Synthesis, topoisomerase I and II inhibitory activity, cytotoxicity, and structure-activity relationship study of hydroxylated 2,4-diphenyl-6-aryl pyridines

Article abstract of DOI:10.1016/j.bmc.2010.03.051

A new series of 2,4-diphenyl-6-aryl pyridines containing hydroxyl group(s) at the ortho, meta, or para position of the phenyl ring were synthesized, and evaluated for topoisomerase I and II inhibitory activity and cytotoxicity against several human cancer cell lines for the development of novel anticancer agents. Structure-activity relationship study revealed that the substitution of hydroxyl group(s) increased topoisomerase I and II inhibitory activity in the order of meta > para > ortho position. Substitution of hydroxyl group on the para position showed better cytotoxicity.

Full text of DOI:10.1016/j.bmc.2010.03.051

Bioorganic & Medicinal Chemistry 18 (2010) 3066–3077
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, topoisomerase I and II inhibitory activity, cytotoxicity, and
structure–activity relationship study of hydroxylated 2,4-diphenyl-6-aryl
pyridines
Radha Karki a, Pritam Thapa a, Mi Jeong Kang a, Tae Cheon Jeong a, Jung Min Nam b, Hye-Lin Kim b,
a,
Younghwa Na c, Won-Jea Cho d, Youngjoo Kwon b, , Eung-Seok Lee
*
*
a College of Pharmacy, Yeungnam University, Kyongsan 712-749, Republic of Korea
b College of Pharmacy, Pharmacy & Division of Life & Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Republic of Korea
c College of Pharmacy, Catholic University of Daegu, Kyongsan 712-702, Republic of Korea
d College of Pharmacy, Chonnam National University, Kwangju 500-757, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of 2,4-diphenyl-6-aryl pyridines containing hydroxyl group(s) at the ortho, meta, or para
position of the phenyl ring were synthesized, and evaluated for topoisomerase I and II inhibitory activity
and cytotoxicity against several human cancer cell lines for the development of novel anticancer agents.
Structure–activity relationship study revealed that the substitution of hydroxyl group(s) increased topo-
isomerase I and II inhibitory activity in the order of meta > para > ortho position. Substitution of hydroxyl
group on the para position showed better cytotoxicity.
Received 11 February 2010
Revised 17 March 2010
Accepted 18 March 2010
Available online 27 March 2010
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Hydroxylated 2,4-diphenyl-6-aryl pyridines
Topoisomerase I and II inhibitor
Cytotoxicity
Anticancer agents
1. Introduction
applications and biochemistry, a wide range of potential uses for
functionalized terpyridines have been found, ranging from colori-
DNA topoisomerases have been established as molecular tar-
gets of anticancer drugs.1,2 They are ubiquitous enzymes that solve
the topological problems associated with DNA replication, tran-
scription, recombination, and other nuclear processes by introduc-
ing temporary single- or double-strand breaks in the DNA. In
addition, these enzymes fine-tune the steady-state level of DNA
supercoiling both to facilitate protein interactions with the DNA
and to prevent excessive supercoiling that is deleterious.3,4 DNA
topoisomerases fall into two categories, topoisomerase I (topo I)
and topoisomerase II (topo II). For topo I, the DNA strands are tran-
siently broken one at a time. For topo II, a pair of strands in a DNA
double helix is transiently broken in concert by a dimeric enzyme
molecule.5 In the course of several years, different agents which are
able to inhibit the topo-DNA cleavable complex have been found
and developed.6
metric metal determination to DNA binding agents9,10 and antitu-
mor research.8 These numerous reports on DNA binding property
and antitumor activity of terpyridine complexes have attracted
many researchers. In addition,
of -terthiophenes, which possess protein kinase C (PKC) inhibi-
tory activities (Fig. 1).11
a-terpyridines are the bioisosteres
a
Our research group has previously reported that terpyridine
derivatives show strong cytotoxicity against several human cancer
cell lines and considerable topo I and II inhibitory activity.12–19
Among prepared compounds, methyl or chloro substituted terpyr-
idine derivatives have shown strong topoisomerase I and II inhib-
itory activities and cytotoxicity compared to the non-substituted
ones.17–19 These results suggested that the substitution of other
functional group on aromatic rings may increase topoisomerase I
and II inhibitory activities and cytotoxicity.
Terpyridines can act as tridentate ligand and form stable com-
plexes by chelating a broad variety of transition metal ions. Plati-
num(II) complexes of terpyridine derivatives have been well
studied and evaluated for their antitumor potential.7,8 In clinical
Many natural and synthetic derivatives bearing the hydroxyl
group or phenol have exhibited a wide spectrum of biological
activities with potential for application as pharmaceutical drugs.
Numerous polyphenolic phytochemicals such as resveratrol, cur-
cumin, flavonoids, epigallocatechin gallate, and chalcones have
been well studied and reported to interfere with specific stages
of the carcinogenic process.20–23 Camptothecin, a well-known topo
I inhibitor, also contains the hydroxyl group. Redinbo et al. and Fan
* Corresponding authors. Tel.: +82 2 3277 4653; fax: +82 2 3277 2851 (Y.K.); tel.:
+82 53 810 2827; fax: +82 53 810 4654 (E.-S.L.).
E-mail addresses: ykwon@ewha.ac.kr (Y. Kwon), eslee@yu.ac.kr (E.-S. Lee).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.051

R. Karki et al. / Bioorg. Med. Chem. 18 (2010) 3066–3077
3067
R2
R1 = H or OH
R2 = H or OH
4
N
2
6
S
N
R3
N
N
S
S
R1
α-terthiophene
α-terpyridine
2,4-diphenyl-6-aryl pyridine
R3 = phenyl
2/ 3 -thienyl
2 -furyl
2/ 3/ 4- pyridyl
o/ m/p- OH phenyl
Figure 1. Structures of a-terthiophene, a-terpyridine, and 2,4-diphenyl-6-aryl pyridine derivatives.
et al. have described the role of the 20-OH group of camptothecin
in the topo I-DNA binding models as they participate in a donor
hydrogen bond.24,25 These reports on the importance of the hydro-
xyl group motivated us to synthesize terpyridine compounds with
hydroxyl moiety.
From the results of the biological activity of the prepared com-
pounds, the structure–activity relationship was established. In
addition, three dihydroxylated terpyridine compounds ( 15, 23,
and 31) were synthesized and evaluated for biological activity to
observe the effect of increase in the number of hydroxyl groups.
In this study, we incorporated the hydroxyl group on phenyl
moiety to investigate the effect on topoisomerase I and II inhibitory
activities and cytotoxicity. We have systematically designed and
synthesized 45 2,4-diphenyl-6-aryl pyridine derivatives containing
hydroxyl groups at different positions (ortho, meta, or para) of the
phenyl ring attached to the central pyridine (Scheme 1 and Fig. 2).
They were then evaluated for topoisomerase I and II inhibitory
activity and cytotoxicity against several human cancer cell lines.
2. Results and discussion
2.1. Chemistry
Synthesis of 2,4-diphenyl-6-aryl pyridine derivatives 8–52 in-
volved three steps as summarized in Scheme 1. Six different
hydroxylated chalcones were synthesized as intermediates using
O
N
R3
I
5 (R3 = a-j)
HO
N
R3
ii
HO
O
O
3 (R1 = h-j, R2 = a)
R2
H
6 (R1 = h-j, R2 = a, R3 = a-j)
O
2 (R2 = a, h-j)
R1
1 (R1 = a, h-j)
CH3
i
O
HO
N
R3
I
OH
5 (R3 = a-g)
N
R3
ii
O
4 (R1 = a, R2 = h-j)
7 (R1 = a, R2 = h-j, R3 = a-g)
S
S
O
N
a
c
e
b
d
OH
HO
N
OH
N
g
j
f
h
i
Scheme 1. General synthetic scheme of 2,4-diphenyl-6-aryl pyridine derivatives. Reagents and conditions: (i) aryl aldehydes (1.0 equiv), KOH (50%), MeOH/H2O (5:1), 2–3 h,
room temperature, 60.7–94.1% yield; (ii) NH4OAc (10.0 equiv), glacial AcOH, 12–24 h, 80–100 °C, 21.4–89.3% yield.

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R. Karki et al. / Bioorg. Med. Chem. 18 (2010) 3066–3077
OH
N
HO
HO
4
3
5
6
OH
2
R3
HO
N
R3
N
R3
N
R3
N
R3
R3
N
1
HO
2'-OH series
3'-OH series
4'-OH series
R3 = a-g
2-OH series
3-OH series
4-OH series
N
HO
OH
di-OH series
Figure 2. Strategy for design of hydroxylated 2,4-diphenyl-6-aryl pyridines.
the Clasien–Schmidt condensation reaction,26 which was not nec-
essary to protect hydroxyl groups. Aryl ketone 1 (R1 = a, h–j) was
added to the solution of 50% KOH in MeOH/H2O (5:1) followed
by aryl aldehydes 2 (R2 = a, h–j) to obtain compounds 3 (R1 = h–j,
R2 = a) and 4 (R1 = a, R2 = h–j) in 60.7–94.1% yield. In the second
step, 10 pyridinium iodide salts 5 (R3 = a–j) were synthesized in
a quantitative yield by the treatment of aryl ketone 1 (R1 = a–j)
with iodine in pyridine. Using modified Kröhnke synthesis,27,28 fi-
nal compounds 6 (R1 = h–j, R2 = a, R3 = a–j) and 7 (R1 = a, R2 = h–
j, R3 = a–g) were synthesized by the reaction of appropriate
hydroxylated chalcones 3 or 4 with pyridinium iodide salt 5 in
the presence of ammonium acetate and glacial acetic acid in
21.4–89.3% yield. Through all the reactions for the preparation of
the final compounds, protection of hydroxyl groups was not re-
quired. It was noticed that the yields were relatively lower in com-
pounds having hydroxyl groups at ortho position compared to meta
or para substitution, which may be from the higher steric hinder-
ance of compounds bearing substitution on ortho position.
Total of 45 compounds were synthesized as shown in Table 1.
All the synthesized compounds contained hydroxyl groups at
ortho, meta, or para position of either 2- or 4- phenyl moiety,
and various aryl groups were attached on the 6-position of cen-
tral pyridine. Among the 45 compounds, 42 were monohydroxy-
lated with substitution at 2- or 4-phenyl moiety, and three were
dihydroxylated with substitution at 2- and 6-phenyl moiety. The
substitutions of hydroxyl groups at various positions of the
phenyl ring allowed us to investigate the effect of the position
of hydroxyl on biological activity. In addition, we are able to
observe the correlation between increase in the number of
hydroxyl groups and biological activity from dihydroxylated
compounds.
plasmid DNA in the presence of ATP. As shown in Figure 4 and
Table 2, compounds 16–19, 23, 31, 39–43, and 45 exhibited signif-
icant topo II inhibition at both 100 and 20 lM concentrations,
which showed stronger or similar inhibitory activity than etopo-
side. Compounds 15, 24–27, 35, 44, and 49 showed considerable
topo II inhibitory activity, although weaker than etoposide. Struc-
ture–activity relationship study revealed that most of the com-
pounds from 3-OH series (39–45) or 30-OH series (16–19)
showed significant topo II inhibitory activity (Fig. 2). The activity
was independent of the aryl group (–R3 in Fig. 2) at 6-position of
central pyridine. Several compounds from 40-OH series (24–27)
and 4-OH series (49) possessed moderate topo II inhibitory activ-
ity, which is weaker than etoposide. All the compounds from 20-
OH and 2-OH series did not show significant topo II inhibitory
activity. These structure–activity relationship results provide valu-
able information that the substitution of hydroxyl groups on the
phenyl ring increase the topo II inhibitory activity of compounds
in the order of meta > para > ortho position.
Compound 23 showed the strongest topo II inhibitory activity,
86.5% and 49.0% at 100 lM and 20 lM, respectively, which is
stronger than that of etoposide (68.2% and 30.4%, respectively).
Similarly, compound 31 had 76.8% and 33.7% topo II inhibition at
100 lM and 20 lM, respectively. It is interesting to note that both
compounds possessed dihydroxyl groups at meta or para position
of 2- and 6- phenyl on central pyridine. In addition, ortho dihydrox-
ylated compound 15 showed moderate topo II inhibitory activity
(42.7% and 12.1% at 100 lM and 20 lM, respectively), which is rel-
atively higher than that of ortho hydroxylated compounds (20-OH
and 2-OH series), which display no significant topo II inhibitory
activity. These results might indicate that increase in number of
hydroxyl groups increase topo II inhibitory activity in the order
of substitution at meta > para > ortho position.
2.2. Topoisomerase I and II inhibitory activity
Figure 5 and Table 2 illustrate the topo I inhibitory activity of
selected compounds. Compounds 22 and 24 showed significant
topo I inhibitory activity, which was similar to the activity of cam-
ptothecin. Compounds 17, 18, 23, 29, 42, 45, and 52 displayed
moderate topo I inhibitory activity. Most of the compounds having
topo I inhibitory activity were from 30-OH (17, 18, and 22) and 3-
OH (42 and 45) series. Several compounds from 40-OH (24 and
29) or 4-OH series (52) also displayed significant topo I inhibitory
activity. None of the compounds from 2-OH or 20-OH series had
topo I inhibitory activity.
The conversion of supercoiled plasmid DNA to relaxed DNA by
topo I and II was examined in the presence of synthesized 2,4-di-
phenyl-6-aryl pyridines (8–52). Camptothecin and etoposide,
well-known topo I and II inhibitors, respectively, were used as po-
sitive controls. The structures of selected compounds having signif-
icant biological activity are displayed in Figure 3.
The effect of synthesized compounds on human DNA topo II
were observed in the relaxation assay using supercoiled pBR322
a

R. Karki et al. / Bioorg. Med. Chem. 18 (2010) 3066–3077
3069
Table 1
Prepared compounds with yield, purity by HPLC, and melting point
R2
N
R1
R3
R3
Entry
R1
R2
Yield (%)
Purity (%)
Mp (°C)
8
9
20-OH phenyl
20-OH phenyl
20-OH phenyl
20-OH phenyl
20-OH phenyl
20-OH phenyl
20 OH phenyl
20-OH phenyl
30-OH phenyl
30-OH phenyl
30-OH phenyl
30-OH phenyl
30-OH phenyl
30-OH phenyl
30-OH phenyl
30-OH phenyl
40-OH phenyl
40-OH phenyl
40-OH phenyl
40-OH phenyl
40-OH phenyl
40-OH phenyl
40-OH phenyl
40-OH phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
2-OH phenyl
2-OH phenyl
2-OH phenyl
2-OH phenyl
2-OH phenyl
2-OH phenyl
2-OH phenyl
3-OH phenyl
3-OH phenyl
3-OH phenyl
3-OH phenyl
3-OH phenyl
3-OH phenyl
3-OH phenyl
4-OH phenyl
4-OH phenyl
4-OH phenyl
4-OH phenyl
4-OH phenyl
4-OH phenyl
4-OH phenyl
Phenyl
57.1
30.4
56.5
45.0
56.4
57.6
46.2
37.7
83.8
88.1
76.8
89.3
58.1
64.6
64.9
58.1
51.0
53.2
71.4
58.1
56.8
75.8
72.7
31.1
21.4
29.6
23.2
40.4
28.7
43.5
40.9
76.5
82.3
79.0
77.5
40.4
57.4
48.0
76.9
70.7
70.5
80.4
41.2
68.8
62.8
99.3
99.3
100.0
100.0
98.7
100.0
100.0
97.7
100.0
100.0
98.0
168.8–169.6
142.3–143.1
134.0–134.9
138.2–139.1
177.9–178.8
169.2–170.3
189.2–190.3
200.1–200.7
180.0–180.7
127.2–127.9
102.1–103.0
168.1–168.9
182.1–182.9
218.0–218.7
278.0–278.6
284.5–285.3
194.5–195.5
164.2–165.0
156.9–157.5
184.5–185.6
259.1–259.7
237.7–238.4
306.0–306.5
194.5–195.2
178.1–178.9
146.1–146.8
121.0–121.7
162.3–163.0
227.6–228.2
210.0–210.7
242.1–242.9
215.8–216.6
172.8–173.5
200.1–200.5
214.2–214.8
198.2–199.0
210.1–210.8
208.1–208.8
230.1–230.7
200.0–200.5
218.1–218.9
212.0–212.8
276.9–277.8
257.6–258.2
325.7–326.6
2-Thienyl
3-Thienyl
2-Furyl
2-Pyridyl
3-Pyridyl
4-Pyridyl
20-OH phenyl
Phenyl
2-Thienyl
3-Thienyl
2-Furyl
2-Pyridyl
3-Pyridyl
4-Pyridyl
30-OH phenyl
Phenyl
2-Thienyl
3-Thienyl
2-Furyl
2-Pyridyl
3-Pyridyl
4-Pyridyl
40-OH phenyl
Phenyl
2-Thienyl
3-Thienyl
2-Furyl
2-Pyridyl
3-Pyridyl
4-Pyridyl
Phenyl
2-Thienyl
3-Thienyl
2-Furyl
2-Pyridyl
3-Pyridyl
4-Pyridyl
Phenyl
2-Thienyl
3-Thienyl
2-Furyl
2-Pyridyl
3-Pyridyl
4-Pyridyl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
98.1
98.6
99.0
98.7
97.6
100.0
100.0
100.0
100.0
100.0
100.0
100.0
95.9
100.0
99.2
95.4
95.3
98.0
100.0
97.0
100.0
100.0
96.2
96.3
98.3
99.2
95.7
100.0
100.0
97.1
98.0
98.6
Phenyl
Phenyl
Phenyl
Phenyl
99.4
97.7
Compound 23, dihydroxylated at meta position of 2- and 6-phe-
nyl on central pyridine, had significant topo I inhibition, 67.2% at
concentrations when compared to the standard. The IC50 values
(lM) of the prepared compounds against those cell lines are shown
100
l
M along with strong topo II inhibitory activity. Several com-
in Table 2. Among the compounds which showed significant topo I
or II inhibitory activity, compounds 24–27, 29, 31, 35, 49, and 52
showed significant cytotoxicity in most of the cell lines, which dis-
played similar or slightly weaker cytotoxicity than positive con-
trols. It is evident that all of these compounds are from 40-OH
and 4-OH series except 35 which is from 2-OH series. Compounds
from 20-OH, 2-OH, 30-OH, or 3-OH series did not show significant
cytotoxicity. It is observed that compounds from 30-OH or 3-OH
series are potent topoisomerase inhibitors but less cytotoxic to
cancer cell lines. Compounds of 40-OH and 4-OH series have shown
positive correlations between topoisomerase inhibition and
cytotoxicity.
pounds (17, 18, 42, and 45) which possessed dual topo I and II
inhibitory activity belonged to 30-OH and 3-OH series. These re-
sults indicate that hydroxyl substitution at 3- or 30-position of
phenyl ring on 2,4-diphenyl-6-aryl pyridines might be required
in order to show significant topo inhibitory activity.
2.3. Cytotoxicity
For the evaluation of cytotoxicity, five different human cancer
cell lines were utilized: MDA-MB231 (human breast adenocarci-
noma cell line), HeLa (human cervix tumor cell line), DU145 (hu-
man prostate tumor cell line), HCT15 (human colorectal
adenocarcinoma cell line), and HL60 (human myeloid leukemic tu-
mor cell line). Adriamycin, camptothecin, and etoposide were used
as positive controls. Most of the compounds displayed moderate
cytotoxicity against those cell lines in relatively lower micromolar
3. Conclusion
We systematically designed and synthesized 45 hydroxylated
2,4-diphenyl-6-aryl pyridines by efficient synthetic routes, and

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R. Karki et al. / Bioorg. Med. Chem. 18 (2010) 3066–3077
OH
OH
HO
HO
HO
HO
S
O
N
N
N
N
N
S
15
16
17
18
19
S
HO
HO
OH
N
N
N
N
N
N
S
HO
HO
HO
22
23
24
25
26
HO
HO
O
O
N
N
N
N
N
HO
HO
N
HO
OH
27
29
31
35
39
HO
HO
HO
HO
HO
S
O
N
N
N
N
N
N
S
N
40
41
42
43
44
OH
OH
HO
O
N
N
N
N
N
45
49
52
Figure 3. Structure of the selected compounds possessing significant biological activity.
were evaluated these for topo I and II inhibitory activity and
cytotoxicity against several human cancer cell lines. Most of
the compounds (16–19, 23, 31, and 39–43, 45) showed potent
topo II inhibitory activity. Compounds 22 and 24 displayed sig-
nificant topo I inhibitory activity. The structure–activity relation-
ship study revealed that the substitution of hydroxyl groups on
the phenyl ring increase the topo I and II inhibitory activity of
compounds in the order of substitution at meta > para > ortho
position. The para-substitution on phenyl ring (40-OH or 4-OH
series) showed relatively stronger cytotoxicity against several
human cancer cell lines. It was observed that the increase in
the number of hydroxyl groups increased the topo I and II inhib-
itory activity. This study may provide valuable information to
researchers working on the development of antitumor agents.
4. Experimental
Compounds used as starting materials and reagents were ob-
tained from Aldrich Chemical Co., Junsei, or other chemical compa-

R. Karki et al. / Bioorg. Med. Chem. 18 (2010) 3066–3077
3071
100 µM
20 µM
Lane D: pBR322 DNA only
Lane T: pBR322 DNA + Topo II
Lane E: pBR322 DNA + Topo II + Etoposide
Lane 15-52 (100 µM): pBR322 DNA + Topo II + Compound 15-52
Lane 15-49 (20 µM): pBR322 DNA + Topo II + Compound 15-49
Figure 4. Human DNA topoisomerase IIa inhibitory activity of the selected compounds.
nies, and utilized without further purification. HPLC grade acetoni-
trile (ACN) and methanol were purchased from Burdick and Jack-
son, USA. Thin-layer chromatography (TLC) and column
chromatography (CC) were performed with Kieselgel 60 F254
(Merck) and silica gel (Kieselgel 60, 230–400 mesh, Merck), respec-
tively. Since all the compounds prepared contained aromatic rings,
they were visualized and detected on TLC plates with UV light
(short wave, long wave, or both). NMR spectra were recorded on
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 (d) were recorded in ppm and coupling constants
(J) in hertz (Hz). Melting points were determined in open capillary
tubes on electrothermal 1A 9100 digital melting point apparatus
and were uncorrected.
ESI LC/MS analyses were performed with a Finnigan LCQ Advan-
tageÒ LC/MS/MS spectrometry utilizing XcaliburÒ program. For ESI
LC/MS, LC was performed with a 2 lL injection volume on Waters
AtlantisÒ T3 reverse-phase C18 column (2.1 ꢀ 50 mm, 3
lM). The
mobile phase consisted of 100% distilled water (A) and 100% ACN
(B). A gradient program was used with a flow rate of 200 lL/min.
The initial composition was 10% B and programmed linearly to
90% B after 5 min and finally 10% B after 15 min. MS ionization con-
ditions were: Sheath gas flow rate: 70 arb, aux gas flow rate:
20 arb, I spray voltage: 4.5 KV, capillary temperature 215 °C, col-
umn temperature 40 °C, capillary voltage: 21 V, tube lens offset:
10 V. Retention time was given in minutes.
4.1. General method for the preparation of 3 and 4
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 vol-
Aryl ketone 1 (R1 = a, h–j) was added to the solution of 50% KOH
in MeOH/H2O (5:1). After complete dissolution, aryl aldehyde 2
(R2 = a, h–j) was added slowly. The mixture was then stirred for
2–3 h at room temperature. Precipitate was formed in most of
the cases which was then filtered, washed with cold MeOH, and
dried to yield 60.70–94.11% solid compound. In those reactions
where no precipitate occured, the reaction mixtures were ex-
tracted with ethyl acetate and washed with water and brine. They
were then further purified by either recrystallization or column
ume of 10 l 5 lM reverse-phase
L was injected in Waters X-TerraÒ
C18 column (4.6 ꢀ 250 mm) with a gradient elution of 60–100% of B
in A for 15 min followed by 100–60% of B in A for 15 min at a flow
rate of 1.0 mL/min at 254 nm UV detection, where mobile phase A
was double distilled water with 20 mM ammonium formate (AF)
and B was 90% ACN in water with 20 mM AF. Purity of compound
is described as percent (%).

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R. Karki et al. / Bioorg. Med. Chem. 18 (2010) 3066–3077
Table 2
Topoisomerase I and II inhibitory activity and cytotoxicity of the selected compounds
a
Compounds
%Inhibition
IC50
(lM)
Topo II
Topo I
MDA-MB231
HeLa
DU145
HCT15
HL60
100
l
M
20
l
M
100
l
M
20 lM
Etoposide
Camptothecin
Adriamycin
15
16
17
18
19
22
23
24
25
26
27
29
31
35
39
40
41
42
43
44
45
49
68.2
30.4
0.70 0.06
0.30 0.01
0.40 0.02
37.3 0.34
19.29 2.87
18.06 2.78
12.06 0.43
13.68 0.2
10.39 1.83
12.78 1.3
1.30 0.10
2.40 0.54
5.70 1.12
1.40 0.07
2.20 0.12
6.70 0.78
4.60 0.50
19.50 0.45
17.73 1.68
18.97 2.97
20.81 1.18
18.73 2.65
22.77 1.93
12.18 1.41
1.00 0.21
2.10 0.05
1.40 0.15
0.50 0.03
0.40 0.16
1.00 0.14
8.0 0.32
1.10 0.02
0.50 0.06
1.20 0.02
>50
1.00 0.01
68.8
40.3
0.40 0.13
1.00 0.07
14.8 0.05
16.07 1.75
16.96 1.9
13.82 0.37
9.43 0.75
28.13 2.55
13.04 0.08
1.70 0.01
1.70 0.10
17.30 0.19
10.90 0.14
13.60 0.46
3.30 0.44
9.70 0.15
15.05 2.08
15.32 0.34
14.90 1.03
10.92 5.15
29.84 2.43
17.52 1.9
14.95 1.37
4.50 0.12
1.60 0.05
0.10 0.00
0.70 0.01
22.4 2.07
11.47 0.86
14.12 0.48
10.3 1.14
9.91 1.65
23.88 0.86
16.44 0.75
1.40 0.01
1.70 0.01
3.60 0.03
3.30 0.07
4.10 0.13
5.70 0.84
1.90 0.01
13.49 3.24
18.92 2.22
11.99 1.45
24.03 1.49
15.13 1.87
12.63 1.39
12.67 1.32
13.70 0.26
1.40 0.00
42.7
48.4
53.8
51.8
51.6
3.1
12.1
32.0
43.5
37.2
38.6
1.8
49.0
NA
1.3
8.3
10.2
NA
33.7
3.1
32.0
34.7
30.8
31.1
21.3
21.3
32.7
10.3
NA
0.0
12.9
51.2
75.8
10.2
70.4
67.2
52.9
4.9
20.4
18.6
44.0
0.3
NA
0.0
5.8
6.8
0.0
25.4
10.5
43.6
NA
NA
NA
0.9
NA
NA
0.0
2.0
3.2
3.5
2.8
1.5
0.9
NA
5.8
17.95 0.46
18.92 1.09
11.7 0.58
14.41 0.95
19.21 1.34
24.93 0.78
3.00 0.42
3.70 0.46
8.50 0.50
3.20 0.22
8.40 0.61
22.0 0.63
4.90 0.35
17.89 0.55
19.68 0.31
11.70 0.58
34.91 3.82
15.51 0.64
18.93 1.64
18.14 4.58
8.60 0.60
2.20 0.09
9.95 0.46
10.85 0.7
7.66 0.53
13.47 0.58
27.44 6.25
18.39 0.61
3.70 0.66
12.50 2.18
1.10 0.08
3.50 0.57
15.10 1.45
3.00 0.50
2.50 0.20
17.95 2.34
14.12 2.83
24.56 2.64
13.86 1.4
20.91 1.71
18.39 2.53
18.71 1.0
5.10 0.82
11.70 1.07
86.5
27.6
35.4
38.3
35.2
18.6
76.8
31.2
68.2
57.5
52.6
47.5
48.1
37.1
42.8
30.2
8.4
1.5
11.5
9.1
6.9
48.8
10.8
8.1
60.8
5.0
52
57.5
NA: not applicable.
Etoposide: positive control for topo II and cytotoxicity.
Camptothecin: positive control for topo I and cytotoxicity.
Adriamycin: positive control for cytotoxicity.
MDA-MB231: human breast adenocarcinoma.
HeLa: human cervix tumor.
DU145: human prostate tumor.
HCT 15: human colorectal adenocarcinoma.
HL60: human myeloid leukemia.
a
Each data represents mean S.D. from three different experiments performed in triplicate.
chromatography. Six different hydroxylated chalcones were syn-
thesized as intermediates following the same method.
J = 7.8 Hz, 1H, 1-phenyl H-6), 7.55 (d, J = 15.7 Hz, 1H, CO–CH@C),
7.44–7.41 (m, 3H, 3-phenyl H-3, H-4, H-5), 7.38 (t, J = 7.8 Hz, 1H,
1-phenyl H-5), 7.15 (dd, J = 8.0, 2.4 Hz, 1H, 1-phenyl H-4), 6.59
(br, 1H, 1-phenyl 3-OH).
4.1.1. Synthesis of 1-(2-hydroxyphenyl)-3-phenylpropenone
(3a)
The procedure described in Section 4.1 was employed with aryl
ketone 1 (R1 = h) (2.40 mL, 20.00 mmol) and aryl aldehyde 2
(R2 = a) (2.02 mL, 20.00 mmol) to yield a yellow solid compound
(2.72 g, 60.7%).
Rf (ethyl acetate/n-hexane 1:2, v/v): 0.46; mp 97.1–97.8 °C.
1H NMR (250 MHz, CDCl3) d 12.84 (s, 1H, 1-phenyl 2-OH), 7.96
(d, J = 15.5 Hz, 1H, CO–C@CH), 7.95 (dd, J = 8.1, 1.6 Hz, 1H, 1-phenyl
6-H), 7.70 (d, J = 15.5 Hz, 1H, CO–CH@C), 7.70–7.66 (m, 2H, 3-phe-
nyl H-2, H-6), 7.51 (dt, J = 8.5, 1.6 Hz, 1H, 1-phenyl 4-H), 7.46–7.43
(m, 3H, 3-phenyl H-3, H-4, H-5), 7.05 (dd, J = 8.5, 1.0 Hz, 1H, 1-phe-
nyl 3-H), 6.95 (dt, J = 8.1, 1.0 Hz, 1H, 1-phenyl 5-H).
4.1.3. Synthesis of 1-(4-hydroxyphenyl)-3-phenylpropenone
(3c)
The procedure described in Section 4.1 was employed with aryl
ketone 1 (R1 = j) (2.72 g, 20.00 mmol) and aryl aldehyde 2 (R2 = a)
(2.02 mL, 20.00 mmol) to yield a light yellow solid compound
(3.85 g, 86.1%).
Rf (ethyl acetate/n-hexane 1:2, v/v): 0.40; mp 180.0–180.7 °C.
1H NMR (250 MHz, CDCl3) d 8.02 (d, J = 8.4 Hz, 2H, 1-phenyl H-
2, H-6), 7.84 (d, J = 15.6 Hz, 1H, CO–C@CH), 7.66–7.63 (m, 2H, 3-
phenyl H-2, H-6), 7.58 (d, J = 15.6 Hz, 1H, CO–CH@C), 7.43–7.40
(m, 3H, 3-phenyl H-3, H-4, H-5), 6.96 (d, J = 8.4 Hz, 2H, 1-phenyl
H-3, H-5), 6.38 (br, 1H, 1-phenyl 4-OH).
4.1.2. Synthesis of 1-(3-hydroxyphenyl)-3-phenylpropenone
(3b)
4.1.4. Synthesis of 3-(2-hydroxyphenyl)-1-phenylpropenone
(4a)
The procedure described in Section 4.1 was employed with aryl
ketone 1 (R1 = i) (4.08 g, 30.00 mmol) and aryl aldehyde 2 (R2 = a)
(3.03 mL, 30.00 mmol) to yield a light yellow solid compound
(5.90 g, 87.7%).
The procedure described in Section 4.1 was employed with aryl
ketone 1 (R1 = a) (2.33 mL, 20.00 mmol) and aryl aldehyde 2
(R2 = h) (2.09 mL, 20.00 mL) to yield a greenish yellow solid com-
pound (3.95 g, 88.2%).
Rf (ethyl acetate/n-hexane 1:2, v/v): 0.35; mp 120.2–120.6 °C.
1H NMR (250 MHz, CDCl3) d 7.86 (d, J = 15.7 Hz, 1H, CO–C@CH),
7.66–7.62 (m, 3H, 3-phenyl H-2, H-6, 1-phenyl H-2), 7.60 (d,
Rf (ethyl acetate/n-hexane 1:2, v/v): 0.37; mp 149.9–150.6 °C.
1H NMR (250 MHz, DMSO) d 10.31 (br, 1H, 3-phenyl 2-OH),
8.10–8.06 (m, 2H, 1-phenyl H-2, H-6), 8.07 (d, J = 15.8 Hz, 1H,

R. Karki et al. / Bioorg. Med. Chem. 18 (2010) 3066–3077
3073
100 µM
20 µM
Lane D: pBR322 DNA only
Lane T: pBR322 DNA + Topo I
Lane C: pBR322 DNA + Topo I + Camptothecin
Lane 17-52 (100 µM): pBR322 DNA + Topo I + Compound 17-52
Lane 22-24 (20 µM): pBR322 DNA + Topo I + Compound 22-24
Figure 5. Human DNA topoisomerase I inhibitory activity of the selected compounds.
CO–C@CH), 7.87 (d, J = 15.8 Hz, 1H, CO–CH@C), 7.85 (dd, J = 7.3,
1.4 Hz, 1H, 3-phenyl H-6), 7.68–7.52 (m, 3H, 1-phenyl H-3, H-4,
H-5), 7.26 (dt, J = 8.2, 1.5 Hz, 1H, 3- phenyl H-4), 6.94 (dd, J = 8.2,
0.8 Hz, 1H, 3-phenyl H-3), 6.89 (t, J = 7.2 Hz, 1H, 3-phenyl H-5).
4.2. General method for the preparation of 5
A mixture of aryl ketone (R1 = a–j), iodine (1.2 equiv) and pyri-
dine was refluxed at 140 °C for 3 h. Precipitate occurred during
reaction, and the mixture was cooled to room temperature. Then
it was filtered and washed with cold pyridine to afford 5 (R3 = a–
j) in quantitative yield. Ten different pyridinium iodide salts were
synthesized by this method.
4.1.5. Synthesis of 3-(3-hydroxyphenyl)-1-phenylpropenone
(4b)
The procedure described in Section 4.1 was employed with aryl
ketone 1 (R1 = a) (1.16 mL, 10.00 mmol) and aryl aldehyde 2 (R2 = i)
(1.22 g, 10.00 mmol) to yield a light yellow solid compound
(2.11 g, 94.1%).
4.3. General method for the preparation of 6 and 7
Rf (ethyl acetate/n-hexane 1:2, v/v): 0.35; mp 170.1–170.9 °C.
1H NMR (250 MHz, DMSO) d 9.67 (br, 1H, 3-phenyl 3-OH), 8.15–
8.11 (m, 2H, 1-phenyl H-2, H-6), 7.87 (d, J = 15.6 Hz, 1H, CO–
C@CH), 7.69–7.53 (m, 3H, 1-phenyl H-3, H-4, H-5), 7.67 (d,
J = 15.6 Hz, 1H, CO–CH@C), 7.33–7.24 (m, 2H, 3-phenyl H-5, H-6),
7.22 (br, 1H, 3-phenyl H-2), 6.87 (ddd, J = 7.7, 2.1, 1.0 Hz, 1H, 3-
phenyl H-4).
A mixture of propenone intermediate 3 (R1 = h–j, R2 = a), or 4
(R1 = a, R2 = h–j), pyridinium iodide salt 5 (R3 = a–j) and anhydrous
ammonium acetate in glacial acetic acid were heated at 80–100 °C
for 12–24 h. The reaction mixture was then extracted with ethyl
acetate, and washed with water and brine. The organic layer was
dried with magnesium sulfate and filtered. The filtrate was evapo-
rated at reduced pressure, which was then purified by silica gel col-
umn chromatography with the gradient elution of ethyl acetate/n-
hexane to afford solid compounds 6 (R1 = h–j, R2 = a, R3 = a–j) and 7
(R1 = a, R2 = h–j, R3 = a–g) in 21.4–89.3% yield. Forty-five 2,4-
diphenyl-6-aryl pyridine compounds were synthesized by this
method.
4.1.6. Synthesis of 3-(4-hydroxyphenyl)-1-phenylpropenone
(4c)
The procedure described in Section 4.1 was employed with aryl
ketone 1 (R1 = a) (2.33 mL, 20.00 mmol) and aryl aldehyde 2 (R2 = j)
(2.44 g, 20.00 mmol) to yield a yellow solid compound (3.78 g,
84.5%).
Rf (ethyl acetate/n-hexane 1:2, v/v): 0.34; mp 174.2–175.0 °C.
1H NMR (250 MHz, DMSO) d 10.14 (br, 1H, 3-phenyl 4-OH),
8.12–8.08 (m, 2H, 1-phenyl H-2, H-6), 7.75 (d, J = 8.6 Hz, 2H, 3-phe-
nyl H-2, H-6), 7.70 (d, J = 15.4 Hz, 1H, CO–C@CH), 7.69–7.51 (m, 4H,
1-phenyl H-3, H-4, H-5, CO-CH@C), 6.84 (d, J = 8.6 Hz, 2H, 3-phenyl
H-3, H-5).
4.3.1. Synthesis of 2,20-(4-phenylpyridine-2,6-diyl)diphenol (15)
The procedure described in Section 4.3 was employed with
3a (0.67 g, 3.00 mmol), anhydrous ammonium acetate (2.31 g,
30.00 mmol), 5 (R3 = h) (1.02 g, 3.00 mmol), and glacial AcOH
(3 mL) to yield a yellow solid (0.38 g, 1.13 mmol).
Rf (ethyl acetate/n-hexane 1:2, v/v): 0.42; LC/MS/MS: retention
time: 7.79 min; [MH]+: 340.27.

3074
R. Karki et al. / Bioorg. Med. Chem. 18 (2010) 3066–3077
1H NMR (250 MHz, DMSO) d 12.31 (s, 2H, 2-phenyl 2-OH, 6-phe-
4.3.5. Synthesis of 3-(6-(furan-2-yl)-4-phenylpyridin-2-yl)-
phenol (19)
The procedure described in Section 4.3 was employed with
3b (0.33 g, 1.50 mmol), anhydrous ammonium acetate (1.15 g,
15.00 mmol), 5 (R3 = d) (0.47 g, 1.50 mmol), and glacial AcOH
(2 mL) to yield a white solid (0.41 g, 1.33 mmol).
nyl 20-OH), 8.20 (s, 2H, pyridine H-3, H-5), 7.98–7.95 (m, 4H, 2-phe-
nyl H-6, 6-phenyl H-6, 4-phenyl H-2, H-6), 7.60–7.52 (m, 3H, 4-
phenyl H-3, H-4, H-5), 7.32 (t, J = 8.2 Hz, 2H, 2-phenyl H-4, 6-phenyl
H-4), 6.99–6.93 (m, 4H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5).
13C NMR (62.5 MHz, DMSO) d 157.51, 155.59, 149.91, 137.76,
131.23, 129.77, 129.44, 128.95, 127.64, 122.26, 119.43, 117.90,
117.51.
Rf (ethyl acetate/n-hexane 1:3, v/v): 0.31; LC/MS/MS: retention
time: 7.77 min; [MH]+: 314.24.
1H NMR (250 MHz, CDCl3) d 7.88 (d, J = 1.3 Hz, 1H, pyridine H-3),
7.77(d, J = 1.3 Hz, 1H, pyridineH-5), 7.76–7.72(m, 2H, 4-phenylH-2,
H-6), 7.70 (t, J = 2.0 Hz, 1H, 2-phenyl H-2), 7.60 (d, J = 7.8 Hz, 1H, 2-
phenyl H-6), 7.55–7.47 (m, 4H, 4-phenyl H-3, H-4, H-5, 6-furan H-
5), 7.34 (t, J = 7.9 Hz, 1H, 2-phenyl H-5), 7.23 (d, J = 3.3 Hz, 1H, 6-fur-
an H-3), 6.91 (dd, J = 8.0, 2.5 Hz, 1H, 2-phenyl H-4), 6.56 (dd, J = 3.3,
1.7 Hz, 1H, 6-furan H-4), 6.12 (br, 1H, 2-phenyl 3-OH).
4.3.2. Synthesis of 3-(4,6-diphenylpyridin-2-yl)phenol (16)
The procedure described in Section 4.3 was employed with
3b (0.44 g, 2.00 mmol), anhydrous ammonium acetate (1.54 g,
20.00 mmol), 5 (R3 = a) (0.65 g, 2.00 mmol), and glacial AcOH
(2 mL) to yield a white solid (0.54 g, 1.67 mmol).
Rf (ethyl acetate/n-hexane 1:2, v/v): 0.44; LC/MS/MS: retention
time: 8.29 min; [MH]+: 324.27.
13C NMR (62.5 MHz, CDCl3) d 157.45, 156.27, 153.74, 150.08,
149.63, 143.29, 140.79, 138.48, 129.91, 129.12, 129.08, 127.08,
119.26, 117.29, 116.28, 115.33, 114.26, 112.13, 109.14.
1H NMR (250 MHz, CDCl3) d 8.18–8.15 (m, 2H, 6-phenyl H-2, H-
6), 7.88 (d, J = 1.1 Hz, 1H, pyridine H-5), 7.86 (d, J = 1.1 Hz, 1H, pyr-
idine H-3), 7.75–7.72 (m, 3H, 2-phenyl H-2, 4-phenyl H-2, H-6),
7.70 (d, J = 8.0 Hz, 1H, 2-phenyl H-6), 7.57–7.43 (m, 6H, 6-phenyl
H-3, H-4, H-5, 4-phenyl H-3, H-4, H-5), 7.36 (t, J = 8.0 Hz, 1H, 2-
phenyl H-5), 6.92 (dd, J = 8.0, 2.5 Hz, 1H, 2-phenyl H-4), 5.56 (br,
1H, 2-phenyl 3-OH).
4.3.6. Synthesis of 3-(4-phenyl-2,40-bipyridin-6-yl)phenol (22)
The procedure described in Section 4.3 was employed with
3b (0.44 g, 2.00 mmol), anhydrous ammonium acetate (1.54 g,
20.00 mmol), 5 (R3 = g) (0.65 g, 2.00 mmol), and glacial AcOH
(2 mL) to yield a white solid (0.42 g, 1.29 mmol).
13C NMR (62.5 MHz, CDCl3) d 157.56, 157.00, 156.05, 150.23,
141.08, 139.44, 138.85, 129.92, 129.11, 129.08, 129.03, 128.70,
127.16, 119.41, 117.49, 117.31, 116.13, 114.11.
Rf (ethyl acetate/n-hexane 1:1, v/v): 0.30; LC/MS/MS: retention
time: 6.90 min; [MH]+: 325.29.
1H NMR (250 MHz, DMSO) d 9.61 (br, 1H, 6-phenyl 3-OH), 8.76
(d, J = 4.3 Hz, 2H, 2-pyridine H-20, H-60), 8.33 (d, J = 1.1 Hz, 1H, pyr-
idine H-3), 8.29 (dd, J = 4.3, 1.4 Hz, 2H, 2-pyridine H-30, H-50), 8.21
(d, J = 1.1 Hz, 1H, pyridine H-5), 8.06–8.02 (m, 2H, 4-phenyl H-2, H-
6), 7.75 (s, 1H, 6-phenyl H-2), 7.72 (d, J = 8.4 Hz, 1H, 6-phenyl H-6),
7.61–7.51 (m, 3H, 4-phenyl H-3, H-4, H-5), 7.33 (t, J = 7.8 Hz, 1H, 6-
phenyl H-5), 6.91 (dd, J = 8.1, 1.7 Hz, 1H, 6-phenyl H-4).
13C NMR (62.5 MHz, DMSO) d 158.08, 157.15, 154.08, 150.59,
150.09, 145.95, 140.01, 137.56, 130.08, 129.77, 129.39, 127.70,
121.37, 118.40, 118.06, 117.69, 116.75, 114.00.
4.3.3. Synthesis of 3-(4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)-
phenol (17)
The procedure described in Section 4.3 was employed with
3b (0.44 g, 2.00 mmol), anhydrous ammonium acetate (1.54 g,
20.00 mmol), 5 (R3 = b) (0.66 g, 2.00 mmol), and glacial AcOH
(2 mL) to yield a white solid (0.58 g, 1.76 mmol).
Rf (ethyl acetate/n-hexane 1:3, v/v): 0.34; LC/MS/MS: retention
time: 8.05 min; [MH]+: 330.24.
1H NMR (250 MHz, CDCl3) d 7.77 (s, 2H, pyridine H-3, H-5),
7.72–7.70 (m, 3H, 2-phenyl H-2, 4-phenyl H-2, H-6), 7.70 (d,
J = 4.4 Hz, 1H, 6-thiophene H-3), 7.68 (d, J = 8.4 Hz, 1H, 2-phenyl
H-6), 7.56–7.47 (m, 3H, 4-phenyl H-3, H-4, H-5), 7.42 (d,
J = 4.9 Hz, 1H, 6-thiophene H-5), 7.35 (t, J = 8.0 Hz, 1H, 2-phenyl
H-5), 7.15 (dd, J = 4.9, 3.7 Hz, 1H, 6-thiophene H-4), 6.93 (dd,
J = 8.0, 2.5 Hz, 1H, 2-phenyl H-4), 5.46 (br, 1H, 2-phenyl 3-OH).
13C NMR (62.5 MHz, CDCl3) d 156.79, 156.06, 152.66, 150.22,
145.16, 140.54, 138.62, 129.93, 129.11, 127.98, 127.73, 127.10,
124.73, 119.30, 117.03, 116.24, 115.60, 114.03.
4.3.7. Synthesis of 3,30-(4-phenylpyridine-2,6-diyl)diphenol (23)
The procedure described in Section 4.3 was employed with
3b (0.44 g, 2.00 mmol), anhydrous ammonium acetate (1.54 g,
20.00 mmol), 5 (R3 = i) (0.68 g, 2.00 mmol), and glacial AcOH
(2 mL) to yield a white solid (0.37 g, 1.16 mmol).
Rf (ethyl acetate/n-hexane 1:2, v/v): 0.35; LC/MS/MS: retention
time: 7.10 min; [MH]+: 340.27.
1H NMR (250 MHz, DMSO) d 9.57 (br, 2H, 2-phenyl 3-OH, 6-
phenyl 30-OH), 8.06 (s, 2H, pyridine H-3, H-5), 8.00–7.98 (m, 2H,
4-phenyl H-2, H-6), 7.73–7.68 (m, 4H, 2-phenyl H-2, H-6, 6-phenyl
H-2, H-6), 7.58–7.50 (m, 3H, 4-phenyl H-3, H-4, H-5), 7.32 (t,
J = 7.9 Hz, 2H, 2-phenyl H-5, 6-phenyl H-5), 6.89 (dd, J = 8.0,
1.7 Hz, 2H, 2-phenyl H-4, 6-phenyl H-4).
4.3.4. Synthesis of 3-(4-phenyl-6-(thiophen-3-yl)pyridin-2-yl)-
phenol (18)
The procedure described in Section 4.3 was employed with
3b (0.44 g, 2.00 mmol), anhydrous ammonium acetate (1.54 g,
20.00 mmol), 5 (R3 = c) (0.66 g, 2.00 mmol), and glacial AcOH
(2 mL) to yield a white solid (0.50 g, 1.53 mmol).
13C NMR (62.5 MHz, DMSO) d 158.01, 156.65, 149.62, 140.46,
138.02, 129.96, 129.51, 129.36, 127.56, 117.94, 116.79, 116.47,
113.96.
Rf (ethyl acetate/n-hexane 1:3, v/v): 0.34; LC/MS/MS: retention
time: 8.01 min; [MH]+: 330.24.
1H NMR (250 MHz, CDCl3) d 8.06 (dd, J = 3.0, 1.2 Hz, 1H, 6-thio-
phene H-2), 7.81 (dd, J = 5.5, 1.2 Hz, 1H, 6-thiophene H-4), 7.79 (d,
J = 1.3, 1H, pyridine H-3), 7.75 (d, J = 1.3 Hz, 1H, pyridine H-5),
7.73–7.69 (m, 3H, 2-phenyl H-2, 4-phenyl H-2, H-6), 7.67 (d,
J = 7.8 Hz, 1H, 2-phenyl H-6), 7.56–7.47 (m, 3H, 4-phenyl H-3, H-
4, H-5), 7.43 (dd, J = 5.0, 3.0 Hz, 1H, 6-thiophene H-5), 7.32 (t,
J = 8.0 Hz, 1H, 2-phenyl H-5), 6.93 (ddd, J = 8.0, 2.5, 0.7 Hz, 1H, 2-
phenyl H-4), 5.92 (br, 1H, 2-phenyl 3-OH)
4.3.8. Synthesis of 4-(4,6-diphenylpyridin-2-yl)phenol (24)
The procedure described in Section 4.3 was employed with
3c (0.56 g, 2.50 mmol), anhydrous ammonium acetate (1.92 g,
25.00 mmol), 5 (R3 = a) (0.81 g, 2.50 mmol), and glacial AcOH
(3 mL) to yield a white solid (0.41 g, 1.27 mmol).
Rf (ethyl acetate/n-hexane 1:2, v/v): 0.40; LC/MS/MS: retention
time: 8.03 min; [MH]+: 324.27.
1H NMR (250 MHz, CDCl3) d 8.19–8.17 (m, 2H, 6-phenyl H-2, H-
6), 8.12 (d, J = 8.6 Hz, 2H, 2-phenyl H-2, H-6), 7.83 (s, 1H, pyridine
H-5), 7.81 (s, 1H, pyridine H-3), 7.75–7.72 (m, 2H, 4-phenyl H-2, H-
6), 7.56–7.41 (m, 6H, 6-phenyl H-3, H-4, H-5, 4-phenyl H-3, H-4, H-
13C NMR (62.5 MHz, CDCl3) d 157.07, 156.16, 153.63, 150.19,
142.28, 140.96, 138.76, 129.88, 129.09, 129.02, 127.10, 126.46,
126.21, 123.91, 119.30, 117.18, 117.08, 116.16, 114.11.

R. Karki et al. / Bioorg. Med. Chem. 18 (2010) 3066–3077
3075
5), 6.96 (d, J = 8.6 Hz, 2H, 2-phenyl H-3, H-5), 5.24 (br, 1H, 2-phenyl
4-OH).
20.00 mmol), 5 (R3 = f) (0.65 g, 2.00 mmol), and glacial AcOH
(2 mL) to yield a light yellow solid (0.49 g, 1.51 mmol).
Rf (ethyl acetate/n-hexane 1:1, v/v): 0.38; LC/MS/MS: retention
time: 6.67 min; [MH]+: 325.27.
13C NMR (62.5 MHz, CDCl3) d 157.38, 157.09, 156.61, 150.13,
139.64, 139.13, 132.37, 129.07, 128.98, 128.92, 128.67, 128.65,
127.15, 127.11, 116.58, 116.38, 115.56.
1H NMR (250 MHz, DMSO) d 9.79 (s, 1H, 6-phenyl 4-OH), 9.48
(s, 1H, 2-pyridine H-20), 8.67–8.63 (m, 2H, 2-pyridine H-60, H-40),
8.20 (d, J = 8.5 Hz, 2H, 6-phenyl H-2, H-6), 8.19 (s, 1H, pyridine
H-3), 8.12 (s, 1H, pyridine H-5), 8.04–8.02 (m, 2H, 4-phenyl H-2,
H-6), 7.58–7.50 (m, 4H, 4-phenyl H-3, H-4, H-5, 2-pyridine H-50),
6.93 (d, J = 8.5 Hz, 2H, 6-phenyl H-3, H-5).
4.3.9. Synthesis of 4-(4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)-
phenol (25)
The procedure described in Section 4.3 was employed with 3c
(0.56 g, 2.50 mmol), anhydrous ammonium acetate (1.92 g,
25.00 mmol), 5 (R3 = b) (0.82 g, 2.50 mmol), and glacial AcOH
(3 mL) to yield a light yellow solid (0.43 g, 1.32 mmol).
Rf (ethyl acetate/n-hexane 1:2, v/v): 0.40; LC/MS/MS: retention
time: 8.03 min; [MH]+: 330.26.
13C NMR (62.5 MHz, DMSO) d 159.03, 157.11, 154.20, 150.09,
149.70, 148.37, 137.84, 134.54, 134.48, 129.65, 129.50, 129.25,
128.65, 127.53, 123.92, 116.13, 116.05, 115.70.
1H NMR (250 MHz, CDCl3) d 8.09 (d, J = 8.3 Hz, 2H, 2-phenyl H-
2, H-6), 7.72 (s, 2H, pyridine H-3, H-5), 7.72–7.70 (m, 3H, 4-phenyl
H-2, H-6, 6-thiophene H-3), 7.55–7.46 (m, 3H, 4-phenyl H-3, H-4,
H-5), 7.42 (d, J = 5.0 Hz, 1H, 6-thiophene H-5), 7.15 (t, J = 4.3, 1H,
6-thiophene H-4), 6.96 (d, J = 8.3 Hz, 2H, 2-phenyl H-3, H-5), 5.22
(br, 1H, 2-phenyl 4-OH).
4.3.13. Synthesis of 4,40-(4-phenylpyridine-2,6-diyl)diphenol (31)
The procedure described in Section 4.3 was employed with 3c
(0.44 g, 2.00 mmol), anhydrous ammonium acetate (1.54 g,
20.00 mmol), 5 (R3 = j) (0.68 g, 2.00 mmol), and glacial AcOH
(2 mL) to yield a yellow solid (0.21 g, 0.62 mmol).
Rf (ethyl acetate/n-hexane 1:2, v/v): 0.24; LC/MS/MS: retention
13C NMR (62.5 MHz, CDCl3) d 156.88, 156.70, 152.56, 150.11,
145.51, 138.90, 131.86, 129.07, 128.99, 128.58, 127.93, 127.56,
127.10, 124.53, 116.06, 115.57, 114.72.
time: 6.72 min; [MH]+: 340.27.
1H NMR (250 MHz, DMSO) d 9.76 (br, s, 2H, 2-phenyl 4-OH, 6-
phenyl 40-OH), 8.16 (d, J = 8.6 Hz, 4H, 2-phenyl H-2, H-6, 6-phenyl
H-2, H-6), 7.98–7.94 (m, 4H, 4-phenyl H-2, H-6, pyridine H-3, H-5),
7.58–7.48 (m, 3H, 4-phenyl H-3, H-4, H-5), 6.91 (d, J = 8.6 Hz, 4H,
2-phenyl H-3, H-5, 6-phenyl H-3, H-5).
4.3.10. Synthesis of 4-(4-phenyl-6-(thiophen-3-yl)pyridin-2-yl)-
phenol (26)
The procedure described in Section 4.3 was employed with
3c (0.44 g, 2.00 mmol), anhydrous ammonium acetate (1.54 g,
20.00 mmol), 5 (R3 = c) (0.66 g, 2.00 mmol), and glacial AcOH
(2 mL) to yield a white solid (0.47 g, 1.42 mmol).
13C NMR (62.5 MHz, DMSO) d 158.80, 156.47, 149.29, 138.36,
130.10, 129.25, 128.49, 127.41, 115.63, 114.48.
4.3.14. Synthesis of 2-(2-(furan-2-yl)-6-phenylpyridin-4-yl)-
phenol (35)
The procedure described in Section 4.3 was employed with 4a
(0.44 g, 2.00 mmol), anhydrous ammonium acetate (1.54 g,
20.00 mmol), 5 (R3 = d) (0.63 g, 2.00 mmol), and glacial AcOH
(2 mL) to yield an orange solid (0.25 g, 0.80 mmol).
Rf (ethyl acetate/n-hexane 1:2, v/v): 0.44; LC/MS/MS: retention
time: 8.00 min; [MH]+: 330.25.
1H NMR (250 MHz, CDCl3) d 8.09–8.05 (m, 3H, 2-phenyl H-2, H-
6, 6-thiophene H-2), 7.83 (dd, J = 5.0, 1.2 Hz, 1H, 6-thiophene H-4),
7.75–7.71 (m, 2H, 4-phenyl H-2, H-6), 7.74 (s, 1H, pyridine H-3),
7.70 (s, 1H, pyridine H-5), 7.56–7.46 (m, 3H, 4-phenyl H-3, H-4,
H-5), 7.43 (dd, J = 5.0, 3.0 Hz, 1H, 6-thiophene H-5), 6.95 (dd,
J = 8.7, 2.0 Hz, 2H, 2-phenyl H-3, H-5), 5.11 (br, 1H, 2-phenyl 4-
OH).
Rf (ethyl acetate/n-hexane 1:2, v/v): 0.44; LC/MS/MS: retention
time: 7.45 min; [MH]+: 314.23.
1H NMR (250 MHz, DMSO) d 9.96 (br, 1H, 4-phenyl 2-OH), 8.19–
8.16 (m, 2H, 6-phenyl H-2, H-6), 7.97 (s, 1H, pyridine H-5), 7.91 (s,
1H, pyridine H-3), 7.87 (s, 1H, 2-furan H-5), 7.55–7.45 (m, 4H, 6-
phenyl H-3, H-4, H-5, 4-phenyl H-6), 7.32 (t, J = 8.0 Hz, 1H, 4-phe-
nyl H-4), 7.26 (d, J = 3.0 Hz, 1H, 2-furan H-3), 7.03 (d, J = 8.0 Hz, 1H,
4-phenyl H-3), 6.99 (t, J = 7.4 Hz, 1H, 4-phenyl H-5), 6.69 (dd,
J = 3.3, 1.7 Hz, 1H, 2-furan H-4).
13C NMR (62.5 MHz, CDCl3) d 157.07, 156.57, 153.53, 150.12,
142.56, 139.06, 132.32, 129.07, 128.93, 128.62, 127.12, 126.48,
126.11, 123.75, 116.33, 116.12, 115.55.
4.3.11. Synthesis of 4-(6-(furan-2-yl)4-phenylpyridin-2-yl)-
phenol (27)
The procedure described in Section 4.3 was employed with
3c (0.44 g, 2.00 mmol), anhydrous ammonium acetate (1.54 g,
20.00 mmol), 5 (R3 = d) (0.63 g, 2.00 mmol), and glacial AcOH
(2 mL) to yield a white solid (0.36 g, 1.16 mmol).
13C NMR (62.5 MHz, DMSO) d 156.17, 155.05, 153.63, 148.46,
148.32, 144.40, 138.85, 130.52, 129.41, 128.99, 127.00, 125.11,
119.95, 119.02, 117.32, 116.59, 112.57, 109.29.
Rf (ethyl acetate/n-hexane 1:2, v/v): 0.38; LC/MS/MS: retention
4.3.15. Synthesis of 3-(2,6-diphenylpyridin-4-yl)phenol (39)
The procedure described in Section 4.3 was employed with
4b (0.44 g, 2.00 mmol), anhydrous ammonium acetate (1.54 g,
20.00 mmol), 5 (R3 = a) (0.65 g, 2.00 mmol), and glacial AcOH
(2 mL) to yield a light yellow solid (0.49 g, 1.52 mmol).
Rf (ethyl acetate/n-hexane 1:3, v/v): 0.35; LC/MS/MS: retention
time: 8.26 min; [MH]+: 324.27.
time: 7.61 min; [MH]+: 314.24.
1H NMR (250 MHz, CDCl3) d 8.03 (dd, J = 8.7, 2.0 Hz, 2H, 2-phe-
nyl H-2, H-6), 7.83 (d, J = 1.4 Hz, 1H, pyridine H-3), 7.76–7.72 (m,
2H, 4-phenyl H-2, H-6), 7.72 (d, J = 1.4 Hz, 1H, pyridine H-5),
7.55–7.45 (m, 4H, 4-phenyl H-3, H-4, H-5, 6-furan H-5), 7.23 (dd,
J = 3.3, 0.5 Hz, 1H, 6-furan H-3), 6.94 (dd, J = 8.7, 2.0 Hz, 2H, 2-phe-
nyl H-3, H-5), 6.57 (dd, J = 3.3, 1.7 Hz, 1H, 6-furan H-4), 5.54 (br,
1H, 2-phenyl 4-OH).
1H NMR (250 MHz, CDCl3) d 8.21–8.17 (m, 4H, 2-phenyl H-2, H-
6, 6-phenyl H-2, H-6), 7.86 (d, J = 1.0 Hz, 2H, pyridine H-3, H-5),
7.55–7.42 (m, 6H, 2-phenyl H-3, H-4, H-5, 6-phenyl H-3, H-4, H-
5), 7.36 (t, J = 7.8 Hz, 1H, 4-phenyl H-5), 7.32–7.29 (m, 1H, 4-phe-
nyl H-6), 7.20 (br, 1H, 4-phenyl H-2), 6.95 (ddd, J = 7.8, 2.3,
1.2 Hz, 1H, 4-phenyl H-4), 5.38 (br, 1H, 4-phenyl 3-OH).
13C NMR (62.5 MHz, CDCl3) d 157.51, 156.16, 149.75, 140.71,
139.46, 130.36, 129.07, 128.71, 127.11, 119.63, 117.11, 115.89,
114.08.
13C NMR (62.5 MHz, CDCl3) d 157.38, 156.76, 154.09, 149.95,
149.56, 143.17, 138.78, 132.06, 129.05, 129.00, 128.66, 127.08,
116.31, 115.61, 114.50, 112.07, 108.94.
4.3.12. Synthesis of 4-(4-phenyl-2,30-bipyridin-6-yl)phenol (29)
The procedure described in Section 4.3 was employed with
3c (0.44 g, 2.00 mmol), anhydrous ammonium acetate (1.54 g,

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R. Karki et al. / Bioorg. Med. Chem. 18 (2010) 3066–3077
4.3.16. Synthesis of 3-(2-phenyl-6-(thiophen-2-yl)pyridin-4-yl)-
phenol (40)
The procedure described in Section 4.3 was employed with
4b (0.33 g, 1.50 mmol), anhydrous ammonium acetate (1.15 g,
15.00 mmol), 5 (R3 = b) (0.49 g, 1.50 mmol), and glacial AcOH
(2 mL) to yield a white solid (0.40 g, 1.23 mmol).
30), 8.56 (s, 1H, pyridine H-3), 8.35–8.32 (m, 2H, 6-phenyl H-2, H-
6), 8.22 (s, 1H, pyridine H-5), 8.04 (dt, J = 7.7, 1.5 Hz, 1H, 2-pyridine
H-40), 7.58–7.48 (m, 4H, 6-phenyl H-3, H-4, H-5, 2-pyridine H-50),
7.41–7.36 (m, 2H, 4-phenyl H-5, H-6), 7.32 (br, 1H, 4-phenyl H-
2), 6.93 (d, J = 6.7 Hz, 1H, 4-phenyl H-4).
13C NMR (62.5 MHz, DMSO) d 158.34, 156.68, 155.85, 155.39,
149.86, 149.59, 139.18, 138.74, 137.73, 130.63, 129.62, 129.05,
127.23, 124.72, 121.09, 118.23, 118.05, 116.65, 113.92.
Rf (ethyl acetate/n-hexane 1:2, v/v): 0.42; LC/MS/MS: retention
time: 8.12 min; [MH]+: 330.24.
1H NMR (250 MHz, DMSO) d 9.73 (br, 1H, 4-phenyl 3-OH), 8.25–
8.22 (m, 2H, 2-phenyl H-2, H-6), 8.10 (s, 1H, pyridine H-3), 8.05 (d,
J = 3.7 Hz, 1H, 6-thiophene H-3), 8.02 (s, 1H, pyridine H-5), 7.68 (d,
J = 5.0 Hz, 1H, 6-thiophene H-5), 7.56–7.46 (m, 3H, 2-phenyl H-3,
H-4, H-5), 7.42–7.35 (m, 2H, 4-phenyl H-5, H-6), 7.33 (br, 1H, 4-
phenyl H-2), 7.20 (dd, J = 5.0, 3.7 Hz, 1H, 6-thiophene H-4), 6.93
(d, J = 7.5 Hz, 1H, 4-phenyl H-4).
4.3.20. Synthesis of 3-(6-phenyl-2,30-bipyridin-4-yl)phenol (44)
The procedure described in Section 4.3 was employed with
4b (0.44 g, 2.00 mmol), anhydrous ammonium acetate (1.54 g,
20.00 mmol), 5 (R3 = f) (0.65 g, 2.00 mmol), and glacial AcOH
(2 mL) to yield a white solid (0.37 g, 1.14 mmol).
Rf (ethyl acetate/n-hexane 1:1, v/v): 0.38; LC/MS/MS: retention
13C NMR (62.5 MHz, DMSO) d 158.24, 156.43, 152.54, 149.99,
145.07, 139.14, 138.47, 130.40, 129.62, 129.04, 128.90, 128.77,
127.04, 126.13, 118.27, 116.56, 116.46, 115.06, 114.31.
time: 6.95 min; [MH]+: 325.29.
1H NMR (250 MHz, DMSO) d 9.74 (br, 1H, 4-phenyl 3-OH), 9.48
(s, 1H, 2-pyridine H-20), 8.68–8.65 (m, 2H, 2-pyridine H-60, H-40),
8.33–8.30 (m, 2H, 6-phenyl H-2, H-6), 8.22 (s, 1H, pyridine H-3),
8.16 (s, 1H, pyridine H-5), 7.58–7.51 (m, 3H, 6-phenyl H-3, H-4,
H-5), 7.47 (t, J = 8.3 Hz, 2H, 2-pyridine H-50, 4-phenyl H-5), 7.38–
7.32 (m, 2H, 4-phenyl H-2, H-6), 6.94 (d, J = 8.0 Hz, 1H, 4-phenyl
H-4).
4.3.17. Synthesis of 3-(2-phenyl-6-(thiophen-3-yl)pyridin-4-yl)-
phenol (41)
The procedure described in Section 4.3 was employed with 4b
(0.33 g, 1.50 mmol), anhydrous ammonium acetate (1.15 g,
15.00 mmol), 5 (R3 = c) (0.49 g, 1.50 mmol), and glacial AcOH
(2 mL) to yield a light yellow solid (0.39 g, 1.18 mmol).
Rf (ethyl acetate/n-hexane 1:2, v/v): 0.42; LC/MS/MS: retention
time: 8.11 min; [MH]+: 330.25.
13C NMR (62.5 MHz, DMSO) d 158.25, 156.98, 154.50, 150.25,
148.46, 139.17, 138.78, 134.64, 134.44, 130.39, 129.62, 129.04,
127.24, 124.04, 118.39, 117.39, 117.28, 116.57, 114.42.
1H NMR (250 MHz, DMSO) d 9.72 (br, 1H, 4-phenyl 3-OH), 8.44
(dd, J = 2.9, 1.1 Hz, 1H, 6-thiophene H-2), 8.30–8.27 (m, 2H, 2-phe-
nyl H-2, H-6), 8.06 (s, 1H, pyridine H-3), 8.03 (s, 1H, pyridine H-5),
7.99 (d, J = 5.0 Hz, 1H, 6-thiophene H-4), 7.69 (dd, J = 5.0, 3.0 Hz,
1H, 6-thiophene H-5), 7.55–7.46 (m, 3H, 2-phenyl H-3, H-4, H-5),
7.42–7.34 (m, 2H, 4-phenyl H-5, H-6), 7.33 (br, 1H, 4-phenyl H-
2), 6.93 (d, J = 7.4 Hz, 1H, 4-phenyl H-4).
4.3.21. Synthesis of 3-(6-phenyl-2,40-bipyridin-4-yl)phenol (45)
The procedure described in Section 4.3 was employed with
4b (0.44 g, 2.00 mmol), anhydrous ammonium acetate (1.54 g,
20.00 mmol), 5 (R3 = g) (0.65 g, 2.00 mmol), and glacial AcOH
(2 mL) to yield a white solid (0.31, 0.96 mmol).
Rf (ethyl acetate/n-hexane 1:1, v/v): 0.38; LC/MS/MS: retention
time: 6.94 min; [MH]+: 325.30.
13C NMR (62.5 MHz, DMSO) d 158.23, 156.53, 153.39, 149.92,
142.35, 139.38, 138.95, 130.38, 129.46, 128.99, 127.15, 127.01,
124.94, 118.26, 116.79, 116.44, 116.30, 114.30.
1H NMR (250 MHz, DMSO) d 9.75 (br, 1H, 4-phenyl 3-OH), 8.75
(d, J = 5.0 Hz, 2H, 2-pyridine H-20, H-60), 8.34–8.28 (m, 4H, 2-pyri-
dine H-30, H-50, 6-phenyl H-2, H-6), 8.28 (s, 1H, pyridine H-3),
8.22 (s, 1H, pyridine H-5), 7.57–7.48 (m, 3H, 6-phenyl H-3, H-4,
H-5), 7.44–7.33 (m, 3H, 4-phenyl H-2, H-5, H-6), 6.94 (d,
J = 6.9 Hz, 1H, 4-phenyl H-4).
4.3.18. Synthesis of 3-(2-(furan-2-yl)-6-phenylpyridin-4-yl)-
phenol (42)
The procedure described in Section 4.3 was employed with 4b
(0.44 g, 2.00 mmol), anhydrous ammonium acetate (1.54 g,
20.00 mmol), 5 (R3 = d) (0.63 g, 2.00 mmol), and glacial AcOH
(2 mL) to yield a white solid (0.48 g, 1.55 mmol).
13C NMR (62.5 MHz, DMSO) d 158.27, 157.06, 154.15, 150.59,
150.41, 145.91, 139.02, 138.64, 130.42, 129.73, 129.07, 127.28,
121.39, 118.42, 117.69, 116.65, 114.45.
Rf (ethyl acetate/n-hexane 1:2, v/v): 0.39; LC/MS/MS: retention
time: 7.74 min; [MH]+: 314.24.
4.3.22. Synthesis of 4-(2-(furan-2-yl)-6-phenylpyridin-4-yl)-
phenol (49)
1H NMR (250 MHz, DMSO) d 9.72 (br, 1H, 4-phenyl 3-OH), 8.28–
8.24 (m, 2H, 6-phenyl H-2, H-6), 8.04 (d, J = 1.3 Hz, 1H, pyridine H-
5), 7.90 (dd, J = 1.6, 0.7 Hz, 1H, 2-furan H-5), 7.87 (d, J = 1.3 Hz, 1H,
pyridine H-3), 7.56–7.47 (m, 3H, 6-phenyl H-3, H-4, H-5), 7.37–
7.34 (m, 2H, 4-phenyl H-5, H-6), 7.33 (dd, J = 3.4, 0.6 Hz, 1H, 2-fur-
an H-3), 7.29 (br, 1H, 4-phenyl H-2), 6.93 (m, 1H, 4-phenyl H-4),
6.71 (dd, J = 3.4, 1.7 Hz, 1H, 2-furan H-4).
The procedure described in Section 4.3 was employed with 4c
(0.44 g, 2.00 mmol), anhydrous ammonium acetate (1.54 g,
20.00 mmol), 5 (R3 = d) (0.63 g, 2.00 mmol), and glacial AcOH
(2 mL) to yield a light orange solid (0.50 g, 0.96 mmol).
Rf (ethyl acetate/n-hexane 1:2, v/v): 0.42; LC/MS/MS: retention
time: 7.44 min; [MH]+: 314.24.
1H NMR (250 MHz, DMSO) d 9.90 (br, 1H, 4-phenyl 4-OH), 8.26–
8.24 (m, 2H, 6-phenyl H-2, H-6), 8.04 (s, 1H, pyridine H-5), 7.88 (s,
2H, 2-furan H-5, pyridine H-3), 7.86 (d, J = 8.6 Hz, 2H, 4-phenyl H-
2, H-6), 7.55–7.43 (m, 3H, 6-phenyl H-3, H-4, H-5), 7.30 (d,
J = 3.2 Hz, 1H, 2-furan H-3), 6.94 (d, J = 8.6 Hz, 2H, 4-phenyl H-3,
H-5), 6.70 (dd, J = 3.2, 1.7 Hz, 1H, 2-furan H-4).
13C NMR (62.5 MHz, DMSO) d 158.27, 156.92, 153.40, 149.75,
149.26, 144.61, 139.04, 138.60, 130.51, 129.60, 128.97, 127.17,
118.09, 116.62, 116.51, 114.56, 114.03, 112.64, 109.76.
4.3.19. Synthesis of 3-(6-phenyl-2,20-bipyridin-4-yl)phenol (43)
The procedure described in Section 4.3 was employed with
4b (0.44 g, 2.00 mmol), anhydrous ammonium acetate (1.54 g,
20.00 mmol), 5 (R3 = e) (0.65 g, 2.00 mmol), and glacial AcOH
(2 mL) to yield a white solid (0.26 g, 0.80 mmol).
13C NMR (62.5 MHz, DMSO) d 159.14, 156.78, 153.58, 149.33,
149.18, 144.41, 138.78, 129.45, 128.89, 128.69, 127.97, 127.12,
116.15, 115.61, 113.68, 112.56, 109.51.
Rf (ethyl acetate/n-hexane 1:2, v/v): 0.39; LC/MS/MS: retention
time: 7.66 min; [MH]+: 325.30.
4.3.23. Synthesis of 4-(6-phenyl-2,40-bipyridin-4-yl)phenol (52)
The procedure described in Section 4.3 was employed with 4c
(0.44 g, 2.00 mmol), anhydrous ammonium acetate (1.54 g,
1H NMR (250 MHz, DMSO) d 9.75 (br, 1H, 4-phenyl 3-OH), 8.74
(d, J = 4.7 Hz, 1H, 2-pyridine H-60), 8.63 (d, J = 8.0, 1H, 2-pyridine H-

R. Karki et al. / Bioorg. Med. Chem. 18 (2010) 3066–3077
3077
20.00 mmol), 5 (R3 = g) (0.65 g, 2.00 mmol), and glacial AcOH
(2 mL) to yield a creamy white solid (0.40 g, 1.25 mmol).
Rf (ethyl acetate/n-hexane 1:1, v/v): 0.22; LC/MS/MS: retention
time: 6.66 min; [MH]+: 325.29.
each well was exchanged with 0.1 mL aliquots of medium contain-
ing graded concentrations of compounds. On day 4, each well was
added with 5 lL of the cell counting kit-8 solution (Dojindo, Japan),
then incubated for additional 4 h under the same condition. The
absorbance of each well was determined by an Automatic Elisa
Reader System (Bio-Rad 3550) at 450 nm wavelength. For determi-
nation of the IC50 values, the absorbance readings at 450 nm were
fitted to the four-parameter logistic equation. Adriamycin, etopo-
side, and camptothecin were purchased from Sigma and used as
positive controls.
1H NMR (250 MHz, DMSO) d 9.92 (br, 1H, 4-phenyl 4-OH), 8.75
(dd, J = 4.7, 1.4 Hz, 2H, 2-pyridine H-20, H-60), 8.34–8.27 (m, 4H, 6-
phenyl H-2, H-6, 2-pyridine H-30, H-50), 8.27 (s, 1H, pyridine H-3),
8.23 (s, 1H, pyridine H-5), 7.96 (d, J = 8.6 Hz, 2H, 4-phenyl H-2, H-
6), 7.57–7.48 (m, 3H, 6-phenyl H-3, H-4, H-5), 6.95 (d, J = 8.6 Hz,
2H, 4-phenyl H-3, H-5).
13C NMR (62.5 MHz, DMSO) d 159.24, 156.96, 154.02, 150.53,
149.93, 146.09, 138.81, 129.59, 129.01, 127.90, 127.22, 121.34,
117.38, 116.67, 116.10.
Acknowledgements
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-
0007620).
4.4. Pharmacology
4.4.1. Assay for DNA topoisomerase I inhibition in vitro
DNA topo I inhibition assay was determined following the
method reported by Fukuda et al.29 with minor modifications.
The test 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 topoisomerase I (TopoGEN INC., USA) was incubated without
and with the prepared compounds at 37 °C for 30 min in the relax-
ation buffer (10 mM Tris–HCl (pH 7.9), 150 mM NaCl, 0.1% bovine
serum albumin, 1 mM spermidine, 5% glycerol). The reaction in the
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USA) was incubated without and with the prepared compounds
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lg/mL bovine
l
l
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4.4.3. Cytotoxicity assay
Five different cancer cell lines were used: MDA-MB231 (human
breast adenocarcinoma 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), and assay was carried out according to
previous method.29 Cancer cells were cultured according to the
supplier’s instructions. Cells were seeded in 96-well plates at a
density of 2–4 ꢀ 104 cells per well and incubated 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