A novel p-terphenyl derivative inducing cell-cycle arrest and apoptosis in MDA-MB-435 cells through topoisomerase inhibition

Article abstract of DOI:10.1016/j.ejmech.2013.07.020

A novel series of p-terphenyl derivatives (1-4, 1a-4a) was successfully synthesized and their in vitro anticancer activities were evaluated. Compound 1, showing the best antiproliferative activity with IC₅₀ < 1 μM against MDA-MB-435 cells, was further investigated. Compound 1 brought about a remarkable accumulation of MDA-MB-435 cells in G2/M phase prior to the induction of apoptosis. Further antitumor mechanism study indicated that compound 1, which inhibited the enzyme activity of Topo I and Topo IIα by interfering predominantly with the enzyme, could be topoisomerase suppressors instead of poisons. We conclude that compound 1 represents a novel class of Topo catalytic suppressors for developing new chemotherapeutic agents.

Full text of DOI:10.1016/j.ejmech.2013.07.020

European Journal of Medicinal Chemistry 68 (2013) 192e202
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A novel p-terphenyl derivative inducing cell-cycle arrest and apoptosis
in MDA-MB-435 cells through topoisomerase inhibition
,
,
a,
**
Jin Qiu a b, Baobing Zhao a, Yan Shen a, Wang Chen a, Yudao Ma c , Yuemao Shen
*
a Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan 250012, Shandong, PR China
b School of Pharmaceutical Sciences, Shandong University of Traditional Chinese Medicine, Jinan 250355, Shandong, PR China
c School of Chemistry and Chemical Engineering, Shandong University, Shanda South Road No. 27, Jinan 250100, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of p-terphenyl derivatives (1e4, 1ae4a) was successfully synthesized and their in vitro
anticancer activities were evaluated. Compound 1, showing the best antiproliferative activity with
Received 13 January 2013
Received in revised form
15 July 2013
Accepted 18 July 2013
Available online 11 August 2013
IC50 < 1
mM against MDA-MB-435 cells, was further investigated. Compound 1 brought about a
remarkable accumulation of MDA-MB-435 cells in G2/M phase prior to the induction of apoptosis.
Further antitumor mechanism study indicated that compound 1, which inhibited the enzyme activity of
Topo I and Topo IIa by interfering predominantly with the enzyme, could be topoisomerase suppressors
instead of poisons. We conclude that compound 1 represents a novel class of Topo catalytic suppressors
for developing new chemotherapeutic agents.
Keywords:
Terphenyls
Topoisomerase
Suppressors
Apoptosis
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
against human hepatocellular carcinoma HepG2 cell line, human
cervical carcinoma HeLa cell line and human breast carcinoma
Topoisomerases (Topo) are the essential enzymes that control
and modify the topological state of DNA during cellular processes,
such as replication, transcription, recombination, and chromatin
remodeling [1,2]. Under normal conditions, the covalent Topo I or
Topo II cleaved DNA intermediates are constitutively transient and,
since the DNA relegation step is much faster than the cleavage one,
they are tolerated by the cell. On the contrary, conditions that
significantly alter the physiological concentration or the life time of
these breaks play a crucial role in inhibiting cell cycle progression
and cell death [3,4]. In this regard, they are now viewed as
important therapeutic targets for cancer chemotherapy [5].
MDA-MB-435 cell line through the generation of ROS, cell cycle
arrest and apoptosis [8]. Nevertheless, the detailed mechanism
responsible for the anti-tumor activity has not been well eluci-
dated. In order to uncover the basic pharmacophore of p-terphenyls
and discover novel derivatives as potential anti-cancer agents, eight
new p-terphenyl derivatives were successfully synthesized. Herein,
preliminary structureeactivity relationship (SAR) of derivatives for
their in vitro cytotoxicity and antitumor mechanism of the com-
pounds were discussed. Our results indicated that compound 1,
with its inhibition of the Topol and Topola activities, could induce
apoptosis and cell cycle arrest.
p-Terphenyl-type chemical entities, mainly found in mycomy-
cetes, include scaffolds with a C-18 tricyclic or polycyclic C-18
skeleton which exhibiting alkylated side chains [6]. Members of
this family of natural products, isolated from A. candidus, have
displayed various degrees of cytotoxicity [7]. In our previous study,
p-terphenyl derivatives, isolated from the marine fungal strain
Aspergillus sp. AF119, were found showing potent cytotoxicity
2. Results
2.1. Chemistry
The compounds 1e4 and 1ae4a (Fig. 1A) were obtained via
two different parallel synthetic methods based on palladium
(0)-catalyzed arylearyl coupling reactions (Scheme 1). As shown
in Scheme 1, the compounds were prepared from 4-hydroxyphenyl
boronic acid (7) or 4-methoxyphenylboronic acid (11) via
sequential Suzuki cross-coupling reactions. Bromide 5 was pur-
* Corresponding author. Tel.: þ86 531 88361869; fax: þ86 531 88565211.
** Corresponding author. Tel.: þ86 531 88382108.
E-mail addresses: ydma@sdu.edu.cn (Y. Ma), yshen@sdu.edu.cn, ahua0966@
sdu.edu.cn (Y. Shen).
chased from Aldrich and bromide
6 was prepared from
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.07.020

J. Qiu et al. / European Journal of Medicinal Chemistry 68 (2013) 192e202
193
2.2.2. Compound 1 induced cell-cycle arrest and apoptosis
A
Cell cycle arrest is an important sign for inhibition of prolifera-
tion. Flow cytometric analysis was applied to analyze the effect on
the cell cycle after being treated with compound 1 for 24 h (Fig. 2A).
After were treated with increasing concentration of compound 1,
the cell number of the cells at the G2/M phase increased signifi-
cantly, while a corresponding decrease of that in the G1 and S phase.
Meanwhile, a slight increase of cell number in the sub-G1 phase was
observed after treatment with 1 mM compound 1. Besides accu-
mulation of cells at the sub-G1 phase was in a time-dependent
manner (Fig. 2D). All these results showed that compound 1 could
induce a G2/M phase arrest and subsequent cell death.
60
50
40
30
20
10
0
B
LNcap
A549
MDA-MB-435
Cell cycle arrest at G2/M phase is a common cellular response to a
variety of DNA-damaging agents [9,10]. Therefore we next investi-
gated whether the anti-tumor activity of compound 1 resulted from
DNA damage response activation. DNA damage in cells evoked a
checkpoint response which moderated cell cycle progression for
repair of the damage. The molecular response to DNA damage began
with the recognition of the DSBs followed by the activation of
phosphatidylinositol 3-kinase-like kinase (PI3K) family members
which were responsible for the phosphorylation of H2AX, such as
ATM, ATR (ATM- and Rad3-related), and DNA-PK. Furthermore, p53
was stabilized and rapidly activated in response to DNA damage
[11].
To examine the activity status of these DNA damage-sensing
proteins, Western blot analysis was performed using phospho-
specific antibodies. As shown in Fig. 2B, Etoposide, a DNA-
damaging agent, increased the phosphorylation levels of Ser1981
at ATM, then led to an opening proceed of downstream target,
Ser139 at H2AX. The level of p53 increased after treatment with
Etoposide for 12 h both in common state and phosphorylation at
Ser15. In contrast, the phosphorylation levels of these proteins did
not change in MDA-MB-435 cells treated with compound 1. These
data suggested that compound 1 did not produce DNA damage.
To study the molecular basis of compound 1 mediated growth
arrest in MDA-MB-435 cells, the expression of known G2/M-
associated proteins were examined in MDA-MB-435 cells after
being treated with compound 1 using Western blot analysis. As
shown in Fig. 2B, after 12 h of compound 1 treatment, the MDA-
MB-435 cells showed a significant increase of cyclin B and cdc2
protein, and the protein level was gradually enhanced up during
1
2
4
Compound3s
Fig. 1. p-Terphenyl derivatives inhibit cancer cells growth. (A) Structure of p-terphenyl
derivatives; (B) Cytotoxicity evaluation of the compounds 1e4 against LNcap, A549 and
MDA-MB-435 cells. The cytotoxicity of compounds was expressed as IC50, which was
defined as the concentration of compound that resulted in a 50% inhibition of growth
rate. Each value represents the mean ꢂ SD of three independent experiments.
cyclohexanone according to the literature procedure described
below. A cross-coupling Suzuki reaction between compound 5
(compound 6) and compound 7 produced the corresponding
biphenyl derivatives, which underwent Suzuki cross-coupling re-
action with the boronic acid 9 to give intermediate compounds 1a
and 4a.
Bromide 10a and bromide 10b were prepared from the
commercially available 2-methoxyphenol and 2,6-dimethoxy
phenol according to the literature described below. A cross-
coupling Suzuki reaction between compound 10a (compound
10b) and compound 11 afforded the corresponding biphenyl de-
rivatives, which reacted with trifluoromethanesulfonic anhydride
and then with compound 9 via Suzuki reaction to give intermediate
compounds 2a and 3a.
24 h. On the contrary, GADD45a, cdc25c, and myt1 markedly
decreased in compound 1-treated MDA-MB-435 cells.
Cell morphological observations were subsequently performed
to investigate the nature of cell death induced by compound 1. With
the gradient enhancement of drug concentration, a large propor-
tion of cells became round in shape and dead, while the untreated
cells displayed normal shapes and clear skeletons under the same
conditions (Fig. 3A). Chromatin condensation and nuclear frag-
mentation were also observed by the DAPI staining, which typically
occurred during apoptosis (Fig. 3A).
To further characterize the apoptosis induced by compound 1,
an Annexin V/PI assay was used to quantitate the apoptotic cells of
MDA-MB-435. As illustrated in Fig. 3B, the proportion of Annexin V
and PI-positive (apoptotic) cells increased from 0.61% to 12.87%
when they were exposed to 2 mM of compound 1 for 24 h. Collec-
tively, these data confirmed that compound 1 induced MDA-MB-
435 cell apoptosis.
We subsequently evaluated whether compound 1 induced
apoptosis in a caspase-dependent manner. To serve this purpose,
the impact of compound 1 on procaspase-3 (an indicator of
caspase-3 activity) and PARP (a downstream target of caspase)
were preferentially examined. The results showed that compound 1
dose-dependently decreased the expression of procaspase-3 and
increased the expression of cleaved-caspase-3 in MDA-MB-435
Finally, the parallel demethylation of compounds 1a, 2a, 3a and
4a with BBr3 at ꢀ78 ꢁC produced the compounds 1, 2, 3 and 4.
2.2. Biological studies
2.2.1. In vitro cytotoxicity of synthetic p-terphenyl derivatives
against cancer cell lines
Compounds 1e4 showed evident anticancer effects against
human prostate cancer cell line LNcap, human lung cancer cell
line A549 and human breast carcinoma cell line MDA-MB-435 by
the MTT assay (Fig. 1B). Compared with the other two ones, MDA-
MB-435 cell line was more sensitive to compounds 1e4. And the
hydroxylation of benzene ring B of compounds 1e4 such as 2, 3
and 4 decreased the cytotoxic activity against MDA-MB-435 cells.
Similar to compound 3a, compound 2a was found to be inactive
up to 100
mM. These data indicated that the phenolic hydroxyl
groups on the benzene ring A and C could be crucial to the
cytotoxicity. Compound 1, which exhibited the best activity
against MDA-MB-435 cells with IC50 < 1 mM, was picked out for
further studies.

194
J. Qiu et al. / European Journal of Medicinal Chemistry 68 (2013) 192e202
Scheme 1. Synthesis route of compounds 1e4 and 1ae4a.
cells after 24 h of incubation time (Fig. 3C). In addition, compound 1
resulted in PARP cleavage in a dose-dependent fashion as evi-
denced by an increase in 85 kDa of inactive intermediate band of
PARP and a concomitant decrease in 116 kDa of the full length
(Fig. 3C).
Induction of apoptosis can be triggered by inhibition of anti-
apoptotic and/or activation of pro-apoptotic mechanisms. Bcl-2
family proteins, including anti-apoptotic members (such as Bcl-2)
and pro-apoptotic members (such as Bax, Bak), play a pivotal role
in apoptotic response [12]. It is well known that the ratio of Bax/Bcl-
xL determines the apoptotic fate of cells and an increase in that
ratio predicts that the cells will undergo apoptosis. As shown in
Fig. 3C, the expression level of anti-apoptotic Bcl-2 was sensibly
affected by the treatment with compound 1 after 12 h, while Bax
and Bak were inversely regulated.
2.2.3. DNA topoisomerases inhibition
In our previous work, natural p-terphenyl derivatives have been
shown to induce generation of ROS in MDA-MB-435 cells [8].
However, the antioxidant N-acetylcysteine (NAC), a direct scav-
enger of ROS, was unable to inhibit the cell cycle arrest and
apoptosis induced by compound 1 (Supplemental Fig. 1). This result
suggested that intracellular ROS generation was not related to
compound 1 inducing cell cycle arrest and apoptosis in MDA-MB-
435 cells.
Topoisomerases are involved in regulating the under- and over-
winding of DNA. This process is essential for removing knots and
tangles in the DNA, without which cells can be dead. It has been
established that strategic interference with enzymes is effective for
cancer therapy [3]. Therefore, a DNA relaxation assay was per-
formed to evaluate the Topo inhibition activity of compound 1.

J. Qiu et al. / European Journal of Medicinal Chemistry 68 (2013) 192e202
195
A
B
Vehicle
0.25 μM
Sub-G1: 1.04%
Time(h)
0.5
1
2
4
6
12
Sub-G1: 0.79%
Compound 1(0.5 μM)
G1: 28.06%
G1: 34.91%
S: 57.11%
S: 47.81%
VP-16(10 μΜ)
G2/M: 14.05%
G2/M: 16.24%
pho-H2AX (Ser139)
pho-ATM (Ser1982)
ATM
1.0 μM
0.5 μM
Sub-G1: 10.50%
Sub-G1: 4.53%
G1: 30.68%
G1: 22.42%
S: 12.58%
pho-p53(Ser15)
p53
S: 26.38%
G2/M: 54.51%
G2/M: 38.41%
β-actin
DNA content
C
D
12 h
24 h
25
Compound 1(μM)
Cyclin B1
0.25 0.5
1
0.25 0.5 1
20
15
10
5
cdc2
cdc25c
GADD45 α
myt1
0
0
β-actin
12
24
48
72
Time (h3o6ur)
Fig. 2. Effect of compound 1 on cell cycle progression. (A) MDA-MB-435 cells were treated with increasing concentrations of compound 1 for 24 h and analyzed for propidium
iodide (PI) stained-DNA content by flow cytometry. Values indicated the percentages of the cell population at the phase of the cell cycle. Results were representative of three
independent experiments. (B) Western blot analysis for the activations of DNA damage-sensing proteins in MDA-MB-435 cells. The phosphorylations of ATM at Ser1981, H2AX at
Ser139, p53 at Ser15, and the expressions of p53 and ATM were determined by Western blotting in MDA-MB-435 cells after the cells were treated with compound 1 for indicated
time. (C) Expression of cell cycle arrest-related proteins in MDA-MB-435 cells treated with compound 1. The levels of cell cycle-related proteins including CDC25, Cyclin B1 and
CDK1 were assessed by western blot assay. Equal loading of protein was confirmed by stripping and reprobing the blots with b-actin. (D) Flow cytometric analysis of compound 1-
induced cell death in MDA-MB-435 cells. MDA-MB-435 cells were treated with compound 1 and analyzed by flow cytometry. The population of cells in the sub-G1 phase rep-
resented cellular fragments due to apoptosis. The data shown were representative of three independent experiments. Data shown are the mean þ SD of three independent
experiments.
In the absence of compound 1, the supercoiled plasmid pBR322
was completely relaxed evidenced by the visualization of its slower
migration rate through agarose gel electrophoresis (Fig. 4A). In
contrast, with the increasing concentration of compound 1, an
increasing amount of supercoiled pBR322 was observed in the
agarose gel, which was distinct from other relaxed plasmid due to
Circular supercoiled plasmid (SC) DNA was used as the substrate
in the inhibition mode assay. If a compound was a Topo suppressor,
the DNA starting material would remain intact because the topo-
isomerase catalyzed cleavage of DNA was blocked by the suppres-
sor. If a compound was a Topo poison, the DNA cleavage would
occur in the presence of the enzyme. Subsequently, the cleaved
DNA products would be stabilized by poisons through the forma-
tion of a cleavable enzymeeinhibitoreDNA complex. Thereafter,
the cleaved DNA products NOC (nicked open circular) DNA for Topo
I and L (linear) DNA for Topo I, could be trapped by sodium dodecyl
sulfate (SDS). Subsequent proteinase K removes the bound en-
zymes. After the resolution of DNA species by gel electrophoresis,
the presence or absence of the NOC DNA band would classify the
inhibitor as a Topo I poison or suppressor, the same as the presence
its faster migration rate. Similar results were obtained for Topo II
activity inhibition (Fig. 4A). These results confirmed that compound
1 inhibited the activity of Topo I and Topo II
Compound 1 was confirmed to inhibit both Topo I and II
a
a.
a
from
the above assays, and we next studied the mode of inhibition.
Topoisomerase inhibitors could be classified as topo poisons and
topo catalytic suppressors. The former acted by stabilizing DNA
cleavable enzyme complexes leading to DNA break. On the con-
trary, the latter acted by stabilizing enzyme, so DNA strands remain
intact and no DNA breaks occur. By detecting the presence or
absence of the DNA breaks, Topo poisons therefore could be
distinguished from Topo catalytic suppressors [13,14].
or absence of the L DNA band for Topo II
a [15]. As illustrated in
Fig. 4B, at a concentration of 100 M, neither NOC DNA nor L DNA
m
band was observed for compound 1. This indicated that compound
1 was a Topo suppressor, not a poison. In contrast, NOC DNA and L

196
J. Qiu et al. / European Journal of Medicinal Chemistry 68 (2013) 192e202
Fig. 3. Compound 1 induced apoptosis in MDA-MB-435 cells. (A) Compound 1 induced apoptosis in MDA-MB-435 cells with DAPI staining. MDA-MB-435 cells were treated with
different concentrations of Compound 1. Then, the cells were harvested and stained with DAPI in PBS and examined by a fluorescence microscopy (scale bar is 10 m). (B) Flow
m
cytometric analysis by Annexin-V/PI double-staining. MDA-MB-435 cells were treated with indicated concentrations of compound 1 for 24 h and stained with annexin V-FITC/PI to
analyze apoptotic and necrotic cell population as described earlier. Data were representative of one of the three similar experiments. (C) Effects of compound 1 on the expression of
apoptotic relating-proteins. MDA-M caspase-3, full-length PARP (116-kDa) and cleaved PARP (85-kDa) indicated by B-435 cells were treated with compound 1 for the indicated
times. Whole cell lysates were prepared and signals of processed proteins were detected by Western blotting with antibodies specific to Bcl-2, Bak, Bad, caspase3, cleaved caspase-3
and PARP. Cleaved caspase-3, full-length PARP (116-kDa) and cleaved PARP (85-kDa) were indicated by arrows.
representative of three independent experiments with similar results.
b-actin was used as an internal loading control. Data shown were
DNA were clearly observed for positive control compounds, OPT
(Fig. 4B, lane 5) for Topo I, and VP-16 (Fig. 4B, lane 6) for Topo II
Fig. 5A and B, after treatment with 100 mM compound 1, 2, 3 and 4
only compound 1 and 3 had been found to inhibit the enzymatic
a
.
DNA intercalation is an important feature of some Topo in-
hibitors such as adriamycin and amsacrine, which can bind to the
minor groove of DNA and produce positively supercoiled DNA in
the presence of topoisomerases [16]. Therefore, the intercalating
ability of compound 1 into DNA was assessed by a Topo I-catalyzed
DNA unwinding assay, which is based on the ability of intercalating
compounds to unwind the DNA duplex and thereby change the
DNA twist [17]. As shown in Fig. 4C, in the presence of a strongly
intercalative drug such as EB and PI, a net negative supercoiling of
relaxed substrate DNA was induced (lanes 7 and 8). Conversely, no
unwinding was observed with the non-intercalative drug OPT
(lanes 3). Compound 1 also showed no unwinding effect on DNA up
activity of Topo I in a way similar to that of known Topo I inhibitors
(OPT, 100
showed no remarkable inhibition of Topo I and Topo II
activity at 100 M. These data suggested there was certain corre-
mM). On the contrary, compound 1a, 2a, 3a and 4a
a
catalytic
m
lation between cytotoxicity and Topo inhibition. The analogs (2 and
4) with lower potency against Topo I still exhibited similar cyto-
toxicity compared to the ones (1 and 3) with higher potency against
Topo I. The fact that the cytotoxicity was mainly related to Topo II
a
inhibition for this class of compounds could be concluded. Addi-
tionally, MDA-MB-435 cell line, in which Topo I was highly
expressed [18,19], was more sensitive to compounds 1e4 than
other two cell lines. This data provided indirect support for the
conclusion. The solvent DMSO has no effect on the results (data not
shown).
to 100 mM (lanes 4e6). The results indicated that compound 1 had
no significant DNA intercalating activity.
The topoisomerases inhibitory activities of other analogs were
also evaluated by the DNA relaxation assay. Hydroxycamptothecine
The Topo II
confirmed by Topo II
shown in Fig. 5C, compound 2 and 4 apparently suppressed Topo II
mediated decatenation of kDNA at the concentration of 100 M.
a
inhibition of compounds 1e4, 1ae4a was further
a
mediated kDNA decatenation assays. As
(OPT) and etoposide (VP-16), two well-known Topo I and Topo II
a
inhibitors, were used as positive control respectively. As shown in
m

J. Qiu et al. / European Journal of Medicinal Chemistry 68 (2013) 192e202
197
A
B
C
Fig. 4. Inhibitory effect of compound 1 on Topo I and Topo II
a
mediated DNA relaxation. (A) Compound 1 inhibited Topo I/II
a
-mediated supercoiled DNA relaxation. Negatively
supercoiled pBR322 (SC) and relaxed DNA (R) were shown by electrophoresis on an agarose gel after compound 1 treatment at the indicated concentration. OPT and VP-16 were
used at 100 M as the positive control. (B) Effects of compound 1 on the Topo I/II -mediated DNA cleavage. Negatively supercoiled pBR322 (lane 1) was shown for reference. The
m
a
position of linear DNA (L) and nicked open circular (NOC) were indicated. (C) Effects of compound 1 on the DNA unwinding assay with Topo I. Lane 1, pBR322 DNA only. Lane 2,
pBR322 DNA and Topo I. Lanes 3e8, pBR322 DNA, Topo I, and various concentrations of compound 1. PI and EB were used as the positive control.
While compound 1 and compound 3 exhibited partial inhibition of
Topo II . Almost no decatenation activity was observed in the
compound 1 with the enzyme prior to the addition of DNA
increased the inhibitory activity.
a
presence of compounds 1a, 2a, 3a and 4a.
In eukaryotes, G2/M phase, a highly complex multistage process,
is a critical phase before cell division during the cell cycle. An
important event prior to mitotic entry is the decatenation of sister
chromatids, which are highly entangled as a consequence of DNA
replication. This process is carried out by DNA topoisomerase
[22,23]. Although compound 1 did not produce DNA strand breaks,
it caused a cell cycle arrest at the G2/M phase on cell cycle just as
Topo I poisons (Fig. 2A). This effect could be due to the fact that
Topo II was perturbed by compound 1, resulting in catenation of
sister chromatids which could then lead to the G2/M delay. Cyclin B,
in association with cdc2 (CDK1), governs cell cycle progression by
enhancing cell cycle distribution in the G2/M fraction. The inhibi-
tion of cyclin B/cdc2 complexes prevents the G2/M transition [24].
However, our data showed that compound 1 increased the level of
cyclin B/cdc2 (Fig. 2C). This property might also be due to Topo I
inhibition, which led to the spindle checkpoint activation and
mitotic exit prevention [25].
3. Discussion
Currently, most of the topoisomerase inhibitors show selective
inhibition against either Topo I or Topo II, and only a small amount
of compounds is capable of inhibiting both the two enzymes above.
Selective inhibition of the Topo I enzyme can, however, induce a
reactive increase in Topo I levels and vice versa. This mechanism is
associated with the development of drug resistance. Therefore,
searching for new compounds that inhibit both Topo I and Topo I is
very important because of the deficiency of specific inhibitors
which can overcome multidrug resistance (MDR) cancer cells [20].
In this study, compound 1, showing significant anticancer activities,
strongly inhibited the enzymatic activity of Topo I and Topo II
(Fig. 4). In contrast, compound 2 and 4, with less anticancer activ-
a
ities than compound 1, showed selective inhibition against Topo II
(Fig. 5).
a
The arrest of cell cycle progression at G2/M phase provides an
opportunity for cells to either undergo repair mechanisms or follow
the apoptosis. In this study, increasing percentages of apoptosis
were observed when the cells were sorted by FACS after exposed to
compound 1 (Fig. 2A), while the time-dependent induction of a
sub-G1 cell population suggested that G2/M-phase arrest might be
an inverse process leading to apoptosis (Fig. 2D). The apoptosis was
further confirmed by nuclear fragmentation staining (DAPI stain-
ing) and Annexin V/PI staining of compound 1-treated cells
(Fig. 3A&B). There are two distinct apoptotic pathways in mammals,
Despite their efficiency in the clinics, current anticancer thera-
pies with topoisomerase inhibitors are limited by certain consid-
erably negative consequences. The most negative consequence
from observation is that the treatment with Topo poisons may
result in secondary malignancies [21]. However, compound 1 did
not induce Topo-mediated DNA cleavage in our experiments
(Fig. 4B). DNA-unwinding assay indicated that compound 1 had no
significant DNA intercalating activity (Fig. 4C). Pre-incubation of

198
J. Qiu et al. / European Journal of Medicinal Chemistry 68 (2013) 192e202
A
B
C
Fig. 5. Effects of p-terphenyls derivatives on the Topo I/II
I/II . Lanes 3e11, pBR322 DNA, Topo I/II , and various compounds (100
was used as substrate, the position of kDNA and decatenation products minicircles was indicated.
a
-mediated supercoiled DNA relaxation. (A): Topo I; (B): Topo II
a
. Lane 1, pBR322 DNA only. Lane 2, pBR322 DNA and Topo
a
a
mM). OPT and VP-16 were used as the positive control; (C) Topo IIa
mediated kDNA decatenation assay. kDNA
the mitochondrial pathway and the death receptor pathway. The
activation of caspase-3 plays a crucial role in the initiation of
apoptosis. Our data showed that compound 1 induced PARP
cleavage and the activation of caspase-3. Furthermore, the ratio of
Bcl-2/Bax (Fig. 3C), which is crucial for the activation of the mito-
chondrial apoptotic pathway, decreased in cells treated with
compound 1. Taken together, these results indicated that com-
pound 1 activated caspase-dependent apoptosis in MDA-MB-435
cells mainly via mitochondrial pathway.
were visualized by iodine vapors or by irradiation with UV light
(254 nm). Flash column chromatography was performed on col-
umn packed with Silica Gel 60 (200e300 mesh). Solvents were
reagent grade and, when necessary, they were purified and dried by
standard methods. Concentration of the reaction solutions involved
the use of rotary evaporator at reduced pressure.
4.1.1. 4-Bromo-2-methoxyphenol (10a) [26]
A solution of NBS (1 eq.) in DMF (50 mL) was added dropwise to
a solution of guaiacol (1 eq.) in DMF (50 mL) at 0 ꢁC. After being
stirred for 30 min, the reaction mixture was quenched with ice
water and extracted with ethyl acetate. The combined organic
layers were washed with brine, dried over anhydrous sodium sul-
fate and then evaporated to dryness under reduced pressure. The
residue was further purified by column chromatography over Silica
Gel 200e300 N (petroleum ether/ethyl acetate 9.5:0.5) to give 10a
as an oil (78%).
In summary, eight new p-terphenyls derivatives were success-
fully synthesized. Some of these compounds exhibited potent
anticancer activity, which were mainly related to Topo II
ability. Our study also identified that compound 1 was a novel kind
of Topo I and Topo II catalytic inhibitor. The biological properties of
a inhibition
a
compound 1 showed its potential as a novel cancer chemothera-
peutic agent.
4. Experimental section
4.1.2. 3,6,6-tribromo-2-hydroxycyclohex-2-enone
4.1. Chemistry
The cyclohexanone (1 eq.) with CuBr2, 30 times as much as that,
in 50% dioxane (v/v, 200 mL) was refluxed for 3 h. Afterward, the
reaction mixture was quenched with water and extracted with
ethyl acetate. The organic layer was washed with brine, dried over
anhydrous sodium sulfate and evaporated to dryness under
reduced pressure. The residue was purified by column chroma-
tography using petroleum ether/ethyl acetate 20:1 to give 3,6,6-
tribromo-2-hydroxycyclohex-2-enone [27] (28%).
All melting points were determined on a micro melting point
apparatus and were uncorrected. 1H NMR and 13C NMR spectra
were obtained on a Brucker Avance-600 NMR-spectrometer or
Inova-600 NMR-spectrometer in the indicated solvents. Chemical
shifts are expressed in ppm (
ternal reference. TLC was performed on Silica Gel GF254 and spots
d units) relative to TMS signal as in-

J. Qiu et al. / European Journal of Medicinal Chemistry 68 (2013) 192e202
199
4.1.3. 3,6-dibromobenzene-1,2-diol
chromatography using CH2Cl2/ethyl acetate 9:1 yielded the corre-
sponding derivatives.
The 3,6,6-tribromo-2-hydroxycyclohex-2-enone (1 eq.) was
stirred with LiCO3 (l.5 eq.) in DMF (20 mL) for 2 h in a nitrogen
atmosphere at 30 ꢁC. After the reaction finished, the mixture was
quenched with water and extracted with ethyl acetate. The organic
layer was washed with 5% HCl and brine, dried over anhydrous
sodium sulfate and evaporated to dryness. The residue was further
purified by column chromatography using petroleum ether/ethyl
acetate 2:1 to give 3,6-dibromobenzene-1,2-diol [28] (65%).
4.1.8. General parallel procedure c (Scheme 1) for the synthesis of
compounds 2a and 3a
Intermediate 12a or 12b was dissolved respectively in a biphasic
mixture of CH2Cl2 and 10% aqueous NaOH followed by slow addi-
tion of triflic anhydride (3 eq.) at 0 ꢁC. After finishing dropping, the
mixture was allowed to warm to room temperature and stirred for
30 min. Two phases were separated and the CH2Cl2 layer, washed
with brine, was evaporated to dryness under reduced pressure, the
crude product of the steps above was used directly in the next step
[35]. Then, the crude product, phenyl boronic acid 2 (2 eq.) and KF
(3.0 eq.) were dissolved in dioxane respectively, and the resulting
mixtures were deoxygenated with a stream of N2. After being
stirred for 10 min, PdCl2(dppf) (0.05 eq.) was added, and each
mixture was brought to reflux, allowed to stir under N2 for 5e28 h
until the reaction, detected by TLC, was complete. Then each
mixture was cooled to the room temperature and treated as fol-
lows. Each solution was poured into a mixture of H2O and ethyl
acetate, and the two phases were separated. The aqueous layer was
washed with ethyl acetate. The organic phases were combined and
washed with brine. The ethyl acetate phase was dried over anhy-
drous sodium sulfate and evaporated to dryness under reduced
pressure. Purification of each crude product by chromatography
using petroleum ether/ethyl acetate 4:1 or 9:1 yielded the corre-
sponding derivatives.
4.1.4. 1,4-dibromo-2,3-dimethoxybenzene (6) [29]
A solution of 3,6-dibromobenzene-1,2-diol (1.0 eq.) in acetone
(10 mL) was added to the mixture of dimethyl sulfate (4.0 eq.) and
K2CO3 (4.0 eq.). After being stirred overnight, the mixture was
quenched with sufficient quantum aqueous ammonia and extrac-
ted with ethyl acetate. The aqueous layer was washed with ethyl
acetate. The organic phases were combined, washed with brine,
dried over anhydrous sodium sulfate and evaporated to dryness
under reduced pressure. The crude product was purified by chro-
matography using petroleum ether/ethyl acetate 20:1 to yield the
corresponding derivatives 1,4-dibromo-2,3-dimethoxybenzene
(95%). 1H NMR (600 MHz, DMSO-d6)
d 7.35 (s, 2H), d 3.83 (s, 6H).
4.1.5. 4-Bromo-2,6-dimethoxyphenol (10b) [30]
2,6-dimethoxyphenol (1 eq.), sodium hydride (0.01 eq., 60% in
mineral oil), and anhydrous MeOH (1.2 mL) in 150 mL of anhydrous
CH2Cl2 were treated with N-bromosuccinimide (1 eq.) at ꢀ45 ꢁC.
The products were evaporated under reduced pressure. The res-
idue was purified by column chromatography over Silica Gel 200e
300 N (petroleum ether/ethyl acetate 9:1) to give white crystals
(50%).
4.1.9. [1,10:40,100-Terphenyl]-400-monol,3,4-dimethoxy (1a)
Mp 250e252 ꢁC; 1H NMR (600 MHz, DMSO-d6)
d 3.77 (s, 3H,
OCH3), 3.92 (s, 3H, OCH3), 6.87 (d, J ¼ 8.4 Hz, 2H, H300, H500), 7.04
(d, J ¼ 8.4 Hz, 1H, H5), 7.23 (d, J ¼ 8.4 Hz, 1H, H6), 7.26 (s, 1H, H2),
7.54 (d, J ¼ 8.4 Hz, 2H, H20, H60), 7.64 (d, J ¼ 8.4 Hz, 2H, H30, H50),
7.71 (d, J ¼ 8.4 Hz, 2H, H200, H600), 9.59 (s, 1H, OH); 13C NMR
4.1.6. General parallel procedure a (Scheme 1) for the synthesis of
Compound 1a, 3a, 8a, 8b, 12a and 12b
The appropriate bromo derivatives (1 eq.), appropriate phenyl
boronic acids 7/9/11 (1.5 eq.) and KF (3.0 eq.) were dissolved
respectively in dioxane, and the three resulting mixtures were
deoxygenated with a stream of N2. After 10 min, PdCl2(dppf)
(0.05 eq.) was added, and each mixture was brought to reflux,
allowed to be stirred under N2 for 5e22 h until the reaction is
complete, which was detected by TLC. Then each solution was
cooled to room temperature. After that, each solution was poured
into a mixture of H2O and ethyl acetate, and the two phases were
separated. The aqueous layer was washed with ethyl acetate, and
the organic phases were combined and washed with brine. The
ethyl acetate layer was dried over anhydrous sodium sulfate and
evaporated to dryness under reduced pressure. Purification of each
crude product by chromatography using petroleum ether/ethyl
acetate 4:1 or 9:1 yielded the corresponding derivatives of 1a, 3a,
(600 MHz, CDCl3) d 55.39 (OCH3), 55.42 (OCH3), 109.99 (C5), 112.0
(C2), 115.6 (C300), 115.6 (C500), 118.4 (C6), 126.1 (C20), 126.1 (C60),
126.6 (C30), 126.6 (C50), 127.5 (C200), 127.5 (C600), 130.3 (C1), 132.4
(C100), 137.9 (C10), 138.4 (C40), 148.3 (C4), 148.9 (C3), 157.0 (C400);
HRMS (ESI) Calcd. for C20H19O3 [M þ H]þ 307.1334, Found:
307.1329.
4.1.10. [1,10:40,100-Terphenyl]-3,4,400-triol (1)
Mp > 260 ꢁC; 1H NMR (600 MHz, DMSO-d6)
d
6.82 (d, J ¼ 8.4 Hz,
1H, H5), 6.85 (d, J ¼ 8.4 Hz, 2H, H300, H500), 6.97 (d, J ¼ 8.4 Hz, 1H,
H6), 7.06 (s, 1H, H2), 7.51 (d, J ¼ 8.4 Hz, 2H, H20, H60), 7.55 (d,
J ¼ 7.8 Hz, 2H, H30, H50), 7.60 (d, J ¼ 7.8 Hz, 2H, H200, H600), 9.02 (s,1H,
13
OH), 9.05 (s, 1H, OH), 9.54 (s, 1H, OH); C NMR (600 MHz,
(CD3)2CO)
d
113.7 (C5), 113.7 (C2), 115.7 (C300), 115.7 (C500), 118.2
(C20),118.2 (C60), 126.5 (C30), 126.5 (C50), 126.6 (C6), 127.7 (C200),
127.7 (C600), 131.9 (C1), 132.7 (C100), 138.99 (C10), 139.1 (C40), 144.9
(C4), 145.4 (C3), 157.1 (C400); HRMS (ESI) Calcd. for C18H15O3
[M þ H]þ 279.1021, Found: 279.1108.
8a [31], 8b, 12a [32]: 1H NMR (600 MHz, DMSO-d6)
7.53 (d, J ¼ 8.4 Hz, 2H), 7.12 (s, 1H), 7.01 (d, J ¼ 8.4 Hz, 1H), 6.97 (d,
J ¼ 8.4 Hz 2H), 6.82 (d, J ¼ 8.4 Hz, 1H), 3.8 (s, 3H), 3.65 (s, 3H), and
12b [33].
d 9.01 (s, 1H),
4.1.11. [1,10:40,100-Terphenyl]-20,3,4,400-tetmethoxy (2a)
4.1.7. General parallel procedure b (Scheme 1) for the synthesis of
compounds 1, 2, 3, and 4
Mp 131e132 ꢁC; 1H NMR (600 MHz, CDCl3)
d 3.87 (s, 3H, OCH3),
3.89 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 6.94 (d,
J ¼ 9 Hz,1H, H5), 7.00 (d, J ¼ 8.4 Hz, 2H, H300, H500), 7.12 (d, J ¼ 1.8 Hz,
1H, H30), 7.14 (s, 1H, H2), 7.15 (d, J ¼ 9 Hz, 1H, H6), 7.21 (dd, J ¼ 7.8,
1.8 Hz, 1H, H50), 7.37 (d, J ¼ 7.8 Hz, 1H, H60), 7.58 (d, J ¼ 8.4 Hz, 2H,
The 1a, 2a, 3a, and 4a (1 eq.) were dissolved respectively in
anhydrous CH2Cl2 (1 mL) at ꢀ78 ꢁC. Then BBr3 (3 eq.) was added to
each solution, and the four resulting reaction mixtures were
allowed to warm to room temperature for 20 h and treated as
follows. Each solution was poured into ice water followed by being
warmed to ambient temperature, and then each solution was
washed twice with ethyl acetate. The combined organic layer was
dried over anhydrous sodium sulfate and evaporated to dryness
under reduced pressure [34]. Purification of each crude product by
H200, H600); 13C NMR (600 MHz, CDCl3)
d
159.2 (C400), 156.6 (C20),
148.3 (C3), 148.1 (C4), 141.2 (C40), 133.5 (C100), 130.93 (C1), 130.87
(C10), 128.8 (C60), 128.1 (C200), 128.1 (C600), 121.8 (C6), 119.2 (C50),
114.2 (C300), 114.2 (C500), 112.9 (C2), 110.8 (C30), 109.8 (C5), 55.9
(OCH3), 55.9 (OCH3), 55.7 (OCH3), 55.4 (OCH3); HRMS (ESI) Calcd.
for C22H23O4 [M þ H]þ 351.1600, Found: 351.1595.

200
J. Qiu et al. / European Journal of Medicinal Chemistry 68 (2013) 192e202
4.1.12. [1,10:40,100-Terphenyl]-20,3,4,400-tetol (2)
Mp > 260 ꢁC; 1H NMR (600 MHz, DMSO-d6)
131.7 (C1), 143.6 (C20), 143.7 (C30), 145.5 (C3),146.0 (C4), 157.4 (C400);
d
6.75 (d, J ¼ 7.8 Hz,
HRMS (ESI) Calcd. for C18H15O5 [M þ H]þ 311.0919, Found: 311.0913.
1H, H5), 6.83 (br, 3H H50 H300, H500), 7.03 (br, 2H, H6, H2), 7.07 (s, 1H,
H30), 7.20 (d, J ¼ 7.2 Hz, 1H, H60), 7.41 (d, J ¼ 7.2 Hz, 2H, H200, H600),
8.86 (s, 2H, OH), 9.41 (s, 1H, OH), 9.54 (s, 1H, OH); 13C NMR
4.2. Biological assay
(600 MHz, DMSO-d6) d
113.8 (C5),115.7 (C2),116.1 (C300), 116.1 (C500),
4.2.1. Cell line and reagents
117.1 (C30), 117.6 (C50), 120.4 (C6), 126.6 (C60), 127.8 (C200), 127.8
(C600), 129.9 (C10), 130.7 (C1), 131.2 (C100), 140.0 (C40), 144.6 (C3),
145.0 (C4), 154.8 (C20), 157.5 (C400); HRMS (ESI) Calcd. for C18H15O4
[M þ H]þ 295.0970, Found: 295.0967.
MDA-MB-435 cell line was obtained from ATCC and cultured in
DMEM/F12 with 10% heat-inactivated fetal bovine serum. Cultures
were maintained in a humidified incubator at 37 ꢁC in an atmo-
sphere of 5% CO2. All the reagents used in this study, unless
otherwise indicated, were purchased from Sigma (St. Louis, MO).
4.1.13. [1,10:40,100-Terphenyl]-20,3,4,400,60-pentamethoxy (3a)
Mp 170e172 ꢁC; 1H NMR (600 MHz, CDCl3)
d 3.81 (s, 6H, OCH3),
4.2.2. In vitro cytotoxicity assays
3.87 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 6.81 (s, 2H,
H30, H50), 6.94 (d, J ¼ 8.4 Hz,1H, H5), 6.96 (d, J ¼ 1.8 Hz,1H, H2), 6.97
(dd, J ¼ 8.4, 1.8 Hz, 1H, H6), 7.00 (d, J ¼ 9 Hz, 2H, H300, H500), 7.58 (d,
The cytotoxicity was measured by the MTT assay as described in
the literature [36,37]. The cells plated in the wells of 96-well plates
(Falcon, USA) were treated in triplicate with various concentrations
of compounds for 72 h in 5% CO2 incubator at 37 ꢁC. After fresh
J ¼ 9 Hz, 2H, H200, H600); 13C NMR (600 MHz, CDCl3)
d55.4 (OCH3),
55.7 (OCH3), 55.8 (OCH3), 56.0 (OCH3), 56.0 (OCH3), 103.1 (C30),
103.1 (C50), 110.5 (C10), 114.2 (C300), 114.2 (C500), 114.3 (C5), 117.6 (C2),
123.2 (C6), 126.2 (C1), 128.2 (C200), 128.2 (C600), 134.0 (C100), 141.6
(C40), 147.8 (C4), 148.1 (C3), 157.9 (C20), 157.9 (C60), 159.3 (C400);
HRMS (ESI) Calcd. for C23H25O5 [M þ H]þ 381.1702, Found:
381.1703.
medium being changed, a 20
m
L aliquot of MTT solution (5 mg/mL)
was added and incubated for 4 h at 37 ꢁC. Then, 100
mL of triplex
solution (10% SDS, 5% isobutanol, 12 mM HCl) was added to each
well and incubated overnight at 37 ꢁC. The absorbance of each well
was determined by a microplate reader (M-3350, Bio-Rad) with a
590 nm wavelength. Growth inhibition rates were calculated with
the following equation:
4.1.14. [1,10:40,100-Terphenyl]-20,3,4,400,60-pentaol (3)
Mp > 260 ꢁC; 1H NMR (600 MHz, DMSO-d6)
d
6.54 (s, 2H, H30,
OD control well ꢀ OD treated well
Inhibition rate ¼
ꢃ 100%
H50), 6.58 (dd, J ¼ 8.4, 1.8 Hz, 1H, H5), 6.69 (d, J ¼ 8.4 Hz, 1H, H6),
6.73 (d, J ¼ 1.8 Hz, 1H, H2), 6.83 (d, J ¼ 8.4 Hz, 2H, H300, H500), 7.33 (d,
J ¼ 8.4 Hz, 2H, H200, H600), 8.69 (s, 2H, OH), 8.96(s, 1H, OH), 9.52 (s,
OD control well ꢀ OD blank well
1H, OH); 13C NMR (600 MHz, DMSO-d6)
d
104.4 (C30), 104.4 (C50),
4.2.3. Cell cycle analysis
114.3 (C10), 114.5 (C5), 115.5 (C300), 115.5 (C500), 118.5 (C2), 121.9 (C6),
125.3 (C1), 127.1 (C200), 127.3 (C600), 131.2 (C100), 139.4 (C40), 143.4
(C3), 143.9 (C4), 155.7 (C20), 155.7 (C60), 156.8 (C400); HRMS (ESI)
Calcd. for C18H15O5 [M þ H]þ 311.0919, Found: 311.0907.
Cell cycle analysis was measured as previously described
[38,39]. Briefly, 5 ꢃ 105 cells were plated in six-well plates. After
drug treatment, the cells were fixed in 75% ethanol at ꢀ22 ꢁC
overnight, resuspended in 1 mL phosphate-buffered saline con-
taining 0.1 mg/mL RNaseA and propidium iodide (40
mg/mL) to
4.1.15. [1,10-Biphenyl]-40-monol, 4-bromo-2,3-dimethoxy (8b)
stain DNA for 30 min at room temperature, and analyzed for DNA
Mp 117e118 ꢁC; 1H NMR (600 MHz, CDCl3)
d
3.65 (s, 3H, OCH3),
contents using a FACScan flow cytometer (Beckman coulter
3.94 (s, 3H, OCH3), 5.57 (s, 1H, OH), 6.88 (d, J ¼ 8.4 Hz, 2H, H30, H50),
6.95 (d, J ¼ 8.4 Hz, 1H, H5), 7.32 (d, J ¼ 8.4 Hz, 1H, H6), 7.38(s,
EPICSxL). Data were analyzed using MultiCycle for windows soft-
ware (Beckman coulter EPICSxL).
J ¼ 8.4 Hz, 2H, H20, H60), 13C NMR (600 MHz, CDCl3)
d 60.8 (OCH3),
60.9 (OCH3),115.2 (C30),115.2 (C50),116.2 (C4),126.4 (C6),127.9 (C1),
129.7 (C5), 130.3 (C20), 130.3 (C60), 135.6 (C10), 150.7 (C2), 151.5 (C3),
155.1 (C40); HRMS (ESI) Calcd. for C14H13BrNaO3 [M þ Na]þ
330.9946, Found: 330.9947.
4.2.4. Analysis of apoptosis
4.2.4.1. Nuclear staining with DAPI. After treatment, cells were
collected, washed once with 2 mL of ice-cold PBS, fixed with 1 mL
4% paraformaldehyde for 20 min, and then washed once again with
2 mL of ice-cold PBS. The cells were incubated in 1 mL of DAPIat
4.1.16. [1,10:40,100-Terphenyl]- 400-monol,20,3,30,4-tetmethoxy (4a)
50 mg/mL containing 100 mg/mL RNaseA. This mixture was incu-
Mp 165e166 ꢁC; 1H NMR (600 MHz, CDCl3)
d
3.69 (s, 3H, OCH3),
bated for 30 min at 37 ꢁC. After having been washed with 2 mL of
PBS three times, the cells were observed using fluorescence mi-
croscopy at 340 nm (excitation) and 488 nm (emission).
3.71 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 5.09 (s, 1H,
OH), 6.91 (d, J ¼ 8.4 Hz, 2H, H300, H500), 6.96 (d, J ¼ 8.4 Hz, 1H, H5),
7.12 (d, J ¼ 7.8 Hz, 1H, H60), 7.13 (d, J ¼ 8.4 Hz, 1H, H6), 7.14 (d,
J ¼ 7.8 Hz, 1H, H50), 7.18 (d, J ¼ 1.8 Hz, 1H, H2), 7.47 (d, J ¼ 8.4 Hz, 2H,
4.2.4.2. Flow cytometric analysis (annexin V-FITC/PI staining) of
phosphatidylserine exposure. At the indicated times and doses of
treatment, MDA-MB-435 cells were assayed for phosphatidylser-
ine exposure, by using the Annexin V-FITC Apoptosis Detection Kit
(Sigma) according to the manufacturer’s instructions. Stained
samples were analyzed by a FACScan flow cytometer (Beckman
coulter EPICSxL). The fractions of cell population in different
quadrants were analyzed using quadrant statistics. Cells in the
lower right quadrant represented apoptosis while cells in the
upper right quadrant represented necrosis or post apoptotic
necrosis.
H200, H600); 13C NMR (600 MHz, CDCl3)
d55.88 (OCH3), 55.9 (OCH3),
60.7 (OCH3), 60.8 (OCH3), 110.3 (C5), 110.9 (C2), 112.5 (C300), 112.5
(C500), 115.1 (C50), 121.4 (C60), 125.3 (C6), 125.4 (C10), 130.4 (C200),
130.4 (C600), 130.5 (C40), 130.8 (C100), 134.8 (C1), 148.2 (C4), 148.5
(C3), 150.9 (C20), 150.9 (C30), 154.9 (C400); HRMS (ESI) Calcd. for
C
22H23O5 [M þ H]þ 367.1545, Found: 367.1547.
4.1.17. [1,10:40,100-Terphenyl]-20,3,30,4,400-pentaol (4)
Mp > 260 ꢁC; 1H NMR (600 MHz, Methanol-d4)
d
6.80 (s, 2H, H30,
H50), 6.85 (d, J ¼ 8.4 Hz, 1H, H5), 6.86 (d, J ¼ 8.4 Hz, 2H, H300, H500),
6.94 (dd, J ¼ 8.4, 1.8 Hz, 1H, H6), 7.09 (d, J ¼ 1.8 Hz, 1H, H2), 7.45 (d,
J ¼ 8.4 Hz, 2H, H200, H600); 13C NMR (600 MHz, Methanol-d4)
d116.0
4.2.5. Western blot analysis
After treatment with or without compounds, cells were har-
vested and lysed in ice-cold lysis buffer (20 mM TriseHCl, pH 7.4,
(C300), 116.0 (C500), 116.2 (C5), 117.4 (C2), 121.7 (C50), 121.7 (C60), 122.1
(C60), 122.2 (C50), 128.9 (C6), 129.1 (C100), 131.2 (C200), 131.3 (C600),

J. Qiu et al. / European Journal of Medicinal Chemistry 68 (2013) 192e202
201
150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium
pyrophosphate, 1 mM - glycerophosphate, 1 mM sodium ortho-
vanadate, 1 mg/mL leupeptin, 1 mM phenylmethylsulfonyl fluo-
ride). The lysates were clarified by centrifugation at 12,000 rpm at
4.2.6.3. DNA-unwinding
assay. DNA-unwinding
assay
was
b
employed to assess the ability of compound 1 of intercalating into
plasmid DNA according to the procedure in previous studies [42].
Relaxed DNA was prepared by treatment of the supercoiled plasmid
pBR322 with excess Topo I(10 units), followed by proteinase K
digestion at 37 ꢁC, phenol-chloroform extraction and ethanol pre-
cipitation. After incubation with testing compounds at 37 ꢁC for
30 min, reactions were terminated by the addition of gel loading
buffer and electrophoresed under the same conditions as described
4
ꢁC for 15 min, mixed with an equal volume of 2ꢃ loading buffer
(4% SDS, 10% 2-mercaptoethanol, 20% glycerol, and 0.2 mg/mL
bromphenol blue in 0.1 M TriseHCl, pH 6.8), and then boiled for
10 min immediately. The boiled lysates were loaded onto 8e12%
SDS-polyacrylamide gels and transferred to Immobilon-P mem-
branes (Millipore). After blocked with 5% skim milk in phosphate-
buffered saline with 0.1% Tween-20 for 1 h, the membranes were
above. The DNA bands were stained with 0.5
bromide and visualized by UV light.
mg/mL of ethidium
incubated with antibodies against
pase3, cleaved caspase-3, PARP, cyclin B1, cdc2, Myt1, GADD45
b
-actin, Bcl-2, Bak, Bad, cas-
and
a
4.2.6.4. Topo II-mediated DNA decatenation assay. Topo II activity
cdc25c (Cell Signaling Technology). Primary antibodies were
detected using either peroxidase-conjugated ImmunoPure goat
anti-rabbit IgG (H þ L) or peroxidase-conjugated ImmunoPure Goat
Anti-Mouse IgG (H þ L) secondary antibody (Jackson ImmunoR-
esearch, USA) and enhanced chemiluminescence.
was measured by the ATP-dependent decatenation of kDNA [43].
Briefly, 0.20
II
at 37 ꢁC for 30 min in a total of 20
(pH 8.0), 120 M KCl, 10 mM MgCl2, 0.5 mM ATP, 0.5 mM DTT, 30
mL BSA]. The reaction was stopped with 2 L of 10% SDS. Samples
m
g of kDNA (TopoGEN, Inc) was incubated 2 unit of topo
L reaction buffer [50 mM Tris
g/
a
m
m
m
were subjected to electrophoresis under the same conditions as
described above.
4.2.6. DNA gel electrophoresis assay of topoisomerases
Plasmid pBR322 DNA and purified calf thymus DNA topoisom-
erase Ⅰ were purchased from TakaRa Biotechnology (Dalian) Co.,
Acknowledgments
Ltd., recombinant human topoisomerase IIa was obtained from
TopoGENINC (USA). All experiments were done at least in duplicate
This work was financially supported by NSFC Projects
to confirm the results.
(81273384, 90913024).
4.2.6.1. DNA relaxation assay. DNA topo I inhibition assay was
performed as described previously [40] with minor modifications.
The test compounds were dissolved in DMSO at 20 mM as stock
solution. The activity of DNA Topo I was determined by assessing
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.07.020.
the relaxation of supercoiled DNA pBR322. The mixture of 0.5 mg of
plasmid pBR322 DNA and 1 units of Topo I was incubated without
and with the prepared compounds at 37 ꢁC for 30 min in the
relaxation buffer (10 mM TriseHCl (pH 7.9), 150 mM NaCl, 0.1%
bovine serum albumin,1 mM spermidine, 5% glycerol). The reaction
References
[1] J.C. Wang, DNA topoisomerases, Annu. Rev. Biochem. 65 (1996) 635e692.
[2] J.J. Champoux, DNA topoisomerases: structure, function, and mechanism,
Annu. Rev. Biochem. 70 (2001) 369e413.
in the final volume of 20 mL was terminated by adding 2.5 mL of the
[3] S. Salerno, F. Da Settimo, S. Taliani, F. Simorini, C. La Motta, G. Fornaciari,
A.M. Marini, Recent advances in the development of dual topoisomerase I and
II inhibitors as anticancer drugs, Curr. Med. Chem. 17 (2010) 4270e4290.
[4] W.A. Denny, Dual topoisomerase I/II poisons as anticancer drugs, Expert Opin.
Invest. Drugs 6 (1997) 1845e1851.
[5] K. Chikamori, A.G. Grozav, T. Kozuki, D. Grabowski, R. Ganapathi,
M.K. Ganapathi, DNA topoisomerase II enzymes as molecular targets for
cancer chemotherapy, Curr. Cancer Drug Targets 10 (2010) 758e771.
[6] V. Calì, C. Spatafora, C. Tringali, Polyhydroxy-p-terphenyls and related p-ter-
phenylquinones from fungi: overview and biological properties, in: R. Atta ur
(Ed.), Studies in Natural Products Chemistry, Elsevier, 2003, pp. 263e307.
[7] T. Kamigauchi, R. Sakazaki, K. Nagashima, Y. Kawamura, Y. Yasuda,
K. Matsushima, H. Tani, Y. Takahashi, K. Ishii, R. Suzuki, K. Koizumi, H. Nakai,
Y. Ikenishi, Y. Terui, Terprenins, novel immunosuppressants produced by
Aspergillus candidus, J. Antibiot. (Tokyo) 51 (1998) 445e450.
[8] S.S. Liu, B.B. Zhao, C.H. Lu, J.J. Huang, Y.M. Shen, Two new p-terphenyl de-
rivatives from the marine fungal strain Aspergillus sp. AF119, Nat. Prod.
Commun. 7 (2012) 1057e1062.
[9] A. Barzilai, K. Yamamoto, DNA damage responses to oxidative stress, DNA
Repair (Amst) 3 (2004) 1109e1115.
[10] G. Iliakis, Y. Wang, J. Guan, H. Wang, DNA damage checkpoint control in cells
exposed to ionizing radiation, Oncogene 22 (2003) 5834e5847.
[11] V.A. Rao, C. Conti, J. Guirouilh-Barbat, A. Nakamura, Z.H. Miao, S.L. Davies,
B. Sacca, I.D. Hickson, A. Bensimon, Y. Pommier, Endogenous gamma-H2AX-
ATM-Chk2 checkpoint activation in Bloom’s syndrome helicase deficient
cells is related to DNA replication arrested forks, Mol. Cancer Res. 5 (2007)
713e724.
stop solution containing 5% sarcosyl, 0.0025% bromophenol blue,
and 25% glycerol. DNA samples were then electrophoresed on a 1%
agarose gel at 5 V/cm for 2 h with a running buffer of TAE. Gels were
stained for 30 min in an aqueous solution of ethidium bromide
(0.5
DNA Topo II
follows [41]. Briefly, the mixture of 0.5
plasmid DNA and 1 units of Topo II was incubated without and
m
g/mL) and photographed under UV light.
inhibitory activity of compounds was measured as
g of supercoiled pBR322
a
m
a
with the prepared compounds in the assay buffer (10 mM TriseHCl
(pH 7.9) containing 50 mM NaCl, 50 mM KCl, 5 mM MgCl2, 1 mM
EDTA, 1 mM ATP, and 15 lg/mL bovine serum albumin) for 30 min at
30 ꢁC. The reaction in a final volume of 20
addition of 3
m
L was terminated by the
m
L of 7 mM EDTA. Reaction products were analyzed on
a 1% agarose gel at 5 V/cm for 2 h with a running buffer of TAE. Gels
were stained for 30 min in an aqueous solution of ethidium bro-
mide (0.5 mg/mL) and photographed under UV light.
4.2.6.2. Cleavage assay of Topo I and Topo II
a. The enzymatic re-
actions were set in the same format as for the Topo I and Topo II
a
inhibition assays except that in the enzymatic reactions an
increased amount of the enzyme (1 units per reaction) was used.
These reaction mixtures were incubated at 37 ꢁC for 30 min and
[12] Y. Tsujimoto, Bcl-2 family of proteins: life-or-death switch in mitochondria,
Biosci. Rep. 22 (2002) 47e58.
[13] A.K. Larsen, A.E. Escargueil, A. Skladanowski, Catalytic topoisomerase II in-
hibitors in cancer therapy, Pharmacol. Ther. 99 (2003) 167e181.
[14] J.M. Fortune, N. Osheroff, Topoisomerase II as a target for anticancer drugs:
when enzymes stop being nice, Prog. Nucleic Acid Res. Mol. Biol. 64 (2000)
221e253.
immediately terminated with 2
(SDS), followed by vigorous mixing. After the enzyme digestion
with proteinase K (50 g/mL) for 20 min, gel loading buffer was
mL of 10% sodium dodecyl sulfate
m
[15] W. Shi, S.L. Marcus, T.L. Lowary, Cytotoxicity and topoisomerase I/II inhibition
of glycosylated 2-phenyl-indoles, 2-phenyl-benzo[b]thiophenes and 2-
phenyl-benzo[b]furans, Bioorg. Med. Chem. 19 (2011) 603e612.
[16] Y. Pommier, J. Covey, D. Kerrigan, W. Mattes, J. Markovits, K.W. Kohn, Role of
DNA intercalation in the inhibition of purified mouse leukemia (L1210) DNA
added to the reaction mixture. Reaction mixture was analyzed on a
1% ethidium bromide-containing agarose gel at 5 V/cm for 2 h with
a running buffer of TAE. Gels were destained in water and photo-
graphed under UV light.

202
J. Qiu et al. / European Journal of Medicinal Chemistry 68 (2013) 192e202
topoisomerase II by 9-aminoacridines, Biochem. Pharmacol. 36 (1987) 3477e
3486.
intermediates by electronic factors: time-resolved laser flash and steady-state
photolysis investigations of photochromic 6- and 7-Arylchromenes, Chem.
Eur. J. 15 (2009) 4289e4300.
[17] A.R. Chowdhury, S. Sharma, S. Mandal, A. Goswami, S. Mukhopadhyay,
H.K. Majumder, Luteolin, an emerging anti-cancer flavonoid, poisons
eukaryotic DNA topoisomerase I, Biochem. J. 366 (2002) 653e661.
[18] H.L. Gomez, J.A. Pinto, M. Olivera, T. Vidaurre, F.D. Doimi, C.E. Vigil,
R.G. Velarde, J.E. Abugattas, E. Alarcon, C.S. Vallejos, Topoisomerase II-alpha as
a predictive factor of response to therapy with anthracyclines in locally
advanced breast cancer, Breast 20 (2011) 39e45.
[19] K. Kawachi, T. Sasaki, A. Murakami, T. Ishikawa, A. Kito, I. Ota, D. Shimizu,
A. Nozawa, Y. Nagashima, R. Machinami, I. Aoki, The topoisomerase II alpha
gene status in primary breast cancer is a predictive marker of the response to
anthracycline-based neoadjuvant chemotherapy, Pathol. Res. Pract. 206
(2010) 156e162.
[32] J.S.F.H.E. Landis, An expeditious aqueous SuzukieMiyaura methodology for
the arylation of bromophenols, Tetrahedron Lett. 47 (2006) 4275e4279.
[33] S.M.N. Widyastuti, Fukuji, Keisuke Watanabe, Nobumichi Sako, Kinji Tanaka,
Isolation and characterization of two aucuparin-related phytoalexins from
Photinia glabra Maxim, Ann. Phytopathol. Soc. Jpn. 58 (1992) 228e233.
[34] M. Roberti, D. Pizzirani, M. Recanatini, D. Simoni, S. Grimaudo, A. Di Cristina,
V. Abbadessa, N. Gebbia, M. Tolomeo, Identification of a terphenyl derivative
that blocks the cell cycle in the G0eG1 phase and induces differentiation in
leukemia cells, J. Med. Chem. 49 (2006) 3012e3018.
[35] D.E. Frantz, D.G. Weaver, J.P. Carey, M.H. Kress, U.H. Dolling, Practical syn-
thesis of aryl triflates under aqueous conditions, Org. Lett. 4 (2002) 4717e
4718.
[36] J. Carmichael, W.G. DeGraff, A.F. Gazdar, J.D. Minna, J.B. Mitchell, Evaluation of
a tetrazolium-based semiautomated colorimetric assay: assessment of che-
mosensitivity testing, Cancer Res. 47 (1987) 936e942.
[37] Y. Huang, J. Wang, G. Li, Z. Zheng, W. Su, Antitumor and antifungal activities in
endophytic fungi isolated from pharmaceutical plants Taxus mairei, Cepha-
lataxus fortunei and Torreya grandis, FEMS Immunol. Med. Microbiol. 31
(2001) 163e167.
[38] T. Ohtsuki, T. Koyano, T. Kowithayakorn, N. Yamaguchi, M. Ishibashi, Isolation
of austroinulin possessing cell cycle inhibition activity from Blumea glomerata
and revision of its absolute configuration, Planta Med. 70 (2004) 1170e1173.
[39] J. Wang, B. Zhao, W. Zhang, X. Wu, R. Wang, Y. Huang, D. Chen, K. Park,
B.C. Weimer, Y. Shen, Mycoepoxydiene, a fungal polyketide, induces cell cycle
arrest at the G2/M phase and apoptosis in HeLa cells, Bioorg. Med. Chem. Lett.
20 (2010) 7054e7058.
[40] M. Fukuda, K. Nishio, F. Kanzawa, H. Ogasawara, T. Ishida, H. Arioka,
K. Bojanowski, M. Oka, N. Saijo, Synergism between cisplatin and topoisom-
erase I inhibitors, NB-506 and SN-38, in human small cell lung cancer cells,
Cancer Res. 56 (1996) 789e793.
[41] J.F. Barrett, J.A. Sutcliffe, T.D. Gootz, In vitro assays used to measure the ac-
tivity of topoisomerases, Antimicrob. Agents Chemother. 34 (1990) 1e7.
[42] Y. Yamashita, N. Fujii, C. Murakata, T. Ashizawa, M. Okabe, H. Nakano, In-
duction of mammalian DNA topoisomerase I mediated DNA cleavage by
antitumor indolocarbazole derivatives, Biochemistry 31 (1992) 12069e12075.
[43] L. Du, H.C. Liu, W. Fu, D.H. Li, Q.M. Pan, T.J. Zhu, M.Y. Geng, Q.Q. Gu, Un-
precedented citrinin trimer tricitinol B functions as a novel topoisomerase
IIalpha inhibitor, J. Med. Chem. 54 (2011) 5796e5810.
[20] R. van Gijn, R. Lendfers, J. Schellens, A. Bult, J. Beijnen, Dual topoisomerase I/II
inhibitors, J. Oncol. Pharm. Pract. 6 (2000) 92e108.
[21] A.M. Azarova, Y.L. Lyu, C.P. Lin, Y.C. Tsai, J.Y. Lau, J.C. Wang, L.F. Liu, Roles of
DNA topoisomerase II isozymes in chemotherapy and secondary malig-
nancies, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 11014e11019.
[22] K. Luo, Z. Lou, Dual roles of topoisomerase II, Cell Cycle 8 (2009) 679.
[23] D.J. Clarke, A.C. Vas, C.A. Andrews, L.A. Diaz-Martinez, J.F. Gimenez-Abian,
Topoisomerase II checkpoints: universal mechanisms that regulate mitosis,
Cell Cycle 5 (2006) 1925e1928.
[24] G.K. Schwartz, M.A. Shah, Targeting the cell cycle: a new approach to cancer
therapy, J. Clin. Oncol. 23 (2005) 9408e9421.
[25] L.A. Diaz-Martinez, D.J. Clarke, Chromosome cohesion and the spindle
checkpoint, Cell Cycle 8 (2009) 2733e2740.
[26] N. Fujikawa, T. Ohta, T. Yamaguchi, T. Fukuda, F. Ishibashi, M. Iwao, Total
synthesis of lamellarins D, L, and N, Tetrahedron 62 (2006) 594e604.
[27] K. Nishizawa, J.Y. Satoh, The reaction of cycloalkanones with copper(II) ha-
lides. II. The reaction of cyclohexanones with copper(II) bromide, Bull. Chem.
Soc. Jpn. 48 (1975) 1875e1877.
[28] K. Nishizawa, J.Y. Satoh, A convenient synthesis of substituted pyrocatechols,
Bull. Chem. Soc. Jpn. 48 (1975) 2215e2216.
[29] M. Albrecht, Synthesis of linear oligo(catechol) ligands for the metal directed
self-assembling of helicates, Synthesis 2 (1996) 230e236.
[30] M.E.L. Jung, Patrick Yuk Sun, Mansuri, M. Muzzamil, Speltz, M. Laurine, Ster-
eoselective synthesis of an analog of podophyllotoxin by an intramolecular
DielseAlder reaction, J. Org. Chem. 50 (1985) 1087e1105.
[31] A.L.K. Jarugu Narasimha Moorthy, Subhas Samanta, Ankur Roy, Werner
M. Nau*[b], Modulation of spectrokinetic properties of o-quinonoid reactive