correlation between their struc-
tures and biological activity could be established. The only
association that could be suggested is that all the substituents
are prone to enhance the lipophylic character around the
phenyl moiety, condition that would facilitate non-polar–
non-polar interactions with vital biomolecules of the para-
site.
For the heterocyclic series two derivatives with the nitro-
gen heteroatom located at a three bonded-distance from the
imidazolic ring, R = 3′-pyridinyl (22) and 3′-quinolyl (23)
(Scheme 1B), were also active against T. cruzi. These results
are in accordance with our previous work that showed that
the naphthoimidazole (3) with a 3′-indolic substituent at the
imidazole moiety, was 34.8 times more active than crystal
violet [19].
In relation to the mechanism of action, we can exclude
damage to the parasite caused by oxidative stress since,
unlike the original b-lapachone, 4–21 do not easily undergo
redox reactions. However, it is possible that the compounds
of in the phenyl series (Scheme 1A), with smaller lipophilic
phenyl substituted groups, could act through intercalation
within a polar regions of proteins. On the other hand, for the
heterocyclic series (Scheme 1B), the planar substituted het-
erocyclic rings at the 2-position could facilitate a polar–polar
interaction of the compounds with macromolecules through
the non-bonding electron pair of the nitrogen atom. The
present results and those previously reported [19] led us to
suggest that for enhancement of the trypanocidal activity, the
nitrogen atom of the heterocyclic substituent should be lo-
cated three bonds-distant from its point of attachment to the
pyran[b-4,3]naphtho[1,2-d]imidazolic skeleton (Scheme 2).
In previous reports [4,18,19], we have already detected
that the cyclofunctionalizations of b-lapachone to imidazolic
structures led to two of the most active derivatives with
phenyl (2) and 3-indolyl (3) linked to the imidazolic ring.
The present work shows another very active naphthoimi-
518.5 ± 78.9
1095.9 ± 92.9
154.9 ± 10.4
190.5 ± 30.3
536.0 ± 3.0
1.03
0.49
3.46
2.81
1.00
CV
a
b
c
d
RA (relative activity) = ED50 CV/ED50 compound.
Ref. [18].
Mean ± standard deviation of at least four separate experiments.
Ref. [4].
of 1,2-quinoidal carbonyls towards nucleophylic reagents
4,14,15,18]. In this work, through the reaction of b-lapa-
[
chone with phenylic and heterocyclic aromatic aldehydes in
the presence of ammonium acetate were synthesized a series
of pyran[b-4,3]naphtho[1,2-d]imidazoles with substituted
aromatic rings at the 2-position. The structural pattern of the
synthesized compounds can be divided according to the
groups appended at the imidazole ring: (A) phenyl with
halogens (F, Cl, Br), methyl, trifluoromethyl or cyane, and
with disubstituted with hydroxy plus nitro (4–21) (Scheme
1
A); (B) pyridinyl and quinolyl rings (22 e23) (Scheme 1B).
The new naphthoimidazoles were assayed against blood-
stream forms of T. cruzi and only six of them showed lower
activity when compared with crystal violet (Table 1). Three
derivatives (o-fluoro, m-fluoro and m-chloro) (4,5,8) have
been previously published [4], but were included in order to
compare with the new related phenyl derivatives here de-
scribed. Among the substituted phenyl derivatives (Scheme
A), those more active against the parasite were p-fluoro (3),
o-chloro (7), p-bromo (11) and the three methyl derivatives
10–12), all with EC /24 below 100 µM. The assays with
trifluoromethyl derivatives (16–18) showed EC50 values in
the range of 128–448 µM. As their synthesis was performed
aiming to find a correlation between trypanocidal activity
and structure, it was possible to verify that only one com-
pound of this series (15), with a methyl group at the para
position in the phenyl ring showed higher activity than the
1
(
50

642
K.C.G. De Moura et al. / European Journal of Medicinal Chemistry 39 (2004) 639–645
dazole, compound 15, with p-methyl as substituent
EC /24 h = 15.5 ± 2.9 µM). In continuity to our work we
4.2.1. 4,5-dihydro-6,6-dimethyl-6H-2-(2′-fluorophenyl)-
pyran[b-4,3]naphth[1,2-d]imidazole (4)
(
50
will investigate in more detail these three naphthoimidazoles,
assaying their effect against intracellular amastigotes, toxic-
ity to mammalian cells, possible alterations in ultrastructural
organization of the parasite, aiming to detect possible or-
ganelle(s) susceptible to these derivatives, and also the direct
effect of these compounds upon selected targets in T. cruzi.
The aldehyde employed was 2-fluorobenzaldehyde after
30 min of reflux. This compound was obtained in 91% yield,
m.p. 195 °C.
UV kmax nm (log e) 348 (4.22), 290 (4.18), 253.0 (4.48),
–
1
223 (4.48). IR (KBr) cm 3423, 3152, 3107, 3064, 2974,
2948, 2929, 1599, 1583, 1462, 1444, 1252, 1223, 1160,
1
120, 1056, 952, 880, 772, 758, 733. MS m/z (%) 346 (29),
3
47 (7), 317 (12), 303 (8), 290 (67), 275 (2): 262 (6), 241 (3).
1
H NMR (CDCl ) d 9.8 (ls, 1H), 8.5 (dd, 1H), 8.3 (d, 1H), 7.4
3
4. Experimental section
(m, 2H), 7.2 (m, 4H), 3.0 (sl, 2H), 1.95 (t, 2H), 1.4 (s, 6H).).
4.1. Chemistry
4
.2.2. 4,5-dihydro-6,6-dimethyl-6H-2-(3′-fluorophenyl)-
pyran[b-4,3]naphth[1,2-d]imidazole (5)
Melting points were determined in a capillary Thomas
Hoover apparatus and are uncorrected. H and C NMR
were recorded using a Varian Gemini 200 and Brucker
The aldehyde employed was 3-fluorobenzaldehyde after
20 min of reflux. This compound was obtained in 90% yield,
m.p. 238 °C.
1
13
2
00 MHz spectrophotometer, in the solvents indicated, with
UV kmax nm (log e) 351 (4.48), 295 (4.38), 255 (3.60): 227
–
1
TMS as internal standard at room temperature. Chemical
shifts (d) are given in ppm and coupling constants (J) in
Hertz. Infrared spectra were recorded on a Perkin–Elmer
(4.62), 208 (4.65). IR (KBr) cm 3447, 3105, 3074, 2975,
2927, 2849, 1615, 1584, 1485, 1473, 1458, 1212, 1159,
1121, 1056, 969, 954, 885, 866, 766, 713. MS m/z (%) 346
7
83 spectrophotometer and Nicolet IRTF in KBr pellets or
liquid films. UV spectra were obtained in Shimadzu UV-
601 spectrophotometer in ethanol or hexane. The mass
(45), 347 (11), 331 (1), 317 (2), 303 (8), 290 (100), 275 (2),
1
261 (5). H NMR ((CD3)
CO) d 8.5 (d, 1H), 8.24 (d, 1H), 8.1
2
1
(d, 1H), 8.01 (m, 1H), 7.5 (m, 2H), 7.4 (dd, 1H), 7.15 (m,
1
3
spectra were obtained at 70 eV in a VG Autospec apparatus.
The fragments were described as a relation between atomic
mass units and the charge (m/z) and the relative abundance in
percentage. Elemental analysis of the compounds was
within ±0.4% of the theoretical values. Column chromatog-
raphy was performed using silica gel (0.063–0.200 mm/70–
1H), 3.1 (t, 2H), 2.0 (t, 2H), 1.4 (s, 6H). C NMR (CD CO)
3
d 1416 (s), 1412 (s), 1404 (s), 1304 (s), 1295 (d), 1294 (s),
1293 (s), 1292 (d), 1291 (d), 1286 (d), 1277 (s), 1231 (d),
1229 (d), 1218 (d), 1218 (d), 1217 (d), 1213 (d), 1038 (s), 915
(d), 745 (s), 325 (t), 260 (q), 220 (t).
4
.2.3. 4,5-dihydro-6,6-dimethyl-6H-2-(4′-fluorophenyl)-
230 mesh). Removal of the solvents during isolation and
pyran[b-4,3]naphth[1,2-d]imidazole (6)
purification of the compounds was done in a Büchi rotatory
evaporator under reduced pressure. When necessary, a high
vacuum system (0.1–10 mm Hg) was also used.Visualization
of the compounds on the chromatographic plates was done
under ultraviolet light, exposure to iodine vapor or spraying
The aldehyde employed was 4-fluorobenzaldehyde after
2
5 min of reflux. This compound was obtained in 90% yield,
m.p. 295 °C, C H N OF.
22 19
2
UV kmax nm (log e) 346 (4.26), 291 (4.26), 254 (4.45), 212
–
1
(
4.50). IR (KBr) cm 3445, 3172, 3134, 3077, 3048, 2978,
2% cerium sulfate in 2 N sulfuric acid followed by gentle
2
1
1
7
2
972, 2955, 2932, 2697, 1631, 1611, 1585, 1548, 1528,
488, 1435, 1379, 1372, 1333, 1287, 1259, 1232, 1197,
160, 1197, 1144, 1054, 1015, 970, 953, 881, 835, 816, 769,
26, 688, 647, 634, 580, 518. MS m/z (%) 346 (2), 290 (4),
heating.
4.2. General procedure for the preparation
of the naphthoimidazoles 4–23
1
52 1, 140 (0,5), 44 (12), 40 (100). H NMR (DMSO) d 8.40
(
d, 1H), 8.30 (m, 2H), 8.10 (d, 1H), 7.50 (t, 1H), 7.40 (m,
To a solution of b-lapachone (1.1 mmoles) in acetic acid
3H), 3.1 (t, 2H), 2.00 (t, 2H), 1.40 (s, 6H).
(6 ml), the proper aldehyde (2.5 mmoles) was added and the
mixture gently heated till 70 °C; at this point ammonium
acetate (16.5 mmoles) was slowly added and reflux was
maintained for a determined time. The course of all the
reactions was monitored by thin layer chromatography. At
the end of the reaction, after addition into water, the precipi-
tate was purified by column chromatography using as eluent
a mixture of hexane/ethyl acetate, with the ratio 9:1, with
increasing polarity gradient, as previously described [18].
Other minor products, of oxazole type, were also isolated and
are under study.
4.2.4. 4,5-dihydro-6,6-dimethyl-6H-2-(2′-chlorophenyl)-
pyran[b-4,3]naphth[1,2-d]imidazole (7)
The aldehyde employed was 2-chlorobenzaldehyde after
30 min of reflux. This compound was obtained in 85% yield,
m.p. 134 °C.
UV kmax nm (log e) 340.8 (3.62), 288.2 (3.58), 253.2
–
1
(4.09), 220.2 (4.12), 202.4 (4.05). IR (KBr) cm 3644, 3445,
3220, 3166, 3123, 3068, 2970, 2939, 2926, 2841, 2657,
1609, 1589, 1567, 1475, 1463, 1449, 1439, 1381, 1367,
1341, 1321, 1260, 1242, 1159, 1121, 1061, 1037, 953, 881,

K.C.G. De Moura et al. / European Journal of Medicinal Chemistry 39 (2004) 639–645
643
7
3
66, 755, 722, 695, 653, 630, 462, 453. MS m/z (%) 364(12),
62 (36), 319 (7), 308 (36), 306 (100), 271 (3), 181 (4), 166
2926, 2849, 1593, 1566, 1446, 1367, 1341, 1259, 1242,
1160, 1145, 1121, 1056, 968, 881, 765, 719, 709, 694, 648.
MS m/z (%) 409 (11), 408 (47), 407 (17), 406 (51), 365 (7),
363 (8), 353 (33), 352 (100), 351 (36), 350 (98), 271 (12),
(7), 148 (8,5), 140 (37), 130 (14), 115 (22), 102 (14), 89 (11),
1
7
7
7 (5). H NMR (CDCl ) d 8.50 (d, 2H), 8.30 (d, 2H),
3
.70–7.30 (m, 4H), 3.10 (t, 2H), 2.00 (t, 2H), 1.10 (s, 6H).
242 (10), 195 (5), 169 (5), 155 (10), 148 (15), 140 (26), 130
1
(
10), 115 (13), 114 (11), 113 (8), 102 (8), 89 (6), 44 (29). H
4
.2.5. 4,5-dihydro-6,6-dimethyl-6H-2-(3′-chlorophenyl)-
NMR (DMSO) d 13.24 (s, 1H), 12.78 (s, 1H), 8.44 (d, 1H),
8.30–8.20 (m, 1H), 8.20–8.10 (m, 1H), 7.70–7.66 (m, 4H),
3.16–2.96 (m, 2H), 2.04–1.86 (m, 2H), 1.40 (s, 6H).
pyran[b-4,3]naphth[1,2-d]imidazole (8)
The aldehyde employed was 3-chlorobenzaldehyde after
25 min of reflux. This compound was obtained in 84% yield,
m.p. 234 °C.
4.2.9. 4,5-dihydro-6,6-dimethyl-6H-2-(4′-bromophenyl)-
pyran[b-4,3]naphth[1,2-d]imidazole (12)
The aldehyde employed was 4-bromo benzaldehyde after
30 min of reflux. This compound was obtained in 85% yield,
m.p. 280 °C.
UV kmax nm (log e) 354 (4.41), 296 (4.31), 253 (4.54), 210
–
1
(4.63). IR (KBr) cm 3095, 3074, 3051, 2973, 2947, 2928,
1
7
3
595,1587, 1446, 1367, 1259, 1159, 1121, 1056, 970, 951,
78, 721. MS m/z (%) 364 (12), 363 (10), 362 (39), 347 (2.5),
19 (6), 306 (100), 292 (1), 271 (4), 242 (7). H NMR
1
UV kmax nm (log e) 354 (4.29), 297 (4.20), 254 (4.41),
–
1
(
CDCl ) d 8.5 (d, 1H), 82 (m, 1H), 8.1 (d, 2H), 7.5 (m, 1H),
227.5 (4.43), 205 (4.52). IR (KBr) cm 3435, 3219, 3183,
3136, 3069, 2975, 2927, 1633, 1585, 1519, 1432, 1381,
3
7.4 (m, 3H), 3.0 (t, 2H), 1.9 (t, 2H), 1.4 (s, 6H).
1
333, 1258, 1242, 1160, 1144, 1119, 1055, 1011, 953, 828,
4
.2.6. 4,5-dihydro-6,6-dimethyl-6H-2-(4′-chlorophenyl)-
771, 722, 646. MS m/z (%) 409 (12), 408 (45), 407 (16), 406
(45), 365 (6), 363 (8), 353 (32), 352 (100), 351 (31), 350
(94), 271 (10), 242 (10), 195 (5), 169 (6), 155 (11), 148 (12),
142 (11), 140 (24), 130 (10), 115 (17), 114 (11), 113 (8), 102
pyran[b-4,3]naphth[1,2-d]imidazole (9)
The aldehyde employed was 4-chlorobenzaldehyde after
20 min of reflux. This compound was obtained in 81% yield,
1
m.p. 288 °C.
(8), 89 (10), 44 (23). H NMR (DMSO) d 8.44 (d, 1H), 8.20
UV kmax nm (log e) 353.2 (4.16), 296.2 (4.06), 254.2
4.28), 227.2 (4.28). IR (KBr) cm 3619, 3432, 3177, 2975,
(m, 3H), 7.76 (d, 2H), 7.60-7.50 (m, 2H), 1.44 (s, 6H).
–1
(
2
1
3
931, 1633, 1587, 1519, 1432, 1377, 1338, 1258, 1159,
4.2.10. 4,5-Dihydro-6,6-dimethyl-6H-2-(2′-methylphenyl)-
pyran[b-4,3]naphth[1,2-d] imidazole (13)
The aldehyde employed was 2-methylbenzaldehyde after
50 min of reflux. This compound was obtained in 70% yield,
m.p. 135 °C.
118, 1054, 1013, 953, 834, 769, 722. MS m/z (%) 364 (2.5),
1
62 (5.7), 319 (0.8), 306 (12), 140 (1,8), 44 (12), 40 (100). H
NMR ((CD3) CO) d 8.50 (d, 1H), 8.30 (d, 2H), 8.10 (d, 1H),
2
7
6
.60 (m, 3H), 7.40 (m, 1H), 3.10 (t, 2H), 1.90 (t, 2H), 1.40 (s,
H).
UV kmax nm (log e) 340 (3.61), 252.2 (4.10), 221.2 (4.10).
–
1
IR (KBr) cm 3654, 3099, 3057, 3012, 2971, 2945, 2923,
2848, 2795, 2707, 2653, 2597, 1633, 1587, 1548, 1519,
1487, 1430, 1402, 1382, 1366, 1322, 1259, 1240, 1205,
1158, 1121, 1091, 1055, 1027, 966, 952, 882, 838, 768, 760,
728, 719, 703, 674, 644, 629, 598, 465, 448. MS m/z (%) 343
(9), 342 (44), 327 (3), 300 (2), 299 (7), 287 (34), 286 (100),
258 (1), 257 (5), 156 (5), 154 (7), 143 (6), 142 (7), 130 (8),
129 (7), 115 (8), 114 (4), 102 (4), 41 (4). H NMR (DMSO)
d 8.40 (d, 1H), 8.20 (d, 1H), 7.80 (m, 1H), 7.50–7.30 (m, 4H),
3.10 (t, 2H), 2.70 (s, 3H), 1.90 (t, 2H), 1.40 (s, 6H).
4
.2.7. 4,5-dihydro-6,6-dimethyl-6H-2-(2′-bromophenyl)-
pyran[b-4,3]naphth[1,2-d]imidazole (10)
The aldehyde employed was 2-bromo benzaldehyde after
30 min of reflux. This compound was obtained in 78% yield,
m.p. 135 °C.
UV kmax nm (log e) 354 (4.36), 296.2 (4.25), 252.6 (4.49,
–1
1
2
2
1
4
12.6 (4.57). IR (KBr) cm 3418, 3071, 3048, 2973, 2928,
847, 1587, 1567, 1444, 1366, 1259, 1242, 1159, 1144,
121, 1056, 969, 765, 720, 709, 693. MS m/z (%) 409 (12),
08 (49), 407 (17), 406 (54), 365 (7), 363 (7), 353 (32), 352
(
(
(
1
(
94), 351 (36), 350 (100), 242 (8), 195 (4,5), 169 (5.6), 155
4.2.11. 4,5-Dihydro-6,6-dimethyl-6H-2-(3′-methylphenyl)-
pyran[b-4,3]naphth[1,2-d] imidazole (14)
The aldehyde employed was 3-methylbenzaldehyde after
45 min of reflux. This compound was obtained in 73% yield,
m.p. 230 °C.
8), 140 (33), 130 (8), 115 (13), 114 (11), 113 (9), 102 (9), 89
1
8), 41 (6). H NMR ((CD3) CO) d 12,42 (sl, 1H), 11,88 (s,
2
H), 8,56 (d, 1H), 8,38 à 8,22 (m, 1H), 7,88 (dd, 1H), 7,76
dd, 1H), 7,62 à 7,32 (m, 4H), 1,44 (s, 6H).
UV kmax nm (log e) 347.4 (4.29), 292.8 (4.28), 254.4
–
1
4
.2.8. 4,5-dihydro-6,6-dimethyl-6H-2-(3′-bromophenyl)-
(4.45), 213 (4.49). IR (KBr) cm 3153, 3093, 3050, 3011,
2972, 2944, 2926, 2848, 2809, 27858, 2702, 2651, 1631,
1604, 1584, 1542, 1444, 1435, 1366, 1322, 1260, 1242,
1208, 1159, 1142, 1121, 1099, 1056, 1039, 1018, 969, 950,
906, 881, 848, 833, 784, 767, 722, 711, 687, 660, 647, 634,
597, 543. MS m/z (%) 343 (11), 342 (43), 341 (9), 327 (3),
300 (3), 299 (7), 287 (36), 286 (100), 258 (2), 257 (4), 162
pyran[b-4,3]naphth[1,2-d]imidazole (11)
The aldehyde employed was 3-bromo benzaldehyde after
0 min of reflux. This compound was obtained in 75% yield,
4
m.p. 251 °C.
UV kmax nm (log e) 354.4 (4.34), 296.2 (4.22), 252.8
–1
(4.48), 212.2 (4.56). IR (KBr) cm 3646, 3434, 3068, 2973,

644
K.C.G. De Moura et al. / European Journal of Medicinal Chemistry 39 (2004) 639–645
(
(
1
1
1
8), 156 (6), 154 (7), 143 (4), 141 (5), 140 (8), 130 (7), 129
3), 115 (8), 114 (4), 102 (4), 441 (11). H NMR (DMSO) d
3.1 (s, 1H), 12.6 (s, 1H), 8.40 (d, 1H), 8.10 (m, 3H), 7.50 (t,
H), 7.40 (t, 2H), 7.10 (d, 1H), 3.10 (sl, 2H), 2.20 (s, 3H),
.90 (t, 2H), 1.40 (s, 6H).
4.2.15. 4,5-dihydro-6,6-dimethyl-6H-2-(4′-trifluorometh-
ylphenyl)-pyran[b-4,3]naphth[1,2-d]imidazole (18)
The aldehyde employed was 4-trifluoromethylbenzal-
dehyde after 15 min of reflux. This compound was obtained
in 92% yield, m.p. 200 °C.
1
UV kmax nm (log e) 359.8 (4.32), 299.2 (4.13), 250.4
–
1
4
.2.12. 4,5-dihydro-6,6-dimethyl-6H-2-(4′-methylphenyl)-
(4.39), 228.8 (4.41). IR (KBr) cm 3624, 3208, 3160, 3128,
3075, 3030, 2976, 2947, 2923, 2851, 2708, 1632, 1617,
pyran[b-4,3]naphth[1,2-d] imidazole (15)
1
1
7
604, 1587, 1576, 1449, 1443, 1370, 1325, 1287, 1260,
245, 1165, 1122, 1066, 1058, 1014, 958, 883, 850, 814, 764,
48, 716, 697, 686, 648, 594, 501. MS m/z (%) 397 (9), 396
The aldehyde employed was 4-methylbenzaldehyde after
5 min of reflux. This compound was obtained in 78% yield,
m.p. 250 °C.
4
(38), 353 (6), 341 (32), 340 (100), 242 (1), 198 (3), 180 (1),
UV kmax nm (log e) 347 (4.21), 293 (4.22), 255 (4.37),
–1
173 (2), 153 (2), 152 (3), 140 (11), 130 (5), 115 (9), 114 (6),
2
2
1
1
9
3
(
(
3
09.5 (4.63). IR (KBr) cm 3201, 3166, 3132, 3075, 3044,
971, 2952, 2930, 2851, 2755, 2693, 1631, 1599, 1585,
574, 1527, 1488, 1459, 1442, 1433, 1379, 1370, 1332,
319, 1283, 1259, 1242, 1159, 1144, 1119, 1054, 1018, 970,
53, 880, 840, 820, 769, 724, 690, 646, 638, 508. MS m/z (%)
1
1
1
13 (4), 102 (3), 44 (17). H NMR ((CD3) CO) d 8.50 (sl,
2
H), 8.40 (d, 2H), 8.30 (d, 1H), 7.80 (t, 1H), 7.40 (t, 1H), 3.10
(tl, 2H), 2.00 (t, 2H), 1.40 (s, 6H).
4
.2.16. 4,5-dihydro-6,6-dimethyl-6H-2-(2′-cyanephenyl)-
42 (4), 287 (4), 286 (13(, 149 (4), 59 (4), 58 (26), 44 (37), 43
1
pyran[b-4,3]naphth[1,2-d]imidazole (19)
100), 42 (9), 41 (5). H NMR (DMSO) d 13.2 (sl, 1H), 12.7
The aldehyde employed was 2-cyanebenzaldehyde after
sl, 1H), 8.40 (d, 1H), 8.20 (d, 3H), 7.50 (t, 1H), 7.30 (t 3H),
.10 (t, 2H), 2.40 (s, 3H), 2.00 (sl, 2H), 1.40 (s, 6H).
1
5 min of reflux. This compound was obtained in 68% yield,
m.p. 192 °C.
UV kmax nm (log e) 363.5 (4.00), 283 (4.00), 253 (4.35),
4.2.13. 4,5-dihydro-6,6-dimethyl-6H-2-(2′-trifluorometh-
–
1
2
3
1
1
7
3
1
2
3
22 (4.44). IR (KBr) cm 3578, 3413, 3222, 3169, 3122,
067, 2973, 2942, 2923, 2224, 1721, 1712, 1665, 1610,
590, 1523, 1485, 1441, 1383, 1368, 1341, 1323, 1296,
260, 1242, 1192, 1161, 1120, 968, 962, 884, 768, 760, 747,
38, 714, 695, 657, 646, 633, 555, 531, 499, 461. MS m/z (%)
54 (3), 353 (11), 336 (4), 298 (8), 297 (33), 140 (5), 115 (5),
02 (3), 44 (100). H NMR (DMSO) d 8,40 (d, 1H), 8,20 (t,
H), 8,00 (d, 1H), 7,80 (m, 1H), 7,60 (m, 2H), 7,50 (d, 1H),
,00 (t, 2H), 2,00 (t, 2H), 1,40 (s, 6H).
ylphenyl)-pyran[b-4,3]naphth[1,2-d]imidazole (16)
The aldehyde employed was 2-trifluoromethylbenzal-
dehyde after 15 min of reflux. This compound was obtained
in 86% yield, m.p. 146 °C.
UV kmax nm (log e) 338.6 (3.81), 289.4 (3.71), 253.4
–1
(
4.39), 221.8 (4.38). IR (KBr) cm 3688, 3645, 3446, 3160,
1
3
1
1
8
108, 3070, 3023, 2971, 2925, 2867, 2823, 1633, 1602,
587, 1548, 1526, 1445, 1434, 1383, 1369, 1316, 1289,
261, 1242, 1161, 1122, 1058, 1054, 1035, 967, 952, 882,
39, 766, 739, 719, 687, 647, 629, 596, 551, 530, 446. MS
4
.2.17. 4,5-dihydro-6,6-dimethyl-6H-2-(3′-cyanephenyl)-
m/z (%) 397 (23), 396 (89), 353 (7), 342 (33), 340 (90), 321
pyran[b-4,3]naphth[1,2-d] imidazole (20)
(
(
4
37), 320 (100), 292 (13), 291 (17), 273 (14), 198 (5), 180
The aldehyde employed was 3-cyanebenzaldehyde after
0 min of reflux. This compound was obtained in 72% yield,
11), 173 (25), 153 (8), 152 (13), 140 (11), 113 (6), 102 (7),
2
1
4 (23). H NMR (CDCl ) d 8.30 (d, 2H), 7.90–7.70 (ml,
3
m.p. 265 °C.
2H), 7.40 (m, 4H), 3.00 (sl, 2H), 1.90 (t, 2H), 1.40 (s, 6H).
UV kmax nm (log e) 361 (4.35), 296 (4.22), 255 (4.51),
–
1
222.5 (4.63). IR (KBr) cm 3433, 3287, 3065, 2967, 2936,
2919, 2849, 2242, 1665, 1594, 1584, 1515, 1486, 1446,
1433. 1385, 1370, 1334, 1286, 1268, 1259, 1244, 1201,
1161, 1146, 1123, 1059, 1016, 975, 967, 952, 886, 800, 761,
714, 680, 661, 648, 596, 587, 475. MS m/z (%) 354 (9), 353
4.2.14. 4,5-dihydro-6,6-dimethyl-6H-2-(3′-trifluorometh-
ylphenyl)-pyran[b-4,3]naphth[1,2-d]imidazole (17)
The aldehyde employed was 3-trifluoromethylbenzal-
dehyde after 15 min of reflux. This compound was obtained
in 88% yield, m.p. 193 °C.
(43), 336 (4), 310 (4), 298 (33), 297 (100), 161 (6), 140 (11),
1
UV kmax nm (log e) 357.8 (4.22), 355.4 (4.23), 295.4
115 (11), 102 (5), 44 (22). H NMR (DMSO) d 8.60 (m, 2H),
8.40 (d, 1H), 8.20 (d, 1H), 7.80 (d, 1H), 7.70 (t, 1H), 7.60 (t,
1H), 7.40 (t, 1H), 3.00 (t, 2H), 1.90 (t, 2H), 1.30 (s, 6H).
(
4.11), 253.8 (4.34), 228.2 (4.34), 212.2 (4.33). IR (KBr)
–1
cm 3647, 3071, 3053, 2974, 2924, 2870, 2848, 2781, 2733,
2
1
1
6
3
696, 1584, 1520, 1485, 1456, 1447, 1353, 1341, 1324,
318, 1305, 1281, 1258, 1239, 1169, 1160, 1122, 1071,
057, 970, 953, 907, 883, 841, 806, 765, 730, 720, 711, 695,
48, 621, 596, 531, 450, 418. MS m/z (%) 397 (10), 396 (45),
53 (7), 341 (35), 340 (100), 292 (1), 291 (1.7), 273 (1), 198
4.2.18. 4,5-dihydro-6,6-dimethyl-6H-2-(4′-cyanephenyl)-
pyran[b-4,3]naphth[1,2-d] imidazole (21)
The aldehyde employed was 4-cyanebenzaldehyde after
25 min of reflux. This compound was obtained in 76% yield,
m.p. 160 °C.
(
(
3), 179 (2), 173 (3), 153 (3), 152 (3), 140 (12), 115 (10), 114
6), 102 (3), 41 (5). H NMR (CDCl ) d 8.50–8.20 (ml, 4H),
1
UV kmax nm (log e) 381 (4.95), 305 (4.01), 293 (4.01), 251
3
–
1
7.60–7.40 (ml, 4H), 3.00 (t, 2H), 1.90 (t, 2H), 1.40 (s, 6H).
(4.37), 234 (4.42), 207.5 (4.37). IR (KBr) cm 3627, 3430,

K.C.G. De Moura et al. / European Journal of Medicinal Chemistry 39 (2004) 639–645
645
3
1
1
1
7
2
405, 3211, 3166, 3130, 3070, 2979, 2925, 2225, 2089,
706, 1601, 1586, 1562, 1525, 1483, 1452, 1440, 1384,
369, 1354, 1344, 1323, 1288, 1280, 1260, 1244, 1200,
182, 1164, 1122, 1060, 1039, 970, 958, 949, 880, 853, 808,
38, 716. MS m/z (%) 354 (10), 353 (42), 336 (4), 298 (31),
DME at twice the desired final concentrations in 96-well
microplates. After incubation for 24 h at 4 °C, the parasites
were counted in a Neubauer chamber. The activity of the
compounds was quantified by the EC50 that corresponds to
the concentration that lyses 50% of the parasite. Untreated
and crystal violet-treated parasites were used as controls
[20].
97 (100), 167 (8), 141 (6), 140 (11)), 130 (5), 115 (10), 114
1
(7), 102 (6), 41 (4). H NMR (DMSO) d 13.50 (sl, 1H), 13.00
(s, 1H), 8.40 (d, 3H), 8.20 (d, 1H), 8.00 (d, 2H), 7.50 (m, 1H),
3.00 (t, 2H), 2.00 (t, 2H), 1.40 (s, 6H).
Acknowledgements
4.2.19. 4,5-dihydro-6,6-dimethyl-6H-2-(3′-pyridinyl)-pyr-
an[b-4,3]naphth[1,2-d]imidazole (22)
This research was supported by grants from the Conselho
Nacional de Desenvolvimento Científico e Tecnológico
(CNPq), FIOCRUZ, Fundação de Amparo a Pesquisa do Rio
de Janeiro (FAPERJ), and Fundação Universitária José
Bonifácio/UFRJ.
The aldehyde employed was 3-pyridine carboxyaldehyde
after 40 min of reflux. This compound was obtained in 77%
yield, m.p. 146 °C.
UV kmax nm (log e) 356 (4.29), 293.5 (4.13), 254 (4.45),
–1
2
2
1
1
6
28.5 (4.44). IR (KBr) cm 3219, 3161, 3104, 3051, 2976,
924, 2843, 2727, 2661, 1588, 1575, 1448, 1422, 1382,
369, 1342, 1322, 1271, 1262, 1245, 1202, 1160, 1147,
120, 1060, 1028, 958, 883., 813, 767, 718, 705, 662, 649,
30, 598. MS m/z (%) 331 (1), 330 (4), 329 (16), 314 (1), 313
References
[1] A. Brunmark, E. Cadenas, Free Rad. Biol. Med 7 (1989) 435.
[2] P. Arenas, J. Ethnopharmacol 21 (1987) 279–295.
(
(
(
(
1), 301 (2), 289 (3), 274 (8), 273 (32), 150 (1), 130 (2), 115
2), 114 (1), 113 (1), 105 (2), 102 (1), 88 (1), 79 (1), 78 (2), 77
2), 55 (1), 51 (1), 44 (16), 41 (4), 40 (100). H NMR
DMSO) d 9.80 (d, 1H), 9.10 (d, 1H), 8.45 (d, 1H), 8.17 (m,
[
[
[
3] O. Gonçalves Lima, J.S. Coelho, I. D’Albuquerque, J.F. Mello,
D.G. Martins, A.L. Lacerda, M.A. Moraes e Souza, Rev. Inst. Anti-
biot. (Univ. Recife) 11 (1971) 21–26.
4] K.C.G. De Moura, F.S. Emery, C. Neves-Pinto, M.C.F.R. Pinto,
A.P. Dantas, K. Salomão, S.L. De Castro, A.V. Pinto, J. Braz. Chem.
Soc. 12 (2001) 325–338.
1
3
1
H), 7.88–7.70 (m, 1H), 7.75–7.56 (m, 2H), 7.54–7.42 (m,
H), 3.10 (t, 2H), 2.00 (t, 2H), 1.42 (s, 6H).
5] WHO, Chagas disease, 1997 13th Programme Report UNDP/TDR,
Genebra.
4.2.20. 4,5-dihydro-6,6-dimethyl-6H-2-(3′-quinolinyl)-pyr-
[
[
6] J.R. Coura, S.L. De Castro, Mem. Inst. Oswaldo Cruz 97 (2002) 3–24.
7] J.N. Lopes, F.S. Cruz, R. DoCampo, M.E. Vasconcellos, M.C.R. Sam-
paio, A.V. Pinto, B. Gilbert, Ann. Trop. Med. Parasitol. 72 (1978)
523–531.
an[b-4,3]naphth[1,2-d]imidazole (22)
The aldehyde employed was 3-quinoline carboxyalde-
hyde after 50 min of reflux. This compound was obtained in
7
5% yield, m.p. 166 °C.
UV kmax nm (log e) 384.5 (4.35), 285.5 (4.43), 255 (4.59),
[8] A. Boveris, A.O.M. Stoppani, R. DoCampo, F.S. Cruz, Comp. Bio-
chem. Physiol. 61C (1978) 327–329.
[9] S.G. Goijman, A.O.M. Stoppani, Arch. Biochem. Biophys. 240
–1
2
2
1
1
7
32 (4.72). IR (KBr) cm 3102, 3059, 2973, 2940, 2926,
849, 1740, 1636, 1587, 1576, 1499, 1484, 1147, 1402,
382, 1366, 1352, 1319, 1273, 1260, 1241, 1203, 1161,
145, 1122, 1097, 1040, 969, 952, 943, 913, 885, 862, 835,
81, 762, 745, 718, 705, 667, 645, 579, 475. MS m/z (%) 379
(
1985) 273–280.
[
10] A.V. Pinto, M.C.F.R. Pinto, B. Gilbert, J. Pelegrino, R.T. Mello, Trans.
Roy. Soc. Trop. Med. Hyg. 71 (1977) 133.
[11] A.V. Pinto, V.F. Ferreira, M.C.F.R. Pinto, L.U. Mayer, Synth. Com-
mun. 15 (1985) 1181–1189.
[
[
[
12] A.V. Pinto, M.C.F.R. Pinto, M.H. Lagrota, M.D. Wigg, A.N. Aguiar,
Rev. Latinoam. Microbiol. 29 (1987) 15–20.
13] A.V. Pinto, V.F. Ferreira, R.S. Capella, B. Gilbert, M.C.F.R. Pinto,
J.S. Da Silva, Trans. Roy. Soc. Trop. Med. Hyg. 81 (1987) 609–610.
14] A.V. Pinto, C. Neves-Pinto, M.C.F.R. Pinto, F.S. Emery, K.C.G. De
Moura, C.E.M. Carvalho, I.M. Brinn, Heterocycles 45 (1997) 2431–
(
(
1), 323 (3), 191 (2), 115 (1), 109(3), 77 92), 58 (9), 46 (5), 45
10), 44 (100), 43 (12), 42 (6). H NMR (DMSO) d 9.44 (s,
1
1
1
3
H), 9.43 (s, 1H), 9.40 (d, 1H), 8.68–8.62 (m, 1H), 8.59 (t,
H), 8.54 (t, 1H), 8.45 (d, 1H), 8.18 (d, 1H), 7.64–7.52 (m,
H), 3.15 (t, 2H), 2.00 (t, 2H), 1.44 (s, 6H).
2
436.
15] J.P. Chaves, M.C.F.R. Pinto, A.V. Pinto, J. Braz. Chem. Soc. 1 (1990)
1–27.
16] M.H. Lagrota, M.D. Wigg, A.N. Aguiar, A.V. Pinto, M.C.F.R. Pinto,
Rev. Latinoam. Microbiol. 28 (1986) 221.
17] C. Neves-Pinto,V.R.S. Malta, M.C.F.R. Pinto, R.H.A. Santos, S.L. De
Castro, A.V. Pinto, J. Med. Chem. 45 (2002) 2112–2115.
[18] A.V. Pinto, C. Neves-Pinto, M.C.F.R. Pinto, R.M. Santa-Rita, C. Pez-
zella, S.L. De Castro, Arzneim-Forsch 47 (I) (1997) 74–79.
[
[
[
4
.3. Trypanocidal assay
2
Stock solutions of the compounds (4–23) were prepared in
dimethylsulfoxide, with the final concentration of the solvent
in the experiments never exceeding 0.1%. Bloodstream
forms were obtained at the peak of parasitaemia from
T. cruzi-infected Swiss mice, isolated by differential cen-
trifugation and resuspended in Dulbecco’s modified Eagle
medium (DME) containing 10% mice blood to a concentra-
[
19] C. Neves-Pinto, A.P. Dantas, K.C.G. De Moura, F.S. Emery, P.F. Pole-
quevitch, M.C.F.R. Pinto, S.L. De Castro,A.V. Pinto,Arzneim-Forsch
5
0 (II) (2000) 1120–1128.
6
tion of 5 × 10 trypomastigotes/ml. This suspension (100 µl)
was added to the same volume of the compounds prepared in
[
20] S.L. De Castro, M.C.F.R. Pinto, A.V. Pinto, Microbios 78 (1994)
83–90.