Synthesis and antiproliferative activity of podocarpane and totarane derivatives

Article abstract of DOI:10.1016/j.ejmech.2018.09.051

The synthesis of podocarpanes, including 12,19-dihydroxy-13-acetyl-8,11,13-podocarpatriene (23), isolated from Gaultheria yunnanensis and not previously synthesized, and totarane-type terpenoids, starting from the natural labdane trans-communic acid (15), is described. Their antiproliferative activities against MCF-7, T-84 and A-549 human tumoural cell lines are studied. An antiproliferative effect was induced by compounds 23, 27 and 28, with IC₅₀ < 10 μM in two (27) or three cell lines (23 and 28). No correlation with log P values was observed. The totarane o-quinone 27, and especially the catechol 28, which is readily oxidisable to compound 27, were the most active compounds, highlighting the functional groups present in C11 and C12. Compound 28 showed limited toxicity in normal peripheral blood mononuclear cells (78.5% cell viability versus non-treated control cultures at 10 μM), and appeared to exert an antiproliferative effect in A-549 cells (IC₅₀ 0.6 μM) through a mechanism that involves the induction of apoptosis mediated by an increased Bax/Bcl-2 ratio. The results of the present study indicate that compound 28, at least, might be useful as an antitumoral agent. Further studies are required to elucidate the cellular and molecular elements involved in its effect, and the activity/toxicity in preclinical models.

Full text of DOI:10.1016/j.ejmech.2018.09.051

Accepted Manuscript
Synthesis and antiproliferative activity of podocarpane and totarane derivatives
Soumicha Mahdjour, Juan J. Guardia, Fernando Rodríguez-Serrano, José Manuel
Garrido, Isabel Blancas López-Barajas, Nuria Mut-Salud, Rachid Chahboun, Enrique
Alvarez-Manzaneda
PII:
S0223-5234(18)30823-7
10.1016/j.ejmech.2018.09.051
EJMECH 10762
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 August 2018
Revised Date: 10 September 2018
Accepted Date: 16 September 2018
Please cite this article as: S. Mahdjour, J.J. Guardia, F. Rodríguez-Serrano, José.Manuel. Garrido, I.B.
López-Barajas, N. Mut-Salud, R. Chahboun, E. Alvarez-Manzaneda, Synthesis and antiproliferative
activity of podocarpane and totarane derivatives, European Journal of Medicinal Chemistry (2018), doi:
https://doi.org/10.1016/j.ejmech.2018.09.051.
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ACCEPTED MANUSCRIPT
The synthesis of podocarpanes, including the acetoxy phenol 23, isolated from Gaultheria yunnanensis and not previously synthesised, and
totarane-type terpenoids, such as the catechol 28 and the ortho-quinone 27, starting from the natural labdane trans-communic acid is described.
Their antiproliferative activities against MCF-7, T-84 and A-549 human tumoural cell lines are studied. The totarane o-quinone 27, and
especially the catechol 28, which is readily oxidisable to compound 27, were the most active compounds. The results of the present study
indicate that compound 28, at least, might be useful as an antitumoural agent.

ACCEPTED MANUSCRIPT
Synthesis and antiproliferative activity of podocarpane and totarane
derivatives
a,b
a
c,d
Soumicha Mahdjour , Juan J. Guardia , Fernando Rodríguez-Serrano , José
d,e
d,f
c
Manuel Garrido , Isabel Blancas López-Barajas , Nuria Mut-Salud , Rachid
a
a
Chahboun* , Enrique Alvarez-Manzaneda*
a
Department of Organic Chemistry, Faculty of Sciences, University of Granada, 18071
Granada, Spain.
b
Laboratory Productions, Plant and Microbial Valuations ( LP2VM ), Department of
Biotechnology, University of Sciences and Technology of Oran Mohamed Boudiaf, BP
1
525, EL M'Naouer. Oran, Algeria.
c
Institute of Biopathology and Regenerative Medicine (IBIMER), University of
Granada, Armilla (Granada), 18100, Spain.
d
Biosanitary Research Institute of Granada (ibs.Granada), Granada, 18012, Spain.
e
Department of Surgery and its Specialities, University of Granada, Granada, 18016,
Spain.
f
Oncology Unit, San Cecilio University Hospital, Granada, 18005, Spain.
KEYWORDS Podocarpane terpenes; Totarane terpenes; Synthesis; Catechol; ortho-
Quinone; Cancer
ABSTRACT
The synthesis of podocarpanes, including 12,19-dihydroxy-13-acetyl-8,11,13-
podocarpatriene (23), isolated from Gaultheria yunnanensis and not previously
synthesised, and totarane-type terpenoids, starting from the natural labdane trans-

ACCEPTED MANUSCRIPT
communic acid (15), is described. Their antiproliferative activities against MCF-7, T-
8
4 and A-549 human tumoural cell lines are studied. An antiproliferative effect was
induced by compounds 23, 27 and 28, with IC <10 µM in two (27) or three cell lines
5
0
(23 and 28). No correlation with log P values was observed. The totarane o-quinone 27,
and especially the catechol 28, which is readily oxidisable to compound 27, were the
most active compounds, highlighting the functional groups present in C11 and C12.
Compound 28 showed limited toxicity in normal peripheral blood mononuclear cells
(78.5% cell viability versus non-treated control cultures at 10 µM), and appeared to
exert an antiproliferative effect in A-549 cells (IC 0.6 µM) through a mechanism that
50
involves the induction of apoptosis mediated by an increased Bax/Bcl-2 ratio. The
results of the present study indicate that compound 28, at least, might be useful as an
antitumoral agent. Further studies are required to elucidate the cellular and molecular
elements involved in its effect, and the activity/toxicity in preclinical models.
1
. Introduction
Totarane-type diterpenes are a family of compounds with a tricyclic skeleton,
isomer from that of abietane derivatives, which are isolated from a wide variety of
vegetal species. The most representative example of these compounds is totarol (1),
which was first isolated from Podocarpus totara [1]. The considerable resistance of this
plant to different types of diseases has long encouraged the investigation of the
components responsible for this behaviour. Totarol (1) protects mitochondrial
respiratory enzyme activities against NADPH-dependent peroxidation injury [2]. Recent
studies have shown that totarol (1) exhibits antiplasmodial, antifungal and antimicrobial
properties [3]. Moreover, it presents a neuroprotective activity, as an activator of the

ACCEPTED MANUSCRIPT
Akt/HO-1 pathway, protecting against ischaemic stroke by reducing oxidative stress [4].
Other totarane-type diterpenes are phenols 2-6, with significant antibacterial activity
against oral pathogenic microorganisms [5]. Podocarpane belongs to a group of tri-nor-
abietane metabolites that are widely distributed, and most frequently found in
Podocarpaceae species [6]. The most important member of this family is podocarpic
acid (7), which was first isolated from Podocarpus cupressinus, P. dacrydioides and
Dacrydium cupressinum [7] and presents a variety of significant biological activities.
For example, it is a potent agonist of liver X receptor and a cytokine release inhibitor,
being potentially useful for the treatment of atherosclerosis or inflammatory diseases
[
8]. Moreover, methyl podocarpate (8) and some O-acyl derivatives, such as compounds
-11, exert activity against Influenza type A viruses [9]. It has recently been reported
9
that podocarpic acid (7) is a TRPA1 activator, and thus a potential therapeutic agent
against pathologies related to diabetes and neurodegenerative diseases [10]. Other
interesting podocarpane-type terpenes include nimbidiol (12), which inhibits intestinal
carbohydrases, and is not only a potential antidiabetic drug [11], but also has an anti-
angiogenesis effect [12], 7-deoxynimbidiol (13), with anti-tumoural [13], analgesic and
anti-inflammatory activities [14], and the aminophenol 14, which is a potent 5-
lypoxygenase inhibitor [15] (Fig. 1).

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Fig. 1. Totarane and podocarpane-type diterpenes with biological properties.
The diverse and important biological properties described above prompted us to
undertake the synthesis of totarane and podocarpane-type terpenoids. In this respect,
total syntheses of totarane derivatives based on the biomimetic cyclisation of aryl
polyenes [16], and aryl epoxypolyenes [17], have been reported previously.
Enantiospecific synthesis starting from totarol (1) has also been described [18], and
more recently processes have been performed based on the α-alkylation of a
podocarpane α,β-unsaturated ketone [19], or via a Pd-catalyzed Stille cross-coupling of
the corresponding o-bromophenol podocarpane [20]. Cationic cyclizations of aryl
polyprenes [21], including enantioselective processes [22], have also been described for
synthesizing podocarpane-type terpenoids. Enantiospecific syntheses of these
compounds have also been reported, utilizing as starting materials podocarpic acid [23]
or abietic acid derivatives; the latter being deisopropylated after rearrangement of the
corresponding 15-hydroperoxide [24]. Labdane diterpenes, such as manool [25] and
sclareol [26], have also been utilised for synthesizing podocarpane terpenes.

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This paper describes the syntheses of some podocarpanes, including 12,19-
dihydroxy-13-acetyl-8,11,13-podocarpatriene (23), isolated from Gaultheria
yunnanensis [27] and not previously synthesised, and totarane-type terpenoids. The
antiproliferative activities of these and of previously-reported synthetic precursors,
against MCF-7, T-84 and A-549 human tumoural cell lines are also discussed.
2
. Results and discussion
2
.1. Chemistry
During our recent first synthesis of a cassane-type benzofurane diterpene [28],
phenols 17 and 18 were synthesized from trans-communic acid (15), a labdane
diterpene that is very abundant in some species of Juniperus and Cupressus [29]
(Scheme 1). Compounds 17 and 18 are suitable intermediates for the synthesis of
totarane, and of abietane or podocarpane diterpenes. Thus, the hydroxy ester 18 has
been transformed into the abietanes sugikurojin A and 19-hydroxyferruginol [30].
Scheme 1. Synthesis of phenols 17 and 18 from trans-communic acid (15).

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In the present study, hydroxy ester 18 was transformed into the podocarpane 23, a
constituent from Gaultheria yunnanensis [27] that has not previously been synthesized
(Scheme 2). The treatment of methoxy ester 19 [28] with MeMgBr and the further
dehydration of the resulting alcohol gave the isopropylene derivative 20. After reduction
of the ester group and ozonolysis of the carbon-carbon double bond, the acetyl
derivative 22 was obtained, which was reacted with AlBr to give the natural hydroxy
3
ketone 23¸whose physical properties were found to be identical to those reported in the
literature [27].
Scheme 2. Synthesis of podocarpane 23 from hydroxy ester 18.
In addition, hydroxy ester 17 was transformed into the totarane o-quinones 26 and
2
7, and the catechol 28 (Scheme 3). The cationic reduction of the isopropylene group of
intermediate 24 with Et SiH and trifluoroacetic acid gave the totarane phenol 25.
3
Treatment of this with (PhSeO) O afforded the mixture of o-quinone 27 and hydroxy
2
dienone 26. Finally, the further reaction of quinone 27 with Raney Ni gave catechol 28.

ACCEPTED MANUSCRIPT
Scheme 3. Synthesis of totarane diterpenes from hydroxy ester 17.
2
2
.2. Biological assays
.2.1. In Vitro Cytotoxicity Assay
Eight newly-synthesised diterpenoids were evaluated to determine their in vitro
antiproliferative activity against selected human cancer cell lines of breast
adenocarcinoma (MCF-7), lung adenocarcinoma (A-549) and colorectal carcinoma
derived from lung metastasis (T-84), using the Sulforhodamine B colorimetric assay
[
31]. 5-fluorouracil (5-Fu), a commercial pharmaceutical product that is widely used to
treat solid tumours [32] was used as the reference standard (Table 1). An
antiproliferative effect was induced by compounds 23, 27 and 28, with an inhibitory
concentration 50 (IC ) <10 µM in two (27) or three cell lines (23 and 28). The most
5
0
active compound was 28. The mean IC values obtained for the three cell lines were 1.5
5
0
µM for compound 28 versus 7.3 µM and 6.4 µM for compounds 23 and 27,
respectively, and 28 was found to be four times more active than 5-Fu (mean IC 6.1
5
0
µM). The best result was observed for compound 28 against the A-549 cells with IC50
.6 µM, whereas the IC of the reference standard 5-Fu was 7.1 µM.
0
5
0
As observed for hydroxy esters 17 and 18, the mean IC value was reduced by at
5
0
least 50% when the ester group was changed from the meta (17) to the ortho position
18) (entries 1 and 2). Podocarpane 23, a constituent of Gaultheria yunnanensis, showed
(
no significant inhibitory activity against LPS-induced nitric oxide (NO), TNF-α, or IL-6
production in RAW 246.7 macrophages, as has been reported previously [27]. However,

ACCEPTED MANUSCRIPT
it did exert an antiproliferative activity against the tumour cell lines used in our study.
The totarane o-quinone 27 and especially the catechol 28, which is readily oxidisable to
compound 27, were the most active compounds. The catechol moiety of compound 28,
compared to the o-quinone fragment in compound 27, improved the mean level of
activity in colon and lung tumour cells. However, phenol 25, lacking the hydroxyl
group in C-11, is not active (IC >10 µM). Interestingly, it has been found that natural
5
0
catechol reduces lung cancer tumour growth in vitro and in vivo [33]. In general, our
results indicate that the catechol or quinone C ring has the greatest influence on the
activity of these compounds.
The different degree of oxidation of compounds 27 and 28 account for the
greater activity of 28 in the T-84 and A-549 lines, for several reasons. The hydroxyl
groups in C11 and C12 may enhance the interactive capacity of the skeleton with
cellular elements that are responsible for the induction of the observed effect of 28 in A-
5
49. Moreover, as in the case of caffeic or ferulic acid, hydroxyl groups could exert a
cellular pro-oxidant activity mediated by reactions with transition metals [34]. As has
been observed experimentally, catechol 28 undergoes rapid oxidation to give o-quinone
2
7, which would lead to the transformation of 28 into 27 in a biological context.
Therefore, the better activity of 28 could be derived from a higher level of intracellular
incorporation. The correlation study between log P and IC50 revealed no significant
association within the series of compounds. However, the log P of 28 was higher than
that of compound 27, which could affect its incorporation. At the cellular level, on the
other hand, the redox cycling of catechol metabolites might contribute to cell death
through the production of ROS and oxidative stress, and through the production of its o-
quinone derivatives, possibly generating ROS once they are reduced by P450
reductases, thus altering the functioning of DNA and proteins [35], and even acting as
Michael acceptors [36].
Table 1: In vitro antiproliferative activity of synthetic compounds against human cancer
cell lines.
IC (µM)ª
5
0
Entry
Compound
MCF-7
T-84
A-549

ACCEPTED MANUSCRIPT
1
2
> 40
> 40
> 40
2
1.7 ± 1.3
20.7 ± 3.0
17.2 ± 1.9
OMe
COOMe
>
40
> 40
> 40
3
4
H
MeOOC
1
9
9.3 ± 0.3
7.8 ± 0.4
30.7 ± 1.0
4.8 ± 0.2
23.7 ± 2.0
2
6.1 ± 1.2
8.8 ± 0.7
5
6
OH
2
30.5 ± 2.4
11.4 ±2.2
18.6 ± 2.9
6.8 ± 0.8
H
MeOOC
2
5
1
.1 ± 0.2
.8 ± 0.2
7
8
2
1.0 ± 0.2
3.4±0.14
0.6 ± 0.02
7.1±0.24
9
7.8±0.19

ACCEPTED MANUSCRIPT
a
IC values expressed as means ± SEM of seven biological replicates in the µM range.
5
0
The ability to inhibit the growth of cancer cells without affecting normal cells is a
desirable characteristic of a chemotherapeutic drug candidate [37]. Accordingly, we
evaluated the effect of the most active compounds, 27 and 28, on normal cells, in order
to assess nonspecific toxicity. Thus, peripheral blood mononuclear cells (PBMC) were
isolated from healthy donor blood samples and induced with increasing concentrations
of compounds. PBMC induced with 27 experienced a dose-dependent reduction of cell
viability ranging from 25 % at 0.5 µM to 43.8 % at 10 µM, while cultures induced with
2
8 only reduced the viability by 21.5 % at 10 µM (Fig. 2).
Fig. 2. Effect of compounds 27 and 28 on the viability of normal human PBMC.
Lipophilicity (log P) plays a significant role in drug discovery and compound
design, because it can determine pharmacokinetic processes such as drug absorption,
distribution and excretion [38], [39]. The log P of the molecules studies was determined
by using Marvin 18.3.8 software (ChemAxon Ltd., 2013) (Table 2). We found no
correlation between the IC50 in the three tumour lines and the log P values of the
compounds (Pearson’s correlation, p >0.05).

ACCEPTED MANUSCRIPT
Table 2: Log P values of synthetic compounds
Compounds
Log P
17
4.5
18
5.2
19
3.9
23
3.8
24
3.8
25
5.6
27
4.8
28
5.3
2
.2.2. Cell Cycle and Apoptosis Analysis
Many cytotoxic compounds exert an inhibitory effect on growth by arresting the
cell cycle at a particular checkpoint of the cell cycle [40], by the induction of apoptosis
41] or by a combined effect on cell cycle and apoptosis [42]. In consequence, the
[
blockage of cell cycle progression by compounds is an interesting area for study [43]. In
the present case, the best antiproliferative result was observed for compound 28 against
A-549 cells (IC 0.6 µM), and as this compound was less toxic in normal cells, we
5
0
analysed the effect of 28 on the A-549 cell cycle (Figure 3). After 24h of induction with
2
8 at IC , we observed a significant increase in cells in the G2/M fraction (14.5 % vs
50
8
.8 % in the control group) and a reduction in the G0/G1 fraction (53.7% vs 60.6 % in
the controls), but in both fractions the changes were of low magnitude. However, at
8h, compound 28 induced a large increase in the sub-G1 phase (70.1 % vs 2.6% in the
4
controls), associated with a reduction in the other phases, which may reflect the
induction of apoptosis [44].

ACCEPTED MANUSCRIPT
Fig. 3. Flow cytometric analysis of A-549 human lung cancer cell cycle after the
treatment with or without (control) compound 28 for 24 and 48h. * Significant
difference with control group, p < 0.05.
Through various molecular effectors, cytotoxic agents may influence cell cycle
progression and apoptosis [45]. Apoptosis is one of the major processes in cell death,
and among its typical characteristics are chromatin condensation and fragmentation [46,
4
7]. We evaluated A-549 cell viability after induction or not (control) for 24h with 28
using an annexin V-FITC/PI kit for flow cytometry [48] and found that 28 induces a
significant reduction in cell viability (54.2% vs 83.6% of the controls) with an increase
in total apoptosis (44.0% vs 13.2%) (Figure 4). The expression of the proteins Bax and
Bcl-2 plays a critical role in the apoptotic process [49, 50]. A-549 cells were induced
with 0x (control), 0.5x or 1x IC50 of compound 28 for 24h. Western blot analysis
showed a decrease and an increase in Bcl-2 and Bax proteins, respectively, which is a
molecular hallmark of apoptosis (Figure 5). In general, members of the Bcl-2 family,
which includes Bcl-2, Bad, and Bax, are important regulators of various apoptotic
pathways [51]. Bax exerts pro-apoptotic effects whereas Bcl-2 presents anti-apoptotic
activities [52, 53]. Collectively, these results suggest that compound 28 promotes

ACCEPTED MANUSCRIPT
apoptosis. This mechanism of action is similar to that reported for various other
compounds, including: 1,8-Cineole, a predominant compound present in the hexane
extract of Callistemon citrinus in A431 human skin carcinoma cells [54]; ovatodiolide,
isolated from the herb Anisomeles indica, in human lung cancer A549 and H1299 cell
lines [55]; [1–9-NαC]-crourorb A1, a cyclic peptide isolated from Croton urucurana, in
Huh-7 human hepatocarcinoma cells [56]; and Glaucocalyxin A (GLA), a major
component isolated from Rabdosia japonica, in the UMUC3 human bladder cancer cell
line [57].
Fig. 4. Flow cytometric analysis of A-549 viability after treatment with or without
(control) compound 28 for 24 h, revealed by annexin V-FITC/PI staining. A) Selected
cytograms of control (left) and treated (right) cultures. B) Histograms representing the
percentages of viable, necrotic and apoptotic cells. * Significant difference with control
group, p < 0.05.

ACCEPTED MANUSCRIPT
Fig. 5. Western blot analysis of Bcl-2 and Bax expression in A-549 cultures induced
with 0x (CNT), 0.5x (1/2IC ) or 1x IC (IC ) of compound 28 for 24h.
5
0
50
50
3. Conclusions
A new series of podocarpane and totarane derivatives, including the natural
podocarpane 23, has been synthesized and evaluated for in vitro antiproliferative
potential against different human cancer cell lines (MCF-7, T-84 and A-549). An
antiproliferative effect was induced by compounds 23, 27 and 28, with IC <10 µM in
5
0
two (27) or three cell lines (23 and 28). No correlation with log P values was observed.
The totarane o-quinone 27, and especially the catechol 28, which is readily oxidisable to
compound 27, were the most active compounds, highlighting the functional groups present
in C11 and C12. Compound 28 showed limited toxicity in normal PBMC, and appeared to
exert an antiproliferative effect in A-549 cells through a mechanism that involves the
induction of apoptosis mediated by an increased Bax/Bcl-2 ratio. The results of the present
study indicate that compound 28 might be useful as antitumoural agent. Further studies
are required to elucidate the cellular and molecular elements involved in its effect, and
the levels of activity/toxicity in preclinical models.

ACCEPTED MANUSCRIPT
4
. Experimental
4
.1 General
Unless stated otherwise, reactions were performed in oven-dried glassware under an
argon atmosphere using dry solvents. Solvents were dried as follows: benzene over Na–
benzophenone, dichloromethane (DCM) over CaH . Thin-layer chromatography (TLC)
2
was performed using F254 precoated plates (0.25 mm) and visualized by UV
fluorescence quenching and phosphomolybdic acid solution staining. Flash
chromatography was performed on silica gel (230-400 mesh). Chromatography
separations were carried out by conventional column on silica gel 60 (230-400 Mesh),
1
13
using hexanes-AcOEt (AcOEt-hexane) mixtures of increasing polarity. H and
C
NMR spectra were recorded at 500 and 400 MHz, and at 150, 125 and 100 MHz,
respectively. Chemical shifts (δ Η) are quoted in parts per million (ppm) referenced to
1
the appropriate residual solvent peak and tetramethylsilane. Data for H NMR spectra
are reported as follows: chemical shift (δ ppm) (multiplicity, coupling constant (Hz),
integration), with the abbreviations s, br s, d, br d, t. dd, ddd, sext and m denoting
singlet, broad singlet, doublet, broad doublet, triplet, double doublet, double double
doublet, sextet and multiplet, respectively. J = coupling constant in Hertz (Hz). Data for
1
3
C NMR spectra are reported in terms of chemical shift relative to Me Si (δ 0.0) and
4
the signals were assigned utilizing DEPT experiments and on the basis of heteronuclear
correlations. Infrared spectra (IR) were recorded as thin films or as solids on a FTIR
spectrophotometer with samples between sodium chloride plates and are reported in
–
1
D
frequency of absorption (cm ). Only selected absorbances (ν ) are reported. ([α] )
max
measurements were carried out in a polarimeter; utilizing a 1dm length cell and CHCl3
as a solvent. Concentration is expressed in mg/mL. HRMS were recorded on a
+
spectrometer, utilizing a Q-TOF analyzer, and ESI ionization.
4
.2. Synthesis
4
.2.1.
(1S,4aS,10aR)-dimethyl
6-methoxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-
octahydrophenanthrene-1,7-dicarboxylate (19)[28]

ACCEPTED MANUSCRIPT
K CO (125 mg, 0.905 mmol) was added to a solution of 18 (190 mg, 0.548 mmol) in
2
3
acetone (8 mL) and the reaction mixture was kept stirring at room temperature for 15
min. Then, dimethyl sulfate (150 mg, 1.19 mmol) was added and the reaction mixture
was stirred at reflux for 12 h. The solvent was evaporated and the crude product was
poured into AcOEt – water (30 : 10 mL). The phases were shaken and separated, and
the organic phase was washed with brine (2 x 10 mL), dried over anh Na SO , filtered
2
4
and the solvent evaporated to give a crude product. Flash chromatograpy on silica gel
using 10% AcOEt/ hexane gave 19 (184 mg, 93%) as a colourless syrup.
4
.2.2
(1S,4aS,10aR)-dimethyl
6-methoxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-
octahydrophenanthrene-1,7-dicarboxylate (20)
To a solution of 19 (161 mg, 0.446 mmol) in dry THF (5 mL) was slowly added 1M
methylmagnesium bromide solution in THF (1.2 mL, 1.2 mmol) at 0°C and under argon
atmosphere. The reaction mixture was stirred at 0 °C for 30 min, at which time TLC
showed no starting material. The reaction was quenched with conc. HCl (2 mL), and the
reaction mixture was stirred at room temperature for an additional 10 min. Then, the
solvent was removed under vacuum and the mixture was diluted with AcOEt - Water
(20 : 5 mL). The phases were shaken and separated. The organic extract was washed
with water (5 mL) and brine (5 mL), dried over anhydrous Na SO , filtered and the
2
4
solvent was evaporated to give a crude product which was purified by flash
chromatography on silica gel (5% AcOEt/hexane), affording 20 (140 mg, 92%) as a
colorless syrup.
2
5
[
α]D = + 108.2 (c = 1.3, CHCl ). IR (film) νmax: 2929, 2849, 1725, 1609, 1498, 1463,
3
1
8
1
403, 1378, 1328, 1305, 1243, 1213, 1190, 1171, 1141, 1111, 1097, 1057, 1033, 973,
-1 1
92, 848, 773 cm . H RMN (CDCl , 500 MHz) δ (ppm): 1.07 (s, 3 H), 1.13 (ddd, J =
3
3.5, 13.5, 4.3 Hz, 1 H), 1.29 (s, 3 H), 1.44 (ddd, J = 13.3, 13.3, 7.1 Hz, 1H), 1.55 (d, J
=
10.49 Hz, 1 H), 1.63 – 1.67 (m, 1 H), 1.93 – 2.04 (m, 1 H), 2.11 (s, 3H), 2.16 – 2.32
(m, 3 H), 2.73 (ddd, J = 14.8, 14.8, 6.3 Hz, 1 H), 2.84 (dd, J = 15.2, 3.6 Hz, 1 H), 3.68
1
3
(
s, 3 H), 3.80 (s, 3 H), 5.07 (s, 1 H), 5.12 (s, 1 H), 6.76 (s, 1 H), 6.88 (s, 1 H). C RMN
(
CDCl , 125 MHz) δ (ppm): 19.9 (CH ), 21.1 (CH ), 22.8 (CH ), 23.3 (CH ), 28.5
3
2
2
3
3
(
CH ), 31.1 (CH ), 37.7 (CH ), 38.6 (C), 39.5 (CH ), 44.0 (C), 51.2 (CH), 52.9 (CH ),
3 2 2 2 3
5
5.6 (CH ), 108.1 (CH), 114.7 (CH ), 127.3 (C), 129.7 (CH), 130.3 (C), 143.9 (C),
3 2

ACCEPTED MANUSCRIPT
+
1
3
47.9 (C), 154.9 (C), 177.9 (C). HRMS (ESI) m/z: calcd for C H O (M+H )
22 31 3
43.2273, found: 343.2276.
4
.2.3
(1S,4aS,10aR)-dimethyl
6-methoxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-
octahydrophenanthrene-1,7-dicarboxylate (21)
LiAlH (60 mg, 1.57 mmol) was added to a stirred solution of 20 (103 mg, 0.3 mmol) in
4
dry Et O (5 mL) cooled to 0°C and the reaction mixture was kept stirred at room
2
temperature under an argon atmosphere for 15 min, at which time TLC showed no
remaining starting material. Then, 2 N HCl (0.5 mL) was slowly added at 0°C, and the
mixture was diluted with Et O – H O (20 : 5 mL). The phases were shaken and
2
2
separated, and the organic phase was washed with brine (2 x 5 mL), dried over anh
Na SO and filtered. Removal of the solvent under vacuum afforded pure 21 (92 mg,
2
4
9
8%) as a colourless syrup.
2
5
[
α]D = + 55.1 (c = 0.9, CHCl ). IR (film) νmax: 3375, 2924, 2851, 1609, 1563, 1497,
3
1
7
463, 1402, 1375, 1327, 1260, 1241, 1207, 1104, 1066, 1027, 987, 970, 891, 848, 802,
-1 1
56, 666, 477 cm . H RMN (CDCl , 500 MHz) δ (ppm): 1.04 (ddd, J = 13.6, 13.6, 4.2
3
Hz, 1 H), 1.07 (s, 3 H), 1.22 (s, 3 H), 1.46 – 1.53 (m, 2 H), 1.58 (d, J =4.9 Hz, 1 H),
.63-1.76 (m 3 H), 1.91 (d, J = 13.7 Hz, 1 H), 1.97 – 2.01 (m, 1 H), 2.11 (s, 3H), 2.31
d, J = 14.7 Hz, 1 H), 2.77 (ddd, J = 10.7, 10.7, 7.1 Hz, 1 H), 2.87 (dd, J = 15.1, 6.7
Hz, 1 H), 3.57 (d, J = 10.9, 1 H), 3.80 (s, 3 H), 3.88 (d, J = 10.9, 1 H), 5.06 (s, 1 H),
1
(
1
3
5
.11 (s, 1 H), 6.77 (s, 1 H), 6.86 (s, 1 H). C RMN (CDCl , 125 MHz) δ (ppm): 19.0
3
(
CH ), 19.3 (CH ), 22.3 (CH ), 25.6 (CH ), 26.8 (CH ), 30.1 (CH ), 35.2 (CH ), 37.9
2 2 3 3 3 2 2
(
C), 38.7 (C), 38.9 (CH ), 51.3 (CH), 55.7 (CH ), 65.3 (CH ), 107.3 (CH), 114.7 (CH ),
2 3 2 2
1
26.7 (C), 129.7 (CH), 130.2 (C), 143.9 (C), 149.6 (C), 154.9 (C). HRMS (ESI) m/z:
+
calcd for C H O (M+H ) 315.2324, found: 315.2326.
2
1
31
2
4
.2.4 1-((4bS,8S,8aR)-8-(hydroxymethyl)-3-methoxy-4b,8-dimethyl-4b,5,6,7,8,8a,9,10-
octahydrophenanthren-2-yl)ethanone (22)
An ozone stream was bubbled into a solution of 21 (81 mg, 0.257 mmol) in CH Cl (10
2
2
mL) cooled at -78 °C for 10 min, at which time TLC showed no starting material. Then

ACCEPTED MANUSCRIPT
an argon stream was bubbled for eliminating the ozone excess, and PPh was added to
3
the cooled solution, and then stirred for 1 h at room temperature. Solvent was
evaporated to afford a crude product that was purified by flash chromatography on silica
gel (30% AcOEt/hexane) to yield 22 (70 mg, 86%) as a colourless syrup
2
5
[
α]D = + 49.3 (c = 0.7, CHCl ). IR (film) νmax: 3447, 2924, 2850, 1666, 1604, 1494,
3
-1
1
1
463, 1402, 1356, 1328, 1264, 1235, 1184, 1034, 989, 969, 850, 752, 612 cm . H
RMN (CDCl , 400 MHz) δ (ppm): 1.03 (ddd, J = 13.6, 13.6, 4.2 Hz, 1 H), 1.06 (s, 3 H),
3
1
1
.19 (s, 3 H), 1.22 – 1.27 (m, 3 H), 1.43-1.51 (m 3 H), 1.61 – 1.76 (m, 2 H), 2.30 (d, J =
4.7 Hz, 1 H), 2.58 (s, 3H), 2.71 – 2.81 (m, 1 H), 2.89 (dd, J = 17.1, 6.8 Hz, 1 H), 3.55
1
3
(
d, J = 10.9, 1 H), 3.85 (d, J = 10.9, 1 H), 3.87 (s, 3 H), 6.83 (s, 1 H), 7.43 (s, 1 H). C
RMN (CDCl , 100 MHz) δ (ppm): 18.9 (CH ), 19.1 (CH ), 25.5 (CH ), 26.8 (CH ), 29.8
3
2
2
3
3
(
CH ), 35.0 (CH ), 38.4 (C), 38.8 (C), 38.9 (CH ), 50.9 (CH), 55.5 (CH ), 65.2 (CH ),
2 2 2 3 2
1
07.6 (CH), 125.7 (C), 127.2 (C), 130.9 (CH), 155.9 (C), 157.3 (C), 199.5 (C). ). HRMS
+
(
ESI) m/z: calcd for C H O (M+H ) 317.2117, found: 317.2117.
2
0
29
3
4
.2.5 1-((4bS,8S,8aR)-3-hydroxy-8-(hydroxymethyl)-4b,8-dimethyl-4b,5,6,7,8,8a,9,10-
octahydrophenanthren-2-yl)ethanone (23)
To a solution of 22 (50 mg, 0.158 mmol) in dry CH Cl (5 mL) was added AlBr (121
2
2
3
mg, 0.454 mmol), and the reaction mixture was stirred at room temperature for 4 h, at
which time TLC showed no starting material. Then, the mixture was poured into ice and
diluted with AcOEt – Water (15 : 5 mL). The phases were shaken and separated. The
organic layer was washed with water and brine, dried over anhydrous Na SO and
2
4
filtered. Removal of the solvent under vacuum afforded a crude product which was
purified by flash silicagel column chromatography using 40% AcOEt/hexanes to give
2
3 (42 mg, 88%) as a white solid.
2
5
[
α]D = + 57.4 (c = 0.8, CHCl ). IR (film) νmax: 3429, 2925, 2851, 1640, 1489, 1373,
3
-1 1
1
333, 1308, 1266, 1220, 1003, 772 cm . H RMN (CDCl , 400 MHz) δ (ppm): 1.02
3
(
ddd, J = 13.6, 13.6, 4.2 Hz, 1 H), 1.06 (s, 3 H), 1.17 (s, 3 H), 1.39 – 1.47 (m, 3 H),
.62-1.76 (m, 3 H), 1.89 (d, J = 14.3 Hz, 1 H), 1.98 – 2.03 (m, 1 H), 2.27 (d, J = 12.5
Hz, 1 H), 2.58 (s, 3H), 2.74 – 2.82 (m, 1 H), 2.91 (dd, J = 17.7, 6.4 Hz, 1 H), 3.56 (d, J
1
1
3
=
10.9, 1 H), 3.84 (d, J = 10.9, 1 H), 6.87 (s, 1 H), 7.38 (s, 1 H), 11.89 (s, 1 H). C

ACCEPTED MANUSCRIPT
RMN (CDCl , 100 MHz) δ (ppm): 18.8 (CH ), 19.1 (CH ), 25.2 (CH ), 26.5 (CH ), 26.8
3
2
2
3
3
(
CH ), 29.6 (CH ), 35.1 (CH), 38.5 (C), 38.6 (CH ), 38.8 (C), 50.6 (CH), 65.3 (CH ),
3 2 2 2
1
13.7 (CH), 117.9 (C), 125.8 (C), 130.9 (CH), 159.6 (C), 160.1 (C), 203.8 (C). HRMS
+
(
ESI) m/z: calcd for C H O (M+H ) 303.1960, found: 303.1952.
1
9
27
3
4
.2.6
(1S,4aS,10aR)-methyl
6-hydroxy-1,4a-dimethyl-8-(prop-1-en-2-yl)-
1
,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxylate (24)
To a solution of 17 (279 mg, 0.805 mmol) in dry THF (10 mL) was slowly added at 0
C 1M methylmagnesium bromide solution in THF (3 ml, 3 mmol) and under argon
°
atmosphere and the reaction mixture was stirred for 8 h. Then, it was quenched at 0 °C
with conc HCl (3 mL), and the reaction mixture was stirred at room temperature for an
additional 10 min. Then, the solvent was removed under vacuum and the mixture was
diluted with AcOEt - Water (30 : 10 mL). The phases were shaken and separated. The
organic extract was washed with water (10 mL) and brine (10 mL), dried over
anhydrous Na SO , filtered and the solvent was evaporated to give 24 (217 mg, 82%) as
2
4
2
5
a yellow syrup. [α]D = + 157.1 (c = 1.1, CHCl ). IR (film) νmax: 3417, 2933, 1723,
3
1
588, 1448, 1378, 1305, 1219, 1192, 114, 1089, 1033, 1219, 1192, 1140, 981, 772, 667
-1 1
cm . H RMN (CDCl , 500 MHz) δ (ppm): 1.03 (s, 3 H), 1.08 (ddd, J = 13.6, 13.6, 4.3
3
Hz, 1 H), 1.27 (s, 3 H), 1.40 (ddd, J = 13.2, 13.2, 4.2 Hz, 1 H), 1.52 (d, J = 10.6, Hz, 1
H), 1.59 – 1.63 (m, 1 H), 1.86 (dddd, J = 12.9, 12.9, 12.9, 5.2 Hz, 1 H), 1.98 (s, 3 H),
2
.16 (d, J = 13.3, Hz, 3 H), 2.27 (d, J = 13.5, Hz, 2 H), 2.57 (ddd, J = 14.7, 14.7, 6.1
Hz, 1 H), 2.81 (dd, J = 16.6, 7.0 Hz, 1 H), 3.66 (s, 3 H), 4.79 (s, 1 H), 5.12 (s, 1 H),
1
3
6
.45 (s, 1 H), 6.69 (s, 1 H). C RMN (CDCl , 125 MHz) δ (ppm): 20.0 (CH ), 21.1
3 2
(
(
(
CH ), 22.9 (CH ), 24.2 (CH ), 28.5 (CH ), 29.2 (CH ), 37.5 (CH ), 39.0 (C), 39.7
2 3 3 3 2 2
CH ), 43.9 (C), 51.3 (CH), 52.7 (CH ), 110.9 (CH), 112.8 (CH), 114.4 (CH ), 124.2
2
3
2
C), 145.2 (C), 146.1 (C), 149.8 (C), 153.3 (C), 178.2 (C). HRMS (ESI) m/z: calcd for
+
C H O (M+H ) 329.2093, found: 329.2104.
21
29
3
4
.2.7 (1S,4aS,10aR)-methyl 6-hydroxy-8-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-
octahydrophenanthrene-1-carboxylate (25)

ACCEPTED MANUSCRIPT
To a solution of 24 (233 mg, 0.709 mmol) in CH Cl (10 mL) were successively added
2
2
Et SiH (0.5 mL) and trifluoroacetic acid (0.2 mL) and the reaction mixture was refluxed
3
for 1 h. Then CH Cl , CF CO H and excess Et SiH were evaporated and the resulting
2
2
3
2
3
crude product, was purified by column chromatography using 20% AcOEt/hexanes to
2
5
give 25 (204 mg, 87%) as colourless syrup. [α]D = + 127.1 (c = 1.6, CHCl ). IR
3
(film) νmax: 3417, 2933, 1723, 1588, 1448, 1378, 1306, 1219, 1192, 1140, 1089, 981,
-
1 1
8
1
98, 862, 773 cm . H RMN (CDCl , 500 MHz) δ (ppm): 1.04 (s, 3 H), 1.07 (ddd, J =
3
3.6, 13.6, 4.3 Hz, 1 H), 1.16 (d, J = 6.8 Hz, 3 H), 1.19 (d, J = 6.8 Hz, 3 H), 1.28 (s, 3
H), 1.36 (ddd, J = 13.5, 13.5, 4.3 Hz, 1 H), 1.50 (dd, J = 12.5, 1.8 Hz, 2 H), 1.59 – 1.63
m, 1 H), 1.90 (ddd, J = 12.9, 12.9, 6.4 Hz, 1 H), 1.94 – 2.02 (m, 1 H), 2.16 (d, J =
2.7, Hz, 1 H), 2.21 – 2.28 (m, 2 H), 2.51 – 2.60 (m, 1 H), 2.91 (dd, J = 16.5, 5.5 Hz, 1
H) 3.09 (h, J = 6.8 Hz, 1 H), 3.67 (s, 3 H), 6.61 (d, J = 2.7 Hz, 1 H), 6.63 (d, J = 2.6 Hz,
(
1
1
3
1
H). C RMN (CDCl , 125 MHz) δ (ppm): 20.0 (CH ), 21.1 (CH ), 22.9 (CH ), 23.1
3 2 2 3
(
CH ), 23.6 (CH ), 28.0 (CH ), 28.4 (CH ), 37.5 (CH ), 38.9 (C), 39.9 (CH ), 43.9 (C),
3 3 2 3 2 2
5
1
3
1.3 (CH), 52.3 (CH ), 109.76 (CH), 109.83 (CH), 124.5 (C), 148.3 (C), 149.8 (C),
3
+
53.7 (C), 178.1 (C). ). HRMS (ESI) m/z: calcd for C H O (M+H ) 331.2273, found:
2
1
31
3
31.2260.
4
.2.8 Treatment of phenol 25 with (PhSeO) O
2
(
PhSeO) O (720 mg,1.99 mmol) was added to a solution of phenol 25 (630 mg, 1.9
2
mmol) in anhydrous THF (10 mL) under an argon atmosphere, and the reaction was
kept stirring at reflux for 10 min. The solvent was evaporated under vacuum to give a
crude product, which after column chromatography on silica gel using 10%
EtOAc/hexane, afforded ortho-quinone 27 (477 mg, 73%) and hydroxy dienone 26 (131
mg, 20%).
(1S,4aS,8aR)-methyl
8a-hydroxy-7-isopropyl-1,4a-dimethyl-6-oxo-
1
,2,3,4,4a,6,8a,9,10,10a-decahydrophenanthrene-1-carboxylate (26)
2
5
[
α]D = + 12.3 (c = 1.8, CHCl ). IR (film) νmax: 3403, 2928, 1726, 1661, 1619, 1463,
3
-
1 1
1
1
387, 1219, 1151, 1044, 773 cm . H RMN (CDCl , 400 MHz) δ (ppm): 1.02 (ddd, J =
3
3.2, 13.2, 3.9J Hz, 1 H), 1.13 (d, J = 6.9 Hz, 3 H), 1.19 (d, J = 6.9 Hz, 3 H), 1.21 (s, 3
H), 1.22 (s, 3 H), 1.50 (ddd, J = 13.0, 13.0, 4.3 Hz, 1 H), 1.61 – 1.67 (m, 1 H), 1.75 (d,
J = 12.0 Hz, 1 H), 1.91 – 2.03 (m, 2 H), 2.22 (d, J = 13.7 Hz, 1 H), 2.41 – 2.52 (m, 2

ACCEPTED MANUSCRIPT
1
3
H), 2.87 (h, J = 6.9 Hz, 1 H), 6.05 (d, J = 1.8 Hz, 1 H), 6.09 (d, J = 1.8 Hz, 1 H). C
RMN (CDCl , 100 MHz) δ (ppm): 18.6 (CH ), 18.9 (CH ), 19.3 (CH ), 24.1 (CH ), 24.7
3
3
2
2
3
(
CH ), 27.8 (CH), 28.7 (CH ), 37.2 (CH ), 37.7 (CH ), 38.3 (CH ), 41.9 (C), 44.4 (C),
3 3 2 2 2
5
1.4 (CH), 55.8 (CH ), 72.1 (C), 122.35 (CH), 122.57 (CH), 169.1 (C), 173.9 (C), 177.3
3
(C), 187.4 (C).
(1S,4aS,10aR)-methyl
8-isopropyl-1,4a-dimethyl-5,6-dioxo-1,2,3,4,4a,5,6,9,10,10a-
decahydrophenanthrene-1-carboxylate (27)
2
5
[
α]D = + 24.3 (c = 0.5, CHCl ). IR (film) νmax: 2928, 2854, 1725, 1679, 1656, 1465,
3
-1
1
1
383, 1308, 1232, 1192, 1159, 1140, 1099, 980, 773 cm . H RMN (CDCl , 400 MHz)
3
δ (ppm): 1.02 (ddd, J = 13.2, 13.2, 3.9J Hz, 1 H), 1.09 (s, 3 H), 1.15 (d, J = 6.7 Hz, 3
H), 1.16 (d, J = 6.7 Hz, 3 H), 1.23 (s, 3 H), 1.49 – 1.56 (m, 3 H), 1.75 – 1.92 (m, 2 H),
2
.17 – 2.22 (m, 2 H), 2.34 – 2.44 (m, 1 H), 2.62 – 2.68 (m, 2 H), 2.82 (h, J = 6.7 Hz, 1
13
H), 3.64 (s, 3 H), 6.21 (s, 1 H). C RMN (CDCl , 100 MHz) δ (ppm): 17.6 (CH ), 19.5
3
3
(
CH ), 19.9 (CH ), 22.6 (CH ), 22.7 (CH ), 28.6 (CH), 29.0 (CH ), 30.4 (CH ), 36.7
2 2 3 3 3 2
(
CH ), 37.4 (CH ), 39.5 (C), 43.5 (C), 51.4 (CH), 52.9 (CH ), 72.1 (C), 122.7 (CH),
2
2
3
1
46.4 (C), 147.7 (C), 164.7 (C), 177.3 (C), 180.4 (C), 180.9 (C). ). HRMS (ESI) m/z:
+
calcd for C H O (M+H ) 345.2066, found: 345.2055.
2
1
29
4
4
.2.9
(1S,4aS,10aR)-methyl
5,6-dihydroxy-8-isopropyl-1,4a-dimethyl-
1
,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxylate (28).
Sodium borohydride (60 mg, 1.58 mmol) was added to a solución of 27 (181 mg, 0.525
mmol) in THF (5 mL) and the yellow reaction mixture was stirred at room temperature
for 10 min, at which time the solution became colourless. Then, Amberlyst A-15 (200
mg) was added carefully and the reaction was stirred for an additional 5 min. Then, the
mixture was filtered and the solvent was removed under vacuum, affording 28 (178 mg,
25
9
8%) as a colourless syrup. [α]D = + 21.5 (c = 0.9, CHCl ). IR (film) νmax: 3419,
3
1
610, 1588, 1452, 1434, 1379, 1305, 1231, 1206, 1161, 1J142, 1087, 1007, 976, 773
-1 1
cm . H RMN (CDCl , 400 MHz) δ (ppm): 1.09 (ddd, J = 13.6, 13.6, 4.3 Hz, 1 H), 1.14
3
(
d, J = 6.8 Hz, 3 H), 1.15 (d, J = 6.8 Hz, 3 H), 1.21 (s, 3 H), 1.28 (s, 3 H), 1.76 (ddd, J =
3.0, 13.0, 4.7 Hz, 2 H), 1.86 – 2.04 (m, 2 H), 2.20 (dd, J = 13.9, 6.4 Hz, 2 H), 2.27 (d,
J = 13.8, Hz, 2 H), 2.57 (ddd, J = 14.6, 14.6, 5.3 Hz, 1 H), 2.87 (d, J = 13.8, Hz, 1 H),
1
13
3
.06 (h, J = 6.8 Hz, 1 H), 3.66 (s, 3 H), 5.30 (s, 1 H), 5.54 (s, 1 H), 6.64 (s, 1 H). C

ACCEPTED MANUSCRIPT
RMN (CDCl , 100 MHz) δ (ppm): 16.6 (CH ), 19.9 (CH ), 21.0 (CH ), 23.4 (CH ), 23.8
3
3
2
2
3
(
CH ), 27.9 (CH), 28.9 (CH ), 29.8 (CH ), 36.1 (CH ), 37.7 (CH ), 40.1 (C), 43.9 (C),
3 3 2 2 2
5
1.2 (CH), 54.7 (CH ), 109.8 (CH), 127.6 (CH), 134.2 (C), 138.0 (C), 140.5 (C), 141.2
3
+
(
C), 178.2 (C). ). HRMS (ESI) m/z: calcd for C H O (M+H ) 347.2222, found:
21
31
4
3
47.2220.
4
4
.3. Biological Assays
.3.1. Cells and culture
Human breast tumor line MCF-7, human colon tumor line T-84 and human lung
tumor line A-549 were cultured at 37°C in 5% CO and 90% humidity with Dulbecco’s
2
modified Eagle medium (DMEM), supplemented with 10% heat inactivated fetal bovine
serum, 10 ml/L penicillin-streptomycin 100x and 2 mM L-glutamine. All cell lines were
provided by the Department of Cell Cultures of the Granada University Scientific
Instrumentation Center. Peripheral blood mononuclear cells (PBMC) were isolated by a
standard method of density gradient centrifugation using Histopaque-1077, and cultured
with RPMI-1640 medium supplemented with 20% fetal bovine serum, 10 ml/L
penicillin-streptomycin 100x and 2mM glutamine at 37°C in 5% CO and 90%
2
humidity. The protocol for the obtention and use of human blood samples had been
approved by the local ethical committee. Culture media and supplements were supplied
by Sigma-Aldrich (St. Louis, MO).
4
.3.2. In vitro antiproliferative assay
In order to quantify the IC of compounds, cells were seeded in 96 well plates at a
50
3
2
density of 5 x 10 cells/cm . At 24h, cells were induced with increasing compound
concentrations for 3 days. Subsequently, cells were fixed with 10% cold trichloroacetic
acid (4°C) and stained with 0.4% sulforhodamine B (SRB) in 1% acetic acid. The
colorant was solubilized with 10 mM Tris-base pH 10.5, and optical density values were
determined by colorimeter at 492 nm (Multiskan EX, Thermo Electron Corporation,
Milford, MA, USA). The inhibitory concentration 50 (IC50), considered as the
concentration of a drug that causes 50% reduction of proliferation and/or cell density
versus control cultures, was calculated from the semilogarithmic dose-response curve

ACCEPTED MANUSCRIPT
by linear interpolation. Alternatively, non-adherent PBMC cultures were quantified
using MTT assay at 570 and 595nm [58,59].
4
.3.3. Cell cycle and Sub-G1 analysis
4
2
A-549 cells were seeded in 6 well plates at a density of 4 x 10 cells/cm , and after
2
4h were induced or not (control) with IC of compound 28 for 24 or 48h. Then the
5
0
cells were collected, fixed with 70% cold ethanol, incubated with a DNA extraction
solution (0.2 M Na HPO , 0.1 M Citric Acid, pH 7.8) for 15 min at 37°C, and stained
2
4
with a solution of propidium iodide/ RNAse for 30 min at 37°C in the dark. Finally,
samples were analyzed using a flow cytometer (BD FACSCalibur) and the FlowJo
software (v 7.6.5, Tree Star, Inc.) [60].
4
.3.4. Apoptosis assays with annexin-V
4
2
A-549 cells were seeded in 6 well plates at a density of 4 x 10 cells/cm , and after
2
4h were induced or not (control) with IC of compound 28 for 24h. Cell viability was
5
0
determined by flow cytometry using the TACS® Annexin V-FITC kit (Trevigen,
Gaithersburg, MD, USA) following the manufacturer's instruction. Samples were
analyzed using a flow cytometer (BD FACSCalibur) and the FlowJo software (v 7.6.5,
Tree Star, Inc.).
4
.3.5. Western blot analysis
4
2
A-549 cells were seeded in 6 well plates at a density of 4 x 10 cells/cm , and after
2
4h were induced with 0x (control), 0.5x or 1x IC50 of compound 28 for 24h. The
medium was then removed, and cells were lysed with sample buffer (62.76 mM Trise
HCl pH 6.8, 5% β-mercaptoethanol, 2% SDS, 10% glycerol, 0.5% bromophenol blue).
Proteins were separated by electrophoresis in 12% acrylamide gels in an Mini Protean
Tetracell (Bio-Rad, CA, USA), transferred to nitrocellulose membranes, and blocked
with a buffer containing 3% non-fat milk powder, 1% bovine serum albumin (Sigma-
Aldrich), and 0.5% Tween-20 in PBS for 1 h. Subsequently, the membranes were
incubated overnight with mouse monoclonal anti-Bax (Santa Cruz Biotechnology, sc-
7
480) or anti-Bcl-2 (Santa Cruz Biotechnology, sc-56015), followed by incubation with
HRP-conjugated anti-mouse IgG. β-actin was detected using a HRP-conjugated mouse
monoclonal anti-β-actin (Sigma, A-3854). All antibodies were purchased from Santa
Cruz Biotechnology (Texas, USA). Protein-antibody complexes were detected by

ACCEPTED MANUSCRIPT
chemoluminiscence using the ECL prime immunodetection reagent (GE Healthcare,
Little Chalfont, UK).
4
.3.6. Statistical analysis
SPSS 24 for Windows (SPSS, Chicago, IL, USA) was used for the statistical
analysis. Results were compared with the Student’s test, one-way ANOVA or Pearson’s
test. p < 0.05 was considered significant. Data were graphically represented by using
Microsoft Excel 2013 software (Microsoft Corporation).
Acknowledgments
This work was supported by grants from Spanish Ministry of Economy and
Competitiveness (Project CTQ2014-56611-R/BQU), the Regional Government of
Andalucia (Project P11-CTS-7651 and assistance for the FQM-348 group), the Ramón
Areces Foundation (Madrid, Spain), and the European Commission for the Erasmus
Mundus – Al Idrisi predoctoral fellowship granted to S. Mahdjour. This paper is a part
of the Ph.D. Thesis of S. Mahdjour.
Appendix A. Supplementary data
1
13
Supplementary data related to this article ( H and C NMR spectra of all new
compounds), can be found at https://doi.org/
REFERENCES
(
1) W. F. Short, H. Stromberg, Totarol. Part I, J. Chem. Soc. (1937) 516-520.
2) H. Haraguchi, H. Ishikawa, I. Kubo, Antioxidative action of diterpenoids from
Podocarpus nagi, Planta Med. 63 (1997) 213-215.
(
(
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