Synthesis and antitumor activity of diterpenylhydroquinone derivatives of natural ent-labdanes

Article abstract of DOI:10.3390/molecules15096502

Two new compounds 2β-acetoxy-15-phenyl-(22,25-acetoxy)-ent-labda-8(17) , 13(E)-diene (9) and 2β-hydroxy-15-phenyl-(22,24,26-trimethoxy)-ent-labda- 8(17),13(E)-diene (10) have been prepared by an Electrophilic Aromatic Substitution (EAS) reaction between diterpenyl allylic alcohols and 1,4-hydroquinone or 1,3,5-trimethoxybenzene using BF₃·Et ₂O as a catalyst. These compounds, along with a series of natural ent-labdanes 3-8, have been evaluated for their in vitro cytotoxic activities against cultured human cancer cells of PC-3 and DU-145 human prostate cancer, MCF-7 and MDA-MB-231 breast carcinoma and dermal human fibroblasts (DHF). Some compounds displayed inhibition at μM IC₅₀ values.

Full text of DOI:10.3390/molecules15096502

Molecules 2010, 15, 6502-6511; doi:10.3390/molecules15096502
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.org/molecules
Article
Synthesis and Antitumor Activity of Diterpenylhydroquinone
Derivatives of Natural Ent-Labdanes
Luis Espinoza Catalán 1,*, Evelyn Baeza Maturana 1,3, Karen Catalán Marín 1, Mauricio Osorio
Olivares 1, Héctor Carrasco Altamirano 2, Mauricio Cuellar Fritis 3 and Joan Villena García 4,*
1
Departamento de Química, Universidad Técnica Federico Santa María, Av. España N 1680,
Valparaíso, Chile
2
Departamento de Ciencias Químicas, Universidad Andrés Bello, Campus Viña del Mar, Los
Fresnos N 52, Viña del Mar, Chile
3
Facultad de Farmacia, Universidad de Valparaíso, Av. Gran Bretaña N 1093, Valparaíso, Chile
4
Facultad de Medicina, Universidad de Valparaíso, Av. Hontaneda N 2664, Centro Regional de
Estudios en Alimentos Saludables (CREAS), Valparaíso, Chile
* Authors to whom correspondence should be addressed; E-Mails: luis.espinozac@usm.cl (L.E.C.);
juan.villena@uv.cl (J.V.G.).
Received: 28 August 2010; in revised form: 8 September 2010 / Accepted: 15 September 2010 /
Published: 17 September 2010
Abstract: Two new compounds 2β-acetoxy-15-phenyl-(22,25-acetoxy)-ent-labda-8(17),
13(E)-diene (9) and 2β-hydroxy-15-phenyl-(22,24,26-trimethoxy)-ent-labda-8(17),13(E)-
diene (10) have been prepared by an Electrophilic Aromatic Substitution (EAS) reaction
between diterpenyl allylic alcohols and 1,4-hydroquinone or 1,3,5-trimethoxybenzene
.
using BF3 Et2O as a catalyst. These compounds, along with a series of natural ent-labdanes
3-8, have been evaluated for their in vitro cytotoxic activities against cultured human
cancer cells of PC-3 and DU-145 human prostate cancer, MCF-7 and MDA-MB-231 breast
carcinoma and dermal human fibroblasts (DHF). Some compounds displayed inhibition at
µM IC50 values.
Keywords: diterpenylhydroquinones; ent-labdanes; antitumoral activity

Molecules 2010, xx
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1. Introduction
Terpenylquinones constitute an interesting group of marine natural products, for which a wide
variety of biological activities have been described, including anti-inflammatory, antifungal, anti-HIV
and most frequently, antineoplastic properties [1,2]. Their cytotoxic activities has been justified by
their ability undergo redox cycles and the generation of reactive oxygen species, which could damage
tumour cells [3,4]. These compounds are generally characterized for having a sesquiterpene skeleton
attached to a (hydro)quinone moiety (Figure 1).
Figure 1. Some examples of natural sesquiterpenylquinones.
OMe
OMe
O
HO
O
O
OH
O
H
OH
OH
OH
H
Ilimaquinone
Yahazunol
Metacromin A
Terpenylquinones are isolated from natural sources in very low yield, and for that reason during the
last few decades, considerable research effort has been focused on obtaining these compounds by
synthesis. The most recurrent strategies used for synthesizing these compounds involve, as a first step,
the separate preparation of the appropriate terpenyl fragments and aromatic nucleus. The crucial step is
the attachment of the aromatic synthon to the terpenyl skeleton. There are many publications that
report different methods for accomplishing these coupling reactions in the synthesis of sesquiterpenyl-
quinones [5-12]. On the other hand, isolation of natural diterpenylquinones has not been reported and
only one work has reported the synthesis of diterpenylnaphthoquinones, which showed an important
activity in the inhibition of the growth of cancerigenous cells [1,2].
Recently we reported the synthesis and the structural determination of a series of natural ent-labdane
derivatives 3-6 (Figure 2) and the diterpenylhydroquinones 7 and 8 (Figure 2) using Electrophilic
Aromatic Substitution (EAS) reactions between an arene nucleus and an allylic alcohol as diterpenyl
synthon [13].
Using this synthetic protocol and as continuation of that research we describe in this paper the
preparation of two new derivatives 9 and 10. Additionally a new compound 11 derived from the
natural ent-labdanes was obtained (Figure 3). Finally the new compounds 9-11 and the previously
reported series of natural ent-labdanes derivatives 3-8 were evaluated in vitro against cultured human
cancer cells in order to analyse the influence of the molecular structure on the antitumoral activity.

Molecules 2010, xx
6504
Figure 2. Series of natural ent-labdanes derivatives.
Figure 3. New diterpenylhydroquinone derivatives of natural ent-labdanes.
2. Results and Discussion
Previously we reported the isolation and structural determination of the mixture of ent-labdanes 1-2
from Calceolaria inamoena and the preparation of derivatives 3 and 8 [13,14]. In the saponification
reaction used for preparation of diterpenyl synthon 4, a new compound 11 was obtained when acidic
conditions were used, presumably due to a transesterification reaction and the presence of ethyl acetate
in the workup procedure, nevertheless the target diol 4 could be obtained under partial neutralization
conditions (Scheme 1).

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6505
The next step was the coupling reaction between compound 4 and 1,4-hydroquinone to give
diterpenylhydroquinone 8, using the synthetic protocol recently reported by us [13]. A subsequent
acetylation reaction, under standard conditions, gave the new compound 9 (Scheme 1). The structural
1
determination of compound 9 was accomplished by H, 13C, DEPT-135, gs-2D HSQC and gs-2D
HMBC NMR techniques and by comparison with the spectral data of compound 8 [13].
Scheme 1. Synthesis of compound 9.
AcO
HO
23
26
OR
22
21
24
25
15
OAc
OH
a
16
11
c
b
3
14
13
12
HO
AcO
HO
2
H
4 R= H
11 R= Ac
H
H
8
9
Conditions and reagents: a) K2CO3/MeOH/AcOEt, r.t., →4 (55.4%) + 11 (10%); b) compound 4
and 1,4-hydroquinone/1,4-dioxane/BF3·Et2O, N2, r.t., →8 (21%). c) Ac2O/CH2Cl2/DMAP,
r.t. 70%.
On the other hand, the reaction between the diol 4 and 1,3,5-trihydroxybenzene (12) was not observed
and compound 11 was unexpectedly obtained again, presumably for the same reasons given above
(Scheme 2). The structure of the new natural ent-labdane derivative 11 was established by comparison
with the previously reported spectral data of compound 4 [13].
The coupling reaction between the diol 4 and 1,3,5-trimethoxybenzene (13, obtained from 12)
1
rendered the new diterpenylhydroquinone 10 (Scheme 2). The H-NMR spectrum of compound 10
showed the existence of three methoxyl groups at δ 3.80 (s, 3H, CH3O); 3.79 (s, 3H, CH3O); 3.78 (s,
3H, CH3O) ppm and the presence of two aromatic hydrogens at δ 6.13 ppm (s, 2H, H-23 and H-25),
furthermore in the 13C-NMR spectrum the presence of six aromatic carbons and three methoxyl groups
at δ 55.7 (CH3O), 55.7 (CH3O), 55.3 (CH3O) ppm were also observed. On the other hand, the point of
coupling between the diterpenyl fragment and the aromatic nuclei was confirmed by the presence of
the signal at 3.25 ppm (dd, J = 10 and 7.5 Hz, 2H) assigned to the H-15 hydrogens, which was
correlated (by 2D HSQC) with the carbon atom at δ 21.7 ppm (C-15) and corroborated by the 2D
HMBC 3J heteronuclear correlations between H-15 and the carbon signals at δ 158.6 (C-22), 158.6 (C-26)
ppm and 2J heteronuclear correlations with δ 111.1 (C-21) ppm (Figure 4a).

Molecules 2010, xx
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Scheme 2. Synthesis of compound 10.
MeO
OMe
23
24
25
22
21
26
15
OMe
OH
16
11
14
OMe
13
12
a
b
c
11
MeO
OMe
HO
OH
13
12
HO
H
10
Conditions and reagents: a) compound 4/BF3·Et2O/1,4-dioxane, N2, r.t., →11 (11%); b) (CH3)2SO4,
K2CO3, acetone, r.t., 80%; c) compound 4/BF3·Et2O/1,4-dioxane, N2, r.t., →10 (10%).
The E-geometry of the trisubstituted C13-C14 double bond was deduced from gs-sel-1H 1D-
NOESY experiments, where H-15 showed a long range interaction with the Me-16 group (1.73 ppm)
(Figure 4b).
Figure 4. Structure of compound 10. (a) HMBC correlations; (b) NOE correlations.
MeO
OMe
MeO
OMe
25
25
22
24
23
24
23
26
21
26
21
22
15
15
14
16
OMe
OMe
16
14
13
12
13
12
HO
HO
H
H
b)
a)
Bioactivity
The antitumor cytotoxicity of the compounds was evaluated in vitro against three different cancer
cell lines: PC-3 and DU-145 human prostate cancer, MCF-7 and MDA-MB-231 breast carcinoma and
one non-tumoral cell line, dermal human fibroblasts (DHF). A conventional colorimetric assay was set
up to estimate the IC50 values, which represent the concentration of a drug that is required for 50%
inhibition in vitro after 72 h of continuous exposure to the test compounds. Four serial dilutions (from
12.5 to 100 µM) for each sample were evaluated in triplicate. The results obtained from these assays
are shown in Table 1 and the following general observations can be made as a result: the natural

Molecules 2010, xx
6507
ent-labdanes derivatives 3-5 (IC50 > 100) were the least cytotoxic of the series. For cell line PC-3
compounds 7-8 were the most active, with IC50 values ranging between 21.45 µM and 28.24 µM,
suggesting that the presence of the hydroquinone moiety significantly increase the bioactivity. The
highest cytotoxicity values were observed for the diterpenylhydroquinone compounds 7 (IC50 21.45 µM,
prostate cancer PC-3), 8 (IC50 28.24 µM, prostate cancer MCF-7) and 9 (IC50 33.83 µM, breast cancer
MDA-MB-231). Additionally, the cytotoxicity of the compounds 7-9 in fibroblast cells is lower than in
the cancer cell lines under study, indicating that the compounds have higher selectivity for cancer cells.
Table 1. Cytotoxicity (IC50 µM) of natural ent-labdane derivatives 3-11.
Compound
DU-145
> 100
> 100
> 100
96,19
44,61
37,58
62,91
> 100
> 100
MCF-7
s
90,56
> 100
74,94
23,9
33,95
41,11
45,62
86,92
MDA-MB-231
> 100
PC-3
> 100
> 100
> 100
>100
21,45
28,24
64,29
91,17
35,58
DHF
> 100
> 100
> 100
>100
>100
>100
72,30
> 100
> 100
3
4
5
6
7
8
9
10
11
> 100
> 100
95,98
75,86
61,78
33,83
85,93
> 100
3. Conclusions
As a conclusion, we have described the synthesis and structural determination of two new
diterpenylhydroquinones 9 and 10 from natural ent-labdane derivatives. These compounds were
obtained by Electrophilic Aromatic Substitution (EAS) coupling reactions between primary allylic
alcohol ent-labdane derivatives with1,4-hydroquinone and 1,3,5-trimethoxybenzene. Furthermore, a
new co-compound 11 derived from a natural ent-labdane was obtained. The new compounds 9 and 10
showed cytotoxic bioactivity against cell lines MCF-7 and MDA-MB-231 breast carcinoma. The
natural ent-labdane derivatives 3-6 and 11 did not affect the bioactivity of the cells lines studied,
however the diterpenylhydroquinones derivatives 7-10 showed IC50 values with significant inhibitory
activity, ranging in the µM level, due to the presence of a hydroquinone moiety. Moreover,
compounds 7-9 showed some selectivity for cancer cells versus fibroblast cells, which could be a way
to access to compounds potentially less toxicity to normal human cells.
4. Experimental
4.1. General
All purchased chemical reagents (Merck or Aldrich) were of the highest commercial available
purity and were used without previous purification. IR spectra were recorded as thin films on a Nicolet
Impact 420 spectrometer and frequencies are reported in cm−1. Optical rotations were measured with a
sodium lamp (λ = 589 nm, D line) on a Perkin Elmer 241 digital polarimeter equipped with 1 dm cells
at the temperature indicated in each case. Low resolution mass spectra were recorded on a Shimadzu
QP-2000 spectrometer at 70eV ionising voltage and are given as m/z (% rel. int.). 1H, 13C (DEPT 135),

Molecules 2010, xx
6508
1
sel. 1D H NOESY, 2D HSQC and 2D HMBC spectra were recorded in CDCl3 solutions on a Bruker
Avance 400 Digital NMR spectrometer, operating at 400.1 MHz for 1H and 100.6 MHz for 13C and are
13
referenced to the residual peaks of CHCl3 at δ 7.26 ppm and δ 77.0 ppm for 1H and C, respectively.
Chemical shifts are reported in δ ppm and coupling constants (J) are given in Hz. Silica gel (Merck
200-300 mesh) was used for column chromatography and HF-254 silica gel plates for TLC. TLC spots
were detected by heating after spraying with 25% H2SO4 in H2O.
4.2. Starting Materials
Natural ent-labdanes 1-2 were isolated from Calceolaria inamoena and transformed into diterpenyl
allylic alcohol derivative 4 by a reported procedure [13,14].
4.3. General Procedure for EAS: Synthesis of Compounds 8, 10
.
BF3 Et2O (0.23 g, 1.6 mmol) was gradually added at room temperature to a solution of 1,4-hydro-
quinone (0.30 g, 2.27 mmol) or 1,3,5-trimethoxybenzene (0.30 g, 1.79 mmol) and compound 3 (0.50 g,
1.6 mmol) in freshly distilled 1,4-dioxane (30 mL). The mixture was stirred at room temperature under
a nitrogen atmosphere for 24 h, when the completion of the reaction was verified by TLC. The mixture
was poured onto crushed ice (about 30 g) and the organic layer was extracted with EtOAc (3 × 30 mL).
The combined organic phase was washed with 5% NaHCO3 (30 mL), then with water (2 × 20 mL) and
dried over anhydrous Na2SO4, filtered and evaporated. The crude residue was redissolved in CH2Cl2
(5 mL) and chromatographed on silica gel with petroleum ether/EtOAc mixtures of increasing polarity
(19.8:0.2→10.2:9.8) for 8 and (19.8:0.2→13.0:7.0) for 10.
2β-Acetoxy-15-phenyl-(22,25-acetoxy)-ent-labda-8(17),13(E)-diene (9). To diterpenylhydro-
quinone 8 (48.8 mg, 0.12 mmol) dissolved in dichloromethane (30 mL), acetic anhydride (0.54 g, 0.5 mL,
5.2 mmol) and dimethylaminopyridine (1.5 mg, 0.01 mmol) were added. The mixture was stirred at
room temperature during 2 h. The organic phase was washed with water and after it was dried over
anhydrous Na2SO4. The solution was evaporated to dryness to afford the crude reaction product, which
gave 45 mg of compound 9 after column chromatography (eluent hexane/ethyl acetate
19.8:0.2→15.8:4.2), Yield 70%. Colorless viscous oil; [α]D23 = −18.1º (c 0.38, CHCl3); 1H-NMR: 7.02
(d, J = 9.5 Hz, 1H, H-23); 6.93 (m, 2H, H-24 and H-26); 5.17 (bt, J = 7.1 Hz, 1H, H-14); 5.01 (ddt,
J = 12.0, 12.0 and 4.2 Hz, 1H, H-2); 4.86 (s, 1H, H-17b); 4.55 (s, 1H, H-17a); 3.22 (d, J = 7.1 Hz, 2H,
H-15); 2.38 (ddd, J = 13.0, 4.9 and 2.2 Hz, 1H, H-7α); 2.30 (s, 3H, CH3CO2-C-22); 2.28 (s, 3H,
CH3CO2-C-25); 2.15 (ddd, J = 14.2, 13.0 and 4.4 Hz, 1H, H-12b); 2.05 (m, 1H, H-1α); 2.03 (s, 3H,
CH3CO2-C-2); 1.92 (ddd, J = 13.5, 13.0 and 4.9 Hz, 1H, H-7β); 1.83 (m, 1H, H-12a); 1.75 (m, 1H, H-3α);
1.71 (m, 1H, H-6α); 1.67 (s, 3H, H-16); 1.62 (bd, J = 11.0 Hz, 1H, H-9); 1.55 (m, 1H, H-11b); 1.46
(ddd, J = 12.2, 9.8 and 4.4 Hz, 1H, H-11a); 1.29 (dd, J = 13.0 and 3.9 Hz, 1H, H-6β); 1.24 (dd, J = 12.0
and 12.0 Hz, 1H, H-3β); 1.08 (dd, J = 13.0 and 2.5 Hz, 1H, H-5); 1.04 (dd, J = 12.0 and 12.0 Hz, 1H,
13
H-1β); 0.93 (s, 3H, H-18); 0.88 (s, 3H, H-19); 0.76 (s, 3H, H-20); C-NMR: 44.1 (C-1), 69.3 (C-2),
46.8 (C-3), 34.9 (C-4), 54.9 (C-5), 23.9 (C-6), 37.9 (C-7), 147.3 (C-8), 55.9 (C-9), 40.9 (C-10), 22.1
(C-11), 38.3 (C-12), 137.8 (C-13), 120.8 (C-14), 28.6 (C-15), 16.2 (C-16), 107.3 (C-17), 33.5 (C-18),
22.4 (C-19), 15.2 (C-20), 134.9 (C-21), 146.2 (C-22), 122.9 (C-23), 119.9 (C-24), 148.2 (C-25), 122.7

Molecules 2010, xx
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(C-26), 170.6 (CH3COC-2), 169.3 (CH3COC-Ar), 169.2 (CH3COC-Ar), 21.5 (CH3CO2C-2), 21.1
(CH3CO2Ar-25), 20.8 (CH3CO2Ar-22); IR (cm−1): 2940, 1767, 1737, 1486, 1363, 1239, 1209, 1168,
1025; MS (m/z, %): M+ 524 (< 1%), 168 (9.5), 167 (100), 132 (6.3), 122 (4.5), 121 (4.3), 113 (25), 112
(14.9), 105 (5.5), 104 (15.2), 93 (4.1), 84 (7.7), 83 (13.8), 82 (2.3), 76 (7.3), 71 (37.3), 70 (31.7), 69
(8.5), 57 (45.5), 55 (17.4).
2β-Hydroxy-15-phenyl-(22,24,26-trimethoxy)-ent-labda-8(17),13(E)-diene (10). colorless viscous
23
1
oil, 70 mg, Yield 10%, [α]D = −6.2º (c 1.66, CHCl3); H-NMR: 6.13 (s, 2H, H-23 and H-25); 5.12
(t, J = 7.2 Hz, 1H, H-14); 4.81 (s, 1H, H-17b); 4.53 (s, 1H, H-17a); 3.83 (ddt, J = 11.5, 11.5 and 4.2 Hz,
1H, H-2); 3.80 (s, 3H, CH3O);3.79 (s, 3H, CH3O); 3.78 (s, 3H, CH3O); 3.25 (dd, J = 10 and 7.5 Hz,
2H, H-15); 2.33 (ddd, J = 12.7, 4.4 and 2.2 Hz, 1H, H-7α); 2.17 (m, 1H, H-12b); 2.10 (m, 1H, H-1a);
1.84 (ddd, J = 13.5, 13.2 and 4.6 Hz, 1H, H-7β); 1.83 (m, 1H, H-12a); 1.75 (m, 1H, H-3α); 1.73 (s, 3H,
H-16); 1.68 (m, 1H, H-6β); 1.57 (bd, J = 9.9 Hz, 1H, H-9); 1.55 (m, 2H, H-11a and H 11-b); 1.26 (m,
1H, H-6α); 1.09 (dd, J = 12.0 and 12.0 Hz, 1H, H-3β); 0.97 (dd, J = 12.6 and 2.6 Hz, 1H, H-5); 0.91 (s,
3H, H-18); 0.82 (s, 3H, H-19); 0.68 (s, 3H, H-20); 13C-NMR: 48.1 (C-1), 65.7 (C-2), 51.1 (C-3), 35.0
(C-4), 54.8 (C-5), 23.9 (C-6), 38.2 (C-7), 147.8 (C-8), 55.5 (C-9), 40.9 (C-10), 21.9 (C-11), 38.0 (C-12),
134.4 (C-13), 123.5 (C-14), 21.7 (C-15), 16.1 (C-16), 107.0 (C-17), 33.6 (C-18), 22.6 (C-19), 15.4 (C-20),
111.1 (C-21), 158.6 (C-22), 90.7 (C-23), 159.1 (C-24), 90.7 (C-25), 158.6 (C-26), 55.7 (CH3O), 55.7
(CH3O), 55.3 (CH3O); IR (cm−1): 2953, 2927, 1727, 1601, 1460, 1207, 1152, 1124, 1036, 750; MS
(m/z, %): M+ 456 (< 1%), 429 (19), 357 (24), 356 (35), 355 (100), 281 (47), 221 (48), 207 (18), 147
(53), 73 (79).
2β-Hydroxy-15-acetoxy-ent-labda-8(17),13(E)-diene (11). To compound 3 (0.22 g, 0.63 mmol)
dissolved in methanol (70 mL), potassium carbonate (0.1 g, 0.72 mmol) was added. The mixture was
stirred at room temperature during 2 h. The solution was evaporated to dryness. Dilution with ethyl
acetate was followed by washing with HCl 5% and organic phase was dried over anhydrous Na2SO4.
The solution was evaporated to dryness to afford the crude reaction product, which gave 22 mg of
compound 11 after column chromatography (eluent hexane/ethyl acetate 19.8:0.2→16.0:4.0), Yield
10%. colorless viscous oil, [α]D23 = −27.6º (c 0.88, CHCl3); 1H-NMR: 5.35 (t, J = 7.3, 1H, H-14); 4.90
(s, 1H, H-17b); 4.58 (s, 1H, H-17a); 4.50 (dd, J = 7.0, and 3.0 Hz, 2H, H-15); 3.87 (ddt, J = 4.2, 11.5 and
11.4 Hz, 1H, H-2); 2.4 (ddd, J = 12.8, 4.1 and 2.4 Hz, 1H, H-7α); 2.17 (m, 1H, H-12b); 2.10 (m, 1H,
H-1α); 2.04 (s, 3H, CH3CO2-C-15); 1.96 (ddd, J = 13.0, 13.0 and 5.0 Hz, 1H, H-7β); 1.83 (m, 1H, H-12a);
1.78 (dd, J = 2.3 and 4.1, 1H, H-3α); 1.75 (s, 3H, H-16); 1.72 (m, 1H, H-6β); 1.61 (bd, J = 10.4 Hz,
1H, H-9); 1.47 (m, 2H, H-11a and H-11b); 1.29 (dd, J = 12.9 and 4.2 Hz, 1H, H-6α); 1.15 (dd, J = 12.0
and 12.0 Hz, 1H, H-3β); 1.07 (dd, J = 12.6 and 2.6 Hz, 1H, H-5); 0.95 (dd, J = 11.5 and 11.5 Hz, 1H,
13
H-1β); 0.93 (s, 3H, H-18); 0.84 (s, 3H, H-19); 0.71 (s, 3H, H-20); C-NMR: 48.2 (C-1), 65.7 (C-2),
51.1 (C-3), 33.7 (C-4), 54.9 (C-5), 23.9 (C-6), 38.3 (C-7), 147.6 (C-8), 56.1 (C-9), 41.0 (C-10), 21.8
(C-11), 38.0 (C-12), 142.8 (C-13), 118.1 (C-14), 61.4 (C-15), 16.5 (C-16), 107.2 (C-17), 35.0 (C-18), 22.6
(C-19), 15.4 (C-20), 21.1 (CH3CO2-C-15), 171.2 (CO2-C-15); IR (cm−1): 2934, 2856, 1725, 1461, 1441,
1366, 1259, 1235, 1029, 737; MS (m/z, %): M+ 348.52 (< 1%), 318 (25), 175 (34), 135 (100), 121
(28), 119 (25), 107 (35), 105 (25), 95 (28), 93 (34), 81 (30), 69 (34), 57 (25).

Molecules 2010, xx
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4.4. Bioactivity: Cell Growth Inhibition Assay
The colorimetric assay using sulforhodamine B (SRB) was realized following an adaptation of the
described method by Skehan et al. [15,16]. Cells were seeded in 96-well microtiter plates, at
5 × 103 cells per well in aliquots of 100 µL of DMEM/F-12 medium, and they were allowed to attach
to the plate surface by growing in drug-free medium for 18 h. Afterward, compounds samples were
added in aliquots (dissolved in EtOH/H2O) to achieve a final concentration of 100, 50, 25 12.5 µM.
After 72 h exposure, the in vitro cytotoxicity was measured by the SRB dye assay. Cells were fixed by
adding cold 50% (wt/vol) trichloroacetic acid (TCA, 25 µL) and incubating for 60 min at 4ºC. Plates
were washed with deionized water and dried; SRB solution (0.1% wt/vol in 1% acetic acid, 50 µL)
was added to each microtiter well and incubated for 30 min at room temperature. Unbound SRB is
removed by washing with 1% acetic acid. Plates were air-dried and bound stain was solubilized with
Tris base (100 µL, 10 mM). Optical densities were read on an automated spectrophotometer plate
reader at a single wavelength of 540 nm. Values shown are the % viability vs. ctrl + SD, n = four
independent experiments in triplicate.
Acknowledgements
The authors thank the Dirección General de Investigación y Postgrado (DGIP) of the Universidad
Técnica Federico Santa María (grant N 13.09.42, PIIC QUI 2009 for E.B.M., PAC 2009-2010 for
M.O.O.) and the Dirección de Investigación y Postgrado (DIPUV) of the Universidad de Valparaiso
(DIPUV N 27/2006 for J.V.G.) for financial support.
References
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Sample Availability: Samples of the compounds 3-11 are available from the authors.
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