Synthetic analogue of rocaglaol displays a potent and selective cytotoxicity in cancer cells: Involvement of apoptosis inducing factor and caspase-12

Article abstract of DOI:10.1021/jm900365v

Flavaglines constitute a family of natural anticancer compounds. We present here 3 (FL3), the first synthetic flavagline that inhibits cell proliferation and viability (IC₅₀≈1 nM) at lower doses than did the parent compound, racemic rocaglaol.

Full text of DOI:10.1021/jm900365v

5176 J. Med. Chem. 2009, 52, 5176–5187
DOI: 10.1021/jm900365v
Synthetic Analogue of Rocaglaol Displays a Potent and Selective Cytotoxicity in Cancer Cells:
Involvement of Apoptosis Inducing Factor and Caspase-12
†
†
‡
†
§
Frederic Thuaud, Yohann Bernard, Gulen Turkeri, Ronan Dirr, Genevieve Aubert, Thierry Cresteil, Aurelie Baguet,
§
ꢀ ꢀ
Catherine Tomasetto, Yuri Svitkin, Nahum Sonenberg, Canan G. Nebigil, and Laurent Desaubry*
ꢁ
ꢀ
^
^
‡
,†
ꢀ
†Therapeutic Innovation Laboratory, UMR7200, CNRS/Universiteꢀ de Strasbourg, Illkirch, France, ‡Institut de Recherche de l’Ecole de
Biotechnologie de Strasbourg, FRE 3211, CNRS/Universiteꢀde Strasbourg, Illkirch, France, §Institut de Chimie des Substances Naturelles, CNRS
UPR 2301, Gif-sur-Yvette, France, Institut de Geꢀneꢀtique et de Biologie Moleꢀculaire et Cellulaire, U 596 INSERM/UMR7104 CNRS/Universiteꢀ
de Strasbourg, Illkirch, France, and ^Department of Biochemistry, McGill Cancer Centre, McGill University, Montreal, QC H3G1Y6, Canada
Received March 23, 2009
Flavaglines constitute a family of natural anticancer compounds. We present here 3 (FL3), the first
synthetic flavagline that inhibits cell proliferation and viability (IC50 ≈ 1 nM) at lower doses than did the
parent compound, racemic rocaglaol. Compound 3 enhanced doxorubicin cytotoxicity in HepG2 cells
and retained its potency against adriamycin-resistant cell lines without inducing cardiomyocyte toxicity.
Compound 3 induced apoptosis of HL60 and Hela cells by triggering the translocation of Apoptosis
Inducing Factor (AIF) and caspase-12 to the nucleus. A fluorescent conjugate of 3 accumulated in
endoplasmic reticulum (ER), suggesting that flavaglines bind to their target in the ER, where it triggers a
cascade of events that leads to the translocation of AIF and caspase-12 to the nucleus and probably
inhibition of eIF4A. Our studies highlight structural features critical to their antineoplastic potential
and suggest that these compounds would retain their activity in cells refractory to caspase activation.
Introduction
Flavaglines strongly activate the stress-response mitogen-
activated protein kinase p38 in leukemia and colorectal tumor
cell lines.6,14 In addition, they potently inhibit protein synth-
esis but not DNA nor RNA synthesis in several cancer cell
lines, suggesting that their cytostatic properties involve in-
hibition of protein synthesis.5,7,8
Pezzuto and colleagues have shown that a naturally occur-
ring flavaglinedelaysfor 23daysthegrowthofa human breast
cancer cell line (BC1) in athymic mice.7 The unique biological
profile of flavaglines has attracted considerable interest these
last 10 years, but the studies of their structure-activity
relationships were limited to natural compounds because of
the difficulty to synthesize them. The specificity of flavaglines’
cytotoxicity toward cancer cells coupled to a unique array of
biological activity and an unknown mode of action prompted
us to pursue the exploration of their structure-activity rela-
tionships and their therapeutic potential in the treatment of
cancer.
Flavaglines are a family of natural compounds extracted
from Asian plants of the genus Aglaia.1,2 King et al. isolated
the first flavagline, rocaglamide, in 1982 on the basis of its
potent in vivo activity against murine P388 lymphocytic
leukemia.3 Since then, many other flavaglines, such as roca-
glaol or silvestrol, have been isolated, mainly by the pharma-
cognosy laboratories of Kinghorn, Pezzuto, and Proksch.1,2
These cyclopenta[b]benzofurans have been shown to inhibit
the proliferation of tumor cells in a low nanomolar range,
either by cytostatic4-8 or cytotoxic effects,9-14 depending on
compound and cell line.
Unlike other anticancer drugs, flavaglines are extremely
effective against cancer cell lines without displaying any
significant toxicity on normal cells, such as human umbilical
vein endothelial cells (HUVECa),15 intestinal epithelial cell
line IEC18,6 normal peripheral blood lymphocytes, and bone
marrow stem cells.14 Moreover, these compounds do not
induce any loss of body weight or any other sign of toxicity
in mice.7 Flavaglines have been reported to induce apoptosis
via the intrinsic pathway.12,16
Herein we report on the preparation and biological evalua-
tion of rocaglaol analogues FL2-4 (2-4), fluorescent probe
5, and affinity ligand 6 (Figure 1). This study led to the
identification of a synthetic compound displaying an en-
hanced in vitro cytotoxicity compared with racemic rocaglaol
1. We also present a model that accounts for the mechanism
by which flavaglines induce apoptosis in cancer cells.
*To whom correspondence should be addressed. Phone: 33-390-244-
141. Fax: 33-390-244-145. E-mail: desaubry@chimie.u-strasbg.fr.
a Abbreviations: HUVEC, human umbilical vein endothelial cells;
MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethonyphenol)-2-(4-
sulfophenyl)-2H-tetrazolium; PI, propidium iodide; FACS, fluores-
cence-activated cell sorting; Z-VAD-FMK, benzyloxycarbonyl-Val-
Ala-Asp(OMe)-fluoromethylketone; ER, endoplasmic reticulum; MLN51,
metastatic lymph node 51 protein; FMRP, fragile X mental retarda-
tion protein; AIF, apoptosis inducing factor; DAPI, 40-6-diamidino-2-
phenylindole; ATAD, benzyloxycarbonyl-Ala-Thr-Ala-Asp(OMe)-
fluoromethylketone; casp-12, caspase-12; MTF, molecular target of
flavaglines.
Chemistry
The structural requirements for the antiproliferative capa-
city tested upon cancer cell lines have been limited to
flavaglines extracted from plants. Flavaglines are cyclopenta-
[b]benzofuran lignans substituted by two aryl groups with
poorly explored structure-activity relationships. Because
r
pubs.acs.org/jmc
Published on Web 08/05/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5177
rocaglaol is particularly active and can be conveniently
synthesized by the approach developed by Dobler and colla-
borators, we selected this flavagline as a reference compound
to examine the effects of substitutions on these two aryl
moieties. The synthesis of racemic rocaglaol (1) and its
analogues 2-4 started with the Michael addition of benzofur-
anones 7 to cinnamaldehyde 8, resulting in a mixture of
adducts 9 and 10 (2:3 ratio) that were separated by silica gel
chromatography (Scheme 1).17 Formation and ring closure of
cyanohydrins 11 afforded the cyclopenta[b]benzofurans 12.
Subsequent deprotection and stereospecific reduction of the
ketones by Me4NBH(OAc)3 provided flavaglines 1-4.
this blue fluorescent dye because it displays suitable photo-
physical properties for fluorescence microscopy, it easily
penetrates into cells, and it does not localize preferentially in
a subcellular compartment.18 The synthesis of flavagline 17
was performed according to the approach developed by Porco
and colleagues19 (Scheme 2). Photocycloaddition [3 þ 2] of
hydroxyflavone 13 with methyl cinnamate 14, followed by an
acyloin rearrangement, gave a mixture of inseparable diaster-
eomers 16. Reduction by Me4NBH(OAc)3, HPLC purifica-
tion and saponification afforded the expected racemic
flavagline 17.
Coupling of coumarin 18 with mono-Boc protected dia-
mine 1920 was mediated by ECDI/HOBT. Subsequent depro-
tection by TFA in CH2Cl2 at room temperature afforded the
amine 20 (72% yield for two steps). ECDI/HOBT-mediated
coupling with 17 afforded the fluorescent probe 5 in a 22%
yield. We also prepared unconjugated coumarin 21 by acyla-
tion of 20, for use as a negative control in confocal microscopy
studies.
To identify the intracellular localization of the molecular
target of flavaglines, we conjugated flavagline 17 to N,N-
dimethyl-7-aminocoumarin 18 through a linker. We selected
To detect the molecular target of flavaglines by pull-down
experiments, weconjugated the carboxylic derivativeof3 onto
Affi-Gel10through a linker. Coupling of23withamino linker
24 was mediated by ECDI/HOBT in CH2Cl2 at 0 ꢀC. Initial
attempts to reduce azide 25 to amine 6 with triphenylpho-
sphine or 1,3-propanedithiol were unsuccessful due to the
formation of many side-products. Finally, the use of thiophe-
nol and SnCl2 according to Fuch’s method21 allowed us to
prepare amine 6 in 75% yield, which was quantitatively
conjugated to Affi-Gel 10 (Scheme 3).
Biological Results
In Vitro Cell Growth Inhibition Assay. The antiprolifera-
tive capacities of racemic rocaglaol (1) and its analogues 2-4
were determined on a variety of human cancer cell lines
from nasopharynx (KB), breast (MCF7, MCF7R), colon
(HCT116 and HCT15), liver (HepG2), and neutrophil
(HL60)-derived neoplasms by the MTS assay after a 72 h
treatment. As shown in Table 1, 1 reduced the cell prolifera-
tion and viability at lower doses (in the low nanomolar
range) than the anticancer drugs doxorubicin, cisplatin,
and camptothecin on a panel of human cancer cell lines.
Removing a methoxy group (R1 = H) decreased potency
more than 3 orders of magnitude (compound 2, Table 1).
Conversely, the replacement of this methoxy group by a
Figure 1. Structures of 1 (racemic rocaglaol, FL1), analogues 2-4
(FL2-4), fluorescent probe 5, and affinity ligand 6.
Scheme 1a
a Reagents and conditions: (a) triton B, THF, rt, 1 h; (b) TMSCN, ZnI2, MeCN, benzene, rt, 12 h ; (c) LDA, THF, -78 ꢀC, 1 h; (d) K2CO3, MeOH, rt,
15 min; (e) Me4NBH(OAc)3, MeCN, AcOH, rt, 18 h.

5178 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Thuaud et al.
Scheme 2a
a Reagents and conditions: (a) hν, CH2Cl2-MeOH, 0 ꢀC, 15 h; (b) MeONa, MeOH, 60 ꢀC, 20 min; (c) Me4NBH(OAc)3, CH3CN; (d) KOH, MeOH,
45 ꢀC, 12 h; (e) ECDI, HOBT, DIPEA, CH2Cl2, rt, 12 h; (f) TFA, CH2Cl2, rt, 12 h; (g) Ac2O, Et3N, CH2Cl2, rt.
Scheme 3a
developed resistance to chemotherapy by expressing the
P-glycoprotein (P-gp), a plasma membrane protein also
named “permeability glycoprotein” and encoded by the
multidrug resistance (MDR1) gene (see Table 1). This was
further confirmed by the direct measurement of drug efflux
from MCF7R cells in the presence of 0.1-1 μM 1-4. Indeed,
under flavaglines 1-4 the efflux of rhodamine 123 from cells
was unchanged, demonstrating the lack of interaction be-
tween flavaglines and P-gp.
Combination of Flavaglines and Doxorubicin on Cancer
Cell Line. Flavaglines have been shown to sensitize therapy-
resistant leukemic T cells to apoptosis induced by TNF-R,
cisplatin, and γ-irradiation,22 but their possible potentiating
effects upon anthracycline-induced cytotoxicity have not
been investigated. Because of the critical need to develop
efficient therapies for hepatocellular carcinoma,23 we se-
lected HepG2 cells to investigate whether 1 and 3 could
potentiate doxorubicin-induced apoptosis. Note that these
slowly growing cells display a low sensitivity toward clini-
cally used anticancer drugs (Table 1). Gratifyingly, both 1
and 3 exhibited additive cytotoxic effects with doxorubicin
(Figure 2). As expected, this enhancement of doxorubicin
cytotoxicity was more pronounced with 3 than 1.
Flavaglines are Not Cardiotoxic. Many anticancer drugs,
such as cisplatin, imatinib mesylate (Gleevec), and trastuzu-
mab (Herceptin) are cardiotoxic.24,25 This cardiotoxicity is
even a major limitation to the use of anthracyclines in clinics.
Therefore, one of the major challenges in anticancer therapy
is to develop therapeutic agents that do not compromise
heart function. This prompted us to investigate whether 1
and 3 are cytotoxic in H9c2 cardiomyoblast cell lines. These
cardiomyoblasts derived from rat heart represent an estab-
lished model to study cardiotoxicity in vitro.26 Cell cycle
progression and apoptosis were measured in H9c2 cells
treated with 1, 3 doxorubicin, or their vehicle, labeled
with annexin and propidium iodide (PI), and analyzed by
a Reagents and conditions: (a) hν, CH2Cl2-MeOH, 0 ꢀC, 30 h;
(b) MeONa, MeOH, 60 ꢀC, 20 min; (c) Me4NBH(OAc)3, CH3CN;
(d) KOH, MeOH, 45 ꢀC, 12 h; (e) ECDI, HOBT, DIPEA, CH2Cl2, rt,
12 h; (f) PhSH, SnCl2, Et3N, CH3CN, rt, 3 h (75%); (g) Affi-Gel 10,
Et3N, CH3CN, rt, 12 h (100%).
bromine atom (R1=Br) improved the cytotoxicity against all
of these cancer cell lines (compound 3). The rank of activity
(H , MeO < Br) suggests a preference for a hydrophobic
substituent in this para position. Introduction of a methoxy
on the other phenyl moiety (R2=OMe) was detrimental for
cytotoxicity (compare 4 with 2).
Interestingly, the cytotoxicity potential of 1 and 3 was
retained in the two cell lines HCT15R and MCF7R that have

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5179
Table 1. Inhibition of Cell Proliferation by Flavaglines 1-4 on Various Human Cancer Cell Lines (IC50, nM)a
cell lines
1
2
3
4
doxorubicin
docetaxel
cisplatin
camptothecin
vinblastine
KB
2
2500
<1
1
>10000
NI
1
0.2
NI
NI
0.5
13
2200
4800
38
10
3
0.8
4.6
NI
1.3
10
MCF7
MCF7R
HCT116
HCT15
HepG2
HL60
4
10000
10000
6000
12
58
6
4
<1
<1
1
>10000
>10000
NI
3100
2700
6
2
6
8000
>10000
6000
81
240
13
2900
>10000
5900
3
70
3
4
<1
NI
>10000
NI
0.5
455
6
NI
1
a IC50 values were determined from dose-response curves performed in duplicate according to methods described under Experimental Section. NI:
noninhibiting at the highest concentration tested.
pathway involved in the cytotoxicity of 3 in these cells.
Next we tested whether 1 and 3 induced apoptosis in a
caspase-dependent manner. To address this question we
investigated caspase 3/7, 8, and 9 activities in HL60 cells
treated for 48 h with 1 or 3. As shown in Figure 4c and
Supplementary Figure S1, neither 1 nor 3 treatment signi-
ficantly activated caspases 3/7 in a concentration-depen-
dent manner, suggesting that caspase activation seen
after treatment with 1 or 3 is a consequence of 1 or 3-induced
apoptosis rather than a direct effect on caspase activity. In
comparison, 1 μM doxorubicin increased caspase 3/7 acti-
vity by 14-fold. Moreover, no cleavage of the upstream
caspase cascade precursors (caspases 8 and 9) was detected
Figure 2. Inhibition of cell proliferation of HepG2 cells exposed to
after treatment with 3 in the same cells (Supplementary
1 and 3 with or without doxorubicin. HepG2 cells were treated with
Figure S2). Taken together, these data suggest that 1 and
1 (() or 3 (2) alone or with 150 nM doxorubicin (1: 1, 3: 9) for 72 h,
3 induce apoptosis in HL60 cells without acting directly
and cell proliferation was measured by MTS assay. Experiments
on caspase 3/7.
were performed in duplicate (n=3).
Inhibitors of Caspases, Necroptosis, and Autophagy do not
fluorescence-activated cell sorting (FACS). Additionally,
cell viability was also assessed by the MTS assay. Remark-
ably, incubation of H9c2 cells with 0.5-50 nM of 1 or 3 for
24 h did not induce apoptosis, indicating that flavaglines are
not deleterious to H9c2 cardiomyocytes (Figure 3a). On the
opposite, doxorubicin (1 μM) treatment for 24 h induced a
3-fold increase in cell death. No clear sign of apoptosis or
alteration of cell viability were observed after 48 and 72 h of
treatment with 3 (20 nM; Figures 3b,c). Next we examined
general toxicity in mice: 3 was administered i.p. twice a day
for four consecutive days at a dose of 0.1 mg/kg, body
weights were measured once a day for 13 days. No overt
sign of acute toxicity, such as diarrhea and body weight loss,
was observed (Figure 3d).
Flavaglines Block HL60 Cells in G2/M Phase. To investi-
gate the effect of flavaglines on cell cycle progression and
apoptosis, HL60 cells were treated for 48 h in a medium
containing varying concentrations of 1 and 3. A similar
experiment was performed with 20 μM doxorubicin as
a positive control. DNA content was determined by flow
cytometry of PI-stained cells (Figure 4a). Incubation of
HL60 cells for 48 h with 1 (200 nM) or 3 (20 nM) resul-
ted in the accumulation of cells in the G2/M phase of the
cell cycle. Note that treatment of cells with 1% DMSO
alone (vehicle control) had no effect on cell cycle profile
compared with untreated controls. An increase in the
percentage of total cells in the G0-G1 peak, characteris-
tic of apoptotic cells, was observed progressively with
both 1 and 3.
Block the Cytotoxicity of 3 in HL60 Cells. To further
characterize molecular mechanisms behind cytotoxicity of
3 in cancer cells, we examined whether this effect could be
blocked by the broad-spectrum caspase inhibitor Z-VAD-
FMK, and also by 3-methyladenine and necrostatin-1, which
respectively inhibit autophagy and necroptosis.27,28 The
antiproliferative activity of both 1 and 3 was unchanged
under cotreatment with these inhibitors (Figure 4d and
Supplementary Figure S3), which confirms that apoptosis
is the main mechanism by which 3 induces the death of HL60
cells and that it does not involve caspases.
Compound 3 does not Inhibit the Proteasome. Some drugs
like bortezomib (MG132) exert their cytotoxic effects
through the inhibition of protease activities mediated by
the proteasome.29 To test whether 1 and 3 could act on the
proteasome, lysates of HL60 cells were treated with 1 or 3
and their capacity to modulate in vitro chymotrypsin- or
trypsin-like activities of these cell lysates was measured.
Neither 1 (20-200 nM) nor 3 (2-20 nM) was capable of
eliciting a reduction of proteasome activity, in contrast with
bortezomib (100 nM), which was used as positive control for
assessing chymotrypsin-like activity of the proteasome
(Supplementary Figure S4).
Compound 3 Inhibits Protein Synthesis, Independently from
eIF2-r Phosphorylation. Rocaglaol (nonracemic 1) has been
reported to inhibit protein translation.8 Given the impor-
tance of translation in cell growth and tumorigenesis, we
checked whether 3 could exert its cytotoxic effect by inhibi-
tion of protein synthesis. Using an in vitro-based translation
assay of capped and polyadenylated luciferase mRNA in a
rabbit reticulocyte lysate (Figure 5A), we observed that 3
inhibited translation in this assay at micromolar concentra-
tions, doses that are 1000 times higher than cytotoxic doses
for cancer cells.
Compound 3 Induces Apoptosis in HL60 Cells Indepen-
dently of Caspases 3/7. We determined by FACS analysis
apoptosis of HL60 cells after 1 or 3 treatment (Figure 4b).
After 72 h of treatment with 3 (20 nM), 43% of HL60 cells
were killed, indicating that apoptosis is the main cell death

5180 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Thuaud et al.
Figure 3. Flavagline 3 is not cardiotoxic. (a) Neither 1 nor 3 induced apoptosis in H9c2 cardiomyocytes. H9c2 cells were treated with 1 and 3 or
their vehicle for 24 h and then labeled with annexin V and PI for FACS analysis. Remarkably, incubation of H9c2 cells with different
concentrations of flavagline for 24 h did not alter the apoptotic cell number as compared to the vehicle treatment, indicating that flavaglines do
not display any adverse sign of apoptosis in H9c2 cardiomyocytes. (b) Compound 3 (20 nM) does not induce apoptosis of H9c2 cells after 24, 48,
and 72 h. (c) Compound 3 (20 nM) does not alter cell viability of H9c2 cells. Cells were treated as previously and viability was assessed by MTS
assay. (d) Flavagline 3 does not induce loss of body weight in mice. Compound 3 was administered i.p. twice a day for four consecutive days at a
dose of 0.1 mg/kg to male B6 mice and body weights were measured once a day for 13 days.
Next, we examined whether 3 induced the phosphoryla-
tion of the R-subunit of the eukaryotic initiation factor 2
(eIF-2R), a key regulator of protein synthesis. Its phosphor-
ylation results in inhibition of translation initiation and the
formation of cytoplasmic aggregates called stress granules.30
Moreover, abrogation of eIF-2R phosphorylation causes
malignant transformation of NIH 3T3 cells.31 Using an
antibody specific for phosphorylated eIF2-R, we found that
eIF2-R phosphorylation was unaffected in HeLa cells treated
for 1 h with 3 (100 and 500 μM; Figure 5B). Arsenite, which is
known to induce eIF2-R phosphorylation, was used as a
positive control.
Stress granules are storage regions for untranslated
mRNAs that appear in the cytoplasm of cells under stress
conditions. To examine whether 3 induces stress granule
formation, immunofluorescence analyses were performed
with stress granules markers such as MLN51 and FMRP
proteins.32,33 Interestingly, 3 treatment induced the forma-
tion of stress granules in HeLa cells at doses with which it
inhibits protein synthesis (concentrations of 3 > 10 μM) and
that are 1000 times higher than the effective cytotoxic con-
centrations (Figure 6).
Fluorescent Probe 5 Accumulates in Endoplasmic Reticu-
lum of Cancer Cells. To identify the intracellular localization
and therefore the potential molecular target of flavaglines,
we examined the cellular uptake of 5 in HeLa cells by
fluorescence microscopy. This probe displayed a cytotoxicity
against HL60 cells with an IC50 of 130 nM, indicating that it
maintained a critical proportion of the activity of 3. Living
HeLa cells were incubated with this fluorescent probe and a
specific signal was localized in the cytoplasm (Figure 7), thus
excluding the nucleus, Golgi apparatus, endolysosomes, and
the plasma membrane as the locus of the potential targets.
Note that no accumulation of the unconjugated coumarin 21
in any specific compartment was observed. To further
analyze the subcellular localization of 5, HeLa cells were
treated with 5 and specific fluorescent probes for the mito-
chondria (Mitotracker), the endoplasmic reticulum (ER-
tracker) and the endoplasmic reticulum (ER), and Golgi
apparatus (BODIPY-brefeldin A). There was no colocaliza-
tion of 5 with the mitochondrial marker (Figure 7A). In
contrast, a clear overlap of both 5 and ER-specific probes
was found (Figure 7B,C). These results suggest that 5
accumulates in the ER and that the molecular target of 5
should reside in the ER rather than in another subcellular
localization.
The recent discovery that flavaglines inhibit the eukaryotic
translation initiation factor 4A (eIF4A) prompted us to
examine by a pull-down experiment whether an immobilized
flavagline binds to this factor.34 First, derivative 6 was
immobilized onto an Affi-Gel 10 matrix through a linker.
Pull down assays did not reveal any interaction between 6
and eIF4A (Supplementary Figure S5), suggesting that
flavaglines might not physically interact with eIF4A.
Compound 3 Induces the Translocation of AIF and Caspase-
12 to the Nucleus. Apoptosis inducing factor (AIF) is a
mitochondrial protein that can induce apoptosis in a cas-
pase-independent manner.35 Upon an excessive calcium
influx, AIF is cleaved by μ-calpain, released in the cytosol,
and translocates to the nucleus where it induces chromatin

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5181
Figure 4. Compound 3 blocks HL60 cells in G2/M phase and induces apoptosis in a caspase-independent manner. (a) Blockade of HL60 cell
cycle progression by 1 and 3. HL60 cells were treated with doxorubicin (20 nM), 1 (200 nM), and 3 (20 nM) for 48 h. Cells were stained with
propidium iodide, and the cell cycle distribution was determined using FACS (n = 4, each sample was duplicated). (b) Compound 3 induces
apoptosis in HL60 cells. HL60 cells were treated with 3 (20 nM) for 72 h and apoptosis was quantified by staining with annexin V followed by
flow cytometry. (c) Compound 3 (10-9-10-6 M) does not activate caspases-3/7 in HL60 cells. HL60 cells were treated with 1, 3, or
doxorubicine as a positive control, for 48 h. Whole cell extracts were prepared and assayed for their ability to cleave the caspase-3-specific
substrate (DEVD-AMC). Relative fluorescence release was measured spectroscopically. Reaction rates were calculated from the slope of the
linear time-dependent reaction and expressed as the fold-activation over the control (HL60 with DMSO alone). (d) Inhibitors of caspases,
necroptosis and autophagy do not block the antiproliferative effects of 1 and 3 in HL60 cells. HL60 cells were treated for 72 h with 1, 3, or
vehicle in the presence or not of Z-VAD-FMK (50 μM), necrostatin-1 (50 μM), and 3-methyladenine (2 mM), which inhibit caspases,
autophagy, and necroptosis, respectively. Growth inhibition was not significantly altered by any of these inhibitors.
Figure 5. Compound 3 inhibitsprotein synthesis, independently from eIF2-R hosphorylation, at micromolar concentrations, i.e. at doses thatare
3 orders of magnitude greater than those observed for cytotoxicity. (a) Compound 3 inhibits translation of capped and polyadenylated luciferase
mRNA in a rabbit reticulocyte lysate (relative light units). Incubation time was 1 h. (b) Compound 3 does not induce the phosphorylation of eIF2-
R in HeLa cells. Samples of total protein (10 μg) obtained from HeLa cells that were untreated, treated for 1 h with arsenite (500 μM), DMSO
(vehicle), or 3 (100 or 500 μM) were analyzed by Western blotting with anti-eIF2-R and antiphospho-eIF2-R antibodies.
condensation and DNA fragmentation.36 We analyzed by
Western blot the effect of 3 on the localization of AIF in
HL60 cells (Figure 8). Note that total AIF level remained
unchanged upon 5 treatment. In nontreated cells, AIF was
exclusively detected in the mitochondrial fraction. By com-
parison, after treatment with 20 nM 3 for 72 h, AIF was
located mainly in the nucleus, indicating that in these cells,
flavaglines induce apoptosis through the translocation of

5182 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Thuaud et al.
Figure 6. High concentrations of 3 induce the formation of stress
granules in HeLa cells. (a) Immunofluorescence analysis of the
stress-granules markers MLN51 and FMRP in HeLa cells after
treatment with 3. Cells were colabeled with anti-MLN51 (green) and
anti-FMRP (red) antibodies. Treatment times are indicated on the
right in hours (h). (b) Immunofluorescence analysis of HeLa cells
treated with DMSO, cycloheximide (CHX; 10 μg/mL), puromycin
(Puro; 100 μg/mL), or 3 (50, 100, or 500 μM) for 1 h. Immuno-
fluorescence was assessed with an antibody to FMRP, a marker of
stress granules. Cells were stained with an anti-FMRP antibody
(red) and nuclei were counterstained with Hoechst 33258 (blue).
Note that 3 induced the formation of stress granules at 50 μM, which
is a concentration 10000 times higher than the effective cytotoxic
doses. The protein synthesis inhibitors cycloheximide and puromy-
cine were used as positive controls.
Figure 7. Subcellular localization of probe 5. Upper panel, HeLa
cells were treated with 50 μM of probe 5 (a, blue) for 2 h and 15 min
with 1 μM of Mito-Tracker Red dye (b, red). Merged confocal
image (c) shows that 5 does not localize in mitochondria. Middle
panel, HeLa cells were treated with 50 μM of 5 (d, blue) for 2 h and
15 min with 1 μM of ER-tracker dye (e, red). Merge image (f) shows
colocalization of both signals. Lower panel, HeLa cells were treated
with 50 μM of 5 (h, blue) for 2 h and 15 min with 1 μM of BODIPY
558/568 brefeldin A (i, red), merge image (j) shows colocalization 5
and the brefeldin A-ER signal and not with the brefeldin A-Golgi
signal. ER-tracker red dye and BODIPY 558/568 are selective for
the endoplasmic reticulum (ER) and both the ER and Golgi
apparatus, respectively. Merged confocal images show that probe
5 colocalizes with ER but not with the Golgi apparatus (yellow
arrows). Inserts in the lower left corner of each image show a 2-fold
magnification. Note that probe 5 does not localize in punctuate
cytoplasmic foci like stress granules. Control probe 21 (uncon-
jugated coumarin) gave no signal (not shown).
AIF to the nucleus. We then examined by immunohisto-
chemistry whether 3 activates AIF in another human cancer
cell line, and whether it also induces the translocation of
caspase-12 from the ER to the nucleus, because this pro-
apoptotic protein is involved in a cross-talk with AIF in ER
stress-induced apoptosis.35,37-39 In Hela cells treated with
the vehicle only, antibodies specific to AIF or caspase-12
diffusely stained the perinuclear region of the cytoplasm. In
contrast, in Hela cells treated with 3 (50 nM) for 72 h, these
antibodies stained specifically the nucleus. The nuclear
localization of AIF and caspase-12 was confirmed by co-
staining with DAPI.
Next we examined the role of caspase-12 and of its
activator, calpain, in the cytotoxicity of 3 in HL60 cells
utilizing inhibitors of these enzymes. Caspase-12 inhibitor
ATAD (Figure 8c) partially but significantly decreases the
cytotoxicity of 3 (20 nM) in HL60 cells. It is noteworthy that
ATAD itself displays some cytotoxicity in HL60 cells. These
data indicate that caspase-12 is partially involved in
3-induced apoptosis in HL60 cells. We did not observe
any diminution of 3 cytotoxicity in presence of m-calpain
inhibitor N-acetyl-Leu-Leu-methioninal, probably because
of the intrinsic cytotoxicity of this compound. All to-
gether these data suggest that 3 may induce apoptosis
through two independent pathways that involve AIF and
caspase-12.
Discussion
All previously reported structure-activity relationships of
flavaglines were performedwithnatural compoundsextracted
from plants. Modifications of the configuration of substituent
at C-1, C-2, C-3, and C-3a positions, and methylation of the
OH group at C-1, abolished cytostatic or cytotoxic activity,
whereas substitution at C-2 by esters or amide groups is well
tolerated (a more exhaustive presentation of the structure-
activity relationships is reviewed in refs 1 and 2). Our present
study extends this knowledge by showing that replacement of
an electron-donating methoxy (R1 =OMe) by a more lipo-
philic bromide significantly improves the cytotoxic potential.
The cognatecompound 3 displays a cytotoxicitythatisatleast
10 times more potent than those of doxorubicin, docetaxel,
cisplatin, camptothecin, or vinblastine on a variety of human
cancer cell lines.
Flavaglines derivatives 1 and 3 offer important advantages
as antineoplastic agents. Flavaglines 1 and 3 are cytotoxic at

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5183
Figure 8. Compound 3 induces the translocation of AIF and caspase-12 to the nucleus. (a) After a 72 h treatment with 3 (20 nM) or its vehicle,
HL60 cells were subjected to subcellular fractionation, and immunoblotting was performed with nuclear and mitochondrial fractions. β-Actin
was used as an internal control. (b) Immunocytochemistry of AIF and caspase-12 in Hela cells treated for 72 h with 3 (50 nM) or its vehicle. Cells
were colabeled with anti-AIF (green) and anti-caspase-12 (red) antibodies. Nuclei were stained by DAPI. (c) Inhibition of caspase-12 partially
but significantly decreases the cytotoxicity of 3 in HL60 cells. HL60 cells were treated for 72 h with 3 (10 nM) or its vehicle in the presence or not
of m-calpain inhibitor (20 μM) and caspase-12 inhibitor ATAD (10 μM).
concentrations in the nanomolar range, they seem insensitive
tothe acquiredMDR1-resistance inMCF7R cell, and they act
synergistically with anthracyclines in HepG2 cells. These
observations are reminiscent of Stockwell’s recent report that
other inhibitors of protein synthesis (cycloheximide, emetine,
anddihydrolycorine) alsopotentiate doxorubicin’slethality.40
Moreover, combined treatment with 1 or 3 and doxorubicin
showed a cumulative cytotoxic effect on HepG2 cells, indicat-
ing that flavaglines could be added to existing antineoplastic
treatments. Another important feature of flavaglines is their
absence of cardiotoxicity, which often occurs with anticancer
drugs and which is the main limitation to the clinical use of
anthracyclines.
Except for silvestrol and episilvestrol that have the pecu-
liarity of a complex dioxanyl ring, flavaglines are generally
consideredtobecytostatic ratherthancytotoxic.4-8 However,
here we found that flavaglines display a strong cytotoxicity
toward many cancer cell lines. We showed that 1 and 3 block
the cell cycle in phase G2/M, which is consistent with previous
reports indicating that flavaglines cause dose-dependent
G2/M phase arrest of various cancerous cell lines.5,6,11 This
G2/Mcell cycle arrestisassociated withthe intrinsic apoptosis
pathway and is induced by many other clinically used cyto-
toxic drugs.38,39 Consistent with this finding, we found that
the cytotoxic effect of 1 and 3 is due to induction of apoptosis
in HL60 cells. Surprisingly, caspase assays indicated that
caspases 3/7 were not specifically activated. This was con-
firmed by the observation that the broad caspase inhibitor
Z-VAD-FMK did not alter the cytotoxicity of these com-
pounds. The weak induction of caspases 3/7 and the absence
of an effect in HeLa cells on caspases 8 and 9 by 1 and 3 are
reminiscent of Kinghorn and Marian’s observation that
natural flavaglines silvestrol and aglaiastatin induce apoptosis
without activating caspase-3 in SW480 and LNCap cells.6,10
In contrast, Swanson showed that rocaglaol (nonracemic 1)
increased the level of cleaved caspase-7 and induced the
intrinsic apoptotic pathway in the same LNCap cell line.11
This discrepancy may be due either to a genetic instability and
divergence of LNCap cells or to the fact that silvestrol and
aglaiastatin do not act through the same molecular targets as
rocaglaol. Also in contrast with our findings, Li-Weber
reported that rocaglamide induces caspases 2, 3, 8, and 9
and triggers apoptosis through the intrinsicpathway in several
leukemia cell lines isolated from patients.14 These data clearly
indicate that flavaglines may trigger apoptosis by different
pathways according to the cancer cell types and may also be
from the chemical structure of the drug.
Although the role of apoptosis in the cytotoxicity of antic-
ancer drugs is well established, it is now evident that alter-
native forms of regulated cell death, such as autophagy or
necroptosis, may also be involved.41,42 Necroptosis occurs
when a death receptor, such as Fas/CD95, TNFR1, death
receptor (DR)4, and DR5, has been activated (e.g., by TNF-
R) in the presence of a broad spectrum caspase inhibitor such
as Z-VAD-FMK.27 This form of death is characterized by
necrotic cell death morphology and activation of autophagy.
Necrostatin-1, which blocks necroptosis by inhibiting the
adaptor kinase RIP1,43 has been shown to prevent the death
of MCF-7 and HEK293 cells induced by shikonin, a naturally
occurring cytotoxic naphthoquinone.44 Similarly, 3-methyla-

5184 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Thuaud et al.
denine, an inhibitor of autophagy reduced the cytotoxicity of
the anticancer drug etoposide in CaSki cells.45 In our case, the
cytotoxicity of 3 in HL60 was not reduced by Z-VAD-FMK,
necrostatin-1, or 3-methyladenine, which indicates that, in
these cells, flavaglines do not induce autophagy or necropto-
sis. However, we showed for the first time that flavagline
3 induces the death of HL60 and Hela cells via activation of
the AIF and caspase-12 pathways, suggesting that 3 induces a
stress in the ER.35-39 This hypothesis is supported by the
localization of probe 5 in the ER and by the formation of
stress granules induced by 3, albeit at high concentration. The
ER fulfills specialized functions that are critical to the cell
(protein translation and maturation, lipid synthesis, Ca2þ
storage, and so on), thus, a dysregulation of ER homeostasis
may jeopardize cell survival. It is now established that cross-
talk between ER and mitochondria is decisive to commit the
cell to apoptosis.46,47 Marigo and collaborators have shown
that ER stress in response to protein misfolding or disruptions
of Ca2þ homeostasis activates at the same time AIF and
caspase-12 apoptotic pathways in fibroblasts and models of
retinitis pigmentosa.38,39 Interestingly, both of these pathways
were shown to reinforce each other.
Figure 9. Proposed model for apoptosis induced by flavaglines in
cancer cells. The binding to the molecular target of flavaglines
(MTF) located in the ER triggers a cascade of events that lead to
translocation of AIF and caspase-12 to the nucleus and also to
inhibition of eIF4A.
The action of AIF has been shown to be critical for the
cytotoxicity of other anticancer agents, such as arsenic tri-
oxide for cervical cancer cells,49 cisplatin for LNCaP prostate
cancer cells,50 taxolforSKOV3ovarian carcinomacells,51 and
flavopiridol for murine glioma cells.52 More importantly,
activation of AIF signaling by staurosporine induces cell
death of nonsmall-cell lung carcinoma cells, which are highly
resistant to conventional anticancer treatments.53 Similarly,
phytosphingosine treatment reversed the resistance of human
T-cell lymphoma to gamma-radiation by induction of AIF-
mediated apoptosis.54 Caspase-12 has been shown to play a
pivotal role in the induction of apoptosis by geldanamycin in
BC-8 histiocytoma cells48 and cisplatin in LLCPK renal
epithelial cells.55 Interestingly, VR3848, a novel cytotoxic
cycloheptapeptide, has been recently shown to induce apop-
tosis in MCF-7 cells also through simultaneous stimulation of
AIF and caspase-12 pathways.56
Previous studies showed that flavaglines are cytostatic or
cytotoxic on all cancer cell lines tested, which suggests that the
molecular target of these drugs is ubiquitous and that it affects
a fundamental cellular pathway whose downstream effects
lead to growth arrest or cell death in cancer cells. We found
that 3 at high concentration induces stress granules and
inhibits protein synthesis. Very recently, Pelletier and collea-
gues have shown that two naturally occurring flavaglines
target protein synthesis through the translation initiation
factor eIF4A by increasing its binding to RNA, thereby
causing its sequestration and inhibiting cap-dependent trans-
lation.34 These authors showed in particular that silvestrol
and 1-O-formylaglafoline, two natural flavaglines, inhibit
cap-dependent translation of firefly luciferase in vitro in cell-
free Krebs-2 extracts, with IC50s of 0.3 and 3 μM. In a similar
assay we observed that 3 inhibited translation in reticulocyte
lysates at comparable concentrations, with an IC50 of 0.2 μM.
We also showed that inhibition of protein synthesis at the high
concentration of 3 was not due to the phosphorylation of the
eukaryotic initiation factor 2R (eIF-2R), consistent with the
findings of Pelletier and colleagues, obtained for other flava-
glines. However, pull-down experiments with immobilized
ligand 6 suggested that flavaglines probably do not bind
directly to eIF4A or that this binding requires another part-
ner, such as mRNA or a scaffold protein.
Our microscopy data with probe 5 suggest that flavaglines
might act on an ER component. The ER is the site of synthesis
for glycoproteins and also membrane-bound and secreted
proteins, which suggests that 3 may impair protein synthesis
at the ER. It is therefore tempting to hypothesize that the
binding of flavaglines to a target located in the ER may induce
apoptosis in cancer cells through a cascade of events that
include the translocation of AIF and caspase-12 to the nucleus
and inhibition of eIF4A (Figure 9). The detailed molecular
mechanism underlying flavagline cytotoxicity is currently
under investigation.
Conclusion
We demonstrated for the first time that a flavagline (3)
induces the death of cancer cells via activation of the AIF and
caspase-12 pathways. This compound offers important ad-
vantages as antineoplastic agents. First, it is cytotoxic at
concentrations in the nanomolar range on cancer cells but
not on cardiomyocytes. Second, it is insensitive to the ac-
quired MDR1-resistance in MCF7R cells. And third, it acts
synergistically with anthracyclines independently of caspases-
3, -7, -8, and -9, suggesting that it would retain its activity in
cells refractory to activation of these caspases.57 Considering
that drug resistance and side effects are the two major
obstacles limiting the efficacy of cancer chemotherapy, our
results reinforce the view that flavaglines hold considerable
potential for the treatment of cancers.
Experimental Section
Chemistry. General Methods. All reagents and solvents for
syntheses were purchased from Sigma-Aldrich, Fluka, or Acros
and used without further purification. Reagent-grade solvents
were purified and dried using standard methods. Reactions were
carried out under an argon atmosphere using flame-dried
glassware with magnetic stirring and degassed solvents. Column
chromatography was carried out on silica gel 60 (Merck, 70-
230 mesh). 1H NMR spectra at 300 MHz and 13C NMR spectra
at 75 MHz were recorded with DPX 300 SY Bruker spectro-
meters with the deuterated solvent as the lock and residual
solvent as the internal reference. Infrared spectra were recorded
using a Perkin-Elmer 881. Purity of target compounds were over

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5185
95% based on reversed-phase HPLC analyses (Hypersil Gold
column 30 ꢀ 1 mm, C18) under the following conditions: flow
rate: 0.3 mL/min; buffer A: CH3CN, buffer B: 0.01% aqueous
TFA; gradient: 98-10% buffer B over 8 min (detection: λ=220/
254 nm).
(CDCl3): 36.1, 53.2, 55.6, 55.7, 79.0, 89.4, 92.5, 94.8, 103.1,
107.4, 121.4, 126.4, 127.7, 127.9, 129.4, 130.2, 134.1, 138.1,
156.8, 160.6, 163.9. IR (thin film): 3486, 2942, 2841, 1624, 1598,
1147 cm-1. HR-MS calcd for C25H23Br1K1O6, 521.0366; found,
521.0360.
3-(2-(4-Bromophenyl)-2,3-dihydro-4,6-dimethoxy-3-oxobenz-
ofuran-2-yl)-3-phenylpropanal (9c). A suspension of benzofur-
anone 7 (R1=Br, 4.7 g, 13.5 mmol) in t-BuOH (300 mL) was
heated to 50 ꢀC under argon. Benzyltrimethylammonium hy-
droxide in MeOH (40%, 306 μL, 0.73 mmol) and, immediately
after, cinnamaldehyde 8 (3.40 mL, 27.0 mmol) were added. The
mixture was stirred for 2 h at 50 ꢀC, cooled to rt, concentrated
and acidified with 1 M HCl (30 mL), extracted with CH2Cl2,
dried over MgSO4, and concentrated to dryness. Purification of
the resulting yellow solid residue by chromatography (Et2O-
pentane 6:4) yielded 1.94 g (33%) of ketoaldehyde 9c as a white
solid: Rf 0.4 (Et2O/hept 9:1). 1H NMR (CDCl3): 2.61 (1H, ddd,
J=0.9, 4.0, 17.3 Hz), 3.07 (1H, ddd, J=2.3, 10.9, 17.3 Hz), 3.70
(3H, s), 3.85 (3H, s), 4.19 (1H, dd, J = 4.0, 10.9 Hz), 5.81 (1H, d,
J=1.8 Hz), 6.21 (1H, d, J=1.8 Hz), 7.08-7.17 (3H, m), 7.29-
7.32 (2H, m), 7.49, 7.63 (4H, AA0BB0, J = 8.7 Hz), 9.41 (1H, d,
J=1.1 Hz). 13C NMR (CDCl3): 44.0, 46.8, 55.9, 55.9, 88.4, 93.0,
103.7, 122.6, 126.9, 127.5, 128.2, 129.5, 131.8, 132.0, 135.6,
136.3, 159.1, 169.9, 174.1, 194.1, 200.0. IR (thin film): 2725,
1720, 1699, 1617, 1590, 1154, 747 cm-1. HR-MS calcd for
C25H21BrLiO5, 487.0732; found, 487.0727.
3a-(4-Bromophenyl)-6,8-dimethoxy-8b-hydroxy-3-phenyl-2,
3-3a,8b-tetrahydro-cyclopenta[b]benzofuran-1-one (12c). Tri-
methylsilyl cyanide (744 mg, 7.5 mmol) was added dropwise to a
solution of aldehyde 9c (1.2 g, 2.5 mmol) in CH3CN (12 mL) at
room temperature under argon. Immediately after, zinc iodide
(10 mg) was added, and the resulting mixture was stirred for
1 h, filtered, and concentrated to afford crude cyanohydrin 11c
(1.74 g, 40%), which was used in the next step without purifica-
tion. LDA (2.7 mmol, 0.6 M) in THF was added dropwise at
-78 ꢀC under argon to a solution of cyanohydrin 11c (1.43 g,
2.46 mmol) in dry THF (12 mL). After stirring for 2 h at -78 ꢀC,
the solution was heated to -50 ꢀC for 10 min. The reaction was
quenched by the addition of saturated aqueous ammonium chlor-
ide (15 mL). Standard extractive workup (CH2Cl2) gave a yellow
solid (1.49 g). This solid was directly treated with tetra-n-butylam-
monium fluoride (2.7 mL, 1 M in THF) added dropwise at room
temperature in dry THF (10 mL). The solution was stirred for 4 h
and quenched by addition of methanol. Standard extractive work-
up (AcOEt) and purification by flash chromatography (Et2O)
afforded tricyclic ketone 12c (240 mg, 20%) as a white solid: Rf 0.35
(Et2O/hept 8:2). 1H NMR (CDCl3): 2.96-3.09 (2H, m), 3.21 (1H,
br s), 3.81 (3H, s), 3.84 (3H, s), 3.90 (1H, dd, J=10.2, 12.1 Hz), 6.10
(1H, d, J=1.9 Hz), 6.33 (1H, d, J=1.9 Hz), 6.89-6.94 (4H, m),
7.10-7.12 (3H, m), 7.24-7.27 (2H, m). 13C NMR (CDCl3): 39.6,
48.6, 55.6, 55.7, 88.6, 89.7, 92.8, 100.8, 106.2, 121.7, 127.1, 127.8,
128.0, 128.3, 130.7, 133.0, 136.6, 158.4, 160.9, 164.8, 210.3. IR (thin
film): 3477, 2942, 2841, 1750, 1621, 1597, 1149 cm-1. HR-MScalcd
for C25H21Br1Na1O6, 503.0470; found, 503.0465.
Biological Assay Methods. Materials. Necrostatin-1 was
synthesized according to the procedure of Degterev et al.27
3-Methyladenine was purchased from Acros Organics,
Z-VAD-FMK from Bachem, and ATAD from PromoCell.
Rhodamine 123 and N-acetyl-Leu-Leu-methioninal were from
Sigma and DEVD-AMC, LEHD-AMC, IETD-AMC, MG132,
LLVY-AMC, and RLR-AMC were purchased from Biomol.
Rabbit antiactive caspase-3 polyclonal antibody was bought
from Chemicon International.
eIF2r Phosphorylation by Western Blotting. HeLa cells were
treated or not with DMSO, sodium arsenite (500 μM), or 3 (100
and 500 μM) for 1 h. Cell lysates were employed to SDS-PAGE.
The blots were incubated with horseradish peroxidase-conju-
gated (HRP) AffiniPure donkey antirabbit at 1/10000 (Jackson
ImmunoResearch). Finally, protein-antibody complexes were
visualized by an enhanced chemiluminescence detection system
(ECL detection reagent, Amersham).
Detection of Stress-Granules by Immunofluorescence. HeLa
cells were seeded on glass coverslips in 24-well plates in DMEM
supplemented with 5% of fetal calf serum. After 24 h of culture,
cells were treated with cycloheximide (10 μg/mL) or puromycin
(100 μg/mL) for 1 h or with different concentrations of 3 (50 nM
to 500 μM) for 30 min to 8 h and examined by immunofluor-
escence using rabbit anti-MLN51 and mouse anti-FMRP anti-
bodies as previously described.32 See Supporting Information
for more details.
Preparation of Nuclear and Mitochondrial Fractions. HL60
cells (105) were seeded and treated with 3 (20 nM) or its vehicle
(DMSO). After 72 h the cells were collected and washed with
PBS, incubated on ice for 10 min, and then resuspended in
isotonic homogenization buffer (250 mM sucrose, 10 mM KCl,
1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothrei-
tol, 10 mM Tris-HCl, pH 7.4, containing protease inhibitor
mixture (Roche)). The cells were sonicated for 1 min at 30%
amplitude. Unbroken cells were removed by centrifugation
(30ꢀg for 5 min). Nuclei and mitochondrial fractions were se-
parated by centrifugation at 750 ꢀ g for 20 min and 14000 ꢀ g for
30 min, respectively. The nuclear fraction was washed three times
with 0.01% (v/v) NP40 containing isotonic homogenization
buffer. The nuclear and mitochondrial fractions were then sub-
jected to Western blot analysis with a rabbit anti-AIF polyclonal
antibody (1: 500, Santa Cruz Biotechnology, U.S.A.).
Acknowledgment. This article is dedicated to our friend
and mentor, Prof. Roger A. Johnson. Generous financial
support for this work was provided to L.D. by the “Associa-
tion pour la Recherche sur le Cancer” (ARC, grant #3940).
We thank ARC (A.B.) and MNESR (F.T. and R.D.) for
fellowships and Dr. Markus R. Dobler (Syngenta, Basel) for
helpful discussions.
3a-(4-Bromophenyl)-6,8-dimethoxy-3-phenyl-1,2,3,3a-tetra-
hydro-cyclopenta[b]benzofuran-1,8b(1H)-diol (3). CH3CN (1.8 mL)
was added at room temperature under argon to Me4NBH(OAc)3
(828 mg, 3.15 mmol) followed by the addition of glacial acetic acid
(1.8 mL). After stirring for 30 min at room temperature a solution
of ketone 12c (170 mg, 0.354 mmol) in CH3CN (4.5 mL) was added
dropwise. The resulting mixture was stirred overnight at room
temperature. The reaction was quenched by the addition of
saturated aqueous ammonium chloride (40 mL). Standard extrac-
tive workup (AcOEt) and purification by flash chromatography
(Et2O) afforded 3 (103 mg, 60%) as a white solid: Rf 0.25 (Et2O/
hept 8:2). 1H NMR (CDCl3): 1.89 (1H, br s), 2.14 (1H, dd, J=7.0,
13.3 Hz), 2.67 (1H, ddd, J=6.4, 13.9, 13.9 Hz), 3.27 (1H, br s), 3.83
(3H, s), 3.86 (3H, s), 3.99 (1H, dd, J=6.5, 14.0 Hz), 4.77 (1H, d,
J=6.0 Hz), 6.10 (1H, d, J=1.8 Hz), 6.28 (1H, d, J=1.8 Hz), 6.95-
6.98 (2H, m), 7.05-7.12 (5H, m), 7.23-7.26 (2H, m). 13C NMR
Supporting Information Available: Supplementary biological
data (effect on the body weight in mice, cell cycle arrest in HL60
cells, activation of caspases 8 and 9, antiproliferative effects in
presence of inhibitors of caspases, necroptosis, and autophagy,
proteasome assay, and 6-agarose pull-down assay), synthesis of
compounds 1, 2, and 4-6, and supplementary biological assay
methods. This material is available free of charge via the Internet
at http://pubs.acs.org.
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