Antiproliferative effect and apoptotic activity of linear geranylphenol derivatives from phloroglucinol and orcinol

Article abstract of DOI:10.1016/j.cbi.2016.01.016

Sixteen synthetic linear derivatives geranylphenols, were obtained from phloroglucinol and orcinol, and cytotoxic activity was evaluated in vitro against cancer cell lines (HT-29, PC-3, MDA-MB231, DU-145) and one non-tumor cell line, human dermal fibrobla

Full text of DOI:10.1016/j.cbi.2016.01.016

Accepted Manuscript
Antiproliferative effect and Apoptotic activity of linear geranylphenol derivatives from
phloroglucinol and orcinol
Lautaro Taborga, Luis Espinoza, Alejandra Moller, Héctor Carrasco, Mauricio Cuellar,
Joan Villena
PII:
S0009-2797(16)30016-3
10.1016/j.cbi.2016.01.016
CBI 7578
DOI:
Reference:
To appear in: Chemico-Biological Interactions
Received Date: 26 August 2015
Revised Date: 23 December 2015
Accepted Date: 21 January 2016
Please cite this article as: L. Taborga, L. Espinoza, A. Moller, H. Carrasco, M. Cuellar, J. Villena,
Antiproliferative effect and Apoptotic activity of linear geranylphenol derivatives from phloroglucinol and
orcinol, Chemico-Biological Interactions (2016), doi: 10.1016/j.cbi.2016.01.016.
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Antiproliferative effect and Apoptotic activity of linear
geranylphenol derivatives from phloroglucinol and orcinol.
Lautaro Taborga 1, †,*, Luis Espinoza 1, , Alejandra Moller 2, Héctor Carrasco 3, , Mauricio
Cuellar 4 and Joan Villena 2, †*.
1 Departamento de Química, Universidad Técnica Federico Santa María, Av. España 1680, Valparaíso,
2390123, Chile. lautaro.taborga@usm.cl (L.T.); luis.espinozac@usm.cl (L.E.)
2
Centro Investigaciones Biomédicas (CIB), Facultad de Medicina, Universidad de Valparaíso,
Hontaneda 2653, Valparaíso, 2341386, Chile. alejandra.moller@uv.cl (A.M); juan.villena@uv.cl
(J.V.)
3
Instituto de Ciencias Químicas Aplicadas, Facultad de Ingeniería, Universidad Autónoma de Chile,
Llano Subercaseaux 2801, San Miguel, Santiago, 8900000, Chile; hector.carrasco@uautonoma.cl
(H.C.)
4 Facultad de Farmacia, Universidad de Valparaíso, Av. Gran Bretaña 1093, Playa Ancha, Valparaíso,
2360102, Chile. mauricio.cuellar@uv.cl (M.C.)
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; lautaro.taborga@usm.cl (L.T.);
juan.villena@uv.cl (J.V.)
Tel.: +56-032-2507354 (ext. 123); Fax: +56-032-2507368.
Abstract: Sixteen synthetic linear derivatives geranylphenols, were obtained from phloroglucinol and
orcinol, and cytotoxic activity was evaluated in vitro against cancer cell lines (HT-29, PC-3, MDA-
MB231, DU-145) and one non-tumor cell line, human dermal fibroblast (HDF). IC50 values were
determined at concentrations of 0–100 µM of each compound for 72 h. Compounds 12, 13, 17, 21, 22
and 25, showed cytotoxic activity. To elucidate whether these compounds reduce cell viability by
inducing apoptosis, cell lines MCF-7, PC-3 and DHF were treated with each active compound 12, 13, 17,
21, 22 and 25 and were examined after Hoechst 33342 staining. The compounds 12, 13 and 17 induced
apoptosis in various cancer cell lines, as shown by nuclear condensation and/or fragmentation. In
addition, it was found that compounds 12 and 13, induced changes in mitochondrial membrane
permeability in those cancer cell lines. Such induction was associated with the depletion of
mitochondrial membrane potential. These activities led to the cleavage of caspases inducing the cell
death process.

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Keywords: cytotoxic activity; cancer cell lines; apoptosis; mitochondrial membrane
permeability; caspase-3 activity; linear geranylphenols.
1. Introduction
Prenylated and polyprenylated 1,4-benzoquinones and hydroquinones such as ubiquinones,
plastoquinones, and tocopherols are widespread in plants and animals, in which they play important
roles in electron transport, photosynthesis, and as antioxidants [1,2]. Prenyl benzoquinones have been
also isolated from brown algae of the order Fucales [3-6], sponges [7-10], alcyonaceans [11],
gorgonaceans [12], and ascidians belonging to the genus Aplidium [13-18]. These substances present a
terpenoid portion ranging from one to nine isoprene units.
It has been extensively documented that multiple metabolites, obtained from species belonging to
the plant kingdom, have special biological properties suitable for controlling several types of animal
and plant pathogen. For instance, linear geranylquinones or geranylhydroquinones, present in higher
plants and in marine urochordates [19], exhibit cytotoxic activity and inhibit larval growth and
development. Some particular compounds such as 2-geranylbenzoquinone (1), isolated from Ascindian
Synoicum castellatum [20], 2-geranylhydroquinone (2) isolated from the Cordia alliodora tree [21],
Phacelia crenulata [22-24], Aplidium antillense [25] and the tunicate Amaroucium multiplicatum [26],
have been related to biological activities including toxicity, cytotoxicity, antimicrobial, anti-cancer
protective and antioxidant effects, among others [23,26-30]. Additionally, linear
geranylmethoxyphenol/acetates (compounds 3-5, see Figure 1), isolated from Phaceliaixodes [25], are
cytotoxic, allergenic and insecticidal, and topical application of 100 µg of geranylbenzoquinone on
pupae of Tenebrio caused severe abnormalities and death [31]. However, considering the activity
against phytophagous insects and pathogens reported for compounds 3-5 (figure 1) [31] and assuming
the presence of this property in other geranylphenol analogs, we have recently reported the synthesis,
structure determination and their effect on mycelial growth of plant pathogen Botrytis cinerea of eight
linear geranylphenols molecules, wherein the compounds 2 and 2-geranylphloroglucinol (6) were the
more active ones, showing an inhibitory effect on the mycelial growth that depends on the applied
concentration [32].

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Figure 1. Structure of some active natural and synthetic linear geranylphenols.
On the other hand, different studies of the structure activity relation (SAR) in a series of non
methoxylated and methoxylated prenylated quinones with side chains containing from one to eight
isoprene units reported that the optimum length of the side-chain is two isoprene units and in the para-
position relative to the methoxy-group [28,33]. Additionally, these authors informed that all tested
quinones (compounds 7-10, figure 2) have inhibited JB6 Cl41 cell transformation and p53 activity
inducing apoptosis and also the activities of AP-1 and NF-κB. Additionally, in previous studies we
have reported the synthesis and cytotoxic activity of 2-geranylbenzoquinone (1) and 2-
geranylhydroquinone (2) and some geranylmethoxyphenol/acetate analogs (figure 1) [34-35].
Figure 2. Structure of some active synthetic linear geranylquinones.

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Therefore, in this research we report the cytotoxic activity of a series of linear geranylphenol,
acetylated and methoxylated derivatives of phloroglucinol (compounds 6, 11-14, see Figure 3) and
orcinol (compounds 15-25, see figure 4). These compounds were obtained by traditional methodology
[33-37] and by a modification of a previously reported synthetic method [32]. Compounds 6 and 11-25
were evaluated in vitro against various human cancer cells lines in order to analyze the cytotoxic
activity.
Figure 3. Structure of synthetic linear derivatives geranylphenols acetylated and methoxilated of
phloroglucinol.

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Figure 4. Structure of synthetic linear derivatives geranylphenols acetylated and methoxilated of
orcinol.
CH3
OH
(16)
(15)
(17)
HO
HO
OH
OH
H3C
OH
CH3
OH
(18)
HO
CH3
CH3
OAc
(20)
(19)
(21)
AcO
AcO
OAc
OAc
H3C
OAc
CH3
OAc
(22)
AcO
CH3
CH3
OCH3
(24)
(23)
H3CO
OCH3
H3C
OCH3
OCH3
(25)
H3CO
CH3
2. Results and Discussion
2.1. Chemical
2-geranylphloroglucinol (6) and derivatives 11-13 were obtained by a previously reported synthetic
method [32]. While the trimethoxylated derivative 14 was obtained by methylation reaction of 6,

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(86.8% yield), in dimethyl sulfate/K2CO3 system under reflux conditions in acetone. The structure of
14 was mainly established by NMR, where the signals at δ =3.92 ppm (s, 3H, OCH3) and δ =3.79
ppm (s, 6H, 2 x OCH3) confirmed the presence of three methoxyl groups. Additionally, in the 13C
H
H
NMR spectrum, the signal at δ
C
= 55.3 ppm was assigned to 2 x OCH3 and δ = 54.9 ppm was assigned
C
to the third methoxy group.
Compounds 15 and 16 were previously synthesized by means of Electrophilic Aromatic Substitutions
reactions, catalyzed by mineral acids, such as orcinol reaction with geraniol in aqueous formic acid
solutions [38], condensation reaction of geraniol and orcinol in methylene chloride in the presence of
p-toluenesulfonic acid [39]. However, the yields obtained with these methodologies were low (2-18%
yield). Subsequently we report the preparation of compounds 15 and 16 from orcinol and geraniol with
.
27.6% and 12.6% yields respectively, using Lewis acid (BF3 Et2O) as catalyst in dioxane [36].
Recently, our research group reported a new synthesis of compounds 15 and 16 with identical yields,
.
by direct geranylation reaction between orcinol and geraniol, using BF3 OEt2 as catalyst and AgNO3 as
secondary catalyst but using acetonitrile instead of dioxane as solvent. Additionally in this reaction the
compounds 17 and 18 were obtained with 6.1% and 9.9 % yields respectively [40]. Subsequently
derivatives 19-22 were obtained by standard acetylation reaction (Ac2O/CH2Cl2/DMAP) from phenols
15-18 respectively [40].
On the other hand the methoxylated compounds 23-25 were obtained by methylation reaction (in
dimethyl sulfate/K2CO3 system under reflux conditions in acetone) from 15, 16 and 18, with 80.1%,
78.7% and 78.1% yield respectively.
1
The structure of 23-25 was mainly established by NMR. For compound 23 in the H NMR spectrum
the signals at δ
H
=3.81 ppm (s, 3H, OCH3) and δ
H
=3.80 ppm (s, 3H, OCH3) confirmed the presence of
=55.5 and 55.1 ppm
= 3.85 ppm (s, 6H,
13
two methoxyl groups. Additionally, in the C NMR spectrum, the signals at δ
C
was assigned to two methoxyl group. For symmetric compound 24, the signal at δ
H
13
OCH3) and the signal at δ
C
= 55.5 ppm (2 x OCH3) in the C NMR spectrum, mainly confirm the
structure 24. Similarly compound 25, showed signals at δ
(s, 3H, OCH3) in the 1H NMR spectrum. While in the 13C NMR spectrum the signals at δ
H
=3.82 ppm (s, 3H, OCH3) and δ
H
=3.72 ppm
= 61.5 and
C
55.5 ppm was assigned to two methoxyl group respectively.
2.1.1. Biological
2.1.1.1. Cell Viability
2.1.1.1.1 In vitro Growth Inhibition Assay
The cytotoxicity of compounds (6 and 11-25) was evaluated in vitro against different cancer cell

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lines: MDA-MB-231 breast cancer, DU-145 and PC-3 prostate cancer, HT-29 colon cancer and one
non-tumor cell line, human dermal fibroblast (HDF). A colorimetric assay was set up to estimate the
IC50 values. The IC50 obtained from these assays are shown in Table 1.
Table 1. Cytotoxicity (IC50 ꢀM) of compounds 6 and 11-25.
MDA-
HDF
HT-29
PC-3
DU-145
N°
MB231
IC50 ± DS
>100
Compound
IC50 ± DS
>100
IC50 ± DS
>100
IC50 ± DS
>100
IC50 ± DS
>100
6
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
>100
>100
>100
>100
>100
23.5 ± 2.3
27.0 ± 5.6
>100
21.7 ± 3.5
19.4 ± 3.1
>100
23.7 ± 3.4
18.8 ± 2.6
>100
18.5 ± 3.5
66.7 ± 10.4
>100
29.9 ± 3.5
82.3 ± 4.9
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
29.9 ± 2.6
>100
46.7 ± 4.1
>100
41.0 ± 4.6
>100
71.1 ± 4.1
>100
62.1 ± 5.6
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
63.2 ± 7.8
>100
60.3 ± 7.8
44.5 ± 6.5
>100
>100
>100
68.1 ± 10.5
>100
91.8 ± 11.8
>100
82.2 ± 7.5
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
68.1 ± 7.3
The highest cytotoxicity values were observed for compounds 12 and 13 in all cell lines tested and
were more active than those of the rest of compounds. The cytotoxicity of compounds in human
dermal fibroblast (HDF) is similar than in the cancer cell lines under study, even so the cytotoxic
activity was not previously described for the compounds 12, 13, 17, 21, 22, and 25 in the literature.
Based on the obtained results, the cytotoxic action against studied cell lines would be explained by
the structure-activity relationship analysis of the results showed in table 1. It is clearly seen, that the
presence of both geranyl chains on the aromatic ring is critical for apoptotic activity, because

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monogeranyl compounds have not activity on any of the studied cell lines. In the same way, by
comparing phloroglucinol with orcinol derivatives it can be seen that introduction of a methyl group
(orcinol derivatives) in replace of acetyl group (phloroglucinol derivatives) decreases the activity, as it
can be seen by comparing the cytotoxicity of compounds 12 and 13 with that exhibited by compounds
17, 21, 22 and 25.
On the other hand, when comparing only orcinol derivatives it can be seen that the presence of a
methyl group between two geranyl chains increases the cytotoxicity on the non-tumor line cells (HDF)
compared with cancer cell lines (compares 17 and 18 compounds). Finally, the differences on
cytotoxic activities between compounds 12 and 13 and the others compounds may be related to the
presence of acetyl and the absence of methyl groups in their structure.
The more elevated log P value for compounds 12, 13 17, 21, 22 and 25 (data not shown) suggests that
these compounds could penetrate membranes more easily. The log P corresponding to partition
coefficient and is the ratio of the equilibrium concentration of a substance between two immiscible
liquids (octanol/water), to major value of log P more affinity with to lipid phase [41].
Additionally, the data in Table 1 show that compound 12 exhibits the highest inhibitory activity
against studied cell lines including non-tumor cells. This results render this compound useless as
anticancer agents. Interestingly, the other active compounds affect selectively some cell lines. For
example, compounds 13 inhibit more efficiently HT-29 and PC-3 cells, whereas compound 22 acts
selectively on PC-3 cell and compound 25 inhibits only DU-145 cells. The reason of these effects is
not clear, and as the active compounds are just a few it is difficult to find a relation with the chemical
structure.
2.1.1.1.2 Morphological Assessment of Cell Apoptosis
In view of the above and given that compounds 12, 13, 17, 21, 22 and 25 had inhibitory effects on
the viability of the cancer cell lines tested, the effect of these compounds deserve to be studied in greater
detail. Moreover, the biological cytotoxic activity was not previously described for the compounds 12, 13,
17, 21, 22, and 25 in the literature.
First, the appearance of morphological changes in the cells treated with 50 ꢀM compound for 24 h
was analyzed. Direct observation, using a phase contrast microscope, revealed that the morphologies
of PC-3 (Figure 5), MDA-MB231, DU-145, HT-29 and HDF (data not shown) cells were distorted and
cells became rounded after treatment with compounds 12, 13, 17, 21, 22 and 25. Moreover, the cells
showed a reduction in number, indicating an increasing progression toward cell death. The control-
treated cells (1% ethanol) displayed normal and healthy shapes (Figure 5).

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Figure 5. Effect of tested compounds on the morphologies of PC-3 cells. Images obtained with an
inverted phase contrast microscope (200×) after 24 h exposure of the cells to 50 ꢀM of compounds 12,

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13,
17,
21,
22
and
25
(1%
ethanol).
To elucidate whether the compounds reduced cell viability in the cell lines tested by inducing apoptosis
(MCF-7, PC-3 and HDF cells), cells treated with each active compound 12, 13, 17, 21, 22 and 25 were
examined after Hoechst 33342 staining. Nuclear changes in PC-3, HT-29, MDA-MB231, DU-145 and
HDF cells were observed under a fluorescence microscope (200×) and chromatin condensation and/or
fragmentation was quantified (Table 2).

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Table 2 Percentage of condensed and/or fragmented nuclei after treatment with
compounds 12, 13, 17, 21, 22, 25 or ethanol (* p < 0.01).
Compound
PC-3
HT-29
MDA-MB 231
23.3 ± 2.1*
13.9 ± 1.3*
12.9 ± 2.0*
5.2 ± 0.8
DU-145
HDF
23.7 ± 3.2*
22.3 ± 2.3*
16.3 ± 1.1*
13.7 ± 1.0*
15.1 ± 1.4*
7.4 ± 0.9
20.1 ± 2.0*
21.8 ± 2.1*
15.3 ± 0.9*
14.3 ± 1.5*
9.3 ± 1.3
22.7 ± 3.5* 19.1 ± 2.0*
14.4 ± 3.3* 18.7 ± 1.1*
14.5 ± 1.4* 18.4 ± 1.3*
12
13
17
6.6 ± 0.9
11.0 ± 1.3
13.8 ± 0.9*
7.6 ± 1.2
6.1 ± 0.9
12.9 ± 1.0*
6.7 ± 1.1
21
8.7 ± 1.2
22
8.9 ± 1.3
8.0 ± 1.0
25
6.9 ± 1.1
7.8 ± 1.0
7.3 ± 1.1
6.4 ± 1.0
EtOH
As shown in Table 2 treatment with compounds 12, 13 and 17 significantly increased the number of
cells with condensed and/or fragmented nuclei versus control-treated cells (* p < 0.01). By other hand
compounds 21, 22 and 25 increased nuclear fragmentation only in some cell lines coinciding with
cellular viability results (Table 1 and 2).
2.1.1.1.3 Analysis of Mitochondrial Membrane Permeability
Mitochondria play a crucial role in the cell death decision, participating in apoptotic cascade by
serving as a convergent center of apoptotic signals originating from both the extrinsic and intrinsic
pathways [42]. Changes induced in the mitochondrial membrane potential (MMP) have been reported
previously to represent a determinant in the execution of cell death [43]. This effect may be determinant
in cell death, we have analyzed the effect of compounds 12, 13, 17, 21, and 22 (50 ꢀM) on the
mitochondrial membrane potential using flow cytometry with rhodamine 123 stain [44]. As shown in
Figure 6B, the percentage of rhodamine 123 stained-cells were decreased significantly after treatments
with compounds 12 and 13 in all cell lines studied. However, compounds 17, 21 and 22 diminished the
rhodamine-stained cells only in one or two cell lines, as compared to 85–90% in the control cells (1%
ethanol).
Figure 6 shows that compounds 12 and 13 increased mitochondrial membrane permeability in cancer
cells with a greater effect than compounds 17, 21 and 22. The compounds have not differences in the
effect on mitochondrial membrane potential between HDF and cancer cell lines. Thus, the effect of
compounds 12 and 13 on the decrease of mitochondrial membrane potential correlated with diminished
viability (see Table 1) and increased nuclear condensation and/or fragmentation (see Table 2).
Moreover, compounds 17, 21 and 22 only changed the mitochondrial membrane potential when
compared to control cells in PC-3 and HDF, MDA-MB231 and PC-3 cell lines, respectively. Probably,

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the difference in the observed activity of these compounds to mitochondrial level, could be associated
with the polarity of these derivatives, since those having lower polarity, corresponding to
phloroglucinol derivatives 12 and 13, exhibit a greater effect and increased mitochondrial membrane
permeability. The reason for this increase in activity is probably due to that the derivatives of minor
polarity might cross cell membranes more easily and lead to an increase in the bioavailability and
activity of these compounds. Specifically, we considered that the passage through the mitochondrial
membrane should lead to an alteration of membrane potential. This change in mitochondrial membrane
potential would cause the release of apoptogenic factors. The precise mechanism by which compounds
12 and 13 are decreasing mitochondrial membrane potential has not been established, though the
structural similarity to diacylphloroglucinol (DAPG) may suggest that the compounds act as
uncouplers in the mitochondrion [45].
Figure 6. A) Compounds 12, 13, 17, 21 and 22 treatment-induced changes in the
mitochondrial membrane permeability’s for MDA-MB231 cells. The cells were stained with

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rhodamine 123, and then analyzed by flow cytometry. B) The table shows the percentage values
of rhodamine 123 stained cells treated without or with compounds 12, 13, 17, 21 and 22 (50
ꢀM) for the different cell lines MDA-MB231, PC-3, DU-145, HT-29 and HDF (* p < 0.05
vs. control treated cells).
MDA-
PC-3
DU-145
HT-29
HDF
MB231
50 µM
50 µM
50 µM
50 µM
50 µM
9.6 ± 1.1*
34.9 ± 3.6*
59.8 ± 3.7*
82.4 ± 6.6
81.7 ± 6.8
80.5 ± 8.4
36.8 ± 4.7*
32.3 ± 2.4*
51.8 ± 5.0*
77.8 ± 6.1
65.2 ± 4.9*
86.1 ± 8.5
60.4 ± 3.9*
50.3 ± 4.1*
79.9 ± 5.9
77.3 ± 5.5
81.9 ± 6.5
85.8 ± 8.2
66.3 ± 2.8*
41.3 ± 6.4*
81.3 ± 6.5
84.9 ± 6.5
87.1 ± 8.8
82.4 ± 6.9
31.3 ± 2.4*
15.2 ± 3.5*
51.6 ± 4.9*
79.3 ± 7.1
80.8 ± 5.3
81.5 ± 10.5
Comp. 12
Comp. 13
Comp. 17
Comp. 21
Comp. 22
Control
2.1.1.1.4 Caspase 3 Activity Assay
Depletion of mitochondrial membrane potential leads to the release of apoptogenic factors, such as
cytochrome c in the apoptotic cascade, and activation of caspases [45, 46]. Then we investigated the
effects of our compounds on caspase-3 activity, the main executor of apoptosis playing a central role in
its biological processing. We analyzed the effect of treatment with compound 12, 13, 17, 21 and 22 on
caspase-3 activation in non-tumor and cancer cell lines (Figure 7).
Figure 7. Effect of compounds 12, 13, 17, 21 and 22 on caspase-3 activity of MDA-MB231 (black

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bar), HDF (white bar), PC-3 (striped bar) and HT-29 (slashed bar) cells. Cells were exposed to compounds
at 50 ꢀM for 48 h. Values are mean ± S.D. (n = 3). All data are reported as the percentage change in
comparison with the vehicle-treated cells (1% ethanol), which were arbitrarily assigned 100%. * p < 0.05,
significantly different from the vehicle-treated cells.
As shown in Figure 7, the activation of caspase-3 in cells exposed to compounds 12, 13 and 17 is
increased versus control-treated cells (1% ethanol). Compound 12 increased the activity of caspase-3 by
4.43 ± 0.11, 2.38 ± 0.16, 2.90 ± 0.15 and 1.61 ± 0.06 times versus control cells in MDA-MB231 , HDF ,
PC-3 and HT-29 cells, respectively. Compound 13 increased the activity of caspase 3 by 2.12 ± 0.30,
3.30 ± 0.21, 2.50 ± 0.26, and 1.86 ± 0.16 times versus control cells in MDA-MB231 (black bar), HDF,
PC-3 and HT-29 cells, respectively. Compound 17 only changed significantly the activity of caspase-3
in HDF and PC-3 cells. Finally, compounds 21 and 22 did not change the activity of caspase-3 when
compared with control cells.

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3. Experimental Section
3.1. Chemical
Unless otherwise stated, all chemical reagents purchased (Merck, Darmstadt, Germany or Aldrich, St.
Louis, MO, USA) were of the highest commercially available purity and were used without previous
purification. IR spectra were recorded as thin films in a FT-IR Nicolet 6700 spectrometer (Thermo
−1
Scientific, San Jose, CA, USA) and frequencies are reported in cm . Low resolution mass spectra were
recorded on an Agilent 5973 spectrometer (Agilent Technologies, Santa Clara, CA, USA) at 70eV
ionizing voltage, in a GC 6890N DB-5 m, 30 m × 0.25 mm × 0.25 ꢀm column, and data are given as
m/z (% rel. int.). High resolution mass spectra were recorded on an LTQ Orbitrap XL spectrometer
(Thermo Scientific, San Jose, CA, USA) by applying a voltage of 1.8 kV in the positive and 1.9 kV in
the negative, ionization mode. The spectra were recorded using full scan mode, covering a mass range
from m/z 100–1,300. The resolution was set to 50,000 and maximum loading time for the ICR cell was
set to 250ms. 1H, 13C, 13C DEPT-135, sel. gs-1D 1H NOESY, gs-2D HSQC and gs-2D HMBC spectra
were recorded in CDCl
3
solutions and are referenced to the residual peaks of CHCl at δ = 7.26 ppm
3
1
13
and δ = 77.0 ppm for H and C, respectively, on a Bruker Avance 400 Digital NMR spectrometer
1
13
(Bruker, Rheinstetten, Germany), operating at 400.1 MHz for H and 100.6 MHz for C. Chemical
shifts are reported in δ ppm and coupling constants (J) are given in Hz. TLC spots were detected by
heating after spraying with 25% H
2
SO4
in H O.
2
3.1.1 Synthesis of Geranylmethoxy Derivatives
(E)-2-(3,7-dimethylocta-2,6-dienyl)-1,3,5-trimethoxybenzene(14). To
a
solution of 2-geranyl
phloroglucinol (6) (360 mg; 1.4 mmol), dimethyl sulfate (0.58 g; 4.6 mmol) and K2CO3 (0.58 g; 4.2
mmol) in acetone (50 mL), was refluxed for 6 hours. The end of the reaction was verified by TLC, and
then the mixture was filtered and the solvent was evaporated under reduced pressure. The crude was
re-dissolved in EtOAc (30 mL) then the organic layer was washed with 5% NaOH (30 mL) and water
(2 x 20 mL), dried over Na
2
SO , filtered and evaporated to dryness. The compound 14 was obtained as
4
a viscous yellow oil, 370 mg (86.8% yield). IR (cm-1): 2935, 2836, 1608, 1596, 1496, 1455, 1437,
1
1417, 1377, 1322. H-NMR (CDCl3, 400.1 MHz) : δ 6.14 (2H, s, H-4 and H-6); 5.19 (1H, t, J = 6.9
Hz, H-2’); 5.09 (1H, t, J = 6.3 Hz, H-6’); 3.92 (3H, s, OCH3); 3.79 (6H, s, OCH3); 3.29 (2H, d, J = 6.8
Hz, H-1’); 2.08-2.05 (2H, m, H-5’); 1.99-1.95 (2H, m, H-4’); 1.78 (3H, s, CH3-C3’); 1.66 (3H, s, H-
8’); 1.59 (3H, s, CH3-C7’). 13C-NMR (CDCl3, 100.6 MHz): δ 159.0 (C-5); 158.4 (C-1 and C-3); 133.6
(C-3’); 130.6 (C-7’); 124.3 (C-6’);123.2 (C-2’); 110.5 (C-2); 90.4 (C-4 and C-6); 55.3 (2 x OCH3);
54.9 (OCH3); 39.6 (C-4’); 26.5 (C-5’); 25.3 (C-8’); 21.4 (C-1’); 17.3 (CH3-C7’); 15.6 (CH3-C3’). MS

+
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(m/z, %): 304 [M] (11%); 235 (24); 181 (100); 167 (15); 69 (13). Spectroscopic data of compound 14
was consistent with those in the literature [36].
(E)-2-(3,7-dimethylocta-2,6-dienyl)-1,5-dimethoxy-3-methylbenzene (23). To a solution of 15 (226 mg;
0.784 mmol), dimethyl sulfate (0.58 g; 4.6 mmol) and K2CO3 (0.58 g; 4.2 mmol) in acetone (50 mL),
was refluxed for 6 hours. The end of the reaction was verified by TLC, and then the mixture was
filtered and the solvent was evaporated under reduced pressure. The crude was re-dissolved in EtOAc
(30 mL) then the organic layer was washed with 5% NaOH (30 mL) and water (2 x 20 mL), dried over
Na2SO4, filtered and evaporated to dryness. The compound 23 was obtained as a viscous dark yellow
1
oil, 201 mg (80.1% yield). H-NMR (CDCl3, 400.1 MHz): 6.36 (2H, s, H-4 and H-6); 5.10-5.08 (2H,
m, H-6’and H-2’); 3.81 (3H, s, OCH3); 3.80 (3H, s, OCH3); 3.32 (2H, d, J = 6.5 Hz, H-1’); 2.29 (3H, s,
CH3-Ar); 2.11-2.06 (2H, m, H-5’); 2.02-1.98 (2H, m, H-4’); 1.79 (3H, s, CH3-C3’); 1.68 (3H, s, H-8’);
1.61 (3H, s, CH3-C7’). 13C-NMR (CDCl3, 100.6 MHz): δ 158.2 (⁺C-5); 158.1 (⁺C-1); 138.1 (C-3);
134.3 (C-3’); 131.1 (C-7’); 124.4 (C-6’); 123.0 (C-2’); 121.1 (C-2); 106.5 (C-4); 96.1 (C-6); 55.5
(OCH3); 55.1 (OCH3); 39.7 (C-4’); 26.7 (C-5’); 25.6 (C-8’); 24.6 (C-1’); 19.9 (CH3-Ar); 17.6 (CH3-
C7’); 16.0 (CH3-C3’). ⁺ Interchangeable signals. HRMS: (M + 1) calcd. for C19H28O2: 289.2089,
found: 289.2097.
(E)-2-(3,7-dimethylocta-2,6-dienyl)-1,3-dimethoxy-5-methylbenzene (24). To a solution of 16 (243 mg;
0.843 mmol), dimethyl sulfate (0.58 g; 4.6 mmol) and K2CO3 (0.58 g; 4.2 mmol) in acetone (50 mL),
was refluxed for 6 hours. The end of the reaction was verified by TLC, and then the mixture was
filtered and the solvent was evaporated under reduced pressure. The crude was re-dissolved in EtOAc
(30 mL) then the organic layer was washed with 5% NaOH (30 mL) and water (2 x 20 mL), dried over
Na2SO4, filtered and evaporated to dryness. The compound 24 was obtained as a viscous dark yellow
oil, 206 mg (78.7% yield). 1H-NMR (CDCl3, 400.1 MHz): δ 6.43 (2H, s, H-4 and H-6); 5.27 (1H, t, J =
7.0 Hz, H-2’); 5.15 (1H, t, J = 6.5 Hz, H-6’); 3.85 (6H, s, OCH3); 3.40 (2H, d, J = 7.0 Hz, H-1’); 2.40
(3H, s, CH3-Ar); 2.14-2.10 (2H, m, H-5’); 2.04-2.01 (2H, m, H-4’); 1.84 (3H, s, CH3-C3’); 1.72 (3H, s,
13
H-8’); 1.65 (3H, s, CH3-C7’). C-NMR (CDCl3, 100.6 MHz): δ 157.9 (C-1 and C-3); 136.5 (C-5);
134.1 (C-3’); 130.8 (C-7’); 124.6 (C-6’); 123.1 (C-2’); 115.4 (C-2); 104.7 (C-4 and C-6); 55.6 (OCH3);
39.8 (C-4’); 26.7 (C-5’); 25.6 (C-8’); 21.9 (CH3-Ar and C-1’); 17.5 (CH3-C7’); 15.9 (CH3-C3’).
HRMS: (M + 1) calcd. for C19H28O2: 289.2089, found: 289.2094.
2,4-bis((E)-3,7-dimethylocta-2,6-dienyl)-1,3-dimethoxy-5-methylbenzene (25). To a solution of 18
(205.6 mg; 0.484 mmol), dimethyl sulfate (0.58 g; 4.6 mmol) and K2CO3 (0.58 g; 4.2 mmol) in
acetone (50 mL), was refluxed for 6 hours. The end of the reaction was verified by TLC, and then the
mixture was filtered and the solvent was evaporated under reduced pressure. The crude was re-

ACCEPTED MANUSCRIPT
dissolved in EtOAc (30 mL) then the organic layer was washed with 5% NaOH (30 mL) and water (2
x 20 mL), dried over Na2SO4, filtered and evaporated to dryness. The compound 25 was obtained as a
viscous dark yellow oil, 178 mg (78.1% yield). 1H-NMR (CDCl3, 400.1 MHz): 6.54 (1H, s, H-6); 5.25
(1H, t, J = 7.0 Hz, H-2’); 5.11-5.10 (3H, m, H-2’’, H-6’ and H-6’’); 3.82 (3H, s, OCH3); 3.72 (3H, s,
OCH3); 3.38 (2H, d, J = 7.0 Hz, H-1’); 3.36 (2H, d, J = 7.4 Hz, H-1’’); 2.29 (3H, s, CH3-Ar); 2.10-2.08
(4H, m, H-5’ and H-5’’); 2.04-2.00 (4H, m, H-4’ and H-4’’); 1.80 (3H, s, CH3-C3’); 1.79 (3H, s, CH3-
13
C3’’); 1.68 (6H, s, H-8’ and H-8’’); 1.61 (6H, s, CH3-C7’ and CH3-C7’’). C-NMR (CDCl3, 100.6
MHz): δ 157.1 (C-3); 156.3 (C-1); 135.8 (C-5); 134.7 (C-3’’); 134.3 (C-3’); 131.2 (⁺C-7’); 131.0 (⁺C-
7’’); 125.6 (C-4); 124.5 (⁺⁺C-6’); 124.3 (⁺⁺C-6’’); 123.8 (⁺⁺⁺C-2’); 123.7 (⁺⁺⁺C-2’’); 120.9 (C-2);
108.8 (C-6); 61.5 (OCH3); 55.5 (OCH3); 39.8 (C-4’); 39.6 (C-4’’); 26.7 (⁺⁺⁺⁺C-5’); 26.6 (⁺⁺⁺⁺C-5’’);
25.6 (C-8’ and C-8’’); 25.5 (C-1’’); 23.2 (C-1’); 19.8 (CH3-Ar); 17.6 (CH3-C7’ and CH3-C7’’); 16.1
(CH3-C3’’); 16.1 (CH3-C3’). ⁺ Interchangeable signals. HRMS: (M + 1) calcd. for C29H44O2:
425.3341, found: 425.3349.
3.2 Biological
3.2.1 Cell Lines
The experimental cell cultures were obtained from the American Type Culture Collection
(Rockville, MD, USA). MDA-MB-231 (breast cancer cell line), HT-29 (colon cancer cell line), PC-3 and
DU-145 (prostate cancer cell lines), and human dermal fibroblast (HDF, non-tumor cell line) were
grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS, 100 U/mL penicillin, 100
ꢀg/mL streptomycin, and 1 mM glutamine. Cells were seeded into 96 well microtiter plates in 100 ꢀL
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.
3.2.2 Cell Viability
3.2.2.1. In vitro Growth Inhibition Assay
The sulforhodamine B assay was used according to the method of Skehan et al. [47] with some
modifications [48]. Briefly, the cells were set up at 3 × 103 cells per well of a 96 200 ꢀL well, flat-
bottomed, microplate. Cells were incubated at 37°C in a humidified 5% CO2/95% air mixture and
treated with the compounds at different 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%

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sulforhodamine B (Sigma-Aldrich, St. Louis, MO, USA), dissolved in 1% acetic acid (50 ꢀL/well) for
30 min, and subsequently washed with 1% acetic acid to remove unbound stain. Protein-bound stain
was solubilized with 100 ꢀL of 10mM 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).
3.2.3. Morphological Assessment of Cell Apoptosis
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 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 1 ꢀM 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 (IX 81 model inverted microscope, Olympus,
Santiago, Chile).
3.2.4. 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 potentials. Exponentially growing
cells were incubated with the compound as indicated in the figure legends. Cells were labelled with 1 ꢀM
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 with rhodamine 123 [44].
3.2.5. Caspase 3 Activity Assay
Caspase activity was measured using a colorimetric assay [49]. Briefly, the cells exposed to compounds
were collected by centrifugation at 1000 rpm and lysed with lysis buffer (1% Triton X-100, 0.32 M
sucrose, 5 mM EDTA, 10 mM Tris–HCl, pH 8.0, 2 mM dithiothreitol, 1 mM 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 ꢀM) specific for caspase-3/7, at 37°C for 1 h. The
intensity of the developed color was read at 405 nm in a microplate reader (SpectraMax, Winooski,
VT, USA). The results are expressed as percentages of the control level.
3.2.6. Statistical Analysis

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Data shown in tables are average results obtained by means of three or 10 replicates and are presented
as average ± standard errors of the mean (SEM). Data were subjected to analysis of variance (ANOVA)
with significant differences between means identified by GLM procedures. Results are given in the
text as probability values, with p < 0.05 adopted as the criterion of significance. Differences between
treatment means were established with a Student–Newman–Keuls (SNK) test. The EC50 values for
each activity were calculated by PROBIT analysis based on percentage of mortality obtained at each
concentration of the samples. EC50 is half maximal effective concentration. Complete statistical analysis
was performed by means of the MicroCal Origin 6.0 statistics and graphs PC program.
4. Conclusions
Different compounds derived from phloroglucinol and orcinol were tested as potential cytotoxic agents
against various cancer cell lines: PC-3 and DU-145 (prostate cancer), MDA-MB 231 (breast cancer),
HT-29 (colon cancer) and one non-tumoral cell line human dermal fibroblasts (HDF). Initial primary
screens indicated IC50 concentration between 0 and 100 ꢀM for compounds 12, 13, 17, 21, 22 and 25.
Among the six, three were found to be effective against most of the cancer cell lines. Compounds 12,
13, 17, 21, 22 and 25, which showed cytotoxicity, were evaluated in an apoptosis assay. Compounds
12, 13 and 17 showed a good apoptotic response against all cell lines studied, whereas the compounds
21, 22, and 25 had minor impact on induction of chromatin nuclear condensation and/or fragmentation,
mitochondrial membrane potential and caspase-3 activity. Moreover the compounds 12, 13 and 17 also
depleted the mitochondrial membrane potential and increased caspase-3 activity. According these
results, the analysis of the relation between the activity and the structure of compounds indicate that
the biological activity depend strongly on, first: mono or disubstitution geranyl chains on the aromatic
(disubstituted compounds geranyl chains are more active than those monosubstituted) second: the
increasing order in biological activity of the oxygen function in the aromatic ring, could be established
as OH > OAc >> OMe. Finally we note that the disubstituted compounds with two geranyl chains and
symmetrical structure (i.e. compounds 12, 13 and 17) are more active than those with asymmetrical
disubstitution (compound 18, 22 and 25). While it is true, compounds 12 and 13 have higher activity
against tumor cells than against normal, this difference is not significant, which it makes little useful as
anticancer drugs. However, these compounds could be used as leads for the search of new derivatives
that would have significantly higher activity against cancer cells.
Acknowledgments
The authors thanks to Yasenka Barrios for her technical support, to DIUV N° 50/2011 (J.V.) of
Universidad de Valparaíso, to FONDECYT (grant No. 1120996) and (DGIP-USM for grants No.
131305, 131512) of Universidad Técnica Federico Santa María for financial support.

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Author Contributions
LE, LT and JV designed research; AM, LE, LT, JV, MC and HC performed research and analyzed the
data; LE, LT, HC and JV wrote the paper. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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Figure 1. Structure of some active natural and synthetic linear geranylphenols.
Figure 2. Structure of some active synthetic linear geranylquinones.

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Figure 3. Structure of synthetic linear derivatives geranylphenols acetylated and methoxilated of
phloroglucinol.
Figure 4. Structure of synthetic linear derivatives geranylphenols acetylated and methoxilated of
orcinol.
CH3
OH
(16)
(15)
(17)
HO
HO
OH
OH
H3C
OH
CH3
OH
(18)
HO
CH3
CH3
OAc
(20)
(19)
(21)
AcO
AcO
OAc
OAc
H3C
OAc
CH3
OAc
(22)
AcO
CH3
CH3
OCH3
(24)
(23)
H3CO
OCH3
H3C
OCH3
OCH3
(25)
H3CO
CH3

ACCEPTED MANUSCRIPT
Figure 5. Effect of tested compounds on the morphologies of PC-3 cells. Images obtained
with an inverted phase contrast microscope (200×) after 24 h exposure of the cells to 50
ꢀM of compounds 12, 13, 17, 21, 22 and 25 (1% ethanol).

ACCEPTED MANUSCRIPT
Figure 6. A) Compounds 12, 13, 17, 21 and 22 treatment-induced changes in the
mitochondrial membrane permeability’s for MDA-MB231 cells. The cells were stained with
rhodamine 123, and then analyzed by flow cytometry. B) The table shows the percentage values
of rhodamine 123 stained cells treated without or with compounds 12, 13, 17, 21 and 22 (50
ꢀM) for the different cell lines MDA-MB231, PC-3, DU-145, HT-29 and HDF (* p < 0.05
vs. control treated cells).
MDA-
PC-3
DU-145
HT-29
HDF
MB231
50 µM
50 µM
50 µM
50 µM
50 µM
9.6 ± 1.1*
34.9 ± 3.6*
59.8 ± 3.7*
82.4 ± 6.6
81.7 ± 6.8
80.5 ± 8.4
36.8 ± 4.7*
32.3 ± 2.4*
51.8 ± 5.0*
77.8 ± 6.1
65.2 ± 4.9*
86.1 ± 8.5
60.4 ± 3.9*
50.3 ± 4.1*
79.9 ± 5.9
77.3 ± 5.5
81.9 ± 6.5
85.8 ± 8.2
66.3 ± 2.8*
41.3 ± 6.4*
81.3 ± 6.5
84.9 ± 6.5
87.1 ± 8.8
82.4 ± 6.9
31.3 ± 2.4*
15.2 ± 3.5*
51.6 ± 4.9*
79.3 ± 7.1
80.8 ± 5.3
81.5 ± 10.5
Comp. 12
Comp. 13
Comp. 17
Comp. 21
Comp. 22
Control