Synthesis and cytotoxicity screening of substituted isobenzofuranones designed from anacardic acids

Article abstract of DOI:10.1016/j.ejmech.2010.05.015

This work is part of a large program,which seeks to discover new antitumor isobenfuranones designed from anacardic acids. The synthetic strategy for the construction of the title compounds takes into consideration the use of inexpensive anacardic acids (2),the major natural cashew (Anacardium occi-dentale) nut-shell phenolic lipid,and features one-pot construction of fused-ring aromatic g-lactones,phthalides. The cytotoxicity screening in different human cancer cell lines (HL-60 leukemia,SF295 glioblastoma and MDA-MB435 melanoma) by the MTT assay showed that acyclic precursor (6),and isobenfuranones (1a and 1b) are active compounds. Interestingly,1a exhibits significant antiproliferative effect against HL-60 cells and moderate activity against SF295 and MDA-MB435 cell lines. Analysis of mechanisms involved in the cytotoxic activity showed that active compounds were leading to DNA damage,triggering apoptosis or necrosis induction.

Full text of DOI:10.1016/j.ejmech.2010.05.015

European Journal of Medicinal Chemistry 45 (2010) 3480e3489
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and cytotoxicity screening of substituted isobenzofuranones designed
from anacardic acids
Lúcio P.L. Logrado a, Camila O. Santos a, Luiz A.S. Romeiro b, Arinice M. Costa c, José R.O. Ferreira c,
Bruno C. Cavalcanti c, O. Manoel de Moraes c, Letícia V. Costa-Lotufo c, Cláudia Pessoa c,
,
Maria L. dos Santos a
*
a Instituto de Química, Universidade de Brasília, Campus Darcy Ribeiro, Brasilia, Brazil
b Faculdade de Ciências da Saúde, Universidade de Brasília, Brasilia, Brazil
c Laboratório de Oncologia Experimental, Universidade Federal do Ceará, Fortaleza, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
This work is part of a large program, which seeks to discover new antitumor isobenfuranones designed
from anacardic acids. The synthetic strategy for the construction of the title compounds takes into
consideration the use of inexpensive anacardic acids (2), the major natural cashew (Anacardium occi-
Received 26 February 2010
Received in revised form
4 May 2010
Accepted 7 May 2010
Available online 12 May 2010
dentale) nut-shell phenolic lipid, and features one-pot construction of fused-ring aromatic g-lactones,
phthalides. The cytotoxicity screening in different human cancer cell lines (HL-60 leukemia, SF295
glioblastoma and MDA-MB435 melanoma) by the MTT assay showed that acyclic precursor (6), and
isobenfuranones (1a and 1b) are active compounds. Interestingly, 1a exhibits significant antiproliferative
effect against HL-60 cells and moderate activity against SF295 and MDA-MB435 cell lines. Analysis of
mechanisms involved in the cytotoxic activity showed that active compounds were leading to DNA
damage, triggering apoptosis or necrosis induction.
Keywords:
Anacardic acids
Isobenzofuranones
Phthalides
Ó 2010 Elsevier Masson SAS. All rights reserved.
Cytotoxicity screening
1. Introduction
Mycophenolic acid, a highly substituted natural phthalide present in
a number of Penicillium spp, exhibits in vitro and in vivo a wide range
Isobenzofuranones, fused-ring aromatic
g
-lactones, have
of biological activities including antifungal, antibacterial, antiviral,
and immunosuppressive effects, has been the subject of several total
and formal syntheses [6]. Vermistatin, a metabolite of Penicillium
species, notably Penicillium vermiculatum [7], Penicillium verruculo-
sum [8], was classified as an antibiotic with cytotoxic effects, while
pestaphthalides A and B, isolated form solid cultures of Pestalotiopsis
foedan, showed modest antifungal activity [9] (Fig. 1).
Interestingly, acetophthalidin (I), an optically inactive (racemic
form) metabolite isolated from broth of fungal strain BM923 [10],
shows a potent inhibitory effect on cell cycle progression of the
attracted the attention of various research groups, regarding their
isolation, structural elucidation, biogenesis, biological activity as
well as synthetic goals. There are few naturally occurring members
of this class of compounds. For instance, isoochracinic acid, a phy-
totoxin isolated from Alternaria kikuchiana, the causative agent of
black leaf spot of pear [1], and Mycospharella fijiensis, the causal
agent of Black Sigatoka disease of bananas and plantains [2], was the
subject of synthetic pathway and also recognized as a suitable
zearelanone intermediate [3]. Isoochracinic acid, isoochracinol, and
other biologically active polyketide metabolites were isolated from
solid-substrate fermentation cultures of an undetermined fungico-
lous hyphomycete that resembles Cladosporium sp [4]. The 5-
hydroxyl derivative of isoochracinic acid, isolated as a metabolite
from sponge-derived fungi Curvularia lunata and Cladosporium
herbarum, namely herbaric acid, showed no activity when tested
against A. salina and the human leukemia cell line HL-60 [5].
tsFT210 mouse cell in the M phase in low concentrations (6.25
mg/
mL) [11]. According to the authors, the fungus produces the 3,4,6,8-
tetrahydroxy-3-methyl-3,4-dihydroisocoumarin (II), that can be
converted into form I by heating in water at pH 1 (Fig. 2). The tri-
hydroxymellein II had been previously isolated as a metabolite in
still cultures of Ceratocystis minor [12], one of the fungal species
associated with the blue stain disease of pine which is caused by
Penicillium brevicompactum [13]. Bioassays on acetophthalidin and
its synthetic analogues, using rat mammary cancer cells (FM3A),
showed that natural racemic acetophthalidin and both synthetic
enantiomers accumulated cells in the G2/M phase but no difference
* Corresponding author. Instituto de Química, Universidade de Brasília, Brasília-
DF 70904-970, PO. Box: 4478, Brazil. Tel.: þ55 61 3107 3855; fax: þ55 61 3273 4149.
E-mail address: mlsantos@unb.br (M.L. dos Santos).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.015

L.P.L. Logrado et al. / European Journal of Medicinal Chemistry 45 (2010) 3480e3489
3481
OH
O
OH
O
O
O
R1
HO2C
MeO
R2
Isoochracinic acid (R1 = H and R2 = CH2CO2H)
Isoochracinol (R1 = H and R2 = CH2CH2OH)
Herbaric acid (R1 = OH and R2 = CH2CO2H)
Mycophenolic acid
OH
OH
O
O
O
O
MeO
O
O
H
O
HO
HO
O
OMe
HO
Pestaphthalide A
HO
Pestaphthalide B
Vermistatin
Fig. 1. Some bioactive naturally occurring isobenzofuranones.
in cell cycle inhibition was observed [14]. To the authors the race-
mization may occur faster than appearance of the activity. All of the
alcohol analogues were inactive, showing that the carbonyl group
seems to be important for the inhibitory activity. Interestingly, the
natural racemic analogue completely inhibited the cell growth, but
it did not cause accumulation of the cells in G2/M. Thus, it was
concluded that acetophthalidin must specifically inhibit the
receptors related to the progression of the M phase. Nevertheless,
the racemic mixture must easily affect the other molecules neces-
sary for cell cycle progression [14,15].
and features one-pot epoxidation and cyclization. First, the
heterogeneous mixture of anacardic acids obtained according to
a protocol previously established [17] was converted to the corre-
sponding methyl esters (3) by dimethyl sulphate under phase
transfer catalysis as described before and than was quantitatively
converted into saturated dimethyl tetrahydroanacardic ester (4) by
conventionally heterogeneous catalytic hydrogenation in a Parr
apparatus [18]. The benzylic bromination of 4 was smoothly
promoted by NBS-benzoyl peroxide to yield compound 5. The
benzylic bromine compound (5), upon treatment with DBU in
refluxing benzene, was quantitatively converted into the trans-
unsaturated compound 6. By distinct treatment with m-CPBA or
performic acid, the latter was directly converted into 3-(1-hydroxy-
tetradecyl)-7-methoxy-3H-isobenzofuran-1-one (1a) in 98% and
74% yield, respectively. The oxidation of the hydroxyl group in 1a
was accomplished in 96% yield by means of the Jones protocol to
give 7-methoxy-3-tetradecanoyl-3H-isobenzofuran-1-one (1b).
The 1H and 13C NMR chemical shifts of the all acyclic precursors
(2 to 6) and phtalides (1a and 1b) were confirmed by COSY
OH
OH
O
O
H3O+
O
O
HO
HO
OH
O
OH
II
I
(correlated
spectroscopy),
HMQC
(heteronuclear
multi-
Acetophthalidin
Trihydroxymellein
pleequantum correlation), HMBC (heteronuclear multipleebond
correlation), and NOESY (nuclear Overhauser effect spectroscopy).
The multiplicities of the carbons signals of all compounds were
determined by DEPT (distortion-less enhancement by polarization
transfer) experiments. The assignments of the 1H and 13C NMR
peaks for the acyclic precursors and isobenzofuranones were
described according to Fig. 4.
Fig. 2. Isomerization of acetophthalidin to the inactive trihydroxymelein.
In continuation of an ongoing program aimed at the develop-
ment of bioactive heterocyclic compounds using as starting mate-
rial non-isoprenoid phenolic lipids from Anacardium occidentale we
became interested in the preparation of isobenzofuranones 1a and
1b from anacardic acids (2), the major cashew nut-shell phenolic
lipid of natural CNSL [16]. These substituted phthalides were
structurally designed for the maintenance of the hydrophobic long
aliphatic chain from the starting lipids and the introduction of both
benzofuran-1-one subunit and oxygenated functions at the side
chain, hydrophilic moieties which should retain the high intrinsic
activity of the natural prototype acetophthalidin (Fig. 3).
2.2. Pharmacology
In order to evaluate antiproliferative effects, the two iso-
benzofuranones (1a and 1b) and their acyclic precursors (4 to 6)
were assayed against different human cancer cell lines (HL-60
leukemia, SF295 glioblastoma and MDA-MB435 melanoma), and
peripheral blood mononuclear cells (PBMC). The cytotoxic potential
of compounds against tumor cells was assessed by the MTT color-
imetric assay and the investigation on the selectivity of these iso-
benzofuranones toward a normal proliferating cell was performed
with PBMC by means of the Alamar Blue test. For both cytotoxic
assays, cells were treated with the test compounds for 72 h using
2. Results and discussion
2.1. Chemistry
As outlined in Scheme 1, our approach for the synthesis of the
title compounds involved the use of inexpensive anacardic acids (2)

3482
L.P.L. Logrado et al. / European Journal of Medicinal Chemistry 45 (2010) 3480e3489
OH
OMe
O
O
COOH
R
2
C13H27
X
8
8
1a: X = OH, H
1b: X = O
R =
11
11
14
8
Fig. 3. Isobenzofuran-1-ones (1a and 1b) and anacardic acids (2).
a concentration range of 0.39e25.00
m
g/mL, and doxorubicin
The antiproliferative effects of the cytotoxic compounds 6, 1a
and 1b were further examined using HL-60 cells, a well established
cell line suitable for examining the effects of test drugs on cell
proliferation, cell cycle, cell differentiation, and apoptosis events
[19,20]. Proliferation capacity was measured by incorporation of
BrdU, a thymidine analog that is incorporated into DNA during the S
phase and can be detected by immunocytochemistry [21]. As
shown in Fig. 5, over a period of 24 h compounds 6 and 1b
decreased the percentage of proliferating BrdU-positive HL-60
cells, while compound 1a did not affect DNA synthesis in HL-60
cells at the concentrations tested. These results corroborate the
data obtained from the MTT assay.
While attempting to determine the mechanism responsible for
their antiproliferative effects, the induction of apoptosis or necrosis,
and DNA integrity of cells treated with the compounds was assayed.
As shown in Fig. 6A and C, after AO/BE staining compounds 6 and
1b reduced the number of viable cells in a concentration-depen-
dent manner after 24 h of exposure. However, the effect on cell
viability was more pronounced in HL-60 cultures treated with 1b.
Unlike the above compounds, 1a slightly reduced cell viability only
at the higher concentration (Fig. 6B). The mechanism of induction
of cell death appears to differ among the compounds. The anti-
proliferative capacity of compound 6 and 1a seem to be related to
the apoptosis activation. On the other hand, in HL-60 cells treated
(DoxolemÒ) as the positive control. The experiments were analyzed
by non-linear regression using the software GraphPad Prism 4.0,
where each sample was tested in triplicate from two independent
experiments.
As shown in Table 1, the acyclic precursors 4 and 5 did not inhibit
the proliferation of any cancer cell line or PBMC in the performed
cytotoxicity screening. Compound 6 showed moderate activity
againstmammalianHL-60(IC50 9.96
mL) cells, significant cytotoxicity against MDA-MB435 (IC50 3.31
mL)cells and lowactivityagainstPBMCcells (IC50 19.80
target compounds, 1a exhibited low activity against HL-60 (IC50
21.00 g/mL)and SF295 (IC50 > 25 g/mL) cells, and moderate activity
against MDA-MB435 cells (IC50 12.27 g/mL), while 1b exhibited
a significant cytotoxic effect against HL-60 cells (IC50 3.24 g/mL),
moderate activity on SF295 (IC50 10.09 g/mL) and MDA-MB435 (IC50
8.70 g/mL)celllines. Doxorubicinstronglyinhibitedtheproliferation
of all three cancer cell lines tested with IC50 values ranging from 0.02
to 0.04 g/mL. The two isobenzofuranones exhibited weak to
moderated activities against PBMC (1a, IC50 > 25 g/mL; 1b, IC50
14.51 g/mL), while doxorubicin displayed a potent antiproliferative
effect on PBMC (IC50 0.96 g/mL). The hemolytic assay showed that
the cytotoxicity of the compounds does not appear to affect the
mg/mL)and SF295 (IC50 12.83
mg/
mg/
mg/mL). Of the
m
m
m
m
m
m
m
m
m
m
integrity of cell membranes (EC50 > 100 mg/mL).
OMe
OR
OMe
CO2Me
C15H31
CO2R
CO2Me
14H29
b
c
C
C15H31-n
Br
4
2: R = H
3: R = Me
5
a
OMe
O
OMe
d
CO2Me
O
f
C
13H27
C13H27
X
6
1a: X = OH, H
1b: X = O
e
Scheme 1. Reagents and conditions: (a) Me2SO4, CH2Cl2, NaOH, Aliquat 336Ò, rt; (b) H2, Pd/C, 4 atm, rt; (c) NBS, benzoyl peroxide, CCl4, reflux, under nitrogen atmosphere; (d) DBU,
PhH, reflux; (e) m-CPBA, CH2Cl2, rt (98%); (f) Jones reagent, ether, rt.

L.P.L. Logrado et al. / European Journal of Medicinal Chemistry 45 (2010) 3480e3489
3483
23
24
80
OMe
OMe
O
70
22
23
1
1
CO2Me
6
5
*
6
5
22
2
3
2
60
50
40
30
20
10
0
O
*
3
*
7
7
4
4
9
9
8
8
*
10-18
10-18
*
X
( )
( )
*
19
19
21
21
20
20
Fig. 4. Atom numbering for spectral data listing.
C
D
1
3
6
1
3
6
1
3
6
μg/mL
with compound 1b there was a large increase in the number of
necrotic cells with increasing concentration.
1a
6
1b
Cell death by apoptosis or necrosis is a process that leads to DNA
degradation and can be detected by flow cytometry analysis. Our
data revealed that all evaluated compounds induce an increase on
the percentage of degraded DNA after 24 h of exposure (Fig. 7A).
During early stages of apoptosis, the cell membrane is impermeable
to vital dyes such as propidium iodide [22], and the opposite situ-
ation occurs during late apoptosis or necrosis. After 24 h of treat-
Fig. 5. Effect of compounds on 5-bromo-2-deoxyuridine (BrdU) incorporation by HL-
60 cells. Negative control (C) was treated with the vehicle (0.1% DMSO) used for
diluting the test substance. Doxorubicin (D) at 0.3 mg/mL was used as positive control.
*p < 0.05 compared to control by ANOVA followed by Newman Keuls test. Data are
presented as mean values ꢀ S.E.M. from three independent experiments in triplicate of
percent of BrdU positivity.
ment, compounds 6 and 1a at 6 mg/mL and 1b (3 and 6 mg/mL)
reduced the percentage of cells with intact cell membranes
(Fig. 7B), which reinforces our morphological analysis (AO/BE
staining method).
results indicated that interference with the mechanisms of DNA
repair may be involved in the cytotoxicity of 1b. Moreover, the
present study provides evidence that the mechanisms of cell growth
inhibition, cell death, and DNA damage of active compounds do not
depend on the production of free radicals (TBARS assay).
Several mechanisms exist by which the compounds could
potentially exert the observed cell killing effects in HL-60 cells.
Herein, we examined whether the generation of free radicals or
direct DNA damage could be related to the activity of the test
compounds. Compound 1b induced DNA damage after a short
incubation period (3 h), as shown in Fig. 8C by the alkaline comet
assay analysis, where longer exposure times led to an increase in
the extent of DNA damage in a time- and concentration-dependent
manner. On the other hand, DNA strand breaks induced by
compounds 6 and 1a occurred only after 12 h of exposure at the
highest concentration (Fig. 8A and B). As shown in Fig. 8D, only 1b
was able to produce DNA double strand breaks (DSBs) after the 24 h
treatment. DSBs are considered the most biologically important
lesions, primarily because of the close relationship with chromo-
some damage, which in turn is closely linked to cell death [23].
Interestingly, DNA breaks induced by compounds 6 and 1a were
repairable to control levels after 1 h, while the DNA lesions induced
by 1b were not repaired (Fig. 9A). Even though the DNA lesions
produced by compounds 6 and 1a could be repaired, no increase in
cell survival was observed after 72 h (data not shown). Repair itself
does not always increase survival, and survival is the outcome of the
actions of several pathways which can be both cell- and tissue-
specific. To determine whether the increase of BrdU incorporation
(UDS detection assay) is due to specific DNA synthesis or to DNA
damage repair, the cells were treated with hydroxyurea. As shown in
Fig. 9B, cells treated with 1b showed significantly less incorporation
of BrdU when compared with cells treated with 6 and 1a. These
According to the cytotoxicity screening, some interesting
conclusions can be drawn. Considering the acyclic precursors, the
data show that the cytotoxic profile can be affected by the intro-
duction of a double bond between C7 and C8. In this case,
compound 6 showed improvements in relation to the activities of
saturated analogue 4 and the enantiomeric mixture of the bromo
derivative 5, having the best activity against the melanoma cells
(MDA-MB435) among all tested compounds, and the second best
profile regarding the other cell lines. These results suggest that the
presence of a planar region linked to the aromatic moiety modu-
lates the activity sketch and suggests that this subunit could reach
a hydrophobic or cationic region which can interact with the pi
electrons of the double bond. Concerning the conformational
aspects, the more rigid analogue would favor a better interaction
with the hydrophobic or cationic region. Moreover, the participa-
tion of free radicals is expected, and therefore, the allylic hydrogen
on carbon C9 can act as a scavenger in an antioxidant fashion with
stabilization of the new radical species by resonance with the allylic
and aromatic conjugated systems.
Taking into account the profile of isobenzofuranones in the
same cell line, in spite of low cytotoxicity, the presence of
a hydrogen bond acceptor (HBA) seems to be more important than
a hydrogen bond donor (HBD), based on the activities of 1b (2.62-
fold less) and 1a (3.70-fold less), respectively. It should be noted
that the ketone group can also act as an electrophilic site, and may
Table 1
Cytotoxicity screening of isobenzofuranones (1a and 1b), and the precursors compounds 4, 5 and 6 based on tumor cell growth inhibition.
Compound
Cell lines CI50 mg/mL (confident range)
HL-60
SF295
MDAMB-435
PBMC
4
>25
>25
>25
>25
5
>25
>25
>25
>25
6
1a
1b
9.96 (8.62e11.51)
21.00 (16.49e26.75)
3.24 (2.93e3.59)
0.02 0.01e0.02
12.83 (10.57e15.58)
>25
10.09 (8.10e12.55)
0.04 0.03e0.05
3.31 (2.35e4.67)
12.27 (9.41e15.99)
8.70 (7.53e10.05)
0.03 0.02e0.04
19.80 (18.55e21.13)
>25
14.51 (10.00e21.05)
0.96 (0.50e1.70)
Doxorubicina
a
Positive control.

3484
L.P.L. Logrado et al. / European Journal of Medicinal Chemistry 45 (2010) 3480e3489
A
300
200
100
0
300
B
Viable
Viable
Apoptotic
Necrotic
*
Apoptotic
Necrotic
*
*
200
*
*
*
*
*
*
100
*
*
*
*
0
C
D
1
3
6
C
D
1
3
6
μg/mL
μg/mL
6
1a
C 300
*
Viable
Apoptotic
Necrotic
*
200
*
*
*
100
*
*
*
6
*
0
C
D
1
3
μg/mL
1b
Fig. 6. Effect of compounds on HL-60 cell viability using acridine orange and ethidium bromide staining as determined by fluorescence microscopy after 24 h of exposure [6 (panel
A), 1a (panel B), and 1c (panel C)]. Negative control (C) was treated with the vehicle (0.1% DMSO) used for diluting the test substance. Doxorubicin (D) at 0.3 g/mL was used as
m
positive control. *p < 0.05 compared to control by ANOVA followed by Newman Keuls test. Data are presented as mean values ꢀ S.E.M. from three independent experiments in
triplicate.
interact with nucleophiles in the biophase. Considering the cyto-
toxic profile against HL-60 cells, it was observed that 1b was 3.1-
fold and 6.5-fold more active than 6 and 1a, respectively. These data
suggest that electronic interaction (HBA) is more important for
activity, followed by hydrophobic interaction, while the HBD group
made a minor contribution not only with this cell line, but also with
SF295 and PBMC cells, while 1a was inactive. With PBMC and SF295
cells, 1b and 6 showed cytotoxicity in the same range, exhibiting
a moderate to low profile.
In conclusion, owing to the improved cytotoxic activity against
MDA-MB435, the planar region of the double bond of 6 was shown
to be important for the cytotoxic profile. Also should be remem-
bered that conformational effects and specific interaction with
complementary regions or even acting as scavenger group may be
involved in the activities. The ketone group in 1b, as an HBA group,
plays an important role in the activity of this compound, particu-
larly against HL-60 cells, which displayed a discriminatory cyto-
toxic profile and may be considered a satisfactory lead that
warrants further studies towards antineoplastic drugs. Taken
together, the results indicate that active compounds studied here
cause DNA damage, triggering apoptosis or necrosis induction.
Furthermore, the present results underscore that the studies
involving isobenfuranones should be continued to access struc-
tureeactivity relationship of therapeutical potentials.
phosphomolybdic acid, sulphuric vanillin or iodine vapours to
visualize the spots. Melting points were obtained on a Köffler
apparatus and are uncorrected. IR-FT spectra were recorded on
a Bomem Hartmann & Braun (MB e 100) spectrometer. 1H and 13
C
NMR uni- and bi-dimensional (COSY, HMQC, HMBC, and DEPT)
were recorded on Mercury plus Varian (7.05 T) spectrometer. The
1H and 13C NMR chemical shifts are reported in parts per million
(ppm) relative to TMS (1H NMR) and CDCl3 13C NMR). Coupling
(
constants were reported as J (Hz) and multiplicities with abbrevi-
ations d, doublet; dd, double doublet; t, triplet, and br, broad.
Elementary analyses were carried out on CNH 2400 PerkineElmer.
3.1.1. Extraction of CNSL and isolation of anacardic acids (2)
Fresh and dry cashew nuts of A. occidentale were deshelling and
the shells were reduced in order to ascertain efficient extraction. The
shell pieces (500 g) were extracted with commercially 95% ethanol
(2000 mL), in a Soxhlet apparatus, for a period of 6 h. The clear
solution was concentrated under reduced pressure using a rotary
evaporator at 50 ꢁC giving a brown oily product (CNSL, 157 g, 31% by
weight). Isolation of anacardic acids (2) was performed according to
Paramashipvappa et al. [17] with slight modification. The procedure
involved dissolving crude CNSL (20 g) in 5% aqueous methanol
(120 mL), and calcium hydroxide (10 g) was added in portions under
stirring. After complete addition of calcium hydroxide, the temper-
ature of reaction mixture was raised to 50 ꢁC and stirring was
continued for 3 h. The supernatant solution was monitored by TLC
(hexaneeethyl acetate, 2:1) for the absence of anacardic acids. After
completion of the reaction, the precipitated calcium anacardates
was vacuum-filtered and washed thoroughly with 5% aqueous
methanol (3 ꢂ 30 mL). The wet crushed cake was carefully trans-
ferred into a flask contained a stirred mixture 6 M HCl (100 mL) and
ethyl acetate(150 mL) and left for 1 h. The organic phasewas washed
with distilled water (3 ꢂ 50 mL), dried over anhydrous sodium
sulphate, filtered, and concentrated at reduced pressure in a rotary
evaporator to yield 11.65 g (c.a. 60%) of heterogeneous mixture of
3. Experimental protocols
3.1. Instrumentation, chemicals and solvents
Fresh and dry cashew nuts of A. occidentale were obtained from
Iracema Indústrias de Caju Ltda (Ceará, Brazil). Catalytic hydroge-
nations were performed in a Parr apparatus. Flash column chro-
matography and dry-column flash chromatography were carried
ꢀ
out using silica gel Merck 60 A (70e230 mesh) and eluting with
a gradient of hexane:ethyl acetate. Analytical thin layer chroma-
tography (TLC) was performed on Merck precoated silica gel
(60F254/0.2 mm) plates using UV light, 5% ethanolic
anacardic acids (monoene, diene, and triene), IR (KBr) ymax/cmꢃ1
3459, 3500e2500, 3010, 2926, 2854, 1646, 1608, 1449, 1303, 1246,
:

L.P.L. Logrado et al. / European Journal of Medicinal Chemistry 45 (2010) 3480e3489
3485
A
B
100
50
40
30
20
10
0
**
**
*
*
*
**
75
**
**
**
**
**
50
25
*
*
*
0
C
D
1
3
6
1
3
6
1
3
6
C
D
1
3
6
1
3
6
1
3 6
μg/mL
μg/mL
6
1a
1b
6
1a
1b
Fig. 7. Effect of compounds on HL-60 DNA fragmentation (panel A), and cell membrane integrity (panel B) determined by flow cytometric analysis after 24 h of incubation. Negative
control (C) was treated with the vehicle (0.1% DMSO) used for diluting the test substance. Doxorubicin (D) at 0.3 g/mL was used as positive control. Five thousand events were
m
analyzed in each experiment. *p < 0.05; **p < 0.001 compared to control by ANOVA followed by Newman Keuls test. Data are presented as mean values ꢀ S.E.M. from three
independent experiments in triplicate.
1211, 1167. 1H NMR (300 MHz, CDCl3)
d
:11.00 (br, mixed Ar-OH/-
pressure, and the brown solid was recrystallized from hexane to
afford 4 as white solid (9.8 g, 96%, mp 31e32 ꢁC). IR (KBr) ymax
cmꢃ1: 2925, 2854, 1736, 1585, 1470, 1431, 1268, 1188, 1110. 1H NMR
(300 MHz, CDCl3) : 7.26 (t, 8.4, H5), 6.82 (d, 7.8, H4), 6.77 (d, 8.7,
H6), 3.91 (s, H22), 3.82 (s, H23), 2.53 (t, 7.5, H7), 1.54 (m, H8), 1.25
(m, H9eH20), 0.879 (t, 6.6; H21). 13C NMR (75 MHz, CDCl3)
: 169.1
COOH), 7.35 (t, 8.2, Ar-H), 6.87 (d, 8.4, Ar-H), 6.77 (d, 7.5, Ar-H),
5.88e5.72 (m, -CH]CH2), 5.48e5.26 (m, ]CH-), 5.10e4.94 (m, ]
CH-), 2.98 (t, 7.8, ArCH2e), 2.86e2.72 (m, ]CHCH2CH]), 2.10e1.94
(m, ]CHeCH2), 1.41e1.23 (br, eCH2e, aliphatic side chain), 0.87(t,
/
d
eCH3). 13C NMR (75 MHz, CDCl3)
d
: 176.2 (C]O), 163.6, 147.8, 135.4,
d
129.8, 112.7 and 115.8 (aromatic carbons), 129.9e110.4 (olefinic
carbons), 36.4 (benzylic carbons), 31.9e22.6, (methylenic carbons),
14.1 (terminal methyl groups).
(C23), 156.4 (C1), 141.6 (C3), 130.4 (C5), 123.7 (C2), 121.7 (C4), 108.5
(C6), 56.0 (C24), 52.2 (C22), 33.7 (C7), 32.1 (C19), 31.3 (C8),
29.5e29.9 (C9eC18), 22.9 (C20), 14.3 (C21). Anal. Calcd. for
C24H40O3: C, 76.55, H, 10.57; Found: C, 75.48, H, 11.21.
3.1.2. 2-Methoxy-6-pentadecylbenzoic acid methyl ester (4)
A solution of 3 (10 g, 29.0 mmol for average molecular wt 376) in
dried ethanol (30 mL), was mixed with 10% palladium on carbon
(0.22 g, 2 mol %) and shaken in a Parr apparatus, under hydrogen
atmosphere (4 atm) until the reaction was shown to be complete by
TLC (hexaneeethyl acetate, 2:1). After 2 h, the reaction mixture was
filtered over CeliteÒ. The filtrate was concentrated under reduced
3.1.3. 2-(1-Bromo-pentadecyl)-6-methoxybenzoicacid methyl ester (5)
A mixture of the ester 4 (2.44 g, 6.48 mmol), NBS (1.39 g,
7.8 mmol) and benzoyl peroxide (0.064 g, 0.26 mmol) in benzene
(100 mL) was refluxed for 6 h, under an argon atmosphere. The
resultant brown mixture was filtered through a glass-fritted funnel.
Water (50 mL) was added to the filtrate and then extracted with
A
B
250
*
250
C
*
C
D
D
200
*
200
*
1 μg/mL
3 μg/mL
6 μg/mL
*
1 μg/mL
3 μg/mL
6 μg/mL
*
150
100
50
150
100
50
*
*
*
*
*
*
*
0
0
3h
6h
12h
24h
3h
6h
12h
24h
1a
6
D
C
250
200
150
100
50
250
200
150
100
50
*
C
D
*
1 μg/mL
3 μg/mL
6 μg/mL
*
*
*
*
*
*
*
*
*
*
*
*
0
0
3h
6h
12h
24h
C
6
1a
1b
compounds
1b
Fig. 8. Effect of compounds on cell damage index by the alkaline version of the comet assay (panels AeC) after different exposure times (3, 6, 12 and 24 h). Panel D showed the
induction of DSBs after 24 h treatment with compounds at 6 g/mL. Negative control (C) was treated with the vehicle (0.1% DMSO) used for diluting the test substance. Doxorubicin
(D) at 0.3 g/mL was used as positive control. *p < 0.05 compared to control by ANOVA followed by Newman Keuls test.
m
m

3486
L.P.L. Logrado et al. / European Journal of Medicinal Chemistry 45 (2010) 3480e3489
A
B
250
200
150
100
50
30
1h
3h
6h
12η
24h
*
20
10
0
*
*
*
*
*
*
0
C
6
1a
1b
C
6
1a
1b
compounds
compounds
Fig. 9. Kinetics of DNA repair (panel A), and UDS detection (panel B) after 12 h exposure with compounds at 6
incorporation test, respectively, after 12 h exposure with compounds at 6 g/mL. Negative control (C) was treated with the vehicle (0.1% DMSO) used for diluting the test substance.
Doxorubicin (D) at 0.3 g/mL was used as positive control. *p < 0.05 compared to control by ANOVA followed by Newman Keuls test.
mg/mL by the alkaline version of the comet assay and BrdU
m
m
ethyl acetate (3 ꢂ 50 mL). The combined organic extracts were
washed with saturated sodium bicarbonate solution (50 mL), brine
(2 ꢂ 50 mL), and dried over sodium sulphate. The organic phase
was concentrated to yield a yellow solid, which was recrystallized
from hexane to afford bromide 5 as white crystals (2.94 g, 100%, mp
57e58 ꢁC). IR (KBr) ymax/cmꢃ1: 2952, 2917, 2848, 1731, 1593, 1474,
3.1.5.2. Method (B). Into a flask (10 mL) were added 30% hydrogen
peroxide (0.12 mL) and formic acid (0.70 mL). The mixture was
stirred with gentle heating, and after 5 min the olefin 7 (0.153 g;
0.42 mmol) dissolved in hexane was added. The mixture was stir-
red with heating until all starting material was consumed (TLC,
dichlorometano), in approximately 1.5 h. At the end of this time,
a solution of sodium bicarbonate (20 mL) was added and the
mixture was extracted with ethyl acetate (2 ꢂ 20 mL). The organic
phase was washed with saturated sodium bicarbonate solution
(20 mL), brine (2 ꢂ 20 mL). After drying over sodium sulphate and
evaporation of the solvent, a white solid was obtained, which was
purified by recrystalization in hexane to yield 1a (0.117 g, 74%; m.p.
79e80ꢁC). IR (KBr) ymax/cmꢃ1: 3378, 2915, 2849, 1777, 1742, 1611,
1488, 1470, 1286, 1202, 1079, 1046, 1008. 1H NMR (300 MHz, CDCl3)
1268, 1109, 1071. 1H NMR (300 MHz, CDCl3)
d: 7.38 (t, 8.1, H5), 7.21
(dd, 8.1, 0.9, H4), 6.84 (dd, 8.4, 0.6, H6), 4.91 (dd, 8.1, 6.9, H7), 3.94 (s,
H22), 3.83 (s, H23), 2.15 (m, H8), 1.44 (m, H9), 1.25 (m, H10-H20),
0.88 (t, 6.6, H21). 13C NMR (75 MHz, CDCl3)
d: 167.9 (C23), 156.0
(C1), 140.7 (C3), 122.6 (C2), 119.8 (C4), 131.1 (C5), 110.6 (C6), 56.3
(C24), 52.7 (C22), 50.9 (C7), 40.2 (C8), 32.2 (C19), 28.3e30.0 (C9-
C18), 23.0 (C20), 14.5 (C21). Anal. Calcd. for C24H39BrO3: C, 63.29, H,
8.63, Br, 17.54, Found: C, 65.15, H, 8.64, Br, 15.71.
d
: 7.61 (dd, 8.4, 7.7, H5), 7.10 (d, 7.7, H6), 6.95 (d, 8.4, H4), 5.32 (d, 4.9,
H7), 3.99 (s, H23), 3.91 (m, H8), 1.56 (bl, H9), 1.25 (m, H10-H20),
0.88 (t, 6.5, H21). 13C NMR (75 MHz, CDCl3)
: 168.8 (C22), 158.8
3.1.4. 2-Methoxy-6-pentadec-1-enylbenzoic acid methyl ester (6)
To a solution of bromide 5 (3.88 g, 8.53 mmol) in benzene (40 mL)
was added DBU (3.8 mL, 25.5 mmol). After stirring under reflux and
an argon atmosphere for 10 h, the reaction mixture was quenched
with 10% hydrochloric acid (50 mL) and then extracted with ethyl
acetate (3 ꢂ 50 mL). The combined organic extracts were washed
with 10% hydrochloric acid (50 mL), brine (2 ꢂ 30 mL), and dried over
sodium sulphate. The organic phase was concentrated to give
a yellow solid, which was recrystallized from hexaneeethyl acetate
to afford the unsaturated compound 6 as white crystals (3.07 g, 96%,
mp 33e34 ꢁC). IR (KBr) ymax/cmꢃ1: 2924, 3853, 1736, 1650, 1597,
1577, 1470, 1431, 1268, 1188, 1112, 1079, 1067. 1H NMR (300 MHz,
d
(C1), 150.1 (C3), 136.3 (C5), 114.9 (C4), 114.1 (C2), 111.1 (C6), 82.8
(C7), 73.0 (C8), 52.1 (C23), 32.3 (C9), 32.0 (C19), 25.7e29.8
(C10eC18), 22.8 (C20), 14.2 (C21). Anal. Calcd. for C23H36O4: C,
73.37, H, 9.64, Found: C, 69.13, H, 9.25.
3.1.6. 7-Methoxy-3-tetradecanoyl-3H-isobenzofuran-1-one (1b)
To a solution of alcohol 1a (0.10 g, 0.265 mmol) in ether (2 mL)
was added Jones reagent (3.0 mL). After stirring for 3 h, the reaction
mixture was quenched with ethanol (about 0.5 mL), diluted with
brine (10 mL) and then extracted with ethyl acetate (3 ꢂ 10 mL). The
combined organic extracts were washed with brine (3 ꢂ10 mL), and
dried over sodium sulphate. The organic phase was concentrated to
give a pale yellow solid which was flash chromatographed on silica
gel (hexaneeethyl acetate, 7:3) affording the pure keto compound
CDCl3)
H7-H8), 3.93 (s, H22), 3.82 (s, H23), 2.18 (q, 6.6, H9), 1.44 (m, H10),
1.26 (m, H11eH20), 0.88 (t, 6.6, H21). 13C NMR (75 MHz, CDCl3)
d: 7.28 (t, 8.4, H5), 7.10 (d, 7.8, H4), 6.77 (d, 8.4, H6), 6.24 (m,
d:
168.6 (C23), 156.3 (C1), 136.6 (C3), 134.7 (C8), 130.1 (C5), 126.0 (C7),
122.1 (C2),117.7 (C4),109.0 (C6), 55.8 (C24), 52.1 (C22), 33.1 (C9), 31.8
(C19), 29.0e29.6 (C10eC18), 22.6 (C20), 14.0 (C21). Anal. Calcd. for
C24H38O3: C, 76.96, H, 10.23, Found: C, 78.62, H, 9.39.
1b as white crystals (97 mg, 96%, mp 84 ꢁC). IR (KBr) ymax/cmꢃ1
2923, 2852,1773,1724,1611,1601,1489,1446,1218,1194,1042,1019.
1H NMR (300 MHz, CDCl3)
: 7.64 (dd, 8.1, 7.5, H5), 7.17 (d, 7.5, H6),
:
d
6.99 (d, 8,4, H4), 5.61 (s, H7), 4.01 (s, H23), 2.68 (ddd, 18.6, 6.3, 8.4,
3.1.5. 3-(1-Hydroxy-tetradecyl)-7-methoxy-3H-isobenzofuran-1-
one (1a)
H9), 2.37 (m, H90), 1.25 (m, H10eH20), 0.88 (t, H21). 13C NMR
(75 MHz, CDCl3) d: 250.6 (C8), 167.7 (C22), 158.4 (C1), 146.2 (C3),
3.1.5.1. Method (A). A solution of m-CPBA (2.63 g, 5.6 mmol) in
dichloromethane (20 mL) was added drop-wise to a solution of
compound 7 (1.0 g, 2.76 mmol) in dichloromethane (10 mL). The
reaction mixture was stirred for 3 h, and then diluted with ethyl
acetate (50 mL). The organic phase was washed with saturated
sodium bicarbonate solution (20 mL), brine (2 ꢂ 20 mL), and dried
over sodium sulphate. The organic phase was concentrated to give
a white solid, which was recrystallized from hexane to afford the
alcohol 1a as white crystals (0.99 g, 98%, mp 79e80 ꢁC).
136.7 (C5),114.3 (C4),111.9 (C2),111.5 (C6), 83.1 (C7), 56.0 (C23), 37.7
(C9), 31.8 (C19), 22.5e29.6 (C10eC18), 22.6 (C20), 14.1 (C21). Anal.
Calcd. for C23H34O4: C, 73.76, H, 9.15, Found: C, 72.54, H, 9.90.
3.2. Biological evaluation
Fetal bovine serum and phytohaemagglutinin were purchased
from Cutilab (Campinas, SP, Brazil), and RPMI 1640 medium, trypsin-
EDTA, penicillin and streptomycin were purchased from GIBCOÒ

L.P.L. Logrado et al. / European Journal of Medicinal Chemistry 45 (2010) 3480e3489
3487
(Invitrogen, Carlsbad, CA, USA). 5-Bromo-20-deoxyuridine and
hydroxyureawere obtained fromSigma Chemical Co. (St, Louis, MO).
Doxorubicin (DoxolemÒ) was from Zodiac Produtos Farmacêuticos
S/A, Brazil. All other chemicals and reagents used were of analytical
grade. The human cell lines, HL-60 (promyelocytic leukemia), MDA-
MB435 (melanoma) and SF295 (glioblastoma), were all obtained
from National Cancer Institute (Bethesda, MD, USA). The cells were
maintained in RPMI 1640 medium supplemented with 10% fetal
some modifications. Each well received 50
containing 10 mM CaCl2. DMSO (0.1%) was used as a negative
control. The substances were tested at concentrations ranging from
m
l of 0.85% NaCl solution
1.56 to 100
(in 0.85% NaCl) to obtain 100% haemolysis. Next, each well received
50 L of a 2% suspension of mouse erythrocytes in 0.85% NaCl
mg/mL. The last well received 50 mL of 0.2% Triton X-100
m
containing 10 mM CaCl2. After incubation at room temperature for
1 h, and centrifugation, the supernatant was removed and the
hemoglobin released was measured spectrophotometrically
(Spectra Count, Packard, Ontario, Canada) as absorbance at 540 nm.
bovine serum, 2 mM glutamine, 100 U/mL penicillin and 100 mg/mL
streptomycin at 37 ꢁC with 5% CO2. The cells were grown in complete
medium one day before each experiment. For all experiments, cells
were seeded at 0.3 ꢂ 106 (HL-60), 0.6 ꢂ 105 (SF295) and 0.1 ꢂ 106
(MDA-MB435) cells/mL. Heparinized blood (from healthy, non-
smoker donors who had not taken any drug for at least 15 days prior
to sampling) was collected, and peripheral blood mononuclear cells
(PBMC) were isolated by a standard method of density-gradient
centrifugation over Histopaque-1077. PBMC were washed and
resuspended in RPMI 1640 medium supplemented with 20% fetal
3.2.2.2. Lipid peroxidation (TBARS) assay. Lipid peroxidation was
measured in cell cultures as previously described [26]. Briefly, after
exposure of cells to test compounds (3, 6, 12 and 24 h), the super-
natant was discarded and HL-60 cells were lysed with Triton X-100.
Next, 250
mixtures were incubated in a water bath at 37ꢁ C for 1 h, followed
by the addition of 400 L of 35% perchloric acid to stop lipid per-
oxidation. The mixture was centrifuged at 1400 g for 10 min, and
200 L of 1.2% sodium 2-thiobarbiturate solutions were added to
the supernatant (600 L). The glass tubes were then placed in
mL of the lysed cells were added to glass tubes and the
m
bovine serum, 2 mM glutamine, 100 U/mL penicillin, 100 mg/mL
streptomycin at 37 ꢁC with 5% CO2. Phytohaemagglutinin (2%) was
added at the beginning of culture. After 24 h of culture, cells were
treated with the test compounds.
m
m
a water bath and heated at 95ꢁ C for 30 min. After cooling, absor-
bance was measured using a multiplate reader (Spectra Count,
Packard, Ontario, Canada) at 535 nm. Experiments were performed
in triplicate in three independent experiments.
3.2.1. Cytotoxic screening
3.2.1.1. Inhibition of tumor cell proliferation. Tumor cell growth was
quantified by the ability of living cells to reduce the yellow dye 3-
(4,5-dimethyl-2-thiozolyl)-2,5-diphenyl-2H-tetrazlium bromide
(MTT) to a purple formazan product [24]. For the experiments, cells
were seeded in 96-well plates. After 24 h, compounds
3.2.2.3. Inhibition of DNA synthesis. HL-60 cells (0.3 ꢂ 106 cells/mL)
were seeded in 24-well tissue culture plates (2 mL/well) and
treated with the test compounds for 24 h. Doxorubicin (0.3 mg/mL)
(0.39e25.00
m
g/mL) dissolved in DMSO (0.1%) were added to each
was used as a positive control. Ten microliters of 5-bromo-20-
deoxyuridine (BrdU, 10 mM) were added to each well and incu-
bated at 37 ꢁC for 3 h before completing the 24 h period of drug
exposure. To assay the amount of BrdU incorporated into DNA [27],
cells were harvested and transferred to cytospin slides, which were
allowed to dry for 2 h at room temperature. Cells that had incor-
porated BrdU were labeled by direct peroxidase immunocyto-
chemistry utilizing the chromogen diaminobenzidine. Slides were
counterstained with haematoxylin, mounted, and coverslipped.
Evaluation of BrdU positivity was performed by light microscopy
(Olympus, Tokyo, Japan). Two hundred cells were counted per
sample to determine the percentage of positive cells.
well and incubated for 72 h. DMSO (0.1%) and doxorubicin were
used as negative and positive controls, respectively. Thereafter, the
plates were centrifuged, and the medium was then replaced by
fresh medium (150
later, the MTT formazan product was dissolved in 150
absorbance was measured using a multiplate reader (Spectra
Count, Packard, Ontario, Canada). Drug effect was quantified as the
percentage of control absorbance of reduced dye at 595 nm.
mL) containing 0.5 mg/mL MTT. Three hours
mL DMSO, and
3.2.1.2. Inhibition of PBMC proliferation e Alamar Blue assay. The
Alamar Blue assay was performed with PBMC after 72 h drug
exposure. After 24 h, compounds (0.39e25.00 mg/mL) dissolved in
DMSO (0.1%) were added to each well and incubated for 72 h.
Doxorubicin was used as positive control. Control groups received
the same amount of DMSO. Twenty-four hours before the end of
3.2.2.4. Morphological characterization of apoptotic and necrotic HL-
60 cells. Apoptotic and necrotic HL-60 cells were determined using
the acridine orange (AO)/ethidium bromide (EB) assay [28]. Briefly,
the incubation, 10
mL of stock solution (0.312 mg/mL) of Alamar
25
mL of the cell suspension were mixed with 1
mL of the staining
Blue (Resazurin, SigmaeAldrich Co) were 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 Alamar 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 alone is subtracted
from the absorbance of medium 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 ꢃ (AHW ꢂ R0) ꢂ 100.
solution (AO 100
a slide, and 300 cells were counted per data point.
m
g/mL þ EB 100
m
g/mL in PBS) and spread on
3.2.2.5. Cell membrane integrity. HL-60 cell membrane integrity
was evaluated by the exclusion of propidium iodide (50 mg/mL).
Aliquots were removed from cultures after 24 h of incubation with
test compounds. Cell fluorescence was then determined by flow
cytometry in Guava EasyCyte Mini (Guava Technologies, Inc., Hay-
ward, CA, USA) using Guava Express Plus software. Five thousand
events were evaluated per experiment.
3.2.2.6. Internucleosomal DNA fragmentation. Aliquots were
removed from HL-60 cell cultures after 24 h of incubation with test
compounds. Next, the aliquots were incubated at 37 ꢁC for 30 min in
the dark in a lysis solution containing 0.1% citrate, 0.1% Triton X-100
and 50 mg/mL propidium iodide. Cell fluorescence was then deter-
3.2.2. Analysis of mechanisms involved in the cytotoxic activity
3.2.2.1. Hemolytic assay. The test was performed in 96-well plates
following the method described by Costa-Lotufo et al. [25] with
mined by flow cytometry in a Guava EasyCyte Mini (Guava Technol-
ogies, Inc., Hayward, CA, USA) using Guava Express Plus software. The
percentage of degraded DNA was determined by the number of cells

3488
L.P.L. Logrado et al. / European Journal of Medicinal Chemistry 45 (2010) 3480e3489
displaying subdiploid(sub-Go/G1)DNAdivided bythetotal numberof
cells examined. Five thousand events were evaluated per experiment.
physical agents. UDS detection was carried out according to the
method of Bootsma et al. [35] with minor modifications. UDS was
assessed through the incorporation of BrdU in the presence of
hydroxyurea, a DNA synthesis inhibitor that does not inhibit repair
mechanisms [36]. In brief, HL-60 cells were treated for 12 h with
test compounds, and then washed twice with ice-cold PBS to
remove the compounds. Afterwards, the cells were immediately
incubated for an additional 30 min with medium containing 3 mM
hydroxyurea, and later, the culture medium was supplemented
with BrdU (10 mM). After 2 h incubation, cells were washed (PBS),
harvested and transferred to cytospin slides which were allowed to
dry for 2 h at room temperature. Cells that had incorporated BrdU
were labeled by direct peroxidase immunocytochemistry utilizing
the chromogen diaminobenzidine. Slides were counterstained with
haematoxylin, mounted, and coverslipped. Evaluation of BrdU
positivity was performed by light microscopy (Olympus, Tokyo,
Japan). Two hundred cells were counted per sample to determine
the percentage of positive cells.
3.2.2.7. DNA strand breaks. The comet assay, used to detect DNA
strand breaks, was conductedunderalkaline conditions as described
by Singh et al. [29] with modifications [30] and following the
recommendations of the International Workshop on Genotoxicity
Test Procedures [31]. HL-60 cells were exposed to compounds at
different times (3, 6, 12 and 24 h). After treatment, cells were
collected and processed for the assay as follows. Briefly, 15
cell suspension were mixed with 90 L of 0.75% low melting point
agarose in PBS at 37 ꢁC. A volume of 100
L of cell suspension was
mL of the
m
m
spread on a glass slide previously coated with a layer of 1.5% normal
melting point agarose in PBS; the slide was covered with a glass
coverslip and placed at 4 ꢁC for 15 min. The coverslip was gently
removed and the slide was submerged in ice-cold lysing solution
(2.5 M NaCl, 10 mM Tris, 0.1 mM EDTA, 1% sodium sarcosinate, 1%
Triton X-100 and 10% DMSO, pH 10) at 4 ꢁC for at least 1 h. After lysis,
the slides were placed in a horizontal gel electrophoresis chamber
with freshly-prepared alkaline buffer (300 mM NaOH and 1 mM
EDTA, pH >13.0). The slides were kept in this solution for 20 min at
4 ꢁC to allow unwinding of the DNA and expression of alkali-labile
sites. The samples were then subjected to electrophoresis in the
same solution at 300 mA and 20 V (0.81 V/cm) for 20 min at 4 ꢁC. The
neutral protocol was carried out following essentially the same
procedure as the alkaline version except for the pH value, and cells
were treated for 24 h. In the neutral version, electrophoresis was
performed in buffer consisting of 100 mM Tris and 300 mM sodium
acetate at pH 8.5 [32]. Electrophoresis was conducted for 60 min,
after a 60-min equilibrium period in neutral electrophoresis buffer,
at 12 mA and 14 V (0.5 V/cm). After electrophoresis, the slides were
rinsed gently three times (5 min each time) with 0.4 M TriseHCl (pH
Acknowledgments
Financial support from CNPq, FUNCAP and FINEP (Process
CTINFRA 970/2001, 1040091/2004 and contract 01.04.0091.00),
Universidade de Brasília, Universidade Federal do Ceará, and Uni-
versidade Católica de Brasília are gratefully acknowledged. CAPES
provided fellowship funds for LPLL. Authors are indebted to
Professor Dr. Inês Sabioni Resck, Universidade de Brasília for NMR
spectra data and also want to acknowledge Claude Bernard Insti-
tute. Dr. A. Levya provided English editing of the manuscript.
References
7.5). Each slide was stained with 50
m
L of ethidium bromide (20
mg/
[1] K. Kameda, M. Namiki, New phthalide from a fungus, Alternaria kikuchiana.
Chem. Lett. (1947) 1491e1942.
mL) and covered with a coverslip. The analysis of the cells was
performed by a visual scoring system [33]. Briefly, fluorescent-
stained nucleoids were scored visually using an epifluorescence
microscope (Olympus, Tokyo, Japan) with an excitation filter of
510e560 nm and a barrier filter of 590 nm at 400ꢂ magnification.
Three hundred randomlyselectedcells (100cells fromeach of the
three replicate slides) were analyzed for each concentration of test
substance. Cells werescored visuallyaccording totail lengthintofive
classes: (1) class 0: undamaged cells having no tail; (2) class 1: cells
having a tail shorter than the diameter of the head (nucleus); (3)
class 2: cells having a tail length 1e2 times the diameter of the head;
(4) class 3: cells having a tail longer than 2 times the diameter of the
head; (5) class 4: comets having no heads. Avalue (damage index, DI)
was assigned to each comet according to its class, using the formula:
[2] A.A. Stierle, R. Upadhyay, J. Hershenhorn, G.A. Strobel, G. Molina, The phyto-
toxins of Mycosphaerella fijiensis, the causative agent of Black Sigatoka disease
of bananas and plantains. Experientia 47 (1991) 853e859.
[3] U. Holler, J.B. Gloer, D.T. Wicklow, Biologically active polyketide metabolites
from an undetermined fungicolous hyphomycete resembling Cladosporium. J.
Nat. Prod. 65 (2002) 876e882.
[4] B.M. Trost, G.T. Rivers, J.M. Gold, Regiocontrolled synthesis of hydroxyph-
thalides. Synthesis of (ꢀ)-isoochracinic acid and a zealeranone intermediate. J.
Org. Chem. 45 (1980) 1835e1845.
[5] R. Jadulco, G. Brauers, R.A. Edrada, R. Ebel, V. Wray, Sudarsono, P. Proksch,
New metabolites from sponge-derived fungi Curvularia lunata and Cladospo-
rium herbarum. J. Nat. Prod. 65 (2002) 730e733.
[6] A. Covarrubias-Zúñiga, A. Gonzáles- Lucas, M.M. Domínguez, Total synthesis
of mycophenolic acid. Tetrahedron 59 (2003) 1989e1994.
[7] J. Fuska, A. Fuskova, P. Nemec, Vermistatin, an antibiotic with cytoyoxic
effects, produced from Penicillium-vermiculatum. Biologia 34 (1979) 735e739.
[8] N. Murtaza, S.A. Husain, T.B. Sarfaraz, B. Tahira, N. Sultana, S. Faizi, Isolation
and identification of vermistatin, ergosterol, esteric acid and mannitol,
metabolic products of Penicillium verruculosum. Planta Med. 63 (1997) 191.
[9] G. Ding, L. Shuchun, L. Guo, Y. Zhou, Y. Che, Antifungal metabolites from the
plant endophytic fungus Pestalotiopsis foedan. J. Nat. Prod. 71 (2008) 615e618.
DI ¼ ð0 ꢂ n0Þ þ ð1 ꢂ n1Þ þ ð2 ꢂ n2Þ þ ð3 ꢂ n3Þ þ ð4 ꢂ n4Þ
where n ¼ number of cells in each class analyzed. The damage
index thus ranged from 0 (completely undamaged: 100 cells ꢂ 0) to
400 (with maximum damage: 100 cells ꢂ 4) [34]. Doxorubicin
[10] C.B. Cui, M. Ubukata, H. Kakeya, Acetophthalidin,
a novel inhibitor of
mammalian cell cycle, produced by a fungus isolated from a sea sediment. J.
Antibiot. 49 (1996) 216e219.
[11] Y. Tominaga, N. Yoshioka, S. Kataoka, Synthesis of amino-pyrimidopyridazines
as chemiluminescent compounds by reaction of functionalized maleimide
with various amine derivatives. Heterocycles 43 (1996) 1597e1600.
[12] W.A. Ayer, S.K. Attah-Poku, L.M. Browne, H. Orszanska, The chemistry of the
blue stain fungi. Part 3. Some metabolites of Ceratocystis minor (Hedgcock)
Hunt. Can. J. Chem. 65 (1987) 765e769.
[13] S. Nomoto, K. Mori, Synthesis of acetophthalidin, a fungal metabolite which
inhibits the progression of the mammalian cell cycle. Liebigs Ann./Recueil
(1997) 721e723.
(0.3 mg/mL) was used as positive control.
3.2.2.8. Kinetics of DNA repair. The alkaline version of the comet
assay was applied to assess whether DNA damage induced by the
active compounds could be repaired. Briefly, HL-60 cells were
exposed to compounds for 12 h. After treatment, the compounds
were removed, and the cultures were then reincubated. Samples of
treated cells were collected at different times (1, 3, 6, 12 and 24 h)
and the comet assay was conducted as described above.
[14] T. Kitahara, K. Uchida, H. Watanabe, H. Usui, H. Osada, Synthesis and bioac-
tivities of acetophthalidin and its derivatives. Heterocycles 48 (1998)
2049e2059.
[15] H. Watanabe, K. Uchida, T. Kitahara, Synthesis of both enantiomers of ace-
tophthalidin. Heterocycle 46 (1997) 45e48.
[16] I.S. Resck, M.L. dos Santos, L.A.S. Romeiro, New application of triphosgene in
a convenient synthesis of 3-aryl-1,3-benzoxazine-2,4-diones from anacardic
acids. Heterocycles 65 (2005) 311e318.
3.2.2.9. Unscheduled DNA synthesis (UDS). The UDS test measures
the DNA repair synthesis after excision and removal of a stretch of
DNA containing the region of damage induced by chemical and

L.P.L. Logrado et al. / European Journal of Medicinal Chemistry 45 (2010) 3480e3489
3489
[17] R. Paramashivappa, P. Phani Kumar, P.J. Vithayathil, A. Srinivasa Rao, Novel
method for isolation of major phenolic constituents from cashew (Anacardium
occidentale L.) nut shell liquid. J. Agric. Food Chem. 49 (2001) 2548e2551.
[18] L.P.L. Logrado, D. Silveira, L.A.S. Romeiro, M.O. de Moraes, B.C. Cavalcanti, L.
V. Costa-Lotufo, C. Pessoa, M.L. dos Santos, Synthesis and biological evaluation
of new salicylate macrolactones from anacardic acids. J. Braz. Chem. Soc. 16
(2005) 1217e1225.
[19] S.J. Collin, The promyelocytic leukemia cell line: proliferation, differentiation,
and cellular oncogene expression. Blood 70 (1987) 1233e1244.
[20] G.C.G. Militão, I.N.F. Dantas, C. Pessoa, M.J.C. Falcão, E.R. Silveira, M.A.S. Lima,
R. Curi, T. Lima, M.O. Moraes, L.V. Costa-Lotufo, Induction of apoptosis by
pterocarpans from Plastymiscium foribundum in human leukemia cells. Life
Sci. 78 (2006) 2409e2417.
[21] M. Holm, M. Thomsen, M. Hoyer, P. Hokland, Optimization of a flow cyto-
metric method for the simultaneous measurement of cell surface antigen,
DNA content, and in vitro BrdU incorporation into normal and malignant
hematopoietic cells. Cytometry 32 (1998) 28e36.
[22] S. van Cruchten, W. van den Broeck, Morphological and biochemical aspects of
apoptosis, oncosis and necrosis. Anat. Histol. Embryol. (2002) 214e223.
[23] T.J. McMillan, S. Tobi, S. Mateos, C. Lemon, The use of DNA double-strand
break quantification in radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 49 (2001)
373e377.
[27] F. Pera, p. Mattias, K. Detzer, Methods for determiningthe proliferation kinetics of
cells by means of 5-bromodeoxyuridine. Cell Tissue Kinet. 10 (1977) 255e264.
[28] B.C. Cavalcanti, D.P. Bezerra, H.I.F. Magalhães, M.O. Moraes, A.S. Lima, E.
R. Silveira, C.A.G. Câmara, V.S. Rao, C. Pessoa, L.V. Costa-Lotufo, Kauren-19-oic
acid induces DNA damage followed by apoptosis in human leukemia cells. J.
Appl. Toxicol. 29 (2009) 560e568.
[29] N.P. Singh, M.T. Maccoy, R.R. Tice, E.L. Schneider, A simple technique for
quantification of low levels of DNA damage in individual cell. Exp. Cell Res.
175 (1988) 184e191.
[30] M. Klaude, S. Erikson, J. Nygren, G. Ahnstrom, The Comet assay: mechanisms
and technical considerations. Mutat. Res. 363 (1996) 89e96.
[31] R.R. Tice, E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi,
Y. Miyamae, E. Rojas, J.C. Ryu, Y.F. Sasaki, Single cell gel/Comet assay:
guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol.
Mutagen. 35 (2000) 206e221.
[32] M. Wojewodzka, I. Buraczewska, M. Kruszewski, A modified neutral comet
assay: elimination of lysis at high temperature and validation of the assay
with anti-single-stranded DNA antibody. Mutat. Res. 518 (2002) 9e20.
[33] Y. Miyamae, M. Yamamoto, Y.F. Sasaki, H. Kobayashi, M. Igarashi-Soga,
K. Shimoi, M. Hayashi, Evaluation of a tissue homogenization that isolates
nucleic for the in vivo single cell gel electrophoresis (Comet) assay: a collab-
orative study by five laboratories. Mutat. Res. 418 (1998) 131e140.
[34] A.R. Collins, M. Ai-guo, S.J. Duthie, The kinetics of repair of oxidative DNA
damage (strand breaks and oxidized pyrimidines) in human cells. Mutat. Res.
336 (1995) 69e77.
[24] T. Mosmann, Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J. Immunol. Methods 16
(1983) 55e63.
[25] L.V. Costa-Lotufo, G.M.A. Cunha, P.A.M. Farias, G.S.B. Viana, K.M.A. Cunha,
C. Pessoa, M.O. Morais, E.R. Silveira, N.V. Gramosa, V.S.N. Rao, The cytotoxic
and embryotoxic effects of kaurenoic acid, a diterpene isolated from Copaifera
langsdorffii oleo-resin. Toxicon 40 (2002) 1231e1234.
[26] H.H. Draper, M. Hadely, Malondialdehyde determination as an index of lipid
peroxidation. Meth. Enzymol. 186 (1990) 421e431.
[35] D. Bootsma, M.P. Mudle, J.A. Cohen, F. Pot, Different inherited levels of DNA
repair replication in xeroderma pigmentosum cell strains after exposure to
ultraviolet irradiation. Mutat. Res. 9 (1970) 507e516.
[36] R.D. Snyder, The role of deoxynucleoside triphosphate pools en the inhibition
of DNA-excision repair and replication in human cells by hydroxyurea. Mutat.
Res. 131 (1984) 163e172.