Dihydroxylated 2,4,6-triphenyl pyridines: Synthesis, topoisomerase i and II inhibitory activity, cytotoxicity, and structure-activity relationship study

Article abstract of DOI:10.1016/j.ejmech.2012.01.015

Twelve dihydroxylated 2,4,6-triphenyl pyridines were designed and synthesized which contain hydroxyl groups at ortho, meta or para position of 2- and 6-phenyl, or 2- and 4-phenyl rings attached to the central pyridine. They were evaluated for topoisomeras

Full text of DOI:10.1016/j.ejmech.2012.01.015

European Journal of Medicinal Chemistry 49 (2012) 219e228
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Dihydroxylated 2,4,6-triphenyl pyridines: Synthesis, topoisomerase I and II
inhibitory activity, cytotoxicity, and structureeactivity relationship study
Radha Karki a, Pritam Thapa a, Han Young Yoo a, Tara Man Kadayat a, Pil-Hoon Park a, Youngwha Na b,
**
,
a,
*
Eunyoung Lee c, Kyung-Hwa Jeon c, Won-Jea Cho d, Heesung Choi e, Youngjoo Kwon c , Eung-Seok Lee
a College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea
b College of Pharmacy, Cha University, Pochon 487-010, Republic of Korea
c College of Pharmacy, Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Republic of Korea
d College of Pharmacy, Chonnam National University, Kwangju 500-757, Republic of Korea
e Eli Lilly Korea Ltd., Seoul 100-958, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Twelve dihydroxylated 2,4,6-triphenyl pyridines were designed and synthesized which contain hydroxyl
groups at ortho, meta or para position of 2- and 6-phenyl, or 2- and 4-phenyl rings attached to the central
pyridine. They were evaluated for topoisomerase I and II inhibitory activity, and cytotoxicity against
several human cancer cell lines for the development of novel anticancer agents. Generally, dihydroxy-
lated 2,4,6-triphenyl pyridines exhibited stronger topoisomerase II inhibitory activity, and cytotoxicity
compared to those of monohydroxylated 2,4,6-triphenyl pyridines. The concrete structureeactivity
relationship was observed that dihydroxylated 2,4,6-triphenyl pyridines with hydroxyl group at meta
or para position of 2-phenyl ring displayed significant topoisomerase II inhibitory activity as well as
cytotoxicity. Positive correlation between topoisomerase II inhibitory activity and cytotoxicity was
observed for compounds 10, 12, 13, 17e20 and 22.
Received 11 October 2011
Received in revised form
3 January 2012
Accepted 6 January 2012
Available online 24 January 2012
Keywords:
Dihydroxylated 2,4,6-triphenyl pyridines
Topoisomerase I
Topoisomerase II
Ó 2012 Elsevier Masson SAS. All rights reserved.
Cytotoxicity
Anticancer agents
1. Introduction
established molecular target since topoisomerases play crucial role
in relieving the torsional stress in the DNA helix that is generated as
a
-Terpyridines are the bioisosteres of
a
-terthiophene which
-Terpyridine
a result of replication, transcription, recombination, and other
nuclear processes. There are two classes of topoisomerases: topo-
isomerase I and topoisomerase II. Topoisomerase I (topo I) enzymes
introduce single strand breaks in DNA whereas topoisomerase II
(topo II) cleaves both strands. Topoisomerase inhibitors such as
camptothecin, doxorubicin and etoposide are highly active anti-
cancer agents in clinical use. However, several side effects such as
hematological toxicity, nausea and vomiting, hair loss, etc are
associated with them. Therefore improving the efficacy of these
drugs is undoubtedly an important topic in anticancer treatment.
Several classes of compounds are under clinical trial for topo-
isomerase inhibitory activity [9e13].
As part of our continuous search for potential anticancer drug
candidates, we previously synthesized several 2,4,6-trisubstituted
pyridine compounds as terpyridine bioisosteres, and evaluated for
topo I and II inhibitory activity, and cytotoxicity [7,14e23]. Several
monohydroxylated 2,4-diphenyl-6-aryl pyridine compounds were
found to possess significant topo I and II inhibitory activity, and
cytotoxicity [22]. Structureeactivity relationship study revealed
that hydroxyl moiety was important for the activity. The prepared
possess protein kinase C (PKC) inhibitory activity [1].
a
contains three nitrogen atoms that enable them to act as tridentate
ligand to form stable complexes by chelating a broad variety of
transition metalions [2]as shown in Fig.1. Platinum (II) complexes of
terpyridine derivatives have been well studied for their antitumor
potential [3e6] and were found to possess DNA-intercalating
activity, and cytotoxicity. They also act as inhibitors of
topoisomerases and human thioredoxin reductase1 enzymes [6]. It
is reported that terpyridine derivatives showed strong cytotoxicity
against several human cancer cell lines [7]. All these reports on DNA
binding property and antitumor activity of terpyridine and their
metal complexes have attracted researchers.
The identification of DNA topoisomerases was a major landmark
in the pharmacology of anticancer drugs [8]. It is now the
* Corresponding author. Tel.: þ82 53 810 2827; fax: þ82 53 810 4654.
** Corresponding author. Tel.: þ82 2 3277 4653; fax: þ82 2 3277 2851.
E-mail addresses: ykwon@ewha.ac.kr (Y. Kwon), eslee@yu.ac.kr (E.-S. Lee).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.015

220
R. Karki et al. / European Journal of Medicinal Chemistry 49 (2012) 219e228
Fig. 1. Structures of
a
-terthiophene,
a
-terpyridine, and terpyridineemetal complex.
compounds were monohydroxylated at 2- or 4-phenyl ring of
central pyridine. Several polyphenolic compounds have exhibited
wide spectrum of biological activities such as antioxidant,
anti-inflammatory, anticancer etc [24,25]. The anticancer effect of
many of the bioflavonoids is attributed to the ability to poison topo II
[26e28]. Epigallocatechin gallate (EGCG), the most abundant poly-
phenol in green tea, is a strong topo II poison [28]. Based on the
previously reported data and diverse biological activities of poly-
phenolic compounds, we herein designed and synthesized twelve
dihydroxylated 2,4,6-triphenyl pyridines.
In this study, we introduced two hydroxyl moieties on each
phenyl ring to investigate the effect of increase in the number of
hydroxyl group on topo I and II inhibitory activity, and cytotoxicity.
We have systematically designed and synthesized twelve
2,4,6-triphenyl pyridine compounds containing two hydroxyl
moieties at different positions (ortho, meta or para) on 2- and
6-phenyl, or 2- and 4-phenyl ring attached to the central pyridine
as shown in Figs. 2 and 3. These compounds were evaluated for
topo I and II inhibitory activity, and cytotoxicity against several
human cancer cell lines.
added BF3eEt2O to obtain compound 4 (R1 ¼ c, R2 ¼ c) in 74% yield.
In the second step, four pyridinium iodide salts 5 (R3 ¼ aed) were
synthesized in quantitative yield by the treatment of aryl ketone 1
(R1 ¼ aed) with iodine in pyridine. Using modified Kröhnke
synthesis [34,35], final compounds 6 (R1, R3 ¼ aec, R2 ¼ d) and 7
(R1, R2 ¼ aec, R3 ¼ d) were synthesized by the reaction of appro-
priate hydroxylated chalcones 3 or 4 with pyridinium iodide salt 5
in the presence of ammonium acetate and glacial acetic acid in
20e97% yield.
Total twelve final compounds 11e22 were synthesized from the
reaction of twelve intermediates and four pyridinium iodide salts
as shown in Fig. 4 and Table 1. All the compounds contain two
hydroxyl moieties substituted at various positions (ortho, meta or
para) of phenyl ring. Six compounds were 2- and 6-hydroxylated
2,4,6-triphenyl pyridines, and nine compounds were 2- and
4-hydroxylated 2,4,6-triphenyl pyridines as shown in Fig. 4,
whereas compounds 8e10 have been synthesized and reported
previously [22]. Synthesis of dihydroxylated compounds allowed us
to investigate the effect of increase in the number of hydroxyl group
on biological activity. Further, substitution of hydroxyl group at
various positions (ortho, meta or para) of phenyl ring helped us to
evaluate the effect of hydroxyl position on biological activity.
2. Chemistry
Dihydroxylated 2,4,6-triphenyl pyridine derivatives 11e22 were
synthesized in three steps as illustrated in Scheme 1. Using
ClaiseneSchmidt condensation reaction [29e32] twelve hydroxyl-
ated propenone intermediates were synthesized. Reactions were
either base [29,30] or acid [31,32] catalyzed, without protection of
hydroxyl groups. In the first method, 50% aqueous solution of KOH
or 6 M NaOH was added to the solution of equimolar amounts of
aryl ketone 1 (R1 ¼ aec) and aryl aldehyde 2 (R2 ¼ aed) in ethanol
to obtain compounds 3 (R1 ¼ aec, R2 ¼ d) and 4 (R1, R2 ¼ aec) in
40e90% yield. Intermediate 4 (R1, R2 ¼ c) was obtained in very low
yield by this method. This may be because of the presence of
hydroxyl substituent in the aromatic aldehyde that hinders the
base catalyze aldol reaction. The basic catalyst decreases the
activity of the aldehyde component because of delocalization of the
anion [33]. To overcome such problem, different methodology was
applied to use Lewis acid, borontrifluoro etherate. To a solution of
aryl ketone 1 (R1 ¼ c) and aryl aldehyde 2 (R2 ¼ c) in dioxane was
3. Results and discussion
Among the fifteen compounds shown in Fig. 4, compounds 8e10
have been reported previously for their topo I and II inhibitory
activity, and cytotoxicity against several human cancer cell lines
[22]. Compounds 8e10, which contain two hydroxyl moieties at
2- and 6-phenyl ring, showed strong topo II and/or topo I inhibitory
activities, and exhibited moderate cytotoxicity against several
human cancer cell lines as shown in Table 2. In general, the effects
of topo inhibitory activity and cytotoxicity for 8e10 were more
potent than those of monohydroxylated 2,4,6-triphenyl pyridines.
From these results we were impelled to design and synthesize
diverse dihydroxylated compounds 11e22, and evaluate their topo
I
and II inhibitory activities, and cytotoxicity. In addition,
structureeactivity relationship was investigated according to the
position of hydroxyl moiety. Compounds 8e13 possess hydroxyl
moiety at 2- and 6-phenyl rings whereas compounds 14e22
contain hydroxyl moiety at 2- and 4-phenyl rings. All the synthe-
sized compounds were evaluated for topo I and II inhibitory
activity, and cytotoxicity against several human cancer cell lines.
The conversion of supercoiled plasmid DNA to relaxed DNA by topo
Fig. 2. Structures of 2,4,6-trisubstituted pyridines and dihydroxylayed 2,4,6-triphenyl
pyridines.
Fig. 3. Strategy for the design of dihydroxylated 2,4,6-triphenyl pyridines.

R. Karki et al. / European Journal of Medicinal Chemistry 49 (2012) 219e228
221
I or II was examined in the presence of the synthesized dihy-
droxylated 2,4,6-triphenyl pyridine compounds 11e22. Campto-
topo II inhibitory activity than the positive control, etoposide. Other
compounds 14e16 and 21 also showed moderate topo II inhibitory
activity. Especially, compound 18 possessed 100.0% topo II inhibi-
thecin and etoposide, well known topo
I and II inhibitors,
respectively, were used as positive controls. Figs. 5 and 6 and
Table 2 illustrate the topo I and II inhibitory activity, and cytotox-
icity of the synthesized compounds 11e22.
tion at 100
100 and 20
m
m
M. Compound 20 displayed significant activity at both
M (87.9% and 31.9%, respectively). Compounds 17, 19,
and 22 possessed 75.9%, 88.1% and 81.7% topo II inhibitory activity
at 100 M, respectively.
m
Two interesting observations were achieved by comparing to
the previously reported results: (a) Introduction of hydroxyl moiety
increases the topo II inhibitory activity (b) Increase in the number
of hydroxyl group generally increases the topo II inhibitory activity.
It was observed that the compounds having structural similarity to
11e22 but without hydroxyl moiety on the structure did not
displayed considerable topoisomerase inhibitory activity [15,16].
Monohydroxylated 2,4-diphenyl-6-aryl pyridines were observed
stronger topo II inhibitors [22] compared to non-hydroxylated
compounds but less potent than the dihydroxylated compounds
8e22. Several other studies also support that introduction of
hydroxyl group at certain precise position enhance topo II inhibi-
tory activity [36,37].
3.1. Topoisomerase I and II inhibitory activity
All the twelve synthesized dihydroxylated 2,4,6-triphenyl pyri-
dines did not show considerable topo I inhibitory activity as shown
in Fig. 6 and Table 2. These results suggest that the prepared
compounds were selective to topo II inhibition, and topo I is not
crucial to exhibit antitumor activities.
The effect of synthesized compounds on human DNA topo II
a
were observed in the relaxation assay using supercoiled pBR322
plasmid DNA in the presence of ATP. Fig. 5 and Table 2 summarizes
the topo II inhibitory activity of the synthesized compounds. Most
of the compounds showed significant inhibitory activity at 100 mM.
Compounds 11e13 which possess hydroxyl moiety at 2- and
6-phenyl rings were potent topo II inhibitors as expected on the
basis of compounds 8e10. Compound 12 displayed 100% and 82.3%
3.2. Cytotoxicity
inhibition at 100 and 20 mM, respectively, which were higher than
the positive control, etoposide. Similarly, compound 11 showed
92.2% and 52.1% inhibition, and compound 13 showed 96.8% and
31% inhibition at 100 and 20 mM, respectively. These results clearly
imply that substitution of hydroxyl moiety at 2- and 6-phenyl ring
increase topo II inhibitory activity.
Compounds 14e22, which possess hydroxyl moiety at 2- and
4-phenyl rings of central pyridine, also displayed significant topo II
inhibitory activity. Compounds 17e20 and 22 showed stronger
For the evaluation of cytotoxicity, five different human cancer cell
lines were utilized: HCT15 (human colorectal adenocarcinoma cell
line), K562 (human myeloid leukemic tumor cell line), DU145
(human prostate tumor cell line), MCF-7 (human breast adenocar-
cinoma cell line), and HeLa (human cervix tumor cell line). The
inhibitory activity (IC50) is expressed as micromolar concentration as
illustrated inTable 2. Among compounds 11e13, compound 12 and 13
showed significant cytotoxicity against most of the tested cell lines
Scheme 1. General synthetic scheme of dihydroxylated 2,4,6-triphenyl pyridines. Reagents and conditions: (i) aryl aldehydes 2 (R2 ¼ aed) (1.0 equiv), KOH/NaOH, EtOH, 2e24 h,
20 ꢀC, 40e90% yield, or aryl aldehyde 2 (R2 ¼ c) (1.5 equiv), BF3Et2O, Dioxane, 2 h, 20 ꢀC, 74% yield; (ii) NH4OAc (10.0 equiv), glacial AcOH, 12e24 h, 90e100 ꢀC, 20e97% yield.

222
R. Karki et al. / European Journal of Medicinal Chemistry 49 (2012) 219e228
Fig. 4. Structures of synthesized dihydroxylated 2,4,6-triphenyl pyridines 8e22.
(<5
m
M). Compound 11 displayed moderate cytotoxicity against
against several human cancer cell lines. Six compounds 8e13,
which possess hydroxyl moiety at 2- and 6-phenyl rings dis-
played significant topo II inhibitory activity and considerable
cytotoxicity against several human cancer cell lines except 8 which
possess ortho- and ortho-hydroxyl at 2- and 6-phenyl positions.
These results indicated that hydroxyl group at meta or para position
on either 2- or 6-phenyl moiety is important to exhibit topo II
inhibitory activity, and/or cytotoxicity. Compounds 14e16, which
possess hydroxyl group at ortho position of 2-phenyl ring showed
comparatively lower topo II inhibition, and cytotoxicity compared
to the compounds which bear hydroxyl group at meta (17e19) and
para (20e22) position. Except compound 21, all other compounds
17e22 displayed topo II inhibitory activity stronger than that of
etoposide. From these results it can be concluded that meta or para
hydroxyl substitution at 2-phenyl ring is more favorable for topo II
inhibitory activity and cytotoxicity than ortho substitution as
shown in Fig. 7.
K562. Compounds 14e16 were less effective against the tested cancer
cell lines with cytotoxicity value 24e50 M except 15 which
possessed cytotoxicity 3.20 M against HCT15. Compounds 17e22
displayed significant cytotoxicity that was comparable to positive
controls adriamycin, etoposide, and camptothecin, in most cell lines,
m
m
especially in HCT15. IC50 values were less than 5 mM in most of the cell
lines for these compounds. Compounds 17e20, and 22 that possessed
significant topo II inhibitory activity also displayed strong cytotox-
icity. The cytotoxicity results indicate that most of the compounds
were effective against HCT15 and K562 cell lines.
3.3. Structureeactivity relationship study
Structureeactivity relationship (SAR) study indicates that the
position of hydroxyl group at the phenyl ring have a variable
influence on the topoisomerase inhibitory activity, and cytotoxicity
Table 1
Prepared compounds with yield, purity by HPLC, and melting point.
Entry
R1
R2
R3
Yield (%)
Purity (%)
Mp (ꢀC)
11
12
13
14
15
16
17
18
19
20
21
22
20 OH Phenyl
20 OH Phenyl
30 OH Phenyl
20 OH Phenyl
20 OH Phenyl
20 OH Phenyl
30 OH Phenyl
30 OH Phenyl
30 OH Phenyl
40 OH Phenyl
40 OH Phenyl
40 OH Phenyl
Phenyl
Phenyl
Phenyl
30 OH Phenyl
40 OH Phenyl
40 OH Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
90.5
31.5
61.7
24.5
97.2
90.9
49.8
72.5
84.3
19.8
87.0
47.9
99
99
240e241
299e300
264e265
221e222
229e230
229e230
191e192
273e274
241e242
209e210
222e223
244e245
100
100
100
100
100
100
100
99
2 OH Phenyl
3 OH Phenyl
4 OH Phenyl
2 OH Phenyl
3 OH Phenyl
4 OH Phenyl
2 OH Phenyl
3 OH Phenyl
4 OH Phenyl
Phenyl
Phenyl
100
98

R. Karki et al. / European Journal of Medicinal Chemistry 49 (2012) 219e228
223
Table 2
Topo II and I inhibitory activity, and cytotoxicity of compounds 8e22.
c
Compounds
%Inhibition
Topo II
IC50
(mM)
Topo I
100
HCT15
K562
DU145
MCF-7
HeLa
100
mM
20
m
M
m
M
Etoposide
Camptothecin
72.6
24.4b/31.6
1.33 ꢁ 0.10
0.26 ꢁ 0.04
1.28 ꢁ 0.07
>50
18.3 ꢁ 0.61
3.0 ꢁ 0.50
39.9 ꢁ 1.49
7.50 ꢁ 1.13
30.52 ꢁ 0.66
41.18 ꢁ 2.80
3.20 ꢁ 0.23
>50
0.93 ꢁ 0.09
1.54 ꢁ 0.02
1.26 ꢁ 0.11
0.86 ꢁ 0.06
0.88 ꢁ 0.03
0.79 ꢁ 0.04
2.09 ꢁ 0.55
1.18 ꢁ 0.14
2.85 ꢁ 1.38
22.4 ꢁ 2.07
16.4 ꢁ 0.75
5.7 ꢁ 0.84
2.94 ꢁ 0.04
2.37 ꢁ 0.55
0.86 ꢁ 0.04
8.0 ꢁ 0.32
24.9 ꢁ 0.78
22.0 ꢁ 0.63
>50
4.40 ꢁ 0.15
1.88 ꢁ 0.17
>50
3.25 ꢁ 0.04
3.91 ꢁ 0.66
3.69 ꢁ 0.13
37.3 ꢁ 0.34
12.7 ꢁ 1.3
6.7 ꢁ 0.78
>50
1.40 ꢁ 0.06
4.99 ꢁ 0.78
>50
3.20 ꢁ 0.57
1.01 ꢁ 0.01
1.45 ꢁ 0.01
14.8 ꢁ 0.05
13.0 ꢁ 0.08
3.3 ꢁ 0.44
>50
3.90 ꢁ 0.44
3.46 ꢁ 0.15
>50
59.9b/63.7
Adriamycin
8a
42.7
86.5
76.8
92.2
100.0
96.8
56.2
56.4
38.7
75.9
100.0
88.1
87.9
41.4
81.7
12.1
49.0
33.7
52.1
82.3
31.0
17.3
3.6
0.0
67.2
0.3
11.0
0.0
0.0
0.5
2.6
4.3
6.7
0.7
0.5
0.0
1.8
7.2
9a
10a
11
12
13
14
15
16
17
18
19
20
21
22
7.45 ꢁ 0.65
15.40 ꢁ 1.48
1.82 ꢁ 0.60
24.81 ꢁ 1.16
33.53 ꢁ 4.72
42.30 ꢁ 2.84
2.86 ꢁ 0.19
5.50 ꢁ 0.21
2.51 ꢁ 0.52
2.25 ꢁ 0.12
4.40 ꢁ 0.37
2.06 ꢁ 0.25
>50
>50
>50
>50
>50
>50
13.9
6.9
37.60 ꢁ 0.14
2.70 ꢁ 0.50
8.95 ꢁ 0.33
7.01 ꢁ 0.14
11.62 ꢁ 1.10
3.60 ꢁ 0.55
10.12 ꢁ 0.15
9.42 ꢁ 1.71
2.92 ꢁ 0.88
3.13 ꢁ 0.10
2.67 ꢁ 0.37
11.19 ꢁ 1.57
1.55 ꢁ 0.28
15.73 ꢁ 1.04
10.98 ꢁ 2.97
2.98 ꢁ 0.09
3.87 ꢁ 0.10
6.75 ꢁ 0.53
17.5
14.6
31.9
21.5
4.2
HCT15: human colorectal adenocarcinoma cell line.
K562: human myeloid leukemic tumor cell line.
DU145: human prostate tumor cell line.
MCF-7: human breast adenocarcinoma cell line.
HeLa: human cervix tumor cell line.
a
Previously reported results[22].
Positive control value for compounds 11e13.
Each data represents mean ꢁ S.D. from three different experiments performed in triplicate.
b
c
Compounds 10, 12, and 13 having para hydroxyl substitution at
2-phenyl ring was more cytotoxic compared to compound 8, 9, and
11 which possessed ortho and meta substitution. In addition,
compounds bearing hydroxyl moiety at para position on 2-phenyl
ring with ortho, meta or para hydroxyl substitution on 4-phenyl
ring of central pyridine (compounds 20e22) displayed the most
significant cytotoxicity, which recorded better or similar cytotox-
icity compared to positive controls, etoposide, camptothecin, and
Fig. 5. Human DNA topo II
a
inhibitory activity of compounds 11e22.

224
R. Karki et al. / European Journal of Medicinal Chemistry 49 (2012) 219e228
Fig. 6. Human DNA topo I inhibitory activity of compounds 11e22.
adriamycin. Compounds having meta hydroxyl substitution at
2-phenyl ring (compounds 17e19) also showed significant cyto-
toxicity but lower than that of compounds bearing hydroxyl moiety
at para position on 2-phenyl ring (compounds 20e22). However,
compounds bearing hydroxyl moiety at ortho position on 2-phenyl
ring (compounds 14e16) did not display considerable cytotoxicity.
These results indicate that the order of substitution for stronger
cytotoxicity is para > meta > ortho on 2-phenyl ring of central
pyridine. Also it was observed that dihydroxylated 2,4,6-triphenyl
pyridines on 2- and 6-phenyl, or 2- and 4-phenyl moiety were
more cytotoxic than those of monohydroxylated and/or non-
substituted 2,4,6-triphenyl pyridines including 2,4,6-triaryl
pyridines.
2-phenyl ring of central pyridine was more favorable than ortho
and/or meta hydroxylated compounds. Positive correlation
between topo II inhibition and cytotoxicity was observed for
compounds 10, 12, 13, 17e20, and 22.
5. Experimental
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
4. Conclusion
Twelve dihydroxylated 2,4,6-triphenyl pyridines were system-
atically designed and synthesized by efficient synthetic route, and
evaluated for topo I and II inhibitory activities, and cytotoxicity
against several human cancer cell lines. The results of biological
evaluation indicate that introduction of hydroxyl moiety on
2,4,6-triphenyl pyridines increases topo II inhibitory activity, and
cytotoxicity. Similarly, increase in the number of hydroxyl group
enhances topo II inhibitory activity, and cytotoxicity. Concrete
structureeactivity relationship for topo II inhibitory activity and
cytotoxicity was observed according to the position of hydroxyl
group on dihydroxylated 2,4,6-triphenyl pyridines. SAR study
revealed that compounds with hydroxyl group at meta or para
position on 2-phenyl ring of central pyridine displayed significant
topo II inhibitory activity as well as cytotoxicity. For cytotoxicity,
compound containing hydroxyl moiety at para position on
(d) were recorded in ppm and coupling constants (J) in hertz (Hz).
Melting points were determined in open capillary tubes on elec-
trothermal 1A 9100 digital melting point apparatus and were
uncorrected.
HPLC analyses were performed using two Shimadzu LC-10AT
pumps gradient-controlled HPLC system equipped with Shimadzu
system controller (SCL-10A VP) and photo diode array detector
(SPD-M10A VP) utilizing Shimadzu Class VP program. Sample
volume of 10 m 5 mM reverse-
L was injected in Waters X-TerraÒ
phase C18 column (4.6 ꢂ 250 mm) with a gradient elutions of
50%e100% of B in A for 15 min followed by 100%e50% of B in A for
15 min at a flow rate of 1.0 mL/min at 254 nm UV detection, where
R2
R2
R2
>
>
OH
HO
N
N
N
R1
R1
R1
HO
R1 = OH / H R2 = H / OH
Fig. 7. Favorable order of substitution for topo II inhibitory activity in dihydroxylated 2,4,6-triphenyl pyridines.

R. Karki et al. / European Journal of Medicinal Chemistry 49 (2012) 219e228
225
mobile phase A was double distilled water with 20 mM ammonium
formate (AF) and B was 90% ACN in water with 20 mM AF. In case of
compounds 16 and 20 gradient elutions of 85%e100% of B in A for
10 min followed by 100%e85% of B in A for 20 min was performed
with the same mobile phase. Purity of compound is described as
percent (%).
1-phenyl H-4), 7.34e7.26 (m, 2H, 3-phenyl H-5, H-6), 7.24 (s, 1H,
3-phenyl H-2), 7.01e6.95 (m, 2H, 1-phenyl H-3, H-5), 6.90 (dt,
J ¼ 7.6, 1.1 Hz, 1H, 3-phenyl H-4). 13C NMR (62.5 MHz, DMSO-d6)
d
193.76, 161.94, 157.96, 145.21, 136.49, 135.93, 131.10, 130.16, 121.92,
121.07, 120.39, 119.41, 118.38, 117.91, 115.69.
ESI LC/MS analyses were performed with a Finnigan LCQ
5.1.3. 1-(2-Hydroxyphenyl)-3-(4-hydroxyphenyl)propenone (4c)
The procedure described in Section 5.1 was employed with aryl
ketone 1 (R1 ¼ a) (2.40 mL, 20.00 mmol) and aryl aldehyde 2
(R2 ¼ c) (2.44 g, 20.00 mmol) to yield a yellow solid (1.91 g, 39.8%,
7.9 mmol). Mp 163e164 ꢀC; Rf (ethyl acetate/n-hexane 1:1 v/v):
AdvantageÒ LC/MS/MS spectrometry utilizing XcaliburÒ program.
For ESI LC/MS, LC was performed with a 2 mL injection volume on
Waters AtlantisÒ T3 reverse-phase C18 column (2.1 ꢂ 50 mm, 3
mm).
The mobile phase consisted of 100% distilled water (A) and 100%
ACN with 20 mM AF (B). A gradient program was used with a flow
0.43. Purity by HPLC: 95%. 1H NMR (250 MHz, DMSO-d6)
d 12.81 (br,
rate of 200
m
L/min. The initial composition was 10% B and
1H, 1-phenyl 20-OH), 10.27 (br, 1H, 3-phenyl 4-OH), 8.26 (d,
J ¼ 7.2 Hz, 1H, 1-phenyl H-6), 7.89 (d, J ¼ 15.7 Hz, 1H, COeCH]CH),
7.83 (d, J ¼ 16.4 Hz, 1H, COeCH]CH), 7.79 (d, J ¼ 8.6 Hz, 2H,
3-phenyl H-2, H-6), 7.54 (td, J ¼ 8.1, 1.0 Hz, 1H, 1-phenyl H-4),
7.01e6.95 (m, 2H, 1-phenyl H-3, H-5), 6.86 (d, J ¼ 8.4 Hz, 2H,
programmed linearly to 90% B after 5 min and finally 10% B after
15 min. MS ionization conditions were: Sheath gas flow rate:
70 arb, aux gas flow rate: 20 arb, I spray voltage: 4.5 kV, capillary
temperature 215 ꢀC, column temperature 40 ꢀC, capillary voltage:
21 V, tube lens offset: 10 V. Retention time was given in minutes.
3-phenyl H-3, H-5). 13C NMR (62.5 MHz, DMSO-d6)
d 193.81, 162.35,
160.89, 145.95, 136.40, 131.82, 130.91, 125.80, 120.75, 119.31, 117.98,
117.86, 116.16.
5.1. General method for the preparation of 3 and 4
Compounds 3 and 4 were synthesized either by KOH/NaOH or
BF3eEt2O catalyzed ClaiseneSchmidt condensation reaction.
KOH or NaOH catalyzed: To a solution of equimolar amounts of
aryl ketone 1 (R1 ¼ aec) and aryl aldehyde 2 (R2 ¼ aed) in EtOH was
added either 50% aqueous solution of KOH or 6 M aqueous NaOH
solution and stirred for 2e24 h at 20 ꢀC. The mixture was
neutralized with 6 M aqueous HCl solution (pH adjusted to 2).
The mixture was extracted with ethyl acetate, and washed with
water and brine. It was further purified by either recrystallization
or column chromatography to yield pure solid compounds.
BF3eEt2O catalyzed: To a solution of aryl ketone 1 (R1 ¼ c) and
aryl aldehyde 2 (R2 ¼ c) in little dioxane was added BF3eEt2O
gradually at 20 ꢀC and stirred for 2 h. The mixture was then
extracted with ethyl acetate and washed with water and brine. It
was further purified by column chromatography to yield pure solid
compound.
5.1.4. 3-(2-Hydroxyphenyl)-1-(3-hydroxyphenyl)propenone (4d)
The procedure described in Section 5.1 was employed with aryl
ketone 1 (R1 ¼ b) (4.08 g, 30.00 mmol) and aryl aldehyde 2 (R2 ¼ a)
(3.13 mL, 30.00 mmol) to yield a green solid (6.35 g, 88.2%,
26.4 mmol). Mp 173e174 ꢀC; Rf (ethyl acetate/n-Hexane 1:1 v/v):
0.47. Purity by HPLC: 100%.1H NMR (250 MHz, DMSO-d6)
d 10.00 (br,
2H, 1-phenyl 30-OH, 3-phenyl 2-OH), 8.02 (d, J ¼ 15.8 Hz, 1H,
COeCH]CH), 7.83 (dd, J ¼ 8.1, 1.3 Hz, 1H, 3-phenyl H-6), 7.79
(d, J ¼ 15.8 Hz, 1H, COeCH]CH), 7.55 (d, J ¼ 7.7 Hz, 1H, 1-phenyl
H-6), 7.40 (t, J ¼ 2.2 Hz, 1H, 1-phenyl H-2), 7.35 (t, J ¼ 7.9 Hz, 1H,
1-phenyl H-5), 7.26 (td, J ¼ 8.5, 1.5 Hz, 1H, 3-phenyl H-4), 7.05 (ddd,
J ¼ 8.0, 2.4, 0.7 Hz, 1H, 1-phenyl H-4), 6.94 (dd, J ¼ 8.2, 0.8 Hz, 1H,
3-phenyl H-3), 6.85 (t, J ¼ 7.7 Hz, 1H, 3-phenyl H-5). 13C NMR
(62.5 MHz, DMSO-d6) d 189.66,157.91,157.46,139.68,139.58,132.23,
130.08, 129.10, 121.58, 121.38, 120.26, 119.66, 119.56, 116.44, 114.73.
Synthetic method of compounds 3 (R1 ¼ aec, R2 ¼ d) was
described previously [22].
5.1.5. 1,3-Bis(3-hydroxyphenyl)propenone (4e)
The procedure described in Section 5.1 was employed with aryl
ketone 1 (R1 ¼ b) (4.08 g, 30.00 mmol) and aryl aldehyde 2 (R2 ¼ b)
(3.66 g, 30.00 mmol) to yield a yellow solid compound (5.90 g,
82.0%, 24.5 mmol). Mp 226e227 ꢀC; Rf (ethyl acetate/n-hexane
1:1 v/v): 0.43. Purity by HPLC: 100%. 1H NMR (250 MHz, DMSO-d6)
5.1.1. 1,3-Bis(2-hydroxyphenyl)propenone (4a)
The procedure described in Section 5.1 was employed with aryl
ketone 1 (R1 ¼ a) (1.80 mL,15.00 mmol) and aryl aldehyde 2 (R2 ¼ a)
(1.57 mL, 15.00 mmol) to yield a yellow solid compound (2.98 g,
82.8%,12.4 mmol). Mp 173e174 ꢀC; Rf (ethyl acetate/n-hexane 1:2 v/
d
9.74 (br, 2H, 1-phenyl 30-OH, 3-phenyl 3-OH), 7.77 (d, J ¼ 15.6 Hz,
1H, COeCH]CH), 7.63 (d, J ¼ 15.4 Hz, 1H, COeCH]CH), 7.62
(d, J ¼ 7.6 Hz, 1H, 1-phenyl H-6), 7.43 (s, 1H, 1-phenyl H-2), 7.35
(t, J ¼ 7.9 Hz, 1H, 1-phenyl H-5), 7.30e7.23 (m, 2H, 3-phenyl H-5,
H-6), 7.19 (s, 1H, 3-phenyl H-2), 7.06 (dd, J ¼ 8.0, 2.3 Hz, 1H,
1-phenyl H-4), 6.87 (d, J ¼ 7.4 Hz, 1H, 1-phenyl H-4). 13C NMR
v): 0.46. Purity by HPLC: 100%. 1H NMR (250 MHz, CDCl3)
d 12.93 (s,
1H,1-phenyl 20-OH), 8.24 (d, J ¼ 15.6 Hz,1H, COeCH]CH), 7.96 (dd,
J ¼ 8.1, 1.3 Hz, 1H, 1-phenyl H-6), 7.89 (d, J ¼ 15.6 Hz, 1H, COeCH]
CH), 7.64 (dd, J ¼ 7.7,1.3 Hz,1H, 3-phenyl H-6), 7.50 (td, J ¼ 8.8,1.4 Hz,
1H, 1-phenyl H-4), 7.30 (td, J ¼ 8.8, 1.5 Hz, 3-phenyl H-4), 7.05 (dd,
J ¼ 8.4, 0.8 Hz, 1H, 1-phenyl H-3), 7.00 (t, J ¼ 7.3 Hz, 1H, 1-phenyl H-
5), 6.95 (td, J ¼ 8.1, 1.0 Hz, 3-phenyl H-5), 6.87 (d, J ¼ 8.0 Hz, 1H,
3-phenyl H-3), 5.73 (s, 1H, 3-phenyl 2-OH). 13C NMR (62.5 MHz,
(62.5 MHz, DMSO-d6) d 189.35,157.97,144.34, 139.24,136.18,130.15,
122.24, 120.55, 120.09, 119.83, 118.04, 115.46, 114.81.
5.1.6. 1-(3-Hydroxyphenyl)-3-(4-hydroxyphenyl)propenone (4f)
The procedure described in Section 5.1 was employed with aryl
ketone 1 (R1 ¼ b) (2.72 g, 20.00 mmol) and aryl aldehyde 2 (R2 ¼ c)
(2.44 g, 20.00 mmol) to yield a yellow solid (2.10 g, 43.8%,
8.7 mmol). Mp 245e246 ꢀC; Rf (ethyl acetate/n-Hexane 1:1 v/v):
CDCl3) d 194.47, 163.52, 155.51, 141.00, 136.31, 132.05, 130.14,129.82,
122.03, 121.26, 121.14, 120.14, 118.85, 118.55, 116.51.
5.1.2. 1-(2-Hydroxyphenyl)-3-(3-hydroxyphenyl)propenone (4b)
The procedure described in Section 5.1 was employed with aryl
ketone 1 (R1 ¼ a) (3.61 mL, 30.00 mmol) and aryl aldehyde 2
(R2 ¼ b) (3.66 g, 30.00 mmol) to yield a yellow solid (6.49 g, 90.2%,
27.0 mmol). Mp 180e181 ꢀC; Rf (ethyl acetate/n-hexane 1:2 v/v):
0.42. Purity by HPLC: 99.6%. 1H NMR (250 MHz, DMSO-d6)
d 9.94
(br, 2H, 1-phenyl 30-OH, 3-phenyl 4-OH), 7.73 (d, J ¼ 8.5 Hz, 2H,
3-phenyl H-2, H-6), 7.63 (d, J ¼ 17.5 Hz, 2H, COeCH]CH), 7.59
(d, J ¼ 7.7 Hz, 1H, 1-phenyl H-6), 7.41 (s, 1H, 1-phenyl H-2), 7.34
(t, J ¼ 7.9 Hz, 1-phenyl H-5), 7.04 (dd, J ¼ 8.0, 2.3 Hz, 1H, 1-phenyl
H-4), 6.83 (d, J ¼ 8.4 Hz, 2H, 3-phenyl H-3, H-5). 13C NMR
0.38. Purity by HPLC: 100%. 1H NMR (250 MHz, DMSO-d6)
d 12.44 (s,
1H, 1-phenyl 20-OH), 9.65 (s, 1H, 3-phenyl 3-OH), 8.23 (dd, J ¼ 8.4,
1.5 Hz,1H,1-phenyl H-6), 7.96 (d, J ¼ 15.5 Hz,1H, COeCH]CH), 7.76
(d, J ¼ 15.5 Hz, 1H, COeCH]CH), 7.55 (td, J ¼ 8.4, 1.3 Hz, 1H,
(62.5 MHz, DMSO-d6)
d 189.14,160.36,157.90,144.57,139.63,131.23,
130.01, 125.99, 120.18, 119.61, 118.82, 116.05, 114.72.

226
R. Karki et al. / European Journal of Medicinal Chemistry 49 (2012) 219e228
5.1.7. 3-(2-Hydroxyphenyl)-1-(4-hydroxyphenyl)propenone (4g)
The procedure described in Section 5.1 was employed with aryl
ketone 1 (R1 ¼ c) (2.72 g, 20.00 mmol) and aryl aldehyde 2 (R2 ¼ a)
(2.09 mL, 20.00 mmol) to yield a red solid (1.74 g, 36.4%, 7.3 mmol).
Mp 197e198 ꢀC; Rf (ethyl acetate/n-hexane 1:1 v/v): 0.33. Purity by
2,4,6-triphenyl pyridine compounds were synthesized by this
method.
5.3.1. 2-(6-(3-Hydroxyphenyl)-4-phenylpyridin-2-yl)phenol (11)
The same procedure described in Section 5.3 was employed
with 3a (0.33 g, 1.50 mmol), dry ammonium acetate (1.15 g,
15.00 mmol), 5 (R3 ¼ b) (0.51 g, 1.50 mmol) and AcOH (2 mL) to
yield a yellow solid (0.46 g, 90.5%, 1.35 mmol). Rf (ethyl acetate/n-
hexane 1:3 v/v): 0.34. LC/MS/MS: retention time: 8.95 min, [MH]þ:
HPLC: 96.9%. 1H NMR (250 MHz, DMSO-d6)
d 10.32 (br, 2H, 1-phenyl
40-OH, 3-phenyl 2-OH), 8.02 (d, J ¼ 8.7 Hz, 2H, 1-phenyl H-2, H-6),
8.01 (d, J ¼ 15.9 Hz, 1H, COeCH]CH), 7.84 (d, J ¼ 15.8 Hz, 1H,
COeCH]CH), 7.83 (d, J ¼ 8.8 Hz, 1H, 3-phenyl H-6), 7.24 (td, J ¼ 8.4,
1.4 Hz, 1H, 3-phenyl H-4), 6.92 (d, J ¼ 7.5 Hz, 1H, 3-phenyl H-3),
6.89e6.81 (m, 3H, 3-phenyl H-5, 1-phenyl H-3, H-5). 13C NMR
340.36. 1H NMR (250 MHz, DMSO-d6)
d 14.43 (s, 1 H, 2-phenyl
2-OH), 9.77 (br, 1 H, 6-phenyl 3-OH), 8.40 (s, 1 H, pyridine H-3),
8.29 (d, J ¼ 7.24 Hz, 1 H, 2-phenyl H-6), 8.10 (s, 1 H, pyridine H-5),
8.07e8.03 (m, 2 H, 4-phenyl H-2, H-6), 7.61e7.52 (m, 4 H, 4-phenyl
H-3, H-4, H-5, 6-phenyl H-6), 7.48 (t, J ¼ 1.76 Hz, 1 H, 6-phenyl H-2),
7.38 (t, J ¼ 7.80 Hz, 1 H, 2-phenyl H-4), 7.35 (t, J ¼ 7.70 Hz, 1 H,
6-phenyl H-5), 6.99e6.91 (m, 3 H, 2-phenyl H-3, H-5, 6-phenyl
(62.5 MHz, DMSO-d6) d 187.62, 162.16, 157.20, 138.37, 131.85, 131.14,
129.58, 128.70, 121.79, 121.12, 119.57, 116.36, 115.54.
5.1.8. 3-(3-Hydroxyphenyl)-1-(4-hydroxyphenyl)propenone (4h)
The procedure described in Section 5.1 was employed with aryl
ketone 1 (R1 ¼ c) (2.72 g, 20.00 mmol) and aryl aldehyde 2 (R2 ¼ b)
(2.44 g, 20.00 mmol) to yield a light yellow solid (3.95 g, 82.3%,
16.4 mmol). Mp 261e262 ꢀC; Rf (ethyl acetate/n-hexane 1:1 v/v):
H-4). 13C NMR (62.5 MHz, DMSO-d6)
d 159.38, 158.28, 157.53,
154.84, 150.74, 139.19, 137.42, 131.74, 130.42, 129.86, 129.31, 127.91,
127.75, 119.29, 119.12, 117.98, 117.86, 117.19, 117.05, 116.32, 113.66.
0.47. Purity by HPLC: 95.3%. 1H NMR (250 MHz, DMSO-d6)
d 10.49
(br, 1H, 1-phenyl 40-OH) 9.64 (br, 1H, 3-phenyl 3-OH), 8.06 (d,
J ¼ 8.7 Hz, 2H, 1-phenyl H-2, H-6), 7.83 (d, J ¼ 15.6 Hz, 1H, COeCH]
CH), 7.59 (d, J ¼ 15.5 Hz, 1H, COeCH]CH), 7.29e7.23 (m, 2H, 3-
phenyl H-5, H-6), 7.18 (s, 1H, 3-phenyl H-2), 6.89 (d, J ¼ 8.7 Hz,
2H, 1-phenyl H-3, H-5), 6.85e6.82 (m, 1H, 3-phenyl H-4). 13C NMR
5.3.2. 2-(6-(4-Hydroxyphenyl)-4-phenylpyridin-2-yl)phenol (12)
The same procedure described in Section 5.3 was employed
with 3a (0.44 g, 2.00 mmol), dry ammonium acetate (1.54 g,
20.00 mmol), 5 (R3 ¼ c) (0.68 g, 2.00 mmol) and AcOH (2 mL) to
yield a yellow solid (0.21 g, 31.5%, 0.62 mmol). Rf (ethyl acetate/n-
hexane 1:2 v/v): 0.42. LC/MS/MS: retention time: 8.84 min, [MH]þ:
(62.5 MHz, DMSO-d6)
d 187.36, 162.43, 157.94, 143.22, 136.40,
131.43, 130.10, 129.33, 122.13, 119.91, 117.74, 115.62, 115.37.
340.35. 1H NMR (250 MHz, DMSO-d6)
d 14.71 (s, 1 H, 2-phenyl
2-OH), 9.97 (s, 1 H, 6-phenyl 4-OH), 8.32 (s, 1 H, pyridine H-3),
8.29 (d, J ¼ 7.39 Hz, 1 H, 2-phenyl H-6), 8.09 (s, 1 H, pyridine H-5),
8.07e8.04 (m, 2 H, 4-phenyl H-2, H-6), 7.98 (d, J ¼ 8.51 Hz, 2 H,
6-phenyl H-2, H-6), 7.58e7.55 (m, 3 H, 4-phenyl H-3, H-4, H-5), 7.34
(t, J ¼ 7.73 Hz, 1 H, 2-phenyl H-4), 6.97e6.94 (m, 4 H, 2-phenyl H-3,
5.1.9. 1,3-Bis(4-hydroxyphenyl)propenone (4i)
To a solution of aryl ketone 1 (R1 ¼ c) (2.72 g, 20.00 mmol) and
aryl aldehyde 2 (R2 ¼ c) (3.66 g, 30.00 mmol) in dioxane (4 mL) was
added BF3eEt2O (1.25 mL, 10.00 mmol) gradually at 20 ꢀC and
stirred for 2 h. The mixture was then extracted with ethyl acetate
and purified by column chromatography to yield a yellow solid
(4.41 g, 74.4%, 18.3 mmol). Mp 226e227 ꢀC; Rf (ethyl acetate:
n-hexane 1:1 v/v): 0.35. Purity by HPLC: 99.4%. 1H NMR (250 MHz,
H-5, 6-phenyl H-3, H-5). 13C NMR (62.5 MHz, DMSO-d6)
d 159.54,
159.44, 157.39, 154.75, 150.66, 137.59, 131.69, 129.84, 129.32, 128.52,
127.83, 127.78, 119.24, 119.06, 118.01, 116.16, 115.18.
DMSO-d6)
d
10.21 (br, 2H, 1-phenyl 40-OH, 3-phenyl 4-OH), 8.04 (d,
5.3.3. 2-(6-(4-Hydroxyphenyl)-4-phenylpyridin-2-yl)phenol (13)
The same procedure described in Section 5.3 was employed
with 3b (0.33 g, 1.50 mmol), dry ammonium acetate (1.15 g,
15.00 mmol), 5 (R3 ¼ c) (0.51 g, 1.50 mmol) and AcOH (2 mL) to
yield a light yellow solid (0.31 g, 61.7%, 0.92 mmol). Rf (ethyl
acetate/n-hexane 1:2 v/v): 0.22. LC/MS/MS: retention time:
J ¼ 8.7 Hz, 2H, 1-phenyl H-2, H-6), 7.71 (d, J ¼ 8.6 Hz, 2H, 3-phenyl
H-2, H-6), 7.68 (d, J ¼ 14.3 Hz, 1H, COeCH]CH), 7.62 (d, J ¼ 15.5 Hz,
1H, COeCH]CH), 6.88 (d, J ¼ 8.7 Hz, 2H, 1-phenyl H-3, H-5), 6.83
(d, J ¼ 8.5 Hz, 2H, 3-phenyl H-3, H-5). 13C NMR (62.5 MHz,
DMSO-d6)
d 187.25, 162.13, 160.04, 143.39, 131.16, 130.96, 129.64,
126.20, 118.73, 115.99, 115.51.
7.93 min, [MH]þ: 340.35. 1H NMR (250 MHz, DMSO-d6)
d 9.73
(br, 1 H, 2-phenyl 3-OH), 9.57 (br, 1 H, 6-phenyl 4-OH), 8.18 (d,
5.2. General method for the preparation of 5
J ¼ 8.61 Hz, 2 H, 6-phenyl H-2, H-6), 8.03 (s, 1 H, pyridine H-3),
7.99e7.95 (m,
3 H, 4-phenyl H-2, H-6, pyridine H-5), 7.73
A mixture of aryl ketone (R3 ¼ aed), iodine (1.2 equiv) and
pyridine was refluxed at 140 ꢀC for 3 h. Precipitate occurred during
reaction which was cooled to room temperature. Then it was
filtered and washed with cold pyridine to afford 5 (R3 ¼ aed) in
quantitative yield. Four different pyridinium iodide salts were
synthesized by this method.
(t, J ¼ 1.57 Hz, 1 H, 2-phenyl H-2), 7.70 (d, J ¼ 7.8 Hz, 1 H, 2-phenyl
H-6), 7.58e7.46 (m, 3 H, 4-phenyl H-3, H-4, H-5), 7.31 (t, J ¼ 7.87 Hz,
1 H, 2-phenyl H-5), 6.93 (d, J ¼ 8.61 Hz, 2 H, 6-phenyl H-3, H-5),
6.88 (dd, J ¼ 8.12, 2.44 Hz, 1 H, 2-phenyl H-4). 13C NMR (62.5 MHz,
DMSO-d6)
d 158.95, 157.98, 156.71, 156.47, 149.50, 140.61, 138.22,
129.99, 129.95, 129.42, 129.34, 128.59, 127.50, 117.90, 116.39, 115.72,
115.67, 115.64, 113.91.
5.3. General method for the preparation of 6 and 7
5.3.4. 2,20-(6-Phenylpyridine-2,4-diyl)diphenol (14)
A mixture of propenone intermediate 3 (R1 ¼ aec, R2 ¼ d) or 4
(R1, R2 ¼ aec), pyridinium iodide salt 5 (R3 ¼ aed) and anhydrous
ammonium acetate in glacial acetic acid were heated at 90e100 ꢀC
for 12e24 h. The reaction mixture was then extracted with ethyl
acetate, washed with water and brine. The organic layer was dried
with magnesium sulfate and filtered. The filtrate was evaporated at
reduced pressure, which was then purified by silica gel column
chromatography with the gradient elution of ethyl acetate/
n-hexane to afford solid compounds 6 (R1, R3 ¼ aec, R2 ¼ d) and
7 (R1, R2 ¼ aec, R3 ¼ d) in 20e97% yield. Twelve dihydroxylated
The same procedure described in Section 5.3 was employed
with 4a (0.48 g, 2.00 mmol), dry ammonium acetate (1.54 g,
20.00 mmol), 5 (R3 ¼ d) (0.65 g, 2.00 mmol) and AcOH (2 mL) to
yield a greenish yellow solid (0.16 g, 24.5%, 0.48 mmol). Rf (ethyl
acetate/n-hexane 1:5 v/v): 0.28. LC/MS/MS: retention time:
9.19 min, [MH]þ: 340.27. 1H NMR (250 MHz, DMSO-d6)
d 14.47
(s, 1H, 2-phenyl 2-OH), 10.06 (br, 1H, 4-phenyl 2-OH), 8.31 (s, 1H,
pyridine H-5), 8.13e8.08 (m, 2H, 2-phenyl H-6, pyridine H-3), 8.04
(d, J ¼ 7.5 Hz, 2H, 6-phenyl H-2, H-6), 7.62e7.53 (m, 4H, 4-phenyl
H-6, 6-phenyl H-3, H-4, H-5), 7.37e7.28 (m, 2H, 2-phenyl H-4,

R. Karki et al. / European Journal of Medicinal Chemistry 49 (2012) 219e228
227
4-phenyl H-4), 7.06e6.92 (m, 4H, 2-phenyl H-3, H-5, 4-phenyl H-3,
H-5). 13C NMR (62.5 MHz, DMSO-d6)
159.35,156.82,155.14,153.81,
149.76, 138.06, 131.65, 130.84, 130.80, 129.93, 129.51, 127.59, 126.89,
125.01, 119.94, 119.92, 119.40, 119.25, 118.90, 118.07, 116.61.
7.57e7.47 (m, 3H, 6-phenyl H-3, H-4, H-5), 7.37 (t, J ¼ 7.8 Hz, 1H,
2-phenyl H-5), 7.34e7.29 (m, 3H, 4-phenyl H-5, H-6, H-2), 6.88 (td,
J ¼ 8.0, 2.0 Hz, 2H, 2-phenyl H-4, 4-phenyl H-4). 13C NMR
d
(62.5 MHz, DMSO-d6)
d 158.25, 158.03, 156.67, 156.55, 149.94,
140.39, 139.46, 139.05, 130.43, 130.03, 129.48, 129.01, 127.15, 118.27,
117.91, 116.81, 116.75, 116.50, 116.46, 114.26, 113.90.
5.3.5. 2-[4-(3-Hydroxyphenyl)-6-phenylpyridin-2-yl]phenol (15)
The same procedure described in Section 5.3 was employed with
4b (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol),
5 (R3 ¼ d) (0.48 g,1.50 mmol) and AcOH (2 mL) to yield a yellow solid
(0.50 g, 97.2%, 1.45 mmol). Rf (ethyl acetate/n-hexane 1:2 v/v): 0.40.
LC/MS/MS: retention time: 8.94 min, [MH]þ: 340.27. 1H NMR
5.3.9. 3-[4-(4-Hydroxyphenyl)-6-phenylpyridin-2-yl]phenol (19)
The same procedure described in Section 5.3 was employed
with 4f (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g,
15.00 mmol), 5 (R3 ¼ d) (0.48 g, 1.50 mmol) and AcOH (2 mL) to
yield a white solid (0.43 g, 84.3%, 1.26 mmol). Rf (ethyl acetate/
n-hexane 1:1 v/v): 0.52. LC/MS/MS: retention time: 7.64 min,
(250 MHz, DMSO-d6)
d 14.38 (s, 1H, 2-phenyl 2-OH), 9.73 (s, 1H,
4-phenyl 3-OH), 8.34 (s, 1H, pyridine H-5), 8.29 (d, J ¼ 7.9 Hz, 1H,
2-phenyl H-6), 8.11 (s, 1H, pyridine H-3), 8.11e8.08 (m, 2H, 6-phenyl
H-2, H-6), 7.62e7.50 (m, 3H, 6-phenyl H-3, H-4, H-5), 7.48
(t, J ¼ 7.6 Hz, 1H, 2-phenyl H-4), 7.40e7.31 (m, 3H, 4-phenyl H-2, H-5,
H-6), 6.98e6.93 (m, 3H, 4-phenyl H-4, 2-phenyl H-3, H-5). 13C NMR
(62.5 MHz, DMSO-d6) d 159.34, 158.23, 157.55, 154.67, 151.02, 138.90,
137.84, 131.75, 130.39, 130.01, 129.41, 127.96, 127.01, 119.37, 119.17,
118.50, 117.99, 117.26, 116.81, 116.43, 114.53.
[MH]þ: 340.26. 1H NMR (250 MHz, DMSO-d6)
d 9.73 (br, 2H,
2-phenyl 3-OH, 4-phenyl 4-OH), 8.30e8.27 (m, 2H, 6-phenyl H-2,
H-6), 8.09 (s, 1H, pyridine H-5), 8.02 (s, 1H, pyridine H-3), 7.91 (d,
J ¼ 8.6 Hz, 2H, 4-phenyl H-2, H-6), 7.73 (d, J ¼ 1.77 Hz, 1H, 2-phenyl
H-2), 7.71 (d, J ¼ 8.1 Hz, 1H, 2-phenyl H-6), 7.56e7.43 (m, 3H,
6-phenyl H-3, H-4, H-5), 7.31 (t, J ¼ 7.8 Hz, 1H, 2-phenyl H-5), 6.94
(d, J ¼ 8.5 Hz, 2H, 4-phenyl H-3, H-5), 6.87 (dd, J ¼ 8.0, 2.1 Hz, 1H,
2-phenyl H-4). 13C NMR (62.5 MHz, DMSO-d6)
d 159.02, 157.98,
5.3.6. 2-[4-(4-Hydroxyphenyl)-6-phenylpyridin-2-yl]phenol (16)
The same procedure described in Section 5.3 was employed
with 4c (0.38 g, 1.60 mmol), dry ammonium acetate (1.23 g,
16.00 mmol), 5 (R3 ¼ d) (0.52 g, 1.60 mmol) and AcOH (2 mL) to
yield a yellow solid (0.50 g, 90.9%, 1.45 mmol). Rf (ethyl acetate/
n-hexane 1:2 v/v): 0.38. LC/MS/MS: retention time: 8.83 min,
156.57, 156.42, 149.49, 140.59, 139.23, 129.94, 129.35, 128.94,
128.86, 128.38, 127.10, 117.88, 116.37, 116.09, 115.88, 115.85, 113.90.
5.3.10. 2-[2-(4-Hydroxyphenyl)-6-phenylpyridin-4-yl]phenol (20)
The same procedure described in Section 5.3 was employed
with 4g (0.40 g, 1.68 mmol), dry ammonium acetate (1.29 g,
16.80 mmol), 5 (R3 ¼ d) (0.54 g, 1.68 mmol) and AcOH (2 mL) to
yield a greenish yellow solid (0.11 g, 19.8%, 0.33 mmol). Rf (ethyl
acetate/n-hexane 1:2 v/v): 0.25. LC/MS/MS: retention time:
[MH]þ: 340.27. 1H NMR (250 MHz, DMSO-d6)
d 14.63 (s, 1H,
2-phenyl 2-OH), 9.95 (s, 1H, 4-phenyl 4-OH), 8.35 (s, 1H, pyridine
H-5), 8.30 (d, J ¼ 7.2 Hz, 1H, 2-phenyl H-6), 8.12 (s, 1H, pyridine
H-3), 8.10e8.07 (m, 2H, 6-phenyl H-2, H-6), 7.99 (d, J ¼ 8.6 Hz, 2H,
4-phenyl H-2, H-6), 7.62e7.53 (m, 3H, 6-phenyl H-3, H-4, H-5), 7.34
(t, J ¼ 7.2 Hz, 1H, 2-phenyl H-4), 6.98e6.92 (m, 4H, 2-phenyl H-3,
7.89 min, [MH]þ: 340.26. 1H NMR (250 MHz, DMSO-d6)
d 9.82 (br,
2H, 2-phenyl 4-OH, 4-phenyl 2-OH), 8.21e8.18 (m, 2H, 6-phenyl
H-2, H-6), 8.09 (d, J ¼ 8.6 Hz, 2H, 2-phenyl H-2, H-6), 7.94 (s, 1H,
pyridine H-5), 7.93 (s, 1H, pyridine H-3), 7.54e7.41 (m, 4H, 6-phenyl
H-3, H-4, H-5, 4-phenyl H-6), 7.27 (td, J ¼ 8.1, 1.3 Hz, 1H, 4-phenyl
H-4), 7.03 (d, J ¼ 8.0 Hz, 1H, 4-phenyl H-3), 6.98 (t, J ¼ 7.7 Hz, 1H,
4-phenyl H-5), 6.91 (d, J ¼ 8.6 Hz, 2H, 2-phenyl H-3, H-5). 13C NMR
H-5, 4-phenyl H-3, H-5). 13C NMR (62.5 MHz, DMSO-d6)
d 159.51,
159.44, 157.50, 154.48, 150.61, 137.97, 131.69, 129.96, 129.40, 129.24,
127.84, 127.73, 126.99, 119.28, 119.08, 118.01, 116.32, 116.10, 115.15.
5.3.7. 2-[2-(3-Hydroxyphenyl)-6-phenylpyridin-4-yl]phenol (17)
The same procedure described in Section 5.3 was employed
with 4d (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g,
15.00 mmol), 5 (R3 ¼ d) (0.48 g, 1.50 mmol) and AcOH (2 mL) to
yield a white solid (0.25 g, 49.8%, 0.74 mmol). Rf (ethyl acetate/n-
hexane 1:1 v/v): 0.57. LC/MS/MS: retention time: 7.97 min, [MH]þ:
(62.5 MHz, DMSO-d6) d 158.77, 155.97, 155.54, 155.00, 148.28,
139.45, 130.63, 130.20, 130.17, 129.15, 128.96, 128.38, 126.93, 125.73,
119.87, 118.30, 118.25, 116.52, 115.74.
5.3.11. 3-[2-(4-Hydroxyphenyl)-6-phenylpyridin-4-yl]phenol (21)
The same procedure described in Section 5.3 was employed
with 4h (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g,
15.00 mmol), 5 (R3 ¼ d) (0.48 g, 1.50 mmol) and AcOH (2 mL) to
yield a white solid (0.44 g, 87.0%, 1.30 mmol). Rf (ethyl acetate/
n-hexane 1:1 v/v): 0.60. LC/MS/MS: retention time: 7.70 min,
340.26. 1H NMR (250 MHz, DMSO-d6)
d 9.86 (br, 1H, 4-phenyl
2-OH), 9.59 (br, 1H, 2-phenyl 3-OH), 8.23e8.20 (m, 2H, 6-phenyl
H-2, H-6), 8.03 (s, 1H, pyridine H-5), 7.98 (s, 1H, pyridine H-3),
7.67 (t, J ¼ 1.9 Hz,1H, 2-phenyl H-2), 7.61 (d, J ¼ 8.0 Hz,1H, 2-phenyl
H-6), 7.57e7.43 (m, 4H, 6-phenyl H-3, H-4, H.-5, 4-phenyl H-6), 7.34
(t, J ¼ 7.9 Hz, 1H, 2-phenyl H-5), 7.25 (t, J ¼ 7.8 Hz, 1H, 4-phenyl
H-4), 7.04 (d, J ¼ 7.7 Hz, 1H, 4-phenyl H-3), 6.96 (t, J ¼ 7.5 Hz, 1H,
4-phenyl H-5), 6.86 (dd, J ¼ 7.9, 2.0 Hz, 1H, 2-phenyl H-4). 13C NMR
[MH]þ: 340.25. 1H NMR (250 MHz, DMSO-d6)
d 9.74 (br, 2H,
2-phenyl 4-OH, 4-phenyl 3-OH), 8.30e8.26 (m, 2H, 6-phenyl H-2,
H-6), 8.18 (d, J ¼ 8.1 Hz, 2H, 2-phenyl H-2, H-6), 7.99 (s, 1H, pyridine
H-5), 7.97 (s, 1H, pyridine H-3), 7.55e7.41 (m, 3H, 6-phenyl H-3,
H-4, H-5), 7.38e7.34 (m, 2H, 4-phenyl H-5, H-6), 7.31 (s, 1H,
4-phenyl H-2), 6.92 (d, J ¼ 8.0 Hz, 3H, 2-phenyl H-3, H-5, 4-phenyl
(62.5 MHz, DMSO-d6)
d 157.93, 155.81, 155.75, 154.95, 148.32,
140.57, 139.25, 130.53, 130.23, 129.90, 129.16, 128.89, 126.88, 125.45,
119.85, 119.24, 119.20, 117.63, 116.53, 116.24, 113.70.
H-4). 13C NMR (62.5 MHz, DMSO-d6)
d 158.95, 158.20, 156.74,
156.35, 149.79, 139.66, 139.20, 130.36, 129.92, 129.34, 128.95,
128.58, 127.09, 118.22, 116.33, 115.72, 115.68, 115.61, 114.22.
5.3.8. 3,30-(6-Phenylpyridine-2,4-diyl)diphenol (18)
The same procedure described in Section 5.3 was employed
with 4e (0.36 g, 1.50 mmol), dry ammonium acetate (1.15 g,
15.00 mmol), 5 (R3 ¼ d) (0.48 g, 1.50 mmol) and AcOH (2 mL) to
yield a white solid (0.37 g, 72.5%, 1.08 mmol). Rf (ethyl acetate/
n-hexane 1:1 v/v): 0.52. LC/MS/MS: retention time: 7.80 min,
5.3.12. 4,40-(6-Phenylpyridine-2,4-diyl)diphenol (22)
The same procedure described in Section 5.3 was employed
with 4i (0.48 g, 2.00 mmol), dry ammonium acetate (1.54 g,
20.00 mmol), 5 (R3 ¼ d) (0.65 g, 2.00 mmol) and AcOH (2 mL) to
yield a yellow solid (0.32 g, 47.9%, 0.95 mmol). Rf (ethyl acetate/
n-hexane 1:2 v/v): 0.17. LC/MS/MS: retention time: 7.55 min,
[MH]þ: 340.26. 1H NMR (250 MHz, DMSO-d6)
d 9.66 (br, 2H,
2-phenyl 3-OH, 4-phenyl 3-OH), 8.31e8.28 (m, 2H, 6-phenyl H-2,
H-6), 8.08 (s, 1H, pyridine H-5), 8.00 (s, 1H, pyridine H-3), 7.73 (d,
J ¼ 1.7 Hz, 1H, 2-phenyl H-2), 7.71 (d, J ¼ 8.0 Hz, 1H, 2-phenyl H-6),
[MH]þ: 340.26. 1H NMR (250 MHz, DMSO-d6)
d
9.82 (br, 2H,
2-phenyl 4-OH, 4-phenyl 4-OH), 8.29e8.26 (m, 2H, 6-phenyl H-2,

228
R. Karki et al. / European Journal of Medicinal Chemistry 49 (2012) 219e228
H-6), 8.17 (d, J ¼ 8.7 Hz, 2H, 2-phenyl H-2, H-6), 8.00 (s, 1H, pyridine
H-5), 7.98 (s, 1H, pyridine H-3), 7.89 (d, J ¼ 8.6 Hz, 2H, 4-phenyl H-2,
H-6), 7.55e7.42 (m, 3H, 6-phenyl H-3, H-4, H-5), 6.93 (d, J ¼ 8.5 Hz,
2H, 2-phenyl H-3, H-5), 6.91 (d, J ¼ 8.6 Hz, 2H, 4-phenyl H-3, H-5).
Acknowledgments
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 (2009-
0066925, 2010-0002646, and 2011-0001170).
13C NMR (62.5 MHz, DMSO-d6)
d 158.91, 158.83, 156.62, 156.23,
149.31, 139.37, 130.11, 129.22, 128.89, 128.78, 128.55, 128.53, 127.05,
116.04, 115.65, 114.80, 114.70.
References
5.4. Pharmacology
[1] D.S.H.L. Kim, C.L. Ashendel, Q. Zhou, C.T. Chang, E.S. Lee, C.J. Chang, Bioorg.
Med. Chem. Lett. 8 (1998) 2695e2698.
[2] U.S. Schubert, C. Eschbaumer, Angew. Chem. Int. Ed. 41 (2002) 2892e2926.
[3] G. Lowe, A.S. Droz, T. Vilaivan, G.W. Weaver, J.J. Park, J.M. Pratt, L. Tweedale,
L.R. Kelland, J. Med. Chem. 42 (1999) 3167e3174.
[4] W.D. McFadyen, L.P.G. Wakelin, I.A.G. Ross, V.A. Leopold, J. Med. Chem. 28
(1985) 1113e1116.
[5] A. McCoubrey, H.C. Latham, P.R. Cook, A. Rodger, G. Lowe, FEBS Lett. 380
(1996) 73e78.
[6] Y.-C. Lo, T.-P. Ko, W.-C. Su, T.-L. Su, A.H.-J. Wang, J. Inorg. Biochem. 103 (2009)
1082e1092.
[7] L.X. Zhao, J. Sherchan, J.K. Park, Y. Jahng, B.S. Jeong, T.C. Jeong, C.S. Lee, E.-
S. Lee, Arch. Pharm. Res. 29 (2006) 1091e1095.
[8] J.C. Wang, Annu. Rev. Biochem. 65 (1996) 635e692.
[9] Y. Pommier, Nat. Rev. Cancer 6 (2006) 789e802.
5.4.1. Assay for DNA topoisomerase I inhibition in vitro
DNA topo I inhibition assay was determined following the
method reported by Fukuda et al. [38] with minor modifications. The
test compounds were dissolved in DMSO at 20 mM as stock solution.
The activity of DNAtopo I was determined byassessing therelaxation
of supercoiled DNA pBR322. The mixture of 100 ng of plasmid
pBR322 DNA and 1 unit 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
TriseHCl (pH 7.9), 150 mM NaCl, 0.1% bovine serum albumin, 1 mM
spermidine, 5% glycerol). The reaction in the final volume of 10
mL
[10] J.L. Nitiss, Nat. Rev. Cancer 9 (2009) 338e350.
[11] J.L. Nitiss, Nat. Rev. Cancer 9 (2009) 327e337.
[12] Y. Pommier, Chem. Rev. 109 (2009) 2894e2902.
was terminated by adding 2.5 L of the stop solution containing 5%
m
sarcosyl, 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 mg/mL). DNA bands were visual-
ized by transillumination with UV light and were quantitated using
[13] J.C. Wang, Nat. Rev. Mol. Cell Biol. 3 (2002) 430e440.
[14] L.X. Zhao, T.S. Kim, S.H. Ahn, T.H. Kim, E.K. Kim, W.J. Cho, H.S. Choi, C.S. Lee,
J.A. Kim, T.C. Jeong, C.-j. Chang, E.S. Lee, Bioorg. Med. Chem. Lett. 11 (2001)
2659e2662.
[15] L.X. Zhao, Y.S. Moon, A. Basnet, E.K. Kim, Y. Jahng, J.G. Park, T.C. Jeong,
W.J. Cho, S.U. Choi, C.O. Lee, S.Y. Lee, C.S. Lee, E.S. Lee, Bioorg. Med. Chem. Lett.
14 (2004) 1333e1337.
AlphaImagerÔ (Alpha Innotech Corporation).
[16] A. Basnet, P. Thapa, R. Karki, Y. Na, Y. Jahng, B.S. Jeong, T.C. Jeong, C.S. Lee,
E.S. Lee, Bioorg. Med. Chem. 15 (2007) 4351e4359.
5.4.2. Assay for DNA topoisomerase II inhibition in vitro
DNA topo II inhibitory activity of compounds were measured as
follows [39]. The mixture of 200 ng of supercoiled pBR322 plasmid
DNA and 1 unit of human DNA topoisomerase IIa (Usb Corp., USA)
was incubated without and with the prepared compounds in the
[17] 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.
[18] 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.
[19] A. Basnet, P. Thapa, R. Karki, H.Y. Choi, J.H. Choi, M. Yun, B.S. Jeong, Y. Jahng,
Y. Na, W.J. Cho, Y. Kwon, C.S. Lee, E.S. Lee, Bioorg. Med. Chem. Lett. 20 (2010)
42e47.
assay buffer (10 mM TriseHCl (pH 7.9) containing 50 mM NaCl,
[20] 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.
5 mM 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
mL
[21] 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.
[22] 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.
[23] R. Karki, P. Thapa, Y. Kwon, E.S. Lee, Bull. Korean Chem. Soc. 31 (2010)
1747e1750.
[24] K. Bisht, K.H. Wagner, A.C. Bulmer, Toxicology 278 (2010) 88e100.
[25] M. Jang, L. Cai, G.O. Udeani, K.V. Slowing, C.F. Thomas, C.W.W. Beecher,
H.H.S. Fong, N.R. Farnsworth, A.D. Kinghorn, R.G. Mehta, R.C. Moon,
J.M. Pezzuto, Science 275 (1997) 218e220.
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-
alized by transillumination with UV light and supercoiled DNA was
quantitated using AlphaImagerÔ (Alpha Innotech Corporation).
[26] O.J. Bandele, N. Osheroff, Biochemistry 46 (2007) 6097e6108.
[27] O.J. Bandele, S.J. Clawson, N. Osheroff, Chem. Res. Toxicol. 21 (2008)
1253e1260.
[28] O.J. Bandele, N. Osheroff, Chem. Res. Toxicol. 21 (2008) 936e943.
[29] Y. Jahng, L.X. Zhao, Y.S. Moon, A. Basnet, E.K. Kim, H.W. Chang, H.K. Ju,
T.C. Jeong, E.-S. Lee, Bioorg. Med. Chem. Lett. 14 (2004) 2559e2562.
[30] D. Batovska, S. Parushev, A. Slavova, V. Bankova, I. Tsvetkova, M. Ninova,
H. Najdenski, Eur. J. Med. Chem. 42 (2007) 87e92.
[31] D.S. Breslow, C.R. Hauser, J. Am. Chem. Soc. 62 (1940) 2385e2388.
[32] T. Narender, K.P. Reddy, Tetrahedron Lett. 48 (2007) 3177e3180.
[33] M.R. Jayapal, N.Y. Sreedhar, Int. J. Curr. Pharm. Res. 2 (2010) 60e62.
[34] F. Kröhnke, Synthesis (1976) 1e24.
5.4.3. Cytotoxicity assay
Cancer cells were cultured according to the supplier’s instruc-
tions. Cells were seeded in 96-well plates at
a density of
2e4 ꢂ 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, after FBS starvation for 4 h, culture
medium in each well was exchanged with 0.1 mL aliquots of
medium containing graded concentrations of compounds. On day
4, each well was added with 5 mL of the cell counting kit-8 solution
[35] F. Kröhnke, Angew. Chem. Int. Ed. 2 (1963) 380e393.
[36] H. Liang, X. Wu, J.C. Yalowich, B.B. Hasinoff, Mol. Pharmacol. 73 (2008)
686e696.
[37] J.F. Riou, P. Fosse, C.H. Nguyen, A.K. Larsen, M.C. Bissery, L. Grondard,
J.M. Saucier, E. Bisagni, F. Lavelle, Cancer Res. 53 (1993) 5987e5993.
[38] M. Fukuda, K. Nishio, F. Kanzawa, H. Ogasawara, T. Ishida, H. Arioka,
K. Bonjanowski, M. Oka, N. Saijo, Cancer Res. 56 (1996) 789e793.
[39] D.H. Kang, J.S. Kim, M.J. Jung, E.-S. Lee, Y. Jhang, Y. Kwon, Y. Na, Bioorg. Med.
Chem. Lett. 18 (2008) 1520e1524.
(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 wave-
length. For determination of the IC50 values, the absorbance read-
ings at 450 nm were fitted to the four-parameter logistic equation.
The compounds like adriamycin, etoposide, and camptothecin were
purchased from Sigma and used as positive controls.