2,4,6-Trisubstituted pyridines: Synthesis, topoisomerase I and II inhibitory activity, cytotoxicity, and structure-activity relationship

Article abstract of DOI:10.1016/j.bmc.2007.04.047

Designed and synthesized were a series of pyridines substituted at 2, 4, and 6 positions with various 5- or 6-memberd heteroaromatics as antitumor agents. They were evaluated their topoisomerase I and II inhibitory activities along with cytotoxicities against several human cancer cell lines. Among the prepared compounds, 10-20 showed significant topoisomerase I or II inhibitory activities, and 21-26 showed considerable cytotoxicities against several human cancer cell lines. Structure-activity relationship study indicates that 4′-pyridine at 6-position of central pyridine plays a key role in biological activity.

Full text of DOI:10.1016/j.bmc.2007.04.047

Bioorganic & Medicinal Chemistry 15 (2007) 4351–4359
2,4,6-Trisubstituted pyridines: Synthesis, topoisomerase I and II
inhibitory activity, cytotoxicity, and structure–activity relationship
Arjun Basnet,a Pritam Thapa,a Radha Karki,a Younghwa Na,b Yurngdong Jahng,a
Byeong-Seon Jeong,a Tae Cheon Jeong,a Chong-Soon Leec and Eung-Seok Leea,*
aCollege of Pharmacy, Yeungnam University, Kyongsan 712-749, Republic of Korea
bCollege of Pharmacy, Catholic University of Daegu, Kyongsan 712-702, Republic of Korea
cDepartment of Biochemistry, Yeungnam University, Kyongsan 712-749, Republic of Korea
Received 27 March 2007; revised 25 April 2007; accepted 26 April 2007
Available online 29 April 2007
Abstract—Designed and synthesized were a series of pyridines substituted at 2, 4, and 6 positions with various 5- or 6-memberd
heteroaromatics as antitumor agents. They were evaluated their topoisomerase I and II inhibitory activities along with cytotoxicities
against several human cancer cell lines. Among the prepared compounds, 10–20 showed significant topoisomerase I or II inhibitory
activities, and 21–26 showed considerable cytotoxicities against several human cancer cell lines. Structure–activity relationship study
indicates that 40-pyridine at 6-position of central pyridine plays a key role in biological activity.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
also reported that terthiophene derivatives, bioisosteres
of terpyridine, showed considerable PKC inhibitory
activities and antitumor cytotoxicities against several hu-
man cancer cell lines.11 Although Topo I inhibitory activ-
ity and cytotoxicity of terpyridine derivatives have been
reported, the effects of terpyridine derivatives on Topo I
and II, and cytotoxicity have not been studied
systematically.
Topoisomerases are nuclear enzymes that play crucial
roles in DNA metabolism such as replication, transcrip-
tion, recombination, repair, chromatin assembly, and
chromosome segregation.1–3 Topoisomerases I (Topo I)
are monomeric and catalyze an ATP-independent relaxa-
tion of DNA supercoils by transiently breaking and reli-
gating single-stranded DNA, whereas topoisomerases II
(Topo II) are dimeric and relax supercoiled DNA through
catalysis of a transient breakage of double-stranded DNA
in an ATP-dependent manner.1,4 Due to the crucial role of
topoisomerases in the maintenance and replication of
DNA during proliferation, cells become highly vulnerable
when these functions are lost.5 Consequently, Topo I and
II have been attractive targets for design of antitumor
agents.6
In an extension to our work, we studied the synthesis of
a series of 2,4,6-trisubstituted pyridine derivatives for
the evaluation of Topo I and II inhibitory activities
and cytotoxicities. In order to study the structure–activ-
ity relationship as a whole, eight different heteroaromat-
ics including phenyl were taken as R1 (a–h) and R2 (a–d)
systematically to prepare compounds 7, 8, and 9 (Fig. 1
and Scheme 1).
a-Terpyridine has been precious due to its ability to form
metal complexes7 and as DNA binding agents since its
discovery in 1932.8 Our research group has previously re-
ported that terpyridine derivatives showed strong cyto-
toxicities against several human cancer cell lines and
considerable Topo I inhibitory activities.9,10 Some of us
2. Results and discussion
We have previously reported the synthesis of various
2,4,6-trisubstituted pyridine derivatives.9,10 A similar
synthetic sequence was employed to prepare the new
2,4,6-trisubstituted pyridine derivatives. Scheme 1 illus-
trates the synthetic method for the preparation of pyri-
dine derivatives. At first, we prepared thirty-two
intermediates (3) in 60.3–97.5% yield by the condensa-
tion of eight different aryl aldehydes (1a–h) with four
Keywords: 2,4,6-Trisubstituted pyridines; Topoisomerase I and II;
Cytotoxicity; Antitumor agent.
*
Corresponding author. Tel.: +82 53 810 2827; fax: +82 53 810
4654; e-mail: eslee@yu.ac.kr
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.04.047

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A. Basnet et al. / Bioorg. Med. Chem. 15 (2007) 4351–4359
R1
4
2
6
4'
N
R2
N
S
S
S
N
N
2'
6'
N
R1, R2 : phenyl
α-terpyridine
α-terthiophene
2- or 3-thienyl
2- or 3-furyl
2- or 3- or 4-pyridyl
Figure 1. Structures of a-terpyridine, a-terthiophene, and 2,4,6-trisubstituted pyridines.
I
O
R1
N
N
4
R2
N
N
7 (R1= a-h, R2= a-d)
I
O
R1
R2
N
R1
O
R1
H
R2
2 (R2= a-d)
5
N
R2
N
i
ii
O
O
N
3 (R1= a-h, R2= a-d)
1 (R1= a-h)
8 (R1= a-h, R2= a-d)
R1
I
O
N
N
6
R2
N
N
9 (R1= a-h, R2= a-d)
N
S
O
N
S
O
N
g
a
c
e
b
d
f
h
Scheme 1. General synthetic scheme of 2,4,6-trisubstituted pyridines. Reagents and conditions: (i) aryl acetyl ketones 2a–d (1.0 eq), KOH (1.1 eq),
MeOH/H2O (5:1), 0 ꢁC, 3 h, 60.3–97.5%; (ii) 4, 5, or 6 (1.2 eq), NH4OAc (10.0 eq), MeOH, 20–80 ꢁC, 12–24 h, 20.5–92.5%.
different aryl methyl ketones (2a–d), respectively. Other
three key intermediates, 1-(2-oxo-2-pyridin-2-yleth-
yl)pyridinium iodide (4), 1-(2-oxo-2-pyridin-3-yleth-
yl)pyridinium iodide (5), and 1-(2-oxo-2-pyridin-4-
ylethyl)pyridinium iodide (6), were prepared in quantita-
tive yield from 2-, 3-, or 4-acetyl pyridine with iodine in
pyridine (scheme not shown). Using modified Kro¨hnke
From the preliminary study, compounds 10 (9, R1 = b,
R2 = d), 11 (7, R1 = c, R2 = b), 12 (9, R1 = c, R2 = b),
13 (9, R1 = c, R2 = d), 14 (9, R1 = g, R2 = d), 15 (9,
R1 = g, R2 = b), 16 (9, R1 = h, R2 = c), 17 (9, R1 = e,
R2 = b), 18 (9, R1 = e, R2 = c), 19 (7, R1 = e, R2 = d),
and 20 (9, R1 = e, R2 = d) were selected for the further
evaluation of Topo I and II inhibitory activities, and
compounds 21 (9, R1, R2 = b), 22 (9, R1 = b, R2 = c),
23 (9, R1, R2 = c), 24 (9, R1 = d, R2 = b), 25 (8, R1 = c,
R2 = b), and 26 (8, R1 = d, R2 = b) were selected for
the evaluation of cytotoxicities. The structures of the se-
lected compounds are shown in Figure 2. The rest of the
compounds were excluded since they did not display
considerable activities.
synthesis,12 ninety-six final products
7
(R1 = a–h,
R2 = a–d), 8 (R1 = a–h, R2 = a–d), and 9 (R1 = a–h,
R2 = a–d) were synthesized by the condensation of thirty
two intermediates 3 with 4, 5, and 6, respectively, in
20.5–92.5% yield. We noted that the yield dramatically
dropped when phenyl moiety was placed in 2-position
of the central pyridine.

A. Basnet et al. / Bioorg. Med. Chem. 15 (2007) 4351–4359
4353
S
S
S
S
N
N
N
N
O
N
S
N
S
N
O
N
10
11
12
13
N
O
N
N
N
N
N
N
S
N
O
N
S
N
N
S
14
15
16
17
O
O
O
S
N
N
N
N
S
N
N
O
N
O
N
S
18
19
20
21
S
S
O
O
S
N
N
N
N
N
N
S
S
N
S
N
S
S
N
N
22
23
24
25
26
Figure 2. Structures of the selected compounds bearing considerable biological activities.
2.1. Topo I and II inhibitory activity of compounds 10–20
2.2. Cytotoxicity of compounds 21–26
Figure 3 shows the conversion of supercoiled plasmid
DNA by calf thymus Topo I in the presence of com-
pounds 10 to 20 with camptothecin, a well-known Topo
I inhibitor, as a positive control. Compounds 10, 11, 12,
and 14 showed significant inhibitory activities at 20 lM
and 100 lM concentration and the rest of the indicated
compounds showed moderate inhibitory activities when
compared to camptothecin. Figure 4 shows human
Topo II inhibitory activities of 2,4,6-trisubstituted pyri-
dine derivatives (10–20) with etoposide as a positive con-
trol. Compounds 13, 15–20 showed significant
inhibitory effects on human DNA Topo II at 20 lM
and 100 lM concentration and the rest of the indicated
compounds displayed weak effects when compared to
etoposide. It is interesting to note that the compounds
which significantly inhibited Topo I displayed weak
inhibitory activities on Topo II, whereas the compounds
which significantly inhibited Topo II displayed moder-
ate inhibitory activities on Topo I.
Cytotoxicities of compounds 21–26 were evaluated on
four different human cancer cell lines A549 (human kid-
ney carcinoma), SK-OV-3 (human ovary adenocarci-
noma), SK-MEL-2 (human malignant melanoma), and
HCT15 (human colon adenocarcinoma). For the evalu-
ation of antitumor activities, cytotoxicities were ex-
pressed in lM using etoposide as a positive control.
Table 1 indicates that the prepared compounds 21–26
show considerable cytotoxicities against several human
cancer cell lines especially on HCT15 when compared
to etoposide. As in the previous study on terpyridine
derivatives,9,10 strong Topo I and II inhibitors have
not displayed strong cytotoxicities and vice versa.
3. Conclusions
We have designed and prepared a series of ninety-six
2,4,6-trisubstituted pyridine derivatives by efficient

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A. Basnet et al. / Bioorg. Med. Chem. 15 (2007) 4351–4359
Lane D: pBR322 DNA only
Lane T: pBR322 DNA + Topo I
Lane C1: pBR322 DNA + Topo I + Camptothecin 20 μM
Lane C2: pBR322 DNA + Topo I + Camptothecin 100 μM
Lane 10–20 Lane a: pBR322 DNA + Topo I + Compound 20 μM
Lane 10–20 Lane b: pBR322 DNA + Topo I + Compound 100 μM
Figure 3. Calf thymus Topo I inhibitory effects of compounds 10–20.
Lane D: pBR322 DNA only
Lane T: pBR322 DNA + Topo II
Lane V1: pBR322 DNA + Topo II + Etoposide 20 μM
Lane V2: pBR322 DNA + Topo II + Etoposide 100 μM
Lane 10 - 20 Lane a: pBR322 DNA + Topo II + Compound 20 μM
Lane 10 - 20 Lane b: pBR322 DNA + Topo II + Compound 100 μM
Figure 4. Human Topo II inhibitory effects of compounds 10–20.
Table 1. Antitumor cytotoxicity of the prepared compounds 21–26
combination of b, c, d, e, g, and h moieties were impor-
tant to display Topo I and II inhibitory activities. For
cytotoxicities, 2,40- and 2,30-bipyridyl moieties with
combination of b, c, and d moieties were important.
There were no direct correlations between antitumor
cytotoxicity and topoisomerase inhibitory activity. This
study may provide valuable information to the research-
ers working on the development of antitumor agents.
Compound
IC50 (lM)
SK-OV-3 SK-MEL-2
A549
HCT15
21
22
4.16
5.69
5.75
14.52
9.86
8.69
7.49
5.90
1.72
3.68
8.94
9.76
5.43
5.61
1.95
0.30
1.72
3.79
2.54
4.18
4.06
5.87
2.43
23
24
27.83
10.94
2.73
25
26
14.51
0.32
Etoposide
4. Experimental
synthetic route, and evaluated their Topo I and II inhib-
itory activity and antitumor cytotoxicity. A structure–
activity relationship study of the 2,4,6-trisubstituted
pyridine derivatives for Topo I and II inhibitory activi-
ties indicates that 2,40- and 2,20-bipyridyl moieties with
Compounds used as starting materials and reagents
were purchased from Aldrich Chemical Co., Sigma,
Fluka, Junsei and used without further purification.
Thin-layer chromatography (TLC) and column chroma-
tography (CC) were performed with Kieselgel 60F254

A. Basnet et al. / Bioorg. Med. Chem. 15 (2007) 4351–4359
4355
(Merck) and silica gel (Kieselgel 60, 230–400 mesh,
Merck), respectively. Since all the compounds prepared
contain aromatic rings, compounds were visualized and
detected on TLC plates with UV light (short wave, long
wave, or both). NMR spectra were recorded on a Bruker
4.2.3. 1-[2-Oxo-(2-pyridin-4-yl-)ethyl]pyridinium iodide
(6). Brown solid; mp 130.0 ꢁC; 1H NMR (250 MHz,
CDCl3) d 8.98 (d, J = 6.4 Hz, 2H, pyridinium H-2, H-
6), 8.95 (dd d, J = 4.8, 1.4, 0.9 Hz, 2H, pyridine H-2,
H-6), 8.76 (dt, J = 7.8, 1.1 Hz, 1H, pyridinium H-4),
8.30 (t, J = 6.7 Hz, 2H, pyridinium H-3, H-5), 7.95 (dd
d, J = 4.8, 1.4, 0.9 Hz, 2H, pyridine H-3, H-5), 6.45 (s,
2H, –CO–CH2–).
1
AMX 250 (250 MHz, FT) for H NMR and 62.5 MHz
for 13C NMR, and TMS (tetramethylsilane) was used
as an internal standard. Chemical shifts (d) were
recorded in ppm and coupling constants (J) in hertz
(Hz). Melting points were determined in open capillary
tubes on electrothermal 1A 9100 digital melting point
apparatus and were uncorrected. ESI LC/MS analyses
were performed with a Finnigan LCQ Advantageꢂ
LC/MS/MS spectrometry utilizing Xcaliburꢂ program.
For ESI LC/MS, LC was performed with a 1 ll injection
volume on a Waters XTerraꢂ 3.5 lm reverse-phase C18
column (2.1 · 100 mm) with a gradient elution from 5%
to 95% of B in A for 20 min followed by 95% B in A for
10 min at a flow rate of 200 lL/min, where mobile phase
A was 100% distilled water with 50 mM ammonium
formate and mobile phase B was 100% acetonitrile. MS
ionization conditions were: Sheath gas flow rate: 70 arb,
aux gas flow rate: 20 arb, I spray voltage: 4.5 KV,
capillary temp.: 215 ꢁC, capillary voltage: 21 V, tube lens
offset: 10 V.
4.3. General method for the preparation of 10–26
A mixture of 3 (R1 = a–h, R2 = a–d), dry ammonium ace-
tate and 4, 5, or 6 in dry MeOH was heated to 80 ꢁC for
12–24 h under nitrogen gas. The solvent was removed
by evaporation under reduced pressure, and the residue
was diluted with ethyl acetate (100 mL), washed with
water (75 mL · 2) and saturated NaCl solution (50 mL).
The organic layer was dried with magnesium sulfate
and filtered. The filtrate was evaporated at reduced
pressure, which was purified by silica gel column chroma-
tography with a gradient elution of ethyl acetate/n-hexane
to afford a white solid in 20.5–92.5% yield. Utilizing the
same procedure, ninety-six compounds were synthesized.
4.3.1.
6-(Furan-2-yl)-4-(thiophene-2-yl)-[2,40]-bipyridyl
(10). The same procedure described at 4.3 was employed
with 3 (R1 = b, R2 = d) (1.02 g, 5.0 mmol), anhydrous
ammonium acetate (3.85 g, 50.0 mmol), 6 (1.63 g,
5.0 mmol), and dry MeOH (30 mL) to yield a white solid
(335 mg, 22.0%). Rf (ethyl acetate/n-hexane 2:1): 0.3; mp
140.5 ꢁC; LC MS/MS: retention time: 18.07 min;
4.1. General method for the preparation of 3
Aryl aldehyde was added to the solution of 85% KOH
(1.2 eq) in MeOH (50 mL) and H2O (10 mL) at 0 ꢁC.
After dissolution, aryl methyl ketone (1.0 eq) was added
over a period of 10 min. The mixture was then stirred
for 3 h at 0 ꢁC. A solid product precipitated was filtered,
washed with cold MeOH, and dried to yield 60.3–
97.5%. Utilizing the same procedure, thirty-two com-
pounds were synthesized.
1
[MH]+: 305.2; H NMR (250 MHz, CDCl3) d 8.77 (dd,
J = 4.6, 1.6 Hz, 2H, 2-pyridine H-20, H-60), 8.01(dd,
J = 4.6, 1.6 Hz, 2H, 2-pyridine H-30, H-50), 7.93 (d,
J = 1.2 Hz, 1H, pyridine H-3), 7.82 (d, J = 1.2 Hz, 1H,
pyridine H-5), 7.48 (dd, J = 3.7, 0.9 Hz, 1H, 4-thiophene
H-5), 7.60 (dd, J = 1.7, 0.9 Hz, 1H, 6-furan H-5), 7.48
(dd, J = 5.0, 0.9 Hz, 1H, 4-thiophene H-3), 7.24 (d,
J = 3.4 Hz, 1H, 6-furan H-3), 7.19 (dd, J = 5.0, 3.7 Hz,
1H, 4-thiophene H-4), 6.60 (dd, J = 3.4, 1.8 Hz, 1H, 6-fur-
an H-4); 13C NMR (62.5 MHz, CDCl3) d 155.04, 153.41,
150.40, 150.23, 146.13, 143.61, 143.29, 141.06, 128.51,
127.46, 125.73, 121.17, 115.25, 114.55, 112.23, 109.61.
4.2. General method for preparation of 4–6
A mixture of acetyl pyridine, iodine (1.2 eq) and pyri-
dine 60 mL was refluxed for 3 h. The solid product pre-
cipitated was cooled to 0 ꢁC and filtered. The product
was washed with cold pyridine and dried to afford 4–6
in quantitative yield.
4.3.2. 4-(Thiophene-3-yl)-6-(thiophene-2-yl)-[2,20]-bipyr-
idyl (11). The same procedure described at 4.3 was em-
ployed with 3 (R1 = c, R2 = b) (1.10 g, 5.0 mmol),
anhydrous ammonium acetate (3.85 g, 50.0 mmol), 4
(1.63 g, 5.0 mmol), and dry MeOH (30 mL) to yield a
white solid (360 mg, 22.5%). Rf (ethyl acetate/n-hexane
2:1): 0.5; mp 156.2 ꢁC; LC MS/MS: retention time:
4.2.1. 1-[2-Oxo-(2-pyridin-2-yl-)ethyl]pyridinium iodide
(4). Grayish black solid; mp >300 ꢁC; 1H NMR
(250 MHz, CDCl3) d 9.00 (d, J = 6.4 Hz, 2H, pyridinium
H-2, H-6), 8.86 (dd, J = 4.8, 0.9 Hz, 1H, pyridine H-6),
8.72 (dt, J = 7.8, 1.2 Hz, 1H, pyridinium H-4), 8.27 (t,
J = 6.6 Hz, 2H, pyridinium H-3, H-5), 8.13–8.02 (m,
2H, pyridine H-3, H-4), 7.83 (ddd, J = 7.2, 4.8, 1.5 Hz,
1H, pyridine H-5), 6.50 (s, 2H, –CO–CH2–).
1
20.12 min; [MH]+: 321.2; H NMR (250 MHz, CDCl3)
d 8.71 (dd d, J = 4.7, 1.6, 0.7 Hz, 1H, 2-pyridine H-60),
8.61 (d, J = 8.0 Hz, 1H, 2-pyridine H-30), 8.53 (d,
J = 1.5 Hz, 1H, pyridine H-3), 7.87 (td, J = 7.7, 1.8 Hz,
1H, 2-pyridine H-40), 7.86 (d, J = 1.5 Hz, 1H, pyridine
H-5), 7.84 (dd, J = 2.8, 1.2 Hz, 1H, 4-thiophene H-2),
7.73 (dd, J = 3.7, 1.0 Hz, 1H, 6-thiophene H-5), 7.61
(dd, J = 5.0, 1.2 Hz, 1H, 4-thiophene H-5), 7.47 (dd,
J = 5.0, 2.9 Hz, 1H, 4-thiophene H-4), 7.44 (dd,
J = 5.1, 1.0 Hz, 1H, 6-thiophene H-3), 7.35 (dd d,
J = 7.5, 4.8, 1.2 Hz, 1H, 2-pyridine H-50), 7.16 (dd,
J = 5.0, 3.7 Hz, 1H, 6-thiophene H-4); 13C NMR
4.2.2. 1-[2-Oxo-(2-pyridin-3-yl-)ethyl]pyridinium iodide
(5). Brown solid; mp 180.2 ꢁC; 1H NMR (250 MHz,
CDCl3) d 9.23 (br, 1H, pyridine H-2), 8.99 (d,
J = 6.5 Hz, 2H, pyridinium H-2, H-6), 8.92 (dd,
J = 4.8, 1.5 Hz, 1H, pyridine H-6), 8.76 (dt, J = 7.8,
1.2 Hz, 1H, pyridinium H-4), 8.42 (dd d, J = 8.0, 1.5,
0.6 Hz, 1H, pyridine H-4) 8.30 (t, J = 6.6 Hz, 2H, pyrid-
inium H-3, H-5), 7.72 (dd, J = 7.7, 4.8 Hz, 1H, pyridine
H-5), 6.48 (s, 2H, –CO–CH2–).

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A. Basnet et al. / Bioorg. Med. Chem. 15 (2007) 4351–4359
(62.5 MHz, CDCl3) d 156.16, 155.80, 152.45, 148.97,
146.39, 145.24, 144.50, 140.83, 139.80, 136.96, 128.02,
127.64, 126.85, 126.07, 124.57, 123.93, 123.41, 121.52,
116.44, 115.75.
idine H-5), 7.29 (d, J = 3.4 Hz, 1H, 60-furan H-3), 6.61
(dd, J = 3.4, 1.8 Hz, 1H, 60-furan H-4); 13C NMR
(62.5 MHz, CDCl3) d 155.64, 153.74, 150.86, 148.57,
147.72, 146.40, 144.22, 134.89, 134.37, 124.29, 121.61,
117.23, 116.74, 112.74, 110.34.
4.3.3. 4-(Thiophene-3-yl)-6-(thiophene-2-yl)-[2,40]-bipyr-
idyl (12). The same procedure described at 4.3 was em-
ployed with 3 (R1 = c, R2 = b) (1.10 g, 5.0 mmol)
anhydrous ammonium acetate (3.85 g, 50.0 mmol), 6
(1.63 g, 5.0 mmol), and dry MeOH (30 mL) to yield a
white solid (328 mg, 20.5%). Rf (ethyl acetate/n-hexane
2:1): 0.3, mp 154.4 ꢁC; LC MS/MS: retention time:
4.3.6. 60-(Thiophene-2-yl)-[3,40;20,400]-terpyridine (15). The
same procedure described at 4.3 was employed with 3
(R1 = g, R2 = b) (0.54 g, 2.5 mmol), anhydrous ammo-
nium acetate (1.93 g, 25.0 mmol), 6 (0.82 g, 2.5 mmol),
and dry MeOH (20 mL) to yield a white solid (320 mg,
40.4%). Rf (ethyl acetate/n-hexane 3:1): 0.21; mp
199.8 ꢁC; LC MS/MS: retention time: 15.04 min;
1
18.52 min; [MH]+: 321.3; H NMR (250 MHz, CDCl3)
1
d 8.76 (dd, J = 4.6, 1.6 Hz, 2H, 2-pyridine H-20, H-60),
8.03(dd, J = 4.6, 1.6 Hz, 2H, 2-pyridine H-30, H-50),
7.82 (br, 2H, pyridine H-3, H-5), 7.76 (dd, J = 2.6,
1.6 Hz, 1H, 4-thiophene H-2), 7.72 (dd, J = 3.7, 1.0 Hz,
1H, 6-thiophene H-5), 7.53 (dd, J = 5.0, 1.6 Hz, 1H, 4-thi-
ophene H-5), 7.50 (dd, J = 5.0, 2.7 Hz, 1 H, 4-thiophene
H-4), 7.45 (dd, J = 5.0, 1.0 Hz, 1H, 6-thiophene H-3),
7.16 (dd, J = 5.0, 3.7 Hz, 1H, 6-thiophene H-4); 13C
NMR (62.5 MHz, CDCl3) d 154.57, 153.28, 150.36,
145.99, 144.74, 144.68, 139.39, 128.14, 128.06, 127.34,
127.04, 126.30, 125.80, 125.16, 125.00, 124.19, 123.48,
121.24, 121.08, 116.20, 115.89.
[MH]+: 316.3; H NMR (250 MHz, CDCl3) d 8.99 (br,
1H, pyridine H-2), 8.76 (br, 2H, pyridine H-200, H-600),
8.76 (br, 1H, pyridine H-6), 8.05 (br, 2H, pyridine H-300,
H-500), 8.00 (dt, J = 7.8, 1.8 Hz, 1H, pyridine H-4), 7.84
(d, J = 1.2 Hz, 1H, pyridine H-30), 7.83 (d, J = 1.2 Hz,
1H, pyridine H-50), 7.76 (dd, J = 3.7, 1.0 Hz, 1H, 60-thio-
phene H-5), 7.51 (br, 1H, pyridine H-5), 7.48 (dd, J = 5.0,
1.0 Hz, 1H, 60-thiophene H-3), 7.18 (dd, J = 5.0, 3.7 Hz,
1H, 60-thiophene H-4); 13C NMR (62.5 MHz, CDCl3) d
154.88, 153.53, 150.46, 148.15, 147.43, 145.62, 144.80,
144.31, 140.90, 134.47, 133.98, 133.30, 128.54, 128.18,
125.87, 125.36, 123.89, 121.31, 116.81, 116.65.
4.3.4.
6-(Furan-2-yl)-4-(thiophene-3-yl)-[2,40]-bipyridyl
4.3.7. 60-(Thiophen-2-yl)-[4,20;40,400]-terpyridine (16). The
same procedure described at 4.3 was employed with 3
(R1 = h, R2 = c) (1.08 g, 5.0 mmol), anhydrous ammo-
nium acetate (3.86 g, 50.0 mmol), 6 (1.63 g, 5.0 mmol),
and dry MeOH (30 mL) to yield a white solid (1.26 g,
79.6%). Rf (ethyl acetate/n-hexane 3:1): 0.12, mp
239.5 ꢁC; LC MS/MS: retention time: 14.95 min;
(13). The same procedure described at 4.3 was employed
with 3 (R1 = c, R2 = d) (1.02 g, 5.0 mmol), anhydrous
ammonium acetate (3.85 g, 50.0 mmol), 6 (1.63 g,
5.0 mmol), and dry MeOH (30 mL) to yield a white solid
(400 mg, 26.8%). Rf (ethyl acetate/n-hexane 2:1): 0.3; mp
120.6 ꢁC; LC MS/MS: retention time: 17.50 min;
[MH]+: 305.3; H NMR (250 MHz, CDCl3) d 8.75 (dd,
[MH]+: 316.3; H NMR (250 MHz, CDCl3) d 8.81 (dd,
1
1
J = 4.6, 1.6 Hz, 2H, 2-pyridine H-20, H-60), 8.00 (dd,
J = 4.6, 1.6 Hz, 2H, 2-pyridine H-30, H-50), 7.91 (d,
J = 1.4 Hz, 1H, pyridine H-3), 7.80 (d, J = 1.4 Hz, 1H,
pyridine H-5), 7.77 (dd, J = 2.8, 1.4 Hz, 1H, 4-thiophene
H-2), 7.57 (dd, J = 1.7, 0.6 Hz, 1H, 6-furan H-5), 7.54
(dd, J = 5.0, 1.4 Hz, 1H, 4-thiophene H-5), 7.48 (dd,
J = 5.0, 2.9 Hz, 1H, 4-thiophene H-4), 7.24 (dd, J = 3.4,
0.5 Hz, 1H, 6-furan H-3), 6.58 (dd, J = 3.4, 1.8 Hz, 1H,
6-furan H-4); 13C NMR (62.5 MHz, CDCl3) d 154.87,
153.54, 151.42, 150.30, 150.17, 146.27, 144.56, 143.48,
140.65, 139.38, 127.22, 126.27, 126.01, 125.84, 125.76,
123.48, 122.60, 121.57, 121.16, 120.69, 116.13, 115.53,
113.03, 112.18, 111.57, 109.42.
J = 4.5, 1.6 Hz, 2H, pyridine H-2, H-6), 8.79 (dd,
J = 4.5, 1.6 Hz, 2H, pyridine H-200, H-600), 8.05 (dd,
J = 4.5, 1.6 Hz, 2H, pyridine H-3, H-5), 7.85 (br, 2H,
pyridine H-30, H-50), 7.76 (dd, J = 3.7, 1.0 Hz, 1H, 60-
thiophene H-5), 7.62 (dd, J = 4.5, 1.6 Hz, 2H, pyridine
H-300, H-500), 7.49 (dd, J = 5.0, 1.0 Hz, 1H, 60-thiophene
H-3), 7.18 (dd, J = 5.0, 3.7 Hz, 1H, 60-thiophene H-4);
13C NMR (62.5 MHz, CDCl3) d 155.03, 153.67,
150.77, 150.51, 147.87, 145.63, 145.49, 144.18, 128.65,
128.21, 125.45, 121.51, 121.05, 116.59, 116.49.
4.3.8.
4-(Furan-3-yl)-6-(thiophene-2-yl)-[2,40]-bipyridyl
(17). The same procedure described at 4.3 was employed
with 3 (R1 = e, R2 = b) (0.71 g, 3.5 mmol), anhydrous
ammonium acetate (2.70 g, 35.0 mmol), 6 (1.14 g,
3.5 mmol), and dry MeOH (20 mL) to yield a white solid
(250 mg, 23.6%). Rf (ethyl acetate/n-hexane 1:2): 0.12;
mp 161.9 ꢁC; LC MS/MS: retention time: 21.47 min;
4.3.5. 60-(Furan-2-yl)-[3,40;20,400]-terpyridine (14). The
same procedure described at 4.3 was employed with 3
(R1 = g, R2 = d) (0.99 g, 5.0 mmol), anhydrous ammo-
nium acetate (3.85 g, 50.0 mmol), 6 (1.63 g, 5.0 mmol),
and dry MeOH (30 mL) to yield a white solid (900 mg,
60.0%). Rf (ethyl acetate/n-hexane 2:1): 0.19; mp
184.3 ꢁC; LC MS/MS: retention time: 13.82 min;
1
[MH]+: 305.2; H NMR (250 MHz, CDCl3) d 8.75 (dd,
J = 4.5, 1.6 Hz, 2H, 2-pyridine H-20, H-60), 8.03 (dd,
J = 4.5, 1.6 Hz, 2H, 2-pyridine H-30, H-50), 7.99 (br,
1H, 4-furan H-2), 7.73 (d, J = 1.3 Hz, 1H, pyridine H-
3), 7.71 (d, J = 1.3 Hz, 1H, pyridine H-5), 7.70 (dd,
J = 3.7, 1.0 Hz, 1H, 6-thiophene H-5), 7.59 (t,
J = 1.7 Hz, 1H, 4-furan H-5), 7.46 (dd, J = 5.0, 1.0 Hz,
1H, 6-thiophene H-3), 7.16 (dd, J = 5.0, 3.7 Hz, 1H, 6-
thiophene H-4), 6.84 (dd, J = 1.8, 0.8 Hz, 1H, 4-furan
H-4); 13C NMR (62.5 MHz, CDCl3) d 154.48, 153.24,
150.54, 150.25, 146.00, 144.57, 144.51, 141.96, 140.53,
1
[MH]+: 300.3; H NMR (250 MHz, CDCl3) d 9.01 (d,
J = 2.2 Hz, 1H, pyridine H-2), 8.77 (dd, J = 4.6, 1.6 Hz,
2H, pyridine H-200, H-600), 8.74 (dd, J = 4.8, 1.6 Hz, 1H,
pyridine H-6), 8.06 (dt, J = 7.9, 1.9 Hz, 1H, pyridine H-
4), 8.03 (dd, J = 4.8, 1.6 Hz, 2H, pyridine H-300, H-500),
7.95 (d, J = 1.4 Hz, 1H, pyridine H-30), 7.84 (d,
J = 1.4 Hz, 1H, pyridine H-50), 7.60 (dd, J = 1.8, 0.7 Hz,
1H, 60-furan H-5), 7.49 (ddd, J = 8.0, 4.8, 0.7 Hz, 1H, pyr-

A. Basnet et al. / Bioorg. Med. Chem. 15 (2007) 4351–4359
4357
128.16, 128.06, 126.88, 125.01, 124.25, 122.84, 121.10,
120.40, 115.60, 115.28, 108.34, 107.63.
4.3.12. 4,6-(Dithiophen-2-yl)-[2,40]-bipyridyl (21). The
same procedure described at 4.3 was employed with 3
(R1, R2 = b) (1.10 g, 5.0 mmol), anhydrous ammonium
acetate (3.85 g, 50.0 mmol), 6 (1.63 g, 5.0 mmol), and
dry MeOH (50 mL) to yield a white solid (347 mg,
21.7%). Rf (ethyl acetate/n-hexane 2:1): 0.3; mp
147.6 ꢁC; LC MS/MS: retention time: 18.12 min;
4.3.9.
4-(Furan-3-yl)-6-(thiophene-3-yl)-[2,40]-bipyridyl
(18). The same procedure described at 4.3 was employed
with 3 (R1 = e, R2 = c) (0.71 g, 3.5 mmol), anhydrous
ammonium acetate (2.70 g, 35.0 mmol), 6 (1.14 g,
3.5 mmol), and dry MeOH (20 mL) to yield a white solid
(400 mg, 37.8%). Rf (ethyl acetate/n-hexane 1:2): 0.12; mp
160.5 ꢁC; LC MS/MS: retention time: 21.28 min; [MH]+:
1
[MH]+: 321.2; H NMR (250 MHz, CDCl3) d 8.77 (dd,
J = 4.6, 1.6 Hz, 2H, 2-pyridine H-20, H-60), 8.03 (dd,
J = 4.5, 1.6 Hz, 2H, 2-pyridine H-30, H-50), 7.84 (d,
J = 1.3 Hz, 1H, pyridine H-3), 7.82 (d, J = 1.3 Hz, 1H,
pyridine H-5), 7.73 (dd, J = 3.7, 1.0 Hz, 1H, 6-thiophene
H-5), 7.63 (dd, J = 3.7, 1.0 Hz, 1H, 4-thiophene H-5),
7.49 (dd, J = 5.0, 1.0 Hz, 1H, 6-thiophene H-3), 7.45
(dd, J = 5.0, 1.0 Hz, 1H, 4-thiophene H-3), 7.20 (dd,
J = 5.0, 3.7 Hz, 1H, 6-thiophene H-4), 7.17 (dd, J = 5.0,
3.7 Hz, 1H, 4-thiophene H-4); 13C NMR (62.5 MHz,
CDCl3) d 154.72, 153.37, 150.42, 145.83, 144.52, 143.38,
140.99, 128.53, 128.28, 128.09, 127.47, 125.70, 125.15,
121.08, 115.26, 114.89.
1
305.2; H NMR (250 MHz, CDCl3) d 8.76 (dd, J = 4.6,
1.5 Hz, 2H, 2-pyridine H-20, H-60), 8.06 (dd, J = 3.0,
1.1 Hz, 1H, 6-thiophene H-3), 8.03 (dd, J = 4.6, 1.5 Hz,
2H, 2-pyridine H-30, H-50), 7.99 (br, 1H, 4-furan H-2),
7.80 (dd, J = 5.0, 1.1 Hz, 1H, 6-thiophene H-5), 7.73 (d,
J = 1.1 Hz, 1H, pyridine H-3), 7.71 (d, J = 1.1 Hz, 1H,
pyridine H-5), 7.59 (t, J = 1.6 Hz, 1H, 4-furan H-5),
7.45 (dd, J = 5.0, 3.0 Hz, 1H, 6-thiophene H-4), 6.84
(dd, J = 1.7, 0.8 Hz, 1H, 4-furan H-4); 13C NMR
(62.5 MHz, CDCl3) d 154.73, 154.14, 150.36, 146.38,
144.51, 141.95, 141.84, 140.45, 126.45, 126.28, 124.42,
124.21, 121.15, 116.85, 115.64, 108.37.
4.3.13. 4-(Thiophene-2-yl)-6-(thiophene-3-yl)-[2,40]-bipyr-
idyl (22). The same procedure described at 4.3 was em-
ployed with 3 (R1 = b, R2 = c) (1.10 g, 5.0 mmol),
anhydrous ammonium acetate (3.85 g, 50.0 mmol), 6
(1.63 g, 5.0 mmol), and dry MeOH (30 mL) to yield a
white solid (0.40 g, 25.0%). Rf (ethyl acetate/n-hexane
2:1): 0.3, mp 165.8 ꢁC; LC MS/MS: retention
time:18.87 min; [MH]+: 321.2; 1H NMR (250 MHz,
CDCl3) d 8.76 (dd, J = 4.6, 1.6 Hz, 2 H, 2-pyridine H-
20, H-60), 8.07 (dd, J = 3.0, 1.2 Hz, 1H, 6-thiophene H-
2), 8.03(dd, J = 4.6, 1.6 Hz, 2H, 2-pyridine H-30, H-50),
7.84 (d, J = 1.4 Hz, 1H, pyridine H-3), 7.81 (d,
J = 1.4 Hz, 1H, pyridine H-5), 7.80 (dd, J = 5.0,
1.2 Hz, 1H, 6-thiophene H-5), 7.61 (dd, J = 3.7, 1.0
Hz, 1H, 4-thiophene H-5), 7.48 (dd, J = 5.0, 1.0 Hz,
1H, 4-thiophene H-3), 7.45 (dd, J = 5.0, 3.0 Hz, 1H, 6-
thiophene H-4), 7.19 (dd, J = 5.0, 3.7 Hz, 1H, 4-thio-
phene H-4); 13C NMR (62.5 MHz, CDCl3) d 154.82,
154.21, 150.34, 146.25, 143.33, 141.73, 141.14, 138.78,
136.97, 128.50, 127.35, 126.43, 126.29, 125.56, 124.33,
121.14, 117.66, 116.45, 115.24.
4.3.10. 6-(Furan-2-yl)-4-(furan-3-yl)-[2,20]-bipyridyl (19).
The same procedure described at 4.3 was employed with 3
(R1 = e, R2 = d) (0.66 g, 3.5 mmol), anhydrous ammo-
nium acetate (2.70 g, 35.0 mmol), 4 (1.14 g, 3.5 mmol),
and dry MeOH (30 mL) to yield a white solid (0.80 g,
79.1%). Rf (ethyl acetate/n-hexane 1:2): 0.28; mp
61.5 ꢁC; LC MS/MS: retention time: 21.89 min; [MH]+:
289.3; 1H NMR (250 MHz, CDCl3) d 8.70 (dd d,
J = 4.8, 1.6, 0.8 Hz, 1H, 2-pyridine H-60), 8.57 (d,
J = 8.0 Hz, 1H, 2-pyridine H-30), 8.40 (d, J = 1.4 Hz,
1H, pyridine H-3), 8.05 (br, 1H, 4-furan H-2), 7.86 (td,
J = 7.7, 1.7 Hz, 1H, 2-pyridine H-40), 7.81 (d, J = 1.4,
1H, pyridine H-5), 7.57 (br, 1H, 6-furan H-5), 7.55 (t,
J = 1.7 Hz, 1H, 4-furan H-5), 7.34 (dd d, J = 7.4, 4.8,
0.8 Hz, 1H, 2-pyridine H-50), 7.22 (d, J = 3.3 Hz, 1H, 6-
furan H-3), 6.92 (dd, J = 1.7, 0.8 Hz, 1H, 4-furan H-4),
6.58 (dd, J = 3.3, 1.7 Hz, 1H, 6-furan H-4); 13C NMR
(62.5 MHz, CDCl3) d 156.26, 155.90, 153.89, 149.33,
148.96, 144.16, 143.22, 141.63, 140.75, 136.91, 124.62,
123.90, 121.42, 115.77, 114.91, 112.08, 108.84, 108.54.
4.3.14. 4, 6-(Dithiophen-2-yl)-[2,40]-bipyridyl (23). The
same procedure described at 4.3 was employed with 3
(R1, R2 = c) (1.10 g, 5.0 mmol), anhydrous ammonium
acetate (3.85 g, 50.0 mmol), 6 (1.63 g, 5.0 mmol), and
dry MeOH (30 mL) to yield a white solid (0.50 g,
31.3%). Rf (ethyl acetate/n-hexane 2:1): 0.3; mp
194.9 ꢁC; LC MS/MS: retention time: 18.30 min;
4.3.11. 6-(Furan-2-yl)-4-(furan-3-yl)-[2,40]-bipyridyl (20).
The same procedure described at 4.3 was employed with
3 (R1 = e, R2 = d) (0.66 g, 3.5 mmol), anhydrous ammo-
nium acetate (2.70 g, 35.0 mmol), 6 (1.14 g, 3.5 mmol),
and dry MeOH (30 mL) to yield a white solid (320 mg,
31.6%). Rf (ethyl acetate/n-hexane 1:2): 0.12; mp 80.0
ꢁC; LC MS/MS: retention time: 20.31 min; [MH]+:
289.2; 1H NMR (250 MHz, CDCl3) d 8.75 (dd,
J = 4.6, 1.5 Hz, 2H, 2-pyridine H-20, H-60), 8.02 (br,
1H, 4-furan H-2), 8.00 (dd, J = 4.6, 1.5 Hz, 2H, 2-pyri-
dine H-30, H-50), 7.82 (d, J = 1.2 Hz, 1H, pyridine H-
3), 7.71 (d, J = 1.2 Hz, 1H, pyridine H-5), 7.58 (br,
1H, 6-furan H-5), 7.57 (t, J = 1.7 Hz, 1H, 4-furan H-
5), 7.23 (d, J = 3.3 Hz, 1H, 6-furan H-3), 6.86 (dd,
J = 1.7, 0.8 Hz, 1H, 4-furan H-4), 6.59 (dd, J = 3.3,
1.7 Hz, 1H, 6-furan H-4); 13C NMR (62.5 MHz, CDCl3)
d 154.78, 153.45, 150.11, 146.44, 144.73, 144.49, 143.53,
143.46, 141.89, 140.62, 138.22, 124.29, 121.25, 115.62,
115.00, 113.22, 112.22, 111.63, 109.47, 109.32, 108.32.
1
[MH]+: 321.3; H NMR (250 MHz, CDCl3) d 8.76 (dd,
J = 4.6, 1.6 Hz, 2H, 2-pyridine H-20, H-60), 8.08 (dd,
J = 3.0, 1.2 Hz, 1H, 6-thiophene H-2), 8.05(dd, J = 4.6,
1.6 Hz, 2H, 2-pyridine H-30, H-50), 7.86 (d, J = 1.2 Hz,
1H, pyridine H-3), 7.83 (d, J = 1.2 Hz, 1H, pyridine H-
5), 7.81 (dd, J = 5.0, 1.2 Hz, 1H, 6-thiophene H-5),
7.45 (dd, J = 2.7, 1.5 Hz, 1H, 4-thiophene H-2), 7.54
(dd, J = 5.0, 1.5 Hz, 1H, 4-thiophene H-5), 7.52 (dd,
J = 5.0, 2.8 Hz, 1H, 4-thiophene H-4), 7.45 (dd,
J = 5.0, 3.0 Hz, 1H, 6-thiophene H-4); 13C NMR
(62.5 MHz, CDCl3) d 154.81, 154.22, 150.39, 149.00,
146.45, 144.81, 141.94, 139.62, 127.33, 126.44, 126.32,
125.84, 124.20, 123.36, 121.17, 117.49, 116.26.

4358
A. Basnet et al. / Bioorg. Med. Chem. 15 (2007) 4351–4359
4.3.15. 4-(Furan-2-yl)-6-(thiophene-2-yl)-[2,40]-bipyridyl
(24). The same procedure described at 4.3 was employed
with 3 (R1 = d, R2 = b) (1.02 g, 5.0 mmol), anhydrous
ammonium acetate (3.85 g, 50.0 mmol), 6 (1.63 g,
5.0 mmol), and dry MeOH (30 mL) to yield a white solid
(360 mg, 24.1%). Rf (ethyl acetate/n-hexane 2:1): 0.28;
mp 156.5 ꢁC; LC MS/MS: retention time: 18.32 min;
4.4. Pharmacology
For the evaluation of cytotoxicity, four different human
cancer cell lines were utilized: A-549 (human lung carci-
noma), SK-OV-3 (human ovary adenocarcinoma), SK-
MEL-2 (human malignant melanoma), and HCT15
(human colon adenocarcinoma). All experimental proce-
dures followed up the NCI’s protocol13,14 based on the
Sulforhodamine B (SRB) method. In brief, tumor cells
were cultured to maintain logarithmic growth by chang-
ing the medium 24 h before cytotoxicity assay. On the
day of the assay, the cells were harvested by trysinization,
counted, diluted in media, and added to 96-well plates.
The concentrations of tumor cells used were 5 · 103
(A549, HCT15), 1 · 104 (SK-MEL-2), and 2 · 104 cells/
well (SK-OV-3). The cells were then preincubated for
24 h in 5% CO2 incubator at 37 ꢁC. The compounds dis-
solved in DMSO were added to the wells in six 3-fold dilu-
tions starting from the highest concentrations, and
incubated for 48 h in 5% CO2 incubator at 37 ꢁC. The final
DMSO concentration was <0.5%. At the termination of
the incubation, the culture medium in each well was re-
moved, and the cells were fixed with cold 10% trichloro-
acetic acid (TCA) for 1 h at 4 ꢁC. The microplates were
washed, dried, and stained with 0.4% SRB in 1% acetic
acid for 30 min at room temperature. The cells were
washed again and the bound stain was solubilized with
10 mM Tris base solution (pH 10.5), and the absorbances
were measured spectrophotometrically at 520 nm on a
microtiter plate reader (Molecular Devices, Sunnyvale,
CA). The data were transformed into MS Excel format
and survival fractions were calculated by regression anal-
ysis (plotting the cell viability versus the concentration of
the test compound). The EC50 values represent concentra-
tions of the compounds that inhibit 50% of cell growth.
All data represent average values for a minimum of three
wells.
1
[MH]+: 305.2; H NMR (250 MHz, CDCl3) d 8.76 (dd,
J = 4.6, 1.6 Hz, 2H, 2-pyridine H-20, H-60), 8.03 (dd,
J = 4.6, 1.6 Hz, 2H, 2-pyridine H-30, H-50), 7.87 (s,
2H, pyridine H-3, H-5), 7.72 (dd, J = 3.7, 1.0 Hz, 1H,
6-thiophene H-5), 7.60 (dd, J = 1.6, 0.5 Hz, 1H, 4-furan
H-5), 7.45 (dd, J = 5.0, 1.0 Hz, 1H, 6-thiophene H-3),
7.15 (dd, J = 5.0, 3.7 Hz, 1H, 6-thiophene H-4), 7.99
(d, J = 3.4 Hz, 1H, 4-furan H-3), 6.58 (dd, J = 3.4,
1.8 Hz, 1H, 4-furan H-4); 13C NMR (62.5 MHz, CDCl3)
d 154.42, 153.20, 151.09, 150.31, 145.91, 144.65, 143.98,
139.28, 128.13, 128.05, 125.07, 121.03, 112.91, 112.60,
112.25, 109.15.
4.3.16. 4-(Thiophene-3-yl)-6-(thiophene-2-yl)-[2,30]-bipyr-
idyl (25). The same procedure described at 4.3 was em-
ployed with 3 (R1 = c, R2 = b) (1.10 g, 5.0 mmol),
anhydrous ammonium acetate (3.85 g, 50.0 mmol), 5
(1.63 g, 5.0 mmol), and dry MeOH (30 mL) to yield a
white solid (525 mg, 32.8%). Rf (ethyl acetate/n-hexane
2:1): 0.3; mp 179.3 ꢁC; LC MS/MS: retention time:
1
18.36 min; [MH]+: 321.2; H NMR (250 MHz, CDCl3)
d 9.34 (d, J = 1.8 Hz, 1H, 2-pyridine H-20), 8.68 (dd,
J = 4.8, 1.5 Hz, 1H, 2-pyridine H-60), 8.47 (dt, J = 8.0,
1.8 Hz, 1H, 2-pyridine H-40), 7.81 (d, J = 1.2 Hz, 1H,
pyridine H-3), 7.79 (d, J = 1.2 Hz, 1H, pyridine H-5),
7.77 (dd, J = 2.8, 1.5 Hz, 1H, 4-thiophene H-2), 7.72
(dd, J = 3.7, 1.0 Hz, 1H, 6-thiophene H-5), 7.54 (dd,
J = 5.0, 1.4 Hz, 1H, 4-thiophene H-5), 7.51 (dd,
J = 5.0, 2.8 Hz, 1H, 4-thiophene H-4), 7.47 (ddd,
J = 7.4, 4.8, 1.0 Hz, 1H, 2-pyridine H-50), 7.45 (dd,
J = 5.0, 1.0 Hz, 1H, 6-thiophene H-3), 7.16 (dd,
J = 5.0, 3.7 Hz, 1H, 6-thiophene H-4); 13C NMR
(62.5 MHz, CDCl3) d 154.83, 153.25, 150.07, 148.32,
144.87, 144.66, 139.56, 134.47, 128.03, 127.27, 125.85,
124.88, 123.57, 123.39, 115.87, 115.01.
The topoisomerase I inhibitory activity was carried out
as follows:15 the activity of DNA topoisomerase I was
determined by measuring the relaxation of supercoiled
DNA pBR322. For measurement of topoisomerase I
activity, the reaction mixture was comprised of 35 mM
Tris–HCl (pH 8.0), 72 mM KCl, 5 mM MgCl2, 5 mM
DTT, 5 mM spermidine, 0.01% bovine serum albumin
(BSA), 1 lg pBR322, and 2 U DNA topoisomerase.
Topoisomerase I inhibitors (prepared compounds) were
added to the above component for measuring the inhibi-
tion of DNA relaxation. The reaction mixture was incu-
bated at 37 ꢁC for 30 min. The reactions were
terminated by adding dye solution comprising 1%
SDS, 0.02% bromophenol blue, and 50% glycerol. The
mixture was applied to 1% agarose gel and electrophore-
sed for 1 h with a running buffer of Tris–acetate EDTA.
The gel was stained with ethidium bromide.
4.3.17. 4-(Furan-2-yl)-6-(thiophene-2-yl)-[2,30]-bipyridyl
(26). The same procedure described at 4.3 was employed
with 3 (R1 = d, R2 = b) (1.02 g, 5.0 mmol), anhydrous
ammonium acetate (3.85 g, 50.0 mmol), 5 (1.63 g,
5.0 mmol), and dry MeOH (30 mL) to yield a white solid
(490 mg, 28.5%). Rf (ethyl acetate/n-hexane 2:1): 0.3, mp
165.8 ꢁC; LC MS/MS: retention time: 18.08 min;
1
[MH]+: 305.3; H NMR (250 MHz, CDCl3) d 9.39 (d,
J = 1.5 Hz, 1H, 2-pyridine H-20), 8.68 (dd, J = 4.7,
1.3 Hz, 1H, 2-pyridine H-60), 8.47 (dt, J = 8.0, 1.9 Hz,
1H, 2-pyridine H-40), 7.85 (br, 2H, pyridine H-3, H-5),
7.73 (dd, J = 3.7, 1.0 Hz, 1H, 6-thiophene H-5), 7.61
(dd, J = 1.7, 0.5 Hz, 1H, 4-furan H-5), 7.45 (dd, J = 5.0,
1.0 Hz, 1H, 6-thiophene H-3), 7.43 (dd d, J = 8.0, 4.9,
0.7 Hz, 1H, 2-pyridine H-50), 7.16 (dd, J = 5.0, 3.7 Hz,
1H, 6-thiophene H-4), 7.00 (d, J = 3.4 Hz, 1H, 4-furan
H-3), 6.59 (dd, J = 3.4, 1.7 Hz, 1H, 4-furan H-4); 13C
NMR (62.5 MHz, CDCl3) d 154.70, 153.17, 151.25,
150.05, 148.29, 144.84, 143.92, 139.21, 134.44, 134.39,
128.02, 124.96, 123.55, 112.58, 112.22, 111.76, 109.04.
Acknowledgments
This work was supported by Korea Research Foundation
Grant (KRF-2006-E00609). The authors thank the Min-
istry of Education and Human Resources, Korea, for the
support of BK21 scholarship of P. Thapa and R. Karki.

A. Basnet et al. / Bioorg. Med. Chem. 15 (2007) 4351–4359
4359
J.; Cheng, C. C.; Thorp, H. H. J. Am. Chem. Soc. 1998,
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