Synthesis of a new ent-cyclozonarone angular analog, and comparison of its cytotoxicity and apoptotic effects with ent-cyclozonarone

Article abstract of DOI:10.3390/molecules18055517

The synthesis of a newangular analog 11 of cyclozonarone was achieved via Diels-Alder reaction between a sesquiterpene-1,3-diene and 1,4-benzoquinone. The cytotoxic activity of ent-cyclozonarone [(+)-10] and the angular (-)-cyclozonarone analog 11 has been determined in three human cancer cell lines and in normal fibroblasts using the sulforhodamine B assay. The analyzed isomers induce cell death in different cancer cell lines by eliciting nuclear condensation and fragmentation, decreasing mitochondrial membrane permeability and increasing caspase-3 activity, all traits indicating apoptosis, with the effects of (+)-10 being stronger than those of 11 in all cases.

Full text of DOI:10.3390/molecules18055517

Molecules 2013, 18, 5517-5530; doi:10.3390/molecules18055517
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of a New ent-Cyclozonarone Angular Analog,
and Comparison of Its Cytotoxicity and Apoptotic Effects with
ent-Cyclozonarone
Natalia Quiñones Sobarzo 1, Iván Montenegro Venegas 1, Cristian Salas Sánchez 2,
Luis Espinoza Catalán 3, Cristóbal Carrasco Rojas 4, Valentina Ulloa Valdivia 4,
Joan Villena García 4,* and Mauricio Cuellar Fritis 1,*
1
Facultad de Farmacia, Universidad de Valparaíso, Av. Gran Bretaña N° 1093, Valparaíso, Chile
2
Facultad de Química, Pontificia Universidad Católica de Chile, Vicuña Mackenna N° 4860, Santiago,
Chile
3
Departamento de Química, Universidad Técnica Federico Santa María, Av. España N° 1680,
Valparaíso, Chile
4
Centro de Investigaciones Biomédicas, Escuela de Medicina, Universidad de Valparaíso,
Av. Hontaneda N° 2664, Valparaíso, Chile
* Authors to whom correspondence should be addressed; E-Mails: juan.villena@uv.cl (J.V.G.);
mauricio.cuellar@uv.cl (M.C.F.).
Received: 9 April 2013; in revised form: 4 May 2013 / Accepted: 6 May 2013 /
Published: 13 May 2013
Abstract: The synthesis of a newangular analog 11 of cyclozonarone was achieved via
Diels-Alder reaction between a sesquiterpene-1,3-diene and 1,4-benzoquinone. The
cytotoxic activity of ent-cyclozonarone [(+)-10] and the angular (−)-cyclozonarone analog
11 has been determined in three human cancer cell lines and in normal fibroblasts using the
sulforhodamine B assay. The analyzed isomers induce cell death in different cancer cell lines
by eliciting nuclear condensation and fragmentation, decreasing mitochondrial membrane
permeability and increasing caspase-3 activity, all traits indicating apoptosis, with the effects
of (+)-10 being stronger than those of 11 in all cases.
Keywords: sesquiterpenequinone; ent-cyclozonarone; cancer cells; cytotoxicity; apoptosis;
caspase 3; mitochondrial membrane permeability

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1. Introduction
Sesquiterpene-substituted quinones and related molecules constitute an important class of bioactive
marine natural products [1], which includes the cytotoxic, antiviral and antifungal sponge metabolites
puupehenone (1) and puupehedione (2), isolated from sponges, mainly of the orders Verongida and
Dictyoceratida [2], and the sesquiterpene quinones spongiaquinone (3) and cyclospongiaquinone-1 (4),
isolated from Stelospongia conulata [3,4] (Figure 1). On the other hand, the sesquiterpene quinines
hydroquinones, zonarone (5), isozonarone (6), zonarol (7), isozonarol (8), yahazunol (9) and
(−)-cyclozonarone (10) have been isolated from the brown alga Dictyopteris undulata [5] (Figure 1).
Natural(−)-cyclozonarone (10) possesses powerful feeding-deterrent activity towards young abalones
and furthermore it shows anti-cancer activity [5,6]. On the other hand, (+)-cyclozonarone shows
antileshmanial activity and was recently shown to be cytotoxic toward different cancer cell lines [7,8].
The absolute configuration of 10 was established through a six-step synthesis route, starting from natural
(−)polygodial, leading us to the synthetic enantiomer (+)-cyclozonarone [9]. Later, (−)-cyclozonarone
was synthesized starting from (+)-albicanol [10]. Both synthetic routes were based on the
Diels-Alder reaction.
Figure 1. Some examples of natural sesquiterpenequinones.
In the literaturequinone/hydroquinone cytotoxicity is usually attributed to two processes: redox
cycling of quinones, resulting in the generation of reactive oxygen species which can damage
biomolecules, and inhibition of mitochondrial function and electrophilic arylation of critical cellular
nucleophiles. Both mechanisms can result in oxidative stress and cell death [11,12].
Apoptosis or programmed cell death not only plays a crucial role in a range of pathological
conditions, but is now recognized as an important component of multi-step carcinogenesis [13,14]. This
physiological phenomenon represents terminal morphological and biochemical events and occurs
through the activation of a cell-intrinsic suicide program [15]. This program is carried out following

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internal, as well as external signals, and is divided into various phases terminating with signals that
initiate chromatin condensation, characterized by DNA fragmentation as well as by the loss of
mitochondrial membrane integrity and release of molecules that initiate the activation of intracellular
protease [14,16,17]. Mitochondria play a crucial role in the apoptotic cascade by serving as a center
where apoptotic signals converge [18], given that changes induced in the mitochondrial membrane
potential (MMP) have been reported previously to represent a determining step in the execution of cell
death [19,20]. Reduction of the mitochondrial membrane potential causes the opening of the
mitochondrial permeability transition pore, which leads to the release of apoptogenic factors and
activation of caspases that are responsible for the morphological and biochemical events that
characterize classical apoptosis [21,22].
Due to the interest of these meroterpenoids and other related compounds several synthetic strategies
have been reported [23], and specifically we decided to synthesize an angular isomer 11 of
(+)-cyclozonarone, easily prepared in a single step by a Diels-Alder reaction of 1,3 diene 12, prepared in
turn from ketone 13, and 1,4-benzoquinone (Scheme 1).
Scheme 1. Retrosynthetic analysis to obtainof (+)-cyclozonarone.
O
O
O
+
O
O
11
12
13
In addition, we have evaluated both sesquiterpene quinones 10 and 11 for theirinvitro cytotoxicity
against cultured human colon and prostate cancer cells and human dermal fibroblasts (DHF). Moreover,
in order to determine if they act as apoptosis inducers, we evaluated the condensation and fragmentation
of chromatin determined by Hoechst staining, the activity of caspase 3 and the permeabilization of the
mitochondrial membrane by rhodamine 123 staining and the activity of caspase 3 [24,25].
2. Results and Discussion
2.1. Chemistry
When polygodial (14), obtained from the hexane extract of the bark of DrimyswinteriForst. [7], was
treated with sodium borohydride in methanol at room temperature it produced diol 15 in excellent yield.
It was previously reported that treatment of diol 15 with one equivalent of tert-butyl-diphenylsilyl
chloride in DMF, in the presence of imidazole, produced silyl ether 16, with specificity towards the
allylic alcohol, and excellent yield [26]. Unsaturated alcohol 16 was subjected to oxidation with Jones
reagent affording enone 17 in moderate yield. Deprotection of silylether 17 with tetrabutylammonium
fluoride in anhydrous tetrahydrofuran furnished alcohol 18 quantitatively. Catalytic hydrogenation of 18
afforded a mixture 19 of epimeric diols which, treated with Jones reagent, afforded the unsaturated
ketone 13, that had been obtained previously from confertifoline [27,28].

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Diene 12 was obtained by dehydration of allyl alcohol 20, prepared by addition of vinylmagnesium
bromide to ketone 13. Finally, according to Scheme 2, diene 12 was condensed with 1,4-benzoquinone
in benzene under thermal activation, and treated subsequently with DBU giving access to the aromatized
product 11 in 33% yield.
Scheme 2. Synthesis of compound 11.
Reagents and Conditions: (a) NaBH4/MeOH, 90%; (b) ClTBDPS, imidazole, DMF, 97%; (c) Jones
reagent, acetone, 49.4%; (d) TBAF, THF,95%; (e) H2, PtO2, 79%; (f) Jones reagent, acetone, 60%;
(g) vinylmagnesium bromide, THF, 27%; (h) thionyl chloride, Py, 52.5%; (i) p-benzoquinone, Bz,
reflux, 12 h; (j) DBU, Bz, 2 h, 33%.
1
The structural confirmation of new compound 11 was based on a careful analysis of its H- and
13C-NMR spectra, with the help of a combination of 1D and 2D experiments, particularlyheteronuclear
multiple-bond correlations (HMBC and HSQC).
In the 1H-NMR spectrum of 11, the signals at δ = 7.98 ppm (1H, d, J = 8.3Hz) and δ = 7.72 ppm (1H,
d, J = 8.3Hz) were to assigned to the H-12 and H-11 hydrogens, respectively. The signal at
1
3
δ = 7.98 ppm showed an HSQC JHC correlation with C-12 at δC = 124.9 ppm and an HMBC JHC
correlation with carbonyl carbon C-1 at δC = 185.6 ppm. Additionally, H-12 showed HMBC correlations
with the quaternary aromatic carbon C-10b (δC = 158.3 ppm; 3JHC) and with C-11 (δC = 129.2 ppm; 2JHC)
(see Figure 2).
Figure 2. Major HMBC correlations observed for compound 11.
On the other hand, the signal at δH  7.72 ppm (H-11) showed an HSQC correlation with C-11 at
δC = 124.9 ppm (1JHC) and HMBC correlations with C-12a (δC = 131.3 ppm) and C-4b (δC = 139.9 ppm;

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3JHC). Finally,the carbonyl carbons C-1 (δC = 185.6 ppm) and C-4 (δC = 187.7 ppm) were unequivocally
3
assigned when H-12 showed an HMBC JHC correlation with C-1. Additionally, in the 13C-NMR
spectrum the signal at δC = 130.2 ppm was assigned to C-4a by observation of HMBC 3JHC correlations
with H-2 and/or H-3 at δ = 6.87 ppm (s, 2H) All these correlations are shown in Figure 2.
2.2. Biology
The cytotoxicity of the compounds was evaluated in vitro against different cancer cell lines: HT29
colon cancer, PC-3 and DU-145 human prostate cancer and one non-tumoral cell line, human dermal
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. The highest
cytotoxicity values were observed for ent-cyclozonarone[(+)-(10)] in all cell lines tested and were
approximately twice more active than those of compound 11. The cytotoxicity of compounds (+)-10 and
11 in fibroblasts is lower than in the cancer cell lines under study, indicating that the compounds are
selectively toxic to cancer cells.
Table 1. Cytotoxicity (IC50 µM) of (+)-10 and 11.
Compound
(+)-10
11
DU-145
6.4 ± 0.8
13.3 ± 1.4
PC-3
HT-29
DHF
9.5 ± 1.2
14.9 ± 1.8
25.0 ± 0.9
45.0 ± 0.5
48.0 ± 2.3
91.0 ± 7.8
Since compounds (+)-10 and 11 had strong inhibitory effects on the growth of the cancer cell types
tested, we decided to study the effect of these compounds in greater detail. First, we analyzed the
appearance of morphological changes in the cells treated with 25 μM compound for 24 h. Direct
observation using a phase contrast microscope revealed that the morphology of PC-3 and HT-29 cells
was severely distorted and cells became rounded, more clearly after treatment with (+)-10. Moreover,
the cells showed a reduction in number, indicating an increasing progression toward cell death. The
control-treated cells displayed normal and healthy shapes (Figure 3).
To elucidate whether compounds (+)-10 and 11 reduced cell viability of HT29, PC-3, DU-145 and
DHF cells by inducing apoptosis, cells treated with each compound were examined after Hoechst 33342
staining. The nuclear changes in HT29, PC-3, DU-145 and HDF cells were also observed under a
fluorescence microscope (200×). Exposure to compounds (+)-10 and 11 significantly affected the
condensation and fragmentation of the nuclei in the treated cells (Figure 4). Following incubation
with the compounds for 24 h, the fluorescent emission was denser and brighter compared to
control-treated cells. Also, the cells showed chromatin condensation and karyopyknosis, which are
typical apoptotic phenomena.

Molecules 2013, 18
Figure 3. Effect of tested compounds on the morphology of HT-29 and PC-3 cells. Images
5522
obtained with an inverted phase contrast microscope (200×) after 24 h exposure of the cells
to 25 μM (+)-10 or 11.
Figure 4. Effect of ent-cyclozonarone and compound 11 on chromatin condensation and
fragmentation. PC-3, HT-29, DU-145 and DHF cells treated with either compound were
stained with Ho33342 (400×). Representative photographs of nuclear morphologic changes
observed by fluorescent microscopy of the treated cells.

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Changes induced in the mitochondrial membrane potential (MMP) have been reported previously
to represent a determinant in the execution of cell death [19,20]. The effect of compounds (+)-10 and 11
on the mitochondrial membrane potential was determined by flow cytometry using rhodamine
123 stain [29]. As shown in Figure 5, the percentage of rhodamine 123 stained-cells were 46.0 ± 3.2%,
3.5 ± 1.1%, 0.5 ± 1.0% and 38.5 ± 3.5% in the DHF, HT-29, PC-3 and DU-145 cells, respectively, after
treatment with (+)-10 (25 μM); and 78.6 ± 5.34%, 70.1 ± 6.42%, 45.3 ± 4.42% and75.2 ± 5.2%,
respectively, after treatment with 11, as compared to 80–85% in the control (ethanol-treated) cells.
Figure 5 showed that compounds (+)-10 and 11 induced increased mitochondrial membrane
permeability in cancer cells with a greater effect of (+)-10 in all cell lines tested. Moreover both
compounds have a weaker effect on mitochondrial membrane potential in the fibroblast cell line, DHF.
Thus, compounds (+)-10 and 11 induced loss of mitochondrial membrane potential correlated with
increased cell death (see Figure 5).
Figure 5. ent-Cyclozonarone [(+)-10] and 11 treatment-induced changes in the
mitochondrial membrane permeability in HDF, HT-29, PC-3 and DU-145 cells. The cells
were stained with rhodamine 123, and then analyzed by flow cytometry. Representative
histograms show changes in mitochondrial membrane permeability in DU-145, HT-29, PC-3
and DHF cells. The values indicate the percentages of the rhodamine 123 stained cells
treated without or with the compounds (+)-10 or 11 (25μM). (* p <0.05 or ** p < 0.001 vs.
control treated cells).
Then we investigated the effects of our compounds on caspase activity. We focused on caspase-3,
which is activated by a great number of apoptotic signals. This enzyme is a main executor of apoptosis
playing a central role in its biological processing. It has been reported that activation of caspase-3 is an

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essential event for the induction of oligonucleosomal DNA fragmentation [30]. We analyzed the effect
of treatment with the (+)-10 and 11 on caspase-3 activation in normal and cancer cells. As shown in
Figure 6, the activation of caspase-3 in cells exposed to compounds (+)-10 and 11 is increased versus
control-treated cells. (+)-10 increased the activity of caspase 3 when compared with control by
3.6 ± 0.3, 3.0 ± 0.1, 4.0 ± 0.3, and 1.6 ± 0.2 times in DU-145(black), PC-3 (white), HT29 (light gray) and
DHF (dark gray) cells, respectively. Compound 11 increased the activity of caspase 3 by 3.4 ± 0.2, 2.8 ± 0.3,
3.8 ± 0.4, 1.5 ± 0.2 times versus control cells in DU-145, PC-3, HT29 and DHF cells, respectively).
Figure 6. Effect of ent-cyclozonarone and compound 11 on caspase-3 activity of DU-145
(black), PC-3 (white), HT-29 (light grey) and DHF (dark grey) cells. Cells were exposed to
compounds at 25 µM and incubated for 24 h. Values are mean ± S.D. (n = 3). All data are
reported as the percentage change in comparison with the vehicle-treated cells, which were
arbitrarily assigned 100%. * p < 0.05, significantly different from the vehicle-treated cells
(1% ethanol in medium, that is, compound concentration = 0) # p < 0.001, significantly
different from the vehicle-treated cells.
3. Experimental
3.1. General
Unless otherwise stated, all chemical reagents purchased (from Merck or Aldrich) were of the highest
purity commercially available and were used without previous purification. Melting points were
determined (in triplicate) on a Stuart-Scientific SMP3 apparatus and are uncorrected. 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. Elemental analyses were obtained in
13
a Fison Instruments EA 1108 microanalyzer.1H-, C- (DEPT 135 and DEPT 90), HSQC and HMBC
spectra were recorded in CDCl3 solutions and are referenced to the residual peaks of CHCl3 at δ 7.26
ppm and δ 77.0 ppm for 1H and 13C, respectively, on a Bruker Avance 400 Digital NMR spectrometer,
1
operating at 400.1 MHz for H and 100.6MHz for 13C. 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

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chromatography and silica gel plates HF-254 for TLC. TLC spots were detected by heating after spraying
with 25% H2SO4 in H2O. Polygodial (14) was obtained by multigram preparative column
chromatography (hexane: ethyl acetate gradient) from the crude hexane extract of the ground stem bark
of Drimyswinteri Forst., as previously described [26]. Compounds 15–19 and 13 were synthesized using
protocols reported in the literatura and the identities were established by comparison with the previously
reported spectral data [26–28]. The syntheses of compounds 11, 12 and 20 are described below.
(1S,4aS,8aS)-5,5,8a-Trimethyl-1-vinyldecahydronaphthalen-1-ol (20). Ketone 13 (140 mg, 0.72 mmol)
was dissolved in anhydrous THF (10 mL) and cooled to −78 °C. Vinylmagnesium bromide (1 mL, of a
1.0 M solution, 1.0 mmol) was added dropwise. The mixture was allowed to warm to room temperature
over 1 h, and a saturated aqueous solution of NH4Cl was added. The resulting slurry was diluted with
ethyl acetate (100 mL) and washed with water (50 mL). The organic phase was dried (MgSO4) and
concentrated. After a column chromatographic purification using a mixture of hexane and ethyl-acetate
with increasing polarity (49:1 and 1:49) alcohol 20 (70 mg, 27%) was obtained as a colourless oil.
1H-NMR (CDCl3) δ: 6.03 (1H, dd, Jtrans = 17.3 Hz; Jcis = 10.9 Hz, vinyl proton), 5.18 (1H, dd,
Jcis = 10.9 Hz; Jgem = 0.7 Hz, vinyl proton), 5.09 (1H, dd, Jtrans =17.3 Hz; Jgem = 0.7 Hz, vinyl proton),
0.97 (3H, s, 8-α-Me), 0.89 (3H, s, 5-α-Me), 0.84 (3H, s, 5--Me). 13C-NMR (CDCl3) δ: 142.8 (=CH),
113.3 (=CH2), 53.5 (C-1), 45.7 (C-4a), 41.9 (C-6), 41.1(C-8a), 33.7 (5-α-Me), 33.4(C-2), 33.2 (C-5),
32.1 (C-8), 21.9 (5--Me), 21.9 (C-3), 21.4 (C-4), 18.5 ( C-7), 16.9 (8a-Me),in accordance with literature [28].
(4αS, 8αS)-4,4,8α-Trimethyl-8-vinyl-1,2,3,4,4a,5,6,8a-octahydronaphthalene (12). Thionyl chloride
(0.10 mL, 1.16 mmol) was added dropwise at −30 °C to a solution of enol20 (70 mg, 0.31 mmol) in
anhydrous pyridine (4 mL). The solution was brought to 0 °C and stirred for 1 h. The reaction mixture
was diluted with hexane and washed with dilute HCl and then with saturated NaHCO3. The organic
phase was dried and concentrated. Purification by column chromatography using a mixture of hexane
and ethyl-acetate with increasing polarity (49:1 and 1:49) afforded diene 12 (40 mg, 52.5%) as a
1
colorless oil. H-NMR (CDCl3) δ: 6.31 (1H, dd, Jcis = 10.7 Hz, Jtrans 17.0 Hz, =CH), 5.63 (1H, t,
J = 3.9 Hz, H-7), 5.23 (1H, dd, Jgem = 2.2 Hz, Jtrans 17.0 Hz, =CH2), 4.89 (1H, dd, Jgem = 2.2 Hz, Jcis = 10.7
Hz, =CH2), 2.12–0.95 (11H, m), 1.06 (3H, s, 8-α-Me), 0.90 (3H, s, 4-α-Me), 0.85 (3H, s, 4--Me),in
accordance with literature[28].
(6aS,10aS)-7,7,10a-Trimethyl-5,6,6a,7,8,9,10,10a-octahydrochrysene-1,4-dione (11). A mixture of diene
12 (40 mg, 0.20 mmol) and p-benzoquinone (32 mg, 0.30 mmol) in benzene (2 mL) was stirred at
refluxfor 12 h, after which TLC analysis showed the disappearance of the starting material. The solution
was allowed to cool to room temperature, and DBU (2 drops) was added with stirring. The reaction
mixture was concentrated (rotary evaporator), and the residue purified by column chromatography using
a mixture of hexane and ethyl-acetate with increasing polarity (49:1 and 1:49) to yield quinine 11 (5.2 mg,
33.0%) as yellow crystals, m.p. 106–108 °C. [α]2D0 = 1,05° (c = 0.3 CHCl3). Analysis: calculated for
C21H24O2, C = 81.78%, H = 7.84%, found:C = 81.52%, H = 7.77%. The assignment of the signals in the
1H-NMR (CDCl3) and 13C-NMR (CDCl3) spectra are shown in the Table 2.

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Table 2. 1H-NMRchemical shifts, δ (ppm), coupling constants J (H,H) (Hz), and 13C-NMR
chemical shifts, δ (ppm), for compound 11.
Position
1H-NMR
13C-NMR
185.6
141.1 *
136.2 *
187.7
129.2
139.9
30.2
1
__
2
6.87 (1H, s)
3
6.87 (1H, s)
4
__
4a
__
4b
__
5
6
3.52 (1H, ddd, 6.0, 6.0, 19.7); 3.29 (1H, ddd, 8.1, 11.2, 19.7)
1.69 (2H, m)
19.3
6a
1.34 (1H, dd, 2.01, 12.4)
49.0
7
__
29.7
8
1.53 (1H, bs, 13.2); 1.3 (1H, m)
41.2
9
2.02 (1H, m); 1.65 (1H, m)
18.8
10
2.34 (1H, bs, 12.7); 1.41 (1H, dd, 3.7, 12.9)
39.2
10a
10b
11
__
33.4
__
158.3
130.2
124.9
131.3
33.1
7.72 (1H, d, 8.3)
7.98 (1H, d, 8.3)
__
12
12a
7-α-Me
7-β-Me
10a-Me
1.00 (3H, s)
0.97 (3H, s)
1.27 (3H, s)
* exchangeable signals.
24.7
21.6
3.2. Cell Lines
The experimental cell cultures were obtained from the American Type Culture Collection (Rockville,
MD, USA). HT-29 cells (colon cancer cell line), PC-3 and DU-145 (prostate cancer cell lines) and
human dermal fibroblasts (DHF) were grown in Dulbecco’s modified Eagle’s medium (DMEM)
containing 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin and 1 mM glutamine. Cells were
seeded into 96 well microtiter plates in 100 µL at a plating density of 5 × 103 cells/well. After 24 h
incubation at 37 °C under a humidified 5% CO2 atmosphere to allow cell attachment, the cells were
treated with different concentrations of drugs and incubated for 72 h under the same conditions. Stock
solutions of compounds were prepared in ethanol and the final concentration of this solvent was kept
constant at 1%. Control cultures received 1% ethanol alone.
3.3. Cell Viability
3.3.1. In Vitro Growth Inhibition Assay
The sulforhodamine B assay was used according to the method of Skehanet al. [29]. Briefly, the cells
were set up at 3 × 103 cells per well of a 96-flat-bottomed, 200 μL well microplate. Cells were incubated
at 37 °C in a humidified 5% CO2/95% air mixture and treated with the compounds at different

Molecules 2013, 18
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concentrations for 72 h. At the end of drug exposure, cells were fixed with 50% trichloroacetic acid at
4 °C. After washing with water, cells were stained with 0.1% sulforhodamine B (Sigma-Aldrich,
St. Louis, MO, USA), dissolved in 1% acetic acid (50 µL/well) for 30 min, and subsequently washed
with 1% acetic acid to remove unbound stain. Protein-bound stain was solubilized with 100 µL of
10 mM unbuffered Tris base, and the cell density was determined using a fluorescence plate reader
(wavelength 540 nm). Values shown are the mean ± SD of three independent experiments in triplicate.
The software used to calculate the IC50 values was GraphPad (GraphPad Software, San Diego,
CA, USA).
3.4. Morphological Assessment of Cell Apoptosis
Hoechst 33342
Morphological changes in the nuclear chromatin of cells undergoing apoptosis were revealed by a
nuclear fluorescent dye, Hoechst 33342. Briefly, on 24-well chamber slides, 1 × 104 cells/mL DHF,
HT-29, PC-3 and DU-145 were cultured and exposed to compounds for 24 h. The control group was also
exposed to 1% ethanol. The cells were washed twice with phosphate buffer solution, fixed with 3.7%
formaldehyde and washed again with phosphate buffer solution. Following the addition of 1µM Hoechst
33342 (Sigma-Aldrich, Santiago, Chile), they were reacted in a dark room at room temperature for 30 min.
After washing, they were examined under an immunofluorescence microscope (Olympus IX 81 model
inverted microscope).
3.5. Analysis of Mitochondrial Membrane Permeability
Rhodamine 123, a cationic voltage-sensitive probe that reversibly accumulates in mitochondria, was
used to detect changes in transmembrane mitochondrial membrane potential. Exponentially growing
cells were incubated with the compound as indicated in the figure legends. Cells were labelled with
1 μM rhodamine 123 at 37 °C in cell medium for 60 min before terminating the experiment. Cells were
detached from the plate, after washing with ice cold PBS, and the samples were analyzed by flow
cytometry. Data are expressed in percentage of cells withrhodamine 123 [29].
3.6. Caspase 3 Activity Assay
Caspase activity was measured using a colorimetric assay. Briefly, the cells exposed to compounds
were collected by centrifugation at 1000 rpm and the cells were lysed with lysis buffer (1% Triton
X-100, 0.32M sucrose, 5 mM EDTA, 10 mM Tris–HCl, pH 8.0, 2mM dithiothreitol, 1mM PMSF,
1 g/mL aprotinin, 1 mg/mL leupeptin). Thereafter, the lysates were transferred to wells in a 96-well
microplate and were incubated with DEVD-pNA (final concentration 200 µM) specific for caspase-3, at
37 °C for 1 h. The intensity of the developed colour was read at 405 nm in a microplate reader
(SpectraMax, Winooski, VT, USA). The results are expressed as percentages of the control level.

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4. Conclusions
In summary, we describe here the synthesis of a new ent-cyclozonarone angular isomer 11
andcompare its cytotoxicity and that of ent-cyclozonarone [(+)-10] toward normal and cancer human
cells. The analyzed compounds induce cell death in different cancer cell lines by inducing dissipation of
the mitochondrial membrane potential and the sequential activation of caspase-3 leading to apoptosis,
the effects of (+)-10 being stronger than those of 11. These compounds displayed promising activity and
could be used as leads in the design and development of new anticancer drugs. However, additional
studies to determine its in vivo biological activities and to identify the anti-tumor potential of these
compounds, which may represent a promising candidate for in vivo studies of anti-tumor therapies, will
be needed.
Acknowledgements
The authors thank the DGIP of Universidad Técnica Federico Santa María (PICC 2012) for
financial support.
Conflict of Interest
The authors declare no conflict of interest.
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