2,6-Dithienyl-4-furyl pyridines: Synthesis, topoisomerase I and II inhibition, cytotoxicity, structure-activity relationship, and docking study

Article abstract of DOI:10.1016/j.bmcl.2009.11.041

For the development of novel antitumor agents, 2,6-dithienyl-4-furyl pyridine derivatives were prepared and evaluated for their topoisomerase I and II inhibitory activity as well as cytotoxicity against several human cancer cell lines. Among the 21 prepared compounds, compound 24 exhibited strong topoisomerase I inhibitory activity. In addition, a docking study with topoisomerase I and compound 24 was performed.

Full text of DOI:10.1016/j.bmcl.2009.11.041

Bioorganic & Medicinal Chemistry Letters 20 (2010) 42–47
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2,6-Dithienyl-4-furyl pyridines: Synthesis, topoisomerase I and II inhibition,
cytotoxicity, structure–activity relationship, and docking study
Arjun Basnet a, Pritam Thapa a, Radha Karki a, Hoyoung Choi a, Jae Hun Choi a, Minho Yun a,
Byeong-Seon Jeong a, Yurngdong Jahng a, Younghwa Na b, Won-Jea Cho c, Youngjoo Kwon d,
Chong-Soon Lee e, Eung-Seok Lee a,
*
a College of Pharmacy, Yeungnam University, Kyongsan 712-749, Republic of Korea
b College of Pharmacy, Catholic University of Daegu, Kyongsan 712-702, Republic of Korea
c College of Pharmacy, Chonnam National University, Kwangju 500-757, Republic of Korea
d College of Pharmacy, Pharmacy & Division of Life & Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Republic of Korea
e Department of Biochemistry, Yeungnam University, Kyongsan 712-749, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
For the development of novel antitumor agents, 2,6-dithienyl-4-furyl pyridine derivatives were prepared
and evaluated for their topoisomerase I and II inhibitory activity as well as cytotoxicity against several
human cancer cell lines. Among the 21 prepared compounds, compound 24 exhibited strong topoisomer-
ase I inhibitory activity. In addition, a docking study with topoisomerase I and compound 24 was
performed.
Received 14 October 2009
Revised 9 November 2009
Accepted 11 November 2009
Available online 14 November 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
2,6-Dithienyl-4-furyl pyridine,
Topoisomerase I and II inhibitor
Cytotoxicity
Antitumor agents
Docking study
Topoisomerases, generally classified as type I and II, are critical
cellular enzymes necessary for cell proliferation by solving topo-
logical hurdles in the process of DNA replication.1–3 Topoisomerase
I mediates the breaking and rejoining of single strand of DNA du-
plex to relax the supercoiled condition of chromosomes. On the
other hand, topoisomerase II produces the relaxation of DNA dou-
ble helices by scissoring and religating the two strands.1,4 Due to
the critical role of these enzymes for the cell proliferative process,
topoisomerases have been one of the major targets in the antican-
cer drug development area.5,6
From the results of the previous studies, it was revealed that the
2-thienyl-4-furylpyridine skeleton (compound 6 in Fig. 2) exhib-
ited strong topoisomerase I inhibitory activity (Fig. 1).10 Such pre-
vious studies prompted us to design 2,6-dithienyl-4-furyl pyridine
derivatives as topoisomerase I or II inhibitors in our endeavor to
develop novel anticancer agents. Since 2,6-dithienyl-4-furyl pyri-
dines possess 2-thienyl-4-furylpyridine skeleton, we expected that
substituents at the 6-position on the central pyridine ring as thie-
nyl group may affect biological activities such as topoisomerase I
or II as well as cytotoxicity. It would be very interesting to observe
the difference of biological activities by systematic substitution on
the 6-position of 2-thienyl-4-furyl pyridine skeletons with thienyl
group and to evaluate cytotoxicity and topoisomerase I and II
inhibitory activities. In addition, we anticipated that we may ob-
tain valuable information on the correlation between positions of
thienyl or furyl groups employed in 2-thienyl-4-furyl pyridine
skeletons and biological activities such as cytotoxicity and topoiso-
merase I or II inhibitory activity, or on the effect of branching on
thienyl or furyl groups such as methyl or chloride. In connection
with previous studies, we designed and prepared 21 2,6-dithie-
nyl-4-furyl pyridine derivatives as terpyridine or terthiophene bio-
isosteres employing 2-thienyl-4-furyl pyridine skeletons, and
Due to its ability to form metal complexes7 and its status as a
DNA binding agent, a-terpyridine has been precious since its dis-
covery in 1932.8 Our research group has previously reported that
terpyridine derivatives showed strong cytotoxicities against sev-
eral human cancer cell lines and considerable topoisomerase I
inhibitory activity.9,10 Some of us also reported that terthiophene
derivatives, bioisosteres of terpyridine, showed considerable pro-
tein kinase C (PKC) inhibitory activity and antitumor cytotoxicities
against several human cancer cell lines.11
* Corresponding author. Tel.: +82 53 810 2827; fax: +82 53 810 4654.
E-mail address: eslee@yu.ac.kr (E.-S. Lee).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.041

A. Basnet et al. / Bioorg. Med. Chem. Lett. 20 (2010) 42–47
43
R2
O
O
4
4
N
S
6
2
6
2
R1
S
R3
S
N
N
N
N
R
S
S
S
α-terthiophene
α-terpyridine
R1, R2, R3: -H, -CH3 or -Cl
2-thienyl-4-furylpyridine
skeleton
Figure 1. Structure of
a-terpyridine,
a-terthiophene and 2-thienyl-4-furylpyridine skeleton.
O
O
O
O
O
O
N
N
N
N
N
N
S
S
S
S
S
S
S
S
S
S
S
S
6
7
8
9
10
11
H3C
Cl
H3C
O
Cl
H3C
Cl
O
O
O
O
O
N
N
N
S
N
N
N
13
O
S
S
S
S
S
S
S
S
S
S
S
12
14
15
16
O
17
H3C
H3C
O
O
O
O
H3C
H3C
H3C
H3C
H3C
H3C
N
N
N
N
N
N
S
S
S
S
S
S
S
S
S
S
S
S
18
19
20
21
22
23
H3C
H3C
O
O
O
O
CH3
CH3
H3C
H3C
N
N
N
N
S
S
S
S
S
H3C
H3C
S
S
S
CH3
CH3
24
25
26
27
Figure 2. The prepared compounds.
evaluated them for their topoisomerase I and II inhibitory activity
and cytotoxicity against several human cancer cell lines, as well as
their structure–activity relationship. In addition, we performed a
docking study with topoisomerase I and the prepared compound
24 exhibited strong topoisomerase I inhibition.
For the design, pyridine moiety was utilized as a basic skeleton,
and thienyl derivatives were attached to the 2,6-position, and furyl
derivatives to the 4 position of the pyridine structure (Fig. 1).
Synthetic methods for the preparation of 2,6-dithienyl-4-furyl
pyridines (6–27) are summarized in Scheme 1. Acetylthiophenes
1a–d were treated with furancarboxaldehydes 2e–h in the pres-
ence of KOH in methanol–water (5:1) to afford intermediates 3
in a 27.1–89.6% yields. Using modified Kröhnke synthesis,12 2,6-
dithienyl-4-furyl pyridines were prepared by treatment of 3 with
1-(2-oxo-2-thieny-ethyl)pyridinium iodide (4a–c) in the presence
of ammonium acetate in methanol to give 6–27 in a 89.4–97.6%

44
A. Basnet et al. / Bioorg. Med. Chem. Lett. 20 (2010) 42–47
R2
H
O
+N
I-
R2
R2
R3
O
R1
2 (R2=e-h)
R1
4 (R3=a-c)
O
KOH
R1
N
R3
O
NH4OAc/MeOH
1 (R1=a-d)
3 (R1=a-d, R2=e-h)
5 (R1=a-d, R2=e-h,
R3=a-c)
CH3
R1 =
H3C
S
CH3
S
S
S
a
b
c
d
CH3
Cl
R2 =
R3 =
O
O
O
O
e
g
f
h
a, b, c
Scheme 1. Synthetic method.
yield. Pyridinium iodides 4a–c were prepared in a quantitative
yield by treatment of 1a–c with iodine in pyridine.
Figure 2 shows the prepared 2,6-dithienyl-4-furyl pyridines
(6–27).
Calf thymus DNA–topoisomerase I inhibitory activities13 for the
21 prepared 2,6-dithienyl-4-furyl pyridines are shown in Figure 3,
and summarized in Tables 1 and 2. Compounds 13, 15, 18, and 21
exhibited moderate topoisomerase I inhibitory activities (42–52%
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 1- 38 Lane a: pBR322 DNA + Topo I + Compounds 20 μM
Lane 1- 38 Lane b: pBR322 DNA + Topo I + Compounds 100 μM
Lane
1
2
3
4
5
Compound
8
10
7
9
11
Lane
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21
Compound 16 14 12 17 15 13 20 23 27 26 18 21 24 19 22 25
Figure 3. Topoisomerase I inhibitory activity of the prepared compounds.

A. Basnet et al. / Bioorg. Med. Chem. Lett. 20 (2010) 42–47
45
Table 1
notice that compounds 15 and 21 exhibited inhibitory activities
in both topoisomerase I and II.
Topoisomerase I and II inhibitory activities of the prepared compounds (7–11)
In structure–activity relationship study of the prepared com-
pounds for topoisomerase I and II inhibition, introduction of
methyl or chloride functionality may increase the inhibitory activ-
ity, since compounds having strong or moderate topoisomerase I or
II inhibitory activities possess chloride functionality on 4-furyl
moiety (compounds 13 and 15), or methyl functionality on 6-thie-
nyl or both on 2- and 6-thienyl moiety (compounds 18, 21 and 24).
However, we could not pull out any evident correlation regarding
the introduction of methyl or chloride functionality. Evaluation of
antitumor cytotoxicity15 for the selected compounds (7, 13, 15,
18, 21, and 24), which possess strong or moderate topoisomerase
I or II inhibitory activities, were performed against five different
human tumor cell lines with adriamycin, etoposide, and campto-
thecin as positive references: DU-145 (human prostate tumor),
MCF-7 (human breast adenocarcinoma), HCT 116 (human colorec-
tal carcinoma), MDA-MB231 (human breast tumor), and HeLa
(human cervix tumor). The cytotoxic activities of the compounds
were not very effective (Table 3). All the compounds exhibited
moderate cytotoxicity in HCT 116, MDA-MB231, and HeLa cell
lines, but they showed lower cytotoxicity than that of the refer-
ences, which we could not pull out the structure correlation of
cytotoxic activities among the prepared compounds. In addition,
Compounds
Topo I (% inhibition)
Topo II (% inhibition)
20
lM
100
l
M
20
lM
100 lM
7
8
9
10
0
5
0
0
12
0
15
0
1
0
4
70
34
18
32
12
0
0
11
0
0
Camptothecin
Etoposide
64
76
36
86
inhibition at 100
l
M), whereas compound 2414 exhibited stronger
M, 92%
M) compared to that of camptothecin (41% inhi-
M, 54% inhibition at 100 M). It is generally recog-
topoisomerase I inhibitory activities (88% inhibition at 20
inhibition at 100
bition at 20
l
l
l
l
nized that such compounds have strong inhibitory activities as
synthetic compounds, even though they have weaker inhibitory
activities than that of camptothecin. Figure 4 shows human
DNA–topoisomerase IIa
inhibitory activities13 for the 21 prepared
2,6-dithienyl-4-furyl pyridines, which are summarized in Tables 1
and 2. Compounds 7, 15, and 21 exhibited moderate topoisomerase
II inhibitory activities (55–70% inhibition at 100
that of etoposide (73% inhibition at 100 M). It is interesting to
lM) compared to
l
Lane D: pBR322 DNA only
Lane T: pBR322 DNA +Topo II
Lane C1: pBR322 DNA + Topo II + Etoposide 20 μM
Lane C2: pBR322 DNA + Topo II + Etoposide 100 μM
Lane 1- 21 Lane a: pBR322 DNA + Topo II + Compounds 20 μM
Lane 1- 21 Lane b: pBR322 DNA + Topo II + Compounds 100 μM
Lane
1
2
3
4
5
6
7
8
9
10 11 12
Compound
8
10
7
9
11 16 14 12 17 15 13 20
Lane
13 14 15 16 17 18 19 20 21
Compound 23 27 26 18 21 24 19 22 25
Figure 4. Topoisomerase II inhibitory activity of the prepared compounds.

46
A. Basnet et al. / Bioorg. Med. Chem. Lett. 20 (2010) 42–47
Table 2
Topoisomerase I and II inhibitory activities of the prepared compounds (12–27)
Compounds
Topo I (% inhibition)
Topo II (% inhibition)
20
lM
100
l
M
20
lM
100 lM
12
13
14
0
12
0
3
42
0
0
0
0
8
35
2
15
16
12
0
50
0
7
0
55
5
17
18
19
0
12
3
14
51
9
0
9
7
8
28
3
20
21
22
23
0
9
0
0
0
15
11
0
25
58
0
52
13
0
0
0
24
25
26
88
15
0
92
30
5
14
0
0
23
0
0
27
0
0
0
7
Camptothecin
Etoposide
41
54
65
73
Figure 6. Space filling model of compound 24 docked in DNA sequence between
C112, A113 and T10, TGP11.
there were no direct correlations between antitumor cytotoxicity
and topoisomerase inhibitory activity.
To verify the binding mode of the potent inhibitor 24, we car-
ried out a docking study using Surflex-Dock in Sybyl version 8.05
by Tripos Associates, operating under Red Hat Linux 4.0 with an
IBM computer (Intel Pentium 4, 2.8 GHz CPU, 1 GB memory) (Figs.
5 and 6). The structure of the inhibitor 24 was drawn into the Sybyl
package and minimized with the Tripos force field and Gasteiger–
Huckel charge on it. We chose the 1SC7 (PDB code) structure in
Protein Data Bank and the structure was polished as follows: the
phosphoester bond of G12 in 1SC7 was rebuilt, and the SH of
G11 on the scissile strand was modified to OH. After running Sur-
flex-Dock, 10 docked conformers were obtained. Among the con-
formations, the best total score (6.07) conformer was selected for
speculating the detailed binding mode in the site. In our model,
thienyl, pyridyl and furanyl rings intercalated between the ꢀ1
and +1 bases, parallel to the plane of the base pairs without having
an H bond with topoisomerase I. In this model, the heterocyclic
rings were considered to work as DNA intercalators to block the
rewinding step of the phosphoester. The binding geometry of CPT
Table 3
Cytotoxicities of selected compounds against various human cancer cell lines
a
IC50
(l
M)
Comp/cells (origin)
DU-145 (prostate)
MCF-7 (breast)
HCT 116 (colon)
MDA-MB231 (breast)
HeLa (cervix)
Adriamycin
Etoposide
Camptothecin
7
13
15
18
21
24
1.30 0.01
21.07 2.58
1.46 0.08
>100
88.11 4.82
>100
>100
>100
98.15 5.09
5.75 0.29
18.01 1.00
7.42 0.81
>100
>100
>100
>100
>100
>100
1.10 0.03
17.92 0.38
2.77 0.29
50.57 2.27
95.53 5.84
>100
56.41 1.33
51.06 1.06
>100
1.16 0.12
16.85 1.87
1.10 0.02
23.60 2.46
54.83 26
26.79 0.981
59.61 4.74
41.79 2.35
38.34 1.03
1.60 0.04
8.43 0.65
1.85 0.28
8.62 0.55
55.18 1.32
43.68 2.44
17.93 1.30
19.19 1.09
35.77 2.38
a
Each data point represents the mean S.D. from three different experiments performed in triplicate.
Figure 5. Model of the binding of compound 24 in the ternary complex consisting of DNA, topo I, and the inhibitor. The diagram is captured for stereoview.

A. Basnet et al. / Bioorg. Med. Chem. Lett. 20 (2010) 42–47
47
S. Bioorg. Med. Chem. 2007, 15, 4351; (d) Son, J. K.; Zhao, L. X.; Basnet, A.; Thapa,
P.; Karki, R.; Na, Y.; Jahng, Y.; Jeong, T. C.; Jeong, B. S.; Lee, C. S.; Lee, E. S. Eur. J.
Med. Chem. 2008, 43, 675; (e) Thapa, P.; Karki, R.; Basnet, A.; Thapa, U.; Choi, H.
Y.; Na, Y.; Jahng, Y.; Lee, C. S.; Kwon, Y.; Jeong, B. S.; Lee, E. S. Bull. Korean Chem.
Soc. 2008, 29, 1605; (f) Thapa, P.; Karki, R.; Thapa, U.; Jahng, Y.; Jung, M.-J.;
Nam, J. M.; Na, Y.; Kwon, Y.; Lee, E. S. Bioorg. Med. Chem. 2009, doi:10.1016/
j.bmc.2009.10.049.
in the DNA–topoisomerase I complex was studied by an ab initio
quantum mechanics calculation to afford the result that the
pꢀp
stacking interactions, DNA intercalating forces were much more
significant than the H bond with the amino acid residues of the
protein.16 In the compound 24, location of oxygen in furan at C-4
and methyl group in thiophene at C-2 carbon of pyridine look enter
into the base stacked space and these tricyclic system stabilize the
DNA-Protein-compound ternary complex. And another 3-methyl-
thiophen part at C-6 of pyridine controls the degree of intercalation
10. Zhao, L.-X.; Moon, Y. S.; Basnet, A.; Kim, E.-K.; Cho, W.-J.; Jahng, Y.; Park, J. G.;
Jeong, T. C.; Cho, W. J.; Choi, S. U.; Lee, C. O.; Lee, S. Y.; Lee, C. S.; Lee, E.-S. Bioorg.
Med. Chem. Lett. 2004, 14, 1333.
11. Kim, D. S. H. L.; Ashendel, C. L.; Zhou, Q.; Chang, C. T.; Lee, E.-S.; Chang, C. J.
Bioorg. Med. Chem. Lett. 1998, 8, 2695.
and optimize the electrostatic
tricylcic core of 24 and DNA base pairs. Our molecular docking
study proved the importance of DNA intercalation of 24.
p-p stacking inter action between
12. (a) Zecher, W.; Kröhnke, F. Chem. Ber. 1961, 94, 690; (b) Zecher, W.; Kröhnke, F.
Chem. Ber. 1961, 94, 698; (c) Zecher, W.; Kröhnke, F. Chem. Ber. 1961, 94, 707;
(d) Kröhnke, F. Angew. Chem., Int. Ed. Engl. 1963, 2, 380; (e) Kröhnke, F. Synthesis
1976, 1.
In conclusion, we have designed and prepared 21 2,6-dithienyl-
4-furyl pyridine derivatives by efficient synthetic routes, and eval-
uated their topoisomerase I and II inhibitory activity and antitumor
cytotoxicity. Among the prepared compounds, 24 exhibited much
stronger topoisomerase I inhibitory activity than camptothecin.
Compounds 7, 13, 15, 18, and 21 exhibited moderate topoisomer-
ase I or II inhibitory activity. The results suggest that introduction
of methyl or chloride functionality on furyl or thienyl moiety may
increase topoisomerase inhibitory activity. A docking study of
compound 24 with topoisomerase I–DNA complex was also per-
13. Fukuda, M.; Nishio, K.; Kanzawa, F.; Ogasawara, H.; Ishida, T.; Arioka, H.;
Bojanowski, K.; Oka, M.; Saijo, N. Cancer Res. 1996, 56, 789. The topoisomerase I
inhibitory activity was carried out as following: 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 dithiothreitol, 2 mM spermidine, 0.01% bovine serum albumin, 200 ng
pBR322, 0.3 U calf thymus DNA–topoisomerase
topoisomerase I inhibitors (prepared compounds) in a final volume of 10
The reaction mixture was incubated at 37 °C for 30 min. The reactions were
terminated by adding 2.5 of solution comprising 10% SDS, 0.2%
I
(Amersham), and
l
L.
lL
bromophenol blue, 0.2% xylene cyanol, and 30% glycerol. The mixture was
applied to 1% agarose gel and electrophoresed for 10 h with a running buffer of
Tris–borate–EDTA. Gels were stained for 30 min in an aqueous solution of
formed, which indicated that the
pꢀp stacking interactions be-
ethidium bromide (0.5 lg/mL). DNA bands were visualized by
tween thienyl and furyl rings with DNA were quite important for
exhibiting topoisomerase I inhibition activities, and these results
proved the importance of DNA intercalation ability of 24 in the
binding site of topoisomerase I–DNA complex. There were no
direct correlations between antitumor cytotoxicity and topoiso-
merase inhibitory activity. This study may provide valuable infor-
mation to researchers working on the development of antitumor
agents.
transillumination with UV light and supercoiled DNA was quantitated by an
image analyzer and LabWork 4.5 software (UVP).The topoisomerase II inhibitory
activity was carried out as following: DNA–topoisomerase II inhibition was
measured by assessing relaxation of supercoiled pBR322 plasmid DNA. The
reaction mixture contained 50 mM Tris–HCl (pH 8.0), 120 mM KCl, 10 mM
MgCl2, 0.5 mM ATP, 0.5 mM dithiothreitol, 30
0.2 g pBR322 plasmid DNA, 0.3 U human DNA–topoisomerase II
and topoisomerase II inhibitors (prepared compounds) in a final volume of
20 L. The reactions were incubated for 30 min at 37 °C and terminated by the
addition of 3 L of solution containing 0.77% sodium dodecyl sulfate, 77 mM
EDTA. Samples were mixed with 2 L of solution containing 30% sucrose, 0.5%
l
g/mL bovine serum albumin,
l
a
(TopoGEN),
l
l
l
bromophenol blue, and 0.5% xylene cyanol, and subjected to electrophoresis on
a 1% agarose gel at 1.5 V/cm for 10 h with a running buffer of Tris–borate–
EDTA. Gels were stained for 30 min in a aqueous solution of ethidium bromide
(0.5 lg/mL). DNA bands were visualized by transillumination with UV light
and supercoiled DNA was quantitated by an image analyzer and LabWork 4.5
software (UVP).
Acknowledgment
This work was supported by the Korea Research Foundation
Grant funded by the Korean Government (MOEHRD, Basic Research
Promotion Fund) (KRF-2008-313-E00762).
14. The spectral data of 24: TLC (EtOAC/n-hexane = 1:5, v:v), Rf = 0.43, mp 101.5–
103.5 °C.1H NMR (250 MHz, CDCl3) d 7.65 (s, 2H, pyridine H-3, H-5), 7.56 (d,
J = 1.39 Hz, 1H, 4-furan H-5), 7.26(d, J = 5.05 Hz, 2H, 2-thiophene H-5, 60-
thiophene H-5), 6.93(d, J = 5.18 Hz, 2H, 2-thiophene H-4, 60-thiophene H-4),
6.94–6.90 (m, 1H, 4-furan H-4), 6.54 (dd, J = 3.08, 1.72 Hz, 1H, 4-furan H-3),
2.61 (s, 6H, 60-thiophene 3-CH3).13C NMR (62.5 MHz, CDCl3) d 153.54, 151.68,
143.72, 138.55, 137.91, 136.06, 132.26, 125.52, 112.78, 112.07, 108.58, 16.56,
ESI LC/MS/MS: Retention time: 20.95 min, [MH]+: 338.HPLC condition:
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5 lm, GL science, flow rate:
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l
l
l
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concentrations of compounds. On day 3, each well was added with 5 lL of the
cell counting kit-8 solution (Dojindo, Japan) then incubated for additional 4 h
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