A new approach for the synthesis of 3-substituted cytotoxic nor-β-lapachones

Article abstract of DOI:10.1590/S0103-50532013000100003

Several studies have demonstrated the cytotoxic potential of nor-β-lapachone derivative against cancer cells. Considering nor-β-lapachone as an important prototype, a set of new 3-substituted nor-β-lapachones was synthesized by a new synthetic route that involves the use of synthetic intermediate generated for coupling with several nucleophiles containing the carbohydrate and 2H-pyrazole substituent moieties. All the compounds were screened against four tumor cell lines. Two of the compounds showed moderate cytotoxicity, while the other compounds strongly inhibit all tested cancer cell lines.

Full text of DOI:10.1590/S0103-50532013000100003

J. Braz. Chem. Soc., Vol. 24, No. 1, 12-16, 2013.
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Article
A
A New Approach for the Synthesis of 3-Substituted Cytotoxic Nor-β-Lapachones
Mariana F. C. Cardoso,a,b Illana M. C. B. da Silva,a Helvécio M. dos Santos Júnior,c
David R. Rocha,a Ana Jérsia Araújo,d Claudia Pessoa,d Manoel O. de Moraes,d
Letícia V. C. Lotufo,d Fernando de C. da Silva,a Wilson C. Santosb and Vitor F. Ferreira*,a
aDepartamento de Química Orgânica, Instituto de Química,
Universidade Federal Fluminense, 24020-141 Niterói-RJ, Brazil
bDepartamento de Farmácia e Administração Farmacêutica, Faculdade de Farmácia,
Universidade Federal Fluminense, 24241-000 Niterói-RJ, Brazil
cDepartamento de Química Orgânica, Instituto de Química, Cidade Universitária,
Universidade Federal do Rio de Janeiro, 21944-970 Rio de Janeiro-RJ, Brazil
dDepartamento de Fisiologia e Farmacologia, Faculdade de Medicina,
Universidade Federal do Ceará, 60430-270 Fortaleza-CE, Brazil
Vários estudos têm mostrado o potencial citotóxico de derivados da nor-β-lapachona contra
células tumorais. Considerando a nor-β-lapachona um importante protótipo, uma série inédita de
nor-β-lapachonas substituídas em C-3 foi sintetizada através de uma nova metodologia sintética
envolvendo intermediário sintético 3-hidróxi-nor-β-lapachona para o acoplamento de alguns
nucleófilos contendo núcleos derivados de carboidratos ou 2H-pirazóis. Todos os derivados
foram avaliados frente a quatro linhagens de células tumorais. Duas das substâncias apresentaram
moderada citotoxicidade, enquanto as demais inibiram fortemente todas as linhagens tumorais
testadas.
Several studies have demonstrated the cytotoxic potential of nor-β-lapachone derivative against
cancer cells. Considering nor-β-lapachone as an important prototype, a set of new 3-substituted
nor-β-lapachones was synthesized by a new synthetic route that involves the use of synthetic
intermediate generated for coupling with several nucleophiles containing the carbohydrate and
2H-pyrazole substituent moieties. All the compounds were screened against four tumor cell lines.
Two of the compounds showed moderate cytotoxicity, while the other compounds strongly inhibit
all tested cancer cell lines.
Keywords: naphthoquinone, heterocycle, cytotoxicity, carbohydrate
Introduction
biological activities, justifying the numerous studies in the
literature on the synthesis and evaluation of either natural
quinones or their analogues as potential antitumor agents.3-7
Despite the broad spectrum of biological activities of
the naphthoquinones, their mechanism of action is not well
understood. Some studies have indicated that the mechanism
involves the nuclear enzymes topoisomerases I and II,8,9 and
other studies have shown that the quinones can be reduced
by one or two electrons, resulting in intermediates that can
induce a redox cycle and generate reactive oxygen species
(ROS), thereby inducing apoptosis.10
Cancer is a leading cause of death worldwide and
accounted for 7.6 million deaths (around 13% of all deaths)
in 2008.1 The development of new chemotherapeutic
agents for the treatment of cancer is urgently needed and
the search for new natural or synthetic substances that
display this activity remains a challenge to medical
science. Drugs containing a quinone moiety, such as
anthracyclines, mitoxantrones and lapachol, show excellent
anticancer activity.2 Among these, it is highlighted the
naphthoquinones, which possess a diverse range of
Figure 1 shows a group of natural and synthetic
naphthoquinones, of which lapachol (1) is the most
well-known member. Lapachol is present as a constituent
*e-mail: cegvito@vm.uff.br

Vol. 24, No. 1, 2013
Cardoso et al.
13
of various plant families, including the Bignoniaceae,
Leguminosae, Sapotaceae, Scrophulariaceae,Verbenaceae,
Malvaceae and Proteaceae. Lapachol exhibits an
impressive list of important biological activities11 such as
trypanocidal,12 antimalarial, antitubercular,13 antitumor,14
antibacterial15 and antileishmanial,16 as well as activity
against the snail Biomphalaria glabrata17 (which is involved
in the transmission of schistosomiasis).18 The structure of
lapachol was the inspiration for other important analogues,
such as atovaquone (2) and buparvaquone (3), both are
important 3-substituted-2-hydroxy-1,4-naphthoquinones
used as drugs for the treatment of Pneumocystis
pneumonia, toxoplasmosis and malaria (Figure 1). The
multiple and varied antimicrobial activity of lapachol and
structurally related compounds highlights the importance
of this class of compounds (Figure 1). An important
derivative of lapachol (1) is the β-lapachone (4), an
ortho-naphthoquinone naturally obtained from a native
SouthAmerica tree Tabebuia avellanedae with a wide range
of pharmacological activities, including anti-bacterial,
antifungal, anti-trypanosomal, anti-retroviral and
anti-inflammatory activities19-26
highlighted substances 9 and 10, which were more active
against cancer cells and Trypanosoma cruzi, respectively
(Scheme 1).
O
O
Br
O
OH
O
O
Br2 (excess)
CHCl3
NaN3
(excess)
N3
O
O
O
8
7
5
ii
i
OMe
O
O
O
O
Ph
N
N
N
N
H
O
O
9
10
(i) Excess 4-methoxyaniline
(ii) CuSO4, ethynylbenzene, sodium ascorbate, CH2Cl2
Scheme 1. Transformation of 5 into naphthoquinones 9 and 10.
O
O
O
O
H
H
N
Br
N
O
O
12, 8%
11, 6%
N
Br
H
O
O
O
O
O
O
O
OH
OH
OH
H
O
N
N
N
N
O
O
NH
N
N
O
O
O
14, 7%
13, 10%
CF3
Lapachol (1)
Atovaquone (2)
Buparvaquone (3)
Cl
O
O
Br
R
O
O
O
O
O
O
O
OH
H
N
N
N
N
N
N
O
O
O
O
O
Br
Br
16, R = Br, 6%
17, R = H, 33%
15, 10%
CH3
CH3
β-Lapachone (4)
Nor-lapachol (5)
Nor-β-lapachone (6)
Figure 2. Naphthoquinones 11-17 obtained with conditions of Scheme 1.
Figure 1. Examples of important natural and synthetic naphthoquinones.
As can be seen in Scheme 1, the methodology used to
prepare the derivatives of 6 substituted in the pyran ring
employed the 2H-naphtho-[1,2-b]-furanylium ion generated
in situ by bromine. However, this protocol has several
disadvantages, and cannot be used as a general method. The
large excess of bromine and nucleophiles (over 50 equiv.)
limit their use.3,5 The excess bromine itself can react with
the nucleophiles, generating undesirable brominated
products and/or degradation of the nucleophile due to
high oxidizing power of bromine. In addition, bromine is
toxic and hazardous to human health. Furthermore, its high
vapor pressure makes it difficult to be easily manipulated.
Continuing with our interest in the synthesis of
3-substituted nor-β-lapachones, our group describes
in this work the synthesis of various 3-substituted
nor-β-lapachones coupled with heterocyclic pyrazole and
carbohydrate derivatives and its cytotoxic potential against
four cancer cell lines.
Another compound, nor-lapachol (5), is a synthetic
derivative that can be obtained from 1 by a degradation
reaction with H2O2, known as the Hooker reaction,27 or by
the condensation of 2-hydroxy-1,4-naphthoquinone with
isobutyraldehyde.28 Compound 5 can be cyclized in a strong
acid, producing nor-β-lapachone (6). Recently, it was
reported that 6 induced a weak in vitro cytotoxic activity in
non-tumor cells (human cultured lymphocytes and Chinese
hamster lung fibroblasts) showing some selectivity for
tumor cells. The cytotoxic effect is related to the compound
ability to decrease the intracellular level of the enzyme
glutathione S-transferase (GSH), leading to an even greater
pro-oxidant condition in cells.29 The elevated oxidative
activity in the cells creates an antiproliferative environment.
Compound 5 was investigated as a starting material in the
synthesis of new substances that would demonstrate better
anticancer and trypanocidal activity. In this respect, it is

14
A New Approach for the Synthesis of 3-Substituted Cytotoxic Nor-β-Lapachones
J. Braz. Chem. Soc.
Results and Discussion
Biological assay
Chemistry
Several studies have demonstrated the potential cytotoxic
of nor-β-lapachone derivatives against cancer cells.3-6,31
The in vitro anticancer activity of new nor-β-lapachone
derivatives (15-17 and 19a-h) was assessed against four
human cancer cell lines, OVCAR-8 (ovarian), HCT-116
(colorectal), SF-295 (central nervous system) and HL-60
(leukaemia), in comparison to doxorubicin, the positive
control, by using MTT (yellow dye 3-(4,5-dimethyl-
2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide) assay.31
Concentrations that induce 50% inhibition of cell growth
(IC50) in µM are reported in Table 1.
Results showed that all compounds are strongly or
moderately cytotoxic against all cancer cell lines with IC50
in the range of 0.16 to 32.29 µM. Based on data collected
from two independent experiments, the compound 16 was
considered less active with IC50 values ranging from 11.7
to 32.29 µM followed by compound 15 that showed IC50
values ranging from 3.09 to 22.17 µM.All other compounds
exhibited similar IC50 values ranging from 0.16 to 2.13
(Table 1).
The nor-β-lapachone compounds were synthesized
using an alternative methodology that utilizes the
nucleophiles in stoichiometric amounts, without the
formation of byproducts like those from the reaction of
bromine and nucleophiles.
Initially, some test reactions were performed under
the same conditions that were previously reported by our
group.3,5 Using tryptamine, aniline, benzotriazol-1-ol and
5-trifluoromethyl-1H-[1,2,4]-triazol-3-ylamine, the
furan-naphthoquinones 11, 12, 13 and 14, respectively,
were obtained in low yield. When 5-methyl-2-phenyl-
2H-pyrazol-3-ylamine was used, the reaction produced
the compounds 15 and 16 or 17. During these reactions,
the excess bromine reacted with heterocyclic and aromatic
rings. Several reactions were also attempted with acetonides
of carbohydrates, but none of them isolated the desired
glycoconjugates due to extensive decomposition of the
carbohydrates under this condition.
To resolve the challenge of bromine reactivity in this
reaction, it was changed the synthetic route (Scheme 2).
First, the compound 5 was converted into 3-hydroxy-
nor-β-lapachone (18),30 which was converted under mild
conditions in the same 2H-naphtho[1,2-b]furanylium
intermediate ion (7). The compound (7) reacted with
various nucleophiles, producing the nor-β-lapachone
(19a-h). With this synthetic route, it was possible to
isolate the pyrazole-naphthoquinones (19a-e) and the
glycoconjugates (19f-h) with moderate yields and recover
the reagent (18).
Conclusions
In summary, this study showed that it is possible to obtain
derivatives of nor-β-lapachone in mild conditions. The
results clearly show that the 3-hydroxy-nor-β-lapachone
(18) is a versatile intermediate in the formation of the
derivatives (19a-h). In relation to the biological studies,
the results presented reaffirm the cytotoxic potential of
nor-β-lapachone derivatives against cancer cell lines.
O
O
O
OH
O
O
Br2/CHCl3
NaHSO3
(i) CH3SO3H, CHCl3
OH
XR
(ii) R-XH
O
O
O
5
18, 60%
19
O
O
O
O
Br
O
O
O
O
H
N
H
H
H
N
N
N
N
N
N
N
N
N
N
O
O
O
O
N
19d, 35%
CH3
19c, 37%
19b, 29%
19a, 52%
OCH3
O
O
O
O
O
O
O
O
O
H
N
O
O
OCH3
N
N
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
Ph
19e, 35%
19f, 62%
19g, R = 14%
19h, R = 11%
CH3
Scheme 2. General scheme used for preparation 3-substituted nor-β-lapachones.

Vol. 24, No. 1, 2013
Cardoso et al.
15
Table 1. Cytotoxic activity expressed by IC50 in µM of nor-β-lapachone derivatives for cancer cell lines, obtained by nonlinear regression for all cell lines
from two independent experimentsa
Compounds
HL-60
HCT-116
SF-295
OVCAR-8
15
3.09 (2.33-4.09)
11.7 (5.76-23.8)
0.8 (0.65-0.98)
0.73 (0.58-0.93)
0.57 (0.53-0.63)
0.43 (0.37-0.5)
0.82 (0.76-0.89)
0.34 (0.29-0.42)
0.35 (0.3-0.4)
8.66 (7.41-10.13)
28.9 (23.8-35.1)
1.59 (1.42-1.76)
1.15 (1.0-1.3)
22.17 (17.18-28.6)
27.12 (21.95-33.5)
1.76 (0.69-4.5)
2.13 nd
8.17 (7.52-8.86)
32.29 (26.6-39.2)
1.32 (1.13-1.53)
1.05 (0.93-1.15)
1.24 (1.08-1.44)
1.08 (0.91-1.26)
1.04 (0.93-1.15)
0.99 (0.74-1.33)
0.88 (0.84-0.93)
0.70 (0.64-0.74)
0.89 (0.83-0.93)
0.45 (0.29-0.52)
16
17
19a
19b
19c
1.87 (1.75-1.97)
1.0 (0.74-1.32)
1.21 (1.11-1.32)
0.72 (0.61-0.82)
0.63 (0.58-0.67)
0.35 (0.31-0.37)
0.51 (0.47-0.55)
0.21 (0.15-0.29)
1.71 (1.59-1.83)
1.06 (0.98-1.15)
1.04 (0.95-1.15)
1.05 (1.01-1.12)
0.33 (0.28-0.35)
0.23 (0.21-0.27)
0.34 (0.32-0.36)
0.41 (0.34-0.46)
19d
19e
19f
19g
0.23 (0.21-0.27)
0.16 (0.14-0.18)
0.03 (0.02-0.03)
19h
Doxorubicin
aData are presented as IC50 values in µM with and 95% confidence intervals obtained by nonlinear regression for all cell lines from two independent
experiments. Doxorubicin was used as positive control. nd: not determined.
Supplementary Information
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Acknowledgments
The authors would like to thank the CNPq (National
Council for Technological and Scientific Development),
CAPES (Brazilian Federal Agency for Support and
Evaluation of Graduate Education) and Fundação de
Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ-
PRONEX, E-26/171.512.2010) for funding this work.V. F.
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Submitted: June 26, 2012
Published online: February 5, 2013