2,4-Diaryl-5,6-dihydro-1,10-phenanthroline and 2,4-diaryl-5,6- dihydrothieno[2,3-h] quinoline derivatives for topoisomerase i and II inhibitory activity, cytotoxicity, and structure-activity relationship study

Article abstract of DOI:10.1016/j.bioorg.2011.09.001

Designed and synthesized thirty-two 2,4-diaryl-5,6-dihydro-1,10- phenanthroline and 2,4-diaryl-5,6-dihydrothieno[2,3-h] quinoline derivatives as rigid analogs of 2,4,6-trisubstituted pyridines were evaluated for topoisomerase I and II inhibitory activitie

Full text of DOI:10.1016/j.bioorg.2011.09.001

Bioorganic Chemistry 40 (2012) 67–78
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
2,4-Diaryl-5,6-dihydro-1,10-phenanthroline and 2,4-diaryl-5,6-dihydrothieno
[2,3-h] quinoline derivatives for topoisomerase I and II inhibitory activity,
cytotoxicity, and structure–activity relationship study
Pritam Thapa a, Radha Karki a, Han Young Yoo a, Pil-Hoon Park a, Eunyoung Lee b, Kyung-Hwa Jeon b,
Younghwa Na c, Won-Jea Cho d, Youngjoo Kwon b, , Eung-Seok Lee a,
⇑
⇑
a College of Pharmacy, Yeungnam University, Kyongsan 712-749, Republic of Korea
b College of Pharmacy, Division of Life & Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Republic of Korea
c College of Pharmacy, Cha University, Pochon 487-010, Republic of Korea
d College of Pharmacy, Chonnam National University, Kwangju 500-757, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 August 2011
Available online 8 September 2011
Designed and synthesized thirty-two 2,4-diaryl-5,6-dihydro-1,10-phenanthroline and 2,4-diaryl-5,6-
dihydrothieno[2,3-h] quinoline derivatives as rigid analogs of 2,4,6-trisubstituted pyridines were evalu-
ated for topoisomerase I and II inhibitory activities as well as cytotoxicities against several human cancer
cell lines. Structure–activity relationship study showed that [2,20;60,200]-terpyridine skeleton is important
for the cytotoxicity against several human cancer cell lines.
Keywords:
Terpyridine
2,4-Diaryl-5,6-dihydro-1,10-phenanthroline
2,4-Diaryl-5,6-dihydrothieno[2,3-h]
quinoline
Ó 2011 Elsevier Inc. All rights reserved.
Topoisomerase I and II
Cytotoxicity
1. Introduction
into the active site of a receptor [5]. Herein we have designed
and synthesized various rigid analogs of flexible 2,4,6-trisubsti-
Our research group has been studying various flexible terpyri-
dine derivatives for the development of anticancer agents. They
have shown moderate to considerable topoisomerase (topo) I
and/or II inhibitory activities, and cytotoxicity against several hu-
man cancer cell lines [1]. Recently, various rigid analogs were re-
ported to possess moderate topoisomerase inhibitory activity and
cytotoxicity against several human cancer cell lines [2].
tuted pyridines. From the previous study it was found that alpha
carbon linkage in terpyridine has important role in displaying cyto-
toxicity against several human cancer cell lines. [2,20;60,200]-Terpyr-
idine (a) was found to have the most significant cytotoxicity [6]
(Fig. 1). This motivate us to synthesize various rigid analogs of
2,4,6-trisubstituted pyridines containing [2,20;60,200]-terpyridine
skeleton and other derivatives.
It is well documented that planarity facilitates the molecules to
intercalate into the DNA in the topo I-DNA ternary complex [3].
Intercalation of planar molecules into DNA helix allows the stabil-
ization of the topo-DNA covalent cleavage intermediates, convert-
ing topo into a lethal DNA-damaging agent. Many clinically used
drugs like topotecan (topo I inhibitor) and doxorubicin (topo II
inhibitor) exert their anticancer action by the similar mechanism
[4]. Generally, a drug to its receptor site is influenced by electronic
or steric factors and these two functions are considered to be
important in the bioactive conformation of a drug. Rigid structures
are commonly considered to have little conformational entropy
compared to flexible structures and can be more efficiently fitted
Moreover, 2,4-diaryl-5,6-dihydro-1,10-phenanthroline (b) and
2,4-diaryl-5,6-dihydrothieno[2,3-h] quinoline derivatives (c) share
common quinoline core in their structure (Fig. 1). Quinoline core is
of great interest in the field of medicinal chemistry as they are
present in a wide range of biologically active compounds fre-
quently condensed with other heterocycles [7]. It is the fact that
well known topo I inhibitor camptothecin bears quinoline moiety.
Modifications or complete replacement of the quinoline ring with
an alternative ring system has been done to find the more potent
compound. Though analogs with replaced quinoline ring system
with other planar heterocycles showed moderate to good activities,
none of these derivatives have surpassed the potency of CPT [8].
In this study, we have designed and synthesized thirty-two 2,4-
diaryl-5,6-dihydro-1,10-phenanthroline (b) and 2,4-diaryl-5,6-
dihydrothieno[2,3-h] quinoline derivatives (c) as rigid analogs of
2,4,6-trisubstituted pyridines (Fig. 1). They were evaluated for
⇑
Corresponding authors. Fax: +82 2 3277 2851 (Y. Kwon), +82 53 810 4654 (E.-S.
Lee).
E-mail addresses: ykwon@ewha.ac.kr (Y. Kwon), eslee@yu.ac.kr (E.-S. Lee).
0045-2068/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2011.09.001

68
P. Thapa et al. / Bioorganic Chemistry 40 (2012) 67–78
white solid. Compounds 7b-d was synthesized according to meth-
R2
N
4'
N
R2
N
quinoline moiety
R1
od B, where aryl aldehydes 3b-d was treated with ketone 5 in the
presence of catalytic amount of AcOH and piperidine at 90 °C for 5–
12 h. This method affords compounds 7b-d in 40.7–68.5% yield as
white or light yellow solids.
2
2"
N
6'
2'
R1
S
N
N
On the basis of modified KrÖhnke synthesis[12], rigid analogs of
2,4,6-trisubstituted pyridine, 8 (R1, R2 = a-d) and 9 (R1, R2 = a-d),
were synthesized by treatment of 6,7-dihydroquinolin-8(5H)-one
derivatives (6a-d) and 6,7-dihydrobenzo[b]thiophen-4(5H)-one
derivatives (7a-d) respectively with pyridinium iodide salts 2a-d
in the presence of NH4OAc in MeOH or glacial acetic acid as
illustrated in Scheme 2. Thirty-two rigid analogs, 2,4-diaryl-5,6-
dihydro-1,10-phenanthrolines (10–25) and 2,4-diaryl-5,6-dihydro-
thieno[2,3-h] quinolines (26–41), of 2,4,6-trisubstituted pyridines,
were synthesized in 11.9–92.4% yield as shown in Fig. 2.
a
b
c
R1 and R2 = phenyl, 2-, 3- or 4- pyridyl
Fig. 1. Structure of 2,20:60,200-terpyridine (a), and rigid analogs of 2,4,6-trisubsti-
tuted pyridines (b, c).
their topoisomerase I and II inhibitory activity, and cytotoxicity
against several human cancer cell lines.
2. Results and discussion
2.2. Topo I and II inhibitory activity
2.1. Synthetic chemistry
The prepared 32 compounds 10–41 were evaluated for their
topo I and II inhibitory activities. Topoisomerase inhibitory activi-
ties of the tested compounds are displayed in Fig. 3 and 4 and
Table 1. Percentage inhibition for compounds 10–25 are not
displayed as they have not shown any topoisomerase I and II inhib-
itory activities.
Pyridinium iodide salts (2a-d) were synthesized by refluxing
aryl methyl ketones (1a-d) with I2 and pyridine at 140 °C for 3 h
in 64.2–99.4% yields. Starting material 6,7-dihydro-5H-quinolin-
8-one (4) was prepared according to previously reported method
[9]. 6,7-Dihydroquinolin-8(5H)-one derivatives (6a-d) were syn-
thesized using the Claisen–Schmidt KOH catalyzed condensation
reaction [10] as illustrated in Scheme 1. Aryl aldehydes (3a-d) were
reacted with 4 in the presence of KOH using a solution of methanol
and water at 0 °C for 3–4 h to get 56.1–90.6% of 6a-d as light yel-
low solid.
6,7-Dihydrobenzo[b]thiophen-4(5H)-one derivatives (7a-d)
were synthesized by condensation of 6,7-dihydrobenzo[b]thio-
phen-4(5H)-one (5) with aryl aldehydes (3a-d) either in the pres-
ence of 40% aqueous KOH in ethanol (Method A) or AcOH/
piperidine as a catalyst (Method B) [11] as shown in Scheme 1.
Compound 7a was synthesized according to method A, where aryl
aldehyde 3a was treated with ketone 5 in the presence of 40%
aqueous KOH and ethanol at 0 °C for 3 h to yield 71.4% of 7a as a
2.2.1. Topoisomerase I inhibitory activity of the evaluated compounds
Synthesized 32 compounds 10–41 were evaluated for topo I
inhibitory activity at 100
ptothecin, a well known topo I inhibitor. Selected compounds, pos-
sessing significant inhibitory activity at 100 M, were evaluated
for topo I inhibitory activity at 20 M. It is found that most of
lM concentration, as compared to cam-
l
l
the compounds were devoid of topo I inhibitory activity except
33 and 35. The percentage of topo I inhibition for 33 and 35 was
60.0% and 25.8%, respectively at 100 lM. The topo I inhibitory
activity of the evaluated compounds is summarized in Fig. 3, and
Table 1.
1
1
2
2
2
2
Scheme 1. Synthesis of pyridinium iodide salts 2a-d, 6,7-dihydroquinolin-8(5H)-one derivatives 6a-d and 6,7-dihydrobenzo[b]thiophen-4(5H)-one derivatives 7a-d.
Reagents and conditions: (i) pyridine, iodine (1.0 equiv.), 3 h, 140 °C, 64.2–99.4%; (ii) KOH (1.2 equiv.), MeOH: H2O (5: 1 v/v), 3–4 h, 0 °C, 56.1–90.6%; (iii) 40% aqueous KOH,
EtOH, 3 h, 0 °C, 71.4%; (iv) AcOH, piperidine, 5–12 h, 90 °C, 40.7–68.5%.

P. Thapa et al. / Bioorganic Chemistry 40 (2012) 67–78
69
R2
N
i
R1
R2
N
O
N
I
R1
+
8
a d
6a d
-
(R1, R2 = - )
N
O
R2
S
2a d
-
ii
R2
R1
N
S
O
7a d
-
9
a d
(R1, R2 = - )
N
R
1 and R2 =
N
N
a
b
c
d
Scheme 2. Synthesis of 2,4-diaryl-5,6-dihydro-1,10-phenanthroline derivatives 8 and 2,4-diaryl-5,6-dihydrothieno[2,3-h] quinoline derivatives 9. Reagents and conditions:
(i) NH4OAc (10.0 equiv.), dry MeOH, 12–18 h, 80–100 °C, 26.2–92.4%; (ii) NH4OAc (10.0 equiv.), glacial AcOH or dry MeOH, 12–48 h, 90–100 °C, 11.9–51.6%.
N
N
N
N
N
N
N
N
N
N
10
N
N
12
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
13
11
14
17
15
16
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
18
21
19
20
22
25
24
23
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
S
S
S
S
S
S
S
S
N
N
N
N
N
N
33
32
27
31
26
29
30
28
N
N
N
N
N
N
N
N
S
S
N
S
S
N
N
N
S
S
S
S
N
N
N
N
N
N
36
37
34
35
41
38
40
39
Fig. 2. Structures of synthesized 2,4-diaryl-5,6-dihydro-1,10-phenanthroline and 2,4-diaryl-5,6-dihydrothieno[2,3-h] quinoline derivatives.
2.2.2. Topoisomerase II inhibitory activity of the evaluated compounds
Synthesized 32 compounds 10–41 were evaluated for topo II
inhibitory activity at 100 lM as compared to etoposide, a well
known topo II inhibitor. Most of the compounds did not show
significant topo II inhibitory activity. Several compounds such as
27–29 and 31 showed mild activity in the range of 11.5–15.7% at
100 lM. The topo II inhibitory activity of the evaluated compounds
is summarized in Fig. 4 and Table 1.
and previously reported data by our laboratory [6]. Previously, it
was found that [2,20;60,200]-terpyridine has shown the most signif-
icant cytotoxicity which indicates that alpha carbon linkage in ter-
pyridine is important for cytotoxicity. Selective compounds were
evaluated for cytotoxicity on five different human cancer cell lines:
human breast adenocarcinoma cell line (MCF-7), human cervix tu-
mor cell line (HeLa), human prostate tumor cell line (DU145), hu-
man colorectal adenocarcinoma cell line (HCT15) and chronic
myelogenous leukemia cell line (K562). Inhibitory activities were
presented as micromolar concentrations of the compounds that
cause 50% inhibition of cell growth (IC50) under the assay condi-
tions and compared with that of adriamycin. The cytotoxicity re-
sults are summarized in Table 2.
2.3. Cytotoxicity
Selected nine compounds were evaluated for cytotoxicity.
Selection was achieved on the basis of topo I inhibitory activity,

70
P. Thapa et al. / Bioorganic Chemistry 40 (2012) 67–78
Lane D: pBR322 only,
Lane T: pBR322 + Topo I,
Lane C: pBR322 + Topo I + Camptothecin,
Lane 10-41: pBR322 + Topo I + samples (10 to 41) at 100 µM
Lane 33 and 35: pBR322 + Topo I + samples (33 and 35) at 20 µM
Fig. 3. Topo I inhibitory activity of synthesized compounds at 100 lM and 20 lM.
Lane D: pBR322 only,
Lane T: pBR322 + Topo II,
Lane C: pBR322 + Topo II + Etoposide,
Lane 10-41: pBR322 + Topo II + samples (10 to 41) at 100 µM
Fig. 4. Topo II inhibitory activity of synthesized compounds at 100 lM.
As anticipated, it is found that compounds (10, 14 and 18) hav-
ing alpha carbons of the pyridine linked to each other showed the
significant cytotoxicity against all five human cancer cell lines.
Table 1
Topoisomerase I and II inhibitory activity of compounds 26–41.
Compounds
%Inhibition
Topo I
2.4. Structure–activity relationship (SAR) study
Topo II
100 lM
100
l
M
SAR was performed according to the results of topo I and II
inhibitory activity, and cytotoxicity of the evaluated compounds.
As most of the compounds were devoid of topo I and II inhibitory
activity, the concrete correlation of the structure of compounds
with topo inhibitory activity could not be determined. However,
some compounds from thienoquinoline derivatives such as 33
Camptothecin
Etoposide
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
60.82/52.39*
67.46/68.28*
5.53
12.45
13.95
11.51
7.44
15.78
5.12
4.95
5.23
8.27
5.90
5.97
1.88
N
N
N
N
has shown significant topo I inhibitory activity at 100
has shown moderate topo I inhibitory activity at 100
l
M, and 35
N
lM as com-
0.94
60.01
3.37
25.84
1.32
0.93
2.04
N
pared to positive control, camptothecin. Moreover, 27–29 and 31
has shown mild topo II inhibitory activity at 100 M. These results,
l
to some extent, indicate that thienoquinoline moiety may have
important role for topo inhibitory activity as compared to phenan-
throline moiety.
From results of cytotoxicity of the selected compounds, it is
found that all compounds possessing [2,20;60,200]-terpyridine skele-
ton has shown significant cytotoxicity against several human can-
cer cell lines. It signify that [2,20;60,200]-terpyridine skeleton is
4.73
4.59
2.80
1.67
N
N
41
*
Value for compounds 34–41, N: none.

P. Thapa et al. / Bioorganic Chemistry 40 (2012) 67–78
71
Table 2
Cytotoxicity of selected nine compounds against five different cancer cell lines.
Compd./cell
aIC50
bMCF-7
(lM)
cHela
dDU145
eHCT15
fK562
Adriamycin
Etoposide
Camptothecin
0.97 0.07
3.56 1.74
0.63 0.05
9.55 0.96
>50
5.67 0.28
>50
4.81 1.30
>50
0.98 0.11
0.94 0.17
1.28 0.25
2.11 0.78
32.40 2.75
0.31 0.19
>50
0.34 0.20
33.72 11.10
>50
0.81 0.07
1.04 0.37
1.17 0.19
1.3 0.078
>50
1.19 0.20
>50
0.12 0.06
>50
1.31 0.06
3.36 1.38
1.19 0.38
0.9 0.03
>50
0.86 0.06
>50
0.97 0.04
>50
0.46 0.12
0.82 0.11
0.71 0.10
1.59 0.40
>50
0.84 0.27
>50
1.85 0.10
>50
10
11
14
15
18
19
33
34
35
>50
>50
>50
>50
>50
>50
>50
13.12 2.48
4.03 1.01
>50
>50
22.89 4.00
50.10 11.76
35.69 7.20
a
b
c
d
e
f
Each data represents mean S.D. from three different experiments performed in triplicate.
MCF: human breast adenocarcinoma.
HeLa: human cervix tumor.
DU145: human prostate tumor.
HCT15: human colorectal adenocarcinoma.
K562: chronic myelogenous leukemia.
important for the cytotoxicity against several human cancer cell
lines as reported earlier [6].
Interruption of resonance between five or six membered ring
with central pyridine due to absence of double bond at 5,6 position
is believed to be the reason behind the inactivity of most com-
pounds as reported in earlier study [13].
volume of 10
l
L, was run in Waters X-TerraÒ
5 lM reverse-phase
C18 column (4.6 ꢀ 250 mm) with a gradient elution of (A) 20–
100% of B in A for 20 min followed by 100–20% of B in A for
10 min; (B) 50–100% of B in A for 10 min followed by 100–50% of
B in A for 20 min at a flow rate of 1.0 mL/min at 254 nm UV detec-
tion, where mobile phase A was doubly distilled water with 20 mM
ammonium formate (AF) and B was 90% ACN in water with 20 mM
AF. Purity of compound is described as percent (%).
3. Conclusion
ESI LC/MS analyses were performed with a Finnigan LCQ Advan-
tageÒ LC/MS/MS spectrometry utilizing XcaliburÒ program. For ESI
We have designed and synthesized thirty-two 2,4-diaryl-5,6-
dihydro-1,10-phenanthroline and 2,4-diaryl-5,6-dihydrothie-
no[2,3-h] quinoline derivatives as rigid analogs of 2,4,6-trisubsti-
tuted pyridines, and evaluated their topo I and II inhibitory
activity, and cytotoxicity against several human cancer cell lines.
Compound 33 showed considerable topo I inhibitory activity of
LC/MS, LC was performed with a 5
Atlantis T3, 3
gradient elution from 20% to 100% of B in A for 6.5 min followed
lL injection volume on a Waters
m reverse-phase C18 column (2.1 ꢀ 50 mm) with a
l
by 100–20% of B in A for 6 min and 20% of B in A for 2.5 min at a
flow rate of 200 lL/min, where mobile phase A was 100% distilled
water with 20 mM ammonium formate (AF) and mobile phase B
was 100% acetonitrile (ACN). MS ionization conditions were:
Sheath gas flow rate: 40 arb, aux gas flow rate: 0 arb, I spray volt-
age: 5.3 kV, capillary temperature: 275 °C, capillary voltage: 27 V,
tube lens offset: 45 V. Retention time was given in minutes.
60.0% at 100
100
lM as compared to camptothecin (60.8% at
l
M). Interestingly, it is found that phenanthroline derivatives
having [2,20;60,200]-terpyridine skeleton has shown significant cyto-
toxicity against all tested human cancer cell lines. This indicates
that
a-carbon linkage in terpyridine is important for possessing
cytotoxicity against several human cancer cell lines. Therefore, in
conclusion, we believe that [2,20;60,200]-terpyridine skeleton has
an important role for displaying the cytotoxic effects.
4.1. General method for preparation of 2a-d
Aryl methyl ketone (1a-d) with equivalent amount of iodine in
pyridine was refluxed at 140 °C for 3 h. Reaction mixture was
cooled to room temperature resulting precipitation. It was filtered
and washed with cold pyridine followed by drying overnight to
yield 64.2–99.4% of 2a-d.
4. Experimental
Compounds used as starting materials and reagents were ob-
tained from Aldrich Chemicals Co., Junsei or other chemical compa-
nies, and utilized without further purification. HPLC grade
acetonitrile (ACN) and methanol were purchased from Burdick and
Jackson, USA. Thin-layer chromatography (TLC) and column chro-
matography (CC) were performed with Kieselgel 60 F254 (Merck)
and silica gel (Kieselgel 60, 230–400 mesh, Merck), respectively.
Since all the compounds prepared contain aromatic ring, they were
visualizedand detected on TLCplates with UV light(short wave, long
wave or both). NMR spectra were recorded on a Bruker AMX 250
(250 MHz, FT) for 1H NMR and 62.5 MHz for 13C NMR, and chemical
shifts were calibrated to solvent peaks. Chemical shifts (d) were re-
corded 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 and were uncorrected.
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
4.2. General method for the preparation of 6a-d
Aryl aldehydes (3a-d) (1.0 equiv.) were reacted with
4
(1.0 equiv.) in the presence of KOH (1.2 equiv.) with methanol
and water at 0 °C for 3–4 h. After completion of reaction as moni-
tored by TLC, methanol was evaporated under reduced pressure.
The mixture was diluted with water, extracted with chloroform,
dried over MgSO4, filtered and concentrated to get yellow solid.
It was further purified by recrystallization in EtOAc and n-hexane
to afford 56.1–90.6% of 6a-d as light yellow solid.
4.2.1. 7-(Pyridin-2-ylmethylene)-6,7-dihydroquinolin-8(5H)-one (6a)
The same procedure described at Section 4.2 was employed
with 3a (0.09 mL, 1.0 mmol) and 4 (0.14 g, 1.0 mmol) to yield
0.16 g (0.67 mmol, 67.7%) of 6a as a yellow solid.

72
P. Thapa et al. / Bioorganic Chemistry 40 (2012) 67–78
Mp 138.1–138.8 °C; Rf (dichloromethane/methanol 20:1 v/v):
0.28.
1H NMR (250 MHz, CDCl3) d 8.78 (dd, J = 4.5, 1.0 Hz, 1H, dihy-
droquinolinone H-2), 8.71 (dd, J = 5.0, 1.2 Hz, 1H, pyridine H-6),
7.82 (s, 1H, @CHA), 7.74 (td, J = 7.7, 1.8 Hz, 1H, pyridine H-4),
7.69 (dd, J = 7.8, 0.8 Hz, 1H, dihydroquinolinone H-4), 7.47 (d,
J = 7.8 Hz, 1H, pyridine H-3), 7.41 (dd, J = 7.8, 4.5 Hz, 1H, dihydro-
quinolinone H-3), 7.24 (ddd, J = 7.6, 4.8, 1.0 Hz, 1H, pyridine H-5),
3.73 (td, J = 6.7, 1.6 Hz, 2H, 6-CH2), 3.05 (t, J = 6.7 Hz, 2H, 5-CH2).
4.3.1. 5-(Pyridin-2-ylmethylene)-6,7-dihydrobenzo[b]thiophen-
4(5H)-one (7a)
The same procedure described at Section 4.3 was employed
with 3a (0.28 mL, 3.00 mmol) and 5 (0.30 g, 2.00 mmol) to yield
0.34 g (1.43 mmol, 71.4%) of 7a as an off-white solid.
Mp 146.5–147.4 °C; Rf (ethyl acetate/n-hexane 3:1 v/v): 0.18.
1H NMR (250 MHz, CDCl3) d 8.70 (d, J = 3.4 Hz, 1H, pyridine H-
6), 7.73 (td, J = 7.6, 1.7 Hz, 1H, pyridine H-4), 7.66 (s, 1H, @CHA),
7.52 (d, J = 5.3 Hz, 1H, dihydrobenzothiophenone H-2), 7.43 (d,
J = 7.7 Hz, 1H, pyridine H-3), 7.22 (dd, J = 7.5, 4.8 Hz, 1H, pyridine
H-5), 7.12 (d, J = 5.2 Hz, 1H, dihydrobenzothiophenone H-3), 3.75
(td, J = 6.3, 1.5 Hz, 2H, 6-CH2), 3.13 (t, J = 6.3 Hz, 2H, 7-CH2).
4.2.2. 7-(Pyridin-3-ylmethylene)-6,7-dihydroquinolin-8(5H)-one (6b)
The same procedure described at Section 4.2 was employed
with 3b (0.09 mL, 1.0 mmol) and 4 (0.14 g, 1.0 mmol) to yield
0.19 g (0.82 mmol, 82.5%) of 6b as a light yellow solid.
Mp 135.8–136.5 °C; Rf (dichloromethane/methanol 20:1 v/v):
0.28.
4.3.2. 5-(Pyridin-3-ylmethylene)-6,7-dihydrobenzo[b]thiophen-
4(5H)-one (7b)
The same procedure described at Section 4.3 was employed
with 3b (0.37 mL, 4.00 mmol) and 5 (0.30 g, 2.00 mmol) to yield
0.19 g (0.81 mmol, 40.7%) of 7b as a white solid.
Mp 113.4–114.8 °C; Rf (ethyl acetate/n-hexane 1:1 v/v): 0.17.
1H NMR (250 MHz, CDCl3) d 8.67 (d, J = 0.8 Hz, 1H, pyridine H-
2), 8.57 (dd, J = 4.7, 1.2 Hz, 1H, pyridine H-6), 7.72 (s, 1H, @CHA),
7.70 (dt, J = 8.2, 1.6 Hz, 1H, pyridine H-4), 7.52 (d, J = 5.3 Hz, 1H,
dihydrobenzothiophenone H-2), 7.35 (dd, J = 7.9, 4.9 Hz, 1H, pyri-
dine H-5), 7.14 (d, J = 5.3 Hz, 1H, dihydrobenzothiophenone H-3),
3.21–3.07 (m, 4H, 6-CH2, 7-CH2).
1H NMR (250 MHz, CDCl3) d 8.79 (d, J = 3.5 Hz, 1H, dihydroquin-
oline H-2), 8.71 (s, 1H, pyridine H-2), 8.60 (d, J = 3.5 Hz, 1H,
pyridine H-6), 7.92 (s, 1H, @CHA), 7.75 (d, J = 7.8 Hz, 1H, pyridine
H-4), 7.68 (dd, J = 7.8, 0.7 Hz, 1H, dihydroquinoline H-4), 7.43
(dd, J = 7.8, 4.5 Hz, 1H, dihydroquinoline H-3), 7.38 (dd, J = 7.9,
4.8 Hz, 1H, pyridine H-5), 3.18 (t, J = 6.5 Hz, 2H, 6-CH2), 3.03
(t, J = 6.2 Hz, 2H, 5-CH2).
4.2.3. 7-(Pyridin-4-ylmethylene)-6,7-dihydroquinolin-8(5H)-one (6c)
The same procedure described at Section 4.2 was employed
with 3c (0.13 mL, 1.36 mmol) and 4 (0.20 g, 1.36 mmol) to yield
0.29 g (1.23 mmol, 90.6%) of 6c as a light yellow solid.
Mp 172.9–173.5 °C; Rf (dichloromethane/methanol 20:1 v/v):
0.33.
4.3.3. 5-(Pyridin-4-ylmethylene)-6,7-dihydrobenzo[b]thiophen-
4(5H)-one (7c)
The same procedure described at Section 4.3 was employed
with 3c (0.34 mL, 3.60 mmol) and 5 (0.30 g, 2.00 mmol) to yield
0.20 g (0.84 mmol, 42.2%) of 7c as a white solid.
Mp 142.5–143.0 °C; Rf (ethyl acetate/n-hexane 1:1 v/v): 0.17.
1H NMR (250 MHz, CDCl3) d 8.67 (dd, J = 4.5, 1.6 Hz, 2H, pyri-
dine H-2, H-6), 7.66 (s, 1H, @CHA), 7.51 (d, J = 5.3 Hz, 1H,
dihydrobenzothiophenone H-2), 7.27 (dd, J = 4.5, 1.0 Hz, 2H, pyri-
dine H-3, H-5), 7.14 (d, J = 5.3 Hz, 1H, dihydrobenzothiophenone
H-3), 3.18–3.09 (m, 4H, 6-CH2, 7-CH2).
1H NMR (250 MHz, CDCl3) d 8.78 (dd, J = 4.5, 1.5 Hz, 1H, dihy-
droquinolinone H-2), 8.69 (dd, J = 4.5, 1.6 Hz, 2H, pyridine H-2,
H-6), 7.84 (s, 1H, @CHA), 7.69 (dd, J = 7.8, 1.3 Hz, 1H, dihydroquin-
olinone H-4), 7.45 (dd, J = 7.8, 4.5 Hz, 1H, dihydroquinoline H-3),
7.29 (dd, J = 5.8, 1.5 Hz, 2H, pyridine H-3, H-5), 3.73 (td, J = 7.0,
1.7 Hz, 2H, 6-CH2), 3.05 (t, J = 6.8 Hz, 2H, 5-CH2).
4.2.4. 7-Benzylidene-6,7-dihydroquinolin-8(5H)-one (6d)
The same procedure described at Section 4.2 was employed
with 3d (0.20 mL, 2.00 mmol) and 4 (0.29 g, 2.00 mmol) to yield
0.26 g (1.12 mmol, 56.1%) of 6d as a light yellow solid.
Mp 125.3–125.9 °C; Rf (ethyl acetate/hexane 3:1 v/v): 0.18.
1H NMR (250 MHz, CDCl3) d 8.77 (dd, J = 4.3, 1.2 Hz, 1H, dihy-
droquinoline H-2), 7.98 (s, 1H, @CHA), 7.65 (d, J = 7.7 Hz, 1H, dihy-
droquinolinone H-4), 7.44–7.36 (m, 5H, Phenyl H-2, H-3, H-4, H-5,
H-6), 7.41 (dd, J = 7.8, 4.4 Hz, 1H, dihydroquinolinone H-3), 3.18
(td, J = 6.7, 1.5 Hz, 2H, 6-CH2), 3.05 (t, J = 6.7 Hz, 2H, 5-CH2).
4.3.4. 5-Benzylidene-6,7-dihydrobenzo[b]thiophen-4(5H)-one (7d)
The same procedure described at Section 4.3 was employed
with 3d (0.36 mL, 3.60 mmol) and 5 (0.30 g, 2.00 mmol) to yield
0.33 g (1.37 mmol, 68.5%) of 7d as a light yellow solid.
Mp 105.7–106.0 °C; Rf (ethyl acetate/n-hexane 1:5 v/v): 0.36.
1H NMR (250 MHz, CDCl3) d 7.77 (s, 1H, @CHA), 7.49 (d,
J = 5.3 Hz, 1H, dihydrobenzothiophenone H-2), 7.40–7.30 (m, 5H,
phenyl H-2, H-3, H-4, H-5, H-6), 7.09 (d, J = 5.3 Hz, 1H,
dihydrobenzothiophenone H-3), 3.20 (td, J = 5.9, 1.5 Hz, 2H, 6-
CH2) 3.05 (t, J = 6.1 Hz, 2H, 7-CH2).
4.3. General method for the preparation of 7a-d
4.4. General method for the preparation of 8 (R1, R2 = a-d) (10–25)
Compounds 7a-d were synthesized by condensation of com-
pound 5 with aryl aldehydes (3a-d) either in the presence of 40%
aqueous KOH in ethanol (method A) or AcOH/piperidine as a cata-
lyst (method B) [11] as shown in Scheme 1.
Compound 7a was synthesized by following method A where
aryl aldehyde 3a was treated with 5 in the presence of 40% aqueous
KOH in ethanol at 0 °C for 3 h, and precipitation was observed. It
was filtered and washed with H2O and cold methanol to yield
71.4% of 7a as an off-white solid.
In method B, aryl aldehydes (3b-d) and 5 were reacted in the
presence of catalytic amount of AcOH and piperidine at 90 °C for
5–12 h. The reaction mixture was diluted with H2O, and extracted
with CHCl3, dried over MgSO4, filtered, and concentrated in re-
duced pressure to yield brown viscous solid. Flash chromatography
with ethyl acetate and n-hexane afford 40.7–68.5% of 7b-d as
white or light yellow solid.
A mixture of 6a-d (1.0 equiv.), 2a-d (1.0 equiv.) and NH4OAc
(10.0 equiv.) in dry methanol was refluxed at 80–100 °C for 12–
18 h under nitrogen gas. Thereafter, MeOH was evaporated under
reduced pressure. The residue was diluted with H2O (30 mL) and
extracted with CHCl3 (15 mL ꢀ 3). The organic layer was collected,
dried over MgSO4 (1 spatula), and filtered. Filtrate was evaporated
at reduced pressure, dried and purified by alumina chromatogra-
phy with a gradient elution of ethyl acetate and n-hexane to yield
26.2–92.4% of compounds 10–25.
4.4.1. 2,4-Di(pyridin-2-yl)-5,6-dihydro-1,10-phenanthroline (10)
Procedure described at Section 4.4 was employed with 6a
(0.10 g, 0.42 mmol), dry ammonium acetate (0.32 g, 4.20 mmol),
2a (0.13 g, 0.42 mmol), and dry methanol (2.5 mL) at 100 °C for
12 h to yield 60 mg (0.19 mmol, 42.6%) of 10 as a light yellow solid.

P. Thapa et al. / Bioorganic Chemistry 40 (2012) 67–78
73
Mp 158.1–159.2 °C; Rf (Al2O3) (ethyl acetate/n-hexane 2:1 v/v):
0.11; purity (condition A): 97.2%.
2d (0.13 g, 0.40 mmol), and dry methanol (2.5 mL) at 100 °C for
12 h to yield 124 mg (0.37 mmol, 92.4%) of 13 as a white solid.
Mp 224.8–225.9 °C; Rf (dichloromethane/methanol 15:1 v/v):
0.34; purity (condition A): 100%.
LC/MS/MS: retention time: 5.99 min; [MH+]: 337.3 (100%).
1H NMR (250 MHz, CDCl3) d 8.80 (dd, J = 4.8, 1.5 Hz, 1H, phe-
nanthroline H-9), 8.76 (d, J = 8.0 Hz, 1H, 2-pyridine H-3), 8.75
(ddd, J = 4.1, 1.7, 0.9 Hz, 1H, 4-pyridine H-6), 8.66 (ddd, J = 4.7,
1.6, 0.8 Hz, 1H, 2-pyridine H-6), 8.54 (s, 1H, phenanthroline H-3),
7.85 (td, J = 7.8, 1.7 Hz, 2H, 2-pyridine H-4, 4-pyridine H-4), 7.60
(d, J = 7.9 Hz, 2H, 4-pyridine H-3, phenanthroline H-7), 7.37 (ddd,
J = 7.5, 4.9, 1.0 Hz, 1H, 2-pyridine H-5), 7.32 (ddd, J = 8.0, 4.7,
1.2 Hz, 1H, 4-pyridine H-5), 7.28 (dd, J = 7.5, 4.8 Hz, 1H, phenan-
throline H-8), 3.14 (t, J = 7.9 Hz, 2H, 5-CH2), 2.95 (t, J = 7.8 Hz, 2H,
6-CH2).
LC/MS/MS: retention time: 7.03 min; [MH+]: 336.3 (100%).
1H NMR (250 MHz, CDCl3) d 8.80–8.78 (m, 2H, phenanthroline
H-9, 4-pyridine H-6), 8.20 (d, J = 8.3 Hz, 2H, 2-phenyl H-2, H-6),
7.86 (s, 1H, phenanthroline H-3), 7.85 (td, J = 7.8, 1.8 Hz, 1H, 4-pyr-
idine H-4), 7.58 (dd, J = 7.5, 0.9 Hz, 1H, phenanthroline H-7), 7.51
(d, J = 7.8 Hz, 1H, 4-pyridine H-3), 7.47–7.36 (m, 4H, 2-phenyl H-
3, H-4, H-5, 4-pyridine H-5), 7.26 (dd, J = 7.5, 4.8 Hz, 1H, phenan-
throline H-8), 3.09–2.95 (m, 2H, 5-CH2), 2.93–2.90 (m, 2H, 6-CH2).
13C NMR (62.5 MHz, CDCl3) d 157.12, 156.23, 152.39, 152.17,
149.69, 149.09, 147.66, 139.23, 136.56, 135.43, 133.96, 130.18,
128.74, 128.49, 127.27, 124.12, 123.46, 122.90, 121.16, 27.52,
24.53.
13C NMR (62.5 MHz, CDCl3) d 157.25, 155.86, 154.82, 152.09,
152.04, 149.42, 149.03, 148.82, 147.93, 136.84, 136.59, 135.54,
134.09, 132.15, 124.22, 123.70, 123.56, 122.82, 121.96, 121.65,
27.45, 24.61.
4.4.5. 2-(Pyridin-2-yl)-4-(pyridin-3-yl)-5,6-dihydro-1,10-
phenanthroline (14)
4.4.2. 4-(Pyridin-2-yl)-2-(pyridin-3-yl)-5, 6-dihydro-1, 10-
phenanthroline (11)
Procedure described at Section 4.4 was employed with 6b
(0.08 g, 0.34 mmol), dry ammonium acetate (0.26 g, 3.40 mmol),
2a (0.11 g, 0.34 mmol), and dry methanol (2.0 mL) at 100 °C for
12 h to yield 52 mg (0.15 mmol, 45.4%) of 14 as a white solid.
Mp 213.4–214.2 °C; Rf (Al2O3) (ethyl acetate/n-hexane 2:1 v/v):
0.13; purity (condition A): 96.3%.
Procedure described at Section 4.4 was employed with 6a
(0.09 g, 0.40 mmol), dry ammonium acetate (0.31 g, 4.00 mmol),
2b (0.13 g, 0.40 mmol), and dry methanol (2.5 mL) at 100 °C for
12 h to yield 92 mg (0.27 mmol, 68.3%) of 11 as a white solid.
Mp 201.2–201.9 °C; Rf (dichloromethane/methanol 15:1 v/v):
0.22; purity (condition A): 100%.
LC/MS/MS: retention time: 5.65 min; [MH+]: 337.3 (100%).
1H NMR (250 MHz, CDCl3) d 8.80 (d, J = 5.4 Hz, 1H, phenanthro-
line H-9), 8.79 (d, J = 1.1 Hz, 1H, 4-pyridine H-2), 8.74 (d, J = 7.8 Hz,
1H, 2-pyridine H-3), 8.71 (dd, J = 4.9, 1.5 Hz, 1H, 4-pyridine H-6),
8.66 (d, J = 4.1 Hz, 1H, 2-pyridine H-6), 8.41 (s, 1H, phenanthroline
H-3), 7.87 (td, J = 7.8, 1.7 Hz, 1H, 2-pyridine H-4), 7.81 (dt, J = 8.1,
1.8 Hz, 1H, 4-pyridine H-4), 7.60 (d, J = 7.4 Hz, 1H, phenanthroline
H-7), 7.45 (dd, J = 7.4, 4.9 Hz, 1H, 4-pyridine H-5), 7.33 (d,
J = 5.0 Hz, 1H, 2-pyridine H-5), 7.28 (dd, J = 7.5, 4.8 Hz, 1H, phenan-
throline H-8), 3.05–2.95 (m, 4H, 5-CH2, 6-CH2).
LC/MS/MS: retention time: 5.57 min; [MH+]: 337.3 (100%).
1H NMR (250 MHz, CDCl3) d 9.28 (d, J = 1.8 Hz, 1H, 2-pyridine H-
2), 8.81–8.77 (m, 2H, phenanthroline H-9, 4-pyridine H-6), 8.65
(dd, J = 4.7, 1.5 Hz, 1H, 2-pyridine H-6), 8.57 (dt, J = 8.0, 2.0 Hz,
1H, 2-pyridine H-4), 7.88 (s, 1H, phenanthroline H-3), 7.87 (td,
J = 7.8, 1.7 Hz, 1H, 4-pyridine H-4), 7.60 (d, J = 7.5 Hz, 1H, phenan-
throline H-7), 7.52 (d, J = 7.8 Hz, 1H, 4-pyridine H-3), 7.44–7.38 (m,
2H, 2-pyridine H-5, 4-pyridine H-5), 7.29 (dd, J = 7.6, 4.8 Hz, 1H,
phenanthroline H-8), 3.11 (t, J = 7.8 Hz, 2H, 5-CH2), 2.95 (t,
J = 7.6 Hz, 2H, 6-CH2).
13C NMR (62.5 MHz, CDCl3) d 155.65, 154.94, 152.02, 151.91,
149.48, 149.43, 149.17, 148.90, 146.10, 136.95, 136.28, 135.63,
134.48, 133.86, 131.74, 123.89, 123.72, 123.29, 122.05, 121.85,
27.44, 24.88.
13C NMR (62.5 MHz, CDCl3) d 156.62, 153.66, 152.91, 151.81,
149.82, 149.75, 149.13, 148.29, 147.97, 136.64, 135.56, 135.01,
134.93, 134.03, 131.00, 124.11, 123.73, 123.49, 123.09, 121.26,
27.38, 24.59.
4.4.6. 2,4-Di(Pyridin-3-yl)-5,6-dihydro-1,10-phenanthroline (15)
Procedure described at Section 4.4 was employed with 6b
(0.07 g, 0.30 mmol), dry ammonium acetate (0.23 g, 3.00 mmol),
2b (0.10 g, 0.30 mmol), and dry methanol (2.0 mL) at 100 °C for
12 h to yield 58 mg (0.17 mmol, 57.5%) of 15 as a white solid.
Mp 217.5–218.2 °C; Rf (dichloromethane/methanol 15:1 v/v):
0.25; purity (condition A): 98.9%.
4.4.3. 4-(Pyridin-2-yl)-2-(pyridin-4-yl)-5,6-dihydro-1,10-
phenanthroline (12)
Procedure described at Section 4.4 was employed with 6a
(0.08 g, 0.34 mmol), dry ammonium acetate (0.26 g, 3.40 mmol),
2c (0.11 g, 0.34 mmol) and dry methanol (2.0 mL) at 100 °C for
12 h to yield 72 mg (0.21 mmol, 63.4%) of 12 as a white solid.
Mp 225.5–226.8 °C; Rf (dichloromethane/methanol 15:1 v/v):
0.22; purity (condition A): 100%.
LC/MS/MS: retention time: 5.29 min; [MH+]: 337.3 (100%).
1H NMR (250 MHz, CDCl3) d 9.27 (d, J = 1.2 Hz, 1H, 2-pyridine H-
2), 8.80 (d, J = 3.5 Hz, 1H, phenanthroline H-9), 8.75 (dd, J = 5.8,
1.3 Hz, 1H, 2-pyridine H-6), 8.73 (br, 1H, 4-pyridine H-2), 8.67
(dd, J = 4.5, 1.1 Hz, 1H, 4-pyridine H-6), 8.55 (dt, J = 7.8, 1.6 Hz,
1H, 2-pyridine H-4), 7.79 (dt, J = 7.7, 1.8 Hz, 1H, 4-pyridine H-4),
7.69 (s, 1H, phenanthroline H-3), 7.61 (d, J = 6.9 Hz, 1H, phenan-
throline H-7), 7.49 (dd, J = 7.6, 4.8 Hz, 1H, 2-pyridine H-5), 7.44
(dd, J = 7.8, 4.8 Hz, 1H, 4-pyridine H-5), 7.30 (dd, J = 7.5, 4.8 Hz,
1H, phenanthroline H-8), 3.03–2.93 (m, 4H, 5-CH2, 6-CH2).
13C NMR (62.5 MHz, CDCl3) d 153.82, 152.90, 151.67, 149.95,
149.69, 149.36, 149.26, 148.25, 146.14, 136.14, 135.67, 135.05,
134.73, 134.22, 133.87, 130.87, 123.91, 123.59, 123.43, 121.47,
27.36, 24.74.
LC/MS/MS: retention time: 5.55 min; [MH+]: 337.3 (100%).
1H NMR (250 MHz, CDCl3) d 8.80 (br, 2H, phenanthroline H-9, 4-
pyridine H-6), 8.72 (d, J = 5.9 Hz, 2H, 2-pyridine H-2, H-6), 8.10 (d,
J = 5.9 Hz, 2H, 2-pyridine H-3, H-5), 7.93 (s, 1H, phenanthroline H-
3), 7.88 (td, J = 7.6, 1.5 Hz, 1H, 4-pyridine H-4), 7.60 (d, J = 7.2 Hz,
1H, phenanthroline H-7), 7.52 (d, J = 7.7 Hz, 1H, 4-pyridine H-3),
7.42 (dd, J = 7.4, 4.9 Hz, 1H, 4-pyridine H-5), 7.30 (dd, J = 7.5,
4.8 Hz, 1H, phenanthroline H-8), 3.13 (d, J = 7.7 Hz, 2H, 5-CH2),
2.94 (t, J = 7.6 Hz, 2H, 6-CH2).
13C NMR (62.5 MHz, CDCl3) d 156.47, 153.41, 152.96, 151.71,
150.26, 149.87, 149.20, 148.04, 146.25, 136.64, 135.59, 134.05,
132.01, 124.13, 123.83, 123.16, 121.46, 121.42, 27.33, 24.71.
4.4.4. 2-Phenyl-4-(pyridin-2-yl)-5,6-dihydro-1,10-phenanthroline
(13)
4.4.7. 4-(Pyridin-3-yl)-2-(pyridin-4-yl)-5,6-dihydro-1,10-
phenanthroline (16)
Procedure described at Section 4.4 was employed with 6a
(0.09 g, 0.40 mmol), dry ammonium acetate (0.31 g, 4.00 mmol),
Procedure described at Section 4.4 was employed with 6b
(0.12 g, 0.50 mmol), dry ammonium acetate (0.38 g, 5.00 mmol),

74
P. Thapa et al. / Bioorganic Chemistry 40 (2012) 67–78
2c (0.16 g, 0.50 mmol), and dry methanol (2.5 mL) at 100 °C for
12 h to yield 58 mg (0.17 mmol, 34.5%) of 16 as a white solid.
Mp 284.2–284.8 °C; Rf (dichloromethane/methanol 15:1 v/v):
0.22; purity (condition A): 99.4%.
LC/MS/MS: retention time: 5.20 min; [MH+]: 337.3 (100%).
1H NMR (250 MHz, CDCl3) d 9.26 (d, J = 1.7 Hz, 1H, 2-pyridine H-
2), 8.80 (d, J = 4.3 Hz, 1H, phenanthroline H-9), 8.78 (dd, J = 4.4,
1.5 Hz, 2H, 4-pyridine H-2, H-6), 8.67 (dd, J = 4.8, 1.6 Hz, 1H, 2-pyr-
idine H-6), 8.55 (dt, J = 8.0, 2.0 Hz, 1H, 2-pyridine H-4), 7.66 (s, 1H,
phenanthroline H-3), 7.61 (dd, J = 7.6, 1.4 Hz, 1H, phenanthroline
H-7), 7.44 (dd, J = 7.9, 4.8 Hz, 1H, 2-pyridine H-5), 7.37 (dd,
J = 4.4, 1.5 Hz, 2H, 4-pyridine H-3, H-5), 7.33 (dd, J = 7.6, 4.8 Hz,
1H, phenanthroline H-8), 2.96 (s, 4H, 5-CH2, 6-CH2).
LC/MS/MS: retention time: 5.26 min; [MH+]: 337.3 (100%).
1H NMR (250 MHz, CDCl3) d 8.80 (dd, J = 4.6, 1.0 Hz, 1H, phe-
nanthroline H-9), 8.74–8.72 (m, 4H, 2-pyridine H-2, H-6, 4-pyri-
dine H-2, H-6), 8.06 (dd, J = 4.5, 1.2 Hz, 2H, 2-pyridine H-3, H-5),
7.76 (dt, J = 7.8, 1.7 Hz, 1H, 4-pyridine H-4), 7.72 (s, 1H, phenan-
throline H-3), 7.61 (d, J = 7.5 Hz, 1H, phenanthroline H-7), 7.48
(dd, J = 7.7, 4.8 Hz, 1H, 4-pyridine H-5), 7.31 (dd, J = 7.4, 4.7 Hz,
1H, phenanthroline H-8), 3.02–2.92 (m, 4H, 5-CH2, 6-CH2).
13C NMR (62.5 MHz, CDCl3) d 153.57, 152.93, 151.51, 150.34,
149.74, 149.31, 149.28, 146.23, 146.06, 136.16, 135.75, 134.13,
133.91, 131.92, 124.05, 123.46, 121.67, 121.41, 27.28, 24.83.
13C NMR (62.5 MHz, CDCl3) d 153.87, 152.90, 151.51, 150.17,
149.97, 149.24, 148.19, 147.00, 146.29, 135.73, 135.06, 134.65,
133.89, 130.30, 124.00, 123.62, 123.50, 120.77, 27.29, 24.66.
4.4.11. 2,4-Di(pyridin-4-yl)-5,6-dihydro-1,10-phenanthroline (20)
Procedure described at Section 4.4 was employed with 6c
(0.08 g, 0.35 mmol), dry ammonium acetate (0.27 g, 3.50 mmol),
2c (0.11 g, 0.35 mmol), and dry methanol (2.5 mL) at 95 °C for
14 h to yield 66 mg (0.19 mmol, 56.1%) of 20 as a white solid.
Mp 284.1–284.6 °C; Rf (dichloromethane/methanol 15:1 v/v):
0.20; purity (condition A): 98.9%.
4.4.8. 2-Phenyl-4-(pyridin-3-yl)-5,6-dihydro-1,10-phenanthroline
(17)
Procedure described at Section 4.4 was employed with 6b
(0.12 g, 0.50 mmol), dry ammonium acetate (0.38 g, 5.00 mmol),
2d (0.16 g, 0.50 mmol), and dry methanol (2.5 mL) at 100 °C for
12 h to yield 127 mg (0.38 mmol, 76.0%) of 17 as a white solid.
Mp 221.2–221.9 °C; Rf (dichloromethane/methanol 15:1 v/v):
0.30; purity (condition A): 100%.
LC/MS/MS: retention time: 5.21 min; [MH+]: 337.3 (100%).
1H NMR (250 MHz, CDCl3) d 8.80–8.78 (m, 3H, phenanthroline
H-9, 4-pyridine H-2, H-6), 8.73 (dd, J = 4.7, 1.4 Hz, 2H, 2-pyridine
H-2, H-6), 8.07 (dd, J = 4.6, 1.4 Hz, 2H, 2-pyridine H-3, H-5), 7.70
(s, 1H, phenanthroline H-3), 7.62 (d, J = 6.5 Hz, 1H, phenanthroline
H-7), 7.36 (dd, J = 4.5, 1.5 Hz, 2H, 4-pyridine H-3, H-5), 7.32 (dd,
J = 7.6, 4.7 Hz, 1H, phenanthroline H-8), 2.97 (s, 4H, 5-CH2, 6-CH2).
13C NMR (62.5 MHz, CDCl3) d 153.72, 153.02, 151.47, 150.38,
150.23, 149.34, 147.11, 146.23, 145.98, 135.69, 133.90, 131.29,
124.06, 123.44, 121.38, 120.93, 27.27, 24.80.
LC/MS/MS: retention time: 6.72 min; [MH+]: 336.3 (100%).
1H NMR (250 MHz, CDCl3) d 8.80 (dd, J = 4.7, 1.3 Hz, 1H, phe-
nanthroline H-9), 8.74–8.71 (m, 2H, 4-pyridine H-2, H-6), 8.17
(dd, J = 8.2, 1.4 Hz, 2H, 2-phenyl H-2, H-6), 7.77 (dt, J = 7.8,
1.7 Hz, 1H, 4-pyridine H-4), 7.67 (s, 1H, phenanthroline H-3),
7.59 (dd, J = 7.5, 0.9 Hz, 1H, phenanthroline H-7), 7.52–7.39 (m,
4H, 4-pyridine H-5, 2-phenyl H-3, H-4, H-5), 7.27 (dd, J = 7.6,
4.8 Hz, 1H, phenanthroline H-8), 3.00–2.90 (m, 4H, 5-CH2, 6-CH2).
13C NMR (62.5 MHz, CDCl3) d 156.36, 152.35, 151.98, 149.45,
149.40, 149.16, 145.76, 138.98, 136.19, 135.56, 134.59, 133.79,
130.01, 128.97, 128.63, 127.24, 123.66, 123.35, 121.41, 27.47,
24.68.
4.4.12. 2-Phenyl-4-(pyridin-4-yl)-5,6-dihydro-1,10-phenanthroline
(21)
Procedure described at Section 4.4 was employed with 6c
(0.08 g, 0.35 mmol), dry ammonium acetate (0.27 g, 3.50 mmol),
2d (0.11 g, 0.35 mmol), and dry methanol (2.5 mL) at 95 °C for
14 h to yield 70 mg (0.21 mmol, 60.2%) of 21 as a white solid.
Mp 173.5–174.5 °C; Rf (dichloromethane/methanol 15:1 v/v):
0.35; purity (condition A): 99.5%.
4.4.9. 2-(Pyridin-2-yl)-4-(pyridin-4-yl)-5,6-dihydro-1,10-
phenanthroline (18)
Procedure described at Section 4.4 was employed with 6c
(0.07 g, 0.30 mmol), dry ammonium acetate (0.23 g, 3.00 mmol),
2a (0.10 g, 0.30 mmol), and dry methanol (2.5 mL) at 100 °C for
12 h to yield 41 mg (0.12 mmol, 40.6%) of 18 as a white solid.
Mp 210.2–210.8 °C; Rf (Al2O3) (ethyl acetate/n-hexane 2:1 v/v):
0.17; purity (condition A): 97.7%.
LC/MS/MS: retention time: 6.67 min; [MH+]: 336.3 (100%).
1H NMR (250 MHz, CDCl3) d 8.80 (dd, J = 4.9, 1.5 Hz, 1H,
phenanthroline H-9), 8.77 (dd, J = 4.4, 1.5 Hz, 2H, 4-pyridine H-2,
H-6), 8.16 (dd, J = 7.8, 1.1 Hz, 2H, 2-phenyl H-2, H-6), 7.64 (s, 1H,
phenanthroline H-3), 7.60 (dd, J = 7.5, 1.3 Hz, 1H, phenanthroline
H-7), 7.52–7.42 (m, 3H, 2-phenyl H-3, H-4, H-5), 7.36 (dd, J = 4.4,
1.3 Hz, 2H, 4-pyridine H-3, H-5), 7.29 (dd, J = 7.6, 4.8 Hz, 1H,
phenanthroline H-8), 2.94 (s, 4H, 5-CH2, 6-CH2).
LC/MS/MS: retention time: 5.62 min; [MH+]: 337.3 (100%).
1H NMR (250 MHz, CDCl3) d 8.80 (dd, J = 4.7, 1.5 Hz, 1H, phe-
nanthroline H-9), 8.77 (d, J = 8.5 Hz, 1H, 2-pyridine H-3), 8.75
(dd, J = 4.4, 1.5 Hz, 2H, 4-pyridine H-2, H-6), 8.65 (d, J = 4.7 Hz,
1H, 2-pyridine H-6), 8.37 (s, 1H, phenanthroline H-3), 7.88 (td,
J = 7.7, 1.7 Hz, 1H, 2-pyridine H-4), 7.61 (dd, J = 7.5, 1.3 Hz, 1H, phe-
nanthroline H-7), 7.37 (dd, J = 4.4, 1.6 Hz, 2H, 4-pyridine H-3, H-5),
7.35 (ddd, J = 7.8, 4.9, 1.0 Hz, 1H, 2-pyridine H-5), 7.32 (dd, J = 7.6,
4.8 Hz, 1H, phenanthroline H-8), 3.03–2.92 (s, 4H, 5-CH2, 6-CH2).
13C NMR (62.5 MHz, CDCl3) d 155.52, 154.99, 151.99, 151.72,
149.97, 149.12, 148.92, 146.92, 146.58, 137.00, 135.69, 133.87,
131.16, 123.95, 123.81, 123.64, 122.00, 121.19, 27.35, 24.79.
13C NMR (62.5 MHz, CDCl3) d 156.50, 152.43, 151.94, 150.09,
149.20, 146.77, 146.68, 138.96, 135.52, 133.79, 129.39, 129.02,
128.64, 127.25, 123.68, 123.56, 120.66, 27.46, 24.65.
4.4.13. 4-Phenyl-2-(pyridin-2-yl)-5,6-dihydro-1,10-phenanthroline
(22)
Procedure described at Section 4.4 was employed with 6d
(0.09 g, 0.40 mmol), dry ammonium acetate (0.30 g, 4.00 mmol),
2a (0.13 g, 0.40 mmol), and dry methanol (2.5 mL) at 100 °C for
16 h to yield 88 mg (0.26 mmol, 65.6%) of 22 as a white solid.
Mp 179.2–179.8 °C; Rf (Al2O3) (ethyl acetate/n-hexane 2:1 v/v):
0.28; purity (condition A): 95.5%.
4.4.10. 2-(Pyridin-3-yl)-4-(pyridin-4-yl)-5,6-dihydro-1,10-
phenanthroline (19)
LC/MS/MS: retention time: 7.58 min; [MH+]: 336.3 (100%).
1H NMR (250 MHz, CDCl3) d 8.80 (dd, J = 4.8, 1.3 Hz, 1H, phe-
nanthroline H-9), 8.75 (d, J = 8.0 Hz, 1H, 2-pyridine H-3), 8.65
(ddd, J = 4.8, 1.6, 0.8 Hz, 1H, 2-pyridine H-6), 8.40 (s, 1H, phenan-
throline H-3), 7.85 (td, J = 7.5, 1.7 Hz, 1H, 2-pyridine H-4), 7.56
(dd, J = 7.5, 0.9 Hz, 1H, phenanthroline H-7), 7.51–7.40 (m, 5H, 4-
phenyl H-2, H-3, H-4, H-5, H-6), 7.31 (ddd, J = 7.4, 4.8, 1.1 Hz, 1H,
Procedure described at Section 4.4 was employed with 6c
(0.07 g, 0.30 mmol), dry ammonium acetate (0.23 g, 3.00 mmol),
2b (0.10 g, 0.30 mmol), and dry methanol (2.5 mL) at 80 °C for
12 h to yield 47 mg (0.14 mmol, 46.6%) of 19 as a yellow solid.
Mp 278.7–279.3 °C; Rf (dichloromethane/methanol 15:1 v/v):
0.29; purity (condition A): 95.3%.

P. Thapa et al. / Bioorganic Chemistry 40 (2012) 67–78
75
2-pyridine H-5), 7.26 (dd, J = 7.3, 5.1 Hz, 1H, phenanthroline H-8),
4.5. General method for the preparation of 9 (R1, R2 = a-d) (26–41)
3.05–2.88 (m, 4H, 5-CH2, 6-CH2).
13C NMR (62.5 MHz, CDCl3) d 156.05, 154.58, 152.23, 151.71,
149.77, 149.01, 148.85, 138.69, 136.85, 135.53, 133.98, 131.76,
128.85, 128.36, 128.10, 123.64, 123.50, 121.99, 27.57, 24.96.
A mixture of 7a-d (1.0 equiv.), 2a-d (1.0 equiv.) and NH4OAc
(10.0 equiv.) in glacial acetic acid or dry MeOH was refluxed at
90–100 °C for 12–48 h under nitrogen gas. Thereafter, mixture
was extracted with EtOAc, washed with H2O, and saturated NaCl.
The organic layer was collected, dried over MgSO4 and filtered.
The filtrate was concentrated at reduced pressure, dried in vacuum
pump and purified by silica gel chromatography with a gradient
elution of ethyl acetate and n-hexane to yield 11.9–51.6% of 26–41.
4.4.14. 4-Phenyl-2-(pyridin-3-yl)-5,6-dihydro-1,10-phenanthroline
(23)
Procedure described at Section 4.4 was employed with 6d
(0.09 g, 0.40 mmol), dry ammonium acetate (0.30 g, 4.00 mmol),
2b (0.13 g, 0.40 mmol), and dry methanol (2.5 mL) at 100 °C for
16 h to yield 81 mg (0.24 mmol, 60.3%) of 23 as a white solid.
Mp 202.1–202.8 °C; Rf (dichloromethane/methanol 15:1 v/v):
0.20; purity (condition A): 99.3%.
4.5.1. 2,4-Di(pyridin-2-yl)-5,6-dihydrothieno[2,3-h]quinoline (26)
Procedure described at Section 4.5 was employed with 7a
(0.10 g, 0.41 mmol), dry ammonium acetate (0.32 g, 4.10 mmol),
2a (0.13 g, 0.41 mmol), and glacial acetic acid (1.5 mL) at 100 °C
for 20 h to yield 43 mg (0.12 mmol, 31.0%) of 26 as an off-white
solid.
LC/MS/MS: retention time: 7.08 min; [MH+]: 336.3 (100%).
1H NMR (250 MHz, CDCl3) d 9.27 (d, J = 1.7 Hz, 1H, 2-pyridine H-
2), 8.77 (dd, J = 4.6, 1.3 Hz, 1H, phenanthroline H-9), 8.65 (dd,
J = 4.7, 1.5 Hz, 1H, 2-pyridine H-6), 8.54 (dt, J = 7.9, 1.9 Hz, 1H, 2-
pyridine H-4), 7.69 (s, 1H, phenanthroline H-3), 7.57 (d,
J = 7.5 Hz, 1H, phenanthroline H-7), 7.54–7.40 (m, 5H, 4-phenyl
H-2, H-3, H-4, H-5, H-6), 7.41 (dd, J = 7.7, 4.3 Hz, 1H, 2-pyridine
H-5), 7.27 (dd, J = 7.5, 4.8 Hz, 1H, phenanthroline H-8), 3.05–2.87
(m, 4H, 5-CH2, 6-CH2).
Mp 128.8–129.3 °C; Rf (ethyl acetate/n-hexane 1:2 v/v): 0.17;
purity (condition B): 96.0%.
LC/MS/MS: retention time: 8.77 min; [MH+]: 342.2 (100%).
1H NMR (250 MHz, CDCl3) d 8.75 (d, J = 4.1 Hz, 1H, 2-pyridine H-
6), 8.65 (d, J = 3.9 Hz, 1H, 4-pyridine H-6), 8.61 (d, J = 8.0 Hz, 1H, 2-
pyridine H-3), 8.33 (s, 1H, thienoquinoline H-3), 7.87 (d, J = 5.0 Hz,
1H, thienoquinoline H-8), 7.84 (td, J = 7.7, 1.8 Hz, 1H, 2-pyridine H-
4), 7.82 (td, J = 7.7, 1.8 Hz, 1H, 4-pyridine H-4), 7.56 (d, J = 7.8 Hz,
1H, 4-pyridine H-3), 7.35 (ddd, J = 7.5, 4.9, 1.0 Hz, 1H, 2-pyridine
H-5), 7.31 (ddd, J = 7.6, 4.9, 1.1 Hz, 1H, 4-pyridine H-5), 7.24 (d,
J = 5.1 Hz, 1H, thienoquinoline H-9), 3.24 (t, J = 8.0 Hz, 2H, 5-CH2),
3.03 (t, J = 8.0 Hz, 2H, 6-CH2).
13C NMR (62.5 MHz, CDCl3) d 153.33, 152.51, 151.91, 149.69,
149.62, 149.01, 148.20, 138.34, 135.48, 134.97, 134.92, 133.89,
130.80, 128.62, 128.52, 128.30, 123.59, 123.43, 121.54, 27.41,
24.73.
4.4.15. 4-Phenyl-2-(pyridin-4-yl)-5,6-dihydro-1,10-phenanthroline
(24)
13C NMR (62.5 MHz, CDCl3) d 157.80, 156.23, 153.23, 151.29,
149.33, 148.95, 147.53, 141.09, 137.48, 136.79, 136.53, 127.37,
124.94, 124.17, 123.46, 122.71, 122.66, 121.00, 119.34, 25.94,
23.08.
Procedure described at Section 4.4 was employed with 6d
(0.07 g, 0.30 mmol), dry ammonium acetate (0.23 g, 3.00 mmol),
2c (0.10 g, 0.30 mmol), and dry methanol (2.5 mL) at 100 °C for
16 h to yield 26 mg (0.08 mmol, 26.2%) of 24 as a white solid.
Mp 215.8–216.3 °C; Rf (dichloromethane/methanol 15:1 v/v):
0.19; purity (condition A): 99.8%.
4.5.2. 4-(Pyridin-2-yl)-2-(pyridin-3-yl)-5,6-dihydrothieno[2,3-h]
quinoline (27)
LC/MS/MS: retention time: 7.10 min; [MH+]: 336.3 (100%).
1H NMR (250 MHz, CDCl3) d 8.80 (dd, J = 4.7, 1.5 Hz, 1H, phe-
nanthroline H-9), 8.72 (dd, J = 4.6, 1.6 Hz, 2H, 2-pyridine H-2, H-
6), 8.05 (dd, J = 4.6, 1.6 Hz, 2H, 2-pyridine H-3, H-5), 7.74 (s, 1H,
phenanthroline H-3), 7.60 (dd, J = 7.6, 1.4 Hz, 1H, phenanthroline
H-7), 7.53–7.48 (m, 3H, 4-phenyl H-3, H-4, H-5), 7.42 (dd, J = 7.8,
1.6 Hz, 2H, 4-phenyl H-2, H-6), 7.29 (dd, J = 7.6, 4.8 Hz, 1H, phe-
nanthroline H-8), 3.04–2.89 (m, 4H, 5-CH2, 6-CH2).
Procedure described at Section 4.5 was employed with 7a
(0.06 g, 0.25 mmol), dry ammonium acetate (0.19 g, 2.50 mmol),
2b (0.08 g, 0.25 mmol), and glacial acetic acid (0.5 mL) at 100 °C
for 20 h to yield 43 mg (0.13 mmol, 51.6%) of 27 as a light yellow
solid.
Mp 155.2–156.0 °C; Rf (ethyl acetate/n-hexane 2:1 v/v): 0.13;
purity (condition B): 96.0%.
LC/MS/MS: retention time: 7.80 min; [MH+]: 342.2 (100%).
1H NMR (250 MHz, CDCl3) d 9.33 (d, J = 1.6 Hz, 1H, 2-pyridine H-
2), 8.79 (d, J = 4.1 Hz, 1H, 4-pyridine H-6), 8.64 (dd, J = 4.7, 1.5 Hz,
1H, 2-pyridine H-6), 8.46 (dt, J = 7.9, 1.9 Hz, 1H, 2-pyridine H-4),
7.88 (td, J = 8.3, 1.7 Hz, 1H, 4-pyridine H-4), 7.84 (d, J = 5.1 Hz,
1H, thienoquinoline H-8), 7.68 (s, 1H, thienoquinoline H-3), 7.49
(d, J = 7.8 Hz, 1H, 4-pyridine H-3), 7.41 (dd, J = 7.9, 5.2 Hz, 1H, 2-
pyridine H-5), 7.39 (dd, J = 7.6, 4.9 Hz, 1H, 4-pyridine H-5), 7.21
(d, J = 5.1 Hz, 1H, thienoquinoline H-9), 3.20 (t, J = 7.9 Hz, 2H, 5-
CH2), 3.00 (t, J = 8.0 Hz, 2H, 6-CH2).
13C NMR (62.5 MHz, CDCl3) d 153.18, 152.64, 151.88, 150.26,
149.87, 149.17, 146.41, 138.34, 135.61, 134.01, 131.91, 128.69,
128.64, 128.46, 123.79, 121.83, 121.43, 27.44, 24.92.
4.4.16. 2,4-Diphenyl-5,6-dihydro-1,10-phenanthroline (25)
Procedure described at Section 4.4 was employed with 6d
(0.08 g, 0.35 mmol), dry ammonium acetate (0.27 g, 3.50 mmol),
2d (0.11 g, 0.35 mmol), and dry methanol (2.5 mL) at 100 °C for
18 h to yield 85 mg (0.25 mmol, 73.0%) of 25 as a white solid.
Mp 210.5–211.0 °C; Rf (dichloromethane/methanol 15:1 v/v):
0.19; purity (condition A): 98.2%.
13C NMR (62.5 MHz, CDCl3) d 157.22, 151.98, 151.82, 149.74,
149.59, 148.19, 147.54, 141.33, 137.30, 136.58, 134.73, 134.13,
126.25, 124.95, 124.05, 123.45, 122.92, 118.69, 25.89, 23.05.
LC/MS/MS: retention time: 8.63 min; [MH+]: 335.3 (100%).
1H NMR (250 MHz, CDCl3) d 8.78 (dd, J = 4.7, 1.5 Hz, 1H, phe-
nanthroline H-9), 8.17 (dd, J = 7.9, 1.6 Hz, 2H, 2-phenyl H-2, H-6),
7.68 (s, 1H, phenanthroline H-3), 7.56 (dd, J = 7.7, 1.7 Hz, 1H, phe-
nanthroline H-7), 7.51–7.39 (m, 6H, 2-phenyl H-3, H-4, H-5, 4-phe-
nyl H-3, H-4, H-5), 7.42 (dd, J = 7.7, 1.7 Hz, 2H, 4-phenyl H-2, H-6),
7.24 (dd, J = 7.5, 4.8 Hz, 1H, phenanthroline H-8), 3.01–2.89 (m, 4H,
5-CH2, 6-CH2).
4.5.3. 4-(Pyridin-2-yl)-2-(pyridin-4-yl)-5,6-dihydrothieno[2,3-h]
quinoline (28)
Procedure described at Section 4.5 was employed with 7a
(0.14 g, 0.60 mmol), dry ammonium acetate (0.46 g, 6.00 mmol),
2c (0.19 g, 0.60 mmol), and glacial acetic acid (1.0 mL) at 100 °C
for 20 h to yield 87 mg (0.25 mmol, 42.5%) of 28 as a light yellow
solid.
13C NMR (62.5 MHz, CDCl3) d 156.02, 152.36, 152.07, 149.42,
149.06, 139.37, 138.88, 135.43, 133.90, 130.04, 128.76, 128.71,
128.54, 128.50, 128.16, 127.26, 123.42, 121.59, 27.63, 24.79.
Mp 161.2–162.1 °C; Rf (ethyl acetate/n-hexane 6:1 v/v): 0.10;
purity (condition B): 97.6%.

76
P. Thapa et al. / Bioorganic Chemistry 40 (2012) 67–78
LC/MS/MS: retention time: 7.84 min; [MH+]: 342.2 (100%).
1H NMR (250 MHz, CDCl3) d 8.80 (d, J = 4.1 Hz, 1H, 4-pyridine H-
1H NMR (250 MHz, CDCl3) d 9.32 (d, J = 1.7 Hz, 1H, 2-pyridine H-
2), 8.72 (dd, J = 4.9, 1.5 Hz, 1H, 4-pyridine H-6), 8.70 (d, J = 1.7 Hz,
1H, 4-pyridine H-2), 8.66 (dd, J = 4.8, 1.5 Hz, 1H, 2-pyridine H-6),
8.43 (dt, J = 8.0, 1.8 Hz, 1H, 2-pyridine H-4), 7.84 (d, J = 5.1 Hz,
1H, thienoquinoline H-8), 7.75 (dt, J = 7.8, 1.8 Hz, 1H, 4-pyridine
H-4), 7.49 (s, 1H, thienoquinoline H-3), 7.46 (dd, J = 7.7, 5.8 Hz,
1H, 2-pyridine H-5), 7.42 (dd, J = 8.0, 7.8 Hz, 1H, 4-pyridine H-5),
7.22 (d, J = 5.1 Hz, 1H, thienoquinoline H-9), 3.09–2.98 (m, 4H, 5-
CH2, 6-CH2).
6), 8.72 (dd, J = 4.6, 1.5 Hz, 2H, 2-pyridine H-2, H-6), 8.04 (dd,
J = 4.6, 1.5 Hz, 2H, 2-pyridine H-3, H-5), 7.87 (td, J = 7.5, 1.7 Hz,
1H, 4-pyridine H-4), 7.84 (d, J = 5.0 Hz, 1H, thienoquinoline H-8),
7.74 (s, 1H, thienoquinoline H-3), 7.49 (d, J = 7.8 Hz, 1H, 4-pyridine
H-3), 7.39 (ddd, J = 7.6, 4.9, 0.8 Hz, 1H, 4-pyridine H-5), 7.21 (d,
J = 5.1 Hz, 1H, thienoquinoline H-9), 3.21 (t, J = 8.0 Hz, 2H, 5-CH2),
3.00 (t, J = 8.0 Hz, 2H, 6-CH2).
13C NMR (62.5 MHz, CDCl3) d 157.10, 152.05, 151.57, 150.29,
149.80, 147.57, 146.30, 141.45, 137.23, 136.58, 127.39, 124.95,
124.04, 122.98, 120.86, 119.08, 26.02, 23.01.
13C NMR (62.5 MHz, CDCl3) d 152.01, 151.93, 149.82, 149.48,
149.34, 148.20, 145.66. 141.36, 137.22, 136.07, 134.84, 134.52,
134.12, 126.19, 124.92, 123.52, 123.38, 123.17, 118.92, 26.08,
23.06.
4.5.4. 2-Phenyl-4-(pyridin-2-yl)-5,6-dihydrothieno[2,3-h]quinoline
(29)
4.5.7. 4-(Pyridin-3-yl)-2-(pyridin-4-yl)-5,6-dihydrothieno[2,3-h]
quinoline (32)
Procedure described at Section 4.5 was employed with 7a
(0.07 g, 0.30 mmol), dry ammonium acetate (0.23 g, 3.00 mmol),
2d (0.10 g, 0.30 mmol), and glacial acetic acid (0.5 mL) at 100 °C
for 12 h to yield 52 mg (0.15 mmol, 50.7%) of 29 as an off-white
solid.
Procedure described at Section 4.5 was employed with 7b
(0.07 g, 0.29 mmol), dry ammonium acetate (0.22 g, 2.90 mmol),
2c (0.09 g, 0.29 mmol), and glacial acetic acid (0.5 mL) at 90 °C
for 14 h to yield 32 mg (0.09 mmol, 31.7%) of 32 as a light yellow
solid.
Mp 168.0–168.8 °C; Rf (ethyl acetate/n-hexane 1:3 v/v): 0.27;
purity (condition B): 96.7%.
Mp 205.2–205.9 °C; Rf (ethyl acetate/n-hexane 3:1 v/v): 0.15;
purity (condition B): 95.0%.
LC/MS/MS: retention time: 9.50 min; [MH+]: 341.2 (100%).
1H NMR (250 MHz, CDCl3) d 8.78 (ddd, J = 4.8, 1.6, 0.9 Hz, 1H, 4-
pyridine H-6), 8.14 (dd, J = 7.9, 1.5 Hz, 2H, 2-phenyl H-2, H-6), 7.87
(d, J = 5.2 Hz, 1H, thienoquinoline H-8), 7.83 (td, J = 7.7, 1.8 Hz, 1H,
4-pyridine H-4), 7.66 (s, 1H, thienoquinoline H-3), 7.48 (d,
J = 7.8 Hz, 1H, 4-pyridine H-3), 7.47–7.42 (m, 3H, 2-phenyl H-3,
H-4, H-5), 7.36 (ddd, J = 7.5, 4.9, 1.0 Hz, 1H, 4-pyridine H-5), 7.19
(d, J = 5.1 Hz, 1H, thienoquinoline H-9), 3.18 (t, J = 8.1 Hz, 2H, 5-
CH2), 2.99 (t, J = 8.0 Hz, 2H, 6-CH2).
LC/MS/MS: retention time: 7.49 min; [MH+]: 342.2 (100%).
1H NMR (250 MHz, CDCl3) d 8.73–8.70 (m, 4H, 2-pyridine H-2,
H-6, 4-pyridine H-2, H-6), 8.01 (d, J = 4.4 Hz, 2H, 2-pyridine H-3,
H-5), 7.85(d, J = 5.0 Hz, 1H, thienoquinoline H-8), 7.74 (d, J = 7.6,
1H, 4-pyridine H-4), 7.55 (s, 1H, thienoquinoline H-3), 7.47 (dd,
J = 6.9, 4.7 Hz, 1H, 4-pyridine H-5), 7.23 (d, J = 4.9 Hz, 1H, thieno-
quinoline H-9), 3.08–3.02 (m, 4H, 5-CH2, 6-CH2).
13C NMR (62.5 MHz, CDCl3) d 151.96, 151.66, 150.30, 149.50,
149.27, 146.08, 145.64, 141.47, 137.08, 136.06, 134.69, 127.34,
124.87, 123.38, 123.23, 120.83, 119.28, 26.16, 22.97.
13C NMR (62.5 MHz, CDCl3) d 157.71, 154.46, 151.58, 149.63,
147.32, 140.94, 139.29, 137.66, 136.51, 128.67, 128.57, 126.76,
125.39, 125.14, 124.06, 122.74, 122.63, 118.67, 25.85, 23.15.
4.5.8. 2-Phenyl-4-(pyridin-3-yl)-5,6-dihydrothieno[2,3-h]quinoline
(33)
4.5.5. 2-(Pyridin-2-yl)-4-(pyridin-3-yl)-5,6-dihydrothieno[2,3-h]
quinoline (30)
Procedure described at Section 4.5 was employed with 7b
(0.10 g, 0.41 mmol), dry ammonium acetate (0.31 g, 4.10 mmol),
2d (0.13 g, 0.41 mmol), and dry methanol (5 mL) at 100 °C for
24 h to yield 36 mg (0.10 mmol, 26.1%) of 33 as a white solid.
Mp 199.0–199.7 °C; Rf (ethyl acetate/n-hexane 1:2 v/v): 0.19;
purity (Condition B): 100.0%.
Procedure described at Section 4.5 was employed with 7b
(0.10 g, 0.40 mmol), dry ammonium acetate (0.30 g, 4.00 mmol),
2a (0.13 g, 0.40 mmol), and dry MeOH (2.5 mL) at 90 °C for 21 h
to yield 45 mg (0.13 mmol, 32.3%) of 30 as a white solid.
Mp 147.6–148.8 °C; Rf (ethyl acetate/n-hexane 1:1 v/v): 0.23;
purity (condition B): 99.2%.
LC/MS/MS: retention time: 9.23 min; [MH+]: 341.2 (100%).
1H NMR (250 MHz, CDCl3) d 8.71–8.69 (m, 2H, 4-pyridine H-2,
H-6), 8.12 (dd, J = 6.7, 1.6 Hz, 2H, 2-phenyl H-2, H-6), 7.87 (d,
J = 5.2 Hz, 1H, thienoquinoline H-8), 7.74 (dt, J = 7.8, 1.8 Hz, 1H,
4-pyridine H-4), 7.52–7.41 (m, 3H, 2-phenyl H-3, H-4, H-5), 7.48
(s, 1H, thienoquinoline H-3), 7.44 (ddd, J = 7.6, 5.3, 0.9 Hz, 1H, 4-
pyridine H-5), 7.20 (d, J = 5.2 Hz, 1H, thienoquinoline H-9), 3.06–
2.96 (m, 4H, 5-CH2, 6-CH2).
LC/MS/MS: retention time: 8.40 min; [MH+]: 342.2 (100%).
1H NMR (250 MHz, CDCl3) d 8.69 (dd, J = 5.0, 1.8 Hz, 1H, 2-pyr-
idine H-6), 8.70–8.63 (m, 2H, 4-pyridine H-2, H-6), 8.61 (d,
J = 8.0 Hz, 1H, 2-pyridine H-3), 8.19 (s, 1H, thienoquinoline H-3),
7.86 (d, J = 5.3 Hz, 1H, thienoquinoline H-8), 7.85 (td, J = 7.9,
1.8 Hz, 1H, 2-pyridine H-4), 7.77 (dt, J = 7.8, 1.9 Hz, 1H, 4-pyridine
H-4), 7.43 (dd, J = 7.8, 4.8 Hz, 1H, 4-pyridine H-5), 7.32 (ddd, J = 7.4,
4.8, 1.04 Hz, 1H, 2-pyridine H-5), 7.22 (d, J = 5.1 Hz, 1H, thieno-
quinoline H-9), 3.10–2.98 (m, 4H, 5-CH2, 6-CH2).
13C NMR (62.5 MHz, CDCl3) d 154.59, 151.49, 149.42, 149.28,
145.36, 140.93, 139.04, 137.55, 136.10, 135.17, 128.88, 128.66,
126.73, 125.28, 125.08, 123.28, 122.88, 118.92, 26.04, 23.12.
13C NMR (62.5 MHz, CDCl3) d 155.99, 153.34, 151.14, 149.43,
149.20, 148.99, 145.64, 141.01, 137.39, 136.88, 136.21, 135.02,
127.02, 124.86, 123.64, 123.24, 123.00, 121.07, 119.55, 26.24,
23.08.
4.5.9. 2-(Pyridin-2-yl)-4-(pyridin-4-yl)-5,6-dihydrothieno[2,3-h]
quinoline (34)
Procedure described at Section 4.5 was employed with 7c
(0.07 g, 0.30 mmol), dry ammonium acetate (0.23 g, 3.00 mmol),
2a (0.10 g, 0.30 mmol), and glacial acetic acid (0.5 mL) at 100 °C
for 12 h to yield 37 mg (0.11 mmol, 36.1%) of 34 as a light yellow
solid.
4.5.6. 2,4-Di(pyridin-3-yl)-5,6-dihydrothieno[2,3-h]quinoline (31)
Procedure described at Section 4.5 was employed with 7b
(0.15 g, 0.62 mmol), dry ammonium acetate (0.48 g, 6.20 mmol),
2b (0.30 g, 0.93 mmol), and dry methanol (5 mL) at 100 °C for
24 h to yield 25 mg (0.07 mmol, 12.0%) of 31 as a white solid.
Mp 208.8–209.4 °C; Rf (ethyl acetate/n-hexane 10:1 v/v): 0.11;
purity (condition B): 96.3%.
Mp 166.8–167.5 °C; Rf (ethyl acetate/n-hexane 1:1 v/v): 0.18;
purity (condition B): 95.8%.
LC/MS/MS: retention time: 8.43 min; [MH+]: 342.2 (100%).
1H NMR (250 MHz, CDCl3) d 8.74 (dd, J = 4.5, 1.5 Hz, 2H, 4-pyr-
idine H-2, H-6), 8.65 (d, J = 3.9 Hz, 1H, 2-pyridine H-6), 8.61 (d,
LC/MS/MS: retention time: 7.50 min; [MH+]: 342.2 (100%).

P. Thapa et al. / Bioorganic Chemistry 40 (2012) 67–78
77
J = 7.9 Hz, 1H, 2-pyridine H-3), 8.17 (s, 1H, thienoquinoline H-3),
7.87 (td, J = 7.7, 1.7 Hz, 1H, 2-pyridine H-4), 7.86 (d, J = 5.1 Hz,
1H, thienoquinoline H-8), 7.37 (dd, J = 4.5, 1.5 Hz, 2H, 4-pyridine
H-3, H-5), 7.33 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H, 2-pyridine H-5), 7.22
(d, J = 5.2 Hz, 1H, thienoquinoline H-9), 3.08–2.98 (m, 4H, 5-CH2,
6-CH2).
13C NMR (62.5 MHz, CDCl3) d 154.74, 151.57, 150.03, 147.42,
146.31, 140.99, 139.01, 137.54, 128.94, 128.67, 126.75, 125.12,
124.64, 123.57, 122.90, 118.12, 26.00, 23.12.
4.5.13. 4-Phenyl-2-(pyridin-2-yl)-5,6-dihydrothieno[2,3-h]quinoline
(38)
13C NMR (62.5 MHz, CDCl3) d 155.92, 153.46, 151.18, 149.94,
149.02, 147.20, 146.52, 141.07, 137.33, 136.91, 126.42, 124.86,
123.70, 123.65, 123.05, 121.07, 118.83, 26.17, 23.05.
Procedure described at Section 4.5 was employed with 7d
(0.12 g, 0.50 mmol), dry ammonium acetate (0.38 g, 5.00 mmol),
2a (0.16 g, 0.50 mmol), and dry methanol (5 mL) at 100 °C for
48 h to yield 45 mg (0.13 mmol, 26.6%) of 38 as a white solid.
Mp 118.5–118.9 °C; Rf (ethyl acetate/n-hexane 1:5 v/v): 0.37;
purity (condition B): 98.8%.
4.5.10. 2-(Pyridin-3-yl)-4-(pyridin-4-yl)-5,6-dihydrothieno[2,3-
h]quinoline (35)
Procedure described at Section 4.5 was employed with 7c
(0.05 g, 0.22 mmol), dry ammonium acetate (0.17 g, 2.20 mmol),
2b (0.07 g, 0.22 mmol), and glacial acetic acid (0.5 mL) at 100 °C
for 14 h to yield 26 mg (0.07 mmol, 34.6%) of 35 as a white solid.
Mp 186.9–187.4 °C; Rf (dichloromethane/methanol 20:1 v/v):
0.13; purity (condition B): 95.1%.
LC/MS/MS: retention time: 10.50 min; [MH+]: 341.2 (100%).
1H NMR (250 MHz, CDCl3) d 8.64 (ddd, J = 4.7, 1.6, 0.8 Hz, 1H, 2-
pyridine H-6), 8.60 (d, J = 8.0 Hz, 1H, 2-pyridine H-3), 8.20 (s, 1H,
thienoquinoline H-3), 7.86 (d, J = 5.1 Hz, 1H, thienoquinoline H-
8), 7.83 (td, J = 7.8, 1.8 Hz, 1H, 2-pyridine H-4), 7.46–7.40 (m, 5H,
4-phenyl H-2, H-3, H-4, H-5, H-6), 7.29 (ddd, J = 7.4, 4.8, 1.1 Hz,
1H, 2-pyridine H-5), 7.20 (d, J = 5.1 Hz, 1H, thienoquinoline H-9),
3.11–2.96 (m, 4H, 5-CH2, 6-CH2).
LC/MS/MS: retention time: 7.47 min; [MH+]: 342.2 (100%).
1H NMR (250 MHz, CDCl3) d 9.31 (d, J = 1.8 Hz, 1H, 2-pyridine H-
2), 8.78 (dd, J = 5.7, 1.3 Hz, 2H, 4-pyridine H-2, H-6), 8.65 (dd,
J = 4.5, 1.1 Hz, 1H, 2-pyridine H-6), 8.42 (dt, J = 7.9, 1.8 Hz, 1H, 2-
pyridine H-4), 7.84 (d, J = 5.1 Hz, 1H, thienoquinoline H-8), 7.47
(s, 1H, thienoquinoline H-3), 7.43 (dd, J = 7.9, 4.8 Hz, 1H, 2-pyridine
H-5), 7.35 (dd, J = 5.8, 1.4 Hz, 2H, 4-pyridine H-3, H-5), 7.22 (d,
J = 5.1 Hz, 1H, thienoquinoline H-9), 3.08–2.97 (m, 4H, 5-CH2, 6-
CH2).
13C NMR (62.5 MHz, CDCl3) d 156.40, 153.00, 150.90, 149.37,
148.96, 140.88, 139.30, 137.66, 136.78, 128.80, 128.30, 127.88,
126.95, 124.92, 123.41, 122.78, 121.03, 119.78, 26.34, 23.19.
4.5.14. 4-Phenyl-2-(pyridin-3-yl)-5,6-dihydrothieno[2,3-h]quinoline
(39)
13C NMR (62.5 MHz, CDCl3) d 152.08, 151.94, 150.13, 149.87,
148.18, 146.92, 146.52, 141.41, 137.13, 134.42, 134.09, 125.55,
124.89, 123.48, 123.20, 118.14, 26.00, 23.02.
Procedure described at Section 4.5 was employed with 7d
(0.09 g, 0.37 mmol), dry ammonium acetate (0.28 g, 3.70 mmol),
2b (0.12 g, 0.37 mmol), and glacial acetic acid (1 mL) at 100 °C
for 20 h to yield 30 mg (0.09 mmol, 24.2%) of 39 as a light yellow
solid.
4.5.11. 2,4-Di(pyridin-4-yl)-5,6-dihydrothieno[2,3-h]quinoline (36)
Procedure described at Section 4.5 was employed with 7c
(0.07 g, 0.30 mmol), dry ammonium acetate (0.23 g, 3.00 mmol),
2c (0.10 g, 0.30 mmol), and glacial acetic acid (0.5 mL) at 100 °C
for 16 h to yield 28 mg (0.08 mmol, 27.6%) of 36 as an off-white
solid.
Mp 163.6–164.2 °C; Rf (ethyl acetate/n-hexane 1:2 v/v): 0.18;
purity (condition B): 95.6%.
LC/MS/MS: retention time: 9.49 min; [MH+]: 341.2 (100%).
1H NMR (250 MHz, CDCl3) d 9.31 (d, J = 1.6 Hz, 1H, 2-pyridine H-
2), 8.64 (dd, J = 4.7, 1.5 Hz, 1H, 2-pyridine H-6), 8.43 (dt, J = 8.0,
1.9 Hz, 1H, 2-pyridine H-4), 7.84 (d, J = 5.2 Hz, 1H, thienoquinoline
H-8), 7.51 (s, 1H, thienoquinoline H-3), 7.47–7.38 (m, 3H, 4-phenyl
H-3, H-4, H-5), 7.42 (dd, J = 7.9, 1.6 Hz, 2H, 4-phenyl H-2, H-6), 7.42
(ddd, J = 8.1, 4.8, 0.9 Hz, 1H, 2-pyridine H-5), 7.20 (d, J = 5.2 Hz, 1H,
thienoquinoline H-9), 3.11–2.94 (m, 4H, 5-CH2, 6-CH2).
13C NMR (62.5 MHz, CDCl3) d 151.64, 151.58, 149.57, 149.31,
148.19, 141.20, 139.08, 137.43, 134.82, 134.09, 128.64, 128.53,
128.14, 126.07, 124.94, 123.46, 122.94, 119.16, 26.14, 23.14.
Mp 224.8–225.5 °C; Rf (dichloromethane/methanol 20:1 v/v):
0.26; purity (condition B): 95.2%.
LC/MS/MS: retention time: 7.46 min; [MH+]: 342.2 (100%).
1H NMR (250 MHz, CDCl3) d 8.78 (dd, J = 4.5, 1.5 Hz, 2H, 2-pyr-
idine H-2, H-6), 8.73 (dd, J = 5.0, 0.9 Hz, 2H, 4-pyridine H-2, H-6),
8.01 (dd, J = 4.5, 1.4 Hz, 2H, 2-pyridine H-3, H-5), 7.85 (d,
J = 5.1 Hz, 1H, thienoquinoline H-8), 7.52 (s, 1H, thienoquinoline
H-3), 7.34 (dd, J = 4.6, 1.4 Hz, 2H, 4-pyridine H-3, H-5), 7.23 (d,
J = 5.1 Hz, 1H, thienoquinoline H-9), 3.09–3.01 (m, 4H, 5-CH2, 6-
CH2).
13C NMR (62.5 MHz, CDCl3) d 152.00, 151.78, 150.36, 150.14,
146.81, 146.54, 145.98, 141.57, 137.01, 126.73, 124.86, 123.47,
123.29, 120.82, 118.53, 26.10, 22.95.
4.5.15. 4-Phenyl-2-(pyridin-4-yl)-5,6-dihydrothieno[2,3-h]quinoline
(40)
Procedure described at Section 4.5 was employed with 7d
(0.09 g, 0.37 mmol), dry ammonium acetate (0.28 g, 3.70 mmol),
2c (0.12 g, 0.37 mmol), and dry methanol (2.5 mL) at 100 °C for
36 h to yield 23 mg (0.06 mmol, 18.0%) of 40 as a white solid.
Mp 165.0–165.3 °C; Rf (ethyl acetate/n-hexane 1:2 v/v): 0.17;
purity: (condition B): 96.0%.
4.5.12. 2-Phenyl-4-(pyridin-4-yl)-5,6-dihydrothieno[2,3-h]quinoline
(37)
Procedure described at Section 4.5 was employed with 7c
(0.08 g, 0.35 mmol), dry ammonium acetate (0.27 g, 3.50 mmol),
2d (0.11 g, 0.35 mmol), and glacial acetic acid (0.5 mL) at 90 °C
for 14 h to yield 25 mg (0.07 mmol, 20.7%) of 37 as a white solid.
Mp 218.0–218.5 °C; Rf (ethyl acetate/n-hexane 1:2 v/v): 0.24;
purity (condition B): 95.3%.
LC/MS/MS: retention time: 9.57 min; [MH+]: 341.2 (100%).
1H NMR (250 MHz, CDCl3) d 8.72 (dd, J = 4.6, 1.4 Hz, 2H, 2-pyr-
idine H-2, H-6), 8.02 (dd, J = 4.5, 1.5 Hz, 2H, 2-pyridine H-3, H-5),
7.85 (d, J = 5.2 Hz, 1H, thienoquinoline H-8), 7.57 (s, 1H, thieno-
quinoline H-3), 7.52–7.46 (m, 3H, 4-phenyl H-3, H-4, H-5), 7.40
(dd, J = 7.8, 1.5 Hz, 2H, 4-phenyl H-2, H-6), 7.22 (d, J = 5.2 Hz, 1H,
thienoquinoline H-9), 3.13–2.95 (m, 4H, 5-CH2, 6-CH2).
13C NMR (62.5 MHz, CDCl3) d 151.72, 151.30, 150.30, 149.35,
146.43, 141.34, 138.97, 137.36, 128.63, 128.57, 128.22, 127.23,
124.93, 123.02, 120.85, 119.55, 26.26, 23.09.
LC/MS/MS: retention time: 9.28 min; [MH+]: 341.2 (100%).
1H NMR (250 MHz, CDCl3) d 8.73 (dd, J = 4.4, 1.5 Hz, 2H, 4-pyr-
idine H-2, H-6), 8.10 (dd, J = 7.9, 1.2 Hz, 2H, 2-phenyl H-2, H-6),
7.86 (d, J = 5.2 Hz, 1H, thienoquinoline H-8), 7.51–7.40 (m, 3H, 2-
phenyl H-3, H-4, H-5), 7.50 (s, 1H, thienoquinoline H-3), 7.30
(dd, J = 4.4, 1.6 Hz, 2H, 4-pyridine H-3, H-5), 7.19 (d, J = 5.2 Hz,
1H, thienoquinoline H-9), 3.06–2.93 (m, 4H, 5-CH2, 6-CH2).

78
P. Thapa et al. / Bioorganic Chemistry 40 (2012) 67–78
4.5.16. 2,4-Diphenyl-5,6-dihydrothieno[2,3-h]quinoline (41)
Procedure described at Section 4.5 was employed with 7d
(0.10 g, 0.41 mmol), dry ammonium acetate (0.32 g, 4.16 mmol),
2d (0.13 g, 0.41 mmol), and dry methanol (5 mL) at 100 °C for
48 h to yield 16 mg (0.05 mmol, 11.9%) of 41 as a white solid.
Mp 174.6–175.0 °C; Rf (ethyl acetate/n-hexane 1:5 v/v): 0.52;
purity (condition B): 98.2%.
ture 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 5 lL of the cell counting kit-8 solution
(Dojindo, Japan) then incubated for additional 4 h under the same
condition. The absorbance of each well was determined by an
Automatic Elisa Reader System (Bio-Rad 3550) with a 450 nm
wavelength. For determination of the IC50 values, the absorbance
readings at 450 nm were fitted to the four-parameter logistic equa-
tion. The compounds of adriamycin, etoposide, and camptothecin
were purchased from Sigma and used as positive controls.
LC/MS/MS: retention time: 11.48 min; [MH+]: 340.2 (100%).
1H NMR (250 MHz, CDCl3) d 8.12 (dd, J = 8.4, 1.5 Hz, 2H, 2-phe-
nyl H-2, H-6), 7.87 (d, J = 5.2 Hz, 1H, thienoquinoline H-8), 7.50 (s,
1H, thienoquinoline H-3), 7.49–7.46 (m, 3H, 2-phenyl H-3, H-4, H-
5), 7.44–7.37 (m, 5H, 4-phenyl H-2, H-3, H-4, H-5, H-6), 7.18 (d,
J = 5.2 Hz, 1H, thienoquinoline H-9), 3.10–2.92 (m, 4H, 5-CH2, 6-
CH2).
Acknowledgment
This work was supported by the 2011 Yeungnam University Re-
search Grant.
13C NMR (62.5 MHz, CDCl3) d 154.23, 151.25, 149.06, 140.80,
139.52, 139.42, 137.81, 128.71, 128.64, 128.59, 128.44, 127.94,
126.74, 125.20, 125.14, 122.66, 119.18, 26.14, 23.24.
References
4.6. Biological assays
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2659–2662;
DNA topo I inhibition assay was determined following the
method reported by Fukuda M. et al. with minor modifications
[14]. 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 0.4 units of recombinant hu-
man DNA topo 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
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volume of 10 lL was terminated by adding 2.5 lL of the stop solu-
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for 30 min in an aqueous solution of ethidium bromide (0.5 lg/
mL). DNA bands were visualized by transillumination with UV light
and were quantitated using AlphaImager™ (Alpha Innotech
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lL
l
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solution of ethidium bromide (0.5 lg/mL). DNA bands were visual-
ized by transillumination with UV light and supercoiled DNA was
quantitated using AlphaImagerTM (Alpha Innotech Corporation).
For the evaluation of cytotoxicity, five different cancer cell lines
were used: human breast adenocarcinoma cell line (MCF-7), hu-
man cervix tumor cell line (HeLa), human prostate tumor cell line
(DU145), human colorectal adenocarcinoma cell line (HCT15) and
chronic myelogenous leukemia cell line (K562). Experiments were
performed as described previously [15]. Cancer cells were cultured
according to the supplier’s instructions. Cells were seeded in 96-
well plates at a density of 2ꢁ4 ꢀ 104 cells per well and incubated
for overnight in 0.1 mL of media supplied with 10% Fetal Bovine
Serum(Hyclone, USA) in 5% CO2 incubator at 37 °C. On day 2, cul-