Synthesis, molecular modeling, and biological evaluation of cinnamic acid metronidazole ester derivatives as novel anticancer agents

Article abstract of DOI:10.1016/j.bmc.2010.06.003

A series of novel cinnamic acid metronidazole ester derivatives have been designed and synthesized, and their biological activities were also evaluated as potential EGFR and HER-2 kinase inhibitors. Compound 3h showed the most potent biological activity (IC₅₀ = 0.62 μM for EGFR and IC₅₀ = 2.15 μM for HER-2). Docking simulation was performed to position compound 3h into the EGFR active site to determine the probable binding model. Antiproliferative assay results demonstrated that some of these compounds possessed good antiproliferative activity against MCF-7. Compound 3h with potent inhibitory activity in tumor growth inhibition may be a potential anticancer agent.

Full text of DOI:10.1016/j.bmc.2010.06.003

Bioorganic & Medicinal Chemistry 18 (2010) 4991–4996
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, molecular modeling, and biological evaluation of cinnamic acid
metronidazole ester derivatives as novel anticancer agents
*
*
*
Yong Qian, Hong-Jia Zhang, Hao Zhang, Chen Xu , Jing Zhao , 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 novel cinnamic acid metronidazole ester derivatives have been designed and synthesized, and
their biological activities were also evaluated as potential EGFR and HER-2 kinase inhibitors. Compound
3h showed the most potent biological activity (IC50 = 0.62 lM for EGFR and IC50 = 2.15 lM for HER-2).
Docking simulation was performed to position compound 3h into the EGFR active site to determine
the probable binding model. Antiproliferative assay results demonstrated that some of these compounds
possessed good antiproliferative activity against MCF-7. Compound 3h with potent inhibitory activity in
tumor growth inhibition may be a potential anticancer agent.
Received 18 April 2010
Revised 2 June 2010
Accepted 3 June 2010
Available online 8 June 2010
Keywords:
Cinnamic acid
Metronidazole
EGFR
Ó 2010 Elsevier Ltd. All rights reserved.
Anticancer
1. Introduction
So nitroimidazoles may provide the attractive possibility of
employing these molecules as carriers for targeted delivery in
Angiogenesis has been intensely investigated as an attractive
cancer therapeutic target during the last decade, as angiogenesis
is the first rate-limiting step for tumor cells to metastasize and is
also essential for cancer growth.1 The rapid progression of this field
is that some important receptors involved in angiogenesis have
been identified, including vascular endothelial growth factor
receptor (VEGFR), epidermal growth factor receptor (EGFR), and
several others.2 These growth factor receptor kinases play impor-
tant roles in the development, progression, aggressiveness, and
metastasis of many solid tumors, such as non small cell lung can-
cer,3 head and neck cancers,4 and glioblastomas.5 Among these ki-
nases, EGFR (also known as erbB-1 or HER-1) and the related
human epidermal growth factor receptor HER-2 (also known as
erbB-2) that have been improved to be relevant for cancer. Erloti-
nilb inhibits EGFR that is overexpressed in tumors and is approved
antitumor agent.6 Thus, these factors are important targets for the
development of new therapeutic antitumor agents.7,8
Nitroimidazoles have been extensively used as antimicrobial
chemotherapeutics and as antiangiogenic hypoxic cell radiosensi-
tizers.9 Nitroimidazole derivatives have attracted considerable
attention as they showed a tendency to penetrate and accumulate
in regions of tumors,10–12 and can undergo bioreduction to yield
electrophilic substances which can damage protein and nucleic
acids.13 Importantly, the toxicology and metabolism of nitroimi-
dazoles, particularly metronidazole, have been characterized.14,15
cancer therapy.12,16 Recently, Swenson et al. synthesized a 10B-en-
riched nitroimidazole by coupling the Cs salt of BSH (Cs2-10B12
11SH) with 1-(2-bromoethyl)-2-methyl-5-nitroimidazole and
used for boron neutron capture therapy of cancer.17
-
H
As a novel class of bioreductively activated nitroimidazole com-
pounds, we designed and synthesized a series of cinnamic acid
metronidazole ester derivatives. We chose cinnamoyl moiety as
it was found in a variety of biologically active substances.18,19 Anti-
tumor activities of various cinnamic acid derivatives were also ex-
plored by many research groups. Particularly, cinnamic acid ester
derivatives shown the potential antitumor activity.20–22 In this
study, we described the synthesis and the SAR of the novel series
of cinnamic acid metronidazole ester derivatives, and the biologi-
cal activity evaluation indicated that some of these compounds
are potent inhibitors of EGFR and HER-2. Docking simulations were
performed using the X-ray crystallographic structure of the EGFR
in complex with an inhibitor to explore the binding modes of these
compounds at the active site.
2. Results and discussion
2.1. Chemistry
The synthetic route for the novel cinnamic acid metronidazole
ester derivatives 3a–s is outlined in Scheme 1. These compounds
were synthesized from cinnamic acids (1) and active metronidaz-
one (2). Compound (1) was prepared according the modified proce-
dure of Davis et al.23 Aromatic aldehydes and malonic acid were
* Corresponding authors. Tel./fax: +86 25 8359 2572.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.003

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Y. Qian et al. / Bioorg. Med. Chem. 18 (2010) 4991–4996
Scheme 1. General synthesis of cinnamic acid metronidazole ester derivatives (3a–s). Reagents and conditions: (a) piperidine, pyridine, 80–90 °C, 24 h; (b) CH2Cl2, NEt3;
(c) K2CO3, DMF, 80 °C, 24 h.
Table 1
dissolved in a mixture of pyridine and piperidine and refluxed for
Structure of cinnamic acid metronidazole ester derivatives (3a–s)
24 h, and cinnamic acids were obtained with yields of 75–85%.
Then, cinnamic acids (1), active metronidazole (2) and K2CO3 were
taken in DMF and refluxed to provide the desired compounds (3a–
s) (Table 1). All of the synthetic compounds gave satisfactory ana-
lytical and spectroscopic data, which were in full accordance with
their depicted structures.
2.2. Biological activity
All the synthesized cinnamic acid metronidazole ester deriva-
tives were evaluated for their ability to inhibit the autophosphory-
Compound
R1
R2
R3
lation of EGFR and HER-2 kinases using a solid-phase ELISA assay.
The results were summarized in Table 2. For the given compounds,
it was observed that the IC50 value for inhibition of HER-2 kinase
was higher than that observed of EGFR kinase, however, they had
the same trends. It was possibly attributed to the fact that higher
concentration of the purified HER-2 kinase was used than EGFR ki-
nase in the enzyme assays. It is evident that there is also a reason-
able correlation between the EGFR and HER-2 inhibitory activities;
thus, this is not surprising in view of the high sequence homology
of the catalytic domains of these two kinases. As shown in Table 2,
a number of cinnamic acid metronidazole ester derivatives exhib-
ited significant EGFR and HER-2 inhibitory activity. Among them,
compound 3h displayed the most potent inhibitory activity
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
30
3p
3q
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NO2
F
OMe
F
Cl
Br
H
Me
OMe
i-Pr
Ph
PhCH2O
H
H
H
H
H
H
H
H
H
F
Cl
Br
NO2
OMe
H
(IC50 = 0.62
lM for EGFR and IC50 = 2.15
l
M for HER-2), compara-
M for EGFR).
H
H
ble to the positive control erlotinib (IC50 = 0.03
l
Structure–activity relationships in these cinnamic acid metroni-
dazole ester derivatives demonstrated that compounds with sub-
stitution at the meta (3o–q) or para (3a–i) position showed more
potent activities than those with substitution at the ortho position
(3j–n). A comparison of the para position substitution on benzene
ring demonstrated that a para halogen group (3a–c) may have
more slightly improved EGFR inhibitory activity and the potency
order is F < Cl < Br, and other para substituents prepared (3e, 3g)
had minimal effects compared with 3d, whereas a methoxy group
substituent (3f) led to a slight loss of activity. Meanwhile, a signif-
icant loss of activity was observed when the halogen substituent
was moved from the para position to the ortho (3j–m) position.

Y. Qian et al. / Bioorg. Med. Chem. 18 (2010) 4991–4996
4993
Table 2
Inhibition (IC50) of EGFR and HER-2 kinases and inhibition (IC50) of cell proliferation
by compound 3a–s
Compounds
Enzyme assay IC50
(lM)
MCF-7 IC50 SD (lM)
EGFR
HER-2
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
4.12
3.62
3.24
7.83
8.11
10.85
6.82
0.62
1.27
26.74
20.81
24.32
25
5.84
4.77
4.65
10.78
9.65
14.32
9.97
2.15
3.46
41.26
36.45
44.85
40
3.76 0.12
3.05 0.15
2.45 0.08
4.41 0.3
4.28 0.5
5.49 0.2
4.96 0.4
0.36 0.04
0.98 0.06
Figure 2. Crystal structure diagrams of compound 3f. Molecule structure diagram
with displacement ellipsoids being at the 30% probability level and H atoms are
shown as small spheres of arbitrary radii.
15
18
16
17
20
2
4
3
3
2
In addition, in vitro antiproliferative activity of these cinnamic
acid metronidazole ester derivatives was studied on a panel of
one human tumor cell line (MCF-7), which overexpressed EGFR
and to a less extent, HER-2. Compounds 3b, 3c, 3h, 3i, 3r, which
have potent inhibitory activity of EGFR and HER-2 showed high
antiproliferative activity against MCF-7 with IC50 ranging from
32
>50
14.53
12.42
13.87
3.88
5.76
0.03
19.34
16.75
18.82
5.03
9.24
0.16
5.68 0.4
5.88 0.8
5.75 0.7
3.4 0.2
4.1 0.3
0.02 0.005
3s
Erlotinib
0.36 0.04 lM to 3.4 0.2 lM, which indicated that these cin-
namic acid metronidazole ester derivatives were potent inhibitor
of EGFR and HER-2 as antitumor agents. Among these compounds,
compound 3h exhibited the most potential inhibitory activity in
tumor growth inhibition and exhibited favored EGFR and HER-2
inhibitory activity.
A methoxy group at ortho position in the 3n structure also led to a
noteworthy loss of activity. Considering the steric effect of the sub-
stitution at the para position of benzene ring, we designed and
evaluated the compounds with phenyl (3h) or benzyloxy (3i)
group substitution, the activity was significantly enhanced up to
12-fold and 4.5-fold by phenyl and benzyloxy group substituent,
respectively, which was compared with the compound 3d. Further-
more, variation of the aromatic ring moiety was also explored.
Compounds 3r and 3s bearing naphthyl moiety exhibited moder-
ate EGFR inhibitory activity.
3. Conclusions
A series of cinnamic acid metronidazole ester derivatives have
been designed and synthesized, and their biological activities were
also evaluated as potent EGFR and HER-2 inhibitory. Compound 3h
In order to gain more understanding of the structure–activity
relationships observed at the EGFR, molecular docking of the most
potent inhibitor 3h into ATP binding site of EGFR kinase was per-
formed on the binding model based on the EGFR complex structure
(1M17.pdb). The binding model of compound 3h and EGFR is de-
picted in Figure 1. In the binding model, compound 3h is nicely
bound to the region with the carbonyl group project toward the
amino hydrogen of Lys828, and the oxygen atom of ester group
of 3h also forms hydrogen bond with the hydroxyl group Leu768,
and oxygen atom of nitro group with amino hydrogen of Lys822.
The enzyme assay data and the molecular docking results showed
that compound 3h was a potential inhibitor of EGFR (Fig. 1).
demonstrated the most potent inhibitory activity (IC50 = 0.62
lM
for EGFR and IC50 = 2.15 M for HER-2), which was compared with
l
the positive control erlotinib. Docking simulation was performed
to position compound 3h into the EGFR active site to determine
the probable binding model. Analysis of the compound 3h’s binding
conformation in active site displayed the compound 3h was stabi-
lized by hydrogen bonding interactions with Lys828, Leu768, and
Lys822. Antiproliferative assay results also showed that these cin-
namic acid metronidazole ester derivatives had the potential to be
developed for antiproliferative agents against MCF-7. Particularly,
compound 3h has demonstrated significant EGFR and HER-2 and tu-
mor growth inhibitory activity as a potential anticancer agent.
4. Experiments
4.1. Materials and measurements
All chemicals and reagents used in current study were analyti-
cal grade. All the 1H NMR spectra were recorded on a Bruker DRX
500 or DPX 300 model spectrometer in CDCl3 and chemical shifts
were reported in ppm (d). ESI-MS spectra were recorded on a Mar-
iner System 5304 Mass spectrometer. Elemental analyses were
performed on a CHN-O-Rapid instrument. TLC was performed on
the glass-backed silica gel sheets (Silica Gel 60 Å GF254) and visu-
alized in UV light (254 nm). Column chromatography was per-
formed using silica gel (200–300 mesh) eluting with ethyl
acetate and petroleum ether.
4.2. General procedure for synthesis of cinnamic acids
A mixture of aromatic aldehydes (3.2 mmol), malonic acid
(3.87 mmol), piperidine (0.387 mmol) was dissolved in pyridine
Figure 1. Docking of compound 3h with EGFR kinase shows intramolecular
hydrogen bonds with Lys828, Leu768, and Lys822.

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Y. Qian et al. / Bioorg. Med. Chem. 18 (2010) 4991–4996
and stirred on 80–90 °C for 24 h. The pyridine was removed at the
vacuum. The reaction mixture was poured in water and washed
with HCl, the precipitate was filtered and washed with hexane
about three times, and dried under vacuum to afford the cinnamic
acids (Scheme 1).
MS: 316.1 (C16H18N3O4, [M+H]+). Anal. Calcd for C16H17N3O4: C,
60.94; H, 5.43; N, 13.33. Found: C, 61.33; H, 5.79; N, 13.65.
4.4.6. (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-(4-meth-
oxyphenyl) acrylate (3f)
1H NMR (CDCl3, 500 MHz): 2.52 (s, 3H), 3.84 (s, 3H), 4.54 (t,
J = 4.8 Hz, 2H), 4.65 (t, J = 5.2 Hz, 2H), 6.20 (d, J = 15.8 Hz, 1H),
6.91 (d, J = 8.5 Hz, 2H), 7.46 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 16.2 Hz,
1H), 7.98 (s, 1H). ESI-MS: 332.1 (C16H18N3O5, [M+H]+). Anal. Calcd
for C16H17N3O5: C, 58.00; H, 5.17; N, 12.68. Found: C, 58.33; H,
5.43; N, 12.95.
4.3. General procedure for synthesis of 2-(2-methyl-5-nitro-1H-
imidazol-1-yl) ethyl methanesulfonate
Equimolar amount of metronidazole and methanesulfonyl chlo-
ride were dissolved in dichloromethane, triethylamine as a catalyst
and stirred in the ice bath for 5 h. The reaction mixture was ex-
tracted with ice water and saturated sodium bicarbonate, respec-
tively. Then, the organic layer was collected and crystallized to
get the product (Scheme 1).
4.4.7. (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-(4-iso-
propylphenyl) acrylate (3g)
1H NMR (CDCl3, 500 MHz): 1.26 (s, 6H), 2.55 (s, 3H), 2.89 (m,
1H), 4.56 (t, J = 5.5 Hz, 2H), 4.66 (t, J = 5.2 Hz, 2H), 6.44 (d,
J = 16.2 Hz, 1H), 6.92 (d, J = 8.2 Hz, 1H), 6.95–6.98 (m, 1H), 7.37
(t, J = 7.0 Hz, 1H), 7.48 (d, J = 6.2 Hz, 1H), 7.95–7.98 (d, J = 16.2 Hz,
1H), 7.99 (s, 1H). ESI-MS: 344.2 (C18H22N3O4, [M+H]+). Anal. Calcd
for C18H21N3O4: C, 62.96; H, 6.16; N, 12.24. Found: C, 63.31; H,
6.48; N, 12.58.
4.4. General procedure for synthesis of cinnamic acid metroni-
dazole ester derivatives
To a stirred solution of cinnamic acids (0.5 mmol), 2-(2-methyl-
5-nitro-1H-imidazol-1-yl) ethyl methanesulfonate (0.5 mmol) in
DMF, potassium carbonate (1 mmol) was added. This mixture
was stirred at 80 °C for 24 h. Then the mixture was filtered and
the filtrate was taken up with EtOAc, washed with saturated so-
dium bicarbonate, dried over Na2SO4, and purified by column chro-
matography on silica gel, to give the target compounds (Scheme 1).
4.4.8. (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-(4-phenyl)
acrylate (3h)
1H NMR (CDCl3, 300 MHz): 2.56 (s, 3H), 4.58 (t, J = 4.9 Hz, 2H),
4.66 (t, J = 5.1 Hz, 2H), 6.37 (d, J = 16.1 Hz, 1H), 7.39 (d, J = 7.1 Hz,
1H), 7.46 (t, J = 7.1 Hz, 2H), 7.57–7.65 (m, 6H), 7.70 (d,
J = 15.9 Hz, 1H), 7.99 (s, 1H). ESI-MS: 378.1 (C21H20N3O4, [M+H]+).
Anal. Calcd for C21H19N3O4: C, 66.83; H, 5.07; N, 11.13. Found: C,
67.14; H, 5.39; N, 11.48.
4.4.1. (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-(4-
fluorophenyl) acrylate (3a)
1H NMR (CDCl3, 500 MHz): 2.52 (s, 3H), 4.55 (t, J = 5.2 Hz, 2H),
4.66 (t, J = 5.2 Hz, 2H), 6.26 (d, J = 16.0 Hz, 1H), 7.01 (t, J = 8.5 Hz,
2H), 7.49–7.51 (m, 2H), 7.61 (d, J = 15.9 Hz, 1H), 7.98 (s, 1H). ESI-
MS: 320.1 (C15H15FN3O4, [M+H]+). Anal. Calcd for C15H14FN3O4: C,
56.43; H, 4.42; N, 13.16. Found: C, 55.77; H, 4.78; N, 13.49.
4.4.9. (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-(4-(ben-
zyloxy) phenyl) acrylate (3i)
1H NMR (CDCl3, 500 MHz): 2.58 (s, 3H), 4.64 (t, J = 5.0 Hz, 2H),
4.72 (t, J = 5.0 Hz, 2H), 5.18 (s, 2H), 7.10 (d, J = 8.5 Hz, 1H), 7.29–
7.47 (m, 7H), 7.71 (d, J = 8.0 Hz, 2H), 7.86 (d, J = 8.5 Hz, 1H), 8.01
(s, 1H). ESI-MS: 407.1 (C22H22N3O5, [M+H]+). Anal. Calcd for
4.4.2. (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-(4-chloro-
phenyl) acrylate (3b)
C22H21N3O: C, 64.86; H, 5.20; N, 10.31. Found: C, 65.23; H, 5.47;
1H NMR (CDCl3, 300 MHz): 2.52 (s, 3H), 4.56 (t, J = 4.9 Hz, 2H),
4.66 (t, J = 4.9 Hz, 2H), 6.31 (d, J = 16.1 Hz, 1H), 7.37 (d, J = 8.4 Hz,
2H), 7.45 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 15.9 Hz, 1H), 7.98 (s, 1H).
ESI-MS: 336.1 (C15H15ClN3O4, [M+H]+). Anal. Calcd for
N, 10.65.
4.4.10. (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-(2-fluo-
rophenyl) acrylate (3j)
C15H14ClN3O4: C, 53.66; H, 4.20; N, 12.52. Found: C, 53.98; H,
1H NMR (CDCl3, 300 MHz): 2.52 (s, 3H), 4.58 (t, J = 5.0 Hz, 2H),
4.66 (t, J = 4.9 Hz, 2H), 6.26 (d, J = 16.1 Hz, 1H), 7.09 (t, J = 8.6 Hz,
2H), 7.48–7.53 (m, 2H), 7.61 (d, J = 15.9 Hz, 1H), 7.98 (s, 1H). ESI-
MS: 320.1 (C15H15FN3O4, [M+H]+). Anal. Calcd for C15H14FN3O4: C,
56.43; H, 4.42; N, 13.16. Found: C, 56.74; H, 4.77; N, 13.45.
4.61; N, 12.84.
4.4.3. (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-(4-bromo-
phenyl) acrylate (3c)
1H NMR (CDCl3, 300 MHz): 2.52 (s, 3H), 4.55 (t, J = 4.9 Hz, 2H),
4.67 (t, J = 4.9 Hz, 2H), 6.32 (d, J = 15.9 Hz, 1H), 7.37 (d, J = 8.6 Hz,
2H), 7.52–7.61 (m, 3H), 7.98 (s, 1H). ESI-MS: 380.0 (C15H15BrN3O4,
[M+H]+). Anal. Calcd for C15H14BrN3O4: C, 47.39; H, 3.71; N, 11.05.
Found: C, 47.70; H, 4.03; N, 11.38.
4.4.11. (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-(2-chlo-
rophenyl) acrylate (3k)
1H NMR (CDCl3, 300 MHz): 2.55 (s, 3H), 4.58 (t, J = 4.9 Hz, 2H),
4.67 (t, J = 4.9 Hz, 2H), 6.35 (d, J = 16.1 Hz, 1H), 7.27–7.36 (m,
2H), 7.43 (d, J = 1.7 Hz, J = 7.9 Hz, 1H), 7.59 (dd, J = 2.0 Hz,
J = 7.3 Hz, 1H), 7.98 (s, 1H), 8.05 (d, J = 15.9 Hz, 1H). ESI-MS:
336.1 (C15H15ClN3O4, [M+H]+). Anal. Calcd for C15H14ClN3O4: C,
53.66; H, 4.20; N, 12.52. Found: C, 53.96; H, 4.55; N, 12.86.
4.4.4. (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl
cinnamate (3d)
1H NMR (CDCl3, 300 MHz): 2.55 (s, 3H), 4.57 (t, J = 4.8 Hz, 2H),
4.67 (t, J = 5.1 Hz, 2H), 6.34 (d, J = 16.1 Hz, 1H), 7.40–7.42 (m,
3H), 7.50–7.53 (m, 2H), 7.67 (d, J = 15.9 Hz, 1H), 8.00 (s, 1H). ESI-
MS: 302.1 (C15H16N3O4, [M+H]+). Anal. Calcd for C15H15N3O4: C,
59.79; H, 5.02; N, 13.95. Found: C, 60.14; H, 5.39; N, 14.41.
4.4.12. (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-(2-bro-
mophenyl)acrylate (3l)
1H NMR (CDCl3, 300 MHz): 2.52 (s, 3H), 4.56 (t, J = 4.9 Hz, 2H),
4.64 (t, J = 4.9 Hz, 2H), 6.37 (d, J = 16.1 Hz, 1H), 7.29–7.35 (m,
2H), 7.44 (d, J = 2.0 Hz, J = 7.8 Hz, 1H), 7.60 (m, 1H), 7.99 (s, 1H),
8.04 (d, J = 16.1 Hz, 1H). ESI-MS: 379.0 (C15H15BrN3O4, [M+H]+).
Anal. Calcd for C15H14BrN3O4: C, 47.39; H, 3.71; N, 11.05. Found:
C, 47.76; H, 4.05; N, 11.36.
4.4.5. (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-p-tolyl-
acrylate (3e)
1H NMR (CDCl3, 500 MHz): 2.38 (s, 3H), 2.52 (s, 3H), 4.55 (m,
2H), 4.66 (m, 2H), 6.29 (d, J = 15.8 Hz, 1H), 7.20 (d, J = 7.8 Hz, 2H),
6.40 (d, J = 8.0 Hz, 2H), 7.62 (d, J = 15.8 Hz, 1H), 7.99 (s, 1H). ESI-

Y. Qian et al. / Bioorg. Med. Chem. 18 (2010) 4991–4996
4995
4.4.13. (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-(2-nitro-
phenyl)acrylate (3m)
ture was solved by direct methods and refined on F2 by full-matrix
least-squares methods using SHELX-97.24 All the non-hydrogen
atoms were refined anisotropically. All the hydrogen atoms were
placed in calculated positions and were assigned fixed isotropic
thermal parameters at 1.2 times the equivalent isotropic U of the
atoms to which they are attached and allowed to ride on their
respective parent atoms. The contributions of these hydrogen
atoms were included in the structure-factors calculations. The
crystal data, data collection, and refinement parameter for the
compound 3f are listed in Table 3.
1H NMR (CDCl3, 500 MHz): 2.57 (s, 3H), 4.61 (t, J = 5.0 Hz, 2H),
4.69 (t, J = 5.0 Hz, 2H), 6.28 (d, J = 15.5 Hz, 1H), 7.58–7.63 (m,
2H), 7.69 (d, J = 7.0 Hz, 2H), 7.99 (s, 1H), 8.09 (d, J = 9.5 Hz, 1H),
8.13 (s, 1H). ESI-MS: 346.1 (C15H15N4O6, [M+H]+). Anal. Calcd for
C15H14N4O6: C, 52.03; H, 4.07; N, 16.18. Found: C, 52.34; H, 4.45;
N, 16.55.
4.4.14. (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-(2-meth-
oxyphenyl) acrylate (3n)
1H NMR (CDCl3, 300 MHz): 2.56 (s, 3H), 3.89 (s, 3H), 4.57 (t,
J = 4.6 Hz, 2H), 4.67 (t, J = 4.4 Hz, 2H), 6.43 (d, J = 16.1 Hz, 1H),
6.91–6.99 (m, 2H), 7.35–7.41 (m, 1H), 7.49 (dd, J = 1.65 Hz,
J = 7.7 Hz, 1H), 7.96 (d, J = 16.3 Hz, 1H), 8.00 (s, 1H). ESI-MS:
332.1 (C16H18N3O5, [M+H]+). Anal. Calcd for C16H17N3O5: C, 58.00;
H, 5.17; N, 12.68. Found: C, 58.37; H, 5.45; N, 12.93.
4.6. General procedure for preparation, purification of HER-2
and EGFR, and inhibitory assay
A 1.7 Kb cDNA encoded for human HER-2 cytoplasmic domain
(HER-2-CD, amino acids 676–1245) and 1.6 kb cDNA encoded for
the EGFR cytoplasmic domain (EGFR-CD, amino acids 645–1186)
were cloned into baculoviral expression vectors pBlueBacHis2B
and pFASTBacHTc (Huakang Company, China), separately. A se-
quence that encodes (His)6 was located at the 50 upstream to the
HER-2 and EGFR sequences. Sf-9 cells were infected for 3 days for
protein expression. Sf-9 cell pellets were solubilized at 0 °C in a buf-
fer at pH 7.4 containing 50 mM HEPES, 10 mM NaCl, 1% Triton,
4.4.15. (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-(3-nitro-
phenyl) acrylate (3o)
1H NMR (CDCl3, 300 MHz): 2.63 (s, 3H), 4.57 (t, J = 5.0 Hz, 2H),
4.70 (t, J = 5.0 Hz, 2H), 6.20 (d, J = 15.9 Hz, 1H), 6.92 (d, J = 8.8 Hz,
2H), 7.48 (d, J = 8.8 Hz, 2H), 7.63 (d, J = 16.1 Hz, 1H), 8.04 (s, 1H).
ESI-MS: 347.1 (C15H15N4O6, [M+H]+). Anal. Calcd for C15H14N4O6:
C, 52.03; H, 4.07; N, 16.18. Found: C, 52.36; H, 4.35; N, 16.43.
10
l
M ammonium molybdate, 100
lM sodium vanadate, 10
l
g/
g/
mL aprotinin, 10
lg/mL leupeptin, 10
lg/mL pepstatin, and 16
l
mL benzamidine HCl for 20 min followed by 20 min centrifugation.
Crude extract supernatant was passed through an equilibrated Ni-
NTA superflow packed column and washed with 10 mM and then
100 mM imidazole to remove nonspecifically bound material. Histi-
dine tagged proteins were eluted with 250 and 500 mM imidazole
and dialyzed against 50 mM NaCl, 20 mM HEPES, 10% glycerol, and
4.4.16. (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-(3-fluo-
rophenyl)acrylate (3p)
1H NMR (CDCl3, 500 MHz): 2.54 (s, 3H), 4.58 (t, J = 5.0 Hz, 2H),
4.67 (t, J = 5.0 Hz, 2H), 6.29 (d, J = 16.0 Hz, 1H), 7.13 (t, J = 8.2 Hz,
2H), 7.51–7.61 (m, 2H), 7.65 (d, J = 16.0 Hz, 1H), 7.99 (s, 1H). ESI-
MS: 320.1 (C15H15FN3O4, [M+H]+). Anal. Calcd for C15H14FN3O4: C,
56.43; H, 4.42; N, 13.16. Found: C, 56.80; H, 4.79; N, 13.53.
1 lg/mL each of aprotinin, leupeptin, and pepstatin for 2 h. The en-
tire purification procedure was performed at 4 °C or on ice.25
Both EGFR and HER-2 kinase assays were set up to assess the le-
vel of autophosphorylation based on DELFIA/Time-Resolved Fluo-
rometry. Compounds 3a–s were dissolved in 100% DMSO and
diluted to the appropriate concentrations with 25 mM HEPES at
4.4.17. (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-(3-meth-
oxyphenyl)acrylate (3q)
1H NMR (CDCl3, 500 MHz): 2.55 (s, 3H), 3.86 (s, 3H), 4.58 (t,
J = 5.0 Hz, 2H), 4.68 (t, J = 5.0 Hz, 2H), 6.35 (d, J = 16.0 Hz, 1H),
6.35 (dd, J = 2.0 Hz, J = 8.0 Hz, 1H), 7.04 (s, 1H), 7.12 (d, J = 8.0 Hz,
2H), 7.33 (t, J = 8.0 Hz, 1H), 7.64 (d, J = 16.0 Hz, 1H), 8.01 (s, 1H).
ESI-MS: 332.1 (C16H18N3O5, [M+H]+). Anal. Calcd for C16H17N3O5:
C, 58.00; H, 5.17; N, 12.68. Found: C, 58.36; H, 5.52; N, 12.99.
pH 7.4. In each well, 10 lL compound was incubated with 10 lL
(12.5 ng for HER-2 or 5 ng for EGFR) recombinant enzyme (1:80
dilution in 100 mM HEPES) for 10 min at room temperature. Then,
10
l
L of 5ꢀ buffer (containing 20 mM HEPES, 2 mM MnCl2, 100
lM
Na3VO4, and 1 mM DTT) and 20
l
L of 0.1 mM ATP–50 mM MgCl2
were added for 1 h. Positive and negative controls were included
4.4.18. (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-(naph-
thalen-1-yl) acrylate (3r)
in each plate by incubation of enzyme with or without ATP–MgCl2.
1H NMR (CDCl3, 500 MHz): 2.56 (s, 3H), 4.61 (t, J = 5.2 Hz, 2H),
4.70 (t, J = 5.2 Hz, 2H), 6.45 (d, J = 15.5 Hz, 1H), 7.49 (t, J = 7.5 Hz,
1H), 7.53–7.56 (m, 1H), 7.58–7.61 (m, 1H), 7.74 (d, J = 7.3 Hz,
1H), 7.89 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 8.2 Hz, 1H), 8.00 (s, 1H),
8.14 (d, J = 8.2 Hz, 1H), 8.51 (d, J = 15.8 Hz, 1H). ESI-MS: 352.1
(C19H18N3O4, [M+H]+). Anal. Calcd for C19H17N3O4: C, 64.95; H,
4.88; N, 11.96. Found: C, 65.30; H, 4.69; N, 12.28.
Table 3
Crystallographical and experimental data for compound 3f
Compounds
3f
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
C
16H17N3O5
331.33
Triclinic
p-1
7.7630(16)
8.7390(17)
13.290(3)
80.94(3)
78.11(3)
64.90(3)
796.4(3)
2
b (Å)
c (Å)
4.4.19. (E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-(naph-
thalen-2-yl) acrylate (3s)
a
(°)
b (°)
(°)
1H NMR (CDCl3, 500 MHz): 2.56 (s, 3H), 4.59 (t, J = 5.0 Hz, 2H),
4.68 (t, J = 5.0 Hz, 2H), 6.47 (d, J = 16.0 Hz, 1H), 7.53–7.56 (m, 2H),
7.65 (d, J = 8.5 Hz, 1H), 7.82–7.89 (m, 4H), 7.94 (s, 1H), 8.01 (s, 1H).
ESI-MS: 352.1 (C19H18N3O4, [M+H]+). Anal. Calcd for C19H17N3O4:
C, 64.95; H, 4.88; N, 11.96. Found: C, 64.96; H, 5.07; N, 12.31.
c
V (Å)
Z
Dcalcd/g cmꢁ3
1.382
h range (°)
F(000)
1.57–25.27
348
Reflections collected/unique
Data/restraints/parameters
Absorption coefficient (mmꢁ1
3126/2894 [Rint = 0.0287]
2894/0/218
0.104
4.5. Crystal structure determination
)
R1; wR2 [I > 2
R1; wR2 (all data)
GOOF
r
(I)]
0.0500/0.1125
0.0817/0.1286
1.019
Crystal structure determination of compound 3f were carried
out on a Nonius CAD4 diffractome0ter equipped with graphite-
monochromated MoKa (k = 0.71073 ÅA) radiation (Fig. 2). The struc-

4996
Y. Qian et al. / Bioorg. Med. Chem. 18 (2010) 4991–4996
At the end of incubation, liquid was aspirated, and plates were
washed three times with wash buffer. A 75 L (400 ng) sample of
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L of 5 mg/mL MTT in
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4.8. Molecular docking modeling
Molecular docking of compound 3h into the three-dimensional
EGFR complex structure (1M17.pdb, downloaded from the PDB)
was carried out using the AutoDock software package (version
4.0) as implemented through the graphical user interface Auto-
DockTools (ADT 1.4.6).
Acknowledgment
This work was supported by the Jiangsu National Science Foun-
dation (No. BK2009239), Anhui National Science Foundation (No.
070416274X), and by a Grant (30772627) from National Natural
Science Foundation of China.