Apoptotic activities in closely related styryllactone stereoisomers toward human tumor cell lines: Investigation of synergism of styryllactone-induced apoptosis with TRAIL

Article abstract of DOI:10.1016/j.bmc.2009.11.045

A related series of styryllactones with small functional and stereochemical variations were compiled for a comparative study of their apoptotic activities toward two tumorigenic and one non-tumorigenic control cell line. While a substantial range of intrinsic activity was observed, the relative order of activity of the different compounds toward the cell types varied somewhat as did the relative ratios of apoptosis and necrosis observed in conjunction with the loss of cell viability. While some of the styryllactones showed substantial activity, a small but significant apoptosis-induced synergism was demonstrated with (-)-altholactone and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand).

Full text of DOI:10.1016/j.bmc.2009.11.045

Bioorganic & Medicinal Chemistry 18 (2010) 849–854
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Apoptotic activities in closely related styryllactone stereoisomers toward
human tumor cell lines: Investigation of synergism of
styryllactone-induced apoptosis with TRAIL
Shuchi Gupta a, Lee Poeppelman a, Channing. L. Hinman a, James Bretz a, Richard A. Hudson a,b
,
L. M. Viranga Tillekeratne a,
*
a Department of Medicinal & Biological Chemistry, College of Pharmacy, University of Toledo, Toledo, OH 43606, USA
b Department of Chemistry, College of Arts & Sciences, University of Toledo, Toledo, OH 43606, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A related series of styryllactones with small functional and stereochemical variations were compiled for a
comparative study of their apoptotic activities toward two tumorigenic and one non-tumorigenic control
cell line. While a substantial range of intrinsic activity was observed, the relative order of activity of the
different compounds toward the cell types varied somewhat as did the relative ratios of apoptosis and
necrosis observed in conjunction with the loss of cell viability. While some of the styryllactones showed
substantial activity, a small but significant apoptosis-induced synergism was demonstrated with (ꢀ)-
altholactone and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand).
Received 3 September 2008
Revised 12 November 2009
Accepted 21 November 2009
Available online 27 November 2009
Key Words:
Styryllactone, TRAIL
Apoptosis
Ó 2009 Elsevier Ltd. All rights reserved.
Synergism
1. Introduction
(+)- and (ꢀ)-altholactone has allowed us to gain access to an in-
creased diversity of closely related styryllactones. This versatile
The styryllactones are a family of natural products derived from
plants of genus Goniothalamus of the family Annonaceae indigenous
to South East Asia.1 Many styryllactones have been isolated and
well-characterized by spectroscopic and by other chemical analy-
sis2–4 including unambiguous stereospecific synthesis of the natu-
rally occurring stereoisomers.5–12
A number of representative styryllactones have been shown to
possess mild to moderate apoptotic activity toward numerous tu-
mor cell lines.13–15 However, little effort has been made to develop
an understanding of either the origin of this activity or whether the
apoptotic effect can be linked synergistically with other apoptotic
agents. The observation of synergism has been previously made
and developed as a useful concept for combination agents in cancer
chemotherapy based on two or more different apoptotic activi-
ties.16,17 A significantly reduced synergism toward normal-non-
targeted cells compared with malignant cells would also need to
be observed to realize the full therapeutic potential in such an
approach.
synthesis has given us an opportunity to expand structure–activity
studies significantly and thus to test the importance of subtle ste-
reochemical alterations on apoptotic activity. Further, the concept
that any or all of the apoptotic styryllactones can act synergisti-
cally with other apoptotic agents acting by different mechanisms
can also be examined. Here we report the baseline apoptotic activ-
ities of a related group of styryllactone stereoisomers and assess
the potential of one of them to act synergistically with TRAIL (tu-
mor necrosis factor-related apoptosis-inducing ligand).19 We have
also assessed baseline apoptotic activities of these styryllactones
toward different tumor cell lines.
2. Results
Figure 1 shows the structures of styryllactones employed in
theses studies. We have previously reported the stereoselective
synthesis of (ꢀ)-gonioheptolide A 1 starting from
L
-(+)-tartrate.18
In the course of that effort we also synthesized and reported the
(+)-enantiomer 2 as well as the C-3 epimer 3 and the naturally-
occurring (ꢀ)-goniofupyrone 4. The synthetic strategy developed
also proved to be useful in the synthesis of other stereoisomers
and closely related stereochemically-defined analogues of 1.
Compound 5 was synthesized from intermediate 818 starting
from (ꢀ)-diethyl tartrate in the Sharpless asymmetric epoxidation
Our recent unambiguous stereochemical synthesis of the styryl-
lactone gonioheptolide A,18 several closely related diastereomers
of gonioheptolide A, and other related styryllactones, including
* Corresponding author. Tel.: +1 419 530 1983; fax: +1 419 530 7946.
E-mail address: ltillek@utnet.utoledo.edu (L.M.V. Tillekeratne).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.045

850
S. Gupta et al. / Bioorg. Med. Chem. 18 (2010) 849–854
OH
O
OH
O
OH
O
H
H
H
O
O
O
OCH3
OCH3
OCH3
OH
(-)-Gonioheptolide A, 1
OH
(+)-Gonioheptolide A, 2
OH
HO
HO
HO
3
OH
O
H
OH
H
H
O
O
O
OCH3
O
HO
O
OH
HO
O
HO
O
O
(-)-Goniofupyrone, 4
5
(+)-Altholactone, 6
H
O
O
HO
(-)-Altholactone, 7
Figure 1. Styryllactones investigated.
protocol to give intermediate 9 (Scheme 1) by a synthetic strategy
similar to that developed earlier.18 Acid-catalyzed intramolecular
cyclization of 9 gave the tetrahydrofuran 10, the secondary hydro-
xyl groups of which were protected as silyl ethers. Removal of the
benzyl ether function followed by DMP oxidation of the resulting
primary alcohol gave the aldehyde 13, stereoselective aldol reac-
tion of which with the lithium enolate of methyl acetate under
kinetically-controlled conditions gave 14. Finally, mild desilylation
using tris(dimethylamino)sulfonium difluorotrimethylsilicate
(TAS-F) gave the target compound 5. The absolute configuration
of the newly-generated secondary alcohol carbon of 14 was con-
firmed as S by Mosher ester analysis, and strong NOE signals
observed between C20 and C30 and between C30 and C40 confirmed
the absolute stereochemistry of 14 (also of 5) as well as the stereo-
chemistry of the epoxide 9.
We also synthesized the natural (+)-altholactone 65–12 from
intermediate 15 which was used in our earlier synthesis of (ꢀ)-
gonioheptolide A.18 Compound 15 was transformed into ester 16
using Still-Gennari type HWE olefination. The Z-ester spontane-
ously cyclized to the lactone upon removal of the TBS groups with
TAS-F to give (+)-altholactone 6 in 85% yield (Scheme 2). (ꢀ)-Altho-
lactone 7 was also synthesized by an identical procedure starting
with the enantiomer of 15.18
The proliferation of both tumorigenic and non-tumorigenic hu-
man breast cell lines, (MCF-7 and MCF 12A, respectively) was
inhibited by (ꢀ)-altholactone 7 in a concentration-dependent
manner. The other agents tested were comparatively less active
inhibitors of cell proliferation, with the exception of (+)-altholac-
tone 6 (Table 1). IC50s for (+)-altholactone were nearly identical
for both MCF-7 and MCF-12A. The other agents (1–5) were unable
HO
OH
H
O
O
Ph
HO
ii
iii
i
O
Ph
Ph
O
Ph
Ph
O
Ph
OH
OH
OH
10
9
8
O
H
H
H
O
O
O
Ph
TBSO
Ph
Ph
OH
O
Ph
iv
O
v
H
OTBS
OTBS
OTBS
TBSO
TBSO
13
12
11
OH
O
H
OH
O
Ph
H
OCH3
2'
O
vii
vi
3'
OCH3
4'
H
TBSO OTBS
H
14
OH
HO
5
Scheme 1. Reagents and conditions: (a)
MeOH, 1 atm, 90%; (e) Dess–Martin periodinane, CH2Cl2, 75%; (f) CH3COOCH3, LDA, THF, ꢀ78 °C, 81%; (g) TAS-F, DMF, 70%.
D
-(ꢀ)-DET, Ti(Oi-Pr)4, TBHP, MS 4 Å, CH2Cl2, ꢀ20 °C, 85%; (b) CSA, CH2Cl2, 85%; (c) TBSOTf, 2,6-luitidine, CH2Cl2, 88%; (d) H2, Pd/C,

S. Gupta et al. / Bioorg. Med. Chem. 18 (2010) 849–854
851
O
H
O
H
O
H
a
b
(+)-Altholactone, 6
CO2CH3
OTBS
OTBS
TBSO
TBSO
15
16
Scheme 2. Reagents and conditions: (a) 18-crown-6-ether, CH3O2CCH2P(O)(OCH2CF3)2, KN(TMS)2, THF, ꢀ78 °C, 85%; (b) TAS-F, DMF, 72%.
Table 1
Figure 2 illustrates in a flow cytometry plot the ability of
(ꢀ)-altholactone to induce apoptosis and necrosis in CEM cells.
Fluorescence intensity of bound annexin-V-fluorescein isothiocya-
nate (FITC) (along the x-axes) reflects the exposure of phosphatidyl
serine. While this negatively charged phospholipid is primarily
located within the inner leaflet of the plasma membrane, during
early stages of apoptosis, the enzymatic activity that keeps it in
place begins to fail, allowing increased binding of annexin-V to
phosphatidyl serine that has moved to the external side of the plas-
ma membrane. Once the integrity of the plasma membrane is suf-
ficiently compromised and necrosis ensues, both annexin-V and
propidium iodide gain access to the interior of the cell, as reflected
by exponentially increased fluorescence along both axes (the upper
right quadrant of each plot), indicating a transition between apop-
tosis and necrosis. While untreated cells displayed minimal bind-
ing of propidium iodide and annexin-V to their targets, a 24-h
exposure of cells to (ꢀ)-altholactone caused a 40% increase in late
apoptosis/necrosis, along with a 10% increase in early apoptosis.
It may also be noted that (+)-altholactone primarily induced
early apoptosis in the leukemic CEM cells (a 33% increase above
than seen in untreated cells), but only a 19% increase in late apop-
tosis/necrosis occurred (data not shown). Exposure of cells to
either (+)- or (ꢀ)-altholactone reduced total viability to only 19–
20%. Figure 3 illustrates the activities of compounds 1–7 toward
Antiproliferative activity of styryllactones on MCF-7 (tumorigenic human breast
cells), MCF-12A (non-tumorigenic human breast cells), and CEM (human leukemic T-
lymphocytes)
a
IC50
(lM)
Compound
MCF-7
MCF-12
CEM
1
2
3
4
5
6
7
Inactive
Inactive
Inactive
Inactive
Inactive
124 40
100 21
Inactive
Inactive
Inactive
Inactive
Inactive
120 90
110 60
Inactive
Inactive
Inactive
Inactive
Inactive
19
24 16
8
a
Inactive means less than 50% inhibition at 250
tested.
lM, the maximum concentration
to achieve 50% inhibition at concentrations up to 250 lM. Never-
theless, the order of decreasing activity was observed to be
5 > 3 > 2 with compounds 1 and 4 demonstrating little or no activ-
ity above control even at 60 lg/ml, for both MCF-7 and MCF-12A
cells. The activities of the more active agents (2, 3, and 5) were,
in general, 1.5–2-fold greater toward the tumorigenic MCF-7 com-
pared with the non-tumorigenic MCF-12A cells. A leukemic cell
line (CEM) was also investigated. CEM cells showed greater suscep-
tibility toward (+)-altholactone 6 (IC50 19
lactone 7 (IC50 24 16 M) than did MCF-7 or MCF-12A cells.
CEM cells at 120
lM (30 lg/ml) after a 24 h incubation. At these
8
l
M) and (ꢀ)-altho-
concentrations only the altholactone enantiomers showed sub-
stantial activity above background. For example, goniofupyrone
produced only a 10% increase in late apoptosis/necrosis.
l
However, compounds 1–5 were essentially inactive toward CEM
cells (Table 1).
Figure 2. Effects of 120
lM (ꢀ)-altholactone (7) upon CEM cells after 24 h incubation. FL1-H reflects Annexin-V-FITC fluorescence; FL2-H reflects propidium iodide
fluorescence. The percentage of cells in each quadrant is indicated. The lower left quadrant contains viable cells; the lower right quadrant contains early apoptotic cells; the
upper right quadrant contains cells that are late apoptotic/necrotic.

852
S. Gupta et al. / Bioorg. Med. Chem. 18 (2010) 849–854
Interestingly, a complete absence of synergism was observed be-
Viable
tween TRAIL and CPT (data not shown). Similarly, (+)-altholactone,
which alone had induced early apoptosis, showed no potentiation
in the presence of TRAIL.
100
80
60
40
20
0
Early Apoptosis
Late Apoptosis/Necrosis
3. Discussion
Among styryl lactone derivatives, dose-dependent apoptosis
was shown by Inayat-Hussain et al.20 to be induced by (+)-altholac-
tone in HL-60 cells, a human promyelocytic leukemia line with
phagocytic activity. With the exception of (+)-altholactone, how-
ever, the apoptotic/necrotic activities of our compounds are de-
scribed here for the first time. Of the compounds studied, (ꢀ)-
altholactone and (+)-altholactone were shown to be nearly equipo-
tent in their ability to inhibit breast cancer (MCF-7) cell prolifera-
tion. Both (ꢀ)-altholactone and (+)-altholactone induced extensive
apoptosis in human T-leukemic (CEM) cells, but the preponderance
of cells in late apoptosis/necrosis due to (ꢀ)-altholactone supports
the proposition that (ꢀ)-altholactone is significantly more cyto-
toxic than its naturally occurring stereoisomer. With regard to
(+)-altholactone cytotoxicity, it may be noted that in mouse T-leu-
kemic cells assayed by changes in the ability of mitochondria to re-
Figure 3. Induction of CEM cell apoptosis/necrosis by styryllactones. Error bars
represent standard deviations from three experiments, 10,000 cells each.
We also treated leukemic (CEM) cells with the most potent of
the derivatives, and then added TRAIL at concentrations that pro-
mote minimal apoptosis. TRAIL is known to activate the extrinsic
pathway of apoptosis through the productive cleavage of procas-
pase-8. Figure 4 illustrates a nominal synergistic activation of
apoptosis produced by (ꢀ)-altholactone in the presence and ab-
sence of TRAIL. At the end of the 24-h incubation and the 2-h
experiment, untreated cells were 81% viable; equal numbers of
the other cells were either apoptotic or had entered the transition
between apoptosis and necrosis. TRAIL alone (added 3.5 h prior to
the end of the 24-h incubation) induced a 5% increase in apoptosis,
with no increase in necrotic cells. By contrast, the combination of
(ꢀ)-altholactone with TRAIL produced increased early apoptosis.
TRAIL as a potential synergistic agent was also used in conjunction
with camptothecin (CPT), which is known to activate the intrinsic
pathway of apoptosis following its internalization by target cells.
duce a tetrazolium dye, an IC50 of 3
l
g/ml was reported by Bermejo
et al.21 This corresponds well with the 10
l
g/ml IC50 we observed
using human T-leukemic cells, particularly in view of the 100-fold
difference in susceptibility among tumor cell lines to (+)-altholac-
tone that was noted in the review by Mereyala and Joe.15 A further
difference between the activities of (ꢀ)-altholactone and (+)-altho-
lactone was observed when these agents were tested in combina-
tion with TRAIL. No potentiation of apoptosis occurred when TRAIL
was added to cells incubated with (+)-altholactone or with cam-
ptothecin. While synergy between TRAIL and (ꢀ)-altholactone
was modest, the increase in the relative contribution to early apop-
tosis suggests that synthetic modifications of the (ꢀ)-altholactone
scaffold could be fruitful in the development of more effective
compounds that will selectively kill cancer cells by TRAIL-potenti-
ated apoptosis.
100
Viable
4. Conclusion
80
60
40
20
Early Apoptosis
In summary, we have synthesized here and elsewhere,18 seven
variant but closely related stereochemically-defined styryllac-
tones, some of which are available from natural sources but some
of which are not available except through a defined chemical syn-
thesis. Each compound has been purified and well-characterized.
The apoptotic activities of each of the synthetic agents were eval-
uated using two tumorigenic cell lines (CEM, leukemic and MCF-7,
breast) and one non-tumorigenic cell line (MCF-12A, breast). A
small but significant synergism in apoptotic activity with one of
the styryllactones ((ꢀ)-altholactone) and TRAIL was demonstrated.
Further improvements in the synergy noted for TRAIL with modi-
fied styryllactones based initially on (ꢀ)-altholactone could pro-
vide additional improvements in both selectivity and efficacy
toward cancer cells. We intend to explore this possibility.
Late Apoptosis/Necrosis
0
5. Experimental section
5.1. Chemical syntheses
Control
TRAIL only
(-)Altholactone
All reactions were carried out under nitrogen atmosphere
using anhydrous conditions, unless stated otherwise. The solvents
used were ACS grade from Fisher. The reagents used were techni-
cal grade purchased from Aldrich and Acros and used without
purification. TLC was carried out on 0.20 mm POLYGRAM SIL silica
gel plates (Art.-Nr. 805 023) with fluorescent indicator. Davisil
(-)Altholactone+TRAIL
Figure 4. Enhanced early apoptosis produced by the combination of (ꢀ)-altholac-
tone (7) and TRAIL. Error bars represent standard deviations. The probability that
the extent of early apoptosis is the same in the presence or absence of TRAIL is P
<0.0005 (Student’s t-test, 4 degrees of freedom).

S. Gupta et al. / Bioorg. Med. Chem. 18 (2010) 849–854
853
(100–200 mesh) silica gel from Fisher was used in normal phase
5.1.6. (S)-Methyl 3-((2S,3R,4S,5S))-3,4-bis(tert-
butyldimethylsilyloxy)-5-phenyl-tetrahydrofuran-2-yl)-3-
hydroxypropanoate (14)
flash column chromatography. NMR spectra were recorded on
INOVA 600, Varian VXRS-40, or Bruker AC-F 300 MHz spectrome-
ters and using residual undeuterated solvent as an internal refer-
ence. Optical rotations were measured on an AUTOPOL III 589/546
polarimeter. High-resolution mass spectra were recorded on a
Micromass LCT electrospray ionization mass spectrometer at the
Mass Spectrometry and Proteomics Facility of the Ohio State
University.
½
a 2D0
ꢁ
¼ þ13:9 (c 0.7, CHCl3); 1H NMR (CDCl3, 600 MHz): d 7.33–
7.25 (m, 5H), 4.83–4.82 (d, 1H, J = 6 Hz), 4.55–4.51 (m, 1H), 4.37–
3.36 (t, 1H, J = 3.6 Hz), 4.00–3.95 (m, 2H), 3.69 (s, 3H), 3.24–3.23
(d, 1H, J = 3 Hz), 2.88–2.85 (dd, 1H, J = 2.4 Hz, 16.2 Hz), 2.51–2.47
(dd, 1H, J = 9.6 Hz, 16.8 Hz), 0.94 (s, 9H), 0.84 (s, 9H), 0.13 (s, 6H),
ꢀ0.11 (s, 3H), ꢀ0.30 (s, 3H).; 13C NMR (CDCl3, 100 MHz): d
173.69, 140.96, 128.59, 128.03, 126.57, 84.53, 81.60, 80.79, 74.20,
67.42, 51.79, 38.48, 26.25, 26.20, 18.62, 18.29, ꢀ3.298, ꢀ4.28,
ꢀ4.61, ꢀ4.80.; HRMS calcd for C23H42O4Si2+Na+ 461.2519; found
461.2522 (M+Na+).
5.1.1. (1R,2S)-3-(Benzyloxy)-1-((2R,3R)-3-phenyloxiran-2-
yl)propane-1,2-diol (9)
1H NMR (CDCl3, 600 MHz): d 7.39–7.20 (m, 10H), 4.59 (d, 1H,
J = 12.0 Hz), 4.54 (d, 1H, J = 12.0 Hz), 3.98–3.93 (m, 1H), 3.92–
3.88 (m, 1H), 3.86–3.82 (m, 1H), 3.70–3.63 (m, 2H), 3.2–3.08 (m,
1H), 2.69–2.65 (m, 2H).; 13C NMR (CDCl3, 100 MHz): d 137.75,
136.73, 128.78, 128.75, 128.59, 128.22, 128.09, 125.93, 73.96,
71.74, 71.45, 70.73, 63.15, 55.60.; HRMS calcd for C18H20O4+Na+
323.1259; found 323.1253 (M+Na+).
5.1.7. (S)-Methyl 3-((2S,3S,4R,5S)-3,4-dihydroxy-5-
phenyltetrahydrofuran-2-yl)-3-hydroxypropanoate (5)
½
a 2D0
ꢁ
¼ ꢀ18:0 (c 0.3, CHCl3); 1H NMR (CDCl3, 600 MHz): d 7.37–
7.27 (m, 5H), 4.81–4.80 (d, 1H, J = 7.8 Hz), 4.48–4.42 (m, 2H), 4.12–
4.03 (m, 2H), 3.72 (s, 3H), 3.65–3.64 (d, 1H, J = 3 Hz), 3.58–3.57 (d,
1H, J = 3.6 Hz), 2.93–2.92 (d, 1H, J = 8.4 Hz), 2.86–2.83 (dd, 1H,
J = 3 Hz, 16.8 Hz), 2.69–2.65 (dd, 1H, J = 9 Hz, 16.8 Hz).; 13C NMR
5.1.2. (2S,3S,4R,5S)-2-(Benzyloxymethyl)-5-
phenyltetrahydrofuran-3,4-diol (10)
(CDCl3, 100 MHz):
d 173.54, 140.55, 128.76, 128.11, 125.87,
a 2D0
ꢁ
¼ ꢀ20:67 (c 0.3, CHCl3); 1H NMR (CDCl3, 600 MHz): d 7.37–
83.61, 81.44, 79.26, 72.52, 68.38, 52.21, 37.76.; HRMS calcd for
½
C14H18O6+Na+ 305.1001; found 305.1002 (M+Na+).
7.32 (m, 10H), 4.93–4.92 (d, 1H, J = 4.8 Hz), 4.66 (d, 1H, J = 12.0 Hz),
4.60 (d, 1H, J = 12.0 Hz), 4.44–4.42 (m, 1H), 4.39–4.37 (m, 1H),
4.04–4.01 (m, 1H), 3.88–3.83 (m, 2H), 3.56–3.55 (d, 1H,
J = 10.2 Hz), 3.31–3.29 (d, 1H, J = 6.6 Hz).; 13C NMR (CDCl3,
5.1.8. (Z)-Methyl 3-((2S,3R,4R,5R)-3,4-bis(tert-
butyldimethylsilyloxy)-5-phenyltetrahydrofuran-2-yl)acrylate
(16)
100 MHz):
d 140.55, 137.11, 128.94, 128.76, 128.51, 128.23,
127.87, 125.52, 125.50, 84.93, 78.80, 78.64, 74.46, 72.62,
69.62.; HRMS calcd for C18H20O4+Na+ 323.1259; found 323.1252
(M+Na+).
To a solution of 18-crown-6-ether (0.273 g, 1.03 mmol) and
bis(2,2,2-trifluoroethyl)(methoxycarbonylmethyl)phosphonate
(0.065 ml, 0.31 mmol) in THF (3.4 ml) at ꢀ78 °C was added
KN(TMS)2 (0.63 ml, 0.32 mmol, 0.5 M in toluene). After stirring at
the same temperature for 5 min., aldehyde 10 (90 mg, 0.21 mmol)
in THF (1 ml) was added. The resulting reaction mixture was stir-
red at ꢀ78 °C for 3 h before quenching it with saturated aqueous
NH4Cl solution. The aqueous layer was extracted with ether and
the combined organic extract was dried over anhydrous Na2SO4,
concentrated and purified by flash chromatography on silica (6%
EtOAc/hexanes) to afford compound 11 (86 mg, 85%) as a colorless
5.1.3. ((2S,3R,4S,5S)-2-(Benzyloxymethyl)-5-
phenyltetrahydrofuran-3,4-diyl)bis(oxy)bis(tert-
butyldimethylsilane) (11)
½
a 2D0
ꢁ
¼ þ12:0 (c 0.7, CHCl3); 1H NMR (CDCl3, 600 MHz): d 7.34–
7.29 (m, 10H), 4.84–4.83 (d, 1H, J = 6.6 Hz), 4.60 (d, 1H, J = 12.0 Hz),
4.52 (d, 1H, J = 12.0 Hz), 4.44–4.41 (m, 1H), 4.21–4.19 (m, 1H),
3.93–3.91 (m, 1H), 3.73–3.68 (m, 2H), 0.89 (s, 9H), 0.80 (s, 9H),
0.08 (s, 3H), 0.03 (s, 3H), ꢀ0.14 (s, 3H).; 13C NMR (CDCl3,
oil. ½a 2D0
¼ þ79:0 (c 1.0, CHCl3); TLC Rf = 0.61 (silica gel, 20% EtOAc/
ꢁ
100 MHz):
d
141.06, 138.49, 128.56, 128.45, 128.26, 127.91,
hexanes); 1H NMR (CDCl3, 600 MHz): d 7.44 (d, 2H, J = 7.2 Hz),
7.29–7.19 (m, 3H), 6.54–6.51 (m, 1H), 5.94 (d, 1H, J = 13.8 Hz),
5.56–5.54 (m, 1H), 4.79 (s, 1H), 4.26 (s, 1H), 4.05 (s, 1H), 3.73 (s,
3H), 0.93 (s, 9H), 0.74 (s, 9H), 0.07 (d, 6H, J = 6 Hz), ꢀ0.07 (s, 3H),
ꢀ0.13 (s, 3H).; 13C NMR (CDCl3, 100 MHz): d 166.3, 147.6, 141.1,
128.3, 127.4, 126.9, 120.8, 90.1, 86.1, 81.8, 80.3, 51.6, 25.9, 25.8,
18.1, 18.1, ꢀ4.2, ꢀ4.3, ꢀ4.5, ꢀ4.8.; HRMS calcd for C26H44O5Si2+Na+
515.2625; found 515.2601 (M+Na+).
127.81, 126.85, 83.89, 80.89, 80.29, 73.69, 73.66, 70.04, 26.24,
26.17, 18.53, 18.29, ꢀ3.97, ꢀ4.20, ꢀ4.51, ꢀ5.13.; HRMS calcd for
C30H48O4Si2+Na+ 551.2989; found 551.2988 (M+Na+).
5.1.4. ((2S,3R,4S,5S)-3,4-Bis(tert-butyldimethylsilyloxy)-5-
phenyltetrahydrofuran-2-yl)methanol (12)
20
½alphaꢁD ¼ ꢀ24:8 (c 0.4, CHCl3); 1H NMR (CDCl3, 600 MHz): d
7.34–7.27 (m, 5H), 4.98–4.97 (d, 1H, J = 3.6 Hz), 4.36–4.31
(m, 2H), 4.00–3.99 (t, 1H, J = 4.2 Hz),), 3.90–3.87 (m, 1H), 3.79–
3.75 (m, 1H), 2.79–2.77 (dd, 1H, J = 1.8 Hz, 9 Hz), 0.91 (s, 9H),
0.89 (s, 9H), 0.07–0.06 (m, 6H), 0.03 (s, 3H), ꢀ0.14 (s, 3H).;
13C NMR (CDCl3, 100 MHz): d 141.06, 138.49, 128.56, 128.45,
128.26, 127.91, 127.81, 126.85, 83.89, 80.89, 80.29, 73.69, 73.66,
70.04, 26.24, 26.17, 18.53, 18.29, ꢀ4.24, ꢀ4.28, ꢀ4.55, ꢀ4.69.;
HRMS calcd for C23H42O4Si2+Na+ 461.2519; found 461.2522
(M+Na+).
5.1.9. (2R,3R,3aS,7aS)-3-Hydroxy-2-phenyl-3,3a-dihydro-2H-
furo[3,2-b]pyran-5(7aH)-one, [(+)-altholactone] (6)
To a solution of the cis olefin 11 (5 mg, 0.01 mmol) in DMF
(0.1 ml) was added dropwise
a solution of TAS-F (13.5 mg,
0.05 mmol) in DMF (0.1 ml). The resulting reaction mixture was
stirred overnight and diluted with a phosphate buffer solution of
pH 7. The organic phase was separated and the aqueous layer
was extracted with EtOAc. The combined organic extracts were
dried over anhydrous Na2SO4, concentrated and purified by flash
chromatography on silica (25% EtOAc/hexanes) to afford (+)-altho-
5.1.5. (2R,3R,4S,5S)-3,4-Bis(tert-butyldimethylsilyloxy)-5-
phenyltetrahydrofuran-2-carbaldehyde (13)
lactone 6 (1.7 mg, 72%) as a white solid. ½a D20
¼ þ180:5 (c 0.3,
ꢁ
1H NMR (CDCl3, 600 MHz): d 9.74–9.73 (d, 1H, J = 2.4 Hz), 7.38–
7.28 (m, 5H), 5.15–5.14 (d, 1H, J = 3.6 Hz), 4.49–4.48 (dd, 1H,
J = 3.6 Hz, 6.6 Hz), 4.39–4.37 (dd, 1H, J = 3 Hz, 6.6 Hz), 3.99–3.97
(m, 1H), 0.87 (s, 18H), 0.07 (s, 3H), 0.06 (s, 3H), ꢀ0.02 (s, 3H),
ꢀ0.15 (s, 3H).
CHCl3); [lit4
½
a 2D5
ꢁ
¼ þ184:7 (c 0.2, EtOH)]; 1H NMR (CDCl3,
600 MHz): d 7.35–7.30 (m, 5H), 7.0 (dd, 1H, J = 5.4, 10.2 Hz), 6.23
(d, 1H, J = 9.6 Hz), 4.94 (dd, 1H, J = 2.4, 5.4 Hz), 4.73 (d, 1H,
J = 6 Hz), 4.65 (t, 1H, J = 5.4 Hz), 4.45 (dd, 1H, J = 2.4, 5.7 Hz).; 13C
NMR (CDCl3, 100 MHz): d 161.0, 140.4, 138.1, 128.7, 128.4, 126.2,

854
S. Gupta et al. / Bioorg. Med. Chem. 18 (2010) 849–854
123.8, 86.4, 85.9, 83.7, 68.2.; HRMS calcd for C13H12O4+Na+
255.0633; found 255.0616 (M+Na+).
the mean absorbance of wells incubated with each test agent. This
value was then divided by the difference between the mean absor-
bance of the untreated cells and that of the blanks in order to cal-
culate a percent inhibition for each concentration of agent.
5.1.10. (2S,3S,3aR,7aR)-3-Hydroxy-2-phenyl-3,3a-dihydro-2H-
furo[3,2-b]pyran-5(7aH)-one (ꢀ)-altholactone (7)
Compound 7 was synthesized as described above for 6.
5.2.3. Flow cytometry
½
a 2D0
ꢁ
¼ ꢀ181:5 (c 0.2, CHCl3); HRMS calcd for C13H12O4+Na+
Following their treatments, human T-leukemic cells (CEM)
255.0633; found 255.0634 (M+Na+).
were washed twice with ice-cold PBS and resuspended in 200
Annexin-V binding buffer (10 mM HEPES, 140 mM NaCl and
2.5 mM CaCl2). Annexin V-FITC (5 l; BD Biosciences Pharmingen,
San Jose, CA) and propidium iodide (PI, Sigma, St. Louis, MO) (10
of a 1 g/ml solution) were added and cells were incubated
shielded from light for 15 min at ambient temperature. After incu-
bation in the dark, additional binding buffer (800 l) was added to
ll
5.2. Cell culture
l
ll
Adherent human breast cell lines MCF-7 (non-multidrug resis-
tant) and MCF-12A (non-tumorigenic) and the human T-leukemic
cell line CEM were obtained from ATCC and cultured in RPMI med-
ium containing 10% fetal calf serum and 2% penicillin/streptomycin
in a humidified atmosphere with 5% CO2 at 37 °C. Cells were main-
tained by passage twice each week and were washed and replated/
resuspended 24 h prior to each experiment. Only cell populations
exhibiting greater than 90% viability by trypan blue exclusion were
used.
l
l
each tube prior to analysis by flow cytometry.22 A FACSCalibur
flow cytometer (BD Biosciences, San Jose, CA) with emission wave-
lengths of 520 nm for FITC and 620 nm for PI was gated on for-
ward light scatter to eliminate subcellular debris. Statistics were
calculated online by CellQuest software. The simultaneous use of
both fluorescent dyes facilitates distinguishing healthy, apoptotic,
and necrotic cells, since PI is normally impermeant and since its
fluorescence is enhanced 20–30-fold upon binding intracellular
nucleic acids.
5.2.1. Treatments
Stock solutions of agents in DMSO at 1 mg/ml were prepared
fresh for each experiment and were diluted in cell culture medium
to achieve desired concentrations upon addition to the target cells.
All three types of cells were incubated 48 h at 37 °C in 96-well
microtiter plates to assess inhibition of cell proliferation by mea-
suring changes in protein content in triplicate wells. For measure-
ment of cell death by apoptosis or necrosis, and for assessment of
potential synergy with the apoptosis-inducing compound TRAIL,
compounds were incubated 24 h at 37 °C with 106 CEM cells/ml
in 15-ml conical centrifuge tubes.
References and notes
1. Blazquez, M. A.; Bermejo, A.; Zafra-Polo, M. C.; Cortes, D. Phytochem. Anal. 1999,
10, 161.
2. Fang, X.; Anderson, J. E.; Qui, X.; Kozlowski, J. F.; Chang, C.; McLaughlin, J. L.
Tetrahedron 1993, 49, 1563.
3. Bermejo, A.; Blazquez, M. A.; Rao, K. S.; Cortes, D. Phytochem. Anal. 1999, 10,
127.
4. Bermejo, A.; Lora, M. J.; Blazquez, M. A.; Rao, K. S.; Cortes, D.; Zafra-Polo, M. C.
Nat. Prod. Lett. 1995, 7, 117.
5. Zhao, G.; Wu, B.; Wu, X. Y.; Zhang, Y. Z. Mini-Rev. Org. Chem. 2005, 2, 333.
6. Mukai, C.; Hiraj, S.; Hanaoka, M. J. Org. Chem. 1997, 62.
7. Tsubuki, M.; Kanai, K.; Nagase, H.; Honda, T. Tetrahedron 1999, 55, 2493.
8. Yadav, J. S.; Rajaiah, G.; Raju, A. K. Tetrahedron Lett. 2003, 44, 5831.
9. Yadav, J. S.; Raju, A. K.; Rao, P. P.; Rajaiah, G. Tetrahedron: Asymmetry 2005, 16,
3283. 6619.
10. Prasad, K. R.; Gholap, S. L. J. Org. Chem. 2008, 73, 2.
11. Favre, A.; Carreaux, F.; Deligny, M.; Carboni, B. Eur. J. Org. Chem. 2008, 4900.
12. Enders, D.; Barbion, J. Chem. Eur. J. 2008, 14, 2842.
13. El-Zayat, A. A.; Ferrigni, N. R.; McCloud, T. G.; McKenzie, A. T.; Byrn, S. R.;
Cassady, J. M.; Chang, C. J.; McLaughlin, J. L. Tetrahedron Lett. 1985, 26, 955.
14. Fang, X. P.; Anderson, J. E.; Chang, C. J.; McLaughlin, J. L. Tetrahedron 1991, 47,
9751.
5.2.2. Cytotoxicity
Cytotoxicity was assessed using sulforhodamine-B (SRB) by
measuring the extent of inhibition of protein synthesis in treated
cells compared with that in untreated cells. Following 48 h treat-
ment, cells were fixed by the addition of cold (4 °C) 50% trichloro-
acetic acid (50
gently washed 5x with deionized water and allowed to dry over-
night. A 0.4% solution of SRB in 1% acetic acid (50 l) was added
ll/well). After a 1-h incubation at 4 °C, plates were
l
to each well, and plates were incubated 10 min at room tempera-
ture. Wells were then rinsed 5ꢂ with 1% acetic acid and allowed to
15. Mereyala, H. B.; Joe, M. Curr. Med. Chem.: Anti-Cancer Agents 2001, 1, 293.
16. Kim, Y.; Seol, D.-W. Mol. Cells 2003, 15, 283.
dry. To solubilize the SRB dye, 150 ll/well of 10 mM Tris base (pH
10.5) was incubated for 20 min at room temperature and plates
were gently vortex-mixed before absorbance at 570 nm was mea-
sured using a spectrophotometer (Spectramax-Pro; Molecular De-
vices, Sunnyvale, CA). Negative control wells (blanks) contained
medium with compound, but no cells. Wells containing untreated
cells served as positive controls (maximal protein synthesis). To
determine the extent of inhibition of protein synthesis, the mean
absorbance of the corresponding set of blanks was subtracted from
17. Zoli, W.; Ricotti, L.; Tesei, A.; Barzanti, F.; Amadori, D. Crit. Rev. Oncol.: Hematol.
2001, 37, 69.
18. Gupta, S.; Rajagopalan, M.; Alhamadsheh, M.; Tillekeratne, L. M. V.; Hudson, R.
A. Synthesis 2007, 3512.
19. LeBlanc, H. N.; Ashkenazi, A. Cell Death Differ. 2003, 10, 66.
20. Inayat-Hussain, S. H.; Osman, A. B.; Din, L. B.; Taniguchi, N. Toxicol. Lett. 2002,
13, 153.
21. Bermejo, A.; Léonce, S.; Cabeo, N.; Andreu, I.; Caignard, D. H.; Atassi, G.; Cortes,
D. J. Nat. Prod. 1999, 62, 1106.
22. Smith, C. A.; Hinman, C. L. J. Biochem. Mol. Toxicol. 2004, 18, 204.