Synthesis, cytotoxic and antioxidant evaluations of amino derivatives from perezone

Article abstract of DOI:10.1016/j.bmc.2012.07.027

A series of eight amino derivatives (3a-h) from perezone 1 were prepared by nucleophilic addition of bioactive amines v.gr. melatonin, acetyl tryptamine, tryptophan and other amino acids esters (valine, leucine and methionine). Their structures were elucidated by spectroscopy data. The cytotoxic evaluation against four human tumor cell lines PC-3, K-562, HCT-15 and SKLU-1 was performed as well as the TBARS assay for antioxidant activity. The results suggest that 1 and its isomer 4 were highly active against all cell lines, 4 was twice as potent than 1 against PC-3 and HCT-15. The derivative 3a (IC₅₀ = 7.5 ± 0.3 μM) was more active than 1 against HCT-15 whereas 3h was selective against K-562 with IC₅₀ = 4.5 ± 0.4 μM. The TBARS assay has shown that 3c with IC₅₀ = 5.564 ± 0.24 μM is a potent antioxidant with superior effect comparing to α-tocopherol and moreover was more active than the precursor molecule 1.

Full text of DOI:10.1016/j.bmc.2012.07.027

Bioorganic & Medicinal Chemistry 20 (2012) 5077–5084
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, cytotoxic and antioxidant evaluations of amino derivatives
from perezone
M. Concepción Lozada a, , Olivia Soria-Arteche a, Ma. Teresa Ramírez Apan b, Antonio Nieto-Camacho b,
⇑
Raúl G. Enríquez b, Teresa Izquierdo a, Arturo Jiménez-Corona a
a Departamento de Sistemas Biológicos, UAM-Xochimilco, Calzada del Hueso 1100, Col. Villa Quietud, Coyoacán, D.F., 04960, Mexico
b Instituto de Química, UNAM, Circuito Exterior, Ciudad Universitaria, Coyoacán , D.F., 04510, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of eight amino derivatives (3a–h) from perezone 1 were prepared by nucleophilic addition of bio-
active amines v.gr. melatonin, acetyl tryptamine, tryptophan and other amino acids esters (valine, leucine
and methionine). Their structures were elucidated by spectroscopy data. The cytotoxic evaluation against
four human tumor cell lines PC-3, K-562, HCT-15 and SKLU-1 was performed as well as the TBARS assay
for antioxidant activity. The results suggest that 1 and its isomer 4 were highly active against all cell lines,
Received 26 April 2012
Revised 30 June 2012
Accepted 10 July 2012
Available online 25 July 2012
4 was twice as potent than 1 against PC-3 and HCT-15. The derivative 3a (IC50 = 7.5 0.3
active than 1 against HCT-15 whereas 3h was selective against K-562 with IC50 = 4.5 0.4
l
M) was more
Keywords:
Perezone
Amino derivatives
Amino acids
Cytotoxic
l
M. The TBARS
assay has shown that 3c with IC50 = 5.564 0.24
ing to -tocopherol and moreover was more active than the precursor molecule 1.
Ó 2012 Elsevier Ltd. All rights reserved.
lM is a potent antioxidant with superior effect compar-
a
Antioxidants
1. Introduction
Also, perezone acts as an electron sink in electron transport in
the mitochondria19 and is considered to be a cardio protective
agent.20 Antifeedant effects and phytotoxic activity21 have been
studied as well as the cytotoxic effect against several cancer cell
lines.22
In a recent investigation the cytotoxic effect of perezone and
isoperezone was evaluated on the K-562 human leukemia cell line,
showing that perezone has greater cytotoxic effect than isoperez-
one.23 On the other hand, perezone was included in the screening
of compounds that induce apoptosis of cancer cell lines24 and for
the screening of agents against neuroblastoma.25
Some amino derivatives have been synthesized previously and
evaluated for biological activity,17 but derivatives of indolamine
type v.gr. melatonin (2a), acetyl tryptamine (2b) and tryptophan
(2c) (Fig. 1) have not been prepared. Therefore we considered it
to be of interest to synthesize these types of derivatives due to
the biological activity such amines have on their own.
Melatonin (N-acetyl-5-methoxytryptamine) (2b) is secreted by
the pineal gland of the vertebrate. Several studies have demon-
strated the role of 2b in physiological regulation of seasonal and cir-
cadian rhythms. Some therapeutic applications such as regulation
of sleep disorders, treatment of depression, as broad-spectrum anti-
oxidant,26 treatment of Alzheimer disease or anticancer agent.27,28
Among the numerous Mexican medicinal species, the genus
Perezia more recently renamed ‘Acourtia’, has received much
attention since the secondary metabolite Perezone (1) was first re-
ported in 1852 by Leopoldo Río de la Loza.1 This natural sesqui-
terpenic quinone was isolated for the first time from Perezia
adnata and was known as ‘raiz del pipitzahuac’ in the traditional
medicine in México.
Compound 1 has been the subject of many chemical studies,
first to elucidate its structure and then to explore its varied reactiv-
ity.2–5 Nevertheless, the correct structure of 1 was determined by
using NMR spectroscopic methods6–8 and its crystal structure
was reported later in 1986.9 Some synthetic methods have been
developed to obtain 1 in racemic form as well as other related qui-
nones.10–12
Mexican traditional medicine employed the roots of perezia for
the preparation of laxative drinks, and also for diuretic, regenera-
tive and analgesic purposes.13 Perezone exhibits diverse biological
profiles, for example, the hypoglycemic effect,14 inhibition of
aggregation of platelet,15 it promotes the release of mitochondrial
Ca2+ maintaining ATP production during reperfusion,16 it relaxes
the basal tonus of the smooth muscle17 and blocks the contractile
response induced by Ach, K+ and Ba2+ 18
.
The conjugation of melatonin with a-lipoic acid was achieved and
the bioconjugate was tested in vitro for radioprotection ability.29
Tryptamine and the essential amino acid tryptophan are analogs
of melatonin and possess biological activities such as neurotrans-
⇑
Corresponding author. Tel.: +52 5 483 7515; fax: +52 5 483 7237.
E-mail address: mclozada@correo.xoc.uam.mx (M. Concepción Lozada).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.027

5078
M. Concepción Lozada et al. / Bioorg. Med. Chem. 20 (2012) 5077–5084
the case of tryptamine, the aliphatic amine was previously acety-
H
N
OH
H
lated in order to avoid a nucleophilic attack of this nitrogen.
First, melatonin or its analogs were set to react with NaH in THF
in order to form the anion, then perezone was added to the reac-
tion mixture and refluxed for 24 h. Although the yield of each reac-
tion is quantitative, the presence of residual secondary products
required their purification by column chromatography. Deep
brown semisolids were obtained. The chemical structures of all
compounds were confirmed by spectroscopy data. For mass spec-
troscopy, in the case of compounds 3a and 3b the acetylation of
the products was necessary to avoid their decomposition. The acet-
ylated compounds are marked with prime, respectively.
In the case of derivatives 3d–f (Scheme 3), the compound 1 re-
acted with valine, leucine and methionine esters 2d–f or trypt-
amine 2a0 and tryptophan 2c (Scheme 4), employing Zn(AcO)2 as
a catalyst in MeOH under mild reaction conditions. All the com-
pounds were characterized by spectroscopy data.
O
O
(R)
(R)
O
OCH3
R
NH2
O
N
H
NH
1
2a R= H
2c
2b R= OCH3
Figure 1. Structures of perezone (1), acetyl tryptamine (2a) melatonin (2b) and
tryptophan methyl ester (2c).
mitter and the building blocks in proteins biosynthesis. The cou-
pling of two biomolecules to obtain bioconjugates, is a strategy to-
wards synthesis of novel derivatives that allow improvement of
biological activities or the modification of physicochemical proper-
ties in comparison with the parent molecules. Natural amino acid
derivatives of naproxen have been prepared to improve analgesic
and anti-inflammatory activity.30 Valine, leucine and methionine
are essential amino acids, which possess important functions and
therefore are good candidates for nucleophilic addition to the quin-
oid system of 1.
The optical rotation was only possible for 3a. Due to the deep
purple color of 3b–h we were unable to get the data in the
polarimeter.
2.3. Cytotoxic activity in human tumor cell lines
In this context, as a continuation of our research on natural
products,31in the present work we prepared some new indole
derivatives 3a–c and amino acid derivatives 3d–h by nucleophilic
addition of indole amines or amino acid esters (valine, leucine,
methionine and tryptophan) to the quinone system of 1. The deriv-
atives have been elucidated by spectroscopic data and assessed for
their cytotoxic activity with respect to four cancer line cells of dif-
ferent histotype (myeloblastoid and carcinomas). The antioxidant
activity was evaluated by TBARS test in rat brain in order to mea-
sure the lipid peroxidation of polyunsaturated fatty acids, which
produce malondialdehyde (MDA) as thiobarbituric acid reactive
substance.
In order to evaluate compounds 1, 3a–3h and 4 as cytotoxic
agents, we performed tests in vitro against three carcinoma type
cell lines: PC-3 (human prostatic), HCT-15 (human colorectal)
and SKLU-1 (non-small cell lung cancer and one myeloblastoid leu-
kaemia cell line: K-562 (human chronic myelogenous leukemia).
The cytotoxicity was determined by the sulforhodamine B (SRB)
assay and Adriamycin was used as a positive control. Table 1 shows
the IC50 data for the compounds obtained.
Although, the compounds 1, 3a–h and 4 showed less cytotoxic
activity than Adriamycin, 1 and 4 presented higher cytotoxic po-
tential against the four cell lines with IC50 < 10 lM. The change
in the position of hydroxyl group present in 1 and 4, leads to
important changes in the cytotoxic activity. Compound 4 was
twice as potent with respect to 1 against PC-3 and HCT-15 cell
lines.
2. Results and discussion
The bioconjugates 3a and 3b are structurally related, since the
presence of a methoxy group in 5 position of indole ring for 3a
leads to an increase of the activity against all cell lines in compar-
ison to 3b. Also 3a was more active than its precursor molecule
against HCT-15 cell line. Derivatives 3g and 3h correspond to bio-
conjugates of 1 with tryptamine and tryptophan methyl ester,
2.1. Isolation of perezone and its isomerization to isoperezone
Perezone was isolated from dry roots of Acourtia cuernavacana
spp. and extracted three times by using maceration with hexane
at room temperature. As a result we obtained orange crystals.
The yield was 2%, but the crystals were not pure. Then their puri-
fication was achieved by recrystallization with acetone-hexane
(1% yield). The non-natural derivative isoperezone 4 was prepared
according to the protocol previously described with our specific
modifications (Scheme 1).32
respectively, bonded by amino group in
a position. The presence
of methyl carboxylate in 3h increases the activity against all cell
lines as compared to 3g. On the other hand, we obtained 3c and
3h as products of addition to each of the nitrogen atoms present
in the molecule of tryptophan methyl ester. For 3c, the IC50 with
K-562, HCT-15 and SKLU-1 cell lines was twice as much as for
3h. For 3h we observed selectivity against the leukemia cell line
and moderate activity for solid tumors.
The addition of aliphatic amino acid esters to perezone pro-
duced derivatives (3d–f), which were less active against the four
cell lines in comparison to the aromatic analog of tryptophan
methyl ester 3 h.
2.2. Synthesis of amino derivatives
Derivatives 3a, 3b and 3c (Scheme 2) were prepared from 1
with melatonin, acetyl tryptamine or tryptophan methyl ester,
respectively, via nucleophilic addition of indole-type nitrogen. In
2.4. Antioxidant activity (TBARS) in rat brain homogenate
SH
N
OH
H
HN
O
O
The antioxidant activity was evaluated by measuring the
amount of plasma concentration of thiobarbituric acid reactive
substances (TBARS) that represent lipid peroxidation. Malondial-
dehyde (MDA) forms complexes with Thiobarbituric Acid (TBA)
to produce a pink coloured complex, spectrophotometrically mea-
sured at a wavelength of 540 nm. TBARS results are expressed as
the amount of free MDA equivalents per 1 mg tissue through the
O
O
H
OH
1
4
Scheme 1. Synthesis of isoperezone.

M. Concepción Lozada et al. / Bioorg. Med. Chem. 20 (2012) 5077–5084
OH
5079
OH
H
O
R1
O
R2
O
NaH / THF
N2
O
N
N
H
R2
R1
R2=CH2CH2NHCOCH3
2a R1= H
R2=CH2CH2NHCOCH3
1
3a R1= H
2b R1=OCH3 R2=CH2CH2NHCOCH3
3b R1=OCH3 R2=CH2CH2NHCOCH3
2c R1=H
R2=CH2CHCO2CH3
NH2
3c R1=H
R2=CH2CHCO2CH3
NH2
Scheme 2. Synthesis of derivatives 3a–b.
OH
O
OH
O
O
Zn (AcO)2
R1
R2O
O
MeOH
O
NH2
HN
CO2R2
H
R1
2d R1= CH(CH3)2
R2=CH3
1
3d R1= CH(CH3)2
R2=CH3
2e R1= CH2CH(CH3)2 R2=CH3
3e R1= CH2CH(CH3)2 R2=CH3
2f R1= CH2CH2SCH3 R2=CH2CH3
3f R1= CH2CH2SCH3 R2=CH2CH3
Scheme 3. Synthesis of derivatives 3d–f.
OH
OH
R
O
O
Zn (AcO)2
MeOH
NH2
O
O
N
R
N
H
H
H
N
H
1
2a´ R= H
3g R= H
3h R= CO2CH3
2c R= CO2CH3
Scheme 4. Synthesis of derivatives 3g and 3h.
and quercetin, which are considered to be as excellent antioxi-
dants. DMSO was used in the blank to correct any possible antiox-
idant effect that it could have.
Table 1
Inhibitory concentration (IC50 in
SKLU-1 for compounds 1, 3a–h and 4.
lM) values obtained in PC-3, K-562, HCT-15 and
IC50
l
M*
With respect to antioxidant activity, the most pronounced
inhibitory effect was found for 3c with superior potency than
a-
Compound
Cell line
tocopherol (Fig. 2), whereas in the case of 3a and 3b analogs the
activity decreased. It is interesting to mention that the bonding site
for tryptophan methyl ester had an important role in the antioxi-
PC-3
6.5 0.6
K-562
HCT-15
SKLU-1
1
3a
3b
3c
3d
3e
3f
3 g
3.0 0.3
6.2 0.6
8.9 1.3
10.0 0.6
13.1 1.0
11.4 0.6
13.0 0.8
12.2 1.0
4.5 0.4
3.4 0.1
14 0.9-
10.3 1.0
7.5 0.3
4.2 0.3
9.5 0.5
9.9 0.5
13.9 1.2
11.6 0.6
24.8 2.0
20.3 1.4
19.7 1.1
23.4 11.3
11.1 0.5
3.3 0.2
dant activity. While 3h is not active, the presence of a-amino acid
9.8 0.48
19.2 0.7
14.3 1.8
15.4 0.3
13.8 1.0
19.4 11.7
8.5 0.8
16.9 0.9
21.1 0.7
16.4 1.8
15.1 0.1
15.0 11.1
22.6 2.2
9.7 0.8
ester in 3c favors antioxidant activity. For compound 4, we ob-
served that the antioxidant activity was twice as high as its isomer
1. The position of hydroxyl group in these compounds is very
important for the antioxidant activity. The results indicate that
derivatives of 4 could further increase its antioxidant activity.
3 h
4
5.9 0.5
4.5 0.5
Adriamycin**
60 1.0
5
0.9
6.7
1
3. Conclusions
*
In DMSO like vehicle.
nM.
**
The new derivatives of perezone obtained in the present work
represent an option of molecules with cytotoxic and antioxidant
activities. Although all compounds were less cytotoxic than Adria-
mycin, all of them exhibited high cytotoxicity predominantly on
leukemia cell line K-562. Compounds 1 and its isomer 4 can be
considered as high cytotoxic agents for the myeloblastoid leukemia
cell line (K-562) and for carcinoma cell lines (PC-3 and SKLU-1)
calibration curve generated from 1,1,3,3-tetramethoxypropane
(TMP). This assay was performed for all compounds because some
amino acids as well as melatonin or its analogs are considered as
antioxidant agents. Thus, the inhibitory concentration IC50 of these
derivatives (Table 2 and Fig. 2) was compared with
a-tocopherol
with IC50 < 10 lM.

5080
M. Concepción Lozada et al. / Bioorg. Med. Chem. 20 (2012) 5077–5084
Table 2
Table 2 (continued)
IC50 for Inhibitory activity of lipid peroxidation in TBARS assay.
Compound
Concentration TBARS
Inhibition (%) IC50 (lM)
Compound
Concentration TBARS
Inhibition (%) IC50 (lM)
(
lM)
(nmol/mg prot.)
(l
M)
(nmol/mg prot.)
5.62
10
0.1
0.32
1
0.49 0.12⁄⁄
0.41 0.12⁄⁄
6.28 0.18
94.90 1.13⁄⁄
95.65 1.24⁄⁄
4.62 0.57
Basal
Control FeSO4 10
1
—
0.29 0.02
7.57 0.22
6.61 0.24
—
—
—
—
a-Tocopherol
6.78 2.16
10
8.48 1.86
16.00 3.97⁄⁄
28.78 4.32
54.21 9.93⁄⁄
89.39 3.14⁄⁄
2.90 1.80
19.00 1.09⁄⁄
49.53 2.36⁄⁄
93.23 0.22⁄⁄
95.63 0.20⁄⁄
49.22 5.66
6.04 0.24⁄
5.21 0.33⁄⁄
3.67 0.56⁄⁄
2.72 0.33
8.26 1.31
21.13 2.56⁄
44.84 6.74⁄⁄
59.00 3.71⁄⁄
72.3 3.87⁄⁄
79.09 4.79⁄⁄
17.78
31.62
56.23
100
10
17.78
31.62
56.23
6.07 0.40⁄⁄
5.14 0.32
3.16
3.30 0.73⁄⁄
0.77 0.23⁄⁄
7.02 0.32⁄⁄
5.85 0.22⁄⁄
3.65 0.27
10
31.62
100
1.84 0.31⁄⁄
1.40 0.36⁄⁄
3a
3b
3c
3d
29.75 0.53
Data are averaged three or four times with independent values standard error on
the mean (X ES). All reported data were subject to variance analysis (ANOVA)
followed by Dunnett tests in order to isolate specific group of significant difference.
The p 6 0.05 (⁄) and p 6 0.01 (⁄⁄) values were considered to be different as com-
pared with the control.
0.49 0.01⁄⁄
0.32 0.01⁄⁄
6.19 0.45⁄⁄
5.10 0.53⁄⁄
3.59 0.58
100
14.85 4.17⁄⁄ 27.44 2.63
17.78
31.62
56.23
29.97 5.76⁄⁄
50.89 6.99
0.68 0.18⁄⁄
0.36 0.03⁄⁄
6.61 0.40
90.78 2.24⁄⁄
100
95.10 0.26⁄⁄
3.16
4.22
5.62
7.5
10
17
19
21
23
25
13.88 4.46⁄
20.57 4.71⁄⁄
46.01 8.90
88.70 2.70⁄⁄
94.13 0.84⁄⁄
5.564 0.24
The addition of the part indole amine or a-amino ester present
in tryptophan methyl ester to perezone produced the derivatives
3c or 3h, which possess very different activities. Compound 3c
has a superior antioxidant activity (IC50 = 5.564 0.24
-tocopherol and exhibits a higher antioxidant activity of 8.8 times
than 1, whereas 3h with IC50 = 4.5 0.4 lM was selective against
myeloblastoid leukemia cell line (K-562).
Taking into account that compound 3c presented a higher anti-
oxidant activity than -tocopherol, we will consider that for future
6.09 0.39⁄
4.15 0.72⁄⁄
0.88 0.24⁄⁄
0.45 0.05⁄⁄
5.45 0.52⁄⁄
4.93 0.47⁄⁄
4.66 0.51⁄⁄
3.38 0.79⁄⁄
2.92 0.53
lM) than
27.28 2.32⁄⁄ 22.44 1.02
a
34.21 2.69⁄⁄
37.82 4.10⁄⁄
55.87 8.08⁄⁄
61.20 6.13
27
29
31
9
1.58 0.44⁄⁄
1.09 0.55⁄⁄
0.37 0.05⁄⁄
7.65 0.41
78.79 5.70⁄⁄
a
85.50 7.28⁄⁄
research its structure as a new leader molecule for the design of
analogs with potent antioxidant activity.
95.04 0.72⁄⁄
3e
7.53 5.07
14.67 0.60
10
11
12
13
14
15
16
17
17
19
21
23
25
27
29
31
17
19
21
23
25
27
29
31
17
19
21
23
25
27
29
31
7.41 0.43
7.07 0.65
6.48 0.50
6.30 0.45
10.45 5.64
14.66 7.58
21.66 6.35
23.94 5.57⁄⁄
42.20 9.99⁄⁄
47.89 9.98
65.42 5.15⁄⁄
85.75 2.69⁄⁄
18.83 2.69⁄
35.82 1.77⁄⁄
57.66 5.86⁄⁄
77.31 5.44⁄⁄
80.92 5.32⁄⁄
89.72 1.28⁄⁄
92.24 1.58⁄⁄
94.56 0.15⁄⁄
4. Experimental
4.75 0.73⁄⁄
430 0.78⁄⁄
2.86 0.40⁄⁄
1.15 0.21⁄⁄
5.98 0.46⁄
4.71 0.29⁄⁄
3.06 0.34⁄⁄
1.62 0.32⁄⁄
1.35 0.30⁄⁄
0.76 0.11⁄⁄
0.56 0.11⁄⁄
0.40 0.02⁄⁄
4.52 0.45⁄⁄
4.25 0.61⁄⁄
2.68 0.79⁄⁄
1.76 0.49⁄⁄
1.17 0.44⁄⁄
0.35 0.06⁄⁄
0.44 0.19⁄⁄
0.28 0.04⁄⁄
5.91 0.62
Melting points (°C) were determined in a Fisher-Jones apparatus
and are uncorrected; IR spectra were recorded in Perkin Elmer
Spectrum RXI in KBr or solution. Optical rotations were measured
in a polarimeter JASCO model DIP360 at 589 nm using a 1 dm cell.
Specific rotations are given in units of 10ꢀ1 deg cm2 gꢀ1 (c g/100
cm3). Mass spectra were obtained in JEOL JMS-AX505HA mass spec-
trometer by electronic impact at 70 eV and JEOL SX 102 A by FAB+.
1D and 2D 1H and 13C spectra were obtained at 500 and
125 MHz (Bruker Avance DMX500) or 400 and 100 MHz (Varian
Mercury 400), respectively, in CDCl3 at room temperature with
TMS as the internal reference, chemical shifts (d) are given in
ppm and coupling constants (J) in Hz.
3f
20.25 0.26
3g
37.12 2.34⁄⁄ 19.33 0.30
38.52 8.36⁄⁄
69.64 9.95⁄⁄
TLC was performed on precoated Kieselgel 60 F254 plates
(Merck, Darmstadt, Germany) and detected with UV light. Flash
chromatography was performed with Merck Kieselgel silica gel
(60, particle size 40–63 mm). All reagents were purified and the
solvents used were of analytical grade.
84.22 7.13⁄⁄
90.34 2.52⁄⁄
95.40 0.83⁄⁄
96.05 0.80⁄⁄
96.83 0.13⁄⁄
3h
20.94 3.44
24.91 1.45
28.6 2.6
25.16 0.08
5.59 0.47
4.1. Plant material
5.32 0.50⁄⁄
4.42 0.50⁄⁄
4.21 0.73⁄⁄
2.72 0.96
40.80 2.73⁄⁄
44.15 5.96⁄⁄
64.82 10.39⁄⁄
78.73 7.79⁄⁄
92.06 2.08⁄⁄
the whole plant of Acourtia cuernavacana (before Perezia cuerna-
vacana) was collected in September–December 2007 in Cuerna-
vaca, Morelos, and identified for comparison with an exemplary
of Herbarium. The roots were separated from the aerial parts,
cleaned and dried for three weeks.
1.64 0.63⁄⁄
0.60 0.17⁄⁄
5.67 0.18⁄⁄
3.78 0.21⁄⁄
1.21 0.21⁄⁄
0.39 0.01⁄⁄
0.33 0.01⁄⁄
9.13 0.62
4
17.78
23.71
31.62
42.17
56.23
0.32
0.56
1
25.20 3.09⁄⁄ 23.26 0.1
47.39 2.83⁄⁄
83.32 2.56⁄⁄
94.62 0.03⁄⁄
95.35 0.18⁄⁄
4.2. Extraction and isolation
Quercetin
5.23 1.17
1.496 0.03
8.58 0.59⁄⁄
10.93 1.20⁄⁄
27.88 2.05
62.68 1.93⁄⁄
85.23 2.81⁄⁄
About 828 g of dried roots of Acourtia cuernavacana were finely
powdered and exhaustively extracted by maceration at room tem-
perature with hexane to give 16 g of orange crystals (2%). Recrys-
tallization of the solid was carried out with acetone-hexane to
give 8 g (1%) of pure perezone.
6.98 0.73
1.78
3.16
3.59 0.33⁄⁄
1.39 0.16⁄⁄

M. Concepción Lozada et al. / Bioorg. Med. Chem. 20 (2012) 5077–5084
5081
was 1.13 g (95%) of 2a. mp 75 °C (lit., mp = 75–76 °C).33 Spectro-
scopic data is according to the previously reported.34
4.5. General procedure for preparation of bioconjugate perezone
derivatives
1.5 mmol of sodium hydride (NaH) were dissolved in 3 mL of
anhydrous tetrahydrofuran (THF) at 0°C. 4.2 mmol of 2a, 2b (acet-
ylated previously) or 2c were added dropwise in a 5 mL THF solu-
tion. This reaction was stirred during 40 minutes in an inert
atmosphere of N2, then 1 mmol of perezone (1) was added drop-
wise in a 5 mL THF solution. The reaction was stirred at room tem-
perature for 24 h. Then the reaction was washed with brine and
extracted with EtOAc. The organic phase was dried with Na2SO4
and filtered. The solvent was evaporated and dried in vacuo. The
residue was purified by flash chromatography (hexane/acetone,
6:1) to obtain compounds 3a, 3b or 3c.
For mass spectroscopy, due to the slow decomposition of 3a and
3b, it was necessary to acetylate them with Ac2O and pyridine, stir-
ring the mixture for 1 h at room temperature. The mixture was
washed with brine and extracted in EtOAc. The organic phase
was dried with Na2SO4 and filtered. The solvent was evaporated
and dried in vacuo. The residue was purified by flash chromatogra-
phy (hexane/acetone, 6:1). The acetylation of 3a and 3b leads to
the more stable products 3a0and 3b0 were, respectively, used for
MS analysis solely.
Figure 2. Inhibitory concentration (IC50) values in the antioxidant test (TBARS) for
compounds 1, 3a–h, 4 and standards (quercetin, Q and -tocopherol,T).
a
4.2.1. Perezone (2-[1,5-dimethyl-4-hexenyl]-3-hydroxy-5-
methyl-1,4-benzoquinone) (1)
Orange crystals, mp:101–102 °C, Rf: 0.44 (hexane/acetone
90:10); IR (cmꢀ1) in KBr: 3413, 2968, 2929, 1654, 1639, 1396,
1377, 1323. 1H NMR (400 MHz, CDCl3): 6.991 (s, 1H), 6.480 (q,
J = 0.9 Hz, 1H), 5.063 (tt, J = 5.4, 0.9 Hz, 1H), 3.004–3.094 (m, 1H),
2.059 (d, J = 1.2 Hz, 3H), 1.758–1.932(m, 3H), 1.64 (d, J = 0.9, 3H),
1.549–1.603 (m, 1H), 1.532 (s, 3H), 1.197 (d, J = 5.4 Hz, 3H). 13C
(100 MHz, CDCl3): 187.419, 184.340, 151.061, 140.671, 135.999,
131.601, 124.699, 124.608, 34.45, 29.687, 27.078, 26.123, 18.660,
18.053, 15.171.
4.5.1. N-(2-(1-(4-Hydroxy-2-methyl-5-((R)-6-methylhept-5-en-
2-yl)-3,6-dioxocyclohexa-1,4-dienyl)-5-methoxy-1H-indol-3-
yl)ethyl)acetamide (3a)
Brown semisolid; 70% yield; Rf: 0.4 (CHCl3–MeOH, 95:5); ½aꢁD:
+35.4 (c 1.00, CHCl3); IR (cmꢀ1) in CHCl3: 3389.31, 3017.98,
1711.63, 1650.21, 1620.1, 1216.0; 1H NMR (500 MHz, CDCl3):
7.397 (sb, 1H), 7.066 (d, J = 2.5 Hz, 1H), 6.915 (s, 1H), 6.877 (dd,
J = 9, 2.5 Hz, 1H), 6.812 (d, J = 8.5 Hz, 1H), 5.751 (sb, 1H), 5.079
(tt, J = 7, 1.5 Hz, 1H), 3.867 (s, 3H), 3.481 (q, J = 7 Hz, 1H), 3.119
(m, 1H), 2.939–2.979 (m, 2H), 1.994 (s, 3H), 1.947 (s, 3H), 1.78–
1.92 (m, 2H), 1.646 (s, 3H), 1.56–1.63 (s, 1H), 1.545 (sb, 3H),
1.231 (d, J = 7 Hz, 3H), 1.194–1.256 (m, 2H). 13C NMR (125 MHz,
CDCl3): 184.133, 182.945, 170.293, 155.048, 151.361, 139.933,
132.823, 131.704, 131.592, 129.129, 128.057, 124.323, 123.745,
115.151, 112.129, 112.256, 101.531, 55.952, 39.215, 34.200,
29.856, 26.733, 25.740, 25.155, 23.389, 18.276, 17.706, 12.437.
3a0 MS (EI, 70 eV): m/z (%) = 520 [M+] (71), 461 (100), 406 (72),
337 (28), 149 (30). HRMS–FAB: m/z [M+H+] calcd for C30H36N2O8:
521.2652; found: 521.2657.
4.3. Isomerization of perezone
Perezone was isomerized by action of 1,2,3,4-tetrahydropyri-
midinethiol with some small modifications as compared to previ-
ously reported protocol.11 4 mmol of perezone (1) were disolved
in 50 ml of MeOH and catalytic quantity of p-TsOH was added
and stirred by 30 min. Then 4 mmol of 3,4,5,6-tetrahydropyrimid-
inethiol in 40 ml of MeOH was added and the mixture was refluxed
for 12 h after evaporation of the solvent the residue was purified by
column chromatography. Orange crystals were obtained.
4.3.1. Isoperezone (2-[1,5-dimethyl-4-hexenyl]-6-hydroxy-5-
methyl-1,4-benzoquinone (4)
4.5.2. N-(2-(1-(4-Hydroxy-2-methyl-5-((R)-6-methylhept-5-en-
2-yl)-3,6-dioxocyclohexa-1,4-dienyl)-1H-indol-3-
yl)ethyl)acetamide (3b)
Orange crystals; 60% yield; Rf: 0.2 (hexane/acetone 90:10); p.f. :
108–109 °C; IR (cmꢀ1) in KBr: 3399.99, 3257.17, 2962.61, 2930.46,
1755.62, 1662.23, 1638.35, 1611.22, 1393.88, 1376.28, 1326.91,
1196.72. 1H NMR (400 MHz, CDCl3): 6.952 (s, 1H), 6.428 (d,
J = 0.8 Hz, 1H), 5.021 (tt, J = 7.2 Hz, 1H), 2.89 (sx, J = 6.8 Hz, 1H),
1.93–2.0 (m, 2H), 1.937 (s, 3H), 1.654 (d, J = 0.8 Hz, 3H), 1.62 (m,
2H), 1.544 (s, 3H), 1.42–1.62 (m, 2H), 1.130 (d, J = 7.2 Hz, 3H). 13C
(100 MHz, CDCl3): 187.761, 182.725, 150.961, 149.232, 132.728,
131.969, 123.505, 116.376, 35.556, 31.339, 25.708, 25.605,
19.356, 17.657, 7. 857.
Brown semisolid; 60% yield; Rf: 0.4 (CHCl3–MeOH, 95:5). IR
(cmꢀ1) in CHCl3: 3296.30, 2964.89, 2930.42, 1651.63, 1458.08,
1380.73, 1269.74. 1H NMR (500 MHz, CDCl3): 7.626 (d, J = 7.5 Hz,
1H), 7.236 (t, J = 7.5 Hz, 1H), 7.186 (t, J = 8 Hz, 1H), 6.943 (s, 1H),
6.916 (d, J = 8.0, 1H), 5.874 (sb, 1H), 5.082 (t, J = 7.0 Hz, 1H), 3.667–
3.717 (m, 1H), 3.549 (sb, 1H), 3.129 (sx, J = 7.0 Hz, 1H), 2.92–3.07
(m, 2H), 1.988 (s, 3H), 1.938 (s, 3H), 1.79–1.971 (m, 3H), 1.644 (s,
3H), 1.590–1.648 (m, 1H), 1.235 (d, J = 7.0 Hz, 3H). 13C NMR
(125 MHz, CDCl3): 184.070, 182.887, 170.411, 151.472, 139.751,
136.600, 131.510, 128.366, 127.252, 124.261, 123.803, 122.91,
120.804, 119.278, 115.188, 111.289, 68.115, 39.271, 34.143,
34.082, 29.770, 26.657, 25.658, 25.017, 23.239, 18.218, 12.319.
Compound 3b0 MS (EI, 70 eV): m/z (%): 490 [M+] (34), 448 (6),
431 (100), 376 (98), 307 (82), 43 (23). HRMS–FAB: m/z [M+H+]
calcd for C29H34N2O5: 490.2468; found: 490.2462.
4.4. N-(2-(1H-Indol-3-yl)ethyl)acetamide (Acetyl triptamine) 2a
Commercial tryptamine (6.2 mmol, 1.0 g) was dissolved in dry
CH2Cl2 (20 mL), and acetic anhydride (1.1 equiv) was added slowly.
The reaction was stirred at room temperature for 40 min. The result-
ing precipitate was filtered and washed with CH2Cl2, and dried. Yield

5082
M. Concepción Lozada et al. / Bioorg. Med. Chem. 20 (2012) 5077–5084
4.5.3. (R)-Methyl 2-amino-3-(1-(4-hydroxy-2-methyl-5-((R)-6-
me thylhept-5-en-2-yl)-3,6-dioxocyclohexa-1,4-dienyl)-1H-
indol-3-yl)propanoate (3c)
124.591, 102.955, 55.366, 52.662, 42.831, 34.138, 29.386, 26.646,
25.678, 24.855, 22.674, 22.578, 18.297, 9.594. MS (EI, 70 eV): m/z
(%): 391 [M+] (58), 359 (30), 332 (57), 330(24), 276 (13), 262
(38), 248 (100), 222 (17), 192 (18), 180 (48), 69 (15), 41 (16).
HRMS–FAB: m/z [M+] calcd for C22H33NO5: 391.2359; found:
391.2350.
Brown semisolid; 20% yield; Rf: 0.4 (CHCl3–MeOH, 95:5). IR
(cmꢀ1) in CHCl3: 3476.78, 3304.54, 3019.39, 2928.07, 2857.13,
1738.52, 1711.15, 1582.65, 1510.23, 1375.43, 1357.84, 1280.37,
1216.22. 1H NMR (500 MHz, CDCl3): 8.21 (br s, 2H), 7.508 (d,
J = 8.0 Hz, 1H), 7.343 (d, J = 8.0 Hz, 1H), 7.189 (td, J = 7.0, 1.0 Hz,
1H), 7.112 (td, J = 7.0, 1.0 Hz, 1H), 7.047 (d, J = 2.0 Hz, 1H), 6.597
(sb, 1H), 5.078 (tt, J = 7.0, 1.0 Hz, 1H), 4.966–5.007 (m, 1H), 3.688
(s, 3H), 3.349–3.388 (m, 2H), 2.960 (sx, J = 7.0 Hz, 1H), 1.904 (s,
3H), 1.827–1.904 (m, 2H), 1.71–1.784 (m, 1H), 1.645 (d, J = 1.0 Hz,
3H), 1.515–1.546 (m, 1H), 1.533 (s, 3H) 1.150 (d, J = 7.5 Hz, 3H).
13C NMR (125 MHz, CDCl3): 182.985, 180.471, 172.051, 153.412,
145.443, 136.249, 131.306, 127.063, 124.637, 123.254, 122.517,
119.871, 118.332, 111.436, 109.024, 56.915, 52.734, 34.171,
29.874, 29.368, 26.685, 25.710, 18.294, 17.655, 9.483. MS (EI,
70 eV): m/z (%): 464 [M+], 432 (4), 405 (2), 334 (10), 276 (18), 250
(21), 192 (5), 130 (100), 69 (5), 28 (5). HRMS-FAB: m/z [M+H+] calcd
for C27H33N2O5: 465.2389; found: 465.2383.
4.6.3. (S)-Methyl 2-(4-hydroxy-2-methyl-5-((R)-6-methylhept-
5-en-2-yl)-3,6-dioxocyclohexa-1,4-dienylamino)-4-(methyl
thio)-butanoate (3f)
Deep purple semisolid; 42% yield; Rf: 0.72 (hex/EtOAc, 95:5).
IR (cmꢀ1) in CHCl3: 3682.07, 3305.19, 3020.13, 2967.59, 2922.06,
2876.12, 1739.39, 1644.90, 1586.21, 1512.24, 1404.01, 1375.75,
1295.56, 1215.58, 1103.16. 1H NMR (500 MHz, CDCl3): 8.062
(bs, 1H), 6.475 (d, J = 9.5 Hz, 1H), 5.042 (tt, J = 7.0, 1.5 Hz, 1H),
4.791 (m, 1H), 2.961 (sx, J = 7.0 Hz, 1H), 2.495–2.595 (m, 2H),
2.113–2.188 (m, 1H), 2.071 (s, 3H), 2.018–2.061 (m, 1H), 2.003
(s, 3H), 1.798–1.919 (m, 2H), 1.716–1.778 (m, 1H), 1.613 (d,
J = 1.0 Hz, 3H), 1.504 (d, J = 0.5 Hz, 3H), 1.477–1.548 (m, 1H),
1.255 (t, J = 7.0 Hz, 3H), 1.142 (d, J = 7.0 Hz, 3H). 13C NMR
(125 MHz, CDCl3): 182.833, 180.772, 171.497, 153.347, 145.377,
131.244, 124.551, 118.658, 103.744, 61.94, 55.460, 34.118,
32.898, 29.805, 29.355, 26.618, 25.653, 18.272, 17.606, 15.432,
9.593. MS (EI, 70 eV): m/z (%): 423 (100) M+, 350 (62), 340
(12), 314 (13), 262 (45), 240 (16), 220 (23), 192 (30), 180 (75),
109 (14), 61 (45), 41(16). HRMS–FAB: m/z [M+H+] calcd for
4.6. General procedure for preparation of perezone derivatives
3d–h
About 0.448 mmol of Zn(AcO)2 in 6 ml of MeOH was mixed with
0.448 mmol of methyl or ethyl ester amino acid and stirred at room
temperature for 1 h. Then, 0.403 mmol of 1 dissolved in 10 ml of
MeOH was added and heated at 40 °C. during 46 h. After that,
the solvent was evaporated and the product extracted with CH2Cl2.
The organic phase was dried over Na2SO4 and evaporated. The res-
idue was purified by chromatography on silica gel eluting with
hexane: EtOAc (90:10 +1 drop of AcOH).
C22H33NO5S: 423.2079; found: 423.2076.
4.6.4. (3g)
Deep purple semisolid; 17% yield; Rf: 0.44 (hex/EtOAc, 80:20).
IR (cmꢀ1) in CHCl3: 3682.18, 3477.29, 3304.18, 3019.85, 2965.75,
2931.39, 1743.18, 1715.06, 1644.64, 1583.24, 1510.54, 1457.58,
1215.53. 1H NMR (500 MHz, CDCl3): 8.144 (bs, 1H), 8.069 (bs,
1H), 7.510 (d, J = 8 Hz, 1H), 7.346 (d, J = 8 Hz, 1H), 7.194 (td,
J = 8.0, 1.0 Hz, 1H), 7.114 (td, J = 8.0, 1.0 Hz, 1H), 7.049 (d,
J = 2.0 Hz, 1H), 5.078 (tt, J = 7.0, 1.0 Hz, 1H), 4.965–5.007 (m,
1H), 3.688 (s, 3H), 3.363 (s, 1H), 3.352 (d, J = 2.0 Hz, 1H), 2.960
(sx, J = 7.0 Hz, 1H), 1.906 (s, 3H), 1.827–1.906 (m, 2H), 1.710–
1.784 (m, 1H), 1.646 (d, J = 1.5 Hz, 3H), 1.489–1.560 (m, 1H),
1.533 (s, 3H), 1.151 (d, J = 7.0 Hz, 3H). 13C NMR (125 MHz,
CDCl3): 182.956, 180.445, 172.012, 153.371, 145.411, 136.209,
131.291, 127.037, 124.607, 123.203, 122.519, 119.870, 118.465,
118.321, 111.399, 109.045, 103.304, 56.886, 52.712, 34.147,
29.850, 29.343, 26.660, 25.689, 18.271, 17.633, 9.464. m/z (%):
464 (14) M+, 334 (8), 276 (18), 250 (15), 130 (100). HRMS–
FAB: m/z [M+] calcd for C27H32N2O5: 464.2311; found: 464.2317.
4.6.1. (S)-Methyl 2-(4-hydroxy-2-methyl-5-((R)-6-methylhept-
5-en-2-yl)-3,6-dioxocyclohexa-1,4-dienylamino)-3-methyl
butano-ate (3d)
Deep purple semisolid; 41% yield; Rf: 0.71 (Hex: EtOAc, 90:10). IR
(cmꢀ1) in CHCl3: 3683.82, 3310.15, 3020.11, 2967.25, 2932.11,
1742.38, 1644.44, 1583.63, 1514.33, 1215.38. 1H NMR (500 MHz,
CDCl3): 8.124 (sb, 1H), 6.681(d, J = 9.5 Hz, 1H), 5.081 (tt, J = 7.0,
1.5 Hz, 1H), 4.481 (dd, J = 10, 5.5 Hz, 1H), 3.775 (s, 3H), 3.036–2.963
(m, 1H), 2.231–2.165 (m, 1H), 2.009 (s, 3H), 1.965–1.837 (m, 2H),
1.820–1.745 (m, 1H), 1.648 (d, J = 1.0 Hz, 3H), 1.574–1.517 (m, 1H),
1.539 (s, 3H), 1.182 (d, J = 7.0 Hz, 3H), 1.046 (d, J = 7.0 Hz, 3H), 0.990
(d, J = 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3): 182.789, 180.631,
171.590, 153.546, 145.445, 131.233, 124.595, 118.507, 102.764,
61.866, 52.395, 34.111, 32.528, 29.386, 26.639, 25.653, 18.496,
18.287, 18.085, 17.599, 9.762. MS (EI, 70 eV): m/z (%): 377 (100) M+,
334 (29), 318 (60), 294 (20), 262 (25), 252 (17), 234 (38), 208 (27),
192 (42), 180 (58), 149 (27), 109 (12), 55 (13), 41 (16). HRMS–FAB:
m/z [M+] calcd for C21H31NO5: 377.2202; found: 377.2199.
4.6.5. (3h)
Deep purple semisolid; 52% yield; Rf: 0.51(Hex:EtOAc, 70:30).
IR (cmꢀ1) in CHCl3: 3474.99, 3404.11, 3019.58, 2958.77, 2928.62,
2858.30, 1745.81, 1645.75, 1584.99, 1508.51, 1457.64, 1373.64,
1215.97, 1092.30. 1H NMR (500 MHz, CDCl3): 8.215 (bs, 1H),
7.575 (d, J = 8 Hz, 1H), 7.378 (d, J = 8 Hz, 1H), 7.244 (br s, 1H),
7.218 (t, J = 8 Hz, 1H), 7.139 (t, J = 7.5 Hz, 1H), 7.092 (s, 1H),
6.552 (s, 1H), 5.066 (tt, J = 7.5, 1.0 Hz, 1H), 3.894 (q, J = 6.5 Hz,
2H), 3.090 (t, J = 7 Hz, 2H), 2.9445 (sx, J = 7 Hz, 1H), 2.168 (s,
3H), 2.099 (s, 2H), 1.905–1.842 (m, 2H), 1.638 (d, J = 1.0 Hz,
3H), 1.523 (d, J = 1.0 Hz, 3H), 1.149 (d, J = 7 Hz, 3H). 13C NMR
(125 MHz, CDCl3): 182.992, 179.746, 154.324, 146.442, 136.505,
131.222, 126.844, 124.638, 122.464, 122.412, 119.639, 118.386,
117.694, 111.430, 101.110, 44.817, 34.117, 31.414, 30.181,
29.330, 26.646, 26.498, 25.665, 18.332, 9.902. m/z (%): 406 (47)
M+, 316 (4), 276 (63), 194 (35), 144 (50), 130 (100). HRMS–
4.6.2. (S)-Methyl 2-(4-hydroxy-2-methyl-5-((R)-6-methylhept-
5-en-2-yl)-3,6-dioxocyclohexa-1,4-dienylamino)-4-methylpen-
tanoate (3e)
Deep purple semisolid; 25% yield; Rf: 0.76 (Hex/EtOAc, 95:5). IR
(cmꢀ1) in CHCl3: 3683.37, 3312.33, 3020.15, 2963.00, 2930.75,
1711.35, 1644.95, 1584.72, 1514.59, 1215.45, 1112.32. 1H NMR
(500 MHz, CDCl3): 6.384 (d, J = 9.0 Hz, 1H), 5.088–5.037 (m, 2H),
4.657–4.612 (m, 1H), 3.737 (s, 3H), 3.010–2.932 (m, 1H), 2.352–
2.244 (m, 1H), 1.988 (s, 3H), 1.899– 1.647 (m, 6H), 1.623 (s, 3H),
1.515 (s, 3H), 0.996 (d, J = 6.5 Hz, 3H), 0.959 (d, J = 6.0 Hz, 3H),
0.916 (d, J = 6.0 Hz, 3H). 13C NMR (125 MHz, CDCl3): 182.799,
180.728, 172.763, 153.506, 145.429, 138.872, 131.294, 124.676,
FAB: m/z [M+H+] calcd for C25H31N2O3: 407.2335; found
:
407.2327.

M. Concepción Lozada et al. / Bioorg. Med. Chem. 20 (2012) 5077–5084
5083
tion) was added to the mixture. After cooling it on ice for 10 min it
was centrifuged at 12 879 xg for 5 min and then incubated at 90 °C
for 30 min. After cooling at room temperature, the absorbance of
5. Biological activity
5.1. Cytotoxic activity
200 lL of supernatant was measured at 540 nm in a Bio-Tek Micro-
plate Reader Elx808. 1,1,3,3-tetramethoxypropane (TMP) was used
as an external standard and the level of lipid peroxidation was ex-
pressed as nmol of malondialdehyde (MDA) per mg of protein.
Inhibition of the test lipid peroxidation was measured as percent-
age against a blank that did not contain any test compounds. Quer-
The cytotoxicity of 1, 3a–c and 4 on tumor cells was determined
using the protein-binding dye sulforhodamine B (SRB) assay in mi-
cro culture to determine cell growth. The National Cancer Institute
(NCI), USA, supplied the cell lines employed in these experiments
(PC-3, K-562, HCT-15 and SKLU-1). The cell lines were cultured in
RPMI-1640 culture medium with 10% fetal bovine serum (FBS),
cetin and
a-tocopherol were used as positive standards. The
inhibition ratio (IR[%]) was calculated using the following formula
IR = (C–E) ꢂ 100/C, where C represent the absorbance of control
and E is the absorbance of the test sample.
2 mM L-glutamine, 10,000 units/ml penicillin G, 10,000 g/ml strep-
tomycin sulfate and 25 g/ml amphotericin B (Gibco). They were
kept at 37 °C in an atmosphere of 5% CO2 with 95% humidity. With
the exception of K-562 cell line, the rest of the adherent cell lines
were removed from the tissue culture flask by adding of 1 mL of
0.05% trypsin-EDTA (GIBCO laboratories) and diluted with fresh
media. The viability of the cells used in the experiments exceeds
95% as determined with trypan blue. For the assay 100 l/well of
5 ꢂ 104 cell/ml (K562), 7.5 ꢂ 104 cell/ml (PC3, SKLU-1) and
10 ꢂ 104 cell/ml (HCT15) of cells suspension were seeded in 96-well
micro-titer plates and incubated to allow for cell attachment. After
24 h of incubation, 100 l of each test compounds (1, 3a–c and 4) and
the reference substance (Adriamycin) were added to each well.
After 48 h, adherent cell culture was fixed in situ by adding 50 L of
cold 50% (wt/vol) trichloro acetic acid (TCA) and incubated for
60 min at 4 °C. For K-562, the suspension cell lines were fixed to
All data were represented as mean standard error (SEM). Data
´
were analyzed by one-way ANOVA followed by Dunnetts test for
comparison against control. Values of p 6 0.05 (⁄) and p 6 0.01(⁄⁄
)
were considered statistically significant. The inhibitory concentra-
tion 50 (IC50), was estimated by means of a linear regression.
Acknowledgments
The authors acknowledge financial support from CONACYT of
México (projects 54530 and 104919) as well as from UAM (project
3410125 in Acuerdos del Rector 11/2007) and project 34610788 in
PROMEP. We are thankful to Claudia Rivera Cerecedo and Héctor
Malagón Rivero (Bioterio, Instituto de Fisiología Celular, UNAM)
for technical assistance.
the bottom of microtiter well by gently adding 50 lL of 80% cold
TCA. The supernatant was discarded and the plates were washed
three times with water and air dried. Cultures fixed with TCA were
stained for 30 min with 100 L of 0.4% SRB solution. Protein-bounded
dye was extracted with 10 mM of non buffered tris base and the
optical densities were measured on an Ultra Microplate Reader
(Elx 808, BIO-TEK Instrument, Inc.) with a tested wavelength of
515 nm. Results were expressed as inhibitory concentration 50
(IC50) values. They were calculated according to the protocol of
Monks,35 where a dose–response curve was plotted for each com-
pound and the concentration (IC50), resulting in an inhibition of
50% was estimated through non-linear regression analysis.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.07.027.
References and notes
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3. Walls, F.; Padilla, J.; Joseph-Nathan, P.; Giral, F.; Escobar, M.; Romo, J.
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4. Sánchez, I. H.; Yañez, R.; Enríquez, R.; Joseph-Nathan, P. J. Org. Chem. 1981, 46,
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The compounds 1, 3a–h and 4 were evaluated as lipid peroxida-
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Animals: Adult male Wistar rats (200–250 g) were provided by
Instituto de Fisiología Celular, UNAM. The procedures with animals
and their care were conducted in conformity with Mexican Official
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L of thiobarbituric Acid (TBA) solution
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lL of the super-
l
l
l
lM
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