Synthesis and evaluation of the cytotoxicities of tetraindoles: Observation that the 5-hydroxy tetraindole (SK228) induces G2 arrest and apoptosis in human breast cancer cells

Article abstract of DOI:10.1021/jm2013425

Current chemical and biological interest in indole-3-carbinol (I3C) and its metabolites has resulted in the discovery of new biologically active indoles. As part of a program aimed at the development of indole analogues, tetraindoles 1-15 were prepared an

Full text of DOI:10.1021/jm2013425

Article
pubs.acs.org/jmc
Synthesis and Evaluation of the Cytotoxicities of Tetraindoles:
Observation that the 5-Hydroxy Tetraindole (SK228) Induces G2
Arrest and Apoptosis in Human Breast Cancer Cells
,
†,§
†,‡,#
†,∥,#
⊥
†
†
Wen-Shan Li,* Chie-Hong Wang,
Shengkai Ko,
Tzu Ting Chang, Ya Ching Jen,
,
∥
†
Ching-Fa Yao,* Shivaji V. More, and Shu-Chuan Jao
†
Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan
‡
Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan
§
Doctoral Degree Program in Marine Biotechnology, National Sun Yat-Sen University, Kaohsiung 804, Taiwan
∥Department of Chemistry, National Taiwan Normal University, Taipei 116, Taiwan
⊥Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan
*
S Supporting Information
ABSTRACT: Current chemical and biological interest in
indole-3-carbinol (I3C) and its metabolites has resulted in
the discovery of new biologically active indoles. As part of a
program aimed at the development of indole analogues,
tetraindoles 1−15 were prepared and their antiproliferative
effects on human breast cancer cells were evaluated. The results
show that the 5-hydroxy-tetraindole 8 (SK228) has optimum
antiproliferative activity against breast adenocarcinoma (MCF 7
and MDA-MB-231) cells and that this activity involves G2-
phase arrest of the cell cycle with a distinctive increase in the
expression of cyclin B1 and phospho-cdc2. Further observa-
tions suggest that 5-hydroxy-tetraindole 8 induces apoptosis through externalization of membrane phosphatidylserine, DNA
fragmentation, and activation of caspase-3. Given the fact that I3C and its metabolites have been shown to improve therapeutic
efficacy and to have a broad range of antitumor activities in human cancer cells, the current findings have important
pharmacological relevance as they open a promising route to the development of a potential chemotherapeutic application of
tetraindoles as agents for the treatment of breast cancer.
INTRODUCTION
The utility of indoles, such as indole-3-carbinol (I3C), 3,3′-
diindolylmethane (DIM), indole-3-carbinol cyclic trimer CTr,
and cyclic tetramer CTet (Chart 1), as chemopreventive and
chemotherapeutic agents has recently been recognized. Indole-
highly likely that multiple mechanisms (e.g., Akt-NFκB
signaling, caspase activiation, cyclin-dependent kinase activity,
estrogen receptor signaling, endoplasmic reticulum stress, and
BRCA gene expression) are operating in different proportions
■
1
2
3
4
1
−4,7
under different conditions within various cell types.
In
contrast, therapeutic applications based on CTet have
developed only slowly. Although I3C has been studied in the
3
-carbinol (I3C) is a dietary compound and an autolysis
product of glucosinolate (glucobrassicin), which was identified
and isolated from Brassica vegetables such as cabbage, broccoli,
and Brussels sprouts. The results of epidemiological and dietary
studies continue to demonstrate that high dietary intake of
5
context of cancer prevention in preclinical trials, it is not
readily amenable to general systemic therapeutic purposes
owing to its poor efficacy and unacceptable pharmacokinetic
8
5
properties. In the stomach, gastric acids convert the inactive
cruciferous vegetables can prevent carcinogenesis. Moreover,
prodrug I3C into the pharmacologically active metabolites
DIM, CTet, or other indole oligomers that possess in vivo
I3C, DIM, and CTet are known to exhibit a broad spectrum of
antiproliferative effects on various tumors (e.g., breast, colon,
prostate, and endometrial cancers) with effective concen-
1
−4
antitumor activity.
1
−4,6
Because they serve as potential chemical building blocks for
construction of different I3C-related antitumor agents
employed in demonstrated apoptotic pathways, indoles have
garnered considerable medicinal interest. For example, a variety
trations in the range of 1−100 μM.
Unlike I3C and its
stomach digest products (DIM and CTet), the cyclic trimer
CTr functions by stimulating the proliferation of estrogen-
responsive MCF-7 cells that contributes to the overall
4c
estrogenic effect of oral I3C.
The exact mechanism(s) of I3C and DIM mediated
suppression of cancer growth remains under studied. It is
Received: October 7, 2011
Published: January 25, 2012
©
2012 American Chemical Society
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Chart 1
RESULTS AND DISCUSSION
■
Design and Synthesis. Tetraindoles 1−4 (Chart 2),
bearing an aromatic central core structure, were prepared in
satisfactory yields by addition reactions of various dialdehydes
1
6−19 with indole in the presence of catalytic amounts of
20
molecular iodine at room temperature (Scheme 1). By
Scheme 1
employing this strategy, tetraindole 1 analogues 5, 7−9, 12, and
of structurally diverse synthetic indole-based compounds, such
20 were synthesized by reacting terephthalaldehyde 16 with
9
10
11
12
13
as BPR0L075, I-387, NSC-741909, SR13668, NB-506,
various substituted indoles under the same condition (Scheme
2, pathway I). Sequential removal of the Fmoc group of 20 gave
10. Tetraindole 11, where the central core structure is replaced
by a 2,2′-oxybisbenzene, was synthesized starting with
isophthalaldehyde (17) and 5-hydroxyindole.
1
4
15
16
17
OSU-A9, ES936, EO9, KW-2189, and their analogues,
display tumor growth inhibition against a wide range of cancer
cell lines and effective antitumor activities in various preclinical
models. We have recently demonstrated that 3-indole (triindole
or 1,1,3-tri[3-indolyl]cyclohexane; Chart 1), possessing a
cyclohexane core structure, induces apoptosis in various lung
Electron-withdrawing (Br) and electron-donating groups
(
OH), were introduced at the C-5 position of the indole ring to
18
generate unsymmetrical tetraindoles 6 and 14, prepared as
outlined in Scheme 2 (pathway II). Under similar conditions,
the intermediate aldehydes 21 and 22 could be isolated and
undergo reactions catalyzed by molecular iodine to afford the
respective unsymmetrical tetraindoles 6 and 14. In contrast, the
unsymmetrical mono-5-hydroxyindole 13 and tri-5-hydroxyin-
dole 15 were prepared by iodine-catalyzed addition reactions of
terephthalaldehyde (16) with different molar ratio of indole
and 5-hydroxyindole (Scheme 2, pathway III). It should be
noted that the studies described above resulted in the
development of a new strategy to prepare unsymmetrical
tetraindoles in high yields.
cancer cells and xenograft models. The apoptosis results from
an intrinsic mitochondrial pathway involving stress-activated
pathways, including reactive oxygen species (ROS) and c-Jun
18
N-terminal kinase (JNK) activities.
In the study described below, we designed and synthesized a
series of tetraindole analogues (Chart 2), derived from the 3-
indole platform, and explored their therapeutic effects against
breast cancer cells. Special interest focused on the identification
of central core structures (benzene, oxidibenzene, or thiophene
in Chart 2), which would support biological activity and whose
substructure optimization would effectively produce substances
with high antitumor activities. In this effort, 5-hydroxy
Growth Inhibition by Tetraindoles against Human
19
Breast Cancer Cells. Inspired by the results of previous
tetraindole 8 (SK228) was identified as a potential strong
lead compound based on its effectively therapeutic action, with
IC50 values falling in the nanomolar range. Observations made
18
studies of 3-indole in lung cancer and the antiproliferative
effects of I3C and its digestion products on breast cancer cells
1−4
(
Table 1),
we postulated that an increase in the number of
in the investigation suggest that 8 (SK228) mediates G -phase
2
active indole rings in a substance would facilitate its cell
cytotoxicity and, hence, its inhibition of tumor cell growth in
vitro. As a first step in exploring this proposal, we designed and
prepared new tetraindoles 1−4 (Chart 2), bearing different
arrest along with apoptosis involving the intrinsic pathway. The
findings lead to a new paradigm for the pharmacological use of
tetraindoles in the treatment of human breast cancer.
Chart 2
1
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Scheme 2
central core structures (e.g., benzene, oxidibenzene, or
thiophene), and evaluated their antiproliferative activities
against human breast cancer cells using an MTT assay. The
results show that unlike 3 and 4, which exhibit less potent
effects against MDA-MB-231, 1 and 2, possessing the
respective 1,4-benzene and 1,3-benzene core structural units,
display improved growth-inhibitory potency with respective
IC50 values of 9.8 and 24.3 μM (Table 1). Furthermore, in
comparison to the other tetraindoles, 1 displays a high potency
toward MCF 7 breast adenocarcinoma cells (16.1 μM). These
observations indicate that a benzene rather than oxidibenzene/
thiophene core structures are required for antiproliferative
activities of tetraindoles against breast cancer cells.
antiproliferative effect of 8 is 2−57 times more potent than
those of the well-known I3C metabolites, DIM and CTet
(Table 1). In contrast, the five other tetraindoles 5−7, 9, and
10 display no apparent effects on MDA-MB-231 cell growth at
20 μM concentrations. To our surprise, tetraindoles 5, 6, 9, and
10, bearing respective 5-bromo, -methoxy, and -amino
substituents on the indole ring, also display dramatically
decreased antiproliferative effects toward MCF-7 cells. It is
important to note that the 5-cyano tetraindole 7 is 2-fold more
potent (IC50 = 9.5 μM) than 1 (Table 1). Interestingly, the
relative IC values of 7 show dissimilar trends against the ER+
cell line MCF-7 and ER− cell line MDA-MB-231, an
observation that indicates that the effects on cell growth
inhibition might be caused by an effect on an estrogen receptor
(ER)-dependent pathway.
In the series explored, 8, possessing a hydroxy group at the
indole C-5 position, was found to be the most active compound
against MDA-MB-231 and MCF-7 (Table 1), suggesting that
the introduction of hydroxy substituents into position 5 on
each indole ring would favor cancer cell growth inhibition.
Indeed, tetraindole 11, bearing a hydroxy group at the C-5
50
Building on these observations, six new tetraindoles 5−10,
having a benzene core structure and electron-withdrawing (Br
and CN) and electron-donating groups (OH, OCH , and NH )
3
2
at C-5 positions of their indole rings, were prepared. Among
these substances, 8 was observed to have the highest
antiproliferative activity, with IC50 values of 0.45−0.88 μM
against two human breast cancer cell lines, which are 18−45-
fold lower than those of 1 and 3-indole (Table 1). Notably, the
1
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2
1
Table 1. Growth Inhibitory Activity of Tetraindoles Against
Breast Cancer Cells
exclusion method. Cells were treated with increasing
concentrations of 8 (0−800 nM) at 24, 48, and 72 h,
respectively (Figure 1). The results demonstrate that 8
a
compd
MDA-MB-231
MCF-7
DIM6
CTet4
10−20
1.0 ± 0.1
50.0 ± 8.0
1.3 ± 0.1
or 8.0 ± 0.5
19.8 ± 1.6
16.1 ± 0.6
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
-indole
20.1 ± 2.2
9.8 ± 1.7
24.3 ± 3.5
b
>20 (32)
b
b
>20 (8)
>20 (48)
b
b
>20 (36)
>20 (34)
b
b
>20 (5)
>20 (25)
b
b
>20 (8)
>20 (17)
b
>20 (8)
9.5 ± 0.9
0.45 ± 0.03
0.88 ± 0.04
b
b
>20 (3)
>20 (43)
b
b
0
1
2
3
4
5
>20 (35)
>20 (25)
b
>20 (0)
17.8 ± 3.7
b
b
>20 (16)
>20 (0)
Figure 1. Time- and dose-dependent antiproliferation effect of 8
(DMSO vehicle; 0.1, 0.2, 0.4, 0.6, 0.8 μM) on MDA-MB-231 cells.
Cells were plated at a density of 200000 cells per well in 6-well culture
plates and treated with the different concentrations of 8 in 10% FBS-
supplemented DMEM/F12 medium. At different time intervals, cells
were harvested by trypsinization and counted using a hemocytometer.
The values plotted are representative of three independent experi-
ments.
13.9 ± 1.6
12.6 ± 1.3
b
b
>20 (36)
>20 (23)
7.8 ± 0.6
6.3 ± 0.7
a
Amount of drug and tetraindoles 1−15 necessary to inhibit the
growth of breast cancer cells (MCF-7 and MDA-MB-231) by 50% in
8 h. Data are means ± SD (n = 3). The percent (%) inhibition, in
b
4
parentheses, at 20 μM is expressed as the percent (%) inhibition of cell
growth.
treatment significantly suppresses cell proliferation of MDA-
MB-231 cells and decreases the cell count in both a time and
dose-dependent manner. This result is in line with the previous
position of each indole ring connected to the 1,3-benzene core
structure, was found to have an improved growth-inhibitory
potency (IC50 = 17.8 μM) against MCF-7 as compared to the
parent compound 2. However, the introduction of a hydroxy
group at the C-6 position of the indole ring as in 12, results in
dramatically decreased potency compared to that of 8 toward
two breast cancer cell lines. This observation suggests that the
location of a hydroxy group at C-5 rather than C-6 on each
indole ring is important in bringing about suppression of breast
cancer cell growth.
The variation in the number of hydroxy groups of 8 raises an
intiguing question about whether or not there is a biological
divergence altering the presence or absence of C-5 hydroxy
group in tetraindoles. The relative order of antiproliferative
potency of 8 analogues is 8 (four hydroxy groups) > 15 (three
hydroxy groups) > 13 (one hydroxy group) ≫ 14 (two
hydroxy groups), implying that modifications of the total
number of 5-hydroxy groups affect the ability of tetraindoles to
modulate antiproliferative potency. The at least 7−44-fold
weaker potency of tetraindoles 13−15, compared with 8,
supports this suggestion.
19
observations.
In addition, in vitro antiproliferative activities of 8 against a
panel of human cancer cell lines, including MDA-MB-361
(
breast adenocarcinoma), MDA-MB-468 (breast adenocarci-
noma), SW480 (colon adenocarcinoma), SW620 (colon
adenocarcinoma), SW48 (colon adenocarcinoma), U-87 MG
(
glioblastoma), and A549 (lung adenocarcinoma), were
determined (Table 2). 8 was found to exhibit similar or greater
growth inhibitory activities against seven human cancer cell
lines (Supporting Information Figures S2−S8) compared to
MCF-7 and MDA-MB-231 cell lines. It is interesting to note
that 8 shows significant inhibition against the SW480 cancer
cell line.
Building on these cytotoxicity results, we extended studies to
evaluate the selectivity of 8 for malignant versus normal cells
(
breast epithelium cell line, M10, and skin embryo fibroblast,
Detroit 551). Selective cytotoxicity against tumor rather than
normal cells is an important factor determining the usefulness
of anticancer agents in the treatment of cancer. As the data in
Tables 1 and 2 show, 8 displays cytotoxicity against nine human
cancer cell lines with IC values ranging from 0.37 to 2.89 μM,
Compound 8 Decreases the Survival Rate of MDA-
MB-231 and Inhibits the Cells’ Growth of a Variety of
Cancer Cell Lines. A greater understanding of the mechanistic
source of the growth inhibitory effects of 8 should provide
insight into its mode of biological action and aid the design of
more effective antitumor agents. In the conventional MTT
assay, 8 was found to inhibit MDA-MB-231 cell viability (IC50
50
and shows a lower activity (2−54-fold less cytotoxic) against
two normal cell lines, with IC values ranging from 5.81−20.0
50
μM. These findings indicate that 8 displays a differential
selectivity for tumor over normal cells (Supporting Information
Figures S9 and S10).
=
0.45 μM) in a dose-dependent manner (Supporting
Compound 8 Induces G Phase Arrest. To determine
2
Information Figure S1). A similar effect was observed in
MCF-7 cells (data not shown), indicating that MDA-MB-231 is
more sensitive to 8-induced cell growth inhibition than is MCF-
whether the suppression of breast cancer cell growth by 8 is
caused by a cell cycle effect, MDA-MB-231 cells were cultured
with 800 nM 8 for 24 and 48 h, respectively, and cell cycle
progression was evaluated by using flow cytometry. The
analysis with MDA-MB-231 cells indicates that the number of
7
(IC = 0.88 μM). The antiproliferative effect of 8 on MDA-
50
MB-231 cells was also examined by using the trypan blue dye
1
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a
Table 2. Growth Inhibitory Activity of 8 against Seven Cultured Human Tumor Cell Lines
IC50 (μM)
compd MDA-MB-361 MDA-MB-468
2.07 ± 0.11 0.83 ± 0.06
SW480
SW620
SW48
U-87 MG
A549
2.89 ± 0.25
M10
Detroit 551
b
8
0.37 ± 0.05
0.98 ± 0.02
0.88 ± 0.05
2.73 ± 0.35
5.81 ± 0.28
>20 (47)
a
b
Amount of 8 necessary to inhibit the growth of a panel of human cancer cells by 50% in 48 h. Data are means ± SD (n = 3). The percent (%)
inhibition, in parentheses, at 20 μM is expressed as the percent (%) inhibition of cell growth.
cells in the G /M phase increases from 30% in a control to 40%
2
and G /M arrest is accompanied by a concomitant decrease in
2
G -phase cells (48−31%) at 24 h (Figure 2). In comparison, 8
1
Figure 3. 8-induced alterations in cell cycle G2/M phase regulatory
1
5
genes. Western blot analysis of cyclin B1 and phospho-cdc2(Tyr ) in
MDA-MB-231 cells treated with indicated concentrations of 8. Actin
was probed as an internal control.
cell population (Figure 2) and later data from an annexin V-
FITC/propidium iodide (PI) binding assay (Figure 4).
Figure 2. Effect of 8 on cell cycle distribution of MDA-MB-231 breast
cancer cells. (a) FACScan analysis of the cell cycle. MDA-MB-231 cells
were treated with DMSO (vehicle control) or 0.8 μM 8 and incubated
for 24 and 48 h, respectively. Cells were then stained with propidium
iodide and analyzed for DNA content by using flow cytometry. A total
of 25000 cells were analyzed from each sample. (b) Graph bars show
the distributions at different portions of the cell cycle at 24 and 48 h.
treated cells display a substantial increase at 48 h in the sub-G1
region, indicated as apoptotic cells (3−21%). Together, these
results demonstrate that 8 treated breast cancer cells are
arrested in G /M phase before cell death occurs.
2
It has been shown previously that cyclin B1, in association
with the cdc2 kinase (known as cdk1), regulates cell cycle
22
progression from the G to the M phase. This observations
2
explains why G /M arrest is associated with overexpression of
2
2
3
cyclin B1. Phosphorylation of Tyr15 of cdc2 is observed to
inactivate cdc2 kinase and suppress activity of the cdc2−cyclin
B1 complex, which remains in the cytoplasm rather than in the
nucleus (only active form of cdc2−cyclin B1 complex).
To gain insight into the molecular mode of action involved in
Figure 4. Fluorescence microscopy analysis of apoptosis. MDA-MB-
2
31 cells were treated with indicated concentrations of 8 in different
time intervals. After treatment, cells were washed twice with PBS then
followed by the commercial kit user’s guide (normal cells, annexin V
negative and PI negative; early apoptotic cells, annexin V positive and
PI negative; late apoptotic cells, annexin V positive and PI positive).
24
G /M arrest, the effect of 8 on the mitosis promoting factors,
2
cyclin B1 and phospho-cdc2, was investigated using Western
blot analysis (Figure 3). Expression levels of cyclin B1 and
phospho-cdc2 were observed to significantly increase upon
treatment with 8 in a dose-dependent manner for 24 and 48 h.
This observation of a high phospho-cdc2 level suggests that
both the activity and level of cdc2−cyclin B1 complex decrease.
Hence, complete termination of the progression of the cells to
mitosis is likely, indicating that 8-treated MDA-MB-231 cells
Compound 8 Induces Apoptosis. To better understand
whether the loss of cancer cell viability promoted by 8 is
associated with apoptosis, an annexin V-FITC/propidium
iodide (PI) binding assay was performed. Because annexin V-
2+
FITC has a high, Ca -dependent affinity for phosphatidylser-
ine (PS) residues, the elements of phospholipid on the inner
leaflet locations of PS residues are easily externalized during the
progression of apoptosis. Three cell populations, including
viable (annexin V-FITC, negative; PI, negative), early apoptotic
are arrested in the G phase but not the M phase. A low cyclin
2
B1 level was also observed after only 48 h treatment with the
highest concentration (0.8 μM) of 8, suggesting perhaps that
MDA-MB-231 cells are in late apoptosis or cell death. This
observation is in excellent agreement with detection of sub-G1
(
annexin V-FITC, positive; PI, negative), and late apoptotic
cells or dead cells (annexin V-FITC, positive; PI, positive),
were utilized for this purpose. After treatment with 0.4 μM 8,
early (annexin V-FITC, positive; PI, negative) and late
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apoptosis (annexin V-FITC, positive; PI, positive) take place
with MDA-MB-231 cells in a time-dependent manner (Figure
found to increase bak (pro-apoptotic protein) levels simulta-
neously at 24 and 48 h. This finding is consistent with well-
documented observations that blockage of Bcl-2 levels and
activation of bak proteins are essential steps in the intrinsic
apoptotic pathway. Furthermore, the result of a Western blot
experiment also demonstrates that 8 causes a dose-dependent
increase in protein levels of cleaved caspase-3 and cleavage of
poly(ADP-ribose)polymerase (cPARP) (Figure 6). Taken
together, the results suggest that perhaps 8 induced apoptosis
follows the intrinsic mitochondrial pathway.
4
b−d), indicating that 8 induces significant apoptotic cell death
in MDA-MB-231 cells.
26
To further confirm that 8-induced formation of a sub-G1
peak is associated with apoptosis, experiments probing DNA
ladder formation were carried out. As the images in in Figure 5
CONCLUSIONS
■
Because of the widespread chemopreventive and chemo-
therapeutic applications of I3C and its metabolites, a strong
demand exists for an efficient strategy to search for other
pharmacologically active indole analogues. The findings arising
from the studies described above open a possible approach to
the design and development of new indole analogues, such as
tetraindoles, that serve as anticancer agents for the treatment of
human breast cancer. In this effort, we have identified 8 as a
potent, nanomolar range anticancer agent against MDA-MB-
2
31 and MCF-7 cells. This substance mediates G -phase arrest
2
via up-regulation of cyclin B1 and phospho-cdc2 and induces
apoptosis involving the intrinsic pathway. Further investigations
to sensitize breast cancer cells to the actions of 8 are underway.
EXPERIMENTAL SECTION
■
Chemistry. All chemicals and reagents were commercially available
and used without further purification unless indicated otherwise. All
solvents were anhydrous grade unless indicated otherwise. Reactions
were monitored by using thin-layer chromatography on silica gel. Flash
chromatography was performed on silica gel of 60−200 μm particle
size. NMR spectra were recorded on Bruker AMX400, AV300, AV400,
or AV500 MHz instruments. Chemical shifts were reported in ppm
and are referenced to the chemical shift of residual solvent. High
resolution mass spectra were recorded on a Bruker Daltonics
spectrometer. Chromatography was performed at room temperature
using a high-performance liquid chromatography (HPLC) consisting
of a Waters model 501 single pump system with a Merck Si-60 (25 cm
Figure 5. Activation of apoptosis by 8 in MDA-MB-231 cells. DNA
ladder formation in MDA-MB-231 cells treated with 0, 0.4, 0.8, 1.2,
and 1.6 μM 8 for 48 h.
show, treatment of MDA-MB-231 cells with different
concentrations of 8 (0.4, 0.8, 1.2, and 1.6 μM) causes
formation of an apoptotic DNA ladder pattern.
Two major mechanisms for the induction of apoptosis exist,
including one that follows an extrinsic apoptotic pathway and
25
×
10 cm) column equilibrated in hexane/EtOAc or methanol as
the other following an intrinsic apoptotic pathway. The
results of a few recent studies show that indole and its
analogues commonly induce apoptosis by the intrinsic pathway
involving the modulated expression of the apoptotic regulatory
proteins, bcl-2/bak. Therefore, in an effort examine the
expression levels of apoptosis-related proteins (Figure 6), we
eluents, at a flow rate of 2 mL/min, and the eluted materials were
monitored using a UV absorbance detector that measured the
absorbance at 254 nm, unless indicated otherwise. The HPLC
fractions containing products were dried in a vacuum concentrator
(
Speedvac SC110, Savant Instruments, Holbrook, NY). The purity of
the tetraindoles was established as being >95% by employing HPLC
1
and H NMR.
Representative Procedure for the Synthesis of Tetraindoles
1
, 5, 7−9, and 12: 1,4-Bis(di(1H-indol-3-yl)methyl)benzene (1).
A round-bottomed flask (10 mL) was charged with terephthalaldehyde
0.13 g, 1.0 mmol), substituted indole (4 mmol), and iodine (0.025 g,
.1 mmol) in ether or MeCN (1 mL). This mixture was then stirred
(
0
for 2 h at room temperature. The reaction mixture was treated with
aqueous Na S O , and the solution was extracted with ethyl acetate (2
2
2
3
×
20 mL). The combined organic layers were dried over MgSO and
4
filtered. The solvent was removed under reduced pressure, and the
residue was subjected to flash column chromatography, followed by
normal-phase HPLC to afford the pure product as a powder (93%),
1
Figure 6. 8-induced apoptosis in MDA-MB-231 cells. Western blot
analysis of bcl-2, bak, cleaved caspase-3, and cleaved PARP (cPARP) in
MDA-MB-231 cells treated with indicated concentrations of 8. Actin
was probed as an internal control.
mp 183−184 °C. H NMR (400 MHz, CDCl ) 7.38−7.35 (m, 8H),
3
7.25−7.21 (m, 8H), 7.14 (td, J = 6.9, 1.1 Hz, 4H), 7.01 (td, J = 7.0, 1.0
13
Hz, 4H), 6.25−6.23 (m, 4H), 5.75 (s, 2H). C NMR (100 MHz,
CDCl ) 141.6, 136.7, 128.6, 127.1, 123.6, 121.8, 120.0, 119.7, 119.0,
3
+
1
11.1, 39.9. HRMS calcd for C H N (M ), 566.2470; found,
40
30
4
observed that treatment of MDA-MB-231 cells with different
concentrations of 8 decreases bcl-2 (antiapoptotic protein)
protein levels in a dose-dependent manner. However, 8 was
566.2469.
Representative Procedure for the Synthesis of Tetraindoles
and 11: 1,3-Bis(di(1H-indol-3-yl)methyl)benzene (2). A round-
2
bottomed flask (10 mL) was charged with isophthalaldehyde (0.13 g,
1
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1
.0 mmol), 5-substituted indole (4 mmol), and iodine (0.025 g, 0.1
126.8, 124.9, 124.5, 123.7, 122.2, 121.8, 119.8, 119.2, 119.0, 118.5,
112.8, 112.2, 111.4, 39.8, 39.4. HRMS calcd for C H N Br Na (M +
Na) , 745.0578; found, 745.0595.
mmol) in ether or MeCN (1 mL). This mixture was then stirred for 2
h at room temperature. The reaction mixture was treated with aqueous
Na S O , and the solution was extracted with ethyl acetate (2 × 20
mL). The combined organic layers were dried over MgSO4 and
filtered. The solvent was removed under reduced pressure, and the
residue was subjected to flash column chromatography, followed by
40
28
4
2
+
1,4-Bis(di(5-cyano-1H-indol-3-yl)methyl)benzene (7). Yield
2
2
3
1
45% (powder), mp 154−155 °C. H NMR (500 MHz, acetone-d )
6
11.44 (br s, 4H), 7.79 (s, 4H), 7.51 (d, J = 8.5 Hz, 4H), 7.38 (m, d, J =
7.5 Hz, 4H), 7.32 (s, 4H), 7.12 (s, 4H), 5.99 (s, 2H). 1 C NMR (125
3
normal-phase HPLC to afford the pure product as a powder (91%),
MHz, DMSO-d ) 142.3, 138.7, 128.6, 126.8, 126.7, 125.0, 124.2, 119.5,
6
1
+
mp 165−166 °C. H NMR (400 MHz, CDCl ) 7.34 (d, J = 7.9 Hz,
113.4, 100.8, 38.6. HRMS calcd for C H N Na (M + Na) ,
3
44 26
8
4
(
H), 7.16−7.06 (m, 16H), 7.00 (t, J = 7.4 Hz, 4H), 5.99 (s, 4H), 5.68
689.2178; found, 689.2185.
1,4-Bis(di(5-hydroxy-1H-indol-3-yl)methyl)benzene (8). Yield
s, 2H). 13C NMR (100 MHz, CDCl ) 143.9, 136.5, 129.3, 128.2,
3
1
1
27.0, 126.6, 123.5, 121.7, 120.0, 119.4, 119.0, 111.1, 40.3. HRMS
89% (powder), mp 239−240 °C. H NMR (400 MHz, CD
(s, 4H), 7.13 (d, J = 8.6 Hz, 4H), 6.73 (d, J = 2.3 Hz, 4H), 6.68−6.58
(m, 8H), 5.63 (s, 2H). 13C NMR (100 MHz, CD
OD) 150.1, 143.1,
3
OD) 7.24
+
calcd for C H N (M ), 566.2470; found, 566.2471.
40
30
4
Representative Procedure for the Synthesis of Tetraindole
3
3
: 3,3′,3″,3‴-(2,2′-Oxybis(2,1-phenylene)bis(methanetriyl)-
133.0, 129.0, 128.7, 125.2, 119.1, 112.0, 111.6, 104.4, 41.0. HRMS
+
tetrakis(1H-indole)) (3). A round-bottomed flask (10 mL) was
charged with 2,2′-oxidibenzaldehyde (0.23 g, 1.0 mmol), indole (0.47
g, 4 mmol), and iodine (0.025 g, 0.1 mmol) in ether (1 mL). This
mixture was then stirred for 2 h at room temperature. The reaction
mixture was treated with aqueous Na S O , and the solution was
calcd for C H N O Na (M + Na) , 653.2165; found, 653.2171.
40
30
4
4
1
,4-Bis(di(5-methoxy-1H-indol-3-yl)methyl)benzene (9).
1
Yield 70% (powder), mp 257−258 °C. H NMR (400 MHz,
DMSO-d ) 10.60 (br s, 4H), 7.25 (s, 4H), 7.21 (d, J = 8.6 Hz, 4H),
6.76 (s, 4H), 6.69−6.65 (m, 8H), 5.69 (s, 2H), 3.52 (s, 12H).
NMR (125 MHz, DMSO-d ) 153.1, 142.7, 132.2, 128.5, 127.5, 124.6,
6
1
3
2
2
3
C
extracted with ethyl acetate (2 × 20 mL). The combined organic layers
6
were dried over MgSO and filtered. The solvent was removed under
118.4, 112.5, 111.1, 101.7, 55.6, 39.5. HRMS calcd for C H N O (M
4
44 38
4
4
reduced pressure, and the residue was subjected to flash column
+), 686.2893; found, 686.2876.
chromatography, followed by normal-phase HPLC to afford the pure
Representative Procedure for the Synthesis of Tetraindole
10: 3,3′,3″,3‴-(1,4-Phenylenebis(methanetriyl)tetrakis(1H-
indol-5-amine)) (10). Step 1. A round-bottomed flask (10
mL) was charged with 5-aminoindole (0.66 g, 5.0 mmol),
Fmoc-OSu (2.00 g, 6.5 mmol), and triethylamine (1.0 mL, 7.5
mmol) in dichlormethane (30 mL). This mixture was then
stirred for 5 h at room temperature. The reaction mixture was
treated with dilute HCl solution and extracted with dichloro-
methane (2 × 30 mL). The combined organic layers were dried
1
product as a powder (93%), mp 169−170 °C. H NMR (400 MHz,
CDCl ) 7.28 (d, J = 8.0 Hz, 4H), 7.23 (dd, J = 7.6, 1.6 Hz, 2H), 7.14−
3
7
8
.06 (m, 12H), 7.06−7.01 (m, 2 H), 6.93−6.84 (m, 6H), 6.67 (d, J =
13
.0 Hz, 2H), 6.28 (br s, 4H), 6.06 (s, 2H). C NMR (100 MHz,
CDCl ) 155.5, 136.7, 134.6, 130.2, 127.6, 127.2, 124.1, 123.0, 121.7,
3
1
−
20.2, 119.1, 118.5, 111.3, 34.4, 34.4. HRMS calcd for C H N O (M
46 33 4
−
H) , 657.2655; found, 657.2654.
Representative Procedure for the Synthesis of Tetraindole
: 2,5-Bis(di(1H-indol-3-yl)methyl)thiophene (4). A round-
4
over MgSO and filtered. The solvent was removed under
4
bottomed flask (10 mL) was charged with 2,5-thiophenedicarbox-
aldehyde (0.14 g, 1.0 mmol), indole (0.47 g, 4 mmol), and iodine
reduced pressure, and the residue was subjected to flash column
chromatography to afford the Fmoc-protected 5-aminoindole
(80%).
(
2
0.025 g, 0.1 mmol) in ether (1 mL). This mixture was then stirred for
h at room temperature. The reaction mixture was treated with
aqueous Na S O , and the solution was extracted with ethyl acetate (2
Step 2. A round-bottomed flask (100 mL) was charged with
phthalaldehyde (0.067 g, 0.5 mmol), Fmoc-protected 5-aminoindole
(0.71 g, 2 mmol), and iodine (0.025 g, 0.1 mmol) in MeCN/DMSO
(5 mL). The mixture was then stirred overnight at room temperature.
The reaction mixture was treated with Na S O solution, extracted
2
2
3
×
20 mL). The combined organic layers were dried over MgSO and
4
filtered. The solvent was removed under reduced pressure, and the
residue was subjected to flash column chromatography, followed by
2
2
3
normal-phase HPLC to afford the pure product as a powder (87%),
with dichloromethane (2 × 30 mL). The combined organic layers
1
mp 180−181 °C. H NMR (400 MHz, CDCl ) 7.41 (d, J = 7.8 Hz,
were dried over MgSO and filtered. The solvent was removed under
3
4
4
4
H), 7.38−7.29 (m, 4H), 7.17−7.10 (m, 8H), 7.01 (td, J = 6.8, 1.4 Hz,
reduced pressure, and the residue was subjected to flash column
chromatography to obtain the protected tetraindole 20 (60%).
Step 3. A round-bottomed flask (100 mL) was charged with the
Fmoc protected tetraindole 20 (0.76 g, 0.5 mmol) and DBU (299 μL,
2 mmol) in dichloromethane (50 mL). The mixture was then stirred
for 30 min at room temperature. The reaction mixture was treated
13
H), 6.63 (s, 2 H), 6.37 (m, 4H), 5.95 (s, 2H). C NMR (100 MHz,
CDCl ) 146.5, 136.4, 126.7, 124.4, 123.3, 121.8, 119.8, 119.4, 119.1,
3
+
1
11.2, 35.6. HRMS calcd for C H N S (M ), 572.2035; found,
38 28 4
5
72.2031.
,4-Bis(di(5-bromo-1H-indol-3-yl)methyl)benzene (5). Yield
1
1
7
5% (powder), mp 195−196 °C. H NMR (300 MHz, CDCl ) 8.08
with Na
mL). The combined organic layers were dried over MgSO
4
2
S
2
O
3
solution and extracted with dichloromethane (2 × 30
3
(
br s, 4H), 7.45 (s, 4H), 7.28−7.18 (m, 8H), 7.10 (s, 4 H), 6.40 (s,
and
4
1
H), 5.65 (s, 2H). 13C NMR (75 MHz, CDCl ) 141.2, 135.3, 128.0,
filtered. The solvent was removed under reduced pressure, and the
residue was subjected to flash column chromatography, followed by
3
25.0, 124.6, 122.1, 118.7, 112.8, 112.3, 39.5. HRMS calcd for
−
C H N Br (M − H) , 876.8813; found, 876.8817.
precipitation from DMSO to afford pure product 10 as a powder
40
25
4
4
1
Representative Procedure for the Synthesis of Tetraindole
: 3,3′-((4-Di(1H-indol-3-yl)methyl)phenyl)methylene)bis(5-
(50%), mp 134−135 °C. H NMR (400 MHz, DMSO-d
6
) 10.22 (br s,
6
4H), 7.14 (s, 4H), 7.01 (d, J = 8.5 Hz, 4H), 6.53 (d, J = 2.1 Hz, 4H),
6.47−6.40 (m, 8H), 5.46 (s, 2H), 4.30 (br s, 8H, NH ). C NMR
(125 MHz, DMSO-d ) 142.4, 140.0, 130.6, 127.8, 127.5, 123.4, 117.2,
1
3
bromo-1H-indole) (6). A round-bottomed flask (10 mL) was
charged with 4-(bis(5-bromo-1H-indol-3-yl)methyl)benzaldehyde 21
2
6
+
(
0.25 g, 0.5 mmol), indole (0.12 g, 1 mmol), and iodine (0.012 g, 0.05
111.9, 111.6, 102.8, 39.9. HRMS calcd for C H N (M + H) ,
40
35
8
mmol) in MeCN (1 mL). The mixture was then stirred for 2 h at
room temperature. The reaction mixture was treated with aqueous
Na S O , and the solution was extracted with ethyl acetate (2 × 20
mL). The combined organic layers were dried over MgSO4 and
filtered. The solvent was removed under reduced pressure, and the
residue was subjected to flash column chromatography, followed by
627.2985; found, 627.2988.
1,3-Bis(di(5-hydroxy-1H-indol-3-yl)methyl)benzene (11).
1
2
2
3
Yield 85% (powder), mp 285−286 °C. H NMR (400 MHz,
CD OD) 7.39 (s, 1H), 7.13 (m, 7H), 6.72 (d, J = 2.4 Hz, 4H),
3
1
3
6.64 (dd, J = 8.6, 2.4 Hz, 4H), 6.49 (s, 4H), 5.60 (s, 2H). C NMR
(100 MHz, CD OD) 150.0, 145.3, 132.9, 130.1, 128.6, 128.2, 126.9,
3
normal-phase HPLC to afford the pure product as a powder (80%),
125.2, 118.9, 112.0, 111.6, 104.4, 41.3. HRMS calcd for C H N O
4
0
31
4
4
1
+
mp 210−211 °C. H NMR (400 MHz, CDCl ) 7.59 (br s, 2H), 7.46−
(M + H) , 631.2345; found, 631.2332.
3
7
4
.38 (m, 5H), 7.31−7.22 (m, 5H), 7.19−7.12 (m, 6H), 7.09−7.02 (m,
3,3′,3″,3‴-(1,4-Phenylenebis(methanetriyl)tetrakis(1H-
H), 6.34 (s, 2 H), 6.20 (s, 2 H), 5.78 (s, 1H), 5.59 (s, 1H). 13C NMR
indol-6-ol)) (12). Yield 55% (powder), mp >300 °C. H NMR (400
1
(
100 MHz, CDCl ) 141.7, 140.6, 136.5, 135.0, 128.6, 128.4, 128.3,
MHz, CD OD) 7.22 (s, 4H), 7.05 (d, J = 8.5 Hz, 4H), 6.73 (s, 4H),
3
3
1
589
dx.doi.org/10.1021/jm2013425 | J. Med. Chem. 2012, 55, 1583−1592

Journal of Medicinal Chemistry
Article
1
3
6
1
.48−6.44 (m, 8H), 5.66 (s, 2H). C NMR (100 MHz, CD OD)
138.1, 132.9, 129.2, 129.2, 128.9, 128.9, 128.2, 125.2, 124.6, 122.0,
120.4, 120.17, 119.3, 112.5, 112.3, 112.2, 112.1, 104.6, 104.5, 41.0,
3
53.0, 143.2, 138.7, 128.9, 122.8, 122.2, 120.6, 119.8, 109.0, 97.0, 40.9.
HRMS calcd for C H N O (M + H) , 631.2345; found, 631.2340.
Representative Procedure for the Synthesis of Tetraindole
3: 3-((4-Di(1H-indol-3-yl)methyl)phenyl)(1H-indol-3-yl)-
methyl)(1H-indol-5-ol) (13). A round-bottomed flask (10 mL)
was charged with terephthalaldehyde (0.13 g, 1.0 mmol), indole (0.28
g, 2.4 mmol), 5-hydroxyindole (0.21 g, 1.6 mmol), and iodine (0.025
g, 0.1 mmol) in MeCN (1 mL). The mixture was then stirred for 2 h at
room temperature. The reaction mixture was treated with Na S O
3
solution and extracted with ethyl acetate (2 × 20 mL). The combined
organic layers were dried over MgSO and filtered. The solvent was
removed under reduced pressure, and the residue was subjected to
flash column chromatography, followed by normal-phase HPLC to
afford the pure product as a powder (30%), mp 139−140 °C. H
NMR (500 MHz, acetone-d ) 9.94 (br s, 3H), 9.67 (br s, 1H), 7.54 (br
+
+
4
0
35
4
4
41.0. HRMS calcd for C H N O (M + H) , 615.2396; found,
40 31
4
3
6
15.2400.
1
27
Biology. Cell viability analysis and fluorescence microscopy
28
analysis were performed by employing previously described
methods.
Cell Culture. Human breast carcinoma cell line MCF-7 and human
adenocarcinoma cells MDA-MB-231 (BCRC-60436; BCRC-60425,
Bioresource Collection and Research Center, Food Industry Research
and Development Institute, Hsinchu, Taiwan) were maintained in
Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% fetal
bovine serum (FBS) and 2 mM L-glutamine at 37 °C in a humidified
2
2
4
1
atmosphere containing 5% CO in air.
2
Cell Growth Inhibition Analysis. MDA-MB-231 cells were
6
5
s, 1H), 7.37−7.33 (m, 10H), 7.19 (d, J = 8.5 Hz, 1H), 7.06 (t, J = 8.0
seeded at a density of 2 × 10 per well in 6-well culture plates. After 24
Hz, 3H), 6.90 (t, J = 7.5 Hz, 3H), 6. 38−6.66 (m, 6H), 5.91 (s, 1H),
h, cells were treated with different concentrations of 8 or DMSO
(vehicle control) in triplicates. At different time intervals, cells were
harvested by trypsinization, stained with 0.4% trypan blue, and
counted using a hemocytometer.
Flow Cytometry Analysis. Cells were incubated in 10% FBS-
supplemented DMEM/F12 with DMSO (vehicle control) or different
concentrations of 8 for 24 h. After treatment, cells were collected by
trypsinization and centrifugation, washed with PBS, and fixed with ice-
cold 70% ethanol at −20 °C overnight. Fixed cells were collected by
centrifugation, washed with PBS, resuspended in 0.5 mL of PBS
containing 20 μg/mL propidium iodine, 200 μg/mL RNase A, and
5
1
1
1
.80 (s, 1H). 13C NMR (100 MHz, CD OD) 151.3, 143.5, 143.4,
3
38.1, 132.9, 129.2, 129.2, 128.9, 128.3, 128.2, 125.2, 124.6, 124.5,
22.0, 122.0, 120.4, 120.4, 120.2, 120.1, 119.4, 119.3, 119.3, 112.5,
12.3, 112.2, 112.1, 104.6, 40.9, 40.9. HRMS calcd for C H N ONa
40
30
4
+
(
M + Na) , 605.2317; found, 605.2324.
Representative Procedure for the Synthesis of Tetraindole
1
4: 3,3′-((4-Di(1H-indol-3-yl)methyl)phenyl)methylene)bis(1H-
indol-5-ol) (14). Step 1. A round-bottomed flask (10 mL) was
charged with terephthalaldehyde (0.13 g, 1.0 mmol), indole
(
0.23 g, 2 mmol), and iodine (0.012 g, 0.05 mmol) in ether (1
mL). The mixture was then stirred for 15 min at room
temperature. The reaction mixture was treated with Na S O
3
solution and extracted with ethyl acetate (2 × 20 mL). The
combined organic layers were dried over MgSO and filtered.
The solvent was removed under reduced pressure, and the
residue was subjected to flash column chromatography to give
-(bis(1H-indol-3-yl)methyl)benzaldehyde 22 (67%).
Step 2. A round-bottomed flask (10 mL) was charged with 22 (0.18
g, 0.5 mmol), indole (0.12 g, 1 mmol), and iodine (0.012 g, 0.05
mmol) in MeCN (1 mL). The mixture was then stirred for 15 min at
room temperature. The reaction mixture was treated with Na S O
3
solution and extracted with ethyl acetate (2 × 20 mL). The combined
organic layers were dried over MgSO and filtered. The solvent was
removed under reduced pressure, and the residue was subjected to
flash column chromatography, followed by normal-phase HPLC to
afford the pure product as a powder (60%), mp 174−175 °C. H
NMR (400 MHz, acetone-d ) 9.92 (br s, 2H), 9.65 (br s, 2H), 7.59 (br
s, 2H, OH), 7.37−7.30 (m, 8H), 7.19 (d, J = 9.0 Hz, 2H), 7.05 (t, J =
.5 Hz, 2H), 6.90 (t, J = 8.0 Hz, 2H), 6.82 (d, J = 1.5 Hz, 2H), 6.77 (d,
0
.1% triton X-100. Samples were incubated on the ice and kept in the
dark for 30 min. The cell cycle distribution was analyzed by using a
FACSCalibur flow cytometer (BD), and the ranges for G , S, G /M,
and sub-G phase cells were validated according to the corresponding
2
2
4
1
2
1
DNA contents, followed by calculated using ModFIT LT software
(BD).
4
Determination of the Apoptotic DNA Ladder. MDA-MB-231
6
2
cells were seeded at a density of 1 × 10 per well in 10 cm culture
dishes. After 24 h, cells were treated with different concentration of 8
or DMSO (vehicle control) and then incubated in 37 °C, 5% CO2
incubator for 48 h. After treatment, the attached and suspension cells
were harvested by trypsinization and centrifugation. The pellets were
washed twice using PBS and lysed in TE lysis buffer (10 mM Tris-HCl
pH 7.5, 1 mM sodium-EDTA, 0.25% NP40). The supernatants were
collected and incubated with 5 μL of RNase A (20 mg/mL) for 1 h at
2
2
4
1
6
3
3
7 °C, followed by adding 5 μL of proteinase K (20 mg/mL) for 1 h at
7 °C. The electrophoresis was performed with 1.8% agarose gel.
Protein Extraction and Western Blot Analysis. MDA-MB-231
7
13
J = 2.0 Hz, 2H), 6.68−6.66 (m, 4H), 5.90 (s, 1H), 5.60 (s, 1H).
NMR (100 MHz, acetone-d ) 151.2, 143.4, 143.3, 138.0, 132.8, 129.2,
29.1, 128.9, 128.2, 125.2, 124.5, 122.0, 120.4, 120.1, 119.3, 119.2,
12.5, 112.2, 112.1, 104.5, 40.9, 40.9. HRMS calcd for C H N O Na
M + Na) , 621.2266; found, 621.2252.
C
cells were treated with 0.2, 0.4, 0.8 μM 8 for 24 and 48 h. After
treatment, the suspended and attached cells were collected by
trypsinization and centrifugation and lysed in lysis buffer (25 mM
Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% SDS, 1% NP-40, 1% sodium
deoxycholate) with proteinase inhibitors. The lysates were incubated
on ice for 10 min and centrifugated at 13000g for 20 min. Protein
concentrations were then determined with BCA protein assay reagents
6
1
1
(
4
0
30
4
2
+
Representative Procedure for the Synthesis of Tetraindole
1
5: 3,3′-((4-Di(5-hydroxy-1H-indol-3-yl)(1H-indol-3-yl)methyl)-
phenyl)methylene)bis(1H-indol-5- ol) (15). A round-bottomed
flask (10 mL) was charged with terephthalaldehyde (0.13 g, 1.0
mmol), indole (0.094 g, 0.8 mmol), 5-hydroxyindole (0.43 g, 3.2
mmol), and iodine (0.025 g, 0.1 mmol) in MeCN (1 mL). The
mixture was then stirred for 2 h at room temperature. The reaction
mixture was treated with Na S O solution and extracted with ethyl
(
Pierce). Equal amounts of total protein were mixed with 4× sample
loading buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, 20% SDS,
.5% bromophenol blue, and 5% 2-mercaptoethanol), boiled for 10
2
min, and then fractionated by electrophoresis on 10% and 15% SDS-
PAGE. Prestained protein ladder (Fermentas) was used as the
molecular weight standard. Proteins were electrically transferred to
nitrocellulose membranes and blocked overnight at 4 °C with 0.05%
TBST and 5% nonfat milk. Blots were subsequently incubated for 1 h
at room temperature for phospho-cdc2 (Santa Cruz), caspase-3
2
2
3
acetate (2 × 20 mL). The combined organic layers were dried over
MgSO and filtered. The solvent was removed under reduced pressure,
and the residue was subjected to flash column chromatography,
followed by normal-phase HPLC to afford the pure product as a
4
1
powder (60%), mp 228−229 °C. H NMR (500 MHz, acetone-d )
6
(
(
IMGENEX), cyclin B1 (Cell Signaling), bak/bcl-2 (AbD), and actin
Chemicon) primary antibodies. Immunoreactive proteins were
9
2
7
6
5
.94 (br s, 1H), 9.68 (br s, 3H), 7.54 (br s, 3H, OH), 7.38−7.36 (m,
H), 7.34−7.30 (m, 4H), 7.20 (dd, J = 8.5 Hz, 1 Hz, 3H), 7.06 (t, J =
.5 Hz, 1H), 6.91 (t, J = 7.7 Hz, 1H), 6.81 (d, J = 1.5 Hz, 1H), 6.76−
.74 (m, 4H), 6.72−6.70 (m, 2H), 6.67−6.65 (m, 3H), 5.79 (s, 1H),
detected after incubation with horseradish peroxidase-conjugated
secondary antibody. The immunoblots were visualized by enhanced
chemiluminescence.
.67 (s, 1H). 13C NMR (100 MHz, CD OD) 151.2, 143.4, 143.4,
3
1
590
dx.doi.org/10.1021/jm2013425 | J. Med. Chem. 2012, 55, 1583−1592

Journal of Medicinal Chemistry
Article
Down-regulation of androgen receptor by 3,3′-diindolylmethane
contributes to inhibition of cell proliferation and induction of
apoptosis in both hormone-sensitive LNCaP and insensitive C4−2B
prostate cancer cells. Cancer Res. 2006, 66, 10064−10072.
ASSOCIATED CONTENT
■
*
S
Supporting Information
Details of HPLC, H and 13C NMR spectra of all new
tetraindols, and effect of 8 on the viability of MDA-MB-231
cells. This material is available free of charge via the Internet at
http://pubs.acs.org.
1
(
3) (a) Xue, L.; Schaldach, C. M.; Janosik, T.; Bergman, J.; Bjeldanes,
L. F. Effects of analogs of indole-3-carbinol cyclic trimerization
product in human breast cancer cells. Chem. Biol. Interact. 2005, 152,
1
19−129. (b) Riby, J. E.; Feng, C.; Chang, Y. C.; Schaldach, C. M.;
AUTHOR INFORMATION
Corresponding Author
For W.-S.L.: phone, +886 (2) 27898662; fax, +886 (2)
7831237; e-mail, wenshan@chem.sinica.edu.tw. For C.-F.Y.:
phone, +886 (2) 77346128; fax, +886 (2) 29324249; e-mail,
cheyaocf@ntnu.edu.tw.
Firestone, G. L.; Bjeldanes, L. F. The major cyclic trimeric product of
indole-3-carbinol is a strong agonist of the estrogen receptor signaling
pathway. Biochemistry 2000, 39, 910−918.
■
*
(4) (a) De Santi, M.; Galluzzi, L.; Lucarini, S.; Paoletti, M. F.;
2
Fraternale, A.; Duranti, A.; De Marco, C.; Fanelli, M.; Zaffaroni, N.;
Brandi, G.; Magnani, M. The indole-3-carbinol cyclic tetrameric
derivative CTet inhibits cell proliferation via overexpression of p21/
CDKN1A in both estrogen receptor-positive and triple-negative breast
cancer cell lines. Breast Cancer Res. 2011, 13, R33. (b) Lucarini, S.; De
Santi, M.; Antonietti, F.; Brandi, G.; Diamantini, G.; Fraternale, A.;
Paoletti, M. F.; Tontini, A.; Magnani, M.; Duranti, A. Synthesis and
biological evaluation of a gamma-cyclodextrin-based formulation of the
anticancer agent 5,6,11,12,17,18,23,24-octahydrocyclododeca[1,2-
b:4,5-b′:7,8-b′′:10,11-b′′′]tetraindole (CTet). Molecules 2010, 15,
Present Address
These authors contributed equally to this work.
#
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
4
085−4093. (c) Brandi, G.; Paiardini, M.; Cervasi, B.; Fiorucci, C.;
Filippone, P.; De Marco, C.; Zaffaroni, N.; Magnani, M. A new indole-
-carbinol tetrameric derivative inhibits cyclin-dependent kinase 6
We are grateful for funding of this work provided by the
Academia Sinica and the National Science Council. Instru-
mentation support was provided by the NMR and Mass
Spectrometry facilities of the Institute of Chemistry at
Academia Sinica, Taiwan.
3
expression, and induces G1 cell cycle arrest in both estrogen-
dependent and estrogen-independent breast cancer cell lines. Cancer
Res. 2003, 63, 4028−4036.
(5) NCI, DCPC Chemoprevention Branch and Agent Development
ABBREVIATIONS USED
■
Committee. Clinical Development Plan: Indole-3-carbinol. J. Cell.
Biochem. 1996, 26, 127−136.
I3C, indole-3-carbinol; cdk1, cdc2 kinase; cPARP, cleavage of
poly(ADP-ribose)polymerase; PS, phosphatidylserine; ER,
estrogen receptor; ROS, reactive oxygen species; JNK, c-Jun
N-terminal kinase; IC , inhibitory concentration 50%; MTT,
(6) (a) Wang, Z.; Yu, B. W.; Rahman, K. M.; Ahmad, F.; Sarkar, F. H.
Induction of growth arrest and apoptosis in human breast cancer cells
by 3,3-diindolylmethane is associated with induction and nuclear
localization of p27kip. Mol. Cancer Ther. 2008, 7, 341−349.
5
0
3
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(b) Vanderlaag, K.; Samudio, I.; Burghardt, R.; Barhoumi, R.; Safe,
S. Inhibition of breast cancer cell growth and induction of cell death by
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