Synthesis and anti-Trypanosoma cruzi activity of β-lapachone analogues

Article abstract of DOI:10.1016/j.ejmech.2011.03.012

The available chemotherapy for Chagas disease, caused by Trypanosoma cruzi, is unsatisfactory; therefore, there is an intense effort to find new drugs for the treatment of this disease. In our laboratory, we have analyzed the effect on bloodstream trypoma

Full text of DOI:10.1016/j.ejmech.2011.03.012

European Journal of Medicinal Chemistry 46 (2011) 3071e3077
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and anti-Trypanosoma cruzi activity of b-lapachone analogues
Sabrina Baptista Ferreira a, Kelly Salomão b, Fernando de Carvalho da Silva c, Antônio Ventura Pinto d,
,
Carlos Roland Kaiser a, Angelo C. Pinto a, Vitor Francisco Ferreira c, Solange L. de Castro b
*
a Departamento de Química Orgânica, Instituto de Química, UFRJ, 21949-900 Rio de Janeiro, RJ, Brazil
b Laboratório de Biologia Celular, Instituto Oswaldo Cruz, FIOCRUZ, 21045-900 Rio de Janeiro, RJ, Brazil
c Departamento de Química Orgânica, Instituto de Química, UFF, 24020-150 Niterói, RJ, Brazil
d Núcleo de Pesquisas em Produtos Naturais, UFRJ, 21944-971 Rio de Janeiro, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
The available chemotherapy for Chagas disease, caused by Trypanosoma cruzi, is unsatisfactory; therefore,
there is an intense effort to find new drugs for the treatment of this disease. In our laboratory, we have
analyzed the effect on bloodstream trypomastigotes of 16 new naphthoquinone analogues of b-lapa-
chone modified in the pyran ring, aiming to find a new prototype with high trypanocidal activity. The
new compounds presented a broad spectrum of activity, and five of them presented IC50/24 h in the
Received 14 January 2011
Received in revised form
2 March 2011
Accepted 7 March 2011
Available online 15 March 2011
range of 22e63
m
M, whereas
b
-lapachone had a higher value of 391.5 ꢀ 16.5
mM.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Lapachones
Naphthoquinones
Trypanosoma cruzi
Chemotherapy
Chagas’ disease
1. Introduction
Naphthoquinones are present in several families of higher
plants and are considered privileged structures in medicinal
chemistry [7]. In folk medicine, plants containing naphthoquinones
are usually employed for the treatment of different diseases [8].
Great interest is present in the study and mechanisms of the action
of compounds with quinoidal structures, in addition to their
widespread presence in nature, for several different roles in
organisms. Naphthoquinones serve as vital links in the electron
transport chains in metabolic pathways, participating in multiple
biological oxidative processes [9], leading to a depletion of the
levels of NADH and/or NAD(P)H and ATP levels [10]. Among the
Trypanosoma cruzi is the etiologic agent of Chagas disease, an
endemic parasitosis in Latin America with 12e14 million people
infected [1]. Acute infections are usually asymptomatic, but the
ensuing chronic T. cruzi infections have been associated with high
rates of morbidity and mortality. Recently, Chagas disease has also
been recognized as an opportunistic disease in HIV-infected indi-
viduals [2] in addition to being reported throughout the world due
to international immigration [3]. At present, the only two nitro
derivative drugs, introduced in the 1960s and 1970s, are available
for the treatment of chagasic patients: nifurtimox (LampitÒ) and
benznidazole (RochaganÒ or RadanilÒ). They are effective for acute
infections, but their undesirable side effects and controversial use
for chronic patients have been forcing the abandonment of the
treatment [4,5]. Due to these inadequacies, an intense research
program has been directed to find alternative drugs for the treat-
ment of chagasic patients [5]. As also noticed in other neglected
sicknesses, drugs for Chagas disease are not of interest for phar-
maceutical industries [6].
natural naphthoquinones, b-lapachone (Fig. 1) has been used in
popular medicine for centuries against several diseases [11]. Its
trypanocidal activity [12] was investigated as a possible candidate
for the treatment of Chagas disease, but research with this
compound has been discarded due to its high human cytoxicity. In
this regard, its adverse cytoxicity was explored at micromolar
concentrations against a variety of in vitro tumour cells revealing
potential compounds for cancer therapy. Indeed, b-lapachone is in
clinical trials with the name ARQ501 for cancer chemotherapy [13].
New derivatives or analogues of this compound are of great
interest. Indeed, it has been demonstrated that trypanocidal
activities change by selectively transforming the ortho-quinone
* Corresponding author. Tel.: þ55 21 2598 4330; fax: þ55 21 2598 4577.
E-mail address: solange@ioc.fiocruz.br (S.L. de Castro).
moieties. Recently, many derivatives and analogues of b-lapachone
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.012

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S.B. Ferreira et al. / European Journal of Medicinal Chemistry 46 (2011) 3071e3077
Fig. 1. Chemical structures of potent trypanocidal compounds structurally related to b-lapachone.
with significant trypanocidal activity have been reported by our
group. Some of them have been obtained by modification by
replacing the ortho-quinone moiety with heterocycles (phenazine,
oxazoles, imidazoles [14], oxyranes [15]) and others by modifica-
tion of the size of the ring and introductory substituents such as
1,2,3-triazole (Fig. 1) [16]. The data described in the literature
clearly show that the ortho-naphthoquinone framework is
important, and the pyran ring has important significance for the
development of new substances with promising trypanocidal
activity [17].
biological processes [20]. They are usually unstable intermediates
that must be generated in situ; however, some of them may be
sufficiently stable to be isolated, depending on their structural
arrangement [21]. The first example of the generation and use of an
o-QM in intramolecular a hetero-DielseAlder reaction was repor-
ted by Brougidou et al. [22]. Following this discovery, many studies
demonstrated that these hetero-diene moieties are suitable for
[4 þ 2] cycloadditions with a wide range of dienophiles. In this
context, our group reported the reaction of citronellal with
2-hydroxy-1,4-naphthoquinone (lawsone), in which the o-QM
intermediate was generated in situ by a Knoevenagel reaction that
upon the hetero-DielseAlder cycloaddition formed tetracyclic
2. Results and discussion
a- and b-pyranonaphthoquinones [23]. The Nair’s group reported
2.1. Chemistry
in a series of papers the study of the reactivity and use of several o-
QMs [24], overcoming the limitation regarding the use of aldehydes
with dienophiles in the same structure, and it was used for the
In a previous communication, we reported the synthesis of
six
b-lapachone analogues by Knoevenagel condensation of 2-
synthesis of several derivatives of a- and b-lapachone and other
hydroxy-1,4-naphthoquinone with paraformaldehyde or arylalde-
hydes followed by a hetero-DielseAlder reaction with substituted
heterocyclic compounds. Despite its scope, this three-component
reaction is still limited to the use of formaldehyde, whereas
aromatic aldehydes do not react under these conditions, and most
of them produce xanthenes instead of DielseAlder adducts.
styrenes in aqueous ethanol media, generating six
b-lapachone
analogues [18]. In the present work, 16 derivatives were synthe-
sized and assayed against the infective bloodstream form of T. cruzi.
Due to the easy access to natural sources of quinones from
Brazilian flora and the facile synthetic routes previously developed
by our group (AVP and VFF) [19], naphthoquinones were used as
the starting points for screening trypanocidal drugs.
The best methodology for the synthesis of pyran-naph-
thoquinones is sequential reactions of ortho-quinone methides
(o-QMs) followed by an intramolecular hetero-DielseAlder reac-
tion. o-QMs are useful reactive intermediates in organic synthesis
and are involved in a large number of chemical reactions and
Following this synthetic protocol, the b-naphthoquinones were
prepared in one step by an improved methodology recently
reported by our group. Compounds 1e16 were obtained from
methylene and aryl o-quinone methides (o-QMs) generated in situ
by the Knoevenagel condensation of lawsone with para-
formaldehyde or arylaldehydes followed by a hetero-DielseAlder
reaction with different dienophiles in ethanol/water (1:1) under
reflux (Scheme 1).
The reaction produced a mixture of
a and b isomers in good
chemical yields (Table 1). The separation of compounds was carried

S.B. Ferreira et al. / European Journal of Medicinal Chemistry 46 (2011) 3071e3077
3073
Scheme 1. Synthetic route for preparing the b-lapachone analogues (1e16).
out on silica gel column chromatography. For reactions that
produced disubstituted naphthoquinones, the crude product was
composed of syn- and anti-diastereoisomers. In most cases, the
proportion of the anti-isomer was higher than the syn-isomer.
The structures of the synthesized compounds were confirmed
by spectroscopic techniques, such as 1H and 13C NMR, infrared
spectroscopy, and HRMS (ESI).
For the reaction that used
a
-methyl styrene as the dienophile (7),
the signal in the region between 5.00 and 6.00 ppm corresponding
to the hydrogen present on the ortho-carbon of the chromogenic
ring was not observed. However, the quaternary carbon was
observed at 100.6 ppm in the NMR of 13C-APT. The cis-configuration
of compounds 12e16 was determined based on the coupling
constants in the range of 3.9 Hz between the pyran hydrogens.
The 1H NMR spectra mainly showed signals between 5.07 and
5.35 ppm for the
b compounds for the hydrogen presented in the
2.2. Trypanocidal activity of prepared compounds
ortho-carbon of the chromogenic ring, which appeared as a doublet
of a doublet. The aromatic region in the 1H NMR spectrum was used
The compounds 1e16 were assayed for the first time against
bloodstream trypomastigotes of T. cruzi and were more active than
for differentiating the
hydrogens of the -naphthoquinones are split into four signals,
while those in the -naphthoquinones are only split into two
a- and b-naphthoquinones. The aromatic
b
b
-lapachone (IC50/24 h ¼ 391.5 ꢀ 16.5
mM), a very well-known
a
signals due to the symmetry of the structures. The 13C NMR spectra
also showed the formation of the desired products by the signals
between 75.0 and 79.9 ppm due to the differences at carbon 1 of the
chromogenic ring.
trypanocidal natural product (Table 1) [25].
In general, naphthoquinones derived from formaldehyde pre-
sented better results than those from arylaldehydes, indicating that
the presence of a large group significantly decreases the trypano-
cidal activity. Analyzing the relative potency of the tested
compounds in comparison to the benznidazole, excellent lytic
activities were noted for compounds 1, 2, 3,12 and 14 (Fig. 2), which
were 2- to 5-fold more active, while 4e7 displayed activity similar
Table 1
Activity of the compounds against bloodstream trypomasti-
gote forms of T. cruzi.
to that of the standard drug (IC50/24 h ¼ 103.6 ꢀ 0.6
mM) [26].
Compound
IC50/24 ha
(mM)
1
2
3
4
5
6
7
8
56.0 ꢀ 5.3
20.2 ꢀ 4.0
51.4 ꢀ 14.9
121.4 ꢀ 24.7
97.6 ꢀ 11.8
132.2 ꢀ 19.2
122.9 ꢀ 25.3
687.5 ꢀ 71.3
>4000
9
10
11
12
13
14
15
16
849.2 ꢀ 79.8
1859.4 ꢀ 201.3
62.6 ꢀ 5.5
161.1 ꢀ 19.6
35.4 ꢀ 12.2
330.9 ꢀ 45.3
880.3 ꢀ 71.9
103.6 ꢀ 0.6
391.5 ꢀ 16.5
Benznidazole
b
-Lapachone
a
Mean ꢀ standard deviation of at least three independent
Fig. 2. Graphic representation of the relative potency of the naphthoquinones to
benznidazole.
experiments.

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S.B. Ferreira et al. / European Journal of Medicinal Chemistry 46 (2011) 3071e3077
4. Experimental section
4.1. General procedures
Melting points were observed on a Fischer Jones and are
uncorrected. Analytical grade solvents were used. Reagents were
purchased from Aldrich or Acros Chemical Co. Column chroma-
tography was performed on silica gel 60 (Merck 70e230 mesh).
Yields refer to purified compounds obtained by chromatography
techniques and confirmed by spectroscopic data. Reactions were
monitored by thin-layer chromatography (TLC) carried out on
0.25 mm. E. Merck silica gel plates (60F-254) using UV light as
visualizing agent and either an ethanolic solution of sulphate.
Infrared spectra were recorded on a PerkineElmer FT-IR Spectrum
One spectrophotometer, calibrated relative to the 1601.8 cmꢁ1
absorbance of polystyrene. NMR spectra were recorded on a Varian
Unity Plus VXR (300 MHz) equipment in DMSO-d6 and CDCl3
solutions and tetramethylsilane was used as the internal standard
50
1
2
3
12
14
0
0
50
100
150
200
250
uM
(
d
¼ 0 ppm). High resolution mass spectra (HRMS) were recorded
Fig. 3. Graphic representation of the effect of the most active naphthoquinones against
trypomastigotes of T. cruzi.
on an MICROMASS Q-TOF MICRO Mass spectrometer using ESI-TOF
(electrospray ionization-time of flight).
4.2. General preparation of 1e16
Fig. 3 depicts the dose-dependent effect of the most active
derivatives against bloodstream trypomastigotes of T. cruzi, dis-
playing compound 2, containing a chlorine atom in position 4 of the
aryl group, with the highest activity. The nonsubstituted (1) and the
methyl (3) analogues were both two times less active than 2, while
the substitution with a fluorine (5), methoxy (4) or ethoxy (6) group
led to a decrease of 4 fold in the trypanocidal activity. Interestingly,
the addition of a methyl group in position 2 of the aryl group led to
a dramatic reduction of such activity. The exchange of hydrogen by
the methyl group in the R4 position decreased the activity, as shown
by the result of compound 7. In addition, when R1 is an aromatic
radical as in 9e11, there was a marked decrease in activity.
However, the synthesis of naphthoquinone fused with two pyran
rings was obtained, and another prototype with excellent activity
was found, as is the case for compound 14. However, the inclusion
of the groups at position 2 in the second pyran ring dramatically
decreased the activity, as seen in 15 (ethoxy) and 16 (methoxy).
Comparing the results of activity of new 1,2-naphthoquinones
obtained in this study with the IC50 previously described for
naphthoquinones of literature, we obtained compounds with
activity similar or better than those described in the literature with
the advantage of obtaining potentially active compounds by
a simple synthetic route in a single step in good yields.
To a round-bottom flask equipped with a magnetic stirring bar
lawsone was dissolved (1 mmol) with water (10 mL) and ethanol
(10 mL). Then, the appropriate aldehyde (8 mmol for para-
formaldehyde or 3 mmol for arylaldehydes) was added. Styrenes
substituted (3 mmol) was added drop-wise and reaction mixture
was stirred under reflux until consumption of the starting material.
The ethanol was removed under reduced pressure, ethyl acetate
was added in the residue and mixture was washed with saturated
aqueous solution of sodium bicarbonate. The combined organic
extracts washed with water, and dried over anhydrous sodium
sulphate, was filtered and concentrated under pressure reduced.
The residual crude product was purified via silica-gel chromatog-
raphy, using gradient mixture of hexaneeethyl acetate.
4.2.1. 2-Phenyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (1)
Compound 1 was obtained as an orange solid, 23% yield,
m.p. ¼ 161e163 ꢂC. IR (KBr, cmꢁ1):
n 1696, 1647, 1605,1573, 1397,
1300, 1280, 1232, 1158, 1093, 922, 700; 1H NMR (CDCl3, 300 MHz):
d
2.08 (1H, dddd, J ¼ 2.7, 3.2, 5.6 and 13.8 Hz, H-3a), 2.33 (1H, dddd,
J ¼ 3.4, 6.3, 7.4 and 13.8 Hz, H-3b), 2.60 (1H, ddd, J ¼ 3.2, 6.3 and
8.8 Hz, H-4a), 2.76 (1H, ddd, J ¼ 3.4, 5.6 and 8.8 Hz, H-4b), 5.27 (1H,
dd, J ¼ 2.7 and 7.4 Hz, H-2), 7.39e7.46 (5H, m, 2-phenyl), 7.53 (1H,
ddd, J ¼ 1.0, 7.4 and 8.6 Hz, H-8), 7.64 (1H, ddd, J ¼ 1.4, 7.6 and
9.1 Hz, H-9), 7.83 (1H, dd, J ¼ 1.0 and 7.6 Hz, H-10), 8.01 (1H, dd,
3. Conclusion
J ¼ 1.4 and 7.6 Hz, H-7). 13C NMR (CDCl3, 75 MHz):
d 18.2 (C-4), 28.2
(C-3), 79.9 (C-2), 113.8 (C-4a), 123.9 (C-10), 125.6 (C-7), 125.7 (C-40-
phenyl), 128.4 (C-8), 128.5 (C-20-phenyl), 128.6 (C-30-phenyl), 129.8
(C-6a), 130.6 (C-9), 131.9 (C-10-phenyl), 139.2 (C-10a), 162.7 (C-10b),
178.7 and 179.0 (C-5 and C-6). HRMS (ESI) calcd for C19H14O3:
290.0943, Found: 290.0944.
Plants containing naphthoquinones are still employed in folk
medicine, and their molecular structure endows them with redox
properties that could interfere in biological oxidative processes.
Due to the previously described lytic effect upon bloodstream
trypomastigotes of T. cruzi, 16 new derivatives were prepared and
assayed against the parasite. The compounds presented a broad
spectrum of activity, presenting five derivatives IC50/24 h in the
4.2.2. 2-(4-Chlorophenyl)-3,4-dihydro-2H-benzo[h]chromene-5,6-
dione (2)
range of 20 to higher than 4000
m
M, compound 2 being the most
Compound 2 was obtained as an orange solid, 25% yield,
active. These analogues appear as interesting new prototype
compounds, and experiments are currently underway in our
laboratory to investigate their mode of action, especially for free
radical generation, possibly opening new perspectives to the
development of more potent and selective trypanocidal drugs. The
most active compounds will be further investigated in relation to
their mode of action and in vivo effect.
m.p. ¼ 143e145 ꢂC. IR (KBr, cmꢁ1):
n
1697, 1647, 1574, 1607, 1393,
2.01 (1H,
1291, 1092, 923, 774, 725; 1H NMR (CDCl3, 300 MHz):
d
dddd, J ¼ 2.4, 3.5, 5.8 and 12.9 Hz, H-3a), 2.32 (1H, dddd, J ¼ 3.3, 6.5,
7.8 and 12.9 Hz, H-3b), 2.63 (1H, ddd, J ¼ 3.5, 6.5 and 8.9 Hz, H-4a);
2.74 (1H, ddd, J ¼ 3.3, 5.8 and 8.8 Hz, H-4b); 5.24 (1H, dd, J ¼ 2.4 and
7.8 Hz, H-2), 7.36e7.45 (4H, m, H-ar), 7.53 (1H, ddd, J ¼ 1.2, 7.5 and
8.5 Hz, H-8), 7.64 (1H, ddd, J ¼ 1.4, 7.5 and 9.0 Hz, H-9), 7.83 (1H, dd,

S.B. Ferreira et al. / European Journal of Medicinal Chemistry 46 (2011) 3071e3077
3075
J ¼ 1.2 and 7.8 Hz, H-10), 8.01 (1H, dd, J ¼ 1.9 and 8.0 Hz, H-7). 13
C
6.2 Hz, OCH2CH3), 2.15 (1H, dddd, J ¼ 2.1, 3.7, 6.1 and 12.5 Hz, H-3a),
2.22 (1H, dddd, J ¼ 3.5, 6.5, 7.3 and 12.5 Hz, H-3b), 2.58 (1H, ddd,
J ¼ 3.7, 6.5 and 9.1 Hz, H-4a), 2.69 (1H, ddd, J ¼ 3.5, 6.1 and 9.1 Hz H-
4b), 4.09 (2H, dd, J ¼ 1.8 and 6.2 Hz, OCH2CH3), 5.11 (1H, dd, J ¼ 2.1
and 7.3 Hz, H-2), 7.05e7.15 (2H, m, H-ar), 7.40e7.47 (2H, m, H-ar),
7.53 (1H, ddd, J ¼ 1.5, 7.7 and 9.2 Hz, H-8), 7.59 (1H, ddd, J ¼ 1.5, 7.7
and 9.2 Hz, H-9), 7.83 (1H, dd, J ¼ 1.2 and 7.7 Hz, H-10), 8.12 (1H, dd,
NMR (CDCl3, 75 MHz):
d 18.2 (C-4), 28.2 (C-3), 79.2 (C-2), 113.8 (C-
4a), 123.8 (C-10), 127.1 (C-7), 128.6 (C-8), 128.8 (C-20), 129.8 (C-6a),
130.7 (C-30), 131.9 (C-90), 134.2 (C-40), 134.8 (C-10), 137.7 (C-10a),
162.5 (10b), 178.3 and 179.2 (C-5 and C-6). HRMS (ESI) calcd for
C21H18O3: 324.0553, Found: 324.8102.
4.2.3. 2-p-tolyl-3,4-Dihydro-2H-benzo[h]chromene-5,6-dione (3)
Compound 3 was obtained as an orange solid, 20% yield,
J ¼ 1.5 e 7.7 Hz, H-7). 13C NMR (CDCl3, 75 MHz):
d 14.4 (CH3), 19.4 (C-
4), 27.0 (C-3), 64.2 (OCH2CH3), 74.5 (C-2), 115.8 (C-4a), 115.0 (C-10),
124.9 (C-7), 125.1 (C-8), 129.2 (C-20), 129.6 (C-6a), 130.2 (C-30), 132.4
(C-90), 131.9 (C-10a), 134.5 (C-10), 160.6 (C-40), 163.1 (C-10b), 179.4
and 180.2 (C-5 and C-6). HRMS (ESI) calcd for C21H18O4: 334.1205,
Found: 334.1502.
m.p. ¼ 165e167 ꢂC. IR (KBr, cmꢁ1):
n 1697, 1647, 1605, 1590, 1572,
1393, 1301, 1280, 1158, 1076, 922, 771; 1H NMR (CDCl3, 300 MHz):
d
2.06 (1H, dddd, J ¼ 2.4, 3.6, 5.6 and 12.7 Hz, H-3a), 2.31 (1H, dddd,
J ¼ 3.1, 6.2, 7.8 and 12.7 Hz, H-3b), 2.40 (3H, s, CH3), 2.59 (1H, ddd,
J ¼ 3.6, 6.2 and 8.7 Hz, H-4a), 2.77 (1H, ddd, J ¼ 3.1, 5.6 and 8.7 Hz,
H-4b), 5.24 (1H, dd, J ¼ 2.4 and 7.8 Hz, H-2), 7.25 (2H, d, J ¼ 7.7 Hz,
H-meta tolyl), 7.32 (2H, d, J ¼ 7.7 Hz, H-ortho tolyl), 7.51 (1H, ddd,
J ¼ 1.4, 7.5 and 8.7 Hz, H-8), 7.62 (1H, ddd, J ¼ 1.4, 7.5 and 9.0 Hz, H-
9), 7.81 (1H, dd, J ¼ 1.2 and 7.8 Hz, H-10), 8.01 (1H, dd, J ¼ 1.4 and
4.2.7. 2-Methyl-2-phenyl-3,4-dihydro-2H-benzo[h]chromene-5,6-
dione (7)
Compound 7 was obtained as an orange solid, 20% yield,
m.p. ¼ 155e157 ꢂC. IR (KBr, cmꢁ1):
n
1606, 1647, 1603, 1387, 1305,
d 1.78 (3H, s, CH3), 2.05 (1H,
7.5 Hz, H-7); 13C NMR (CDCl3, 75 MHz):
d
18.7 (C-4), 21.4 (CH3), 28.6
1359, 919. 1H NMR (CDCl3, 300 MHz):
(C-3), 80.4 (C-2), 114.3 (C-4a), 124.3 (C-10), 126.1 (C-7), 128.9 (C-8),
129.7 (C-20-phenyl), 130.2 (C-6a), 131.0 (C-30-phenyl), 132.4 (C-90),
135.1 (C-10-phenyl), 136.7 (C-40-phenyl), 138.7 (C-10a), 163.2 (C-
10b), 178.8 and 179.8 (C-5 and C-6). HRMS (ESI) calcd for C20H16O3:
304.1099, Found: 304.1012.
ddd, J ¼ 3.1, 5.3 and 12.9 Hz, H-3a), 2.24 (1H, ddd, J ¼ 3.0, 6.0 and
12.9 Hz, H-3b), 2.42 (1H, ddd, J ¼ 3.1, 6.0 and 7.9 Hz, H-4a), 2.60 (1H,
ddd, J ¼ 3.0, 5.3 and 7.9 Hz, H-4b), 7.29e7.38 (5H, m, 2-phenyl), 7.55
(1H, ddd, J ¼ 1.2, 7.5 and 9.0 Hz, H-8), 7.71 (1H, ddd, J ¼ 1.2, 7.5 and
9.0 Hz, H-9), 8.00 (1H, dd, J ¼ 1.0 and 7.5 Hz, H-10), 8.10 (1H, dd,
J ¼ 1.0 and 7.5 Hz, H-7). 13C NMR (CDCl3, 75 MHz):
d 16.5 (C-4), 29.4
4.2.4. 2-(4-Methoxyphenyl)-3,4-dihydro-2H-benzo[h]chromene-
5,6-dione (4)
(CH3), 31.1 (C-3), 82.9 (C-2), 114.2 (C-4a), 124.1 (C-10), 124.2 (C-7),
127.8 (C-40-phenyl), 129.0 (C-8), 129.1 (C-20-phenyl), 131.0 (C-30-
phenyl), 135.2 (C-10-phenyl), 130.3 (C-6a), 132.4 (C-10a), 143.2 (C-9),
161.9 (10b), 178.6 and 179.7 (C-5 and C-6). HRMS (ESI) calcd for
C20H16O3: 304.1099, Found: 304.3501.
Compound 4 was obtained as an orange solid, 17% yield,
m.p. ¼155e157 ꢂC.IR(KBr, cmꢁ1):
n
1605,1572,1525,1280,1249,1170,
2.11 (1H, dddd, J ¼ 2.4, 3.6, 5.7
921, 774; 1H NMR (CDCl3, 300 MHz):
d
and 13.0 Hz, H-3a); 2.31 (1H, dddd, J ¼ 3.2, 6.2, 7.8 and 13.0 Hz, H-3b);
2.61 (1H, ddd, J ¼ 3.6, 6.2 and 8.7 Hz, H-4a), 2.76 (1H, ddd, J ¼ 3.2, 5.7
and 8.7 Hz, H-4b), 3.85 (3H, s, OCH3), 5.20 (1H, dd, J ¼ 2.4 and 7.8 Hz,
C-2), 6.96e6.99 (2H, m, H-ar), 7.34e7.37 (2H, m, H-ar), 7.51 (1H, ddd,
J ¼ 1.4, 7.5 and 8.7 Hz, H-8), 7.60 (1H, ddd, J ¼ 1.4, 7.5 and 9.0 Hz, H-9),
7.80 (1H, dd, J ¼ 1.2 and 7.8 Hz, H-10), 8.08 (1H, dd, J ¼ 1.4 and 7.5 Hz,
4.2.8. 2-(2,4-Dimethylphenyl)-3,4-dihydro-2H-benzo[h]chromene-
5,6-dione (8)
Compound 8 was obtained as an orange solid, 27% yield,
m.p. ¼ 164e167 ꢂC. IR (KBr, cmꢁ1):
n
1694, 1646, 1603, 1574, 1394,
2.07 (1H,
1283, 1231, 1160, 914, 728. 1H NMR (CDCl3, 300 MHz):
d
H-7). 13C NMR (CDCl3, 75 MHz):
d
18.4 (C-4), 28.0 (C-3), 55.2 (OCH3),
dddd, J ¼ 2.4, 2.8, 5.6 and 11.8 Hz, H-3a), 2.31 (1H, dddd, J ¼ 3.7, 6.5,
7.8 and 11.8 Hz, H-3b), 2.36 (3H, s, CH3), 2.38 (3H, s, CH3), 2.63 (1H,
ddd, J ¼ 2.8, 6.5 and 8.0 Hz, H-4a), 2.85 (1H, ddd, J ¼ 3.7, 5.6 and
8.0 Hz, H-4b), 5.35 (1H, dd, J ¼ 2.4 and 7.8 Hz, C-2), 7.08e7.13 (2H,
m, H-ar), 7.33 (1H, d, J ¼ 7.8 Hz, H-ar), 7.51 (1H, ddd, J ¼ 0.9, 7.5 and
8.7 Hz, H-8), 7.61 (1H, ddd, J ¼ 1.4, 7.5 and 9.0 Hz, H-9), 7.71 (1H, dd,
J ¼ 0.9 and 7.8 Hz, H-10), 8.06 (1H, dd, J ¼ 1.4 and 7.5 Hz, H-7). 13C
79.9 (C-2), 113.8 (C-4a), 114.0 (C-10), 123.9 (C-7), 127.2 (C-8), 128.5
(C-20), 129.8 (C-6a), 130.6 (C-30), 131.2 (C-90), 132.0 (C-10a), 134.7
(C-10),159.6 (C-40),162.9(C-10b),178.4and 179.4(C-5 and C-6). HRMS
(ESI) calcd for C21H18O3: 320.1049, Found: 320.3596.
4.2.5. 2-(4-Fluorophenyl)-3,4-dihydro-2H-benzo[h]chromene-5,6-
dione (5)
NMR (CDCl3, 75 MHz): d 19.2 (CH3), 19.2 (C-4), 21.2 (CH3), 27.5 (C-3),
Compound 5 was obtained as an orange solid, 26% yield,
77.9 (C-2),114.2 (C-4a),124.3 (C-10),125.8 (C-7),127.3 (C-40-phenyl),
128.9 (C-8), 130.9 (C-20-phenyl), 131.8 (C-30-phenyl), 134.6 (C-10-
phenyl),130.2 (C-6a),132.4 (C-10a),163.5 (10b),178.7 and 179.8 (C-5
and C-6). HRMS (ESI) calcd for C21H18O3: 318.1256, Found:
318.3792.
m.p. ¼ 142e147 ꢂC. IR (KBr, cmꢁ1):
n
1573, 1607, 1513, 1394, 1280,
2.07 (1H, dddd,
1229, 1156, 922; 1H NMR (CDCl3, 300 MHz):
d
J ¼ 2.4, 3.2, 5.6 and 12.8 Hz, H-3a), 2.34 (1H, dddd, J ¼ 3.1, 6.5, 7.8
and 12.8 Hz, H-3b), 2.61 (1H, ddd, J ¼ 3.2, 6.5 and 8.7 Hz, H-4a), 2.75
(1H, ddd, J ¼ 3.1, 5.6 and 8.7 Hz, H-4b), 5.24 (1H, dd, J ¼ 2.4 and
7.8 Hz, H-2), 7.15 (2H, t, J ¼ 8.5 Hz, 2CHar-F), 7.39e7.44 (2H, m, H-
ar), 7.53 (1H, ddd, J ¼ 1.2, 7.2 and 8.7 Hz, H-8), 7.64 (1H, ddd, J ¼ 1.4,
7.8 and 9.2 Hz, H-9), 7.83 (1H, dd, J ¼ 1.1 and 6.5 Hz, H-10), 8.01 (1H,
4.2.9. 4-(4-Nitrophenyl)-2-p-tolyl-3,4-dihydro-2H-benzo[h]
chromene-5,6-dione (9)
Compound 9 was obtained as an orange solid, 24% yield,
m.p. ¼ 232e235 ꢂC. IR (KBr, cmꢁ1): 1696, 1645, 1600, 1570, 1513,
1344, 1287, 1233, 1168, 1091, 912, 736, 702. 1H NMR (300 MHz,
dd, J ¼ 1.1 and 6.3 Hz, H-7). 13C NMR (CDCl3, 75 MHz):
d 18.3 (C-4),
28.3 (C-3), 79.4 (C-2), 113.8 (C-4a), 115.4 and 115.7 (d, J ¼ 21.4 Hz, C-
30), 123.8 (C-10), 127.5 (C-7), 127.6 (C-8), 128.6 (C-10), 129.8 (C-6a),
130.7 (C-30), 131.8 (C-20), 134.7 (C-90), 135.7 (C-40), 137.7 (C-10a),
162.6 (C-10b), 178.4 and 179.2 (C-5 and C-6). HRMS (ESI) calcd for
C21H18O3: 308.0849, Found: 308.3243.
CDCl3):
d
2.19 (1H, dt, J ¼ 2.4 and 14.4 Hz, H-3a syn isomer) and 2.27
(1H, dt, J ¼ 11.2 and 14.4 Hz, H-3a anti isomer), 2.38 and 2.39 (3H, s,
CH3), 2.55 (1H, ddd, J ¼ 5.9, 12.0 and 14.4 Hz, H-3b syn isomer) and
2.61 (1H, ddd, J ¼ 2.2, 7.1 and 14.4 Hz, H-3b anti isomer), 4.31 (1H,
dd, J ¼ 7.1 and 11.0 Hz, H-4 anti isomer) and 4.48 (1H, dd, J ¼ 2.4 and
5.9 Hz, H-4 syn isomer), 5.07 (1H, dd, J ¼ 2.4 and 12.0 Hz, H-2 syn
isomer) and 5.31 (1H, dd, J ¼ 2.2 and 11.2 Hz, H-2 anti isomer), 7.25
(1H, d, J ¼ 8.1 Hz, H-meta p-tolyl), 7.35 (1H, d, J ¼ 8.1 Hz, H-ortho p-
tolyl), 7.38 and 7.47 (1H, d, J ¼ 8.8 Hz, H-ortho 4-nitrophenyl),
7.56e7.94 (1H, m, H-7), 7.56e7.94 (1H, m, H-8), 8.11 (1H, d,
4.2.6. 2-(4-Ethoxyphenyl)-3,4-dihydro-2H-benzo[h]chromene-5,6-
dione (6)
Compound 6 was obtained as an orange solid, 22% yield,
m.p. ¼ 145e147 ꢂC. IR (KBr, cmꢁ1):
n 1610, 1642, 1601, 1390, 1300,
1365, 939. 1H NMR (CDCl3, 300 MHz): 1.45 (3H, dd, J ¼ 1.8 and

3076
S.B. Ferreira et al. / European Journal of Medicinal Chemistry 46 (2011) 3071e3077
J ¼ 8.8 Hz, H-meta 4-nitrophenyl), 8.09e8.23 (1H, m, H-9),
8.09e8.23 (1H, m, H-6). 13C NMR (75 MHz, CDCl3):
21.1 (CH3), 34.9
5.48e5.50 (1H, m, H-1a), 6.28e6.31 (1H, m H-3), 6.18e6.21 (1H, m
H-2), 7.68e7.73 (1H, m, ArH), 7.85e7.87 (2H, m, ArH), 8.01e8.08
d
and 38.4 (C-4), 36.7 and 39.8 (C-3), 80.0 (C-2), 115.9 (C-4a), 123.8
and 123.9 (C-30 4-nitrophenyl), 124.0 (C-20 4-nitrophenyl), 125.9 and
126.0 (C-30 p-tolyl), 127.7 (C-8), 128.6 (C-7), 128.8 and 129.0 (C-20 p-
tolyl), 129.5 (C-10), 130.3 (C-6a), 130.4 (C-10 p-tolyl), 131.3 (C-9),
131.7 (C-40 p-tolyl), 135.0 (C-10a), 138.9 (C-40 4-nitrophenyl), 151.1
(C-10 4-nitrophenyl), 164.9 (C-10b), 177.6 (C-5), 178.7 (C-6). HRMS
(ESI) calcd for C26H19NO5Hþ: 426.1336, Found: 426.4483.
(1H, m, ArH). 13C NMR (75 MHz, CDCl3):
d 19.7 (C-5), 34.5 (C-4a),
37.9 (C-4), 85.0 (C-1a), 112.5 (C-5a), 124.4 (C-3), 135.7 (C-2), 128.5,
131.2, 131.5 and 135.7 (C-ar), 130.3 (C-7a), 132.6 (C-11a), 163.6 (C-
11b), 178.4 and 179.8 (C-5 and C-6). HRMS (ESI) calcd for C16H12O3:
252.0786, Found: 252.0897.
4.2.13. (7aS,11aR)-7,7a,8,9-Tetrahydro-5H-benzo[c]xanthene-
5,6(11aH)-dione (13)
4.2.10. Phenyl-2-p-tolyl-3,4-dihydro-2H-benzo[h]chromene-5,6-
dione (10)
Compound 10 was obtained as an orange solid, 33% yield,
m.p. ¼ 92e95 ꢂC. IR (KBr, cmꢁ1): 1696, 1653, 1600, 1568, 1382, 1284,
1231, 1164, 1086, 909, 767, 699. 1H NMR (300 MHz, CDCl3):
Compound 13 was obtained as an orange solid, 20% yield,
m.p. ¼ 142e143 ꢂC. IR (KBr, cmꢁ1): 1604, 1649, 1500, 1204, 1033,
1012, 930, 715. 1H NMR (300 MHz, CDCl3):
d 1.62e1.69 (3H, m, H-4
and H-5), 2.17e2.30 (2H, m, H-5 and H-5a), 2.66 (1H, dd, J ¼ 6.5 and
11.0 Hz, H-6), 2.50 (1H, dd, J ¼ 6.5 and 11.0 Hz, H-6), 4.77 (1H, t,
J ¼ 3.9 Hz, H-1a), 5.93e5.99 (1H, m, H-3), 6.04e6.09 (1H, m, H-3),
7.49 (1H, ddd, J ¼ 1.2, 7.5 and 9.0 Hz, H-8), 7.63 (1H, ddd, J ¼ 1.2, 7.5
and 9.0 Hz, H-9), 7.80 (1H, dd, J ¼ 0.9 and 7.5 Hz, H-10), 8.05 (1H, dd,
d
2.13e2.37 (1H, m, H-3a), 2.38 and 2.39 (3H, s, CH3), 2.59 (1H, ddd,
J ¼ 2.0, 7.3 and 14.4 Hz, H-3b), 4.20 (1H,dd, J ¼ 7.1 and 11.2 Hz, H-4
anti isomer) and 4.42 (1H, dd, J ¼ 1.7 and 5.4 Hz, H-4 syn isomer),
5.15 (1H, dd, J ¼ 2.7 and 12.0 Hz, H-2 syn isomer) and 5.24 (1H, dd,
J ¼ 1.7 and 12.0 Hz, H-2 anti isomer), 7.13e7.38 (4H, m, p-tolyl),
7.13e7.38 (5H, m, Ph), 7.52e7.60 (1H, m, H-7), 7.64e7.70 (1H, m, H-
8), 7.88e7.92 (1H, m, H-9), 8.07e8.10 (1H, m, H-6 anti isomer) and
J ¼ 0.9 and 7.5 Hz, H-7). 13C NMR (75 MHz, CDCl3):
d 22.5 (C-4), 23.6
(C-5), 24.7 (C-6), 29.3 (C-5a), 73.8 (C-1a), 112.2 (C-6a), 124.3 (C-3),
124.7 (C-2), 128.8, 130.8, 134.1 and 135.0 (C-ar), 130.2 (C-8a), 132.4
(C-12a), 161.6 (C-12b), 179.9 (C-7 and C-8). HRMS (ESI) calcd for
C17H14O3: 266.0943, Found: 266.3040.
8.12e8.15 (1H, m, H-6 syn isomer). 13C NMR (75 MHz, CDCl3):
d 21.1
(CH3), 34.6 and 38.5 (C-4), 40.6 (C-3), 80.1 (C-2), 117.5 (C-4a), 124.5
(C-30 Ph), 126.0 and 126.1 (C-30 p-tolyl), 126.3 (C-20 Ph), 126.7 and
126.8 (C-40 Ph), 128.4 (C-8), 128.5 and 128.6 (C-7), 129.2 and 129.3
(C-20 p-tolyl), 130.2 (C-6a),130.8 (C-10),132.1 (C-10 p-tolyl),134.8 (C-
9), 135.6 (C-40 p-tolyl), 138.6 (C-10a), 143.3 (C40Ph), 164.1 (C-10b),
178.0 (C-5), 179.1 (C-6). HRMS (ESI) calcd for C26H20O3Hþ: 381.1485,
Found: 381.4503.
4.2.14. (7aS,11aR)-7a,8,9,10-Tetrahydrobenzo[h]pyrano[2,3-b]
chromene-5,6(7H,11aH)-dione (14)
Compound 14 was obtained as an orange solid, 12% yield,
m.p. ¼ 153e155 ꢂC. IR (KBr, cmꢁ1): 1606, 1646, 1588, 1278, 1040,
1020, 950. 1H NMR (300 MHz, CDCl3):
d 1.54e1.88 (5H, m, H-6, H-5
and H-4), 2.30 (1H, d, J ¼ 2.6 Hz, H-5a), 2.52e2.67 (1H, m, H-6a),
3.82e4.02 (2H, m, H-3), 5.58 (1H, d, J ¼ 2.6 Hz, H-1a), 7.53 (1H, ddd,
J ¼ 1.2, 7.5 and 8.7 Hz, H-8), 7.67 (1H, ddd, J ¼ 1.2, 7.5 and 9.0 Hz, H-
9), 7.90 (1H, dd, J ¼ 0.9 and 7.5 Hz, H-10), 8.08 (1H, dd, J ¼ 0.9 and
4.2.11. 4-(Thiophen-2-yl)-2-p-tolyl-3,4-dihydro-2H-benzo[h]
chromene-5,6-dione (11)
Compound 11 was obtained as an orange solid, 33% yield,
m.p. ¼ 83e87 ꢂC. IR (KBr, cmꢁ1): 1696, 1652, 1599, 1568, 1383, 1284,
1231, 1170, 1086, 907, 818, 722, 697. 1H NMR (300 MHz, CDCl3):
7.5 Hz, H-7). 13C NMR (75 MHz, CDCl3):
d 23.3 (C-4 and C-5), 23.6 (C-
6), 30.4 (C-5a), 62.1 (C-3), 98.7 (C-1a), 111.5 (C-6a), 129.9 (C-8a),
131.4 (C-12a), 123.9, 128.6, 130.7 and 134.8 (C-ar), 160.9 (C-12b),
178.6 and 179.1 (C-7 and C-8). HRMS (ESI) calcd for C16H14O4:
270.0892, Found: 270.1897.
d
2.28e2.43 (1H, m, H-3a), 2.40 (3H, s, CH3), 2.68 (1H, ddd, 2.1, 7.1
and 14.4 Hz, H-3b), 4.57 (1H, dd, J ¼ 7.1 and 11.0 Hz) and 4.65 (1H,
dd, J ¼ 1.0 and 3.2 Hz) H-4 conformers, 5.23 (1H, dd, J ¼ 1.9 and
11.5 Hz) and 5.35 (1H, dd, J ¼ 4.4 and 10.0 Hz) H-2 conformers, 6.89
and 6.90 (1H, dd, J ¼ 1.0 and 3.4 Hz, H-30 thiophen-2-yl conformers),
6.86 and 6.95 (1H, dd, J ¼ 3.7 and 5.1 Hz, H-40 thiophen-2-yl
conformers), 7.06 and 7.18 (1H, dd, J ¼ 1.2 and 5.1 Hz, H-50 thiophen-
2-yl conformers), 7.24 and 7.25 (1H, d, J ¼ 8.0 Hz, H-meta
conformers), 7.30 and 7.36 (1H, d, J ¼ 8.0 Hz, H-ortho conformers),
7.54 and 7.57 (1H, td, J ¼ 1.2 and 7.6 Hz, H-8 conformers), 7.65 and
7.67 (1H, dt, J ¼ 1.2 and 7.6 Hz, H-9 conformers), 7.90 (1H, d,
J ¼ 7.6 Hz, H-10), 8.09 and 8.13 (1H, dd, J ¼ 1.5 and 7.6 Hz, H-7
4.2.15. (7aS,11aS)-10-Ethoxy-7a,8,9,10-tetrahydrobenzo[h]pyrano
[2,3-b]chromene-5,6(7H,11aH)-dione (15)
Compound 15 was obtained as an orange solid, 20% yield,
m.p. ¼ 150e152 ꢂC. IR (KBr, cmꢁ1): 1604, 1653, 1580, 1269, 1032,
920. 1H NMR (300 MHz, CDCl3):
d
1.35 (1H, 3H, dd, J ¼ 1.9 and
6.5 Hz, OCH2CH3), 1.55e1.91 (5H, m, H-6, H-5 and H-4), 2.26 (1H, d,
J ¼ 2.5 Hz, H-5a), 2.50e2.57 (1H, m, H-6a), 3.79e4.00 (2H, m, H-3),
4.05 (2H, dd, J ¼ 1.9 and 6.5 Hz, OCH2CH3), 5.65 (1H, d, J ¼ 2.7 Hz, H-
1a), 7.55 (1H, ddd, J ¼ 1.3, 7.5 and 8.9 Hz, H-8), 7.67 (1H, ddd, J ¼ 1.3,
7.5 and 8.9 Hz, H-9), 7.92 (1H, dd, J ¼ 0.9 and 7.5 Hz, H-10), 8.12 (1H,
conformers). 13C NMR (75 MHz, CDCl3):
d 21.0 (CH3), 30.1 and 33.4
(C-4), 37.1 and 40.9 (C-3), 76.6 and 80.0 (C-2),115.2 and 117.2 (C-4a),
122.7 and 123.9 (C-50 thiophen-2-yl), 124.5 and 124.6 (C-30 p-tolyl),
125.0 (C-8), 126.0 and 126.1 (C-20 p-tolyl), 126.4 and 126.9 (C-10),
128.6 and 128.7 (C-7), 129.3 (C-40 thiophen-2-yl), 130.2 and 130.3
(C-6a),130.9 and 131.0 (C-9),131.7 and 131.9 (C-10 p-tolyl), 134.7 and
134.8 (C-30 thiophen-2-yl), 135.3 and 135.7 (C-40 p-tolyl), 138.5 and
138.6 (C-10a), 146.2 and 146.7 (C-20 thiophen-2-yl), 163.0 and 163.4
(C-10b), 177.7 and 178.0 (C-5), 179.0 (C-6). HRMS (ESI) calcd for
C24H18O3SHþ: 387.1049, Found: 387.4789.
dd, J ¼ 0.9 and 7.5 Hz, H-7). 13C NMR (75 MHz, CDCl3):
d 22.1 (C-4
and C-5), 23.5 (C-6), 32.0 (C-5a), 60.0 (C-3), 95.7 (C-1a), 111.7 (C-6a),
130.9 (C-8a), 131.3 (C-12a), 120.9, 126.6, 132.7 and 133.9 (C-ar),
162.0 (C-12b), 178.5 and 179.5 (C-7 and C-8). HRMS (ESI) calcd for
C18H18O5: 314.1154, Found: 314.2209.
4.2.16. (7aS,11aS)-10-Methoxy-7a,8,9,10-tetrahydrobenzo[h]
pyrano[2,3-b]chromene-5,6(7H,11aH)-dione (16)
Compound 16 was obtained as an orange solid, 16% yield,
m.p. ¼ 149e152 ꢂC. IR (KBr, cmꢁ1): 1606, 1650, 1563, 1245, 1020,
4.2.12. (7aS,10aR)-7a,8-Dihydrobenzo[h]cyclopenta[b]chromene-
5,6(7H,10aH)-dione (12)
919. 1H NMR (300 MHz, CDCl3):
d 1.65e1.98 (5H, m, H-6, H-5 and H-
4), 2.23 (1H, d, J ¼ 2.1 Hz, H-5a), 2.55e2.61 (1H, m, H-6a), 3.80e4.03
(2H, m, H-3), 3.86 (3H, s, OCH3), 5.68 (1H, d, J ¼ 2.1 Hz, H-1a), 7.51
(1H, ddd, J ¼ 1.1, 7.5 and 9.1 Hz, H-8), 7.70 (1H, ddd, J ¼ 1.1, 7.5 and
9.1 Hz, H-9), 7.95 (1H, dd, J ¼ 0.8 and 7.5 Hz, H-10), 8.15 (1H, dd,
Compound 12 was obtained as an orange solid, 15% yield,
m.p. ¼ 146e147 ꢂC. IR (KBr, cmꢁ1): 1738, 1716, 1599, 1284, 1036,
1024, 942, 720. 1H NMR (300 MHz, CDCl3):
d 2.23e2.32 (1H, m, H-
5), 2.47e2.54 (1H, m, H-5), 2.66e2.95 (3H, m, H-4 and H-4a),
J ¼ 0.8 and 7.5 Hz, H-7). 13C NMR (75 MHz, CDCl3):
d 23.1 (C-4 and

S.B. Ferreira et al. / European Journal of Medicinal Chemistry 46 (2011) 3071e3077
3077
[13] S.B. Ferreira, C.R. Kaiser, V.F. Ferreira, Org. Prep. Proced. Int. 41 (2009)
211e215.
[14] (a) K.C.G. 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)
325e338;
C-5), 23.5 (C-6), 35.0 (C-5a), 58.3 (C-3), 96.8 (C-1a), 110.7 (C-6a),
131.7 (C-8a),132.3 (C-12a),121.6,125.6,133.7 and 133.9 (C-ar),165.0
(C-12b), 178.3 and 178.9 (C-7 and C-8). HRMS (ESI) calcd for
C17H16O5: 300.0998, Found: 300.1006.
(b) C. Neves-Pinto, V.R. Malta, M.C.F.R. Pinto, R.H. Santos, S.L. de Castro,
A.V. Pinto, J. Med. Chem. 45 (2002) 2112e2115;
(c) K.C.G. Moura, K. Salomão, R.F.S. Menna-Barreto, F.S. Emery,
M.C.F.R. Pinto, A.V. Pinto, S.L. de Castro, Eur. J. Med. Chem. 39 (2004)
639e645;
4.3. Trypanocidal assays
Stock solutions of the compounds were prepared in dime-
thylsulfoxide (DMSO), with the final concentration of the latter in
the experiments never exceeding 0.1%. Preliminary experiments
showed that at concentrations up to 0.5%, DMSO has no deleterious
effect on the parasites. Bloodstream trypomastigotes from the Y
strain were obtained at the peak of parasitaemia from infected
albino mice, isolated by differential centrifugation and resuspended
in Dulbecco’s modified Eagle’s medium (DMEM) to a parasite
concentration of 107 cells/mL in the presence of 10% of mouse
(d) R.F.S. Menna-Barreto, A. Henriques-Pons, A.V. Pinto, J.A. Morgado-Diaz,
M.J. Soares, S.L. de Castro, J. Antimicrob. Chemother. 56 (2005) 1034e1041.
[15] (a) V.F. Ferreira, A. Jorqueira, A.M. Souza, M.N. da Silva, M.C. de Souza,
R.M. Gouvea, C.R. Rodrigues, A.V. Pinto, H.C. Castro, D.O. Santos, H.P. Araujo,
S.C. Bourguignon, Bioorg. Med. Chem. 14 (2006) 5459e5466;
(b) A. Jorqueira, R.M. Gouvea, V.F. Ferreira, M.N. da Silva, M.C. de Souza,
A.A. Zuma, D.F. Cavalcanti, H.P. Araujo, D.O. Santos, S.C. Bourguignon, Para-
sitol. Res. 99 (2006) 429e433.
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A.J. Araújo, C. Pessoa, L.V. Costa-Lotufo, R.C. Montenegro, M.O. Moraes,
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blood. This suspension (100 mL) was added in the same volume of
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Acknowledgements
This research was supported by grants from the CNPq, FIOCRUZ,
Fundação Carlos Chagas Filho de Amparo à Pesquisa do Rio de
Janeiro (FAPERJ), and PRONEX/E-26/110.574/2010. The authors
thank Prof. Dr. Claudia Moraes de Rezende from the Universidade
Federal do Rio de Janeiro for the mass spectra analysis.
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