A new phenolic series of indenopyridinone as topoisomerase inhibitors: Design, synthesis, and structure-activity relationships

Article abstract of DOI:10.1016/j.bmc.2018.09.021

DNA Topoisomerase IIα (topo IIα) is one of the most effective therapeutic targets to control cancer. In an effort to develop novel and effective topo IIα targeting anti-proliferative agent, a phenolic series of indenopyridinone and indenopyridinol were designed and prepared using efficient multi-component one pot synthetic method. Total twenty-two synthesized compounds were assessed for topo I and IIα inhibition, and anti-proliferation in three different human cancer cell lines. Overall structure-activity relationship study explored the significance of meta-phenolic group at 4-position and para-phenolic group at 2- and/or 4-position of indenopyridinone skeleton for strong topo IIα-selective inhibition and anti-proliferative activity against human cervix (HeLa) and colorectal (HCT15) cell lines. Compound 12 with excellent topo IIα inhibition (93.7%) was confirmed as a DNA intercalator that could be a new promising lead to develop effective topo IIα-targeted anticancer agents.

Full text of DOI:10.1016/j.bmc.2018.09.021

Accepted Manuscript
A new phenolic series of indenopyridinone as topoisomerase inhibitors: Design,
synthesis, and structure-activity relationships
Aarajana Shrestha, Seojeong Park, Hae Jin Jang, Pramila Katila, Ritina Shrestha,
Youngjoo Kwon, Eung-Seok Lee
PII:
S0968-0896(18)31513-X
https://doi.org/10.1016/j.bmc.2018.09.021
BMC 14546
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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27 August 2018
18 September 2018
19 September 2018
Please cite this article as: Shrestha, A., Park, S., Jin Jang, H., Katila, P., Shrestha, R., Kwon, Y., Lee, E-S., A new
phenolic series of indenopyridinone as topoisomerase inhibitors: Design, synthesis, and structure-activity
relationships, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.09.021
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A new phenolic series of indenopyridinone as topoisomerase inhibitors: Design,
synthesis, and structure-activity relationships
Aarajana Shrestha,1,# Seojeong Park,2,# Hae Jin Jang,2 Pramila Katila,1 Ritina Shrestha,1
Youngjoo Kwon2,* and Eung-Seok Lee1,*
1College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea
2College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Global Top 5
Program, Ewha Womans University, Seoul, 120-750, Republic of Korea.
* Corresponding Authors:
email: ykwon@ewha.ac.kr (Y.K.)
email: eslee@yu.ac.kr (E.-S.L.)
#Authors are equal contributors
1

Abstract
DNA Topoisomerase IIα (topo IIα) is one of the most effective therapeutic targets to control
cancer. In an effort to develop novel and effective topo IIα targeting anti-proliferative agent, a
phenolic series of indenopyridinone and indenopyridinol were designed and prepared using
efficient multi-component one pot synthetic method. Total twenty-two synthesized
compounds were assessed for topo I and IIα inhibition, and anti-proliferation in three
different human cancer cell lines. Overall structure-activity relationship study explored the
significance of meta-phenolic group at 4-position and para-phenolic group at 2- and/or 4-
position of indenopyridinone skeleton for strong topo IIα-selective inhibition and anti-
proliferative activity against human cervix (HeLa) and colorectal (HCT15) cell lines.
Compound 12 with excellent topo IIα inhibition (93.7%) was confirmed as a DNA
intercalator that could be a new promising lead to develop effective topo IIα-targeted
anticancer agents.
Keywords: Indenopyridinone; Phenolic group; Topoisomerase inhibitor; DNA intercalator;
Anti-proliferative activity
2

1. Introduction
Despite of decrease in prevalence of risk factors, sophisticated treatments and early detection,
cancer is still leading cause of death with intolerable burden worldwide which is expected to
increase due to adoption of unhealthy lifestyle behaviors and an increase in the aged
population. Cancer is a malignant tumor that occurs due to deviation in normal cell
development.1 In cellular metabolic process like DNA replication, transcription, cell
segregation and recombination, topoisomerase (topo) plays a key mechanistic role to relieve
topological stress of supercoiled DNA.2-4 Topo act by transiently breaking either only single
strand (topo I) or both the strands (topo II) of double stranded DNA helix. Because of distinct
functions, expression of topo I remain constant during cell cycle whereas concentration of
topo IIα is found high in S phase and significantly alter in amount and stability across cell
cycle.5 The vital role of topo IIα during cell proliferation has made it attractive nuclear target
where interference of topo IIα function or generation of topo IIα-mediated cell damages have
become one of the major strategies for anticancer therapy. Similarly, topo IIα is also cellular
targets for many DNA intercalating agents such as adriamycin and mitoxantrone, and DNA
non-intercalating agents like teniposide and etoposide.6
In the recent years, studies on synthesis and biological activity of fused N-heterocyclic
compounds have become an important scope in the medicinal chemistry field to develop
novel pharmacological active agents.7 Moreover, as shown in Figure 1, many research on
compounds containing indenopyridinone ring have been reported for their cytotoxic activity
on various cancer cell lines which act by inhibiting topo enzymes as well as intercalating with
DNA.8-10 Recently, our group studied importance of compound (A) containing
indenopyridinone ring in Figure 1 (a) as a topo IIα catalytic inhibitor with DNA intercalative
property.11 Further, we explored many compounds which consist of fused nitrogen containing
3

heterocyclic scaffold as a main pharmacophore with excellent topo-targeted anti-proliferative
activity.12,13 In addition, we found importance of phenol moiety substituted at 2- and/or 4-
position of 5H-indeno[1,2-b]pyridines, benzofuro[3,2-b]pyridines and benzofuro[3,2-
b]pyridinols.14-16 These positive results prompted us to modify the core structure of fused
pyridine ring by substituting ketone (-C=O) functional group at C-5 position including phenol
moiety substituted at 2- and/or 4- position and study their effect on topo inhibitory and anti-
proliferative property (Figure 1). In this study, we designed and synthesized systematically a
new series of non-, mono- and di-hydroxylated 2,4- diphenyl indenopyridinones (1-16) and
evaluated their biological activities. In addition, further reduction of ketone group to alcohol
form was carried out to synthesize novel scaffold of indenopyridinols (1R, 4R, 9R, 11R, 14R
and 15R) to compare and investigate SAR study based on topo inhibitory and anti-
proliferative activity. The most active compound 12 was further tested for its DNA
intercalating property.
4

Figure 1. Design strategy of the target compounds based on (a) previously reported bio-
active 5H-indeno[1,2-b]pyridin-5-one derivatives (b) modification of fused N-heterocyclic
compounds.
5

2. Results and discussion
2.1. Chemistry
As outlined in Scheme 1, three different types of multicomponent one-pot synthetic methods
were used to synthesize the designed compounds 1-16.17-19 Previously, our group reported
conventional Kröhnke method to synthesize pyridine derivatives that includes three-steps
reaction which was difficult to synthesize and purify novel indenopyridinone derivatives with
very low yield. Therefore, as a new alternative synthetic method, we used 2,2,2-
trifluoroethanol (TFE) or L-proline-catalyzed or microwave-assisted one-pot method for the
synthesis of novel non-, mono- and di- hydroxylated 2,4-diphenyl indenopyridineone
derivatives. Here we found this new one-pot synthetic method comparatively simple, efficient,
rapid and inexpensive to conventional Kröhnke pyridine synthesis. Compounds 1-16 were
synthesized by three components one-pot reaction using equivalent amount of aryl methyl
ketones, aryl aldehydes and indene-1,3-dione in presence of dry ammonium acetate
(NH4OAc). The reaction was carried out either in presence of L-proline as a catalyst using
ethanol (EtOH) or N,N-dimethylformamide (DMF) as a solvent by refluxing at 100-120°C
for 2-24 hours or by refluxing with TFE at 80°C for 2-4 hours or under microwave irradiation
using DMF as a solvent at power 200 W at 120°C for 1-2 hours (Scheme 1A). Further, we
synthesized six novel indenopyridinols (1R, 4R, 9R, 11R, 14R and 15R), selected based on
number of hydroxyl group (non-, mono-OH and di-OH), by reducing corresponding
indenopyridinones using sodium borohydride (NaBH4) in presence of aqueous
tetrahydrofuran (THF) at 25°C. All the prepared compounds were characterized by measuring
1
13
their purity (>95%), H and C NMR, LCMS and melting point (°C) which are presented in
experimental and supplementary data. The structures of synthesized indenopyridinones and
their reduced indenopyridinol compounds are shown in Figure 2.
6

Scheme 1. Synthetic route of target compounds. Reagents and conditions: (i) NH4OAc, L-
proline, EtOH/DMF, reflux at 100-120 °C, 2-24 h, 2-26% yield; (ii) NH4OAc, TFE, reflux at
80 °C, 2-4 h, 2-10% yield; (iii) NH4OAc, DMF, 200 W, 120 °C, 1-2 h Microwave, 7-12%
yield; (iv) NaBH4, THF, H2O, 25°C, 15-30 min, 85-97% yield.
7

Figure 2. Structures of the synthesized non-, mono- and di-hydroxylated 2,4 diphenyl (A)
indenopyridinones, and (B) indenopyridinols.
8

2.2. Evaluation of topo I and IIα inhibition
As shown in Figure 3 and Table 1, non-hydroxylated 2,4-diphenyl indenopyridinone
(compound 1) showed weak topo I inhibitory activity (5.8%) at 100 µM. Similarly, among
mono-hydroxylated indenopyridinones, compounds 2, 3, 5, 9 and 13 exhibited negligible to
null topo I inhibition at 100 µM. Only compound 4 exhibited moderate topo I inhibition (50%)
at 100 µM as compared to reference compound, camptothecin (84.4%). In case of di-
hydroxylated indenopyridinones, only compounds 14 and 15 exhibited considerable topo I
inhibition (60% and 75.5%, respectively) at 100 µM as compared to camptothecin.
Compounds 6-8, 10-12 and 16 exhibited weak to null topo I inhibition at 100 µM. Similarly,
almost all the di-hydroxylated indenopyridinones did not show topo I inhibition at 20 µM.
Out of all indenopyridinones, only compound 12 displayed weaker (35.4%) topo I inhibition
than positive control, camptothecin (45.8%) at 20 µM. Moreover, we analyzed topo IIα
inhibition of prepared compounds 1-16. Except compound 4, all the non- and mono-
hydroxylated indenopyridinones showed very weak to negligible topo IIα inhibition at 100
µM. Compound 4 displayed moderate (63.3%) and weak (15.2%) topo IIα inhibition at 100
and 20 µM, respectively. Among the di-hydroxylated indenopyridinones, compounds 7, 11,
12, 14-16 exhibited significant topo IIα inhibition ranging from 68.9% to 100% as compared
to reference compound, etoposide at 100 µM. Compounds 12 and 15 showed potent topo IIα
inhibition (93.7% and 60.5%, respectively) as compared to etoposide at 20 µM, whereas at
100 µM, compounds 12 and 16 displayed 100% topo IIα inhibition.
9

Figure 3. Topoisomerase I and IIα inhibitory activities of compounds. (A) Lane D: pBR322
DNA only; Lane T: pBR322 DNA + topoisomerase I; Lane C: pBR322 DNA +
topoisomerase I + camptothecin; Lane 1-16: pBR322 DNA + topoisomerase I + compound 1-
16; (B) Lane D: pBR322 DNA only; Lane T: pBR322 DNA + topoisomerase IIα; Lane E:
pBR322 DNA + topoisomerase IIα + etoposide; Lane 1-16: pBR322 DNA + topoisomerase
IIα + compound 1-16.
In addition, Table 2 and Figure 4 illustrate the topo I and topo IIα inhibition by six non-,
mono- and di-hydroxylated indenopyridinols. Interestingly, upon reduction of compound 1 to
1R which does not consist hydroxyl moiety at 2- and 4- phenyl group of indenopyridinols
showed selective and strong topo I inhibition (81%) as compared to camptothecin (74.5%) at
100 µM. Similarly, only compounds 4R with mono-hydroxyl moiety and 15R with di-
10

hydroxyl group exhibited considerable topo I inhibition (63.9% and 71.9%, respectively) at
100 µM. And compound 15R exhibited potent topo I inhibition (61.7%) as compared to
camptothecin (45.8%) in 20 µM. Other compounds (9R, 11R and 14R) showed very weak to
negligible topo I inhibition at both 100 and 20 µM. In case of topo IIα inhibitory property,
compound 1R displayed very weak topo IIα inhibition at 100 µM whereas increase in
hydroxyl moiety at 2- and/or 4- phenyl ring, compounds showed moderate topo IIα inhibition
(40.7% to 60.9%) at 100 µM. Whereas, compounds exhibited very weak to considerable topo
IIα inhibition (0.3% to 26.7%) at 20 µM.
Figure 4. Topoisomerase I and IIα inhibitory activities of indeno[1,2-b]pyridin-5-ol
compounds. (A) Lane D: pBR322 DNA only; Lane T: pBR322 DNA + topoisomerase I; Lane
C: pBR322 DNA + topoisomerase I + camptothecin; Lane 1R, 4R, 9R, 11R, 14R, 15R:
pBR322 DNA + topoisomerase I + compound 1R, 4R, 9R, 11R, 14R, 15R; (B) Lane D:
pBR322 DNA only; Lane T: pBR322 DNA + topoisomerase IIα; Lane E: pBR322 DNA +
topoisomerase IIα + etoposide; Lane 1R, 4R, 9R, 11R, 14R, 15R: pBR322 DNA +
topoisomerase IIα + compound 1R, 4R, 9R, 11R, 14R, 15R.
11

Table 1. Topo I and IIα inhibitory activity and antiproliferative activity of the prepared indenopyridinones (1-16).
Topo I
Topo IIα
Compounds
IC50 *(µM)
(% Inhibition)
(% Inhibition)
HCT15
T47D
HeLa
100 µM
84.4
20 µM
45.8
100 µM
20 µM
49.8
Camptothecin
0.05±0.01
2.94±0.10
0.51±0.09
>50
0.07±0.02
0.40±0.03
0.31±0.04
6.50±0.57
1.33±0.05
1.18±0.17
>50
1.37±0.14
9.21±0.16
1.50±0.01
>50
Etoposide
70.2
Adriamycin
NT
1
2
5.8
NT
NT
NT
25.0
NT
NT
0.0
7.4
50.0
0.0
0.0
0.0
0.0
0.5
0.0
25.7
23.8
9.3
2.32±0.18
2.48±0.13
>50
2.58±0.05
2.24±0.02
>50
3
4
0.0
NT
63.3
4.9
15.2
NT
9.9
5
>50
10.53±0.42
>50
31.65±0.69
>50
NT
NT
NT
NT
NT
NT
6
32.8
86.5
5.2
>50
7
12.8
NT
6.09±0.44
>50
>50
4.84±0.43
>50
8
>50
NT
NT
9
22.5
2.6
>50
>50
>50
10
11
1.36±0.15
>50
0.11±0.02
1.24±0.10
1.52±0.04
>50
68.9
20.8
12

12
13
14
15
16
42.6
0.0
35.4
NT
0.0
100.0
23.3
93.7
NT
9.51±0.42
2.18±0.30
2.82±0.22
1.96±0.17
1.87±0.07
1.54±0.04
1.4±0.01
>50
2.25±0.10
2.30±0.47
4.32±0.03
4.72±0.17
60.0
75.5
0.0
72.8
12.0
60.5
30.5
0.42±0.01
1.49±0.05
0.37±0.01
7.5
71.9
NT
100.0
NT: Not tested, Compound 1 (no-OH); Compounds 2-5, 9, 13 (mono-OH); and Compounds 6-8, 10-12, 14-16 (di-OH).
HCT15: human colorectal adenocarcinoma cell line; T47D: human ductal breast cancer cell line; HeLa: human cervix tumor cell line
Camptothecin: positive control for topo I and anti-proliferative activity; Etoposide: positive control for topo IIα and anti-proliferative
activity; Adriamycin: positive control for anti-proliferative activity.
Each datum represents the mean ± S.D. of three different experiments performed in triplicate.
13

Table 2. Topo I and IIα inhibitory activity and antiproliferative activity of the prepared indenopyridinols (1R, 4R, 9R, 11R, 14R and 15R).
Topo I
Topo IIα
Compounds
IC50 *(µM)
(% Inhibition)
(% Inhibition)
HCT15
T47D
HeLa
100 µM
20 µM
45.8
100 µM
20 µM
Camptothecin
74.5
81.0
0.05±0.01
2.94±0.10
0.51±0.09
>50
0.07±0.02
0.40±0.03
0.31±0.04
>50
1.37±0.14
9.21±0.16
1.50±0.01
>50
Etoposide
Adriamycin
1R
80.1
21.5
49.1
0.0
NT
4R
63.9
0.0
0.0
NT
NT
NT
53.6
40.7
55.1
57.7
22.6
0.3
>50
>50
3.25±0.38
>50
>50
>50
9R
11R
20.1
11.5
17.8
12.1
6.20±0.57
13.02±0.04
1.61±0.12
4.28±0.53
6.91±0.14
10.80±0.62
14R
15R
71.9
61.7
60.9
26.7
9.76±0.18
1.70±0.12
7.08±0.25
NT: Not tested; HCT15: human colorectal adenocarcinoma cell line; T47D: human ductal breast cancer cell line; HeLa: human
cervix tumor cell line; Camptothecin: positive control for topo I and anti-proliferative activity; Etoposide: positive control for topo
IIα and anti-proliferative activity; Adriamycin: positive control for anti-proliferative activity.
Each datum represents the mean ± S.D. of three different experiments performed in triplicate.
14

2.3. Anti-proliferative activity
We evaluated the half maximal inhibitory concentration (IC50) of synthesized
indenopyridinones (1-16) and indenopyridinols (1R, 4R, 9R, 11R, 14R and 15R) against
human cancer cell lines, including breast (T47D), cervix (HeLa) and colorectal (HCT15).
Adriamycin, etoposide and camptothecin were used as reference compounds for anti-
proliferative effect. The evaluated IC50 values are listed in Tables 1 and 2. Compound 1
without hydroxyl moiety in 2,4-diphenyl indenopyridinone showed weak anti-proliferative
effect in all three tested cell lines. Interestingly, in case of mono-hydroxylated
indenopyridinones, compounds 2, 3 and 13 exhibited strong anti-proliferative activity in both
HCT15 (IC50 = 2.32 µM, 2.48 µM and 2.18 µM, respectively) and HeLa (IC50 = 2.58 µM,
2.24 µM and 2.25 µM, respectively) cell lines in comparison to etoposide. In addition, these
compounds (2, 3 and 13) showed moderate anti-proliferative activity in T47D cell line (IC50 =
1.33 µM, 1.18 µM and 1.40 µM, respectively). In other hand, compounds 4, 5 and 9 showed
weak anti-proliferative effect against all three cell lines.
Among di-hydroxylated indenopyridinones, compounds 7, 10 and 14-16 showed significant
anti-proliferative activity in HeLa cell line (IC50 = 1.52 µM to 4.84 µM) than etoposide (IC50
= 9.21 µM). Similarly, compounds 10 and 14-16 exhibited stronger anti-proliferative activity
in HCT15 cell line (IC50 = 1.36 µM to 2.82 µM) than etoposide (IC50 = 2.94 µM). In T47D
cell line, all the di-hydroxylated indenopyridinones (6-8) with ortho- phenolic group at 2-
position showed weak anti-proliferative activity whereas, compounds (10-12 and 14-16) with
meta- and para- phenolic group at 2-position showed moderate to strong anti-proliferative
activity. Compound 10 displayed significant anti-proliferative activity (IC50 = 0.11 µM) than
etoposide (IC50 = 0.40 µM) and adriyamycin (IC50 = 0.31 µM), while compound 16 showed
strong anti-proliferative activity (IC50 = 0.37 µM) than etoposide in T47D cell line. Moreover,
15

on evaluation of IC50 value of selected 2,4-diphenyl indenopyridinols (1R, 4R, 9R, 11R, 14R
and 15R) almost all of the non- and mono- hydroxylated indenopyridinols showed weaker
anti-proliferative activity than any of the reference compounds in all three human cancer cell
lines. Only compound 4R displayed moderate anti-proliferative effect in T47D cell line. In
contrast, di-hydroxylated indenopyridinols (11R, 14R and 15R) displayed moderate anti-
proliferative activity in HCT15 and T47D cancer cell lines as compared to reference
etoposide. Compounds 11R and 15R exhibited significant anti-proliferative activity (IC50 =
6.91 µM and 7.08 µM, respectively) in HeLa cell line as compared to etoposide (IC50 = 9.21
µM).
2.4. Structure-activity relationships
Structure-activity relationships of non-, mono- and di-hydroxylated 2,4-diphenyl
indenopyridinone derivatives was studied based on the results of topo I and IIα inhibition and
anti-proliferative activity. Interestingly, none of the compounds (1-16) showed better topo I
inhibition than reference camptothecin at both 100 and 20 µM. Similarly, all the mono-
hydroxylated indenopyridinones exhibited poor topo IIα inhibition, whereas majority of the
di-hydroxylated indenopyridinones showed significantly better topo IIα inhibition than
etoposide. This indicates the significance of phenolic groups at 2- and 4-position of
indenopyridinone for selectivity of topo IIα inhibition. Moreover, comparing the anti-
proliferative results with etoposide, compounds containing indenopyridinone scaffold can
inhibit proliferation of HCT15 and HeLa cell lines rather than T47D cell line. As shown in
Table 2, we found that indenopyridinols (1R, 4R, 9R, 11R, 14R, 15R), showed similar topo I
and IIα inhibition, whereas weaker anti-proliferative activity as compared with corresponding
indenopyridinones (1, 4, 9, 11, 14, 15). This result might indicate that keto-group (-C=O) at
16

C-5 is important for anti-proliferative activity as compared to hydroxyl group (-C–OH). The
overall SAR study of indenopyridinone derivatives and its reduced form indenopyridinols are
illustrated in Figure 5.
Figure 5. SAR study based on (A) importance of hydroxyl group at 2- and 4-phenyl ring of
5H-indeno[1,2-b]pyridin-5-ones (B) importance of hydroxyl group at 2- and 4-phenyl ring of
5H-indeno[1,2-b]pyridin-5-ols and (C) di-hydroxylated 5H-indeno[1,2-b]pyridin-5-ones and
5H-indeno[1,2-b]pyridin-5-ols.
17

In addition, relative potencies of topo IIα inhibition of indenopyridinones (1-16) were
calculated as compared to etoposide at 100 µM in order to study the effect of positions of the
phenolic groups at indenopyridinones (Table 3 and Figure 6).
Figure 6. Structure of compounds with comparable to stronger topo IIα inhibitory activity
than positive control, etoposide, at 100 µM.
18

Table 3. Relative topo IIα inhibitory potencies of synthesized compounds compared to etoposide.
Relative potency* for topoisomerase IIα inhibitory activity compared to positive control (Etoposide)
Topo IIα
Topo IIα
20 µM
Mono-hydroxylated
indenopyridineones
Di-hydroxylated
indenopyridineones
100 µM
20 µM
100 µM
0.44
1.15
0.07
0.03
0.98
1.33
1.04
1.02
1.33
2: R= H, R1= ortho- OH
5: R= ortho- OH, R1= H
0.32
0.06
NT
6: R= ortho- OH, R1=ortho- OH
7: R= ortho- OH, R1= meta- OH
8: R= ortho- OH, R1= para- OH
10: R= meta-OH, R1=ortho- OH
11: R= meta-OH, R1= meta- OH
12: R= meta-OH, R1= para- OH
14: R= para-OH, R1=ortho- OH
15: R= para-OH, R1= meta- OH
16: R= para-OH, R1= para- OH
0.20
0.26
NT
NT
3: R= H, R1= meta- OH
9: R= meta- OH, R1= H
0.12
0.32
NT
NT
NT
0.47
1.88
0.27
1.38
0.61
4: R= H, R1= para- OH
13: R= para- OH, R1= H
0.90
0.31
0.35
NT
*
(RP) : % inhibition of compound % inhibition of positive control, NT : Not Tested
Relative potency
/
19

As shown in Table 3 and Figure 6, compounds 14-16 with para- phenolic group at 2-position
of indenopyridinone displayed significant topo IIα inhibition than etoposide (Relative
potency > 1). Similarly, compounds 4, 12 and 16 with para-phenolic group at 4-position of
indenopyridinone showed comparable to better topo IIα inhibition than etoposide (Relative
potency ≥ 0.9). This reveals that para- phenolic group at both 2- and 4- position is very
essential for selective topo IIα inhibition. Likewise, compounds 7, 11 and 15 with meta-
phenolic group at 4-position of indenopyridinone exhibited comparable to better topo IIα
inhibition than etoposide (Relative potency ≥ 0.98). Whereas, only compound 12 with meta-
phenolic group at 2-position of indenopyridinone displayed significant and potent topo IIα
inhibition (Relative potency >1) than etoposide at both 100 and 20 µM. This implies that
meta-phenolic group at 4-position is more favorable for topo IIα inhibition. In contrast,
except compound 7 and 14, none of the ortho-phenolic group at both 2- and 4-position
showed significant topo IIα inhibition than etoposide (Relative potency < 0.45). This
indicates that ortho- phenolic group is least important than meta- and para-phenolic group for
topo IIα inhibitory activity.
Figure 7. Summary of SAR based on importance of position of phenolic group at 2- and 4-
position of indenopyridinone.
20

As demonstrated in Figure 7, the overall SAR study revealed that meta-phenolic group at 4-
position and para-phenolic group at both 2- and 4-position of indenopyridinone are essential
for topo IIα-selective inhibition. Result showed that compound 12 showed the strongest and
potent topo IIα inhibition than etoposide (Relative potency = 1.89) at 20 µM.
2.5. DNA-intercalating property of compound 12
In order to elucidate DNA intercalative property and rationalize the introduction of
indenopyridinone scaffold, we performed ethidium bromide (EtBr) displacement test of
compound 12 which was most active against topo IIα activity (93.7%) at 20 µM. EtBr is a
fluorophore containing well-known DNA intercalator. DNA intercalative property can be
determined by assessing the intensity of fluorescence of free form of displaced EtBr as an
ability of compound to replace the DNA intercalated EtBr from DNA.20 Amasacrine (m-
AMSA), a DNA intercalative topo II poison, was used as reference compound. As shown in
Figure 8, both m-AMSA and compound 12 increase the fluorescence intensity of EtBr
displaced from calf thymus DNA (cDNA) in dose dependent manner indicating compound 2
as a DNA intercalator. However, previously, our group reported a indenopyridine scaffold,14
as a non-intercalative topo IIα catalytic inhibitor. The findings suggest that ketone group
introduced at C-5 position of indenopyridine may results in change of intercalative property
of compound 12. Similar to the previous results of compounds containing indenopyridinone
(A and B in Figure 1), this results supports that indenopyridinone skeleton might be a
significant pharmacophore for DNA intercalative property of the compound.
21

80000
60000
40000
20000
0
80000
60000
40000
20000
0
m-AMSA
Compound 12
0 M
5 M
10 M
20 M
40 M
Wave length (nm)
Figure 8. Competitive EtBr displacement assay of compound 12. Intercalating ability of
compound 12 was measured using a fluorescence spectroscopy and m-AMSA, a well-known
DNA intercalative topo II poison as a positive control.21 The fluorescence intensity of EtBr-
calf thymus DNA (ctDNA) complex was scanned at emission wavelength range of 550 nm to
690 nm using excitation at 471 nm in the presence of m-AMSA and compound 12 at the
concentration range designated in the figure. The fluorescence of EtBr, a fluorophore-
containing DNA intercalator20 was quenched by the addition of m-AMSA and compound 12,
because both compounds displaced the intercalated EtBr in ctDNA to make EtBr free from
the DNA.
22

3. Conclusion
In conclusion, we justify the systematic design of a novel phenolic series of
indenopyridinones and synthesize using a simple and efficient multi-component one pot
synthetic methods. Further as an introduction of new scaffold, selected indenopyridinones
were reduced to indenopyridinols. The SAR study based on in vitro topo I and IIα inhibition,
and anti-proliferative effect of tested compounds was investigated. In case of
indenopyridinones, potency of topo IIα inhibitory activity was found to increase in
substitution of phenolic group in 2- and 4- position along with increase in anti-proliferative
effect against HCT15 and HeLa cell lines. However, mono- and di-hydroxylated
indenopyridinols exhibited weak to moderate topo I and IIα inhibition, and anti-proliferation.
Overall comparison of the bio-activity of indenopyridinones and indenopyridinols showed
significant topo IIα inhibition and anti-proliferation of di-phenolic indenopyridinones. In
addition, SAR study based on relative potencies of topo IIα inhibition of indenopyridinones
(1-16) revealed the importance of meta-phenolic group at 4-position and para-phenolic at
both 2- and/or 4-position for selective and potent topo IIα inhibition. Moreover, EtBr
displacement assay of the strong and selective topo IIα inhibitor, compound 12 showed DNA
intercalative property. Findings from this study would provide valuable information for
further optimization of scaffold 12 to develop a viable drug candidate for cancer therapy.
23

4. Experimental
4.1. Chemistry section
All the starting materials and reagents were purchased from Sigma-Aldrich Chemical Co.
(USA), and Junsei Chemical Co. (Japan), and used for synthesis without further purification.
HPLC grade acetonitrile (ACN) and methanol for purity test were obtained from Burdick and
Jackson, USA. Thin-layer chromatography (TLC) and column chromatography (CC) were
conducted with Kieselgel 60 F254 (Merck), silica gel (Kieselgel 60, 230-400 mesh, Merck),
respectively. Flash column chromatography was conducted using Biotage Isolera One
equipped with an internal variable dual wavelength dioade array detector (200 nm – 400 nm)
Purity of the synthesized compounds were recorded using HPLC (High performance liquid
chromatography) using two Shimadzu LC-10AT pumps, photo diode array detector (SPD-
M10A VP), and a gradient-controlled HPLC system equipped with Shimadzu system
controller (SCL-10A VP) utilizing the Shimadzu Class VP program. A sample volume of 10
μL was injected into a Waters X-Terra® 5 μM reverse-phase C18 column (4.6 ⅹ250 mm).
Both proton and carbon NMR (Nuclear Magnetic Resonance) spectra were performed on a
1
Bruker AMX 250 MHz spectrometer (250 MHz H NMR and 62.5 MHz 13C NMR). The
chemical shifts value (δ) were recorded in ppm (parts per million) and were calibrated
according to TMS and relative to residual peak of solvent used and, the coupling constants (J
values) in Hertz (Hz). ESI LC/MS analysis was performed to calculate mass with a Finnigan
LCQ Advantage® LC/MS/MS spectrometry utilizing the Xcalibur® program. The melting
points were examined in open capillary tubes on an electrothermal 1A 9100 digital melting
point apparatus.
4.2. General method for the synthesis of 1-16
24

4.2.1. General method for Microwave-assisted synthetic pathway 17
In a 5 mL microwave vial, aryl methyl ketone (1.0 equiv), aryl aldehyde (1.0 equiv), indene-
1,3-dione (1.0 equiv) and anhydrous ammonium acetate (5 equiv) were mixed together with
DMF (3 mL). The mixture was irradiated at power of 200 W and 120°C for 1-2 h. Upon
completion as indicated by TLC monitoring. The reaction mixture was cooled to room
temperature and then poured into cold water. Then the mixture was diluted and extracted with
ethylacetate (140 mL), washed with distilled water (30 mL x 4) and saturated sodium chloride
solution (40 mL x 3). The organic layer was dried with magnesium sulfate and filtered. The
filtrate was evaporated with the help of rotatory evaporator. Then flash column
chromatography (Biostage Isolera) was performed to purify using the solvent system of
ethylacetate and hexanes or methylene chloride, ethylacetate and hexanes. Rotary evaporation
of the pure fraction of elutes afforded to 7-12% of desired compounds (1, 2, 5, 6, and 16).
4.2.2. General Method for L-proline catalyzed synthetic pathway 18
In a 25 mL reaction vial, aryl methyl ketone (1.0 equiv), aryl aldehyde (1.0 equiv), indene-
1,3-dione (1.0 equiv), anhydrous ammonium acetate (2.5 equiv) and L-proline (0.15 equiv)
were stirred together in DMF (2 mL) at 100-120°C for 2-24 h. Upon completion as indicated
by TLC monitoring. The reaction mixture was cooled to room temperature and then poured
into cold water. Then the mixture was diluted and extracted with ethylacetate (140 mL),
washed with distilled water (30 mL x 4) and saturated sodium chloride solution (40 mL x 3).
The organic layer was dried with magnesium sulfate and filtered. The filtrate was evaporated
with the help of rotatory evaporator. Then flash column chromatography (Biostage Isolera)
was performed to purify using the solvent system of ethylacetate and hexanes or methylene
25

chloride, ethylacetate and hexanes. Rotary evaporation of the pure fraction of elutes afforded
to 2-26% of desired compounds (4, 8, 9 and 11- 15).
4.2.3. General method for TFE-catalyzed synthetic pathway 19
In a 25 mL reaction vial, aryl methyl ketone (1.0 equiv), aryl aldehyde (1.0 equiv), indene-
1,3-dione (1.0 equiv), anhydrous ammonium acetate (2.5 equiv) were stirred together in TFE
(4 mL) at 80°C for 2-4 h. Upon completion as indicated by TLC monitoring. The reaction
mixture was cooled to room temperature and then poured into cold water. Then the mixture
was diluted and extracted with ethylacetate (140 mL), washed with distilled water (30 mL x 4)
and saturated sodium chloride solution (40 mL x 3). The organic layer was dried with
magnesium sulfate and filtered. The filtrate was evaporated with the help of rotatory
evaporator. Then flash column chromatography (Biostage Isolera) was performed to purify
using the solvent system of ethylacetate and hexanes or methylene chloride, ethylacetate and
hexanes. Rotary evaporation of the pure fraction of elutes afforded to 2-10% of desired
compounds (3, 7 and 10).
Synthesis of 2,4-diphenyl-5H-indeno[1,2-b]pyridin-5-one (1)
Yellow solid, Yield 12.0 %, TLC (ethyl acetate / n-hexane = 1:5) Rf = 0.24, mp: 167.8-
169.3oC, HPLC: Retention time: 9.31 min, purity: 99.4 %; ESI-MS: m/z calcd for C24H15NO
1
[M+H]+ 333.1; found 333.3. H NMR (250 MHz, CDCl3) δ 8.11 – 7.96 (m, 2H), 7.73 – 7.58
(m, 5H), 7.58 – 7.41 (m, 8H). 13C NMR (62.5 MHz, CDCl3) δ 189.67, 171.09, 141.63, 135.62,
134.94 (3C), 132.09 (3C), 130.12, 129.71, 129.27 (3C), 127.49 (3C), 123.83 (3C), 121.78
(3C).
26

Synthesis of 4-(2-hydroxyphenyl)-2-phenyl-5H-indeno[1,2-b]pyridin-5-one (2)
Yellow solid, Yield 7.3 %, TLC (ethyl acetate / n-hexane = 1:3) Rf = 0.23, mp: 196.8-197.6°C,
HPLC: Retention time: 10.28 min, purity: 98.4 %; ESI-MS: m/z calcd for C24H15NO2 [M+H]+
350.1; found 350.8. 1H NMR (250 MHz, DMSO-d6) δ 9.77 (s, 1H), 8.29 – 8.25 (m, 2H), 7.98
(d, J = 7.3 Hz, 1H), 7.79 (s, 1H), 7.74 (td, J = 7.4, 1.2 Hz, 1H), 7.64 (d, J = 7.0 Hz, 1H), 7.60
– 7.48 (m, 4H), 7.37 (dd, J = 7.5, 1.6 Hz, 1H), 7.30 (td, J = 8.4, 8.2, 1.6 Hz, 1H), 6.97 – 6.88
(m, 2H). 13C NMR (62.5 MHz, DMSO-d6) δ 190.24, 165.06, 159.89, 155.18, 146.31, 142.58,
137.89, 135.59, 135.23, 131.69, 130.82, 130.72, 130.50, 129.21 (2C), 127.55 (2C), 123.86,
123.66, 122.93, 122.35, 120.94, 119.00, 115.85.
Synthesis of 4-(3-hydroxyphenyl)-2-phenyl-5H-indeno[1,2-b]pyridin-5-one (3)
Yellow solid, Yield 9.7 %, TLC (ethyl acetate / n-hexane = 1:2) Rf = 0.28, mp: 217.1-218.5°C,
HPLC: Retention time: 11.43 min, purity: 98.9 %; ESI-MS: m/z calcd for C24H15NO2 [M+H]+
350.1; found 350.2. 1H NMR (250 MHz, DMSO-d6) δ 9.69 (s, 1H), 8.39 – 8.27 (m, 4H), 7.97
(d, J = 7.3 Hz, 2H), 7.80 (s, 2H), 7.74 (td, J = 7.4, 1.2 Hz, 2H), 7.65 (d, J = 7.1 Hz, 2H), 7.62
– 7.51 (m, 8H), 7.31 (t, J = 7.8 Hz, 2H), 7.20 – 7.09 (m, 4H), 6.97 – 6.88 (m, 2H). 13C NMR
(62.5 MHz, DMSO-d6) δ 190.39, 165.86, 160.20, 157.28, 149.49, 142.39, 137.69, 136.46,
135.66, 135.16, 131.79, 130.63, 129.43, 129.18 (2C), 127.66 (2C), 123.81, 122.49, 121.15,
120.94, 120.23, 116.73, 116.45.
Synthesis of 4-(4-hydroxyphenyl)-2-phenyl-5H-indeno[1,2-b]pyridin-5-one (4)
Yellow solid, Yield 20.3 %, TLC (ethyl acetate / n-hexane = 1:3) Rf = 0.25, mp: 296.1-
297.5°C, HPLC: Retention time: 9.23 min, purity: 99.3 %; ESI-MS: m/z calcd for C24H15NO2
1
[M+H]+ 350.1; found 350.9. H NMR (250 MHz, DMSO-d6) δ 9.95 (s, 1H), 8.39 – 8.25 (m,
27

2H), 7.96 (d, J = 7.3 Hz, 1H), 7.79 (s, 1H), 7.78 – 7.63 (m, 4H), 7.62 – 7.50 (m, 4H), 6.88 (d,
J = 8.6 Hz, 2H). 13C NMR (62.5 MHz, DMSO-d6) δ 190.67, 166.03, 159.95, 159.40, 149.49,
145.49, 144.44, 142.37, 137.85, 135.52, 135.21, 131.67, 131.51 (2C), 130.51, 129.13 (2C),
127.62 (2C), 125.59, 123.73, 122.03, 120.84, 120.81, 115.12 (2C).
Synthesis of 2-(2-hydroxyphenyl)-4-phenyl-5H-indeno[1,2-b]pyridin-5-one (5)
Yellow solid, Yield 7.9 %, TLC (ethyl acetate / n-hexane = 1:10) Rf = 0.31, mp: 223.5-
226.1°C, HPLC: Retention time: 9.25 min, purity: 98.0 %; ESI-MS: m/z calcd for
C24H15NO2Na [M+Na]+ 372.1; found 372.2. 1H NMR (250 MHz, DMSO-d6) δ 12.73 (s, 1H),
8.18 (d, J = 7.9 Hz, 1H), 8.03 (s, 1H), 7.87 (d, J = 7.3 Hz, 1H), 7.77 – 7.65 (m, 4H), 7.60 –
7.52 (m, 4H), 7.38 (t, J = 7.7 Hz, 1H), 7.01 – 6.93 (m, 2H). 13C NMR (62.5 MHz, DMSO-d6)
δ 189.66, 163.83, 160.23, 158.89, 149.46, 141.13, 135.53, 134.96, 134.91, 132.57, 132.01,
129.81, 129.54 (2C), 129.10, 128.21 (2C), 123.86, 121.93, 121.77, 120.62, 120.20, 119.55,
118.02.
Synthesis of 2,4-bis(2-hydroxyphenyl)-5H-indeno[1,2-b]pyridin-5-one (6)
Yellow solid, Yield 7.4 %, TLC (ethyl acetate / n-hexane = 1:3) Rf = 0.22, mp: 258.0-261.2°C,
HPLC: Retention time: 10.49 min, purity: 99.0 %; ESI-MS: m/z calcd for C24H15NO3 [M+H]+
366.1; found 366.5. 1H NMR (250 MHz, DMSO-d6) δ 12.77 (s, 1H), 9.72 (s, 1H), 8.11 (dd, J
= 8.0, 1.5 Hz, 1H), 7.99 (s, 1H), 7.88 (d, J = 7.3 Hz, 1H), 7.74 (td, J = 7.3, 1.3 Hz, 1H), 7.65
(d, J = 6.9 Hz, 1H), 7.57 (td, J = 7.3, 0.8 Hz, 1H), 7.43 – 7.27 (m, 3H), 7.04 – 6.87 (m, 4H).
13C NMR (62.5 MHz, DMSO-d6) δ 189.62, 163.10, 160.01, 158.90, 155.21, 146.73, 141.38,
135.67, 135.10, 132.63, 132.12, 130.91, 130.82, 129.07, 123.87, 123.37, 122.95, 122.81,
120.82, 120.38, 119.78, 119.05, 118.17, 115.89.
28

Synthesis of 2-(2-hydroxyphenyl)-4-(3-hydroxyphenyl)-5H-indeno[1,2-b]pyridin-5-one (7)
Yellow solid, Yield 2.0 %, TLC (ethyl acetate / n-hexane = 1:3) Rf = 0.20, mp: 299.4-301.6°C,
HPLC: Retention time: 7.59 min, purity: 98.5 %; ESI-MS: m/z calcd for C24H15NO3 [M+H]+
366.1; found 366.5. 1H NMR (250 MHz, DMSO-d6) δ 12.65 (s, 1H), 9.70 (s, 1H), 8.19 (d, J =
7.8 Hz, 1H), 8.02 (s, 1H), 7.89 (d, J = 7.4 Hz, 1H), 7.80 – 7.65 (m, 2H), 7.62 – 7.56 (m, 1H),
7.38 (t, J = 7.5 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.19 – 7.09 (m, 2H), 7.01 (d, J = 8.1 Hz,
1H), 6.98 – 6.88 (m, 2H). 13C NMR (62.5 MHz, DMSO-d6) δ 189.63, 163.89, 160.15, 158.83,
157.21, 149.67, 141.17, 136.26, 135.55, 134.96, 132.56, 132.03, 129.33, 129.12, 123.84,
121.88, 121.73, 120.64, 120.29, 120.15, 119.60, 118.02, 116.76, 116.34.
Synthesis of 2-(2-hydroxyphenyl)-4-(4-hydroxyphenyl)-5H-indeno[1,2-b]pyridin-5-one (8)
Yellow solid, Yield 2.0 %, TLC (ethyl acetate / n-hexane = 1:2) Rf = 0.28, mp: 325.6-327.5°C,
HPLC: Retention time: 6.25 min, purity: 95.1 %; ESI-MS: m/z calcd for C24H15NO3Na
1
[M+Na]+ 388.1; found 388.9. H NMR (250 MHz, DMSO-d6) δ 12.87 (s, 1H), 9.94 (s, 1H),
8.17 (d, J = 7.9 Hz, 1H), 7.97 (s, 1H), 7.85 (d, J = 7.5 Hz, 1H), 7.79 – 7.62 (m, 4H), 7.57 (t, J
13
= 7.3 Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H), 7.10 – 6.94 (m, 2H), 6.94 – 6.83 (m, 2H). C NMR
(62.5 MHz, DMSO-d6) δ 189.98, 164.02, 159.97, 159.56, 159.02, 149.85, 141.10, 135.57,
135.07, 132.62, 132.09, 131.58 (2C), 129.05, 125.41, 123.91, 121.55, 121.19, 120.63, 120.15,
119.66, 118.14, 115.16 (2C).
Synthesis of 2-(3-hydroxyphenyl)-4-phenyl-5H-indeno[1,2-b]pyridin-5-one (9)
Yellow solid, Yield 26.1 %, TLC (ethyl acetate / n-hexane = 1:3) Rf = 0.28, mp: 222.7-
224.9°C, HPLC: Retention time: 9.47 min, purity: 97.3 %; ESI-MS: m/z calcd for C24H15NO2
29