Cytotoxic activity of naphthoquinones with special emphasis on juglone and its 5-O-methyl derivative

Article abstract of DOI:10.1016/j.cbi.2010.01.041

The cytotoxicity of nine naphthoquinones (NQ) was assayed against HL-60 (leukaemia), MDA-MB-435 (melanoma), SF-295 (brain) and HCT-8 (colon), all human cancer cell lines, and peripheral blood mononuclear cells (PBMC), as representatives of normal cells, after 72h of incubation. 5-Methoxy-1,4-naphthoquinone was the most active compound, showing IC₅₀ values in the range of 0.31 (1.7μM) in HL-60 to 0.88μg/mL (4.7μM) in SF-295 and IC₅₀ of 0.69μg/mL (3.7μM) against PBMC. With the introduction of a bromo-substituent in position 2 or 3 of juglone, the IC₅₀ significantly decreased, regardless of the position on the NQ moiety. However, compared with juglone methyl ether, the halogen substitution decreased the activity. To further understand the mechanism underlying the cytotoxicity of 5-methoxy-1,4-naphthoquinone, studies involving DNA fragmentation, cell cycle analysis, phosphatidyl serine externalization, mitochondrial depolarization and activation of caspases 8 and 3/7 were performed in HL-60 cell line, using doxorubicin as a positive control. The results indicate that the cytotoxic 5-methoxy-1,4-naphthoquinone activates caspases 8 and 3/7 and thus induces apoptosis independent of mitochondria.

Full text of DOI:10.1016/j.cbi.2010.01.041

Chemico-Biological Interactions 184 (2010) 439–448
Contents lists available at ScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Cytotoxic activity of naphthoquinones with special emphasis on juglone and its
5-O-methyl derivative
Raquel Carvalho Montenegroa,∗, Ana Jérsia Araújoa, María Teresa Molinab,
José Delano Barreto Marinho Filhoa, Danilo Damasceno Rochaa, Eulogio Lopéz-Monterob,
Marília O.F. Goulartc, E.S. Bentoc, Ana Paula Nunes Negreiros Alvesd, Cláudia Pessoaa,
Manoel Odorico de Moraesa, Letícia Veras Costa-Lotufoa
a Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, Campus do Porangabussu, Caixa Postal 3157, Fortaleza, Ceará, 60430-270, Brazil
b Instituto de Química Médica – Consejo Superior de Investigaciones Científicas (CSIC), 28006 Madrid, Spain
c Instituto de Química e Biotecnologia, Universidade Federal de Alagoas, Tabuleiro do Martins, Maceió, Alagoas, 57072-970, Brazil
d Departamento de Odontologia Clínica, Universidade Federal de Ceará, Fortaleza, Ceará, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
The cytotoxicity of nine naphthoquinones (NQ) was assayed against HL-60 (leukaemia), MDA-MB-
435 (melanoma), SF-295 (brain) and HCT-8 (colon), all human cancer cell lines, and peripheral blood
mononuclear cells (PBMC), as representatives of normal cells, after 72 h of incubation. 5-Methoxy-1,4-
naphthoquinone was the most active compound, showing IC50 values in the range of 0.31 (1.7 ␮M) in
HL-60 to 0.88 ␮g/mL (4.7 ␮M) in SF-295 and IC50 of 0.69 ␮g/mL (3.7 ␮M) against PBMC. With the intro-
duction of a bromo-substituent in position 2 or 3 of juglone, the IC50 significantly decreased, regardless
of the position on the NQ moiety. However, compared with juglone methyl ether, the halogen sub-
stitution decreased the activity. To further understand the mechanism underlying the cytotoxicity of
5-methoxy-1,4-naphthoquinone, studies involving DNA fragmentation, cell cycle analysis, phosphatidyl
serine externalization, mitochondrial depolarization and activation of caspases 8 and 3/7 were performed
in HL-60 cell line, using doxorubicin as a positive control. The results indicate that the cytotoxic 5-
methoxy-1,4-naphthoquinone activates caspases 8 and 3/7 and thus induces apoptosis independent of
mitochondria.
Received 21 November 2009
Received in revised form 26 January 2010
Accepted 27 January 2010
Available online 4 February 2010
Keywords:
Naphthoquinones
Juglone
Cytotoxicity
Apoptosis
Caspases
Cell cycle
© 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
shown cytotoxic effects against various tumor cells [7–11] and
also has antifungal, antibacterial and antiviral activities, as well
Quinones still account for oneofthelargest families ofantitumor
agents. Although most current anticancer drugs were discovered
empirically, considerable insight has been gained into the mecha-
nisms by which many of these compounds affect cellular growth
[1–3]. The basic knowledge on quinone studies has been used to
design new anticancer drugs [4–6], improving selectivity and pro-
viding a more rational therapeutic application of these agents.
as allelophatic activities [12]. Sugie et al. [13] found that juglone
reduced the formation of azoxymethane induced intestinal tumors
in F344 rats and concluded that juglone could be a promising
chemopreventive agent. Several proposals have been made to
explain the mode of action underlying cytotoxicity of juglone [9,11].
Although this molecule has multiple effects, including apoptosis
[9,14,15], it is well known that these effects are cell-type-specific
[14] and the exact mechanism remains unclear. Very recently,
Aithal et al. [16] demonstrated cytotoxic and genotoxic potentials
of juglone, which can be attributed to mechanisms including the
induction of oxidative stress, cell membrane damage, and a clasto-
genic action leading to cell death by both apoptosis and necrosis.
Bromo-juglone derivatives are very useful from the synthetic
standpoint because they have been used as synthons for the regios-
elective preparation of anthraquinones [17], angucyclines [18] and
benz[b]phenantridines [19], among other classes of natural prod-
ucts. To the best of our knowledge, so far, only one recent study on
their biological properties has been published [20].
Juglone (5-hydroxy-1,4-naphthoquinone) is
a natural 1,4-
naphthoquinone found in the Juglandaceae family, particularly
in the roots, leaves, bark and wood of Juglans regia (English
walnut, Persian walnut and California walnut), J. cinerea (butter-
nut and white walnut), and J. nigra (black walnut). Juglone has
∗
Corresponding author at: Departamento de Fisiologia e Farmacologia, Univer-
sidade Federal do Ceará, Rua Cel. Nunes de Melo, 1127, P.O. Box 3157, 60430-270
Fortaleza, CE, Brazil. Tel.: +55 85 3366 8255; fax: +55 85 3366 8333.
E-mail address: rc montenegro@yahoo.com.br (R.C. Montenegro).
0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2010.01.041

440
R.C. Montenegro et al. / Chemico-Biological Interactions 184 (2010) 439–448
Fig. 1. Chemical structures of juglone (1) and its derivatives (2–9).
In the search for new quinone-derived anticancer compounds,
to juglone (1). To further understand the mechanism under-
lying the cytotoxicity of compound 3, studies involving DNA
fragmentation, cell cycle analysis, phosphatidyl serine externaliza-
tion and mitochondrial depolarization were performed. In search
for selectivity, the cytotoxicity of compounds 1 and 3 was also
evaluated in human peripheral blood mononuclear cells (PBMC)
(Table 1).
in this paper, the cytotoxic effects of juglone (1) and some
derivatives (2–9) (Fig. 1) toward four tumor cell lines are
reported, using the anthraquinone doxorubicin as a positive con-
trol (Table 1). Also, in a second set of experiments, the effects
of the most active derivative, 5-methoxy-1,4-naphthoquinone
(3), were assessed using HL-60 cell line and then compared
Table 1
Cytotoxic activity of juglone and its derivatives on tumor cell lines, on peripheral blood mononuclear cells and erythrocytes.
MTT IC50 ␮g/mL (␮M)*
Hemolysis EC50 (␮g/mL)
HL-60
MDAMB 435
>5 (28.7)
SF-295
HCT-8
PBMC
>5 (28.7)
nd
2.97 (17.0)
2.01–4.38
1.33 (7.6)
0.74–2.39
>200
>200
>200
>200
>200
>200
>200
>200
>200
nd
>5 (28.7)
1
2
3
4
5
6
7
8
9
1.98 (9.2)
1.69–2.33
1.35 (6.2)
1.03–1.77
1.82 (8.4)
1.6–2.07
1.11 (5.1)
0.77–1.6
0.31 (1.7)
0.23–0.43
0.72 (3.8)
0.62–0.95
0.88 (4.7)
0.72–1.07
0.85 (4.5)
0.71–1.05
0.69 (3.7)
0.49–0.99
1.08 (4.3)
0.93–1.23
1.39 (5.5)
1.06–1.82
1.6 (6.3)
1.29–1.99
1.62 (6.4)
1.12–2.35
nd
nd
nd
nd
nd
nd
2.31 (7.8)
1.86–2.89
1.85 (6.3)
1.37–2.5
1.81 (6.1)
1.47–2.23
1.58 (5.3)
1.32–1.86
2.99 (11.2)
2.4–3.7
2.36 (8.8)
1.84–3.03
2.2 (8.2)
1.02–4.71
2.35 (8.8)
1.59–3.46
1.52 (6.0)
1.04–2.25
2.26 (8.9)
1.48–3.42
2.92 (11.5)
2.31–3.69
2.23 (8.8)
1.78–2.79
2.33 (7.9)
1.79–3.04
2.48 (8.4)
1.84–3.35
2.30 (8.6)
1.82–2.91
2.61 (7.8)
1.81–3.77
1.93 (7.2)
1.54–2.42
1.73 (6.5)
1.38–2.16
1.41 (5.3)
1.18–1.67
1.54 (5.8)
1.08–2.19
0.02 (0.03)
0.01–0.02
0.48 (0.8)
0.34–0.66
0.23 (0.4)
0.19–0.25
0.01 (0.02)
0.01–0.02
0.97 (1.7)
0.52–1.80
Dox
*
Data are presented as IC50 values and 95% of confidence interval for leukaemia (HL-60), melanoma (MDAMB-435), nervous system (SF-295), colon (HCT-8) cancer cells
and peripheral blood mononuclear cells (PBMC). Doxorubicin (Dox) was used as a positive control. Experiments were performed in triplicate. nd – not determined.

R.C. Montenegro et al. / Chemico-Biological Interactions 184 (2010) 439–448
441
1255, 1176, 1097, 905, 815 cm−1. EI-MS m/z (%): 254 (Mꢀ, 100.0),
252 (Mꢀ, 98.1), 173 (90.31, 145 (44.1), 89 (35.5), 63 (54.0), 62 (22.2),
53 (29.1) [28]. 1H NMR (400 MHz, CDCl3): ı 7.32 (dd, J = 8.3, J = 1.4,
1 H, H-6), 7.50 (s, 1 H, H-3), 7.64 (dd, J = 8.3, J = 7.4, 1 H, H-7), 7.75
(dd, J = 7.4, J = 1.4, 1 H, 8-H), 11.77 (s, 1 H, OH), 13C NMR (100 MHz,
CDCl3): ı 114.6 (C-4a), 121.0 (C-8), 125.1 (C-6), 130.7 (C-8a), 136.4
(C-7), 140.3 (C-3), 140.9 (C-2), 161.7 (C-5), 176.0 (C-1), 187.5 (C-4).
2. Materials and methods
Melting points were measured with a Reichert-Jung Kofler
hot-stage microscope and are uncorrected. Infrared spectra were
recorded with a Shimadzu IR-435 and the 1H and 13C NMR spectra
with Varian Gemini 200 (200 MHz), Bruker 300 (300 MHz), Varian
Inova 400 (400 MHz) and/or Bruker Avance 400 (400 MHz) spec-
trometers. Chemical shifts are reported in ppm on the delta scale,
using chloroform-d as internal standard, and coupling constants
(J) are given in Hz. Data processing was performed off-line with
the TOPSPIN (Bruker) program package. Mass spectra (electronic
impact) were recorded with a Hewlett-Packard 5973 MSD instru-
ment.
2.1.4. 5-Acetoxy-2-bromo-1,4-naphthoquinone (5)
Yellow needles mp 159–161 ◦C, lit. 155–156 ◦C [27].
Obtained from 4 by standard acetylation [29,30]. IR (KBr) ꢀ:
1682,1600,1370,1330,1270,1190, 1098, EI-MS m/z (%): 296 (1.7).
(M+), 294 (0.8) (M+), 254 (70.9), 252 (67.5), 173 (35.5), 145 (10.6),
63 (12.9), 43 (100.0). Data are compatible with those already
published [27].
2.1. Synthesis of juglone derivatives
All the naphthoquinone derivatives 1–9 used in this study are
known compounds and were prepared according to the methods
described in the literature.
2.1.5. 2-Bromo-5-methoxy-1,4-naphthoquinone (6)
Yellow needles, mp 127–130 ◦C, lit.132–133 ◦C [31] were
synthesized by standard methylation of 4 that led to the corre-
sponding methyl ether 6 [31,32]. IR (KBr) ꢀ: 2975, 1680, 1650,
1595,1475,1458,1293,1245,1120, 1042, 908, 813 cm−1. EI-MS m/z
(%): 268 (100.0) (M+), 266 (98.0) (M+), 187 (35.9), 159 (24.9), 157
(43.5), 131 (27.4), 129 (40.9), 116 (37.9), 101 (31.2), 76 (33.0), 75
(28.0), 74 (25.2), 63 (23.2), 62 (31.6), 53 (23.7). 1H NMR (400 MHz,
CDCl3): ı 4.03 (s, 3 H, OCH3), 7.36 (dd, J = 7.68, J = 1.13, 1 H, H-6),
7.42 (s, 1 H, H-3), 7.71 (t, J = 7.68, H-7), 7.85 (dd, J = 7.68, J = 113,
1 H, H-8), 13C NMR (100 MHz, CDCl3): ı 56.81 (OCH3) 118.49 (C-
6), 118.67 (C-4a), 120.88 (C-8), 135.08 (C-7), 135.34 (C-8a), 137.11
(C-2), 142.54 (C-3), 160.24 (C-5), 178.54 (C-4), 182.78 (C-1).
The 3-bromo-juglone derivatives were prepared by selective
bromination of juglone according to Brimble [28], which yielded
7 as the major isomer.
Juglone (1) is a commercial material (Sigma–Aldrich, St. Louis,
USA) and, when needed in large scale, was prepared according to
the method by Tietze [21] and was purified by flash chromatog-
raphy (chloroform). The spectroscopic data are compatible with
those already reported [21]. Orange-red needles; 154–162 ◦C, lit.
[21] 154–161 ◦C. IR (KBr) ꢀ: 3062, 1665 (free C O), 1643 (chelated
C
O), 1600, 1451, 1363, 1337, 1290 (C–O), 1225. 1H NMR (400 MHz,
CDCl3): ı 6.96 (s, 2 H, 2-H, 3-H), 7.29 (dd, J = 6.3, J = 2.9 Hz, 1 H, 6-H),
7.64 (m, 2 H, 7-H, 8-H), 12.01 (s, 1 H, OH) ppm. 13C NMR (100 MHz,
CDCl3): ı 114.92 (C-4a), 119.13 (C-6), 124.47 (C-8), 131.70 (C-8a),
138.62 (C-7), 136.54, 139.56 (C-2, C-3), 161.40 (C-5), 184.22, 190.25
(C-1, C-4) ppm. EI-MS m/z (%) 174.1 (M+,100), 158.1 (4), 146.1 (8),
118.1 (22), 92.0 (16), 63.0 (10) [21].
2.1.1. 5-Acetoxy-1,4-naphthoquinone (2)
2.1.6. 3-Bromo-5-hydroxy-1,4-naphthoquinone (7)
Dark red needles; mp 171–174 ◦C, lit. mp 172 ◦C [27]. Data are
compatible to those reported [27,28].
Yellow prisms; mp 145–151 ◦C, lit., mp 150–151 ◦C [22]; mp
153–154 ◦C [23]. Acetylation of juglone under standard condi-
tions afforded juglone acetate (2) [23]. IR (KBr) ꢀ: 1758 (ester),
1662, 1610,1594, 1444, 1367, 1331, 1296, 1274. 1H NMR (400 MHz,
CDCl3): ı 2.46 (s, 3 H, –CH3), 6.88 (d, J = 10.26 Hz, 1 H, H-3), 6.95
(d, J = 10.26 Hz, 1 H, H-2), 7.41 (dd, J = 7.78, J = 1.26, 1 H, H-6), 7.78
(t, J = 7.78, 1 H, H-7), 8.07 (dd, J = 7.78, J = 1.26, 1 H, H-8). 13C NMR
(100 MHz, CDCl3): ı 21.30 (COCH3), 124.27 (C-4a), 125.23 (C-8),
129.98 (C-6), 133.78 (C-8a), 135.09 (C-7), 137.58 (C-2), 140.13 (C-3),
149.79 (C-5), 169.68 (COCH3), 183.89 (C-4), 184.43 (C-1). EI-MS m/z
(%) 216 (M+, 6); 174 (100); 146 (7); 118 (12); 92 (7). Mass spectrum
was similar to the one reported by Bowie et al. [24].
2.1.7. 5-Acetoxy-3-bromo-1,4-naphthoquinone (8)
Yellow needles, mp 145–149 ◦C, lit. 149–150 [27] obtained from
7 by standard acetylation [29,30]. Data match those already pub-
lished [27]. EI-MS m/z (%): 253.9, 251.9 (42,40), 173.0 (32), 145.0
(30), 95.1 (22), 81.1 (53), 69.1 (100), 57.1 (52). 1H NMR (400 MHz,
CDCl3): ı 2.45 (s, 3 H, –COCH3), 7.42 (dd, J = 8, J = 1.5, 1 H), 7.53 (s, 1
H, H-2), 7.75 (t, J = 8, 1 H, H-7), 8.01 (dd, J = 8, J = 1.5, 1 H, H-8). 13
C
NMR (100 MHz, CDCl3): ı 20.9 (COCH3), 122.9 (s), 125.4 (d), 130.3
(d), 133.9 (s), 135.7 (d), 139.5 (d), 141.2 (s), 150.9 (s), 169.3 (s), 176.3
(s), 181.6 (s).
2.1.2. 5-Methoxy-1,4-naphthoquinone (3)
Bright yellow prisms; mp 186–190 ◦C (lit. mp 182–185 ◦C [23];
184–189 ◦C [21]) was prepared by methylation of juglone (methyl
iodide, silver (I) oxide) [21,23,26]. IR (KBr) ꢀ: 1655 (C O), 1614,
1583, 1470, 1443, 1377, 1336, 1298 (C–O), 1275, 1253. 1H NMR
(400 MHz, CDCl3): ı 4.02 (s, 3 H, –OCH3), 6.88 (s, 2 H, H-2, H-3), 7.33
(dd, J = 8.01, J = 1.28 Hz, 1 H, H-6), 7.70 (t, J = 8.01, 1 H, H-7), 7.75 (dd,
J = 8.01, J = 1.28 Hz, 1 H, H-8), 13C NMR (100 MHz, CDCl3): ı 56.70
(OCH3), 118.14 (C-6), 119.15 (C-8), 119.58 (C-4A), 134.25 (C-8A),
135.22 (C-7), 136.43 (C-2), 141.11 (C-3), 159.84 (C-9), 184.59 (C-4),
185.44 (C-1). EI-MS m/z (%): 188.1 (M+,100), 174.1 (20), 159.1 (18),
130 (16), 104 (18), 76 (20), 63 (12). Mass spectrum corresponds to
data reported by Bowie et al. [24].
2.1.8. 3-Bromo-5-methoxy-1,4-naphthoquinone (9)
Yellow needles, mp 150–153 ◦C, lit. 154–155 ◦C [25,33],
obtained from 7 by standard methylation (methyl iodide, silver (I)
oxide) [33]. IR (KBr) ꢀ: 3043, 1673,1652,1581, 1470, 1444, 1336,
1311, 1276, 1212. 1H NMR (400 MHz, CDCl3): ı 4.04 (s, 3 H, –OCH3),
7.34 (dd, J = 2.37, J = 7.25, 1 H, H-6), 7.46 (s, 1 H, H-2), 7.72 (t, J = 7.25,
H-7), 7.75 (m, 1 H H-8). 13C NMR (100 MHz, CDCl3): ı 56.78 (OCH3),
118.31 (C-4a), 118,31 (C-6), 119,73 (C-8), 133.74 (C-8a), 135.80 (C-
7), 138.56 (C-2), 142.84 (C-3), 160.65 (C-5), 176.20 (C-4), 182.87
(C-1). The spectral data are in agreement with those already pub-
lished [33].
Compounds 1–3 and several bromo-derivatives were recently
used as starting materials for the synthesis of benzo-fused 1,4-
quinones [34].
Each compound was dissolved in DMSO to obtain a concentra-
tion of 5 mg/mL. For the experiments, the vehicle concentration,
DMSO, was kept at a maximum of 0.1%.
2.1.3. 2-Bromo-5-hydroxy-1,4-naphthoquinone (4)
Orange needles; mp 130–135 ◦C, lit. 135–136 ◦C [27] was
prepared according to reported procedure by hydrolysis of the cor-
responding acetate 5 [27]. The data are compatible to those already
reported [27,28]. IR (KBr) ꢀ: 1683, 1644, 1590, 1460, 1365, 1308,

442
R.C. Montenegro et al. / Chemico-Biological Interactions 184 (2010) 439–448
2.2. Cell line and cell culture
pounds (1 and 3). Compound 1 was tested at 4 ␮M and compound 3
at 0.5, 1.0, 2.0, 4.0, 8.0 and 10 ␮M. In these experiments, doxorubicin
(0.5 ␮M) was used as a positive control.
The tumor cell lines used in this work were HL-60 (leukaemia),
HCT-8 (colon carcinoma), MDA-MB 435 (melanoma) and SF-295
(nervous system) kindly provided by the National Cancer Insti-
tute (Bethesda, MD, USA). Also, peripheral blood mononuclear cells
(PBMC) were tested, for chosen compounds. Heparinized blood
(from healthy, non-smoker donors who had not taken any drug
at least 15 days prior to sampling) was collected and PBMC were
isolated by a standard method of density-gradient centrifugation
over Ficoll-Hypaque. PBMC were washed and resuspended at a
concentration of 3 × 105 cells/mL and platted in a 96-well plate
with RPMI 1640 medium supplemented with 20% fetal bovine
serum, 2 mM glutamine, 100 U/mL penicillin, 100 ␮g/mL strepto-
mycin at 37 ◦C with 5% CO2. Phytohemagglutinin (3%) was added at
the beginning of culture. After 24 h, the compound (0.01–10 ␮M)
dissolved in DMSO 1% was added to each well and incubated
for 72 h. All cancer cells were maintained in RPMI 1640 medium
supplemented with 10% fetal bovine serum, 2 mM glutamine,
100 U/mL penicillin, 100 ␮g/mL streptomycin at 37 ◦C with 5%
CO2.
2.5.1. Analysis of morphological changes
Untreated and treated HL-60 cells were examined for mor-
phological changes by light microscopy (Olympus, Tokyo, Japan).
To evaluate cell morphology, cells were harvested, transferred to
cytospin slides, fixed with methanol for 1 min and stained with
hematoxylin and eosin (HE) (Doles, Brazil). To evaluate nuclear
morphology, HL-60 cells were stained with May-Grunwald-Giemsa
(Bioclin, Brazil).
2.5.2. Acridine orange/ethidium bromide assay to determine cell
death
Cell death pattern was determined by differential staining
with acridine orange/ethidium bromide (AO/BE) (Sigma–Aldrich).
Briefly, cells were pelleted and resuspended in 25 ␮L in PBS.
Afterwards, 1 ␮L of aqueous solution of acridine orange/ethidium
bromide (AO/EB, 100 ␮g/mL) was added and the cells were
observed under
a fluorescence microscope (Olympus, Tokyo,
Japan). Three hundred cells were analyzed using a fluorescence
microscope with filter for 470/40 nm. The cells were then classified
as follows: live cells, apoptotic cells and necrotic cells. The percent-
age of apoptotic and necrotic cells was then calculated as described
[37].
2.3. MTT assay
The cytotoxicity of all compounds was tested against four tumor
cell lines using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT) (Sigma Aldrich Co., St. Louis, MO/USA)
reduction assay [35]. For all experiments, cells were plated in 96-
well plates (105 cells/well for adherent cells or 3 × 105 cells/well
for suspended cells in 100 ␮L of medium). Compounds 1–9
(0.01–5 ␮g/mL) dissolved in DMSO 1% were added to each well
(using the HTS – high-throughput screening – biomek 3000 – Beck-
man Coulter, Inc. Fullerton, California, USA) and incubated for 72 h.
Control groups received the same amount of DMSO. After 69 h of
incubation, the supernatant was replaced by fresh medium con-
taining MTT (0.5 mg/mL). Three hours later, the MTT formazan
product was dissolved in 150 ␮L of DMSO, and absorbance was
measured at 595 nm (DTX 880 Multimode Detector, Beckman Coul-
ter, Inc. Fullerton, CA, USA). Doxorubicin (0.01–5 ␮g/mL) was used
as positive control.
2.5.3. Flow cytometry analysis
For all tested compounds, five thousand events were evaluated
per experiment and cellular debris was omitted from the analysis.
HL-60 cell fluorescence was then determined by flow cytometry in
a Guava EasyCyte Mine using Guava Express Plus software. Inter-
nucleosomal DNA fragmentation and cell cycle were analyzed by
ModFit LT for Win32 version 3.1.
2.5.3.1. Cell membrane integrity. The cell membrane integrity was
evaluated by the exclusion of propidium iodide (50 ␮g/mL, Sigma
Aldrich Co., St. Louis, MO/USA). Briefly, 100 ␮L of treated and
untreated cells were incubated with propidium iodide (50 ␮g/mL).
The cells were, then, incubated for 5 min. Fluorescence was mea-
sured and analyzed for cell morphology, granularity and membrane
integrity [38].
2.4. Alamar Blue assay
In order to investigate the selectivity of compounds 1 and 3
toward a normal proliferating cell, the Alamar Blue assay was
performed with peripheral blood mononuclear cells (PBMC) after
72 h drug exposure [36]. Briefly, PBMC were plated in 96-well
plates (2 × 104 cells/well in 100 ␮L of medium). After 24 h, com-
pounds 1 and 3 (0.01–25 ␮g/mL) dissolved in DMSO 1% were
added to each well using the HTS and incubated for 72 h. Dox-
orubicin (0.009–5 ␮g/mL) was used as a positive control. Control
groups received the same amount of DMSO. Twenty-four hours
before the end of incubation, 10 ␮L of stock solution (0.436 mg/mL)
of the Alamar Blue (resazurin – Sigma Aldrich Co., St. Louis,
MO/USA) was added to each well. The absorbance was measured
using a multiplate reader (DTX 880 Multimode Detector, Beckman
Coulter, Inc. Fullerton, California, USA). The drug effect was quan-
tified as the percentage of control absorbance at 570 and 595 nm
[36].
2.5.3.2. Internucleosomal DNA fragmentation and cell cycle anal-
ysis. DNA fragmentation was analyzed by flow cytometry after
DNA staining with propidium iodide. Briefly, 100 ␮L of treated and
untreated cells were incubated for 30 min, in the dark, with hypo-
tonic solution containing 50 ␮g/mL propidium iodide, 0.1% sodium
citrate, and 0.1% Triton X-100. Fluorescence was measured and DNA
fragmentation and cell cycle were analyzed [39].
2.5.3.3. Phosphatidylserine (PS) externalization. PS externalization
was analyzed by flow cytometry after PS staining with Annexin V
according to the method described by Vermes et al. [40]. Guava
Nexin Assay Kit was used to determine early apoptosis. Cells were
washed twice with cold PBS and then resuspended in 135 ␮L
of PBS with 5 ␮L of 7-amino-actinomycin D (7AAD) and 10 ␮L
of Annexin V-PE. The cells were gently vortexed and incubated
for 20 min at room temperature (20–25 ◦C) in the dark. After-
wards, the cells were analyzed by flow cytometry (EasyCyte from
Guava Technologies). Annexin V is a phospholipid-binding pro-
tein that has a high affinity for PS. 7-AAD, a cell impermeant
dye, is used as an indicator of membrane structural integrity.
Fluorescence of Annexin V-PE was measured: yellow fluorescence-
583 nm and 7-AAD in red fluorescence-680 nm. The percentage of
2.5. Analysis of possible mechanisms involved in the cytotoxic
activity of 5-methoxy-1,4-naphthoquinone
In order to study the mechanism involved in the activity of 1 and
3, a second set of experiments was performed, where leukaemia
cells (HL-60 cell line) were incubated, for 24 h, with the tested com-

R.C. Montenegro et al. / Chemico-Biological Interactions 184 (2010) 439–448
443
early and late apoptotic cells and necrotic cells was then calcu-
3. Results
lated.
3.1. Cytotoxicity of juglone (1) and its derivatives (2–9)
2.5.3.4. Mitochondrial transmembrane potential. Mitochondrial
depolarization was evaluated by incorporation of Rhodamine 123
(Sigma Aldrich Co., St. Louis, MO/USA). Rhodamine 123 is a cell-
permeable, cationic, fluorescent dye that is readily sequestered
by active mitochondria without inducing cytotoxic effects. Briefly,
cells were centrifuged at 2000 rpm for 5 min and the pellet
was resuspended in 500 ␮L of 1 ␮g/mL of rhodamine 123 for
15 min in the dark. After incubation, cells were centrifuged at
2000 rpm for 5 min and the pellet was resuspended in 500 ␮L in
phosphate-buffered saline (PBS) and incubated for 30 min in the
dark. Fluorescence was measured and percentage mitochondrial
depolarization was analyzed [37].
The MTT analysis showed that all compounds (1–9) exhibited
cytotoxic activity against all tested cancer cell lines. Based on data
collected from two independent experiments in triplicate, the IC50
values ranged from 1.7 to over 28.7 ␮M between cells after 72 h
of incubation (Table 1). Compound 3 was the most active among
all, with IC50 ranging from 1.7 to 4.7 ␮M in HL-60 and SF-295,
respectively, whereas for the prototype (compound 1), IC50 ranged
from 7.6 to over 28.7 ␮M in HCT-8 and HL-60 and/or MDA-MB435,
respectively. For normal PBMC, IC50 values for compounds 1 and
3 were over 28.7 and 3.7 ␮M, respectively. Compound 3 was, in
fact, generally more active than compound 1 for all tested cells,
including normal ones. While compound 3 was active more than
20 times in HL-60 cells, it was only 1.56 times more active against
HCT-8 cells. Additionally, the selectively was hardly affected, since
compound 3 was even more active to PMBC than to adherent tumor
cells (HCT-8, MDA-MB-435 and SF-295).
2.5.3.5. Caspases activity. Caspase 3/7 and 8 activities were ana-
lyzed by flow cytometry, using Guava® EasyCyte Caspase Kit, after
24 h of incubation. HL-60 cells (3 × 105 cells/mL) were incubated
with Fluorescent Labeled Inhibitor of Caspases (FLICA) and main-
tained for 1 h at 37 ◦C and 5% CO2. After incubation, 80 ␮L of washing
buffer were added and cells were centrifuged at 2000 rpm for 5 min.
The resulting pellet was resuspended in 200 ␮L of washing buffer
and centrifuged again. Then the cells were resuspended in the
working solution (propidium iodide 1:200 in 1× washing buffer)
and analyzed immediately using flow cytometry.
Based on these results, all subsequent experiments were con-
ducted to compare the cytotoxicity of juglone (compound 1) and
5-methoxy-1,4-naphthoquinone (compound 3) on the HL-60 cell
line.
3.2. Morphological changes and cell death pattern induced by
juglone (1) and its 5-O-methyl derivative (3)
2.5.4. Statistical analysis
Analysis by light microscopy of treated and untreated HL-60
cells revealed several drug-induced morphological changes. Con-
trol cells exhibited a typical non-adherent and vacuolization round
morphology after 24 h in culture (Fig. 2 A). Juglone (1) at 4 ␮M
Data obtained from experiments are presented as means SEM
from at least three independent experiments and evaluated by
Newman–Keuls test and the significance level was also set at 1%.
Fig. 2. Microscopic analysis of hematoxylin/eosin-stained. HL-60 cells untreated (A) or treated with compound 1 (4 ␮M, C) and compound 3 (0.5 ␮M, D, 1 ␮M, E, 2 ␮M, F, 4 ␮M,
G, 8 ␮M, H or 10 ␮M, I) were analyzed by light microscopy (400×). Doxorubicin (0.5 ␮M) was used as positive control (B). At 2 ␮M (F) cells stained with May-Grunwald-Giemsa
demonstrated chromatin condensation. Black arrows show smaller cells with intact membranes.

444
R.C. Montenegro et al. / Chemico-Biological Interactions 184 (2010) 439–448
did not induce any noticeable changes in treated cells (Fig. 2C).
On the other hand, HL-60 cells treated with 3 showed intense
chromatin condensation, internuclear vacuoles, cytoplasm protu-
berances, reduction of the cell and nuclear volume, chromatolysis
that became more evident for incubations at higher concentra-
tions (Fig. 2E–I). Despite these typical apoptotic features, it was
also observed an increasing number of cells with round shape, sug-
gesting a preserved membrane, but smaller than a viable cell and
with an intense staining, different from the viable ones (Fig. 2E–I)
which were considered apoptotic cells. Doxorubicin (0.5 ␮M)
induced cell shrinkage, chromatin condensation and nuclear frag-
mentation, on HL-60 cells, being all them, features of apoptosis
(Fig. 2B).
Fluorescence microscopy confirmed the results found by light
microscopy. After 24 h, HL-60 treated with 3 showed a decrease
in cell viability in a dose-dependent manner, while the number of
apoptotic cells increased with the concentration of compound 3,
corroborating the results reported above (Fig. 3). At the concentra-
tion of 1 and 2 ␮M of compound 3, the viable cells were reduced by
Fig. 3. Effects of compounds 1 and 3, in HL-60 cell viability by acridine orange and
ethidium bromide-stained (AO/EB) determined fluorescence microscope, after 24 h
incubation. The negative control (C) was the vehicle used for diluting the tested
substances. Doxorubicin (0.5 ␮M) was used as positive control (D). *p < 0.05 com-
pared to control by ANOVA, followed by Newman–Keuls multiple comparison test.
Data are presented as mean values S.E.M. from three independent experiments
performed in duplicate (n = 6).
Fig. 4. Effects of juglone (1) and its 5-O-methyl derivative (3) at 2 and 4 ␮M in HL-60 cell population determined by flow cytometry using propidium iodide, after 24 h
incubation. The negative control (C) was the vehicle used for diluting the tested substances. Doxorubicin (D) (0.5 ␮M) was used as a positive control. (A) The forward light
scatter and side light scatter of the laser were used as indices of cell size and granularity, respectively. (B) Bars correspond to the means SEM of the percentage of viable
and non-viable cells obtained from 3 independent experiments. Five thousand events were analyzed in each experiment. *p< 0.05 compared to control by ANOVA, followed
by Newman–Keuls multiple comparison test.

R.C. Montenegro et al. / Chemico-Biological Interactions 184 (2010) 439–448
445
19.3% and 37.7%, respectively, followed by a significant reduction
of 58.7% at 4 ␮M. No viable cells were observed at 8 or 10 ␮M, only
the apoptotic and necrotic cells previously described. Also, apopto-
sis starts to occur, significantly, as early as 1 ␮M (29.1%) and it rises
until 10 ␮M (77.7%). In respect to necrosis, it was shown a discrete
increase in a dose-dependent manner, beginning at 4 ␮M (12.93%)
following by 8 and 10 ␮M (12.9; 21% and 22.2%, respectively). No
difference was observed after treatment with compound 1 at 4 ␮M
and the negative control.
HL-60 cells treated for 24 h with 3 showed a decrease on cell
size and granularity in a dose-dependent manner as observed by
forward scatter and side scatter, respectively, but the membrane
integrity was essentially preserved (Fig. 4A), confirming the pre-
vious results described above. Disruption of membrane integrity
was observed only at concentrations equal or above 4 ␮M (Fig. 4B).
No difference was observed for doxorubicin or compound 1 when
compared with the negative control.
Fig. 5. Effects of compounds 1 and 3 in internucleosomal DNA fragmentation in HL-
60 cells determined by flow cytometry using propidium iodide, Triton X-100 and
citrate after 24 h incubation. The negative control (C) was the vehicle used for dilut-
ing the tested substance. Doxorubicin (0.5 ␮M) was used as positive control (D).
Data are presented as mean values S.E.M. from three independent experiments
performed in triplicate. Five thousand events were analyzed in each experiment.
*p < 0.01 compared to control by ANOVA, followed by Newman–Keuls multiple com-
parison test.
3.3. DNA fragmentation, cell cycle, PS externalization,
mitochondrial depolarization and caspases activity
Since morphological changes indicated that treated cells are
undergoing a possible apoptotic process, DNA fragmentation, PS
externalization, mitochondrial depolarization and caspases acti-
vation were analyzed. The percentage of HL-60 cells with DNA
fragmentation was significantly increased by the treatment with
3 as early as 2 ␮M (21.29%) (p < 0.05), in a dose-dependent manner,
while no difference was observed for 1 at 4 ␮M (Fig. 5). Doxoru-
bicin at 0.5 ␮M, used as a positive control, induced 54.14% of DNA
fragmentation, comparable with data from compound 3 at 8 and
10 ␮M (68.10% and 70.68%, respectively). No cell cycle changes
were observed (data not shown).
classified in the late apoptosis state (Fig. 6). Since all previous
results indicate that compound 3 induces cell death by apoptosis,
the pathway (intrinsic or extrinsic) triggering apoptosis was evalu-
ated. Except for compound 3 at 10 ␮M (12.57% depolarization), no
mitochondrial depolarization was observed for compound 1 and
3, at all tested concentrations, while doxorubicin showed 51.27%
of mitochondrial depolarization (Fig. 7). Activation of caspases 8
and 3/7 was also evaluated. The results showed that 66.07% and
82.57% of caspase 8 was activated at both concentrations tested (4
and 8 ␮M, respectively) (Fig. 8A) and 53.36% and 74.02% of caspase
3/7 was activated (Fig. 8B). All results presented herein, suggest
that compound 3 induces apoptosis independent of mitochondria
depolarization.
Based on PS externalization analysis, it can be demonstrated that
compound 3, at all tested concentrations, induces significant apop-
tosis. At the highest tested concentration (8 ␮M), 40.27% of treated
cells presented initial apoptotic features, while 52.78% could be
Fig. 6. Effects of compound 3 at 1 ␮M (C), 4 ␮M (D) and 8 ␮M (E) in HL-60 PS externalization determined by flow cytometry, using Annexin V-PE and 7-AAD after 24 h
incubation. The negative control (C) was the vehicle used for diluting the tested substance. Doxorubicin at 0.5 ␮M was used as positive control (D). Data are presented as dot
plot in which cell membrane integrity (7-AAD-y-axis) is plotted against PS externalization (Annexin V-PE-x-axis). Percentages of cells in early and late apoptosis and necrosis
are indicated.

446
R.C. Montenegro et al. / Chemico-Biological Interactions 184 (2010) 439–448
series I, methylation of juglone, furnishing 3, led to a significant
increase of activity toward all cell lines. The acetyl derivative 2
was also more active than juglone but less than juglone methyl
ether 3. Previous studies by Phillips [41] in a set of cancer cell lines,
demonstrated that 1,4-naphthoquinone presented IC50 range from
0.6 to 690.4 ␮M in HT-29 (colon cancer) and H-460 (lung cancer),
respectively. In the same study, 5-methoxy-1,4-naphthoquinone
presented IC50 ranged from 2.5 to 12.9 ␮M in A459 and H460 (lung
cancer), respectively.
With the introduction of a bromo-atom in position 2 or 3 on
the juglone (1 vs. 4 vs. 7, Table 1), the IC50 significantly decreased,
regardless the position on the NQ moiety. Comparing the methyl
and acetyl derivatives (2 vs. 5 vs. 8), there is some similarity. Com-
paring 3, 6 and 9, the presence of bromo was ineffective, removing
the original activity. In the series IIA (4 vs. 5 vs. 6), the –OH group
is the most active, except for SF295 (similar activity) and HCT-8,
whereas 5 presented a lower IC50. In IIB (7 vs. 8 vs. 9), the methoxy
9 is more active, but still less active than 3. Concerning to PBMC,
compound 1 showed IC50 over 28 ␮M whereas for compound 3,
the IC50 was 3.7 ␮M, which could suggest an increase in the unspe-
cific mechanisms of cytotoxicity. The selectivity index, obtained as
Fig. 7. Effects of compound 1 (4 and 8 ␮M, respectively) and compound 3 (0.5, 1, 2,
4, 8 and 10 ␮M) in HL-60 mitochondrial membrane depolarization, determined by
flow cytometry using rhodamine123 after 24 h incubation. Negative control (C) was
treated with the vehicle used for diluting the tested substance. Doxorubicin (0.5 ␮M)
was used as positive control (D). Data are presented as histograms in which the cell
number (y-axis) is plotted against cell membrane integrity (x-axis). Percentages of
cells with mitochondrial membrane depolarization are indicated at each histogram.
the ratio between the IC50 (␮M) value against PBMC and different
∼
cancer cell lines, ranged from 1 (PBMC vs. HL-60 and PBMC vs.
=
MDA-MB-435) to 3.77 (PBMC vs. SF-295) for compound 1, and from
0.78 (PBMC vs. SF-295) to 2.17 (PBMC vs. HL-60) for compound 3.
These data suggested that compound 3 presented a low selectivity
to cancer cells.
Several studies have been performed to elucidate the mecha-
nism of action of juglone (1) and it is well known that juglone
exerts its effects by modifying sulphidryl groups [42] inhibiting
different kinds of enzymes. In 2001, Chao et al. [43] demon-
strated that juglone is an effective and rapid inhibitor of RNA
polymerase II transcription and that juglone treatment causes cells
to be refractory to lysis during stimuli. However, several studies
demonstrated that juglone can induce apoptosis and/or necrosis,
depending on dose-treatment [8,16,44]. Interestingly, compound
3 leads to severe morphological changes, markedly characterized
by smaller cells and intrinsic fluorescence, which could be related
to the refractoriness of membrane to lysis, keeping cell integrity
even in the late stages of apoptosis process. In spite of these alter-
ations, treated cells also presented morphological characteristics
of apoptotic cells, such as chromatin condensation, a reduction in
cell volume and fragmentation of the nuclei. Some authors con-
sider the distinction between apoptosis and necrosis difficult and
controversial, and the morphological definition of apoptosis and
necrosis could be based on membrane integrity [45]. According to
Dartsch et al. [46], there are two crucial points for the distinction
between apoptosis and necrosis: the former is characterized by the
occurrence of DNA and nuclear fragmentation with concomitant
maintenance of membrane integrity and involves the triggering
of phagocytic elimination of apoptotic bodies by the externaliza-
tion of phosphatidyl serine. Our data are in agreement with this
definition, since compound 3 leads to DNA fragmentation without
affecting membrane integrity, and also induces phosphatidyl serine
externalization.
Fig. 8. Effects of compounds 1 and 3 in HL-60 caspase 3/7 (A) and caspase 8 (B)
activity, determined by flow cytometry after 24 h incubation. Negative control (C)
was treated with the vehicle used for diluting the tested substances. Doxorubicin
(0.5 ␮M) was used as positive control (D). Data are presented as mean values S.E.M.
from three independent experiments performed in triplicate. Five thousand events
were analyzed in each experiment. *p < 0.01 compared to control by ANOVA, fol-
lowed by Student–Newman–Keuls test.
4. Discussion
The two major apoptosis signaling pathways in mammalian
systems are the “extrinsic” pathway initiated by activation of
membrane-bound death receptors leading to cleavage of caspase
[47] and the “intrinsic” pathway characterized by mitochondrial
depolarization, release of cytochrome c, and subsequent activa-
tion of caspases [48,49]. Thus, mitochondrial depolarization and
the type of activated caspase may indicate the pathway involved
in apoptosis induction. Some naphthoquinones can induce apop-
tosis via caspase activation [44,50]. In a study by Li and co-workers
[44] it was demonstrated that a benzobijuglone induces apoptosis
via activation of caspases 8 and 3. Our results corroborates those
The quinone moiety is present in many anticancer drugs such as
anthracyclines (daunorubicin, doxorubicin), mitomycin and mitox-
antrone, which are used clinically in the therapy of solid tumors [1].
In structural terms, the nine compounds tested can be divided into
two series, depending on the absence (I, 1–3) or presence of the
bromo-substituent (II, 4-9) in their structures. In II, they can be clas-
sified in IIA (2-bromo-substituted) and IIB (3-bromo-substituted).
Comparing with juglone (1), the prototype, all the compounds
(2–9), showed higher toxicity except for HCT-8 (colon cancer), for
which juglone presented its higher activity (7.6 ␮M) (Table 1). In

R.C. Montenegro et al. / Chemico-Biological Interactions 184 (2010) 439–448
447
of Li and co-workers [44]. HL-60 cells treated with compound 3
induce DNA fragmentation, activation of caspase 8 and 3/7 and lack
of mitochondrial depolarization even in the higher dose, all known
features of extrinsic apoptosis pathway, induced by death receptors
that are at the cell surface. Also, apoptosis began as early as 0.5 ␮M
with compound 3, supporting all findings. It is clear that the recep-
tors transmit apoptotic signals initiated by specific ligands and they
play an important role in apoptosis activating the caspase cascade
within seconds of ligand binding. Induction of apoptosis via this
mechanism is therefore very rapid [51].
Death receptors have been proposed as a potential target for
cancer therapy. They can be activated and induce tumor cell to com-
mit apoptotic suicide independently of tumor suppressor p53 being
useful in tumors that have lost p53 function. Targeting the death
receptors in tumor cells might be a useful tool in cancer therapy,
especially in tumors that have been resistant to conventional ther-
apy [52]. Interestingly, HL-60 cell, the model used in this work, lacks
p53 [53] and this cell line was the most sensitive to compound 3.
In conclusion, the results presented herein reaffirm the cyto-
toxicity of simple quinones and demonstrate that the introduction
of a methoxy group at C-5, strongly increases the cytotoxicity
of juglone. Moreover, it was demonstrated that 5-methoxy-1,4-
naphthoquinone induces apoptosis by an extrinsic pathway, and
is, thus, independent of mitochondrial depolarization.
[12] P. Babula, V. Adam, R. Kizek, Z. Sladky, L. Havel, Naphthoquinones as alle-
lochemical triggers of programmed cell death, Environ. Exp. Bot. 65 (2009)
330–337.
[13] S. Sugie, K. Okamoto, K.M.W. Rahman, T. Tanaka, K. Kawai, J. Yamahara, H.
Mori, Inhibitory effects of plumbagin and juglone on azoxymethane-induced
intestinal carcinogenesis in rats, Cancer Lett. 127 (1998) 177–183.
[14] M.T. Paulsen, M. Ljungman, The natural toxin juglone causes degradation of p53
and induces rapid H2AX phosphorylation and cell death in human fibroblasts,
Toxicol. Applied Pharmacol. 209 (2005) 1–9.
[15] S.K. Sandur, H. Ichikawa, G. Sethi, K.S. Ahn, B.B. Aggarwal, Plumba-
gin (5-hydroxy-2-methyl-1,4-naphthoquinone) suppresses NF-␬B activation
and NF-␬B-regulated gene products through modulation of p65 and I␬
B␣ kinase activation, leading to potentiation of apoptosis induced by
cytokine and chemotherapeutic agents, J. Biol. Chem. 281 (2006) 17023–
17033.
[16] B.K. Aithal, M.R.S. Kumar, B.N. Rao, N. Udupa, B.S.S. Rao, Juglone, a naphtho-
quinone from walnut, exerts cytotoxic and genotoxic effects against cultured
melanoma tumor cells, Cell Biol. Int. 33 (2009) 1039–1049.
[17] K. Krohn, N. Böker, 1,2-, 1,4-, 2,9- and 9,0-anthraquinones and higher fused
polycyclic quinone derivatives, in: A.G. Griesbeck (Ed.), Science in Synthesis,
Thieme Verlag, Stuttagart, 2006, pp. 367–506.
[18] M.C. Carren˜o, A. Urbano, Recent advances in the synthesis of angucyclines,
Synlett 1 (2005) 1–25.
[19] J.A. Valderrama, M.F. González, C. Valderrama, Studies on quinones. Part 32.
Regioselective synthesis of benz[b]phenantridines related to phenantroviri-
done, Tetrahedron 55 (1999) 6039–6050.
[20] K.A.L. Ribeiro, C.M. de Carvalho, M.T. Molina, E.P. Lima, E. López-Montero, J.R.M.
Reys, M.B.F. de Oliveira, A.V. Pinto, A.E.G. Santana, M.O.F. Goulart, Activities
of naphthoquinones against Aedes aegypti, vector of dengue and Biomphalaria
glabrata, intermediate host of Schistosoma mansoni, Acta Trop. 111 (2009)
44–50.
[21] L.F. Tietze, C. Güntner, K.M. Gericke, A Diels-Alder reaction for the total syn-
thesis of the novel antibiotic antitumor agent mensacarcin, Eur. J. Org. Chem.
(2005) 2459–2467.
[22] M.P. Sibi, J.W. Dankwardt, V. Snieckus, Anionic aromatic ring annelation of o-
allylbenzamides. Regiospecific synthesis of naphthols and naphthoquinones, J.
Org. Chem. 51 (1986) 271–273.
Conflict of interest statement
No conflict of interest.
[23] L.F. Fieser, J.T. Dunn, Addition of dienes to halogenated and hydroxylated naph-
thoquinones, J. Am. Chem. Soc. 59 (1937) 1016–1021.
[24] J.H. Bowie, D.W. Cameron, D.H. Williams, Studies in mass spectrometry. I.
Mass spectra of substituted naphthoquinones, J. Am. Chem. Soc. 87 (1965)
5094–5099.
[25] R.L. Hannan, R.B. Barber, H. Rapoport, Synthesis of bromonaphthoquinones
from 1,3-dimethoxynaphthalene, J. Org. Chem. 44 (1979) 2153–2158.
[26] J.F. Garden, R.H. Thomson, Quinones. Part VI. Some new polyhydroxynaphtho-
quinones, J. Chem. Soc. (1957) 2483–2489.
[27] J.R. Grunwell, A. Karipides, C.T. Wigal, S.W. Heinzman, J. Parlow, J.A. Surso,
L. Clayton, F.J. Fleitz, M. Daffner, J.E. Stevens, The formal oxidative addition
of electron-rich transoid dienes to bromonaphthoquinones, J. Org. Chem. 56
(1991) 91–95.
[28] M.A. Brimble, T.J. Brenstrum, C-Glycosylation of tri-O-benzyl-2-deoxy-D-
glucose: synthesis of naphthyl substituted 3,6-dioxabicyclo[3.2.2]nonanes, J.
Chem. Soc. [Perkin 1] (2001) 1612–1623.
Acknowledgments
We wish to thank CNPq, CSIC-CNPq (2005 BR0046, to
M.T.M and M.O.F.G), DGICYT (Spain, grant BQU2003-00813 and
SAF2006-04698 to M.T.M.)), CSIC (for a I3P contract, to E.L.-
M.), CAPES, FUNCAP, FINEP, BNB/FUNDECI, FAPEAL, MCT/DECIT,
MCT/MS/Neoplasias, IM-INOFAR/CNPq and PRONEX/FAPEAL for
financial support in the form of grants and fellowship awards. We
are grateful to Silvana Franc¸ a dos Santos and Luciana Franc¸ a for
technical assistance.
[29] R.H. Thomson, Studies in the juglone series. I. Some halogen derivatives and
their reaction with aniline, J. Org. Chem. 13 (1948) 377–383.
[30] K.A. Parker, M.E. Sworin, Assignment of regiochemistry to substituted naphtho-
quinones by bromo juglone derivatives chemical and spectroscopic methods
amino-, hydroxy-, and bromojuglone derivatives, J. Org. Chem. 46 (1981)
3218–3223.
[31] M.E. Jung, J.A. Hagenah, Synthetic approach to aklavinone using 2-oxo-
2H-pyran-5-carboxylate (coumalate) intermediates, J. Org. Chem. 52 (1987)
1889–1902.
[32] A.M. Echavarren, N. Tamayo, O. de Frutos, A. Garcia, Synthesis of
benzo[b]carbazoloquinones by coupling of organostannanes with bromo-
quinones, Tetrahedron 53 (1997) 16835–16846.
[33] L.F. Tietze, K.M. Gericke, I. Schuberth, Synthesis of highly functionalized
anthraquinones and evaluation of their antitumor activity, Eur. J. Org. Chem.
(2007) 4563–4577.
[34] M.T. Molina, C. Navarro, A. Moreno, A.G. Csaky, Arylation of benzo-fused 1,4-
quinones by the addition of boronic acids under dicationic Pd(II)-catalysis, Org.
Lett. 11 (2009) 4938–4941.
[35] T.J. Mosmann, Rapid colorimetric assay for cellular growth and survivor: appli-
cation to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983)
55–63.
[36] S.A. Ahmed, R.M. Gogal, J.E. Walsh, A new rapid and simple non-radioactive
assay to monitor and determine the proliferation of lymphocytes an alterna-
tive to [3H]thymidine incorporation assay, J. Immunol. Methods 170 (1994)
211–224.
[37] M.F. Cury-Boaventura, C. Pompeia, R. Curi, Comparative toxicity of oleic acid
and linoleic acid on Jurkat cells, Clin. Nutr. 23 (2004) 721–732.
[38] Z. Darzynkiewicz, S. Bruno, G. Del Bino, W. Gorczyca, M.A. Hotz, P. Lassota, F.
Traganos, Features of apoptotic cells measured by flow cytometry, Cytometry
13 (1992) 795–808.
References
[1] W.O. Foye (Ed.), Cancer Chemotherapeutic Agents, American Chemical Society,
Washington, D.C., 1995, pp. 205–239.
[2] C. Asche, Antitumor quinones, Mini Rev. Med. Chem. 5 (2005) 449–467.
[3] R. Boutros, V. Lobjois, B. Ducommun, CDC25 phosphatases in cancer cells: key
players? Good targets? Nat. Rev. Cancer 7 (2007) 495–507.
[4] M.D. Garrett, P. Workman, Discovering novel chemotherapeutic drugs for the
third millennium, Eur. J. Cancer 35 (1999) 2010–2030.
[5] F.H. Fry, C. Jacob, Sensor/effector drug design with potential relevance to cancer,
Curr. Pharm. Des. 12 (2006) 4479–4499.
[6] E.N. da Silva Júnior, C.F. de Deus, B.C. Cavalcanti, C. Pessoa, L.V. Costa-Lotufo,
R.C. Montenegro, M.O. de Moraes, M.C.F.R. Pinto, C.A. de Simone, V.F. Ferreira,
M.O.F. Goulart, C.K.Z. Andrade, A.V. Pinto, 3-Arylamino and 3-alkoxy-nor-␤-
lapachone derivatives: synthesis and cytotoxicity against cancer cell lines, J.
Med. Chem. 53 (2010) 504–508.
[7] H. Kamei, T. Koide, Y. Hashimoto, M. Hasegawa, Inhibition of cell growth in
culture by quinones, Cancer Biother. Radiopharm. 13 (1998) 185–188.
[8] J.F. Rippmann, S. Hobbie, C. Daiber, B. Guilliard, M. Bauer, J. Birk, H. Nar, P.
Garin-Chesa, W.J. Rettig, A. Schnapp, Phosphorylation-dependent proline iso-
merization catalyzed by Pin1 is essential for tumor cell survival and entry into
mitosis, Cell Growth Differ. 11 (2000) 409–416.
[9] N. Cenas, S. Prast, H. Nivinskas, J. Sarlauskas, E.S.J. Arnér, Interactions of
nitroaromatic compounds with the mammalian selenoprotein thioredoxin
reductase and the relation to induction of apoptosis in human cancer cells,
J. Biol. Chem. 281 (2006) 5593–5603.
[10] E. Dickancaité, N. Cenas, A. Kalvelyté, N. Serapiniené, Toxicity of daunoru-
bicin and naphthoquinones to HL-60 cells: an involvement of oxidative stress,
Biochem. Mol. Biol. Int. 41 (1997) 987–994.
[39] I. Nicoletti, G. Migliorati, M.C. Pagliacci, F. Grignani, C. Riccardi, A rapid and sim-
ple method for measuring thymocyte apoptosis by propidium iodide staining
and flow cytometry, J. Immunol. Methods 139 (1991) 271–279.
[11] J. Segura-Aguilar, K. Jönsson, U. Tidefelt, C. Paul, The cytotoxic effects of 5-
OH-1,4-naphthoquinone and 5,8-di-OH-1,4-naphthoquinone on doxorubicin-
resistant human Leukemia cells (HL-60), Leuk. Res. 16 (1992) 631–637.

448
R.C. Montenegro et al. / Chemico-Biological Interactions 184 (2010) 439–448
[40] I. Vermes, C. Haanen, H. Steffens-Nakken, C. Reutelingsperger, A novel assay
for apoptosis. Flow cytometric detection of phosphatidylserine expression on
early apoptotic cells using fluorescein labelled Annexin V, J. Immunol. Methods
184 (1995) 39–51.
[41] R.M. Phillips, M. Jaffar, D.J. Maitland, P.M. Loadman, S.D. Shnyder, G. Steans, P.A.
Cooper, A. Race, A.V. Patterson, I.J. Stratford, Pharmacological and biological
evaluation of a series of substituted 1,4-naphthoquinone bioreductive drugs,
Biochem. Pharmacol. 68 (2004) 2107–2116.
[42] L. Henning, C. Christner, M. Kipping, B. Schelbert, K.P. Rucknagel, S. Grabley,
G. Kullertz, G. Fischer, Selective inactivation of parvulin-like peptidyl-prolyl
cis/trans isomerases by juglone, Biochemistry 37 (1998) 5953–5960.
[43] S.H. Chao, A.L. Greenleaf, D.H. Price, Juglone, an inhibitor of the peptidyl-prolyl
isomerase Pin1, also directly blocks transcription, Nucl. Acids Res. 29 (2001)
767–773.
[46] D.C. Dartsch, A. Schaefer, S. Boldt, W. Kolch, H. Marquardt, Comparison of
anthracycline-induced death of human leukemia cells: programmed cell death
versus necrosis, Apoptosis 7 (2002) 537–548.
[47] A. Ashkenazi, V.M. Dixit, Death receptors: signaling and modulation, Science
281 (1998) 1305–1308.
[48] D.R. Green, J.C. Reed, Mitochondria and apoptosis, Science 281 (1998)
1309–1312.
[49] R. Khosravi-Far, M.D. Esposti, Death receptor signals to mitochondria, Cancer
Biol. Ther. 3 (2004) 1051–1057.
[50] K.H. Kang, K.H. Lee, M.Y. Kim, K.H. Choi, Caspase-3-mediated cleavage of the
NF-␬B subunit p65 at the NH2 terminus potentiates naphthoquinone analog-
induced apoptosis, J. Biol. Chem. 276 (2001) 24638–24644.
[51] I.M. Ghobrial, T.E. Witzig, A.A. Adjei, Targeting apoptosis pathways in cancer
therapy, Cancer J. Clin. 55 (2005) 178–194.
[44] Z.B. Li, J.Y. Wang, B. Jiang, X.L. Zhang, L.J. An, Y.M. Bao, Benzobijuglone, a novel
cytotoxic compound from Juglans mandshurica, induced apoptosis in HeLa cer-
vical cancer cells, Phytomedicine 14 (2007) 846–852.
[45] M. Masquelier, Q.F. Zhou, A. Gruber, S. Vitols, Relationship between daunoru-
bicin concentration and apoptosis induction in leukemic cells, Biochem.
Pharmacology 67 (2004) 1047–1056.
[52] H.N. LeBlanc, A. Ashkenazi, Apo2L/TRAIL and its death and decay receptors, Cell
Death Differ. 10 (2003) 66–75.
[53] H. Berglind, Y. Pawitan, S. Kato, C. Ishioka, T. Soussi, Analysis of p53 mutation
status in human cancer cell lines, Cancer Biol. Ther. 7 (2008) 701–710.