Synthesis of 2,4-diaryl chromenopyridines and evaluation of their topoisomerase i and II inhibitory activity, cytotoxicity, and structure-activity relationship

Article abstract of DOI:10.1016/j.ejmech.2011.04.029

Designed and synthesized were a series of 5H-chromeno[4,3-b]pyridines with substitution at 2- and 4-positions with various 5- or 6-membered heteroaromatics as antitumor agents. They were evaluated for topoisomerase I and II inhibitory activities as well a

Full text of DOI:10.1016/j.ejmech.2011.04.029

European Journal of Medicinal Chemistry 46 (2011) 3201e3209
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of 2,4-diaryl chromenopyridines and evaluation of their topoisomerase
I and II inhibitory activity, cytotoxicity, and structureeactivity relationship
Uttam Thapa a, Pritam Thapa a, Radha Karki a, Minho Yun a, Jae Hun Choi a, Yurngdong Jahng a,
,
Eunyoung Lee b, Kyung-Hwa Jeon b, Younghwa Na c, Eun-Mi Ha c, Won-Jea Cho d, Youngjoo Kwon b
**
,
*
,
Eung-Seok Lee a
a College of Pharmacy, Yeungnam University, Kyongsan 712-749, Republic of Korea
b College of Pharmacy, Division of Life & Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Republic of Korea
c College of Pharmacy, 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:
Designed and synthesized were a series of 5H-chromeno[4,3-b]pyridines with substitution at 2- and
4-positions with various 5- or 6-membered heteroaromatics as antitumor agents. They were evaluated
for topoisomerase I and II inhibitory activities as well as cytotoxicities against several human cancer cell
lines. Structureeactivity relationship study showed that 2-furyl or 2-thienyl at 2- or 4-position of central
pyridine is crucial in displaying topo I or II inhibitory activity and cytotoxicity.
Received 16 September 2010
Received in revised form
18 March 2011
Accepted 9 April 2011
Available online 29 April 2011
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Terthiophene
Terpyridine
4-chromanone
Topoisomerase I and II
Cytotoxicity
1. Introduction
inhibitory activities as well as a strong cytotoxicity against several
human cancer cell lines [5]. From the previous study it was found
DNA is one of the most important pharmacological targets of
many drugs. DNA topoisomerases are enzymes that transiently
break one or two strands of DNA, which allow to solve various DNA
topological problems generated during vital cellular processes [1,2].
Because of the crucial role of topoisomerases for the maintenance
and replication of DNA during proliferation, topoisomerase inhib-
itors are among the most potent anticancer agents to date.
that number of aryl groups in terpyridine derivatives has important
role in determining the antitumor cytotoxicity [5e]. Furthermore, it
has been reported by our research group that conformationally
constrained rigid analogs of 2,4,6-trisubstituted pyridine contain-
ing 5,6-dihydrobenzo[h]quinoline moiety showed considerable
topo I and II inhibitory activities, and cytotoxicity against several
human cancer cell lines [6]. Various compounds possessing chro-
meno[4,3-b]pyridine and chromene moiety were reported as
a-Terthiophene has been reported to possess various biological
activities such as anti HIV-1 activity, protein kinase C (PKC) inhibitory
estrogen receptor b-selective ligands [7], TNF-a inhibitors [8], and
activity, nematocidal, and phototoxic to the pathogenic yeast Candida
anti-inflammatory agents [9]. 2,3-Dimethyl-4-chromanol skeleton
of natural Callophyllum coumarins which consist in 4-chromanone
containing pyridine bioisosteres of our synthesized compound is
responsible for anti HIV-1 activity [10].
In connection with the previous results, it would be very
interesting to observe biological activities by introduction of con-
formationally constrained rigid molecule in terpyridine skeleton
such as 2,4-diaryl chromenopyridines, which possess three fused
rings and two non-fused rings. Generally, a drug to its receptor site
is influenced by electronic or steric factors and these two functions
are considered to be important in the bioactive conformation of
albicans [3]. Terpyridine, bioisosteres of
a-terthiophene, acts as
metal-based pseudonuclease (photonuclease/chemical nuclease) [4].
Previously, our research group has reported that terpyridine
derivatives showed strong topoisomerase I (topo I) and II (topo II)
* Corresponding author. Tel.: þ82 2 3277 4653; fax: þ82 2 3277 2851.
** Corresponding author. Tel.: þ82 53 810 2827; fax: þ82 53 810 4654.
E-mail addresses: ykwon@ewha.ac.kr (Y. Kwon), eslee@yu.ac.kr (E.-S. Lee).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.04.029

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U. Thapa et al. / European Journal of Medicinal Chemistry 46 (2011) 3201e3209
a drug. Rigid structures are commonly considered to have little
conformational entropy compared to flexible structures and can be
more efficiently fitted into the active site of a receptor [11]. In here,
the rigidification of the flexible ring flattened the molecules so that
they could work as DNA intercalators in the topoisomerase IeDNA
ternary complex [12]. In this study, twenty-three rigid analogs of
2,4-diaryl chromenopyridines were designed and synthesized as
shown in Fig. 1 and Scheme 1. They were evaluated for topo I and II
inhibitory activities, cytotoxicity, and studied for structureeactivity
relationships.
(14, 15, 26, and 36) having moderate topo II inhibitory activity also
possess 2-furyl or 2-thienyl at 2-position or 4-position of central
pyridine.
2.3. Cytotoxicity
For the evaluation of cytotoxicity, 5 different human cancer cell
lines were utilized: MDA-MB231 (human breast tumor cell line),
HeLa (human cervix tumor cell line), DU145 (human prostate
tumor cell line), HCT15 (human colorectal adenocarcinoma cell
line), and HL60 (human myeloid leukemic tumor cell line). Cyto-
toxic evaluation was performed for the selected compounds having
significant topo I or II inhibitory activity. The IC50 values of
4-chromanone containing pyridine bioisosteres (14e36) against
several human cancer cell lines are as shown in Table 2. Most of the
compounds showed moderate cytotoxicity, generally IC50 values of
2. Results and discussion
2.1. Synthetic chemistry
Synthetic method for the preparation of 4-chromanone con-
taining pyridine bioisosteres are summarized in Scheme 1. We
prepared 23 final compounds of 4-chromanone containing pyridine
bioisosteres. 4-Chromanone (1) was condensed with aryl aldehydes
(2aeg) to prepare intermediates (3aeg) in the presence of EtOH
and 5% aqueous NaOH with the yield of 46.1e90.5%. Five pyr-
idinium iodide salts 4e8 were prepared by reacting aryl acetyls,
iodine and pyridine in 72.5e96.2% yield. By treatment of interme-
diates (3aeg) with 4e8, using modified Kröhnke synthesis [13],
final compounds 9 (R1 ¼ a, c, eeg),10 (R1 ¼ aeg), 11 (R1 ¼ a, c, eeg),
12 (R1 ¼ aec, e, f), and 13 (R1 ¼ d) were synthesized with the yield
of 29.4e53.5%. Structures of the prepared compounds are shown in
Fig. 2, and physical properties of the prepared compounds are
shown in Table 1.
0.1e25 mM. Among them, Compounds 24 and 28 which exhibited
strong topo I inhibitory activity, and compound 26 which exhibi-
ted strong topo II inhibitory activity showed significant cytotoxic-
ities, as strong as positive controls, camptothecin, etoposide, and
adriamycin.
3. Conclusions
We have designed and synthesized 23 compounds by efficient
synthetic route and evaluated for topo I and II inhibitory activities
along with cytotoxicity against several human cancer cell lines.
Among them, 15, 24, 27, and 28 displayed significant topo I inhib-
itory activity, and 14, 15, 26, and 36 exhibited moderate topo II
inhibitory activity. Compounds 24, 26, and 28 exhibited strong
cytotoxicity. A structureeactivity relationship study of 2,4-diaryl
chromenopyridines for topo I and II inhibitory activities and cyto-
toxicity indicates that 2-furyl or 2-thienyl at 2- or 4-position of
central pyridine is crucial in displaying topo I or II inhibitory
activity and cytotoxicity. In this study, we investigated conforma-
tionally constrained rigid molecules such as 2,4-diaryl chromeno-
pyridines for the development of potent antitumor agents, and
obtained positive results. This study may provide valuable infor-
mation to researchers working on the development of antitumor
agents.
2.2. Topo I and II inhibitory activities
The conversion of supercoiled plasmid DNA to relaxed DNA by
topo I and II was examined in the presence of the prepared
2,4-diaryl chromenopyridines 14e36. Camptothecin and etoposide,
well-known topo I and II inhibitors, respectively, were used as
positive controls. The effect of the prepared compounds on human
DNA topo I was evaluated by the topo I relaxation assays. The
reaction products were analyzed by electrophoretic mobility and
developed in ethidium bromide in the presence of UV light.
As shown in Fig. 3, compounds 15, 24, 27, and 28 exhibited
4. Experimental
significant topo I inhibitory activity at 100
mM concentration.
Especially, compounds 15, 24, and 28 displayed the most significant
topo I inhibitory activity as strong as a positive control, campto-
thecin (Table 2). The compounds (15, 24, 27, and 28) having
significant topo I inhibitory activity possess 2-furyl or 2-thienyl at
2-position or 4-position of central pyridine, which supports the
idea that 2-furyl or 2-thienyl at 2- or 4-position on central pyridine
play the crucial role for topo I or II inhibitory activity as reported
previously [5b,d,gej]. Compounds 14, 15, 26, and 36 exhibited
Compounds used as starting materials and reagents were ob-
tained from Aldrich Chemical Co., Junsei or other chemical
companies, and utilized without further purification. HPLC grade
acetonitrile (ACN) and methanol were purchased from Burdick and
Jackson, USA. Thin-layer chromatography (TLC) and column chro-
matography (CC) were performed with Kieselgel 60 F254 (Merck)
and silica gel (Kieselgel 60, 230e400 mesh, Merck) respectively.
Since all the compounds prepared contain aromatic ring, they were
visualized and detected on TLC plates with UV light (short wave,
long wave or both). NMR spectra were recorded on a Bruker AMX
250 (250 MHz, FT) for 1H NMR and 62.5 MHz for 13C NMR, and
chemical shifts were calibrated according to TMS. Chemical shifts
moderate topo II inhibitory activity at 100
mM concentration as
shown in Fig. 4 and Table 2. In addition, compound 15 showed
both considerable topo I and II inhibitory activities. The compounds
(d) were recorded in ppm and coupling constants (J) in hertz (Hz).
R1
Melting points were determined in open capillary tubes on elec-
trothermal 1A 9100 digital melting point apparatus and were
uncorrected.
R1,R 2 = phenyl,
Chromene moiety
O
4
O
2- or 3- thienyl,
2- or 3- furyl,
2- or 3- pyridiyl
R2
N
2 N
HPLC analyses were performed using two Shimadzu LC-10AT
pumps gradient-controlled HPLC system equipped with Shimadzu
system controller (SCL-10A VP) and photo diode array detector
(SPD-M10A VP) utilizing Shimadzu Class VP program. Sample
5H-chromeno[4,3-b]pyridine
2,4-diaryl chromenopyridines
volume of 10
phase C18 column (4.6 ꢀ 250 mm) with a gradient elution of
m 5 mM reverse-
L was injected in Waters X-TerraÒ
Fig. 1. Structure of 5H-chromeno[4,3-b]pyridine and 2,4-diaryl chromenopyridines.

U. Thapa et al. / European Journal of Medicinal Chemistry 46 (2011) 3201e3209
R1
3203
I
O
N
N
N
O
N
4
9 (R1=a, c, e-g)
ii
R1
I
O
N
O
5
N
ii
10 (R1=a-g)
I
R1
N
O
O
O
O
O
O
R1
H
6
i
N
+
R1
O
ii
O
11 (R1=a, c, e-g)
O
3 (R1=a-g)
R1
I
2 (a-g)
1
N
S
O
O
7
N
S
ii
12 (R1=a-c, e, f)
I
R1
S
N
O
O
8
N
ii
S
13 (R1=d)
R1
=
S
O
N
O
S
N
a
b
c
d
e
f
g
Scheme 1. General synthetic scheme of 2,4-diaryl chromenopyridines Reagents and conditions: i) aq. NaOH (0.5 g in 10 ml), EtOH, room temperature, 46.1e90.5% yield ii) 4e8
(1.0 eq.), NH4OAc (10.0 eq.), glacial AcOH, 12e24 h, 100e110 ꢁC, 29.4e53.5% yield.
i) 40e100% of B in A for 10 min followed by 100e40% of B in A for
20 min ii) 75e100% of B in A for 10 min followed by 100e75% of B in
A for 20 min and iii)85e100% of B in A for 10 min followed by
100e85% of B in A for 20 min at a flow rate of 1.0 mL/min at 254 nm
UV detection, where mobile phase A was doubly distilled water
with 50 mM ammonium formate (AF) and B was 90% ACN in water
with 20 mM AF. Purity of compound is described as percent (%).
ESI LC/MS analyses were performed with a Finnigan LCQ
AdvantageÒ LC/MS/MS spectrometry utilizing XcaliburÒ program.
observed, cooled for 30 min, filtered, and washed with water and
cold methanol. The precipitate was dried to give compound 3
(R1 ¼ aeg) in the yield of 46.1e90.5%.
4.2. General method for the preparation of 4e8
Aryl acetyl was mixed with pyridine followed by addition of
equivalent amount of iodine. The mixture was then refluxed at
140 ꢁC for 3 h. Precipitate occurred during reaction was cooled to
room temperature. Then it was filtered and washed with cold
pyridine followed by drying overnight to yield 72.5e96.2% of 4e8.
For ESI LC/MS, LC was performed with 8
a Waters X-TerraÒ 3.5
m reverse-phase C18 column (2.1 ꢀ100 mm)
with a gradient elution from 10 to 95% of B in A for 10 min followed
by 95e10% of B in A for 10 min at a flow rate of 200 L/min, where
mL injection volume on
m
m
4.3. General method for the preparation of 14e36
mobile phase A was 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 temp.: 275 ꢁC, capillary voltage: 27 V, tube lens offset:
45 V. Retention time is given in minutes.
Anhydrous ammonium acetate (10.0 equiv) was mixed with
glacial acetic acid followed by addition of the rigid chalcones of
4-chromanone (1.0 equiv), 3 (R1 ¼ aeg) and pyridinium iodide salts
4e8 (1.0 equiv). The mixture was then refluxed at 100e110 ꢁC for
12e24 h. The reaction mixture was then extracted with ethyl
acetate, washed with water and brine solution. The organic layer
was dried with magnesium sulfate and filtered. The filtrate was
evaporated at reduced pressure, which was then purified with silica
gel column chromatography with the gradient elution of ethyl
acetate/n-hexane to afford solid compounds 14e36 in 29.4e53.5%
yield.
4.1. General method for preparation of 3
4-Chromanone (1) was dissolved in ethanol followed by the
addition of equivalent amount of aryl aldehydes (2aeg). The 5%
aqueous solution of NaOH was added dropwise to the mixture at
room temperature. On addition of NaOH, precipitation was

3204
U. Thapa et al. / European Journal of Medicinal Chemistry 46 (2011) 3201e3209
N
O
S
N
O
O
O
O
O
N
N
N
N
N
N
N
N
N
N
18
16
17
14
15
N
S
O
N
O
O
O
O
O
N
N
N
N
N
19
20
21
22
23
N
N
O
S
O
O
O
O
O
N
N
N
N
N
O
O
O
O
O
28
27
24
25
26
O
O
S
O
S
O
O
O
O
O
N
N
N
N
N
S
S
S
32
33
29
30
31
N
N
O
O
O
N
N
N
S
S
S
34
35
36
Fig. 2. Structure of the prepared compounds.
4.3.1. 4-(furan-2-yl)-2-(pyridin-2-yl)-5H-chromeno[4,3-b]pyridine
(14)
4.3.2. 2-(pyridin-2-yl)-4-(thiophen-2-yl)-5H-chromeno[4,3-b]
pyridine (15)
A white solid, Yield: 52.0%.
A light yellow solid, Yield: 50.3%.
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.50.
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.36.
LC/MS/MS: retention time: 7.62 min, [MH]þ: 327.27.
LC/MS/MS: retention time: 6.30 min, [MH]þ: 343.27.
1H NMR (250 MHz, CDCl3)
d
8.70 (dd, J ¼ 4.7, 2.2 Hz, 1H,
1H NMR (250 MHz, CDCl3)
d
8.68 (ddd, J ¼ 3.9, 1.7, 0.8 Hz, 1H,
2-pyridine H-6), 8.64 (d, J ¼ 7.6 Hz, 1H, 2-pyridine H-3), 8.59 (s, 1H,
pyridine H-3), 8.42 (d, J ¼ 7.7, 1.5 Hz, 1H, chromeno H-5), 7.86 (td,
J ¼ 7.7, 1.6 Hz, 1H, 2-pyridine H-4), 7.63 (dd, J ¼ 1.4, 0.5 Hz, 1H, furan
H-5), 7.37e7.31 (m, 2H, chromeno H-7 and 2-pyridine H-5), 7.16
(t, J ¼ 7.2 Hz, 1H, chromeno H-6), 6.99 (d, J ¼ 8.0, 1H, chromeno
H-8), 6.84 (d, J ¼ 3.4, 1H, furan H-3), 6.59 (dd, J ¼ 3.3, 1.7 Hz, 1H,
furan H-4), 5.62 (s, 2H, chromeno H-2).
2-pyridine H-6), 8.64 (dd, J ¼ 7.9, 0.6 Hz, 1H, 2-pyridine H-3), 8.44
(s, 1H, pyridine H-3), 8.42 (dd, J ¼ 7.6, 1.5 Hz, 1H, chromeno H-5),
7.82 (td, J ¼ 7.7, 1.7 Hz, 1H, 2-pyridine H-4), 7.50 (dd, J ¼ 5.0, 1.2 Hz,
1H, 4-thiophene H-5), 7.36e7.30 (m, 2H, 4-thiophene H-3 and
chromeno H-7), 7.19e7.12 (m, 3H, 4-thiophene H-4, 2-pyridine H-5
and chromeno H-6), 7.01 (dd, J ¼ 8.0, 0.8 Hz, 1H, chromeno H-8),
5.47 (s, 2H, chromeno H-2).
13C NMR (62.5 MHz, CDCl3)
d
156.22, 156.87, 154.94, 150.81,
13C NMR (62.5 MHz, CDCl3)
d 156.28, 155.80, 155.01, 149.08,
149.06, 148.72, 143.94, 136.89, 134.87, 131.21, 125.12, 123.86, 123.39,
122.19, 121.97, 121.22, 116.73, 116.63, 112.03, 111.60, 66.32.
148.69, 139.92, 138.52, 136.89, 131.30, 127.80, 127.51, 125.19, 123.89,
123.55, 12.53, 122.31, 121.29, 120.43, 116.80, 66.04.

U. Thapa et al. / European Journal of Medicinal Chemistry 46 (2011) 3201e3209
3205
Table 1
chromeno H-7), 6.97 (d, J ¼ 7.6 Hz, 1H, chromeno H-6), 5.05 (s, 2H,
Prepared compounds with yield, purity by HPLC, and melting point.
chromeno H-2).
13C NMR (62.5 MHz, CDCl3)
d 156.42, 156.01, 154.89, 149.05,
147.59, 137.36, 136.89, 131.21, 128.62, 128.54, 125.15, 123.81, 123.75,
123.69, 122.28, 121.30, 120.58, 116.83, 66.03.
4.3.4. 2-(pyridin-2-yl)-4-(pyridin-3-yl)-5H-chromeno[4,3-b]
pyridine (17)
A light yellow solid, Yield: 29.4%.
Rf (ethyl acetate/n-hexane 1:2 v/v): 0.36.
LC/MS/MS: retention time: 3.73 min, [MH]þ: 338.34.
Entry
R1
R2
Yield (%)
Purity (%)
mp (ꢁC)
1H NMR (250 MHz, CDCl3)
d
8.95 (d, J ¼ 2.2 Hz, 1H, 4-pyridine
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
2-furyl
2-thienyl
phenyl
3-pyridyl
2-pyridyl
2-furyl
2-thienyl
phenyl
2-pyridyl
3-pyridyl
2-furyl
2-thienyl
phenyl
2-pyridyl
3-pyridyl
3-furyl
3-thienyl
2-furyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
phenyl
phenyl
phenyl
phenyl
phenyl
2-furyl
2-furyl
2-furyl
2-furyl
2-furyl
phenyl
phenyl
52.0
50.3
36.5
29.4
41.4
38.2
31.7
30.9
32.7
29.4
43.9
35.3
41.6
43.0
48.8
43.0
34.5
33.9
33.9
32.4
53.5
35.3
52.5
100.0
100.0
100.0
100.0
100.0
100.0
96.1
96.4
97.8
99.3
97.8
97.6
98.8
100.0
97.5
95.5
96.2
99.3
98.9
96.6
96.3
99.1
99.0
153.3e155.2
167.6e168.2
147.1e148.9
177.5e180.6
178.6e182.4
164.9e165.7
172.2e174.6
116.9e117.6
165.3e167.1
146.2e147.8
140.4e142.4
130.4e131.3
107.8e109.9
170.6e171.5
128.3e130.2
152.8e161.1
142.6e144.6
184.3e185.7
147.1e148.7
149.5e150.7
127.9e128.2
164.6e165.8
181.6e182.5
H-2), 8.75 (d, J ¼ 5.1 Hz, 1H, 2-pyridine H-6), 8.73e8.67 (m, 2H,
2-pyridine H-3 and 4-pyridine H-6), 8.42 (d, J ¼ 7.9 Hz, 1H, 4-pyri-
dine H-4), 8.20 (d, J ¼ 7.7, 1.6 Hz 1H, chromeno H-5), 7.89e7.82 (m,
2H, 2-pyridine H-4 and pyridine H-3), 7.50 (dd, J ¼ 5.1, 1.8 Hz, 1H, 2-
pyridine H-5), 7.39e7.32 (m, 2H, 4-pyridine H-5 and chromeno H-7),
7.14 (dt, J ¼ 7.6, 1.12 Hz, 1H, chromeno H-6), 6.90 (dd, J ¼ 8.1, 0.8 Hz,
1H, chromeno H-8), 5.31(s, 2H, chromeno H-2).
13C NMR (62.5 MHz, CDCl3)
d 156.95, 156.52, 155.69, 149.94,
149.27, 149.18, 147.75, 145.78 137.07, 132.58, 131.79, 130.50, 126.18,
124.85, 124.05, 122.62, 122.55, 121.32, 121.00, 118.57, 117.10, 67.79.
4.3.5. 2,4-di(pyridin-2-yl)-5H-chromeno[4,3-b]pyridine (18)
A white solid, Yield: 41.4%.
Rf (ethyl acetate/n-hexane 1:2 v/v): 0.36.
2-thienyl
2-thienyl
2-thienyl
2-thienyl
2-thienyl
3-thienyl
LC/MS/MS: retention time: 5.76 min, [MH]þ: 338.32.
2-thienyl
3-furyl
phenyl
2-pyridyl
2-pyridyl
1H NMR (250 MHz, CDCl3)
d 8.74e8.67 (m, 3H, 2-pyridine H-3,
H-6 and 4-pyridine H-6), 8.53 (s, 1H, pyridine H-3), 8.43 (dd, J ¼ 7.7,
1.6 Hz, 1H, chromeno H-5), 7.88e7.83(m, 2H, 4-pyridine H-3 and
2-pyridine H-4), 7.74 (d, J ¼ 8.0, 1.5 Hz, 1H, 4-pyrridine H-4),
7.38e7.31 (m, 3H, 2-pyridine H-5, 4-pyridine H-5 and chromeno
H-7), 7.10 (dt, J ¼ 7.5, 1.0 Hz, 1H, chromeno H-6), 6.90 (d, J ¼ 8.0 Hz,
1H, chromeno H-8), 5.53 (s, 2H, chromeno H-2).
4.3.3. 4-phenyl-2-(pyridin-2-yl)-5H-chromeno[4,3-b]pyridine (16)
A white solid, Yield: 36.5%.
13C NMR (62.5 MHz, CDCl3)
d 156.62, 156.23, 155.88, 154.93,
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.35.
149.17, 149.01, 148.96, 145.40, 136.95, 136.93, 131.20, 125.12, 124.92,
123.86, 123.71, 123.60, 123.07, 122.11, 121.26, 119.64, 116.81, 66.50.
LC/MS/MS: retention time: 8.37 min, [MH]þ: 337.32.
1H NMR (250 MHz, CDCl3)
d 8.69e8.65 (m, 2H, 2-pyridine H-3,
H-6), 8.47 (dd, J ¼ 7.7, 1.63 Hz, 1H, chromeno H-5), 8.34 (s, 1H,
pyridine H-3), 7.86 (td, J ¼ 7.8,1.5 Hz,1H, 2-pyridine H-4), 7.49e7.45
(m, 3H, 4-phenyl H-3, H-4, H-5), 7.40e7.30 (m, 4H, 4-phenyl H-2,
H-6, 2-pyridine H-5 and chromeno H-8), 7.16 (t, J ¼ 7.7 Hz, 1H,
4.3.6. 4-(furan-2-yl)-2-phenyl-5H-chromeno[4,3-b]pyridine (19)
A light yellow solid, Yield: 38.2%.
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.50.
LC/MS/MS: retention time: 9.06 min, [MH]þ: 326.28.
1H NMR (250 MHz, CDCl3)
d
8.40 ( dd, J ¼ 7.7, 1.6 Hz, 1H, chro-
meno H-5), 8.15e8.19 (m, 2H, 2-phenyl H-2,6), 7.89 (s, 1H, pyridine
H-3), 7.60 (d, J ¼ 1.9 Hz, 1H, 4-furan H-5), 7.51e7.44 (m, 3H,
2-phenyl H-3, H-4, H-5), 7.34 (dt, J ¼ 8.0, 1.8 Hz, 1H, chromeno H-7),
7.10 (dt, J ¼ 7.6, 1.1 Hz, 1H, chromeno H-6), 6.90 (dd. J ¼ 8.0, 0.6 Hz,
1H, chromeno H-8), 6.68 (d, J ¼ 3.4 Hz, 1H, 4-furan H-3), 6.60 (dd,
J ¼ 3.4, 1.8 Hz, 1H, 4-furan H-4), 5.57 (s, 2H, chromeno H-2).
13C NMR (62.5 MHz, CDCl3)
d 156.22, 156.17, 150.50, 148.96,
145.09, 143.88, 139.03, 134.67, 131.17, 129.12, 128.69, 126.86, 125.28,
123.47, 122.22, 120.41, 116.63, 125.98, 112.07, 111.42, 66.13.
4.3.7. 2-phenyl-4-(thiophen-2-yl)-5H-chromeno[4,3-b]pyridine
(20)
A white solid, Yield: 31.7%.
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.51.
LC/MS/MS: retention time: 9.84 min, [MH]þ: 342.27.
1H NMR (250 MHz, CDCl3)
d
8.46 (dd, J ¼ 7.6, 1.5 Hz, 1H, chro-
meno H-5), 8.17e8.14 (m, 2H, 2-phenyl H-2, H-6), 7.71 (s, 1H,
pyridine H-3), 7.54e4.43 (m, 4H, 2-phenyl H-3, H-4, H-5 and
4-thiophene H-5), 7.34 (dt, J ¼ 7.7, 1.6 Hz, 1H, chromeno H-6),
7.20e7.14 (m, 2H, 4-thiophene H-3 and chromeno H-6), 7.09
(dt, J ¼ 4.2, 0.7 Hz, 1H, 4-thiophene H-4) 6.98 (d, J ¼ 8.1 Hz, 1H,
chromeno H-8), 5.43 (s, 2H, chromeno H-2).
Fig. 3. Topoisomerase I inhibitory activity of the prepared compounds 14e36.

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U. Thapa et al. / European Journal of Medicinal Chemistry 46 (2011) 3201e3209
Table 2
Topo I and II inhibitory activities, and cytotoxicity of the prepared compounds.
Compounds
Topo I (% Inhibition)
Topo II (% Inhibition)
IC50 (mM)
100
m
M
20
m
M
100
m
M
20
m
M
MDA-MB231a
HeLab
DU145c
HCT15d
HL60e
Adriamycinf
0.4 ꢂ 0.0
0.7 ꢂ 0.1
0.3 ꢂ 0.0
3.3 ꢂ 0.2
2.4 ꢂ 0.1
11.1 ꢂ 0.2
7.2 ꢂ 0.7
4.7 ꢂ 0.3
11.6 ꢂ 0.7
3.9 ꢂ 0.1
1.8 ꢂ 0.6
1.9 ꢂ 0.0
NA
1.0 ꢂ 0.1
1.4 ꢂ 0.2
0.4 ꢂ 0.1
>50
6.2 ꢂ 0.2
5.0 ꢂ 0.1
1.3 ꢂ 0.0
>50
1.4 ꢂ 0.0
16.0 ꢂ 0.2
14.2 ꢂ 0.3
2.8 ꢂ 0.0
NA
1.0 ꢂ 0.1
0.5 ꢂ 0.0
0.4 ꢂ 0.2
22.9 ꢂ 0.3
13.1 ꢂ 1.0
7.6 ꢂ 0.8
4.4 ꢂ 0.4
>50
18.6 ꢂ 3.1
2.6 ꢂ 0.3
6.9 ꢂ 2.1
1.9 ꢂ 0.1
NA
1.2 ꢂ 0.0
1.1 ꢂ 0.0
0.5 ꢂ 0.1
>50
14.0 ꢂ 0.8
15.6 ꢂ 1.1
3.3 ꢂ 0.3
23.1 ꢂ 0.3
12.3 ꢂ 2.0
1.2 ꢂ 0.4
5.1 ꢂ 1.4
12.1 ꢂ 2.9
NA
0.7 ꢂ 0.0
1.0 ꢂ 0.0
0.1 ꢂ 0.0
18.6 ꢂ 0.5
13.7 ꢂ 0.5
14.5 ꢂ 0.6
>50
0.2 ꢂ 0.0
7.3 ꢂ 0.3
>50
Etoposideg
40.5
22.3
Camptothecinh
58.0
4.4
48.2
3.1
2.6
0.6
0.0
0.0
0.0
0.0
0.0
46.4
0.0
4.9
11.5
41.6
1.9
3.7
9.2
0.7
0.0
5.5
1.8
3.3
20.9
NA
4.1
NA
NA
NA
NA
NA
NA
NA
NA
22.9
NA
NA
NA
15.5
NA
NA
NA
NA
NA
NA
NA
NA
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
23.4
19.4
7.2
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
3.2
8.7
10.7
11.5
10.8
15.4
8.7
14.0
11.6
6.8
28.4
2.0
5.6
10.5
17.6
14.1
7.9
18.8 ꢂ 0.3
>50
NA
0.6 ꢂ 0.2
1.3 ꢂ 0.0
0.9 ꢂ 0.2
0.6 ꢂ 0.2
3.4 ꢂ 0.1
NA
NA
NA
NA
NA
0.2 ꢂ 0.1
NA
1.1 ꢂ 0.1
10.0 ꢂ 0.1
11.9 ꢂ 0.2
NA
NA
NA
NA
NA
2.3 ꢂ 0.0
NA
2.2 ꢂ 0.1
39.6 ꢂ 5.7
>50
NA
NA
NA
NA
0.1 ꢂ 0.0
NA
0.4 ꢂ 0.2
NA
0.6 ꢂ 0.2
31.1 ꢂ 2.6
>50
NA
NA
NA
NA
1.4 ꢂ 0.0
NA
1.8 ꢂ 0.1
6.6 ꢂ 1.0
>50
NA
NA
NA
NA
4.6 ꢂ 0.3
>50
>50
NA
NA
NA
NA
NA
5.0 ꢂ 1.9
8.4
7.5
8.5
25.3
NA
>50
NA
8.3 ꢂ 0.8
NA
>50
25.8 ꢂ 0.2
Each data represents mean ꢂ S.D. from three different experiments performed in triplicate.
NA: not applicable.
a
MDA-MB231: human breast adenocarcinoma.
HeLa: human cervix tumor.
DU145: human prostate tumor.
HCT15 human colorectal adenocarcinoma.
HL60: chronic myelogenous leukemia.
Adriamycin; positive control for cytotoxicity.
Etoposide: positive control for topo II and cytotoxicity.
Camptothecin: positive control for topo I and cytotoxicity.
b
c
d
e
f
g
h
4.3.8. 2,4-diphenyl-5H-chromeno[4,3-b]pyridine (21)
A white solid, Yield: 30.9%.
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.50.
LC/MS/MS: retention time: 9.84 min, [MH]þ: 336.31.
1H NMR (250 MHz, CDCl3)
d
8.48 (dd, J ¼ 7.7, 1.6 Hz, 1H, chro-
meno H-5), 8.18e8.14 (m, 2H, 2-phenyl H-2 and H-6), 7.63 (s, 1H,
pyridine H-3), 7.54e7.43 (m, 6H, 2-phenyl H-3, H-4, H-5 and
4-phenyl H-3, H-4, H-5), 7.37e7.34 (m, 3H, 4-phenyl H-2, H-6 and
chromeno H-7), 7.15 (t, J ¼ 7.5, 1.0 Hz, 1H, chromeno H-6), 6.96 (dd,
J ¼ 8.0, 0.9 Hz, 1H, chromeno H-8) 5.25 (s, 2H, chromeno H-2).
13C NMR (62.5 MHz, CDCl3)
d 156.40, 156.12, 148.71, 147.27,
139.08, 137.49, 131.17, 129.08, 128.75, 128.69, 128.59, 128.43, 127.10,
126.90, 125.32, 123.77, 122.28, 122.12, 119.94, 116.73, 65.90.
4.3.9. 2-phenyl-4-(pyridin-2-yl)-5H-chromeno[4,3-b]pyridine (22)
A yellow solid, Yield: 32.7%.
Rf (ethyl acetate/n-hexane 1:2 v/v): 0.50.
LC/MS/MS: retention time: 7.45 min, [MH]þ: 337.30.
1H NMR (250 MHz, CDCl3)
d
8.75 ( dd, J ¼ 4.0, 0.8 Hz, 1H,
4-pyridine H-6), 8.47 (dd, J ¼ 7.7, 1.6 Hz, 1H, 4-pyridine H-5),
8.20e8.17 (m, 2H, 2-phenyl H-2, H-6), 7.87 (dt, J ¼ 7.6, 1.6 Hz, 1H,
4-pyridine H-4), 7.80 (s, 1H, pyridine H-3), 7.62 (d, J ¼ 7.8 Hz, 1H,
chromeno H-8), 7.54e7.44 (m, 3H, 2-phenyl H-3, H-4, H-5),
7.40e7.31 (m, 2H, 4-pyridine H-3, H-5), 7.15 (d, J ¼ 7.5 Hz, 1H,
chromeno H-7), 6.98 (d, J ¼ 8.06 Hz, 1H, chromeno H-6), 5.05 (s, 2H,
chromeno H-2).
Fig. 4. Topoisomerase II inhibitory activity of the prepared compounds 14e36.
13C NMR (62.5 MHz, CDCl3)
d 156.26, 149.04, 139.60, 138.88,
138.54, 131.26, 129.18, 128.71, 128.00, 127.92, 127.39, 126.91, 125.36,
125.11, 123.61, 122.31, 121.98, 119.82, 116.71, 111.42, 65.89.

U. Thapa et al. / European Journal of Medicinal Chemistry 46 (2011) 3201e3209
3207
13C NMR (62.5 MHz, CDCl3)
d
156.57, 156.23, 156.17, 149.46,
4.3.14. 2-(furan-2-yl)-4-(pyridin-2-yl)-5H-chromeno[4,3-b]
149.23, 145.24, 139.03, 136.97, 131.18, 129.11, 128.69, 126.91, 125.32,
123.69, 123.45, 123.14, 123.02, 122.15, 119.12, 116.72, 66.26.
pyridine (27)
A light green soild, Yield: 43.0%.
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.50.
LC/MS/MS: retention time: 6.01 min, [MH]þ: 327.23.
4.3.10. 2-phenyl-4-(pyridin-3-yl)-5H-chromeno[4,3-b]pyridine
(23)
1H NMR (250 MHz, CDCl3)
d
8.70 (dd, J ¼ 3.6, 0.8 Hz, 1H,
A white solid, Yield: 29.4%.
4-pyridine H-6), 8.38 (dd, J ¼ 7.7, 1.6 Hz, 1H, chromeno H-5), 7.81
(dt, J ¼ 7.7, 1.7 Hz, 1H, 4-pyridine H-3), 7.76 (s, 1H, pyridine H-3),
7.62 (d, J ¼ 7.8 Hz, 1H, 4-pyridine H-4), 7.51 (d, J ¼ 0.9 Hz, 1H,
2-furan H-5), 7.39e7.23 (m, 2H, 4-pyridine H-5 and chromeno H-7),
7.23 (d, J ¼ 3.3 Hz, 1H, 2-furan H-3), 7.15 (dt, J ¼ 7.5, 1 Hz, 1H,
chromeno H-6), 6.90 (dd, J ¼ 8.6, 0.7 Hz, 1H, chromeno H-8), 6.5
(dd, J ¼ 3.3, 1.7 Hz, 1H, 2-furan H-4), 5.44 (s, 2H, chromeno H-2).
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.50.
LC/MS/MS: retention time: 5.22 min, [MH]þ: 337.36.
1H NMR (250 MHz, CDCl3)
d
8.90 ( d, J ¼ 2.1 Hz, 1H, 4-pyridine
H-2), 8.80 ( d, J ¼ 5.1 Hz, 1H, 4-pyridine, H-6), 8.20 (dd, J ¼ 7.7,
1.6 Hz, 1H, chromeno H-5), 8.00 ( dd, J ¼ 9.4, 1.2 Hz, 2H, 2-phenyl
H-2, H-6), 7.93 (s, 1H, pyridine H-3), 7.70 (d, J ¼ 2.0 Hz, 1H,
4-pyridine H-4), 7.56e7.45 (m, 4H, 4-pyridine H-5 and 2-phenyl
H-3, H-4, H-5), 7.30 (dt, J ¼ 7.7, 1.6 Hz, 1H, chromeno H-7), 7.10
(dt, J ¼ 8.0, 1.0 Hz, 1H, chromeno H-6), 6.90 (dt, J ¼ 8.0 Hz, 1H,
chromeno H-8), 5.32 (s, 2H, chromeno H-2).
13C NMR (62.5 MHz, CDCl3)
d 156.60, 156.02, 153.77, 149.36,
148.52 145.11, 143.24, 136.99, 131.27, 125.26, 123.49, 123.42, 123.17,
123, 122.16, 117.12, 116.73, 112.13, 108.96, 66.32.
13C NMR (62.5 MHz, CDCl3)
d 158.50, 156.17, 156.53, 150.41,
4.3.15. 2-(furan-2-yl)-4-(pyridin-3-yl)-5H-chromeno[4,3-b]
pyridine (28)
147.75, 145.78, 139.06, 132.81, 131.88, 130.44, 129.32, 128.87, 127.05,
126.26, 124.85, 122.62, 119.73, 118.31, 117.15, 111.42, 67.82.
A light yellow solid, Yield: 48.8%.
Rf (ethyl acetate/n-hexane 1:1 v/v): 0.24.
LC/MS/MS: retention time: 4.41 min, [MH]þ: 327.26.
4.3.11. 2,4-di(furan-2-yl)-5H-chromeno[4,3-b]pyridine (24)
A yellow solid, Yield: 43.9%.
1H NMR (250 MHz, CDCl3)
d
8.74 (td, J ¼ 4.6, 1.3, 1.1 Hz, 1H,
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.50.
4-pyridine H-6), 8.63 (t, J ¼ 1.1 Hz, 1H, 4-pyridine H-2), 8.37
(dd, J ¼ 7.1, 1.1 Hz, 1H, chromeno H-5), 7.71 (dd, J ¼ 5.4, 2.3 Hz, 1H,
2-furan H-5), 7.57e7.54 (m, 2H, 4-pyridine H-4 and pyridine H-3),
7.46 (dd, J ¼ 7.7, 4.8 Hz, 1H, 4-pyridine H-5), 7.35 (tt, J ¼ 6.6, 1.5,
1.1 Hz, 1H, chromeno H-7), 7.25 (d, J ¼ 4.3 Hz, 1H, 2-furan H-3), 7.15
(tt, J ¼ 7.5, 1.0 Hz, 1H, chromeno H-6), 6.97 (d, J ¼ 8.1 Hz, 1H,
chromeno H-8), 6.57 (dd, J ¼ 3.0, 1.4 Hz, 1H, furan H-4), 5.20 (s, 2H,
chromeno H-2).
LC/MS/MS: retention time: 13.14 min, [MH]þ: 316.22.
1H NMR (250 MHz, CDCl3)
d
8.30 (dd, J ¼ 7.7, 1.4 Hz, 1H, chro-
meno H-5), 7.84 (s, 1H, pyridine H-3), 7.63 (t, J ¼ 0.8 Hz 1H, 2-furan
H-5), 7.55 (t, J ¼ 0.8 Hz, 1H, 4-furan H-5), 7.33 (tt, J ¼ 7.0, 0.8 Hz,
1H, chromeno H-7), 7.20 (d, J ¼ 3.3 Hz, 1H, 2-furan H-3), 7.10
(t, J ¼ 7.4 Hz, 1H, chromeno H-6), 6.92 ( d, J ¼ 8.0 Hz, 1H, chromeno
H-8), 6.70 (d, J ¼ 3.4 Hz,1H, 4-furan H-3), 6.59e6.56 (m, 2H, 2-furan
H-4 and 4-furan H-4), 5.54 (s, 2H, chromeno H-2).
13C NMR (62.5 MHz, CDCl3)
d 156.36, 153.48, 149.93,
13C NMR (62.5 MHz, CDCl3)
d 156.19, 153.73, 150.45, 149.08,
149.16 149.04, 148.73, 143.46, 135.81, 133.12, 131.55, 125.32, 123.51,
123.20, 122.45, 121.96, 117.75, 116.81, 112.18, 109.33, 65.59.
148.50 143.94, 143.23, 134.52, 131.25, 125.23, 123.19, 122.22, 120.23,
116.63, 113.99, 112.09, 112.06, 111.55, 108.90, 35.73.
4.3.16. 4-(Furan-3-yl)-2-phenyl-5H-chromeno[4,3-b]pyridine (29)
A white solid, Yield: 43.0%.
4.3.12. 2-(furan-2-yl)-4-(thiophen-2-yl)-5H-chromeno[4,3-b]
pyridine (25)
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.50.
A white solid, Yield: 35.3%.
LC/MS/MS: retention time: 8.18 min, [MH]þ: 326.27.
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.50.
1H NMR (250 MHz, CDCl3)
d
8.44 (dd, J ¼ 8.0, 1.5 Hz, 1H, chro-
LC/MS/MS: retention time: 5.85 min, [MH]þ: 332.22.
meno H-5), 8.16e8.12 (m, 2H, 2-phenyl H-2, H-6), 7.63 (s, 1H,
pyridine H-3), 7.59e7.56 (m, 2H, 4-furan H-2, H-5), 7.52e7.43
(m, 3H, 2-phenyl H-3, H-4, H-5), 7.30 (ddd, J ¼ 6.8, 1.7, 0.7 Hz, 1H,
chromeno H-7), 7.14 (dt, J ¼ 7.5, 1.1 Hz, 1H, chromeno H-6), 6.90 (dd,
J ¼ 7.6, 0.7 Hz, 1H, chromeno H-8), 6.60 (dd, J ¼ 1.8, 0.9 Hz, 1H,
4-furan H-4), 5.36 (s, 2H, chromeno H-2).
1H NMR (250 MHz, CDCl3)
d
8.30 (dd, J ¼ 7.7, 1.5 Hz, 1H, chro-
meno H-5), 7.68 (s, 1H, pyridine H-3), 7.50 (d, J ¼ 0.8 Hz, 1H, 2-furan
H-5), 7.49 (dd, J ¼ 5.1, 1.0 Hz, 1H, 4-thiophene H-5), 7.30 (dt, J ¼ 7.7,
1.6 Hz, 1H, chromeno H-6), 7.22e7.13 (m, 3H, 4-thiophene H-4, 2-
furan H-3 and chromeno H-7), 7.00 (dd, J ¼ 3.5, 1 Hz, 1H, 4-thio-
phene H-3), 6.90 (dd, J ¼ 8.0, 0.7 Hz, 1H, chromeno H-8), 6.50 (dd,
J ¼ 3.3, 1.7 Hz, 1H, 2-furan H-4), 5.41 (s, 2H, chromeno H-2).
13C NMR (62.5 MHz, CDCl3)
d 156.32, 156.24, 148.73, 143.84,
140.92, 139.03, 137.80, 131.19, 129.11, 128.70, 126.88, 125.29, 123.57,
122.30, 122.11, 116.69, 110.57, 65.84.
13C NMR (62.5 MHz, CDCl3)
d 156.29, 153.60, 149.13,
148.51 143.34, 139.52, 138.36, 131.34, 128.07, 127.89, 127.49, 125.30,
123.35, 122.33, 121.80, 117.75, 116.71, 112.10, 109.10, 65.92.
4.3.17. 2-phenyl-4-(thiophen-3-yl)-5H-chromeno[4,3-b]pyridine
(30)
4.3.13. 2-(furan-2-yl)-4-phenyl-5H-chromeno[4,3-b]pyridine (26)
A white solid, Yield: 41.6%.
A white solid, Yield: 34.5%.
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.50.
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.50.
LC/MS/MS: retention time: 9.22 min, [MH]þ: 342.27.
LC/MS/MS: retention time: 8.57 min, [MH]þ: 326.25.
1H NMR (250 MHz, CDCl3)
d
8.45 (dd, J ¼ 7.6, 1,5 Hz, 1H, chro-
1H NMR (250 MHz, CDCl3)
d
8.32 (dd, J ¼ 7.6,1.4 Hz,1H, chromeno
meno H-5), 8.17e8.13 (m, 2H, 2-phenyl H-2,6) 7.66 (S, 1H, pyridine
H-3), 7.53e7.43 (m, 4H, 2-phenyl H-3,4,5 and 4-thiophene H-2),
7.37e7.30 (m, 2H, 4-thiophene H-4 and chromeno H-7), 7.20e7.11
(m, 2H, chromeno H-6,8), 6.97 (dd, J ¼ 8.1, 0.8 Hz, 1H, chromeno
H-8), 5.05 (s, 2H, chromeno H-2).
H-5), 7.59 (s,1H, pyridine H-3), 7.53e7.46 (m, 4H, 4-phenyl H-2, H-6,
furan H-5 and chromeno H-6), 7.36e7.30 (m, 3H, 4-phenyl, H-3, H-4,
H-5), 7.20 (d, J ¼ 4.3 Hz, 1H, furan H-3), 7.10 (t, J ¼ 7.4 Hz, 1H, chro-
meno H-7), 6.90 (d, J ¼ 8.0 Hz, 1H, chromeno H-8), 6.50 (dd, J ¼ 3.1,
1.7 Hz, 1H, furan H-4), 3.98 (s, 2H, chromeno H-2).
13C NMR (62.5 MHz, CDCl3)
d 156.33, 156.21, 148.79, 141.78,
13C NMR (62.5 MHz, CDCl3)
d 156.41, 153.81, 148.82,
139.04, 137.95, 131.18, 129.10, 128.70, 127.82, 126.89, 126.70, 125.31,
125.46, 123.71, 122.30, 122.12, 119.56, 116.72,65.93.
148.43 147.20, 143.25, 137.27, 131.26, 128.27, 128.63, 128.42, 125.27,
123.50, 122.32, 121.99, 117.99, 116.74, 112.08, 108.92, 65.93.

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U. Thapa et al. / European Journal of Medicinal Chemistry 46 (2011) 3201e3209
4.3.18. 4-(furan-2-yl)-2-(thiophen-2-yl)-5H-chromeno [4,3-b]
pyridine (31)
4.3.22. 4-(pyridin-2-yl)-2-(thiophen-2-yl)-5H-chromeno[4,3-b]
pyridine (35)
A white solid, Yield: 33.9%.
A yellow solid, Yield: 35.3%.
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.40.
Rf (ethyl acetate/n-hexane 1:2 v/v): 0.40.
LC/MS/MS: retention time: 8.56 min, [MH]þ: 332.24.
LC/MS/MS: retention time: 7.01 min, [MH]þ: 343.25.
1H NMR (250 MHz, CDCl3)
d
8.30 (dd, J ¼ 7.7, 1.6 Hz, 1H, chro-
1H NMR (250 MHz, CDCl3)
d
8.70 (d, J ¼ 4.6 Hz, 1H, 4-pyridine
meno H-5), 7.76 (s, 1H, pyridine H-3), 7.68 (dd, J ¼ 3.6, 1.1 Hz, 1H,
2-thiophene H-3), 7.61 (d, J ¼ 1.2 Hz, 1H, 4-furan H-5), 7.40 (dd,
J ¼ 5.0, 1.0 Hz, 1H, 2-thiophene H-5), 7.30 (dt, J ¼ 8.0, 1.7 Hz, 1H,
chromeno H-7), 7.16e7.10 (m, 2H, 4-furan H-3 and chromeno H-6),
6.90 (dd, J ¼ 8.1, 0.8 Hz, 1H, chromeno H-8), 6.70 (d, J ¼ 3.1 Hz, 1H,
2-thiophene H-4), 6.63 (dd, J ¼ 3.4, 1.8 Hz, 1H, 4-furan H-4), 5.53
(s, 2H, chromeno H-2).
H-6), 8.40 (dd, J ¼ 7.6, 1.5 Hz, 1H, chromeno H-5), 7.86 (dt, J ¼ 7.7,
1.7 Hz, 1H, 4-pyridine H-4), 7.67 (d, J ¼ 2.3 Hz, 2H, pyridine H-3 and
2-thiophene H-3), 7.60 (d, J ¼ 7.8 Hz, 1H, 4-pyridine H-3), 7.43e7.30
(m, 3H, chromeno H-6,7 and 2-thiophene H-5), 7.17e7.11 (m, 2H,
4-pyridine H-5 and 2-thiophene H-4), 6.98 (d, J ¼ 7.6 Hz, 1H,
chromeno H-8), 5.42 (s, 2H, chromeno H-2).
13C NMR (62.5 MHz, CDCl3)
d 156.54, 155.89, 151.63, 149.49,
13C NMR (62.5 MHz, CDCl3)
d
156.17, 151.65, 150.25,
149.16 145.25, 144.92, 136.99, 131.30, 127.96, 127.69, 125.35, 124.67,
123.44, 123.28, 123.22, 122.80, 122.20, 117.39, 116.69, 66.23.
148.91, 144.94, 143.91, 134.64, 131.29, 127.94, 127.62, 125.33, 124.63,
123.07, 122.25, 120.18, 116.59, 114.24, 112.07, 111.55, 66.12.
4.3.23. 4-(pyridin-2-yl)-2-(thiophen-3-yl)-5H-chromeno[4,3-b]
pyridine (36)
4.3.19. 2,4-di(thiophen-2-yl)-5H-chromeno[4,3-b]pyridine (32)
A white solid, Yield: 33.9%.
A yellow solid, Yield: 52.5%.
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.40.
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.50.
LC/MS/MS: retention time: 6.87 min, [MH]þ: 343.26.
LC/MS/MS: retention time: 9.35 min, [MH]þ: 348.23.
1H NMR (250 MHz, CDCl3)
d
8.70 (dd, J ¼ 3.8, 1.0 Hz, 1H,
1H NMR (250 MHz, CDCl3)
d
8.30 (dd, J ¼ 8.7, 1.7 Hz, 1H, chro-
4-pyridine H-6), 8.40 (dd, J ¼ 8.7 Hz, 1H, chromeno H-5), 8.00 (d,
J ¼ 2.8 Hz, 1H, 2-thiophene H-2), 7.90e7.78 (m, 2H, 4-pyridine H-3
and 2-thiophene H-5), 7.66 (s, 1H, pyridine H-3), 7.59 (d, J ¼ 7.8 Hz,
1H, chromeno H-7), 7.43e7.31 (m, 3H, 2-thiophene H-4 and
4-pyridine H-4,5), 7.10 (t, J ¼ 7.4 Hz, 2H, chromeno H-6), 6.98
(d, J ¼ 8.0 Hz, 1H, chromeno H-8), 5.42 (s, 1H, chromeno H-2).
meno H-5), 7.64 (dd, J ¼ 4.7, 1.0 Hz, 1H, 2-thiophene H-3), 7.59
(s, 1H, pyridine H-3), 7.48 (dd, J ¼ 5.1, 1.1 Hz, 1H, 4-thiophene H-3),
7.41 (dd, J ¼ 5.0,1.0 Hz,1H, 2-thiophene H-5), 7.32 (dt, J ¼ 8.4,1.7 Hz,
1H, chromeno H-7), 7.18e7.11 (m, 2H, 4-thiophene H-5 and chro-
meno H-6), 7.10 (dd, J ¼ 3.4, 1.3 Hz, 1H, 2-thiophene H-4), 7.02 (dd,
J ¼ 3.6, 1.0 Hz, 1H, 4-thiophene H-4), 6.97 (dd, J ¼ 8.0, 1.8 Hz, 1H,
chromeno H-8), 5.38 (s, 2H, chromeno H-2).
13C NMR (62.5 MHz, CDCl3)
d 156.57, 156.06, 152.50,
13C NMR (62.5 MHz, CDCl3)
d 156.29, 151.65, 149, 144.76, 139.62,
138.26, 131.39, 128.03, 127.97, 127.93, 127.78, 127.44, 125.42, 124.76,
123.22, 122.35, 121.79, 118.05, 116.68, 65.89.
149.49 145.25, 142.06, 136.97, 131.19, 126.33, 126.21, 125.24,
123.477 123.59, 123.345, 123.16, 122.68, 122.13, 118.91, 116.73,
66.25.
4.4. Biological assays
4.3.20. 4-(furan-3-yl)-2-(thiophen-2-yl)-5H-chromeno[4,3-b]
pyridine (33)
DNA topo I inhibition assay was determined following the
previously reported method [14]. The prepared compounds were
dissolved in DMSO at 20 mM as stock solution. The activity of DNA
topo I was determined by assessing the relaxation of supercoiled
DNA pBR322. The mixture of 100 ng of plasmid pBR322 DNA and
0.4 units of recombinant human DNA topo I (TopoGEN INC., USA)
was incubated without and with the prepared compounds at 37 ꢁC
for 30 min in the relaxation buffer (10 mM TriseHCl (pH 7.9),
150 mM NaCl, 0.1% bovine serum albumin, 1 mM spermidine, 5%
A white solid, Yield: 32.4%.
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.50.
LC/MS/MS: retention time: 7.78 min, [MH]þ: 332.32.
1H NMR (250 MHz, CDCl3)
d
8.30 (dd, J ¼ 7.7, 1.6 Hz, 1H, chro-
meno H-5), 7.64 (dd, J ¼ 3.6, 0.9 Hz, 1H, 2-thiophene H-3), 7.58
(d, J ¼ 2.7 Hz, 1H, 4-furan H-5), 7.57 (d, J ¼ 0.8 Hz, 1H, 4-furan H-2),
7.59 (s, 1H, pyridine H-3), 7.41 (dd, J ¼ 5.0, 0.9 Hz, 1H, thiophene
H-5), 7.32 (dt, J ¼ 7.7, 1.6 Hz, 1H, chromeno H-7), 7.17e7.11 (m, 2H,
2-thiophene H-4 and chromeno H-6), 6.97 (dd, J ¼ 7.65, 0.8 Hz, 1H,
chromeno H-8), 6.63 (dd, J ¼ 1.5, 0.8 Hz, 1H, 4-furan H-4), 5.32
(s, 2H, chromeno H-2).
glycerol). The reaction in the final volume of 10
mL was terminated
by adding 2.5 L of the stop solution containing 5% sarcosyl,
m
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
13C NMR (62.5 MHz, CDCl3)
144.87, 143.86, 140.96, 137.81, 131.30, 127.95, 127.67, 125.31, 124.58,
123.16, 122.34, 122.11, 117.27, 116.65, 110.53, 65.81.
d 156.22, 151.67, 148.66,
solution of ethidium bromide (0.5 mg/mL). DNA bands were visu-
alized by transillumination with UV light and were quantitated
using AlphaImagerÔ (Alpha Innotech Corporation).
DNA topo II inhibitory activity of compounds was measured as
follows [15]. The mixture of 200 ng of supercoiled pBR322 plasmid
DNA and 1 unit of human DNA topo IIa (TopoGEN INC., USA) was
4.3.21. 4-phenyl-2-(thiophen-2-yl)-5H-chromeno[4,3-b]pyridine
(34)
A white solid, Yield: 53.5%.
Rf (ethyl acetate/n-hexane 1:5 v/v): 0.50.
LC/MS/MS: retention time: 9.50 min, [MH]þ: 342.27.
incubated without and with the prepared compounds in the assay
buffer (10 mM TriseHCl (pH 7.9) containing 50 mM NaCl, 5 mM
1H NMR (250 MHz, CDCl3)
d
8.42 (dd, J ¼ 7.6, 1.5 Hz, 1H, chro-
meno H-5), 7.64 (d, J ¼ 3.6 Hz, 1H, 2-thiophene H-3), 7.51e7.49
(m, 4H, pyridine H-3, 6-phenyl H-2,4,6), 7.40 (d, J ¼ 5.0 Hz, 1H,
2-thiophene H-5), 7.36e7.30 (m, 3H, chromeno H-7 and 6-phenyl
H-3,5), 7.18e7.10 (m, 2H, chromeno H-6 and 2-thiophene H-4), 6.97
(d, J ¼ 8.0 Hz, 1H, chromeno H-8), 5.21 (s, 2H, chromeno H-2).
MgCl2, 1 mM EDTA, 1 mM ATP, and 15
albumin) 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
mg/mL bovine serum
m
L
m
products were analyzed on 1% agarose gel at 25 V for 4 h with
a running buffer of TAE. Gels were stained for 30 min in an aqueous
solution of ethidium bromide (0.5 mg/mL). DNA bands were visu-
13C NMR (62.5 MHz, CDCl3)
d 160.94, 156.56, 155.51,
148.98 143.70, 143.55, 141.63, 140.91, 137.04, 136.93, 132.96, 128.79,
127.28, 125.01, 123.57, 121.11, 116.22, 109.38, 35.49.
alized by transillumination with UV light and supercoiled DNA was
quantitated using AlphaImagerÔ (Alpha Innotech Corporation).

U. Thapa et al. / European Journal of Medicinal Chemistry 46 (2011) 3201e3209
(1979) 1841e1844;
3209
The added amount of DMSO for each reaction mixture was same in
both of enzyme only and enzyme with designated compound for
topo I and II assays.
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845e850;
For the evaluation of cytotoxicity, five different cancer cell lines
were used: human breast adenocarcinoma cell line (MCF-7),
human prostate tumor cell line (DU145), human cervix tumor cell
line (HeLa), chronic myelogenous leukemia cell line (K562), and
human colorectal adenocarcinoma cell line (HCT15). Experiments
were performed by methods previously described [15]. Cancer cells
were cultured according to the supplier’s instructions. Cells were
seeded in 96-well plates at a density of 2e4 ꢀ 104 cells per well and
incubated for overnight in 0.1 mL of media supplied with 10% Fetal
Bovine Serum (Hyclone, USA) in 5% CO2 incubator at 37 ꢁC. On day
2, culture medium in each well was exchanged with 0.1 mL aliquots
of medium containing graded concentrations of compounds. On
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E.S. Lee, Bioorg. Med. Chem. 15 (2007) 4351e4359;
(e) J.K. Son, L.X. Zhao, A. Basnet, P. Thapa, R. Karki, Y. Na, Y. Jahng, T.C. Jeong,
B.S. Jeong, C.S. Lee, E.S. Lee, Eur. J. Med. Chem. 43 (2008) 675e682;
(f) P. Thapa, R. Karki, A. Basnet, U. Thapa, H.Y. Choi, Y. Na, Y. Jahng, C.S. Lee,
Y. Kwon, B.S. Jeong, E.S. Lee, Bull. Korean Chem. Soc. 29 (2008) 1605e1608;
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day 4, each well was added with 5 mL 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) at 450 nm
wavelength. For determination of the IC50 values, the absorbance
readings at 450 nm were fitted to the four-parameter logistic
equation. Adriamycin, etoposide, and camptothecin were pur-
chased from Sigma and used as positive controls.
(h) P. Thapa, R. Karki, U. Thapa, Y. Jahng, M.J. Jung, J.M. Nam, Y. Na, Y. Kwon,
E.S. Lee, Bioorg. Med. Chem. 18 (2010) 377e386;
(i) P. Thapa, R. Karki, H.Y. Choi, J.H. Choi, M. Yun, B.S. Jeong, M.J. Jung, J.M. Nam,
Y. Na, W.J. Cho, Y. Kwon, E.S. Lee, Bioorg. Med. Chem. 18 (2010) 2245e2254;
(j) R. Karki, P. Thapa, M.J. Kang, T.C. Jeong, J.M. Nam, H.L. Kim, Y. Na, W.J. Cho,
Y. Kwon, E.S. Lee, Bioorg. Med. Chem. 18 (2010) 3066e3077;
(k) R. Karki, P. Thapa, Y. Kwon, E.S. Lee, Bull. Korean Chem. Soc. 31 (2010)
1747e1750.
Acknowledgments
[6] B.S. Jeong, H.Y. Choi, P. Thapa, R. Karki, M.J. Jung, J.M. Nam, Y. Na, E.M. Ha,
Y. Kwon, E.S. Lee, Bull. Korean Chem. Soc. 32 (2011) 303e306.
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Bioorg. Med. Chem. Lett. 17 (2007) 4053e4056.
[8] J.F. Cheng, A. Ishikawa, Y. Ono, T. Arrhenius, A. Nadzan, Bioorg. Med. Chem.
Lett. 13 (2003) 3647e3650.
This research was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded
by the Ministry of Education, Science and Technology (2010-
0001661, 2009-0066925, 2010-0005506 and 2010-0002646).
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