Hydroxychalcones induce apoptosis in B16-F10 melanoma cells via GSH and ATP depletion

Article abstract of DOI:10.1016/j.ejmech.2008.09.009

Searching for leading compounds of new drugs for cancer therapy, we studied the toxicity of 13 hydroxychalcones never tested before toward melanoma cell line (B16-F10). The compounds were obtained by aldolic condensation between aldehydes and hydroxylated

Full text of DOI:10.1016/j.ejmech.2008.09.009

European Journal of Medicinal Chemistry 44 (2009) 1630–1637
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Hydroxychalcones induce apoptosis in B16-F10 melanoma cells via GSH
and ATP depletion
a
,
,
,1
Andreia Lilian Formento Navarini 1, Louise Domeneghini Chiaradia b 1, Alessandra Mascarello b
,
´
a
b
b
a,
*
´
´
ˆ
Marcio Fritzen , Ricardo Jose Nunes , Rosendo Augusto Yunes , Tania Beatriz Creczynski-Pasa
a Departamento de Cieˆncias Farmaceˆuticas, Universidade Federal de Santa Catarina, Centro de Cieˆncias da Sau´de, Campus Trindade, CEP 88040-900, Floriano´polis, SC, Brazil
b Departamento de Qu´ımica, Universidade Federal de Santa Catarina, Campus Trindade, CEP 88040-900, Floriano´polis, SC, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 April 2008
Received in revised form 31 July 2008
Accepted 4 September 2008
Available online 19 September 2008
Searching for leading compounds of new drugs for cancer therapy, we studied the toxicity of 13
hydroxychalcones never tested before toward melanoma cell line (B16-F10). The compounds were
obtained by aldolic condensation between aldehydes and hydroxylated acetophenones, in alkaline
conditions. Three of them showed cytotoxicity to the cell line. Two of them induced mitochondrial GSH
and ATP depletion and promoted cell death through apoptosis in melanoma cells. One of the compounds
induced cell death through necrosis but did not significantly decrease the intracellular mitochondrial
GSH and ATP levels in melanoma cells. The results suggest that the predominant factor for the activity is
the molecule shape, and secondarily the number of hydroxyl groups.
Keywords:
Hydroxychalcones
Melanoma
Antitumoral
Apoptosis
GSH
Ó 2008 Published by Elsevier Masson SAS.
ATP
1. Introduction
the most invasive and also the most virulent in the subcutaneous
implantation model [3].
Malignant melanoma is a highly lethal disease, and the incidence
and mortality associated to this kind of cancer are increasing world-
wide. Currently, between 2 and 3 millions non-melanoma skin cancers
and 132.000 melanoma skin cancers occur globally each year [1]. Many
factors predispose an individual to skin cancer including personal and
family histories of the disease and geographic location [2]. The
metastasis and growth of tumors depend on the cell capability to
migrate, implant, adhere and penetrate into the membrane, reaching
the blood vessel and the tissue [3,4]. Selected B16 murine cell lines
(B16-F10 and B16-F1) showed a different metastatic potential: the F10
line colonized the lungs more efficiently than F1; the F10 cell line was
Among the drugs exerting anticancer activity against malignant
melanoma, dacarbazine has been extensively used, however, with
response rates of 11–25% and with short survival time. Thus, the
development of chemotherapy agents against malignant mela-
noma is justified [5].
Chalcones are essential intermediate compounds in the flavo-
noid biosynthesis in plants. Naturally occurring chalcones are
mainly in hydroxylated form and several reports have documented
their biological properties [6]. Many studies have demonstrated
antitumoral [7–11], anti-inflammatory [12], antibacterial [13], anti-
ulcerogenic [14], antioxidant [6,15], antimalarial [16] and anti-
leishmaniosis [16,17] activities for hydroxychalcones, aside from
other pharmacological effects. Concerning antitumoral activity,
Kobori et al. [7] described that the 20,40,6,4-tetrahydroxidihy-
drochalcone (phloretin) induced cell death by apoptosis in B16
mouse melanoma 4A5 and in HL-60 human leukaemia cell lines.
Iwashita et al. [9] showed that the isoliquiritigenin (20,40,4-trihy-
droxychalcone) and butein (20,40,3,4-tetrahydroxychalcone)
inhibited cell proliferation and induced apoptosis in B16 melanoma
4A5 cells; also, Hsu et al. [11] described that isoliquiritigenin
induced apoptosis and blocked the cell cycle progression in Hep G2
(human hepatoma cells). For new antitumoral molecules it is very
important to demonstrate cell toxicity through apoptosis. The
Abbreviations: DNA, deoxyribonucleic acid; mDNA, mitochondrial DNA; ATP,
adenosine triphosphate; GSH, glutathione; GSSG, oxidized gluthathione; DMEM,
Dulbecco’s modified Eagle’s medium; NMR, nuclear magnetic resonance spectros-
copy; MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide; PBS,
phosphate-buffered saline; DMSO, dimethylsulfoxide; HEPES, 4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; DTNB,
5,50-dithiobis(2-nitrobenzoic acid); NADPH, nicotinamide adenine dinucleotide
phosphate.
* Corresponding author. Tel.: þ55 48 3721 8057; fax: þ55 48 3721 5642.
E-mail addresses: taniac@hu.ufsc.br, taniacbcp@gmail.com (T.B. Creczynski-Pasa).
These authors contributed equally to this study.
1
0223-5234/$ – see front matter Ó 2008 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2008.09.009

A.L.F. Navarini et al. / European Journal of Medicinal Chemistry 44 (2009) 1630–1637
1631
death through apoptosis is characterized by nuclear condensation
that induces DNA fragmentation caused by endonucleolytic
cleavage of genomic DNA [18]. The apoptosis or programmed cell
death is a mechanism to control the cell proliferation, thus the
resistance of malignant cell to apoptosis is hallmark of cancer and
constitutes an important clinical problem [19].
The mitochondria is responsible for the production of 80–90% of
ATP needed for cell respiration and survival and play an important
role in the pro-and anti-apoptotic stimuli [20]. Skulachev [21]
showed the inter-relations between bioenergetic processes and
programmed death phenomena, and concluded that ATP is
required for certain steps of apoptosis and necrosis. Usually the
malfunctioning of mitochondria results in strong decrease in the
initial cellular content of ATP, however, in some cases the apoptosis
is accompanied by the increase of ATP concentration at early stages
followed by a decrease at final stage. The energy production is also
affected by mitochondrial glutathione (GSH) depletion. Changes in
mitochondrial GSH content have also been related with apoptosis
regulation, cell differentiation and growth [22,23].
The objective of this research is based on the fact that melanoma
cells have a relatively poor response to the apoptotic stimulus, [24] and
that solid tumor cells develop resistance to antineoplasic drugs and
that multidrug resistance is a major cause of chemotherapy failures by
human malignances [25]. The B16-F10 melanoma cell line has high
metastatic potential, and in general doesnot respond easily to cytotoxic
agents. Thus, in this work we studied and characterized the murine
B16-F10 melanoma cell death via apoptosis, induced by hydrox-
ychalcones. The results suggest that the principal causes were mito-
chondrial GSH and ATP depletion. An analysis of the structure was
performed to find out a relationship between the cytotoxic effects.
1-naphtaldehyde; chalcones 4 and 5 are derived from 2-naph-
taldehyde, in order to observe the influence of hydrophobic and
voluminous groups in the B-ring in the activity of the hydrox-
ychalcones. Chalcones 2 and 7 are derived from xanthoxyline (2-
hydroxy-4,6-dimethoxyacetofenone), to evaluate the influence of
two methoxyl groups in the A-ring beyond the presence of
a hydroxyl group and to observe the influence of the aldehydes
substituted with electron donors or electron acceptor groups;
chalcones 8–13 are derived from monobromide xanthoxyline (2-
hydroxy-3-bromo-4,6-dimethoxychalcone), to verify how much
the activity would be affected by the addition of bromide in the A-
ring and to observe the influence of the electron donor or electron
acceptor substituents in the B-ring on the activity of the
compounds. The obtained yields were between 36% and 66%.
The structures of the compounds 5, 6, 8, 9, 10 and 13, (new
compounds), were confirmed by chemical identification data: 1H
NMR, 13C NMR, IR and elemental analysis. For chalcone 1, the cor-
responding peak of the hydroxyl group does not appear because
probably during analysis the hydroxyl group was exchanged with
a deutered hydrogen atom of the chloroform, the solvent used. 1H
NMR spectra revealed that all structures were geometrically pure
and configured E (JH
¼ 15.2–16.4 Hz).
a
-Hb
3. Biological results and discussion
Initially, a selection of the hydroxychalcone toxicity for mela-
noma B16-F10 cells was done. The compounds 1, 3 and 13 reduced
cell viability by 97.7 ꢀ 0.7%, 75.2 ꢀ 1.5% and 50 ꢀ 1.9%, respectively,
when compared with the control (non-treated cells) (Fig. 2). All
other chalcones presented a non-significant effect at the tested
concentration. A total of 100 mM was chosen as maximal concen-
2. Chemistry
tration because much higher doses do not normally reach the blood
plasma [26,27] and we are looking for compounds with high
activity (low IC50) which could eventually be used in the future as
a drug for chemotherapy with very low to no side effects.
Thirteen hydroxychalcones were prepared by aldolic conden-
sation as presented in Fig. 1. Chalcones 1, 3 and 6 are derived from
Fig. 1. Synthesis of chalcones. The reaction consists of an aldolic condensation as described in Section 4 (a) methanol, KOH 50%, r.t., 24 h. *New compounds.

1632
A.L.F. Navarini et al. / European Journal of Medicinal Chemistry 44 (2009) 1630–1637
determined from concentration–response curve, which are
M, respectively (Fig. 3).
12 ꢀ 0.7
m
M, 17 ꢀ 1.5
m
M and 30 ꢀ 1.9
m
In order to elucidate the mechanism of death of B16-F10 cells
induced by hydroxychalcones we investigated if these compounds
could induce DNA fragmentation,
a biochemical marker of
apoptosis [28]. The lanes (B and C) that correspond to the DNA of
the cells incubated with compounds 1 and 3, respectively, showed
fragmented DNA distributed in bands with defined sizes, charac-
teristic of DNA fragmentation by apoptosis (Fig. 4). The lanes that
correspond to the DNA from the cells incubated without hydrox-
ychalcones (A) as well as the lane which corresponds to DNA from
cells incubated with hydroxychalcone 13 (D), showed uniform
chromatin. Since the hydroxychalcone 13 induced cell death
(Fig. 1), it seems to be through another mechanism, not apoptosis.
The mitochondria is a central organelle involved in apoptosis,
with the capacity to directly activate the execution pathways [29]
a process in which ATP is directly involved. Thus, the compounds
were incubated with the cells and the ATP depletion profile was
analyzed. The hydroxychalcones 1 and 3 decreased the ATP content
of the melanoma cells in a concentration-dependent manner
(Fig. 5B); the amount of ATP depletion was higher than the
percentage of cell death (Fig. 5A). The cell death through apoptosis
can be a consequence of cell energy loss, caused by the compounds
[31]. Possibly the hydroxychalcone 13 induced cell death through
necrosis, since DNA breakage induced by this compound is not
characteristic of apoptosis (Fig. 4). However, it did not significantly
alter the ATP intracellular content of melanoma cells as expected
for death induced by necrosis. These results are in part, in accor-
dance with the data from the literature by Hu et al. [30] which
demonstrated that IN6CPBD induces apoptosis in human mela-
noma through a mitochondrial dysfunction. Sabzevari et al. [8]
suggested that the cytotoxic activity of the phloretin, iso-
liquiritigenin, and of 10 other hydroxylated chalcones, toward K562
leukaemia or melanoma cells are due to their ability to mitochon-
drial uncoupling. Cao et al. [29] also detected the induction of
apoptosis through mitochondrial hyperpolarization and mtDNA
Fig. 2. Toxicity of hydroxychalcones for B16-F10 cell line. The cells (1 ꢁ105) were
incubated with 100 mM of the compounds, for 24 h. The cell viability was monitored
through MTT assay. Optical density of control groups (C) was taken as 100% of cell
viability. Each point represents the mean ꢀ S.E.M. of three experiments in triplicates.
*P < 0.05, **P < 0.01 and ***P < 0.001 compared to control groups.
Recently, Cabrera et al. [10] have investigated the relation
between the structure of some chalcones, flavonones and flavones
and their activity against human kidney carcinoma cells TK-40,
human mammary adenocarcinoma cells MCF-7 and human colon
adenocarcinoma cells HT-29. In general, no cytotoxic selectivity
between the different cellular lines was observed. However, in the
same substituted series the activity decreased following the
sequence chalcones > flavonones > flavones. Some other require-
ments for activity are the presence of the hydroxyl group that has
hydrogen bond donor capability and the presence of the conjugated
enone group, since the compounds lose their activity when
changed to an aldol structure.
The importance of the presence of a hydroxyl substituent was
demonstrated previously by Sabzevari et al. [8] via measurements of
the cytotoxic activity of 12 hydroxylated chalcones toward K562
leukaemia or melanoma cells. They observed that an increase in the
numberof hydroxyl groups on an aromatic ring decreases the pKa value
of the first hydroxyl group suggesting that chalcones with lower pKa
values are less toxic to hepatocytes. However, it should be noted that
the arrangement of the hydroxyl groups on the A-ring or B-ring plays
a crucial role in determining the mechanism of cytotoxicity toward
isolated rat hepatocytes. Thus, if the hydroxyl group is in ortho position
the chalcone can be metabolized to a quinone intermediate which
could react with cell nucleophile and deplete GSH.
In this case, the importance of the hydroxyl group in A-ring for
activity is apparent as can be observed in chalcones 1 and 3.
However, this activity is conditioned by the conformation of the
molecule as observed with chalcones 4 and 5 where the B-ring
consists of 1-naphtyl or 2-naphtyl groups. This is also observed
with chalcone 6 where the presence of a methoxyl group in posi-
tion 6 also changes the conformation of the molecule. This detail
may also explain the lack of strong activity of chalcones 2, 7–12,
xanthoxyline derivatives. The presence of a methoxy group in
position 6 should give a steric effect forcing the carbonyl group out
of the plane of the A-ring and then decrease the hydrogen bond
strength with the 2-hydroxyl groups. This conformation change
appears to be important to maintain the activity of these
compounds. Chalcone 13 with a carboxyl substituent in B-ring
exhibits a good activity that may be explained by the probable
formation of a nine-membered ring between this group and the
carbonyl group. Thus, the planarity and the rigidity of the molecular
structure seem to be important factors to the activity in these cases.
We further investigated the cytotoxic effect of the more active
compounds which were 1, 3 and 13 and the IC50 values were
Fig. 3. Concentration–response curves of hydroxychalcones 1, 3 and 13 on B16-F10 cell
line. The cells (1 ꢁ104) were incubated with the compounds (0–40
mM) for 72 h.
Optical density of control groups was taken as 100% of cell viability. Each bar repre-
sents the mean ꢀ S.E.M. of three experiments. **P < 0.01 and #P < 0.001 compared to
control groups.

A.L.F. Navarini et al. / European Journal of Medicinal Chemistry 44 (2009) 1630–1637
1633
Fig. 4. DNA fragmentation of B16-F10 cells induced by hydroxychalcones. The cells
(5 ꢁ105) were incubated for 24 h with: 0
m
M, Lane A – Control; Lane B – 12
mM of
compound 1; Lane C – 17 mM of compound 3; Lane D – 30 mM of compound 13 (IC50
concentrations for cell toxicity).
damage in human hepatoma G2 cells by curcumin. Garland and
Halestrap [22] also showed that the depletion of intracelular ATP,
by ATPase mitochondrial uncoupling capabilities, induced cell
death through apoptosis, exhibiting DNA fragmentation in cell lines
pro-B interleukin-3 (IL-3)-dependent.
We further analyzed a possible effect of hydroxychalcones on
mitochondria function measuring mitochondrial GSH content in
melanoma cells after incubation with the compounds. Fig. 6 shows
that the compounds 1 and 3 reduced the amount of mitochondrial
GSH after 24 h of incubation, this reduction may be related to ATP
depletion induced by these compounds. Kachadouriam and Day
[32] showed that low GSH content in the mitochondria of human
lung epithelial cells A549, treated with 20,50-hydroxychalcone was
related to mitochondrial dysfunction. The GSH, present in mito-
chondria, plays an important role in the control of reactive oxygen
species generation, in the integrity of mitochondrial membranes,
release of proapoptotic factors and in the modulation of cell death
by several pathways [33]. Moreover, the GSH in mitochondria
protects the integrity of mitochondrial components, thus playing
a determinant role in response to apoptosis.
Fig. 5. Effect of hydroxychalcones 1, 3 and 13 on ATP amounts, compared to cytotoxic
effect, in B16-F10 cells. The cells (1 ꢁ105) were incubated with the compounds (0–
100
m
M) for 24 h to analyze the cell viability (A). 3 ꢁ 105 cells were incubated with the
compounds at the same concentrations for 24 h, for ATP concentration measurements
(B). Each bar represents the mean ꢀ S.E.M. of three experiments in triplicate. *P < 0.05,
**P < 0.01 and #P < 0.001 compared to control groups.
The hydroxychalcones 1 and 3 may be considered promising
molecules, based on the fact that mitochondrial GSH and ATP
depletion cause mitochondrial dysfunction and the rapid growth of
tumor cells is highly energy dependent.
In summary, 13 hydroxychalcones were synthesized, and 1, 3
and 13 exhibited effective cytotoxicity against melanoma B16-F10
Compounds 1 and 3 were selected to continue the research. Further
studies are being developed by our group to elucidate the mechanism
of action. The analyses of the molecule structures suggest that the
predominant factor for activity is the molecule shape, and secondarily,
the number of hydroxyl groups in the chalcone.
cells. Compound 1 was the most active, with an IC50 value of 12 mM.
Compounds 1 and 3 induced cell apoptosis, suggested to occur
through mitochondrial GSH and ATP depletion. Compound 13 did
not induce cell death through apoptosis nor did it induce ATP
depletion. The main similarity between chalcones 1 and 3 is the
presence of a unit of 1-naphtyl at B-ring. The higher activity of
compound 1 seems to be due to the free hydroxyl group at A-ring.
This is an interesting compound since it occurs naturally on the
chalcones in this same position [34].
4. Experimental
4.1. Preparation of the compounds
The compounds were prepared by aldolic condensation. All
reagents used were obtained commercially (Merck, Sigma–Aldrich),

1634
A.L.F. Navarini et al. / European Journal of Medicinal Chemistry 44 (2009) 1630–1637
and dichloromethane. 1H NMR spectra revealed that all the struc-
tures were geometrically pure and configured E (JH
¼ 15.2–
a
-Hb
16,4 Hz).
4.3. Physico-chemical data of synthesized compounds
4.3.1. (2E)-1-(30-Hydroxyphenyl)-3-(1-naphtyl)-2-propen-1-one
Yellow solid, m.p. 174–175. 1H NMR (CDCl3)
d 7.10 (d, 1H,
J ¼ 8.0 Hz, H40), 7.40 (dd, 1H, H50), 7.51–7.65 (m, 5H, H3, H4, H6, H7,
H20), 7.60 (d, 1H, J ¼ 15.6 Hz, H
a), 7.90 (d, 2H, H5, H8), 7.94 (d, 1H,
J ¼ 8.4 Hz, H60), 8.26 (d, 1H, J ¼ 8.0 Hz, H2), 8.67 (d, 1H, J ¼ 15.6 Hz,
Hb d
). 13C NMR (CDCl3) 115.07 (C20), 120.22 (C40), 120.32 (C60),
123.41 (C
a), 124.97 (C2), 125.56 (C3), 125.89 (C8), 126.52 (C6),
127.30 (C7), 129.07 (C4), 130.08 (C5), 130.93 (C50), 132.01 (C9),
132.34 (C10), 134.16 (C1), 139.91 (C10), 140.39 (C ), 158.06 (C30),
b
189.09 (C]O). IR nmax/cmꢂ1 3176, 1642, 1351, 1571, 1273, 1475, 1172,
962, 772, 707 (KBr). Anal. Calcd for C19H14O2: C 83.19, H 5.14. Found:
C 83.42, H 5.21. Yield: 51%.
4.3.2. (2E)-1-(20-Hydroxy-40,60-dimethoxyphenyl)-3-(3-
nitrophenyl)-2-propen-1-one
Orange solid, m.p. 171–172 ꢃC. 1H NMR (CDCl3)
d
3.85 (s, 3H,
OCH3), 3.94 (s, 3H, OCH3), 5.98 (s, 1H, H50), 6.12 (s, 1H, H30), 7.74 (d,
1H, J ¼ 15.6 Hz, H ), 7.84–7.88 (m, 2H, H4, H5), 7.98 (d, 1H,
J ¼ 15.6 Hz, H ), 8.22 (d, 1H, J ¼ 7.75 Hz, H6), 8.46 (s, 1H, H2), 14.09
(s, 1H, OH). 13C NMR (CDCl3)
55.56 (OCH3), 55.82 (OCH3), 91.37
(C50), 93.84 (C30), 106.20 (C10), 122.08 (C2), 123.99 (C4), 129.78 (C
),
), 148.71 (C3),162.45
a
Fig. 6. Effect of hydroxychalcones 1, 3 and 13 on mitochondrial GSH content. The cells
b
(3 ꢁ 105) were incubated with the compounds (0–100
mM) for 24 h. Mitochondrial
d
glutathione (GSH þ GSSG) was measured using the glutathione reductase method, as
described in Section 4. Each bar represents the mean ꢀ S.E.M. of three experiments in
triplicate. *P < 0.05, **P < 0.01 and #P < 0.001 compared to control groups.
a
130.51 (C5), 134.01 (C6),137.43 (C1), 138.68 (C
b
(C20), 166.45 (C60), 168.46 (C40), 191.78 (C]O). IR nmax/cmꢂ1 1640,
4580 (KBr). Anal. Calcd for C17H15NO6: C 62.00, H 4.59, N 4.59.
Found: C 62.08, H 4.26. Yield: 51%.
except the xanthoxyline (2-hydroxy-4,6-dimethoxyacetofenone)
and the monobromide xanthoxyline (2-hydroxy-3-bromo-4,6-
dimethoxychalcone), that were prepared as previously described
[35]. All chalcones were prepared by magnetic agitation of hydrox-
ylated acetophenone (2 mmol), methanol (30 mL), KOH 50% w/v
(5 mL) and the corresponding aldehyde (2 mmol), at room temper-
ature for 24 h. Distilled water and chloric acid 10% were added in the
reaction for total precipitation of the compounds. The compounds
were then obtained by vacuum filtration and later recrystallized in
dichloromethane and hexane. The chalcones 1, 3, 4, 7 and 12 were
previously cited, respectively, by Hollinshead [36], Subbanwad and
Vibhute [37], Liu et al. [16], Hsu et al. [38] and by Bora et al. [39]. The
compounds 2 and 11 were described by Boeck and co-workers [17]
and the structures 5, 6, 8–10 and 13 are new chalcones.
4.3.3. (2E)-1-(20-Hydroxyphenyl)-3-(1-naphtyl)-2-propen-1-one
Yellow solid, m.p. 106–108 ꢃC. 1H NMR (CDCl3)
d 6.97 (dd, 1H,
J ¼ 7.4 Hz, H50), 7.06 (d, 1H, J ¼ 8.4 Hz, H30), 7.51–7.62 (m, 4H, H3,
H6, H7, H40), 7.76 (d, 1H, J ¼ 15.2 Hz, H
a
), 7.90–7.99 (m, 4H, H60, H4,
H5, H8), 8.29 (d, 1H, J ¼ 8.4 Hz, H2), 8.79 (d, 1H, J ¼ 15.2 Hz, H
b
),
118.93 (C30),119.17 (C50),122.99
), 123.67 (C2), 125.59 (C8), 125.68 (C10), 126.67 (C3), 127.41 (C6),
129.07 (C4, C5), 130.00 (C9, C60), 131.48 (C10), 136.74 (C1, C40),
142.66 (C
), 163.93 (C20), 194.12 (C]O). IR nmax/cmꢂ1 3451, 1635,
12.88 (s, 1H, OH). 13C NMR (CDCl3)
(C
d
a
b
1351, 1576, 1203, 1015, 3047, 1435, 1162, 972, 760 (KBr). Anal. Calcd
for C19H14O2: C 83.19, H 5.14. Found: C 83.88, H 5.18. Yield: 57%.
4.3.4. (2E)-1-(20-Hydroxyphenyl)-3-(2-naphtyl)-2-propen-1-one
Yellow solid, m.p.: 135–137 ꢃC (lit. p.f.: 146–148 ꢃC)132. 1H NMR
4.2. Physico-chemical data of the compounds
(CDCl3)
d
6.96 (d, 1H, J ¼ 8.0 Hz, H30), 7.03–7.10 (m, 1H, H50), 7.48–
7.54 (m, 3H, H3, H4, H40), 7.75 (d, 1H, J ¼ 16.0 Hz, H
a), 7.78–7.89 (m,
The purified chalcones were obtained in yields between 36% and
66%. The structures were identified using melting points (m.p.),
infrared spectroscopy (IR), 1H and 13C nuclear magnetic resonance
spectroscopy (NMR), and to the unpublished ones, also elementary
analyses. Melting points were determined with a Microquı´mica
MGAPF-301 apparatus and are uncorrected. IR spectra were
recorded with an Abb Bomen FTLA 2000 spectrometer on KBr disks.
NMR (1H and 13C NMR) were recorded on Varian Oxford AS-400
(400 MHz), using tetramethylsilane as an internal standard.
5H, H5, H6, H7, H8, H60), 7.93 (d, 1H, J ¼ 16.0 Hz, H
b), 8.04 (s, 1H,
H1), 12.89 (s, 1H, OH). 13C NMR (CDCl3)
d
118.89 (C30), 120.41 (C50),
121.93 (C3), 123.89 (C
127.33 (C1), 128.40 (C5), 129.08 (C8), 129.94 (C4), 131.39 (C60),
a
), 125.65 (C10), 126.80 (C6), 127.13 (C7),
132.31 (C10), 133.57 (C9), 134.78 (C2), 136.65 (C40), 145.79 (C
b),
163.86 (C20), 193.91 (C]O). IR nmax/cmꢂ1 3195 (OH), 1689 (C]O),
1568 (C]C), 3046, 1482, 1432, 1021, 985, 819, 752 (Ar) (KBr). Anal.
Calcd for C19H14O2: C 83.19, H 5.14. Found: C 82.04, H 5.18. Yield:
66%.
Elementary analyses were obtained with
a CHNS EA 1110.
Percentages of C and H were in agreement with the product
formula (within ꢀ0.4% of theoretical values to C). The purity of the
synthesized chalcones was analyzed by thin-layer chromatography
(TLC) using Merck silica pre-coated aluminum plates 200
thickness with several solvent systems of different polarities.
Compounds were visualized with ultraviolet light (
4.3.5. (2E)-1-(30-Methoxy-40-hydroxyphenyl)-3-(2-naphtyl)-2-
propen-1-one
Light yellow solid, m.p.: 166–168 ꢃC. 1H NMR (CDCl3)
d 4.01 (s,
mm in
3H, OCH3), 6.10 (s, 1H, OH), 7.02 (d, 1H, J ¼ 8.0 Hz, H50), 7.29 (s, 1H,
H20), 7.52–7.54 (m, 1H, H60), 7.66–7.72 (m, 3H, H3, H6, H7), 7.83 (d,
l
¼ 254 and
1H, J ¼ 15.6 Hz, H
J ¼ 15.6 Hz, H
), 8.05 (s, 1H, H1). 13C NMR (DMSO-d6)
(m-OCH3), 110.74 (C20), 114.05 (C50), 121.99 (C3), 123.96 (C
a
), 7.80–7.87 (m, 3H, H4, H5, H8), 7.97 (d, 1H,
56.37
), 126.97
360 nm) and using sulfuric anisaldehyde solution followed by heat
as developing agent and purified by recrystallization from hexane
b
d
a

A.L.F. Navarini et al. / European Journal of Medicinal Chemistry 44 (2009) 1630–1637
1635
(C60), 127.51 (C6), 128.03 (C7), 128.85 (C1), 128.91 (C8), 130.68 (C4,
H2, H6), 7.74 (d, 1H, J ¼ 16.0 Hz, H
a
), 7.93 (d, 1H, J ¼ 160 Hz, H
b),
C5), 131.33 (C10), 132.81 (C10), 133.63 (C9), 134.54 (C2), 144.30 (C
b
),
8.24 (d, 2H, J ¼ 8.0 Hz, H3, H5), 14.73 (s, 1H, OH). 13C NMR (CDCl3)
147.15 (C40), 150.64 (C30), 188.74 (C]O). IR nmax/cmꢂ1 3265 (OH),
1643, 1202 (C]O), 1280, 1025 (C–O), 1563 (C]C), 2950, 2835, 1522,
1445, 970, 844, 816, 779 (Ar) (KBr). Anal. Calcd for C20H16O3: C
78.93, H 5.30. Found: C 78.86, H 5.76. Yield: 39%.
d
55.74 (OCH3), 56.32 (OCH3), 86.69 (C50), 91.63 (C30), 106.55 (C10),
109.77 (C3, C5), 123.78 (C
(C
a), 126.66 (C2, C6), 128.82 (C1), 131.03
b
), 150.47 (C4), 161.92 (C60), 162.62 (C20), 162.68 (C40), 203.27
(C]O). IR nmax/cmꢂ1 3450, 2943, 1600, 1509, 1345, 1093, 976, 852
(KBr). Anal. Calcd for C17H14BrNO6: C 50.02, H 3.46, N 3.43. Found: C
49.57, H 3.02, N 4.53. Yield: 42%.
4.3.6. (2E)-1-(20-Hydroxy-40,60-dimethoxyphenyl)-3-(1-naphtyl)-
2-propen-1-one
Yellow solid, m.p. 116–117 ꢃC. 1H NMR (CDCl3)
d
3.85 (s, 3H,
4.3.11. (2E)-1-(20-Hydroxy-30-bromo-40,60-dimethoxyphenyl)-3-(2-
chlorophenyl)-2-propen-1-one
OCH3), 3.92 (s, 3H, OCH3), 5.98 (s, 1H, H30), 6.14 (s, 1H, H50), 7.50–
7.61 (m, 3H, H3, H6, H7), 7.83–7.92 (m, 3H, H4, H5, H8), 7.97 (d, 1H,
Light yellow solid, m.p. 210–212 ꢃC. 1H NMR (CDCl3)
d
3.98 (s,
3H, OCH3), 4.04 (s, 3H, OCH3), 6.21 (s,1H, H50), 7.29–7.32 (m, 2H, H4,
H5), 7.43 (d, 1H, H3), 7.67 (d, 1H, H6), 7.83 (d, 1H, J ¼ 15.2 Hz, H ),
8.18 (d, 1H, J ¼ 15.2 Hz, H
), 14.67 (s, 1H, OH). 13C NMR (acetone-d6)
56.85 (OCH3), 57.08 (OCH3), 87.80 (C50), 92.80 (C30), 106.00 (C10),
127.72 (C ), 128.60 (C5), 130.22 (C6), 131.06 (C3), 131.62 (C4), 134.33
(C2),136.21 (C1),139.49 (C
J ¼ 15.2 Hz, H
(OH). 13C NMR (CDCl3)
94.03 (C30), 101.63 (C10), 124.00 (C
126.46 (C3), 127.03 (C6), 128.93 (C7), 130.46 (C4), 130.55 (C5),
a
), 8.31 (d, 1H, H2), 8.61 (d, 1H, J ¼ 15.2 Hz, H
55.87 (OCH3), 56.15 (OCH3), 91.54 (C50),
), 125.39 (C2), 125.71 (C8),
b), 12.83
d
a
a
b
d
132.02 (C9), 133.32 (C10), 133.99 (C1), 139.49 (C
b
), 162.79 (C20),
a
166.55 (C60), 168.68 (C40), 192.79 (C]O). IR nmax/cmꢂ1 3450, 1627,
1341, 1571, 1215, 1109, 1440, 1153, 975, 810, 768 (KBr). Anal. Calcd
for C21H18O4: C 75.43, H 5.43. Found: C 75.66, H 5.94. Yield: 58%.
b
),162.90 (C20),163.00 (C60),163.93 (C40),
193.25 (C]O). IR nmax/cmꢂ1 3450, 2934, 1618, 1557, 1216, 1125, 965,
788 (KBr). Anal. Calcd for C17H14BrClO4: C 51.35, H 3.55. Found: C
51.45, H 3.58. Yield: 62%.
4.3.7. (2E)-1-(20-Hydroxy-40,60-dimethoxyphenyl)-3-(3-
chlorophenyl)-2-propen-1-one
4.3.12. (2E)-1-(20-Hydroxy-30-bromo-40,60-dimethoxyphenyl)-3-(4-
Yellow solid, m.p.: 104–106 ꢃC. 1H NMR (CDCl3)
d
3.84 (s, 3H,
methoxyphenyl)-2-propen-1-one
OCH3), 3.93 (s, 3H, OCH3), 5.97 (s, 1H, H30), 6.11 (s, 1H, H50), 7.33–
Yellow solid, m.p. 144–146 ꢃC. 1H NMR (CDCl3)
d 3.86 (s, 3H,
7.35 (m, 3H, H4, H5, H6), 7.57 (s, 1H, H2), 7.68 (d, 1H, J ¼ 15.6 Hz,
OCH3), 3.97 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 6.07 (s, 1H, H50), 6.93
H
d
a
), 7.87 (d, 1H, J ¼ 15.6 Hz, H
b
), 13.95 (s, 1H, OH). 13C NMR (CDCl3)
(d, 2H, J ¼ 8.8 Hz, H3, H5), 7.56 (d, 2H, J ¼ 8.8 Hz, H2, H6), 7.79 (d,
55.89 (OCH3), 56.19 (OCH3), 91.58 (C50), 94.02 (C30), 106.50 (C10),
), 128.09 (C6), 129.10 (C2), 130.07 (C4), 130.35 (C5),
), 162.73 (C20), 166.68 (C60),
1H, J ¼ 16.4 Hz, H
a
), 7.89 (d, 1H, J ¼ 16.4 Hz, H
b
), 14.81 (OH). 13C
126.88 (C
135.06 (C3), 137.68 (C1), 140.70 (C
a
NMR (acetone-d6)
d
55.17 (OCH3), 56.25 (OCH3), 56.37 (OCH3),
),
128.10 (C1), 130.68 (C2, C6), 143.59 (Cb
), 162.17 (C4), 162.92 (C20,
b
88.34 (C50), 94.76 (C30), 106.66 (C10), 114.70 (C3, C5), 124.61 (C
a
168.68 (C40), 192.51 (C]O). IR nmax/cmꢂ1 3451 (OH), 1624, 1215
(C]O), 1029 (C–O), 1576 (C]C), 2936, 1439, 977, 909, 822 (Ar)
(KBr). Anal. Calcd for C17H15ClO4: C 64.06, H 4.74. Found: C 64.61, H
5.39. Yield: 36%.
C60), 163.28 (C40), 205.72 (C]O). IR nmax/cmꢂ1 3443, 1615, 1557,
1220, 1177, 824 (KBr). Anal. Calcd for C18H17BrO5: C 54.98, H 4.36.
Found: C 51.95, H 4.46. Yield: 40%.
4.3.8. (2E)-1-(20-Hydroxy-30-bromo-40,60-dimethoxyphenyl)-3-
4.3.13. (2E)-1-(20-Hydroxy-30-bromo-40,60-dimethoxyphenyl)-3-(2-
(2,6-imethoxyphenyl)-2-propen-1-one
carboxyphenyl)-2-propen-1-one
Orange solid, m.p. 193–194 ꢃC. 1H NMR (CDCl3)
d
3.91 (s, 6H,
Yellow solid, m.p. 230 ꢃC. 1H NMR (acetone-d6)
d 3.79 (s, 3H,
OCH3), 3.96 (s, 6H, OCH3), 6.04 (s, 1H, H50), 6.57 (d, 2H, J ¼ 8.4 Hz,
OCH3), 3.85 (s, 3H, OCH3), 6.00 (s, 1H, H50), 7.64 (t, 1H, J ¼ 8.0 Hz,
H3, H5), 7.28 (t, 1H, H4), 8.29 (d, 1H, J ¼ 15.6 Hz, H
a
), 8.34 (d, 1H,
55.72
(2CH3), 56.06 (OCH3), 56.32 (OCH3), 88.35 (C50), 94.76 (C30), 104.21
(C10, C3, C5),112.81 (C1),129.11 (C
),132.35 (C4),134.85 (C ),160.81
H4), 7.64 (d, 1H, J ¼ 16.0 Hz, H ), 7.79 (d, 1H, J ¼ 8.0 Hz, H6), 7.87 (t,
a
J ¼ 15.6 Hz, H
b
), 14.65 (s, 1H, OH). 13C NMR (acetone-d6)
d
1H, J ¼ 8.0 Hz, H5), 7.87 (d, 1H, J ¼ 16.0 Hz, H
b), 7.92 (d, 1H,
J ¼ 8.0 Hz, H3), 14.23 (COOH), 14.57 (OH). 13C NMR (acetone-d6)
a
b
d
57.25 (OCH3), 57.74 (OCH3), 88.12 (C50), 94.78 (C30), 106.14 (C10),
(C2, C6), 162.35 (C60), 162.91 (C20), 166.31 (C40), 205.56 (C]O). IR
nmax/cmꢂ1 3442, 2941, 1616, 1552, 1325, 1253, 1193, 916, 782 (KBr).
Anal. Calcd for C19H19BrO6: C 53.92, H 4.52. Found: C 53.67, H 4.44.
Yield: 42%.
124.40 (C
134.88 (C5), 135.66 (C1), 136.76 (C
a), 126.49 (C6), 130.09 (C4), 130.73 (C2), 132.61 (C3),
b
), 156.72 (C60), 165.62 (C20),
166.90 (C40), 180.67 (COOH), 206.97 (C]O). IR nmax/cmꢂ1 3441,
2942, 2640, 1627, 1559, 1216, 1125, 964, 781 (KBr). Anal. Calcd for
C18H15BrO6: C 53.09, H 3.71. Found: C 49.08, H 3.71. Yield: 59%.
4.3.9. (2E)-1-(20-Hydroxy-30-bromo-40,60-dimethoxyphenyl)-3-(4-
buthoxyphenyl)-2-propen-1-one
4.4. Biological assays
Yellowsolid, m.p.172–173 ꢃC.1H NMR (CDCl3)
d 0.98 (t, 3H, –CH3),
1.47–1.60 (m, 2H, –CH2CH3),1.75–1.81 (m, 2H, –CH2CH2CH3), 3.98 (s,
3H, OCH3), 3.99 (s, 3H, OCH3), 4.01 (t, 2H, –OCH2–), 6.06 (s, 1H, H50),
6.91 (d, 2H, J ¼ 8.4 Hz, H3, H5), 7.54 (d, 2H, J ¼ 8.4 Hz, H2, H6), 7.75 (d,
4.4.1. Reagents
Tissue culture media and fetal bovine serum were purchased
from CULTILAB (Sa˜o Paulo, Brazil), penicillin/streptomycin were
purchased from GIBCO (Grand Island, NY, USA), isopropanol was
purchased from Merck (Darmstadt, Germany), 3-(4,5-dimethylth-
iazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were
purchased from Sigma (St. Louis, MO, USA). The luciferin–luciferase
kit was purchased from Bio-OrbitÒ (Turku, Finland).
1H, J ¼ 15.6 Hz, H
(acetone-d6)
56.25 (OCH3), 67.86 (–OCH2–), 88.37 (C50), 94.61 (C30), 104.43 (C10),
a
), 7.83 (d,1H, J ¼ 15.6 Hz, H
b
),14.96 (OH).13C NMR
d
13.42 (CH3), 19.18 (–CH2CH3), 31.28 (–CH2CH2CH3),
115.19 (C3, C5),124.46 (Ca), 128.04 (C1),130.71 (C2, C6),143.72 (Cb),
160.02 (C4), 168.46 (C20), 168.82 (C60), 176.12 (C40), 198.07 (C]O). IR
nmax/cmꢂ1 3450, 2945, 1615, 1554, 1221, 963 (KBr). Anal. Calcd for
C21H23BrO5: C 57.94, H 5.33. Found: C 57.27, H 5.27. Yield: 47%.
4.4.2. Cell culture
4.3.10. (2E)-1-(20-Hydroxy-30-bromo-40,60-dimethoxyphenyl)-3-(4-
Murine B16-F10 melanoma cell lines were obtained from
American Type Culture Cell (ATCC). The cells were cultured in
DMEM supplemented with 10% heat-inactivated fetal bovine
nitrophenyl)-2-propen-1-one
Yellow solid, m.p. 230–231 ꢃC. 1H NMR (CDCl3)
d 3.94 (s, 3H,
OCH3), 3.97 (s, 3H, OCH3), 6.03 (s, 1H, H50), 7.61 (d, 2H, J ¼ 8.0 Hz,
serum, 100 U/mL penicillin, 100 mg/mL streptomycin and 10 mM

1636
A.L.F. Navarini et al. / European Journal of Medicinal Chemistry 44 (2009) 1630–1637
HEPES. The cell cultures were maintained at 37 ꢃC in a 5% CO2
humidified atmosphere and pH 7.4. Once the cells reached 90% of
confluence, a cell suspension was obtained by trypsinization. In all
experiments, viable cells were checked in the beginning of the
experiment by Trypan Blue exclusion.
(Organon Teknika). Values were expressed as percentage of control
GSH values.
4.4.7. Statistical analysis
The statistical program GraphPad PrismÒ 4.0 Windows
(GraphPad software, Inc., San Diego, CA, 2003) [42] was used to
calculate the IC50 and the statistical significance. The results were
presented as mean ꢀ S.E.M. of triplicates from three independent
experiments. Statistical significance was assessed by ANOVA fol-
lowed by Bonferroni’s t-test, p values <0.05 were taken as statis-
tically significant.
4.4.3. Cytotoxicity
The cytotoxic effect of synthetic hydroxychalcones was evalu-
ated using an MTT assay. Briefly, 1 ꢁ104 cells/well were incubated
for 72 h at 37 ꢃC, in triplicate, with compounds dissolved in DMSO
(0.1% final concentration) and diluted with culture medium to their
final concentrations, in 96-well microplates. After the incubation
time, the cells were washed with new culture medium and 10
MTT (5 mg/mL) were added followed by 3 h of incubation at 37 ꢃC.
The precipitated formazan was dissolved in 100 L of acid iso-
mL
Acknowledgments
m
This research was supported by grants and fellowships from
propanol solution (isopropanol containing HCl 0.04 M) and the
absorbance was measured at 540 nm using microwell system
reader (Organon Teknika, Belgium). The cells were incubated with
´
CNPq (Conselho Nacional de Desenvolvimento Cientıfico e Tecno-
´
˜
´
logico), CAPES (Coordenaçao de Pessoal de Nıvel Superior) and
FAPESC (Fundaça˜o de Amparo a Ciencia e Tecnologia de Santa
Catarina). This work is part of the thesis of Andreia L.F. Navarini and
`
ˆ
the hydroxychalcones in
a concentration range of 0–40 mM,
´
following with MTT assay. The IC50 values (a concentration that
produces 50% reduction of viable cell number) were calculated
from the concentration–response curves. The cell viability was
checked in the beginning of experiment by Trypan Blue exclusion.
Alessandra Mascarello, who are M.Sc. students in Pharmacy and
Chemistry, respectively. We would like to thank the Department of
Chemistry and the Hospital of the Universidade Federal de Santa
Catarina for some facilities.
4.4.4. DNA fragmentation analysis by gel electrophoresis
References
Briefly, 5 ꢁ105 cells/well were incubated for 24 h with
compounds dissolved in DMSO (0.1% final concentration) and
diluted with culture medium to final concentrations (the IC50 for
cell viability of each compound). To isolate DNA fragments, the cells
were harvested and washed twice with cold phosphate-buffered
saline (PBS). Cell pellets were then incubated in a lyses buffer
containing EDTA 10 mM, Tris–HCl 50 mM, at pH 8.0, NP-40 – 0.25%,
proteinase K 0.5 g/L at 50 ꢃC for 2 h. The DNA was then precipitated
with 2.5 vol of ethanol–NaCl at ꢂ25 ꢃC overnight and dried in air.
After washing with ice-cold 70% ethanol, the pellets were then
dissolved in TE buffer containing Tris-HCl 10 mM, at pH 8.0, EDTA
1 mM and RNase A 0.6 mg/mL and incubated at 37 ꢃC for 1 h. DNA
solutions were then separated in agarose gels 1.5%, stained with
ethidium bromide and visualized by 2UV Transilluminator (Mac-
roVue UV-20 Hoefer) for ladder formation.
[1] WHO. World Health Organization, Available at: <www.who.int/cancer>,
Accessed in 30 March 2008.
[2] L.Y. Xu, J. Koo, Int. J. Dermatol. 45 (2006) 1275–1283.
[3] I.J. Fidler, Cancer Res. 38 (1978) 2651–2660;
I.J. Fidler, Cancer Res. 35 (1975) 218–224.
[4] Z.F.O. Freitas, E.G. Rodrigues, V. Oliveira, A.K. Carmona, L.R. Travassos, Mela-
noma Res. 14 (2004) 333–344.
[5] M.F. Avril, S. Aamdal, J.J. Grob, A. Hauschild, P. Mohr, J.J. Bonerandi,
M. Weichenthal, K. Neuber, T. Bieber, K. Gilde, V.G. Porta, J. Fra, J. Bonneterre,
P. Sa¨ıag, D. Kamanabrou, H. Pehamberger, J. Sufliarsky, J.L.G. Larriba,
A. Scherrer, Y. Menu, J. Clin. Oncol. 22 (2004) 1118–1125.
[6] L.M. Ni, C.Q. Meng, J.A. Sikorski, Expert Opin. Ther. Pat. 14 (2004) 1669–1691.
[7] M. Kobori, K. Iwashita, H. Shinmoto, T. Tsushida, Biosci. Biotechnol. Biochem.
63 (1999) 719–725.
[8] O. Sabzevari, G. Galati, M.Y. Moridani, A. Siraki, P.J.O. Brien, Chem. Biol.
Interact. 148 (2004) 57–67.
[9] K. Iwashita, M. Kobori, K. Yamaki, T. Tsushida, Biosci. Biotechnol. Biochem. 64
(2000) 1813–1820.
[10] M. Cabrera, M. Simoens, G. Falchi, M.L. Lavaggi, O.E. Piro, E.E. Castellano,
A. Vidal, A. Azqueta, A. Monge, A.L. Cera´in, G. Sagrera, G. Seoane, H. Cerecetto,
M. Gonza´lez, Bioorg. Med. Chem. 15 (2007) 3356–3367.
[11] Y.L. Hsu, P.L. Kuo, C.C. Lin, Life Sci. 77 (2005) 279–292.
[12] Y.C. Liu, C.W. Hsieh, C.C. Wu, B.S. Wung, Life Sci. 80 (2007) 1420–1430.
[13] Y.M. Lin, Y. Zhou, M.T. Flavin, L.M. Zhou, W. Nie, F.C. Chen, Bioorg. Med. Chem.
10 (2002) 2795–2802.
[14] K. Yamamoto, H. Kakegawa, H. Ueda, H. Matsumoto, T. Sudo, T. Miki, T. Satoh,
Planta Med. 58 (1992) 389–393.
[15] C. Zhan, J. Yang, Pharmacol. Res. 53 (2006) 303–309.
[16] M. Liu, P. Wilairat, S.L. Croft, A.L. Tan, M.L. Go, Bioorg. Med. Chem. 11 (2003)
2729–2738.
[17] P. Boeck, C.A.B. Falca˜o, P.C. Leal, R.A. Yunes, V. Cechinel, E.C. Torres-Santos,
B. Rossi-Bergamann, Bioorg. Med. Chem. 14 (2006) 1538–1545.
[18] S.W. Lowe, H.E. Ruley, T. Jacks, D.E. Housman, Cell 74 (1993) 957–967.
[19] I.M. Ghobrial, T.E. Witzig, A.A. Adjei, CA Cancer J. Clin. 55 (2005) 178–194.
[20] N. Dias, C. Bailly, Biochem. Pharmacol. 70 (2005) 1–12.
[21] V.P. Skulachev, Apoptosis 11 (2006) 473–485.
4.4.5. Bioluminescent assay for ATP
The amount of intracellular ATP was determined by biolumi-
nescent assay measuring the light output of the luciferin–luciferase
reaction. Approximately 3 ꢁ 105 cells were incubated in the pres-
ence of hydroxychalcones at different concentrations then pelleted
and extracted with 40
mL of trichloroacetic acid 1.25%. The cell
extracts were kept on ice for 30 min and neutralized with 20
mL of
Tris-acetate 1 M, at pH 7.5. After centrifugation, the supernatants
were diluted with the same buffer (ꢁ4) and used for ATP
quantification.
4.4.6. Mitochondrial glutathione measurement
Mitochondria enriched preparation was obtained by centrifu-
gation [40]. Total glutathione (GSH þ GSSG, reduced and disulphide
forms, respectively) was measured through the glutathione
reductase method [41]. Approximately 3 ꢁ 105 cells were washed in
PBS and homogenized in a buffer containing Tris-sacarose 10 mM/
[22] J.M. Garland, A. Halestrap, J. Biol. Chem. 272 (1997) 4680–4688.
[23] L.H. Lash, Chem. Biol. Interact. 163 (2006) 54–67.
´
´
[24] J. Kluza, A. Lansiaux, N. Wattez, M.P. Hildebrand, S. Leonce, A. Pierre,
J.A. Hickman, C. Bailly, Biochem. Pharmacol. 63 (2002) 1443–1452.
[25] T. Ozben, FEBS Lett. 580 (2006) 2903–2909.
[26] P. Cos, M. Calomme, J.B. Sindambiwe, T. De Bruyne, K. Cimanga, L. Pieters,
A.J. Vlietinck, D. Vanden-Berghe, Planta Med. 67 (2001) 515–519.
[27] P. Cos, P. Rajan, I. Vedernikova, M. Calomme, L. Pieters, A.J. Vlietinck,
K. Augustyns, A. Haemers, D. Vanden Berghe, Free Radic. Res. 36 (2002) 711–716.
[28] A.H. Wyllie, J.F. Kerr, A.R. Currie, Int. Rev. Cytol. 68 (1980) 251–306.
[29] J. Cao, Y. Liu, L. Jia, H.M. Zhou, Y. Kong, G. Yang, L.P. Jiang, Q.J. Li, L.F. Zhong, Free
Radic. Biol. Med. 43 (2007) 968–975.
[30] W.P. Hu, H.S. Yu, P.J. Sung, F.Y. Tsai, Y.K. Shen, L.S. Chang, J.J. Wang, Chem. Res.
Toxicol. 20 (2007) 905–912.
[31] K.M. Regula, K. Ens, L.A. Kirshenbaum, J. Mol. Cell. Cardiol. 35 (2003) 559–567.
[32] R. Kachadouriam, B.J. Day, Free Radic. Biol. Med. 41 (2006) 65–76.
250 mM, resuspended in 100
EDTA 1 mM and sonified for 10 s. Then, 20
were transferred to a 96-well plate followed by addition of 180
of the reaction medium containing DTNB 75 M; NADPH 120 mM,
mL chilled Milli-Q water containing
mL of each homogenate
mL
m
glutathione reductase 1 U/mL and EDTA 10 mM in phosphate buffer
100 mM, at pH 7.4. A standard curve was made with GSH (0.001–
1
mM). The absorbance was measured immediately and then every
minute during 5 min at 412 nm using Microwell Systems
a

A.L.F. Navarini et al. / European Journal of Medicinal Chemistry 44 (2009) 1630–1637
1637
[33] C. Garcia-Ruiz, J.C. Fe´rnandez-Checa, Gastroenterol. Hepatol. 3 (2006) 3–6.
[34] K.B.G. Torssell (Ed.), Natural Product Chemistry, Swedish Pharmaceutical
Press, Stockholm, 1997.
[38] K.K. Hsu, M. Hung, F.C. Chen, Taiwan Kexue 13 (1959) 92–94.
[39] U. Bora, G. Bose, M.K. Chaudhuri, S.S. Dhar, R. Gopinath, A.T. Khan, B.K. Patel,
Org. Lett. 2 (2000) 247–249.
[35] V.F. Cechinel, Z.R. Vaz, L. Zunino, J.B. Calixto, R.A. Yunes, Eur. J. Med. Chem. 31
(1996) 833–839.
[36] S.P. Hollinshead, Tetrahedron Lett. 51 (1996) 9157–9160.
[37] G.R. Subbanwad, Y.B. Vibhute, Asian J. Chem. 5 (1993) 352–357.
[40] J.B. Schenkman, D.L. Cinti, Methods Enzymol. 52 (1978) 83–89.
[41] F. Tietze, Anal. Biochem. 27 (1969) 502–522.
[42] H.J. Motulsky, A. Christopoulos (Eds.), GraphPad Software Inc., 2003, pp.
257–275.