2-Thienyl-4-furyl-6-aryl pyridine derivatives: Synthesis, topoisomerase I and II inhibitory activity, cytotoxicity, and structure-activity relationship study

Article abstract of DOI:10.1016/j.bmc.2009.10.049

Designed and synthesized 60 2-thienyl-4-furyl-6-aryl pyridine derivatives were evaluated for their topoisomerase I and II inhibitory activities at 20 μM and 100 μM and cytotoxicity against several human cancer cell lines. Compounds 8, 9, 11-29 showed sign

Full text of DOI:10.1016/j.bmc.2009.10.049

Bioorganic & Medicinal Chemistry 18 (2010) 377–386
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2-Thienyl-4-furyl-6-aryl pyridine derivatives: Synthesis, topoisomerase I and
II inhibitory activity, cytotoxicity, and structure–activity relationship study
Pritam Thapa a, Radha Karki a, Uttam Thapa a, Yurngdong Jahng a, Mi-Ja Jung b, Jung Min Nam b,
a,
Younghwa Na c, Youngjoo Kwon b, , Eung-Seok Lee
*
*
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, Catholic University of Daegu, Kyongsan 712-702, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Designed and synthesized 60 2-thienyl-4-furyl-6-aryl pyridine derivatives were evaluated for their topo-
Received 11 September 2009
Revised 23 October 2009
Accepted 24 October 2009
Available online 29 October 2009
isomerase I and II inhibitory activities at 20 lM and 100 lM and cytotoxicity against several human can-
cer cell lines. Compounds 8, 9, 11–29 showed significant topoisomerase II inhibitory activity and
compounds 10 and 11 showed significant topoisomerase I inhibitory activity. Most of the compounds
(7–21) possessing 2-(5-chlorothiophen-2-yl)-4-(furan-3-yl) moiety showed higher or similar cytotoxicity
against HCT15 cell line as compared to standards. Most of the selected compounds displayed moderate
cytotoxicity against MCF-7, HeLa, DU145, and K562 cell lines. Structure–activity relationship study
revealed that 2-(5-chlorothiophen-2-yl)-4-(furan-3-yl) moiety has an important role in displaying bio-
logical activities.
Keywords:
2-Thienyl-4-furyl-6-aryl pyridine
Topoisomerase I and II
Cytotoxicity
Ó 2009 Elsevier Ltd. All rights reserved.
2-(5-Chlorothiophen-2-yl)-4-(furan-3-yl)
1. Introduction
cancer cell lines and topoisomerase inhibitory activity. Recently,
our research group has reported that terpyridine derivatives show
DNA topoisomerases, first discovered in 1971 by Wang (the
omega protein,
, from Escherichia coli),1 are nuclear enzymes that
a strong cytotoxicity against several human cancer cell lines and
considerable topoisomerase I (topo I) and II (topo II) inhibitory
activities.10 From previous structure–activity relationship studies,
we found 2-thienyl-4-furyl-pyridine skeleton exhibited strong
topo I and II inhibitory activities.10b,g Further exploration on this
skeleton prompted us to design and synthesize a series of 2-thie-
nyl-4-furyl-pyridine skeletons containing compounds with sub-
stituents like chlorine or methyl at different aryl moieties as
shown in Figure 1. They were evaluated for topo I and II inhibitory
activities and cytotoxicity against several human cancer cell lines.
In order to study the structure–activity relationship, 60 com-
pounds in six different series were synthesized systematically as
shown in Scheme 1 and Figure 2.
x
alter the topology of DNA by transiently breaking one or two
strands of DNA, passing a single or double-stranded DNA through
the break and again resealing the breaks. They are involved in a
number of vital cellular processes like replication, transcription,
recombination, repair, chromatin assembly, and chromosome seg-
regation.2 Topoisomerases have been basically classified into type I
and II depending on their mechanism of action, making either sin-
gle- or double-stranded breaks, respectively.3 Because of the cru-
cial role of topoisomerase for the maintenance and replication of
DNA during proliferation, cells become highly vulnerable when
these functions are lost,4 so they are now viewed as important
therapeutic targets for cancer chemotherapy.5,6
2,20:60,200-Terpyridine, bioisostere of
a-terthiophene, was first
2. Results and discussion
2.1. Chemistry
discovered in 1932 and has been precious due to its ability to form
metal complexes7 and as RNA/DNA binding agents8 (Fig. 1). Since
the terthiophene derivatives show considerable PKC inhibitory
activity and cytotoxicity against human cancer cell lines,9 a ratio-
nale evolved in our research group that terpyridine derivatives as
bioisosteres of terthiophene may have cytotoxicity against human
Synthesis of various 2,4,6-trisubstituted pyridine derivatives
have already been reported by our research group.10 Our strategy
to synthesize 2,4,6-trisubstituted pyridine derivatives was based
on previously proposed methods11,12 as shown in Scheme 1. At
first, using the Claisen–Schmidt KOH catalyzed condensation reac-
tion,11 we synthesized six propenone intermediates (3) in 65.7–
94.2% yield by the condensation of two aryl acetyls (1a, b) with
* Corresponding authors. Tel.: +82 2 3277 4653; fax: +82 2 3277 2851 (Y.K.); tel.:
+82 53 810 2827; fax: +82 53 810 4654 (E.-S.L.).
E-mail addresses: ykwon@ewha.ac.kr (Y. Kwon), eslee@yu.ac.kr (E.-S. Lee).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.049

378
P. Thapa et al. / Bioorg. Med. Chem. 18 (2010) 377–386
O
4
2
6
N
S
N
R3
S
S
N
N
S
α- terpyridine
α- terthiophene
R3
=
H3C
O
O
CH3
or Cl
N
2 or 3
Figure 1. Structure of
a-terthiophene,
a-terpyridine and 2-thienyl-4-furyl-6-aryl pyridine derivatives.
O
R2
R2
H
O
2 (R2 = c-e)
R1
R2
R1
CH3
i
O
3 (R1 = a,b; R2 = c-e)
1 (R1 = a, b)
R1
N
R3
iii
6
O
O
N
(R1 = a,b; R2 = c-e; R3 = e-n)
R3
CH3
R3
I-
ii
5 (R3 = e-n)
4 (R3 = e-n)
H3C
H3C
O
CH3
O
Cl
O
Cl
O
S
S
a
c
e
g
b
d
f
Cl
CH3
Cl
Cl
CH3
N
N
m
n
j
h
i
k
l
Scheme 1. General synthetic scheme of 2,4,6-trisubstituted pyridines. Reagents and conditions: (i) aryl aldehyde 2c–e (1.0 equiv), KOH (1.2 equiv), MeOH/H2O (5:1), 10 min
to 3 h, 0 °C, 65.7–94.2%; (ii) pyridine, iodine (1.0 equiv), 3 h, 140 °C, 42.2–99.4%; (iii) 3 (1.0 equiv), 5 (1.0 equiv), NH4OAc (10.0 equiv), glacial AcOH, 16–22 h, 80–95 °C, 20.6–
71.6%.
Cl
Cl
O
O
O
O
O
O
H3C
H3C
H3C
N
R3
N
R3
N
R3
N
R3
N
R3
N
R3
S
S
S
S
S
S
Cl
Cl
Cl
Series 1
Series 2
Series 3
R3 = e-n
Series 4
Series 5
Series 6
Figure 2. Strategy and design of six different series of 2-thienyl-4-furyl-6-aryl pyridine derivatives.
three different aryl aldehydes (2c–e). Secondly, 10 pyridinium io-
dide salts (5) were prepared in 42.2–99.4% yield by refluxing 10
different aryl acetyls (4e–n) with iodine in pyridine. Finally, on
the basis of modified Kröhnke synthesis method,12 60 2-thienyl-
4-furyl-6-aryl pyridine derivatives (6) were synthesized by react-
ing 6 propenone intermediates (3) with 10 different pyridinium io-
dide salts (5) in 20.6–71.6% yield. It was noticed that the yield of
compounds having substituents Cl or CH3 at ortho position of phe-
nyl ring was lower than those having substituents at meta and para
position of phenyl ring. That is likely due to the steric hindrance by
substituents when placed at ortho position.
Sixty 2-thienyl-4-furyl-6-aryl pyridine derivatives were synthe-
sized systematically in six different series as shown in Figure 2.
2.2. Topoisomerase I and II inhibitory activity
Sixty 2-thienyl-4-furyl-6-aryl pyridine derivatives were pre-
pared and evaluated for their topo I and II inhibitory activities.

P. Thapa et al. / Bioorg. Med. Chem. 18 (2010) 377–386
379
The structure of selected compounds was displayed in Figure 3.
The rest of the compounds were excluded since they did not show
considerable activities.
control. Most of all compounds were devoid of topo I inhibitory
activity. However, compound 10 has shown significant inhibitory
activity at 100 lM and compound 11 has shown considerable
Figure 4 and Table 1 depict the human DNA topo II inhibitory
activities of 2-thienyl-4-furyl-6-aryl pyridine derivatives (7–29)
with etoposide as a positive control. Compounds 8, 9, 11–29
inhibitory activity at both 20
camptothecin.
lM and 100 lM compared to
From the structure–activity relationship study, most of the
compounds with significant topo II inhibitory activity, which have
stronger inhibitory activity than etoposide, possess 2-(5-chloro-
thiophen-2-yl)-4-(furan-3-yl) moiety (series 5 in Fig. 2) in com-
mon. It was also noticed that there is not much variation in
activity within a series.
showed significant topo II inhibitory activity at 20
lM and
100 M whereas compounds 7 and 10 showed weaker inhibitory
l
activity as compared to etoposide. Similarly, Figure 5 and Table 1
depict human topo I inhibitory activities of 2-thienyl-4-furyl-6-
aryl pyridine derivatives (7–29) with camptothecin as a positive
O
O
O
O
Cl
H3C
H3C
H3C
H3C
N
N
N
N
S
S
S
S
O
Cl
Cl
7
8
9
10
O
O
O
O
CH3
N
N
N
N
S
O
S
S
Cl
S
O
Cl
H3C
Cl
Cl
Cl
11
12
13
14
O
O
O
O
Cl
Cl
CH3
N
N
N
N
S
S
S
S
CH3
Cl
Cl
Cl
Cl
18
17
16
15
Cl
O
O
O
O
H3C
N
N
N
N
S
S
N
S
O
S
N
Cl
Cl
Cl
Cl
19
20
21
22
Cl
Cl
Cl
Cl
O
O
O
O
H3C
H3C
H3C
Cl
N
N
N
N
S
S
O
S
S
Cl
N
Cl
23
24
25
26
Cl
Cl
Cl
O
O
O
Cl
N
N
N
S
S
S
Cl
CH3
Cl
Cl
Cl
27
28
29
Figure 3. Structures of selected compounds possessing significant biological activities.

380
P. Thapa et al. / Bioorg. Med. Chem. 18 (2010) 377–386
Lane D: pBR322 DNA only
Lane T: pBR322 DNA + Topo II
Lane E: pBR322 DNA + Topo II + Etoposide
Lane 7 – 29: pBR322 DNA + Topo II + Compound 7 - 29
Figure 4. Human DNA topoisomerase IIa inhibitory activity of compounds 7–29.
Table 1
Inhibitory effects of compounds 7–29 on topoisomerase I and II (% inhibition ratio of relaxation) and their IC50 values against MCF-7, HeLa, DU145, HCT15, K562 cell lines
Compounds
% Inhibition
aIC50
dDU145
(lM)
Topo II
Topo I
bMCF-7
cHeLa
eHCT15
fK562
100
l
M
20
lM
100
lM
20 lM
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
33.2
56.3
59.9
33.8
53.3
49.1
60.5
60.7
50.2
50.3
49.5
54.0
62.7
49.6
60.8
61.1
51.5
59.8
57.2
53.8
58.6
53.8
59.8
3.5
17.8
36.4
8.1
2.0
1.0
5.4
85.9
53.1
9.6
13.2
4.6
2.6
0.0
0.0
2.1
2.3
3.1
2.0
7.8
5.9
5.1
NA
NA
NA
14.21 3.31
0.89 0.32
18.25 1.91
20.33 0.24
1.52 0.58
4.74 1.27
1.90 0.44
1.43 0.10
3.99 0.77
5.34 1.49
7.36 1.37
2.92 0.79
2.60 0.55
3.36 0.37
3.48 0.54
5.57 0.42
18.83 2.31
15.28 7.87
5.81 0.98
16.85 0.70
3.04 1.02
15.09 0.00
12.55 2.33
0.06 0.02
1.89 0.34
2.10 0.75
24.02 2.95
24.49 1.46
30.5 2.2
29.87 1.58
26.68 4.37
30.44 2.72
20.09 0.78
14.75 4.29
17.23 2.28
33.44 3.48
18.51 3.72
20.34 2.68
25.32 2.37
16.41 0.66
22.96 1.80
29.47 3.15
20.52 2.43
18.49 2.38
27.31 2.79
20.52 1.18
21.93 3.30
30.81 5.55
27.67 2.20
>50
0.54 0.05
0.71 0.07
0.64 0.02
2.99 2.75
1.08 0.27
1.28 0.23
0.79 0.10
0.99 0.21
0.88 0.06
1.17 0.27
1.10 0.17
0.95 0.18
0.64 0.02
1.27 0.29
0.72 0.06
26.31 0.07
26.43 4.63
30.34 2.85
>50
25.41 0.17
20.69 5.81
23.30 1.23
19.60 3.97
10.45 1.15
16.17 1.93
12.66 4.18
12.29 0.45
13.71 1.65
17.86 4.91
15.76 2.75
22.05 1.19
19.60 4.20
19.96 2.84
18.69 2.83
10.59 2.13
13.36 2.01
15.58 2.72
12.33 0.67
22.48 0.78
21.4 0.53
10.6
42.9
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
41.2
17.43 2.52
16.98 3.08
22.69 4.96
26.52 1.16
18.96 3.49
23.63 1.88
28.71 3.35
19.01 0.85
23.66 1.83
16.51 1.47
21.56 2.5
19.99 2.39
22.22 2.17
24.83 2.4
29.32 1.45
>50
48.4
48.6
54.0
49.2
37.6
53.6
37.6
50.6
56.9
38.9
40.9
30.7
35.7
40.4
25.5
39.9
41.1
40.7
42.7
21.4
23.1
14.2
13.3
2.2
>50
>50
>50
>50
>50
25.27 5.17
0.76 0.08
1.09 0.04
2.12 0.05
25.17 0.91
25.08 0.46
0.63 0.05
1.29 0.16
1.13 0.27
21.57 0.40
22.98 0.66
0.66 0.01
1.30 0.20
1.50 0.37
19.61 2.85
19.32 3.21
0.08 0.00
2.41 0.70
2.50 0.48
gCamptothecin (C)
hEtoposide (E)
iAdriamycin
79.0
54.9
28.9
NA: not applicable.
a
Each data represents mean S.D. from three different experiments performed in triplicate.
MCF-7: human breast adenocarcinoma.
HeLa: human cervix tumor.
DU145: human prostate tumor.
HCT15: human colorectal adenocarcinoma.
b
c
d
e
f
K562: chronic myelogenous leukemia.
g
h
i
Camptothecin; positive control for topo I and cytotoxicity.
Etoposide: positive control for topo II and cytotoxicity.
Adriamycin: positive control for cytotoxicity.
2.3. Cytotoxicity
noma cell line (HCT15), and chronic myelogenous leukemia cell
line (K562). Inhibitory activities (IC50) were presented as micromo-
lar concentrations of the compounds as shown in Table 1. It was
found that compounds 7–21 displayed stronger or similar cytotox-
icity against HCT15 cell line compared to positive controls. And
several compounds exhibit similar or moderate cytotoxicity
Synthesized compounds were evaluated for cytotoxicity on five
different human cancer cell lines: human breast adenocarcinoma
cell line (MCF-7), human cervix tumor cell line (HeLa), human
prostate tumor cell line (DU145), human colorectal adenocarci-

P. Thapa et al. / Bioorg. Med. Chem. 18 (2010) 377–386
381
Lane D: pBR322 DNA only
Lane T: pBR322 DNA + Topo I
Lane C: pBR322 DNA + Topo I + Camptothecin
Lane 7 – 29 (100 M): pBR322 DNA + Topo I + Compound 7 – 29
Lane 10 and 11 (20 M): pBR322 DNA + Topo I + Compound 10 and 11
Figure 5. Human DNA topoisomerase I inhibitory activity of compounds 7–29.
against the MCF-7 cell line. Most of the compounds exhibit moder-
ate cytotoxicity against HeLa, DU145, and K562 cell lines but weak-
er than those of positive controls.
column (4.6 ꢀ 250 mm) with a gradient elution of 80–100% of B in
A for 10 min followed by 100–80% of B in A for 20 min at a flow rate
of 1.0 mL/min at 254 nm UV detection, in which 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
We have designed and synthesized 60 2-thienyl-4-furyl-6-aryl
pyridine derivatives systematically in a total of six series by effi-
cient synthetic routes and evaluated them for topo I and II inhibi-
tory activities and antitumor cytotoxicity. Structure–activity
relationship study revealed that 2-(5-chlorothiophen-2-yl)-4-(fur-
an-3-yl) moiety was important to display topo II inhibitory activ-
ity. As almost all of the compounds were devoid of topo I
inhibitory activity, it was difficult to infer the structure–activity
relationship. However, two compounds (10 and 11), which showed
considerable topo I inhibitory activity, have 4-chlorophenyl in
common. That indicates, to some extent, that 4-chlorophenyl
might have an important role in displaying topo I inhibitory activ-
ity. For cytotoxicity, 4-(furan-2 or 3-yl)-6-(3-methylthiophen-2-yl)
and 2-(5-chlorothiophen-2-yl)-4-(furan-3-yl) moieties with com-
bination of j, l, e, and l, n, respectively, were important.
AdvantageÒ LC/MS/MS spectrometry utilizing XcaliburÒ program.
For ESI LC/MS, LC was performed with 8
l
L injection volume on
a
Waters X-TerraÒ 3.5
lm
reverse-phase C18 column (2.1 ꢀ
100 mm) with a gradient elution: (A) from 70% to 90% of B in A
for 6 min followed by 90% to 70% of B in A for 1 min, and 70% of
B in A for 8 min (B) from 70% to 90% of B in A for 6 min followed
by 90% to 70% of B in A for 1 min, and 70% to 30% of B in A for
8 min (C) from 80% to 95% of B in A for 5 min followed by 95%
to 80% of B in A for 1 min, and 80% of B in A for 9 min at a flow
rate of 200 lL/min, in which mobile phase A was 100% distilled
water with 20 mM AF and mobile phase B was 100% ACN. MS ion-
ization conditions were: Sheath gas flow rate: 40 arb, aux gas flow
rate: 0 arb, I spray voltage: 5.3 KV, capillary temp.: 275 °C, capil-
lary voltage: 27 V, tube lens offset: 45 V. Retention time is given
in minutes.
4. Experimental
4.1. General method for preparation of 3
Compounds used as starting materials and reagents were ob-
tained from Aldrich Chemicals Co., or Junsei, and utilized without
further purification. HPLC grade acetonitrile (ACN) and methanol
were purchased from Burdick and Jackson, USA. Thin-layer chro-
matography (TLC) and column chromatography (CC) were per-
formed with Kieselgel 60 F254 (Merck) and silica gel (Kieselgel 60,
230–400 mesh, Merck) respectively. Since all the compounds pre-
pared contained aromatic rings, 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) for 1H
NMR and 62.5 MHz for 13C NMR, and chemical shifts were cali-
brated to TMS (tetramethylsilane). 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 vol-
To an ice cold solution of 85% KOH (1.2 equiv) in methanol
(10 mL) and H2O (2 mL) aryl acetyl 1 (1.0 equiv) was added. After
dissolution, aryl aldehyde 2 (1.0 equiv) was added slowly. The
reaction mixture was then stirred for 10 min to 3 h at 0 °C. Precip-
itate formed was filtered, washed with cold MeOH and dried to get
solid compound 3 in 65.7–94.2% yield. Following the same proce-
dure, 6 compounds were synthesized.
4.1.1. 3-Furan-2-yl-1-(3-methyl-thiophen-2-yl)-propenone 3
(R1 = b, R2 = e)
The same procedure described in Section 4.1 was employed
with 1 (R1 = b) and 2 (R2 = e) to yield yellow solid 65.70%.
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.35; mp 69.6 °C.
1H NMR (250 MHz, CDCl3) d 7.50 (d, J = 14.88 Hz, 1H, –CO–
C@CH–), 7.47 (br, 1H, 3-furan H-5), 7.43 (d, J = 4.92 Hz, 1H, 1-thio-
phene H-5), 7.20 (d, J = 15.01 Hz, 1H, –CO–CH@C–), 6.96 (d,
J = 4.88 Hz, 1H, 1-thiophene H-4), 6.67 (d, J = 3.26 Hz, 1H, 3-furan
H-3), 6.46 (dd, J = 3.19, 1.71 Hz, 1H, 3-furan H-4), 2.60 (s, 3H, 1-thi-
ophene 3-CH3).
ume of 10 l 5 lM reverse-phase C18
L, was run in Waters X-TerraÒ

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P. Thapa et al. / Bioorg. Med. Chem. 18 (2010) 377–386
4.1.2. 3-Furan-3-yl-1-(3-methyl-thiophen-2-yl)-propenone 3
(R1 = b, R2 = d)
22 h under nitrogen gas. The solvent was evaporated and the resi-
due extracted with ethyl acetate (80 mL), washed with water
(50 mL ꢀ 3), saturated NaCl solution (30 mL) and dried over
MgSO4. After filtration, the filtrate was concentrated and purified
by silica gel column chromatography with a gradient elution of
ethyl acetate/n-hexane to afford a white solid compound 6 in
20.6–71.6% yield. Following the same procedure, 60 compounds
were synthesized.
The same procedure described in Section 4.1 was employed
with 1 (R1 = b) and 2 (R2 = d) to yield creamy white solid 92.85%.
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.35; mp 68.6 °C.
1H NMR (250 MHz, CDCl3) d 7.73 (br, 1H, 3-furan H-2), 7.69 (d,
J = 15.46 Hz, 1H, –CO–C@CH–), 7.46 (br, 1H, 3-furan H-5), 7.45 (d,
J = 5.04 Hz, 1H, 1-thiophene H-5), 7.06 (d, J = 15.20 Hz, 1H, –CO–
CH @C–), 6.99 (d, J = 4.93 Hz, 1H, 1-thiophene H-4), 6.69 (d,
J = 1.06 Hz, 1H, 3-furan H-4).
4.3.1. 2-(2-Chlorophenyl)-4-(furan-2-yl)-6-(3-methylthiophen-
2-yl) pyridine (7)
4.1.3. 3-(5-Chlorofuran-2-yl)-1-(3-methyl-thiophen-2-yl)-
propenone 3 (R1 = b, R2 = c)
The same procedure described in Section 4.3 was employed
with 3 (R1 = b, R2 = e) (0.76 g, 3.50 mmol), dry ammonium acetate
(2.69 g, 35.00 mmol), 5 (R3 = j) (1.25 g, 3.50 mmol) and glacial ace-
tic acid (8.00 mL) to yield a white solid (284 mg, 23.10%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.31; mp 89.5 °C, purity:
99.2%.
The same procedure described in Section 4.1 was employed
with 1 (R1 = b) and 2 (R2 = c) to yield light yellow solid 92.43%.
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.34; mp 95.6 °C.
1H NMR (250 MHz, CDCl3) d 7.47 (d, J = 4.98 Hz, 1H, 1-thiophene
H-5), 7.43 (d, J = 15.10 Hz, 1H, –CO–C@CH–), 7.20 (d, J = 15.07 Hz,
1H, –CO–CH@C–), 6.99 (d, J = 4.96 Hz, 1H, 1-thiophene H-4), 6.67
(d, J = 3.45 Hz, 1H, 3-furan H-3), 6.30 (d, J = 3.45 Hz, 1H, 3-furan
H-4).
LC/MS/MS (condition A): retention time: 6.74 min; [MH+]:
352.2 (100%), [MH+2]: 354.2 (38%).
1H NMR (250 MHz, CDCl3) d 7.77 (d, J = 1.32 Hz, 1H, pyridine H-
3), 7.76 (d, J = 1.34 Hz, 1H, pyridine H-5), 7.73–7.69 (m, 1H, 2-phe-
nyl H-6), 7.56 (dd, J = 1.72, 0.60 Hz, 1H, 4-furan H-5), 7.49–7.46 (m,
1H, 2-phenyl H-3), 7.40–7.32 (m, 2H, 2-phenyl H-4, H-5), 7.27 (d,
J = 5.04 Hz, 1H, 6-thiophene H-5), 6.94 (d, J = 5.01 Hz, 1H, 6-thio-
phene H-4), 6.92 (dd, J = 3.38, 0.63 Hz, 1H, 4-furan H-3), 6.54 (dd,
J = 3.44, 1.79 Hz, 1H, 4-furan H-4), 2.59 (s, 3H, 6-thiophene 3-CH3).
13C NMR (62.5 MHz, CDCl3) d 156.91, 153.92, 151.61, 143.82,
139.08, 138.00, 137.76, 135.95, 132.31, 132.07, 131.80, 130.18,
129.58, 126.95, 125.48, 116.66, 114.26, 112.12, 108.80, 16.28.
4.1.4. 1-(5-Chlorothiophen-2-yl)-3-(furan-2-yl)-propenone 3
(R1 = a, R2 = e)
The same procedure described in Section 4.1 was employed
with 1 (R1 = a) and 2 (R2 = e) to yield light yellow solid 94.21%.
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.36; mp 100.3 °C.
1H NMR (250 MHz, CDCl3) d 7.61 (d, J = 4.09 Hz, 1H, 1-thiophene
H-3), 7.56 (d, J = 15.33 Hz, 1H, –CO–C@CH–), 7.52 (d, J = 1.41 Hz,
1H, 3-furan H-5), 7.20 (d, J = 15.27 Hz, 1H, –CO–CH@C–), 6.98 (d,
J = 4.05 Hz, 1H, 1-thiophene H-4), 6.71 (d, J = 3.40 Hz, 1H, 3-furan
H-3), 6.50 (dd, J = 3.42, 1.80 Hz, 1H, 3-furan H-4).
4.3.2. 2-(4-Chlorophenyl)-4-(furan-2-yl)-6-(3-methylthiophen-
2-yl) pyridine (8)
The same procedure described in Section 4.3 was employed
with 3 (R1 = b, R2 = e) (0.43 g, 2.00 mmol), dry ammonium acetate
(1.54 g, 20.00 mmol), 5 (R3 = l) (0.72 g, 2.00 mmol) and glacial ace-
tic acid (5.00 mL) to yield a white solid (250 mg, 35.58%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.34; mp 97.6 °C, purity:
99.4%.
4.1.5. 1-(5-Chlorothiophen-2-yl)-3-(furan-3-yl)-propenone 3
(R1 = a, R2 = d)
The same procedure described in Section 4.1 was employed
with 1 (R1 = a) and 2 (R2 = d) to yield white solid 85.56%.
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.28; mp 124.5 °C.
1H NMR (250 MHz, CDCl3) d 7.75 (d, J = 0.39 Hz, 1H, 3-furan H-
2), 7.74 (d, J = 15.34 Hz, –CO–C@CH–), 7.61 (d, J = 4.07 Hz, 1H, 1-
thiophene H-3), 7.48 (br, 1H, 3-furan H-5), 7.03 (d, J = 14.94 Hz,
1H, –CO–CH@C–), 6.99 (d, J = 3.79 Hz, 1H, 1-thiophene H-4), 6.69
(dd, J = 1.24, 0.43 Hz, 1H, 3-furan H-4).
LC/MS/MS (condition A): retention time: 8.62 min; [MH+]:
352.2 (100%), [MH+2]: 354.2 (38%).
1H NMR (250 MHz, CDCl3) d 8.09 (d, J = 8.55 Hz, 2H, 2-phenyl H-
2, H-6), 7.80 (d, J = 1.01 Hz, Hz, pyridine H-3), 7.73 (d, J = 0.93 Hz,
1H, pyridine H-5), 7.57 (d, J = 1.69 Hz, 1H, 4-furan H-5), 7.46 (d,
J = 8.53 Hz, 2H, 2-phenyl H-3, H-5), 7.30 (d, J = 5.05 Hz, 1H, 6-thio-
phene H-5), 6.96 (d, J = 5.40 Hz, 1H, 6-thiophene H-4), 6.95 (d,
J = 3.13 Hz, 1H, 4-furan H-3), 6.55 (dd, J = 3.43, 1.79 Hz, 1H, 4-furan
H-4), 2.62 (s, 3H, 6-thiophene 3-CH3).
4.1.6. 3-(5-Chlorofuran-2-yl)-1-(5-chlorothiophen-2-yl)-
propenone 3 (R1 = a, R2 = c)
The same procedure described in Section 4.1 was employed
with 1 (R1 = a) and 2 (R2 = c) to yield yellow solid 93.99%.
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.29; mp 109.4 °C.
1H NMR (250 MHz, CDCl3) d 7.65 (d, J = 4.08 Hz, 1H, 1-thiophene
H-3), 7.46 (d, J = 15.18 Hz, 1H, –CO–C@CH–), 7.19 (d, J = 15.18 Hz, 1H,
–CO–CH @C–), 7.00 (d, J = 4.07 Hz, 1H, 1-thiophene H-4), 6.70 (d,
J = 3.47 Hz, 1H, 3-furan H-3), 6.32 (d, J = 3.47 Hz, 1H, 3-furan H-4).
13C NMR (62.5 MHz, CDCl3) d 156.02, 153.90, 151.65, 143.79,
138.97, 137.96, 137.53, 136.03, 135.25, 132.25, 128.85, 128.24,
125.56, 114.04, 112.14, 111.69, 108.70, 16.52.
4.3.3. 2-(Furan-2-yl)-4-(furan-3-yl)-6-(3-methylthiophen-2-yl)
pyridine (9)
The same procedure described in Section 4.3 was employed
with 3 (R1 = b, R2 = d) (0.39 g, 1.80 mmol), dry ammonium acetate
(1.38 g, 18.00 mmol), 5 (R3 = e) (0.56 g, 1.80 mmol) and glacial ace-
tic acid (2.00 mL) to yield a white solid (396 mg, 71.60%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.37; mp 94.6 °C, purity:
99.2%.
4.2. General method for preparation of 5
A mixture of aryl acetyl, iodine (1.0 equiv) and pyridine (60 mL)
was refluxed at 140 °C for 3 h. The reaction mixture was cooled to
0 °C, precipitate formed was filtered, washed with cold pyridine
and dried to get solid compound 5 in 42.2–99.4% yield. Following
the same procedure, 10 compounds were synthesized.
LC/MS/MS (condition B): retention time: 4.91 min; [MH+]: 308.2
(100%).
1H NMR (250 MHz, CDCl3) d 7.95 (dd, J = 1.36, 0.89 Hz, 1H, 4-
furan H-2), 7.65 (d, J = 1.36 Hz, 1H, pyridine H-3), 7.55 (t,
J = 1.66 Hz, 1H, 4-furan H-5), 7.54 (dd, J = 1.65, 0.84 Hz, 1H, 2-furan
H-5), 7.48 (d, J = 1.35 Hz, 1H, pyridine H-5), 7.28 (d, J = 5.05 Hz, 1H,
6-thiophene H-5), 7.17 (dd, J = 3.36, 0.68 Hz, 1H, 2-furan H-3), 6.95
4.3. General method for preparation of 6
A mixture of 3 (R1 = a, b, R2 = c–e), 5 (R3 = e–n) and dry ammo-
nium acetate in glacial acetic acid was heated to 80–95 °C for 16–

P. Thapa et al. / Bioorg. Med. Chem. 18 (2010) 377–386
383
(d, J = 5.05 Hz, 1H, 6-thiophene H-4), 6.82 (dd, J = 1.85, 0.85 Hz, 1H,
4-furan H-4), 6.55 (dd, J = 3.38, 1.76 Hz, 1H, 2-furan H-4), 2.60 (s,
3H, 6-thiophene 3-CH3).
13C NMR (62.5 MHz, CDCl3) d 153.38, 151.99, 149.52, 144.35,
143.53, 143.35, 141.47, 140.53, 132.58, 127.07, 124.38, 123.58,
113.42, 112.67, 112.16, 109.40, 108.37.
13C NMR (62.5 MHz, CDCl3) d 153.92, 153.81, 149.48, 144.28,
143.19, 141.18, 140.43, 137.40, 136.17, 132.16, 125.29, 124.62,
116.43, 112.55, 112.11, 109.17, 108.45, 16.28.
4.3.7. 2-(5-Chlorothiophen-2-yl)-4-(furan-3-yl)-6-(5-
methylfuran-2-yl) pyridine (13)
The same procedure described in Section 4.3 was employed
with 3 (R1 = a, R2 = d) (0.35 g, 1.50 mmol), dry ammonium acetate
(1.15 g, 15.00 mmol), 5 (R3 = f) (0.49 g, 1.50 mmol) and glacial ace-
tic acid (5.00 mL) to yield a white solid (180 mg, 35.20%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.35; mp 166.9 °C, purity:
100%.
4.3.4. 2-(4-Chlorophenyl)-4-(furan-3-yl)-6-(3-methylthiophen-
2-yl) pyridine (10)
The same procedure described in Section 4.3 was employed
with 3 (R1 = b, R2 = d) (0.39 g, 1.80 mmol), dry ammonium acetate
(1.38 g, 18.00 mmol), 5 (R3 = l) (0.64 g, 1.80 mmol) and glacial ace-
tic acid (2.00 mL) to yield a white solid (450 mg, 71.11%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.35; mp 89.30 °C, purity:
99.2%.
LC/MS/MS (condition B): retention time: 8.52 min; [MH+]:
342.1 (100%), [MH+2]: 344.1 (38%).
1H NMR (250 MHz, CDCl3) d 7.95 (dd, J = 1.22, 0.90 Hz, 1H, 4-
furan H-2), 7.57 (d, J = 1.32 Hz, 1H, pyridine H-5), 7.55 (t,
J = 1.60 Hz, 1H, 4-furan H-5), 7.42 (d, J = 1.35 Hz, 1H, pyridine
H-3), 7.39 (d, J = 3.96 Hz, 1H, 2-thiophene H-3), 7.06 (d,
J = 3.21 Hz, 1H, 6-furan H-3), 6.92 (d, J = 3.96 Hz, 1H, 2-thio-
phene H-4), 6.82 (dd, J = 1.82, 0.82 Hz, 1H, 4-furan H-4), 6.15
(dd, J = 3.24, 0.93 Hz, 1H, 6-furan H-4), 2.42 (s, 3H, 6-furan 5-
CH3).
LC/MS/MS (condition B): retention time: 8.69 min; [MH+]:
352.2 (100%), [MH+2]: 354.2 (38%).
1H NMR (250 MHz, CDCl3) d 8.06 (d, J = 8.53 Hz, 2H, 2-phenyl H-
2, H-6), 7.94 (br, 1H, 4-furan H-2), 7.63 (d, J = 1.16 Hz, 1H, pyridine
H-3), 7.56 (d, J = 1.45 Hz, 1H, pyridine H-5), 7.55 (t, J = 2.01 Hz, 1H,
4-furan H-5), 7.45 (d, J = 8.54 Hz, 2H, 2-phenyl H-3, H-5), 7.30 (d,
J = 5.06 Hz, 1H, 6-thiophene H-5), 6.96 (d, J = 5.06 Hz, 1H, 6-thio-
phene H-4), 6.81 (dd, J = 1.81, 0.81 Hz, 1H, 4-furan H-4), 2.62 (s,
3H, 6-thiophene 3-CH3).
13C NMR (62.5 MHz, CDCl3) d 153.64, 151.88, 151.75, 149.68,
144.25, 143.70, 141.32, 140.49, 132.41, 127.02, 124.46, 123.46,
112.89, 112.17, 110.57, 108.46, 108.43, 13.94.
13C NMR (62.5 MHz, CDCl3) d 156.09, 153.95, 144.38, 141.50,
140.33, 137.83, 137.53, 135.98, 135.22, 132.24, 128.85, 128.23,
125.52, 124.70, 116.76, 114.35, 108.46, 16.48.
4.3.8. 2-(5-Chlorothiophen-2-yl)-4-(furan-3-yl)-6-o-tolyl
pyridine (14)
4.3.5. 2-(4-Chlorophenyl)-6-(5-chlorothiophen-2-yl)-4-(furan-
2-yl) pyridine (11)
The same procedure described in Section 4.3 was employed
with 3 (R1 = a, R2 = d) (0.35 g, 1.50 mmol), dry ammonium acetate
(1.15 g, 15.00 mmol), 5 (R3 = g) (0.50 g, 1.50 mmol) and glacial ace-
tic acid (5.00 mL) to yield a white solid (114 mg, 21.60%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.33; mp 93.3 °C, purity:
99.1%.
The same procedure described in Section 4.3 was employed
with 3 (R1 = a, R2 = e) (0.47 g, 2.00 mmol), dry ammonium acetate
(1.54 g, 20.00 mmol), 5 (R3 = l) (0.72 g, 2.00 mmol) and glacial ace-
tic acid (5.00 mL) to yield a white solid (320 mg, 43.03%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.25; mp 187.4 °C, purity:
97.8%.
LC/MS/MS (condition B): retention time: 8.75 min; [MH+]:
352.2 (100%), [MH+2]: 354.2 (38%).
LC/MS/MS (condition C): retention time: 7.23 min; [M+]: 372.2
(100%), [M+2]: 374.2 (71%), [M+4]: 376.2 (15%).
1H NMR (250 MHz, CDCl3) d 7.93 (br, 1H, 4-furan H-2), 7.57 (d,
J = 1.27 Hz, 1H, pyridine H-3), 7.55 (t, J = 1.54 Hz, 1H, 4-furan H-5),
7.49–7.46 (m, 1H, 6-phenyl H-6), 7.42 (d, J = 3.96 Hz, 1H, 2-thio-
phene H-3), 7.36 (d, J = 1.23 Hz, 1H, pyridine H-5), 7.32–7.28 (m,
3H, 6-phenyl H-3, H-4, H-5), 6.93 (d, J = 3.96 Hz, 1H, 2-thiophene
H-4), 6.79 (dd, J = 1.80, 0.84 Hz, 1H, 4-furan H-4), 2.49 (s, 3H, 6-
phenyl 2-CH3).
1H NMR (250 MHz, CDCl3) d 8.04 (d, J = 8.62 Hz, 2H, 2-phenyl H-
2, H-6), 7.77 (d, J = 1.18 Hz, 1H, pyridine H-3), 7.70 (d, J = 1.18 Hz,
1H, pyridine H-5), 7.57 (d, J = 1.40 Hz, 1H, 4-furan H-5), 7.44 (d,
J = 8.61 Hz, 2H, 2-phenyl H-3, H-5), 7.42 (d, J = 3.99 Hz, 1H, 6-thio-
phene H-3), 6.94 (d, J = 3.49 Hz, 1H, 4-furan H-3), 6.93 (d,
J = 3.98 Hz, 1H, 6-thiophene H-4), 6.55 (dd, J = 3.43, 1.79 Hz, 1H,
4-furan H-4).
13C NMR (62.5 MHz, CDCl3) d 160.40, 151.44, 144.40, 143.93,
141.13, 140.38, 139.57, 136.48, 132.48, 131.06, 129.56, 128.55,
127.07, 125.90, 124.44, 123.34, 119.24, 112.37, 108.36, 20.70.
13C NMR (62.5 MHz, CDCl3) d 155.97, 152.00, 151.26, 143.87,
143.76, 139.13, 136.92, 135.41, 132.73, 128.86, 128.14, 127.14,
123.63, 112.48, 112.21, 110.49, 108.95.
4.3.9. 2-(5-Chlorothiophen-2-yl)-4-(furan-3-yl)-6-m-tolyl
pyridine (15)
4.3.6. 2-(5-Chlorothiophen-2-yl)-6-(furan-2-yl)-4-(furan-3-yl)
pyridine (12)
The same procedure described in Section 4.3 was employed
with 3 (R1 = a, R2 = d) (0.35 g, 1.50 mmol), dry ammonium acetate
(1.15 g, 15.00 mmol), 5 (R3 = h) (0.50 g, 1.50 mmol) and glacial ace-
tic acid (5.00 mL) to yield a white solid (133 mg, 25.33%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.35; mp 103.7 °C, purity:
100%.
The same procedure described in Section 4.3 was employed
with 3 (R1 = a, R2 = d) (0.35 g, 1.50 mmol), dry ammonium acetate
(1.15 g, 15.00 mmol), 5 (R3 = e) (0.47 g, 1.50 mmol) and glacial ace-
tic acid (5.00 mL) to yield a white solid (298 mg, 60.71%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.30; mp 105.0 °C, purity:
100%.
LC/MS/MS (condition C): retention time: 5.59 min; [MH+]:
352.2 (100%), [MH+2]: 354.2 (38%).
LC/MS/MS (condition B): retention time: 7.67 min; [MH+]:
328.1 (100%), [MH+2]: 330.1 (38%).
1H NMR (250 MHz, CDCl3) d 7.96 (d, J = 0.79 Hz, 1H, 4-furan H-
2), 7.90 (s, 1H, 6-phenyl H-2), 7.89 (d, J = 8.09 Hz, 1H, 6-phenyl H-
6), 7.65 (d, J = 1.12 Hz, 1H, pyridine H-5), 7.56 (t, J = 1.75 Hz, 1H, 4-
furan H-5), 7.54 (d, J = 1.14 Hz, 1H, pyridine H-3), 7.42 (d,
J = 3.91 Hz, 1H, 2-thiophene H-3), 7.39 (t, J = 7.48 Hz, 1H, 6-phenyl
H-5), 7.26 (d, J = 7.08 Hz, 1H, 6-phenyl H-4), 6.95 (d, J = 3.95 Hz, 1H,
2-thiophene H-4), 6.82 (dd, J = 1.83, 0.87 Hz, 1H, 4-furan H-4), 2.47
(s, 3H, 6-phenyl 3-CH3).
1H NMR (250 MHz, CDCl3) d 7.96 (dd, J = 1.26, 0.97 Hz, 1H, 4-
furan H-2), 7.63 (d, J = 1.36 Hz, 1H, pyridine H-5), 7.56 (t,
J = 1.90 Hz, 1H, 4-furan H-5), 7.55 (br, 1H, 6-furan H-5), 7.47 (d,
J = 1.38 Hz, 1H, pyridine H-3), 7.40 (d, J = 3.96 Hz, 1H, 2-thiophene
H-3), 7.17 (dd, J = 3.38, 0.70 Hz, 1H, 6-furan H-3), 6.93 (d,
J = 3.96 Hz, 1H, 2-thiophene H-4), 6.82 (dd, J = 1.88, 0.88 Hz, 1H,
4-furan H-4), 6.56 (dd, J = 3.39, 1.77 Hz, 1H, 6-furan H-4).

384
P. Thapa et al. / Bioorg. Med. Chem. 18 (2010) 377–386
13C NMR (62.5 MHz, CDCl3) d 157.54, 151.91, 144.35, 144.05,
141.52, 140.37, 138.55, 138.34, 132.50, 130.09, 128.62, 127.57,
127.07, 124.58, 124.06, 123.36, 115.59, 112.87, 108.45, 21.62.
4.3.13. 2-(4-Chlorophenyl)-6-(5-chlorothiophen-2-yl)-4-(furan-
3-yl) pyridine (19)
The same procedure described in Section 4.3 was employed
with 3 (R1 = a, R2 = d) (0.35 g, 1.50 mmol), dry ammonium acetate
(1.15 g, 15.00 mmol), 5 (R3 = l) (0.54 g, 1.50 mmol) and glacial ace-
tic acid (5.00 mL) to yield a white solid (232 mg, 41.54%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.26; mp 152.6 °C, purity:
100%.
4.3.10. 2-(5-Chlorothiophen-2-yl)-4-(furan-3-yl)-6-p-tolyl
pyridine (16)
The same procedure described in Section 4.3 was employed
with 3 (R1 = a, R2 = d) (0.35 g, 1.50 mmol), dry ammonium acetate
(1.15 g, 15.00 mmol), 5 (R3 = i) (0.50 g, 1.50 mmol) and glacial ace-
tic acid (5.00 mL) to yield a white solid (241 mg, 45.74%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.41; mp 118.4 °C, purity:
100%.
LC/MS/MS (condition C): retention time: 6.23 min; [M+]: 372.1
(100%), [M+2]: 374.1 (71%), [M+4]: 376.1 (15%).
1H NMR (250 MHz, CDCl3) d 8.04 (d, J = 8.72 Hz, 2H, 2-phenyl H-
2, H-6), 7.95 (dd, J = 1.30, 0.98 Hz, 1H, 4-furan H-2), 7.62 (d,
J = 1.28 Hz, 1H, pyridine H-3), 7.57 (t, J = 1.67 Hz, 1H, 4-furan H-
5), 7.55 (d, J = 1.27 Hz, 1H, pyridine H-5), 7.46 (d, J = 8.70 Hz, 2H,
2-phenyl H-3, H-5), 7.42 (d, J = 3.97 Hz, 1H, 6-thiophene H-3),
6.94 (d, J = 3.97 Hz, 1H, 6-thiophene H-4), 6.81 (dd, J = 1.87,
0.88 Hz, 1H, 4-furan H-4).
LC/MS/MS (condition C): retention time: 5.67 min; [MH+]:
352.2 (100%), [MH+2]: 354.2 (38%).
1H NMR (250 MHz, CDCl3) d 8.00 (d, J = 8.20 Hz, 2H, 6-phenyl H-
2, H-6), 7.94 (br, 1H, 4-furan H-2), 7.64 (d, J = 1.22 Hz, 1H, pyridine
H-5), 7.56 (t, J = 1.63 Hz, 1H, 4-furan H-5), 7.52 (d, J = 1.23 Hz, 1H,
pyridine H-3), 7.41 (d, J = 3.95 Hz, 1H, 2-thiophene H-3), 7.30 (d,
J = 8.03 Hz, 2H, 6-phenyl H-3, H-5), 6.94 (d, J = 3.95 Hz, 1H, 2-thio-
phene H-4), 6.81 (dd, J = 1.80, 0.81 Hz, 1H, 4-furan H-4), 2.42 (s, 3H,
6-phenyl 4-CH3).
13C NMR (62.5 MHz, CDCl3) d 156.08, 152.05, 144.44, 143.74,
141.79, 140.43, 136.96, 135.42, 132.75, 128.88, 128.15, 127.13,
124.43, 123.55, 115.19, 113.13, 108.38.
13C NMR (62.5 MHz, CDCl3) d 157.33, 151.86, 144.33, 144.15,
141.49, 140.33, 139.39, 135.77, 132.48, 129.43, 127.05, 126.76,
124.62, 123.28, 115.16, 112.63, 108.45, 21.33.
4.3.14. 6-(5-Chlorothiophen-2-yl)-4-(furan-3-yl)-2, 20-
bipyridine (20)
The same procedure described in Section 4.3 was employed
with 3 (R1 = a, R2 = d) (0.35 g, 1.50 mmol), dry ammonium acetate
(1.15 g, 15.00 mmol), 5 (R3 = m) (0.49 g, 1.50 mmol) and glacial
acetic acid (5.00 mL) to yield a white solid (275 mg, 54.11%).
Rf (ethyl acetate/n-hexane 1:2 v/v): 0.56; mp 166.7 °C, purity:
100%.
4.3.11. 2-(2-Chlorophenyl)-6-(5-chlorothiophen-2-yl)-4-(furan-
3-yl) pyridine (17)
The same procedure described in Section 4.3 was employed
with 3 (R1 = a, R2 = d) (0.47 g, 2.00 mmol), dry ammonium acetate
(1.54 g, 20.00 mmol), 5 (R3 = j) (0.72 g, 2.00 mmol) and glacial ace-
tic acid (5.00 mL) to yield a white solid (153 mg, 20.64%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.35; mp 160.4 °C, purity:
100%.
LC/MS/MS (condition B): retention time: 7.67 min; [MH+]:
339.2 (100%), [MH+2]: 341.2 (38%).
1H NMR (250 MHz, CDCl3) d 8.69 (d, J = 4.74 Hz, 1H, 2-pyridine
H-60), 8.53 (d, J = 7.98 Hz, 1H, 2-pyridine H-30), 8.40 (d, J = 1.34 Hz,
1H, pyridine H-3), 8.03 (br, 1H, 4-furan H-2), 7.86 (dt, J = 7.70,
1.73 Hz, 1H, 2-pyridine H-40), 7.63 (d, J = 1.35 Hz, 1H, pyridine H-
5), 7.55 (t, J = 1.61 Hz, 1H, 4-furan H-5), 7.43 (d, J = 3.95 Hz, 1H,
6-thiophene H-3), 7.35 (ddd, J = 7.44, 4.80, 0.98 Hz, 1H, 2-pyridine
H-50), 6.95 (d, J = 3.94 Hz, 1H, 6-thiophene H-4), 6.88 (dd, J = 1.02,
0.80 Hz, 1H, 4-furan H-4).
LC/MS/MS (condition C): retention time: 4.49 min; [M+]: 372.2
(100%), [M+2]: 374.1 (71%), [M+4]: 376.1 (15%).
1H NMR (250 MHz, CDCl3) d 7.94 (d, J = 0.91 Hz, 1H, 4-furan H-
2), 7.72–7.68 (m, 1H, 2-phenyl H-6), 7.63 (d, J = 1.31 Hz, 1H, pyri-
dine H-3), 7.60 (d, J = 1.33 Hz, 1H, pyridine H-5), 7.55 (t,
J = 1.71 Hz, 1H, 4-furan H-5), 7.52–7.48 (m, 1H, 2-phenyl H-3),
7.43 (d, J = 3.94 Hz, 1H, 6-thiophene H-3), 7.40–7.35 (m, 2H, 2-phe-
nyl H-4, H-5), 6.94 (d, J = 3.95 Hz, 1H, 6-thiophene H-4), 6.80 (dd,
J = 1.80, 0.83 Hz, 1H, 4-furan H-4).
13C NMR (62.5 MHz, CDCl3) d 156.04, 155.43, 151.57, 148.96,
144.27, 143.85, 141.85, 140.75, 136.99, 132.52, 127.14, 124.45,
124.05, 123.40, 121.46, 116.02, 114.25, 108.48.
13C NMR (62.5 MHz, CDCl3) d 156.95, 152.06, 144.42, 143.53,
140.78, 140.52, 138.54, 132.60, 132.24, 131.75, 130.26, 129.76,
127.10, 127.03, 124.35, 123.57, 120.11, 113.22, 108.40.
4.3.15. 6-(5-Chlorothiophen-2-yl)-4-(furan-3-yl)-2,30-bipyridine
(21)
The same procedure described in Section 4.3 was employed
with 3 (R1 = a, R2 = d) (0.47 g, 2.00 mmol), dry ammonium acetate
(1.54 g, 20.00 mmol), 5 (R3 = n) (0.65 g, 2.00 mmol) and glacial ace-
tic acid (5.00 mL) to yield a white solid (315 mg, 46.51%).
Rf (ethyl acetate/n-hexane 1:2 v/v): 0.28; mp 178.6 °C, purity:
100%.
4.3.12. 2-(3-Chlorophenyl)-6-(5-chlorothiophen-2-yl)-4-(furan-
3-yl) pyridine (18)
The same procedure described in Section 4.3 was employed
with 3 (R1 = a, R2 = d) (0.35 g, 1.50 mmol), dry ammonium acetate
(1.15 g, 15.00 mmol), 5 (R3 = k) (0.54 g, 1.50 mmol) and glacial ace-
tic acid (5.00 mL) to yield a white solid (225 mg, 40.29%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.35; mp 138.3 °C, purity:
100%.
LC/MS/MS (condition B): retention time: 6.22 min; [MH+]:
339.2 (100%), [MH+2]: 341.2 (38%).
1H NMR (250 MHz, CDCl3) d 9.28 (dd, J = 2.22, 0.72 Hz, 1H, 2-
pyridine H-20), 8.68 (dd, J = 4.80, 1.63 Hz, 1H, 2-pyridine H-6’),
8.43 (td, J = 8.02, 1.72 Hz, 1H, 2-pyridine H-4’), 7.97 (dd, J = 1.34,
0.96 Hz, 1H, 4-furan H-2), 7.67 (d, J = 1.26 Hz, 1H, pyridine H-3),
7.60 (d, J = 1.30 Hz, 1H, pyridine H-5), 7.58 (t, J = 1.67 Hz, 1H, 4-
furan H-5), 7.45 (ddd, J = 7.92, 4.75, 0.80 Hz, 1H, 2-pyridine H-
5’), 7.44 (d, J = 3.97 Hz, 1H, 6-thiophene H-3), 6.96 (d,
J = 3.97 Hz, 1H, 6-thiophene H-4), 6.83 (dd, J = 1.87, 0.90 Hz, 1H,
4-furan H-4).
LC/MS/MS (condition C): retention time: 6.22 min; [M+]: 372.2
(100%), [M+2]: 374.1 (71%), [M+4]: 376.1 (15%).
1H NMR (250 MHz, CDCl3) d 8.08–8.07 (m, 1H, 2-phenyl H-2),
7.99–7.97 (m, 1H, 2-phenyl H-6), 7.96 (dd, J = 1.31, 1.06 Hz, 1H,
4-furan H-2), 7.62 (d, J = 1.24 Hz, 1H, pyridine H-3), 7.57 (t,
J = 1.70 Hz, 1H, 4-furan H-5), 7.56 (d, J = 1.26 Hz, 1H, pyridine H-
5), 7.42 (d, J = 3.89 Hz, 1H, 6-thiophene H-3), 7.42–7.40 (m, 2H,
2-phenyl H-4, H-5), 6.95 (d, J = 3.96 Hz, 1H, 6-thiophene H-4),
6.81 (dd, J = 1.81, 0.83 Hz, 1H, 4-furan H-4).
13C NMR (62.5 MHz, CDCl3) d 154.77, 152.38, 150.18, 148.17,
144.54, 143.44, 141.97, 140.54, 134.43, 134.11, 132.96, 127.18,
124.26, 123.76, 123.59, 115.43, 113.54, 108.34.
13C NMR (62.5 MHz, CDCl3) d 155.84, 152.11, 144.47, 143.64,
141.85, 140.49, 140.37, 134.79, 132.82, 129.95, 129.27, 127.14,
127.03, 124.99, 124.38, 123.64, 115.49, 113.43, 108.38.

P. Thapa et al. / Bioorg. Med. Chem. 18 (2010) 377–386
385
4.3.16. 4-(5-Chlorofuran-2-yl)-2-(furan-2-yl)-6-(3-methylthio-
phen-2-yl) pyridine (22)
(0.77 g, 10.00 mmol), 5 (R3 = n) (0.32 g, 1.00 mmol) and glacial ace-
tic acid (2.00 mL) to yield a white solid (195 mg, 55.26%).
Rf (ethyl acetate/n-hexane 1:2 v/v): 0.21; mp 168.0 °C, purity:
98.5%.
The same procedure described in Section 4.3 was employed
with 3 (R1 = b, R2 = c) (0.25 g, 1.00 mmol), dry ammonium acetate
(0.77 g, 10.00 mmol), 5 (R3 = e) (0.31 g, 1.00 mmol) and glacial ace-
tic acid (2.00 mL) to yield a white solid (152 mg, 44.61%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.38; mp 115.0 °C, purity:
99.6%.
LC/MS/MS (condition B): retention time: 6.98 min; [MH+]:
353.2 (100%), [MH+2]: 355.2 (38%).
1H NMR (250 MHz, CDCl3) d 9.34 (d, J = 1.58 Hz, 1H, 2-pyridine
H-2’), 8.68 (dd, J = 4.77, 1.54 Hz, 1H, 2-pyridine H-6’), 8.43 (td,
J = 8.01, 1.75 Hz, 1H, 2-pyridine H-4’), 7.79 (d, J = 1.23 Hz, 1H, pyr-
idine H-3), 7.70 (d, J = 1.18 Hz, 1H, pyridine H-5), 7.43 (ddd,
J = 7.98, 4.82, 0.70 Hz, 1H, 2-pyridine H-5’), 7.32 (d, J = 5.04 Hz,
1H, 6-thiophene H-5), 6.97 (d, J = 4.88 Hz, 1H, 6-thiophene H-4),
6.95 (d, J = 3.44 Hz, 1H, 4-furan H-3), 6.37 (d, J = 3.49 Hz, 1H, 4-fur-
an H-4), 2.64 (s, 3H, 6-thiophene 3-CH3).
LC/MS/MS (condition B): retention time: 8.76 min; [MH+]:
342.1 (100%), [MH+2]: 344.1 (38%).
1H NMR (250 MHz, CDCl3) d 7.73 (d, J = 1.36 Hz, 1H, pyridine H-
3), 7.58 (d, J = 1.35 Hz, 1H, pyridine H-5), 7.55 (dd, J = 1.70, 0.78 Hz,
1H, 2-furan H-5), 7.29 (d, J = 5.05 Hz, 1H, 6-thiophene H-5), 7.17
(dd, J = 3.38, 0.70 Hz, 1H, 2-furan H-3), 6.95 (d, J = 5.10 Hz, 1H, 6-
thiophene H-4), 6.93 (d, J = 3.45 Hz, 1H, 4-furan H-3), 6.55 (dd,
J = 3.39, 1.76 Hz, 1H, 2-furan H-4), 6.34 (d, J = 3.48 Hz, 1H, 4-furan
H-4), 2.61 (s, 3H, 6-thiophene 3-CH3).
13C NMR (62.5 MHz, CDCl3) 154.79, 154.31, 150.78, 150.13,
148.35, 138.39, 138.06, 137.39, 136.47, 134.39, 132.36, 125.82,
123.54, 113.77, 111.32, 110.89, 108.94, 16.60.
13C NMR (62.5 MHz, CDCl3) d 153.98, 153.65, 151.03, 149.49,
143.32, 138.13, 137.67, 137.27, 136.41, 132.21, 125.45, 113.13,
112.13, 110.69, 109.47, 109.34, 108.84, 16.35.
4.3.20. 4-(5-Chlorofuran-2-yl)-2-(5-chlorothiophen-2-yl)-6-
(furan-2-yl) pyridine (26)
The same procedure described in Section 4.3 was employed
with 3 (R1 = a, R2 = c) (0.54 g, 2.00 mmol), dry ammonium acetate
(1.54 g, 20.00 mmol), 5 (R3 = e) (0.63 g, 2.00 mmol) and glacial ace-
tic acid (5.00 mL) to yield a light yellow solid (232 mg, 32.09%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.34; mp 148.5 °C, purity:
100%.
4.3.17. 4-(5-Chlorofuran-2-yl)-2-(3-chlorophenyl)-6-(3-
methylthiophen-2-yl) pyridine (23)
The same procedure described in Section 4.3 was employed
with 3 (R1 = b, R2 = c) (0.30 g, 1.20 mmol), dry ammonium acetate
(0.92 g, 12.00 mmol), 5 (R3 = k) (0.43 g, 1.20 mmol) and glacial ace-
tic acid (2.00 mL) to yield a white solid (250 mg, 53.95%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.39; mp 114.0 °C, purity:
98.9%.
LC/MS/MS (condition B): retention time: 9.30 min; [M+]: 362.1
(100%), [M+2]: 364.1 (71%), [M+4]: 366.1 (15%).
1H NMR (250 MHz, CDCl3) d 7.69 (d, J = 1.31 Hz, 1H, pyridine H-
3), 7.57 (d, J = 1.34 Hz, 1H, pyridine H-5), 7.55 (dd, J = 1.60, 0.65 Hz,
1H, 6-furan H-5), 7.43 (d, J = 3.97 Hz, 1H, 2-thiophene H-3), 7.16
(dd, J = 3.35, 0.46 Hz, 1H, 6-furan H-3), 6.94 (d, J = 3.99 Hz, 1H, 2-
thiophene H-4), 6.93 (d, J = 3.43 Hz, 1H, 4-furan H-3), 6.56 (dd,
J = 3.38, 1.75 Hz, 1H, 6-furan H-4), 6.35 (d, J = 3.49 Hz, 1H, 4-furan
H-4).
LC/MS/MS (condition C): retention time: 7.23 min; [M+]: 386.1
(100%), [M+2]: 388.1 (71%), [M+4]: 390.1 (15%).
1H NMR (250 MHz, CDCl3) d 8.13–8.12 (m, 1H, 2-phenyl H-2),
8.04–7.99 (m, 1H, 2-phenyl H-6), 7.75 (d, J = 1.26 Hz, 1H, pyridine
H-3), 7.67 (d, J = 1.23 Hz, 1H, pyridine H-5), 7.43–7.40 (m, 2H, 2-
phenyl H-4, H-5), 7.31 (d, J = 5.06 Hz, 1H, 6-thiophene H-5), 6.97
(d, J = 5.09 Hz, 1H, 6-thiophene H-4), 6.94 (d, J = 3.49 Hz, 1H, 4-fur-
an H-3), 6.36 (d, J = 3.48 Hz, 1H, 4-furan H-4), 2.63 (s, 3H, 6-thio-
phene 3-CH3).
13C NMR (62.5 MHz, CDCl3) d 153.20, 152.08, 150.70, 149.49,
143.46, 143.33, 138.25, 137.83, 132.76, 127.12, 123.86, 112.18,
110.93, 110.23, 109.55, 109.51, 108.90.
13C NMR (62.5 MHz, CDCl3) d 155.87, 154.03, 150.95, 140.68,
138.27, 137.99, 137.60, 136.27, 134.76, 132.29, 129.93, 129.20,
127.11, 125.73, 125.05, 113.70, 111.39, 110.74, 108.90, 16.53.
4.3.21. 4-(5-Chlorofuran-2-yl)-2-(5-chlorothiophen-2-yl)-6-p-
tolyl pyridine (27)
The same procedure described in Section 4.3 was employed
with 3 (R1 = a, R2 = c) (0.54 g, 2.00 mmol), dry ammonium acetate
(1.54 g, 20.00 mmol), 5 (R3 = i) (0.67 g, 2.00 mmol) and glacial ace-
tic acid (5.00 mL) to yield a white solid (195 mg, 25.26%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.41; mp 157.9 °C, purity:
100%.
4.3.18. 4-(5-Chlorofuran-2-yl)-2-(4-chlorophenyl)-6-(3-
methylthiophen-2-yl) pyridine (24)
The same procedure described in Section 4.3 was employed
with 3 (R1 = b, R2 = c) (0.30 g, 1.20 mmol), dry ammonium acetate
(0.92 g, 12.00 mmol), 5 (R3 = l) (0.43 g, 1.20 mmol) and glacial ace-
tic acid (2.00 mL) to yield a white solid (221 mg, 47.82%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.39; mp 101.8 °C, purity:
99.2%.
LC/MS/MS (condition C): retention time: 7.94 min; [M+]: 386.1
(100%), [M+2]: 388.1 (71%), [M+4]: 390.1 (15%).
1H NMR (250 MHz, CDCl3) d 8.01 (d, J = 8.19 Hz, 2H, 6-phenyl H-
2, H-6), 7.72 (d, J = 1.20 Hz, 1H, pyridine H-5), 7.61 (d, J = 1.20 Hz,
1H, pyridine H-3), 7.43 (d, J = 3.97 Hz, 1H, 2-thiophene H-3), 7.30
(d, J = 7.99 Hz, 2H, 6-phenyl H-3, H-5), 6.94 (d, J = 3.97 Hz, 1H, 2-
thiophene H-4), 6.92 (d, J = 3.49 Hz, 1H, 4-furan H-3), 6.34 (d,
J = 3.48 Hz, 1H, 4-furan H-4), 2.42 (s, 3H, 6-phenyl 4-CH3).
13C NMR (62.5 MHz, CDCl3) d 157.33, 151.93, 151.00, 143.98,
139.53, 138.11, 137.92, 135.55, 132.66, 129.43, 127.09, 126.75,
123.54, 111.83, 110.61, 109.47, 108.86, 21.34.
LC/MS/MS (condition C): retention time: 7.24 min; [M+]: 386.1
(100%), [M+2]: 388.1 (71%), [M+4]: 390.1 (15%).
1H NMR (250 MHz, CDCl3) d 8.08 (d, J = 8.57 Hz, 2H, 2-phenyl H-
2, H-6), 7.74 (d, J = 1.07 Hz, 1H, pyridine H-3), 7.65 (d, J = 1.00 Hz,
1H, pyridine H-5), 7.46 (d, J = 8.56 Hz, 2H, 2-phenyl H-3, H-5),
7.31 (d, J = 5.05 Hz, 1H, 6-thiophene H-5), 6.96 (d, J = 5.07 Hz, 1H,
6-thiophene H-4), 6.93 (d, J = 3.48 Hz, 1H, 4-furan H-3), 6.35 (d,
J = 3.46 Hz, 1H, 4-furan H-4), 2.62 (s, 3H, 6-thiophene 3-CH3).
13C NMR (62.5 MHz, CDCl3) d 156.10, 153.94, 151.02, 138.20,
137.94, 137.68, 137.27, 136.19, 135.34, 132.29, 128.86, 128.22,
125.67, 113.40, 111.09, 110.65, 108.87, 16.54.
4.3.22. 4-(5-Chlorofuran-2-yl)-2-(3-chlorophenyl)-6-(5-
chlorothiophen-2-yl) pyridine (28)
The same procedure described in Section 4.3 was employed
with 3 (R1 = a, R2 = c) (0.27 g, 1.00 mmol), dry ammonium acetate
(0.77 g, 10.00 mmol), 5 (R3 = k) (0.36 g, 1.00 mmol) and glacial ace-
tic acid (5.00 mL) to yield a creamy white solid (102 mg, 25.20%).
4.3.19. 4-(5-Chlorofuran-2-yl)-6-(3-methylthiophen-2-yl)-2,30-
bipyridine (25)
The same procedure described in Section 4.3 was employed
with 3 (R1 = b, R2 = c) (0.25 g, 1.00 mmol), dry ammonium acetate

386
P. Thapa et al. / Bioorg. Med. Chem. 18 (2010) 377–386
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.39; mp 177.4 °C, purity:
98.4%.
LC/MS/MS (condition C): retention time: 9.05 min; [M+]: 406.1
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 g/mL).
DNA bands were visualized 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.14 Cancer cells were cultured
according to the supplier’s instructions. Cells were seeded in 96-
well plates at a density of 2–4 ꢀ 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-
ture medium in each well was exchanged with 0.1 mL aliquots of
medium containing graded concentrations of compounds. On day
l
(97%), [M+2]: 408.1 (100%), [M+4]: 410.1 (36%), [M+6]: 412.1 (5%).
1H NMR (250 MHz, CDCl3) d 8.09 (br, 1H, 2-phenyl H-2), 8.01–
7.97 (m, 1H, 2-phenyl H-6), 7.72 (d, J = 1.20 Hz, 1H, pyridine H-
3), 7.67 (d, J = 1.20 Hz, 1H, pyridine H-5), 7.46 (d, J = 3.96 Hz, 1H,
6-thiophene H-3), 7.44–7.41 (m, 2H, 2-phenyl H-4, H-5), 6.96 (d,
J = 3.61 Hz, 2H, 4-furan H-3, 6-thiophene H-4), 6.35 (d,
J = 3.49 Hz, 1H, 4-furan H-4).
13C NMR (62.5 MHz, CDCl3) d 155.91, 152.22, 150.65, 143.45,
140.17, 138.45, 138.21, 134.82, 133.03, 129.97, 129.40, 127.20,
127.03, 125.00, 123.93, 112.22, 110.99, 110.28, 108.98.
4.3.23. 4-(5-Chlorofuran-2-yl)-2-(4-chlorophenyl)-6-(5-
chlorothiophen-2-yl) pyridine (29)
The same procedure described in Section 4.3 was employed
with 3 (R1 = a, R2 = c) (0.32 g, 1.20 mmol), dry ammonium acetate
(0.92 g, 12.00 mmol), 5 (R3 = l) (0.43 g, 1.20 mmol) and glacial ace-
tic acid (5.00 mL) to yield a creamy white solid (133 mg, 27.43%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.37; mp 182.0 °C, purity:
98.1%.
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 adriamycin, etoposide, and camptothecin
were purchased from Sigma and used as positive controls.
LC/MS/MS (condition A): retention time: 6.41 min; [M+]: 406.1
(100%), [M+2]: 408.1 (99%), [M+4]: 410.1 (36%), [M+6]: 412.1 (5%).
1H NMR (250 MHz, CDCl3) d 8.07 (d, J = 8.70 Hz, 2H, 2-phenyl H-
2, H-6), 7.72 (d, J = 1.24 Hz, 1H, pyridine H-3), 7.65 (d, J = 1.25 Hz,
1H, pyridine H-5), 7.46 (d, J = 8.70 Hz, 2H, 2-phenyl H-3, H-5),
7.45 (d, J = 3.93 Hz, 1H, 6-thiophene H-3), 6.95 (d, J = 3.90 Hz, 1H,
6-thiophene H-4), 6.94 (d, J = 3.42 Hz, 1H, 4-furan H-3), 6.36 (d,
J = 3.50 Hz, 1H, 4-furan H-4).
Acknowledgments
This work was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded
by the Ministry of Education, Science and Technology (R11-2007-
040-02004-0).
13C NMR (62.5 MHz, CDCl3) d 156.08, 152.10, 150.71, 143.56,
138.36, 138.12, 136.72, 135.55, 132.93, 128.88, 128.14, 127.17,
123.80, 111.85, 110.88, 109.93, 108.94.
References and notes
4.4. Biological assays
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DNA topo I inhibition assay was determined following the
method reported by Fukuda et al.13 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 0.4 units 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 relax-
ation 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
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2001, 11, 2659; (b) Zhao, L. X.; Moon, Y. S.; Basnet, A.; Kim, E. K.; Jahng, Y.; Park,
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final volume of 10 lL was terminated by adding 2.5 lL of the stop
solution containing 5% sarcosyl, 0.0025% bromophenol blue, and
25% glycerol. DNA samples were then electrophoresed on a 1% aga-
rose 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 lg/mL). DNA bands were visualized by transillumination with
UV light and were quantitated using AlphaImagerTM (Alpha Inno-
tech Corporation).
DNA topo II inhibitory activity of compounds was measured as
follows.14 The mixture of 200 ng of supercoiled pBR322 plasmid
DNA and 1 units of human DNA topoisomerase II
a (Usb 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, 50 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1 mM ATP, and
12. KrÖhnke, F. Synthesis 1976, 1.
13. Fukuda, M.; Nishio, K.; Kanzawa, F.; Ogasawara, H.; Ishida, T.; Arioka, H.;
Bonjanowski, K.; Oka, M.; Saijo, N. Cancer Res. 1996, 56, 789.
14. Kang, D. H.; Kim, J. S.; Jung, M. J.; Lee, E. S.; Jahng, Y.; Kwon, Y.; Na, Y. Bioorg.
Med. Chem. Lett. 2008, 18, 1520.
15
in a final volume of 20
7 mM EDTA. Reaction products were analyzed on a 1% agarose gel
lg/mL bovine serum albumin) for 30 min at 30 °C. The reaction
l
L was terminated by the addition of 3 L of
l