Synthesis and evaluation of quinonoid compounds against tumor cell lines

Article abstract of DOI:10.1016/j.ejmech.2010.11.006

Thirty two compounds were synthesized in moderate to high yields and showed activity against cancer cells HL-60 (leukemia), MDA-MB435 (melanoma), HCT-8 (colon) and SF295 (central nervous system), with IC₅₀ below 2 μM for some compounds. The β-lapachone-based 1,2,3-triazoles showed the best cytoxicity profile and emerge as promising anticancer prototypes. Insights about the reactive oxygen species (ROS) mechanism of anticancer action for some compounds were obtained by addition of 1-bromoheptane that deplete reduced glutathione (GSH) content and by using N-acetylcysteine that protects cells against apoptotic cellular death, as well by analysis of thiobarbituric acid reactive substances (TBARS) formation, and oxidative DNA damage after treatment detected by the comet assay with the bacterial enzymes formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (ENDOIII).

Full text of DOI:10.1016/j.ejmech.2010.11.006

European Journal of Medicinal Chemistry 46 (2011) 399e410
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and evaluation of quinonoid compounds against tumor cell lines
,
c
,
b
a
a
Eufrânio N. da Silva Jr. a , Bruno C. Cavalcanti , Tiago T. Guimarães , Maria do Carmo F.R. Pinto ,
Igor O. Cabral b, Cláudia Pessoa b, Letícia V. Costa-Lotufo b, Manoel O. de Moraes b,
Carlos K.Z. de Andrade c, Marcelo R. dos Santos c, Carlos A. de Simone e,
*
Marilia O.F. Goulart d, Antonio V. Pinto a
,
1
a Núcleo de Pesquisas de Produtos Naturais, UFRJ, 21941-971, Rio de Janeiro, RJ, Brazil
b Universidade Federal do Ceará, Departamento de Fisiologia e Farmacologia, Campus do Porangabussu, 60430-270 Fortaleza, CE, Brazil
c Instituto de Química, Universidade de Brasília, 70910-970 Brasília, DF, Brazil
d Instituto de Química e Biotecnologia, UFAL, Tabuleiro do Martins, 57072-970 Maceió, AL, Brazil
e Departamento de Física e Informática, Instituto de Física, USP, 13560-970 São Carlos, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Thirty two compounds were synthesized in moderate to high yields and showed activity against cancer
cells HL-60 (leukemia), MDA-MB435 (melanoma), HCT-8 (colon) and SF295 (central nervous system),
Received 29 September 2010
Received in revised form
30 October 2010
Accepted 2 November 2010
Available online 9 November 2010
with IC50 below 2
mM for some compounds. The b-lapachone-based 1,2,3-triazoles showed the best
cytoxicity profile and emerge as promising anticancer prototypes. Insights about the reactive oxygen
species (ROS) mechanism of anticancer action for some compounds were obtained by addition of
1-bromoheptane that deplete reduced glutathione (GSH) content and by using N-acetylcysteine that
protects cells against apoptotic cellular death, as well by analysis of thiobarbituric acid reactive
substances (TBARS) formation, and oxidative DNA damage after treatment detected by the comet assay
with the bacterial enzymes formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (ENDOIII).
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Naphthoquinones
Cancer
Cytotoxicity
Lapachol
1. Introduction
nor-b-lapachone-based 1,2,3-triazoles [14], arylamino and alkoxy
derivatives [15,16].
Naphthoquinones (NQs) have been the subject of much research
due to their pharmacological activities [1e4].
In continuity of our program for obtaining new substances from
NQs with pharmacological activity, we prepared three classes of
compounds. Recently, our research group, based on the studies of
Ettlinger [17] and Anufriev [18], described the correct structure for
Hooker’s lapachol peroxide by X-ray crystallography [19]. Upon the
use of this reaction, we synthesized the first class of compounds.
Anthraquinone imidazoles constitute the second group and finally,
Recently, shikonin, rhinacanthin and juglone derivatives [4e6],
lapachol analogues [7,8], copper(II) complexes [9] obtained from
lawsone have been described as antitumoral and are examples of
the importance of natural products analogues as source of antitu-
moral compounds.
Several strategies are used to obtain new active compounds from
natural products, as for instance, bioisosterism [10] and molecular
hybridization [11] and in the last years the employment of click
reactions [12] along with those strategies made possible the pre-
paration of several heterocycles with antitumor activities [13].
Aiming to discover cytotoxic NQs, in the last few years, we have
synthesized and evaluated the antitumor activity of naphthodi-
hydrofuranquinones obtained from nor-lapachol, as for instance,
using the approach of molecular hybridization [11], b-lapachone-
based 1,2,3-triazoles, the third group, were prepared [20]. The
trypanocidal activity against Trypanosoma cruzi of few of them has
already been reported [20].
2. Chemistry
Lapachol (1) (2-hydroxy-3-(30-methyl-20-butenyl)-1,4-naphtho-
quinone) was extracted from the heartwood of Tabebuia sp. (Tecoma)
and purified by a series of recrystallizations [21]. From this quinone,
nor-lapachol (2) (2-hydroxy-3-(20-methyl-propenyl)-1,4-naphtho-
quinone) was obtained by Hooker oxidation method [22].
* Corresponding author. Núcleo de Pesquisas de Produtos Naturais, UFRJ, 21944-
971, Rio de Janeiro, RJ, Brazil. Tel.: þ55 21 25626794; fax: þ55 21 25626512.
E-mail address: eufranio.junior@yahoo.com.br (E.N. da Silva).
1
The reduced quinones 7 and 9 synthesized from lapachol (1), 3
and 5 from nor-lapachol (2) and 13 and 15 from C-allyl lawsone (11)
In the memory of Professor Antonio Ventura Pinto due to his wonderful
contribution for the accomplishment of this paper.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.11.006

400
E.N. da Silva Jr. et al. / European Journal of Medicinal Chemistry 46 (2011) 399e410
O
O
O
OH
O
O
i
iii
O
O
O
7, 90%
8, 80%
O
O
O
O
OH
OH
O
O
O
O
iii
iii
O
O
O
O
O
O
i
OH
OH
O
O
4, 70%
3, 95%
O
O
1
ii
2
O
O
5, 100%
6, 70%
O
O
O
O
O
OH
iii
ii
O
O
O
9, 100%
10, 75%
Scheme 1. Reagents and conditions: (i) EtOH, 10 mol % Pd/C, 30 psi of H2, 15 min; (ii) AcOH, 10 mol % Pd/C, 55 psi of H2, 6 h; (iii) AcOH, PbO2.
O
O
spectroscopic techniques, yellow crystals were obtained and the
structure was reconfirmed by crystallographic methods. Ortep-3
diagram of the molecule is shown in Fig. 1, and Table 1 lists main
crystallographic parameters.
O
OH
O
O
iii
i
O
O
O
13, 90%
14, 70%
The second series is constituted by anthraquinone imidazoles
18e28. All the substances are herein described for the first time,
except compounds 18 and 24 [20,23]. All of them are easily
obtained from the reaction of 1,2-diaminoanthracene-9,10-dione
(17a) and substituted aldehydes (Scheme 4).
O
O
O
O
O
O
O
OH
iii
O
11
12, 75%
The
as previously described [20]. These compounds were obtained from
-lapachone (29), synthesized by acid cyclisation of lapachol (1).
N-bromosuccinimide (NBS) in CCl4 with benzoyl peroxide as initi-
ator were used to obtain 3,4-dibromo- -lapachone (30) [24]. Sub-
b-lapachone-based 1,2,3-triazoles 33e36 were synthesized
O
O
O
O
OH
O
O
iii
ii
b
O
O
15, 98%
16, 75%
b
stances 31 and 32 were separated by column chromatography and
compound 32 was submitted to azide-alkyne Huisgen’s cyclo-
addition catalysed by Cu(I) [25], producing the 1,2,3-triazoles
33e36 (Fig. 2).
Scheme 2. Reagents and conditions: (i) EtOH, 10 mol % Pd/C, 30 psi of H2, 15 min; (ii)
AcOH, 10 mol % Pd/C, 55 psi of H2, 6 h; (iii) AcOH, PbO2.
The structures of compounds were confirmed by techniques such
as 1H and 13C NMR, IR, high-resolution (electrospray ionization)
mass spectra and spectroscopic data in comparison with related
compounds [19,26]. Spectral data for the new ones are included.
were obtained by catalytic reduction with Pd/C used as catalyst.
Using the Hooker’s reaction with PbO2 [19], these reduced
compounds were used to prepare ether derivatives 4, 6, 8, 10, 12, 14
and 16, in good yields (Schemes 1 and 2). Syntheses of these ethers
are easily accomplished by heating glacial acetic acid and lead
dioxide. The reaction is finished in 2 min and after filtration to
remove lead oxide, the products were purified by recrystallization
and obtained as yellow crystals. Silica gel column chromatography
should be avoided, once it favours the cleavage of the CeOeC
bonds.
Finally, we applied the reaction of oxidative coupling with PbO2
in phthiocol (Scheme 3) and an unexpected product 17 was
obtained. Besides elucidating the structure by the use of
O
O
O
Me
OH
Me
O
PbO2
AcOH
Me
O
O
O
Phthiocol
17
Fig. 1. An ORTEP-3 projection, showing the atomic labelling and the 50% probability
ellipsoids of the compound 17.
Scheme 3. Synthesis of compound 17.

E.N. da Silva Jr. et al. / European Journal of Medicinal Chemistry 46 (2011) 399e410
401
Table 1
cell lines was evidenced forall 1,2,3-triazole derivatives (33e36) and
intermediates 31 and 32. The most significant activity was observed
toward HL-60 and the compounds 32, 33 and 35 were highly active
Crystal data and structure refinement for compound 17.
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
C21H14O5$2H2O
355.95
298(2) K
0.71073 Å
Monoclinic
P 21/a
with IC50 values of 1.43, 1.01 and 1.20
this cell line, these substances are as active as the prototype,
lapachone (29) (IC50 ¼ 1.65 M).
For MDA-MB435, the activity of compounds 33e36 was signi-
ficant with IC50 values below 2 M. Only substance 33 was
considered highly active against HCT-8 cell lines with IC50 value of
1.37 M. Against SF295 all the substances presented values within
the range 1.55e3.01 M.
mM, respectively. Considering
b
-
m
Unit cell dimensions
a ¼ 7.7078(3) Å
b ¼ 19.9585(11) Å
c ¼ 13.0416(6) Å
1978.02(10) Å3
4
m
m
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta range for data collection
Index ranges
Reflections collected
Independent reflections
Completeness to theta
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2sigma(I)]
R indices (all data)
Largest diff. peak and hole
m
1.19 mg/m3
The most important cytotoxic mechanisms of quinonoid
compounds involve drug bioreduction, followed by reaction with
molecular oxygen and generation of ROS [1,29,30], and DNA
damage is considered to be the most serious ROS-induced cellular
modifications [31].
0.086 mmꢁ1
747
(0.242 ꢂ 0.225 ꢂ 0.132) mm3
3.0e25.7ꢃ
9 ꢄ h ꢄ 9
15300
In fact, many of the effects of the chemical and physical agents
that are commonly used in the treatment of human malignancies
are mediated by induction of apoptosis. Nowadays, apoptosis has
currently been a target for developing antitumor drugs [32]. To
determine whether ROS are involved with quinonoid compounds-
induced cell death, cultures were pre-treated with 1-bromo-
heptane, a non-toxic reduced glutathione (GSH) depleting agent
[33] or pre-exposed to N-acetylcysteine (NAC), a widely used thiol-
containing antioxidant that is a precursor of GSH which protects
against oxidative stress-induced cell death by interacting with HOꢀ
and H2O2 [34]. Cell viability, morphological characterization of
apoptotic cancer cells, lipid peroxidation, and DNA-damaging
activities (i.e. DNA strand breaks and oxidized bases at the single-
cell level) were monitored after cell treatment with some repre-
sentatives of the above described three classes of compounds.
Cell membranes are often permeable to NQs, which, once,
present in cell, may form adducts with DNA, leading to abasic sites
and single or double strand breaks [35]. Initially, we correlated the
3680 [R(int) ¼ 0.115]
97.0%
None
0.9913 and 0.9913
Full matrix least
2171/0/255
1.024
R1 ¼ 0.079, wR2 ¼ 0.130
R1 ¼ 0.222, wR2 ¼ 0.272
0.378 and ꢁ0.255 e Åꢁ3
3. Results and discussion
All the substances herein described, except compounds 1, 2, 11,
17a and 30, were evaluated in vitro against four cancer cell lines HL-
60 (leukemia), MDA-MB435 (melanoma), HCT-8 (colon) and SF295
(central nervous system). Doxorubicin was used as the positive
control, based on the MTT assay (Table 2) [27]. The selectivity of
compounds toward a normal proliferating cell was investigated
using the Alamar Blue assay performed with peripheral blood
mononucluear cells (PBMC) after 72 h of drug exposure. Following
Pérez-Sacau et al. [7] the compounds were classified according to
ability of the precursor molecules
b- and nor-b-lapachone to
induce DNA strand breaks via intracellular ROS generation
(Table 3), highlighting the great importance of the production of
ROS on the antiproliferative effects of these NQs. Structure
modifications at the level of nucleotidic bases can also occur as
a consequence of oxidative stress. Oxidation of nucleotidic bases
is likely to be as important as DNA strand breaks to overall cellular
function and survival [36]. The most commonly used endonucle-
ases in the modified comet assay are formamidopyrimidine
their activity as highly active (IC50 < 2
(2 M < IC50 < 10 M), or inactive (IC50 > 10
Despite positive results in terms of antitumor activity [7,28,29]
mM), moderately active
m
m
mM).
reduced compounds 3, 5, 7, 9, 13 and hybrid imidazoles were
inactive. However, antitumoral activity against the evaluated cancer
R3
18 -
R1 = R2 = R3 = H
19 - R2 = R3 = H, R1 = F
R2
20 - R1 = R3 = H, R2 = F
CHO
R1
21 -
R1 = R2 = H, R3 = F
R1
O
NH2
O
O
HN
22 - R2 = R3 = H, R1 = CH3
23 - R1 = R3 = H, R2 = CH3
N
NH2
R2
24 -
R1 = R2 = H, R3 = CH3
R3
25 - R2 = R3 = H, R1 = OH
26 - R1 = R3 = H, R2 = OH
AcO-Na+, AcOH
Reflux
O
17a
27 -
R1 = R2 = H, R3 = OH
28 - R1 = H, R2 = OCH3, R3 = OH
Scheme 4. Synthetic route for the preparation of imidazolic anthraquinones 18e28.
Fig. 2. Compounds 30e32 and b-Lapachone-based 1,2,3-triazoles 33e36 obtained from b-lapachone (29).

402
E.N. da Silva Jr. et al. / European Journal of Medicinal Chemistry 46 (2011) 399e410
Table 2
Cytotoxic activity expressed by IC50
(m
M) (95% CI) of compounds for cancer cell lines, obtained by nonlinear regression for all cell lines from three independent experiments.a
Compounds
IC50
(
mM) (CI 95%)
PBMC
HL-60
MDA-MB435
HCT-8
SF295
3
4
5
6
7
8
9
10
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
31
32
33
34
35
36
>21.71
>10.91
>21.35
>10.72
>20.46
>10.27
>20.13
>10.10
>11.72
>23.12
>11.61
>22.70
>11.40
>14.43
>15.41
>14.60
>14.60
>14.60
>14.77
>14.77
>14.77
>14.69
>14.69
>14.69
>21.71
>10.91
>21.35
>10.72
>20.46
>10.27
>20.13
>10.10
>11.72
>23.12
>11.61
>22.70
>11.40
>14.43
>15.41
>14.60
>14.60
>14.60
>14.77
>14.77
>14.77
>14.69
>14.69
>14.69
>13.61
>21.71
>10.91
>21.35
>10.72
>20.46
>10.27
>20.13
>10.10
>11.72
>23.12
>11.61
>22.70
>11.40
>14.43
>15.41
>14.60
>14.60
>14.60
>14.77
>14.77
>14.77
>14.69
>14.69
>14.69
>21.71
>10.91
>21.35
>10.72
>20.46
>10.27
>20.13
>10.10
>11.72
>23.12
>11.61
>21.71
>10.91
>21.35
>10.72
>20.46
>10.27
>20.13
>10.10
>11.72
>23.12
>11.61
>22.70
>11.40
>14.43
>15.41
>14.60
>14.60
>14.60
>14.77
>14.77
>14.77
3.99 (2.54e6.31)
>11.40
>14.43
>15.41
>14.60
>14.60
>14.60
>14.77
>14.77
>14.77
3.64 (2.90e4.55)
>14.69
>14.69
>13.61
12.39 (11.20e13.72)
2.42 (2.31e2.54)
1.37 (1.27e1.50)
2.24 (2.11e2.41)
2.26 (2.13e2.40)
3.21(2.89e3.60)
3.87 (2.99e5.02)
>14.69
>14.69
>13.61
>13.80
>13.61
>13.61
11.3 (9.74e13.17)
3.58 (3.34e3.86)
2.08 (1.80e2.41)
4.01 (3.53e4.49)
3.37 (3.00e3.81)
2.52 (2.16e2.95)
5.68 (5.13e7.97)
3.97 (3.23e4.91)
1.54 (1.46e1.62)
1.22 (0.88e1.74)
1.99 (1.74e2.31)
1.20 (1.03e1.40)
1.92 (1.61e2.28)
1.43 (1.32e1.54)
1.01 (0.92e1.11)
1.67 (1.56e1.82)
1.20 (1.03e1.38)
1.88 (1.69e2.08)
1.93 (1.76e2.12)
1.55 (1.20e1.55)
2.33 (2.02e2.72)
1.76 (1.47e2.11)
3.01 (2.32e3.88)
Hooker’s ‘lapachol peroxide’b
O
O
O
O
O
>10.36
>10.36
>10.36
9.76 (7.10e13.40)
>10.36
O
b
-lapachonec
>20.6
>21.9
0.42 (0.18e0.69)
1.65 (1.49e1.78)
1.75 (nd)
0.03 (0.02e0.04)
0.25 (0.16e0.33)
0.31 (0.22e0.39)
0.88 (0.62e1.21)
0.83 (0.74e0.87)
1.36 (1.18e1.53)
0.06 (0.04e0.08)
0.91 (0.74e1.11)
1.58(1.31e1.88)
0.41 (0.29e0.44)
Nor-
b
-lapachonec
Doxorubicinc
a
Alamar Blue assay was performed with human peripheral blood mononuclear cells (PBMC) after 72 h of drug exposure. Nd: not determined.
Ref. [19].
Refs. [15,16].
b
c
DNA-glycosylase (FPG, also known as MutM), and endonuclease III
results with the enzymes clearly show increased DNA migration of
the compounds-treated human cancer cells cultures. The results
indicate that the extent of oxidative DNA damage caused by
compounds, as recognized by ENDOIII and FPG in cancer cells, was
significantly higher. The present quinonoid compounds-generated
ROS can attack DNA at both pyrimidine and purine bases, and lead
to strand breaks. Based on these results, we suggest that active
compounds facilitate DNA oxidative damage by ROS, such as
(ENDOIII, also known as NTH). FPG is specific for oxidized purines,
specially for 8-oxo-7,8-dihydroguanine (8-oxoGua), and ENDOIII
recognizes oxidized pyrimidines, including thymine glycol and
uracil glycol [37].
Fig. 3 shows mean DNA damage caused by tested compounds,
expressed as DNA damage index after treatment with DNA-repair
enzymes ENDOIII and FPG. It was noted that the post-incubation
Table 3
Effects of
b-lapachone and nor-b-lapachone on DNA damage index for 24 h using alkaline version of comet assay in the presence or absence of 5 mM NAC.
Compoundsa
NACb
DNA Damage index ꢅ S.E.M
HL-60
MDA-MB435
HCT-8
SF295
b
-lapachone
ꢁ
þ
61.36 ꢅ 4.15*
8.33 ꢅ 0.25
83.17 ꢅ 3.25*
5.16 ꢅ 0.10
72.41 ꢅ 2.56*
11.41 ꢅ 1.15
58.75 ꢅ 1.15*
8.72 ꢅ 2.25
Nor-b
-lapachone
ꢁ
þ
67.19 ꢅ 0.96*
12.36 ꢅ 0.86
78.52 ꢅ 1.11*
7.49 ꢅ 0.33
63.25 ꢅ 3.25*
10.48 ꢅ 2.15
63.81 ꢅ 0.75*
9.18 ꢅ 0.56
*p < 0.05 compared to cultures treated in the presence of NAC by ANOVA/Tukey’s test.
a
Compounds were tested at 5
N-acetylcysteine (NAC).
mM.
b

E.N. da Silva Jr. et al. / European Journal of Medicinal Chemistry 46 (2011) 399e410
403
Fig. 3. Effects of FPG and ENDOIII on compounds (5 mM)-induced oxidative DNA damage after 12 h exposure by the alkaline version of the comet assay: HL-60 (A), MDA-MB435 (B),
HCT-8 (C), and SF295 (D). Bars represent the mean ꢅ S.E.M. of three independent experiments. *p < 0.05 vs. DMSO (0.1%) by ANOVA/Tukey’s test.
superoxide radical (O2 ꢁ) and hydrogen peroxide (H2O2). H2O2 is
not toxic by itself, but its high in vivo reactivity, through the Fenton
reaction, where it reacts with partially reduced metal ions, gener-
ates hydroxyl radical (HOꢀ), the most important radical in ROS-
related DNA damage [38]. These radicals cause a variety of base
lesions in DNA. In the case of tested compounds, the lesion
produced is probably 8-oxoGua, the preferred substrate for FPG
[39], and modified bases thymine glycol which is the more
commonly recognized lesion by ENDOIII [40]. NQs also react with
proteins and lipids, destroying their functionality, thereby causing
cell death [41]. Therefore, in order to get additional evidences of the
oxidative damage triggered by the present quinonoid compounds,
ꢀ
Fig. 4. Determination of TBARS of cancer cells [HL-60 (A), MDA-MB435 (B), HCT-8 (C), and SF295 (D)] exposed to compounds (5
(DMSO; 0.1%), H2O2 (10 M for 2 h), or treatment with compounds. Filled bars represent the effect of pre-treatment with 5 mM N-acetylcysteine (NAC) added before compounds
exposure. Bars represent the mean ꢅ S.E.M. of three independent experiments. *p < 0.05 vs. DMSO (0.1%) by ANOVA/Tukey’s test.
mM) for 24 h. Open bars represent negative control
m

404
E.N. da Silva Jr. et al. / European Journal of Medicinal Chemistry 46 (2011) 399e410
Fig. 5. Effects of compounds 33 (A), 35 (B),
b
-lapachone (C), and nor-
b-lapachone (D) at 5
mM on cancer cell viability using trypan blue dye exclusion after 24 h of incubation in the
presence or absence of 5 mM N-acetylcysteine (NAC) and 50
(0.1%) by ANOVA/Tukey’s test.
m
M 1-bromoheptane (BH). Bars represent the mean ꢅ S.E.M. of three independent experiments. *p < 0.05 vs DMSO
we examined the lipid peroxidation induced by these compounds.
As seen in Fig. 4, the treatment of cancer cells with active
substances resulted in an increase of TBARS formation. Pre-incu-
bation with NAC prevented the quinonoid compounds-generated
oxidative damage, as seen by the reduced level of TBARS.
These data corroborate other reports, which showed that
-lapachone, related compounds and other NQs induce ROS
production and oxidative DNA damage [42].
Compounds 33 and 35 promoted cell death by apoptosis and
genotoxicity [DNA strand breaks were reduced after pre-treatment
b
Fig. 6. Effect of compounds (33 and 35) at 5
the presence or absence of 5 mM N-acetylcysteine (NAC) and 50
of three independent experiments. *p < 0.05 vs DMSO (0.1%) by ANOVA/Tukey’s test.
mM on the induction of DNA strand breaks in human cancer cell lines after 24 h exposure by the alkaline version of the comet assay in
mM 1-bromoheptane (BH): HL-60 (A), MDA-MB435 (B), HCT-8 (C), and SF295 (D). Bars represent the mean ꢅ S.E.M.

E.N. da Silva Jr. et al. / European Journal of Medicinal Chemistry 46 (2011) 399e410
405
Fig. 7. Induction of cancer cells apoptosis by compounds (33 and 35) at 5
microscopy count after 24 h of incubation in the presence or absence of 5 mM N-acetylcysteine (NAC) and 50
SF295 (D). Bars represent the mean ꢅ S.E.M. of three independent experiments. *p < 0.05 vs DMSO (0.1%) by ANOVA/Tukey’s test.
mM using acridine-orange and ethidium-bromide staining (AO/EB), and determined by fluorescence
mM 1-bromoheptane (BH): HL-60 (A), MDA-MB435 (B), HCT-8 (C), and
with NAC, which increase the percentage of viable cells (trypan
Table 4
blue stain)], suggesting that tested quinones-induced apoptosis is
associated with ROS production (Figs. 5e7). Moreover, emphasizing
the ROS contribution on the cellular toxicity, the GSH content was
depleted by using a non-toxic concentration of 1-bromoheptane
(trypan blue stain). In GSH-depleted cell cultures, an increase in cell
death (% apoptotic nuclei) as well as in the levels of DNA strand
breaks (comet assay), was observed.
The effects of anthraquinone imidazoles 18 and 22e27, ethers
quinones 8,10, 12,16 and Hooker’s ‘lapachol peroxide’ [19] (inactive
compounds) on cancer cell apoptosis at 5 mM using DNA-binding
fluorescent dye (AO/EB) staining (Table 4) showed that these
substances do not generate ROS in this concentration, since the pre-
treatment with 1-bromoheptane did not show any influence on the
activity of these compounds. These data corroborate with the
inactivity in cancer cell lines.
Effects of anthraquinone imidazoles, ethers quinones and Hooker’s ‘lapachol
peroxide’ on cancer cell apoptosis at 5
mM using acridine-orange and ethidium-
bromide staining (AO/EB). Apoptotic nuclei were determined by fluorescence
microscopy analysis after 24 h of exposure in the presence or absence 50 mM BH.
Compoundsa BHb Apoptotic nuclei (%) ꢅ S.E.M.
HL-60
MDA-MB435 HCT-8
SF295
c
H2O2
18
ꢁ
þ
ꢁ
þ
ꢁ
þ
ꢁ
þ
ꢁ
þ
ꢁ
þ
ꢁ
þ
ꢁ
þ
ꢁ
þ
ꢁ
þ
ꢁ
þ
ꢁ
þ
ꢁ
83.17 ꢅ 4.15* 74.59 ꢅ 3.75* 80.66 ꢅ 5.16* 85.03 ꢅ 7.15*
8.37 ꢅ 2.45 11.44 ꢅ 0.81
8.11 ꢅ 0.10
9.17 ꢅ 0.96
7.32 ꢅ 0.11
12.59 ꢅ 4.10
9.02 ꢅ 0.75 10.35 ꢅ 0.10
22
23
24
25
26
27
8
10.75 ꢅ 0.56 10.38 ꢅ 0.10
9.17 ꢅ 1.45 13.95 ꢅ 3.45
5.33 ꢅ 0.33 11.85 ꢅ 0.81
11.47 ꢅ 1.75
6.18 ꢅ 0.81
7.33 ꢅ 1.17
6.33 ꢅ 0.33
6.17 ꢅ 0.56
7.28 ꢅ 0.11
7.38 ꢅ 1.15
9.81 ꢅ 0.11 12.01 ꢅ 0.10 10.05 ꢅ 0.10
9.62 ꢅ 0.56 12.27 ꢅ 3.15
9.31 ꢅ 0.33
8.72 ꢅ 1.17
4.19 ꢅ 0.10
4.17 ꢅ 0.10
5.82 ꢅ 0.10
9.91 ꢅ 0.75
5.27 ꢅ 0.11
12.59 ꢅ 2.45 10.04 ꢅ 0.21
8.21 ꢅ 0.75 7.39 ꢅ 0.33
7.49 ꢅ 0.25
6.48 ꢅ 0.56 11.73 ꢅ 1.17
8.17 ꢅ 0.33 13.75 ꢅ 0.56
7.25 ꢅ 0.45
9.66 ꢅ 0.10
4. Conclusions
10.49 ꢅ 1.15 12.51 ꢅ 0.10 12.73 ꢅ 2.45 13.51 ꢅ 3.15
14.26 ꢅ 3.45 13.07 ꢅ 1.15 11.16 ꢅ 0.11 10.18 ꢅ 0.56
10.70 ꢅ 2.08
4.17 ꢅ 0.11
8.13 ꢅ 0.17
9.56 ꢅ 1.75
8.51 ꢅ 0.75
5.93 ꢅ 0.11
8.01 ꢅ 0.10
7.94 ꢅ 0.56
In this paper, we listed a series of new NQs that were evaluated
7.44 ꢅ 1.05 11.46 ꢅ 0.10
9.51 ꢅ 1.25 8.71 ꢅ 1.05
against tumor cell lines. Only the
b-lapachone-based 1,2,3-triazoles
were highly active (IC50 < 2 M) for cancer cell lines, for instance,
m
10
12
16
10.62 ꢅ 0.25 13.72 ꢅ 2.45 10.33 ꢅ 0.11 12.06 ꢅ 2.25
10.21 ꢅ 1.15
8.55 ꢅ 0.11 11.36 ꢅ 1.15
3.91 ꢅ 0.11
8.38 ꢅ 0.75
5.85 ꢅ 0.25
8.33 ꢅ 0.75
HL-60 and MDA-MB435, inducing apoptotic cell death mediated by
ROS generation, similarly to the precursor molecules. Thus, the
9.37 ꢅ 0.15 11.47 ꢅ 0.11
8.30 ꢅ 0.10
7.94 ꢅ 0.10 12.52 ꢅ 0.75
b-lapachone-based 1,2,3-triazoles can be considered promising
11.04 ꢅ 0.15 12.09 ꢅ 2.45 11.70 ꢅ 1.15 13.97 ꢅ 0.33
prototypes for cancer therapy.
9.13 ꢅ 3.15
8.46 ꢅ 0.15
11.17 ꢅ 0.33
8.56 ꢅ 0.10
5.82 ꢅ 0.11
4.72 ꢅ 0.25
9.27 ꢅ 0.11 11.73 ꢅ 1.15
9.16 ꢅ 0.33 7.30 ꢅ 0.21
8.52 ꢅ 0.10 11.45 ꢅ 0.15
Hooker’s
‘lapachol
peroxide’d
5. Experimental
*p < 0.05 compared to cultures treated in the presence or absence of BH by ANOVA/
5.1. Chemistry
Tukey’s test.
a
Compounds were tested at 5
1-Bromoheptane (BH).
mM.
Melting points were obtained on Thomas Hoover and are
uncorrected. Analytical grade solvents were used. Column chro-
matography was performed on silica gel (Acros Organics 0.035e
b
c
H2O2 at 10
Ref. [19].
mM during 2 h.
d

406
E.N. da Silva Jr. et al. / European Journal of Medicinal Chemistry 46 (2011) 399e410
0.070 mm, pore diameter ca 6 nm). Infrared spectra were recorded
on a PerkineElmer FT-IR Spectrometer. 1H and 13C NMR were
recorded at room temperature using a Varian Mercury Plus 300 and
Varian MR 400 instrument, in the solvents indicated, with TMS as
5.2.4. 3-Isobutyl-3-(3-isobutyl-1,4-dioxo-1,4,5,6,7,8-hexahydro-
naphthalen-2-yloxy)-5,6,7,8-tetrahydro-naphthalene-1,2,4-trione 6
The reaction of 2-hydroxy-3-isobutyl-5,6,7,8-tetrahydro-[1,4]
naphthoquinone (5), (234 mg,1 mmol), 2.5 mL of AcOH and 234 mg
of PbO2 yielded product 6, (326 mg, 0.7 mmol, 70% yield) as an
orange solid; mp 78e82 ꢃC; IR (KBr) 1749 (C]O), 1683 (C]O), 1641
internal standard. Chemical shifts (d) are given in ppm and coupling
constants (J) in Hertz. High-resolution mass spectra (electrospray
ionization) were obtained using a MicroTOF Ic - Bruker Daltonics.
All the compounds were nominated using the program CS Chem-
Draw Ultra version 5.0 and 10.0.
(C]O), 1602 (C]O) cmꢁ1 1H NMR (300 MHz, CDCl3)
; d 2.84e2.61
(2H, m), 2.51e2.32 (5H, m), 2.20e2.04 (2H, m), 1.91e1.77 (6H, m),
1.66e1.51 (5H, m), 0.98 (3H, d, J ¼ 1.8 Hz), 0.96 (3H, d, J ¼ 1.8 Hz),
0.93 (3H, d, J ¼ 6.5 Hz), 0.90 (3H, d, J ¼ 6.5 Hz); 13C NMR (75 MHz,
5.2. General procedures to prepare the ether-compounds
CDCl3) d 190.0 (C]O),187.1 (C]O),184.0 (C]O),183.8 (C]O),182.1
(C]O), 149.2, 146.6, 142.7, 139.5, 128.1, 92.7, 46.6, 32.1, 28.2, 24.3,
23.9, 23.8, 23.0, 22.9, 22.0, 21.0, 20.8, 20.7; EI/HRMS (m/z) [M þ H]þ
467.2441. Calcd for [C28H34O6H]þ: 467.2434.
The hydroxyquinone in glacial acetic acid was heated to boiling,
the source of heat was removed, and lead dioxide was added. The
hot mixture was shaken for 2 min and filtered to remove lead oxide,
and after precipitation on cooling, very slowly, and filtration,
crystals of the ether were obtained [19].
5.2.5. 3-Allyl-3-(3-allyl-1,4-dioxo-1,4-dihydro-naphthalen-2-
yloxy)-naphthalene-1,2,4-trione 12
The reaction of 2-allyl-3-hydroxy-[1,4]naphthoquinone (11),
(214 mg, 1 mmol), 2.5 mL of AcOH and 214 mg of PbO2 yielded
product 12, (319 mg, 0.7 mmol, 70% yield) as an orange solid; mp
93e95 ꢃC; IR (KBr) 1747 (C]O), 1699 (C]O), 1651 (C]O), 1591 (C]
5.2.1. 3-(3-Methyl-butyl)-3-[3-(3-methyl-butyl)-1,4-dioxo-1,4-
dihydro-naphthalen-2-yloxy]-naphthalene-1,2,4-trione 8
The reaction of 2-hydroxy-3-(3-methyl-butyl)-[1,4]naph-
thoquinone (7), (244 mg, 1 mmol), 2.5 mL of AcOH and 244 mg of
PbO2 yielded product 8, (388 mg, 0.8 mmol, 80% yield) as an orange
solid; mp 77e81 ꢃC; IR (KBr) 1747 (C]O), 1697 (C]O), 1649 (C]O),
O), 1577 (C]O) cmꢁ1; 1H NMR (300 MHz, CDCl3)
d 8.45e8.39 (1H,
m), 8.28e8.17 (1H, m), 8.06e8.01 (1H, m), 7.97e7.91 (2H, m),
7.70e7.63 (2H, m), 7.59e7.49 (1H, m), 6.08e5.95 (1H, m), 5.78e5.64
(1H, m), 5.35e5.27 (1H, m), 5.19e5.13 (2H, m), 5.12e5.05 (1H, m),
3.58e3.55 (2H, m), 2.85e2.82 (2H, m); 13C NMR (75 MHz, CDCl3)
1593 (C]O), 1577 (C]O) cmꢁ1
;
1H NMR (300 MHz, CDCl3)
d
8.44e8.41 (1H, m), 8.24e8.21 (1H, m), 8.04e8.00 (1H, m),
7.96e7.92 (2H, m), 7.67e7.62 (2H, m), 7.53e7.48 (1H, m), 2.82e2.77
(2H, m), 2.06e1.99 (2H, m), 1.78e1.25 (6H, m), 1.02 (6H, d,
d 188.5 (C]O), 184.0 (C]O), 183.6 (C]O), 182.0 (C]O), 180.0 (C]
O), 150.0, 136.6, 134.9, 134.6, 133.8, 133.0, 131.3, 130.3, 130.0, 129.0,
128.3,128.2,126.4,126.1,126.1,116.8, 92.7, 42.0, 27.9; EI/HRMS (m/z)
[M þ H]þ 427.1175. Calcd for [C26H18O6H]þ: 427.1182.
J ¼ 6.4 Hz), 0.85e0.81 (6H, m); 13C NMR (75 MHz, CDCl3)
d 189.0
(C]O),184.3 (C]O),184.2 (C]O),182.0 (C]O), 180.7 (C]O), 150.8,
136.1, 134.8, 134.4, 133.9, 133.6, 132.9, 131.6, 130.6, 129.0, 128.5,
126.5, 126.1, 93.1, 36.9, 35.5, 31.5, 28.8, 27.9, 22.5, 22.4, 22.2; EI/
HRMS (m/z) [M þ H]þ 487.2124. Calcd for [C30H30O6H]þ: 487.2121.
5.2.6. 3-(1,4-Dioxo-3-propyl-1,4-dihydro-naphthalen-2-yloxy)-3-
propyl-naphthalene-1,2,4-trione 14
The reaction of 2-hydroxy-3-propyl-[1,4]naphthoquinone (13),
(216 mg,1 mmol), 2.5 mL of AcOH and 216 mg of PbO2 yielded product
14, (301 mg, 0.7 mmol, 70% yield) as an orange solid; mp 89e91 ꢃC; IR
(KBr) 1747 (C]O),1701 (C]O),1651 (C]O),1593 (C]O),1577 (C]O)
5.2.2. 3-(3-Methyl-butyl)-3-[3-(3-methyl-butyl)-1,4-dioxo-
1,4,5,6,7,8-hexahydro-naphthalen-2-yloxy]-5,6,7,8-tetrahydro-
naphthalene-1,2,4-trione 10
The reaction of 2-hydroxy-3-(3-methyl-butyl)-5,6,7,8-tetrahy-
dro-[1,4]naphthoquinone (9), (248 mg, 1 mmol), 2.5 mL of AcOH
and 248 mg of PbO2 yielded producꢃt 10, (370 mg, 0.7 mmol, 75%
yield) as an orange solid; mp 74e78 C; IR (KBr) 1749 (C]O), 1683
(C]O), 1639 (C]O), 1604 (C]O) cmꢁ1; 1H NMR (300 MHz, CDCl3)
cmꢁ1; 1H NMR (300 MHz, CDCl3)
d 8.43e7.48 (8H, m), 2.81e1.39 (8H,
m), 1.08 (3H, t, J ¼ 7.0 Hz), 0.91 (3H, t, J ¼ 7.0 Hz); 13C NMR (75 MHz,
CDCl3) d: 192.3 (C]O),189.0 (C]O),184.4 (C]O),184.3 (C]O),182.1
(C]O),151.0,136.1,135.0,134.4,134.0,133.1, 133.0,133.0,131.6, 130.6,
129.0, 128.4, 126.5, 126.2, 93.2, 39.5, 26.1, 21.6, 16.6, 14.5, 14.0.
d
2.85e2.53 (4H, m), 2.20e1.15 (22H, m), 0.95 (6H, d, J ¼ 6.4 Hz),
0.85 (6H, d, J ¼ 6.4 Hz); 13C NMR (75 MHz, CDCl3)
d
190.5 (C]O),
5.2.7. 3-(1,4-Dioxo-3-propyl-1,4,5,6,7,8-hexahydro-naphthalen-2-
yloxy)-3-propyl-5,6,7,8-tetrahydro-naphthalene-1,2,4-trione 16
The reaction of 2-hydroxy-3-propyl-5,6,7,8-tetrahydro-[1,4]
naphthoquinone (15), (220 mg, 1 mmol), 2.5 mL of AcOH and
220 mg of PbO2 yielded product 16, (328 mg, 0.7 mmol, 70% yield)
as an orange solid; mp 71e94 ꢃC; IR (KBr) 1749 (C]O), 1683 (C]O),
187.1 (C]O), 184.4 (C]O), 184.1 (C]O), 182.1 (C]O), 149.3, 147.1,
143.2, 139.7, 129.5, 92.2, 37.2, 36.3, 32.0, 29.0, 28.2, 24.5, 23.4, 23.2,
23.0, 22.7, 22.5, 22.4, 22.3, 21.8, 21.2, 21.1, 21.0; EI/HRMS (m/z)
[M þ H]þ 495.2731. Calcd for [C30H38O6H]þ: 495.2747.
5.2.3. 3-Isobutyl-3-(3-isobutyl-1,4-dioxo-1,4-dihydro-naphthalen-
2-yloxy)-naphthalene-1,2,4-trione 4
1639 (C]O), 1604 (C]O) cmꢁ1 1H NMR (300 MHz, CDCl3)
;
d
2.87e2.27 (8H, m), 2.16e1.25 (16H, m), 0.99 (3H, t, J ¼ 7.0 Hz), 0.92
The reaction of 2-hydroxy-3-isobutyl-[1,4]-naphthoquinone (3),
(230 mg, 1 mmol), 2.5 mL of AcOH and 230 mg of PbO2 yielded
product 4, (320 mg, 0.70 mmol, 70% yield) as an orange solid; mp
79e80 ꢃC; IR (KBr) 1749 (C]O), 1701 (C]O), 1651 (C]O), 1593 (C]
(3H, t, J ¼ 7.0 Hz); 13C NMR (75 MHz, CDCl3)
d 190.5 (C]O), 187.2
(C]O), 184.1 (C]O), 183.0 (C]O), 182.0 (C]O), 149.4, 147.2, 143.1,
139.7, 129.1, 92.3, 40.2, 34.2, 25.6, 24.5, 23.4, 23.0, 22.3, 21.9, 21.2,
21.1, 21.0, 17.1, 14.6, 14.2; EI/HRMS (m/z) [M þ H]þ 439.2126. Calcd
for [C26H30O6H]þ: 439.2121.
O), 1575 (C]O) cmꢁ1; 1H NMR (300 MHz, CDCl3)
d 8.43e8.40 (1H,
m), 8.23e8.20 (1H, m), 8.05e8.01 (1H, m), 7.96e7.92 (2H, m),
7.68e7.62 (2H, m), 7.54e7.48 (1H, m), 2.74 (2H, d, J ¼ 7.4 Hz),
2.31e1.94 (2H, m), 1.94 (2H, d, J ¼ 7.4 Hz), 1.07 (3H, d, J ¼ 1.6 Hz),
1.07 (3H, d, J ¼ 1.6 Hz), 0.97 (3H, d, J ¼ 6.5 Hz), 0.91 (3H, d,
5.2.8. 2-Methyl-3-(2-methyl-1,3-dioxo-indan-2-yloxy)-[1,4]
naphthoquinone 17
The reaction of 188 mg of phithiocol, 18.8 mL of AcOH and
188 mg of PbO2 yielded the compound 17, (311 mg, 0.9 mmol, 90%
yield) as a yellow prisms; mp 152e153 ꢃC; IR (KBr) 1754 (C]O),
J ¼ 6.5 Hz); 13C NMR (75 MHz, CDCl3)
d 188.8 (C]O), 184.5 (C]O),
184.3 (C]O), 182.1 (C]O), 181.0 (C]O), 151.3, 136.1, 134.7, 134.4,
134.1, 132.9, 132.8, 132.5, 131.5, 130.7, 129.0, 128.5, 126.5, 126.2, 94.0,
45.8, 32.8, 28.4, 24.0, 23.9, 23.1, 23.0; EI/HRMS (m/z) [M þ H]þ
459.1802. Calcd for [C28H26O6H]þ: 459.1808.
1720 (C]O), 1655 (C]O), 1612 (C]O) cmꢁ1 1H NMR (300 MHz,
;
CDCl3)
d 8.07e8.00 (3H, m), 7.96e7.89 (2H, m), 7.67e7.60 (2H, m),
7.55e7.48 (1H, m), 2.27 (3H, s), 1.74 (3H, s); 13C NMR (75 MHz,

E.N. da Silva Jr. et al. / European Journal of Medicinal Chemistry 46 (2011) 399e410
407
CDCl3)
d
195.5 (C]O), 184.9 (C]O), 181.1(C]O), 152.8, 138.6, 136.0,
(d, J ¼ 8.42 Hz, 1H), 8.14 (d, J ¼ 8.37 Hz, 1H), 7.96e7.89 (m, 2H),
7.86e7.79 (m, 2H), 7.57 (dd, J ¼ 13.61, 7.92 Hz, 1H), 7.31e7.27 (m,
134.2, 133.0, 132.0, 130.5, 130.3, 126.2, 124.1, 84.1, 23.4, 9.8. EI/HRMS
(m/z) [M þ Na]þ 369.0717. Calcd for [C21H14O5Na]þ: 369.0739.
1H). 13C NMR (75 MHz, DMSO-d6)
d 183.5, 182.8, 164.5, 156.8, 134.9,
134.8, 133.4, 131.7, 131.6, 131.5, 131.4, 131.3, 128.7, 127.2, 126.7, 124.7,
121.5, 118.3, 115.3.
5.3. General procedures to prepare the reduced compounds
3-Hydroxy-naphthoquinone was dissolved in the appropriate
solvent and submitted to catalytic reduction over 10 mol% of the
Pd/C as a catalyst. The reaction was monitored by TLC and after the
complete consumption of the quinone reactant the reaction
mixture was filtered and the product was purified by column
chromatography in silica gel, eluted with an increasing polarity
gradient mixture of hexane and ethyl acetate.
5.4.3. 2-(4-Fluorophenyl)-1H-anthra[1,2-d]imidazole-6,11-dione 21
From 4-fluoro-benzaldehyde (136 mg,1.1 mmol), 21 was obtained
as a yellow solid (226 mg, 0.66 mmol, 66% yield, mp 280e283 ꢃC).
IR (KBr) 3429, 1670, 1589, 1489, 1330, 1301, 1290, 717 cmꢁ1 1H
NMR (400 MHz, DMSO-d6)
d 11.38e11.24 (m, 1H), 8.39e8.33 (m,
1H), 8.32e8.28 (m, 1H), 8.26 (d, J ¼ 8.36 Hz, 1H), 8.22e8.16 (m, 2H),
8.13 (d, J ¼ 8.43 Hz, 1H), 7.86e7.80 (m, 2H), 7.32e7.28 (m, 2H). 13C
NMR (75 MHz, DMSO-d6)
d 183.0, 182.2, 156.6, 164.5, 134.9, 134.8,
5.3.1. 2-Hydroxy-3-isobutyl-5,6,7,8-tetrahydro-[1,4]
naphthoquinone 5
134.3, 134.2, 132.9, 130.6, 130.5, 127.9, 126.6, 126.3, 126.1, 125.4,
120.9, 115.8, 115.5.
Nor-lapachol (2), (228 mg, 1 mmol) in 15 mL of AcOH, 55 psi of
H2 and 10% of Pd/C during 6 h, yielded product 5, (234 mg, 1 mmol,
100% yield) as a yellow solid; mp 88e90 ꢃC; IR (KBr) 1647 (C]O),
5.4.4. 2-o-Tolyl-1H-anthra[1,2-d]imidazole-6,11-dione 22
From 2-methyl-benzaldehyde (132 mg, 1.1 mmol), 22 was
obtained as a yellow solid (271 mg, 0.80 mmol, 80% yield, mp
235e239 ꢃC).
1631 (C]O), 3369 (-OH) cmꢁ1 1H NMR (300 MHz, CDCl3)
; d 6.93
(OH, sl), 2.49e2.39 (4H, m), 2.31 (1H, d, J ¼ 7.2 Hz), 1.92e1.79 (1H,
m), 1.71e1.67 (4H, m), 0.89 (6H, d, J ¼ 6.5 Hz); 13C NMR (75 MHz,
IR (KBr) 3346, 1666, 1649, 1575, 1519, 1483, 1438, 1323, 1296,
CDCl3)
d 187.5 (C]O), 183.4 (C]O), 151.0, 145.0, 137.7, 120.0, 31.6,
1008, 840, 773, 727, 713, 648 cmꢁ1 1H NMR (300 MHz, DMSO-d6)
27.9, 23.1, 22.6, 21.8, 21.1, 21.0; MS [70 eV, m/z (%)]: 115 (8), 192 (11),
65 (13), 91 (17), 234 (17), 79 (27), 41 (100).
d
8.27e8.19 (m,1H), 8.17e8.10 (m, 2H), 8.04 (d, J ¼ 8.38 Hz,1H), 7.77
(d, J ¼ 7.42 Hz, 1H), 7.72e7.66 (m, 2H), 7.38e7.23 (m, 3H), 2.63 (s,
3H). 13C NMR (75 MHz, DMSO-d6)
183.5, 183.0, 159.0, 137.9, 134.9,
d
5.3.2. 2-Hydroxy-3-propyl-5,6,7,8-tetrahydro-[1,4]
naphthoquinone 15
134.7, 133.6, 131.3, 131.1, 130.6, 129.9, 128.4, 127.2, 126.7, 126.1, 125.5,
121.1, 21.1.
C-Allyl lawsone (11), (214 mg, 1 mmol) in 15 mL of AcOH, 55 psi
of H2 and 10% of Pd/C during 6 h, yielded product 15, (220 mg,
0.98 mmol, 98% yield) as a yellow solid; mp 65e67 ꢃC; IR (KBr) 1647
5.4.5. 2-m-Tolyl-1H-anthra[1,2-d]imidazole-6,11-dione 23
From 3-methyl-benzaldehyde (132 mg, 1.1 mmol), 23 was
obtained as a yellow solid (251 mg, 0.74 mmol, 74% yield, mp
229e231 ꢃC).
(C]O), 1618 (C]O) cmꢁ1 1H NMR (300 MHz, CDCl3)
; d 2.51e2.35
(6H, m), 1.74e1.65 (4H, m), 1.48 (2H, sextet, J ¼ 7.5 Hz), 0.93 (1H, t,
J ¼ 7.4 Hz); 13C NMR (75 MHz, CDCl3)
d
187.7 (C]O), 183.8 (C]O),
IR (KBr) 3444, 1670, 1581, 1525, 1489, 1330, 1290, 713, 684,
151.0, 145.2, 137.9, 120.8, 25.0, 23.2, 22.1, 21.8, 21.4, 21.2, 14.3; MS
354 cmꢁ1 1H NMR (400 MHz, DMSO-d6)
d 8.34e8.29 (m, 1H),
[70 eV, m/z (%)]: 85 (70), 220 (80), 83 (100).
8.28e8.23 (m, 1H), 8.21 (d, J ¼ 8.37 Hz, 1H), 8.09 (d, J ¼ 8.39 Hz, 1H),
7.98 (s, 1H), 7.92 (d, J ¼ 7.65 Hz, 1H), 7.82e7.76 (m, 2H), 7.45 (t,
J ¼ 7.62 Hz, 1H), 7.37 (d, J ¼ 7.60 Hz, 1H), 2.49 (s, 3H). 13C NMR
5.4. General procedure for the synthesis of the antraquinones
(100 MHz, DMSO-d6) d 182.8, 183.5, 158.2, 138.5, 134.8, 134.7, 133.5,
A solution of 1,2-diamino-anthraquinone (17a) (238 mg,1 mmol)
in 5 mL of AcOH, sodium acetate (106 mg,1.3 mmol) and the desired
aldehyde (see below) were stirred in reflux. TLC was used to monitor
the end of the reaction. After the addition of water, the formed
precipitate was filtered, and the product was purified by column
chromatography using as eluent a mixture of dichloromethane/
ethyl acetate (5:1).
132.1, 129.2, 129.1, 128.4, 127.2, 126.6, 125.7, 121.4, 21.4.
5.4.6. 2-(2-Hydroxyphenyl)-1H-anthra[1,2-d]imidazole-6,11-
dione 25
From 2-hydroxy-benzaldehyde (150 mg, 1.1 mmol), 25 was
obtained as a yellow solid (174 mg, 0.51 mmol, 51% yield, mp
>300 ꢃC).
IR (KBr) 3352, 1664, 1589, 1521, 1487, 1327, 1290, 752, 713 cmꢁ1
5.4.1. 2-(2-Fluorophenyl)-1H-anthra[1,2-d]imidazole-6,11-dione 19
From 2-fluoro-benzaldehyde (136 mg,1.1 mmol),19 was obtained
as a yellow solid (207 mg, 0.61 mmol, 61% yield, mp 290e291 ꢃC).
IR (KBr) 3450, 1668, 1583, 1489, 1465, 1436, 1328, 1290, 1271,
1H NMR (300 MHz, DMSO-d6)
d 12.68 (s, 1H), 12.11 (s, 1H), 8.32 (d,
J ¼ 7.4Hz, 1H), 8.14e8.10 (m, 2H), 7.99 (s, 2H), 7.85e7.82 (m, 2H),
7.43e7.37 (ddd, J ¼ 7.4 and 1.7Hz, 1H), 7.07e6.97 (m, 2H). 13C NMR
(75 MHz, DMSO-d6)
d 183.6, 182.3, 157.3, 156.3, 134.6, 134.3, 133.2,
765, 715, 605 cmꢁ1 1H NMR (400 MHz, DMSO-d6)
d
11.81e11.64 (m,
133.1, 132.9, 128.8, 127.9, 126.9, 126.3, 123.8, 121.4, 119.8, 117.2, 113.4.
1H), 8.54 (t, J ¼ 7.79 Hz, 1H), 8.38e8.34 (m, 1H), 8.34e8.30 (m, 1H),
8.27 (d, J ¼ 8.40 Hz, 1H), 8.16 (d, J ¼ 8.39 Hz, 1H), 7.85e7.79 (m, 2H),
7.61e7.51 (m, 1H), 7.42e7.29 (m, 2H). 13C NMR (75 MHz, DMSO-d6)
5.4.7. 2-(3-Hydroxyphenyl)-1H-anthra[1,2-d]imidazole-6,11-
dione 26
d
183.5, 182.8, 164.5, 156.8, 134.9, 134.8, 133.4, 131.7, 131.6, 131.5,
From 3-hydroxy-benzaldehyde (150 mg, 1.1 mmol), 26 was
obtained as a yellow solid (200 mg, 0.59 mmol, 59% yield, mp
>300 ꢃC).
131.4, 131.3, 128.7, 127.2, 126.6, 124.8, 121.5, 118.3, 118.0.
5.4.2. 2-(3-Fluorophenyl)-1H-anthra[1,2-d]imidazole-6,11-dione 20
From 3-fluoro-benzaldehyde (136 mg, 1.1 mmol), 20 was
obtained as a yellow solid (177 mg, 0.52 mmol, 52% yield, mp
250e255 ꢃC).
IR (KBr) 3530, 3410, 3020, 1658, 1580, 1535, 1488, 1462, 1440,
1325, 1290, 1264, 1234, 1153, 890, 784, 714, 680, 595, 568 cmꢁ1 1H
NMR (300 MHz, DMSO-d6)
d 12.92 (s, 1H), 9.71 (s, 1H), 8.20e8.16
(m, 2H), 8.09e8.02 (m, 2H), 7.91e7.80 (m, 4H), 7.38 (t, J ¼ 7.8 Hz,
IR (KBr) 3414, 1670, 1647, 1587, 1577, 1529, 1485, 1458, 1328,
1H), 6.99 (dd, J ¼ 7.96 and 1.92 Hz, 1H). 13C NMR (75 MHz, DMSO-
1294, 1274, 1188, 713 cmꢁ1 1H NMR (400 MHz, DMSO-d6)
d6) d 183.0, 182.3, 157.8, 157.6, 149.2, 134.4, 134.2, 133.0, 132.9, 132.8,
d
11.38e11.29 (m, 1H), 8.38e8.33 (m, 1H), 8.32e8.28 (m, 1H), 8.26
130.1, 129.8, 127.9, 126.7, 126.2, 124.9, 120.9, 119.1, 118.6, 118.2, 114.9.

408
E.N. da Silva Jr. et al. / European Journal of Medicinal Chemistry 46 (2011) 399e410
5.4.8. 2-(4-Hydroxyphenyl)-1H-anthra[1,2-d]imidazole-6,11-
dione 27
From 4-hydroxy-benzaldehyde (150 mg, 1.1 mmol), 27 was
obtained as a yellow solid (187 mg, 0.55 mmol, 55% yield, mp
>300 ꢃC).
GIBCOÒ (Invitrogen, Carlsbad, CA, USA). 5-bromo-20-deoxyuridine,
thiobarbituric acid, hydrolyzed 1,1,3,3-tetramethoxypropan, 1-bro-
moheptane, N-acetylcysteine and colchicine were obtained from
Sigma Chemical Co. (St, Louis, MO). Hydrogen peroxide was from
Vetec (Brazil). Doxorubicin (DoxolemÒ) was from Zodiac Produtos
Farmacêuticos S/A, Brazil. Low melting point agarose and agarose
were obtained from Invitrogen (Carlsbad, CA, USA). All other chem-
icals and reagents used were of analytical grade.
IR (KBr) 3431, 3134,1668,1608,1585,1490,1475,1458,1440,1328,
1295, 1246, 1174, 715 cmꢁ1 1H NMR (300 MHz, DMSO-d6)
d 12.78 (s,
1H), 10.06 (s, 1H), 8.27 (d, J ¼ 8.37 Hz, 1H), 8.22e8.17 (m, 3H),
8.06e7.98 (m, 2H), 7.91e7.88 (m, 2H), 6.93 (d, J ¼ 8.78, 2H). 13C NMR
(75 MHz, DMSO-d6)
d 183.2, 182.3, 160.3, 158.2, 149.7, 134.4, 134.2,
6.2. Cytotoxicity against cancer cell lines
133.2,133.0, 103.1, 127.4, 126.8, 126.2, 124.2, 121.0, 119.8, 118.2, 115.6.
Compounds (0.01e5 mg/mL) were tested for cytotoxic activity
5.4.9. 2-(4-Hydroxy-3-methoxyphenyl)-1H-anthra[1,2-d]
imidazole-6,11-dione 28
From 4-hydroxy-3-methoxy-benzaldehyde (167 mg, 1.1 mmol),
28 was obtained as a yellow solid (258 mg, 0.77 mmol, 77% yield,
mp >300 ꢃC).
against four cancer cell lines: SF295 (central nervous system), HCT-
8 (colon), MDA-MB435 (breast), HL-60 (leukemia) from National
Cancer Institute (Bethesda, MD). All cell lines were maintained in
RPMI 1640 medium supplemented with 10% fetal bovine serum,
2 mM glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin at
37 ꢃC with 5% CO2. Each compound was dissolved with DMSO to
obtain a concentration of 1 mg/mL. They were incubated with the
cells for 72 h. The negative control received the same amount of
DMSO (0.1% in the highest concentration). Doxorubicin
IR (KBr) 3431, 3331, 1662, 1589, 1490, 1438, 1327, 1290, 1270,
1188, 1165, 1026, 721 cmꢁ1 1H NMR (400 MHz, DMSO-d6)
d
11.25e11.25 (s, 1H), 8.37 (dd, J ¼ 6.94, 2.11 Hz, 1H), 8.31 (dd,
J ¼ 6.88, 2.14 Hz, 1H), 8.25 (d, J ¼ 8.38 Hz, 1H), 8.11 (d, J ¼ 8.39 Hz,
1H), 7.85e7.80 (m, 3H), 7.62 (dd, J ¼ 8.27, 1.98 Hz, 1H), 7.11 (d,
J ¼ 8.24 Hz, 1H), 6.04e6.03 (m, 1H), 4.09e4.07 (s, 3H). 13C NMR
(0.1e0.58 mg/mL) was used as a positive control. The cell viability
was determined by reduction of the yellow dye 3-(4,5-dimethyl-2-
thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a blue
formazan product as described by Mosmann [27].
(100 MHz, DMSO-d6)
d 183.6, 182.6, 158.6, 150.1, 148.1, 134.8,
134.6, 133.5, 133.4, 127.8, 127.1, 126.5, 122.4, 121.4, 120.4, 116.0,
112.1, 56.3.
6.3. Inhibition of PBMC proliferation e Alamar Blue assay
5.5. X-ray analysis
To investigate the selectivity of compounds toward a normal
proliferating cell, the Alamar Blue assay was performed with
peripheral blood mononuclear cells (PBMC) after 72 h of drug
A yellow crystal (0.242 ꢂ 0.225 ꢂ 0.132 mm3) of compound 17
was selected for X-ray diffraction. Intensity data were collected at
room temperature (T ¼ 298K) using a diffractometer Kappa CCD of
exposure. After 24 h, compounds (0.048e25
DMSO (0.1%) were added to each well and incubated for 72 h.
Doxorubicin (0.01e1.06 M) was used as positive control. Twenty-
four h before the end of the incubation, 10 L of stock solution
mg/mL) dissolved in
Enraf Nonius with MoK
a
monochromatic radiation (
l
¼ 0.71073 Å)
and using the Collect [43] software, as well as Scalepack [44] for
m
cell refinement. For compound 17 a total of 15300 reflections to
m
a maximum 2q
of 25.76ꢃ were measured. The crystal structure for
(0.312 mg/mL) of the Alamar Blue (Resazurin, SigmaeAldrich Co)
was added to each well. The absorbance was measured using
a multiplate reader (DTX 880 Multimode Detector, Beckman
CoulterÒ) and the drug effect was quantified as the percentage of
control absorbance at 570 nm and 595 nm. The absorbance of Ala-
mar Blue in culture medium is measured at a higher wavelength and
a lower wavelength. The absorbance of the medium is also measured
at the higher and lower wavelengths. The absorbance of the medium
aloneis subtractedfromtheabsorbance ofmedium plus Alamar Blue
at the higher wavelength. This value is called AOHW. The absorbance
of the medium alone is subtracted from the absorbance of medium
plus Alamar Blue at the lower wavelength. This value is called AOLW.
A correction factor, R0, can be calculated from AOHW and AOLW,
where R0 ¼ AOLW/AOHW. The percent Alamar Blue reduced is then
expressed as follows: % reduced ¼ ALW ꢁ (AHW ꢂ R0) ꢂ 100.
compound 17 was solved by direct methods and refined aniso-
tropically with full matrix least square on F2 using SHELXL-97
program [45]. H atoms attached to C atoms were located on
stereochemical grounds placed (CeH ¼ 0.93e0.98 Å) and refined
as riding with Uiso(H) ¼ 1.5 Ueq(C-methyl) or 1.2 Ueq(other) times
the value of the equivalent isotropic displacement parameter of
atoms to which they are bonded. Compound 17 crystallized with
two water molecules. The software used were: data collection:
COLLECT [43]; cell refinement: HKL SCALEPACK [44]; data reduc-
tion: HKL DENZO and SCALEPACK [44]. The program(s) used to
solve structure: SHELXS-97 [46]. The program(s) used to refine
structure: SHELXL-97 [46] molecular graphics: ORTEP-3 [47],
software used to prepare material for publication: WinGX [47].
The main crystallographic parameters were inserted in the sup-
porting information.
Crystallographic data for compound 17 have been deposited
with the Cambridge Crystallographic Data Center as Supplemen-
tary Publication No. CCDC 742743. Copies of the data can be
obtained, free of charge, on application to CCDC, 12 Union Road,
Cambridge CH21EZ, UK (fax: þ44 1223 336 033 or e-mail: deposit@
ccdc.cam.ac.uk).
6.4. Cells treatments
Cancer cell lines were treated with compounds (5 mM) during
24 h. Moreover, in order to evaluate the contribution of ROS on the
cytotoxicity and genotoxicity of tested compounds, cells were pre-
treated for 24 h with 5 mM N-acetylcysteine (NAC) or 50
bromoheptane (BH), and after they were exposed to tested
compounds (5 M) during 24 h.
mM 1-
6. Anticancer assay
m
6.1. Chemicals
6.5. Cell viability
Fetal bovine serum and phytohaemagglutinin were purchased
from Cutilab (Campinas, SP, Brazil), and RPMI 1640 medium,
trypsineEDTA, penicillin and streptomycin were purchased from
Cell viability was determined by the trypan blue dye exclusion
test in human cancer cell lines. After treatment trypan blue-

E.N. da Silva Jr. et al. / European Journal of Medicinal Chemistry 46 (2011) 399e410
409
excluding cells were counted in a Neubauer chamber, in cell
aliquots removed from cultures after 24 h [48].
The assays were performed by according to Salgo and Pryor (1996)
[54], with minor modifications. Cancer cells were incubated with
compounds (5 mM) for 24 h, and after lysis with TriseHCl (15 mM for
6.6. Morphological characterization of apoptotic cancer cells
1 h). Two milliliters 0.4 mg/mL TCA, 0.25 M HCl were added to the
lysate, which was then incubated with 6.7 mg/mL thrichloroacetic
acid for 15 min at 100 ꢃC. The mixture was centrifuged at 750ꢂg for
10 min. As TBA reacts with other products of lipid peroxidation in
addition to MDA, results are expressed in terms of thiobarbituric
reactive species (TBARS), which are determined by absorbance at
532 nm. Hydrolyzed 1,1,3,3-tetramethoxypropan was used as the
standard. The results were normalized by protein content [55].
Apoptotic cells were determined at the end of each treatment by
use of the acridine-orange (AO)/ethidium-bromide (EB) assay:
25
mL of the cell suspension were mixed with 1
mL of the staining
solution (AO 100
a slide, and 300 cells were counted per data point [49]. The
percentage of apoptotic cells was then calculated.
m
g/mL þ EB 100
m
g/mL in PBS) and spread on
6.7. Alkaline comet assay (single cell gel electrophoresis)
Conflict of interest
Authors declare no conflict of interest.
The alkaline comet assay was performed as described by Singh
et al. (1988) [50] with minor modifications [51], and following the
recommendations of the International Workshop on Genotoxicity
Test Procedures [52]. At the end of the treatment, cells were
Acknowledgments
This research was supported by grants from the Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq),
Fundação Carlos Chagas Filho de Amparo à Pesquisa do Rio de
Janeiro (FAPERJ), CAPES, UnB, UFAL, UFRJ and National Cancer
Institute. The authors are also indebted to the PRONEX-FAPERJ
(E-26/110.574/2010) and to PRONEX-CNPq-FAPEAL. Dr. Eufrânio N.
da Silva Júnior thanks the CNPq for post doctoral financial support
(project no. 151558/2009-4).
washed with ice-cold PBS, detached with 100
mL trypsin (0.15%) and
resuspended in complete RPMI medium. Next, 20
mL of cell
suspension (w106 cells/mL) were mixed with 0.75% low melting
point agarose and immediately spread onto a glass microscope
slide precoated with a layer of 1% normal melting point agarose.
The agarose was allowed to set at 4 ꢃC for 5 min. The slides were
incubated in ice-cold lysis solution (2.5 M NaCl,10 mM Tris,100 mM
EDTA, 1% Triton X-100 and 10% DMSO, pH 10.0) at 4 ꢃC for
a minimum of 1 h to remove cellular proteins, leaving the DNA as
“nucleoids.” After the lysis procedure, the slides were placed on
a horizontal electrophoresis unit. The unit was filled with fresh
buffer (300 mM NaOH and 1 mM EDTA, pH > 13.0) to cover the
slides for 20 min at 4 ꢃC to allow DNA unwinding and expression of
alkali-labile sites. Electrophoresis was conducted for 20 min at 25 V
and 300 mA (0.86 V/cm). After electrophoresis, the slides were
neutralized (0.4 M Tris, pH 7.5), stained with ethidium-bromide
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