3-Arylamino and 3-alkoxy-nor-β-lapachone derivatives: Synthesis and cytotoxicity against cancer cell lines

Article abstract of DOI:10.1021/jm900865m

Several 3-arylamino and 3-alkoxy-nor-β-lapachone derivatives were synthesized in moderate to high yields and found to be highly potent against cancer cells SF295 (central nervous system),HCT8 (colon), MDA-MB435 (melanoma), and HL60 (leukemia), with IC

Full text of DOI:10.1021/jm900865m

504 J. Med. Chem. 2010, 53, 504–508
DOI: 10.1021/jm900865m
3-Arylamino and 3-Alkoxy-nor-β-lapachone Derivatives: Synthesis and Cytotoxicity
against Cancer Cell Lines
†
†
‡
‡
Eufranio N. da Silva Junior, Clara F. de Deus, Bruno C. Cavalcanti, Claudia Pessoa, Letı
´
cia V. Costa-Lotufo,‡
^
ꢀ
ꢀ
Raquel C. Montenegro,‡ Manoel O. de Moraes,‡ Maria do Carmo F. R. Pinto,§ Carlos A. de Simone, ,^ Vitor F. Ferreira,#
Marilia O. F. Goulart, Carlos Kleber Z. Andrade,*,† and Antonio V. Pinto*
,§
^
†
‡
Instituto de Quımica, Universidade de Brasılia, 70910-970 Brasılia, DF, Brazil, Departamento de Fisiologia e Farmacologia, Universidade
´
´
Federal do Ceara, Campus do Porangabussu, 60430-270 Fortaleza, CE, Brazil, Nucleo de Pesquisas em Produtos Naturais, UFRJ, P.O. Box
´
§
ꢀ
69035, 21941-971, Rio de Janeiro, RJ, Brazil, Instituto de Quımica e Biotecnologia, UFAL, Tabuleiro do Martins, 57072-970 Maceio, AL, Brazil,
ꢀ
´
ꢀ
Departamento de Fısica e Informatica, Instituto de Fısica, USP, 13560-970 Sao Carlos, SP, Brazil, and Departamento de Quımica Organica,
^
#
´
Instituto de Quımica, Universidade Federal Fluminense, Campus do Valonguinho, 24020-150 Niteroi, RJ, Brazil
ꢀ
´
~
´
^
´
ꢀ
Received June 12, 2009
Several 3-arylamino and 3-alkoxy-nor-β-lapachone derivatives were synthesized in moderate to high
yields and found to be highly potent against cancer cells SF295 (central nervous system), HCT8 (colon),
MDA-MB435 (melanoma), and HL60 (leukemia), with IC50 below 2 μM. The arylamino para-nitro and
the 2,4-dimethoxy substituted naphthoquinones showed the best cytoxicity profile, while the ortho-
nitro and the 2,4-dimethoxy substituted ones were more selective than doxorubicin and similar to the
precursor lapachones, thus emerging as promising new lead compounds in anticancer drug develop-
ment.
Introduction
on evaluated cancer cell lines (Scheme 1),8 mainly upon
insertion of an electron-poor arylamino ring modified, for
instance, by nitro, fluorine, and bromine groups, with IC50
below 1.76 μM.8
Quinones have been the subject of much interest because of
their various biological activities.1 Reports concerning the
biological evaluation of new naturally occurring naphthoqui-
nones (NQsa) and semisynthetic analogues containing naphth-
alenic type structures are constantly increasing.2 Nearly 300
NQs of related structural types have been isolated from plants,
bacteria, and fungi. These naturally occurring compounds have
long been used in folk medicine, and more recent studies have
proved the therapeutic value of natural and semisynthetic NQs.3
In the past few decades, a large number of natural NQs have
been studied extensively because of their antitumor activity.4
Many clinically important antitumor drugs containing quinone
nuclei, such as anthracyclines, mitoxantrones and saintopin,
show excellent anticancer activity.5 Among the cytotoxic
naphthoquinones, β-lapachone 1 has been the most extensively
studied in recent years.6 The semisynthetic nor-β-lapachone 2
and its amino derivatives have been the subject of several studies
related to Chagas’s disease7 and as a cytotoxic agent against
several cancer cell lines.8
Following this strategy, we synthesized the first series of
substances, new arylaminonaphthoquinones (Scheme 2),
where the arylamino ring was substituted by at least one of
the following groups: nitro, chlorine, or bromine, together
with disubstituted arylaminoquinones, using the methoxy
group (resonance electron donating group), halides, and nitro
(electron withdrawing group) in different chemical environ-
ments. The trypanocidal activity against Trypanosoma cruzi
(T. cruzi) of a few of them, 5-8, has already been reported.7
The second series of compounds was planned on the basis
of bioisosteric replacement, i.e., NQs-NH-aryl by NQs-O-
alkyl. The arylamino and alkoxy-nor-β-lapachone deriva-
tives, described here for the first time, were easily obtained.
The methodology employed enabled us to prepare a variety of
related analogues with good to excellent yield (Scheme 2).
Chemistry
To discover cytotoxic naphthoquinones, in the past few
years we have synthesized and evaluated the pharmacological
activity of naphthodihydrofuranquinones obtained from nor-
lapachol 3, for instance, heterocyclic9 and arylamino deriva-
tives of nor-β-lapachone 2,8 (Scheme 1). We have proved that
the insertion of groups in the C-3 position of the dihydrofuran
ring intensifies the pharmacological activity of nor-β-lapachone 2
All the arylamino-nor-β-lapachone derivatives were synthe-
sized as described in the literature.8 Synthesis was carried out in
a one-pot reaction. The first step involved the in situ prepara-
tion of 3-bromo-nor-β-lapachone 4 from nor-lapachol 3,10
which was converted into the arylamino derivatives 5-16
(Scheme 2) in moderate to high yields (from 65% to 95%).
The syntheses of 5-9 have been previously described.7,11
A similar route was used to synthesize the alkoxy-nor-β-
lapachone derivatives. Initially, nor-lapachol 3 was prepared
using the classical methodology described by Fieser,12 fol-
lowed by cyclization in the presence of molecular bromine.
Freshly obtained 3-bromo-nor-β-lapachone 4 was subjected
to nucleophilic substitution with the respective alcohols. The
*To whom correspondence should be addressed. For C.K.Z.A.:
phone, þ55 61 3107-3861; fax, þ55 61 32734149; e-mail, ckleber@
unb.br. For A.V.P.: phone, þ55 21 25626794; fax, þ55 21 25626512;
e-mail, avpinto@globo.com.
a Abbreviations: NQs, naphthoquinones; T. cruzi, Trypanosoma cruzi;
DOXO, doxorubicin; PBMC, peripheral blood mononuclear cells;
DMSO, dimethyl sulfoxide.
r
pubs.acs.org/jmc
Published on Web 11/30/2009
2009 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 505
Scheme 1. Arylamino and Naphthoquinone 1,2,3-Triazole Derivatives with Activity against Cancer8,9
Scheme 2. Synthetic Route To Obtain Compounds 5-16 and 17-24
alcohols were chosen according to how easily they were
removed at the end of the reaction. In the case of alcohols
with high boiling point, such as benzyl alcohol, the isola-
tion of the product was not possible. The compound
obtained was unstable when heated, and its distillation
and recrystallization were not feasible. Attempts to isolate
the product by silica gel column chromatography led to
quinone 22. Because 18 degrades after a few days, it was not
pharmacologically evaluated. Substances 24 and 25, which
were subjected to antitumor studies, were obtained from
lapachol and phythiocol, respectively, as described in the
literature13 (Schemes 2 and 3). Substance 22 was obtained
as described in the literature10 and used for antitumor
screening.
Scheme 3. Synthetic Route To Obtain Compound 25
The structures of the compounds were confirmed by
1
techniques such as H and 13C NMR, IR, high-resolution
(electrospray ionization) mass spectrometry, elemental
analysis, and literature data.14 The structures of 11, 12,
18, and 20 were further confirmed by X-ray crystallo-
graphy.

506 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
da Silva Juꢀnior et al.
Table 1. Cytotoxic Activity Expressed by IC50 (μM) (95% CI) of Compounds for Cancer Cell Lines, Obtained by Nonlinear Regression for All Cell
Lines from Three Independent Experimentsa
IC50 (μM) (CI 95%)
compd
HL60
MDA-MB435
HCT8
SF295
PBMC
5
0.96(0.79- 1.15)
1.24 (1.07-1.44)
3.76 (3.21-4.41)
1.97 (1.20-2.08)
0.67 (0.54-0.81)
0.54 (0.49-0.64)
1.34 (1.14-1.57)
>13.21
0.19 (0.13-0.30)
0.31 (0.28-0.33)
0.45 (0.37-0.57)
0.48 (0.42-0.51)
0.67 (0.62-0.69)
1.28 (1.05-1.57)
0.78 (0.73-0.81)
2.85 (2.35-3.51)
0.39 (0.36-0.41)
0.74 (0.63-0.85)
0.52 (0.47-0.58)
0.39 (0.21-0.42)
0.59 (0.2-16.20)
0.49 (0.03-5.52)
0.27 (0.21-0.30)
1.26 (nd)
0.76 (0.54-0.87)
1.13 (0.70-1.46)
1.48 (1.15-1.58)
1.74 (1.60-1.94)
1.06 (0.88-1.27)
3.42 (2.52-4.66)
1.57 (0.48-5.12)
>13.21
0.82 (0.38-1.70)
1.32 (1.15-1.55)
1.83 (1.53-2.20)
1.11 (0.88-1.45)
1.82 (1.57-2.13)
4.89 (4.30-5.56)
1.77 (1.29-2.56)
>13.21
5.02 (1.70-5.54)
3.90 (2.74-5.53)
3.16 (1.18-7.35)
4.06 (1.57-5.52)
2.45 (2.30-2.59)
2.49 (2.26-2.75)
3.06 (2.71-3.44)
>13.21
6
7
8
9
10
11
12
13
14
15
16
19
20
21
22
23
24
25
0.28 (0.23-0.36)
1.18 (0.82-1.70)
1.87 (1.66-2.11)
0.28 (0.26-0.31)
2.51 (2.20-2.89)
1.66 (1.46-1.89)
1.04 (0.82-1.34)
4.62 (nd)
1.56 (1.30-1.85)
1.59 (1.39-1.81)
1.32 (1.13-1.53)
0.15 (0.02-0.23)
2.61 (2.02-3.38)
4.39 (3.99-4.82)
2.02 (1.71-2.35)
4.74 (nd)
1.04 (0.88-1.25)
1.78 (1.61-1.97)
1.85 (1.66-2.06)
0.39 (0.26-0.44)
4.33 (1.81-10.30)
3.39 (2.69-4.22)
2.29 (1.19-4.35)
4.62 (nd)
2.11 (1.90-2.35)
12.02 (10.31-13.99)
10.65 (8.82-12.87)
>13.17
5.93 (5.20-6.81)
5.19 (4.49-5.99)
4.65 (4.04-5.36)
nd
>22.0
>20.72
>26.71
>22.0
>20.72
>26.71
>22.0
>20.72
>26.71
>22.0
>20.72
>26.71
>22.0
>20.72
>26.71
doxorubicin
β-lapachone (1)
nor-β-lapachone(2)
0.03 (0.02-0.04)
1.65 (1.49 - 1.78)
1.75 (nd)
0.88 (0.62-1.21)
0.25 (0.16 - 0.33)
0.31 (0.22- 0.39)
0.06 (0.04-0.08)
0.83 (0.74 - 0.87)
1.36 (1.18 - 1.53)
0.41 (0.29-0.44)
0.91 (0.74 -1.11)
1.58 (1.31 - 1.88)
0.42 (0.18-0.69)
>20.6
>21.9
a Alamar blue assay was performed with human peripheral blood mononuclear cells (PBMC) after 72 h of drug exposure. nd, not determined.
Results and Discussion
respectively) and 5 (IC50 =0.76 μM and 0.82 μM, respectively)
(Table 1), being more potent than nor-β-lapachone 2 (IC50=1.36
and 1.58 μM) and β-lapachone 1 (IC50 = 0.83 and 0.91 μM).
Despite its mild activity, 12 displays important selectivity
against the cancer cell line MDA-MB435 (IC50 = 2.85 μM).
As for the hydroxyquinone 22 and alkoxy derived quinones
19-21, the results indicated that the bioisosteric replacement did
not enhance the cytotoxicity of the compounds but these sub-
stances present an apparent selectivity for MDA-MB435 cell line.
For the normal cell PBMC, the IC50 values were generally
higher than those presented for cancer cell lines (Table 1,
column 6). Table 2 lists the selectivity index, represented by
the ratio of cytotoxicities between normal cells and different
lines of cancer cells.
For 5, 6, 14-16, and 19-21, the values of the selectivity
index (PMBC vs MDA-MB-435) were significant (>10).
In terms of cytotoxicity, 5 and 16 showed the best profile
and 14 and 16 the highest selectivity, with a much better
selectivity index than the one for DOXO and similar to that of
the lapachones. Compounds 5, 14, and 16 can be considered
interesting prototypes for further studies.
All the arylamino and alkoxy-nor-β-lapachone derivatives
5-16, 19-21, and the substances 22-25 were tested in
vitro against four cancer cell lines to compare them to doxo-
rubicin (DOXO), the positive control, based on an MTT assay
(Table 1).15 To investigate the selectivity of compounds toward
a normal proliferating cell, the Alamar blue assay was per-
formed with peripheral blood mononucluear cells (PBMC) after
72 h of drug exposure. The compounds were classified according
to their activity as highly active (IC50 < 2 μM), moderately
active (2 μM < IC50 < 10 μM), or inactive (IC50 > 10 μM).16
The majority of the arylamino-nor-β-lapachone derivatives
present potent activity against all cancer cell lines, but 12,
which showed only reasonable activity against MDA-MB435
(IC50 = 2.85 μM) and is inactive toward HL60, HCT8, and
SF295. Thetwo most active compounds withIC50 below 1 μM
against all cancer cells are 5 and 16, with even better activities
than the standard doxorubicin (toward MDA-MB435) and
the precursor 2 and superior to analogue 1 (Table 1).
The most sensitive cell toward quinones is MDA-MB435,
as recently observed;8 for instance, 5-9, 11, 13-21 are more
potent than doxorubicin (Table 1), despite being less active
than analogues 1 (IC50 = 0.25 μM) and 2 (IC50 = 0.31 μM),
with the exception of 5 (IC50 = 0.19 μM) and 21 (IC50 = 0.27
μM). Compound 13 is more active toward HL60 (IC50 = 0.28
μM), and 16 [HCT8 (IC50 = 0.15 μM), HL60 (IC50 = 0.28
μM)] and 10 [HL60 (IC50 = 0.54 μM)] also show a different
toxicity profile.
The 2-amino-1,4-naphthoquinones 23-25 were included as
representatives of inactive quinones.
The absence of hemolytic activity (EC50 > 50 μg/mL),
tested with erythrocyte suspensions, suggests that the cyto-
toxicity of the compounds was not related to membrane
damage of mouse erythrocytes.
Conclusions
Doxorubicin is by far the most active (IC50 = 0.03 μM)
toward HL60 cancer cell line; however, 5, 6, 9-11, 13, 14, 16,
and 21 are superior in activity when compared to nor-β-
lapachone 2 (IC50 = 1.75 μM) and β-lapachone 1 (IC50
1.65 μM). Compounds 13 and 16 present similar and signifi-
cant activity (IC50 = 0.28 μM).
Concerning HCT8 and SF295 cancer cell lines, doxorubicin is
again the most active (IC50=0.06 and 0.41 μM, respectively), with
two compounds of the series, 16 (IC50 = 0.15 μM and 0.39 μM,
In this paper, we listed a series of important substances with
potent antitumor activity. In general, all the compounds were
highly active (IC50 < 2 μM) and several substances were more
active than β-lapachone 1, an antiprostate cancer prototype
currently in phase II studies. Compound 16 presented the best
antitumoral profile and selectivity index, even better than
doxorubicin, the positive control. Thus, this derivative presents
a promising profile for further experimental investigations.
=

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 507
Table 2. Selectivity Index [Ratio between the Cytoxicities, Expressed as
IC50 (μM), against PBMC and Referenced Cancer Cell Line]
alcohol was evaporated to yield a solid and the product was
recrystallized in appropriate solvent.
3-Isopropoxy-2,2-dimethyl-2,3-dihydronaphtho[1,2-b]furan-
4,5-dione 19. The reaction of nor-lapachol 3 (228 mg, 1 mmol),
2 mL of bromine (6 g, 38 mmol), and an excess of propan-2-ol
produced 19 as an orange solid (170 mg, 0.6 mmol, 60% yield,
mp 114-115 ꢀC). 1H NMR (300 MHz, CDCl3) δ: 8.10 (1H, dd,
J = 6.8, 1.3 Hz), 7.70-7.57 (3H, m), 4.62 (1H, s), 6.53 (1H, q,
J = 6.1 Hz), 1.61 (3H, s), 1.48 (3H, s), 1.22 (6H, q, J = 6.1 Hz);
13C NMR (75 MHz, CDCl3) δ: 181.1, 175.5, 169.9, 134.4,
132.2, 131.2, 129.2, 127.6, 124.8, 116.7, 95.4, 80.7, 72.9, 26.6,
22.4, 22.2, 21.1; EI-HRMS (m/z) [M þ Na]þ 309.1108. Calcd.
for [C17H18O4Na]þ: 309.1102.
Biology. Cytotoxicity against Cancer Cell Lines. Compounds
(0.009-5 μg/mL) were tested for cytotoxic activity against four
cancer cell lines: SF-295 (glioblastoma), HCT-8 (colon),
MDAMB-435 (melanoma), and HL60 (leukemia) (National
Cancer Institute, Bethesda, MD). For details see Supporting
Information.
Cell Membrane Disruption. The test was performed in 96-well
plates using a 2% mouse erythrocyte suspension in 0.85% NaCl
containing 10 mM CaCl2, following the method described by
Jimenez et al.17 For details see Supporting Information.
Inhibition of PBMC Proliferation. Alamar Blue Assay. To
investigate the selectivity of compounds toward a normal pro-
liferating cell, the Alamar blue assay was performed with
peripheral blood mononuclear cells (PBMC) after 72 h of drug
exposure. For details see Supporting Information.
X-ray Crystallographic Analysis. Crystallographic data for
11, 12, 18, and 20 have been deposited with the Cambridge
Crystallographic Data Center as Supplementary Publication
Nos. CCDC 730149, 730151, 730147, and 730148, respectively.
Copies of the data can be obtained, free of charge, on applica-
tion to CCDC, 12 Union Road, Cambridge CH21EZ, U.K.
(fax, þ44 1223 336 033; e-mail, deposit@ccdc.cam.ac.uk). Fig-
ure 1 (in Supporting Information) shows an ORTEP-3 diagram
of each molecule.
PBMC vs
HL 60
PBMC vs
MDA MB435
PBMC vs
HCT 8
PBMC vs
SF 295
compd
5
5.23
3.14
0.84
2.06
3.66
4.61
2.28
∼1.00
7.53
10.18
5.69
47.03
2.36
3.13
4.47
14
26.42
12.58
7.00
6.60
3.45
2.13
2.33
2.31
0.79
1.95
∼1.00
1.35
8.07
7.55
87.80
2.27
1.18
2.30
7
6.12
2.95
1.73
3.66
1.35
0.51
1.73
∼1.00
2.03
5.76
6.74
33.77
1.37
1.53
2.03
1.02
6
7
8
8.46
3.66
9
10
11
12
13
14
15
16
19
20
21
DOXO
1.94
3.92
∼ 4.62
5.41
20.48
16.20
33.77
10.05
10.59
17.22
0.48
Experimental Section
Chemistry. The purity of the compounds was tested by
combustion analysis, and it is g95%. For details see Supporting
Information.
Synthetic Procedures. Lapachol (2-hydroxy-3-(30-methyl-2-
butenyl)-1,4-naphthoquinone) was extracted from the hardwood
Tabebuia sp. (Tecoma) and purified by a series of recrystallizations.
Nor-lapachol 3 (2-hydroxy-3-(2-methylpropenyl)[1,4]naphthoqui-
none) was obtained from lapachol by Hooker oxidation.12
The syntheses of 10 and 19 are described below as represen-
tatives of the 3-arylamino and 3-alkoxy-nor-β-lapachone series.
Detailed syntheses of all other compounds are to be found in the
Supporting Information.
General Procedures for Preparing 3-Arylamino-nor-β-lapa-
chones. An amount of 2 mL of bromine (6 g, 38 mmol) was
added to a solution of nor-lapachol 3 (228 mg, 1 mmol) in 25
mL of chloroform. The bromo intermediate 4 (3-bromo-2,2-
dimethyl-2,3-dihydronaphtho[1,2-b]furan-4,5-dione) precipi-
tated immediately as an orange solid. After removal of the
bromine, an excess of the appropriate arylamine was added to
this mixture and stirred overnight, after which the crude
product was poured into 50 mL of water. The organic phase
was separated and washed with 10% HCl (3 ꢀ 50 mL), dried
over sodium sulfate, filtered, and evaporated under reduced
pressure to yield a solid, which was purified by column
chromatography in silica gel and eluted with an increas-
ing polarity gradient mixture of hexane and ethyl acetate
(9/1 to 7/3).
3-(3,4-Dichlorophenylamino)-2,2-dimethyl-2,3-dihydronaphtho-
[1,2-b]furan-4,5-dione 10. The reaction of nor-lapachol 3 (228 mg,
1 mmol), 2 mL of bromine (6 g, 38 mmol), and 3,4-dichloroben-
zenamine (320 mg, 2 mmol) produced 10 (193 mg, 0.5 mmol, 50%
yield, mp 218-219 ꢀC). 1H NMR (300 MHz, CDCl3) δ: 8.10 (1H,
dd, J = 6.7, 1.3 Hz), 7.75-7.62 (3H, m), 7.19 (1H, d, J = 8.8 Hz),
6.66 (1H, d, J = 2.7 Hz), 6.42 (1H, dd, J = 8.8, 2.7 Hz), 4.74 (1H,
d, J = 6.7Hz), 4,04 (NH, d, J = 6.7 Hz), 1.67 (3H, s), 1.56 (3H, s);
13C NMR (75 MHz, CDCl3) δ: 180.9, 175.5, 170.0, 147.0, 134.6,
132.8, 132.7, 131.1, 130.7, 129.6, 127.1, 125.1, 120.7, 114.5, 114.2,
112.6, 95.5, 61.4, 27.4, 21.7; EI-HRMS (m/z) [M þ Na]þ
410.0321. Calcd for [C20H15Cl2NO3Na]þ: 410.0326.
Acknowledgment. This research was supported by grants
from the Conselho Nacional de Desenvolvimento Cientıfico e
´
ꢀ
^
~
Tecnologico (CNPq), Instituto do Milenio;Inovac-ao em
Farmaco/CNPq, FAPERJ, CAPES, UFRJ, UFAL, UFC,
ꢀ
and UnB. The authors are also indebted to the FINEP-CT
INFRA Project No. 0970/01, PRONEX-FAPERJ (E-26/
171.512.2006), and PRONEX-CNPq-FAPEAL.
Supporting Information Available: Synthesis details of all
compounds (except 19 and 10); X-ray diffraction data, including
a file of crystallographic information in Microsoft Word for-
mat, for 11, 12, 18, and 20; HRMS data; elemental analysis data;
biology assay. This material is available free of charge via the
Internet at http://pubs.acs.org.
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General Procedures for Preparing 3-Alkoxy-nor-β-lapa-
chones. An amount of 2 mL of bromine (6 g, 38 mmol) was
added to a solution of nor-lapachol 3 (228 mg, 1 mmol) in 25 mL
of chloroform. The bromo intermediate 4 (3-bromo-2,2-dimeth-
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508 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
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