Metronidazole acid acyl sulfonamide: A novel class of anticancer agents and potential EGFR tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.08.038

A series of novel metronidazole derivatives were recently reported as potent anticancer agents targeting EGFR and HER-2 by our group [Qian, Y.; Zhang, H. J.; Zhang, H.; Xu, C.; Zhao, J.; Zhu, H. L. Bioorg. Med. Chem. 2010, 18, 4991]. Based on the previous

Full text of DOI:10.1016/j.bmc.2011.08.038

Bioorganic & Medicinal Chemistry 19 (2011) 6069–6076
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Metronidazole acid acyl sulfonamide: A novel class of anticancer agents
and potential EGFR tyrosine kinase inhibitors
Yin Luo a, Yao Li a, Ke-Ming Qiu a, Xiang Lu a, Jie Fu b, Hai-Liang Zhu a,
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
b State Key Laboratory of Pollution Control & Resource Reuse, Nanjing University (Xianlin Campus), Nanjing 210046, 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 metronidazole derivatives were recently reported as potent anticancer agents targeting
EGFR and HER-2 by our group [Qian, Y.; Zhang, H. J.; Zhang, H.; Xu, C.; Zhao, J.; Zhu, H. L. Bioorg. Med.
Chem. 2010, 18, 4991]. Based on the previous results, we designed and synthesized a new series of met-
ronidazole acid acyl sulfonamide derivatives and a new series of phenylacetyl benzenesulfonamide deriv-
atives and their anticancer activities were evaluated as potential EGFR and HER-2 kinase inhibitors.
Among all the compounds, compound 12 displayed the most potent inhibitory activity EGFR and HER-
Received 14 July 2011
Revised 18 August 2011
Accepted 18 August 2011
Available online 26 August 2011
Keywords:
Metronidazole
Sulfonamide
2 (IC50 = 0.39
inhibitory activity against A549 and B16-F10 cancer cell line in vitro, with an IC50 value of 1.26
for A549 and 0.35 g/mL for B16-F10. Docking simulation was further performed to position compound
12 into the EGFR active site to determine the probable binding model.
l
M for EGFR and IC50 = 1.53
lM for HER-2) and it also showed the most potent growth
l
g/mL
l
EGFR
Antiproliferative activity
Molecular docking
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
attracted considerable attention as they showed a tendency to pen-
etrate 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 metab-
olism of nitroimidazoles, particularly metronidazole, have been
characterized.14,15 Therefore, nitroimidazoles may provide the
attractive possibility of employing these molecules as carriers for
targeted delivery in cancer therapy.12,16 Based on the results, a ser-
ies of novel metronidazole derivatives were recently reported as
potent anticancer agents targeting EGFR and HER-2 in our group
and compound 3h (Fig. 1) was the most active agent with IC50 va-
Epidermal growth factor receptor (EGFR) is a kind of tyrosine ki-
nase firstly reported in the literature.2,3 It has become one of the
targets of anticancer drug research and development because of
its widely distribution in the cell and important role in cell life.
EGFRs are distributed in mammalian epithelial cell membranes
and have relationships with cell proliferation, death, and differen-
tiation. They are junctions to deliver extracellular growth signals
intracellular. EGFR family comprise four members, including: EGFR
(HER1/ErbB-1), ErbB-2 (HER2/neu), ErbB-3 (HER3), and ErbB-4
(HER4).4 EGFR tyrosine kinase-mediated cell growth signaling
pathway plays an important role in the formation and develop-
ment of many types of solid tumors, such as non small cell lung
cancer,5 head and neck cancer,6 and glioblastomas.7 Overexpres-
sion of EGFR family receptors have always been observed in these
tumors, approximately in 60% of all tumors.5 EGFR and ErbB-2 are
the hottest targets in current research and their overexpression or
abnormal activation often cause cell malignant transformation.
Also they have relationship with postoperative adverse, radiother-
apy and chemotherapy resistance and tumor angiogenesis.8
In previous study, metronidazole (Fig. 1), a kind of nitroimidaz-
ole, has been selected as starting material for the preparation of
99mTc radiopharmaceuticals due to its affinity for hypoxic tu-
mors.9 On the mechanism level, nitroimidazole derivatives have
lue of 0.62 lM for EGFR and 2.15 l
M for HER-2.4
In addition, many acyl sulfonamides displayed potent biological
activities and low toxicities in previous reports. Among them, some
compounds showed their anticancer effects by inhibiting vascular
endothelial growth factor receptor (VEGFR), such as compound 1
and 2 shown in Figure 1.17 The two compounds had curative activ-
ity against colon adenocarcinoma, human colon, human prostate,
and human breast cancer lines.18 All of these encouraged us to con-
tinuously design and screen new metronidazole acyl sulfonamides
as potential EGFR inhibition agents.
In this paper, the metronidazole derivatives were further com-
bined with sulfonamides by amide bond, which was usually used
to connect substructures in our laboratory. Meanwhile, we also
synthesized the analogs (phenylacetyl benzenesulfonamide deriv-
atives) of compound 1 and 2 to contrast with metronidazole deriv-
atives because phenylacetic acid and metronidazole had the same
length of side chain. The two series of compounds, metronidazole
⇑
Corresponding author. Tel./fax: +86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.038

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Y. Luo et al. / Bioorg. Med. Chem. 19 (2011) 6069–6076
N
N
OH
O
N
N
NO2
O
O2N
Metronidazole
3h
O
Cl
O
Cl
O
O
O
O
S
S
N
N
H
H
Cl
Cl
Cl
1
2
Figure 1. Chemical structures of some reported compounds.
acid benzenesulfonamide and phenylacetyl benzenesulfonamide
derivatives, might exhibit synergistic effect in anticancer activities.
The objectives of present work are to synthesize new acyl benzene-
sulfonamides; to evaluate their EGFR inhibition activities; to test
their anticancer activities and to investigate the inhibitor interac-
tion with EGFR by docking study.
was obtained with yield of 65%. Then, synthesized compound 4,
active sulfonamides 5–9, 4-dimethyaminopyridine (DMAP) and
1-[3-(dimethyamino)-propyl]-3-ethylcarbodiimide hydrochloride
(EDCI) were dissolved in dichloromethane and refluxed overnight
to give the desired compounds 10–14 (Table 1).
A series of novel phenylacetyl benzenesulfonamide derivatives
(20–44) were also synthesized by the routes outlined in Scheme
2. Phenylacetic acids 15–19, active sulfonamides 5–9, DMAP and
EDCI were dissolved in CH2Cl2 and refluxed to give the desired
compounds 20–44.
2. Results and discussion
2.1. Chemistry
2.2. In vitro enzyme inhibition activity
A series of novel metronidazole–sulfonamide derivatives (10–
14) were synthesized by the routes outlined in Scheme 1. Com-
pound 4 was synthesized from metronidazole (3) according to
the modified procedure of Mirzaei et al.19 Metronidazole was dis-
solved in water and sodium dichromate aqueous solution was
added followed by drop wise addition sulfuric acid 50%. The mix-
ture was stirred overnight and then neutralized. Compound 4
The two series of benzenesulfonamide derivatives (10–14 and
20–44) were evaluated for their abilities to inhibit the autophos-
phorylation of EGFR kinases using a solid-phase ELISA assay. The
inhibition constants (IC50) of the compounds were summarized
in Tables 2. It was observed that benzenesulfonamide derivatives
OH
N
OH
N
a
N
N
O
NO2
NO2
3
4
R1
H
O
OH
N
N
N
b
N
S
O
N
+
O
O
S
O
NO2
NH2
NO2
O
R1
3
5-9
10--14
Scheme 1. General synthesis of sulfonamide derivatives (10–14). Reagents and conditions: (a) sodium dichromate, 50% sulfuric acid, 25 °C, overnight; (b) CH2Cl2, EDCI,
DMAP, 25 °C, overnight.

Y. Luo et al. / Bioorg. Med. Chem. 19 (2011) 6069–6076
6071
Table 1
bromine. Compounds 20 and 21 have the lowest activities. The
explanation theory of B ring can be also applied on A ring. Com-
Chemical structures of benzenesulfonamide derivatives 10–14 and 20–44
pounds (40, IC50 = 24.68
HER-2) with meta-substituted group showed slightly less potent
activities than those of para-substituted (30, IC50 = 17.81 M for
EGFR and IC50 = 19.05 M for HER-2).
Among all the compounds 22–44, 22 (IC50 = 5.17
and IC50 = 7.02 M for HER-2) showed the best activity while the
substituents in the A ring and B ring are both fluorine and fluorine
atom is para-substituted in the A ring. The results showed that
electron-withdrawing groups could enhance the activities of com-
pounds. Compound 22 could be a promising lead for the further
development of novel EGFR inhibition agents.
The above results indicated that the metronidazole skeleton in
the benzenesulfonamide derivatives 10–14 might play an impor-
tant role in the EGFR inhibitory activity which might be related
with the electron-withdrawing nitro-group in metronidazole. This
result was similar with the reported synthesized metronidazole
derivatives1 and conformed to our estimate. However, the metro-
nidazole–benzenesulfonamide derivatives different with classical
EGFR inhibitors (e.g., Gefitinib and Erlotinib) were reported EGFR
inhibitors.
For the synthesized compounds, it was observed that the IC50
value for inhibiting HER-2 was higher than EGFR, however, they
had the same trends. This might be attributed to the concentration
of purified kinase HER-2 is higher than that of EGFR kinase in the
experiments. Obviously, there was a reasonable correlation be-
tween sensitivities of EGFR and HER-2 to these synthesized com-
pounds and this result was the same view that the highly
homologous sequence of these two kinase catalytic domains
decided their sensitivities.
lM for EGFR and IC50 = 26.33 lM for
Compound
R1
R2
R3
Compound
R1
R2
R3
10
11
12
13
14
20
21
22
23
24
25
26
27
28
29
H
CH3
F
Cl
Br
H
CH3
F
Cl
Br
H
CH3
F
—
—
—
—
—
F
F
F
F
F
Cl
Cl
Cl
Cl
Cl
—
—
—
—
—
H
H
H
H
H
H
H
H
H
H
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
H
CH3
F
Cl
Br
H
CH3
F
Cl
Br
H
CH3
F
Br
Br
Br
Br
H
H
H
H
H
H
H
H
l
l
lM for EGFR
Br
l
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
Br
Br
Br
Br
Br
Cl
Br
Cl
Br
containing metronidazole skeleton (compounds 10–14) showed
fairly good inhibiting EGFR activities displaying IC50 values be-
tween 0.39 and 38.52
substituted (12–14) (IC50 = 0.39, 1.51, 2.17
IC50 = 1.53, 3.41, 3.59 M for HER-2) exhibited significant inhibi-
tion activities in the order of fluorine > chlorine > bromine. Com-
pound 11 with the electron donating substitute methyl had the
lowest activity (IC50 = 3.43 lM). These results demonstrated that
a hydrophobic and electron-withdrawing halogen group might
have slightly promotion function to EGFR and HER-2 inhibition
activities, however, electron donating substitutes had negative
effects.
l
M. The compounds with para halogen
lM for EGFR and
l
As illustrated in Table 2, compounds 20–44 displayed moderate
EGFR inhibitory activities with IC50 ranging from 5.17 to 38.52 lM.
2.3. Antiproliferative activity and molecular docking study
In the case of constant B ring (Scheme 2) substituents, change of
substituents on A ring can affect the activities of these compounds.
Among the compounds 20–24, these compounds with the halogen
atom substituent (22–24) show stronger anticancer activity and
the strength order is similar with A ring: fluorine > chlorine >
The in vitro anticancer activities of 10 compounds (10–14 and
20–24) with better inhibition activities against EGFR were tested
using adenocarcinomic human alveolar basal epithelial cells
H
O
R1
R3
R2
OH
R3
R2
N
S
c
O
A
+
O
O
O
B
S
NH2
R1
O
20-44
15-19
5-9
Scheme 2. General synthesis of sulfonamide derivatives (20–44). Reagents and conditions: (c) CH2Cl2, EDCI, DMAP, overnight.
Table 2
Inhibition activities of compounds 10–14 and 20–44 against EGFR and HER-2 (IC50
, lM)
Compounds
EGFR (lM)
HER-2 (lM)
Compounds
EGFR (
lM)
HER-2 (lM)
10
11
12
13
14
20
21
22
23
24
25
26
27
28
29
Erlotinib
2.94 0.23
3.43 0.22
0.39 0.02
1.51 0.11
2.17 0.32
6.74 0.46
8.28 0.72
5.17 0.64
5.93 0.53
6.41 0.67
11.91 2.33
13.73 1.57
9.59 0.85
10.34 1.92
11.29 1.52
0.032 0.002
4.21 0.35
4.76 0.42
1.53 0.18
3.41 0.32
3.59 0.28
8.41 0.76
10.53 1.41
7.02 0.65
7.56 0.82
8.18 0.94
13.07 1.24
15.57 1.63
11.05 1.09
13.32 1.46
13.56 1.47
0.16 0.02
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
17.81 2.13
18.35 1.56
14.53 1.7
15.92 0.78
17.26 1.29
26.2 3.44
38.52 3.59
21.84 2.48
32.85 3.96
24.15 2.12
24.68 1.94
26.17 2.75
18.72 1.88
21.28 2.55
24.33 2.39
19.05 1.57
20.33 1.78
16.26 1.38
18.94 1.93
19.83 1.56
29.07 3.02
>50
23.45 2.38
37.93 3.15
25.03 2.04
26.33 2.92
27.89 2.46
20.45 1.65
23.46 2.47
27.38 2.33

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Y. Luo et al. / Bioorg. Med. Chem. 19 (2011) 6069–6076
Table 3
Antiproliferative activities and docking parameters of compounds 10–14 and 20–24
Compounds
A549 (IC50
,
l
g/mL)
B16-F10 (IC50
,
lg/mL)
Binding energy DGb (kcal/mol)
10
11
12
13
14
20
21
22
3.96 0.34
4.38 0.43
1.26 0.16
2.47 0.26
3.03 0.37
10.11 0.94
12.04 1.36
5.64 0.51
6.28 0.67
7.94 0.79
0.13 0.01
2.93 0.27
3.55 0.36
0.35 0.03
0.92 0.08
2.37 0.25
9.07 0.94
9.94 1.36
4.23 0.45
6.36 0.68
8.2 0.92
ꢀ12.56
ꢀ11.09
ꢀ14.14
ꢀ13.78
ꢀ13.04
ꢀ8.17
ꢀ7.95
ꢀ10.06
ꢀ9.34
23
24
Erlotinib
ꢀ8.25
0.022 0.001
ꢀ8.83
benzenesulfonamide derivatives were potent anticancer agents as
inhibitor of EGFR. In particular, compound 12 has shown signifi-
cant inhibitory activity in tumor growth and displayed favorable
EGFR inhibitory activity.
To gain better understanding on the potency of the 10 com-
pounds and guide further SAR studies, we proceeded to examine
the interaction of these compounds with EGFR (PDB code: 1M17)
by molecular docking, which was performed by simulation of the
10 compounds into the ATP binding site in EGFR. All docking runs
were applied the Lamarckian genetic algorithm of Auto-Dock 4.0.20
The binding model of compound 12 and EGFR is depicted in Figure
2. The amino acid residues which had interaction with EGFR were
labeled. In the binding mode, compound 12 was nicely bound to
the ATP binding site of EGFR hydrophobic interaction and binding
was stabilized by a hydrogen bond and a p–cation interaction.
The predicted binding free energy that includes the intermolec-
ular energy and torsional free energy was used as the criterion for
ranking. The estimated free energy of other compounds (10–14
and 20–24) were ranging from ꢀ14.14 to ꢀ7.95 kcal/mol. The se-
lected pose of 12 had an estimated binding free energy of
ꢀ14.14 kcal/mol (binding free energy of control compound, erloti-
nib is ꢀ8.83 kcal/mol). The oxygen atom of the sulfonamide system
formed one hydrogen bond with the amino hydrogen of LYS828
(bond length: LYS828 N–Hꢁ ꢁ ꢁO = 2.744 Å; bond angle: LYS828 N–
Hꢁ ꢁ ꢁO = 130.98°). This residue (LYS828) was also found to be
involved in the binding of other EGFR inhibitors.4 The amino
Figure 2. Binding mode of compound 12 (colored by atom: carbons: gray; oxygens:
red) with EGFR enzyme (entry 1M17 in the Protein Data Bank). The dotted lines
show the hydrogen bonds and the p–cation interaction is shown as yellow column.
hydrogen of LYS828 was also formed a
p–cation interaction with
the benzene ring of compound 12, which enhanced the binding
A549 and mouse melanoma cells B16-F10, in both of which EGFR
are overexpressed. As shown in Table 3, compounds 12 and 22,
which had potent inhibitory activity of EGFR showed high antipro-
action between receptor EGFR and ligand compound 12. The elec-
tron donating substituent amino –NH2 strengthen the
p–cation
binding and the results indicated that it was primarily due to direct
through-space interaction between the substituent and the
cation.21
liferative activities (IC50 = 1.26 and 5.64
lg/mL for A549 and
IC50 = 0.35 and 4.23 g/mL for B16-F10), indicating that these
l
Figure 3A. 3D model of the interaction between compound 12 and the ATP binding site. The protein is represented by molecular surface. Left: 12 is depicted by sticks and
balls. Right: 12 is depicted by molecular surface, sticks and balls.

Y. Luo et al. / Bioorg. Med. Chem. 19 (2011) 6069–6076
6073
Figure 3B. 3D model of the interaction between compound 22 and the ATP binding site. The protein is represented by molecular surface. Left: 22 is depicted by sticks and
balls. Right: 22 is depicted by molecular surface, sticks and balls.
Figure 3C. 3D model of the interaction between erlotinib and the ATP binding site. The protein is represented by molecular surface. Left: erlotinib is depicted by sticks and
balls. Right: erlotinib is depicted by molecular surface, sticks and balls.
3D model of the interaction between compound 12 or 22 and
the ATP binding site was depicted in Figure 3A and 3B. The model
was similar with the models between erlotinib and the ATP bind-
ing site (Fig. 3C). Comparing these models, it was found that the
hydrophobic pockets of ATP binding site were occupied by these
compounds, and the difference was the arrangement on the en-
zyme surface.
Meanwhile, we chose 22 as the positive control in the docking
procedure. In Figure 3B, it was found that there was only one
hydrogen bond in the binding pocket. The sulfonamide oxygen
atom of compound 22 formed one hydrogen bond with the amino
hydrogen of LEU768 (bond length: LEU768 N–Hꢁ ꢁ ꢁO = 2.741 Å;
bond angle: LEU768 N–Hꢁ ꢁ ꢁO = 148.07°). Furthermore, in the com-
parison to Erlotinib’s binding mode (Fig. 3C), the formation of
exhibited potent EGFR and HER-2 inhibitory activities and antipro-
liferative activities against A549 cell lines and B16-F10 cell lines.
Compound 12 showed the most potent EGFR inhibition activities
(IC50 = 0.39
lM for EGFR and IC50 = 1.53
lM for HER-2) and anti-
cancer activities (IC50 = 1.26
lg/mL for A549 and IC50 = 0.35
l
g/
mL for B16-F10). Molecular docking was further performed to
study the inhibitor-EGFR protein interactions. After analysis of
the binding model of compound 12 with EGFR, it was found that
a hydrogen bond and a p–cation interaction with the protein resi-
dues in the ATP binding site might play a crucial role in its EGFR
inhibition and antiproliferative activities. Among these com-
pounds, it could be concluded that compound 12 had been demon-
strated to show significant EGFR and tumor growth inhibitory
activity as a potential anticancer agent. The result of this work
might be helpful for the design and synthesis of EGFR inhibitors
with stronger activities.
hydrogen bond with LYS828 and the p–cation interaction seemed
significant for 12 and the binding energy was up to ꢀ14.14 kcal/
mol, better than ꢀ8.83 kcal/mol from the optimal binding confor-
mation of Erlotinib (Fig. 3C). This molecular docking result, along
with the biological assay data, suggesting that compound 12 is a
potential inhibitor of EGFR.
4. Experiments
4.1. Materials and measurements
3. Conclusion
All chemicals and reagents used in current study were of analyt-
ical grade. All the 1H NMR spectra were recorded on a Bruker DPX
300 model Spectrometer in CDCl3 or DMSO-d6 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
In this paper, a series of novel EGFR inhibitors (10–14 and 20–
44) bearing metronidazole (or substituted phenylacetic acid skele-
ton) and benzenesulfonamide moiety had been synthesized and
their biological activities were evaluated. These compounds

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Y. Luo et al. / Bioorg. Med. Chem. 19 (2011) 6069–6076
the glass-backed silica gel sheets (Silica Gel 60 GF254) and visual-
ized in UV light (254 nm). Column chromatography was performed
using silica gel (200–300 mesh) eluting with ethyl acetate and
petroleum ether.
4.2.5. N-(4-Bromophenylsulfonyl)-2-(2-methyl-5-nitro-1H-
imidazol-1-yl) acetamide (14)
Yield 81%; mp 275 °C. 1H NMR (300 Hz, DMSO-d6): d 2.33 (s,
3H); 5.06 (s, 2H); 7.71 (d, J = 8.58 Hz, 2H); 7.90 (d, J = 6.79 Hz,
2H); 7.96 (s, 1H). ESI-MS: 402.96 (C12H12BrN4O5S, [M+H]+). Anal.
Calcd for C12H11BrN4O5S: C, 35.75; H, 2.75; N, 13.90. Found: C,
35.64; H, 2.73; N, 13.95.
4.2. General procedure for synthesis of metronidazole–sulfona
mide derivatives (10–14)
Compound 4 was synthesized from metronidazole (3) according
the modified procedure of Javad et al.19 To a stirring suspension of
metronidazole (3, 8.55 g, 0.05 mol) in water (20 mL) at room tem-
perature was added a solution of 14.25 g of sodium dichromate in
water (69 mL) followed by drop wise addition of sulfuric acid
(28.5 mL 50%). The mixture was stirred overnight. The reaction
mixture was carefully neutralized with 4 N NaOH aqueous solution
(250 mL). It was extracted with ethyl acetate/THF (1:1, 4 ꢂ 30 mL).
The combined organic layer was washed with brine (100 mL), dried
under Na2SO4 and evaporated under reduced pressure. The crude
residue was recrystallized from CH2Cl2 to give compound 4.
To a round-bottom flask (500 mL) that contained a solution of
aryl sulfonamide (6 mmol), 4-dimethyaminopyridine (DMAP,
13 mmol), and 1-[3-(dimethyamino)-propyl]-3-ethylcarbodiimide
hydrochloride (EDCI, 13 mmol) in CH2Cl2 (150 mL) was added the
synthesized compound 4 (6 mmol) at room temperature. The result-
ing mixture was stirred at room temperature for 12 h, then cooled to
5 °C, and acidified to pH 1 with addition of HCl aqueous solution
(10%), which was followed by extraction with CH2Cl2/MeOH (9:1,
3 ꢂ 100 mL). The combined organic layers were washed with H2O
and brine, dried over using Na2SO4, and concentrated in vacuo. The
residue was subjected to silica gel chromatographyor crystallization
if necessary to afford the compounds (11–14) (Scheme 1).
4.3. General procedure for synthesis of phenylacetyl bezenesu
lfonamides (20–44)
To a round-bottom flask (500 mL) that contained a solution of
aryl sulfonamide (6 mmol), 4-dimethyaminopyridine (DMAP,
13 mmol), and 1-[3-(dimethyamino)-propyl]-3-ethylcarbodiimide
hydrochloride (EDCI, 13 mmol) in CH2Cl2 (150 mL) was added the
synthesized phenylacetic acid (6 mmol) at room temperature. The
resulting mixture was stirred at room temperature for 12 h, then
cooled to 5 °C, and acidified to pH 1 with addition of HCl aqueous
solution (10%), which was followed by extraction with
CH2Cl2/MeOH (9:1, 3 ꢂ 100 mL). The combined organic layers were
washed with H2O and brine, dried over Na2SO4, and concentrated in
vacuo. The residue was subjected to silica gel chromatography or
crystallization if necessary to afford the compounds (20–44)
(Scheme 2).
4.3.1. 2-(4-Fluorophenyl)-N-(phenylsulfonyl)acetamide (20)
Yield 82%; mp 115–117 °C. 1H NMR (300 MHz, DMSO-d6): d
3.56 (s, 2H); 7.25–7.28 (m, 3H); 7.33–7.37 (m, 2H); 7.39–7.42 (m,
1H); 7.50–7.56 (m, 1H); 7.70 (d, J = 8.22 Hz, 1H); 7.97 (d,
J = 8.4 Hz, 1H); 13.54 (s, 1 Hz); ESI-MS: 294.05 (C14H13FNO3S,
[M+H]+). Anal. Calcd for C14H12FNO3S: C, 57.33; H, 4.12; N, 4.78.
Found: C, 57.13; H, 4.15; N, 4.75.
4.2.1. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-N-(phenylsulfonyl
)acetamide (10)
4.3.2. 2-(4-Fluorophenyl)-N-tosylacetamide (21)
Yield 82%; mp 96 °C. 1H NMR (300 MHz, DMSO-d6): d 2.32 (s,
3H); 3.56 (s, 2H); 7.26 (d, J = 6.36, 2H); 7.34–7.37 (m, 2H); 7.50–
7.55 (m, 1H); 7.70 (d, J = 8.43 Hz, 1H); 7.96 (d, J = 8.4 Hz, 1H);
13.54 (s, 1 Hz). ESI-MS: 308.07 (C15H15FNO3S, [M+H]+). Anal. Calcd
for C15H14FNO3S: C, 58.62; H, 4.59; N, 4.56. Found: C, 58.45; H,
4.55; N, 4.58.
Yield 82%; mp 262 °C. 1H NMR (300 Hz, DMSO-d6): d 2.31
(s, 3H); 5.08 (s, 2H); 7.59–7.64 (t, J = 7.41 Hz, 2H); 7.69–7.74
(t, J = 7.5 Hz, 1H); 7.89–7.92 (t, J = 4.2 Hz, 2H); 7.98 (s, 1H) ppm.
ESI-MS: 325.31 (C12H13N4O5S, [M+H]+). Anal. Calcd for
C12H12N4O5S: C, 44.44; H, 3.73; N, 17.28. Found: C, 44.29; H,
3.79; N, 17.32.
4.3.3. 2-(4-Fluorophenyl)-N-(4-fluorophenylsulfonyl)acetamide
(22)
4.2.2. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-N-tosylacetamide
(11)
Yield 82%; mp 89–90 °C. 1H NMR (300 MHz, DMSO-d6): d 3.57
(s, 2H); 7.17 (d, J = 8.13 Hz, 2H); 7.35 (d, J = 8.83, 2H); 7.54–7.59
(d, J = 8.70 Hz, 2H); 7.90–7.94 (m, 2H); 12.45 (s, 1H). ESI-MS:
312.04 (C14H12F2NO3S, [M+H]+). Anal. Calcd for C14H11F2NO3S: C,
54.01; H, 3.56; N, 4.50. Found: C, 54.21; H, 3.54; N, 4.53.
Yield 85%; mp 246 °C. 1H NMR (300 Hz, DMSO-d6): d 2.51 (s,
3H); 5.07 (s, 2H); 7.41(d, J = 8.22 Hz, 2H); 7.78 (d, J = 8.22 Hz,
2H); 7.99 (s, 1H). ESI-MS: 339.07 (C13H15N4O5S, [M+H]+). Anal.
Calcd for C13H14N4O5S: C, 46.15; H, 4.17; N, 16.56; Found: C,
46.25; H, 4.23; N, 16.45.
4.3.4. N-(4-Chlorophenylsulfonyl)-2-(4-fluorophenyl)acetamide
(23)
4.2.3. N-(4-Fluorophenylsulfonyl)-2-(2-methyl-5-nitro-1H-
imidazol-1-yl) acetamide (12)
Yield 82%; mp 102–104 °C. 1H NMR (300 MHz, DMSO-d6): d 3.57
(s, 2H); 7.15 (d, J = 8.4 Hz, 2H); 7.34 (d, J = 8.43, 2H); 7.42–7.48 (d,
J = 8.76 Hz, 2H); 7.94–7.99 (m, 2H); 12.46 (s, 1H). ESI-MS: 328.01
(C14H12ClFNO3S, [M+H]+). Anal. Calcd for C14H11ClFNO3S: C,
51.30; H, 3.38; N, 4.27. Found: C, 51.41; H, 3.43; N, 4.25.
Yield 84%; mp 232–234 °C. 1H NMR (300 Hz, DMSO-d6): d 2.32
(s, 3H); 5.07 (s, 2H); 7.43–7.49 (t, J = 8.77 Hz, 2H); 7.95–7.99 (m,
3H). ESI-MS: 343.04 (C12H12FN4O5S, [M+H]+). Anal. Calcd for
C12H11FN4O5S: C, 42.11; H, 3.24; N, 16.37. Found: C, 42.23; H,
3.12; N, 16.49.
4.2.4. N-(4-Chlorophenylsulfonyl)-2-(2-methyl-5-nitro-1H-imid
azol-1-yl) acetamide (13)
4.3.5. N-(4-Bromophenylsulfonyl)-2-(4-fluorophenyl)acetamide
(24)
Yield 81%; mp 252 °C. 1H NMR (300 Hz, DMSO-d6): d 2.32 (s,
3H); 5.06 (s, 2H); 7.73 (d, J = 8.61 Hz, 2H); 7.89 (d, J = 6.78 Hz,
2H); 7.99(s, 1H). ESI-MS: 359.01 (C12H12ClN4O5S, [M+H]+). Anal.
Calcd for C12H11ClN4O5S: C, 40.17; H, 3.09; N, 15.62. Found: C,
40.23; H, 3.29; N, 15.45.
Yield 85%; mp 143–145 °C. 1H NMR (300 MHz, DMSO-d6): d
3.56 (s, 2H); 7.11 (d, J = 8.22 Hz, 2H); 7.47 (d, J = 8.25 Hz, 2H);
7.78–7.85 (m, 4H); 12.46 (s, 1H). ESI-MS: 371.96 (C14H12BrFNO3S,
[M+H]+). Anal. Calcd for C14H11BrFNO3S: C, 45.18; H, 2.98; N,
3.76. Found: C, 45.29; H, 2.95; N, 3.75.

Y. Luo et al. / Bioorg. Med. Chem. 19 (2011) 6069–6076
6075
4.3.6. 2-(4-Chlorophenyl)-N-(phenylsulfonyl)acetamide (25)
4.3.14. 2-(4-Bromophenyl)-N-(4-bromopheny lsulfonyl )
acetamide (34)
Yield 85%; mp 125–127 °C. 1H NMR(300 MHz, DMSO-d6): 3.56
(s, 2H); 7.25–7.28 (m, 3H); 7.33–7.37 (m, 2H); 7.39–7.42 (m,
1H); 7.50–7.56 (m, 1H); 7.70 (d, J = 8.22 Hz, 1H); 7.97 (d,
J = 8.4 Hz, 1H); 13.54 (s, 1 Hz); ESI-MS: 310.02 (C14H13ClNO3S,
[M+H]+). Anal. Calcd for C14H12ClNO3S: C, 54.28; H, 3.90; N, 4.52.
Found: C, 54.37; H, 3.76; N, 4.83.
Yield 81%; mp 218–221 °C. 1H NMR (300 MHz, DMSO-d6): d
3.56 (s, 2H); 7.10 (d, J = 8.28 Hz, 2H); 7.32 (d, J = 6.54 Hz, 2H);
7.72–7.9 (m, 4H); 12.47 (s, 1H); ESI-MS: 431.88 (C14H12Br2NO3S,
[M+H]+). Anal. Calcd for C14H11Br2NO3S: C, 38.82; H, 2.56; N,
3.23; Found: C, 38.91; H, 2.60; N, 3.12.
4.3.15. N-(4-Fluorophenylsulfonyl)-2-(4-methoxy phenyl)
acetamide (37)
4.3.7. 2-(4-Chlorophenyl)-N-tosylacetamide (26)
Yield 82%; mp 128–130 °C. 1H NMR (300 MHz, DMSO-d6): d
2.32 (s, 3H); 3.56 (s, 2H); 7.26 (d, J = 6.36, 2H); 7.34–7.37 (m,
2H); 7.50–7.55 (m, 1H); 7.70 (d, J = 8.43 Hz, 1H); 7.96 (d,
J = 8.4 Hz, 1H); 13.54 (s, 1 Hz). ESI-MS: 324.04 (C15H15ClNO3S,
[M+H]+). Anal. Calcd for C15H14ClNO3S: C, 55.64; H, 4.36; N, 4.33.
Found: C, 55.49; H, 4.45; N, 4.51.
Yield 87%; mp 130–132 °C. 1H NMR (300 MHz, DMSO-d6): d 3.60
(s, 2H); 3.73 (s, 3H); 7.27 (d, J = 4.40, 1H); 7.28–7.34 (m, 2H); 7.39–
7.42 (t, J = 4.50 Hz, 1H); 7.51–7.54 (t, J = 4.60 Hz, 1H); 7.70 (d,
J = 5.11 Hz, 1H); 7.97 (d, J = 5.73 Hz, 1H); 13.01 (s, 1H). ESI-MS:
324.06 (C15H15FNO4S, [M+H]+). Anal. Calcd for C15H14FNO4S: C,
55.72; H, 4.36; N, 4.33. Found: C, 55.34; H, 4.35; N, 4.30.
4.3.8. 2-(4-Chlorophenyl)-N-(4-fluorophenylsulfonyl)acetamide
(27)
4.3.16. N-(4-Bromophenylsulfonyl)-2-(4-methoxyphenyl)
acetamide (39)
Yield 80%; mp 144 °C. 1H NMR (300 MHz, DMSO-d6): d 3.57 (s,
2H); 7.17 (d, J = 8.4 Hz, 2H); 7.34 (d, J = 8.43, 2H); 7.42–7.48 (d,
J = 8.76 Hz, 2H); 7.94–7.99 (m, 2H); 12.46 (s, 1H). ESI-MS: 328.01
(C14H12ClFNO3S, [M+H]+). Anal. Calcd for C14H11ClFNO3S: C,
51.30; H, 3.38; N, 4.27. Found: C, 52.41; H, 3.43; N, 4.25.
Yield 87%; mp 172–173 °C. 1H NMR (300 MHz, DMSO-d6): d
3.61 (s, 2H); 3.75 (s, 3H); 7.23 (d, J = 4.59, 1H); 7.30–7.36 (m,
2H); 7.40–7.43 (t, J = 4.58 Hz, 1H); 7.53–7.56 (t, J = 4.58 Hz, 1H);
7.72 (d, J = 5.13 Hz, 1H); 7.98 (d, J = 5.76 Hz, 1H); 13.59 (s, 1H).
ESI-MS: 382.98 (C15H15BrNO4S, [M+H]+). Anal. Calcd for
C
15H14BrNO4S: C, 46.89; H, 3.67; N, 3.65. Found: C, 46.75; H,
4.3.9. N-(4-Bromophenylsulfonyl)-2-(4-chlorophenyl)acetamide
(29)
3.65; N, 3.66.
Yield 83%; mp 228–230 °C. 1H NMR (300 MHz, DMSO-d6): d
3.56 (s, 2H); 7.11 (d, J = 8.22 Hz, 2H); 7.47 (d, J = 8.25 Hz, 2H);
7.78–7.85 (m, 4H); 12.46 (s, 1H). ESI-MS: 387.93 (C14H12BrClNO3S,
[M+H]+). Anal. Calcd for C14H11ClFNO3S: C, 43.26; H, 2.85; N, 3.60.
Found: C,43.19; H, 2.89; N, 3.67.
4.3.17. 2-(3-Bromophenyl)-N-(phenylsulfonyl)acetamide (40)
Yield 84%; mp 84–86 °C. 1H NMR (300 MHz, DMSO-d6): d 3.60
(s, 2H); 7.26–7.30 (m, 3H); 7.40–7.45 (m, 2H); 7.48 (s, 1H); 7.53–
7.56 (m, 1H); 7.71 (d, J = 5.13 Hz, 1H); 7.98 (d, J = 5.13 Hz, 1H);
13.58 (s, 1H). ESI-MS: 353.97 (C14H13BrNO3S, [M+H]+). Anal. Calcd
for C14H12BrNO3S: C, 47.47; H, 3.41; N, 3.95. Found: C, 47.32; H,
3.56; N, 3.87.
4.3.10. 2-(4-Bromophenyl)-N-(phenylsulfonyl)acetamide (30)
Yield 85%; mp 139–140 °C. 1H NMR (300 MHz, DMSO-d6): d
3.57 (s, 2H); 7.22 (d, J = 8.25 Hz, 2H); 7.39-.7.44 (t, J = 7.5 Hz,
1H); 7.49–7.57 (m, 4H); 7.72 (d, J = 8.22 Hz, 1H); 7.98 (d,
J = 8.61 Hz, 1H); 13.52 (s, 1H). ESI-MS: 353.97 (C14H13BrNO3S,
[M+H]+). Anal. Calcd for C14H12BrNO3S: C, 47.47; H, 3.41; N, 3.95.
Found: C, 47.32; H, 3.56; N, 4.09.
4.3.18. 2-(3-Bromophenyl)-N-tosylacetamide (41)
Yield 84%; mp 71–71 °C. 1H NMR(300 MHz, DMSO-d6): d 2.45 (s,
3H); 3.61 (s, 2H); 7.26–7.29 (m, 2H); 7.41–7.47 (m, 2H); 7.50 (s,
1H); 7.54–7.57 (m, 1H); 7.75 (d, J = 5.10 Hz, 1H); 7.98 (d,
J = 5.25 Hz, 1H); 13.58 (s, 1H). ESI-MS: 367.99 (C15H15BrNO3S,
[M+H]+). Anal. Calcd for C14H12BrNO3S: C, 48.92; H, 3.83; N, 3.80.
Found: C, 48.70; H, 3.80; N, 3.82.
4.3.11. 2-(4-Bromophenyl)-N-tosylacetamide (31)
Yield 82%; mp 137 °C. 1H NMR (300 MHz, DMSO-d6): d 2.32 (s,
3H); 3.56 (s, 2H); 7.22 (d, J = 5.13 Hz, 2H); 7.40–7.43 (t,
J = 7.40 Hz, 1H); 7.49–7.51 (m, 2H); 7.53–7.56 (t, J = 4.58 Hz, 1H);
7.72 (d, J = 4.95 Hz, 1H); 7.98 (d, J = 5.13 Hz, 1H); 13.59 (s, 1H).
ESI-MS: 367.99 (C15H15BrNO3S, [M+H]+). Anal. Calcd for
4.3.19. 2-(3-Bromophenyl)-N-(4-fluorophenylsulfonyl)
acetamide (42)
Yield 84%; mp 67–69 °C. 1H NMR (300 MHz, DMSO-d6): d 3.59
(s, 2H); 7.24–7.28 (m, 2H); 7.45–7.49 (m, 2H); 7.52 (s, 1H); 7.57–
7.61 (m, 1H); 7.74 (d, J = 5.19 Hz, 1H); 7.93 (d, J = 5.70 Hz, 1H);
13.52 (s, 1H). ESI-MS: 371.96 (C14H12BrFNO3S, [M+H]+). Anal. Calcd
for C14H11BrFNO3S: C, 45.18; H, 2.98; N, 3.76. Found: C, 45.02; H,
2.95; N, 3.74.
C15H14BrNO3S: C, 48.92; H, 3.83; N, 3.80. Found: C, 48.75; H,
3.96; N, 3.72.
4.3.12. 2-(4-Bromophenyl)-N-(4-fluorophenylsulfonyl) aceta
mide (32)
4.3.20. 2-(3-Bromophenyl)-N-(4-chlorophenylsulfonyl)
acetamide (43)
Yield 83%; mp 160–161 °C. 1H NMR (300 MHz, DMSO-d6): d
3.55 (s, 2H); 7.11 (d, J = 8.22 Hz, 2H); 7.42–7.48 (m, 4H); 7.94–
7.99 (m, 2H); 12.41 (s, 1H). ESI-MS: 371.96 (C14H12BrFNO3S,
[M+H]+). Anal. Calcd for C14H11BrFNO3S: C, 45.18; H, 2.98; N,
3.76. Found: C, 45.29; H, 2.88; N, 3.84.
Yield 84%; mp 80–83 °C. 1H NMR (300 MHz, DMSO-d6): d 3.63
(s, 2H); 7.36–7.41 (m, 2H); 7.46–7.49 (m, 2H); 7.53 (s, 1H); 7.59–
7.63 (m, 1H); 7.77 (d, J = 5.20 Hz, 1H); 8.02 (d, J = 5.31 Hz, 1H);
13.73 (s, 1H). ESI-MS: 387.93 (C14H12BrClNO3S, [M+H]+). Anal.
Calcd for C14H11BrClNO3S: C, 43.26; H, 2.85; N, 3.60. Found: C,
43.32; H, 2.81; N, 3.63.
4.3.13. 2-(4-Bromophenyl)-N-(4-chlorophe nylsulfonyl )
acetamide (33)
Yield 83%; mp 187–188 °C. 1H NMR (300 MHz, DMSO-d6): d 3.57
(s, 2H); 7.17 (d, J = 8.61 Hz, 2H); 7.34 (d, J = 6.6 Hz, 2H); 7.79–7.85
(m, 4H); 12.46 (s, 1H); ESI-MS: 387.93 (C14H12BrClNO3S,
[M+H]+). Anal. Calcd for C14H11BrClNO3S: C, 43.26; H, 2.85; N, 3.60.
Found: C, 43.12; H, 2.83; N, 3.62%.
4.3.21. 2-(3-Bromophenyl)-N-(4-bromophenylsulfonyl)
acetamide (44)
Yield 84%; mp 99–102 °C. 1H NMR(300 Hz, DMSO-d6): d 3.62 (s,
2H); 7.40–7.44 (m, 2H); 7.52–7.56 (m, 2H); 7.64 (s, 1H); 7.73–7.78
(m, 1H); 7.82 (d, J = 5.14 Hz, 1H); 8.12 (d, J = 5.16 Hz, 1H); 14.02 (s,

6076
Y. Luo et al. / Bioorg. Med. Chem. 19 (2011) 6069–6076
1H). ESI-MS: 431.88 (C14H12Br2NO3S, [M+H]+). Anal. Calcd for
14H11Br2NO3S: C, 38.82; H, 2.56; N, 3.23; Found: C, 38.19; H,
2.52; N, 3.26.
plates. The subsequent incubation was permitted at 37 °C, 5%
CO2 atmosphere for 24 h before the cytotoxicity assessments.
Tested samples at pre-set concentrations were added to six wells
with erlotinib assayed as positive reference. After 48 h exposure
C
4.4. General procedure for preparation, purification of HER-2
and EGFR, and inhibitory assay
period, 40
dimethylthiazol–2-yl)-2,5-diphenyltetrazolium bromide)) was
added to each well. Four hours later, 100 L extraction solution
l
L of PBS containing 2.5 mg mLꢀ1 of MTT (3-(4,5-
l
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 three days
for protein expression. Sf-9 cell pellets were solubilized at 0 °C in a
buffer at pH 7.4 containing 50 mM HEPES, 10 mM NaCl, 1% Triton,
(10% SDS–5% isobutyl alcohol–0.01 M HCl) was added. After an
overnight incubation at 37 °C, the optical density was measured
at a wavelength of 570 nm on an ELISA microplate reader. In all
experiments three replicate wells were used for each drug concen-
tration. Each assay was carried out for at least three times. The re-
sults were summarized in Table 3.
4.6. Molecular docking modeling
10
mL aprotinin, 10
16 g/mL benzamidine HCl for 20 min followed by 20 min centri-
fugation. Crude extract supernatant was passed through an equili-
brated 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
l
M ammonium molybdate, 100
l
M sodium vanadate, 10
l
g/
Molecular docking of compounds into the 3D EGFR complex
structure (1M17.pdb, downloaded from the PDB) was carried out
using the AutoDock software package (version 4.0) as imple-
mented through the graphical user interface Auto-DockTools
(ADT 1.4.6).
lg/mL leupeptin, 10
lg/mL pepstatin, and
l
Acknowledgments
HEPES, 10% glycerol, and 1
l
g/mL each of aprotinin, leupeptin,
This work was supported by Jiangsu National Science Founda-
tion (No. BK2009239) and the Fundamental Research Funds for
the Central Universities (No. 1092020804 & 1106020824).
and pepstatin for 2 h. The entire purification procedure was per-
formed at 4 °C or on ice.22
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 10–14 and 20–44 were dissolved in 100%
DMSO and diluted to the appropriate concentrations with 25 mM
References and notes
1. Qian, Y.; Zhang, H. J.; Zhang, H.; Xu, C.; Zhao, J.; Zhu, H. L. Bioorg. Med. Chem.
2010, 18, 4991.
2. Slamon, D. J.; Clark, G. M.; Wong, S. G.; Levin, W. J.; Ullrich, A.; McGuire, W. L.
Science 1987, 235, 177.
3. Slamon, D. J.; Godolphin, W.; Jones, L. A. Science (Washington DC) 1989, 244,
707.
4. Kolibaba, K. S.; Druker, B. J. Biochim. Biophys. Acta. 1997, 1333, 217.
5. Tateishi, M.; Ishida, T.; Mitsudomi, T.; Kaneko, S.; Sugimachi, K. Cancer Res.
1990, 50, 7077.
6. Fleming, T. P.; Saxena, A.; Clark, W. C.; Robertson, J. T.; Oldfield, E. H.; Aaronson,
S. A.; Ali, I. U. Cancer Res. 1992, 52, 4550.
7. Shin, D. M.; Ro, J. Y.; Hong, W. K.; Hittelamn, W. N. Cancer Res. 1994, 54, 3153.
8. Ciardiello, F.; Tortola, G. Clin. Cancer Res. 2001, 7, 2958.
9. Giglio, J.; Fernández, S.; Rey, A.; Cerecetto, H. Bioorg. Med. Chem. Lett. 2011, 21,
394.
10. Mallia, M. B.; Mathur, A.; Subramanian, S.; Banerjee, S.; Sarma, H. d.;
Venkatesh, M. Bioorg. Med. Chem. Lett. 2005, 15, 3398.
11. Born, J. L.; Smith, B. R.; Harper, N.; Koch, C. J. Biochem. Pharmacol. 1992, 43,
1337.
12. Brown, J. M. Int. J. Radiat. Oncol. Biol. Phys. 1989, 16, 987.
13. Lord, E. M.; Harwell, L.; Koch, C. J. Cancer Res. 1993, 53, 5721.
14. Webster, L. T. Drugs Used in the Chemotherapy of Protozoal Infections In
Gilman, A., Rall, T. W., Nies, A. S., Taylor, P., Eds.; The pharmacological basis of
therapeutics; Pergamon Press: New York, 1990; pp 999–1007.
15. Alvaro, R. F.; Wislocki, P. G.; Miwa, G. T.; Lu, A. Y. Chem. Biol. Interact. 1992, 82,
21.
HEPES at pH 7.4. In each well, 10
10 L (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 of 5 mM buffer (containing 20 mM HEPES, 2 mM
MnCl2, 100 M Na3VO4, and 1 mM DTT) and 20 L of 0.1 mM
lL compound was incubated with
l
l
l
l
ATP–50 mM MgCl2 were added for 1 h. Positive and negative con-
trols 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 anti-
body was added to each well for another 1 h of incubation. After
washing, enhancement solution was added and the signal was de-
tected by Victor (Wallac Inc.) with excitation at 340 nm and emis-
sion at 615 nm. The percentage of autophosphorylation inhibition
by the compounds was calculated using the following formula:
100% ꢀ [(negative control)/(positive control ꢀ negative control)].
The IC50 was obtained from curves of percentage inhibition with
eight concentrations of compound. As the contaminants in the en-
zyme preparation are fairly low, the majority of the signal detected
by the anti-phosphotyrosine antibody is from EGFR or HER-2.
16. Chapman, J. D. N. Eng. J. Med. 1979, 301, 1429.
17. Lobb, K. L.; Hipskind, P. A.; Aikins, J. A.; Alvarez, E.; Cheung, Y. Y., et al J. Med.
Chem. 2004, 47, 5367.
18. Corbett, T. H.; White, K.; Polin, L.; Kushner, J.; Paluch, J.; Shih, C.; Grossman, C.
S. Invest. New. Drugs. 2003, 21, 33.
4.5. Antiproliferation assay
19. Mirzaei, J.; Amini, M.; Pirelahi1, H.; Shafiee, A. J. Heterocycl. Chem. 2008, 45,
921.
20. Huey, R.; Morris, G. M.; Olson, A. J.; Goodsell, D. S. J. Comput. Chem. 2007, 28,
1145.
21. Wheeler, S. E.; Houk, K. N. J. Am. Chem. Soc. 2009, 131, 3126.
22. Tsou, H. R.; Mamuya, N.; Johnson, B. D.; Reich, M. F.; Gruber, B. C.; Ye, F.;
Nilakantan, R.; Shen, R.; Discafani, C.; DeBlanc, R.; Davis, R.; Koehn, F. E.;
Greenberger, L. M.; Wang, Y. F.; Wissner, A. J. Med. Chem. 2001, 44, 2719.
23. Boumendjel, A.; Boccard, J.; Carrupt, P. A.; Nicolle, E.; Blanc, M.; Geze, A.;
Choisnard, L.; Wouessidjewe, D.; Matera, E. L.; Dumontet, C. J. Med. Chem. 2008,
51, 2307.
The antiproliferative activities of the prepared compounds (10–
14 and 20–24) against A549 adenocarcinomic human alveolar ba-
sal epithelial cell line and B16-F10 mouse melanoma cell line were
evaluated as described elsewhere with some modifications.23 Tar-
get tumor cell lines were grown to log phase in RPMI 1640 medium
supplemented with 10% fetal bovine serum. After diluting to
2 ꢂ 104 cells mLꢀ1 with the complete medium, 100
lL of the ob-
tained cell suspension was added to each well of 96-well culture