Synthesis of 2-(thienyl-2-yl or -3-yl)-4-furyl-6-aryl pyridine derivatives and evaluation of their topoisomerase I and II inhibitory activity, cytotoxicity, and structure-activity relationship

Article abstract of DOI:10.1016/j.bmc.2010.01.065

A series of 2-(thienyl-2-yl or -3-yl)-4-furyl-6-aryl pyridine derivatives were designed, synthesized, and evaluated for their topoisomerase I and II inhibition and cytotoxic activity against several human cancer cell lines. Compounds 10-19 showed moderate

Full text of DOI:10.1016/j.bmc.2010.01.065

Bioorganic & Medicinal Chemistry 18 (2010) 2245–2254
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 2-(thienyl-2-yl or -3-yl)-4-furyl-6-aryl pyridine derivatives
and evaluation of their topoisomerase I and II inhibitory activity, cytotoxicity,
and structure–activity relationship
Pritam Thapa a, Radha Karki a, Hoyoung Choi a, Jae Hun Choi a, Minho Yun a, Byeong-Seon Jeong a,
a,
Mi-Ja Jung b, Jung Min Nam b, Younghwa Na c, Won-Jea Cho d, Youngjoo Kwon b, , Eung-Seok Lee
*
*
a College of Pharmacy, Yeungnam University, Kyongsan 712-749, Republic of Korea
b College of Pharmacy, 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
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:
A series of 2-(thienyl-2-yl or -3-yl)-4-furyl-6-aryl pyridine derivatives were designed, synthesized, and
evaluated for their topoisomerase I and II inhibition and cytotoxic activity against several human cancer
cell lines. Compounds 10–19 showed moderate topoisomerase I and II inhibitory activity and 20–29
showed significant topoisomerase II inhibitory activity. Structure–activity relationship study revealed
that 4-(5-chlorofuran-2-yl)-2-(thiophen-3-yl) moiety has an important role in displaying topoisomerase
II inhibition.
Received 12 November 2009
Revised 22 January 2010
Accepted 23 January 2010
Available online 4 February 2010
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
2-(Thienyl-2-yl)-4-furyl-6-aryl pyridine
2-(Thienyl-3-yl)-4-furyl-6-aryl pyridine
Cytotoxicity
Topoisomerase I and II inhibition
1. Introduction
inhibitory activity.8h It would be very interesting to observe the dif-
ference of biological activities by systematic substitution on the
Topoisomerases have been one of the most promising targets
for the design of various anticancer agents.1 They are nuclear en-
zymes that play an important role in various key cellular processes
such as replication, transcription, recombination, repair, and chro-
matin assembly.2 They are basically classified into two major clas-
ses: type I enzymes that make single stranded cuts in DNA, and
type II enzymes that cut and pass double stranded DNA to allow
a cell to change its topology.3 Drugs from the camptothecin family4
and etoposide5 are clinically used to target topoisomerase I (topo I)
and topoisomerase II (topo II), respectively.
2-position of 2-(thienyl-2-yl or -3-yl)-4-furyl-6-aryl pyridine skele-
tons with various aromatic groups, and to evaluate cytotoxicity and
topoisomerase I and II inhibitory activities. In addition, we antici-
pated that we may obtain valuable information on the correlation
between positions of 2-(thienyl-2-yl or -3-yl)-4-furyl-6-aryl pyri-
dine skeletons and biological activities such as cytotoxicity and
topoisomerase I or II inhibitory activity, or on the effect of branching
on furyl or phenyl groups such as methyl or chloride. In continuation
of previous work,8h we designed and systematically prepared six dif-
ferent series of 2-(thienyl-2-yl or -3-yl)-4-furyl-6-aryl pyridine
derivatives with chloride or methyl substituents at different aryl
moieties for the purpose of pursuing more potent topo I and II inhib-
itory activity as shown in Figure 1. The compounds were evaluated
for topo I and II inhibitory activity and cytotoxicity against several
human cancer cell lines.
a
-Terpyridine is able to form metal complexes6 and binds with
RNA/DNA7 (Fig. 1). Our research group has reported that terpyridine
derivatives show strong cytotoxicity against several human cancer
cell lines and considerable topo I and II inhibitory activity.8 From
previous structure–activity relationship studies, we found that the
2-thienyl-4-furyl-pyridine skeleton exhibited considerable topo I
and II inhibitory activity.8b,g,h In addition, substitution with chloride
or methyl on thienyl and/or furyl moiety increased topoisomerase
2. Results and discussion
2.1. Chemistry
* 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.).
2-(Thienyl-2-yl or -3-yl)-4-furyl-6-aryl pyridine derivatives
were synthesized on the basis of previously reported methods9,10
E-mail addresses: ykwon@ewha.ac.kr (Y. Kwon), eslee@yu.ac.kr (E.-S. Lee).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.065

2246
P. Thapa et al. / Bioorg. Med. Chem. 18 (2010) 2245–2254
R = H, Cl
R
S
O
R1
=
S
4
R2=
H3C
O
N
O
2
6
R1
N
R2
CH3
or Cl
N
N
N
N
α-terpyridine
(2,2 :6',2''-terpyridine)
2-thienyl-4-furyl-6-aryl pyridine derivatives
Figure 1. Structure of a-terpyridine and 2-thienyl-4-furyl-6-aryl pyridine derivatives.
as shown in Scheme 1. At first, 10 pyridinium iodide salts were pre-
pared in 42.2–99.4% yield by refluxing 10 different aryl acetyls
(1a–j) with iodine in pyridine. Propenone intermediates were syn-
thesized by applying Claisen–Schmidt KOH catalyzed condensation
reaction.9 Two aryl ketones (3k, l) were reacted with three differ-
ent aryl aldehydes to get six propenone intermediates (4a, b; 5a,
b; 6a, b) in 74.2–91.8% yield. Finally, as per the modified Kröhnke
synthesis method,10 previously synthesized propenone intermedi-
ates and pyridinium iodide salts were reacted to give 2-thienyl-4-
furyl-6-aryl pyridine derivatives in 15.1–82.4% yield. Only seven
pyridinium salts 2 (R1 = b–h) were employed for four series, 7a,
7b, 8a, and 8b, to give 28 compounds. Compounds for the remain-
O
O
Step I
i
N
I-
R1
CH3
R1
2 (R1 = a-j)
1 (R1 = a-j)
O
O
2 (R1 = b-h)
H
R2
iii
O
ii
R1
N
R2
O
O
7a (R1 = b-h; R2 = k)
7b (R1 = b-h; R2 = l)
2-furaldehyde
4a (R2 = k)
4b (R2 = l)
O
Step III
Step II
ii
O
O
2 (R1 = b-h)
R2
CH3
R2
iii
O
H
R1
N
R2
O
3 (R2 = k, l)
O
5a (R2 = k)
5b (R2 = l)
8a (R1 = b-h; R2 = k)
8b (R1 = b-h; R2 = l)
3-furaldehyde
Cl
O
Cl
O
2 (R1 = a-j)
R2
H
O
iii
Cl
ii
R1
N
R2
O
O
5-chloro-2-furaldehyde
6a (R2 = k)
9a (R1 = a-j; R2 = k)
9b (R1 = a-j; R2 = l)
6b (R2 = l)
CH3
Cl
H3C
CH3
R1 and R2
=
CH3
O
O
a
e
b
c
d
f
Cl
Cl
S
N
S
N
l
k
j
h
i
g
Scheme 1. General synthetic scheme. Reagents and conditions: (i) pyridine, iodine (1.0 equiv), 3 h, 140 °C, 42.2–99.4%; (ii) aryl aldehydes (1.0 equiv), KOH (1.2 equiv), MeOH/
H2O (5:1), 1–3 h, 0 °C, 74.2–91.8%; (iii) NH4OAc (10.0 equiv), dry methanol or glacial AcOH, 7–21 h, 80–100 °C, 15.1–82.4%.

P. Thapa et al. / Bioorg. Med. Chem. 18 (2010) 2245–2254
2247
ing pyridinium salts have already been reported by our research
inhibitory activity at 100
l
M whereas others had very low or no
group.8d Similarly, 10 pyridinium salts 2 (R1 = a–j) were used for
two series, 9a, 9b, to give 20 compounds. As a whole, 48 com-
pounds were synthesized. It was noticed that the yield was re-
duced when Cl or CH3 substituents were placed at the ortho
position of phenyl ring.8h The structures of compounds bearing
considerable biological activities are displayed in Figure 2.
activity. Figure 4 and Table 1 illustrate the human DNA topo II
inhibitory activity of 2-(thienyl-2-yl or -3-yl)-4-furyl-6-aryl pyri-
dine derivatives (10–29) with etoposide as a positive control. Com-
pounds 20–29 showed significant topo II inhibitory activity both at
100 lM and 20 lM ranges from the 59.6–86.5% and 34.7–75.4%
inhibition ratio, respectively. Compounds 10–19 showed moderate
topo II inhibitory activity.
2.2. Topoisomerase I and II inhibitory activity
Structure–activity relationship study revealed that compounds
15–19, which bore moderate topo I and II inhibitory activity, pos-
sessed 4-(5-chlorofuran-2-yl)-2-(thiophen-2-yl) moiety in common.
Interestingly, compounds 20–29, which bore 4-(5-chlorofuran-2-
yl)-2-(thiophen-3-yl) moiety, all exhibited strong topo II inhibitory
activity, whereas topo I inhibitory activity was reduced or completely
lost, the exception being 27. This suggest that 4-(5-chlorofuran-2-yl)-
2-(thiophen-3-yl) moiety is important for displaying significant topo
Forty-eight 2-(thienyl-2-yl or -3-yl)-4-furyl-6-aryl pyridine
derivatives were prepared and evaluated for their topo I and II
inhibitory activity. Figure 3 and Table 1 illustrate the human
DNA topo I inhibitory activity of 2-(thienyl-2-yl or -3-yl)-4-furyl-
6-aryl pyridine derivatives (10–29) with camptothecin as a posi-
tive control. Compounds 15–19 and 27 showed moderate topo I
O
O
O
O
N
N
N
N
S
S
S
Cl
S
CH3
Cl
Cl
Cl
10
11
12
13
Cl
Cl
Cl
Cl
O
O
O
O
Cl
N
N
N
N
S
S
S
S
O
CH3
14
15
16
17
Cl
Cl
Cl
Cl
O
O
O
O
N
N
N
N
O
S
S
O
S
S
Cl
N
CH3
18
19
20
21
Cl
Cl
Cl
Cl
O
O
O
O
Cl
CH3
CH3
N
N
N
N
S
S
CH3
S
S
22
23
24
25
Cl
Cl
Cl
Cl
O
O
O
O
Cl
N
N
N
N
N
S
S
S
S
Cl
N
26
27
28
29
Figure 2. Structures of compounds displaying significant biological activities.

2248
P. Thapa et al. / Bioorg. Med. Chem. 18 (2010) 2245–2254
100 µM
T
2.3. Cytotoxicity
D
C
10 11 12 13 14 15 16
17 18 19
The prepared 2-(thienyl-2-yl or -3-yl)-4-furyl-6-aryl pyridine
derivatives 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 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 per unit of enzyme
(IC50) under the assay conditions and compared with that of adri-
amycin. Most of the compounds exhibited moderate cytotoxicity
against human cancer cell lines (Table 1). Compounds 12, 13, 19,
and 24 showed the most significant cytotoxicity against HeLa,
K562, HCT15, and MCF-7 cell lines, respectively, as compared to
standard. Similarly, compounds 15, 21–23, 25, 27 showed consid-
erable cytotoxicity against MCF-7.
D
T
C
20 21 22 23 24 25 26 27 28 29
20 µM
D
T
C
27
D
T
C 15 16 17 18
3. Conclusion
Lane D: pBR322 DNA only
Forty-eight 2-(thienyl-2-yl or -3-yl)-4-furyl-6-aryl pyridine
derivatives were designed and synthesized systematically in a total
of six series by efficient synthetic routes and evaluated for topo I
and II inhibitory activity and cytotoxicity against several human
cancer cell lines. Structure–activity relationship study for topo
inhibitory activities indicated that 4-(5-chlorofuran-2-yl)-2-(thio-
phen-3-yl) moiety was important to display significant topo II
inhibitory activity. 4-(5-Chlorofuran-2-yl)-2-(thiophen-2-yl) moi-
ety was also important to display moderate topo I and II inhibitory
activity. Most of the compounds showed moderate cytotoxicity
against several human cancer cell lines. However, we could not
reach a conclusion on the concrete correlation between cytotoxic
activity and topo I or II inhibitory activities, or between cytotoxic
activity and structures of compounds. However, 4-(furan-3-yl)-2-
(thiophen-3-yl), 4-(5-chlorofuran-2-yl)-2-(thiophen-3-yl), and 4-
Lane T: pBR322 DNA + Topo I
Lane C: pBR322 DNA + Topo I + Camptothecin
Lane 10-29: pBR322 DNA + Topo I + Compound 10-29 (100 µM)
Lane 15-18, 27: pBR322 DNA + Topo I + Compound 15-18, 27 (20 µM)
Figure 3. Human DNA topoisomerase I inhibitory activity of compounds 10–29.
II inhibitoryactivity (Fig. 5). Although the compounds with 4-(5-chlo-
rofuran-2-yl)-2-(thiophen-2-yl) moiety showed moderate topo I and
II inhibitoryactivity, further explorationon this moiety may providea
promising skeleton for the development of dual topo I and II
inhibitors.
Table 1
Inhibitory activity of compounds 10–29 against topoisomerase I and II (% inhibition ratio of relaxation) and their IC50 values against MCF-7, HeLa, DU145, HCT15 and K562 cell
lines
a
Compound
% Inhibition
IC50
(l
M)
Topo I
20
Topo II
20 lM
MCF-7
HeLa
DU145
HCT15
K562
100
lM
l
M
100
lM
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
2.7
0
13.2
0
16.0
41.8
41.3
42.0
38.9
25.8
8.2
9.1
6.7
7.5
2.6
0.0
0.0
29.8
0.0
NA
NA
NA
NA
NA
9.3
8.0
8.5
6.3
NA
NA
NA
NA
NA
NA
NA
NA
5.2
NA
NA
31.4
30.2
14.2
20.5
38.5
21.8
35.0
44.7
42.5
51.5
78.6
62.6
59.6
64.1
78.9
64.1
75.9
75.9
63.1
86.5
NA
NA
NA
NA
25.5
NA
15.4
32.7
38.1
30.9
63.8
34.7
46.3
56.7
70.2
59.5
75.4
65.7
60.9
60.8
24.60 1.20
29.09 2.34
9.32 0.87
56.14 20.30
5.27 0.90
1.33 0.40
1.93 0.64
2.86 1.67
2.45 1.41
2.87 0.90
21.28 2.67
1.42 0.56
1.23 0.10
1.28 0.87
0.63 0.39
1.71 0.80
4.26 1.51
1.77 0.56
20.99 1.08
>50
32.72 10.65
5.53 1.10
1.47 0.07
4.14 0.88
18.80 2.77
16.3 3.56
18.53 2.14
12.76 1.82
21.34 2.94
14.64 3.42
>50
>100
>100
>100
21.07 9.21
13.01 2.16
37.59 9.13
21.84 4.59
19.27 3.68
16.82 2.33
18.10 2.72
19.18 2.80
12.98 1.62
18.86 0.87
18.50 1.95
24.76 3.78
17.92 2.25
21.23 0.96
19.30 1.12
10.42 0.10
19.53 3.30
10.94 1.38
21.57 1.42
0.94 0.19
50.01 6.77
44.82 5.20
>100
21.12 3.54
47.81 1.58
0.73 0.04
16.03 3.38
21.50 0.94
17.18 1.69
19.75 3.70
20.88 2.04
22.30 0.53
22.35 0.6
23.29 3.55
18.38 3.25
20.16 2.22
19.86 3.04
17.24 2.76
23.04 2.77
18.74 2.73
21.88 1.52
21.69 2.49
0.08 0.01
2.84 0.19
2.45 0.23
>50
9.89 0.08
6.19 2.98
15.50 5.50
14.20 3.51
1.24 0.15
>50
>50
>50
9.77 2.73
20.3 2.72
16.23 4.27
8.07 1.88
8.71 2.89
8.41 2.34
22.46 1.11
>50
4.63 1.78
21.71 2.37
7.41 1.45
4.57 0.79
13.67 0.97
5.19 3.25
>50
26
27
28
29
0.0
>50
Camptothecin (C)
Etoposide (E)
Adriamycin
75.3/81.7*
53.4/39.5*
0.05 0.02
1.66 0.25
1.25 0.47
0.63 0.02
1.57 0.43
1.82 0.84
0.64 0.03
1.29 0.53
2.32 1.35
51.0/55.1*
31.3/24.0*
2.45 1.05
1.98 0.92
NA: not applicable.
a
Each data represents mean S.D. from three different experiments performed in triplicate.
*
Control value for compounds 20–29.

P. Thapa et al. / Bioorg. Med. Chem. 18 (2010) 2245–2254
2249
a Bruker AMX 250 (250 MHz, FT) for 1H NMR and 62.5 MHz for
13C NMR, and chemical shifts were calibrated to TMS (tetramethyl-
silane). 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.
100 µM
D
T
E
10 11 12 13 14 15 16 17 18 19
D
T
E
20 21 22 23 24 25 26 27 28 29
HPLC analyses were performed using a gradient-controlled
HPLC system with two Shimadzu LC-10AT pumps, equipped with
Shimadzu system controller (SCL-10A VP) and photo diode array
detector (SPD-M10A VP) that utilized the Shimadzu Class VP pro-
gram. Sample volume of 10 l 5 lM
L was run in Waters X-TerraÒ
reverse-phase C18 column (4.6 ꢀ 250 mm) with a gradient elution
of 75–100% of B in A for 10 min followed by 100–75% of B in A
for 20 min at flow rate of 1.0 mL/min at 254 nm UV detection,
where mobile phase A was double distilled water with 20 mM
ammonium formate (AF), and B was 90% ACN in water with
20 mM AF. The purity of the compound is described as percent (%).
ESI LC/MS analyses were performed with a Finnigan LCQ Advan-
tageÒ LC/MS/MS spectrometry that utilized the XcaliburÒ program.
20 µM
D
T
E
14 16 17 18 19
D
T
E
20 21 22 23 24 25 26 27 28 29
For ESI LC/MS, LC was performed with 8
lL injection volume on a
Waters X-TerraÒ 3.5
l
m reverse-phase C18 column (2.1 ꢀ 100 mm)
with a gradient elution: (A) from 10% to 95% of B in A for 10 min fol-
lowed by 95% to 10% of B in A for 10 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% of B in A
for 8 min at a flow rate of 200 lL/min, where mobile phase A was
Lane D: pBR322 DNA only
100% distilled water with 20 mM AF and mobile phase B was 100%
ACN. MS ionization conditions were: Sheath gas flow rate: 40 arb,
aux gas flow rate: 0 arb, I spray voltage: 5.3 KV, capillary tempera-
ture: 275 °C, capillary voltage: 27 V, tube lens offset: 45 V. Retention
time is given in minutes.
Lane T: pBR322 DNA + Topo II
Lane E: pBR322 DNA + Topo II + Etoposide
Lane 10-29: pBR322 DNA + Topo II + Compound 10-29 (100 µM)
Lane 14, 16-29: pBR322 DNA + Topo II + Compound 14, 16-29 (20 µM)
4.1. General method for preparation of 2
Figure 4. Human DNA topoisomerase II
a
inhibitory activity of compounds 10–29.
A mixture of aryl acetyl 1, iodine (1.0 equiv) and pyridine (50 mL)
was refluxed at 140 °C for 3 h. The reaction mixture was cooled to
0 °C, the precipitate formed was filtered, washed with cold pyridine,
and dried to get solid compound 2 in 42.2–99.4% yield. Following the
same procedure, 10 compounds were synthesized.
Cl
O
Cl
O
4.2. General method for preparation of 4, 5, 6
N
R2
N
R2
To an ice cold solution of 85% KOH (1.2 equiv) in methanol
(10 mL) and H2O (2 mL), aryl acetyl 3 (1.0 equiv) was added. After
dissolution, aryl aldehyde (1.0 equiv) was added slowly. The reac-
tion mixture was then stirred for 1–3 h at 0 °C. The precipitation
formed was filtered, washed with cold MeOH, and dried to get solid
compound 4, 5, and 6 in 74.2–91.8% yield. Following the same pro-
cedure, 6 compounds were synthesized.
S
S
4-(5-chlorofuran-2-yl)-2-(thiophen-2-yl)
moiety
4-(5-chlorofuran-2-yl)-2-(thiophen-3-yl)
moiety
Figure 5. Structure of 4-(5-chlorofuran-2-yl)-2-(thiophen-3-yl) and 4-(5-chlorofu-
ran-2-yl)-2-(thiophen-2-yl) moiety.
(5-chlorofuran-2-yl)-2-(thiophen-2-yl) moieties, in combination
with e, h, and j moieties, were important for cytotoxic effect. These
results may provide valuable information to researchers working
on the development of antitumor agents.
4.2.1. 3-(Furan-2-yl)-1-(thiophen-2-yl)-propenone (4a)
The same procedure described in Section 4.2 was employed
with 3 (R2 = k) and 2-furaldehyde to yield yellow solid (81.1%).
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.30; mp 68.2–69.3 °C.
1H NMR (250 MHz, CDCl3) d 7.83 (dd, J = 3.8, 1.0 Hz, 1H, 1-thio-
phene H-3), 7.64 (dd, J = 4.8, 1.0 Hz, 1H, 1-thiophene H-5), 7.58 (d,
J = 15.3 Hz, 1H, –CO–C@CH–), 7.51 (d, J = 1.4 Hz, 1H, 3-furan H-5),
7.30 (d, J = 15.3 Hz, 1H, –CO–CH@C–), 7.15 (dd, J = 4.9, 3.9 Hz, 1H,
1-thiophene H-4), 6.71 (d, J = 3.4 Hz, 1H, 3-furan H-3), 6.50 (dd,
J = 3.4, 1.8 Hz, 1H, 3-furan H-4).
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
chromatography (CC) were performed with Kieselgel 60 F254
(Merck) and silica gel (Kieselgel 60, 230–400 mesh, Merck), respec-
tively. Since all the compounds prepared 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
4.2.2. 3-(Furan-2-yl)-1-(thiophen-3-yl)-propenone (4b)
The same procedure described in Section 4.2 was employed
with 3 (R2 = l) and 2-furaldehyde to yield a yellow solid (82.5%).
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.30; mp 51.7–52.1 °C.
1H NMR (250 MHz, CDCl3) d 8.15 (dd, J = 2.8, 1.2 Hz, 1H, 1-thio-
phene H-2), 7.63 (dd, J = 5.2, 1.2 Hz, 1H, 1-thiophene H-4), 7.56 (d,

2250
P. Thapa et al. / Bioorg. Med. Chem. 18 (2010) 2245–2254
J = 15.3 Hz, 1H, –CO–C@CH–), 7.50 (br, 1H, 3-furan H-5), 7.32 (dd,
J = 5.2, 2.8 Hz, 1H, 1-thiophene H-5), 7.29 (d, J = 15.3 Hz, 1H, –
CO–CH@C–), 6.69 (d, J = 3.4 Hz, 1H, 3-furan H-3), 6.49 (dd, J = 3.4,
1.8 Hz, 1H, 3-furan H-4).
30.00 mmol), 2 (R1 = h) (1.07 g, 3.00 mmol), and glacial acetic acid
(5.00 mL) to yield a white solid (204 mg, 20.1%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.26; mp 125.8–127.2 °C,
purity: 100%.
LC/MS/MS (condition A): retention time: 13.76 min; [MH+]:
338.2 (100%), [MH+2]: 340.2 (38%).
4.2.3. 3-(Furan-3-yl)-1-(thiophen-2-yl)-propenone (5a)
The same procedure described in Section 4.2 was employed
with 3 (R2 = k) and 3-furaldehyde to yield a creamy white solid
(74.2%).
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.30; mp 83.3–84.5 °C.
1H NMR (250 MHz, CDCl3) d 7.81 (d, J = 3.8 Hz, 1H, 1-thiophene
H-3), 7.73 (d, J = 15.4 Hz, 1H, –CO–C@CH–), 7.72 (br, 1H, 3-furan H-
2), 7.65 (d, J = 4.8 Hz, 1H, 1-thiophene H-5), 7.45 (br, 1H, 3-furan H-
5), 7.17 (dd, J = 5.7, 2.9 Hz, 1H, 1-thiophene H-4), 7.11 (d,
J = 15.7 Hz, 1H, –CO–CH@C–), 6.68 (br, 1H, 3-furan H-4).
1H NMR (250 MHz, CDCl3) d 8.10 (dd, J = 8.5, 2.0 Hz, 2H, 2-phe-
nyl H-2, H-6), 7.81 (s, 1H, pyridine H-3), 7.80 (s, 1H, pyridine H-5),
7.71 (dd, J = 3.7, 1.0 Hz, 1H, 6-thiophene H-3), 7.59 (dd, J = 1.7,
0.6 Hz, 1H, 4-furan H-5), 7.47 (dd, J = 8.5, 2.0 Hz, 2H, 2-phenyl H-
3, H-5), 7.43 (dd, J = 5.0, 1.0 Hz, 1H, 6-thiophene H-5), 7.15 (dd,
J = 5.0, 3.7 Hz, 1H, 6-thiophene H-4), 6.97 (d, J = 3.4 Hz, 1H, 4-furan
H-3), 6.57 (dd, J = 3.4, 1.7 Hz, 1H, 4-furan H-4).
13C NMR (62.5 MHz, CDCl3) d 156.46, 153.26, 151.88, 145.51,
144.22, 139.53, 137.70, 135.71, 129.28, 128.66, 128.44, 128.28,
125.23, 112.80, 112.62, 111.80, 109.26.
4.2.4. 3-(Furan-3-yl)-1-(thiophen-3-yl)-propenone (5b)
The same procedure described in Section 4.2 was employed
with 3 (R2 = l) and 3-furaldehyde to yield a creamy white solid
(79.5%).
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.35; mp 96.3–97.4 °C.
1H NMR (250 MHz, CDCl3) d 8.11 (dd, J = 2.8, 1.2 Hz, 1H, 1-thio-
phene H-2), 7.70 (d, J = 15.4 Hz, 1H, –CO–C@CH–), 7.71 (br, 1H,
3-furan H-2), 7.63 (d, J = 5.0 Hz, 1H, 1-thiophene H-4), 7.45 (br,
1H, 3-furan H-5), 7.33 (dd, J = 5.0, 2.8 Hz, 1H, 1-thiophene H-5),
7.09 (d, J = 15.5 Hz, 1H, –CO–CH@C), 6.67 (br, 1H, 3-furan H-4).
4.3.2. 2-(4-Chlorophenyl)-4-(furan-3-yl)-6-(thiophen-2-yl)
pyridine (11)
The same procedure described in Section 4.3 was employed
with 5a (0.51 g, 2.50 mmol), dry ammonium acetate (1.92 g,
25.00 mmol), 2 (R1 = h) (0.90 g, 2.50 mmol), and glacial acetic acid
(5.00 mL) to yield a white solid (428 mg, 50.7%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.29; mp 119.2–120.0 °C,
purity: 96.9%.
LC/MS/MS (condition A): retention time: 13.24 min; [MH+]:
338.2 (100%), [MH+2]: 340.2 (38%).
4.2.5. 3-(5-Chlorofuran-2-yl)-1-(thiophen-2-yl)-propenone (6a)
The same procedure described in Section 4.2 was employed
with 3 (R2 = k) and 5-chloro-2-furaldehyde to yield a yellow solid
(91.8%).
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.33; mp 109.4–110.5 °C.
1H NMR (250 MHz, CDCl3) d 7.87 (dd, J = 3.8, 1.1 Hz, 1H, 1-thio-
phene H-3), 7.69 (dd, J = 4.9, 1.1 Hz, 1H, 1-thiophene H-5), 7.49
(d, J = 15.1 Hz, 1H, –CO–C@CH–), 7.29 (d, J = 15.0 Hz, 1H, –CO–
CH@C–), 7.18 (dd, J = 4.9, 3.8 Hz, 1H, 1-thiophene H-4), 6.69 (d,
J = 3.5 Hz, 1H, 3-furan H-3), 6.32 (d, J = 3.5 Hz, 1H, 3-furan H-4).
1H NMR (250 MHz, CDCl3) d 8.06 (dd, J = 8.7, 1.9 Hz, 2H, 2-phe-
nyl H-2, H-6), 7.95 (dd, J = 1.3, 1.0 Hz, 1H, 4-furan H-2), 7.67 (dd,
J = 3.7, 1.0 Hz, 1H, 6-thiophene H-3), 7.63 (d, J = 1.3 Hz, 1H, pyri-
dine H-3), 7.61 (d, J = 1.3 Hz, 1H, pyridine H-5), 7.55 (t, J = 1.8 Hz,
1H, 4-furan H-5), 7.45 (dd, J = 8.7, 2.0 Hz, 2H, 2-phenyl H-3, H-5),
7.42 (dd, J = 5.0, 1.0 Hz, 1H, 6-thiophene H-5), 7.13 (dd, J = 5.0,
3.7 Hz, 1H, 6-thiophene H-4), 6.81 (dd, J = 1.8, 0.9 Hz, 1H, 4-furan
H-4).
13C NMR (62.5 MHz, CDCl3) d 156.15, 152.91, 145.08, 144.38,
141.70, 140.40, 137.35, 135.29, 128.86, 128.24, 127.97, 127.83,
124.72, 124.59, 115.05, 114.03, 108.46.
4.2.6. 3-(5-Chlorofuran-2-yl)-1-(thiophen-3-yl)-propenone (6b)
The same procedure described in Section 4.2 was employed
with 3 (R2 = l) and 5-chloro-2-furaldehyde to yield a yellow solid
(90.8%).
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.33; mp 109.8–110.8 °C.
1H NMR (250 MHz, CDCl3) d 8.19 (dd, J = 2.9, 1.2 Hz, 1H, 1-thio-
phene H-2), 7.67 (dd, J = 5.1, 1.2 Hz, 1H, 1-thiophene H-4), 7.47 (d,
J = 15.3 Hz, 1H, –CO–C@CH–), 7.36 (dd, J = 5.1, 2.9 Hz, 1H, 1-thio-
phene H-5), 7.29 (d, J = 15.3 Hz, 1H, –CO–CH@C–), 6.68 (d,
J = 3.5 Hz, 1H, 3-furan H-3), 6.31 (d, J = 3.5 Hz, 1H, 3-furan H-4).
4.3.3. 4-(Furan-3-yl)-2-(thiophen-3-yl)-6-p-tolyl pyridine (12)
The same procedure described in Section 4.3 was employed
with 5b (1.02 g, 5.00 mmol), dry ammonium acetate (3.85 g,
50.00 mmol),
2 (R1 = e) (1.69 g, 5.00 mmol), and methanol
(30.00 mL) to yield a white solid (912 mg, 57.5%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.35; mp 93.2–94.1 °C,
purity: 97.9%.
LC/MS/MS (condition A): retention time: 13.50 min; [MH+]:
318.2 (100%).
1H NMR (250 MHz, CDCl3) d 8.03 (d, J = 7.9 Hz, 2H, 6-phenyl H-
2, H-6), 8.02 (br, 1H, 2-thiophene H-2), 7.94 (br, 1H, 4-furan H-2),
7.79 (dd, J = 5.0, 1.0 Hz, 1H, 2-thiophene H-4), 7.65 (d, J = 1.1 Hz,
1H, pyridine H-5), 7.58 (d, J = 1.1 Hz, 1H, pyridine H-3), 7.54 (t,
J = 1.5 Hz, 1H, 4-furan H-5), 7.40 (dd, J = 5.0, 3.0 Hz, 1H, 2-thio-
phene H-5), 7.30 (d, J = 7.9 Hz, 2H, 6-phenyl H-3, H-5), 6.82 (d,
J = 0.9 Hz, 1H, 4-furan H-4), 2.41 (s, 3H, 6-phenyl 4-CH3).
13C NMR (62.5 MHz, CDCl3) d 157.50, 153.58, 144.23, 142.44,
141.33, 140.20, 139.06, 136.52, 129.36, 126.85, 126.41, 126.10,
124.84, 123.73, 115.09, 115.03, 108.49, 21.31.
4.3. General method for preparation of 7, 8, 9
A mixture of 2 (R1 = a–j), 4, 5 or 6 (R2 = k, l), and dry ammonium
acetate in glacial acetic acid or dry methanol was heated to 80–
100 °C for 7–21 h under nitrogen gas. The solvent was evaporated
and the residue was extracted with ethyl acetate (80 mL), washed
with water (50 mL ꢀ 3) and 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 or yellow solid
compound 7, 8, or 9 in 15.1–82.4% yield. Following the same pro-
cedure, 48 compounds were synthesized.
4.3.4. 2-(4-Chlorophenyl)-4-(furan-3-yl)-6-(thiophen-3-yl)
pyridine (13)
The same procedure described in Section 4.3 was employed
with 5b (1.02 g, 5.00 mmol), dry ammonium acetate (3.85 g,
4.3.1. 2-(4-Chlorophenyl)-4-(furan-2-yl)-6-(thiophen-2-yl)
pyridine (10)
The same procedure described in Section 4.3 was employed
with 4a (0.61 g, 3.00 mmol), dry ammonium acetate (2.31 g,
50.00 mmol),
2 (R1 = h) (1.79 g, 5.00 mmol), and methanol
(30.00 mL) to yield a white solid (683 mg, 40.4%).

P. Thapa et al. / Bioorg. Med. Chem. 18 (2010) 2245–2254
2251
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.36; mp 130.8–132.0 °C,
purity: 100%.
1H NMR (250 MHz, CDCl3) d 7.80 (d, J = 1.4 Hz, 1H, pyridine H-
3), 7.74–7.71 (m, 1H, 2-phenyl H-6), 7.73 (dd, J = 3.7, 1.1 Hz, 1H,
6-thiophene H-3), 7.69 (d, J = 1.4 Hz, 1H, pyridine H-5), 7.51–7.48
(m, 1H, 2-phenyl H-3), 7.41 (dd, J = 5.1, 1.1 Hz, 1H, 6-thiophene
H-5), 7.39–7.34 (m, 2H, 2-phenyl H-4, H-5), 7.14 (dd, J = 5.1,
3.7 Hz, 1H, 6-thiophene H-4), 6.92 (d, J = 3.5 Hz, 1H, 4-furan H-3),
6.35 (d, J = 3.5 Hz, 1H, 4-furan H-4).
LC/MS/MS (condition A): retention time: 13.86 min; [MH+]:
338.2 (100%), [MH+2]: 340.2 (38%).
1H NMR (250 MHz, CDCl3) d 8.08 (d, J = 8.6 Hz, 2H, 2-phenyl H-
2, H-6), 8.02 (dd, J = 2.9, 1.0 Hz, 1H, 6-thiophene H-2), 7.94 (s, 1H,
4-furan H-2), 7.76 (dd, J = 5.0, 0.8 Hz, 1H, 6-thiophene H-4), 7.63 (s,
1H, pyridine H-3), 7.61 (s, 1H, pyridine H-5), 7.55 (t, J = 1.5 Hz, 1H,
4-furan H-5), 7.46 (d, J = 8.6 Hz, 2H, 2-phenyl H-3, H-5), 7.40 (dd,
J = 5.0, 3.0 Hz, 1H, 6-thiophene H-5), 6.81 (br, 1H, 4-furan H-4).
13C NMR (62.5 MHz, CDCl3) d 156.27, 153.77, 144.36, 142.17,
141.63, 140.31, 137.73, 135.13, 128.82, 128.26, 126.34, 126.27,
124.66, 123.93, 115.58, 115.07, 108.44.
13C NMR (62.5 MHz, CDCl3) d 157.02, 153.01, 150.88, 144.67,
138.70, 138.21, 137.14, 132.28, 131.77, 130.22, 129.74, 127.99,
127.87, 127.01, 125.03, 116.70, 110.95, 110.82, 108.89.
4.3.8. 4-(5-Chlorofuran-2-yl)-2-(3-chlorophenyl)-6-(thiophen-
2-yl) pyridine (17)
The same procedure described in Section 4.3 was employed
with 6a (0.47 g, 2.00 mmol), dry ammonium acetate (1.54 g,
20.00 mmol), 2 (R1 = g) (0.72 g, 2.00 mmol), and glacial acetic acid
(5.00 mL) to yield a white solid (298 mg, 40.0%).
4.3.5. 4-(5-Chlorofuran-2-yl)-2-(furan-2-yl)-6-(thiophen-2-yl)
pyridine (14)
The same procedure described in Section 4.3 was employed
with 6a (0.59 g, 2.50 mmol), dry ammonium acetate (1.92 g,
25.00 mmol), 2 (R1 = a) (0.78 g, 2.50 mmol), and glacial acetic acid
(5.00 mL) to yield a white solid (336 mg, 41.0%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.29; mp 147.0–147.8 °C,
purity: 99.3%.
LC/MS/MS (condition B): retention time: 9.40 min; [M+]: 372.1
(100%), [M+2]: 374.1 (38%), [M+4]: 376.1 (15%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.30; mp 142.5–143.5 °C,
purity: 100%.
1H NMR (250 MHz, CDCl3) d 8.12 (br, 1H, 2-phenyl H-2), 8.03–
7.99 (m, 1H, 2-phenyl H-6), 7.74 (d, J = 5.9 Hz, 1H, 6-thiophene
H-3), 7.72 (d, J = 1.3 Hz, 2H, pyridine H-3, H-5), 7.45–7.41 (m, 2H,
2-phenyl H-4, H-5), 7.43 (dd, J = 4.3, 1.1 Hz, 1H, 6-thiophene H-
5), 7.15 (dd, J = 4.9, 3.7 Hz, 1H, 6-thiophene H-4), 6.95 (d,
J = 3.5 Hz, 1H, 4-furan H-3), 6.36 (d, J = 3.5 Hz, 1H, 4-furan H-4).
13C NMR (62.5 MHz, CDCl3) d 155.87, 152.99, 150.81, 144.77,
140.46, 138.27, 138.10, 134.76, 129.92, 129.25, 128.05, 128.02,
127.08, 125.03, 112.04, 111.08, 110.82, 108.92.
LC/MS/MS (condition B): retention time: 6.49 min; [MH+]:
328.2 (100%), [MH+2]: 330.1 (38%).
1H NMR (250 MHz, CDCl3) d 7.69 (d, J = 1.2 Hz, 1H, pyridine H-
3), 7.67 (dd, J = 3.5, 1.1 Hz, 1H, 6-thiophene H-3), 7.65 (d,
J = 1.4 Hz, 1H, pyridine H-5), 7.54 (dd, J = 1.7, 0.8 Hz, 1H, 2-furan
H-5), 7.41 (dd, J = 5.0, 1.1 Hz, 1H, 6-thiophene H-5), 7.19 (dd,
J = 3.4, 0.7 Hz, 1H, 2-furan H-3), 7.12 (dd, J = 5.0, 3.7 Hz, 1H, 6-thi-
ophene H-4), 6.93 (d, J = 3.5 Hz, 1H, 4-furan H-3), 6.54 (dd, J = 3.4,
1.7 Hz, 1H, 2-furan H-4), 6.33 (d, J = 3.5 Hz, 1H, 4-furan H-4).
13C NMR (62.5 MHz, CDCl3) d 153.43, 152.92, 150.88, 149.53,
144.67, 143.37, 138.13, 137.79, 127.96, 127.88, 125.00, 112.14,
110.79, 110.41, 110.11, 109.42, 108.87.
4.3.9. 4-(5-Chlorofuran-2-yl)-2-(4-chlorophenyl)-6-(thiophen-
2-yl) pyridine (18)
The same procedure described in Section 4.3 was employed
with 6a (0.47 g, 2.00 mmol), dry ammonium acetate (1.54 g,
20.00 mmol), 2 (R1 = h) (0.72 g, 2.00 mmol), and glacial acetic acid
(5.00 mL) to yield a white solid (190 mg, 25.5%).
4.3.6. 4-(5-Chlorofuran-2-yl)-2-(thiophen-2-yl)-6-p-tolyl
pyridine (15)
The same procedure described in Section 4.3 was employed
with 6a (0.47 g, 2.00 mmol), dry ammonium acetate (1.54 g,
20.00 mmol), 2 (R1 = e) (0.67 g, 2.00 mmol), and glacial acetic acid
(5.00 mL) to yield a white solid (106 mg, 15.1%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.32; mp 134.1–134.9 °C,
purity: 99.6%.
LC/MS/MS (condition B): retention time: 9.44 min; [M+]: 372.1
(100%), [M+2]: 374.1 (38%), [M+4]: 376.1 (15%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.34; mp 121.7–122.3 °C,
purity: 99.1%.
1H NMR (250 MHz, CDCl3) d 8.09 (d, J = 8.7 Hz, 2H, 2-phenyl H-
2, H-6), 7.73 (d, J = 1.2 Hz, 1H, pyridine H-3), 7.72 (d, J = 1.2 Hz, 1H,
pyridine H-5), 7.70 (dd, J = 4.2, 1.1 Hz, 1H, 6-thiophene H-3), 7.46
(d, J = 8.7 Hz, 2H, 2-phenyl H-3, H-5), 7.42 (dd, J = 5.1, 1.1 Hz, 1H,
6-thiophene H-5), 7.15 (dd, J = 5.1, 3.7 Hz, 1H, 6-thiophene H-4),
6.94 (d, J = 3.5 Hz, 1H, 4-furan H-3), 6.36 (d, J = 3.5 Hz, 1H, 4-furan
H-4).
LC/MS/MS (condition B): retention time: 8.80 min; [MH+]: 352.2
(100%), [MH+2]: 354.2 (38%).
1H NMR (250 MHz, CDCl3) d 8.05 (d, J = 8.2 Hz, 2H, 6-phenyl H-
2, H-6), 7.74 (d, J = 1.2 Hz, 1H, pyridine H-5), 7.71 (dd, J = 3.4,
1.4 Hz, 1H, 2-thiophene H-3), 7.70 (d, J = 2.0 Hz, 1H, pyridine H-
3), 7.42 (dd, J = 5.0, 1.0 Hz, 1H, 2-thiophene H-5), 7.30 (d,
J = 8.2 Hz, 2H, 6-phenyl H-3, H-5), 7.14 (dd, J = 5.0, 3.7 Hz, 1H, 2-
thiophene H-4), 6.93 (d, J = 3.5 Hz, 1H, 4-furan H-3), 6.35 (d,
J = 3.5 Hz, 1H, 4-furan H-4), 2.42 (s, 3H, 6-phenyl 4-CH3).
13C NMR (62.5 MHz, CDCl3) d 157.38, 152.74, 151.19, 145.27,
139.35, 137.97, 137.87, 135.89, 129.41, 127.93, 127.76, 126.81,
124.71, 111.71, 110.47, 110.33, 108.82, 21.34.
13C NMR (62.5 MHz, CDCl3) d 156.12, 152.92, 150.88, 144.88,
138.21, 138.06, 137.07, 135.39, 128.86, 128.21, 128.01, 127.99,
124.93, 111.74, 110.77, 110.74, 108.90.
4.3.10. 4-(5-Chlorofuran-2-yl)-6-(thiophen-2-yl)-2,30-
bipyridine (19)
The same procedure described in Section 4.3 was employed
with 6a (0.32 g, 1.35 mmol), dry ammonium acetate (1.04 g,
13.50 mmol), 2 (R1 = j) (0.44 g, 1.35 mmol), and glacial acetic acid
(4.00 mL) to yield a yellow solid (181 mg, 39.6%).
Rf (ethyl acetate/n-hexane 1:2 v/v): 0.24; mp 178.6–179.6 °C,
purity: 100%.
4.3.7. 4-(5-Chlorofuran-2-yl)-2-(2-chlorophenyl)-6-(thiophen-
2-yl) pyridine (16)
The same procedure described in Section 4.3 was employed
with 6a (0.71 g, 3.00 mmol), dry ammonium acetate (2.31 g,
30.00 mmol), 2 (R1 = f) (1.07 g, 3.00 mmol), and glacial acetic acid
(5.00 mL) to yield a light yellow solid (172 mg, 15.4%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.31; mp 122.0–123.0 °C,
purity: 100%.
LC/MS/MS (condition B): retention time: 5.23 min; [MH+]:
339.2 (100%), [MH+2]: 341.2 (38%).
1H NMR (250 MHz, CDCl3) d 9.33 (d, J = 1.7 Hz, 1H, 2-pyridine H-
20), 8.68 (d, J = 4.7 Hz, 1H, 2-pyridine H-60), 8.46 (td, J = 8.0, 1.7 Hz,
1H, pyridine H-40), 7.78 (d, J = 0.8 Hz, 1H, pyridine H-3), 7.77 (d,
J = 0.8 Hz, 1H, pyridine H-5), 7.73 (dd, J = 3.7, 0.9 Hz, 1H, 6-thio-
LC/MS/MS (condition B): retention time: 7.44 min; [M+]: 372.1
(100%), [M+2]: 374.1 (38%), [M+4]: 376.1 (15%).

2252
P. Thapa et al. / Bioorg. Med. Chem. 18 (2010) 2245–2254
phene H-3), 7.45 (dd, J = 5.1, 1.0 Hz, 1H, 6-thiophene H-5), 7.43 (t,
J = 7.8 Hz, 1H, pyridine H-50), 7.16 (dd, J = 4.9, 3.7 Hz, 1H, 6-thio-
phene H-4), 6.97 (d, J = 3.5 Hz, 1H, 4-furan H-3), 6.37 (d,
J = 3.5 Hz, 1H, 4-furan H-4).
J = 3.5 Hz, 1H, 4-furan H-3), 6.34 (d, J = 3.5 Hz, 1H, 4-furan H-4),
2.48 (s, 3H, 6-phenyl 2-CH3).
13C NMR (62.5 MHz, CDCl3) d 160.54, 153.35, 151.17, 142.15,
140.20, 138.00, 137.50, 136.21, 130.89, 129.58, 128.43, 126.40,
126.17, 125.88, 124.04, 115.75, 111.66, 110.44, 108.81, 20.60.
13C NMR (62.5 MHz, CDCl3) d 154.88, 153.32, 150.71, 150.18,
148.33, 144.65, 138.23, 134.43, 134.24, 128.20, 128.06, 125.14,
123.55, 112.01, 111.23, 110.98, 108.97.
4.3.14. 4-(5-Chlorofuran-2-yl)-2-(thiophen-3-yl)-6-m-tolyl
pyridine (23)
The same procedure described in Section 4.3 was employed
with 6b (0.23 g, 1.00 mmol), dry ammonium acetate (0.77 g,
10.00 mmol), 2 (R1 = d) (0.34 g, 1.00 mmol), and glacial acetic acid
(2.50 mL) to yield a white solid (162 mg, 46.0%).
4.3.11. 4-(5-Chlorofuran-2-yl)-2-(furan-2-yl)-6-(thiophen-3-yl)
pyridine (20)
The same procedure described in Section 4.3 was employed
with 6b (0.31 g, 1.30 mmol), dry ammonium acetate (1.00 g,
13.00 mmol), 2 (R1 = a) (0.40 g, 1.30 mmol), and glacial acetic acid
(3.00 mL) to yield a white solid (351 mg, 82.4%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.35; mp 121.7–122.5 °C,
purity: 99.8%.
LC/MS/MS (condition B): retention time: 8.56 min; [MH+]:
352.2 (100%), [MH+2]: 354.2 (38%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.34; mp 155.5–156.3 °C,
purity: 99.7%.
1H NMR (250 MHz, CDCl3) d 8.07 (dd, J = 3.0, 1.2 Hz, 1H, 2-thio-
phene H-2), 7.96 (br, 1H, 6-phenyl H-2), 7.94 (d, J = 8.2 Hz, 1H, 6-
phenyl H-6), 7.81 (dd, J = 5.0, 1.2 Hz, 1H, 2-thiophene H-4), 7.77
(d, J = 1.3 Hz, 1H, pyridine H-5), 7.71 (d, J = 1.3 Hz, 1H, pyridine
H-3), 7.42 (dd, J = 5.0, 3.0 Hz, 1H, 2-thiophene H-5), 7.39 (t,
J = 7.6 Hz, 1H, 6-phenyl H-5), 7.25 (d, J = 7.6 Hz, 1H, 6-phenyl H-
4), 6.94 (d, J = 3.5 Hz, 1H, 4-furan H-3), 6.35 (d, J = 3.5 Hz, 1H, 4-fur-
an H-4), 2.47 (s, 3H, 6-phenyl 3-CH3).
LC/MS/MS (condition B) retention time: 6.23 min; [MH+]: 328.1
(100%), [MH+2]: 330.1 (38%).
1H NMR (250 MHz, CDCl3) d 8.03 (dd, J = 3.0, 1.2 Hz, 1H, 6-thio-
phene H-2), 7.77 (dd, J = 5.2, 1.2 Hz, 1H, 6-thiophene H-4), 7.75 (d,
J = 1.3 Hz, 1H, pyridine H-3), 7.65 (d, J = 1.3 Hz, 1H, pyridine H-5),
7.56 (dd, J = 1.6, 0.7 Hz, 1H, 2-furan H-5), 7.42 (dd, J = 5.0, 3.0 Hz,
1H, 6-thiophene H-5), 7.20 (d , J = 3.4 Hz, 1H, 2-furan H-3), 6.94
(d, J = 3.5 Hz, 1H, 4-furan H-3), 6.57 (dd, J = 3.4, 1.75 Hz, 1H, 2-fur-
an H-4), 6.35 (d, J = 3.5 Hz, 1H, 4-furan H-4).
13C NMR (62.5 MHz, CDCl3) d 157.79, 153.75, 151.34, 142.29,
139.12, 138.29, 137.95, 137.92, 129.96, 128.57, 127.68, 126.45,
126.15, 124.15, 124.02, 112.20, 112.15, 110.37, 108.81, 21.60.
13C NMR (62.5 MHz, CDCl3) d 153.84, 153.73, 151.06, 149.68,
143.29, 141.92, 138.04, 137.82, 126.36, 126.21, 124.13, 112.10,
111.98, 110.63, 110.15, 109.12, 108.84.
4.3.15. 4-(5-Chlorofuran-2-yl)-2-(thiophen-3-yl)-6-p-tolyl
pyridine (24)
4.3.12. 4-(5-Chlorofuran-2-yl)-2-(5-methylfuran-2-yl)-6-
(thiophen-3-yl) pyridine (21)
The same procedure described in Section 4.3 was employed
with 6b (0.31 g, 1.30 mmol), dry ammonium acetate (1.00 g,
13.00 mmol), 2 (R1 = b) (0.42 g, 1.30 mmol), and glacial acetic acid
(3.00 mL) to yield a white solid (228 mg, 51.3%).
The same procedure described in Section 4.3 was employed
with 6b (0.31 g, 1.30 mmol), dry ammonium acetate (1.00 g,
13.00 mmol), 2 (R1 = e) (0.44 g, 1.30 mmol), and glacial acetic acid
(3.00 mL) to yield a white solid (198 mg, 43.3%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.39; mp 147.2–148.0 °C,
purity: 100%.
LC/MS/MS (condition B): retention time: 8.67 min; [MH+]:
352.2 (100%), [MH+2]: 354.2 (38%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.37; mp 129.7–130.5 °C,
purity: 100%.
LC/MS/MS (condition B): retention time: 7.30 min; [MH+]: 342.1
(100%), [MH+2]: 344.1 (38%).
1H NMR (250 MHz, CDCl3) d 8.06 (br, 1H, 2-thiophene H-2), 8.04
(d, J = 8.3 Hz, 2H, 6-phenyl H-2, H-6), 7.82 (dd, J = 5.0, 1.1 Hz, 1H, 2-
thiophene H-4), 7.76 (d, J = 1.1 Hz, 1H, pyridine H-5), 7.69 (d,
J = 1.1 Hz, 1H, pyridine H-3), 7.42 (dd, J = 5.0, 3.0 Hz, 1H, 2-thio-
phene H-5), 7.30 (d, J = 8.3 Hz, 2H, 6-phenyl H-3, H-5), 6.92 (d,
J = 3.5 Hz, 1H, 4-furan H-3), 6.34 (d, J = 3.5 Hz, 1H, 4-furan H-4),
2.42 (s, 3H, 6-phenyl 4-CH3).
1H NMR (250 MHz, CDCl3) d 8.02 (dd, J = 3.0, 1.2 Hz, 1H, 6-thio-
phene H-2), 7.75 (dd, J = 5.1, 1.2 Hz, 1H, 6-thiophene H-4), 7.68 (d,
J = 1.4 Hz, 1H, pyridine H-3), 7.61 (d, J = 1.4 Hz, 1H, pyridine H-5),
7.41 (dd, J = 5.1, 3.0 Hz, 1H, 6-thiophene H-5), 7.09 (d, J = 3.2 Hz,
1H, 2-furan H-3), 6.94 (d, J = 3.5 Hz, 1H, 4-furan H-3), 6.34 (d,
J = 3.5 Hz, 1H, 4-furan H-4), 6.15 (dd, J = 3.2, 0.9 Hz, 1H, 2-furan
H-4), 2.43 (s, 3H, 2-furan 5-CH3).
13C NMR (62.5 MHz, CDCl3) d 157.57, 153.67, 151.39, 142.33,
139.22, 137.90, 136.35, 129.38, 126.86, 126.44, 126.11, 123.95,
111.92, 111.78, 110.30, 108.78, 21.31.
13C NMR (62.5 MHz, CDCl3) d 153.75, 153.59, 152.11, 151.22,
149.87, 142.05, 137.90, 137.73, 126.38, 126.13, 124.03, 111.49,
110.52, 110.28, 109.64, 108.79, 108.39, 13.96.
4.3.16. 4-(5-Chlorofuran-2-yl)-2-(2-chlorophenyl)-6-(thiophen-
3-yl) pyridine (25)
4.3.13. 4-(5-Chlorofuran-2-yl)-2-(thiophen-3-yl)-6-o-tolyl
pyridine (22)
The same procedure described in Section 4.3 was employed
with 6b (0.47 g, 2.00 mmol), dry ammonium acetate (1.54 g,
20.00 mmol), 2 (R1 = c) (0.67 g, 2.00 mmol), and glacial acetic acid
(3.00 mL) to yield a white solid (171 mg, 24.3%).
The same procedure described in Section 4.3 was employed
with 6b (0.41 g, 1.75 mmol), dry ammonium acetate (1.34 g,
17.50 mmol), 2 (R1 = f) (0.63 g, 1.75 mmol), and glacial acetic acid
(3.00 mL) to yield a white solid (249 mg, 38.3%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.35; mp 101.0–101.6 °C,
purity: 100%.
LC/MS/MS (condition B): retention time: 7.47 min; [M+]: 372.1
(100%), [M+2]: 374.1 (71%), [M+4]: 376.1 (15%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.35; mp 110.7–111.4 °C,
purity: 98.9%.
LC/MS/MS (condition B): retention time: 7.59 min; [MH+]:
352.2 (100%), [MH+2]: 354.2 (38%).
1H NMR (250 MHz, CDCl3) d 8.02 (dd, J = 3.0, 1.3 Hz, 1H, 6-thio-
phene H-2), 7.78 (d, J = 1.4 Hz, 1H, pyridine H-3), 7.75 (dd, J = 5.1,
1.3 Hz, 1H, 6-thiophene H-4), 7.72–7.69 (m, 1H, 2-phenyl H-6),
7.68 (d, J = 1.3 Hz, 1H, pyridine H-5), 7.52–7.48 (m, 1H, 2-phenyl
H-3), 7.40 (dd, J = 5.1, 3.0 Hz, 1H, 6-thiophene H-5), 7.39–7.34
(m, 2H, 2-phenyl H-4, H-5), 6.91 (d, J = 3.5 Hz, 1H, 4-furan H-3),
6.34 (d, J = 3.5 Hz, 1H, 4-furan H-4).
1H NMR (250 MHz, CDCl3) d 8.01 (dd, J = 3.0, 1.2 Hz, 1H, 2-thio-
phene H-2), 7.76 (dd, J = 4.9, 1.2 Hz, 1H, 2-thiophene H-4), 7.75 (d,
J = 1.3 Hz, 1H, pyridine H-3), 7.51–7.47 (m, 1H, 6-phenyl H-6), 7.46
(d, J = 1.3 Hz, 1H, pyridine H-5), 7.40 (dd, J = 5.0, 3.0 Hz, 1H, 2-thi-
ophene H-5), 7.34–7.28 (m, 3H, 6-phenyl H-3, H-4, H-5), 6.90 (d,

P. Thapa et al. / Bioorg. Med. Chem. 18 (2010) 2245–2254
2253
13C NMR (62.5 MHz, CDCl3) d 157.25, 153.93, 151.06, 141.98,
139.13, 138.14, 137.21, 132.33, 131.67, 130.19, 129.67, 126.94,
126.41, 126.23, 124.20, 116.56, 112.44, 110.67, 108.86.
13C NMR (62.5 MHz, CDCl3) d 156.18, 155.86, 153.45, 151.23,
148.99, 142.10, 138.21, 137.98, 136.91, 126.39, 126.26, 123.97,
121.38, 113.65, 112.71, 110.88, 108.87.
4.3.20. 4-(5-Chlorofuran-2-yl)-6-(thiophen-3-yl)-2,30-
bipyridine (29)
The same procedure described in Section 4.3 was employed
with 6b (0.28 g, 1.20 mmol), dry ammonium acetate (0.92 g,
12.00 mmol), 2 (R1 = j) (0.39 g, 1.20 mmol), and glacial acetic acid
(2.50 mL) to yield a yellow solid (238 mg, 58.6%).
4.3.17. 4-(5-Chlorofuran-2-yl)-2-(3-chlorophenyl)-6-(thiophen-
3-yl) pyridine (26)
The same procedure described in Section 4.3 was employed
with 6b (0.31 g, 1.30 mmol), dry ammonium acetate (1.00 g,
13.00 mmol), 2 (R1 = g) (0.46 g, 1.30 mmol), and glacial acetic acid
(3.00 mL) to yield a white solid (302 mg, 62.4%).
Rf (ethyl acetate/n-hexane 1:2 v/v): 0.23; mp 188.6–189.0 °C,
purity: 100%.
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.35; mp 153.8–154.6 °C,
purity: 99.6%.
LC/MS/MS (condition B): retention time: 4.29 min; [MH+]:
339.2 (100%), [MH+2]: 341.2 (38%).
LC/MS/MS (condition B): retention time: 9.26 min; [M+]: 372.1
(100%), [M+2]: 374.1 (71%), [M+4]: 376.1 (15%).
1H NMR (250 MHz, CDCl3) d 9.34 (d, J = 1.6 Hz, 1H, 2-pyridine H-
20), 8.68 (dd, J = 4.7, 1.3 Hz, 1H, 2-pyridine H-60), 8.45 (td, J = 8.0,
1.9 Hz, 1H, 2-pyridine H-40), 8.07 (dd, J = 3.0, 1.2 Hz, 1H, 6-thio-
phene H-2), 7.81 (dd, J = 5.1, 1.2 Hz, 1H, 6-thiophene H-4), 7.79
(d, J = 1.3 Hz, 1H, pyridine H-3), 7.76 (d, J = 1.2 Hz, 1H, pyridine
H-5), 7.45 (ddd, J = 8.0, 4.7, 1.8 Hz, 1H, 2-pyridine H-50), 7.42 (dd,
J = 5.0, 2.9 Hz, 1H, 6-thiophene H-5), 6.96 (d, J = 3.5 Hz, 1H, 4-furan
H-3), 6.37 (d, J = 3.5 Hz, 1H, 4-furan H-4).
1H NMR (250 MHz, CDCl3) d 8.15–8.14 (m, 1H, 2-phenyl H-2),
8.07 (dd, J = 3.0, 1.2 Hz, 1H, 6-thiophene H-2), 8.02–7.98 (m, 1H,
2-phenyl H-6), 7.81 (dd, J = 5.1, 1.2 Hz, 1H, 6-thiophene H-4),
7.74 (d, J = 1.3 Hz, 1H, pyridine H-3), 7.73 (d, J = 1.2 Hz, 1H, pyri-
dine H-5), 7.43 (dd, J = 5.1, 3.0 Hz, 1H, 6-thiophene H-5), 7.45–
7.41 (m, 2H, 2-phenyl H-4, H-5), 6.95 (d, J = 3.5 Hz, 1H, 4-furan
H-3), 6.36 (d, J = 3.5 Hz, 1H, 4-furan H-4).
13C NMR (62.5 MHz, CDCl3) d 156.02, 153.86, 150.97, 141.92,
140.89, 138.16, 138.11, 134.74, 129.88, 129.13, 127.13, 126.34,
126.31, 125.03, 124.23, 112.62, 112.08, 110.66, 108.89.
13C NMR (62.5 MHz, CDCl3) d 154.98, 154.14, 150.85, 150.04,
148.35, 141.82, 138.33, 138.22, 134.63, 134.42, 126.39, 126.29,
124.30, 123.51, 112.75, 112.06, 110.82, 108.93.
4.4. Pharmacology
4.3.18. 4-(5-Chlorofuran-2-yl)-2-(4-chlorophenyl)-6-(thiophen-
3-yl) pyridine (27)
DNA topo I inhibition assay was determined by following the
method previously reported.11 The test compounds were dissolved
in DMSO at 20 mM as the stock solution. The activity of DNA topoi-
somerase I was determined by assessing the relaxation of super-
coiled 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 relaxation buffer (10 mM Tris–
HCl (pH 7.9), 150 mM NaCl, 0.1% bovine serum albumin, 1 mM sper-
The same procedure described in Section 4.3 was employed
with 6b (0.31 g, 1.30 mmol), dry ammonium acetate (1.00 g,
13.00 mmol), 2 (R1 = h) (0.46 g, 1.30 mmol), and glacial acetic acid
(3.00 mL) to yield a white solid (316 mg, 65.4%).
Rf (ethyl acetate/n-hexane 1:10 v/v): 0.35; mp 128.4–130.0 °C,
purity: 99.4%.
LC/MS/MS (condition B): retention time: 9.23 min; [M+]: 372.1
(100%), [M+2]: 374.1 (71%), [M+4]: 376.1 (15%).
1H NMR (250 MHz, CDCl3) d 8.09 (d, J = 8.7 Hz, 2H, 2-phenyl H-
2, H-6), 8.05 (dd, J = 3.0, 1.2 Hz, 1H, 6-thiophene H-2), 7.80 (dd,
J = 5.1, 1.2 Hz, 1H, 6-thiophene H-4), 7.75 (d, J = 1.3 Hz, 1H, pyri-
dine H-3), 7.72 (d, J = 1.3 Hz, 1H, pyridine H-5), 7.46 (d, J = 8.7 Hz,
2H, 2-phenyl H-3, H-5), 7.42 (dd, J = 5.1, 3.0 Hz, 1H, 6-thiophene
H-5), 6.94 (d, J = 3.5 Hz, 1H, 4-furan H-3), 6.36 (d, J = 3.5 Hz, 1H,
4-furan H-4).
midine, 5% glycerol). The reaction in the final volume of 10
lL was
terminated by adding 2.5 L of the stop solution containing 5% sar-
l
cosyl, 0.0025% bromophenol blue, and 25% glycerol. DNA samples
were then electrophoresed on a 1% agarose gel at 15 V for 7 h with
a running buffer of TAE. Gels were stained for 30 min in an aqueous
solution of ethidium bromide (0.5 lg/ml). DNA bands were visual-
ized by transillumination with UV light and were quantitated using
AlphaImager™ (Alpha Innotech Corporation).
DNA topo II inhibitory activity of compounds was measured as
follows.11 The mixture of 200 ng of supercoiled pBR322 plasmid
13C NMR (62.5 MHz, CDCl3) d 156.29, 153.81, 151.05, 142.01,
138.12, 138.08, 137.51, 135.27, 128.83, 128.25, 126.33, 126.29,
124.13, 112.36, 111.81, 110.58, 108.87.
DNA and two units of human DNA topo IIa (Amersham, USA) was
incubated with and without the prepared compounds in the assay
buffer (10 mM Tris–HCl (pH 7.9) containing 50 mM NaCl, 5 mM
4.3.19. 4-(5-Chlorofuran-2-yl)-6-(thiophen-3-yl)-2,20-
bipyridine (28)
The same procedure described in Section 4.3 was employed
with 6b (0.23 g, 1.00 mmol), dry ammonium acetate (0.77 g,
10.00 mmol), 2 (R1 = i) (0.32 g, 1.00 mmol), and glacial acetic acid
(2.00 mL) to yield a white solid (173 mg, 51.3%).
MgCl2, 1 mM EDTA, 1 mM ATP, and 15
min) for 30 min at 30 °C. The reaction in a final volume of 20
was terminated by the addition of 3 L of 7 mM EDTA. Reaction
lg/mL bovine serum albu-
lL
l
productswereanalyzedon a1%agarosegelat25 Vfor4 hwitha run-
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.28; mp 167.6–168.3 °C,
purity: 100%.
ning buffer of TAE. Gels were stained for 30 min in an aqueous solu-
tionofethidiumbromide(0.5 lg/mL). DNAbandswerevisualizedby
transillumination with UV light and supercoiled DNA was quanti-
tated using AlphaImager™ (Alpha Innotech Corporation).
LC/MS/MS (condition B): retention time: 6.18 min; [MH+]:
339.2 (100%), [MH+2]: 341.2 (38%).
1H NMR (250 MHz, CDCl3) d 8.72 (td, J = 4.8, 1.2 Hz, 1H, 2-pyri-
dine H-60), 8.60 (d, J = 8.0 Hz, 1H, 2-pyridine H-30), 8.45 (d,
J = 1.5 Hz, 1H, pyridine H-3), 8.08 (dd, J = 3.0, 1.2 Hz, 1H, 6-thio-
phene H-2), 7.86 (dt, J = 8.0, 1.8 Hz, 1H, 2-pyridine H-40), 7.84 (d,
J = 1.4 Hz, 1H, pyridine H-5), 7.83 (dd, J = 5.0, 1.2 Hz, 1H, 6-thio-
phene H-4), 7.44 (dd, J = 5.0, 3.0 Hz, 1H, 6-thiophene H-5), 7.35
(ddd, J = 7.5, 4.8, 1.2 Hz, 1H, 2-pyridine H-50), 7.03 (d, J = 3.5 Hz,
1H, 4-furan H-3), 6.35 (d, J = 3.5 Hz, 1H, 4-furan H-4).
For the evaluation of cytotoxicity, five different cancer cell lines
were used: human breast adenocarcinoma cell line (MCF-7), human
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 by methods previously described.11 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

2254
P. Thapa et al. / Bioorg. Med. Chem. 18 (2010) 2245–2254
2. Nitiss, J. L. Biochim. Biophys. Acta 1998, 1400, 63.
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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, the culture medium in each well was exchanged with 0.1 mL ali-
quots 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 absor-
bance readings at 450 nm were fitted to a four-parameter logistic
equation. The compounds adriamycin, etoposide, and camptothecin
were purchased from Sigma and used as positive controls.
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).
9. Jahng, Y.; Zhao, L. X.; Moon, Y. S.; Basnet, A.; Kim, E. K.; Chang, H. W.; Ju, H. K.;
Jeong, T. C.; Lee, E.-S. Bioorg. Med. Chem. Lett. 2004, 14, 2559.
10. Kröhnke, F. Synthesis 1976, 1.
11. 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.
References and notes
1. Holden, J. A. Curr. Med. Chem.-Anti-Cancer Agents 2001, 1, 1.