Design, synthesis and biological evaluation of chrysin long-chain derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.bmc.2009.12.048

A series of long-chain derivatives of chrysin (compounds 3-22) were synthesized to evaluate for their antiproliferative activities against the human liver cancer cell line HT-29 and EGFR inhibitory activity. Among the compounds tested, compounds hexadecyl

Full text of DOI:10.1016/j.bmc.2009.12.048

Bioorganic & Medicinal Chemistry 18 (2010) 1117–1123
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of chrysin long-chain derivatives
as potential anticancer agents
*
Peng-Cheng Lv, Kai-Rui Wang, Qing-Shan Li, Jin Chen, Juan Sun, Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of 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 long-chain derivatives of chrysin (compounds 3–22) were synthesized to evaluate for their
antiproliferative activities against the human liver cancer cell line HT-29 and EGFR inhibitory activity.
Among the compounds tested, compounds hexadecyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetate (10) and N-hexadecyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yloxy)acetamide (20) dis-
Received 12 November 2009
Revised 15 December 2009
Accepted 16 December 2009
Available online 24 December 2009
played potent EGFR inhibitory activity with IC50 values of 0.048 lM and 0.035 lM), comparable to the
positive control erlotinib. Docking simulation of compounds 10 and 20 was carried out to illustrate the
binding mode of the molecular into the EGFR active site, and the result suggested that compound 10
and 20 can bind the EGFR kinase well. Thus, compounds 10 and 20 with potent EGFR inhibitory activity
would be potential anticancer agents.
Keywords:
Chrysin
Long-chain derivatives
Anticancer
HT-29
Ó 2009 Elsevier Ltd. All rights reserved.
EGFR
1. Introduction
our knowledge, few reports have been dedicated to the synthesis
and EGFR inhibitory activity of chrysin long-chain derivatives.19
Cancer chemotherapy has entered a new era of molecularly tar-
geted therapeutics, which is highly selective and not associated
with the serious toxicities of conventional cytotoxic drugs.1 Recep-
tor protein tyrosine kinases play a key role in signal transduction
pathways that regulate cell division and differentiation. Among
the growth factor receptor kinases that have been identified as
being important in cancer is epidermal growth factor receptor
(EGFR) kinase. Activation of EGFR may be because of overexpres-
sion, mutations resulting in constitutive activation, or autocrine
expression of ligand.2,3 The role of EGFR has been most thoroughly
studied in breast cancer, where it is overexpressed in 25–30% of
cases and is correlated with a poor prognosis. EGFR overexpression
is also seen in ovarian cancer,4 lung cancer (especially lung adeno-
carcinomas)5–7 and in hormone-refractory prostate cancer.8
Compounds that inhibit the kinase activity of EGFR and/or HER-
2 after binding of its cognate ligand are of potential interest as new
therapeutic antitumor agents.9,10
Flavonoids are natural polyphenolic phytochemicals that are
ubiquitous in plants and present in the average human diet. Chry-
sin is a natural flavonoid found in many plant extracts, honey, and
propolis, which has been reported to have many different biologi-
cal activities such as anti-viral, anti-cancer, anti-bactericidal, anti-
inflammatory, anti-allergic, DNA cleavage, vasodilator, anti-muta-
genic, anti-anxiolytic and anti-oxidant effects.11–18 However, to
Herein, in continuation to extend our research on anticancer com-
pounds with EGFR inhibitory activity,20,21 in this study, we de-
scribe the synthesis and the SAR of a series of chrysin long-chain
derivatives. Biological evaluation indicated that compounds 10
and 20 showed the most potent EGFR inhibitory activities with
IC50 values of 0.048 lM and 0.035 lM. Docking simulations were
performed using the X-ray crystallographic structure of the EGFR
in complex with inhibitors to explore the binding modes of these
compounds at the active site.
2. Chemistry
The routes adapted for the synthesis of chrysin long-chain deriv-
atives are outlined in Scheme 1. To a stirring solution of chrysin in
acetone, K2CO3 was added and then a mixture of ethyl bromoacetate
and acetone was added dropwise to give compound 2. Compound 2
was treated in Na2CO3 solution (DMSO) and after that most of the
volatiles were evaporated. The residue was dissolved in water and
the solution was adjusted to pH 2 by using HCl solution to give com-
pound 3. Compound 3 was suspended in DMF with stirring at room
temperature, then long-chain alcohol or long-chain amine, 1-ethyl-
3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDCꢀHCl)
were added. The reaction solution was stirred for 12 h at room
temperature. Then, compounds 3–22 were obtained by subsequent
purification with flash chromatography. All of the synthetic
compounds gave satisfactory analytical and spectroscopic data,
which were in full accordance with their depicted structures.
* Corresponding author. Tel./fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.048

1118
P.-C. Lv et al. / Bioorg. Med. Chem. 18 (2010) 1117–1123
O
O
K2CO3
HO
O
O
O
O
O
EtO
Br
+
OEt
acetone
OH
OH
1
O
5%Na2CO3
DMSO
O
O
O
HO
OH
2
O
EDC
DMF
O
O
O
R
+
OH
RO
OH
8
R=C12H25
R=C14H29
3
4
5
6
7
R=C6H13
R=C7H15
R=C8H17
R=C9H19
9
10 R=C16 33
H
11 R=C18 37
H
12 R=C20 41
H
R=C10H21
O
EDC
DMF
O
O
R
+
NH2
RHN
OH
O
18 R=C12H25
19 R=C14H29
13 R=C6H13
14 R=C7H15
15 R=C8H17
16 R=C9H19
20 R=C16 33
H
21 R=C18 37
H
22 R=C20 41
H
17 R=C10H21
Scheme 1. Synthesis routes of compounds 1–22.
(Table 1). Compounds 10 and 20 displayed potent inhibitory activ-
ity (IC50 = 0.048 M and 0.035 M, respectively).
3. Result and discussion
3.1. Biological activity
l
l
EGFR kinase inhibition IC50 values of long-chain ester and amide
derivatives are similarly parabolic curves with the increase in the
number of carbon atoms (Fig. 1). As the chain length increases,
the inhibitory activities of those derivatives are increased. When
the number of carbon atoms of long-chain part is more than 16,
the activity is no further increase. The results indicated that, chry-
sin coupled with long-chain could get good antiproliferative activ-
ity. However, when the carbon number of long-chain part is more
than 16 it gives a negative impact on the antiproliferative activity.
The in vitro antiproliferative activities of the chrysin long-chain
derivatives 3–22 was studied on the human liver cancer cell line
HT-29 by applying the MTT colorimetric assay. Compounds were
tested over a range of concentrations from 0.1 to 100
the calculated IC50 values, that is, the concentration (
compound able to cause 50% of cell death with respect to the con-
trol culture, are reported in Table 1. As shown in Table 1, among
the ester and amide derivatives, compounds 10 and 20 were found
l
g/mL, and
l
g/mL) of a
3.2. Binding mode of 20 into EGFR kinase
to show the most potent activity with IC50 values of 8.7 and 4.2
mL, which is comparable to the positive control Iressa
(IC50 = 9.8 g/mL).
Furthermore, compounds 3–22 were also evaluated for their
ability to inhibit the autophosphorylation of EGFR kinases using
a solid-phase ELISA assay. A number of synthesized compounds
displayed potent EGFR kinase inhibitory activity. The results have
the same trends with the antiproliferative activities against HT-
29 due to that EGFR is an important factor in colon cancer
lg/
The ICM Suite of Software provides a way to dock flexible ligand
and receptor, and to give intuitionistic structure insights of ligand/
enzyme interactions. Molecular docking of the most potent activity
compound 20 into the ATP binding site of EGFR kinase was per-
formed on the binding model based on the EGFR complex structure
(PDB code: 1M17,9) giving an explanation and understanding of
good activity observed. The binding models of compounds 10
l

P.-C. Lv et al. / Bioorg. Med. Chem. 18 (2010) 1117–1123
1119
Table 1
Antiproliferative activities data, inhibition of EGFR kinase and yields of compounds 3–
22
Compound
HT-29 IC50
(lg/mL)
EGFR Inhibition IC50
(lM)
Yield (%)
3
4
5
6
7
8
9
77 14
58 13
9.12 0.18
7.23 0.14
5.84 0.12
4.96 0.10
3.11 0.08
1.72 0.05
1.01 0.07
0.048 0.003
0.692 0.017
1.589 0.024
8.71 0.15
6.28 0.12
5.33 0.10
3.58 0.08
2.14 0.09
1.27 0.06
0.316 0.012
0.035 0.002
0.464 0.014
1.35 0.09
0.033 0.001
40
51
44
38
50
43
43
49
45
33
38
47
30
37
42
35
46
44
45
40
—
51
43 16
31
24 12
16
8.7 0.4
6
8
6
10
11
12
13
14
15
16
17
18
19
20
21
22
Iressa
15
20
3
5
67 17
65 14
51 12
39 21
25 11
16
8
7
4
Figure 2. Binding mode of compound 10 with EGFR kinase. For clarity, only
interacting residues are displayed. Ligand and interacting key residues are
represented as stick models, while the proteins are represented as ribbons. The
H-bond (purple) is displayed as line, and the green dots show the binding sites of
EGFR.
4.2 0.5
11
19
4
8
9.8 0.3
Figure 1. The curves of EGFR inhibition IC50 value and the number of carbon atoms.
Figure 3. Binding mode of compound 20 with EGFR kinase. For clarity, only
interacting residues are displayed. Ligand and interacting key residues are repre-
sented as stick models, while the proteins are represented as ribbons. The H-bond
(purple) is displayed as line, and the green dots show the binding sites of EGFR.
and 20 into EGFR are depicted in Figures 2 and 3. Docking studies
of compound 10 and 20 into the active site of EGFR provided well-
clustered solutions. In the binding model of compound 10 and
EGFR, the hydroxyl group and the carbonyl group of compound
10 form three hydrogen bonds with Met 769. Moreover, in the
binding model of compound 20 and EGFR, there are two hydrogen
bonds. Compound 20 is nicely bound to the region with the hydro-
xyl group project toward Thr766, with the hydroxyl group forming
a more optimal H-bond interaction, and carbonyl group of com-
pound 20 also forms hydrogen bond with Met 769. The molecular
docking results, along with the enzyme assay data, suggesting that
compound 10 and 20 are potential inhibitors of EGFR.
a solid-phase ELISA assay, the results have the same trend as the
antiproliferative activities assay. Moreover, docking simulations
were performed to give the probable binding modes of compound
10 and 20 into the ATP binding site of EGFR kinase.
5. Experimental section
5.1. Chemistry
All chemicals (reagent grade) used were purchased from Aldrich
(U.S.A) and Sinopharm Chemical Reagent Co., Ltd (China). Melting
points (uncorrected) were determined on a XT4 MP apparatus (Tai-
ke Corp., Beijing, China). TLC was run on the silica gel coated alu-
minum sheets (Silica Gel 60 GF254, E. Merk, Germany) and
visualized in UV light (254 nm). EI mass spectra were obtained
on a Waters GCT mass spectrometer, and 1H NMR spectra were re-
corded on a Bruker DPX-300, AV-300 or AV-500 spectrometer at
25 °C with TMS and solvent signals allotted as internal standards.
Chemical shifts were reported in ppm (d). Elemental analyses were
4. Conclusions
A series of long-chain derivatives of chrysin were synthesized to
evaluate for antiproliferative activities against the human cancer
cell line HT-29 and EGFR inhibitory activity. The results showed
that chrysin coupling with long-chain can get good antiprolifera-
tive activity and the compounds with 16 carbon atoms chain were
found to display potent antiproliferative activity. EGFR inhibitory
ability of these synthesized compounds was also evaluated using

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P.-C. Lv et al. / Bioorg. Med. Chem. 18 (2010) 1117–1123
performed on a CHN–O-rapid instrument and were within 0.4% of
the theoretical values.
5.2.3. Octyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetate (5)
Light yellow powder, yield: 44%. Mp: 58–60 °C. 1H NMR
(300 MHz, DMSO-d6, d ppm): 0.81 (t, J = 6.7 Hz, 3H); 1.22 (m,
10H); 1.56 (m, 2H); 4.13 (t, J = 6.2 Hz, 2H); 4.98 (s, 2H); 6.43 (d,
J = 2.2 Hz, 1H); 6.85 (d, J = 2.2 Hz, 1H); 7.07 (s, 1H); 7.62 (m, 3H);
8.10 (m, 2H); 12.81 (s, 1H). 13C NMR (DMSO-d6, d ppm): 182.4,
168.5, 164.0, 163.9, 163.6, 157.6, 132.5, 130.9, 129.5, 126.8,
105.7, 105.6, 98.9, 93.6, 65.4, 65.0, 31.5, 28.9, 28.8, 28.4, 25.6,
22.4, 14.2. MS (ESI): 424.2 ([M+H]+). Anal. Calcd for C25H28O6: C,
70.74; H, 6.65. Found: C, 70.95; H, 6.52.
5.1.1. Ethyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetate (1)
Chrysin (2.54 g 0.01 mol) and anhydrous potassium carbonate
(1.66 g 0.011 mol) were added in one portion to a stirred solution
of 100 mL acetone, then drop ethyl bromoacetate (1.84 g
0.011 mol) into the solution. The reaction mixture was stirred at
50–60 °C for 6 h. On cooling, the precipitate was removed by filtra-
tion, the filtrate was concentrated and the residue was purified by
column chromatography on silica gel, eluting with petroleum
ether/EtOAc (3:1) to give a light yellow solid 2.95 g (yield: 87%).
Mp: 145–148 °C. 1H NMR (300 MHz, DMSO-d6, d ppm): 1.21 (t,
J = 7.0 Hz, 3H); 4.20 (p, J = 2.2 Hz, 2H); 4.96 (s, 2H); 6.44 (d,
J = 2.2 Hz, 1H); 6.86 (d, J = 2.2 Hz, 1H); 7.05(s, 1H); 7.62 (m, 3H);
8.10 (m, 2H); 12.81 (s, 1H). MS (ESI):340.1 ([M+H]+). Anal. Calcd
for C19H16O6: C, 67.05; H, 4.74. Found: C, 66.77; H, 4.91.
5.2.4. Nonyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetate (6)
Light yellow powder, yield: 38%. Mp: 61–63 °C. 1H NMR
(300 MHz, DMSO-d6, d ppm): 0.82 (t, J = 6.7 Hz, 3H); 1.23 (m,
12H); 1.56 (m, 2H); 4.13 (t, J = 6.4 Hz, 2H); 4.98 (s, 2H); 6.44 (d,
J = 2.2 Hz, 1H); 6.85 (d, J = 2.2 Hz, 1H); 7.06 (s, 1H); 7.61 (m, 3H);
8.10 (m, 2H); 12.82 (s, 1H). 13C NMR (DMSO-d6, d ppm): 182.3,
167.8, 164.1, 163.5, 162.3, 157.5, 131.8, 131.4, 131.1, 129.0,
128.9, 126.2, 105.8, 98.4, 93.3, 65.7, 65.2, 31.5, 30.7, 29.5, 28.7,
28.4, 25.6, 22.4, 13.9. MS (ESI): 438.2 ([M+H]+). Anal. Calcd for
C26H30O6: C, 71.21; H, 6.90. Found: C, 71.49; H, 6.76.
5.1.2. 2-(5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetic acid (2)
A mixture of compound 1(0.680 g 0.002 mol) and 10 mL 5% so-
dium carbonate solution in 40 mL DMSO was stirred at 80–90 °C
for 10 h. Then the mixture was pour into 10% 300 mL dilute hydro-
chloric acid solution. After standing overnight, The yellow solid
was filtered off, washed with water, and dried in vacuo to give a
yellow solid 0.441 g (yield: 71%). Mp: 279–282 °C. 1H NMR
(300 MHz, DMSO-d6, d ppm): 4.96 (s, 2H); 6.41 (d, J = 2.2 Hz, 1H);
6.83 (d, J = 2.1 Hz, 1H); 7.05(s, 1H); 7.62 (t, J = 7.2 Hz, 3H); 8.10
(m, 2H); 12.81 (s, 1H). MS (ESI):312.1 ([M+H]+). Anal. Calcd for
C17H12O6: C, 65.39; H, 3.87%. Found: C, 65.14; H, 4.02.
5.2.5. Decyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetate (7)
Light yellow powder, yield: 50%. Mp: 64–66 °C. 1H NMR
(300 MHz, DMSO-d6, d ppm): 0.82 (t, J = 6.8 Hz, 3H); 1.23 (m,
14H); 1.56 (m, 2H); 4.13 (t, J = 6.4 Hz, 2H); 4.98 (s, 2H); 6.43 (d,
J = 2.4 Hz, 1H); 6.85 (d, J = 2.4 Hz, 1H); 7.07 (s, 1H); 7.61 (m, 3H);
8.10 (m, 2H); 12.81 (s, 1H). 13C NMR (DMSO-d6, d ppm): 182.5,
168.1, 165.3, 164.1, 162.8, 157.4, 132.2, 131.7, 131.2, 130.8,
129.2, 127.6, 106.7, 99.1, 94.5, 66.7, 65.3, 31.6, 30.7, 29.8, 29.4,
28.6, 28.2, 25.5, 22.3, 13.9. MS (ESI): 452.2 ([M+H]+). Anal. Calcd
for C27H32O6: C, 71.66; H, 7.13. Found: C, 71.91; H, 7.07.
5.2. General method of synthesis 3–22
Equimolar quantities (1.0 mmol) of (2) and long-chain alcohol
or long-chain amine were dissolved in DMF (10 mL), and 1.2 mmol
EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) were also
added to the solution as catalyst. The solution was then stirred at
80–90 °C for approximately 12 h. The product was extracted by
EtOAc. Then evaporated of the solvent, the residue was purified
by column chromatography on silica gel, eluting with petroleum
ether/EtOAc (6:1) to give a yellow solid.
5.2.6. Dodecyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetate (8)
Light yellow powder, yield: 43%. Mp: 67–69 °C. 1H NMR
(300 MHz, DMSO-d6, d ppm): 0.84 (t, J = 7.2 Hz, 3H); 1.24 (m,
18H); 1.56 (m, 2H); 4.13 (t, J = 6.3 Hz, 2H); 4.98 (s, 2H); 6.44 (d,
J = 2.2 Hz, 1H); 6.85 (d, J = 2.2 Hz, 1H); 7.07 (s, 1H); 7.61 (t, 3H);
8.10 (m, 2H); 12.82 (s, 1H). 13C NMR (DMSO-d6, d ppm): 182.5,
167.3, 164.8, 163.4, 162.1, 158.6, 132.5, 131.9, 129.8, 129.5,
128.7, 126.4, 106.2, 99.1, 92.8, 65.7, 65.1, 31.8, 30.6, 29.9, 29.5,
29.2, 28.3, 28.1, 25.6, 22.5, 13.9. MS (ESI): 480.2 ([M+H]+). Anal.
Calcd for C29H36O6: C, 72.48; H, 7.55. Found: C, 72.66; H, 7.39%.
5.2.1. Hexyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetate (3)
Light yellow powder, yield: 40%. Mp: 56–57 °C. 1H NMR
(300 MHz, DMSO-d6, d ppm): 0.79 (t, J = 6.9 Hz, 3H); 1.23 (m,
6H); 1.56 (m, 2H); 4.13 (t, J = 6.4 Hz, 2H); 4.98 (s, 2H); 6.43 (d,
J = 2.2 Hz, 1H); 6.85 (d, J = 2.2 Hz, 1H); 7.06 (s, 1H); 7.61 (m, 3H);
8.10 (m, 2H); 12.81 (s, 1H). 13C NMR (DMSO-d6, d ppm): 182.3,
168.1, 164.2, 163.7, 161.2, 157.1, 132.0, 131.1, 129.7, 126.4,
105.8, 104.9, 98.7, 94.2, 65.1, 64.8, 31.5, 28.8, 25.5, 22.3, 14.0. MS
(ESI):396.2 ([M+H]+). Anal. Calcd for C23H24O6: C, 69.68; H, 6.10.
Found: C, 69.45; H, 6.17.
5.2.7. Tetradecyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetate (9)
Light yellow powder, yield: 43%. Mp: 70–72 °C. 1H NMR
(300 MHz, DMSO-d6, d ppm): 0.85 (t, J = 6.8 Hz, 3H); 1.24 (m,
22H); 1.56 (m, 2H); 4.14 (t, J = 6.4 Hz, 2H); 4.98 (s, 2H); 6.44 (d,
J = 2.2 Hz, 1H); 6.85 (d, J = 2.4 Hz, 1H); 7.06(s, 1H); 7.62 (m, 3H);
8.10 (m, 2H); 12.81 (s, 1H). 13C NMR (DMSO-d6, d ppm): 182.4,
168.2, 165.3, 163.4, 162.8, 158.4, 132.4, 131.4, 131.1, 129.8,
128.4, 126.1, 105.4, 98.5, 93.1, 66.1, 65.4, 31.6, 31.2, 30.8, 30.1,
29.7, 29.4, 29.0, 28.2, 28.0, 25.4, 22.4, 14.1. MS (ESI): 508.3
([M+H]+). Anal. Calcd for C31H40O6: C, 73.20; H, 7.93. Found: C,
73.51; H, 7.82.
5.2.2. Heptyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetate (4)
Light yellow powder, yield: 51%. Mp: 56–58 °C. 1H NMR
(300 MHz, DMSO-d6, d ppm): 0.80 (t, J = 6.6 Hz, 3H); 1.17 (m,
8H); 1.57 (m, 2H); 4.14 (t, J = 6.4 Hz, 2H); 4.98 (s, 2H); 6.44 (d,
J = 2.4 Hz, 1H); 6.85 (d, J = 2.4 Hz, 1H); 7.07 (s, 1H); 7.62 (m, 3H);
8.10 (m, 2H); 12.82 (s, 1H). 13C NMR (DMSO-d6, d ppm): 182.5,
168.7, 164.1, 163.7, 160.8, 156.6, 133.2, 131.5, 129.8, 127.1,
125.8, 105.9, 98.5, 94.3, 65.1, 64.7, 31.6, 28.8, 28.3, 25.6, 22.5,
14.1. MS (ESI): 410.2 ([M+H]+). Anal. Calcd for C24H26O6: C, 70.23;
H, 6.38. Found: C, 70.53; H, 6.12.
5.2.8. Hexadecyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetate (10)
Light yellow powder, yield: 49%. Mp: 71–72 °C. 1H NMR
(300 MHz, DMSO-d6, d ppm): 0.85 (t, J = 6.4 Hz, 3H); 1.25 (m,
26H); 1.57 (m, 2H); 4.14 (t, J = 6.3 Hz, 2H); 4.99 (s, 2H); 6.45 (d,

P.-C. Lv et al. / Bioorg. Med. Chem. 18 (2010) 1117–1123
1121
J = 2.2 Hz, 1H); 6.86 (d, J = 2.2 Hz, 1H); 7.09 (s, 1H); 7.63 (m, 3H);
8.11 (m, 2H); 12.82 (s, 1H). 13C NMR (DMSO-d6, d ppm): 182.7,
168.1, 164.3, 163.4, 162.1, 157.4, 132.4, 131.8, 131.7, 129.5,
128.7, 126.8, 126.2, 98.7, 93.2, 65.8, 65.4, 32.1, 31.5, 31.1, 30.8,
30.2, 29.7, 29.5, 29.1, 28.9, 28.4, 28.2, 25.4, 22.3, 14.0. MS (ESI):
536.3 ([M+H]+). Anal. Calcd for C33H44O6: C, 73.85; H, 8.26. Found:
C, 74.09; H, 8.19.
93.1, 65.1, 65.5, 31.7, 28.3, 28.0, 27.3, 26.4, 22.2, 14.3. MS (ESI):
423.2 ([M+H]+). Anal. Calcd for C25H29NO5: C, 70.90; H, 6.90; N,
3.31. Found: C, 71.11; H, 6.99; N, 3.22.
5.2.14. N-Nonyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetamide (16)
Yellow powder, yield: 37%. Mp: 128–130 °C. 1H NMR (300 MHz,
DMSO-d6, d ppm): 0.83 (t, J = 6.6 Hz, 3H); 1.17 (m, 12H); 1.42 (m,
2H); 3.12 (d, J = 6.0 Hz, 2H); 4.63 (s, 2H); 6.44 (d, J = 2.2 Hz, 1H);
6.81 (d, J = 2.2 Hz, 1H); 7.06 (s, 1H); 7.62 (m, 3H); 8.11 (m, 3H);
12.81 (s, 1H). 13C NMR (DMSO-d6, d ppm): 182.4, 166.8, 165.1,
164.3, 163.8, 157.6, 132.1, 131.8, 130.6, 129.5, 128.1, 126.0,
106.3, 97.6, 94.5, 65.1, 66.3, 31.7, 30.1, 29.8, 28.8, 28.2, 25.7,
22.3, 13.8. MS (ESI): 437.2 ([M+H]+). Anal. Calcd for C26H31NO5:
C, 71.37; H, 7.14; N, 3.20. Found: C, 71.74; H, 7.03; N, 3.02.
5.2.9. Octadecyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetate (11)
Light yellow powder, yield: 45%. Mp: 73–75 °C. 1H NMR
(300 MHz, DMSO-d6, d ppm): 0.85 (t, J = 7.0 Hz, 3H); 1.22 (m,
30H); 1.57 (m, 2H); 4.13 (t, J = 6.5 Hz, 2H); 4.97 (s, 2H); 6.43 (d,
J = 2.2 Hz, 1H); 6.84 (d, J = 2.2 Hz, 1H); 7.05 (s, 1H); 7.60 (m, 3H);
8.10 (m, 2H); 12.80 (s, 1H). 13C NMR (DMSO-d6, d ppm): 182.3,
168.2, 164.5, 163.8, 162.4, 157.4, 132.5, 131.8, 131.2, 129.4,
128.6, 126.4, 105.9, 98.8, 93.6, 65.8, 65.1, 32.4, 32.2, 31.7, 31.4,
30.6, 30.1, 29.6, 29.3, 29.0, 28.7, 28.4, 28.1, 25.7, 22.3, 14.1. MS
(ESI): 564.4 ([M+H]+). Anal. Calcd for C35H48O6: C, 74.44; H, 8.57.
Found: C, 74.70; H, 8.38.
5.2.15. N-Decyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetamide (17)
Yellow powder, yield: 42%. Mp: 129–131 °C. 1H NMR (300 MHz,
DMSO-d6, d ppm): 0.85 (t, J = 6.2 Hz, 3H); 1.19 (m, 14H); 1.41 (m,
2H); 3.13 (d, J = 6.4 Hz, 2H); 4.63 (s, 2H); 6.45 (d, J = 2.2 Hz, 1H);
6.81 (d, J = 2.2 Hz, 1H); 7.07 (s, 1H); 7.62 (m, 3H); 8.11 (m, 3H);
12.80 (s, 1H). 13C NMR (DMSO-d6, d ppm): 182.4, 169.3, 166.4,
163.7, 161.5, 157.1, 133.5, 132.2, 131.7, 130.4, 128.6, 127.3,
107.6, 99.4, 94.8, 66.2, 65.1, 31.9, 31.4, 29.5, 29.0, 28.4, 28.1,
25.7, 22.4, 14.1. MS (ESI): 451.2 ([M+H]+). Anal. Calcd for
C27H33NO5: C, 71.82; H, 7.37; N, 3.10. Found: C, 72.15; H, 7.02;
N, 2.99.
5.2.10. Icosyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetate (12)
Light yellow powder, yield: 33%. Mp: 74–75 °C. 1H NMR
(300 MHz, DMSO-d6, d ppm): 0.85 (t, J = 6.8 Hz, 3H); 1.25 (m,
34H); 1.57 (m, 2H); 4.13 (t, J = 6.5 Hz, 2H); 4.98 (s, 2H); 6.45 (d,
J = 2.2 Hz, 1H); 6.85 (d, J = 2.2 Hz, 1H); 7.07 (s, 1H); 7.60 (m, 3H);
8.11 (m, 2H); 12.80 (s, 1H). 13C NMR (DMSO-d6, d ppm): 182.4,
168.4, 165.1, 163.8, 162.4, 157.4, 131.8, 131.1, 129.4, 128.6,
126.4, 105.9, 98.6, 93.6, 65.9, 65.1, 32.3, 31.9, 31.6, 31.2, 30.9,
30.5, 30.2, 29.8, 29.4, 29.0, 28.8, 28.6, 28.3, 25.7, 22.3, 14.1. MS
(ESI): 592.4 ([M+H]+). Anal. Calcd for C37H52O6: C, 74.96; H, 8.84.
Found: C, 75.21; H, 8.45.
5.2.16. N-Dodecyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-
7-yloxy)acetamide (18)
Yellow powder, yield: 35%. Mp: 135–137 °C. 1H NMR (300 MHz,
DMSO-d6, d ppm): 0.84 (t, J = 6.2 Hz, 3H); 1.19 (m, 18H); 1.41 (m,
2H); 3.12 (d, J = 6.2 Hz, 2H); 4.63 (s, 2H); 6.45 (d, J = 1.8 Hz, 1H);
6.81 (d, J = 1.8 Hz, 1H); 7.07 (s, 1H); 7.62 (m, 3H); 8.11 (m, 3H);
12.81 (s, 1H). 13C NMR (DMSO-d6, d ppm): 182.3, 168.4, 165.3,
164.0, 163.2, 159.1, 133.6, 132.5, 129.7, 128.3, 127.4, 126.1,
107.8, 99.6, 92.1, 65.3, 65.0, 31.9, 30.2, 29.8, 29.5, 29.0, 28.4,
28.1, 25.3, 22.6, 14.0. MS (ESI): 479.3 ([M+H]+). Anal. Calcd for
C29H37NO5: C, 72.62; H, 7.78; N, 2.92. Found: C, 72.98; H, 7.52;
N, 2.74.
5.2.11. N-Hexyl-2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetamide (13)
Yellow powder, yield: 38%. Mp: 120–122 °C. 1H NMR (300 MHz,
DMSO-d6, d ppm): 0.80 (t, J = 6.8 Hz, 3H); 1.11 (m, 6H); 1.41 (m,
2H); 3.12 (d, J = 6.4 Hz, 2H); 4.63 (s, 2H); 6.45 (d, J = 2.2 Hz, 1H);
6.81 (d, J = 2.2 Hz, 1H); 7.06 (s, 1H); 7.62 (m, 3H); 8.11 (m, 3H);
12.81 (s, 1H). 13C NMR (DMSO-d6, d ppm): 182.6, 168.3, 164.9,
162.5, 160.8, 158.3, 133.4, 132.4, 128.7, 125.2, 106.5, 105.2, 98.5,
94.8, 65.6, 64.1, 32.2, 29.1, 25.7, 22.6, 14.1. MS (ESI): 395.2
([M+H]+). Anal. Calcd for C23H25NO5: C, 69.86; H, 6.37; N, 3.54.
Found: C, 70.12; H, 6.48; N, 3.37.
5.2.17. N-Tetradecyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-
chromen-7-yloxy)acetamide (19)
Yellow powder, yield: 46%. Mp: 135–137 °C. 1H NMR (300 MHz,
DMSO-d6, d ppm): 0.83 (t, J = 6.6 Hz, 3H); 1.17 (m, 22H); 1.42 (m,
2H); 3.13 (d, J = 6.2 Hz, 2H); 4.63 (s, 2H); 6.45 (d, J = 2.2 Hz, 1H);
6.81 (d, J = 2.2 Hz, 1H); 7.07(s, 1H); 7.62 (m, 3H); 8.11 (m, 3H);
12.80 (s, 1H). 13C NMR (DMSO-d6, d ppm): 182.6, 167.5, 166.1,
162.8, 161.3, 158.7, 131.8, 131.1, 130.7, 129.4, 127.8, 126.7, 105.1,
98.8, 93.4, 66.5, 65.1, 31.9, 31.4, 30.5, 30.0, 29.5, 29.3, 29.0, 28.7,
28.1, 25.2, 22.6, 14.1. MS (ESI): 507.3 ([M+H]+). Anal. Calcd for
C31H41NO5: C, 73.34; H, 8.14; N, 2.76. Found: C, 73.53; H, 8.39;N, 2.88.
5.2.12. N-Heptyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetamide (14)
Yellow powder, yield: 47%. Mp: 121–123 °C. 1H NMR (300 MHz,
DMSO-d6, d ppm): 0.80 (t, J = 6.6 Hz, 3H); 1.08 (m, 8H); 1.42 (m,
2H); 3.12 (d, J = 6.2 Hz, 2H); 4.63 (s, 2H); 6.45 (d, J = 2.2 Hz, 1H);
6.81 (d, J = 2.2 Hz, 1H); 7.05 (s, 1H); 7.62 (m, 3H); 8.11 (m, 3H);
12.81 (s, 1H). 13C NMR (DMSO-d6, d ppm): 182.3, 167.4, 165.8,
164.2, 161.3, 155.3, 134.1, 131.8, 129.4, 127.1, 126.8, 106.3, 98.7,
95.4, 65.1, 64.5, 31.8, 29.1, 28.5, 25.3, 22.4, 14.0. MS (ESI): 409.2
([M+H]+). Anal. Calcd for C24H27NO5: C, 70.40; H, 6.65; N, 3.42.
Found: C, 70.73; H, 6.82; N, 3.24.
5.2.18. N-Hexadecyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-
chromen-7-yloxy)acetamide (20)
Yellow powder, yield: 44%. Mp: 137–139 °C. 1H NMR (300 MHz,
DMSO-d6, d ppm): 0.82 (t, J = 6.8 Hz, 3H); 1.22 (m, 26H); 1.41 (m,
2H); 3.12 (d, J = 6.0 Hz, 2H); 4.63 (s, 2H); 6.45 (d, J = 2.2 Hz, 1H);
6.81 (d, J = 2.2 Hz, 1H); 7.06(s, 1H); 7.62 (m, 3H); 8.11 (m, 3H);
12.81 (s, 1H). 13C NMR (DMSO-d6, d ppm): 182.4, 168.7, 165.6,
164.5, 163.7, 158.2, 133.5, 132.4, 131.8, 129.1, 127.3, 126.2,
125.4, 98.1, 93.7, 65.6, 65.2, 32.5, 31.7, 31.3, 30.5, 30.1, 29.7,
29.5, 29.3, 28.6, 28.4, 28.0, 25.3, 22.1, 13.9. MS (ESI): 535.3
([M+H]+). Anal. Calcd for C33H45NO5: C, 73.99; H, 8.47; N, 2.61.
Found: C, 74.42; H, 8.63; N, 2.44.
5.2.13. N-Octyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetamide (15)
Yellow powder, yield: 30%. Mp: 124–126 °C. 1H NMR (300 MHz,
DMSO-d6, d ppm): 0.82 (t, J = 6.8 Hz, 3H); 1.17 (m, 10H); 1.41 (m,
2H); 3.14 (d, J = 6.6 Hz, 2H); 4.64 (s, 2H); 6.45 (d, J = 2.2 Hz, 1H);
6.81 (d, J = 2.2 Hz, 1H); 7.07 (s, 1H); 7.62 (m, 3H); 8.11 (m, 3H);
12.81 (s, 1H). 13C NMR (DMSO-d6, d ppm): 182.7, 167.5, 165.2,
164.2, 163.6, 158.1, 133.2, 131.3, 128.7, 125.6, 106.1, 106.0, 98.4,

1122
P.-C. Lv et al. / Bioorg. Med. Chem. 18 (2010) 1117–1123
5.2.19. N-Octadecyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-
7-yloxy)acetamide (21)
passed through an equilibrated Ni-NTA superflow packed column
and washed with 10 mM and then 100 mM imidazole to remove
nonspecifically bound material. Histidine tagged proteins were
eluted with 250 and 500 mM imidazole and dialyzed against
50 mM NaCl, 20 mM HEPES, 10% glycerol, and 1 lg/mL each of
aprotinin, leupeptin, and pepstatin for 2 h. The entire purification
Yellow powder, yield: 45%. Mp: 138–140 °C. 1H NMR (300 MHz,
DMSO-d6, d ppm): 0.85 (t, J = 6.4 Hz, 3H); 1.22 (m, 30H); 1.42 (m,
2H); 3.13 (d, J = 6.2 Hz, 2H); 4.63 (s, 2H); 6.44 (d, J = 2.0 Hz, 1H);
6.81 (d, J = 2.0 Hz, 1H); 7.05 (s, 1H); 7.61 (m, 3H); 8.10 (m, 3H);
12.80 (s, 1H). 13C NMR (DMSO-d6, d ppm): 182.6, 167.6, 163.7,
163.2, 162.1, 158.5, 131.4, 131.0, 130.5, 129.1, 127.6, 125.3,
105.4, 98.3, 93.1, 65.7, 64.6, 32.7, 32.4, 31.5, 31.2, 30.9, 30.4,
29.4, 29.2, 28.7, 28.5, 28.4, 27.6, 25.1, 22.5, 14.0. MS (ESI): 563.3
([M+H]+). Anal. Calcd for C35H49NO5: C, 74.57; H, 8.76; N, 2.48.
Found: C, 74.93; H, 9.02; N, 2.23.
procedure was performed at 4 °C or on ice.
The EGFR kinase assay was set up to assess the level of auto-
phosphorylation based on DELFIA/Time-Resolved Fluorometry.
Compounds 3–22 were dissolved in 100% DMSO and diluted to
the appropriate concentrations with 25 mM HEPES at pH 7.4. In
each well, 10
for HER-2 or 5 ng for EGFR) of recombinant enzyme (1:80 dilution
in 100 mM HEPES) for 10 min at room temperature. Then, 10 L of
5 mM buffer (containing 20 mM HEPES, 2 mM MnCl2, 100
Na3VO4, and 1 mM DTT) and 20 L of 0.1 mM ATP-50 mM MgCl2
lL of compound was incubated with 10 lL (12.5 ng
5.2.20. N-Icosyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy) acetamide (22)
l
l
M
Yellow powder, yield: 40%. Mp: 137–139 °C. 1H NMR (300 MHz,
DMSO-d6, d ppm): 0.85 (t, J = 6.4 Hz, 3H); 1.22 (m, 34H); 1.42 (m,
2H); 3.12 (d, J = 6.0 Hz, 2H); 4.63 (s, 2H); 6.44 (d, J = 2.0 Hz, 1H);
6.81 (d, J = 2.2 Hz, 1H); 7.06 (s, 1H); 7.61 (m, 3H); 8.11 (m, 3H);
12.81 (s, 1H). 13C NMR (DMSO-d6, d ppm): 182.6, 169.4, 166.5,
164.3, 162.7, 156.5, 132.3, 130.8, 129.1, 127.7, 126.5, 105.3, 98.5,
94.6, 65.8, 64.3, 32.6, 31.7, 31.4, 31.2, 30.8, 30.4, 30.0, 29.6, 29.2,
29.0, 28.7, 28.5, 28.1, 25.8, 22.6, 14.1. MS (ESI): 591.4 ([M+H]+).
Anal. Calcd for C37H53NO5: C, 75.09; H, 9.03; N, 2.37. Found: C,
74.76; H, 9.17; N, 2.19.
l
was added for 1 h. Positive and negative controls were included
in each plate by incubation of enzyme with or without ATP-MgCl2.
At the end of incubation, liquid was aspirated, and plates were
washed three times with wash buffer. A 75 lL (400 ng) sample of
europium labeled anti-phosphotyrosine antibody was added to
each well for another 1 h of incubation. After washing, enhance-
ment solution was added and the signal was detected by Victor
(Wallac Inc.) with excitation at 340 nm and emission at 615 nm.
The percentage of autophosphorylation inhibition by the com-
pounds was calculated using the following equation: 100% ꢂ [(neg-
ative control)/(positive control ꢂ negative control)]. The IC50 was
obtained from curves of percentage inhibition with eight concen-
trations of compound. As the contaminants in the enzyme prepara-
tion are fairly low, the majority of the signal detected by the anti-
phosphotyrosine antibody is from EGFR.
5.3. Antiproliferative activities assay
The antiproliferative activities of compounds 3–22 were deter-
mined using a standard (MTT)-based colorimetric assay (Sigma).
Briefly, cell lines were seeded at a density of 7 ꢁ 103 cells/well in
96-well microtiter plates (Costar). After 24 h, exponentially grow-
ing cells were exposed to the indicated compounds at final concen-
5.6. Docking simulations
trations ranging from 0.1 to 100
was determined by the addition of an MTT solution (10
5 mg/mL MTT in PBS). After 4 h, 100 L of 10% SDS in 0.01 N HCl
was added, and the plates were incubated at 37 °C for a further
18 h; optical absorbance was measured at 570 nm on an LX300 Ep-
son Diagnostic microplate reader. Survival ratios are expressed in
percentages with respect to untreated cells. IC50 values were deter-
mined from replicates of six wells from at least two independent
experiments.
l
g/mL. After 48 h, cell survival
Molecular docking of compounds 10 and 20 into the three-
dimensional EGFR complex structure (PDB code: 1M17, download
from the PDB) was carried out using the Molsoft ICM-Pro software
package (version 3.5-0a).22,23
lL of
l
Acknowledgements
The work was financed by a grant (Project 30772627) from Na-
tional Natural Science Foundation of China.
5.4. Cell culture
References and notes
Colorectal cancer cell line, HT-29, was obtained from Jiangsu
Institute of Hematology and Cells were cultured in Mccoy’s 5A cul-
ture medium (Gibco BRL, USA) supplemented with 10% fetal bovine
serum (FBS) (Sigma, USA). The cultures were incubated at 37 °C in
humidified 5% CO2 incubator. When experiments were performed
in the absence of FBS, FBS was eliminated 24 h before initiating
the experiments.
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was located at the 50 upstream to the EGFR sequence. Sf-9 cells
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