Amyrin esters induce cell death by apoptosis in HL-60 leukemia cells

Article abstract of DOI:10.1016/j.bmc.2010.12.016

Four derivatives of an α,β-amyrin mixture were synthesized by acylation with appropriate anhydrides. The structures of the compounds were confirmed by means of IR and ¹H and ¹³C NMR. The compounds were screened for cytotoxic activity using four human tumor cell lines (HL-60, MDAMB-435, SF-295 and HCT-8) and normal peripheral blood mononuclear cells (PBMC). 3-O-Carboxymaleinate of α,β-amyrin (3a/3b) were found to be the only active compounds of the series (high cytotoxicity), showing IC ₅₀ values ranging from 1.8 to 3 μM. In PBMC, 3a/3b were not toxic, suggesting selectivity for tumor cells. To better understand the mechanism of action involved in the cytotoxicity of 3a/3b, HL-60 cells treated with 3a/3b were examined for morphological changes, DNA fragmentation, cell cycle perturbation, externalization of phosphatidylserine and activation of caspases 3/7, with doxorubicin serving as the positive control. The results indicate that the cytotoxicity of 3a/3b involves the induction of cell death by apoptosis.

Full text of DOI:10.1016/j.bmc.2010.12.016

Bioorganic & Medicinal Chemistry 19 (2011) 1268–1276
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Amyrin esters induce cell death by apoptosis in HL-60 leukemia cells
Francisco W. A. Barros a, Paulo N. Bandeira b, Daisy J. B. Lima a, Assuero S. Meira a, Silvana S. de Farias b,
Maria Rose J. R. Albuquerque b, Hélcio S. dos Santos b, Telma L. G. Lemos c, Manoel Odorico de Morais a,
Letícia Veras Costa-Lotufo a, Claudia do Ó Pessoa a,
⇑
a Departament of Physiology and Pharmacology, Federal University of Ceará, Fortaleza, Ceará 60430-270, Brazil
b Laboratory of Organic Chemistry, State University Vale do Acarau, Sobral, Ceará 62040-370, Brazil
c Departament of Organic and Inorganic Chemistry, Ceara Federal University, Fortaleza, Ceará 60431-970, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Four derivatives of an
a,b-amyrin mixture were synthesized by acylation with appropriate anhydrides.
The structures of the compounds were confirmed by means of IR and 1H and 13C NMR. The compounds
were screened for cytotoxic activity using four human tumor cell lines (HL-60, MDAMB-435, SF-295 and
Received 13 September 2010
Revised 29 November 2010
Accepted 4 December 2010
Available online 9 December 2010
HCT-8) and normal peripheral blood mononuclear cells (PBMC). 3-O-Carboxymaleinate of
(3a/3b) were found to be the only active compounds of the series (high cytotoxicity), showing IC50 values
ranging from 1.8 to 3 M. In PBMC, 3a/3b were not toxic, suggesting selectivity for tumor cells. To better
a,b-amyrin
l
Keywords:
Protium heptaphyllum
Derivatives of a,b-amyrin
Cytotoxic activity
understand the mechanism of action involved in the cytotoxicity of 3a/3b, HL-60 cells treated with 3a/3b
were examined for morphological changes, DNA fragmentation, cell cycle perturbation, externalization of
phosphatidylserine and activation of caspases 3/7, with doxorubicin serving as the positive control. The
results indicate that the cytotoxicity of 3a/3b involves the induction of cell death by apoptosis.
Ó 2010 Elsevier Ltd. All rights reserved.
Apoptosis
1. Introduction
triterpenes a,b-amyrin have weak anticancer activity, many other
triterpene compounds, in contrast, have shown strong cytotoxic
effects against various human tumor cells.11 Investigations of the
cytotoxic effects of these compounds have been conducted, and
studies show that this effect is related to the induction of
apoptosis.11,16,17
Triterpenoids are compounds that have attracted much interest
because of their structural diversity and the discovery of a broad
spectrum of pharmacological activities, such as anti-inflammatory,
gastroprotective, antimicrobial, antinociceptive, antioxidant, anal-
gesic, anticancinorgenic, cytotoxic and antidepressive effects.1–11
In the last ten years, a large number of synthetic derivatives of nat-
ural triterpenoids have contributed significantly to the pharmaco-
logical study of these compounds.12,13
Taking into account that small changes in a molecule can signif-
icantly alter its pharmacological activity,18 the objective of this
work was the synthesis of new derivatives of
a,b-amyrin, specifi-
cally esters modified at C-3 of the A ring, and evaluation of their
cytotoxic activities against four human cancer cell lines and mono-
nuclear cells from human peripheral blood (PBMC), used as a mod-
el of normal cells
In this respect, the pentacyclic triterpenes a,b-amyrin, naturally
occurring in many species, especially in the resin of species of
Protium, possess anti-inflammatory, gastoprotective, antinociceptive,
antidepressant, antiplatelet and analgesic activities.6,9,10,14 Studies on
Protium heptaphyllum resin showed that it also has anti-inflamma-
tory, gastroprotective, anti-allergic and antinociceptive properties.
These and other biological effects have been attributed to the pres-
2. Results and discussion
ence of pentacyclic triterpenes, especially
The triterpenoid mixture known as ,b-amyrin has a basic skel-
a
,b-amyrin.14,15
2.1. Chemistry
a
In order, to obtain derivatives 1a/1b, 2a/2b, 3a/3b and 4a/4b,
eton of the ursan and olean type, respectively. Whose main differ-
ence is in the position of the methyl group in the E ring located at
C-20. Its structural similarity with steroidal drugs may explain its
broad pharmacological activity.14 Although the pentacyclic
the
a,b-amyrin (a/b) mixture was isolated from the resin of
Protium heptaphyllum.19 The synthesis of the derivatives is outlined
in Figure 1.
2.1.1. Isolation and structural elucidation of the mixture of
⇑
Corresponding author at present address: Departmento de Fisiologia e Farmac-
a
- and b-amyrin (a/b)
The mixture of triterpenoid a,b-amyrin (a/b) was isolated from
ologia, Universidade Federal do Ceará, Rua Cel. Nunes de Melo, 1127, PO Box 3157,
60430-270 Fortaleza, Ceará, Brazil. Tel.: +55 85 3366 8341; fax: +55 85 3366 8333.
E-mail address: cpessoa@ufc.br (Claudia do Ó Pessoa).
the resin of Protium heptaphyllum19 and analyzed by 1H NMR,
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.016

F. W. A. Barros et al. / Bioorg. Med. Chem. 19 (2011) 1268–1276
1269
29
derivative and d = 5.21 ppm of the b-amyrin derivative. Signal for
30
R1
3
H-10 is at d = 2.69 ppm as doublet with J = 5.65 Hz and H-20 at
R2
20
17
d = 2.64 ppm as dublet with 3J = 5.85 Hz. In the 13C NMR, the signal
for C-3 is at d = 81.8 ppm, while C-12 appears at d = 124.5 ppm of
the a-amyrin derivative and d = 121.8 ppm of the b-amyrin deriv-
19
12
26
22
28
11
R1 = H, R2 = CH3 - α-amyrin
R1 = CH3, R2 = H - β-amyrin
25
1
9
14
27
16
ative, C-13 appears at d = 139.9 ppm of the
a-amyrin derivative
10
A
and d = 145.4 ppm of the b-amyrin derivative. Signal for H-10 is at
3
5
RO
3
d = 2.69 ppm as dublet with J = 5.65 Hz and H-20 at d = 2.64 ppm
as dublet with 3J = 5.85 Hz. The C@O carbon atoms appear at
d = 172.0 ppm for esters and d = 168.1 ppm for acids. MS (70 eV)
m/z (%) 526 (44, M+), 525.39 (100), 325.19 (12).
24
(a/b) R = H α,- β-amyrin
In the 1H NMR spectra of 3a/3b, d 0.80–2.10 ppm (characteristic
profile of the substrate). H-3 appears as a double–doublet at
O
C
O
(3a/3b) R = CO2HCH=CHC
d = 4.69 ppm and H-12 as a triplet at d = 5.14 ppm of the a-amyrin
(1a/1b) R =
derivative and d = 5.20 ppm of the b-amyrin derivative. H-10 appear
C
O
OH
O
3
as a doublet at d = 6.39 ppm with J = 12.8 Hz and H-20 as a
doublet at d = 6.46 ppm with 3J = 12.8 Hz, which agrees with a
trans-configuration. In the 13C NMR, the signal for C-3 is at
O
(4a/4b) R = CO2HCH2CH2CH2C
(2a/2b) R = CO2HCH2CH2C
d = d = 85.3 ppm, while C-12 appears at d = 124.3 ppm of the
rin derivative and d = 121.7 ppm of the b-amyrin derivative, C-13 at
d = 139.9 ppm of the -amyrin derivative and d = 145.5 ppm of the
a-amy-
Figure 1. Derivatives of ,b-amyrin: (1a/1b) phthalic anhydride, py, reflux (50 °C),
a
a
ethyl ether, 8 h. (2a/2b) succinic anhydride, DMAP, reflux (50 °C), CH2Cl2, 8 h.
(3a/3b) maleic anhydride, reflux (50 °C), CH2Cl2, 8 h. (4a/4b) glutaric anhydride,
DMAP, reflux (50 °C), CH2Cl2, 8 h.
b-amyrin derivative. The signal for C-10 is at d = 136.9 ppm and for
C-20 at d = 129.9 ppm. The C@O carbon atoms appear at
d = 164.6 ppm for esters and d = 167.9 for acids. MS (70 eV) m/z (%)
524 (37, M+), 523.37 (100).
enabling the determination of a 2:1 ratio, respectively. The struc-
ture elucidation was based on spectral data (IR, 1H NMR, 13C
NMR). The IR spectra showed mainly absorptions bands at
3307 cmꢀ1 assigned to hydroxyl group (OH) and 1460–
1383 cmꢀ1 characterisitics of C@C. In the 1H NMR spectra of a/b,
The IR spectra of 4a/4b showed the expected absorption band
for (C@O) of esters at 1727 cmꢀ1, and also showed an absorption
band at 3461 cmꢀ1 corresponding to (OH) of acids. In the 1H
NMR spectra of 4a/4b, d 0.80–2.10 ppm (characteristic profile of
the substrate). H-3 appears as a double–doublet at d = 4.52 ppm
d 0.80–2.10 ppm (characteristic profile of the
pears as a double–doublet at d = 3.22 ppm with 3J = 10.4 and 5.0 Hz
of the
-amyrin and as a double–doublet at d = 3.23 with 3J = 10.4
and 5.0 Hz of the b-amyrin. H-12 appears as triplet at
-amyrin and d = 5.10 ppm
a
,b-amyrin). H-3 ap-
and H-12 as a triplet at d = 5.13 ppm of the a-amyrin derivative
and d = 5.21 ppm of the b-amyrin derivative. The signals of H-10
and H-30 appear as a double–triplet at d = 2.44 and 2.41 ppm with
3J = 7.40 Hz and 7.45 Hz respective and H-20 as multiplet at
d = 1.98 ppm. In the 13C NMR, the signal for C-3 is at
a
a
d = 5.10 ppm with 3J = 3.6 Hz of the
a
with 3J = 3.6 Hz of the b-amyrin. In the 13C NMR, the signal for C-
3 is at d = 79.1 ppm, while C-12 appears at d = 124.4 ppm of the
d = 81.4 ppm, while C-12 appears at d = 124.5 of the
derivative and d = 121.8 ppm of the b-amyrin derivative, C-13 at
d = 139.8 ppm of the -amyrin derivative and d = 145.4 ppm de
a-amyrin
a-amyrin and d = 21.7 ppm of the b-amyrin and C-13 appears at
a
d = 139.6 ppm of the -amyrin and d = 145.2 ppm of the b-amyrin.
a
b-amyrin derivative. Signals for C-10, C-20 and C-30 are at d = 33.2,
20.2 and 33.87 ppm, respectively. The C@O carbon atoms appear
at d = 172.9 ppm for esters and d = 178.7 ppm for acids. MS
(70 eV) m/z (%) 540.41 (37, M+), 539.41 (100), 339.19 (12). Analysis
of DEPT - 135° nd 2D-heteronuclear NMR spectra (HSQC and
HMBC) provided the final structural elucidation of compounds
1a/1b, 2a/2b, 3a/3b and 4a/4b.
2.1.2. Structural elucidation of the a,b-amyrin derivatives
The IR spectra of the mixture 1a/1b showed one absorption
band at 1701 cmꢀ1 assigned to (C@O) of esters, typical absorption
band of (OH) of acids, and absorption bands between 1583 and
1404 cmꢀ1 of (C@C) of the aromatic ring.
In the 1H NMR spectra of 1a/1b, d 0.80–2.10 ppm (characteristic
profile of the substrate), H-3 appears as a double–doublet at
d = 4.77 ppm with 3J = 8.1 and 3.5 Hz and H-12 as a triplet at
2.2. Cytotoxic activity of ,b-amyrin derivatives
a
d = 5.13 ppm of the
a
-amyrin derivative and d = 5.19 ppm of the
The cytotoxic activity of a,b-amyrin derivatives was determined
b-amyrin derivative with 3J = 3.6 Hz. Signals for aromatic hydrogen
by the MTT assay using four human tumor cell lines (HL-60,
MDAMB-435, SF-295 and HCT-8). The results after 72 h of incuba-
tion (Table 1) show that only the derivatives 3a/3b were cytotoxic
against the cell lines used. The derivatives 3a/3b showed IC50 val-
were identified at d 7.27–7.88. In the 13C NMR, the signal for C-3 is
at d = 83.3 ppm, while C-12 appears at d = 124.5 ppm of the
rin derivative and d = 121.9 ppm of the b-amyrin derivative, C-13
appears at d = 139.8 ppm of the -amyrin derivative and
a-amy-
a
ues ranging from 0.94 (1.8 lM) to 1.84 lg/mL (3.0 lM). From a
d = 145.4 of the b-amyrin derivative. Signals for aromatic carbon
are in the range of d 129.1–133.4 ppm. The C@O carbon atoms ap-
pears at d = 168.1 ppm for esters and d = 172.1 for acids. MS (70 eV)
m/z (%) 574 (37, M+), 573.39 (100).
structure–activity point of view, the high cytotoxicity of 3a/3b
can be attributed to the presence of the double bond in the
maleinate group attached to carbon 3 of the A ring, which is absent
in the other derivatives lacking cytotoxicity. Chemically, the pres-
ence of the double bond between carbons C-10 and C-20 in the acyl
group of the derivatives 3a/3b generates an electron-rich center
likely to interact with centers of low electron density. Furthermore,
this double bond is in a conjugated system susceptible to reso-
nance that can create positive and negative centers partially able
to promote chemical interactions with centers rich or deficient in
electrons, which is not observed in the acyl groups of other deriv-
atives (without activity). In fact, many studies have shown a
Analysis of the IR spectra of 2a/2b allowed absorption bands for
(C@O) of esters at 1715 cmꢀ1 and for (C@O) of acids at 1731 cmꢀ1
,
one absorption band at 2925 cmꢀ1 identified as the (OH) of acids
and absorption bands between 1633 and 1376 cmꢀ1 of (C@C),
characteristic of alkenes.
In the 1H NMR spectra of 2a/2b, d 0.80–2.10 ppm (characteristic
profile of the substrate), H-3 appears as a double–doublet at
d = 4.54 ppm and H-12 as a triplet at d = 5.13 ppm of the a-amyrin

1270
F. W. A. Barros et al. / Bioorg. Med. Chem. 19 (2011) 1268–1276
Table 1
Cytotoxic effect of derivatives of
a
- and b-amyrin on human tumor cell lines and peripheral blood mononuclear cells
Compound
HL-60
IC50 l (lM)
g/mLa
MDAMB-435
SF-295
HCT-8
PBMC
1a/1b
2a/2b
3a/3b
4a/4b
Doxb
>5 (8.5)
>5 (9.2)
0.94 (1.8) 0.72–1.22
>5 (9.2)
>5
>5
>5
>5
>5
>5
>5
>5
>5 (9.5)
>5
1.52 (2.9) 1.33–1.73
>5
0.48 (0.8) 0.34–0.66
1.84 (3.5) 1.53–2.21
>5
0.24 (0.4) 0.17–0.36
1.58 (3.0) 1.33–1.88
>5
0.01 (0.02) 0.01–0.02
0.02 (0.03) 0.01–0.02
0.97 (1.7) 0.52–1.80
a
Concentration able to inhibit 50% of cell growth.
Positive control, doxorubicin.
b
positive relationship between the presence of double bond (unsat-
uration) in the structure of the compound and the potential cyto-
toxic effect.20,21 Cavalcanti et al. demonstrated the importance of
the exocyclic double bond in karen-19-oic acid in the induction
of DNA damage and subsequent apoptosis of HL-60 cells. Also in
this work, karen-19-oic acid was able to interact with cellular
DNA and interfere with the catalytic activity of the enzyme topoi-
somerase I.22 Alternatively, the presence of the double bond be-
tween carbons C-10 and C-20 in the derivatives 3a/3b may be
related to ROS production. Its oxidation leads to the production
of lipid hydroperoxides and non-radical intermediates. These by-
products can interact with DNA and altered gene expression,
resulting in cell death.23
(Fig. 3B), characteristic of apoptosis. The cells treated with 3a/3b at a
concentration of 1.5 M showed no striking morphological changes
(Fig. 3C). On the other hand, at 3 and 6 M, 3a/3b induced changes
typical of apoptosis, including reduction in cell volume, pyknotic nu-
clei and chromatolysis,23 which was more intense at the higher con-
centration. Moreover, there was an increase in the number of small
cells with an intact cell membrane (Fig. 3D and E).
To confirm the light microscopy findings, we performed a mor-
phological analysis of cells treated with 3a/3b by staining with
AO/EB and examining cells by fluorescence microscopy. The per-
centages of viable, apoptotic and necrotic cells were calculated.
After 24 h, HL-60 cells treated with 3a/3b showed a reduction in
l
l
cell viability only at concentrations of 3 and 6 lM. At these concen-
These results confirm that small changes in the molecule can
significantly alter the biological effect of a compound, being valid,
thus, the production of derived by chemical techniques for semi-
synthesis in the search for new drugs, including anticancer.18 Cragg
et al. have been reported that compounds from natural sources
have high anticancer potential, they need to be optimized for re-
moval, modification, introduction of functional groups and stereo-
centers to improve its properties.24 Combinatorial biosynthetic
methods are limited, however, there are numerous examples
including the taxanes, camptothecins and combretastatins.25–27
Interestingly, in the Alamar Blue assay using human peripheral
blood mononuclear cells (PBMC, normal cells), the derivatives
trations, the number of apoptotic cells significantly increased,
corroborating the results described above (Fig. 4). The compounds
3a/3b reduced the number of viable cells by 46.61% and 96.17% at
concentrations of 3 and 6
age of apoptotic cells at 3 and 6
respectively. With respect to necrosis, a slight increase in number
was observed at a concentration of 6 M (16.22%).
lM, respectively. In parallel, the percent-
lM was 35.59% and 71.84%,
l
In flow cytometry, treatment of HL-60 cells for 24 h with 3a/3b
showed a reduction in cell size and granularity in a concentration-
dependent manner. This may be seen in Figure 5 and corroborated
by the reduced number of cells with viable normal morphology
(note the panel A gates and panel B). Additionally, membrane
integrity was essentially preserved (Fig. 5, panel C), supporting
the results indicative of cell death by apoptosis. At a concentration
3a/3b were not cytotoxic at the concentrations tested (>5
suggesting selectivity for tumor cells. Additionally, none of the
derivatives tested showed hemolytic activity (>50 g/mL) using
lg/mL),
l
of 6
may be indicative of cells in the final process of death or secondary
necrosis as observed in microscopic analysis.28 Similarly, 0.5
lM, 3a/3b caused disruption of membrane integrity, which
mouse erythrocytes, which makes us suspect the activation of a
more specific cell death which does not involve the immediate lysis
of the cell membrane.
l
M
doxorubicin caused a reduction in cell viability and increased num-
The cytotoxicity results for the derivatives 3a/3b are interesting
because the prototype molecule is devoid of anticancer effect, but
has instead gastroprotective and antioxidant properties.11 There-
fore, the following experiments were carried out aimed at under-
standing the mechanism of action of the cytotoxic derivatives
3a/3b in the HL-60 cell line.
ber of apoptotic cells.
2.4. DNA fragmentation, phosphatidylserine (PS)
externalization and activation of caspases 3/7 induced by 3a/3b
Morphological analysis of HL-60 cells indicated that the proba-
ble process of cell death induced by 3a/3b occurs by induction of
apoptosis. We therefore proceeded with the determination of
DNA fragmentation, cell cycle changes, PS externalization and the
activity of the effector caspases 3/7 by flow cytometry, which are
also considered marker events of apoptosis.28,29 After 24 h, DNA
fragmentation of HL-60 cells was significantly increased by com-
pounds 3a/3b in a concentration-dependent manner (Table 2).
2.3. Morphological changes typical of apoptosis induced by
3a/3b
All experiments to investigate the mechanism of action of deriv-
atives 3a/3b in HL-60 cells were accompanied by the trypan blue
test to verify cell viability and ensure that the compounds tested
maintained their activity. As seen in Figure 2 3a/3b reduced the
number of viable cells and increased the number of non-viable
cells in a concentration-dependent manner.
Light microscopy was used to verify the morphology of HL-60 cells
untreated or treated with 3a/3b and stained with May Grünwald–
Giemsa. After 24 h incubation, cells from the negative control
exhibited typical non-adherent and round morphology with vacuoli-
zation (Fig. 3A). Chromatin condensation and nuclear and cellular
The positive control, doxorubicin (0.5
(p <0.0001) DNA fragmentation, comparable to the data for the
compounds 3a/3b at 3 and 6 M (61.54% and 84.24%, respectively).
Regarding cell cycle analysis, 3a/3b caused the accumulation of
cells in G0/G1 phase at 3 M (30.52%) (p <0.05) and in S phase at
M (88.63%) (p <0.001). Cell cycle arrest could be related to an
lM), induced 77.67%
l
l
6
l
attempt by the cell to repair the DNA damage caused by 3a/3b.
Since the damage appears to be very intense, repair does not occur
and apoptosis is triggered.28
fragmentation were observed in the presence of 0.5 lM doxorubicin

F. W. A. Barros et al. / Bioorg. Med. Chem. 19 (2011) 1268–1276
1271
Figure 2. Effect of 3a/3b on HL-60 cell viability determined by trypan blue exclusion after 24 h of incubation. Negative control (C) was treated with the vehicle used for
diluting the test compound. Doxorubicin (0.5 M) was used as the positive control (D). (A) Number of viable cells. (B) Number of non viable cells. Results are expressed as
mean standard error of mean (SEM) for multiple independent experiments. ⁄p <0.05; ⁄⁄p <0.001 compared to control by ANOVA followed by Dunnett’s test.
l
Figure 3. Microscopic analysis (May Grünwald/Giemsa-stained) of effect of 3a/3b on HL-60 cell after 24 h of incubation. Cells untreated (A) or treated with 3a/3b (1.5
lM, C;
3
l
M, D; or 6 M) was used as the positive control (B). Black arrows show reduction of cell volume,
lM, E) were analyzed by light microscopy (200ꢁ). Doxorubicin (0.5 l
nuclear fragmentation and cellular debris.
The detection of PS externalization showed that 3a/3b signifi-
cantly induced apoptosis (Fig. 6A), especially at 6 M (44.22%)
l
(p <0.0001). In parallel, it was found that 3a/3b was also able to
activate caspases 3/7 in a significant and concentration-dependent
manner. The results showed that 7.88% and 21.58% of cells had
activated caspases 3/7 at 3 and 6 lM, respectively (Fig. 6B). All re-
sults presented herein suggest that compounds 3a/3b induce cell
death by apoptosis in HL-60 leukemia cells.
3. Conclusions
Despite all the technological advances, the treatment of can-
cer is still a challenge and cases of cure are rare. Natural prod-
ucts and chemical modification of antitumor substances are
among the most important strategies used in the search of
new antineoplastic drugs.30 Therefore, the objective of this study
Figure 4. Effect of 3a/3b on HL-60 cell death pattern determined by acridine orange
and ethidium bromide-staining (AO/EB) after 24 h of incubation. Negative control
(C) was treated with the vehicle used for diluting the test compound. Doxorubicin
was to extract and identify the triterpenoid mixture of
a- and
(0.5 lM) was used as the positive control (D). Results are expressed as mean stan-
b-amyrin, as well as, to produce new derivatives and determine
their cytotoxic effects on human tumor cell lines and normal
cells.
dard error of mean (SEM) for three independent experiments performed in
duplicate (n = 6). ⁄p <0.05; ⁄⁄p <0.001; ⁄** p <0.0001 compared to control by ANOVA
followed by Dunnett’s test.

1272
F. W. A. Barros et al. / Bioorg. Med. Chem. 19 (2011) 1268–1276
Figure 5. Effects of 3a/3b (1.5, 3 and 6
lM) on HL-60 cell population determined by flow cytometry using propidium iodide, after 24 h of incubation. The negative control (C)
was treated with the vehicle used for diluting the test compound. Doxorubicin (0.5 lM) was used as the positive control (D). (Panel A) The forward light scatter and side light
scatter of the laser were used as indices of cell size and granularity, respectively. (Panel B) Number of viable cells by size and granularity (gated cells in panel A). (Panel C)
Percentage of cells with intact membrane. Results are expressed as mean standard error of mean (SEM) for three independent experiments performed in duplicate (n = 6).
**p <0.001; ***p <0.0001 compared to control by ANOVA followed by Dunnett’s test.
Table 2
Effect of 3a/3b on the nuclear DNA content of HL-60 cells determined by flow cytometry after 24 h of incubation
Compound
Concentration (
l
M)
DNAb (%)
Sub-G0/G1
G0/G1
S
G2/M
Control
—
8.25 0.15
77.67 2.41***
17.57 1.17**
61. 54 0.52***
84.24 1.16***
22.07 0.43
19.67 3.72
25.17 2.92
67.13 1.09
78.91 4.84
67.41 3.27
64.92 1.95
88.63 3.65**
11.83 1.08
1.41 1.11***
7.41 0.62*
4.57 1.29**
0.04 0.04***
Doxorubicina
3a/3b
0.5
1.5
3.0
6.0
30.52 2.66**
7.11 0.56**
*p <0.05; **p <0.001; ***p <0.0001, compared to control by ANOVA followed by Dunnett’s test.
a
Doxorubicin (0.5
l
M) was used as the positive control.
b
Results are expressed as mean standard error of mean (SEM) for three independent experiments performed in duplicate (n = 6).
In this study, we demonstrated that among the mixtures of
derivatives of ,b-amyrin tested, the mixture 3a/3b stood out be-
and altered gene expression.22,23 Additionally, 3a/3b seemed to
be selective for tumor cells, since they were not toxic to normal
PBMC cells. Regarding the mechanism of action, 3a/3b induced
the death of HL-60 cells by the induction of apoptosis, which was
demonstrated by morphological analysis (volume reduction, main-
tenance of cell membrane integrity and nuclear fragmentation),
DNA fragmentation, externalization of PS and activation of caspas-
es 3/7.29,31,32
a
cause of its high cytotoxic effect on human tumor cell lines, with
highest activity in HL-60 cells. This activity is possibly related to
the presence of the maleinate group linked to C-3 of the A ring of
the triterpenoid skeleton. There is a positive correlation between
the IC50 and the presence of the double bond in this group, due
the possibility to interact with cellular DNA, inhibiting the enzyme
topoisomerase I, or lipid peroxidation generates a large amount of
by-products and adduct formation, which can damage the DNA
The esterases could be a critical point in this work, but another
researches concluded that some of the non-natural ester analogues

F. W. A. Barros et al. / Bioorg. Med. Chem. 19 (2011) 1268–1276
1273
Figure 6. Effect of 3a/3b on PS externalization (Panel A) and caspase 3/7 activation (Panel B) of HL-60 cells by flow cytometry after 24 h of incubation. Negative control (C)
was treated with the vehicle used for diluting the test compound. Doxorubicin (0.5 M) was used as the positive control (D). Results are expressed as mean standard error of
mean (SEM) for two independent experiments performed in triplicate (n = 6). *p <0.05; **p <0.001; ***p <0.0001, compared to control by ANOVA followed by Dunnett’s test.
l
of the naturally occurring
ing than the naturally isolated
a
-amyrin are better glucose ameliorat-
20 mL of CH2Cl2) and DMAP in catalytic amounts, with subsequent
refluxing for 8 h at a temperature of 50 °C. The solution was con-
centrated and the resulting solid was chromatographed on a silica
gel column eluted with EtOAc, yielding 45.84 mg (37%).
a-amyrin acetate and almost as
equipotent as metformin.33 The by-products in this organic reac-
tion could be more effective than the original compound. To mea-
sure this reaction in vivo will be the next step in this work.
Nanoscience and nanotechnology strategies for imaging and treat-
ing cancer could be useful to design a method to avoid the
esterases action, like another works that find a combinatorial drug
delivery nanocapsule with sequential delivery feature for effective
antivasculature and anticancer activities in vitro and in vivo.34
Among the various approaches considered, nanotechnology offers
the best promise for the targeted delivery of drugs and genes to
a tumor site and for alleviating side effects of chemotherapeutic
agents. Renowned industrial and academic researchers describe
the advantages of nanoparticles as sensing, delivery, image
enhancement agents for cancer treatments, and various types of
useful nanoparticles such as polymeric nanoparticles and
dendrimers.35,36
4.1.1.3. 3-O-Carboxymaleinate of
a,b-amyrin (3a/3b). The mix-
ture of ,b-amyrin (a/b), 200 mg, was dissolved in 20 mL of CH2Cl2
a
and then added to a solution of maleic anhydride (184 mg in 20 mL
of CH2Cl2). The mixture was then refluxed for 8 h at a temperature
of 50 °C. The solution was concentrated under vacuum and the
resulting solid chromatographed on a silica gel column eluted with
EtOAc, yielding 76.27 mg (31%).
4.1.1.4. 3-O-Carboxyglutarate of
a,b-amyrin (4a/4b). The mix-
ture of ,b-amyrin (a/b), 100 mg, was dissolved in 20 mL of CH2Cl2
a
and then added to a solution of glutaric anhydride (26.76 mg in
20 mL of CH2Cl2) and DMAP in catalytic amounts, with subsequent
refluxing for 8 h at a temperature of 50 °C. The reaction product
was concentrated under vacuum and the resulting solid chromato-
graphed on a silica gel column eluted with EtOAc, yielding 92 mg
(35%).
4. Experimental
4.1. Chemistry
4.1.2. 3-O-Carboxybenzoate of
White solid, 43% yield; mp: 135 °C. FTIR (KBr)
2925, 1701, 1583, 1404. 1H NMR (500 MHz, CDCl3) d 4.77 (dd, H-
3), 5.13 (t, H-12 of the -amyrin derivative), 5.19 (t, H-12 of the
a,b-amyrin (1a/1b)
(cmꢀ1): 3461,
The melting point was determined using a Mettler Toledo
FP82HT micromelting point apparatus. The IR spectra were mea-
sured in KBr pellets using a Perkin-Elmer FT-IR Spectrum 1000.
All NMR data were recorded using a Bruker Avance DPX 300 and
Avance DRX-500 spectrometer operating at the frequency of the
hydrogen at 300.13 and 500.13 MHz in the frequency of the carbon
for 75.47 and 125.75 MHz, using CDCl3 as the solvent. High resolu-
tion data were obtained in a MS-IT-TOF mass spectrometer.
m
a
b-amyrin derivative). 13C (125 MHz, CDCl3) d 38.7/38.2 (C-1),
26.8 (C-2), 83.3 (C-3), 38.2 (C-4), 55, 6 (C-5), 18.4 (C-6), 33.1/33.5
(C-7), 40.0/39.8 (C-8), 47.9/47.8 (C-9), 38.0 (C-10), 23.5/23.6
(C-11), 124.5/121.9 (C-12), 139.8/145.4 (C-13), 41.8/40.0 (C-14),
28.3/26.8 (C-15), 26.8 (C-16), 33.6/33.1 (C-17), 59.3/47.5 (C-18),
39.8/47.5 (C-19), 39.2/31.5 (C-20), 31.5/33.7 (C- 21), 41.8/38.0
(C-22), 28.3/28.4 (C-23), 15.9 (C-24), 15.9 (C-25), 17.1/17.0
(C-26), 23.1/26.8 (C-27), 28.9/28.4 (C-28), 17.8/33.6 (C-29), 21. 6
(C-30), 168.1/172.1 (C@O), 131.9 (C-10), 129.1 (C-20), 133.4 (C-30),
131.1 (C-40), 130.1 (C-50), 131.1 (C-60).
4.1.1. Procedure for the synthesis of the derivatives of
amyrin
a,b-
4.1.1.1. 3-O-Carboxybenzoate of
a,b-amyrin (1a/1b). The mix-
ture of ,b-amyrin (a/b), 100 mg, was dissolved in 20 mL of ethyl
a
ether and added to a solution of phthalic anhydride (139 mg in
20 mL of ethyl ether) in pyridine (0.5 mL); the reaction proceeded
under reflux for 8 h at a temperature of 50 °C. The final solution
was added to 15 mL of a concentrated solution of copper sulfate,
and the organic phase was separated and evaporated to dryness
at room temperature. The reaction material was chromatographed
on a silica gel column eluted with EtOAc, yielding 58.4 mg (43%).
4.1.3. 3-O-Carboxysuccinate of
White solid, 37% yield; mp: 200 °C. FTIR (KBr)
2925, 1731, 1715, 1450. 1H NMR (500 MHz, CDCl3) d 4.54 (dd,
H-3), 5.13 (t, H-12 of the -amyrin derivative), 5.21 (t, H-12 of
a,b-amyrin (2a/2b)
m
(cmꢀ1): 3446,
a
the b-amyrin derivative), 2.64 (dd, J = 5.40 and 5.85 Hz, H-10),
2.69 (dd, J = 5.40 and 5.85 Hz, H-20). 13C (125 MHz, CDCl3) d
38.7/38.0 (C-1), 26.8 (C-2), 81.8 (C-3), 38.0 (C-4), 55, 5 (C-5), 18.4
(C-6), 31.7 (C-7), 39.9 (C-8), 47.8/47.6 (C-9), 37.9 (C-10),
23.1/23.7 (C-11), 124.5/121.8 (C-12), 139.9/145.4 (C-13), 41.8/40.0
(C-14), 28.2/26.8 (C-15), 26.8 (C-16), 33.1/31.5 (C-17), 59.3/ 47.5
4.1.1.2. 3-O-Carboxysuccinate of
ture of ,b-amyrin (a/b), 100 mg, was dissolved in 20 mL of CH2Cl2
and then added a solution of succinic anhydride (43.67 mg in
a,b-amyrin (2a/2b). The mix-
a

1274
F. W. A. Barros et al. / Bioorg. Med. Chem. 19 (2011) 1268–1276
(C-18), 39.8/47.8 (C-19), 39.5/31.0 (C-20), 31.5/33.1 (C-21),
41.6/38.0 (C-22), 28.2/28.3 (C-23), 16.0/15.9 (C-24), 15.9 (C-25),
16.9/16.0 (C-26), 23.5/26.0 (C-27), 28.3/28.2 (C-28), 17.7/33.5
(C-29), 21.6 (C-30), 172.9/178.7 (C@O), 28.2 (C-10), 28.2 (C-20).
exposure. 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 den-
sity-gradient centrifugation over Ficoll-Hypaque. PBMC were
washed and resuspended at a concentration of 3 ꢁ 105 cells/mL in
RPMI 1640 medium supplemented with 20% fetal bovine serum,
4.1.4. 3-O-Carboxymaleinate of
White solid, 31% yield; mp: 188 °C FTIR (KBr)
2924, 1722, 1633, 1454. 1H NMR (500 MHz, CDCl3) d 4.69 (dd,
H-3), 5.14 (t, H-12 of the -amyrin derivative), 5.20 (t, H-12 of
a
,b-amyrin (3a/3b)
m
(cmꢀ1): 3422,
2 mM glutamine, 100 U/mL penicillin and 100
at 37 °C with 5% CO2. Phytohemagglutinin (3%) was added at the
beginning of culture. After 24 h, the compounds (0.078–5 g/mL)
lg/mL streptomycin,
a
l
the b-amyrin derivative), 6.39 (d, J = 12.8 Hz, H-10), 6.47 (d,
J = 12.8 H, H-20). 13C (125 MHz, CDCl3) d C 38.6/38.4 (C-1), 27.1
(C-2), 85.3 (C-3), 37.3 (C-4), 55.5 (C-5), 18.4 (C-6), 32.7
(C-7), 40.2/40.0 (C-8), 47.9/47.8 (C-9), 37.0 (C-10), 23.6/23.7
(C-11), 124.3/121.7 (C-12), 139.9/145.3 (C-13), 42.3/41.9 (C-14),
28.3/26.3 (C-15), 26.8 (C-16), 33.9/32.7 (C-17), 59.3/47.4 (C-18),
39.8/47.0 (C-19), 39.8/31.0 (C-20), 31.4/35.0 (C-21), 41.7/37.3
(C-22), 28.3 (C-23), 15.9/13.7 (C-24), 15.9/15.7 (C-25), 16.9/16.8
(C-26), 23.5/26.2 (C-27), 28.6/28.3 (C-28), 17.7/33.5 (C-29), 21.6
(C-30), 164.6/167.9 (C@O), 136.9 (C-10), 129.9 (C-20).
dissolved in DMSO were added to each plate well, and the cells were
incubated for 72 h. Control groups received the same amount of
vehicle and doxorubicin was used as the positive control. The final
concentration of DMSO in the culture medium was kept constant,
below 0.1% (v/v). Twenty-four hours before the end of the incuba-
tion, 10 lL of stock solution (0.312 mg/mL) of the Alamar Blue (Res-
azurin, Sigma–Aldrich 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 expressed as
the percentage of control absorbance at 570 nm and 595 nm.
4.1.5. 3-O-Carboxyglutarate of
White solid, 35% yield; mp: 209 °C. FTIR (KBr)
2924, 1727, 1455. 1H NMR (500 MHz, CDCl3) d 4.52 (dd, H-3),
5.13 (t, H-12 of the -amyrin derivative), 5.21 (t, H-12 of the
a
,b-amyrin (4a/4b)
4.2.3. Hemolytic assay
m
(cmꢀ1): 3461,
The test was performed in 96-well plates using a 2% mouse
erythrocyte suspension in 0.85% NaCl containing 10 mM CaCl2, fol-
lowing the method described by Jimenez et al.39 The compounds
a
b-amyrin derivative), 2.40 (t, J = 7.3 Hz, H-10), 2.44 (t, J = 7.4 Hz,
H-30), 1.98 (m, H-20). 13C (125 MHz, CDCl3) d 38.6/38.5 (C-1), 27.1
(C-2), 81.4 (C-3), 37.9 (C-4), 55.5 (C-5), 18.4 (C-6), 32.7 (C-7),
40.2/40.0 (C-8), 47.8/47.7 (C-9), 37.1 (C-10), 23.6/23.7 (C-11),
124.5/121.8 (C-12), 139.8/145.4 (C-13), 42.3/41.9 (C-14),
28.3/26.3 (C-15), 26.8 (C-16), 33.9/32.7 (C-17), 59.3/47.4 (C-18),
39.8/47.0 (C-19), 39.9/31.2 (C-20), 31.5/34.9 (C-21), 41.8/37.4
(C-22), 28.3 (C-23), 15.9/15.8 (C-24), 15.9/15.8 (C-25), 17.0/16.9
(C-26), 23.5/26.2 (C-27), 28.6/28.3 (C-28), 17.7/33.5 (C-29), 21.6
(C-30), 172.9/178.7 (C@O), 33.2 (C-10), 20.2 (C-20), 33.8 (C-30).
were tested at concentrations ranging from 0.38 to 200 lg/mL.
After incubation (1 h) at room temperature, the supernatant was
removed and the hemoglobin released was measured spectropho-
tometrically at 540 nm. DMSO at 10% was used as the negative
control and Triton X-100 (1%) was used as the positive control.
4.2.4. Study of the mechanism of cytotoxicity induced by 3-O-
carboximaleinate of a- and b-amyrin (3a/3b)
In the second stage of the study aimed at determining the cyto-
toxicity induced by 3a/3b, the HL-60 promyelocytic leukemia cell
line was chosen based on preliminary cytotoxicity screening
(MTT assay).
4.2. Biology
The HL-60 cell line is one of the most often used model for
studying cytotoxic substances.40,41 Therefore, the following exper-
iments were performed using HL-60 cells as a model and 3a/3b
were dissolved in sterile DMSO (vehicle control). The cells were
incubated with the test compounds for 24 h at three different con-
4.2.1. Cytotoxicity against cancer cell lines—MTT assay
The cytotoxic effect of compounds was evaluated by the MTT
assay,37 using four human cancer cell lines: HL-60 (promyelocytic
leukemia), SF-295 (glioblastoma), HCT-8 (colon cancer) and
MDAMB-435 (melanoma), all obtained from the National Cancer
Institute (Bethesda, MD, USA). All cell lines were maintained in
RPMI 1640 medium supplemented with 10% fetal bovine serum,
centrations (1.5, 3.0 and 6.0 lM), chosen based on IC50 values ob-
tained by the MTT assay. Doxorubicin (0.5
lM) was used as the
positive control.
2 mM glutamine, 100 U/mL penicillin and 100 lg/mL streptomy-
cin, at 37 °C with 5% CO2. Cancer cell growth was quantified by
the ability of living cells to reduce the yellow dye 3-(4,5-di-
methyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)
(Sigma Aldrich Co., St. Louis, MO/USA) to a purple formazan
product. Briefly, cells were seeded in 96-well plates (0.7 ꢁ 105
cells/well for adherent cells and 0.3 ꢁ 106 cells/well for suspended
4.2.4.1. Cell viability by trypan blue exclusion. Cell viability was
determined by the trypan blue dye exclusion test42 after treat-
ment. Cells (0.3 ꢁ 106 cells/mL) were incubated with 3a/3b, and
trypan blue-excluding cells were counted in a Neubauer chamber,
using an aliquot of cells removed from cultures after incubation.
cells), and compounds (0.078–5
lg/mL) dissolved in DMSO were
4.2.4.2. Analysis of morphological changes. Untreated and trea-
ted HL-60 cells were examined for morphological changes by light
microscopy (Olympus, Tokyo, Japan). Cells from cultures after 24 h
were harvested, transferred to cytospin slides, fixed with methanol
for 1 min and stained with May Grünwald–Giemsa (Bioclin, Brazil).
added to each plate well. Control groups received the same amount
of vehicle. The final concentration of DMSO in the culture medium
was kept constant, below 0.1% (v/v). After 72 h of incubation, the
supernatant was replaced by fresh medium containing MTT
(0.5 mg/mL). Three hours later, the MTT formazan product was dis-
solved in 150
l
L DMSO, and absorbance was measured at 595 nm
4.2.4.3. Morphological analysis with fluorescence micros-
copy. Acridine orange/ethidium bromide (AO/EB) (Sigma
Aldrich) staining43 of HL-60 cells was performed to evaluate the
cell death pattern induced by increasing concentrations of com-
pounds. After incubation, cells were pelleted and each sample
(DTX-880 Multimode Detector, Beckman CoulterÒ). All cell treat-
ments were carried out in triplicate and doxorubicin was used as
the positive control.
4.2.2. Inhibition of PBMC proliferation—Alamar Blue assay
In order to investigate the effect of the test compounds on normal
proliferating cells, the Alamar Blue assay38 was performed with hu-
man peripheral blood mononuclear cells (PBMC) after 72 h drug
was mixed with 1
lL of aqueous AO/EB solution (100 lg/mL of
AO in PBS; 100 g/mL EB in PBS) just prior to fluorescence micros-
l
copy and quantification (Olympus, Tokyo, Japan). Three hundred
cells were counted per sample and scored as follows: viable cells,

F. W. A. Barros et al. / Bioorg. Med. Chem. 19 (2011) 1268–1276
1275
apoptotic cells and necrotic cells.44,45 The percentage of apoptotic
and necrotic cells was calculated.
presented as means SEM for at least three independent
experiments and evaluated by ANOVA followed by the Student
Newman–Keuls test.
4.2.4.4. Flow cytometry analysis. HL-60 cell fluorescence was
determined by flow cytometry in a Guava EasyCyte Mine using
Guava Express Plus software. Five thousand events were analyzed
for each replicate in three independent experiments, and cellular
debris was omitted from the analysis. Internucleosomal DNA frag-
mentation and cell cycle were determined by ModFit LT for Win32
version 3.1.
5. Conflict of interest statement
None
Acknowledgments
This research received financial support from FUNCAP, CNPq
and CAPES. The authors thank National Cancer Institute (Bethesda,
MD, USA) for donating the tumor cell lines used in this study and
CENAUREM for conducting the NMR spectra. Dr. A. Leyva provided
English editing of the manuscript.
4.2.5. Cell membrane integrity
The cell membrane integrity was evaluated by the exclusion of
propidium iodide (50
Briefly, a 100 L suspension of treated and untreated cells was
incubated with propidium iodide (50 g/mL). The cells were then
lg/mL, Sigma Aldrich Co., St. Louis, MO/USA).
l
l
incubated for 5 min. Fluorescence was measured and analyzed
References and notes
for cell morphology, granularity and membrane integrity.46
1. Mahato, S. B.; Nandy, A. K.; Roy, G. Phytochemistry 1992, 31, 2199.
2. Mahato, S. B.; Sen, S. Phytochemistry 1997, 44, 1185.
3. Oliveira, F. A.; Costa, C. L. S.; Chaves, M. H.; Almeida, F. R. C.; Cavalcante, I. J. M.;
Lima, A. F.; Lima, R. C. P., Jr.; Silva, R. M.; Campos, A. R.; Santos, F. A.; Rao, V. S. N.
Life Sci. 2005, 77, 2942.
4. Keterere, D. R.; Gray, A. I.; Nash, R. J.; Waigh, R. D. Phytochemistry 2003, 63, 81.
5. Oliveira, F. A.; Lima, R. C. P., Jr.; Cordeiro, W. M.; Vieira, G. M., Jr.; Chaves, M. H.;
Almeida, F. R. C.; Silva, R. M.; Santos, F. A.; Rao, V. S. N. Pharmacol. Biochem.
Behav. 2004, 78, 719.
6. Oliveira, F. A.; Vieira, H. M., Jr.; Chaves, M. H.; Almeida, F. R. C.; Florêncio, M. G.;
Lima,R. C. P., Jr.;Silva, R. M.;Santos, F. A.;Rao, V. S. N. Pharmacol. Res. 2004, 49, 105.
7. Pinto, S. A. H.; Pinto, L. M. S.; Cunha, G. M. A.; Chaves, M. H.; Santos, F. A.; Rao, V.
S. N. Inflammopharmacology 2008, 16, 48.
4.2.6. Internucleosomal DNA fragmentation and cell cycle
analysis
DNA fragmentation and cell cycle were analyzed by flow cytom-
etry after DNA staining with propidium iodide. Briefly, a 100
suspension of treated and untreated cells was incubated for
30 min, in the dark, with hypotonic solution containing 50 g/mL
lL
l
propidium iodide, 0.1% sodium citrate, and 0.1% Triton X-100. Fluo-
rescence was measured and DNA fragmentation and cell cycle
phases were determined.47
8. Pinto, S. A. H.; Pinto, L. M. S.; Guedes, G. M. A.; Cunha, G. M. A.; Chaves, M. H.;
Santos, F. A.; Rao, V. S. N. Phytomedicine 2008, 15, 630.
9. Aragão, G. F.; Carneiro, L. M. V.; Junior, A. P. F.; Vieira, L. C.; Bandeira, P. N.;
Lemos, T. L. G.; Viana, G. S. B. Pharmacol. Biochem. Behav. 2006, 85, 827.
10. Aragão, G. F.; Cristhiany, C. P.; Bandeira, P. N.; Lemos, T. L. G.; Viana, G. S. B. J.
Herbal Pharmacother. 2007, 7, 31.
11. Laszczyk, M. N. Planta Med. 2009, 75, 1549.
12. Bandeira, P. N.; Lemos, T. L. G.; Costa, S. M. O.; Santos, H. S. Rev. Bras. Farmacogn.
2007, 17, 204.
4.2.7. 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.48 The Guava Nexin Assay Kit was used to determine
early apoptosis. Cells were washed twice with cold PBS and then
13. Soldi, C.; Latti, M. G. P.; Luiz, A. P.; Marcon, R.; Meotti, F. C.; Mioto, L. A.; Santos,
A. R. S. Bioorg. Med. Chem. 2008, 1.
14. Aragão, G. F.; Carneiro, L. M. V.; Junior, A. P. F.; Bandeira, P. N.; Lemos, T. L. G.;
Viana, G. S. B. Pharm. Biol. 2007, 45, 343.
15. Oliveira, F. A.; Lima, R. C. P., Jr.; Cordeira, W. M.; Vieira, G. M., Jr.; Chaves, M. H.;
Almeida, F. R. C.; Silva, R. M.; Santos, F. A.; Rao, V. S. N. Pharmacol. Biochem.
Behav. 2004, 78, 718.
16. Fernandes, J.; Castilho, R. O.; Costa, M. R.; Wagner-Souza, K.; Kaplan, M. A. C.;
Gattass, C. R. Cancer Lett. 2003, 190, 165.
17. Sun, H. X.; Ye, Y. P.; Pan, Y. J. J. Ethnopharmacol. 2004, 90, 261.
18. Lee, K. H. J. Nat. Prod. 2010, 73, 500.
19. Bandeira, P. N.; Pessoa, O. D. L.; Trevisan, M. T. S.; Lemos, T. L. G. Quim. Nova
2002, 25, 1078.
20. Amslinger, S. ChemMedChem 2010, 5, 351.
21. Roh, E. M.; Jin, Q.; Jin, H. G.; Shin, J. E.; Choi, E. J.; Moon, Y. H.; Woo, E. R. Arch.
Pharmacol. Res. 2010, 33, 1347.
resuspended in 135
lL of PBS with 5 lL of 7-amino-actinomycin
D (7AAD) and 10 L of Annexin V-PE. The cells were gently vor-
l
texed and incubated for 20 min at room temperature (20–25 °C)
in the dark. Afterward, the cells were analyzed by flow cytometry
(EasyCyte from Guava Technologies). Annexin V is a phospholipid-
binding protein that has a high affinity for PS. 7-AAD, a cell
impermeant dye, is used as an indicator of membrane structural
integrity. Annexin V-PE was measured as a yellow fluorescence
at 583 nm and 7-AAD as a red fluorescence at 680 nm. The percent-
age of early and late apoptotic cells and necrotic cells was then
calculated.
22. Cavalcanti, B. C.; Bezerra, D. P.; Magalhães, H. I.; Moraes, M. O.; Lima, M. A.;
Silveira, E. R.; Câmara, C. A.; Rao, V. S.; Pessoa, C.; Costa-Lotufo, L. V. J. Appl.
Toxicol. 2009, 7, 560.
23. Pillon, N. J.; Soulère, L.; Vella, R. E.; Croze, M.; Caré, B. R.; Soula, H. A.; Doutheau,
A.; Lagarde, M.; Soulage, C. O. Chem. Biol. Interact. 2010, 188, 171.
24. Cragg, G. M.; Grothaus, P. G.; Newman, D. J.; Cragg, G. M. Chem. Rev. 2009, 109,
3012.
4.2.8. Activity of caspases 3 and 7
Caspase 3/7 activity was analyzed by flow cytometry, using
GuavaÒ EasyCyte Caspase Kit, after 24 h of incubation. HL-60 cells
(3 ꢁ 105 cells/mL) were incubated with Fluorescent Labeled Inhib-
itor of Caspases (FLICA) and maintained for 1 h at 37 °C and 5% CO2.
After incubation, 80
were centrifuged at 2000 rpm for 5 min. The resulting pellet was
resuspended in 200 L of washing buffer and centrifuged again.
lL of washing buffer were added and cells
25. Coseri, S. Mini-Rev. Med. Chem. 2009, 9, 560.
26. Kingston, D. G. I. J. Org. Chem. 2008, 73, 3975.
27. Rahier, N. J.; Thomas, C. J.; Hecht, S. M. In Anticancer Agents from Natural
Products; Cragg, G. M., Kingston, D. G. I., Newman, D. J., Eds.; CRC Press LLC:
Boca Raton, FL, 2005; p 5.
28. Maia, D. P.; Wilke, D. V.; Mafezoli, J.; Júnior, J. N. S.; Moraes, M. O.; Pessoa, C.;
Costa-Lotufo, L. V. Chem. Biol. Interact. 2009, 180, 220.
29. Bezerra, D. P.; Militão, G. C. G.; Castro, F. O.; Pessoa, C.; Moraes, M. O.; Silveira,
E. R.; Lima, M. A. S.; Elmiro, F. J. M.; Costa-Lotufo, L. V. Toxicol. In Vitro 2007,
21, 1.
30. Pinkerton, D. M.; Banwell, M. G.; Garson, M. J.; Kumar, N.; Moraes, M. O.;
Cavalcanti, B. C.; Barros, F. W. A.; Pessoa, C. Chem. Biodiv. 2010, 7, 1311.
31. Esteves-Souza, A.; Lucio, K. A.; Da Cunha, A. S.; Da Cunha Pinto, A.; Da Silva
Lima, E. L.; Camara, C. A.; Vargas, M. D.; Gattass, C. R. Oncol. Rep. 2008, 20, 225.
32. Yedjou, C. G.; Milner, J. N.; Howard, C. B.; Tchounwou, P. B. Int. J. Environ. Res.
Public Health. 2010, 7, 2008.
l
The cells were then resuspended in the working solution (propidi-
um iodide 1:200 in 1ꢁ washing buffer) and analyzed immediately
using flow cytometry.
4.3. Statistical analysis
For cytotoxicity assays, the IC50 or EC50 values and their 95%
confidence intervals were obtained by nonlinear regression using
the GraphPad program (Intuitive Software for Science, San Diego,
CA). Data obtained from the studies of mechanism of action are

1276
F. W. A. Barros et al. / Bioorg. Med. Chem. 19 (2011) 1268–1276
33. Narender, T.; Khaliq, T.; Singh, A. B.; Joshi, M. D.; Mishra, P.; Chaturvedi, J. P.;
Srivastava, A. K.; Maurya, R.; Agarwal, S. C. Eur. J. Med. Chem. 2009, 44, 1215.
34. Wang, Z.; Ho, P. C. Biomaterials 2010, 31, 7115.
35. Nassar, T.; Rom, A.; Nyska, A.; Benita, S. Pharm. Res. 2008, 25, 2019.
36. Nassar, T.; Rom, A.; Nyska, A.; Benita, S. J. Controlled Release 2009, 133, 77.
37. Mosman, T. J. Immunol. Methods 1983, 65, 55.
42. Renzi, D.; Valtolina, M.; Foster, R. Altern. Lab. Anim. 1993, 21, 89.
43. McGahon, A. J.; Martin, S. M.; Bissonnette, R. P.; Mahboubi, A.; Shi, Y.; Mogil, R.
J.; Nishioka, W. K.; Green, D. R. Methods Cell Biol. 1995, 46, 153.
44. Geng, C. X.; Zeng, Z. C.; Wang, J. Y. World J. Gastroenterol. 2003, 9,
696.
45. Cury-Boaventura, M. F.; Pompeia, C.; Curi, R. Clin. Nutr. 2004, 23, 721.
46. Darzynkiewicz, Z.; Bruno, S.; Del Bino, G.; Gorczyca, W.; Hotz, M. A.; Lassota, P.;
Traganos, F. Cytometry 1992, 13, 795.
38. Zhi-Jun, Y.; Sriranganathan, N.; Vaught, T.; Arastu, S. K.; Ansar-Ahmed, S. J.
Immunol. Methods 1997, 210, 25.
39. Jimenez, P. C.; Fortier, S. C.; Lotufo, T. M. C.; Pessoa, C.; Moraes, M. E. A.; Moraes,
M. O.; Costa-Lotufo, L. V. J. Exp. Marine Biol. Ecol. 2003, 287, 93.
40. Collins, S. J. Blood 1987, 70, 1233.
41. Militão, G. C. G.; Dantas, I. N. F.; Pessoa, C.; Falcão, M. J. C.; Silveira, E. R.; Lima,
M. A. S.; Curi, R.; Lima, T.; Moraes, M. O.; Costa-Lotufo, L. V. Life Sci. 2006, 78,
2409.
47. Nicoletti, I.; Migliorati, G.; Pagliacci, M. C.; Grignani, F.; Riccardi, C. J. Immunol.
Methods 1991, 139, 271.
48. Vermes, I.; Haanen, C.; Steffens-Nakken, H.; Reutlingsperger, C. J. Immunol.
Methods 1995, 184, 39.