Synthesis and molecular docking studies of novel 2-chloro-pyridine derivatives containing flavone moieties as potential antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2010.05.080

A series of novel 2-chloro-pyridine derivatives containing flavone, chrome or dihydropyrazole moieties as potential telomerase inhibitors were synthesized. The bioassay tests showed that compounds 6e and 6f exhibited some effect against gastric cancer cell SGC-7901 with IC₅₀ values of 22.28 ± 6.26 and 18.45 ± 2.79 μg/mL, respectively. All title compounds were assayed for telomerase inhibition by a modified TRAP assay, the results showed that compound 6e can strongly inhibit telomerase with IC₅₀ value of 0.8 ± 0.07 μM. Docking simulation was performed to position compound 6e into the active site of telomerase (3DU6) to determine the probable binding model.

Full text of DOI:10.1016/j.bmcl.2010.05.080

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4163–4167
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and molecular docking studies of novel 2-chloro-pyridine
derivatives containing flavone moieties as potential antitumor agents
Xin-Hua Liu a,b, Hui-Feng Liu b, Xu Shen a, Bao-An Song c, Pinaki S. Bhadury c, Hai-Liang Zhu a,b,
,
*
Jin-Xing Liu b, Xing-Bao Qi d
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
b School of Chemistry and Chemical Engineering, Anhui University of Technology, Maanshan 243002, PR China
c Key Laboratory of Green Pesticide and Agriculture Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, PR China
d School of Pharmacy, China Pharmaceutical University, Nanjing 210009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 2-chloro-pyridine derivatives containing flavone, chrome or dihydropyrazole moieties as
potential telomerase inhibitors were synthesized. The bioassay tests showed that compounds 6e and 6f
exhibited some effect against gastric cancer cell SGC-7901 with IC50 values of 22.28 6.26 and
Received 28 February 2010
Revised 16 April 2010
Accepted 14 May 2010
Available online 24 May 2010
18.45 2.79
ified TRAP assay, the results showed that compound 6e can strongly inhibit telomerase with IC50 value of
0.8 0.07 M. Docking simulation was performed to position compound 6e into the active site of telome-
rase (3DU6) to determine the probable binding model.
lg/mL, respectively. All title compounds were assayed for telomerase inhibition by a mod-
l
Keywords:
Synthesis
2-Chloro-pyridine
Flavone
Ó 2010 Elsevier Ltd. All rights reserved.
Molecular docking
Antitumor agent
Telomerase remains active in the early stages of life maintain-
ing telomere length and the chromosomal integrity of frequently
dividing cells. It turns dormant in most somatic cells during adult-
hood.1 In cancer cells, however, telomerase gets reactivated and
works tirelessly to maintain the short length of telomeres of rap-
idly dividing cells, leading to their immortality.2 The essential role
of telomerase in cancer and aging makes it an important target for
the development of therapies to treat cancer and other age-associ-
ated disorders. Telomere and telomerase are closely related to the
occurrence and development of gastric cancer.3
Pyridine derivatives find several applications in pharmaceutical
and in agrochemical fields.4 Kim et al. have reported bioisosteres of
terpyridine with considerable protein kinase C (PKC) inhibitory
activity and antitumor cytotoxicities against several human cancer
cell lines.5 Furthermore, 2-pyridine, a small bioactive molecule, is
an important pharmacophore that can form hydrogen-bonded
structures similar to those encountered with the base-pairing
mechanism in DNA and RNA.6,7 In view of their importance as
drugs, biologically active natural products, and in other related
applications, extensive studies have been carried out on the syn-
thesis of pyridine compounds in recent years. On the other hand,
flavonoids belong to an important class of compounds consisting
of more than 5000 polyphenolic compounds that occur in nature
in several foods of plant origin. These compounds are often charac-
terized by the presence of a common phenylbenzopyrone linkage
(C6–C3–C6) in their structures. They have a wide range of biolog-
ical activities, for example, antimutagenic and antiproliferative
activities, can act as antioxidants and are usually involved in cell
signaling, cell cycle regulation, and angiogenesis.8–11 A large num-
bers of in vitro studies have been conducted on the potential anti-
cancer activity of flavonoids in various cells including human oral
cancer system.12 Significant contribution was made in this field by
Elattar and Virji who reported appreciable inhibitory effects of tea
polyphenols, curcumin, genistein, quercetin, and cisplatin on the
growth of oral cancer cell lines SCC-25.13
Based on these reports, we considered the possibility of intro-
ducing heterocyclic flavone moiety into the parent 2-chloro-pyri-
dine unit to design novel structures with enhanced anticancer
activities. Since there are only a very few systematic reports on
the synthetic methodology and evaluation of anticancer activities
of these compounds, we prepared herein a series 2-chloro-pyridine
derivatives containing flavone and other heteroaromatic derivatives
Abbreviations: MTT, 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2 h-tetrazolium
bromide; DMSO, dimethyl sulfoxide; DMF, dimethyl formamide; MH, Mueller–
Hinton; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent
assay; TRAP, telomere repeat amplification protocol.
* Corresponding author. Tel./fax: +86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.080

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X.-H. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4163–4167
and tested their activities against gastric cancer cell SGC-7901. In
order to elucidate the potential mechanism by which the title com-
pounds induce anticancer activity, docking simulation was per-
formed to position selected compounds into the active site of
telomerase 3DU6.
13C NMR (CDCl3, 125 MHz): d 20.9, 24.1, 70.8, 40.3, 55.3, 113.7,
120.4, 124.7, 127.5, 128.8, 130.1, 131.2, 138.0, 146.6, 149.9,
152.1, 158.7, 169.9; ESI-MS: 343.1 (C18H18ClN3O2, [M+H]+); Anal.
Calcd for C18H18ClN3O2: C, 62.88; H, 5.28; N, 12.22. Found: C,
63.05; H, 5.21; N, 12.44.
The synthetic routes to intermediates 1, 10, and 3 are shown in
Scheme 1. Compound 1 was prepared according to the literature
method as described.14 Compounds 10 and 3 were prepared by fol-
lowing the procedures published previously.15,16
The single-crystal structure of compound 4b was determined by
X-ray crystallography. Colorless crystals, yield, 82%; mp 164–
165 °C; crystal data of 4b: C18H18ClN3O2, M = 343.8, monoclinic,
space group P2(1)/c; a = 12.2753(12), b = 8.0425(8), c = 18.1766(19)
The compounds 2a and 2b (Scheme 2) were synthesized by the
reaction of 2-chloro-5-(chloromethyl) pyridine 1 with chromen 10
in presence of catalyst Et3N in chloroform at 40 °C. For the prepa-
ration of title compounds 4a, 4b, and 6a–6f, 2-chloro-5-(chloro-
methyl) pyridine was slowly added to a well-stirred mixture of
5-aryl-dihydropyrazole, flavone, KCO3, and KI in DMF at 40 °C.
The temperature of the system was raised and the reaction mixture
was refluxed for 5 h, the solvent was removed in vacuo and the
crude residue was purified by chromatography on SiO2 (acetone/
petroleum, v/v = 3:1) to give the compounds as colorless solids.
The spectral data can be found in the Supporting information.17
Compound 2a: 3-((6-Chloropyridin-3-yl)methoxy)-2-phenyl-4H-
chromen-4-one: Colorless crystals, yield, 71%; mp 235–236 °C; 1H
NMR (CDCl3, 300 MHz): d 5.05 (s, 2H, –CH2–), 7.09–8.20 (m, 10H,
flavone-H and pyridine-H), 8.21 (s, 1H, pyridine, 6-H), 8.24 (dd,
1H, J = 7.86 and 1.44 Hz, flavone, 5-H); 13C NMR (CDCl3,
125 MHz): d 71.2, 121.4, 123.0, 123.7, 124.0, 124.2, 131.8, 132.0,
135.5, 139.1, 140.6, 142.4, 146.7, 150.1, 151.5, 152.3, 158.7,
160.4, 182.3; ESI-MS: 364.2 (C21H14ClNO3, [M+H]+); Anal. Calcd
for C21H14ClNO3: C, 69.33; H, 3.88; N, 3.85. Found: C, 69.71; H,
4.11; N, 3.77.
(Å); a = 90, b = 109.74, c
= 90(°), V = 1689.1(3) nm3, T = 173(2) K,
Z = 4, Dc = 1.352 g/cm3, F(0 0 0) = 720. Reflections collected/unique:
10,914/2941, fine, R1 = 0.0431, wR(F2) = 0.1123.
The molecular structure of the compound 4b is shown in Fig-
ure 1. Crystallographic data (excluding structure factors) for the
structure have been deposited with the Cambridge Crystallographic
Data Center as supplementary publication No. CCDC-762238.
Compound 6a: 7-((6-Chloropyridin-3-yl)methoxy)-5-hydroxy-2-
phenyl-4H-chromen-4-one: Colorless crystals, yield, 64%; mp 221–
222 °C; 1H NMR (DMSO, 300 MHz): d 5.32 (s, 2H, –CH2–), 6.53 (s,
1H, flavone, 6-H), 6.95 (s, 1H, flavone, 8-H), 7.07 (s, 1H, flavone,
3-H), 7.58–8.09 (m, 5H, flavone, 20,30,40,50,60-H), 8.10–8.56 (m, 2H,
pyridine, 3,4-H), 8.57 (s, 1H, pyridine, 6-H), 12.83 (s, 1H, flavone,
5-OH); 13C NMR (CDCl3, 125 MHz): d 72.0, 95.0, 96.9, 104.8,
105.1, 123.2, 126.1, 128.4, 129.0, 131.1, 132.4, 139.2, 148.1,
150.3, 150.9, 163.7, 163.9, 167.8, 183.1; ESI-MS: 380.0
(C21H14ClNO4, [M+H]+); Anal. Calcd for C21H14ClNO4: C, 66.41; H,
3.72; N, 3.69. Found: C, 66.31; H, 4.04; N, 3.77.
In the screening assay studies, all the compounds were evalu-
ated for their cytotoxic activity against gastric SGC-7901 cell line.18
The cell was allowed to proliferate in presence of tested material
for 48 h, and the results are reported in terms of IC50 values (Ta-
ble 1). It is obvious from the data that compounds 6e and 6f exhib-
ited inhibitory activities to a certain degree against the gastric cell
Compound 4a: 1-(5-(2-((6-Chloropyridin-3-yl)methoxy)phenyl)-
3-methyl-4,5-dihydropyrazol-1-yl): Ethanone, Colorless crystals,
yield, 75%; mp 156–157 °C; 1H NMR (CDCl3, 300 MHz): d 1.94 (s,
3H, Me), 2.32 (s, 3H, Me), 2.58 (dd, J = 18.1 and 4.7 Hz, 1H, pyrazole,
4-Ha), 3.28 (dd, J = 11.8 and 18.2 Hz, pyrazole, 1H, 4-Hb), 5.08 (s,
2H, CH2–), 5.68 (dd, J = 4.7 and 11.7 Hz, 1H, pyrazole, 5-H), 6.89–
7.79 (m, 6H, ArH and pyridine, 3,4-H), 8.46 (s, 1H, pyridine, 6-H);
SGC-7901 with the IC50 values of 22.28 6.26 and 18.45 2.79
mL, respectively.
lg/
Among the different groups of synthesized compounds, 2-
chloro-pyridine derivatives 2a,b and 4a,b containing chrome and
a
c
b
CH3
Cl
CH2Cl
CH3
Cl
CH3
N
N
N
N
1
O
O
O
OH
O
H
CH3
d
R1
+
R
OH
R1
O
O
e
OH
1'
H3C
O
O
N
f
g
N
OH
H
HO
HO
3
H3C
O
1'a: R1=H
1'b: R1=4'-OMe
Scheme 1. Synthesis of intermediate compounds. Reagent and conditions: (a) H2O2, CH3COOH, 15–20 atm, 115–190 °C, 6 h; (b) POCl3, CH2Cl2, ꢀ10 °C, 3 h; (c) Cl2, KCO3, CCl4,
60 °C, 6 h; (d) KOH, CH3OH, HCl, 25 °C, 10 h; (e) KOH, CH3OH, H2O2, HCl, 25 °C, 4 h; (f) NaOH, CH3COCH3, HCl, 20 °C, 15 h; (g) 80% NH2–NH2ꢁH2O, 98% CH3COOH, reflux, 2 h.

X.-H. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4163–4167
4165
R1
O
h
1
1'
+
2a: R1=H
O
2b: R1=4'-OMe
O
N
Cl
N
H3C
O
Cl
N
4a
N
H3C
H3C
O
I
1
3
+
N
4b
N
O
Cl
N
H3C
O
O
HO
OH
HO
O
HO
O
O
OH
O
O
O
OH
5a
5b
5c
HO
O
O
HO
O
O
HO
O
OH
OH
O
+
OH
5e
O
5f
5d
J
1
6
5
Cl
N
Cl
N
O
O
HO
N
O
O
O
O
O
O
O
N
Cl
OH
O
OH
6a
6c
O
6b
O
N
Cl
N
Cl
Cl
N
O
O
O
O
O
O
O
N
O
OH
6e
O
Cl
O
Cl
6d
6f
Scheme 2. Synthesis of title compounds. Reagent and conditions: (h) CHCl3, Et3N, 40 °C, 4 h; (I) KCO3, KI, DMF, reflux 5 h; (J) KCO3, KI, DMF, reflux 5–8 h.
Table 1
Cytotoxic activity of the synthesized compounds against SGC-7901 cell linea
Compound
IC50
SGC-7901
(
l
g/mL)b
Compound
IC50
SGC-7901
(l
g/mL)b
d
2a
2b
4a
4b
—
—
—
—
6b
6c
6d
6e
25.94 2.41
31.93 4.49
35.17 4.28
22.28 6.26
18.45 2.79
7.38 0.98
d
d
d
d
6a
—
6f
5-Fluorouracilc
7.38 0.98
5-Fluorouracilc
Figure 1. ORTEP drawing of 4b.
Negative control DMSO, no activity.
a
The data represented the mean of three experiments in triplicate and were
expressed as means SD; only descriptive statistics were done in the text.
b
The IC50 value was defined as the concentration at which 50% survival of cells
5-aryl-dihydropyrazole moieties, respectively, displayed poor
activity against gastric cell SGC-7901, whereas 2-chloro-pyridine
derivatives 6a–6f containing flavones, in general, showed rela-
was observed. The results are listed in the table.
c
Used as a positive control.
d
No significant activity.

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X.-H. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4163–4167
Table 2
Biological properties of test compounds
Compound
IC50
(
lM) Taq polymerase
IC50
(
lM) telomerase
Selectivity indexa (SI)
2a
2b
4a
4b
6a
6b
6c
6d
Noc
Noc
Noc
Noc
46.7 1.1
20.9 0.78
3.5 0.16
2.5 0.06
3.1 0.09
0.8 0.07
3.0 0.13
2.4 0.8
—
—
—
—
1.3
1.9
3.0
2.1
2.5
1.6
Noc
53.2 2.2
Noc
27.5 2.2
6.8 2.2
7.5 2.2
6.4 2.2
2.0 2.2
5.0 2.2
6e
6f
Ethidium bromideb
No, means were not observed in the tested concentration range 0–60 lM.
a
Ratio between the drug concentrations at which 50% Taq polymerase/telomerase inhibition was observed.
Ethidium bromide is reported as a control. The inhibition constant of ethidium toward telomerase has been reported previously.
Indicates the highest concentration the compounds were tested.
b
c
cially the IC50 value of compound 6e was as low as 0.8 0.07
which was even better than that of the ethidium bromide.
lM,
In an effort to elucidate the mechanism by which the title com-
pounds can induce anticancer activity in the gastric cell SGC-7901
and to establish an SAR based on our experimental studies, molec-
ular docking of the potent inhibitors 6e into ATP binding site of tel-
omerase was performed to simulate a binding model derived from
telomerase structure (3DU6 PDB). See Figure 2. Visual inspection of
the pose of 6e into the ATP-site revealed that four more optimal
intramolecular hydrogen bonds are observed (N–Hꢁ ꢁ ꢁO: 2.98 Å,
with amino hydrogen group of Arg 194; N–Hꢁ ꢁ ꢁO: 2.67 Å, with ami-
no hydrogen group of Arg 194, Oꢁ ꢁ ꢁNꢁ ꢁ ꢁO, 59.49°; N–Hꢁ ꢁ ꢁO: 2.86 Å,
with amino hydrogen group of Asp 254; N–Hꢁ ꢁ ꢁO: 2.29 Å, with ami-
no hydrogen group of Gln 308). Also the 2-chloro-pyridine ring
projects into a hydrophobic region, which is comprised of the side
chains of Lys 189, Asn 369, Ile 252, Arg 253, that is important for
the potent inhibitory activity of 6e. These residues influenced the
accessibility of the hydrophobic pocket that flanks the ATP binding
site, and their size can be a key factor in controlling telomerase
selectivity. In the other end of the ATP-binding pocket, the N of an-
other 2-chloro-pyridine interacted with the residue Gly 314, which
made the 3D structure more stable.
In summary, we prepared a series of novel 2-chloro-pyridine
derivatives containing flavone, chrome or dihydropyrazole unit as
potential telomerase inhibitors. A systematic SAR study illustrated
that the optimal activity could be obtained with a selected class of
compounds. Thus, the best potency was achieved when the 2-
chloro-pyridine core was attached with flavone. The results
showed that compounds 6e and 6f had certain activity against gas-
tric cell SGC-7901, compound 6e can strongly inhibit telomerase
Figure 2. Molecular docking modeling of compound 6e (A) with telomerase; the
small molecule and the critical interaction of 3DU6 are represented by sticks. Panel
is a view into the active site cavity. (B) Schematic representation of the binding
mode of 6e in the ATP binding site of 3DU6.
with IC50 value of 0.8 0.07 lM. Docking simulation was per-
formed to position compound 6e into the telomerase 3DU6 active
site. The results show that compound 6e can bind well with the tel-
omerase active site and act as potential telomerase inhibitor.
Supporting information: CCDC-762238 contains the supplemen-
tary crystallographic data for this paper. These data can be ob-
tained free of charge via the URL http://www.ccdc.cam.ac.uk/
conts/retrieving.html (or from the CCDC, 12 Union Road, Cam-
bridge CB2 1EZ, UK; fax: (+44) 1223 336033; e-mail: deposit@ccdc.
cam.ac.uk).
tively higher inhibitory potency against the gastric SGC-7901 cell
(Table 1).
All purified title compounds were assayed for telomerase inhibi-
tion by a modified TRAP20 assay, using a SGC-7901 cell extract. Mod-
ified TRAP is a powerful technique and could give us some
information about small molecules inhibiting telomere elongation
qualitatively and quantitatively.21 To avoid false positive results
due to drug interference with the amplification step, Taq polymer-
ase inhibition was additionally monitored. The results are summa-
rized in Table 2, where a selectivity index (SI, ratio between IC50
for Taq polymerase vs telomerase inhibition) is also included. The
results suggested that the telomerase inhibitory abilities of certain
2-chloro-pyridine derivatives containing flavone were potent. Espe-
Acknowledgments
The authors thank the National Natural Science Foundation of
China (No. 20902003), the Key Research Projects of University Nat-
ural Science, Anhui Province (No. KJ2009A011Z), the Young College
Teachers Research Projects of Anhui Province (No. 2008JQ1030).

X.-H. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4163–4167
4167
125 MHz): d 56.3, 70.8, 103.2, 110.1, 113.8, 117.7, 123.9, 124.1, 125.2, 128.0,
132.3, 132.6, 137.8, 146.4, 150.9, 153.7, 158.6, 160.3, 166.5, 177.6; ESI-MS:
393.4 (C22H16ClNO4, [M+H]+); Anal. Calcd for C22H16ClNO4: C, 67.10; H, 4.10; N,
3.56. Found: C, 66.84; H, 4.41; N, 3.25. Compound 6d: Colorless crystals, yield,
81%; mp 205–206 °C; 1H NMR (DMSO, 300 MHz): d 3.84 (s, 3H, OMe), 5.16 (s,
2H, –CH2–), 6.91–8.50 (m, 11H, flavone-H and pyridine-H); 13C NMR (DMSO,
125 MHz): d 56.1, 71.2, 103.3, 103.7, 109.6, 113.4, 117.6, 123.0, 124.1, 128.0,
131.3, 131.9, 138.1, 144.7, 151.8, 155.2, 160.1, 163.3, 166.9, 182.9; ESI-MS:
394.2 (C22H16ClNO4, [M+H]+); Anal. Calcd for C22H16ClNO4: C, 67.10; H, 4.10; N,
3.56. Found: C, 66.99; H, 4.05; N, 3.83. Compound 6e: Colorless crystals, yield,
42%; mp 202–203 °C; 1H NMR (DMSO, 300 MHz): d 5.29 (s, 4H, 2CH2–), 6.50–
8.55 (m, 13H, flavone-H and pyridine-H), 12.91 (s, 1H, flavone, 5-OH); 13C NMR
(DMSO, 125 MHz): d 71.1, 97.3, 98.2, 103.7, 104.8, 113.7, 123.1, 123.9, 128.0,
132.1, 138.9, 146.6, 151.4, 161.5, 162.3, 163.8, 164.7, 169.7, 182.9; ESI-MS:
522.0 (C27H18Cl2N2O5, [M+H]+); Anal. Calcd for C27H18Cl2N2O5: C, 62.20; H,
3.48; N, 5.37. Found: C, 62.04; H, 3.17; N, 5.65. Compound 6f: Colorless crystals,
yield, 39%; mp 214–215 °C; 1H NMR (DMSO, 300 MHz): d 5.11 (s, 2H, –CH2–),
5.16 (s, 2H, –CH2–), 6.90–8.54 (m, 14H, flavone-H and pyridine-H); 13C NMR
(DMSO, 125 MHz): d 71.2, 103.7, 104.2, 108.8, 113.2, 117.1, 121.9, 124.1, 128.0,
132.3, 132.5, 138.7, 146.4, 151.5, 155.7, 163.2, 163.7, 168.2, 182.9; ESI-MS:
506.4 (C27H18Cl2N2O4, [M+H]+); Anal. Calcd for C27H18Cl2N2O4: C, 64.17; H,
3.59; N, 5.54. Found: C, 64.00; H, 3.81; N, 5.79.
References and notes
1. Wright, W. E.; Shay, J. W. Curr. Opin. Genet. Dev. 2001, 11, 98.
2. Bodnar, A. G.; Ouellette, M.; Frolkis, M.; Holt, S. E.; Chiu, C. P.; Morin, G. B.;
Harley, C. B.; Shay, J. W.; Lichtsteiner, S.; Wright, W. E. Science 1998, 279,
349.
3. Andrew, J. G.; Anthony, P. S.; Emmanuel, S. Nature 2008, 455, 633.
4. (a) Hanket, T.; Brunne, R. M.; Miller, H.; Reichel, F. Angew. Chem., Int. Ed. 1999,
38, 643; (b) Gavin, D. H. Tetrahedron 2004, 60, 6043.
5. Kim, D. S. H. L.; Ashendel, C. L.; Zhou, Q.; Chang, C. T.; Lee, E. S.; Chang, C. J.
Bioorg. Med. Chem. Lett. 1998, 8, 2695.
6. Tipparaju, S. K.; Joyasawal, S. Bioorg. Med. Chem. Lett. 2008, 18, 3565.
7. Hanessian, S.; Simard, D.; Bayrakdarian, M.; Therrien, E.; Nilsson, I. Bioorg. Med.
Chem. Lett. 2008, 18, 1972.
8. Neuhouser, M. L. Nutr. Cancer 2004, 50, 1.
9. Min, L. W. Cancer Treat. Rev. 2009, 35, 57.
10. Yukihiro, A.; Tomohiro, I.; Kenji, O.; Munekazu, I.; Yoshinori, N. Bioorg. Med.
Chem. 2008, 16, 2803.
11. Jared, R.; Mays, S. A.; Hill, J. T.; Moyers, B. S.; Blagg, J. Bioorg. Med. Chem. 2010,
18, 249.
12. Hsu, S.; Singh, B.; Schuster, G. Oral Oncol. 2004, 40, 461.
13. Elattar, T. M.; Virji, A. S. Anticancer Res. 2000, 20, 3459.
14. Werbitzky, O.; Studer. P. U.S. Patent 6022974, February 8, 2000; Chem. Abstr.
1997, 120, 134302.
15. Liu, X. H.; Cui, P.; Song, B. A.; Bhadury, P. S.; Zhu, H. L.; Wang, Sh. F. Bioorg. Med.
Chem. 2008, 16, 4075.
18. The cytotoxicity evaluation was conducted by using a modified procedure as
described in the literature.19 Briefly, target tumor cells were grown to log
phase in RPMI 1640 medium supplemented with 10% fetal bovine serum. After
reaching a dilution of 3 ꢂ 104 cells mLꢀ1 with the medium, 100
lL of the
obtained cell suspension was added to each well of 96-well culture plates.
Subsequently, incubation was performed at 37 °C in 5% CO2 atmosphere for
24 h before subjecting to cytotoxicity assessment. Tested samples at pre-set
concentrations were added to six-wells with 5-fluorouracil being employed as
16. Prakash, O.; Kumar, R.; Parkash, V. Eur. J. Med. Chem. 2008, 43, 435.
17. Compound 2b: Colorless crystals, yield, 76%; mp 226–227 °C; 1H NMR (CDCl3,
300 MHz): d 3.89 (s, 3H, –OMe), 5.31 (s, 2H, –CH2–), 6.89–8.48 (m, 11H,
flavone-H and pyridine-H); 13C NMR (CDCl3, 125 MHz): d 56.2, 69.4, 118.5,
123.8, 124.0, 125.3, 125.5, 128.0, 131.3, 131.8, 135.0, 140.1, 140.7, 144.3, 146.2,
150.9, 157.5, 160.2, 161.3, 181.7; ESI-MS: 393.1 (C22H16ClNO4, [M+H]+); Anal.
Calcd for C22H16ClNO4: C, 67.10; H, 4.10; N, 3.56. Found: C, 66.89; H, 4.39; N,
3.21. Compound 6b: Colorless crystals, yield, 60%; mp 190–192 °C; 1H NMR
(DMSO, 300 MHz): d 5.31 (s, 2H, –CH2–), 6.52–8.56 (m, 10H, flavone-H and
pyridine-H), 9.60 (s, 1H, flavone, 7-OH), 12.97 (s, 1H, flavone, 5-OH); 13C NMR
(DMSO, 125 MHz): d 70.7, 98.1, 98.5, 104.3, 113.7, 123.0, 123.3, 124.7, 128.2,
133.0, 138.3, 146.2, 151.1, 154.4, 155.9, 160.9, 163.2, 167.1, 179.9; ESI-MS:
396.9 (C21H14ClNO5, [M+H]+); Anal. Calcd for C21H14ClNO5: C, 63.73; H, 3.57; N,
3.54. Found: C, 64.01; H, 3.35; N, 3.72. Compound 6c: Colorless crystals, yield,
78%; mp 188–189 °C; 1H NMR (DMSO, 300 MHz): d 3.84 (s, 3H, OMe), 5.16 (s,
2H, –CH2–), 6.91–8.49 (m, 11H, flavone-H and pyridine-H); 13C NMR (DMSO,
a positive reference. After 48 h exposure period, 25
2.5 mg mLꢀ1 of MTT was added to each well. After 4 h, the medium was
replaced by 150 L DMSO to dissolve the purple formazan crystals produced.
lL of PBS containing
l
The absorbance at 570 nm of each well was measured on an ELISA plate reader.
The data represented the mean of three experiments in triplicate and were
expressed as means SD using Student’s t test. The IC50 value was defined as
the concentration at which 50% of the cells could survive.
19. Chen, X. Y.; Plasencia, C.; Hou, Y.; Neamati, N. J. Med. Chem. 2005, 48, 1098.
20. Kim, N. W.; Piatyszek, M. A.; Prowse, K. R.; Harley, C. B.; West, M. D.; Ho, P. L.;
Coviello, G. M.; Wright, W. E.; Weinrich, S. L.; Shay, J. W. Science 1994, 266,
2011.
21. Han, H.; Hurly, L. H.; Salazar, M. Nucleic Acids Res. 1999, 27, 537.