Modified 2,4-diaryl-5H-indeno[1,2-b]pyridines with hydroxyl and chlorine moiety: Synthesis, anticancer activity, and structure-activity relationship study

Article abstract of DOI:10.1016/j.bioorg.2015.07.002

As a part of ongoing studies in developing novel anticancer agents, a series of modified 2,4-diaryl-5H-indeno[1,2-b]pyridines were designed, and synthesized by introducing hydroxyl and chlorine moieties. They were evaluated for topoisomerase inhibitory ac

Full text of DOI:10.1016/j.bioorg.2015.07.002

Accepted Manuscript
Modified 2,4-Diaryl-5H-indeno[1,2-b] pyridines with Hydroxyl and Chlorine
Moiety: Synthesis, Anticancer Activity, and Structure-activity Relationship
Study
Tara Man Kadayat, Chanju Song, Youngjoo Kwon, Eung-Seok Lee
PII:
S0045-2068(15)30006-7
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.07.002
YBIOO 1829
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
7 April 2015
8 June 2015
10 July 2015
Please cite this article as: T.M. Kadayat, C. Song, Y. Kwon, E-S. Lee, Modified 2,4-Diaryl-5H-indeno[1,2-b]
pyridines with Hydroxyl and Chlorine Moiety: Synthesis, Anticancer Activity, and Structure-activity Relationship
Study, Bioorganic Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bioorg.2015.07.002
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‘
REVISED’: Clean Version
Modified 2,4-Diaryl-5H-indeno[1,2-b]pyridines with Hydroxyl and Chlorine Moiety:
Synthesis, Anticancer Activity, and Structure-activity Relationship Study
[
a]
[b]
[b]
[a]
Tara Man Kadayat, Chanju Song , Youngjoo Kwon* , Eung-Seok Lee,*
[
[
a]
b]
College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea
College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Global Top 5
program Ewha Womans University, Seoul 120-750, Republic of Korea
*Corresponding Authors:
Tel: +82 2 3277-4653, fax: +82 2 3277-2851, email: ykwon@ewha.ac.kr (Y.K.)
Tel: +82 53 810-2827, fax: +82 53 810-4654, email: eslee@yu.ac.kr (E.-S.L.)

Modified 2,4-Diaryl-5H-indeno[1,2-b]pyridines with Hydroxyl and Chlorine Moiety:
Synthesis, Anticancer Activity, and Structure-activity Relationship Study
[
a]
[b]
[b]
[a]
Tara Man Kadayat, Chanju Song , Youngjoo Kwon* , Eung-Seok Lee,*
Abstract
As a part of ongoing studies in developing novel anticancer agents, a series of modified 2,4-
diaryl-5H-indeno[1,2-b]pyridines were designed, and synthesized by introducing hydroxyl and
chlorine moieties. They were evaluated for topoisomerase inhibitory activity and cytotoxicity
against HCT15, T47D, and HeLa cancer cell lines. This modification allowed us to demonstrate
structure-activity relationship (SAR) study with respect to the non-substituted 2,4-diaryl-5H-
indeno[1,2-b]pyridines. Compounds (2, 3, 4, 5, 8, and 9) with meta or para hydroxyl group on 2
or 4-phenyl ring have enhanced topo I and II inhibitory activity and cytotoxicity. However,
additional substitution of chlorine group on furyl or thienyl ring (11, 12, 14, 16-18) generally
reduced topo I and II inhibitory activity but improved cytotoxicity. The observation of cytotoxic
properties and SAR study according to the position of hydroxyl and chlorine group will provide
valuable insight for further study of development of novel anticancer agents with related
scaffolds.
Keywords: 2,4-Diaryl-5H-indeno[1,2-b]pyridines; Hydroxyl group, chlorine group substitution;
Antitumor agents; Cytotoxicity; Topoisomerase I and II inhibition

1
. Introduction
The difference in expression of Human DNA topoisomerases (topos) more in tumor tissues than
normal tissues mainly in colorectal, breast, cervix, and prostate cancers suggests therapeutic
efficacy of topos inhibitors [1-4]. Therefore, inhibition of topoisomerase enzymes (topo I and/or
topo II), which are involved in solving the topological problems associated with nuclear
processes, is a promising approach for the design of anticancer agents [5, 6]. Topoisomerase
inhibitors such as camptothecin, etoposide, and doxorubicin are the widely used anticancer drugs
that interfere with the function of these enzymes [7, 8]. However, the severe side effects
associated with the existing topoisomerase inhibitors obligate the investigation and development
of novel topoisomerase inhibitors [9]. For a continuous effort to search a potent topoisomerase
inhibitors, our research group has been trying to design compounds containing indenopyridine
moiety of which biological activities were reported to be anticancer and anti-inflammatory
activity [10-12]. Recently we reported 2,4-diaryl-5H-indeno[1,2-b]pyridines with or without
hydroxyl group on 2-phenyl ring as conformationally constrained rigid molecule in terpyridine
skeleton (Fig. 1). Among them, several compounds with meta or para-hydroxyl group at phenyl
ring in combination with furyl or thieny moiety exhibited considerable topo inhibitory activity
and cytotoxicity [13, 14].
For the optimization of lead compounds, several researches are ongoing to introduce chlorine
moiety because chlorine can accommodate in tight and deep cavities, as well as providing in
hydrophobic pockets of the biological targets which contribute to improve the potency, stability
and specificity of the binding site [15, 16]. It would be interesting to observe and compare the
difference in biological activities by systematic substitution of chlorine group at furyl or thienyl

ring on 5H-indeno[1,2-b]pyridines. Therefore, in extension to previous work, we incorporated
the chlorine functionality at furyl or thieny group at 2 or 4-position of central pyridine and
examined their topo inhibitory activity and cytotoxicity.
In this article, we report the systematic design (Fig. 2), synthesis, and structure-activity
relationship (SAR) study for modified 2,4-diaryl-5H-indeno[1,2-b]pyridines containing hydroxyl
and chlorine moiety. This modification allowed us to investigate the effect of hydroxyl and
chlorine substitution on cytotoxicity and topo inhibition with respect to the non-substituted 2,4-
diaryl-5H-indeno[1,2-b]pyridines (A-C).
Figure 1
Figure 2
2
. Design and synthesis
We systematically designed eighteen compounds (1-18) in six different series introducing
hydroxyl and chlorine groups at phenyl, thienyl or furyl ring of 2,4-diaryl-5H-indeno[1,2-
b]pyridines (Fig. 2). Compounds 1-9 shown in Figure 3a contain a hydroxyl group at phenyl ring
whereas compounds 10-18 contain one hydroxyl group at phenyl ring and one chlorine group at
furyl or thienyl ring on 2- or 4-position of the central pyridine (Fig. 3b). Synthesis of compounds
1-6 was reported earlier [14]. The synthetic route with three different steps for the target
compounds 7–18 is outlined in Scheme 1. At first, 1-indanone (I) was condensed with aryl
1
1
aldehydes II (R =a-e) to prepare indanone intermediates III (R =a-e) in the presence of 5%
aqueous NaOH in ethanol using Clasien-Schmidt condensation reaction [17]. In the second step,

2
four pyridinium iodide salts V (R =a-d, f) were synthesized by refluxing acetophenones IV
2
(
R =a-d, f) with iodine in pyridine. Finally, using modified Kröhnke synthesis [18, 19], indanone
1
2
intermediates III (R =a-e) and pyridinium iodide salts V (R =a-d, f) were reacted in the presence
of ammonium acetate in methanol or acetic acid to give final compounds 7-18 in the yields of
2
4.9-58.2%. Structures, yields (%), and HPLC purities (%) of the prepared compounds are
depicted in Figure 3 and Table 1.
Scheme 1
Figure 3
Table 1
3. Results and Discussion
All the prepared compounds were evaluated for topo I and II inhibitory activity and cytotoxicity
against several human cancer cell lines. The conversion of supercoiled plasmid DNA to relaxed
DNA by topo I and II was examined in the presence of prepared compounds (7-18).
Camptothecin and etoposide, well known topo I and II inhibitors, respectively, were used as
positive controls. Inhibitory activities were evaluated both at 100 µM and 20 µM. The reaction
products were analyzed by electrophoretic mobility and developed in ethidium bromide in the
presence of UV light. Figure 4 and Table 3 and 4 illustrate the topo I and II inhibitory activity
and cytotoxicity of the prepared compounds 7–18.
Figure 4
Table 2

Table 3
Table 4
3
.1. Cytotoxicity of the compounds
We performed cytotoxicity assay using 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). Adriamycin, etoposide and camptothecin, widely used
anticancer drugs, were used as positive controls. The inhibitory activity (IC50) of the synthesized
compounds (7-18) is expressed as micromolar concentration as shown in Table 3 and 4.
Most of the prepared compounds showed significant or moderate cytotoxicity, generally 0.68-
1
0.28 µM. Among prepared hydroxylated compounds (7-9) compounds 8 and 9 showed strong
cytotoxicity against all the tested cell lines (Table 3). Compound 10-12, 14, and 16-18 showed
similar or considerable cytotoxicity than positive controls in all the tested HCT15 colon cancer
cells. Among the compounds tested against T47D breast cancer cells, compounds 11, 12, 14, and
16-18 displayed stronger cytotoxicity (IC50 < 11 µM) than positive controls, etoposide (57.9 µM)
and camptothecin (11.2 µM), but weaker than adriamycin (0.47 µM). Similarly, for the HeLa
cervix cancer cells, compounds 11, 12, 14, and 16-18 displayed stronger cytotoxicity than
etoposide but considerable cytotoxicity as compared to adriamycin and camptothecin.
Compounds 11, 12, 14, and 16-18 exhibited strong to moderate cytotoxicity against all the tested
cancer cell lines. Compounds 18 with para-hydroxyl group at 4-phenyl and chlorine group at 2-
thienyl ring of central pyridine showed the most significant cytotoxicity with IC values of 0.68
5
0
µM, 2.30 µM, and 2.08 µM against HCT15, T47D, and HeLa cells, respectively. Compound 13

and 15 did not display any cytotoxicity against all the tested cancer cells. Interestingly, except
compound 15, it was observed that combination of meta or para-hydroxyl group at phenyl ring
and chlorine group at furyl or thienyl ring is favorable for the cytotoxic activity.
3.2. Topo I and II inhibitory activities
As shown in Figure 4 and Table 3 and 4, all the synthesized compounds 7-18 were evaluated for
topo I and II inhibitory activity at 100 and 20 µM concentrations. Most of the compounds
exhibited mild or considerable topo I inhibitory activities (6.8-53.5% inhibition at 100 µM) as
compared to that of camptothecin (80.6% inhibition at 100 µM). However, all of the prepared
compounds were devoid of topo I inhibitory activity at 20 µM. It is interesting to note that only
compound 10 and 12 exhibited both topo I and II inhibitory activity at 100 µM (Fig. 4A).
Table 3 displayed that hydroxylated compounds 8 and 9 showed significant or moderate topo II
inhibition at both 100 and 20 µM. Figure 4B and Table 4 summarize the topo II inhibitory
activity of the synthesized compounds. Except compound 10 and 12, all of the compounds (10-
1
8) containing one hydroxyl group at phenyl ring and one chlorine group at furyl or thienyl ring
on 2- or 4-position of the central pyridine showed mild or very weak topo II inhibitory activities
3.4-19.5% inhibition at 100 µM) as compared to positive control etoposide (71.1% inhibition at
00 µM). Compound 10 exhibited 54.5% inhibition, whereas compound 12 displayed stronger
(
1
topo II inhibitory activity (74.2%) than etoposide at 100 µM concentration. Interestingly, none of
the compounds show topo II inhibitory activity at 20 µM.
3.3. Structure-activity relationship study

Structure-activity relationship (SAR) was studied based on results of topo I and II inhibitory
activity and cytotoxicity with respect to the non-substituted 2,4-diaryl-5H-indeno[1,2-b]pyridines
(A, B, C), one hydroxylated 2,4-diaryl-5H-indeno[1,2-b]pyridines (1-9), and one chlorinated 2,4-
diaryl-5H-indeno[1,2-b]pyridines containing one hydroxyl moiety (10-18). Figure 5 illustrates
the overall SAR study. Compounds A and C were reported to have strong and moderate topo I
inhibitory activity, respectively, whereas compound B showed very weak topo I activity at 100
µM. Compounds (4-6) with one hydroxyl group at 2-phenyl ring and thienyl ring on 4-position
exhibited strong topo I inhibitory activity, however further substitution of one chlorine group at
4-thienyl ring (13-15) decreased the topo I inhibitory activity. Similarly, compounds 1-3, and 7-9
containing one hydroxyl group at phenyl ring exhibited moderate or strong topo I inhibitory
activity. But introduction of one chlorine group at furyl or thienyl moieties (compounds 10-12
and 16-18) at 2 or 4 position resulted decrease in topo I inhibitory activity. It is interesting to
note that compounds 10-18 with one hydroxyl group at phenyl ring in combination with one
chlorine group at furyl or thienyl rings are less active for topo I inhibition than non-chlorinated
compounds with one hydroxyl group at phenyl ring (1-9).
Among non-substituted compounds A, B, and C which contain neither hydroxyl or chlorine
moiety, only compound A was reported strong topo II inhibitory activity at both 100 and 20 µM
and moderate cytotoxicity against tested cancer cells (Table 2). However, introduction of one
hydroxyl group at phenyl ring on 2 or 4-position (compounds 1-9) increased topo II inhibitory
activity and cytotoxicity. Compounds with meta-hydroxyl group at phenyl ring (2, 5, 8) are
found to be important for both topo II inhibitory and cytotoxicity.

Furthermore, introduction of one chlorine group at furyl or thienyl ring on 2 or 4-position of
central pyridine resulted in increase of cytotoxicity but decrease in topo II inhibitory activity.
Therefore, chlorinated compounds (10-18) are less active for topo II inhibition but more active
against the tested cancer cells. Chlorinated compounds are generally more active against HCT15
cells among all the tested cancer cells. Compounds 16-18 possessing a hydroxyl group at 4-
phenyl ring and a chlorine group at 2-thienyl ring displayed significant cytotoxicity against all
the tested cells as compared to etoposide, and moderate activity than that of adriamycin and
camptothecin. From these results it might be implied that introduction of hydroxyl group at 4-
phenyl ring and chlorine group at 2-thienyl ring is important for cytotoxic activity. However, a
positive correlation between cytotoxicity and topo inhibitory activity was not observed. Further
effort to explore the mechanistic pathway for the significant cytotoxicity by introduction of
chlorine group is a future scope for our research group.
Figure 5
4. Conclusion
We systematically designed and synthesized twelve modified 2,4-diaryl-5H-indeno[1,2-
b]pyridines containing hydroxyl and chlorine moiety (7-18) by efficient synthetic route. They
were evaluated for topo I and II inhibitory activity and cytotoxicity against three types of human
cancer cell lines. Compounds with a chlorine group at furyl or thienyl ring on 2 or 4-position at
central pyridine were found to be less active for topo I and II inhibition but more cytotoxic
against the tested cancer cell lines. A SAR study, overall, showed that introduction of a hydroxyl
group at phenyl ring on 2 or 4-position is favorable for topo I and II inhibitory activity and
cytotoxicity. However, additional substitution of a chlorine group at furyl or thienyl ring on 2,4-

diaryl-5H-indeno[1,2-b]pyridines generally reduced topo I and II inhibitory activity but
enhanced cytotoxicity. In conclusion, we believe that chlorinated 2,4-diaryl-5H-indeno[1,2-
b]pyridine skeleton with hydroxyl group has important role for displaying the significant
cytotoxic effects. The results from this study may provide valuable information for the medicinal
chemist studying for hydroxylated or halogenated compounds as novel anticancer agents.
5
. Experimental
Compounds used as starting materials and reagents were obtained from Aldrich Chemical Co.,
Junsei or other chemical companies, and used without further purification. HPLC grade
acetonitrile (ACN) was purchased from Burdick and Jackson, USA. Thin-layer chromatography
(
TLC) and column chromatography (CC) were performed with Kieselgel 60 F254 (Merck) and
silica gel (Kieselgel 60, 230-400 mesh, Merck), respectively. Since all the prepared compounds
contained aromatic ring, they were visualized and detected on TLC plates with UV light (short
wave, long wave or both). NMR spectra were recorded on a Bruker AMX 250 (250 MHz, FT)
1
13
for H NMR and 62.5 MHz for C NMR, and chemical shifts were calibrated according to TMS.
Chemical shifts (δ) were recorded in ppm and coupling constants (J) in hertz (Hz). Melting
points were determined in open capillary tubes on electrothermal 1A 9100 digital melting point
apparatus without correction.
HPLC analyses were performed using two Shimadzu LC-10AT pumps gradient-controlled
HPLC system equipped with Shimadzu system controller (SCL-10A VP) and photo diode array
detector (SPD-M10A VP) utilizing Shimadzu Class VP program. Sample volume of 10 μL was
®
injected in Waters X-Terra 5 μM reverse-phase C18 column (4.6 ⅹ250 mm) with a gradient

elution of 70% to 100% of B in A for 10 min followed by 100% to 70% of B in A for 20-25 min
at a flow rate of 0.5 mL/min at 254 nm UV detection, where mobile phase A was the 20 mM
ammonium formate (AF) solution in doubly distilled water and B was 75% acetonitrile (ACN).
Purity of compounds was described as percent (%) and retention time was given in minutes. ESI
®
LC/MS analyses were performed with a Finnigan LCQ Advantage LC/MS/MS spectrometry
®
utilizing Xcalibur program. For ESI LC/MS, LC was performed with a 8 μL injection volume
®
on a Waters Atlantis T
3
reverse-phase C18 column (2.1ⅹ 50 mm, 3 μm) with a gradient elution
from; (A) 50% to 90% of B in A for 4 min and remained linear 90% of B in A for 1 min
followed by 90% to 50% of B in A for 3 min and remained linear 50% of B in A for 7 min at
flow rate of 200 μL/min; (B) 50% to 90% of B in A for 5 min followed by 90% to 50% of B in A
for 5 min and remained linear 50% of B in A for 5 min at flow rate of 200 μL/min, where mobile
phase A was the 20 mM ammonium formate (AF) solution in doubly distilled water and mobile
phase B was 100% ACN. MS ionization conditions were: Sheath gas flow rate: 40 arb, aux gas
flow rate: 0 arb, I spray voltage: 5.3 KV, capillary temperature: 275°C, capillary voltage: 27 V,
tube lens offset: 45 V.
1
5
. 1. General method for the preparation of III (R = a-e)
1
Total five 1H-inden-1-one derivatives III (R = a-e) were synthesized by base catalyzed Claisen-
Schmidt condensation reaction as reported previously. 1-Indanone (1) was added in ethanol
1
followed by the addition of equivalent amount of aryl aldehydes II (R = a-e). The 5% aqueous
solution of NaOH was added dropwise to the mixture at room temperature which resulted in
precipitation. The mixture was then cooled for 30 min, filtered, washed with cold methanol, and
dried to yield 61.9.1-96.6% solid compound.

5
.1.1. Synthesis of 2-(2-hydroxybenzylidene)-2,3-dihydro-1H-inden-1-one (IIIa)
The procedure described in Section 5.1 was employed with 1-indanone (I, 3.96 g, 30 mmol) and
salicylaldehyde (IIa, 3.14 mL, 30 mmol) in presence of 1.25 N NaOH to yield 6.85 g (28.99
mmol, 96.6%) as a red solid.
R
f
(ethyl acetate / n-hexane 1:3, v / v): 0.23; mp 203.4-204.9 °C
) δ 8.27 (s, 1H, =CH-), 7.69 (d, J = 7.55 Hz, 1H, indeno H-7),
1
H NMR (250 MHz, DMSO-d
6
7.61-7.56 (m, 2H, indeno H-4, H-5), 7.48 (d, J = 7.82 Hz, 1H, phenyl H-6), 7.41 (t, J = 5.4 Hz,
1H, indeno H-6), 6.93 (t, J = 7.35 Hz, 1H, phenyl H-4), 6.45 (d, J = 8.5 Hz, 1H, phenyl H-3),
6.20 (t, J = 7.25 Hz, 1H, phenyl H-5), 3.93 (s, 2H, indeno H-3).
1
3
C NMR (62.5 MHz, DMSO-d ) δ 193.72, 172.46, 150.09, 139.72, 134.23, 134.01, 132.80,
6
1
30.65, 127.93, 127.29, 127.14, 123.60, 123.47, 121.66, 111.56, 33.30.
5
.1.2. Synthesis of 2-(3-hydroxybenzylidene)-2, 3-dihydro-1H-inden-1-one (IIIb)
The procedure described in Section 5.1 was employed with 1-indanone (I, 3.96 g, 30 mmol) and
-hydroxybenzaldehyde (IIb, 3.66 g, 30 mmol) in presence of 1.25 N NaOH to yield 4.81 g
20.33 mmol, 67.8 %) as an orange solid.
3
(
R
f
(ethyl acetate / n-hexane 1:3, v / v): 0.21; mp 264.8-265.4 °C
) δ 7.72 (d, J = 7.55 Hz, 1H, indeno H-7), 7.63 (t, J = 7.50 Hz,
1
H NMR (250 MHz, DMSO-d
6
1H, indeno H-5), 7.56 (d, J = 7.75 Hz, 1H, indeno H-4) 7.42 (t, J = 7.32 Hz, 1H, indeno H-6),
7.35 (s, 1H, =CH-), 7.12 (t, J = 7.72 Hz, 1H, phenyl H-5), 7.01 (s, 1H, phenyl H-2), 6.82 (d, J =
7.45 Hz, 1H, phenyl H-4), 6.69 (d, J = 7.85 Hz, 1H, phenyl H-6), 3.88 (s, 2H, indeno H-3).

1
3
C NMR (62.5 MHz, DMSO-d ) δ 193.78, 164.81, 150.25, 137.67, 135.73, 135.18, 134.91,
6
1
33.92, 129.81, 127.88, 126.93, 123.75, 119.91, 119.11, 117.93. 32.25.
5
.1.3. Synthesis of 2-(4-hydroxybenzylidene)-2, 3-dihydro-1H-inden-1-one (IIIc)
The procedure described in Section 5.1 was employed with 1-indanone (I, 2.64 g, 20 mmol) and
-hydroxybenzaldehyde (IIc, 2.44 g, 20 mmol) in presence of 1.25 N NaOH to yield 2.93 g
12.39 mmol, 61.9%) as a light greenish solid.
4
(
R
f
(ethyl acetate / n-hexane 1:2, v / v): 0.30; mp 235.4-236.0 °C
) δ 10.13 (s, 1H, phenyl 4-OH), 7.75 (d, J = 7.27 Hz, 1H, indeno
1
H NMR (250 MHz, DMSO-d
6
H-7), 7.67-7.60 (m, 4H, phenyl H-2, H-6, indeno H-4, H-5), 7.46-7.42 (m, 2H, =CH-, indeno H-
), 6.88 (d, J = 8.35 Hz, 2H, phenyl H-3, H-5), 4.04 (s, 2H, indeno H-3).
6
1
3
C NMR (62.5 MHz, DMSO-d ) δ 194.11, 160.30, 150.66, 138.45, 135.36, 134.22, 133.87 (2C),
6
1
32.40, 128.45, 127.47, 126.85, 124.28, 116.90 (2C), 32.83.
5
.1.4. Synthesis of 2-((5-chlorothiophen-2-yl)methylene)-2,3-dihydro-1H-inden-1-one (IIId)
The procedure described in Section 5.1 was employed with 1-indanone (I, 0.66 g, 5 mmol) and
-chloro-2-thiophencarboxaldehyde (IId, 0.53 mL, 5 mmol) in presence of 1.25 N NaOH to
yield 1.24 g (4.76 mmol, 95.2%) as a light yellow solid.
5
R
f
(ethyl acetate / n-hexane 1:7, v / v): 0.23; mp 163.8-164.6°C
) δ 7.87 (d, J = 7.57 Hz, 1H, indeno H-7), 7.72 (s, 1H, =CH-), 7.62 (t,
1
H NMR (250 MHz, CDCl
3
J = 7.70 Hz, 1H, indeno H-5), 7.54 (d, J = 7.35 Hz, 1H, indeno H-4), 7.42 (t, J = 7.02 Hz, 1H,
indeno H-6), 7.20 (d, J = 3.95 Hz, 1H, chlorothienyl H-3), 7.99 (d, J = 3.95 Hz, 1H,
chlorothienyl H-4), 3.85 (s, 2H, indeno H-3).

1
3
3
C NMR (62.5 MHz, CDCl ) δ 193.46, 148.77, 138.64, 138.36, 135.30, 134.66, 132.82, 132.40,
1
27.74, 127.43, 126.21, 126.01, 124.34, 32.10.
5
.1.5. Synthesis of 2-((5-chlorofuran-2-yl)methylene)-2,3-dihydro-1H-inden-1-one (IIIe)
The procedure described in Section 5.1 was employed with 1-indanone (I, 0.66 g, 5 mmol) and
-chloro-2-furaldehyde (IIe, 0.65 g, 5 mmol) in presence of 1.25 N NaOH to yield 1.17 g (4.79
mmol, 95.9%) as a creamy-white solid.
5
R
f
(ethyl acetate / n-hexane 1:7, v / v): 0.20; mp 140.1-140.7 °C
) δ 7.86 (d, J = 7.44652 Hz, 1H, indeno H-7), 7.62-7.52 (m, 2H,
1
H NMR (250 MHz, CDCl
3
indeno H-5, H-4), 7.39 (t, J = 6.92 Hz, 1H, indeno H-6), 7.30 (s, 1H, =CH-), 6.71 (d, J = 3.47
Hz, 1H, chlorofuryl H-3), 6.32 (d, J = 3.4 Hz, 1H, chlorofuryl H-4), 3.99 (s, 2H, indeno H-3).
1
3
3
C NMR (62.5 MHz, CDCl ) δ 193.75, 151.79, 149.68, 139.85, 138.29, 134.58, 132.90,. 127.52,
1
26.21, 124.24, 118.85, 118.38, 109.52, 32.20.
2
5
. 2. General method for the preparation of V (R = a-d, f)
2
A mixture of equivalent amount of acetophenones IV (R = a-d, f) and iodine in pyridine (15
equiv.) was refluxed for 3 h. After cooling the mixture to room temperature, the precipitate was
filtered and washed with cold pyridine followed by drying overnight to yield 55.4-93.1% of V
2
(
R = a-d, f).
5
.3. General method for the preparation of 7-18
Anhydrous ammonium acetate (10.0 equiv.) was mixed with glacial acetic acid or methanol
1
followed by addition of the indanone intermediates (1.0 equiv.), III (R = a-e) and pyridinium

2
iodide salts V (R = a-d, f) (1.0 equiv.). The mixture was then refluxed for 24-36 h. The reaction
mixture was then extracted with ethyl acetate, and washed with water and brine solution. The
organic layer was dried with magnesium sulfate and filtered. The filtrate was evaporated at
reduced pressure, which was then purified with silica gel column chromatography with the
gradient elution of ethyl acetate/n-hexane to afford solid compounds 7-18 in 24.9-58.2% yield.
5
.3.1. Synthesis of 2-(2-(thiophen-2-yl)-5H-indeno[1,2-b]pyridin-4-yl)phenol (7)
The same procedure described in section 5.3 was employed with IIIa (0.71 g, 3 mmol),
anhydrous ammonium acetate (2.31 g, 30 mmol), Vf (1.49 g, 4.5 mmol) and methanol (15 mL) at
1
00°C for 24 h to yield 378 mg (1.12 mmol, 37.3 %) as a white solid.
TLC (ethyl acetate / n-hexane = 1:7) R
f
= 0.29, mp: 292.6-293.9 °C, HPLC: Retention time:
+
10.12 min, purity: 98.5%; ESI LC/MS (condition A): m/z calcd for C22
H
15NOS [MH] 342.09;
found 342.41.
1
6
H NMR (250 MHz, DMSO-d ) δ 9.86 (s, 1H, 4-phenyl 2-OH), 8.01 (d, J = 6.27 Hz, 1H,
indenopyridine H-9), 7.87 (d, J =3.5 Hz, 1H, 2-thiophene H-3), 7.76 (s,1H, indenopyridine H-3),
.64-7.60 (m, 2H, 2-thiophene H-5 and indenopyridine H-6), 7.51-7.44 (m, 2H, indenopyridine
H-7, H-8), 7.39 (d, J =7.55 Hz, 1H, 4-phenyl H-6), 7.31 (t, J =7.25Hz, 1H, 4-phenyl H-4), 7.16
d, J =3.97 Hz, 1H, 2-thiophene H-4), 7.03 (d, J =8.1 Hz, 1H, 4-phenyl H-3), 6.96 (t, J = 7.4 Hz,
7
(
1
H, 4-phenyl H-5), 3.87 (s, 2H, indenopyridine H-5).
13
6
C NMR (62.5 MHz, DMSO-d ) δ 159.93, 155.15, 151.75, 145.81, 145.46, 145.12, 141.01,
1
1
35.47, 131.28, 130.79, 129.64, 129.23, 128.67, 127.95, 126.33, 125.96, 125.74, 121.32, 120.21,
18.58, 116.90, 34.98.

5
.3.2. Synthesis of 3-(2-(thiophen-2-yl)-5H-indeno[1,2-b]pyridin-4-yl)phenol (8)
The same procedure described in section 5.3 was employed with IIIb (0.71 g, 3 mmol),
anhydrous ammonium acetate (2.31 g, 30 mmol), Vf (1.49 g, 4.5 mmol) and methanol (15 mL) at
1
00°C for 30 h to yield 473 mg (1.39 mmol, 46.2 %) as a light yellow solid.
TLC (ethyl acetate / n-hexane = 1:5) R
f
= 0.22, mp: 211.4-212.8 °C, HPLC: Retention time:
+
10.29 min, purity: 97.9%; ESI LC/MS (condition A): m/z calcd for C22
H
15NOS [MH] 342.09;
found 342.42.
1
6
H NMR (250 MHz, DMSO-d ) δ 9.71 (s, 1H, 4-phenyl 3-OH), 8.03 (m, 1H, indenopyridine H-
9), 7.96 (dd, J =3.6, 0.97 Hz, 1H, 2-thiophene H-3), 7.85 (s,1H, indenopyridine H-3), 7.66-7.46
(m, 2H, 2-thiophene H-5 and indenopyridine H-6), 7.50-7.46 (m, 2H, indenopyridine H-7, H-8),
7
2
.36 (t, J =7.77 Hz, 1H, 4-phenyl H-5), 7.25 (t, J =7.82Hz, 1H, 4-phenyl H-6), 7.20-7.16 (m, 2H,
-thiophene H-4 and 4-phenyl H-2), 6.89 (d, J = 7.95 Hz, 1H, 4-phenyl H-4), 4.07 (s, 2H,
indenopyridine H-5).
13
6
C NMR (62.5 MHz, DMSO-d ) δ 160.85, 158.62, 152.47, 147.07, 145.66, 145.29, 140.82,
1
1
39.87, 133.62, 130.86, 129.82, 129.29, 128.91, 128.08, 126.34 (2 C), 121.42, 119.97, 116.68,
16.63, 116.07, 35.16.
5
.3.3. Synthesis of 4-(2-(thiophen-2-yl)-5H-indeno[1,2-b]pyridin-4-yl)phenol (9)
The same procedure described in section 5.3 was employed with IIIc (0.71 g, 3 mmol),
anhydrous ammonium acetate (2.31 g, 30 mmol), Vf (1.49 g, 4.5 mmol) and methanol (15 mL) at
1
00°C for 24 h to yield 314 mg (0.92 mmol, 30.7 %) as a light yellow solid.

TLC (ethyl acetate / n-hexane = 1:5) R
f
= 0.2, mp: 268.9-269.9 °C, HPLC: Retention time: 10.08
+
min, purity: 96.1%; ESI LC/MS (condition A): m/z calcd for C22
H
15NOS [MH] 342.09; found
3
42.42.
1
6
H NMR (250 MHz, DMSO-d ) δ 9.88 (s, 1H, 4-phenyl 4-OH), 8.01-7.98 (m, 1H,
indenopyridine H-9), 7.93 (d, J =3.0 Hz, 1H, 2-thiophene H-3), 7.83 (s,1H, indenopyridine H-3),
.71 (d, J =8.52 Hz, 2H, 4-phenyl H-2, H-6), 7.63-7.62 (m, 2H, 2-thiophene H-5 and
7
indenopyridine H-6), 7.47-7.45 (m, 2H, indenopyridine H-7, H-8), 7.17 (t, J =3.77 Hz, 1H, 2-
thiophene H-4), 6.95 (d, J = 8.55 Hz, 2H, 4-phenyl H-3, H-5), 4.09 (s, 2H, indenopyridine H-5).
13
C NMR (62.5 MHz, DMSO-d
6
) δ 160.83, 159.14, 152.32, 146.81, 145.82, 145.31, 140.90.,
1
37.53, 133.21, 130.77 (2 C), 129.74, 129.26, 129.05, 128.04, 126.30, 126.19, 121.40, 116.60 (2
C), 116.27, 35.6.
5
.3.4. Synthesis of 2-(4-(5-chlorofuran-2-yl)-5H-indeno[1,2-b]pyridin-2-yl)phenol (10)
The same procedure described in section 5.3 was employed with IIIe (0.48 g, 2 mmol),
anhydrous ammonium acetate (1.54 g, 20 mmol), Va (1.02 g, 3 mmol) and methanol (15 mL) at
1
00°C for 24 h to yield 188 mg (0.49 mmol, 24.9 %) as a light yellow solid.
TLC (ethyl acetate / n-hexane = 1:7) R
f
= 0.3, mp: 181.4-181.8 °C, HPLC: Retention time: 15.91
+
min, purity: 98.4%; ESI LC/MS (condition A): m/z calcd for C22
H
14ClNO
2
[MH] 360.08; found
360.12 (100%), [MH+2]: 362.12 (32.6%).
1
H NMR (250 MHz, CDCl
3
) δ 14.51 (s, 1H, 2-phenyl 2-OH), 8.06 (s, 1H, indenopyridine H-3),
.01-7.98 (m, 1H, indenopyridine H-9), 7.91 (dd, J = 7.95, 0.95 Hz, 1H, 2-phenyl H-6), 7.65 (br,
H, 4-furan H-5), 7.60-7.57 (m, 1H, indenopyridine H-6), 7.46-7.43 (m, 2H, indenopyridine H-8,
8
1
H-7), 7.33 (td, J = 8.27, 1.3 Hz, 1H, 2-phenyl H-4), 7.06 (d, J = 8 Hz, 1H, 2-phenyl H-3), 6.98

(d, J = 3.47 Hz, 1H, 4-furan H-3), 6.92 (t, J = 7.77 Hz, 1H, 2-phenyl H-5), 6.62-6.60 (m, 1H, 4-
furan H-4), 4.02 (s, 2H, indenopyridine H-5).
1
3
3
C NMR (62.5 MHz, CDCl ) δ 159.94, 158.09, 156.89, 150.98, 144.01, 143.98, 139.18, 135.26,
1
1
31.12, 129.31, 128.87, 127.41, 126.43, 125.06, 120.89, 119.44, 118.80, 118.55, 112.30, 111.45,
11.22, 35.69.
5
.3.5. Synthesis of 3-(4-(5-chlorofuran-2-yl)-5H-indeno[1,2-b]pyridin-2-yl)phenol (11)
The same procedure described in section 5.3 was employed with IIIe (0.48 g, 2 mmol),
anhydrous ammonium acetate (1.54 g, 20 mmol), Vb (1.02 g, 3 mmol) and methanol (15 mL) at
1
00°C for 24 h to yield 240 mg (0.69 mmol, 36.3 %) as a brownish green solid.
TLC (ethyl acetate / n-hexane = 1:3) R
f
= 0.28, mp: 171.9-172.5 °C, HPLC: Retention time: 9.65
+
min, purity: 98.3%; ESI LC/MS (condition A): m/z calcd for C22
H
14ClNO
2
[MH] 360.08; found
360.11 (100%), [MH+2]: 362.12 (32.6%).
1
3
H NMR (250 MHz, CDCl ) δ 8.19 (d, J = 7.25 Hz, 1H, indenopyridine H-9), 7.84 (s, 1H,
indenopyridine H-3), 7.79 (s, 1H, phenyl H-6), 7.60-7.57 (m, 2H, phenyl H-2, indenopyridine H-
), 7.46-7.40 (m, 2H, phenyl H-2, indenopyridine H-8), 7.34 (t, J = 7.9 Hz, 1H, indenopyridine
H-7), 6.96 (d, J = 3.5 Hz, 1H, chlorofuryl H-3), 6.88 (dd, J = 8.07, 2.47 Hz, 1H, phenyl H-4),
.39 (d, J = 3.5 Hz, 1H, chlorofuryl H-4), 4.04 (s, 2H, indenopyridine H-5).
6
6
1
3
3
C NMR (62.5 MHz, CDCl ) δ 156.67, 156.26, 155.75, 150.96, 143.88, 140.96, 140.34, 129.96,
1
1
29.66, 129.21, 129.11, 127.41, 124.97, 121.95, 121.52, 120.16, 119.53, 116.32, 114.43, 112.93,
08.95, 35.38.
5
.3.6. Synthesis of 4-(4-(5-chlorofuran-2-yl)-5H-indeno[1,2-b]pyridin-2-yl)phenol (12)

The same procedure described in section 5.3 was employed with IIIe (0.48 g, 2 mmol),
anhydrous ammonium acetate (1.54 g, 20 mmol), Vc (1.02 g, 3 mmol) and methanol (15 mL) at
1
00°C for 24 h to yield 240 mg (0.66 mmol, 32.8 %) as a brownish orange solid.
TLC (ethyl acetate / n-hexane = 1:3), R
f
= 0.23, mp: 258.4-258.7 °C, HPLC: Retention time: 9.91
+
min, purity: 96.9%; ESI LC/MS (condition A): m/z calcd for C22
H
14ClNO
2
[MH] 360.08; found
360.22 (100%), [MH+2]: 362.23 (32.6%).
1
6
H NMR (250 MHz, DMSO-d ) δ 9.83 (s, 1H, phenyl 4-OH), 8.11 (d, J = 8.67 Hz, 2H, phenyl
H-2, H-6), 8.07-8.03 (m, 1H, indenopyridine H-9), 7.95 (s, 1H, indenopyridine H-3), 7.70-7.67
m, 1H, indenopyridine H-6), 7.49 (d, J = 3.67 Hz, 1H, chlorofuryl H-3), 7.48-7.46 (m, 2H,
(
phenyl H-7, H-8), 6.91 (d, J = 8.62 Hz, 2H, phenyl H-3, H-5), 6.80 (d, J = 3.55 Hz, 1H,
chlorofuryl H-4), 4.14 (s, 2H, indenopyridine H-5).
1
3
6
C NMR (62.5 MHz, DMSO-d ) δ 161.05, 159.00, 156.32, 151.11, 144.66, 140.41, 137.18,
1
1
33.36, 129.89, 129.38 (2C), 128.56, 128.30, 127.58, 125.73, 120.85, 115.96 (2C), 114.25,
10.95, 110.05, 34.78.
5
.3.7. Synthesis of 2-(4-(5-chlorothiophen-2-yl)-5H-indeno[1,2-b]pyridin-2-yl)phenol (13)
The same procedure described in section 5.3 was employed with IIId (0.52 g, 2 mmol),
anhydrous ammonium acetate (1.54 g, 20 mmol), Va (1.02 g, 3 mmol) and glacial acetic acid (4
mL) at 100°C for 30 h to yield 282 mg (0.74 mmol, 37.4 %) as a yellow solid.
TLC (ethyl acetate / n-hexane = 1:10) R
f
=0.3, mp: 235.4-235.9 °C, HPLC: Retention time:
+
16.45 min, purity: 100%; ESI LC/MS (condition B): m/z calcd for C22
H
14ClNOS [MH] 376.06;
found 376.10 (100%), [MH+2]: 378.12 (32.6%).

1
3
H NMR (250 MHz, CDCl ) δ 14.37 (s, 1H, phenyl 2-OH), 7.97 (d, J=6.82 Hz, 1H,
indenopyridine H-9), 7.82 (dd, J = 7.87, 1.42 Hz, 1H, phenyl H-6), 7.81(s, 1H, indenopyridine
H-3), 7.58-7.55 (m, 1H, indenopyridine H-6), 7.48-7.42 (m, 2H, indenopyridine H-7, H-8),7.40
(d, J = 4.0 Hz, 1H, chlorothienyl H-3), 7.31 (td, J = 8.45, 1.5 Hz, 1H, phenyl H-4), 7.05 (br, 1H,
phenyl H-2), 7.01 (d, J = 4.01, 1.17 Hz, 1H, chlorothienyl H-4), 6.91 (td, J = 8.12, 1.2 Hz, 1H,
phenyl H-5), 3.98 (s, 2H, indenopyridine H-5).
1
3
3
C NMR (62.5 MHz, CDCl ) δ 159.83, 158.38, 156.98, 143.64, 138.95, 138.77, 138.70, 132.81,
1
1
31.34, 130.09, 129.55, 127.58, 127.31, 126.79, 126.25, 125.06, 121.01, 119.04, 118.89, 118.60,
13.30, 35.62.
5
.3.8. Synthesis of 3-(4-(5-chlorothiophen-2-yl)-5H-indeno[1,2-b]pyridin-2-yl)phenol (14)
The same procedure described in section 5.3 was employed with IIId (0.52 g, 2 mmol),
anhydrous ammonium acetate (1.54 g, 20 mmol), Vb (1.02 g, 3 mmol) and glacial acetic acid (4
mL) at 100°C for 24 h to yield 228 mg (0.61 mmol, 30.3 %) as a yellow solid.
TLC (ethyl acetate / n-hexane = 1:3) R
f
= 0.3, mp: 206.8-207.4 °C, HPLC: Retention time: 11.89
+
min, purity: 100%; ESI LC/MS (condition A): m/z calcd for C22
H
14ClNOS [MH] 376.06; found
376.27 (100%), [MH+2]: 378.28 (32.6%).
1
6
H NMR (250 MHz, DMSO-d ) δ 9.62 (s, 1H, phenyl 3-OH), 8.06 (d, J = 7.0Hz, 1H,
indenopyridine H-9), 7.98 (s, 1H, indenopyridine H-3), 7.95 (d, J = 4.05 Hz, 1H, chlorothienyl
H-3), 7.78-7.66 (m, 3H, phenyl H-2, H-6 indenopyridine H-6), 7.52-7.49 (m, 2H, indenopyridine
H-7, H-8), 7.37 (d, J = 4.1 Hz, 1H, chlorothienyl H-4), 7.30 (d, J = 7.8 Hz, 1H, phenyl H-5), 6.86
(d, J = 7.6 Hz, 1H, phenyl H-4), 4.20 (s, 2H, indenopyridine H-5).

1
3
6
C NMR (62.5 MHz, DMSO-d ) δ 161.26, 158.00, 156.21, 144.31, 140.24, 138.91, 137.57,
1
30.91, 130.80, 129.99, 129.31 (2C), 128.49, 128.43, 127.66, 125.82, 120.80, 118.13, 116.35,
14.76, 113.93, 35.13.
1
5
.3.9. Synthesis of 4-(4-(5-chlorothiophen-2-yl)-5H-indeno[1,2-b]pyridin-2-yl)phenol (15)
The same procedure described in section 5.3 was employed with IIId (0.52 g, 2 mmol),
anhydrous ammonium acetate (1.54 g, 20 mmol), Vc (1.02 g, 3 mmol) and methanol (20 mL) at
1
00°C for 24 h to yield 207 mg (0.54 mmol, 27.5%) as a light yellow solid.
TLC (ethyl acetate / n-hexane = 1:3) R
f
= 0.25, mp: 241.2-241.8 °C, HPLC: Retention time:
+
1
2.64 min, purity: 97.5%; ESI LC/MS (condition B): m/z calcd for C22
H
14ClNOS [MH] 376.06;
found 376.20 (100%), [MH+2]: 378.22(32.6%).
1
6
H NMR (250 MHz, DMSO-d ) δ 9.80 ( s, 1H, phenyl 4-OH), 8.13 (d, J = 8.62 Hz, 2H, phenyl
H-2, H-6), 7.93 (s, 1H, indenopyridine H-3), 7.90 (d, J = 4.05 Hz, 1H, chlorothienyl H-3), 7.68
m, 1H, indenopyridine H-6), 7.50-7.46 (m, 2H, indenopyridine H-7, H-8), 7.34 (d, J = 3.97 Hz,
(
1
5
H, chlorothienyl H-4), 6.90 (d, J = 8.6 Hz, 2H, pheny H-3, H-5), 4.15 (s, 2H, indenopyridine H-
).
1
3
6
C NMR (62.5 MHz, DMSO-d ) δ 160.99, 158.86, 156.30, 144.28, 140.42, 139.10, 137.47,
1
3
30.73, 129.72, 129.65 (3C), 129.27, 128.54, 128.20, 127.52, 120.84, 115.73 (3C), 113.55,
5.13.
5
.3.10. Synthesis of 2-(2-(5-chlorothiophen-2-yl)-5H-indeno[1,2-b]pyridin-4-yl)phenol (16)

The same procedure described in section 5.3 was employed with IIIa (0.47 g, 2 mmol),
anhydrous ammonium acetate (1.54 g, 20 mmol), Vd (1.09 g, 3 mmol) and glacial acetic acid (4
mL) at 110 °C for 36 h to yield 201 mg (0.51 mmol, 26.8%) as a yellow solid.
o
TLC (ethyl acetate / n-hexane = 1:4) R
f
= 0.23, mp : 218.6-219.2 C, HPLC: Retention time:
+
1
2.55 min, purity: 95.3%; ESI LC/MS m/z calcd for C22
H
14ClNOS [MH] 376.06; found 376.16
(100%), [MH+2]: 378.16 (32.6%).
1
6
H NMR (250 MHz, DMSO-d ) δ 9.91 (s,1H, phenyl 2-OH), 7.98 (d, J = 6.05 Hz, 1H,
indenopyridine H-9), 7.78 (s, 1H, indenopyridine H-3), 7.76 (s, J = 4.0 Hz, 1H, chlorothienyl H-
3
), 7.62 (d, J = 6.1 Hz, 1H, indenopyridine H-6), 7.47-7.45 (m, 2H, indenopyridine H-7, H-8),
.38 (d, J = 7.42 Hz, 1H, phenyl H-6), 7.31 (t, J = 8.02 Hz, 1H, phenyl H-4), 7.17 (d, J = 3.95
7
Hz, 1H, chlorothienyl H-4), 7.01 (d, J = 8.07 Hz, 1H, phenyl H-3), 6.95 (t, J = 7.45 Hz, 1H,
phenyl H-5), 3.86 (s, 2H, indenopyridine H-5).
1
3
6
C NMR (62.5 MHz, DMSO-d ) δ 159.33, 154.57, 150.20, 145.07, 144.60, 144.39, 140.10,
1
35.47, 130.74, 130.35, 130.17, 129.28, 128.54, 127.45, 125.82, 124.99 (2C), 120.78, 119.68,
17.43, 116.30, 34.44.
1
5
.3.11. Synthesis of 3-(2-(5-chlorothiophen-2-yl)-5H-indeno[1,2-b]pyridin-4-yl)phenol (17)
The same procedure described in section 5.3 was employed with IIIb (0.71 g, 2 mmol),
anhydrous ammonium acetate (2.31 g, 30 mmol), Vd (1.09 g, 3 mmol) and methanol (20 mL) at
110 °C for 36 h to yield 400 mg (1.04 mmol, 35.4%) as a light brown solid.
o
TLC (ethyl acetate / n-hexane = 1:3) R
f
= 0.28, mp : 194.6-195.1 C, HPLC: Retention time:
+
1
3.28 min, purity: 100%; ESI LC/MS m/z calcd for C22
H
14ClNOS [MH] 376.06; found 376.15
(100%), [MH+2]: 378.15 (32.6%).

1
H NMR (250 MHz, DMSO-d
indenopyridine H-9), 7.88 (s, 1H, indenopyridine H-3), 7.86 (d, J = 4.0 Hz, 1H, chlorothienyl H-
), 7.67-7.64 (m,1H, indenopyridine H-6), 7.49-7.46 (m, 2H, indenopyridine H-7, H-8), 7.36 (t, J
7.7 Hz, 1H, phenyl H-5), 7.25-7.22 (m, 2H, phenyl H-2, H-6), 7.19 (d, J = 4.17 Hz, 1H,
chlorothienyl H-4), 6.91 (d, J = 7.77 Hz, 1H, phenyl H-4), 4.07 (s, 2H, indenopyridine H-5).
6
) δ 9.76 (s, 1H, phenyl 3-OH), 7.99 (d, J = 7.05 Hz, 1H,
3
=
1
3
6
C NMR (62.5 MHz, DMSO-d ) δ 160.19, 158.03, 150.92, 146.60, 144.73, 144.20, 139.88,
1
1
39.07, 133.59, 130.35, 130.24, 129.37, 128.55, 127.50, 125.77, 125.29, 120.83, 119.39, 116.17,
15.54 (2C), 34.67.
5
.3.12. Synthesis of 4-(2-(5-chlorothiophen-2-yl)-5H-indeno[1,2-b]pyridin-4-yl)phenol (18)
The same procedure described in section 5.3 was employed with IIIc (0.71 g, 2 mmol),
anhydrous ammonium acetate (2.31 g, 30 mmol), Vd (1.09 g, 3 mmol) and methanol (20 mL) at
110 °C for 36 h to yield 656 mg (1.72 mmol, 58.2%) as an off-white solid.
o
TLC (ethyl acetate / n-hexane = 1:2) R
f
= 0.23, mp : 237.9-238.5 C, HPLC: Retention time:
+
1
3.11 min, purity: 99.4%; ESI LC/MS m/z calcd for C22
H
14ClNOS [MH] 376.06; found 376.13
(100%), [MH+2]: 378.13 (32.6%).
1
6
H NMR (250 MHz, DMSO-d ) δ 9.91 (s, 1H, phenyl 4-OH), 9.98 (d, J = 7.62 Hz, 1H,
indenopyridine H-9), 7.86 (s, 1H, indnopyridine H-3), 7.83 (d, J= 3.97Hzz, 1H, chlorothienyl H-
), 7.71 (d, J = 8.35 Hz, 2H, phenyl H-2, H-6), 7.66-7.63 (m, 1H, indenopyridine H-6), 7.48-7.45
m, 2H, indenopyridine H-7, H-8), 7.18 (d, J = 4.0 Hz, 1H, chlorothienyl H-4), 6.94 (d, J = 8.15
Hz, 2H, phenyl H-3, H-5), 4.10 (s, 2H, indenopyridine H-5).
3
(

1
3
6
C NMR (62.5 MHz, DMSO-d ) δ 160.19, 158.60, 150.78, 146.34, 144.55, 144.75, 139.98,
1
3
33.10, 130.23 (3C), 129.28, 128.52, 128.24, 125.71, 125.16, 120.80, 115.97 (3C), 115.13,
4.83.
5
.4. Pharmacology
5
.4.1. Assay for DNA topoisomerase I inhibition
DNA topo I inhibition assay was determined following the method reported by Fukuda et al. [20]
with minor modifications. The test compounds were dissolved in DMSO at 20 mM as stock
solution. The activity of DNA topo I was determined by assessing the relaxation of supercoiled
DNA pBR322. The mixture of 100 ng of plasmid pBR322 DNA and 1 unit of recombinant
human DNA topoisomerase I (TopoGEN INC., USA) was incubated without and with the
prepared compounds at 37 °C for 30 min in the relaxation buffer (10 mM Tris-HCl (pH 7.9), 150
mM NaCl, 0.1% bovine serum albumin, 1 mM spermidine, 5% glycerol). The reaction in the
final volume of 10 μL was terminated by adding 2.5 μL of the stop solution containing 5%
sarcosyl, 0.0025% bromophenol blue and 25% glycerol. DNA samples were then
electrophoresed on a 1% agarose gel at 15 V for 7 h with a running buffer of TAE. Gels were
stained for 30 min in an aqueous solution of ethidium bromide (0.5 μg/mL). DNA bands were
TM
visualized by transillumination with UV light and were quantitated using AlphaImager (Alpha
Innotech Corporation).
5
.4.2. Assay for DNA Topoisomerase II Inhibition
DNA topo II inhibitory activity of compounds were measured as follows [21]. The mixture of
00 ng of supercoiled pBR322 plasmid DNA and 1 unit of human DNA topoisomerase IIα (Usb
2

Corp., USA) was incubated without and with the prepared compounds in the assay buffer (10
mM Tris-HCl (pH 7.9) containing 50 mM NaCl, 5 mM MgCl , 1 mM EDTA, 1 mM ATP, and
5 μg/mL bovine serum albumin) for 30 min at 30 °C. The reaction in a final volume of 20 μL
2
1
was terminated by the addition of 3 μL of 7 mM EDTA. Reaction products were analyzed on 1%
agarose gel at 25 V for 4 h with a running buffer of TAE. Gels were stained for 30 min in an
aqueous solution of ethidium bromide (0.5 μg/mL). DNA bands were visualized by
TM
transillumination with UV light and supercoiled DNA was quantitated using AlphaImager
(Alpha Innotech Corporation).
5.4.3. Assay for Cytotoxicity
Cancer cells were cultured according to the supplier's instructions. Cells were seeded in 96-well
4
plates at a density of 2~4 x 10 cells per well and incubated for overnight in 0.1 mL of media
supplied with 10% Fetal Bovine Serum (Hyclone, USA) in 5% CO
, after FBS starvation for 4 h, culture medium in each well was exchanged with 0.1 mL aliquots
of medium containing graded concentrations of compounds. On day 4, each well was added with
μL of the cell counting kit-8 solution (Dojindo, Japan) then incubated for additional 4 h under
2
incubator at 37 °C. On day
2
5
the same condition. The absorbance of each well was determined by an Automatic Elisa Reader
System (Bio-Rad 3550) at 450 nm wavelength. For determination of the IC50 values, the
absorbance readings at 450 nm were fitted to the four-parameter logistic equation. Adriamycin,
etoposide, and camptothecin were purchased from Sigma and used as positive controls.
Acknowledgements
This research was supported by the 2012 Yeungnam University Research Grant.

References
[
[
[
[
1] H.L. McLeod, F. Douglas, M. Oates, R.P. Symonds, D. Prakash, A.G. Van Der Zee, S.B.
Kaye, R. Brown, W.N. Keith, Topoisomerase I and II activity in human breast, cervix, lung and
colon cancer, Int. J. Cancer 59 (1994) 607-611.
2] A. Codegoni, S. Castagna, C. Mangioni, A. Scovassi, M. Broggini, M. D'lncalci, DNA-
topoisomerase I activity and content in epithelial ovarian cancer, Ann. Oncol. 9 (1998) 313-
3
19.
3] I. Husain, J.L. Mohler, H.F. Seigler, J.M. Besterman, Elevation of topoisomerase I messenger
RNA, protein, and catalytic activity in human tumors: demonstration of tumor-type specificity
and implications for cancer chemotherapy, Cancer Res. 54 (1994) 539-546.
4] B.C. Giovanella, J.S. Stehlin, M.E. Wall, M.C. Wani, A.W. Nicholas, L.F. Liu, R. Silber, M.
Potmesil, DNA topoisomerase I--targeted chemotherapy of human colon cancer in xenografts,
Science 246 (1989) 1046-1048.
[
[
5] J.L. Nitiss, Investigating the biological functions of DNA topoisomerases in eukaryotic cells,
Biochim. Biophys. Acta 1400 (1998) 63-81.
6] J.C. Wang, Cellular roles of DNA topoisomerases: a molecular perspective, Nat. Rev. Mol.
Cell. Biol. 3 (2002) 430-440.

[
[
[
7] Y. Pommier, Topoisomerase I inhibitors: camptothecins and beyond, Nat. Rev. Cancer, 6
2006) 789-802.
(
8] J.L. Nitiss, Targeting DNA topoisomerase II in cancer chemotherapy, Nat. Rev. Cancer 9
2009) 338-350.
(
9] M.B. Sporn, N. Suh, Chemoprevention of cancer, Carcinogenesis 21 (2000) 525-530.
[
10] T. Utsugi, K. Aoyagi, T. Asao, S. Okazaki, Y. Aoyagi, M. Sano, K. Wierzba, Y. Yamada,
Antitumor activity of a novel quinoline derivative, TAS-103, with inhibitory effects on
topoisomerases I and II, Jpn J Cancer Res, 88 (1997) 992-1002.
[
11] M. Manpadi, P.Y. Uglinskii, S.K. Rastogi, K.M. Cotter, Y.S. Wong, L.A. Anderson, A.J.
Ortega, S. Van Slambrouck, W.F. Steelant, S. Rogelj, P. Tongwa, M.Y. Antipin, I.V.
Magedov, A. Kornienko, Three-component synthesis and anticancer evaluation of polycyclic
indenopyridines lead to the discovery of a novel indenoheterocycle with potent apoptosis
inducing properties, Org. Biomol. Chem. 5 (2007) 3865-3872.
[
12] M.M. Ghorab, M.S. Al-Said, Anticancer activity of novel indenopyridine derivatives, Arch.
Pharm. Res. 35 (2012) 987-994.

[
13] T.M. Kadayat, C. Park, K.-Y. Jun, T.B.T. Magar, G. Bist, H.Y. Yoo, Y. Kwon, E.-S. Lee,
Design and synthesis of novel 2,4-diaryl-5H-indeno[1,2-b]pyridine derivatives, and their
evaluation of topoisomerase inhibitory activity and cytotoxicity, Bioorg. Med. Chem. 23
(2015 ) 160-173.
[
14] T.M. Kadayat, C. Song, S. Shin, T.B.T. Magar, G. Bist, A. Shrestha, P. Thapa, Y. Na, Y.
Kwon, E.-S. Lee, Synthesis, topoisomerase I and II inhibitory activity, cytotoxicity, and
structure-activity relationship study of 2-phenyl- or hydroxylated 2-phenyl-4-aryl-5H-
indeno[1,2-b]pyridines, Bioorg. Med. Chem. 23 (2015 ) 3499-3512.
[
[
[
15] C.M. Harris, R. Kannan, H. Kopecka, T.M. Harris, The role of the chlorine substituents in the
antibiotic vancomycin: preparation and characterization of mono-and didechlorovancomycin, J.
Am. Chem. Soc. 107 (1985) 6652-6658.
16] M.Z. Hernandes, S.M.T. Cavalcanti, D.R.M. Moreira, J. de Azevedo, W. Filgueira, A.C.L.
Leite, Halogen atoms in the modern medicinal chemistry: hints for the drug design, Curr. Drug
Targets 11 (2010) 303-314.
17] D. Batovska, S. Parushev, A. Slavova, V. Bankova, I. Tsvetkova, M. Ninova, H. Najdenski,
Study on the substituents' effects of a series of synthetic chalcones against the yeast Candida
albicans, Eur. J. Med. Chem. 42 (2007) 87-92.

[
18] F. Kröhnke, Syntheses Using Pyridinium Salts (IV), Angew. Chem. Int. Ed. 2 (1963) 380-
93.
3
[
[
19] F. Kröhnke, The specific synthesis of pyridines and oligopyridines, Synthesis (1976) 1-24.
20] M. Fukuda, K. Nishio, F. Kanzawa, H. Ogasawara, T. Ishida, H. Arioka, K. Bojanowski, M.
Oka, N. Saijo, Synergism between cisplatin and topoisomerase I inhibitors, NB-506 and SN-38,
in human small cell lung cancer cells, Cancer Res. 56 (1996) 789-793.
[
21] H.B. Kwon, C. Park, K.-H. Jeon, E. Lee, S.-E. Park, K.-Y. Jun, T.M. Kadayat, P. Thapa, R.
Karki, Y. Na, A series of novel terpyridine-skeleton molecule derivants inhibit tumor growth
and metastasis by targeting topoisomerases, J. Med. Chem. 58 (2015) 1100-1122.