Novel 2-aryl-4-(4′-hydroxyphenyl)-5H-indeno[1,2-b]pyridines as potent DNA non-intercalative topoisomerase catalytic inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.09.019

On the basis of previous reports on the importance of thienyl, furyl or phenol group substitution on 5H-indeno[1,2-b]pyridine skeleton, a new series of rigid 2-aryl-4-(4′-hydroxyphenyl)-5H-indeno[1,2-b]pyridine derivatives were systematically designed and

Full text of DOI:10.1016/j.ejmech.2016.09.019

European Journal of Medicinal Chemistry 125 (2017) 14e28
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
0
Novel 2-aryl-4-(4 -hydroxyphenyl)-5H-indeno[1,2-b]pyridines as
potent DNA non-intercalative topoisomerase catalytic inhibitors
Seojeong Park a , Tara Man Kadayat b , Kyu-Yeon Jun , Til Bahadur Thapa Magar ,
,
1
,
1
a
b
b
b
b,
**
, Youngjoo Kwon a
, *
Ganesh Bist , Aarajana Shrestha , Eung-Seok Lee
College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Republic of Korea
College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea
a
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 June 2016
Received in revised form
On the basis of previous reports on the importance of thienyl, furyl or phenol group substitution on 5H-
indeno[1,2-b]pyridine skeleton, a new series of rigid 2-aryl-4-(4 -hydroxyphenyl)-5H-indeno[1,2-b]pyr-
idine derivatives were systematically designed and synthesized. Topoisomerase inhibitory activity and
antiproliferative activity of all the synthesized compounds were determined using human colorectal
0
5
September 2016
Accepted 6 September 2016
Available online 8 September 2016
(
HCT15), breast (T47D), prostate (DU145) and cervix (HeLa) cancer cells. Compounds 9, 10, 12, 13, 15, 16,
8 and 19 with thienyl or furyl moiety at the 2-position and hydroxyl group at the meta or para positions
of 4-phenyl ring displayed strong to moderate topoisomerase II (topo II ) inhibitory activity and sig-
nificant antiproliferative activity. The evaluation of compound 16 to determine its mechanism of action
was performed with topo II -DNA cleavable complex, topo II -mediated ATPase assay, DNA unwinding
and in vitro and ex vivo topo II relaxation assay. Compound 16 functioned as a DNA non-intercalative
topo II catalytic inhibitor with better potency than etoposide in T47D breast cancer cells. Molecular
docking study revealed that compound 16 cannot intercalate into regularly stacked base-pairs of DNA
duplex but can interact or intercalate to topo II -bound DNA.
2016 Elsevier Masson SAS. All rights reserved.
1
a
a
Keywords:
Anticancer agents
0
a
a
2
-Aryl-4-(4 -hydroxyphenyl)-5H-indeno
[1,2-b]pyridine
a
Antiproliferative activity
Docking study
Dual topoisomerase I and II inhibitors
a
a
©
1
. Introduction
important biological activities such as anticancer, anti-
inflammatory, antidepressant and coronary dilating activities
[6e9]. There are more reports that some similar pharmacore
compounds consisting of imidazo-pyridine [10,11], 2-
arylquinazolinone [12], thiophenylmethylene-thiohydantoin [13],
2-phenylnaphthalenoids and 2-phenylbenzofuranoids [14] have
human topo I and/or II catalytic inhibitory activity. In our previous
studies, conformationally constrained 2-hydroxyphenyl-4-aryl-5H-
indeno[1,2-b]pyridines (aryl ¼ 2- or 3-furyl or 2- or 3-thienyl)
(Fig. 1a) showed dual topo I and II inhibition [15]. In addition, subtle
modification in the position of hydroxyl group on the phenyl ring
and aryl group attached to central pyridine altered the mechanism
of action of compounds, for instance, compound Y (Fig. 1c) func-
tioned as a topo II poison [16] whereas compound Z (Fig. 1c) was
defined to be a topo II catalytic inhibitor [17]. Compounds con-
taining thienyl or furyl groups at the 4-position of 5H-indeno[1,2-b]
pyridine (Fig. 1b) exhibited strong topo II inhibitory activity [18].
Thus, we put efforts continuously to design and synthesize a new
Despite several rationale strategies for the treatment of cancer,
chemotherapy remains as one of the well-established approach for
cancer therapy [1]. The human DNA topoisomerases consisting of
two subtypes, I (topo I) and II (topo II), have been vitally important
molecular targets for the development of anticancer drugs [2,3].
Human topo II has been considered as more important molecular
target than topo I for designing anticancer agents because of its
ability to cleave both strands of one DNA duplex (G-segment) to
make the other DNA duplex (T-segment) pass through transient
break of G-segment and thus to solve the DNA topological prob-
lems. This simultaneous cleavage function of topo II on DNA double
strand endows topo II to play an essential role for DNA chromosome
condensation and segregation in mitosis [4,5].
Several compounds containing indenopyridine skeleton showed
0
*
Corresponding author.
series of rigid analogs of 2-aryl-4-(4 -hydroxyphenyl)-5H-indeno
[1,2-b]pyridines with strategy described in Fig. 3. Hydroxylated
rigid compounds 8e28 (Fig. 4) were newly synthesized in the
*
* Corresponding author.
E-mail addresses: eslee@yu.ac.kr (E.-S. Lee), ykwon@ewha.ac.kr (Y. Kwon).
Authors are equal contributors.
1
http://dx.doi.org/10.1016/j.ejmech.2016.09.019
223-5234/© 2016 Elsevier Masson SAS. All rights reserved.
0

S. Park et al. / European Journal of Medicinal Chemistry 125 (2017) 14e28
15
Ar = 2- or 3- furyl,
2- or 3-thienyl,
Ar
Ar
4
4
5H-indeno [1,2 b]pyridines
2
2
N
Ar
N
HO
(
Topo II inhibitor)
N
(Dual topo I and II inhibitor)
a) 2-hydroxyphenyl-4-aryl-
b) 2,4-diaryl-5H-indeno
[1,2-b]pyridines
5
H-indeno[1,2-b]pyridines
OH
HO
4
4
2
6
2
6
O
S
N
N
HO
Y
HO
Z
(
Topo II poison)
(Topo II catalytic inhibitor)
c) 2,4,6-trisubstituted pyridines
Fig. 1. Structures of previously synthesized a) 2-hydroxyphenyl-4-aryl-5H-indeno[1,2-b]pyridines, b) 2,4-diaryl-5H-indeno[1,2-b]pyridines, and c) 2,4,6-trisubstituted pyridines.
current study and their biological activities were examined. In
addition, structure-activity relationship study of compounds 8e28
was compared with non-hydroxylated 2-aryl-4-phenyl-5H-indeno
8e10 was reported earlier [12,19]. The synthetic route for com-
pounds 4, 7e28 is outlined in Scheme 1. In the first step (i), 1-
indanone (I) was condensed with aryl aldehydes II (R ¼ a-d) in
the presence of 5% NaOH in ethanol (EtOH) to obtain indanone
intermediates III (R ¼ a-d) using Clasien-Schmidt condensation
[1,2-b]pyridines (Fig. 2, compounds 1e7). The most active com-
pound 16 in in vitro and ex vivo anticancer efficacy was further
carried out for molecular docking study to clarify its mechanism of
action, and turned out to be a novel DNA non-intercalative topo
catalytic inhibitor.
1
reaction [13,20]. In the second step (ii), acetophenones IV (R ¼ e-k)
were refluxed with iodine in pyridine to yield seven pyridinium
1
iodide salts V (R ¼ e-k). In the last step (iii), indanone in-
termediates III (R ¼ a-d) were reacted with pyridinium iodide salts
1
V (R ¼ e-k) in the presence of ammonium acetate in methanol or
2
. Results and discussion
acetic acid using modified Kr o€ hnke synthesis [21,22] to obtain final
compounds 4, 7e28 in the yields of 20e83%. The yields (%), melting
2.1. Design and synthesis
ꢀ
points ( C), purities (%), and retention time of the prepared com-
pounds are listed in Table S1 (Supplementary Data).
Twenty-eight compounds (1e28) in seven different series were
On the basis of the previously reported biological results, the
present investigation was undertaken in order to determine the
effect of thienyl or furyl moieties at the 2-position and phenol
moiety at the 4-position of 5H-indeno[1,2-b]pyridine skeleton
prepared (Fig. 3). Compounds 1e7 (Fig. 2) are non-hydroxylated 2-
aryl-4-phenyl-5H-indeno[1,2-b]pyridines whereas each of com-
pounds 8e28 has a hydroxyl group at ortho, meta or para position of
the 4-phenyl ring (Fig. 4). Synthesis of compounds 1e3, 5, 6, and
Fig. 2. Structures of non-hydroxylated 2-aryl-4-phenyl-5H-indeno[1,2-b]pyridines.

16
S. Park et al. / European Journal of Medicinal Chemistry 125 (2017) 14e28
Fig. 3. Strategy for the design of compounds 2-aryl-4-phenyl or hydroxyphenyl-5H-indeno[1,2-b]pyridines.
0
Fig. 4. Structures of prepared 2-aryl-4-(4 -hydroxyphenyl)-5H-indeno[1,2-b]pyridines.

S. Park et al. / European Journal of Medicinal Chemistry 125 (2017) 14e28
17
1
Scheme 1. General synthetic scheme of indanone intermediates III (R ¼ a-d), pyridinium iodide salts V (R ¼ e-k), and 2-aryl-4-hydroxyphenyl-5H-indeno[1,2-b]pyridines (4,
ꢀ
7
e28); Reagents and conditions: i) aq. NaOH (0.5 g in 10 ml), EtOH, 1e12 h, room temperature, 62e97% yield; ii) iodine (1.0 equiv.), pyridine (15.0 equiv.), 3 h, 140 C, 38e90% yield;
ꢀ
iii) NH
4
OAc (10.0 equiv.), glacial acetic acid or methanol, 24e36 h, 100e110 C, 20e83% yield.
(
compounds 8e19). In addition, nine compounds 20e28 containing
respectively. After agarose gel electrophoresis of reaction product,
the gel was stained in ethidium bromide containing solution then
the extent of remained relaxed DNA was quantitated. As shown in
Fig. 5 and Table 1, compounds 4 and 7 possessing no hydroxy in 4-
pyridyl moiety at the 2-position were additionally included for the
evaluation of biological activity to perform the SAR study.
phenyl ring had almost no inhibition on both topo I and II
00 M (less than 6.5%). Most of the hydroxylated compounds with
furyl or thienyl moiety (8e19) at the 2-position, belonging to series
e4 (Fig. 3), inhibited topo II much strongly than topo I except for
compounds 14, 16, and 17. Compounds 14 and 16 showed potent
dual topo I and II inhibitory activity at both concentrations of
00 M and 20 M. Compound 14 inhibited topo I by 62.4% and
7.1% and inhibited topo II by 58.7% and 20.0% at 100 M and
M, respectively. While the percent inhibition of compound 16
M and 20 M,
a at
2
2
.2. Biological evaluation
1
m
.2.1. Topo I and topo II
The effect of prepared compounds on recombinant topo I and
topo II was evaluated by measuring the extent of those enzyme-
a inhibitory activity
1
a
a
a
mediated relaxed DNA remained after treatment of each com-
pound (Figs. 5 and 6). Inhibitory activities were first evaluated at
1
2
2
m
m
a
m
1
00
than 30% at 100
etoposide were used as positive controls for topo I and II,
mM and compound of which the percent inhibition was more
0
m
mM was further tested at 20 M. Camptothecin and
m
against topo I are 69.7% and 53.5% at 100
m
m
Fig. 5. Topoisomerase I inhibitory activities of prepared compounds 4, 7e28. Lane D: pBR322 only, Lane T: pBR322 þ Topo I, Lane C: pBR322 þ Topo I þ Camptothecin, and (A) Lane
, 7e28: pBR322 þ Topo I þ 100 M of each of compounds 4, 7e28. (B) Lane 12e14, 16: pBR322 þ Topo I þ 20 M of each of compounds 12e14, 16.
4
m
m

18
S. Park et al. / European Journal of Medicinal Chemistry 125 (2017) 14e28
Fig. 6. Topoisomerase II
a
inhibitory activities of prepared compounds 4, 7e28. Lane D: pBR322 only, Lane T: pBR322 þ Topo II
a
, Lane E: pBR322 þ Topo II
a
þ Etoposide, and (A)
Lane 4, 7e28: pBR322 þ Topo II
a
þ 100
m
M of each of compounds 4, 7e28. (B) Lane 9e16, 18, 19, 23, 26, 28: pBR322 þ Topo II
a
þ 20 M of each of compounds 9e16, 18, 19, 23, 26,
m
28.
Table 1
Topo I and II
a
inhibitory activities, and antiproliferative activity of the prepared compounds 4, 7e28.
Topo I (% inhibition) Topo II (% inhibition) (m
M)b
Compounds
a
IC50
HCT15
100
mM
20
m
M
100
mM
20
m
M
T47D
DU145
HeLa
Adriamycin
Etoposide
Camptothecin
4
7
8
9
e
e
e
e
e
e
1.23 ± 0.001
2.82 ± 0.24
18.87 ± 0.34
7.05 ± 0.11
16.35 ± 0.27
>50
2.81 ± 0.14
5.19 ± 0.15
>50
2.49 ± 0.05
2.45 ± 0.03
>50
1.58 ± 0.07
3.12 ± 0.04
>50
1.24 ± 0.06
1.83 ± 0.02
>50
>50
>50
3.52 ± 0.05
1.87 ± 0.09
>50
2.18 ± 0.05
1.94 ± 0.06
>50
1.34 ± 0.03
1.84 ± 0.44
13.7 ± 0.81
17.41 ± 0.55
>50
0.52 ± 0.28
0.02 ± 0.001
2.09 ± 0.11
1.09 ± 0.02
>50
0.88 ± 0.08
0.18 ± 0.02
7.32 ± 0.15
10.83 ± 0.74
>50
a
a
61.5/80.1
e
20.1/32.3
e
a
58.3/67.5
6.4
0.0
21.3
e
c
3.9
2.3
e
e
e
24.3
22.3
15.7
12.8
33.1
45.5
62.4
28.9
69.7
0.4
4.5
1.4
0.5
2.4
0.0
0.0
1.5
0.0
e
22.6
75.6
71.9
50.2
75.5
74.8
58.7
77.5
95.1
1.8
82.2
61.4
15.2
3.5
14.8
65.5
18.5
4.0
e
>50
>50
>50
e
49.1
15.1
4.9
22.0
65.9
20.0
36.9
92.2
e
1.43 ± 0.05
6.53 ± 0.05
>50
1.52 ± 0.07
4.52 ± 0.05
>50
1.07 ± 0.03
0.95 ± 0.03
>50
2.91 ± 0.06
2.60 ± 0.01
29.62 ± 2.96
>50
4.49 ± 0.02
4.23 ± 0.02
>50
3.12 ± 0.09
3.35 ± 0.02
>50
3.75 ± 0.06
3.74 ± 0.01
>50
1.51 ± 0.01
6.01 ± 0.02
>50
4.10 ± 0.10
3.94 ± 0.04
>50
1.74 ± 0.43
4.90 ± 0.12
>50
6.27 ± 0.07
3.96 ± 0.19
>50
4.48 ± 0.02
5.21 ± 0.16
>50
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
e
e
0.0
2.5
27.1
e
53.5
e
e
29.6
28.0
e
e
e
e
e
>50
>50
>50
>50
e
e
>50
e
10.5
e
5.15 ± 0.04
1.17 ± 0
>50
2.44 ± 0.01
1.05 ± 0.03
>50
7.46 ± 0.21
2.80 ± 0.02
>50
4.70 ± 0.07
4.36 ± 0.02
>50
7.80 ± 0.12
5.19 ± 0.01
>50
5.78 ± 0.02
2.41 ± 0.16
>50
e
e
e
0.0
1.3
0.0
e
75.2
49.5
1.14
0.9
e
e
e
e
HCT15: human colorectal adenocarcinoma; T47D: Human breast ductal carcinoma; DU145: human prostate tumor; HeLa: human cervix adenocarcinoma cell line; Adria-
mycin: positive control for antiproliferative activity; Etoposide: positive control for topo II
antiproliferative activity.
a and antiproliferative activity; Camptothecin: positive control for topo I and
For compounds 8e10, topo I and II
a
inhibitory and antiproliferative activity results reported earlier [26].
a
Control value for compounds 4, 7, and 17e28.
b
c
Each data represents mean ± S.D. from three different experiments performed in triplicate.
Not determined.

S. Park et al. / European Journal of Medicinal Chemistry 125 (2017) 14e28
19
respectively and those against topo II
pound 16 showed the strongest topo II
concentrations of 100 M and 20
a
a
are 95.1% and 92.2%. Com-
inhibitory activity at both
Table 3
The cytotoxicity of compound 16 against normal mammalian cells.
m
mM among all the prepared
Compounds
IC50 (mM)
compounds. Compound 17 showed no inhibitory activity for both
enzymes. All of the compounds 20e28 possessing pyridyl moiety at
the 2-position, belonging to series 5e7 (Fig. 3), did not inhibit topo I
HEK293
HFL-1
Adriamycin
Etoposide
Camptothecin
16
4.96 ± 0.001
1.74 ± 0.05
0.83 ± 0.02
3.47 ± 0.06
2.31 ± 0.01
87.63 ± 9.29
1.06 ± 0.001
7.99 ± 0.14
nor II
3, 15, 16, 18, 19 possessing 2-thienyl or 2-furyl ring in combination
with meta- or para-hydroxy-4-phenyl ring showed greater or
comparable topo II inhibitory activity at both 100 M and 20
than etoposide (61.5% at 100 M and 20.1% at 20 M) (Fig. 6 and
a except for compounds 23, 26, and 27. Compounds 9, 10, 12,
1
HEK293: Derived from human healthy embryonic kidney cell grown in tissue cul-
ture; HFL-1: Human normal embryonic lung cell.
a
m
mM
m
m
Table 2). Compounds 23 (65.5%) and 26 (75.2%) possessing 2-
pyridyl ring in combination with ortho-hydroxy-4-phenyl ring
addition, topo IIa enables to transiently cleave both strands of a
DNA duplex resulted in decatenation which is a requirement pro-
cess for progression toward mitosis [25]. There are two types of
showed stronger topo II
a inhibitory activity at 100 mM than eto-
poside without topo I inhibition.
topo IIa inhibitors; one is to stabilize transiently formed topo IIa-
DNA complex leading to blockage of the DNA double strand breaks
from being religated. Then undesired truncated DNAs are accu-
mulated, which causes a severe genetic toxicity. By this reason, the
2
.2.2. Antiproliferative activity of compounds
Antiproliferative potency of all the synthesized compounds was
evaluated using HCT15 (human colorectal adenocarcinoma cell
line), T47D (human breast ductal carcinoma cell line), DU145 (hu-
man prostate tumor cell line), and HeLa (human cervix adenocar-
cinoma cell line). The antiproliferative activities of compounds 4
and 7e28 against used cancer cells are listed as the IC50 values (
in Table 2. Compounds 9, 10, 12, 13, 15, 16, 18, 19, 23, 26, and 27
possessing significant topo II inhibitory activity showed similar or
stronger antiproliferative activities compared to adriamycin, eto-
poside, and camptothecin. This result demonstrates the propensity
that the effective inhibition on topo IIa mediated by compound is
topo II
named topo II
itor, is to inhibit any another step of topo II
blocking the religation step. Thus, the current trend is more
focusing on developing topo II catalytic inhibitor as an anticancer
agent [17,26e29]. Therefore, the mechanism of action of compound
6 was inspected whether to act as a topo II poison or topo II
catalytic inhibitor. First, topo II -DNA cleavable complex assay was
performed. As shown in Fig. 7A etoposide, a well-known topo II
poison, induced the linear truncated DNA but compound 16 did not
generate linear form even at much higher concentration (500 M).
It is evident that the compound 16 functioned as a topo II catalytic
inhibitor not a topo II poison. Second, DNA intercalating character
of compound 16 was tested with DNA unwinding assay. Compound
6 did not unwind DNA in the presence of excess amount of topo I.
a
inhibitors stabilizing the cleavable DNA-topo II
poison. The other, called a topo II catalytic inhib-
catalytic cycle besides
a complex
a
a
a
mM)
a
a
1
a
a
a
a
positively correlated with its antiproliferative activities. Only
compounds 24 showed certain cytotoxicity against used cancer
cells but no inhibitory activity against topo I and IIa.
m
a
a
2
.2.3. Study on mechanism of action of compound 16
Compound 16 exhibited strongest potency as dual topo I and II
1
a
While amsacrine, an eminent DNA intercalating topo II poison,
inhibited topo I-mediated DNA unwinding in dose dependent
manner, which is consistent with the previous report (Fig. 7B)
inhibitor with significant tumor cell growth inhibition as listed in
Table 2. First of all, the cytotoxicity against a healthy mammalian
cell lines of the most active compound 16 was checked using pos-
itive controls, HEK293 (derived from human healthy embryonic
kidney cell grown in tissue culture) and HFL-1 (human embryonic
lung cell). As shown in Table 3, compound 16 inhibited HEK293 cell
with less potency than etoposide and camptothecin. While the
cytotoxic efficacy against HFL-1 cell of compound 16 was much
stronger than that of etoposide, and weaker than those of adria-
mycin and camptothecin. Especially at low concentration, com-
[
(
28,29]. The high concentration treatment of compound 16
500 M) in the last lane of Fig. 7A showed the supercoiled DNA
M). It might be
m
unlike low concentration treatment (10 or 50
m
explained that compound 16 blocked enzymatic activity of topo II
a
at high concentration by interfering DNA binding to topo II
a
through either induction of tertiary conformational change in
enzyme or DNA leading to blockage of the relaxation process before
starting of catalytic cycle of topo IIa. Compound 16 can be further
analogized as a topo I catalytic inhibitor not a topo I poison based
on its functions such as not inducing linear DNA and no inter-
pound 16 has the selectivity for topo II
overexpressed in highly proliferative cancer cells and the expres-
sion level of topo II is in accordance with cell cycle of cancer cells;
a over topo I. Since topo IIa is
a
calating. Third, since compound 16 catalytically inhibited topo II
a
it is highest in the late S and G2/M phase of cell cycle [23,24]. In
Table 2
Topo I and II
a
inhibitory activities, and antiproliferative activity of previously synthesized compounds 1e3, 5, 6 [25].
Topo I (% inhibition) Topo II (% inhibition)
M)c
20 HCT15
Compounds
a
IC50 (m
100
m
M
20
mM
100
m
M
mM
DU145
0.95 ± 1.05 /1.0 ± 0.14
HeLa
a
b
b
b
a
b
a
b
b
b
Adriamycin
Etoposide
Camptothecin
1
2
3
5
6
1.08 ± 0.10 /1.21 ± 0.02
1.20 ± 0.20 /1.00 ± 0.07
1.26 ± 0.03 /1.40 ± 0.15
1.30 ± 0.14 /0.40 ± 0.13
>50
>50
16.60 ± 1.22
a
b
46.7 /20.2b
e
a
a
a
b
b
a
e
e
87.8 /32.4
e
2.6 ± 1.681 /1.10 ± 0.02
13.1 ± 1.71 /0.50 ± 0.03
a
b
a
b
a
a
a
62.9 /45.2
56.0
72.5
18.7
12.3
19.6 /25.9
0.0
0.31 ± 0.09 /0.50 ± 0.06
0.11 ± 0.05 /0.42 ± 0.16
5.8
0.5
41.3
0.0
ND
e
>50
>50
>50
e
>50
>50
7.74 ± 0.85
e
0.0
e
d
-
5.7
e
e
e
ND
e
e
e
e
e
a
Control value for previously reported compounds 1e2.
Control value for previously reported compounds 3, 5, 6.
Each data represents mean ± S.D. from three different experiments performed in triplicate.
b
c
d
Not determined.

20
S. Park et al. / European Journal of Medicinal Chemistry 125 (2017) 14e28
Fig. 7. Mechanism of action of compound 16 was determined by systematic assays including (A) DNA-topo II
and (C) topo II ATPase assay. Compound 16 functions as a DNA non-intercalative topo catalytic inhibitor.
a cleavable complex assay, (B) topo I-mediated DNA unwinding assay
a
without intercalation into DNA, it was necessitated to evaluate
whether to inhibit ATPase activity of topo II . As detected in Fig. 7C,
compound 16 did not inhibit ATPase activity and ATP hydrolysis of
topo II unlike novobiocin which is reported to hinder topo II cat-
showed weak topo IIa inhibitory activity and less cytotoxic than the
a
hydroxylated compounds as shown in Tables 1 and 2. Among the
hydroxylated compounds, 2- or 3-thienyl compounds (9, 10, 12, and
13) and 2- or 3-furyl compounds (15, 16, 18, and 19) have a pref-
erence with meta- or para-hydroxy-4-phenyl ring for strong topo
a
alytic activity through inhibiting ATPase activity of topo II [30].
Taken together, compound 16 may work on the other catalytic steps
IIa inhibition and noticeable cytotoxicity over ortho-hydroxy-4-
of topo II
catalytic inhibitor.
a
reaction cycle besides its religation step as a topo II
a
phenyl ring (8, 11, 14 and 17). However, 2-, 3- or 4-pyridyl com-
pounds 20e28 showed the different trend from compounds 8e19.
2-Pyridyl compounds 20e22 are almost inactive. 3- or 4-Pyridyl
2
.2.4. Effect of compound 16 on endogenous topoisomerase
The effect of compound 16 was finally evaluated against
endogenous topo in T47D human breast tumor cells. After T47D
cells were treated with 20 and 50 M of compound 16 followed by
compounds with ortho-hydroxy-4-phenyl ring (23 and 26)
showed better potency than compounds 24, 25, 27, and 28 con-
taining meta- or para-hydroxy-4-phenyl ring. The preference for
positioning of hydroxy group in 4-phenyl rings of thienyl (9, 10, 12,
and 13) and furyl compounds (15, 16, 18, and 19) differs from that of
pyridyl compounds (23 and 26). However, introduction of hydroxy
m
nuclear extraction, the each of prepared nuclear extract was reacted
with the supercoiled pBR322 DNA. Compound 16 attenuated the
intracellular topo activity by 34.3% and 97.0% inhibition at 20 and
to 4-phenyl ring of 5H-indeno[1,2-b]pyridines enhanced topo II
a
5
0
m
M treatments, respectively. While etoposide almost did not
inhibit the intracellular topo at 20 M and inhibited it by 61.1% at
M which are much lower than compound 16 (Fig. 8). This result
is consistent what observed in cell free relaxation assay system
inhibitory activity and cytotoxicity which is similar tendency to
previous results [31].
m
50 m
2.3. Molecular docking study
compound 16 inhibited both recombinant topo I and IIa with much
stronger activity than camptothecin and etoposide.
The biological studies clarified that compound 16 potently
inhibited both topo I and topo IIa (Figs. 5 and 6 and Table 2) but it
2
.2.5. Structure-activity relationship (SAR) studies
Most of all the synthesized compounds inhibited recombinant
topo II with stronger intensity than topo I, thus SAR of compounds
e28 was studied based on the extent of inhibition on both topo II
did not block DNA from re-ligation (Fig. 7A). In addition, the com-
pound 16 neither intercalated into DNA (Fig. 7B) nor inhibited topo
II ATP hydrolysis (Fig. 7C). Therefore, docking studies were carried
out to inspect the mechanism of action of compound 16 with topo I
a
1
a
and cell proliferation. The non-hydroxylated compounds (1e7)
and topo II
a
. Fig. 9 shows the compound 16 binding site of topo I
(Fig. 9B). First, analysis of interaction between
(Fig. 9A) and topo II
a
topo I and compound 16 showed that it interacts with single-strand
cleavage site. Especially, the oxygen of furyl group of compound 16
has hydrogen bonding interaction with Arg364. This is an impor-
tant residue where it also has interactions with other inhibitors
such as camptothecin, indolocarbazole, and indenoisoquinoline
[32]. Compound 16 also bound to the topo IIa DNA cleavage site
where etoposide is positioned. It had interaction with Lys456 and
Asp479 with the hydroxyl group of the phenyl ring. The hydrogen
bonding interaction of Asp479 is also observed in the hydroxyl
group of E ring of etoposide [33]. The 5H-indeno [1,2-b]pyridine
moiety of compound 16 overlaps with the polycyclic aglycone core
of etoposide. The total Surflex-Dock scores of compound 16 for topo
I and topo IIa were 8.9012 and 6.7948, respectively while that of
camptothecin was 10.3861 and that of etoposide 11.7508. The much
higher Surflex-Dock scores meant that camptothecin or etoposide
interacted very strongly with the nicked DNA, leading to its
Fig. 8. DNA non-intercalative topo catalytic inhibition of compound 16 attenuated
endogenouse topo-mediated DNA relaxation with better potency than etoposide.

S. Park et al. / European Journal of Medicinal Chemistry 125 (2017) 14e28
21
Fig. 9. Binding mode of compound 16. (A) Binding of compound 16 with topo I and (B) topo IIa. Compound 16 and the interacting residues of the receptor are shown in sticks
colored by atom types except for carbon atoms of compound 16 are colored in orange. The DNA phosphate backbone and bases are shown in cartoons in yellow. The hydrogen
bonding are represented in yellow dashed lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
function as a topo I or topo II poison. Merbarone under clinical trial
is a non-intercalative topo II catalytic inhibitor through binding to
the topo II active cleavage site including Tyr805 which resulted in
inhibition of DNA cleavage. The Tyr805 residue of topo II is required
for DNA cutting and religation. Etoposide also interacts with
Tyr805. Merbarone induces neither changes on DNA-topo II com-
plex nor binding to DNA [10,34]. From these binding modes and
diverse assay results, it can be suggested that similar to merbarone,
compound 16 could not intercalate into regularly stacked base-
pairs of DNA duplex but could interact with topo-bound DNA
without causing changes on topo-DNA complex and then could
designing novel effective anticancer agents by substituting other
functionalities at rigid 5H-indeno[1,2-b]pyridine skeleton.
4. Experimental
Compounds used as starting materials and reagents were ob-
tained from Aldrich Chemical Co., Junsei or other chemical com-
panies, and used without further purification. HPLC grade
acetonitrile (ACN) and methanol were purchased from Burdick and
Jackson, USA. Thin-layer chromatography (TLC) and column chro-
matography (CC) were performed with Kieselgel 60 F254 (Merck)
and silica gel (Kieselgel 60, 230e400 mesh, Merck) respectively.
Since all the prepared compounds contained aromatic ring, they
were visualized and detected on TLC plates with UV light (short
wave, long wave or both). NMR spectra were recorded on a Bruker
inhibit catalytic activity of both topo I and topo II
DNA religation.
a without blocking
3
. Conclusion
A total of twenty-eight new rigid analogs of 5H-indeno[1,2-b]
1
13
AMX 250 (250 MHz, FT) for H NMR and 62.5 MHz for C NMR, and
chemical shifts were calibrated according to TMS. Chemical shifts
pyridine substituted with thienyl, furyl, or pyridyl at the 2-position
and phenyl or phenolic moiety at the 4-position were prepared by
modified Kr o€ hnke synthesis. The biological activity of these newly
(d) were recorded in ppm and coupling constants (J) in hertz (Hz).
Melting points were determined in open capillary tubes on elec-
trothermal 1A 9100 digital melting point apparatus and were
uncorrected.
synthesized compounds was verified using recombinant human
DNA topo I and II
DU145, and HeLa). Among tested compounds, compound 16
inhibited most strongly both topo I and II with comparable or
a
, and human cancer cell lines (HCT15, T47D,
HPLC analyses were performed using two Shimadzu LC-10AT
pumps gradient-controlled HPLC system equipped with Shimadzu
system controller (SCL-10A VP) and photo diode array detector
a
stronger cytotoxicity in compared with positive controls. The
structure-activity relationship study showed that compounds 9, 10,
(SPD-M10A VP) using Shimadzu Class VP program. Sample volume
®
of 10
m
L was injected in Waters X-Terra
5
mM reverse-phase C18
1
2, 13, 15, 16, 18 and 19 containing 2- or 3-thienyl and 2- or 3-furyl
column (4.6ⅹ250 mm) with a gradient elution of 70%e100% of B in
A for 10 min followed by 100%e70% of B in A for 20 min at a flow
rate of 0.5 mL/min at 254 nm UV detection, where mobile phase A
was solution (20 mM) of ammonium formate (AF) in doubly
distilled water and B was 100% acetonitrile (ACN). Purity of com-
pounds was described as percent (%). ESI LC/MS analyses were
moiety at 2-position with and hydroxyl group at meta or para po-
sitions of the 4-phenyl ring are important for exhibiting potent topo
II
mechanism of action of compound 16, the most potent topo II
inhibitor with significant antiproliferative activity, was further
evaluated with various assays such as topo II -DNA cleavable
complex, topo II -mediated ATPase assays, topo I-mediated DNA
unwinding, in vitro topo I and topo II relaxation, and ex vivo topo
a inhibitory activity and significant antiproliferative activity. The
a
®
a
performed with a Finnigan LCQ Advantage LC/MS/MS spectrom-
a
etry utilizing Xcalibur® program. For ESI LC/MS, LC was performed
®
a
with an 8
mL injection volume on a Waters Atlantis
T
3
reverse-
relaxation assays. Compound 16 was determined as a non-
intercalative topo catalytic inhibitor with better potency than eto-
poside in T47D cells. Molecular docking study of compound 16 with
phase C18 column (2.1ⅹ 50 mm, 3
from; (A) 50%e90% of B in A for 4 min and remained linear 90% of B
in A for 1 min followed by 90%e50% of B in A for 3 min and
mm) with a gradient elution
topo I and topo II
a
revealed that the binding sites of compound 16
remained linear 50% of B in A for 7 min at flow rate of 200 mL/min;
partially overlapped with those of camptothecin and etoposide,
respectively. However the total binding scores of compound 16
were much lower than those of camptothecin and etoposide. The
new information obtained from these efforts provide a basis for
(B) 50%e90% of B in A for 5 min followed by 90%e50% of B in A for
5 min and remained linear 50% of B in A for 5 min at flow rate of
200
mL/min, where mobile phase A was solution (20 mM) of AF in
distilled water and mobile phase B was ACN. MS ionization

22
S. Park et al. / European Journal of Medicinal Chemistry 125 (2017) 14e28
conditions were: Sheath gas flow rate: 40 arb, aux gas flow rate:
13C NMR (62.5 MHz, DMSO-d
) d 193.78, 164.81, 150.25, 137.67,
6
135.73, 135.18, 134.91, 133.92, 129.81, 127.88, 126.93, 123.75, 119.91,
119.11, 117.93. 32.25.
ꢀ
0
arb, I spray voltage: 5.3 KV, capillary temperature: 275 C,
capillary voltage: 27 V, tube lens offset: 45 V. Retention time was
given in minutes.
4.1.4. Synthesis of 2-(4-hydroxybenzylidene)-2, 3-dihydro-1H-
4.1. General method for preparation of III (R ¼ a-d)
inden-1-one (IIId)
The procedure described in Section 4.1 was employed with 1-
indanone (I, 2.64 g, 20 mmol) and 4-hydroxybenzaldehyde (IId,
2.44 g, 20 mmol) to yield 2.93 g (12.39 mmol, 62%) as a light
1
-Indanone (I) was added in ethanol followed by the addition of
equivalent amount of aryl aldehydes II (R ¼ a-d). The 5% aqueous
solution of NaOH was added drop wise to the mixture at room
temperature which resulted in precipitation. The mixture was then
cooled for 30 min, filtered, washed with cold methanol, and dried
to yield 62e97% solid compounds. In those reactions where no
precipitation occurred, the reaction mixtures were extracted with
ethyl acetate and washed with water and saturated solution of NaCl
greenish solid.
ꢀ
TLC (ethyl acetate/n-hexane ¼ 1:2) R
f
¼ 0.3, mp 235.4e236.0 C.
1
6
H NMR (250 MHz, DMSO-d ) d 10.13 (s, 1H, phenyl 4-OH), 7.75
(d, J ¼ 7.27 Hz, 1H, indeno H-7), 7.67e7.60 (m, 4H, phenyl H-2, H-6,
indeno H-4, H-5), 7.46e7.42 (m, 2H, ]CHe, indeno H-6), 6.88 (d,
J ¼ 8.35 Hz, 2H, phenyl H-3, H-5), 4.04 (s, 2H, indeno H-3).
13
(
brine). Crude products were further purified by column chroma-
6
C NMR (62.5 MHz, DMSO-d ) d 194.11, 160.30, 150.66, 138.45,
tography. Four different indanone intermediates III (R ¼ a-d) were
135.36, 134.22, 133.87 (2C), 132.40, 128.45, 127.47, 126.85, 124.28,
116.90 (2C), 32.83.
synthesized following the same method.
1
4
(
.1.1. Synthesis of 2-Benzylidene-2,3-dihydro-1H-inden-1-one
IIIa)
The procedure described in Section 4.1 was employed with 1-
indanone (I, 2.64 g, 20 mmol) and benzaldehyde (IIa, 2.03 mL,
0 mmol) to yield 4.29 g (19.46 mmol, 97%) as an off-white solid.
4.2. General method for the preparation of V (R ¼ e-k)
A mixture of equivalent amounts of acetophenones and iodine
in pyridine (15.0 equiv) was refluxed at 140 C for 3 h. After cooling
the mixture to room temperature, the precipitate was filtered and
ꢀ
2
TLC (ethyl acetate/n-hexane
¼
1:10)
R
f
¼
0.27, mp
washed with cold pyridine followed by drying overnight to yield
38e90% of V (R ¼ e-k).
ꢀ
1
1
32.1e133.2 C.
1
H NMR (250 MHz, CDCl
3
)
d
7.9 (d, J ¼ 7.6 Hz, 1H, indeno H-7),
7
.68e7.64 (m, 3H, phenyl H-2, H-6, indeno H-5), 7.61e7.53 (m, 2H,
4.3. General method for the preparation of 4, 7e28
indeno H-4, ¼CH-), 7.49e7.36 (m, 4H, indeno H-6, phenyl H-3, H-4,
H-5), 4.04 (s, 2H, indeno H-3).
Anhydrous ammonium acetate (10.0 equiv.) was mixed with
glacial acetic acid or methanol followed by addition of the indanone
intermediates (1.0 equiv.), III (R ¼ a-d) and pyridinium iodide salts
13
3
C NMR (62.5 MHz, CDCl ) d 194.23, 145.61, 138.04, 135.43,
134.76, 134.57, 133.88, 130.68, 129.61, 128.90, 127.65, 126.14, 124.43,
1
3
2.42.
V (R ¼ e-k) (1.5 equiv.). The reaction mixture was then refluxed at
ꢀ
1
00e110 C for 24e36 h followed by extraction with ethyl acetate
4
.1.2. Synthesis of 2-(2-hydroxybenzylidene)-2,3-dihydro-1H-
and washing with water and brine solution. The organic layer was
dried with magnesium sulfate and filtered. The filtrate was evap-
orated at reduced pressure, which was purified with silica gel col-
umn chromatography with the gradient elution of ethyl acetate/n-
hexane to afford solid compounds 4, 7e28 in 20e83% yields.
inden-1-one (IIIb)
The procedure described in Section 4.1 was employed with 1-
indanone (I, 3.96 g, 30 mmol) and salicylaldehyde (IIb, 3.14 mL,
3
0 mmol) to yield 6.85 g (28.99 mmol, 97%) as a dark-red solid.
TLC (ethyl acetate/n-hexane
03.4e204.9 C.
¼
1:3)
R
f
¼
0.23, mp
ꢀ
2
4.3.1. Synthesis of 2-(furan-3-yl)-4-phenyl-5H-indeno[1,2-b]
pyridine (4)
1H NMR (250 MHz, DMSO-d
)
d
8.27 (s, 1H, ¼CH-), 7.69 (d,
J ¼ 7.55 Hz, 1H, indeno H-7), 7.61e7.56 (m, 2H, indeno H-4, H-5),
.48 (d, J ¼ 7.82 Hz,1H, phenyl H-6), 7.41 (t, J ¼ 5.4 Hz,1H, indeno H-
6
The same procedure described in section 4.3 was employed with
IIIa (0.33 g, 1.5 mmol), anhydrous ammonium acetate (1.15 g,
7
6
ꢀ
), 6.93 (t, J ¼ 7.35 Hz, 1H, phenyl H-4), 6.45 (d, J ¼ 8.5 Hz, 1H,
15 mmol), Vh (0.63 g, 2 mmol) and methanol (8 mL) at 100 C for
phenyl H-3), 6.20 (t, J ¼ 7.25 Hz, 1H, phenyl H-5), 3.93 (s, 2H, indeno
24 h to yield 196 mg (0.63 mmol, 42%) as a white solid.
ꢀ
H-3).
TLC (ethyl acetate/n-hexane ¼ 1:5) R
f
¼ 0.3, mp: 144.4e145.9 C,
13C NMR (62.5 MHz, DMSO-d
)
d
193.72, 172.46, 150.09, 139.72,
34.23, 134.01, 132.80, 130.65, 127.93, 127.29, 127.14, 123.60, 123.47,
21.66, 111.56, 33.30.
HPLC: Retention time: 7.52 min, purity: 96.5%; ESI LC/MS (condition
6
þ
1
1
1
A): m/z calcd for C22
(250 MHz, DMSO-d
H
15NO [MH] 310.12; found 310.28. H NMR
6
)
d
8.49 (s, 1H, 2-furyl H-2), 8.06 (d, J ¼ 8.35 Hz,
1H, indenopyridine H-9), 7.85e7.80 (m, 3H, 4-phenyl H-2, H-6, 2-
4.1.3. Synthesis of 2-(3-hydroxybenzylidene)-2, 3-dihydro-1H-
furyl H-4), 7.73 (s, 1H, indenopyridine H-3), 7.66e7.45 (m, 6H,
inden-1-one (IIIc)
The procedure described in Section 4.1 was employed with 1-
indanone (I, 3.96 g, 30 mmol) and 3-hydroxybenzaldehyde (IIc,
indenopyridine H-6, H-7, H-8, 4-phenyl H-3, H-4, H-5), 7.24 (br, 1H,
13
2-furyl H-5), 4.04 (s, 2H, indneopyridine H-5). C NMR (62.5 MHz,
DMSO-d 160.19, 150.82, 145.93, 144.45, 144.40, 142.01, 140.32,
6
) d
3
.66 g, 30 mmol) to yield 4.81 g (20.33 mmol, 68%) as an orange
137.91, 132.45, 128.98 (2C), 128.89 (2C), 128.52 (2C), 127.20, 127.16,
125.53, 120.65, 117.46, 109.22, 34.19.
solid.
TLC (ethyl acetate/n-hexane
¼
1:3)
R
f
¼
0.21, mp
ꢀ
2
64.8e265.4 C.
4.3.2. Synthesis of 4-phenyl-2-(pyridin-4-yl)-5H-indeno[1,2-b]
pyridine (7)
1H NMR (250 MHz, DMSO-d
d
7.72 (d, J ¼ 7.55 Hz, 1H, indeno
6
)
H-7), 7.63 (t, J ¼ 7.50 Hz, 1H, indeno H-5), 7.56 (d, J ¼ 7.75 Hz, 1H,
indeno H-4) 7.42 (t, J ¼ 7.32 Hz, 1H, indeno H-6), 7.35 (s, 1H, ¼CH-),
The same procedure described in section 4.3 was employed with
IIIa (0.33 g, 1.5 mmol), anhydrous ammonium acetate (1.15 g,
ꢀ
7
.12 (t, J ¼ 7.72 Hz, 1H, phenyl H-5), 7.01 (s, 1H, phenyl H-2), 6.82 (d,
15 mmol), Vk (0.81 g, 2.5 mmol) and methanol (8 mL) at 100 C for
J ¼ 7.45 Hz, 1H, phenyl H-4), 6.69 (d, J ¼ 7.85 Hz, 1H, phenyl H-6),
24 h to yield 156 mg (0.48 mmol, 33%) as a white solid.
3
.88 (s, 2H, indeno H-3).
TLC (ethyl acetate/n-hexane
¼
1:1)
R
f
¼
0.26, mp:

S. Park et al. / European Journal of Medicinal Chemistry 125 (2017) 14e28
23
ꢀ
1
82.7e183.5 C, HPLC: Retention time: 7.68 min, purity: 99.3%; ESI
8.01e7.98 (m, 1H, indenopyridine H-9), 7.93 (d, J ¼ 3.0 Hz, 1H, 2-
thiophene H-3), 7.83 (s,1H, indenopyridine H-3), 7.71 (d,
J ¼ 8.52 Hz, 2H, 4-phenyl H-2, H-6), 7.63e7.62 (m, 2H, 2-thiophene
H-5 and indenopyridine H-6), 7.47e7.45 (m, 2H, indenopyridine H-
7, H-8), 7.17 (t, J ¼ 3.77 Hz, 1H, 2-thiophene H-4), 6.95 (d,
J ¼ 8.55 Hz, 2H, 4-phenyl H-3, H-5), 4.09 (s, 2H, indenopyridine H-
þ
LC/MS (condition A): m/z calcd for C23
21.40. H NMR (250 MHz, DMSO-d
H
16
N
2
[MH] 321.14; found
1
3
6
)
d
8.73 (d, J ¼ 6.02 Hz, 2H, 2-
pyrisyl H-2, H-6), 8.28 (d, J ¼ 6.05 Hz, 2H, 2-pryridyl H-3, H-5), 8.14
(
d, J ¼ 8.45 Hz, 1H, indneopyridine H-9), 8.09 (s, 1H, indenopyridine
H-3), 7.91 (d, J ¼ 6.65 Hz, 2H, 4-phenyl H-2, H-6), 7.67 (d,
J ¼ 8.02 Hz, 1H, indenopyridine H-6), 7.63e7.49 (m, 5H, indneo-
pyridine H-7, H-8, 4-phenyl H-3, H-4, H-5), 4.19 (s, 2H, inden-
13
6
5). C NMR (62.5 MHz, DMSO-d ) d 160.83, 159.14, 152.32, 146.81,
145.82, 145.31, 140.90, 137.53, 133.21, 130.77 (2C), 129.74, 129.26,
129.05, 128.04, 126.30, 126.19, 121.40, 116.60 (2C), 116.27, 35.6.
opyridine H-5). 13C NMR (62.5 MHz, DMSO-d
)
d
160.77, 153.15,
50.34 (2C), 146.27, 145.85, 144.58, 139.97, 137.62, 134.91, 129.26,
29.04, 128.98 (2C), 128.62 (2C), 127.32, 125.59, 121.08 (2C), 120.79,
18.44, 34.29.
6
1
1
1
4.3.6. Synthesis of 2-(2-(thiophen-3-yl)-5H-indeno[1,2-b]pyridin-
4-yl)phenol (11)
The same procedure described in section 4.3 was employed with
IIIb (0.71 g, 3 mmol), anhydrous ammonium acetate (2.31 g,
30 mmol), Vf (1.49 g, 4.5 mmol) and methanol (20 mL) at 100 C for
4
4
.3.3. Synthesis of 2-(2-(thiophen-2-yl)-5H-indeno[1,2-b]pyridin-
-yl)phenol (8)
ꢀ
The same procedure described in section 4.3 was employed with
24 h to yield 669 mg (1.96 mmol, 66%) as a light brown solid.
IIIb (0.71 g, 3 mmol), anhydrous ammonium acetate (2.31 g,
TLC (ethyl acetate/n-hexane
297.0e298.7 C, HPLC: Retention time: 9.98 min, purity: 100%; ESI
¼
1:5)
R
f
¼
0.19, mp:
ꢀ
ꢀ
3
0 mmol), Ve (1.49 g, 4.5 mmol) and methanol (15 mL) at 100 C for
þ
2
4 h to yield 378 mg (1.12 mmol, 37%) as a white solid.
LC/MS (condition A): m/z calcd for C22
H
15NOS [MH] 342.09; found
9.85 (s, 1H, 4-phenyl 2-OH),
1
TLC (ethyl acetate/n-hexane
92.6e293.9 C, HPLC: Retention time: 10.12 min, purity: 98.5%; ESI
¼
1:7)
R
f
¼
0.29, mp:
342.41. H NMR (250 MHz, DMSO-d
6
)
d
ꢀ
2
8.29e8.28 (m, 1H, 2-thiophene H-2), 8.08e8.05 (m, 1H, inden-
opyridine H-9), 7.92 (dd, J ¼ 4.92, 0.9 Hz, 1H, 2-thiophene H-4), 7.72
(s, 1H, indenopyridine H-3), 7.67e7.60 (m, 2H, 2-thiophene H-5 and
indenopyridine H-6), 7.51e7.43 (m, 2H, indenopyridine H-7, H-8),
7.34 (d, J ¼ 7.57 Hz, 1H, 4-phenyl H-6), 7.30 (t, J ¼ 8.05 Hz, 1H, 4-
phenyl H-4), 7.02 (d, J ¼ 8.0 Hz, 1H, 4-phenyl H-3), 6.95 (t,
þ
LC/MS (condition A): m/z calcd for C22
H
15NOS [MH] 342.09; found
9.86 (s, 1H, 4-phenyl 2-OH),
1
3
8
2
2
42.41. H NMR (250 MHz, DMSO-d
6
)
d
.01 (d, J ¼ 6.27 Hz, 1H, indenopyridine H-9), 7.87 (d, J ¼ 3.5 Hz, 1H,
-thiophene H-3), 7.76 (s,1H, indenopyridine H-3), 7.64e7.60 (m,
H, 2-thiophene H-5 and indenopyridine H-6), 7.51e7.44 (m, 2H,
1
3
indenopyridine H-7, H-8), 7.39 (d, J ¼ 7.55 Hz, 1H, 4-phenyl H-6),
.31 (t, J ¼ 7.25 Hz, 1H, 4-phenyl H-4), 7.16 (d, J ¼ 3.97 Hz, 1H, 2-
thiophene H-4), 7.03 (d, J ¼ 8.1 Hz, 1H, 4-phenyl H-3), 6.96 (t,
J ¼ 7.37 Hz, 1H, 4-phenyl H-5), 3.87 (s, 2H, indenopyridine H-5).
C
7
NMR (62.5 MHz, DMSO-d ) d 159.96, 155.11, 152.60, 145.37, 145.03,
6
143.07, 141.40, 135.07, 131.29, 130.65, 129.45, 127.85, 127.63, 127.47,
126.26, 125.93, 124.51, 121.26, 120.13 (2C), 116.83, 34.88.
13
J ¼ 7.4 Hz, 1H, 4-phenyl H-5), 3.87 (s, 2H, indenopyridine H-5).
NMR (62.5 MHz, DMSO-d 159.93, 155.15, 151.75, 145.81, 145.46,
45.12, 141.01, 135.47, 131.28, 130.79, 129.64, 129.23, 128.67, 127.95,
26.33, 125.96, 125.74, 121.32, 120.21, 118.58, 116.90, 34.98.
C
6
) d
1
1
4.3.7. Synthesis of 2-(2-(thiophen-3-yl)-5H-indeno[1,2-b]pyridin-
4-yl)phenol (12)
The same procedure described in section 4.3 was employed with
IIIc (0.71 g, 3 mmol), anhydrous ammonium acetate (2.31 g,
30 mmol), Vf (1.49 g, 4.5 mmol) and methanol (15 mL) at 100 C for
4
4
.3.4. Synthesis of 3-(2-(thiophen-2-yl)-5H-indeno[1,2-b]pyridin-
-yl)phenol (9)
ꢀ
The same procedure described in section 4.3 was employed with
36 h to yield 586 mg (1.7 mmol, 57%) as a light brown solid.
IIIc (0.71 g, 3 mmol), anhydrous ammonium acetate (2.31 g,
TLC (ethyl acetate/n-hexane
225.9e226.6 C, HPLC: Retention time: 9.47 min, purity: 100%; ESI
¼
1:4)
R
f
¼
0.21, mp:
ꢀ
ꢀ
3
0 mmol), Ve (1.49 g, 4.5 mmol) and methanol (15 mL) at 100 C for
þ
3
0 h to yield 473 mg (1.39 mmol, 46%) as a light yellow solid.
LC/MS (condition A): m/z calcd for C22
H
15NOS [MH] 342.09; found
9.71 (s, 1H, 4-phenyl 3-OH),
1
TLC (ethyl acetate/n-hexane
11.4e212.8 C, HPLC: Retention time: 10.29 min, purity: 97.9%; ESI
¼
1:5)
R
f
¼
0.22, mp:
342.42. H NMR (250 MHz, DMSO-d
6
)
d
ꢀ
2
8.35 (s, 1H, 2-thiophene H-2), 8.07 (d, J ¼ 6.45 Hz, 1H, indenopyr-
idine H-9), 7.95 (d, J ¼ 4.85 H, 1H, 2-thiophene H-4), 7.82 (s,1H,
indenopyridine H-3), 7.66 (br, 2H, 2-thiophene H-5 and inden-
opyridine H-6), 7.48 (br, 2H, indenopyridine H-7, H-8), 7.36 (t,
J ¼ 7.8 Hz, 1H, 4-phenyl H-5), 7.25 (d, J ¼ 7.17 Hz, 1H, 4-phenyl H-6),
7.19 (s,1H, 4-phenyl H-2), 6.90 (d, J ¼ 7.6 Hz,1H, 4-phenyl H-4), 4.07
þ
LC/MS (condition A): m/z calcd for C22
H
15NOS [MH] 342.09; found
9.71 (s, 1H, 4-phenyl 3-OH),
1
3
42.42. H NMR (250 MHz, DMSO-d
6
)
d
8
.03 (m, 1H, indenopyridine H-9), 7.96 (dd, J ¼ 3.6, 0.97 Hz, 1H, 2-
thiophene H-3), 7.85 (s,1H, indenopyridine H-3), 7.66e7.46 (m,
H, 2-thiophene H-5 and indenopyridine H-6), 7.50e7.46 (m, 2H,
indenopyridine H-7, H-8), 7.36 (t, J ¼ 7.77 Hz, 1H, 4-phenyl H-5),
.25 (t, J ¼ 7.82 Hz, 1H, 4-phenyl H-6), 7.20e7.16 (m, 2H, 2-
thiophene H-4 and 4-phenyl H-2), 6.89 (d, J ¼ 7.95 Hz, 1H, 4-
2
13
(s, 2H, indenopyridine H-5). C NMR (62.5 MHz, DMSO-d
6
)
7
d 160.93, 158.58, 153.32, 146.97, 145.81, 142.99, 141.21, 140.07,
133.24, 190.80, 129.65, 127.98, 127.70, 127.58, 125.30, 124.36, 121.39,
119.95, 118.21, 116.56, 116.36, 35.07.
13
phenyl H-4), 4.07 (s, 2H, indenopyridine H-5).
62.5 MHz, DMSO-d 160.85, 158.62, 152.47, 147.07, 145.66,
45.29,140.82, 139.87, 133.62,130.86, 129.82,129.29, 128.91,128.08,
26.34 (2C), 121.42, 119.97, 116.68, 116.63, 116.07, 35.16.
C NMR
(
1
1
6
) d
4.3.8. Synthesis of 2-(2-(thiophen-3-yl)-5H-indeno[1,2-b]pyridin-
4-yl)phenol (13)
The same procedure described in section 4.3 was employed with
IIId (0.71 g, 3 mmol), anhydrous ammonium acetate (2.31 g,
30 mmol), Vf (1.49 g, 4.5 mmol) and methanol (15 mL) at 100 C for
4
4
.3.5. Synthesis of 4-(2-(thiophen-2-yl)-5H-indeno[1,2-b]pyridin-
-yl)phenol (10)
ꢀ
The same procedure described in section 4.3 was employed with
30 h to yield 470 mg (1.37 mmol, 46%) as a light brown solid.
IIId (0.71 g, 3 mmol), anhydrous ammonium acetate (2.31 g,
TLC (ethyl acetate/n-hexane
291.2e292.4 C, HPLC: Retention time: 9.47 min, purity: 96.1%; ESI
¼
1:4)
R
f
¼
0.25, mp:
ꢀ
ꢀ
3
2
0 mmol), Ve (1.49 g, 4.5 mmol) and methanol (15 mL) at 100 C for
4 h to yield 314 mg (0.92 mmol, 31%) as a light yellow solid.
þ
LC/MS (condition A): m/z calcd for C22
H
15NOS [MH] 342.09; found
9.84 (s, 1H, 4-phenyl 4-OH),
1
TLC (ethyl acetate/n-hexane
68.9e269.9 C, HPLC: Retention time: 10.08 min, purity: 96.1%; ESI
¼
1:5)
R
f
¼
0.2, mp:
342.42. H NMR (250 MHz, DMSO-d
6
)
d
ꢀ
2
8.33 (br, 1H, 2-thiophene H-2), 8.05 (br, 1H, indenopyridine H-9),
7.94 (d, J ¼ 6.7 Hz, 1H, 2-thiophene H-4), 7.81 (s,1H, indenopyridine
H-3), 7.74e7.66 (m, 4H, 4-phenyl H-2, H-6, 2-thiophene H-5 and
þ
LC/MS (condition A): m/z calcd for C22
H
d
15NOS [MH] 342.09; found
9.88 (s, 1H, 4-phenyl 4-OH),
1
3
42.42. H NMR (250 MHz, DMSO-d
6
)

24
S. Park et al. / European Journal of Medicinal Chemistry 125 (2017) 14e28
indenopyridine H-6), 7.47 (br, 2H, indenopyridine H-7, H-8), 6.95 (d,
4.3.12. Synthesis of 2-(2-(furan-3-yl)-5H-indeno[1,2-b]pyridin-4-
yl)phenol (17)
J ¼ 7.47 Hz, 2H, 4-phenyl H-3, H-5), 4.10 (s, 2H, indenopyridine H-
13
5
). C NMR (62.5 MHz, DMSO-d
6
)
d
160.88, 159.02, 153.24, 146.69,
The same procedure described in section 4.3 was employed with
IIIb (0.35 g, 1.5 mmol), anhydrous ammonium acetate (1.15 g,
145.23, 143.14, 141.30, 132.82, 130.71 (2C), 129.53, 129.23, 127.93,
ꢀ
127.62 (2C), 126.23, 124.69, 121.35, 117.87, 116.51 (2 C), 35.26.
15 mmol), Vh (0.63 g, 2 mmol) and methanol (8 mL) at 100 C for
24 h to yield 403 mg (1.24 mmol, 83%) as a white solid.
4
.3.9. Synthesis of 2-(2-(furan-2-yl)-5H-indeno[1,2-b]pyridin-4-yl)
phenol (14)
The same procedure described in section 4.3 was employed with
TLC (ethyl acetate/n-hexane
288.2e288.8 C, HPLC: Retention time: 5.83 min, purity: 98.6%; ESI
¼
1:5)
R
f
¼
0.21, mp:
ꢀ
þ
LC/MS (condition A): m/z calcd for C22
H
15NO
2
[MH] 326.12; found
1
IIIb (0.71 g, 3 mmol), anhydrous ammonium acetate (2.31 g,
3
2
6
326.28. H NMR (250 MHz, DMSO-d ) d 9.79 (s, 1H, 4-phenyl 2-OH),
ꢀ
0 mmol), Vg (1.41 g, 4.5 mmol) and methanol (15 mL) at 100 C for
8.42 (s, 1H, 2-furyl H-2), 8.05 (d, J ¼ 7.32 Hz, 1H, indenopyridine H-
9), 7.77 (br, 1H, 2-furyl H-4), 7.61 (d, J ¼ 6.55 Hz, 1H, indenopyridine
H-6), 7.57 (s, 1H, indenopyridine H-3), 7.50e7.39 (m, 2H, inden-
opyridine H-7, H-8), 7.34 (dd, J ¼ 7.52, 1.25 Hz, 1H, 4-phenyl H-6),
7.29 (t, J ¼ 7.95 Hz, 1H, 4-phenyl H-4), 7.18 (br, 1H, 2-furyl H-5), 7.02
(d, J ¼ 8.1 Hz, 1H, 4-phenyl H-3), 6.95 (t, J ¼ 7.43 Hz, 1H, 4-phenyl H-
4 h to yield 391 mg (1.2 mmol, 40%) as a brown solid.
TLC (ethyl acetate/n-hexane
¼
1:5)
R
f
¼
0.19, mp:
ꢀ
313.8e314.4 C, HPLC: Retention time: 8.37 min, purity: 97.6%; ESI
þ
LC/MS (condition A): m/z calcd for C22
3
8
H
15NO
2
[MH] 326.12; found
9.87 (s, 1H, 4-phenyl 2-OH),
.05e8.02 (m, 1H, indenopyridine H-9), 7.84 (s,1H, indenopyridine
1
6
26.36. H NMR (250 MHz, DMSO-d ) d
1
3
5), 3.85 (s, 2H, indenopyridine H-5). C NMR (62.5 MHz, DMSO-d
6
)
H-3), 7.60 (br, 2H, 2-furan H-5 and indenopyridine H-6), 7.50e7.44
d
159.15, 154.24, 149.97, 144.46, 144.20, 144.14, 141.60, 140.52,
(
m, 2H, indenopyridine H-7, H-8), 7.40 (dd, J ¼ 7.47, 1.47 Hz, 1H, 4-
134.13, 130.39, 129.79, 128.59, 127.18, 126.99, 125.40, 125.08, 120.43,
119.31, 119.11, 116.02, 109.07, 34.05.
phenyl H-6), 7.30 (t, J ¼ 8.05 Hz, 1H, 4-phenyl H-4), 7.23 (d,
J ¼ 3.25 Hz, 1H, 2-furan H-3), 7.02 (d, J ¼ 7.97 Hz, 1H, 4-phenyl H-3),
6
3
d
.95 (t, J ¼ 7.37 Hz, 1H, 4-phenyl H-5), 6.67 (br, 1H, 2-furan H-4),
4.3.13. Synthesis of 3-(2-(furan-3-yl)-5H-indeno[1,2-b]pyridin-4-
yl)phenol (18)
13
.89 (s, 2H, indenopyridine H-5). C NMR (62.5 MHz, DMSO-d
160.35, 155.14, 154.36, 148.42, 145.24, 145.12, 145.74, 141.10,
35.36, 131.18, 130.84, 129.69, 127.95, 126.32, 125.63, 121.40, 120.23,
18.26, 116.97, 113.13, 109.46, 34.99.
6
)
The same procedure described in section 4.3 was employed with
IIIc (0.23 g, 1 mmol), anhydrous ammonium acetate (0.77 g,
1
1
ꢀ
10 mmol), Vh (0.63 g, 2 mmol) and methanol (8 mL) at 110 C for
24 h to yield 126 mg (0.39 mmol, 39%) as a white solid.
4
.3.10. Synthesis of 3-(2-(furan-2-yl)-5H-indeno[1,2-b]pyridin-4-
TLC (ethyl acetate/n-hexane
243.9e244.7 C, HPLC: Retention time: 6.03 min, purity: 97.3%; ESI
¼
1:3)
R
f
¼
0.24, mp:
ꢀ
yl)phenol (15)
þ
The same procedure described in section 4.3 was employed with
LC/MS (condition B): m/z calcd for C22
H
15NO
2
[MH] 326.12; found
1
IIIc (0.71 g, 3 mmol), anhydrous ammonium acetate (2.31 g,
3
2
6
326.28. H NMR (250 MHz, DMSO-d ) d 9.66 (s, 1H, 4-phenyl 3-OH),
ꢀ
0 mmol), Vg (1.41 g, 4.5 mmol) and methanol (15 mL) at 100 C for
8.48 (s, 1H, 2-furyl H-2), 8.05 (d, J ¼ 7.37 Hz, 1H, indenopyridine H-
9), 7.79 (br, 1H, 2-furyl H-4), 7.68 (s, 1H, indenopyridine H-3),
7.66e7.64 (m, 1H, indenopyridine H-6), 7.51e7.42 (m, 2H, inden-
opyridine H-7, H-8), 7.35 (t, J ¼ 7.95 Hz, 1H, 4-phenyl H-5),
7.23e7.21 (m, 2H, 4-phenyl H-6, 2-furyl H-5), 7.16 (br, 1H, 4-phenyl
H-2), 6.90 (dd, J ¼ 7.05, 2.27 Hz, 1H, 4-phenyl H-4), 4.06 (s, 2H,
4 h to yield 416 mg (1.28 mmol, 43%) as a light yellow solid.
TLC (ethyl acetate/n-hexane
25.1e226.3 C, HPLC: Retention time: 8.32 min, purity: 99.6%; ESI
¼
1:4)
R
f
¼
0.29, mp:
ꢀ
2
þ
LC/MS (condition A): m/z calcd for C22
3
8
H
15NO
2
[MH] 326.12; found
1
26.36. H NMR (250 MHz, DMSO-d
.04 (m, 1H, indenopyridine H-9), 7.86 (s,1H, indenopyridine H-3),
6
) d 9.72 (s, 1H, 4-phenyl 3-OH),
13
6
indenopyridine H-5). C NMR (62.5 MHz, DMSO-d ) d 160.22,
7
2
7
.66 (br, 2H, 2-furan H-5 and indenopyridine H-6), 7.49e7.46 (m,
H, indenopyridine H-7, H-8), 7.36 (t, J ¼ 7.85 Hz,1H, 4-phenyl H-5),
.26 (br, 2H, 2-furan H-3 and 4-phenyl H-6), 7.17 (s, 1H, 4-phenyl H-
157.86, 150.85, 146.16, 144.45 (2C), 142.03, 140.46, 139.29, 132.39,
130.07, 128.92, 127.26 (2C), 125.58, 120.67, 119.21, 117.34, 115.84,
115.33, 109.26, 34.37.
2
4
d
), 6.89 (d, J ¼ 7.25 Hz, 1H, 4-phenyl H-4), 6.69 (br, 1H, 2-furan H-4),
13
.09 (s, 2H, indenopyridine H-5). C NMR (62.5 MHz, DMSO-d
161.22, 158.64, 154.18, 149.13, 146.83, 145.27, 144.91, 140.86,
36.81, 133.56, 130.92, 129.87, 128.05, 126.31, 121.50, 119.82, 116.75,
16.17, 115.87, 113.19, 109.78, 35.13.
6
)
4.3.14. Synthesis of 4-(2-(furan-3-yl)-5H-indeno[1,2-b]pyridin-4-
yl)phenol (19)
1
1
The same procedure described in section 4.3 was employed with
IIId (0.35 g, 1.5 mmol), anhydrous ammonium acetate (1.15 g,
ꢀ
1
5 mmol), Vh (0.63 g, 2 mmol) and methanol (10 mL) at 100 C for
4.3.11. Synthesis of 4-(2-(furan-2-yl)-5H-indeno[1,2-b]pyridin-4-
24 h to yield 138 mg (0.42 mmol, 28%) as a white solid.
yl)phenol (16)
TLC (ethyl acetate/n-hexane
266.8e267.3 C, HPLC: Retention time: 6.42 min, purity: 97.6%; ESI
¼
1:3)
R
f
¼
0.21, mp:
ꢀ
The same procedure described in section 4.3 was employed with
IIId (0.71 g, 3 mmol), anhydrous ammonium acetate (2.31 g,
þ
LC/MS (condition B): m/z calcd for C22
H
15NO
2
[MH] 326.12; found
ꢀ
1
3
2
0 mmol), Vg (1.41 g, 4.5 mmol) and methanol (15 mL) at 100 C for
4 h to yield 312 mg (0.96 mmol, 32%) as a light yellow solid.
6
326.26. H NMR (250 MHz, DMSO-d ) d 9.79 (s, 1H, 4-phenyl 4-OH),
8.46 (s, 1H, 2-furyl H-2), 8.04 (d, J ¼ 7.17 Hz, 1H, indenopyridine H-
9), 7.78 (br, 1H, 2-furyl H-4), 7.69 (d, J ¼ 8.7 Hz, 2H, 4-phenyl H-2, H-
6), 7.67 (s, 1H, indenopyridine H-3), 7.63 (br, 1H, indenopyridine H-
6), 7.50e7.44 (m, 2H, indenopyridine H-7, H-8), 7.22 (br, 1H, 2-furyl
H-5), 6.93 (d, J ¼ 8.75 Hz, 2H, 4-phenyl H-3, H-5), 4.09 (s, 2H,
TLC (ethyl acetate/n-hexane
¼
1:4)
R
f
¼
0.19, mp:
ꢀ
2
82.2e283.7 C, HPLC: Retention time: 8.29 min, purity: 98.5%; ESI
þ
LC/MS (condition A): m/z calcd for C22
H
15NO
2
[MH] 326.12; found
6
) d 9.82 (s, 1H, 4-phenyl 4-OH),
1
3
26.27. H NMR (250 MHz, DMSO-d
13
8
.04 (m, 1H, indenopyridine H-9), 7.84 (s,1H, indenopyridine H-3),
6
indenopyridine H-5). C NMR (62.5 MHz, DMSO-d ) d 160.17,
7
.70e7.66 (m, 4H, 4-phenyl H-2, H-6, 2-furan H-5 and indenopyr-
158.32, 150.74, 145.86, 144.49, 144.37, 141.91, 140.56, 131.99, 129.97
(2C), 128.79, 128.46, 127.36, 127.19, 125.51, 120.64, 116.99, 115.80
(2C), 109.30, 34.58.
idine H-6), 7.48e7.45 (m, 2H, indenopyridine H-7, H-8), 7.24 (d,
J ¼ 3.03 Hz, 1H, 2-furan H-3), 6.95 (d, J ¼ 8.42 Hz, 2H, 4-phenyl H-3,
1
3
H-5),6.68 (br, 1H, 2-furan H-4), 4.11 (s, 2H, indenopyridine H-5).
C
NMR (62.5 MHz, DMSO-d
45.23, 144.69, 140.945, 133.09, 130.52 (2C), 129.65, 128.97, 127.92,
26.18, 121.40, 116.58 (2C), 115.80, 113.06, 109.51, 35.26.
6
)
d
161.08, 159.08, 154.33, 149.02, 146.58,
4.3.15. Synthesis of 2-(2-(pyridin-2-yl)-5H-indeno[1,2-b]pyridin-4-
yl)phenol (20)
1
1
The same procedure described in section 4.3 was employed with

S. Park et al. / European Journal of Medicinal Chemistry 125 (2017) 14e28
25
IIIb (0.47 g, 2 mmol), anhydrous ammonium acetate (1.54 g,
IIIb (0.47 g, 2 mmol), anhydrous ammonium acetate (1.54 g,
20 mmol), Vj (0.97 g, 3 mmol) and methanol (10 mL) at 100 C for
24 h to yield 279 mg (0.82 mmol, 42%) as a white solid.
ꢀ
ꢀ
2
2
0 mmol), Vi (0.97 g, 3 mmol) and methanol (10 mL) at 110 C for
4 h to yield 242 mg (0.72 mmol, 36%) as a white solid.
TLC (ethyl acetate/n-hexane
63.2e263.8 C, HPLC: Retention time: 7.72 min, purity: 98.5%; ESI
¼
1:2)
R
f
¼
0.26, mp:
TLC (ethyl acetate/n-hexane
238.5e239.2 C, HPLC: Retention time: 6.26 min, purity: 98.8%; ESI
¼
1:1)
R
f
¼
0.23, mp:
ꢀ
ꢀ
2
þ
þ
LC/MS (condition A): m/z calcd for C23
H
16
N
2
O [MH] 337.14; found
LC/MS (condition A): m/z calcd for C23
H
16
N
2
O [MH] 337.14; found
1
1
3
8
37.46. H NMR (250 MHz, DMSO-d
6
)
d
9.89 (s, 1H, 4-phenyl 2-OH),
6
337.36. H NMR (250 MHz, DMSO-d ) d 9.94 (s, 1H, 4-phenyl 2-OH),
.67 (d, J ¼ 4.73 Hz, 1H, 2-pyridyl H-3), 8.62 (d, J ¼ 7.95 Hz, 1H,
9.41 (s, 1H, 2-pyridyl H-2), 8.64 (d, J ¼ 4.7 Hz, 1H, 2-pyridyl H-4),
8.59(dd, J ¼ 8.02, 1.82 Hz,1H, 2-pyridyl H-6), 8.12 (d, J ¼ 6.72 Hz,1H,
indenopyridine H-9), 7.88 (s, 1H, indenopyridine H-3), 7.63 (d,
J ¼ 7.65 Hz, 1H, 4-phenyl H-6), 7.56e7.43 (m, 4H, 2-pyridyl H-5,
indenopyridine H-6, H-7, H-8), 7.32 (td, J ¼ 8.25, 1.37 Hz, 1H, 4-
phenyl H-4),7.03 (d, J ¼ 8.07 Hz, 1H, 4-phenyl H-3), 6.96 (t,
indenopyridine H-9), 8.32 (s, 1H, indenopyridine H-3), 8.12 (d,
J ¼ 6.85 Hz, 1H, 2-pyridyl H-6), 7.99 (td, J ¼ 7.85, 1.62 Hz, 1H, 2-
pyridyl H-4), 7.64 (d, J ¼ 7.1 Hz, 1H, 4-phenyl H-6), 7.53e7.40 (m,
4
1
6
H, 2-pyridyl H-5, indenopyridine H-6, H-7, H-8), 7.32 (td, J ¼ 7.47,
.37 Hz, 1H, 4-phenyl H-4), 7.03 (d, J ¼ 8.17 Hz, 1H, 4-phenyl H-3),
1
3
.96 (t, J ¼ 7.42 Hz, 1H, 4-phenyl H-5), 3.95 (s, 2H, indenopyridine
J ¼ 7.42 Hz, 1H, 4-phenyl H-5), 3.93 (s, 2H, indenopyridine H-5).
C
13
H-5). C NMR (62.5 MHz, DMSO-d
54.34, 149.51, 144.72, 144.53, 140.57, 137.58, 136.60, 130.57, 130.23,
29.10, 127.38, 125.78, 125.22, 124.25, 120.74 (2C), 119.90, 119.65,
16.35, 34.43.
6
)
d
159.62, 155.83, 154.52,
NMR (62.5 MHz, DMSO-d ) d 159.96, 154.58, 152.98, 149.91, 148.24,
6
1
1
1
145.07,144.55,140.58, 135.68,134.74,134.42,130.88, 130.29, 129.22,
127.43, 125.80, 125.15, 124.09, 120.89, 119.96, 119.66, 116.31, 34.43.
4.3.19. Synthesis of 3-(2-(pyridin-3-yl)-5H-indeno[1,2-b]pyridin-4-
4.3.16. Synthesis of 3-(2-(pyridin-2-yl)-5H-indeno[1,2-b]pyridin-4-
yl)phenol (24)
yl)phenol (21)
The same procedure described in section 4.3 was employed with
IIIc (0.23 g, 1 mmol), anhydrous ammonium acetate (0.77 g,
10 mmol), Vj (0.65 g, 2 mmol) and methanol (10 mL) at 110 C for
The same procedure described in section 4.3 was employed with
IIIc (0.47 g, 2 mmol), anhydrous ammonium acetate (1.54 g,
ꢀ
ꢀ
2
2
0 mmol), Vi (0.97 g, 3 mmol) and methanol (10 mL) at 100 C for
4 h to yield 196 mg (0.58 mmol, 29%) as a creamy white solid.
36 h to yield 136 mg (0.41 mmol, 41%) as a creamy white solid.
TLC (ethyl acetate/n-hexane
263.5e264.1 C, HPLC: Retention time: 6.81 min, purity: 97.1%; ESI
¼
2:1)
R
f
¼
0.24, mp:
ꢀ
TLC (ethyl acetate/n-hexane
¼
1:2)
R
f
¼
0.18, mp:
ꢀ
þ
2
72.8e273.6 C, HPLC: Retention time: 7.94 min, purity: 98.4%; ESI
LC/MS (condition A): m/z calcd for C23
H
16
N
2
O [MH] 337.14; found
þ
1
LC/MS (condition A): m/z calcd for C23
H
16
N
2
O [MH] 337.13; found
9.76 (s, 1H, 4-phenyl 3-OH),
6
337.36. H NMR (250 MHz, DMSO-d ) d 9.65 (s, 1H, 4-phenyl 3-OH),
1
3
8
37.46. H NMR (250 MHz, DMSO-d
6
)
d
9.45 (s, 1H, 2-pyridyl H-2), 8.66e8.61 (m, 2H, 2-pyridyl H-4, H-6),
8.13 (d, J ¼ 8.67 Hz, 1H, indenopyridine H-9), 7.96 (s, 1H, inden-
opyridine H-3), 7.67 (d, J ¼ 7.95 Hz, 1H, indenopyridine H-6),
7.57e7.45 (m, 3H, 2-pyridyl H-5, indenopyridine H-7, H-8), 7.37 (t,
J ¼ 7.65 Hz, 1H, 4-phenyl H-5), 7.29 (d, J ¼ 7.75 Hz, 1H, 4-phenyl H-
6), 7.23 (s, 1H, 4-phenyl H-2), 6.91 (d, J ¼ 7.8 Hz, 1H, 4-phenyl H-4),
.71 (d, J ¼ 4.73 Hz, 1H, 2-pyridyl H-3), 8.62 (d, J ¼ 7.95 Hz, 1H,
indenopyridine H-9), 8.39 (s, 1H, indenopyridine H-3), 8.14 (d,
J ¼ 5.55 Hz, 1H, 2-pyridyl H-6), 8.01 (t, J ¼ 7.67 Hz, 1H, 2-pyridyl H-
4
5
), 7.68 (br, 1H, indenopyridine H-6), 7.54e7.45 (m, 3H, 2-pyridyl H-
, indenopyridine H-7, H-8), 7.37 (t, J ¼ 7.75 Hz, 1H, 4-phenyl H-5),
13
7
.26 (d, J ¼ 7.62 Hz, 1H, 4-phenyl H-6), 7.20 (s, 1H, 4-phenyl H-2),
6
4.13 (s, 2H, indenopyridine H-5). C NMR (62.5 MHz, DMSO-d )
6
5
.91 (d, J ¼ 7.9 Hz, 1H, 4-phenyl H-4), 4.15 (s, 2H, indenopyridine H-
d 160.71, 157.87, 153.61, 149.85, 148.20, 146.46, 144.53, 140.27,
139.13, 134.49, 134.31, 133.66, 130.06, 129.16, 127.34, 125.60, 123.85,
120.80, 119.31, 117.88, 115.96, 115.42, 34.42.
13
). C NMR (62.5 MHz, DMSO-d
49.64, 146.28, 144.76, 140.37, 139.46, 137.75, 134.92, 130.49, 129.37,
27.57, 125.86, 124.53, 120.95, 120.87, 119.27, 117.79, 116.24, 115.23,
6
)
d
160.60, 158.12, 155.65, 155.15,
1
1
3
4.64.
4.3.20. Synthesis of 4-(2-(pyridin-3-yl)-5H-indeno[1,2-b]pyridin-4-
yl)phenol (25)
4
.3.17. Synthesis of 4-(2-(pyridin-2-yl)-5H-indeno[1,2-b]pyridin-4-
The same procedure described in section 4.3 was employed with
IIId (0.23 g, 1 mmol), anhydrous ammonium acetate (0.77 g,
10 mmol), Vj (0.65 g, 2 mmol) and methanol (10 mL) at 110 C for
yl)phenol (22)
ꢀ
The same procedure described in section 4.3 was employed with
IIId (0.47 g, 2 mmol), anhydrous ammonium acetate (1.54 g,
36 h to yield 114 mg (0.33 mmol, 34%) as a light yellow solid.
ꢀ
2
0 mmol), Vi (0.97 g, 3 mmol) and methanol (10 mL) at 110 C for
TLC (ethyl acetate/n-hexane
290.8e291.7 C, HPLC: Retention time: 6.67 min, purity: 95.3%; ESI
¼
2:1)
R
f
¼
0.29, mp:
ꢀ
2
4 h to yield 272 mg (0.81 mmol, 41%) as a white solid.
þ
TLC (ethyl acetate/n-hexane
78.3e279.0 C, HPLC: Retention time: 8.02 min, purity: 99.6%; ESI
¼
1:2)
R
f
¼
0.13, mp:
LC/MS (condition A): m/z calcd for C23
H
16
N
2
O [MH] 337.14; found
ꢀ
1
2
6
337.46. H NMR (250 MHz, DMSO-d ) d 9.88 (s, 1H, 4-phenyl 4-OH),
þ
LC/MS (condition A): m/z calcd for C23
H
16
N
2
O [MH] 337.13; found
9.6 (s, 1H, 2-pyridyl H-2), 8.65e8.61 (m, 2H, 2-pyridyl H-4, H-6),
8.11 (d, J ¼ 8. 7 Hz, 1H, indenopyridine H-9), 7.97 (s, 1H, inden-
opyridine H-3), 7.78 (d, J ¼ 8.57 Hz, 2H, 4-phenyl H-2, H-6), 7.67, (d,
J ¼ 8.02 Hz, 1H, indenopyridine H-6), 7.58e7.48 (m, 3H, 2-pyridyl
H-5, indenopyridine H-7, H-8), 6.95 (d, J ¼ 8.57 Hz, 2H, 4-phenyl
1
3
8
6
37.46. H NMR (250 MHz, DMSO-d ) d 9.93 (s, 1H, 4-phenyl 4-OH),
.70 (d, J ¼ 4.72 Hz, 1H, 2-pyridyl H-3), 8.61 (d, J ¼ 7.9 Hz, 1H,
indenopyridine H-9), 8.37 (s, 1H, indenopyridine H-3), 8.12 (d,
J ¼ 5.42 Hz, 1H, 2-pyridyl H-6), 8.0 (td, J ¼ 7.7, 1.72 Hz, 1H, 2-pyridyl
H-4), 7.71 (d, J ¼ 8.64 Hz, 2H, 4-phenyl H-2, H-6), 7.66 (br, 1H,
indenopyridine H-6), 7.53e7.44 (m, 3H, 2-pyridyl H-5, indenopyr-
idine H-7, H-8), 6.95 (d, J ¼ 8.6 Hz, 2H, 4-phenyl H-3, H-5), 4.16 (s,
13
H-3, H-5), 4.17 (s, 2H, indenopyridine H-5). C NMR (62.5 MHz,
6
DMSO-d ) d 159.64, 158.51, 155.63, 155.09, 150.57, 146.12, 144.71,
139.64, 136.92, 135.42, 129.89 (2C), 129.11, 128.92, 127.32, 124.73,
124.12, 121.32, 120.83, 117.96, 116.32 (2C), 34.09.
13
2
6
H, indenopyridine H-5). C NMR (62.5 MHz, DMSO-d ) d 160.46,
1
1
1
58.54, 155.81, 155.02, 149.57, 146.10, 144.76, 140.47, 137.66, 134.45,
30.02 (2C), 129.19, 128.65, 127.47, 125.76, 124.40, 120.88, 120.83,
17.44, 116.12 (2C), 34.77.
4.3.21. Synthesis of 2-(2-(pyridin-4-yl)-5H-indeno[1,2-b]pyridin-4-
yl)phenol (26)
The same procedure described in section 4.3 was employed with
IIIb (0.23 g, 1 mmol), anhydrous ammonium acetate (0.77 g,
10 mmol), Vk (0.65 g, 2 mmol) and methanol (10 mL) at 110 C for
4
.3.18. Synthesis of 2-(2-(pyridin-3-yl)-5H-indeno[1,2-b]pyridin-4-
ꢀ
yl)phenol (23)
The same procedure described in section 4.3 was employed with
36 h to yield 78 mg (0.24 mmol, 24%) as a light yellow solid.

26
S. Park et al. / European Journal of Medicinal Chemistry 125 (2017) 14e28
TLC (ethyl acetate/n-hexane
82.6e283.3 C, HPLC: Retention time: 6.73 min, purity: 96.8%; ESI
¼
1:1)
R
f
¼
0.12, mp:
enzyme was re-determined and set as one unit when supercoiled
pBR322 DNA was fully relaxed by reaction. The mixture of 100 ng of
plasmid pBR322 DNA and 1 unit of recombinant human DNA topo I
(TopoGEN INC., USA) was incubated without and with the prepared
each compound at 37 C for 30 min in the relaxation buffer (10 mM
Tris-HCl (pH 7.9), 150 mM NaCl, 0.1% bovine serum albumin, 1 mM
ꢀ
2
þ
LC/MS (condition A): m/z calcd for C23
H
16
N
2
O [MH] 337.14; found
1
3
8
2
7
1
6
5
(
1
3
6
37.47. H NMR (250 MHz, DMSO-d ) d 9.86 (s, 1H, 4-phenyl 2-OH),
ꢀ
.71 (d, J ¼ 5.82 Hz, 2H, 2-pyridyl H-2, H-6), 8.21 (d, J ¼ 6.02 Hz, 2H,
-pyridyl H-3, H-5), 8.13 (d, J ¼ 7.77 Hz, 1H, indenopyridine H-9),
.53e7.42 (m, 3H, indenopyridine H-6, H-7, H-8), 7.32 (td, J ¼ 8.12,
spermidine, 5% glycerol). The reaction in the final volume of 10
was terminated by adding 2.5
sarcosyl, 0.0025% bromophenol blue and 25% glycerol. DNA topo II
m
L
.47 Hz, 1H, 4-phenyl H-4),7.05 (d, J ¼ 8.07 Hz, 1H, 4-phenyl H-3),
mL of the stop solution containing 5%
.97 (t, J ¼ 7.4 Hz, 1H, 4-phenyl H-5), 3.95 (s, 2H, indenopyridine H-
a
13
). C NMR (62.5 MHz, DMSO-d
6
)
d
160.02, 154.51, 152.50, 150.46
inhibitory activity of compounds were measured as follows [36].
The mixture of 200 ng of supercoiled pBR322 plasmid DNA and 1
2C), 146.14, 145.02, 144.49, 140.35, 136.70, 130.71, 130.21, 129.22,
27.35, 125.71, 124.93, 121.09 (2C), 120.80, 120.26, 119.53, 116.27,
4.39.
unit of human DNA topo II
and with the prepared compounds in the assay buffer (10 mM Tris-
HCl (pH 7.9) containing 50 mM NaCl, 5 mM MgCl , 1 mM EDTA,
g/mL bovine serum albumin) for 30 min at
L was terminated by the
L of 7 mM EDTA. Reaction products after each of topo
relaxation assay were analyzed on 1% agarose gel at 15 V
for 7 h for topo I and 25 V for 4 h for topo II , respectively, with a
running buffer of TAE. Gels were stained for 30 min in an aqueous
solution of ethidium bromide (0.5 g/mL). DNA bands were visu-
a (Usb Corp., USA) was incubated without
2
4
.3.22. Synthesis of 3-(2-(pyridin-4-yl)-5H-indeno[1,2-b]pyridin-4-
yl)phenol (27)
The same procedure described in section 4.3 was employed with
IIIc (0.23 g, 1 mmol), anhydrous ammonium acetate (0.77 g,
0 mmol), Vk (0.65 g, 2 mmol) and methanol (10 mL) at 100 C for
6 h to yield 108 mg (0.32 mmol, 32%) as a creamy white solid.
1 mM ATP, and 15
30 C. The reaction in a final volume of 20
m
ꢀ
m
addition of 3
I and II
m
a
ꢀ
1
3
a
TLC (ethyl acetate/n-hexane
¼
2:1)
R
f
¼
0.24, mp:
m
ꢀ
2
85.8e286.5 C, HPLC: Retention time: 8.51 min, purity: 99.4%; ESI
alized by transillumination with UV light and supercoiled DNA was
quantitated using AlphaImager™ (Alpha Innotech Corporation).
þ
LC/MS (condition A): m/z calcd for C23
H
16
N
2
O [MH] 337.14; found
1
3
8
2
8
6
37.47. H NMR (250 MHz, DMSO-d ) d 9.72 (s, 1H, 4-phenyl 3-OH),
.72 (d, J ¼ 5.55 Hz, 2H, 2-yridyl H-2, H-6), 8.26 (d, J ¼ 5.65 Hz, 2H,
-pyridyl H-3, H-5), 8.13 (d, J ¼ 8.05 Hz, 1H, indenopyridine H-9),
.04 (s, 1H, indenopyridine H-3), 7.68 (d, J ¼ 7.8 Hz, 1H, inden-
4.4.2. Antiproliferative activity assay
Cancer and normal cells were cultured according to the sup-
plier's instructions. Cells were seeded in 96-well plates at a density
4
opyridine H-6), 7.55e7.47 (m, 2H, indenopyridine H-7, H-8), 7.37 (t,
J ¼ 7.7 Hz, 1H, 4-phenyl H-5), 7.29 (d, J ¼ 7.77 Hz, 1H, 4-phenyl H-6),
of 2e4 ꢁ 10 cells per well and incubated for overnight in 0.1 mL of
media supplied with 10% Fetal Bovine Serum (Hyclone, USA) in 5%
ꢀ
7
(
d
.23 (s,1H, 4-phenyl H-2), 6.93 (d, J ¼ 8.0 Hz,1H, 4-phenyl H-4), 4.15
CO
2
incubator at 37 C. On the next day, after FBS starvation for 4 h,
13
s, 2H, indenopyridine H-5). C NMR (62.5 MHz, DMSO-d
160.89, 157.93, 157.22, 153.29, 150.46 (2C), 146.56, 146.04, 144.66,
40.14, 139.09, 134.97, 130.15, 129.44, 127.49, 125.76, 121.17 (2C),
6
)
culture medium in each well was exchanged with 0.1 mL aliquots of
medium containing graded concentrations of compounds followed
by incubation for 72 h. Each well was then added with 5 mL of the
1
1
20.89, 119.37, 118.34, 115.52, 34.46.
cell counting kit-8 solution (Dojindo, Japan) then incubated for
additional 4 h under the same condition. The absorbance of each
well was determined by an Automatic Elisa Reader System (Bio-Rad
3550) at 450 nm wavelength. For determination of the IC50 values,
the absorbance readings at 450 nm were fitted to the four-
parameter logistic equation. Adriamycin, etoposide, and campto-
thecin were purchased from Sigma and used as positive controls.
4.3.23. Synthesis of 3-(2-(pyridin-4-yl)-5H-indeno[1,2-b]pyridin-4-
yl)phenol (28)
The same procedure described in section 4.3 was employed with
IIId (0.23 g, 1 mmol), anhydrous ammonium acetate (0.77 g,
ꢀ
10 mmol), Vk (0.65 g, 2 mmol) and methanol (10 mL) at 100 C for
3
6 h to yield 68 mg (0.19 mmol, 20%) as a light yellow solid.
TLC (ethyl acetate/n-hexane
01.2e302.7 C, HPLC: Retention time: 8.64 min, purity: 95.6%; ESI
¼
1:1)
R
f
¼
0.23, mp:
4.4.3. Topo IIa-DNA cleavable complex assay
Cleavage complex assay was carried out by the previously re-
ported methods with minor modification [37]. 100 ng supercoiled
DNA pBR322 (Fermentas, USA) was reacted with 3 units of topo IIa
(Usb Corp., USA) for 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 and 7 M EDTA fol-
ꢀ
3
þ
LC/MS (condition B): m/z calcd for C23
H
16
N
2
O [MH] 337.14; found
1
3
8
2
37.47. H NMR (250 MHz, DMSO-d
6
)
d
9.82 (s, 1H, 4-phenyl 4-OH),
.76 (d, J ¼ 6.05 Hz, 2H, 2-pyridyl H-2, H-6), 8.27 (d, J ¼ 6.03 Hz, 2H,
ꢀ
-pyridyl H-3, H-5), 8.15 (d, J ¼ 7.62 Hz, 1H, indenopyridine H-9),
7
.99 (s, 1H, indenopyridine H-3), 7.73 (d, J ¼ 7.92 Hz, 2H, 4-phenyl
ꢀ
H-2, H-6), 7.62 (d, J ¼ 46 Hz, 1H, indenopyridine H-6), 7.54e7.46 (m,
lowed by digestion with proteinase K at 45 C for 30 min. After
2
H, indenopyridine H-7, H-8), 6.94 (d, J ¼ 8.43 Hz, 2H, 4-phenyl H-3,
addition of loading buffer, the reaction mixture was heated for
13
ꢀ
H-5), 4.14 (s, 2H, indenopyridine H-5). C NMR (62.5 MHz, DMSO-
159.46, 157.92, 144.51 (2C), 143.34, 140.98 (2C), 133.57 (2C),
30.29 (2C), 128.50 (2C), 127.32 (2C), 127.14, 125.66 (2C), 120.53
2C), 115.74 (2C), 34.11.
2 min at 70 C and electrophoresed with a 0.8% agarose gel in TAE
d
6
)
d
buffer containing 0.5 mL/mL ethidium bromide, followed by
destaining the gel with water for 20 min. The gel was visualized
using an AlphaImager™ (Alpha Innotech Corporation).
1
(
4
4
.4. Pharmacology
4.4.4. Topo II
ATPase assays were performed using ATP dehydrogenase reac-
tion with topo II . Reaction was performed in a 100 L of mixture
containing reaction buffer (10 mM tris-HCl, pH 7.9, 50 mM NaCl,
a ATPase activity assay
.4.1. DNA topoisomerase I and II
DNA topo I inhibition assay was determined following the pre-
a
inhibition assay in vitro
a
m
viously reported method [35]. The test compounds were dissolved
in DMSO at 20 mM as a stock solution. The activities of DNA topo I
50 mM KCl, 5 mM MgCl
supercoiled DNA pBR322 100 ng, ATP 250
USA) 4 unit per well in the 96-well plate and incubated at 37 C for
1 h. After reaction mixture was incubated for 30 min at room
2
, 0.1 mM EDTA, and 15
m
a
g/mL BSA),
mM, Topo II
(Usb Corp.,
ꢀ
and II
a
were checked periodically by assessing the relaxation of
supercoiled pBR322 DNA according to each reaction protocol
described below because both enzymes are very labile during
storage. Whenever both enzymes were used, the unit of each
temperature with 20
mL of working reagent of malachite green
phosphate assay kit (BioAssay Systems, USA) per well, and then

S. Park et al. / European Journal of Medicinal Chemistry 125 (2017) 14e28
27
analyzed by ELISA Microplate reader (VERSAmax, Molecular De-
vices) at a wavelength of 620 nm.
parameters were used except for 270,000 population size and 50
docking runs. The ligand was redocked to the receptor and analyzed
with the original ligand bound to the receptor to validate the
method.
4.4.5. DNA unwinding assay
The DNA unwinding capacity of compound 16 was analyzed
using a DNA unwinding kit (TopoGEN, Port Orange, FL, USA) ac-
cording to the manufacturer's instructions. Briefly, 100 ng pHOT1
plasmid DNA was treated with 3 units of topo I (TopoGEN, Port
Acknowledgments
This research was supported by the grant of the Korean Health
Technology R&D Project, Ministry of Health & Welfare, Republic of
Korea (HI14C2469).
Orange, FL, USA) in 20
m
L of reaction buffer (10 mM Tris-HCl, pH 7.5,
ꢀ
1
mM EDTA) for 30 min at 37 C. Relaxed plasmids were then
ꢀ
incubated in the presence of compound 16 at 37 C for an additional
0 min. Amsacrine (100, 200, 500, and 1000 M) was used as a
3
m
Appendix A. Supplementary data
positive control. The reaction was terminated by adding 1% SDS and
loading dye at the end of the incubation and reaction product was
resolved on 1% agarose gels at 15 V/cm for 12e15 h. After electro-
phoresis, gels were stained in TAE buffer with ethidium bromide for
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.09.019.
3
0 min and visualized using an AlphaImager™ (Alpha Innotech
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4
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[
1
a
b
23 (2015) 3499e3512.
a
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