Discovery and Biological Evaluations of Halogenated 2,4-Diphenyl Indeno[1,2- b]pyridinol Derivatives as Potent Topoisomerase IIα-Targeted Chemotherapeutic Agents for Breast Cancer

Article abstract of DOI:10.1021/acs.jmedchem.9b00970

With the aim of developing new effective topoisomerase IIα-targeted anticancer agents, we synthesized a series of hydroxy- and halogenated 2,4-diphenyl indeno[1,2-b]pyridinols using a microwave-assisted single step synthetic method and investigated structure-activity relationships. The majority of compounds with chlorophenyl group at 2-position and phenol group at the 4-position of indeno[1,2-b]pyridinols exhibited potent antiproliferative activity and topoisomerase IIα-selective inhibition. Of the 172 compounds tested, 89 showed highly potent and selective topoisomerase IIα inhibition and antiproliferative activity in the nanomolar range against human T47D breast (2.6 nM) cancer cell lines. In addition, mechanistic studies revealed compound 89 is a nonintercalative topoisomerase II poison, and in vitro studies showed it had promising cytotoxic effects in diverse breast cancer cell lines and was particularly effective at inducing apoptosis in T47D cells. Furthermore, in vivo administration of compound 89 had significant antitumor effects in orthotopic mouse model of breast cancer.

Full text of DOI:10.1021/acs.jmedchem.9b00970

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Article
Discovery and Biological Evaluations of Halogenated 2,4-Diphenyl
Indeno[1,2-b]pyridinol Derivatives as Potent Topoisomerase
II#-targeted Chemotherapeutic Agents for Breast Cancer
Tara Man Kadayat, Seojeong Park, Aarajana Shrestha, Hyunji Jo, Soo-Yeon Hwang, Pramila
Katila, Ritina Shrestha, Mahesh Raj Nepal, Keumhan Noh, Sang Kyoon Kim, Woo-Suk Koh,
Kil Soo Kim, Yong Hyun Jeon, Tae Cheon Jeong, Youngjoo Kwon, and Eung-Seok Lee
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00970 • Publication Date (Web): 09 Aug 2019
Downloaded from pubs.acs.org on August 10, 2019
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Discovery and Biological Evaluations of Halogenated 2,4-Diphenyl Indeno[1,2-
b]pyridinol Derivatives as Potent Topoisomerase II-targeted Chemotherapeutic
Agents for Breast Cancer
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Tara Man Kadayat,†,#,⊥ Seojeong Park,‡,⊥ Aarajana Shrestha,†,⊥ Hyunji Jo,‡ Soo-Yeon
‡
†
†
†
†
Hwang, Pramila Katila, Ritina Shrestha, Mahesh Raj Nepal, Keumhan Noh, Sang Kyoon
§
§
§,∥
§
†
Kim, Woo-Suk Koh, Kil Soo Kim, Yong Hyun Jeon, Tae Cheon Jeong, Youngjoo
‡
,*
†,*
Kwon, Eung-Seok Lee
†College of Pharmacy, Yeungnam University, Gyeongsan 38541, Republic of Korea
‡College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans
University, Seoul 120-750, Republic of Korea
#New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu
4
1061, Republic of Korea
§Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu
4
1061, Republic of Korea
∥College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Republic of
Korea
⊥These authors contributed equally to this study.
*
*
*
Corresponding Authors:
E-mail: ykwon@ewha.ac.kr. Tel: +82 2 3277-4653. Fax: +82 2 3277-2851 (Y.K.)
E-mail: eslee@yu.ac.kr. Tel: +82 53 810-2827. Fax: +82 53 810-4654 (E.-S.L.)
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Abstract
With the aim of developing new effective topoisomerase II-targeted anticancer agents, we
synthesized a series of hydroxy- and halogenated 2,4-diphenyl indeno[1,2-b]pyridinols using
a microwave-assisted single step synthetic method, and investigated structure-activity
relationships. The majority of compounds with chlorophenyl group at 2-position and phenol
group at 4-position of indeno[1,2-b]pyridinols exhibited potent anti-proliferative activity and
topoisomerase II-selective inhibition. Of the one hundred seventy-two compounds tested,
0
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9 showed highly potent and selective topoisomerase II inhibition and anti-proliferative
activity in the nanomolar range against human T47D breast (2.6 nM) cancer cell lines. In
addition, mechanistic studies revealed compound 89 is a non-intercalative topoisomerase II
poison, and in vitro studies showed it had promising cytotoxic effects in diverse breast cancer
cell lines and was particularly effective at inducing apoptosis in T47D cells. Furthermore, in
vivo administration of compound 89 had significant antitumor effects in orthotopic mouse
model of breast cancer.
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Introduction
Cancers are heterogeneous malignant diseases caused by abnormal cell division, and cancer
cells have the ability to disseminate through the blood or lymphatic systems and invade
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distant organs. DNA topoisomerase (topo) has the unique ability to alleviate the DNA
torsional stress that occurs during replication, transcription, recombination, and chromosomal
segregation and is an important clinical target for the treatment of cancer.2-4 There are many
clinically approved topo inhibiting drugs, such as doxorubicin and teniposide, but due to the
complexities of cancer, drug resistance, and systemic side-effects, the selectivity for topo is
5
being scrutinized. Of the major types of clinically important human topo enzymes, topo I
transiently breaks only one strand of DNA, whereas, topo II, (which has α and β isoforms)
cuts both strands in the presence of magnesium and ATP binding.6-8 Despite similar catalytic
properties and 70% similarly between the amino acid sequences of two different topo II
isoforms, only the expression level of topo II varies significantly during cell growth.9-11 This
unique characteristic makes topo II an efficient and safe target as its use minimizes topo II-
mediated toxicities such as cardiotoxicity and leukemia.12-14 Therefore, research efforts are
being directed toward developing safer and more effective topo II -targeting cancer
chemotherapies.15
Over the past few decades, several studies on drug discovery and development have
revealed the importance of hydroxyl and halogen moieties on molecular scaffolds of potential
interest.16-18 With the aim of identifying novel topo-targeted anticancer agents, our group
previously designed and synthesized several flexible 2,4,6-trisubstituted pyridines with
phenolic and chlorophenyl groups at the 2- and/or 4-positions, and thienyl, furyl, or phenyl
groups at the 6-position.19-21 The promising results obtained using these flexible pyridine
derivatives prompted us to design and synthesize rigid benzofuro[3,2-b]pyridines and 5H-
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indeno[1,2-b]pyridines incorporating phenolic, chlorophenyl, and other aryl groups at 2- and
4
-positions (Fig. 1). Introductions of various functionalities in rigid benzofuro[3,2-b]pyridine
and 5H-indeno[1,2-b]pyridine allowed us to explore structure-activity relationships based on
their topo inhibitory and anticancer activities.22, 23 In particular, we found that a phenol or
chlorophenyl ring at the 2- or 4-positions of the benzofuro[3,2-b]pyridine skeleton (Fig. 2,
compounds A and B) were crucial for the dual inhibitions of topo I and II.22, 24 However,
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benzofuro[3,2-b]pyridin-7-ol (compound C) showed potent and selective topo II
2
5
inhibition. On the other hand, 5H-indeno[1,2-b]pyridine compounds D and E with a phenol
or chlorophenyl group at the 2- or 4-positions acted as a non-intercalative topo II catalytic
inhibitor and as a strong cytotoxic agent, respectively.23, 26
R
4
R
Y
Modification to
rigid analogs
Z
X
R =
2
6
Z
R
N
R
R
N
Where, Y= H, OH or Cl
Z= S or O
Flexible
,4,6-trisubstituted pyridines
Rigid analogs
X= O: benzofuro[3,2-b]pyridine
X= CH : 5H-indeno[1,2-b]pyridine
2
2
Figure 1. Structures of previously reported rigid analogs of 2,4,6-trisubstituted pyridines.
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OH
HO
benzofuro[3,2-b]pyridine
4
O
O
O
O
HO
HO
2
N
N
N
N
OH
A
Cl
B
C
Non-intercalative dual topo I
and II catalytic inhibitor
Non-intercalative dual topo I
and II catalytic inhibitor
Potent and selective
topo II inhibitor
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OH
HO
4
Hydroxylation at
R
6-, 7-, 8- & 9- position
2
N
Cl
N
N
6
OH
5H-indeno[1,2-b]pyridine
D
E
N
7
R1
9
8
Non-intercalative topo
II catalytic inhibitor
Strong cytotoxic for HCT15 colon
cancers cell (IC50 =0.31 M)
R, R = H, OH, Cl, F, CF
1
3
5
H-indeno[1,2-b]pyridinol
scaffold (1-172)
Figure 2. Strategy for the design of 2,4-diphenyl-substituted-5H-indeno[1,2-b]pyridinols.
Compounds A-E are previously synthesized active compounds, and compounds 1-172 are
newly prepared in the present study.
Based on these reported findings, our major focus was to maximize topo II-targeting
anticancer activity by introducing a hydroxyl group at positions 6-, 7-, 8- or 9- of the 5H-
indeno[1,2-b]pyridine skeleton. We designed 172 novel rigid 2,4-disubstituted 5H-
indeno[1,2-b]pyridinol compounds incorporating moieties such as phenyl, phenolic,
chlorophenyl, fluorophenyl and trifluoromethylphenyl at the 2- or 4-positions (Fig. 2). Here,
for the first time we have introduced fluorine (-F) and trifluoromethyl (-CF ) functionalities
3
at the 2- phenyl rings anticipating to discover the novel and more potent topo II inhibitors.
Compounds were synthesized using a microwave-assisted single step, three-components
strategy, which proved to be rapid, effective, and environmental-friendly. In addition, as
compared to the previously reported Kröhnke synthetic method, the devised method does not
involve additional synthetic steps for the preparations of intermediates and pyridinium iodide
salts. Compound 89 exhibited highly potent topo IIselective inhibition and anti-
proliferative activity in the nanomolar range (~2.6 nM) against T47D human breast cancer
cell lines and was selected for further mechanistic and in vivo ADME profile studies. In
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addition, bioluminescent imaging and in vivo studies were performed to investigate the
potential of compound 89 for breast cancer therapy.
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Results and Discussion
Chemistry. Initially, we utilized the previously reported three-step Kröhnke synthetic
23
method (Scheme 1A) to prepare 2,4-diphenyl 5H-indeno[1,2-b]pyridinols (compounds 1 to
4
) by adding hydroxy-indanone intermediates (Va-d) (1.0 equiv) and pyridinium iodide salt
(II) (1.5 equiv) to glacial acetic acid and then adding anhydrous ammonium acetate
(
NH OAc; 10.0 equiv). This mixture was then refluxed at 100 °C for 24 h, and compounds 1
4
to 4 were synthesized in yields of 35.7 to 51.6% as described in the Supporting Information.
However, as shown in Scheme 1B and detailed in the Experimental Section the use of
microwave radiation27 and three-component reactions between equivalent amounts of
benzaldehyde, hydroxy-1-indanone, and acetophenone in the presence of NH OAc (2.5
4
o
equiv.) in DMF at 120 C for 20-30 min resulted relatively higher yields (38.5 to 63.6%) of
target compounds 1 to 4. Thus, to synthesize a series of 2,4-disubstituted 5H-indeno[1,2-
b]pyridinols (compounds 5-172), we utilized this microwave-assisted synthetic method
(Scheme 2). The observed range of compounds yield (11.1 to 58.6%) depends on substitution
of hydroxyl and halogen groups at 2- and 4-phenyl ring of 5H-indeno[1,2-b]pyridinols.
Compounds 1-4 without any substitution at 2- and 4-phenyl ring have highest range of yield
(
38-63%), while on substituting hydroxyl group at 2- or 4-phenyl position, compounds 5-28
except 18) resulted decrease in yield ranging from 23-58%. Similarly, on addition of halogen
(
group, chlorinated compounds 29-100, most of the compounds have moderate yield ranging
from 20-43%. However, most of fluorine containing compounds (101-172) have low yield
(below 20%). Purity and characterization of all compounds synthesized were established by
HPLC, MS, NMR and TLC.
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a
Scheme 1. Synthesis of compounds 1-4 , (A) Using conventional Kröhnke synthetic
approach, and (B) Microwave-assisted single-step synthetic method.
A
O
HO
CH3
1
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1
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O
I (Acetophenone)
III (Hydroxy-indanone)
O
R = 6-OH 7-OH 8-OH
9-OH
H
a
b
c
d
(i)
(ii)
N
IV (Benzaldehyde)
O
N
I-
(iii)
HO
R
N
O
Va-d
II
1-4
(
Pyridinium iodide Salt)
(Hydroxy-indanone Intermediate)
(35.7-51.6%)
B
HO
(iv)
H
+
CH3
OH
+
N
O
O
O
Benzaldehyde
hydroxy-1-indanone
Acetophenone
1-4
(38.5-63.6%)
aReagents and conditions: (i) iodine (1.0 equiv.), pyridine, 3 h at 140 °C, 66% yield; (ii) aq.
NaOH, EtOH, 1-3 h at room temperature, 28.8- 81.7% yield; (iii) NH OAc (10.0 equiv.),
glacial acetic acid, 24 h at 100 C, 35.7-51.6% yield; (iv) NH OAc (2.5 equiv.), DMF, 200 W,
4
o
4
o
20-30 min at 120 C, 38.5-63.6% yield.
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Scheme 2. Synthesis of compounds 5-172a
R
R = H/OH
4
6
2
OH
7
0
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(i)
R
R
N
H
+ HO
CH3
R
+
9
8
5
-28
O
O
O
(
2,4-phenol series)
Cl
4
6
2
OH
7
(i)
Cl
HO
N
H
+ HO
CH3
HO
9
8
+
29-64
O
O
O
(
2-phenol-4-chlorophenyl series)
OH
R1 = Cl, F, CF3
4
6
2
OH
7
(i)
HO
R1
N
H
+ HO
CH3
R1
9
8
+
6
5-172
O
O
O
(2-halophenyl-4-phenol series)
a
o
Reagents and conditions: (i) NH OAc (2.5 equiv.), DMF, 200 W, 30-180 min at 120-150 C,
4
1
1.1-58.6% yield.
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In vitro Cell Proliferation Inhibitory Activities. First, we evaluated the abilities of
the 172 synthesized compounds to inhibit the four human cancer cell lines HCT15, T47D,
DU145, and HeLa (IC values are provided in Tables 1-6). Most of the compounds potently
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inhibited cell proliferation as compared with etoposide (the reference compound).
Camptothecin, etoposide and adriamycin were used as positive control for antiproliferative
activity.
In the 2,4-diphenyl series (compounds 1-4), all four compounds inhibited HCT15,
T47D and HeLa proliferation more potently than etoposide. Compound 3 had greater
antiproliferative activity (IC = 0.19 µM) than all three reference compounds in the T47D
5
0
cell line. Interestingly, almost all the 2-phenyl, 4-phenol series (compounds 5-16)
significantly inhibited the proliferations of HCT15, T47D, and DU145 cell lines, whereas
half of the compounds inhibited HeLa proliferation more than etoposide. Compounds 5, 7-10,
1
2 inhibited DU145 proliferation significantly more (IC < 0.81 µM) than the three reference
50
compounds. Similarly, most compounds in the 2-phenol, 4-phenyl series (compounds 17-28)
inhibited the proliferation of DU145 (IC = 0.27-0.71 µM) significantly more than the three
50
reference compounds. However, only compounds 21, 22, 25-27 potently inhibited the
proliferation of HCT15 cells (IC = 1.02-1.58 µM) lines. Compounds 21, 24, 26, and 27
5
0
inhibited T47D cell proliferation, and compounds 17, 26-28 inhibited HeLa proliferation
(IC = 1.83-2.74 µM) better than etoposide (Table 1).
5
0
Likewise, in the 2-phenol, 4-chlorophenyl series (compounds 29-64), with the
exceptions of compounds 30-32 in the HeLa cell line, compounds 29-40, which contained a
chlorine group at the ortho-position of 4-phenyl ring, exhibited antiproliferative activities
similar to/or greater than etoposide. However, when the chlorine group was in the meta- or
para-positions, most compounds showed weak to moderate antiproliferative activity,
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2
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2
2
2
2
2
2
2
3
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4
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4
4
4
4
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5
5
5
5
5
5
5
5
5
6
especially in the HCT15, T47D, and HeLa cell lines. Only compounds 44, 45, 49, and 57
inhibited HCT15 proliferation more than etoposide, whereas compounds 44, 49-51, 57, 60,
62, and 63 inhibited T47D proliferation more than etoposide. Similarly, only compounds 46,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
47, 49-51, 57, and 62 inhibited HeLa proliferation more etoposide. Compounds, 44, 47, 49-
51, 56, 57, and 60-63, inhibited DU145 proliferation significantly more than etoposide (Table
2).
Interestingly, interchanging the positions of chlorine and hydroxyl groups altered
antiproliferative activities. For example, the 2-chlorophenyl, 4-phenol series (compounds 65-
00) had better antiproliferative activities than the 2-phenol, 4-chlorophenyl series in all four
1
cell lines. With the exception of compound 100, all compounds in the 2-chlorophenyl, 4-
phenol series displayed greater antiproliferative activity (IC50 = 0.00267-3.5 µM) than
etoposide (IC = 4.85 µM) in the T47D cell line. Similarly, in the DU145 cell line, almost all
5
0
compounds in the 2-chlorophenyl, 4-phenol series showed greater antiproliferative activity
IC = 0.072-2.03 µM) than etoposide (IC = 2.19 µM). Likewise, with the exception of
(
5
0
50
compound 100 in the HCT15 cell line and of compounds 69, 79, 95, and 100 in the HeLa cell
line, all compounds displayed greater or antiproliferative activities similar to etoposide in
these two cell lines. As shown in Table 3, compound 78 exhibited highly potent
antiproliferative activity (IC50 = 1.33 nM) against HCT15 cells, whereas compound 89
showed IC50 value 2.67 nM against T47D cells. It is very interesting to note that both
compounds (78 and 89) showed antiproliferative activity at nanomolar levels.
In the 2-fluorophenyl, 4-phenol series (compounds 101-136) (Table 4), with the
exceptions of compounds 104, 110, 127, 128, and 136, all compounds inhibited T47D
proliferation more than or to extents similar (IC = 0.19 to 2.97 µM) as the three positive
5
0
controls, and compound 111 had the greatest effect (IC = 0.19 µM). Moreover, half of the
5
0
compounds in this series inhibited HeLa proliferation (IC = 2.25 µM to 4.94 µM) more than
5
0
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etoposide (IC = 5.76 µM). However, in DU145 cell line, only compounds 107, 115, 120,
5
0
1
21, and 123, which contained ortho- or meta-phenolic groups inhibited (IC = 0.40 µM to
50
1.06 µM) proliferation greater than etoposide (IC = 1.15 µM). The other compounds showed
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
only moderate anti-proliferative activity. In the case of the HCT15 cell line, only compounds
19 (IC = 1.19 µM) and 123 (IC = 1.46 µM) exhibited greater anti-proliferative activity
1
5
0
50
than etoposide (IC = 1.72 µM). The other compounds inhibited HCT15 less than or at levels
50
similar to the three positive controls.
The IC values of members of the 2-trifluorophenyl, 4-phenol series (compounds
5
0
1
37 - 172) for inhibitions of the proliferations of HCT15, T47D, DU145, and HeLa cell lines
are provided in Table 5. With the exceptions of compounds 157, 158, 167, 168, and 172, all
of these compounds inhibited T47D proliferation (IC = 0.51 µM to 4.2 µM) more than
5
0
etoposide (IC = 4.26 µM). In HeLa cells, all compounds containing an ortho-phenolic group
5
0
(137 to 148) displayed stronger anti-proliferative activity (IC = 1.99 µM to 7.87 µM) than
5
0
etoposide (IC = 9.75 µM). Similarly, with the exceptions of six compounds (156, 158, 161,
5
0
1
64, 168, and 172), all members of the meta- and para- phenolic series inhibited HeLa cell
proliferation more (IC = 2.03 µM to 6.66 µM) than etoposide. Likewise, with the exceptions
50
of six compounds (148, 157, 161, 164, 168, and 172) all compounds inhibited DU145
proliferation (IC = 1.14 µM to 3.46 µM) more than etoposide (IC = 4.49 µM). Finally, in
5
0
50
the case of the HCT15 cell line, with the exceptions of compounds (145, 146, 155 - 158, 160,
164, 168, 169, 171, and 172), all compounds inhibited proliferation (IC = 1.32 µM to 4.04
5
0
µM) more than etoposide (IC = 4.06 µM).
5
0
1
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9
1
1
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
Table 1. Structures, topo I and II inhibitory activities, and IC50 values for compounds 1-28 in the four cancer cell lines.
%
Inhibition of
Compound
R
1
R
2
*IC50 (µM)
Topo I
00 µM 20 µM
Topo II
100 µM
1
20 µM
31.1
1.3
HCT15
T47D
DU145
HeLa
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
Camptothecin
Etoposide
71.6
NT
1
.6±0.03
.97±0.17
.78±0.06
1.98±0.01
4.02±0.04
0.81±0.02
1.93±0.03
1.24±0.02
4.96±0.03
76.2
6
1
Adriamycin
0.34±0
1.04±0.01
2.71±0.05
0.82±0.01
4.18±0.07
1
2
3
4
-
-
-
-
6’-OH
7’-OH
8’-OH
9’-OH
4
.6
2.1
.6
0.0
.0
8.2
.0
0.1
.6
20.0
.5
1.6
.4
11.1
.0
10.2
.6
NT
75.7
3.39±0.18
1.77±0.03
NT
NT
54.2
79.9
2.1
5.5
3.37±0.03
3.13±0.03
0.7±0.01
3.82±0.02
3.49±0.02
4.94±0.1
2
0.19±0.01
4.38±0.01
R2
N
NT
NT
57.5
78.5
0.0
0.0
2.07±0.05
3.47±0.21
0.74±0.01
2.33±0.07
1.4±0.02
0.8±0.02
3.08±0.08
6.32±0.02
5
6
7
8
9
2-OH 6’-OH
2-OH 7’-OH
2-OH 8’-OH
2-OH 9’-OH
3-OH 6’-OH
0
NT
NT
74.8
97.4
0.0
0.0
2.08±0.08
3.01±0.04
2.44±0.05
2.33±0.07
1±0.16
27.4±0.06
3.74±0.07
0
0.15±0.01
NT
NT
NT
NT
NT
NT
NT
NT
95.8
95.4
96.8
1.2
0.1
1.36±0.02
2.52±0.08
1.48±0.01
1.37±0.01
1.48±0.03
2.78±0.04
>50
2.44±0.05
2.89±0.1
0.17±0.03
0.19±0.02
0.6±0.01
2.95±0.07
6.74±0.35
5.64±0.05
1.38±0.01
2.03±0.08
1.94±0.03
2.75±0.01
>50
4
0.0
0.0
NT
NT
NT
NT
NT
R1
10 3-OH 7’-OH
11 3-OH 8’-OH
2.66±0.12
0.84±0.01
2.01±0.06
2.89±0.07
3.55±0.29
2.99±0.08
0
1.57±0.02
0.72±0.01
1.28±0.03
2.48±0.01
1.38±0.01
R2
N
1
2
3-OH 9’-OH
13 4-OH 6’-OH
4-OH 7’-OH
15 4-OH 8’-OH
4-OH 9’-OH
17 2-OH 6’-OH
6.3
4
1.5
1
4
2.5
6
2.4
>50
1
6
NT
NT
4.0
1.1
NT
NT
>50
>50
>50
4.13±0.09
2.49±0.12
>50
6
6.43±0.06
1.83±0.01
1
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9
1
1
2
2
2
2
2
2
8
9
0
1
2
3
4
5
2-OH 7’-OH
2-OH 8’-OH
2-OH 9’-OH
3-OH 6’-OH
3-OH 7’-OH
3-OH 8’-OH
3-OH 9’-OH
4-OH 6’-OH
2
.0
0.0
.0
0.4
NT
NT
19.0
2.8
NT
NT
>50
>50
>50
>50
1.62±0.02
1.87±0.02
>50
>50
NT
1
NT
NT
NT
NT
NT
NT
NT
NT
NT
1.2
4.5
>50
1.58±0.05
1.3±0.03
>50
>50
9.61±0.33
0.52±0.03
0.27±0.09
0.42±0
>50
R3
N
NT
3.3
NT
NT
NT
8.3
9.3
NT
3.92±0.05
4.12±0.02
>50
6.2±0.02
5.7±0.02
>50
R1
1
0.2
36.0
3.5
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
0.0
0
.0
5.3
.8
20.9
.1
0.0
>50
3.68±0.03
6.23±0.02
0.73±0.01
3.43±0.05
11.56±0.45
0.43±0.02
0.37±0.03
0.71±0.01
0.57±0.02
0.66±0.06
>50
4.4
1.02±0.05
1.27±0.04
1.43±0.04
>50
5.74±0.24
2.67±0.12
2.74±0.09
2.06±0.02
26 4-OH 7’-OH
4-OH 8’-OH
28 4-OH 9’-OH
8
42.8
54.8
3.7
2
7
1
NT: Not Tested; HCT-15: human colorectal adenocarcinoma; T47D: Human breast ductal carcinoma; DU145 (human prostate
tumor cell line; HeLa: human cervix adenocarcinoma cell line; Adriamycin: positive control for antiproliferative activity;
Etoposide: positive control for topo IIα and antiproliferative activity; Camptothecin: positive control for topo I and
antiproliferative activity. *Results are presented as the means±SDs of three independent experiments performed in triplicate.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
Table 2. Structures, topo I and II inhibitory activities, and IC50 values of compounds 29-64 in the four cancer cell lines.
%
Inhibition
Compound
R
1
R
2
*IC50 (µM)
DU145
Topo I
Topo II
1
00 µM
20 µM
100 µM
20 µM
HCT15
T47D
HeLa
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
Camptothecin
Etoposide
78.6
38.2
0
.16±0.001
1.64±0.07
5.54±0.21
0.05±0.004
4.13±0.46
1.24±0.02
8.09±0.12
77.9
38.3
5.25±0.03
.02±0.01
Adriamycin
2
1.12±0.07
3.03±0.01
2.96±0.01
4.03±0.03
1.89±0.01
4.59±0.04
2
3
3
3
3
9
0
1
2
3
2-OH 6’-OH
2-OH 7’-OH
2-OH 8’-OH
2-OH 9’-OH
3-OH 6’-OH
3-OH 7’-OH
3-OH 8’-OH
3-OH 9’-OH
4-OH 6’-OH
4-OH 7’-OH
4-OH 8’-OH
4-OH 9’-OH
2-OH 6’-OH
2-OH 7’-OH
2-OH 8’-OH
2-OH 9’-OH
3-OH 6’-OH
23.7
NT
79.2
1.8
4.12±0.08
3
7.0
59.1
3.9
7.4
55.9
74.1
3.5
3.13±0.02
7.98±0.1
3.82±0.07
3.28±0.05
4.43±0.008
3.99±0.02
17.89±0.06
>50
14.8
38.2
2
NT
NT
6.3
6.3
0.2
0
56.5
67.5
91.3
93.2
80.2
97.0
80.0
74.8
1.6
11.4
20.0
13.8
3.6
2.03±0.02
8.28±0.09
3.82±0.02
2.5±0.03
2.93±0.05
2.88±0.06
2.84±0.29
2.91±0.01
2.51±0.04
1.73±0.01
2.7±0.06
5.67±0.02
5.63±0.01
2.35±0.02
2.03±0.01
3.21±0.01
5.84±0.14
3.25±0.06
4.65±0.005
>50
28.1
77.4
55.9
7.36±0.03
6.48±0.04
5±0.01
34
Cl
3
5
R2
N
R1
36
7
9.3
62.9
1.9
4.0
2.43±0.01
2.42±0.01
4.27±0.04
2.91±1.46
3.64±0.02
3.72±0.01
4.92±0.11
6.5±0.01
3
7
13.0
7.0
38
1
NT
NT
3
9
2.0
1.64±0.1
40
1
0.3
0
NT
NT
66.9
75.4
0.8
1.5
1.05±0.1
0.96±0.03
12.03±0.1
0.86±0.002
6.01±0.02
2.79±0.01
4
1
2.45±0.03
20.53±0.95
42
1
.6
.9
NT
NT
74.3
67.2
3.4
4.0
>50
>50
>50
>50
4
3
1
7
29.77±0.44
7.69±0.06
37.14±0.59
19.98±0.24
44
0
NT
NT
74.5
64.6
2.4
2.3
2.46±0.01
4.79±0.04
1.97±0.06
2.82±0.02
21.3±0.1
14.34±0.58
35.49±0.15
4
5
.4
33.09±0.02
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9
Cl
4
4
4
4
5
5
5
5
6
7
8
9
0
1
2
3
3-OH 7’-OH
3-OH 8’-OH
3-OH 9’-OH
4-OH 6’-OH
4-OH 7’-OH
4-OH 8’-OH
4-OH 9’-OH
2-OH 6’-OH
2-OH 7’-OH
2-OH 8’-OH
2-OH 9’-OH
3-OH 6’-OH
3-OH 7’-OH
3-OH 8’-OH
3-OH 9’-OH
4-OH 6’-OH
4-OH 7’-OH
4-OH 8’-OH
4-OH 9’-OH
3
.7
NT
NT
NT
NT
NT
NT
83.0
76.3
71.9
75.3
73.9
73.6
27.0
3.5
0.3
2.2
3.3
1.2
25.31±0.34
6.6±0.01
>50
22.63±0.75
7.2±0.03
>50
6.29±0.04
3.22±0.02
17.12±1.33
1.64±0.02
2.25±0.02
2.1±0.004
7.74±0.54
6.24±0.04
>50
R2
11.6
N
R1
8
6
8
.9
.1
.8
2.39±0.02
5.69±0.15
7.59±0.05
1.92±0.14
1.86±0.01
2.76±0.03
1.66±0.08
5.94±0.03
8.06±0.02
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
8.8
3
8
.2
.4
NT
NT
71.6
71.6
13.3
1.5
>50
>50
19.03±0.71
>50
6.04±0.055
12.82±0.26
43.78±0.17
>50
54
2
1.4
.9
NT
NT
70.8
21.4
0
0
>50
>50
>50
>50
> 50
> 50
>50
>50
5
5
2
56
0
NT
NT
NT
NT
NT
NT
NT
NT
NT
70.5
86.3
74.3
80.4
95.7
97
6.4
4.4
18.79±2.23
2.58±0.04
>50
13.04±0.21
2.11±0.03
>50
1.95±0.01
3.94±0.004
> 50
>50
4.63±0.05
>50
Cl
5
7
0
58
59
60
2
.1
7.3
.3
1.3
.7
6.2
R2
N
R1
12.4
8.5
>50
>50
32.18±0.19
4.07±0.02
1.81±0.12
1.43±0.01
1.74±0.01
> 50
>50
9
14.67±0.13
9.1±0.03
18.48±0.41
12.01±0.21
>50
2.22±0.01
12.75±0.16
1.51±0.02
2.67±0.03
>50
9.01±0.01
18.32±0.19
6.83±0.09
10.05±0.02
>50
6
1
7.5
62
7
98.1
95.3
96.8
8.9
6
3
1
4.2
0
10.8
9.3
64
*
Results are presented as the means±SDs of three independent experiments performed in triplicate.
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2
3
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
Table 3. Structures, topo I and II inhibitory activities, and IC50 values of 65-100 in the four cancer cell lines
%
Inhibition
Compound
R
1
R
2
*IC50 (µM)
DU145
Topo I
Topo II
1
00 µM
20 µM
100 µM
20 µM
HCT15
T47D
HeLa
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
Camptothecin
Etoposide
72.5
NT
0
.35±0.018
0.59±0.008
4.85±0.017
0.38±0.07
2.19±0.14
1.39±0.07
4.13±0.012
75.0
35.6
1.27±0.007
.74±0.007
Adriamycin
0
1.5±0.063
1.59±0.06
0.83±0.01
2.04±0.06
6
6
6
6
6
5
6
7
8
9
2-Cl 6’-OH
2-Cl 7’-OH
2-Cl 8’-OH
2-Cl 9’-OH
3-Cl 6’-OH
3-Cl 7’-OH
3-Cl 8’-OH
3-Cl 9’-OH
4-Cl 6’-OH
4-Cl 7’-OH
4-Cl 8’-OH
4-Cl 9’-OH
2-Cl 6’-OH
2-Cl 7’-OH
0
NT
82.0
0
0.77±0.001
0.92±0.002
0.68±0.001
1
1.7
NT
NT
70.5
76.8
8.2
5.8
2.34±0.23
4.94±0.06
2.86±0.07
2.84±0.06
1.51±0.04
0.77±0.02
0.84±0.01
6.71±0.13
0.0
1
3.4
NT
NT
NT
NT
NT
NT
NT
NT
80.9
83.5
80.8
87.9
80.0
80.3
97.45
86.6
3.7
6.9
3.17±0.22
7.47±0.07
2.76±0.01
1.76±0.03
1.31±0.08
2.31±0.14
3.97±0.01
1.78±0.02
0.87±0.01
0.64±0.04
2.77±0.05
1.16±0.01
0.96±0
0.29±0.01
0.56±0.02
0.36±0.01
0.32±0.02
0.22±0.02
0.61±0.01
0.65±0.02
0.49±0.03
1.12±0.04
>50
0.0
70
OH
14.6
22.9
2.6
2.69±0.05
2.77±0.05
3.62±0.04
1.96±0.01
3.84±0.06
4.61±0.03
7
1
R2
10.8
3.2
N
R1
72
1
3.1
14.5
6.8
8.0
.0
7
3
7.0
1.25±0.07
0.62±0.03
0.72±0.04
74
2
4.4
7
5
25.7
76
0
NT
NT
83.9
25.3
24.9
1.23±0.03
0.8±0.002
2.41±0.06
0.55±0.02
0.92±0.01
5.31±0.01
7
7
0
100.0
1.53±0.015
0.59±0.004
78
0
.00133±0.0
023
5.6
3.9
NT
100.0
32.3
0.78±0.002
0.81±0.002
0.91±0.03
0
7
8
9
0
2-Cl 8’-OH
2-Cl 9’-OH
NT
NT
88.7
89.6
38.3
10.9
3.04±0.01
0.55±0.02
0.12±0.01
0.57±0.01
0.24±0.02
>50
6
.3
3.82±0.08
5.25±0.03
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Journal of Medicinal Chemistry
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9
OH
8
8
8
8
8
8
8
8
1
2
3
4
5
6
7
8
3-Cl 6’-OH
3-Cl 7’-OH
3-Cl 8’-OH
3-Cl 9’-OH
4-Cl 6’-OH
4-Cl 7’-OH
4-Cl 8’-OH
4-Cl 9’-OH
2-Cl 6’-OH
0
.5
.5
NT
NT
NT
NT
NT
NT
NT
NT
NT
100.0
80.2
90.5
88.6
76.8
95.0
87.8
85.2
100.0
24.1
37.8
14.3
3.4
1.13±0.01
1.62±0.01
0.84±0.02
2.49±0.02
1.82±0.001
0.85±0.002
0.86±0.01
3.05±0.06
0.014±0.001
1.52±0.001
0.86±0.004
1.37±0.02
1.63±0.02
1.96±0.01
1.5±0.01
1.49±0.02
3.21±0.037
3.28±0.45
3.48±0.62
2.03±0.019
1.28±0.026
4.43±0.28
0.36±0.01
0.072±0.001
3.31±0.071
5.99±0.04
5.44±0.14
4.59±0.03
4.32±0.005
1.09±0.02
1.73±0.17
6.74±0.02
2.46±0.049
0
R2
N
R1
1
1
1
6.9
8.6
0
42
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
11.2
51.9
26.5
78.9
1
6.7
3.5±0.08
7.4
0
3.5±0.08
0.00267±0.0
0021
89
90
2-Cl 7’-OH
2-Cl 8’-OH
2-Cl 9’-OH
3-Cl 6’-OH
3-Cl 7’-OH
3-Cl 8’-OH
3-Cl 9’-OH
4-Cl 6’-OH
4-Cl 7’-OH
4-Cl 8’-OH
4-Cl 9’-OH
7
.6
NT
NT
86.6
80.9
53.6
19.2
1.85±0.07
3.04±0.08
1.87±0
0.43±0.02
0.54±0.03
0.7±0.01
9
1
0
0.95±0.02
3.22±0.04
92
0
NT
30.3
3.6
2.97±0.04
0.61±0
0.19±0.01
2.85±0.01
OH
9
3
1.2
0
NT
NT
NT
NT
NT
NT
NT
NT
72.8
100.0
71.3
24.4
59.9
100.0
74.5
26.2
36.9
31.9
56.4
NT
1.84±0.01
0.75±0.003
2.36±0.05
1.3±0.44
3±0.13
0.62±0.01
1.2±0.01
0.6±0
0.22±0.01
1.2±0.006
0.48±0.07
0.03±0.02
1.88±0.01
1.91±0.009
2.25±0.06
2.8±0.11
3.56±0.14
1.17±0.01
>50
94
R2
9
5
N
8.7
R1
96
0
6
.0
.7
0.83±0
6.23±0.03
4.08±0.01
1.02±0.01
1.73±0.03
>50
9
7
0.0
1.15±0.01
1.84±0.004
1.15±0.02
>50
98
0
50.2
0.0
0.77±0.003
3.22±0.06
>50
9
9
0
1
00
0
.2
NT
*
Results are presented as the means±SDs of three independent experiments performed in triplicate.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
Table 4. Structures, topo I and II inhibitory activities, and IC50 values for compounds 101-136 in the four cancer cell lines
%
Inhibition
Compound
R
1
R
2
*IC50 (µM)
DU145
Topo I
Topo II
1
00 µM
7.5
20 µM
100 µM
20 µM
HCT15
0.049±0.03
1.72±0.11
T47D
HeLa
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
Camptothecin
Etoposide
6
NT
0.33±0.02
1.64±0.12
0.18±0.01
1.15±0.08
0.74±0.05
5.76±0.55
82.1
30.5
Adriamycin
0
.94±0.03
0.74±0.07
1.77±0.03
0.7±0.01
0.87±0.05
2.71±0.05
3.82±0.02
4.07±0.02
9.9±0.1
0.8±0.05
4.18±0.07
4.94±0.1
1
1
1
1
1
01
02
03
04
05
2-F
2-F
2-F
2-F
3-F
3-F
3-F
3-F
4-F
4-F
4-F
4-F
2-F
2-F
2-F
2-F
3-F
6’-OH
7’-OH
8’-OH
9’-OH
6’-OH
7’-OH
8’-OH
9’-OH
6’-OH
7’-OH
8’-OH
9’-OH
6’-OH
7’-OH
8’-OH
9’-OH
6’-OH
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
24.1
13.4
27.0
0.6
3.39±0.18
3.37±0.03
2.98±0.01
3.76±0.07
2.35±0.21
4.23±0.21
2.9±0.02
0.61±0.01
4.56±0.09
1.25±0.02
0.86±0.05
0.68±0.01
0.74±0.01
0.61±0.01
3.14±0.03
0.19±0.01
0.74±0.01
2.71±0.05
2.24±0.08
2.97±0.02
0.83±0.02
0.89±0.01
4.36±0.06
7.63±0.17
3.92±0.05
2.43±0.05
4.22±0.01
7.9±0.07
80.7
28.0
28.3
8.4
4.8
NT
3.94±0.04
3.13±0.01
0.86±0.01
9.97±0.17
2.36±0.01
4.76±0.1
106
07
108
09
110
11
112
13
114
15
116
17
OH
1
NT
NT
R2
N
R1
5.03±0.06
4.31±0.16
4.61±0.13
3.13±0.03
2.07±0.05
4.98±0.11
3.58±0.04
2.96±0.04
8.33±0.32
4.9±0.2
1
67.0
6.0
0.0
NT
31.95±0.91
14.33±0.31
4.38±0.01
3.08±0.08
6.75±0.05
3.98±0.11
3.74±0.24
12.12±0.02
7.04±0.49
1
NT
23.3
34.4
22.4
44.6
28.1
8.7
3.49±0.02
1.4±0.02
NT
NT
0.0
NT
1
9
9
.7
.1
2.03±0.03
1.62±0.03
0.4±0.02
NT
NT
NT
NT
4.9
NT
1
1
6.3
NT
5.6
4
.0
.8
1.88±0.08
2.37±0.04
1
9
43.2
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
OH
1
1
1
1
1
1
1
18
19
20
21
22
23
24
3-F
3-F
3-F
4-F
4-F
4-F
4-F
7’-OH
8’-OH
9’-OH
6’-OH
7’-OH
8’-OH
9’-OH
NT
NT
NT
NT
NT
NT
NT
1
7.5
66.2
44.2
17.8
27.8
65.1
48.3
27.3
0.0
2.71±0.05
1.19±0.03
>50
0.79±0.01
0.54±0.02
1.16±0.03
1.72±0.04
1.72±0.04
0.68±0.01
2.24±0.08
1.41±0.02
2.17±0.01
0.94±0.01
0.56±0.03
2.94±0.02
1.06±0.05
3.3±0.03
3.85±0.23
3.45±0.12
15±0.2
17.1
14.0
26.2
4.9
NT
R2
N
R1
NT
2.68±0.06
1.92±0.03
1.46±0.02
4.04±0.04
6.71±0.02
3.74±0.05
3.82±0.04
8.81±0.04
2
2.3
28.7
5.4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
11.7
2
5
.3
.4
NT
NT
NT
NT
125
26
127
28
129
2-F
2-F
2-F
2-F
3-F
3-F
3-F
3-F
4-F
4-F
4-F
4-F
6’-OH
7’-OH
8’-OH
9’-OH
6’-OH
7’-OH
8’-OH
9’-OH
6’-OH
7’-OH
8’-OH
9’-OH
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
15.3
10.6
6.4
2.81±0.07
3.01±0.04
>50
0.87±0.01
1.39±0.01
29.25±0.88
47.25±0.68
0.57±0.01
1.34±0.03
0.7±0.02
1.81±0.01
1.26±0.03
14.12±0.18
10.28±0.29
2.04±0.04
1.44±0.03
4.83±0.26
5.78±0.07
1.84±0.01
1.59±0.04
2.34±0.03
>50
6.19±0.03
4.84±0.27
>50
1
1
2.2
3
.3
1
1
1.7
47.1
0.7
1.4
NT
>50
>50
OH
8
.6
.7
4.75±0.05
3.57±0.02
4.49±0.2
>50
7.3±0.06
6.764±0.02
8.63±0.03
7.2±0.53
4.88±0.02
3.84±0.07
2.25±0.05
>50
1
1
1
1
1
1
1
30
31
32
33
34
35
36
NT
NT
8
4.5
R2
3.0
.1
14.7
.0
0.9
.2
6.2
N
R1
1
67.0
50.8
17.1
47.6
75.3
3.9
3.6
NT
1.4
1.4
0.75±0.04
1.37±0.09
1.85±0.02
1.14±0.01
24.48±0.35
2.23±0.02
4.4±0.05
2.54±0.05
>50
8
1
*
Results are presented as the means±SDs of independent experiments performed in triplicate.
1
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Journal of Medicinal Chemistry
Page 20 of 130
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
Table 5. Structures, topo I and II inhibitory activities, and IC50 values of compounds 137-172 in the four cancer cell lines
%
Inhibition
Compound
R
1
R
2
*IC50 (µM)
DU145
Topo I
Topo II
1
00 µM
3.0
20 µM
100 µM
20 µM
HCT15
0.16±0.001
4.06±0.02
T47D
HeLa
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
Camptothecin
Etoposide
7
NT
0.25±0.003
4.26±0.03
0.62±0.02
4.49±0.03
0.5±0.01
9.75±0.08
72.9
39.6
Adriamycin
0
.94±0.003
1.18±0.01
3.02±0.02
1.14±0.02
1.41±0.02
0.75±0.001
0.77±0.005
0.7±0.002
0.51±0.001
0.98±0.01
1.33±0.01
0.83±0.001
0.67±0.01
4.2±0.03
1.04±0.01
1.38±0.03
2.02±0.02
2.86±0.02
2±0.26
1.98±0.01
3.95±0.01
2.92±0.01
2.96±0.02
1.99±0.01
4.25±0.03
3.1±0.02
1
1
1
1
1
37 2-CF
38 2-CF
39 2-CF
40 2-CF
41 3-CF
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
6’-OH
7’-OH
8’-OH
9’-OH
6’-OH
7’-OH
8’-OH
9’-OH
6’-OH
7’-OH
8’-OH
9’-OH
6’-OH
7’-OH
8’-OH
9’-OH
6’-OH
3.5
.6
0.4
.0
0.3
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
0.0
NT
NT
NT
NT
83.7
90.2
86.1
84.8
94.2
85.4
98.9
95.8
98.9
99.9
100.0
92.2
89.5
80.6
79
1.8
3.7
3.4
4.0
5.3
4.1
2.8
4.3
2.4
11.9
3.0
0.9
2.1
2.4
3.1
3.3
40.5
1.74±0.01
1.61±0.01
4.04±0.004
1.64±0.01
3.34±0.01
3.89±0.002
2.95±0.02
2.84±0.01
4.54±0.02
4.33±0.02
3.25±0.07
3.49±0.01
3.39±0.03
2.82±0.02
1.67±0.01
2.1±0.01
1
0
1.87±0.01
1.72±0.05
1.63±0.02
3.38±0.06
3.46±0.04
2.09±0.01
2.8±0.02
4.5±0.03
1.8±0.01
2.5±0.41
1.83±0.02
1.14±0.01
1.62±0.02
142 3-CF
43 3-CF
144 3-CF
45 4-CF
146 4-CF
47 4-CF
148 4-CF
49 2-CF
150 2-CF
51 2-CF
152 2-CF
53 3-CF
0
0
2
0
0
0
.0
.0
.7
.0
.0
.0
OH
1
5.25±0.04
5.73±0.04
6.14±0.02
7.87±0.02
2.23±0.01
5.24±0.02
3.79±0.05
4±0.26
R2
N
R1
1
1
1
6
1
3.6
5.7
1.7
1
1.46±0.05
1.25±0.01
1.22±0.002
1.75±0.02
1.91±0.01
1
1
.8
3.71±0.03
4.69±0.01
4.17±0.02
1
5.9
65.2
67.3
1
5
.6
2.69±0.001
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OH
1
1
1
1
1
1
1
54 3-CF
55 3-CF
56 3-CF
57 4-CF
58 4-CF
59 4-CF
60 4-CF
3
3
3
3
3
3
3
7’-OH
8’-OH
9’-OH
6’-OH
7’-OH
8’-OH
9’-OH
1
0.2
NT
NT
NT
NT
NT
NT
NT
85.2
90.7
71.6
10.9
100
100
100
29.0
10.5
0.7
2.22±0.01
8.61±0.02
14.16±0.02
6.98±0.34
6.82±0.08
2.95±0.04
11.27±0.07
1.33±0.01
1.33±0.002
3.63±0.02
6.15±0.05
8.9±0.12
1.42±0.04
1.54±0.01
2.77±0.05
5.01±0.04
1.59±0.03
3.18±0.04
2.22±0.02
2.03±0.02
4.7±0.09
>50
5.2
0.2
0.7
R2
N
R1
NT
6.3±0.17
11.39±0.06
3.63±0.01
6.66±0.08
2
.8
14.9
5.1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
0.0
1.8±0.004
2.14±0.01
0
7
.0
.1
0.0
161 2-CF
62 2-CF
163 2-CF
64 2-CF
165 3-CF
3
3
3
3
3
3
3
3
3
3
3
3
6’-OH
7’-OH
8’-OH
9’-OH
6’-OH
7’-OH
8’-OH
9’-OH
6’-OH
7’-OH
8’-OH
9’-OH
NT
NT
NT
NT
NT
0.3
NT
NT
0.0
0.0
0.0
NT
30.1
85.9
81.0
2.9
0.6
0
3.82±0.09
2.43±0.02
1.32±0.01
>50
2.66±0.1
1.05±0.004
1.47±0.01
1.44±0.01
1.23±0.003
1.43±0.01
4.69±0.05
9.57±0.21
2.01±0.02
1.91±0.01
1.36±0.01
>50
5.34±0.03
2.31±0.01
2.78±0.01
>50
>50
1
1
3.9
2.07±0.02
1.52±0.004
>50
6
.7
.0
.4
1.2
NT
0
1
3
OH
9
84.4
88.7
87.1
12.9
91.3
83.2
83.1
82.3
2.62±0.003
3.47±0.01
2.77±0.02
4.77±0.04
>50
1.75±0.02
1.9±0.02
2.15±0.02
44.14±0.2
2.11±0.02
1.14±0.04
2.98±0.03
13.16±0.1
4.57±0.04
4.52±0.04
3.06±0.03
16.8±0.02
6.54±1.55
4.8±0.03
5.51±0.02
>50
1
1
1
1
1
1
1
66 3-CF
67 3-CF
68 3-CF
69 4-CF
70 4-CF
71 4-CF
72 4-CF
5
5.8
8.5
.3
32.8
7.4
100
8.5
4.6
11.1
NT
11.1
1.1
0.5
0
R2
N
R1
2
8
2.63±0.02
5.03±0.02
>50
1
*
Results are presented as the means±SDs of three independent experiments performed in triplicate.
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2
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6
In Vitro Topo Inhibitory Activities. In view of their potent antiproliferative
activities, we investigated topoisomerase I and II inhibitory activities by detecting
conversion of supercoiled pBR322 plasmid DNA to its relaxed form in the presence of the
synthesized compounds 1-172. Camptothecin and etoposide were used as reference
compounds for topo I and II inhibition, respectively. Topo inhibitory activities evaluated at
100 and 20 µM are listed in Tables 1-5 and depicted in Figures 3-7. Interestingly, almost all
compounds showed weak topo I inhibitory activity. Only compounds 36, 170, and 171
exhibited greater topo I inhibitory activity than reference compound, camptothecin, whereas
compounds 31, 34, 35, 37, 149, and 166 inhibited topo I moderately at 100 µM.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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7
8
9
0
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2
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9
0
1
2
3
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
As regards of topo II inhibition, only compound 3 (member of the 2,4-diphenyl
series) inhibited topo II (79.9%) more than etoposide (76.2%) at 100 µM, but at 20 µM its
inhibitory effect was weak (5.5%). The other compounds of this series exhibited moderate
topo II inhibition (54.2-75.7%) at 100 µM and negligible inhibition at 20 µM (Figure 3).
(A)
(B)
Figure 3. Topoisomerase I and IIα inhibitory activities of the compounds investigated. (A)
Lane D: pBR322 DNA only; Lane T: pBR322 DNA + topoisomerase I; Lane C: pBR322
DNA + topoisomerase I + camptothecin; Lane 1-28: pBR322 DNA + topoisomerase I +
compound 1 – 28; (B) Lane D: pBR322 DNA only; Lane T: pBR322 DNA + topoisomerase
IIα; Lane E: pBR322 DNA + topoisomerase IIα + etoposide; Lane 1-28 : pBR322 DNA +
topoisomerase IIα + compound 1 – 28.
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(
B)
(A)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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4
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0
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2
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9
0
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2
3
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8
9
0
1
2
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Topoisomerase I and IIα inhibitory activities of the investigated compounds. (A)
Lane D: pBR322 DNA only; Lane T: pBR322 DNA + topoisomerase I; Lane C: pBR322
DNA + topoisomerase I + camptothecin; Lane 29-64: pBR322 DNA + topoisomerase I +
compound 29 – 64; (B) Lane D: pBR322 DNA only; Lane T: pBR322 DNA + topoisomerase
IIα; Lane E: pBR322 DNA + topoisomerase IIα + etoposide; Lane 29-64 : pBR322 DNA +
topoisomerase IIα + compound 29 – 64.
In the 2-phenyl, 4-phenol series (5-16), compounds 5 and 7-10 inhibited topo II
(
78.5-97.4%) significantly more than etoposide at 100 µM. However, with the exception of
compound 6, all compounds exhibited little topo II inhibitory activity at 100 µM, and no
compound inhibited topo II at 20 µM. Likewise, no compound in the 2-phenol, 4-phenyl
series inhibited topo II more than etoposide at 100 and 20 µM (Table 1). However, in the 2-
phenol, 4-chlorophenyl series (29-64), most of compounds inhibited better or considerable
topo II activity than etoposide at 100 µM, whereas at 20 µM none of the compounds in this
series inhibited better than etoposide (Figure 4). Of the compounds with a chlorine group at
the ortho- and para-positions, a half (29, 34-38, 57, and 59-64) significantly inhibited topo
II (79.2-98.1%) at 100 µM. However, only compound 46 with chlorine group at the meta-
position inhibited topo II (83.0%) more than etoposide at 100 µM (Table 2).
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
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5
5
5
5
5
6
(
A)
(B)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Topoisomerase I and IIα inhibitory activities of the investigated compounds. (A)
Lane D: pBR322 DNA only; Lane T: pBR322 DNA + topoisomerase I; Lane C: pBR322
DNA + topoisomerase I + camptothecin; Lane 65-100: pBR322 DNA + topoisomerase I +
compound 65 – 100; (B) Lane D: pBR322 DNA only; Lane T: pBR322 DNA +
topoisomerase IIα; Lane E: pBR322 DNA + topoisomerase IIα + etoposide; Lane 65-100 :
pBR322 DNA + topoisomerase IIα + compound 65 – 100.
As shown in Figure 5 and Table 3, it was interesting to note the topo II inhibitory
results of 2-chlorophenyl, 4-phenol series (65-100). With the exception of compound 66,
compounds with a hydroxyl at the ortho- or meta-positions of 4-phenyl ring (65-88)
displayed significantly better topo II inhibition (76.8-100%) than etoposide (75.0%) at 100
µM, whereas at 20 µM concentration, only compounds 79, 82, 85, and 87 with a hydroxyl at
the meta-position inhibited topo II (37.8-51.9%) more than etoposide (35.6%). In addition,
half of the compounds (89-91, 94, 95, and 98) with a para-positioned hydroxyl inhibited topo
II (80.9-100%) significantly more than etoposide at 100 µM, and compounds 89, 90, 93, 95,
and 98 inhibited topo II (36.9-78.9%) more than etoposide at 20 µM. Among all, compound
8
9 was the most potent topo II inhibitor (at 100 µM: 100% inhibition; at 20 µM: 78.9%
inhibition).
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In the 2-fluorophenyl, 4-phenol series (101-136), none of the compounds exhibited
better topo IIα inhibition than etoposide at 100 and 20 µM (Figure 6, Table 4). Interestingly,
with the exception of compounds 152, 153, 156, 157, 161, 164, and 168, all compounds in
the 2-trifluoromethyl, 4-phenol series inhibited topo IIα better than etoposide (72.9%) at 100
µM. However, at 20 µM, only 153 inhibited topo IIα (40.5% inhibition) greater than
etoposide (Figure 7, Table 5).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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5
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5
5
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5
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0
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8
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0
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0
1
2
3
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5
6
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(A)
Figure 6. Topoisomerase I and IIα inhibitory activities of the investigated compounds. (A)
Lane D: pBR322 DNA only; Lane T: pBR322 DNA + topoisomerase I; Lane C: pBR322
DNA + topoisomerase I + camptothecin; Lane 101-136: pBR322 DNA + topoisomerase I +
compound 101 – 136; (B) Lane D: pBR322 DNA only; Lane T: pBR322 DNA +
topoisomerase IIα; Lane E: pBR322 DNA + topoisomerase IIα + etoposide; Lane 101-136 :
pBR322 DNA + topoisomerase IIα + compound 101 – 136.
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2
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2
2
3
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(
A)
(B)
0
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9
0
1
2
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Topoisomerase I and IIα inhibitory activities of the investigated compounds. (A)
Lane D: pBR322 DNA only; Lane T: pBR322 DNA + topoisomerase I; Lane C: pBR322
DNA + topoisomerase I + camptothecin; Lane 137-172: pBR322 DNA + topoisomerase I +
compound 137 – 172; (B) Lane D: pBR322 DNA only; Lane T: pBR322 DNA +
topoisomerase IIα; Lane E: pBR322 DNA + topoisomerase IIα + etoposide; Lane 137-172 :
pBR322 DNA + topoisomerase IIα + compound 137 – 172.
Structure-Activity Relationship (SAR) Study. One of the major aims of this study
was to investigate SARs among the synthesized compounds with respect to their topo
inhibitory and antiproliferative activities. This investigation was conducted by exploring the
effects of hydroxyls at C-(6, 7, 8, and 9) in the indenopyridine ring, and by assessing the
biological effects caused by introducing phenyl, phenol, and halophenyl moieties at the 2- or
4
-positions of the indenopyridine ring.
Of the compounds containing phenyl and phenol moieties (compounds 1-28), only
ortho- or meta-phenol at the 4-position showed selective, strong topo II inhibitory activity.
Interestingly. all the compounds showed significant antiproliferative activity in DU145 cell
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line, suggesting their potential use in human prostate cancer. In addition, when we
incorporated a chlorine functionality at 4-phenyl ring, we found that all the compounds in the
2-phenol, 4-chlorophenyl series (compounds 29-64) exhibited moderate to strong topo II
inhibition. SAR study revealed a meta- or para- phenol group at position 2, and ortho- or
para-chlorophenyl group at position 4, resulted in stronger topo II inhibition at 100 µM.
Comparatively, compounds with ortho-chlorophenyl exhibited stronger antiproliferative
activity in HCT15, T47D and DU145 cells. Interestingly, interconversion of chlorine and
hydroxyl group at 2- and 4- phenyl ring respectively, improves potency and selectivity
towards topo II enzyme. Moreover, most of the meta- and para-phenolic group at 4-
position showed potent topo II inhibitory activity at 20 μM. With few exceptions,
interconversion results in remarkable improvement in antiproliferative activity in all the
tested cancer cell lines (HCT15, T47D, DU145 and HeLa). Noticeably, compounds 78 and 89
exhibited strongest antiproliferative activity in HCT15 and T47D, respectively in nanomolar
range. In case of the 2-fluorophenyl, 4-phenolic series almost all compounds (101 to 136)
with a fluorine at 2-phenyl ring showed weak topo I and IIα inhibitory activities. This result
indicates that unlike chlorine functionality, fluorine substitution at 2-phenyl ring does not
favor topo IIα inhibition. However, compounds showed significant anti-proliferative activity
in T47D and DU145 cell lines. Interestingly, replacement of fluorine group by
trifluoromethyl group in the 2-phenyl ring enhanced topo IIα inhibitory activity at 100 μM.
SAR study revealed comparable antiproliferative activity of 2-trifluoromethylphenyl, 4-
phenolic series with 2-fluorophenyl, 4-phenolic series.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In summary, from SAR study we observed that most of the compounds with a
chlorine group at 2-phenyl ring and a meta- and para-hydroxyl group in the 4-phenyl ring of
indenopyridinol (78, 79, 82, 87, 89, 90, 93, 94, 95 and 98) displayed strong and potent topo
II inhibitory activity at 20 μM. These results highlight the potencies and significance of 2-
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9
1
1
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1
1
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
chlorophenyl, 4-phenolic series with regards to position and functional groups as compared to
2
-phenolic, 4-chlorophenyl series and other compounds containing phenyl, fluorine and
trifluoromethyl groups (Figure 8). Observed increases in antiproliferative activity achieved
by the substitution of chlorophenyl and phenolic moieties in the 2- and 4- positions with a
hydroxyl group at the C-(6, 7, 8, and 9) positions of the indenopyridine ring encouraged us to
investigate the mechanisms responsible. Compounds 78 and 89 potently inhibited the
proliferations of HCT15 and T47D cells at nanomolar levels (IC = 1.33 and 2.67 nM,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
respectively). However, compound 89 was selected for the mechanistic study because it most
potently inhibited topo II (100% inhibition at 100 µM and 78.9% at 20 µM), significantly
inhibited T47D proliferation (IC = 2.67 nM), and was cytotoxic to several breast cancer cell
5
0
lines (Table 6).
(A)
HO
HO
HO
Cl
R1
<
>
>
<
OH
OH
OH
OH
OH
N
N
N
N
N
F
F3C
Cl
HO
R
R= H / OH
Selective and Potent topo II inhibition,
R1=OH / H
Strong antiproliferative inhibition
(B)
OH
R
R= meta-OH or para-OH
R
OH
Cl-group substitution
at 2-phenyl ring
OH-group substitution
at 6, 7, 8, 9-posititon
4
OH
Cl
Cl
6
2
N
7
N
N
OH
N
Cl
9
8
Strong topo II inhibition
Moderate Antiproliferation
Loss of topo II inhibition
Strong Antiproliferation
(HCT15 and T47D cells)
Comp. 78
IC50 =1.3 nM (HCT15 cells)
Selective and Potent topo II inhibition)
Comp. 89
IC50 =2.6 nM (T47D cells)
(HCT15 and T47D cells)
(
Figure 8. Summary of SAR study, (A) effects of substitution in the 2-phenyl ring; and (B)
effects of the position of hydroxyl substitution in the indeno ring.
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Table 6. IC values for compound 89 in different breast cancer cell lines.
5
0
IC50
Compound
MDA-MB-231 MDA-MB-436
HCC70
1.45±0.02
>50
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
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3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Adriamycin
Etoposide
5.18±0.29
>50
2.03±0.07
39.42±4.74
31.86±4.66
2.64±0.03
Camptothecin
Compound 89
23.98±2.27
2.7±0.01
0.044±0.001
2.083±0.015
*
Results are presented as the means±SDs of three independent experiments
performed in triplicate.
2
9
ACS Paragon Plus Environment