Rational design, synthesis, and evaluation of novel 2,4-Chloro- and Hydroxy-Substituted diphenyl Benzofuro[3,2-b]Pyridines: Non-intercalative catalytic topoisomerase I and II dual inhibitor

European Journal of Medicinal Chemistry 127 (2017) 318e333

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Research paper

Rational design, synthesis, and evaluation of novel 2,4-Chloro- and

Hydroxy-Substituted diphenyl Benzofuro[3,2-b]Pyridines:

Non-intercalative catalytic topoisomerase I and II dual inhibitor

,

Seojeong Park ^{a 1}, Til Bahadur Thapa Magar ^{b 1}, Tara Man Kadayat ^b, Hwa Jong Lee ^a,

Ganesh Bist ^b, Aarajana Shrestha ^b, Eung-Seok Lee ^b^*, Youngjoo Kwon ^a

,

, **

^aCollege of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Republic of Korea

^bCollege of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history:

Novel series of conformationally constrained 2,4-chloro- and hydroxy-substituted diphenyl benzofuro

[3,2-b]pyridines were rationally designed and synthesized. Their biological activities were evaluated for

topoisomerase I and II inhibitory activity, and antiproliferative activity against several human cancer cell

lines for the development of novel anticancer agents. Most of the compounds with phenol moiety at 4-

position of central pyridine exhibited signiﬁcant dual topoisomerase I and II inhibitory activities, and

strong antiproliferative activity in low micromolar range. Structure activity relationship study suggested

that phenol moiety at 4-position of the central pyridine regardless of chlorophenyl moiety at 2-position

of the central pyridine has an important role in dual topoisomerase inhibitory activity as well as anti-

proliferative activity. For investigation of mode of action for compound 14 which displayed the most

strong dual topoisomerase I and II inhibitory activity and antiproliferative activity against HCT15 cell, we

performed cleavable complex assay, band depletion assay, comet assay, and competitive EtBr displace-

ment assay. Compound 14 functioned as non-intercalative catalytic topo I and II dual inhibitor. In

addition, compound 14 induced apoptosis in HCT15 cells through increase of Bax, decrease of Bcl-2 and

increase of PARP cleavage.

Received 28 September 2016

Received in revised form

28 November 2016

Accepted 1 January 2017

Available online 2 January 2017

Keywords:

2,4-chloro- and hydroxy-substituted

diphenyl benzofuro[3,2-b]pyridines

Dual topoisomerase I and II inhibition

Antiproliferative activity

Non-intercalative catalytic inhibiton

Anticancer agent

1. Introduction

transcription, recombination, repair, chromatin assembly and

chromosome segregation [3,4]. Since the discovery of topo by J. C.

It has been generally considered that caner is a very complicated

disease caused by multiple pathogenic factors. The characteristics

of cancer are an accelerated rate of tumor cell proliferation and

numerous genetic (inherent) and/or epigenetic mutations to

extend their survival [1]. Despite of diverse and heterogeneous

characters of tumors, all types of tumors have in common the

accelerated cell proliferation rate which is positively related to the

overexpression of DNA topoisomerase (topo) in tumors [1,2].

Human DNA topo, a nuclear enzyme, is the key component of

the cell which can solve all topological problems of DNA associated

with several vital cellular processes like DNA replication,

Wang in 1971, it has been shown to be one of the promising mo-

lecular targets of anticancer agents [5,6]. There are two types of

human DNA topo; one is topo I which makes DNA single strand

cleavage at a time. The other is topo II which induces concurrent

cleavage on DNA double strands. During DNA cleavage process by

topo I or topo II, the covalent bond is transiently formed between

topo and DNA giving rise to DNA-topo cleavable complex followed

by going back to DNA religation which peruses DNA relaxation or

unwinding processes to solve DNA topological problems [7]. Those

compounds, which stabilize the cleavable complex by forming

topo-DNA-compound ternary complex leading to disturbance on

this speciﬁc religation step in the catalytic cycle of topo function

mechanism, are termed as topo poisons. On the other hand, com-

pounds, which function on the other steps beside prevention from

the religation step, are named as topo catalytic cycle inhibitors. Just

as predicted in the nomenclature, topo poisons generate more

* Corresponding author.

** Corresponding author.

E-mail addresses: eslee@ynu.ac.kr (E.-S. Lee), ykwon@ewha.ac.kr (Y. Kwon).

Authors are equal contributors.

1

http://dx.doi.org/10.1016/j.ejmech.2017.01.003

S. Park et al. / European Journal of Medicinal Chemistry 127 (2017) 318e333

319

detrimental DNA truncation and severe genotoxicity [8,9]. At the

present, camptothecin, topotecan, irinotecan, etoposide, and teni-

poside are under clinical use as topo-targeting anticancer drugs.

Although extensive investigation on topo inhibitors in the last four

decades has offered several potent anticancer drugs, they have

undesirable side effects and other limitations such as an induction

of secondary malignancy during medication [10]. Further investi-

gation is needed for the development of better topo-targeting

anticancer agent. Depending upon the cell types, the expression

of topo I and topo II is markedly different. Topo I is highly expressed

in colon cancer cell lines [11], while topo II expression is high in

breast and ovarian cancer cell lines [12]. It is also observed that the

time-course of topo I and topo II expression differs during cell cycle.

Topo I expression is relatively constant throughout the cell cycle,

whereas topo II expression is maximum at S phase [13]. Either of

the enzyme expression is sufﬁcient for cell division and the resis-

tance to topo I inhibitor is developed due to concomitant increase

in topo II expression and vice versa [14]. Therefore, dual topo I and

II inhibitors have the advantage and the interest of medicinal

chemists is growing towards research for the development of dual

topo I and II-targeting anticancer drugs [15,16].

pyridine for 3 h at 140 ^ꢀC generated corresponding pyridinium

iodide salts 2 (R ¼ a-f) in 52.2e93.0% yield. Secondly, benzofuran-

3(2H)-one intermediates 5 (R¹¼ a-f) were synthesized using

benzofuran-3(2H)-one (3) and aryl aldehyde 4 (R¹¼ a-f) as starting

material in the presence of activated, basic aluminum oxide and

methylene chloride for 3 h at 25 ^ꢀC. Total six benzofuran-3(2H)-one

intermediates 5 (R¹¼ a-f) were synthesized in the yield of

51.0e81.6%. Finally, treatment of pyridinium iodide salts 2 (R ¼ a-f)

and benzofuran-3(2H)-one intermediates 5 (R¹¼ a-f) in the pres-

ence of NH₄OAc and glacial acetic acid for 12e24 h at 100 ^ꢀC

afforded the corresponding 2,4-chloro- and hydroxy-substituted

€

diphenyl benzofuro[3,2-b]pyridines (6e23) via modiﬁed Krohnke

pyridine synthesis method [34] in the yield of 8.9e36.5%. Yield (%),

purity (%), retention time (min) obtained from HPLC analysis, and

melting point (^ꢀC) of each of synthesized compounds are summa-

rized in Table S1 (Supplementary Data).

2.2. Topoisomerase I and II inhibitory activity

We measured the extent of topo I or II-mediated conversion of

supercoiled DNA to relaxed DNA in the absence and presence of the

prepared 2,4-chloro- and hydroxy-substituted diphenyl benzofuro

[3,2-b]pyridines (6e23) as shown in Fig. 4, and their % inhibitory

activities are summarized in Table 1. Camptothecin and etoposide,

selective topo I and II inhibitors, respectively, were used as positive

Many researchers have reported that several natural and syn-

thetic benzofuran containing compounds exhibit potent anticancer

activities via wide variety of mechanisms including aromatase in-

hibition [17], hypoxia-inducible factor 1-alpha (HIF-1a) inhibition

[18], topo inhibition [19], farnesyltransferase inhibition [20], proto-

oncogene serine/threonine-protein kinase (Pim-1) inhibition [21],

controls. Inhibitory activities were evaluated at 100

compounds showing more than 30% inhibitory activity were

further examined at 20 M.

mM in which

HDAC inhibition [22], GSK-3

b

inhibition [23], and mTOR inhibition

m

[24]. Moreover, it has been reported that halogen containing

compounds improve the drug-target binding afﬁnity by forming

halogen bonds [25e27]. Especially, chlorination of 2- or 4-phenyl

ring increased the topo I and topo II inhibitory activity as well as

antiproliferative activity against several human cancer cell lines

[28,29]. Furthermore, hydroxylation is important for the formation

of H-bond leading to enhanced topo I and II inhibitory activity and

antiproliferative activity [30]. In addition, previously reported

conformationally constrained 2-phenol-4-phenylbenzofuro[3,2-b]

pyridine (compound I, Fig. 1) which contains benzofuran moiety

displayed dual topo I and topo II inhibitory activity [31]. It has been

considered that rigid structure has planar conﬁguration and less

conformational entropy facilitating compound to be ﬁtted more

efﬁciently into the active site of target enzyme [32].

2.2.1. Topoisomerase I inhibitory activity

As shown in Fig. 4A, majority of the compounds displayed the

considerable to signiﬁcant topo I inhibitory activity. Among the

tested compounds, 14 possesses the most signiﬁcant topo I inhib-

itory activity (95.6% at 100

mM, 47.9% at 20

m

M) which is more active

than a positive control, camptothecin (59.0% at 100

mM, 34.1% at

20 m

M). Compounds 8e14 containing 2⁰-, 3⁰-, or 4⁰-chlorophenyl at

2-position and 2⁰-, 3⁰-, or 4⁰-hydroxyphenyl group at 4-position of

central pyridine showed signiﬁcant topo I inhibitory activity at both

100 mM and 20 mM concentrations. On the other hand, compounds

15e23 possessing 2⁰-, 3⁰-, or 4⁰-hydroxylpheny group at 2-poisition

and 2⁰-, 3⁰-, or 4⁰-chlorphenyl group at 4-position of central pyri-

dine showed moderate to signiﬁcant topo I inhibitory activity

On the basis of previous reports and results, we introduced

conformationally rigid novel benzofuran moiety bearing chloride

and hydroxyl functionality at 2- or 4-phenyl ring, which expected

to display dual topo I and topo II inhibitory activity as well as to

improve their biological activities. The strategy for design of 2,4-

chloro- and hydroxy-substituted diphenyl benzofuro[3,2-b]pyri-

dines is shown in Fig. 2. Benzofuropyridine is the novel rigid moiety

ﬁrst discovered in our research group having dual topo I and topo II

inhibitory activity, and antiproliferative activity against several

human cancer cell lines. In this study, we have designed and syn-

thesized eighteen novel series of benzofuro[3,2-b]pyridines pos-

sessing 2⁰-, 3⁰-, or 4⁰-chlorophenyl and 2⁰-, 3⁰-, or 4⁰-phenol moiety

at 2- and 4-position of the central pyridine ring as shown in Fig. 3.

(41.7%e87.5%) only at 100 mM.

2.2.2. Topoisomerase II inhibitory activity

Compounds 6e14 containing 2⁰-, 3⁰-, or 4⁰-chlorophenyl at 2-

position and 2⁰-, 3⁰-, or 4⁰-hydroxyphenyl group at 4-position of

central pyridine displayed signiﬁcant topo II inhibitory activity

(82.9%e100%) as compared to etoposide (86.9%) at 100 mM. Espe-

cially, compound 14 with 4⁰-chlorophenyl at 2-position and 4⁰-

hydroxyphenyl at 4-position of central pyridine showed the most

potent topo II inhibitory activity of 100% (100

(20 M) which are more active than etoposide (86.9% at 100

34.2% at 20

m

M) and 59.8%

m

mM,

m

M). However, compounds 15e23 possessing 2⁰-, 3⁰-, or

4⁰-hydroxylpheny group at 2-poisition and 2⁰-, 3⁰-, or 4⁰-chlor-

phenyl group at 4-position of central pyridine did not show sig-

2. Results and discussion

niﬁcant topo II inhibitory activity (0%e65.6% at 100 mM). Among the

compounds 15e23, compounds 15, 16, 17, and 23 displayed com-

2.1. Synthetic chemistry

parable topo II inhibitory activity.

As illustrated in Scheme 1, benzofuro[3,2-b]pyridines containing

chlorophenyl or phenol moiety at 2- and 4-position of central

pyridine were synthesized in three steps based on the previously

reported methods [31,33]. At ﬁrst, reaction of appropriate aryl

methyl ketones 1 (R ¼ a-f) with equivalent amount of iodine in

2.3. Antiproliferative activity

Three different human cancer cell lines were used for the

evaluation of antiproliferative activity: human colorectal adeno-

carcinoma cell line (HCT15), human breast cancer cell line (T47D),

320

S. Park et al. / European Journal of Medicinal Chemistry 127 (2017) 318e333

Fig. 1. Structures of previously synthesized rigid analogs of (a) 2,4,6-trisubstituted pyridines, (b) Chloro- and hydroxy-substituted 2,4-diphenyl indenopyridines, and (c) 2-phenyl-

or hydroxyphenyl-4-phenyl benzofuro[3,2-b]pyridines.

and human cervix tumor cell line (HeLa). IC₅₀values determined for

compounds 6e23 are listed in Table 1. Most of the compounds

exhibited signiﬁcant antiproliferative activity against tested cell

lines. Among all the tested compounds, compound 6, 7 and 10e15

displayed strong antiproliferative activity against HCT15 as

compared to positive controls. Compound 7 showed the strongest

controls. All of the compounds except 8 which containing 2⁰-, 3⁰-, or

4⁰-chlorophenyl at 2-position and 2⁰-, 3⁰-, or 4⁰-hydroxyphenyl

group at 4-position of central pyridine (compounds 6e14) showed

signiﬁcant antiproliferative activity against all the tested cancer cell

lines. However, more than half compounds (17, 18, 21, 22, and 23)

which possessing 2⁰-, 3⁰-, or 4⁰-hydroxylpheny group at 2-poisition

and 2⁰-, 3⁰-, or 4⁰-chlorphenyl group at 4-position of central pyri-

dine (compounds 15e23) did not displayed considerable anti-

proliferative activity. Among the compounds 15e23 only 15,16, and

19 displayed signiﬁcant antiproliferative activity against all the

tested cancer cell lines. As illustrated by results of antiproliferative

activity, compounds bearing phenol on 4-position of central pyri-

dine ring showed stronger antiproliferative activity than those of

bearing phenol on 2-position. In addition, antiproliferative activity

was not depending on the position of chlorophenyl on 2-position of

central pyridine ring.

antiproliferative activity (0.01 mM) especially against HCT15 which

is 60, 540 and 100 fold more potent than positive controls camp-

tothecin, etoposide, and adriamycin, respectively although the topo

I and II inhibitory activity of compound 7 was less potent than

compound 14. The better antiproliferative activity of compound 7

with less topo I and II inhibitory than compound 14 reﬂected

compound 7 is likely to have another targets related to cell prolif-

eration process in cells. Compound 14 possessing excellent dual

topo I and II inhibitory activity also showed strong antiproliferative

activity against HCT15 cell line as compared to all three positive

S. Park et al. / European Journal of Medicinal Chemistry 127 (2017) 318e333

321

Fig. 2. Strategy for the design of 2,4-chloro- and hydroxy-substituted diphenyl benzofuro[3,2-b]pyridines.

Fig. 3. Structures of the prepared 2,4-chloro- and hydroxy-substituted diphenyl benzofuro[3,2-b]pyridines.

2.4. Structure-activity relationship study (SAR)

compared with hydroxyl or chloride substituted analogs. On the

other hand, introduction of chloride functionality (6e23) on 2-

hydroxyphenyl-4-phenylbenzofuro[3,2-b]pyridine derivatives (H-

J) displayed mixed results. Among tested compounds in the current

study, compounds 15e23 were obtained by introduction of 2⁰-, 3⁰-,

or 4⁰-chloride at 4-phenylring of compounds H-J. Compounds 15

and 21 having 4-(2⁰-chlorophenyl) and 4-(4⁰-chorophenyl) moiety,

respectively, showed better topo I and topo II inhibitory activity

than respective non-chlorinated analog (H). In contrast, 4-(3⁰-

chlorophenyl)-containing compound 18 displayed stronger topo I

inhibitory activity but weaker topo II inhibitory activity. Similar

results were observed in the case of 2-(4⁰-hydroxyphenyl)-con-

taining compound (J) and respective chlorinated derivatives 17, 20

and 23. However, Compound I having 2-(3⁰-hydroxyphenyl) moiety

displayed better relative potency for topo II than respective chlo-

rinated analogs 16,19 and 22 while compounds 19 and 20 exhibited

slightly stronger relative potency for topo I than compound I. The

We performed the structure-activity relationship study utilizing

the results of topo I and topo II inhibitory activity, and anti-

proliferative activity of 2- or 4-chlorophenyl-2- or 4-

hydroxyphenyl-5H-indeno[1,2-b]pyridines

diphenylbenzofuro[3,2-b]pyridines (G),

phenylbenzofuro[3,2-b]pyridines (H-J), and 2,4-chloro- and

hydroxy-substituted diphenyl benzofuro[3,2-b]pyridines (6e23).

Fig. 1 shows the structures of previously reported compounds and

Table 2 summarizes the relative topo I and II inhibitory potencies of

(A-F),

2-hydroxyphenyl-4-

2,4-

the representative compounds at 100 mM and 20 mM concentra-

tions. Interestingly, replacement of conventional indenopyridine

moiety (A-F) with 2,4-chloro- and hydroxy-substituted diphenyl

benzofuro[3,2-b]pyridines (6e23) signiﬁcantly enhanced the topo I

and topo II inhibitory activity as well as antiproliferative activity.

Relative potency of non-substituted compound G is very low as

322

S. Park et al. / European Journal of Medicinal Chemistry 127 (2017) 318e333

Scheme 1. General synthetic method of 2,4-chloro- and hydroxy-substituted diphenyl benzofuro[3,2-b]pyridines 6e23; Reagents and conditions: i) iodine (1.0 equiv.), pyridine, 3 h,

140 ^ꢀC, 52.2e93.0% yield; ii) Al₂O₃, CH₂Cl₂, 3 h, rt, 51.0e81.6% yield; iii) NH₄OAc (10.0 equiv.), glacial acetic acid, 12e24 h, 100 ^ꢀC, 8.9e36.5% yield.

topo inhibitory activity and antiproliferative activity would be

affected by position of hydroxyl and chloride groups. Compounds

6e14 possessing hydroxyl group at 4-phenyl ring displayed sig-

niﬁcant dual topo I and II inhibitory activity, and strong anti-

proliferative activity. On the other hand, compounds 15e23

possessing hydroxyl group at 2-phenyl group showed compara-

tively low topo I and II inhibitory activity and/or antiproliferative

activity. These results indicate the crucial role of hydroxyl group at

4-phenyl ring for displaying dual topo I and II inhibitory activity as

well as antiproliferative activity. Generally, we observed positive

correlation between dual topo I and II inhibitory activity and

antiproliferative activity. Compounds bearing phenol on 4-position

of central pyridine ring showed stronger antiproliferative activity

than those of bearing phenol on 2-position (Fig. 5). In addition, dual

topo I and II inhibitory activity and antiproliferative activity was not

depending on the position of chlorophenyl on 2-position of central

pyridine ring.

inhibitory activity was chosen for further analysis to determine its

mode of action. Moreover, compound 14 inhibited most potently

HCT15 cell growth (Table 1). Therefore, cell-based experiments to

clarify the mode of action of compound 14 were performed with

HCT15 cells.

2.5.1. Compound 14 acted as a catalytic topo I and II dual inhibitor

not a topo poison

Cleavable complex assay was ﬁrst performed to assess whether

compound 14 generated a truncated linear DNA which could be a

surrogate marker for a topo poison. Since the topo poison like

etoposide has ability to stabilize the transiently formed topo II-DNA

cleavable complex leading to prevention of the transiently cleaved

DNA double strand from being religated and consequently induc-

tion of undesirable short DNA [35]. As shown in Fig. 6A, etoposide

made the truncated linear DNA while compound 14 did not.

Kinetoplast DNA (kDNA) decatenation assay is a method to spe-

ciﬁcally evaluate the topo II inhibitory activity [36]. In order to

conﬁrm the topo II inhibitory activity of compound 14, we per-

formed the assay with compound 14 using kDNA as a substrate.

kDNA is a catenated network of thousands of interlocked closed

2.5. Mode of action of compound 14

Compound 14 showing the most potent dual topo I and II

S. Park et al. / European Journal of Medicinal Chemistry 127 (2017) 318e333

323

Fig. 4. DNA topo I (A) and topo II (B) inhibitory activity of the prepared compounds 6e23 at the concentrations of 100

mM and 20

m

M. (A) Lane D: pBR322 only, Lane T:

pBR322 þ Topo I, Lane C: pBR322 þ Topo I þ Camptothecin, Lane 6e23: pBR322 þ Topo I þ compounds 6e23 at 100

mM or 20

m

M. (B) Lane D: pBR322 only, Lane T: pBR322 þ Topo

II, Lane E: pBR322 þ Topo II þ Etoposide, Lane 6e23: pBR322 þ Topo II þ compounds 6e23 at 100

mM or 20 mM.

Table 1

Topo I and II inhibitory and antiproliferative activity of the prepared compounds 6e23.

Compounds

%Inhibition

Topo II

IC₅₀

(mM)

Topo I

100

HCT15

T47D

HeLa

100

mM

20

m

M

m

M

20

m

M

Camptothecin

Etoposide

Adriamycin

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

59.0^a/55.8^b

34.1^a/22^b

0.6 0.02

5.4 0.5

1.0 0.02

0.4 0.02

0.01 0.001

>50

1.6 0.2

0.4 0.01

0.2 0.02

0.4 0.02

0.2 0.01

0.2 0.03

0.3 0.02

1.4 0.03

46.2 0.8

>50

2.1 0.001

7.0 0.05

0.3 0.02

3.9 0.2

0.4 0.04

>50

1.5 0.1

1.4 0.01

2.1 0.03

6.1 0.06

1.9 0.001

5.9 0.1

3.0 0.1

0.1 0.02

0.8 0.1

>50

0.09 0.01

9.1 0.2

1.2 0.01

2.8 0.1

5.2 0.4

9.5 0.1

4.4 0.04

4.3 0.1

5.7 0.03

3.9 0.01

0.9 0.001

4.3 0.2

10.9 0.3

4.3 0.03

15.9 0.1

>50

86.9^a/56.3^b

34.2^a/30^b

85.6

84.0

86.7

82.9

85.4

87.9

92.8

95.4

100

58.2

60.2

52.7

4.7

21.2

31.2

21.6

14.4

22.1

9.3

19.1

23.1

59.8

20.7

17.2

47.8

NT

11.2

59.8

82.1

71.8

66.3

78.1

82.2

92.1

95.6

41.7

42.7

63.5

79.6

83.8

76.6

64.5

69.5

87.5

NT

13.0

31.7

33.4

69.0

71.3

67.8

44.1

47.9

0

0.6

10.0

14.4

0

3.2

0

35.0

4.9

55.8

N/A

65.6

6.2

NT

4.7

NT

5.9 0.01

>50

21.3 0.9

1.2 0.03

>50

2.0 0.01

11.6 0.1

6.4 0.04

>50

11.1 0.04

11.8 0.4

15.1

0

28.4

Each data represents mean S.D. from three different experiments performed in triplicate.

a

NT: Not tested, NA; not active, value for compounds 6e14; ^bvalue for compounds 15e23.

HCT15: human colorectal adenocarcinoma cell line; T47D: human breast cancer cell line; HeLa: human cervix tumor cell line.

Camptothecin: positive control for topo I and cytotoxicity; Etoposide: positive control for topo II and cytotoxicity; Adriamycin: positive control for cytotoxicity.

circular DNA. Ellipticin and etoposide were used as positive con-

positive controls. However, its suppressive effect on topo II

a

trols. The inhibitory activity of compound 14 was not as effective as

showed nice concentration dependency as shown in Fig. 6B. This

324

S. Park et al. / European Journal of Medicinal Chemistry 127 (2017) 318e333

Table 2

Relative topo I and II inhibitory potencies of representative compounds compared to etoposide or camptothecin.

Relative potency^afor % inhibition compared to positive control

(Topo II: Etoposide and Topo I: Camptothecin)

Compounds

Topo II

Topo I

Compounds

Topo II

Topo I

100

mM

20

m

M

100

m

M

20

mM

100

m

M

20

mM

100

m

M

20 mM

A

B

C

D

E

F

G

H

I

0.08

0.05

0.10

0.03

0.04

0.08

0.66

1.30

1.02

0.98

0.97

0.99

0.95

NT

NA

0.05

1.77

0.90

0.62

0.91

0.63

0.42

NA

0.1

0.26

0.01

NA

0.02

0.63

1.19

0.50

0.18

1.01

1.39

1.22

NT

NA

0.05

NT

0.38

0.93

0.98

10

11

12

13

14

15

16

17

18

19

20

21

22

23

0.98

1.01

1.07

1.10

1.15

1.03

1.07

0.94

0.08

0.62

0.09

0.99

NA

0.65

0.27

0.56

0.77

1.75

0.69

0.57

1.59

NT

0.21

NT

0.16

NT

1.12

1.32

1.39

1.56

1.62

0.75

0.77

1.14

1.43

1.50

1.37

1.16

1.25

1.57

2.02

2.09

1.99

1.29

1.40

NA

0.03

0.45

0.65

NA

J

6

7

8

9

0.15

NA

0.69

NA

1.17

0.95

a

Relative potency: % inhibition of compound/% inhibition of positive control, NT: not tested, NA: not active.

Fig. 5. Structure-activity relationship study of 2,4-chloro- and hydroxy-substituted diphenyl benzofuro[3,2-b]pyridines.

implies that the compound 14 indisputably holds considerable topo

II inhibitory activity. Band depletion assay was accomplished to

conﬁrm that compound 14 did not induce topo II-DNA complex in

cancer cells. After compound 14 and etoposide were treated at

indicates the DNA strand breaks. Fig. 6D indicates that compound

14 (6.3 1.3% at 30 M) almost did not induce tail compared to

untreated control (3.0 1.1%) like ICRF-187 (5.5 1.9% at 30 M),

a

m

well-known topo II catalytic inhibitor [37], while etoposide, a well-

concentration of 50

mM, respectively, with HCT15 cells for 2 h, and

known topo II poison, induced a signiﬁcant DNA damage (the

then cell lysates were electrophoresed and western blotted to

check the remained free topo II. The topo II-DNA covalent complex

induced by etoposide was depleted during DNA precipitation pro-

cess as described in method. As shown in Fig. 6C, free topo II was

not detected in etoposide-treated HCT15 cell lysate but it was

detected in compound 14-treated one. Comet assay was further

utilized to determine the DNA damage caused by compound 14,

etoposide and ICRF-187. The extent of comet (tail) formation

percent of tail DNA induced by etoposide: 46.3 9.2% at 10

65.5 9.8% at 30 M) as expected. When DNA was damaged such as

DNA double strand break induced by etoposide in mammalian cells,

histones are phosphorylated and become -H2AX. -H2AX is

widely used as a marker for DNA damage [38]. After 30 M treat-

ment of each of etoposide, ICRF-187 and compound 14, the changes

on -H2AX expression was analyzed by western blotting. As shown

in Fig. 6E, the signiﬁcant increase of -H2AX protein level in

mM and

m

g

m

g

S. Park et al. / European Journal of Medicinal Chemistry 127 (2017) 318e333

325

Fig. 6. (A) Cleavable complex assay: To determine whether topo II poison or catalytic inhibitor, 250 ng of pBR322 DNA was treated with 3 units of topo II for 10 min prior to

treatment of etoposide (100 M) or compound 14 (100 M and 500

M). After incubation for additional 30 min at 37 ^ꢀC, the agarose gel electrophoresis was performed. A linear

band on the gel for compound 14 was not observed reﬂecting that it worked as a catalytic inhibitor. (B) kDNA decatenation assay: The assay was performed in a total reaction

volume of 10 L containing 75 ng of kDNA, each of compounds in ﬁnal concentration of 250 or 500

M and 3 units of top II. The reaction mixture was incubated for 30 min at 37 ^ꢀ

and then terminated by the addition of 2.5

L of stop solution (5% SDS, 25% ﬁcoll and 0.05% bromphenol blue) followed by treatment with 0.25 mg/ml proteinase K at 37 ^ꢀC for

30 min. Samples were resolved by electrophoresis on a 1.2% (w/v) agarose gel containing 0.5 g/mL ethidium bromide in TAE buffer (upper panel). The quantiﬁcation of product

m

C

m

formed in the upper panel was graphed in lower panel. (C) Band depletion assay: Whether functioning as a topo poison or a catalytic inhibitor can be deﬁned by looking at free topo

II band. The topo II covalently bound to DNA was depleted during DNA precipitation process after treatment of topo II poison like etoposide. The cells seeded in a density of 2 ꢁ 10⁵

were treated with each of etoposide and compound 14 for 2 h at 37 ^ꢀC and then were harvested and lysed by denaturing agent of 62.5 mM Tris-HCl (pH 6.8) containing 1 mM EDTA

and 2% SDS. The prepared lysates were detected by Western blotting. (D) Comet assay: To evaluate whether compound 14 induced DNA damage like a topo poison, etoposide, each

of compound 14, etoposide and ICRF-187 was treated with HCT15 cells for 24 h in ﬁnal concentrations of 10 or 30 mM. Then harvested cells were executed for alkaline unwinding

process and electrophoresis under the alkaline condition followed by staining with SYBR Green. The percent DNA in tail was analyzed by Komet 5.0 software. Data were represented

both by imaging (upper panel) and vertical bar graph (lower panel) by randomly selecting comet lengths of HCT15 cells. Etoposide generated tail signiﬁcantly at both concentration

of 10 and 30

and compound 14, was treated with HCT15 cells in concentrations of 30

by western blot. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

mM while ICRF-187 and compound 14 almost did not at 30

mM. (E) The effect of compounds on the expression level of

g-H2AX: each of compounds, etoposide, ICRF-187

m

M for 24 h followed by lysis. 60 g of protein per sample was resolved by 12% SDS-PAGE and then analyzed

m

compared to the untreated control was detected in etoposide-

treated HCT15 cells while not observed in ICRF-187- and com-

pound 14-treated cells. This result is consistent with the degree of

comet formation (Fig. 6D). Based on results of cleavable complex

assay and band depletion assay, it could be concluded that the

compound 14 did not act as topo poison unlike etoposide, in

contrast compound 14 functioned as topo II catalytic inhibitor. In

addition, since compound 14 inhibited both topo I and II with

almost no generation of tail in comet assay, compound 14 can be

reasonably ratiocinated as a catalytic topo I and II dual inhibitor.

concentration of compound 14 and amsacrine (m-AMSA), well-

known DNA intercalative topo II poison [8]. The ﬂuorescence of

EtBr was decreased by addition of m-AMSA in dose-dependent

manner (Fig. 7A) because m-AMSA displaced EtBr through inter-

calation into DNA. The released EtBr from ctDNA lost ﬂuorescence

due to its free rotation in the solution. The addition of compound 14

did not reduce the ﬂuorescence intensity of EtBr (Fig. 7B) reﬂecting

that compound 14 could not intercalate into DNA. Taken together

compound 14 is non-intercalative catalytic topo I and II dual

inhibitor.

2.5.2. Compound 14 did not intercalate into DNA

2.5.3. Compound 14 induced apoptosis in HCT15 cells

To check whether compound 14 intercalate into DNA, ethidium

bromide (EtBr) displacement assay was performed with calf

thymus DNA (ctDNA) intercalated with EtBr by increasing

Topo plays important roles in fast tumor cell proliferation rate

[1,2], therefore inhibition of endogenous topo I and II as well as

326

S. Park et al. / European Journal of Medicinal Chemistry 127 (2017) 318e333

Fig. 7. Competitive EtBr displacement assay: The mixture of ctDNA (30 mM) and EtBr (20 mM) in the 10 mM tris buffer solution (pH 7.2) was incubated for 30 min at room

temperature followed by addition of m-AMSA (A) and compound 14 (B) at the designated concentrations with continuous incubation for additional 30 min. The ﬂuorescence

intensity of each well was measured with excitation at 471 nm. The emission spectrum was collected at 500e700 nm. Addition of m-AMSA, well known DNA intercalative topo II

poison, decreased EtBr-induced ﬂuorescence in dose-dependent manner but compound 14 did not.

apoptosis induction of cancer cells might be efﬁcient strategy for

development of anticancer agent [39]. To evaluate the effect of

compound 14 on endogenous topo I and II, HCT15 cells were pre-

treated for 24 h with camptothecin, etoposide and compound 14

followed by preparation of nuclear fraction of each compound-

treated cells. Camptothecin and etoposide were used as positive

controls as topo I and topo II inhibitors, respectively. The effect of

compound 14 was relatively insigniﬁcant than the positive controls

in cells (Fig. 8A and B). We assume that this phenomenon is due to

the poor solubility of compound 14, which made it difﬁcult to

penetrate through the cell membranes. However, it still showed

inhibitory activity in a concentration dependent manner against

both topo I and topo II in the cellular system. Thus, compound 14 is

a valid candidate as a dual inhibitor of topo I and topo II. It has been

proposed that a ratio of Bax (an apoptosis promoter) to Bcl-2 (an

apoptosis inhibitor) rules apoptosis in various types of cancer cells

as the response to chemotherapy; chemotherapeutic drugs induce

an increase of Bax and decrease of Bcl-2 leading to apoptosis and

cell death [40,41]. The upregulated ratio of Bax to Bcl-2 activates

caspase which consequently makes poly (ADP-ribose) polymerase

(PARP) to be cleaved. Cleavage of PARP by caspase is reﬂected as a

feature of apoptosis [42]. Compound 14 was evaluated for its ability

to induce apoptosis of HCT15 cells. Compound 14 treatment

increased apoptotic proteins such as cleaved PARP and Bax and

simultaneously decreased anti-apoptotic protein, Bcl-2 in

HCT15 cells in dose-dependent manner (Fig. 8C).

potent non-intercalative catalytic topo I and II dual inhibitor and

induced apoptosis in HCT15 cell through increase of Bax, decrease

of Bcl-2 and increase of PARP cleavage. This study might provide

valuable information for the researchers working on benzofuran

derivatives and topoisomerase as potential anticancer drug target.

4. Experimental

Commercially available starting materials and reagents were

purchased from Sigma-Aldrich, TCI Chemicals, Alfa-Aesar, and

Junsei, and were used without further puriﬁcation. HPLC grade

acetonitrile (ACN) and methanol were purchased from Burdick and

Jackson, USA. Column chromatography was carried out using silica

gel (Kieselgel 60, 230e400 mesh, Merck). Thin layer chromatog-

raphy (TLC) was performed on silica gel plates (Kieselgel 60 F_254,

Merck) having layer thickness of 0.25 mm. Melting points were

recorded using open capillary tube on electrothermal 1A 9100

digital melting point apparatus and were uncorrected. ¹H NMR

(250 MHz) and ¹³C NMR (62.5 MHz) spectra were recorded in

Bruker AMX 250 (250 MHz, FT) spectrometer using CDCl₃and

DMSO-d₆as solvent, and tetramethylsilane (TMS) as internal

standard. All the chemical shifts values (d) were recorded in parts

per million (ppm) and coupling constants (J) in hertz (Hz).

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

(SPD-M10A VP) utilizing Shimadzu LC Solution program. Sample

volume of 10 m 5 mM reverse-phase

L, was run in Waters X- Terra^®

3. Conclusion

C₁₈column (4.6 ꢁ 250 mm) with a gradient elution of 85%e100%

of B in A for 10 min followed by 100%e85% of B in A for 10 min at a

ﬂow rate of 0.7 mL/min at 254 nm UV detection, where mobile

phase A was doubly distilled water with 50 mM ammonium

formate (AF) and B was 100% ACN. Purity of compound is stated as

percent (%) and retention time in minutes.

Mass spectra of the compound were recorded with Advion

Expression CMS^®ESI-MS spectrometry (Advion, Ithaca, NY, USA),

using methanol: water: formic acid (80: 20: 0.1) as a mobile phase.

Total eighteen 2,4-chloro- and hydroxy-substituted diphenyl

benzofuro[3,2-b]pyridines were designed and synthesized sys-

tematically and evaluated for their topo I and II inhibitory activity,

and antiproliferative activity against several human cancer cell

lines. Synthesized compounds were found to be more anti-

proliferative to HCT15 cancer cell line. Most of the tested com-

pounds displayed dual topo I and topo II inhibitory activity. SAR

study revealed that the introduction of novel benzofuropyridine

moiety signiﬁcantly enhanced the topo I and II inhibitory activity as

well as antiproliferative activity as compared to corresponding

indenopyridine derivatives. Introduction of hydroxyphenyl moiety

at 4-position of central pyridine ring would be crucial to exhibit

dual topo I and II inhibitory activity as well as antiproliferative

activity. Compound 14 was selected to study the mode of action

since it showed the most potent dual topo I and II inhibition and

antiproliferative activity in HCT15 cell. Compound 14 functioned as

4.1. General method for preparation of 2 (R ¼ a-f)

Pyridinium iodide salts 2 (R ¼ a-f) were synthesized by reacting

aryl methyl ketones 1 (R ¼ a-f) with equivalent amount of iodine in

pyridine at 140 ^ꢀC for 3 h at reﬂux condition. The reaction mixture

was cooled to room temperature which resulted precipitation.

Precipitate formed was ﬁltered and washed with cold pyridine

S. Park et al. / European Journal of Medicinal Chemistry 127 (2017) 318e333

327

Fig. 8. (A) Endogenous topo inhibition assay: Nuclear lysates of HCT15 cells, prepared after treatment with etoposide, camptothecin and compound 14 in concentrations of 25, 50 or

100

M for 24 h, were incubated for 30 min with 100 ng pBR322 plasmid at 37 ^ꢀC to evaluate the inhibitory activity of each compound against endogenous topo I and II. DNA band

m

visualized by transillumination with UV light (A) is a representative image, obtained from three different experiments. (B) The average inhibition percent of each compound was

quantiﬁed and plotted from three experiments. (C)The effect of compound 14 on apoptotic proteins: After compound 14 was treated with HCT15 cells in concentrations of 10 and

30

mM for 24 h, cells were harvested and lysed. The supernatants of compound 14-treated cell lysates after lysis were electrophoresed on 12% SDS-PAGE gels. Western blotting

analyses were then performed in according to the method described in the experimental. Cleaved PARP, Bcl-2, and Bax were detected in compound 14-treated HCT15 cells.

followed by drying overnight to yield 52.2e93.0% of 2 (R ¼ a-f).

Compounds were used without further puriﬁcation.

as a yellow solid.

TLC (ethyl acetate/n-hexane

¼

1:4 v/v) R_f

¼

0.27; Mp:

136.2e136.7 ^ꢀ

C

¹H NMR (250 MHz, DMSO-d₆)

d 8.01e7.93 (m, 2H, benzofuro H-

4.2. General method for preparation of 5 (R¹¼ a-f)

4 and H-6), 7.83e7.77 (m, 2H, 2-phenyl H-4 and H-6), 7.59e7.50 (m,

3H, benzofuro H-7, 2-phenyl H-2 and H-5), 7.32 (t, J ¼ 7.35 Hz, 1H,

benzofuro H-5), 6.93 (s, 1H, ¼CH-).

To the mixture of benzofuran-3(2H)-one (3) and aryl aldehydes

(1.5 equivalent) 4 (R¹¼ a-f) in methylene chloride was added

aluminum oxide and the reaction mixture was stirred at 25 ^ꢀC for

3 h. The mixture was then ﬁltered and washed with ethyl acetate.

Silica gel column chromatography was used to purify the com-

pounds. Total six benzofuran-3(2H)-one intermediates 5 (R¹¼ a-f)

were synthesized at the yield of 51.0e81.6%.

¹³C NMR (62.5 MHz, DMSO-d₆)

d 183.61, 165.42, 146.41, 137.25,

135.06, 133.16, 130.01, 129.92, 129.20, 125.26, 124.56, 120.98, 121.72,

113.43, 111.01.

4.2.3. Synthesis of 2-(4-chlorobenzylidene)benzofuran-3(2H)-one

(5c)

The procedure described in Section 4.2 was employed with

4.2.1. Synthesis of 2-(2-chlorobenzylidene)benzofuran-3(2H)-one

(5a)

benzofuran-3(2H)-one

(1.34

g,

10.0

mmol)

and

4-

chlorobenzahdehyde (2.11 mL, 15.0 mmol) to yield 1.48 g (57.7%)

as a yellow solid.

The procedure described in Section 4.2 was employed with

benzofuran-3(2H)-one

(1.34

g,

10.0

mmol)

and

2-

TLC (ethyl acetate/n-hexane

¼

1:4 v/v) R_f

¼

0.25; Mp:

chlorobenzahdehyde (1.69 mL, 15.0 mmol) to yield 2.02 g (78.5%)

as a yellow solid.

180.7e181.2 ^ꢀC

¹H NMR (250 MHz, DMSO-d₆)

d

8.01 (d, J ¼ 8.55 Hz, 2H, 2-phenyl

TLC (ethyl acetate/n-hexane

144.6e145.1 ^ꢀ

¹H NMR (250 MHz, CDCl₃)

zofuro H-4), 7.79 (d, J ¼ 7.64 Hz, 1H, benzofuro H-7), 7.64 (td,

J ¼ 7.22, 1.42 Hz, 1H, benzofuro H-6), 7.46e7.18 (m, 6H, benzofuro

H-5, 2-phenyl H-3, H-4, H-5, H-6 and ¼ CH-).

¼

1:4 v/v) R_f

¼

0.28; Mp:

H-2 and H-6), 7.79 (d, J ¼ 7.45 Hz, 2H, 2-phenyl H-3 and H-5),

7.59e7.54 (m, 3H, benzofuro H-4, H-6 and H-7), 7.32 (t, J ¼ 7.5 Hz,

1H, benzofuro H-5), 6.96 (s, 1H, ¼CH-).

C

d

8.33 (dd, J ¼ 7.7, 1.82 Hz, 1H, ben-

¹³C NMR (62.5 MHz, DMSO-d₆)

d 183.74, 165.54, 146.58, 137.96,

134.76, 133.03 (2C), 130.94, 129.24 (2C), 124.48, 124.23, 120.83,

113.34, 110.92).

¹³C NMR (62.5 MHz, CDCl₃)

d 184.75, 166.40, 147.82, 137.28,

136.16, 132.46, 130.81, 130.62, 130.23, 127.26, 125.06, 123.91, 121.72,

113.14, 108.21.

4.2.4. Synthesis of 2-(2-hydroxybenzylidene)benzofuran-3(2H)-one

(5d)

4.2.2. Synthesis of 2-(3-chlorobenzylidene)benzofuran-3(2H)-one

(5b)

The procedure described in Section 4.2 was employed with

benzofuran-3(2H)-one (0.80 g, 6.0 mmol) and salicylaldehyde

(0.96 mL, 9.0 mmol) to yield 0.98 g (69.9%) as a yellow solid.

The procedure described in Section 4.2 was employed with

benzofuran-3(2H)-one

chlorobenzahdehyde (1.70 mL, 15.0 mmol) to yield 2.14 g (81.6%)

(1.34

g,

10.0

mmol)

and

3-

TLC (ethyl acetate/n-hexane

¼

1:2 v/v) R_f

¼

0.29; Mp:

248.2e248.7 ^ꢀC

328

S. Park et al. / European Journal of Medicinal Chemistry 127 (2017) 318e333

¹H NMR (250 MHz, DMSO-d₆)

d

10.43 (s, 1H, 2-phenyl 2-OH),

ESI-MS: m/z calcd for

[MþH]^þ: 372.98.

C

₂₃H₁₄ClNO₂[MþH]^þ: 372.08; found

8.15 (d, J ¼ 7.02 Hz, 1H, 2-phenyl H-6), 7.80e7.76 (m, 2H, benzofuro

H-4 and H-6), 7.55 (d, J ¼ 8.7 Hz, 1H, benzofuro H-7), 7.34e7.24 (m,

3H, benzofuro H-5, 2-phenyl H-5 and ¼ CH-), 6.97e6.93 (m, 2H, 2-

phenyl H-3 and H-4).

¹H NMR (250 MHz, CDCl₃)

d

8.31 (d, J ¼ 7.65 Hz, 1H, benzofuro

H-4), 7.82 (s, 1H, pyridine H-3), 7.72e7.69 (m, 1H, benzofuro H-7),

7.62e7.28 (m, 8H, 2-phenyl H-3, H-4, H-5, H-6, 4-phenyl H-4, H-6,

benzofuro H-5 and H-6), 7.09 (td, J ¼ 7.53, 1.13 Hz, 1H, 4-phenyl H-

5), 7.01 (dd, J ¼ 8.15, 0.93 Hz, 1H, 4-phenyl H-3).

¹³C NMR (62.5 MHz, DMSO-d₆)

d 183.41, 165.24, 157.51, 145.82,

137.37, 131.89, 131.15, 124.17, 123.81, 121.03, 119.68, 118.67, 115.82,

113.19, 106.55.

¹³C NMR (62.5 MHz, CDCl₃)

d 157.72, 153.85, 153.33, 146.50,

144.59, 139.18, 132.69, 132.17, 131.39, 131.25, 130.29, 129.91, 129.71,

129.55, 127.33, 124.15, 124.09, 123.53, 122.00, 121.40, 121.20, 117.62,

112.49.

4.2.5. Synthesis of 2-(3-hydroxybenzylidene)benzofuran-3(2H)-one

(5e)

The procedure described in Section 4.2 was employed with

benzofuran-3(2H)-one

hydroxybenzahdehyde (1.83 g, 15.0 mmol) to yield 1.26 g (51.0%)

as a yellow solid.

(1.34

g,

10.0

mmol)

and

3-

4.3.2. Synthesis of 2-(2-(3-chlorophenyl)benzofuro[3,2-b]pyridin-

4-yl)phenol (7)

The compound was synthesized as described in section 4.3 with

5d (0.24 g, 1.0 mmol), dry ammonium acetate (0.77 g, 10.0 mmol),

2b (0.36 g, 1.0 mmol) with glacial acetic acid (2 mL) to yield 70 mg

(20.0%) of a yellow solid.

TLC (ethyl acetate/n-hexane

275.8e276.3 ^ꢀ

¹H NMR (250 MHz, DMSO-d₆)

¼

1:2 v/v) R_f

¼

0.24; Mp:

C

d

9.75 (s, 1H, 2-phenyl 3-OH),

7.83e7.77 (m, 2H, benzofuro H-4 and H-6), 7.59 (d, J ¼ 8.35 Hz, 1H,

benzofuro H-7), 7.43e7.26 (m, 4H, benzofuro H-5, 2-phenyl H-2, H-

5 and H-6), 6.89e6.84 (m, 2H, 2-phenyl H-4 and ¼ CH-).

TLC (ethyl acetate/n-hexane

¼

2:5 v/v) R_f

¼

0.32; Mp:

224.5e224.8 ^ꢀC; HPLC: Retention time: 9.18 min, purity: 98.2%; ESI-

MS: m/z calcd for C₂₃H₁₄ClNO₂[MþH]^þ: 372.08; found [MþH]^þ:

372.19.

¹³C NMR (62.5 MHz, DMSO-d₆)

d 183.76, 165.50, 157.69, 156.27,

137.82, 133.0, 130.09, 124.42, 124.08, 122.81, 120.94, 117.67, 117.62,

113.24, 112.65.

¹H NMR (250 MHz, DMSO-d₆)

d 9.97 (br S, 1H, 4-phenyl 2-OH)

8.29 (br, 2H, benzofuro H-4 and 2-phenyl H-2), 8.18 (br, 2H, pyri-

dine H-3 and 2-phenyl H-6), 7.78 (d, J ¼ 8.27 Hz, 1H, benzofuro H-

7), 7.69e7.49 (m, 5H, 2-phenyl H-4, H-5, 4-phenyl H-4, H-6 and

benzofuro H-6), 7.36 (td, J ¼ 7.22, 1.55 Hz, 1H, benzofuro H-5),

7.08e6.98 (m, 2H, 4-phenyl H-3 and H-5).

4.2.6. Synthesis of 2-(4-hydroxybenzylidene)benzofuran-3(2H)-one

(5f)

The procedure described in Section 4.2 was employed with

benzofuran-3(2H)-one

(0.80

g,

6.0

mmol)

and

4-

¹³C NMR (62.5 MHz, DMSO-d₆)

d 157.32, 155.28, 150.89, 147.11,

hydroxybenzahdehyde (1.10 g, 9.0 mmol) to yield 1.06 g (74.1%)

as a yellow solid.

143.54, 141.14, 133.97, 131.61, 131.39, 130.90, 130.84, 129.93, 128.69,

126.70, 125.65, 124.14, 123.07, 121.27, 120.71, 120.52, 119.48, 116.34,

112.72.

TLC (ethyl acetate/n-hexane

¼

1:2 v/v) R_f

¼

0.27; Mp:

204.9e205.6 ^ꢀC

¹H NMR (250 MHz, DMSO-d₆)

d

10.26 (br s, 1H, 2-phenyl 4-OH),

4.3.3. Synthesis of 2-(2-(4-chlorophenyl)benzofuro[3,2-b]pyridin-

4-yl)phenol (8)

The compound was synthesized as described in section 4.3 with

5d (0.24 g, 1.0 mmol), dry ammonium acetate (0.77 g, 10.0 mmol),

2c (0.36 g, 1.0 mmol) with glacial acetic acid (2 mL) to yield 0.11 g

(30.4%) of a white solid.

7.86 (d, J ¼ 8.76 Hz, 2H, 2-phenyl H-2 and H-6), 7.79e7.73 (m, 2H,

benzofuro H-4 and H-6), 7.52 (d, J ¼ 7.37 Hz, 1H, benzofuro H-7),

7.28 (t, J ¼ 7.37 Hz, 1H, benzofuro H-5), 6.91e6.88 (m, 3H, 2-phenyl

H-3 and H-5 and ¼ CH-).

¹³C NMR (62.5 MHz, DMSO-d₆)

d 183.72, 165.20, 159.98, 144.90,

137.36, 133.95 (2C), 124.29, 123.95, 123.03, 121.53, 116.38 (2C),

113.64, 113.35.

TLC (ethyl acetate/n-hexane

¼

2:5 v/v) R_f

¼

0.32; Mp:

316.4e316.8 ^ꢀC; HPLC: Retention time: 9.05 min, purity: 95.7%; ESI-

MS: m/z calcd for C₂₃H₁₄ClNO₂[MþH]^þ: 372.08; found [MþH]^þ:

372.13.

4.3. General method for preparation of 6e23

¹H NMR (250 MHz, DMSO-d₆)

d 10.02 (br S, 1H, 4-phenyl 2-OH)

€

Based upon the modiﬁed Krohnke pyridine synthesis, 2,4-

8.27e8.22 (m, 3H, benzofuro H-4, 2-phenyl H-2 and H-6), 8.13 (s,

1H, pyridine H-3), 7.77 (d, J ¼ 8.15 Hz, 1H, benzofuro H-7),

7.68e7.48 (m, 5H, 2-phenyl H-3, H-5, 4-phenyl H-4, H-6 and ben-

zofuro H-6), 7.36 (td, J ¼ 7.50, 1.55 Hz, 1H, benzofuro H-5),

7.07e6.97 (m, 2H, 4-phenyl H-3 and H-5).

chloro- and hydroxy-substituted diphenyl benzofuro[3,2-b]pyri-

dines (6e23) were synthesized. To the mixture of benzofuran-

3(2H)-one intermediates 5 (R¹¼ a-f) and aryl pyridinium iodide

salts 2 (R ¼ a-f) in glacial acetic acid, was added anhydrous

ammonium acetate (10.0 equiv.). The mixture was stirred at 100 ^ꢀC

for 12e24 h. The mixture was then extracted with ethyl acetate,

washed with water and brine. The anhydrous magnesium sulfate

was used to dry the organic layer and ﬁltered. For the puriﬁcation,

silica gel column chromatography was utilized with the gradient

elution of ethyl acetate and n-hexane to afford solid compounds

6e23 in 8.9e36.5% yield.

¹³C NMR (62.5 MHz, DMSO-d₆)

d 157.39, 155.37, 151.43, 147.05,

143.60, 137.94, 133.89, 131.69, 131.48, 130.97, 130.01, 129.13 (2C),

128.93 (2C), 124.26, 123.19, 121.30, 120.83, 120.35, 119.63, 116.44,

112.85.

4.3.4. Synthesis of 3-(2-(2-chlorophenyl)benzofuro[3,2-b]pyridin-

4-yl)phenol (9)

The compound was synthesized as described in section 4.3 with

5e (0.24 g, 1.0 mmol), dry ammonium acetate (0.77 g, 10.0 mmol),

2a (0.36 g, 1.0 mmol) with glacial acetic acid (2 mL) to yield 0.14 g

(36.5%) of a white solid.

4.3.1. Synthesis of 2-(2-(2-chlorophenyl)benzofuro[3,2-b]pyridin-4-

yl)phenol (6)

The compound was synthesized as described in section 4.3 with

5d (0.24 g, 1.0 mmol), dry ammonium acetate (0.77 g, 10.0 mmol),

2a (0.36 g, 1.0 mmol) with glacial acetic acid (2 mL) to yield 66 mg

(17.7%) of a yellow solid.

TLC (ethyl acetate/n-hexane

¼

2:5 v/v) R_f

¼

0.25; Mp:

271.8e272.4 ^ꢀC; HPLC: Retention time: 7.99 min, purity: 98.4%; ESI-

MS: m/z calcd for C₂₃H₁₄ClNO₂[MþH]^þ: 372.08; found [MþH]^þ:

372.22.

TLC (ethyl acetate/n-hexane

¼

2:5 v/v) R_f

¼

0.28; Mp:

192.7e193.3 ^ꢀC; HPLC: Retention time: 9.27 min, purity: 100.0%;

¹H NMR (250 MHz, DMSO-d₆)

d 9.86 (s, 1H, 4-phenyl 3-OH) 8.21

S. Park et al. / European Journal of Medicinal Chemistry 127 (2017) 318e333

329

(d, J ¼ 7.65 Hz, 1H, benzofuro H-4), 7.89e7.85 (m, 2H, pyridine H-3

and benzofuro H-7), 7.76e7.49 (m, 8H, 2-phenyl H-3, H-4, H-5, H-6,

4-phenyl H-2, H-5, H-6 and benzofuro H-6), 7.41 (t, J ¼ 8.02 Hz, 1H,

benzofuro H-5), 6.95 (d, J ¼ 7.90 Hz, 1H, 4-phenyl H-4).

¹³C NMR (62.5 MHz, DMSO-d₆)

143.68, 139.10, 131.91, 131.47, 131.35, 130.21 (2C), 129.94, 129.77,

129.65, 127.26, 124.00, 123.36, 122.70, 120.96, 120.17, 115.99 (2C),

112.53.

d

159.01, 156.99, 153.01, 145.31,

¹³C NMR (62.5 MHz, DMSO-d₆)

d 157.85, 157.11, 152.93, 145.51,

143.91, 138.86, 134.16, 132.01, 131.44, 130.25, 130.07, 129.85, 127.35,

124.12, 122.62, 121.09, 119.39, 116.74, 115.48, 112.56.

4.3.8. Synthesis of 4-(2-(3-chlorophenyl)benzofuro[3,2-b]pyridin-

4-yl)phenol (13)

The compound was synthesized as described in section 4.3 with

5f (0.71 g, 3.0 mmol), dry ammonium acetate (2.31 g, 30.0 mmol),

2b (1.08 g, 3.0 mmol) with glacial acetic acid (5 mL) to yield 0.10 g

(8.9%) of a yellow solid.

4.3.5. Synthesis of 3-(2-(3-chlorophenyl)benzofuro[3,2-b]pyridin-

4-yl)phenol (10)

The compound was synthesized as described in section 4.3 with

5e (0.24 g, 1.0 mmol), dry ammonium acetate (0.77 g, 10.0 mmol),

2b (0.36 g, 1.0 mmol) with glacial acetic acid (2 mL) to yield 0.11 g

(29.6%) of a whitish yellow solid.

TLC (ethyl acetate/n-hexane

¼

2:5 v/v) R_f

¼

0.33; Mp:

270.5e271.1 ^ꢀC; HPLC: Retention time: 9.07 min, purity: 98.7%; ESI-

MS: m/z calcd for C₂₃H₁₄ClNO₂[MþH]^þ: 372.08; found [MþH]^þ:

372.42.

TLC (ethyl acetate/n-hexane

¼

2:5 v/v) R_f

¼

0.31; Mp:

223.9e224.5 ^ꢀC; HPLC: Retention time: 8.88 min, purity: 100.0%;

¹H NMR (250 MHz, DMSO-d₆)

d 10.09 (s, 1H, 4-phenyl 4-OH),

ESI-MS: m/z calcd for

C

₂₃H₁₄ClNO₂[MþH]^þ: 372.08; found

8.34 (br, 1H, benzofuro H-4), 8.27e8.19 (m, 3H, 2-phenyl H-2, H-6

and pyridine H-3), 8.1 (d, J ¼ 8.67 Hz, 2H, 4-phenyl H-2 and H-6),

7.83 (d, J ¼ 8.25 Hz, 1H, benzofuro H-7), 7.67 (td, J ¼ 7.2, 1.25 Hz, 1H,

benzofuro H-6), 7.58e7.48 (m, 3H, 2-phenyl H-3, H-5 and benzo-

furo H-5), 7.01 (d, J ¼ 8.7 Hz, 2H, 4-phenyl H-3 and H-5).

[MþH]^þ: 372.55.

¹H NMR (250 MHz, DMSO-d₆)

d 9.87 (s, 1H, 4-phenyl 3-OH)

8.32e8.22 (m, 4H, 2-phenyl H-2, H-6, pyridine H-3 and benzofuro

H-4), 7.82 (d, J ¼ 8.23 Hz, 1H, benzofuro H-7), 7.67 (t, J ¼ 7.5 Hz, 1H,

benzofuro H-6), 7.58e7.48 (m, 5H, 2-phenyl H-4, H-5, 4-phenyl H-2,

H-5 and H-6), 7.42 (t, J ¼ 7.62 Hz, 1H, benzofuro H-5), 6.97 (d,

J ¼ 8.42 Hz, 1H, 4-phenyl H-4).

¹³C NMR (62.5 MHz, DMSO-d₆)

d 159.30, 157.43, 151.80, 146.22,

144.27, 141.23, 134.00, 132.60, 130.87, 130.78 (2C), 130.12, 128.80,

126.89, 125.89, 124.48, 123.84, 123.01, 121.38, 116.88, 116.18 (2C),

112.89.

¹³C NMR (62.5 MHz, DMSO-d₆)

d 158.09, 157.58, 151.83, 146.43,

144.45, 141.09, 134.65, 134.10, 132.83, 130.98, 130.49, 130.31, 128.96,

126.96, 125.97, 124.50, 122.96, 121.51, 120.03, 117.90, 117.06, 116.07,

112.91.

4.3.9. Synthesis of 4-(2-(4-chlorophenyl)benzofuro[3,2-b]pyridin-

4-yl)phenol (14)

The compound was synthesized as described in section 4.3 with

5f (0.60 g, 2.5 mmol), dry ammonium acetate (1.93 g, 25.0 mmol),

2c (1.08 g, 3.0 mmol) with glacial acetic acid (5 mL) to yield 0.14 g

(14.7%) of a yellow solid.

4.3.6. Synthesis of 3-(2-(4-chlorophenyl)benzofuro[3,2-b]pyridin-

4-yl)phenol (11)

The compound was synthesized as described in section 4.3 with

5e (0.48 g, 2 mmol), dry ammonium acetate (1.54 g, 20.0 mmol), 2c

(0.72 g, 2.0 mmol) with glacial acetic acid (5 mL) to yield 0.20 g

(26.4%) of a pale yellow solid.

TLC (ethyl acetate/n-hexane

¼

2:5 v/v) R_f

¼

0.34; Mp:

285.8e286.2 ^ꢀC; HPLC: Retention time: 9.03 min, purity: 100.0%;

ESI-MS: m/z calcd for

C

₂₃H₁₄ClNO₂[MþH]^þ: 372.08; found

TLC (ethyl acetate/n-hexane

¼

2:5 v/v) R_f

¼

0.31; Mp:

[MþH]^þ: 372.13.

282.7e283.4 ^ꢀC; HPLC: Retention time: 8.83 min, purity: 97.9%; ESI-

MS: m/z calcd for C₂₃H₁₄ClNO₂[MþH]^þ: 372.08; found [MþH]^þ:

372.39.

¹H NMR (250 MHz, DMSO-d₆)

d 10.06 (s, 1H, 4-phenyl 4-OH),

8.31 (d, J ¼ 8.52 Hz, 2H, 2-phenyl H-2 and H-6), 8.24e8.21 (m, 2H,

benzofuro H-4 and pyridine H-3), 8.08 (d, J ¼ 8.60 Hz, 2H, 4-phenyl

H-2 and H-6), 7.83 (d, J ¼ 8.22 Hz, 1H, benzofuro H-7), 7.66 (t,

J ¼ 7.3 Hz, 1H, benzofuro H-6), 7.59e7.47 (m, 3H, benzofuro H-5, 2-

phenyl H-3 and H-5), 7.01 (d, J ¼ 8.57 Hz, 2H, 4-phenyl H-3 and H-

5).

¹H NMR (250 MHz, DMSO-d₆)

d 9.86 (s, 1H, 4-phenyl 3-OH)

8.34e8.22 (m, 4H, 2-phenyl H-2, H-6, pyridine H-3 and benzofuro

H-4), 7.85 (d, J ¼ 8.25 Hz, 1H, benzofuro H-7), 7.69 (t, J ¼ 7.3 Hz, 1H,

benzofuro H-6), 7.60e7.50 (m, 5H, 2-phenyl H-3, H-5, 4-phenyl H-2,

H-5 and H-6), 7.43 (t, J ¼ 7.87 Hz, 1H, benzofuro H-5), 6.97 (d,

J ¼ 8.07 Hz, 1H, 4-phenyl H-4).

¹³C NMR (62.5 MHz, DMSO-d₆)

d 159.28, 157.38, 152.09, 146.06,

144.19, 137.92, 133.88, 132.54, 131.43 (2C), 130.72 (2C), 129.03,

128.97 (2C), 124.29, 123.87, 123.05, 121.28, 116.63, 116.17 (2C),

112.85.

¹³C NMR (62.5 MHz, DMSO-d₆)

d 157.78, 157.22, 151.88, 145.94,

144.07, 137.50, 134.37, 133.69, 132.46, 130.10, 129.84, 128.73 (2C),

128.71 (2C), 124.07, 122.71, 121.04, 119.57, 117.25, 116.67, 115.70,

112.56.

4.3.10. Synthesis of 2-(4-(2-chlorophenyl)benzofuro[3,2-b]pyridin-

2-yl)phenol (15)

4.3.7. Synthesis of 4-(2-(2-chlorophenyl)benzofuro[3,2-b]pyridin-

4-yl)phenol (12)

The compound was synthesized as described in section 4.3 with

5f (0.48 g, 2.0 mmol), dry ammonium acetate (1.54 g, 20.0 mmol),

2a (0.72 g, 2.0 mmol) with glacial acetic acid (5 mL) to yield 86 mg

(11.6%) of a yellow solid.

The compound was synthesized as described in section 4.3 with

5a (0.51 g, 2.0 mmol), dry ammonium acetate (1.54 g, 20.0 mmol),

2d (0.85 g, 2.5 mmol) with glacial acetic acid (5 mL) to yield 0.24 g

(32.8%) of a yellow solid.

TLC (ethyl acetate/n-hexane ¼ 1:20 v/v) R_f¼ 0.20; Mp:

189.9e190.5 ^ꢀC; HPLC: Retention time: 7.63 min, purity: 97.7%; ESI-

MS: m/z calcd for C₂₃H₁₄ClNO₂[MþH]^þ: 372.08; found [MþH]^þ:

372.37.

TLC (ethyl acetate/n-hexane

¼

2:5 v/v) R_f

¼

0.29; Mp:

195.6e196.1 ^ꢀC; HPLC: Retention time: 8.25 min, purity: 98.63%;

ESI-MS: m/z calcd for

C

₂₃H₁₄ClNO₂[MþH]^þ: 372.08; found

¹H NMR (250 MHz, CDCl₃)

d 14.02 (s, 1H, 2-phenyl 2-OH), 8.21

[MþH]^þ: 372.44.

(d, J ¼ 7.6 Hz, 1H, benzofuro H-4), 8.01 (s, 1H, pyridine H-3), 7.86

(dd, J ¼ 8.02, 1.47 Hz, 1H, benzofuro H-7), 7.65e7.43 (m, 7H, ben-

zofuro H-5, H-6, 2-phenyl H-6, 4-phenyl H-3, H-4, H-5 and H-6),

7.32 (td, J ¼ 7.07, 1.55 Hz, 1H, 2-phenyl H-5), 7.09 (dd, J ¼ 8.22,

1.2 Hz, 1H, 2-phenyl H-3), 6.9 (td, J ¼ 8.07, 1.27 Hz, 1H, 2-phenyl H-

4).

¹H NMR (250 MHz, DMSO-d₆)

d 9.98 (s, 1H, 4-phenyl 4-OH), 8.2

(d, J ¼ 7.6 Hz, 1H, benzofuro H-4), 8.02 (d, 2H, d, J ¼ 8.6 Hz, 2H, 4-

phenyl H-2 and H-6), 7.89e7.85 (m, 2H, pyridine H-3 and benzofuro

H-7), 7.75e7.46 (m, 6H, 2-phenyl H-3, H-4, H-5, H-6, benzofuro H-5

and H-6), 7.01 (d, J ¼ 8.65 Hz, 2H, 4-phenyl H-3 and H-5).

330

S. Park et al. / European Journal of Medicinal Chemistry 127 (2017) 318e333

¹³C NMR (62.5 MHz, CDCl₃)

d

159.50, 158.09, 154.03, 146.48,

4.3.14. Synthesis of 3-(4-(3-chlorophenyl)benzofuro[3,2-b]pyridin-

141.38, 133.48, 133.19, 133.00, 131.72, 131.42, 130.93, 130.62, 130.10,

127.42, 126.95, 124.25, 122.49, 121.56, 119.80, 119.43, 119.09, 118.85,

112.82.

2-yl)phenol (19)

The compound was synthesized as described in section 4.3 with

5b (0.51 g, 2.0 mmol), dry ammonium acetate (1.54 g, 20.0 mmol),

2e (0.85 g, 2.5 mmol) with glacial acetic acid (5 mL) to yield 0.26 g

(33.8%) of a yellow solid.

4.3.11. Synthesis of 3-(4-(2-chlorophenyl)benzofuro[3,2-b]pyridin-

2-yl)phenol (16)

The compound was synthesized as described in section 4.3 with

5a (0.77 g, 3.0 mmol), dry ammonium acetate (2.31 g, 30.0 mmol),

2e (1.36 g, 4.0 mmol) with glacial acetic acid (5 mL) to yield 0.22 g

(20.1%) of a white solid.

TLC (ethyl acetate/n-hexane

¼

2:5 v/v) R_f

¼

0.28; Mp:

220.9e221.5 ^ꢀC; HPLC: Retention time: 8.92 min, purity: 97.9%; ESI-

MS: m/z calcd for C₂₃H₁₄ClNO₂[MþH]^þ: 372.08; found [MþH]^þ:

372.53.

¹H NMR (250 MHz, DMSO-d₆)

d 9.52 (br s, 1H, 2-phenyl 3-OH),

8.26e8.11 (m, 4H, benzofuro H-4, 4-phenyl H-2, H-6 and pyridine

H-3), 7.85 (d, J ¼ 8.25 Hz, 1H, benzofuro H-7), 7.72e7.63 (m, 5H,

benzofuro H-6, 2-phenyl H-2, H-6, 4-phhenyl H-4 and H-5), 7.53

(td, J ¼ 7.9, 0.77 Hz, 1H, benzofuro H-5), 7.33 (t, J ¼ 7.87 Hz, 1H, 2-

phenyl H-5), 6.89e6.85 (m, 1H, 2-phenyl H-4).

TLC (ethyl acetate/n-hexane

¼

2:5 v/v) R_f

¼

0.32; Mp:

210.2e210.9 ^ꢀC; HPLC: Retention time: 7.66 min, purity: 99.1%; ESI-

MS: m/z calcd for C₂₃H₁₄ClNO₂[MþH]^þ: 372.08; found [MþH]^þ:

372.82.

¹H NMR (250 MHz, CDCl₃)

d

8.34 (d, J ¼ 7.82 Hz, 1H, benzofuro

¹³C NMR (62.5 MHz, DMSO-d₆)

d 157.96, 157.43, 153.69, 145.68,

H-4), 7.82e7.79 (m, 2H, 2-phenyl H-2 and pyridine H-3), 7.62e7.37

(m, 8H, benzofuro H-5, H-6, H-7, 2-phenyl H-6, 4-phenyl H-3, H-4,

H-5 and H-6), 7.32 (t, J ¼ 7.8 Hz, 1H, 2-phenyl H-5), 6.89 (dd,

J ¼ 8.02, 2.47 Hz, 1H, 2-phenyl H-4).

144.24, 140.19, 135.59, 134.03, 131.06, 130.77, 130.01, 129.90, 129.63,

128.63, 127.83, 124.35, 122.94, 121.14, 118.19, 117.59, 116.15, 114.10,

112.85.

¹³C NMR (62.5 MHz, CDCl₃)

d 158.13, 156.90, 153.86, 147.04,

144.69, 141.16, 133.50, 133.45, 131.76, 131.64, 130.62, 130.51, 130.32,

129.65, 127.27, 123.97, 123.51, 122.01, 120.83, 119.71.116.39, 114.93,

112.51.

4.3.15. Synthesis of 4-(4-(3-chlorophenyl)benzofuro[3,2-b]pyridin-

2-yl)phenol (20)

The compound was synthesized as described in section 4.3 with

5b (0.51 g, 2.0 mmol), dry ammonium acetate (1.54 g, 20.0 mmol),

2f (0.85 g, 2.5 mmol) with glacial acetic acid (5 mL) to yield 0.11 g

(23.5%) of a yellow solid.

4.3.12. Synthesis of 4-(4-(2-chlorophenyl)benzofuro[3,2-b]pyridin-

2-yl)phenol (17)

The compound was synthesized as described in section 4.3 with

5a (0.77, 3.0 mmol), dry ammonium acetate (2.30 g, 30.0 mmol), 2f

(1.36 g, 4.0 mmol) with glacial acetic acid (5 mL) to yield 0.12 g

(12.3%) of a yellow solid.

TLC (ethyl acetate/n-hexane

¼

2:5 v/v) R_f

¼

0.28; Mp:

241.6e242.1 ^ꢀC; HPLC: Retention time: 8.09 min, purity: 99.9%; ESI-

MS: m/z calcd for C₂₃H₁₄ClNO₂[M]^þ: 371.07; found [M]^þ: 371.49.

¹H NMR (250 MHz, DMSO-d₆)

d 9.82 (br s, 1H, 2-phenyl 4-OH),

TLC (ethyl acetate/n-hexane

¼

2:5 v/v) R_f

¼

0.29; Mp:

260.1e260.7 ^ꢀC; HPLC: Retention time: 6.88 min, purity: 97.1%; ESI-

MS: m/z calcd for C₂₃H₁₄ClNO₂[MþH]^þ: 372.08; found [MþH]^þ:

372.98.

8.25e8.09 (m, 6H, benzofuro H-4, 2-phenyl H-2, H-6, 4-phenyl H-2,

H-6 and pyridine H-3), 7.84 (d, J ¼ 8.25 Hz, 1H, benzofuro H-7),

7.69e7.60 (m, 3H, benzofuro H-6, 4-phenyl H-4 and H-5), 7.51 (t,

J ¼ 7.22 Hz, 1H, benzofuro H-5), 6.91 (d, J ¼ 8.67 Hz, 2H, 2-phenyl H-

3 and H-5).

¹H NMR (250 MHz, DMSO-d₆)

d 9.87 (s, 1H, 2-phenyl 4-OH), 8.25

(d, J ¼ 7.65 Hz,1H, benzofuro H-4), 8.06 (d, J ¼ 8.47 Hz, 2H, 2-phenyl

H-2 and H-6), 7.91 (s, 1H, pyridine H-3), 7.74e7.47 (m, 7H, benzo-

furo H-5, H-6, H-7, 4-phenyl H-3, H-4, H-5 and H-6), 6.89 (d,

J ¼ 8.5 Hz, 2H, 2-phenyl H-3 and H-5).

¹³C NMR (62.5 MHz, DMSO-d₆)

d 158.72, 157.39, 153.93, 145.42,

144.09, 135.81, 134.11, 131.16, 130.86, 129.94, 129.83, 129.69, 128.81

(2C), 128.70, 127.94, 124.34, 123.11, 121.26, 116.63, 115.77 (2C),

112.92.

¹³C NMR (62.5 MHz, DMSO-d₆)

d 158.82, 157.53, 153.57, 145.99,

143.55, 138.30, 133.31, 132.57, 132.25, 131.41, 130.26, 130.17, 129.86,

128.82 (2C), 1.28.13, 124.51, 123.28, 121.47, 119.08, 116.07 (2C),

112.97.

4.3.16. Synthesis of 2-(4-(4-chlorophenyl)benzofuro[3,2-b]pyridin-

2-yl)phenol (21)

4.3.13. Synthesis of 2-(4-(3-chlorophenyl)benzofuro[3,2-b]pyridin-

2-yl)phenol (18)

The compound was synthesized as described in section 4.3 with

5b (0.77 g, 3.0 mmol), dry ammonium acetate (2.31 g, 30.0 mmol),

2d (1.36 g, 4.0 mmol) with glacial acetic acid (5 mL) to yield 0.22 g

(20.1%) of a white solid.

The compound was synthesized as described in section 4.3 with

5c (0.51 g, 2.0 mmol), dry ammonium acetate (1.54 g, 20.0 mmol),

2d (0.85 g, 2.5 mmol) with glacial acetic acid (5 mL) to yield 0.17 g

(23.4%) of a yellow solid.

TLC (ethyl acetate/n-hexane ¼ 1:20 v/v) R_f¼ 0.23; Mp:

188.9e189.5 ^ꢀC; HPLC: Retention time: 9.74 min, purity: 97.2%; ESI-

MS: m/z calcd for C₂₃H₁₄ClNO₂[MþH]^þ: 372.08; found [MþH]^þ:

372.26.

TLC (ethyl acetate/n-hexane ¼ 1:20 v/v) R_f¼ 0.21; Mp:

173.8e174.4 ^ꢀC; HPLC: Retention time: 12.20 min, purity: 98.1%;

ESI-MS: m/z calcd for C₂₃H₁₄ClNO₂[M]^þ: 371.07; found [M]^þ: 371.4.

¹H NMR (250 MHz, CDCl₃)

d 13.95 (s, 1H, 2-phenyl 2-OH), 8.16

¹H NMR (250 MHz, DMSO-d₆)

d

13.02 (br s, 1H, 2-phenyl 2-OH),

(d, J ¼ 7.67 Hz, 1H, benzofuro H-4), 8.04 (s, 1H, pyridine H-3), 7.96

(d, J ¼ 8.45 Hz, 2H, 4-phebyl H-2 and H-6), 7.88 (d, J ¼ 8.0 Hz, 1H,

benzofuro H-7), 7.64e7.54 (m, 4H, benzofuro H-6, 2-phenyl H-6, 4-

phenyl H-3 and H-5), 7.45 (td, J ¼ 7.75, 1.5 Hz, 1H, benzofuro H-5),

7.32 (td, J ¼ 8.2, 1.05 Hz, 1H, 2-phenyl H-5), 7.07 (d, J ¼ 8.05 Hz, 1H,

2-phenyl H-3), 6.94 (t, J ¼ 7.8 Hz, 1H, 2-phenyl H-4).

8.37 (s, 1H, pyridine H-3), 8.19e8.07 (m, 4H, benzofuro H-4, 2-

phenyl H-6, 4-phenyl H-2 and H-6), 7.83 (d, J ¼ 8.27 Hz, 1H, ben-

zofuro H-7), 7.71e7.63 (m, 3H, benzofuro H-6, 4-phenyl H-4 and H-

5), 7.52 (t, J ¼ 7.32 Hz, 1H, benzofuro H-5), 7.31 (t, J ¼ 7.62 Hz, 1H, 2-

phenyl H-5), 6.98e6.92 (m, 2H, 2-phenyl H-3 and H-4).

¹³C NMR (62.5 MHz, DMSO-d₆)

d

158.06, 157.34, 153.92, 145.48,

¹³C NMR (62.5 MHz, CDCl₃)

d 159.52, 158.01, 154.60, 146.00,

141.57, 135.21, 134.13, 132.24, 132.19, 131.31, 131.23, 130.57, 130.05,

128.81, 128.67, 128.03, 124.70, 121.70, 121.15, 120.61, 119.53, 117.90,

113.03.

141.83, 136.31, 133.31, 132.30, 131.47, 130.41 (2C), 130.13, 129.67

(2C), 126.81, 124.33, 122.32, 121.58, 119.73, 119.41, 118.91, 116.10,

112.73.

S. Park et al. / European Journal of Medicinal Chemistry 127 (2017) 318e333

331

4.3.17. Synthesis of 3-(4-(4-chlorophenyl)benzofuro[3,2-b]pyridin-

2-yl)phenol (22)

The compound was synthesized as described in section 4.3 with

5c (0.51 g, 2.0 mmol), dry ammonium acetate (1.54 g, 20.0 mmol),

2e (0.85 g, 2.5 mmol) with glacial acetic acid (5 mL) to yield 0.22 mg

(29.1%) of a pale yellow solid.

compound 14 on intracellular topo-mediated DNA relaxation

in vivo, we prepared nuclear fractionations of compound-treated

and untreated cells according to the method reported previously

[31]. The DNA relaxation activity of the prepared nuclear extract

was deﬁned through the determination of the amount of each

nuclear extraction product that could completely relax 100 ng

pBR322 DNA (Fermentas, USA) by incubating at 37 ^ꢀC for 20 min.

The DNA reacted with nuclear extract was separated via electro-

phoresis on a 1.0% agarose gel at 50 V for 50 min with TAE as the

running buffer. The gel staining and DNA band visualizing were

performed as the same method used for recombinant DNA topo I

and II in vitro relaxation assay.

TLC (ethyl acetate/n-hexane

¼

2:5 v/v) R_f

¼

0.34; Mp:

223.7e224.3 ^ꢀC; HPLC: Retention time: 8.26 min, purity: 96.7%;

ESI-MS: m/z calcd for

C

₂₃H₁₄ClNO₂[MþH]^þ: 372.08; found

[MþH]^þ: 372.33.

¹H NMR (250 MHz, DMSO-d₆)

d 9.65 (s, 1H, 2-phenyl 3-OH),

8.26e8.18 (m, 4H, benzofuro H-4, 4-phenyl H-2, H-6 and pyridine

H-3), 7.84 (d, J ¼ 8.3 Hz, 1H, benzofuro H-7), 7.71e7.64 (m, 5H,

benzofuro H-6, 2-phenyl H-2, H-6, 4-phhenyl H-3 and H-5), 7.53 (t,

J ¼ 7.57, Hz, 1H, benzofuro H-5), 7.33 (t, J ¼ 7.75 Hz, 1H, 2-phenyl H-

5), 6.87 (dd, 1H, J ¼ 6.77, 2.3 Hz, 1H, 2-phenyl H-4).

4.4.2. kDNA decatenation assay

The assay was performed by the method previously published

[31]. kDNA 75 ng was added in the reaction buffer containing

50 mM Tris-HCl (pH 8.0), 120 mM KCl, 10 mM MgCl2, 0.5 mM

¹³C NMR (62.5 MHz, DMSO-d₆)

d 158.05, 157.47, 153.71, 145.93,

144.28, 140.30, 134.77, 132.38, 131.05, 130.97 (2C), 130.11, 130.06,

129.39 (2C), 124.45, 123.01, 121.28, 118.21, 117.48, 116.22, 114.11,

112.92.

dithiothreitol, 0.5 mM ATP, 30

lowed by addition of compound as designated concentration in

ﬁgure legend. A total reaction volume was 10 L. Reactions were

mg/mL bovine serum albumin fol-

m

initiated by the addition of 3 units of topo II, and mixtures were

4.3.18. Synthesis of 4-(4-(4-chlorophenyl)benzofuro[3,2-b]pyridin-

2-yl)phenol (23)

incubated for 30 min at 37 ^ꢀC. The reaction was terminated by the

addition of 2.5 mL of stock solution (5% SDS, 25% ﬁcoll, and 0.05%

The compound was synthesized as described in section 4.3 with

5c (0.62 g, 2.5 mmol), dry ammonium acetate (1.93 g, 25.0 mmol),

2f (1.02 g, 3.0 mmol) with glacial acetic acid (5 mL) to yield 0.15 g

(16.2%) of a yellow solid.

bromphenol blue) followed by treatment with 0.25 mg/ml pro-

teinase K (Sigma, USA) at 37 ^ꢀC for 30 min to eliminate the protein.

Samples were resolved by electrophoresis on a 1.2% (w/v) agarose

gel containing 0.5 mg/ml ethidium bromide in TAE buffer (100 mM

TLC (ethyl acetate/n-hexane

¼

2:5 v/v) R_f

¼

0.30; Mp:

Tris-acetate and 2 mM Na2EDTA, pH 8.3). DNA bands were visu-

alized as followed DNA topo II relaxation assay.

285.2e286.1 ^ꢀC; HPLC: Retention time: 8.27 min, purity: 96.1%; ESI-

MS: m/z calcd for C₂₃H₁₄ClNO₂[MþH]^þ: 372.08; found [MþH]^þ:

372.56.

4.4.3. Antiproliferative assay

¹H NMR (250 MHz, DMSO-d₆)

d

9.82 (s, 1H, 2-phenyl 4-OH),

Cancer cells were cultured according to the supplier's in-

structions. Cells were seeded in 96-well plates at a density of 2e4 x

10⁴cells per well with incubation for overnight in 0.1 mL of media

supplied with 10% fetal bovine serum (FBS, Hyclone, USA) in 5% CO₂

incubator at 37 ^ꢀC. After FBS starvation for 4 h, culture medium in

each well was exchanged with 0.1 mL aliquots of medium con-

taining graded concentrations of compounds followed by addi-

8.25e8.11 (m, 6H, benzofuro H-4, 2-phenyl H-2, H-6, 4-phenyl H-2,

H-6 and pyridine H-3), 7.81 (d, J ¼ 8.25 Hz, 1H, benzofuro H-7),

7.7e7.62 (m, 3H, benzofuro H-6, 4-phenyl H-3 and H-5), 7.51 (t,

J ¼ 7.42 Hz,1H, benzofuro H-5), 6.91 (d, J ¼ 8.67 Hz, 2H, 2-phenyl H-

3 and H-5).

¹³C NMR (62.5 MHz, DMSO-d₆)

d 158.72, 157.39, 153.96, 145.44,

144.11, 134.72, 132.55, 131.10, 130.95 (2C), 129.96, 129.90, 129.40

(2C), 128.78 (2C), 124.35, 123.15, 121.30, 116.44, 115.82 (2C), 112.87.

tional incubation for 72 h. Then each well was added with 5 mL of

the cell counting kit-8 solution (Dojindo, Japan) followed by addi-

tional incubation for 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

IC₅₀values, the absorbance readings at 450 nm were ﬁtted to the

four-parameter logistic equation using Table Curve 2D (SPSS Inc.,

USA). The reference compounds such as adriamycin, etoposide and

camptothecin were purchased from Sigma (USA).

4.4. Pharmacology

4.4.1. DNA topoisomerase I and II relaxation assay

All the test compounds were dissolved in DMSO at a concen-

tration of 20 mM as a stock solution and stored under ꢂ20 ^ꢀC until

needed. The DNA topoisomerase I and II inhibitory activity of each

compound was measured as follows according to the manufac-

turer's protocol. A mixture comprising of 100 ng supercoiled

pBR322 plasmid DNA (Thermo Scientiﬁc, USA) and 1 unit of re-

combinant human DNA topo I (TopoGEN INC., USA) or topo II (Usb

Corp., USA) was incubated with and without the prepared com-

pounds in the assay buffer (For topo I, 10 mM Tris-HCl (pH 7.9)

containing 150 mM NaCl, 0.1% bovine serum albumin (BSA), 0.1 mM

spermidine and 5% glycerol; For topo II, 10 mM Tris-HCl (pH 7.9)

containing 50 mM NaCl, 50 mM KCl, 5 mM MgCl₂, 1 mM EDTA,

4.4.4. Cleavable complex assay

The mixture of 250 ng of supercoiled DNA pBR322 and 3 units of

human topoisomerase II with or without compound at the desig-

nated concentrations in the ﬁgure legend was incubated at 37 ^ꢀC for

30 min in the topo II relaxation assay buffer. The reaction was then

terminated by the addition of 2.5

mL of stop solution (5% SDS, 25%

ﬁcoll and 0.05% bromophenol blue) followed by treatment of 2

mL of

0.25 mg/mL proteinase K (Sigma, USA) with continuous incubation

1 mM ATP and 15

ﬁnal volume of 10

m

g/mL BSA) for 30 min at 37 ^ꢀC. The reaction in a

L was quenched by adding 1 L of the stop

at 45 ^ꢀC for 30 min to eliminate the protein. Samples were elec-

m

trophoresed on a 1% (w/v) agarose gel containing 0.5 mg/mL EtBr at

solution (For topo I, 10% SDS solution containing 0.2% bromophenol

blue, 0.2% xylene cyanol and 30% glycerol; for topo II, 7 mM EDTA).

The reaction products were analyzed on 0.8% agarose gel at 50 V for

1 h with TAE as the running buffer. The gels were stained in an EtBr

30 V for 6 h in TAE running buffer. DNA bands on the gel were

detected by UV and visualized by AlphaImager™.

4.4.5. Band depletion assay

solution (0.5

mg/mL). DNA bands were visualized by trans-

HCT15 cells were seeded overnight at a density of 2 ꢁ 10⁵cells

per well. The cells were treated for 2 h at 37 ^ꢀC with each of eto-

poside and compound 14, and co-treated with etoposide and

illumination with UV light and were quantitated using Alpha Tech

Imager (Alpha Innotech Corporation). To analyze the effect of

332

S. Park et al. / European Journal of Medicinal Chemistry 127 (2017) 318e333

compound 14 followed by cell harvesting. The cell pellet after

centrifugation (4 ^ꢀC, 3200 rpm, and 3 min) was washed with 1 mL

of PBS and then ice-incubated for 1.5 h, followed by lysis with

denaturing agent (62.5 mM Tris-HCl (pH 6.8), 1 mM EDTA and 2%

SDS) and sonication (10e20 bursts, 2 s). The samples were ice-

incubated for 20 min and centrifuged for 20 min (4 ^ꢀC and

12,000 rpm) again. Finally the supernatant of sample was taken to

perform Western blot analysis on 10% SDS-PAGE gels.

Acknowledgements

This research was supported by the Basic Science Research

Program through the National Research Foundation of Korea (NRF)

funded by the Ministry of Education, Science and Technology (NRF-

2015R1A2A1A15054445 and NRF-2013R1A1A2060408) and by the

Korean Health Technology R&D Project funded by Ministry of

Health & Welfare, Republic of Korea (HI14C2469).

4.4.6. Comet assay

Appendix A. Supplementary data

To evaluate DNA damage, comet assay was performed using

single-cell gel electrophoresis with a Trevigen kit (Gaithersburg,

USA) according to the method previously reported [43]. Brieﬂy,

HCT15 cells, seeded in a density of 1 ꢁ 10⁵cells per well in six-well

plates were treated with compound 14 and etoposide for 24 h at the

designated concentration in ﬁgure legend and harvested by tryp-

sinization followed by resuspending cells in 1 mL of ice-cold PBS.

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.ejmech.2017.01.003.

References

[1] G.I. Evan, K.H. Vousden, Proliferation, cell cycle and apoptosis in cancer, Na-

ture 411 (2001) 342e348.

[2] S.Z. Millis, S. Ejadi, M.J. Demeure, Molecular pro ﬁ ling of refractory adreno-

cortical cancers and predictive biomarkers to therapy, Biomark. Cancer 7

(2015) 69e76.

[3] J.C. Wang, Cellular roles of DNA topoisomerases: a molecular perspective, Nat.

Rev. Mol. Cell Biol. 3 (2002) 430e440.

[4] J.C. Wang, DNA topoisomerases, Annu. Rev. Biochem. 65 (1996) 635e692.

[5] J.C. Wang, DNA topoisomerases, Annu. Rev. Biochem. 54 (1985) 665e697.

[6] A.Y. Chen, L.F. Liu, DNA Topoisomerases: essential enzymes and lethal targets,

Annu. Rev. Pharmacol. Toxicol. 34 (1994) 191e218.

[7] P. Forterre, S. Gribaldo, D. Gadelle, M.-C. Serre, Origin and evolution of DNA

topoisomerases, Biochimie 89 (2007) 427e446.

[8] J.L. Nitiss, Targeting DNA topoisomerase II in cancer chemotherapy, Nat. Rev.

Cancer 9 (2009) 338e350.

[9] M. Li, Y. Liu, Topoisomerase I in human disease pathogenesis and treatments,

Genomics Proteomics Bioinforma. 14 (2016) 166e171.

[10] D. Gewirtz, A critical evaluation of the mechanisms of action proposed for the

antitumor effects of the anthracycline antibiotics adriamycin and daunoru-

bicin, Biochem. Pharmacol. 57 (1999) 727e741.

Then, 8

mL of resuspened cells were mixed with 80 mL of low-

melting agarose at 37 ^ꢀC, spread on slides and solidiﬁed in the

dark for 40 min at 4 ^ꢀC. Slides were lysed in ice-cold lysis solution in

the dark for 30 min at 4 ^ꢀC and then submerged in a fresh alkaline

solution (pH > 13) at room temperature for 30 min to allow alkaline

unwinding. Electrophoresis was performed under alkaline condi-

tions for 20 min for 15 V. Slides were rinsed twice with distilled

water, once with 70% ethanol and stained with SYBR Green (Tre-

vigen INC., USA) in a TE buffer for 5 min in the dark at 4 ^ꢀC. Comet

images were obtained using an inverted ﬂuorescence microscope

(Zeiss, Axiovert 200) at 10ꢁ magniﬁcation and percent DNA in tail

was analyzed by Komet 5.0 software (Kinetic imaging Ltd, UK). Data

were represented both by imaging and graphically by randomly

selecting comet lengths of HCT15 cells.

[11] I. Husain, J.L. Mohler, H.F. Seigler, J.M. Besterman, Elevation of topoisomerase I

messenger RNA, protein, and catalytic activity in human tumors: demon-

stration of tumor-type speci ﬁ city and implications for cancer chemotherapy,

Cancer Res. 54 (1994) 539e546.

[12] J.A. Holden, D.H. Rolfson, C.T. Wittwer, Human DNA topoisomerase II: eval-

uation of enzyme activity in normal and neoplastic tissues, Biochem. (Mosc.)

29 (1990) 2127e2134.

[13] M.M. Heck, W.N. Hittelman, W.C. Earnshaw, Differential expression of DNA

topoisomerases I and II during the eukaryotic cell cycle, Proc. Natl. Acad. Sci.

85 (1988) 1086e1090.

4.4.7. Competitive EtBr displacement assay

ctDNA (30 mM) was mixed with EtBr (20 mM) in the 10 mM tris

buffer solution (pH 7.2), followed by incubation for 30 min at room

temperature with shaking. Each compound (m-AMSA and com-

pound 14) was added to the well containing the mixture of EtBr and

ctDNA with increased concentrations up to 40 mM as designated in

the ﬁgure legend. The DMSO was added for untreated control in the

same amount as compound addition and the ﬁnal DMSO amount

was equally 1% for all samples. After addition of compound, the

plate was continuously incubated for 30 min at room temperature

with shaking. The ﬂuorescence intensity of EtBr was measured by

TECAN multimode microplate reader (Swizerland) with excitation

at 471 nm. The emission spectrum was collected at 500e700 nm.

[14] C.M. Whitacre, E. Zborowska, N.H. Gordon, W. Mackay, N.A. Berger, Topotecan

increases topoisomerase II a levels and sensitivity to treatment with etoposide

in schedule-dependent process, Cancer Res. 57 (1997) 1425e1428.

[15] W.A. Denny, Emerging DNA topoisomerase inhibitors as anticancer drugs,

Expert Opin. Emerg. Drugs 9 (2004) 105e133.

[16] S. Wang, W. Miller, J. Milton, N. Vicker, A. Stewart, P. Charlton, P. Mistry,

D. Hardick, W.A. Denny, Structureeactivity relationships for analogues of the

phenazine-based dual topoisomerase I/II inhibitor XR11576, Bioorg. Med.

Chem. Lett. 12 (2002) 415e418.

[17] C. Palma, M. Criscuoli, A. Lippi, M. Muratori, S. Mauro, C.A. Maggi, Effect of the

aromatase inhibitor, MEN 11066, on growth of two different MCF-7 sublines,

Eur. J. Pharmacol. 409 (2000) 93e101.

4.4.8. Western blotting

HCT15 cells were grown on 60 mm tissue culture dishes at

1 ꢁ 10⁶cells until reaching 80% conﬂuency. The cells were then

[18] Y. Xia, Y. Jin, N. Kaur, Y. Choi, K. Lee, HIF-1 a inhibitors: synthesis and biological

evaluation of novel moracin O and P analogues, Eur. J. Med. Chem. 46 (2011)

2386e2396.

[19] K. Suzuki, T. Okawara, T. Higashijima, K. Yokomizo, T. Mizushima, M. Otsuka,

Inhibitory activities against topoisomerase I and II by isoaurostatin derivatives

and their structureeactivity relationships, Bioorg. Med. Chem. Lett. 15 (2005)

2065e2068.

treated with compound 14 in concentrations of 10 and 30 mM for

24 h followed by lysis with a lysis buffer solution containing 50 mM

Tris HCl, 300 mM NaCl, 1% Triton X-100, 10% glycerol, 1.5 mM MgCl₂,

1 mM CaCl₂, 1 mM PMSF and 1% protease inhibitor cocktail. 60 mg of

protein per sample was resolved by 12% SDS-PAGE and transferred

to a PVDF membrane (Millipore, USA). The membranes were

blocked with 5% skim milk in Tris buffered saline containing 0.1%

tween 20 (TBST) and probed with primary antibodies in a dilution

ratio of 1:1000 for 2e3 h. The blots were washed, exposed to HRP-

conjugated anti-rabbit IgG (Cell Signaling Technology Inc. USA) in a

dilution ratio of 1:2000 for 2 h, and detected with ECL western

blotting detection reagent (Animal genetics Inc, Korea). All primary

antibodies used were purchased from Cell Signaling Technology

Inc. (USA). Western blot images were taken by LAS-3000 (Fuji Photo

Film Co., Ltd, Japan) and analyzed using Multi-Gauge Software (Fuji

Photo Film Co. Ltd., Japan).

[20] K. Asoh, M. Kohchi, I. Hyoudoh, T. Ohtsuka, M. Masubuchi, K. Kawasaki,

H. Ebiike, Y. Shiratori, T.A. Fukami, O. Kondoh, T. Tsukaguchi, N. Ishii, Y. Aoki,

N. Shimma, M. Sakaitani, Synthesis and structureeactivity relationships of

novel benzofuran farnesyltransferase inhibitors, Bioorg. Med. Chem. Lett. 19

(2009) 1753e1757.

[21] Y. Xiang, B. Hirth, G. Asmussen, H.-P. Biemann, K.A. Bishop, A. Good,

M. Fitzgerald, T. Gladysheva, A. Jain, K. Jancsics, J. Liu, M. Metz, A. Papoulis,

R. Skerlj, J.D. Stepp, R.R. Wei, The discovery of novel benzofuran-2-carboxylic

acids as potent Pim-1 inhibitors, Bioorg. Med. Chem. Lett. 21 (2011)

3050e3056.

[22] J.J. Buggy, Z.A. Cao, K.E. Bass, E. Verner, S. Balasubramanian, L. Liu, B.E. Schultz,

P.R. Young, S.A. Dalrymple, CRA-024781: a novel synthetic inhibitor of histone

deacetylase enzymes with antitumor activity in vitro and in vivo, Am. Assoc.

Cancer Res. 5 (2006) 1309e1317.

[23] I.N. Gaisina, F. Gallier, A.V. Ougolkov, K.H. Kim, T. Kurome, S. Guo, D. Holzle,

S. Park et al. / European Journal of Medicinal Chemistry 127 (2017) 318e333

333

D.N. Luchini, S.Y. Blond, D.D. Billadeau, A.P. Kozikowski, From

product lead to the identi ﬁ cation of potent and selective benzofuran-3-yl-

(indol-3-yl)maleimides as glycogen synthase kinase 3 inhibitors that sup-

a

natural

camptothecin ꢂ DNA ꢂ topoisomerase

Chem. Soc. 127 (2005) 9960e9961.

I

ternary cleavage complexes, J. Am.

b

[33] P. Thapa, Y. Jahng, P.-H. Park, J.-G. Jee, Y. Kwon, E.-S. Lee, 2,4-diaryl benzofuro

[3,2-b]pyridine derivatives: design, synthesis, and evaluation of topoisomer-

ase inhibitory activity and cytotoxicity, Bull. Korean Chem. Soc. 34 (2013)

3073e3082.

press proliferation and survival of pancreatic cancer cells, J. Med. Chem. 52

(2009) 1853e1863.

ꢀ

[24] C. Salome, N. Ribeiro, T. Chavagnan, F. Thuaud, M. Serova, A. de Gramont,

ꢀ

€

S. Faivre, E. Raymond, L. Desaubry, Benzofuran derivatives as anticancer in-

[34] F. KroHnke, The speci ﬁ c synthesis of pyridines and oligopyridines 1, Synthesis

hibitors of mTOR signaling, Eur. J. Med. Chem. 81 (2014) 181e191.

[25] R. Wilcken, M.O. Zimmermann, A. Lange, A.C. Joerger, F.M. Boeckler, Principles

and applications of halogen bonding in medicinal chemistry and chemical

biology, J. Med. Chem. 56 (2013) 1363e1388.

[26] Z. Xu, Z. Yang, Y. Liu, Y. Lu, K. Chen, W. Zhu, Halogen bond: its role beyond

drugetarget binding af ﬁ nity for drug discovery and development, J. Chem. Inf.

Model 54 (2014) 69e78.

[27] M.O. Zimmermann, A. Lange, R. Wilcken, M.B. Cieslik, T.E. Exner, A.C. Joerger,

P. Koch, F.M. Boeckler, Halogen-enriched fragment libraries as chemical

probes for harnessing halogen bonding in fragment-based lead discovery,

Future Med. Chem. 6 (2014) 617e639.

[28] T.M. Kadayat, S. Park, K.-Y. Jun, T.B.T. Magar, G. Bist, A. Shrestha, Y. Na,

Y. Kwon, E.-S. Lee, Effect of chlorine substituent on cytotoxic activities: design

and synthesis of systematically modi ﬁ ed 2,4-diphenyl-5H-indeno[1,2-b]pyr-

idines, Bioorg. Med. Chem. Lett. 26 (2016) 1726e1731.

[29] T.M. Kadayat, C. Song, Y. Kwon, E.-S. Lee, Modi ﬁ ed 2,4-diaryl-5H-indeno[1,2-

b]pyridines with hydroxyl and chlorine moiety: synthesis, anticancer activity,

and structureeactivity relationship study, Bioorg. Chem. 62 (2015) 30e40.

[30] R. Karki, K.-Y. Jun, T.M. Kadayat, S. Shin, T.B. Thapa Magar, G. Bist, A. Shrestha,

Y. Na, Y. Kwon, E.-S. Lee, A new series of 2-phenol-4-aryl-6-chlorophenyl

pyridine derivatives as dual topoisomerase I/II inhibitors: synthesis, biolog-

ical evaluation and 3D-QSAR study, Eur. J. Med. Chem. 113 (2016) 228e245.

[31] H.-B. Kwon, C. Park, K.-H. Jeon, E. Lee, S.-E. Park, K.-Y. Jun, T.M. Kadayat,

P. Thapa, R. Karki, Y. Na, M.S. Park, S.B. Rho, E.-S. Lee, Y. Kwon, A series of novel

terpyridine-skeleton molecule derivants inhibit tumor growth and metastasis

by targeting topoisomerases, J. Med. Chem. 58 (2015) 1100e1122.

[32] X. Xiao, M. Cushman, An Ab initio quantum mechanics calculation that cor-

relates with ligand orientation and DNA cleavage site selectivity in

1976 (1976) 1e24.

[35] S.J. Froelich-Ammon, N. Osheroff, Topoisomerase poisons: harnessing the dark

side of enzyme mechanism, J. Biol. Chem. 270 (1995) 21429e21432.

[36] J.C. Marini, K.G. Miller, P.T. Englund, Decatenation of kinetoplast DNA by

topoisomerases, J. Biol. Chem. 255 (1980) 4976e4979.

[37] B.B. Hasinoff, M.E. Abram, N. Barnabe, T. Khelifa, W.P. Allan, J.C. Yalowich, The

catalytic DNA topoisomerase II inhibitor dexrazoxane (ICRF-187) induces

differentiation and apoptosis in human leukemia K562 cells, Mol. Pharmacol.

59 (2001) 453e461.

[38] A. Ivashkevich, C.E. Redon, A.J. Nakamura, R.F. Martin, O.A. Martin, Use of the

gamma-H2AX assay to monitor DNA damage and repair in translational

cancer research, Cancer Lett. 327 (2012) 123e133.

[39] P. Majumdar, C. Bathula, S.M. Basu, S.K. Das, R. Agarwal, S. Hati, A. Singh,

S. Sen, B.B. Das, Design, synthesis and evaluation of thiohydantoin derivatives

as potent topoisomerase I (Top1) inhibitors with anticancer activity, Eur. J.

Med. Chem. 102 (2015) 540e551.

ꢀ

[40] J.C. Reed, Balancing cell life and death: bax, apoptosis, and breast cancer,

J. Clin. Invest 97 (1996) 2403e2404.

[41] A. Basu, S. Haldar, The relationship between BcI2, Bax and p53: consequences

for cell cycle progression and cell death, Mol. Hum. Reprod.

1099e1109.

4 (1998)

[42] S.H. Kaufmann, S. Desnoyers, Y. Ottaviano, N.E. Davidson, G.G. Poirier, Speci ﬁ c

proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of

chemotherapy-induced apoptosis, Cancer Res. 53 (1993) 3976e3985.

[43] R.R. Tice, E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi,

Y. Miyamae, E. Rojas, J.C. Ryu, Y.F. Sasaki, Single cell gel/comet assay: guide-

lines for in vitro and in vivo genetic toxicology testing, Environ. Mol. Mutagen

35 (2000) 206e221.

Article Doi

Rational design, synthesis, and evaluation of novel 2,4-Chloro- and Hydroxy-Substituted diphenyl Benzofuro[3,2-b]Pyridines: Non-intercalative catalytic topoisomerase I and II dual inhibitor

DOI: 10.1016/j.ejmech.2017.01.003

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