Enhancement of antiproliferative activity by molecular simplification of catalpol

Article abstract of DOI:10.1016/j.bmc.2010.02.044

Two iridoid scaffolds were synthesized enantioselectively using as key step an l-proline-catalyzed α-formyl oxidation. The in vitro antiproliferative activities were evaluated against a representative panel of human solid tumor cell lines. Both iridoids induced considerably growth inhibition in the range 0.38-1.86 μM. Cell cycle studies for these compounds showed the induction of cell cycle arrest at the G₁ phase. This result was consistent with a decrease in the expression of cyclin D1. Damaged cells underwent apoptosis as indicated by specific Annexin V staining.

Full text of DOI:10.1016/j.bmc.2010.02.044

Bioorganic & Medicinal Chemistry 18 (2010) 2515–2523
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Enhancement of antiproliferative activity by molecular simplification
of catalpol
Celina García a, , Leticia G. León a,b, Carlos R. Pungitore c, Carla Ríos-Luci a, Antonio H. Daranas a,d
,
*
Juan C. Montero e, Atanasio Pandiella e, Carlos E. Tonn c, Víctor S. Martín a, , José M. Padrón
a,b,
*
*
a Instituto Universitario de Bio-Orgánica ‘Antonio González’ (IUBO-AG), Universidad de La Laguna, C/Astrofísico Francisco Sánchez 2, 38206 La Laguna, Spain
b BioLab, Instituto Canario de Investigación del Cáncer (ICIC), C/Astrofísico Francisco Sánchez 2, 38206 La Laguna, Spain
c INTEQUI-CONICET, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera-5700-San Luis, Argentina
d Departamento de Ingeniería Química y Tecnología Farmacéutica, Universidad de La Laguna, Avda. Astrofísico Francisco Sánchez s/n, 38206 La Laguna, Spain
e Centro de Investigación del Cáncer, IBMCC/CSIC-Universidad de Salamanca, Campus Miguel de Unamuno. 37007 Salamanca, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Two iridoid scaffolds were synthesized enantioselectively using as key step an
myl oxidation. The in vitro antiproliferative activities were evaluated against a representative panel of
human solid tumor cell lines. Both iridoids induced considerably growth inhibition in the range 0.38–
1.86 lM. Cell cycle studies for these compounds showed the induction of cell cycle arrest at the G1 phase.
This result was consistent with a decrease in the expression of cyclin D1. Damaged cells underwent apop-
L
-proline-catalyzed a-for-
Received 9 September 2009
Revised 19 February 2010
Accepted 22 February 2010
Available online 1 March 2010
tosis as indicated by specific Annexin V staining.
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Antitumor agents
Catalpol
Cell cycle
Molecular simplification
Structure elucidation
1. Introduction
occurring sugiol (1)5 and catalpol (2)6 can be transformed into
antiproliferative drugs in a straightforward manner. Catalpol (2)
Molecular simplification represents a drug design strategy to
shorten synthetic routes while keeping or enhancing the biological
activity of any given natural or synthetic template. Initially, this
concept was applied empirically to natural products with complex
structures in order to obtain active derivatives with a simplified
molecular structure. Classical examples of molecular simplification
are the natural alkaloids morphine and quinine, which led to sev-
eral drugs currently used in therapy. The molecular simplification
approach is particularly interesting in the development of new
antitumor drugs, since a large number of current anticancer che-
motherapeutics is derived from natural products that have com-
shows significant in vitro inhibition of Taq DNA polymerase.7
Structurally this metabolite illustrates a certain resemblance with
a nucleoside framework (Fig. 1). The bicyclic aglycone is alike the
purine scaffold present in nucleosides and that could explain the
observed Taq DNA polymerase inhibitory activity.47b DNA poly-
merase inhibitors represent important drugs in anticancer or anti-
viral therapy, such as the deoxycytidine analogs ara-C (1-b-D-
R2O
H
OH
plex chemical structures.1 Recently,
a series of simplified
O
ecteinascidin2 and epothilone3 analogs have been synthesized
and evaluated, showing promising results. In this particular con-
text, we have reported novel antiproliferative acetogenin analogs
synthesized in a limited number of chemical steps.4
OR1
O
R1O
H
O
O
O
HO
OH
H
Within our research program directed toward the discovery of
novel antiproliferative molecules, we have shown that naturally
OH
1
2a R1 = R2 = H
2b R1 = TBS, R2 = H
2c R1 = TBDPS, R2 = H
2d R1 = R2 = TBDPS
* Corresponding authors. Tel.: +34 922316502x6126; fax: +34 922318571.
E-mail addresses: cgargon@ull.es (C. García), vmartin@ull.es (V.S. Martín),
jmpadron@ull.es (J.M. Padrón).
Figure 1. Structure of naturally occurring sugiol (1) and catalpol (2).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.044

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C. García et al. / Bioorg. Med. Chem. 18 (2010) 2515–2523
arabinofuranosyl-cytosine) and gemcitabine (20,20-difluorodeoxy-
cytidine, dFdC).8 These drugs belong to the group of antimetabo-
lites. Examples of molecular simplified antimetabolites (lacking
the sugar moiety) are the purine analogs 6-mercaptopurine and
thioguanine, and the pyrimidine analog 5-fluorouracil.
observed between H-1 and H-7a, H-7, methyl group at C-7 position
and a weak and intriguing one with the methyl groups of the tert-
butyl moiety. However, with the exception of the last one, these
NOEs could be possible for both C-1 epimers.
A solution for this problem is suggested based on the results of a
molecular mechanics approach to determine the relative configu-
ration of these chiral centers in combination with NMR data.
Once the planar structure and the stereochemistry of most chi-
ral centers of 9a were determined, a conformational study was car-
ried out in order to determine the configuration of carbon C-1.
Herein we report on the synthesis and biological evaluation of a
simplified analog of the bicyclic aglycone of catalpol (2).
2. Results and discussion
2.1. Chemistry
*
Therefore, we decided to study the two possible isomers 1R and
1S*. For that reason, Monte Carlo Multiple Minimum (MCMM) con-
formational searches were undertaken.
Inspired by the chemical architecture of this iridoid, we envi-
sioned the bicyclic core construction similar to that using by Man-
gion and MacMillan in the total synthesis of brasoside and
littoralisone.9 The synthesis of the compounds reported in this
study began from b-citronellol (3) and is outlined in Scheme 1.
Thus, benzoylation of the primary alcohol group led to ester 4,
which was converted into aldehyde 5 by ozonolysis. Applying the
These consisted of two independent searches with the MM2*11
force field as implemented in MacroModel 8.512 using the general-
ized Born/surface area (GBSA) chloroform solvent model.13 Ran-
dom searches of 5000 MCMM steps were undertaken for each
diastereoisomer to insure that the potential energy surface was
thoroughly explored using the TNCG algorithm. All local minima
within 50 kJ of the global minimum were saved. The global mini-
mum was found 97 times for each search, indicating that a thor-
ough search of the molecule’s potential energy surface was done.12
For the 1R* (9a) isomer, the conformational search resulted in
two conformational families with some differences into the bicy-
clic ring (Fig. 2). The orientation of H-1 in these molecules changes
from being located in axial position in the best energy structure to
an equatorial position in the best energy representative of the
proline-catalyzed
aldehyde 5,10 followed by olefination of the resultant aldehyde
using Horner–Wadsworth–Emmons conditions, produced (E)- ,b-
a-formyl oxidation protocol using L-proline over
a
unsaturated ester 6. The secondary hydroxyl group was protected
as its tert-butyldiphenylsilyl (TBDPS) ether and the resulting struc-
ture 7 was subsequently treated with DIBAL-H providing us the
diol 8. Dess–Martin oxidation of compound 8, exposure of the dial-
dehyde intermediate to L-proline in DMSO, and subsequently acet-
ylation provided the bicyclic derivative 9.
other conformational family (DE = +3.5 kJ/mol). In the same way,
for the 1S* epimer, the results obtained were quite similar. Again
two conformational families where the orientation of H-1 changes
from equatorial in the best energy solution to an axial position in
2.2. Relative configuration
the other family (
However, some important clues to find the solution of our prob-
lem were found. The conformational search conducted for the 1S
DE = +3.5 kJ/mol) were found.
The relative configuration of the chiral centers in 9a was deter-
mined mainly by the observed dipolar correlations in the NOESY
spectrum, since the J-based configurational analysis was inconclu-
sive. Considering that the relative configuration of the chiral cen-
ters in C-5 and C-7 is clear as resulted from the aforementioned
asymmetric synthesis10 and from the starting material respec-
tively, we will refer our results to them. Therefore, H-5 (dH 4.25)
showed strong NOE cross-correlation peaks with H-4a (dH 2.5),
H-7a (dH 1.6) and the methyl group at C-7 position (dH 0.85), indi-
cating that all of them are in the same side of the bicyclic system.
In the same way, H-4a (dH 2.5) showed a strong connectivity with
H-7a (dH 1.6), confirming that the existence of a cis-type ring clo-
sure. These data allowed us to determine relative stereochemist-
ries at C-4a, C-7a, C-5, and C-7 as 4aS,5R,7R,7aS. Likewise, the
relative configuration of C-1 was determined mainly by analysis
of the NOESY experiment. This time, NOE cross-correlations were
*
epimer resulted in conformations were either H-1 and H-4a (2.8 Å)
or the methyl groups of the acetate and the tert-butyl groups
(3.3 Å) should be close enough to expect a clear but unobserved
NOE correlations. On the other hand, the results obtained for the
1R* epimer did not show such inconsistencies and at the same time
could explain the intriguing NOE between H-1 and the methyls of
the tert-butyl group, as one set of structures showed appropriated
distances (2.35 Å).
The results of these simulations fully agree with experimentally
3
measured JH–H. In fact, it should to be noted that the resulting
ensemble-averaged (derived from the estimated populations by a
Boltzmann distribution at 300 K) structures obtained from the
MCMM searches and the experimentally measured values matched
OH
O
a
c
b
CO2Me
OBz
R
OH
R
OBz
R
OBz
R
3a R = α-Me, (+)-β-Citronellol
3b R = β-Me, (-)-β-Citronellol
4a R = α-Me
5a R = α-Me
6a R = α-Me
6b R = β-Me
4b R = β-Me
5b R = β-Me
TBDPSO
R1
OTBDPS
OTBDPS
d
e
f
5
4a
7a
OH
CO2Me
7
O
1
R1
R
OBz
R
OH
R2
R
9a R = α-Me, R1 = α-H, R2 = α-OAc
9b R = β-Me, R1 = β-H, R2 = β-OAc
7a R = α-Me
7b R = β-Me
8a R = α-Me
8b R = β-Me
Scheme 1. Reagents and conditions: (a) BzCl, Et3N, CH2Cl2, 99%; (b) (i) O3, Py, CH2Cl2/MeOH (9:1), ꢀ78 °C; (ii) PPh3, ꢀ78 °C to 0 °C, 91%; (c) (i)
(MeO)2P(O)CH2CO2Me, DBU, LiCl, CH3CN, ꢀ15 °C; (iii) NH4Cl, MeOH, ꢀ15 °C to rt, 24%; (d) TBDPSCl, imidazole, CH2Cl2, rt, 94%; (e) DIBAL-H, Et2O, ꢀ78 °C, 45%; (f) (i) DMP,
CH2Cl2, rt; (ii) -proline, DMSO, 40 °C, 60 h; (iii) Ac2O, DMAP, Py, 0 °C, 15%.
L-proline, PhNO, DMSO; (ii)
L

C. García et al. / Bioorg. Med. Chem. 18 (2010) 2515–2523
2517
*
*
Figure 2. Comparison of the two different structural motifs found out of the MCMM conformational search conducted for the 1R and 1S epimers of 9a. In clear gray the best
*
*
energy conformer and in dark gray the best energy representative of the second conformational motif for the 1R (left) and 1S (right) epimers of 9a.
very well. Even though 9a is a rigid molecule, it shows some flex-
ibility along the five and six membered-rings. Next, the results ob-
tained from the simulations were compared to the NMR data
available for 9a and the outcome of such comparisons was that
the energy-averaged structure matched appropriately with the
experimental NMR data. In this way, the calculated values for those
couplings using the Díez–Altona–Donders equations, as
2.3. Antiproliferative activity
Prior to the synthesis of the compounds reported in this article,
we prepared the same set of derivatives but starting from racemic
citronellol using the same synthetic strategy described in Scheme
1. In this unreported work, we prepared as a diastereomeric mix-
ture the desilylated derivatives of compounds 9a-b. The antiprolif-
erative activity of this mixture is listed in Table 1. We observed
that the unsilylated bicycle was less active when compared to
the silylated precursor. Therefore, for the study reported in our
manuscript when we started the synthesis using both enantiomers
of citronellol we finished the synthesis with compounds 9a-b.
The antiproliferative activity of bicyclic analogs 9a-b was stud-
ied using the NCI protocol.15 The effect, defined as 50% growth
inhibition (GI50),16 was determined against the panel of represen-
tative human solid tumor cell lines A2780 (ovarian), HBL-100
(breast), HeLa (cervix), SW1573 (non-small cell lung), T-47D
(breast), and WiDr (colon). Compounds 9a-b showed very active
against all cell lines with GI50 values in the range 0.38-1.86 lM
(Table 1). Interestingly, the data reveals a similar pattern of activity
of both stereoisomers.
When comparing the growth inhibition data of the simplified
aglycones 9a-b (Table 1) with those obtained for catalpol (2) and
its silylated derivatives,6 an important consequence can be in-
ferred. The aglycone fragment alone is more potent than when it
is attached to a sugar moiety. In fact, catalpol (2) showed inactive
3
implemented in the software MSpin,14 were JH4a–H7a = 6.2 Hz,
3
3
3JH7a–H1 = 3.4 Hz, JH7a–H7 = 8.1 Hz, and JH4a–H5 = 5.1 Hz, in excel-
lent agreement with the corresponding experimentally measured
3
3
values (3JH4a–H7a = 6.3 Hz, JH7a–H1 = 2.7 Hz, JH7a–H7 = 7.5 Hz, and
3JH4a–H5 = 4.2 Hz).
3
Consequently, based on the previous analysis (NOESY, JH–H
couplings and MCMM simulations) we propose the 1R,4aS,5R,7-
R,7aS stereochemistry for 9a.
On the other hand, the stereochemistry assignment was clearer
for 9b. Analysis of 1D and 2D NOESY experiments allowed us to
find strong dipolar correlations between H-7a and both H-4a and
the methyl protons at C-7 position that indicated they are all on
the same face of the molecule. In addition, medium and weak
NOE effects were observed for H-7 and H-1 as well as between
H-7 and H-5, respectively; data that together with a strong NOE ef-
fect of H-5 with H-4, located all these protons on the opposite face
of the molecule. As a result, we now propose the 1S,4aR,5R,7S,7aR
stereochemistry for 9b.
In order to confirm the previously described observations, a con-
formational search using the same conditions applied for 9a was
undertaken (Fig. 3). The result was also similar, as one major confor-
mational family appeared including the global minimum that was
found 65 times. The best energy structure fully reproduces the
NMR experimental observations (NOESY and 3JH–H couplings). This
time, the calculated proton–proton coupling constants showed
excellent agreement with the experimentally observed values.
Therefore, calculated values for 3JH4a–H7a = 6.1 Hz, 3JH7a–H1 = 2.1 Hz,
3JH7a–H7 = 8.3 Hz, and 3JH4a–H5 = 1.6 Hz perfectly matched the exper-
(GI50 >100
lM) against the same panel of cell lines used in this
study.6
2.4. Cell cycle disturbances
In addition to growth inhibition, we studied cell cycle phase dis-
tribution by flow cytometry to determine if cell growth inhibition
involved cell cycle changes. DNA polymerase inhibitors are known
to interfere with the cell cycle and cause arrest in G0/G1 phase.17 To
study the influence on the cell cycle, we selected HBL-100 and
SW1573 cells as representative examples. The cells were exposed
3
3
3
imental ones of JH4a–H7a = 6.1 Hz, JH7a–H1 = 2.6 Hz, and JH4a–H5
ꢁ
1 Hz, confirming our proposal.

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C. García et al. / Bioorg. Med. Chem. 18 (2010) 2515–2523
Figure 3. From left to right: Best energy structure found for 9b and superimposition of the 10 best energy structures found in the conformational search of 9b (DE = +6.5 kJ/
mol).
Table 1
Antiproliferative activity (GI50) against human solid tumor cellsa
HO
H
Cell line
9a
9b
O
H
OAc
A2780 (ovarian)
HBL-100 (breast)
HeLa (cervix)
SW1573 (lung)
T-47D (breast)
WiDr (colon)
1.24 ( 0.05)
0.38 ( 0.04)
1.68 ( 0.31)
0.43 ( 0.02)
1.62 ( 0.07)
1.13 ( 0.02)
1.59 ( 0.13)
0.50 ( 0.14)
1.86 ( 0.07)
0.59 ( 0.11)
1.46 ( 0.35)
1.26 ( 0.34)
15 ( 0.5)
16 ( 1.2)
n.d.b
18 ( 1.5)
16 ( 1.4)
16 ( 1.8)
a
Values are given in
lM
standard deviation and are means of three to five
experiments.
b
n.d. = not done.
to compound 9b for 24 h at four different doses (1, 2, 3, and 5 lM),
which were chosen based on the GI50 values of the drug.
The results of cell cycle distributions of samples collected from
control and treated cells are summarized in Figure 4. Overall, the
same effect is obtained for the cell cycle distribution of HBL-100
and SW1573 cells exposed to analog 9b. At the low doses 1 and
2 lM, an increase in the percentage of cells in the G0/G1 phase
was observed. The rise is concomitant with a decrease in the S
phase compartment and consistent with DNA polymerase inhibi-
Figure 4. Cell cycle phase distribution of untreated (control) SW1573 and HBL-100
cells, and cells treated with compound 9b for 24 h at 1, 2, 3, and 5 M drug dose.
l
tion. The exposure to the higher drug doses 3 and 5 lM induces cell
kill in a dose dependent manner, as shown by the appearance of a
sub-G1 peak. Based on the results of cell cycle distribution, we
speculate that the sub-G1 peak originates from cells arrested in
G0/G1 phase. The percentage of cells in both G0/G1 and sub-G1
phase represent in all drug treatments more than the amount of
untreated cells in G0/G1. This implies that compound-induced cell
death occurs for those cells arrested in G0/G1 phase.
exert their anticancer activity by inducing apoptosis. In the early
stages of apoptosis, the phosphatidyl choline residues of the cell
membrane are translocated from the inner to the outer membrane
surface. The residues can be labeled with Annexin V-FITC and de-
tected by flow cytometry.
To study the induction of apoptosis, we selected HeLa and WiDr
cells as representative examples. The cells were exposed to com-
pounds 9a-b for 24 h at 3 lM. Flow cytometry analysis of samples
2.5. Annexin V binding
labeled with Annexin V confirmed that both compounds were able
to induce apoptosis (Fig. 5).
Our next efforts were directed to determine if cell death was
occurring through apoptosis. Apoptosis is a highly regulated form
of cell death involving multiple signaling pathways that is trig-
gered when a cell has been damaged and cannot recover. Several
studies have shown that most, if not all, chemotherapeutic agents
2.6. Immunoblotting
In eukaryotic cells, progression of the cell cycle is controlled by
checkpoints. One of these checkpoints is cyclin D1, which acts as a

C. García et al. / Bioorg. Med. Chem. 18 (2010) 2515–2523
2519
Figure 5. Annexin V and PI staining of untreated cells (Control) and cells treated with compounds 9a–b for 24 h at 3
apoptotic cells are AnnexinV+ and PIꢀ, and late apoptotic cells are AnnexinV+ and PI+. Quantification of apoptosis showed the percentage of cells that were apoptotic.
l
M drug dose. Viable cells are AnnexinVꢀ and PIꢀ, early
regulator of G1/S-phase progression.18 Cyclin D1 levels begin to rise
early in G1 and continue to accumulate until the G1/S-phase
boundary when levels rapidly decline. Immunoblotting of HeLa
cells exposed to 9b reduced cyclin D1 levels in a time dependent
manner (Fig. 6). This result is consistent with the observed cell cy-
cle arrest on G1 (Fig. 4).
from CaH2 and Na/benzophenone, respectively, under N2 prior to
use. Other solvents or chemicals were purified by standard tech-
niques.19 Thin-layer chromatography was carried out on Merck
aluminium sheets coated with Silica Gel 60 F254. Plates were visu-
alized by use of UV light and/or phosphomolybdic acid 20wt %
solution in ethanol with heating. Anhydrous magnesium sulfate
was used for drying solutions. Flash chromatography was per-
formed on Silica Gel Merck grade 9385, 60 Å. Optical rotations
were determined for solutions in chloroform on a Perkin Elmer
343 polarimeter. NMR spectra were measured at 500, 400 or
300 MHz (1H NMR) and 75 MHZ (13C NMR), and chemical shifts
are reported relative to internal Me4Si (d = 0). IR spectra were re-
corded on a Bruker IFS 55 spectrometer model.
In summary, we have synthesized a bicyclic derivative by means
of a cyclization catalyzed by L-proline. The title compound repre-
sents a simplified scaffold of the aglycone framework of naturally
occurring iridoids. These compounds show remarkable biological
activity towards human cancer cell lines, including cell cycle arrest
and apoptosis induction. More experiments are ongoing to corrobo-
rate the scope of these simplified analogs as potential therapeutics
for the treatment of cancer, either alone or in combination.
3.1.1. (R)-3,7-Dimethyloct-6-enyl benzoate (4a)
3. Experimental
3.1. General
To a solution of (+)-b-citronellol (3a) (2.95 g, 18.87 mmol) in
dry CH2Cl2 (188 mL) under argon atmosphere were sequentially
added Et3N (3.9 mL, 28.3 mmol) and benzoyl chloride (2.6 mL,
22.65 mmol) at 0 °C. The reaction was stirred for 2 h, after which
time TLC showed that the starting material had disappeared. Then
saturated aqueous solution of NaCl was added and the resulting
mixture was extracted with CH2Cl2. The combined organic phases
were dried, filtered, and evaporated in vacuum. The residue was
purified by column chromatography on silica gel to give 4a
Reactions requiring anhydrous conditions were performed un-
der nitrogen. Dichloromethane and ethylic ether were distilled
(4.86 g, 99% yield) as a colorless oil: ½a D20
ꢂ
= +3.47 (c 3.8 in CHCl3);
1H NMR (300 MHz, CDCl3), d (ppm): 0.97 (d, J = 6.3 Hz, 3H), 1.20–
1.29 (m, 1H), 1.35–1.46 (m, 1H), 1.54–1.85 (m, 9H), 1.96–2.06
(m, 2H), 4.32–4.38 (m, 2H), 5.10 (dd, J = 6.9, 6.9 Hz, 1H), 7.40–
7.45 (m, 2H), 7.52–7.56 (m, 1H), 8.03–8.05 (m, 2H); 13C NMR
(75 MHz, CDCl3) d: 17.4 (q), 19.2 (q), 25.1 (t), 25.4 (q), 29.2 (d),
35.2 (t), 36.7 (t), 63.2 (t), 124.3 (d), 128.0 (d), 129.2 (d), 130.2 (s),
Figure 6. Western blot analysis of protein extracts from HeLa cells exposed to
compound 9b at 10 lM after 12 and 24 h of drug treatment.
131.1 (s), 132.5 (d), 166.4 (s); IR (film)
m
cmꢀ1 2962, 2958, 1721,

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C. García et al. / Bioorg. Med. Chem. 18 (2010) 2515–2523
1273, 1112, 711; MS (EI+) m/z (%): 138 (72) [MꢀBzOH]+, 123 (66),
123 (75), 109 (26), 105 (100), 95 (61); HRMS-EI m/z [MꢀBzOH]+
calcd for C10H18: 138.1409, found:138.1404.
NH4Cl, NaHCO3, and NaCl. The aqueous layers were extracted with
CH2Cl2, and the combined organic layers were dried over MgSO4
and concentrated in vacuum. Flash chromatography (Hexanes/
AcOEt = 9:1–6:4) afforded 1.13 g of 6a as oil (24% yield):
3.1.2. (S)-3,7-Dimethyloct-6-enyl benzoate (4b)
The procedure used above to obtain 4a from (+)-b-citronellol
(3a) was applied to (ꢀ)-b-citronellol (3b) on a 4.9 g (31.35 mmol)
½
a 2D0 = +16.5 (c 2.0 in CHCl3); 1H NMR (300 MHz, CDCl3), d (ppm):
ꢂ
1.00 (d, J = 6.5 Hz, 3H), 1.37 (ddd, J = 13.5, 9.3, 4.0 Hz, 1H), 1.59–
1.68 (m, 2H), 1.73–1.84 (m, 1H), 1.87–1.94 (m, 1H), 2.43 (br s,
1H), 3.70 (s, 3H), 4.27–4.43 (m, 3H), 6.03 (d, J = 15.6 Hz, 1H), 6.94
(dd, J = 15.6, 4.7 Hz, 1H), 7.33–7.43 (m, 2H), 7.50–7.55 (m, 1H),
7.99–8.01 (m, 2H); 13C NMR (75 MHz, CDCl3): d 18.8 (q), 26.1 (d),
35.8 (t), 43.3 (t), 51.4 (q), 62.8 (t), 68.5 (d), 119.2 (d), 128.1 (d),
129.3 (d), 129.9 (s), 132.7 (d), 150.2 (d), 166.5 (s), 166.8 (s); IR
scale, yielding 4b (8.08 g, 99% yield) as
a colorless oil:
½
a 2D0
ꢂ
= ꢀ3.19 (c 3.7 in CHCl3); 1H NMR (300 MHz, CDCl3), d (ppm):
0.97 (d, J = 6.4 Hz, 3H), 1.20–1.30 (m, 1H), 1.35–1.70 (m, 9H),
1.77–1.86 (m, 1H), 1.96–2.06 (m, 2H), 4.32–4.39 (m, 2H), 5.10
(dd, J = 7.0, 7.0 Hz, 1H), 7.40–7.45 (m, 2H), 7.52–7.57 (m, 1H),
8.02–8.05 (m, 2H); 13C NMR (75 MHz, CDCl3): d 17.4 (q), 19.2 (q),
25.1 (t), 25.4 (q), 29.3 (d), 35.2 (t), 36.7 (t), 63.2 (t), 124.3 (d),
128.0 (d), 129.2 (d), 130.2 (s), 131.1 (s), 132.5 (d), 166.4 ppm (s);
(film):
m
cmꢀ1 3490, 2957, 1718, 1452, 1276, 1115, 714; MS (EI+)
m/z (%): 274 (1) [MꢀCH3OH]+, 256 (2), 184 (3), 166 (9), 123 (20),
122 (10), 107 (13), 105 (100), 92 (33); HRMS-EI m/z [MꢀCH3OH]+
calcd for C16H18O4: 274.1205, found: 274.1210.
IR (film)
m
cmꢀ1 2962, 2920, 1721, 1274, 1112, 711; MS (EI+) m/z
(%): 261 (0.2) [M+1]+, 138 (63) [MꢀBzOH]+, 123 (66), 109 (23),
105 (100), 95 (56); HRMS-EI m/z [M]+ calcd for C17H24O2:
260.1779, found: 260.1776.
3.1.6. (E,3R,5R)-7-(Methoxycarbonyl)-5-hydroxy-3-methylhept-
6-enyl benzoate (6b)
The procedure used above to obtain 6a from 5a was applied to
5b on a 6.8 g (28.93 mmol) scale, yielding 6b (2.1 g, 24% yield) as
3.1.3. (R)-5-Formyl-3-methylpentyl benzoate (5a)
A solution of 4a (4.46 g, 17.15 mmol) and pyridine (2.1 mL,
25.72 mmol) in CH2Cl2/MeOH (77 mL/8.5 mL) was cooled to
ꢀ78 °C. Ozone was bubbled through the solution until a dark blue
color developed. At this time triphenylphosphine (5.0 g,
18.86 mmol) was added and the resulting mixture was stirred for
3 h allowing it to reach 0 °C. After concentration, flash chromatog-
raphy (Hexanes/AcOEt = 95:5) afforded the title compound as a
oil: ½a 2D0
ꢂ
= ꢀ10.69 (c 2.1 in CHCl3); 1H NMR (300 MHz, CDCl3), d
(ppm): 1.02 (d, J = 6.6 Hz, 3H), 1.53–1.61 (m, 3H), 1.84–1.91 (m,
2H), 2.14 (br s, 1H), 3.71 (s, 3H), 4.29–4.43 (m, 3H), 6.0 (dd,
J = 15.6, 1.5 Hz, 1H), 6.94 (dd, J = 15.6, 5.1 Hz, 1H), 7.39–7.44 (m,
2H), 7.51–7.57 (m, 1H), 7.99–8.02 (m, 2H); 13C NMR (75 MHz,
CDCl3): d 19.8 (q), 26.4 (d), 34.7 (t), 43.4 (t), 51.4 (q), 62.8 (t),
68.9 (d), 119.5 (d), 128.1 (d), 129.2 (d), 130.0 (s), 132.7 (d), 150.2
clear, colorless oil in 95% yield (3.8 g): ½a D20
ꢂ
= +3.13 (c 4.1 in CHCl3);
(d), 166.4 (s), 166.6 (s); IR (film): m
cmꢀ1 3477, 2956, 2927, 1718,
1H NMR (300 MHz, CDCl3), d (ppm): 0.93 (d, J = 6.1 Hz, 3H), 1.41–
1.81 (m, 5H), 2.31–2.49 (m, 2H), 4.33 (ddd, J = 6.3, 6.3, 3.1 Hz,
2H), 7.36–7.41 (m, 2H), 7.48–7.53 (m, 1H), 7.97–8.00 (m, 2H),
9.72 (s, 1H); 13C NMR (75 MHz, CDCl3): d 18.8 (q), 28.4 (t), 29.3
(d), 35.0 (t), 41.2 (t), 62.8 (t), 128.1 (d), 129.2 (d), 130.0 (s), 132.5
1452, 1276, 1115, 714; MS (EI+) m/z (%): 274 (4) [MꢀCH3OH]+,
184 (4) [MꢀBzOH]+, 152 (12), 123 (34), 122 (10), 105 (100), 92
(16); HRMS-EI m/z [MꢀBzOH]+ calcd for C10H16O3: 184.1099,
found: 184.1098.
(d), 166.3 (s), 202.1 (d); IR (film):
m
cmꢀ1 2960, 2930, 2361, 2338,
3.1.7. (E,3S,5R)-5-(tert-Butyl-diphenyl-silanyloxy)-7-methoxy-
carbonyl-3-methyl-6-heptenyl benzoate (7a)
To a stirred solution of the alcohol 6a (1.0 g, 3.26 mmol) in dry
CH2Cl2 (32 mL) under nitrogen were added imidazole (673 mg,
1719, 1275, 1112, 713; MS (EI+) m/z (%) 233 (0.2) [Mꢀ1]+, 128
(95), 123 (32), 122 (10), 110 (42), 105 (100); HRMS-EI m/z
[Mꢀ1]+ calcd for C14H17O3: 233.1178, found: 233.1172.
9.79 mmol)
and
tert-butylchlorodiphenylsilane
(1.3 mL,
3.1.4. (S)-5-Formyl-3-methylpentyl benzoate (5b)
4.89 mmol) at 0 °C. The reaction was allowed to warm to room
temperature and was stirred overnight. Then it was poured into
H2O and extracted with CH2Cl2. The combined organic phases were
washed with saturated solutions of NaCl, dried, filtered, and con-
centrated. The crude obtained was purified by flash chromatogra-
The procedure used above to obtain 5a from 4a was applied to
4b on a 8.0 g (30.72 mmol) scale, yielding 5b (6.9 g, 96% yield) as a
colorless oil: ½a D20
ꢂ
= ꢀ3.09 (c 4.1 in CHCl3); 1H NMR (300 MHz,
CDCl3), d (ppm): 0.93 (d, J = 6.1 Hz, 3H), 1.44–1.78 (m, 5H), 2.31–
2.43 (m, 2H), 4.29–4.34 (m, 2H), 7.35–7.40 (m, 2H), 7.46–7.51
(m, 1H), 7.97–8.00 (m, 2H), 9.71 (s, 1H); 13C NMR (75 MHz, CDCl3):
d 18.8 (q), 28.4 (t), 29.3 (d), 35.0 (t), 41.2 (t), 62.8 (t), 128.0 (d),
phy yielding 7b (1.67 g, 94% yield) as an oil: ½a D20
ꢂ
= +17.82 (c 1.7
in CHCl3); 1H NMR (300 MHz, CDCl3), d (ppm): 0.74 (d, J = 6.1 Hz,
3H), 1.05 (s, 9H), 1.25–1.34 (m, 2H), 1.58–1.69 (m, 3H), 3.68 (s,
3H), 4.19 (dd, J = 6.3, 6.3 Hz, 2H), 4.35–4.37 (m, 1H), 5.83 (d,
J = 15.6 Hz, 1H), 6.88 (dd, J = 15.6, 5.8 Hz, 1H), 7.30–7.44 (m, 7H),
7.52–7.72 (m, 6H), 7.97–8.00 (m, 2H); 13C NMR (75 MHz, CDCl3):
d 19.1 (s), 19.4 (q), 25.8 (d), 26.3 (q), 26.8 (q), 35.3 (t), 44.7 (t),
51.3 (d), 62.7 (t), 70.8 (d), 119.6 (d), 127.3 (d), 127.4 (d), 128.1
(d), 129.3 (d), 129.5 (d), 129.6 (d), 130.1 (s), 132.6 (d), 133.1 (s),
133.4 (s), 134.6 (d), 135.6 (d), 135.7 (d), 150.1 (d), 166.3 (s),
129.2 (d), 130.0 (s), 132.6 (d), 166.3 (s), 202.2 (d); IR (film):
m
cmꢀ1 2960, 2361, 1716, 1276, 1112, 713; MS (EI+) m/z (%) 233
(0.2) [Mꢀ1]+, 128 (86), 123 (32), 105 (100); HRMS-EI m/z [Mꢀ1]+
calcd for C14H17O3: 233.1178, found: 233.1165.
3.1.5. (E,3S,5R)-7-(Methoxycarbonyl)-5-hydroxy-3-methylhept-
6-enyl benzoate (6a)
L-Proline (0.71 g, 6.16 mmol) was added to a stirring solution of
166.6 (s); IR (film): m
cmꢀ1 3508, 2956, 2932, 2857, 1721, 1274,
5a (3.6 g, 15.40 mmol) and nitrosobenzene (1.7 g, 15.40 mmol) in
DMSO (62 mL). After 0.5 h the solution became a bright orange,
at which time it was cooled to ꢀ15 °C. A premixed solution of
methyl diethyl phosphonoacetate (7.5 mL, 46.22 mmol), 1,8-diaza-
bicyclo[5.4.0]undec-7-ene (7 mL, 46.22 mmol) and lithium chlo-
ride (1.96 g, 46.22 mmol) in CH3CN (62 mL) was added over
5 min via cannula. After 15 min the solution was diluted with
MeOH (204 mL) and NH4Cl (2.47 g, 46.22 mmol) was added. The
resulting mixture was allowed to warm to room temperature and
stand for 2 d. At this time the solution was diluted with Et2O
(1000 mL), and washed successively with saturated solutions of
1110, 706; MS (EI+) m/z (%): 514 (0.3) [Mꢀ2Me]+, 513 (0.8), 488
(14), 487 (39) [MꢀtBu]+, 304 (25), 303 (100), 243 (37), 199 (19),
135 (14); HRMS-EI m/z [MꢀtBu]+ calcd for C29H31O5Si: 487.1941,
found: 487.1949.
3.1.8. (E,3R,5R)-5-(tert-Butyl-diphenyl-silanyloxy)-7-methoxy-
carbonyl-3-methyl-6-heptenyl benzoate (7b)
The procedure used above to obtain 7a from 6a was applied to
6b on a 2.0 g (6.52 mmol) scale, yielding 7b (3.34 g, 94% yield) as
oil: ½a 2D0
ꢂ
= +9.26 (c 1.4 in CHCl3); 1H NMR (300 MHz, CDCl3), d
(ppm): 0.68 (d, J = 4.7 Hz, 3H), 1.06 (s, 9H), 1.39–1.48 (m, 2H),

C. García et al. / Bioorg. Med. Chem. 18 (2010) 2515–2523
2521
1.54–1.61 (m, 3H), 3.68 (s, 3H), 4.19 (d, J = 6.8 Hz, 2H), 4.37 (br s,
1H), 5.88 (d, J = 15.6 Hz, 1H), 6.89 (dd, J = 15.6, 5.5 Hz, 1H), 7.34–
7.44 (m, 8H), 7.52–7.72 (m, 5H), 7.97–7.99 (m, 2H); 13C NMR
(75 MHz, CDCl3): d 18.7 (s), 25.8 (d), 26.3 (q), 26.7 (q), 35.6 (t),
44.2 (t), 51.3 (q), 62.7 (t), 70.7 (d), 119.7 (d), 127.3 (d), 127.4 (d),
127.4 (d), 128.1 (d), 129.2 (d), 129.4 (d), 129.5 (d), 129.6 (d),
130.0 (s), 132.6 (d), 133.0 (s), 133.4 (s), 134.5 (d), 135.6 (d),
to 40 °C and stirred at this temperature for 60 h. The reaction was
then cooled to 0 °C, and acetic anhydride (0.17 mL, 1.79 mmol) was
added, followed by pyridine (0.07 mL, 0.91 mmol) and DMAP
(2 mg, 0.01 mmol). After 15 min the reaction was diluted with
Et2O and washed with saturated solutions of NH4Cl, NaHCO3, and
NaCl. The aqueous layers were then extracted with CH2Cl2 and
Et2O. The combined organic layers were dried over MgSO4 and con-
centrated in vacuum. Flash chromatography (Hexanes/AcOEt =
98:2) afforded 9a as a clear, colorless oil in 15% yield (13 mg):
149.7 (d), 166.3 (s), 166.6 (s); IR (film):
m
cmꢀ1 3476, 2956, 2931,
1721, 1275, 1111, 705; MS (EI+) m/z (%): 487 (41) [MꢀtBu]+, 304
(24), 303 (98), 243 (36), 135 (17), 105 (100); HRMS-EI m/z
[MꢀtBu]+ calcd for C29H31O5Si: 487.1941, found: 487.1935.
½
a 2D0 = +80.44 (c 1.2 in CHCl3); 1H NMR (500 MHz, CDCl3), d
ꢂ
(ppm): 0.85 (d, J = 6.8 Hz, 3H, CHCH3), 1.09 (s, 9H, tBu), 1.67
(ddd, J = 7.3, 7.3, 4.1 Hz, 1H, H7a), 1.85–1.90 (m, 2H, 2 ꢃ H6), 2.10
(br s, 4H, H7, OCOCH3), 2.58 (ddd, J = 7.6, 5.2, 2.5 Hz, 1H, H4a),
4.25 (ddd, J = 6.6, 6.6, 1.8 Hz, 1H, H5), 5.04 (dd, J = 6.4, 2.6 Hz, 1H,
H4), 6.07 (d, J = 4.2 Hz, 1H, H1), 6.33 (dd, J = 6.3, 2.2 Hz, 1H, H3),
7.38–7.45 (m, 6H, Ar), 7.52–7.68 (m, 4H, Ar); 13C NMR (75 MHz,
CDCl3): d 19.0 (s, tBu), 20.9 (q, OCOCH3), 21.0 (q, CHCH3), 26.6 (q,
tBu), 31.1 (d, C7), 38.3 (d, C4a), 40.4 (t, C6), 45.5 (d, C7a), 74.8 (d,
C5), 91.1 (d, C1), 100.8 (d, C4), 127.3 (d, Ar), 127.3 (d, Ar), 129.4
(d, Ar), 133.6 (s, Ar), 133.9 (s, Ar), 135.5 (d, Ar), 135.6 (d, Ar),
3.1.9. (E,4R,6S)-6-Methyl-4-(tert-butyl-diphenyl-silanyloxy)-2-
octene-1,8-diol (8a)
To a cooled solution of the compound 7a (358 mg, 0.65 mmol)
in dry Et2O (6.5 mL) under argon atmosphere was added dropwise
DIBAL-H (3.9 mL 1 M in cyclohexane, 3.94 mmol) at ꢀ78 °C. The
mixture was stirred at ꢀ78 °C until no starting material was pre-
sented by TLC. The reaction was quenched by the addition of
0.1 mL of H2O and then, to the same flask, was added MgSO4.
The gelatinous solution was filtered through a pad of Celite and
washed with Et2O. The solvent was removed under reduced pres-
sure, and the residue was purified by flash chromatography on a
silica gel column yielding 8a (122 mg, 45% yield) as a colorless
140.5 (d, C3), 169.8 (s, OCOCH3); IR (film):
m
cmꢀ1 3446, 2928,
2856, 1750, 1650, 1211, 1110, 703, 505 cmꢀ1; MS (EI+) m/z (%):
393 (4) [MꢀtBu]+, 333 (40) [MꢀtBuꢀAcOH]+, 319 (16), 245 (10),
241 (26), 199 (100), 181 (13); HRMS-EI m/z [MꢀtBuꢀAcOH]+ calcd
for C21H21O2Si: 333.1311, found: 333.1297.
oil: ½a 2D0
ꢂ
= +13.05 (c 1.6 in CHCl3); 1H NMR (300 MHz, CDCl3), d
(ppm): 0.77 (d, J = 6.5 Hz, 3H), 1.04 (s, 9H), 1.17–1.32 (m, 2H),
1.36–1.49 (m, 2H), 1.53–1.78 (m, 3H), 3.54 (m, 2H), 3.86 (d,
J = 5.5 Hz, 2H), 4.23 (dd, J = 13.2, 6.6 Hz, 1H), 5.37 (ddd, J = 15.4,
11.0, 5.5 Hz, 1H), 5.55 (dd, J = 15.4, 7.2 Hz, 1H), 7.32–7.45 (m,
6H), 7.63–7.69 (m, 4H); 13C NMR (75 MHz, CDCl3): d 19.0 (s),
19.8 (q), 25.2 (d), 26.8 (q), 39.48 (t), 45.1 (t), 60.5 (t), 62.7 (t),
72.0 (d), 127.0 (d), 127.2 (d), 129.1 (d), 129.2 (d), 129.4 (d), 133.9
3.1.12. (1S,4aR,5R,7S,7aR)-5-(tert-Butyl-diphenyl-silanyloxy)-7-
methyl-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-1-yl acetate
(9b)
The procedure used above to obtain 9a from 8a was applied to
8b on a 247 mg (0.59 mmol) scale, yielding 9b (40 mg, 15% yield)
as
a
colorless oil:
½
a 2D0
ꢂ
= ꢀ76.45 (c 1.1 in CHCl3); 1H NMR
(s), 134.2 (s), 134.5 (d), 135.7 (d), 135.8 (d); IR (film):
m
cmꢀ1
(500 MHz, CDCl3), d (ppm): 1.00 (s, 9H, tBu), 1.14 (d, J = 6.7 Hz,
3H, CHCH3), 1.91 (dddd, J = 13.6, 6.8, 6.8, 2.2 Hz, 1H, H7), 2.04–
2.15 (m, 6H, H7a, 2xH6, OCOCH3), 2.62 (d, J = 6.0 Hz, 1H, H4a), 4.00
(dd, J = 5.4, 3.1 Hz, 1H, H5), 4.34 (ddd, J = 6.3, 2.5, 1.1 Hz, 1H, H4),
6.02 (dd, J = 6.3, 2.3 Hz, 1H, H3), 6.09 (d, J = 2.6 Hz, 1H, H1), 7.38–
7.47 (m, 6H, Ar), 7.67–7.69 (m, 4H, Ar); 13C NMR (75 MHz, CDCl3):
d 18.8 (s, tBu), 20.4 (q, CHCH3), 21.0 (q, OCOCH3), 26.6 (q, tBu), 32.8
(d, C7), 41.2 (d, C4a), 42.1 (t, C6), 45.8 (d, C7a), 79.1 (d, C5), 90.1 (d,
C1), 103.2 (d, C4), 127.3 (d, Ar), 129.4 (d, Ar), 133.7 (s, Ar), 133.9
(s, Ar), 135.4 (d, Ar), 135.5 (d, Ar), 139.4 (d, C3), 170.1 (s, OCOCH3);
3382, 2931, 1645, 1427, 1109, 1059, 740, 703; MS (EI+) m/z (%)
337 (32) [MꢀtBuꢀH2O]+, 325 (3), 200 (19), 199 (100), 197 (11),
139 (18); HRMS-EI m/z [MꢀtBuꢀH2O]+ calcd for C21H25O2Si:
337.1624, found: 337.1615.
3.1.10. (E,4R,6R)-6-Methyl-4-(tert-butyl-diphenyl-silanyloxy)-
2-octene-1,8-diol (8b)
The procedure used above to obtain 8a from 7a was applied to
7b on a 1.6 g (2.99 mmol) scale, yielding 8b (545 mg, 45% yield) as
a colorless oil: ½a D20
ꢂ
= +0.27 (c 3.6 in CHCl3); 1H NMR (300 MHz,
IR (film): m
cmꢀ1 3450, 2957, 2930, 2857, 1750, 1654, 1215, 1110,
CDCl3), d (ppm): 0.67 (d, J = 6.2 Hz, 3H), 1.05 (s, 9H), 1.20–1.29
(m, 1H), 1.32–1.47 (m, 4H), 1.68 (br s, 2H), 3.56 (ddd, J = 17.0,
10.6, 6.5 Hz, 2H), 3.89 (d, J = 5.4 Hz, 2H), 4.19 (dd, J = 13.0, 7.3 Hz,
1H), 5.38 (ddd, J = 15.4, 11.0, 5.4 Hz, 1H), 5.55 (dd, J = 15.4,
7.3 Hz, 1H), 7.32–7.43 (m, 6H), 7.63–7.68 (m, 4H); 13C NMR
(75 MHz, CDCl3): d 19.0 (s), 19.4 (q), 25.2 (d), 26.7 (q), 39.8 (t),
45.0 (t), 60.3 (t), 62.6 (t), 72.3 (d), 127.1 (d), 127.2 (d), 129.2 (d),
129.3 (d), 129.4 (d), 134.0 (s), 134.1 (d), 134.2 (s), 135.7 (d),
703, 507; MS (EI+) m/z (%) 393 (4) [MꢀtBu]+, 351 (3)
[MꢀtBuꢀAc+1]+, 334 (10), 333 (37) [MꢀtBuꢀAcOH]+, 319 (16),
315 (11), 255 (10), 199 (100); HRMS-EI m/z [MꢀtBuꢀAcOH]+ calcd
for C21H21O2Si: 333.1311, found: 333.1287.
3.2. Biological tests
All starting materials were commercially available research-
grade chemicals and used without further purification. RPMI
1640 medium was purchased from Flow Laboratories (Irvine,
UK), fetal calf serum (FCS) was from Gibco (Grand Island, NY), tri-
chloroacetic acid (TCA) and glutamine were from Merck (Darms-
tadt, Germany), and penicillin G, streptomycin, dimethyl
sulfoxide (DMSO), and sulforhodamine B (SRB) were from Sigma
(St Louis, MO).
135.8 (d); IR (film):
m
cmꢀ1 3391, 2930, 2360, 1646, 1427, 1109,
822, 703; MS (EI+) m/z (%): 337 (14) [MꢀtBuꢀH2O]+, 335 (3), 323
(3), 239 (1), 200 (17), 199 (100), 139 (11); HRMS-EI m/z
[MꢀtBuꢀH2O]+ calcd for C21H25O2Si: 337.1624, found: 337.1622.
3.1.11. (1R,4aS,5R,7R,7aS)-5-(tert-Butyl-diphenyl-silanyloxy)-7-
methyl-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-1-yl acetate
(9a)
Dess–Martin periodinane (194 mg, 0.46 mmol) was added to a
stirred solution of 8a (82 mg, 0.20 mmol) in CH2Cl2 (2 mL). After
40 min the reaction was concentrated and extracted with pentane.
The combined organics were concentrated in vacuum, providing
the corresponding dialdehyde, which was immediately redissolved
3.2.1. Cells, culture and plating
The human solid tumor cell lines A2780, HBL-100, HeLa,
SW1573, T-47D, and WiDr were used in this study. These cell lines
were a kind gift from Professor Godefridus J. Peters (VU Medical
Center, Amsterdam, The Netherlands). Cells were maintained in
25 cm2 culture flasks in RPMI 1640 supplemented with 5% heat
in DMSO (5 mL).
L-proline (7 mg, 0.06 mmol) was added to this
stirred solution in one portion. After 5 h, the reaction was warmed
inactivated fetal calf serum and 2 mM -glutamine in a 37 °C, 5%
L

2522
C. García et al. / Bioorg. Med. Chem. 18 (2010) 2515–2523
CO2, 95% humidified air incubator. Exponentially growing cells
were trypsinized and resuspended in antibiotic containing med-
ium (100 units penicillin G and 0.1 mg of streptomycin per mL).
Single cell suspensions displaying >97% viability by trypan blue
dye exclusion were subsequently counted. After counting, dilu-
tions were made to give the appropriate cell densities for inocula-
tion onto 96-well microtiter plates. Cells were inoculated in a
volume of 100 lL per well at densities of 15,000 (WiDr, T-47D
and HeLa) and 10,000 (A2780, SW1573 and HBL-100) cells per
well, based on their doubling times.
Annexin staining protocol was performed according to the man-
ufacturer’s protocol (Annexin V-FITC apoptosis detection Kit I, Bec-
ton Dickinson, San José, CA), with minor modifications. Cells were
stained by the addition of both 5
solution. Samples were gently vortexed and incubated for 15 min
at rt. in the dark. Then, 400 L of 1x binding buffer was added to
lL Annexin V-FITC and 5 lL PI
l
each tube. The samples were analyzed by flow cytometry using Cell
Quest Pro software (Becton Dickinson, San José, CA). Typically,
10,000 events were collected using excitation/emission wave-
lengths of 488/525 and 488/675 nm for Annexin V and PI, respec-
tively. Results were processed using WinMDI 2.8 free software.
3.2.2. Antiproliferative tests
Chemosensititvity tests were performed using the SRB assay of
the NCI with slight modifications. Briefly, pure compounds were
initially dissolved in DMSO at 400 times the desired final maxi-
mum test concentration. Control cells were exposed to an equiva-
lent concentration of DMSO (0.25% v/v, negative control). Each
agent was tested in triplicates at different dilutions in the range
3.2.5. Immunoblotting
Protein extracts were prepared using a lysis buffer freshly sup-
plemented with protease and phosphatase inhibitors [50 mM Tris
(pH 8.0), 250 mM NaCl, 1% NP-40, 0.1% SDS, 5 mM EDTA, 2 mM
Na3VO4, 10 mM Na2P2O7, 10 mM NaF, 10
lg/mL aprotinin, 10 lg/
mL leupeptin, 0.5 g/mL pepstatin, and 1 mM PMSF]. Cells were
l
1–100
Drug incubation times were 48 h, after which time cells were pre-
cipitated with 25 L ice-cold 50% (w/v) trichloroacetic acid and
l
M. The drug treatment was started on day 1 after plating.
incubated on ice for 10 min and then cell debris was spun down
at 10,000g for 10 min. Proteins were separated by SDS–PAGE and
electrotransferred to ImmunoBlot PVDF membrane (BioRad). Cy-
clin D1 (Santa Cruz, USA) primary antibody was used. After being
washed with TBST, membranes were incubated with horseradish
peroxidase-conjugated secondary antibodies for 30 min and bands
were visualized by a luminal-based detection system with p-iodo-
phenol enhancement.
l
fixed for 60 min at 4 °C. Then the SRB assay was performed. The
optical density (OD) of each well was measured at 492 nm, using
BioTek’s PowerWave XS Absorbance Microplate Reader. Values
were corrected for background OD from wells only containing
medium. The percentage growth (PG) was calculated with respect
to untreated control cells (C) at each of the drug concentration lev-
els based on the difference in OD at the start (T0) and end of drug
exposure (T), according to NCI formulas. Therefore, if T is greater
than or equal to T0 the calculation is 100 ꢃ [(TꢀT0)/(CꢀT0)]. If T
is less than T0 denoting cell killing the calculation is
100 ꢃ [(TꢀT0)/(T0)]. The effect is defined as percentage of growth,
where 50% growth inhibition (GI50) represents the concentration
at which PG is +50. With these calculations a PG value of 0 corre-
sponds to the amount of cells present at the start of drug exposure,
while negative PG values denote net cell kill.
Acknowledgments
This research was supported by the Spanish MEC co-financed by
the European Regional Development Fund (CTQ2008-06806-C02-
01/BQU), and the Canary Islands ACIISI (PI 2007/021) co-financed
by the European FEDER; the Spanish MSC (RTICC RD06/0020/1046
and RD06/0020/0041) and FUNCIS (REDESFAC PI 01/06 and 35/
06); the Argentinian UNSL (22/Q505), CONICET (PIP 6228) and AN-
PCyT (PICT-10714, 9759). C.R.P. and C.E.T. belong to CIC-CONICET.
L.G.L. thanks the Spanish MSC-FIS for a postdoctoral contract. C.G.
and J.M.P. thank the Spanish MEC-FSE for Ramón y Cajal contracts.
3.2.3. Cell cycle analysis
Cells were seeded in a six well plates at a density of 2.5–5 ꢃ 105
cells/well. After 24 h the products were added to the respective
well and incubated for an additional period of 24 h. Cells were
trypsinized, harvested, transferred to test tubes (12 ꢃ 75 mm)
and centrifuged at 1,500 rpm for 10 minutes at 5 °C. The superna-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.02.044.
tant was discarded and the cell pellets were resuspended in 200 lL
of cold PBS and fixed by the addition of 1 mL ice-cold 70% ethanol.
Fixed cells were incubated overnight at ꢀ20 °C after which time
was centrifuged at 1,500 rpm for 10 minutes. The cell pellets were
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