Synthesis and screening of novel vitamin e derivatives for anticancer functions

Article abstract of DOI:10.1016/j.ejmech.2012.09.045

α-TEA, RRR-α-tocopherol ether linked acetic acid, exhibits potent anticancer actions in vitro and in vivo; whereas, the parent molecule has no anticancer activity. In this study, we incorporated fluorine at the chroman head and/or ether linkage between the chroman head and phytyl tail of α-TEA as well as RRR-α-tocopherol to synthesize 6 vitamin E derivatives, and evaluated the anticancer actions in vitro for ability to induce cell death by apoptosis of human MCF-7 and MDA-MB-231 breast cancer cell lines and mouse mammary cancer cell line 66cl-4GFP. All derivatives, with the exception of compound 12, exhibited anticancer properties. The modified α-TEA ether-type phytyl group exhibited the highest pro-apoptotic activity in comparison with α-TEA as well as other vitamin E derivatives.

Full text of DOI:10.1016/j.ejmech.2012.09.045

European Journal of Medicinal Chemistry 58 (2012) 72e83
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and screening of novel vitamin E derivatives for anticancer functions
,
,
,
Wenbin Chen a 1, Sook Kyung Park a 2, Weiping Yu a, Ailian Xiong b, Bob G. Sanders a, Kimberly Kline b
*
a School of Biological Sciences/C0900, University of Texas at Austin, Austin 78712, TX, USA
b Department of Nutritional Sciences/A2703, University of Texas at Austin, Austin 78712, TX, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2012
Received in revised form
24 September 2012
Accepted 27 September 2012
Available online 4 October 2012
a
-TEA, RRR-
whereas, the parent molecule has no anticancer activity. In this study, we incorporated fluorine at the
chroman head and/or ether linkage between the chroman head and phytyl tail of -TEA as well as RRR-a-
a-tocopherol ether linked acetic acid, exhibits potent anticancer actions in vitro and in vivo;
a
tocopherol to synthesize 6 vitamin E derivatives, and evaluated the anticancer actions in vitro for ability
to induce cell death by apoptosis of human MCF-7 and MDA-MB-231 breast cancer cell lines and mouse
mammary cancer cell line 66cl-4GFP. All derivatives, with the exception of compound 12, exhibited
anticancer properties. The modified
activity in comparison with -TEA as well as other vitamin E derivatives.
Ó 2012 Elsevier Masson SAS. All rights reserved.
a-TEA ether-type phytyl group exhibited the highest pro-apoptotic
Keywords:
a
a
-TEA
Vitamin E derivative
Chemical synthesis
Anticancer activity
1. Introduction
effectiveness of chemotherapy, highlighting an urgent need for
development of potent anticancer drugs with reduced toxicity as
new treatment strategies. In recent years, selected tocopherol and
tocotrienol forms as well as metabolites [5e7] and synthetic
derivatives [8e11] have been reported to have antitumor and anti-
Vitamin E is a general term that describes a family of eight
chemically related compounds that are subdivided into two
subgroups called tocopherols and tocotrienols, as well as synthetic
vitamin E forms [1]. Tocopherols have a basic chemical structure
characterized by a long phytyl chain attached at the 2-position of
a chromane ring, while tocotrienols differ from that of tocopherols
by the presence of three E (trans) double bonds in the phytyl tail
inflammatory activities. Of these compounds, the monoester of
a-
tocopherol, -tocopheryl succinate, a representative vitamin E
a
analog, has been demonstrated to exhibit anticancer properties in
several cancer models and low toxicity to normal cells [12,13]. We
[2,3]. Moreover, the tocopherols and the tocotrienols have
and forms, named on the basis of the number and position of the
methyl groups on the chromanol ring. Vitamin E form RRR-
tocopherol has traditionally been recognized for its free radical-
scavenging antioxidant properties that are important for protect-
ing polyunsaturated fats from peroxidation. The antioxidant
property of vitamin E is exerted through the phenolic hydroxyl
group, which readily donates its hydrogen atom (H) to the lipid
peroxyl radical, resulting in the formation of a stable lipid
species [4].
a
,
b
,
g
,
developed another vitamin E derivative,
a-tocopherol ether-linked
d
acetic acid ( -TEA), that exhibits potent anticancer activity in
a
a
-
a wide variety of epithelial cancer cell types, including breast,
prostate, lung, colon, ovarian, cervical, and endometrial in cell
culture, and significantly reduces tumor burden and metastasis in
a syngeneic mouse mammary tumor model, as well as in xenografts
of human breast cancer cells [14e21]. Studies also show that a-TEA,
in combination with the cyclooxygenase-2 inhibitor celecoxib and
the chemotherapeutic drug 9-nitro-camptothecin, significantly
decreases tumor burden and metastasis in comparison to single
Cancer remains a leading cause of deaths worldwide, and
despite intense studies the incidence of cancer remains high.
Toxicity and resistance to standard drug treatments limit the
agents [22]. Dietary administration of a-TEA has been shown to
significantly reduce tumor volume and lung metastasis in a highly
metastatic murine syngeneic model, and to suppress tumor growth
and multiplicity of spontaneous murine mammary cancer [20,21].
Chemical modifications of vitamin E, to generate new deriva-
tives can in principle occur at the following distinguishable regions
of the molecule (Fig. 1): (I) side chain length and saturation [23e
25], (II) the position and geometry (E or Z) of the double bonds
[24], (III) heteroatom member of the saturated ring [26e28], (IV)
* Corresponding author. Tel.: þ1 512 471 8911; fax: þ1 512 232 7040.
E-mail address: k.kline@mail.utexas.edu (K. Kline).
1
Present address: The State Key Laboratory of Elemento-Organic Chemistry,
Nankai University, Tianjin 300071, PR China.
2
Present address: CJ Cheil Jedang Corporation/Pharmaceutical Research Center,
South Korea.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.045

W. Chen et al. / European Journal of Medicinal Chemistry 58 (2012) 72e83
73
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of fluorinated vitamin E analogs
Fluorine (F) is a small atom with high electronegativity [36] and
the carbonefluorine bond (CeF) is often used in place of strategic
carbonehydrogen bonds (CeH) in drug design strategies, since F is
the smallest atom that can replace H [37]. Recent examination of
structureeactivity relationships during drug discovery and lead
compound optimization have shown that fluorinated compounds
have advantageous properties in the design of small molecules
[36,38]. For example, introduction of fluorine atoms can increase
metabolic stability of a compound by blocking the metabolically
labile site of the liver enzymes, in particular the P450 cytochromes;
replacing H with F can lead to a change in the molecular confor-
mation which may be preferable; and fluorinated compounds can
have a significant impact on increasing binding affinity and selec-
tivity of proteineligand complexes either directly or indirectly by
increasing the lipophilicity in pharmaceuticals [36,38]. Since there
are limited reports of vitamin E compounds containing fluorine
[39], we introduced a fluorine atom on the benzene ring instead of
a methyl group to synthesize the title compound 14 in an effort to
enhance anticancer properties. The synthetic pathways to generate
title compound 14 are depicted in Scheme 1.
Fig. 1. Modification positions of vitamin E.
numbers of methyl substitutions on the benzene ring [23,29], and
(V) esterification and amidation of hydroxyl on the benzene ring
[23,24,29e31]. Esterification of hydroxyl on the benzene ring
enhances water solubility and potency [32e34]. Amides derived
a- and d-tocopheramines have been reported to improve
anticancer activity [31,32,35].
In this study, six novel tocopherol-based derivatives were
synthesized and shown to exhibit anticancer properties when
tested using human MCF-7 and MDA-MB-231 breast cancer cell
lines as well as mouse mammary cancer cell line 66cl-4GFP. Data
are presented in two sections for clarity: synthesis of new
tocopherol derivatives and testing for anticancer properties.
from
Scheme 1. Synthesis routes to compound 14.

74
W. Chen et al. / European Journal of Medicinal Chemistry 58 (2012) 72e83
To obtain the fluorinated vitamin E analog 14, the compound 3
2.1.2. Synthesis of ether-type phytyl group-modified vitamin E
derivatives
In our group, we found
a wide variety of epithelial cancer cell types, in an effort to enhance
the anticancer properties of RRR- -tocopherol derivative and find
a better lead tocopherol derivative, we introduced an oxygen atom
in the side chain of -TEA to obtain compounds 22a and 22b
(Scheme 2). This change may improve the molecule’s hydrophilicity
which affect bioavailability, and then enhance anticancer activities.
Trolox methyl ester 16a and 16b are readily prepared from the
commercial free acid via methyl esterification. The intermediates 18
were obtained according to the identical procedure. In order to
generate the ether side chain molecule 22 series, 18a,b were treated
with bromo-3,7,11-trimethyldodecane 9 in the presence of NaH in
THF under reflux to afford the corresponding 19a,b in 62e76% yield,
which were subjected to hydrogen over palladium catalyst to give
the de-protected hydroxyl group 20a,b, and then followed the above
procedure to afford the target compounds 22a,b in good yields.
is a key intermediate, which can be generated by the hetero-
DielseAlder reaction of methacrylate with in situ-generated o-
quinone methides produced from the reaction of para-
formaldehyde, dibutylamine and acetic acid [40]. This procedure
avoids the use of the high-pressure conditions used to prepare
Trolox methyl ester patented by Kazuo [41]. o-Fluorophenol 1 was
treated with potassium persulfate in sodium hydroxide and then
hydrolyzed to give the fluorohydroquinone 2 in 52% yield in two
steps via an Elbs oxidation [42,43]. The hetero-DielseAlder reac-
tion of fluorohydroquinone 2, methyl methacrylate and para-
formaldehyde under reflux conditions in the presence of
dibutylamine and acetic acid should have three different products
3a, 3b and 3c (Fig. 2), but we only isolated one product 3a, where
the fluorine atom is located at 6-position on the benzene ring
(Fig. 2). The DielseAlder reaction of o-quinone methide and
methyl methacrylate provided the ortho regioisomer selectively
[44], and the structure of compound 3a was also confirmed by
single crystal X-ray diffraction.
a
-TEA is a potent inducer of apoptosis in
a
a
The hydroxyl group at the 6-position on chroman head was
protected by benzyl group to give the compound 4 which were
reduced by LiAlH4 to obtain the 6-fluorochroman-2-methol, 5, in
95% yield. The desired intermediate 6 was afforded by Desse
Martin oxidization of 5 in 90% yield. The phytyl side chain was
appended via a Wittig coupling with 15-carbon phosphonium
bromide 10 [45] in the presence of lithium hexamethyldisilyla-
mide (Li-HMDS) [46e48] to produce the Z, E mixture 11 in 80%
yield. The mixture of alkenes was subjected to catalytic hydroge-
nation to afford fluorinated vitamin E derivative 12 in 85% yield.
The target fluorinated tocopherol ether-linked acetic acid 14 was
synthesized according to the reported method [49] with minor
modifications.
2.1.3. Synthesis of ether-type phytyl group-modified fluorinated
vitamin E derivatives
After obtaining the ether-type phytyl group-modified
a-TEA, it
is easy to get the fluorinated ethyl-type phytyl -TEA derivatives.
a
Compound 5 was alkylated with compound 9 (Scheme 1) in the
presence of NaH and THF under reflux to afford the compound 23 in
62% yield. The target compound 26 was obtained by the identical
procedure to the synthesis of compounds 22 in good yields
(Scheme 3).
In the above reactions, the phytyl group or ether-type phytyl
group was obtained from the mixture isomers of farnesol, which was
reduced and brominated to the all-rac-bromotrimethyldodecane, so
the final compounds have two or three chiral carbon centers, and
Fig. 2. The intermediate 3 generation procedure and its crystal structure.

W. Chen et al. / European Journal of Medicinal Chemistry 58 (2012) 72e83
75
Scheme 2. Synthesis routes to compounds 22a and 22b.
there is a racemic mixture (all-rac) of four or eight stereoisomers,
which was confirmed by 13C NMR spectrum, and the side chain
carbons (2-CH3, 20, 30, 40, 40-CH3, 50, 80, and 90) in all-racemic mixture
of eight stereoisomers give more than one resonance signal [50].
In summary, the compounds prepared in this work can be
divided into three groups (Fig. 3): (i) fluorinated RRR- -tocopherol
(compound 12) and -TEA (compound 14) derivative, (ii) ether-type
phytyl group modified -TEA (compound 22a and 22b), and (iii)
a
a
a
Scheme 3. Synthesis routes to compound 26.

76
W. Chen et al. / European Journal of Medicinal Chemistry 58 (2012) 72e83
and MCF-7 breast cancer cells in comparison with
24 exhibited increased PARP cleavage only in MCF-7 cells in
comparison with -TEA (Fig. 4A). Compound 12 did not induce
a-TEA (Fig. 4A).
a
apoptosis as verified by inability to cleave PARP (Fig. 4A).
To further confirm the pro-apoptotic activity of the analogs that
exhibited increased PARP cleavage in comparison with a-TEA, we
conducted Annexin V/PI/FACS assay to quantify apoptotic induction
in MCF-7 and MDA-MB-231 human breast cancer cell lines. Data
show that compound 22a, 22b and 26 at 20
significantly induced increased levels of apoptosis in MDA-MB-231
cells (Fig. 4B) and, MCF-7 cells at 20 and 40 M for 22a and 40
for 22b and 26 (Fig. 4C), respectively, in comparison with -TEA. EC-
50 values of apoptosis as determined by Annexin V assay, for -TEA,
14, 22a, 22b and 26 were 35, 34, 18, 16 and 27 M in MDA-MB-231
cells and 51, 60, 21, 29 and 35 M in MCF-7 cells, respectively
(Table 1). Based on structure modifications, data demonstrated
that; (i) fluorinated RRR- -tocopherol (compound 12) did not
exhibit pro-apoptotic activity (no PARP cleavage) and ether-type
phytyl group-modified fluorinated RRR- -tocopherol (compound
24) induced PARP cleavage only in MCF-7 cells, (ii) fluorinated
mM and 40 mM
m
mM
a
a
m
m
a
a
a
-
TEA (compound 14) showed enhanced PARP cleavage, but did not
show enhanced apoptosis detected by Annexin V in MCF-7 cells in
comparison to
apoptosis in comparison with
a
-TEA. Compound 14 did not show increased
-TEA by either PARP cleavage or
a
Annexin V assay in MDA-MB-231 cells, leading to the conclusion
that compound 14 is not a potential candidate as an effective novel
anticancer compound, (iii) ether-type phytyl group-modified a-TEA
(S and R forms) (22a and 22b) significantly enhanced apoptosis
tested by both PARP cleavage and Annexin V, and are the best
vitamin E analogs based on pro-apoptotic activity among the tested
derivatives, also the apoptosis isn’t affected by the configuration of
the 2-position and (v) based on EC-50 apoptotic values (Table 1),
ether-type phytyl group-modified fluorinated
26) exhibit less pro-apoptotic property than fluorinated
suggesting that the inclusion of fluorine in the ether-type phytyl
a-TEA (compound
a
-TEA,
group modified a-TEA in such a way as to reduce the pro-apoptotic
activity.
a
-TEA (RRR-
tocopherol analog, derived from RRR-
cancer properties in vitro and in vivo; while, parent compound RRR-
-tocopherol does not. Goal of these studies was to develop new
and more effective vitamin E derivatives, in comparison to -TEA,
a
-tocopherol ether linked acetic acid), a RRR-
a-
a
-tocopherol exhibits anti-
a
a
for anticancer therapeutics. To achieve this goal, we incorporated
fluorine at the chroman head and/or ether linkage between chro-
man head and phytyl tail of
a-TEA and RRR-a-tocopherol, and
tested these derivatives for their pro-apoptotic action in vitro using
human breast cancer cells as well as a mouse mammary cancer cell
line. Data presented here demonstrated that ether-type phytyl
Fig. 3. Structures and molecular weights of novel fluorinated and ether linked phytyl
tail vitamin E compounds (Compound 5 as a control).
group modified
property in comparison to other vitamin E derivatives and
Data show that ether-type phytyl group modified -TEA is a novel
a
-TEA exhibited the most potent pro-apoptotic
ether-type phytyl group modified fluorinated RRR-
a-tocopherol
a
-TEA.
(compound 24) and -TEA (compound 26).
a
a
vitamin E derivative with potent anticancer activity that merits
further investigation.
2.2. Screening of novel vitamin E derivatives for pro-apoptotic
properties
Modification of a compound to enhance its biological functions
can be achieved via different ways (mechanisms) including
improving its bioavailability (uptake and stability) and binding
activity as well as reaching new targets. Our in vitro data show that
addition of an ether linkage between the chroman head and phytyl
The pro-apoptotic properties of prepared vitamin E derivatives
depicted in Fig. 4 were evaluated by Western blot analyses for
polyADP-ribose polymerase (PARP) cleavage, a caspase-dependent
apoptotic marker, following treatments of murine mammary (66cl-
4GFP) cells and human breast cancer MCF-7 and MDA-MB-231
cells. As shown in Fig. 4A, novel vitamin E derivatives 22a, 22b
and 26 exhibited markedly increased PARP cleavage in comparison
tail (Compound 22a and 22b) of
apoptotic property of -TEA. However, the mechanism (s)
whereby ether-type phytyl group modified -TEA enhances pro-
apoptotic activity in comparison with -TEA is not known.
a-TEA can improve the pro-
a
a
a
with
possess superior pro-apoptotic activity in comparison to
Compound 14 also exhibited increased PARP cleavage in 66cl-4GFP
a
-TEA in all three cell lines, suggesting that these derivatives
Although this modification has been reported to reduce the anti-
oxidant activity of vitamin E compounds [25], it may not apply for
the increased anticancer activity, because a-TEA has no antioxidant
a
-TEA.

W. Chen et al. / European Journal of Medicinal Chemistry 58 (2012) 72e83
77
A
B
C
Fig. 4. Evaluating the ability of novel vitamin E analogs to induce apoptosis. (A) Western blot analyses were used to evaluate efficacy of different compounds to cleave PARP on
MDA-MB-231, MCF-7 and 66cl-4GFP cells. Cells were treated with different compounds at 20 M or vehicle control (VEH) for 15 h. Data are representative of two separate
m

78
W. Chen et al. / European Journal of Medicinal Chemistry 58 (2012) 72e83
Table 1
EC-50 values of apoptosis (
All water-sensitive syntheses were performed under an inert
atmosphere of argon using standard Schlenk techniques. 1H and 13
NMR data were collected on Varian Mercury 300 MHz and 600 MHz
spectrometers at 300 MHz, 600 MHz and 75 MHz, 125 MHz,
m
M).a
Compound
-TEA
C
Cancer cell line
a
14
22a
22b
26
respectively. Chemical shifts (d) are reported in ppm and are
MDA-MB-231
MCF-7
35
51
34
60
18
21
16
29
27
35
referenced downfield from (CH3)4Si using the residual solvent peak
as an internal standard. High resolution mass spectra (HRMS) were
obtained with a VG analytical ZAB2-E instrument (ESI). Melting
point was measured on the Buchi Melting Point B-540, and the
temperature was not corrected. TLC analyses were performed on
glass sheets pre-coated with silica gel (Silica Gel 60, F254, 0.25 mm
Layer Thickness). Column chromatographic separations were
carried out on silica gel (Silica Gel 60, 200 ꢀ 400 mesh) under
pressure.
a
The cells were treated with different concentrations of vitamin E analogs fol-
lowed by Annexin V assay to determine apoptosis. EC-50 values were determined
using commercially available software (Calcusyn; Biosoft, Manchester, UK).
activity. Based on the modified structure, addition of an ether
linkage between the chroman head and phytyl tail may further
increase the hydrophilicity of the total molecule, leading to more
uptake. It is also possible that the polar change alternates the
structure of the modified
a-TEA, leading to increased binding
4.1.1. Preparation of 2-fluoro-1,4-hydroquinone 2
activity to its target or triggering a new target. Further study will be
definitely needed to address these questions.
The number of chroman head methyl group exhibits inverse
correlation with anticancer efficacy [17], reduction in number of
methyl groups will increase anticancer efficacy. So we hypothesized
that decrease of the electron density on the chroman head will
To the 2-fluorophenol 1 (5.6 g, 50.0 mmol) dissolved in 6%
aqueous NaOH (200 mL), was added solid potassium persulfate
(13.5 g, 50.0 mmol) with stirring in several portions over 10 min to
result in a dark solution. This dark solution was stirred overnight at
room temperature, then concentrated to ca. one third of its original
volume under reduced pressure. The solution was cooled to 0 ꢁC,
neutralized to pH 6.5 with concentrated HCl and extracted once
with diethyl ether (100 mL). This organic phase was dried (MgSO4)
and concentrated under reduced pressure to reisolate the unreac-
ted starting material, 2-fluorophenol, using flash silica gel column
chromatography with hexane/ethyl acetate (V/V 6:1) as eluent to
give 2.0 g 2-fluorophenol.
improve the anticancer properties of a-TEA derivatives by reduc-
tion of methyl group and addition of a fluorine in vitamin E
compounds. Fluorine is a small molecule and has been reported to
increase the bioavailability when cooperated into some pharma-
ceutical compounds [51]. In this regard, we developed fluorinated
vitamin
(compound 12), fluorinated
-TEA with an ether linkage between the chroman head and phytyl
E
compounds such as fluorinated RRR-a-tocopherol
a
-TEA (compound 14) and fluorinated
The aqueous solution was acidified with concentrated HCl
(50 mL), refluxed for 1 h, then concentrated to ca. 50 mL under
reduced pressure. Addition of acetone (100 mL) precipitated the
inorganic salts, which were removed by filtration. The filtrate was
taken to dryness on the rotary evaporator and the dark residue,
dissolved in acetone, was coated on silica gel (10 g). The dry
material was subjected to column chromatography (hexane/ethyl
acetate 3:1) to give the product 2.2 g, white solid, m.p. 120e
121 ꢁC, yield 52%, Rf 0.30 (20% EtOAc in hexane).
a
tail (Compound 26) derivatives. To our surprising, we did not see
the better anticancer activity of these fluorinated vitamin E
compounds in comparison with their parental compounds by
testing in vitro pro-apoptotic property. These data suggest that
cooperation of fluorine may increase the bio-functions in some
compounds, but not all. Why cooperation of fluorine into vitamin E
compound cannot improve their in vitro anticancer activity is still
unknown. Since the difference in bioavailability of vitamin E
between in vitro and in vivo, further in vivo anticancer test is needed
to confirm if fluorinated vitamin E compounds possess better
anticancer property.
1H NMR (300 M, CDCl3): 6.51e6.56 (m, 1H), 6.64e6.69 (m, 1H),
6.85e6.92 (m, 1H), 4.77 (brs, 2H, 2ꢀ OH).
EI-MS Calcd. for C6H5FO2 128 (Mþ), found 129 (M þ 1)þ,128 (Mþ).
4.1.2. Preparation of methyl 7-fluoro-6-hydroxychroman-2-
carboxylate 3
3. Conclusions
To the mixture of paraformaldehyde (0.23 g, 6.87 mmol, 1.2 eq),
methyl methacrylate (3.2 g, 3.4 mL, 31.25 mmol, 5.0 eq), and acetic
acid (3.5 mL), was added 2-fluoro-1, 4-hydroquinone 2 (0.8 g,
6.25 mmol, 1.0 eq), then dibutylamine (0.096 g, 0.127 mL,
0.75 mmol, 0.12 eq) was added. After the mixture was heated to
120 ꢁC for 24 h, the excess acetic acid was evaporated and diluted
with ethyl acetate (50 mL), washed with saturated NaHCO3, NH4Cl
and water, successively, dried over Na2SO4. Most of solvent was
evaporated and silica gel (5.0 g) was added, then the total solvent
was evaporated and the residue was separated by flash silica gel
column chromatography using hexane/ethyl acetate (3:1 to 2:1) as
eluent to give the title compound 0.31 g, colorless oil, stand at room
temperature overnight to give the white solid, m.p. 78e79 ꢁC, yield
21%, Rf 0.36 (20% EtOAc in hexane).
In this study we synthesized 6 novel vitamin E derivatives by
incorporating fluorine at the chroman head and/or ether linkage
between the chroman head and phytyl tail of
-tocopherol and tested their in vitro anticancer property on
human breast cancer and mouse mammary cancer cell lines. Data
suggest that ether-type phytyl group modified -TEA (22a and 22b)
-TEA and merit further
a-TEA as well as RRR-
a
a
possess better anticancer potential than
a
in vivo evaluation and mechanistic study.
4. Experimental section
4.1. Chemistry
1H NMR (
d/ppm, 300 M, CDCl3): 1.61 (s, 3H, CH3), 1.82e1.92 (m,
Dichloromethane was distilled from CaH2. THF was distilled from
Na/benzophenone. All other chemicals were purchased from
SigmaeAldrich, Fluka, and Acros and used without further
purification.
1H, 3-CH2), 2.34e2.43 (m, 1H, 3-CH2), 2.61e2.67 (m, 2H, 4-CH2),
3.73 (s, 3H, COOCH3), 4.90 (brs, 1H, OH), 6.64e6.71 (dd, 2H,
J ¼ 11.27, 8.36 Hz).
experiments. (B and C) MDA-MB-231 and MCF-7 cells were treated with different concentrations of tested compounds or VEH for 15 h. Apoptosis was determined by FACS/Annexin
V analyses. Data are presented as the mean þ SD of three independent experiments. *Significantly different from -TEA at 40 M (P < 0.05) and **significantly different from -TEA
at 20 M.
a
m
a
m

W. Chen et al. / European Journal of Medicinal Chemistry 58 (2012) 72e83
79
13C NMR (
d
/ppm, 75 M, CDCl3): 22.31, 25.37, 30.55, 52.88, 78.29,
the reactant was concentrated and the residue was separated by
silica gel column chromatography (hexane/ethyl acetate V/V 5:1)
to give the title aldehyde compound, oil, stand to be white solid,
m.p. 89e90 ꢁC, 0.15 g, yield 90%. Rf 0.15 (hexane/ethyl acetate V/V
25:1).
104.41, 104.69, 116.75, 116.79, 116.96, 116.99, 137.44, 137.63, 147.00,
147.14, 148.54, 151.67, 174.29.
HREI-MS Calcd. for C12H14FO4 (M þ 1) 241.0876, found 241.0871.
To determine the exact position of fluorine atom at the chroman
ring, we grew the single crystal of compound 3, and found the
fluorine atom is located at the 7-position of chroman head.
1H NMR (
d/ppm, 300 M, CDCl3): 1.41 (s, 3H, CH3), 1.77e1.87 (m,
1H, 3-CH2), 2.19e2.77 (dt, 1H, J ¼ 5.54 Hz, 13.67 Hz, 3-CH2), 2.62e
2.66 (m, 2H, 4-CH2), 5.06 (s, 2H, CH2Ph), 6.67e6.76 (dd, 2H, J ¼ 11.9,
9.0 Hz), 7.34e7.46 (m, 5H, C6H5).
4.1.3. Preparation of methyl 7-fluoro-6-benzylchroman-2-
carboxylate 4
HR-EIMS Calcd. for C18H17FO3 300.1162 (Mþ), found 300.1163.
To the mixture of 7-fluoro-6-hydroxychroman-2-carboxylate 3
(0.17 g, 0.71 mmol) in 5 mL of DMF, was added K2CO3 (0.147 g,
1.06 mmol, 1.5 eq) under ice-bath. After the mixture was stirred for
20 min, benzyl bromide (0.146 g, 0.85 mmol, 1.2 eq) was added and
the mixture was stirred at room temperature overnight. The reac-
tant was diluted with 10 mL of water and 15 mL of ethyl acetate, the
aqueous was extracted with ethyl acetate (15 mL ꢀ 2), the
combined ethyl acetate phase were washed with water (20 mL ꢀ 2),
dried over Na2SO4. The solvent was evaporated and the residue was
separated by silica gel column chromatography using eluent (10%
ethyl acetate in hexane) to give white solid, m.p. 125e126 ꢁC, 0.21 g,
yield 92%, Rf 0.34 (10% EtOAc in hexane).
4.1.6. 3,7,11-Trimethyldodecanol 8
A mixture of farnesol 7 (20.0 g, 89.94 mmol) and 10% Pd/C (3.0 g)
in ethyl acetate (100 mL) was hydrogenated under 40 PSI for 5.5 h.
The catalyst was removed by filtering on a short pad of celite, the
filtrate was evaporated and the residue was purified by flash
column chromatography on silica gel to give 19.0 g, yield 95%,
colorless liquid, Rf 0.6 (20 %EtOAc in hexane), 1H NMR (
d/ppm,
300 MHz, CDCl3) 3.66 (m, 2H), 1.49e1.59 (m, 4H), 1.01e1.40 (m,
14H), 0.81e0.88 (m, 12H).
4.1.7. 1-Bromo-3,7,11-trimethyldodecane 9
1H NMR (
d/ppm, 300 M, CDCl3): 1.62 (s, 3H, CH3), 1.82e1.92 (m,
To a solution of 3,7,11-trimethyldodecanol 8 (4.56 g, 0.02 mmol)
in CH2Cl2 (10 mL), was added triphenyl phosphine (5.8 g,
0.022 mmol). The clear solution was cooled in ice batch and N-
bromosuccinimide was added in portions, keeping the temperature
less than 30 ꢁC. The mixture was subsequently stirred for 1 h at
25 ꢁC, evaporated in vacuo, and treated with hexane (20 mL ꢀ 3),
filtered and concentrated to give crude product. It was purified by
flash column chromatography on silica gel to give colorless liquid
4.67 g, yield 81%,, Rf 0.9 (100% hexane).
1H, 3-CH2), 2.36e2.44 (dt, 1H, J ¼ 4.5, 13.5 Hz, 1H, 3-CH2), 2.60e
2.64 (dd, 2H, J ¼ 4.5, 8.4 Hz, 4-CH2), 3.73 (s, 3H, COOCH3), 5.04 (s,
2H, CH2Ph), 6.65e6.73 (dd, 2H, J ¼ 12.1, 9.1 Hz).
13C NMR (
d/ppm, 75 M, CDCl3): 22.45, 25.41, 30.54, 52.82, 72.75,
78.32, 105.39, 105.67, 115.87, 115.92, 117.11, 117.14, 127.77, 128.23,
128.76, 137.20, 140.82, 140.97, 148.08, 148.22, 150.81, 154.05, 174.03.
EI-MS for C19H19FO4 Calcd. 330.4 (Mþ), found 330, 271, 253, 279,
171.
1H NMR (
d/ppm, 300 MHz, CDCl3) 3.37e3.52 (m, 2H), 1.83e1.96
4.1.4. Preparation of (6-benzyloxy-7-fluoro-2-methyl-chroman-2-
yl)methanol 5
(m, 1H), 1.47e1.74 (m, 3H), 1.03e1.44 (m, 13H), 0.85e0.91 (dd, 12H,
J ¼ 6.48 Hz).
In 100 mL of flask, was placed LiAlH4 (0.17 g, 4.5 mmol) in 10 mL
of dry THF, then methyl 7-fluoro-6-benzylchroman-2-carboxylate 4
(0.30 g, 0.9 mmol) in 10 mL of dry THF was added dropwise under
0 ꢁC with stirring. After addition, the mixture was stirred at 0 ꢁC for
1 h, then warmed to room temperature for 2 h. After the TLC show
there is no starting material, the reactant was quenched with
saturated NH4Cl aqueous under ice-bath, filtered and concentrated,
then diluted with 40 mL of ethyl acetate, washed with brine
(20 mL ꢀ 2), water (20 mL ꢀ 1), successively, dried over Na2SO4. The
solvent was evaporated and the residue was separated by silica gel
column chromatography using hexane and ethyl acetate (V/V 4:1)
as eluent to give an oil, 0.27 g, yield 95%, stood at room temperature
for overnight to become a white solid, m.p. 74e75 ꢁC, Rf 0.31 (20%
EtOAc in hexane).
13C NMR (
d/ppm, 75 M, CDCl3): 40.35, 40.27, 39.59, 37.59, 37.51,
37.49, 37.46, 37.07, 37.01, 33.00, 32.97, 32.48, 31.92, 31.88, 28.22,
25.05, 25.03, 24.46, 22.96, 22.86, 19.97, 19.90, 19.24, 19.17.
EI-MS Calcd. for C15H31Br 291 (Mþ), found 291, 289, 249, 207,
177, 141, 127.
4.1.8. Triphenyl (3,7,11-trimethyldodecyl)phosphonium bromide 10
The mixture of triphenyl phosphine (2.88 g, 11.0 mmol) and 1-
bromo-3,7,11-trimethyldodecane 9 (2.9 g, 10 mmol) was heated to
210 ꢁC under Argon for 6 h, then cooled to room temperature and
diluted with ethyl ether (30 mL), centrifuged, and repeated the step
for 3 times until no triphenyl phosphine, dried under vacuum to
give 1.8 g viscous solid, yield 82%.
1H NMR (
d/ppm, 300 MHz, CDCl3) 3.71e3.84 (m, 2H), 2.33e2.42
(m, 2H), 1.75e1.87 (m, 1H), 1.45e1.57 (m, 2H), 1.05e1.35 (m, 12H),
0.80e1.01 (ddd, 12H, J ¼ 6.43, 6.59, 6.32 Hz).
1H NMR (
d
/ppm, 300 M, CDCl3): 1.25 (s, 3H, CH3), 1.65e1.73 (m,
1H, 3-CH2), 1.95e2.05 (m, 1H, 3-CH2), 2.62e2.82 (m, 2H, 4-CH2),
3.56e3.66 (q, 2H, J ¼ 11.48 Hz, CH2OH), 5.06 (s, 2H, CH2Ph), 6.58e
6.74 (dd, 2H, J ¼ 12.1, 9.2 Hz), 7.34e7.47 (m, 5H, C6H5).
4.1.9. Preparation of 6-benzyloxy-7-fluoro-2-methyl-2-(40,80,120-
trimethyl-tridecenyl)-2, 3-dihydro-benzofuran (11)
13C NMR (
d/ppm, 75 M, CDCl3): 20.74, 21.67, 27.72, 69.20, 72.89,
76.90, 105.58, 105.86, 116.32, 116.36, 117.47, 117.51, 127.82, 128.25,
128.78, 137.24, 140.42, 140.57, 147.99, 148.12, 150.82, 154.06.
EI-MS Calcd. for C18H19FO3 302 (Mþ), found 302, 285, 231, 225,
211, 195, 141.
To the mixture of triphenyl (3,7,11-trimethyldodecyl)phospho-
nium bromide 10 (0.6 g, 1.32 mmol) in dry THF (10 mL), was added
LiHMDS (1 M in THF, 2 mL, 2.0 mmol) under ꢂ78 ꢁC. After addition,
the mixture was stirred for 30 min and then 6-benzyloxy-7-fluoro-
2-methyl-chroman-2-carbaldehyde 6 (0.6 g, 1.32 mmol) in dry THF
(10 mL) was added under ꢂ78 ꢁC. After the mixture was stirred for
30 min at ꢂ78 ꢁC and another 30 min at 0 ꢁC, then refluxed for 1.5 h,
then solvent was evaporated and diluted with saturated NH4Cl,
extracted with CH2Cl2 (20 mL ꢀ 3), the combined extracts were
dried over MgSO4, and separated by silica gel column chromatog-
raphy (100% hexane to 4% ethyl acetate in hexane) to obtain the
colorless oil, 0.32 g, yield 81%.
4.1.5. Preparation of 6-benzyloxy-7-fluoro-2-methyl-chroman-2-
carbaldehyde 6
To the mixture of (6-benzyloxy-7-fluoro-2-methyl-chroman-
2-yl)methanol 5 (0.17 g, 0.56 mmol) in 20 mL of dry CH2CH2, was
added DesseMartin periodinane (15 wt.%, 2.5 mL) at room
temperature. After addition, the mixture was stirred for 1 h at
room temperature. After the starting material was disappeared,

80
W. Chen et al. / European Journal of Medicinal Chemistry 58 (2012) 72e83
1H NMR (
d
/ppm, 600 M, CDCl3): 0.77e0.87 (m, 12H, 4ꢀ CH3),
4.1.12. Preparation of 2-(7-fluoro-2-methyl-2-(40,80,120-trimethyltride-
cyl)-chroman-6-yloxy) acetic acid 14
1.03e1.09 (m, 3H), 1.11e1.15 (m, 2H), 1.20e1.31 (m, 7H), 1.34e1.39
(m, 2H), 1.46 (d, J ¼ 1.96 Hz, 3H, 2-CH3), 1.49e1.54 (m, 1H), 1.71e
1.75 (m, 1H), 1.91e2.04 (m, 1H), 2.11e2.23 (m, 1H), 2.27e2.40 (m,
1H), 2.55e2.59 (dt, J ¼ 4.9, 16.3 Hz, 1H), 2.68e2.73 (m, 1H), 5.02 (s,
2H, CH2Ph), 5.31e5.38 (m, 2H, CH]CH), 6.55e6.67 (dd, 2H, J ¼ 9.22,
12.27 Hz, 5, 8-phenyl H), 7.28e7.43 (m, 5H, C6H5).
The mixture of ethyl 2-(7-fluoro-2-methyl-2-(40,80,120-trime-
thyltridecyl)-chroman-6-yloxy)-acetate 13 (0.22 g, 0.44 mmol) in
THF (5 mL) and 10% KOH (15 mL) was stirred at room temperature
for about 3 h, then the THF was evaporated, and the residue was
neutralized with HCl to pH 1e2, extracted with CH2Cl2 (20 mL ꢀ 3),
the combined CH2Cl2 were washed with water (20 mL ꢀ 2), and
dried over MgSO4. The solvent was evaporated and the residue was
separated by silica gel column chromatography (19% ethyl acetate,
80% hexane, 1% HAc) to give the oil, 0.19 g, yield 95%. Rf 0.17 (19%
ethyl acetate, 80% hexane, 1% HAc).
13C NMR (
d/ppm, 125 M, CDCl3): 19.57, 19.58, 19.63, 19.64, 19.67,
19.70, 19.74, 19.77, 22.34, 22.65, 22.74, 24.55, 24.56, 24.63, 24.82,
24.83, 24.84, 27.39, 27.42, 28.00, 32.78, 32.80, 32.81, 32.82, 33.04,
33.07, 33.58, 33.60, 33.61, 35.09, 35.18, 35.30, 37.05, 37.10, 37.28,
37.31, 37.32, 37.35, 37.39, 37.41, 37.42, 37.43, 39.39, 72.71, 72.74,
77.10, 77.13, 105.28, 105.29, 105.42, 105.43, 116.59, 116.61, 116.64,
117.20, 117.22, 117.25, 117.27, 127.55, 127.57, 127.93, 127.94, 128.49,
132.01, 132.03, 132.06, 132.08, 132.87, 132.90, 137.16, 137.17, 140.02,
142.05, 142.10, 142.13, 148.35, 148.42, 151.32, 151.33, 152.94, 152.95.
HR-EIMS Calcd. for C33H47FO2 494.3560 (Mþ), found 494.3565.
1H NMR (
d
/ppm, 600 M, CDCl3), 0.82e0.85 (m, 12H, 4ꢀ CH3),
1.02e1.17 (m, 7H), 1.18e1.42 (m, 14H), 1.47e1.59 (m, 3H), 1.69e1.73
(m, 1H), 1.76e1.81 (m, 1H), 2.61e2.69 (m, 2H), 4.62 (s, 2H, OCH2Ph),
6.53 (d, 1H, J ¼ 12.40 Hz), 6.70 (d, 1H, J ¼ 9.21 Hz).
13C NMR (
d/ppm, 125 M, CDCl3): 172.95, 152.96, 151.34, 149.80,
149.73, 138.55, 138.47, 118.28, 118.26, 116.55, 116.53, 105.72, 105.59,
76.57, 67.94, 67.93, 65.86, 39.94, 39.90, 39.38, 37.55, 37.54, 37.46,
37.45,37.44,37.41, 37.39,37.36,37.35,37.29,32.79,32.78,32.72,32.70,
30.68, 30.65, 27.98, 25.58, 24.81, 24.80, 24.46, 23.99, 22.72, 22.62,
21.82, 21.02, 21.01, 21.00, 19.75, 19.68, 19.67, 19.66, 19.61, 19.60, 15.17.
HR-EIMSCalcd. forC28H46FO4 465.3380(Mþ 1)þ, found465.3374.
4.1.10. Preparation of 6-hydroxy-7-fluoro-2-methyl-2-(40,80,120-
trimethyl-tridecenyl)-2,3-dihydro-benzofuran (12)
The mixture of 6-benzyloxy-7-fluoro-2-methyl-2-(40,80,120-tri-
methyltridecenyl)-2,3-dihydro-benzofuran 11 (0.4 g, 0.8 mmol)
and 10% Pd/C (0.1 g) in ethyl acetate (20 mL) was hydrogenated
under 45 PSI hydrogen atmosphere for 24 h. The mixture was
filtered, and concentrated, the residue was separated by silica gel
column chromatography (hexane/ethyl acetate V/V 10:1) to give
the oil, 0.28 g, yield 85%, Rf 0.3 (hexane/ethyl acetate V/V 10:1).
4.1.13. Preparation of methyl 6-hydroxy-2,5,7,8-tetramethylchroman-
2-carboxylate 16a, 16b
To the mixture of (R)-Trolox 15b (5.0 g, 20.0 mmol) in methanol
(100 mL) and CH2Cl2 (20 mL), was added 4-methylbenzenesulfonic
acid (PTSA) (1.0 g) at room temperature. Then the mixture was
heated to reflux for 6 h. After cooling to room temperature, the
reactant was concentrated to give a white solid, dissolved in ethyl
acetate (120 mL), washed with saturated NaHCO3 (80 mL ꢀ 2),
water (80 mL ꢀ 1), successively, dried over MgSO4. After the solvent
was evaporated, the white solid was filtered and washed with
hexane to give pure product 16b, m.p. 131e132 ꢁC, 5.1 g, yield 96%.
1H NMR (
d
/ppm, 600 M, CDCl3): 0.84e0.87 (m, 12H, 4ꢀ CH3),
1.03e1.15 (m, 6H), 1.20e1.42 (m, 14H), 1.48e1.59 (m, 4H), 1.69e1.81
(m, 2H), 2.65e2.67 (m, 2H, 4-CH2), 4.59 (brs, 1H, OH), 6.51e6.67
(dd, 2H, J ¼ 9.80, 11.82 Hz, 5, 8-phenyl H).
13C NMR (
d/ppm, 125 M, CDCl3): 19.57, 19.58, 19.63, 19.64, 19.65,
19.71, 20.97, 20.98, 20.99, 21.00, 22.58, 22.68, 23.94, 23.95, 24.41,
24.42, 24.76, 24.77, 27.95, 30.78, 30.81, 32.64, 32.66, 32.73, 32.74,
32.75, 37.30, 37.31, 37.35, 37.40, 37.42, 37.43, 39.73, 39.78, 60.36,
76.16, 104.28, 104.41, 116.58, 116.60, 117.07, 117.09, 136.27, 136.37,
147.35, 147.41, 148.95, 150.52.
1H NMR (
d/ppm, 300 M, CDCl3),1.63 (s, 3H, 2-CH3),1.84e1.96 (m,
1H), 2.06 (s, 3H, CH3), 2.18 (s, 3H, CH3), 2.21 (s, 3H, CH3), 2.41e2.72
HR-EIMS Calcd. for C26H43FO2 406.3247 (Mþ), found 406.3253.
(m, 3H), 3.70 (s, 3H, CH3), 4.33 (m, 1H, OH).
13C NMR (
d/ppm, 75 M, CDCl3) 174.76, 145.74, 145.54, 122.79,
4.1.11. Preparation of ethyl 2-(7-fluoro-2-methyl-2-(40,80,120-
trimethyl-tridecyl)-chroman-6-yloxy) acetate (13)
121.54, 118.69, 117.10, 77.27, 52.62, 30.86, 25.68, 21.20, 12.46, 12.07,
11.49.
To the mixture of 6-hydroxy-7-fluoro-2-methyl-2-(40,80,120-tri-
methyltridecenyl)-2,3-dihydro-benzofuran 12 (0.1 g, 0.25 mmol) in
DMF (5 mL), was added ethyl bromoacetate (50 mg, 0.3 mmol), and
then powder NaOH (15 mg, 0.375 mmol) was added, the mixture
was stirred at room temperature until there is no starting material
and diluted with ethyl acetate (20 mL) and brine (10 mL), the
aqueous was extracted with ethyl acetate (10 mL ꢀ 2), the
combined ethyl acetate were washed with brine (10 mL ꢀ 2), water
(10 mL ꢀ 1), dried over MgSO4. After removal of the solvent, the
residue was separated by silica gel column chromatography (10%
ethyl acetate in hexane) to give the colorless oil, 0.26 g, yield 89%.
16a was obtained under the identical conditions with 15a, white
solid, m.p. 130e131 ꢁC, yield 95%, 1H NMR (
d/ppm, 300 M, CDCl3),
1.62 (s, 3H, 2-CH3), 1.83e1.94 (m, 1H), 2.08 (s, 3H, CH3), 2.18 (s, 3H,
CH3), 2.20 (s, 3H, CH3), 2.41e2.71 (m, 3H), 3.69 (s, 3H, CH3).
13C NMR (
d/ppm, 75 M, CDCl3) 174.74, 145.71, 145.58, 122.73,
121.58, 118.62, 117.15, 77.22, 52.68, 30.83, 25.64, 21.23, 12.49, 12.02,
11.47.
4.1.14. Preparation of methyl 6-benzylchroman-2-carboxylate 17a,
17b
To the mixture of 2-(R)-6-hydroxy-2,5,7,8-tetramethylchroman-
2-carboxylate 16b (5.0 g, 19 mmol) in 50 mL of DMF, was added
K2CO3 (3.93 g, 28.5 mmol, 1.5 eq) under ice-bath. After the mixture
was stirred for 20 min, benzyl bromide (3.89 g, 22.8 mmol, 1.2 eq)
was added and the mixture was stirred at room temperature
overnight. The reactant was diluted with 50 mL of water and
150 mL of ethyl acetate, the aqueous was extracted with ethyl
acetate (50 mL ꢀ 2), the combined ethyl acetate phase were washed
with water (1000 mL ꢀ 2), dried over Na2SO4. The solvent was
removed off and the residue was separated by silica gel using eluent
(10% ethyl acetate in hexane) to give white solid 17b, m.p. 101e
102 ꢁC, 6.7 g, yield 95%, Rf 0.50 (10% EtOAc in hexane).
1H NMR (
d
/ppm, 600 M, CDCl3): 0.81e0.85 (m, 12H, 4ꢀ CH3),
1.00e1.05 (m, 6H), 1.18e1.40 (m, 18H), 1.46e1.56 (m, 3H), 1.67e1.72
(m, 1H), 1.75e1.80 (m, 1H), 2.59e2.67 (m, 2H), 4.22e4.25 (q, 2H,
J ¼ 7.14 Hz), 4.59 (s, 2H, OCH2Ph), 6.51 (d, 1H, J ¼ 12.45 Hz), 6.68 (d,
1H, J ¼ 9.24 Hz).
13C NMR (
d/ppm, 125 M, CDCl3): 169.13, 153.04, 151.42, 149.42,
149.35, 139.01, 138.93, 118.31, 118.30, 116.31, 116.29, 105.58, 105.44,
76.42, 68.37, 68.36, 61.19, 39.90, 39.87, 39.36, 37.53, 37.52, 37.45,
37.44, 37.42, 37.39, 37.38, 37.379, 37.374, 37.34, 37.33, 37.27, 32.77,
32.75, 32.70, 32.68, 30.71, 30.69, 27.96, 24.79, 24.78, 24.44, 23.99,
23.98, 22.70, 22.61, 21.81, 21.00, 20.99, 19.73, 19.66, 19.65, 19.64,
19.59, 19.58, 14.14.
1H NMR (
1H, 3-CH2), 2.18 (s, 3H, CH3), 2.24 (s, 3H, CH3), 2.28 (s, 3H, CH3),
d/ppm, 300 M, CDCl3): 1.67 (s, 3H, CH3), 1.88e1.98 (m,
HR-EIMS Calcd. for C30H49FO4 492.3615 (Mþ), found 492.3616.

W. Chen et al. / European Journal of Medicinal Chemistry 58 (2012) 72e83
81
2.45e2.72 (m, 3H, 3-CH2, 4-CH2), 3.74 (s, 3H, COOCH3), 4.74 (s, 2H,
CH2Ph), 7.38e7.56 (m, 5H, C6H5).
over MgSO4. After the solvent was evaporated, the residue was
separated by flash column chromatography on silica gel (hexane/
EtOAc V/V 25:1) to give a colorless oil 19b, 1.0 g, yield 76%, Rf 0.5
(hexane/EtOAc V/V 20:1).
13C NMR (
d/ppm, 75 M, CDCl3): 174.61, 149.12, 148.06, 138.22,
128.74, 128.56, 128.07, 127.96, 126.20, 123.20, 117.45, 77.44, 74.91,
52.64, 30.74, 25.71, 21.16, 13.17, 12.26, 12.16.
EI-MS for C22H26O4 Calcd. 354 (Mþ), found 356, 354, 295, 264,
263, 231.
1H NMR (
d
/ppm, 300 MHz, CDCl3) 0.86e0.92 (m, 12H, 4ꢀ CH3),
1.07e1.67 (m, 20H), 1.75e1.84 (m, 1H), 1.97e2.06 (m, 1H), 2.13 (s,
3H, CH3), 2.21 (s, 3H, CH3), 2.25 (s, 3H, CH3), 2.63 (t, 2H, J ¼ 6.75 Hz,
4-CH2), 3.39e3.62 (m, 4H, 2ꢀ CH2O), 4.73 (s, 2H, CH2Ph), 7.34e7.54
(m, 5H, C6H5).
17a, white solid, m.p. 101e102 ꢁC, yield 94%, Rf 0.50 (10% EtOAc
in hexane).
1H NMR (
d
/ppm, 300 M, CDCl3): 1.63 (s, 3H, CH3), 1.84e1.95 (m,
13C NMR (
d/ppm, 75 M, CDCl3): 148.53, 147.96, 138.23, 128.71,
1H, 3-CH2), 2.14 (s, 3H, CH3), 2.19 (s, 3H, CH3), 2.24 (s, 3H, CH3),
2.41e2.69 (m, 3H, 3-CH2, 4-CH2), 3.70 (s, 3H, COOCH3), 4.70 (s, 2H,
CH2Ph), 7.35e7.52 (m, 5H, C6H5).
128.27, 128.03, 127.96, 126.27, 123.08, 117.97, 76.37, 75.25, 74.97,
70.64, 70.59, 39.63, 37.71, 37.69, 37.64, 37.61, 37.54, 37.04, 36.98,
36.96, 33.04, 30.18, 30.15, 28.79, 28.24, 25.08, 25.06, 24.64, 22.99,
22.89, 22.76, 20.60, 20.04, 20.01, 19.98, 19.93, 13.12, 12.27, 12.16.
13C NMR (
d/ppm, 75 M, CDCl3): 174.67, 149.15, 148.09, 138.28,
128.71, 128.53, 128.05, 127.98, 126.26, 123.23, 117.48, 77.47, 74.95,
52.66, 30.78, 25.72, 21.18, 13.15, 12.24, 12.18.
EI-MS for C22H26O4 Calcd. 354 (Mþ), found 355 (M þ 1), 295,
264, 263.
HR-EIMS Calcd. for
537.4296.
C
36H57O3 537.4308 (M
þ
1)þ, found
19a and 23 were prepared by the identical procedure instead of
18a and 5 as starting materials separately.
19a: colorless oil, yield 62%, Rf 0.45 (hexane/EtOAc V/V 20:1).
4.1.15. Preparation of (6-benzyloxy-2-methyl-chroman-2-yl)
methanol 18a, 18b
1H NMR (
d
/ppm, 300 MHz, CDCl3) 0.91e0.95 (m, 12H, 4ꢀ CH3),
1.11e1.48 (m, 17H),1.52e1.81 (m, 4H),1.97e2.06 (m,1H), 2.70 (t, 2H,
J ¼ 6.71 Hz, 4-CH2), 3.39e3.62 (m, 4H, 2ꢀ CH2O), 5.08 (s, 2H, CH2Ph),
6.62e6.75 (dd, 2H, J ¼ 12.31, 9.23 Hz), 7.32e7.50 (m, 5H, C6H5).
To the precipitate of LiAlH4 (0.67 g, 17.0 mmol) in 50 mL of dry
THF, was added dropwise methyl 6-benzylchroman-2-carboxylate
17b (2.0 g, 5.6 mmol) in 50 mL of dry THF under 0 ꢁC with stir-
ring. After addition, the mixture was stirred at 0 ꢁC for 1 h, then
warmed to room temperature for 2 h. After the TLC show there is no
starting material, the reactant was quenched with saturated NH4Cl
aqueous under ice-bath, filtered and concentrated, then diluted
with 80 mL of ethyl acetate, washed with brine (40 mL ꢀ 2), water
(40 mL ꢀ 1), successively, dried over Na2SO4. The solvent was
evaporated and the residue was separated by silica gel chromatog-
raphy using hexane and ethyl acetate (V/V 3.5:1) as eluent to give an
oil 18b, 1.7 g, yield 93%, stood at room temperature for overnight to
become a white solid, m.p. 71e72 ꢁC, Rf 0.38 (25% EtOAc in hexane).
13C NMR (
d/ppm, 75 M, CDCl3): 154.10, 150.86, 148.52, 148.38,
140.38, 140.23, 137.37, 128.76, 128.21, 127.80, 117.52, 117.48, 116.47,
116.42, 105.88, 105.60, 76.34, 76.04, 72.94, 70.60, 39.67, 37.73, 37.68,
37.64, 37.58, 36.94, 36.86, 33.07, 30.17, 28.64, 28.27, 25.12, 25.10,
24.67, 23.04, 22.94, 22.73, 22.71, 21.91, 20.05, 19.98.
HR-EIMS Calcd. for C33H49FO3 512.3666 (M þ 1)þ, found
512.3663.
23: colorless oil, yield 67%, Rf 0.5 (hexane/EtOAc V/V 20:1), 1
NMR (
H
d
/ppm, 600 MHz, CDCl3) 0.83e0.88 (m, 12H, 4ꢀ CH3), 1.04e
1.09 (m, 2H), 1.11e1.55 (m, 2H), 1.21e1.41 (m,12H), 1.48e1.64 (m,
4H), 1.74e1.78 (m, 1H), 1.95e2.00 (m, 1H), 2.09 (s, 3H, CH3), 2.16 (s,
3H, CH3), 2.21 (s, 3H, CH3), 2.59 (t, 2H, J ¼ 6.79 Hz, 4-CH2), 3.36e
3.39 (dd, 1H, J ¼ 7.63 Hz), 3.45e3.48 (dd, 1H, J ¼ 7.45, 7.49 Hz),
3.49e3.58 (m, 2H), 4.69 (s, 2H, CH2Ph), 7.31e7.50 (m, 5H, C6H5).
1H NMR (
d/ppm, 300 M, CDCl3): 1.29 (s, 3H, CH3), 1.75e1.83 (m,
1H, 3-CH2), 2.02e2.12 (m, 1H, 3-CH2), 2.16 (s, 3H, CH3), 2.23 (s, 3H,
CH3), 2.28 (s, 3H, CH3), 2.68e2.73 (m, 2H, 4-CH2), 3.63e3.75 (q, 2H,
J ¼ 11.19 Hz, CH2OH), 4.75 (s, 2H, CH2Ph), 7.36e7.57 (m, 5H, C6H5).
13C NMR (
d/ppm, 125 M, CDCl3): 148.34, 147.74, 138.03, 128.46,
13C NMR (
d
/ppm, 75 M, CDCl3): 148.88, 147.55, 138.14, 128.76,
128.03, 127.78, 127.72, 126.01, 122.86, 117.75, 117.74, 76.17, 76.16,
75.02, 75.02, 74.74, 70.42, 70.37, 39.40, 37.50, 37.49, 37.47, 37.45,
37.42, 37.38, 37.32, 36.84, 36.82, 36.77, 36.74, 32.82, 32.81, 29.98,
29.97, 29.95, 29.93, 28.60, 28.00, 24.84, 24.82, 24.82, 24.42, 24.41,
24.40, 22.74, 22.65, 22.54, 22.53, 20.37, 19.81, 19.78, 19.77, 19.74,
19.72, 19.70, 19.70, 12.88, 12.02, 11.91.
128.52, 128.12, 128.01, 126.51, 123.19, 117.86, 75.64, 75.04, 69.64,
27.93, 20.83, 20.46, 13.16, 12.32, 12.20.
HR-EIMS for C21H26O Calcd. 326.1882 (Mþ), found 326.1883.
18a was obtained by the identical procedure.
18a: oil, stood at room temperature for a long time to become
a white solid, m.p. 71e72 ꢁC, yield 92%, Rf 0.29 (hexane/EtOAc V/V
5:1).
HR-EIMS Calcd. for
537.4294.
C
36H57O3 537.4308 (M
þ
1)þ, found
1H NMR (
d/ppm, 300 M, CDCl3): 1.27 (s, 3H, CH3), 1.73e1.81 (m,
1H, 3-CH2), 1.91e2.09 (m, 1H, 3-CH2), 2.13 (s, 3H, CH3), 2.20 (s, 3H,
CH3), 2.25 (s, 3H, CH3), 2.65e2.71 (m, 2H, 4-CH2), 3.59e3.72 (m, 2H,
CH2OH), 4.72 (s, 2H, CH2Ph), 7.36e7.54 (m, 5H, C6H5).
4.1.17. Preparation of 6-hydroxy-2-(30,70,110-trimethyl-dodecyloxy
methyl)-chroman 20a, 20b and 24
The mixture of 6-benzyloxy-2,5,7,8-tetramethyl-2-(30,70,110-tri-
methyl-dodecyloxymethyl)-chroman 19b (0.8 g, 1.5 mmol) and 10%
Pd/C in 25 mL of ethyl acetate was hydrogenated under 45 PSI for
24 h at room temperature. Then the reactant was filtered,
concentrated, and the residue was purified by column chroma-
tography on silica gel (hexane/EtOAc V/V 10:1) to give an oil 20b,
0.56 g, yield 85%, Rf 0.40 (hexane/EtOAc V/V 10:1).
13C NMR (
d/ppm, 75 M, CDCl3): 148.78, 147.50, 138.12, 128.66,
128.51, 128.13, 128.02, 126.48, 123.15, 117.85, 75.62, 75.01, 69.67,
27.96, 20.84, 20.47, 13.21, 12.31, 12.25.
HR-EIMS for C21H26O Calcd. 326.1882 (Mþ), found 326.1879.
4.1.16. Preparation of 6-benzyloxy-2-methyl-2-(3, 7, 11-trimethyl-
dodecyloxy-methyl)-chroman 19a, 19b and 23
1H NMR (
d
/ppm, 300 M, CDCl3), 0.88e0.92 (m, 12H, 4ꢀ CH3),
After the mixture of 6-(benzyloxy-2-methyl-chroman-2-yl)
methanol 18b (0.8 g, 2.45 mmol) and NaH (60%, 0.5 g, 12.25 mmol,
5.0 eq) in 40 mL of dry THF was refluxed for 30 min, 1-bromo-
3,7,11-trimethyldodecane 9 (0.87 g, 3.0 mmol) in 10 mL of dry THF
was added and the mixture was constituted to reflux for 48 h. Then
the mixture was cooled at room temperature and the solvent was
evaporated under the reduced pressure. The residue was diluted
with ethyl acetate (50 mL), washed with water (30 mL ꢀ 2), dried
1.07e1.43 (m, 17H), 1.51e1.71 (m, 3H), 1.75e1.84 (m, 1H), 1.96e2.07
(m, 1H), 2.15 (s, 6H, 2ꢀ CH3), 2.19 (s, 3H, CH3), 2.65 (t, 2H,
J ¼ 6.79 Hz, 4-CH2), 3.38e3.65 (m, 4H, 2ꢀ CH2O), 4.28 (brs, 1H, OH).
13C NMR (
d/ppm, 75 M, CDCl3): 145.60, 144.97, 122.76, 121.37,
118.78, 117.71, 76.31, 75.00, 70.63, 70.59, 39.64, 37.74, 37.71, 37.66,
37.62, 37.55, 37.07, 37.05, 36.99, 36.96, 33.06, 30.19, 30.16, 28.98,
28.25, 25.09, 25.08, 24.66, 23.00, 22.90, 22.65, 20.69, 20.02, 19.96,
19.94, 12.48, 12.12, 11.56.

82
W. Chen et al. / European Journal of Medicinal Chemistry 58 (2012) 72e83
HR-EIMS Calcd. for C29H50O3 446.3760 (Mþ), found 446.3764.
20a and 24 were prepared by the identical procedure.
20a: yield 81%, Rf 0.22 (hexane/EtOAc V/V 10:1).
1H NMR (
d
/ppm, 300 M, CDCl3), 0.85e0.91 (m, 12H, 4ꢀ CH3),
1.05e1.43 (m, 20H),1.48e1.66 (m, 3H), 1.73e1.82 (m, 1H), 1.94e2.03
(m, 1H), 2.09 (s, 3H, CH3), 2.17 (s, 3H, CH3), 2.21 (s, 3H, CH3), 2.59 (t,
2H, J ¼ 6.79 Hz, 4-CH2), 3.36e3.60 (m, 4H, 2ꢀ CH2O), 4.27e4.36 (m,
4H).
1H NMR (
d
/ppm, 300 M, CDCl3), 0.86e0.91 (m, 12H, 4ꢀ CH3),
1.05e1.45 (m, 17H), 1.49e1.78 (m, 4H), 1.94e2.03 (m, 1H), 2.68 (t,
2H, J ¼ 6.67 Hz, 4-CH2), 3.37e3.58 (m, 4H, 2ꢀ CH2O), 5.18 (brs, 1H,
OH), 6.54e6.70 (dd, 2H, J ¼ 9.76, 11.82 Hz).
13C NMR (
d/ppm, 75 M, CDCl3): 169.61, 148.25, 148.20, 127.89,
125.93, 123.17, 118.00, 76.34, 75.29, 70.62, 70.58, 70.25, 61.31, 39.62,
37.70, 37.67, 37.63, 37.59, 37.52, 37.02, 36.96, 36.93, 33.03, 30.16,
30.14, 28.69, 28.22, 25.07, 25.05, 24.63, 22.97, 22.88, 22.72, 20.52,
20.02, 19.99, 19.95, 19.92, 14.47,, 12.98, 12.12, 12.10.
13C NMR (
d/ppm, 75 M, CDCl3): 151.64, 148.51, 147.30, 147.16,
137.01, 136.82, 117.34, 117.29, 117.06, 117.02, 104.86, 104.58, 76.27,
76.04, 70.65, 39.62, 37.67, 37.63, 37.59, 37.53, 36.84, 36.76, 33.03,
30.11, 28.61, 28.23, 25.07, 25.05, 24.62, 22.98, 22.89, 22.53, 22.51,
21.74, 19.98, 19.92.
HR-EIMS Calcd. for C33H56O5 532.4128 (Mþ), found 532.4126.
HR-EIMS Calcd. for C26H43FO3 423.3274 (M þ 1) þ, found
4.1.19. Preparation of 2-(2-methyl-2-((30,70,110-trimethyldodecyloxy)-
methyl)-chroman-6-yloxy)-acetic acid 22a, 22b an 26
423.3268.
24: yield 96%, Rf 0.40 (hexane/EtOAc V/V 10:1).
The mixture of ethyl 2-(2-methyl-2-(40,80,120-trimethyl-
tridecyl)-chroman-6-yloxy)-21b (0.45 g, 0.84 mmol) in THF
(10 mL) and 10% KOH (30 mL) was stirred at room temperature for
about 3 h, then the THF was removed off, and the residue was
neutralized with HCl to pH 1e2, extracted with CH2Cl2
(20 mL ꢀ 3), the combined CH2Cl2 were washed with water
(20 mL ꢀ 2), and dried over MgSO4. The solvent was evaporated,
and the residue was separated by column chromatography (19%
ethyl acetate, 80% hexane, 1% HAc) to give the oil. The oil was
dissolved in 30 mL of CH2Cl2 again, washed with water
(10 mL ꢀ 30), dried over MgSO4, then the solvent was removed off
to give the oil 22b, 0.4 g, yield 95%, Rf 0.27 (19% ethyl acetate, 80%
hexane, 1% HAc).
1H NMR (
d
/ppm, 300 M, CDCl3), 0.86e0.91 (m, 12H, 4ꢀ CH3),
1.05e1.45 (m, 17H), 1.49e1.68 (m, 3H), 1.74e1.83 (m, 1H), 1.95e2.04
(m, 1H), 2.14 (s, 3H, CH3), 2.15 (s, 3H, CH3), 2.19 (s, 3H, CH3), 2.64 (t,
2H, J ¼ 6.82 Hz, 4-CH2), 3.37e3.63 (m, 4H, 2ꢀ CH2O), 4.24 (brs, 1H,
OH).
13C NMR (
d/ppm, 75 M, CDCl3): 145.59, 144.95, 122.77, 121.32,
118.74, 117.72, 76.30, 75.00, 70.62, 70.58, 39.63, 37.72, 37.69, 37.64,
37.61, 37.54, 37.06, 37.04, 36.98, 36.95, 33.04, 30.17, 30.14, 28.97,
28.24, 25.06, 25.07, 24.63, 22.99, 22.89, 22.63, 20.68, 20.00, 19.96,
19.93, 12.47, 12.11, 11.55.
HR-EIMS Calcd. for C29H50O3 446.3760 (Mþ), found 446.3764.
4.1.18. Preparation of ethyl 2-(2-methyl-2-((30,70,110-trimethyldodecyl-
oxy)-methyl) chroman-6-yloxy)acetate 21a, 21b and 25
1H NMR (
d
/ppm, 300 M, CDCl3), 0.85e0.91 (m, 12H, 4ꢀ CH3),
1.03e1.44 (m, 17H), 1.48e1.69 (m, 3H), 1.74e1.82 (m, 1H), 1.95e
2.04 (m, 1H), 2.10 (s, 3H, CH3), 2.16 (s, 3H, CH3), 2.20 (s, 3H,
CH3), 2.61 (t, 2H, J ¼ 6.78 Hz, 4-CH2), 3.38e3.58 (m, 4H, 2ꢀ CH2O),
4.38 (s, 2H).
To the mixture of 6-hydroxy-2,5,7,8-tetramethyl-2-(30,70,110-
trimethyl-dodecyloxymethyl)-chroman 20b (0.36 g, 0.8 mol) in
DMF (10 mL), was added ethyl bromoacetate (0.16 g, 0.97 mmol),
and then powder NaOH (48 mg, 1.2 mmol) was added, the mixture
was stirred at room temperature until there is no starting material
and diluted with ethyl acetate (50 mL) and brine (30 mL), the
aqueous was extracted with ethyl acetate (20 mL ꢀ 2), the
combined ethyl acetate were washed with brine (30 mL ꢀ 2), water
(30 mL ꢀ 1), dried over MgSO4. After removal of the solvent, the
residue was purified by column chromatography (hexane/ethyl
acetate V/V 10:1) to give the colorless oil 21b, 0.4 g, yield 95%, Rf
0.45 (hexane/EtOAc V/V 10:1).
13C NMR (
d/ppm, 75 M, CDCl3): 173.56, 148.56, 147.44, 127.68,
125.76, 123.43, 118.17, 76.33, 75.42, 70.68, 70.65, 69.41, 39.62, 37.69,
37.66, 37.63, 37.60, 37.53, 36.99, 36.92, 36.90, 33.03, 30.16, 30.14,
28.62, 28.23, 25.07, 25.05, 24.62, 22.97, 22.88, 22.62, 20.52, 19.99,
19.92, 12.98, 12.12.
HR-EIMS Calcd. for C31H52O5 504.3815 (Mþ), found 504.3817.
22a and 26 were prepared by the identical procedure.
22a, oil, yield 96%, Rf 0.20 (19% ethyl acetate, 80% hexane, 1%
HAc).
1HNMR(
d
/ppm, 300M, CDCl3), 0.86e0.92(m,12H,4ꢀ CH3),1.07e
1H NMR (
d
/ppm, 300 M, CDCl3), 0.85e0.89 (m, 12H, 4ꢀ CH3),
1.42 (m, 20H), 1.48e1.67 (m, 3H), 1.73e1.82 (m, 1H), 1.95e2.04 (m,
1H), 2.09 (s, 3H, CH3), 2.17 (s, 3H, CH3), 2.21 (s, 3H, CH3), 2.61 (t, 2H,
J ¼ 6.85 Hz, 4-CH2), 3.37e3.61 (m, 4H, 2ꢀ CH2O), 4.25e4.36 (m, 4H).
1.04e1.43 (m, 17H), 1.49e1.66 (m, 3H), 1.71e1.78 (m, 1H), 1.94e2.03
(m, 1H), 2.69 (t, 2H, J ¼ 6.67 Hz, 4-CH2), 3.37e3.57 (m, 4H, 2ꢀ
CH2O), 4.65 (s, 2H), 6.56e6.74 (dd, 2H, J ¼ 12.38, 9.19 Hz), 9.44 (brs,
1H, COOH).
13C NMR (
d/ppm, 75 M, CDCl3): 169.60, 148.25, 148.21, 127.89,
125.93, 123.17, 118.00, 76.34, 75.29, 70.62, 70.57, 70.26, 61.31, 39.62,
37.70, 37.67, 37.63, 37.60, 37.53, 37.02, 36.96, 36.94, 33.03, 30.16,
30.14, 28.70, 28.23, 25.07, 25.05, 24.63, 22.98, 22.88, 22.73, 20.53,
20.02, 20.00, 19.96, 19.93, 14.48, 12.99, 12.13, 12.11.
13C NMR (
d/ppm, 75 M, CDCl3): 174.29, 153.92, 150.68, 149.59,
149.45, 139.09, 138.94, 118.32, 118.29, 116.83, 116.78, 106.04, 105.76,
76.58, 75.99, 70.67, 67.94, 39.60, 37.67, 37.64, 37.61, 37.58, 37.51,
36.83, 36.74, 33.01, 30.11, 28.43, 28.22, 25.05, 25.04, 24.61, 22.97,
22.88, 22.54, 22.51, 21.77, 19.98, 19.91.
HR-EIMS Calcd. for C33H56O5 532.4128 (Mþ), found 532.4128.
21a and 25 were prepared by the identical procedure.
HR-EIMS Calcd. for C28H45FO5 480.3251 (Mþ), found 480.3250.
26, oil, yield 93%, Rf 0.27 (19% ethyl acetate, 80% hexane,1% HAc).
21a: oil, yield 89%, Rf 0.60 (hexane/EtOAc V/V 10:1).
1H NMR (
d
/ppm, 300 M, CDCl3), 0.85e0.89 (m, 12H, 4ꢀ CH3),
1H NMR (
d
/ppm, 300 M, CDCl3), 0.86e0.92 (m, 12H, 4ꢀ CH3),
1.00e1.43 (m, 20H),1.46e1.67 (m, 3H),1.69e1.78 (m,1H),1.93e2.02
(m, 1H), 2.67 (t, 2H, J ¼ 6.6 Hz, 4-CH2), 3.34e3.56 (m, 4H, 2ꢀ CH2O),
4.23e4.30 (q, 2H, J ¼ 7.14 Hz, OCH2CH3), 4.60 (s, 2H, CH2O), 6.55e
6.73 (dd, 2H, J ¼ 12.42, 9.23 Hz).
1.05e1.46 (m, 17H), 1.49e1.70 (m, 3H), 1.75e1.83 (m, 1H), 1.96e2.05
(m, 1H), 2.11 (s, 3H, CH3), 2.17 (s, 3H, CH3), 2.21 (s, 3H, CH3), 2.61 (t,
2H, J ¼ 6.57 Hz, 4-CH2), 3.40e3.62 (m, 4H, 2ꢀ CH2O), 4.39 (s, 2H),
9.80 (brs, 1H, COOH).
13C NMR (
d
/ppm, 75 M, CDCl3), 169.34, 154.01, 150.77, 149.35,
13C NMR (
d/ppm, 75 M, CDCl3): 174.12, 148.49, 147.59, 127.73,
149.22, 139.46, 139.31, 118.40, 118.36, 116.67, 116.62, 105.92, 105.64,
76.46, 75.98, 70.58, 68.49, 68.46, 61.47, 39.60, 37.67, 37.64, 37.61,
37.58, 37.51, 36.87, 36.78, 33.01, 30.11, 28.49, 28.21, 25.05, 25.03,
24.60, 22.96, 22.87, 22.65, 22.63, 21.80, 19.97, 19.91, 14.39.
HR-EIMS Calcd. for C30H49FO5 508.3564 (Mþ), found 508.3568.
25: oil, yield 73%, Rf 0.36 (hexane/EtOAc V/V 15:1).
125.81, 123.38, 118.11, 76.33, 75.41, 70.72, 70.68, 69.45, 39.63, 37.70,
37.64, 37.60, 37.54, 36.98, 36.91, 36.89, 33.03, 30.16, 30.13, 28.63,
28.23, 25.08, 25.06, 24.63, 22.99, 22.89, 22.58, 20.52, 20.00, 19.93,
12.98, 12.12.
HR-EIMS Calcd. for
505.3893.
C
31H52O5 505.3893 (M
þ
1)þ, found

W. Chen et al. / European Journal of Medicinal Chemistry 58 (2012) 72e83
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4.2.2. Evaluation of apoptosis
Apoptosis was quantified using the Annexin V-PE assay
following the manufacturer’s instructions (Invitrogen, Carlsbad, CA,
USA). This assay measures amount of phosphatidylserine on the
outer surface of the plasma membrane (a biochemical alteration
unique to membranes of apoptotic cells). Fluorescence was
measured using FACS Calibur flow cytometry and data were
analyzed using CellQuest software (BD Biosciences, San Jose, CA,
USA). Cells displaying phosphatidylserine on their surface (positive
for Annexin-V fluorescence) were considered to be apoptotic [53].
4.2.3. Western blot analyses
Western blot analyses to assess protein levels in whole cell
extracts were performed as described previously [19]. Antibodies to
poly (ADP-ribose) polymerase (PARP) and Survivin were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Following
transfer, blots were reacted with primary antibody in 0.1% BSA/
TBST overnight at 4 ꢁC, washed three times with TBST, and reacted
with horseradish peroxidase-conjugated goat anti-rabbit or rabbit
anti-mouse (Jackson Immunoresearch, Rockford, IL, USA)
secondary antibodies.
Acknowledgments
The authors would like to acknowledge grant support from the
Clayton Foundation for Research and Tianjin Natural Science
Foundation (12JCZDJC22000) in China.
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