Discovery of dihydroxylated 2,4-diphenyl-6-thiophen-2-yl-pyridine as a non-intercalative DNA-binding topoisomerase II-specific catalytic inhibitor

Article abstract of DOI:10.1016/j.ejmech.2014.04.066

We describe our rationale for designing specific catalytic inhibitors of topoisomerase II (topo II) over topoisomerase I (topo I). Based on 3D-QSAR studies of previously published dihydroxylated 2,4-diphenyl-6-aryl pyridine derivatives, 9 novel dihydroxyl

Full text of DOI:10.1016/j.ejmech.2014.04.066

European Journal of Medicinal Chemistry 80 (2014) 428e438
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of dihydroxylated 2,4-diphenyl-6-thiophen-2-yl-pyridine as
a non-intercalative DNA-binding topoisomerase II-specific catalytic
inhibitor
,
1
Kyu-Yeon Jun a, Hanbyeol Kwon a, So-Eun Park a, Eunyoung Lee a, Radha Karki b
,
,
,
a,
*
Pritam Thapa b 1, Jun-Ho Lee c, Eung-Seok Lee b , Youngjoo Kwon
*
a College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Global Top5, Ewha Womans University, Seoul 120-750, Republic of Korea
b College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea
c Department of Emergency Medical Technology, Daejeon University, Daejeon 300-716, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe our rationale for designing specific catalytic inhibitors of topoisomerase II (topo II) over
topoisomerase I (topo I). Based on 3D-QSAR studies of previously published dihydroxylated 2,4-
diphenyl-6-aryl pyridine derivatives, 9 novel dihydroxylated 2,4-diphenyl-6-thiophen-2-yl pyridine
compounds were designed, synthesized, and their biological activities were evaluated. These compounds
have 2-thienyl ring substituted on the R3 group on the pyridine ring and they all showed excellent
specificity toward topo II compared to topo I. In vitro experiments were performed for compound 13 to
determine the mechanism of action for this series of compounds. Compound 13 inhibited topoisomerase
II specifically by non-intercalative binding to DNA and did not stabilize enzyme-cleavable DNA complex.
Compound 13 efficiently inhibited cell viability, cell migration, and induced G1 arrest. Also from 3D-
QSAR studies, the results were compared with other previously published dihydroxylated 2,4-diphenyl-
6-aryl pyridine derivatives to explain the structureeactivity relationships.
Received 13 December 2013
Received in revised form
28 March 2014
Accepted 23 April 2014
Available online 24 April 2014
Keywords:
Topoisomerase I
Topoisomerase II
Cytotoxicity
Anticancer agents
Dihydroxylated 2,4-diphenyl-6-thiophen-2-
yl-pyridines
Ó 2014 Elsevier Masson SAS. All rights reserved.
3D-QSAR
1. Introduction
whereby topo II
expressed in late S and G2-M phase cells [6].
-Terpyridine is a heterocyclic compound derived from pyridine.
-Terpyridine possesses three nitrogen atoms, and thus is able to
a, among two subtypes of topo II, is primarily
DNA topoisomerases are enzymes that manipulate the topology
of DNA. Topoisomerases are one of the most promising targets for
development of anticancer agents because they are highly over-
expressed in proliferating cancer cells [1]. Topoisomerases play an
important role in various key cellular processes including replica-
tion, transcription, recombination, repair, and chromatin assembly
[2,3]. There are two types of topoisomerases in humans, namely,
topoisomerase type I (topo I) and type II (topo II). Topo I breaks and
rejoins single-strand of a double helix while topo II breaks and
rejoins double-strand DNA [4,5]. Because topo II breaks double-
strand DNA, it can decatenate or catenate double-strand DNA
rings that form during cell replication. The biological role of topo II
is consistent with its expression pattern across the cell cycle,
a
a
form metal complexes [7] that bind to RNA/DNA [8,9]. We previ-
ously reported various terpyridine derivatives that possess topo I
and II inhibitory activity and exhibit strong cytotoxicity against
several human cancer cell lines [10e16]. Previous structureeac-
tivity relationship studies have shown that the 2-thienyl-4-furyl-
pyridine skeleton has considerable topo I inhibitory activity [17].
Further, 2,6-dithienyl-4-furyl pyridine derivatives inhibit topo I and
induce cytotoxicity, especially in the case of compounds having a
methyl or chloride substitution on the thienyl and/or furyl ring [16].
A series of 2,4,6-triaryl pyridine derivatives containing chlor-
ophenyl and phenolic moieties at the 2- and 4-position of the
central pyridine were shown to inhibit both topo I and II [18].
Evaluation of these series of compounds indicated that when the 6-
position of the central pyridine is substituted with a furyl, thienyl,
or phenyl group instead of a pyridine group, specific inhibition of
topo II is achieved. Furthermore, dihydroxylated 2,4,6-triphenyl
pyridine derivatives exhibit stronger topo II inhibition compared
* Corresponding author.
E-mail addresses: eslee@ynu.ac.kr (E.-S. Lee), ykwon@ewha.ac.kr (Y. Kwon).
Present address: Division of Hematology & Oncology, Department of Internal
1
Medicine, Kansas University Medical Center, Kansas City, KS 66103, USA.
http://dx.doi.org/10.1016/j.ejmech.2014.04.066
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

K.-Y. Jun et al. / European Journal of Medicinal Chemistry 80 (2014) 428e438
429
to topo I [19]. The position of the hydroxyl group appears to be
important for these series of compounds, because hydroxyl groups
at the meta or para position on either a 2- or 6-phenyl group result
in cytotoxicity associated with specific inhibition of topo II. Despite
these findings, a clear mechanism of action and mode of binding of
these classes of compounds have not been established. While the
skeletons of the terpyridine derivatives were very similar, the
specificities for topo I and topo II varied widely. In this paper,
dihydroxylated 2,4-diphenyl-6-thiophen-2-yl pyridine derivatives
were evaluated for topo I and II inhibitory activity and cytotoxicity
against several human cancer cell lines. In vitro experiments were
performed to determine the mechanism of action for these series of
compounds. In addition, we compared our 3D-QSAR results with
those of previously published dihydroxylated 2,4-diphenyl-6-aryl
pyridine derivatives to identify structureeactivity relationships.
pyridine. Using modified Krohnke synthesis [24,25], final com-
pounds 7e15 (R1, R2 ¼ aec) were synthesized by the reaction of
appropriate dihydroxylated chalcones 3e5 with pyridinium iodide
salt 6 in the presence of ammonium acetate and glacial acetic acid
(AcOH) in 49e92% yield.
The reaction gave a total of nine final compounds as shown in
Scheme 1. All the compounds contain two hydroxyl moieties, one in
each of the 2- and 4-phenyl ring of central pyridine. The hydroxyl
moieties were substituted at various positions (ortho, meta or para)
of the phenyl ring. Synthesis of dihydroxylated compounds allowed
us to investigate the effect of the increase in the number of hy-
droxyl group on biological activity. Further, substitution of hydroxyl
group at various positions (ortho, meta or para) of the phenyl ring
helped us to evaluate the effect of hydroxyl position on biological
activity.
2. Chemistry
3. Result and discussion
Dihydroxylated 2,4-diphenyl-6-thiophen-2-yl-pyridine de-
rivatives 7e15 were synthesized in three steps as illustrated in
Scheme 1. Using ClaiseneSchmidt condensation reaction [20e23]
nine dihydroxylated propenone intermediates were synthesized.
Reactions were either base [20,21] or acid [22,23] catalyzed,
without protection of hydroxyl groups. In the first method, 50%
aqueous solution of KOH or 6 M NaOH was added to the solution of
equimolar amounts of aryl ketone 1 (R1 ¼ aec) and aryl aldehyde 2
(R2 ¼ aec) in ethanol to obtain compounds 3e5 (R1, R2 ¼ aec) in
36e90% yield. Another method utilized Lewis acid, borontrifluoro
etherate (BF3eEt2O). To a solution of aryl ketone 1 (R1 ¼ c) and aryl
aldehyde 2 (R2 ¼ c) in dioxane was added BF3eEt2O to obtain
compound 5 (R1 ¼ c, R2 ¼ c) in 74% yield. In the second step, 2-
thienyl pyridinium iodide salt 6 was synthesized in quantitative
yield by the treatment of 2-acetyl thiophene with iodine in
3.1. Evaluation of topoisomerase I and II inhibition
We synthesized nine dihydroxylated 2,4-diphenyl-6-thiophen-
2-yl-pyridine compounds 7e15 that contained a hydroxyl group on
each of the 2- and 4-phenyl rings in all possible positions (Scheme
1). The derivatives were then screened for their topo I and II
inhibitory activities (Fig. S1 & Table 1). Topoisomerase inhibitory
activities were determined by observation of relaxation of super-
coiled pBR322 plasmid DNA. None of the synthesized compounds
exhibited topo I inhibitory activity, because the supercoiled DNA
(lower band marked SC) was transformed to a relaxed form (upper
band marked Rel) by topo I with treatment of compounds (Fig. S1B).
However, in the topo II relaxation assay, all of the compounds
showed significant inhibitory effects at 100
mM. Compounds 7e15
exhibited superior inhibition of topo II compared with the positive
Scheme 1. General synthetic scheme of dihydroxylated 2,4-diphenyl-6-thiophen-2-yl-pyridine derivatives. Reagents and conditions: (i) aryl aldehydes 2 (R1 ¼ aec) (1.0 equiv),
KOH/NaOH/BF3Et2O, EtOH, 2e24 h, 20 ꢀC, 36e90% yield; (ii) NH4OAc (10.0 equiv), glacial AcOH, 12e24 h, 90e100 ꢀC, 49e97% yield.

430
K.-Y. Jun et al. / European Journal of Medicinal Chemistry 80 (2014) 428e438
Table 1
Topo II and I inhibitory activity and cytotoxicity of compounds 7e15.
a
Compounds
% Inhibition
Topo II
IC50
(mM)
Topo I
HCT15
K562
DU145
MCF-7
HeLa
100
m
M
20
m
M
100 mM
Etoposide
Camptothecin
Adriamycin
7
8
9
10
11
12
13
14
15
87.1
45.4
1.33 ꢁ 0.10
0.26 ꢁ 0.04
1.28 ꢁ 0.07
4.32 ꢁ 0.58
2.96 ꢁ 0.09
2.66 ꢁ 0.07
1.76 ꢁ 0.35
1.12 ꢁ 0.17
3.09 ꢁ 0.02
0.84 ꢁ 0.15
1.24 ꢁ 0.14
0.73 ꢁ 0.03
2.09 ꢁ 0.55
1.18 ꢁ 0.14
2.85 ꢁ 1.38
7.58 ꢁ 0.47
1.52 ꢁ 0.47
1.31 ꢁ 0.15
3.62 ꢁ 0.22
10.48 ꢁ 2.35
1.80 ꢁ 0.65
3.67 ꢁ 0.10
4.12 ꢁ 0.15
0.85 ꢁ 0.04
2.94 ꢁ 0.04
2.37 ꢁ 0.55
0.86 ꢁ 0.04
35.30 ꢁ 2.92
10.12 ꢁ 0.19
>50
4.29 ꢁ 0.04
2.70 ꢁ 0.28
1.69 ꢁ 0.18
3.12 ꢁ 0.39
10.40 ꢁ 0.46
1.53 ꢁ 0.14
3.25 ꢁ 0.04
3.91 ꢁ 0.66
3.69 ꢁ 0.13
>50
>50
>50
12.01 ꢁ 1.02
8.67 ꢁ 0.10
5.72 ꢁ 1.34
5.52 ꢁ 0.13
2.70 ꢁ 0.18
10.24 ꢁ 0.24
3.20 ꢁ 0.57
58.4
1.01 ꢁ 0.01
1.45 ꢁ 0.01
16.06 ꢁ 0.75
4.69 ꢁ 0.54
>50
97.5
97.9
96.8
100
100
100
98.0
100
100
40.1
42.4
18.1
9.4
24.3
14.2
37.3
18.8
19.4
0.0
0.0
0.0
0.0
1.0
0.0
0.0
0.6
5.4
2.32 ꢁ 0.25
15.98 ꢁ 2.60
9.13 ꢁ 1.61
1.87 ꢁ 0.33
8.27 ꢁ 1.24
2.08 ꢁ 0.23
a
Each data represents mean ꢁ S.D. from three different experiments performed in triplicate.
control etoposide (Table 1). Furthermore, compounds 7, 8, and 13
had either greater or comparable inhibition at both 100 and 20
and ellipticine at 100 mM (Fig. 1A). Etoposide does not affect the
mM
catalytic activity of topo II, since its cleavage step is reversible [26].
Conversely, treatment with ellipticine strongly inhibits topo II
decatenation. These behaviors can be observed with gel electro-
phoresis, as decatenated products are the major product in samples
treated with etoposide while the majority of kDNA remains in
ellipticine treated samples (Fig. 1A). Compound 13 partially
compared to etoposide. These results indicated that the dihy-
droxylated 2,4-diphenyl-6-thiophen-2-yl-pyridine compounds
were specific to topo II, which was similar to previously reported
results for dihydroxylated 2,4,6-triphenyl pyridines [19]. With
respect to the dihydroxylated 2,4,6-triphenyl pyridines, the com-
pounds with a hydroxyl group substituted at the 2- and 4-phenyl
groups could be directly compared with 7e15 for the difference
in properties between the phenyl group and thienyl group at the 6-
position. Specifically, compounds with a 2-thienyl group exhibited
superior topo II inhibition activity compared with compounds with
a phenyl group on the 6-position of pyridine.
inhibited topo II decatenation at 50
mM, and fully inhibited topo II at
100 M. This result indicated that compound 13 exhibited com-
m
parable or better efficacy than ellipticine. Together, these results
confirmed that compound 13 is specific inhibitor of topo II.
3.2. Cytotoxicity of the compounds
Five human cancer cell lines were used to evaluate the cyto-
toxicity of dihydroxylated 2,4-diphenyl-6-thiophen-2-yl-pyridine
compounds: HCT15 (human colorectal adenocarcinoma cell line),
K562 (human myeloid leukemic tumor cell line), DU145 (human
prostate tumor cell line), MCF-7 (human breast adenocarcinoma
cell line), and HeLa cells (human cervix tumor cell line). The
inhibitory activities (IC50) of the tested compounds were expressed
in micromolar concentration as illustrated in Table 1. Cells were
treated with each compound for 48 h after which cytotoxicity was
measured. The compounds were generally cytotoxic against most
of the cell lines except for 9, which was cytotoxic to HCT15 and
K562 cells only. Among the tested compounds, 13 exhibited the
highest and most uniform level of cytotoxicity, with an IC50 value
<6 mM for all of the cell lines tested. Based on these results, 13 was
selected for further evaluation.
3.3. Compound 13 is topo II specific inhibitor
The results of screening the dihydroxylated 2,4-diphenyl-6-
thiophen-2-yl-pyridine compounds indicated that they all inhibi-
ted topo II rather than topo I. Among the synthesized compounds,
compound 13 exhibited the strongest potency for inhibition of topo
II relaxation assay and produced the highest level of cytotoxicity. To
further confirm the topo II inhibitory activity of compound 13, we
used a kinetoplast DNA (kDNA) decatenation assay. Through the
specific catalytic activity of topo II, kDNA forms several decatenated
forms of DNA including nicked (Nck), relaxed (Rel), and supercoiled
(SC) DNA. Because of its size, catenated kDNA is unable to enter into
agarose gels unless it is decatenated by topo II. Thus, the inhibitory
activity of compound 13 was compared with the controls etoposide
Fig. 1. Compound 13 is a topo II inhibitor. (A) kDNA decatenation assay with com-
pound 13. Topo II-mediated kDNA decatenation was inhibited by compound 13. The
positions of kDNA and other forms of DNA are indicated. (B) Cleavage complex assay.
Three units of topo II were added to supercoiled pBR322 DNA 10 min before adding
either etoposide or compound 13. The formation of a linear band indicated that a
cleavage complex was formed. As expected, treatment with etoposide formed a linear
band, while compound 13 did not. Thus, compound 13 does not act as a topo II poison.

K.-Y. Jun et al. / European Journal of Medicinal Chemistry 80 (2014) 428e438
431
3.4. Compound 13 is catalytic inhibitor of topo II
The results of the kDNA decatenation assay showed that com-
pound 13 is a topo II inhibitor. To further investigate the mechanism
of action of compound 13, we performed several in vitro experi-
ments including unwinding, cleavage complex stabilization, and
ATP competition assays. Topo II inhibitors can be categorized into
two types, namely, topo II poisons and catalytic inhibitors [27].
Cleavage complex stabilization assays can be used to differentiate
topo II poisons and catalytic inhibitors based on whether or not
linear DNA is formed when topo II is added to supercoiled pBR322
DNA 10 min prior to addition of the test compound to initiate DNA
relaxation. Electrophoresis can then be carried out in the presence
of ethidium bromide to clearly visualize the linear form of DNA.
Etoposide is a well-known topo II poison, and thus the linear form
of DNA can be observed as a control (Fig. 1B). Conversely, treatment
with compound 13 did not generate linear DNA at 100 and 300 mM,
and thus is not a topo II poison. Based on these data, compound 13
was deduced as a catalytic inhibitor of topo II.
3.5. Compound 13 does not intercalate into DNA
Fig. 3. Compound 13 inhibits the migration of HeLa cells. A clean wound was made in a
90% confluent monolayer of HeLa cells with a sterile micro tip. Cells were exposed to
Topo II catalytic inhibitors can be classified as DNA intercalators
and non-intercalators [28]. In topo II-catalyzed ATP hydrolysis and
ATP competition assays, compound 13 exhibited neither inhibition
of ATP hydrolysis nor any change in inhibition rate, indicating that it
does not compete with ATP (data not shown) in the ATPase domain.
Based on these results, compound 13 was next tested for its ability
to intercalate into DNA. When DNA intercalators such as m-AMSA
are briefly incubated with relaxed plasmid DNA, subsequent
removal of the compound and enzyme results in re-supercoiling
[29]. Thus, by using this assay, it is possible to differentiate be-
tween intercalators and non-intercalators. Negatively supercoiled
DNA was first incubated with excess topo I to fully relax supercoiled
DNA. Next, either compound 13 or m-AMSA, which was used as a
positive control, was incubated with relaxed DNA and topo I. The
results of this experiment indicated that compound 13 does not
compound 13 at 5 mM and 10 mM for 24 h and were allowed to migrate in the medium.
intercalate with DNA (Fig. 2A). However, in a separate assay to
measure intercalation by evaluating retardation of migration in gel
electrophoresis, compound 13 was found to interact with DNA
(Fig. 2B). Therefore, compound 13 was determined to inhibit topo II
catalytically by binding to DNA in a non-intercalative mode.
3.6. Compound 13 induced apoptosis
Cell migration and proliferation can be easily evaluated by
wound healing assay. HeLa cells were treated with various com-
pounds at 5 or 10 mM after making a wound. In untreated control
cells, the gap wound narrowed over 24 h. However, in cells treated
with compound 13, the gap did not decrease after 24 h of treatment
at 5 mM (Fig. 3). For cells treated with compound 13 at 10 mM, the
edges of cells became rough and the gap width increased, indi-
cating the presence of cell death. Thus, compound 13 inhibited cell
migration in a dose-dependent manner, with higher doses result-
ing in apoptosis.
The mechanism of action of compound 13 was further investi-
gated by observing its effects on cell cycle progression in HCT15
cells (Fig. 4A, B). Specifically, HCT15 cells were treated with com-
pound 13 and cell cycle progression was analyzed by florescence
activated cell sorting (FACS). The G0/G1 population increased as the
concentration of compound 13 increased (0, 0.05, 0.1, 0.5, and
1
m
M). In addition, a time dependent effect of the drug was
observed after treating cells with 0.1 M of compound 13 at various
time points (0, 4, 8, 12, and 24 h). This also increased the G0/G1
population as the time of treatment increased, reaching
m
a
maximum at 12 h. Therefore, compound 13 was determined to
induce G0/G1 arrest in a both a time- and dose-dependent manner.
In order to determine whether the cytotoxic effects of com-
pound 13 towards HCT15 cells were due to apoptosis, we next
evaluated PARP cleavage, a marker of apoptosis, by western blot
analysis (Fig. 4C). The amount of cleaved PARP increased as the
Fig. 2. Compound 13 is a topo II inhibitor. (A) Compound 13 does not intercalate into
DNA as determined by
a DNA unwinding assay. Lane D: pBR322 only; Lane T:
pBR322 þ Topo I; Lane 1e4: pBR322 þ Topo I þ m-AMSA in indicated concentrations;
concentration of drug treated increased (0, 0.5, 1, 2, and 4
mM),
Lanes 5e8: pBR322 þ Topo I þ compound 13 at the indicated concentrations. (B) DNA
while increasing amounts of 13 had no effect on -tubulin
a
intercalation assay. 1.25
m
L of negatively supercoiled DNA pBR322 (100 ng/
m
m
L Fer-
L was
expression, which was used as a loading control. Thus, our results
indicated that compound 13 induced apoptosis in HCT15 cells in a
dose dependent manner.
mentas) and 100 M or 300
m
mM of the test compound in a volume of 10
incubated at 37 ꢀC for 20 min. Ethidium bromide at 0.4
as positive controls.
mg/mL and 1 mg/mL were used

432
K.-Y. Jun et al. / European Journal of Medicinal Chemistry 80 (2014) 428e438
Fig. 4. Compound 13 induces apoptosis in HCT15 colon cancer cells. (A) Compound 13 induces G1 arrest in HCT15 colon cancer cells in a concentration dependent manner. Cell cycle
distribution was evaluated by flow cytometric DNA content analysis. HCT15 cells were treated with 13 at 0.05, 0.1, 0.5 and 1 M for 24 h (B) HCT15 cells were treated with 13 at
0.1 M for various time points. (C) Western blot analysis of HCT15 whole cell lysates. Treatment of HCT15 cells with 13 resulted in increased PARP cleavage in a dose-dependent
manner. The graph results and error bars show the mean ꢁ SD of triplicate experiments. *p < 0.05 and **p < 0.01, and ***p < 0.001 compared with the vehicle control.
m
m
3.7. Analysis of the 3D structureeactivity relationship
and H-bonding factors that influence the cytotoxicity of small
molecules [30,31]. According to CoMSIA, the cross-validated r2 (q2)
of the training set was 0.709 with 2 components, while the non-
cross-validated r2 was 0.842 with a standard error of estimate of
0.168. The donor and acceptor descriptors produced the most
contributions (41.4% and 33.9%). Fig. 5 shows the contour maps for
the H-bonding donor and acceptor fields based on the most active
compound 13. In the donor contour map (Fig. 5A), cyan represents
regions that favor donor groups and purple the opposite. The H-
bond donor groups were favored in meta and para site on R1 and
ortho and meta site on R2 of the phenyl ring as it is represented as
We next evaluated the structure activity relationships for the
compounds synthesized in this study and those previously pub-
lished, all of which shared a common pyridine core ring with
substitutions at the 2, 4, and 6 positions [18,19]. The common
structure for the 34 compounds used in the study was 2,4-
diphenylpyridine ring and compound 13, one of the most active
inhibitors was selected as the template for alignment. Comparative
molecular similarity index analysis (CoMSIA) can be used to
quantify specific contributions of steric, electrostatic, hydrophobic,
Fig. 5. CoMSIA contour maps of H-bond donor and acceptor fields based on compound 13; (A) donor map. Cyan and purple indicate regions that favor and disfavor donor groups,
respectively. (B) acceptor map. Magenta and red represents regions that favor and disfavor acceptor groups, respectively. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

K.-Y. Jun et al. / European Journal of Medicinal Chemistry 80 (2014) 428e438
433
cyan contours. When the H-bond donors were in the ortho of R1 and
Table 2
para of R2, the activity decreases as it is indicated by purple con-
tours. Fig. 5B shows the acceptor field contour map where H-bond
acceptor are favorable in magenta colored region and unfavorable
in red. As it is indicated by magenta, the acceptor groups are
favored in meta and para site on R1. When the acceptor groups are
situated on the ortho of R1 and para of R2, it is unfavorable according
to the red contours. In addition, the red contour next to the R3 aryl
group represents that when acceptor atom is located on this region
such as 3-pyridinyl group, it is unfavorable. The model was vali-
dated by a test set that included five compounds, indicated by as-
terisks in Table 2. The 3D-QSAR model gave good statistical results
and good prediction using the test set. In summary, H-bonding was
the most important factor for the activity of the compounds ac-
cording to the CoMSIA model.
Structures and biological activities of compounds used for CoMSIA.
No. R1
R2
R3
Actual CoMSIA
CoMSIA
pIC50
predicted residual
pIC50
A1a 20 OH phenyl
A2 30 OH phenyl
3 OH phenyl 2-furanyl
3 OH phenyl 2-furanyl
5.4101 5.1650
5.6737 5.8251
5.0958 5.1263
5.6091 5.5269
4.9914 5.3800
6.0315 5.9563
5.8996 5.7551
6.0655 6.0226
6.0555 6.0328
5.2147 5.2015
5.1599 5.2318
0.2448
ꢃ0.1514
ꢃ0.0305
0.0822
A3 30 chlorophenyl 4 OH phenyl 2-furanyl
A4a 30 chlorophenyl 3 OH phenyl 2-furanyl
A5a 40 chlorophenyl 3 OH phenyl 2-furanyl
ꢃ0.3886
B1 30 OH phenyl
B2 30 OH phenyl
B3 40 OH phenyl
B4 40 OH phenyl
B5 30 chlorophenyl 4 OH phenyl phenyl
B6 40 chlorophenyl 4 OH phenyl phenyl
2 OH phenyl phenyl
4 OH phenyl phenyl
2 OH phenyl phenyl
3 OH phenyl phenyl
0.0752
0.1445
0.0429
0.0227
0.0131
4. Conclusions
In this study we designed, synthesized and tested 2,4-diphenyl-
6-thiophen-2-yl-pyridine compounds for their ability to inhibit
topoisomerases in an attempt to develop targeted anti-cancer
drugs. The series of compounds showed excellent specificity to-
wards topo II over topo I, and compound 13 was determined to be
the most effective. Compound 13 was further analyzed to deter-
mine its mechanism of action, and was found to be a specific in-
hibitor of topo II through non-intercalative binding to DNA, and
was able to inhibit cell viability and cell migration. We also per-
formed a 3D-QSAR for the 2, 4, 6-aryl pyridine compounds. Based
on five CoMSIA fields, H-bonding was found to be the highest
contributing factor towards the cytotoxic effect of the compounds,
with the position of the H-bond donor and acceptor being espe-
cially important. According to the contour plots, the H-bond donors
and acceptors such as hydroxyl group was favored on the meta and
para of R1 and ortho of R2 phenyl ring. The donor and acceptor
groups on the ortho of R1 and para of R2 phenyl ring was unfavor-
able. Importantly, the insights gained from CoMSIA method pro-
vided valuable information that will be useful for designing new
lead compounds exhibiting specificity towards topo II and anti-
tumor activity.
ꢃ0.0719
C1 30 OH phenyl
C2a 30 OH phenyl
C3 20 OH phenyl
C4 30 OH phenyl
C5 30 OH phenyl
C6 40 OH phenyl
C7 40 OH phenyl
2 OH phenyl 2-pyridinyl 5.9706 5.9128
3 OH phenyl 2-pyridinyl 5.8996 5.8970
4 OH phenyl 3-pyridinyl 4.9739 4.9401
4 OH phenyl 3-pyridinyl 5.5591 5.6434
2 OH phenyl 4-pyridinyl 5.7423 5.9670
2 OH phenyl 4-pyridinyl 5.983 6.0746
3 OH phenyl 4-pyridinyl 6.1427 6.0869
0.0578
0.0026
0.0338
ꢃ0.0843
ꢃ0.2247
ꢃ0.0917
0.0557
C8 40 chlorophenyl 3 OH phenyl 4-pyridinyl 5.58
5.6134
ꢃ0.0334
8
20 OH phenyl
3 OH phenyl 2-thienyl
2 OH phenyl 2-thienyl
3 OH phenyl 2-thienyl
4 OH phenyl 2-thienyl
2 OH phenyl 2-thienyl
3 OH phenyl 2-thienyl
5.5287 5.4610
5.7545 5.8540
5.9508 5.8649
0.1191
10 30 OH phenyl
11 30 OH phenyl
12 30 OH phenyl
13 40 OH phenyl
14 40 OH phenyl
ꢃ0.0995
0.0859
5.51
5.6356
ꢃ0.1256
6.0757 5.9597
5.9066 5.9707
5.0325 5.3444
5.1891 5.1155
4.8652 5.1695
5.4908 5.1826
6.0177 5.8483
5.5528 5.6350
6.0706 5.9535
5.2343 5.1626
5.0531 5.3395
0.1160
ꢃ0.0641
ꢃ0.3120
0.0736
D1 40 chlorophenyl 3 OH phenyl 2-thienyl
D2 40 chlorophenyl 4 OH phenyl 2-thienyl
D3 20 OH phenyl
D4 20 OH phenyl
D5 30 OH phenyl
D6a 30 OH phenyl
D7 40 OH phenyl
2 OH phenyl 3-thienyl
3 OH phenyl 3-thienyl
2 OH phenyl 3-thienyl
4 OH phenyl 3-thienyl
2 OH phenyl 3-thienyl
ꢃ0.3043
0.3081
0.1694
ꢃ0.0825
0.1171
D8 20 chlorophenyl 4 OH phenyl 3-thienyl
D9 40 chlorophenyl 3 OH phenyl 3-thienyl
0.0717
ꢃ0.2865
a
Prediction set.
5. Experimental section
of 50%e100% of B in A for 15 min followed by 100%e50% of B in A
for 15 min at a flow rate of 1.0 mL/min. UV detection was performed
at 254 nm. The mobile phase A consisted of double distilled water
with 20 mM ammonium formate (AF) and B was 90% ACN in water
with 20 mM AF. Compound purities are described as percentages
(%).
5.1. Chemistry
Compounds used as starting materials and reagents were ob-
tained from Aldrich and/or Junsei and were used without further
purification. HPLC grade acetonitrile (ACN) and methanol were
purchased from Burdick and Jackson, USA. Thin-layer chromatog-
raphy (TLC) and column chromatography (CC) were performed
using Kieselgel 60 F254 (Merck) and silica gel (Kieselgel 60, 230e
400 mesh, Merck), respectively. Because all of the prepared com-
pounds contained aromatic rings, they were visualized and detec-
ted on TLC plates by UV light (short wave, long wave or both). NMR
spectra were recorded on a Bruker AMX 250 at 250 MHz (FT) for 1H
NMR and 62.5 MHz for 13C NMR; chemical shifts were calibrated
ESI LC/MS analyses were performed with a Finnigan LCQ
AdvantageÒ LC/MS/MS utilizing XcaliburÒ software. For ESI LC/MS,
LC was performed with an injection volume of 2 mL on a Waters
AtlantisÒ T3 reverse-phase C18 column (2.1 ꢂ 50 mm, 3
mm). The
mobile phase consisted of 100% distilled water (A) and 100% ACN
with 20 mM AF (B). A gradient program was used at a flow rate of
200
mL/min. The initial composition was 10% B and was pro-
grammed linearly to 90% B after 5 min and finally 10% B after
15 min. MS ionization conditions were as follows: Sheath gas flow
rate, 70 arb; aux gas flow rate, 20 arb; I spray voltage, 4.5 kV;
capillary temperature, 215 ꢀC; column temperature, 40 ꢀC; capillary
voltage, 21 V; and tube lens offset, 10 V. Retention times are re-
ported in minutes.
according to TMS. Chemical shifts (d) and coupling constants (J)
were recorded in ppm and hertz (Hz), respectively. Melting points
were determined in open capillary tubes with an electrothermal 1A
9100 digital melting point apparatus and were uncorrected.
HPLC analyses were performed using two Shimadzu LC-10AT
pumps gradient-controlled HPLC system equipped with a Shi-
madzu system controller (SCL-10A VP) and photo diode array de-
tector (SPD-M10A VP) utilizing Shimadzu Class VP software. A
5.1.1. General method for the preparation of 3
Compound 3 was synthesized either by a KOH/NaOH or BF3e
Et2O catalyzed ClaiseneSchmidt condensation reactions.
sample volume of 10
reverse-phase C18 column (4.6 ꢂ 250 mm) with a gradient elution
m
L was injected into a Waters X-TerraÒ
5
mM

434
K.-Y. Jun et al. / European Journal of Medicinal Chemistry 80 (2014) 428e438
KOH/NaOH catalyzed: To a solution of equimolar amounts of
[MH]þ ¼ 346.22, and 100% purity by HPLC. 1H NMR (250 MHz,
aryl ketone 1 (R1 ¼ aec) and aryl aldehyde 2 (R1 ¼ aec) in EtOH, a
solution of either 50% aqueous KOH or 6 M NaOH was added and
stirred for 2e24 h at 20 ꢀC. The mixture was then neutralized with a
solution of 6 M aqueous HCl (pH adjusted to 2). Lastly, the mixture
was extracted with ethyl acetate, and washed with water and brine.
Compound 3 was further purified by either recrystallization or
column chromatography to yield pure solid compound.
BF3eEt2O catalyzed: To a solution of aryl ketone 1 (R1 ¼ c) and
aryl aldehyde 2 (R1 ¼ c) with minimal dioxane, BF3eEt2O was
added gradually at 20 ꢀC and stirred for 2 h. The mixture was then
extracted with ethyl acetate and washed with water and brine. 3
was further purified by column chromatography to yield pure solid
compound.
DMSO-d6) d 13.47 (br, 1H, 2-phenyl 2-OH), 9.73 (br, 1H, 4-phenyl 3-
OH), 8.24 (s,1H, pyridine H-3), 8.21 (d, J ¼ 8.9 Hz,1H, 2-phenyl H-6),
8.17 (s,1H, pyridine H-5), 8.11 (dd, J ¼ 3.8,1.1 Hz,1H, 6-thiophene H-
3), 7.77 (dd, J ¼ 4.9, 1.0 Hz, 1H, 6-thiophene H-5), 7.45 (t, J ¼ 7.7 Hz,
1H, 2-phenyl H-4), 7.40e7.31 (m, 3H, 4-phenyl H-5, H-6, H-2), 7.26
(dd, J ¼ 4.9, 3.8 Hz, 1H, 6-thiophene H-4), 6.98e6.92 (m, 3H, 4-
phenyl H-4, 2-phenyl H-3, H-5). 13C NMR (62.5 MHz, DMSO-d6)
d
158.84, 158.20, 157.15, 150.83, 149.90, 142.70, 138.69, 131.76,
130.36, 129.21, 129.07, 128.09, 127.23, 119.38, 119.32, 118.42, 117.97,
116.84, 116.37, 115.17, 114.47.
5.1.6. Synthesis of 2-[4-(4-Hydroxyphenyl)-6-(thiophen-2-yl)
pyridin-2-yl]phenol (9)
The synthesis of 3 (R1, R2 ¼ aec) have been described previously
[19].
Using the procedure described above, 1-(2-hydroxyphenyl)-3-
(4-hydroxyphenyl)propenone (264.27 mg, 1.10 mmol), dry ammo-
nium acetate (847.88 mg, 11.00 mmol), 6 (364.28 mg, 1.10 mmol),
and AcOH (2 mL) were reacted to yield a yellow solid (216.70 mg,
57.0%, 0.62 mmol). mp ¼ 230e231 ꢀC, Rf (ethyl acetate/n-hexane
5.1.2. General method for the preparation of 6
Thiophen-2-yl ketone, iodine (1.2 equiv) and pyridine were
refluxed at 140 ꢀC for 3 h. A precipitate formed during the reaction,
which was cooled to room temperature. The precipitate was then
filtered and washed with cold pyridine to afford 6 in quantitative
yield.
1:1 v/v)
¼
0.58. LC/MS/MS retention time
¼
8.60 min,
[MH]þ ¼ 346.23, and 100% purity by HPLC. 1H NMR (250 MHz,
DMSO-d6)
d 13.68 (s, 1H, 2-phenyl 2-OH), 9.94 (s, 1H, 4-phenyl 4-
OH), 8.24 (s, 1H, pyridine H-3), 8.21 (m, 1H, 2-phenyl H-6), 8.17 (s,
1H, pyridine H-5), 8.09 (dd, J ¼ 3.6, 1.3 Hz, 1H, 6-thiophene H-3),
7.96 (d, J ¼ 8.2 Hz, 2H, 4-phenyl H-2, H-6), 7.76 (d, J ¼ 5.0 Hz, 1H, 6-
thiophene H-5), 7.33 (t, J ¼ 7.9 Hz, 1H, 2-phenyl H-4), 7.25 (dd,
J ¼ 5.0, 3.9 Hz, 1H, 6-thiophene H-4), 6.97 (d, J ¼ 8.2 Hz, 2H, 4-
phenyl H-3, H-5), 6.95e6.92 (m, 2H, 2-phenyl H-3, H-5). 13C NMR
5.1.3. General method for the preparation of 7e15
A mixture of the propenone intermediates 3e5 (R1, R2 ¼ aec),
pyridinium iodide salt 6, and anhydrous ammonium acetate in
glacial acetic acid was heated at 90e100 ꢀC for 12e24 h. The re-
action mixture was then extracted with ethyl acetate and washed
with water and brine. The organic layer was dried with magnesium
sulfate and filtered. The resulting filtrate was evaporated at reduced
pressure and purified by silica gel column chromatography with
gradient elution of ethyl acetate/n-hexane to afford solid com-
pounds 7e15 in 49e92% yield. Nine dihydroxylated 2,4-diphenyl-
6-thiophene-2-yl pyridine compounds were synthesized by this
method.
(62.5 MHz, DMSO-d6)
d 159.53, 159.03, 157.15, 150.38, 149.72,
142.84, 131.74, 129.20, 129.18, 128.96, 128.00, 127.45, 127.13, 119.29,
119.26, 118.03, 116.11, 115.09, 114.18.
5.1.7. Synthesis of 2-[2-(3-Hydroxyphenyl)-6-(thiophen-2-yl)
pyridin-4-yl]phenol (10)
Using the procedure described above, 3-(2-hydroxyphenyl)-1-
(3-hydroxyphenyl)propenone (360.37 mg, 1.50 mmol), dry
ammonium acetate (1.15 g, 15.00 mmol), 6 (496.75 mg, 1.50 mmol),
and AcOH (2 mL) were reacted to yield a white solid (252.10 mg,
48.6%, 0.72 mmol). mp ¼ 150e151 ꢀC, Rf (ethyl acetate/n-hexane
5.1.4. Synthesis of 2,20-[6-(Thiophen-2-yl)pyridine-2,4-diyl]
diphenol (7)
Using
the
procedure
described
above,
1,3-bis(2-
1:1 v/v)
¼
0.56. LC/MS/MS retention time
¼
7.88 min,
hydroxyphenyl)-propenone (345.96 mg, 1.44 mmol), dry ammo-
nium acetate (1.10 g, 14.40 mmol), 6 (476.88 mg, 1.44 mmol) and
AcOH (2 mL) were reacted to yield a yellow solid (248.00 mg, 49.9%,
0.71 mmol). mp ¼ 223e224 ꢀC, Rf (ethyl acetate/n-hexane 1:2 v/
v) ¼ 0.44, LC/MS/MS retention time ¼ 8.90 min, [MH]þ ¼ 346.22,
[MH]þ ¼ 346.21, and 100% purity by HPLC. 1H NMR (250 MHz,
DMSO-d6)
d 9.90 (br, 1H, 4-phenyl 2-OH), 9.61 (br, 1H, 2-phenyl 3-
OH), 7.98 (s, 1H, pyridine H-3), 7.88 (s, 1H, pyridine H-5), 7.87 (dd,
J ¼ 3.5, 1.1 Hz, 1H, 6-thiophene H-3), 7.66 (dd, J ¼ 5.0, 0.8 Hz, 1H, 6-
thiophene H-5), 7.58 (t, J ¼ 1.8 Hz, 1H, 2-phenyl H-2), 7.55 (d,
J ¼ 7.4 Hz, 1H, 2-phenyl H-6), 7.52 (dd, J ¼ 7.3, 1.4 Hz, 1H, 4-phenyl
H-6), 7.30 (t, J ¼ 7.8 Hz, 1H, 2-phenyl H-5), 7.27 (td, J ¼ 7.5, 1.5 Hz,
1H, 4-phenyl H-4), 7.18 (dd, J ¼ 4.9, 3.8 Hz, 1H, 6-thiophene H-4),
7.02 (d, J ¼ 8.2 Hz,1H, 4-phenyl H-3), 6.95 (t, J ¼ 7.5 Hz,1H, 4-phenyl
H-5), 6.86 (dd, J ¼ 8.1,1.7 Hz,1H, 2-phenyl H-4). 13C NMR (62.5 MHz,
and 97% purity by HPLC. 1H NMR (250 MHz, DMSO-d6)
d 13.56 (s,
1H, 2-phenyl 2-OH), 10.03 (br, 1H, 4-phenyl 2-OH), 8.19 (s, 1H,
pyridine H-3), 8.09 (s, 1H, pyridine H-5), 8.07 (d, J ¼ 7.3 Hz, 1H, 2-
phenyl H-6), 7.94 (dd, J ¼ 3.6, 0.7 Hz, 1H, 6-thiophene H-3), 7.76
(dd, J ¼ 4.9, 0.7 Hz,1H, 6-thiophene H-5), 7.56 (dd, J ¼ 7.5,1.3 Hz,1H,
4-phenyl H-6), 7.36e7.27 (m, 2H, 2-phenyl H-4, 4-phenyl H-4), 7.24
(dd, J ¼ 4.9, 3.8 Hz, 1H, 6-thiophene H-4), 7.04e6.91 (m, 4H, 2-
phenyl H-3, H-5, 4-phenyl H-3, H-5). 13C NMR (62.5 MHz, DMSO-
DMSO-d6)
d 157.98, 155.65, 155.03, 151.66, 148.41, 145.25, 140.09,
130.55, 130.39, 130.01, 128.65, 128.54, 125.50, 125.26, 119.85, 119.12,
117.60, 117.55, 116.54, 116.44, 113.67.
d6)
d 158.86, 156.40, 155.10, 149.65, 149.04, 142.83, 131.68, 130.80,
130.73, 129.24, 128.92, 127.73, 126.75, 124.91, 119.91, 119.41, 119.00,
118.06, 117.89, 116.58.
5.1.8. Synthesis of 3,30-[6-(Thiophen-2-yl)pyridine-2,4-diyl]
diphenol (11)
Using the procedure described above 1,3-bis(3-hydroxyphenyl)
propenone (360.37 mg, 1.50 mmol), dry ammonium acetate (1.15 g,
15.00 mmol), 6 (496.75 mg, 1.50 mmol) and AcOH (2 mL) were
reacted to yield a light yellow solid (443.50 mg, 85.6%, 1.28 mmol).
mp ¼ 114e115 ꢀC, Rf (ethyl acetate/n-hexane 1:1 v/v) ¼ 0.48, LC/
MS/MS retention time ¼ 7.69 min, [MH]þ ¼ 346.21, and 100% purity
5.1.5. Synthesis of 2-[4-(3-Hydroxyphenyl)-6-(thiophen-2-yl)
pyridin-2-yl]phenol (7)
Using the procedure described above, 1-(2-hydroxyphenyl)-3-
(3-hydroxyphenyl)propenone (360.37 mg, 1.50 mmol), dry
ammonium acetate (1.15 g, 15.00 mmol), 6 (496.75 mg, 1.50 mmol),
and AcOH (2 mL) were reacted to yield a yellow solid (449.60 mg,
86.8%, 1.30 mmol). mp ¼ 224e225 ꢀC, Rf (ethyl acetate/n-hexane
by HPLC. 1H NMR (250 MHz, DMSO-d6)
d 9.63 (br, 2H, 2-phenyl 3-
OH, 4-phenyl 3-OH), 8.07 (s, 1H, pyridine H-3), 8.02 (dd, J ¼ 3.6,
1:1 v/v)
¼
0.63, LC/MS/MS retention time
¼
8.71 min,
0.9 Hz, 1H, 6-thiophene H-3), 7.91 (s, 1H, pyridine H-5), 7.68 (dd,

K.-Y. Jun et al. / European Journal of Medicinal Chemistry 80 (2014) 428e438
435
J ¼ 5.3, 0.8 Hz, 1H, 6-thiophene H-5), 7.65 (d, J ¼ 1.9 Hz, 1H, 2-
phenyl H-2), 7.62 (d, J ¼ 7.0 Hz, 1H, 2-phenyl H-6), 7.37 (t,
J ¼ 7.8 Hz, 1H, 2-phenyl H-5), 7.34e7.28 (m, 3H, 4-phenyl H-5, H-6,
H-2), 7.20 (dd, J ¼ 4.9, 3.7 Hz, 1H, 6-thiophene H-4), 6.93e6.85 (m,
2H, 2-phenyl H-4, 4-phenyl H-4). 13C NMR (62.5 MHz, DMSO-d6)
(d, J ¼ 7.9 Hz, 3H, 2-phenyl H-3, H-5, 4-phenyl H-4). 13C NMR
(62.5 MHz, DMSO-d6)
d 159.05, 158.18, 156.52, 152.22, 149.73,
145.32, 139.35, 130.34, 129.35, 128.65, 128.47, 125.80, 118.17, 116.42,
115.73, 115.25, 114.20, 113.91.
d
158.23, 158.01, 156.51, 152.45, 149.86, 145.11, 139.88, 139.16,
5.1.12. Synthesis of 4,40-[6-(Thiophen-2-yl)pyridine-2,4-diyl]
diphenol (15)
130.42, 130.02, 128.86, 128.74, 126.07, 118.23, 117.82, 116.63, 116.54,
116.41, 115.02, 114.23, 113.84.
Using the procedure described above, 1,3-bis(4-hydroxyphenyl)
propenone (240.25 mg, 1.00 mmol), dry ammonium acetate
(770.80 mg, 10.00 mmol), 6 (331.17 mg, 1.00 mmol), and AcOH
(2 mL) were reacted to yield a light yellow solid (258.40 mg, 74.8%,
0.74 mmol). mp ¼ 229e230 ꢀC, Rf (ethyl acetate/n-hexane 1:1 v/
v) ¼ 0.45, LC/MS/MS retention time ¼ 7.48 min, [MH]þ ¼ 346.21,
5.1.9. Synthesis of 3-[4-(4-Hydroxyphenyl)-6-(thiophen-2-yl)
pyridin-2-yl]phenol (12)
Using the procedure described above, 1-(3-hydroxyphenyl)-3-
(4-hydroxyphenyl)propenone (360.37 mg, 1.50 mmol), dry
ammonium acetate (1.15 g, 15.00 mmol), 6 (496.75 mg, 1.50 mmol),
and AcOH (2 mL) were reacted to yield a white solid (433.60 mg,
83.7%, 1.25 mmol). mp ¼ 249e250 ꢀC, Rf (ethyl acetate/n-hexane
and 100% purity by HPLC. 1H NMR (250 MHz, DMSO-d6)
d 9.83 (br,
2H, 2-phenyl 4-OH, 4-phenyl 4-OH), 8.10 (d, J ¼ 8.5 Hz, 2H, 2-
phenyl H-2, H-6), 7.99 (s, 1H, pyridine H-3), 7.97 (d, J ¼ 3.6 Hz,
1H, 6-thiophene H-3), 7.90 (s, 1H, pyridine H-5), 7.87 (d, J ¼ 8.5 Hz,
2H, 4-phenyl H-2, H-6), 7.64 (d, J ¼ 4.9 Hz, 1H, 6-thiophene H-5),
7.18 (t, J ¼ 4.7 Hz, 1H, 6-thiophene H-4), 6.93 (d, J ¼ 8.2 Hz, 2H, 2-
phenyl H-3, H-5), 6.90 (d, J ¼ 7.9 Hz, 2H, 4-phenyl H-3, H-5). 13C
1:1 v/v)
¼
0.52, LC/MS/MS retention time
¼
7.55 min,
[MH]þ ¼ 346.22, and 100% purity by HPLC. 1H NMR (250 MHz,
DMSO-d6)
d 9.84 (br, 1H, 4-phenyl 4-OH), 9.59 (br, 1H, 2-phenyl 3-
OH), 8.08 (s, 1H, pyridine H-3), 8.01 (dd, J ¼ 3.8, 1.0 Hz, 1H, 6-
thiophene H-3), 7.93 (s, 1H, pyridine H-5), 7.88 (d, J ¼ 8.6 Hz, 2H,
4-phenyl H-2, H-6), 7.66 (dd, J ¼ 4.7, 0.9 Hz, 1H, 6-thiophene H-5),
7.64 (s, 1H, 2-phenyl H-2), 7.62 (d, J ¼ 7.1 Hz, 1H, 2-phenyl H-6), 7.31
(t, J ¼ 8.1 Hz, 1H, 2-phenyl H-5), 7.20 (dd, J ¼ 4.9, 3.8 Hz, 1H, 6-
thiophene H-4), 6.94 (d, J ¼ 8.6 Hz, 2H, 4-phenyl H-3, H-5), 6.88
(dd, J ¼ 7.2, 1.8 Hz, 1H, 2-phenyl H-4). 13C NMR (62.5 MHz, DMSO-
NMR (62.5 MHz, DMSO-d6)
d 159.02, 158.95, 156.43, 152.15, 149.21,
145.57, 129.55, 128.76, 128.62, 128.47, 128.45, 128.21, 125.61, 116.05,
115.69, 114.32, 112.98.
5.2. Biological assays
d6)
d
159.10, 157.95, 156.41, 152.32, 149.34, 145.34, 140.06, 129.92,
5.2.1. In vitro DNA topoisomerase I-mediated relaxation assay
A DNA topo I inhibition assay was performed as previously
described by Fukuda et al. [32] with minor modifications. Briefly,
compounds were dissolved in DMSO as 20 mM stock solutions. The
activity of DNA topo I was determined by assessing the relaxation of
supercoiled DNA pBR322. To this end, a mixture of 100 ng of
plasmid pBR322 DNA and 1 unit of recombinant human DNA topo I
(TopoGEN INC., USA) was incubated with or without the as-
prepared compounds at 37 ꢀC for 30 min in relaxation buffer
(10 mM TriseHCl (pH 7.9), 150 mM NaCl, 0.1% bovine serum albu-
min, 1 mM spermidine, and 5% glycerol). The reaction was termi-
128.80, 128.64, 128.01, 125.82, 117.79, 116.48, 116.07, 115.49, 114.02,
113.84.
5.1.10. Synthesis of 2-[2-(4-Hydroxyphenyl)-6-(thiophen-2-yl)
pyridin-4-yl]phenol (13)
Using the procedures described in the SI, 3-(2-hydroxyphenyl)-
1-(4-hydroxyphenyl)propenone (480.50 mg, 2.00 mmol), dry
ammonium acetate (1.54 g, 20.00 mmol), 6 (662.34 mg, 2.00 mmol)
and AcOH (2 mL) were reacted to yield a yellow solid (336.50 mg,
48.7%, 0.97 mmol) with a mp ¼ 237e238 ꢀC, Rf (ethyl acetate/n-
hexane 1:1 v/v) ¼ 0.51, LC/MS/MS retention time ¼ 7.79 min,
[MH]þ ¼ 346.21, and purity of 100% determined by HPLC. 1H NMR
nated by adding 2.5
bromophenol blue and 25% glycerol) to a final volume of 10
m
L of a stop solution (5% sarcosyl, 0.0025%
L. DNA
m
(250 MHz, DMSO-d6)
d
9.83 (br, 2H, 2-phenyl 4-OH, 4-phenyl 2-
samples were then electrophoresed on a 1% agarose gel at 50 V for
OH), 8.02 (d, J ¼ 8.7 Hz, 2H, 2-phenyl H-2, H-6), 7.90 (d,
J ¼ 0.8 Hz, 1H, pyridine H-3), 7.85 (dd, J ¼ 3.6, 0.8 Hz, 1H, 6-
thiophene H-3), 7.84 (d, J ¼ 0.6 Hz, 1H, pyridine H-5), 7.64 (dd,
J ¼ 5.0, 0.8 Hz, 1H, 6-thiophene H-5), 7.50 (dd, J ¼ 7.6, 1.5 Hz, 1H, 4-
phenyl H-6), 7.27 (td, J ¼ 8.6, 1.6 Hz, 1H, 4-phenyl H-4), 7.17 (dd,
J ¼ 5.0, 3.7 Hz, 1H, 6-thiophene H-4), 7.02 (d, J ¼ 8.2 Hz, 1H, 4-
phenyl H-3), 6.94 (t, J ¼ 7.6 Hz, 1H, 4-phenyl H-5), 6.91 (d,
J ¼ 8.7 Hz, 2H, 2-phenyl H-3, H-5). 13C NMR (62.5 MHz, DMSO-d6)
1 h in TriseacetateeEDTA (TAE) running buffer. Gels were stained
for 30 min in an aqueous solution of ethidium bromide (0.5 mg/mL).
DNA bands were visualized by UV-transillumination and quanti-
tated using AlphaImagerÔ software (Alpha Innotech Corporation).
5.2.2. In vitro DNA topoisomerase II-mediated inhibition assay
The DNA topo II inhibitory activity of compounds were
measured as follows [33]. A mixture of 200 ng of supercoiled
d
158.77, 155.69, 154.88, 151.32, 148.22, 145.39, 130.38, 130.12,
pBR322 plasmid DNA and 1 unit of human DNA topoisomerase IIa
129.55, 128.44, 128.14, 125.47, 125.10, 119.72, 117.88, 116.46, 115.66.
(USB, USA) was incubated with or without the as-prepared com-
pounds in assay buffer (10 mM TriseHCl (pH 7.9) containing 50 mM
5.1.11. Synthesis of 3-[2-(4-Hydroxyphenyl)-6-(thiophen-2-yl)
pyridin-4-yl]phenol (14)
NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM ATP, and 15
serum albumin) for 30 min at 30 ꢀC. The reaction was terminated by
the addition of 3 L of 7 mM EDTA to a final volume of 20 L. Re-
action products were analyzed on a 1% agarose gel running at 50 V
for 1 h in TAE buffer. Gels were stained for 30 min in an aqueous
solution of ethidium bromide (0.5 mg/mL). DNA bands were visu-
alized by UV-transillumination and supercoiled DNA was quanti-
tated using AlphaImagerÔ software (Alpha Innotech Corporation).
mg/mL bovine
Using the procedure described above, 3-(3-hydroxyphenyl)-1-
(4-hydroxyphenyl)propenone (360.37 mg, 1.50 mmol), dry
ammonium acetate (1.15 g, 15.00 mmol), 6 (496.75 mg, 1.50 mmol),
and AcOH (2 mL) were reacted to yield a white solid (475.70 mg,
91.8%, 1.37 mmol). mp ¼ 212e213 ꢀC, Rf (ethyl acetate/n-hexane 1:1
v/v) ¼ 0.50, LC/MS/MS retention time ¼ 7.63 min, [MH]þ ¼ 346.21,
m
m
and 100% purity by HPLC. 1H NMR (250 MHz, DMSO-d6)
d 9.73 (br,
2H, 2-phenyl 4-OH, 4-phenyl 3-OH), 8.11 (d, J ¼ 7.9 Hz, 2H, 2-phenyl
H-2, H-6), 8.00 (dd, J ¼ 4.0, 0.9 Hz, 1H, 6-thiophene H-3), 7.98 (s, 1H,
pyridine H-3), 7.88 (s, 1H, pyridine H-5), 7.65 (dd, J ¼ 5.0, 1.2 Hz, 1H,
6-thiophene H-5), 7.36e7.33 (m, 2H, 4-phenyl H-5, H-6), 7.29 (s, 1H,
4-phenyl H-2), 7.19 (dd, J ¼ 4.8, 3.8 Hz, 1H, 6-thiophene H-4), 6.91
5.2.3. Cytotoxicity assay
Cancer cells were cultured according to the supplier’s in-
structions. For the cytotoxicity assay, cells were seeded in 96-well
plates at a density of 2e4 ꢂ 104 cells per well and incubated
overnight in 0.1 mL of media supplemented with 10% Fetal Bovine

436
K.-Y. Jun et al. / European Journal of Medicinal Chemistry 80 (2014) 428e438
Serum (Hyclone, USA) in an incubator at 37 ꢀC with a 5% CO2 at-
mosphere. The following day the culture medium in each well was
exchanged with 0.1 mL aliquots of medium containing varying
gels at 15 V/cm for 12e15 h. After electrophoresis, gels were stained
in TAE buffer with ethidium bromide for 30 min and visualized
using an Alpha Tech Imager.
concentrations of compounds. On the fourth day, 5
mL of cell
In a separate assay, we determined inhibition of DNA unwinding
counting kit-8 solution (Dojindo, Japan) was added to each well,
and plates were incubated for an additional 4 h under the same
conditions [34]. The absorbance of each well was then determined
with an Automatic Elisa Reader System (Bio-Rad 3550) at a wave-
length of 450 nm. To determine IC50 values, absorbance readings at
450 nm were fitted to a four-parameter logistic equation. Adria-
mycin, etoposide, and camptothecin were purchased from Sigma
and used as positive controls.
activity as retardation of migration of DNA [36]. Briefly, 1.25
negatively supercoiled DNA pBR322 (100 ng/ L Fermentas) and
100 M or 300 M of investigational compounds in a total volume
of 10
L was incubated at 37 ꢀC for 20 min. Positive controls con-
sisted of 0.4 g/mL and 1 g/mL ethidium bromide. Next, mixtures
were resolved on 1% agarose gels at 20 vol/cm for 12e16 h. After
electrophoresis, gels were stained in 1ꢂ TAE buffer with ethidium
bromide for 30 min and visualized using an Alpha Tech Imager.
mL of
m
m
m
m
m
m
5.2.4. kDNA decatenation assay
Topo II specificity was determined using a kDNA decatenation
assay [35]. Briefly, the assay was performed in a total reaction
5.2.7. Cell cycle analysis
HCT15 cells were seeded in 60 mm dishes at a density of
5 ꢂ 105 cells per dish. When cells reached 80% confluency, they
were treated with the test compounds at concentrations of 0.05, 0.1,
0.5, and 1 mM. Cells were then washed with ice-cold PBS (pH 7.4)
and harvested by centrifugation at 2000 rpm for 5 min. The
resulting pellets were fixed with 70% ethanol, and fixed cells were
then washed with PBS before incubation with 50
iodide (Sigma, USA) and 2.5 g/ml RNase (Sigma, USA). Fluores-
volume of 10
10 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM ATP, 30
serum albumin, and 75 ng of kDNA. Compounds were added to a
final concentration of 50, 100 M in 1% DMSO. Reactions were
m
L containing 50 mM TriseHCl (pH 8.0), 120 mM KCl,
mg/mL bovine
m
initiated by addition of 2e4 units of topo II, followed by incubation
mg/mL propidium
with the compound mixtures for 30 min at 37 ꢀC. Reactions were
m
terminated by the addition of 2.5 mL of stop solution (5% SDS, 25%
cence was measured with a Fluorescence-Activated Cell Sorting
(FACS)-Caliber flow cytometer (BD Biosciences, USA). At least
10,000 cells were measured for each sample [37].
ficoll, and 0.05% bromphenol blue) followed by treatment with
0.25 mg/mL proteinase K (Roche) at 55 ꢀC for 30 min to eliminate
protein. Samples were resolved by electrophoresis on a 1.2% (w/v)
agarose gel containing 0.5 mg/mL ethidium bromide in TAE buffer
(100 mM Triseacetate and 2 mM Na2EDTA, pH 8.3). DNA bands
were visualized by UV and photographed and documented with
AlphaImagerÔ software (Alpha Innotech Corporation). For quan-
titation of the substrate (kDNA) and products of the topo II reaction
(Nck, SC, and catenanes), the numerical values of intensity profiles
of the different lanes were obtained after background subtraction
and exported to Microsoft Excel. For each lane, the overall intensity
was used to obtain the fraction of the intensity for the different
bands, namely, kDNA, Nck, and SC. We subsequently determined
topo II activity based on the relative amounts of the final products
(nicked plus supercoiled minicircles). A positive control (1% DMSO,
topo II) and negative control (1% DMSO) were used to establish
limit values (1 and 0, respectively).
5.2.8. Wound healing assay
HeLa cells were cultured in 6-well plates to 90% confluency. A
clean wound in the cell monolayer then created across the center of
the well with a sterile micro tip. After starvation with low serum
media (1% FBS in DMEM) for 4 h, cells were exposed to the com-
pound at either 5 or 10 mM and allowed to migrate in the medium.
The wound was assessed by microscopy at 5ꢂ magnification at
different time points (0, 12 and 24 h).
5.2.9. Western blot assay
HCT15 cells were grown on 60 mm tissue culture dishes at
1 ꢂ 106 cells until reaching 80% confluency. The cells were then
treated with compound 13 at various concentrations (0, 0.5, 1, 2,
4 mM) for 24 h. Cells were lysed in lysis buffer solution containing
5.2.5. Cleavage complex stabilization assay
50 mM TriseHCl, 300 mM NaCl, 1% Triton X-100, 10% glycerol,
1.5 mM MgCl2, 1 mM CaCl2, 1 mM PMSF, and 1% protease inhibitor
cocktail. Protein concentrations of soluble extracts of lysates were
then determined by BCAÔ Protein Assay Kit (Pierce, USA) and a
For the cleavage complex assay, supercoiled DNA pBR322 (Fer-
mentas) was used as a substrate, and 3 units of topo II (TopoGEN,
USA) were added 10 min before the addition of test compounds.
The reaction mixture was incubated at 37 ꢀC for 20 min and the
reaction was stopped by addition of 10% SDS followed by digestion
with proteinase K at 45 ꢀC for 30 min. After addition of loading
buffer, the reaction mixture was heated for 2 min at 70 ꢀC. Finally,
electrophoresis was carried out with a 0.8% agarose gel in TAE
total of 60
mg protein per sample was loaded on a 12% poly-
acrylamide gel, which was then transferred to a poly vinylidene
fluoride membrane (Millipore Corporation, USA). Next, membranes
were blocked for 1 h at room temperature in TBST with 5% skim
milk. Membranes were incubated with antibodies for either of
tubulin or PARP (1:1000) in TBST with 5% skim milk overnight at
ꢀC. Anti-rabbit IgG horseradish peroxidase was used as the sec-
a-
buffer containing 0.5
mL/mL ethidium bromide, followed by
destaining the gel with water for 20 min.
4
ondary antibody. After incubation antibodies, membranes were
washed three times with TBST and detected with ECL plus western
blotting detection reagent (Ab Frontier, Korea). Western blot im-
ages were analyzed using Multi-Gauge Software (Fuji Photo Film
Co., Ltd., Japan).
5.2.6. DNA unwinding assay
The DNA-unwinding capacity of compound 13 was analyzed
using a DNA unwinding kit (TopoGEN, Port Orange, FL, USA) ac-
cording to the manufacturer’s instructions. Briefly, 100 ng plasmid
(pHOT1) was treated with 2 U of topo I (TopoGEN, Port Orange, FL,
USA) in 20 ml of buffer (10 mM TriseHCl, pH 7.5, 1 mM EDTA) for
30 min at 37 ꢀC. Relaxed plasmids were then incubated in the
presence of various test compounds at 37 ꢀC for an additional
5.2.10. Statistical analysis
Data were expressed as the mean ꢁ standard deviation (SD),
with at least 3 replicates per experimental group. Comparison of
differences was conducted using unpaired, two-tailed Student t-
test. Differences with p values ꢄ0.05 were considered statistically
significant.
30 min m-AMSA (100, 200, 500, and 1000
mM) was used as a
positive control for DNA unwinding activity. At the end of the in-
cubation, 1% SDS and loading dye were added to terminate the
reactions. The resulting aqueous phase was resolved on 1% agarose

K.-Y. Jun et al. / European Journal of Medicinal Chemistry 80 (2014) 428e438
437
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5.3. Computational analysis
The datasets generated in this study as well as those curated
from published studies comprised 34 compounds. The structures of
compounds and their biological data are shown in Table 2. In order
to analyze the structure activity relationships, we used the com-
mon biological data, which consisted of the IC50 values for the
HCT15 cell line. IC50 values in units of molarity (M) were trans-
formed to negative log values. The 3D compound structures were
prepared using Sybyl X-2.0 [38]. The lowest energy Gasteigere
Hückel charges were applied to each molecule and energy mini-
mization was performed using the Tripos force field. Compound 13
was used as a template and the remaining compounds were
manually aligned using the common core ring structure. The tor-
sion angles the 2, 4, and 6-aryl groups were adjusted to match those
of compound 13. When adjusting the torsion angles, all the possible
combinations of rotational conformations were checked to find the
lowest energy conformer and each compounds were energetically
minimized using the same method as above. The final alignment of
3D-QSAR data for compounds was analyzed using the CoMSIA
descriptors. The CoMSIA model generated using the pIC50 data from
HCT15 cells was validated by predicting a test set consisting of 5
compounds.
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their antitumor activities, European Journal of Medicinal Chemistry 43 (2008)
675e682.
[14] P. Thapa, R. Karki, A. Basnet, U. Thapa, H. Choi, Y. Na, Y. Jahng, C.-S. Lee,
Y. Kwon, B.-S. Jeong, E.-S. Lee, 2,6-Dithienyl-4-arylpyridines: synthesis,
topoisomerase I and II inhibition and structure-activity relationship, Bulletin
of the Korean Chemical Society 29 (2008) 1605e1608.
[15] P. Thapa, R. Karki, U. Thapa, Y. Jahng, M.J. Jung, J.M. Nam, Y. Na, Y. Kwon,
E. Lee, S. 2-Thienyl-4-furyl-6-aryl pyridine derivatives: synthesis, topoisom-
erase I and II inhibitory activity, cytotoxicity, and structure-activity relation-
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Acknowledgments
This research was supported by the Basic Science Research
Program through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education, Science and Technology
(2011e0011007). K.-Y. Jun was supported by RP-Grant 2012 of Ewha
Womans University.
Abbreviations
[17] L.X. Zhao, Y.S. Moon, A. Basnet, E.K. Kim, Y. Jahng, J.G. Park, T.C. Jeong,
W.J. Cho, S.U. Choi, C.O. Lee, S.Y. Lee, C.S. Lee, E.S. Lee, Synthesis, topoisom-
Topo
kDNA
Nck
Rel
topoisomerase
kinetoplast DNA
nicked
relaxed
supercoiled
erase
I inhibition and structure-activity relationship study of 2,4,6-
trisubstituted pyridine derivatives, Bioorganic & Medicinal Chemistry Let-
ters 14 (2004) 1333e1337.
[18] P. Thapa, R. Karki, M. Yun, T.M. Kadayat, E. Lee, H.B. Kwon, Y. Na, W.J. Cho,
N.D. Kim, B.S. Jeong, Y. Kwon, E.S. Lee, Design, synthesis, and antitumor
evaluation of 2,4,6-triaryl pyridines containing chlorophenyl and phenolic
moiety, European Journal of Medicinal Chemistry 52 (2012) 123e136.
[19] R. Karki, P. Thapa, H.Y. Yoo, T.M. Kadayat, P.H. Park, Y. Na, E. Lee, K.H. Jeon,
W.J. Cho, H. Choi, Y. Kwon, E.S. Lee, Dihydroxylated 2,4,6-triphenyl pyridines:
SC
ACN
TLC
CC
acetonitrile
thin-layer chromatography
column chromatography
ammonium formate
AF
synthesis, topoisomerase
I and II inhibitory activity, cytotoxicity, and
DMSO dimethylsulfoxide
structure-activity relationship study, European Journal of Medicinal Chemis-
try 49 (2012) 219e228.
[20] Y. Jahng, L.X. Zhao, Y.S. Moon, A. Basnet, E.K. Kim, H.W. Chang, H.K. Ju,
T.C. Jeong, E.S. Lee, Simple aromatic compounds containing propenone moiety
show considerable dual COX/5-LOX inhibitory activities, Bioorganic & Me-
dicinal Chemistry Letters 14 (2004) 2559e2562.
[21] D. Batovska, S. Parushev, A. Slavova, V. Bankova, I. Tsvetkova, M. Ninova,
H. Najdenski, Study on the substituents’ effects of a series of synthetic chal-
cones against the yeast Candida albicans, European Journal of Medicinal
Chemistry 42 (2007) 87e92.
TAE
IC50
SDS
SD
TriseacetateeEDTA
50% inhibitory concentration
sodium dodecyl sulfate
standard deviation.
Appendix A. Supplementary data
[22] D.S. Breslow, C.R. Hauser, Condensations. XI. Condensations of certain active
hydrogen compounds effected by borontrifluoride and aluminum chloride,
Journal of the American Chemical Society 62 (1940) 2385e2388.
[23] T. Narender, K.P. Reddy, A simple and highly efficient method for the syn-
thesis of chalcones by using borontrifluoride-etherate, Tetrahedron Letters 48
(2007) 3177e3180.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.066.
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