Effect of chlorine substituent on cytotoxic activities: Design and synthesis of systematically modified 2,4-diphenyl-5H-indeno[1,2-b]pyridines

Article abstract of DOI:10.1016/j.bmcl.2016.02.053

In continuation of our previous work, six hydroxylated 2,4-diphenyl-5H-indeno[1,2-b]pyridine analogs were modified by introducing one chlorine functionality at ortho, meta or para position of the 2- or 4-phenyl ring. Eighteen new chlorinated compounds wer

Full text of DOI:10.1016/j.bmcl.2016.02.053

Accepted Manuscript
Effect of chlorine substituent on cytotoxic activities: Design and synthesis of
systematically modified 2,4-diphenyl-5H-indeno[1,2-b]pyridines
Tara Man Kadayat, Seojeong Park, Kyu-Yeon Jun, Til Bahadur Thapa Magar,
Ganesh Bist, Aarajana Shrestha, Younghwa Na, Youngjoo Kwon, Eung-Seok
Lee
PII:
S0960-894X(16)30165-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.02.053
BMCL 23607
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 October 2015
29 January 2016
19 February 2016
Please cite this article as: Kadayat, T.M., Park, S., Jun, K-Y., Magar, T.B.T., Bist, G., Shrestha, A., Na, Y., Kwon,
Y., Lee, E-S., Effect of chlorine substituent on cytotoxic activities: Design and synthesis of systematically modified
2,4-diphenyl-5H-indeno[1,2-b]pyridines, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/
10.1016/j.bmcl.2016.02.053
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Effect of chlorine substituent on cytotoxic activities: Design and synthesis of systematically
modified 2,4-diphenyl-5H-indeno[1,2-b]pyridines
Tara Man Kadayata,#, Seojeong Parkb,#, Kyu-Yeon Junb, Til Bahadur Thapa Magara, Ganesh Bista,
Aarajana Shresthaa, Younghwa Nac, Youngjoo Kwonb,*, Eung-Seok Lee a,*
aCollege of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea
bCollege of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Global Top 5
program Ewha Womans University, Seoul 120-750, Republic of Korea
cCollege of Pharmacy, Cha University, Pochon 487-010, Republic of Korea
#Authors are equal contributors.
*Corresponding Authors:
Tel: +82 53 810-2827, fax: +82 53 810-4654, email: eslee@yu.ac.kr (E.-S.L.)
Tel: +82 2 3277-4653, fax: +82 2 3277-2851, email: ykwon@ewha.ac.kr (Y.K.)

Effect of chlorine substituent on cytotoxic activities: Design and synthesis of systematically
modified 2,4-diphenyl-5H-indeno[1,2-b]pyridines
Tara Man Kadayata,#, Seojeong Parkb,#, Kyu-Yeon Junb, Til Bahadur Thapa Magara, Ganesh Bista,
Aarajana Shresthaa, Younghwa Nac, Youngjoo Kwonb,*, Eung-Seok Lee a,*
Abstract
In continuation of our previous work, six hydroxylated 2,4-diphenyl-5H-indeno[1,2-b]pyridine
analogues were modified by introducing one chlorine functionality at ortho, meta or para
position of the 2- or 4 phenyl ring. Eighteen new chlorinated compounds were thus prepared and
assessed for topoisomerase inhibitory activity and cytotoxicity against HCT15, T47D, and HeLa
cancer cell lines. All of the chlorinated compounds displayed significant cytotoxic effect,
revealing potent anticancer activity against T47D breast cancer cells. This functional group
modification allowed us to explore the importance of chlorine group substitution for the
cytotoxic properties. The information reported here provides valuable insight for further study to
develop new anticancer agents using related scaffolds.
Keywords: 2,4-Diphenyl-5H-indeno[1,2-b]pyridine analogue; Anticancer agents; Breast cancer;
Cytotoxicity

Functional group modification of biologically active compounds is one of the most efficient
strategies in the field of medicinal chemistry. In the search of novel anticancer agents, our
research group has been designing compounds containing indenopyridine skeleton which are
reported to have anti-inflammatory activity and cytotoxicity against different human cancer cell
lines.1-4 We recently reported a series of analogues of 2,4-diphenyl 5H-indeno[1,2-b]pyridine (1)
containing ortho, meta or para hydroxyl group at 2- or 4-phenyl ring (Figure 1). The structure-
activity relationship study revealed that introduction of hydroxyl group at meta or para positions
of 2- or 4-phenyl ring is important for displaying significant topoisomerase inhibitory activity
and cytotoxicity.5 Topoisomerase (topo) plays an important role in solving various DNA
topological problems occurred during DNA replication and other vital cellular processes, and
consequently is considered one of the attractive molecular targets for the development of
anticancer agents. 6, 7

Figure 1. Structures of previously synthesized 5H-indeno[1,2-b]pyridine derivatives; a) non-
substituted, b) 2-phenol-4-phenyl substituted, and c) 4-phenol-2-phenyl substituted -5H-
indeno[1,2-b]pyridines.
Over the past few years, substitution of halogen group on the several compounds is attempted for
modifying their chemical and pharmacological activities.8-10 Furthermore, introduction of
chlorine atom is reported to improve the stability, potency and specificity of the binding site due
to its ability to accommodate in tight and deep hydrophobic pockets of the biological targets.11, 12
Therefore, with an aim to further explore structure-activity relationships, we decided to

incorporate a chlorine functionality at ortho, meta or para positions of the 2- or 4-phenyl ring of
hydroxylated 2,4-diphenyl 5H-indeno[1,2-b]pyridine compounds (2-7).
Herein, we report the systematic design (Figure 2), synthesis, and topo inhibitory activity and
cytotoxicity of novel 2,4-diphenyl-5H-indeno[1,2-b]pyridines containing hydroxyl and chlorine
moiety at 2- or 4-phenyl ring of 5H-indeno[1,2-b]pyridine. Total eighteen modified 2,4-
diphenyl-5H-indeno[1,2-b]pyridine compounds (8–25) were synthesized in six different series
containing one phenolic ring and one chlorophenyl ring on 2- or 4- position of the central
pyridine (Figure 3).
Figure 2. Strategy for the design of chloro- and hydroxy-substituted 2,4-diphenyl-5H-
indeno[1,2-b]pyridines.

Cl
Cl
Cl
Cl
Cl
Cl
OH
OH
OH
HO
HO
HO
N
N
N
N
N
N
8
9
10
11
12
13
Cl
OH
Cl
OH
Cl
OH
Cl
Cl
Cl
N
N
N
N
N
N
HO
HO
HO
14
15
16
17
18
19
OH
OH
OH
OH
OH
OH
Cl
Cl
N
N
N
Cl
N
N
N
Cl
Cl
Cl
20
21
23
24
25
22
Figure 3. Structures of the prepared chloro- and hydroxy-substituted 2,4-diphenyl-5H-
indeno[1,2-b]pyridines.
The synthetic route with three different steps for the target compounds 8–25 is outlined in
Scheme 1. In the first step, 1-indanone (I) was condensed with aryl aldehydes II (R1=a-f) to
prepare indanone intermediates III (R1=a-f) in the presence of 5% aqueous NaOH in ethanol
using Clasien-Schmidt condensation reaction.13 Then, six pyridinium iodide salts V (R2=a-f)
were synthesized by refluxing acetophenones IV (R2=a-f) with iodine in pyridine. Finally, using
modified Kröhnke synthesis,14, 15 indanone intermediates III (R1=a-f) and pyridinium iodide salts
V (R2=a-f) were reacted in the presence of dry ammonium acetate in methanol or acetic acid to
give final compounds 8-25 in the yields of 23.3-69.0%. Although, protection of hydroxyl groups
was not needed for the preparation of the final compounds, yields of the compounds 8-10 having
hydroxyl groups at ortho position of 2-phenyl ring was relatively lower than meta or para
hydroxyl substituted compounds. This may be due to the formation of intermolecular H-bonding,

and the higher steric hindrance of compounds bearing ortho hydroxyl substitution at 2- phenyl
ring. Structures, yields (%), HPLC purities (%), and melting point (°C) of the prepared
compounds are shown in Table S1 of Supporting Information.
R1
R1
H
i
+
O
O
O
R1
II (R1=a-f)
III (R1=a-f)
I
iii
R2
N
I
R2
CH3
R2
8-25
N
ii
O
O
IV (R2=a-f)
V (R2=a-f)
Cl
OH
Cl
OH
Cl
HO
2
R1
R =
,
a
b
c
d
e
f
Scheme 1. General synthetic method of chloro- and hydroxy-substituted 2,4-diphenyl-5H-
indeno[1,2-b]pyridines. Reagents and conditions: (i) aq. NaOH (5%), EtOH, 1-12 h, room
temperature, 61.9-99.7% yield; (ii) pyridine (15 equiv.), iodine, 3 h, 140 °C, 64.7-93.2% yield;
(iii) NH4OAc (10 equiv.), methanol, 24–36 h, 100°C, 23.3-69.0% yield.
All the prepared compounds were evaluated for topo I and II inhibitory activity and cytotoxicity
against several human cancer cell lines. Figure 4A and Table 1 summarize the topo I inhibitory
activity of the synthesized compounds (8–25). Except compound 11 and 15, all of the
compounds containing one hydroxyl and one chlorine group at 2- or 4-phenyl ring of the central

pyridine showed very weak topo I inhibitory activities (0.8-19.1% inhibition at 100 µM) as
compared to the positive control, camptothecin (74.5% inhibition at 100 µM). Compound 11 and
15 displayed moderate topo I inhibitory activity (29.3% and 35.5%, respectively) at 100 µM
concentration. None of the compounds were active at 20 µM. Similarly, all of the compounds
showed very weak topo II inhibitory activity as compared to the positive control, etoposide at
100 µM (Figure 4B and Table 1).
Figure 4. Human DNA topo I (A) and topo II (B) inhibitory activity of the prepared compounds
8-25 at the concentration of 100 µM and 20 µM, respectively. (A) Lane D: pBR322 DNA only;
Lane T: pBR322 DNA + Topo I; Lane C: pBR322 DNA + Topo I + Camptothecin; Lane 8-25:
pBR322 DNA + Topo I + each of compounds 8-25. (B) Lane D: pBR322 DNA only; Lane T:
pBR322 DNA + Topo II; Lane E: pBR322 DNA + Topo II + Etoposide; Lane 8-25: pBR322
DNA + Topo II + each of compounds 8-25.

Table 1. Topo I and II inhibitory activity and cytotoxicity of the prepared compounds.
Compounds
Adriamycin
Etoposide
Topo I (% Inhibition)
Topo II (% Inhibition)
IC50 *(µM)
100 µM
20 µM
100 µM
HCT15
1.23 0.00a/
0.09 0.01b
2.82 0.24a/
6.28 0.19b
18.87 0.34a/
1.29 0.01b
> 50
T47D
HeLa
1.34 0.03a/ 0.88 0.08a/
1.47 0.02b 0.82 0.00b
1.84 0.44a/ 0.18 0.02a/
57.98 0.25b 15.03 0.17b
13.7 0.81a/ 7.32 0.15a/
11.17 0.58b 0.59 0.02b
58.6a/
81.5b
58.7a/
74.5b
0.0
27.6a/
33.5b
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Camptothecin
17.8
36.4
76.0
80.6
69.6
70.5
65.9
4.8
26.8 0.26
> 50
17.3 0.56
> 50
1
> 50
1.3
2
2.71 0.12
2.01 0.18
> 50
3.93 0.17
2.10 0.04
> 50
2.99 0.31
3.93 0.07
> 50
2.3
3
5.2
4
15.1
20.6
25.9
0.0
5
1.35 0.03
2.15 0.12
>50
1.55 0.04
1.36 0.04
>50
4.00 0.15
4.84 0.05
>50
6
7
8
1.1
2.8
>50
>50
>50
9
8.3
2.8
>50
>50
>50
10
11
12
29.3
7.5
6.7
1.17 0.02
1.59 0.04
2.19 0.02
1.85 0.31
2.65 0.16
1.91 0.01
4.4

5.8
15.5
35.5
19.1
0.8
ND
ND
2.8
3.8
1.6
4.9
8.5
1.9
3.1
3.5
5.4
3.2
3.0
2.7
3.9
3.2
2.09 0.01
0.84 0.00
0.81 0.00
0.93 0.0
6.23 0.13
0.40 0.09
1.82 0.02
30.26 0.97
3.09 0.03
0.31 0.01
>50
1.49 0.04
1.63 0.01
1.63 0.03
0.88 0.02
6.64 0.04
0.31 0.04
0.79 0.05
>50
2.27 0.02
3.41 0.12
3.68 0.05
2.45 0.01
7.01 0.09
2.01 0.26
2.70 0.06
17.07 0.56
4.59 0.02
5.04 0.14
>50
13
14
15
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
16
17
0.0
18
0.0
19
0.0
20
0.0
1.32 0.03
1.42 0.09
>50
21
0.0
22
0.0
23
0.0
1.88 0.01
0.87 0.01
1.36 0.02
1.27 0.06
6.58 0.06
4.34 0.16
24
1.4
25
ND: Not determined
HCT15: human colorectal adenocarcinoma; T47D: Human breast ductal carcinoma; HeLa: human cervix adenocarcinoma
cell line; Adriamycin: positive control for cytotoxicity; Etoposide: positive control for topo II and cytotoxicity;
Camptothecin: positive control for topo I and cytotoxicity.
aControl value for previously reported compounds 1-7. 5
bControl value for the compounds 8-25.
*Each data represents mean S.D. from three different experiments performed in triplicate.

Evaluation of anticancer activity for the prepared compounds was performed against three
different human cancer cell lines: human colorectal adenocarcinoma cell line (HCT15), human
ductal breast epithelial tumor cell line (T47D), and human cervix tumor cell line (HeLa) with
adriamycin, etoposide, and camptothecin as positive controls. The inhibitory activity (IC50) of
the synthesized compounds (8-25) is expressed in micromolar concentration as summarized in
Table 1. Except compounds 8-10, 20, and 23, all the compounds showed stronger or moderate
cytotoxicity (IC50 < 7.01 µM) than positive controls. Among the compounds tested against
HCT15 colon cancer cells, compounds 11, 14-16, 18, 22, and 25 displayed stronger cytotoxicity
(IC50 < 1.17 µM) than etoposide (6.28 µM) and camptothecin (1.29 µM), but weaker than
adriamycin (0.09 µM). Similarly, among the compounds tested against T47D breast cancer cells,
compounds 11-19, 21, 22, 24, and 25 displayed stronger cytotoxicity (IC50 < 6.64 µM) than
etoposide (57.9 µM) and camptothecin (11.2 µM), and similar or moderate cytotoxicity
compared to adriamycin (1.47 µM). Likewise, for the HeLa cervix cancer cells, most of the
compounds displayed significant cytotoxicity than etoposide but moderate as compared to
adriamycin and camptothecin.
Compounds 11-16 with meta- or para- phenolic ring at 2-position and chlorophenyl moiety at 4-
position of central pyridine showed the significant cytotoxicity with a range of IC50 values of
0.81-3.68 µM against HCT15, T47D and HeLa cells. Similarly, compounds 18, 19, 21, 22, 24,
and 25 with meta or para- phenolic ring at 4-position and chlorophenyl moiety at 2-position of
central pyridine showed significant cytotoxicity with a range of IC50 values of 0.31-4.59 µM
against HCT15, T47D and HeLa cells. Compounds 8-10, 17, 20, and 23 displayed weak

cytotoxicity against all the tested cancer cells. Interestingly, it was observed that combination of
meta- or para- phenolic group with chlorophenyl ring is favorable for the cytotoxic activity.
We investigated the structure-activity relationship study by assessing the role of substituted
chlorine group in the biological activity of modified 2,4-diphenyl-5H-indeno[1,2-b]pyridines.
Introduction of hydroxyl moiety at 2- or 4-phenyl ring (compounds 2-7) of the 2,4-diphenyl-5H-
indeno[1,2-b]pyridine (1) significantly increased topo II inhibitory activity. However, further
substitution of a chlorine moiety at 2- or 4-phenyl ring (compounds 8-25) of the corresponding
hydroxylated 2,4-diphenyl-5H-indeno[1,2-b]pyridines (2-7) resulted decrease in the topo II
inhibitory activity. From the results, reported in Table 1, it is evident that chlorine moiety
substitution in 2- or 4-phenyl ring of 5H-indeno[1,2-b]pyridine skeleton is not favorable for
displaying topo II inhibitory activity.
As expected from the result of previously reported compound (2 and 5), compounds 8-10, 17,
20, and 23 that contain ortho- hydroxyl moiety at 2- or 4-phenyl ring were less active against
tested cancer cells. However, compounds 11-16, 18, 19, 21, 22, 24, and 25 containing meta or
para- phenolic ring on 2 or 4-position displayed significant cytotoxicity. It is interesting to note
that introduction of chlorine group on meta- or para- hydroxylated 2,4-diphenyl-5H-indeno[1,2-
b]pyridine resulted in significant cytotoxicity against all the tested cells as compared to etoposide,
and moderate activity than that of adriamycin and camptothecin. From this result, it can be
implied that introduction of meta- or para- phenolic ring and chlorophenyl ring at the 2- or 4-
position is important for cytotoxic activity. Chlorinated compounds are generally more active
against T47D breast cancer cells among all the tested cancer cells.

Table 2 illustrates relative potencies for cytotoxicity (against HCT15, T47D, and HeLa cell lines)
of the representative compounds 11-16, 18, 19, 21, 22, 24, and 25 compared to adriamycin, a
widely used anticancer drug as positive control, for easier comparison with previously
synthesized compounds 3, 4, 6 and 7. Interestingly, compared with the corresponding meta- or
para-hydroxylated 2,4-diphenyl-5H-indeno[1,2-b]pyridines (3, 4, 6 and 7), the presence of
chlorine moiety enhanced the cytotoxicity against T47D breast cancer cells. Chlorinated
compounds were less active against HCT15 cells. However, these chlorinated compounds had
similar or greater cytotoxic activity than their non-chlorinated analogues against HeLa cell lines.
Moreover, compounds 16, 18, 19, 21, 22, 24, and 25 exhibited stronger activities (Relative
Potency > 1) compared to positive control, adriamycin against T47D breast cancer cells. This
result suggests that introduction of chlorine group at 2-phenyl ring and meta or para-hydroxyl
group at 4-phenyl ring of 5H-indeno[1,2-b]pyridine skeleton is important for potent cytotoxic
effect.

Table 2. Relative potency of representative compounds showing effect of position of meta or para- phenolic moiety at 2 or 4- position
of 5H-indeno[1,2-b]pyridines.
Relative potency* for cytotoxicity compared to positive control (Adriamycin)
IC50 (µM)
IC50 (µM)
Compound
Compound
HCT15
T47D
HeLa
HCT15
T47D
HeLa
Adriamycina (IC50)
1.23 0.0 1.34 0.3 0.88 0.1
Adriamycinb (IC50)
0.09 0.0 1.47 0.0 0.82 0.0
11: R= ortho- Cl, R1= meta- OH
12: R= meta- Cl, R1= meta- OH
13: R= para- Cl, R1= meta- OH
14: R= ortho- Cl, R1= para- OH
15: R= meta- Cl, R1= para- OH
16: R= para- Cl, R1= para- OH
18: R= meta- OH, R1= ortho- Cl
19: R= para- OH, R1= ortho- Cl
21: R= meta- OH, R1= meta- Cl
22: R= para- OH, R1= meta- Cl
24: R= meta- OH, R1= para- Cl
25: R= para- OH, R1= para- Cl
0.08
0.06
0.05
0.12
0.12
0.11
0.25
0.05
0.03
0.32
0.05
0.11
0.67
0.79
0.99
0.90
0.90
1.67
4.74
1.86
1.11
1.03
1.08
1.16
0.31
0.43
0.36
0.24
0.22
0.33
0.41
0.30
0.18
0.16
0.12
0.19
3:R= H, R1= meta- OH
4:R= H, R1= para- OH
6: R= meta- OH, R1= H
7: R= para- OH, R1= H
0.45
0.61
0.91
0.57
0.34
0.63
0.86
0.98
0.29
0.22
0.22
0.18
* Relative potency: IC of positive control Adriamycin IC of the compound
/
50
50

aIC50 control value of Adriamycin for the previously reported compounds 3, 4, 6 and
bIC50 control value of Adriamycin for newly synthesized compounds 11-16, 18, 19, 21, 22, 24, and 25.
7.

In conclusion, we systematically designed and prepared a new series of eighteen chloro- and
hydroxy-substituted 2,4-diphenyl-5H-indeno[1,2-b]pyridines using modified Kröhnke synthesis,
and evaluated their topo I and II inhibitory activity and cytotoxicity against HCT15, T47D, and
HeLa cancer cell lines. Introduction of chlorine group on 2- or 4-phenyl ring of their
corresponding hydroxylated analogues resulted in decrease of topo I and II inhibitory activity.
However, these chlorinated compounds displayed significant cytotoxic activity against tested
cancer cell lines. All of the chlorinated compounds exhibited stronger cytotoxic effect compared
to corresponding non-chlorinated compounds against T47D breast cancer cells. Particularly,
compounds 16, 18, 19, 21, 22, 24, and 25 found to be more cytotoxic than adriamycin against
T47D cells. These results suggest that introduction of chlorine and hydroxyl moiety on 2,4-
diphenyl-5H-indeno[1,2-b]pyridine skeleton is important for displaying the significant cytotoxic
activity. Thus, this study may provide valuable information to medicinal chemist working with
indenopyridines containing chlorine or hydroxyl functionalities. Experiment to investigate the
mechanistic pathway of these chlorinated compounds is a future scope of our research group to
develop novel and potent anticancer agents.
Acknowledgements
This study 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-2015 R1A2A1A15054445 and NRF-2007-0056420) and by a grant of the Korean Health
Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI14C2469).

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Figure Captions
Figure 1. Structures of previously synthesized 5H-indeno[1,2-b]pyridine derivatives; a) non-
substituted, b) 2-phenol-4-phenyl substituted, and c) 4-phenol-2-phenyl substituted 5H-
indeno[1,2-b]pyridines.
Figure 2. Strategy for the design of chloro- and hydroxy-substituted 2,4-diphenyl-5H-
indeno[1,2-b]pyridines.
Figure 3. Structures of the prepared chloro- and hydroxy-substituted 2,4-diphenyl-5H-
indeno[1,2-b]pyridines.
Figure 4. Human DNA topo I (A) and topo II (B) inhibitory activity of the prepared compounds
8-25 at the concentration of 100 µM and 20 µM, respectively. (A) Lane D: pBR322 DNA only;
Lane T: pBR322 DNA + Topo I; Lane C: pBR322 DNA + Topo I + Camptothecin; Lane 8-25:
pBR322 DNA + Topo I + each of compounds 8-25. (B) Lane D: pBR322 DNA only; Lane T:
pBR322 DNA + Topo II; Lane E: pBR322 DNA + Topo II + Etoposide; Lane 8-25: pBR322
DNA + Topo II + each of compounds 8-25.

Scheme Caption
Scheme 1. General synthetic method of chloro- and hydroxy-substituted 2,4-diphenyl-5H-
indeno[1,2-b]pyridines. Reagents and conditions: (i) aq. NaOH (5%), EtOH, 1-12 h, room
temperature, 61.9-99.7% yield; (ii) pyridine (15 equiv.), iodine, 3 h, 140 °C, 64.7-93.2% yield;
(iii) NH4OAc (10.0 equiv.), methanol, 24–36 h, 100°C, 23.3-69.0% yield.

Table Captions
Table 1. Topo I and II inhibitory activity and cytotoxicity of the prepared compounds.
Table 2. Relative potency of representative compounds showing effect of position of meta or
para- phenolic moiety at 2 or 4- position of 5H-indeno[1,2-b]pyridines.

Graphical Abstract