Synthesis and preliminary pharmacological evaluation of new (+/-) 1,4-naphthoquinones structurally related to lapachol.

Article abstract of DOI:10.1016/S0968-0896(02)00100-1

Seven new 1,4-naphthoquinones structurally related to lapachol were synthesized from lawsone and oxygenated arylmercurials. These compounds can also be seen as pterocarpan derivatives where the A-ring was substituted by the 1,4-naphthoquinone nucleus. Pha

Full text of DOI:10.1016/S0968-0896(02)00100-1

Bioorganic & Medicinal Chemistry 10 (2002) 2731–2738
Synthesis and Preliminary Pharmacological Evaluation of New
(Æ) 1,4-Naphthoquinones Structurally Related to Lapachol
Alcides J. M. da Silva,a Camilla D. Buarque,a Flavia V. Brito,a Laure Aurelian,d
Luciana F. Macedo,c,d Linda H. Malkas,d Robert J. Hickey,d,e Daniele V. S. Lopes,b
Francois Noel,b Yugo L. B. Murakami,b Noelson M. V. Silva,b Paulo A. Melo,b
Rodrigo R. B. Caruso,b Newton G. Castrob and Paulo R. R. Costaa,*
a
´
Laboratorio de Quımica Bioorganica (LQB), Nucleo de Pesquisas de Produtos Naturais, Centro de Ciencias da Saude, Bloco H,
´
ˆ
´
Universidade Federal do Rio de Janeiro RJ 21941-590, Brazil
ˆ
´
b
´
Departamento de Farmacologia Basica e Clınica, Centro de Ciencias da Saude, Bloco J, Universidade Federal do Rio de Janeiro
´
ˆ
RJ 21941-590, Brazil
´
c
´
Instituto de Biofısica Carlos Chagas Filho, Centro de Cieˆncias da Sau´de, Bloco G, Universidade Federal do Rio de Janeiro
RJ 21941-590, Brazil
dDepartment of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore,
MD 21210, USA
eGreenebaum Cancer Center, University Hospital, Baltimore, MD 21210, USA
Received 19 October 2001; accepted 12 February 2002
Abstract—Seven new 1,4-naphthoquinones structurally related to lapachol were synthesized from lawsone and oxygenated aryl-
mercurials. These compounds can also be seen as pterocarpan derivatives where the A-ring was substituted by the 1,4-naphtho-
quinone nucleus. Pharmacological screening provided evidence of significant biological activities, including effects against
proliferation of the MCF-7 human breast cancer cell line, against Herpes Simplex Virus type 2 infection, and against snake poison-
induced myotoxicity. One derivative displaced flunitrazepam binding and showed benzodiazepine-like activity, suggesting novel
neuroactive structural motifs. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
MCF-7, HT-29)6,7 and antitumor activity, inhibiting the
growth of KB cells in culture.8 Pyrano-1,4-naphthoqui-
nones are also active against Gram-positive bacteria,
fungi and mycoplasma.9
Naphthoquinones are widespread in nature and play
important physiological roles in animals and plants.1,2
Secondary metabolites bearing in their structure the 1,4-
and 1,2-naphthoquinone moieties have been isolated
from plants and exhibit interesting biological activities.
Lapachol
(2-hydroxy-3-prenyl-1,4-naphthoquinone),
found in species of Tabebuia, was shown to be active
against the Walker-256 carcinoma and Yoshida sar-
coma.3,4 Several synthetic derivatives of lapachol
have been prepared. Its 2b-O-glycoside-tetraacetate
derivative, for example, was shown to be active against
leukemia,5 while the tricyclic derivatives furano-1,4-
naphthoquinones and pyrano-1,4-naphthoquinones
showed, respectively, potent cytotoxicity against cell
lines derived from human solid tumors in vitro (A-549,
Among the derivatives of lapachol bearing the 1,2-
naphthoquinone nucleus, b-allyl lapachone shows a
trypanocidal activity in contaminated blood samples,10
while the combination of taxol and b-lapachone induced
death of cultured cancer cells from ovary, breast, pros-
tate, melanoma, lung, colon and pancreas.11,12 As part
of a program aimed at synthesizing anti-virus and anti-
tumor compounds,13 we report the synthesis and pre-
liminary pharmacological evaluation of new lapachol
*Corresponding author. Tel.: +55-21-2270-2683; fax: +55-21-2523-
5938; e-mail: lqb@nppn.ufrj.br
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00100-1

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A. J. M. da Silva et al. / Bioorg. Med. Chem. 10 (2002) 2731–2738
derivatives 1a–g where the conformationally restricted
aromatic substituents at C3 were designed to mimic the
prenyl side chain of the natural product. These com-
pounds can also be seen as pterocarpan derivatives
where the A-ring was substituted by the 1,4-naphtho-
quinone moiety. The presence of different patterns
of oxygenation at the D-ring in compounds 1a–g pro-
vides new potential sites for interaction with biological
macromolecules.
Cancer cell assays
The antiproliferative activity of derivatives 1a–g was
studied using MCF-7 breast cancer cells (Fig. 1). Cells
in exponential growth were incubated for 24 h in the
absence (control, 100%) or presence of increasing con-
centrations of the derivatives. The inhibition of cell
growth was determined using an in vitro cytotoxicity
assay. Stock solutions of the compounds were prepared
in dimethylsulfoxide (DMSO) and final solvent con-
centrations were below the cytotoxic level for MCF-7
cells. The results of three independent experiments indi-
cated that compounds 1e and 1f have comparable and
significant activities and are the most potent prolifera-
tion inhibitors in this series, with mean inhibitory con-
centrations (IC50) of 5.3 and 7.8 mM, respectively
(Table 1). The growth of MCF-7 cells was also inhibited
by compounds 1a and 1d but less potently (IC50 values
of 27.7 and 29.0 mM, respectively). The other compounds
Results and Discussion
Chemistry
The lapachol derivatives 1a–g were prepared as shown
in Scheme 1. Using the protocol described by Snieckus
et al. for the synthesis of chromens from phenols,14
chromen 3 was obtained from commercially available
lawsone (2), in a one-pot procedure, through the reac-
tion with acrolein in the presence of boronic acid, in
50% yield after chromatographic purification.
The o-cloromercuriophenol derivatives 4a–d were pre-
pared from piperonal, vanillin and isovanillin, as pre-
viously described by our group.15 These compounds
were allowed to react with chromen 3 in the presence of
lithium tetrachloropalladate II and acetone,16,17 leading
to the desired new naphthoquinones 1a–d through a
Heck-type coupling reaction, in around 50% yield.
These coupling reactions could be accomplished starting
from crude 3, without any loss in the total chemical
yield and purity. All naphthoquinones obtained are
red solids and could be purified by crystallization.
Compounds 1a–c were allowed to react with hydrogen,
leading to the phenolic derivatives 1e,f and the cathe-
cholic derivative 1g.
Figure 1. Effect of the derivatives on intact MCF-7 cell proliferation
after 24 h of treatment with the compounds. Cells were plated at a
density of 1.4Â105 cells/mL per well using 96-well plates. The cell
growth was assessed by the MTT assay and is expressed relative to
untreated control cultures (100%). Each point represents the average
of at least three independent experiments in which four replicate
determinations were made; error bars represent the SEM. The curves
were determined by non-linear fitting to the Hill equation. Six to eight
concentrations of each compound were used. Some data points (below
1 mM) are not shown, for clarity.
Table 1. Effects of compounds on the growth of MCF-7 cells in
culture
Comp.
Cytotoxicity IC50 (mM)a
1a
1b
1c
1d
1e
1f
27.7Æ3.3
>100
>100
29.0Æ1.3
5.3Æ0.7
7.8Æ0.6
115.1Æ24.3
1g
aData represent the meanÆSEM of the IC50 obtained from at least
three independent experiments, each with four replicates. At least five
concentrations of each compound were tested to determine the IC50 by
fitting the Hill equation to the data.
Scheme 1.

A. J. M. da Silva et al. / Bioorg. Med. Chem. 10 (2002) 2731–2738
2733
studied in this series were also able to inhibit cell growth
in a concentration-dependent manner, but their full
effect apparently required higher concentrations that
could not be tested due to the limited solubility in
DMSO and aqueous solutions.
The ortho-quinone b-lapachone was previously shown
to kill MCF-7 cells mainly by apoptosis, in a manner
independent of cell cycle stage and of the p53/p21
pathway.18À20 The cytotoxic effect of the quinones has
often been ascribed to their redox reactive properties,
which may lead to covalent modification of key
enzymes, particularly DNA topoisomerases.21,22 How-
ever, the differences in potency among the derivatives
shown here may indicate that their antiproliferative
effect is not solely due to the reactive quinone group,
since their activity was influenced by the pattern of
oxygenation in ring D. Furthermore, a recent in vitro
study suggested that the 1,4-naphthoquinone a-lapa-
chone inhibits topoisomerase II through a mechanism
distinct from that of b-lapachone.23 Thus, the mechan-
ism of action of the novel series of compounds might be
different from that of other quinones.
Figure 2. Effect of compounds 1b, 1d, and 1e on HSV-2 growth. Vero
cells were infected with HSV-2 (strain G) at m.o.i.=10 p.f.u./cell in the
presence of increasing concentrations of the compounds and virus
growth was determined by plaque assay at 24 h p.i. Results are
expressed as% inhibition relative to virus titers in cells grown in the
absence of the compounds. Results obtained in three independent
experiments did not differ by more than 6–9% (* is 1d; * is 1b and !
is 1e).
Viral infection assay
The incidence of resistance to acyclovir (ACV), which is
commonly used for the treatment of HSV infections, is
increasing rapidly. This is particularly true among
immunocompromised individuals in whom resistant
strains can cause severe and debilitating disease. Ongo-
ing research is focused on the development of agents
that may be effective against ACV-resistant strains,28À30
most of which are mutated in thymidine kinase (TK).31
Compound 1d is particularly promising for the treat-
ment of infections due to ACV-resistant TK mutants
because it is unlikely to target the HSV TK. Indeed,
b-lapachone is a DNA topoisomerase I inhibitor that
induces cell cycle delay at the G1 to S phase.11,12
Topoisomerase I was previously implicated in HSV-2
replication.32 However, additional studies are needed
in order to determine the mechanism responsible for
HSV-2 growth inhibition by the lapachol derivatives 1d
and 1b and define the spectrum of their antiviral activity.
The antiviral activity of derivatives 1a–e was studied
using infection with Herpes Simplex Virus type 2 (HSV-
2) as a model system. African green monkey (Vero) cells
were infected with HSV-2 (strain G) at a multiplicity of
infection (m.o.i.) of 10 plaque-forming units (p.f.u.) per
cell in the presence or absence of increasing concentra-
tions of the respective compounds. Cell extracts were
collected at 24 h post infection (p.i.) and assayed for
virus titers by plaque assay, as described.24À26 Duplicate
cultures were mock-infected with phosphate buffered
saline instead of virus, exposed to increasing concentra-
tions of compounds 1a–e or DMSO (used as vehicle for
compound solubilization) and examined for cytotoxic
effects 24 h later. Cytotoxicity was seen in the mock-
infected cultures treated with 20–100 mg/mL of DMSO
or compounds 1a–e. The results of three independent
experiments that did not differ by more than 6–9%
indicated that compound 1d caused a significant con-
centration-dependent decrease in HSV-2 growth (Fig. 2).
The IC50 calculated from these data was 25 ng/mL and
the IC90 was 400 ng/mL. The therapeutic index, expres-
sed as the minimal toxic concentration divided by the
minimal concentration inhibiting virus growth was
2000. This value is within the range of most antiviral
compounds with clinical effectiveness. HSV-2 growth
was also inhibited by compound 1b, albeit less efficiently
(Fig. 2). The approximate IC50 for 1b, obtained from
only two drug concentrations, was 180 ng/mL. Virus
growth was not inhibited by the other compounds
studied in this series.
Muscle toxicity assay
The activity against muscle damage induced by snake
venom was tested in order to identify mixed enzyme-
inhibiting properties of the novel naphthoquinones. The
crude venom of Bothrops jararacussu is myotoxic,
mainly due to the presence of proteolytic and phospho-
lipase A2 activities which interact with tissues inducing
hemorrhage and myonecrosis.33À35 Sarcolemmal damage
and myotoxicity induced by B. jararacussu crude venom
were assessed in vitro by the rate of loss of creatine
kinase (CK) from mice isolated extensor digitorum
longus (EDL) muscles.36,37 At 30 mM, six compounds
(1a, 1b, 1d, 1e, 1f, and 1g) antagonized the increase of
CK release induced by the venom (Fig. 3A). The com-
pound bearing two hydroxyls in ring D (1g) was the
most potent and was able to fully inhibit the venom’s
myotoxic activity with an IC50 of 1.3 mM (Fig. 3B).
Interestingly, coumestans with the same pattern of
We interpret these findings to indicate that compound
1d (and to a lower extent 1b) has antiviral activity, at
least against HSV-2. This is particularly significant since
HSV-2 is one of the most widely distributed sexually
transmitted infections and its prevalence has recently
reached epidemic proportions.27

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A. J. M. da Silva et al. / Bioorg. Med. Chem. 10 (2002) 2731–2738
Figure 4. (A) Effect of compounds 1d–1g on 0.2 nM [3H]-fluni-
trazepam binding to rat brain synaptosomes. (B) Full inhibition curve
for compound 1e, which yielded an IC50 of 2 mM. Each point repre-
sents the meanÆSEM of 2–4 independent experiments performed in
triplicate.
Figure 3. Effect of compounds 1a, 1b, 1d, 1e, 1f, and 1g on CK release
induced by Bothrops jararacussu venom. (A) Bars represent the rate of
CK release (U gÀ1 hÀ1) of mouse EDL muscles exposed during 60 min
to B. jararacussu venom (V; 25 mg/mL) either alone or in the presence
of 30 mM of compounds 1a–g. The basal rate of CK release in physi-
ological saline was 0.33Æ0.03 U gÀ1 hÀ1 (n=58). (B) Concentration–
effect curve for compound 1g (0.1–10 mM) plus 25 mg/mL of B. jarar-
acussu crude venom. Each point represents the meanÆSEM (n=3–7).
oxygenation in ring D were previously shown to inhibit
the cell damage due to various crude venoms or purified
protease and phospholipase A2 toxins, including the
natural coumestan wedelolactone38,39 and some of its
derivatives recently described by our group.15
Benzodiazepine-like activity assays
In a preliminary pharmacological screening of the novel
compounds, their ability to compete with [3H]-fluni-
trazepam for the binding to benzodiazepine (BZ) recep-
tors in rat brain synaptosomes was assessed. As shown
in Fig. 4A, compounds 1d–1g inhibited the binding of
[3H]-flunitrazepam in a concentration-dependent man-
ner when tested at 3–30 mM. A complete inhibition
curve of the most active compound 1e (Fig. 4B) yielded
an IC50 of 2 mM. These results indicate a potential
modulatory effect on the g-aminobutyric acid A
(GABAA)/chloride ion channel involved in inhibitory
transmission in the central nervous system.40,41
Figure 5. Sample traces showing effects of 1e in rat hippocampal neu-
rons voltage-clamped at 0 mV. (A) Tonic outward ClÀ currents
induced by endogenous GABA were reversibly potentiated by 1e
30 mM and by midazolam 0.1 mM (MID). (B) Blockade by bicuculline
10 mM (BIC) reveals the magnitude of the tonic GABA-induced cur-
rent which was potentiated by MID and 1e in an additive manner. The
indicated drugs were applied during the periods corresponding to the
thick lines under the traces.
The effects of compound 1e on the GABAA-BZ receptor
complex were further investigated using electro-
physiological techniques. When applied alone, com-
pound 1e at 30 mM potentiated the bicuculline-sensitive
tonic whole-cell currents due to endogenous GABA in
28 out of 32 rat hippocampal neurons (Fig. 5), but had
no effect when currents elicited by GABA were absent
or blocked. The augmented current elicited by 1e was
inhibited by the competitive GABAA antagonist
bicuculline 10 mM. The average potentiating effect of 1e
30 mM was 84Æ6.7% of that of the full BZ agonist
midazolam at 0.1 mM (n=6 cells). Addition of 1e 30 mM
to midazolam 0.1 mM caused further current increases
(n=4). These data suggest that compound 1e is an
agonist at the BZ modulatory site of the GABAA

A. J. M. da Silva et al. / Bioorg. Med. Chem. 10 (2002) 2731–2738
2735
receptor-channel. Although the potency and the affinity
of 1e seem to be much lower than that of the benzodia-
zepines, their structures are unrelated, and the investi-
gation of new derivatives will be of significant interest in
view of the potential applications of subtype-selective
BZ ligands.41
(dd, J=3.5, 2.0 Hz, 2H), 5.87 (dt, J=10.0, 3.5 Hz, 1H),
6.72 (dt, J=10.0, 2.0 Hz, 1H), 7.48–8.27 (m, 4H). Anal.
calcd for C13H8O3: C, 73.58; H, 3.80. Found: C, 73.56;
H, 3.81.
General procedure for the preparation of naphthoqui-
nones (1a–d). Example: naphthoquinone (1d). To a
mixture of PdCl2 (0.194 g, 1.1 mmol) and LiCl (0.047 g,
1.1 mmol) in acetone (10 mL) was added chromen 3
(0.250 g, 1.1 mmol) in acetone (10 mL). This mixture
was stirred for 15 min at 0 ꢀC and then 2-chloro-
mercurio-4,5-methylenedioxyphenol 4 days (0.414 g,
1.1 mmol) in acetone (10 mL) was added to it. The sus-
pension thus obtained was stirred for 12 h at 25 ꢀC.
After this time, brine (30 mL) was added to it and the
mixture was extracted with acetyl acetate (100 mL), the
organic extract dried (Na2SO4), and subjected to column
chromatography to give theꢀcompound 1d as a red solid
(0.218 g, 57%), mp 136–138 C. 1H NMR (CDCl3) d 3.44
(m, 1H), 3.76 (t, J=11.2 Hz, 1H), 4.51 (dd, J=11.2,
5.1 Hz, 1H), 5.63 (d, J=6.7 Hz, 1H), 5.91 (d, J=5.6 Hz,
1H), 5.92 (d, J=5.6 Hz, 1H), 6.58 (s, 1H), 6.72 (s, 1H),
7.71–7.78 (m, 2H), 8.10–8.25 (m, 2H); 13C NMR
(CDCl3) d 38.47 (C), 67.30 (C), 73.07 (CH), 94.34 (CH),
101.33 (CH2), 104.36 (CH), 115.86 (C), 118.18 (C),
126.52 (CH), 127.83 (CH), 130.57 (C), 131.85 (C), 133.41
(CH), 134.58 (CH), 142.02 (C), 148.42 (C), 153.74 (C),
157.05 (C), 179.33 (C), 183.32 (C). Anal. calcd for
C20H12O6: C, 68.97; H, 3.47. Found: C, 68.95; H, 3.48.
Conclusion
In summary, the newly synthesized 1,4-naphthoqui-
nones exhibited a range of significant biological activ-
ities, suggesting potential therapeutic value as anticancer
and antiviral agents, as well as against the myotoxic
effects of crotalid venoms. Compounds 1b and 1d were
very active against HSV-2 growth, showing IC50 values
in the submicromolar range. In vivo studies using ani-
mal models of viral infections and additional chemical
alterations that may enhance their activity and widen
their antiviral spectrum are under consideration. Com-
pound 1e emerged as the most potent inhibitor of breast
cancer cell proliferation, but also as a modulator at the
BZ binding site, an effect not yet reported for quinones.
Considering the known activity of the ortho- and para-
quinones b-lapachone and a-lapachone,11,12 the naphtho-
quinone moiety may be essential for the antiproliferative
and antiviral effects. However, the substituents in ring D
may confer selectivity at the target(s) involved in these
effects and may be the main determinants of the activity
against cytotoxic snake venom enzymes and of the
binding affinity to BZ receptors. The observed differ-
ences in potency and selectivity of the compounds may
help clarify the structural requirements for binding to
their respective molecular targets, and thus deserve fur-
ther investigation.
The following naphthoquinones were prepared accord-
ingly.
Naphthoquinone (1a). This compound was obtained by
column chromatography as a solid in 55% yield, mp
ꢀ
1
65–67 C. H NMR (CDCl3) d 3.43 (m, 1H), 3.73 (t,
J=11.1 Hz), 3.82 (s, 3H), 4.48 (dd, J=11.3, 4.6 Hz, 1H),
5.61 (d, J=6.6 Hz, 1H), 5.06 (s, 2H), 6.60 (s, 1H), 6.68
(s, 1H), 7.38–7.60 (m, 5H), 7.73–7.82 (m, 2H), 8.11–8.26
(2H); 13C NMR (CDCl3) d 38.75 (CH), 56.01 (CH3),
67.34 (CH2), 72.76 (CH2), 72.92 (CH), 96.34 (CH),
112.54 (CH), 114.93 (C), 118.32 (C), 127.50 (CH),
127.85 (CH), 128.37 (CH), 130.64 (CH), 131.09 (CH),
131.93 (CH), 132.53 (CH), 133.36 (CH), 134.58 (CH),
135.48 (CH), 137.15 (C), 142.73 (C), 151.68 (C), 154.06
(C), 157.06 (C), 179.41 (C), 183.38 (C). Anal. calcd for
C27H20O6: C, 73.63; H, 4.58. Found: C, 73.64; H, 4.57.
Experimental
Chemistry
Melting points were determined with a Thomas-Hoover
apparatus and are uncorrected. Column chromato-
graphy was performed on silica gel 230–400 mesh
1
(Aldrich). H NMR spectra were recorded on a Varian
Gemini (200 MHz) instrument using tetramethylsilane
(TMS) as standard and CDCl3 as solvent. J Values are
given in Hz. 13C NMR spectra were obtained at
50 MHz.
Naphthoquinone (1b). This compound was obtained by
column chromatography as a solid in 58% yield, mp
96–98 ꢀC. H NMR (CDCl3) d 3.46 (m, 1H), 3.75 (t,
1
2H-Benzo[g]chromene-5,10-dione (3). A solution of law-
sone (2) (1,0 g, 5.74 mmol), acrolein (13.2 mL), phe-
nylboronic acid (0.7 g, 5.74 mmol) and glacial HOAc
(26.4 mL) in toluene (210.0 mL) was refluxed for 1 h.
Optimization of product formation was effected by TLC
monitoring. The mixture was cooled, concentrated in
vacuo, and the residue was extracted with several por-
tions of EtOAc. The combined extract was washed suc-
cessively with H2O, NaHCO3, and brine, dried
(Na2SO4), the solvent was evaporated in vacuo, and the
crude product was purified by flash chromatography on
silica gel (EtOAc–hexane 1:9 eluent) to give a red solid
J=11.7 Hz, 1H), 3.84 (s, 3H), 4.52 (d, J=11.0, 5.0 Hz,
1H), 5.08 (s, 2H), 6.57 (s, 1H), 6.83 (s, 1H), 7.25–7.58
(m, 5H), 7.65–7.78 (m,2H), 8.08–8.24 (m, 2H); 13C
NMR (CDCl3) d 38.71 (CH3), 57.11 (CH), 67.29
(CH2), 71.00 (CH2), 72.80 (CH), 98.43 (CH), 109.28
(CH), 115.58 (C), 118.31 (C), 126.44 (CH), 127.02 (CH),
127.76 (CH), 128.41 (CH), 130.58 (C), 131.85 (C),
132.46 (CH), 133.34 (CH), 133.53 (CH), 134.49 (CH),
135.43 (CH), 135.43 (CH), 136.54 (C), 144.50 (C),
149.64 (C), 153.16 (C), 157.00 (C), 179.29 (C), 183.29
(C). Anal. calcd for C27H20O6: C, 73.63; H, 4.58.
Found: C, 73.65; H, 4.57.
(0.6 g, 50%), mp 215–217 ꢀC. H NMR (CDCl3) dn 5.1
1

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A. J. M. da Silva et al. / Bioorg. Med. Chem. 10 (2002) 2731–2738
Naphthoquinone (1c). This compound was obtained by
column chromatography as a solid in 50% yield, mp
pounds in DMSO (stored frozen) were freshly diluted in
culture medium and filter-sterilized before addition to
the cells. The final concentrations of DMSO had no
effect on cellular proliferation. The cells were incubated
for 24 h in the absence and presence of the test com-
pounds. After the incubation period, the medium was
removed and cells were incubated with 1 mg/mL MTT
at 37 ꢀC for 3 h. Reduced MTT was measured at 560 nm
using a 96-well plate reader (Victor 1420 Multilabel
Counter—Wallac) after solubilization with DMSO and
homogenization.
ꢀ
1
147–149 C. H NMR (CDCl3) d 3.39 (m, 1H), 3.70 (t,
J=10.8 Hz, 1H), 4.45 (dd, J=11.2, 5.1 Hz, 1H), 5.05 (s,
2H), 5.07 (s, 2H), 5.57 (d, J=6.5 Hz, 1H), 6.60 (s, 1H),
6.84 (s, 1H), 7.23–7.74 (m, 10H), 8.06–8.24 (m, 4H); 13C
NMR (CDCl3) d 38.55 (CH), 67.29 (CH2), 71.18 (CH2),
72.91 (CH), 73.34 (CH2), 98.78 (CH), 113.90 (CH),
116.00 (C), 118.29 (C), 126.45 (CH), 126.51 (CH),
127.16 (CH), 127.63 (CH), 128.28 (CH), 130.67 (CH),
130.96 (CH), 131.93 (CH), 132.53 (CH), 133.38 (CH),
134.51 (CH), 135.50 (CH), 136.67 (C), 137.31 (C),
143.38 (C), 151.05 (C), 154.18 (C), 157.07 (C), 179.31
(C), 183.32 (C). Anal. calcd for C33H24O6: C, 76.73; H,
4.68. Found: C, 76.75; H, 4.69.
In vitro myotoxicity was evaluated at room temperature
as described.36 Briefly, EDL muscles were blotted,
weighed rapidly and then transferred to sample collect-
ing units of 2.5 mL capacity, where they were superfused
continuously at a rate of 3 mL/min with physiological
saline solution (PSS) equilibrated with 95% O2–5%
CO2. At 40-min intervals, the solutions perfusing the
muscles were collected and replaced with fresh media.
The collected samples were stored at 4 ꢀC until their CK
activity was determined using a diagnostic kit (Sigma).
The rate of CK release from the isolated muscles is
expressed as international enzyme units released into the
medium per gram of muscle per h of collection
(U gÀ1 hÀ1). The basal release rate refers to the enzyme
loss from the muscles into the PSS at the beginning of
the experiment, after the preparations had been moun-
ted in the sample collecting units for at least 1 h. The
composition of the PSS was (in mM) NaCl 135, KCl 5,
CaCl2 2, MgCl2 1, NaHCO3 15, NaH2PO4 1, and glu-
cose 11 (pH 7.4Æ0.02 after equilibration with 95% O2–
5% CO2). Bothrops jararacussu venom (Instituto Vital
Brazil, Niteroi, RJ, Brazil) alone or associated to the
different naphthoquinones was added to the nutrient
solution which superfused the isolated muscles.
General procedure for the preparation of naphthoqui-
nones (1e–g). Example: naphthoquinone (1e). Naphtho-
quinone (1a) (0.440 g, 0.1 mmol) in acetone was
hydrogenated (3 atm) in the presence of Pd/C (10%).
After 3 h the catalyst was filtered and the product pur-
ified by chromatography on silica to give 1e in 65%
yield, mp 119–121 ꢀC. 1H NMR (CDCl3) d 3.47 (m,
1H), 3.76 (t, J=11.0 Hz, 1H), 3.84 (s, 3H), 4.54 (dd,
J=11.0, 5.1 Hz, 1H), 5.61 (d, J=6.7 Hz, 1H), 6.57 (s,
1H), 6.85 (s, 1H), 7.69–7.83 (m, 2H), 8.09–8.22 (m, 2H).
Anal. calcd for C20H14O6: C, 68.57; H, 4.03. Found: C,
68.56; H, 4.01.
The following phenolic derivatives were prepared
accordingly.
Naphthoquinone (1f). This compound was obtained by
column chromatography as solid in 70% yield, mp 117–
120 ꢀC. 1H NMR (CDCl3) d 3.48 (m, 1H), 3.79 (t,
J=11.1 Hz, 1H), 3.87 (s, 3H), 4.54 (dd, J=11.1, 4.0 Hz,
1H), 5.62 (d, J=6.8 Hz, 1H), 6.60 (s, 1H), 6.81 (s, 1H),
7.73–7.83 (m, 2H), 8.12–8.22 (m, 2H). Anal. calcd for
C20H14O6: C, 68.57; H, 4.03. Found: C, 68.55; H, 4.00.
Specific binding of flunitrazepam was studied in crude
synaptosomes prepared from rat brain (without cere-
bellum and brainstem). Briefly, brains were homo-
genized in a Potter homogenizer with a motor-driven
teflon pestle at 4 ꢀC in 15 volumes of ice-cold 0.32 M
buffered sucrose (pH 7.4) per gram of organ. After cen-
trifuging at 1000gmax for 10 min, the supernatant was
centrifuged at 48,000gav for 20 min to obtain the crude
synaptosomes that were resuspended in buffered Krebs
solution and stored at À80 ꢀC until use. Synaptosomes
(200 mg protein) were incubated at 4 ꢀC for 90 min in a
buffered Krebs solution containing 0.2 nM [3H]-fluni-
trazepam (85 Ci/mmol, New England Nuclear Life
Science Products, USA). After incubation, samples were
rapidly diluted with 3 mL of ice-cold Tris–HCl buffer
and instantaneously filtered on glass fibre filters (GMF
3 type from Filtrak, Germany) under vacuum. Filters
were then washed once more with 3 mL buffer, dried,
immersed in a scintillation cocktail and the radioactivity
retained in the filters was counted with a Packard Tri-
Carb1600 TR liquid scintillation analyser. Nonspecific
binding was estimated in the presence of 5 mM unlabeled
flunitrazepam.
Naphthoquinone (1g). This compound was obtained by
column chromatography as solid in 60% yield, mp 195–
197 ꢀC. 1H NMR (CDCl3) d 3.56 (m, 1H), 3.77 (t,
J=10.1 Hz, 1H), 4.56 (dd, J=10.1, 4.8 Hz, 1H), 5.54 (d,
J=6.8 Hz, 1H), 6.36 (s, 1H), 6.88 (s, 1H), 7.28–7.45 (m,
2H), 7.83–8.13 (m, 2H). Anal. calcd for C19H12O6: C,
67.86; H, 3.60. Found: C, 67.87; H, 3.62.
Pharmacology
The effect of the test compounds on MCF-7 cell pro-
liferation was examined using the 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT)
colorimetric assay.42À44 MCF-7 cells were grown in
MEM complete medium containing 1 mM sodium pyr-
uvate (90%), 10% fetal bovine serum and 1% penicillin/
streptomycin (all from GibcoBRL) at 37 ꢀC in a humi-
dified atmosphere of 5% CO2–95% air. Cells were see-
ded in 96-well flat-bottom plates (Falcon, Becton
Dickinson Labware) at 1.4Â105 cells/mL (100 mL) on
day zero. Twenty-four hours later, when the cells were
in exponential growth, several concentrations of the test
compounds were added. Stock solutions of the com-
Hippocampal neurons were isolated from rat fetuses at
18–20 days of gestation, maintained in culture for 14–35

A. J. M. da Silva et al. / Bioorg. Med. Chem. 10 (2002) 2731–2738
2737
days, and used in whole-cell patch-clamp membrane
current recordings as previously described.45 Com-
pounds were dissolved in the standard bath saline,
which consisted of (in mM) NaCl 165, KCl 5, CaCl2
2, HEPES 5, NaOH 2 (pH 7.35), and solutions were
applied to the neurons in fast-switching pulses with
a motorized parallel tube array system. Chloride
currents induced by endogenous GABA were recor-
ded in the presence of the sodium channel blocker
tetrodotoxin (0.3 mM) at a fixed membrane potential
of 0 mV, in order to eliminate action potential-
dependent activity and spontaneous excitatory synaptic
currents.
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The concentration–response and binding curves were
fitted by either a single-site equation (rectangular
hyperbola) or the Hill equation, using nonlinear regres-
sion. Data from assays of viral growth, venom-induced
CK release and [3H]-flunitrazepam binding were well
fitted by a single-site model, whereas cancer cell pro-
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Acknowledgements
Our research was supported by grants from PRONEX
(No. 41.96.0888.00), FAPERJ, FUJB-UFRJ, and
CAPES, in Brazil, and from NIH/NCI (5RO1 CA83199–
02) to L.H.M., and NIH/NCI (CA74904–03) to R.J.H.,
in the USA. A.J.M.S is a postdoctoral fellow of FAPERJ
(No. 26/151.081/97). F.N., N.G.C. and P.R.R.C are
CNPq fellows. F.V.B, C.D.B., D.V.S., V.P.R. and
R.R.B.C. are recipients of CNPq (PIBIC) fellowships.
E.S.C.P. is recipient of a FAPERJ fellowship. L.F.M.
was supported in part by CAPES and by US Army
Medical Research and Materiel Command’s Breast
Cancer Predoctoral grant. We thank Produtos Roche,
Brazil, for supplying flunitrazepam and midazolam.
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Kwong, A. D.; Rozhon, E. J.; O’Connell, J. F. Antiviral Res.
1997, 35, 139.
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