Dihydroxylated 2,6-diphenyl-4-chlorophenylpyridines: Topoisomerase I and IIα dual inhibitors with DNA non-intercalative catalytic activity

Article abstract of DOI:10.1016/j.ejmech.2017.03.048

With the aim to develop novel antiproliferative agents, a new series of eighteen dihydroxylated 2,6-diphenyl-4-chlorophenylpyridines were systematically designed, prepared, and investigated for their topoisomerase (topo) I and IIα inhibitory properties an

Full text of DOI:10.1016/j.ejmech.2017.03.048

Accepted Manuscript
Dihydroxylated 2,6-diphenyl-4-chlorophenylpyridines: Topoisomerase I and IIα dual
inhibitors with DNA non-intercalative catalytic activity
Ganesh Bist, Seojung Park, Chanju Song, Til Bahadur Thapa Magar, Aarajana
Shrestha, Youngjoo Kwon, Eung-Seok Lee
PII:
S0223-5234(17)30215-5
10.1016/j.ejmech.2017.03.048
EJMECH 9308
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 March 2017
Revised Date: 22 March 2017
Accepted Date: 23 March 2017
Please cite this article as: G. Bist, S. Park, C. Song, T.B. Thapa Magar, A. Shrestha, Y. Kwon, E.-S.
Lee, Dihydroxylated 2,6-diphenyl-4-chlorophenylpyridines: Topoisomerase I and IIα dual inhibitors with
DNA non-intercalative catalytic activity, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.03.048.
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Graphical Abstract

ACCEPTED MANUSCRIPT
Dihydroxylated 2,6-diphenyl-4-chlorophenylpyridines: Topoisomerase I and IIα dual
inhibitors with DNA non-intercalative catalytic activity
a #
b,#
b
a
,
Ganesh Bist, Seojung Park, Chanju Song, Til Bahadur Thapa Magar, Aarajana
a
b,*
a,*
Shrestha, Youngjoo Kwon, Eung-Seok Lee
a
b
College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea
College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Global Top5
program, Ewha Womans University, Seoul 120-750, Republic of Korea
#
Authors are equal contributors.
*
Corresponding Authors:
Tel: +82 2 3277-4653, fax: +82 2 3277-2851, email: ykwon@ewha.ac.kr (Y.K.)
Tel: +82 53 810-2827, fax: +82 53 810-4654, email: eslee@yu.ac.kr (E.-S.L.)
1

ACCEPTED MANUSCRIPT
Dihydroxylated 2,6-diphenyl-4-chlorophenylpyridines: Topoisomerase I and IIα dual
inhibitors with DNA non-intercalative catalytic activity
a #
b #
b
a
,
Ganesh Bist, Seojung Park, Chanju Song, Til Bahadur Thapa Magar, Aarajana
a
b,*
a,*
Shrestha, Youngjoo Kwon, Eung-Seok Lee
Abstract
With the aim to develop novel antiproliferative agents, a new series of eighteen
dihydroxylated 2,6-diphenyl-4-chlorophenylpyridines were systematically designed, prepared,
and investigated for their topoisomerase (topo) I and IIα inhibitory properties and
antiproliferative effect in three different human cancer cell lines (HCT15, T47D, and HeLa).
Compounds 22-30 which possess a meta- or para-phenol on 2-, or 6-position of central
pyridine ring showed significant dual topo I and topo IIα inhibitory activities with strong
antiproliferative activities against all the tested human cancer cell lines. However,
compounds 13-21 which possess an ortho-phenol on 2-, or 6-position of central pyridine ring
did not show significant topo I and topo IIα inhibitory activities but displayed moderate
antiproliferative activities against all the tested human cancer cell lines. Compound 23
exhibited the highest antiproliferative potency as much as 348.5 and 105 times compared to
etoposide and camptothecin, respectively, in T47D cancer cell line. The structure-activity
relationship study revealed that the para position of a hydroxyl group at 2-and 6-phenyl ring
and chlorine atom at the para position of 4-phenyl ring of the central pyridine exhibited the
most significant topo I and topo IIα inhibition, which might indicate introduction of the
2

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chlorine atom at the phenyl ring of 4-pyridine have an important role as dual inhibitors of
topo I and topo IIα. Compound 30 which showed the most potent dual topo I and topo IIα
inhibition with strong antiproliferative activity in T47D cell line was selected to perform
further study on the mechanism of action, which revealed that compound 30 functions as a
potent DNA non-intercalative catalytic topo I and IIα dual inhibitor.
Keywords: Antiproliferative agents; Dihydroxylated 2,6-diphenyl-4-chlorophenylpyridines;
Dual inhibitors of topo I and topo IIα; Antiproliferative activity; DNA non-intercalative
catalytic inhibitor
3

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1
. Introduction
Although there has been a significant amount of development in the anticancer therapy, DNA
still remains to be the most important target for the cancer treatment [1]. Antiproliferative
compounds show their activity by interacting directly with DNA either by forming covalent
complexes or by damaging the function of DNA-processing enzymes, thereby initiating the
cell cycle arrest and apoptosis. Topoisomerase (topo) inhibitors have a significant part in
impairing the function of DNA processing enzymes [2]. Topos solve the topological
problems of the DNA double helix that occur during the metabolic processes of transcription,
replication, recombination, repair, and chromatin assembly [3, 4]. Therefore, topos have
become a popular target in the antiproliferative therapy because of their greater over-
expression in the tumor cells [5].
Based on the molecular mechanism of cleavage, topos are categorized as topo I and topo II
[
6]. Topo I breaks a single-strand of DNA double helix by the trans-esterification reactions
and re-ligates DNA strands during the cell cycle process to maintain genomic integrity [7-9].
Topo II works by making a double-strand break in an ATP-dependent reaction. It is mainly
involved in the DNA complex processes such as recombination, splitting of daughter
chromosomes, and condensation of chromosomes [10]. The topo-directed agents have
different mechanism of actions: Topo poisons stabilize the cleavage complex of DNA and
topo to restrict DNA breaking-rejoining mechanism of the enzyme, whereas catalytic
inhibitors impair either one or more steps within the catalytic cycle of topo [10-12].
4

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Recently, the camptothecin analogs, the target of which is topo I, have been widely used for
the treatment of colon cancer [13], whereas drugs such as etoposide, teniposide, doxorubicin,
and mitoxantrone which specifically target topo II are usually used for the treatment of
various malignancies [14]. However, these topo inhibitors have several problems such as
dose limiting side effects, chemical instability, and drug-efflux mediated resistance [15, 16].
Due to these limitations, there is a continuous need for the progressive development and
innovation of novel topo I as well as topo II inhibitors. Topo I and topo II have shown to be
good targets, so dual inhibition of topo I and topo II can synergistically enhance their
antiproliferative property as well as reduce drug resistance. Therefore, development of a sole
unique compound which can mutually obstruct topo I and topo II at the same time, will have
the combinational advantages of the individual inhibitor and thus can result in an increased
antiproliferative property [4]. Recently, dual topo I and topo II inhibitors have received a lot
of interest in the development of anticancer drugs because of their greater antiproliferative
effects and lower side effects [4, 17, 18]. Therefore, with the aim to search for and develop
potent dual topoisomerase inhibitors, our research group has previously synthesized a series
of 2,4,6-triaryl pyridine derivatives and discovered that several compounds exhibited strong
dual inhibitory activities of topo I and II with significant antiproliferative activities against
different types of human cancer cell lines [19-23]. Likewise, various drugs and compounds
such as acridine DACA [24], indenoquinolinone TAS-103 [25], benzopyridoindole
intoplicine [26], tafluposide (F-11782) [27], and pyrazoloacridine NSC 366140 [28] also
have been reported as topo I and II dual inhibitors [17, 20]
.
From various studies, it has been found that most phenolic and polyphenolic derivatives show
antioxidant [29], anti-inflammatory [30], anti-angiogenic, and antiproliferative properties [31,
5

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3
2]. Most bioflavonoids show antiproliferative property because of their function as topo II
poison [33-35]. Epigallocatechin gallate, a polyphenolic compound, which is isolated from
green tea is one of the potent topo II poisons [35]. Camptothecin, a very popular topo I
inhibitor which exhibits a potent antiproliferative property and also possesses a hydroxyl
group in its structure [36, 37]. Additionally, as reported from previous studies in our research
group, mono and dihydroxylated 2,4,6-triarylpyridines justified the necessity of the hydroxyl
group in order to show their greater topo inhibition and antiproliferative properties. Structure-
activity relationship (SAR) studies demonstrated that the inhibition of both topo I and II and
antiproliferative properties can be enhanced either by the replacement or by the addition of
hydroxyl group on the phenyl ring [38, 39]. Moreover, selective topo II inhibition and
antiproliferative properties were observed in the compounds that had hydroxyl moiety at the
meta or para position on 2-phenyl ring of the central pyridine [39]. Additionally, a bulky
group like halogen atoms tends to occupy most of the deeper pockets as well as the reacting
regions of the molecular targets thereby inducing either antagonistic or agonistic activity. In
recent years, much research has been performed by introducing chlorine atom into structures
because of its capacity to enhance potency and bioavailability, to increase brain blood barrier
penetrability, and also to enhance chemical and metabolic stability through increasing
lipophilicity and to acting as a hydrogen bond acceptor [40]. Further studies of terpyridine
derivatives with the chlorine substituents have displayed stronger topo I and II inhibitory
properties and antiproliferative activities than those with the non-substituted ones [21, 41].
From these various reports exhibiting the importance of the phenolic group together with the
introduction of the chlorine atom has inspired us to integrate these functional groups into
2
,4,6-triaryl pyridine compounds to synthesize dihydroxylated 2,6-diphenyl-4-
chlorophenylpyridines as shown in Figure 1.
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Figure 1. Structures of 2,4,6-triarylpyridines, monohydroxylated 2-phenyl-4,6-
diarylpyridines, mono- and dihydroxylated 2,4,6-triphenylpyridines, and dihydroxylated 2,6-
diphenyl-4-chlorophenylpyridines
In this study, we have systematically designed and prepared eighteen dihydroxylated 2,6-
diphenyl-4-chlorophenylpyridine derivatives. A hydroxyl group at 2- and 6-phenyl ring
together with the chlorine atom at 4-phenyl ring attached to the central pyridine was prepared
for investigating their topo I and IIα inhibitory property and antiproliferative activity against
three different types of human cancer cell lines. Subsequently, ortho, meta, and para
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substitution of the hydroxyl and chlorine atom would give us an evidence to further verify the
effect of these positions on topo inhibitory activity and antiproliferative activity.
2
. Results and discussion
2
.1. Synthetic Chemistry
According to the method reported by W. Zecher and F. Kröhnke in 1961, dihydroxylated 2,6-
diphenyl-4-chlorophenylpyridines were prepared [42]. Synthesis of compounds involved
1
three basic methods as outlined in scheme 1. At first, aryl methyl ketone I (R =a-c) was
o
refluxed in pyridine in the presence of iodine for 3 h at 140 C to synthesize aryl pyridinium
iodide salts 1-3 to yield 65.4-99.5% by using Ortoleva-King reaction [43]. In parallel, aryl
2
3
aldehyde II (R =d-f) and aryl methyl ketone III (R =a-c) were reacted in methanol in the
presence of NaOH to synthesize propenone intermediates 4-12 to yield 57.2-83.1% by using
Claisen-Schmidt condensation reaction [44, 45]. Finally, aryl pyridinium iodide salts 1-3 and
propenone intermediates 4-12 were refluxed either in glacial acetic acid or methanol for 2-36
o
h at 90-100 C in the presence of ammonium acetate (NH OAc) to give dihydroxylated 2,6-
4
diphenyl-4-chlorophenylpyridines (13-30) in 11.4-63.8% yield by using modified Kröhnke
pyridine synthesis. The compounds 13-30 were prepared in nine separate series as shown in
Figure 2. Structure of previously synthesized dihydroxylated 2,4,6-triphenylpyridines (A-F)
which were reported earlier [39] and newly synthesized dihydroxylated 2,6-diphenyl-4-
chlorophenylpyridines (13-30) are shown in Figure 3, and their physical properties are shown
in Table S1 (Supplementary Data).
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I
R1
R1
(i)
N
O
O
1
-3
1
I (R =a-c)
I
R1
R2
N
O
OH
OH R2
1-3
iii)
R1
(ii)
N
(
1
3-21
O
4
-6
I
R2
R1
N
OH
O
R2
HO
R1
R3
2-3
iii)
N
R2
H
(ii)
+
(
O
2
O
3
2
2-27
O
II (R =d-f) III (R =a-c)
7
-9
I
R1
R2
N
N
O
R2
HO
3
(ii)
R1
(iii)
HO
O
28-30
1
0-12
OH
Cl
e
OH
HO
Cl
Cl
1
3
2
R , R =
R =
b
c
f
a
d
Scheme 1. Schematic representation for synthesis of pyridinium iodide salts 1-3, propenone
intermediates 4-12 and 2,6-diphenyl-6-chlorophenylpyridines 13-30. Reagents and
o
conditions: (i) Iodine (1.0 equivalent), pyridine, 3 h, 140 C, 90.1- 99.8% yield, (ii) aq.
NaOH (3.0 equivalent), MeOH, 1-3 h, room temperature, 90.1- 99.8% yield, (iii) NH OAc
4
o
(
10.0 equivalent), glacial acetic acid or methanol, 12-36 h, 95-100 C, 22.3-67.5% yield.
9

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Figure 2. Strategic design of dihydroxylated 2,6-diphenyl-4-chlorophenylpyridines
1
0

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Figure 3. Structures of a) previously synthesized dihydroxylated 2,4,6-triphenylpyridines,
and b) newly synthesized dihydroxylated 2,6-diphenyl-4-chlorophenylpyridines
1
1

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2
. 2. Topo I and IIα inhibitory property
The inhibition on transformation of supercoiled pBR322 DNA to the relaxed form was
examined and analyzed in the presence of the prepared dihydroxylated 2,6-diphenyl-4-
chlorophenylpyridines (13-30) to measure their inhibitory activity of topo I and IIα.
Camptothecin and etoposide, selective topo I and II inhibitors, respectively, were used as
positive controls. The inhibitory properties were measured at 100 µM and 20 µM
concentration.
Figure 4 and Table 1 illustrates the topo I inhibitory property of prepared compounds 13-30.
Among the synthesized compounds, compounds 18, 23, 24, 26-30 and compounds 18, 19, 24,
2
5, 27, 30 have shown significant topo I inhibitory property at 100 µM and 20 µM,
respectively, which were stronger or comparable to the positive control, camptothecin.
Compounds 27 and 30 showed the most potent topo I inhibitory property compared to
camptothecin at both 100 µM and 20 µM in which compound 27 displayed 70.6% and 28.2%
inhibition, and compound 30 displayed 82.7% and 23.7% inhibition, while camptothecin
displayed 64.4% and 21.6% inhibition at 100 µM and 20 µM, respectively. Compounds 19,
2
2, and 23 exhibited moderate topo I inhibition (35.0%, 44.2%, and 42.3%, respectively) at
00 µM. Similarly, compound 24 exhibited moderate topo I inhibition (13.2%) at 20 µM.
1
On the other hand, as shown in Figure 5 and Table 1, compounds 22-30 except 28 (45.7%
inhibition) and compounds 19, 21-30 except 22 and 23 (19.3% and 15.5% inhibition)
exhibited significant inhibition of topo IIα at the concentration of 100 µM and 20 µM,
respectively, which were stronger or comparable to the positive control, etoposide. Among
them, compound 30 showed the strongest inhibition of topo IIα at both 100 µM and 20 µM
1
2

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(100% and 57%, respectively) whereas the positive control etoposide displayed 67.2% and
2
2.8% inhibition, respectively, at 100 µM and 20 µM. Similarly, compounds 22, 25, 27, and
9 displayed 71.6%, 68.5%, 69.2 %, and 82.1% inhibition, respectively, at 100 µM whereas
2
compounds 24-29 displayed 31.0%, 30.4%, 55.0%, 49.0%, 44.5%, and 45.5%, respectively,
at 20 µM.
Figure 4. Human DNA topo I inhibitory activity of prepared compounds 13-30. (A) Lane D:
pBR322 only. (B) Lane T: pBR322 + Topo I. (C) Lane C: pBR322 + Topo I + Camptothecin.
(D) Lane 13-30(100 µM): pBR322 + Topo I + compounds 13-30. (E) Lane 18, 19, 22-30 (20
µM): pBR322 + Topo I + compounds 18, 19, 22-30.
1
3

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Figure 5. Human DNA topo IIα inhibitory activity of compounds 13-30. (A) Lane D:
pBR322 only. (B) Lane T: pBR322 + Topo IIα. (C) Lane C: pBR322 + Topo IIα + Etoposide
(D) Lane 13-30 (100 µM): pBR322 + Topo IIα + compounds 13-30. (E) Lane 19, 21-30 (20
µM): pBR322 + Topo IIα + compounds 19, 21-30.
1
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Table 1. Topoisomerase I and II inhibitory properties and antiproliferative activities of compounds 13-30
*
*
Compounds
Topo I (% Inhibition)
00 µM 20 µM
Topo II (% Inhibition)
IC50 (µM)
1
100 µM
20 µM
HCT15
T47D
HeLa
1
1
.82±0.02/
.28±0.07
0.25±0.02/
3.69 ± 0.13
6.97±0.05/
3.25 ± 0.04
2.10±0.01/
3.91 ± 0.66
1.20±0.03/
1.45 ± 0.01
9.10±0.20/
3.20 ± 0.57
0.06±0.02/
1.01 ± 0.01
Adriamycin
d
d
d
d
d
a
5.29±0.28/
1
11.96±0.81/
0
Etoposide
69.7
32.7/23.5
d
d
.33±0.10
b
c
Camptothecin
63.0
42.1 /21.6
d
d
.26±0.04
A
B
C
D
E
F
0
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
41.0
41.6
42.7
92.2
100.0
86.5
96.8
76.8
3.0
12.1
52.1
82.3
49.0
31.0
33.7
NT
>50
37.3 ± 0.34
>50
14.8 ± 0.05
>50
11.0
0
39.90 ± 1.49
7.50 ± 1.13
18.30 ± 0.61
30.52 ± 0.66
1.40 ± 0.06
12.70 ± 1.30
4.99 ± 0.78
3.90 ± 0.44
13.0 ± 0.08
3.46 ± 0.15
67.2
0
0.3
7.2
3.00 ± 0.50
3.88±0.03
>50
6.70 ± 0.78
1.22±0.02
>50
3.30 ± 0.44
3.53±0.01
>50
1
1
1
1
1
1
1
3
4
5
6
7
8
9
5
0
2
9
.0
.8
.2
.0
NA
NT
>50
>50
>50
NA
NT
4.19±0.12
2.21±0.07
4.70±0.05
5.56±0.36
1.62±0.03
0.83±0.01
1.01±0.01
10.81±0.98
5.24±0.01
5.51±0.07
8.17±0.06
18.82±0.13
2.5
NT
2.1
NT
70.0
35.0
NA
NT
34.8
32.7
1
5

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2
0
1.67±0.13
>50
>50
NA
8.6
NT
NT
0
NA
NT
2
2
2
2
2
2
2
2
2
3
1
2
3
4
5
6
7
8
9
0
38.5
71.6
63.7
65.2
68.5
63.6
69.2
45.7
82.1
100.0
30.9
19.3
15.5
31.0
30.4
55.0
49.0
44.5
45.5
57.0
>50
>50
>50
3.50±0.02
3.44±0.13
5.97±0.09
3.80±0.19
4.27±0.05
5.35±0.04
5.08±0.07
4.18±0.02
4.89±0.04
0.07±0.01
0.02±0.01
0.15±0.01
0.79±0.01
0.07±0.01
0.84±0.01
0.78±0.22
1.65±0.01
0.93±0.01
6.34±0.05
2.63±0.08
5.47±0.01
2.47±0.03
0.07±0.01
0.58±0.13
0.69±0.01
1.51±0.01
5.06±0.01
44.2
61.4
0
6
4
6
7
8.2
2.3
2.3
0.6
18.4
18.3
1.5
28.2
13.2
0
66.9
79.1
82.7
23.7
*
*
Each data represents mean ± S.D. from three different experiments performed in triplicate.
NT: Not Tested, NA: Not Active, Control value for compounds B, C, E and 22-30; Control value for compounds 13-21
Control value for compounds 22-30; Control value for compounds A-F against HCT15, T47D and HeLa cell lines
a
b
c
d
HCT15: human colorectal adenocarcinoma; T47D: human breast ductal carcinoma; HeLa: human cervix adenocarcinoma; Adriamycin:
positive control for antiproliferative activity; Etoposide: positive control for topo II and antiproliferative activity; Camptothecin: positive
control for topo I and antiproliferative activity
1
6

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2
. 3. Antiproliferative activity
Antiproliferative activities of all the prepared compounds were examined against three
human cancer cell lines; human breast ductal carcinoma cell line (T47D), human colorectal
adenocarcinoma cell line (HCT15), and human cervix adenocarcinoma cell line (HeLa).
Camptothecin, etoposide, and adriamycin were used as positive controls. The inhibitory
properties (IC50 values) of the synthesized dihydroxylated 2,6-diphenyl-4-
chlorophenylpyridines (13-30) against three different human cancer cell lines are shown in
Table 1. Compared to positive controls, many of the prepared compounds exhibited strong
antiproliferative properties at low micromolar ranges against all the tested human cancer cell
lines. Especially, compounds 22-30 displayed submicromolar antiproliferative property in
T47D cell line. Compounds possessing stronger or comparable topo I and/or topo II
inhibitory properties also showed significant antiproliferative activities against all the tested
human cancer cell lines. Particularly, compounds 22-24, and 26 displayed stronger
antiproliferative activities against T47D cell line as compared to all three positive controls
whereas compounds 13, 16-18, 25, and 27-30 displayed stronger antiproliferative activities
compared to the two positive controls, etoposide and camptothecin against T47D cell line. In
HCT15 cell line, only compound 20 displayed strong antiproliferative activity compared to
all the three positive controls whereas compounds 13, 16-18, 22, 23, 25, 26, and 28-30
showed strong antiproliferative activity compared to two positive controls, camptothecin and
etoposide. Likewise, in HeLa cell line, compounds 26-29 showed stronger antiproliferative
activity compared to two positive controls, adriamycin and etoposide whereas compounds 13,
1
6-18, 22-25, 29, and 30 showed stronger antiproliferative activity against single positive
control, etoposide. Compound 30 exhibiting the highest topo I and topo IIα inhibitory activity
and stronger antiproliferative activity in HCT15 and T47D cancer cell lines compared to
1
7

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camptothecin and etoposide was selected for further study to determine the mode of action.
2
.4. Compound 30 functions as a DNA non-intercalative topo IIα catalytic inhibitor
Compound 30 which displayed the most strong topo IIα inhibitory property at both 100 µM
and 20 µM was selected to determine the mode of action. Several experiments were
accomplished such as cleavage complex stabilization assay, band depletion assay, and EtBr
displacement assay for investigating the mode of action of compound 30. In cleavage
complex stabilization assay, etoposide, a popular topo II poison, produced the linear DNA,
while compound 30 did not produce any linear DNA up to 500 µM as shown in Figure 6A,
which indicated compound 30 did not act as a topo IIα poison. Similarly, in band depletion
assay, etoposide treatment depleted the free topo IIα due to its stabilizing action on the
covalent topo IIα-DNA complex whereas compound 30 treatment showed an intense band of
free topo IIα as shown in Figure 6B. These results from cleavage complex stabilization and
band depletion assay propose that compound 30 acts as a topo IIα catalytic inhibitor. Further,
fluorescence titrimetric method of EtBr-ctDNA complex was performed to check the DNA
intercalative activity of compound 30. The EtBr-ctDNA complex emission intensity was
recorded from 500-700 nm with excitation at 471 nm. As displayed in Figure 6C, the intense
fluorescence emission of EtBr was detected because of intercalation of EtBr into the base
pairs of DNA. Addition of m-AMSA, known as DNA intercalative topo II poison, caused a
progressive reduction in the fluorescence emission intensity of EtBr as increased the
concentration of m-AMSA. It reflected that the intercalated EtBr was displaced with m-
AMSA and then the free EtBr could not generate the fluorescence emission as much as the
bound EtBr. However, the addition of compound 30 did not affect the fluorescence intensity
1
8

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of EtBr-ctDNA complex. This result suggests that the compound 30 acts as a DNA non-
intercalator.
(A)
(B)
(C)
Figure 6. DNA non-intercalative topo IIα catalytic activity of compound 30. (A) Cleavage
complex assay. (B) Band depletion assay in T47D cells. (C) EtBr displacement assay.
2
.5. Compound 30 induced apoptosis with very low DNA damage in T47D cells
Since compound 30 functioned as a topo IIα catalytic inhibitor, the degree of the induction of
DNA damage by compound 30 was further analyzed. In general, topo II poison such as
etoposide produces serious DNA damage by stabilizing the cleavage complex, resulting in
1
9

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blocking the re-ligation of DNA double strand breaks [46]. Comet assay was used to examine
whether or not compound 30 and etoposide caused the aggregation of broken DNA strand in
T47D cancer cells. Comet assay is a simple but an effective way for evaluating the DNA
toxicity caused by the buildup of broken DNA strands in cells. The greater the percentile of
comet (tail) formation, more is the breakage of DNA strand [47]. Figure 7 indicated that
compound 30 (11.6 ± 2.4% at 5 µM and 12.5 ± 4.2% at 10 µM) did not induce comet tail as
compared to etoposide (62.7 ± 9.2% at 5 µM and 66.3 ± 6.9% at 10 µM). In other words,
compound 30 caused less DNA toxicity when compared to etoposide in T47D cells.
In other to determine whether the T47D cell death by compound 30 was due to apoptosis, we
further examined the expression levels of proteins involved in apoptosis, such as poly ADP-
ribose polymerase (PARP), Bax (pro-apoptotic protein), and Bcl-2 (anti-apoptotic protein), in
T47D cells through western blot analysis. As shown in Figure 8, the amount of cleaved
PARP is directly proportional to the concentration of treated compound 30. In addition, the
expression level of Bcl-2 decreased and that of Bax increased by the increase of compound
3
0 treatment amount. Increase of PARP cleavage, reduction of anti-apoptotic Bcl-2, and
increase of pro-apoptotic Bax by compound 30 treatment were reflected as characteristics of
induction of apoptosis [48, 49].
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0

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***
***
Figure 7. Comet assay for assessment of DNA damage. (A) The images of control (non-
treated), etoposide (topo poison) and compound 30 treated T47D cells showing comet
formation. (B) Graphical representation of the selected comet tail lengths of untreated- and
treated-T47D cells using Komet 5.0 software. Columns and error bars indicate mean ± SD (n
¼
50). ***P < 0.001, for significant differences from the vehicle control.
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Figure 8. Representative western blots of apoptosis-related proteins including cleaved PARP,
Bcl-2 and Bax after T47D cells were treated with compound 30 for 24 h.
2
.6. SAR study
SAR study was performed in accordance with the results of topo I and topo IIα inhibitory
property and antiproliferative activity of the prepared compounds 13-30 and previously
synthesized non-chlorinated compounds A-F. As shown in Table 1, it was revealed that most
compounds 13-21, containing a hydroxyl moiety at the ortho-position of 2-, or 6-phenyl ring
displayed either a very weak or no topo I and/or topo IIα inhibitory property at both 100 µM
and 20 µM. On the other hand, compounds 22-30 which possess hydroxyl moiety either at
meta- or para-position of 2-, or 6-phenyl ring showed significantly strong to moderate topo I
and/or topo IIα inhibitory activities at 100 µM and/or 20 µM. These results indicated that the
meta- or para-hydroxyl group of 2-, or 6-phenyl ring rather than ortho-hydroxyl group is
crucial for topo I and topo IIα inhibition. Furthermore, most compounds 22-30 have an
increasing order of topo I and topo IIα inhibitory activities with respect to the ortho-, meta-,
or para-chlorophenyl substituents, respectively. This revealed that the para position of the
2
2

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chlorine at the 4-phenyl ring is important for exhibiting the most significant topo I and topo
IIα inhibition, as displayed in Figure 9.
Figure 9. SAR study showing the favorable substitution order on dihydroxylated 2,6-
diphenyl-4-chlorophenylpyridines for topo I and IIα inhibition.
As shown in Table 1, previously synthesized representative compounds A-F displayed
significant topo IIα inhibitory property with the exception of compound A whereas these
compounds showed very weak or no topo I inhibitory activity with the exception of
compound D compared to the positive controls, etoposide and camptothecin, respectively.
Interestingly, substitution of the chlorine atom in the phenyl ring of 4-pyridine with the
ortho-hydroxyl group on 2-, or 6-phenyl ring of the previously synthesized compounds A-F
did not show considerable topo I and topo IIα inhibitory properties (compounds 13-21).
However, substitution of the chlorine atom in the phenyl ring of 4-pyridine with the meta- or
para-hydroxyl group on 2-, or 6-phenyl ring of the previously synthesized compounds A-F
displayed both topo I and topo IIα inhibitory properties in compounds 22-30. These results
2
3

ACCEPTED MANUSCRIPT
indicated that the addition of the chlorine atom in the phenyl ring of 4-pyridine in case of
meta- or para-hydroxyl group on 2-, or 6-phenyl ring is essential for acting as a dual
inhibitors of topo I and topo IIα by disrupting the topo IIα selectivity of compounds A-F.
For the comparative potency of topo inhibition study, Table 2 illustrates the relative topo I
and IIα inhibition potencies of newly synthesized compounds 13-30 and previously
synthesized compounds A-F compared to positive controls, camptothecin and etoposide.
Since, most of the compounds 13-21, and their corresponding non-chlorinated compounds A-
C displayed either very weak or no topo I inhibitory property at both100 µM and 20 µM, it
was difficult to correlate their relative topo I inhibition study. However, the relative topo IIα
inhibition potency of compounds A-C were significantly higher compared to compounds 13-
3
0. This signifies that at least one ortho-phenol moiety at either 2-, or 6-position and chloro-
phenyl moiety at 4-position of central pyridine is responsible for neither topo I nor topo II α
inhibitory potency whereas removal of chlorine atom from the 4-phenyl ring significantly
enhances only the topo IIα inhibitory potency without having any effect on the topo I
inhibitory potency. On the other hand, among the newly synthesized compounds 22-30, the
relative topo I inhibitory potency of compounds 25-30 were significantly higher compared to
their corresponding non-chlorinated compounds E-F whereas compounds 23 and 24 showed
similar relative potency like that of corresponding compound D. This signifies that the meta-
or para-phenol moiety at 2-position, para-phenol moiety at 6-position and chloro-phenyl
moiety at 4-position of the central pyridine is responsible for their better topo I inhibitory
potency than that of corresponding compound E-F. In contrast, the relative topo IIα inhibition
potency of these newly synthesized compounds 22-30 displayed mixed results compared with
their corresponding non-chlorinated compounds D-F. Compounds 22, 23, and 24 that possess
meta-phenol moiety at both 2- and 6-position with chloro-phenyl moiety at 4-position of the
2
4

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central pyridine, displayed relatively low potency for inhibition of topo IIα at both 100 µM
and 20 µM than that of corresponding non-chlorinated compound D. However, it was
observed that compounds 25, 26, 27 and their corresponding non-chlorinated compounds E
showed relatively high potency at 20 µM. This signifies that the meta-phenol moiety at 2-
position, para-phenol moiety at 6-position and chloro-phenyl moiety at 4-position of the
central pyridine is responsible for their higher topo IIα inhibitory potency than that of
corresponding compound E at 20 µM. In case of compounds 28, 29, 30 and their
corresponding non-chlorinated compound F, compounds 29 and 30 showed relatively higher
topo IIα inhibition potency at both 100 µM and 20 µM. However, compound 28 displayed
low potency at 100 µM but showed higher relative potency at 20 µM compared to
corresponding non-chlorinated compounds F. This signifies that the para-phenol moiety at 2-
and 6-position and chloro-phenyl moiety at 4-position of the central pyridine is responsible
for their higher topo IIα inhibitory potency than the corresponding compound E.
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5

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Table 2. Relative topo I and topo II potencies of newly synthesized compounds 13-30 and previously synthesized compounds A-F compared to
positive controls, camptothecin and etoposide.
Relative potency* for % inhibition compared to positive controls
(Topo II: Etoposide; Topo I: Camptothecin)
Compounds
Topo I
Compounds
Topo I
Compounds
Topo II
Compounds
Topo II
100 µM 20 µM
100 µM
20µM
100 µM 20 µM
100 µM
20 µM
1
3
13
0
.11
NT
NT
NT
N
NT
NT
NT
A
B
C
D
NA
0.17
NA
NT
NT
NT
NT
14
N
A
B
C
D
0.61
1.32
1.43
1.24
0.37
2.22
3.50
1.50
14
15
NA
NA
1
1
5
6
N
16
N
NT
NT
N
N
NT
NT
NT
17
0.14
17
18
1
1
8
9
1
.11
.56
0.97
NA
19
0
0.99
NT
0.47
NA
1.00
NT
20
NA
20
21
2
2
1
2
0.14
NT
0.55
0.94
22
23
0.70
NA
NA
1.03
0.91
0.82
0.66
1.07
23
0.97
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6

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2
2
4
5
24
25
1
.08
.67
0.85
0.85
0.94
1.32
0
0.98
0.91
0.99
1.29
2.34
2.08
E
F
NA
N
NT
NT
26
0.99
N
E
F
1.39
1.10
1.32
1.03
26
27
2
2
7
8
1
.12
.06
1.31
28
1
0.61
NA
0.66
1.18
1.43
1.89
1.94
2.43
29
1.26
.31
29
30
3
0
1
1.10
*
Relative potency: % inhibition of compounds / % inhibition of positive control, NT: Not Tested, NA: Not Active, N: Value less than 0.1
Results of compounds A-F are reported earlier [39]
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Table 3. Relative antiproliferative potencies of newly synthesized compounds 13-30 and previously synthesized compounds A-F compared to
positive controls, etoposide and camptothecin, against HCT15, HeLa, and T47D cancer cell lines.
Relative potency* for IC (µM) compared to positive controls
5
0
Etoposide(ETP); Camptothecin(CPT)
HeLa HCT15
HCT15
T47D
T47D
HeLa
Compounds
Compounds
ETP
CPT
ETP
CPT
0.10
ETP
0.22
CPT
0.07
ETP
CPT
3.08
ETP
5.71
CPT
1.72
ETP
.58
<0.18
0.18
.74
1.65
CPT
0.02
13
1.36
2
A
<0.03 <0.01
0.09
14
<0.11
<0.11
<0.24
<0.24
<0.14
<0.14
<0.04
<0.04
<0.01
<0.01
1
1
5
6
<
1.26
2.85
4.30
1.30
0.01
1
B
0.03
0.01
<0.07 <0.08
<0.06 <0.02
17
2.39
1.13
5.41
2.54
8.40
6.90
2.53
2.08
0.01
0.01
1
1
8
9
1
0
.11
.48
0.95
2.15
0.64
0.19
0.01
2
8